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WO2024218296A1 - Combination treatments comprising administration of leucine-rich repeat kinase 2 (lrrk2) inhibitors - Google Patents

Combination treatments comprising administration of leucine-rich repeat kinase 2 (lrrk2) inhibitors Download PDF

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Publication number
WO2024218296A1
WO2024218296A1 PCT/EP2024/060716 EP2024060716W WO2024218296A1 WO 2024218296 A1 WO2024218296 A1 WO 2024218296A1 EP 2024060716 W EP2024060716 W EP 2024060716W WO 2024218296 A1 WO2024218296 A1 WO 2024218296A1
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Prior art keywords
compound
mmol
methyl
nitro
pharmaceutically acceptable
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French (fr)
Inventor
Thomas Jensen
Thomas Andersen
Mikkel JESSING
Wanwan YU
David Rodriguez DIAZ
Jacob Nielsen
Christopher Richard Jones
Mikkel Fog Jacobsen
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H Lundbeck AS
Vernalis R&D Ltd
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H Lundbeck AS
Vernalis R&D Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention provides a combination treatment comprising administration of 5 a first compound which is a Leucine-rich repeat kinase 2 (LRRK2) inhibitor and a second com- pound, which compound is useful in the treatment of synucleinopathies, such as Parkinson’s disease.
  • the combination treatment of the invention is for the treatment of a patient suffering from a synucleinopathy disorders, such as Parkinson’s disease.
  • Background of the Invention 10 Parkinson’s disease (PD) is a neurodegenerative disease. It is the second most common neurodegenerative disease after Alzheimer’s disease and affects more than 1% of the population above the age of 65.
  • Parkinson’s disease is clinically characterised by resting tremor, bradykinesia, muscular rigidity and postural instability. In addition to motor symptoms, other symptoms such as neuropsychiatric symptoms are also present in many patients, and in late 15 stages of the disease, Parkinson’s disease dementia commonly develops.
  • Pathologically the disease is characterised by loss of dopaminergic neurons with consequent decrease in dopamine levels in the brain and by aggregation of the protein ⁇ -synuclein in the dopaminergic neurons. These aggregations, called Lewy bodies, are composed of insoluble ⁇ -synuclein phosphorylated at serine-129 and ubiquitin.
  • LRRK2 Leucine-rich repeat kinase 2
  • LRRK2 Parkinsonism Several such pathogenic variants have been identified including G2019S, I2020T, N1437H, R1441C, R1441G, R1441H and Y1699C (Shu et al., A Comprehensive Analysis of Population Differences in LRRK2 Variant Distribution in Parkinson's Disease, Front Aging Neurosci., 11:13, 2019; Chittoor-Vinod et al., Genetic and Environmental Factors Influence the Pleomorphy of LRRK2 Parkinsonism, Int. J. Mol. Sci., 2021, 22, 1045).
  • the most common 5 pathogenic form of LRRK2-associated Parkinson’s disease is the amino acid substitution G2019S in the kinase domain of the LRRK2 protein.
  • G2019S Parkinson’s disease is inherited in an autosomal dominant fashion suggesting a gain-of-function mutation of the LRRK2 protein.
  • biochemical studies have shown that both G2019S and other pathogenic LRRK2 variants lead to an increased kinase activity of LRRK2 (West et al, Parkinson’s disease- 10 associated mutations in leucine-rich repeat kinase 2 augment kinase activity, Proc. Nat. Acad. Sci, 102, 16842-16847, 2005; Chittoor-Vinod et al., Genetic and Environmental Factors Influence the Pleomorphy of LRRK2 Parkinsonism, Int. J. Mol. Sci., 2021, 22, 1045).
  • Parkinson’s disease associated with LRRK2 mutations are very similar to those of idiopathic Parkinson’s disease (Trinh et al., A comparative study of Parkinson's disease 15 and leucine-rich repeat kinase 2 p.G2019S parkinsonism, Neurobiol. Aging., 35(5), 1125-31, 2014). This strongly suggests a causal involvement of overactive LRRK2 in the pathogenesis of Parkinson’s disease in patients with such activating mutations in LRRK2 and that inhibitors of LRRK2 could be used as disease modifying treatment in familiar Parkinson’s disease.
  • LRRK2 variants with lower but significant association with Parkinson’s disease showing that LRRK2 also contributes to idiopathic Parkinson’s disease.
  • LRRK2 promotor region where the Parkinson’s disease associated variants appear to be associated with increased LRRK2 expression at least in some cell types.
  • Nalls et al. Identification of novel risk loci, causal insights, and heritable risk for 25 Parkinson's disease: a meta-analysis of genome-wide association studies, Lancet Neurol,2019, 18, 1091–1102; Sun et al., Genetic Variants Associated With Neurodegenerative Diseases Regulate Gene Expression in Immune Cell CD14+ Monocytes, Front Genet., 2018, 18, 9:666; Langston et al., Association of a Common Genetic Variant with Parkinson’s Disease is Propagated through Microglia, bioRxiv, 20
  • LRRK2 Parkinson’s disease risk variants including the A419V and G2385R that are common in the Asian population (Shu et al., A Comprehensive Analysis of Population Differences in LRRK2 Variant Distribution in Parkinson's Disease, Front Aging Neurosci., 11:13, 2019).
  • LRRK2 LRRK2 N551K R1398H variant
  • Wang et al. Understanding LRRK2 kinase activity in preclinical models and human subjects through quantitative analysis of LRRK2 and pT73 Rab10, Scientific Reports, 2021, 11:12900
  • LRRK2 affects trafficking of lysosomes and other vesicles through phosphorylation of RAB GTPases, and PD-associated genes are enriched for genes involved in lysosomal function and autophagy (Chang et al., A meta-analysis of genome-wide association studies identifies 17 new Parkinson's disease risk loci, Nat Genet., 2017, 49(10): 1511–1516).
  • LRRK2 activity may impact ⁇ -synuclein pathology development after seeding with ⁇ -synuclein (O’Hara et al., LRRK2 and ⁇ -Synuclein: Distinct or Synergistic Players in Parkinson's Disease?, Front Neurosci., 2020, 17;14:577).
  • LRRK2 inhibitors for treatment of 20 Parkinson’s disease
  • LRRK2 is highly expressed in white blood cells and spleen suggesting a potential for LRRK2 inhibitors for treatment of aberrant immune responses.
  • LRRK2 inflammatory bowel diseases 25 including Crohn’s disease and leprosy
  • diseases particularly inflammatory bowel diseases 25 including Crohn’s disease and leprosy
  • LRRK2 inhibitors may have potential for treatment of these diseases.
  • LRRK2 inhibitors The historic development of LRRK2 inhibitors is well described in the literature (Delgado et al., N-bridged 5,6-bicyclic pyridines: Recent applications in central nervous system disorders, European Journal of Medicinal Chemistry 97 (2015) 719-731); Xiao Ding & Feng Ren (2020) Leucine-rich repeat kinase 2 inhibitors: a patent review (2014-present), Expert Opinion on 5 Therapeutic Patents, 30:4, 275-286).
  • the current standard of care treatment paradigms for this type of diseases are merely symptomatic treatments which are not efficacious in all patients, and which does not modify the 20 disease progression. Hence, there remains a need for alternative methods of treatment of such diseases.
  • Summary of the invention The invention provides the combined use of certain 5,7-(azenometheno)dipyrazolo[3,4- b:5',1'-g][1]oxa[4,6,8]triazacycloundecine compounds which are LRRK2 inhibitors, and as such 25 are useful to treat synucleinopathies, such as Parkinson’s disease, and a second compound that is useful in the treatment of the same diseases.
  • a first compound of formula I or a pharmaceutically acceptable salt thereof, wherein: R 1 is CH 2 R 4 or R 4 ; R 2 is a C 1 -C 3 alkyl, an isotopically labelled C 1 -C 3 alkyl, a C 3 -C 6 cycloalkyl, or a C 1 -C 3 haloalkyl; R 3 is halogen, cyano, a O-C 1 -C 3 haloalkyl, a C 1 -C 3 haloalkyl, a C 3 -C 6 cycloalkyl, or a C 1 -C 3 alkyl; 5 R 4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen; a C 1 -C 3 alkyl, a C 1 -C 3 cyanoalkyl, a C 1 -C 3 haloal
  • the invention provides a pharmaceutical composition comprising a first compound of formula I as disclosed herein or pharmaceutically acceptable salts thereof, a second compound or a pharmaceutically acceptable salt thereof, which second compound is20 useful in the treatment of synucleinopathies, and one or more pharmaceutically acceptable car- riers.
  • the invention provides a first compound of formula I as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceuti-25 cally acceptable salt thereof, which second compound is useful in the treatment of synucleinop- athies, for combined use in the treatment of a synucleinopathy, such as Parkinson’s disease.
  • the invention provides combined use of a first compound of formula I as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, in the manufacture of a medicament for the treatment of a synucleinopathy, such as Parkinson’s disease.
  • the invention provides combined use of a first compound of formula I as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, in the treatment of a synucleinopathy, such as Parkinson’s disease.
  • the invention relates to a method for the treatment of synucleinop- athies such as Parkinson disease, the method comprising the administration of a therapeutically effective amount of a first compound of formula I as disclosed herein or pharmaceutically ac- ceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, to a patient in need15 thereof.
  • a first compound which is a LRRK2 inhibitor and a second compound or a pharmaceutically acceptable salt thereof, which combined use is for the treatment of synucleinopathies selected from Lewy body dementia, multiple system at-20 rophy or Parkinson’s disease.
  • Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl and the like.
  • isotopically labelled alkyl group means that either the carbon or the hydrogen atom(s) in the alkyl group is replaced with a corresponding isotope such as 13 C 5 and/or 14 C for carbon atom(s), or deuterium or tritium for hydrogen atom(s).
  • the hydrogen atoms of the alkyl group are all replaced by deuterium.
  • Repre- sentative examples of isotopically labelled alkyl include but are not limited to -CD 3 , -CD 2 CD 3 .
  • the isotopically labelled C 1- C 3 -alkyl is -CD 3 .
  • alkylene refers to a divalent group derived from a straight 10 or branched chain hydrocarbon of 1 to 10 carbon atoms, for example, of 2 to 5 carbon atoms.
  • alkylene examples include, but are not limited to, -CH 2 -, -CH 2 CH 2 -, - CH 2 CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 CH 2 -, -CH(CH 3 )- and - CH 2 CH 2 CH 2 CH 2 CH 2 -.
  • alkoxy refers to a group of formula -O-alkyl, wherein alkyl is 15 defined as above.
  • C 1 -C 3 -alkoxy is intended to indicate a hydrocarbon having 1, 2 or 3 carbon atoms.
  • alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, isobutoxy, t-butoxy and the like.
  • haloalkyl or “haloalkoxy” is intended to refer to an alkyl or alkoxy group as defined hereinabove with 1, 2 or 3 hydrogens replaced by a halogen.
  • Representative examples 20 include but are not limited to CH 2 F, CHF 2 CF 3 , OCF 3 , OCH 2 F, and OCHF 2 .
  • haloalkoxy may also be referred to as “O-haloalkyl”, such as for example “O-C 1 -C 3 haloalkyl”.
  • fluoroalkyl is intended to refer to an alkyl group as defined here- inabove, with 1, 2, or 3 hydrogens replaced by fluoro. Representative examples include but are not limited to -CF 3 . 25
  • halogen is intended to indicate members of the 7 th main group of the periodic table of the elements, such as fluoro, bromo and chloro.
  • heteroatom is intended to mean sulfur, oxygen or nitrogen.
  • cyano as used herein, means at least one -CN group is appended to the par- ent molecular moiety.
  • cyanoalkyl as used herein is intended to indicate an alkyl group as defined herein, wherein at least one -CN group is appended to the parent molecular moiety.
  • cyclic as used herein refers to any cyclic structure, including heterocyclic, aromatic and heteroaromatic non-fused ring systems.
  • membered is meant to denote the number of skeletal atoms that constitute the ring.
  • pyridyl, pyranyl, and pyrimidinyl are six-membered rings and pyrrolyl
  • tetrahydrofuranyl are five-membered rings.
  • cycloalkyl refers to a carbocyclic ring system containing 5 three to ten carbon atoms, zero heteroatoms and zero double bonds.
  • the cycloalkyl may be monocyclic or bicyclic, wherein the bicyclic ring is joined bridged, fused, or spirocyclic.
  • heterocycle refers to saturated or unsaturated nonaromatic rings containing from 4 to 7 ring atoms where one or more of the ring atoms are heteroatoms.
  • the heterocycle may be monocy-10 protest or bicyclic, wherein the bicyclic ring is joined bridged, fused, or spirocyclic.
  • the term "heterocycle”, “heterocyclic” and “heterocyclyl” may refer to a saturated or unsaturated nonaromatic ring containing 8 ring atoms where one or more of the ring atoms are heteroatoms.
  • Such 8-membered heterocycle is a bicyclic ring, wherein the bicyclic ring is joined bridged, fused, or spirocyclic.
  • the term "therapeutically effective amount" of a compound is intended to indicate an amount sufficient to alleviate or partially arrest the clinical manifesta- tions of a given disease and its complications in a therapeutic intervention comprising the ad- ministration of said compound.
  • An amount adequate to accomplish this is defined as “therapeu- tically effective amount”.
  • Effective amounts for each purpose will depend on the severity of the 20 disease or injury as well as the weight and general state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, e.g. by con- structing a matrix of values and testing different points in the matrix, which is all within the ordinary skills of a trained physician.
  • treatment means the management and 25 care of a patient for the purpose of combating a disease.
  • the term is intended to include the full spectrum of treatments for a given disease from which the patient is suffering, such as admin- istration of the active compound to alleviate the symptoms or complications, to delay the pro- gression of the disease, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease.
  • the patient to be treated is preferably a mammal, in particular a human30 being.
  • “disease” can be used synonymous with disorder, condition, mal- function, dysfunction and the like.
  • first and second compounds may be administered simultaneously or with a time gap between the administrations of the two com- pounds.
  • the first and second compounds may be administered either as part of the same phar- maceutical formulation or composition, or in separate pharmaceutical formulations or compo- sitions.
  • the first and second compounds may be administered on the same day or on different 20 days.
  • the two compounds may be administered by the same route, such as by oral administration, or by depot, or by intramuscular or intraperitoneal injection, or by intravenous injection; or by different routes wherein one compound is for example administered orally or placed by depot and the other compound is injected, or wherein one compound is for example placed by depot and the other is administered orally or injected.
  • the two compounds may be administered by the same25 dosage regimes or interval, such as once or twice daily, weekly, or monthly; or by different dos- age regimes or intervals for example wherein one is administered once daily and the other is administered twice daily, weekly or monthly.
  • a patient to be treated may already be in treatment with the second compound or a pharmaceutically acceptable salt thereof, which is useful in the treatment of a synucleinopathy when the treatment with a first 30 compound according to formula I, or a pharmaceutically acceptable salt thereof, is initiated.
  • the patient may already be in treatment with a first compound according to formula I or a pharmaceutically acceptable salt thereof when treatment with the second com- pound or a pharmaceutically acceptable salt thereof, which compound is useful in the treatment of a synucleinopathy is initiated.
  • a first compound of formula I or a pharmaceutically acceptable salt thereof, wherein: 10 R 1 is CH 2 R 4 or R 4 ; R 2 is a C 1 -C 3 alkyl, an isotopically labelled C 1 -C 3 alkyl, a C 3 -C 6 cycloalkyl, or a C 1 -C 3 haloalkyl; R 3 is halogen, cyano, a O-C 1 -C 3 haloalkyl, a C 1 -C 3 haloalkyl, a C 3 -C 6 cycloalkyl, or a C 1 -C 3 alkyl; R 4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox-15 ygen and nitrogen; a C 1 -C 3 alkyl, a C 1 -C 3 cyanoalkyl, a C 1 -C 3 haloalkyl, wherein: 10 R 1 is CH 2 R 4 or R
  • R 1 is CH 2 R 4 . In some embodiments of the invention, R 1 is R 4 . In some embodiments of the invention, the first compound is a compound of formula Ia, or a pharmaceutically acceptable salt thereof, wherein: .
  • R 2 is selected from a C 1 -C 3 alkyl, an isotopically 5 labelled C 1 -C 3 alkyl or a C 3 -C 6 cycloalkyl. In some embodiments of the invention, R 2 is selected from -CH 3 , -CH 2 CH 3 , -CD 3 , or cyclo- propyl. In some embodiments of the invention, R 2 is C 1 -C 3 alkyl.
  • R 2 is methyl. 10 In some embodiments of the invention, R 2 is ethyl. In some embodiments of the invention, R 2 is an isotopically labelled C 1 -C 3 alkyl. In some embodiments of the invention, R 2 is selected from the group consisting of -CD 3 or -CD 2 CD 3 . 15 In some embodiments of the invention, R 2 is -CD 3 . In some embodiments of the invention, R 2 is cyclopropyl. In some embodiments of the invention, R 3 is C 1 -C 3 haloalkyl. 20 In some embodiments of the invention, R 3 is CF 3 . In some embodiments of the invention, R 3 is halogen.
  • R 3 is chloro. In some embodiments of the invention, R 3 is bromo. 25 In some embodiments of the invention, R 3 is cyano. In some embodiments of the invention, R 3 is a C 3 -C 6 cycloalkyl. In some embodiments of the invention, R 3 is cyclopropyl.
  • R 4 is a 4- to 6-membered heterocycle having one oxygen atom, wherein the 4- to 6-membered heterocycle is unsubstituted or substituted with one group selected from the group consisting of cyano, deuterium, halogen, C 1 -C 3 alkyl, an isotopically labelled C 1 -C 3 alkyl, O-C 1 -C 3 haloalkyl, O-C 1 -C 3 alkyl, or C 1 -C 3 haloalkyl.
  • R 4 is a 4- to 6-membered heterocycle having one oxygen atom, wherein the 4- to 6-membered heterocycle is unsubstituted or substituted with one group selected from the group consisting of deuterium, halogen, C 1 -C 3 alkyl, or an iso- topically labelled C 1 -C 3 alkyl.
  • R 4 is 6-membered heterocycle having one oxy- gen atom, wherein the 6-membered heterocycle is unsubstituted or substituted with one group selected from the group consisting of deuterium, halogen, C 1 -C 3 alkyl, or an isotopically labelled C 1 -C 3 alkyl.
  • R 4 is 6-membered heterocycle having one oxy- gen atom, wherein the 6-membered heterocycle is unsubstituted or substituted with two groups independently selected from the group consisting of deuterium, halogen, C 1 -C 3 alkyl, or an iso- topically labelled C 1 -C 3 alkyl.
  • R 4 is selected from the group consisting of: 25 In some embodiments of the invention, R 4 is selected from the group consisting of: CH 3 ,
  • R 4 is tetrahydropyran. In some embodiments of the invention, R 4 is unsubstituted tetrahydropyran. 5 In some embodiments of the invention, R 4 is tetrahydro-2H-pyran-4-yl. In some embodiments of the invention, R 4 is tetrahydro-2H-pyran-3-yl. In some embodiments of the invention, R 4 is 4-oxaspiro[2.5]octan-7-yl. In some embodiments of the invention, the 8-membered heterocycle is an unsubsti-10 tuted 8-membered spirocyclic heterocycle.
  • the bicyclic 8-membered heterocycle contains one oxygen. In some embodiments of the invention, the bicyclic 8-membered heterocycle contains one oxygen and is a spirocyclic heterocycle. 15
  • the first compound is a compound of formula Ib, or a pharmaceutically acceptable salt thereof, wherein: R 2 is a C 1 -C 3 alkyl, an isotopically labelled C 1 -C 3 alkyl, a C 3 -C 6 cycloalkyl, or a C 1 -C 3 haloalkyl; R 4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen, a C 1 -C 3 alkyl, a C 1 -C 3 cyanoalkyl, a C 1 -C 3 haloalkyl, or a C 3 -C 6 cycloalkyl; wherein each heterocycle or cycloal
  • R 2 is selected from -CH 3 , -CH 2 CH 3 , -CD 3 , or cy- clopropyl.
  • the first compound is a compound of formula Ic, or a pharmaceutically acceptable salt thereof, wherein: R 4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen, a C 1 -C 3 alkyl, a C 1 -C 3 cyanoalkyl, a C 1 -C 3 haloalkyl, or a C 3 -C 6 cycloalkyl;15 wherein each heterocycle or cycloalkyl is unsubstituted or substituted with 1 group se- lected from the groups selected from cyano, deuterium, halogen, a C 1 -C 3 alkyl, an isotopically labelled C 1 -C 3 alkyl, a O-C 1 -C 3
  • the first compound is a compound of formula 20 Id, or a pharmaceutically acceptable salt thereof, wherein: R 4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen, a C 1 -C 3 alkyl, a C 1 -C 3 cyanoalkyl, a C 1 -C 3 haloalkyl, or a C 3 -C 6 cycloalkyl; wherein each heterocycle or cycloalkyl is unsubstituted or substituted with 1 group se- 25 lected from the groups selected from cyano, deuterium, halogen, a C 1 -C 3 alkyl, an isotopically labelled C 1 -C 3 alkyl, a O-C 1 -C 3 haloalkyl, a O-C 1 -C 3 alkyl, or a C 1 -C 3 haloalkyl.
  • R 4 is a 4- to 7-membered
  • the 4- to 7-membered heterocycle is an unsub- stituted 6-7 membered spirocyclic heterocycle. In some embodiments of the invention, the 4- to 7-membered heterocycle is an unsub- stituted 6-7 membered bridged heterocycle.
  • the first compound is selected from the group consisting of : , , 10 , , , , 1 In some embodiments of the invention, the first compound is selected from the group consisting of: 8-Chloro-2,3-dimethyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo-[3,4- b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 5 8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; (R)-8-chloro-3-methyl-2-(tetrahydro-2H-pyran-3-yl
  • the first compound is , or a pharmaceutically acceptable salt. In some embodiments of the invention, the first compound is . In some embodiments of the invention, the first compound is , or a pharmaceutically acceptable salt. In some embodiments of the invention, the first compound is , or a pharmaceutically acceptable salt. In some embodiments of the invention, the first compound is 5 . In some embodiments of the invention, the first compound is , or a pharmaceutically acceptable salt thereof. In some embodiments of the invention, the first compound is , or a pharmaceutically acceptable salt thereof. 10 In some embodiments of the invention, the first compound is , or a pharmaceutically acceptable salt thereof. In some embodiments of the invention, the first compound is ,or a pharmaceutically acceptable salt thereof.
  • the first compound is ,or a pharmaceutically acceptable salt thereof. 5 In some embodiments of the invention, the first compound is , or a pharmaceutically acceptable salt thereof. In some embodiments of the invention, the first compound is 10 , or a pharmaceutically acceptable salt thereof. In some embodiments of the invention, the first compound is . 15 In some embodiments of the invention, the first compound is . In some embodiments of the invention, the first compound is . In some embodiments of the invention, the first compound is 5 In some embodiments of the invention, the first compound is . In some embodiments of the invention, the first compound is . 10 In some embodiments of the invention, the first compound is . In some embodiments of the invention, the first compound is .
  • the first compound is . In some embodiments of the invention, the first compound is 5 . In some embodiments of the invention, the first compound is . 10 In a further aspect, the invention provides a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treat- ment of synucleinopathies, for combined use in the treatment of a synucleinopathy, such as Parkinson’s disease.
  • the invention provides a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treat- ment of Parkinson’s disease, for combined use in the treatment of Parkinson’s disease.
  • the invention provides combined use of a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, in the manufacture of a medicament for the treat- 5 ment of a synucleinopathy, such as Parkinson’s disease.
  • the invention provides combined use of a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of Parkinson’s disease, in the manufacture of a medicament for the 10 treatment of Parkinson’s disease.
  • the invention provides combined use of a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is 15 useful in the treatment of synucleinopathies, in the treatment of a synucleinopathy, such as Parkinson’s disease.
  • the invention provides combined use of a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is 20 useful in the treatment of Parkinson’s disease, in the treatment of Parkinson’s disease.
  • the invention relates to a method for the treatment of synucleinop- athies such as Parkinson disease, the method comprising the administration of a therapeutically effective amount of a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or25 pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically ac- ceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, to a patient in need thereof.
  • the invention relates to a method for the treatment of Parkinson disease, the method comprising the administration of a therapeutically effective amount of a30 first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically accepta- ble salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of Parkinson’s disease, to a patient in need thereof.
  • the invention relates to a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, for use in therapy.
  • the synucleinopathy is selected from the group consisting of Lewy body dementia, multiple system atrophy, and Parkinson’s disease.
  • the synucleinopathy is parkinson’s disease.
  • the Parkinson’s disease is idiopathic Parkinson’s disease, sporadic Parkinson’s disease, Parkinson’s disease in patients carrying a G2019S mutation in LRRK2, or 10 Parkinson’s disease in patients carrying one or more LRRK2 non-coding variants selected from rs76904798-T and Rs1491942-G.
  • the Parkinson’s disease is idiopathic Parkinson’s disease, sporadic Parkinson’s disease or Parkinson’s disease in patients carrying one or more mutated forms of LRRK2 selected from G2019S, I2020T, M1646T, G2385R, A419V, N551K, R1398H, K1423K,15 R1441G, R1441H, R1441C, R1628P, S1647T, Y1699C, I2020T and Y2189C.
  • the Parkinson’s disease is idiopathic Parkinson’s disease, sporadic Parkinson’s disease or Parkinson’s disease in patients carrying a G2019S mutation in LRRK2.
  • Parkinson’s disease is idiopathic Parkinson’s disease, sporadic Parkinson’s disease or Parkinson’s disease in patients carrying one or more LRRK2 non-coding20 variants selected from rs76904798-T and Rs1491942-G.
  • the synucleinopathy is characterized by increased LRRK2 kinase ac- tivity or by expression of one or more mutated forms of LRRK2 selected from G2019S, I2020T, M1646T, G2385R, A419V, N551K, R1398H, K1423K, R1441G, R1441H, R1441C, R1628P, S1647T, Y1699C, I2020T and Y2189C or one or more LRRK2 non-coding variants alone or in combination25 selected from rs76904798-T and Rs1491942-G.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a first compound of formula I as disclosed herein or pharmaceutically acceptable salts thereof, a second compound or a pharmaceutically acceptable salt thereof, which second compound is30 useful in the treatment of synucleinopathies, and one or more pharmaceutically acceptable car- riers.
  • the second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is symptomatic drugs or disease modifying compounds. 5
  • the second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is selected from anti- cholinergic agents, dopaminergic agents, and adenosine receptor antagonists.
  • the anticholinergic agent is selected from the group comprising tri- hexyphenidyl, biperiden, metixene, procyclidine, profenamine, dexetimide, phenglutarimide,10 mazaticol, bornaprine, tropatepine, etanautine, orphenadrine, benzatropine, and etybenzatro- pine.
  • the dopaminergic agent is selected from the group comprising dopa- mine, levodopa, carbidopa, levodopa/carbidopa, melevodopa, etilevodopa, foslevodopa, aman- tadine, bromocriptine, pergolide, dihydroergocryptine mesylate, ropinirole, pramipexole, caber-15 goline, apomorphine, piribedil, rotigotine, selegiline, rasagiline, safinamide, tolcapone, entaca- pone, budipine, and opicapone.
  • the adenosine receptor antagonist is istradefylline.
  • the second compound or a pharmaceutically acceptable salt 20 thereof, which second compound is useful in the treatment of synucleinopathies is selected from caspase inhibitors, calpain inhibitors, LRRK2 inhibitors, BACE1 inhibitors, antibodies against any form of A ⁇ peptides, inflammation inhibitors, LAG-3 antibodies, molecules inhib- iting alpha-synuclein aggregation, nicotine, caffeine, Monoamine oxidase B inhibitor, Levo- dopa/carbidopa, Dopamine agonists, COMT inhibitors, A2A antagonists, anti-alphasynuclein25 antibodies, Mitogen-activated protein kinase 14 inhibitors, USP30 inhibitors, Beta adrenore- ceptor agonists, dopamine D1 PAMs, Toll-like receptor 2 antagonists, Carnitine palmitoyl transferase 1 inhibitors, Glutamate 5
  • the second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is selected from group comprising pralnacasan, bapineuzemab, solanezumab, gantenerumab, crenezumab, aducanu- mab, glunozumab, prasinezumab, miglustat, eliglustat, ropinerole, neflamapimod, bosutinib, 10 oxafuramine, nadolol, nilotinib, vodobatinib, clenbuterol, mevidalen, emeramide, tomaralimab, mitometin, dipraglurant, ezeprogind, blarcamesine, buntanetap, renzapride, tavapadon, prami- prexole, salbutamol, Infudopa, alirinetide, antraquinolol,
  • the first compounds of the present invention may have one or more asymmetric centres and it is intended that any optical isomers (i.e. enantiomers or diastereomers) as separated, 20 pure or partially purified optical isomers and any mixtures thereof including racemic mixtures, i.e. a mixture of stereoisomers, are included within the scope of the invention.
  • any optical isomers i.e. enantiomers or diastereomers
  • any optical isomers i.e. enantiomers or diastereomers
  • any mixtures thereof including racemic mixtures i.e. a mixture of stereoisomers
  • one embodiment of the invention relates to a first compound of the invention having an enantiomeric excess (ee) 25 of at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 96%, preferably at least 98%.
  • Racemic forms can be resolved into the optical antipodes by known methods, for exam- ple by separation of diastereomeric salts thereof with an optically active acid and liberating the optically active amine compound by treatment with a base.
  • Another method for resolving race- 30 mates into the optical antipodes is based upon chromatography on an optically active matrix.
  • the first compounds of the present invention may also be resolved by the formation of diastere- omeric derivatives.
  • optical isomers may be prepared from optically active starting materials. Abso- lute stereochemistry may be determined by methods known to the skilled person, such as vi- brational circular dichroism (VCD) Spectroscopic analysis. 5
  • VCD vi- brational circular dichroism
  • molecules having a bond with restricted rotation may form geometric isomers. These are also intended to be included within the scope of the present in-10 vention.
  • some of the first compounds of the present invention may exist in differ- ent tautomeric forms and it is intended that any tautomeric forms that the compounds are able to form are included within the scope of the present invention. Included in this invention are also isotopically labelled first compounds, wherein one or 15 more atoms are represented by an atom of the same element having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (e.g., 2 H, 11 C, 1 3 C, 15 N, 18 F and the like). Particular mention is made of 2 H substituted compounds i.e.
  • com- pounds wherein one or more H atoms are represented by deuterium.
  • a first compound of formula I, Ia, Ib, Ic, Id, A or Aa 20 is isotopically labelled.
  • one or more of the hydrogen atoms of the first compound of formula I, Ia, Ib, Ic, Id, A or Aa are represented by deuterium. It is recognized that elements are present in natural isotopic abundances in most synthetic com- pounds and result in inherent incorporation of deuterium. However, the natural isotopic abun- dance of hydrogen isotopes such as deuterium is immaterial (about 0.015%) relative to the de-25 gree of stable isotopic substitution of compounds indicated herein.
  • desig- nation of an atom as deuterium at a position indicates that the abundance of deuterium is sig- nificantly greater than the natural abundance of deuterium.
  • Any atom not designated as a par- ticular isotope is intended to represent any stable isotope of that atom, as will be apparent to the ordinarily skilled artisan.
  • designation of a position as “D” in a compound has a minimum deuterium incorporation of greater than about 60% at that position such as greater than about 70% at that position such as greater than about 80% at that position such as greater than about 85% at that position.
  • first and second compounds of this invention are generally utilized as the free sub- stance or as a pharmaceutically acceptable salt thereof.
  • a first compound of formula I, Ia, Ib, Ic, Id, A or Aa contains a free base
  • such salts may be prepared in a conventional manner by treating a solution or suspension of a free base of formula I, Ia, Ib, Ic, Id, A or Aa with a molar equivalent of a pharmaceutically acceptable acid.
  • Pharmaceutically acceptable salts in the present context are intended to indicate non- toxic, i.e. physiologically acceptable salts.
  • the term pharmaceutically acceptable salts includes salts formed with inorganic and/or organic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitrous acid, sulphuric acid, benzoic acid, citric acid, gluconic acid, lactic acid,15 maleic acid, succinic acid, tartaric acid, acetic acid, propionic acid, oxalic acid, maleic acid, fu- maric acid, glutamic acid, pyroglutamic acid, salicylic acid, salicylic acid and sulfonic acids, such as methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid and benzene-sulfonic acid.
  • Di- or tri-acids i.e. acids containing two or three acidic hydro- gens, such as phosphoric acid, sulphuric acid, fumaric acid and maleic acid.
  • Di- and tri-acids may 20 form 1:1, 1:2 or 1:3 (tri-acids) salts, i.e. a salt formed between two or three molecules of the compound of the present invention and one molecule of the acid.
  • pharmaceutically acceptable salts include salts formed with inorganic and/or organic bases, such as alkali metal bases, such as sodium hydroxide, lithium hydroxide, potas- sium hydroxide, alkaline earth bases, such as calcium hydroxide and magnesium hydroxide, and 25 organic bases, such as trimethylamine.
  • alkali metal bases such as sodium hydroxide, lithium hydroxide, potas- sium hydroxide
  • alkaline earth bases such as calcium hydroxide and magnesium hydroxide
  • 25 organic bases such as trimethylamine.
  • Some of the bases listed above are di- or tri-bases, i.e. bases able to receive two or three acidic hydrogens, such as calcium hydroxide and magnesium hydroxide.
  • Di- and tri-bases may form 1:1 or 1:2 salts, i.e. a salt formed between two molecules of the compound of the present invention and one molecule of the base. Additional examples of useful acids and bases to form pharmaceutically acceptable salts 30 can be found e.g.
  • first or second compounds or pharmaceutically acceptable salts thereof may be in a composition as the sole active ingredient or in combination with other active ingredients. Additionally, one or more pharmaceutically acceptable carriers or diluents may be 5 in the composition.
  • compositions may be specifically formulated for administration by any suitable route such as the oral, rectal, nasal, pulmonary, topical (including buccal and sub- lingual), transdermal, intracisternal, intraperitoneal, vaginal and parenteral (including subcuta- neous, intramuscular, intrathecal, intravenous and intradermal) route, the oral route being pre- 10 ferred. It will be appreciated that the preferred route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingre- dominant chosen.
  • Pharmaceutical compositions for oral administration include solid dosage forms such as capsules, tablets, dragées, pills, lozenges, powders and granules. Where appropriate, they can15 be prepared with coatings.
  • Liquid dosage forms for oral administration include solutions, emulsions, suspensions, syrups and elixirs.
  • Pharmaceutical compositions for parenteral administration include sterile aqueous and nonaqueous injectable solutions, dispersions, suspensions or emulsions as well as sterile pow- 20 ders to be reconstituted in sterile injectable solutions or dispersions prior to use.
  • Other suitable administration forms include suppositories, sprays, ointments, creams, gels, inhalants, dermal patches, implants, etc.
  • the first compounds of the invention are administered in a unit dosage form containing said compound in an amount of about 0.1 to 500 mg, such as 10 mg, 50 mg, 100 25 mg, 150 mg, 200 mg or 250 mg of a first compound of the present invention.
  • solutions of the compound of the invention in sterile aqueous solution, aqueous propylene glycol, aqueous vitamin E or sesame or peanut oil may be employed.
  • Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • the aqueous solutions are particularly 30 suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents.
  • solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and lower alkyl ethers of cellulose.
  • liquid carriers are syrup, peanut oil, olive oil, phosphor lipids, 5 fatty acids, fatty acid amines, polyoxyethylene and water.
  • compositions formed by combining the first and/or second compound of the invention and the pharmaceuti- cally acceptable carriers are then readily administered in a variety of dosage forms suitable for the disclosed routes of administration.
  • Formulations of the present invention suitable for oral administration may be presented 10 as discrete units such as capsules or tablets, each containing a predetermined amount of the active ingredient, and which may include a suitable excipient.
  • the orally available formulations may be in the form of a powder or granules, a solution or suspension in an aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsion.
  • the preparation may be tablet, e.g.
  • the amount of solid carrier may vary but will usually be from about 25 mg to about 1 g. If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatine capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.
  • 20 Tablets may be prepared by mixing the active ingredient with ordinary adjuvants and/or diluents followed by compression of the mixture in a conventional tabletting machine.
  • adjuvants or diluents comprise corn starch, potato starch, talcum, magnesium stearate, gela- tine, lactose, gums, and the like. Any other adjuvants or additives usually used for such purposes such as colourings, flavourings, preservatives etc. may be used provided that they are compati-25 ble with the active ingredients.
  • Treating diseases As established above, LRRK2 inhibitors may be used in the treatment of Parkinson’s dis- ease and particular mention is made of Parkinson’s disease associated with mutations in LRRK2, such as Gly2019Ser. Moreover, LRRK2 inhibitors are also expected to be useful in the treatment30 of other diseases which are associated with LRRK2.
  • LRRK2 has been identified as a core compo- nent in Lewy bodies and is thus expected to be useful in the treatment of Lewy body dementia [Neuropathol. Appl. Neurobiol., 34, 272-283, 2008].
  • Expression of LRRK2 mRNA is highly enriched in brain, lungs, kidney, spleen and blood suggesting that functional impact of increased LRRK2 activity is likely to be most relevant in pathogenic and pathologic conditions associated with those regions. Support for that notion can be found in studies showing an increased risk of non- skin cancer in LRRK2 Gly2019Ser mutation carriers and especially for renal and lung cancer [Mov. 5 Disorder, 25, 2536-2541, 2010].
  • LRRK2 Over-expression of LRRK2 by chromosomal amplification has also been identified in papillary renal and thyroid carcinomas. Also, genetic association of LRRK2 has been reported for diseases where aberrant responses of the immune system are involved. This is the case for inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis as well as for leprosy [Nat. Genet.42, 1118-1125, 2010; Inflamm. Bowel Dis.16, 557-558, 2010; N. 10 Engl. J. Med.361, 2609-2618, 2009; Inflamm. Bowel Dis].
  • a first compound, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the invention for use in the treat- ment of a disease in the central nervous system selected from Lewy body dementia, multiple system atrophy or Parkinson’s disease.
  • the disease in the central nervous system is Parkinson’s disease.
  • the Parkinson’s disease is idiopathic Parkinson’s disease, sporadic Parkinson’s disease or Parkinson’s disease in patients carrying a G2019S mutation in LRRK2.
  • the Parkinson’s disease is idiopathic Parkinson’s disease.
  • the Parkinson’s disease is sporadic Parkinson’s disease.
  • the Parkinson’s disease is in patients carrying a G2019S mutation in LRRK2.
  • the first compounds, or pharmaceutically acceptable salt thereof as outlined in formula I, Ia, Ib, Ic, Id, A or Aa hereinabove, or compositions comprising said first compounds may be used in the treatment of cancer or an immune related disorder.
  • the cancer diseases may reside in the brain, lungs, kidney and spleen or blood organs such as renal cancer, lung cancer, skin cancer, and papillary renal and thyroid carcinomas.
  • the immune related disorder may in one embodiment be Crohn’s disease, ulcerative colitis, tuberculosis or leprosy.
  • the treatment may be in a patient with an increased LRRK2 kinase activity or carrying one or more mutated forms of LRRK2 such as G2019S, I2020T, M1646T, G2385R, A419V, N551K, R1398H, K1423K, R1441G, R1441H, R1441C, R1628P, S1647T, Y1699C, I2020T or Y2189C.
  • the first compound of the present invention is administered in an amount from about 0.001 mg/kg body weight to about 100 mg/kg body weight per day.
  • daily dosages may be in the range of 0.01 mg/kg body weight to about 50 mg/kg body weight per day. The exact dosages will depend upon the frequency and mode of administration, 5 the sex, the age, the weight, and the general condition of the subject to be treated, the nature and the severity of the condition to be treated, any concomitant diseases to be treated, the desired effect of the treatment and other factors known to those skilled in the art.
  • a typical oral dosage for adults will be in the range of 1-1000 mg/day of a first com- pound, or pharmaceutically acceptable salt thereof of the present invention, such as 1-500 10 mg/day.
  • the first compounds, or pharmaceutically acceptable salt thereof of the present inven- tion may be administered alone as a pure compound or in combination with pharmaceutically acceptable carriers or excipients, in either single or multiple doses.
  • the pharmaceutical compo- sitions according to the invention may be formulated with pharmaceutically acceptable carriers 15 or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 22 nd Edition, Pharmaceutical Press, 2012.
  • excipient”, “carrier”, “diluent”, “adjuvant” and the like are used synonymously and are intended to mean the same.
  • a first compound of formula I or a pharmaceutically acceptable salt thereof, wherein: R 1 is CH 2 R 4 or R 4 ; R 2 is a C 1 -C 3 alkyl, an isotopically labelled C 1 -C 3 alkyl, a C 3 -C 6 cycloalkyl, or a C 1 -C 3 haloalkyl; R 3 is halogen, cyano, a O-C 1 -C 3 haloalkyl, a C 1 -C 3 haloalkyl, a C 3 -C 6 cycloalkyl, or a C 1 -C 3 alkyl; 5 R 4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen; a C 1 -C 3 alkyl, a C 1 -C 3 cyanoalkyl, a C 1 -C 3 haloalkyl, or a C 3 -C 6 cycloalky
  • R 4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen, a C 1 -C 3 alkyl, a C 1 -C 3 cyanoalkyl, a C 1 -C 3 haloalkyl, or a C 3 -C 6 cycloalkyl; wherein each heterocycle or cycloalkyl is unsubstituted or substituted with 1 group se- 5 lected from the groups selected from cyano, deuterium, halogen, a C 1 -C 3 alkyl, an isotopically labelled C 1 -C 3 alkyl, a O-C 1 -C 3 haloalkyl, a O-C 1 -C 3 alkyl, or a C 1 -C 3 haloalky
  • R 1 is CH 2 R 4 or R 4 ;
  • R 2 is a C 1 -C 3 alkyl, an isotopically labelled C 1 -C 3 alkyl, a C 3 -C 6 cycloalkyl, or a C 1 -C 3 haloalkyl;
  • R 3 is halogen, cyano, a O-C 1 -C 3 haloalkyl, a C 1 -C 3 haloalkyl, a C 3 -C 6 cycloalkyl, or a C 1 -C 3 alkyl;
  • R 4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen; a C 1 -C 3 alkyl, a C 1 -C 3 cyanoalkyl, a C 1 -
  • R 2 is selected from a C 1 -C 3 alkyl, an isotopically labelled C 1 -C 3 alkyl or a C 3 -C 6 cycloalkyl.
  • the first and second compound of any of embodiment E1-E9, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa25 and R 2 is methyl.
  • the first and second compound of any of embodiment E1-E9, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R 2 is an isotopically labelled C 1 -C 3 alkyl.
  • the first and second compound of any of embodiment E1-E9, wherein the first compound 10 or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R 2 is selected from the group consisting of -CD 3 or -CD 2 CD 3 .
  • the first and second compound of any of embodiment E1-E9, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa15 and R 2 is -CD 3 .
  • the first and second compound of any of embodiment E1-E18, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R 3 is C 1 -C 3 haloalkyl. 25 E20.
  • the first and second compound of any of embodiment E1-E18, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R 3 is CF 3 .
  • the first and second compound of any of embodiment E1-E18, wherein the first compound 30 or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R 3 is halogen.
  • the first and second compound of any of embodiment E1-E18, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and, R 3 is chloro. 5 E23.
  • the first and second compound of any of embodiment E1-E18, wherein the first compound 10 or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R 3 is cyano.
  • the first and second compound of any of embodiment E1-E18, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R 3 is cyclopropyl. 20 E27.
  • the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R 4 is a 4- to 6-membered heterocycle having one oxygen atom, wherein the 4- to 6-mem- bered
  • the first and second compound of any of embodiment E1-E26 wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R 4 is 6-membered heterocycle having one oxygen atom, wherein the 6-membered hetero- cycle is unsubstituted or substituted with one group selected from the group consisting of deu- 5 terium, halogen, C 1 -C 3 alkyl, or an isotopically labelled C 1 -C 3 alkyl. E30.
  • the first and second compound of any of embodiment E1-E26, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R 4 is selected from the group consisting of: 20 25 E32.
  • the first and second compound of any of embodiment E1-E26, wherein the first com- pound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R 4 is selected from the group consisting of: -CH 3 , 5
  • E33 The first and second compound of any of embodiment E1-E26, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa 5 and R 4 is tetrahydropyran.
  • E34 The first and second compound of any of embodiment E1-E26, wherein the first com- pound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R 4 is unsubstituted tetrahydropyran. 10 E35.
  • the first and second compound of any of embodiment E1-E26, wherein the first compound 10 or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and the bicyclic 8-membered heterocycle contains one oxygen and is a spirocyclic heterocycle.
  • the first and second compound of any of embodiment E1-E41, wherein the second com- pound or a pharmaceutically acceptable salt thereof, which second compound is useful in the 10 treatment of synucleinopathies is selected from anticholinergic agents, dopaminergic agents, and adenosine receptor antagonists.
  • the first and second compound of embodiment E43, wherein the second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of15 synucleinopathies is an anticholinergic agent selected from the group comprising trihex- yphenidyl, biperiden, metixene, procyclidine, profenamine, dexetimide, phenglutarimide, maza- ticol, bornaprine, tropatepine, etanautine, orphenadrine, benzatropine, and etybenzatropine.
  • an anticholinergic agent selected from the group comprising trihex- yphenidyl, biperiden, metixene, procyclidine, profenamine, dexetimide, phenglutarimide, maza- ticol, bornaprine, tropatepine, etanautine, orphenadrine, benzatropine, and etybenzatropine.
  • the first and second compound of embodiment E43, wherein the second compound or a 20 pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is a dopaminergic agent selected from the group comprising dopamine, levo- dopa, carbidopa, levodopa/carbidopa, melevodopa, etilevodopa, foslevodopa, amantadine, bromocriptine, pergolide, dihydroergocryptine mesylate, ropinirole, pramipexole, cabergoline, apomorphine, piribedil, rotigotine, selegiline, rasagiline, safinamide, tolcapone, entacapone, 25 budipine, and opicapone.
  • a dopaminergic agent selected from the group comprising dopamine, levo- dopa, carbidopa, levodopa/carbidop
  • the first and second compound of embodiment E43, wherein the second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is istradefylline. 5 E47.
  • the first and second compound of any of embodiments E1-E41, wherein the second com- pound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is selected from caspase inhibitors, calpain inhibitors, LRRK2 in- hibitors, BACE1 inhibitors, antibodies against any form of A ⁇ peptides, inflammation inhibitors, LAG-3 antibodies, molecules inhibiting alpha-synuclein aggregation, nicotine, caffeine, Mono-10 amine oxidase B inhibitor, Levodopa/carbidopa, Dopamine agonists, COMT inhibitors, A2A an- tagonists, anti-alphasynuclein antibodies, Mitogen-activated protein kinase 14 inhibitors, USP30 inhibitor
  • the first and second compound of any of embodiments E1-E41, wherein the second com- pound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is selected from the group comprising pralnacasan, bapi- neuzemab, solanezumab, gantenerumab, crenezumab, aducanumab, glunozumab, 30 prasinezumab, miglustat, eliglustat, ropinerole, neflamapimod, bosutinib, oxafuramine, nadolol, nilotinib, vodobatinib, clenbuterol, mevidalen, emeramide, tomaralimab, mitometin, diprag- lurant, ezeprogind, blarcamesine, buntanetap, renzapride, tavapadon, pramiprexole
  • the first and second compound of any of embodiments E1-E41, wherein the second com- pound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is selected from the list consisting of trihexyphenidyl, biperiden,10 metixene, procyclidine, profenamine, dexetimide, phenglutarimide, mazaticol, bornaprine, tro- patepine, etanautine, orphenadrine, benzatropine, etybenzatropine, dopamine, levodopa, car- bidopa, levodopa/carbidopa, melevodopa, etilevodopa, foslevodopa, amantadine, bromocrip- tine, pergolide, dihydroergocryptine mesylate, ropinirole, pramipexole, cabergoline, apomor
  • the first and second compound of any of embodiments E1-E41, wherein the second com- pound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is selected from the list consisting of trihexyphenidyl, biperiden, metixene, procyclidine, profenamine, dexetimide, phenglutarimide, mazaticol, bornaprine, tro- patepine, etanautine, orphenadrine, benzatropine, etybenzatropine, dopamine, levodopa, car-30 bidopa, levodopa/carbidopa, melevodopa, etilevodopa, foslevodopa, amantadine, bromocrip- tine, pergolide, dihydroergocryptine mesylate, ropinirole, pramipexole, cabergoline, apomor
  • a pharmaceutical composition comprising a first compound for use according to any one of the previous embodiments E1-E50 and one or more pharmaceutically acceptable carriers.
  • 5 E52. A first and second compound of any of embodiments E1-E50, or a pharmaceutically ac- ceptable salt thereof, for combined use in the treatment of a synucleinopathy, wherein the synu- cleinopathy is selected from Lewy body dementia, multiple system atrophy, or Parkinson’s dis- ease.
  • 10 E53 A first and second compound of any of embodiments E1-E50, or a pharmaceutically ac- ceptable salt thereof, for combined use in the treatment of a synucleinopathy, wherein the synu- cleinopathy is parkinson’s disease.
  • the Parkinson’s dis- ease is idiopathic Parkinson’s disease, sporadic Parkinson’s disease or Parkinson’s disease in pa- tients carrying a G2019S mutation in LRRK2.
  • E56 Use of a first and second compound of any one of embodiments E1-E50, or a pharmaceuti- cally acceptable salt thereof, in the manufacture of a medicament for the treatment of a disease or disorder in the central nervous system selected from Lewy body dementia, multiple system30 atrophy, or Parkinson’s disease.
  • E57 The use according to embodiment E56 wherein the Parkinson’s disease is idiopathic Parkin- son’s disease, sporadic Parkinson’s disease or Parkinson’s disease in patients carrying a G2019S mutation in LRRK2, or Parkinson’s disease in patients carrying one or more LRRK2 non-coding variants selected from rs76904798-T and Rs1491942-G.
  • the Parkinson’s disease is idiopathic Parkin- son’s disease, sporadic Parkinson’s disease or Parkinson’s disease in patients carrying a G2019S mutation in LRRK2, or Parkinson’s disease in patients carrying one or more LRRK2 non-coding variants selected from rs76904798
  • a method for the treatment of a disease or disorder in the central nervous system selected 5 from Lewy body dementia, multiple system atrophy or Parkinson’s disease comprising the ad- ministration of a therapeutically effective amount of the first and second compound of any one of embodiments E1-E50, or a pharmaceutically acceptable salt thereof, to a patient in need thereof.
  • the method according to embodiment E58 wherein the Parkinson’s disease is idiopathic Parkinson’s disease, sporadic Parkinson’s disease, Parkinson’s disease in patients carrying a G2019S mutation in LRRK2, or Parkinson’s disease in patients carrying one or more LRRK2 non- coding variants selected from rs76904798-T and Rs1491942-G. 15
  • the compounds of formula I, Ia, Ib, Ic, Id, A or Aa may be prepared by methods described below, together with synthetic methods known in the art of organic chemistry, or modifications that are familiar to those of ordinary skill in the art. For example, the methods describe the use of selective protecting groups during the synthesis of the compounds of the invention. One skilled in the art would be able to select the appropriate protecting group for a particular reaction. Methods 10 for protection and deprotection of such groups are well known in the art and may be found in Watts and Green et al., Protective Groups in Organic Synthesis, 2006, 4 th Edition, Wiley Interscience, New York.
  • the starting materials used herein are available commercially or may be prepared by rou- tine methods known in the art, such as those method described in standard reference books such as “Compendium of Organic Synthetic Methods, Vol. I-XII” (published by Wiley Inter- 15 science). Preferred methods include, but are not limited to, those described below.
  • the schemes are representative of methods useful in synthesizing the compounds of the present invention. They are not to constrain the scope of the invention in any way.
  • Scheme 1 5 compounds of formula I can be prepared according to scheme 1.
  • the com- pounds of formula I can for example be prepared through a one pot procedure by reducing an intermediate of type III with iron and ammonium chloride in a suitable solvent e.g. a mixture of ethanol and water as described for Example 1.
  • a suitable solvent e.g. a mixture of ethanol and water
  • an intermediate of type III can be reduced using e.g. sodium dithionite in the presence of a base such as potassium hydrogen car-10 bonate in a solvent such as a mixture of water and tetrahydrofuran to afford an aminopyrazole intermediate of type II.
  • An intermediate of type II can be cyclized to afford compounds of for- mula I in the presence of a base e.g.
  • the compounds of formula I can be prepared by employing a cross-coupling reaction between an intermediate of type I’ and an organometallic alkyl intermediate IV (wherein M is for example Bpin, B(OH) 2 , Sn(n-Bu) 3 or SnMe 3 , ZnBr or ZnCl).
  • M is for example Bpin, B(OH) 2 , Sn(n-Bu) 3 or SnMe 3 , ZnBr or ZnCl.
  • the coupling is exemplified by but not limited to a Negishi- type cross-coupling reaction.
  • a catalyst like di- ⁇ -iodobis(tri-t-butylphosphino)dipalladium(I) and a suitable solvent such as a mixture of tol- uene and tetrahydrofuran as described for Example 7.
  • a suitable solvent such as a mixture of tol- uene and tetrahydrofuran as described for Example 7.
  • an alcohol such as IX (wherein Pg 1 is for example TBS) under Mitsunobu-type conditions employing for example diisopropyl azodicarboxylate and triphenylphosphine in a solvent such as tetrahydrofuran to afford an intermediate of type VIII.
  • Intermediates of type VIII (wherein Pg1 is for example TBS) can be converted into an alcohol intermediate of type VII using for ex- ample an acid such as aqueous hydrochloric acid in an appropriate solvent such as a mixture of15 tetrahydrofuran and water, as described in the synthesis of 3-(3,6-dichloro-1H-pyrazolo[3,4- d]pyrimidin-1-yl)propan-1-ol.
  • An alcohol intermediate of type VII can be reacted with an intermediate of type V under Mitsunobu-type conditions employing e.g. diisopropyl azodicarboxylate and triphenylphosphine in a solvent such as tetrahydrofuran to afford an intermediate of type III, as detailed in for ex-20 ample the synthesis of 3,6-dichloro-1-(3-((5-cyclopropyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)- 1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine.
  • an intermediate of type VII can be converted into an intermediate of type VI (wherein LG1 is for example OTs) using p-toluenesulfonyl chloride in the presence of an ap-litiste base such as triethylamine in a suitable solvent for example dichloromethane.
  • An in- 25 termediate of type VI can be reacted with intermediates of type V in the presence of base for example potassium carbonate in a solvent such as N,N-dimethylformamide to afford an inter- mediate of type III as for example described in the synthesis of 3,6-dichloro-1-(3-((5-methyl-1- (3-methyloxetan-3-yl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine.
  • Scheme 4 An alternative method to prepare a nitropyrazole intermediate of type III is described in scheme 4 starting from a pyrazole intermediate of type V.
  • Intermediates of type XIII can be pre- 10 pared from an intermediate of type V through a Mitsunobu-type reaction with an alcohol like XIV (wherein PG2 is for example TBS) using for example diisopropyl azodicarboxylate and tri- phenylphosphine in a suitable solvent such as tetrahydrofuran.
  • an intermediate of type XIII can be prepared from intermediates of type V through alkylation with an intermedi- ate of type XV (wherein for example LG2 is OTs and PG2 is TBS) in the presence of a base such as15 cesium carbonate in a suitable solvent e.g. N,N-dimethylformamide.
  • an intermediate of type XIII can be converted into an intermediate of type XI wherein R 2 has been modified:
  • an intermediate of type XI can be reacted with a pyrazolopyrimidine intermedi- 5 ate of type X using a Mitsunobu-type reaction employing for example 1-(azodicarbon
  • an intermediate of type XI can be reacted with for example methanesul-10 fonyl chloride in the presence of a base such as triethylamine in a suitable solvent e.g. dichloro- methane to afford and intermediate of type XII (wherein LG3 is OMs).
  • a base such as triethylamine
  • a suitable solvent e.g. dichloro- methane
  • Intermediates of type XII can be converted into a an intermediate of type III through reaction with a pyrazolopyrimidine intermediate of type X in the presence of a base for example N,N-diisopropylethylamine, an additive such as sodium iodide in a suitable solvent e.g.
  • Intermediates of type XIX can be converted into intermediates of type XVI in the presence of an alcohol intermediate such as XVII using for example Mitsunobu-type reaction conditions employing di-tert-butyl azodicarboxylate 25 and triphenylphosphine in a solvent such as tetrahydrofuran, as described in the synthesis of 3,6-dichloro-1-(3-((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)- 1H-pyrazolo[3,4-d]pyrimidine.
  • Mitsunobu-type reaction conditions employing di-tert-butyl azodicarboxylate 25 and triphenylphosphine in a solvent such as tetrahydrofuran, as described in the synthesis of 3,6-dichloro-1-(3-((5-methyl-4-nitro-1-(tetrahydro
  • an intermediate of type XIX can be reacted with and intermediate like XVIII (wherein LG 4 is for example a halogen) in the presence of a suitable base such as potas- 30 sium carbonate in a solvent like acetonitrile to afford an intermediate of type XVI, as described in the synthesis of 1-(3-((1-((2-oxabicyclo[2.1.1]hexan-4-yl)methyl)-5-methyl-4-nitro-1H-pyra- zol-3-yl)oxy)propyl)-3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine.
  • Intermediates of type XXIII can be reacted with an alkylating agent of type XVIII (wherein LG4 is for example a halogen) in the presence of a base such as cesium carbonate in a solvent such as N,N-dimethylformamide to afford an intermediate of type XXII.
  • Intermediates of type XXII can 15 be modified into an intermediate of type XXI employing for example iodine and a base such as lithium bis(trimethylsilyl)amide in a suitable solvent such as tetrahydrofuran.
  • reaction can be converted into an intermediate of type XVI through reaction with a reagent of type XX (wherein M is for example Bpin, B(OH)2, Sn(n-Bu)3 or SnMe3, ZnBr).
  • M is for example Bpin, B(OH)2, Sn(n-Bu)3 or SnMe3, ZnBr.
  • the coupling is ex- emplified by but not limited to a Suzuki-Miyaura-type cross-coupling reaction.
  • Intermediates of type XIII (wherein Pg2 is for example TBS) can be prepared from a pyrazole intermediate of type XXIV through reaction with an alcohol such as XVII under Mitsunobu-like reaction conditions employing 2-(tributyl- ⁇ 5 -phosphanylidene)ace- tonitrile in a solvent like toluene as described in the synthesis of intermediate cis-3,6-Dichloro-10 1-(3-((5-methyl-1-(2-methyltetrahydro-2H-pyran-4-yl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H- pyrazolo[3,4-d]pyrimidine.
  • intermediates if type XIII can be prepared through re- action of pyrazole intermediates such as XXIV employing an alkylating agent of type XVIII (wherein, LG4 is for example OTs or Br) in the presence of a base like potassium carbonate in an appropriate solvent for example N,N-dimethylformamide, as detailed in the synthesis of trans- 15 3-((1-(4-fluorocyclohexyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propan-1-ol.
  • a base like potassium carbonate in an appropriate solvent for example N,N-dimethylformamide
  • TBS can be synthesized from an alcohol such as XXV using for example tert-butyldimethylsilyl chloride in the presence of a base such as imidazole and a catalyst such as 4-dimethylaminopyridine in an appropriate solvent such as di- chloromethane.
  • An alcohol intermediate such as XXV can be prepared from a nitropyazole such 20 as XXVI (wherein Pg3 is for example, 2-tetrahydropyranyl) by treatment with an acid such as aqueous hydrochloric acid in a suitable solvent for example methanol.
  • Alcohol intermediates of type XXVI can be prepared by reacting a chloro intermediate XXVIII with propane-1,3-diol XXVII in the presence of a base for example cesium fluoride in a solvent such as N,N-dimethylacetamide.
  • Chloro intermediates such as XXVIII (wherein Pg 3 is for example, 2-tetrahydropyranyl) can be prepared from a nitropyrazole intermediate XXIX (wherein Pg 3 is for example, 2-tetrahy- dropyranyl) using a strong base for example lithium bis(trimethylsilyl)amide, and an electrophile for example hexachloroethane in a suitable solvent such as tetrahydrofuran.
  • a nitropyazole intermediate of type XXIX (wherein Pg3 is for example, 2-tetrahydropy- ranyl) can be prepared from a pyrazole such as XXX employing 3,4-dihydro-2H-pyran in the pres- ence of an acid for example para-toluenesulfonic acid monohydrate in an appropriate solvent for example tetrahydrofuran.
  • the general synthetic sequence to prepare XXIV is for example described in the preparation of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-10 pyrazole.
  • Scheme 8 15 compounds of formula I can be prepared from a compound of formula XXXII according to scheme 8.
  • the compounds of formula I can be prepared by employing e.g. a copper-mediated coupling between an intermediate of type XXXII and an intermediate of type XXXI (wherein M is for example B(OH) 2 ).
  • the coupling is exemplified by but not limited to a Chan- Lam type coupling.
  • Method 9 Scheme 9 An intermediate such as XXXII can be prepared according to scheme 9.
  • the intermedi- ates of type XXXII can for example be prepared through a one pot procedure by reducing an intermediate of type XXXIII (wherein Pg3 is for example, 2-tetrahydropyranyl) with iron and am- monium chloride in a suitable solvent e.g. a mixture of ethanol and water.
  • a suitable solvent e.g. a mixture of ethanol and water.
  • Nitropyrazole intermediates like XXXIII can be prepared by reaction of a pyrazolopyrim- idine like X with an alcohol such as XXVI (wherein Pg3 is for example, 2-tetrahydropyranyl) em- ploying Mitsunobu-like reaction conditions for example diisopropyl azodicarboxylate and tri- phenylphosphine in a solvent such as tetrahydrofuran.
  • an alcohol such as XXVI (wherein Pg3 is for example, 2-tetrahydropyranyl) em- ploying Mitsunobu-like reaction conditions for example diisopropyl azodicarboxylate and tri- phenylphosphine in a solvent such as tetrahydrofuran.
  • the coupling is 20 exemplified by but not limited to a Negishi-type cross-coupling.
  • the reaction can be performed by reacting an intermediate of type XXXIV with an organozinc species such as (CD 3 ) 2 Zn in the presence of a catalyst like bis[tris(tert-butyl)phosphine]palladium, a base such as lithium bis(tri- methylsilyl)amide in an appropriate solvent such as tetrahydrofuran as described for Example 50.
  • a bromo intermediate such as XXXIV can be prepared from a pyrazole like XXXV using a brominating agent for example N-bromosuccinimide in a suitable solvent such as tetrahydrofu- ran.
  • Intermediates of type XXXV can in turn be synthesized from a nitropyrazole intermediate such as XXXVI employing a one-pot reduction and cyclization procedure.
  • the reduction and sub- 5 sequent cyclization can be performed using for example iron in the presence of ammonium chlo- ride in a suitable solvent such as a mixture of ethanol and water.
  • Nitropyrazole intermediates such as XXXVI can for example be synthesized through reaction of a pyrazolopyrimidine such as X and an alcohol like XXXVII employing Mitsunobu-like reaction conditions.
  • the Mitsunobu-like coupling can for example be conducted using diisopropyl azodicarboxylate in the presence of10 triphenylphosphine in an appropriate solvent such as tetrathydrofuran.
  • the synthesis of an in- termediate like XXXIV is exemplified in the preparation of 3-bromo-8-chloro-2-((1r,4r)-4-meth- oxycyclohexyl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine.
  • Method 11 A method to prepare an intermediate of type XXXVII is depicted in scheme 11.
  • Alcohol intermediates of type XXXVII can be prepared from a nitro pyrazole intermediate of type XXXVIII 20 (wherein Pg3 is e.g. TBS) using tetra-n-butyl ammonium fluoride in a suitable solvent such as tetrahydrofuran.
  • Nitropyrazole intermediates of type XXXVIII can synthesized from pyrazole in- termediates such as XXXIX and an alcohols such as XVII under Mitsunobu-like reaction condi- tions employing 2-(tributyl- ⁇ 5 -phosphanylidene)acetonitrile in a solvent like toluene as de- scribed in the synthesis of intermediate 3-((1-((1r,4r)-4-methoxycyclohexyl)-4-nitro-1H-pyrazol- 3-yl)oxy)propan-1-ol Intermediates of type XXXIX (wherein Pg3 is e.g.
  • TBS can be synthesized from an alcohol 5 such as XL using for example tert-butyldimethylsilyl chloride in the presence of a base such as imidazole and a catalyst such as 4-dimethylaminopyridine in an appropriate solvent such as di- chloromethane.
  • An alcohol intermediate such as XL can be prepared from a nitropyazole such as XLI (wherein Pg 4 is for example, 2-tetrahydropyranyl) by treatment with an acid such as aque- ous hydrochloric acid in a suitable solvent for example methanol.
  • Alcohol intermediates of type XLI can be prepared by reacting a chloro intermediate XLII with propane-1,3-diol XXVII in the presence of a base for example cesium fluoride in a solvent such as N,N-dimethylacetamide.
  • Chloro intermediates such as XLII (wherein Pg4 is for example, 2-tetrahydropyranyl) can be pre- pared from a nitropyrazole intermediate XLIII (wherein Pg 4 is for example, 2-tetrahydropyranyl) 15 using a strong base for example lithium bis(trimethylsilyl)amide, and an electrophile for example hexachloroethane in a suitable solvent such as tetrahydrofuran.
  • ABBREVIATIONS Abbreviations used in the experimental may include, but are not limited to the follow-20 ing: Boc: tert-butyloxycarbonyl; BRIJ-35: polyoxyethylene (23) lauryl ether; C: Celsius; CPME: cyclo- pentyl methyl ether; cPr: cyclopropyl; dba: dibenzylideneacetone; DBAD: di-tert-butyl azodicar- boxylate; DCM: dichloromethane; DIAD: diisopropyl azodicarboxylate; DIPEA: N,N-diisopro- pylethylamine; DMA: N,N-dimethylacetamide; DMF: N’N-dimethylformamid; DMSO: dimethyl-25 sulfoxide; DTT: dithiothreitol; EGTA: ethylene glycol-bis( ⁇ -aminoethyl ether)-N
  • LC-MS were run on Agilent LC1200-MS6110 UPLC-MS or a consisting of Ag- ilent LC1200 including column manager, binary solvent manager, sample organizer, PDA detec- tor (operating at 220&254 nM), ELS detector, and MS6110 equipped with APPI-source operating 10 in positive ion mode.
  • LC-conditions The column was Xtimate C182.1 ⁇ 30mm, 3 ⁇ m operating at 50oC with 1.2 mL/min of a binary gradient consisting of water + 0.037 % trifluoroacetic acid (A) and acetonitrile + 0.018 % trifluoroacetic acid(B). The retention times (tR) are expressed in minutes based on UV-trace at 220&254 nm.
  • Method B LC-MS were run on Agilent LC1200-MS6150 or LC1200-MS6110 UPLC-MS consisting of Agilent LC1200 including column manager, binary solvent manager, sample organ- izer, PDA detector (operating at 220&254 nM), ELS detector, and MS6150 or MS6110 equipped with APPI-source operating in positive ion mode.
  • LC-conditions The column was MERCK, RP-18e 25 25 ⁇ 3.0 mm operating at 50oC with 1.5 mL/min of a binary gradient consisting of water + 0.037 % trifluoroacetic acid (A) and acetonitrile + 0.018 % trifluoroacetic acid(B).
  • the retention times (tR) are expressed in minutes based on UV-trace at 220&254 nm.
  • Method C LC-MS were run on Agilent Prime-6125B UPLC-MS consisting of Agilent Prime including column manager, binary solvent manager, sample organizer, PDA detector (operating at 254 nM), ELS detector, and 6125B equipped with APPI-source operating in positive ion mode.
  • LC-conditions The column was Agilent Poroshell 120 EC-C181.9 ⁇ m; 3.0 ⁇ 30mm operating at 50oC with 1.5 mL/min of a binary gradient consisting of water + 0.037 % trifluoroacetic acid (A) and acetonitrile + 0.018 % trifluoroacetic acid(B).
  • the retention times (tR) are expressed in minutes based on UV-trace at 254 nm.
  • LC-MS were run on Waters Aquity UPLC-MS consisting of Waters Aquity in- cluding column manager, binary solvent manager, sample organizer, PDA detector (operating at 254 nM), ELS detector, and SQD-MS equipped with APPI-source operating in positive ion mode.
  • LC-conditions The column was Acquity UPLC BEH C181.7 ⁇ m ; 2.1 ⁇ 50mm operating at 30 60oC with 1.2 ml/min of a binary gradient consisting of water + 0.05 % trifluoroacetic acid (A) and acetonitrile + 5% water + 0.035 % trifluoroacetic acid.
  • LC-conditions The column was Acquity UPLC BEH C181.7 ⁇ m; 2.1 ⁇ 50mm operating at 60oC with 5 1.2 ml/min of a binary gradient consisting of water + 0.05 % trifluoroacetic acid (A) and acetoni- trile + 5% water + 0.05 % trifluoroacetic acid.
  • the retention times (tR) are expressed in minutes based on UV-trace at 254 nm.
  • LC-MS were run on Agilent Prime-6125B UPLC-MS consisting of Agilent Prime 15 including column manager, binary solvent manager, sample organizer, PDA detector (operating at 254 nM), ELS detector, and 6125B equipped with APPI-source operating in positive ion mode.
  • LC-conditions The column was Agilent Poroshell 120 EC-C181.9 ⁇ m; 3.0 ⁇ 30mm operating at 30oC with 1.5 mL/min of a binary gradient consisting of water + 0.05 % NH 3 .H 2 O (A) and acetoni- trile (B).
  • LC-MS were run on Shimadzu LC-20AD;LCMS-2020 consisting of Shimadzu LC-20AD including column manager, binary solvent manager, sample organizer, PDA detector (operating at 220&254 nM), ELS detector, and MS6150 equipped with APPI-source operating in positive ion mode.
  • LC-conditions The column was Chromolith® Flash RP-18e 25-3 mm operat- 5 ing at 50oC with 1.5 mL/min of a binary gradient consisting of water + 0.037 % trifluoroacetic acid (A) and acetonitrile + 0.018 % trifluoroacetic acid(B). The retention times (tR) are ex- pressed in minutes based on UV-trace at 220&254 nm.
  • LC-conditions The column was Xtimate, C18, 2.1 ⁇ 30 mm, 3 ⁇ m operating at 50oC with 0.8 mL/min of a binary gradient consisting of water + 0.037 % trifluoroacetic acid (A) and ace- 20 tonitrile + 0.018 % trifluoroacetic acid(B).
  • the retention times (t R ) are expressed in minutes based on UV-trace at 220&254 nm.
  • LC-MS were run on Shimadzu LC20-MS2010 UPLC-MS consisting of Shimadzu LC20 including column manager, binary solvent manager, sample organizer, PDA detector (operating at 220&254 nm), ELS detector, and MS2010 equipped with APPI-source operating in positive ion mode.
  • LC-conditions The column was Xtimate, C18, 2.1 ⁇ 30 mm, 3 ⁇ m operating at 50oC with 0.8 mL/min of a binary gradient consisting of water + 0.037 % trifluoroacetic acid (A) and ace- 35 tonitrile + 0.018 % trifluoroacetic acid(B).
  • the retention times (tR) are expressed in minutes based on UV-trace at 220&254 nm.
  • PPh3 (2.55 g, 9.71 mmol) was added portionwise and the mixture was stirred for 15 minutes.3- Bromo-6-chloro-1H-pyrazolo[3,4-d]pyrimidine (1.60 g, 6.85 mmol) was then added in portions followed by a solution of 3-((tert-butyldimethylsilyl)oxy)propan-1-ol (1.09 g, 5.71 mmol) in THF (10 mL). The cooling bath was allowed to expire upon overnight stirring. The reaction mixture25 was concentrated under reduced pressure.
  • the res- 10 idue was purified by chromatography on silica gel (eluent: heptane:EtOAc 100:0 ⁇ 25:75) to afford ( ⁇ )-3-chloro-5-methyl-4-nitro-1-(tetrahydro-2H-pyran-3-yl)-1H-pyrazole (600 mg) of suffi- cient purity for the subsequent step.
  • the reaction mixture was concentrated under reduced pressure.
  • the reac-15 tion mixture was diluted with water (10 mL) and saturated aqueous potassium carbonate ad- justing pH ⁇ 11.
  • the mixture was extracted with EtOAc (3 x 50 mL), and the combined organics were washed with brine, and dried over sodium sulfate, filtered and concentrated under re- symbolized pressure.
  • reaction mixture was concen- trated under reduced pressure.
  • the mixture was diluted with water (30 mL) and extracted with EtOAc (3 ⁇ 30 mL).
  • EtOAc 3 ⁇ 30 mL
  • the combined organic layers were washed with brine (3 ⁇ 30 mL), dried over Na2SO4 and concentrated under reduced pressure.
  • the residue was purified by chromatography10 on silica gel (eluent: petroleum ether:EtOAc 80:20 ⁇ 75:25) to afford 3-chloro-5-methyl-1-((3- methyloxetan-3-yl)methyl)-4-nitro-1H-pyrazole (6.3 g) of sufficient purity for the subsequent step.
  • PPh 3 ( ⁇ 3 mmol/g on resin) (777 mg, 2.96 mmol, employed 1.0 g resin) was added in portions and the mixture was stirred for 15 minutes.5-Cyclopropyl-4-nitro-1-(tetrahy- 20 dro-2H-pyran-4-yl)-1H-pyrazol-3-ol (500 mg, 1.97 mmol, prepared as described previously) was then added followed by a solution of 3-((tert-butyldimethylsilyl)oxy)propan-1-ol (376 mg, 1.97 mmol) in THF (1 mL). The cooling bath was removed and the reaction mixture was stirred at room temperature for 3 h. The mixture was filtered and concentrated under reduced pressure.
  • 1,1,1,2,2,2-hexachloroethane 14.80 g, 62.5 mmol dissolved in THF (50 mL) was added and the mixture was stirred at -65 oC for 60 minutes and then warmed to 20 oC 15 for 10 minutes. The mixture was quenched with saturated aqueous NH 4 Cl (200 mL) at 0 oC and then warmed to 20 oC. After 20 min stirring the mixture was extracted with EtOAc (3 ⁇ 150 mL). The combined organic layers were washed with brine (80 mL) and concentrated under reduced pressure.
  • reaction mixture was quenched with saturated aqueous NH4Cl (100 mL) at 0 oC, extracted with EtOAc (3 ⁇ 100 mL). The combined organic layers were washed with brine (3 ⁇ 100 25 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 ⁇ 90:10) to afford 5-chloro- 4-nitro-1-tetrahydropyran-2-yl-pyrazole (11.4 g) of sufficient purity for the subsequent step.
  • the reaction mix- ture was diluted with water (100 mL) and extracted with DCM (3 ⁇ 100 mL). The combined organic layers were washed with brine (3 ⁇ 100 mL), dried over Na 2 SO 4 , and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum 10 ether:EtOAc 60:40 ⁇ 50:50) to afford 4-(oxiran-2-yl)butan-1-ol (8 g) of sufficient purity for the subsequent step. To a solution of 4-(oxiran-2-yl)butan-1-ol (7.5 g, 65 mmol) in THF (100 mL) was added t- BuOK (8.69 g, 77.5 mmol).
  • reaction mixture was stirred at 50 oC for 16 h.
  • the reaction mix- ture was added water (50 mL) and extracted with EtOAc (3 ⁇ 50 mL).
  • EtOAc 3 ⁇ 50 mL
  • the combined organic 15 layers were washed with brine (3 ⁇ 50 mL), dried over Na2SO4, and concentrated under reduced pressure.
  • the residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 30:70 ⁇ 20:80) to give a mixture of oxepan-3-ol and (tetrahydro-2H-pyran-2- yl)methanol (4 g).
  • the mixture of tetrahydropyran-2-ylmethanol and oxepan-3-ol was dissolved in DCM (40 mL).
  • DIAD (1.37 g, 6.80 mmol) was added to a solution of 3-(3,6-dichloro-1H-pyrazolo[3,4- d]pyrimidin-1-yl)propan-1-ol (280 mg, 1.13 mmol), 5-methyl-4-nitro-1-(oxepan-4-yl)-1H-pyrazol- 20 3-ol (273 mg, 1.13 mmol) and PPh3 on resin (2.26 g, 6.80 mmol, 3 mmol/g) in THF (20 mL) at 0 oC. The mixture was stirred at 20 oC for 15 h. The reaction mixture was concentrated under reduced pressure.
  • reaction mixture was diluted with aqueous NH4Cl (50 mL) and extracted with DCM (3 ⁇ 100 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 ⁇ 80:20) to afford 3-(3-(3-((tert-butyldimethylsi- 15 lyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyrazol-1-yl)bicyclo[1.1.1]pentane-1-carboxylate (300 mg) of sufficient purity for the subsequent step.
  • reaction mixture was concentrated under reduced pressure.
  • residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 ⁇ 70:30) to afford (1s,4s)-4-methoxycyclo- hexyl 4-methylbenzenesulfonate (350 mg) of sufficient purity for the subsequent step.
  • the mixture was stirred at 80 oC for 16 h.
  • the reaction mixture was diluted with EtOAc (50 mL) and extracted with H 2 O (3 ⁇ 40 mL).
  • the com- bined organic layers were washed with brine (50 mL), dried over Na 2 SO 4 , filtered, and concen- trated under reduced pressure.
  • NBS 470 mg, 2.64 mmol
  • THF 5 mL
  • Example 2 8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 20
  • the compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- ((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4- d]pyrimidine (840 mg, 1.84 mmol), Fe (514 mg, 9.20 mmol), and NH4Cl (492 mg, 9.20 mmol) in EtOH (40 mL) and H 2 O (4 mL) at 100 oC for 15 h, followed by work-up, and preparative
  • Example 3 (R) or (S)-8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine peak 1 and
  • Example 4 (R) or (S)-8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine peak 2
  • the racemic mixture of Example 3 and Example 4 was prepared in a manner similar to Example 1 using ( ⁇ )-3,6-dichloro-1-(3-((5-methyl-4-nitro
  • Example 4 (R) or (S)-8-chloro-3-methyl-2-(tetrahydro-2H-pyran-3- 5 yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triaza- cycloundecine (50 mg).
  • Example 5 3-Methyl-2-(tetrahydro-2H-pyran-4-yl)-8-(trifluoromethyl)-2,4,12,13-tetrahydro-15 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • the compound was prepared in a manner similar to Example 1 using 6-chloro-1-(3-((5- methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)-3-(trifluoromethyl)- 1H-pyrazolo[3,4-d]pyrimidine (95 mg, 0.19 mmol), Fe (54 mg, 0.97 mmol), and NH4Cl (52 mg, 520 Eq, 0.97 mmol) in EtOH (15 mL) and H 2 O (1.5 mL) at
  • Example 6 8-Bromo-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 5
  • DIAD 87 ⁇ L, 0.45 mmol
  • THF 5 cooled to 0 oC.
  • Example 7 8-Cyclopropyl-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 25
  • Example 6 60 mg, 0.14 mmol
  • the vial was capped and the atmosphere was ex- changed for argon before ⁇ DMA (0.35 mL) ⁇ was added.
  • the solution was purged with argon for 1 minute before ⁇ DIPEA (1.8 ⁇ L, 0.010 mmol) ⁇ was added. ⁇ The mixture was stirred at 85 ⁇ oC over- 10 night.
  • the reaction mixture was concentrated ⁇ under reduced pressure.
  • the residue was purified by chromatography on silica gel (eluent: heptane:EtOAc (w.2% MeOH) 90:10 ⁇ 0:100) to afford the title compound (2.3 mg).
  • Example 10 8-Chloro-3-cyclopropyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • the compound was prepared in a manner similar to Example 1 using of 3,6-dichloro-1-20 (3-((5-cyclopropyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyra- zolo[3,4-d]pyrimidine (140 mg, 0.29 mmol), Fe (81 mg, 1.5 mmol), and NH 4 Cl (78 mg, 1.5 mmol) in EtOH (70 mL) and H 2 O (10 mL) at 100 oC for 16 h, followed by work-
  • Example 11 2-((2-Oxabicyclo[2.1.1]hexan-1-yl)methyl)-8-chloro-3-methyl-2,4,12,13-tetrahy- 5 dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • the compound was prepared in a manner similar to Example 1 using 1-(3-((1-((2-oxabi- cyclo[2.1.1]hexan-1-yl)methyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-3,6-dichloro-1H- pyrazolo[3,4-d]pyrimidine (230 mg, 0.49 mmol), Fe (137 mg, 2.5 mmol), and NH4Cl (131 mg, 2.5 10 mmol) in EtOH (93 mL) and H 2 O (13 mL) at
  • Example 12 8-Chloro-3-methyl-2-((3-methyloxetan-3-yl)methyl)-2,4,12,13-tetrahydro-11H-20 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • the compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- ((5-methyl-1-((3-methyloxetan-3-yl)methyl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyra- zolo[3,4-d]pyrimidine (50 mg, 0.11 mmol), Fe (31 mg, 0.55 mmol), and NH4Cl (29 mg, 0.55 mmol)25 in EtOH (10 mL) and H 2 O (10 mL) at 80 oC for 5 h, followed by work-up, and
  • Example 13 8-Chloro-3-(methyl-d 3 )-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H- 10 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 3,6-Dichloro-1-(3-((5-(methyl-d 3 )-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3- yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (100 mg, 0.217 mmol) ⁇ was dissolved in ⁇ Etha- nol (15 mL) ⁇ and ⁇ deuterium oxide (1.5 mL) in a vial. ⁇ Fe (48.5 mg, 0.869 mmol) ⁇ and NH4Cl (46.5 15 mg, 0.869
  • Example 14 8-Chloro-3-methyl-2-(3-methyloxetan-3-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • the compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- 5 ((5-methyl-1-(3-methyloxetan-3-yl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]py- rimidine (190 mg, 0.43 mmol), Fe (120 mg, 2.15 mmol), and NH4Cl (115 mg, 2.15 mmol) in EtOH (40 mL) and H 2 O (40 mL) at 80 oC for 16 h, followed by work-up, and preparative HP
  • Example 15 8-Chloro-3-(methyl-d3)-2-(tetrahydro-2H-pyran-4-yl-4-d)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • the compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3-20 ((5-(methyl-d3)-4-nitro-1-(tetrahydro-2H-pyran-4-yl-4-d)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyra- zolo[3,4-d]pyrimidine (520 mg, 1.13 mmol), Fe (315 mg, 5.65 mmol), and NH4Cl (302 mg, 5.65 mmol) in EtOH (100 mL) and H 2 O (100 mL) at 90
  • Example 16 8-Chloro-3-(1-fluorocyclopropyl)-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahy- 5 dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • potassium hydrogen carbonate 120 mg, 1.20 mmol
  • water 6.4 mL
  • the vial was cooled to 0 oC and the solution was purged with argon before sodium dithionite (120 mg, 0.688 mmol) was added.
  • Example 17 8-Chloro-3-ethyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • the compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- 5 ((5-ethyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4- d]pyrimidine (50 mg, 0.11 mmol), Fe (30 mg, 0.53 mmol), and NH 4 Cl (28 mg, 0.53 mmol) in EtOH (2 mL) and H 2 O (0.2 mL) at 100 oC for 3 h, followed by work-up
  • Example 18 (+)-8-Chloro-3-methyl-2-((2R,4R)-or-(2S,4S)-2-methyltetrahydro-2H-pyran-4-yl)- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine and Example 19: (–)-8-Chloro-3-methyl-2-((2R,4R)-or-(2S,4S)-2-methyltetrahydro-2H-pyran-4-yl)- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy-20 cloundecine
  • the racemic mixture of Example 18 and Example 19 was prepared in a
  • Example 20 8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine-11,11,12,12,13,13- d 6 5
  • the compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- ((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl-1,1,2,2,3,3-d 6 )-1H- pyrazolo[3,4-d]pyrimidine (270 mg, 0.584 mmol), Fe (163 mg, 2.92 mmol), and NH4Cl (156 mg, 2.92 mmol) in EtOH (27 mL) and
  • Example 25 8-Chloro-2-((1r,4r)-4-fluorocyclohexyl)-3-methyl-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 15
  • the compound was prepared in a manner similar to Example 1 using trans-3,6-dichloro- 1-(3-((1-(4-fluorocyclohexyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4- d]pyrimidine (110 mg, 0.27 mmol) , Fe (65 mg, 1.2 mmol), and NH4Cl (62 mg, 1.2 mmol) in EtOH (14 mL) and H 2 O (7 mL) at 80 oC for 16 h, followed by work-
  • Example 27 (–)-(R) or (S)-8-Chloro-3-methyl-2-(oxepan-3-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine and 15
  • Example 28 (+)-8-(R) or (S)-Chloro-3-methyl-2-(oxepan-3-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • the racemic mixture of Example 27 and Example 28 was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3-((5-methyl-4-nitro-1-(oxepan-3-yl)-1H
  • Example 27 (–)-(R) or (S)-8-Chloro-3-methyl-2-(oxepan-3-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacyclo
  • Example 29 (–)-(R) or (S)-8-Chloro-3-methyl-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine and Example 30 (+)-8-(R) or (S)-Chloro-3-methyl-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7-30 (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • the racemic mixture of Example 29 and Example 30 was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3-((5-methyl-4-
  • Example 31 (–)-8-Chloro-2-((3S,4R) or (3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)-3-methyl- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy-10 cloundecine and
  • Example 32 (+)-8-Chloro-2-((3S,4R) or (3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)-3-methyl- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine
  • Example 32 (+)-8-Chloro-2-((3S,4R) or
  • reaction mixture was stirred at 0 oC for 1 h. Then additional TFAA (106 mg, 0.50 mmol) and TEA (70 mg, 0.69 mmol) was added to the mixture and the mixture was stirred at 0 oC for 1 h.
  • the reaction mixture was diluted with water (20 mL) and extracted with DCM (3 ⁇ 20 mL). The combined organic layers were washed with brine (3 ⁇ 20 mL), dried 5 over Na2SO4, filtered, and concentrated under reduced pressure.
  • Example 34 8-Chloro-3-methyl-2-(2-oxaspiro[3.3]heptan-6-yl)-2,4,12,13-tetrahydro-11H-5,7-15 (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • the compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- ((5-methyl-4-nitro-1-(2-oxaspiro[3.3]heptan-6-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4- d]pyrimidine (70 mg, 0.15 mmol), Fe (42 mg, 0.75 mmol), and NH4Cl (42 mg, 0.78 mmol) in EtOH 20 (7 mL) and H 2 O (3.5 mL) at 54 oC for 16 h, followed by work
  • Example 35 8-Chloro-3-(methyl-d 3 )-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine- 11,11,12,12,13,13-d6 5
  • the compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- ((5-(methyl-d3)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl-1,1,2,2,3,3-d 6 )- 1H-pyrazolo[3,4-d]pyrimidine (100 mg, 0.215 mmol), Fe (60.0mg, 1.07 mmol), and NH4Cl (57.5 mg, 1.07 mmol)
  • Example 36 1-(8-Chloro-3-methyl-12,13-dihydro-11H-5,7-(azenometheno)dipyrazolo[3,4- b:5',1'-g][1]oxa[4,6,8]triazacycloundecin-2(4H)-yl)cyclopropane-1-carbonitrile 20
  • the compound was prepared in a manner similar to Example 33 using 1-(8-chloro-3- methyl-4,11,12,13-tetrahydro-2H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1,4,6,8]ox- atriazacycloundecin-2-yl)cyclopropanecarboxamide (35 mg, 0.090 mmol), TFAA (28 mg, 0.135 mmol) and TEA (36.4 mg, 0.360 mmol) in DCM (14 mL) at 0 oC for 1
  • Example 37 8-Chloro-2-((1r,4r)-4-methoxycyclohexyl)-3-methyl-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • the compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3-10 ((1-((1r,4r)-4-methoxycyclohexyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyra- zolo[3,4-d]pyrimidine (160 mg, 0.33 mmol), Fe (92 mg, 1.7 mmol), and NH 4 Cl (88 mg, 1.7 mmol) in EtOH (16 mL) and H 2 O (4 mL) at 80 oC for 16 h, followed by work
  • Example 38 2-((1R,5S,6r)-3-Oxabicyclo[3.1.0]hexan-6-yl)-8-chloro-3-methyl-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • the compound was prepared in a manner similar to Example 1 using 1-(3-((1-(3-oxabi- cyclo[3.1.0]hexan-6-yl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-3,6-dichloro-1H-pyra- zolo[3,4-d]pyrimidine (230 mg, 0.51 mmol), Fe (142 mg, 2.54 mmol), and NH4Cl (136 mg, 2.54 mmol) in EtOH (10 mL) and H 2 O (
  • Example 39 (–)-(R) or (S)-8-Chloro-2-(2,2-difluorocyclopropyl)-3-methyl-2,4,12,13-tetrahydro-15 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine and
  • Example 40 (+)-(R) or (S)-8-Chloro-2-(2,2-difluorocyclopropyl)-3-methyl-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • Example 41 8-chloro-2-((2R,4r,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-3-methyl-2,4,12,13- tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 25
  • the compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- ((1-((2R,4r,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-5-methyl-4-nitro-1H-pyrazol-3- yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (140 mg, 0.29 mmol), Fe (129 mg, 2.31mmol), and
  • Example 42 (+)-(R) or (S)-8-Chloro-2-(2,2-dimethyltetrahydro-2H-pyran-4-yl)-3-methyl- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine and
  • Example 43 (–)-(R) or (S)-8-Chloro-2-(2,2-dimethyltetrahydro-2H-pyran-4-yl)-3-methyl- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy-10 cloundecine
  • the racemic mixture of Example 42 and Example 43 was prepared in a manner similar to Example 1 using 3,6-dichloro
  • Example 44 2-((1R,3s,5S)-8-Oxabicyclo[3.2.1]octan-3-yl)-8-chloro-3-methyl-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • the compound was prepared in a manner similar to Example 1 using 1-(3-((1-(8-oxabi-20 cyclo[3.2.1]octan-3-yl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-3,6-dichloro-1H-pyra- zolo[3,4-d]pyrimidine (0.62 g, 1.3 mmol), Fe (162 mg, 2.89 mmol), and NH 4 Cl (155 mg, 2.89 mmol) in EtOH (24 mL) and
  • Example 45 8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine-11,11-d2 5
  • the compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-[1,1- dideuterio-3-(5-methyl-4-nitro-1-tetrahydropyran-4-yl-pyrazol-3-yl)oxy-propyl]pyrazolo[3,4- d]pyrimidine (680 mg, 1.48 mmol), Fe (415 mg, 7.43 mmol), and NH4Cl (397 mg, 7.42 mmol) in EtOH (20 mL) and H 2 O (2 mL) at 80 oC for 16 h, followed by work-up, and pur
  • Example 46 8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine-13,13-d2 20
  • the compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-[3,3- dideuterio-3-(5-methyl-4-nitro-1-tetrahydropyran-4-yl-pyrazol-3-yl)oxy-propyl]pyrazolo[3,4- d]pyrimidine (700 mg, 1.5 mmol), Fe (427 mg, 7.64 mmol), and NH 4 Cl (409 mg, 7.64 mmol) in EtOH (30 mL) and H 2 O (5 mL) at 80 oC for 15 h, followed by work-up, and purification by
  • Example 47 (+)-8-Chloro-2-((3R,4S) or (3S,4R)-3-Fluoro-3-methyltetrahydro-2H-pyran-4-yl)-3-10 methyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine and
  • Example 48 (–)-8-Chloro-2-((3R,4S) or (3S,4R)-3-Fluoro-3-methyltetrahydro-2H-pyran-4-yl)-3- methyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine 15
  • Example 50 8-Chloro-2-((1r,4r)-4-methoxycyclohexyl)-3-(methyl-d 3 )-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 10
  • 3-bromo-8-chloro-2-((1r,4r)-4-methoxycyclohexyl)-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 235 mg, 0.487 mmol
  • bis[tris(tert-butyl)phosphine]palladium 3.3 mg, 0.0065 mmol
  • Example 51 (+)-8-Chloro-3-(methyl-d 3 )-2-((2R,4R) or (2S,4S)-2-methyltetrahydro-2H-pyran-4- yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triaza- cycloundecine 5
  • the compound was prepared in a manner similar to Example 50 using 3-bromo-8- chloro-2-((2R,4R) or (2S,4S)-2-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak1 (559 mg, 1.13 m
  • Example 53 (+)-(R) or (S)-8-Chloro-3-methyl-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro- 10 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine and
  • Example 54 (–)-(R) or (S)-8-Chloro-3-methyl-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • Example 54 (–)-(R) or (S)-8-Chloro-3-methyl-2-(4-oxaspir
  • Example 55 (+)-8-Chloro-3-ethyl-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-20 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine and
  • Example 56 (–)-8-Chloro-3-ethyl-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine 25
  • the racemic mixture of Example 55 and Example 56 was prepared in
  • Example 58 (+)-(R) or (S)-8-Chloro-3-(methyl-d3)-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine and
  • Example 59 (–)-(R) or (S)-8-Chloro-3-(methyl-d 3 )-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • the reaction mixture was heated at 100 oC for 16 h.
  • the reaction mixture was concentrated under25 reduced pressure.
  • the residue was purified by chromatography on silica gel (eluent: hep- tane:EtOAc (10 v% MeOH and 10 v% DCM) 100:0 ⁇ 50:50) to afford a racemic mixture of Exam- ple 58 and Example 59 (0.29 g).
  • Example 60 (R) or (S)-8-Chloro-3-(methyl-d 3 )-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak 1 and
  • Example 61 (R) or (S)-8-Chloro-3-(methyl-d3)-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahy- 5 dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak 2
  • the racemic mixture of Example 60 and Example 61 was prepared in a manner similar to Example 50 using 3-
  • Example 62 (-)-8-Chloro-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-3-(methyl-d3)- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine 15
  • the compound was prepared in a manner similar to Example 50 using (-)-3-bromo-8- chloro-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (101 mg, 0.214
  • Example 63 (+)-8-Chloro-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-3-(methyl-d 3 )- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine 5
  • the compound was prepared in a manner similar to Example 50 using (+)-3-bromo-8- chloro-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (127 mg, 0.268
  • Example 64 (-)-(R) or (S)-8-Chloro-3-(methyl-d3)-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetra- hydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
  • the compound was prepared in a manner similar to Example 50 using (-)-(R) or (S)-3- bromo-8-chloro-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- 25 theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (102 mg, 0.213 mmol), bis[tris(tert
  • Example 65 (+)-(R) or (S)-8-Chloro-3-(methyl-d3)-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetra- hydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 15
  • the compound was prepared in a manner similar to Example 50 using (+)-(R) or (S)-3- bromo-8-chloro-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyra
  • the assay is a homogeneous time resolved-flu- orescence resonance energy transfer (TR-FRET) assay that measures phosphorylation of a fluo- 5 rescein-labelled peptide substrate Fluorescein-ERM LRRKtide obtainable from Life Technologies Corporation as a result of LRRK2 kinase activity.
  • TR-FRET time resolved-flu- orescence resonance energy transfer
  • the phosphorylated peptide is recognized by a terbium-labelled phospho-specific anti-LRRKtide antibody (pLRRKtide antibody), obtainable from Life Technologies Corporation and, subsequently, the phosphorylated LRRKtide can be quantified by the extent of TR-FRET between the terbium donor and fluorescein acceptor.
  • the LRRK2 kinase was obtained from Invitrogen (Life Technologies Corporation) and comprises residue 970 to 2527 of the full length human wild-type LRRK2 kinase, or a similar sequence with the G2019S mutation. As discussed above, this mutation increases the kinase activity relative to the wild type.
  • the kinase reactions were performed in a 20 ⁇ L volume in 384- well plates.
  • the kinase reaction buffer consisted of 50 mM Tris pH 8.5, 0.01% BRIJ-35, 10 mM15 MgCl2, 1 mM EGTA, and 2 mM DTT.
  • the reaction mixture (20 ⁇ l total volume) was incubated for 3.5 h (for LRRK2 WT) and 3 h (for LRRK2 G2019S)20 at 30 ⁇ C, before the reaction was terminated by addition of 10 mM EDTA and 1 nM terbium- labelled anti-phospho-LRRKtide antibody (final volume 20 ⁇ l).
  • the mixture was further incu- bated for 30 minutes at RT.
  • TR-FRET was measured by excitation of the terbium-donor with 340 nm light and subsequent (delay time 100 ⁇ s) measurement of terbium and fluorescein emission at 495 nm and 520 nm, respectively, over a time window of 1000 ⁇ s.
  • TR-FRET measurements were performed on a Biotek Synergy plate.
  • the TR- FRET signal was calculated as the emission-ratio at 520 nm over 495 nm.
  • the TR-FRET ratio readout for test compounds was normalized to 0% inhibition corre- sponding to TR-FRET ratio measured in control wells with no inhibition of the kinase activity and 30 100% inhibition corresponding to TR-FRET ratio measured in control wells with inhibitor.
  • Test compound potency was estimated by nonlinear regression using the sigmoidal dose-re- sponse (variable slope) using Xlfit 4 (IDBS, Guildford, Surrey, UK, model 205). Were the IC 50 could not be determined the % inhibition at the highest tested concentration is given by equation 1.
  • y (A+((B-A)/(1+((C/x) ⁇ D)))) (1) 5 where y is the normalized TR-TRET ratio measurement for a given concentration of test compound, x is the concentration of test compound, A is the estimated efficacy (% inhibition) at infinite compound dilution, and B is the maximal efficacy (% inhibition).
  • C is the IC50 value and D is the Hill slope coefficient.
  • Table 2 shows the IC50 values in nM obtained as described above for the exemplified first compounds of the invention, data is based on n ⁇ 2 tests.
  • Table 2 LRRK2 wild-type and G2019S kinase activity Method I Broad Kinase Selectivity Protein kinase profiling of the inhibitors (Fabian, M.A. et al. inhibitors. Nat. Biotechnol. 5 23, 329-336 (2005)) were undertaken at a concentration of 0.1 ⁇ M and carried out Eurofins Dis- coverX scanMAX panel of 403 wild type kinases (primarily of human origin).
  • AGC PKG, PKC family kinases
  • CAMK Calcium/calmodulin-dependent protein kinases
  • CK1 Casein kinase 1 kinases
  • CMGC CDK, MAPK, GSK3, CLK families
  • STE homologs of yeast Sterile 7, Ster- ile 11, Sterile 20 kinases
  • TK Tyrosine kinases
  • TKL Tyrosine kinase-like kinases
  • lipid and atyp-10 ical kinase families Selectivity Score or S-score is a quantitative measure of compound selectivity.
  • IV intravenous
  • Equation 4 5 Where [F] is the analyte concentration on the buffer (receiver) side of the membrane; [T] is the analyte concentration on the plasma or brain (donor) side of the membrane; [T0] is the analyte concentration in the plasma or brain sample at time zero; D is matrix dilution fac- tor which is determined as 4 for brain matrix and 1 for plasma matrix in these assays.
  • the slices were homogenized in 9 volumes (w/v) of buffer using an ultrasonic probe (GeneReady ultra cool (BSH-C2)).
  • the buffer (aECF) was15 sampled directly from the dish (150 ⁇ L) into an eppendorf tube containing control rat brain ho- mogenate (150 ⁇ L) that had been prepared with 4 volumes buffer.
  • the matrix matched samples were extracted with cold solvent (4 volumes acetonitrile) containing an appropriate bioanalyti- cal internal standard. After centrifugation (20 min, 3200 g, 4 oC) the supernatants were diluted with appropriate volumes of water and compound concentrations were quantified by LC/MS-20 MS against matrix matched calibration standards.
  • the brain Kp,uu values shown in table 7 are further substantiated in table 8 using a more physio- logically relevant brain free fraction model supporting that Example 13 is moderately high brain penetrant. 10

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Abstract

The present invention is directed to combination treatment comprising administration of a first compound of formula I which is a Leucine-rich repeat kinase 2 (LRRK2) inhibitor and a second compound, which compound is useful in the treatment of synucleinopathies, such as Parkinson's disease; wherein formula (I) is.

Description

Combination treatments comprising administration of Leucine-rich repeat kinase 2 (LRRK2) inhibitors Field of the Invention The present invention provides a combination treatment comprising administration of 5 a first compound which is a Leucine-rich repeat kinase 2 (LRRK2) inhibitor and a second com- pound, which compound is useful in the treatment of synucleinopathies, such as Parkinson’s disease. The combination treatment of the invention is for the treatment of a patient suffering from a synucleinopathy disorders, such as Parkinson’s disease. Background of the Invention 10 Parkinson’s disease (PD) is a neurodegenerative disease. It is the second most common neurodegenerative disease after Alzheimer’s disease and affects more than 1% of the population above the age of 65. Parkinson’s disease is clinically characterised by resting tremor, bradykinesia, muscular rigidity and postural instability. In addition to motor symptoms, other symptoms such as neuropsychiatric symptoms are also present in many patients, and in late 15 stages of the disease, Parkinson’s disease dementia commonly develops. Pathologically, the disease is characterised by loss of dopaminergic neurons with consequent decrease in dopamine levels in the brain and by aggregation of the protein α-synuclein in the dopaminergic neurons. These aggregations, called Lewy bodies, are composed of insoluble α-synuclein phosphorylated at serine-129 and ubiquitin. 20 Current Parkinson’s disease therapeutic intervention strategies aim at increasing the dopamine levels by administration of levodopa or monoamine oxidase B inhibitors. As an alternative, dopamine agonists are administered to stimulate dopaminergic receptors, an effect similar to that obtained by increasing the dopamine levels. Although these therapies provide significant symptomatic benefit to the patient, they are also associated with adverse side effects 25 and often become ineffective after prolonged treatment and progression of the underlying disease. Importantly, none of the existing therapies addresses the underlying and disease- causing problem, i.e. the progressive loss of dopaminergic neurons. Leucine-rich repeat kinase 2 (LRRK2) is a 2527 amino acid protein involved in catalysing protein phosphorylation. Evidence is mounting for a relationship between LRRK2 and the 30 pathogenesis of Parkinson’s disease. Single nucleotide polymorphisms that alter amino acids in functional domains of LRRK2 have been shown to cause familiar and sporadic Parkinson’s disease. Several such pathogenic variants have been identified including G2019S, I2020T, N1437H, R1441C, R1441G, R1441H and Y1699C (Shu et al., A Comprehensive Analysis of Population Differences in LRRK2 Variant Distribution in Parkinson's Disease, Front Aging Neurosci., 11:13, 2019; Chittoor-Vinod et al., Genetic and Environmental Factors Influence the Pleomorphy of LRRK2 Parkinsonism, Int. J. Mol. Sci., 2021, 22, 1045). The most common 5 pathogenic form of LRRK2-associated Parkinson’s disease is the amino acid substitution G2019S in the kinase domain of the LRRK2 protein. G2019S Parkinson’s disease is inherited in an autosomal dominant fashion suggesting a gain-of-function mutation of the LRRK2 protein. In support of this notion, biochemical studies have shown that both G2019S and other pathogenic LRRK2 variants lead to an increased kinase activity of LRRK2 (West et al, Parkinson’s disease- 10 associated mutations in leucine-rich repeat kinase 2 augment kinase activity, Proc. Nat. Acad. Sci, 102, 16842-16847, 2005; Chittoor-Vinod et al., Genetic and Environmental Factors Influence the Pleomorphy of LRRK2 Parkinsonism, Int. J. Mol. Sci., 2021, 22, 1045). The clinical and pathological features of Parkinson’s disease associated with LRRK2 mutations are very similar to those of idiopathic Parkinson’s disease (Trinh et al., A comparative study of Parkinson's disease 15 and leucine-rich repeat kinase 2 p.G2019S parkinsonism, Neurobiol. Aging., 35(5), 1125-31, 2014). This strongly suggests a causal involvement of overactive LRRK2 in the pathogenesis of Parkinson’s disease in patients with such activating mutations in LRRK2 and that inhibitors of LRRK2 could be used as disease modifying treatment in familiar Parkinson’s disease. In addition to the rare high-penetrance exonic LRRK2 variants mentioned above, there 20 are also common LRRK2 variants with lower but significant association with Parkinson’s disease showing that LRRK2 also contributes to idiopathic Parkinson’s disease. These include very common single-nucleotide polymorphisms in the LRRK2 promotor region, where the Parkinson’s disease associated variants appear to be associated with increased LRRK2 expression at least in some cell types (Nalls et al., Identification of novel risk loci, causal insights, and heritable risk for 25 Parkinson's disease: a meta-analysis of genome-wide association studies, Lancet Neurol,2019, 18, 1091–1102; Sun et al., Genetic Variants Associated With Neurodegenerative Diseases Regulate Gene Expression in Immune Cell CD14+ Monocytes, Front Genet., 2018, 18, 9:666; Langston et al., Association of a Common Genetic Variant with Parkinson’s Disease is Propagated through Microglia, bioRxiv, 2021) suggesting that LRRK2 inhibition might be relevant. Further, 30 investigations of common exonic polymorphic variants have highlighted several LRRK2 Parkinson’s disease risk variants including the A419V and G2385R that are common in the Asian population (Shu et al., A Comprehensive Analysis of Population Differences in LRRK2 Variant Distribution in Parkinson's Disease, Front Aging Neurosci., 11:13, 2019). There is also a protective variant of LRRK2 with reduced kinase activity such as the LRRK2 N551K R1398H variant (Wang et al., Understanding LRRK2 kinase activity in preclinical models and human subjects through quantitative analysis of LRRK2 and pT73 Rab10, Scientific Reports, 2021, 11:12900), which lends further support to the potential of LRRK2 inhibition in idiopathic 5 Parkinson’s disease by showing that wild-type LRRK2 activity is not optimal in a Parkinson’s disease context. Functionally, LRRK2 affects trafficking of lysosomes and other vesicles through phosphorylation of RAB GTPases, and PD-associated genes are enriched for genes involved in lysosomal function and autophagy (Chang et al., A meta-analysis of genome-wide association studies identifies 17 new Parkinson's disease risk loci, Nat Genet., 2017, 49(10): 1511–1516). 10 Two genes associated with Parkinson’s disease, VPS35 and RAB29, have been shown to directly interact with LRRK2 biology as they increase LRRK2 activity (Taylor et al., Advances in elucidating the function of leucine-rich repeat protein kinase-2 in normal cells and Parkinson's disease, Curr Opin Cell Biol., 2020, 63:102-113), and as mentioned above, LRRK2 associated Parkinson’s disease is very similar to idiopathic PD. Together this strongly supports the relevance of LRRK215 inhibition in treatment of idiopathic PD. Several lines of evidence suggest that LRRK2 activity may impact α-synuclein pathology development after seeding with α-synuclein (O’Hara et al., LRRK2 and α-Synuclein: Distinct or Synergistic Players in Parkinson's Disease?, Front Neurosci., 2020, 17;14:577). In addition to strengthening the case for potential of LRRK2 inhibitors for treatment of 20 Parkinson’s disease, this indicates potential of LRRK2 inhibitors for treatment of other synucleinopathies including Lewy body dementia and multiple system atrophy. LRRK2 is highly expressed in white blood cells and spleen suggesting a potential for LRRK2 inhibitors for treatment of aberrant immune responses. This is further supported by genetic association of LRRK2 with such diseases particularly inflammatory bowel diseases 25 including Crohn’s disease and leprosy (Liu et al., Association analyses identify 38 susceptibility loci for inflammatory bowel disease and highlight shared genetic risk across populations, Nat Genet., 2015, 47(9):979-986; Rastegar et al., Leucine Rich Repeat Kinase 2 and Innate Immunity, Front Neurosci., 2020, 10;14:193). Thus, LRRK2 inhibitors may have potential for treatment of these diseases. 30 Both from the pharmaceutical industry and academic labs there has been a high interest in developing potent selective LRRK2 inhibitors due to its great promise in treating Parkinson’s disease. The historic development of LRRK2 inhibitors is well described in the literature (Delgado et al., N-bridged 5,6-bicyclic pyridines: Recent applications in central nervous system disorders, European Journal of Medicinal Chemistry 97 (2015) 719-731); Xiao Ding & Feng Ren (2020) Leucine-rich repeat kinase 2 inhibitors: a patent review (2014-present), Expert Opinion on 5 Therapeutic Patents, 30:4, 275-286). Even though a lot of focus has been on designing new LRRK2 inhibitors from the pharmaceutical industry and academic labs, the task of designing a brain penetrant, potent, selective LRRK2 inhibitor remains a challenging task for the medicinal chemistry community (Delgado et al., N-bridged 5,6-bicyclic pyridines: Recent applications in central nervous system disorders, European Journal of Medicinal Chemistry 97 (2015) 719-731). 10 Despite the tremendous efforts from pharmaceutical industry and academic labs only two molecules (DNL201 and DNL151) from Denali Therapeutics have reached clinical phase (Xiao Ding & Feng Ren (2020) Leucine-rich repeat kinase 2 inhibitors: a patent review (2014-present), Expert Opinion on Therapeutic Patents, 30:4, 275-286). Against this background there is still a highly unmet need to provide a LRRK2 inhibitor15 with good pharmacokinetic properties, whilst maintaining high potency and good selectivity. The combined use and pharmaceutical compositions of the invention may offer alternatives to current treatment paradigms for synucleinopathies, such as parkinson’s disease. The current standard of care treatment paradigms for this type of diseases are merely symptomatic treatments which are not efficacious in all patients, and which does not modify the 20 disease progression. Hence, there remains a need for alternative methods of treatment of such diseases. Summary of the invention The invention provides the combined use of certain 5,7-(azenometheno)dipyrazolo[3,4- b:5',1'-g][1]oxa[4,6,8]triazacycloundecine compounds which are LRRK2 inhibitors, and as such 25 are useful to treat synucleinopathies, such as Parkinson’s disease, and a second compound that is useful in the treatment of the same diseases. Accordingly, in a first aspect of the invention provides: (i) a first compound of formula I, or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000006_0001
R1 is CH2R4 or R4; R2 is a C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, a C3-C6 cycloalkyl, or a C1-C3 haloalkyl; R3 is halogen, cyano, a O-C1-C3 haloalkyl, a C1-C3 haloalkyl, a C3-C6 cycloalkyl, or a C1-C3 alkyl; 5 R4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen; a C1-C3 alkyl, a C1-C3 cyanoalkyl, a C1-C3 haloalkyl, or a C3-C6 cycloalkyl; or R4 is a bicyclic 8-membered heterocycle having 1-2 heteroatoms independently selected from oxygen and nitrogen; wherein each heterocycle or cycloalkyl is unsubstituted or substituted with 1, 2, or 3 groups 10 independently selected from the group consisting of cyano, deuterium, halogen, C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, a O-C1-C3 haloalkyl, a O-C1-C3 alkyl, or a C1-C3 haloalkyl; and (ii) a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, wherein the first and the15 second compound are for combined use in the treatment of synucleinopathies. In a further aspect, the invention provides a pharmaceutical composition comprising a first compound of formula I as disclosed herein or pharmaceutically acceptable salts thereof, a second compound or a pharmaceutically acceptable salt thereof, which second compound is20 useful in the treatment of synucleinopathies, and one or more pharmaceutically acceptable car- riers. In a further aspect, the invention provides a first compound of formula I as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceuti-25 cally acceptable salt thereof, which second compound is useful in the treatment of synucleinop- athies, for combined use in the treatment of a synucleinopathy, such as Parkinson’s disease. In a further aspect, the invention provides combined use of a first compound of formula I as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, in the manufacture of a medicament for the treatment of a synucleinopathy, such as Parkinson’s disease. 5 In a further aspect, the invention provides combined use of a first compound of formula I as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, in the treatment of a synucleinopathy, such as Parkinson’s disease. 10 In a further aspect, the invention relates to a method for the treatment of synucleinop- athies such as Parkinson disease, the method comprising the administration of a therapeutically effective amount of a first compound of formula I as disclosed herein or pharmaceutically ac- ceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, to a patient in need15 thereof. Detailed description of the invention The invention provides the combined use of a first compound which is a LRRK2 inhibitor and a second compound or a pharmaceutically acceptable salt thereof, which combined use is for the treatment of synucleinopathies selected from Lewy body dementia, multiple system at-20 rophy or Parkinson’s disease. The first compounds of the invention are listed in table 1. As can be seen from the ex- amples of the present disclosure, the compounds were demonstrated to possess a low clearance and a high brain penetrance, while maintaining a high potency and selectivity for LRRK2 and having good pharmacokinetics. As has been demonstrated in the historic literature of LRRK2 25 inhibitors, as presented above, identification of compound having such characteristics is by no means trivial. Table 1: First compounds of the invention
Figure imgf000007_0001
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Definitions In the present context, “alkyl” is intended to indicate a straight or branched saturated hydrocarbon. In particular, C1-C3-alkyl is intended to indicate such hydrocarbon having 1, 2 or 3 carbon atoms in the longest continuous carbon chain. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl and the like. As used herein, the term "isotopically labelled alkyl group" means that either the carbon or the hydrogen atom(s) in the alkyl group is replaced with a corresponding isotope such as 13C 5 and/or 14C for carbon atom(s), or deuterium or tritium for hydrogen atom(s). In an embodiment of the invention the hydrogen atoms of the alkyl group are all replaced by deuterium. Repre- sentative examples of isotopically labelled alkyl include but are not limited to -CD3, -CD2CD3. In a preferred embodiment, the isotopically labelled C1-C3-alkyl is -CD3. The term “alkylene,” as used herein, refers to a divalent group derived from a straight 10 or branched chain hydrocarbon of 1 to 10 carbon atoms, for example, of 2 to 5 carbon atoms. Representative examples of alkylene include, but are not limited to, -CH2-, -CH2CH2-, - CH2CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH(CH3)- and - CH2CH2CH2CH2CH2-. The term “alkoxy” as used herein refers to a group of formula -O-alkyl, wherein alkyl is 15 defined as above. In particular, C1-C3-alkoxy is intended to indicate a hydrocarbon having 1, 2 or 3 carbon atoms. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, isobutoxy, t-butoxy and the like. The term “haloalkyl” or “haloalkoxy” is intended to refer to an alkyl or alkoxy group as defined hereinabove with 1, 2 or 3 hydrogens replaced by a halogen. Representative examples 20 include but are not limited to CH2F, CHF2 CF3, OCF3, OCH2F, and OCHF2. In the present application “haloalkoxy” may also be referred to as “O-haloalkyl”, such as for example “O-C1-C3 haloalkyl”. Similarly, the term “fluoroalkyl” is intended to refer to an alkyl group as defined here- inabove, with 1, 2, or 3 hydrogens replaced by fluoro. Representative examples include but are not limited to -CF3. 25 In the present context, “halogen” is intended to indicate members of the 7th main group of the periodic table of the elements, such as fluoro, bromo and chloro. The term “heteroatom” is intended to mean sulfur, oxygen or nitrogen. The term “cyano” as used herein, means at least one -CN group is appended to the par- ent molecular moiety. 30 The term “cyanoalkyl” as used herein is intended to indicate an alkyl group as defined herein, wherein at least one -CN group is appended to the parent molecular moiety. The term "cyclic" as used herein refers to any cyclic structure, including heterocyclic, aromatic and heteroaromatic non-fused ring systems. The term "membered" is meant to denote the number of skeletal atoms that constitute the ring. Thus, for example, pyridyl, pyranyl, and pyrimidinyl are six-membered rings and pyrrolyl, and tetrahydrofuranyl are five-membered rings. The term “cycloalkyl,” as used herein, refers to a carbocyclic ring system containing 5 three to ten carbon atoms, zero heteroatoms and zero double bonds. The cycloalkyl may be monocyclic or bicyclic, wherein the bicyclic ring is joined bridged, fused, or spirocyclic. The terms "heterocycle", "heterocyclic" and "heterocyclyl" as used herein, alone or in combination, refers to saturated or unsaturated nonaromatic rings containing from 4 to 7 ring atoms where one or more of the ring atoms are heteroatoms. The heterocycle may be monocy-10 clic or bicyclic, wherein the bicyclic ring is joined bridged, fused, or spirocyclic. In some embodi- ments if explicitly stated, the term "heterocycle", "heterocyclic" and "heterocyclyl" may refer to a saturated or unsaturated nonaromatic ring containing 8 ring atoms where one or more of the ring atoms are heteroatoms. Such 8-membered heterocycle is a bicyclic ring, wherein the bicyclic ring is joined bridged, fused, or spirocyclic. 15 In the present context, the term "therapeutically effective amount" of a compound is intended to indicate an amount sufficient to alleviate or partially arrest the clinical manifesta- tions of a given disease and its complications in a therapeutic intervention comprising the ad- ministration of said compound. An amount adequate to accomplish this is defined as "therapeu- tically effective amount". Effective amounts for each purpose will depend on the severity of the 20 disease or injury as well as the weight and general state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, e.g. by con- structing a matrix of values and testing different points in the matrix, which is all within the ordinary skills of a trained physician. In the present context, the term "treatment" and "treating" means the management and 25 care of a patient for the purpose of combating a disease. The term is intended to include the full spectrum of treatments for a given disease from which the patient is suffering, such as admin- istration of the active compound to alleviate the symptoms or complications, to delay the pro- gression of the disease, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease. The patient to be treated is preferably a mammal, in particular a human30 being. In the present context, “disease” can be used synonymous with disorder, condition, mal- function, dysfunction and the like. The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. For example, the phrase "the compound" is to be understood as referring to various "compounds" of the invention or particular described aspect, unless otherwise indicated. The description herein of any aspect or aspect of the invention using terms such as “compris- 5 ing”, “having,” “including,” or “containing” with reference to an element or elements is intended to provide support for a similar aspect or aspect of the invention that “consists of”, “consists essentially of”, or “substantially comprises” that particular element or elements, unless other- wise stated or clearly contradicted by context (e.g., a composition described herein as compris- ing a particular element should be understood as also describing a composition consisting of10 that element, unless otherwise stated or clearly contradicted by context). The terms “combined”, “combined use” “in combination with” and “a combination of” and the like as used herein is intended to indicate the administration of a first compound according to formula (I) or a pharmaceutically acceptable salt thereof simultaneously or sequentially, in any order, with a second compound or a pharmaceutically acceptable salt thereof, which second 15 compound is useful in the treatment of a synucleinopathy. The first and second compounds may be administered simultaneously or with a time gap between the administrations of the two com- pounds. The first and second compounds may be administered either as part of the same phar- maceutical formulation or composition, or in separate pharmaceutical formulations or compo- sitions. The first and second compounds may be administered on the same day or on different 20 days. They may be administered by the same route, such as by oral administration, or by depot, or by intramuscular or intraperitoneal injection, or by intravenous injection; or by different routes wherein one compound is for example administered orally or placed by depot and the other compound is injected, or wherein one compound is for example placed by depot and the other is administered orally or injected. The two compounds may be administered by the same25 dosage regimes or interval, such as once or twice daily, weekly, or monthly; or by different dos- age regimes or intervals for example wherein one is administered once daily and the other is administered twice daily, weekly or monthly. In some instances, a patient to be treated may already be in treatment with the second compound or a pharmaceutically acceptable salt thereof, which is useful in the treatment of a synucleinopathy when the treatment with a first 30 compound according to formula I, or a pharmaceutically acceptable salt thereof, is initiated. In other instances, the patient may already be in treatment with a first compound according to formula I or a pharmaceutically acceptable salt thereof when treatment with the second com- pound or a pharmaceutically acceptable salt thereof, which compound is useful in the treatment of a synucleinopathy is initiated. In other instances, the treatment with a first compound accord- ing to formula I or a pharmaceutically acceptable salt thereof and treatment with a second com- pound or a pharmaceutically acceptable salt thereof, which compound is useful in the treatment of a synucleinopathy is initiated at the same time. 5 Embodiments of the invention In the following embodiments of the invention are disclosed. In a first aspect of the invention provides: (i) a first compound of formula I, or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000034_0001
10 R1 is CH2R4 or R4; R2 is a C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, a C3-C6 cycloalkyl, or a C1-C3 haloalkyl; R3 is halogen, cyano, a O-C1-C3 haloalkyl, a C1-C3 haloalkyl, a C3-C6 cycloalkyl, or a C1-C3 alkyl; R4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox-15 ygen and nitrogen; a C1-C3 alkyl, a C1-C3 cyanoalkyl, a C1-C3 haloalkyl, or a C3-C6 cycloalkyl; or R4 is a bicyclic 8-membered heterocycle having 1-2 heteroatoms independently selected from oxygen and nitrogen; wherein each heterocycle or cycloalkyl is unsubstituted or substituted with 1, 2, or 3 groups independently selected from the group consisting of cyano, deuterium, halogen, C1-C3 alkyl, an 20 isotopically labelled C1-C3 alkyl, a O-C1-C3 haloalkyl, a O-C1-C3 alkyl, or a C1-C3 haloalkyl; and (ii) a second compound, which second compound is useful in the treatment of synucle- inopathies, wherein the first and the second compound are for combined use in the treatment of synucleinopathies. 25 In some embodiments of the invention, R1 is CH2R4. In some embodiments of the invention, R1 is R4. In some embodiments of the invention, the first compound is a compound of formula Ia, or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000035_0001
. In some embodiments of the invention, R2 is selected from a C1-C3 alkyl, an isotopically 5 labelled C1-C3 alkyl or a C3-C6 cycloalkyl. In some embodiments of the invention, R2 is selected from -CH3, -CH2CH3, -CD3, or cyclo- propyl. In some embodiments of the invention, R2 is C1-C3 alkyl. In some embodiments of the invention, R2 is methyl. 10 In some embodiments of the invention, R2 is ethyl. In some embodiments of the invention, R2 is an isotopically labelled C1-C3 alkyl. In some embodiments of the invention, R2 is selected from the group consisting of -CD3 or -CD2CD3. 15 In some embodiments of the invention, R2 is -CD3. In some embodiments of the invention, R2 is cyclopropyl. In some embodiments of the invention, R3 is C1-C3 haloalkyl. 20 In some embodiments of the invention, R3 is CF3. In some embodiments of the invention, R3 is halogen. In some embodiments of the invention, R3 is chloro. In some embodiments of the invention, R3 is bromo. 25 In some embodiments of the invention, R3 is cyano. In some embodiments of the invention, R3 is a C3-C6 cycloalkyl. In some embodiments of the invention, R3 is cyclopropyl. In some embodiments of the invention, R4 is a 4- to 6-membered heterocycle having one oxygen atom, wherein the 4- to 6-membered heterocycle is unsubstituted or substituted with one group selected from the group consisting of cyano, deuterium, halogen, C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, O-C1-C3 haloalkyl, O-C1-C3 alkyl, or C1-C3 haloalkyl. 5 In some embodiments of the invention, R4 is a 4- to 6-membered heterocycle having one oxygen atom, wherein the 4- to 6-membered heterocycle is unsubstituted or substituted with one group selected from the group consisting of deuterium, halogen, C1-C3 alkyl, or an iso- topically labelled C1-C3 alkyl. 10 In some embodiments of the invention, R4 is 6-membered heterocycle having one oxy- gen atom, wherein the 6-membered heterocycle is unsubstituted or substituted with one group selected from the group consisting of deuterium, halogen, C1-C3 alkyl, or an isotopically labelled C1-C3 alkyl. 15 In some embodiments of the invention, R4 is 6-membered heterocycle having one oxy- gen atom, wherein the 6-membered heterocycle is unsubstituted or substituted with two groups independently selected from the group consisting of deuterium, halogen, C1-C3 alkyl, or an iso- topically labelled C1-C3 alkyl. 20 In some embodiments of the invention, R4 is selected from the group consisting of:
Figure imgf000036_0001
25 In some embodiments of the invention, R4 is selected from the group consisting of: CH3,
Figure imgf000037_0001
Figure imgf000038_0001
In some embodiments of the invention, R4 is tetrahydropyran. In some embodiments of the invention, R4 is unsubstituted tetrahydropyran. 5 In some embodiments of the invention, R4 is tetrahydro-2H-pyran-4-yl. In some embodiments of the invention, R4 is tetrahydro-2H-pyran-3-yl. In some embodiments of the invention, R4 is 4-oxaspiro[2.5]octan-7-yl. In some embodiments of the invention, the 8-membered heterocycle is an unsubsti-10 tuted 8-membered spirocyclic heterocycle. In some embodiments of the invention, the bicyclic 8-membered heterocycle contains one oxygen. In some embodiments of the invention, the bicyclic 8-membered heterocycle contains one oxygen and is a spirocyclic heterocycle. 15 In some embodiments of the invention, the first compound is a compound of formula Ib, or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000038_0002
R2 is a C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, a C3-C6 cycloalkyl, or a C1-C3 haloalkyl; R4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen, a C1-C3 alkyl, a C1-C3 cyanoalkyl, a C1-C3 haloalkyl, or a C3-C6 cycloalkyl; wherein each heterocycle or cycloalkyl is unsubstituted or substituted with 1 group se- lected from the groups selected from cyano, deuterium, halogen, a C1-C3 alkyl, an isotopically 5 labelled C1-C3 alkyl, a O-C1-C3 haloalkyl, a O-C1-C3 alkyl, or a C1-C3 haloalkyl. In some embodiments of the invention, R2 is selected from -CH3, -CH2CH3, -CD3, or cy- clopropyl. 10 In some embodiments of the invention, the first compound is a compound of formula Ic, or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000039_0001
R4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen, a C1-C3 alkyl, a C1-C3 cyanoalkyl, a C1-C3 haloalkyl, or a C3-C6 cycloalkyl;15 wherein each heterocycle or cycloalkyl is unsubstituted or substituted with 1 group se- lected from the groups selected from cyano, deuterium, halogen, a C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, a O-C1-C3 haloalkyl, a O-C1-C3 alkyl, or a C1-C3 haloalkyl. In some embodiments of the invention, the first compound is a compound of formula 20 Id, or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000039_0002
R4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen, a C1-C3 alkyl, a C1-C3 cyanoalkyl, a C1-C3 haloalkyl, or a C3-C6 cycloalkyl; wherein each heterocycle or cycloalkyl is unsubstituted or substituted with 1 group se- 25 lected from the groups selected from cyano, deuterium, halogen, a C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, a O-C1-C3 haloalkyl, a O-C1-C3 alkyl, or a C1-C3 haloalkyl. In some embodiments of the invention, the 4- to 7-membered heterocycle is an unsub- stituted 6-7 membered spirocyclic heterocycle. In some embodiments of the invention, the 4- to 7-membered heterocycle is an unsub- stituted 6-7 membered bridged heterocycle. 5 In some embodiments of the invention, the first compound is selected from the group consisting of : , , 10 , , ,
Figure imgf000040_0001
,
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
1
Figure imgf000044_0001
In some embodiments of the invention, the first compound is selected from the group consisting of: 8-Chloro-2,3-dimethyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo-[3,4- b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 5 8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; (R)-8-chloro-3-methyl-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; (S)-8-chloro-3-methyl-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahydro-11H-5,7-10 (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 3-Methyl-2-(tetrahydro-2H-pyran-4-yl)-8-(trifluoromethyl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Bromo-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 15 8-Cyclopropyl-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 3,8-Dimethyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 3-Methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome-20 theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine-8-carbonitrile; 8-Chloro-3-cyclopropyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 2-((2-Oxabicyclo[2.1.1]hexan-1-yl)methyl)-8-chloro-3-methyl-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 25 8-Chloro-3-methyl-2-((3-methyloxetan-3-yl)methyl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-(methyl-d3)-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-methyl-2-(3-methyloxetan-3-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome-30 theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-(methyl-d3)-2-(tetrahydro-2H-pyran-4-yl-4-d)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; and 8-Chloro-3-(1-fluorocyclopropyl)-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-ethyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 5 8-Chloro-3-methyl-2-((2R,4R)-2-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-methyl-2-((2S,4S)-2-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-10 (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine-11,11,12,12,13,13- d6; 8-Chloro-3-methyl-2-((3R,4S) -3-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-methyl-2- ((3S,4R) -3-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahy-15 dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-methyl-2-((3S,4S) -3-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-methyl-2-((3R,4R)-3-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 20 8-Chloro-2-((1r,4r)-4-fluorocyclohexyl)-3-methyl-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-2-(4,4-difluorocyclohexyl)-3-methyl-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; (R)-8-Chloro-3-methyl-2-(oxepan-3-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome-25 theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; (S)-8-Chloro-3-methyl-2-(oxepan-3-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; (R)-8-Chloro-3-methyl-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; (S)-8-Chloro-3-methyl-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 5 8-Chloro-2-((3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-3-methyl-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-2-((3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)-3-methyl-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 3-(8-Chloro-3-methyl-12,13-dihydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-10 g][1]oxa[4,6,8]triazacycloundecin-2(4H)-yl)bicyclo[1.1.1]pentane-1-carbonitrile; 8-Chloro-3-methyl-2-(2-oxaspiro[3.3]heptan-6-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-(methyl-d3)-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine-11,11,12,12,13,13-15 d6; 1-(8-Chloro-3-methyl-12,13-dihydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecin-2(4H)-yl)cyclopropane-1-carbonitrile; 8-Chloro-2-((1r,4r)-4-methoxycyclohexyl)-3-methyl-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 20 2-((1R,5S,6r)-3-Oxabicyclo[3.1.0]hexan-6-yl)-8-chloro-3-methyl-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; (R)-8-Chloro-2-(2,2-difluorocyclopropyl)-3-methyl-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; (S)-8-Chloro-2-(2,2-difluorocyclopropyl)-3-methyl-2,4,12,13-tetrahydro-11H-5,7-25 (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-chloro-2-((2R,4r,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-3-methyl-2,4,12,13-tet- rahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; (R)-8-Chloro-2-(2,2-dimethyltetrahydro-2H-pyran-4-yl)-3-methyl-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; (S)-8-Chloro-2-(2,2-dimethyltetrahydro-2H-pyran-4-yl)-3-methyl-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 5 2-((1R,3s,5S)-8-Oxabicyclo[3.2.1]octan-3-yl)-8-chloro-3-methyl-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine-11,11-d2; 8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-10 (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine-13,13-d2; 8-Chloro-2-((3R,4S)-3-Fluoro-3-methyltetrahydro-2H-pyran-4-yl)-3-methyl-2,4,12,13- tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-2-((3S,4R)-3-Fluoro-3-methyltetrahydro-2H-pyran-4-yl)-3-methyl-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 15 8-Chloro-3-methyl-2-((tetrahydro-2H-pyran-4-yl)methyl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-2-((1r,4r)-4-methoxycyclohexyl)-3-(methyl-d3)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-(methyl-d3)-2-((2R,4R )-2-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetra-20 hydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-(methyl-d3)-2-((2S,4S )-2-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetra- hydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; (R)-8-Chloro-3-methyl-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 25 (S)-8-Chloro-3-methyl-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-ethyl-2-((3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-ethyl-2-((3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-30 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-3-ethyl-2-((1r,4r)-4-methoxycyclohexyl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; (R)-8-Chloro-3-(methyl-d3)-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 5 (S)-8-Chloro-3-(methyl-d3)-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; (R)-8-Chloro-3-(methyl-d3)-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; (S)-8-Chloro-3-(methyl-d3)-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahydro-11H-10 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-Chloro-2-((3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)-3-(methyl-d3)-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 8-chloro-2-((3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-3-(methyl-d3)-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; 15 (R)-8-chloro-3-(methyl-d3)-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; and (S)-8-chloro-3-(methyl-d3)-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine; or a pharmaceuti- cally acceptable salt thereof. 20 In some embodiments of the invention, the first compound is
Figure imgf000049_0002
, or a pharmaceutically acceptable salt. In some embodiments of the invention, the first compound is
Figure imgf000049_0001
. In some embodiments of the invention, the first compound is , or a pharmaceutically acceptable salt. In some embodiments of the invention, the first compound is , or a pharmaceutically acceptable salt. In some embodiments of the invention, the first compound is 5
Figure imgf000050_0001
. In some embodiments of the invention, the first compound is , or a pharmaceutically acceptable salt thereof. In some embodiments of the invention, the first compound is , or a pharmaceutically acceptable salt thereof. 10 In some embodiments of the invention, the first compound is , or a pharmaceutically acceptable salt thereof. In some embodiments of the invention, the first compound is
Figure imgf000051_0001
,or a pharmaceutically acceptable salt thereof. In some embodiments of the invention, the first compound is
Figure imgf000051_0002
,or a pharmaceutically acceptable salt thereof. 5 In some embodiments of the invention, the first compound is , or a pharmaceutically acceptable salt thereof. In some embodiments of the invention, the first compound is 10 , or a pharmaceutically acceptable salt thereof. In some embodiments of the invention, the first compound is
Figure imgf000051_0003
. 15 In some embodiments of the invention, the first compound is
Figure imgf000052_0001
. In some embodiments of the invention, the first compound is
Figure imgf000052_0002
. In some embodiments of the invention, the first compound is 5
Figure imgf000052_0003
In some embodiments of the invention, the first compound is
Figure imgf000052_0004
. In some embodiments of the invention, the first compound is
Figure imgf000052_0005
. 10 In some embodiments of the invention, the first compound is
Figure imgf000052_0006
. In some embodiments of the invention, the first compound is
Figure imgf000053_0001
. In some embodiments of the invention, the first compound is
Figure imgf000053_0002
. In some embodiments of the invention, the first compound is 5
Figure imgf000053_0003
. In some embodiments of the invention, the first compound is
Figure imgf000053_0004
. 10 In a further aspect, the invention provides a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treat- ment of synucleinopathies, for combined use in the treatment of a synucleinopathy, such as Parkinson’s disease. 15 In an embodiment, the invention provides a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treat- ment of Parkinson’s disease, for combined use in the treatment of Parkinson’s disease. In a further aspect, the invention provides combined use of a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, in the manufacture of a medicament for the treat- 5 ment of a synucleinopathy, such as Parkinson’s disease. In an embodiment, the invention provides combined use of a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of Parkinson’s disease, in the manufacture of a medicament for the 10 treatment of Parkinson’s disease. In a further aspect, the invention provides combined use of a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is 15 useful in the treatment of synucleinopathies, in the treatment of a synucleinopathy, such as Parkinson’s disease. In an embodiment, the invention provides combined use of a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is 20 useful in the treatment of Parkinson’s disease, in the treatment of Parkinson’s disease. In a further aspect, the invention relates to a method for the treatment of synucleinop- athies such as Parkinson disease, the method comprising the administration of a therapeutically effective amount of a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or25 pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically ac- ceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, to a patient in need thereof. In an embodiment, the invention relates to a method for the treatment of Parkinson disease, the method comprising the administration of a therapeutically effective amount of a30 first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically accepta- ble salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of Parkinson’s disease, to a patient in need thereof. In an aspect, the invention relates to a first compound of formula I, Ia, Ib, Ic, Id, A or Aa as disclosed herein or pharmaceutically acceptable salt thereof and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, for use in therapy. 5 In an embodiment the synucleinopathy is selected from the group consisting of Lewy body dementia, multiple system atrophy, and Parkinson’s disease. In an embodiment the synucleinopathy is parkinson’s disease. In an embodiment the Parkinson’s disease is idiopathic Parkinson’s disease, sporadic Parkinson’s disease, Parkinson’s disease in patients carrying a G2019S mutation in LRRK2, or 10 Parkinson’s disease in patients carrying one or more LRRK2 non-coding variants selected from rs76904798-T and Rs1491942-G. In an embodiment the Parkinson’s disease is idiopathic Parkinson’s disease, sporadic Parkinson’s disease or Parkinson’s disease in patients carrying one or more mutated forms of LRRK2 selected from G2019S, I2020T, M1646T, G2385R, A419V, N551K, R1398H, K1423K,15 R1441G, R1441H, R1441C, R1628P, S1647T, Y1699C, I2020T and Y2189C. In an embodiment the Parkinson’s disease is idiopathic Parkinson’s disease, sporadic Parkinson’s disease or Parkinson’s disease in patients carrying a G2019S mutation in LRRK2. In an embodiment the Parkinson’s disease is idiopathic Parkinson’s disease, sporadic Parkinson’s disease or Parkinson’s disease in patients carrying one or more LRRK2 non-coding20 variants selected from rs76904798-T and Rs1491942-G. In an embodiment the synucleinopathy is characterized by increased LRRK2 kinase ac- tivity or by expression of one or more mutated forms of LRRK2 selected from G2019S, I2020T, M1646T, G2385R, A419V, N551K, R1398H, K1423K, R1441G, R1441H, R1441C, R1628P, S1647T, Y1699C, I2020T and Y2189C or one or more LRRK2 non-coding variants alone or in combination25 selected from rs76904798-T and Rs1491942-G. In a further aspect, the invention provides a pharmaceutical composition comprising a first compound of formula I as disclosed herein or pharmaceutically acceptable salts thereof, a second compound or a pharmaceutically acceptable salt thereof, which second compound is30 useful in the treatment of synucleinopathies, and one or more pharmaceutically acceptable car- riers. In an embodiment the second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is symptomatic drugs or disease modifying compounds. 5 In an embodiment the second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is selected from anti- cholinergic agents, dopaminergic agents, and adenosine receptor antagonists. In an embodiment the anticholinergic agent is selected from the group comprising tri- hexyphenidyl, biperiden, metixene, procyclidine, profenamine, dexetimide, phenglutarimide,10 mazaticol, bornaprine, tropatepine, etanautine, orphenadrine, benzatropine, and etybenzatro- pine. In an embodiment the dopaminergic agent is selected from the group comprising dopa- mine, levodopa, carbidopa, levodopa/carbidopa, melevodopa, etilevodopa, foslevodopa, aman- tadine, bromocriptine, pergolide, dihydroergocryptine mesylate, ropinirole, pramipexole, caber-15 goline, apomorphine, piribedil, rotigotine, selegiline, rasagiline, safinamide, tolcapone, entaca- pone, budipine, and opicapone. In an embodiment the adenosine receptor antagonist is istradefylline. In an embodiment the second compound or a pharmaceutically acceptable salt 20 thereof, which second compound is useful in the treatment of synucleinopathies is selected from caspase inhibitors, calpain inhibitors, LRRK2 inhibitors, BACE1 inhibitors, antibodies against any form of Aβ peptides, inflammation inhibitors, LAG-3 antibodies, molecules inhib- iting alpha-synuclein aggregation, nicotine, caffeine, Monoamine oxidase B inhibitor, Levo- dopa/carbidopa, Dopamine agonists, COMT inhibitors, A2A antagonists, anti-alphasynuclein25 antibodies, Mitogen-activated protein kinase 14 inhibitors, USP30 inhibitors, Beta adrenore- ceptor agonists, dopamine D1 PAMs, Toll-like receptor 2 antagonists, Carnitine palmitoyl transferase 1 inhibitors, Glutamate 5 receptor antagonists, Muscarinic M2 receptor antago- nists, Muscarinic M3 receptor antagonists, Muscarinic M4 receptor antagonists, 5 Hydroxy tryptamine 1A receptor agonists, 5 Hydroxy tryptamine 4 receptor agonists, 5 Hydroxy tryp-30 tamine 3 receptor agonists, 5 Hydroxy tryptamine 2A receptor antagonists, Alpha 1a adreno- receptor antagonists, Sigma 1 receptor agonists, Sigma 2 receptor antagonists, Dopamine D1 receptor agonists, Dopamine D2 receptor agonists, Dopamine D3 receptor agonists, Dopa- mine D5 receptor antagonists, dopamine reuptake inhibitors, DOPA decarboxylase inhibitors, Insulin-like growth factor 1 antagonists, Insulin-like growth factor 2 antagonists, Tyrosine ki- nase inhibitors, Ras inhibitors, Leucotriene D4 antagonists, Leucotriene receptor antagonists, PDE1 inhibitors, T-type calcium channel antagonists, Peroxiredoxin inhibitors, GLP-1R ago- nists, NMDA receptor blockers, Lipoxygenase inhibitors, and Peroxisome proliferator-acti- 5 vated receptor gamma agonists. In an embodiment the second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is selected from group comprising pralnacasan, bapineuzemab, solanezumab, gantenerumab, crenezumab, aducanu- mab, glunozumab, prasinezumab, miglustat, eliglustat, ropinerole, neflamapimod, bosutinib, 10 oxafuramine, nadolol, nilotinib, vodobatinib, clenbuterol, mevidalen, emeramide, tomaralimab, mitometin, dipraglurant, ezeprogind, blarcamesine, buntanetap, renzapride, tavapadon, prami- prexole, salbutamol, Infudopa, alirinetide, antraquinolol, a rasagiline prodrug, apomorphine, pramiprexole, radotinib, talineuren, montelukast, lenrispodun, pirepemat, suvecaltamide, sonlicromanol, L-Demethyl phencynonate hydrochloride, mesocarb, roluperidone, exenatide,15 befiradol, ketamine, pridopidine, utroloxestat, aplindore, finamine, phenlarmide, (R)-trolox- amide quinone, cannibigerol, and kenterin. The first compounds of the present invention may have one or more asymmetric centres and it is intended that any optical isomers (i.e. enantiomers or diastereomers) as separated, 20 pure or partially purified optical isomers and any mixtures thereof including racemic mixtures, i.e. a mixture of stereoisomers, are included within the scope of the invention. In this context is understood that when specifying the enantiomeric form, the com- pound is in enantiomeric excess, e.g. essentially in a pure form. Accordingly, one embodiment of the invention relates to a first compound of the invention having an enantiomeric excess (ee) 25 of at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 96%, preferably at least 98%. Racemic forms can be resolved into the optical antipodes by known methods, for exam- ple by separation of diastereomeric salts thereof with an optically active acid and liberating the optically active amine compound by treatment with a base. Another method for resolving race- 30 mates into the optical antipodes is based upon chromatography on an optically active matrix. The first compounds of the present invention may also be resolved by the formation of diastere- omeric derivatives. Additional methods for the resolution of optical isomers, known to those skilled in the art, may be used. Such methods include those discussed by J. Jaques, A. Collet and S. Wilen in “Enantiomers, Racemates, and Resolutions”, John Wiley and Sons, New York (1981). Optically active compounds can also be prepared from optically active starting materials. Abso- lute stereochemistry may be determined by methods known to the skilled person, such as vi- brational circular dichroism (VCD) Spectroscopic analysis. 5 Furthermore, when a double bond or a fully or partially saturated ring system is present in the molecule geometric isomers may be formed. It is intended that any geometric isomers as separated, pure or partially purified geometric isomers or mixtures thereof are included within the scope of the invention. Likewise, molecules having a bond with restricted rotation may form geometric isomers. These are also intended to be included within the scope of the present in-10 vention. Furthermore, some of the first compounds of the present invention may exist in differ- ent tautomeric forms and it is intended that any tautomeric forms that the compounds are able to form are included within the scope of the present invention. Included in this invention are also isotopically labelled first compounds, wherein one or 15 more atoms are represented by an atom of the same element having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (e.g., 2H, 11C, 13C, 15N, 18F and the like). Particular mention is made of 2H substituted compounds i.e. com- pounds wherein one or more H atoms are represented by deuterium. In one embodiment of the invention a first compound of formula I, Ia, Ib, Ic, Id, A or Aa 20 is isotopically labelled. In a further embodiment of the invention, one or more of the hydrogen atoms of the first compound of formula I, Ia, Ib, Ic, Id, A or Aa are represented by deuterium. It is recognized that elements are present in natural isotopic abundances in most synthetic com- pounds and result in inherent incorporation of deuterium. However, the natural isotopic abun- dance of hydrogen isotopes such as deuterium is immaterial (about 0.015%) relative to the de-25 gree of stable isotopic substitution of compounds indicated herein. Thus, as used herein, desig- nation of an atom as deuterium at a position indicates that the abundance of deuterium is sig- nificantly greater than the natural abundance of deuterium. Any atom not designated as a par- ticular isotope is intended to represent any stable isotope of that atom, as will be apparent to the ordinarily skilled artisan. 30 In one embodiment, designation of a position as “D” in a compound has a minimum deuterium incorporation of greater than about 60% at that position such as greater than about 70% at that position such as greater than about 80% at that position such as greater than about 85% at that position. In a further embodiment, designation of a position as “D” in a compound has a minimum deuterium incorporation of greater than about 90% at that position such as greater than about 95% at that position such as greater than about 97% at that position such as greater than about 99% at that position. Pharmaceutically Acceptable Salts 5 The first and second compounds of this invention are generally utilized as the free sub- stance or as a pharmaceutically acceptable salt thereof. When a first compound of formula I, Ia, Ib, Ic, Id, A or Aa contains a free base, such salts may be prepared in a conventional manner by treating a solution or suspension of a free base of formula I, Ia, Ib, Ic, Id, A or Aa with a molar equivalent of a pharmaceutically acceptable acid. Representative examples of suitable organic10 and inorganic acids are described below. Pharmaceutically acceptable salts in the present context are intended to indicate non- toxic, i.e. physiologically acceptable salts. The term pharmaceutically acceptable salts includes salts formed with inorganic and/or organic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitrous acid, sulphuric acid, benzoic acid, citric acid, gluconic acid, lactic acid,15 maleic acid, succinic acid, tartaric acid, acetic acid, propionic acid, oxalic acid, maleic acid, fu- maric acid, glutamic acid, pyroglutamic acid, salicylic acid, salicylic acid and sulfonic acids, such as methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid and benzene-sulfonic acid. Some of the acids listed above are di- or tri-acids, i.e. acids containing two or three acidic hydro- gens, such as phosphoric acid, sulphuric acid, fumaric acid and maleic acid. Di- and tri-acids may 20 form 1:1, 1:2 or 1:3 (tri-acids) salts, i.e. a salt formed between two or three molecules of the compound of the present invention and one molecule of the acid. The term pharmaceutically acceptable salts include salts formed with inorganic and/or organic bases, such as alkali metal bases, such as sodium hydroxide, lithium hydroxide, potas- sium hydroxide, alkaline earth bases, such as calcium hydroxide and magnesium hydroxide, and 25 organic bases, such as trimethylamine. Some of the bases listed above are di- or tri-bases, i.e. bases able to receive two or three acidic hydrogens, such as calcium hydroxide and magnesium hydroxide. Di- and tri-bases may form 1:1 or 1:2 salts, i.e. a salt formed between two molecules of the compound of the present invention and one molecule of the base. Additional examples of useful acids and bases to form pharmaceutically acceptable salts 30 can be found e.g. in Stahl and Wermuth (Eds.) “Handbook of Pharmaceutical salts. Properties, selection, and use”, Wiley-VCH, 2008. Pharmaceutical Composition The above-mentioned first or second compounds or pharmaceutically acceptable salts thereof may be in a composition as the sole active ingredient or in combination with other active ingredients. Additionally, one or more pharmaceutically acceptable carriers or diluents may be 5 in the composition. The pharmaceutical compositions may be specifically formulated for administration by any suitable route such as the oral, rectal, nasal, pulmonary, topical (including buccal and sub- lingual), transdermal, intracisternal, intraperitoneal, vaginal and parenteral (including subcuta- neous, intramuscular, intrathecal, intravenous and intradermal) route, the oral route being pre- 10 ferred. It will be appreciated that the preferred route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingre- dient chosen. Pharmaceutical compositions for oral administration include solid dosage forms such as capsules, tablets, dragées, pills, lozenges, powders and granules. Where appropriate, they can15 be prepared with coatings. Liquid dosage forms for oral administration include solutions, emulsions, suspensions, syrups and elixirs. Pharmaceutical compositions for parenteral administration include sterile aqueous and nonaqueous injectable solutions, dispersions, suspensions or emulsions as well as sterile pow- 20 ders to be reconstituted in sterile injectable solutions or dispersions prior to use. Other suitable administration forms include suppositories, sprays, ointments, creams, gels, inhalants, dermal patches, implants, etc. Conveniently, the first compounds of the invention are administered in a unit dosage form containing said compound in an amount of about 0.1 to 500 mg, such as 10 mg, 50 mg, 100 25 mg, 150 mg, 200 mg or 250 mg of a first compound of the present invention. For parenteral administration, solutions of the compound of the invention in sterile aqueous solution, aqueous propylene glycol, aqueous vitamin E or sesame or peanut oil may be employed. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. The aqueous solutions are particularly 30 suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art. Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phosphor lipids, 5 fatty acids, fatty acid amines, polyoxyethylene and water. The pharmaceutical compositions formed by combining the first and/or second compound of the invention and the pharmaceuti- cally acceptable carriers are then readily administered in a variety of dosage forms suitable for the disclosed routes of administration. Formulations of the present invention suitable for oral administration may be presented 10 as discrete units such as capsules or tablets, each containing a predetermined amount of the active ingredient, and which may include a suitable excipient. Furthermore, the orally available formulations may be in the form of a powder or granules, a solution or suspension in an aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsion. If a solid carrier is used for oral administration, the preparation may be tablet, e.g. 15 placed in a hard gelatine capsule in powder or pellet form or in the form of a troche or lozenge. The amount of solid carrier may vary but will usually be from about 25 mg to about 1 g. If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatine capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution. 20 Tablets may be prepared by mixing the active ingredient with ordinary adjuvants and/or diluents followed by compression of the mixture in a conventional tabletting machine. Examples of adjuvants or diluents comprise corn starch, potato starch, talcum, magnesium stearate, gela- tine, lactose, gums, and the like. Any other adjuvants or additives usually used for such purposes such as colourings, flavourings, preservatives etc. may be used provided that they are compati-25 ble with the active ingredients. Treating diseases As established above, LRRK2 inhibitors may be used in the treatment of Parkinson’s dis- ease and particular mention is made of Parkinson’s disease associated with mutations in LRRK2, such as Gly2019Ser. Moreover, LRRK2 inhibitors are also expected to be useful in the treatment30 of other diseases which are associated with LRRK2. LRRK2 has been identified as a core compo- nent in Lewy bodies and is thus expected to be useful in the treatment of Lewy body dementia [Neuropathol. Appl. Neurobiol., 34, 272-283, 2008]. Expression of LRRK2 mRNA is highly enriched in brain, lungs, kidney, spleen and blood suggesting that functional impact of increased LRRK2 activity is likely to be most relevant in pathogenic and pathologic conditions associated with those regions. Support for that notion can be found in studies showing an increased risk of non- skin cancer in LRRK2 Gly2019Ser mutation carriers and especially for renal and lung cancer [Mov. 5 Disorder, 25, 2536-2541, 2010]. Over-expression of LRRK2 by chromosomal amplification has also been identified in papillary renal and thyroid carcinomas. Also, genetic association of LRRK2 has been reported for diseases where aberrant responses of the immune system are involved. This is the case for inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis as well as for leprosy [Nat. Genet.42, 1118-1125, 2010; Inflamm. Bowel Dis.16, 557-558, 2010; N. 10 Engl. J. Med.361, 2609-2618, 2009; Inflamm. Bowel Dis]. Thus, in an embodiment is provided a first compound, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the invention for use in the treat- ment of a disease in the central nervous system selected from Lewy body dementia, multiple system atrophy or Parkinson’s disease. 15 In an embodiment the disease in the central nervous system is Parkinson’s disease. In an embodiment the Parkinson’s disease is idiopathic Parkinson’s disease, sporadic Parkinson’s disease or Parkinson’s disease in patients carrying a G2019S mutation in LRRK2. In an embodiment the Parkinson’s disease is idiopathic Parkinson’s disease. In an embodiment the Parkinson’s disease is sporadic Parkinson’s disease. 20 In an embodiment the Parkinson’s disease is in patients carrying a G2019S mutation in LRRK2. In a further embodiment, the first compounds, or pharmaceutically acceptable salt thereof as outlined in formula I, Ia, Ib, Ic, Id, A or Aa hereinabove, or compositions comprising said first compounds may be used in the treatment of cancer or an immune related disorder. 25 In some embodiments, the cancer diseases may reside in the brain, lungs, kidney and spleen or blood organs such as renal cancer, lung cancer, skin cancer, and papillary renal and thyroid carcinomas. In some embodiments, the immune related disorder may in one embodiment be Crohn’s disease, ulcerative colitis, tuberculosis or leprosy. 30 According to an embodiment of the invention, the treatment may be in a patient with an increased LRRK2 kinase activity or carrying one or more mutated forms of LRRK2 such as G2019S, I2020T, M1646T, G2385R, A419V, N551K, R1398H, K1423K, R1441G, R1441H, R1441C, R1628P, S1647T, Y1699C, I2020T or Y2189C. In one embodiment, the first compound of the present invention is administered in an amount from about 0.001 mg/kg body weight to about 100 mg/kg body weight per day. In par- ticular, daily dosages may be in the range of 0.01 mg/kg body weight to about 50 mg/kg body weight per day. The exact dosages will depend upon the frequency and mode of administration, 5 the sex, the age, the weight, and the general condition of the subject to be treated, the nature and the severity of the condition to be treated, any concomitant diseases to be treated, the desired effect of the treatment and other factors known to those skilled in the art. A typical oral dosage for adults will be in the range of 1-1000 mg/day of a first com- pound, or pharmaceutically acceptable salt thereof of the present invention, such as 1-500 10 mg/day. The first compounds, or pharmaceutically acceptable salt thereof of the present inven- tion may be administered alone as a pure compound or in combination with pharmaceutically acceptable carriers or excipients, in either single or multiple doses. The pharmaceutical compo- sitions according to the invention may be formulated with pharmaceutically acceptable carriers 15 or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press, 2012. In the present context, “excipient”, “carrier”, “diluent”, “adjuvant” and the like are used synonymously and are intended to mean the same. All references, including publications, patent applications, and patents, cited herein are 20 hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of any separately provided incorporation of particular documents made elsewhere herein. Specific Embodiments of the Invention 25 In the following specific embodiments of the invention are disclosed. The first embodiment is denoted E1, the second embodiment is denoted E2 and so forth. E1. A first compound of formula I, or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000064_0001
R1 is CH2R4 or R4; R2 is a C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, a C3-C6 cycloalkyl, or a C1-C3 haloalkyl; R3 is halogen, cyano, a O-C1-C3 haloalkyl, a C1-C3 haloalkyl, a C3-C6 cycloalkyl, or a C1-C3 alkyl; 5 R4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen; a C1-C3 alkyl, a C1-C3 cyanoalkyl, a C1-C3 haloalkyl, or a C3-C6 cycloalkyl; or R4 is a bicyclic 8-membered heterocycle having 1-2 heteroatoms independently selected from oxygen and nitrogen; wherein each heterocycle or cycloalkyl is unsubstituted or substituted with 1, 2, or 3 groups 10 independently selected from the group consisting of cyano, deuterium, halogen, C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, a O-C1-C3 haloalkyl, a O-C1-C3 alkyl, or a C1-C3 haloalkyl; and a second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, wherein the first and the second compound are15 for combined use in the treatment of synucleinopathies. E2. The first and second compound of embodiment E1, wherein the first compound is a com- pound of formula Ia, or a pharmaceutically acceptable salt thereof: 20
Figure imgf000064_0002
. E3. The first and second compound of embodiment E1, wherein the first compound is a com- pound of formula Ib, or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000065_0001
R2 is a C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, a C3-C6 cycloalkyl, or a C1-C3 haloalkyl; R4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen, a C1-C3 alkyl, a C1-C3 cyanoalkyl, a C1-C3 haloalkyl, or a C3-C6 cycloalkyl; 5 wherein each heterocycle or cycloalkyl is unsubstituted or substituted with 1 group se- lected from the groups selected from cyano, deuterium, halogen, a C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, a O-C1-C3 haloalkyl, a O-C1-C3 alkyl, or a C1-C3 haloalkyl. E4. The first and second compound of embodiment E1, wherein the first compound is a com- 10 pound of formula Ic, or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000065_0002
R4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen, a C1-C3 alkyl, a C1-C3 cyanoalkyl, a C1-C3 haloalkyl, or a C3-C6 cycloalkyl;15 wherein each heterocycle or cycloalkyl is unsubstituted or substituted with 1 group se- lected from the groups selected from cyano, deuterium, halogen, a C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, a O-C1-C3 haloalkyl, a O-C1-C3 alkyl, or a C1-C3 haloalkyl. E5. The first and second compound of embodiment E1, wherein the first compound is a com- 20 pound of formula Id, or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000066_0001
R4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen, a C1-C3 alkyl, a C1-C3 cyanoalkyl, a C1-C3 haloalkyl, or a C3-C6 cycloalkyl; wherein each heterocycle or cycloalkyl is unsubstituted or substituted with 1 group se- 5 lected from the groups selected from cyano, deuterium, halogen, a C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, a O-C1-C3 haloalkyl, a O-C1-C3 alkyl, or a C1-C3 haloalkyl. E6. The first and second compound of embodiment E1, wherein the first compound is a com- pound of formula A, or a pharmaceutically acceptable salt thereof, wherein: 10
Figure imgf000066_0002
R1 is CH2R4 or R4; R2 is a C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, a C3-C6 cycloalkyl, or a C1-C3 haloalkyl; R3 is halogen, cyano, a O-C1-C3 haloalkyl, a C1-C3 haloalkyl, a C3-C6 cycloalkyl, or a C1-C3 alkyl;15 R4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected from ox- ygen and nitrogen; a C1-C3 alkyl, a C1-C3 cyanoalkyl, a C1-C3 haloalkyl, or a C3-C6 cycloalkyl; wherein each heterocycle or cycloalkyl is unsubstituted or substituted with 1, 2, or 3 groups independently selected from the group consisting of cyano, deuterium, halo, C1-C3 alkyl, an iso- topically labelled C1-C3 alkyl, a O-C1-C3 haloalkyl, a O-C1-C3 alkyl, or a C1-C3 haloalkyl. 20 E7. The first and second compound of embodiment E6, wherein the first compound is a com- pound of formula Aa, or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000067_0001
. E8. The first and second compound of any of embodiment E1-E7, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa 5 and R1 is CH2R4. E9. The first and second compound of any of embodiment E1-E7, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R1 is R4. 10 E10. The first and second compound of any of embodiment E1-E9, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R2 is selected from a C1-C3 alkyl, an isotopically labelled C1-C3 alkyl or a C3-C6 cycloalkyl. 15 E11. The first and second compound of any of embodiment E1-E9, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R2 is selected from -CH3, -CH2CH3, -CD3, or cyclopropyl. E12. The first and second compound of any of embodiment E1-E9, wherein the first compound 20 or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R2 is C1-C3 alkyl. E13. The first and second compound of any of embodiment E1-E9, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa25 and R2 is methyl. E14. The first and second compound of any of embodiment E1-E9, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R2 is ethyl. 5 E15. The first and second compound of any of embodiment E1-E9, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R2 is an isotopically labelled C1-C3 alkyl. E16. The first and second compound of any of embodiment E1-E9, wherein the first compound 10 or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R2 is selected from the group consisting of -CD3 or -CD2CD3. E17. The first and second compound of any of embodiment E1-E9, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa15 and R2 is -CD3. E18. The first and second compound of any of embodiment E1-E9, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R2 is cyclopropyl. 20 E19. The first and second compound of any of embodiment E1-E18, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R3 is C1-C3 haloalkyl. 25 E20. The first and second compound of any of embodiment E1-E18, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R3 is CF3. E21. The first and second compound of any of embodiment E1-E18, wherein the first compound 30 or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R3 is halogen. E22. The first and second compound of any of embodiment E1-E18, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and, R3 is chloro. 5 E23. The first and second compound of any of embodiment E1-E18, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R3 is bromo. E24. The first and second compound of any of embodiment E1-E18, wherein the first compound 10 or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R3 is cyano. E25. The first and second compound of any of embodiment E1-E18, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa 15 and R3 is a C3-C6 cycloalkyl. E26. The first and second compound of any of embodiment E1-E18, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R3 is cyclopropyl. 20 E27. The first and second compound of any of embodiment E1-E26, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R4 is a 4- to 6-membered heterocycle having one oxygen atom, wherein the 4- to 6-mem- bered heterocycle is unsubstituted or substituted with one group selected from the group con-25 sisting of cyano, deuterium, halogen, C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, O-C1-C3 haloalkyl, O-C1-C3 alkyl, or C1-C3 haloalkyl. E28. The first and second compound of any of embodiment E1-E26, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa30 and R4 is a 4- to 6-membered heterocycle having one oxygen atom, wherein the 4- to 6-mem- bered heterocycle is unsubstituted or substituted with one group selected from the group con- sisting of deuterium, halogen, C1-C3 alkyl, or an isotopically labelled C1-C3 alkyl. E29. The first and second compound of any of embodiment E1-E26, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R4 is 6-membered heterocycle having one oxygen atom, wherein the 6-membered hetero- cycle is unsubstituted or substituted with one group selected from the group consisting of deu- 5 terium, halogen, C1-C3 alkyl, or an isotopically labelled C1-C3 alkyl. E30. The first and second compound of any of embodiment E1-E26, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa10 and R4 is 6-membered heterocycle having one oxygen atom, wherein the 6-membered hetero- cycle is unsubstituted or substituted with two groups independently selected from the group consisting of deuterium, halogen, C1-C3 alkyl, or an isotopically labelled C1-C3 alkyl. 15 E31. The first and second compound of any of embodiment E1-E26, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R4 is selected from the group consisting of:
Figure imgf000070_0001
20 25 E32. The first and second compound of any of embodiment E1-E26, wherein the first com- pound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R4 is selected from the group consisting of: -CH3, 5
Figure imgf000071_0001
Figure imgf000072_0001
E33. The first and second compound of any of embodiment E1-E26, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa 5 and R4 is tetrahydropyran. E34. The first and second compound of any of embodiment E1-E26, wherein the first com- pound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R4 is unsubstituted tetrahydropyran. 10 E35. The first and second compound of any of embodiment E1-E26, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R4 is tetrahydro-2H-pyran-4-yl. 15 E36. The first and second compound of any of embodiment E1-E26, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R4 is tetrahydro-2H-pyran-3-yl. E37. The first and second compound of any of embodiment E1-E26, wherein the first compound 20 or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and R4 is 4-oxaspiro[2.5]octan-7-yl. E38. The first and second compound of any of embodiment E1-E26, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and the 8-membered heterocycle is an unsubstituted 8-membered spirocyclic heterocycle. 5 E39. The first and second compound of any of embodiment E1-E26, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and the bicyclic 8-membered heterocycle contains one oxygen. E40. The first and second compound of any of embodiment E1-E26, wherein the first compound 10 or a pharmaceutically acceptable salt thereof is a compound of formula I, Ia, Ib, Ic, Id, A or Aa and the bicyclic 8-membered heterocycle contains one oxygen and is a spirocyclic heterocycle. E41. The first and second compound of any of embodiment E1-E40, wherein the first com- pound is selected from the list of: 15 , , ,
Figure imgf000073_0001
,
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
, , and ; or a pharmaceutically acceptable salt thereof. E42. The first and second compound of any of embodiment E1-E41, wherein the second com- 5 pound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is a symptomatic drug or a disease modifying compound. E43. The first and second compound of any of embodiment E1-E41, wherein the second com- pound or a pharmaceutically acceptable salt thereof, which second compound is useful in the 10 treatment of synucleinopathies is selected from anticholinergic agents, dopaminergic agents, and adenosine receptor antagonists. E44. The first and second compound of embodiment E43, wherein the second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of15 synucleinopathies is an anticholinergic agent selected from the group comprising trihex- yphenidyl, biperiden, metixene, procyclidine, profenamine, dexetimide, phenglutarimide, maza- ticol, bornaprine, tropatepine, etanautine, orphenadrine, benzatropine, and etybenzatropine. E45. The first and second compound of embodiment E43, wherein the second compound or a 20 pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is a dopaminergic agent selected from the group comprising dopamine, levo- dopa, carbidopa, levodopa/carbidopa, melevodopa, etilevodopa, foslevodopa, amantadine, bromocriptine, pergolide, dihydroergocryptine mesylate, ropinirole, pramipexole, cabergoline, apomorphine, piribedil, rotigotine, selegiline, rasagiline, safinamide, tolcapone, entacapone, 25 budipine, and opicapone. E46. The first and second compound of embodiment E43, wherein the second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is istradefylline. 5 E47. The first and second compound of any of embodiments E1-E41, wherein the second com- pound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is selected from caspase inhibitors, calpain inhibitors, LRRK2 in- hibitors, BACE1 inhibitors, antibodies against any form of Aβ peptides, inflammation inhibitors, LAG-3 antibodies, molecules inhibiting alpha-synuclein aggregation, nicotine, caffeine, Mono-10 amine oxidase B inhibitor, Levodopa/carbidopa, Dopamine agonists, COMT inhibitors, A2A an- tagonists, anti-alphasynuclein antibodies, Mitogen-activated protein kinase 14 inhibitors, USP30 inhibitors, Beta adrenoreceptor agonists, dopamine D1 PAMs, Toll-like receptor 2 antagonists, Carnitine palmitoyl transferase 1 inhibitors, Glutamate 5 receptor antagonists, Muscarinic M2 receptor antagonists, Muscarinic M3 receptor antagonists, Muscarinic M4 receptor antagonists,15 5 Hydroxy tryptamine 1A receptor agonists, 5 Hydroxy tryptamine 4 receptor agonists, 5 Hy- droxy tryptamine 3 receptor agonists, 5 Hydroxy tryptamine 2A receptor antagonists, Alpha 1a adrenoreceptor antagonists, Sigma 1 receptor agonists, Sigma 2 receptor antagonists, Dopa- mine D1 receptor agonists, Dopamine D2 receptor agonists, Dopamine D3 receptor agonists, Dopamine D5 receptor antagonists, dopamine reuptake inhibitors, DOPA decarboxylase inhibi- 20 tors, Insulin-like growth factor 1 antagonists, Insulin-like growth factor 2 antagonists, Tyrosine kinase inhibitors, Ras inhibitors, Leucotriene D4 antagonists, Leucotriene receptor antagonists, PDE1 inhibitors, T-type calcium channel antagonists, Peroxiredoxin inhibitors, GLP-1R agonists, NMDA receptor blockers, Lipoxygenase inhibitors, and Peroxisome proliferator-activated recep- tor gamma agonists. 25 E48. The first and second compound of any of embodiments E1-E41, wherein the second com- pound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is selected from the group comprising pralnacasan, bapi- neuzemab, solanezumab, gantenerumab, crenezumab, aducanumab, glunozumab, 30 prasinezumab, miglustat, eliglustat, ropinerole, neflamapimod, bosutinib, oxafuramine, nadolol, nilotinib, vodobatinib, clenbuterol, mevidalen, emeramide, tomaralimab, mitometin, diprag- lurant, ezeprogind, blarcamesine, buntanetap, renzapride, tavapadon, pramiprexole, salbuta- mol, Infudopa, alirinetide, antraquinolol, a rasagiline prodrug, apomorphine, pramiprexole, ra- dotinib, talineuren, montelukast, lenrispodun, pirepemat, suvecaltamide, sonlicromanol, L-De- methyl phencynonate hydrochloride, mesocarb, roluperidone, exenatide, befiradol, ketamine, pridopidine, utroloxestat, aplindore, finamine, phenlarmide, (R)-troloxamide quinone, cannibig- 5 erol, and kenterin. E49. The first and second compound of any of embodiments E1-E41, wherein the second com- pound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is selected from the list consisting of trihexyphenidyl, biperiden,10 metixene, procyclidine, profenamine, dexetimide, phenglutarimide, mazaticol, bornaprine, tro- patepine, etanautine, orphenadrine, benzatropine, etybenzatropine, dopamine, levodopa, car- bidopa, levodopa/carbidopa, melevodopa, etilevodopa, foslevodopa, amantadine, bromocrip- tine, pergolide, dihydroergocryptine mesylate, ropinirole, pramipexole, cabergoline, apomor- phine, piribedil, rotigotine, selegiline, rasagiline, safinamide, tolcapone, entacapone, budipine,15 opicapone, istradefylline, pralnacasan, bapineuzemab, solanezumab, gantenerumab, cren- ezumab, aducanumab, glunozumab, prasinezumab, miglustat, eliglustat, ropinerole, neflamapi- mod, bosutinib, oxafuramine, nadolol, nilotinib, vodobatinib, clenbuterol, mevidalen, emera- mide, tomaralimab, mitometin, dipraglurant, ezeprogind, blarcamesine, buntanetap, ren- zapride, tavapadon, pramiprexole, salbutamol, Infudopa, alirinetide, antraquinolol, a rasagiline 20 prodrug, apomorphine, pramiprexole, radotinib, talineuren, montelukast, lenrispodun, pirepemat, suvecaltamide, sonlicromanol, L-Demethyl phencynonate hydrochloride, mesocarb, roluperidone, exenatide, befiradol, ketamine, pridopidine, utroloxestat, aplindore, finamine, phenlarmide, (R)-troloxamide quinone, cannibigerol, and kenterin. 25 E50. The first and second compound of any of embodiments E1-E41, wherein the second com- pound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies is selected from the list consisting of trihexyphenidyl, biperiden, metixene, procyclidine, profenamine, dexetimide, phenglutarimide, mazaticol, bornaprine, tro- patepine, etanautine, orphenadrine, benzatropine, etybenzatropine, dopamine, levodopa, car-30 bidopa, levodopa/carbidopa, melevodopa, etilevodopa, foslevodopa, amantadine, bromocrip- tine, pergolide, dihydroergocryptine mesylate, ropinirole, pramipexole, cabergoline, apomor- phine, piribedil, rotigotine, selegiline, rasagiline, safinamide, tolcapone, entacapone, budipine, opicapone, istradefylline, pralnacasan, prasinezumab, miglustat, eliglustat, and neflamapimod. E51. A pharmaceutical composition comprising a first compound for use according to any one of the previous embodiments E1-E50 and one or more pharmaceutically acceptable carriers. 5 E52. A first and second compound of any of embodiments E1-E50, or a pharmaceutically ac- ceptable salt thereof, for combined use in the treatment of a synucleinopathy, wherein the synu- cleinopathy is selected from Lewy body dementia, multiple system atrophy, or Parkinson’s dis- ease. 10 E53. A first and second compound of any of embodiments E1-E50, or a pharmaceutically ac- ceptable salt thereof, for combined use in the treatment of a synucleinopathy, wherein the synu- cleinopathy is parkinson’s disease. E54. A first and second compound of embodiment E53, or a pharmaceutically acceptable salt15 thereof, for combined use in the treatment of a synucleinopathy, wherein the Parkinson’s dis- ease is idiopathic Parkinson’s disease, sporadic Parkinson’s disease or Parkinson’s disease in pa- tients carrying a G2019S mutation in LRRK2. E55. A first and second compound of any of embodiments E1-E50, or a pharmaceutically ac-20 ceptable salt thereof, for combined use in the treatment of a synucleinopathy, wherein the synu- cleinopathy is a disease or disorder characterized by increased LRRK2 kinase activity; or by ex- pression of one or more mutated forms of LRRK2 selected from G2019S, I2020T, M1646T, G2385R, A419V, N551K, R1398H, K1423K, R1441G, R1441H, R1441C, R1628P, S1647T, Y1699C, I2020T and Y2189C; or by one or more a LRRK2 non-coding variants selected from rs76904798-25 T and Rs1491942-G. E56. Use of a first and second compound of any one of embodiments E1-E50, or a pharmaceuti- cally acceptable salt thereof, in the manufacture of a medicament for the treatment of a disease or disorder in the central nervous system selected from Lewy body dementia, multiple system30 atrophy, or Parkinson’s disease. E57. The use according to embodiment E56 wherein the Parkinson’s disease is idiopathic Parkin- son’s disease, sporadic Parkinson’s disease or Parkinson’s disease in patients carrying a G2019S mutation in LRRK2, or Parkinson’s disease in patients carrying one or more LRRK2 non-coding variants selected from rs76904798-T and Rs1491942-G. E58. A method for the treatment of a disease or disorder in the central nervous system selected 5 from Lewy body dementia, multiple system atrophy or Parkinson’s disease comprising the ad- ministration of a therapeutically effective amount of the first and second compound of any one of embodiments E1-E50, or a pharmaceutically acceptable salt thereof, to a patient in need thereof. 10 E59. The method according to embodiment E58 wherein the Parkinson’s disease is idiopathic Parkinson’s disease, sporadic Parkinson’s disease, Parkinson’s disease in patients carrying a G2019S mutation in LRRK2, or Parkinson’s disease in patients carrying one or more LRRK2 non- coding variants selected from rs76904798-T and Rs1491942-G. 15
EXPERIMENTAL SECTION Preparation of the First Compounds of the Invention
Figure imgf000083_0001
5 The compounds of formula I, Ia, Ib, Ic, Id, A or Aa may be prepared by methods described below, together with synthetic methods known in the art of organic chemistry, or modifications that are familiar to those of ordinary skill in the art. For example, the methods describe the use of selective protecting groups during the synthesis of the compounds of the invention. One skilled in the art would be able to select the appropriate protecting group for a particular reaction. Methods 10 for protection and deprotection of such groups are well known in the art and may be found in Watts and Green et al., Protective Groups in Organic Synthesis, 2006, 4th Edition, Wiley Interscience, New York. The starting materials used herein are available commercially or may be prepared by rou- tine methods known in the art, such as those method described in standard reference books such as “Compendium of Organic Synthetic Methods, Vol. I-XII” (published by Wiley Inter- 15 science). Preferred methods include, but are not limited to, those described below. The schemes are representative of methods useful in synthesizing the compounds of the present invention. They are not to constrain the scope of the invention in any way.
Method 1:
Figure imgf000084_0001
Scheme 1 5 In brief, compounds of formula I can be prepared according to scheme 1. The com- pounds of formula I can for example be prepared through a one pot procedure by reducing an intermediate of type III with iron and ammonium chloride in a suitable solvent e.g. a mixture of ethanol and water as described for Example 1. Alternatively, an intermediate of type III can be reduced using e.g. sodium dithionite in the presence of a base such as potassium hydrogen car-10 bonate in a solvent such as a mixture of water and tetrahydrofuran to afford an aminopyrazole intermediate of type II. An intermediate of type II can be cyclized to afford compounds of for- mula I in the presence of a base e.g. potassium fluoride and a solvent such as dimethylsulfoxide as detailed for Example 16. 15 Method 2:
Figure imgf000084_0002
Scheme 2 Alternatively, compounds of formula I can be prepared from a compound of formula I’ 20 (i.e. a compound of type I wherein R3 = halogen e.g. Br) according to scheme 2. The compounds of formula I can be prepared by employing a cross-coupling reaction between an intermediate of type I’ and an organometallic alkyl intermediate IV (wherein M is for example Bpin, B(OH)2, Sn(n-Bu)3 or SnMe3, ZnBr or ZnCl). The coupling is exemplified by but not limited to a Negishi- type cross-coupling reaction. The reaction can be performed by reacting an intermediate of type I’ with an organozinc intermediate IV in which M= ZnBr or ZnCl in the presence of a catalyst like di-µ-iodobis(tri-t-butylphosphino)dipalladium(I) and a suitable solvent such as a mixture of tol- uene and tetrahydrofuran as described for Example 7. 5 Method 3:
Figure imgf000085_0001
Scheme 3 A nitropyrazole intermediate of type III can be prepared according to scheme 3 starting from a pyrazolopyrimidine intermediate of type X. An intermediate of type X can be reacted10 with e.g. an alcohol such as IX (wherein Pg1 is for example TBS) under Mitsunobu-type conditions employing for example diisopropyl azodicarboxylate and triphenylphosphine in a solvent such as tetrahydrofuran to afford an intermediate of type VIII. Intermediates of type VIII (wherein Pg1 is for example TBS) can be converted into an alcohol intermediate of type VII using for ex- ample an acid such as aqueous hydrochloric acid in an appropriate solvent such as a mixture of15 tetrahydrofuran and water, as described in the synthesis of 3-(3,6-dichloro-1H-pyrazolo[3,4- d]pyrimidin-1-yl)propan-1-ol. An alcohol intermediate of type VII can be reacted with an intermediate of type V under Mitsunobu-type conditions employing e.g. diisopropyl azodicarboxylate and triphenylphosphine in a solvent such as tetrahydrofuran to afford an intermediate of type III, as detailed in for ex-20 ample the synthesis of 3,6-dichloro-1-(3-((5-cyclopropyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)- 1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine. Alternatively, an intermediate of type VII can be converted into an intermediate of type VI (wherein LG1 is for example OTs) using p-toluenesulfonyl chloride in the presence of an ap- propriate base such as triethylamine in a suitable solvent for example dichloromethane. An in- 25 termediate of type VI can be reacted with intermediates of type V in the presence of base for example potassium carbonate in a solvent such as N,N-dimethylformamide to afford an inter- mediate of type III as for example described in the synthesis of 3,6-dichloro-1-(3-((5-methyl-1- (3-methyloxetan-3-yl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine. 5 Method 4:
Figure imgf000086_0001
Scheme 4 An alternative method to prepare a nitropyrazole intermediate of type III is described in scheme 4 starting from a pyrazole intermediate of type V. Intermediates of type XIII can be pre- 10 pared from an intermediate of type V through a Mitsunobu-type reaction with an alcohol like XIV (wherein PG2 is for example TBS) using for example diisopropyl azodicarboxylate and tri- phenylphosphine in a suitable solvent such as tetrahydrofuran. Alternatively, an intermediate of type XIII can be prepared from intermediates of type V through alkylation with an intermedi- ate of type XV (wherein for example LG2 is OTs and PG2 is TBS) in the presence of a base such as15 cesium carbonate in a suitable solvent e.g. N,N-dimethylformamide. An intermediate of type XIII (wherein PG2 is for example TBS) can be converted into intermediates of type XI employing for example aqueous HCl in a solvent such as tetrahydrofuran. It is understood that an intermediate of type XIII (wherein PG2 is for example TBS) can be transformed into another intermediate of type XIII wherein R2 has been modified: For exam- 20 ple an intermediate of XIII (wherein R2=cyclopropyl and PG2=TBS) can be reacted with a base such as lithium diisopropylamide and N-Fluorobis(phenylsulfonyl)amine in a suitable solvent such as tetrahydrofuran to afford an intermediate of type XIII (wherein R2= 1-fluorocyclopropyl and PG2=TBS), as detailed in the synthesis of 3,6-dichloro-1-(3-((5-(1-fluorocyclopropyl)-4-nitro- 1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine. 25 It is furthermore understood that an intermediate of type XIII can be converted into an intermediate of type XI wherein R2 has been modified: For example an intermediate of type XIII (wherein R2=CH3 and PG2=TBS) can be transformed into an intermediate of type XI (wherein R2=CD3) through reaction with a base such as potassium tert-butoxide in hexadeuterodimethyl sulfoxide, as described in the synthesis of 3,6-dichloro-1-(3-((5-(methyl-d3)-4-nitro-1-(tetrahy- dro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine Finally, an intermediate of type XI can be reacted with a pyrazolopyrimidine intermedi- 5 ate of type X using a Mitsunobu-type reaction employing for example 1-(azodicarbonyl)-dipiper- idine and tributylphosphine in a solvent such as tetrahydrofuran to afford intermediates of type III, as detailed in the synthesis of (±)-3,6-dichloro-1-(3-((5-methyl-4-nitro-1-(tetrahydro-2H-py- ran-3-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine. Alternatively, an intermediate of type XI can be reacted with for example methanesul-10 fonyl chloride in the presence of a base such as triethylamine in a suitable solvent e.g. dichloro- methane to afford and intermediate of type XII (wherein LG3 is OMs). Intermediates of type XII can be converted into a an intermediate of type III through reaction with a pyrazolopyrimidine intermediate of type X in the presence of a base for example N,N-diisopropylethylamine, an additive such as sodium iodide in a suitable solvent e.g. N,N-dimethylformamide, as described15 in the synthesis of 3,6-dichloro-1-(3-((1,5-dimethyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-py- razo-lo[3,4-d]pyrimidine. Method 5:
Figure imgf000087_0001
20 Scheme 5 An intermediate of type V can be prepared according to scheme 5 through a two-step sequence employing alkylation and subsequent hydrolysis. Intermediates of type XIX can be converted into intermediates of type XVI in the presence of an alcohol intermediate such as XVII using for example Mitsunobu-type reaction conditions employing di-tert-butyl azodicarboxylate 25 and triphenylphosphine in a solvent such as tetrahydrofuran, as described in the synthesis of 3,6-dichloro-1-(3-((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)- 1H-pyrazolo[3,4-d]pyrimidine. Alternatively, an intermediate of type XIX can be reacted with and intermediate like XVIII (wherein LG4 is for example a halogen) in the presence of a suitable base such as potas- 30 sium carbonate in a solvent like acetonitrile to afford an intermediate of type XVI, as described in the synthesis of 1-(3-((1-((2-oxabicyclo[2.1.1]hexan-4-yl)methyl)-5-methyl-4-nitro-1H-pyra- zol-3-yl)oxy)propyl)-3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine. Intermediates of type XVI can be transformed into intermediates of type V us- ing a base like potassium hydroxide in a suitable solvent such as water, as detailed in the syn- 5 thesis of 3,6-dichloro-1-(3-((5-(methyl-d3)-4-nitro-1-(tetrahydro-2H-pyran-4-yl-4-d)-1H-pyrazol- 3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine Method 6:
Figure imgf000088_0001
10 Scheme 6 An alternative way to prepare an intermediate of type XVI is described in scheme 6. Intermediates of type XXIII can be reacted with an alkylating agent of type XVIII (wherein LG4 is for example a halogen) in the presence of a base such as cesium carbonate in a solvent such as N,N-dimethylformamide to afford an intermediate of type XXII. Intermediates of type XXII can 15 be modified into an intermediate of type XXI employing for example iodine and a base such as lithium bis(trimethylsilyl)amide in a suitable solvent such as tetrahydrofuran. Intermediates of type XXI can be converted into an intermediate of type XVI through reaction with a reagent of type XX (wherein M is for example Bpin, B(OH)2, Sn(n-Bu)3 or SnMe3, ZnBr). The coupling is ex- emplified by but not limited to a Suzuki-Miyaura-type cross-coupling reaction. The reaction can 20 be mediated employing a boronic acid in which M=B(OH)2 in the presence of a catalyst system consisting of e.g. tris(dibenzylideneacetone)dipalladium(0) and tricyclohexylphosphine, a base such as potassium carbonate in a suitable solvent e.g. N,N-dimethylformamide, as described for 3,6-dichloro-1-(3-((5-cyclopropyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)pro- pyl)-1H-pyrazolo[3,4-d]pyrimidine. 25 Method 7:
Figure imgf000089_0001
Scheme 7 5 An alternative method to prepare an intermediate of type XIII (wherein R2 is e.g. Me or Et) is depicted in scheme 7. Intermediates of type XIII (wherein Pg2 is for example TBS) can be prepared from a pyrazole intermediate of type XXIV through reaction with an alcohol such as XVII under Mitsunobu-like reaction conditions employing 2-(tributyl-λ5-phosphanylidene)ace- tonitrile in a solvent like toluene as described in the synthesis of intermediate cis-3,6-Dichloro-10 1-(3-((5-methyl-1-(2-methyltetrahydro-2H-pyran-4-yl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H- pyrazolo[3,4-d]pyrimidine. Alternatively, intermediates if type XIII can be prepared through re- action of pyrazole intermediates such as XXIV employing an alkylating agent of type XVIII (wherein, LG4 is for example OTs or Br) in the presence of a base like potassium carbonate in an appropriate solvent for example N,N-dimethylformamide, as detailed in the synthesis of trans- 15 3-((1-(4-fluorocyclohexyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propan-1-ol. Intermediates of type XXIV (wherein Pg2 is e.g. TBS) can be synthesized from an alcohol such as XXV using for example tert-butyldimethylsilyl chloride in the presence of a base such as imidazole and a catalyst such as 4-dimethylaminopyridine in an appropriate solvent such as di- chloromethane. An alcohol intermediate such as XXV can be prepared from a nitropyazole such 20 as XXVI (wherein Pg3 is for example, 2-tetrahydropyranyl) by treatment with an acid such as aqueous hydrochloric acid in a suitable solvent for example methanol. Alcohol intermediates of type XXVI can be prepared by reacting a chloro intermediate XXVIII with propane-1,3-diol XXVII in the presence of a base for example cesium fluoride in a solvent such as N,N-dimethylacetamide. Chloro intermediates such as XXVIII (wherein Pg3 is for example, 2-tetrahydropyranyl) can be prepared from a nitropyrazole intermediate XXIX (wherein Pg3 is for example, 2-tetrahy- dropyranyl) using a strong base for example lithium bis(trimethylsilyl)amide, and an electrophile for example hexachloroethane in a suitable solvent such as tetrahydrofuran. 5 A nitropyazole intermediate of type XXIX (wherein Pg3 is for example, 2-tetrahydropy- ranyl) can be prepared from a pyrazole such as XXX employing 3,4-dihydro-2H-pyran in the pres- ence of an acid for example para-toluenesulfonic acid monohydrate in an appropriate solvent for example tetrahydrofuran. The general synthetic sequence to prepare XXIV is for example described in the preparation of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-10 pyrazole. Method 8:
Figure imgf000090_0001
Scheme 8 15 Alternatively, compounds of formula I can be prepared from a compound of formula XXXII according to scheme 8. The compounds of formula I can be prepared by employing e.g. a copper-mediated coupling between an intermediate of type XXXII and an intermediate of type XXXI (wherein M is for example B(OH)2). The coupling is exemplified by but not limited to a Chan- Lam type coupling. The reaction can be performed by reacting an intermediate of type XXXII 20 with a boronic acid intermediate XXXI in which M= B(OH)2 in the presence of a copper salt such as copper(II) acetate, a base for example pyridine, a drying agent such as 4Å molecular sieves under an oxygen atmosphere and a suitable solvent such as 1,2-dichloroethane as described for Example 39 and 40. 25 Method 9:
Figure imgf000090_0002
Scheme 9 An intermediate such as XXXII can be prepared according to scheme 9. The intermedi- ates of type XXXII can for example be prepared through a one pot procedure by reducing an intermediate of type XXXIII (wherein Pg3 is for example, 2-tetrahydropyranyl) with iron and am- monium chloride in a suitable solvent e.g. a mixture of ethanol and water. 5 Nitropyrazole intermediates like XXXIII can be prepared by reaction of a pyrazolopyrim- idine like X with an alcohol such as XXVI (wherein Pg3 is for example, 2-tetrahydropyranyl) em- ploying Mitsunobu-like reaction conditions for example diisopropyl azodicarboxylate and tri- phenylphosphine in a solvent such as tetrahydrofuran. The general synthetic sequence in scheme 9 is for example described in the synthesis of10 intermediate 8-chloro-3-methyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4- b:5',1'-g][1]oxa[4,6,8]triazacycloundecine. Method 10:
Figure imgf000091_0001
15 Scheme 10 Alternatively, compounds of formula I can be prepared according to scheme 10. A bromo intermediate of type XXXIV can be reacted with an organometallic species such as XX (wherein M is for example Bpin, B(OH)2, Sn(n-Bu)3 or SnMe3, Zn, ZnBr or ZnCl). The coupling is 20 exemplified by but not limited to a Negishi-type cross-coupling. The reaction can be performed by reacting an intermediate of type XXXIV with an organozinc species such as (CD3)2Zn in the presence of a catalyst like bis[tris(tert-butyl)phosphine]palladium, a base such as lithium bis(tri- methylsilyl)amide in an appropriate solvent such as tetrahydrofuran as described for Example 50. A bromo intermediate such as XXXIV can be prepared from a pyrazole like XXXV using a brominating agent for example N-bromosuccinimide in a suitable solvent such as tetrahydrofu- ran. Intermediates of type XXXV can in turn be synthesized from a nitropyrazole intermediate such as XXXVI employing a one-pot reduction and cyclization procedure. The reduction and sub- 5 sequent cyclization can be performed using for example iron in the presence of ammonium chlo- ride in a suitable solvent such as a mixture of ethanol and water. Nitropyrazole intermediates such as XXXVI can for example be synthesized through reaction of a pyrazolopyrimidine such as X and an alcohol like XXXVII employing Mitsunobu-like reaction conditions. The Mitsunobu-like coupling can for example be conducted using diisopropyl azodicarboxylate in the presence of10 triphenylphosphine in an appropriate solvent such as tetrathydrofuran. The synthesis of an in- termediate like XXXIV is exemplified in the preparation of 3-bromo-8-chloro-2-((1r,4r)-4-meth- oxycyclohexyl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine. 15 Method 11:
Figure imgf000092_0001
Scheme 11 A method to prepare an intermediate of type XXXVII is depicted in scheme 11. Alcohol intermediates of type XXXVII can be prepared from a nitro pyrazole intermediate of type XXXVIII 20 (wherein Pg3 is e.g. TBS) using tetra-n-butyl ammonium fluoride in a suitable solvent such as tetrahydrofuran. Nitropyrazole intermediates of type XXXVIII can synthesized from pyrazole in- termediates such as XXXIX and an alcohols such as XVII under Mitsunobu-like reaction condi- tions employing 2-(tributyl-λ5-phosphanylidene)acetonitrile in a solvent like toluene as de- scribed in the synthesis of intermediate 3-((1-((1r,4r)-4-methoxycyclohexyl)-4-nitro-1H-pyrazol- 3-yl)oxy)propan-1-ol Intermediates of type XXXIX (wherein Pg3 is e.g. TBS) can be synthesized from an alcohol 5 such as XL using for example tert-butyldimethylsilyl chloride in the presence of a base such as imidazole and a catalyst such as 4-dimethylaminopyridine in an appropriate solvent such as di- chloromethane. An alcohol intermediate such as XL can be prepared from a nitropyazole such as XLI (wherein Pg4 is for example, 2-tetrahydropyranyl) by treatment with an acid such as aque- ous hydrochloric acid in a suitable solvent for example methanol. 10 Alcohol intermediates of type XLI can be prepared by reacting a chloro intermediate XLII with propane-1,3-diol XXVII in the presence of a base for example cesium fluoride in a solvent such as N,N-dimethylacetamide. Chloro intermediates such as XLII (wherein Pg4 is for example, 2-tetrahydropyranyl) can be pre- pared from a nitropyrazole intermediate XLIII (wherein Pg4 is for example, 2-tetrahydropyranyl) 15 using a strong base for example lithium bis(trimethylsilyl)amide, and an electrophile for example hexachloroethane in a suitable solvent such as tetrahydrofuran. ABBREVIATIONS Abbreviations used in the experimental may include, but are not limited to the follow-20 ing: Boc: tert-butyloxycarbonyl; BRIJ-35: polyoxyethylene (23) lauryl ether; C: Celsius; CPME: cyclo- pentyl methyl ether; cPr: cyclopropyl; dba: dibenzylideneacetone; DBAD: di-tert-butyl azodicar- boxylate; DCM: dichloromethane; DIAD: diisopropyl azodicarboxylate; DIPEA: N,N-diisopro- pylethylamine; DMA: N,N-dimethylacetamide; DMF: N’N-dimethylformamid; DMSO: dimethyl-25 sulfoxide; DTT: dithiothreitol; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,Nʹ,Nʹ- tetraacetic acid; Et: ethyl;EtOAc: ethyl acetate ; g: gram; h: hour(s); HMDS: hexamethyldisilaz- ane; L: liter; LDA: lithium diisopropylamide; M: molar; Me: methyl ; mg: milligram; mL: milliliter; mmol: millimole; Ms: methanesulfonyl; n-Bu: n-butyl; NFSI: N-fluorobenzenesulfonimide; NMR: nucear magnetic resonance; Ph: phenyl; Pin: pinacol; PG: protecting group; SFC: supercritical fluid 30 chromatography; TBS: tert-butyl(dimethyl)silyl; TEA: triethylamine; THF: tetrahydrofuran ; THP: tetrahydropyran; TMS: trimethylsilyl; Tris: trisaminomethane; Ts: toluenesulfonyl. CHEMICAL NAMES The chemical names for the Examples of the invention were generated using ChemDraw, version 20.0.0.41 by PerkinElmer Informatics, Inc. 5 ANALYTICAL METHODS LC-MS Methods Method A: LC-MS were run on Agilent LC1200-MS6110 UPLC-MS or a consisting of Ag- ilent LC1200 including column manager, binary solvent manager, sample organizer, PDA detec- tor (operating at 220&254 nM), ELS detector, and MS6110 equipped with APPI-source operating 10 in positive ion mode. LC-conditions: The column was Xtimate C182.1×30mm, 3µm operating at 50ºC with 1.2 mL/min of a binary gradient consisting of water + 0.037 % trifluoroacetic acid (A) and acetonitrile + 0.018 % trifluoroacetic acid(B). The retention times (tR) are expressed in minutes based on UV-trace at 220&254 nm. Gradient: 0.00 min 10% B 15 0.9 min 80% B 1.5 min 80% B 1.51 min 10% B 2.00 min 10% B Total run time: 2.0 min 20 Method B: LC-MS were run on Agilent LC1200-MS6150 or LC1200-MS6110 UPLC-MS consisting of Agilent LC1200 including column manager, binary solvent manager, sample organ- izer, PDA detector (operating at 220&254 nM), ELS detector, and MS6150 or MS6110 equipped with APPI-source operating in positive ion mode. LC-conditions: The column was MERCK, RP-18e 25 25 × 3.0 mm operating at 50ºC with 1.5 mL/min of a binary gradient consisting of water + 0.037 % trifluoroacetic acid (A) and acetonitrile + 0.018 % trifluoroacetic acid(B). The retention times (tR) are expressed in minutes based on UV-trace at 220&254 nm. Gradient: 0.00 min 5% B 0.7 min 95% B 30 1.1 min 95% B 1.11 min 5% B 1.5 min 5% B Total run time: 1.5 min 35 Method C: LC-MS were run on Agilent Prime-6125B UPLC-MS consisting of Agilent Prime including column manager, binary solvent manager, sample organizer, PDA detector (operating at 254 nM), ELS detector, and 6125B equipped with APPI-source operating in positive ion mode. LC-conditions: The column was Agilent Poroshell 120 EC-C181.9µm; 3.0×30mm operating at 50ºC with 1.5 mL/min of a binary gradient consisting of water + 0.037 % trifluoroacetic acid (A) and acetonitrile + 0.018 % trifluoroacetic acid(B). The retention times (tR) are expressed in minutes based on UV-trace at 254 nm. 5 Gradient: 0.00 min 5% B 1.20 min 80% B 2.5 min 95% B 2.51 min 5% B 3.00 min 5% B 10 Total run time: 3.0 min Method D: LC-MS were run on Agilent Prime-6125B UPLC-MS consisting of Agilent Prime including column manager, binary solvent manager, sample organizer, PDA detector (operating at 254 nM), ELS detector, and 6125B equipped with APPI-source operating in positive ion mode. 15 LC-conditions: The column was Agilent Poroshell 120 EC-C181.9µm; 3.0×30mm operating at 50ºC with 1.5 mL/min of a binary gradient consisting of water + 0.037 % trifluoroacetic acid (A) and acetonitrile + 0.018 % trifluoroacetic acid(B). The retention times (tR) are expressed in minutes based on UV-trace at 254 nm. Gradient: 0.00 min 0% B 20 1.20 min 30% B 2.5 min 95% B 2.51 min 0% B 3.00 min 0% B Total run time: 3.0 min 25 Method E: LC-MS were run on Waters Aquity UPLC-MS consisting of Waters Aquity in- cluding column manager, binary solvent manager, sample organizer, PDA detector (operating at 254 nM), ELS detector, and SQD-MS equipped with APPI-source operating in positive ion mode.LC-conditions: The column was Acquity UPLC BEH C181.7µm ; 2.1×50mm operating at 30 60ºC with 1.2 ml/min of a binary gradient consisting of water + 0.05 % trifluoroacetic acid (A) and acetonitrile + 5% water + 0.035 % trifluoroacetic acid. The retention times (tR) are expressed in minutes based on UV-trace at 254 nm. Gradient: 0.00 min 10% B 1.00 min 100% B 35 1.01 min 10% B 1.15 min 10% B Total run time: 1.15 min Method F: LC-MS were run on Waters Aquity UPLC-MS consisting of Waters Aquity in- cluding column manager, binary solvent manager, sample organizer, PDA detector (operating at 254 nM), ELS detector, and TQD-MS equipped with APPI-source operating in positive ion mode. LC-conditions: The column was Acquity UPLC BEH C181.7µm; 2.1×50mm operating at 60ºC with 5 1.2 ml/min of a binary gradient consisting of water + 0.05 % trifluoroacetic acid (A) and acetoni- trile + 5% water + 0.05 % trifluoroacetic acid. The retention times (tR) are expressed in minutes based on UV-trace at 254 nm. Gradient: 0.00 min 10% B 1.00 min 100% B 10 1.01 min 10% B 1.15 min 10% B Total run time: 1.15 min Method G: LC-MS were run on Agilent Prime-6125B UPLC-MS consisting of Agilent Prime 15 including column manager, binary solvent manager, sample organizer, PDA detector (operating at 254 nM), ELS detector, and 6125B equipped with APPI-source operating in positive ion mode. LC-conditions: The column was Agilent Poroshell 120 EC-C181.9µm; 3.0×30mm operating at 30ºC with 1.5 mL/min of a binary gradient consisting of water + 0.05 % NH3.H2O (A) and acetoni- trile (B). The retention times (tR) are expressed in minutes based on UV-trace at 254 nm. 20 Gradient: 0.00 min 5% B 1.20 min 80% B 2.5 min 95% B 2.51 min 5% B 3.00 min 5% B 25 Total run time: 3.0 min Method H: method PHQA: LC-MS were run on a Shimadzu LCMS-2020. PDA detector operating at 190-400 nM, ESI+ with a mass scan range 90-900 (m/z). The column was a EVO C18 30 1.9µm; 3.0×50mm operating at 40ºC with 1.2 mL/min of a binary gradient consisting of water + 5 mM NH4HCO3 in water (A) and acetonitrile (B). The retention times (tR) are expressed in minutes based on UV-trace at 254 nm. Gradient: 0.0 min 10% B 2.0 min 95% B 35 2.6 min 95% B 2.7 min 10% B Total run time: 2.8 min Method I: LC-MS were run on Shimadzu LC-20AD；LCMS-2020 consisting of Shimadzu LC-20AD including column manager, binary solvent manager, sample organizer, PDA detector (operating at 220&254 nM), ELS detector, and MS6150 equipped with APPI-source operating in positive ion mode. LC-conditions: The column was Chromolith® Flash RP-18e 25-3 mm operat- 5 ing at 50ºC with 1.5 mL/min of a binary gradient consisting of water + 0.037 % trifluoroacetic acid (A) and acetonitrile + 0.018 % trifluoroacetic acid(B). The retention times (tR) are ex- pressed in minutes based on UV-trace at 220&254 nm. Gradient: 0 min 5% B 0.4 min 95% B 10 0.7 min 95% B 0.71 min 5% B 1.0 min 5% B Total run time: 1.0 min 15 Method J: LC-MS were run on Shimadzu LC20-MS2010 UPLC-MS consisting of Shimadzu LC20 including column manager, binary solvent manager, sample organizer, PDA detector (operating at 220&254 nm), ELS detector, and MS2010 equipped with APPI-source operating in positive ion mode. LC-conditions: The column was Xtimate, C18, 2.1 × 30 mm, 3μm operating at 50ºC with 0.8 mL/min of a binary gradient consisting of water + 0.037 % trifluoroacetic acid (A) and ace- 20 tonitrile + 0.018 % trifluoroacetic acid(B). The retention times (tR) are expressed in minutes based on UV-trace at 220&254 nm. Gradient: 0.00 min 10% B 1.35 min 80% B 2.25 min 80% B 25 2.26 min 10% B 3 min 10% B Total run time: 3 min 30 Method K: LC-MS were run on Shimadzu LC20-MS2010 UPLC-MS consisting of Shimadzu LC20 including column manager, binary solvent manager, sample organizer, PDA detector (operating at 220&254 nm), ELS detector, and MS2010 equipped with APPI-source operating in positive ion mode. LC-conditions: The column was Xtimate, C18, 2.1 × 30 mm, 3μm operating at 50ºC with 0.8 mL/min of a binary gradient consisting of water + 0.037 % trifluoroacetic acid (A) and ace- 35 tonitrile + 0.018 % trifluoroacetic acid(B). The retention times (tR) are expressed in minutes based on UV-trace at 220&254 nm. Gradient: 0.00 min 0% B 1.35 min 60% B 2.25 min 60% B 2.26 min 0% B 5 3 min 0% B Total run time: 3 min NMR 10 1H NMR spectra were recorded at 600 MHz on a Bruker 600-Avance-III spectrometer, at 500 MHz on a Bruker 500-Avance DRX spectrometer, at 400 MHz on Bruker Avance AV-III-400 or a Varian MR400 spectrometers or at 300 MHZ using a Bruker Avance III HD. Chemical shift values are expressed in ppm-values relative to tetramethylsilane. The following abbreviations or their combinations are used for multiplicity of NMR signals: br =15 broad, d = doublet, m = multiplet, q = quartet, quint = quintet, s = singlet and t = triplet. Preparation of Reagents Reagent: Bis(methyl-d3)zinc in THF-dibutyl ether-Toluene 20
Figure imgf000098_0001
To a mixture of magnesium turnings (3.51 g, 145 mmol) and n-butyl ether (39.0 mL) was added DIBAL-H in toluene (1.00 mL, 1.00 molar, 1.00 mmol) at room temperature. The mixture was stirred for 15 min then iodomethane-d3 (0.62 mL, 10 mmol) was added. The mixture was then heated to 40 ºC and stirred for 15 minutes. Then additional iodomethane-d3 (5.6 mL, 9025 mmol) was added dropwise over a period of 30 minutes. The mixture was further stirred at 40 ºC for 1.5 h. The mixture was cooled, and an aliquot was subjected to iodometric titration (I2 in 2-MeTHF; slow addition of Grignard reagent) to determine the concentration = 1.38 M. To dried zinc(II) bromide (2.20 g, 9.77 mmol) under N2 was added THF (4.40 mL). This was stirred for 10 min. Then n-Butyl ether (4.40 mL) was slowly added to form a white suspen- 30 sion. Then D3CMgI (1.38 M in n-Bu2O) (14.2 mL, 1.38 M, 19.6 mmol) was added slowly over a period of 5 minutes at ~5ºC (ice-water cooling) to form a white suspension. The mixture was stirred for 30 minutes at room temperature. Then toluene (5.52 mL) was added, and the sus- pension was stirred for 15 min. The mixture was filtered under an inert atmosphere, and the filtrate was used in the subsequent reaction. Iodometric titration (I2 in 2-MeTHF) to determine the concentration = 0.71 M. Preparation of Intermediates 5 Intermediate: 3,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000099_0001
Three reactions were run in parallel: To a solution of 6-chloro-1H-pyrazolo[3,4-d]pyrim- idine (8 g, 51.8 mmol) in MeCN (240 mL) was added 1,3-dichloro-5,5-dimethylimidazolidine-2,4- 10 dione (7.14 g, 36.23 mmol). The mixture was stirred at 85 ºC for 24 h. The reaction mixture was concentrated under reduced pressure. The residues from the three reactions were combined and purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 80:20) to afford 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (4.3 g) of sufficient purity for the subsequent step.1H NMR (DMSO-d6, 400MHz) δ 14.55 (br s, 1H), 9.29 (s, 1H). 15 Intermediate : 3-(3,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propan-1-ol
Figure imgf000099_0002
To a solution of 3-((tert-butyldimethylsilyl)oxy)propan-1-ol (4.4 g, 23.1 mmol), 3,6-di- chloro-1H-pyrazolo[3,4-d]pyrimidine (5.24 g, 27.7 mmol), and PPh3 (12.1 g, 46.2 mmol) in THF 20 (45 mL) was added DIAD (9.35 g, 46.2 mmol) in a dropwise manner at 0 ºC. The mixture was stirred at 20 ºC for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 95:5 → 90:10) to afford 1-(3-((tert-butyldimethylsilyl)oxy)propyl)-3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (4.4 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.97 (s, 1H), 4.53 (t, J25 = 6.8 Hz, 2H), 3.66 (t, J = 5.6 Hz, 2H), 2.20-2.12 (m, 2H), 0.88 (s, 9H), 0.01 (s, 6H). To a solution of 1-(3-((tert-butyldimethylsilyl)oxy)propyl)-3,6-dichloro-1H-pyrazolo[3,4- d]pyrimidine (7 g, 19 mmol) in THF (20 mL) was added HCl (16 mL, 12M in H2O). The mixture was stirred at 20 ºC for 16 h. The mixture was concentrated under reduced pressure. The residue was treated with saturated aqueous NaHCO3, final pH ~ 7 and extracted with EtOAc (3×30 mL). The combined organic layers were washed with brine (3×30 mL), dried over Na2SO4, and con- centrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 80:20 → 50:50) to afford 3-(3,6-dichloro-1H-pyrazolo[3,4-d]py- 5 rimidin-1-yl)propan-1-ol (2.1 g) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 400MHz) δ 9.00 (s, 1H), 4.59 (t, J = 6.8 Hz, 2H), 3.66 (q, J = 5.6 Hz, 2H), 2.20-2.10 (m, 3H). Intermediate: 3-(3,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propyl 4-methylbenzenesul- fonate 10
Figure imgf000100_0001
To a solution of 3-(3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propan-1-ol (580 mg, 2.35 mmol) in DCM (10 mL) was added TEA (713 mg, 7.04 mmol) and TsCl (671 mg, 3.52 mmol). The mixture was stirred at 15 ºC for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:015 → 70:30) to afford 3-(3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propyl 4-methylbenzene- sulfonate (800 mg).1H NMR (CDCl3, 400MHz) δ 8.95 (s, 1H), 7.75 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 4.47 (t, J = 6.8 Hz, 2H), 4.09 (t, J = 6.0 Hz, 2H), 2.45 (s, 3H), 2.37-2.29 (m, 2H). Intermediate: 3-(3-Bromo-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propan-1-ol
Figure imgf000100_0002
20 A solution of DIAD (1.96 g, 1.89 mL, 9.71 mmol) in THF (65 mL) was cooled to 0 ºC. PPh3 (2.55 g, 9.71 mmol) was added portionwise and the mixture was stirred for 15 minutes.3- Bromo-6-chloro-1H-pyrazolo[3,4-d]pyrimidine (1.60 g, 6.85 mmol) was then added in portions followed by a solution of 3-((tert-butyldimethylsilyl)oxy)propan-1-ol (1.09 g, 5.71 mmol) in THF (10 mL). The cooling bath was allowed to expire upon overnight stirring. The reaction mixture25 was concentrated under reduced pressure. The residue was purified by chromatography on sil- ica gel (eluent: petroleum ether:EtOAc 100:0 → 70:30) to afford 3-bromo-1-(3-((tert-butyldime- thylsilyl)oxy)propyl)-6-chloro-1H-pyrazolo[3,4-d]pyrimidine (2.17 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 600MHz) δ 8.89 (s, 1H), δ 4.54 (t, J = 6.9 Hz, 2H), 3.65 (t, J = 5.8 Hz, 2H), 2.15 (m, 2H), 0.88 (s, 9H), 0.01 (s, 6H). To a dry flask was added 3-bromo-1-(3-((tert-butyldimethylsilyl)oxy)propyl)-6-chloro- 1H-pyrazolo[3,4-d]pyrimidine (1.00 g, 2.46 mmol) and THF (25 mL). The flask was capped and 5 the atmosphere was exchanged for argon. HCl (3M in CPME) (3.02 mL, 9.1 mmol) was added and the mixture was stirred at 50 ºC overnight. The reaction mixture was diluted with water (10 mL), and pH adjusted using saturated aqueous potassium carbonate, pH~11, and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by chromatography10 on silica gel (eluent: petroleum ether:EtOAc 80:20 → 20:80) to afford 3-(3-bromo-6-chloro-1H- pyrazolo[3,4-d]pyrimidin-1-yl)propan-1-ol (650 mg) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 600MHz) δ 8.91 (s, 1H), 4.60 (t, J = 6.6 Hz, 2H), 3.65 (q, J = 5.8 Hz, 2H), 2.18-2.13 (m, 2H), 2.11 (t, J = 5.8 Hz, 1H). 15 Intermediate: 3,6-Dichloro-1-(3-((1,5-dimethyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyra- zolo[3,4-d]pyrimidine
Figure imgf000101_0001
To a solution of 1,5-dimethyl-1H-pyrazol-3-ol hydrochloride (5 g, 33.7 mmol, HCl salt) in 20 H2SO4 (40 mL) was added KNO3 (4.08 g, 40.4 mmol) at 0 ºC. The mixture was stirred at 0 ºC for 1 h. The reaction mixture was poured onto ice water (200 mL) and adjusted to pH 5-6 with satu- rated aqueous NaOH. The mixture was extracted with EtOAc (3×200 mL) and the combined or- ganic layers were concentrated to afford 1,5-dimethyl-4-nitro-1H-pyrazol-3-ol (2.8 g) as a solid of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 3.72 (s, 3H), 2.61 (s, 3H). 25 To a solution of 1,5-dimethyl-4-nitro-pyrazol-3-ol (500 mg, 3.18 mmol), 3-[tert-butyl(di- methyl)silyl]oxypropan-1-ol (727 mg, 3.82 mmol), and PPh3 (1.09 g, 4.14 mmol) in THF (10 mL) was added dropewise DIAD (837 mg, 4.14 mmol) at 0 ºC. The mixture was stirred at 15ºC for 16 h. The mixture was concentrated. The residue was purified by chromatography on silica gel (el- uent: petroleum ether:EtOAc 70:30 → 60:40) to afford give 3-(3-((tert-butyldimethylsi- lyl)oxy)propoxy)-1,5-dimethyl-4-nitro-1H-pyrazole (700 mg) of sufficient purity for the subse- quent step.1H NMR (CDCl3, 400MHz) δ 4.38 (t, J = 6.0 Hz, 2H), 3.82 (t, J = 6.0 Hz, 2H), 3.69 (s, 3H), 5 2.60 (s, 3H), 2.07-1.97 (m, 2H), 0.89 (s, 9H), 0.05 (s, 6H). To a solution of tert-butyl-[3-(1,5-dimethyl-4-nitro-pyrazol-3-yl)oxypropoxy]-dimethyl- silane (560 mg, 1.70 mmol) in THF (5 mL) were added saturated aqueous HCl (12 M, 1.42 mL). The reaction mixture was stirred at 10 ºC for 16 h. The solvent was removed under reduced pressure to afford 3-((1,5-dimethyl-4-nitro-1H-pyrazol-3-yl)oxy)propan-1-ol (365.7 mg) of suffi-10 cient purity for the subsequent step. LC-MS (method A) (m/z) = 216.2 (MH)+ tR = 0.42 minutes. To a solution of 3-(1,5-dimethyl-4-nitro-pyrazol-3-yl)oxypropan-1-ol (200 mg, 0.93 mmol) in DCM (5 mL) were added MsCl (266 mg, 2.32 mmol) and TEA (282 mg, 2.79 mmol) at 0 ºC. The reaction mixture was stirred at 15 ºC for 16 h. The reaction mixture was diluted with water (10 mL) and extracted with DCM (3 × 10 mL). The combined organic layers were washed15 with brine (3 × 10 mL), dried over Na2SO4, and concentrated under reduced pressure. The resi- due was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 70:30 → 60:40) to afford 3-((1,5-dimethyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl methanesulfonate (150 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.67-4.40 (m, 4H), 3.69 (s, 3H), 3.03 (s, 3H), 2.60 (s, 3H), 2.30-2.24 (m, 2H). 20 To a solution of 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (170 mg, 0.90 mmol) in DMF (2 mL) were added 3-((1,5-dimethyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl methanesulfonate (264 mg, 0.90 mmol), NaI (135 mg, 0.90 mmol), and DIPEA (581 mg, 4.50 mmol). The reaction mixture was stirred at 80 ºC for 16 h. The reaction mixture was diluted with water (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (3 × 10 mL), dried 25 over Na2SO4, and concentrated under reduced. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 60:40 → 50:50) to afford 3,6-dichloro-1-(3-((1,5-dime- thyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (100 mg) of sufficient pu- rity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.95 (s, 1H), 4.65 (t, J = 6.0 Hz, 2H), 4.32 (t, J = 6.0 Hz, 2H), 3.64 (s, 3H), 2.58 (s, 3H), 2.50-2.44 (m, 2H). 30 Intermediate: 3,6-Dichloro-1-(3-((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3- yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000103_0001
To a solution of 5-chloro-3-methyl-4-nitro-1H-pyrazole (10.8 g, 66.85 mmol), tetrahy- dro-2H-pyran-4-ol (13.66 g, 133.7 mmol) and PPh3 (21.04 g, 80.22 mmol) in THF (100 mL) was added a solution of DBAD (18.47 g, 80.22 mmol) in THF (40 mL) dropwise at 0 ºC. The resulting 5 mixture was stirred at 20 ºC for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: heptane:EtOAc 100:0 → 20:80) twice to afford 3-chloro-5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole (11 g) of suf- ficient purity for the next step.1H NMR (CDCl3, 400MHz) δ 4.35-4.26 (m, 1H), 4.17-4.13 (m, 2H), 3.56 - 3.49 (m, 2H), 2.62 (s, 3H), 2.38-2.26 (m, 2H), 1.83-1.78 (m, 2H). 10 3-Chloro-5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole (2.5 g, 10.2 mmol) and KOH (9 g, 160 mmol) in H2O (40 mL) was heated at 140ºC for 8 hours. The mixture was adjusted to pH ~3 with aqueous HCl (12M). The precipitate was filtered. The filter cake was dried to afford 5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-ol (2.2 g) of suffi- cient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.44 (s, 1H), 4.23-4.16 (m, 1H),15 4.13-4.09 (m, 2H), 3.52-3.45 (m, 2H), 2.64 (s, 3H), 2.37-2.26 (m, 2H), 1.76-1.72 (m, 2H). A solution of DIAD (4.16 g, 20.6 mmol) was added to a solution of 5-methyl-4-nitro-1- (tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-ol (3.6 g, 15 mmol), 3-((tert-butyldimethylsilyl)oxy)pro- pan-1-ol (3.62 g, 19.0 mmol) and PPh3 (5.40 g, 20.6 mmol) in THF (50 mL) at 0 ºC. Then the mixture was stirred at 20 ºC for 40 h. The reaction mixture was concentrated under reduced 20 pressure. The residue was purified by chromatography on silica gel (eluent: heptane:EtOAc 100:0 → 80:20) to afford 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1-(tetra- hydro-2H-pyran-4-yl)-1H-pyrazole (4 g) of sufficient purity for the next step. 1H NMR (CDCl3, 400MHz) δ 4.40 (t, J = 6.4 Hz, 2H), 4.27-4.17 (m, 1H), 4.16-4.11 (m, 2H), 3.52 (t, J = 6.4 Hz, 2H), 3.59-3.43 (m, 2H), 2.63 (s, 3H), 2.38-2.20 (m, 2H), 2.04-1.99 (m, 2H), 1.80-1.70 (m, 2H), 0.88 (s,25 9H), 0.04 (s, 6H). To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1-(tetrahy- dro-2H-pyran-4-yl)-1H-pyrazole (4 g, 10 mmol) in THF (40 mL) was added HCl (12 M, 8.34 mL, 100 mmol). The mixture was stirred at 20 ºC for 16 h. The reaction mixture was concentrated under reduced pressure. The reaction mixture was diluted with water (50 mL) and the pH ad- justed to 9~10 using saturated aqueous Na2CO3 and extracted with EtOAc (3×100 mL). The com- bined organics were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: heptane:EtOAc 100:0 → 20:80) to 5 afford 3-((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propan-1-ol (1.4 g) of sufficient purity for the next step.1H NMR (CDCl3, 400MHz) δ 4.50 (t, J = 6.0 Hz, 2H), 4.28- 4.17 (m, 1H), 4.17-4.12 (m, 2H), 3.91-3.78 (m, 2H), 3.57-3.46 (m, 2H), 2.65 (s, 3H), 2.36-2.23 (m, 2H), 2.11-2.05 (m, 2H), 1.81-1.73 (m, 2H). To a solution of 3-((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3- 10 yl)oxy)propan-1-ol (1.4 g, 4.91 mmol) in DCM (20 mL) was added MsCl (1.41 g, 12.31 mmol) and TEA (1.49 g, 14.7 mmol) at 0ºC. The mixture was stirred at 20 ºC for 16 h. The reaction mixture was diluted with water (50 mL) and extracted with DCM (3×100 mL). The combined organic lay- ers were washed with brine (3×100 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: heptane:EtOAc 100:015 → 20:80) to afford 3-((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3- yl)oxy)propylmethanesulfonate (1.5 g) of sufficient purity for the next step. 1H NMR (CDCl3, 400MHz) δ 4.49-4.42 (m, 4H), 4.27-4.17 (m, 1H), 4.16-4.11 (m, 2H), 3.58-3.47 (m, 2H), 3.04 (s, 3H), 2.65 (s, 3H), 2.36-2.22 (m, 4H), 1.80-1.72 (m, 2H). To a solution of 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (810 mg, 4.29 mmol) in DMF20 (20 mL) were added 3-((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)pro- pyl methanesulfonate (1.5 g, 4.13 mmol), NaI (619 mg, 4.13 mmol) and DIPEA (2.67 g, 20.6 mmol). The mixture was stirred at 80 ºC for 16 h. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine (3×100 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue25 was purified by chromatography on silica gel (eluent: heptane:EtOAc 100:0 → 60:40) to afford 3,6-dichloro-1-(3-((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)- 1H-pyrazolo[3,4-d]pyrimidine (840 mg) of sufficient purity for the next step. 1H NMR (CDCl3, 400MHz) δ 8.96 (s, 1H), 4.65 (t, J = 6.4 Hz, 2H), 4.35 (t, J = 6.4 Hz, 2H), 4.23-4.15 (m, 1H), 4.12- 4.07 (m, 2H), 3.54-3.46 (m, 2H), 2.63 (s, 3H), 2.51-2.43 (m, 2H), 2.25-2.14 (m, 2H), 1.76-1.69 (m,30 2H). Intermediate: (±)-3,6-Dichloro-1-(3-((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-3-yl)-1H-pyra- zol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000105_0001
To a dry flask fitted with a stirbar and capped with a septum was added DIAD (2.69 mL, 13.8 mmol) and the atmosphere was exchanged for argon. THF (200 mL) was added and the so- lution was cooled to 0 ºC. Then, PPh3 (on resin, loading: 1.6 mmol/g) (3.62 g, 13.8 mmol, 8.625 5 g resin used), was added and the solution was stirred under argon for 5 minutes. 3-Chloro-5- methyl-4-nitro-1H-pyrazole (1.80 g, 11.1 mmol) was added followed by the addition of tetrahy- dro-2H-pyran-3-ol (1.71 g, 16.7 mmol) as a solution in THF (10 mL). The mixture was allowed to reach room temperature and was stirred for 4 h. The reaction was diluted with THF and was filtered while washing with THF. The filtrate was concentrated under reduced pressure. The res- 10 idue was purified by chromatography on silica gel (eluent: heptane:EtOAc 100:0 → 25:75) to afford (±)-3-chloro-5-methyl-4-nitro-1-(tetrahydro-2H-pyran-3-yl)-1H-pyrazole (600 mg) of suffi- cient purity for the subsequent step.1H NMR (CDCl3, 600MHz) δ 4.27-4.22 (m, 1H), 4.01-3.92 (m, 2H), 3.73 (t, J = 11.1 Hz, 1H), 3.48-3.42 (m, 1H), 2.68 (s, 3H), 2.32-2.23 (m, 1H), 2.11-2.05 (m, 1H), 1.89-1.78 (m, 2H). 15 To a vial was added (±)-3-chloro-5-methyl-4-nitro-1-(tetrahydro-2H-pyran-3-yl)-1H-py- razole (725 mg, 2.95 mmol), potassium hydroxide (1.16 g, 20.7 mmol), and water (11.7 mL, 649 mmol). The vial was capped, and the reaction mixture was heated using a microwave reactor at 152 ºC for 3 h. The mixture was cooled to room temperature and was diluted with water (50 mL). The mixture was washed with EtOAc (10 mL). The aqueous phase was then acidified by slow20 addition of 2 M aqueous HCl and the mixture was extracted with EtOAc (3 x 100 mL). The com- bined organic phases were washed with saturated NaCl, dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford (±)-5-methyl-4-nitro-1-(tetrahydro-2H-pyran-3- yl)-1H-pyrazol-3-ol (510 mg) as a solid of sufficient purity for the next step.1H NMR (CDCl3, 600MHz) δ 4.21-4.15 (m, 1H), 3.99-3.95 (m, 1H), 3.93-3.89 (m, 1H), 3.75 (t, J = 10.7 Hz, 1H), 3.46-25 3.40 (m, 1H), 2.69 (s, 1H), 2.64 (s, 3H), 2.31-2.22 (m, 1H), 2.06-1.99 (m, 1H), 1.88-1.79 (m, 2H). To a dry flask was added 3-((tert-butyldimethylsilyl)oxy)propyl 4-methylbenzenesul- fonate (1.45 g, 4.20 mmol), DMF (15 mL), and (±) 5-methyl-4-nitro-1-(tetrahydro-2H-pyran-3-yl)- 1H-pyrazol-3-ol (510 mg, 2.24 mmol). The flask was flushed with argon before cesium carbonate (1.40 g, 4.30 mmol) was added in a portionwise manner. The reaction was stirred at room tem- perature for 16 h. The mixture was concentrated under reduced pressure. The residue was pu- rified by chromatography on silica gel (eluent: heptane:EtOAc 100:0 → 25:75) twice to afford (±)- 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1-(tetrahydro-2H-pyran-3-yl)-1H- 5 pyrazole (401 mg) of sufficient purity for the next step.1H NMR (CDCl3, 600MHz) δ 4.38 (t, J = 6.3 Hz, 2H), 4.18-4.11 (m, 1H), 3.99-3.95 (m, 1H), 3.93-3.88 (m, 1H), 3.80 (t, J = 6.0 Hz, 2H), 3.70 (t, J = 10.7 Hz, 1H), 3.46-3.40 (m, 1H), 2.62 (s, 3H), 2.26-2.17 (m, 1H), 2.05-1.97 (m, 3H), 1.85- 1.79 (m, 2H), 0.88 (s, 9H), 0.04 (s, 6H). To a dry flask was added (±)-3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-ni- 10 tro-1-(tetrahydro-2H-pyran-3-yl)-1H-pyrazole (400 mg, 1.00 mmol) and DCM (10 mL). The flask was capped and the atmosphere was exchanged for argon. HCl (3M in CPME) (2.67 mL, 8.01 mmol) was added and the mixture was briefly heated and then stirred for 1 h at room temper- ature. HCl (3M in CPME) (2.67 mL, 8.01 mmol) was added and the flask was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure. The reac-15 tion mixture was diluted with water (10 mL) and saturated aqueous potassium carbonate ad- justing pH ~ 11. The mixture was extracted with EtOAc (3 x 50 mL), and the combined organics were washed with brine, and dried over sodium sulfate, filtered and concentrated under re- duced pressure. The residue was purified by chromatography on silica gel (eluent: Hep- tane:EtOAc 100:0 → 20:80) to afford (±)-3-((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-3-yl)-1H- 20 pyrazol-3-yl)oxy)propan-1-ol (248 mg) of sufficient purity for the next step. 1H NMR (CDCl3, 600MHz) δ 4.52-4.44 (m, 2H), 4.16 (tt, J = 11.0, 4.3 Hz, 1H), 3.99-3.95 (m, 1H), 3.94-3.88 (m, 1H), 3.86-3.80 (m, 2H), 3.69 (t, J = 11.2 Hz, 1H), 3.47-3.41 (m, 1H), 2.63 (s, 3H), 2.55-2.50 (m, 1H), 2.24-2.16 (m, 1H), 2.09-2.00 (m, 3H), 1.88-1.79 (m, 2H). A solution of 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (201 mg, 1.06 mmol) and (±)-3-25 ((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-3-yl)-1H-pyrazol-3-yl)oxy)propan-1-ol (243 mg, 0.852 mmol) in THF (25 mL) was degassed thoroughly with argon in a dry vial. The vial was capped and 1,1-(Azodicarbonyl)-Dipiperidine (484 mg, 1.92 mmol), and tributylphosphine (476 µL, 1.92 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was quenched with MeOH (0.5 mL) and diluted with EtOAc (25 mL). 30 Celite was added and the mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: heptane:EtOAc 100:0 → 20:80) twice to af- ford (±)-3,6-dichloro-1-(3-((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-3-yl)-1H-pyrazol-3- yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (244 mg) as a solid of sufficient purity for the next step.1H NMR (CDCl3, 600MHz) δ 8.95 (s, 1H), 4.68-4.60 (m, 2H), 4.34 (t, J = 5.8 Hz, 2H), 4.16-4.10 (m, 1H), 3.99-3.94 (m, 1H), 3.91-3.86 (m, 1H), 3.62 (t, J = 10.7 Hz, 1H), 3.43-3.37 (m, 1H), 2.61 (s, 3H), 2.46 (p, J = 6.2 Hz, 2H), 2.17 - 2.09 (m, 1H), 2.01-1.96 (m, 1H), 1.84-1.78 (m, 2H). 5 Intermediate: 6-Chloro-1-(3-((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3- yl)oxy)propyl)-3-(trifluoromethyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000107_0001
A solution of DIAD (218 µL, 1.12 mmol) in THF (1 mL) was cooled to 0 ºC under an at- mosphere of argon. PPh3 (294 mg, 1.12 mmol) was added and the mixture was stirred for 10 10 minutes.6-Chloro-3-(trifluoromethyl)-1H-pyrazolo[3,4-d]pyrimidine (156 mg, 0.701 mmol) was then added followed by a solution of 3-((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-py- razol-3-yl)oxy)propan-1-ol (160 mg, 0.561 mmol, prepared as previously described) in THF (0.25 mL). The reaction mixture was stirred at 0 ºC for 2 minutes and then at room temperature over- night. The reaction mixture was concentrated under reduced pressure. The residue was purified15 by chromatography on silica gel (eluent: heptane:EtOAc 100:0 → 20:80) to afford 6-chloro-1-(3- ((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)-3-(trifluorome- thyl)-1H-pyrazolo[3,4-d]pyrimidine (190 mg) as a solid of sufficient purity for the subsequent step.1H NMR (CDCl3, 600MHz) δ 9.12 (s, 1H), 4.76 (t, J = 6.5 Hz, 2H), 4.38 (t, J = 5.8 Hz, 2H), 4.21- 4.14 (m, 1H), 4.12-4.07 (m 2H), 3.52-3.46 (m, 2H), 2.61 (s, 3H), 2.52 (p, J = 6.2 Hz, 2H), 2.23-2.1620 (m, 2H), 1.75-1.69 (m, 2H). Intermediate: 3,6-Dichloro-1-(3-((5-cyclopropyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyra- zol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000107_0002
A mixture of 3-chloro-4-nitro-1H-pyrazole (10 g, 68 mmol), 4-bromotetrahydro-2H-py- ran (22.37 g, 135.6 mmol) and Cs2CO3 (55.22 g, 169.5 mmol) in DMF (100 mL) was degassed and purged with N2 (× 3), and then the mixture was stirred at 80 ºC for 16 h. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (4×200 mL). The combined organic 5 layers were washed with brine (3×300 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 80:20 → 70:30) to afford 3-chloro-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole (8.7 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.22 (s, 1H), 4.38- 4.28 (m, 1H), 4.15 (dd, J = 3.2, 10.8 Hz, 2H), 3.59-3.49 (m, 2H), 2.19-2.00 (m, 4H). 10 Three reactions were run in parallel on the same scale. To a solution of 3-chloro-4-nitro- 1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole (2.2 g, 9.5 mmol) in THF (100 mL) was added LiHMDS (28.49 mL, 1M in THF) in a dropwise manner at -78 ºC. After stirring for 0.5 h at -78 ºC a solution of I2 (7.23 g, 28.5 mmol) in THF (20 mL) was added in a dropwise manner. The reaction mixture was stirred at -78ºC for 1.5 h. The reaction was quenched with saturated aqueous NH4Cl (50 mL) 15 and extracted with EtOAc (5×50 mL). The combined organic layers were washed with saturated aqueous Na2SO3 (3×50 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue from the three reactions were combined and purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 80:20 → 70:30) to afford 3-chloro-5-iodo-4-nitro-1-(tetrahydro- 2H-pyran-4-yl)-1H-pyrazole (8.6 g) for the three reactions of sufficient purity for the subsequent20 step.1H NMR (CDCl3, 400MHz) δ 4.68-4.58 (m, 1H), 4.17-4.14 (m, 2H), 3.60-3.51 (m, 2H), 2.36- 2.23 (m, 2H), 1.90 (dd, J = 2.4, 12.8 Hz, 2H). Four reactions were run in parallel on the same scale. A mixture of 3-chloro-5-iodo-4- nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole (1.15 g, 3.22 mmol), cyclopropylboronic acid (553 mg, 6.43 mmol), Pd2(dba)3 (185 mg, 0.20 mmol), PCy3 (90 mg, 0.32 mmol), and K2CO3 (1.3325 g, 9.65 mmol) in DMF (10 mL) was stirred at 140 ºC for 2 h using a microwave reactor. The reac- tion mixture was quenched with water (40 mL) and extracted with EtOAc (3×50 mL). The com- bined organic layers were washed with brine (3×60 mL), dried over Na2SO4, and concentrated under reduced pressure. The residues from the four reactions were combined and purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 80:20 → 70:30) followed by pre-30 parative HPLC (Instrument: Gilson GX-281 Liquid Handler, SHIMADZU LC-8A; Column: Phenom- enex C18150×40mm×5μm; Mobile Phase A: water (NH3H2O+NH4HCO3), Mobile phase B: MeCN; Gradient: B from 26% to 56% in 10 min then hold at 100% for 2 min; Flow Rate (mL/min): 60; Column temperature: 30ºC; Wavelength: 220nm 254nm basic condition) to afford 3-chloro-5- cyclopropyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole (2 g) for the four reactions of suf- ficient purity for the next step.1H NMR (CDCl3, 400MHz) δ 4.77-4.68 (m, 1H), 4.15 (dd, J = 4.0, 11.2 Hz, 2H), 3.58-3.49 (m, 2H), 2.41-2.28 (m, 2H), 1.92-1.83 (m, 1H), 1.83-1.75 (m, 2H), 1.34- 1.27 (m, 2H), 0.81-0.75 (m, 2H). 5 A mixture of 3-chloro-5-cyclopropyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole (2.9 g, 10.67 mmol), and KOH (8.98 g, 160.10 mmol) in H2O (50 mL) was degassed and purged with N2 (× 3), and then the mixture was stirred at 140 ºC for 16 h. The mixture was acidified with concentrated HCl to pH = 3 and extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine (3×150 mL), dried over Na2SO4, and concentrated under reduced pres-10 sure to afford 5-cyclopropyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-ol (1.13 g) of suf- ficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.62 (br s, 1H), 4.71-4.61 (m, 1H), 4.14 (dd, J = 5.2, 12.0 Hz, 2H), 3.57-3.47 (m, 2H), 2.42-2.29 (m, 2H), 1.87-1.78 (m, 1H), 1.74 (dd, J = 3.2, 12.8 Hz, 2H), 1.35-1.28 (m, 2H), 0.91-0.84 (m, 2H). LC-MS (method C) (m/z) = 254.2 (MH)+ tR = 1.59 minutes. 15 To a solution of 5-cyclopropyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-ol (200 mg, 0.79 mmol), 3-(3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propan-1-ol (195 mg, 0.79 mmol, prepared as described previously), and PPh3 (621 mg, 2.37 mmol) in THF (10 mL) was added DIAD (479 mg, 2.37 mmol) in a dropwise manner at 0 ºC. The mixture was stirred at 20 ºC for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by20 chromatography on silica gel (eluent: petroleum ether:EtOAc 80:20 → 70:30) to afford 3,6-di- chloro-1-(3-((5-cyclopropyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)- 1H-pyrazolo[3,4-d]pyrimidine (140 mg) of sufficient purity for the subsequent step. LC-MS (method B) (m/z) = 428.1 (MH)+ tR = 0.87 minutes.
Intermediate: 1-(3-((1-((2-Oxabicyclo[2.1.1]hexan-1-yl)methyl)-5-methyl-4-nitro-1H-pyrazol-3- yl)oxy)propyl)-3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000110_0001
A mixture of 1-(iodomethyl)-2-oxabicyclo[2.1.1]hexane (17.93 g, 80.02 mmol), 3-chloro- 5 5-methyl-4-nitro-1H-pyrazole (10.8 g, 66.9 mmol), and K2CO3 (27.7 g, 201 mmol) in MeCN (350 mL) was stirred at 70 ºC for 16 h. The mixture was filtered and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 70:30) to afford 1-((2-oxabicyclo[2.1.1]hexan-1-yl)methyl)-3-chloro-5-me- thyl-4-nitro-1H-pyrazole (8.4 g) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 10 400MHz) δ 4.40 (s, 2H), 3.74 (s, 2H), 2.98-2.92 (m, 1H), 2.71 (s, 3H), 1.87-1.79 (m, 2H), 1.48-1.44 (m, 2H). To a solution of 1-((2-oxabicyclo[2.1.1]hexan-1-yl)methyl)-3-chloro-5-methyl-4-nitro- 1H-pyrazole (1 g, 3.88 mmol) in H2O (80 mL) was added KOH (4.35 g, 77.6 mmol). The mixture was stirred at 120 ºC for 23 h. Then additional KOH (1.58 g, 28.2 mmol) was added and the 15 mixture was stirred at 140 ºC for 40 h. The mixture was adjusted to pH ~ 2 with concentrated aqueous HCl and extracted with EtOAc (3×50 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to afford 1-((2-oxabicy- clo[2.1.1]hexan-1-yl)methyl)-5-methyl-4-nitro-1H-pyrazol-3-ol (880 mg) of sufficient purity for the subsequent step.1H NMR (DMSO-d6, 400MHz) δ 11.37 (s, 1H), 4.30 (s, 2H), 3.63 (s, 2H), 2.8620 (t, J = 6.4 Hz, 1H), 2.53 (s, 3H), 1.875-1.75 (m, 2H), 1.40-1.27 (m, 2H). To a solution of 1-((2-oxabicyclo[2.1.1]hexan-1-yl)methyl)-5-methyl-4-nitro-1H-pyrazol- 3-ol (200 mg, 0.84 mmol) and 3-(3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propan-1-ol (248 mg, 1.00 mmol, prepared as described previously) in THF (6 mL) was added DIAD (686 mg, 3.39 mmol) and PPh3 (877 mg, 3.34 mmol). The mixture was stirred at 20 ºC for 16 h. The mixture was 25 concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 50:50) to afford 1-(3-((1-((2-oxabicyclo[2.1.1]hexan-1- yl)methyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-3,6-dichloro-1H-pyrazolo[3,4-d]pyrimi- dine (230 mg) of sufficient purity for the subsequent step. Intermediate: 3,6-Dichloro-1-(3-((5-methyl-1-((3-methyloxetan-3-yl)methyl)-4-nitro-1H-pyra- zol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000111_0001
To a solution of 3-chloro-5-methyl-4-nitro-1H-pyrazole (10 g, 61.9 mmol) in MeCN (100 5 mL) were added 3-(iodomethyl)-3-methyl-oxetane (14.44 g, 68.1 mmol) and K2CO3 (25.7 g, 186 mmol). The reaction mixture was stirred at 80 ºC for 16 h. The reaction mixture was concen- trated under reduced pressure. The mixture was diluted with water (30 mL) and extracted with EtOAc (3 × 30 mL). The combined organic layers were washed with brine (3 × 30 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by chromatography10 on silica gel (eluent: petroleum ether:EtOAc 80:20 → 75:25) to afford 3-chloro-5-methyl-1-((3- methyloxetan-3-yl)methyl)-4-nitro-1H-pyrazole (6.3 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.73 (d, J = 6.4 Hz, 2H), 4.44 (d, J = 6.4 Hz, 2H), 4.29 (s, 2H), 2.67 (s, 3H), 1.28 (s, 3H). To a solution of 3-chloro-5-methyl-1-((3-methyloxetan-3-yl)methyl)-4-nitro-1H-pyrazole 15 (500 mg, 2.04 mmol) in H2O (25 mL) was added NaOH (2.44 g, 61.1 mmol). The mixture was stirred at 140 ºC for 1.5 h. The reaction mixture was extracted with EtOAc (3×100 mL), the com- bined organic layers were discarded. The water phase was adjusted to pH=3 with aqueous HCl (12M), extracted with EtOAc (3×100 mL), dried over Na2SO4, and concentrated under reduced pressure to afford 5-methyl-1-((3-methyloxetan-3-yl)methyl)-4-nitro-1H-pyrazol-3-ol (100 mg) 20 of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.78 (d, J = 6.4 Hz, 2H), 4.37 (d, J = 6.4 Hz, 2H), 4.22 (s, 2H), 2.59 (s, 3H), 1.22 (s, 3H). To a solution of 5-methyl-1-((3-methyloxetan-3-yl)methyl)-4-nitro-1H-pyrazol-3-ol (140 mg, 0.62 mmol), 3-(3,6-dichloropyrazolo[3,4-d]pyrimidin-1-yl)propan-1-ol (152 mg, 0.62 mmol, prepared as described previously), and PPh3 (485 mg, 1.85 mmol) in THF (4 mL) was added DIAD 25 (374 mg, 1.85 mmol) at 0 ºC. The mixture was stirred at 20 ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 40:60) and further purified by preparative HPLC (Instru- ment: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV Detector; Column: Xtimate C18150×40mm×5μm; Mobile Phase A: water (NH3H2O), Mobile phase B: MeCN;Gradient: B from 38% to 68% in 8 min then hold at 100% for 1 min; Flow Rate (mL/min): 60; Column temperature: 30ºC; Wavelength: 220nm 254nm) to afford 3,6-dichloro-1-(3-((5-methyl-1-((3-methyloxetan-3- yl)methyl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (75 mg) of suffi- cient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.96 (s, 1H), 4.70 (d, J = 6.4 Hz, 5 2H), 4.64 (t, J = 6.4 Hz, 2H), 4.37 (d, J = 6.4 Hz, 2H), 4.30 (t, J = 5.6 Hz, 2H), 4.11 (s, 2H), 2.59 (s, 3H), 2.49-2.44 (m, 2H), 1.22 (s, 3H). Intermediate: 3,6-Dichloro-1-(3-((5-(methyl-d3)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyra- zol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine 10
Figure imgf000112_0001
To a dry vial containing 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1- (tetrahydro-2H-pyran-4-yl)-1H-pyrazole (200 mg, 0.501 mmol) in DMSO-d6 (8 mL) under an at- mosphere of argon was added^potassium tert-butoxide (28.1 mg, 0.250 mmol). The solution turned dark orange. The reaction mixture was stirred at 30 ºC for 72 h. The mixture was diluted 15 with^deuterium oxide (10 mL) and was extracted with EtOAc (3 x 25 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated^under reduced pressure. The residue was dried thoroughly via freeze drying to remove residual solvent to afford 3-((5-(methyl-d3)-4- nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propan-1-ol (140 mg) of sufficient purity for the next step. 1H NMR(CDCl3,600MHz) δ 4.49 (t, J = 5.9 Hz, 2H), 4.23-4.17 (m, 1H), 4.13- 20 4.08(m, 2H), 3.86-3.80 (m, 2H), 3.54-3.47 (m, 2H), 2.55 (t, J = 6.0 Hz, 1H), 2.33-2.23 (m, 2H), 2.10-2.03 (m, 2H), 1.78-1.73 (m, 2H). A solution of DIAD (113 µL, 0.583 mmol)^in^THF (7 mL) was cooled to 0 ºC under an at- mosphere of argon. PPh3 (153 mg, 0.583 mmol)^(on resin, ~3 mmol/g: employed 194 mg = 0.583 mmol) was added and the mixture was stirred for 10 minutes.3,6-Dichloro-1H-pyrazolo[3,4-25 d]pyrimidine (118 mg, 0.624 mmol)^was then added followed by a solution of 3-((5-(methyl-d3)- 4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propan-1-ol (120 mg, 0.416 mmol)inTHF (1.5 mL). The reaction mixture was stirred at 0 ºC for 2 minutes and then at room temperature overnight. The reaction mixture was filtered and the filtrate was concentrated un- der reduced pressure. The residue was purified by chromatography on silica gel (eluent: petro- leum ether:EtOAc 100:0 → 20:80) to afford^3,6-dichloro-1-(3-((5-(methyl-d3)-4-nitro-1-(tetrahy- dro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (133 mg)^as a solid of sufficient purity for the subsequent step.1H NMR^(CDCl3,^600MHz) δ 8.95 (s, 1H), 4.64 (t, J = 6.4 Hz, 2H), 4.35 (t, J = 5.8 Hz, 2H), 4.20-4.13 (m, 1H), 4.12-4.07 (m, 2H), 3.53- 5 3.44 ( m, 2H), 2.51 - 2.44 (m, 2H), 2.23-2.14 (m, 2H), 1.76-1.68 (m, 2H). Intermediate: 3,6-Dichloro-1-(3-((5-methyl-1-(3-methyloxetan-3-yl)-4-nitro-1H-pyrazol-3- yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000113_0001
10 To a solution of 3-chloro-4-nitro-1H-pyrazole (5 g, 33.9 mmol) in DMF (50 mL) was added diethyl 2-bromo-2-methylmalonate (17.16 g, 67.79 mmol) and K2CO3 (9.39 g, 68.0 mmol) and the reaction was stirred at 100 ºC for 12 h. The mixture was diluted with H2O (50 mL) and ex- tracted with EtOAc (2×50 mL). The organic phase was washed with brine (2×50 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by chromatography15 on silica gel (eluent: petroleum ether:EtOAc 100:0 → 70:30) to afford diethyl 2-(3-chloro-4-nitro- 1H-pyrazol-1-yl)-2-methylmalonate (7.25 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.60 (s, 1H), 4.33 (q, J = 7.2 Hz, 4H), 2.12 (s, 3H), 1.31 (t, J = 7.2 Hz, 6H). To a solution of diethyl 2-(3-chloro-4-nitro-1H-pyrazol-1-yl)-2-methylmalonate (3.6 g, 11.3 mmol) in MeOH (80 mL) was added NaBH4 (854 mg, 22.6 mmol) in portions at 0 ºC and the 20 reaction was stirred at 0 ºC for 1 h. The mixture was concentrated under reduced pressure. The residue was diluted with H2O (10 mL), saturated aqueous NH4Cl (10 mL), and extracted with EtOAc (3×20 mL). The organic phase was washed with brine (20 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 20:80) to afford 2-(3-chloro-4-nitro-1H-pyrazol-1-yl)-2- 25 methylpropane-1,3-diol (1.55 g) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 400MHz) δ 8.48 (s, 1H), 4.01 (d, J = 4.4 Hz, 4H), 2.83-2.72 (m, 2H), 1.53 (s, 3H). To a solution of 2-(3-chloro-4-nitro-1H-pyrazol-1-yl)-2-methylpropane-1,3-diol (1.55 g, 6.58 mmol) in DCM (20 mL) was added TsCl (1.32 g, 6.91 mmol) and TEA (998 mg, 9.87 mmol), the reaction was stirred at 20 ºC for 12 h. The reaction was concentrated under reduced pres- sure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 5 100:0 → 70:30) to afford 2-(3-chloro-4-nitro-1H-pyrazol-1-yl)-3-hydroxy-2-methylpropyl 4- methylbenzenesulfonate (1.5 g) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 400MHz) δ 8.22 (s, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 4.40 (d, J = 10.8 Hz, 1H), 4.27 (d, J = 10.8 Hz, 1H), 3.95 (s, 2H), 2.64 (br s, 1H), 2.46 (s, 3H), 1.62 (s, 3H). To a solution of 2-(3-chloro-4-nitro-1H-pyrazol-1-yl)-3-hydroxy-2-methylpropyl 4- 10 methylbenzenesulfonate (1.1 g, 2.8 mmol) in THF (20 mL) was added NaH (170 mg, 4.25 mmol, 60% in mineral oil) at 0 ºC and the reaction was stirred at 70 ºC for 2 h. The mixture was quenched with H2O (20 mL) at 0 ºC and extracted with EtOAc (2×20 mL). The combined organic phases were washed with brine (20 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 15 → 60:40) to afford 3-chloro-1-(3-methyloxetan-3-yl)-4-nitro-1H-pyrazole (360 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.32 (s, 1H), 5.13 (d, J = 6.4 Hz, 2H), 4.68 (d, J = 6.8 Hz, 2H), 1.99 (s, 3H). To a solution of trimethylsulfoxonium iodide (1.76 g, 8.00 mmol) in DMSO (7 mL) was added t-BuOK (897 mg, 7.99 mmol) at 25 ºC and the reaction was stirred at 25 ºC for 1 h. Then a 20 solution of 3-chloro-1-(3-methyloxetan-3-yl)-4-nitro-1H-pyrazole (870 mg, 4.00 mmol) in DMSO (3 mL) was added dropwise at 35 ºC and the mixture was stirred at 35 ºC for 12 h. The mixture was diluted with H2O (10 mL) and extracted with EtOAc (4×10 mL). The organic phase was washed with brine (10 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 →25 60:40) to afford 3-chloro-5-methyl-1-(3-methyloxetan-3-yl)-4-nitro-1H-pyrazole (605 mg) of suf- ficient purity for the subsequent step. LC-MS (method B) (m/z) = 232.0 (MH)+ tR = 0.69 minutes. A mixture of 3-chloro-5-methyl-1-(3-methyloxetan-3-yl)-4-nitro-1H-pyrazole (600 mg, 2.59 mmol) in a solution of NaOH (1.20 g, 30 mmol) in H2O (15 mL) was stirred at 100 ºC for 4 h. The reaction was cooled to room temperature (20 ºC) and acidified with saturated aqueous30 KHSO4 to pH 5. The solid was filtered, washed with H2O (2×1 mL) and dried to afford 5-methyl- 1-(3-methyloxetan-3-yl)-4-nitro-1H-pyrazol-3-ol (405 mg) of sufficient purity for the subsequent step, which was used in the next step directly.1H NMR (CDCl3, 400MHz) δ 8.40 (br s, 1H), 5.19 (d, J = 6.8 Hz, 2H), 4.59 (d, J = 7.2 Hz, 2H), 2.46 (s, 3H), 1.87 (s, 3H). 5-methyl-1-(3-methyloxetan-3-yl)-4-nitro-1H-pyrazol-3-ol (150 mg, 0.7 mmol), 3-(3,6- dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propyl 4-methylbenzenesulfonate (283 mg, 0.71 mmol, prepared as described previously) and K2CO3 (292 mg, 2.11 mmol) were taken up into a sealed tube in DMF (3 mL) and the reaction was stirred at 85 ºC for 2 h. The mixture was diluted 5 with H2O (3 mL) and extracted with EtOAc (2×3 mL). The organic phase was washed with brine (2×3 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was puri- fied by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 50:50) to afford 3,6-dichloro-1-(3-((5-methyl-1-(3-methyloxetan-3-yl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H- pyrazolo[3,4-d]pyrimidine (201 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 10 400MHz) δ 8.96 (s, 1H), 5.11 (d, J = 6.4 Hz, 2H), 4.65 (t, J = 6.4 Hz, 2H), 4.54 (d, J = 7.2 Hz, 2H), 4.31 (t, J = 5.6 Hz, 2H), 2.51-2.44 (m, 2H), 2.41 (s, 3H), 1.80 (s, 3H). Intermediate: 3,6-Dichloro-1-(3-((5-(methyl-d3)-4-nitro-1-(tetrahydro-2H-pyran-4-yl-4-d)-1H- pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine 15
Figure imgf000115_0001
A mixture of dihydro-2H-pyran-4(3H)-one (17 g, 170 mmol) in THF (200 mL) was de- gassed and purged with N2 (×3), lithium aluminum deuteride (9.67 g, 255 mmol) was added por- tionwise at 0 ºC and then the mixture was stirred at 0 ºC for 1 h. The reaction mixture was quenched with water (10 mL), followed by aqueous 10% NaOH (30 mL) and water (10 mL). The 20 mixture was filtered, and the filtrate was concentrated to afford tetrahydro-2H-pyran-4-d-4-ol (17.5 g) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 400MHz) δ 3.99-3.91 (m, 2H), 3.48-3.39 (m, 2H), 1.94-1.86 (m, 2H), 1.61-1.49 (m, 2H). To a solution of tetrahydro-2H-pyran-4-d-4-ol (15 g, 145 mmol), TEA (22.08 g, 218 mmol) and N,N-dimethylmethanamine hydrochloride (1.39 g, 14.5 mmol) in DCM (450 mL) was added 25 TsCl (33.3 g, 175 mmol) at 0 ºC. The mixture was stirred at 20 ºC for 17 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on sil- ica gel (eluent: petroleum ether:EtOAc 100:0 → 80:20) to afford tetrahydro-2H-pyran-4-yl-4-d 4-methylbenzenesulfonate (30.9 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 7.81 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 3.93-3.80 (m, 2H), 3.52-3.43 (m, 2H), 5 2.45 (s, 3H), 1.89-1.82 (m, 2H), 1.79-1.70 (m, 2H). To a solution of 5-chloro-3-methyl-4-nitro-1H-pyrazole (16 g, 99.0 mmol) and tetrahy- dro-2H-pyran-4-yl-4-d 4-methylbenzenesulfonate (30.9 g, 120 mmol) in DMF (300 mL) was added K2CO3 (41.1 g, 297 mmol). The mixture was stirred at 80 ºC for 20 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on sil-10 ica gel (eluent: petroleum ether:EtOAc 100:0 → 80:20) three times to afford 3-chloro-5-methyl- 4-nitro-1-(tetrahydro-2H-pyran-4-yl-4-d)-1H-pyrazole (11 g) of sufficient purity for the subse- quent step.1H NMR (CDCl3, 400MHz) δ 4.18-4.12 (m, 2H), 3.57-3.49 (m, 2H), 2.70 (s, 3H), 2.38- 2.28 (m, 2H), 1.85-1.79 (m, 2H). To a solution of 3-chloro-5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl-4-d)-1H-pyra- 15 zole (11 g, 44.6 mmol) in H2O (150 mL) was added KOH (25 g, 446 mmol). The mixture was stirred at 140 ºC for 22 h. The mixture was acidified with aqueous HCl (12 M), pH=3. The precipitate was filtered. The filter cake was dried to afford 5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl-4-d)- 1H-pyrazol-3-ol (8 g). The filtrate was extracted with EtOAc (3×300 mL). The combined organic layers were washed with brine (3×300 mL), dried over Na2SO4, and concentrated to afford 5-20 methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl-4-d)-1H-pyrazol-3-ol (2 g). The two crops of mate- rial were combined to afford 5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl-4-d)-1H-pyrazol-3-ol (10 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 11.37 (br s, 1H), 3.97-3.90 (m, 2H), 3.50-3.42 (m, 2H), 2.59 (s, 3H), 1.99-1.89 (m, 2H), 1.79-1.70 (m, 2H). To a solution of 5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl-4-d)-1H-pyrazol-3-ol (6.0 25 g, 26 mmol) in DMSO-d6 (240 mL) was added t-BuOK (3.25 g, 28.9 mmol). The mixture was stirred at 30 ºC for 64 h. The mixture was diluted with deuterium oxide (100 mL) and adjusted to pH=3 with aqueous HCl (2M). The aqueous phase was extracted with EtOAc (6×300 mL). The combined organic layers were washed with brine (2×200 mL), dried over Na2SO4, and concentrated. Water (100 mL) was added to the residue and the mixture was lyophilized to afford 5-(methyl-d3)-4-30 nitro-1-(tetrahydro-2H-pyran-4-yl-4-d)-1H-pyrazol-3-ol (5 g) of sufficient purity for the subse- quent step. 1H NMR (CDCl3, 400MHz) δ 11.36 (br s, 1H), 3.97-3.89 (m, 2H), 3.51-3.41 (m, 2H), 1.99-1.89 (m, 2H), 1.79-1.69 (m, 2H). LC-MS: tR = 1.653 min (Method D), m/z = 232.1 [M + H]+. To a solution of 3-(3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propyl 4-methylben- zenesulfonate (760 mg, 1.89 mmol, prepared as described previously) in DMSO-d6 (20 mL) was added K2CO3 (288 mg, 2.08 mmol) and 5-(methyl-d3)-4-nitro-1-(tetrahydro-2H-pyran-4-yl-4-d)- 1H-pyrazol-3-ol (438 mg, 1.89 mmol). The mixture was stirred at 80 ºC for 1 h. The reaction mix- 5 ture was diluted with aqueous HCl (2 M), pH=6~7, and extracted with EtOAc (3×30 mL). The combined organic layers were washed with brine (3×40 mL), dried over Na2SO4, and concen- trated under reduced pressure. The residue was purified by chromatography on silica gel (elu- ent: petroleum ether:EtOAc 100:0 → 50:50) to afford 3,6-dichloro-1-(3-((5-(methyl-d3)-4-nitro- 1-(tetrahydro-2H-pyran-4-yl-4-d)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine 10 (540 mg) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 400MHz) δ 8.94 (s, 1H), 4.64 (t, J = 6.4 Hz, 2H), 4.34 (t, J = 6.0 Hz, 2H), 4.10-4.05 (m, 2H), 3.53-3.44 (m, 2H), 2.50-2.42 (m, 2H), 2.22-2.12 (m, 2H), 1.76-1.67 (m, 2H). Intermediate: 3,6-Dichloro-1-(3-((5-(1-fluorocyclopropyl)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-15 1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000117_0001
A solution of DIAD (576 µL, 2.96 mmol) in THF (60 mL) was cooled to 0 ºC under an at- mosphere of argon. PPh3 (~3 mmol/g on resin) (777 mg, 2.96 mmol, employed 1.0 g resin) was added in portions and the mixture was stirred for 15 minutes.5-Cyclopropyl-4-nitro-1-(tetrahy- 20 dro-2H-pyran-4-yl)-1H-pyrazol-3-ol (500 mg, 1.97 mmol, prepared as described previously) was then added followed by a solution of 3-((tert-butyldimethylsilyl)oxy)propan-1-ol (376 mg, 1.97 mmol) in THF (1 mL). The cooling bath was removed and the reaction mixture was stirred at room temperature for 3 h. The mixture was filtered and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:025 → 65:35) to afford 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-cyclopropyl-4-nitro-1-(tetrahy- dro-2H-pyran-4-yl)-1H-pyrazole (518 mg) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 600MHz) δ 4.66 - 4.59 (m, 1H), 4.38 (t, J = 6.3 Hz, 2H), 4.15-4.09 (m, 2H), 3.79 (t, J = 6.1 Hz, 2H), 3.55-3.48 (m, 2H), 2.34-2.25 (m, 2H), 2.04-1.98 (m, 2H), 1.85-1.79 (m, 1H), 1.74- 1.68 (m, 2H), 1.26-1.21 (m, 2H), 0.88 (s, 9H), 0.78-0.73 (m, 2H), 0.04 (s, 6H). A solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-cyclopropyl-4-nitro-1-(tetra- hydro-2H-pyran-4-yl)-1H-pyrazole (318 mg, 0.747 mmol), lithium chloride (158 mg, 3.74 mmol, 5 dried overnight at 90 ºC in vacuum oven) and N-Fluorobis(phenylsulfonyl)amine (530 mg, 1.68 mmol) in THF (20 mL) was cooled to -78 ºC under an atmosphere of argon. LDA (1.5M in hex- anes) (1.74 mL, 2.62 mmol) was added dropwise by syringe to form a dark orange solution. Im- mediately following, was added a solution of N-Fluorobis(phenylsulfonyl)amine (707 mg, 2.24 mmol) in THF (2.5 mL) over the course of 20 seconds. The orange color was quenched during 10 the addition. The mixture was stirred for 5 min. The mixture was quenched with water (~0.1 mL) and was concentrated to dryness. The residue was suspended DCM, filtered, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: pe- troleum ether:EtOAc 100:0 → 50:50) to afford 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-(1- fluorocyclopropyl)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole (204 mg) of sufficient pu- 15 rity for the subsequent step. 1H NMR (CDCl3, 500MHz) δ 4.78-4.70 (m, 1H), 4.43 (t, J = 6.3 Hz, 2H), 4.17-4.10 (m, 2H), 3.80 (t, J = 6.0 Hz, 2H), 3.57-3.49 (m, 2H), 2.39-2.28 (m, 2H), 2.05-1.98 (m, 2H), 1.83-1.76 (m, 2H), 1.74-1.65 (m, 2H), 1.14-1.07 (m, 2H), 0.88 (s, 9H), 0.05 (s, 6H). To a dry flask was added 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-(1-fluorocyclo- propyl)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole (204 mg, 0.460 mmol), DCM (20 20 mL) and HCl (3M in diethyl ether) (1.61 mL, 3.22 mmol). The flask was flushed with argon and stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 0:100) to afford 3-((5-(1-fluorocyclopropyl)-4-nitro-1-(tetrahydro-2H-py- ran-4-yl)-1H-pyrazol-3-yl)oxy)propan-1-ol (108 mg) of sufficient purity for the subsequent step. 25 1H NMR (CDCl3, 600MHz) δ 4.78 - 4.71 (m, 1H), 4.52 (t, J = 5.9 Hz, 2H), 4.17 - 4.11 (m, 2H), 3.86 (t, J = 5.6 Hz, 2H), 3.57-3.51 (m, 2H), 2.38-2.28 (m, 2H), 2.22 (br s, 1H), 2.11-2.05 (m, 2H), 1.84- 1.77 (m, 2H), 1.73-1.66 (m, 2H), 1.15-1.08 (m, 2H). A solution of DIAD (121 µL, 0.623 mmol) in THF (15 mL) was cooled to 0 ºC under an atmosphere of argon. PPh3 (on resin, ~3 mmol/g) (163 mg, 0.623 mmol, 207 mg resin used), was 30 added and the mixture was stirred for 2 minutes.3,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidine (77.5 mg, 0.410 mmol) was then added followed by a solution of 3-((5-(1-fluorocyclopropyl)-4- nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propan-1-ol (108 mg, 1.0 Eq, 328 µmol) in THF (1 mL). The cooling bath was removed and the reaction mix- ture was stirred for 2 h. The reaction mixture was filtered and the filtrate was concentrated un- der reduced pressure. The residue was purified by chromatography on silica gel (eluent: petro- leum ether:EtOAc 100:0 → 0:100) to afford 3,6-dichloro-1-(3-((5-(1-fluorocyclopropyl)-4-nitro-1- 5 (tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (129 mg) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 600MHz) δ 8.95 (s, 1H), 4.75- 4.68 (m, 1H), 4.65 (t, J = 6.4 Hz, 2H), 4.41-4.36 (m, 2H), 4.13-4.08 (m, 2H), 3.56-3.49 (m, 2H), 2.50-2.44 (m, 2H), 2.27-2.18 (m, 2H), 1.81-1.75 (m, 2H), 1.73-1.66 (m, 2H), 1.15-1.09 (m, 2H). 10 Intermediate: 3,6-Dichloro-1-(3-((5-ethyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3- yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000119_0001
To a dry flask were added 3-bromo-4-nitro-1H-pyrazole (14.00 g, 72.93 mmol) and DMF 15 (anhydrous) (150 mL). Cs2CO3 (59.4 g, 182 mmol) was added followed by 4-bromooxane (24.07 g, 146 mmol). The mixture was stirred at 80 ºC for 16 h. The mixture was cooled to room tem- perature and water (120 mL) and EtOAc (150 mL) were added. The phases were separated, and the organic phase was washed with aqueous NH4Cl (2 x 150 mL), dried over Na2SO4, and concen- trated under reduced pressure. The residue was purified by chromatography on silica gel (elu- 20 ent: petroleum ether:EtOAc 83:17) to afford 3-bromo-4-nitro-1-(oxan-4-yl)pyrazole (9.49 g) of sufficient purity for the subsequent step. A mixture of 3-bromo-4-nitro-1-(oxan-4-yl)pyrazole (8.44 g, 30.6 mmol) and KOH (33.76 g, 601.7 mmol) in H2O (330 mL) was stirred at 100 ºC for 16 hours. The mixture was acidified to pH = 3 with HCl (aq.), extracted with EtOAc (130 mLx3) and concentrated under reduced pressure. 25 The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 33:67) to afford 4-nitro-1-(oxan-4-yl)pyrazol-3-ol (5 g) of sufficient purity for the subsequent step. To a stirred solution of 4-nitro-1-(oxan-4-yl)pyrazol-3-ol (5 g, 23 mmol) and 3-[(tert-butyl- dimethylsilyl)oxy]propan-1-ol (8.93 g, 46.9 mmol) in THF (100 mL) was added PPh3 (12.30 g, 46.9 mmol) in one portion at room temperature under argon atmosphere. Then DIAD (9.48 g, 46.9 mmol) was added at 0 ºC. The resulting mixture was stirred for 2 h at room temperature. The 5 reaction was quenched with water (15 mL) and extracted with EtOAc (2 x 25 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 83:17) to afford 3- {3-[(tert-butyldimethylsilyl)oxy]propoxy}-4-nitro-1-(oxan-4-yl)pyrazole (6 g) of sufficient purity for the subsequent step. 10 To a stirred solution of 3-{3-[(tert-butyldimethylsilyl)oxy]propoxy}-4-nitro-1-(oxan-4-yl)py- razole (500 mg, 1.30 mmol) and 15-crown-5 (3.43 g, 15.6 mmol) in THF (25 mL) was added LDA (6.48 mL, 13.0 mmol, 2 M) dropwise at -78 ºC. The mixture was stirred for 60 minutes at -78 ºC. Then ethyl iodide (1.01 g, 6.49 mmol) was added and the resulting mixture was stirred for 2 h at -78ºC. The reaction was quenched with aqueous NH4Cl (25 mL) and the resulting mixture was 15 extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative TLC (eluent: petroleum ether:EtOAc 83:17) to afford 3-{3-[(tert-butyldimethylsilyl)oxy]propoxy}-5-ethyl-4-ni- tro-1-(oxan-4-yl)pyrazole (320 mg) of sufficient purity for the subsequent step. To a stirred solution of 3-{3-[(tert-butyldimethylsilyl)oxy]propoxy}-5-ethyl-4-nitro-1-(oxan- 20 4-yl)pyrazole (50 mg, 0.12 mmol) in DCM (0.8 mL) was added HCl in 1,4-dioxane (0.10 mL, 0.40 mmol, 4 M) dropwise at room temperature. The resulting mixture was stirred for 2 h at room temperature. The residue was purified by preparative TLC (eluent: petroleum ether:EtOAc 33:67) to afford 3-{[5-ethyl-4-nitro-1-(oxan-4-yl)pyrazol-3-yl]oxy}propan-1-ol (23 mg) of suffi- cient purity for the subsequent step. 25 To a stirred mixture of 3-{[5-ethyl-4-nitro-1-(oxan-4-yl)pyrazol-3-yl]oxy}propan-1-ol (170 mg, 0.57 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (215 mg, 1.14 mmol) and PPh3 (298 mg, 1.14 mmol) in THF (12 mL) was added DIAD (230 mg, 1.14 mmol) dropwise at 0 ºC under argon atmosphere. The resulting mixture was stirred for 2 h at room temperature and concentrated under reduced pressure. The residue was purified by preparative TLC (eluent: pe-30 troleum ether:EtOAc 80:20) to afford 3,6-dichloro-1-(3-((5-ethyl-4-nitro-1-(tetrahydro-2H-py- ran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine desired product (230 mg) of sufficient purity for the subsequent step. Intermediate: 3-(3-((tert-Butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyrazole
Figure imgf000121_0001
To a solution of 5-methyl-4-nitro-1H-pyrazole (12 g, 94 mmol) in THF (140 mL) was 5 added TsOH·H2O (898 mg, 4.7 mmol) and the reaction was stirred at 20 ºC for 30 minutes. The reaction was cooled to 0 ºC and 3,4-dihydro-2H-pyran (10.2 g, 121 mmol) was added. The mix- ture was stirred at 20 ºC for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 80:20) to afford 3-methyl-4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (12 g) of sufficient10 purity for the subsequent step. To a solution of 3-methyl-4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (12 g, 57 mmol) in THF (100 mL) was added LiHMDS (1 M in THF, 62.5 mL) at -65 ºC and stirred at -65 ºC for 30 minutes. Then 1,1,1,2,2,2-hexachloroethane (14.80 g, 62.5 mmol) dissolved in THF (50 mL) was added and the mixture was stirred at -65 ºC for 60 minutes and then warmed to 20 ºC 15 for 10 minutes. The mixture was quenched with saturated aqueous NH4Cl (200 mL) at 0 ºC and then warmed to 20 ºC. After 20 min stirring the mixture was extracted with EtOAc (3×150 mL). The combined organic layers were washed with brine (80 mL) and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 90:10) to afford 5-chloro-3-methyl-4-nitro-1-tetrahydropyran-2-yl-pyra-20 zole (7.9 g) of sufficient purity for the subsequent step. To a solution of 5-chloro-3-methyl-4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (19 g, 77 mmol) in DMA (150 mL) was added CsF (41.1 g, 271 mmol) and propane-1,3-diol (29.4 g, 387 mmol). The mixture was stirred at 50 ºC for 16 h. The mixture was added water (200 mL) and extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine25 (3×100 mL), and concentrated under reduced pressure. The residue was purified by chromatog- raphy on silica gel (eluent: petroleum ether:EtOAc 100:0 → 69:31) to afford 3-((3-methyl-4-nitro- 1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)oxy)propan-1-ol (13.7 g) of sufficient purity for the subsequent step. To a solution of 3-((3-methyl-4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5- yl)oxy)propan-1-ol (4 g, 14 mmol) in MeOH (20 mL) was added aqueous HCl (12 M, 14 mL). The 5 mixture was stirred at 20 ºC for 16 h and then heated to 60 ºC for 2 h. The mixture was concen- trated under reduced pressure and pH adjusted to 10 with 4M aqueous NaOH. The resulting mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 80:20) to afford 3-((5-methyl-4-nitro-1H- pyrazol-3-yl)oxy)propan-1-ol (2.7 g) of sufficient purity for the subsequent step.1H NMR (DMSO- 10 d6, 400MHz) δ 12.94 (br s, 1H), 4.55 (t, J = 4.8 Hz, 1H), 4.28 (t, J = 6.4 Hz, 2H), 3.60 - 3.49 (m, 2H), 2.47 (s, 3H), 1.92 - 1.82 (m, 2H). To a solution of 3-((5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propan-1-ol (2.7 g, 13 mmol) in DCM (30 mL) was added imidazole (1.46 g, 21.5 mmol), TBSCl (4.05 g, 26.8 mmol) and DMAP (820 mg, 6.71 mmol). The mixture was stirred at 20 ºC for 16 h. The mixture was concentrated15 under reduced pressure. The residue was purified by chromatography on silica gel (eluent: pe- troleum ether:EtOAc 100:0 → 79:21 to afford 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-me- thyl-4-nitro-1H-pyrazole (3.7 g) of sufficient purity for the subsequent step. 1H NMR (CD3OD, 400MHz) δ 4.40 (t, J = 6.4 Hz, 2H), 3.84 (t, J = 6.0 Hz, 2H), 2.61 (s, 3H), 2.03 (t, J = 6.0 Hz, 2H), 0.89 (s, 9H), 0.05 (s, 6H). 20 Intermediate: 3-(3-((tert-Butyldimethylsilyl)oxy)propoxy)-5-ethyl-4-nitro-1H-pyrazole 5
Figure imgf000122_0001
To a solution of 5-ethyl-4-nitro-1H-pyrazole (12.5 g, 88.6 mmol) in THF (150 mL) was added TsOH·H2O (843 mg, 4.43 mmol). The mixture was stirred at 25 ºC for 15 minute and then 25 3,4-dihydro-2H-pyran (9.54 g, 113 mmol) was added at 0 ºC. The mixture was stirred at 25 ºC for 16 h. The mixture was concentrated. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 90:10) to afford 3-ethyl-4-nitro-1-tetrahydropyran-2- yl-pyrazole (19 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.33 (s, 1H), 5.34 - 5.30 (m, 1H), 4.13 - 4.06 (m, 1H), 3.76 - 3.66 (m, 1H), 2.98 (q, J = 7.6 Hz, 2H), 2.19 - 2.09 (m, 1H), 2.02 - 1.96 (m, 1H), 1.77 - 1.63 (m, 4H), 1.28 (t, J = 7.6 Hz, 3H). 5 To a solution of 3-ethyl-4-nitro-1-tetrahydropyran-2-yl-pyrazole (19 g, 84.4 mmol) in THF (80 mL) was added LiHMDS (1 M in THF, 92.8 mL) at -65 ºC and stirred at -65 ºC for 30 minutes. Then 1,1,1,2,2,2-hexachloroethane (22 g, 93 mmol) dissolved in THF (60 mL) was added and the mixture was stirred at -65 ºC for 2 h and then warmed to 20 ºC for 10 minutes. The mixture was quenched with saturated aqueous NH4Cl solution (200 mL) at 0 ºC then warmed to 10 20 ºC for 20 minutes and extracted with EtOAc (3×200 mL). The combined organic layers were washed with brine (3×80 mL) and concentrated under reduced pressure. The residue was puri- fied by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 95:5) to afford 5- chloro-3-ethyl-4-nitro-1-tetrahydropyran-2-yl-pyrazole (17 g) of sufficient purity for the subse- quent step.1H NMR (CDCl3, 400MHz) δ 5.52 (dd, J = 2.8, 10.0 Hz, 1H), 4.13 - 4.03 (m, 1H), 3.76 - 15 3.64 (m, 1H), 2.98(q, J = 7.2 Hz, 2H), 2.51 - 2.36 (m, 1H), 2.22 - 2.10 (m, 1H), 1.95 - 1.85 (m, 1H), 1.81 - 1.60 (m, 3H), 1.29 (t, J = 7.2 Hz, 3H). To a solution of 5-chloro-3-ethyl-4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (12 g, 46 mmol) and propane-1,3-diol (17.58 g, 231.1 mmol) in DMA (150 mL) was added CsF (21.06 g, 138.6 mmol). The mixture was stirred at 50 ºC for 16 h. The mixture was diluted with water (200 20 mL) and extracted with EtOAc (4×150 mL). The combined organic layers were washed with brine (4×100 mL) and concentrated under reduced pressure. The residue was purified by chromatog- raphy on silica gel (eluent: petroleum ether:EtOAc 100:0 → 60:40) to afford 3-((3-ethyl-4-nitro- 1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)oxy)propan-1-ol (14 g) of sufficient purity for the subsequent step. 25 To a solution of 3-((3-ethyl-4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5- yl)oxy)propan-1-ol (14 g, 47 mmol) in MeOH (140 mL) was added concentrated aqueous HCl (24 mL, 12 M). The mixture was stirred at 60 ºC for 3 h. The mixture was cooled to 0 ºC, pH was adjusted to 8 using NaOH and then the mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 30 10:90) to afford 3-((5-ethyl-4-nitro-1H-pyrazol-3-yl)oxy)propan-1-ol (8.5 g) of sufficient purity for the subsequent step.1H NMR (DMSO-d6, 400MHz) δ 12.95 (br s, 1H), 4.54 (br, 1H), 4.29 (t, J = 6.8 Hz, 2H), 3.54 (t, J = 6.0 Hz, 2H), 2.89 (q, J = 7.6 Hz, 2H), 1.92 - 1.83 (m, 2H), 1.20 (t, J = 7.6 Hz, 3H). To a solution of 3-((5-ethyl-4-nitro-1H-pyrazol-3-yl)oxy)propan-1-ol (8.5 g, 40 mmol) in DCM (70 mL) was added DMAP (1.45 g, 11.9 mmol) and imidazole (4.30 g, 63.2 mmol), and TBSCl (11.9 g, 79.0 mmol). The mixture was stirred at 25 ºC for 16 h. The mixture was con- centrated under reduced pressure. The residue was purified by chromatography on silica gel (el- 5 uent: petroleum ether:EtOAc 100:0 → 10:90) to afford 3-(3-((tert-butyldimethylsi- lyl)oxy)propoxy)-5-ethyl-4-nitro-1H-pyrazole 5 (8.5 g) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 400MHz) δ 4.40 (t, J = 6.4 Hz, 2H), 3.83 (t, J = 6.0 Hz, 2H), 3.04 (q, J = 7.6 Hz, 2H), 2.09 - 1.99 (m, 2H), 1.33 (t, J = 7.6 Hz, 3H), 0.88 (s, 9H), 0.05 (s, 6H). 10 Intermediate: 3-(3-((tert-Butyldimethylsilyl)oxy)propoxy)-4-nitro-1H-pyrazole
Figure imgf000124_0001
To a solution of 4-nitro-1H-pyrazole (10 g, 88 mmol) in THF (100 mL) was added 3,4-di- hydro-2H-pyran (22.3 g, 265 mmol) and TsOH·H2O (841 mg, 4.4 mmol). The mixture was stirred at 20 ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The residue 15 was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 90:10) to afford 4-nitro-1-tetrahydropyran-2-yl-pyrazole (17 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.37 (s, 1H), 8.10 (s, 1H), 5.41 (dd, J = 9.2 Hz, 2.8Hz, 1H), 4.10 - 4.06 (m, 1H), 3.80 – 3.70 (m, 1H), 2.16 -2.10 (m, 1H), 2.05 - 1.90 (m, 2H), 1.80 - 1.60 (m, 3H). To a solution of 4-nitro-1-tetrahydropyran-2-yl-pyrazole (15 g, 76 mmol) in THF (150 20 mL) was added dropwise LiHMDS (1 M in THF, 83.7 mL) at -78 ºC. After addition, the mixture was stirred at this temperature for 0.5 h, and then 1,1,1,2,2,2-hexachloroethane (19.81 g, 83.7 mmol) in THF (50 mL) was added dropwise at -78 ºC. The resulting mixture was stirred at 20 ºC for 1.5 h. The reaction mixture was quenched with saturated aqueous NH4Cl (100 mL) at 0 ºC, extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine (3×100 25 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 90:10) to afford 5-chloro- 4-nitro-1-tetrahydropyran-2-yl-pyrazole (11.4 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.21 (s, 1H), 5.57 (dd, J = 2.8, 9.6 Hz, 1H), 4.15 - 3.98 (m, 1H), 3.77 - 3.64 (m, 1H), 2.50 - 2.35 (m, 1H), 2.24 - 2.10 (m, 1H), 1.99 - 1.91 (m, 1H), 1.80 – 1.60 (m, 2H), 1.60 - 1.52 (m, 1H). To a solution of 5-chloro-4-nitro-1-tetrahydropyran-2-yl-pyrazole (10 g, 43 mmol) in DMA (150 mL) was added CsF (26.23 g, 172.7 mmol) and propane-1,3-diol (16.42 g, 5 215.9 mmol). The mixture was stirred at 50 ºC for 15 h. The reaction mixture was diluted with water (100 mL), extracted with EtOAc (3×200 mL). The combined organic layers were washed with brine (5×300 mL), dried over Na2SO4, and concentrated under reduced pressure. The resi- due was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 60:40) to afford 3-((4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)oxy)propan-1-ol (6.7 g)10 of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.03 (s, 1H), 5.45 (dd, J = 2.8, 9.2 Hz, 1H), 4.68 - 4.62 (m, 1H), 4.55 - 4.48 (m, 1H), 4.10 - 4.03 (m, 1H), 3.92 (t, J = 6.0 Hz, 2H), 3.74 - 3.65 (m, 1H), 2.47 - 2.29 (m, 1H), 2.18 - 2.13 (m, 1H), 2.13 - 2.07 (m, 2H), 1.76 - 1.57 (m, 4H). To a solution of 3-((4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)oxy)propan-1- 15 ol (19.8 g, 73.0 mmol) in MeOH (200 mL) was added concentrated aqueous HCl (12 M, 73 mL). The mixture was stirred at 60 ºC for 16 h. The mixture was concentrated under reduced pressure and pH adjusted to 10 with 4M aqueous NaOH. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc (10 v% MeOH) 100:0 → 50:50) to afford 3-((4-nitro-1H-pyrazol-5-yl)oxy)propan-1-20 ol (10.5 g) of sufficient purity for the subsequent step. LC-MS (method K) (m/z) = 187.7 (MH)+ tR = 0.96 minutes. To a solution of 3-((4-nitro-1H-pyrazol-5-yl)oxy)propan-1-ol (10.5 g, 56.1 mmol), imidaz- ole (6.11 g, 89.8 mmol) and DMAP (3.43 g, 28.1 mmol) in DCM (200 mL) was added TBSCl (16.91 g, 112.2 mmol). The mixture was stirred at 20 ºC for 12 hours. The mixture was concentrated. 25 The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc (10 v% MeOH) 100:0 → 50:50) to afford 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-4-nitro-1H-pyrazole (13.6 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.20 (s, 1H), 4.46 (t, J = 6.0 Hz, 2H), 3.86 (t, J = 5.6 Hz, 2H), 2.11 - 2.07 (m, 2H), 0.91 (s, 9H), 0.11 - 0.03 (m, 6H). 30 Intermediate: cis-3,6-Dichloro-1-(3-((5-methyl-1-(2-methyltetrahydro-2H-pyran-4-yl)-4-nitro- 1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000126_0001
A solution of DIAD (5.22 g, 25.8 mmol) was added to a solution of cis-2-methyltetrahy- dro-2H-pyran-4-ol (2 g, 17.2 mmol), 4-nitrobenzoic acid (3.31 g, 19.8 mmol) and PPh3 (6.77 g, 25.8 mmol) in THF (50 mL) at 0 ºC. The mixture was stirred at 20 ºC for 15 h. The reaction mixture 5 was concentrated under reduced pressure. The residue was purified by chromatography on sil- ica gel (eluent: petroleum ether:EtOAc 100:0 → 90:10) to afford trans-2-methyltetrahydro-2H- pyran-4-yl 4-nitrobenzoate (6.5 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.32 (d, J = 6.8 Hz, 2H), 8.24 (d, J = 6.8 Hz, 2H), 5.52 - 5.41 (m, 1H), 3.99 - 3.84 (m, 3H), 2.04 - 1.91 (m, 2H), 1.90 - 1.81 (m, 1H), 1.71 - 1.63 (m, 1H), 1.22 (d, J = 6.4 Hz, 3H). 10 To a solution of trans-2-methyltetrahydro-2H-pyran-4-yl 4-nitrobenzoate (6.5 g, 24 mmol) in THF (50 mL) and MeOH (50 mL) was added aqueous NaOH (2 M, 37 mL). The mixture was stirred at 20 ºC for 2 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 40:60) to afford trans-2-methyltetrahydro-2H-pyran-4-ol (1.3 g) of sufficient purity for the sub- 15 sequent step.1H NMR (CDCl3, 400MHz) δ 4.25 - 4.19 (m, 1H), 3.93 - 3.84 (m, 2H), 3.81 - 3.74 (m, 1H), 1.89 - 1.80 (m, 1H), 1.66 - 1.63 (m, 1H), 1.58 - 1.50 (m, 2H), 1.15 (d, J = 6.4 Hz, 3H). To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyra- zole (1 g, 3.17 mmol) in toluene (20 mL) was added trans-2-methyltetrahydro-2H-pyran-4-ol (553 mg, 4.76 mmol) and 2-(tributyl-λ5-phosphanylidene)acetonitrile (3.06 g, 12.68 mmol). The 20 mixture was stirred at 110 ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 40:60) to afford cis-3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl- 1-(2-methyltetrahydro-2H-pyran-4-yl)-4-nitro-1H-pyrazole (1 g) of sufficient purity for the sub- sequent step. LC-MS (method B) (m/z) = 414.3 (MH)+ tR = 1.10 minutes. 25 A solution of cis-3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-1-(2-methyltetra- hydro-2H-pyran-4-yl)-4-nitro-1H-pyrazole (1.0 g, 2.4 mmol) in HCl/1,4-dioxane (4 M, 10 mL) was stirred at 20 ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 50:50) to afford cis-3-((5-methyl-1-(2-methyltetrahydro-2H-pyran-4-yl)-4-nitro-1H-pyrazol-3- yl)oxy)propan-1-ol (430 mg) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 5 400MHz,) δ 4.50 (t, J = 6.0 Hz, 2H), 4.29 - 4.19 (m, 1H), 4.13 - 4.05 (m, 1H), 3.85 (t, J = 5.2 Hz, 2H), 3.61 - 3.51 (m, 2H), 2.65 (s, 3H), 2.61 - 2.49 (m, 1H), 2.30 - 2.16 (m, 1H), 2.12 - 2.06 (m, 2H), 2.01 - 1.89 (m, 1H), 1.84 - 1.70 (m, 2H), 1.28 (d, J = 6.0 Hz, 3H). To a solution of cis-3-((5-methyl-1-(2-methyltetrahydro-2H-pyran-4-yl)-4-nitro-1H-pyra- zol-3-yl)oxy)propan-1-ol (412 mg, 1.38 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (260 10 mg, 1.38 mmol), and PPh3 (1.08 g, 4.13 mmol) in THF (20 mL) was added DIAD (835 mg, 4.13 mmol) at 0 ºC. The mixture was stirred at 20 ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: pe- troleum ether:EtOAc 100:0 → 60:40) to afford cis-3,6-dichloro-1-(3-((5-methyl-1-(2-methyltet- rahydro-2H-pyran-4-yl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (500 15 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.95 (s, 1H), 4.65 (t, J = 6.4 Hz, 2H), 4.35 (t, J = 6.0 Hz, 2H), 4.25 - 4.15 (m, 1H), 4.12 - 4.07 (m, 1H), 3.61 - 3.48 (m, 2H), 2.62 (s, 3H), 2.52 - 2.42 (m, 2H), 2.20 - 2.08 (m, 1H), 1.91 - 1.80 (m, 1H), 1.79 - 1.73 (m, 1H), 1.72 - 1.67 (m, 1H), 1.27 (d, J = 6.0 Hz, 3H). 20 Intermediate: 3,6-Dichloro-1-(3-((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3- yl)oxy)propyl-1,1,2,2,3,3-d6)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000127_0001
To a stirred mixture of 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (200 mg, 1.05 mmol) and propane-d6-1,3-diol (130 mg, 1.58 mmol) in THF (2 mL) were added PPh3 (555 mg, 2.11 mmol) 25 and DIAD (427 mg, 2.11 mmol) portionwise at 0 ºC under an argon atmosphere. The resulting mixture was stirred for 2 h at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 50:50) to afford 3-(3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propan- 1,1,2,2,3,3-d6-1-ol (124.8 mg) of sufficient purity for the subsequent step. 1H NMR (DMSO-d6, 400 MHz) δ 9.30 (s, 1H), 4.53 (s, 1H). To a stirred mixture of 3-(3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propan- 1,1,2,2,3,3-d6-1-ol (700 mg, 2.76 mmol) and 5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H- 5 pyrazol-3-ol (754 mg, 3.31 mmol) in THF (10 mL)^were added PPh3 (1.45 g, 5.53 mmol) and DIAD (1.12 g, 5.53 mmol)^at 0^ºC under an argon atmosphere. The resulting mixture was stirred for 2 h at room temperature. The residue was purified by chromatography on silica gel (eluent: pe- troleum ether:EtOAc 50:50) to afford 3,6-dichloro-1-(3-((5-methyl-4-nitro-1-(tetrahydro-2H-py- ran-4-yl)-1H-pyrazol-3-yl)oxy)propyl-1,1,2,2,3,3-d6)-1H-pyrazolo[3,4-d]pyrimidine (750 mg) of10 sufficient purity for the subsequent step. Intermediate: 3,6-Dichloro-1-(3-((5-methyl-1-(3-methyltetrahydro-2H-pyran-4-yl)-4-nitro-1H- pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine 15
Figure imgf000128_0001
To a solution of 2-(2-methyl-1,3-dioxolan-2-yl)acetohydrazide (5 g, 31 mmol) in MeOH (50 mL) was added 3-methyltetrahydro-4H-pyran-4-one (3.57 g, 31.3 mmol) at 25 ºC. The mix- ture was stirred at 25 ºC for 1 h. NaBH3CN (5.89 g, 93.7 mmol) was added into the mixture and the mixture was stirred at 25 ºC for 1 h. The mixture was concentrated under reduced pressure. 20 The residue was diluted with EtOAc (100 mL) and washed with saturated aqueous NH4Cl (2×50 mL), H2O (50 mL) and brine (50 mL), dried over Na2SO4, filtered and concentrated to give 2-(2- methyl-1,3-dioxolan-2-yl)-N'-(3-methyltetrahydro-2H-pyran-4-yl)acetohydrazide (8.06 g) of suf- ficient purity for the subsequent step. To a solution of 2-(2-methyl-1,3-dioxolan-2-yl)-N'-(3-methyltetrahydro-2H-pyran-4- 25 yl)acetohydrazide (8.06 g, 31 mmol) in EtOH (100 mL) was added TFA (4.63 mL, 62.6 mmol) and the mixture was stirred at 90 ºC 12 h. Additional TFA (4.63 mL) was added at 25 ºC and the reaction was stirred at 90 ºC for another 12 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 20:80) to afford 5-methyl-1-(3-methyltetrahydro-2H-pyran-4-yl)-1H-pyra- zol-3-ol (3.8 g) of sufficient purity for the subsequent step. To a solution of 5-methyl-1-(3-methyltetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-ol (3.8 g, 5 19 mmol) in H2SO4 (40 mL) was added KNO3 (5.97 g, 59.1 mmol) slowly at 0 ºC. The mixture was stirred at 0 ºC for 0.5 h. The mixture was poured into ice-water (150 mL) and extracted with EtOAC (4×50 mL). The organic phase was washed with saturated aqueous NaHCO3 (100 mL), brine (2×100 mL), dried over Na2SO4 and concentrated under reduced pressure to give 5-methyl- 1-(3-methyltetrahydro-2H-pyran-4-yl)-4-nitro-1H-pyrazol-3-ol (2.9 g) of sufficient purity for the10 subsequent step. A mixture of 3-(3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propan-1-ol (1.53 g, 6.19 mmol), 5-methyl-1-(3-methyltetrahydro-2H-pyran-4-yl)-4-nitro-1H-pyrazol-3-ol (1.5 g, 6.22 mmol), DIAD (3.76 g, 18.6 mmol), and PPh3 on resin (6.18 g, 18.58 mmol, 3 mmol/g) in THF (30 mL) was stirred at 20 ºC for 16 h. The mixture was concentrated under reduced pressure. The 15 residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 40:60) to afford 3,6-dichloro-1-(3-((5-methyl-1-(3-methyltetrahydro-2H-pyran-4-yl)-4-nitro-1H- pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (1.7 g) of sufficient purity for the subse- quent step. 20 Intermediate: trans-3,6-Dichloro-1-(3-((1-(4-fluorocyclohexyl)-5-methyl-4-nitro-1H-pyrazol-3- yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000129_0001
To a solution of 4-fluorocyclohexanol (500 mg, 4.23 mmol, cis:trans=3:1) in DCM (7 mL) was added TEA (727 mg, 7.18 mmol) and DMAP (52 mg, 0.43 mmol) followed by TsCl (88825 mg, 4.66 mmol) at 0 ºC. The mixture was stirred at 20 ºC for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: pe- troleum ether:EtOAc 100:0 → 80:20) to afford cis-4-fluorocyclohexyl 4-methylbenzenesul- fonate(720 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 7.80 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 4.72 - 4.50 (m, 2H), 2.45 (s, 3H), 2.02 - 1.87 (m, 4H), 1.74 - 1.57 (m, 4H). 5 To a solution of cis-(4-fluorocyclohexyl) 4-methylbenzenesulfonate (342 mg, 1.25 mmol) and 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyrazole (350 mg, 1.11 mmol) in DMF (6 mL) was added K2CO3 (467 mg, 3.38 mmol). The mixture was stirred at 80 ºC for 24 h. The mixture was diluted with water (20 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (3×20 mL) and concentrated under re- 10 duced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 60:40) to afford trans-3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-1-(4-flu- orocyclohexyl)-5-methyl-4-nitro-1H-pyrazole (220 mg) of sufficient purity for the subsequent step.1H NMR (400MHz, CDCl3) δ 4.75 - 4.53 (m, 1H), 4.38 (t, J = 6.4 Hz, 2H), 4.12 - 3.97 (m, 1H), 3.81 (t, J = 6.0 Hz, 2H), 2.62 (s, 3H), 2.37 - 2.23 (m, 2H), 2.13 - 1.98 (m, 4H), 1.97 - 1.88 (m, 2H),15 1.76 - 1.64 (m, 2H), 0.89 (s, 9H), 0.05 (s, 6H). To a solution of trans-3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-1-(4-fluorocyclohexyl)- 5-methyl-4-nitro-1H-pyrazole (220 mg, 0.53 mmol) in DCM (1 mL) was added HCl/dioxane (4 M, 5 mL). The mixture was stirred at 20 ºC for 2 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum20 ether:EtOAc 100:0 → 60:40) to afford trans-3-((1-(4-fluorocyclohexyl)-5-methyl-4-nitro-1H-py- razol-3-yl)oxy)propan-1-ol (110 mg) of sufficient purity for the subsequent step. 1H NMR (400MHz, CDCl3) δ 4.76 - 4.54 (m, 1H), 4.48 (t, J = 5.6 Hz, 2H), 4.11 - 3.99 (m, 1H), 3.84 (t, J = 5.2 Hz, 2H), 2.63 (s, 3H), 2.35 - 2.25(m, 2H), 2.10 - 1.90 (m, 6H), 1.71 - 1.63(m, 2H). A mixture of trans-3-[1-(4-fluorocyclohexyl)-5-methyl-4-nitro-pyrazol-3-yl]oxypropan-1- 25 ol (100 mg, 0.4 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (63 mg, 0.4 mmol), PPh3 (261 mg, 1.0 mol), and DIAD (201 mg, 1.0 mmol) in THF (1 mL) was stirred at 20 ºC for 16 h. The mix- ture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 60:40) to afford trans-3,6-dichloro-1-(3-((1- (4-fluorocyclohexyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimi-30 dine (170 mg) of sufficient purity for the subsequent step. Intermediate: 3,6-Dichloro-1-(3-((1-(4,4-difluorocyclohexyl)-5-methyl-4-nitro-1H-pyrazol-3- yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000131_0001
To a mixture of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyra- zole (0.3 g, 0.95 mmol) and 4-bromo-1,1-difluoro-cyclohexane (1.33 g, 6.66 mmol) in DMF (6 mL) was added Cs2CO3 (620 mg, 1.90 mmol) at 20 ºC. The reaction mixture was stirred at 80 ºC for 5 16 h. The mixture was concentrated under reduced pressure. The residue was purified by chro- matography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 70:30) to afford 3-(3-((tert- butyldimethylsilyl)oxy)propoxy)-1-(4,4-difluorocyclohexyl)-5-methyl-4-nitro-1H-pyrazole (0.32 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.41 (t, J = 6.4 Hz, 2H), 4.19-4.12 (m, 1H), 3.83 (t, J = 6.0 Hz, 2H), 2.65 (s, 3H), 2.41 - 2.24 (m, 4H), 2.08 - 2.03 (m, 2H),10 2.01 - 1.86 (m, 4H), 0.90 (s, 9H), 0.06 (s, 6H). To a mixture of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-1-(4,4-difluorocyclohexyl)- 5-methyl-4-nitro-1H-pyrazole (0.31 g, 0.72 mmol) in THF (5 mL) was added HCl/dioxane (4 M, 5 mL) and then the mixture was stirred at 25 ºC for 1 h. The mixture was concentrated under re- duced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum15 ether:EtOAc 100:0 → 50:50) to afford 3-((1-(4,4-difluorocyclohexyl)-5-methyl-4-nitro-1H-pyra- zol-3-yl)oxy)propan-1-ol (0.21 g) of sufficient purity for the subsequent step. LC-MS (method B) (m/z) = 320.2 (MH)+ tR = 0.81 minutes. To a mixture of 3-((1-(4,4-difluorocyclohexyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)pro- pan-1-ol (0.2 g, 0.63 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (118 mg, 0.6320 mmol), and PPh3 (329 mg, 1.25 mmol) in THF (20 mL) was added DIAD (253 mg, 1.25 mmol) at 0 ºC. The mixture was stirred at 25 ºC for 16 h. The mixture was concentrated under reduced pres- sure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 70:30) to afford 3,6-dichloro-1-(3-((1-(4,4-difluorocyclohexyl)-5-methyl-4-nitro-1H-py- razol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (0.28 g) of sufficient purity for the subse- 25 quent step. 1H NMR (CDCl3, 400MHz) δ 8.97 (s, 1H), 4.66 (t, J = 6.8 Hz, 2H), 4.36 (t, J = 5.6 Hz, 2H), 4.13 - 4.04 (m, 1H), 2.63 (s, 3H), 2.53 - 2.43 (m, 2H), 2.36 - 2.17 (m, 4H), 2.00 - 1.83 (m, 4H). Intermediate: 3,6-Dichloro-1-(3-((5-methyl-4-nitro-1-(oxepan-3-yl)-1H-pyrazol-3-yl)oxy)pro- pyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000132_0001
To a solution of hex-5-en-1-ol (9.0 g, 89 mmol) in DCM (150 mL) were added m-CPBA 5 (29.08 g, 135 mmol, 80% purity) at 0 ºC. The reaction mixture was stirred at 20 ºC for 16 h. The reaction was cooled to 0 ºC and quenched with aqueous NaOH (1 M, 140 ml). The reaction mix- ture was diluted with water (100 mL) and extracted with DCM (3 × 100 mL). The combined organic layers were washed with brine (3 × 100 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum 10 ether:EtOAc 60:40 → 50:50) to afford 4-(oxiran-2-yl)butan-1-ol (8 g) of sufficient purity for the subsequent step. To a solution of 4-(oxiran-2-yl)butan-1-ol (7.5 g, 65 mmol) in THF (100 mL) was added t- BuOK (8.69 g, 77.5 mmol). The reaction mixture was stirred at 50 ºC for 16 h. The reaction mix- ture was added water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic 15 layers were washed with brine (3 × 50 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 30:70 → 20:80) to give a mixture of oxepan-3-ol and (tetrahydro-2H-pyran-2- yl)methanol (4 g). The mixture of tetrahydropyran-2-ylmethanol and oxepan-3-ol was dissolved in DCM (40 mL). TEA (5.23 g, 51.65 mmol) and N,N-dimethylmethanamine;hydrochloride (329 20 mg, 3.44 mmol) were added followed by TsCl (7.88 g, 41.3 mmol) at 0 ºC. The mixture was stirred at 20 ºC for 16 h. The solvent was removed under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 80:20 → 70:30) to afford a mixture of oxepan-3-yl 4-methylbenzenesulfonate and (tetrahydro-2H-pyran-2-yl)methyl 4-methylben- zenesulfonate (5 g). The mixture of oxepan-3-yl 4-methylbenzenesulfonate and (tetrahydro-2H-25 pyran-2-yl)methyl 4-methylbenzenesulfonate (4 g) was added to a solution of 3-chloro-5-methyl- 4-nitro-1H-pyrazole (2 g, 12.38 mmol) in DMF (10 mL). K2CO3 (5.13 g, 37.1 mmol) was added and the reaction mixture was stirred at 80 ºC for 16 h. The reaction mixture was diluted with water (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (3 × 10 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 85:15) to af- ford 3-chloro-5-methyl-4-nitro-1-(oxepan-3-yl)pyrazole (700 mg) of sufficient purity for the sub- 5 sequent step.1H NMR (CDCl3, 400MHz) δ 4.42 - 4.32 (m, 1H), 4.02 - 3.94 (m, 1H), 3.92 - 3.77 (m, 3H), 2.67 (s, 3H), 2.13 - 2.04 (m, 2H), 1.99 - 1.86 (m, 3H), 1.72 - 1.63 (m, 1H). To a solution of 3-chloro-5-methyl-4-nitro-1-(oxepan-3-yl)pyrazole (200 mg, 0.77 mmol) in H2O (10 mL) was added KOH (1.1 g, 20 mmol). The reaction mixture was stirred at 155ºC for 16 h using a microwave reactor. The reaction mixture was washed with EtOAc (10 mL). 10 The aqueous phase was adjusted to pH=2~3, and extracted with EtOAc (3×10 mL), dried over Na2SO4, and concentrated under reduced pressure to afford 5-methyl-4-nitro-1-(oxepan-3-yl)py- razol-3-ol (150 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.37 - 4.24 (m, 1H), 4.02 - 3.94 (m, 1H), 3.91 - 3.84 (m, 1H), 3.84 - 3.78 (m, 2H), 2.63 (s, 3H), 2.15 - 2.01 (m, 2H), 1.99 - 1.84 (m, 3H), 1.72 - 1.62 (m, 1H). 15 To a solution of 5-methyl-4-nitro-1-(oxepan-3-yl)pyrazol-3-ol (150 mg, 0.62 mmol) in THF (10 mL) were added 3-(3,6-dichloropyrazolo[3,4-d]pyrimidin-1-yl)propan-1-ol (169 mg, 0.68 mmol), DIAD (754 mg, 3.73 mmol) and PPh3 on resin (975 mg, 3.73 mmol) at 0 ºC. The reaction mixture was stirred at 25 ºC for 16 h. The solvent was removed. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 70:30 → 60:40) to afford 3,6-di-20 chloro-1-(3-((5-methyl-4-nitro-1-(oxepan-3-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4- d]pyrimidine (250 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.95 (s, 1H), 4.65 (t, J = 6.4 Hz, 2H), 4.34 (t, J = 6.0 Hz, 2H), 4.31 - 4.23 (m, 1H), 3.86 (d, J = 7.2 Hz, 2H), 3.82 - 3.78 (m, 2H), 2.60 (s, 3H), 2.50 - 2.44 (m, 2H), 2.02 - 1.96 (m, 2H), 1.92 - 1.79 (m, 3H), 1.69 - 1.62 (m, 1H). 25 Intermediate: 3,6-Dichloro-1-(3-((5-methyl-4-nitro-1-(oxepan-4-yl)-1H-pyrazol-3-yl)oxy)pro- pyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000133_0001
To a solution of 3-chloro-5-methyl-4-nitro-1H-pyrazole (2 g, 12 mmol) and 4-bromo- oxepane (2.44 g, 13.6 mmol) in MeCN (50 mL) was added K2CO3 (5.13 g, 37.1 mmol). The mixture was stirred at 80 ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 5 → 80:20) to afford 3-chloro-5-methyl-4-nitro-1-(oxepan-4-yl)-1H-pyrazole (1.2 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.49 - 4.39 (m, 1H), 3.98 - 3.91 (m, 1H), 3.90 - 3.77 (m, 2H), 3.69 - 3.61 (m, 1H), 2.68 (s, 3H), 2.45 - 2.35 (m, 1H), 2.35 - 2.24 (m, 1H), 2.19 - 2.08 (m, 1H), 2.04 - 1.96 (m, 1H), 1.96 - 1.88 (m, 1H), 1.87 - 1.73 (m, 1H). To a solution of 3-chloro-5-methyl-4-nitro-1-(oxepan-4-yl)-1H-pyrazole (200 mg, 770 10 μmol) in H2O (10 mL) was added KOH (1.12 g, 20.02 mmol). The mixture was stirred at 155 ºC for 3 h using a microwave reactor. The reaction mixture was washed with EtOAc (50 mL×3). The aqueous phase was adjusted to pH=3 using HCl (12M) and extracted with EtOAc (100 mL×3). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to afford 5-methyl-4-nitro-1-(oxepan-4-yl)-1H-pyrazol-3-ol (200 mg) of sufficient purity for the sub- 15 sequent step.1H NMR (CDCl3, 400MHz) δ 11.31 (br s, 1H), 4.57 - 4.35 (m, 1H), 3.80 - 3.69 (m, 2H), 3.66 - 3.54 (m, 2H), 2.57 (s, 3H), 2.14 - 2.06 (m, 1H), 2.06 - 1.93 (m, 2H), 1.92 - 1.84 (m, 1H), 1.83 - 1.73 (m, 2H). DIAD (1.37 g, 6.80 mmol) was added to a solution of 3-(3,6-dichloro-1H-pyrazolo[3,4- d]pyrimidin-1-yl)propan-1-ol (280 mg, 1.13 mmol), 5-methyl-4-nitro-1-(oxepan-4-yl)-1H-pyrazol- 20 3-ol (273 mg, 1.13 mmol) and PPh3 on resin (2.26 g, 6.80 mmol, 3 mmol/g) in THF (20 mL) at 0 ºC. The mixture was stirred at 20 ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 50:50) to afford 3,6-dichloro-1-(3-((5-methyl-4-nitro-1-(oxepan-4-yl)-1H- pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (330 mg) of sufficient purity for the sub- 25 sequent step.1H NMR (CDCl3, 400MHz) δ 8.99 (s, 1H), 4.65 (t, J = 6.4 Hz, 2H), 4.35 (t, J = 6.0 Hz, 2H), 4.32 - 4.23 (m, 1H), 3.96 - 3.81 (m, 2H), 3.79 - 3.71 (m, 1H), 3.69 - 3.59 (m, 1H), 2.61 (s, 3H), 2.53 - 2.42 (m, 2H), 2.34 - 2.24 (m, 1H), 2.24 - 2.14 (m, 1H), 2.04 - 1.98 (m, 1H), 1.95 - 1.86 (m, 2H), 1.83 - 1.73 (m, 1H). 30 Intermediate: trans-3,6-Dichloro-1-(3-((1-(3-fluorotetrahydro-2H-pyran-4-yl)-5-methyl-4-nitro- 1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000135_0001
To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyra- zole (1.6 g, 5.1 mmol) in toluene (20 mL) was added cis-3-fluorotetrahydropyran-4-ol (914 mg, 7.61 mmol) and 2-(tributyl-λ5-phosphanylidene)acetonitrile (4.90 g, 20.3 mmol). The mixture 5 was stirred at 110ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 80:20) to afford trans-3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-1-(3-fluorotetrahydro-2H- pyran-4-yl)-5-methyl-4-nitro-1H-pyrazole (1.6 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 5.00 - 4.75 (m, 1H), 4.41 (t, J = 6.4 Hz, 2H), 4.31 - 4.18 (m, 2H), 4.11 -10 4.04 (m, 1H), 3.82 (t, J = 6.0 Hz, 2H), 3.56 - 3.46 (m, 1H), 3.43 - 3.34 (m, 1H), 2.66 (s, 3H), 2.57 - 2.45 (m, 1H), 2.05 - 1.99 (m, 2H), 1.98 - 1.89 (m, 1H), 0.89 (s, 9H), 0.05 (s, 6H). A solution of trans-3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-1-(3-fluorotetrahydro- 2H-pyran-4-yl)-5-methyl-4-nitro-1H-pyrazole (1.6 g, 3.8 mmol) in HCl/dioxane (4 M, 10 mL) was stirred at 20 ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The 15 residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 40:60) to afford trans-3-((1-(3-fluorotetrahydro-2H-pyran-4-yl)-5-methyl-4-nitro-1H-pyrazol-3- yl)oxy)propan-1-ol (850 mg) of sufficient purity for the subsequent step. LC-MS (method B) (m/z) = 304.2 (MH)+ tR = 0.73 minutes. DIAD (800 mg, 3.96 mmol) was added to a solution of trans-3-((1-(3-fluorotetrahydro-20 2H-pyran-4-yl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propan-1-ol (400 mg, 1.32 mmol) 3,6-di- chloro-1H-pyrazolo[3,4-d]pyrimidine (249 mg, 1.32 mmol), and PPh3 (1.04 g, 3.96 mmol) in THF (20 mL) at 0 ºC. Then the mixture was stirred at 20 ºC for 15 h. The reaction mixture was con- centrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 70:30) to afford trans-3,6-dichloro-1-(3-((1-(3-fluoro-25 tetrahydro-2H-pyran-4-yl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]py- rimidine (450 mg) of sufficient purity for the subsequent step. LC-MS (method B) (m/z) = 474.2 (MH)+ tR = 0.92 minutes. Intermediate: 3-(8-Chloro-3-methyl-4,11,12,13-tetrahydro-2H-5,7-(azenometheno)dipyra- zolo[3,4-b:5',1'-g][1,4,6,8]oxatriazacycloundecin-2-yl)bicyclo[1.1.1]pentane-1-carboxamide
Figure imgf000136_0001
5 A mixture of O3-[(3-methoxycarbonylbicyclo[1.1.1]pentane-1-carbonyl)oxy-(2,4,6-tri- methylphenyl)-λ3-iodanyl] O1-methyl bicyclo[1.1.1]pentane-1,3-dicarboxylate (600 mg, 1.03 mmol), 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyrazole (300 mg, 0.951 mmol), pentane-2,4-dione (114 mg, 1.14 mmol), thiophene-2-carbonyloxycopper (218 mg, 1.14 mmol), 4,7-diphenyl-1,10-phenanthroline (632 mg, 1.90 mmol), and DBU (145 mg, 0.951 mmol) 10 in 1,4-dioxane (15 mL) was degassed and purged with N2 for 3 times and then the mixture was stirred at 20 ºC for 60 h in the dark. The reaction mixture was diluted with aqueous NH4Cl (50 mL) and extracted with DCM (3×100 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 80:20) to afford 3-(3-(3-((tert-butyldimethylsi- 15 lyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyrazol-1-yl)bicyclo[1.1.1]pentane-1-carboxylate (300 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.39 (t, J = 6.4 Hz, 2H), 3.80 (t, J = 6.4 Hz, 2H), 3.75 (s, 3H), 2.67 (s, 3H), 2.63 (s, 6H), 2.04 - 1.98 (m, 2H), 0.89 (s, 9H), 0.05 (s, 6H). A solution of methyl 3-(3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H- 20 pyrazol-1-yl)bicyclo[1.1.1]pentane-1-carboxylate (570 mg, 1.30 mmol) in HCl/MeOH (4M, 10 mL) was stirred at 20 ºC for 1 hour. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 60:40) to afford methyl-3-(3-(3-hydroxypropoxy)-5-methyl-4-nitro-1H-pyrazol-1-yl)bicy- clo[1.1.1]pentane-1-carboxylate (250 mg) of sufficient purity for the subsequent step.1H NMR 5 (DMSO-d6, 400MHz) δ 4.53 (s, 1H), 4.28 (t, J = 6.4 Hz, 2H), 3.66 (s, 3H), 3.53 (t, J = 6.0 Hz, 2H), 2.63 (s, 3H), 2.61 (s, 6H), 1.94 - 1.81 (m, 2H). DIAD (448 mg, 2.21 mmol) was added to a solution of methyl-3-(3-(3-hydroxypropoxy)- 5-methyl-4-nitro-1H-pyrazol-1-yl)bicyclo[1.1.1]pentane-1-carboxylate (240 mg, 0.738 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (139 mg, 0.738 mmol), and PPh3 on resin (736 mg, 10 2.21 mmol, 3 mmol/g) in THF (10 mL) at 0 ºC. The mixture was stirred at 20 ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chro- matography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 40:60) to afford methyl 3-(3- (3-(3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propoxy)-5-methyl-4-nitro-1H-pyrazol-1-yl)bi- cyclo[1.1.1]pentane-1-carboxylate (270 mg) of sufficient purity for the subsequent step.1H NMR 15 (CDCl3, 400MHz) δ 8.96 (s, 1H), 4.65 (t, J = 6.4 Hz, 2H), 4.33 (t, J = 6.0 Hz, 2H), 3.75 (s, 3H), 2.66 (s, 3H), 2.60 (s, 6H), 2.51 - 2.41 (m, 2H). To a solution of methyl 3-(3-(3-(3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propoxy)- 5-methyl-4-nitro-1H-pyrazol-1-yl)bicyclo[1.1.1]pentane-1-carboxylate (260 mg, 0.524 mmol) in EtOH (40 mL) and H2O (8 mL) was added Fe (146 mg, 2.61 mmol) and NH4Cl (140 mg, 2.62 mmol). 20 The mixture was stirred at 80 ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 50:50) to afford methyl 3-(8-chloro-3-methyl-4,11,12,13-tetrahydro-2H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1,4,6,8]oxatriazacycloundecin-2-yl)bicy- clo[1.1.1]pentane-1-carboxylate (100 mg) of sufficient purity for the subsequent step.1H NMR 25 (CDCl3, 400MHz) δ 8.65 (s, 1H), 6.82 (br s, 1H), 4.54 - 4.46 (m, 2H), 4.46 - 4.38 (m, 2H), 3.74 (s, 3H), 2.61 (s, 6H), 2.33 (s, 3H), 2.01 - 1.85 (m, 2H). NH3/MeOH (7 M, 15 mL) was added to methyl 3-(8-chloro-3-methyl-4,11,12,13-tetrahy- dro-2H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1,4,6,8]oxatriazacycloundecin-2-yl)bicy- clo[1.1.1]pentane-1-carboxylate (90 mg, 0.209 mmol) in a sealed flask. The reaction mixture was 30 stirred at 80 ºC for 15 h. The reaction mixture was concentrated under reduced pressure to afford 3-(8-chloro-3-methyl-4,11,12,13-tetrahydro-2H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1,4,6,8]oxatriazacycloundecin-2-yl)bicyclo[1.1.1]pentane-1-carboxamide (80 mg) of suffi- cient purity for the subsequent step. Intermediate: 3,6-Dichloro-1-(3-((5-methyl-4-nitro-1-(2-oxaspiro[3.3]heptan-6-yl)-1H-pyrazol- 3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000138_0001
A mixture of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyrazole 5 (350 mg, 1.11 mmol), 2-oxaspiro[3.3]heptan-6-ol (152 mg, 1.33 mmol), PPh3 on resin (553 mg, 1.66 mmol, 3 mmol/g), and DIAD (337 mg, 1.66 mmol) in THF (5 mL) was stirred at 20 ºC for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by chroma- tography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 70:30) to afford 3-(3-((tert-butyl- dimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1-(2-oxaspiro[3.3]heptan-6-yl)-1H-pyrazole (295 10 mg) of sufficient purity for the subsequent step. H NMR (CDCl3, 400MHz) δ 4.77 (d, J = 4.8 Hz, 4H), 4.56 - 4.49 (m, 1H), 4.41 (t, J = 6.0 Hz, 2H), 3.82 (t, J = 6.0 Hz, 2H), 2.87 - 2.79 (m, 2H), 2.77 - 2.69 (m, 2H), 2.56 (s, 3H), 2.07 - 2.02 (m, 2H), 0.89 (s, 9H), 0.05 (s, 6H). To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1-(2-ox- aspiro[3.3]heptan-6-yl)-1H-pyrazole (260 mg, 0.63 mmol) in THF (5 mL) was added TBAF (1 M, 1 15 mL). The mixture was stirred at 20 ºC for 2 h. The mixture was diluted with water (10 mL) and extracted with EtOAc (15 mL×3). The combined organic layers were concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 0:100) to afford 3-((5-methyl-4-nitro-1-(2-oxaspiro[3.3]heptan-6-yl)-1H- pyrazol-3-yl)oxy)propan-1-ol (179 mg) of sufficient purity for the subsequent step. 1H NMR 20 (CDCl3, 400MHz) δ 4.77 (d, J = 9.2 Hz, 4H), 4.56 - 4.47 (m, 3H), 3.86 (t, J = 5.6 Hz, 2H), 2.86 - 2.78 (m, 2H), 2.77 - 2.70 (m, 2H), 2.57 (s, 3H), 2.14 - 2.06 (m, 2H). A mixture of 3-((5-methyl-4-nitro-1-(2-oxaspiro[3.3]heptan-6-yl)-1H-pyrazol-3- yl)oxy)propan-1-ol (160 mg, 0.54 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (101.71 mg, 0.54 mmol), PPh3 on resin (304 mg, 0.92 mmol) (3 mmol/g), and DIAD (185 mg, 0.9225 mmol) in THF (5 mL) was stirred at 20 ºC for 16 h. The mixture was concentrated under re- duced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 20:80) to afford 3,6-dichloro-1-(3-((5-methyl-4-nitro-1-(2-ox- aspiro[3.3]heptan-6-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (170 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.96 (s, 1H), 4.77 (s, 2H), 4.72 (s, 2H), 4.66 (t, J = 6.4 Hz, 2H), 4.52 - 4.45 (m, 1H), 4.37 (t, J = 5.6 Hz, 2H), 2.78 - 2.69 (m, 5 4H), 2.55 (s, 3H), 2.50 - 2.44 (m, 2H). Intermediate: 3,6-Dichloro-1-(3-((5-(methyl-d3)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyra- zol-3-yl)oxy)propyl-1,1,2,2,3,3-d6)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000139_0001
10 To a stirred mixture of 4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-ol (100 mg, 0.469 mmol) and K2CO3 (259 mg, 1.87 mmol) in DMF (3 mL) was added bromo(methoxy)methane (117 mg, 0.938 mmol) in portions at 0 ºC. The resulting mixture was stirred for 16 h at room temper- ature. The reaction was quenched by the addition of water (100 mL) and extracted with EtOAc (3x50 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, and 15 concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 50:50) afford 3-(methoxymethoxy)-4-nitro-1-(tetrahydro-2H- pyran-4-yl)-1H-pyrazole (40 mg) of sufficient purity for the subsequent step. To a stirred solution of 3-(methoxymethoxy)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-py- razole (200 mg, 0.777 mmol) in THF (2 mL) was added LiHMDS (1.55 mL, 1.55 mmol, 1M in THF) 20 dropwise at -78 ºC under. The resulting mixture was stirred for 2 h at -78 ºC. D2O (4 mL) was added at -78 ºC. The resulting mixture was diluted with water (100 mL) and extracted with EtOAc (3x50 mL). The combined organic layers were dried over Na2SO4 and concentrated under re- duced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 50:50) to afford 3-(methoxymethoxy)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-py- 25 razole-5-d (180 mg) of sufficient purity for the subsequent step. To a stirred solution 3-(methoxymethoxy)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyra- zole-5-d (50 mg) in THF (1.5 mL) was added LiHMDS (0.39 mL, 0.39 mmol, 1M in THF) in a drop- wise manner at -78 ºC. The resulting mixture was stirred for 1 h at -78 ºC. CD3I (113 mg, 0.776 mmol) was added in a dropwise manner at -78 ºC. The resulting mixture was stirred for 2 h at - 5 78 ºC followed by dropwise addition of D2O (2 mL) dropwise at -78 ºC. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 67:33) to afford 3-(methox- ymethoxy)-5-(methyl-d3)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazole (40 mg) of sufficient purity for the subsequent step. A solution of 3-(methoxymethoxy)-5-(methyl-d3)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H- 10 pyrazole (150 mg, 0.547 mmol) and HCl in 1,4-dioxane (4 mL, 16 mmol, 4M) in DCM (2 mL) was stirred at room temperature for 2 h. The resulting mixture was concentrated under reduced pressure to afford 5-(methyl-d3)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-ol (101 mg) of sufficient purity for the subsequent step.1H NMR (DMSO-d6, 400 MHz) δ 11.35 (s, 1H), 4.47 (tt, J = 11.2, 4.0 Hz, 1H), 3.96-3.92 (m, 2H), 3.46 (t, J = 12.0 Hz, 2H), 1.97-1.93 (m, 2H), 1.77-15 1.74(m, 2H). To a stirred solution of 5-(methyl-d3)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-ol (95.5 mg, 0.416 mmol) in THF (5 mL) were added 3-(3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin- 1-yl)propan-1,1,2,2,3,3-d6-1-ol (70 mg, 0.277 mmol) and PPh3 (145 mg, 0.554 mmol) in portions at room temperature under argon atmosphere. To the above mixture was added DIAD (0.11 mL,20 0.554 mmol) dropwise at 0 ºC. The resulting mixture was stirred for 2 h at room temperature and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 50:50) to afford 3,6-dichloro-1-(3-((5-(methyl-d3)-4-nitro-1- (tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl-1,1,2,2,3,3-d6)-1H-pyrazolo[3,4-d]pyrim- idine (110 mg) of sufficient purity for the subsequent step. 25 Intermediate: 1-(8-Chloro-3-methyl-4,11,12,13-tetrahydro-2H-5,7-(azenometheno)dipyra- zolo[3,4-b:5',1'-g][1,4,6,8]oxatriazacycloundecin-2-yl)cyclopropanecarboxamide
Figure imgf000141_0001
To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyra- zole (600 mg, 1.90 mmol) in DMF (24 mL) was added Cs2CO3 (2.48 g, 7.62 mmol) and methyl 2,4- dibromobutanoate (552 mg, 2.12 mmol). The mixture was stirred at 45 ºC for 16 h. The reaction 5 mixture was diluted with water (15 mL) and extracted with EtOAc (3×15 mL). The combined organic layers were washed with brine (3×15 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 90:10) to afford 1-(3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4- nitro-1H-pyrazol-1-yl)cyclopropanecarboxylate (440 mg) of sufficient purity for the subsequent 10 step.1H NMR (DMSO-d6, 400MHz) δ 4.28 (t, J = 6.0 Hz, 2H), 3.73 (t, J = 6.0 Hz, 2H), 3.65 (s, 3H), 2.54 (s, 3H), 1.95 - 1.85 (m, 2H), 1.86 - 1.78 (m, 2H), 1.77 - 1.70 (m, 2H), 0.84 (s, 9H), 0.01 (s, 6H). To methyl 1-(3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyrazol-1-yl)cy- clopropanecarboxylate (440 mg, 1.06 mmol) was added HCl/dioxane (4 M, 5 mL) and the mix- ture was stirred at 20 ºC for 16 h. The reaction mixture was concentrated under reduced pres- 15 sure. The residue was purified by chromatography on silica gel (eluent: DCM:MeOH 100:0 → 97:3) to afford a residue that was suspended in water (10 mL), extracted with EtOAc (3×40 mL) and the combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to afford methyl 1-(3-(3-hydroxypropoxy)-5-methyl-4-nitro-1H-pyrazol-1-yl)cyclopro- panecarboxylate (320 mg) of sufficient purity for the subsequent step. 20 To a solution of methyl 1-(3-(3-hydroxypropoxy)-5-methyl-4-nitro-1H-pyrazol-1-yl)cy- clopropanecarboxylate (300 mg, 1.00 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (202 mg, 1.07 mmol), and DIAD (608 mg, 3.01 mmol) in THF (5 mL) was added PPh3 (789 mg, 3.01 mmol) at 0 ºC. The mixture was stirred at 20 ºC for 16 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: pe- troleum ether:EtOAc 100:0 → 70:30) to afford methyl 1-(3-(3-(3,6-dichloro-1H-pyrazolo[3,4- d]pyrimidin-1-yl)propoxy)-5-methyl-4-nitro-1H-pyrazol-1-yl)cyclopropanecarboxylate (230 mg) of sufficient purity for the subsequent step.1H NMR (DMSO-d6, 400MHz) δ 9.27 (s, 1H), 4.55 (t, 5 J = 6.4 Hz, 2H), 4.26 (t, J = 5.6 Hz, 2H), 3.65 (s, 3H), 2.54 (s, 3H), 2.35 - 2.25 (m, 2H), 1.85 - 1.77 (m, 2H), 1.70 - 1.65 (m, 2H). A mixture of methyl 1-(3-(3-(3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propoxy)-5- methyl-4-nitro-1H-pyrazol-1-yl)cyclopropanecarboxylate (190 mg, 0.340 mmol), Fe (95 mg, 1.70 mmol), and NH4Cl (91 mg, 1.7 mmol) in EtOH (4 mL) and H2O (1 mL) was degassed and purged 10 with N2 for 3 times, and then the mixture was stirred at 80 ºC for 16 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 50:50) to afford methyl 1-(8-chloro-3-methyl- 4,11,12,13-tetrahydro-2H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1,4,6,8]oxatriazacy- cloundecin-2-yl)cyclopropanecarboxylate (63 mg) of sufficient purity for the subsequent step. 15 LC-MS (method B) (m/z) = 404.2 (MH)+ tR = 0.79 minutes. A mixture of NH3/MeOH (7 M, 8 mL) and methyl 1-(8-chloro-3-methyl-4,11,12,13-tetra- hydro-2H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1,4,6,8]oxatriazacycloundecin-2-yl)cy- clopropanecarboxylate (60 mg, 0.15 mmol) in a sealed tube was heated at 80 ºC for 2 h using a microwave reactor. The reaction mixture was concentrated under reduced pressure. The resi- 20 due was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 80:20) to afford 1-(8-chloro-3-methyl-4,11,12,13-tetrahydro-2H-5,7-(azenometheno)dipyra- zolo[3,4-b:5',1'-g][1,4,6,8]oxatriazacycloundecin-2-yl)cyclopropanecarboxamide (40 mg) of suf- ficient purity for the subsequent step. LC-MS (method B) (m/z) = 389.2 (MH)+ tR = 0.70 minutes. 25 Intermediate: 3,6-Dichloro-1-(3-((1-((1r,4r)-4-methoxycyclohexyl)-5-methyl-4-nitro-1H-pyra- zol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000143_0001
To a solution of (1s,4s)-cyclohexane-1,4-diol (2 g, 17 mmol) in water (10 mL) was added KOH (1.06 g, 18.94mmol) and MeI (3.67 g, 25.8 mmol). The mixture was stirred at 100 ºC for 5 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by 5 chromatography on silica gel (eluent: petroleum ether:EtOAc 95:5 → 50:50) to afford (1s,4s)-4- methoxycyclohexanol (500 mg) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 400MHz) δ 3.79 - 3.69 (m, 1H), 3.38 - 3.21 (m, 4H), 1.89 - 1.75 (m, 2H), 1.70 - 1.62 (m, 4H), 1.58 - 1.50 (m, 2H). To a solution of (1s,4s)-4-methoxycyclohexanol (400 mg, 3.07 mmol) in DCM (6 mL) at 0 10 ºC were successively added Et3N (684 mg, 6.76 mmol), DMAP (38 mg, 0.31 mmol), and TsCl (820 mg, 4.30 mmol). The mixture was allowed to warm to 15 ºC and stirred for 16 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 70:30) to afford (1s,4s)-4-methoxycyclo- hexyl 4-methylbenzenesulfonate (350 mg) of sufficient purity for the subsequent step.1H NMR 15 (CDCl3, 400MHz) δ 7.80 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 4.65 - 4.56 (m, 1H), 3.30 (s, 3H), 3.26 - 3.18 (m, 1H), 2.45 (s, 3H), 1.94 - 1.82 (m, 2H), 1.80 - 1.70 (m, 2H), 1.63 - 1.56 (m, 4H). A mixture of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyrazole (400 mg, 1.27 mmol), (1s,4s)-4-methoxycyclohexyl 4-methylbenzenesulfonate (400 mg, 1.41 mmol), and Cs2CO3 (826 mg, 2.54 mmol) in DMF (10 mL) was degassed and purged with N2×3, 20 and then stirred at 80 ºC for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 95:5 → 70:30) to afford 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-1-((1r,4r)-4-fluorocyclohexyl)-5-me- thyl-4-nitro-1H-pyrazole (240 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.39 (t, J = 6.0 Hz, 2H), 4.03 - 3.94 (m, 1H), 3.81 (t, J = 6.0 Hz, 2H), 3.39 (s, 3H), 3.30 - 25 3.20 (m, 1H), 2.62 (s, 3H), 2.28 - 2.19 (m, 2H), 2.06 - 2.04 (m, 2H), 2.02 - 1.97 (m, 2H), 1.95 - 1.84 (m, 2H), 1.42 - 1.30 (m, 2H), 0.89 (s, 9H), 0.05 (s, 6H). A mixture of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-1-((1r,4r)-4-fluorocyclohexyl)- 5-methyl-4-nitro-1H-pyrazole (240 mg, 0.56 mmol) in HCl/dioxane (4 M, 5 mL) was degassed and purged with N2×3, and then the mixture was stirred at 15 ºC for 1 h. The mixture was concen- trated under reduced pressure. The residue was purified by chromatography on silica gel (elu- 5 ent: petroleum ether:EtOAc 95:5 → 0:100) to afford 3-((1-((1r,4r)-4-methoxycyclohexyl)-5-me- thyl-4-nitro-1H-pyrazol-3-yl)oxy)propan-1-ol (130 mg) of sufficient purity for the subsequent step.1H NMR (400MHz, CDCl3) δ 4.50 (t, J = 6.4 Hz, 2H), 4.06 - 3.95 (m, 1H), 3.82 (t, J = 5.6 Hz, 2H), 3.38 (s, 3H), 3.30 - 3.18 (m, 1H), 2.63 (s, 3H), 2.31 - 2.17 (m, 2H), 2.10 - 2.05 (m, 2H), 2.04 - 1.95 (m, 2H), 1.94 - 1.82 (m, 2H), 1.43 - 1.30 (m, 2H). 10 To a solution of 3-((1-((1r,4r)-4-methoxycyclohexyl)-5-methyl-4-nitro-1H-pyrazol-3- yl)oxy)propan-1-ol (130 mg, 0.41 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (78 mg, 0.41 mmol) and PPh3 (218 mg, 0.83 mmol) in THF (10 mL) was added DIAD (168 mg, 0.83 mmol) in a dropwise manner at 0 ºC. The mixture was stirred at 15 ºC for 16 h. The mixture was con- centrated under reduced pressure. The residue was purified by chromatography on silica gel15 (eluent: petroleum ether:EtOAc 80:20 → 50:50) to afford 3,6-dichloro-1-(3-((1-((1r,4r)-4-meth- oxycyclohexyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (160 mg) of sufficient purity for the subsequent step. LC-MS (method B) (m/z) = 484.2 (MH)+ tR = 0.95 minutes. 20 Intermediate: 1-(3-((1-(3-Oxabicyclo[3.1.0]hexan-6-yl)-5-methyl-4-nitro-1H-pyrazol-3- yl)oxy)propyl)-3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000144_0001
To 2-(3-oxabicyclo[3.1.0]hexan-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (950 mg, 4.52 mmol) in THF (20 mL) and H2O (5 mL) was added sodium periodate (2.91 g, 13.6 mmol) and 25 the reaction was stirred at 20 ºC for 30 minutes. Then aqueous HCl (1 M, 5 mL) was added and the reaction was stirred at 20 ºC for 12 h. The reaction was filtered and the filter cake was washed with THF (20 mL×2). The filtrate was concentrated under reduced pressure. The residue was diluted with EtOAc (30 mL), washed with brine (10 mL), dried over Na2SO4, and concentrated under reduced pressure to afford 3-oxabicyclo[3.1.0]hexan-6-ylboronic acid (579 mg) of suffi- 5 cient purity for the subsequent step. A mixture of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyrazole (700 mg, 2.22 mmol), 3-oxabicyclo[3.1.0]hexan-6-ylboronic acid (579 mg, 4.53 mmol), Cu(OAc)2 (613 mg, 3.37 mmol) and 4Å MS (700 mg) in DCE (50 mL) was stirred at 65 ºC under oxygen (15 psi) for 36 h. The reaction mixture was filtered and concentrated under reduced pressure. The 10 residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 70:30) to afford 1-(3-oxabicyclo[3.1.0]hexan-6-yl)-3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5- methyl-4-nitro-1H-pyrazole (730 mg) of sufficient purity for the subsequent step. LC-MS (method B) (m/z) = 398.2 (MH)+ tR = 0.96 minutes. To a solution of 1-(3-oxabicyclo[3.1.0]hexan-6-yl)-3-(3-((tert-butyldimethylsi- 15 lyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyrazole (700 mg, 1.76 mmol) in THF (5 mL) was added TBAF (1 M in THF, 2.64 mL) at 0 ºC and the reaction was stirred at 20 ºC for 1 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 0:100) to afford 3-((1-(3-oxabicyclo[3.1.0]hexan-6-yl)- 5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propan-1-ol (310 mg) of sufficient purity for the subse- 20 quent step.1H NMR (CDCl3, 400MHz) δ 4.46 (t, J = 5.6 Hz, 2H), 4.18 - 4.11 (m, 2H), 3.87 - 3.79 (m, 4H), 3.18 (t, J = 2.0 Hz, 1H), 2.66 (s, 3H), 2.64 - 2.59 (m, 1H), 2.39 - 2.35 (m, 2H), 2.09 - 2.05 (m, 2H). To a mixture of 3-((1-(3-oxabicyclo[3.1.0]hexan-6-yl)-5-methyl-4-nitro-1H-pyrazol-3- yl)oxy)propan-1-ol 300 mg, 1.06 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (210 mg, 25 1.11 mmol) and PPh3 (555 mg, 2.12 mmol) in THF (10 mL) was added DIAD (428 mg, 2.12 mmol) at 0 ºC and the reaction was stirred at 20 ºC for 12 h. The reaction was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 50:50) to afford 1-(3-((1-(3-oxabicyclo[3.1.0]hexan-6-yl)-5-methyl-4-nitro- 1H-pyrazol-3-yl)oxy)propyl)-3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (250 mg) of sufficient30 purity for the subsequent step. LC-MS (method B) (m/z) = 454.1 (MH)+ tR = 0.78 minutes. Intermediate: 8-Chloro-3-methyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyra- zolo[3,4-b:5',1'-g][1]oxa[4,6,8]-triazacycloundecine
Figure imgf000146_0001
A mixture of 3-((3-methyl-4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5- yl)oxy)propan-1-ol (2 g, 7.0 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (1.32 g, 7.01 mmol), PPh3 (2.76 g, 10.52 mmol), and DIAD (2.13 g, 10.52 mmol) in THF (20 mL) was stirred 5 at 15 ºC for 16 h. The mixture was concentrated under reduced pressure. The residue was puri- fied by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 70:30). The crude product was dissolved in a mixture of EtOAc 40 mL and Petroleum ether 20 mL at 50 ºC for 20 minutes. The mixture was allowed to cool to 20 ºC, filtered, and the filter cake was dried to afford 3,6-dichloro-1-(3-((3-methyl-4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5- 10 yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (2.3 g) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 400MHz) δ 9.01 (s, 1H), 5.43 (dd, J = 2.8, 10.4 Hz, 1H), 4.70 (t, J = 6.8 Hz, 2H), 4.59 - 4.46(m, 1H), 4.44 - 4.34 (m, 1H), 4.08 - 4.00 (m, 1H), 3.74 - 3.60 (m, 1H), 2.57 - 2.47 (m, 5H), 2.43 - 2.31 (m, 1H), 2.17 - 2.06 (m, 1H), 1.93 - 1.79 (m, 1H), 1.78 - 1.67 (m, 2H), 1.27 (d, J = 6.4 Hz, 1H). 15 To a solution of 3,6-dichloro-1-(3-((3-methyl-4-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H- pyrazol-5-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (2.3 g, 5.0 mmol) in EtOH (300 mL) was added Fe (1.41 g, 25.2 mmol) and then a solution of NH4Cl (1.35 g, 25.2 mmol) in H2O (20 mL). The mixture was stirred at 80 ºC for 32 h. The mixture was concentrated under reduced pres- sure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc20 100:0: → 0:100 followed by DCM:MeOH 100:0 → 90:10) to afford 8-chloro-3-methyl-2,4,12,13- tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]-triazacycloundecine (500 mg) of sufficient purity for the subsequent step.1H NMR (DMSO-d6, 400MHz) δ 11.68 (br s, 1H), 9.33 (s, 1H), 8.77 (s, 1H), 4.44 - 4.24 (m, 4H), 2.18 (s, 3H), 1.82 - 1.72(m, 2H). LC-MS (method C) (m/z) = 306.1 (MH)+ tR = 1.19 minutes. 25 Intermediate: 3,6-Dichloro-1-(3-((1-((2R,4r,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-5-me- thyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000147_0001
To a solution of (2R,4r,6S)-2,6-dimethyltetrahydro-2H-pyran-4-ol (5 g, 38 mmol) in DCM (150 mL) was added PPh3 (30.2 g, 115 mmol) and CBr4 (19.11 g, 57.61 mmol). The mixture was stirred at 20 ºC for 16 h. The reaction mixture was poured into MTBE (100 mL) and filtered. The 5 filter cake was washed with MTBE (50 mL) and the filtrate was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 91:9) to afford (2R,4s,6S)-4-bromo-2,6-dimethyltetrahydro-2H-pyran (2.6 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.73 - 4.68 (m, 1H), 4.05 - 4.01 (m, 2H), 1.99 - 1.94 (m, 2H), 1.72 - 1.67 (m, 2H), 1.22 (d, J = 6.0 Hz, 6H). 10 To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyra- zole (500 mg, 1.59 mmol) in DMF (15 mL) was added (2R,4s,6S)-4-bromo-2,6-dimethyltetrahy- dro-2H-pyran (461 mg, 2.38 mmol), and K2CO3 (659 mg, 4.77 mmol). The mixture was stirred at 80 ºC for 16 h. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (3×40 mL). The combined organic layers were washed with brine (3×40 mL), dried over Na2SO4, 15 and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 40:60) to afford 3-(3-((tert-butyldimethylsi- lyl)oxy)propoxy)-1-((2R,4r,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-5-methyl-4-nitro-1H-py- razole (410 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.40 (t, J = 6.4 Hz, 2H), 4.29 - 4.18 (m, 1H), 3.81 (t, J = 6.0 Hz, 2H), 3.63 - 3.58 (m, 2H), 2.62 (s, 3H), 2.05 20 - 1.99 (m, 2H), 1.95 - 1.83 (m, 2H), 1.79 - 1.71 (m, 2H), 1.28 (d, J = 6.4 Hz, 6H), 0.89 (s, 9H), 0.05 (s, 6H). 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-1-((2R,4r,6S)-2,6-dimethyltetrahydro-2H- pyran-4-yl)-5-methyl-4-nitro-1H-pyrazole (400 mg, 0.935 mmol) was dissolved in HCl/dioxane (4 M, 3 mL) and stirred at 20 ºC for 2 h. The reaction mixture was concentrated under reduced 25 pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 55:45) to afford 3-((1-((2R,4r,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-5- methyl-4-nitro-1H-pyrazol-3-yl)oxy)propan-1-ol (223 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.51 (t, J = 6.0 Hz, 2H), 4.32 - 4.22 (m, 1H), 3.86 (t, J = 5.6 Hz, 2H), 3.70 - 3.55 (m, 2H), 2.65 (s, 3H), 2.14 - 2.06 (m, 2H), 1.95 - 1.73 (m, 4H), 1.30 (d, J = 6.0 Hz, 5 6H). To a solution of 3-((1-((2R,4r,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-5-methyl-4-ni- tro-1H-pyrazol-3-yl)oxy)propan-1-ol (208 mg, 0.664 mmol) and 3,6-dichloro-1H-pyrazolo[3,4- d]pyrimidine (125 mg, 0.664 mmol) in THF (6 mL) was added PPh3 (522 mg, 1.99 mmol). The mixture was cooled to 0 ºC then DIAD (403 mg, 1.99 mmol) was added. The resulting mixture 10 was stirred at 20 ºC for 12 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: pe- troleum ether:EtOAc 100:0 → 75:25) to afford 3,6-dichloro-1-(3-((1-((2R,4r,6S)-2,6-dimethyltet- rahydro-2H-pyran-4-yl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrim- idine (170 mg) of sufficient purity for the subsequent step. 15 Intermediate: 3,6-Dichloro-1-(3-((1-(2,2-dimethyltetrahydro-2H-pyran-4-yl)-5-methyl-4-nitro- 1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000148_0001
20 To a solution of 2,2-dimethyltetrahydropyran-4-ol (250 mg, 1.92 mmol) in toluene (20 mL) was added 2-(tributyl-λ5-phosphanylidene)acetonitrile (1.68 g, 6.97 mmol) and 3-(3-((tert- butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyrazole (550 mg, 1.74 mmol). The mix- ture was stirred at 110 ºC for 15 h. The solvent was removed under reduced pressure. The resi- due was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 70:30) to afford 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-1-(2,2-dimethyltetrahydro-2H-py- ran-4-yl)-5-methyl-4-nitro-1H-pyrazole (230 mg) of sufficient purity for the subsequent step. LC- MS (method B) (m/z) = 428.2 (MH)+ tR = 1.11 minutes. To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-1-(2,2-dimethyltetrahydro- 5 2H-pyran-4-yl)-5-methyl-4-nitro-1H-pyrazole (450 mg, 1.05 mmol) in THF (8 mL) was added TBAF (1 M, 1.58 mL) at 25 ºC. The resulting solution was stirred at 25 ºC for 2 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on sil- ica gel (eluent: petroleum ether:EtOAc 80:20 → 30:70) to afford 3-((1-(2,2-dimethyltetrahydro- 2H-pyran-4-yl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propan-1-ol (285 mg) of sufficient purity 10 for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.51 (t, J = 6.0 Hz, 2H), 4.45 - 4.33 (m, 1H), 3.99 - 3.75 (m, 4H), 2.66 (s, 3H), 2.61 - 2.52 (m, 1H), 2.31 - 2.16 (m, 1H), 2.12 - 2.07 (m, 3H), 1.81 - 1.67 (m, 2H), 1.33 (s, 6H). To a solution of 3-((1-(2,2-dimethyltetrahydro-2H-pyran-4-yl)-5-methyl-4-nitro-1H-py- razol-3-yl)oxy)propan-1-ol (275 mg, 0.878 mmol) and 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine 15 (166 mg, 0.878 mmol) in THF (15 mL) was added PPh3 (691 mg, 2.63 mmol) followed by DIAD (532 mg, 2.63 mmol) at 5 ºC. The resulting mixture was stirred at 25 ºC for 12 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 40:60) to afford 3,6-dichloro-1-(3-((1-(2,2-dimethyltetrahydro-2H-pyran-4-yl)-5-methyl-4-nitro-1H-pyrazol-3- 20 yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (210 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.96 (s, 1H), 4.66 (t, J = 6.4 Hz, 2H), 4.42 - 4.34 (m, 3H), 3.97 - 3.87 (m, 1H), 3.83 - 3.71 (m, 1H), 2.64 (s, 3H), 2.53 - 2.45 (m, 2H), 2.20 - 2.09 (m, 1H), 2.03 - 1.93 (m, 1H), 1.77 - 1.63 (m, 2H), 1.35 - 1.30 (m, 6H). 25 Intermediate: 1-(3-((1-(8-Oxabicyclo[3.2.1]octan-3-yl)-5-methyl-4-nitro-1H-pyrazol-3- yl)oxy)propyl)-3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000150_0001
A mixture of 8-oxabicyclo[3.2.1]octan-3-ol (609 mg, 4.76 mmol), tert-butyl-dimethyl-[3- [(5-methyl-4-nitro-1H-pyrazol-3-yl)oxy]propoxy]silane (1.0 g, 3.17 mmol) and 2-(tributyl-λ5- phosphanylidene)acetonitrile (3.06 g, 12.7 mmol) in toluene (20 mL) was stirred at 110 ºC for 16 5 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 75:25) to afford 1-(8-oxa- bicyclo[3.2.1]octan-3-yl)-3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyra- zole (1.7 g) of sufficient purity for the subsequent step. To 1-(8-oxabicyclo[3.2.1]octan-3-yl)-3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-me- 10 thyl-4-nitro-1H-pyrazole (1.5 g, 2.5 mmol, ~70 mol% purity) in THF (35 mL) was slowly added TBAF (1 M in THF, 6.17 mL). The mixture was stirred at 25 ºC for 16 h. The reaction mixture was extracted with EtOAc (50 mL×3). The combined organic layers were washed with brine (50 mL×2), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 50:50) to afford 15 3-((1-(8-oxabicyclo[3.2.1]octan-3-yl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propan-1-ol (370 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.57-4.56 (m, 1H), 4.51-4.46 (m, 3H), 4.45-4.38 (m, 1H), 3.86-3.83 (m, 2H), 2.63-2.61 (m, 3H), 2.46-2.37 (m, 2H), 2.13-2.06 (m, 2H), 2.04-1.98 (m, 1H), 1.96-1.93 (m, 2H), 1.91-1.86 (m, 1H), 1.84-1.80 (m, 1H), 1.70-1.66 (m, 1H). 20 To a solution of 3-((1-(8-oxabicyclo[3.2.1]octan-3-yl)-5-methyl-4-nitro-1H-pyrazol-3- yl)oxy)propan-1-ol (325 mg, 1.04 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (197 mg, 1.04 mmol), and PPh3 (821 mg, 3.13 mmol) in THF (14 mL) was added DIAD (633 mg, 3.13 mmol) at 0 ºC. The mixture was then stirred at 25 ºC for 16 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: pe-25 troleum ether:EtOAc 100:0 → 50:50) to afford 1-(3-((1-(8-oxabicyclo[3.2.1]octan-3-yl)-5-methyl- 4-nitro-1H-pyrazol-3-yl)oxy)propyl)-3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (620 mg) of suf- ficient purity for the subsequent step. LC-MS (method I) (m/z) = 482.0 (MH)+ tR = 0.55 minutes. Intermediate: 3,6-Dichloro-1-[1,1-dideuterio-3-(5-methyl-4-nitro-1-tetrahydropyran-4-yl-pyra- 5 zol-3-yl)oxy-propyl]pyrazolo[3,4-d]pyrimidine
Figure imgf000151_0001
To a solution of ethyl 3-hydroxypropanoate (5 g, 42 mmol) in THF (60 mL) was added TsOH.H2O (403 mg, 2.12 mmol) and stirred at 20 ºC for 30 minutes. Then 3,4-dihydro-2H-pyran (4.56 g, 54.2 mmol) was added at 0 ºC and the mixture was stirred at 20 ºC for 16 h. The mixture 10 was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 92:8) to afford ethyl 3-((tetrahydro-2H-pyran-2- yl)oxy)propanoate (7.3 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.64 (t, J = 2.8 Hz, 1H), 4.20 - 4.13 (m, 2H), 4.05 - 3.95 (m, 1H), 3.89-3.65 (m, 1H), 3.55 - 3.45 (m, 1H), 3.57 - 3.47 (m, 1H), 2.61 (t, J = 6.4 Hz, 2H), 1.87 - 1.75 (m, 1H), 1.74 - 1.65 (m, 1H), 1.6215 - 1.48 (m, 4H), 1.27 (t, J = 7.2 Hz, 3H). A solution of ethyl 3-((tetrahydro-2H-pyran-2-yl)oxy)propanoate (7.85 g, 38.8 mmol) in THF (50 mL) was added dropwise to a solution of LiAlD4 (2.95 g, 77.6 mmol) in THF (80 mL) at 0 ºC and then the mixture was stirred at 20 ºC for 2 h. The reaction mixture was quenched with water (2 mL), followed by aqueous 10% NaOH (2 mL) and water (2 mL). Then the mixture was20 filtered, and the filtrate was concentrated under reduced pressure to afford 1,1-dideuterio-3- tetrahydropyran-2-yloxy-propan-1-ol (6.28 g) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 400MHz) δ 4.60 (t, J = 2.8 Hz, 1H), 3.99 - 3.84 (m, 2H), 3.65 - 3.50 (m, 2H), 2.49 - 2.23 (m, 1H), 1.89 - 1.70 (m, 4H), 1.62 - 1.52 (m, 4H). To a solution of 1,1-dideuterio-3-tetrahydropyran-2-yloxy-propan-1-ol (3.09 g, 19.125 mmol) in THF (180 mL) was added 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (3 g, 16 mmol), PPh3 (12.49 g, 47.62 mmol) and DIAD (9.63 g, 47.6 mmol) at 0 ºC. The mixture was stirred at 20 ºC for 16 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 80:20) to afford 3,6-dichloro-1-(1,1-dideuterio-3-tetrahydropyran-2-yloxy-propyl)pyrazolo[3,4-d]pyrimidine (4.2 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.97 (s, 1H), 4.50 (t, J = 3.6 Hz, 1H), 3.82 - 3.74 (m, 2H), 3.51 - 3.41 (m, 1H), 3.38 - 3.25 (m, 1H), 2.22 (t, J = 6.0 Hz, 5 2H), 1.79 - 1.65 (m, 2H), 1.61 - 1.49 (m, 4H). To 3,6-dichloro-1-(1,1-dideuterio-3-tetrahydropyran-2-yloxy-propyl)pyrazolo[3,4-d]py- rimidine (4.1 g, 12 mmol) in MeOH (100 mL) was added concentrated aqueous HCl (12 M, 10 mL) and stirred at 20 ºC for 16 h. The reaction mixture was concentrated under reduced pres- sure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc10 100:0 → 79:21) to afford 3,3-dideuterio-3-(3,6-dichloropyrazolo[3,4-d]pyrimidin-1-yl)propan-1- ol (2.6 g) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 400MHz) δ 8.99 (s, 1H), 3.65 (t, J = 6.0 Hz, 2H), 2.30 (br, 1H), 2.14 (t, J = 6.0 Hz, 2H). To a solution of 3,3-dideuterio-3-(3,6-dichloropyrazolo[3,4-d]pyrimidin-1-yl)propan-1- ol (200 mg, 0.803 mmol) in THF (15 mL) was added 5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4- 15 yl)-1H-pyrazol-3-ol (182 mg, 0.803 mmol), DIAD (487 mg, 2.41 mmol), and PPh3 (632 mg, 2.41 mmol) at 0 ºC. The mixture was stirred at 20 ºC for 16 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: pe- troleum ether:EtOAc 100:0 → 35:65) to afford 3,6-dichloro-1-[1,1-dideuterio-3-(5-methyl-4-ni- tro-1-tetrahydropyran-4-yl-pyrazol-3-yl)oxy-propyl]pyrazolo[3,4-d]pyrimidine (680 mg) of suffi-20 cient purity for the subsequent step. Intermediate: 3,6-Dichloro-1-[3,3-dideuterio-3-(5-methyl-4-nitro-1-tetrahydropyran-4-yl-pyra- zol-3-yl)oxy-propyl]pyrazolo[3,4-d]pyrimidine
Figure imgf000152_0001
25 DIAD (1.33 g, 6.60 mmol) was added to a solution of 5-methyl-4-nitro-1-(tetrahydro-2H- pyran-4-yl)-1H-pyrazol-3-ol (500 mg, 2.20 mmol), 1,1-dideuterio-3-tetrahydropyran-2-yloxy- propan-1-ol (428 mg, 2.64 mmol), and PPh3 (1.73 g, 6.60 mmol) in THF (20 mL) at 0 ºC. Then the mixture was stirred at 20 ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 65:35) to afford 3-(1,1-dideuterio-3-tetrahydropyran-2-yloxy-propoxy)-5- 5 methyl-4-nitro-1-tetrahydropyran-4-yl-pyrazole (1 g) of sufficient purity for the subsequent step. All of the material was dissolved in MeOH (10 mL) and aqueous HCl (12 M, 5 mL) was added. The mixture was stirred at 20 ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 0:100) and then preparative HPLC (instrument: Gilson GX-281 Liquid Han- 10 dler, Gilson 322 Pump, Gilson 156 UV detector, column: Xtimate C18150 × 40 mm × 10 μm, mobile Phase: A: water (NH3·H2O+NH4HCO3), mobile phase B: MeCN, gradient: B from 25% to 55% in 8 min, flow rate (mL/min): 55, column temperature: 35 ºC, wavelengths: 220nm, 254nm) to afford 3,3-dideuterio-3-(5-methyl-4-nitro-1-tetrahydropyran-4-yl-pyrazol-3-yl)oxy-propan-1- ol (260 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.28 - 4.18 15 (m, 1H), 4.17 - 4.09 (m, 2H), 3.92 - 3.78 (m, 2H), 3.58 - 3.45 (m, 2H), 2.65 (s, 3H), 2.57 - 2.48 (m, 1H), 2.38 - 2.22 (m, 2H), 2.06 (t, J = 5.6 Hz, 2H), 1.81 - 1.72 (m, 2H). DIAD (450 mg, 2.23 mmol) was added to a solution of 3,6-dichloro-1H-pyrazolo[3,4- d]pyrimidine (140 mg, 0.741 mmol), 3,3-dideuterio-3-(5-methyl-4-nitro-1-tetrahydropyran-4-yl- pyrazol-3-yl)oxy-propan-1-ol(235 mg, 0.812 mmol), and PPh3 (583 mg, 2.22 mmol) in THF (2020 mL) at 0 ºC. Then the mixture was stirred at 20 ºC for 15 h. The reaction mixture was concen- trated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 60:40) to afford 3,6-dichloro-1-[3,3-dideuterio-3-(5-methyl-4- nitro-1-tetrahydropyran-4-yl-pyrazol-3-yl)oxy-propyl]pyrazolo[3,4-d]pyrimidine (700 mg) of suf- ficient purity for the subsequent step. 25 Intermediate: 3,6-Dichloro-1-(3-((1-(3-fluoro-3-methyltetrahydro-2H-pyran-4-yl)-5-methyl-4- nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000153_0001
To a solution of 3-methyltetrahydropyran-4-one (5 g, 44 mmol) and TEA (8.87 g, 87.6 mmol) in DCM (50 mL) was added TMSOTf (14.6 g, 65.7 mmol) at 0 ºC. The mixture was stirred at 0 ºC for 2 h. The reaction mixture was quenched with saturated aqueous NaCl (15 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were dried over Na2SO4 and con- 5 centrated under reduced pressure. The residue was purified by distillation (temperature:80 ºC, pressure: 0.26 mbar) to afford trimethyl((5-methyl-3,6-dihydro-2H-pyran-4-yl)oxy)silane (6.7 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.03 - 3.98 (m, 2H), 3.84 - 3.80 (m, 2H), 2.20 - 2.11 (m, 2H), 1.51 (s, 3H), 0.20 (s, 9H). To a solution of trimethyl((5-methyl-3,6-dihydro-2H-pyran-4-yl)oxy)silane (6.7 g, 36 10 mmol) in CH3CN (70 mL) was added Selectfluor (15.29 g, 43.15 mmol) at 0 ºC. The mixture was stirred at 0 ºC for 2 h. The reaction mixture was diluted with saturated aqueous NaCl (150 mL) and extracted with Et2O (4×50 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by distillation (temperature: 25 ºC, pressure: 20 mBar) to afford 3-fluoro-3-methyltetrahydro-4H-pyran-4-one (3.9 g) of suffi- 15 cient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.00 - 3.93 (m, 2H), 3.92 - 3.88 (m, 1H), 3.73 - 3.69 (m, 1H), 2.89 - 2.80 (m, 1H), 2.62 - 2.58 (m, 1H), 1.55 - 1.46 (m, 3H). To a solution of 2-(2-methyl-1,3-dioxolan-2-yl)acetohydrazide (1.3 g, 8.12 mmol) and 3- fluoro-3-methyltetrahydro-4H-pyran-4-one (2.15 g, 16.2 mmol) in MeOH (30 mL) was added AcOH (975 mg, 16.2 mmol). The reaction mixture was stirred at 20 ºC for 1 h. NaBH3CN 20 (1.53 g, 24.4 mmol) was added, and the reaction was stirred at 60 ºC for 15 h. The mixture was concentrated under reduced pressure and the residue was diluted with EtOAc (50 mL) and washed with H2O (20 mL) and brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to afford N'-(3-fluoro-3-methyltetrahydro-2H-pyran-4-yl)-2-(2-methyl-1,3-di- oxolan-2-yl)acetohydrazide (1.65 g) of sufficient purity for the subsequent step. LC-MS (method25 B) (m/z) = 277.2 (MH)+ tR = 0.57 minutes. To a solution of N'-(3-fluoro-3-methyltetrahydro-2H-pyran-4-yl)-2-(2-methyl-1,3-dioxo- lan-2-yl)acetohydrazide (1.62 g, 5.86 mmol) in EtOH (20 mL) was added TFA (2.67 g, 23.5 mmol), and the reaction was stirred at 90 ºC for 15 h. TFA (2.7 g, 24 mmol) was added to the mixture and the reaction was stirred at 90ºC for another 16 h. The mixture was concentrated under re-30 duced pressure to afford 1-(3-fluoro-3-methyltetrahydro-2H-pyran-4-yl)-5-methyl-1H-pyrazol- 3-ol (1.9 g). All of the material was dissolved in H2SO4 (20 mL) and KNO3 (2.95 g, 29.2 mmol) was added at 0 ºC, and the reaction was stirred at 0 ºC for 0.5 h. The mixture was poured into ice- water (100 mL) and extracted with EtOAc (4×50 mL). The combined organic phases were washed with brine (50 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc (10 v% MeOH) 100:0 → 70:30) to afford 1-(3-fluoro-3-methyltetrahydro-2H-pyran-4-yl)-5-methyl-4-nitro-1H- pyrazol-3-ol (0.14 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.47 5 (br s, 1H), 4.46 - 4.37 (m, 1H), 4.21 - 4.14 (m, 1H), 3.94 - 3.89 (m, 1H), 3.60 - 3.52 (m, 1H), 3.51 - 3.46 (m, 1H), 2.70 (d, J = 1.6 Hz, 3H), 2.67 - 2.65 (m, 1H), 1.99 - 1.92 (m, 1H), 1.36 (d, J = 23.6 Hz, 3H). To a mixture of 3-(3,6-dichloropyrazolo[3,4-d]pyrimidin-1-yl)propan-1-ol (0.12 g, 0.49 mmol), 1-(3-fluoro-3-methyltetrahydro-2H-pyran-4-yl)-5-methyl-4-nitro-1H-pyrazol-3-ol (126 10 mg, 0.49 mmol), and PPh3 (382 mg, 1.46 mmol) in THF (20 mL) was added DIAD (295 mg, 1.46 mmol) at 0 ºC. The mixture was stirred at 20 ºC for 12 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 60:40, repeated twice) to afford 3,6-dichloro-1-(3-((1-(3-fluoro-3-methyl- tetrahydro-2H-pyran-4-yl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]py-15 rimidine (0.2 g) of sufficient purity for the subsequent step. LC-MS (method J) (m/z) = 488.0 (MH)+ tR = 1.64 minutes. Intermediate: 3,6-Dichloro-1-(3-((5-methyl-4-nitro-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H- pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine 20
Figure imgf000155_0001
To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyra- zole (600 mg, 1.90 mmol) and Cs2CO3 (1.86 g, 5.71 mmol) in DMF (10 mL) was added 4-(bromo- methyl)tetrahydro-2H-pyran (511 mg, 2.85 mmol). The mixture was stirred at 80 ºC for 16 h. The reaction mixture was diluted with EtOAc (50 mL) and extracted with H2O (3×40 mL). The com- bined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concen- trated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 70:30) to afford 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5- methyl-4-nitro-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazole (480 mg) of sufficient purity 5 for the subsequent step.1H NMR (CDCl3, 400 MHz) δ 4.38 (t, J = 6.0Hz, 2H), 4.01 - 3.97 (m, 2H), 3.85 - 3.79 (m, 4H), 3.41 - 3.33 (m, 2H), 2.61 (s, 3H), 2.23 - 2.11 (m, 1H), 2.07 - 1.99 (m, 2H), 1.59 - 1.49 (m, 2H), 1.44 - 1.32 (m, 2H), 0.88 (s, 9H), 0.04 (s, 6H). To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1-((tetra- hydro-2H-pyran-4-yl)methyl)-1H-pyrazole (470 mg, 1.14 mmol) in THF (7 mL) was added TBAF10 (1 M in THF, 1.70 mL). The mixture was stirred at 25 ºC for 1 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: pe- troleum ether:EtOAc 100:0 → 40:60) to afford 3-((5-methyl-4-nitro-1-((tetrahydro-2H-pyran-4- yl)methyl)-1H-pyrazol-3-yl)oxy)propan-1-ol (300 mg) of sufficient purity for the subsequent step.1H NMR (400 MHz, CDCl3) δ 4.50 (t, J = 6.0 Hz, 2H), 4.01 - 3.97 (m, 2H), 3.87 - 3.79 (m, 4H), 15 3.40 - 3.34 (m, 2H), 2.61 (s, 3H), 2.22 - 2.12 (m, 1H), 2.10 - 2.05 (m, 2H), 1.55 - 1.48 (m, 2H), 1.44 - 1.32 (m, 2H). To a solution of 3-((5-methyl-4-nitro-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazol- 3-yl)oxy)propan-1-ol (290 mg, 0.97 mmol) and 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (183 mg, 0.97 mmol,) in THF (7 mL) was added PPh3 (762 mg, 2.91 mmol), and DIAD (588 mg, 2.9120 mmol). The mixture was stirred at 25 ºC for 16 h. The mixture was filtered and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: pe- troleum ether:EtOAc 100:0 → 50:50) to afford 3,6-dichloro-1-(3-((5-methyl-4-nitro-1-((tetrahy- dro-2H-pyran-4-yl)methyl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (690 mg) of sufficient purity for the subsequent step. 25 Intermediate: 3-Bromo-8-chloro-2-((1r,4r)-4-methoxycyclohexyl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000157_0001
To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-4-nitro-1H-pyrazole (2.8 g, 9.3 mmol) and cis-4-methoxycyclohexan-1-ol (1.81 g, 13.9 mmol) in toluene (60 mL) was added 2-(tributyl-λ5-phosphanylidene)acetonitrile (8.97 g, 37.2 mmol). The mixture was stirred at 110 5 ºC for 14 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 70:30) to afford 3-(3-((tert- butyldimethylsilyl)oxy)propoxy)-1-((1r,4r)-4-methoxycyclohexyl)-4-nitro-1H-pyrazole (2.54 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.01 (s, 1H), 4.42 (t, J = 6.4 Hz, 2H), 4.01-3.91 (m, 1H), 3.82 (t, J = 6.0 Hz, 2H), 3.40 (s, 3H), 3.30 - 3.20 (m, 1H), 2.27 - 2.28 10 (m, 4H), 2.07 - 2.01 (m, 2H), 1.83 - 1.70 (m, 2H), 1.48 - 1.34 (m, 2H), 0.89 (s, 9H), 0.05 (s, 6H). To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-1-((1r,4r)-4-methoxycyclo- hexyl)-4-nitro-1H-pyrazole (2.4 g, 5.8 mmol) in THF (50 mL) was added TBAF (1 M in THF, 8.7 mL) and then stirred at 20 ºC for 1 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc (MeOH15 10v%) 100:0 → 50:50) to afford 3-((1-((1r,4r)-4-methoxycyclohexyl)-4-nitro-1H-pyrazol-3- yl)oxy)propan-1-ol (1.7 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.03 (s, 1H), 4.51 (t, J = 6.0 Hz, 2H), 4.02 - 3.93 (m, 1H), 3.85 (t, J = 5.6 Hz, 2H), 3.39 (s, 3H), 3.29 - 3.20 (m, 1H), 2.28 - 2.18 (m, 4H), 2.14 - 2.06 (m, 2H), 1.84 - 1.71 (m, 2H), 1.47 - 1.36 (m, 2H). To a mixture of 3-((1-((1r,4r)-4-methoxycyclohexyl)-4-nitro-1H-pyrazol-3-yl)oxy)propan-20 1-ol (1.7 g, 5.7 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (1.07 g, 5.68 mmol) and PPh3 (4.47 g, 17.0 mmol) in THF (120 mL) was added DIAD (3.45 g, 17.0 mmol) at 0 ºC. The mixture was stirred at 20 ºC for 11 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 60:40) to afford 3,6-dichloro-1-(3-((1-((1r,4r)-4-methoxycyclohexyl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)- 25 1H-pyrazolo[3,4-d]pyrimidine (1.24 g) of sufficient purity for the subsequent step. LC-MS (method J) (m/z) = 470.0 (MH)+ tR = 1.60 minutes. To a mixture of 3,6-dichloro-1-(3-((1-((1r,4r)-4-methoxycyclohexyl)-4-nitro-1H-pyrazol- 3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (1.24 g, 2.64 mmol) and NH4Cl (705 mg, 13.2 mmol) in a mixture of EtOH (150 mL) and H2O (15 mL) was added Fe (736 mg, 13.2 mmol) at 20 ºC. The reaction was stirred at 80 ºC for 16 h. The mixture was filtered and concentrated under 5 reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc (MeOH 10v%) 100:0 → 50:50) to afford 8-chloro-2-((1r,4r)-4-methoxycyclohexyl)- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine (0.8 g) of sufficient purity for the subsequent step. LC-MS (method J) (m/z) = 404.0 (MH)+ tR = 1.40 minutes. 10 A solution of NBS (463 mg, 2.60 mmol) in THF (10 mL) was added dropwise to a solution of 8-chloro-2-((1r,4r)-4-methoxycyclohexyl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)di- pyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (0.7 g, 1.73 mmol) in THF (70 mL) over 0.5 h at 5 ºC. The mixture was stirred for 0.5 hat 5 ºC and concentrated under reduced pres- sure. The residue was diluted with EtOAc (100 mL) and washed with brine (50 mL×3), dried, and 15 concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc (DCM 10v%) 100:0 → 50:50) and preparative SFC (instrument: Thar 200, column: DAICEL CHIRALCEL OJ-H 250 × 30 mm, 5 μm, mobile phase: supercritical CO2/EtOH (0.1% NH3.H2O, v%)= 80/20, flow rate: 60 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60ºC, evaporator temperature: 20 ºC, trimmer tempera-20 ture: 25 ºC, wavelength: 220 nm) to afford 3-Bromo-8-chloro-2-((1r,4r)-4-methoxycyclohexyl)- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine (0.26 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.62 (s, 1H), 6.79 (s, 1H), 4.43 (t, J = 4.4 Hz, 2H), 4.36 (t, J = 4.8 Hz, 2H), 4.15 - 4.04 (m, 1H), 3.31 (s, 3H), 3.24 - 3.12 (m, 1H), 2.26 - 2.09 (m, 2H), 2.04 - 1.80 (m, 6H), 1.39 - 1.24 (m, 2H). LC-MS25 (method C) (m/z) = 484.1 (MH)+ tR = 1.69 minutes. Intermediate: (+)-cis-8-Chloro-2-(2-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine and (–)-cis- 8-Chloro-2-(2-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome-30 theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000159_0001
To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-4-nitro-1H-pyrazole (5 g, 16.6 mmol) and trans-2-methyltetrahydro-2H-pyran-4-ol (2.31 g, 19.9 mmol) in toluene (150 mL) was added 2-(tributyl-λ5-phosphanylidene)acetonitrile (16.01 g, 66.35 mmol). The resulting 5 mixture was stirred at 110 ºC for 16 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 65:35) to afford cis-3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-1-(2-me- thyltetrahydro-2H-pyran-4-yl)-4-nitro-1H-pyrazole (4.6 g) of sufficient purity for the subsequent step. 10 To a solution of cis-3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-1-(2-methyltetrahydro- 2H-pyran-4-yl)-4-nitro-1H-pyrazole (4.6 g, 11.5 mmol) in THF (90 mL) was added TBAF (1 M in THF, 17.3 mL). The mixture was stirred at 20 ºC for 2 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 0:100) to afford cis- 3-((1-(2-methyltetrahydro-2H-pyran-4-yl)-4-nitro-1H- 15 pyrazol-3-yl)oxy)propan-1-ol (3 g) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 400MHz) δ 8.05 (s, 1H), 4.51 (t, J=6.0 Hz, 2H), 4.25 - 4.13 (m, 2H), 3.85 (t, J=5.6 Hz, 2H), 3.63 - 3.47 (m, 2H), 2.19 - 2.13 (m, 1H), 2.11 - 2.06 (m, 3H), 2.00 - 1.88 (m, 1H), 1.71 - 1.59 (m, 1H), 1.29 (d, J=6.4 Hz, 3H). To a solution of cis-3-((1-(2-methyltetrahydro-2H-pyran-4-yl)-4-nitro-1H-pyrazol-3- 20 yl)oxy)propan-1-ol (3.25 g, 11.4 mmol) and 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (2.15 g, 11.4 mmol) in THF (250 mL) was added PPh3 (8.96 g, 34.2 mmol) followed by DIAD (6.91 g, 34.2 mmol) at 5 ºC. The resulting mixture was stirred at 20 ºC for 16 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 50:50) to afford cis-3,6-25 dichloro-1-(3-((1-(2-methyltetrahydro-2H-pyran-4-yl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H- pyrazolo[3,4-d]pyrimidine (2.75 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.97 (s, 1H), 8.03 (s, 1H), 4.67 (t, J=6.4 Hz, 2H), 4.38 (t, J=6.0 Hz, 2H), 4.19 - 4.12 (m, 2H), 3.62 - 3.51 (m, 2H), 2.53 – 2.45 (m, 2H), 2.15 – 2.10 (m, 1H), 2.08 - 2.01 (m, 1H), 1.97 - 1.84 (m, 1H), 1.67 - 1.62 (m, 1H), 1.30 (d, J=6.8 Hz, 3H). To a suspension of cis-3,6-dichloro-1-(3-((1-(2-methyltetrahydro-2H-pyran-4-yl)-4-ni- 5 tro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (1.87 g, 4.10 mmol) in EtOH (200 mL) and H2O (50 mL) was added Fe (1.14 g, 20.5 mmol) and NH4Cl (1.10 g, 20.5 mmol). The mix- ture was stirred at 80 ºC for 16 h. The reaction mixture was filtered and the filtrate was concen- trated under reduced pressure. The material was suspended in DCM (100 mL), filtered and con- centrated under reduced pressure. The residue was purified by chromatography on silica gel (el-10 uent: petroleum ether:EtOAc (MeOH 10 v%) 80:20 → 20:80) to afford and cis-8-chloro-2-(2-me- thyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4- b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (1.1 g). cis-8-Chloro-2-(2-methyltetrahydro-2H-pyran- 4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triaza- cycloundecine (1.5 g) was separated using preparative SFC (instrument: Berger MultiGram II, 15 column: DAICEL CHIRALPAK AD 250 × 30 mm, 10 μm, mobile phase: supercritical CO2/EtOH (0.1% NH3.H2O, v%)= 45/55, flow rate: 80 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator temperature: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to afford: (+)-cis-8-Chloro-2-(2-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- 20 (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (500 mg). 1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 7.27 (s, 1H), 7.06 (br s, 1H), 4.54 - 4.47 (m, 2H), 4.47 - 4.41 (m, 2H), 4.23 - 4.10 (m, 2H), 3.62 - 3.51 (m, 2H), 2.18 - 2.12 (m, 1H), 2.10 - 2.04 (m, 1H), 2.03 - 1.87 (m, 3H), 1.71 - 1.60 (m, 1H), 1.28 (d, J = 6.0 Hz, 3H). LC-MS (method C) (m/z) = 390.2 (MH)+ tR = 1.43 minutes. Chiral analytical SFC conditions (instrument: Waters UPCC with PDA Detector, col-25 umn: Chiralpak AD-350 × 4.6mm I.D., 3μm, mobile phase: A: CO2 B: ethanol (0.05% DEA), gradi- ent: from 5% to 40% of B in 2 min and hold 40% for 1.2 min, then 5% of B for 0.8 min, flow rate: 4 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 4 min, wavelength: 220 nm), ee > 99 %, tR = 2.39 minutes. (c = 0.5 g/100 mL, CHCl3)
Figure imgf000160_0001
and the corresponding enantiomer 30 (–)-cis-8-Chloro-2-(2-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (550 mg). 1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 7.28 (s, 1H), 7.01 (br s, 1H), 4.55 - 4.47 (m, 2H), 4.47 - 4.42 (m, 2H), 4.22 - 4.11 (m, 2H), 3.61 - 3.53 (m, 2H), 2.20 - 2.11 (m, 1H), 2.10 - 2.04 (m, 1H), 2.03 - 1.88 (m, 3H), 1.65 - 1.60 (m, 1H), 1.28 (d, J = 6.0 Hz, 3H). LC-MS (method C) (m/z) = 390.2 (MH)+ tR = 1.42 minutes. Chiral analytical SFC conditions (instrument: Waters UPCC with PDA Detector, col- umn: Chiralpak AD-350 × 4.6mm I.D., 3μm, mobile phase: A: CO2 B: ethanol (0.05% DEA), gradi- ent: from 5% to 40% of B in 2 min and hold 40% for 1.2 min, then 5% of B for 0.8 min, flow rate: 5 4 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 4 min, wavelength: 220 nm), ee > 99 %, tR = 2.85 minutes. [^]^ = -12 (c = 0.2 g/100 mL, CHCl3). Intermediate: 3-Bromo-8-chloro-2-((2R,4R) or (2S,4S)-2-methyltetrahydro-2H-pyran-4-yl)- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy-10 cloundecine, peak 1
Figure imgf000161_0001
To a solution of (+)-cis-8-chloro-2-(2-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetra- hydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (520 mg, 1.33 mmol) in THF (19 mL,) was added, over a period of 13 min, a solution of NBS (268 mg, 15 1.51 mmol) in THF (4 mL) at room temperature. The mixture was stirred at room temperature for 1 h. Water (5 mL) was slowly added at room temperature, and the reaction mixture was concentrated under reduced pressure to remove most of the THF. The mixture was filtered and the filter cake was washed with water and dried before being concentrated under reduced pres- sure to afford 3-bromo-8-chloro-2-((2R,4R) or (2S,4S)-2-methyltetrahydro-2H-pyran-4-yl)-20 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine, peak1 (559 mg) of sufficient purity for the next step. Intermediate: 3-Bromo-8-chloro-2-((2R,4R) or (2S,4S)-2-methyltetrahydro-2H-pyran-4-yl)- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy-25 cloundecine, peak 2
Figure imgf000161_0002
To a solution of (–)-cis-8-chloro-2-(2-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetra- hydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (475 mg, 1.22 mmol) in THF (19 mL,) was added, over a period of 13 min, a solution of NBS (239 mg, 1.34 mmol) in THF (4 mL) at room temperature. The mixture was stirred at room temperature 5 for 1 h. Water (5 mL) was slowly added at room temperature, and the reaction mixture was concentrated on to remove most of the THF. The mixture was filtered and the filter cake was washed with water and dried before being concentrated to afford 3-bromo-8-chloro-2-((2R,4R) or (2S,4S)-2-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak2 (473 mg) of sufficient10 purity for the next step. Intermediate: 3,6-Dichloro-1-(3-((5-methyl-4-nitro-1-(4-oxaspiro[2.5]octan-7-yl)-1H-pyrazol-3- yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000162_0001
To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1H-pyra-15 zole (900 mg, 2.85 mmol) in toluene (30 mL) was added 2-(tributyl-λ5-phosphanylidene)acetoni- trile (2.75 g, 11.4 mmol) and 4-oxaspiro[2.5]octan-7-ol (439 mg, 3.42 mmol, prepared in a man- ner similar to Mathur, A. et al. J. Org. Chem., 2017, 82, 10376-10387). The mixture was stirred at 110 ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 80:20) to20 afford 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1-(4-oxaspiro[2.5]octan-7- yl)-1H-pyrazole (600 mg) of sufficient purity for the subsequent step. LC-MS (method B) (m/z) = 426.2 (MH)+ tR = 1.11 minutes. To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-methyl-4-nitro-1-(4-ox- aspiro[2.5]octan-7-yl)-1H-pyrazole (590 mg, 1.39 mmol) in THF (10 mL) was added TBAF (1 M in25 THF, 2.08 mL). The mixture was stirred at 20 ºC for 1 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: pe- troleum ether:EtOAc 100:0 → 50:50) to afford give 3-((5-methyl-4-nitro-1-(4-oxaspiro[2.5]octan- 7-yl)-1H-pyrazol-3-yl)oxy)propan-1-ol (400 mg) of sufficient purity for the subsequent step. LC- MS (method B) (m/z) = 312.1 (MH)+ tR = 0.77 minutes. 5 DIAD (760 mg, 3.76 mmol) was added to a solution of 3-((5-methyl-4-nitro-1-(4-ox- aspiro[2.5]octan-7-yl)-1H-pyrazol-3-yl)oxy)propan-1-ol (390 mg, 1.25 mmol), 3,6-dichloro-1H- pyrazolo[3,4-d]pyrimidine (237 mg, 1.25 mmol), and PPh3 (986 mg, 3.76 mmol) in THF (20 mL) at 0 ºC. Then the mixture was stirred at 20 ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: pe-10 troleum ether:EtOAc 100:0 → 60:40) to afford 3,6-dichloro-1-(3-((5-methyl-4-nitro-1-(4-ox- aspiro[2.5]octan-7-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (500 mg) of sufficient purity for the subsequent step. Intermediate: trans-3,6-Dichloro-1-(3-((5-ethyl-1-(3-fluorotetrahydro-2H-pyran-4-yl)-4-nitro-15 1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000163_0001
A solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-ethyl-4-nitro-1H-pyrazole (1 g, 3.0 mmol), cis-3-fluorotetrahydro-2H-pyran-4-ol (547 mg, 4.55 mmol) and 2-(tributylphospho- ranylidene)acetonitrile (2.93 g, 12.1 mmol) in toluene (40 mL) was stirred at 110 ºC for 15 h. The 20 mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 95:5 → 80:20) to afford trans-3-(3-((tert-butyldime- thylsilyl)oxy)propoxy)-5-ethyl-1-(3-fluorotetrahydro-2H-pyran-4-yl)-4-nitro-1H-pyrazole (950 mg) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 5.07 - 4.83 (m, 1H), 4.42 (t, J = 6.4 Hz, 2H), 4.34 - 4.15 (m, 2H), 4.13 - 4.02 (m, 1H), 3.82 (t, J = 6.0 Hz, 2H), 3.55 - 3.4625 (m, 1H), 3.44 - 3.34 (m, 1H), 3.15 - 2.98 (m, 2H), 2.55 - 2.42 (m, 1H), 2.09 - 2.00 (m, 2H), 1.95 - 1.86 (m, 1H), 1.28 (t, J = 7.6 Hz, 3H), 0.89 (s, 9H), 0.05 (s, 6H). To a solution of trans-3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-ethyl-1-(3-fluorotet- rahydro-2H-pyran-4-yl)-4-nitro-1H-pyrazole (950 mg, 2.20 mmol) in THF (15 mL) was added TBAF (3.30 mL, 1M in THF) at 0 ºC and the reaction was stirred at 25 ºC for 2 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel 5 (eluent: petroleum ether:EtOAc 95:5 → 50:50) to afford trans-3-((5-ethyl-1-(3-fluorotetrahydro- 2H-pyran-4-yl)-4-nitro-1H-pyrazol-3-yl)oxy)propan-1-ol (600 mg) of sufficient purity for the sub- sequent step.1H NMR (CDCl3, 400MHz) δ 5.06 - 4.83 (m, 1H), 4.50 (t, J = 6.0 Hz, 2H), 4.34 - 4.16 (m, 2H), 4.13 - 4.04 (m, 1H), 3.88 (t, J = 5.6 Hz, 2H), 3.56 - 3.46 (m, 1H), 3.44 - 3.34 (m, 1H), 3.21 - 2.94 (m, 2H), 2.55 - 2.40 (m, 1H), 2.18 - 2.02 (m, 2H), 1.97 - 1.87 (m, 1H), 1.28 (t, J = 7.6 Hz, 3H). 10 To a solution of trans-3-((5-ethyl-1-(3-fluorotetrahydro-2H-pyran-4-yl)-4-nitro-1H-pyra- zol-3-yl)oxy)propan-1-ol (600 mg, 1.89 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (357 mg, 1.89 mmol) and PPh3 (992 mg, 3.78 mmol) in THF (20 mL) was added DIAD (765 mg, 3.78 mmol) in a dropwise manner at 0 ºC. The mixture was stirred at 25 ºC for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel15 (eluent: petroleum ether:EtOAc 80:20 → 50:50) to afford trans-3,6-dichloro-1-(3-((5-ethyl-1-(3- fluorotetrahydro-2H-pyran-4-yl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimi- dine (830 mg) of sufficient purity for the subsequent step. LC-MS (method B) (m/z) = 488.1 (MH)+ tR = 0.94 minutes. 20 Intermediate: 3,6-Dichloro-1-(3-((5-ethyl-1-((1r,4r)-4-methoxycyclohexyl)-4-nitro-1H-pyrazol-3- yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine
Figure imgf000164_0001
A mixture of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-ethyl-4-nitro-1H-pyrazole (125 g, 3.0 mmol), cis-4-methoxycyclohexanol (790 mg, 6.07 mmol) and 2-(tributylphosphoranyli- dene)acetonitrile (2.93 g, 12.1 mmol) in toluene (40 mL) was stirred at 110 ºC for 15 h. The mix- ture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 95:5 → 80:20) to afford 3-(3-((tert-butyldimethylsi- lyl)oxy)propoxy)-5-ethyl-1-((1r,4r)-4-methoxycyclohexyl)-4-nitro-1H-pyrazole (700 mg) of sufficient 5 purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 4.39 (t, J = 6.4 Hz, 2H), 4.03 - 3.91 (m, 1H), 3.82 (t, J = 6.4 Hz, 2H), 3.39 (s, 3H), 3.33 - 3.20 (m, 1H), 3.03 (q, J = 7.6 Hz, 2H), 2.30 - 2.19 (m, 2H), 2.15 - 2.01 (m, 4H), 1.91 - 1.81 (m, 2H), 1.46 - 1.34 (m, 2H), 1.26 (t, J = 6.8 Hz, 3H), 0.89 (s, 9H), 0.05 (s, 6H). To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-ethyl-1-((1r,4r)-4-methoxycy- 10 clohexyl)-4-nitro-1H-pyrazole (700 mg, 1.59 mmol) in THF (15 mL) was added TBAF (2.38 mL, 1M in THF) at 0 ºC and the reaction was stirred at 30 ºC for 2 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 95:5 → 50:50) to afford 3-((5-ethyl-1-((1r,4r)-4-methoxycyclohexyl)-4-nitro-1H-pyrazol- 3-yl)oxy)propan-1-ol (500 mg) of sufficient purity for the subsequent step. 1H NMR (CDCl3,15 400MHz) δ 4.50 (t, J = 6.0 Hz, 2H), 4.05 - 3.93 (m, 1H), 3.83 (t, J = 5.6 Hz, 2H), 3.39 (s, 3H), 3.31 - 3.19 (m, 1H), 3.04 (q, J = 7.6 Hz, 2H), 2.30 - 2.20 (m, 2H), 2.09 - 2.04 (m, 4H), 1.93 - 1.83 (m, 2H), 1.42 - 1.32 (m, 2H), 1.27 (t, J = 7.2 Hz, 3H). To a solution of 3-((5-ethyl-1-((1r,4r)-4-methoxycyclohexyl)-4-nitro-1H-pyrazol-3-yl)oxy)pro- pan-1-ol (500 mg, 1.53 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (289 mg, 1.53 mmol), 20 and PPh3 (801 mg, 3.05 mmol) in THF (20 mL) was added dropewise DIAD (618 mg, 3.05 mmol) at 0 ºC. The mixture was stirred at 25 ºC for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 80:20 → 50:50) to afford 3,6-dichloro-1-(3-((5-ethyl-1-((1r,4r)-4-methoxycyclo- hexyl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (1 g) of sufficient purity25 for the subsequent step. LC-MS (method B) (m/z) = 498.1 (MH)+ tR = 0.97 minutes. Intermediate: 3-Bromo-8-chloro-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 30
Figure imgf000166_0001
To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-4-nitro-1H-pyrazole (6 g, 20 mmol) and oxepan-4-ol (3.47 g, 29.9 mmol) in toluene (120 mL) was added 2-(tributyl-λ5-phos- phanylidene)acetonitrile (19.22 g, 79.6 mmol). The mixture was stirred at 110 ºC for 15 h. The 5 mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 30:70) to afford 3-(3-((tert-butyldimethylsi- lyl)oxy)propoxy)-4-nitro-1-(oxepan-4-yl)-1H-pyrazole (7.2 g) of sufficient purity for the subse- quent step.1H NMR (CDCl3, 400MHz) δ 8.04 (s, 1H), 4.47 - 4.37 (m, 2H), 4.29 - 4.20 (m, 1H), 3.94 - 3.79 (m, 4H), 3.77 - 3.67 (m, 2H), 2.30 - 2.10 (m, 4H), 2.05 - 1.99 (m, 2H), 1.95 - 1.79 (m, 2H),10 0.91 - 0.87 (m, 9H), 0.09 - 0.02 (m, 6H) To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-4-nitro-1-(oxepan-4-yl)-1H- pyrazole (7.4 g, 18.5 mmol) in THF (150 mL) was added TBAF (1 M in THF, 28 mL) and then stirred at 20 ºC for 1 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc (MeOH 10 v%) 100:0 → 50:50) 15 to afford 3-((4-nitro-1-(oxepan-4-yl)-1H-pyrazol-3-yl)oxy)propan-1-ol (5.1 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.06 (s, 1H), 4.51 (t, J = 6.0 Hz, 2H), 4.31 - 4.19 (m, 1H), 3.94 - 3.80 (m, 4H), 3.77 - 3.67 (m, 2H), 2.58 (br s, 1H), 2.31 - 2.06 (m, 6H), 1.94 - 1.79 (m, 2H). To a mixture of 3-((4-nitro-1-(oxepan-4-yl)-1H-pyrazol-3-yl)oxy)propan-1-ol (1.7 g, 6.0 20 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (1.13 g, 6.0 mmol) and PPh3 (4.69 g, 17.9 mmol) in THF (100 mL) was added DIAD (3.61 g, 17.9 mmol) at 0 ºC. The mixture was stirred at 20 ºC for 16 h. The mixture was concentrated under reduced pressure. The residue was puri- fied by chromatography on silica gel (eluent: petroleum ether:EtOAc 100:0 → 40:60) to afford 3,6-dichloro-1-(3-((4-nitro-1-(oxepan-4-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]py-25 rimidine (3.3 g) of sufficient purity for the subsequent step. LC-MS (method J) (m/z) = 455.9 (MH)+ tR = 1.54 minutes. To a mixture of 3,6-dichloro-1-(3-((4-nitro-1-(oxepan-4-yl)-1H-pyrazol-3-yl)oxy)propyl)- 1H-pyrazolo[3,4-d]pyrimidine (3.5 g, 7.7 mmol) and NH4Cl (1.02 g, 19.1 mmol) in EtOH (150 mL) and H2O (30 mL) was added Fe (1.07 g, 19.1 mmol) at 20 ºC. The mixture was stirred at 80 ºC for 16 h, filtered, and concentrated under reduced pressure. The residue was purified 5 by chromatography on silica gel (eluent: petroleum ether:EtOAc (DCM 10v%) 100:0 → 50:50) to afford 8-chloro-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4- b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (1.5 g) of sufficient purity for the subsequent step.1H NMR (DMSO-d6, 400MHz) δ 9.63 (s, 1H), 8.84 (s, 1H), 7.53 (s, 1H), 4.53 - 4.37 (m, 4H), 4.35 - 4.25 (m, 1H), 3.88 - 3.74 (m, 2H), 3.74 - 3.59 (m, 2H), 2.21 - 2.11 (m, 2H), 2.09 - 2.01 (m, 2H), 2.0 -10 1.88 (m, 2H), 1.87 - 1.75 (m, 2H). A solution of NBS (685 mg, 3.85 mmol) in THF (5 mL) was added dropwise to a solution of 8-chloro-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4- b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (1 g, 2.6 mmol) in THF (50 mL) at 25 ºC. The mixture was stirred for 1 h at 25 ºC. The mixture was concentrated under reduced pressure. The residue 15 was diluted with EtOAc (50 mL). The organic phase was washed with brine (20 mL×3), dried and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether:EtOAc (DCM 10v%) 100:0 → 80:20) to afford 3-bromo-8-chloro-2-(ox- epan-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine (0.8 g) of sufficient purity for the subsequent step.1H NMR 20 (400MHz, CDCl3) δ 8.63 (s, 1H), 6.90 - 6.71 (m, 1H), 4.50 - 4.39 (m, 3H), 4.38 - 4.32 (m, 2H), 3.88 - 3.76 (m, 2H), 3.75 - 3.66 (m, 1H), 3.65 - 3.56 (m, 1H), 2.36 - 2.23 (m, 1H), 2.23 - 2.13 (m, 1H), 2.13 - 2.02 (m, 1H), 2.0-1.9 (m, 3H), 1.88 - 1.73 (m, 2H). Intermediate: 3-Bromo-8-chloro-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahydro-11H-5,7-25 (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000167_0001
To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-4-nitro-1H-pyrazole (0.56 g, 1.84 mmol) in toluene (11 mL) was added tetrahydro-2H-pyran-3-ol (0.261 mL, 2.76 mmol) and 2-(tributyl-λ5-phosphanylidene)acetonitrile (1.98 g, 8.20 mmol). The mixture was stirred at 110 ºC for 15 h. The reaction mixture was concentrated under reduced pressure. The 5 residue was purified by chromatography on silica gel (eluent: heptane:EtOAc 100:0 → 0:100) to afford 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-4-nitro-1-(tetrahydro-2H-pyran-3-yl)-1H-pyra- zole (412 mg) of sufficient purity for the subsequent step. To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-4-nitro-1-(tetrahydro-2H- pyran-3-yl)-1H-pyrazole (412 mg, 0.930 mmol) in THF (15.1 mL) was added aqueous HCl (0.80 10 mL, 12 M, 9.6 mmol). The mixture was stirred at room temperature for 30 minutes. The mixture was concentrated under reduced pressure and saturated aqueous NaHCO3 was added to adjust pH=8-9, extracted with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: heptane:EtOAc 100:0 → 0:100) to afford 3-((4-nitro-1-(tetrahydro-2H-pyran-15 3-yl)-1H-pyrazol-3-yl)oxy)propan-1-ol (261 mg) of sufficient purity for the subsequent step. To a reaction vessel was added DIAD (355 µL, 1.83 mmol) and the atmosphere was ex- changed for argon. THF (12 mL) was added, and the solution was cooled to 0 ºC. Then, tri- phenylphosphine (on resin, loading: 1.6 mmol/g) (1.18 g, 1.89 mmol), was added and the solu- tion was stirred under argon for 5 minutes. At this point 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimi-20 dine (223 mg, 1.09 mmol) was added followed by the addition of 3-((4-nitro-1-(tetrahydro-2H- pyran-3-yl)-1H-pyrazol-3-yl)oxy)propan-1-ol (261 mg, 0.914 mmol) as a solution in THF (4 mL). The mixture was allowed to reach room temperature and was stirred overnight at room tem- perature. The mixture was filtered and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: heptane:EtOAc 100:0 → 0:100) to afford 3,6-25 dichloro-1-(3-((4-nitro-1-(tetrahydro-2H-pyran-3-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyra- zolo[3,4-d]pyrimidine (498 mg) of sufficient purity for the subsequent step. To a solution of 3,6-dichloro-1-(3-((4-nitro-1-(tetrahydro-2H-pyran-3-yl)-1H-pyrazol-3- yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (430 mg, 0.914 mmol) in EtOH (130 mL) and H2O (10 mL,) was added Fe (570 mg, 10.2 mmol) and NH4Cl (500 mg, 9.35 mmol). The mixture was 30 stirred at 60 ºC overnight, followed by 80 ºC for 23 h. The reaction mixture was filtered through celite and then concentrated under reduced pressure. The residue was purified by chromatog- raphy on silica gel (eluent: heptane:EtOAc 100:0 → 0:100) to afford 8-chloro-2-(tetrahydro-2H- pyran-3-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine (214 mg) of sufficient purity for the subsequent step. To a solution of 8-chloro-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (532 mg, 1.37 5 mmol) in THF (19 mL) was added NBS (276 mg, 1.55 mmol) as a solution in THF (4 mL) over a period of 13 minutes at room temperature. The mixture was stirred at room temperature for 1 h. Water (5 mL) was slowly added at room temperature, and the reaction mixture was concen- trated under reduced pressure to remove most of the THF. The mixture was filtered and the filter cake was washed with water and dried to afford 3-bromo-8-chloro-2-(tetrahydro-2H-py-10 ran-3-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine (492 mg) of sufficient purity for the subsequent step. Intermediate: 8-Chloro-2-((3R,4S) or (3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)-2,4,12,13-tetra- 15 hydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak 1 and 8-Chloro-2-((3R,4S) or (3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak 2
Figure imgf000169_0001
20 To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-4-nitro-1H-pyrazole (6 g, 20 mmol) and cis-3-fluorotetrahydro-2H-pyran-4-ol:trans-3-fluorotetrahydro-2H-pyran-4-ol (cis/trans=3:1, 3.59 g, 29.9 mmol) in toluene (120 mL) was added 2-(tributyl-λ5-phosphanyli- dene)acetonitrile (19.22 g, 79.6 mmol). The mixture was stirred at 110 ºC for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on sil-25 ica gel (eluent: Petroleum ether:EtOAc 100:0 → 30:70) to afford trans-3-(3-((tert-butyldime- thylsilyl)oxy)propoxy)-1-(3-fluorotetrahydro-2H-pyran-4-yl)-4-nitro-1H-pyrazole (5.6 g) of suffi- cient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.12 (s, 1H), 4.93 - 4.72 (m, 1H), 4.44 (t, J = 6.4 Hz, 2H), 4.27 (dd, J = 5.6, 11.6 Hz, 1H), 4.12 - 4.04 (m, 2H), 3.83 (t, J = 5.6 Hz, 2H), 3.55 - 3.45 (m, 1H), 3.43 - 3.34 (m, 1H), 2.44 - 2.33 (m, 1H), 2.18 - 2.09 (m, 1H), 2.06 - 2.00 5 (m, 2H), 0.89 (s, 9H), 0.05 (s, 6H). To a solution of trans-3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-1-(3-fluorotetrahydro- 2H-pyran-4-yl)-4-nitro-1H-pyrazole (5.47 g, 13.6 mmol) in THF (100 mL) was added TBAF (1 M in THF, 20 mL) and then stirred at 20 ºC for 1 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: Petroleum10 ether:EtOAc (MeOH, 10 v%) 100:0 → 50:50) to afford trans-3-((1-(3-fluorotetrahydro-2H-pyran- 4-yl)-4-nitro-1H-pyrazol-3-yl)oxy)propan-1-ol (3.6 g) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 400MHz) δ 8.14 (s, 1H), 4.93 - 4.71 (m, 1H), 4.51 (t, J = 6.0 Hz, 2H), 4.27 (dd, J = 5.2, 11.2 Hz, 1H), 4.21 - 4.14 (m, 1H), 4.10 - 4.04 (m, 1H), 3.87 (t, J = 6.0 Hz, 2H), 3.55 - 3.45 (m, 1H), 3.43 - 3.33 (m, 1H), 2.43 - 2.33 (m, 1H), 2.18 - 2.13 (m, 1H), 2.12 - 2.07 (m, 2H). 15 To a mixture of trans-3-((1-(3-fluorotetrahydro-2H-pyran-4-yl)-4-nitro-1H-pyrazol-3- yl)oxy)propan-1-ol (3.6 g, 135 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (2.35 g, 12.5 mmol), and PPh3 (9.79 g, 37.3 mmol) in THF (200 mL) was added DIAD (7.55 g, 37.3 mmol) at 0 ºC. The mixture was stirred at 20 ºC for 11 h. The mixture was concentrated under reduced pres- sure. The residue was purified by chromatography on silica gel (eluent: Petroleum ether:EtOAc20 (MeOH, 10 v%) 100:0 → 50:50) to afford trans-3,6-dichloro-1-(3-((1-(3-fluorotetrahydro-2H-py- ran-4-yl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (3 g) of sufficient pu- rity for the subsequent step. LC-MS (method J) (m/z) = 482.0 (M+Na)+ tR = 1.54 minutes. To a mixture of trans-3,6-dichloro-1-(3-((1-(3-fluorotetrahydro-2H-pyran-4-yl)-4-nitro- 1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (3 g, 6.5 mmol) and NH4Cl (1.74 g, 25 32.6 mmol) in a mixture of EtOH (600 mL) and H2O (100 mL) was added Fe (1.82 g, 32.6 mmol) at 20 ºC under. The mixture was stirred at 80 ºC for 16 h. Additional Fe (1.82 g) and NH4Cl (1.74 g) were added to the mixture and stirred at 80 ºC for another 16 h. The mixture was filtered and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: Petroleum ether:EtOAc (DCM, 10 v%) 100:0 → 50:50) to afford trans-8-chloro-2-(3-30 fluorotetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4- b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (1.6 g). trans-8-Chloro-2-(3-fluorotetrahydro-2H-py- ran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine (1.6 g) was separated using preparative SFC (instrument: Thar SFC Prep 200, column: DAICEL CHIRALPAK AD 250 × 30 mm, 10 μm, mobile phase: super- critical CO2/ETOH (0.1% NH3.H2O, v%)= 50/50, flow rate: 200 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator temperature: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to afford: 5 8-Chloro-2-((3R,4S) or (3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak 1 (0.62 g) of sufficient purity for the subsequent step. LC-MS (method B) (m/z) = 394.1 (MH)+ tR = 0.77 minutes. Chiral analytical SFC conditions (instrument: Waters UPCC with PDA Detector, column: Chiralpak AD-350 × 4.6mm I.D., 3μm, mobile phase: A: CO2 B: ethanol (0.05% DEA), isocratic:10 40% B, flow rate: 4 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 4 min, wave- length: 220 nm), ee > 99 %, tR = 1.42 minutes. and the corresponding enantiomer 8-Chloro-2-((3R,4S) or (3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak 2 (0.53 15 g) of sufficient purity for the next step. LC-MS (method B) (m/z) = 394.1 (MH)+ tR = 0.77 minutes. Chiral analytical SFC conditions (instrument: Waters UPCC with PDA Detector, column: Chiralpak AD-350 × 4.6mm I.D., 3μm, mobile phase: A: CO2 B: ethanol (0.05% DEA), isocratic: 40% B, flow rate: 4 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 4 min, wavelength: 220 nm), ee > 99 %, tR = 1.97 minutes. 20 Intermediate: (+)-3-Bromo-8-chloro-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine 25
Figure imgf000171_0001
A solution of NBS (470 mg, 2.64 mmol) in THF (5 mL) was added dropwise to a solution of 8-chloro-2-((3R,4S) or (3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak 1 (0.52 g, 1.32 mmol) in THF (50 mL) at 20 ºC. The mixture was stirred for 1 h at 20 ºC. The mixture was concentrated under reduced pressure. The residue was diluted with EtOAc (10 mL). The organic phase was washed with brine (5 mL×2), dried, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: Petroleum ether:EtOAc (DCM 10v%) 100:0 → 50:50) and preperative SFC (instrument: Thar SFC Prep 80, column: DAICEL CHIRALCEL 5 OD-H 250 × 30 mm, 10 μm, mobile phase: supercritical CO2/EtOH (0.1% NH3.H2O, v%)= 70/30,flow rate: 80 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle tem- perature: 60 ºC, evaporator temperature: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to afford (+)-3-bromo-8-chloro-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- 10 cloundecine (0.2 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.73 (s, 1H), 6.82 (br s, 1H), 5.09 - 4.87 (m, 1H), 4.60 - 4.40 (m, 5H), 4.34 - 4.26 (m, 1H), 4.12 - 4.04 (m, 1H), 3.60 - 3.50 (m, 1H), 3.49 - 3.39 (m, 1H), 2.43-2.30 (m, 1H), 2.12 - 1.95 (m, 3H). LC-MS (method C) (m/z) = 474.1 (MH)+ tR = 1.60 minutes. Chiral analytical SFC conditions (instrument: Waters UPCC with PDA Detector, column: Chiralpak AD-350 × 4.6mm I.D., 3μm, mobile phase: 15 A: CO2 B: ethanol (0.05% DEA), gradient: from 5% to 40% of B in 2 min and hold 40% for 1.2 min, then 5% of B for 0.8 min, flow rate: 4 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 4 min, wavelength: 254 nm), ee > 99 %, tR = 0.78 minutes. = +18.7 (c = 0.3 g/100 mL, CHCl3). Intermediate: (-)-3-Bromo-8-chloro-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-20 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine
Figure imgf000172_0001
The intermediate was prepared in a manner similar to (+)-3-Bromo-8-chloro-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)di- 25 pyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine using NBS (434 mg, 2.44 mmol) in THF (5 mL), 8-chloro-2-((3R,4S or 3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak 2 (0.48 g, 1.22 mmol) in THF (50 mL) at 20 ºC for 1 h, followed by work-up, and purification by chromatog- raphy on silica gel (eluent: Petroleum ether:EtOAc (DCM 10v%) 100:0 → 50:50) and preperative SFC (instrument: Thar SFC Prep 80, column: DAICEL CHIRALCEL OD-H 250 × 30 mm, 10 μm, mo- bile phase: supercritical CO2/EtOH (0.1% NH3.H2O, v%)= 70/30, flow rate: 80 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator tempera- ture: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to afford (-)-3-bromo-8-chloro-2- 5 ((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (0.23 g) of sufficient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 8.72 (s, 1H), 6.91 (br s, 1H), 5.09 - 4.87 (m, 1H), 4.60 - 4.41 (m, 5H), 4.34 - 4.25 (m, 1H), 4.12 - 4.04 (m, 1H), 3.56 - 3.50 (m, 1H), 3.49 - 3.39 (m, 1H), 2.43 - 2.29 (m, 1H), 2.12-1.95 (m, 3H). LC-MS (method C) (m/z) = 474.0 (MH)+ tR = 1.60 10 minutes. Chiral analytical SFC conditions (instrument: Waters UPCC with PDA Detector, column: Chiralpak AD-350 × 4.6mm I.D., 3μm, mobile phase: A: CO2 B: ethanol (0.05% DEA), gradient: from 5% to 40% of B in 2 min and hold 40% for 1.2 min, then 5% of B for 0.8 min, flow rate: 4 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 4 min, wavelength: 254 nm), ee > 99 %, tR = 0.99 minutes. = -20 (c = 0.3 g/100 mL, CHCl3). 15 Intermediate: (-)-(R) or (S)-3-bromo-8-chloro-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine and (+)- (R) or (S)-3-bromo-8-chloro-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 20
Figure imgf000173_0001
To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-4-nitro-1H-pyrazole (7.6 g, 25 mmol) and 4-oxaspiro[2.5]octan-7-ol (4.2 g, 33 mmol, prepared in a manner similar to Ma- thur, A. et al. J. Org. Chem., 2017, 82, 10376-10387) in toluene (220 mL) was added 2-(tributyl- λ5-phosphanylidene)acetonitrile (24.34 g, 100.9 mmol). The mixture was stirred at 110 ºC for 15 5 h. The mixture was concentrated under reduced pressure. The residue was purified by chroma- tography on silica gel (eluent: Petroleum ether:EtOAc 100:0 → 70:30) to afford 3-(3-((tert-butyl- dimethylsilyl)oxy)propoxy)-4-nitro-1-(4-oxaspiro[2.5]octan-7-yl)-1H-pyrazole (6.3 g) of suffi- cient purity for the subsequent step.1H NMR (CDCl3, 400MHz) δ 7.99 (s, 1H), 4.37 (t, J = 6.4 Hz, 2H), 4.32 - 4.22 (m, 1H), 4.0 - 3.93 (m, 1H), 3.77 (t, J = 6.4 Hz, 2H), 3.64 - 3.55 (m, 1H), 2.47 - 2.39 10 (m, 1H), 2.14 - 2.02 (m, 2H), 2.00 - 1.94 (m, 2H), 1.57 - 1.52 (m, 1H), 0.96 - 0.90 (m, 1H), 0.84 (s, 9H), 0.75 - 0.66 (m, 1H), 0.60 - 0.53 (m, 1H), 0.49 - 0.40 (m, 1H), 0.03 (s, 6H). To a solution of 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-4-nitro-1-(4-ox- aspiro[2.5]octan-7-yl)-1H-pyrazole (6.3 g, 15 mmol) in THF (120 mL) was added TBAF (1 M in THF, 23 mL) and then stirred at 25 ºC for 1 h. The reaction mixture was concentrated under 15 reduced pressure. The residue was purified by chromatography on silica gel (eluent: Petroleum ether:EtOAc (MeOH 10 v%) 100:0 → 50:50) to afford 3-((4-nitro-1-(4-oxaspiro[2.5]octan-7-yl)- 1H-pyrazol-3-yl)oxy)propan-1-ol (4.4 g) of sufficient purity for the subsequent step. 1H NMR (CDCl3, 400MHz) δ 8.07 (s, 1H), 4.51 (t, J = 6.0 Hz, 2H), 4.39-4.28 (m, 1H), 4.06 - 3.99 (m, 1H), 3.86 (t, J = 5.6 Hz, 2H), 3.70 - 3.60 (m, 1H), 2.53 - 2.45 (m, 1H), 2.22 - 2.08 (m, 4H), 1.70 (br s, 1H), 20 1.63 - 1.57 (m, 1H), 1.01 - 0.95 (m, 1H), 0.82 - 0.71 (m, 1H), 0.68 - 0.58 (m, 1H), 0.54-0.44 (m, 1H). To a mixture of 3-((4-nitro-1-(4-oxaspiro[2.5]octan-7-yl)-1H-pyrazol-3-yl)oxy)propan-1- ol (4.3 g, 14.5 mmol), 3,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (2.73 g, 14.5 mmol) and PPh3 (11.4 g, 43.4 mmol) in THF (180 mL) was added DIAD (8.77 g, 43.4 mmol) at 0 ºC. The mixture 25 was stirred at 25 ºC for 14 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: Petroleum ether:EtOAc 100:0 → 60:40) to afford 3,6-dichloro-1-(3-((4-nitro-1-(4-oxaspiro[2.5]octan-7-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H- pyrazolo[3,4-d]pyrimidine (3.3 g) of sufficient purity for the subsequent step. LC-MS (method B) (m/z) = 468.1 (MH)+ tR = 0.93 minutes. 30 To 3,6-dichloro-1-(3-((4-nitro-1-(4-oxaspiro[2.5]octan-7-yl)-1H-pyrazol-3-yl)oxy)pro- pyl)-1H-pyrazolo[3,4-d]pyrimidine (3.2 g, 6.8 mmol) and NH4Cl (3.20 g, 59.8 mmol) in a mixture of EtOH (320 mL) and H2O (60 mL) was added Fe (3.20 g, 57.3 mmol) at 20 ºC. The mixture was stirred at 80 ºC for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: Petroleum ether:EtOAc (DCM 10 v%) 100:0 → 50:50) to afford 8-chloro-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (1.5 g) of sufficient purity for the subsequent step. LC-MS (method B) (m/z) = 402.1 (MH)+ tR = 0.70 minutes. 5 A solution of NBS (1.24 g, 6.97 mmol) in THF (20 mL) was added dropwise to a solution of 8-chloro-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyra- zolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (1.4 g, 3.5 mmol) in THF (130 mL) at 25 ºC. The mixture was stirred for 1 h at 25ºC. The mixture was concentrated under reduced pressure. The residue was diluted with EtOAc (20 mL). The organic phase was washed with brine (3×1010 mL), dried, and concentrated under reduced pressure. The residue was purified by chromatog- raphy on silica gel (eluent: Petroleum ether:EtOAc (DCM 10 v%) 100:0 → 80:20) to afford 3- bromo-8-chloro-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (0.7 g) of sufficient purity for the subsequent step. 3-Bromo-8-chloro-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro- 15 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (0.7 g) was separated using preparative SFC (instrument: SFC150AP, column: DAICEL CHIRALPAK AD 250 × 30 mm, 10 μm, mobile phase: supercritical CO2/EtOH (0.1% NH3·H2O, v%)= 40/60, flow rate: 100 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60ºC, evap- orator temperature: 20ºC, trimmer temperature: 25ºC, wavelength: 220 nm) to afford 20 (-)-(R) or (S)-3-bromo-8-chloro-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (0.24 g). 1H NMR (CDCl3, 400MHz) δ 8.71 (s, 1H), 7.02 (br s, 1H), 4.60 - 4.50 (m, 3H), 4.49 - 4.42 (m, 2H), 4.06 - 3.99 (m, 1H), 3.72 - 3.63 (m, 1H), 2.78 - 2.69 (m, 1H), 2.40 - 2.27 (m, 1H), 2.10 - 2.01 (m, 2H), 1.98 - 25 1.90 (m, 1H), 1.41 - 1.33 (m, 1H), 1.00 - 0.93 (m, 1H), 0.81 - 0.73 (m, 1H), 0.63 - 0.56 (m, 1H), 0.52 - 0.45 (m, 1H). LC-MS (method C) (m/z) = 482.0 (MH)+ tR = 1.68 minutes. Chiral analytical SFC conditions (instrument: Waters UPCC with PDA Detector, column: Chiralpak AD-350 × 4.6mm I.D., 3μm, mobile phase: A: CO2 B: ethanol (0.05% DEA), gradient: from 5% to 40% of B in 2 min and hold 40% for 1.2 min, then 5% of B for 0.8 min, flow rate: 4 mL/min, column temperature: 30 35 ºC, ABPR: 1500psi, run time: 4 min, wavelength: 254 nm), ee > 99 %, tR = 1.18 minutes. = -48 (c = 0.35 g/100 mL, CHCl3). and the corresponding enantiomer (+)-(R) or (S)-3-bromo-8-chloro-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (0.21 g) 1H NMR (CDCl3, 400MHz) δ 8.71 (s, 1H), 6.95 (br s, 1H), 4.61 - 4.50 (m, 3H), 4.48 - 4.43 (m, 2H), 4.06 - 3.98 (m, 1H), 3.72 - 3.64 (m, 1H), 2.78 - 2.69 (m, 1H), 2.40 - 2.27 (m, 1H), 2.10 - 2.01 (m, 2H), 1.98 - 5 1.90 (m, 1H), 1.41 - 1.33 (m, 1H), 1.00 - 0.92 (m, 1H), 0.81 - 0.74 (m, 1H), 0.63 - 0.56 (m, 1H), 0.52 - 0.44 (m, 1H). LC-MS (method C) (m/z) = 482.0 (MH)+ tR = 1.68 minutes. Chiral analytical SFC conditions (instrument: Waters UPCC with PDA Detector, column: Chiralpak AD-350 × 4.6mm I.D., 3μm, mobile phase: A: CO2 B: ethanol (0.05% DEA), gradient: from 5% to 40% of B in 2 min and hold 40% for 1.2 min, then 5% of B for 0.8 min, flow rate: 4 mL/min, column temperature: 10 35 ºC, ABPR: 1500psi, run time: 4 min, wavelength: 254 nm), ee > 99 %, tR = 3.95 minutes. = +49.4 (c = 0.35 g/100 mL, CHCl3).
COMPOUNDS OF THE INVENTION Example 1: 8-Chloro-2,3-dimethyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyra- zolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 5
Figure imgf000177_0001
To a solution of 3,6-dichloro-1-(3-((1,5-dimethyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)- 1H-pyrazolo[3,4-d]pyrimidine (90 mg, 0.23 mmol) in H2O (1 mL) and EtOH (5 mL) was added Fe (65 mg, 1.17 mmol) and NH4Cl (62 mg, 1.17 mmol). The reaction mixture was stirred at 100 ºC for 16 h. The reaction mixture was filtered and concentrated under reduced pressure. The 10 residue was purified by preparative HPLC ((Instrument: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV Detector; Column: Phenomenex Gemini-NX C1875×30mm×3μm; Mobile Phase: A: water (0.05%NH3.H2O v/v+10mM NH4HCO3), Mobile phase B: MeCN; Gradient: B from 30% to 60% in 7 min then hold at 100 % for 2 min; Flow Rate (mL/min): 25; Column temperature: 25ºC; Wavelength: 220nm 254nm) to afford the title compound (30 mg). 1H NMR (CDCl3, 15 400MHz) δ 8.67 (s, 1H), 6.76 (br s, 1H), 4.54-4.42 (m, 4H), 3.70 (s, 3H), 2.29 (s, 3H), 1.99-1.95 (m, 2H). LC-MS (method C) (m/z) = 320.2 (MH)+ tR = 1.67 minutes. Example 2: 8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 20
Figure imgf000177_0002
The compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- ((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4- d]pyrimidine (840 mg, 1.84 mmol), Fe (514 mg, 9.20 mmol), and NH4Cl (492 mg, 9.20 mmol) in EtOH (40 mL) and H2O (4 mL) at 100 ºC for 15 h, followed by work-up, and preparative HPLC 25 (Instrument: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV Detector; Column: Xtimate C18150×40mm×5μm; Mobile Phase A: water (0.05%NH3H2O) Mobile phase B: MeCN; Gradient: B from 28% to 53% in 6 min then hold at 100% for 1 min Flow Rate (mL/min); Column temperature: 25 ºC; Wavelength: 220nm 254nm) to afford the title compound.1H NMR (CDCl3, 400MHz) δ 8.65 (s, 1H), 6.85 (br s, 1H), 4.57-4.35 (m, 4H), 4.17-4.06 (m, 3H), 3.59-3.46 (m, 2H), 2.36-2.21 (m, 5H), 2.01-1.90 (m, 2H), 1.85-1.77 (m, 2H). LC-MS (method C) (m/z) = 390.2 (MH)+ tR = 1.79 minutes. 5 Example 3: (R) or (S)-8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine peak 1 and Example 4: (R) or (S)-8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine peak 2 10
Figure imgf000178_0001
The racemic mixture of Example 3 and Example 4 was prepared in a manner similar to Example 1 using (±)-3,6-dichloro-1-(3-((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-3-yl)-1H-pyra- zol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (218 mg, 0.478 mmol), Fe (133 mg, 2.3915 mmol), and NH4Cl (128 mg, 2.39 mmol) in EtOH (17 mL) and H2O ((1.7 mL) at 94 ºC for 4 h, fol- lowed by work-up and purification by chromatography on silica gel (eluent: Heptane:EtOAc 80:20 → 10:90) to afford (±)-8-chloro-3-methyl-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (140 mg). The racemic mixture (140 mg) was separated into the two enantiomers by preparative chi-20 ral SFC (instrument: Berger Multigram II, column: Chiralpak-IA, 250 mm x 20 mm, particle size 5µm, mobile phase: CO2/EtOH (96% containing 0.1% DEA, v/v) = 60/40, flow rate 50 mL/min, column temperature: 35 ºC, pressure: 100 bar) to afford: Example 3, Peak 1: (R) or (S)-8-chloro-3-methyl-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13- tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 25 (35 mg).1H NMR (600 MHz, CDCl3) δ 8.66 (s, 1H), 6.52 (br s, 1H), 4.53-4.37 (m, 4H), 4.07 (tt, J = 11.1, 4.4 Hz, 1H)), 3.98-3.91 (m, 2H), 3.69 (t, J = 10.8 Hz, 1H), 3.48-3.40 (m, 1H), 2.28 (s, 3H), 2.28-2.18 (m, 1H), 2.09-2.04 (m, 1H), 2.00-1.88 (m, 2H), 1.86-1.78 (m, 2H). LC-MS (method E) (m/z) = 390.4 (MH)+ tR = 0.59 minutes. Chiral analytical SFC conditions (instrument: Aurora SFC Fusion5/Agilent, column: Chiralpak-IA, 150×4.6 mm, 5 µm particle size, mobile phase; CO2/EtOH (96% containing 0.1% DEA, v/v) = 60/40, isocratic elution 7 minutes, flow rate 4 mL/min, column temperature 40 ºC, UV detection: 254 nm), ee > 99 %, tR = 1.92 minutes. and the corresponding enantiomer Example 4, Peak 2: Example 4: (R) or (S)-8-chloro-3-methyl-2-(tetrahydro-2H-pyran-3- 5 yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triaza- cycloundecine (50 mg). 1H NMR (600 MHz, CDCl3) δ 8.66 (s, 1H), 6.52 (br s, 1H), 4.53-4.37 (m, 4H), 4.07 (tt, J = 11.1, 4.4 Hz, 1H)), 3.98-3.91 (m, 2H), 3.69 (t, J = 10.8 Hz, 1H), 3.48-3.40 (m, 1H), 2.28 (s, 3H), 2.28-2.18 (m, 1H), 2.09-2.04 (m, 1H), 2.00-1.88 (m, 2H), 1.86-1.78 (m, 2H). LC-MS (method E) (m/z) = 390.4 (MH)+ tR = 0.59 minutes. Chiral analytical SFC conditions (instrument: 10 Aurora SFC Fusion5/Agilent, column: Chiralpak-IA, 150×4.6 mm, 5 µm particle size, mobile phase; CO2/EtOH (96% containing 0.1% DEA, v/v) = 60/40, isocratic elution 7 minutes, flow rate 4 mL/min, column temperature 40 ºC, UV detection: 254 nm), ee > 99 %, tR = 2.53 minutes. Example 5: 3-Methyl-2-(tetrahydro-2H-pyran-4-yl)-8-(trifluoromethyl)-2,4,12,13-tetrahydro-15 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000179_0001
The compound was prepared in a manner similar to Example 1 using 6-chloro-1-(3-((5- methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)-3-(trifluoromethyl)- 1H-pyrazolo[3,4-d]pyrimidine (95 mg, 0.19 mmol), Fe (54 mg, 0.97 mmol), and NH4Cl (52 mg, 520 Eq, 0.97 mmol) in EtOH (15 mL) and H2O (1.5 mL) at 85 ºC for 72 h, followed by work-up, and purification by chromatography on silica gel (eluent: Heptane:EtOAc 80:20 → 10:90) to afford the title compound (42 mg).1H NMR (CDCl3, 600MHz) δ 8.81 (s, 1H), 6.57 (br s, 1H), 4.60-4.42 (m, 4H), 4.17-4.06 (m, 3H), 3.52 (td, J = 12.1, 2.1 Hz, 2H), 2.33-2.24 (m, 2H), 2.30 (s, 3H), 2.08- 1.94 (m, 2H), 1.84-1.78 (m, 2H).19F NMR (CDCl3, 470MHz) δ -61.5 (s). LC-MS (method E) (m/z) =25 424.4 (MH)+ tR = 0.64 minutes. Example 6: 8-Bromo-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000180_0001
5 A solution of DIAD (87 µL, 0.45 mmol) in THF (5 mL) was cooled to 0 ºC. PPh3 (on resin, ~3 mmol/g) (118 mg, 0.449 mmol)(employed 150 mg resin (~3mmol/g) = 0.449 mmol)), was added portionwise and the mixture was stirred for 10 minutes.5-methyl-4-nitro-1-(tetrahydro- 2H-pyran-4-yl)-1H-pyrazol-3-ol (60.0 mg, 0.264 mmol, prepared as previously described) was then added followed by a solution of 3-(3-bromo-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-10 yl)propan-1-ol (77.0 mg, 0.26 mmol) in THF (1 mL). The cooling bath was removed and the reac- tion mixture was stirred for 2 h. The mixture was filtered while washing with THF, and the filtrate was concentrated to dryness under reduced pressure. The residue obtained was re-dissolved in ethanol (5 mL) and water (0.5 mL). Fe (73.7 mg, 1.32 mmol) and NH4Cl (70.6 mg, 1.32 mmol) was added and the flask was briefly flushed with argon and stirred while heating to 90 ºC 15 for 2 h and then at 60 ºC overnight. The reaction mixture was filtered while washing with EtOAc and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by chromatography on silica gel (eluent: heptane:EtOAc (w.1% MeOH) 100:0 → 10:90) to afford the title compound (32 mg).1H NMR (CDCl3, 600MHz) δ 8.57 (s, 1H), 6.57 (br s, 1H), 4.60-4.34 (m, 4H), 4.17 - 4.00 (m, 3H), 3.51 (t, J = 11.9, 2H), 2.36-2.19 (m, 5H), 2.02-1.86 (m, 2H), 1.85-1.7420 (m, 2H). LC-MS (method E) (m/z) = 420.3 (MH)+ tR = 0.56 minutes. Example 7: 8-Cyclopropyl-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000180_0002
25 To a dry vial was added Example 6 (60 mg, 0.14 mmol) and the vial was evacuated and backfilled with argon. Toluene (0.25 mL) and THF (2.5 mL) were added and the mixture stirred for a few minutes before di-µ-iodobis(tri-t-butylphosphino)dipalladium(I) (18 mg, 0.021 mmol) was added. The mixture was stirred for an additional 2 minutes at which point cyclo- propylzinc(II) bromide (0.5 M in THF)(1.1 mL, 0.55 mmol) was added in one portion. The reaction was stirred for 1 h at room temperature. The mixture was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: heptane:EtOAc (w.1% MeOH) 5 100:0 → 10:90) to afford the title compound (48 mg).1H NMR (CDCl3, 600MHz) δ 8.97 (br s, 1H), 8.80 (s, 1H), 4.55-4.28 (m, 4H), 4.16-4.05 (m, 3H), 3.57-3.45 (m, 2H), 2.36 (s, 3H), 2.31-2.21 (m, 2H), 2.03 (tt, J = 8.3, 5.0 Hz, 1H), 2.00-1.88 (m, 2H), 1.83-1.75 (m, 2H), 1.06-0.93 (m, 4H). LC-MS (method E) (m/z) = 396.4 (MH)+ tR = 0.51 minutes. 10 Example 8: 3,8-Dimethyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000181_0001
To a dry vial containing THF (2 mL) under argon atmosphere was added bromo-methyl- magnesium (3M in diethylether) (537 µL, 1.61 mmol) and zinc chloride (1.9M in THF) (1.02 mL, 15 1.93 mmol). The mixture was stirred for 5 minutes at room temperature which led to formation of a white precipitate. The solids were allowed to settle and the top solution (approx.2 mL, clear solution) was taken out by syringe. The above prepared solution was added to a second vial containing Example 6 (70.0 mg, 0.161 mmol) and di-µ-iodobis(tri-t-butylphosphino)dipalla- dium(I) (17.6 mg, 0.0201 mmol) in a mixture of toluene (0.4 mL) and THF (10 mL). The mixture20 was stirred at room temperature overnight. The reaction mixture was concentrated under re- duced pressure. The residue was purified by chromatography on silica gel (eluent: hep- tane:EtOAc (w.2% MeOH) 90:10 → 0:100) to afford the title compound (13 mg).1H NMR (CDCl3, 600MHz) δ 8.71 (s, 1H), 4.52-4.34 (m, 4H), 4.15-4.09 (m, 2H), 3.52 (t, J = 12.1 Hz, 2H), 2.46 (s, 3H), 2.33 (s, 3H), 2.32-2.23 (m, 2H), 2.01-1.90 (m, 2H), 1.84-1.77 (m, 2H). LC-MS (method E) (m/z)25 = 370.4 (MH)+ tR = 0.41 minutes. Example 9: 3-Methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine-8-carbonitrile
Figure imgf000182_0001
To a dry vial fitted with a stirbar under an atmosphere of argon was added^Example 5 6 (15 mg, 0.035 mmol),^Zinc cyanide (4.1 mg, 0.035 mmol)^and^methanesulfonato[9,9-dimethyl- 4,5-bis(diphenylphosphino)xanthene][2'-amino-1,1'-biphenyl]palladium(II) dichloro-me- thane adduct (2.5 mg, 0.0026 mmol). The vial was capped and the atmosphere was ex- changed for argon before^DMA (0.35 mL)^was added. The solution was purged with argon for 1 minute before^DIPEA (1.8 µL, 0.010 mmol)^was added.^The mixture was stirred at 85^ºC over- 10 night. The reaction mixture was concentrated^under reduced pressure. The residue was purified by chromatography on silica gel (eluent: heptane:EtOAc (w.2% MeOH) 90:10 → 0:100) to afford the title compound (2.3 mg).1H NMR (CDCl3, 600MHz) δ 8.84 (s, 1H), 6.69 (s, 1H), 4.59 - 4.42 (m, 4H), 4.15 - 4.07 (m, 3H), 3.56 - 3.48 (m, 2H), 2.32 - 2.23 (m, 5H), 2.07 - 1.91 (m, 2H), 1.84 - 1.77 (m, 2H).. LC-MS (method F) (m/z) = 381.2 (MH)+ tR = 0.60 minutes. 15 Example 10: 8-Chloro-3-cyclopropyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000182_0002
The compound was prepared in a manner similar to Example 1 using of 3,6-dichloro-1-20 (3-((5-cyclopropyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyra- zolo[3,4-d]pyrimidine (140 mg, 0.29 mmol), Fe (81 mg, 1.5 mmol), and NH4Cl (78 mg, 1.5 mmol) in EtOH (70 mL) and H2O (10 mL) at 100 ºC for 16 h, followed by work-up, and preparative HPLC ((Instrument: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV Detector; Column: Boston Prime C18150×30mm×5μm; Mobile Phase: A: water (0.05%NH3.H2O+10mM NH4HCO3 25 v/v), Mobile phase B: MeCN; Gradient: B from 40% to 70% in 7 min then hold at 100% for 2 min; Flow Rate (mL/min): 25; Column temperature: 35ºC; Wavelength: 220nm 254nm) to afford the title compound (50 mg).1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 6.76 (br s, 1H), 4.56-4.39 (m, 5H), 4.13 (dd, J = 3.6, 10.8 Hz, 2H), 3.54 (t, J = 10.4 Hz, 2H), 2.36-2.23 (m, 2H), 2.04-1.93 (m, 2H), 1.79 (dd, J = 2.0, 12.8 Hz, 2H), 1.70-1.63 (m, 1H), 1.12-1.04 (m, 2H), 0.87-0.79 (m, 2H). LC-MS (methodC) (m/z) = 416.1 (MH)+ tR = 1.56 minutes. Example 11: 2-((2-Oxabicyclo[2.1.1]hexan-1-yl)methyl)-8-chloro-3-methyl-2,4,12,13-tetrahy- 5 dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000183_0001
The compound was prepared in a manner similar to Example 1 using 1-(3-((1-((2-oxabi- cyclo[2.1.1]hexan-1-yl)methyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-3,6-dichloro-1H- pyrazolo[3,4-d]pyrimidine (230 mg, 0.49 mmol), Fe (137 mg, 2.5 mmol), and NH4Cl (131 mg, 2.5 10 mmol) in EtOH (93 mL) and H2O (13 mL) at 100 ºC for 16 h, followed by work-up, and preparative HPLC (Instrument: Gilson GX-215 Liquid Handler, SHIMADZU LC-20AP, SHIMADZU SPD-20A; Col- umn: Welch Xtimate C18 150×30mm×5μm; Mobile Phase A: water (0.05%NH3H2O +10mM NH4HCO3) Mobile phase B: MeCN; Gradient: B from 27% to 57% in 9 min then hold at 100% for 3 min Flow Rate (mL/min): 30; Column temperature: 30 ºC; Wavelength: 220nm 254nm) to af-15 ford the title compound (37 mg).1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 6.68 (br s, 1H), 4.51- 4.38 (m, 4H), 4.27 (s, 2H), 3.77 (s, 2H), 2.89 (t, J = 3.2 Hz, 1H), 2.31 (s, 3H), 2.01-1.96 (m, 2H), 1.82-1.75 (m, 2H), 1.51-1.43 (m, 2H). LC-MS (method C) (m/z) = 402.1 (MH)+ tR = 1.45 minutes. Example 12: 8-Chloro-3-methyl-2-((3-methyloxetan-3-yl)methyl)-2,4,12,13-tetrahydro-11H-20 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000183_0002
The compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- ((5-methyl-1-((3-methyloxetan-3-yl)methyl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyra- zolo[3,4-d]pyrimidine (50 mg, 0.11 mmol), Fe (31 mg, 0.55 mmol), and NH4Cl (29 mg, 0.55 mmol)25 in EtOH (10 mL) and H2O (10 mL) at 80 ºC for 5 h, followed by work-up, and preparative HPLC (Instrument: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV; Detector Column: Boston Prime C18150×30mm×5μm; Mobile Phase A: water (0.05%NH3H2O +10mM NH4HCO3) Mobile phase B: MeCN; Gradient: B from 30% to 60% in 7 min then hold at 100% for 2 min; Flow Rate (mL/min): 25; Column temperature: 30 ºC; Wavelength: 220nm 254nm) to afford the title 5 compound (15 mg).1H NMR (CDCl3, 400MHz) δ 8.71 (br s, 1H), 7.30 (s, 1H), 4.78 (d, J = 6.0 Hz, 2H), 4.46-4.39 (m, 6H), 4.10 (s, 2H), 2.28 (s, 3H), 2.02-1.93 (m, 2H), 1.29 (s, 3H). LC-MS (method G) (m/z) = 390.3 (MH)+ tR = 1.75 minutes. Example 13: 8-Chloro-3-(methyl-d3)-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H- 10 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000184_0001
3,6-Dichloro-1-(3-((5-(methyl-d3)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3- yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (100 mg, 0.217 mmol)^was dissolved in^Etha- nol (15 mL)^and^deuterium oxide (1.5 mL) in a vial.^Fe (48.5 mg, 0.869 mmol)^and NH4Cl (46.5 15 mg, 0.869 mmol)^was added and the flask was thoroughly flushed with argon and stirred while heating to 85 ºC overnight.^The reaction mixture was cooled and filtered while washing with EtOAc and DCM and the filtrate was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: heptane:EtOAc (w.2% MeOH) 90:10 → 0:100) to afford the title compound (49.7 mg). 1H NMR (CDCl3, 600MHz) δ 8.66 (s, 1H), 6.54 (s, 1H), 20 4.53-4.38 (m, 4H), 4.14-4.07 (m, 3H), 3.52 (td, J = 12.1, 2.1 Hz, 2H), 2.32-2.23 (m, 2H), 2.02-1.87 (m, 2H), 1.84-1.77 (m, 2H). LC-MS (method F) (m/z) = 393.2 (MH)+ tR = 0.55 minutes
Example 14: 8-Chloro-3-methyl-2-(3-methyloxetan-3-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000185_0001
The compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- 5 ((5-methyl-1-(3-methyloxetan-3-yl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]py- rimidine (190 mg, 0.43 mmol), Fe (120 mg, 2.15 mmol), and NH4Cl (115 mg, 2.15 mmol) in EtOH (40 mL) and H2O (40 mL) at 80 ºC for 16 h, followed by work-up, and preparative HPLC (Instru- ment: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156; UV Detector; Column: Boston Prime C18150×30mm×5μm; Mobile Phase A: water (0.05%NH3H2O+10mM NH4HCO3) Mobile 10 phase B: MeCN; Gradient: B from 30% to 60% in 7 min then hold at 100% for 2 min; Flow Rate (mL/min): 25; Column temperature: 30 ºC; Wavelength: 220nm 254nm) to afford the title com- pound (51 mg).1H NMR (CDCl3, 400MHz) δ 8.67 (s, 1H), 6.55 (s, 1H), 5.25 (d, J = 6.4 Hz, 2H), 4.59 (d, J=6.8 Hz, 2H), 4.48-4.43 (m, 4H), 2.15 (s, 3H), 2.01-1.91 (m, 2H), 1.80 (s, 3H). LC-MS (method C) (m/z) = 376.1 (MH)+ tR = 1.37 minutes. 15 Example 15: 8-Chloro-3-(methyl-d3)-2-(tetrahydro-2H-pyran-4-yl-4-d)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000185_0002
The compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3-20 ((5-(methyl-d3)-4-nitro-1-(tetrahydro-2H-pyran-4-yl-4-d)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyra- zolo[3,4-d]pyrimidine (520 mg, 1.13 mmol), Fe (315 mg, 5.65 mmol), and NH4Cl (302 mg, 5.65 mmol) in EtOH (100 mL) and H2O (100 mL) at 90 ºC for 16 h, followed by work-up, and prepara- tive HPLC (Instrument: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV Detector; Column: Boston Prime C18150×30mm×5μm; Mobile Phase: A: water (0.05%NH3.H2O+10mM 25 NH4HCO3 v/v), Mobile phase B: MeCN; Gradient: B from 30% to 60% in 7 min then hold at 100% for 2 min; Flow Rate (mL/min): 25; Column temperature: 35ºC;Wavelength: 220nm 254nm) to afford the title compound (250 mg).1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 6.74 (s, 1H), 4.55- 4.37 (m, 4H), 4.12 (dd, J = 4.0, 11.2 Hz, 2H), 3.58-3.47 (m, 2H), 2.35-2.23 (m, 2H), 2.01-1.88 (m, 2H), 1.84-1.77 (m, 2H). LC-MS (method G) (m/z) = 394.2 (MH)+ tR = 1.39 minutes. Example 16: 8-Chloro-3-(1-fluorocyclopropyl)-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahy- 5 dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000186_0001
To a flask was added potassium hydrogen carbonate (120 mg, 1.20 mmol) and water (6.4 mL). The vial was cooled to 0 ºC and the solution was purged with argon before sodium dithionite (120 mg, 0.688 mmol) was added. The solution was purged with argon for 5 minutes10 before 3,6-dichloro-1-(3-((5-(1-fluorocyclopropyl)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-py- razol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (80.0 mg, 0.160 mmol) was added along with THF (6.4 mL). The reaction mixture was stirred at 0 ºC for 2.5 h, and then from 0 - 10 ºC for an additional 1 h. The mixture was diluted with water (10 mL) and EtOAc (20 mL). The phases were separated, and the aqueous phase was extracted with EtOAc (3 x 20 mL). The combined 15 organics washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was transferred to a dry vial and DMSO (7 mL), 1,4-Dioxane (7 mL) and po- tassium fluoride (37.2 mg, 0.640 mmol) were added. The vial was flushed with argon, capped and stirred at 77 ºC for 16 h. The reaction mixture was diluted with water (5 mL) and extracted with EtOAc (3 x 20 mL). The combined organics were washed with brine repeatedly, dried over 20 sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: heptane:EtOAc 100:0 → 10:90) to afford the title com- pound (2.2 mg).1H NMR (CDCl3, 600MHz) δ 8.69 (s, 1H), 7.09 (s, 1H), 4.58 (tt, J = 11.6, 4.3 Hz, 1H), 4.49-4.40 (m, 4H), 4.15-4.09 (m, 2H), 3.58-3.51 (m, 2H), 2.36-2.25 (m, 2H), 2.02-1.92 (m, 2H), 1.88-1.81 (m, 2H), 1.35-1.24 (m, 2H), 1.19-1.13 (m, 2H).19F NMR (CDCl3, 470MHz) δ -164.9 25 (s). LC-MS (method E) (m/z) = 434.5 (MH)+ tR = 0.67 minutes. Example 17: 8-Chloro-3-ethyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000187_0001
The compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- 5 ((5-ethyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4- d]pyrimidine (50 mg, 0.11 mmol), Fe (30 mg, 0.53 mmol), and NH4Cl (28 mg, 0.53 mmol) in EtOH (2 mL) and H2O (0.2 mL) at 100 ºC for 3 h, followed by work-up, and preparative HPLC (Column: YMC-Actus Triart C18 ExRS, 30×150 mm, particle size 5μm, mobile phase A: Water(10 mmol/L NH4HCO3), mobile phase B: ACN; flow rate: 25 mL/min, gradient: 34% B to 50% B in 7 minutes, 10 50% B; wave length: 254/220 nm) to afford the title compound (15 mg). 1H NMR (CDCl3, 300 MHz) δ 8.67 (s, 1H), 6.93 (s, 1H), 4.51-4.43 (m, 4H), 4.17-4.07 (m, 3H), 3.61-3.50 (m, 2H), 2.74 (q, J = 7.6 Hz, 2H), 2.35 (qd, J = 12.5, 4.6 Hz, 2H), 2.08-1.92 (m, 2H), 1.92-1.71 (m, 2H), 1.28 (t, J = 7.6 Hz, 3H). LC-MS (method H) (m/z) = 404.2 (MH)+ tR = 1.28 minutes. 15 Example 18 (+)-8-Chloro-3-methyl-2-((2R,4R)-or-(2S,4S)-2-methyltetrahydro-2H-pyran-4-yl)- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine and Example 19: (–)-8-Chloro-3-methyl-2-((2R,4R)-or-(2S,4S)-2-methyltetrahydro-2H-pyran-4-yl)- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy-20 cloundecine
Figure imgf000187_0002
The racemic mixture of Example 18 and Example 19 was prepared in a manner similar to Example 1 using cis-3,6-dichloro-1-(3-((5-methyl-1-(2-methyltetrahydro-2H-pyran-4-yl)-4-nitro- 1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (500 mg, 1.1 mmol), Fe (297 mg, 5.32 25 mmol), and NH4Cl (284 mg, 5.32 mmol) in EtOH (30 mL) and H2O (5 mL) at 80 ºC for 15 h, followed by work-up and purification by chromatography on silica gel (eluent: Petroleum ether:EtOAc 100:0 → 30:70) to afford 8-chloro-3-methyl-2-((2R,4R and 2S,4S )-2-methyltetrahydro-2H-pyran-4- yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine (350 mg). The racemic mixture (350 mg) was separated into the two enantiomers by preparative chiral SFC (instrument: Berger MultiGram II, column: DAICEL CHIRALPAK AS 250 × 5 30 mm, particle size 10 μm, mobile phase: supercritical CO2/MeOH (0.1% NH3.H2O, v%)= 70/30, flow rate: 80 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator temperature: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to af- ford: Example 18 (+)-8-Chloro-3-methyl-2-((2R,4R)-or-(2S,4S)-2-methyltetrahydro-2H-pyran-4-10 yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triaza- cycloundecine (70 mg).1H NMR (CDCl3, 400MHz) δ 8.68 (s, 1H), 6.70 (br s, 1H), 4.59 - 4.36 (m, 4H), 4.23 - 4.04 (m, 2H), 3.66 - 3.48 (m, 2H), 2.30 (s, 3H), 2.27 - 2.16 (m, 1H), 2.07 - 1.89 (m, 3H), 1.88 - 1.82 (m, 1H), 1.81 - 1.76 (m, 1H), 1.27 (d, J = 6.4 Hz, 3H). LC-MS (method C) (m/z) = 404.1 (MH)+ tR = 1.46 minutes. Chiral analytical SFC (instrument: Waters UPCC with PDA Detector, col- 15 umn: Chiralpak AS-3150 × 4.6mm I.D., particle size 3μm, mobile phase: A: CO2 B: methanol (0.05% DEA), gradient: from 5% to 40% of B in 5 min and hold 40% for 2.5 min, then 5% of B for 2.5 min, flow rate: 2.5 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 10 min, wavelength: 220 nm), ee = >99%, tR = 4.05 minutes. = +16 (c = 0.1 g/100 mL, CHCl3) and the corresponding enantiomer 20 Example 19: (–)-8-Chloro-3-methyl-2-((2R,4R)-or-(2S,4S)-2-methyltetrahydro-2H-pyran-4- yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triaza- cycloundecine (80 mg).1H NMR (CDCl3, 400MHz) δ 8.67 (s, 1H), 6.69 (br s, 1H), 4.64 - 4.31 (m, 4H), 4.21 - 4.04 (m, 2H), 3.66 - 3.47 (m, 2H), 2.30 (s, 3H), 2.28 - 2.15 (m, 1H), 2.07 - 1.89 (m, 3H), 1.88 - 1.82 (m, 1H), 1.80 - 1.76 (m, 1H), 1.27 (d, J = 6.0 Hz, 3H). LC-MS (method C) (m/z) = 404.225 (MH)+ tR = 1.46 minutes. Chiral analytical SFC (instrument: Waters UPCC with PDA Detector, col- umn: Chiralpak AS-3150 × 4.6mm I.D., particle size 3μm, mobile phase: A: CO2 B: methanol (0.05% DEA), gradient: from 5% to 40% of B in 5 min and hold 40% for 2.5 min, then 5% of B for 2.5 min, flow rate: 2.5 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 10 min, wavelength: 220 nm), ee = 99%, tR = 4.34 minutes. = -17.3 (c = 0.15 g/100 mL, CHCl3). 30 Example 20: 8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine-11,11,12,12,13,13- d6
Figure imgf000189_0001
5 The compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- ((5-methyl-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl-1,1,2,2,3,3-d6)-1H- pyrazolo[3,4-d]pyrimidine (270 mg, 0.584 mmol), Fe (163 mg, 2.92 mmol), and NH4Cl (156 mg, 2.92 mmol) in EtOH (27 mL) and D2O (2.7 mL) at 100 ºC for 16 h, followed by work-up, and pre- parative HPLC ((instrument: waters sample manager 2767, waters 2545 pump, waters UV De- 10 tector 2545, Column: YMC-Actus Triart C18 ExRS, 30×150 mm, particle size 5μm, mobile phase A: water, mobile phase B: ACN, gradient: B from 28% to 41% in 9 min, flow rate: 25 mL/min, column temperature: 30 ºC, wavelength: 220 nm, 254 nm) to afford the title compound (112 mg).1H NMR (DMSO-d6, 300 MHz) δ 9.37 (s, 1H), 8.78 (s, 1H), 4.30-4.22 (m, 1H), 3.98-3.92 (m, 2H), 3.50-3.41 (m, 2H), 2.26 (s, 3H), 2.05-1.90 (m, 2H), 1.76-1.71 (m, 2H). LC-MS (method H)15 (m/z) = 396.2 (MH)+ tR = 1.15 minutes. Example 218-Chloro-3-methyl-2-((3R,4S) or (3S,4R) or (3S,4S) or (3R,4R)-3-methyltetrahydro- 2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine peak 1 and
Example 228-Chloro-3-methyl-2-((3R,4S) or (3S,4R) or (3S,4S) or (3R,4R)-3-methyltetrahydro- 2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine peak 3 and Example 238-Chloro-3-methyl-2-((3R,4S) or (3S,4R) or (3S,4S) or (3R,4R)-3-methyltetrahydro- 5 2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine peak 4 and Example 248-Chloro-3-methyl-2-((3R,4S) or (3S,4R) or (3S,4S) or (3R,4R)-3-methyltetrahydro- 2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine peak 2 10
Figure imgf000190_0001
The mixture of Example 21, Example 22, Example 23, and Example 24 was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3-((5-methyl-1-(3-methyltetrahydro-2H-py- ran-4-yl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (1.1 g, 2.34 mmol), 15 Fe (660 mg, 11.82 mmol), and NH4Cl (625 mg, 11.69 mmol) in EtOH (40 mL) and H2O (20 mL) at 80 ºC for 16 h, followed by work-up and purification by chromatography on silica gel (eluent: Petroleum ether:EtOAc 100:0 → 0:100) to afford 8-chloro-3-methyl-2-(3-methyltetrahydro-2H-py- ran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triaza- cycloundecine (590 mg). The mixture (580 mg) was separated into the four isomers by prepara- 20 tive chiral SFC (instrument: Berger MultiGram II, column: DAICEL CHIRALPAK AD 250 × 30 mm, 10 μm, mobile phase: supercritical CO2/IPA (0.1% NH3.H2O, v%)= 65/35, flow rate: 80 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator temperature: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to afford: 25 Example 218-Chloro-3-methyl-2-((3R,4S) or (3S,4R) or (3S,4S) or (3R,4R)-3-methyltetra- hydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine peak 1 (100 mg). 1H NMR (CDCl3 , 400MHz) δ 8.67 (s, 1H), 6.56 (br s, 1H), 4.57 - 4.40 (m, 4H), 4.12 (dd, J = 4.8, 11.6 Hz, 1H), 4.00 (dd, J = 4.4, 12.0 Hz, 1H), 3.69 - 3.61(m, 1H), 3.57 - 3.46 (m, 1H), 3.14 (t, J = 11.6 Hz, 1H), 2.50 - 2.37 (m, 1H), 2.35 - 2.25 (m, 4H), 2.09 - 1.98(m, 1H), 1.96 - 1.86 (m, 1H), 1.81 - 1.73 (m, 1H), 0.68 (d, J = 6.8 Hz, 3H). LC- MS (method C) (m/z) = 404.2 (MH)+ tR = 1.44 minutes. Chiral analytical SFC (instrument: Waters 5 UPCC with PDA Detector, Column: Chiralpak AD-3150 × 4.6mm I.D., particle size 3μm, mobile phase: A: CO2 B: Methanol (0.05% DEA), gradient: 40% of iso-propanol (0.05% DEA) in CO2, flow rate: 2.5 mL/min, column temp.: 35 ºC, ABPR: 1500 psi, run time: 6 min, wavelength: 220 nm), ee > 99%, tR = 3.08 minutes. and isomer 10 Example 228-Chloro-3-methyl-2-((3R,4S) or (3S,4R) or (3S,4S) or (3R,4R)-3-methyltetra- hydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine peak 3 (110 mg).1H NMR (CDCl3, 400MHz) δ 8.67 (s, 1H), 6.56 (br s, 1H), 4.57 - 4.40 (m, 4H), 4.12 (dd, J = 4.8, 11.6 Hz, 1H), 4.00 (dd, J = 4.4, 12.0 Hz, 1H), 3.69 - 3.61(m, 1H), 3.57 - 3.46 (m, 1H), 3.14 (t, J = 11.6 Hz, 1H), 2.50 - 2.37 (m, 1H), 2.35 - 2.25 (m, 15 4H), 2.09 - 1.98(m, 1H), 1.96 - 1.86 (m, 1H), 1.81 - 1.73 (m, 1H), 0.68 (d, J = 6.8 Hz, 3H). LC-MS (method C) (m/z) = 404.2 (MH)+ tR = 1.44 minutes. Chiral analytical SFC (instrument: Waters UPCC with PDA Detector, Column: Chiralpak AD-3150 × 4.6mm I.D., particle size 3μm, mobile phase: A: CO2 B: Methanol (0.05% DEA), gradient: 40% of iso-propanol (0.05% DEA) in CO2, flow rate: 2.5 mL/min, column temp.: 35 ºC, ABPR: 1500 psi, run time: 6 min, wavelength: 220 nm),20 ee > 99%, tR = 3.90 minutes. and isomer Example 238-Chloro-3-methyl-2-((3R,4S) or (3S,4R) or (3S,4S) or (3R,4R)-3-methyltetra- hydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine peak 4 (120 mg). 1H NMR (CDCl3 , 400MHz) δ 8.67 (s, 1H), 25 6.56 (br s, 1H), 4.52 - 4.47(m, 2H), 4.45 - 4.40 (m, 2H), 4.33 - 4.22 (m, 2H), 3.93 (dd, J = 4.4, 11.2 Hz, 1H), 3.68 - 3.58 (m, 2H), 2.54 - 2.43 (m, 1H), 2.30 (s, 3H), 2.15 - 2.05(m, 1H), 2.03 - 1.95 (m, 2H), 1.89 - 1.81 (m, 1H), 0.88 (d, J = 7.2 Hz, 3H). LC-MS (method C) (m/z) = 404.2 (MH)+ tR = 1.48 minutes. Chiral analytical SFC (instrument: Waters UPCC with PDA Detector, Column: Chiralpak AD-3150 × 4.6 mm I.D., particle size 3μm, mobile phase: A: CO2 B: Methanol (0.05% DEA), gra- 30 dient: 40% of iso-propanol (0.05% DEA) in CO2, flow rate: 2.5 mL/min, column temp.: 35 ºC, ABPR: 1500 psi, run time: 6 min, wavelength: 220 nm), ee > 99%, tR = 4.31 minutes. and isomer Example 248-Chloro-3-methyl-2-((3R,4S) or (3S,4R) or (3S,4S) or (3R,4R)-3-methyltetra- hydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine peak 2 (110 mg).1H NMR (400MHz, CDCl3) δ 8.66 (s, 1H), 6.59 (br s, 1H), 4.52 - 4.47(m, 2H), 4.45 - 4.40 (m, 2H), 4.33 - 4.22 (m, 2H), 3.93 (dd, J = 4.4, 11.2 Hz, 5 1H), 3.68 - 3.58 (m, 2H), 2.54 - 2.43 (m, 1H), 2.30 (s, 3H), 2.15 - 2.05(m, 1H), 2.03 - 1.95 (m, 2H), 1.89 - 1.81 (m, 1H), 0.88 (d, J = 7.2 Hz, 3H). LC-MS (method C) (m/z) = 404.2 (MH)+ tR = 1.48 minutes. Chiral analytical SFC (instrument: Waters UPCC with PDA Detector, Column: Chiralpak AD-3150 × 4.6mm I.D., particle size 3μm, mobile phase: A: CO2 B: Methanol (0.05% DEA), gradi- ent: 40% of iso-propanol (0.05% DEA) in CO2, flow rate: 2.5 mL/min, column temp.: 35 ºC, ABPR:10 1500psi, run time: 6 min, wavelength: 220 nm), ee > 99%, tR = 3.48 minutes. Example 25: 8-Chloro-2-((1r,4r)-4-fluorocyclohexyl)-3-methyl-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000192_0001
15 The compound was prepared in a manner similar to Example 1 using trans-3,6-dichloro- 1-(3-((1-(4-fluorocyclohexyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4- d]pyrimidine (110 mg, 0.27 mmol) , Fe (65 mg, 1.2 mmol), and NH4Cl (62 mg, 1.2 mmol) in EtOH (14 mL) and H2O (7 mL) at 80 ºC for 16 h, followed by work-up, and purification by chromatog- raphy on silica gel (eluent: Petroleum ether:EtOAc 100:0 → 0:100) and them preparative SFC (in-20 strument: SFC150AP, column: DAICEL CHIRALPAK AD(250mm×30mm, particle size 10μm), mo- bile phase: supercritical CO2/EtOH (0.1% NH3`H2O, v%) = 65/35, flow rate: 80 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator tempera- ture: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to afford the title compound (25 mg) 1H NMR (CDCl3 , 400MHz) δ 8.67 (br s, 1H), 6.71 (br s, 1H), 4.77 - 4.53 (m, 1H), 4.52 - 4.3725 (m, 4H), 4.03 - 3.86 (m, 1H), 2.29 - 2.23 (m, 5H), 2.13 - 2.01 (m, 2H), 2.01 - 1.91(m, 4H), 1.73 - 1.68 (m, 1H), 1.65 - 1.59 (m, 1H). LC-MS (method C) (m/z) = 406.1 (MH)+ tR = 1.58 minutes. Example 26: 8-Chloro-2-(4,4-difluorocyclohexyl)-3-methyl-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000193_0001
The compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- 5 ((1-(4,4-difluorocyclohexyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4- d]pyrimidine (0.26 g, 0.53 mmol) , Fe (148 mg, 2.65 mmol), and NH4Cl (142 mg, 2.65 mmol) in EtOH (50 mL) and H2O (10 mL) at 80 ºC for 16 h, followed by work-up, and purification by chro- matography on silica gel (eluent: Petroleum ether:EtOAc (10v% MeOH) 100:0 → 50:50) to afford the title compound (90 mg).1H NMR (CDCl3 , 400MHz) δ 8.67 (s, 1H), 6.68 (s, 1H), 4.55 - 4.38 (m,10 4H), 4.11 - 4.00 (m, 1H), 2.40 - 2.23 (m, 7H), 2.03 - 1.82 (m, 6H). LC-MS (method C) (m/z) = 424.2 (MH)+ tR = 1.64 minutes. Example 27: (–)-(R) or (S)-8-Chloro-3-methyl-2-(oxepan-3-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine and 15 Example 28 (+)-8-(R) or (S)-Chloro-3-methyl-2-(oxepan-3-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000193_0002
The racemic mixture of Example 27 and Example 28 was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3-((5-methyl-4-nitro-1-(oxepan-3-yl)-1H-pyrazol-3- 20 yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (240 mg, 0.51 mmol), Fe (143 mg, 2.55 mmol), and NH4Cl (137 mg, 2.55 mmol) in EtOH (15 mL) and H2O (5 mL) at 80 ºC for 16 h, followed by work- up and purification by chromatography on silica gel (eluent: Petroleum ether:EtOAc 30:70 → 20:80) to afford 8-Chloro-3-methyl-2-(oxepan-3-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (120 mg). The racemic mixture 25 (120 mg) was separated into the two enantiomers by preparative chiral SFC (instrument: Berger MultiGram II, column: DAICEL CHIRALPAK AD(250mm×30mm, particle size 10µm), mobile phase: supercritical CO2/IPA(0.1% ammonium hydroxide) = 55/45, flow rate: 80 mL/min, column tem- perature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator temperature: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to afford: 5 Example 27 (–)-(R) or (S)-8-Chloro-3-methyl-2-(oxepan-3-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (55 mg). 1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 6.62 (br s, 1H), 4.49 - 4.45 (m, 2H), 4.45 - 4.41 (m, 2H), 4.27 - 4.18 (m, 1H), 3.97 - 3.90 (m, 2H), 3.85 - 3.80 (m, 2H), 2.28 (s, 3H), 2.14 - 2.03 (m, 2H), 1.98-1.92 (m, 3H), 1.91 - 1.85 (m, 2H), 1.70 - 1.64 (m, 1H). LC-MS (method G) (m/z) = 404.2 (MH)+ tR = 1.50 10 minutes. Chiral analytical SFC (instrument: Agilent 1260 with DAD detector, column: ChiralPak AD-350 × 4.6mm I.D., particle size 3μm, mobile phase: A: CO2 B: iso-propanol (0.05% DEA), gra- dient: hold 40% for 3 min, flow rate: 4 mL/min, column temperature: 40 ºC, ABPR: 1500psi, run time: 3 minutes, wavelength: 220 nm) ee = >99%, tR = 0.88 minutes. = -10.0 (c = 0.1 g/100 mL, MeOH) 15 and the corresponding enantiomer Example 28 (+)-(R) or (S)-8-Chloro-3-methyl-2-(oxepan-3-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (50 mg). 1H NMR (CDCl3, 400MHz) δ 8.65 (s, 1H), 6.69 (br s, 1H), 4.49 - 4.45 (m, 2H), 4.45 - 4.41 (m, 2H), 4.27 - 4.18 (m, 1H), 3.97 - 3.90 (m, 2H), 3.85 - 3.80 (m, 2H), 2.28 (s, 3H), 2.14 - 2.03 (m, 2H), 1.98-1.92 (m,20 3H), 1.91 - 1.85 (m, 2H), 1.70 - 1.64 (m, 1H). LC-MS (method G) (m/z) = 404.2 (MH)+ tR = 1.50 minutes. Chiral analytical SFC (instrument: Agilent 1260 with DAD detector, column: ChiralPak AD-350 × 4.6mm I.D., particle size 3μm, mobile phase: A: CO2 B: iso-propanol (0.05% DEA), gra- dient: hold 40% for 3 min, flow rate: 4 mL/min, column temperature: 40 ºC, ABPR: 1500psi, run time: 3 minutes, wavelength: 220 nm) ee = >99%, tR = 1.98 minutes. = +8.0 (c = 0.1 g/10025 mL, MeOH) Example 29: (–)-(R) or (S)-8-Chloro-3-methyl-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine and Example 30 (+)-8-(R) or (S)-Chloro-3-methyl-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7-30 (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000195_0001
The racemic mixture of Example 29 and Example 30 was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3-((5-methyl-4-nitro-1-(oxepan-4-yl)-1H-pyrazol-3- yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (330 mg, 0.702 mmol) Fe (196 mg, 3.51 mmol), and 5 NH4Cl (188 mg, 3.51 mmol) in EtOH (20 mL) and H2O (4 mL) at 80 ºC for 15 h, followed by work- up and purification by chromatography on silica gel (eluent: Petroleum ether:EtOAc 100:0 → 0:100) to afford 8-chloro-3-methyl-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (120 mg). The racemic mixture (120 mg) was separated into the two enantiomers by preparative chiral SFC (instrument: SFC-10 80Q, column: DAICEL CHIRALPAK AD 250 × 30 mm, particle size 10 μm, mobile phase: supercriti- cal CO2/IPA (0.1% NH3.H2O, v%)= 55/45, flow rate: 80 mL/min, column temperature: 38 ºC, noz- zle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator temperature: 20 ºC, trimmer tem- perature: 25 ºC, wavelength: 220 nm) to afford: Example 29: (–)-(R) or (S)-8-Chloro-3-methyl-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H- 15 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (45 mg). 1H NMR (CDCl3, 400MHz) δ 8.64 (s, 1H), 6.85 (br s, 1H), 4.59 - 4.36 (m, 4H), 4.32 - 4.20 (m, 1H), 3.97 - 3.84 (m, 2H), 3.83 - 3.74 (m, 1H), 3.71 - 3.61 (m, 1H), 2.45 - 2.32 (m, 1H), 2.27 (s, 3H), 2.26 - 2.18 (m, 1H), 2.17 - 2.07 (m, 1H), 2.04 - 1.92 (m, 3H), 1.92 - 1.85 (m, 1H), 1.84 - 1.75 (m, 1H). LC-MS (method C) (m/z) = 404.2 (MH)+ tR = 1.45 minutes. Chiral analytical SFC (instrument: Waters 20 UPCC with PDA Detector, column: Chiralpak AD-350 × 4.6mm I.D., particle size 3μm, mobile phase: A: CO2 B: iso-propanol (0.05% DEA), gradient: from 5% to 40% of B in 2 min and hold 40% for 1.2 min, then 5% of B for 0.8 min, flow rate: 4 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 4 min, wavelength: 220 nm) ee = 98%, tR = 2.48 minutes. = -4.0 (c = 0.1 g/100 mL, MeOH) 25 and the corresponding enantiomer Example 30 (+)-(R) or (S)-8-Chloro-3-methyl-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (50 mg). 1H NMR (CDCl3, 400MHz) δ 8.65 (s, 1H), 6.71 (br s, 1H), 4.57 - 4.35 (m, 4H), 4.32 - 4.18 (m, 1H), 3.96 - 3.84 (m, 2H), 3.83 - 3.74 (m, 1H), 3.71 - 3.62 (m, 1H), 2.45 - 2.32 (m, 1H), 2.28 (s, 3H), 2.27 - 2.18 (m, 30 1H), 2.17 - 2.08 (m, 1H), 2.06 - 1.92 (m, 3H), 1.92 - 1.85 (m, 1H), 1.84 - 1.72 (m, 1H). LC-MS (method C) (m/z) = 404.2 (MH)+ tR = 1.45 minutes. Chiral analytical SFC (instrument: Waters UPCC with PDA Detector, column: Chiralpak AD-350 × 4.6mm I.D., particle size 3μm, mobile phase: A: CO2 B: iso-propanol (0.05% DEA), gradient: from 5% to 40% of B in 2 min and hold 40% for 1.2 min, then 5% of B for 0.8 min, flow rate: 4 mL/min, column temperature: 35 ºC, ABPR: 5 1500psi, run time: 4 min, wavelength: 220 nm) ee = >99%, tR = 2.34 minutes. = +4.0 (c = 0.1 g/100 mL, MeOH). Example 31 (–)-8-Chloro-2-((3S,4R) or (3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)-3-methyl- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy-10 cloundecine and Example 32 (+)-8-Chloro-2-((3S,4R) or (3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)-3-methyl- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine 15
Figure imgf000196_0001
The racemic mixture of Example 31 and Example 32 was prepared in a manner similar to Example 1 using trans-3,6-dichloro-1-(3-((1-(3-fluorotetrahydro-2H-pyran-4-yl)-5-methyl-4- nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (450 mg, 0.95 mmol), Fe (265 mg, 4.74 mmol), and NH4Cl (254 mg, 4.74 mmol) in EtOH (40 mL) and H2O (4 mL) at 80 ºC for 15 20 h, followed by work-up and purification by chromatography on silica gel (eluent: Petroleum ether:EtOAc 100:0 → 50:50) to afford 8-Chloro-2-((3S,4R and 3R,4S)-3-fluorotetrahydro-2H-py- ran-4-yl)-3-methyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine (200 mg). The racemic mixture (200 mg) was separated into the two enantiomers by preparative chiral SFC (instrument: Berger MultiGram II, col- 25 umn: DAICEL CHIRALPAK AS 250 × 30 mm, particle size 10 μm, mobile phase: supercritical CO2/IPA (0.1% NH3.H2O, v%)= 75/25, flow rate: 70 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator temperature: 20 ºC, trimmer temper- ature: 25 ºC, wavelength: 220 nm) to afford: Example 31 (–)-8-Chloro-2-((3S,4R) or (3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)-3-methyl- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- 5 cloundecine (60 mg).1H NMR (CDCl3, 400MHz) δ 8.67 (s, 1H), 6.57 (br s, 1H), 4.99 - 4.76 (m, 1H), 4.58 - 4.35 (m, 4H), 4.31 - 4.23 (m, 1H), 4.22 - 3.98 (m, 2H), 3.58 - 3.45 (m, 1H), 3.44 - 3.32 (m, 1H), 2.59 - 2.43 (m, 1H), 2.31 (s, 3H), 2.13 - 1.81 (m, 3H). LC-MS (method G) (m/z) = 408.1 (MH)+ tR = 1.43 minutes. Chiral analytical SFC (instrument: Waters UPCC with PDA Detector, column: Chiralpak AS-3100 × 4.6mm I.D., particle size 3μm, mobile phase: A: CO2 B: ethanol (0.05% DEA), 10 gradient: from 5% to 40% of B in 4 min and hold 40% for 0.5 min, then 5% of B for 1.5 min, flow rate: 2.8 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 6 min, wavelength: 220 nm), ee > 99%, tR = 2.77 minutes. = -42 (c = 0.5 g/100 mL, CHCl3). and the corresponding enantiomer Example 32 (+)-8-Chloro-2-((3S,4R) or (3R,4S)-3-fluorotetrahydro-2H-pyran-4-yl)-3-methyl-15 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine (60 mg).1H NMR (CDCl3, 400MHz) δ 8.67 (s, 1H), 6.64 (br s, 1H), 5.03 - 4.75 (m, 1H), 4.59 - 4.37 (m, 4H), 4.31 - 4.23 (m, 1H), 4.20 - 4.01 (m, 2H), 3.59 - 3.46 (m, 1H), 3.44 - 3.33 (m, 1H), 2.61 - 2.41 (m, 1H), 2.31 (s, 3H), 2.11 - 1.84 (m, 3H). LC-MS (method G) (m/z) = 408.1 (MH)+ tR = 1.43 minutes. Chiral analytical SFC (instrument: Waters UPCC with PDA Detector, column: 20 Chiralpak AS-3100 × 4.6mm I.D., particle size 3μm, mobile phase: A: CO2 B: ethanol (0.05% DEA), gradient: from 5% to 40% of B in 4 min and hold 40% for 0.5 min, then 5% of B for 1.5 min, flow rate: 2.8 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 6 min, wavelength: 220 nm), ee = 98.5%, tR = 3.30 minutes. = +48.5 (c = 0.8 g/100 mL, CHCl3) 25 Example 333-(8-Chloro-3-methyl-12,13-dihydro-11H-5,7-(azenometheno)dipyrazolo[3,4- b:5',1'-g][1]oxa[4,6,8]triazacycloundecin-2(4H)-yl)bicyclo[1.1.1]pentane-1-carbonitrile
Figure imgf000197_0001
To a solution of 3-(8-chloro-3-methyl-4,11,12,13-tetrahydro-2H-5,7-(azenometheno)di-30 pyrazolo[3,4-b:5',1'-g][1,4,6,8]oxatriazacycloundecin-2-yl)bicyclo[1.1.1]pentane-1-carbox- amide (70 mg, 0.17 mmol) in DCM (10 mL) were added TFAA (53.16 mg, 0.253 mmol) and TEA (68 mg, 0.68 mmol) at 0 ºC. The reaction mixture was stirred at 0 ºC for 1 h. Then additional TFAA (106 mg, 0.50 mmol) and TEA (70 mg, 0.69 mmol) was added to the mixture and the mixture was stirred at 0 ºC for 1 h. The reaction mixture was diluted with water (20 mL) and extracted with DCM (3×20 mL). The combined organic layers were washed with brine (3×20 mL), dried 5 over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified using preparative SFC (instrument: Berger MultiGram II, column: DAICEL CHIRALCEL OD-H 250 × 30 mm, particle size 5 μm, mobile phase: supercritical CO2/ETOH (0.1% NH3.H2O, v%)= 60/40, flow rate: 80 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator temperature: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to afford 10 the title compound (35 mg).1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 6.70 (br s, 1H), 4.53 - 4.36 (m, 4H), 2.79 (s, 6H), 2.32 (s, 3H), 2.03 - 1.85 (m, 2H). LC-MS (method G) (m/z) = 397.1 (MH)+ tR = 1.47 minutes. Example 34: 8-Chloro-3-methyl-2-(2-oxaspiro[3.3]heptan-6-yl)-2,4,12,13-tetrahydro-11H-5,7-15 (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000198_0001
The compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- ((5-methyl-4-nitro-1-(2-oxaspiro[3.3]heptan-6-yl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4- d]pyrimidine (70 mg, 0.15 mmol), Fe (42 mg, 0.75 mmol), and NH4Cl (42 mg, 0.78 mmol) in EtOH 20 (7 mL) and H2O (3.5 mL) at 54 ºC for 16 h, followed by work-up, and purification by preparative HPLC (instrument: Gilson GX-215, Gilson 322 Pump, Gilson 156 UV Detector, column: Welch Xti- mate C18150×30mm×5μm, mobile Phase A: water (0.05%NH3H2O+10mM NH4HCO3), mobile phase B: MeCN, gradient: B from 28% to 58% in 9 min then hold at 100% for 2 min, flow rate (mL/min): 35, column temperature: 30 ºC, wavelengths: 220 nm and 254nm) to afford the title 25 compound (12 mg).1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 6.59 (br s, 1H), 4.80 (s, 2H), 4.75 (s, 2H), 4.54 - 4.47(m, 2H), 4.45 - 4.36 (m, 3H), 2.89 - 2.80 (m, 2H), 2.73 - 2.66 (m, 2H), 2.23 (s, 3H), 1.98 - 1.85(m, 2H). LC-MS (method G) (m/z) = 402.1 (MH)+ tR = 1.38 minutes. Example 35: 8-Chloro-3-(methyl-d3)-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine- 11,11,12,12,13,13-d6
Figure imgf000199_0001
5 The compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- ((5-(methyl-d3)-4-nitro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-3-yl)oxy)propyl-1,1,2,2,3,3-d6)- 1H-pyrazolo[3,4-d]pyrimidine (100 mg, 0.215 mmol), Fe (60.0mg, 1.07 mmol), and NH4Cl (57.5 mg, 1.07 mmol) in EtOH (1 mL) and H2O (0.1 mL) at 100 ºC for 2 h, followed by work-up, and purification by preparative HPLC (instrument: Waters sample manager 2767; Waters 2545 10 pump, Waters UV Detector 2545, column: Sunfire prep C18 column, 30×150 mm, particle size 5μm, mobile phase A: Water(0.1%FA), mobile phase B: CAN, gradient: 18% B to 53% B in 7 min, flow rate: 60 mL/min, column temperature: 30 ºC, wavelengths: 220 nm, 254 nm) to afford the title compound (20 mg).1H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 7.74 (s, 1H), 4.14-4.08 (m, 3H), 3.52 (t, J = 11.2 Hz, 2H), 2.27 (tt, J = 12.4, 8.0 Hz, 2H), 1.80 (d, J = 11.6 Hz, 2H). LC-MS (method15 E) (m/z) = 399.7 (MH)+ tR = 0.56 minutes. Example 36: 1-(8-Chloro-3-methyl-12,13-dihydro-11H-5,7-(azenometheno)dipyrazolo[3,4- b:5',1'-g][1]oxa[4,6,8]triazacycloundecin-2(4H)-yl)cyclopropane-1-carbonitrile
Figure imgf000199_0002
20 The compound was prepared in a manner similar to Example 33 using 1-(8-chloro-3- methyl-4,11,12,13-tetrahydro-2H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1,4,6,8]ox- atriazacycloundecin-2-yl)cyclopropanecarboxamide (35 mg, 0.090 mmol), TFAA (28 mg, 0.135 mmol) and TEA (36.4 mg, 0.360 mmol) in DCM (14 mL) at 0 ºC for 1 h, followed by work-up and preparative SFC (instrument: SFC-80Q, column: DAICEL CHIRALCEL OD-H(250mm × 30mm, par- 25 ticle size 5μm), mobile phase: supercritical CO2 /EtOH (0.1% NH3.H2O, v %) = 30/30, flow rate: 80 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evap- orator temperature: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to afford the title compound (12 mg). 1H NMR (CDCl3, 400MHz) δ 8.67 (s, 1H), 7.01 (br s, 1H), 4.63 - 4.28 (m, 4H), 2.44 (s, 3H), 1.95 - 1.90 (m, 2H), 1.85 - 1.75 (m, 4H). LC-MS (method G) (m/z) = 371.1 (MH)+ tR = 5 1.40 minutes. Example 37: 8-Chloro-2-((1r,4r)-4-methoxycyclohexyl)-3-methyl-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000200_0001
The compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3-10 ((1-((1r,4r)-4-methoxycyclohexyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyra- zolo[3,4-d]pyrimidine (160 mg, 0.33 mmol), Fe (92 mg, 1.7 mmol), and NH4Cl (88 mg, 1.7 mmol) in EtOH (16 mL) and H2O (4 mL) at 80 ºC for 16 h, followed by work-up, and purification by chro- matography on silica gel (eluent: Petroleum ether:EtOAc 83:17 → 0:100) and preparative SFC (instrument: Thar SFC Prep 80, column: DAICEL CHIRALCEL OJ (250mm×30mm, 10μm), mobile15 phase: supercritical CO2/EtOH (0.1% NH3.H2O, v %) = 70/30, flow rate: 70 mL/min, column tem- perature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator temperature: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to afford the title compound (80 mg). 1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 6.63 (br, 1H), 4.59 - 4.36 (m, 4H), 4.03 - 3.82 (m, 1H), 3.39 (s, 3H), 3.31 - 3.16 (m, 1H), 2.28 (s, 3H), 2.26 - 2.17 (m, 2H), 2.09 - 1.86 (m, 6H), 1.42 - 1.2620 (m, 2H). LC-MS (method C) (m/z) = 418.2 (MH)+ tR = 1.52 minutes. Example 38: 2-((1R,5S,6r)-3-Oxabicyclo[3.1.0]hexan-6-yl)-8-chloro-3-methyl-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000200_0002
The compound was prepared in a manner similar to Example 1 using 1-(3-((1-(3-oxabi- cyclo[3.1.0]hexan-6-yl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-3,6-dichloro-1H-pyra- zolo[3,4-d]pyrimidine (230 mg, 0.51 mmol), Fe (142 mg, 2.54 mmol), and NH4Cl (136 mg, 2.54 mmol) in EtOH (10 mL) and H2O (2.5 mL) at 80 ºC for 12 h, followed by work-up, and purification 5 by chromatography on silica gel (eluent: Petroleum ether:EtOAc 100:0 → 0:100) and preparative (instrument: Gilson GX-215, Gilson 322 Pump, Gilson 156 UV Detector, column: Welch Xtimate C18150 × 30mm × 5μm, mobile Phase A: water (NH3H2O + NH4HCO3), mobile phase B: MeCN, gradient: B from 30% to 60% in 7 min, flow rate (mL/min): 25 mL/min, column temperature: 30 ºC, wavelengths: 220nm 254nm) to afford the title compound (61 mg). 1H NMR (DMSO-d6, 10 400MHz) δ 9.41 (br s, 1H), 8.78 (s, 1H), 4.35 - 4.31 (m, 2H), 4.29 - 4.25 (m, 2H), 3.98 (d, J =8.4 Hz, 2H), 3.70 (d, J =8.4 Hz, 2H), 3.10 - 3.04 (m, 1H), 2.33 - 2.28 (m, 2H), 2.28 (s, 3H), 1.79 - 1.75 (m, 2H). LC-MS (method G) (m/z) = 388.1 (MH)+ tR = 1.37 minutes. Example 39 (–)-(R) or (S)-8-Chloro-2-(2,2-difluorocyclopropyl)-3-methyl-2,4,12,13-tetrahydro-15 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine and Example 40 (+)-(R) or (S)-8-Chloro-2-(2,2-difluorocyclopropyl)-3-methyl-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000201_0001
A mixture of 8-chloro-3-methyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyra-20 zolo[3,4-b:5',1'-g][1]oxa[4,6,8]-triazacycloundecine (300 mg, 0.981 mmol), (2,2-difluorocyclo- propyl)boronic acid (120mg, 0.981 mmol), pyridine (315 mg, 3.98 mmol), Cu(OAc)2 (270 mg, 1.49 mmol) and 4Å MS (300 mg) in DCE (10 mL) was stirred at 65 ºC under oxygen (15 psi) for 12 h. The mixture was filtered, the filter cake was washed with DCM (3×10 mL), and the combined filtrate was concentrated under reduced pressure. The residue was purified by chromatography25 on silica gel (eluent: Petroleum ether:EtOAc 100:0 → 40:60) to afford 8-Chloro-2-(2,2-difluoro- cyclopropyl)-3-methyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine (80 mg). The racemic mixture (80 mg) was separated into the two enantiomers by preparative chiral SFC (instrument: Berger MultiGram II, column: Phenom- enex-Cellulose-2250 × 30 mm, 10 μm, mobile phase: supercritical CO2/EtOH (0.1% NH3.H2O, 30 v%)= 70/30, flow rate: 80 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator temperature: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to afford: Example 39 (–)-(R) or (S)-8-Chloro-2-(2,2-difluorocyclopropyl)-3-methyl-2,4,12,13-tetra- hydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (30 5 mg).1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 6.85 (br s, 1H), 4.63 - 4.34 (m, 4H), 3.84 - 3.69 (m, 1H), 2.41 - 2.24 (m, 4H), 2.13 - 1.95 (m, 2H), 1.94 - 1.81 (m, 1H). LC-MS (method C) (m/z) = 382.1 (MH)+ tR = 1.51 minutes. Chiral analytical SFC (instrument: Waters UPCC with PDA Detector and QDa Detector, column: Cellulose-2100 × 4.6mm I.D., particle size 3μm, mobile phase: A: CO2 B: ethanol (0.05% DEA), gradient: from 5% to 40% of B in 4 min and hold 40% for 0.5 min, then 5% 10 of B for 1.5 min, flow rate: 2.8 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 6 min, wavelength: 220 nm), ee > 99%, tR = 2.62 minutes. = -68 (c = 0.1 g/100 mL, MeOH). and the corresponding enantiomer Example 40 (+)-(R) or (S)-8-Chloro-2-(2,2-difluorocyclopropyl)-3-methyl-2,4,12,13-tetra- hydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (30 15 mg).1H NMR (CDCl3, 400MHz) δ 8.68 (s, 1H), 6.66 (br s, 1H), 4.58 - 4.36 (m, 4H), 3.83 - 3.71 (m, 1H), 2.40 - 2.26 (m, 4H), 2.13 - 1.95 (m, 2H), 1.95 - 1.77 (m, 1H). LC-MS (method C) (m/z) = 382.1 (MH)+ tR = 1.51 minutes. Chiral analytical SFC (instrument: Waters UPCC with PDA Detector and QDa Detector, column: Cellulose-2100 × 4.6mm I.D., particle size 3μm, mobile phase: A: CO2 B: ethanol (0.05% DEA), gradient: from 5% to 40% of B in 4 min and hold 40% for 0.5 min, then 5% 20 of B for 1.5 min, flow rate: 2.8 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 6 min, wavelength: 220 nm), ee = 95.3%, tR = 2.97 minutes. = +68 (c = 0.1 g/100 mL, MeOH) Example 41: 8-chloro-2-((2R,4r,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-3-methyl-2,4,12,13- tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 25
Figure imgf000202_0001
The compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- ((1-((2R,4r,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-5-methyl-4-nitro-1H-pyrazol-3- yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (140 mg, 0.29 mmol), Fe (129 mg, 2.31mmol), and NH4Cl (124 mg, 2.31 mmol) in EtOH (20 mL) and H2O (5 mL) at 80 ºC for 12 h, followed by work- up, and purification by chromatography on silica gel (eluent: Petroleum ether:EtOAc70:30 → 70:30) to afford the title compound (40 mg).1H NMR (CDCl3, 400MHz) δ 8.68 (s, 1H), 6.68 (br s, 1H), 4.49 - 4.45 (m, 4H), 4.18 - 4.15 (m, 1H), 3.63 (m, 2H), 2.30 (s, 3H), 2.05 - 1.76 (m, 6H), 1.30 (d, J = 3.6 Hz, 6H). LC-MS (method C) (m/z) = 418.2 (MH)+ tR = 1.56 minutes. 5 Example 42 (+)-(R) or (S)-8-Chloro-2-(2,2-dimethyltetrahydro-2H-pyran-4-yl)-3-methyl- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine and Example 43 (–)-(R) or (S)-8-Chloro-2-(2,2-dimethyltetrahydro-2H-pyran-4-yl)-3-methyl- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy-10 cloundecine
Figure imgf000203_0001
The racemic mixture of Example 42 and Example 43 was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3-((1-(2,2-dimethyltetrahydro-2H-pyran-4-yl)-5-methyl-4-ni- tro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (210 mg, 0.43 mmol), Fe (121 mg, 15 2.17 mmol), and NH4Cl (116 mg, 2.17 mmol) in EtOH (25 mL) and H2O (6 mL) at 80 ºC for 12 h, followed by work-up and purification by chromatography on silica gel (eluent: Petroleum ether:EtOAc 80:20 → 25:75) to afford 8-Chloro-2-(2,2-dimethyltetrahydro-2H-pyran-4-yl)-3-me- thyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triaza- cycloundecine (112 mg). The racemic mixture (112 mg) was separated into the two enantiomers 20 by preparative chiral SFC (instrument: Berger MultiGram II, column: DAICEL CHIRALCEL OD 250 × 30 mm, 10 μm, mobile phase: supercritical CO2/EtOH (0.1% NH3.H2O, v%)= 70/30, flow rate: 80 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evap- orator temperature: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to afford: Example 42 (+)-(R) or (S)-8-Chloro-2-(2,2-dimethyltetrahydro-2H-pyran-4-yl)-3-methyl-25 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine (34 mg).1H NMR (CDCl3, 400MHz) δ 8.67 (s, 1H), 6.66 (br s, 1H), 4.60 - 4.38 (m, 4H), 4.36 - 4.22 (m, 1H), 3.99 - 3.88 (m, 1H), 3.86 - 3.76 (m, 1H), 2.31 (s, 3H), 2.27 - 2.15 (m, 1H), 2.15 - 2.06 (m, 1H), 2.04 - 1.90 (m, 2H), 1.84 - 1.71 (m, 2H), 1.33 (d, J = 4.0 Hz, 6H). LC-MS (method C) (m/z) = 418.2 (MH)+ tR = 1.52 minutes. Chiral analytical SFC (instrument: Waters UPCC with PDA Detector, column: Chiralcel OD-3150 × 4.6mm I.D., 3μm, mobile phase: 40% of ethanol (0.05% DEA) in CO2, flow rate: 2.5 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 3.5 min, wavelength: 220 nm), ee > 99%, tR = 2.14 minutes. = +27.5 (c = 0.24 g/100 mL, MeOH) and the corresponding enantiomer 5 Example 43 (–)-(R) or (S)-8-Chloro-2-(2,2-dimethyltetrahydro-2H-pyran-4-yl)-3-methyl- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine (37 mg). 1H NMR (CDCl3, 400MHz) δ 8.70 (s, 1H), 6.66 (br s, 1H), 4.56 - 4.42 (m, 4H), 4.34 - 4.25 (m, 1H), 3.97 - 3.90 (m, 1H), 3.86 - 3.76 (m, 1H), 2.31 (s, 3H), 2.27 - 2.15 (m, 1H), 2.14 - 2.06 (m, 1H), 2.04 - 1.90 (m, 2H), 1.84 - 1.72 (m, 2H), 1.33 (d, J = 4.010 Hz, 6H). LC-MS (method C) (m/z) = 418.2 (MH)+ tR = 1.52 minutes. Chiral analytical SFC (instru- ment: Waters UPCC with PDA Detector, column: Chiralcel OD-3150 × 4.6mm I.D., 3μm, mobile phase: 40% of ethanol (0.05% DEA) in CO2, flow rate: 2.5 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 3.5 min, wavelength: 220 nm), ee > 99%, tR = 1.55 minutes. = -24 (c = 0.2 g/100 mL, MeOH). 15 Example 44: 2-((1R,3s,5S)-8-Oxabicyclo[3.2.1]octan-3-yl)-8-chloro-3-methyl-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000204_0001
The compound was prepared in a manner similar to Example 1 using 1-(3-((1-(8-oxabi-20 cyclo[3.2.1]octan-3-yl)-5-methyl-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-3,6-dichloro-1H-pyra- zolo[3,4-d]pyrimidine (0.62 g, 1.3 mmol), Fe (162 mg, 2.89 mmol), and NH4Cl (155 mg, 2.89 mmol) in EtOH (24 mL) and H2O (6 mL) at 80 ºC for 16 h, followed by work-up, and purification by preparative HPLC (SHIMADZU LH-40, column: Xtimate C18 100×30mm×10 um), mobile phase A: water (FA), mobile phase B: MeCN, gradient: B from 50% to 80% in 10 min, hold 25 100% B for 2 min, flow rate: 25 mL/min, column temperature: 25 ºC, wavelengths: 220 nm, 254 nm) to afford the title compound (30 mg).1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 6.89 (br s, 1H), 4.58 - 4.54 (m, 2H), 4.52 - 4.40 (m, 4H), 4.39 - 4.27 (m, 1H), 2.47 - 2.34 (m, 2H), 2.29 (s, 3H), 2.15 - 2.04 (m, 2H), 2.00 - 1.92 (m, 2H), 1.85 - 1.82 (m, 2H), 1.78-1.73 (m, 2H). LC- MS (method C) (m/z) = 416.2 (MH)+ tR = 1.45 minutes. Example 45: 8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine-11,11-d2
Figure imgf000205_0001
5 The compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-[1,1- dideuterio-3-(5-methyl-4-nitro-1-tetrahydropyran-4-yl-pyrazol-3-yl)oxy-propyl]pyrazolo[3,4- d]pyrimidine (680 mg, 1.48 mmol), Fe (415 mg, 7.43 mmol), and NH4Cl (397 mg, 7.42 mmol) in EtOH (20 mL) and H2O (2 mL) at 80 ºC for 16 h, followed by work-up, and purification by chro- matography on silica gel (eluent: Petroleum ether:EtOAc 100:0 → 0:100) and preparative HPLC 10 (instrument: Gilson GX-215, Gilson 322 Pump, Gilson 156 UV Detector, column: Welch Xtimate C18150 × 30mm × 5μm, mobile phase A: water (NH3H2O + NH4HCO3), mobile phase B: MeCN, gradient: B from 28% to 58% in 25 min then hold at 100% for 2 min, flow rate (mL/min): 35 mL/min, column temperature: 30 ºC, wavelengths: 220nm, 254nm) to afford the title compound (65 mg).1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 6.67 (br s, 1H), 4.62 - 4.45 (m, 2H), 4.16 - 4.08 15 (m, 3H), 3.62 - 3.44 (m, 2H), 2.34 - 2.24 (m, 5H), 1.97 - 1.92 (m, 2H), 1.84 - 1.78 (m, 2H). LC-MS (method G) (m/z) = 392.2 (MH)+ tR = 1.38 minutes. Example 46: 8-Chloro-3-methyl-2-(tetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine-13,13-d2 20
Figure imgf000205_0002
The compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-[3,3- dideuterio-3-(5-methyl-4-nitro-1-tetrahydropyran-4-yl-pyrazol-3-yl)oxy-propyl]pyrazolo[3,4- d]pyrimidine (700 mg, 1.5 mmol), Fe (427 mg, 7.64 mmol), and NH4Cl (409 mg, 7.64 mmol) in EtOH (30 mL) and H2O (5 mL) at 80 ºC for 15 h, followed by work-up, and purification by chro- 25 matography on silica gel (eluent: Petroleum ether:EtOAc 100:0 → 30:70) and preparative HPLC (instrument: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV Detector, column: Welch Xtimate C18150 × 30 mm × 5 μm, mobile phase: A: water (NH3H2O+NH4HCO3), mobile phase B: MeCN, gradient: B from 28% to 58% in 25 min then hold at 100% for 2 min, flow rate (mL/min): 35, column temperature: 35 ºC, wavelengths: 220nm, 254nm) to afford the title com- 5 pound (50 mg).1H NMR (CDCl3, 400MHz) δ 8.65 (s, 1H), 6.89 (br s, 1H), 4.43 (t, J = 4.8 Hz, 2H), 4.17 - 4.06 (m, 3H), 3.52 (t, J = 10.8 Hz, 2H), 2.35 - 2.22 (m, 5H), 2.01 - 1.88 (m, 2H), 1.85 - 1.77 (m, 2H). LC-MS (method G) (m/z) = 392.1 (MH)+ tR = 1.38 minutes. Example 47 (+)-8-Chloro-2-((3R,4S) or (3S,4R)-3-Fluoro-3-methyltetrahydro-2H-pyran-4-yl)-3-10 methyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine and Example 48 (–)-8-Chloro-2-((3R,4S) or (3S,4R)-3-Fluoro-3-methyltetrahydro-2H-pyran-4-yl)-3- methyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine 15
Figure imgf000206_0001
The racemic mixture of Example 47 and Example 48 was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3-((1-(3-fluoro-3-methyltetrahydro-2H-pyran-4-yl)-5-methyl-4- nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (0.16 g, 0.33mmol), Fe (91 mg, 1.64 mmol), and NH4Cl (88 mg, 1.64 mmol) in EtOH (40 mL) and H2O (4 mL) at 80 ºC for 14 h, 20 followed by work-up and purification by chromatography on silica gel (eluent: Petroleum ether:EtOAc (10 v% MeOH) 100:0 → 50:50) to afford 8-chloro-2-(3-fluoro-3-methyltetrahydro- 2H-pyran-4-yl)-3-methyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine (65 mg). The mixture (65 mg) was separated using prepara- tive chiral SFC (instrument: Waters 150, column: DAICEL CHIRALPAK AD 150 × 30 mm, 10 μm,25 mobile phase: supercritical CO2/EtOH (0.1% NH3.H2O, v%)= 50/50, flow rate: 130 mL/min, col- umn temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator tem- perature: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to afford: Example 47 (+)-8-Chloro-2-((3R,4S) or (3S,4R)-3-Fluoro-3-methyltetrahydro-2H-pyran- 4-yl)-3-methyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine (20 mg).1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 6.75 (br s, 1H), 4.53 - 4.39 (m, 4H), 4.34 - 4.24 (m, 1H), 4.22 - 4.13 (m, 1H), 3.96 - 3.88 (m, 1H), 3.63 - 3.53 (m, 1H), 3.52 - 3.46 (m, 1H), 2.66 - 2.50 (m, 1H), 2.32 (s, 3H), 2.07 - 1.90 (m, 3H), 1.32 (d, J = 23.6 Hz, 3H). LC-MS (method C) (m/z) = 422.2 (MH)+ tR = 1.53 minutes. Chiral analytical SFC ((instru- 5 ment: Waters UPCC with PDA Detector, column: Chiralpak AD-350 × 4.6mm I.D., 3μm, mobile phase: A: CO2 B: ethanol (0.05% DEA), gradient: isocratic 40% B, flow rate: 4 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 5 min, wavelength: 220 nm), ee > 99%, tR = 1.06 minutes. = +39.3 (c = 0.3 g/100 mL, MeOH) and the corresponding enantiomer 10 Example 48 (–)-8-Chloro-2-((3R,4S) or (3S,4R)-3-Fluoro-3-methyltetrahydro-2H-pyran- 4-yl)-3-methyl-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine (23 mg).1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 6.76 (br s, 1H), 4.53 - 4.39 (m, 4H), 4.34 - 4.24 (m, 1H), 4.22 - 4.13 (m, 1H), 3.96 - 3.88 (m, 1H), 3.63 - 3.53 (m, 1H), 3.52 - 3.46 (m, 1H), 2.66 - 2.50 (m, 1H), 2.32 (s, 3H), 2.07 - 1.90 (m, 3H), 1.32 (d, J = 24.015 Hz, 3H). LC-MS (method C) (m/z) = 422.2 (MH)+ tR = 1.53 minutes. Chiral analytical SFC ((instru- ment: Waters UPCC with PDA Detector, column: Chiralpak AD-350 × 4.6mm I.D., 3μm, mobile phase: A: CO2 B: ethanol (0.05% DEA), gradient: isocratic 40% B, flow rate: 4 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 5 min, wavelength: 220 nm), ee > 99%, tR = 3.00 minutes. = -38 (c = 0.3 g/100 mL, MeOH). 20 Example 498-Chloro-3-methyl-2-((tetrahydro-2H-pyran-4-yl)methyl)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000207_0001
The compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3-25 ((5-methyl-4-nitro-1-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazol-3-yl)oxy)propyl)-1H-pyra- zolo[3,4-d]pyrimidine (670 mg, 1.4 mmol), Fe (398 mg, 7.12 mmol), and NH4Cl (381 mg, 7.12 mmol) in EtOH (10 mL) and H2O (1 mL) at 80 ºC for 16 h, followed by work-up, and purification by chromatography on silica gel (eluent: Petroleum ether:EtOAc 100:0 → 0:100) and preparative SFC (instrument: Thar SFC Prep 80, column: DAICEL CHIRALCEL OD-H (250 × 30 mm, 5 μm), mo- bile phase: supercritical CO2/ETOH (0.1% NH3.H2O, v%)= 70/30, flow rate: 80 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60ºC, evaporator tempera- ture: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to afford the title compound (50 5 mg).1H NMR (DMSO-d6, 400MHz) δ 9.36 (s, 1H), 8.78 (br s, 1H), 4.39 - 4.21 (m, 4H), 3.91 - 3.74 (m, 4H), 3.28 - 3.21 (m, 2H), 2.22 (s, 3H), 2.04 - 1.96 (m, 1H), 1.93 - 1.65 (m, 2H), 1.47 - 1.38 (m, 2H), 1.33 - 1.20 (m, 2H). LC-MS (method C) (m/z) = 404.2 (MH)+ tR = 1.39 minutes. Example 50: 8-Chloro-2-((1r,4r)-4-methoxycyclohexyl)-3-(methyl-d3)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 10
Figure imgf000208_0001
To a mixture of 3-bromo-8-chloro-2-((1r,4r)-4-methoxycyclohexyl)-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (235 mg, 0.487 mmol) and bis[tris(tert-butyl)phosphine]palladium (3.3 mg, 0.0065 mmol) was added THF (3 mL) at room temperature. Then LiHMDS in THF (490 µL, 1 M, 0.490 mmol) was added at room 15 temperature. The mixture was stirred for 20 minutes. Then bis(methyl-d3)zinc in THF-dibutyl ether-Toluene (1.40 mL, 0.47 M, 0.658 mmol) was added. The reaction mixture was heated at 50 ºC for 20 h. Additional bis(methyl-d3)zinc in THF-dibutyl ether-Toluene (0.25 mL, 0.83 molar, 0.21 mmol) was added and then reaction was stirred at 50 ºC overnight. The mixture was cooled to room temperature, saturated aqueous NH4Cl (5 mL) and water (5 mL) were added. The mix- 20 ture was stirred for 15 minutes at room temperature. The mixture was extracted with EtOAc. The organic phase was washed with saturated aqueous NaCl, dried over Na2SO4, and concen- trated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: heptane:EtOAc 100:0 → 0:100) to afford the title compound (152 mg). 1H NMR (DMSO-d6, 600MHz) δ 9.34 (s, 1H), 8.77 (s, 1H), 4.44 – 4.12 (m, 4H), 4.02 (td, J = 10.0, 4.7 Hz, 1H), 3.25 (s, 25 3H), 3.19 (td, J = 10.8, 5.2 Hz, 1H), 2.52 (m, 1H), 2.08 (m, 2H), 1.80 (m, 5H), 1.29 (qd, J = 11.9, 5.2 Hz, 2H). LC-MS (method F) (m/z) = 421.3 (MH)+ tR = 0.64 minutes. Example 51 (+)-8-Chloro-3-(methyl-d3)-2-((2R,4R) or (2S,4S)-2-methyltetrahydro-2H-pyran-4- yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triaza- cycloundecine 5
Figure imgf000209_0001
The compound was prepared in a manner similar to Example 50 using 3-bromo-8- chloro-2-((2R,4R) or (2S,4S)-2-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak1 (559 mg, 1.13 mmol), bis[tris(tert-butyl)phosphine]palladium (6.7 mg, 0.013 mmol), LiHMDS (1.20 mL, 1 M, 10 1.20 mmol), and bis(methyl-d3)zinc in THF-dibutyl ether-Toluene (3.20 mL, 0.47 M, 1.5 mmol) in THF (5 mL) at 50 ºC for 20 h followed by additional bis(methyl-d3)zinc in THF-dibutyl ether-Tolu- ene (1.5 mL, 0.83 M, 1.2 mmol) and stirring at 50 ºC overnight, work-up and purification by chro- matography on silica gel (eluent heptane:EtOAc 100:0 → 0:100) to afford the title compound (213 mg).1H NMR (DMSO-d6, 600MHz) δ 9.37 (s, 1H), 8.78 (s, 1H), 4.44 – 4.19 (m, 5H), 3.94 (dd, 15 J = 11.5, 4.6 Hz, 1H), 3.57 – 3.52 (m, 1H), 3.52 – 3.46 (m, 1H), 2.52 – 2.48 (m, 2H), 1.90 (qd, J = 12.4, 4.8 Hz, 1H), 1.85 – 1.76 (m, 1H), 1.76 – 1.69 (m, 1H), 1.65 (q, J = 11.8 Hz, 1H), 1.14 (dd, J = 6.3, 1.2 Hz, 3H). LC-MS (method F) (m/z) = 407.3 (MH)+ tR = 0.60 minutes. = +16.1 (c = 1.0 g/100 mL, CHCl3). Example 52 (–)-8-Chloro-3-(methyl-d3)-2-((2R,4R) or (2S,4S)-2-methyltetrahydro-2H-pyran-4-20 yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triaza- cycloundecine
Figure imgf000209_0002
The compound was prepared in a manner similar to Example 50 using 3-bromo-8- chloro-2-((2R,4R) or (2S,4S)-2-methyltetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- 25 (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak2 (473 mg, 0.868 mmol), bis[tris(tert-butyl)phosphine]palladium (6.6 mg, 0.013 mmol), LiHMDS (0.99 mL, 1 M, 0.99 mmol), and bis(methyl-d3)zinc in THF-dibutyl ether-Toluene (2.40 mL, 0.47 molar, 1.13 mmol) in THF (5 mL) at 50 ºC for 22 h followed by additional bis(methyl-d3)zinc in THF-dibutyl ether-Toluene (1.2 mL, 0.83 molar, 1.0 mmol) and stirring at 50 ºC overnight, work-up and puri- fication by chromatography on silica gel (eluent heptane:EtOAc 100:0 → 0:100) to afford the title compound (192 mg).1H NMR (DMSO-d6, 600MHz) δ 9.37 (s, 1H), 8.78 (s, 1H), 4.44 – 4.13 5 (m, 5H), 3.94 (m, 1H), 3.59 – 3.45 (m, 2H), 2.52 – 2.48 (m, 2H), 1.90 (qd, J = 12.4, 4.7 Hz, 1H), 1.84 – 1.76 (m, 1H), 1.74 – 1.70 (m, 1H), 1.65 (q, J = 11.8 Hz, 1H), 1.14 (dd, J = 6.2, 1.0 Hz, 3H). LC-MS (method F) (m/z) = 407.3 (MH)+ tR = 0.61 minutes. = -25.0 (c = 1.0 g/100 mL, CHCl3). Example 53 (+)-(R) or (S)-8-Chloro-3-methyl-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro- 10 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine and Example 54 (–)-(R) or (S)-8-Chloro-3-methyl-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000210_0001
The racemic mixture of Example 53 and Example 54 was prepared in a manner similar15 to Example 1 using 3,6-dichloro-1-(3-((5-methyl-4-nitro-1-(4-oxaspiro[2.5]octan-7-yl)-1H-pyra- zol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (490 mg, 1.0 mmol), Fe (490 mg, 8.77 mmol), and NH4Cl (490 mg, 9.16 mmol) in EtOH (30 mL) and H2O (3 mL) at 80 ºC for 15 h, followed by work-up and purification by chromatography on silica gel (eluent: Petroleum ether:EtOAc 100:0 → 40:60) to afford 8-chloro-3-methyl-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro-20 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (270 mg). The racemic mixture (270 mg) was separated into the two enantiomers by preparative chiral SFC (instrument: SFC150AP, column: DAICEL CHIRALCEL OD 250 × 30 mm, 10 μm, mobile phase: su- percritical CO2/IPA (0.1% NH3.H2O, v%)= 65/35, flow rate: 100 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator temperature: 20 ºC, trimmer25 temperature: 25 ºC, wavelength: 220 nm) to afford: Example 53 (+)-(R) or (S)-8-Chloro-3-methyl-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tet- rahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (90 mg).1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 6.80 (br s, 1H), 4.60 - 4.37 (m, 4H), 4.36 - 4.22 (m, 1H), 4.06 - 3.94 (m, 1H), 3.77 - 3.53 (m, 1H), 2.89 - 2.65 (m, 1H), 2.43 - 2.24 (m, 4H), 2.05 - 1.91 (m, 2H), 1.89 - 1.83 (m, 1H), 1.32 - 1.25 (m, 1H), 0.98 - 0.90 (m, 1H), 0.80 - 0.69 (m, 1H), 0.63 - 0.53 (m, 1H), 0.49 - 0.36 (m, 1H). LC-MS (method C) (m/z) = 416.2 (MH)+ tR = 1.54 minutes. Chiral analytical SFC ((instrument: Waters UPCC with PDA Detector, column: Chiralcel OD-3 150 × 4.6mm I.D., 3μm, mobile phase: 40% of iso-propanol (0.05% DEA) in CO2, flow rate: 2.5 mL/min, 5 column temperature: 35 ºC, ABPR: 1500psi, run time: 7 min, wavelength: 220 nm), ee = 98.3%, tR = 2.99 minutes. = +68.0 (c = 0.1 g/100 mL, MeOH) and the corresponding enantiomer Example 54 (–)-(R) or (S)-8-Chloro-3-methyl-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tet- rahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (90 10 mg).1H NMR (CDCl3, 400MHz) δ 8.65 (s, 1H), 6.80 (br s, 1H), 4.54 - 4.39 (m, 4H), 4.35 - 4.20 (m, 1H), 4.07 - 3.95 (m, 1H), 3.71 - 3.58 (m, 1H), 2.84 - 2.71 (m, 1H), 2.40 - 2.24 (m, 4H), 2.04 - 1.91 (m, 2H), 1.90 - 1.83 (m, 1H), 1.33 - 1.25 (m, 1H), 0.98 - 0.90 (m, 1H), 0.78 - 0.69 (m, 1H), 0.62 - 0.53 (m, 1H), 0.47 - 0.38 (m, 1H). LC-MS (method C) (m/z) = 416.2 (MH)+ tR = 1.54 minutes. Chiral analytical SFC ((instrument: Waters UPCC with PDA Detector, column: Chiralcel OD-3 150 × 15 4.6mm I.D., 3μm, mobile phase: 40% of iso-propanol (0.05% DEA) in CO2, flow rate: 2.5 mL/min, column temperature: 35 ºC, ABPR: 1500psi, run time: 7 min, wavelength: 220 nm), ee > 99%, tR = 2.04 minutes. = -72 (c = 0.25 g/100 mL, MeOH). Example 55 (+)-8-Chloro-3-ethyl-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-20 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine and Example 56 (–)-8-Chloro-3-ethyl-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine 25
Figure imgf000211_0001
The racemic mixture of Example 55 and Example 56 was prepared in a manner similar to Example 1 using trans-3,6-dichloro-1-(3-((5-ethyl-1-(3-fluorotetrahydro-2H-pyran-4-yl)-4-ni- tro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4-d]pyrimidine (830 mg, 1.70 mmol), Fe (475 mg, 8.50 mmol), and NH4Cl (455 mg, 8.50 mmol) in EtOH (45 mL) and H2O (6 mL) at 80 ºC for 16 h, 30 followed by work-up and purification by chromatography on silica gel (eluent: Petroleum ether:EtOAc 83:17 → 50:50) to afford 8-chloro-3-ethyl-2-((3R,4S and 3S,4R)-3-fluorotetrahydro- 2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'- g][1]oxa[4,6,8]triazacycloundecine (310 mg). The racemic mixture (310 mg) was separated into the two enantiomers by preparative chiral SFC ((Instrument: SFC-80Q, column: DAICEL CHI- 5 RALPAK AD (250mm×30mm, 10μm), mobile phase: supercritical CO2/EtOH (0.1% NH3.H2O, v %) = 45/55, flow rate: 80 mL/min, column temperature: 38 ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator temperature: 20 ºC, trimmer temperature: 25 ºC, wavelength: 220 nm) to afford: Example 55 (+)-8-Chloro-3-ethyl-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-10 yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triaza- cycloundecine (90 mg).1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 6.69 (br s, 1H), 5.14 - 4.79 (m, 1H), 4.71 - 4.36 (m, 4H), 4.28 (dd, J = 5.2, 11.2 Hz, 1H), 4.21 - 3.98 (m, 2H), 3.57 - 3.45 (m, 1H), 3.44 - 3.35 (m, 1H), 2.85 - 2.60 (m, 2H), 2.55 - 2.38 (m, 1H), 2.02 - 1.79 (m, 3H), 1.26 (t, J = 7.6 Hz, 3H). LC-MS (method C) (m/z) = 422.2 (MH)+ tR = 1.54 minutes. Chiral analytical SFC (instru- 15 ment: Waters UPCC with PDA Detector and QDa Detector, column: Chiralpak AD-350 × 4.6mm I.D., 3μm, mobile phase: A: CO2 B: ethanol (0.05% DEA), gradient: 40% B, column temperature: 35 ºC, flow rate: 4 mL/min, ABPR: 1500 psi, wavelength: 220 nm, run time: 3 min), ee > 99%, tR = 0.98 minutes. = +26.0 (c = 0.1 g/100 mL, MeOH) and the corresponding enantiomer 20 Example 56 (–)-8-Chloro-3-ethyl-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4- yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triaza- cycloundecine (85 mg).1H NMR (CDCl3, 400MHz) δ 8.66 (s, 1H), 6.75 (br s, 1H), 5.08 - 4.79 (m, 1H), 4.60 - 4.35 (m, 4H), 4.28 (dd, J = 5.2, 11.2 Hz, 1H), 4.21 - 4.00 (m, 2H), 3.57 - 3.46 (m, 1H), 3.45 - 3.35 (m, 1H), 2.83 - 2.61 (m, 2H), 2.55 - 2.40 (m, 1H), 2.04 - 1.90 (m, 3H), 1.26 (t, J = 7.625 Hz, 3H). LC-MS (method C) (m/z) = 422.2 (MH)+ tR = 1.54 minutes. Chiral analytical SFC (instru- ment: Waters UPCC with PDA Detector and QDa Detector, column: Chiralpak AD-350 × 4.6mm I.D., 3μm, mobile phase: A: CO2 B: ethanol (0.05% DEA), gradient: 40% B, column temperature: 35 ºC, flow rate: 4 mL/min, ABPR: 1500 psi, wavelength: 220 nm, run time: 3 min), ee > 99%, tR = 1.71 minutes. = -28.0 (c = 0.1 g/100 mL, MeOH) 30 Example 57: 8-Chloro-3-ethyl-2-((1r,4r)-4-methoxycyclohexyl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000213_0001
The compound was prepared in a manner similar to Example 1 using 3,6-dichloro-1-(3- ((5-ethyl-1-((1r,4r)-4-methoxycyclohexyl)-4-nitro-1H-pyrazol-3-yl)oxy)propyl)-1H-pyrazolo[3,4- d]pyrimidine (1 g, 2 mmol), Fe (560 mg, 10.0 mmol), and NH4Cl (537 mg, 10.0 mmol) in EtOH (40 5 mL) and H2O (6 mL) at 80 ºC for 16 h, followed by work-up, and purification by chromatography on silica gel (eluent: Petroleum ether:EtOAc 83:17 → 50:50) and preparative SFC (instrument: SFC-80Q, column: DAICEL CHIRALCEL OD (250mm×30mm, 10μm), mobile phase: supercritical CO2/EtOH (0.1% NH3.H2O, v %) = 75/25, flow rate: 100 mL/min, column temperature: 38 ºC, noz- zle pressure: 100 bar, nozzle temperature: 60 ºC, evaporator temperature: 20 ºC, trimmer tem- 10 perature: 25 ºC, wavelength: 220 nm) to afford the title compound (60 mg). 1H NMR (CDCl3, 400MHz) δ 8.65 (s, 1H), 6.72 (br s, 1H), 4.52 - 4.37 (m, 4H), 3.98 - 3.81 (m, 1H), 3.39 (s, 3H), 3.33 - 3.23 (m, 1H), 2.69 (q, J = 7.6 Hz, 2H), 2.28 - 2.18 (m, 2H), 2.14 - 1.86 (m, 6H), 1.41 - 1.30 (m, 2H), 1.25 (t, J = 7.6 Hz, 3H). LC-MS (method C) (m/z) = 432.2 (MH)+ tR = 1.61 minutes. 15 Example 58 (+)-(R) or (S)-8-Chloro-3-(methyl-d3)-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine and Example 59 (–)-(R) or (S)-8-Chloro-3-(methyl-d3)-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000213_0002
20 To a mixture of 3-bromo-8-chloro-2-(oxepan-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (0.48 g, 1.02 mmol), trideuteriomethylboronic acid (129 mg, 2.05 mmol) and K2CO3 (283 mg, 2.05 mmol) in a mixture of 1,4-dioxane (50 mL) and H2O (12.5 mL) at 20 ºC was added Pd(PPh3)4 (118 mg, 0.1 mmol). The reaction mixture was heated at 100 ºC for 16 h. The reaction mixture was concentrated under25 reduced pressure. The residue was purified by chromatography on silica gel (eluent: hep- tane:EtOAc (10 v% MeOH and 10 v% DCM) 100:0 → 50:50) to afford a racemic mixture of Exam- ple 58 and Example 59 (0.29 g). The racemic material (330 mg) was separated into the two enantiomers by preparative chiral SFC (instrument: SFC-80Q, column: CHIRALPAK AD 250 × 30 mm, 10 μm, mobile phase: supercritical CO2/IPA (0.1% NH3·H2O, v%)= 55/45, flow Rate: 80 5 mL/min, column temperature: 38, ºC, nozzle pressure: 100 bar, nozzle temperature: 60 ºC, evap- orator temperature: 20 ºC, trimmer temperature: 25ºC, wavelength: 220 nm) to afford: Example 58 (+)-(R) or (S)-8-Chloro-3-(methyl-d3)-2-(oxepan-4-yl)-2,4,12,13-tetrahydro- 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (128 mg).1H 10 NMR (DMSO-d6, 400MHz) δ 9.37 (s, 1H), 8.78 (s, 1H), 4.38 - 4.22 (m, 5H), 3.80 - 3.70 (m, 2H), 3.68 - 3.56 (m, 2H), 2.20 - 2.09 (m, 1H), 2.08 - 2.00 (m, 1H), 1.99 - 1.92 (m, 1H), 1.91 - 1.68 (m, 5H). LC-MS (method C) (m/z) = 407.2 (MH)+ tR = 1.45 minutes. Chiral analytical SFC (instrument: Agilent 1260 with DAD Detector, column: Chiralpak AD-3100 × 4.6mm I.D., 3μm, mobile phase: A: CO2 B: IPA (0.05% DEA), gradient: from 5% to 40% of B in 1.5 min and hold 40% for 3 min, 15 then 5% of B for 1.5 min, flow rate: 2.5 mL/min, column temperature: 40 ºC, ABPR: 1500psi, run time: 10 min, wavelength: 220 nm) ee >99%, tR = 8.03 minutes. = +6.5 (c = 0.4 g/100 mL, CHCl3) and the corresponding enantiomer Example 59: (-)-(R) or (S)-8-chloro-3-(methyl-d3)-2-(oxepan-4-yl)-2,4,12,13-tetrahydro- 20 11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (137 mg).1H NMR (DMSO-d6, 400MHz) δ 9.37 (s, 1H), 8.78 (s, 1H), 4.37 - 4.23 (m, 5H), 3.78 - 3.70 (m, 2H), 3.67 - 3.56 (m, 2H), 2.20 - 2.10 (m, 1H), 2.08 - 2.00 (m, 1H), 1.99 - 1.92 (m, 1H), 1.92 - 1.69 (m, 5H). LC-MS (method C) (m/z) = 407.2 (MH)+ tR = 1.45 minutes. Chiral analytical SFC (instrument: Agilent 1260 with DAD Detector, column: Chiralpak AD-3100 × 4.6mm I.D., 3μm, mobile phase: 25 A: CO2 B: IPA (0.05% DEA), gradient: from 5% to 40% of B in 1.5 min and hold 40% for 3 min, then 5% of B for 1.5 min, flow rate: 2.5 mL/min, column temperature: 40 ºC, ABPR: 1500psi, run time: 10 min, wavelength: 220 nm) ee = 98.3%, tR = 8.57 minutes. = -7.5 (c = 0.4 g/100 mL, CHCl3). Example 60 (R) or (S)-8-Chloro-3-(methyl-d3)-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahy- dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak 1 and Example 61 (R) or (S)-8-Chloro-3-(methyl-d3)-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahy- 5 dro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak 2
Figure imgf000215_0001
The racemic mixture of Example 60 and Example 61 was prepared in a manner similar to Example 50 using 3-bromo-8-chloro-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahydro-11H- 5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (492 mg, 0.909 10 mmol), bis[tris(tert-butyl)phosphine]palladium (5.5 mg, 0.011 mmol), LiHMDS (1.05 mL, 1 M, 1.05 mmol), and bis(methyl-d3)zinc in THF-dibutyl ether-toluene (2.60 mL, 0.47 M, 1.22 mmol) in THF (5 mL) at 50 ºC for 20 h followed by additional bis(methyl-d3)zinc in THF-dibutyl ether- toluene (1 mL, 0.83 M, 0.8 mmol) and stirring at 50 ºC overnight, work-up and purification by chromatography on silica gel (eluent heptane:EtOAc 100:0 → 0:100) to afford 8-chloro-3-(me-15 thyl-d3)-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyra- zolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (243 mg). The racemic mixture was sepa- rated using preparative SFC (instrument: Shimadzu Nexera Prep SFC, column: Chiralpack-IA 50 × 250 mm, 5 μm, mobile phase: supercritical CO2/EtOH (0.1% DEA, v%)= 60/40, flow rate: 60 mL/min, column temperature: 40 ºC, nozzle pressure: 100 bar, nozzle temperature: 40 ºC, wave-20 length: 254 nm) to afford: Example 60 (R) or (S)-8-Chloro-3-(methyl-d3)-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13- tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak 1 (94 mg). 1H NMR (DMSO-d6, 600 MHz) δ 9.49 (s, 1H), 8.89 (s, 1H), 4.53 – 4.33 (m, 4H), 4.21 (m, 1H), 3.96 (td, J = 10.4, 3.7 Hz, 2H), 3.58 (t, J = 10.6 Hz, 1H), 3.44-3.41 (m, 1H), 2.65 (m, 25 2H), 2.20 – 2.01 (m, 2H), 1.97 – 1.69 (m, 2H). LC-MS (method F) (m/z) = 393.3 (MH)+ tR = 0.59 minutes. Chiral analytical SFC (instrument: AuroraSFC Fusion5/Agilent, column: Chiralpack-IA 6 × 150 mm, 5 μm, mobile phase: supercritical CO2/EtOH (96% containing 0.1% DEA, v%)= 60/40, flow rate: 4.0 mL/min, column temperature: 35 ºC, ABPR: 150 bar, run time: 5 min, wavelength: 254 nm) ee = 96.8%, tR = 1.85 minutes. 30 and the corresponding enantiomer Example 61 (R) or (S)-8-Chloro-3-(methyl-d3)-2-(tetrahydro-2H-pyran-3-yl)-2,4,12,13- tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine, peak 2 (90 mg). 1H NMR (DMSO-d6, 600 MHz) δ 9.39 (s, 1H), 8.78 (s, 1H), 4.46 – 4.26 (m, 4H), 4.11 (tt, J = 10.1, 4.6 Hz, 1H), 3.86 (td, J = 10.3, 3.6 Hz, 2H), 3.48 (t, J = 10.6 Hz, 1H), 3.34-3.30 (m, 5 1H), 2.55 (m, 2H), 2.11 – 1.95 (m, 2H), 1.91 – 1.63 (m, 2H). LC-MS (method F) (m/z) = 393.3 (MH)+ tR = 0.61 minutes.. Chiral analytical SFC (instrument: AuroraSFC Fusion5/Agilent, column: Chi- ralpack-IA 6 × 150 mm, 5 μm, mobile phase: supercritical CO2/EtOH (96% containing 0.1% DEA, v%)= 60/40, flow rate: 4.0 mL/min, column temperature: 35 ºC, ABPR: 150 bar, run time: 5 min, wavelength: 254 nm) ee > 99%, tR = 2.52 minutes. 10 Example 62: (-)-8-Chloro-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-3-(methyl-d3)- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine
Figure imgf000216_0001
15 The compound was prepared in a manner similar to Example 50 using (-)-3-bromo-8- chloro-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (101 mg, 0.214 mmol), bis[tris(tert-butyl)phosphine]palladium (14 mg, 0.027 mmol), LiHMDS (0.220 mL, 1 M, 0.220 mmol), and bis(methyl-d3)zinc in THF-dibutyl ether-toluene (0.40 mL, 0.81 M, 0.324 mmol)20 in THF (2.0 mL) at 50 ºC for 20 h followed by additional bis(methyl-d3)zinc in THF-dibutyl ether- toluene (1.5 mL, 0.83 M, 1.2 mmol) and stirring at 50 ºC overnight, work-up and purification by chromatography on silica gel (eluent heptane:EtOAc 100:0 → 0:100) and preparative SFC (instru- ment: Shimadzu Nexera Prep SFC, column: 2-ethylpyridin 21.2 × 150 mm, 3 μm, mobile phase: supercritical CO2/EtOH (0.1% DEA, v%)= 90/10, flow rate: 60 mL/min, column temperature: 40 25 ºC, nozzle pressure: 100 bar, nozzle temperature: 40 ºC, wavelength: 254 nm) to afford the title compound (20 mg). 1H NMR (CDCl3, 600MHz) δ 8.67 (s, 1H), 6.59 (s, 1H), 4.98 – 4.75 (m, 1H), 4.62 – 4.37 (m, 4H), 4.27 (m, 1H), 4.20 – 4.03 (m, 2H), 3.51 (m, 1H), 3.39 (td, J = 10.5, 9.0, 5.3 Hz, 1H), 2.49 (m, 1H), 2.09 – 1.84 (m, 3H). LC-MS (method F) (m/z) = 411.2 (MH)+ tR = 0.59 minutes. = -11.4 (c = 1.0 g/100 mL, CHCl3). Example 63: (+)-8-Chloro-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-3-(methyl-d3)- 2,4,12,13-tetrahydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacy- cloundecine
Figure imgf000217_0001
5 The compound was prepared in a manner similar to Example 50 using (+)-3-bromo-8- chloro-2-((3R,4S) or (3S,4R)-3-fluorotetrahydro-2H-pyran-4-yl)-2,4,12,13-tetrahydro-11H-5,7- (azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (127 mg, 0.268 mmol) , bis[tris(tert-butyl)phosphine]palladium (16 mg, 0.031 mmol), LiHMDS (0.28 mL, 1 M, 0.28 mmol), and bis(methyl-d3)zinc in THF-dibutyl ether-toluene (0.500 mL, 0.81 M, 0.41 mmol)10 in THF (2.5 mL) at 50 ºC for 18 h, work-up and purification by chromatography on silica gel (elu- ent heptane:EtOAc 100:0 → 0:100) and preparative SFC (instrument: Shimadzu Nexera Prep SFC, column: Chiralcel-OD-H 21.2 × 250 mm, 5 μm, mobile phase: supercritical CO2/EtOH (0.1% DEA, v%)= 80/20, flow rate: 60 mL/min, column temperature: 40 ºC, nozzle pressure: 100 bar, nozzle temperature: 40 ºC, wavelength: 254 nm) to afford the title compound (15 mg).1H NMR 15 (CDCl3, 600 MHz) δ 8.67 (s, 1H), 6.58 (s, 1H), 4.98 – 4.75 (m, 1H), 4.65 – 4.34 (m, 4H), 4.26 (m, 1H), 4.21 – 4.02 (m, 2H), 3.51 (ddd, J = 14.2, 11.1, 2.1 Hz, 1H), 3.38 (m, 1H), 2.49 (qd, J = 11.3, 9.5, 6.4 Hz, 1H), 2.12 – 1.84 (m, 3H). LC-MS (method F) (m/z) = 411.2 (MH)+ tR = 0.59 minutes. = +42 (c = 1.0 g/100 mL, CHCl3). 20 Example 64: (-)-(R) or (S)-8-Chloro-3-(methyl-d3)-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetra- hydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine
Figure imgf000217_0002
The compound was prepared in a manner similar to Example 50 using (-)-(R) or (S)-3- bromo-8-chloro-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- 25 theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (102 mg, 0.213 mmol), bis[tris(tert-butyl)phosphine]palladium (16 mg, 0.031 mmol), LiHMDS (0.22 mL, 1 M, 0.22 mmol), and bis(methyl-d3)zinc in THF-dibutyl ether-toluene (0.400 mL, 0.80 M, 0.32 mmol) in THF (2.0 mL) at 50 ºC for 18 h, followed by additional bis(methyl-d3)zinc in THF-dibutyl ether- toluene (0.45 mL, 0.80 M, 0.36 mmol), stirring at 50 ºC overnight, followed by a third batch of bis(methyl-d3)zinc in THF-dibutyl ether-toluene (0.45 mL, 0.80 M, 0.36 mmol) and stirring over- night at 50 ºC, work-up and purification by chromatography on silica gel (eluent heptane:EtOAc 5 100:0 → 0:100) to afford the title compound (35 mg).1H NMR (CDCl3, 600 MHz) δ 8.66 (s, 1H), 6.60 (br s, 1H), 4.63 – 4.35 (m, 4H), 4.27 (m, 1H), 3.99 (ddd, J = 11.8, 4.7, 2.6 Hz, 1H), 3.64 (m, 1H), 2.77 (m, 1H), 2.33 (m, 1H), 1.96 (m, 2H), 1.86 (dq, J = 13.2, 2.2 Hz, 1H), 1.28 (ddt, J = 14.9, 4.2, 2.0 Hz, 1H), 0.93 (td, J = 10.0, 6.1 Hz, 1H), 0.74 (ddq, J = 8.6, 4.5, 2.2 Hz, 1H), 0.57 (m, 1H), 0.42 (dt, J = 9.5, 3.5 Hz, 1H). LC-MS (method F) (m/z) = 419.2 (MH)+ tR = 0.64 minutes. = -10 78.4 (c = 0.98 g/100 mL, CHCl3). Example 65: (+)-(R) or (S)-8-Chloro-3-(methyl-d3)-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetra- hydro-11H-5,7-(azenometheno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine 15
Figure imgf000218_0001
The compound was prepared in a manner similar to Example 50 using (+)-(R) or (S)-3- bromo-8-chloro-2-(4-oxaspiro[2.5]octan-7-yl)-2,4,12,13-tetrahydro-11H-5,7-(azenome- theno)dipyrazolo[3,4-b:5',1'-g][1]oxa[4,6,8]triazacycloundecine (102 mg, 0.21 mmol), bis[tris(tert-butyl)phosphine]palladium (21 mg, 0,041 mmol), LiHMDS (0.22 mL, 1 M, 0.22 20 mmol), and bis(methyl-d3)zinc in THF-dibutyl ether-toluene (0.400 mL, 0.80 M, 0.32 mmol) in THF (2.0 mL) at 50 ºC for 18 h, followed by additional bis(methyl-d3)zinc in THF-dibutyl ether- toluene (0.45 mL, 0.80 M, 0.36 mmol), stirring at 50 ºC overnight, followed by a third batch of bis(methyl-d3)zinc in THF-dibutyl ether-toluene (0.45 mL, 0.80 M, 0.36 mmol) and stirring over- night at 50 ºC, work-up and purification by chromatography on silica gel (eluent heptane:EtOAc 25 100:0 → 0:100) to afford the title compound (30 mg).1H NMR (CDCl3, 600 MHz) δ 8.66 (s, 1H), 6.59 (br s, 1H), 4.55 – 4.36 (m, 4H), 4.27 (td, J = 9.8, 8.2, 5.8 Hz, 1H), 4.00 (ddd, J = 11.5, 4.5, 2.4 Hz, 1H), 3.64 (m, 1H), 2.77 (t, J = 12.4 Hz, 1H), 2.33 (m, 1H), 1.96 (m, 2H), 1.86 (dq, J = 13.2, 2.2 Hz, 1H), 1.28 (m, 1H), 0.93 (m, 1H), 0.74 (ddd, J = 8.6, 4.4, 2.1 Hz, 1H), 0.57 (dt, J = 6.8, 2.7 Hz, 1H), 0.41 (m, 1H). LC-MS (method F) (m/z) = 419.3 (MH)+ tR = 0.64 minutes. = +70.4 (c =30 0.98 g/100 mL, CHCl3). LRRK2 wild-type and G2019S kinase activity assay LRRK2 kinase activity was measured using a LanthaScreen kinase activity assay available from Invitrogen (Life Technologies Corporation). The assay is a homogeneous time resolved-flu- orescence resonance energy transfer (TR-FRET) assay that measures phosphorylation of a fluo- 5 rescein-labelled peptide substrate Fluorescein-ERM LRRKtide obtainable from Life Technologies Corporation as a result of LRRK2 kinase activity. The phosphorylated peptide is recognized by a terbium-labelled phospho-specific anti-LRRKtide antibody (pLRRKtide antibody), obtainable from Life Technologies Corporation and, subsequently, the phosphorylated LRRKtide can be quantified by the extent of TR-FRET between the terbium donor and fluorescein acceptor. 10 The LRRK2 kinase was obtained from Invitrogen (Life Technologies Corporation) and comprises residue 970 to 2527 of the full length human wild-type LRRK2 kinase, or a similar sequence with the G2019S mutation. As discussed above, this mutation increases the kinase activity relative to the wild type. The kinase reactions were performed in a 20 μL volume in 384- well plates. The kinase reaction buffer consisted of 50 mM Tris pH 8.5, 0.01% BRIJ-35, 10 mM15 MgCl2, 1 mM EGTA, and 2 mM DTT. In the assay, 1 nM LRRK2 WT or 250 pM LRRK2 G2019S kinase in kinase reaction buffer was incubated with the test compound (typically at 0 to 30 ^M) for 30 minutes before the kinase reaction was initiated by addition of 1.3 mM ATP and 0.4 ^M fluorescein-LRRKtide. The reaction mixture (20 ^l total volume) was incubated for 3.5 h (for LRRK2 WT) and 3 h (for LRRK2 G2019S)20 at 30 ^C, before the reaction was terminated by addition of 10 mM EDTA and 1 nM terbium- labelled anti-phospho-LRRKtide antibody (final volume 20 ^l). The mixture was further incu- bated for 30 minutes at RT. TR-FRET was measured by excitation of the terbium-donor with 340 nm light and subsequent (delay time 100 μs) measurement of terbium and fluorescein emission at 495 nm and 520 nm, respectively, over a time window of 1000 μs. The measurement was 25 repeated 30 times for fluorescein and 30 times for terbium emission with a 1000 μs time window between repeats. TR-FRET measurements were performed on a Biotek Synergy plate. The TR- FRET signal was calculated as the emission-ratio at 520 nm over 495 nm. The TR-FRET ratio readout for test compounds was normalized to 0% inhibition corre- sponding to TR-FRET ratio measured in control wells with no inhibition of the kinase activity and 30 100% inhibition corresponding to TR-FRET ratio measured in control wells with inhibitor. Test compound potency (IC50) was estimated by nonlinear regression using the sigmoidal dose-re- sponse (variable slope) using Xlfit 4 (IDBS, Guildford, Surrey, UK, model 205). Were the IC50 could not be determined the % inhibition at the highest tested concentration is given by equation 1. y = (A+((B-A)/(1+((C/x)^D)))) (1) 5 where y is the normalized TR-TRET ratio measurement for a given concentration of test compound, x is the concentration of test compound, A is the estimated efficacy (% inhibition) at infinite compound dilution, and B is the maximal efficacy (% inhibition). C is the IC50 value and D is the Hill slope coefficient. IC50 estimates were obtained from independent experiment and the logarithmic average was calculated. 10 Table 2 below shows the IC50 values in nM obtained as described above for the exemplified first compounds of the invention, data is based on n ≥ 2 tests. Table 2: LRRK2 wild-type and G2019S kinase activity
Figure imgf000220_0001
Figure imgf000221_0001
Method I Broad Kinase Selectivity Protein kinase profiling of the inhibitors (Fabian, M.A. et al. inhibitors. Nat. Biotechnol. 5 23, 329-336 (2005)) were undertaken at a concentration of 0.1 μM and carried out Eurofins Dis- coverX scanMAX panel of 403 wild type kinases (primarily of human origin). covering AGC (PKA, PKG, PKC family kinases), CAMK (Calcium/calmodulin-dependent protein kinases), CK1 (Casein kinase 1 kinases), CMGC (CDK, MAPK, GSK3, CLK families), STE (homologs of yeast Sterile 7, Ster- ile 11, Sterile 20 kinases), TK (Tyrosine kinases), TKL (Tyrosine kinase-like kinases), lipid and atyp-10 ical kinase families. Selectivity Score or S-score is a quantitative measure of compound selectivity. It was calculated by dividing the number of kinases that compounds bind to by the total number of distinct kinases tested, excluding mutant variants. S = Number of hits / Number of assays. This value can be calculated using %Ctrl as a potency threshold (below) and provides a quantitative 15 method of describing compound selectivity to facilitate comparison of different compounds. Table 3: Kinase selectivity
Figure imgf000222_0001
*LRRK2 WT; S(35) = (number of non-mutant kinases with %Ctrl <35)/(number of non-mutant kinases tested); S(10) = (number of 20 non-mutant kinases with %Ctrl <10)/(number of non-mutant kinases tested); S(1) = (number of non-mutant kinases with %Ctrl <1)/(number of non-mutant kinases tested) Method II Hepatocyte intrinsic clearance assay: 25 Test compounds (final concentration 0.1 µM, 0.05% organic) were incubated for 2 h at 37ºC, with shaking, in supplemented Leibovitz L-15 media (pH7.4) containing commercially sourced, pooled donor, cryopreserved hepatocytes (final concentration 1x106 hepatocytes/mL). The CLint reactions (350 µL) were initiated by addition of test compound. Aliquots (25 µL) were taken at 1, 5, 10, 15, 30, 60, 90 and 120 minutes and then protein crashed with ice-cold acetoni- trile containing internal standard (150 µL) then centrifuged (1960 g for 20 minutes at 4ºC). Su- pernatant was diluted (1:4) with deionized water then analyzed by liquid chromatography (LC)– tandem mass spectrometry (MS/MS). The intrinsic clearances (CLint) were calculated from the 5 slope (k) of the linear regressions of percentages of compound remaining in incubation against incubation time, according to equations 2 and 3. Equation 2: Equation 3:
Figure imgf000223_0001
10 CLint (L/h/kg body weight) = Ln(2) × V (L/106 hepatocytes) / t1/2 (h) × hepatocellularity (106 hepatocytes / g liver) × liver weight (g liver / kg body weight) (3) V = incubation volume = 0.001 L/106 hepatocytes, Human hepatocellularity = 120 x 106 hepato- cytes/g, Human liver weight = 20 g/kg body weight. 15 Table 4: Hepatocyte intrinsic clearance for first compounds of the invention
Figure imgf000223_0002
Figure imgf000224_0001
*CLint measured on 3 separate occasions; **CLint measured on a single occasion Summary/Conclusion: Based on the hepatocyte CLint values the test compounds are classified as having moderately-low to low rates of metabolism. 5 Method III GSH adduct screening Test compounds (final concentration 1 µM, 0.25% organic) were incubated for 3 h at 37ºC in commercially sourced, pooled donor, human liver microsomes (HLM, final concentration 0.5 mg/mL) with and without addition of glutathione (GSH; final concentration 1 µM). The met- 10 abolic reaction was initiated by addition of microsomal solution to the mixture of cofactor NADPH (final concentration 1 mM), test compound and GSH. After 3 h of incubation in a water bath at 37ºC, the reaction was stopped by addition of 0.2% formic acid in acetonitrile, followed by mixing and centrifugation (16000 g for 5 minutes). Supernatant was diluted (1:1) with HPLC water then analyzed by liquid chromatography (LC)–quadrupole time-of-flight mass spectrome- 15 try (QTOF-MS). Accurate mass MS and MS/MS spectra from samples with GSH and without GSH, respectively, were compared to identify detectable GSH and/or cysteine related adducts. Table 5: GSH adduct measurements
Figure imgf000224_0002
Figure imgf000225_0001
*Measured on two separate occasions. It was found that Examples 13, 17 and 50 did not form GSH adduct based on in vitro experi- ments. Method IV 5 Minipig B/P: Brain disposition of Example 13 was evaluated in female Göttingen minipigs (n=3, body weight range 13-14 kg). Briefly, test compound was formulated in vehicle (5%DMSO/95% of 20% hydroxypropyl-β-cyclodextrin solution, pH3.0) then administered as an intravenous (IV) bolus (0.28 mg/kg, 1 mL/kg) followed immediately by a constant rate IV infusion (0.60 mg/kg, 2.5 10 mL/kg) via an ear vein. Serial bloods were collected from the jugular vein at designated time points (0.083, 0.50, 1.00, 1.50 and 1.83 h, n=3 per time point) after IV bolus injection. At the end of infusion (2 h) the minipigs were sacrificed and terminal blood as well as brain samples were taken (n=3). Isolated plasma and brain homogenates were extracted by standard protein pre- cipitation in acetonitrile, containing internal standard, followed by LC-MS/MS analysis using an15 optimized analytical method. Concentrations of test compound in plasma and brain were quan- tified against matrix matched calibration standards. The total plasma and brain concentration time data are presented in Table 6 alongside the calculated brain Kp (total brain concentra- tion:total plasma concentration ratio). Table 6: Total concentrations, brain Kp are presented (mean ±stdev from n=3 minipigs)
Figure imgf000225_0002
Figure imgf000226_0001
Method V Rat B/P: Brain disposition was evaluated for each compound in male Sprague-Dawley rats (n=3, 5 standard body weight). Briefly, test compound was formulated as a simple suspension (0.5% HPMC in water) then administered by oral gavage (10 mg/kg, 10 mL/kg or 1 mg/kg, 1 mL/kg). At the designated time point (1 h post dose) rats were sacrificed and terminal blood and brain samples taken. Isolated plasma and brain homogenates were extracted by standard protein pre- cipitation in acetonitrile, containing internal standard, followed by LC-MS/MS analysis using an10 optimized analytical method. Concentrations of test compound in plasma and brain were quan- tified against matrix matched calibration standards. The total plasma and brain concentration data are presented in Table 7 alongside the calculated brain Kp (total brain concentration:total plasma concentration ratio). 15 Method VI Free fraction in plasma and brain homogenate methodology: The free fractions in male Sprague-Dawley rat or female Göttingen minipig plasma (fu- plasma) and rat brain homogenate (fubrain) were determined by equilibrium dialysis using 96-well HTD-dialysis plates with dialysis membranes (molecular weight cut off 12–14 KDa). One side of 20 the HTD-dialysis plate was loaded with matrix (plasma or brain homogenate) and the other side with buffer (100 mM sodium phosphate buffer, pH 7.4). Test compounds were dissolved in DMSO then spiked (5 µL of 0.2 mM) into blank (995 µL) plasma or diluted brain homogenate (1:4 ratio in phosphate buffer) giving a final nominal concentration 1 µM (≤0.5% DMSO). The matri- ces were loaded into respective chambers and equilibrated against phosphate buffer for 5 h at 25 37 ºC (in a humidified air incubator with 5% CO2 with shaking). Samples from both chambers (buffer and plasma or brain homogenate) were aliquoted to fresh 96-well polypropylene plates then matrix matched using an equal volume of opposite blank matrix before extraction with cold solvent (3 volumes acetonitrile) containing an appropriate bioanalytical internal standard. After centrifugation (20 min, 3200 g, 4 ºC) the supernatants were diluted with appropriate volumes of water and compound concentrations were quantified by LC/MS-MS against matrix matched cal- ibration standards. The fuplasma and fubrain were calculated as a percent free according to equation 4 below. Equation 4: 5
Figure imgf000227_0001
Where [F] is the analyte concentration on the buffer (receiver) side of the membrane; [T] is the analyte concentration on the plasma or brain (donor) side of the membrane; [T0] is the analyte concentration in the plasma or brain sample at time zero; D is matrix dilution fac- tor which is determined as 4 for brain matrix and 1 for plasma matrix in these assays. 10 Table 7: Total plasma and brain concentrations, brain Kp, fubrain / fuplasma and brain Kp,uu are presented (mean ±stdev from n=3 rats by oral gavage)
Figure imgf000227_0002
Based on the calculated rat brain Kp,uu values (parameter describing the extent of brain penetra- tion) Compounds listed in table 7 are classified as being moderately or highly brain penetrant. Method VII 5 Free fraction in rat brain slice methodology: For each test compound, the free fraction in brain was assessed using the more physiologically relevant brain slice model (Loryan et al., 2013; The brain slice method for studying drug distri- bution in the CNS; Fluids and Barriers of the CNS; 10(6): 1-9). In brief, freshly prepared 300 μm brain slices (n=6 slices per incubation dish) from striatum region of male Sprague-Dawley rat 10 brain were incubated (5 h, 37ºC) in an artificial extracellular fluid (aECF) buffer (129 mM NaCl, 10 mM D-glucose, 3 mM KCl, 1.4 mM CaCl2, 1.2 mM Mg2SO4, 0.4 mM K2HPO4 and 25 mM HEPES) containing compound 13 (10 nM). The slices were removed, dried on filter paper, individually weighed then transferred into 2 mL eppendorf tubes. The slices were homogenized in 9 volumes (w/v) of buffer using an ultrasonic probe (GeneReady ultra cool (BSH-C2)). The buffer (aECF) was15 sampled directly from the dish (150 µL) into an eppendorf tube containing control rat brain ho- mogenate (150 µL) that had been prepared with 4 volumes buffer. The matrix matched samples were extracted with cold solvent (4 volumes acetonitrile) containing an appropriate bioanalyti- cal internal standard. After centrifugation (20 min, 3200 g, 4 ºC) the supernatants were diluted with appropriate volumes of water and compound concentrations were quantified by LC/MS-20 MS against matrix matched calibration standards. The Vu,brain was calculated according to equa- tion 5 and fu,brain in turn from equation 6. Equation 5: Vu,brain = Aslice – Vi x Cbuffer / Cbuffer x (1-Vi) (5) 25 Vu,brain (mL/g brain) = unbound brain volume of distribution, Aslice = compound amount in the slice (mg), Cbuffer = compound concentration (μM) in the aESF buffer, Vi = buffer adhesion to the brain slice (value = 0.0931 mL/g slice). Equation 6: 30 fu,brain,slice = (1 / Vu,brain) x 100 (6) fu,brain,slice = free fraction in brain calculated as a percent free. Table 8: Total plasma and brain concentrations, brain Kp, fubrain,slice / fuplasma and brain Kp,uu are presented for Example 13 (mean ±stdev from n=3 rats by oral gavage)
Figure imgf000229_0001
*The brain free fraction was determined in the rat brain slice model which is a more physiolog- 5 ically relevant, mechanistic in vitro model. The brain Kp,uu values shown in table 7 are further substantiated in table 8 using a more physio- logically relevant brain free fraction model supporting that Example 13 is moderately high brain penetrant. 10

Claims

Claims 1. A first compound of formula I, or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000230_0001
R1 is CH2R4 or R4; 5 R2 is a C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, a C3-C6 cycloalkyl, or a C1-C3 haloal- kyl; R3 is halogen, cyano, a O-C1-C3 haloalkyl, a C1-C3 haloalkyl, a C3-C6 cycloalkyl, or a C1-C3 alkyl; R4 is a 4- to 7-membered heterocycle having 1-2 heteroatoms independently selected 10 from oxygen and nitrogen; a C1-C3 alkyl, a C1-C3 cyanoalkyl, a C1-C3 haloalkyl, or a C3-C6 cycloalkyl; or R4 is a bicyclic 8-membered heterocycle having 1-2 heteroatoms independently se- lected from oxygen and nitrogen; wherein each heterocycle or cycloalkyl is unsubstituted or substituted with 1, 2, or 3 15 groups independently selected from the group consisting of cyano, deuterium, halogen, C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, a O-C1-C3 haloalkyl, a O-C1-C3 alkyl, or a C1- C3 haloalkyl; and a second compound or a pharmaceutically acceptable salt thereof, which second com- 20 pound is useful in the treatment of synucleinopathies, wherein the first and the second compound are for combined use in the treatment of synucleinopathies.
2. The first and second compound of claim 1, wherein the first compound or a pharmaceu- tically acceptable salt thereof is a compound of formula I and R1 is CH2R4 or R1 is R4. 25
3. The first and second compound of any of claims 1-2, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I and R2 is selected from a C1-C3 alkyl, an isotopically labelled C1-C3 alkyl or a C3-C6 cycloalkyl.
4. The first and second compound of any of claims 1-3, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I and R2 is selected from -CH3, -CH2CH3, -CD3, or cyclopropyl.
5 5. The first and second compound of any of claims 1-4, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I and R3 is C1-C3 haloalkyl, a C3-C6 cycloalkyl, cyano or halogen.
6. The first and second compound of any of claims 1-5, wherein the first compound or a 10 pharmaceutically acceptable salt thereof is a compound of formula I and R3 is CF3, chloro, bromo, cyano, or cyclopropyl.
7. The first and second compound of any of claims 1-6, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I and R4 is a 4- to 6-15 membered heterocycle having one oxygen atom, wherein the 4- to 6-membered heter- ocycle is unsubstituted or substituted with one group selected from the group consisting of cyano, deuterium, halogen, C1-C3 alkyl, an isotopically labelled C1-C3 alkyl, O-C1-C3 haloalkyl, O-C1-C3 alkyl, or C1-C3 haloalkyl. 20
8. The first and second compound of any of claims 1-7, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I and R4 is a 6-mem- bered heterocycle having one oxygen atom, wherein the 6-membered heterocycle is unsubstituted or substituted with two groups independently selected from the group consisting of deuterium, halogen, C1-C3 alkyl, or an isotopically labelled C1-C3 alkyl. 25
9. The first and second compound of any of claims 1-6, wherein the first compound or a pharmaceutically acceptable salt thereof is a compound of formula I and R4 is selected from the group consisting of: -CH3,
Figure imgf000232_0001
Figure imgf000233_0001
10. The first and second compound of any of claims 1-9, wherein the first compound is se- lected from the list of: 5 , , ,
Figure imgf000233_0002
,
Figure imgf000234_0001
Figure imgf000235_0001
Figure imgf000236_0001
Figure imgf000237_0001
Figure imgf000238_0001
and or a pharmaceutically acceptable salt thereof.
11. The first and second compound of any of claims 1-10, wherein the second compound or 5 a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, is selected from caspase inhibitors, calpain inhibitors, LRRK2 inhibitors, BACE1 inhibitors, antibodies against any form of Aβ peptides, inflam- mation inhibitors, LAG-3 antibodies, molecules inhibiting alpha-synuclein aggregation, nicotine, caffeine, Monoamine oxidase B inhibitor, Levodopa/carbidopa, Dopamine ag-10 onists, COMT inhibitors, A2A antagonists, anti-alphasynuclein antibodies, Mitogen-acti- vated protein kinase 14 inhibitors, USP30 inhibitors, Beta adrenoreceptor agonists, do- pamine D1 PAMs, Toll-like receptor 2 antagonists, Carnitine palmitoyl transferase 1 in- hibitors, Glutamate 5 receptor antagonists, Muscarinic M2 receptor antagonists, Mus- carinic M3 receptor antagonists, Muscarinic M4 receptor antagonists, 5 Hydroxy trypta-15 mine 1A receptor agonists, 5 Hydroxy tryptamine 4 receptor agonists, 5 Hydroxy trypta- mine 3 receptor agonists, 5 Hydroxy tryptamine 2A receptor antagonists, Alpha 1a adre- noreceptor antagonists, Sigma 1 receptor agonists, Sigma 2 receptor antagonists, Dopa- mine D1 receptor agonists, Dopamine D2 receptor agonists, Dopamine D3 receptor ag- onists, Dopamine D5 receptor antagonists, dopamine reuptake inhibitors, DOPA decar- 20 boxylase inhibitors, Insulin-like growth factor 1 antagonists, Insulin-like growth factor 2 antagonists, Tyrosine kinase inhibitors, Ras inhibitors, Leucotriene D4 antagonists, Leu- cotriene receptor antagonists, PDE1 inhibitors, T-type calcium channel antagonists, Peroxiredoxin inhibitors, GLP-1R agonists, NMDA receptor blockers, Lipoxygenase inhib- itors, and Peroxisome proliferator-activated receptor gamma agonists. 25
12. The first and second compound of any of claims 1-10, wherein the second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, is an anticholinergic agent selected from the group 5 comprising trihexyphenidyl, biperiden, metixene, procyclidine, profenamine, dexe- timide, phenglutarimide, mazaticol, bornaprine, tropatepine, etanautine, orphenadrine, benzatropine, and etybenzatropine.
13. The first and second compound of any of claims 1-10, wherein the second compound or 10 a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, is a dopaminergic agent selected from the group com- prising dopamine, levodopa, carbidopa, levodopa/carbidopa, melevodopa, etilevodopa, foslevodopa, amantadine, bromocriptine, pergolide, dihydroergocryptine mesylate, ropinirole, pramipexole, cabergoline, apomorphine, piribedil, rotigotine, selegiline, 15 rasagiline, safinamide, tolcapone, entacapone, budipine, and opicapone.
14. The first and second compound of any of claims 1-10, wherein the second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, is selected from the list consisting of trihexyphenidyl,20 biperiden, metixene, procyclidine, profenamine, dexetimide, phenglutarimide, maza- ticol, bornaprine, tropatepine, etanautine, orphenadrine, benzatropine, etybenzatro- pine, dopamine, levodopa, carbidopa, levodopa/carbidopa, melevodopa, etilevodopa, foslevodopa, amantadine, bromocriptine, pergolide, dihydroergocryptine mesylate, ropinirole, pramipexole, cabergoline, apomorphine, piribedil, rotigotine, selegiline, 25 rasagiline, safinamide, tolcapone, entacapone, budipine, opicapone, istradefylline, pralnacasan, bapineuzemab, solanezumab, gantenerumab, crenezumab, aducanumab, glunozumab, prasinezumab, miglustat, eliglustat, ropinerole, neflamapimod, bosutinib, oxafuramine, nadolol, nilotinib, vodobatinib, clenbuterol, mevidalen, emeramide, tomaralimab, mitometin, dipraglurant, ezeprogind, blarcamesine, buntanetap, ren- 30 zapride, tavapadon, pramiprexole, salbutamol, Infudopa, alirinetide, antraquinolol, a rasagiline prodrug, apomorphine, pramiprexole, radotinib, talineuren, montelukast, len- rispodun, pirepemat, suvecaltamide, sonlicromanol, L-Demethyl phencynonate hydro- chloride, mesocarb, roluperidone, exenatide, befiradol, ketamine, pridopidine, utrolox- estat, aplindore, finamine, phenlarmide, (R)-troloxamide quinone, cannibigerol, and kenterin. 5 15. The first and second compound of any of claims 1-10, wherein the second compound or a pharmaceutically acceptable salt thereof, which second compound is useful in the treatment of synucleinopathies, is selected from the list consisting of trihexyphenidyl, biperiden, metixene, procyclidine, profenamine, dexetimide, phenglutarimide, maza- ticol, bornaprine, tropatepine, etanautine, orphenadrine, benzatropine, etybenzatro- 10 pine, dopamine, levodopa, carbidopa, levodopa/carbidopa, melevodopa, etilevodopa, foslevodopa, amantadine, bromocriptine, pergolide, dihydroergocryptine mesylate, ropinirole, pramipexole, cabergoline, apomorphine, piribedil, rotigotine, selegiline, rasagiline, safinamide, tolcapone, entacapone, budipine, opicapone, istradefylline, pralnacasan, prasinezumab, miglustat, eliglustat, and neflamapimod. 15 16. The first and second compound of any of claims 1-15, or a pharmaceutically acceptable salt thereof, for combined use in the treatment of a synucleinopathy, wherein the synu- cleinopathy is selected from Lewy body dementia, multiple system atrophy, or Parkin- son’s disease. 20 17. The first and second compound of any of claims 1-15, or a pharmaceutically acceptable salt thereof, for combined use in the treatment of a synucleinopathy, wherein the synu- cleinopathy is parkinson’s disease. 25 18. Use of a first and second compound of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a dis- ease or disorder in the central nervous system selected from Lewy body dementia, mul- tiple system atrophy, or Parkinson’s disease. 30 19. A method for the treatment of a disease or disorder in the central nervous system se- lected from Lewy body dementia, multiple system atrophy or Parkinson’s disease com- prising the administration of a therapeutically effective amount of the first and second compound of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, to a patient in need thereof. 5
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