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WO2024218296A1 - Traitements combinés comprenant l'administration d'inhibiteurs de kinase 2 à répétition riche en leucine (lrrk2) - Google Patents

Traitements combinés comprenant l'administration d'inhibiteurs de kinase 2 à répétition riche en leucine (lrrk2) 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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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

La présente invention concerne un traitement combiné comprenant l'administration d'un premier composé de formule I qui est un inhibiteur de la kinase 2 à répétition riche en leucine (LRRK2) et d'un second composé, lequel composé est utile dans le traitement de synucléinopathies, telles que la maladie de Parkinson ; la formule (I) étant .
PCT/EP2024/060716 2023-04-20 2024-04-19 Traitements combinés comprenant l'administration d'inhibiteurs de kinase 2 à répétition riche en leucine (lrrk2) Pending WO2024218296A1 (fr)

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US20160031905A1 (en) * 2013-03-15 2016-02-04 Ipsen Pharma S.A.S. Macrocyclic LRRK2 Kinase Inhibitors
US20170240565A1 (en) * 2014-09-17 2017-08-24 Oncodesign S.A. Macrocyclic lrrk2 kinase inhibitors
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WO2023073013A1 (fr) * 2021-10-27 2023-05-04 H. Lundbeck A/S Inhibiteurs de lrrk2
WO2024056775A1 (fr) * 2022-09-15 2024-03-21 H. Lundbeck A/S Inhibiteurs macrocycliques de kinase 2 à répétition riche en leucine (lrrk2)

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US20160031905A1 (en) * 2013-03-15 2016-02-04 Ipsen Pharma S.A.S. Macrocyclic LRRK2 Kinase Inhibitors
US20170240565A1 (en) * 2014-09-17 2017-08-24 Oncodesign S.A. Macrocyclic lrrk2 kinase inhibitors
WO2018137607A1 (fr) * 2017-01-25 2018-08-02 Glaxosmithkline Intellectual Property Development Limited Composés
WO2019012093A1 (fr) * 2017-07-14 2019-01-17 Glaxosmithkline Intellectual Property Development Limited Inhibiteurs de la kinase 2 à répétition riche en leucine
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