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WO2025016899A1 - Spirocyclic compounds for the treatment of cancer - Google Patents

Spirocyclic compounds for the treatment of cancer Download PDF

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Publication number
WO2025016899A1
WO2025016899A1 PCT/EP2024/069791 EP2024069791W WO2025016899A1 WO 2025016899 A1 WO2025016899 A1 WO 2025016899A1 EP 2024069791 W EP2024069791 W EP 2024069791W WO 2025016899 A1 WO2025016899 A1 WO 2025016899A1
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WIPO (PCT)
Prior art keywords
pyrimidin
azetidine
amino
spiro
dihydro
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PCT/EP2024/069791
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French (fr)
Inventor
Volker Schulze
Susanne Röhrig
Patrik Lukas BRAUN
Holger Siebeneicher
Arwed Cleve
Wolfgang Schwede
G Xavier Jeremie MORTIER
Norbert Gary HERMANN
Stefan BÄURLE
Philip Lienau
Lisa CANDISH
Roman Hillig
Christian Lechner
Charles AWORTWE
Matthias Arlt
Marisa FÜRST
Jan Kramer
Benjamin Bader
Lydia FARACK
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Bayer AG
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Bayer AG
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Publication of WO2025016899A1 publication Critical patent/WO2025016899A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/10—Spiro-condensed systems

Definitions

  • the present invention covers spirocyclic compounds of general formula (I) as described and defined herein, methods of preparing said compounds, intermediate compounds useful for preparing said compounds, pharmaceutical compositions and combinations comprising said compounds, and the use of said compounds for manufacturing pharmaceutical compositions for the treatment or prophylaxis of diseases, in particular for neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling, as a sole agent or in combination with other active ingredients.
  • KRAS mutations display a frequency of 97% and 32% respectively.
  • Other indications with frequently mutated KRAS include colorectal carcinoma (CRC) (52%), and multiple myeloma (43%) (Cox, 2014).
  • RAS proteins act as molecular switches that cycle between an active, GTPbound state and an inactive, GDP-bound state. Activated by guanine nucleotide exchange factors (GEFs), RAS in its GTPbound state interacts with a number of effectors (Hillig, 2019).
  • GEFs guanine nucleotide exchange factors
  • GAPs GTPase- activating proteins
  • the GAP activity is impaired or greatly reduced, resulting in permanent activation, which is the basis of oncogenic RAS signaling (Haigis, 2017); for example, through the RAS-RAF-MEK-ERK and RAS-PI3K-PDK1-AKT pathways, both essential to cell survival and proliferation (Downward 2003).
  • mutant KRAS has been considered “undruggable” with classical pharmacological small molecule inhibitors.
  • KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP) (Oestrem, 2013; Janes, 2018).
  • Covalent KRASG12C inhibitors as described by Shokat et al. (Ostrem JM, Shokat KM (2016) Direct small-molecule inhibitors of KRAS: From structural insights to mechanism-based design. Nat Rev Drug Discov 15:771–785.) occupy the so-called switch-II pocket and bind with their Michael acceptor system covalently to the cysteine mutation at G12 in this specific KRAS mutant. Occupation of this pocket with the covalent inhibitor results in a locked inactive GDP-bound protein conformation.
  • mutant KRAS has been considered “undruggable” via classical pharmacological small molecule inhibitors.
  • S-IIP switch II pocket
  • Biaryl derivatives were mentioned as KRAS G12C covalent inhibitors (WO2014152588, WO2016049524 and WO 2016044772).
  • anilinoacetamide and biaryl derivatives include anilinoacetamide and biaryl derivatives (WO2016049565, WO 2017058768, WO 2017058792), naphthalene or hexahydrofurofurane derivatives (WO 2014143659), quinazolinone (WO2017015562), phenylpyrazine derivatives (WO 2017058728).
  • Benzimidazol, (aza)indole, imidazopyridine derivatives were disclosed as KRAS covalent inhibitors in WO2018145013, benzothiazole, benzothiophene, benzisoxazole derivatives in WO2018140599, pyridopyrimidone, benzothiazole in WO2018119183 and tetrahydropyridopyrimidine in WO2017201161.
  • Compounds of the following general formula BHC233018 - FC - 3 / 152 - are described in US 2018/0201610 (NantBio) which selectively inhibit mutant K-Ras, especially G12V and/or G12D over wild type K-Ras or other mutant K-Ras forms.
  • Substituted quinazoline compounds of the following general formula R2a are described as inhibitors of Ras-protein in WO 2017/172979 (Araxes).
  • Compounds of general formula are described as inhibitors of KRAS G12D in WO 2021/041671 (Mirati).
  • Compounds of general formula are described as to inhibit mutant KRAS in CN 112047948 (Xuanzhu).
  • Reversible, non-covalent inhibitors of KRAS G12D have been described in patent applications (WO2021041671 and WO2017172979A1). However, so far compounds of general formula (I) have not been disclosed as reversible, non-covalent KRAS G12D inhibitors.
  • the compounds of the present invention have surprisingly been found to effectively inhibit KRAS, especially KRAS G12D, and may therefore be used for the treatment or prophylaxis of neoplastic disorders, repectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling, for example.
  • the present invention covers compounds of general formula (I): in which X represents CH, C-F, or N; Y represents CH, C-F, C-Cl, C-CN, C-CH3, or N; Z represents -CH2-, or -CH2-CH2-; R 1 a selected from the i i i i i , , , , , , BHC233018 - FC - 5 / 152 - , , , or ; wherein * indicates the point of attachment of said group with the rest of the molecule; R 2 a selected from the , , , , , , BHC233018 - FC - 6 / 152 - H N , , , , or , wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate,
  • substituted means that one or more hydrogen atoms on the designated atom or group are replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded. Combinations of substituents and/or variables are permissible.
  • optionally substituted means that the number of substituents can be equal to or different from zero. Unless otherwise indicated, it is possible that optionally substituted groups are substituted with as many optional substituents as can be accommodated by replacing a hydrogen atom with a non-hydrogen substituent on any available carbon or nitrogen or atom. Commonly, it is possible for the number of optional substituents, when present, to be 1, 2, 3, 4 or 5, in particular 1, 2 or 3.
  • the term “one or more”, e.g. in the definition of the substituents of the compounds of general formula (I) of the present invention, means “1, 2, 3, 4 or 5, particularly 1, 2, 3 or 4, more particularly 1, 2 or 3, even more particularly 1 or 2”.
  • groups in the compounds according to the invention are substituted, it is possible for said groups to be mono-substituted or poly-substituted with substituent(s), unless otherwise specified.
  • the meanings of all groups which occur repeatedly are independent from one another. It is possible that groups in the compounds according to the invention are substituted with one, two or three identical or different substituents, particularly with one substituent.
  • an oxo substituent represents an oxygen atom, which is bound to a carbon atom or to a sulfur atom via a double bond.
  • the term “comprising” when used in the specification includes “consisting of”. If within the present text any item is referred to as “as mentioned herein”, it means that it may be mentioned anywhere in the present text.
  • the terms as mentioned in the present text have the following meanings:
  • the term “C1-C3-alkyl” means a linear or branched, saturated, monovalent hydrocarbon group having 1, 2, or 3 carbon atoms, e.g. a methyl, ethyl, propyl, or isopropyl group.
  • the term “leaving group” means an atom or a group of atoms that is displaced in a chemical reaction as stable species taking with it the bonding electrons.
  • a leaving group is selected from the group comprising: halide, in particular fluoride, chloride, bromide or iodide, (methylsulfonyl)oxy, [(trifluoromethyl)sulfonyl]oxy, [(nonafluorobutyl)sulfonyl]oxy, (phenylsulfonyl)oxy, [(4-methylphenyl)sulfonyl]oxy, [(4-bromophenyl)sulfonyl]oxy, [(4-nitrophenyl)sulfonyl]oxy, [(2-nitrophenyl)sulfonyl]oxy, [(4-isopropylphenyl)sulfonyl]oxy, [(2,4,6-triisopropylphen
  • the invention therefore includes one or more isotopic variant(s) of the compounds of general formula (I), particularly deuterium- containing compounds of general formula (I).
  • the term “Isotopic variant” of a compound or a reagent is defined as a compound exhibiting an unnatural proportion of one or more of the isotopes that constitute such a compound.
  • the term “Isotopic variant of the compound of general formula (I)” is defined as a compound of general formula (I) exhibiting an unnatural proportion of one or more of the isotopes that constitute such a compound.
  • the expression “unnatural proportion” means a proportion of such isotope which is higher than its natural abundance.
  • isotopes to be applied in this context are described in “Isotopic Compositions of the Elements 1997”, Pure Appl. Chem., 70(1), 217-235, 1998.
  • isotopes include stable and radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, such as 2 H (deuterium), 3 H (tritium), 11 C, 13 C, 14 C, 15 N, 17 O, 18 O, 32 P, 33 P, 33 S, 34 S, 35 S, 36 S, 18 F, 36 Cl, 82 Br, 123 I, 124 I, 125 I, 129 I and 131 I, respectively.
  • the isotopic variant(s) of the compounds of general formula (I) preferably contain deuterium (“deuterium-containing compounds of general formula (I)”).
  • deuterium-containing compounds of general formula (I) Isotopic variants of the compounds of general formula (I) in which one or more radioactive isotopes, such as 3 H or 14 C, are incorporated are useful e.g. in drug and/or substrate tissue distribution studies. These isotopes are particularly preferred for the ease of their incorporation and detectability.
  • Positron emitting isotopes such as 18 F or 11 C may be incorporated into a compound of general formula (I). These isotopic variants of the compounds of general formula (I) are useful for in vivo imaging applications.
  • Deuterium-containing and 13 C-containing compounds of general formula (I) can be used in mass spectrometry analyses in the context of preclinical or clinical studies.
  • BHC233018 - FC - 8 / 152 - Isotopic variants of the compounds of general formula (I) can generally be prepared by methods known to a person skilled in the art, such as those described in the schemes and/or examples herein, by substituting a reagent for an isotopic variant of said reagent, preferably for a deuterium-containing reagent.
  • deuterium from D2O can be incorporated either directly into the compounds or into reagents that are useful for synthesizing such compounds.
  • Deuterium gas is also a useful reagent for incorporating deuterium into molecules. Catalytic deuteration of olefinic bonds and acetylenic bonds is a rapid route for incorporation of deuterium. Metal catalysts (i.e. Pd, Pt, and Rh) in the presence of deuterium gas can be used to directly exchange deuterium for hydrogen in functional groups containing hydrocarbons.
  • Pd, Pt, and Rh metal catalysts in the presence of deuterium gas can be used to directly exchange deuterium for hydrogen in functional groups containing hydrocarbons.
  • a variety of deuterated reagents and synthetic building blocks are commercially available from companies such as for example C/D/N Isotopes, Quebec, Canada; Cambridge Isotope Laboratories Inc., Andover, MA, USA; and CombiPhos Catalysts, Inc., Princeton, NJ, USA.
  • deuterium-containing compound of general formula (I) is defined as a compound of general formula (I), in which one or more hydrogen atom(s) is/are replaced by one or more deuterium atom(s) and in which the abundance of deuterium at each deuterated position of the compound of general formula (I) is higher than the natural abundance of deuterium, which is about 0.015%.
  • the abundance of deuterium at each deuterated position of the compound of general formula (I) is higher than 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, preferably higher than 90%, 95%, 96% or 97%, even more preferably higher than 98% or 99% at said position(s).
  • the abundance of deuterium at each deuterated position is independent of the abundance of deuterium at other deuterated position(s).
  • the selective incorporation of one or more deuterium atom(s) into a compound of general formula (I) may alter the physicochemical properties (such as for example acidity [C. L. Perrin, et al., J. Am. Chem. Soc., 2007, 129, 4490], basicity [C. L. Perrin et al., J. Am. Chem. Soc., 2005, 127, 9641], lipophilicity [B. Testa et al., Int. J.
  • deuterium substitution reduces or eliminates the formation of an undesired or toxic metabolite and enhances the formation of a desired metabolite (e.g. Nevirapine: A. M. Sharma et al., Chem. Res. Toxicol., 2013, 26, 410; Efavirenz: A. E. Mutlib et al., Toxicol. Appl. Pharmacol., 2000, 169, 102).
  • the major effect of deuteration is to reduce the rate of systemic clearance.
  • BHC233018 - FC - 9 / 152 - the biological half-life of the compound is increased.
  • the potential clinical benefits would include the ability to maintain similar systemic exposure with decreased peak levels and increased trough levels.
  • Deuterated drugs showing this effect may have reduced dosing requirements (e.g. lower number of doses or lower dosage to achieve the desired effect) and/or may produce lower metabolite loads.
  • a compound of general formula (I) may have multiple potential sites of attack for metabolism. To optimize the above-described effects on physicochemical properties and metabolic profile, deuterium- containing compounds of general formula (I) having a certain pattern of one or more deuterium-hydrogen exchange(s) can be selected.
  • the deuterium atom(s) of deuterium-containing compound(s) of general formula (I) is/are attached to a carbon atom and/or is/are located at those positions of the compound of general formula (I), which are sites of attack for metabolizing enzymes such as e.g. cytochrome P450.
  • metabolizing enzymes such as e.g. cytochrome P450.
  • the plural form of the word compounds, salts, polymorphs, hydrates, solvates and the like, is used herein, this is taken to mean also a single compound, salt, polymorph, isomer, hydrate, solvate or the like.
  • stable compound' or “stable structure” is meant a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
  • the compounds of the present invention optionally contain one or more asymmetric centres, depending upon the location and nature of the various substituents desired. It is possible that one or more asymmetric carbon atoms are present in the (R) or (S) configuration, which can result in racemic mixtures in the case of a single asymmetric centre, and in diastereomeric mixtures in the case of multiple asymmetric centres. In certain instances, it is possible that asymmetry also be present due to restricted rotation about a given bond, for example, the central bond adjoining two substituted aromatic rings of the specified compounds. Preferred compounds are those which produce the more desirable biological activity.
  • the optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example, by the formation of diastereoisomeric salts using an optically active acid or base or formation of covalent diastereomers.
  • appropriate acids are tartaric, diacetyltartaric, ditoluoyltartaric and camphorsulfonic acid.
  • Mixtures of diastereoisomers can be separated into their individual diastereomers on the basis of their physical and/or chemical differences by methods known in the art, for example, by chromatography or fractional crystallisation.
  • the optically active bases or acids are then liberated from the separated diastereomeric salts.
  • a different process for separation of optical isomers involves the use of chiral chromatography (e.g., HPLC columns using a chiral phase), with or without conventional derivatisation, optimally chosen to maximise the separation of the enantiomers.
  • Suitable HPLC columns using a chiral phase are commercially available, such as those manufactured by Daicel, e.g., Chiracel OD and Chiracel OJ, for example, among many others, which are all routinely selectable.
  • Enzymatic separations, with or without derivatisation are also useful.
  • the optically active compounds of the present invention can likewise be obtained by chiral syntheses utilizing optically active starting materials.
  • the present invention includes all possible stereoisomers of the compounds of the present invention as single stereoisomers, or as any mixture of said stereoisomers, e.g. (R)- or (S)- isomers, in any ratio.
  • Isolation of a single stereoisomer, e.g. a single enantiomer or a single diastereomer, of a compound of the present invention is achieved by any suitable state of the art method, such as chromatography, especially chiral chromatography, for example. Further, it is possible for the compounds of the present invention to exist as tautomers.
  • any compound of the present invention which contains an imidazopyridine moiety as a heteroaryl group for example can exist as a 1H tautomer, or a 3H tautomer, or even a mixture in any amount of the two tautomers, namely : H 1H tautomer 3H tautomer
  • the present invention includes all possible tautomers of the compounds of the present invention as single tautomers, or as any mixture of said tautomers, in any ratio.
  • the compounds of the present invention can exist as N-oxides, which are defined in that at least one nitrogen of the compounds of the present invention is oxidised.
  • the present invention includes all such possible N-oxides.
  • the present invention also covers useful forms of the compounds of the present invention, such as metabolites, hydrates, solvates, prodrugs, salts, in particular pharmaceutically acceptable salts, and/or co- precipitates.
  • the compounds of the present invention can exist as a hydrate, or as a solvate, wherein the compounds of the present invention contain polar solvents, in particular water, methanol or ethanol for example, as structural element of the crystal lattice of the compounds. It is possible for the amount of polar solvents, in particular water, to exist in a stoichiometric or non-stoichiometric ratio. In the case of stoichiometric solvates, e.g.
  • a hydrate, hemi-, (semi-), mono-, sesqui-, di-, tri-, tetra-, penta- etc. solvates or hydrates, respectively, are possible.
  • the present invention includes all such hydrates or solvates.
  • the compounds of the present invention to exist in free form, e.g. as a free base, or as a free acid, or as a zwitterion, or to exist in the form of a salt.
  • Said salt may be any salt, either an organic or inorganic addition salt, particularly any pharmaceutically acceptable organic or inorganic addition salt, which is customarily used in pharmacy, or which is used, for example, for isolating or purifying the compounds of the present invention.
  • pharmaceutically acceptable salt refers to an inorganic or organic acid addition salt of a compound of the present invention.
  • pharmaceutically acceptable salt refers to an inorganic or organic acid addition salt of a compound of the present invention.
  • S. M. Berge, et al. “Pharmaceutical Salts,” J. Pharm. Sci.1977, 66, 1-19.
  • a suitable pharmaceutically acceptable salt of the compounds of the present invention may be, for example, an acid-addition salt of a compound of the present invention bearing a nitrogen atom, in a chain or in a ring, for example, which is sufficiently basic, such as an acid-addition salt with an inorganic acid, or “mineral acid”, such as hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfamic, bisulfuric, phosphoric, or nitric acid, for example, or with an organic acid, such as formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic, 2-(4- hydroxybenzoyl)-benzoic, camphoric, cinnamic, cyclopentanepropionic, digluconic, 3-hydroxy-2- naphthoic, nicot
  • an alkali metal salt for example a sodium or potassium salt
  • an alkaline earth metal salt for example a calcium, magnesium or strontium salt, or an aluminium or a zinc salt
  • acid addition salts of the claimed compounds to be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods.
  • alkali and alkaline earth metal salts of acidic compounds of the present invention are prepared by reacting the compounds of the present invention with the appropriate base via a variety of known methods.
  • the present invention includes all possible salts of the compounds of the present invention as single salts, or as any mixture of said salts, in any ratio.
  • in vivo hydrolysable ester means an in vivo hydrolysable ester of a compound of the present invention containing a carboxy or hydroxy group, for example, a pharmaceutically acceptable ester which is hydrolysed in the human or animal body to produce the parent acid or alcohol.
  • Suitable pharmaceutically acceptable esters for carboxy include for example alkyl, cycloalkyl and optionally substituted phenylalkyl, in particular benzyl esters, C 1 -C 6 alkoxymethyl esters, e.g. BHC233018 - FC - 13 / 152 - methoxymethyl, C1-C6 alkanoyloxymethyl esters, e.g. pivaloyloxymethyl, phthalidyl esters, C3-C8 cycloalkoxy-carbonyloxy-C1-C6 alkyl esters, e.g.
  • esters 1-cyclohexylcarbonyloxyethyl ; 1,3-dioxolen-2- onylmethyl esters, e.g. 5-methyl-1,3-dioxolen-2-onylmethyl ; and C1-C6-alkoxycarbonyloxyethyl esters, e.g.1-methoxycarbonyloxyethyl, it being possible for said esters to be formed at any carboxy group in the compounds of the present invention.
  • An in vivo hydrolysable ester of a compound of the present invention containing a hydroxy group includes inorganic esters such as phosphate esters and [alpha]-acyloxyalkyl ethers and related compounds which as a result of the in vivo hydrolysis of the ester breakdown to give the parent hydroxy group.
  • inorganic esters such as phosphate esters and [alpha]-acyloxyalkyl ethers and related compounds which as a result of the in vivo hydrolysis of the ester breakdown to give the parent hydroxy group.
  • [alpha]-acyloxyalkyl ethers include acetoxymethoxy and 2,2-dimethylpropionyloxymethoxy.
  • a selection of in vivo hydrolysable ester forming groups for hydroxy include alkanoyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl, alkoxycarbonyl (to give alkyl carbonate esters), dialkylcarbamoyl and N-(dialkylaminoethyl)-N-alkylcarbamoyl (to give carbamates), dialkylaminoacetyl and carboxyacetyl.
  • the present invention covers all such esters.
  • the present invention includes all possible crystalline forms, or polymorphs, of the compounds of the present invention, either as single polymorph, or as a mixture of more than one polymorph, in any ratio.
  • the present invention also includes prodrugs of the compounds according to the invention.
  • prodrugs here designates compounds which themselves can be biologically active or inactive, but are converted (for example metabolically or hydrolytically) into compounds according to the invention during their residence time in the body.
  • the present invention covers compounds of general formula (I), supra, in which: X represents CH, C-F, or N; Y represents CH, C-F, C-CN, C-CH 3 , or N; Z represents -CH 2 -, or -CH 2 -CH 2 -; R 1 represents a group selected from the group: i i * * * , , , , , , , BHC233018 - FC - 14 / 152 - , , or ; wherein * indicates the point of attachment of said group with the rest of the molecule; R 2 represents a group selected from the group: , , , or , wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • R 1 represents a group selected from the group: i i * * * , ,
  • the present invention covers compounds of general formula (I), supra, in which: X represents N; Y represents CH, C-F, or C-CN; Z represents -CH2-CH2-; BHC233018 - FC - 15 / 152 - R 1 a selected from the CH3 , , , , , or ; wherein * indicates the point of attachment of said group with the rest of the molecule; R 2 represents a group selected from the group: , wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • the present invention also covers the following compounds of general formula (I), supra, 2'-Amino-1-[6-(3,8-diazabicyclo[3.2.1]octan-3-yl)-2- ⁇ [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy ⁇ pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer); 2'-Amino-1-[6-(3,8-diazabicyclo[3.2.1]octan-3-yl)-2- ⁇ [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy ⁇ pyrimidin-4-yl]-5',6'-dihydrospiro[azetidine
  • the present invention covers compounds of formula (I), supra, in which: BHC233018 - FC - 19 / 152 - X represents CH, C-F, or N; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • the present invention covers compounds of formula (I), supra, in which: X represents N; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • the present invention covers compounds of formula (I), supra, in which: Y represents CH, C-F, C-Cl, C-CN, C-CH3, or N; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • the present invention covers compounds of formula (I), supra, in which: Y represents CH, C-F, C-CN, C-CH3, or N; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • the present invention covers compounds of formula (I), supra, in which: Y represents CH, C-F, or C-CN; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • the present invention covers compounds of formula (I), supra, in which: Y represents C-CN; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • the present invention covers compounds of formula (I), supra, in which: Z represents -CH 2 -, or -CH 2 -CH 2 -; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • the present invention covers compounds of formula (I), supra, in which: Z represents -CH 2 -CH 2 -; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • the present invention covers compounds of formula (I), supra, in which: BHC233018 - FC - 20 / 152 - represents a group selected from the group: ( ) , , , , , or ; wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • the present invention covers compounds of formula (I), supra, in which: BHC233018 - FC - 21 / 152 - R 1 represents a group selected from the group: , , , , or ; wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • the present invention covers compounds of formula (I), supra, in which: R 1 a selected from the , , , , , or ; wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • the present invention covers compounds of formula (I), supra, in which: R 2 a selected from the , , , , BHC233018 - FC - 22 / 152 - , , , , or , wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • the present invention covers compounds of formula (I), supra, in which: R 2 represents a group selected from the group: , , , or , wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • the present invention covers compounds of formula (I), supra, in which: BHC233018 - FC - 23 / 152 - R 2 a selected from the H , wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
  • the present invention covers combinations of two or more of the above mentioned embodiments under the heading “further embodiments of the first aspect of the present invention”. The present invention covers any sub-combination within any embodiment or aspect of the present invention of compounds of general formula (I), supra.
  • the present invention covers any sub-combination within any embodiment or aspect of the present invention.
  • the present invention covers the compounds of general formula (I) which are disclosed in the Example Section of this text, infra.
  • the compounds according to the invention of general formula (I) can be prepared according to the following schemes 1 and 2.
  • the schemes and procedures described below illustrate synthetic routes to the compounds of general formula (I) of the invention and are not intended to be limiting. It is clear to the person skilled in the art that the order of transformations as exemplified in schemes 1 and 2 can be modified in various ways. The order of transformations exemplified in these schemes is therefore not intended to be limiting.
  • interconversion of any of the substituents, R 1 , R 2 or R 3 can be achieved before and/or after the exemplified transformations.
  • modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art.
  • transformations include those which introduce a functionality which allows for further interconversion of substituents.
  • Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3 rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs.
  • the compounds of general formula (I) of the present invention can be converted to any salt, preferably pharmaceutically acceptable salts, as described herein, by any method which is known to the person skilled BHC233018 - FC - 24 / 152 - in the art.
  • any salt of a compound of general formula (I) of the present invention can be converted into the free compound, by any method which is known to the person skilled in the art.
  • Compounds of general formula (I) of the present invention demonstrate a valuable pharmacological spectrum of action which could not have been predicted.
  • Compounds of the present invention have surprisingly been found to effectively inhibit KRAS and it is possible therefore that said compounds be used for the treatment or prophylaxis of diseases, preferably neoplasic disorders in humans and animals.
  • Compounds of the present invention can be utilized to inhibit, block, reduce, decrease, etc., cell proliferation and/or cell division, and/or produce apoptosis.
  • This method comprises administering to a mammal in need thereof, including a human, an amount of a compound of general formula (I) of the present invention, or a pharmaceutically acceptable salt, isomer, polymorph, metabolite, hydrate, solvate or ester thereof, which is effective to treat the disorder.
  • Hyperproliferative disorders include, but are not limited to, for example : psoriasis, keloids, and other hyperplasias affecting the skin, benign prostate hyperplasia (BPH), solid tumours, such as cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid and their distant metastases. Those disorders also include lymphomas, sarcomas, and leukaemias. Examples of breast cancers include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.
  • cancers of the respiratory tract include, but are not limited to, small-cell and non-small-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma.
  • brain cancers include, but are not limited to, brain stem and hypophtalmic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumour.
  • Tumours of the male reproductive organs include, but are not limited to, prostate and testicular cancer.
  • Tumours of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus.
  • Tumours of the digestive tract include, but are not limited to, anal, colon, colorectal, oesophageal, gallbladder, gastric, pancreatic, rectal, small-intestine, and salivary gland cancers.
  • Tumours of the urinary tract include, but are not limited to, bladder, penile, kidney, renal pelvis, ureter, urethral and human papillary renal cancers.
  • Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma.
  • liver cancers include, but are not limited to, hepatocellular carcinoma (liver cell carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma.
  • Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi’s sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.
  • Head-and-neck cancers include, but are not limited to, laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancer, lip and oral cavity cancer and squamous cell.
  • Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin’s lymphoma, cutaneous T-cell lymphoma, Burkitt lymphoma, Hodgkin’s disease, and lymphoma of the central nervous system.
  • Sarcomas include, but are not limited to, sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.
  • Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.
  • the present invention also provides methods of treating angiogenic disorders including diseases associated with excessive and/or abnormal angiogenesis. Inappropriate and ectopic expression of angiogenesis can be deleterious to an organism.
  • a number of pathological conditions are associated with the growth of extraneous blood vessels. These include, for example, diabetic retinopathy, ischemic retinal-vein occlusion, and retinopathy of prematurity [Aiello et al., New Engl. J.
  • compounds of general formula (I) of the present invention can be utilized to treat and/or prevent any of the aforementioned angiogenesis disorders, for example by inhibiting and/or reducing blood vessel formation; by inhibiting, blocking, reducing, decreasing, etc. endothelial cell proliferation, or other types involved in angiogenesis, as well as causing cell death or apoptosis of such cell types.
  • angiogenesis disorders for example by inhibiting and/or reducing blood vessel formation; by inhibiting, blocking, reducing, decreasing, etc. endothelial cell proliferation, or other types involved in angiogenesis, as well as causing cell death or apoptosis of such cell types.
  • treating or “treatment” as stated throughout this document is used conventionally, for example the management or care of a subject for the purpose of combating, alleviating, reducing, relieving, improving the condition of a disease or disorder, such as a carcinoma.
  • the compounds of the present invention can be used in particular in therapy and prevention, i.e. prophylaxis, of tumour growth and metastases, especially in solid tumours of all indications and stages with or without pre-treatment of the tumour growth.
  • chemotherapeutic agents and/or anti-cancer agents in combination with a compound or pharmaceutical composition of the present invention will serve to: 1.
  • the compounds of general formula (I) of the present invention can also be used in combination with radiotherapy and/or surgical intervention.
  • the compounds of general formula (I) of the present invention may be used to sensitize a cell to radiation, i.e. treatment of a cell with a compound of the present invention prior to radiation treatment of the cell renders the cell more susceptible to DNA damage and cell death than the cell would be in the absence of any treatment with a compound of the present invention.
  • the cell is treated with at least one compound of general formula (I) of the present invention.
  • the present invention also provides a method of killing a cell, wherein a cell is administered one or more compounds of the present invention in combination with conventional radiation therapy.
  • the present invention also provides a method of rendering a cell more susceptible to cell death, wherein the cell is treated with one or more compounds of general formula (I) of the present invention prior to the BHC233018 - FC - 27 / 152 - treatment of the cell to cause or induce cell death.
  • the cell is treated with at least one compound, or at least one method, or a combination thereof, in order to cause DNA damage for the purpose of inhibiting the function of the normal cell or killing the cell.
  • a cell is killed by treating the cell with at least one DNA damaging agent, i.e.
  • DNA damaging agents useful in the present invention include, but are not limited to, chemotherapeutic agents (e.g. cis platin), ionizing radiation (X-rays, ultraviolet radiation), carcinogenic agents, and mutagenic agents.
  • a cell is killed by treating the cell with at least one method to cause or induce DNA damage.
  • Such methods include, but are not limited to, activation of a cell signalling pathway that results in DNA damage when the pathway is activated, inhibiting of a cell signalling pathway that results in DNA damage when the pathway is inhibited, and inducing a biochemical change in a cell, wherein the change results in DNA damage.
  • a DNA repair pathway in a cell can be inhibited, thereby preventing the repair of DNA damage and resulting in an abnormal accumulation of DNA damage in a cell.
  • a compound of general formula (I) of the present invention is administered to a cell prior to the radiation or other induction of DNA damage in the cell.
  • a compound of general formula (I) of the present invention is administered to a cell concomitantly with the radiation or other induction of DNA damage in the cell.
  • a compound of general formula (I) of the present invention is administered to a cell immediately after radiation or other induction of DNA damage in the cell has begun.
  • the cell is in vitro.
  • the cell is in vivo.
  • the present invention covers compounds of general formula (I), as described supra, or stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, particularly pharmaceutically acceptable salts thereof, or mixtures of same, for use in the treatment or prophylaxis of diseases, in particular neoplastic disorders.
  • the pharmaceutical activity of the compounds according to the invention can be explained by their activity as KRAS inhibitors.
  • the present invention covers the use of compounds of general formula (I), as described supra, or stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, particularly pharmaceutically acceptable salts thereof, or mixtures of same, for the treatment or BHC233018 - FC - 28 / 152 - prophylaxis of diseases, in particular neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling.
  • the present invention covers the use of a compound of formula (I), described supra, or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, for the prophylaxis or treatment of diseases, in particular neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling.
  • the present invention covers the use of compounds of general formula (I), as described supra, or stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, particularly pharmaceutically acceptable salts thereof, or mixtures of same, in a method of treatment or prophylaxis of diseases, in particular neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling.
  • the present invention covers use of a compound of general formula (I), as described supra, or stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, particularly pharmaceutically acceptable salts thereof, or mixtures of same, for the preparation of a pharmaceutical composition, preferably a medicament, for the prophylaxis or treatment of diseases, in particular neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling.
  • a pharmaceutical composition preferably a medicament, for the prophylaxis or treatment of diseases, in particular neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling.
  • the present invention covers a method of treatment or prophylaxis of diseases, in particular neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling, using an effective amount of a compound of general formula (I), as described supra, or stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, particularly pharmaceutically acceptable salts thereof, or mixtures of same.
  • diseases in particular neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling
  • the present invention covers pharmaceutical compositions, in particular a medicament, comprising a compound of general formula (I), as described supra, or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, a salt thereof, particularly a pharmaceutically acceptable salt, or a mixture of same, and one or more excipients), in particular one or more pharmaceutically acceptable excipient(s).
  • a compound of general formula (I) as described supra, or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, a salt thereof, particularly a pharmaceutically acceptable salt, or a mixture of same, and one or more excipients), in particular one or more pharmaceutically acceptable excipient(s).
  • excipients in particular one or more pharmaceutically acceptable excipient(s).
  • Conventional procedures for preparing such pharmaceutical compositions in appropriate dosage forms can be utilized.
  • the present invention furthermore covers pharmaceutical compositions, in particular medicaments, which comprise at
  • the compounds according to the invention can have systemic and/or local activity.
  • they can be administered in a suitable manner, such as, for example, via the oral, parenteral, BHC233018 - FC - 29 / 152 - pulmonary, nasal, sublingual, lingual, buccal, rectal, vaginal, dermal, transdermal, conjunctival, otic route or as an implant or stent.
  • a suitable manner such as, for example, via the oral, parenteral, BHC233018 - FC - 29 / 152 - pulmonary, nasal, sublingual, lingual, buccal, rectal, vaginal, dermal, transdermal, conjunctival, otic route or as an implant or stent.
  • the compounds according to the invention can be administered in suitable administration forms.
  • the compounds according to the invention for oral administration, it is possible to formulate the compounds according to the invention to dosage forms known in the art that deliver the compounds of the invention rapidly and/or in a modified manner, such as, for example, tablets (uncoated or coated tablets, for example with enteric or controlled release coatings that dissolve with a delay or are insoluble), orally-disintegrating tablets, films/wafers, films/lyophylisates, capsules (for example hard or soft gelatine capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions. It is possible to incorporate the compounds according to the invention in crystalline and/or amorphised and/or dissolved form into said dosage forms.
  • Parenteral administration can be effected with avoidance of an absorption step (for example intravenous, intraarterial, intracardial, intraspinal or intralumbal) or with inclusion of absorption (for example intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal).
  • absorption step for example intravenous, intraarterial, intracardial, intraspinal or intralumbal
  • absorption for example intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal.
  • Administration forms which are suitable for parenteral administration are, inter alia, preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophylisates or sterile powders.
  • Examples which are suitable for other administration routes are pharmaceutical forms for inhalation [inter alia powder inhalers, nebulizers], nasal drops, nasal solutions, nasal sprays; tablets/films/wafers/capsules for lingual, sublingual or buccal administration; suppositories; eye drops, eye ointments, eye baths, ocular inserts, ear drops, ear sprays, ear powders, ear-rinses, ear tampons; vaginal capsules, aqueous suspensions (lotions, mixturae agitandae), lipophilic suspensions, emulsions, ointments, creams, transdermal therapeutic systems (such as, for example, patches), milk, pastes, foams, dusting powders, implants or stents.
  • inhalation inter alia powder inhalers, nebulizers
  • nasal drops nasal solutions, nasal sprays
  • tablets/films/wafers/capsules for lingual, sublingual or buccal
  • compositions according to the invention can be incorporated into the stated administration forms. This can be effected in a manner known per se by mixing with pharmaceutically suitable excipients.
  • Pharmaceutically suitable excipients include, inter alia, • fillers and carriers (for example cellulose, microcrystalline cellulose (such as, for example, Avicel ® ), lactose, mannitol, starch, calcium phosphate (such as, for example, Di-Cafos ® )), • ointment bases (for example petroleum jelly, paraffins, triglycerides, waxes, wool wax, wool wax alcohols, lanolin, hydrophilic ointment, polyethylene glycols), • bases for suppositories (for example polyethylene glycols, cacao butter, hard fat), • solvents (for example water, ethanol, isopropanol, glycerol, propylene glycol, medium chain- length triglycerides, fatty oils, liquid polyethylene glycols, paraffins
  • the present invention furthermore relates to a pharmaceutical composition which comprise at least one compound according to the invention, conventionally together with one or more pharmaceutically suitable excipient(s), and to their use according to the present invention.
  • the compounds according to the invention can have systemic and/or local activity.
  • they can be administered in a suitable manner, such as, for example, via the oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, vaginal, dermal, transdermal, conjunctival, otic route or as an implant or stent.
  • a suitable manner such as, for example, via the oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, vaginal, dermal, transdermal, conjunctival, otic route or as an implant or stent.
  • the compounds according to the invention can be administered in suitable administration forms.
  • the compounds according to the invention for oral administration, it is possible to formulate the compounds according to the invention to dosage forms known in the art that deliver the compounds of the invention rapidly and/or in a modified manner, such as, for example, tablets (uncoated or coated tablets, for example with enteric or controlled release coatings that dissolve with a delay or are insoluble), orally-disintegrating tablets, films/wafers, films/lyophylisates, capsules (for example hard or soft gelatine capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions. It is possible to incorporate the compounds according to the invention in crystalline and/or amorphised and/or dissolved form into said dosage forms.
  • Parenteral administration can be effected with avoidance of an absorption step (for example intravenous, intraarterial, intracardial, intraspinal or intralumbal) or with inclusion of absorption (for example intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal).
  • absorption step for example intravenous, intraarterial, intracardial, intraspinal or intralumbal
  • absorption for example intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal.
  • Administration forms which are suitable for parenteral administration are, inter alia, preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophylisates or sterile powders.
  • Examples which are suitable for other administration routes are pharmaceutical forms for inhalation [inter alia powder inhalers, nebulizers], nasal drops, nasal solutions, nasal sprays; tablets/films/wafers/capsules for lingual, sublingual or buccal administration; suppositories; eye drops, eye ointments, eye baths, ocular inserts, ear drops, ear sprays, ear powders, ear-rinses, ear tampons; vaginal capsules, aqueous suspensions (lotions, mixturae agitandae), lipophilic suspensions, emulsions, ointments, creams, transdermal BHC233018 - FC - 32 / 152 - therapeutic systems (such as, for example, patches), milk, pastes, foams, dusting powders, implants or stents.
  • inhalation inter alia powder inhalers, nebulizers
  • nasal drops nasal solutions, nasal sprays
  • compositions according to the invention can be incorporated into the stated administration forms. This can be effected in a manner known per se by mixing with pharmaceutically suitable excipients.
  • Pharmaceutically suitable excipients include, inter alia, • fillers and carriers (for example cellulose, microcrystalline cellulose (such as, for example, Avicel ® ), lactose, mannitol, starch, calcium phosphate (such as, for example, Di-Cafos ® )), • ointment bases (for example petroleum jelly, paraffins, triglycerides, waxes, wool wax, wool wax alcohols, lanolin, hydrophilic ointment, polyethylene glycols), • bases for suppositories (for example polyethylene glycols, cacao butter, hard fat), • solvents (for example water, ethanol, isopropanol, glycerol, propylene glycol, medium chain- length triglycerides fatty oils, liquid polyethylene glycols, paraffins),
  • the present invention furthermore relates to a pharmaceutical composition which comprise at least one compound according to the invention, conventionally together with one or more pharmaceutically suitable excipient(s), and to their use according to the present invention.
  • the present invention covers pharmaceutical combinations, in particular medicaments, comprising at least one compound of general formula (I) of the present invention and at least one or more further active ingredients, in particular for the treatment and/or prophylaxis of a neoplastic disorder, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling.
  • the present invention covers a pharmaceutical combination, which comprises: • one or more first active ingredients, in particular compounds of general formula (I) as defined supra, and • one or more further active ingredients, in particular cancer agents.
  • a “fixed combination” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein, for example, a first active ingredient, such as one or more compounds of general formula (I) of the present invention, and a further active ingredient are present together in one unit dosage or in one single entity.
  • a “fixed combination” is a pharmaceutical composition wherein a first active ingredient and a further active ingredient are present in admixture for simultaneous administration, such as in a formulation.
  • a “fixed combination” is a pharmaceutical combination wherein a first active ingredient and a further active ingredient are present in one unit without being in admixture.
  • a non-fixed combination or “kit-of-parts” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein a first active ingredient and a further active ingredient are present in more than one unit.
  • One example of a non-fixed combination or kit-of-parts is a combination wherein the first active ingredient and the further active ingredient are present separately. It is possible for the components of the non-fixed combination or kit-of-parts to be administered separately, sequentially, simultaneously, concurrently or chronologically staggered.
  • the compounds of the present invention can be administered as the sole pharmaceutical agent or in combination with one or more other pharmaceutically active ingredients where the combination causes no unacceptable adverse effects.
  • the present invention also covers such pharmaceutical combinations.
  • the compounds of the present invention can be combined with known cancer agents.
  • cancer agents include: 131I-chTNT, abarelix, abemaciclib, abiraterone, acalabrutinib, aclarubicin, adalimumab, ado-trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alectinib, alemtuzumab, alendronic acid, alitretinoin, alpharadin, altretamine, amifostine, aminoglutethimide, hexyl aminolevulinate, amrubicin, amsacrine, anastrozole, ancestim, anethole dithiolethione, anetuma
  • the effective dosage of the compounds of the present invention can readily be determined for treatment of each desired indication.
  • the amount of the active ingredient to be administered in the treatment of one of these conditions can vary widely according to such considerations as the particular compound and dosage unit employed, the mode of administration, the period of treatment, the age and sex of the patient treated, and the nature and extent of the condition treated.
  • the total amount of the active ingredient to be administered will generally range from about 0.001 mg/kg to about 200 mg/kg body weight per day, and preferably from about 0.01 mg/kg to about 20 mg/kg body weight per day.
  • Clinically useful dosing schedules will range from one to three times a day dosing to once every four weeks dosing.
  • drug holidays in which a patient is not dosed with a drug for a certain period of time, to be beneficial to the overall balance between pharmacological effect and tolerability. It is possible for a unit dosage to contain from about 0.5 mg to about 1500 mg of active ingredient, and can be administered one or more times per day or less than once a day.
  • the average daily dosage for administration by injection will preferably be from 0.01 to 200 mg/kg of total body weight.
  • the average daily rectal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight.
  • the average daily vaginal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight.
  • the average daily topical dosage regimen will preferably be from 0.1 to 200 mg administered between one to four times daily.
  • the transdermal concentration will preferably be that required to maintain a daily dose of from 0.01 to 200 mg/kg.
  • the average daily inhalation dosage regimen will preferably be from 0.01 to 100 mg/kg of total body weight.
  • Trichloropyrimidines of general formula (II) can be coupled to nucleophiles of formula (III), where the shown H is directly connected to a heteratom like N or O, using a base (like BHC233018 - FC - 38 / 152 - triethylamine) in a solvent (such as dichloromethane) at temperatures between 25°C and 100°C to get the dichloropyrimidine of general formula (IV).
  • a base like BHC233018 - FC - 38 / 152 - triethylamine
  • a solvent such as dichloromethane
  • intermediates of general formula (IV) can be converted in a second SNAr reaction with a further ⁇ -heteroatom (such as O or N) containing nucleophile of general formula (V) in the presence of a strong base (like sodium hydride)(usually in case of an ⁇ -oxygen bearing nucleophile) or an inorganic base (such as sodium carbonate)(usually in case of an ⁇ -nitrogen bearing nucleophile) in a polar aprotic solvent (such as N,N-dimethylformamide or dimethyl sulfoxide) at temperatures between -80°C and 100°C.
  • a strong base like sodium hydride
  • an inorganic base such as sodium carbonate
  • a polar aprotic solvent such as N,N-dimethylformamide or dimethyl sulfoxide
  • X and Y are different from each other, usually a mixture of two regioisomeric products of general formula (VI) is obtained, wherein the second nitrogen of the corresponding pyrimidine core can be either represented by X or Y of general formula (VI) and the other position is represented by either a CH or a substituted quarternary carbon.
  • These regioisomers can be separated by column chromatography or preparative HPLC. To avoid the formation of regioisomers or at least increase the regioisomeric ratio dichloro-methylthio-pyrimidines of general formula (VIII)(like Cas No 6299-25-8) can be used as starting materials.
  • the obtained product of general formula (IX) is activated for the second SNAr reaction at the methylthio position via oxidation to the methyl sulfonyl compound of general formula (X).
  • This oxidation is usually done with at least 2 equivalents of an oxidizing reagent like meta- chloroperoxybenzoic acid or oxone in a solvent such as dichloromethane at temperatures between -20°C and 40°C.
  • the second SNAr reaction can be performed regioselectively via substitution of the methyl sulfone at the pyrimidine core by using a strong base like sodium bis(trimethylsilyl)amide to quantitatively deprotonate the nucleophile of general formula (V) in a solvent like tetrahydrofuran at temperatures ranging between -80°C and 100°C.
  • this lower pathway leads also to intermediates of general formula (VI) that can be reacted in a final third S N Ar reaction with a tricyclic cyano-aminothiophene containing a spiroazetidine (general formula (VII)).
  • an inorganic base such as sodium or caesium carbonate is employed in a dipolar aprotic solvent like dimethyl sulfoxide or N,N-dimethylacetamide at temperatures between 50°C and 150°C to obtain the desired compounds of general formula (I).
  • X,Y, Z, R 1 , R 2 represent (hetero)atoms or substituents according to claim 1;
  • HQ represents an acid able to form a salt with the azetidine of compounds (VII) like HCl, formic acid or trifluoroacetic acid;
  • reaction conditions in scheme 1 steps 1, 2, 3, 5, and 6: base, solvent; step 4: oxidizing agent, solvent.
  • compounds of the general formula (I) can be obtained via a changed order of addition of the three substituents at the pyrimidine core again employing three S N Ar reactions.
  • dichloro-methylthio-pyrimidines of general formula (VIII) (like Cas No 6299-25-8) can first be reacted with the tricyclic thienospiroazetidines BHC233018 - FC - 40 / 152 - of general formula (VII) using an inorganic base like sodium carbonate in a dipolar aprotic solvent such as dimethyl sulfoxide at temperatures between 25°C and 100°C.
  • a dipolar aprotic solvent such as dimethyl sulfoxide
  • R 4 represents C1 to C4 alkyl preferably t-butyl.
  • the other protecting group described as general PG can also be a more labile second carbamate or a more stable benzyl group like para-methoxybenzyl.
  • Double protection with two t-butyloxycarbonyl groups (Boc) can be done using two equivalents of di-tert-butyl dicarbonate together with an organic base like N,N-diisopropylethylamine and a nucleophilic catalyst like 4-(dimethylamino)pyridine in a solvent like tetrahydrofuran at temperatures ranging from 0°C to 40°C.
  • the amino intermediate of general formula (XI) can first be mono-protected with for example one equivalent of di-tert-butyl decarbonate and subsequently protected with a benzylic group like para-methoxybenzyl (PMB) using para-methoxybenzyl chloride together with an inorganic base like caesium carbonate in an aprotic solvent such as N,N-dimethylformamide at temperatures between 50°C and 120°C.
  • PMB para-methoxybenzyl
  • the double protected intermediate of general formula (XI) can then be oxidized at the methylthiol group using at least 2 equivalents of an oxidizing reagent like meta-chloroperoxybenzoic acid or oxone under conditions already described at scheme 1 for intermediates of general formula (X).
  • the subsequent SNAr reaction with nucleophiles of general formula (V) is usually performed via employment of a strong base like sodium bis(trimethylsilyl)amide in a solvent like tetrahydrofuran at temperatures ranging between 25°C and 100°C.
  • a strong base like sodium bis(trimethylsilyl)amide
  • a solvent like tetrahydrofuran at temperatures ranging between 25°C and 100°C.
  • this strong basic conditions described for the subsequent S N Ar can lead in situ to the loss of the more labile second Boc-protecting group.
  • the more stable carbamate group can be selectively removed using acidic conditions like trifluoroacetic acid in dichloromethane or HCl in dioxane to get the mono-protected intermediates of general formula (XIV).
  • the final S N Ar reaction is usually performed with a basic ⁇ -nitrogen bearing nucleophile of general formula (III) using an inorganic base like caesium carbonate in an aprotic solvent such as dimethyl sulfoxide at temperatures between 50°C and 170°C.
  • the obtained compounds of general formula (XV) are finally deproteced at the amino-position of the thiophene.
  • this final deprotection can be performed using acidic conditions like trifluoroacetic acid in dichloromethane or HCl in dioxane.
  • the final protecting group is a benzylic group deprotection can be performed using hydrogenolytic conditions (like hydrogen atmosphere between 1 and 100 bar together with a catalyst like palladium on charcoal in a protic solvent like ethanol at temperatures between 20°C and 100°C) or oxidative conditions (like ceric ammonium nitrate in a solvent or mixture like aqueous acetonitrile at temperatures between 0°C and 50°C).
  • hydrogenolytic conditions like hydrogen atmosphere between 1 and 100 bar together with a catalyst like palladium on charcoal in a protic solvent like ethanol at temperatures between 20°C and 100°C
  • oxidative conditions like ceric ammonium nitrate in a solvent or mixture like aqueous acetonitrile at temperatures between 0°C and 50°C.
  • X,Y, Z, R 1 , R 2 represent (hetero)atoms or substituents according to claim 1;
  • HQ represents an acid able to form a salt with the azetidine of compounds (VII) like HCl, formic acid or trifluoroacetic acid;
  • R 4 represents a C1 to C4 alkyl preferably t-butyl;
  • PG represents any protecting group, preferably para-methoxybenzyl or t-butyloxycarbonyl; reaction conditions in scheme 2: steps 1, and 5: base, solvent; step 2: double protection; step 3: oxidizing agent, solvent; step 4: base, solvent, then acid; step 6: deprotection.
  • the described head group R 1 as well as the described side chain R 2 might contain additional amine functional groups that are linked via a cycle or chain to the attaching atom A connected to the central heterocycle, wherein A represents nitrogen, oxygen, or sulfur preferably nitrogen.
  • additional amine functional groups are usually protected as carbamates to avoid regioselectivity issues during the SNAr reactions at the central heterocycle, wherein this heterocycle is preferably pyrimidine (scheme 3).
  • Additional carbamate protected amines in the head group R 1 as indicated for compounds of general formula (XVI) might be deprotected in a final reaction step using acidic conditions like trifluoroacetic acid in dichloromethane or HCl in dioxane as already described in scheme 1 and 2 to get compounds of general formula (XVII).
  • acidic conditions like trifluoroacetic acid in dichloromethane or HCl in dioxane as already described in scheme 1 and 2 to get compounds of general formula (XVII).
  • the same conditions can be used to deprotect carbamate protected amines in the side chain R 2 as indicated for compounds of general formula (XVIII).
  • This deprotection of the side chain R 2 can be done at an intermediate state (A is still a halogen usually chloride) or at the final state (A is then the usually tricyclic thienoazetidine).
  • Typical reaction conditions include a mild reducing agent such as sodium cyanoborohydride, sodium triacetoxyborohydride or 2-picoline borane complex, that chemoselectively prefers to reduce the in situ BHC233018 - FC - 42 / 152 - formed imine species from compounds of general formula (XIX) and the carbonyl or acetal compound corresponding to substituent R 5 over the direct reduction of the carbonyl or acetal compound.
  • an acidic additive like acetic acid can be employed in a protic (e.g. methanol) or aprotic solvent (e.g. acetonitrile) with temperatures ranging from 0°C to 100°C.
  • X,Y, Z, R 1 , R 2 represent (hetero)atoms or substituents according to claim 1;
  • A represents an attaching atom nitrogen optionally substituted with R 5 , oxygen or sulfur preferably nitrogen;
  • E represents the shown tricyclic thienospiroazetidine or halogen preferably chlorine;
  • R 4 represents a C1 to C4 alkyl preferably t-butyl;
  • R 5 represents C1 to C6 alkyl preferably methyl or cyclopropyl;
  • the continuous half circle represents any chain connection between the attaching atom A and the carbamate protected amine;
  • the dashed half circle that might be present or not represents a possible second chain connection between the attaching atom A and the carbamate protected amine (in case of a present second chain connection the formed cycle has an unsubstituted nitrogen as attaching atom A
  • Typical reaction conditions require elementary sulfur as source to thiophene ring construction and further include a nuclephilic catalyst to activate the carbonyl moiety such as (S)-(-)-proline in an aprotic solvent such as N,N-dimethylformamide at temperatures between 50°C and 150°C.
  • Ionic liquids such as 1-butyl-3- methyl-1H-imidazol-3-ium chloride (BMIMCl) can be added to increase the reaction rates.
  • BMIMCl 1-butyl-3- methyl-1H-imidazol-3-ium chloride
  • This final deprotection step can be performed using acidic conditions like trifluoroacetic acid in dichloromethane or HCl in dioxane already described in scheme 1 and 2.
  • Z represents CH 2 or CH 2 CH 2 according to claim 1;
  • HQ represents an acid able to form a salt with the azetidine of compounds (VII) like HCl, formic acid or trifluoroacetic acid;
  • R 4 represents a C 1 to C 4 alkyl preferably t-butyl; reaction conditions in scheme 4: step 1: sulfur, BMIMCl, (S)-(-)-proline, N,N- dimethylformamide; step 2: deprotection.
  • a 1 H-NMR peaklist BHC233018 - FC - 44 / 152 - is similar to a classical 1 H-NMR readout, and thus usually contains all the peaks listed in a classical NMR interpretation.
  • peaklists can show solvent signals, signals derived from stereoisomers of the particular target compound, peaks of impurities, 13 C satellite peaks, and/or spinning sidebands.
  • the peaks of stereoisomers, and/or peaks of impurities are typically displayed with a lower intensity compared to the peaks of the target compound (e.g., with a purity of >90%).
  • Such stereoisomers and/or impurities may be typical for the particular manufacturing process, and therefore their peaks may help to identify a reproduction of the manufacturing process on the basis of "by- product fingerprints".
  • Table 1 lists the abbreviations used in this paragraph and in the Examples section as far as they are not explained within the text body. Other abbreviations have their meanings customary per se to the skilled person.
  • Table 1 Abbreviations Table 1 lists the abbreviations used in this paragraph and in the Intermediates and Examples sections as far as they are not explained within the text body. BHC233018 - FC - 45 / 152 - Table 1 Abbreviation Meaning ACN acetonitrile AcOH acetic acid (ethanoic acid) aq.
  • impurities may be stirred out using a suitable solvent.
  • the compounds may be purified by chromatography, particularly flash column chromatography, using for example prepacked silica gel cartridges, e.g. Biotage BHC233018 - FC - 49 / 152 - SNAP cartidges KP-Sil ® or KP-NH ® in combination with a Biotage autopurifier system (SP4 ® or Isolera Four ® ) and eluents such as gradients of hexane/ethyl acetate or DCM/methanol.
  • SP4 ® or Isolera Four ® Biotage autopurifier system
  • eluents such as gradients of hexane/ethyl acetate or DCM/methanol.
  • the compounds may be purified by preparative HPLC using for example a Waters autopurifier equipped with a diode array detector and/or on-line electrospray ionization mass spectrometer in combination with a suitable prepacked reverse phase column and eluents such as gradients of water and acetonitrile which may contain additives such as trifluoroacetic acid, formic acid or aqueous ammonia.
  • a Waters autopurifier equipped with a diode array detector and/or on-line electrospray ionization mass spectrometer in combination with a suitable prepacked reverse phase column and eluents such as gradients of water and acetonitrile which may contain additives such as trifluoroacetic acid, formic acid or aqueous ammonia.
  • purification methods as described above can provide those compounds of the present invention which possess a sufficiently basic or acidic functionality in the form of a salt, such as, in the case of a compound of the present invention which is sufficiently basic, a trifluoroacetate or formate salt for example, or, in the case of a compound of the present invention which is sufficiently acidic, an ammonium salt for example.
  • a salt of this type can either be transformed into its free base or free acid form, respectively, by various methods known to the person skilled in the art, or be used as salts in subsequent biological assays. It is to be understood that the specific form (e.g.
  • a 1 H-NMR peaklist is similar to a classical 1 H-NMR readout, and thus usually contains all the peaks listed in a classical NMR interpretation.
  • peaklists can show solvent signals, signals derived from stereoisomers of the particular target compound, peaks of impurities, 13 C satellite peaks, and/or spinning sidebands.
  • the peaks of stereoisomers, and/or peaks of impurities are typically displayed with a lower intensity compared to the peaks of the target compound (e.g., with a purity of >90%).
  • Such stereoisomers and/or impurities may be typical for the particular manufacturing process, and therefore their peaks may help to identify a reproduction of the manufacturing process on the basis of "by- product fingerprints".
  • the parameter "MinimumHeight" can be adjusted between 1% and 4%. However, depending on the chemical structure and/or depending on the concentration of the measured compound it may be reasonable to set the parameter "MinimumHeight" ⁇ 1%.
  • Reactions employing microwave irradiation may be run with a Biotage Initiator ⁇ microwave oven optionally equipped with a robotic unit.
  • the reported reaction times employing microwave heating are intended to be understood as fixed reaction times after reaching the indicated reaction temperature.
  • the compounds and intermediates produced according to the methods of the invention may require purification. Purification of organic compounds is well known to the person skilled in the art and there may be several ways of purifying the same compound. In some cases, no purification may be necessary.
  • the compounds may be purified by crystallization. In some cases, impurities may be stirred out using a suitable solvent. In some cases, the compounds may be purified by chromatography, particularly flash column chromatography, using for example pre-packed silica gel cartridges, e.g. from Separtis such as Isolute® Flash silica gel or Isolute® Flash NH2 silica gel in combination with a Isolera® autopurifier (Biotage) and eluents such as gradients of e.g. hexane/ethyl acetate or DCM/methanol.
  • Separtis such as Isolute® Flash silica gel or Isolute® Flash NH2 silica gel in combination with a Isolera® autopurifier (Biotage)
  • eluents such as gradients of e.g. hexane/ethyl acetate or DCM/methanol.
  • the compounds may be purified by preparative HPLC using for example a Waters autopurifier equipped with a diode array detector and/or on-line electrospray ionization mass spectrometer in combination with a suitable prepacked reverse phase column and eluents such as gradients of water and acetonitrile which may contain additives such as trifluoroacetic acid, formic acid or aqueous ammonia.
  • a Waters autopurifier equipped with a diode array detector and/or on-line electrospray ionization mass spectrometer in combination with a suitable prepacked reverse phase column and eluents such as gradients of water and acetonitrile which may contain additives such as trifluoroacetic acid, formic acid or aqueous ammonia.
  • purification methods as described above can provide those compounds of the present invention which possess a sufficiently basic or acidic functionality in the form of a salt, such as, in the case of a compound of the present invention which is sufficiently basic, a trifluoroacetate or formate salt for example, or, in the case of a compound of the present invention which is sufficiently acidic, an ammonium salt for example.
  • a salt of this type can either be transformed into its free base or free acid form, respectively, by various methods known to the person skilled in the art, or be used as salts in subsequent biological assays. It is to be understood that the specific form (e.g.
  • LC-MS Analytical Method 1: Instrument: Thermo Scientific FT-MS; UHPLC: Thermo Scientific UltiMate 3000; column: Waters HSS T3 C181.8 ⁇ m, 75 mm ⁇ 2.1 mm; eluent A: water + 0.01% formic acid; eluent B: acetonitrile + 0.01% formic acid; gradient: 0.0 min 10% B ⁇ 2.5 min 95% B ⁇ 3.5 min 95% B; temperature: 50°C; flow rate: 0.90 mL/min; UV detection: 210-400 nm.
  • LC-MS Analytical Method 3 Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C181.7 ⁇ m, 50x2.1mm; eluent A: water + 0.1 vol % formic acid (99%), eluent B: acetonitrile; gradient: 0-1.6 min 1- 99% B, 1.6-2.0 min 99% B; flow 0.8 ml/min; temperature: 60 °C; DAD scan: 210-400 nm.
  • the first eluting product with target molecular mass as formic acid salt (519 mg) was dissolved in ethyl acetate, washed two times with saturated aqueous sodium bicarbonate solution, concentrated under reduced pressure and dried in vacuo. Yield: 402 mg (34% of theory).
  • the reaction mixture was stirred at 95°C for 7 h in a closed microwave vial, diluted with water and aqueous hydrochloric acid solution (1 N, 912 ⁇ L).
  • the suspension was dissolved in a mixture of acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 101 mg (66% of theory).
  • the second eluting product with target molecular mass as formic acid salt (208 mg) was dissolved in ethyl acetate, washed two times with saturated aqueous sodium bicarbonate solution, concentrated under reduced pressure and dried in vacuo. Yield: 185 mg (16% of theory).
  • reaction mixture was stirred at 100°C for 2.5 h in a closed microwave vial, diluted with water and aqueous hydrochloric acid solution (1 N, 300 ⁇ L).
  • the mixture was dissolved in a mixture of acetonitrile / water / dimethyl sulfoxide and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 26 mg (58% of theory).
  • reaction mixture was stirred at 95°C for 3 h in a BHC233018 - FC - 61 / 152 - closed microwave vial, mixed with additional 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile trifluoroacetate (41 mg, 78% purity, 0.10 mmol, 0.8 eq.) and stirred for another 6 h.
  • the reaction mixture was stirred at reflux for 2 h and concentrated under reduced pressure.
  • the residue was mixed with aqueous sodium hydroxide solution (1 N) and extracted with ethyl acetate. After phase separation, the aqueous phase was extracted with ethyl acetate.
  • the combined organic phases were washed with brine, dried over phase separation filter paper and concentrated under reduced pressure.
  • the aqueous phase still contained desired product and was again extracted three times with ethyl acetate.
  • the BHC233018 - FC - 67 / 152 - combined organic phases were washed with brine, dried over phase separation filter paper and concentrated under reduced pressure.
  • reaction mixture was stirred at RT for 1 h, combined with a batch of a previously performed test reaction (0.11 mmol scale) and concentrated under reduced pressure.
  • the residue was purified by flash silica gel chromatography (dichloromethane / methanol gradient) and subsequent RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 293 mg (57% of theory for both reactions).
  • reaction mixture was stirred at 95°C overnight in a closed microwave vial, mixed with additional 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile trifluoroacetate (30 mg, 72% purity, 0.06 mmol, 0.4 eq.), stirred at 95°C for another 2 h, mixed with additional 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile trifluoroacetate (30 mg, 72% purity, 0.06 mmol, 0.4 eq.) and sodium carbonate (17 mg, 0.16 mmol, 1.0 eq.), stirred at 95°C for another 6 h, mixed with additional 2'-amino- 6',7'-dihydro-5'H-spiro[azetidine-3,4'
  • reaction mixture was stirred at RT overnight, at 50°C for 1 h, mixed with additional sodium hydride (60% in mineral oil, 11 mg, 0.28 mmol, 1.0 eq.), stirred for another 1 h, mixed with additional tert-butyl 6- (hydroxymethyl)-6,7-dihydropyrazolo[1,5-a]pyrazine-5(4H)-carboxylate (racemate) (35 mg, 0.14 mmol, 0.5 eq.) and stirred at RT for another 2 h.
  • sodium hydride 50% in mineral oil, 11 mg, 0.28 mmol, 1.0 eq.
  • sodium hydride 46.8 mg, 60 % purity, 1.17 mmol; CAS-RN:[7646-69-7]
  • sodium hydride 46.8 mg, 60 % purity, 1.17 mmol; CAS-RN:[7646-69-7]
  • the reaction mixture was heated at 70°C for 1 h.
  • the reaction was allowed to cool to room temperature and quenched with water and the aqueous phase was extracted with ethyl acetate.
  • the combined organic layers were washed with brine, filtered through a water repellant filter and concentrated in vacuo.
  • the crudeoch was taken up in 4 ml acetonitrile / water (7:3).
  • the precipitate thus obtained was collected by vacuum filtration, washed with a little amount of acetonitrile and dried in vacuo.
  • the residue was diluted with 2 ml acetonitrile and purified by preparative HPLC (Method A; gradient: 40% B to 85% B).
  • the reaction mixture was stirred for 16 h at RT.
  • the reaction was quenched with water and the aqueous phase was extracted with ethyl acetate.
  • the combined organics were washed with brine, filtered through a water repellant filter and concentrated.
  • the crude product was taken up in 5 ml methyl tert-butyl ether, sonicated and added to 50 ml of hexane.
  • the suspension was intensively stirred for 10 minutes, then the solid was collected by vacuum filtration and dried to afford 673 mg (76% yield, 94% purity) of the title compound as a beige solid.
  • reaction mixture was stirred for 1 hour at RT.
  • the mixture was diluted with dichloromethane and extracted with sodium hydroxide solution (2M).
  • the organic layer was filtered through a water repellant filter and concentrated to afford 22 mg (68% yield, 98% purity) of the title compound as a white solid.
  • the crude material was purified by Biotage IsoleraTM chromatography (Sfär Silica HC D – 20 ⁇ m 10 g), eluting with hexane - ethyl acetate, 9:1 to 0:1, then with ethyl acetate – ethanol, 1:0 to 9:1) to afford 8.0 mg (40% yield, 97% purity) of the title compound as a white foam.
  • the reaction mixture was heated at 140°C for 1,5 h under microwave irradiation. To the mixture were added further 4,4-difluoropiperidine (30.8 mg, 255 ⁇ mol) and the reaction mixture was heated at 150°C for 1 h under microwave irradiation. The mixture was filtered and driectly purified by preparative HPLC (Method A; gradient: 25% B to 70% B). The product fractions were pooled and concentrated in vacuo to afford 10.0 mg (17% yield, 93% purity) of the title compound as a white powder.
  • the reaction mixture was heated at 150°C for 2 h under microwave irradiation.
  • the reaction was diluted with ethyl acetate and water was added.
  • the phases were separated and the aqueous phase was extracted with ethyl acetate.
  • the combined organic phases were washed with brine, filtered through a water repellant filter and concentrated.
  • the crude product was purified by preparative HPLC (Method A, gradient C). The product fractions were pooled and concentrated in vacuo to afford 23 mg (38% yield, 92% purity) of the BHC233018 - FC - 95 / 152 - title compound as a white powder.
  • the reaction mixture was heated at 70°C for 2 h. The reaction was allowed to cool to room temperature, then the mixture was poured into water (20 ml). The mixture was intensively stirred for 10 minutes and the solid was collected by vacuum filtration. The solid was washed with hexane and dried to afford 222 mg (77% yield, 97% purity) of the title compound as a white solid.
  • reaction mixture was stirred at RT for 4 h. Upon completion to the mixture was added sodium hydroxide solution (5 ml; 2M). The aqueous phase was extracted with dichloromethane and the combined organic phases were washed with brine. Then the phase was filtered through a water repellant filter and concentrated to afford 121 mg (crude) of the title compound as a white solid.
  • the reaction mixture was stirred at RT for 2 h and concentrated under reduced pressure.
  • the residue was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient).
  • the resulting salt was dissolved in methanol and filtered in four portions through four SPE cartridges (200 mg PL-HCO3 MP 6 mL tubes, gravity filtration, washed each with 6 mL of methanol).
  • the combined filtrates were concentrated under reduced pressure and dried in vacuo. Yield: 55 mg (64% of theory).
  • the reaction mixture was stirred at 95°C for 6 h in a closed microwave vial and diluted with water and aqueous hydrochloric acid solution (1 N, 300 ⁇ L).
  • the suspension was dissolved in a mixture of acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 21 mg (51% of theory).
  • Example 02-02 2'-Amino-1-(2- ⁇ [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy ⁇ -6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]pyrimidin-4-yl)-5',6'-dihydrospiro[azetidine-3,4'- cyclopenta[b]thiophene]-3'-carbonitrile (single stereoisomer) Sodium carbonate (39 mg, 0.37 mmol, 4.0 eq.) was added to a solution of (3R)-1-(6-chloro-2- ⁇ [(2R,7aS)- 2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy ⁇ pyrimidin-4-yl)-3-methylpiperidin-3-ol formate (single stereoisomer) (40 mg, 0.09 mmol) and
  • reaction mixture was stirred at 95°C for 3 h in a closed microwave vial, mixed with additional 2'-amino-5',6'-dihydrospiro[azetidine-3,4'-cyclopenta[b]thiophene]-3'-carbonitrile BHC233018 - FC - 104 / 152 - trifluoroacetate (15 mg, 78% purity, 0.04 mmol, 0.4 eq.), stirred for another 6 h at 95°C and quenched with aqueous hydrochloric acid solution (1 N, 400 ⁇ L).
  • reaction mixture was stirred at 95°C overnight in a closed microwave vial, mixed with additional 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (35 mg, 76% purity, 0.08 mmol, 1.0 eq.), stirred at 95°C for another 4 h and diluted with water and aqueous hydrochloric acid solution (1 N, 321 ⁇ L).
  • the suspension was dissolved in a mixture of acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 16 mg (36% of theory).
  • Example 02-04 2'-Amino-1-(2- ⁇ [(2S)-4,4-difluoro-1-methylpyrrolidin-2-yl]methoxy ⁇ -6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- BHC233018 - FC - 105 / 152 - carbonitrile (single stereoisomer) Sodium carbonate (180 mg, 1.70 mmol, 4.0 eq.) was added to a solution of (3R)-1-(6-chloro-2- ⁇ [(2S)-4,4- difluoro-1-methylpyrrolidin-2-yl]methoxy ⁇ pyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (160 mg, 0.43 mmol) and 2
  • reaction mixture was stirred at 95°C overnight in a closed microwave vial, quenched with aqueous hydrochloric acid solution (1 N, 1.7 mL) and diluted with water (0.5 mL) and acetonitrile (0.5 mL).
  • aqueous hydrochloric acid solution (1 N, 1.7 mL) and diluted with water (0.5 mL) and acetonitrile (0.5 mL).
  • the solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 172 mg (73% of theory).
  • reaction mixture was stirred at 95°C overnight in a closed microwave vial, mixed with additional sodium carbonate (10 mg, 0.09 mmol, 1.5 eq.), stirred for another 1 h at 95°C, mixed with additional 2'-amino- BHC233018 - FC - 106 / 152 - 6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (11 mg, 72% purity, 0.03 mmol, 0.4 eq.) and sodium carbonate (3 mg, 0.03 mmol, 0.4 eq.) and stirred at 95°C overnight.
  • Example 02-06 2'-Amino-1-(2- ⁇ [(2S,4R)-1-cyclopropyl-4-fluoropyrrolidin-2-yl]methoxy ⁇ -6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer) Sodium carbonate (92 mg, 0.87 mmol, 5.0 eq.) was added to a solution of (3R)-1-(6-chloro-2- ⁇ [(2S,4R)- 1-cyclopropyl-4-fluoropyrrolidin-2-yl]methoxy ⁇ pyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (67 mg, 0.17 mmol) and 2'-amino-6',7'
  • reaction mixture was stirred at 95°C overnight in a closed microwave vial, diluted with water and quenched with aqueous hydrochloric acid solution (1 N, 870 ⁇ L).
  • the suspension was diluted with acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 58 mg (56% of theory).
  • Example 02-08 2'-Amino-1- ⁇ 2-[(3-cyano-1-methylpyrrolidin-3-yl)methoxy]-6-[(3R)-3-hydroxy-3-methylpiperidin- 1-yl]pyrimidin-4-yl ⁇ -6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile (mixture of two enantiopure diastereomers) CH3 Sodium carbonate (89 mg, 0.84 mmol, 4.0 eq.) was added to a solution of 3-[( ⁇ 4-chloro-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl ⁇ oxy)methyl]-1-methylpyrrolidine-3-carbonitrile (mixture of two enantiopure diastereomers) (77 mg, 0.21 mmol) and 2'-amino-6',7'-dihydro-5
  • the reaction mixture was stirred at 95°C for 7 h in a closed microwave vial, kept over the weekend at RT, stirred at 95°C for another 24 h and quenched with aqueous hydrochloric acid solution (1 N, 842 ⁇ L).
  • the mixture was purified by RP-HPLC (acetonitrile / 0.1% BHC233018 - FC - 108 / 152 - formic acid in water gradient). Yield: 45 mg (38% of theory).
  • reaction mixture was stirred at 95°C overnight in a closed microwave vial, diluted with water and quenched with aqueous hydrochloric acid solution (1 N, 881 ⁇ L).
  • the suspension was dissolved in acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 37 mg (38% of theory).
  • reaction mixture was stirred at 95°C overnight in a closed microwave vial, combined with a batch of a previously performed test reaction (0.05 mmol scale) and quenched with aqueous hydrochloric acid solution (1 N, 792 ⁇ L).
  • aqueous hydrochloric acid solution (1 N, 792 ⁇ L).
  • the mixture was dissolved with water (0.5 mL) and acetonitrile (1.0 mL) and purified by RP- HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 63 mg (48% of theory for both reactions).
  • reaction mixture was stirred at 95°C overnight in a closed microwave vial, quenched with aqueous hydrochloric acid solution (1 N, 416 ⁇ L) and diluted with water (0.5 mL) and N,N-dimethylformamide (0.5 mL).
  • aqueous hydrochloric acid solution (1 N, 416 ⁇ L) and diluted with water (0.5 mL) and N,N-dimethylformamide (0.5 mL).
  • the solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 9 mg (13% of theory).
  • Example 03-04 2'-amino-1-[2- ⁇ [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy ⁇ -6-(3- oxopiperazin-1-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer)
  • reaction mixture was stirred for 1 h at 50°C.
  • the reaction mixture was heated at 100°C for further 11 h.
  • the reaction mixture was diluted with 2 ml acetonitrile / water (7:3) and purified by preparative HPLC (Method A; gradient: 20% B to 60% B).
  • the product fractions were pooled and concentrated in vacuo to afford 12.3 mg (11% yield, 93% purity) of the title compound as a white fluffy solid.
  • Example 03-06 2'-amino-1-[6-(3,3-difluoropiperidin-1-yl)-2- ⁇ [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy ⁇ pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer) BHC233018 - FC - 114 / 152 - To a stirred suspension of (2R,7aS)-7a-( ⁇ [4-chloro-6-(3,3-difluoropiperidin-1-yl)pyrimidin-2- yl]oxy ⁇ methyl)-2-fluorohexahydro-1H-pyrrolizine (60.0 mg, 154 ⁇ mol; intermediate 6-14) and sodium carbonate (65.1 mg, 614 ⁇ mol;
  • the reaction mixture was heated at 100°C for further 10 h.
  • the mixture was filtered through a pad of celite, rinsed with ethyl acetate and the filtrate was washed with water.
  • the organic phase was filtered through a water repellant filter and concentrated.
  • the crude product was purified by by preparative HPLC (Method A, gradient C). The product fractions were pooled and concentrated in vacuo to afford 35.4 mg (37% yield, 91% purity) of the title compound as a white fluffy solid.
  • Example 03-08 tert-butyl 1-[6-(2'-amino-3'-cyano-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1-yl)- 2- ⁇ [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy ⁇ pyrimidin-4-yl]-1,6- diazaspiro[3.4]octane-6-carboxylate (mixture of two enantiopure diastereomers) To a stirred suspension of tert-butyl 1-(6-chloro-2- ⁇ [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy ⁇ pyrimidin-4-yl)-1,6-diazaspiro[3.4]octane-6-carboxylate (150
  • the reaction mixture was heated at 100°C for further 10 h.
  • the reaction was allowed to cool to RT, then ethyl acetate and water were added.
  • the aqueous phase was extreacted with ethyl acetat and the combined organic phases were washed with brine, filtered through a water repellant filter and concentrated.
  • the crude product was purified by preparative HPLC (Method A; gradient: 20% B to 60% B). The product fractions were pooled and concentrated in vacuo to afford 56.3 mg (27% yield, 99% purity) of the title compound as a white solid.
  • Example 03-10 1-(6-[(4RS)-6-acetyl-1,6-diazaspiro[3.4]octan-1-yl]-2- ⁇ [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy ⁇ pyrimidin-4-yl)-2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (mixture of two enantiopure diastereomers) BHC233018 - FC - 117 / 152 - To a solution of 2’-amino-1-(6-[(4RS)-1,6-diazaspiro[3.4]octan-1-yl]-2- ⁇ [(2R,7aS)-2-fluorotetrahydro- 1H-pyrrolizin-7a(5H)-yl
  • reaction mixture was stirred at 95°C overnight in a closed microwave vial, diluted BHC233018 - FC - 118 / 152 - with water and quenched with aqueous hydrochloric acid solution (1 N, 533 ⁇ L).
  • the suspension was dissolved in acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 13 mg (20% of theory).
  • Example 04-02 2'-Amino-1-[2- ⁇ [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy ⁇ -5-methyl-6-(3- oxopiperazin-1-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer) Sodium carbonate (86.7 mg, 0.82 mmol, 4.0 eq.) and molecular sieve (3 ⁇ , 20 mg)were added to a solution of 4-(6-chloro-2- ⁇ [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy ⁇ -5- methylpyrimidin-4-yl)piperazin-2-one (single stereoisomer) (78.5 mg,
  • the reaction mixture was stirred at 95°C overnight in a closed microwave vial, diluted with water and quenched with aqueous hydrochloric acid solution (1 N, 1.2 mL).
  • the suspension was dissolved in acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient), followed by a second RP-HPLC (acetonitrile / 1% ammonia in water gradient) to separate both regioisomers. Yield: 13 mg (9% of theory).
  • the reaction mixture was stirred at RT for 3 h, mixed with additional trifluoroacetic acid (30 ⁇ L, 0.39 mmol, 10 eq.), stirred at RT for 1.5 h and concentrated under reduced pressure.
  • the residue was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient).
  • the resulting salt was dissolved in methanol and filtered through a SPE cartridge (200 mg PL-HCO3 MP 6 mL tube, gravity filtration, washed with 3.0 mL of methanol). The filtrate was concentrated under reduced pressure and dried in vacuo. Yield: 12 mg (54% of theory).
  • the reaction mixture was stirred at 95°C for 3 h in a closed microwave vial, diluted with water and aqueous hydrochloric acid solution (1 N, 300 ⁇ L).
  • the suspension was dissolved in a BHC233018 - FC - 125 / 152 - mixture of acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 9 mg (22% of theory).
  • reaction mixture was stirred at 95°C overnight in a closed microwave vial, mixed with additional 2'-amino- 6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (8 mg, 76% purity, 0.02 mmol, 1.0 eq.), stirred at 95°C for another 4 h and diluted with water and aqueous hydrochloric acid solution (1 N, 69 ⁇ L).
  • the suspension was dissolved in a mixture of acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 3 mg (90% purity, 32% of theory).
  • the reaction mixture was stirred at 95°C overnight in a closed microwave vial, diluted with water and quenched with aqueous hydrochloric acid solution (1 N, 1.2 mL).
  • the suspension was dissolved in acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient), followed by a second RP-HPLC (acetonitrile / 1% ammonia in water gradient) to separate both regioisomers. Yield: 9 mg (6% of theory).
  • BHC233018 - FC - 127 / 152 - BIOLOGICAL ASSAYS Examples were tested in selected biological assays one or more times. When tested more than once, data are reported as either average values or as median values, wherein • the average value, also referred to as the arithmetic mean value, represents the sum of the values obtained divided by the number of times tested, and • the median value represents the middle number of the group of values when ranked in ascending or descending order. If the number of values in the data set is odd, the median is the middle value. If the number of values in the data set is even, the median is the arithmetic mean of the two middle values. Examples were synthesized one or more times.
  • test compound dilutions Preparation of test compound dilutions.
  • a 100-fold concentrated solution of the test compound (50 nL) in DMSO was transferred to microtiter test plates (384 or 1,536 wells, Greiner Bio-One, Germany) using either a Hummingbird liquid handler (Digilab, MA, USA) or an Echo acoustic system (Labcyte, CA, USA). Plates were sealed with adhesive foil or heat-sealed and stored at –20 C until use.
  • Detection of successful loading was achieved by measuring resonance energy transfer from anti-GST-terbium (FRET donor) bound to GST-KRAS G12D to the loaded fluorescent GTP analogue (FRET acceptor).
  • the fluorescent GTP analogue EDA–GTP–DY- 647P1 [2'/3'-O-(2-aminoethyl-carbamoyl)guanosine-5'-triphosphate labelled with DY-647P1 (Dyomics BHC233018 - FC - 128 / 152 - GmbH, Germany)] was synthesized by Jena Bioscience (Germany) and supplied as a 1 mM aqueous solution.
  • a KRAS G12D working solution was prepared in assay buffer [10 mM HEPES pH 7.4 (AppliChem), 150 mM NaCl (Sigma), 5 mM MgCl2 (Sigma), 1 mM DTT (Thermo Fisher), 0.05% BSA Fraction V pH 7.0 (ICN Biomedicals), 0.0025% (v/v) Igepal (Sigma)] containing GST-KRAS G12D (final concentration in assay 2 nM) and anti-GST-terbium (Cisbio, France) (final concentration in assay 1 nM).
  • a SOS1 cat working solution was prepared in assay buffer containing SOS1 cat (final concentration 5 nM) and EDA–GTP–DY-647P1 (final concentration 100 nM).
  • An inhibitor control solution was prepared in assay buffer containing EDA–GTP–DY-647P1 (final concentration 100 nM) without SOS1 cat but with addition of GDP (final concentration 20 ⁇ M, Jena Bioscience, prepared from 100 mM stock solution). All steps of the assay were performed at 20 °C.
  • a volume of 3 ⁇ L of the KRAS G12D working solution was added to all wells of the test plate using a Multidrop dispenser (Thermo LabSystems).
  • the fluorescent GTP analogue EDA–GTP–DY- 647P1 [2'/3'-O-(2-aminoethyl-carbamoyl)guanosine-5'-triphosphate labelled with DY-647P1 (Dyomics GmbH, Germany)] was synthesized by Jena Bioscience (Germany) and supplied as a 1 mM aqueous solution.
  • a KRAS WT working solution was prepared in assay buffer [10 mM HEPES pH 7.4 (AppliChem), 150 mM NaCl (Sigma), 5 mM MgCl 2 (Sigma), 1 mM DTT (Thermo Fisher), 0.05% BSA Fraction V pH 7.0 (ICN Biomedicals), 0.0025% (v/v) Igepal (Sigma)] containing GST-KRAS WT (final concentration in assay 2 nM) and anti-GST-terbium (Cisbio, France) (final concentration in assay 1 nM).
  • a SOS1 cat working solution was prepared in assay buffer containing SOS1 cat (final concentration 5 nM) and EDA– GTP–DY-647P1 (final concentration 100 nM).
  • An inhibitor control solution was prepared in assay buffer containing EDA–GTP–DY-647P1 (final concentration 100 nM) without SOS1 cat but with addition of GDP (final concentration 20 ⁇ M, Jena Bioscience, prepared from 100 mM stock solution). All steps of the assay were performed at 20 °C.
  • a volume of 3 ⁇ L of the KRAS WT working solution was added to all wells of the test plate using a Multidrop dispenser (Thermo LabSystems).
  • the fluorescent GTP analogue EDA–GTP–DY-647P1 [2'/3'-O-(2-aminoethyl-carbamoyl)guanosine-5'-triphosphate labelled BHC233018 - FC - 129 / 152 - with DY-647P1 (Dyomics GmbH, Germany)] was synthesized by Jena Bioscience (Germany) and supplied as a 1 mM aqueous solution.
  • a HRAS WT working solution was prepared in assay buffer [10 mM HEPES pH 7.4 (AppliChem), 150 mM NaCl (Sigma), 5 mM MgCl2 (Sigma), 1 mM DTT (Thermo Fisher), 0.05% BSA Fraction V pH 7.0 (ICN Biomedicals), 0.0025% (v/v) Igepal (Sigma)] containing GST- HRAS WT (final concentration in assay 2 nM) and anti-GST-terbium (Cisbio, France) (final concentration in assay 1 nM).
  • a SOS1 cat working solution was prepared in assay buffer containing SOS1 cat (final concentration 1 nM) and EDA–GTP–DY-647P1 (final concentration 100 nM).
  • An inhibitor control solution was prepared in assay buffer containing EDA–GTP–DY-647P1 (final concentration 100 nM) without SOS1 cat but with addition of GDP (final concentration 20 ⁇ M, Jena Bioscience, prepared from 100 mM stock solution). All steps of the assay were performed at 20 °C.
  • a volume of 3 ⁇ L of the HRAS WT working solution was added to all wells of the test plate using a Multidrop dispenser (Thermo LabSystems).
  • NRAS WT activation by SOS1 cat assay (“On-assay”). This assay quantifies SOS1 cat mediated loading of NRAS WT –GDP with a fluorescent GTP analogue. Detection of successful loading was achieved by measuring resonance energy transfer from anti-GST-terbium (FRET donor) bound to GST-NRAS WT (Reaction Biology) to the loaded fluorescent GTP analogue (FRET acceptor).
  • the fluorescent GTP analogue EDA–GTP–DY-647P1 [2'/3'-O-(2-aminoethyl-carbamoyl)guanosine-5'-triphosphate labelled with DY-647P1 (Dyomics GmbH, Germany)] was synthesized by Jena Bioscience (Germany) and supplied as a 1 mM aqueous solution.
  • a NRAS WT working solution was prepared in assay buffer [10 mM HEPES pH 7.4 (AppliChem), 150 mM NaCl (Sigma), 5 mM MgCl 2 (Sigma), 1 mM DTT (Thermo Fisher), 0.05% BSA Fraction V pH 7.0 (ICN Biomedicals), 0.0025% (v/v) Igepal (Sigma)] containing GST- NRAS WT (final concentration in assay 2 nM) and anti-GST-terbium (Cisbio, France) (final concentration in assay 1 nM).
  • a SOS1 cat working solution was prepared in assay buffer containing SOS1 cat (final concentration 1 nM) and EDA–GTP–DY-647P1 (final concentration 100 nM).
  • An inhibitor control solution was prepared in assay buffer containing EDA–GTP–DY-647P1 (final concentration 100 nM) without SOS1 cat but with addition of GDP (final concentration 20 ⁇ M, Jena Bioscience, prepared from 100 mM stock solution). All steps of the assay were performed at 20 °C.
  • a volume of 3 ⁇ L of the NRAS WT working solution was added to all wells of the test plate using a Multidrop dispenser (Thermo LabSystems).
  • KRAS G12V activation by SOS1 cat assay (“On-assay”). This assay quantifies SOS1 cat mediated loading of KRAS G12V –GDP with a fluorescent GTP analogue. Detection of successful loading was achieved by measuring resonance energy transfer from anti-GST-terbium (FRET donor) bound to GST-KRAS G12V (Reaction Biology) to the loaded fluorescent GTP analogue (FRET acceptor).
  • the fluorescent GTP analogue EDA–GTP–DY-647P1 [2'/3'-O-(2-aminoethyl-carbamoyl)guanosine-5'-triphosphate labelled BHC233018 - FC - 130 / 152 - with DY-647P1 (Dyomics GmbH, Germany)] was synthesized by Jena Bioscience (Germany) and supplied as a 1 mM aqueous solution.
  • a KRAS G12V working solution was prepared in assay buffer [10 mM HEPES pH 7.4 (AppliChem), 150 mM NaCl (Sigma), 5 mM MgCl2 (Sigma), 1 mM DTT (Thermo Fisher), 0.05% BSA Fraction V pH 7.0 (ICN Biomedicals), 0.0025% (v/v) Igepal (Sigma)] containing GST-KRAS G12V (final concentration in assay 2 nM) and anti-GST-terbium (Cisbio, France) (final concentration in assay 1 nM).
  • a SOS1 cat working solution was prepared in assay buffer containing SOS1 cat (final concentration 20 nM) and EDA–GTP–DY-647P1 (final concentration 100 nM).
  • An inhibitor control solution was prepared in assay buffer containing EDA–GTP–DY-647P1 (final concentration 100 nM) without SOS1 cat but with addition of GDP (final concentration 20 ⁇ M, Jena Bioscience, prepared from 100 mM stock solution). All steps of the assay were performed at 20 °C.
  • a volume of 3 ⁇ L of the KRAS G12V working solution was added to all wells of the test plate using a Multidrop dispenser (Thermo LabSystems).
  • KRAS G12C activation by SOS1 cat assay (“OFF-assay”). This assay quantifies SOS1 cat mediated deloading of KRAS G12C pre-loaded with a fluorescent GDP analogue at excess GTP. Detection of successful deloading was achieved by measuring resonance energy transfer from anti-GST-terbium (FRET donor) bound to GST-KRAS G12C to the loaded fluorescent GDP analogue (FRET acceptor).
  • the fluorescent GDP analogue EDA–GDP–DY-647P1 [2'/3'-O-(2-aminoethyl-carbamoyl)guanosine-5'- diphosphate labelled with DY-647P1 (Dyomics GmbH, Germany)] was synthesized by Jena Bioscience (Germany).
  • a KRAS G12C working solution was prepared in assay buffer [10 mM HEPES pH 7.4 (AppliChem), 150 mM NaCl (Sigma), 5 mM MgCl 2 (Sigma), 1 mM DTT (Thermo Fisher), 0.05% BSA Fraction V pH 7.0 (ICN Biomedicals), 0.0025% (v/v) Igepal (Sigma)] containing GST-KRAS G12C preloaded with EDA–GDP–DY-647P1 (final concentration in assay 5 nM) and anti-GST-terbium (Cisbio, France) (final concentration in assay 1 nM).
  • a SOS1 cat working solution was prepared in assay buffer containing SOS1 cat (final concentration 1 nM) and GTP (final concentration 50 ⁇ M).
  • An inhibitor control solution was prepared in assay buffer containing SOS1 cat (final concentration 1 nM). All steps of the assay were performed at 20 ⁇ °C. A volume of 3 ⁇ L of the KRAS G12C working solution was added to all wells of the test plate using a Multidrop dispenser (Thermo LabSystems). After 15 min, 2 ⁇ L of the SOS1 cat working solution was added to all wells, except for the inhibitor control solution wells. After 30 min incubation, HTRF was measured. Table 2. Biochemical KRAS/SOS1 activation assay results.
  • Biochemical KRAS/SOS1 activation assay results KRAS WT GDP NRAS WT GDP HRAS WT GDP
  • SOS1 on- activation by SOS1 (on- activation by SOS1 (on- No assay) assay) assay
  • IC50 [mol/L] IC50 [mol/L] 01-01 6.12 E-7 > 2.00 E-5 > 2.00 E-5 01-02 7.56 E-7 > 2.00 E-5 > 2.00 E-5 01-03 1.53 E-5 > 2.00 E-5 > 2.00 E-5 02-01 1.45 E-7 > 2.00 E-5 > 2.00 E-5 1.77 E-5 02-02 5.78 E-6 > 2.00 E-5 > 2.00 E-5 02-03 1.01 E-7 > 2.00 E-5 > 2.00 E-5 02-04 4.46 E-6 > 2.00 E-5 > 2.00 E-5 3.97 E-6 4.89 E-6 02-05 2.13 E-6 > 2.00 E-5 > 2.00 E-5 02-06 8.40 E-6 > 2.00 E-5 > 2.00 E-5 > 2.00 E-5 > 2.00 E
  • the at NUVISAN ICB GmbH produced biotinylated recombinant KRAS variants (WT, G12D, G12V, G12C constructs, for details see section “KRAS Protein Production for SPR”) or purchased biotinylated recombinant KRAS G12V (MSC-11-536, kRas_P_2-169_G12V), NRAS (MSC-11-543, nRas_P_WT_2- 169) and HRAS (MSC-11-547, hRas_P_WT_2-169) from Reaction Biology were used for immobilization at a concentration of 5 ⁇ g/mL or 50 ⁇ g/mL on SA-Chip (Cytiva Europe GmbH) in 10 mM Hepes pH7.5, 150 mM NaCl, 5 mM MgCl 2 , 0.05% BSA, 1 mM DTT, 0.0025% Igepal (NP40) with a flow rate of 5 ⁇ L
  • KD titrations were performed in multi- cycle mode using running buffer of 10 mM Hepes pH7.5, 150 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 0.05% BSA, 0.0025% Igepal (NP40), 2% DMSO in a flow rate of 30 ⁇ L/min with a contact time of 90s and a dissociation time of 150s.
  • KD titrations were performed in BHC233018 - FC - 133 / 152 - single-cycle mode in the same running buffer in a flow rate of 100 ⁇ L/min with a contact time of 60s and dissociation time of 3500s.
  • KRAS WT Aa 1-169; C118S
  • KRAS G12D Aa 1-169; G12D; C118S
  • KRAS G12C Aa 1-169; G12C; C118S
  • KRAS G12V Aa 1-169; G12V; C118S
  • KRAS WT Aa 1-169; C118S
  • KRAS G12D Aa 1-169; G12D; C118S
  • KRAS G12C Aa 1-169; G12C; C118S
  • KRAS G12V Aa 1-169; G12V; C118S
  • This vectors were co-transfected with pBirAcm and expressed into E. coli BL21(DE3) using LB 184 medium or Terrific Broth media in the presence of 200 ⁇ g/mL Ampicillin and 34 ⁇ g/mL Chloramphenicol.
  • the BHC233018 - FC - 134 / 152 - cells were grown at 37°C until the OD550 reached 1, at which point 0.1 mM or 0.5 mM IPTG and 50 ⁇ M Biotin were added and the temperature was lowered to 27°C. The cells were harvested after 24 hours.
  • coli cell pellet was resuspended in 3.5 mL buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10 mM Imidazole, 0.5% CHAPS, Complete-EDTAfree protease inhibitor, 2 ⁇ g Benzonase) per gram wet weight and lysed by sonication or microfluidizer.
  • the soluble protein was separated by centrifugation at 24000 xg for an hour at 4°C.
  • the protein was purified via Ni-NTA affinity chromatography using buffer (50 mM Tris HCl pH 7.5, 300 mM NaCl) with 10 mM Imidazole for washing and 300 mM Imidazole for elution.
  • KRAS SOS1 Protein Production for Biochemical activation assays
  • KRAS WT Aa 1-169
  • KRAS G12D Aa 1-169; G12D; C118S
  • KRAS G12V Aa 1-169; G12V
  • hSOS1 human SOS1
  • the cultures were grown in Terrific Broth media with 200 ug/mL ampicillin at a temperature of 37 °C to a density of 0.6 (OD600), shifted to a temperature of 27 °C (for hK-Ras expression vectors) or 17 °C (for hSOS expression vectors), induced for expression with 0.1 mM IPTG and further cultivated for 24 hours.
  • Purification After cultivation the transformed E. coli were harvested by centrifugation and the resulting pellet was suspended in a lysis buffer (see below) and lysed by passing three-times through a high pressure device (Microfluidics). The lysate was centrifuged at 4 °C and the supernatant used for further purification.
  • the Glutathione Agarose 4B lados with protein was transferred to a chromatography column connected to an ⁇ kta chromatography system.
  • the column was washed with wash buffer (50mM Tris HCl 7.5, 500mM NaCl, 1mM DTT) and the bound protein eluted with elution buffer (50mM Tris HCl 7.5, 500mM NaCl, 1mM DTT, 15 or 20 mM Glutathione).
  • wash buffer 50mM Tris HCl 7.5, 500mM NaCl, 1mM DTT, 15 or 20 mM Glutathione.
  • the main fractions of the elution peak (monitored by OD280) were pooled.
  • IMAC immobilized metal ion affinity chromatography
  • the centrifuged lysate was incubated with 30mL Ni-NTA (Macherey-Nagel; #745400.100) in a spinner flask (16 h, 4°C) and subsequently transferred to a chromatography column connected to an ⁇ kta chromatography system.
  • the column was rinsed with wash buffer (25mM Tris HCl 7.5, 500mM NaCl, 20mM Imidazol) and the bound protein eluted with a linear gradient (0-100%) of elution buffer (25mM Tris HCl 7.5, 500mM NaCl, 300mM Imidazol).
  • the main fractions of the elution peak (monitored by OD280) containing homogenous His10-hSOS were pooled.
  • the final yield of His10-hSOS1 was about 110 mg purified protein per L culture and the final product concentration was about 2 mg/mL.
  • HTRF® Homogenous Time-Resolved Fluorescence phospho-ERK1/2 (THR202/TYR204) assay with AGS, ASPC-1, GP2d, HPAC, LK2, NCI-H441 and PANC10.05 cells 2500 AGS cells were seeded in a HTRF 384-well low volume plate (Greiner bio-one #784080) in DMEM+F12 (GIBCO) complemented with L-Alanyl-L-Glutamine (final: 2 mM, Gibco), FCS Superior (final: 10%, Sigma).
  • 5000 ASPC-1 cells were seeded in a HTRF 384-well low volume plate (Greiner bio-one #784080) in RPMI-1640 (GIBCO) complemented with L-Alanyl-L-Glutamine (final: 2 mM, Gibco), FCS Superior (final: 10%, Sigma).
  • 2500 GP2d cells were seeded in a HTRF 384-well low volume plate (Greiner bio-one #784080) in RPMI- 1640 (GIBCO) complemented with L-Alanyl-L-Glutamine (final: 2 mM, Gibco), FCS Superior (final: 10%, Sigma).
  • BHC233018 - FC - 136 / 152 - 2500 HPAC cells were seeded in a HTRF 384-well low volume plate (Greiner bio-one #784080) in DMEM+F12 including 15 mM HEPES (GIBCO) complemented with L-Alanyl-L-Glutamine (final: 2 mM, Gibco), FCS Superior (final: 5%, Sigma), E2 (final: 10 nM), hEGF (final: 10 ng/ml, Invitrogen), Hydrocortisone (final: 40 ng/ml, Sigma), hInsulin (final: 2 ⁇ g/ml, Sigma), Sodium pyruvate (final: 0.5 mM, Gibco), Transferrin (final: 5 ⁇ g/ml, Sigma).
  • LK2 cells were seeded in a HTRF 384-well low volume plate (Greiner bio-one #784080) in RPMI- 1640 (GIBCO) complemented with L-Alanyl-L-Glutamine (final: 2 mM, Gibco), FCS Superior (final: 10%, Sigma).
  • 2500 NCI-H441 cells were seeded in a HTRF 384-well low volume plate (Greiner bio-one #784080) in RPMI-1640 (GIBCO) complemented with L-Alanyl-L-Glutamine (final: 2 mM, Gibco), FCS Superior (final: 10%, Sigma).
  • Panc10.05 cells were seeded in a HTRF 384-well low volume plate (Greiner bio-one #784080) in RPMI-1640 (GIBCO) complemented with L-Alanyl-L-Glutamine (final: 2 mM, Gibco), FCS Superior (final: 10%, Sigma), hInsulin (final: 10 units/ml). After 24 h in a standard incubator (37°C, 5% CO2) cells were treated with varying concentrations of the different test compounds dissolved in DMSO (1% final DMSO concentration per well) using a HP- dispenser followed by incubation for 1.5 h (at 37°C 5% CO2).
  • DMSO 1% final DMSO concentration per well
  • NCI-H441 were stimulated for 3 min with hEGF (final concentration 200 ng/ml), while the other cell lines were processed without stimulation.
  • the following steps were performed for all cell lines as described in the supplier's manual (Cisbio one-plate assay protocol; https://www.cisbio.eu/) for the HTRF ADVANCED PHOSPHO- ERK1/2 (THR202/TYR204) DETECTION KITS (#64AERPEH) .
  • the content of pERK/ERK was measured with a PHERAstar (BMG Labtech) using the HTRF module. Calculation of the pERK/ERK Ratio was done as described in the assay protocol.
  • the resulting IC50 values for the test compounds reflect the inhibition of ERK phosphorylation compared to DMSO-treated control cells.
  • the ratio of cells solely treated with 1% DMSO was set as 100% and the ratio of maximal pERK inhibition obtained with the internal control compound (Trametinib) was set as 0%.
  • Table 5 Cellular pERK assay results.
  • TEER transepithelial electrical resistance
  • the efflux ratio (ER) basolateral (B) to apical (A) was calculated by dividing Papp B ⁇ A BHC233018 - FC - 138 / 152 - by Papp A ⁇ B. In addition, the compound recovery was calculated. As assay control, reference compounds were analyzed in parallel. Table 7. Caco permeability and Efflux ratio.
  • hepatocytes from male Wistar rats were isolated via a two-step perfusion method. After perfusion, the liver was carefully removed from the rat, the liver capsule was opened, and the hepatocytes were gently shaken out into a Petri dish with ice-cold Williams’ medium E (WME). The resulting cell suspension was filtered through sterile gauze into 50 mL Falcon tubes and centrifuged at 50 ⁇ g for 3 min at rt. The cell pellet was resuspended in 30 mL of WME and centrifuged through a Percoll gradient two times at 100 ⁇ g. The hepatocytes were washed again and resuspended with WME. Cell viability was determined by trypan blue exclusion.
  • cryopreserved or freshly isolated hepatocytes were distributed in WME to glass vials at a density of 1.0 ⁇ 10e6 vital cells/mL.
  • the test compound was added to a final concentration of 1 ⁇ M.
  • Organic solvent in the incubations was limited to ⁇ 0.01% DMSO and ⁇ 1% acetonitrile.
  • the hepatocyte suspensions were continuously shaken at 580 BHC233018 - FC - 139 / 152 - rpm and aliquots were taken at 2, 8, 16, 30, 45, and 90 min, to which an equal volume of cold acetonitrile was immediately added.
  • liver blood flow 4.2 L/h/kg, specific liver weight: 32 g/kg body weight; liver cells in vivo, 1.1 ⁇ 10e8 cells/g liver; liver cells in vitro, 1.0 ⁇ 10e6/mL.
  • liver blood flow 4.2 L/h/kg, specific liver weight: 32 g/kg body weight; liver cells in vivo, 1.1 ⁇ 10e8 cells/g liver; liver cells in vitro, 1.0 ⁇ 10e6/mL.
  • Cryopreserved human hepatocytes were thawed according to the suppliers information.
  • cryopreserved hepatocytes were distributed in Williams’ medium E to glass vials at a density of 1.0 ⁇ 10e6 vital cells/mL. The test compound was added to a final concentration of 1 ⁇ M.
  • Organic solvent in the incubations was limited to ⁇ 0.01% DMSO and ⁇ 1% acetonitrile.
  • the hepatocyte suspensions were continuously shaken at 580 rpm and aliquots were taken at 2, 8, 16, 30, 45, and 90 min, to which an equal volume of cold acetonitrile was immediately added. Samples were frozen at –20 °C overnight, and subsequently centrifuged at 3000 rpm for 15 min. The supernatant was analyzed with an Agilent 1200 HPLC system with MS/MS detection. The half-life of a test compound was determined from the concentration–time plot.
  • liver blood flow 1.32 L/h/kg, specific liver weight: 21 g/kg body weight; liver cells in vivo, 1.1 ⁇ 10e8 cells/g liver; liver cells in vitro, 1.0 ⁇ 10e6/mL.
  • test compounds were determined by incubation at 1 ⁇ M in a suspension of liver microsomes in 100 mM phosphate buffer pH 7.4 (NaH2PO4 ⁇ H2O + Na2HPO4 ⁇ 2H2O) and at a protein concentration of 0.5 mg/mL at 37 °C.
  • the microsomes were activated by adding a cofactor mix containing 8 mM glucose-6-phosphate, 0.5 mM NADP, and 1 IU/mL glucose-6-phos ⁇ phate dehydro- genase in phosphate buffer pH 7.4.
  • the metabolic assay was started shortly afterwards by adding the test com-pound to the incubation at a final volume of 1 mL. During incubation, the microsomal suspensions were continuously shaken at 580 rpm and aliquots were taken at 2, 8, 16, 30, 45, and 60 min. Further handling and analysis as per the hepatocyte method described above. BHC233018 - FC - 140 / 152 - Table 8.
  • Metabolic stability data Example Rat Hepatocytes Human Liver Microsomes No Fmax [%] Fmax [%] 01-01 99 59 01-02 96 71 01-03 85 44 02-01 71 31 02-02 84 44 02-03 91 41 02-04 31 6 02-05 75 16 02-06 10 02-07 100 75 02-08 73 14 02-09 60 11 02-11 53 18 03-01 94 35 03-02 99 60 03-03 29 03-04 86 03-07 46 17 03-08 30 7 03-09 97 76 03-10 79 10 04-02 85 45 04-03 67 22 05-01 84 30 05-02 33 05-03 44 8 05-04 73 7 05-05 41 4 06-01 84 31 07-01 37 14 07-02 85 In Vivo Pharmacokinetics in Rats All animal experiments were conducted in accordance with the German Animal Welfare Law and were approved by local authorities.
  • test compounds were administered to male Wistar rats intravenously at doses of 0.1 to 0.5 mg/kg as solutions potentially using solubilizers such as PEG 400 in well-tolerated amounts. Concerning iv administration, test compounds were given as bolus injection. Blood samples were taken at various time points after dosing, including 2 min and 30 min timepoints. Blood was collected into K3-EDTA tubes and centrifuged at 1811 g for min. 5 min or at 14000 g for 1.5 min. An aliquot of 62.5 ⁇ L from the supernatant (plasma) was taken and BHC233018 - FC - 141 / 152 - precipitated by the addition of 250 ⁇ L of MeOH.
  • iv rat PK Plasma levels after 2 min and 30 min and calculation of the recovery of the parent compound concentration after 30 min in relation to 2 min.

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Abstract

The present invention covers spirocyclic compounds of general formula (I), in which R1, R2, X, Y, and Z are as defined herein, methods of preparing said compounds, intermediate compounds useful for preparing said compounds, pharmaceutical compositions and combinations comprising said compounds and the use of said compounds for manufacturing pharmaceutical compositions for the treatment or prophylaxis of diseases, in particular of neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling, as a sole agent or in combination with other active ingredients.

Description

BHC233018 - FC - 1 / 152 - SPIROCYCLIC COMPOUNDS FOR THE TREATMENT OF CANCER The present invention covers spirocyclic compounds of general formula (I) as described and defined herein, methods of preparing said compounds, intermediate compounds useful for preparing said compounds, pharmaceutical compositions and combinations comprising said compounds, and the use of said compounds for manufacturing pharmaceutical compositions for the treatment or prophylaxis of diseases, in particular for neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling, as a sole agent or in combination with other active ingredients. BACKGROUND Mutant KRAS is a well-understood oncogenic driver and has a wide-spread prevalence in various human cancer indications (Bos, 1989). In 1982, mutationally activated RAS genes were detected in human cancer, marking the first discovery of mutated genes in this disease (Cox, 2010). The frequent mutation of RAS in three of the four most lethal cancers (lung, colon and pancreatic cancers) in the United States has spurred intense interest and effort in developing RAS inhibitors (Cox, 2014). Overall, RAS mutations have been detected in 9–30% of all tumor samples sequenced. In pancreatic ductal adenocarcinoma (PDAC; ~90% of all pancreatic cancers) and lung adenocarcinoma (LAC; 30–35% of all lung cancers) KRAS mutations display a frequency of 97% and 32% respectively. Other indications with frequently mutated KRAS include colorectal carcinoma (CRC) (52%), and multiple myeloma (43%) (Cox, 2014). RAS proteins act as molecular switches that cycle between an active, GTPbound state and an inactive, GDP-bound state. Activated by guanine nucleotide exchange factors (GEFs), RAS in its GTPbound state interacts with a number of effectors (Hillig, 2019). Return to the inactive state is driven by GTPase- activating proteins (GAPs), which down-regulate active RAS by accelerating the weak intrinsic GTPase activity by up to 5 orders of magnitude. For oncogenic RAS mutants, however, the GAP activity is impaired or greatly reduced, resulting in permanent activation, which is the basis of oncogenic RAS signaling (Haigis, 2017); for example, through the RAS-RAF-MEK-ERK and RAS-PI3K-PDK1-AKT pathways, both essential to cell survival and proliferation (Downward 2003). For decades, mutant KRAS has been considered “undruggable” with classical pharmacological small molecule inhibitors. However, KRASG12C was recently identified to be potentially druggable by allele- specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP) (Oestrem, 2013; Janes, 2018). Covalent KRASG12C inhibitors as described by Shokat et al. (Ostrem JM, Shokat KM (2016) Direct small-molecule inhibitors of KRAS: From structural insights to mechanism-based design. Nat Rev Drug Discov 15:771–785.) occupy the so-called switch-II pocket and bind with their Michael acceptor system covalently to the cysteine mutation at G12 in this specific KRAS mutant. Occupation of this pocket with the covalent inhibitor results in a locked inactive GDP-bound protein conformation. Captured in this BHC233018 - FC - 2 / 152 - conformation, cycling of the mutated protein into the active GTP-bound state is prevented and thereby activity of the mutant KRASG12C is shut down. For decades, mutant KRAS has been considered “undruggable” via classical pharmacological small molecule inhibitors. However, in 2013 the G12C mutant of KRAS was found to be potentially druggable by covalent targeting of Cys-12 in vicinity to an inducible so-called “switch II pocket” (S-IIP) of KRAS G12C (Oestrem, 2013; Janes, 2018). There have since been significant efforts by the pharmaceutical industry to develop KRas inhibitors targeting the SII-P for cancer therapy and several agents have entered clinical trials. However, no such therapies have yet won regulatory approval (McCormick, 2015). Besides G12C, other oncogenic mutants of KRAS include G12D, G12V and G12R, all of which represent attractive drug targets, with the G12D mutation being most prevalent across tumor types (Kashofer, 2020). There is therefore a clear and continued desire for therapies targeting KRas mutants and especially G12D for the treatment of cancer. STATE OF THE ART Covalent inhibitors of KRAS G12C have been described in literatures and patent applications. Biaryl derivatives were mentioned as KRAS G12C covalent inhibitors (WO2014152588, WO2016049524 and WO 2016044772). WO2016164675, WO2015054572, WO2016044772, WO2016049568, WO2016168540, WO20170070256, WO2017087528, WO2017100546, WO2017172979, WO2018064510, WO2018145012, WO2018145014 disclosed quinazoline, quinoline, dihydrobenzo- naphthyridinone, quinazolinone, dihydropyrimidoquinolinone, isoquinoline derivatives. Further disclosures include anilinoacetamide and biaryl derivatives (WO2016049565, WO 2017058768, WO 2017058792), naphthalene or hexahydrofurofurane derivatives (WO 2014143659), quinazolinone (WO2017015562), phenylpyrazine derivatives (WO 2017058728). bezoimidazolsulfone, dihydroquinoxaline or dihydroquinoxalinone (WO 2017058805), phenylpiperazine-1-carbohydrazide (WO 2017058807), tetrahydronaphthyridine (WO 2017058902), imidazolopyridine (WO 2017058915), various chemical entities (WO2018068017), bicyclic 6,5-aryl, hetaryl rings containing compounds (WO2018140600). Benzimidazol, (aza)indole, imidazopyridine derivatives were disclosed as KRAS covalent inhibitors in WO2018145013, benzothiazole, benzothiophene, benzisoxazole derivatives in WO2018140599, pyridopyrimidone, benzothiazole in WO2018119183 and tetrahydropyridopyrimidine in WO2017201161. Compounds of the following general formula BHC233018 - FC - 3 / 152 -
Figure imgf000004_0001
are described in US 2018/0201610 (NantBio) which selectively inhibit mutant K-Ras, especially G12V and/or G12D over wild type K-Ras or other mutant K-Ras forms. Substituted quinazoline compounds of the following general formula
Figure imgf000004_0002
R2a are described as inhibitors of Ras-protein in WO 2017/172979 (Araxes). Compounds of general formula
Figure imgf000004_0003
are described as inhibitors of KRAS G12D in WO 2021/041671 (Mirati). Compounds of general formula
Figure imgf000004_0004
are described as to inhibit mutant KRAS in CN 112047948 (Xuanzhu). Reversible, non-covalent inhibitors of KRAS G12D have been described in patent applications (WO2021041671 and WO2017172979A1). However, so far compounds of general formula (I) have not been disclosed as reversible, non-covalent KRAS G12D inhibitors. BHC233018 - FC - 4 / 152 - Further related KRAS inhibitors are described in: Nature, volume 619, pages 160–166 (2023) WO2023/099624, WO2023/099623, WO2023/099608, WO2023/099620, WO2023/099612, WO2023/099592, WO 2023/018809, and WO2023/001141. It has now been found, and this constitutes the basis of the present invention, that the compounds of the present invention have surprising and advantageous properties. In particular, the compounds of the present invention have surprisingly been found to effectively inhibit KRAS, especially KRAS G12D, and may therefore be used for the treatment or prophylaxis of neoplastic disorders, repectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling, for example. DESCRIPTION of the INVENTION In accordance with a first aspect, the present invention covers compounds of general formula (I):
Figure imgf000005_0001
in which X represents CH, C-F, or N; Y represents CH, C-F, C-Cl, C-CN, C-CH3, or N; Z represents -CH2-, or -CH2-CH2-; R1 a selected from the
Figure imgf000005_0002
i i i i i , , , , , , BHC233018 - FC - 5 / 152 -
Figure imgf000006_0001
, , , , or ; wherein * indicates the point of attachment of said group with the rest of the molecule; R2 a selected from the
Figure imgf000006_0002
, , , , , , BHC233018 - FC - 6 / 152 -
Figure imgf000007_0001
H N , , , , or , wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. DEFINITIONS The term “substituted” means that one or more hydrogen atoms on the designated atom or group are replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded. Combinations of substituents and/or variables are permissible. The term “optionally substituted” means that the number of substituents can be equal to or different from zero. Unless otherwise indicated, it is possible that optionally substituted groups are substituted with as many optional substituents as can be accommodated by replacing a hydrogen atom with a non-hydrogen substituent on any available carbon or nitrogen or atom. Commonly, it is possible for the number of optional substituents, when present, to be 1, 2, 3, 4 or 5, in particular 1, 2 or 3. As used herein, the term “one or more”, e.g. in the definition of the substituents of the compounds of general formula (I) of the present invention, means “1, 2, 3, 4 or 5, particularly 1, 2, 3 or 4, more particularly 1, 2 or 3, even more particularly 1 or 2”. When groups in the compounds according to the invention are substituted, it is possible for said groups to be mono-substituted or poly-substituted with substituent(s), unless otherwise specified. Within the scope of the present invention, the meanings of all groups which occur repeatedly are independent from one another. It is possible that groups in the compounds according to the invention are substituted with one, two or three identical or different substituents, particularly with one substituent. As used herein, an oxo substituent represents an oxygen atom, which is bound to a carbon atom or to a sulfur atom via a double bond. The term “comprising” when used in the specification includes “consisting of”. If within the present text any item is referred to as “as mentioned herein”, it means that it may be mentioned anywhere in the present text. BHC233018 - FC - 7 / 152 - The terms as mentioned in the present text have the following meanings: The term “C1-C3-alkyl” means a linear or branched, saturated, monovalent hydrocarbon group having 1, 2, or 3 carbon atoms, e.g. a methyl, ethyl, propyl, or isopropyl group. As used herein, the term “leaving group” means an atom or a group of atoms that is displaced in a chemical reaction as stable species taking with it the bonding electrons. In particular, such a leaving group is selected from the group comprising: halide, in particular fluoride, chloride, bromide or iodide, (methylsulfonyl)oxy, [(trifluoromethyl)sulfonyl]oxy, [(nonafluorobutyl)sulfonyl]oxy, (phenylsulfonyl)oxy, [(4-methylphenyl)sulfonyl]oxy, [(4-bromophenyl)sulfonyl]oxy, [(4-nitrophenyl)sulfonyl]oxy, [(2-nitrophenyl)sulfonyl]oxy, [(4-isopropylphenyl)sulfonyl]oxy, [(2,4,6-triisopropylphenyl)sulfonyl]oxy, [(2,4,6-trimethylphenyl)sulfonyl]oxy, [(4-tert-butyl- phenyl)sulfonyl]oxy and [(4-methoxyphenyl)sulfonyl]oxy. It is possible for the compounds of general formula (I) to exist as isotopic variants. The invention therefore includes one or more isotopic variant(s) of the compounds of general formula (I), particularly deuterium- containing compounds of general formula (I). The term “Isotopic variant” of a compound or a reagent is defined as a compound exhibiting an unnatural proportion of one or more of the isotopes that constitute such a compound. The term “Isotopic variant of the compound of general formula (I)” is defined as a compound of general formula (I) exhibiting an unnatural proportion of one or more of the isotopes that constitute such a compound. The expression “unnatural proportion” means a proportion of such isotope which is higher than its natural abundance. The natural abundances of isotopes to be applied in this context are described in “Isotopic Compositions of the Elements 1997”, Pure Appl. Chem., 70(1), 217-235, 1998. Examples of such isotopes include stable and radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, such as 2H (deuterium), 3H (tritium), 11C, 13C, 14C, 15N, 17O, 18O, 32P, 33P, 33S, 34S, 35S, 36S, 18F, 36Cl, 82Br, 123I, 124I, 125I, 129I and 131I, respectively. With respect to the treatment and/or prophylaxis of the disorders specified herein the isotopic variant(s) of the compounds of general formula (I) preferably contain deuterium (“deuterium-containing compounds of general formula (I)”). Isotopic variants of the compounds of general formula (I) in which one or more radioactive isotopes, such as 3H or 14C, are incorporated are useful e.g. in drug and/or substrate tissue distribution studies. These isotopes are particularly preferred for the ease of their incorporation and detectability. Positron emitting isotopes such as 18F or 11C may be incorporated into a compound of general formula (I). These isotopic variants of the compounds of general formula (I) are useful for in vivo imaging applications. Deuterium-containing and 13C-containing compounds of general formula (I) can be used in mass spectrometry analyses in the context of preclinical or clinical studies. BHC233018 - FC - 8 / 152 - Isotopic variants of the compounds of general formula (I) can generally be prepared by methods known to a person skilled in the art, such as those described in the schemes and/or examples herein, by substituting a reagent for an isotopic variant of said reagent, preferably for a deuterium-containing reagent. Depending on the desired sites of deuteration, in some cases deuterium from D2O can be incorporated either directly into the compounds or into reagents that are useful for synthesizing such compounds. Deuterium gas is also a useful reagent for incorporating deuterium into molecules. Catalytic deuteration of olefinic bonds and acetylenic bonds is a rapid route for incorporation of deuterium. Metal catalysts (i.e. Pd, Pt, and Rh) in the presence of deuterium gas can be used to directly exchange deuterium for hydrogen in functional groups containing hydrocarbons. A variety of deuterated reagents and synthetic building blocks are commercially available from companies such as for example C/D/N Isotopes, Quebec, Canada; Cambridge Isotope Laboratories Inc., Andover, MA, USA; and CombiPhos Catalysts, Inc., Princeton, NJ, USA. The term “deuterium-containing compound of general formula (I)” is defined as a compound of general formula (I), in which one or more hydrogen atom(s) is/are replaced by one or more deuterium atom(s) and in which the abundance of deuterium at each deuterated position of the compound of general formula (I) is higher than the natural abundance of deuterium, which is about 0.015%. Particularly, in a deuterium- containing compound of general formula (I) the abundance of deuterium at each deuterated position of the compound of general formula (I) is higher than 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, preferably higher than 90%, 95%, 96% or 97%, even more preferably higher than 98% or 99% at said position(s). It is understood that the abundance of deuterium at each deuterated position is independent of the abundance of deuterium at other deuterated position(s). The selective incorporation of one or more deuterium atom(s) into a compound of general formula (I) may alter the physicochemical properties (such as for example acidity [C. L. Perrin, et al., J. Am. Chem. Soc., 2007, 129, 4490], basicity [C. L. Perrin et al., J. Am. Chem. Soc., 2005, 127, 9641], lipophilicity [B. Testa et al., Int. J. Pharm., 1984, 19(3), 271]) and/or the metabolic profile of the molecule and may result in changes in the ratio of parent compound to metabolites or in the amounts of metabolites formed. Such changes may result in certain therapeutic advantages and hence may be preferred in some circumstances. Reduced rates of metabolism and metabolic switching, where the ratio of metabolites is changed, have been reported (A. E. Mutlib et al., Toxicol. Appl. Pharmacol., 2000, 169, 102). These changes in the exposure to parent drug and metabolites can have important consequences with respect to the pharmacodynamics, tolerability and efficacy of a deuterium-containing compound of general formula (I). In some cases deuterium substitution reduces or eliminates the formation of an undesired or toxic metabolite and enhances the formation of a desired metabolite (e.g. Nevirapine: A. M. Sharma et al., Chem. Res. Toxicol., 2013, 26, 410; Efavirenz: A. E. Mutlib et al., Toxicol. Appl. Pharmacol., 2000, 169, 102). In other cases the major effect of deuteration is to reduce the rate of systemic clearance. As a result, BHC233018 - FC - 9 / 152 - the biological half-life of the compound is increased. The potential clinical benefits would include the ability to maintain similar systemic exposure with decreased peak levels and increased trough levels. This could result in lower side effects and enhanced efficacy, depending on the particular compound’s pharmacokinetic/ pharmacodynamic relationship. ML-337 (C. J. Wenthur et al., J. Med. Chem., 2013, 56, 5208) and Odanacatib (K. Kassahun et al., WO2012/112363) are examples for this deuterium effect. Still other cases have been reported in which reduced rates of metabolism result in an increase in exposure of the drug without changing the rate of systemic clearance (e.g. Rofecoxib: F. Schneider et al., Arzneim. Forsch. / Drug. Res., 2006, 56, 295; Telaprevir: F. Maltais et al., J. Med. Chem., 2009, 52, 7993). Deuterated drugs showing this effect may have reduced dosing requirements (e.g. lower number of doses or lower dosage to achieve the desired effect) and/or may produce lower metabolite loads. A compound of general formula (I) may have multiple potential sites of attack for metabolism. To optimize the above-described effects on physicochemical properties and metabolic profile, deuterium- containing compounds of general formula (I) having a certain pattern of one or more deuterium-hydrogen exchange(s) can be selected. Particularly, the deuterium atom(s) of deuterium-containing compound(s) of general formula (I) is/are attached to a carbon atom and/or is/are located at those positions of the compound of general formula (I), which are sites of attack for metabolizing enzymes such as e.g. cytochrome P450. Where the plural form of the word compounds, salts, polymorphs, hydrates, solvates and the like, is used herein, this is taken to mean also a single compound, salt, polymorph, isomer, hydrate, solvate or the like. By "stable compound' or "stable structure" is meant a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. The compounds of the present invention optionally contain one or more asymmetric centres, depending upon the location and nature of the various substituents desired. It is possible that one or more asymmetric carbon atoms are present in the (R) or (S) configuration, which can result in racemic mixtures in the case of a single asymmetric centre, and in diastereomeric mixtures in the case of multiple asymmetric centres. In certain instances, it is possible that asymmetry also be present due to restricted rotation about a given bond, for example, the central bond adjoining two substituted aromatic rings of the specified compounds. Preferred compounds are those which produce the more desirable biological activity. Separated, pure or partially purified isomers and stereoisomers or racemic or diastereomeric mixtures of the compounds of the present invention are also included within the scope of the present invention. The purification and the separation of such materials can be accomplished by standard techniques known in the art. Preferred isomers are those which produce the more desirable biological activity. These separated, pure or partially purified isomers or racemic mixtures of the compounds of this invention are also included BHC233018 - FC - 10 / 152 - within the scope of the present invention. The purification and the separation of such materials can be accomplished by standard techniques known in the art. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example, by the formation of diastereoisomeric salts using an optically active acid or base or formation of covalent diastereomers. Examples of appropriate acids are tartaric, diacetyltartaric, ditoluoyltartaric and camphorsulfonic acid. Mixtures of diastereoisomers can be separated into their individual diastereomers on the basis of their physical and/or chemical differences by methods known in the art, for example, by chromatography or fractional crystallisation. The optically active bases or acids are then liberated from the separated diastereomeric salts. A different process for separation of optical isomers involves the use of chiral chromatography (e.g., HPLC columns using a chiral phase), with or without conventional derivatisation, optimally chosen to maximise the separation of the enantiomers. Suitable HPLC columns using a chiral phase are commercially available, such as those manufactured by Daicel, e.g., Chiracel OD and Chiracel OJ, for example, among many others, which are all routinely selectable. Enzymatic separations, with or without derivatisation, are also useful. The optically active compounds of the present invention can likewise be obtained by chiral syntheses utilizing optically active starting materials. In order to distinguish different types of isomers from each other reference is made to IUPAC Rules Section E (Pure Appl Chem 45, 11-30, 1976). The present invention includes all possible stereoisomers of the compounds of the present invention as single stereoisomers, or as any mixture of said stereoisomers, e.g. (R)- or (S)- isomers, in any ratio. Isolation of a single stereoisomer, e.g. a single enantiomer or a single diastereomer, of a compound of the present invention is achieved by any suitable state of the art method, such as chromatography, especially chiral chromatography, for example. Further, it is possible for the compounds of the present invention to exist as tautomers. For example, any compound of the present invention which contains an imidazopyridine moiety as a heteroaryl group for example can exist as a 1H tautomer, or a 3H tautomer, or even a mixture in any amount of the two tautomers, namely :
Figure imgf000011_0001
H 1H tautomer 3H tautomer The present invention includes all possible tautomers of the compounds of the present invention as single tautomers, or as any mixture of said tautomers, in any ratio. BHC233018 - FC - 11 / 152 - Further, the compounds of the present invention can exist as N-oxides, which are defined in that at least one nitrogen of the compounds of the present invention is oxidised. The present invention includes all such possible N-oxides. The present invention also covers useful forms of the compounds of the present invention, such as metabolites, hydrates, solvates, prodrugs, salts, in particular pharmaceutically acceptable salts, and/or co- precipitates. The compounds of the present invention can exist as a hydrate, or as a solvate, wherein the compounds of the present invention contain polar solvents, in particular water, methanol or ethanol for example, as structural element of the crystal lattice of the compounds. It is possible for the amount of polar solvents, in particular water, to exist in a stoichiometric or non-stoichiometric ratio. In the case of stoichiometric solvates, e.g. a hydrate, hemi-, (semi-), mono-, sesqui-, di-, tri-, tetra-, penta- etc. solvates or hydrates, respectively, are possible. The present invention includes all such hydrates or solvates. Further, it is possible for the compounds of the present invention to exist in free form, e.g. as a free base, or as a free acid, or as a zwitterion, or to exist in the form of a salt. Said salt may be any salt, either an organic or inorganic addition salt, particularly any pharmaceutically acceptable organic or inorganic addition salt, which is customarily used in pharmacy, or which is used, for example, for isolating or purifying the compounds of the present invention. The term “pharmaceutically acceptable salt" refers to an inorganic or organic acid addition salt of a compound of the present invention. For example, see S. M. Berge, et al. “Pharmaceutical Salts,” J. Pharm. Sci.1977, 66, 1-19. A suitable pharmaceutically acceptable salt of the compounds of the present invention may be, for example, an acid-addition salt of a compound of the present invention bearing a nitrogen atom, in a chain or in a ring, for example, which is sufficiently basic, such as an acid-addition salt with an inorganic acid, or “mineral acid”, such as hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfamic, bisulfuric, phosphoric, or nitric acid, for example, or with an organic acid, such as formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic, 2-(4- hydroxybenzoyl)-benzoic, camphoric, cinnamic, cyclopentanepropionic, digluconic, 3-hydroxy-2- naphthoic, nicotinic, pamoic, pectinic, 3-phenylpropionic, pivalic, 2-hydroxyethanesulfonic, itaconic, trifluoromethanesulfonic, dodecylsulfuric, ethanesulfonic, benzenesulfonic, para-toluenesulfonic, methanesulfonic, 2-naphthalenesulfonic, naphthalinedisulfonic, camphorsulfonic acid, citric, tartaric, stearic, lactic, oxalic, malonic, succinic, malic, adipic, alginic, maleic, fumaric, D-gluconic, mandelic, ascorbic, glucoheptanoic, glycerophosphoric, aspartic, sulfosalicylic, or thiocyanic acid, for example. BHC233018 - FC - 12 / 152 - Further, another suitably pharmaceutically acceptable salt of a compound of the present invention which is sufficiently acidic, is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium, magnesium or strontium salt, or an aluminium or a zinc salt, or an ammonium salt derived from ammonia or from an organic primary, secondary or tertiary amine having 1 to 20 carbon atoms, such as ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, diethylaminoethanol, tris(hydroxymethyl)aminomethane, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, 1,2- ethylenediamine, N-methylpiperidine, N-methyl-glucamine, N,N-dimethyl-glucamine, N-ethyl- glucamine, 1,6-hexanediamine, glucosamine, sarcosine, serinol, 2-amino-1,3-propanediol, 3-amino-1,2- propanediol, 4-amino-1,2,3-butanetriol, or a salt with a quarternary ammonium ion having 1 to 20 carbon atoms, such as tetramethylammonium, tetraethylammonium, tetra(n-propyl)ammonium, tetra(n- butyl)ammonium, N-benzyl-N,N,N-trimethylammonium, choline or benzalkonium. Those skilled in the art will further recognise that it is possible for acid addition salts of the claimed compounds to be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods. Alternatively, alkali and alkaline earth metal salts of acidic compounds of the present invention are prepared by reacting the compounds of the present invention with the appropriate base via a variety of known methods. The present invention includes all possible salts of the compounds of the present invention as single salts, or as any mixture of said salts, in any ratio. In the present text, in particular in the Experimental Section, for the synthesis of intermediates and of examples of the present invention, when a compound is mentioned as a salt form with the corresponding base or acid, the exact stoichiometric composition of said salt form, as obtained by the respective preparation and/or purification process, is, in most cases, unknown. Unless specified otherwise, suffixes to chemical names or structural formulae relating to salts, such as "hydrochloride", "trifluoroacetate", "sodium salt", or "x HCl", "x CF3COOH", "x Na+", for example, mean a salt form, the stoichiometry of which salt form not being specified. This applies analogously to cases in which synthesis intermediates or example compounds or salts thereof have been obtained, by the preparation and/or purification processes described, as solvates, such as hydrates, with (if defined) unknown stoichiometric composition. As used herein, the term “in vivo hydrolysable ester” means an in vivo hydrolysable ester of a compound of the present invention containing a carboxy or hydroxy group, for example, a pharmaceutically acceptable ester which is hydrolysed in the human or animal body to produce the parent acid or alcohol. Suitable pharmaceutically acceptable esters for carboxy include for example alkyl, cycloalkyl and optionally substituted phenylalkyl, in particular benzyl esters, C1-C6 alkoxymethyl esters, e.g. BHC233018 - FC - 13 / 152 - methoxymethyl, C1-C6 alkanoyloxymethyl esters, e.g. pivaloyloxymethyl, phthalidyl esters, C3-C8 cycloalkoxy-carbonyloxy-C1-C6 alkyl esters, e.g. 1-cyclohexylcarbonyloxyethyl ; 1,3-dioxolen-2- onylmethyl esters, e.g. 5-methyl-1,3-dioxolen-2-onylmethyl ; and C1-C6-alkoxycarbonyloxyethyl esters, e.g.1-methoxycarbonyloxyethyl, it being possible for said esters to be formed at any carboxy group in the compounds of the present invention. An in vivo hydrolysable ester of a compound of the present invention containing a hydroxy group includes inorganic esters such as phosphate esters and [alpha]-acyloxyalkyl ethers and related compounds which as a result of the in vivo hydrolysis of the ester breakdown to give the parent hydroxy group. Examples of [alpha]-acyloxyalkyl ethers include acetoxymethoxy and 2,2-dimethylpropionyloxymethoxy. A selection of in vivo hydrolysable ester forming groups for hydroxy include alkanoyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl, alkoxycarbonyl (to give alkyl carbonate esters), dialkylcarbamoyl and N-(dialkylaminoethyl)-N-alkylcarbamoyl (to give carbamates), dialkylaminoacetyl and carboxyacetyl. The present invention covers all such esters. Furthermore, the present invention includes all possible crystalline forms, or polymorphs, of the compounds of the present invention, either as single polymorph, or as a mixture of more than one polymorph, in any ratio. Moreover, the present invention also includes prodrugs of the compounds according to the invention. The term “prodrugs” here designates compounds which themselves can be biologically active or inactive, but are converted (for example metabolically or hydrolytically) into compounds according to the invention during their residence time in the body. In accordance with a second embodiment of the first aspect, the present invention covers compounds of general formula (I), supra, in which: X represents CH, C-F, or N; Y represents CH, C-F, C-CN, C-CH3, or N; Z represents -CH2-, or -CH2-CH2-; R1 represents a group selected from the group:
Figure imgf000014_0001
i i * * * , , , , , , , , BHC233018 - FC - 14 / 152 -
Figure imgf000015_0001
, , or ; wherein * indicates the point of attachment of said group with the rest of the molecule; R2 represents a group selected from the group: ,
Figure imgf000015_0002
, , or , wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In accordance with a third embodiment of the first aspect, the present invention covers compounds of general formula (I), supra, in which: X represents N; Y represents CH, C-F, or C-CN; Z represents -CH2-CH2-; BHC233018 - FC - 15 / 152 - R1 a selected from the
Figure imgf000016_0001
CH3 , , , , , or ; wherein * indicates the point of attachment of said group with the rest of the molecule; R2 represents a group selected from the group:
Figure imgf000016_0002
, wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. The present invention also covers the following compounds of general formula (I), supra, 2'-Amino-1-[6-(3,8-diazabicyclo[3.2.1]octan-3-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer); 2'-Amino-1-[6-(3,8-diazabicyclo[3.2.1]octan-3-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy}pyrimidin-4-yl]-5',6'-dihydrospiro[azetidine-3,4'-cyclopenta[b]thiophene]-3'- carbonitrile (single stereoisomer); 2'-Amino-1-[6-(3,8-diazabicyclo[3.2.1]octan-3-yl)-2-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-6- ylmethoxy)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile formate (racemate); 2'-Amino-1-(2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer); 2'-Amino-1-(2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-4-yl)-5',6'-dihydrospiro[azetidine-3,4'-cyclopenta[b]thiophene]-3'- carbonitrile (single stereoisomer); 2'-Amino-1-(6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]-2-{[(2S)-5-oxopyrrolidin-2- BHC233018 - FC - 16 / 152 - yl]methoxy}pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile (single stereoisomer); 2'-Amino-1-(2-{[(2S)-4,4-difluoro-1-methylpyrrolidin-2-yl]methoxy}-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer); 2'-Amino-1-(2-{[(2S,4R)-4-fluoro-1-methylpyrrolidin-2-yl]methoxy}-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer); 2'-Amino-1-(2-{[(2S,4R)-1-cyclopropyl-4-fluoropyrrolidin-2-yl]methoxy}-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer); 2'-Amino-1-{2-[(3-cyanopyrrolidin-3-yl)methoxy]-6-[(3R)-3-hydroxy-3-methylpiperidin-1- yl]pyrimidin-4-yl}-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile formate (mixture of two enantiopure diastereomers); 2'-Amino-1-{2-[(3-cyano-1-methylpyrrolidin-3-yl)methoxy]-6-[(3R)-3-hydroxy-3-methylpiperidin-1- yl]pyrimidin-4-yl}-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile (mixture of two enantiopure diastereomers); 2'-Amino-1-{6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]-2-[2-(1-methyl-1H-imidazol-2- yl)ethoxy]pyrimidin-4-yl}-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile (single stereoisomer); 2'-Amino-1-(6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]-2-{[(2S)-2-methylpyrrolidin-2- yl]methoxy}pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile (single stereoisomer); 2'-Amino-1-(2-{[1-(dimethylamino)cyclopropyl]methoxy}-6-[(3R)-3-hydroxy-3-methylpiperidin-1- yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile (single stereoisomer); 2'-Amino-1-[2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-(2-oxo-1,6- diazaspiro[3.5]nonan-6-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile formate (mixture of two enantiopure diastereomers); 2'-Amino-1-[2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-(3-oxa-7,9- diazabicyclo[3.3.1]nonan-7-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile formate (single stereoisomer); 2'-amino-1-[6-(4,4-difluoropiperidin-1-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- BHC233018 - FC - 17 / 152 - yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile (single stereoisomer); 2'-amino-1-[2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-(3-oxopiperazin-1- yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile (single stereoisomer); N-{1-[6-(2'-amino-3'-cyano-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1-yl)-2- {[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]piperidin-4- yl}acetamide (single stereoisomer); 2'-amino-1-[6-(3,3-difluoropiperidin-1-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile (single stereoisomer); 2'-amino-1-[6-(4-fluoropiperidin-1-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile (single stereoisomer); tert-butyl 1-[6-(2'-amino-3'-cyano-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1-yl)-2- {[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-1,6- diazaspiro[3.4]octane-6-carboxylate (mixture of two enantiopure diastereomers); 2'-amino-1-[6-(1,6-diazaspiro[3.4]octan-1-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile (mixture of two enantiopure diastereomers); 1-(6-[(4RS)-6-acetyl-1,6-diazaspiro[3.4]octan-1-yl]-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy}pyrimidin-4-yl)-2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]- 3'-carbonitrile (mixture of two enantiopure diastereomers); 2'-Amino-1-(2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]-5-methylpyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (single stereoisomer); 2'-Amino-1-[2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-5-methyl-6-(3- oxopiperazin-1-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer); 2'-Amino-1-(5-fluoro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (single stereoisomer); BHC233018 - FC - 18 / 152 - 2'-amino-1-[5-cyano-6-(3,8-diazabicyclo[3.2.1]octan-3-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H- pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (single stereoisomer); 2'-amino-1-(5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (single stereoisomer); 2'-amino-1-[5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-(1,4- oxazepan-4-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile (single stereoisomer); 2'-amino-1-(5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(6 RS)-6- hydroxy-6-methyl-1,4-oxazepan-4-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (mixture of two enantiopure diastereomers); 2'-amino-1-(5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(6 R)-6- hydroxy-6-methyl-1,4-oxazepan-4-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (single stereoisomer); 2'-amino-1-(5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(6 S)-6- hydroxy-6-methyl-1,4-oxazepan-4-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (single stereoisomer); 2'-Amino-1-[4-(3,8-diazabicyclo[3.2.1]octan-3-yl)-6-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy}pyrimidin-2-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer); 2'-Amino-1-(4-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-2-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer); 2'-Amino-1-(4-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]-6-{[(2S)-5-oxopyrrolidin-2- yl]methoxy}pyrimidin-2-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile (single stereoisomer) and 2'-Amino-1-(5-fluoro-4-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (single stereoisomer) or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In a further embodiment of the first aspect, the present invention covers compounds of formula (I), supra, in which: BHC233018 - FC - 19 / 152 - X represents CH, C-F, or N; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In a further embodiment of the first aspect, the present invention covers compounds of formula (I), supra, in which: X represents N; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In a further embodiment of the first aspect, the present invention covers compounds of formula (I), supra, in which: Y represents CH, C-F, C-Cl, C-CN, C-CH3, or N; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In a further embodiment of the first aspect, the present invention covers compounds of formula (I), supra, in which: Y represents CH, C-F, C-CN, C-CH3, or N; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In a further embodiment of the first aspect, the present invention covers compounds of formula (I), supra, in which: Y represents CH, C-F, or C-CN; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In a further embodiment of the first aspect, the present invention covers compounds of formula (I), supra, in which: Y represents C-CN; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In a further embodiment of the first aspect, the present invention covers compounds of formula (I), supra, in which: Z represents -CH2-, or -CH2-CH2-; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In a further embodiment of the first aspect, the present invention covers compounds of formula (I), supra, in which: Z represents -CH2-CH2-; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In a further embodiment of the first aspect, the present invention covers compounds of formula (I), supra, in which: BHC233018 - FC - 20 / 152 - represents a group selected from the group: ( ) ,
Figure imgf000021_0001
, , , , or ; wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In a further embodiment of the first aspect, the present invention covers compounds of formula (I), supra, in which: BHC233018 - FC - 21 / 152 - R1 represents a group selected from the group: , ,
Figure imgf000022_0001
, , or ; wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In a further embodiment of the first aspect, the present invention covers compounds of formula (I), supra, in which: R1 a selected from the
Figure imgf000022_0002
, , , , , or ; wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In a further embodiment of the first aspect, the present invention covers compounds of formula (I), supra, in which: R2 a selected from the
Figure imgf000022_0003
, , , , BHC233018 - FC - 22 / 152 -
Figure imgf000023_0001
, , , , or , wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In a further embodiment of the first aspect, the present invention covers compounds of formula (I), supra, in which: R2 represents a group selected from the group: ,
Figure imgf000023_0002
, , or , wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In a further embodiment of the first aspect, the present invention covers compounds of formula (I), supra, in which: BHC233018 - FC - 23 / 152 - R2 a selected from the
Figure imgf000024_0001
H , wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. In a particular further embodiment of the first aspect, the present invention covers combinations of two or more of the above mentioned embodiments under the heading “further embodiments of the first aspect of the present invention”. The present invention covers any sub-combination within any embodiment or aspect of the present invention of compounds of general formula (I), supra. The present invention covers any sub-combination within any embodiment or aspect of the present invention. The present invention covers the compounds of general formula (I) which are disclosed in the Example Section of this text, infra. The compounds according to the invention of general formula (I) can be prepared according to the following schemes 1 and 2. The schemes and procedures described below illustrate synthetic routes to the compounds of general formula (I) of the invention and are not intended to be limiting. It is clear to the person skilled in the art that the order of transformations as exemplified in schemes 1 and 2 can be modified in various ways. The order of transformations exemplified in these schemes is therefore not intended to be limiting. In addition, interconversion of any of the substituents, R1, R2 or R3, can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. The compounds of general formula (I) of the present invention can be converted to any salt, preferably pharmaceutically acceptable salts, as described herein, by any method which is known to the person skilled BHC233018 - FC - 24 / 152 - in the art. Similarly, any salt of a compound of general formula (I) of the present invention can be converted into the free compound, by any method which is known to the person skilled in the art. Compounds of general formula (I) of the present invention demonstrate a valuable pharmacological spectrum of action which could not have been predicted. Compounds of the present invention have surprisingly been found to effectively inhibit KRAS and it is possible therefore that said compounds be used for the treatment or prophylaxis of diseases, preferably neoplasic disorders in humans and animals. Compounds of the present invention can be utilized to inhibit, block, reduce, decrease, etc., cell proliferation and/or cell division, and/or produce apoptosis. This method comprises administering to a mammal in need thereof, including a human, an amount of a compound of general formula (I) of the present invention, or a pharmaceutically acceptable salt, isomer, polymorph, metabolite, hydrate, solvate or ester thereof, which is effective to treat the disorder. Hyperproliferative disorders include, but are not limited to, for example : psoriasis, keloids, and other hyperplasias affecting the skin, benign prostate hyperplasia (BPH), solid tumours, such as cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid and their distant metastases. Those disorders also include lymphomas, sarcomas, and leukaemias. Examples of breast cancers include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ. Examples of cancers of the respiratory tract include, but are not limited to, small-cell and non-small-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma. Examples of brain cancers include, but are not limited to, brain stem and hypophtalmic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumour. Tumours of the male reproductive organs include, but are not limited to, prostate and testicular cancer. Tumours of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus. Tumours of the digestive tract include, but are not limited to, anal, colon, colorectal, oesophageal, gallbladder, gastric, pancreatic, rectal, small-intestine, and salivary gland cancers. Tumours of the urinary tract include, but are not limited to, bladder, penile, kidney, renal pelvis, ureter, urethral and human papillary renal cancers. Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma. BHC233018 - FC - 25 / 152 - Examples of liver cancers include, but are not limited to, hepatocellular carcinoma (liver cell carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma. Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi’s sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head-and-neck cancers include, but are not limited to, laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancer, lip and oral cavity cancer and squamous cell. Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin’s lymphoma, cutaneous T-cell lymphoma, Burkitt lymphoma, Hodgkin’s disease, and lymphoma of the central nervous system. Sarcomas include, but are not limited to, sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia. The present invention also provides methods of treating angiogenic disorders including diseases associated with excessive and/or abnormal angiogenesis. Inappropriate and ectopic expression of angiogenesis can be deleterious to an organism. A number of pathological conditions are associated with the growth of extraneous blood vessels. These include, for example, diabetic retinopathy, ischemic retinal-vein occlusion, and retinopathy of prematurity [Aiello et al., New Engl. J. Med., 1994, 331, 1480 ; Peer et al., Lab. Invest., 1995, 72, 638], age-related macular degeneration (AMD) [Lopez et al., Invest. Opththalmol. Vis. Sci., 1996, 37, 855], neovascular glaucoma, psoriasis, retrolental fibroplasias, angiofibroma, inflammation, rheumatoid arthritis (RA), restenosis, in- stent restenosis, vascular graft restenosis, etc. In addition, the increased blood supply associated with cancerous and neoplastic tissue, encourages growth, leading to rapid tumour enlargement and metastasis. Moreover, the growth of new blood and lymph vessels in a tumour provides an escape route for renegade cells, encouraging metastasis and the consequence spread of the cancer. Thus, compounds of general formula (I) of the present invention can be utilized to treat and/or prevent any of the aforementioned angiogenesis disorders, for example by inhibiting and/or reducing blood vessel formation; by inhibiting, blocking, reducing, decreasing, etc. endothelial cell proliferation, or other types involved in angiogenesis, as well as causing cell death or apoptosis of such cell types. These disorders have been well characterized in humans, but also exist with a similar etiology in other mammals, and can be treated by administering pharmaceutical compositions of the present invention. BHC233018 - FC - 26 / 152 - The term “treating” or “treatment” as stated throughout this document is used conventionally, for example the management or care of a subject for the purpose of combating, alleviating, reducing, relieving, improving the condition of a disease or disorder, such as a carcinoma. The compounds of the present invention can be used in particular in therapy and prevention, i.e. prophylaxis, of tumour growth and metastases, especially in solid tumours of all indications and stages with or without pre-treatment of the tumour growth. Generally, the use of chemotherapeutic agents and/or anti-cancer agents in combination with a compound or pharmaceutical composition of the present invention will serve to: 1. yield better efficacy in reducing the growth of a tumour or even eliminate the tumour as compared to administration of either agent alone, 2. provide for the administration of lesser amounts of the administered chemotherapeutic agents, 3. provide for a chemotherapeutic treatment that is well tolerated in the patient with fewer deleterious pharmacological complications than observed with single agent chemotherapies and certain other combined therapies, 4. provide for treating a broader spectrum of different cancer types in mammals, especially humans, 5. provide for a higher response rate among treated patients, 6. provide for a longer survival time among treated patients compared to standard chemotherapy treatments, 7. provide a longer time for tumour progression, and/or 8. yield efficacy and tolerability results at least as good as those of the agents used alone, compared to known instances where other cancer agent combinations produce antagonistic effects. In addition, the compounds of general formula (I) of the present invention can also be used in combination with radiotherapy and/or surgical intervention. In a further embodiment of the present invention, the compounds of general formula (I) of the present invention may be used to sensitize a cell to radiation, i.e. treatment of a cell with a compound of the present invention prior to radiation treatment of the cell renders the cell more susceptible to DNA damage and cell death than the cell would be in the absence of any treatment with a compound of the present invention. In one aspect, the cell is treated with at least one compound of general formula (I) of the present invention. Thus, the present invention also provides a method of killing a cell, wherein a cell is administered one or more compounds of the present invention in combination with conventional radiation therapy. The present invention also provides a method of rendering a cell more susceptible to cell death, wherein the cell is treated with one or more compounds of general formula (I) of the present invention prior to the BHC233018 - FC - 27 / 152 - treatment of the cell to cause or induce cell death. In one aspect, after the cell is treated with one or more compounds of general formula (I) of the present invention, the cell is treated with at least one compound, or at least one method, or a combination thereof, in order to cause DNA damage for the purpose of inhibiting the function of the normal cell or killing the cell. In other embodiments of the present invention, a cell is killed by treating the cell with at least one DNA damaging agent, i.e. after treating a cell with one or more compounds of general formula (I) of the present invention to sensitize the cell to cell death, the cell is treated with at least one DNA damaging agent to kill the cell. DNA damaging agents useful in the present invention include, but are not limited to, chemotherapeutic agents (e.g. cis platin), ionizing radiation (X-rays, ultraviolet radiation), carcinogenic agents, and mutagenic agents. In other embodiments, a cell is killed by treating the cell with at least one method to cause or induce DNA damage. Such methods include, but are not limited to, activation of a cell signalling pathway that results in DNA damage when the pathway is activated, inhibiting of a cell signalling pathway that results in DNA damage when the pathway is inhibited, and inducing a biochemical change in a cell, wherein the change results in DNA damage. By way of a non-limiting example, a DNA repair pathway in a cell can be inhibited, thereby preventing the repair of DNA damage and resulting in an abnormal accumulation of DNA damage in a cell. In one aspect of the invention, a compound of general formula (I) of the present invention is administered to a cell prior to the radiation or other induction of DNA damage in the cell. In another aspect of the invention, a compound of general formula (I) of the present invention is administered to a cell concomitantly with the radiation or other induction of DNA damage in the cell. In yet another aspect of the invention, a compound of general formula (I) of the present invention is administered to a cell immediately after radiation or other induction of DNA damage in the cell has begun. In another aspect, the cell is in vitro. In another embodiment, the cell is in vivo. In accordance with a further aspect, the present invention covers compounds of general formula (I), as described supra, or stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, particularly pharmaceutically acceptable salts thereof, or mixtures of same, for use in the treatment or prophylaxis of diseases, in particular neoplastic disorders. The pharmaceutical activity of the compounds according to the invention can be explained by their activity as KRAS inhibitors. In accordance with a further aspect, the present invention covers the use of compounds of general formula (I), as described supra, or stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, particularly pharmaceutically acceptable salts thereof, or mixtures of same, for the treatment or BHC233018 - FC - 28 / 152 - prophylaxis of diseases, in particular neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling. In accordance with a further aspect, the present invention covers the use of a compound of formula (I), described supra, or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, for the prophylaxis or treatment of diseases, in particular neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling. In accordance with a further aspect, the present invention covers the use of compounds of general formula (I), as described supra, or stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, particularly pharmaceutically acceptable salts thereof, or mixtures of same, in a method of treatment or prophylaxis of diseases, in particular neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling. In accordance with a further aspect, the present invention covers use of a compound of general formula (I), as described supra, or stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, particularly pharmaceutically acceptable salts thereof, or mixtures of same, for the preparation of a pharmaceutical composition, preferably a medicament, for the prophylaxis or treatment of diseases, in particular neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling. In accordance with a further aspect, the present invention covers a method of treatment or prophylaxis of diseases, in particular neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling, using an effective amount of a compound of general formula (I), as described supra, or stereoisomers, tautomers, N-oxides, hydrates, solvates, and salts thereof, particularly pharmaceutically acceptable salts thereof, or mixtures of same. In accordance with a further aspect, the present invention covers pharmaceutical compositions, in particular a medicament, comprising a compound of general formula (I), as described supra, or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, a salt thereof, particularly a pharmaceutically acceptable salt, or a mixture of same, and one or more excipients), in particular one or more pharmaceutically acceptable excipient(s). Conventional procedures for preparing such pharmaceutical compositions in appropriate dosage forms can be utilized. The present invention furthermore covers pharmaceutical compositions, in particular medicaments, which comprise at least one compound according to the invention, conventionally together with one or more pharmaceutically suitable excipients, and to their use for the above mentioned purposes. It is possible for the compounds according to the invention to have systemic and/or local activity. For this purpose, they can be administered in a suitable manner, such as, for example, via the oral, parenteral, BHC233018 - FC - 29 / 152 - pulmonary, nasal, sublingual, lingual, buccal, rectal, vaginal, dermal, transdermal, conjunctival, otic route or as an implant or stent. For these administration routes, it is possible for the compounds according to the invention to be administered in suitable administration forms. For oral administration, it is possible to formulate the compounds according to the invention to dosage forms known in the art that deliver the compounds of the invention rapidly and/or in a modified manner, such as, for example, tablets (uncoated or coated tablets, for example with enteric or controlled release coatings that dissolve with a delay or are insoluble), orally-disintegrating tablets, films/wafers, films/lyophylisates, capsules (for example hard or soft gelatine capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions. It is possible to incorporate the compounds according to the invention in crystalline and/or amorphised and/or dissolved form into said dosage forms. Parenteral administration can be effected with avoidance of an absorption step (for example intravenous, intraarterial, intracardial, intraspinal or intralumbal) or with inclusion of absorption (for example intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal). Administration forms which are suitable for parenteral administration are, inter alia, preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophylisates or sterile powders. Examples which are suitable for other administration routes are pharmaceutical forms for inhalation [inter alia powder inhalers, nebulizers], nasal drops, nasal solutions, nasal sprays; tablets/films/wafers/capsules for lingual, sublingual or buccal administration; suppositories; eye drops, eye ointments, eye baths, ocular inserts, ear drops, ear sprays, ear powders, ear-rinses, ear tampons; vaginal capsules, aqueous suspensions (lotions, mixturae agitandae), lipophilic suspensions, emulsions, ointments, creams, transdermal therapeutic systems (such as, for example, patches), milk, pastes, foams, dusting powders, implants or stents. The compounds according to the invention can be incorporated into the stated administration forms. This can be effected in a manner known per se by mixing with pharmaceutically suitable excipients. Pharmaceutically suitable excipients include, inter alia, • fillers and carriers (for example cellulose, microcrystalline cellulose (such as, for example, Avicel®), lactose, mannitol, starch, calcium phosphate (such as, for example, Di-Cafos®)), • ointment bases (for example petroleum jelly, paraffins, triglycerides, waxes, wool wax, wool wax alcohols, lanolin, hydrophilic ointment, polyethylene glycols), • bases for suppositories (for example polyethylene glycols, cacao butter, hard fat), • solvents (for example water, ethanol, isopropanol, glycerol, propylene glycol, medium chain- length triglycerides, fatty oils, liquid polyethylene glycols, paraffins), BHC233018 - FC - 30 / 152 - • surfactants, emulsifiers, dispersants or wetters (for example sodium dodecyl sulfate), lecithin, phospholipids, fatty alcohols (such as, for example, Lanette®), sorbitan fatty acid esters (such as, for example, Span®), polyoxyethylene sorbitan fatty acid esters (such as, for example, Tween®), polyoxyethylene fatty acid glycerides (such as, for example, Cremophor®), polyoxethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, glycerol fatty acid esters, poloxamers (such as, for example, Pluronic®), • buffers, acids and bases (for example phosphates, carbonates, citric acid, acetic acid, hydrochloric acid, sodium hydroxide solution, ammonium carbonate, trometamol, triethanolamine), • isotonicity agents (for example glucose, sodium chloride), • adsorbents (for example highly-disperse silicas), • viscosity-increasing agents, gel formers, thickeners and/or binders (for example polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, carboxymethylcellulose-sodium, starch, carbomers, polyacrylic acids (such as, for example, Carbopol®); alginates, gelatine), • disintegrants (for example modified starch, carboxymethylcellulose-sodium, sodium starch glycolate (such as, for example, Explotab®), cross- linked polyvinylpyrrolidone, croscarmellose- sodium (such as, for example, AcDiSol®)), • flow regulators, lubricants, glidants and mould release agents (for example magnesium stearate, stearic acid, talc, highly-disperse silicas (such as, for example, Aerosil®)), • coating materials (for example sugar, shellac) and film formers for films or diffusion membranes which dissolve rapidly or in a modified manner (for example polyvinylpyrrolidones (such as, for example, Kollidon®), polyvinyl alcohol, hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, hydroxypropylmethylcellulose phthalate, cellulose acetate, cellulose acetate phthalate, polyacrylates, polymethacrylates such as, for example, Eudragit®)), • capsule materials (for example gelatine, hydroxypropylmethylcellulose), • synthetic polymers (for example polylactides, polyglycolides, polyacrylates, polymethacrylates (such as, for example, Eudragit®), polyvinylpyrrolidones (such as, for example, Kollidon®), polyvinyl alcohols, polyvinyl acetates, polyethylene oxides, polyethylene glycols and their copolymers and blockcopolymers), • plasticizers (for example polyethylene glycols, propylene glycol, glycerol, triacetine, triacetyl citrate, dibutyl phthalate), • penetration enhancers, BHC233018 - FC - 31 / 152 - • stabilisers (for example antioxidants such as, for example, ascorbic acid, ascorbyl palmitate, sodium ascorbate, butylhydroxyanisole, butylhydroxytoluene, propyl gallate), • preservatives (for example parabens, sorbic acid, thiomersal, benzalkonium chloride, chlorhexidine acetate, sodium benzoate), • colourants (for example inorganic pigments such as, for example, iron oxides, titanium dioxide), • flavourings, sweeteners, flavour- and/or odour-masking agents. The present invention furthermore relates to a pharmaceutical composition which comprise at least one compound according to the invention, conventionally together with one or more pharmaceutically suitable excipient(s), and to their use according to the present invention. It is possible for the compounds according to the invention to have systemic and/or local activity. For this purpose, they can be administered in a suitable manner, such as, for example, via the oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, vaginal, dermal, transdermal, conjunctival, otic route or as an implant or stent. For these administration routes, it is possible for the compounds according to the invention to be administered in suitable administration forms. For oral administration, it is possible to formulate the compounds according to the invention to dosage forms known in the art that deliver the compounds of the invention rapidly and/or in a modified manner, such as, for example, tablets (uncoated or coated tablets, for example with enteric or controlled release coatings that dissolve with a delay or are insoluble), orally-disintegrating tablets, films/wafers, films/lyophylisates, capsules (for example hard or soft gelatine capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions. It is possible to incorporate the compounds according to the invention in crystalline and/or amorphised and/or dissolved form into said dosage forms. Parenteral administration can be effected with avoidance of an absorption step (for example intravenous, intraarterial, intracardial, intraspinal or intralumbal) or with inclusion of absorption (for example intramuscular, subcutaneous, intracutaneous, percutaneous or intraperitoneal). Administration forms which are suitable for parenteral administration are, inter alia, preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophylisates or sterile powders. Examples which are suitable for other administration routes are pharmaceutical forms for inhalation [inter alia powder inhalers, nebulizers], nasal drops, nasal solutions, nasal sprays; tablets/films/wafers/capsules for lingual, sublingual or buccal administration; suppositories; eye drops, eye ointments, eye baths, ocular inserts, ear drops, ear sprays, ear powders, ear-rinses, ear tampons; vaginal capsules, aqueous suspensions (lotions, mixturae agitandae), lipophilic suspensions, emulsions, ointments, creams, transdermal BHC233018 - FC - 32 / 152 - therapeutic systems (such as, for example, patches), milk, pastes, foams, dusting powders, implants or stents. The compounds according to the invention can be incorporated into the stated administration forms. This can be effected in a manner known per se by mixing with pharmaceutically suitable excipients. Pharmaceutically suitable excipients include, inter alia, • fillers and carriers (for example cellulose, microcrystalline cellulose (such as, for example, Avicel®), lactose, mannitol, starch, calcium phosphate (such as, for example, Di-Cafos®)), • ointment bases (for example petroleum jelly, paraffins, triglycerides, waxes, wool wax, wool wax alcohols, lanolin, hydrophilic ointment, polyethylene glycols), • bases for suppositories (for example polyethylene glycols, cacao butter, hard fat), • solvents (for example water, ethanol, isopropanol, glycerol, propylene glycol, medium chain- length triglycerides fatty oils, liquid polyethylene glycols, paraffins), • surfactants, emulsifiers, dispersants or wetters (for example sodium dodecyl sulfate), lecithin, phospholipids, fatty alcohols (such as, for example, Lanette®), sorbitan fatty acid esters (such as, for example, Span®), polyoxyethylene sorbitan fatty acid esters (such as, for example, Tween®), polyoxyethylene fatty acid glycerides (such as, for example, Cremophor®), polyoxethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, glycerol fatty acid esters, poloxamers (such as, for example, Pluronic®), • buffers, acids and bases (for example phosphates, carbonates, citric acid, acetic acid, hydrochloric acid, sodium hydroxide solution, ammonium carbonate, trometamol, triethanolamine), • isotonicity agents (for example glucose, sodium chloride), • adsorbents (for example highly-disperse silicas), • viscosity-increasing agents, gel formers, thickeners and/or binders (for example polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, carboxymethylcellulose-sodium, starch, carbomers, polyacrylic acids (such as, for example, Carbopol®); alginates, gelatine), • disintegrants (for example modified starch, carboxymethylcellulose-sodium, sodium starch glycolate (such as, for example, Explotab®), cross- linked polyvinylpyrrolidone, croscarmellose- sodium (such as, for example, AcDiSol®)), • flow regulators, lubricants, glidants and mould release agents (for example magnesium stearate, stearic acid, talc, highly-disperse silicas (such as, for example, Aerosil®)), BHC233018 - FC - 33 / 152 - • coating materials (for example sugar, shellac) and film formers for films or diffusion membranes which dissolve rapidly or in a modified manner (for example polyvinylpyrrolidones (such as, for example, Kollidon®), polyvinyl alcohol, hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, hydroxypropylmethylcellulose phthalate, cellulose acetate, cellulose acetate phthalate, polyacrylates, polymethacrylates such as, for example, Eudragit®)), • capsule materials (for example gelatine, hydroxypropylmethylcellulose), • synthetic polymers (for example polylactides, polyglycolides, polyacrylates, polymethacrylates (such as, for example, Eudragit®), polyvinylpyrrolidones (such as, for example, Kollidon®), polyvinyl alcohols, polyvinyl acetates, polyethylene oxides, polyethylene glycols and their copolymers and blockcopolymers), • plasticizers (for example polyethylene glycols, propylene glycol, glycerol, triacetine, triacetyl citrate, dibutyl phthalate), • penetration enhancers, • stabilisers (for example antioxidants such as, for example, ascorbic acid, ascorbyl palmitate, sodium ascorbate, butylhydroxyanisole, butylhydroxytoluene, propyl gallate), • preservatives (for example parabens, sorbic acid, thiomersal, benzalkonium chloride, chlorhexidine acetate, sodium benzoate), • colourants (for example inorganic pigments such as, for example, iron oxides, titanium dioxide), • flavourings, sweeteners, flavour- and/or odour-masking agents. The present invention furthermore relates to a pharmaceutical composition which comprise at least one compound according to the invention, conventionally together with one or more pharmaceutically suitable excipient(s), and to their use according to the present invention. In accordance with another aspect, the present invention covers pharmaceutical combinations, in particular medicaments, comprising at least one compound of general formula (I) of the present invention and at least one or more further active ingredients, in particular for the treatment and/or prophylaxis of a neoplastic disorder, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling. Particularly, the present invention covers a pharmaceutical combination, which comprises: • one or more first active ingredients, in particular compounds of general formula (I) as defined supra, and • one or more further active ingredients, in particular cancer agents. BHC233018 - FC - 34 / 152 - The term “combination” in the present invention is used as known to persons skilled in the art, it being possible for said combination to be a fixed combination, a non-fixed combination or a kit-of-parts. A “fixed combination” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein, for example, a first active ingredient, such as one or more compounds of general formula (I) of the present invention, and a further active ingredient are present together in one unit dosage or in one single entity. One example of a “fixed combination” is a pharmaceutical composition wherein a first active ingredient and a further active ingredient are present in admixture for simultaneous administration, such as in a formulation. Another example of a “fixed combination” is a pharmaceutical combination wherein a first active ingredient and a further active ingredient are present in one unit without being in admixture. A non-fixed combination or “kit-of-parts” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein a first active ingredient and a further active ingredient are present in more than one unit. One example of a non-fixed combination or kit-of-parts is a combination wherein the first active ingredient and the further active ingredient are present separately. It is possible for the components of the non-fixed combination or kit-of-parts to be administered separately, sequentially, simultaneously, concurrently or chronologically staggered. The compounds of the present invention can be administered as the sole pharmaceutical agent or in combination with one or more other pharmaceutically active ingredients where the combination causes no unacceptable adverse effects. The present invention also covers such pharmaceutical combinations. For example, the compounds of the present invention can be combined with known cancer agents. Examples of cancer agents include: 131I-chTNT, abarelix, abemaciclib, abiraterone, acalabrutinib, aclarubicin, adalimumab, ado-trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alectinib, alemtuzumab, alendronic acid, alitretinoin, alpharadin, altretamine, amifostine, aminoglutethimide, hexyl aminolevulinate, amrubicin, amsacrine, anastrozole, ancestim, anethole dithiolethione, anetumab ravtansine, angiotensin II, antithrombin III, apalutamide, aprepitant, arcitumomab, arglabin, arsenic trioxide, asparaginase, atezolizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, basiliximab, belotecan, bendamustine, besilesomab, belinostat, bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin, blinatumomab, bortezomib, bosutinib, buserelin, brentuximab vedotin, brigatinib, busulfan, cabazitaxel, cabozantinib, calcitonine, calcium folinate, calcium levofolinate, capecitabine, capromab, carbamazepine carboplatin, carboquone, carfilzomib, carmofur, carmustine, catumaxomab, celecoxib, celmoleukin, cemiplimab, ceritinib, cetuximab, chlorambucil, chlormadinone, chlormethine, cidofovir, cinacalcet, cisplatin, cladribine, clodronic acid, clofarabine, cobimetinib, copanlisib, crisantaspase, crizotinib, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daratumumab, darbepoetin alfa, dabrafenib, darolutamide, dasatinib, daunorubicin, decitabine, degarelix, denileukin diftitox, denosumab, depreotide, BHC233018 - FC - 35 / 152 - deslorelin, dianhydrogalactitol, dexrazoxane, dibrospidium chloride, dianhydrogalactitol, diclofenac, dinutuximab, docetaxel, dolasetron, doxifluridine, doxorubicin, doxorubicin + estrone, dronabinol, durvalumab, eculizumab, edrecolomab, elliptinium acetate, elotuzumab, eltrombopag, enasidenib, endostatin, enocitabine, enzalutamide, epirubicin, epitiostanol, epoetin alfa, epoetin beta, epoetin zeta, eptaplatin, eribulin, erlotinib, esomeprazole, estradiol, estramustine, ethinylestradiol, etoposide, everolimus, exemestane, fadrozole, fentanyl, filgrastim, fluoxymesterone, floxuridine, fludarabine, fluorouracil, flutamide, folinic acid, formestane, fosaprepitant, fotemustine, fulvestrant, gadobutrol, gadoteridol, gadoteric acid meglumine, gadoversetamide, gadoxetic acid, gallium nitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, Glucarpidase, glutoxim, GM-CSF, goserelin, granisetron, granulocyte colony stimulating factor, histamine dihydrochloride, histrelin, hydroxycarbamide, I-125 seeds, lansoprazole, ibandronic acid, ibritumomab tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib, imiquimod, improsulfan, indisetron, incadronic acid, ingenol mebutate, inotuzumab ozogamicin, interferon alfa, interferon beta, interferon gamma, iobitridol, iobenguane (123I), iomeprol, ipilimumab, irinotecan, Itraconazole, ixabepilone, ixazomib, lanreotide, lansoprazole, lapatinib, Iasocholine, lenalidomide, lenvatinib, lenograstim, lentinan, letrozole, leuprorelin, levamisole, levonorgestrel, levothyroxine sodium, lisuride, lobaplatin, lomustine, lonidamine, lutetium Lu 177 dotatate, masoprocol, medroxyprogesterone, megestrol, melarsoprol, melphalan, mepitiostane, mercaptopurine, mesna, methadone, methotrexate, methoxsalen, methylaminolevulinate, methylprednisolone, methyltestosterone, metirosine, midostaurin, mifamurtide, miltefosine, miriplatin, mitobronitol, mitoguazone, mitolactol, mitomycin, mitotane, mitoxantrone, mogamulizumab, molgramostim, mopidamol, morphine hydrochloride, morphine sulfate, mvasi, nabilone, nabiximols, nafarelin, naloxone + pentazocine, naltrexone, nartograstim, necitumumab, nedaplatin, nelarabine, neratinib, neridronic acid, netupitant/palonosetron, nivolumab, pentetreotide, nilotinib, nilutamide, nimorazole, nimotuzumab, nimustine, nintedanib, niraparib, nitracrine, nivolumab, obinutuzumab, octreotide, ofatumumab, olaparib, olaratumab, omacetaxine mepesuccinate, omeprazole, ondansetron, oprelvekin, orgotein, orilotimod, osimertinib, oxaliplatin, oxycodone, oxymetholone, ozogamicine, p53 gene therapy, paclitaxel, palbociclib, palifermin, palladium-103 seed, palonosetron, pamidronic acid, panitumumab, panobinostat, pantoprazole, pazopanib, pegaspargase, PEG-epoetin beta (methoxy PEG-epoetin beta), pembrolizumab, pegfilgrastim, peginterferon alfa-2b, pembrolizumab, pemetrexed, pentazocine, pentostatin, peplomycin, Perflubutane, perfosfamide, Pertuzumab, picibanil, pilocarpine, pirarubicin, pixantrone, plerixafor, plicamycin, poliglusam, polyestradiol phosphate, polyvinylpyrrolidone + sodium hyaluronate, polysaccharide-K, pomalidomide, ponatinib, porfimer sodium, pralatrexate, prednimustine, prednisone, procarbazine, procodazole, propranolol, quinagolide, rabeprazole, racotumomab, radium-223 chloride, radotinib, raloxifene, raltitrexed, ramosetron, ramucirumab, ranimustine, rasburicase, razoxane, refametinib, regorafenib, ribociclib, risedronic acid, rhenium-186 etidronate, rituximab, rolapitant, romidepsin, romiplostim, romurtide, rucaparib, samarium (153Sm) lexidronam, sargramostim, sarilumab, BHC233018 - FC - 36 / 152 - satumomab, secretin, siltuximab, sipuleucel-T, sizofiran, sobuzoxane, sodium glycididazole, sonidegib, sorafenib, stanozolol, streptozocin, sunitinib, talaporfin, talimogene laherparepvec, tamibarotene, tamoxifen, tapentadol, tasonermin, teceleukin, technetium (99mTc) nofetumomab merpentan, 99mTc- HYNIC-[Tyr3]-octreotide, tegafur, tegafur + gimeracil + oteracil, temoporfin, temozolomide, temsirolimus, teniposide, testosterone, tetrofosmin, thalidomide, thiotepa, thymalfasin, thyrotropin alfa, tioguanine, tisagenlecleucel, tislelizumab, tocilizumab, topotecan, toremifene, tositumomab, trabectedin, trametinib, tramadol, trastuzumab, trastuzumab emtansine, treosulfan, tretinoin, trifluridine + tipiracil, trilostane, triptorelin, trametinib, trofosfamide, thrombopoietin, tryptophan, ubenimex, valatinib, valrubicin, vandetanib, vapreotide, vemurafenib, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, vismodegib, vorinostat, vorozole, yttrium-90 glass microspheres, zinostatin, zinostatin stimalamer, zoledronic acid, zorubicin. Based upon standard laboratory techniques known to evaluate compounds useful for the treatment of neoplastic disorders, by standard toxicity tests and by standard pharmacological assays for the determination of treatment of the conditions identified above in mammals, and by comparison of these results with the results of known active ingredients or medicaments that are used to treat these conditions, the effective dosage of the compounds of the present invention can readily be determined for treatment of each desired indication. The amount of the active ingredient to be administered in the treatment of one of these conditions can vary widely according to such considerations as the particular compound and dosage unit employed, the mode of administration, the period of treatment, the age and sex of the patient treated, and the nature and extent of the condition treated. The total amount of the active ingredient to be administered will generally range from about 0.001 mg/kg to about 200 mg/kg body weight per day, and preferably from about 0.01 mg/kg to about 20 mg/kg body weight per day. Clinically useful dosing schedules will range from one to three times a day dosing to once every four weeks dosing. In addition, it is possible for "drug holidays", in which a patient is not dosed with a drug for a certain period of time, to be beneficial to the overall balance between pharmacological effect and tolerability. It is possible for a unit dosage to contain from about 0.5 mg to about 1500 mg of active ingredient, and can be administered one or more times per day or less than once a day. The average daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous and parenteral injections, and use of infusion techniques will preferably be from 0.01 to 200 mg/kg of total body weight. The average daily rectal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight. The average daily vaginal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight. The average daily topical dosage regimen will preferably be from 0.1 to 200 mg administered between one to four times daily. The transdermal concentration will preferably be that required to maintain a daily dose of from 0.01 to 200 mg/kg. The average daily inhalation dosage regimen will preferably be from 0.01 to 100 mg/kg of total body weight. BHC233018 - FC - 37 / 152 - Of course the specific initial and continuing dosage regimen for each patient will vary according to the nature and severity of the condition as determined by the attending diagnostician, the activity of the specific compound employed, the age and general condition of the patient, time of administration, route of administration, rate of excretion of the drug, drug combinations, and the like. The desired mode of treatment and number of doses of a compound of the present invention or a pharmaceutically acceptable salt or ester or composition thereof can be ascertained by those skilled in the art using conventional treatment tests. General Procedures The compounds according to the invention of general formula (I) can be prepared according to the following schemes 1 to 4. The schemes and procedures described below illustrate synthetic routes to the compounds of general formula (I) of the invention and are not intended to be limiting. It is clear to the person skilled in the art that the order of transformations as exemplified in schemes 1 to 4 can be modified in various ways. The order of transformations exemplified in these schemes is therefore not intended to be limiting. In addition, interconversion of any of the substituents R1 and R2 can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (e. g. Greene′s Protective Groups in Organic Synthesis, 4th edition, 2006, P. Wuts, T. Greene, John Wiley & Sons or Protecting Groups, 3rd edition, 2003, P.J. Kocienski, Thieme). Specific examples are described in the subsequent paragraphs. The starting materials are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the Experimental Section. Four routes for the preparation of compounds of general formula (I) are described in schemes 1 to 4. Synthesis of compounds of general formula (I) of the present invention Compounds of general formula (I) with the meaning of X, Y, Z, R1, and R2 as defined in general formula (I), can be synthesised according to a general procedure depicted in scheme 1 starting from trichorosynthons of the formula (II), by methods known to those skilled in the art. Trichloropyrimidines of general formula (II)(like CAS No: 3764-01-0) can be coupled to nucleophiles of formula (III), where the shown H is directly connected to a heteratom like N or O, using a base (like BHC233018 - FC - 38 / 152 - triethylamine) in a solvent (such as dichloromethane) at temperatures between 25°C and 100°C to get the dichloropyrimidine of general formula (IV). These intermediates of general formula (IV) can be converted in a second SNAr reaction with a further α-heteroatom (such as O or N) containing nucleophile of general formula (V) in the presence of a strong base (like sodium hydride)(usually in case of an α-oxygen bearing nucleophile) or an inorganic base (such as sodium carbonate)(usually in case of an α-nitrogen bearing nucleophile) in a polar aprotic solvent (such as N,N-dimethylformamide or dimethyl sulfoxide) at temperatures between -80°C and 100°C. If X and Y are different from each other, usually a mixture of two regioisomeric products of general formula (VI) is obtained, wherein the second nitrogen of the corresponding pyrimidine core can be either represented by X or Y of general formula (VI) and the other position is represented by either a CH or a substituted quarternary carbon. These regioisomers can be separated by column chromatography or preparative HPLC. To avoid the formation of regioisomers or at least increase the regioisomeric ratio dichloro-methylthio-pyrimidines of general formula (VIII)(like Cas No 6299-25-8) can be used as starting materials. After the already described first SNAr reaction with the first nuclephile of general formula (III) the obtained product of general formula (IX) is activated for the second SNAr reaction at the methylthio position via oxidation to the methyl sulfonyl compound of general formula (X). This oxidation is usually done with at least 2 equivalents of an oxidizing reagent like meta- chloroperoxybenzoic acid or oxone in a solvent such as dichloromethane at temperatures between -20°C and 40°C. After this oxidative activation, the second SNAr reaction can be performed regioselectively via substitution of the methyl sulfone at the pyrimidine core by using a strong base like sodium bis(trimethylsilyl)amide to quantitatively deprotonate the nucleophile of general formula (V) in a solvent like tetrahydrofuran at temperatures ranging between -80°C and 100°C. Like the upper reaction pathway shown in scheme 1, this lower pathway leads also to intermediates of general formula (VI) that can be reacted in a final third SNAr reaction with a tricyclic cyano-aminothiophene containing a spiroazetidine (general formula (VII)). For this final conversion usually an inorganic base such as sodium or caesium carbonate is employed in a dipolar aprotic solvent like dimethyl sulfoxide or N,N-dimethylacetamide at temperatures between 50°C and 150°C to obtain the desired compounds of general formula (I). Scheme 1 BHC233018 - FC - 39 / 152 -
Figure imgf000040_0001
In Scheme 1, X,Y, Z, R1, R2 represent (hetero)atoms or substituents according to claim 1; HQ represents an acid able to form a salt with the azetidine of compounds (VII) like HCl, formic acid or trifluoroacetic acid; reaction conditions in scheme 1: steps 1, 2, 3, 5, and 6: base, solvent; step 4: oxidizing agent, solvent. In an alternative approach outlined in scheme 2, compounds of the general formula (I) can be obtained via a changed order of addition of the three substituents at the pyrimidine core again employing three SNAr reactions. In contrast to the reaction sequence outlined in scheme 1, dichloro-methylthio-pyrimidines of general formula (VIII)(like Cas No 6299-25-8) can first be reacted with the tricyclic thienospiroazetidines BHC233018 - FC - 40 / 152 - of general formula (VII) using an inorganic base like sodium carbonate in a dipolar aprotic solvent such as dimethyl sulfoxide at temperatures between 25°C and 100°C. To avoid N-oxide formation or self- reaction in the subsequent steps the intermediate of general formula (XI) is first double protected at the primary amine in position 2 of the thiophene. As one protecting group a carbamate is employed wherein R4 represents C1 to C4 alkyl preferably t-butyl. The other protecting group described as general PG can also be a more labile second carbamate or a more stable benzyl group like para-methoxybenzyl. Double protection with two t-butyloxycarbonyl groups (Boc) can be done using two equivalents of di-tert-butyl dicarbonate together with an organic base like N,N-diisopropylethylamine and a nucleophilic catalyst like 4-(dimethylamino)pyridine in a solvent like tetrahydrofuran at temperatures ranging from 0°C to 40°C. Alternatively, the amino intermediate of general formula (XI) can first be mono-protected with for example one equivalent of di-tert-butyl decarbonate and subsequently protected with a benzylic group like para-methoxybenzyl (PMB) using para-methoxybenzyl chloride together with an inorganic base like caesium carbonate in an aprotic solvent such as N,N-dimethylformamide at temperatures between 50°C and 120°C. The double protected intermediate of general formula (XI) can then be oxidized at the methylthiol group using at least 2 equivalents of an oxidizing reagent like meta-chloroperoxybenzoic acid or oxone under conditions already described at scheme 1 for intermediates of general formula (X). The subsequent SNAr reaction with nucleophiles of general formula (V) is usually performed via employment of a strong base like sodium bis(trimethylsilyl)amide in a solvent like tetrahydrofuran at temperatures ranging between 25°C and 100°C. In case of a double Boc-protected intermediate of general formula (XIII) this strong basic conditions described for the subsequent SNAr can lead in situ to the loss of the more labile second Boc-protecting group. In case of mixed protected intermediate (XIII) the more stable carbamate group can be selectively removed using acidic conditions like trifluoroacetic acid in dichloromethane or HCl in dioxane to get the mono-protected intermediates of general formula (XIV). The final SNAr reaction is usually performed with a basic α-nitrogen bearing nucleophile of general formula (III) using an inorganic base like caesium carbonate in an aprotic solvent such as dimethyl sulfoxide at temperatures between 50°C and 170°C. The obtained compounds of general formula (XV) are finally deproteced at the amino-position of the thiophene. In case a second carbamate protecting group this final deprotection can be performed using acidic conditions like trifluoroacetic acid in dichloromethane or HCl in dioxane. If the final protecting group is a benzylic group deprotection can be performed using hydrogenolytic conditions (like hydrogen atmosphere between 1 and 100 bar together with a catalyst like palladium on charcoal in a protic solvent like ethanol at temperatures between 20°C and 100°C) or oxidative conditions (like ceric ammonium nitrate in a solvent or mixture like aqueous acetonitrile at temperatures between 0°C and 50°C). Scheme 2 BHC233018 - FC - 41 / 152 -
Figure imgf000042_0001
In scheme 2, X,Y, Z, R1, R2 represent (hetero)atoms or substituents according to claim 1; HQ represents an acid able to form a salt with the azetidine of compounds (VII) like HCl, formic acid or trifluoroacetic acid; R4 represents a C1 to C4 alkyl preferably t-butyl; PG represents any protecting group, preferably para-methoxybenzyl or t-butyloxycarbonyl; reaction conditions in scheme 2: steps 1, and 5: base, solvent; step 2: double protection; step 3: oxidizing agent, solvent; step 4: base, solvent, then acid; step 6: deprotection. In compounds of general formula (I) the described head group R1 as well as the described side chain R2 might contain additional amine functional groups that are linked via a cycle or chain to the attaching atom A connected to the central heterocycle, wherein A represents nitrogen, oxygen, or sulfur preferably nitrogen. These additional amine functional groups are usually protected as carbamates to avoid regioselectivity issues during the SNAr reactions at the central heterocycle, wherein this heterocycle is preferably pyrimidine (scheme 3). Additional carbamate protected amines in the head group R1 as indicated for compounds of general formula (XVI) might be deprotected in a final reaction step using acidic conditions like trifluoroacetic acid in dichloromethane or HCl in dioxane as already described in scheme 1 and 2 to get compounds of general formula (XVII). The same conditions can be used to deprotect carbamate protected amines in the side chain R2 as indicated for compounds of general formula (XVIII). This deprotection of the side chain R2 can be done at an intermediate state (A is still a halogen usually chloride) or at the final state (A is then the usually tricyclic thienoazetidine). After deprotection of the side chain R2 the free NH of intermediates of general formula (XIX) can be alkylated using reductive amination conditions known to the person skilled in the art. The substituent R5 in compounds of general formula (XX) shown in scheme 3 can be introduced from a corresponding carbonyl compound like an aldehyde such as formaldehyde or a ketone in case of an α-branched R5 such as acetone or it can be introduced from an acetal such as [(1-ethoxycyclopropyl)oxy](trimethyl)silane in case R5 is a cyclopropyl moiety. Typical reaction conditions include a mild reducing agent such as sodium cyanoborohydride, sodium triacetoxyborohydride or 2-picoline borane complex, that chemoselectively prefers to reduce the in situ BHC233018 - FC - 42 / 152 - formed imine species from compounds of general formula (XIX) and the carbonyl or acetal compound corresponding to substituent R5 over the direct reduction of the carbonyl or acetal compound. Further to the mild reducing agent an acidic additive like acetic acid can be employed in a protic (e.g. methanol) or aprotic solvent (e.g. acetonitrile) with temperatures ranging from 0°C to 100°C. To shift the equilibrium of in situ imine formation towards the imine species the byproduct water can be trapped using scavengers such as mol sieves (3 Å). Scheme 3
Figure imgf000043_0001
In scheme 3, X,Y, Z, R1, R2 represent (hetero)atoms or substituents according to claim 1; A represents an attaching atom nitrogen optionally substituted with R5, oxygen or sulfur preferably nitrogen; E represents the shown tricyclic thienospiroazetidine or halogen preferably chlorine; R4 represents a C1 to C4 alkyl preferably t-butyl; R5 represents C1 to C6 alkyl preferably methyl or cyclopropyl; the continuous half circle represents any chain connection between the attaching atom A and the carbamate protected amine; the dashed half circle that might be present or not represents a possible second chain connection between the attaching atom A and the carbamate protected amine (in case of a present second chain connection the formed cycle has an unsubstituted nitrogen as attaching atom A to the heterocyclic core); reaction conditions in scheme 3: steps 1, and 2: deprotection; step 3: alkylation. The synthesis towards the tricyclic thienospiroazetidines of general formula (VII) is exemplified in BHC233018 - FC - 43 / 152 - scheme 4. The commercially available carbamate protected spiroazetidines of general formula (XXI)(like e. g. CAS No: 2306272-86-4) can be converted in a Gewald reaction using malononitrile (general formula (XXII)) to build the annelated cyano-aminothiophene compounds of general formula (XXIII). Typical reaction conditions require elementary sulfur as source to thiophene ring construction and further include a nuclephilic catalyst to activate the carbonyl moiety such as (S)-(-)-proline in an aprotic solvent such as N,N-dimethylformamide at temperatures between 50°C and 150°C. Ionic liquids such as 1-butyl-3- methyl-1H-imidazol-3-ium chloride (BMIMCl) can be added to increase the reaction rates. The carbamate groups of the intermediates of general formula (XXIII) need to be removed to yield the deprotected tricyclic thienospiroazetidines of general formula (VII). This final deprotection step can be performed using acidic conditions like trifluoroacetic acid in dichloromethane or HCl in dioxane already described in scheme 1 and 2. Scheme 4
Figure imgf000044_0001
In scheme 4, Z represents CH2 or CH2CH2 according to claim 1; HQ represents an acid able to form a salt with the azetidine of compounds (VII) like HCl, formic acid or trifluoroacetic acid; R4 represents a C1 to C4 alkyl preferably t-butyl; reaction conditions in scheme 4: step 1: sulfur, BMIMCl, (S)-(-)-proline, N,N- dimethylformamide; step 2: deprotection. EXPERIMENTAL SECTION NMR peak forms are stated as they appear in the spectra, possible higher order effects have not been considered. The 1H-NMR data of selected compounds are listed in the form of 1H-NMR peaklists. Therein, for each signal peak the δ value in ppm is given, followed by the signal intensity, reported in round brackets. The δ value-signal intensity pairs from different peaks are separated by commas. Therefore, a peaklist is described by the general form: δ1 (intensity1), δ2 (intensity2), ... , δi (intensityi), ... , δn (intensityn). The intensity of a sharp signal correlates with the height (in cm) of the signal in a printed NMR spectrum. When compared with other signals, this data can be correlated to the real ratios of the signal intensities. In the case of broad signals, more than one peak, or the center of the signal along with their relative intensity, compared to the most intense signal displayed in the spectrum, are shown. A 1H-NMR peaklist BHC233018 - FC - 44 / 152 - is similar to a classical 1H-NMR readout, and thus usually contains all the peaks listed in a classical NMR interpretation. Moreover, similar to classical 1H-NMR printouts, peaklists can show solvent signals, signals derived from stereoisomers of the particular target compound, peaks of impurities, 13C satellite peaks, and/or spinning sidebands. The peaks of stereoisomers, and/or peaks of impurities are typically displayed with a lower intensity compared to the peaks of the target compound (e.g., with a purity of >90%). Such stereoisomers and/or impurities may be typical for the particular manufacturing process, and therefore their peaks may help to identify a reproduction of the manufacturing process on the basis of "by- product fingerprints". An expert who calculates the peaks of the target compound by known methods (MestReC, ACD simulation, or by use of empirically evaluated expectation values), can isolate the peaks of the target compound as required, optionally using additional intensity filters. Such an operation would be similar to peak-picking in classical 1H-NMR interpretation. A detailed description of the reporting of NMR data in the form of peaklists can be found in the publication "Citation of NMR Peaklist Data within Patent Applications" (cf. http://www.researchdisclosure.com/searching-disclosures, Research Disclosure Database Number 605005, 2014, 01 Aug 2014). In the peak picking routine, as described in the Research Disclosure Database Number 605005, the parameter "MinimumHeight" can be adjusted between 1% and 4%. However, depending on the chemical structure and/or depending on the concentration of the measured compound it may be reasonable to set the parameter "MinimumHeight" <1%. Chemical names were generated using the ACD/Name software from ACD/Labs. In some cases generally accepted names of commercially available reagents were used in place of ACD/Name generated names. Optical rotations were measured using a JASCO P2000 Polarimeter. Typical, a solution of the compound with a concentration of 1 mg/mL to 15 mg/mL was used for the measurement. The specific rotation [α]D was calculated according to the following formula:
Figure imgf000045_0001
In this equation, α is the measured rotation in degrees; d is the path length in decimetres and β is the concentration in g/mL. The following table 1 lists the abbreviations used in this paragraph and in the Examples section as far as they are not explained within the text body. Other abbreviations have their meanings customary per se to the skilled person. Table 1: Abbreviations Table 1 lists the abbreviations used in this paragraph and in the Intermediates and Examples sections as far as they are not explained within the text body. BHC233018 - FC - 45 / 152 - Table 1 Abbreviation Meaning ACN acetonitrile AcOH acetic acid (ethanoic acid) aq. Aqueous BINAP 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl Boc tert-butoxycarbonyl BOP (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate br Broad br s broad singlet (in NMR) br d broad doublet (in NMR) br t broad triplet (in NMR) CI chemical ionization Cs2CO3 cesium carbonate d day(s), doublet (in NMR) DAD diode array detector DBU 1,8-diazabicyclo(5.4.0)undec-7-ene DCC N,N‘-dicyclohexylcarbodiimide DCI direct chemical ionization (in MS) DCM Dichloromethane dd doublet of doublets (in NMR) DIBALH diisobutylaluminium hydride DIC N,N'-diisopropylcarbodiimide DIPEA Diisopropylethylamine DMA Dimethylacetamide DMF N,N-dimethylformamide DMSO dimethyl sulfoxide dt double-triplet EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide BHC233018 - FC - 46 / 152 - ELSD Evaporative Light Scattering Detector EtOAc ethyl acetate EtOH Ethanol eq. Equivalent(s) ESI electrospray (ES) ionization (in MS) h Hour(s) HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5- b]pyridinium 3-oxid hexafluorophosphate HBTU (o-benzotriazole-10yl)-N,N,N',N,-tetramethyluronium hexafluorophosphate HCl hydrochloric acid HPLC high-pressure / high performance liquid chromatography K2CO3 potassium carbonate LC-MS liquid chromatography-coupled mass spectrometry LiHMDS lithium bis(trimethylsilyl)amide m multiplet (in NMR) mCPBA meta-Chloroperbenzoic acid min Minute(s) MeCN Acetonitrile MeOH Methanol MS mass spectrometry / mass spectroscopy MTBE methyl-tert-butyl ether n-BuLi n-butyllithium NaCl sodium chloride NaH sodium hydride NH3 ammonia NH4Cl ammonium chloride NBS 1-bromopyrrolidine-2,5-dione; N-Bromsuccinimid NaHCO3 sodium hydrogen carbonate or sodium bicarbonate BHC233018 - FC - 47 / 152 - NMR nuclear magnetic resonance spectroscopy: chemical shifts (δ) are given in ppm. The chemical shifts were corrected by setting the DMSO signal to 2.50 ppm unless otherwise stated. Na2SO4 sodium sulfate PDA Photo Diode Array Pd/C palladium on activated charcoal Pd2(dba)3 tris(dibenzylideneacetone)dipalladium(0) Pd(OAc)2 Palladium(II) acetate PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate q quartet or quadruplet (in NMR) quin quintet (in NMR) RP reverse phase (in HPLC) r.t. or rt or RT room temperature Rt retention time (as measured either with HPLC or UPLC) in minutes s singlet (in NMR) sat. Saturated SEM (2-methoxyethyl)(trimethyl)silyl group SEM-Cl [2-(chloromethoxy)ethyl](trimethyl)silane SFC supercritical fluid chromatography SIBX stabilized 2-iodoxybenzoic acid SM starting material SQD Single-Quadrupole-Detector T3P propylphosphonic anhydride t triplet (in NMR) tBuBrettPhos di-tert-butyl(2',4',6'-triisopropyl-3,6-dimethoxy[biphenyl]-2- yl)phosphine (CAS 1160861-53-9) tBuBrettPhos Pd G3 (2'-amino[biphenyl]-2-yl)(methanesulfonato- kappaO)palladium - di-tert-butyl(2',4',6'-triisopropyl-3,6- BHC233018 - FC - 48 / 152 - dimethoxy[biphenyl]-2-yl)phosphine (1:1) (CAS: 1536473- 72-9) td triple-doublet TEA Triethylamine TFA trifluoroacetic acid TFAA trifluoroacetic anhydride THF Tetrahydrofuran tR retention time (in HPLC) UPLC ultra performance liquid chromatography XPhos or Dicyclohexyl[2′,4′,6′-tris(propan-2-yl)[1,1′-biphenyl]-2- X-Phos yl]phosphane; Dicyclohexyl(2',4',6'-triisopropyl[biphenyl]-2-yl)phosphine; CAS-RN:[564483-18-7] XPhos Pd G3; or (2-Dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′- XPhos-Pd-G3 biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate Other abbreviations have their meanings customary per se to the skilled person. The various aspects of the invention described in this application are illustrated by the following examples which are not meant to limit the invention in any way. The example testing experiments described herein serve to illustrate the present invention and the invention is not limited to the examples given. GENERAL PART All reagents, for which the synthesis is not described in the experimental part, are either commercially available, or are known compounds or may be formed from known compounds by known methods by a person skilled in the art. The compounds and intermediates produced according to the methods of the invention may require purification. Purification of organic compounds is well known to the person skilled in the art and there may be several ways of purifying the same compound. In some cases, no purification may be necessary. In some cases, the compounds may be purified by crystallization. In some cases, impurities may be stirred out using a suitable solvent. In some cases, the compounds may be purified by chromatography, particularly flash column chromatography, using for example prepacked silica gel cartridges, e.g. Biotage BHC233018 - FC - 49 / 152 - SNAP cartidges KP-Sil® or KP-NH® in combination with a Biotage autopurifier system (SP4® or Isolera Four®) and eluents such as gradients of hexane/ethyl acetate or DCM/methanol. In some cases, the compounds may be purified by preparative HPLC using for example a Waters autopurifier equipped with a diode array detector and/or on-line electrospray ionization mass spectrometer in combination with a suitable prepacked reverse phase column and eluents such as gradients of water and acetonitrile which may contain additives such as trifluoroacetic acid, formic acid or aqueous ammonia. In some cases, purification methods as described above can provide those compounds of the present invention which possess a sufficiently basic or acidic functionality in the form of a salt, such as, in the case of a compound of the present invention which is sufficiently basic, a trifluoroacetate or formate salt for example, or, in the case of a compound of the present invention which is sufficiently acidic, an ammonium salt for example. A salt of this type can either be transformed into its free base or free acid form, respectively, by various methods known to the person skilled in the art, or be used as salts in subsequent biological assays. It is to be understood that the specific form (e.g. salt, free base etc.) of a compound of the present invention as isolated and as described herein is not necessarily the only form in which said compound can be applied to a biological assay in order to quantify the specific biological activity. NMR peak forms in the following specific experimental descriptions are stated as they appear in the spectra, possible higher order effects have not been considered. The 1H-NMR data of selected compounds are listed in the form of 1H-NMR peaklists. Therein, for each signal peak the δ value in ppm is given, followed by the signal intensity, reported in round brackets. The δ value-signal intensity pairs from different peaks are separated by commas. Therefore, a peaklist is described by the general form: δ1 (intensity1), δ2 (intensity2), ... , δi (intensityi), ... , δn (intensityn). The intensity of a sharp signal correlates with the height (in cm) of the signal in a printed NMR spectrum. When compared with other signals, this data can be correlated to the real ratios of the signal intensities. In the case of broad signals, more than one peak, or the center of the signal along with their relative intensity, compared to the most intense signal displayed in the spectrum, are shown. A 1H-NMR peaklist is similar to a classical 1H-NMR readout, and thus usually contains all the peaks listed in a classical NMR interpretation. Moreover, similar to classical 1H-NMR printouts, peaklists can show solvent signals, signals derived from stereoisomers of the particular target compound, peaks of impurities, 13C satellite peaks, and/or spinning sidebands. The peaks of stereoisomers, and/or peaks of impurities are typically displayed with a lower intensity compared to the peaks of the target compound (e.g., with a purity of >90%). Such stereoisomers and/or impurities may be typical for the particular manufacturing process, and therefore their peaks may help to identify a reproduction of the manufacturing process on the basis of "by- product fingerprints". An expert who calculates the peaks of the target compound by known methods (MestReC, ACD simulation, or by use of empirically evaluated expectation values), can isolate the peaks BHC233018 - FC - 50 / 152 - of the target compound as required, optionally using additional intensity filters. Such an operation would be similar to peak-picking in classical 1H-NMR interpretation. A detailed description of the reporting of NMR data in the form of peaklists can be found in the publication "Citation of NMR Peaklist Data within Patent Applications" (cf. http://www.researchdisclosure.com/searching-disclosures, Research Disclosure Database Number 605005, 2014, 01 Aug 2014). In the peak picking routine, as described in the Research Disclosure Database Number 605005, the parameter "MinimumHeight" can be adjusted between 1% and 4%. However, depending on the chemical structure and/or depending on the concentration of the measured compound it may be reasonable to set the parameter "MinimumHeight" <1%. Reactions employing microwave irradiation may be run with a Biotage Initiator ^ microwave oven optionally equipped with a robotic unit. The reported reaction times employing microwave heating are intended to be understood as fixed reaction times after reaching the indicated reaction temperature. The compounds and intermediates produced according to the methods of the invention may require purification. Purification of organic compounds is well known to the person skilled in the art and there may be several ways of purifying the same compound. In some cases, no purification may be necessary. In some cases, the compounds may be purified by crystallization. In some cases, impurities may be stirred out using a suitable solvent. In some cases, the compounds may be purified by chromatography, particularly flash column chromatography, using for example pre-packed silica gel cartridges, e.g. from Separtis such as Isolute® Flash silica gel or Isolute® Flash NH2 silica gel in combination with a Isolera® autopurifier (Biotage) and eluents such as gradients of e.g. hexane/ethyl acetate or DCM/methanol. In some cases, the compounds may be purified by preparative HPLC using for example a Waters autopurifier equipped with a diode array detector and/or on-line electrospray ionization mass spectrometer in combination with a suitable prepacked reverse phase column and eluents such as gradients of water and acetonitrile which may contain additives such as trifluoroacetic acid, formic acid or aqueous ammonia. In some cases, purification methods as described above can provide those compounds of the present invention which possess a sufficiently basic or acidic functionality in the form of a salt, such as, in the case of a compound of the present invention which is sufficiently basic, a trifluoroacetate or formate salt for example, or, in the case of a compound of the present invention which is sufficiently acidic, an ammonium salt for example. A salt of this type can either be transformed into its free base or free acid form, respectively, by various methods known to the person skilled in the art, or be used as salts in subsequent biological assays. It is to be understood that the specific form (e.g. salt, free base etc) of a compound of the present invention as isolated as described herein is not necessarily the only form in which said compound can be applied to a biological assay in order to quantify the specific biological activity. The percentage yields reported in the following examples are based on the starting component that was used in the lowest molar amount. Air and moisture sensitive liquids and solutions were transferred via syringe or cannula and introduced into reaction vessels through rubber septa. Commercial grade reagents BHC233018 - FC - 51 / 152 - and solvents were used without further purification. The term “concentrated in vacuo” refers to the use of a Buchi rotary evaporator at a minimum pressure of approximately 15 mm of Hg. All temperatures are reported uncorrected in degrees Celsius (°C). In order that this invention may be better understood, the following examples are set forth. These examples are for the purpose of illustration only, and are not to be construed as limiting the scope of the invention in any manner. All publications mentioned herein are incorporated by reference in their entirety. UPLC-MS Standard Procedures UPLC-MS-data given in the subsequent specific experimental descriptions refer (unless otherwise noted) to the following conditions: LC-MS, Analytical Method 1: Instrument: Thermo Scientific FT-MS; UHPLC: Thermo Scientific UltiMate 3000; column: Waters HSS T3 C181.8 µm, 75 mm × 2.1 mm; eluent A: water + 0.01% formic acid; eluent B: acetonitrile + 0.01% formic acid; gradient: 0.0 min 10% B → 2.5 min 95% B → 3.5 min 95% B; temperature: 50°C; flow rate: 0.90 mL/min; UV detection: 210-400 nm. LC-MS, Analytical Method 2: Instrument: Thermo Scientific FT-MS; UHPLC+: Thermo Scientific Vanquish; column: Waters HSS T3 C181.8 µm, 2.1 mm × 75 mm; eluent A: 1 L water + 0.01% formic acid; eluent B: 1 L acetonitrile + 0.01% formic acid; gradient: 0.0 min 10% B → 2.5 min 95% B → 3.5 min 95% B; temperature: 50°C; flow rate: 0.90 mL/min; UV detection: 210 nm. LC-MS, Analytical Method 3 Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C181.7 µm, 50x2.1mm; eluent A: water + 0.1 vol % formic acid (99%), eluent B: acetonitrile; gradient: 0-1.6 min 1- 99% B, 1.6-2.0 min 99% B; flow 0.8 ml/min; temperature: 60 °C; DAD scan: 210-400 nm. LC-MS, Analytical Method 4 Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C181.7 µm, 50x2.1mm; eluent A: water + 0.2 vol % aqueous ammonia (32%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow 0.8 ml/min; temperature: 60 °C; DAD scan: 210-400 nm. Purification Methods: Flash column chromatography conditions BHC233018 - FC - 52 / 152 - Biotage IsoleraTM chromatography system (http://www.biotage.com/product-area/flash-purification) using pre-packed silica and pre-packed modified silica cartridges. Purification by (flash) column chromatography” as stated in the subsequent specific experimental descriptions refers to the use of a Biotage Isolera purification system. For technical specifications see “Biotage product catalogue” on www.biotage.com. Preparative HPLC, Method A: Instrument: pump: Labomatic HD-5000 or HD-3000, head HDK 280, lowpressure gradient module ND-B1000; manual injection valve: Rheodyne 3725i038; detector: Knauer Azura UVD 2.15; collector: Labomatic Labocol Vario-4000; column: Chromatorex RP C-1810 µm, 125x30mm; eluent A: water + 0.1 vol % formic acid (99%), eluent B: acetonitrile; gradient A: 0 - 15 min 1 – 25% B; flow: 60 mL/min; gradient B: 0 - 15 min 10 – 50% B; flow: 60 mL/min; gradient C: 0 - 15 min 15 – 55% B; flow: 60 mL/min; gradient D: 0 - 15 min 30 – 70% B; flow: 60 mL/min; gradient E: 0 - 15 min 40 – 80% B; flow: 60 mL/min; gradient F: 0 - 15 min 65 – 100% B; flow: 60 mL/min; temperature: 25 °C; solution: max.250 mg / 2ml dimethyl sulfoxide; injection: 1 x 2 ml; Detection: UV 254 nm; Software: SCPA PrepCon5. Preparative HPLC, Method B: Instrument: pump: Labomatic HD-5000 or HD-3000, head HDK 280, lowpressure gradient module ND-B1000; manual injection valve: Rheodyne 3725i038; detector: Knauer Azura UVD 2.15; collector: Labomatic Labocol Vario-4000; column: Chromatorex RP C-1810 µm, 125x30mm; eluent A: water + 0.2 vol-% ammonia (32%), eluent B: acetonitrile; gradient A: 0 - 15 min 1 – 25% B; flow: 60 mL/min; gradient B: 0 - 15 min 10 – 50% B; flow: 60 mL/min; gradient C: 0 - 15 min 15 – 55% B; flow: 60 mL/min; gradient D: 0 - 15 min 30 – 70% B; flow: 60 mL/min; gradient E: 0 - 15 min 40 – 80% B; flow: 60 mL/min; gradient F: 0 - 15 min 65 – 100% B; flow: 60 mL/min; temperature: 25 °C; solution: max.250 mg / 2ml dimethyl sulfoxide; injection: 1 x 2 ml; Detection: UV 254 nm; Software: SCPA PrepCon5. Preparative HPLC, Method C: Instrument: Waters Autopurification MS SingleQuad; Column: Waters XBrigde C185µ 100x30mm; eluent A: water + 0.1 vol % formic acid (99%), eluent B: acetonitrile; gradient: 0-5.5 min 5-100% B; flow 70 ml/min; temperature: 25 °C; DAD scan: 210-400 nm. Preparative HPLC, Method D: Instrument: Waters Autopurificationsystem; column: XBrigde C185 μm 100x30 mm; mobile phase A: water + 0.2 vol % aqueous ammonia (32%); mobile phase B: acetonitril; BHC233018 - FC - 53 / 152 - gradient: 0.0-0.5 min 35% B (35-70 ml/min), 0.5-5.5 min 35-65% B; flow: 70 ml/min; temperature: 25°C; wavelength: 210-400 nm
BHC233018 - FC - 54 / 152 - EXAMPLES AND INTERMEDIATES Intermediates Intermediate 1-1A tert-Butyl 2'-amino-3'-cyano-6',7'-dihydro-1H,5'H-spiro[azetidine-3,4'-[1]benzothiophene]-1- carboxylate
Figure imgf000055_0001
1-Butyl-3-methylimidazolium chloride (BMIMCl, 73 mg, 0.42 mmol, 0.2 eq.), sulfur (201 mg, 6.27 mmol, 3.0 eq.), malononitrile (304 mg, 4.60 mmol, 2.2 eq.) and (S)-(-)-proline (241 mg, 2.09 mmol, 1.0 eq.) were added at RT to a solution of tert-butyl 5-oxo-2-azaspiro[3.5]nonane-2-carboxylate (500 mg, 2.09 mmol) in N,N-dimethylformamide (2.5 mL). The reaction mixture was stirred for 5.5 h at 78°C, cooled to RT, mixed with water and extracted with ethyl acetate. The organic phase was washed with water and brine, dried over phase separation filter paper and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (cyclohexane / ethyl acetate gradient). Yield: 587 mg (84% of theory). LC/MS (method 1): tR = 1.87 min, MS (ESIneg): m/z = 318 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.04 (s, 2H), 4.20 (br d, 1H), 4.08 (br d, 1H), 3.67-3.54 (m, 2H), 2.39 (t, 2H), 1.98-1.89 (m, 2H), 1.71-1.62 (m, 2H), 1.38 (s, 9H). Intermediate 1-1B 2'-Amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate
Figure imgf000055_0002
Trifluoroacetic acid (1.34 mL, 17.46 mmol, 10 eq.) was added at 0°C to a solution of tert-butyl 2'-amino- 3'-cyano-6',7'-dihydro-1H,5'H-spiro[azetidine-3,4'-[1]benzothiophene]-1-carboxylate (587 mg, 1.75 mmol) in dichloromethane (17 mL). The reaction mixture was stirred at RT for 1.5 h and concentrated under reduced pressure. The residue was co-evaporated three times with dichloromethane, dried in vacuo and used without further purification. Yield: 803 mg (76% purity, 100% of theory). LC/MS (method 1): tR = 0.51 min, MS (ESIpos): m/z = 220 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 9.40 (br s, 1H), 8.80 (br s, 1H), 7.23 (br s, 2H), 4.47-4.36 (m, 2H), 3.74-3.65 (m, 2H), 2.42 (t, 2H), 2.14-2.06 (m, BHC233018 - FC - 55 / 152 - 2H), 1.76-1.66 (m, 2H). Intermediate 1-2A tert-Butyl 2'-amino-3'-cyano-5',6'-dihydro-1H-spiro[azetidine-3,4'-cyclopenta[b]thiophene]-1- carboxylate
Figure imgf000056_0001
1-Butyl-3-methylimidazolium chloride (BMIMCl, 39 mg, 0.22 mmol, 0.2 eq.), sulfur (107 mg, 3.33 mmol, 3.0 eq.), malononitrile (161 mg, 2.44 mmol, 2.2 eq.) and (S)-(-)-proline (128 mg, 1.11 mmol, 1.0 eq.) were added at RT to a solution of tert-butyl 5-oxo-2-azaspiro[3.4]octane-2-carboxylate (250 mg, 1.11 mmol) in N,N-dimethylformamide (1.5 mL). The reaction mixture was stirred for 5.5 h at 78°C, cooled to RT, mixed with water and extracted with ethyl acetate. The organic phase was washed with water and brine, dried over phase separation filter paper and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (cyclohexane / ethyl acetate gradient). Yield: 126 mg (37% of theory). LC/MS (method 1): tR = 1.77 min, MS (ESIneg): m/z = 304 [M-H]-. Intermediate 1-2B 2'-Amino-5',6'-dihydrospiro[azetidine-3,4'-cyclopenta[b]thiophene]-3'-carbonitrile trifluoroacetate
Figure imgf000056_0002
Trifluoroacetic acid (318 µL, 4.13 mmol, 10 eq.) was added at 0°C to a solution of tert-butyl 2'-amino-3'- cyano-5',6'-dihydro-1H-spiro[azetidine-3,4'-cyclopenta[b]thiophene]-1-carboxylate (126 mg, 0.41 mmol) in dichloromethane (4.0 mL). The reaction mixture was stirred at RT for 45 min and concentrated under reduced pressure. The residue was co-evaporated several times with dichloromethane, dried in vacuo and used without further purification. Yield: 180 mg (78% purity, 100% of theory). LC/MS (method 1): tR = 0.36 min, MS (ESIpos): m/z = 206 [M+H]+. Intermediate 3-1A [1-(Dimethylamino)cyclopropyl]methanol BHC233018 - FC - 56 / 152 -
Figure imgf000057_0001
A mixture of (1-aminocyclopropyl)methanol (250 mg, 2.87 mmol), formaldehyde solution (37% in water, 2.49 mL, 33.29 mmol, 11.6 eq.) and formic acid (205 µL, 5.42 mmol, 1.89 eq.) was stirred at 100°C overnight, concentrated under reduced pressure and mixed with aqueous sodium hydroxide solution to become alkaline. The solution was diluted with dichloromethane and 2 drops of methanol and purified flash silica gel chromatography (Biotage SNAP cartridge KP-NH, dichloromethane / methanol gradient). Yield: 56 mg (17% of theory).1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 4.33 (t, 1H), 3.47 (d, 2H), 2.34 (s, 6H), 0.51-0.44 (m, 2H), 0.42-0.35 (m, 2H). Intermediate 3-2A 2-[(1H-Pyrazol-5-ylmethyl)amino]propane-1,3-diol
Figure imgf000057_0002
1H-Pyrazole-5-carbaldehyde (61.0 g, 635 mmol, 1.0 eq.) and magnesium sulfate (76.4 g, 635 mmol, 1.0 eq.) were added at 20-30°C to a solution of 2-aminopropane-1,3-diol (57.8 g, 635 mmol, 1.0 eq.) in methanol (610 mL). After addition, the mixture was stirred at this temperature for 18 h, and then sodium borohydride (26.4 g, 698 mmol, 1.1 eq.) was added by portions at 20-30°C. The resulting mixture was stirred at 20-30°C for 2 h, filtered, and the filtrate was used for next step directly. Intermediate 3-2B tert-Butyl (1,3-dihydroxypropan-2-yl)(1H-pyrazol-5-ylmethyl)carbamate
Figure imgf000057_0003
Di-tert-butyl dicarbonate (207 g, 947 mmol, 218 mL) was added at 20-30°C to a solution of 2-[(1H- pyrazol-5-ylmethyl)amino]propane-1,3-diol. After addition, the mixture was stirred at this temperature for 15 h and concentrated under reduced pressure. TLC (ethyl acetate / methanol 10/1, Rf = 0.4). The residue was purified by silica gel column (ethyl acetate / methanol 1 to 10/1). Yield: 90.0 g (crude). Intermediate 3-2C BHC233018 - FC - 57 / 152 - tert-Butyl 6-(hydroxymethyl)-6,7-dihydropyrazolo[1,5-a]pyrazine-5(4H)-carboxylate (racemate)
Figure imgf000058_0001
CH30 A mixture of crude tert-butyl (1,3-dihydroxypropan-2-yl)(1H-pyrazol-5-ylmethyl)carbamate (156 g), tributylphosphine (184 g, 864 mmol, 213 mL) and tetramethylazodicarboxamide (149 g, 864 mmol) in tetrahydrofuran (780 mL) was degassed and purged with nitrogen for three times. The mixture was stirred at 25-30°C for 1 h under nitrogen atmosphere and filtered. The filtrate was concentrated under reduced pressure. TLC (petroleum ether / ethyl acetate 1/1, Rf = 0.4). The residue was purified by column chromatography (petroleum ether / ethyl acetate 3/1 to 1/1). Yield: 100 g (crude). Intermediate 4-1A tert-Butyl 3-(2,6-dichloropyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate
Figure imgf000058_0002
tert-Butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate (609 mg, 2.73 mmol, 1.0 eq.) was added under argon atmosphere at 0°C to a solution of 2,4,6-trichloropyrimidine (500 mg, 2.73 mmol) and triethylamine (380 µL, 2.73 mmol, 1.0 eq.) in dichloromethane (17.5 mL). The reaction mixture was stirred at RT for 20 min and concentrated under reduced pressure. The residue was mixed with dichloromethane (4.0 mL). The formed precipitated was filtered and dried in vacuo to give a first batch of desired product. Yield: 233 mg (24% of theory). The filtrate was purified by flash silica gel chromatography (cyclohexane / ethyl acetate gradient). Yield: 635 mg (65% of theory). LC/MS (method 1): tR = 2.19 min, MS (ESIpos): m/z = 359 [M+H]+. Intermediate 4-1B tert-Butyl 3-(6-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (single stereoisomer) BHC233018 - FC - 58 / 152 -
Figure imgf000059_0001
Sodium hydride (60% in mineral oil, 109 mg, 2.71 mmol, 1.1 eq.) was added under argon atmosphere at 0°C to a solution of [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (single stereoisomer) (401 mg, 2.47 mmol, 1.0 eq.) in N,N-dimethylformamide (5.0 mL). The mixture was stirred at 0°C for 15 min and added at 0°C to a solution of tert-butyl 3-(2,6-dichloropyrimidin-4-yl)-3,8- diazabicyclo[3.2.1]octane-8-carboxylate (886 mg, 2.47 mmol) in N,N-dimethylformamide (10 mL). The reaction mixture was stirred at RT overnight and quenched with water and aqueous hydrochloric acid solution (1 N). The resulting solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). The first eluting product with target molecular mass as formic acid salt (519 mg) was dissolved in ethyl acetate, washed two times with saturated aqueous sodium bicarbonate solution, concentrated under reduced pressure and dried in vacuo. Yield: 402 mg (34% of theory). LC/MS (method 1): tR = 1.33 min, MS (ESIpos): m/z = 482 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 6.53 (s, 1H), 5.32/5.18 (d, 1H), 4.46-4.32 (br s, 1H), 4.20 (br s, 2H), 3.96 (d, 1H), 3.86 (d, 1H), 3.81-3.64 (br s, 1H), 3.11-2.93 (m, 5H), 2.85-2.77 (m, 1H), 2.10-2.04 (m, 1H), 2.01-1.97 (m, 1H), 1.96-1.91 (m, 1H), 1.88-1.68 (m, 5H), 1.61-1.51 (m, 2H), 1.42 (s, 9H). Intermediate 4-1C tert-Butyl 3-[6-(2'-amino-3'-cyano-6',7'-dihydro-1H,5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1- yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-3,8- diazabicyclo[3.2.1]octane-8-carboxylate (single stereoisomer)
Figure imgf000059_0002
Sodium carbonate (97 mg, 0.91 mmol, 4.0 eq.) was added to a solution of tert-butyl 3-(6-chloro-2- {[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl)-3,8- diazabicyclo[3.2.1]octane-8-carboxylate (single stereoisomer) (110 mg, 0.23 mmol) and 2'-amino-6',7'- dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (100 mg, 76% purity, BHC233018 - FC - 59 / 152 - 0.23 mmol, 1.0 eq.) in dimethyl sulfoxide (1.0 mL). The reaction mixture was stirred at 95°C for 7 h in a closed microwave vial, diluted with water and aqueous hydrochloric acid solution (1 N, 912 µL). The suspension was dissolved in a mixture of acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 101 mg (66% of theory). LC/MS (method 1): tR = 1.61 min, MS (ESIneg): m/z = 663 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.04 (s, 2H), 5.31/5.18 (d, 1H), 5.14 (s, 1H), 4.26-4.13 (m, 4H), 4.01-3.69 (m, 6H), 3.12-3.03 (m, 2H), 3.02-2.97 (m, 1H), 2.93-2.85 (m, 2H), 2.85-2.76 (m, 1H), 2.43 (t, 2H), 2.10-1.91 (m, 5H), 1.88-1.65 (m, 7H), 1.64-1.53 (m, 2H), 1.42 (s, 9H). Intermediate 4-2A tert-Butyl 3-(2-chloro-6-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (single stereoisomer)
Figure imgf000060_0001
Sodium hydride (60% in mineral oil, 109 mg, 2.71 mmol, 1.1 eq.) was added under argon atmosphere at 0°C to a solution of [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (single stereoisomer) (401 mg, 2.47 mmol, 1.0 eq.) in N,N-dimethylformamide (5.0 mL). The mixture was stirred at 0°C for 15 min and added at 0°C to a solution of tert-butyl 3-(2,6-dichloropyrimidin-4-yl)-3,8- diazabicyclo[3.2.1]octane-8-carboxylate (886 mg, 2.47 mmol) in N,N-dimethylformamide (10 mL). The reaction mixture was stirred at RT overnight and quenched with water and aqueous hydrochloric acid solution (1 N). The resulting solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). The second eluting product with target molecular mass as formic acid salt (208 mg) was dissolved in ethyl acetate, washed two times with saturated aqueous sodium bicarbonate solution, concentrated under reduced pressure and dried in vacuo. Yield: 185 mg (16% of theory). LC/MS (method 1): tR = 1.42 min, MS (ESIpos): m/z = 482 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 6.03 (s, 1H), 5.32/5.19 (d, 1H), 4.20 (br s, 2H), 4.08-3.82 (br s, 2H), 3.95 (d, 1H), 3.87 (d, 1H), 3.12-2.93 (m, 5H), 2.85-2.77 (m, 1H), 2.08-2.03 (m, 1H), 2.00-1.96 (m, 1H), 1.95-1.89 (m, 1H), 1.89-1.69 (m, 5H), 1.61-1.51 (m, 2H), 1.42 (s, 9H). Intermediate 4-2B tert-Butyl 3-[2-(2'-amino-3'-cyano-6',7'-dihydro-1H,5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1- yl)-6-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-3,8- BHC233018 - FC - 60 / 152 - diazabicyclo[3.2.1]octane-8-carboxylate (single stereoisomer)
Figure imgf000061_0001
Sodium carbonate (29 mg, 0.27 mmol, 4.0 eq.) was added to a solution of tert-butyl 3-(2-chloro-6- {[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl)-3,8- diazabicyclo[3.2.1]octane-8-carboxylate (single stereoisomer) (33 mg, 0.07 mmol) and 2'-amino-6',7'- dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (30 mg, 76% purity, 0.07 mmol, 1.0 eq.) in dimethyl sulfoxide (330 µL). The reaction mixture was stirred at 100°C for 2.5 h in a closed microwave vial, diluted with water and aqueous hydrochloric acid solution (1 N, 300 µL). The mixture was dissolved in a mixture of acetonitrile / water / dimethyl sulfoxide and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 26 mg (58% of theory). LC/MS (method 1): tR = 1.66 min, MS (ESIneg): m/z = 663 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.00 (s, 2H), 5.34 (s, 1H), 5.31/5.18 (d, 1H), 4.24 (d, 2H), 4.15 (m, 2H), 4.08-3.78 (m, 4H), 3.74 (d, 2H), 3.10-3.03 (m, 2H), 3.03-2.97 (m, 1H), 2.91-2.83 (m, 2H), 2.83-2.76 (m, 1H), 2.43 (t, 2H), 2.08-1.90 (m, 5H), 1.87-1.65 (m, 7H), 1.63-1.53 (m, 2H), 1.42 (s, 9H). Intermediate 4-3A tert-Butyl 3-[6-(2'-amino-3'-cyano-5',6'-dihydro-1H-spiro[azetidine-3,4'-cyclopenta[b]thiophen]-1- yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-3,8- diazabicyclo[3.2.1]octane-8-carboxylate (single stereoisomer)
Figure imgf000061_0002
Sodium carbonate (53 mg, 0.50 mmol, 4.0 eq.) was added to a solution of tert-butyl 3-(6-chloro-2- {[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl)-3,8- diazabicyclo[3.2.1]octane-8-carboxylate (single stereoisomer) (60 mg, 0.12 mmol) and 2'-amino-6',7'- dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (61 mg, 78% purity, 0.15 mmol, 1.2 eq.) in dimethyl sulfoxide (1.8 mL). The reaction mixture was stirred at 95°C for 3 h in a BHC233018 - FC - 61 / 152 - closed microwave vial, mixed with additional 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile trifluoroacetate (41 mg, 78% purity, 0.10 mmol, 0.8 eq.) and stirred for another 6 h. The mixture was combined with a batch of a previously performed test reaction (0.03 mmol scale), quenched with aqueous hydrochloric acid solution (1 N, 600 µL) and diluted with water (500 µL) and acetonitrile (500 µL). The solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 34 mg (92% purity, 31% of theory for both reactions). LC/MS (method 1): tR = 1.51 min, MS (ESIneg): m/z = 649 [M-H]-. Intermediate 4-4A (3R)-1-(2,6-Dichloropyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer)
Figure imgf000062_0001
(3R)-3-Methylpiperidin-3-ol hydrochloride (single stereoisomer) (1.27 g, 8.18 mmol, 1.0 eq.) was added under argon atmosphere at 0°C to a solution of 2,4,6-trichloropyrimidine (1.50 g, 8.18 mmol) and triethylamine (2.28 mL, 16.36 mmol, 2.0 eq.) in dichloromethane (51 mL). The reaction mixture was stirred at RT for 1.5 h and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (cyclohexane / ethyl acetate gradient). Yield: 1.52 g (71% of theory). LC/MS (method 1): tR = 1.48 min, MS (ESIpos): m/z = 262 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 6.96 (s, 1H), 4.50 (br s, 1H), 4.40-4.23 / 3.95-3.56 (2x m, 2H), 3.31-2.99 (m, 2H), 1.80-1.65 (m, 1H), 1.65-1.52 (m, 2H), 1.52-1.42 (m, 1H), 1.12 (s, 3H). Intermediate 4-4B (3R)-1-(6-Chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4- yl)-3-methylpiperidin-3-ol formate (single stereoisomer)
Figure imgf000062_0002
x HCOOH Sodium hydride (60% in mineral oil, 38 mg, 0.95 mmol, 1.1 eq.) was added under argon atmosphere at 0°C to a solution of [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (single stereoisomer) (138 mg, 0.87 mmol, 1.0 eq.) in N,N-dimethylformamide (2.0 mL). The mixture was stirred at 0°C for 15 min and added at 0°C to a solution of (3R)-1-(2,6-dichloropyrimidin-4-yl)-3-methylpiperidin-3-ol (single BHC233018 - FC - 62 / 152 - stereoisomer) (227 mg, 0.87 mmol) in N,N-dimethylformamide (3.0 mL). The reaction mixture was stirred at RT overnight, quenched with aqueous hydrochloric acid solution (1 N, 1.0 mL) and diluted with dimethyl sulfoxide (2.0 mL), acetonitrile (1.0 mL) and water (1.0 mL). The resulting solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). The first eluting product with target molecular mass gave the desired compound. Yield: 230 mg (62% of theory). LC/MS (method 1): tR = 0.89 min, MS (ESIpos): m/z = 385 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 8.14 (s, 1H), 6.60 (s, 1H), 5.50/5.36 (d, 1H), 4.47 (br s, 1H), 4.27-4.11 (m, 2H), 3.72-3.3 (m, 6H, partially concealed), 3.12- 2.98 (m, 2H), 2.36-2.20 (m, 2H), 2.18-2.08 (m, 1H), 2.07-1.84 (m, 3H), 1.77-1.65 (m, 1H), 1.65-1.50 (m, 2H), 1.50-1.39 (m, 1H), 1.11 (s, 3H). Intermediate 4-5A (3R)-1-(2-Chloro-6-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4- yl)-3-methylpiperidin-3-ol formate (single stereoisomer)
Figure imgf000063_0001
x HCOOH Sodium hydride (60% in mineral oil, 38 mg, 0.95 mmol, 1.1 eq.) was added under argon atmosphere at 0°C to a solution of [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (single stereoisomer) (138 mg, 0.87 mmol, 1.0 eq.) in N,N-dimethylformamide (2.0 mL). The mixture was stirred at 0°C for 15 min and added at 0°C to a solution of (3R)-1-(2,6-dichloropyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (227 mg, 0.87 mmol) in N,N-dimethylformamide (3.0 mL). The reaction mixture was stirred at RT overnight, quenched with aqueous hydrochloric acid solution (1 N, 1.0 mL) and diluted with dimethyl sulfoxide (2.0 mL), acetonitrile (1.0 mL) and water (1.0 mL). The resulting solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). The second eluting product with target molecular mass gave the desired compound. Yield: 31 mg (8% of theory). LC/MS (method 1): tR = 0.94 min, MS (ESIpos): m/z = 385 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 8.15 (s, 1H), 6.02 (s, 1H), 5.34/5.20 (d, 1H), 4.45 (br s, 1H), 3.94 (d, 1H), 3.87 (d, 1H), 2.87-2.78 (m, 1H), 2.13-1.63 (m, 7H), 1.63-1.38 (m, 3H), 1.10 (s, 3H), 7H partially concealed. Intermediate 4-6A (5S)-5-[({4-Chloro-6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-2- yl}oxy)methyl]pyrrolidin-2-one formate (single stereoisomer) BHC233018 - FC - 63 / 152 -
Figure imgf000064_0001
x Sodium hydride (60% in mineral oil, 17 mg, 0.42 mmol, 2.2 eq.) was added under argon atmosphere at 0°C to a solution of (5S)-5-(hydroxymethyl)pyrrolidin-2-one (single stereoisomer) (22 mg, 0.19 mmol, 1.0 eq.) in N,N-dimethylformamide (400 µL). The mixture was stirred at 0°C for 15 min and added at 0°C to a solution of (3R)-1-(2,6-dichloropyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (50 mg, 0.19 mmol) in N,N-dimethylformamide (600 µL). The reaction mixture was stirred at RT overnight, combined with a batch of a previously performed test reaction (0.19 mmol scale), quenched with aqueous hydrochloric acid solution (1 N, 1.0 mL) and diluted with dimethyl sulfoxide (2.0 mL), acetonitrile (1.0 mL) and water (1.0 mL). The resulting solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). The first eluting product with target molecular mass gave the desired compound. Yield: 63 mg (43% of theory for both reactions). LC/MS (method 1): tR = 1.12 min, MS (ESIpos): m/z = 341 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 8.14 (s, 1H), 7.79 (s, 1H), 6.56 (s, 1H), 4.45 (br s, 1H), 4.18-4.08 (m, 2H), 3.90-3.81 (m, 1H), 3.73-3.3 (m, 4H, partially concealed), 2.26-2.05 (m, 3H), 1.87- 1.78 (m, 1H), 1.78-1.64 (m 1H), 1.64-1.49 (m, 2H), 1.49-1.38 (m, 1H), 1.10 (s, 3H). Intermediate 4-7A (5S)-5-[({2-Chloro-6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-4- yl}oxy)methyl]pyrrolidin-2-one formate (single stereoisomer)
Figure imgf000064_0002
x Sodium hydride (60% in mineral oil, 17 mg, 0.42 mmol, 2.2 eq.) was added under argon atmosphere at 0°C to a solution of (5S)-5-(hydroxymethyl)pyrrolidin-2-one (single stereoisomer) (22 mg, 0.19 mmol, 1.0 eq.) in N,N-dimethylformamide (400 µL). The mixture was stirred at 0°C for 15 min and added at 0°C to a solution of (3R)-1-(2,6-dichloropyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (50 mg, 0.19 mmol) in N,N-dimethylformamide (600 µL). The reaction mixture was stirred at RT overnight, combined with a batch of a previously performed test reaction (0.19 mmol scale), quenched with aqueous hydrochloric acid solution (1 N, 1.0 mL) and diluted with dimethyl sulfoxide (2.0 mL), acetonitrile (1.0 mL) and water (1.0 mL). The resulting solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid BHC233018 - FC - 64 / 152 - in water gradient). The second eluting product with target molecular mass gave the desired compound. Yield: 7 mg (94% purity, 4% of theory for both reactions). LC/MS (method 1): tR = 1.19 min, MS (ESIpos): m/z = 341 [M+H]+. Intermediate 4-8A (3R)-1-(6-Chloro-2-{[(2S)-4,4-difluoro-1-methylpyrrolidin-2-yl]methoxy}pyrimidin-4-yl)-3- methylpiperidin-3-ol (single stereoisomer)
Figure imgf000065_0001
Sodium hydride (60% in mineral oil, 50 mg, 1.18 mmol, 2.2 eq.) was added under argon atmosphere at 0°C to a solution of [(2S)-4,4-difluoro-1-methylpyrrolidin-2-yl]methanol (single stereoisomer) (81 mg, 0.53 mmol, 1.0 eq.) in N,N-dimethylformamide (1.0 mL). The mixture was stirred at 0°C for 15 min and added at 0°C to a solution of (3R)-1-(2,6-dichloropyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (140 mg, 0.53 mmol) in N,N-dimethylformamide (2.0 mL). The reaction mixture was stirred at RT for 1 h, quenched with aqueous hydrochloric acid solution (1 N, 1.2 mL) and diluted with water. The resulting solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). The first eluting product with target molecular mass gave the desired compound. Yield: 132 mg (66% of theory). LC/MS (method 1): tR = 1.13 min, MS (ESIpos): m/z = 377 [M+H]+. Intermediate 4-9A tert-Butyl (2S,4R)-2-[({4-chloro-6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-2- yl}oxy)methyl]-4-fluoropyrrolidine-1-carboxylate (single stereoisomer)
Figure imgf000065_0002
Sodium hydride (60% in mineral oil, 101 mg, 2.52 mmol, 2.2 eq.) was added under argon atmosphere at 0°C to a solution of tert-butyl (2S,4R)-4-fluoro-2-(hydroxymethyl)pyrrolidine-1-carboxylate (single stereoisomer) (251 mg, 1.14 mmol, 1.0 eq.) in N,N-dimethylformamide (3.0 mL). The mixture was stirred at 0°C for 15 min and added at 0°C to a solution of (3R)-1-(2,6-dichloropyrimidin-4-yl)-3- BHC233018 - FC - 65 / 152 - methylpiperidin-3-ol (single stereoisomer) (300 mg, 1.14 mmol) in N,N-dimethylformamide (4.0 mL). The reaction mixture was stirred at RT overnight, added to saturated aqueous ammonium chloride solution, diluted with water and extracted with ethyl acetate. After phase separation, the aqueous phase was two times extracted with ethyl acetate. The combined organic phases were washed with brine, dried over phase separation filter paper and concentrated under reduced pressure. The residue was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 238 mg (47% of theory). LC/MS (method 1): tR = 1.90 min, MS (ESIpos): m/z = 445 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 6.56 (s, 1H), 5.34/5.20 (d, 1H), 4.45 (br s, 1H), 4.40-4.28 (m, 2H), 4.22-4.12 (m, 1H), 3.84-3.65 (m, 1H), 3.65-3.49 (m, 1H), 3.49-3.04 (m, 4H, partially concealed), 2.42-2.24 (m, 1H), 2.20-2.11/2.10-2.00 (2x m, 1H), 1.77-1.64 (m, 1H), 1.64-1.49 (m, 2H), 1.49-1.4 (m, 1H, partially concealed), 1.39 (s, 9H), 1.10 (s, 3H). Intermediate 4-9B (3R)-1-(6-Chloro-2-{[(2S,4R)-4-fluoropyrrolidin-2-yl]methoxy}pyrimidin-4-yl)-3-methylpiperidin- 3-ol (single stereoisomer)
Figure imgf000066_0001
chloride solution (4 M in 1,4-dioxane, 2.66 mL, 10.65 mmol, 20 eq.) was added to a solution tert-butyl (2S,4R)-2-[({4-chloro-6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-2- yl}oxy)methyl]-4-fluoropyrrolidine-1-carboxylate (single stereoisomer) (237 mg, 0.53 mmol) in dichloromethane (20 mL). The reaction mixture was stirred at RT for 2.5 h and concentrated under reduced pressure. The residue was co-evaporated two times with dichloromethane, dried in vacuo and used without further purification. Yield: 241 mg (84% purity, 100% of theory). LC/MS (method 1): tR = 0.77 min, MS (ESIpos): m/z = 345 [M+H]+. Intermediate 4-9C (3R)-1-(6-Chloro-2-{[(2S,4R)-4-fluoro-1-methylpyrrolidin-2-yl]methoxy}pyrimidin-4-yl)-3- methylpiperidin-3-ol (single stereoisomer) BHC233018 - FC - 66 / 152 -
Figure imgf000067_0001
Formaldehyde solution (35% in water, 44 µL, 0.56 mmol, 5.0 eq.) was added under argon atmosphere at RT to a solution of (3R)-1-(6-chloro-2-{[(2S,4R)-4-fluoropyrrolidin-2-yl]methoxy}pyrimidin-4-yl)-3- methylpiperidin-3-ol (single stereoisomer) (41 mg, 94% purity, 0.11 mmol) in acetonitrile (0.7 mL). The mixture was stirred for 20 min, followed by addition of sodium cyanoborohydride (11 mg, 0.18 mmol, 1.6 eq.) in portions. The reaction mixture was stirred at RT for another 30 min, combined with a batch of a previously performed test reaction (0.08 mmol scale) and concentrated under reduced pressure. The residue was mixed with aqueous sodium hydroxide solution (1 N), and the solution was extracted three times with dichloromethane. The combined organic phases were washed with water and brine, dried over phase separation filter paper and concentrated under reduced pressure. The residue was purified by RP- HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 22 mg (32% of theory for both reactions). LC/MS (method 1): tR = 0.79 min, MS (ESIpos): m/z = 359 [M+H]+. Intermediate 4-10A (3R)-1-(6-Chloro-2-{[(2S,4R)-1-cyclopropyl-4-fluoropyrrolidin-2-yl]methoxy}pyrimidin-4-yl)-3- methylpiperidin-3-ol (single stereoisomer)
Figure imgf000067_0002
Sodium cyanoborohydride (150 mg, 2.38 mmol, 4.5 eq.) was added at RT to a mixture of (3R)-1-(6-chloro- 2-{[(2S,4R)-4-fluoropyrrolidin-2-yl]methoxy}pyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (240 mg, 84% purity, 0.53 mmol), molecular sieve (3 Å, 191 mg), acetic acid (94 µL, 1.64 mmol, 3.1 eq.) and [(1-ethoxycyclopropyl)oxy](trimethyl)silane (638 µL, 3.17 mmol, 6.0 eq.) in methanol (3.9 mL). The reaction mixture was stirred at reflux for 2 h and concentrated under reduced pressure. The residue was mixed with aqueous sodium hydroxide solution (1 N) and extracted with ethyl acetate. After phase separation, the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with brine, dried over phase separation filter paper and concentrated under reduced pressure. The aqueous phase still contained desired product and was again extracted three times with ethyl acetate. The BHC233018 - FC - 67 / 152 - combined organic phases were washed with brine, dried over phase separation filter paper and concentrated under reduced pressure. The combined raw materials were dissolved in dimethyl sulfoxide (6.0 mL) and acetonitrile (1.0 mL), and the solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 137 mg (67% of theory). LC/MS (method 2): tR = 1.07 min, MS (ESIpos): m/z = 385 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 6.54 (s, 1H), 5.24/5.10 (d, 1H), 4.47 (d, 1H), 4.44 (d, 1H), 4.27 (br s, 1H), 4.05 (dd, 1H), 3.58 (br s, 1H), 3.40 (dd, 1H), 3.3-3.08 (m, 3H, partially concealed), 2.81 (dd, 1H), 2.22-2.06 (m, 1H), 1.99-1.77 (m, 2H), 1.77-1.64 (m, 1H), 1.64-1.49 (m, 2H), 1.49-1.38 (m, 1H), 1.10 (s, 3H), 0.51-0.43 (m, 1H), 0.43-0.32 (m, 2H), 0.32-0.23 (m, 1H). Intermediate 4-11A tert-Butyl 3-[({4-chloro-6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl}oxy)methyl]-3- cyanopyrrolidine-1-carboxylate (mixture of two enantiopure diastereomers)
Figure imgf000068_0001
H3C CH3 Sodium hydride (60% in mineral oil, 122 mg, 3.06 mmol, 2.2 eq.) was added under argon atmosphere at 0°C to a solution of tert-butyl 3-cyano-3-(hydroxymethyl)pyrrolidine-1-carboxylate (racemate) (314 mg, 1.39 mmol, 1.0 eq.) in N,N-dimethylformamide (3.5 mL). The mixture was stirred at 0°C for 15 min and added at 0°C to a solution of (3R)-1-(2,6-dichloropyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (364 mg, 1.39 mmol) in N,N-dimethylformamide (4.5 mL). The reaction mixture was stirred at RT overnight, added to saturated aqueous ammonium chloride solution, diluted with water and extracted with ethyl acetate. After phase separation, the aqueous phase was two times extracted with ethyl acetate. The combined organic phases were washed with brine, dried over phase separation filter paper and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (cyclohexane / ethyl acetate gradient). Yield: 436 mg (69% of theory). LC/MS (method 1): tR = 1.87 min, MS (ESIpos): m/z = 452 [M+H]+. Intermediate 4-11B tert-Butyl 3-[({4-(2'-amino-3'-cyano-6',7'-dihydro-1H,5'H-spiro[azetidine-3,4'-[1]benzothiophen]- 1-yl)-6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl}oxy)methyl]-3-cyanopyrrolidine-1- carboxylate (mixture of two enantiopure diastereomers) BHC233018 - FC - 68 / 152 -
Figure imgf000069_0001
Sodium carbonate (89 mg, 0.84 mmol, 4.0 eq.) was added to a solution of tert-butyl 3-[({4-chloro-6-[(3R)- 3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl}oxy)methyl]-3-cyanopyrrolidine-1-carboxylate (mixture of two enantiopure diastereomers) (95 mg, 0.21 mmol) and 2'-amino-6',7'-dihydro-5'H- spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (146 mg, 72% purity, 0.32 mmol, 1.5 eq.) in dimethyl sulfoxide (1.8 mL). The reaction mixture was stirred at 95°C overnight in a closed microwave vial and quenched with aqueous hydrochloric acid solution (1 N, 841 µL). The mixture was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 58 mg (43% of theory). LC/MS (method 1): tR = 1.80 min, MS (ESIpos): m/z = 635 [M+H]+. Intermediate 4-12A 3-[({4-Chloro-6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl}oxy)methyl]pyrrolidine-3- carbonitrile hydrochloride (mixture of two enantiopure diastereomers)
Figure imgf000069_0002
x Hydrogen chloride solution (4 M in 1,4-dioxane, 2.61 mL, 10.44 mmol, 20 eq.) was added to a solution of tert-butyl 3-[({4-chloro-6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl}oxy)methyl]-3- cyanopyrrolidine-1-carboxylate (mixture of two enantiopure diastereomers) (236 mg, 0.52 mmol) in dichloromethane (5.0 mL). The reaction mixture was stirred at RT for 1.5 h and concentrated under reduced pressure. The residue was co-evaporated several times with dichloromethane, dried in vacuo and used without further purification. Yield: 234 mg (100% of theory). LC/MS (method 1): tR = 0.79 min, MS (ESIpos): m/z = 352 [M+H]+. Intermediate 4-12B 3-[({4-Chloro-6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl}oxy)methyl]-1- methylpyrrolidine-3-carbonitrile (mixture of two enantiopure diastereomers) BHC233018 - FC - 69 / 152 -
Figure imgf000070_0001
Formaldehyde solution (35% in water, 227 µL, 2.86 mmol, 5.0 eq.) was added under argon atmosphere at RT to a solution of 3-[({4-chloro-6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-2- yl}oxy)methyl]pyrrolidine-3-carbonitrile hydrochloride (mixture of two enantiopure diastereomers) (234 mg, 0.57 mmol) in acetonitrile (4.0 mL). The mixture was stirred for 20 min, followed by addition of sodium cyanoborohydride (58 mg, 0.92 mmol, 1.6 eq.) in portions. The reaction mixture was stirred at RT for another 30 min, mixed with aqueous sodium hydroxide solution (1 N), and the solution was extracted three times with dichloromethane. The combined organic phases were washed with water and brine, dried over phase separation filter paper and concentrated under reduced pressure. The residue was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 77 mg (37% of theory). LC/MS (method 1): tR = 0.81 min, MS (ESIpos): m/z = 366 [M+H]+. Intermediate 4-13A (3R)-1-{6-Chloro-2-[2-(1-methyl-1H-imidazol-2-yl)ethoxy]pyrimidin-4-yl}-3-methylpiperidin-3-ol (single stereoisomer)
Figure imgf000070_0002
Sodium hydride (60% in mineral oil, 34 mg, 0.84 mmol, 2.2 eq.) was added under argon atmosphere at 0°C to a solution of 2-(1-methyl-1H-imidazol-2-yl)ethanol (48 mg, 0.38 mmol, 1.0 eq.) in N,N- dimethylformamide (1.0 mL). The mixture was stirred at 0°C for 15 min and added at 0°C to a solution of (3R)-1-(2,6-dichloropyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (100 mg, 0.38 mmol) in N,N-dimethylformamide (1.0 mL). The reaction mixture was stirred at RT overnight, quenched with aqueous hydrochloric acid solution (1 N) and diluted with dimethyl sulfoxide (2.0 mL), acetonitrile (1.0 mL) and water (1.0 mL). The resulting solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). The resulting product was dissolved in ethyl acetate and washed with saturated aqueous sodium bicarbonate solution. After phase separation, the aqueous phase was two times extracted with ethyl acetate. The combined organic phases were washed with brine, dried over phase separation filter paper, concentrated under reduced pressure and dried in vacuo. Yield: 436 mg (69% of theory). BHC233018 - FC - 70 / 152 - LC/MS (method 1): tR = 0.80 min, MS (ESIpos): m/z = 352 [M+H]+. Intermediate 4-14A tert-Butyl (2S)-2-[({4-chloro-6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-2- yl}oxy)methyl]-2-methylpyrrolidine-1-carboxylate (single stereoisomer)
Figure imgf000071_0001
Sodium hydride (60% in mineral oil, 18 mg, 0.50 mmol, 1.5 eq.) was added under argon atmosphere at 0°C to a solution of tert-butyl (2S)-2-(hydroxymethyl)-2-methylpyrrolidine-1-carboxylate (single stereoisomer) (108 mg, 0.50 mmol, 1.0 eq.) in N,N-dimethylformamide (1.0 mL). The mixture was stirred at 0°C for 15 min and added at 0°C to a solution of (3R)-1-(2,6-dichloropyrimidin-4-yl)-3- methylpiperidin-3-ol (single stereoisomer) (131 mg, 0.50 mmol) in N,N-dimethylformamide (2.0 mL). The reaction mixture was stirred at RT overnight, combined with a batch of a previously performed test reaction (0.10 mmol scale), quenched with saturated aqueous ammonium chloride solution, diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried over phase separation filter paper and concentrated under reduced pressure. The residue was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 99 mg (89% purity, 33% of theory for both reactions). LC/MS (method 1): tR = 2.15 min, MS (ESIpos): m/z = 441 [M+H]+. Intermediate 4-14B tert-Butyl (2S)-2-[({4-(2'-amino-3'-cyano-6',7'-dihydro-1H,5'H-spiro[azetidine-3,4'- [1]benzothiophen]-1-yl)-6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl}oxy)methyl]-2- methylpyrrolidine-1-carboxylate (single stereoisomer)
Figure imgf000071_0002
Sodium carbonate (26 mg, 0.24 mmol, 4.0 eq.) was added to a solution of tert-butyl (2S)-2-[({4-chloro-6- [(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl}oxy)methyl]-2-methylpyrrolidine-1-carboxylate BHC233018 - FC - 71 / 152 - (single stereoisomer) (30 mg, 89% purity, 0.06 mmol) and 2'-amino-6',7'-dihydro-5'H-spiro[azetidine- 3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (37 mg, 71% purity, 0.08 mmol, 1.3 eq.) in dimethyl sulfoxide (1.0 mL). The reaction mixture was stirred at 95°C overnight in a closed microwave vial and quenched with aqueous hydrochloric acid solution (1 N, 242 µL). The mixture was diluted with water (1.0 mL) and acetonitrile (1.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 32 mg (83% of theory). LC/MS (method 1): tR = 1.73 min, MS (ESIpos): m/z = 624 [M+H]+. Intermediate 4-15A (3R)-1-(6-Chloro-2-{[1-(dimethylamino)cyclopropyl]methoxy}pyrimidin-4-yl)-3-methylpiperidin- 3-ol (single stereoisomer)
Figure imgf000072_0001
Sodium hydride (60% in mineral oil, 38 mg, 0.94 mmol, 2.0 eq.) was added under argon atmosphere at 0°C to a solution of [1-(dimethylamino)cyclopropyl]methanol (54 mg, 0.47 mmol, 1.0 eq.) in N,N- dimethylformamide (1.0 mL). The mixture was stirred at 0°C for 15 min and added at 0°C to a solution of (3R)-1-(2,6-dichloropyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (123 mg, 0.47 mmol) in N,N-dimethylformamide (2.0 mL). The reaction mixture was stirred at RT for 3.5 h, quenched with saturated aqueous ammonium chloride solution, diluted with water and extracted three times with ethyl acetate. The combined organic phases were washed with brine, dried over phase separation filter paper, concentrated under reduced pressure and dried in vacuo. The residue was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 65 mg (41% of theory). LC/MS (method 2): tR = 0.85 min, MS (ESIpos): m/z = 341 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 6.52 (s, 1H), 4.45 (br s, 1H), 4.28 (s, 2H), 3.65-5.50 (m, 2H), 2.34 (s, 6H), 1.76-1.38 (m, 4H), 1.10 (s, 3H), 0.69-0.62 (m, 2H), 0.62-0.56 (m, 2H), 2H concealed. Intermediate 4-16A 6-(2,6-Dichloropyrimidin-4-yl)-1,6-diazaspiro[3.5]nonan-2-one (racemate)
Figure imgf000072_0002
BHC233018 - FC - 72 / 152 - 1,6-Diazaspiro[3.5]nonan-2-one (racemate) (235 mg, 1.68 mmol, 1.0 eq.) was added under argon atmosphere at 0°C to a solution of 2,4,6-trichloropyrimidine (307 mg, 1.68 mmol) and triethylamine (0.35 mL, 2.52 mmol, 1.5 eq.) in dichloromethane (7.0 mL). The reaction mixture was stirred at RT for 1 h, combined with a batch of a previously performed test reaction (0.11 mmol scale) and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (dichloromethane / methanol gradient) and subsequent RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 293 mg (57% of theory for both reactions). LC/MS (method 1): tR = 1.26 min, MS (ESIpos): m/z = 287 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 8.37 (s, 1H), 7.15 (br s, 1H), 4.20-3.69 (m, 2H), 3.69-3.53 (m, 1H), 3.44-3.3 (m, 1H, partially concealed), 2.70-2.5 (m, 2H, partially concealed), 1.93-1.82 (m, 1H), 1.80-1.70 (m, 1H), 1.67-1.55 (m, 2H). Intermediate 4-16B 6-(6-Chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl)- 1,6-diazaspiro[3.5]nonan-2-one (mixture of two enantiopure diastereomers)
Figure imgf000073_0001
Sodium hydride (60% in mineral oil, 31 mg, 0.77 mmol, 2.2 eq.) was added under argon atmosphere at 0°C to a solution of [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (single stereoisomer) (55 mg, 0.35 mmol, 1.0 eq.) in N,N-dimethylformamide (0.8 mL). The mixture was stirred at 0°C for 15 min and added at 0°C to a solution of 6-(2,6-dichloropyrimidin-4-yl)-1,6-diazaspiro[3.5]nonan-2-one (racemate) (100 mg, 0.35 mmol) in N,N-dimethylformamide (1.2 mL). The reaction mixture was stirred at RT overnight, combined with a batch of a previously performed test reaction (0.09 mmol scale) and quenched with aqueous hydrochloric acid solution (1 N, 0.45 mL). Purification of the resulting mixture by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient) failed, and all product containing fractions were combined and concentrated under reduced pressure. The residue was stirred in a mixture of dichloromethane / methanol (4:1, 5.0 mL), and the insoluble solid was filtered and discarded. The filtrate was concentrated under reduced pressure and dried in vacuo. Yield: 52 mg (82% purity, 24% of theory). LC/MS (method 1): tR = 0.80 min, MS (ESIpos): m/z = 410 [M+H]+. Intermediate 4-17A tert-Butyl 7-(2,6-dichloropyrimidin-4-yl)-3-oxa-7,9-diazabicyclo[3.3.1]nonane-9-carboxylate BHC233018 - FC - 73 / 152 -
Figure imgf000074_0001
tert-Butyl 3-oxa-7,9-diazabicyclo[3.3.1]nonane-9-carboxylate (685 mg, 3.00 mmol, 1.0 eq.) was added under argon atmosphere at 0°C to a solution of 2,4,6-trichloropyrimidine (550 mg, 3.00 mmol) and triethylamine (0.63 mL, 4.50 mmol, 1.5 eq.) in dichloromethane (15 mL). The reaction mixture was stirred at RT for 1.5 h, mixed with additional tert-butyl 3-oxa-7,9-diazabicyclo[3.3.1]nonane-9-carboxylate (171 mg, 0.75 mmol, 0.25 eq.), stirred at RT for another 3 h and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (cyclohexane / ethyl acetate gradient). Yield: 636 mg (56% of theory). LC/MS (method 1): tR = 1.95 min, MS (ESIpos): m/z = 375 [M+H]+. Intermediate 4-17B tert-Butyl 7-(6-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl)-3-oxa-7,9-diazabicyclo[3.3.1]nonane-9-carboxylate (single stereoisomer)
Figure imgf000074_0002
Sodium hydride (60% in mineral oil, 35 mg, 0.88 mmol, 2.2 eq.) was added under argon atmosphere at 0°C to a solution of [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (single stereoisomer) (64 mg, 0.40 mmol, 1.0 eq.) in N,N-dimethylformamide (1.0 mL). The mixture was stirred at 0°C for 15 min and added at 0°C to a solution of tert-butyl 7-(2,6-dichloropyrimidin-4-yl)-3-oxa-7,9- diazabicyclo[3.3.1]nonane-9-carboxylate (150 mg, 0.40 mmol) in N,N-dimethylformamide (2.0 mL). The reaction mixture was stirred at RT for 4 h, quenched with aqueous hydrochloric acid solution (1 N, 0.88 mL) and diluted with water (0.5 mL) and acetonitrile (0.5 mL). The solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 81 mg (41% of theory). LC/MS (method 1): tR = 1.20 min, MS (ESIpos): m/z = 498 [M+H]+. Intermediate 4-17C tert-Butyl 7-[6-(2'-amino-3'-cyano-6',7'-dihydro-1H,5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1- yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-3-oxa-7,9- BHC233018 - FC - 74 / 152 - diazabicyclo[3.3.1]nonane-9-carboxylate (single stereoisomer)
Figure imgf000075_0001
Sodium carbonate (68 mg, 0.64 mmol, 4.0 eq.) was added to a solution of tert-butyl 7-(6-chloro-2- {[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl)-3-oxa-7,9- diazabicyclo[3.3.1]nonane-9-carboxylate (single stereoisomer) (80 mg, 0.16 mmol) and 2'-amino-6',7'- dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (85 mg, 76% purity, 0.19 mmol, 1.2 eq.) in dimethyl sulfoxide (2.0 mL). The reaction mixture was stirred at 95°C overnight in a closed microwave vial, mixed with additional 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile trifluoroacetate (30 mg, 72% purity, 0.06 mmol, 0.4 eq.), stirred at 95°C for another 2 h, mixed with additional 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile trifluoroacetate (30 mg, 72% purity, 0.06 mmol, 0.4 eq.) and sodium carbonate (17 mg, 0.16 mmol, 1.0 eq.), stirred at 95°C for another 6 h, mixed with additional 2'-amino- 6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (22 mg, 72% purity, 0.05 mmol, 0.3 eq.) and sodium carbonate (9 mg, 0.08 mmol, 0.5 eq.) and stirred at 95°C for another 6 h. The mixture was quenched with aqueous hydrochloric acid solution (1 N, 884 µL) and diluted with water (0.5 mL) and acetonitrile (0.5 mL). The solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 54 mg (91% purity, 45% of theory). LC/MS (method 1): tR = 1.38 min, MS (ESIneg): m/z = 679 [M-H]-. Intermediate 4-19A (3R)-1-(2,6-Dichloro-5-methylpyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer)
Figure imgf000075_0002
(3R)-3-Methylpiperidin-3-ol hydrochloride (single stereoisomer) (118 mg, 0.76 mmol, 1.0 eq.) was added under argon atmosphere at 0°C to a solution of 2,4,6-trichloro-5-methylpyrimidine (150 mg, 0.76 mmol) and triethylamine (212 µL, 1.52 mmol, 2.0 eq.) in dichloromethane (4.5 mL). The reaction mixture was stirred at RT for 1.5 h and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (cyclohexane / ethyl acetate gradient). Yield: 181 mg (86% of theory). LC/MS BHC233018 - FC - 75 / 152 - (method 1): tR = 1.67 min, MS (ESIpos): m/z = 276 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 4.45 (s, 1H), 3.79-3.71 (m, 1H), 3.45 (d, 1H), 3.19 (d, 1H), 3.11-3.02 (m, 1H), 2.20 (s, 3H), 1.95-1.83 (m, 1H), 1.64-1.44 (m, 3H), 1.09 (s, 3H). Intermediate 4-19B (3R)-1-(6-Chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-5- methylpyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer)
Figure imgf000076_0001
Sodium hydride (60% in mineral oil, 29 mg, 0.71 mmol, 1.1 eq.) was added under argon atmosphere at 0°C to a mixture of [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (single stereoisomer) (103 mg, 0.65 mmol, 1.0 eq.) and molecular sieve (3 Å, 20 mg) in N,N-dimethylformamide (1.5 mL). The mixture was stirred at 0°C for 15 min and added at 0°C to a mixture of (3R)-1-(2,6-dichloro-5- methylpyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (179 mg, 0.65 mmol) and molecular sieve (3 Å, 20 mg) in N,N-dimethylformamide (2.5 mL). The reaction mixture was stirred at RT for 4 h, quenched with aqueous hydrochloric acid solution (1 N, 713 µL) and diluted with dimethyl sulfoxide (2.0 mL), acetonitrile (1.0 mL) and water (1.0 mL). The resulting solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). The first eluting product with target molecular mass gave the desired compound. Yield: 86 mg (33% of theory). LC/MS (method 1): tR = 0.95 min, MS (ESIpos): m/z = 399 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 5.41/5.28 (d, 1H), 4.46 (br s, 1H), 4.09 (d, 1H), 4.02 (d, 1H), 3.67-3.3 (m, 3H, partially concealed), 3.3-3.04 (m, 4H, partially concealed), 3.01-2.90 (m, 1H), 2.28-2.09 (m, 2H), 2.15 (s, 3H), 2.09-1.75 (m, 5H), 1.63-1.45 (m, 3H), 1.09 (s, 3H). Intermediate 4-20A (3R)-1-(2,6-Dichloro-5-fluoropyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer)
Figure imgf000076_0002
(3R)-3-Methylpiperidin-3-ol hydrochloride (single stereoisomer) (115 mg, 0.75 mmol, 1.0 eq.) was added under argon atmosphere at 0°C to a solution of 2,4,6-trichloro-5-fluoropyrimidine (150 mg, 0.75 mmol) BHC233018 - FC - 76 / 152 - and triethylamine (208 µL, 1.49 mmol, 2.0 eq.) in dichloromethane (4.5 mL). The reaction mixture was stirred at RT for 1.5 h and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (cyclohexane / ethyl acetate gradient). Yield: 200 mg (96% of theory). LC/MS (method 1): tR = 1.68 min, MS (ESIpos): m/z = 280 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 4.61 (s, 1H), 4.13-4.02 (m, 1H), 3.80 (d, 1H), 3.27-3.16 (m, 1H), 1.89-1.74 (m, 1H), 1.67-1.44 (m, 3H), 1.11 (s, 3H), 1H concealed. Intermediate 4-20B Mixture of (3R)-1-(6-Chloro-5-fluoro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) and (3R)-1-(2-chloro-5- fluoro-6-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl)-3- methylpiperidin-3-ol (single stereoisomer)
Figure imgf000077_0001
Sodium hydride (60% in mineral oil, 31 mg, 0.78 mmol, 1.1 eq.) was added under argon atmosphere at 0°C to a mixture of [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (single stereoisomer) (113 mg, 0.71 mmol, 1.0 eq.) and molecular sieve (3 Å, 20 mg) in N,N-dimethylformamide (1.5 mL). The mixture was stirred at 0°C for 15 min and added at 0°C to a mixture of (3R)-1-(2,6-dichloro-5- fluoropyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (198 mg, 0.71 mmol) and molecular sieve (3 Å, 20 mg) in N,N-dimethylformamide (2.5 mL). The reaction mixture was stirred at RT for 4 h, quenched with aqueous hydrochloric acid solution (1 N, 777 µL) and diluted with dimethyl sulfoxide (2.0 mL), acetonitrile (1.0 mL) and water (1.0 mL). The resulting solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient) resulting in a mixture of both regioisomers (ca. 1:6) which was used without further separation. Yield: 100 mg (33% of theory). LC/MS (method 1): tR = 0.93 min, MS (ESIpos): m/z = 403 [M+H]+ and tR = 0.97 min, MS (ESIpos): m/z = 403 [M+H]+. Intermediate 4-21A tert-Butyl 3-[6-chloro-2-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-6-ylmethoxy)pyrimidin-4-yl]- 3,8-diazabicyclo[3.2.1]octane-8-carboxylate (racemate) BHC233018 - FC - 77 / 152 -
Figure imgf000078_0001
Sodium hydride (60% in mineral oil, 24 mg, 0.61 mmol, 2.2 eq.) was added under argon atmosphere at 0°C to a solution of tert-butyl 6-(hydroxymethyl)-6,7-dihydropyrazolo[1,5-a]pyrazine-5(4H)-carboxylate (racemate) (71 mg, 0.28 mmol, 1.0 eq.) in N,N-dimethylformamide (1.0 mL). The mixture was stirred at 0°C for 15 min and added at 0°C to a solution of tert-butyl 3-(2,6-dichloropyrimidin-4-yl)-3,8- diazabicyclo[3.2.1]octane-8-carboxylate (100 mg, 0.28 mmol) in N,N-dimethylformamide (1.0 mL). The reaction mixture was stirred at RT overnight, at 50°C for 1 h, mixed with additional sodium hydride (60% in mineral oil, 11 mg, 0.28 mmol, 1.0 eq.), stirred for another 1 h, mixed with additional tert-butyl 6- (hydroxymethyl)-6,7-dihydropyrazolo[1,5-a]pyrazine-5(4H)-carboxylate (racemate) (35 mg, 0.14 mmol, 0.5 eq.) and stirred at RT for another 2 h. The mixture was quenched with aqueous hydrochloric acid solution (1 N, 891 µL) and diluted with dimethyl sulfoxide (2.0 mL), acetonitrile (1.0 mL) and water (1.0 mL). The resulting solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 43 mg (33% of theory). LC/MS (method 2): tR = 1.37 min, MS (ESIpos): m/z = 476 [M+H]+; 1H- NMR (400 MHz, DMSO-d6): δ [ppm] = 7.37 (d, 1H), 6.58 (s, 1H), 5.99 (d, 1H), 4.51-4.3 (br s, 1H, partially concealed), 4.38-4.27 (m, 2H), 4.21 (br s, 2H), 4.16 (dd, 1H), 4.04 (d, 1H), 3.92-3.69 (br s, 1H, partially concealed), 3.86 (d, 1H), 3.76 (t, 1H), 3.51-3.3 (m, 2H), 3.17-2.90 (m, 2H), 1.92-1.75 (m, 2H), 1.64-1.52 (m, 2H), 1.42 (s, 9H). Intermediate 4-21B tert-Butyl 3-[6-(2'-amino-3'-cyano-6',7'-dihydro-1H,5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1- yl)-2-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-6-ylmethoxy)pyrimidin-4-yl]-3,8- diazabicyclo[3.2.1]octane-8-carboxylate (racemate)
Figure imgf000078_0002
Sodium carbonate (48 mg, 0.46 mmol, 5.0 eq.) was added to a solution of tert-butyl 3-[6-chloro-2-(4,5,6,7- tetrahydropyrazolo[1,5-a]pyrazin-6-ylmethoxy)pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8- carboxylate (racemate) (43 mg, 0.09 mmol) and 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'- BHC233018 - FC - 78 / 152 - [1]benzothiophene]-3'-carbonitrile trifluoroacetate (56 mg, 71% purity, 0.12 mmol, 1.3 eq.) in dimethyl sulfoxide (440 µL). The reaction mixture was stirred at 95°C for 4 h in a closed microwave vial and quenched with aqueous hydrochloric acid solution (1 N, 456 µL). The mixture was diluted with dimethyl sulfoxide (4.0 mL), water (1.0 mL) and acetonitrile (1.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 30 mg (84% purity, 43% of theory). LC/MS (method 2): tR = 1.57 min, MS (ESIpos): m/z = 659 [M+H]+. Intermediate 04-22A 4-(4,6-Dichloro-5-methylpyrimidin-2-yl)piperazin-2-one
Figure imgf000079_0001
To a stirred solution of 2,4,6-trichloro-5-methylpyrimidine (3.00 g, 15.19 mmol) and triethylamine (2.1 mL, 15.19 mmol; CAS-RN:[121-44-8]) in dichloromethane (25 mL) was added a suspension of piperazin-2-one (1.52 g, 15.19 mmol; CAS-RN:[5625-67-2]) in dichloromethane (38 mL) at 0° C and the mixture was allowed to warm up to ambient temperature over 1 h. The solution was concentrated in vacuo. Silica gel chromatography of the residue (gradient: ethyl acetate / ethanol 0-10 %) gave 2.06 g (51 % yield) of the title compound. LC-MS (method 3): Rt = 0.83 min; MS (ESIpos): m/z = 261 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 8.15 (br s, 1H), 4.02 (s, 2H), 3.69 (br t, 2H), 3.30 (m, 2H), 2.23 (s, 3H) Intermediate 04-22B 4-(6-Chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-5- methylpyrimidin-4-yl)piperazin-2-one (single stereoisomer) BHC233018 - FC - 79 / 152 -
Figure imgf000080_0001
Sodium hydride (60% in mineral oil, 60.3 mg, 1.51 mmol, 2.0 eq.) was added under argon atmosphere at 0 °C to a mixture of [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (single stereoisomer) (120 mg, 0.75 mmol, 1.0 eq., CAS-RN:[2097518-76-6]) and molecular sieve (3 Å, 20 mg) in N,N-dimethylformamide (3.0 mL). The mixture was stirred at 0 °C for 15 min. At 0 °C, a solution of 4- (4,6-dichloro-5-methylpyrimidin-2-yl)piperazin-2-one (200 mg, 0.75 mmol) in N,N-dimethylformamide (3.0 mL) was added. The reaction mixture was stirred at RT for 2 h, added to saturated aqueous ammonium chloride solution, and extracted three times with ethyl acetate. The combined organic phases were washed with brine, dried over phase separation filter paper, and concentrated in vacuo. The residue was purified by Biotage Isolera™ chromatography (Biotage Sfär KP-NH2 – 50 µm 28 g, gradient: ethyl acetate / ethanol 0-50 %) to afford 85.4 mg (12 % yield) of the title compound as a mixture of regioisomers (40 : 60). LC/MS (method 4): tR = 1.01 min, MS (ESIpos): m/z = 384 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 8.04 (br s, 1H), 5.26 (br d, 1H), 4.01 (d, 1H), 3.94 (d, 1H), 3.89 (s, 2H), 3.55 (br t, 2H), 3.26 (m, 2H), 3.08 (m, 2H), 3.00 (m, 1H), 2.82 (m, 1H), 2.16-1.90 (m, 3H), 2.00 (s, 3H), 1.90-1.62 (m, 3H) Intermediate 05-01 tert-butyl 3-[6-chloro-5-cyano-2-(methylsulfanyl)pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8- carboxylate BHC233018 - FC - 80 / 152 -
Figure imgf000081_0001
CI N S' - To a stirred solution of 4,6-dichloro-2-(methylsulfanyl)pyrimidine-5-carbonitrile (500 mg, 2.27 mmol) and triethylamine (950 µl, 6.8 mmol; CAS-RN:[121-44-8]) in dichloromethane dichloromethane (35 ml) was added tert-butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate (434 mg, 2.04 mmol) at 0° C and the mixture was stirred at 0° C for 1 h. A half-concentrated solution of sodium bicarbonate was added and the mixture was extracted with dichloromethane. The organic phase was dried (sodium sulfate), filtered and the solvent was removed in vacuum. Silicagel chromatography (Gradient: hexanes / ethyl acetate 20- 100%) gave 735 mg (81 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 1.42 min; MS (ESIpos): m/z = 396 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 4.43 (br d, 2H), 4.25 (br s, 2H), 3.33-3.27 (m, 2H), 2.49 (br s, 3H), 1.83 (br d, 2H), 1.67 (br d, 2H), 1.43 (s, 9H) Intermediate 05-02 tert-butyl 3-[6-chloro-5-cyano-2-(methanesulfonyl)pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8- carboxylate
Figure imgf000081_0002
To a stirred solution of tert-butyl 3-[6-chloro-5-cyano-2-(methylsulfanyl)pyrimidin-4-yl]-3,8- diazabicyclo[3.2.1]octane-8-carboxylate (630 mg, 1.59 mmol) in dichloromethane (31 ml) was added BHC233018 - FC - 81 / 152 - mCPBA (1.07 g, 77 % purity, 4.77 mmol; CAS-RN: [937-14-4]) at 0° C and the mixture was stirred at rt for 5 h. The mixture was combined with a second equal reaction starting with tert-butyl 3-[6-chloro-5- cyano-2-(methylsulfanyl)pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (100 mg, 0.25 mmol). An aqueous solution of sodium thiosulfate (Na2S2O3, CAS-RN: [10102-17-7]) was added and the mixture was extracted with a mixture of dichloromethane and methanol. The organic phase was dried (sodium sulfate), filtered and the solvent was removed in vacuum. Silicagel chromatography (Gradient: hexanes / ethyl acetate 20-60%) gave 660 mg of the title compound. LC-MS (Analytical Method 3): Rt = 1.16 min; MS (ESIpos): m/z = 426 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 4.59-4.35 (m, 2H), 4.28 (br s, 2H), 3.39 (br, m, 5H), 1.84 (br d, 2H), 1.69 (br d, 2H), 1.44 (s, 9H) Intermediate 05-03 tert-butyl 3-(6-chloro-5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate
Figure imgf000082_0001
To a strirred solution of [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (734 mg, 4.61 mmol) in THF THF (5.0 ml) was added sodium hydride (123 mg, 60 % purity, in oil, 3.08 mmol; CAS- RN:[7646-69-7]) at rt. The mixture was stirred for 30 minutes at rt and then cooled down to -78° C. tert- butyl 3-[6-chloro-5-cyano-2-(methanesulfonyl)pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8- carboxylate (658 mg, 1.54 mmol) dissolved in THF (2 mL) was added at -78° C and the reaction mixture was stirred at -78° C for 15 min. Ice cold water was added, the mixture was allowed to warm up to rt, and the mixture was extracted with ethyl acetate. The organic phase was dried (sodium sulfate), filtered and the solvent was removed in vacuum. Silicagel chromatography (Gradient: dichloromethane / methanol 0- 10%) gave 425 mg (54 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 0.94 min; MS (ESIpos): m/z = 507 [M+H]+ BHC233018 - FC - 82 / 152 - 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 5.41-5.09 (m, 1H), 4.41 (br d, 2H), 4.25 (br s, 2H), 4.10-3.90 (m, 2H), 3.27 (br d, 2H), 3.06 (br s, 2H), 3.01-2.91 (m, 1H), 2.86-2.76 (m, 1H), 2.08 (br s, 1H), 2.03-1.98 (m, 1H), 1.95 (br d, 1H), 1.89-1.70 (m, 5H), 1.66 (br d, 2H), 1.43 (s, 9H) Intermediate 05-04 tert-butyl 3-[6-(2'-amino-3'-cyano-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1-yl)- 5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-3,8- diazabicyclo[3.2.1]octane-8-carboxylate
Figure imgf000083_0001
To a stirred solution of tert-butyl 3-(6-chloro-5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy}pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (100 mg, 197 µmol) and 2’-amino-6’,7’-dihydro-5’H-spiro[azetidine-3,4’-[1]benzothiophene]-3’-carbonitrile trifluoroacetate (106 mg, 237 µmol) in N,N-dimethylacetamide (4.0 ml) was added cesium carbonate (257 mg, 789 µmol; CAS-RN:[534-17-8]) and the mixture was stirred at 50° C for 1 h. Water was added and the mixture was extracted with ethyl acetate. The organic phase was washed with half-saturated sodium chloride solution, dried (sodium sulfate), filtered and the solvent was removed in vacuum. Aminophase-silicagel chromatography (Gradient: dichloromethane / methanol 0-10%) gave 88.0 mg (65 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 1.10 min; MS (ESIpos): m/z = 690 [M+H]+ 1H-NMR (500 MHz, DMSO-d6): δ [ppm] = 7.10 (s, 2H), 5.35-5.12 (m, 1H), 4.64-4.28 (m, 2H), 4.27- 4.16 (m, 5H), 4.04-3.90 (m, 2H), 3.89-3.77 (m, 1H), 3.22-3.12 (m, 2H), 3.09-2.93 (m, 3H), 2.84-2.75 (m, 1H), 2.42 (t, 2H), 2.07-1.89 (m, 5H), 1.87-1.75 (m, 3H), 1.75-1.58 (m, 6H), 1.43 (s, 9H) Intermediate 05-05 4-chloro-6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]-2-(methylsulfanyl)pyrimidine-5-carbonitrile BHC233018 - FC - 83 / 152 -
Figure imgf000084_0001
. To a stirred solution of 4,6-dichloro-2-(methylsulfanyl)pyrimidine-5-carbonitrile (2.00 g, 9.09 mmol) and triethylamine (3.8 ml, 27 mmol; CAS-RN:[121-44-8]) in dichloromethane (140 ml) was added (3R)-3- methylpiperidin-3-ol hydrogen chloride salt (1.24 g, 8.18 mmol) at 0° C and the mixture was stirred at 0° C for 1 h. A half-concentrated solution of sodium bicarbonate was added and the mixture was extracted with dichloromethane. The organic phase was dried (sodium sulfate), filtered and the solvent was removed in vacuum. Silicagel chromatography (Gradient: hexanes / ethyl acetate 20-60%) gave 1.93 g (71 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 1.09 min; MS (ESIpos): m/z = 299 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 4.66 (s, 1H), 4.28 (br d, 1H), 4.12 (d, 1H), 3.51 (d, 1H), 3.43- 3.36 (m, 1H), 2.50 (s, 3H), 1.96-1.77 (m, 1H), 1.68-1.49 (m, 3H), 1.13 (s, 3H) Intermediate 05-06 4-chloro-6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]-2-(methanesulfonyl)pyrimidine-5-carbonitrile
Figure imgf000084_0002
, ‘10 To a stirred solution of 4-chloro-6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]-2- (methylsulfanyl)pyrimidine-5-carbonitrile (500 mg, 1.67 mmol) in dichloromethane (33 ml) was added mCPBA (1.13 g, 77 % purity, 5.02 mmol; CAS-RN:[937-14-4]) at 0° C and the mixture was stirred at rt for 3 h. A solution of sodium thiosulfate (Na2S2O3) was added and the mixture was extracted with dichloromethane. The organic phase was dried (sodium sulfate), filtered and the solvent was removed in vacuum. Silicagel chromatography (Gradient: hexanes / ethyl acetate 20-60%) gave 436 mg (78 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 0.84 min; MS (ESIpos): m/z = 331 [M+H]+ BHC233018 - FC - 84 / 152 - 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 4.88-4.61 (m, 1H), 4.54-4.30 (m, 1H), 4.29-4.12 (m, 1H), 3.54 (d, 1H), 3.37 (s, 3H), 3.36-3.35 (m, 1H), 1.95-1.80 (m, 1H), 1.72-1.53 (m, 3H), 1.15 (s, 3H) Intermediate 05-07 4-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidine-5-carbonitrile
Figure imgf000085_0001
To a strirred solution of [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (152 mg, 952 µmol) in THF (2.0 ml) was added molecular sives (4A) and the mixture was stirred for 15 min. Then sodium hydride (50.8 mg, 60 % purity, 1.27 mmol; CAS-RN:[7646-69-7]) was added at rt. The mixture was stirred for 30 minutes at rt and then cooled down to -78° C. 4-chloro-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]-2-(methanesulfonyl)pyrimidine-5-carbonitrile (210 mg, 635 µmol) dissolved in THF (2 mL) was added at -78° C and the reaction mixture was stirred at -78° C for 15 min. Ice cold water was added, the mixture was allowed to warm up to rt, and the mixture was extracted with ethyl acetate. The organic phase was dried (sodium sulfate), filtered and the solvent was removed in vacuum. Silicagel chromatography (Gradient: dichloromethane / methanol 0-10%) gave 135 mg (52 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 0.63 min; MS (ESIpos): m/z = 410 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 5.37-5.09 (m, 1H), 4.64 (s, 1H), 4.20 (br d, 1H), 4.11-3.88 (m, 3H), 3.50 (d, 1H), 3.44-3.35 (m, 1H), 3.12-3.02 (m, 2H), 3.01-2.92 (m, 1H), 2.86-2.76 (m, 1H), 2.08 (br s, 1H), 2.03-1.91 (m, 2H), 1.89-1.68 (m, 4H), 1.65-1.46 (m, 3H), 1.11 (s, 3H) Intermediate 05-08 4-chloro-2-(methylsulfanyl)-6-(1,4-oxazepan-4-yl)pyrimidine-5-carbonitrile BHC233018 - FC - 85 / 152 -
Figure imgf000086_0001
To a stirred solution of 4,6-dichloro-2-(methylsulfanyl)pyrimidine-5-carbonitrile (2.00 g, 9.09 mmol) and triethylamine (3.8 ml, 27 mmol; CAS-RN:[121-44-8]) in dichloromethane (140 ml) was added 1,4- oxazepane (919 mg, 9.09 mmol; CAS-RN:[5638-60-8]) at 0° C and the mixture was stirred at 0° C for 1 h. A half-concentrated solution of sodium bicarbonate was added and the mixture was extracted with dichloromethane. The organic phase was combined with a second equal reaction starting with 500 mg of 4,6-dichloro-2-(methylsulfanyl)pyrimidine-5-carbonitrile. The combined organic phases were dried (sodium sulfate), filtered and the solvent was removed in vacuum. Silicagel chromatography (Gradient: hexanes / ethyl acetate 20-60%) gave 2.63 g (72 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 1.12 min; MS (ESIpos): m/z = 285 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 4.05-3.95 (m, 4H), 3.79 (t, 2H), 3.71-3.65 (m, 2H), 2.48 (s, 3H), 1.94 (quin, 2H) Intermediate 05-09 4-chloro-2-(methanesulfonyl)-6-(1,4-oxazepan-4-yl)pyrimidine-5-carbonitrile
Figure imgf000086_0002
To a stirred solution of 4-chloro-2-(methylsulfanyl)-6-(1,4-oxazepan-4-yl)pyrimidine-5-carbonitrile (1.50 g, 5.27 mmol) in dichloromethane (100 ml) was added mCPBA (5.90 g, 77 % purity, 26.3 mmol; CAS- RN:[937-14-4]) at rt and the mixture was stirred at rt for 3 h. A solution of sodium thiosulfate (Na2S2O3) was added and the mixture was extracted with a mixture of dichloromethane and methanol. The organic phase was dried (sodium sulfate), filtered and the solvent was removed in vacuum. Silicagel chromatography (Gradient: hexanes / ethyl acetate 20-60%) gave 1.44 g (86 % yield) of the title compound. BHC233018 - FC - 86 / 152 - LC-MS (Analytical Method 3): Rt = 0.83 min; MS (ESIpos): m/z = 317 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 4.14-3.99 (m, 4H), 3.81 (br s, 2H), 3.74-3.68 (m, 2H), 3.37 (s, 3H), 2.06-1.89 (m, 2H) Intermediate 05-10 4-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-(1,4-oxazepan-4- yl)pyrimidine-5-carbonitrile
Figure imgf000087_0001
To a strirred solution of [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (503 mg, 3.16 mmol) in THF (5 ml) was added sodium hydride (126 mg, 60 % purity, 3.16 mmol; CAS-RN:[7646-69- 7]) at rt. The mixture was stirred for 30 minutes at rt and then cooled down to -78° C. 4-chloro-2- (methanesulfonyl)-6-(1,4-oxazepan-4-yl)pyrimidine-5-carbonitrile (500 mg, 1.58 mmol) dissolved in THF (2 mL) was added at -78° C and the reaction mixture was stirred at -78° C for 15 min. Ice cold water was added, the mixture was allowed to warm up to rt, and the mixture was extracted with ethyl acetate. The organic phase was dried (sodium sulfate), filtered and the solvent was removed in vacuum. Silicagel chromatography (Gradient: dichloromethane / methanol 0-10%) gave 517 mg (82 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 0.61 min; MS (ESIpos): m/z = 396 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 5.37-5.08 (m, 1H), 4.06-3.91 (m, 6H), 3.78 (t, 2H), 3.71-3.63 (m, 2H), 3.11-2.90 (m, 3H), 2.85-2.75 (m, 1H), 2.07 (d, 1H), 2.01-1.97 (m, 1H), 1.96-1.88 (m, 3H), 1.87- 1.66 (m, 3H) Intermediate 05-11 4-chloro-6-[(6R or S)-6-hydroxy-6-methyl-1,4-oxazepan-4-yl]-2-(methylsulfanyl)pyrimidine-5- carbonitrile (single stereoisomer) BHC233018 - FC - 87 / 152 -
Figure imgf000088_0001
CI N S". To a stirred solution of 4,6-dichloro-2-(methylsulfanyl)pyrimidine-5-carbonitrile (1.86 g, 8.47 mmol) and triethylamine (3.5 ml, 25 mmol; CAS-RN:[121-44-8]) in dichloromethane (130 ml) was added (6R or S)- (-)-6-methyl-1,4-oxazepan-6-ol (1.00 g, 7.62 mmol) (WO 2023/018809, p112-113; optical rotation: [a]D = - 11,67 ° (from solution in DMSO)) at 0° C and the mixture was stirred at 0° C for 1 h. A half- concentrated solution of sodium bicarbonate was added and the mixture was extracted with dichloromethane. The organic phase was combined with a second equal reaction starting with 112 mg of 4,6-dichloro-2-(methylsulfanyl)pyrimidine-5-carbonitrile. The combined organic phases were dried (sodium sulfate), filtered and the solvent was removed in vacuum. Silicagel chromatography (Gradient: hexanes / ethyl acetate 20-60%) gave 2.27 g of the title compound. LC-MS (Analytical Method 3): Rt = 1.00 min; MS (ESIpos): m/z = 315 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 4.89 (s, 1H), 4.30-4.07 (m, 2H), 4.00-3.80 (m, 4H), 3.54-3.41 (m, 2H), 2.50 (s, 3H), 1.08 (s, 3H) Intermediate 05-12 4-chloro-6-[(6R or S)-6-hydroxy-6-methyl-1,4-oxazepan-4-yl]-2-(methanesulfonyl)pyrimidine-5- carbonitrile (single stereoisomer)
Figure imgf000088_0002
To a stirred solution of 4-chloro-6-[(6R or S)-6-hydroxy-6-methyl-1,4-oxazepan-4-yl]-2- (methylsulfanyl)pyrimidine-5-carbonitrile (Intermediate 05-11) (500 mg, 1.59 mmol) in dichloromethane (31 ml) was added mCPBA (1.07 g, 77 % purity, 4.76 mmol; CAS-RN:[937-14-4]) at 0° C and the mixture was stirred at rt for 3 h. A solution of sodium thiosulfate (Na2S2O3) was added and the mixture was extracted with a mixture of dichloromethane and methanol. The organic phase was dried (sodium sulfate), BHC233018 - FC - 88 / 152 - filtered and the solvent was removed in vacuum. Silicagel chromatography (Gradient: hexanes / ethyl acetate 40-100%) gave 454 mg (82 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 0.76 min; MS (ESIpos): m/z = 347 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 5.13-4.40 (m, 1H), 4.36-4.11 (m, 2H), 4.00-3.69 (m, 4H), 3.56-3.43 (m, 2H), 3.37 (s, 3H), 1.09 (s, 3H) Intermediate 05-13 4-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(6R or S)-6- hydroxy-6-methyl-1,4-oxazepan-4-yl]pyrimidine-5-carbonitrile (single stereoisomer)
Figure imgf000089_0001
To a strirred solution of [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (304 mg, 1.91 mmol) in THF (6 ml) was added molecular sives (4A) and the mixture was stirred for 15 min. Then sodium hydride (102 mg, 60 % purity, 2.55 mmol; CAS-RN:[7646-69-7]) was added at rt. The mixture was stirred for 30 minutes at rt and then cooled down to -78° C. 4-chloro-6-[(6R or S)-6-hydroxy-6-methyl-1,4- oxazepan-4-yl]-2-(methanesulfonyl)pyrimidine-5-carbonitrile (Intermediate 05-12) (442 mg, 1.27 mmol) dissolved in N,N-dimethylacetamide (1.4 ml) was added at -78° C and the reaction mixture was stirred at -78° C for 15 min. Ice cold water was added, the mixture was allowed to warm up to rt, and the mixture was extracted with ethyl acetate. The organic phase was dried (sodium sulfate), filtered and the solvent was removed in vacuum. Silicagel chromatography (Gradient: dichloromethane / methanol 0-10%) gave 243 mg (44 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 0.64 min; MS (ESIpos): m/z = 426 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 5.36-5.13 (m, 1H), 4.87 (s, 1H), 4.24-3.77 (m, 8H), 3.50-3.39 (m, 2H), 3.10-2.95 (m, 3H), 2.85-2.78 (m, 1H), 2.10-2.03 (m, 1H), 2.02-1.98 (m, 1H), 1.97-1.92 (m, 1H), 1.87-1.80 (m, 1H), 1.80-1.67 (m, 2H), 1.08 (s, 3H) Intermediate 05-14 4-chloro-6-[(6R or S)-6-hydroxy-6-methyl-1,4-oxazepan-4-yl]-2-(methylsulfanyl)pyrimidine-5- carbonitrile (single stereoisomer) BHC233018 - FC - 89 / 152 - (
Figure imgf000090_0001
CI N S' To a stirred solution of 4,6-dichloro-2-(methylsulfanyl)pyrimidine-5-carbonitrile (1.95 g, 8.60 mmol) and (6R or S)-(+)-6-methyl-1,4-oxazepan-6-ol (1.01 g, 7.74 mmol) (WO 2023/018809, p112-113; optical rotation: [a]D = + 8,93 ° (from solution in DMSO)) in dichloromethane (130 ml) was added triethylamine (3.6 ml, 26 mmol; CAS-RN:[121-44-8]) at 0° C and the mixture was stirred at 0° C for 1 h. A half- concentrated solution of sodium bicarbonate was added and the mixture was extracted with dichloromethane. The organic phase was dried (sodium sulfate), filtered and the solvent was removed in vacuum. Silicagel chromatography (Gradient: hexanes / ethyl acetate 30-80%) gave 2.36 g (87 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 1.00 min; MS (ESIpos): m/z = 315 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 4.90 (s, 1H), 4.27-4.08 (m, 2H), 3.99-3.81 (m, 4H), 3.53-3.41 (m, 2H), 2.50 (s, 3H), 1.08 (s, 3H) Intermediate 05-15 4-chloro-6-[(6R or S)-6-hydroxy-6-methyl-1,4-oxazepan-4-yl]-2-(methanesulfonyl)pyrimidine-5- carbonitrile (single stereoisomer) (
Figure imgf000090_0002
To a stirred solution of 4-chloro-6-[(6R or S)-6-hydroxy-6-methyl-1,4-oxazepan-4-yl]-2- (methylsulfanyl)pyrimidine-5-carbonitrile (Intermediate 05-14) (62.3 mg, 198 µmol) in dichloromethane dichloromethane (3.9 ml) was added mCPBA (133 mg, 77 % purity, 594 µmol; CAS-RN:[937-14-4]) at 0° C and the mixture was stirred at rt for 3 h. A solution of sodium thiosulfate (Na2S2O3) was added and the mixture was extracted with a mixture of dichloromethane. The organic phase was washed with an aqueous solution of sodium bicarbonate and with a saturated solution of sodium chloride, dried (sodium BHC233018 - FC - 90 / 152 - sulfate), filtered and the solvent was removed in vacuum. Silicagel chromatography (Gradient: hexanes / ethyl acetate 40-100%) gave 41.7 mg (61 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 0.76 min; MS (ESIpos): m/z = 347 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 4.97 (br d, 1H), 4.37-3.69 (m, 6H), 3.59-3.41 (m, 2H), 3.37 (s, 3H), 1.09 (s, 3H) Intermediate 05-16 4-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(6R or S)-6- hydroxy-6-methyl-1,4-oxazepan-4-yl]pyrimidine-5-carbonitrile (single stereoisomer)
Figure imgf000091_0001
To a strirred solution of [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (143 mg, 877 µmol) in THF (2.0 ml) was added molecular sives (4A) and the mixture was stirred for 10 min. Then sodium hydride (46.8 mg, 60 % purity, 1.17 mmol; CAS-RN:[7646-69-7]) was added at rt. The mixture was stirred for 30 minutes at rt and then cooled down to -78° C. 4-chloro-6-[(6R or S)-6-hydroxy-6- methyl-1,4-oxazepan-4-yl]-2-(methanesulfonyl)pyrimidine-5-carbonitrile (Intermediate 05-15) (203 mg, 585 µmol) dissolved in a mixture of THF (2 mL), dichloromethane (1 mL) and DMA (0.5 mL) was added at -78° C and the reaction mixture was stirred at -78° C for 15 min. Ice cold water was added, the mixture was allowed to warm up to rt, and the mixture was extracted with ethyl acetate. The organic phase was dried (sodium sulfate), filtered and the solvent was removed in vacuum. Silicagel chromatography (Gradient: dichloromethane / methanol 0-10%) gave 161 mg (65 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 0.64 min; MS (ESIpos): m/z = 426 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 5.40-5.11 (m, 1H), 4.87 (s, 1H), 4.23-3.74 (m, 8H), 3.51-3.39 (m, 2H), 3.10-2.92 (m, 3H), 2.86-2.76 (m, 1H), 2.08 (d, 1H), 2.02-1.99 (m, 1H), 1.98-1.88 (m, 1H), 1.88- 1.80 (m, 1H), 1.79-1.67 (m, 2H), 1.08 (s, 3H) Intermediate 6-1 2'-amino-1-[6-chloro-2-(methylsulfanyl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile BHC233018 - FC - 91 / 152 -
Figure imgf000092_0002
To a stirred suspension of 4,6-dichloro-2-(methylsulfanyl)pyrimidine (100 mg, 513 µmol; CAS- RN:[6299-25-8]) and sodium carbonate (217 mg, 2.05 mmol) in dry dimethyl sulfoxide (2.0 ml) was added 2’-amino-6’,7’-dihydro-5’H-spiro[azetidine-3,4’-[1]benzothiophene]-3’-carbonitrile trifluoroacetate salt (1/1) (214 mg, 80 % purity, 513 µmol). The reaction mixture was heated at 70°C for 1 h. The reaction was allowed to cool to room temperature and quenched with water and the aqueous phase was extracted with ethyl acetate. The combined organic layers were washed with brine, filtered through a water repellant filter and concentrated in vacuo. The crude produkt was taken up in 4 ml acetonitrile / water (7:3). The precipitate thus obtained was collected by vacuum filtration, washed with a little amount of acetonitrile and dried in vacuo. The residue was diluted with 2 ml acetonitrile and purified by preparative HPLC (Method A; gradient: 40% B to 85% B). The product fractions were pooled and concentrated in vacuo to afford 109 mg (53% yield, 95% purity) of the title compound as a light brown solid. LC-MS (Method 3): Rt = 1.33 min; MS (ESIpos): m/z = 378 [M+H]+ ; 1H NMR (400 MHz, DMSO- d6) δ [ppm]: 1.69 (br d, 2H), 1.97-2.05 (m, 2H), 2.42 (s, 3H), 2.44 (br s, 1H), 3.91 (dd, 2H), 4.29 (d, 2H), 6.28 (s, 1H), 7.11 (s, 2H). Intermediate 6-2 di-tert-butyl {1-[6-chloro-2-(methylsulfanyl)pyrimidin-4-yl]-3'-cyano-6',7'-dihydro-5'H- spiro[azetidine-3,4'-[1]benzothiophen]-2'-yl}-2-imidodicarbonate
Figure imgf000092_0001
To a solution of 2’-amino-1-[6-chloro-2-(methylsulfanyl)pyrimidin-4-yl]-6’,7’-dihydro-5’H- spiro[azetidine-3,4’-[1]benzothiophene]-3’-carbonitrile (550 mg, 1.46 mmol; intermediate 6-1) in tetrahydrofuran (28 ml) was added N,N-diisopropylethylamine (510 µl, 2.9 mmol) and DMAP (17.8 mg, 146 µmol; CAS-RN:[1122-58-3]) followed by di-tert-butyl dicarbonate (910 µl, 4.4 mmol; CAS- BHC233018 - FC - 92 / 152 - RN:[24424-99-5]). The reaction mixture was stirred for 16 h at RT. The reaction was quenched with water and the aqueous phase was extracted with ethyl acetate. The combined organics were washed with brine, filtered through a water repellant filter and concentrated. The crude product was taken up in 5 ml methyl tert-butyl ether, sonicated and added to 50 ml of hexane. The suspension was intensively stirred for 10 minutes, then the solid was collected by vacuum filtration and dried to afford 673 mg (76% yield, 94% purity) of the title compound as a beige solid. LC-MS (Method 3): Rt = 1.60 min; MS (ESIpos): m/z = 578 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.43 (s, 18H), 1.73-1.83 (m, 2H), 2.13 (br d, 2H), 2.42 (s, 3H), 2.75 (t, 2H), 4.00-4.13 (m, 2H), 4.19-4.32 (m, 2H), 6.36 (s, 1H). Intermediate 6-3 di-tert-butyl {1-[6-chloro-2-(methanesulfonyl)pyrimidin-4-yl]-3'-cyano-6',7'-dihydro-5'H- spiro[azetidine-3,4'-[1]benzothiophen]-2'-yl}-2-imidodicarbonate
Figure imgf000093_0001
Di-tert-butyl {1-[6-chloro-2-(methylsulfanyl)pyrimidin-4-yl]-3’-cyano-6’,7’-dihydro-5’H- spiro[azetidine-3,4’-[1]benzothiophen]-2’-yl}-2-imidodicarbonate (30.0 mg, 51.9 µmol; intermediate 6- 2) was dissolved in dry dichloromethane (600 µl) under nitrogen atomsphere, then 3-chlorobenzene-1- carboperoxoic acid (18.8 mg, 109 µmol; CAS-RN:[937-14-4]) was added. The reaction mixture was stirred for 1 hour at RT. The mixture was diluted with dichloromethane and extracted with sodium hydroxide solution (2M). The organic layer was filtered through a water repellant filter and concentrated to afford 22 mg (68% yield, 98% purity) of the title compound as a white solid. LC-MS (Method 3): Rt = 1.42 min; MS (ESIpos): m/z = 610 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 1.43 (s, 18H), 1.79 (br d, 2H), 2.12-2.19 (m, 2H), 2.73-2.80 (m, 2H), 3.29-3.31 (m, 3H), 4.14-4.25 (m, 2H), 4.32 (d, 1H), 4.40 (d, 1H), 6.94 (s, 1H). Intermediate 6-4 tert-butyl [1-(6-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl)-3'-cyano-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophen]-2'- yl]carbamate BHC233018 - FC - 93 / 152 -
Figure imgf000094_0001
Di-tert-butyl {1-[6-chloro-2-(methanesulfonyl)pyrimidin-4-yl]-3’-cyano-6’,7’-dihydro-5’H- spiro[azetidine-3,4’-[1]benzothiophen]-2’-yl}-2-imidodicarbonate (20.0 mg, 32.8 µmol; intermediate 6- 3) was dissolved in dry tetrahydrofuran (500 µl). The solution was flushed with argon for 2 minutes, then [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (5.48 mg, 34.4 µmol) was added, followed by a solution of sodium bis(trimethylsilyl)amide (2M in tetrahydrofuran)(66 µl, 1.0 M, 66 µmol; CAS-RN:[1070-89-9]). The reaction mixture was stirred for 30 minutes at RT. The reaction was quenched with water and the mixture was extracted by ethyl acetate. The combined organic phases were washed with brine, filtered through a water repellant filter and concentrated. The crude material was purified by Biotage Isolera™ chromatography (Sfär Silica HC D – 20 µm 10 g), eluting with hexane - ethyl acetate, 9:1 to 0:1, then with ethyl acetate – ethanol, 1:0 to 9:1) to afford 8.0 mg (40% yield, 97% purity) of the title compound as a white foam. LC-MS (Method 3): Rt = 1.01 min; MS (ESIpos): m/z = 590 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ [ppm]: 1.47 (s, 9H), 1.65-1.85 (m, 5H), 1.92 (br d, 1H), 1.99 (s, 1H), 2.07 (br s, 3H), 2.60 (br t, 2H), 2.75-2.86 (m, 1H), 2.99 (br s, 1H), 3.01-3.11 (m, 2H), 3.81-3.90 (m, 1H), 3.90- 3.99 (m, 3H), 4.30 (br d, 2H), 5.25 (br d, 1H), 6.22 (s, 1H), 10.95 (br s, 1H). Intermediate 6-5 tert-butyl {3'-cyano-1-[6-(4,4-difluoropiperidin-1-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H- pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophen]-2'-yl}carbamate
Figure imgf000094_0002
BHC233018 - FC - 94 / 152 - To a stirred suspension of tert-butyl [1-(6-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl)-3’-cyano-6’,7’-dihydro-5’H-spiro[azetidine-3,4’-[1]benzothiophen]-2’- yl]carbamate (50.0 mg, 84.9 µmol) and caesium carbonate (138 mg, 424 µmol; intermediate 6-4) in dry dimethyl sulfoxide (800 µl) was added 4,4-difluoropiperidine (30.8 mg, 255 µmol; CAS-RN:[21987-29- 1]). The reaction mixture was heated at 140°C for 1,5 h under microwave irradiation. To the mixture were added further 4,4-difluoropiperidine (30.8 mg, 255 µmol) and the reaction mixture was heated at 150°C for 1 h under microwave irradiation. The mixture was filtered and driectly purified by preparative HPLC (Method A; gradient: 25% B to 70% B). The product fractions were pooled and concentrated in vacuo to afford 10.0 mg (17% yield, 93% purity) of the title compound as a white powder. LC-MS (Method 3): Rt = 1.19 min; MS (ESIpos): m/z = 674 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.47 (s, 9H), 1.73 (br s, 4H), 1.84 (br s, 1H), 1.88-2.09 (m, 9H), 2.57-2.64 (m, 2H), 2.82 (br d, 1H), 3.01 (br s, 1H), 3.09 (br s, 2H), 3.65 (br s, 4H), 3.81 (br s, 3H), 3.93 (br s, 1H), 4.25 (br d, 2H), 5.26 (br d, 1H), 5.37 (s, 1H). Intermediate 6-6 tert-butyl {3'-cyano-1-[2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-(3- oxopiperazin-1-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophen]-2'- yl}carbamate
Figure imgf000095_0001
To a stirred suspension of tert-butyl [1-(6-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl)-3’-cyano-6’,7’-dihydro-5’H-spiro[azetidine-3,4’-[1]benzothiophen]-2’- yl]carbamate (50.0 mg, 84.9 µmol; intermediate 6-4) and caesium carbonate (138 mg, 424 µmol) in dry dimethyl sulfoxide (800 µl) was added piperazin-2-one (25.5 mg, 255 µmol; CAS-RN:[5625-67-2]). The reaction mixture was heated at 150°C for 2 h under microwave irradiation. The reaction was diluted with ethyl acetate and water was added. The phases were separated and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with brine, filtered through a water repellant filter and concentrated. The crude product was purified by preparative HPLC (Method A, gradient C). The product fractions were pooled and concentrated in vacuo to afford 23 mg (38% yield, 92% purity) of the BHC233018 - FC - 95 / 152 - title compound as a white powder. LC-MS (Method 3): Rt = 0.92 min; MS (ESIpos): m/z = 654 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.47 (s, 9H), 1.75 (br s, 3H), 1.84-1.93 (m, 1H), 1.94-2.13 (m, 5H), 2.17 (br s, 1H), 2.57-2.63 (m, 2H), 2.89 (br s, 1H), 3.03-3.20 (m, 2H), 3.20-3.29 (m, 3H), 3.69 (br t, 2H), 3.83 (br d, 2H), 3.97 (s, 2H), 4.25 (br s, 2H), 5.25 (s, 1H), 5.31 (br d, 1H), 8.12 (s, 1H), 10.88 (br s, 1H). Intermediate 6-7 N-{1-[6-chloro-2-(methylsulfanyl)pyrimidin-4-yl]piperidin-4-yl}acetamide
Figure imgf000096_0001
C I N * S ' To a stirred suspension of 4,6-dichloro-2-(methylsulfanyl)pyrimidine (200 mg, 1.03 mmol; CAS- RN:[6299-25-8]) and sodium carbonate (435 mg, 4.10 mmol) in dry dimethyl sulfoxide (4.0 ml) was added N-(piperidin-4-yl)acetamide hydrogen chloride (1/1) (201 mg, 1.13 mmol; CAS-RN:[58083-34- 4]). The reaction mixture was heated at 70°C for 2 h. The reaction was allowed to cool to room temperature, then the mixture was poured into water (20 ml). The mixture was intensively stirred for 10 minutes and the solid was collected by vacuum filtration. The solid was washed with hexane and dried to afford 250 mg (79% yield, 97% purity) of the title compound as a white solid. LC-MS (Method 3): Rt = 0.94 min; MS (ESIpos): m/z = 301 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.21-1.36 (m, 2H), 1.77-1.81 (m, 2H), 1.79 (s, 3H), 2.42 (s, 3H), 3.03-3.15 (m, 2H), 3.79-3.88 (m, 1H), 6.70 (s, 1H), 7.83 (d, 1H). Intermediate 6-8 4-chloro-6-(3,3-difluoropiperidin-1-yl)-2-(methylsulfanyl)pyrimidine
Figure imgf000096_0002
C I N S ' BHC233018 - FC - 96 / 152 - To a stirred suspension of 4,6-dichloro-2-(methylsulfanyl)pyrimidine (200 mg, 1.03 mmol; CAS- RN:[6299-25-8]) and sodium carbonate (435 mg, 4.10 mmol; CAS-RN:[497-19-8]) in dry dimethyl sulfoxide (4.0 ml) was added 3,3-difluoropiperidine hydrogen chloride (1/1) (178 mg, 1.13 mmol). The reaction mixture was heated at 70°C for 2 h. The reaction was allowed to cool to room temperature, then the mixture was poured into water (20 ml). The mixture was intensively stirred for 10 minutes and the solid was collected by vacuum filtration. The solid was washed with hexane and dried to afford 222 mg (77% yield, 97% purity) of the title compound as a white solid. LC-MS (Method 3): Rt = 1.25 min; MS (ESIpos): m/z = 280 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.66-1.73 (m, 2H), 1.98-2.17 (m, 2H), 2.44 (s, 3H), 3.70 (br s, 2H), 4.05 (br t, 2H), 6.86 (s, 1H). Intermediate 6-9 4-chloro-6-(4-fluoropiperidin-1-yl)-2-(methylsulfanyl)pyrimidine
Figure imgf000097_0001
C I N S ' To a stirred suspension of 4,6-dichloro-2-(methylsulfanyl)pyrimidine (200 mg, 1.03 mmol; CAS- RN:[6299-25-8]) and sodium carbonate (435 mg, 4.10 mmol) in dry dimethyl sulfoxide (4.0 ml) was added 4-fluoropiperidine hydrogen chloride (1/1) (157 mg, 1.13 mmol; CAS-RN:[57395-89-8]). The reaction mixture was heated at 70°C for 2 h. The reaction was allowed to cool to room temperature, then the mixture was poured into water (20 ml). The mixture was intensively stirred for 10 minutes and the solid was collected by vacuum filtration. The solid was washed with hexane and dried to afford 235 mg (86% yield, 98% purity) of the title compound as a white solid. LC-MS (Method 3): Rt = 1.23 min; MS (ESIpos): m/z = 262 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.71 (tq, 2H), 1.82-1.97 (m, 2H), 2.43 (s, 3H), 3.71 (br s, 4H), 4.91 (dtt, 1H), 6.74 (s, 1H). Intermediate 6-10 N-{1-[6-chloro-2-(methanesulfonyl)pyrimidin-4-yl]piperidin-4-yl}acetamide BHC233018 - FC - 97 / 152 -
Figure imgf000098_0001
o To a stirred solution of N-{1-[6-chloro-2-(methylsulfanyl)pyrimidin-4-yl]piperidin-4-yl}acetamide (100 mg, 332 µmol; intermediate 6-7) in dichloromethane (1.5 ml) was added 3-chlorobenzene-1- carboperoxoic acid (126 mg, 731 µmol; CAS-RN:[937-14-4]). The reaction mixture was stirred at RT for 4 h. Upon completion to the mixture was added sodium hydroxide solution (5 ml; 2M). The aqueous phase was extracted with dichloromethane and the combined organic phases were washed with brine. Then the phase was filtered through a water repellant filter and concentrated to afford 121 mg (crude) of the title compound as a white solid. LC-MS (Method 3): Rt = 0.68 min; MS (ESIpos): m/z = 333 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.34 (br d, 2H), 1.80 (s, 3H), 1.84 (br dd, 2H), 3.14-3.28 (m, 2H), 3.31 (s, 3H), 3.78-3.96 (m, 1H), 3.97-4.11 (m, 1H), 4.52 (br s, 1H), 7.25 (s, 1H), 7.87 (d, 1H). Intermediate 6-11 4-chloro-6-(3,3-difluoropiperidin-1-yl)-2-(methanesulfonyl)pyrimidine
Figure imgf000098_0002
0 To a stirred solution of 4-chloro-6-(3,3-difluoropiperidin-1-yl)-2-(methylsulfanyl)pyrimidine (220 mg, 786 µmol; intermediate 6-8) in dichloromethane (2.5 ml) was added 3-chlorobenzene-1-carboperoxoic acid (407 mg, 2.36 mmol; CAS-RN:[937-14-4]). The reaction mixture was stirred at RT for 4 h. The reaction was basified with sodium hydroxide solution (2M) and extracted with ethyl acetate. The combined organic layers were washed with brine, filtered through a water repellant filter and concentrated to afford 268 mg (crude) of the title compound as a white solid. LC-MS (Method 3): Rt = 0.93 min; MS BHC233018 - FC - 98 / 152 - (ESIpos): m/z = 312 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 1.74 (br s, 2H), 2.08-2.20 (m, 2H), 3.34 (s, 3H), 3.77 (br s, 2H), 4.14 (br s, 2H), 7.43 (br s, 1H). Intermediate 6-12 4-chloro-6-(4-fluoropiperidin-1-yl)-2-(methanesulfonyl)pyrimidine
Figure imgf000099_0001
To a stirred solution of 4-chloro-6-(4-fluoropiperidin-1-yl)-2-(methylsulfanyl)pyrimidine (220 mg, 840 µmol; intermediate 6-9) in dichloromethane (3.0 ml) was added 3-chlorobenzene-1-carboperoxoic acid (435 mg, 2.52 mmol; CAS-RN:[937-14-4]). The reaction mixture was stirred at RT for 3 h. The reaction was basified with sodium hydroxide solution (2M) and extracted with ethyl acetate. The combined organic layers were washed with brine, filtered through a water repellant filter and concentrated to afford 214 mg (crude) of the title compound as a white solid. LC-MS (Method 3): Rt = 0.89 min; MS (ESIpos): m/z = 294 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.73-1.84 (m, 2H), 1.87-2.02 (m, 2H), 3.32 (s, 3H), 3.60-3.96 (m, 4H), 4.95 (dtt, 1H), 7.29 (s, 1H). Intermediate 6-13 N-[1-(6-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4- yl)piperidin-4-yl]acetamide
Figure imgf000099_0002
BHC233018 - FC - 99 / 152 - To a stirred solution of N-{1-[6-chloro-2-(methanesulfonyl)pyrimidin-4-yl]piperidin-4-yl}acetamide (100 mg, 300 µmol; intermediate 6-10) in dry tetrahydrofuran (2.0 ml) under argon atmosphere was added [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (71.8 mg, 451 µmol). The mixture was cooled by an ice-bath to 4°C, then sodium bis(trimethylsilyl)amide (2M in tetrahydrofuran)(300 µl, 2.0 M, 600 µmol) was added dropwise into the cold solution. The ice-bath was removed and the reaction mixture was stirred for 1 h at RT. The reaction was quenched with water and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with brine, filtered through a water repellant filter and concentrated. The obtained residue was purified by preparative HPLC (Method A; gradient: 5% B to 45% B). The product fractions were pooled and concentrated in vacuo to afford 73 mg (57% yield, 97% purity) of the title compound as a yellow foam. LC-MS (Method 3): Rt = 0.58 min; MS (ESIpos): m/z = 412 [M+H]+;1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.21-1.35 (m, 2H), 1.70- 1.78 (m, 3H), 1.79 (s, 3H), 1.83 (br s, 1H), 1.90-2.04 (m, 2H), 2.06-2.09 (m, 1H), 2.77-2.86 (m, 1H), 2.90- 3.13 (m, 6H), 3.77-3.83 (m, 1H), 3.85 (d, 1H), 3.92-3.98 (m, 1H), 5.24 (br d, 1H), 5.30-5.35 (m, 1H), 6.62 (s, 1H), 7.82 (d, 1H), 8.15 (s, 1H). Intermediate 6-14 (2R,7aS)-7a-({[4-chloro-6-(3,3-difluoropiperidin-1-yl)pyrimidin-2-yl]oxy}methyl)-2- fluorohexahydro-1H-pyrrolizine
Figure imgf000100_0001
To a stirred solution of 4-chloro-6-(3,3-difluoropiperidin-1-yl)-2-(methanesulfonyl)pyrimidine (100 mg, 321 µmol; intermediate 6-11) in dry tetrahydrofuran (2.1 ml) under argon atmosphere was added [(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (76.6 mg, 481 µmol). The mixture was cooled by an ice-bath to 4°C, then sodium bis(trimethylsilyl)amide (2M in tetrahydrofuran)(320 µl, 2.0 M, 640 µmol) was added dropwise into the cold solution. The ice-bath was removed and the reaction mixture was stirred for 1 h at RT. The reaction was quenched with water and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with brine, filtered through a water repellant filter and concentrated. The obtained residue was purified by preparative HPLC (Method A; gradient: 5% B to 45% B). The product fractions were pooled and concentrated in vacuo to afford 62 mg (46% yield, 94% purity) of the title compound as a white foam. LC-MS (Method 3): Rt = 0.74 min; BHC233018 - FC - 100 / 152 - MS (ESIpos): m/z = 391 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.66-1.87 (m, 4H), 1.91- 2.03 (m, 2H), 2.07-2.15 (m, 2H), 2.76-2.86 (m, 1H), 2.97-3.10 (m, 3H), 3.68 (br s, 2H), 3.86-3.90 (m, 1H), 3.97 (d, 1H), 3.99-4.05 (m, 2H), 5.24 (br d, 1H), 5.32 (br s, 1H), 6.77 (s, 1H), 8.15 (s, 1H). Intermediate 6-15 (2R,7aS)-7a-({[4-chloro-6-(4-fluoropiperidin-1-yl)pyrimidin-2-yl]oxy}methyl)-2-fluorohexahydro- 1H-pyrrolizine
Figure imgf000101_0001
To a stirred solution of 4-chloro-6-(4-fluoropiperidin-1-yl)-2-(methanesulfonyl)pyrimidine (150 mg, 511 µmol; intermediate 6-12) in dry tetrahydrofuran (3.5 ml) under argon atmosphere, was added [(2R,7aS)- 2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methanol (122 mg, 766 µmol). The mixture was cooled by an ice-bath to 4°C, then sodium bis(trimethylsilyl)amide (2M in tetrahydrofuran)(510 µl, 2.0 M, 1.0 mmol; CAS-RN:[1070-89-9]) was added dropwise into the cold solution. The ice-bath was removed and the reaction mixture was stirred for 1 h at RT. The reaction was quenched with water and the aqueous phase was extracted with ethyl acetate. The combined orgnic phases were washed with brine, filtered through a water repellant filter and concentrated. The obtained residue was purified by preparative HPLC (Method A, gradient C). The product fractions were pooled and concentrated in vacuo to afford 122 mg (62% yield, 97% purity) of the title compound as a white solid.by HPLC (gradient C; formic acid). LC-MS (Method 3): Rt = 0.69 min; MS (ESIpos): m/z = 373 [M+H]+;1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.67-1.79 (m, 4H), 1.80-1.97 (m, 4H), 1.99-2.01 (m, 1H), 2.07-2.09 (m, 1H), 2.76-2.87 (m, 1H), 2.96-3.12 (m, 3H), 3.58-3.78 (m, 4H), 3.86 (d, 1H), 3.96 (d, 1H), 4.91 (ddq, 1H), 5.26 (br dd, 1H), 6.66 (s, 1H). BHC233018 - FC - 101 / 152 - Examples Example 01-01 2'-Amino-1-[6-(3,8-diazabicyclo[3.2.1]octan-3-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer)
Figure imgf000102_0001
Trifluoroacetic acid (229 µL, 2.98 mmol, 20 eq.) was added at 0°C to a solution of tert-butyl 3-[6-(2'- amino-3'-cyano-6',7'-dihydro-1H,5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1-yl)-2-{[(2R,7aS)-2- fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8- carboxylate (single stereoisomer) (99 mg, 0.15 mmol) in dichloromethane (1.5 mL). The reaction mixture was stirred at RT for 2 h and concentrated under reduced pressure. The residue was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). The resulting salt was dissolved in methanol and filtered in four portions through four SPE cartridges (200 mg PL-HCO3 MP 6 mL tubes, gravity filtration, washed each with 6 mL of methanol). The combined filtrates were concentrated under reduced pressure and dried in vacuo. Yield: 55 mg (64% of theory). LC/MS (method 1): tR = 0.81 min, MS (ESIneg): m/z = 563 [M- H]; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.04 (s, 2H), 5.31/5.17 (d, 1H), 5.03 (s, 1H), 4.19 (br d, 2H), 3.90 (br d, 1H), 3.83-3.67 (m, 5H), 3.43 (br s, 2H), 3.11-3.02 (m, 2H), 3.01-2.95 (m, 1H), 2.86-2.75 (m, 3H), 2.43 (t, 2H), 2.09-1.90 (m, 5H), 1.87-1.78 (m, 1H), 1.76-1.66 (m, 4H), 1.66-1.48 (m, 4H), 1H concealed. Example 01-02 2'-Amino-1-[6-(3,8-diazabicyclo[3.2.1]octan-3-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy}pyrimidin-4-yl]-5',6'-dihydrospiro[azetidine-3,4'-cyclopenta[b]thiophene]-3'- carbonitrile (single stereoisomer)
Figure imgf000102_0002
BHC233018 - FC - 102 / 152 - Hydrogen chloride solution (4 M in 1,4-dioxane, 233 µL, 0.93 mmol, 20 eq.) was added to a solution of tert-butyl 3-[6-(2'-amino-3'-cyano-5',6'-dihydro-1H-spiro[azetidine-3,4'-cyclopenta[b]thiophen]-1-yl)-2- {[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-3,8- diazabicyclo[3.2.1]octane-8-carboxylate (single stereoisomer) (33 mg, 92% purity, 0.05 mmol) in dichloromethane (2.0 mL). The reaction mixture was stirred at RT for 1 h. The formed precipitate was filtered, dissolved in N,N-dimethylformamide and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient), followed by a second RP-HPLC (acetonitrile / 2% ammonia in water gradient). Yield: 4 mg (14% of theory). LC/MS (method 1): tR = 0.67 min, MS (ESIneg): m/z = 549 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.19 (s, 2H), 5.31/5.17 (d, 1H), 5.06 (s, 1H), 4.09 (d, 2H), 3.94 (d, 2H), 3.90 (d, 1H), 3.79 (br s, 1H), 3.75 (d, 2H), 3.44 (br s, 2H), 3.11-3.02 (m, 2H), 3.02-2.95 (m, 1H), 2.88-2.75 (m, 3H), 2.73-2.60 (m, 4H), 2.13-1.88 (m, 3H), 1.88-1.48 (m, 8H). Example 01-03 2'-Amino-1-[6-(3,8-diazabicyclo[3.2.1]octan-3-yl)-2-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-6- ylmethoxy)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile formate (racemate)
Figure imgf000103_0001
x HCOOH Trifluoroacetic acid (57 µL, 0.74 mmol, 20 eq.) was added at RT to a solution of tert-butyl 3-[6-(2'-amino- 3'-cyano-6',7'-dihydro-1H,5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1-yl)-2-(4,5,6,7- tetrahydropyrazolo[1,5-a]pyrazin-6-ylmethoxy)pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8- carboxylate (racemate) (29 mg, 0.04 mmol) in dichloromethane (1.5 mL). The reaction mixture was stirred at RT for 4 h and concentrated under reduced pressure. The residue purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 15 mg (67% of theory). LC/MS (method 1): tR = 0.76 min, MS (ESIneg): m/z = 557 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 8.20 (s, 1H), 7.36 (d, 1H), 7.05 (s, 2H), 5.98 (d, 1H), 5.19 (s, 1H), 4.32-4.18 (m, 4H), 4.14 (dd, 1H), 4.04 (d, 2H), 3.99 (br s, 1H), 3.88 (br s, 2H), 3.86 (d, 1H, partially concealed), 3.80-3.70 (m, 4H, partially concealed), 3.44-3.3 (m, 2H, partially concealed), 3.05 (d, 2H), 2.44 (t, 2H), 2.04-1.95 (m, 2H), 1.89-1.79 (m, 2H), 1.79-1.62 (m, 4H). Example 02-01 2'-Amino-1-(2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- BHC233018 - FC - 103 / 152 - [1]benzothiophene]-3'-carbonitrile (single stereoisomer)
Figure imgf000104_0001
Sodium carbonate (30 mg, 0.28 mmol, 4.0 eq.) was added to a solution of (3R)-1-(6-chloro-2-{[(2R,7aS)- 2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl)-3-methylpiperidin-3-ol formate (single stereoisomer) (30 mg, 0.07 mmol) and 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile trifluoroacetate (31 mg, 76% purity, 0.07 mmol, 1.0 eq.) in dimethyl sulfoxide (340 µL). The reaction mixture was stirred at 95°C for 6 h in a closed microwave vial and diluted with water and aqueous hydrochloric acid solution (1 N, 300 µL). The suspension was dissolved in a mixture of acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 21 mg (51% of theory). LC/MS (method 1): tR = 1.19 min, MS (ESIneg): m/z = 566 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.00 (s, 2H), 5.33 (s, 1H), 5.31/5.18 (d, 1H), 4.37 (br s, 1H), 4.24 (d, 2H), 3.93 (br s, 1H), 3.78 (br s, 1H), 3.74 (br d, 2H), 3.64-3.3 (m, 3H, partially concealed), 3.23 (d, 1H), 3.13-3.03 (m, 2H), 3.03-2.96 (m, 1H), 2.85-2.75 (m, 1H), 2.43 (t, 2H), 2.14-1.89 (m, 5H), 1.88-1.60 (m, 6H), 1.60-1.46 (m, 2H), 1.46-1.36 (m, 1H), 1.07 (s, 3H). Example 02-02 2'-Amino-1-(2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]pyrimidin-4-yl)-5',6'-dihydrospiro[azetidine-3,4'- cyclopenta[b]thiophene]-3'-carbonitrile (single stereoisomer)
Figure imgf000104_0002
Sodium carbonate (39 mg, 0.37 mmol, 4.0 eq.) was added to a solution of (3R)-1-(6-chloro-2-{[(2R,7aS)- 2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl)-3-methylpiperidin-3-ol formate (single stereoisomer) (40 mg, 0.09 mmol) and 2'-amino-5',6'-dihydrospiro[azetidine-3,4'- cyclopenta[b]thiophene]-3'-carbonitrile trifluoroacetate (46 mg, 78% purity, 0.11 mmol, 1.2 eq.) in dimethyl sulfoxide (1.5 mL). The reaction mixture was stirred at 95°C for 3 h in a closed microwave vial, mixed with additional 2'-amino-5',6'-dihydrospiro[azetidine-3,4'-cyclopenta[b]thiophene]-3'-carbonitrile BHC233018 - FC - 104 / 152 - trifluoroacetate (15 mg, 78% purity, 0.04 mmol, 0.4 eq.), stirred for another 6 h at 95°C and quenched with aqueous hydrochloric acid solution (1 N, 400 µL). The mixture was diluted with water (0.5 mL) and acetonitrile (0.5 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 3 mg (6% of theory). LC/MS (method 1): tR = 1.12 min, MS (ESIneg): m/z = 552 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.18 (s, 2H), 5.32/5.18 (d, 1H), 5.17 (s, 1H), 4.46 (s, 1H), 4.09 (dd, 2H), 3.99-3.88 (m, 3H), 3.78 (d, 1H), 3.13-3.04 (m, 2H), 3.04-2.97 (m, 1H), 2.87-2.77 (m, 1H), 2.73-2.61 (m, 4H), 2.16-1.62 (m, 8H), 1.61-1.49 (m, 2H), 1.49-1.35 (m, 1H), 1.08 (s, 3H), 3H concealed. Example 02-03 2'-Amino-1-(6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]-2-{[(2S)-5-oxopyrrolidin-2- yl]methoxy}pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer)
Figure imgf000105_0001
Sodium carbonate (34 mg, 0.32 mmol, 4.0 eq.) was added to a solution of (5S)-5-[({4-chloro-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl}oxy)methyl]pyrrolidin-2-one formate (single stereoisomer) (31 mg, 0.08 mmol) and 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile trifluoroacetate (35 mg, 76% purity, 0.08 mmol, 1.0 eq.) in dimethyl sulfoxide (390 µL). The reaction mixture was stirred at 95°C overnight in a closed microwave vial, mixed with additional 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (35 mg, 76% purity, 0.08 mmol, 1.0 eq.), stirred at 95°C for another 4 h and diluted with water and aqueous hydrochloric acid solution (1 N, 321 µL). The suspension was dissolved in a mixture of acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 16 mg (36% of theory). LC/MS (method 1): tR = 1.16 min, MS (ESIpos): m/z = 524 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.78 (s, 1H), 7.03 (s, 2H), 5.17 (s, 1H), 4.43 (br s, 1H), 4.21 (d, 2H), 4.14-3.99 (m, 2H), 3.87-3.78 (m, 1H), 3.78-3.69 (m, 2H), 3.60-3.17 (m, 4H, partially concealed), 2.43 (t, 2H), 2.24-2.05 (m, 3H), 2.04-1.92 (m, 2H), 1.87-1.77 (m, 1H), 1.77-1.62 (m, 3H), 1.62-1.48 (m, 2H), 1.48-1.36 (m, 1H), 1.08 (s, 3H). Example 02-04 2'-Amino-1-(2-{[(2S)-4,4-difluoro-1-methylpyrrolidin-2-yl]methoxy}-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- BHC233018 - FC - 105 / 152 - carbonitrile (single stereoisomer)
Figure imgf000106_0001
Sodium carbonate (180 mg, 1.70 mmol, 4.0 eq.) was added to a solution of (3R)-1-(6-chloro-2-{[(2S)-4,4- difluoro-1-methylpyrrolidin-2-yl]methoxy}pyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (160 mg, 0.43 mmol) and 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile trifluoroacetate (295 mg, 72% purity, 0.64 mmol, 1.5 eq.) in dimethyl sulfoxide (4.5 mL). The reaction mixture was stirred at 95°C overnight in a closed microwave vial, quenched with aqueous hydrochloric acid solution (1 N, 1.7 mL) and diluted with water (0.5 mL) and acetonitrile (0.5 mL). The solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 172 mg (73% of theory). LC/MS (method 1): tR = 1.32 min, MS (ESIneg): m/z = 524 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.03 (s, 2H), 5.17 (s, 1H), 4.42 (s, 1H), 4.35-4.25 (m, 1H), 4.25-4.16 (m, 2H), 4.13- 4.02 (m, 1H), 3.74 (br d, 2H), 3.58-3.44 (m, 1H), 3.44-3.3 (m, 2H, partially concealed), 2.91-2.81 (m, 1H), 2.70-2.58 (m, 1H), 2.43 (t, 2H), 2.34 (s, 3H), 2.22-2.05 (m, 1H), 2.03-1.94 (m, 2H), 1.77-1.63 (m, 3H), 1.61-1.48 (m, 2H), 1.48-1.36 (m, 1H), 1.08 (s, 3H), 3H concealed. Example 02-05 2'-Amino-1-(2-{[(2S,4R)-4-fluoro-1-methylpyrrolidin-2-yl]methoxy}-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer)
Figure imgf000106_0002
Sodium carbonate (26 mg, 0.25 mmol, 4.0 eq.) was added to a solution of (3R)-1-(6-chloro-2-{[(2S,4R)- 4-fluoro-1-methylpyrrolidin-2-yl]methoxy}pyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (22 mg, 0.06 mmol) and 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile trifluoroacetate (43 mg, 72% purity, 0.09 mmol, 1.5 eq.) in dimethyl sulfoxide (0.5 mL). The reaction mixture was stirred at 95°C overnight in a closed microwave vial, mixed with additional sodium carbonate (10 mg, 0.09 mmol, 1.5 eq.), stirred for another 1 h at 95°C, mixed with additional 2'-amino- BHC233018 - FC - 106 / 152 - 6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (11 mg, 72% purity, 0.03 mmol, 0.4 eq.) and sodium carbonate (3 mg, 0.03 mmol, 0.4 eq.) and stirred at 95°C overnight. The mixture was quenched with aqueous hydrochloric acid solution (1 N, 362 µL). The solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 11 mg (31% of theory). LC/MS (method 1): tR = 1.14 min, MS (ESIneg): m/z = 540 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.04 (s, 2H), 5.22/5.11 (d, 1H), 5.16 (s, 1H), 4.43 (br s, 1H), 4.32-4.24 (m, 1H), 4.24-4.16 (m, 2H), 4.08-3.95 (m, 1H), 3.79-3.68 (m, 2H), 3.56-3.20 (m, 6H, partially concealed), 2.89-2.80 (m, 1H), 2.43 (t, 2H), 2.37 (s, 3H), 2.15-2.03 (m, 1H), 2.03-1.95 (m, 2H), 1.93-1.77 (m, 1H), 1.75-1.64 (m, 3H), 1.60-1.49 (m, 2H), 1.47-1-37 (m, 1H), 1.08 (s, 3H). Example 02-06 2'-Amino-1-(2-{[(2S,4R)-1-cyclopropyl-4-fluoropyrrolidin-2-yl]methoxy}-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer)
Figure imgf000107_0001
Sodium carbonate (92 mg, 0.87 mmol, 5.0 eq.) was added to a solution of (3R)-1-(6-chloro-2-{[(2S,4R)- 1-cyclopropyl-4-fluoropyrrolidin-2-yl]methoxy}pyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (67 mg, 0.17 mmol) and 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile trifluoroacetate (121 mg, 72% purity, 0.26 mmol, 1.5 eq.) in dimethyl sulfoxide (800 µL). The reaction mixture was stirred at 95°C overnight in a closed microwave vial, diluted with water and quenched with aqueous hydrochloric acid solution (1 N, 870 µL). The suspension was diluted with acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 58 mg (56% of theory). LC/MS (method 1): tR = 1.26 min, MS (ESIneg): m/z = 566 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.03 (s, 2H), 5.24/5.11 (d, 1H), 5.17 (s, 1H), 4.51 (br d, 1H), 4.44 (s, 1H), 4.21 (br d, 2H), 3.95 (br s, 1H), 3.74 (br d, 2H), 3.57-3.19 (m, 6H, partially concealed), 2.92-2.72 (m, 1H), 2.44 (t, 2H), 2.23-2.08 (m, 1H), 2.05-1.78 (m, 4H), 1.77-1.63 (m, 3H), 1.61-1.49 (m, 2H), 1.49-1.37 (m, 1H), 1.08 (s, 3H), 0.57-0.46 (m, 1H), 0.46-0.35 (m, 2H), 0.35- 0.24 (m, 1H). Example 02-07 2'-Amino-1-{2-[(3-cyanopyrrolidin-3-yl)methoxy]-6-[(3R)-3-hydroxy-3-methylpiperidin-1- BHC233018 - FC - 107 / 152 - yl]pyrimidin-4-yl}-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile formate (mixture of two enantiopure diastereomers)
Figure imgf000108_0001
HCOOH Trifluoroacetic acid (141 µL, 1.83 mmol, 20 eq.) was added at RT to a solution of tert-butyl 3-[({4-(2'- amino-3'-cyano-6',7'-dihydro-1H,5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1-yl)-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-2-yl}oxy)methyl]-3-cyanopyrrolidine-1-carboxylate (mixture of two enantiopure diastereomers) (58 mg, 0.09 mmol) in dichloromethane (4.0 mL). The reaction mixture was stirred at RT for about 1 h and concentrated under reduced pressure. The residue was co-evaporated with dichloromethane, dried in vacuo and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 30 mg (53% of theory). LC/MS (method 1): tR = 1.12 min, MS (ESIneg): m/z = 533 [M- H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 8.16 (s, 2H), 7.04 (s, 2H), 5.19 (s, 1H), 4.31-4.17 (m, 5H), 3.45-3.25 (m, 6H), 3.18 (d, 1H), 3.12 (d, 1H), 3.05-2.89 (m, 2H), 2.44 (t, 2H), 2.22-2.12 (m, 1H), 2.09-1.90 (m, 3H), 1.79-1.63 (m, 3H), 1.62-1.48 (m, 2H), 1.48-1.37 (m, 1H), 1.09 (s, 3H), 1H concealed. Example 02-08 2'-Amino-1-{2-[(3-cyano-1-methylpyrrolidin-3-yl)methoxy]-6-[(3R)-3-hydroxy-3-methylpiperidin- 1-yl]pyrimidin-4-yl}-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile (mixture of two enantiopure diastereomers)
Figure imgf000108_0002
CH3 Sodium carbonate (89 mg, 0.84 mmol, 4.0 eq.) was added to a solution of 3-[({4-chloro-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl}oxy)methyl]-1-methylpyrrolidine-3-carbonitrile (mixture of two enantiopure diastereomers) (77 mg, 0.21 mmol) and 2'-amino-6',7'-dihydro-5'H- spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (148 mg, 71% purity, 0.32 mmol, 1.5 eq.) in dimethyl sulfoxide (1.8 mL). The reaction mixture was stirred at 95°C for 7 h in a closed microwave vial, kept over the weekend at RT, stirred at 95°C for another 24 h and quenched with aqueous hydrochloric acid solution (1 N, 842 µL). The mixture was purified by RP-HPLC (acetonitrile / 0.1% BHC233018 - FC - 108 / 152 - formic acid in water gradient). Yield: 45 mg (38% of theory). LC/MS (method 1): tR = 1.15 min, MS (ESIneg): m/z = 547 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.03 (s, 2H), 5.19 (s, 1H), 4.45 (br s, 1H), 4.30-4.18 (m, 4H), 3.81-3.71 (m, 2H), 2.85 (d, 1H), 2.70-2.60 (m, 2H), 2.44 (t, 2H), 2.27 (s, 3H), 2.25-2.17 (m, 1H, partially concealed), 2.06-1.95 (m, 3H), 1.77-1.63 (m, 3H), 1.61-1.49 (m, 2H), 1.49-1.37 (m, 1H), 1.09 (s, 3H), 5H concealed. Example 02-09 2'-Amino-1-{6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]-2-[2-(1-methyl-1H-imidazol-2- yl)ethoxy]pyrimidin-4-yl}-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer)
Figure imgf000109_0001
Sodium carbonate (93 mg, 0.88 mmol, 5.0 eq.) was added to a solution of (3R)-1-{6-chloro-2-[2-(1- methyl-1H-imidazol-2-yl)ethoxy]pyrimidin-4-yl}-3-methylpiperidin-3-ol (single stereoisomer) (62 mg, 0.18 mmol) and 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (124 mg, 71% purity, 0.26 mmol, 1.5 eq.) in dimethyl sulfoxide (900 µL). The reaction mixture was stirred at 95°C overnight in a closed microwave vial, diluted with water and quenched with aqueous hydrochloric acid solution (1 N, 881 µL). The suspension was dissolved in acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 37 mg (38% of theory). LC/MS (method 1): tR = 1.08 min, MS (ESIneg): m/z = 533 [M-H]-; 1H- NMR (400 MHz, DMSO-d6): δ [ppm] = 7.18 (s, 1H), 7.03 (s, 2H), 6.98 (s, 1H), 5.17 (s, 1H), 4.50-4.39 (m, 3H), 4.20 (br d, 2H), 3.73 (d, 2H), 3.64 (s, 3H), 3.12 (t, 2H), 2.43 (t, 2H), 2.03-1.95 (m, 2H). 1.76- 1.63 (m, 3H), 1.61-1.48 (m, 2H), 1.48-1.36 (m, 1H), 1.08 (s, 3H), 4H concealed. Example 02-10 2'-Amino-1-(6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]-2-{[(2S)-2-methylpyrrolidin-2- yl]methoxy}pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer) BHC233018 - FC - 109 / 152 -
Figure imgf000110_0001
Trifluoroacetic acid (57 µL, 0.75 mmol, 15 eq.) was added at RT to a solution of tert-butyl (2S)-2-[({4- (2'-amino-3'-cyano-6',7'-dihydro-1H,5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1-yl)-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl}oxy)methyl]-2-methylpyrrolidine-1-carboxylate (single stereoisomer) (31 mg, 0.05 mmol) in dichloromethane (3.0 mL). The reaction mixture was stirred at RT for 3 h and concentrated under reduced pressure. The residue was co-evaporated with dichloromethane and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 11 mg (43% of theory). LC/MS (method 1): tR = 1.14 min, MS (ESIneg): m/z = 522 [M-H]-; 1H-NMR (400 MHz, DMSO- d6): δ [ppm] = 8.36 (s, 1H), 7.04 (s, 2H), 5.17 (s, 1H), 4.21 (d, 2H), 4.09-3.95 (m, 2H), 3.74 (d, 2H), 3.25 (d, 2H), 3.06-2.88 (m, 2H), 2.44 (t, 2H), 2.06-1.93 (m, 2H), 1.86-1.63 (m, 6H), 1.61-1.49 (m, 3H), 1.49- 1.36 (m, 1H), 1.21 (s, 3H), 1.08 (s, 3H), 3H concealed. Example 02-11 2'-Amino-1-(2-{[1-(dimethylamino)cyclopropyl]methoxy}-6-[(3R)-3-hydroxy-3-methylpiperidin-1- yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile (single stereoisomer)
Figure imgf000110_0002
Sodium carbonate (84 mg, 0.79 mmol, 4.0 eq.) was added to a solution of (3R)-1-(6-chloro-2-{[1- (dimethylamino)cyclopropyl]methoxy}pyrimidin-4-yl)-3-methylpiperidin-3-ol (single enantiomer) (51 mg, 0.20 mmol) and 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (139 mg, 71% purity, 0.30 mmol, 1.5 eq.) in dimethyl sulfoxide (1.2 mL). The reaction mixture was stirred at 95°C overnight in a closed microwave vial, combined with a batch of a previously performed test reaction (0.05 mmol scale) and quenched with aqueous hydrochloric acid solution (1 N, 792 µL). The mixture was dissolved with water (0.5 mL) and acetonitrile (1.0 mL) and purified by RP- HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 63 mg (48% of theory for both reactions). LC/MS (method 1): tR = 1.05 min, MS (ESIneg): m/z = 522 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.03 (s, 2H), 5.16 (s, 1H), 4.43 (s, 1H), 4.35 (br s, 2H), 4.21 (d, 2H), 3.74 (d, 2H), 3.57-3.3 (m, BHC233018 - FC - 110 / 152 - 2H, partially concealed), 3.3-3.15 (m, 2H, partially concealed), 2.64-2.5 (br s, 6H, partially concealed), 2.44 (t, 2H), 2.04-1.94 (m, 2H), 1.76-1.63 (m, 3H), 1.61-1.48 (m, 2H), 1.48-1.37 (m, 1H), 1.08 (s, 3H), 0.80 (br s, 4H). Example 03-01 2'-Amino-1-[2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-(2-oxo-1,6- diazaspiro[3.5]nonan-6-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile formate (mixture of two enantiopure diastereomers)
Figure imgf000111_0001
x HCOOH Sodium carbonate (44 mg, 0.42 mmol, 4.0 eq.) was added to a solution of 6-(6-chloro-2-{[(2R,7aS)-2- fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl)-1,6-diazaspiro[3.5]nonan-2-one (mixture of two enantiopure diastereomers) (52 mg, 82% purity, 0.10 mmol) and 2'-amino-6',7'-dihydro- 5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (68 mg, 76% purity, 0.16 mmol, 1.5 eq.) in dimethyl sulfoxide (2.0 mL). The reaction mixture was stirred at 95°C overnight in a closed microwave vial, quenched with aqueous hydrochloric acid solution (1 N, 416 µL) and diluted with water (0.5 mL) and N,N-dimethylformamide (0.5 mL). The solution was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 9 mg (13% of theory). LC/MS (method 1): tR = 1.12 min, MS (ESIneg): m/z = 591 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 8.29 (s, 1H), 7.04 (s, 2H), 5.31/5.18 (d, 1H), 5.27 (s, 1H), 4.21 (d, 2H), 3.98-3.87 (m, 1H), 3.82-3.71 (m, 2H), 3.71-3.63 (m, 1H), 3.11-3.03 (m, 2H), 3.03-2.95 (m, 1H), 2.85-2.74 (m, 1H), 2.64-2.5 (m, 2H, partially concealed), 2.44 (t, 2H), 2.10-1.90 (m, 5H), 1.85-1.49 (m, 11H), 2H concealed. Example 03-02 2'-Amino-1-[2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-(3-oxa-7,9- diazabicyclo[3.3.1]nonan-7-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile formate (single stereoisomer) BHC233018 - FC - 111 / 152 -
Figure imgf000112_0001
HCOOH Trifluoroacetic acid (111 µL, 1.44 mmol, 20 eq.) was added at RT to a solution of tert-butyl 7-[6-(2'- amino-3'-cyano-6',7'-dihydro-1H,5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1-yl)-2-{[(2R,7aS)-2- fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-3-oxa-7,9- diazabicyclo[3.3.1]nonane-9-carboxylate (single stereoisomer) (54 mg, 91% purity, 0.07 mmol) in dichloromethane (2.3 mL). The reaction mixture was stirred at RT for about 1 h and concentrated under reduced pressure. The residue was co-evaporated several times with dichloromethane and purified by RP- HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 26 mg (54% of theory). LC/MS (method 1): tR = 0.72 min, MS (ESIpos): m/z = 581 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 10.72 (br s, 1H), 9.61 (br s, 1H), 9.51 (br s, 1H), 7.06 (br s, 2H), 5.62/5.49 (d, 1H), 5.32 (s, 1H), 4.69-4.19 (m, 6H), 3.99 (d, 2H), 3.93 (d, 2H), 3.89-3.66 (m, 6H), 3.64 (br s, 2H), 3.3-3.21 (m, 2H, partially concealed), 2.5- 2.39 (m, 3H, partially concealed), 2.29-1.93 (m, 7H), 1.81-1.65 (m, 2H). Example 03-03 2'-amino-1-[6-(4,4-difluoropiperidin-1-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer)
Figure imgf000112_0002
To a solution of tert-butyl {3’-cyano-1-[6-(4,4-difluoropiperidin-1-yl)-2-{[(2R,7aS)-2-fluorotetrahydro- 1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-6’,7’-dihydro-5’H-spiro[azetidine-3,4’- [1]benzothiophen]-2’-yl}carbamate (25.0 mg, 37.1 µmol; intermediate 6-5) in dichloromethane (320 µl) and methanol (200 µl) was added hydrochloric acid (4M in dioxane) (190 µl, 4.0 M, 740 µmol; CAS- RN:[7647-01-0]). The reaction mixture was stirred for 3 h at RT. The mixture was concentrated and the BHC233018 - FC - 112 / 152 - residue was taken up in dichloromethane and some methanol. Then solid sodium bicarbonate was added, the mixture was adsorbed on silica gel and purified by Biotage Isolera™ chromatography (Biotage Sfär KP-NH2 – 50 µm 5 g, eluting with dichloromethane - ethanol, 1:0 to 1:1) to afford 7.4 mg (33% yield, 95% purity) of the title compound as a white solid. LC-MS (Method 3): Rt = 0.92 min; MS (ESIpos): m/z = 574 [M+H]+;1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.71 (br s, 2H), 1.78 (br s, 2H), 1.87-2.03 (m, 8H), 2.41-2.46 (m, 2H), 2.89 (br s, 1H), 3.04-3.30 (m, 2H), 3.43-3.53 (m, 3H), 3.62-3.81 (m, 8H), 4.22 (br d, 2H), 5.24 + 5.37 (br d, 1H), 5.37 (s, 1H), 7.06 (s, 2H). Example 03-04 2'-amino-1-[2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-(3- oxopiperazin-1-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer)
Figure imgf000113_0001
To a solution of tert-butyl {3’-cyano-1-[2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}-6-(3-oxopiperazin-1-yl)pyrimidin-4-yl]-6’,7’-dihydro-5’H-spiro[azetidine-3,4’- [1]benzothiophen]-2’-yl}carbamate (25.0 mg, 38.3 µmol; intermediate 6-6) in dichloromethane (330 µl) and methanol (210 µl) was added hydrochloric acid (4M in dioxane)(190 µl, 4.0 M, 770 µmol; CAS- RN:[7647-01-0]). The reaction mixture was stirred for 4 h at RT. The mixture was concentrated and the residue was taken up in acetonitrile, filtered and purified by preparative HPLC (Method A; gradient: 5% B to 30% B). The product fractions were pooled and concentrated in vacuo to afford 8.0 mg (37% yield, 07% purity) of the title compound as a white solid. LC-MS (Method 3): Rt = 0.74 min; MS (ESIpos): m/z = 554 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.72 (br s, 4H), 1.85 (br s, 1H), 1.93-2.05 (m, 4H), 2.10 (br s, 1H), 2.40-2.46 (m, 2H), 2.84 (br s, 1H), 2.99-3.14 (m, 2H), 3.18-3.30 (m, 2H), 3.69 (br t, 2H), 3.76 (br d, 3H), 3.96 (s, 3H), 4.21 (br s, 2H), 5.21 (s, 1H), 5.27 (br d, 1H), 7.06 (s, 2H), 8.09-8.14 (m, 1H). Example 03-05 BHC233018 - FC - 113 / 152 - N-{1-[6-(2'-amino-3'-cyano-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1-yl)-2- {[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]piperidin-4- yl}acetamide (single stereoisomer)
Figure imgf000114_0001
To a stirred suspension of N-[1-(6-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl)piperidin-4-yl]acetamide (70.0 mg, 170 µmol; intermediate 6-13) and caesium carbonate (221 mg, 680 µmol; CAS-RN:[534-17-8]) in dry N,N-dimethylacetamide (2.5 ml) was added 2’-amino-6’,7’-dihydro-5’H-spiro[azetidine-3,4’-[1]benzothiophene]-3’-carbonitrile trifluoroacetate (1/1) (68.0 mg, 204 µmol). The reaction mixture was stirred for 1 h at 50°C. The reaction mixture was heated at 100°C for further 11 h. After cooling to RT, the reaction mixture was diluted with 2 ml acetonitrile / water (7:3) and purified by preparative HPLC (Method A; gradient: 20% B to 60% B). The product fractions were pooled and concentrated in vacuo to afford 12.3 mg (11% yield, 93% purity) of the title compound as a white fluffy solid. LC-MS (Method 3): Rt = 0.82 min; MS (ESIneg): m/z = 593 [M-H]-; 1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.19-1.33 (m, 3H), 1.66-1.77 (m, 6H), 1.78 (s, 3H), 1.89-2.10 (m, 5H), 2.39-2.47 (m, 2H), 2.60-2.66 (m, 1H), 2.77-2.83 (m, 1H), 2.86-2.95 (m, 2H), 2.99 (br s, 1H), 3.00-3.10 (m, 2H), 3.68-3.85 (m, 4H), 3.91 (br d, 1H), 4.09-4.29 (m, 4H), 5.24 (br d, 1H), 5.22 (s, 1H), 7.06 (s, 1H), 7.81 (d, 1H). Example 03-06 2'-amino-1-[6-(3,3-difluoropiperidin-1-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer) BHC233018 - FC - 114 / 152 -
Figure imgf000115_0001
To a stirred suspension of (2R,7aS)-7a-({[4-chloro-6-(3,3-difluoropiperidin-1-yl)pyrimidin-2- yl]oxy}methyl)-2-fluorohexahydro-1H-pyrrolizine (60.0 mg, 154 µmol; intermediate 6-14) and sodium carbonate (65.1 mg, 614 µmol; CAS-RN:[497-19-8]) in N,N-dimethylacetamide (2.2 ml) was added 2’- amino-6’,7’-dihydro-5’H-spiro[azetidine-3,4’-[1]benzothiophene]-3’-carbonitrile trifluoroacetate (1/1) (61.4 mg, 184 µmol). The reaction mixture was heated at 100°C for further 10 h. The mixture was filtered through a pad of celite, rinsed with ethyl acetate and the filtrate was washed with water. The organic phase was filtered through a water repellant filter and concentrated. The crude product was purified by by preparative HPLC (Method A, gradient C). The product fractions were pooled and concentrated in vacuo to afford 35.4 mg (37% yield, 91% purity) of the title compound as a white fluffy solid. LC-MS (Method 3): Rt = 0.90 min; MS (ESIpos): m/z = 575 [M+H]+;1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.64-1.87 (m, 8H), 1.92-2.12 (m, 8H), 2.39-2.46 (m, 2H), 2.75-2.86 (m, 1H), 2.99 (br s, 1H), 3.02-3.11 (m, 2H), 3.53 (br s, 2H), 3.66-3.82 (m, 2H), 3.85-3.97 (m, 2H), 4.21 (br d, 2H), 5.24 (br d, 1H), 5.31 (s, 1H), 7.06 (s, 2H). Example 03-07 2'-amino-1-[6-(4-fluoropiperidin-1-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer)
Figure imgf000115_0002
BHC233018 - FC - 115 / 152 - To a stirred suspension of (2R,7aS)-7a-({[4-chloro-6-(4-fluoropiperidin-1-yl)pyrimidin-2- yl]oxy}methyl)-2-fluorohexahydro-1H-pyrrolizine (80.0 mg, 215 µmol; intermediate 6-15) and sodium carbonate (91.0 mg, 858 µmol) in dry N,N-dimethylacetamide (2.0 ml) was added 2’-amino-6’,7’-dihydro- 5’H-spiro[azetidine-3,4’-[1]benzothiophene]-3’-carbonitrile trifluoroacetate (1/1) (85.8 mg, 257 µmol). The reaction mixture was heated at 100°C for further 10 h. The reaction was filtered and the filtrate was concentrated. The crude product was purified by preparative HPLC (Method A, gradient C). The product fractions were pooled and concentrated in vacuo to afford 8.0 mg (7% yield, 97% purity) of the title compound as a white solid. LC-MS (Method 3): Rt = 0.97 min; MS (ESIpos): m/z = 556 [M+H]+;1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.64-1.76 (m, 6H), 1.78-2.01 (m, 7H), 2.03-2.11 (m, 1H), 2.41-2.46 (m, 2H), 2.75-2.85 (m, 1H), 2.98 (br s, 1H), 3.01-3.10 (m, 2H), 3.38-3.51 (m, 2H), 3.64-3.81 (m, 5H), 3.91 (br d, 1H), 4.21 (br d, 2H), 4.77-4.85 + 4.89-4.97 (m, 1H), 5.24 (br d, 1H), 5.26 (s, 1H), 7.06 (s, 2H). Example 03-08 tert-butyl 1-[6-(2'-amino-3'-cyano-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1-yl)- 2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-1,6- diazaspiro[3.4]octane-6-carboxylate (mixture of two enantiopure diastereomers)
Figure imgf000116_0001
To a stirred suspension of tert-butyl 1-(6-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl)-1,6-diazaspiro[3.4]octane-6-carboxylate (150 mg, 311 µmol) and sodium carbonate (132 mg, 1.24 mmol) in dry N,N-dimethylacetamide (2.9 ml) was added 2’-amino-6’,7’- dihydro-5’H-spiro[azetidine-3,4’-[1]benzothiophene]-3’-carbonitrile trifluoroacetate (1/1) (124 mg, 373 µmol). The reaction mixture was heated at 100°C for further 10 h. The reaction was allowed to cool to RT, then ethyl acetate and water were added. The aqueous phase was extreacted with ethyl acetat and the combined organic phases were washed with brine, filtered through a water repellant filter and concentrated. The crude product was purified by preparative HPLC (Method A; gradient: 20% B to 60% B). The product fractions were pooled and concentrated in vacuo to afford 56.3 mg (27% yield, 99% purity) of the title compound as a white solid. LC-MS (Method 3): Rt = 1.07 min; MS (ESIneg): m/z = 663 [M-H]-;1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.39 (s, 9H), 1.64-1.84 (m, 5H), 1.87-2.12 (m, BHC233018 - FC - 116 / 152 - 6H), 2.18-2.38 (m, 2H), 2.43 (br t, 2H), 2.52-2.62 (m, 1H), 2.73-2.84 (m, 1H), 2.93-3.08 (m, 3H), 3.13- 3.27 (m, 1H), 3.36-3.40 (m, 1H), 3.42-3.56 (m, 1H), 3.68-3.96 (m, 7H), 4.18 (br s, 2H), 4.73 (br s, 1H), 5.23 (br d, 1H), 7.06 (s, 2H). Example 03-09 2'-amino-1-[6-(1,6-diazaspiro[3.4]octan-1-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (mixture of two enantiopure diastereomers)
Figure imgf000117_0001
tert-butyl 1-[6-(2’-amino-3’-cyano-6’,7’-dihydro-5’H-spiro[azetidine-3,4’-[1]benzothiophen]-1-yl)-2- {[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-1,6- diazaspiro[3.4]octane-6-carboxylate (50.0 mg, 75.2 µmol; example 3-8) was dissolved in dichloromethane (550 µl) and methanol (55 µl), then hydrochloric acid (4M in dioxane)(380 µl, 4.0 M, 1.5 mmol; CAS- RN:[7647-01-0]) was added. The reaction mixture was stirred for 3 h at RT. The volatiles were removed under reduced pressure and the crude product was purified by preparative HPLC (Method A, gradient B). The product fractions were pooled and concentrated in vacuo to afford 20.4 mg (43% yield, 90% purity) of the title compound as a white fluffy solid. LC-MS (Method 3): Rt = 0.74 min; MS (ESIpos): m/z = 565 [M+H]+;1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.66-1.90 (m, 5H), 1.91-2.13 (m, 6H), 2.29-2.37 (m, 1H), 2.40-2.47 (m, 3H), 2.75-2.85 (m, 1H), 2.96-3.15 (m, 4H), 3.52 (br d, 1H), 3.67-3.94 (m, 6H), 4.19 (br s, 3H), 4.78 (br s, 1H), 5.18 (br s, 1H), 5.32 (br s, 1H), 7.05 (s, 2H), 8.29 (s, 2H). Example 03-10 1-(6-[(4RS)-6-acetyl-1,6-diazaspiro[3.4]octan-1-yl]-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy}pyrimidin-4-yl)-2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (mixture of two enantiopure diastereomers) BHC233018 - FC - 117 / 152 -
Figure imgf000118_0001
To a solution of 2’-amino-1-(6-[(4RS)-1,6-diazaspiro[3.4]octan-1-yl]-2-{[(2R,7aS)-2-fluorotetrahydro- 1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl)-6’,7’-dihydro-5’H-spiro[azetidine-3,4’- [1]benzothiophene]-3’-carbonitrile (18.0 mg, 31.9 µmol; example 3-10) in dichloromethane (250 µl) and N,N-diisopropylethylamine (11 µl, 64 µmol) was added acetyl chloride (2.5 µl, 35 µmol; CAS-RN:[75- 36-5]). The reaction mixture was stirred for 2 h at RT. The solvent was evaporated and the crude product purified by preparative HPLC (Method A, gradient C). The product fractions were pooled and concentrated in vacuo to afford 12.0 mg (60% yield, 97% purity) of the title compound as a white solid. LC-MS (Method 3): Rt = 0.88 min; MS (ESIpos): m/z = 607 [M+H]+;1H NMR (400 MHz, DMSO-d6) δ [ppm] = 1.70 (br s, 4H), 1.82 (br s, 1H), 1.89-2.09 (m, 8H), 2.43 (br t, 2H), 2.52-2.68 (m, 2H), 2.80 (br d, 1H), 2.94-3.03 (m, 1H), 3.05 (br s, 2H), 3.17-3.26 (m, 1H), 3.38-3.62 (m, 3H), 3.71-3.88 (m, 6H), 4.02 (br dd, 1H), 4.18 (br s, 2H), 4.70-4.76 (m, 1H), 5.23 (br d, 1H), 7.04 (s, 2H), 8.14 (s, 1H). Example 04-01 2'-Amino-1-(2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]-5-methylpyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (single stereoisomer)
Figure imgf000118_0002
Sodium carbonate (56 mg, 0.53 mmol, 5.0 eq.) was added to a solution of (3R)-1-(6-chloro-2-{[(2R,7aS)- 2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-5-methylpyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (43 mg, 0.11 mmol) and 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile trifluoroacetate (64 mg, 72% purity, 0.14 mmol, 1.3 eq.) in dimethyl sulfoxide (0.5 mL). The reaction mixture was stirred at 95°C overnight in a closed microwave vial, diluted BHC233018 - FC - 118 / 152 - with water and quenched with aqueous hydrochloric acid solution (1 N, 533 µL). The suspension was dissolved in acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 13 mg (20% of theory). LC/MS (method 1): tR = 1.35 min, MS (ESIneg): m/z = 580 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.01 (s, 2H), 5.31/5.18 (d, 1H), 4.54 (br s, 1H), 4.35 (br t, 2H), 3.99-3.87 (m, 3H), 3.78 (d, 1H), 3.13-3.04 (m, 4H), 3.04-2.95 (m, 3H), 2.85-2.75 (m, 1H), 2.43 (t, 2H), 2.13-1.88 (m, 5H), 1.92 (s, 3H), 1.86-1.65 (m, 6H), 1.58-1.46 (m, 3H), 1.12 (s, 3H). Example 04-02 2'-Amino-1-[2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-5-methyl-6-(3- oxopiperazin-1-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer)
Figure imgf000119_0001
Sodium carbonate (86.7 mg, 0.82 mmol, 4.0 eq.) and molecular sieve (3 Å, 20 mg)were added to a solution of 4-(6-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-5- methylpyrimidin-4-yl)piperazin-2-one (single stereoisomer) (78.5 mg, 0.21 mmol) and 2'-amino-6',7'- dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (88.5 mg, 77 % purity, 0.21 mmol, 1.0 eq.) in dimethyl sulfoxide (3 mL). The reaction mixture was stirred at 95°C for 24 h in a closed microwave vial, then diluted with ethyl acetate and water, and extracted three times with ethyl acetate. The combined organic phases were washed with brine, dried over phase separation filter paper, and concentrated in vacuo. The residue was separated by preparative HPLC (method D). Yield: 2.4 mg (2% yield). LC/MS (method 4): tR = 1.13 min, MS (ESIpos): m/z = 567 [M-H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.91 (br s, 1H), 7.03 (s, 2H), 5.24 (br d, 1H), 4.37 (m, 2H), 3.96 (m, 2H), 3.92 (br d, 1H), 3.80 (br d, 1H), 3.75 (s, 2H), 3.40 (m, 2H), 3.23 (m, 2H), 3.06 (m, 2H), 2.98 (m, 1H), 2.79 (m, 1H), 2.43 (br t, 2H), 2.10-1.89 (m, 5H), 1.92 (s, 3H), 1.86-1.65 (m, 5H) BHC233018 - FC - 119 / 152 - Example 04-03 2'-Amino-1-(5-fluoro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)- 3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (single stereoisomer)
Figure imgf000120_0001
Sodium carbonate (123 mg, 1.16 mmol, 5.0 eq.) was added to a solution of (3R)-1-(6-chloro-5-fluoro-2- {[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl)-3-methylpiperidin-3- ol (single stereoisomer), (3R)-1-(2-chloro-5-fluoro-6-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy}pyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (99 mg, 0.23 mmol) and 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (140 mg, 72% purity, 0.30 mmol, 1.3 eq.) in dimethyl sulfoxide (1.0 mL). The reaction mixture was stirred at 95°C overnight in a closed microwave vial, diluted with water and quenched with aqueous hydrochloric acid solution (1 N, 1.2 mL). The suspension was dissolved in acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient), followed by a second RP-HPLC (acetonitrile / 1% ammonia in water gradient) to separate both regioisomers. Yield: 13 mg (9% of theory). LC/MS (method 1): tR = 1.37 min, MS (ESIpos): m/z = 586 [M+H]+; 1H-NMR (500 MHz, DMSO-d6): δ [ppm] = 7.05 (s, 2H), 5.29/5.18 (d, 1H), 4.39 (br s, 2H), 3.95-3.81 (m, 3H), 3.72 (d, 2H), 3.41 (d, 2H), 3.31 (d, 1H), 3.11-2.93 (m, 3H), 2.87-2.71 (m, 1H), 2.42 (t, 2H), 2.11-1.89 (m, 5H), 1.87- 1.64 (m, 6H), 1.61-1.37 (m, 3H), 1.07 (s, 3H), 1H concealed. Example 05-01 2'-amino-1-[5-cyano-6-(3,8-diazabicyclo[3.2.1]octan-3-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H- pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (single stereoisomer) BHC233018 - FC - 120 / 152 -
Figure imgf000121_0001
To a stirred solution of tert-butyl 3-[6-(2’-amino-3’-cyano-6’,7’-dihydro-5’H-spiro[azetidine-3,4’- [1]benzothiophen]-1-yl)-5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (85.0 mg, 123 µmol) in dichloromethane (0.63 mL) and methanol (0.31 mL) was added HCl in dioxane (310 µl, 4.0 M, 1.2 mmol; CAS-RN:[7647-01-0]). The mixture was stirred at rt for 3 h. The solvent was removed in vacuum. The residue was dissolved in a mixture of dichloromethane and methanol, sodium bicarbonate was added and the mixture was stirred for 5 min and then concentrated in vacuum. The residue was subjected to aminophase-silicagel chromatography (Gradient: dichloromethane / methanol 0-10%) and gave 60.0 mg (82 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 0.69 min; MS (ESIneg): m/z = 588 [M-H]- 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.09 (s, 2H), 5.36-5.10 (m, 1H), 4.67-4.24 (m, 2H), 4.23-4.06 (m, 3H), 4.03-3.88 (m, 2H), 3.87-3.74 (m, 1H), 3.43 (br s, 2H), 3.17-3.02 (m, 4H), 3.01-2.90 (m, 1H), 2.84-2.74 (m, 1H), 2.42 (br t, 2H), 2.05 (br s, 1H), 2.04-1.87 (m, 4H), 1.86-1.77 (m, 1H), 1.77-1.65 (m, 4H), 1.64-1.51 (m, 4H) (1H not detected) Example 05-02 2'-amino-1-(5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)- 3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (single stereoisomer)
Figure imgf000121_0002
BHC233018 - FC - 121 / 152 - To a stirred solution of 4-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6- [(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidine-5-carbonitrile (65.0 mg, 159 µmol) and 2'-amino- 6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (85.1 mg) in N,N-dimethylacetamide (2.5 ml) was added cesium carbonate (207 mg, 634 µmol; CAS-RN:[534-17-8]) and the mixture was stirred at 50° C for 1 h. Water was added and the mixture was extracted with ethyl acetate. The organic phase was washed with half-saturated sodium chloride solution, dried (sodium sulfate), filtered and the solvent was removed in vacuum. Aminophase-silicagel chromatography (Gradient: dichloromethane / methanol 0-20%) gave 62.0 mg (63 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 0.85 min; MS (ESIpos): m/z = 594 [M+H]+ 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.09 (s, 2H), 5.36-5.10 (m, 1H), 4.73-4.49 (m, 2H), 4.42-4.09 (m, 2H), 4.05-3.67 (m, 4H), 3.65-3.45 (m, 3H), 3.10-3.03 (m, 2H), 3.02-2.91 (m, 1H), 2.84-2.75 (m, 1H), 2.42 (br t, 2H), 2.06 (br s, 1H), 2.04-1.88 (m, 4H), 1.86-1.65 (m, 6H), 1.63-1.43 (m, 3H), 1.08 (s, 3H) Example 05-03 2'-amino-1-[5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-(1,4- oxazepan-4-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer)
Figure imgf000122_0001
To a stirred solution of 4-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6- (1,4-oxazepan-4-yl)pyrimidine-5-carbonitrile (70.0 mg, 177 µmol) and 2'-amino-6',7'-dihydro-5'H- spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (70.7 mg, 212 µmol) in N,N- dimethylacetamide (2.5 ml) was added cesium carbonate (230 mg, 707 µmol; CAS-RN:[534-17-8]) and the mixture was stirred at 50° C for 1 h. Water was added and the mixture was extracted with ethyl acetate. The organic phase was washed with half-saturated sodium chloride solution, dried (sodium sulfate), filtered and the solvent was removed in vacuum. Aminophase-silicagel chromatography (Gradient: dichloromethane / methanol 0-6%) (performed twice) gave 41.0 mg (38 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 0.84 min; MS (ESIpos): m/z = 579 [M+H]+ BHC233018 - FC - 122 / 152 - 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.09 (s, 2H), 5.46-5.04 (m, 1H), 4.73-4.51 (m, 1H), 4.41-4.12 (m, 2H), 4.05-3.90 (m, 2H), 3.85 (br d, 5H), 3.78-3.71 (m, 2H), 3.67-3.59 (m, 2H), 3.04 (s, 2H), 3.01-2.90 (m, 1H), 2.85-2.73 (m, 1H), 2.45-2.37 (m, 2H), 2.08-1.95 (m, 4H), 1.91 (quin, 3H), 1.87-1.77 (m, 1H), 1.77-1.63 (m, 4H) Example 05-04 2'-amino-1-(5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(6R or S)-6-hydroxy-6-methyl-1,4-oxazepan-4-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (single stereoisomer)
Figure imgf000123_0001
To a stirred solution of 4-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6- [(6R or S)-6-hydroxy-6-methyl-1,4-oxazepan-4-yl]pyrimidine-5-carbonitrile (Intermediate 05-13) (105 mg, 247 µmol) and 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (98.6 mg, 296 µmol) in N,N-dimethylacetamide (3.5 ml) was added cesium carbonate (321 mg, 986 µmol; CAS-RN:[534-17-8]) and the mixture was stirred at 50° C for 1 h. Water was added and the mixture was extracted with ethyl acetate. The organic phase was washed with half-saturated sodium chloride solution, dried (sodium sulfate), filtered and the solvent was removed in vacuum. Aminophase-silicagel chromatography (Gradient: dichloromethane / methanol 0-20%) gave 58.0 mg (39 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 0.87 min; MS (ESIneg): m/z = 607 [M-H]- 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.10 (s, 2H), 5.37-5.13 (m, 1H), 4.97 (br s, 1H), 4.73-4.51 (m, 1H), 4.40-4.28 (m, 1H), 4.27-4.16 (m, 1H), 4.10 (br t, 1H), 4.03-3.77 (m, 7H), 3.69 (d, 1H), 3.46-3.39 (m, 1H), 3.36 (br s, 1H), 3.09-3.02 (m, 2H), 3.02-2.90 (m, 1H), 2.83-2.74 (m, 1H), 2.42 (br t, 2H), 2.10-1.95 (m, 4H), 1.92 (br s, 1H), 1.86-1.78 (m, 1H), 1.77-1.65 (m, 4H), 1.07 (s, 3H) Example 05-05 BHC233018 - FC - 123 / 152 - 2'-amino-1-(5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(6R or S)-6-hydroxy-6-methyl-1,4-oxazepan-4-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (single stereoisomer)
Figure imgf000124_0001
To a stirred solution of 4-chloro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6- [(6R or S)-6-hydroxy-6-methyl-1,4-oxazepan-4-yl]pyrimidine-5-carbonitrile (Intermediate 05-16) (99.7 mg, 234 µmol) and 2’-amino-6’,7’-dihydro-5’H-spiro[azetidine-3,4’-[1]benzothiophene]-3’-carbonitrile trifluoroacetate (126 mg, 281 µmol) in N,N-dimethylacetamide (3.7 ml) was added cesium carbonate (305 mg, 936 µmol; CAS-RN:[534-17-8]) and the mixture was stirred at 50° C for 1 h. Water was added and the mixture was extracted with ethyl acetate. The organic phase was washed with half-saturated sodium chloride solution, dried (sodium sulfate), filtered and the solvent was removed in vacuum. Aminophase- silicagel chromatography (Gradient: dichloromethane / methanol 0-20%) followed by aminophase- silicagel chromatography (Gradient: dichloromethane / methanol 0-5%) gave 65.5 mg (44 % yield) of the title compound. LC-MS (Analytical Method 3): Rt = 0.86 min; MS (ESIneg): m/z = 607 [M-H]- 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.08 (s, 2H), 5.36-5.12 (m, 1H), 4.96 (br s, 1H), 4.73-4.51 (m, 1H), 4.39-4.28 (m, 1H), 4.23 (br d, 1H), 4.10 (br d, 1H), 4.04-3.77 (m, 7H), 3.69 (d, 1H), 3.46-3.39 (m, 1H), 3.37 (br s, 1H), 3.11-3.02 (m, 2H), 3.02-2.90 (m, 1H), 2.84-2.74 (m, 1H), 2.42 (t, 2H), 2.10-1.95 (m, 4H), 1.95-1.87 (m, 1H), 1.86-1.78 (m, 1H), 1.77-1.65 (m, 4H), 1.07 (s, 3H) Example 06-01 2'-Amino-1-[4-(3,8-diazabicyclo[3.2.1]octan-3-yl)-6-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy}pyrimidin-2-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer) BHC233018 - FC - 124 / 152 -
Figure imgf000125_0001
Trifluoroacetic acid (30 µL, 0.39 mmol, 10 eq.) was added at 0°C to a solution of tert-butyl 3-[2-(2'- amino-3'-cyano-6',7'-dihydro-1H,5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1-yl)-6-{[(2R,7aS)-2- fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8- carboxylate (single stereoisomer) (26 mg, 0.04 mmol) in dichloromethane (0.4 mL). The reaction mixture was stirred at RT for 3 h, mixed with additional trifluoroacetic acid (30 µL, 0.39 mmol, 10 eq.), stirred at RT for 1.5 h and concentrated under reduced pressure. The residue was purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). The resulting salt was dissolved in methanol and filtered through a SPE cartridge (200 mg PL-HCO3 MP 6 mL tube, gravity filtration, washed with 3.0 mL of methanol). The filtrate was concentrated under reduced pressure and dried in vacuo. Yield: 12 mg (54% of theory). LC/MS (method 1): tR = 0.86 min, MS (ESIneg): m/z = 563 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.00 (s, 2H), 5.31/5.17 (d, 1H), 5.23 (s, 1H), 4.23 (br d, 2H), 3.99-3.75 (m, 4H), 3.73 (d, 2H), 3.40 (br s, 2H), 3.11-3.02 (m, 2H), 3.01-2.95 (m, 1H), 2.86-2.75 (m, 3H), 2.42 (t, 2H), 2.36-2.23 (m, 1H), 2.11-1.89 (m, 5H), 1.86-1.65 (m, 5H), 1.65-1.47 (m, 4H). Example 07-01 2'-Amino-1-(4-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (single stereoisomer)
Figure imgf000125_0002
Sodium carbonate (30 mg, 0.28 mmol, 4.0 eq.) was added to a solution of (3R)-1-(2-chloro-6-{[(2R,7aS)- 2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl)-3-methylpiperidin-3-ol formate (single stereoisomer) (30 mg, 0.07 mmol) and 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile trifluoroacetate (31 mg, 76% purity, 0.07 mmol, 1.0 eq.) in dimethyl sulfoxide (340 µL). The reaction mixture was stirred at 95°C for 3 h in a closed microwave vial, diluted with water and aqueous hydrochloric acid solution (1 N, 300 µL). The suspension was dissolved in a BHC233018 - FC - 125 / 152 - mixture of acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 9 mg (22% of theory). LC/MS (method 1): tR = 1.30 min, MS (ESIneg): m/z = 566 [M-H]-; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.03 (s, 2H), 5.33/5.20 (d, 1H), 5.15 (s, 1H), 4.44 (br s, 1H), 4.20 (d, 2H), 3.95 (br d, 1H), 3.80 (br d, 1H), 3.73 (br d, 2H), 3.52-3.18 (m, 4H), 3.17-2.98 (m, 3H), 2.88-2.78 (m, 1H), 2.43 (t, 2H), 2.16-1.92 (m, 5H), 1.89-1.62 (m, 6H), 1.60-1.48 (m, 2H), 1.47-1.37 (m, 1H), 1.08 (s, 3H). Example 07-02 2'-Amino-1-(4-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]-6-{[(2S)-5-oxopyrrolidin-2- yl]methoxy}pyrimidin-2-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile (single stereoisomer)
Figure imgf000126_0001
Sodium carbonate (7 mg, 0.07 mmol, 4.0 eq.) was added to a solution of (5S)-5-[({2-chloro-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]pyrimidin-4-yl}oxy)methyl]pyrrolidin-2-one formate (single stereoisomer) (7 mg, 0.02 mmol) and 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]- 3'-carbonitrile trifluoroacetate (8 mg, 76% purity, 0.02 mmol, 1.0 eq.) in dimethyl sulfoxide (85 µL). The reaction mixture was stirred at 95°C overnight in a closed microwave vial, mixed with additional 2'-amino- 6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (8 mg, 76% purity, 0.02 mmol, 1.0 eq.), stirred at 95°C for another 4 h and diluted with water and aqueous hydrochloric acid solution (1 N, 69 µL). The suspension was dissolved in a mixture of acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient). Yield: 3 mg (90% purity, 32% of theory). LC/MS (method 1): tR = 1.26 min, MS (ESIpos): m/z = 524 [M+H]+; 1H- NMR (400 MHz, DMSO-d6): δ [ppm] = 7.75 (s, 1H), 7.00 (s, 2H), 5.36 (s, 1H), 4.40 (br s, 1H), 4.31-4.20 (m, 2H), 4.15-4.03 (m, 2H), 3.86-3.70 (m, 3H), 3.68-3.16 (m, 4H, partially concealed), 2.43 (t, 2H), 2.28- 1.91 (m, 5H), 1.86-1.61 (m, 4H), 1.59-1.48 (m, 2H), 1.46-1.35 (m, 1H), 1.07 (s, 3H). Example 07-03 2'-Amino-1-(5-fluoro-4-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)- 3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile (single stereoisomer) BHC233018 - FC - 126 / 152 -
Figure imgf000127_0001
Sodium carbonate (123 mg, 1.16 mmol, 5.0 eq.) was added to a solution of (3R)-1-(6-chloro-5-fluoro-2- {[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl)-3-methylpiperidin-3- ol (single stereoisomer), (3R)-1-(2-chloro-5-fluoro-6-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy}pyrimidin-4-yl)-3-methylpiperidin-3-ol (single stereoisomer) (99 mg, 0.23 mmol) and 2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile trifluoroacetate (140 mg, 72% purity, 0.30 mmol, 1.3 eq.) in dimethyl sulfoxide (1.0 mL). The reaction mixture was stirred at 95°C overnight in a closed microwave vial, diluted with water and quenched with aqueous hydrochloric acid solution (1 N, 1.2 mL). The suspension was dissolved in acetonitrile / dimethyl sulfoxide (1:1, 5.0 mL) and purified by RP-HPLC (acetonitrile / 0.1% formic acid in water gradient), followed by a second RP-HPLC (acetonitrile / 1% ammonia in water gradient) to separate both regioisomers. Yield: 9 mg (6% of theory). LC/MS (method 1): tR = 1.40 min, MS (ESIpos): m/z = 586 [M+H]+; 1H-NMR (400 MHz, DMSO-d6): δ [ppm] = 7.00 (s, 2H), 5.31/5.18 (d, 1H), 4.47 (s, 1H), 4.27-4.18 (m, 2H), 4.07-3.83 (m, 2H), 3.75-3.68 (m, 2H), 3.57-3.43 (m, 3H), 3.12-3.02 (m, 2H), 3.02-2.95 (m, 1H), 2.84-2.75 (m, 1H), 2.43 (t, 2H), 2.14-1.89 (m, 5H), 1.87-1.64 (m, 6H), 1.59-1.40 (m, 3H), 1.07 (s, 3H), 1H concealed.
BHC233018 - FC - 127 / 152 - BIOLOGICAL ASSAYS Examples were tested in selected biological assays one or more times. When tested more than once, data are reported as either average values or as median values, wherein • the average value, also referred to as the arithmetic mean value, represents the sum of the values obtained divided by the number of times tested, and • the median value represents the middle number of the group of values when ranked in ascending or descending order. If the number of values in the data set is odd, the median is the middle value. If the number of values in the data set is even, the median is the arithmetic mean of the two middle values. Examples were synthesized one or more times. When synthesized more than once, data from biological assays represent average values or median values calculated utilizing data sets obtained from testing of one or more synthetic batch. The in vitro activity of the compounds of the present invention can be demonstrated in the following assays: Biochemical KRAS/SOS1 activation assays Preparation of test compound dilutions. A 100-fold concentrated solution of the test compound (50 nL) in DMSO was transferred to microtiter test plates (384 or 1,536 wells, Greiner Bio-One, Germany) using either a Hummingbird liquid handler (Digilab, MA, USA) or an Echo acoustic system (Labcyte, CA, USA). Plates were sealed with adhesive foil or heat-sealed and stored at –20 C until use. Serial dilutions of test compounds were prepared in 100% DMSO using a Precision Pipetting System (BioTek, USA). Measurement and evaluation of inhibition data, calculation of IC50 values. Homogeneous time- resolved fluorescence (HTRF) was measured with a PHERAstar reader (BMG, Germany) using the HTRF module (excitation: 337 nm; emission 1: 620 nm, emission 2: 665 nm). The ratio of the emissions at 665 and 620 nm was used as the specific signal for further evaluation. The data were normalized using the controls: DMSO = 0% inhibition, inhibition control wells with inhibitor control solution = 100% inhibition. Compounds were tested in duplicates at up to 11 concentrations (e.g.20 µM, 5.7 µM, 1.6 µM, 0.47 µM, 0.13 µM, 38 nM, 11 nM, 3.1 nM, 0.89 nM, 0.25 nM and 0.073 nM). IC50 values were calculated using a four-parameter fit, with a commercial software package (Genedata Screener, Switzerland). KRASG12D activation by SOS1cat assay (“On-assay”). This assay quantifies SOS1cat mediated loading of KRASG12D–GDP with a fluorescent GTP analogue. Detection of successful loading was achieved by measuring resonance energy transfer from anti-GST-terbium (FRET donor) bound to GST-KRASG12D to the loaded fluorescent GTP analogue (FRET acceptor). The fluorescent GTP analogue EDA–GTP–DY- 647P1 [2'/3'-O-(2-aminoethyl-carbamoyl)guanosine-5'-triphosphate labelled with DY-647P1 (Dyomics BHC233018 - FC - 128 / 152 - GmbH, Germany)] was synthesized by Jena Bioscience (Germany) and supplied as a 1 mM aqueous solution. A KRASG12D working solution was prepared in assay buffer [10 mM HEPES pH 7.4 (AppliChem), 150 mM NaCl (Sigma), 5 mM MgCl2 (Sigma), 1 mM DTT (Thermo Fisher), 0.05% BSA Fraction V pH 7.0 (ICN Biomedicals), 0.0025% (v/v) Igepal (Sigma)] containing GST-KRASG12D (final concentration in assay 2 nM) and anti-GST-terbium (Cisbio, France) (final concentration in assay 1 nM). A SOS1cat working solution was prepared in assay buffer containing SOS1cat (final concentration 5 nM) and EDA–GTP–DY-647P1 (final concentration 100 nM). An inhibitor control solution was prepared in assay buffer containing EDA–GTP–DY-647P1 (final concentration 100 nM) without SOS1cat but with addition of GDP (final concentration 20µM, Jena Bioscience, prepared from 100 mM stock solution). All steps of the assay were performed at 20 °C. A volume of 3 µL of the KRASG12D working solution was added to all wells of the test plate using a Multidrop dispenser (Thermo LabSystems). After 15 min, 2 µL of the SOS1cat working solution was added to all wells, except for the inhibitor control solution wells. After 10 min incubation, HTRF was measured. KRASWT activation by SOS1cat assay (“On-assay”). This assay quantifies SOS1cat mediated loading of KRASWT–GDP with a fluorescent GTP analogue. Detection of successful loading was achieved by measuring resonance energy transfer from anti-GST-terbium (FRET donor) bound to GST-KRASWT to the loaded fluorescent GTP analogue (FRET acceptor). The fluorescent GTP analogue EDA–GTP–DY- 647P1 [2'/3'-O-(2-aminoethyl-carbamoyl)guanosine-5'-triphosphate labelled with DY-647P1 (Dyomics GmbH, Germany)] was synthesized by Jena Bioscience (Germany) and supplied as a 1 mM aqueous solution. A KRASWT working solution was prepared in assay buffer [10 mM HEPES pH 7.4 (AppliChem), 150 mM NaCl (Sigma), 5 mM MgCl2 (Sigma), 1 mM DTT (Thermo Fisher), 0.05% BSA Fraction V pH 7.0 (ICN Biomedicals), 0.0025% (v/v) Igepal (Sigma)] containing GST-KRASWT (final concentration in assay 2 nM) and anti-GST-terbium (Cisbio, France) (final concentration in assay 1 nM). A SOS1cat working solution was prepared in assay buffer containing SOS1cat (final concentration 5 nM) and EDA– GTP–DY-647P1 (final concentration 100 nM). An inhibitor control solution was prepared in assay buffer containing EDA–GTP–DY-647P1 (final concentration 100 nM) without SOS1cat but with addition of GDP (final concentration 20µM, Jena Bioscience, prepared from 100 mM stock solution). All steps of the assay were performed at 20 °C. A volume of 3 µL of the KRASWT working solution was added to all wells of the test plate using a Multidrop dispenser (Thermo LabSystems). After 15 min, 2 µL of the SOS1cat working solution was added to all wells, except for the inhibitor control solution wells. After 10 min incubation, HTRF was measured. HRASWT activation by SOS1cat assay (“On-assay”). This assay quantifies SOS1cat mediated loading of HRASWT–GDP with a fluorescent GTP analogue. Detection of successful loading was achieved by measuring resonance energy transfer from anti-GST-terbium (FRET donor) bound to GST-HRASWT (Reaction Biology) to the loaded fluorescent GTP analogue (FRET acceptor). The fluorescent GTP analogue EDA–GTP–DY-647P1 [2'/3'-O-(2-aminoethyl-carbamoyl)guanosine-5'-triphosphate labelled BHC233018 - FC - 129 / 152 - with DY-647P1 (Dyomics GmbH, Germany)] was synthesized by Jena Bioscience (Germany) and supplied as a 1 mM aqueous solution. A HRASWT working solution was prepared in assay buffer [10 mM HEPES pH 7.4 (AppliChem), 150 mM NaCl (Sigma), 5 mM MgCl2 (Sigma), 1 mM DTT (Thermo Fisher), 0.05% BSA Fraction V pH 7.0 (ICN Biomedicals), 0.0025% (v/v) Igepal (Sigma)] containing GST- HRASWT (final concentration in assay 2 nM) and anti-GST-terbium (Cisbio, France) (final concentration in assay 1 nM). A SOS1cat working solution was prepared in assay buffer containing SOS1cat (final concentration 1 nM) and EDA–GTP–DY-647P1 (final concentration 100 nM). An inhibitor control solution was prepared in assay buffer containing EDA–GTP–DY-647P1 (final concentration 100 nM) without SOS1cat but with addition of GDP (final concentration 20µM, Jena Bioscience, prepared from 100 mM stock solution). All steps of the assay were performed at 20 °C. A volume of 3 µL of the HRASWT working solution was added to all wells of the test plate using a Multidrop dispenser (Thermo LabSystems). After 15 min, 2 µL of the SOS1cat working solution was added to all wells, except for the inhibitor control solution wells. After 10 min incubation, HTRF was measured. NRASWT activation by SOS1cat assay (“On-assay”). This assay quantifies SOS1cat mediated loading of NRASWT–GDP with a fluorescent GTP analogue. Detection of successful loading was achieved by measuring resonance energy transfer from anti-GST-terbium (FRET donor) bound to GST-NRASWT (Reaction Biology) to the loaded fluorescent GTP analogue (FRET acceptor). The fluorescent GTP analogue EDA–GTP–DY-647P1 [2'/3'-O-(2-aminoethyl-carbamoyl)guanosine-5'-triphosphate labelled with DY-647P1 (Dyomics GmbH, Germany)] was synthesized by Jena Bioscience (Germany) and supplied as a 1 mM aqueous solution. A NRASWT working solution was prepared in assay buffer [10 mM HEPES pH 7.4 (AppliChem), 150 mM NaCl (Sigma), 5 mM MgCl2 (Sigma), 1 mM DTT (Thermo Fisher), 0.05% BSA Fraction V pH 7.0 (ICN Biomedicals), 0.0025% (v/v) Igepal (Sigma)] containing GST- NRASWT (final concentration in assay 2 nM) and anti-GST-terbium (Cisbio, France) (final concentration in assay 1 nM). A SOS1cat working solution was prepared in assay buffer containing SOS1cat (final concentration 1 nM) and EDA–GTP–DY-647P1 (final concentration 100 nM). An inhibitor control solution was prepared in assay buffer containing EDA–GTP–DY-647P1 (final concentration 100 nM) without SOS1cat but with addition of GDP (final concentration 20µM, Jena Bioscience, prepared from 100 mM stock solution). All steps of the assay were performed at 20 °C. A volume of 3 µL of the NRASWT working solution was added to all wells of the test plate using a Multidrop dispenser (Thermo LabSystems). After 15 min, 2 µL of the SOS1cat working solution was added to all wells, except for the inhibitor control solution wells. After 10 min incubation, HTRF was measured. KRASG12V activation by SOS1cat assay (“On-assay”). This assay quantifies SOS1cat mediated loading of KRASG12V–GDP with a fluorescent GTP analogue. Detection of successful loading was achieved by measuring resonance energy transfer from anti-GST-terbium (FRET donor) bound to GST-KRASG12V (Reaction Biology) to the loaded fluorescent GTP analogue (FRET acceptor). The fluorescent GTP analogue EDA–GTP–DY-647P1 [2'/3'-O-(2-aminoethyl-carbamoyl)guanosine-5'-triphosphate labelled BHC233018 - FC - 130 / 152 - with DY-647P1 (Dyomics GmbH, Germany)] was synthesized by Jena Bioscience (Germany) and supplied as a 1 mM aqueous solution. A KRASG12V working solution was prepared in assay buffer [10 mM HEPES pH 7.4 (AppliChem), 150 mM NaCl (Sigma), 5 mM MgCl2 (Sigma), 1 mM DTT (Thermo Fisher), 0.05% BSA Fraction V pH 7.0 (ICN Biomedicals), 0.0025% (v/v) Igepal (Sigma)] containing GST-KRASG12V (final concentration in assay 2 nM) and anti-GST-terbium (Cisbio, France) (final concentration in assay 1 nM). A SOS1cat working solution was prepared in assay buffer containing SOS1cat (final concentration 20 nM) and EDA–GTP–DY-647P1 (final concentration 100 nM). An inhibitor control solution was prepared in assay buffer containing EDA–GTP–DY-647P1 (final concentration 100 nM) without SOS1cat but with addition of GDP (final concentration 20µM, Jena Bioscience, prepared from 100 mM stock solution). All steps of the assay were performed at 20 °C. A volume of 3 µL of the KRASG12V working solution was added to all wells of the test plate using a Multidrop dispenser (Thermo LabSystems). After 15 min, 2 µL of the SOS1cat working solution was added to all wells, except for the inhibitor control solution wells. After 30 min incubation, HTRF was measured. KRASG12C activation by SOS1cat assay (“OFF-assay”). This assay quantifies SOS1cat mediated deloading of KRASG12C pre-loaded with a fluorescent GDP analogue at excess GTP. Detection of successful deloading was achieved by measuring resonance energy transfer from anti-GST-terbium (FRET donor) bound to GST-KRASG12C to the loaded fluorescent GDP analogue (FRET acceptor). The fluorescent GDP analogue EDA–GDP–DY-647P1 [2'/3'-O-(2-aminoethyl-carbamoyl)guanosine-5'- diphosphate labelled with DY-647P1 (Dyomics GmbH, Germany)] was synthesized by Jena Bioscience (Germany). A KRASG12C working solution was prepared in assay buffer [10 mM HEPES pH 7.4 (AppliChem), 150 mM NaCl (Sigma), 5 mM MgCl2 (Sigma), 1 mM DTT (Thermo Fisher), 0.05% BSA Fraction V pH 7.0 (ICN Biomedicals), 0.0025% (v/v) Igepal (Sigma)] containing GST-KRASG12C preloaded with EDA–GDP–DY-647P1 (final concentration in assay 5 nM) and anti-GST-terbium (Cisbio, France) (final concentration in assay 1 nM). A SOS1cat working solution was prepared in assay buffer containing SOS1cat (final concentration 1 nM) and GTP (final concentration 50 µM). An inhibitor control solution was prepared in assay buffer containing SOS1cat (final concentration 1 nM). All steps of the assay were performed at 20^°C. A volume of 3 µL of the KRASG12C working solution was added to all wells of the test plate using a Multidrop dispenser (Thermo LabSystems). After 15 min, 2 µL of the SOS1cat working solution was added to all wells, except for the inhibitor control solution wells. After 30 min incubation, HTRF was measured. Table 2. Biochemical KRAS/SOS1 activation assay results. KRAS G12D GDP KRAS G12V GDP KRAS G12C GDP Example activation by SOS1 (on- activation by SOS1 (on- activation by SOS1 (off- No assay) assay) assay) IC50 [mol/L] IC50 [mol/L] IC50 [mol/L] 01-01 2.06 E-8 3.44 E-6 1.07 E-6 01-02 1.60 E-8 1.07 E-6 5.53 E-7 BHC233018 - FC - 131 / 152 - 01-03 3.47 E-7 1.19 E-5 02-01 2.22 E-7 1.04 E-7 8.68 E-8 02-02 8.39 E-6 1.07 E-5 5.65 E-6 02-03 1.06 E-7 5.01 E-8 7.60 E-8 02-04 3.73 E-6 5.87 E-6 > 2.00 E-5 02-05 3.82 E-6 1.60 E-6 02-06 9.82 E-6 7.05 E-6 02-07 8.56 E-6 5.53 E-6 02-08 9.05 E-6 4.66 E-6 02-09 1.60 E-5 7.28 E-6 02-10 9.47 E-7 8.40 E-7 02-11 8.82 E-6 3.06 E-6 03-01 2.99 E-7 1.11 E-7 7.83 E-8 03-02 4.58 E-7 3.96 E-7 6.95 E-7 03-03 7.15 E-6 5.53 E-7 03-04 4.55 E-6 3.60 E-7 03-05 9.95 E-6 1.22 E-6 03-06 1.09 E-6 3.65 E-7 03-07 3.30 E-6 4.11 E-7 03-08 3.76 E-6 6.03 E-7 03-09 8.27 E-6 6.20 E-6 03-10 3.35 E-7 9.73 E-8 04-01 7.29 E-6 2.53 E-6 04-02 7.08 E-6 5.95 E-7 04-03 4.81 E-7 3.39 E-7 05-01 3.29 E-9 4.29 E-7 1.78 E-7 05-02 6.74 E-7 2.00 E-7 05-03 2.19 E-7 3.99 E-8 05-04 3.40 E-7 1.01 E-7 05-05 4.18 E-7 2.57 E-8 06-01 1.30 E-7 3.88 E-6 1.61 E-6 07-01 1.90 E-7 1.99 E-7 2.16 E-7 07-02 4.31 E-6 5.65 E-6 1.69 E-6 07-03 > 2.00 E-5 1.34 E-5 Table 3. Biochemical KRAS/SOS1 activation assay results KRAS WT GDP NRAS WT GDP HRAS WT GDP Example activation by SOS1 (on- activation by SOS1 (on- activation by SOS1 (on- No assay) assay) assay) IC50 [mol/L] IC50 [mol/L] IC50 [mol/L] 01-01 6.12 E-7 > 2.00 E-5 > 2.00 E-5 01-02 7.56 E-7 > 2.00 E-5 > 2.00 E-5 01-03 1.53 E-5 > 2.00 E-5 > 2.00 E-5 02-01 1.45 E-7 > 2.00 E-5 > 2.00 E-5 1.77 E-5 02-02 5.78 E-6 > 2.00 E-5 > 2.00 E-5 02-03 1.01 E-7 > 2.00 E-5 > 2.00 E-5 02-04 4.46 E-6 > 2.00 E-5 > 2.00 E-5 3.97 E-6 4.89 E-6 02-05 2.13 E-6 > 2.00 E-5 > 2.00 E-5 02-06 8.40 E-6 > 2.00 E-5 > 2.00 E-5 BHC233018 - FC - 132 / 152 - 02-07 6.39 E-6 > 2.00 E-5 > 2.00 E-5 02-08 5.99 E-6 > 2.00 E-5 > 2.00 E-5 02-09 8.85 E-6 > 2.00 E-5 > 2.00 E-5 02-10 8.05 E-7 > 2.00 E-5 > 2.00 E-5 02-11 5.37 E-6 > 2.00 E-5 > 2.00 E-5 03-01 1.63 E-7 > 2.00 E-5 > 2.00 E-5 03-02 9.36 E-7 > 2.00 E-5 > 2.00 E-5 03-03 6.38 E-7 > 2.00 E-5 > 2.00 E-5 03-04 8.72 E-7 > 2.00 E-5 > 2.00 E-5 03-05 8.77 E-7 > 2.00 E-5 > 2.00 E-5 03-06 3.36 E-7 5.70 E-6 5.98 E-6 03-07 5.63 E-7 9.18 E-6 9.31 E-6 03-08 2.43 E-6 > 2.00 E-5 > 2.00 E-5 03-09 7.42 E-6 > 2.00 E-5 > 2.00 E-5 03-10 3.07 E-7 > 2.00 E-5 > 2.00 E-5 04-01 2.05 E-6 > 2.00 E-5 > 2.00 E-5 04-02 2.76 E-6 > 2.00 E-5 > 2.00 E-5 04-03 3.18 E-7 > 2.00 E-5 > 2.00 E-5 05-01 8.51 E-8 > 2.00 E-5 1.75 E-5 > 2.00 E-5 05-02 2.22 E-7 > 2.00 E-5 1.66 E-5 2.49 E-6 > 2.00 E-5 05-03 5.24 E-8 > 2.00 E-5 1.09 E-5 05-04 1.74 E-7 > 2.00 E-5 > 2.00 E-5 05-05 4.69 E-8 1.35 E-5 8.28 E-6 06-01 3.72 E-6 > 2.00 E-5 > 2.00 E-5 07-01 2.71 E-7 > 2.00 E-5 > 2.00 E-5 07-02 4.64 E-6 > 2.00 E-5 > 2.00 E-5 07-03 1.54 E-5 > 2.00 E-5 > 2.00 E-5 KRAS Surface plasmon resonance assay The surface plasmon resonance experiments were performed at 20°C using a Biacore 8K or 8K+ system (Cytiva Europe GmbH). The at NUVISAN ICB GmbH produced biotinylated recombinant KRAS variants (WT, G12D, G12V, G12C constructs, for details see section “KRAS Protein Production for SPR”) or purchased biotinylated recombinant KRASG12V (MSC-11-536, kRas_P_2-169_G12V), NRAS (MSC-11-543, nRas_P_WT_2- 169) and HRAS (MSC-11-547, hRas_P_WT_2-169) from Reaction Biology were used for immobilization at a concentration of 5 µg/mL or 50 µg/mL on SA-Chip (Cytiva Europe GmbH) in 10 mM Hepes pH7.5, 150 mM NaCl, 5 mM MgCl2, 0.05% BSA, 1 mM DTT, 0.0025% Igepal (NP40) with a flow rate of 5 µL/min to reach target ligand density between 1000 and 4000 RU. KD titrations were performed in multi- cycle mode using running buffer of 10 mM Hepes pH7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.05% BSA, 0.0025% Igepal (NP40), 2% DMSO in a flow rate of 30 µL/min with a contact time of 90s and a dissociation time of 150s. For selected high-affinity compounds, KD titrations were performed in BHC233018 - FC - 133 / 152 - single-cycle mode in the same running buffer in a flow rate of 100 µL/min with a contact time of 60s and dissociation time of 3500s. Starting concentration of the dilution series for each compound was adjusted based on the estimated KD or results of a test run. In general, for multi cycle experiments a 1:2 dilution series with 10 concentrations was prepared, while for single cycle experiments a 1:3 dilutions dilution series with 6 concentrations was used. All SPR sensograms were analysed using the Biacore Insight Evaluation Software (Version: 3.0.12.15655 or 4.0.8.20368 GE.Healthcare Bio-Sciences Corp.). Multi cycle experiments were analysed by steady state affinity or by 1:1 binding kinetics model, while single cycle data was analysed by 1:1 binding kinetics model after double referencing and solvent correction hat been applied. All measurements were performed at least as duplicates. Final KD-value were prepared by taking the mean value of the independently determined KD-values. Table 4. SPR assay results Example Kd [mol/L] KRAS Kd [mol/L] KRAS Kd [mol/L] KRAS G12V G Kd [mol/L] KRAS No G12D 12V (Assay version 1) (Assay version 2) wt 01-01 2.39 E-9 3.84 E-6 4.03 E-7 02-01 1.01 E-7 9.86 E-8 7.78 E-8 02-03 9.89 E-9 4.65 E-8 3.29 E-8 02-05 6.96 E-7 9.90 E-7 8.77 E-7 03-06 9.22 E-7 5.56 E-7 3.11 E-7 04-01 4.49 E-6 4.25 E-6 2.09 E-6 04-03 1.18 E-7 1.76 E-7 9.11 E-8 05-02 1.52 E-7 1.78 E-7 7.11 E-8 05-03 5.19 E-8 2.27 E-8 06-01 3.54 E-8 4.54 E-6 1.41 E-6 07-01 1.52 E-7 3.14 E-7 2.06 E-7 07-03 1.43 E-5 1.02 E-5 KRAS, NRAS, HRAS Protein Production for SPR Cloning, in vivo biotinylating expression and purification DNA sequences encoding for human KRAS (Acc. No P01116-2) variants: KRASWT (Aa 1-169; C118S), KRASG12D (Aa 1-169; G12D; C118S), KRASG12C (Aa 1-169; G12C; C118S), KRASG12V (Aa 1-169; G12V; C118S) were synthesized by the GeneArt Technology at Life Technologies as codon optimized version for expression in E. coli with N-terminal enhanced TEV cleavage site as well as additionally encoded att-site sequences at the 5´and 3´ ends for subcloning into various destination vectors using the Gateway Technology. DNA fragments were cloned into N-terminal His-StrepII-Avi-tag vector using Gateway technologies. This vectors were co-transfected with pBirAcm and expressed into E. coli BL21(DE3) using LB 184 medium or Terrific Broth media in the presence of 200 µg/mL Ampicillin and 34 µg/mL Chloramphenicol. The BHC233018 - FC - 134 / 152 - cells were grown at 37°C until the OD550 reached 1, at which point 0.1 mM or 0.5 mM IPTG and 50 µM Biotin were added and the temperature was lowered to 27°C. The cells were harvested after 24 hours. E. coli cell pellet was resuspended in 3.5 mL buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10 mM Imidazole, 0.5% CHAPS, Complete-EDTAfree protease inhibitor, 2 µg Benzonase) per gram wet weight and lysed by sonication or microfluidizer. The soluble protein was separated by centrifugation at 24000 xg for an hour at 4°C. The protein was purified via Ni-NTA affinity chromatography using buffer (50 mM Tris HCl pH 7.5, 300 mM NaCl) with 10 mM Imidazole for washing and 300 mM Imidazole for elution. The eluted protein was then concentrated and further purified by size exclusion chromatography (Superdex 200) in 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2. KRAS, SOS1 Protein Production for Biochemical activation assays Cloning, in vivo biotinylating expression and purification DNA sequences encoding for human KRAS (Acc. No P01116-2) variants: KRASWT (Aa 1-169), KRASG12D (Aa 1-169; G12D; C118S), KRASG12V (Aa 1-169; G12V), and human SOS1 (hSOS1) (Acc. No Q07889, Aa 564-1049) were synthesized by the GeneArt Technology at Life Technologies as codon optimized version for expression in E. coli with N-terminal TEV/ enhanced TEV (Tobacco Etch Virus) protease site as well as additionally encoded att-site sequences at the 5´and 3´ ends for subcloning into various destination vectors using the Gateway Technology. All above mentioned KRAS DNA fragments were cloned into N-terminal GST-tag vector (an in-house derivate of the pET vector series from Novagen with ampicillin resistance gene) using Gateway technologies, while hSOS1 was cloned into N-terminal His10-tag vector (an in-house derivate of the pET vector series from Novagen with ampicillin resistance gene). This vectors were transformed into E. coli strain BL21 (DE3). Cultivation of the transformed strains for expression was done in 10 L , 2L, 1 L fermenters or shaker flasks. The cultures were grown in Terrific Broth media with 200 ug/mL ampicillin at a temperature of 37 °C to a density of 0.6 (OD600), shifted to a temperature of 27 °C (for hK-Ras expression vectors) or 17 °C (for hSOS expression vectors), induced for expression with 0.1 mM IPTG and further cultivated for 24 hours. Purification After cultivation the transformed E. coli were harvested by centrifugation and the resulting pellet was suspended in a lysis buffer (see below) and lysed by passing three-times through a high pressure device (Microfluidics). The lysate was centrifuged at 4 °C and the supernatant used for further purification. An Äkta chromatography system was used for all further chromatography steps. Purification of GST Ras constrcuts for biochemical assays E. coli culture was lysed in lysis buffer (50mM Tris HCl 7.5, 500mM NaCl,1mM DTT, 0,5% CHAPS, Complete Protease Inhibitor Cocktail-(Roche)). As a first chromatography step the centrifuged lysate BHC233018 - FC - 135 / 152 - was incubated with Glutathione Agarose 4B (Macherey-Nagel; 745500.100) in a spinner flask (16 h, 10°C). The Glutathione Agarose 4B loased with protein was transferred to a chromatography column connected to an Äkta chromatography system. The column was washed with wash buffer (50mM Tris HCl 7.5, 500mM NaCl, 1mM DTT) and the bound protein eluted with elution buffer (50mM Tris HCl 7.5, 500mM NaCl, 1mM DTT, 15 or 20 mM Glutathione). The main fractions of the elution peak (monitored by OD280) were pooled. For further purification by size-exclusion chromatography the above eluate volume was applied to a column Superdex 200 HR prep grade (GE Healthcare) running SEC buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT) and the resulting peak fractions of the eluted fusion protein were collected. Purification of His10-hSOS1 for biochemical assays E. coli transformed with pD-ECO5_hSOS1 were cultured and induced in a fermenter, harvested and lysed in lysis buffer (25mM Tris HCl 7.5, 500mM NaCl, 20mM Imidazol, Complete EDTA-free (Roche)). For immobilized metal ion affinity chromatography (IMAC) the centrifuged lysate was incubated with 30mL Ni-NTA (Macherey-Nagel; #745400.100) in a spinner flask (16 h, 4°C) and subsequently transferred to a chromatography column connected to an Äkta chromatography system. The column was rinsed with wash buffer (25mM Tris HCl 7.5, 500mM NaCl, 20mM Imidazol) and the bound protein eluted with a linear gradient (0-100%) of elution buffer (25mM Tris HCl 7.5, 500mM NaCl, 300mM Imidazol). The main fractions of the elution peak (monitored by OD280) containing homogenous His10-hSOS were pooled. The final yield of His10-hSOS1 was about 110 mg purified protein per L culture and the final product concentration was about 2 mg/mL. HTRF® (Homogenous Time-Resolved Fluorescence) phospho-ERK1/2 (THR202/TYR204) assay with AGS, ASPC-1, GP2d, HPAC, LK2, NCI-H441 and PANC10.05 cells 2500 AGS cells were seeded in a HTRF 384-well low volume plate (Greiner bio-one #784080) in DMEM+F12 (GIBCO) complemented with L-Alanyl-L-Glutamine (final: 2 mM, Gibco), FCS Superior (final: 10%, Sigma). 5000 ASPC-1 cells were seeded in a HTRF 384-well low volume plate (Greiner bio-one #784080) in RPMI-1640 (GIBCO) complemented with L-Alanyl-L-Glutamine (final: 2 mM, Gibco), FCS Superior (final: 10%, Sigma). 2500 GP2d cells were seeded in a HTRF 384-well low volume plate (Greiner bio-one #784080) in RPMI- 1640 (GIBCO) complemented with L-Alanyl-L-Glutamine (final: 2 mM, Gibco), FCS Superior (final: 10%, Sigma). BHC233018 - FC - 136 / 152 - 2500 HPAC cells were seeded in a HTRF 384-well low volume plate (Greiner bio-one #784080) in DMEM+F12 including 15 mM HEPES (GIBCO) complemented with L-Alanyl-L-Glutamine (final: 2 mM, Gibco), FCS Superior (final: 5%, Sigma), E2 (final: 10 nM), hEGF (final: 10 ng/ml, Invitrogen), Hydrocortisone (final: 40 ng/ml, Sigma), hInsulin (final: 2 µg/ml, Sigma), Sodium pyruvate (final: 0.5 mM, Gibco), Transferrin (final: 5 µg/ml, Sigma). 20000 LK2 cells were seeded in a HTRF 384-well low volume plate (Greiner bio-one #784080) in RPMI- 1640 (GIBCO) complemented with L-Alanyl-L-Glutamine (final: 2 mM, Gibco), FCS Superior (final: 10%, Sigma). 2500 NCI-H441 cells were seeded in a HTRF 384-well low volume plate (Greiner bio-one #784080) in RPMI-1640 (GIBCO) complemented with L-Alanyl-L-Glutamine (final: 2 mM, Gibco), FCS Superior (final: 10%, Sigma). 2500 Panc10.05 cells were seeded in a HTRF 384-well low volume plate (Greiner bio-one #784080) in RPMI-1640 (GIBCO) complemented with L-Alanyl-L-Glutamine (final: 2 mM, Gibco), FCS Superior (final: 10%, Sigma), hInsulin (final: 10 units/ml). After 24 h in a standard incubator (37°C, 5% CO2) cells were treated with varying concentrations of the different test compounds dissolved in DMSO (1% final DMSO concentration per well) using a HP- dispenser followed by incubation for 1.5 h (at 37°C 5% CO2). Subsequently, NCI-H441 were stimulated for 3 min with hEGF (final concentration 200 ng/ml), while the other cell lines were processed without stimulation. The following steps were performed for all cell lines as described in the supplier's manual (Cisbio one-plate assay protocol; https://www.cisbio.eu/) for the HTRF ADVANCED PHOSPHO- ERK1/2 (THR202/TYR204) DETECTION KITS (#64AERPEH) .The content of pERK/ERK was measured with a PHERAstar (BMG Labtech) using the HTRF module. Calculation of the pERK/ERK Ratio was done as described in the assay protocol. The resulting IC50 values for the test compounds reflect the inhibition of ERK phosphorylation compared to DMSO-treated control cells.The ratio of cells solely treated with 1% DMSO was set as 100% and the ratio of maximal pERK inhibition obtained with the internal control compound (Trametinib) was set as 0%. Table 5, Cellular pERK assay results. Example IC50 [mol/L] IC50 [mol/L] No PANC10.05 IC50 [mol/L] AGS ASPC-1 IC50 [mol/L] GP2d 01-01 1.33 E-6 4.44 E-7 2.12 E-6 1.55 E-6 01-02 6.54 E-7 3.52 E-7 1.96 E-6 7.15 E-7 01-03 BHC233018 - FC - 137 / 152 - 02-01 4.45 E-6 2.59 E-6 6.49 E-6 1.41 E-6 02-02 9.97 E-11 > 2.99 E-5 > 2.99 E-5 > 2.99 E-5 03-01 > 2.99 E-5 1.26 E-5 2.36 E-5 05-01 5.05 E-8 1.94 E-8 5.91 E-8 6.71 E-8 06-01 3.11 E-6 1.18 E-6 1.30 E-6 07-01 3.03 E-6 5.08 E-6 1.43 E-6 Table 6, Cellular pERK assay results. Example IC50 [mol/L] IC50 [mol/L] IC50 [mol/L] No HPAC NCI-H441 LK2 01-01 1.19 E-5 > 2.99 E-5 > 2.99 E-5 01-02 5.90 E-6 > 2.99 E-5 > 2.99 E-5 01-03 02-01 1.16 E-5 1.17 E-5 1.60 E-6 > 2.99 E-5 02-02 > 2.99 E-5 03-01 3.42 E-6 05-01 3.02 E-7 1.53 E-5 5.41 E-7 > 2.99 E-5 06-01 > 2.99 E-5 07-01 3.37 E-6 Bidirectional Permeability in Caco-2 Cells Caco-2 cells (purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany) were seeded at a density of 4.5 × 10e4 cells/well on 24-well insert plates, 0.4 μm pore size (Costar) and grown for 14-21 days in DMEM supplemented with 10% FCS, 1% GlutaMAX (100×, Gibco), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), and 1% nonessential amino acids (100×, Thermo Fischer Scientific). Cells were maintained at 37 °C in a humidified 5% CO2 atmosphere. Medium was changed every 2−3 days. Before the assay was run, the culture medium was replaced by FCS-free transport buffer. For the assessment of monolayer integrity, the transepithelial electrical resistance (TEER) was measured. Test compounds were pre-dissolved in DMSO and added either to the apical or basolateral compartment at a final concentration of 2 or 30 μM. The organic solvent in the incubations was limited to ≤1% dimethylsulfoxide (DMSO). Before and after incubation for 2 h at 37 °C, samples were taken from both compartments and analyzed by LC−MS/MS detection after precipitation with MeOH. The apparent permeability coefficient (Papp) was calculated both for the apical to basolateral (A → B) and the basolateral to apical (B → A) direction using the following equation: Papp = (Vr/P0)(1/S)(P2/t), where Vr is the volume of medium in the receiver chamber, P0 is the measured peak area of the test compound in the donor chamber at t = 0, S is the surface area of the monolayer, P2 is the measured peak area of the test compound in the acceptor chamber after incubation for 2 h, and t is the incubation time. The efflux ratio (ER) basolateral (B) to apical (A) was calculated by dividing Papp B−A BHC233018 - FC - 138 / 152 - by Papp A−B. In addition, the compound recovery was calculated. As assay control, reference compounds were analyzed in parallel. Table 7. Caco permeability and Efflux ratio. Papp A-B [nm/s] Efflux ratio Papp A-B [nm/s] Efflux ratio Example at concentration of at concentration of at concentration of at concentration of No 2 µM 2 µM 30 µM 30 µM 01-01 0.37 156.01 01-02 0.40 81.97 0.23 380.93 02-01 0.69 224.13 02-02 0.35 264.83 02-03 0.61 239.31 02-04 11.33 17.74 02-05 1.01 211.30 25.11 6.15 02-06 48.52 3.34 02-09 0.87 218.29 03-01 0.40 242.42 1.99 61.54 03-02 0.61 42.24 03-03 3.67 39.84 03-04 0.81 91.70 03-07 16.98 6.87 03-08 0.89 93.88 03-09 1.48 14.93 04-01 195.12 0.59 04-03 2.45 71.70 78.69 1.37 05-01 0.29 265.00 05-02 2.10 89.00 128.13 0.93 05-03 6.42 34.19 279.33 0.39 06-01 0.18 538.66 07-02 0.92 152.06 In Vitro Metabolic Stability in Rat Hepatocytes Cryopreserved rat hepatocytes were thawed according to the suppliers information. Alternetively, hepatocytes from male Wistar rats were isolated via a two-step perfusion method. After perfusion, the liver was carefully removed from the rat, the liver capsule was opened, and the hepatocytes were gently shaken out into a Petri dish with ice-cold Williams’ medium E (WME). The resulting cell suspension was filtered through sterile gauze into 50 mL Falcon tubes and centrifuged at 50 × g for 3 min at rt. The cell pellet was resuspended in 30 mL of WME and centrifuged through a Percoll gradient two times at 100 × g. The hepatocytes were washed again and resuspended with WME. Cell viability was determined by trypan blue exclusion. For the metabolic stability assay, cryopreserved or freshly isolated hepatocytes were distributed in WME to glass vials at a density of 1.0 × 10e6 vital cells/mL. The test compound was added to a final concentration of 1 µM. Organic solvent in the incubations was limited to ≤0.01% DMSO and ≤1% acetonitrile. During incubation, the hepatocyte suspensions were continuously shaken at 580 BHC233018 - FC - 139 / 152 - rpm and aliquots were taken at 2, 8, 16, 30, 45, and 90 min, to which an equal volume of cold acetonitrile was immediately added. Samples were frozen at –20 °C overnight, and subsequently centrifuged at 3000 rpm for 15 min. The supernatant was analyzed with an Agilent 1200 HPLC system with MS/MS detection. The half-life of a test compound was determined from the concentration–time plot. From the half-life, the intrinsic and the in vitro predicted blood clearances were calculated, as well as the hepatic extraction ratio with EH = CLb/LBF and Fmax as 1 – EH *100%,. In combination with the standardized liver blood flow (LBF) and amount of liver cells in vivo and in vitro, the in vitro blood clearance (CLb, in vitro) was calculated according to the ‘well-stirred’ liver model. The following parameter values were used: liver blood flow: 4.2 L/h/kg, specific liver weight: 32 g/kg body weight; liver cells in vivo, 1.1 × 10e8 cells/g liver; liver cells in vitro, 1.0 × 10e6/mL. In Vitro Metabolic Stability in Human Hepatocytes Cryopreserved human hepatocytes were thawed according to the suppliers information. For the metabolic stability assay, cryopreserved hepatocytes were distributed in Williams’ medium E to glass vials at a density of 1.0 × 10e6 vital cells/mL. The test compound was added to a final concentration of 1 µM. Organic solvent in the incubations was limited to ≤0.01% DMSO and ≤1% acetonitrile. During incubation, the hepatocyte suspensions were continuously shaken at 580 rpm and aliquots were taken at 2, 8, 16, 30, 45, and 90 min, to which an equal volume of cold acetonitrile was immediately added. Samples were frozen at –20 °C overnight, and subsequently centrifuged at 3000 rpm for 15 min. The supernatant was analyzed with an Agilent 1200 HPLC system with MS/MS detection. The half-life of a test compound was determined from the concentration–time plot. From the half-life, the intrinsic and the in vitro predicted blood clearances were calculated, as well as the hepatic extraction ratio with EH = CLb/LBF and Fmax as 1 – EH *100%. In combination with the standardized liver blood flow (LBF) and amount of liver cells in vivo and in vitro, the in vitro blood clearance (CLb, in vitro) was calculated according to the ‘well-stirred’ liver model. The following parameter values were used: liver blood flow: 1.32 L/h/kg, specific liver weight: 21 g/kg body weight; liver cells in vivo, 1.1 × 10e8 cells/g liver; liver cells in vitro, 1.0 × 10e6/mL. In Vitro Metabolic Stability in Liver Microsomes The in vitro metabolic stability of test compounds was determined by incubation at 1 µM in a suspension of liver microsomes in 100 mM phosphate buffer pH 7.4 (NaH2PO4·H2O + Na2HPO4·2H2O) and at a protein concentration of 0.5 mg/mL at 37 °C. The microsomes were activated by adding a cofactor mix containing 8 mM glucose-6-phosphate, 0.5 mM NADP, and 1 IU/mL glucose-6-phos¬phate dehydro- genase in phosphate buffer pH 7.4. The metabolic assay was started shortly afterwards by adding the test com-pound to the incubation at a final volume of 1 mL. During incubation, the microsomal suspensions were continuously shaken at 580 rpm and aliquots were taken at 2, 8, 16, 30, 45, and 60 min. Further handling and analysis as per the hepatocyte method described above. BHC233018 - FC - 140 / 152 - Table 8. Metabolic stability data: Example Rat Hepatocytes Human Liver Microsomes No Fmax [%] Fmax [%] 01-01 99 59 01-02 96 71 01-03 85 44 02-01 71 31 02-02 84 44 02-03 91 41 02-04 31 6 02-05 75 16 02-06 10 02-07 100 75 02-08 73 14 02-09 60 11 02-11 53 18 03-01 94 35 03-02 99 60 03-03 29 03-04 86 03-07 46 17 03-08 30 7 03-09 97 76 03-10 79 10 04-02 85 45 04-03 67 22 05-01 84 30 05-02 33 05-03 44 8 05-04 73 7 05-05 41 4 06-01 84 31 07-01 37 14 07-02 85 In Vivo Pharmacokinetics in Rats All animal experiments were conducted in accordance with the German Animal Welfare Law and were approved by local authorities. For in vivo pharmacokinetic experiments, test compounds were administered to male Wistar rats intravenously at doses of 0.1 to 0.5 mg/kg as solutions potentially using solubilizers such as PEG 400 in well-tolerated amounts. Concerning iv administration, test compounds were given as bolus injection. Blood samples were taken at various time points after dosing, including 2 min and 30 min timepoints. Blood was collected into K3-EDTA tubes and centrifuged at 1811 g for min. 5 min or at 14000 g for 1.5 min. An aliquot of 62.5 µL from the supernatant (plasma) was taken and BHC233018 - FC - 141 / 152 - precipitated by the addition of 250 µL of MeOH. Samples were frozen at –20 °C overnight and subsequently thawed and centrifuged at 3000 rpm, 4 °C for 15 min. Aliquots of the supernatants were analyzed by LC−MS/MS detection. Measured concentrations after 30 min were put into relation with those after 2 min and percent recovery after 30 min was calculated. MRTX1133 (J. Med. Chem.2022, 65, 4, 3123–3133) was characterized in this assay for comparison. Table 9. iv rat PK: Plasma levels after 2 min and 30 min and calculation of the recovery of the parent compound concentration after 30 min in relation to 2 min. Plasma Reco centration after 2 Plasma co very [%] after 30 Example No con ncentration after 30 min (µg min related to 2 min min (µg/L) /L) value MRTX1133 396 3.7 0.93 01-01 627 26 4.1 02-01 219 13 5.9 02-04 249 108 43.0 05-01 456 6.7 1.5

Claims

BHC233018 - FC - 142 / 152 - CLAIMS 1. A compound of general formula (I)
Figure imgf000143_0001
in which: X represents CH, C-F, or N; Y represents CH, C-F, C-Cl, C-CN, C-CH3, or N; Z represents -CH2-, or -CH2-CH2-; R1 represents a group selected from the group: ,
Figure imgf000143_0002
, , , , , , , BHC233018 - FC - 143 / 152 -
Figure imgf000144_0001
, , , , or ; wherein * indicates the point of attachment of said group with the rest of the molecule; R2 represents a group selected from the group: ,
Figure imgf000144_0002
, , , , or , wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. 2. The compound of formula (I) according to claim 1, wherein: BHC233018 - FC - 144 / 152 - X represents CH, C-F, or N; Y represents CH, C-F, C-CN, C-CH3, or N; Z represents -CH2-, or -CH2-CH2-; R1 represents a group selected from the group: , ,
Figure imgf000145_0001
, , or ; wherein * indicates the point of attachment of said group with the rest of the molecule; R2 a selected from the ,
Figure imgf000145_0002
, , or , wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. BHC233018 - FC - 145 / 152 - 3. The compound of formula (I) according to claim 1, or claim 2, wherein: X represents N; Y represents CH, C-F, or C-CN; Z represents -CH2-CH2-; R1 a selected from the
Figure imgf000146_0001
, , , , , or ; wherein * indicates the point of attachment of said group with the rest of the molecule; R2 represents a group selected from the group:
Figure imgf000146_0002
, wherein * indicates the point of attachment of said group with the rest of the molecule; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same 4. The compound according to claim 1 which is selected from the group consisting of: 2'-Amino-1-[6-(3,8-diazabicyclo[3.2.1]octan-3-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H- pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile; 2'-Amino-1-[6-(3,8-diazabicyclo[3.2.1]octan-3-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H- pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-5',6'-dihydrospiro[azetidine-3,4'- cyclopenta[b]thiophene]-3'-carbonitrile; 2'-Amino-1-[6-(3,8-diazabicyclo[3.2.1]octan-3-yl)-2-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-6- ylmethoxy)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile formate; BHC233018 - FC - 146 / 152 - 2'-Amino-1-(2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile; 2'-Amino-1-(2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]pyrimidin-4-yl)-5',6'-dihydrospiro[azetidine-3,4'- cyclopenta[b]thiophene]-3'-carbonitrile; 2'-Amino-1-(6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]-2-{[(2S)-5-oxopyrrolidin-2- yl]methoxy}pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile; 2'-Amino-1-(2-{[(2S)-4,4-difluoro-1-methylpyrrolidin-2-yl]methoxy}-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]- 3'-carbonitrile; 2'-Amino-1-(2-{[(2S,4R)-4-fluoro-1-methylpyrrolidin-2-yl]methoxy}-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]- 3'-carbonitrile; 2'-Amino-1-(2-{[(2S,4R)-1-cyclopropyl-4-fluoropyrrolidin-2-yl]methoxy}-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]- 3'-carbonitrile; 2'-Amino-1-{2-[(3-cyanopyrrolidin-3-yl)methoxy]-6-[(3R)-3-hydroxy-3-methylpiperidin-1- yl]pyrimidin-4-yl}-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'-carbonitrile formate; 2'-Amino-1-{2-[(3-cyano-1-methylpyrrolidin-3-yl)methoxy]-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-4-yl}-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]- 3'-carbonitrile; 2'-Amino-1-{6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]-2-[2-(1-methyl-1H-imidazol-2- yl)ethoxy]pyrimidin-4-yl}-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile; 2'-Amino-1-(6-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]-2-{[(2S)-2-methylpyrrolidin-2- yl]methoxy}pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile; 2'-Amino-1-(2-{[1-(dimethylamino)cyclopropyl]methoxy}-6-[(3R)-3-hydroxy-3- methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]- 3'-carbonitrile; BHC233018 - FC - 147 / 152 - 2'-Amino-1-[2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-(2-oxo-1,6- diazaspiro[3.5]nonan-6-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile formate; 2'-Amino-1-[2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-(3-oxa-7,9- diazabicyclo[3.3.1]nonan-7-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile formate; 2'-amino-1-[6-(4,4-difluoropiperidin-1-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]- 3'-carbonitrile; 2'-amino-1-[2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-(3- oxopiperazin-1-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile; N-{1-[6-(2'-amino-3'-cyano-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1-yl)-2- {[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]piperidin-4- yl}acetamide; 2'-amino-1-[6-(3,3-difluoropiperidin-1-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]- 3'-carbonitrile; 2'-amino-1-[6-(4-fluoropiperidin-1-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)- yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile; tert-butyl 1-[6-(2'-amino-3'-cyano-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophen]-1- yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-1,6- diazaspiro[3.4]octane-6-carboxylate; 2'-amino-1-[6-(1,6-diazaspiro[3.4]octan-1-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin- 7a(5H)-yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]- 3'-carbonitrile; 1-(6-[(4RS)-6-acetyl-1,6-diazaspiro[3.4]octan-1-yl]-2-{[(2R,7aS)-2-fluorotetrahydro-1H- pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl)-2'-amino-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile; 2'-Amino-1-(2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]-5-methylpyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine- 3,4'-[1]benzothiophene]-3'-carbonitrile; BHC233018 - FC - 148 / 152 - 2'-Amino-1-[2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-5-methyl-6- (3-oxopiperazin-1-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]- 3'-carbonitrile; 2'-Amino-1-(5-fluoro-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6- [(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile; 2'-amino-1-[5-cyano-6-(3,8-diazabicyclo[3.2.1]octan-3-yl)-2-{[(2R,7aS)-2-fluorotetrahydro-1H- pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile; 2'-amino-1-(5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6- [(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile; 2'-amino-1-[5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6- (1,4-oxazepan-4-yl)pyrimidin-4-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]- 3'-carbonitrile; 2'-amino-1-(5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(6 RS)-6-hydroxy-6-methyl-1,4-oxazepan-4-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine- 3,4'-[1]benzothiophene]-3'-carbonitrile; 2'-amino-1-(5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(6 R)-6-hydroxy-6-methyl-1,4-oxazepan-4-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine- 3,4'-[1]benzothiophene]-3'-carbonitrile; 2'-amino-1-(5-cyano-2-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(6 S)-6-hydroxy-6-methyl-1,4-oxazepan-4-yl]pyrimidin-4-yl)-6',7'-dihydro-5'H-spiro[azetidine- 3,4'-[1]benzothiophene]-3'-carbonitrile; 2'-Amino-1-[4-(3,8-diazabicyclo[3.2.1]octan-3-yl)-6-{[(2R,7aS)-2-fluorotetrahydro-1H- pyrrolizin-7a(5H)-yl]methoxy}pyrimidin-2-yl]-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile; 2'-Amino-1-(4-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6-[(3R)-3- hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile; 2'-Amino-1-(4-[(3R)-3-hydroxy-3-methylpiperidin-1-yl]-6-{[(2S)-5-oxopyrrolidin-2- yl]methoxy}pyrimidin-2-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'-[1]benzothiophene]-3'- carbonitrile and BHC233018 - FC - 149 / 152 - 2'-Amino-1-(5-fluoro-4-{[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy}-6- [(3R)-3-hydroxy-3-methylpiperidin-1-yl]pyrimidin-2-yl)-6',7'-dihydro-5'H-spiro[azetidine-3,4'- [1]benzothiophene]-3'-carbonitrile or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same. 5. A compound of general formula (I) according to any one of claims 1 or 2 for use in the treatment or prophylaxis of a disease. 6. A pharmaceutical composition comprising a compound of general formula (I) according to any one of claims 1 or 2 and one or more pharmaceutically acceptable excipients. 7. A pharmaceutical combination comprising: • one or more first active ingredients, in particular compounds of general formula (I) according to any one of claims 1 or 2, and • one or more further active ingredients, in particular cancer agents like: 131I-chTNT, abarelix, abemaciclib, abiraterone, acalabrutinib, aclarubicin, adalimumab, ado- trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alectinib, alemtuzumab, alendronic acid, alitretinoin, alpharadin, altretamine, amifostine, aminoglutethimide, hexyl aminolevulinate, amrubicin, amsacrine, anastrozole, ancestim, anethole dithiolethione, anetumab ravtansine, angiotensin II, antithrombin III, apalutamide, aprepitant, arcitumomab, arglabin, arsenic trioxide, asparaginase, atezolizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, basiliximab, belotecan, bendamustine, besilesomab, belinostat, bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin, blinatumomab, bortezomib, bosutinib, buserelin, brentuximab vedotin, brigatinib, busulfan, cabazitaxel, cabozantinib, calcitonine, calcium folinate, calcium levofolinate, capecitabine, capromab, carbamazepine carboplatin, carboquone, carfilzomib, carmofur, carmustine, catumaxomab, celecoxib, celmoleukin, cemiplimab, ceritinib, cetuximab, chlorambucil, chlormadinone, chlormethine, cidofovir, cinacalcet, cisplatin, cladribine, clodronic acid, clofarabine, cobimetinib, copanlisib, crisantaspase, crizotinib, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daratumumab, darbepoetin alfa, dabrafenib, darolutamide, dasatinib, daunorubicin, decitabine, degarelix, denileukin diftitox, denosumab, depreotide, deslorelin, dianhydrogalactitol, dexrazoxane, dibrospidium chloride, dianhydrogalactitol, diclofenac, dinutuximab, docetaxel, dolasetron, doxifluridine, doxorubicin, doxorubicin + estrone, dronabinol, durvalumab, eculizumab, edrecolomab, elliptinium acetate, elotuzumab, eltrombopag, enasidenib, endostatin, enocitabine, enzalutamide, epirubicin, epitiostanol, epoetin alfa, epoetin beta, epoetin zeta, eptaplatin, eribulin, erlotinib, esomeprazole, estradiol, estramustine, ethinylestradiol, etoposide, everolimus, exemestane, fadrozole, fentanyl, BHC233018 - FC - 150 / 152 - filgrastim, fluoxymesterone, floxuridine, fludarabine, fluorouracil, flutamide, folinic acid, formestane, fosaprepitant, fotemustine, fulvestrant, gadobutrol, gadoteridol, gadoteric acid meglumine, gadoversetamide, gadoxetic acid, gallium nitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, Glucarpidase, glutoxim, GM-CSF, goserelin, granisetron, granulocyte colony stimulating factor, histamine dihydrochloride, histrelin, hydroxycarbamide, I-125 seeds, lansoprazole, ibandronic acid, ibritumomab tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib, imiquimod, improsulfan, indisetron, incadronic acid, ingenol mebutate, inotuzumab ozogamicin, interferon alfa, interferon beta, interferon gamma, iobitridol, iobenguane (123I), iomeprol, ipilimumab, irinotecan, Itraconazole, ixabepilone, ixazomib, lanreotide, lansoprazole, lapatinib, Iasocholine, lenalidomide, lenvatinib, lenograstim, lentinan, letrozole, leuprorelin, levamisole, levonorgestrel, levothyroxine sodium, lisuride, lobaplatin, lomustine, lonidamine, lutetium Lu 177 dotatate, masoprocol, medroxyprogesterone, megestrol, melarsoprol, melphalan, mepitiostane, mercaptopurine, mesna, methadone, methotrexate, methoxsalen, methylaminolevulinate, methylprednisolone, methyltestosterone, metirosine, midostaurin, mifamurtide, miltefosine, miriplatin, mitobronitol, mitoguazone, mitolactol, mitomycin, mitotane, mitoxantrone, mogamulizumab, molgramostim, mopidamol, morphine hydrochloride, morphine sulfate, mvasi, nabilone, nabiximols, nafarelin, naloxone + pentazocine, naltrexone, nartograstim, necitumumab, nedaplatin, nelarabine, neratinib, neridronic acid, netupitant/palonosetron, nivolumab, pentetreotide, nilotinib, nilutamide, nimorazole, nimotuzumab, nimustine, nintedanib, niraparib, nitracrine, nivolumab, obinutuzumab, octreotide, ofatumumab, olaparib, olaratumab, omacetaxine mepesuccinate, omeprazole, ondansetron, oprelvekin, orgotein, orilotimod, osimertinib, oxaliplatin, oxycodone, oxymetholone, ozogamicine, p53 gene therapy, paclitaxel, palbociclib, palifermin, palladium-103 seed, palonosetron, pamidronic acid, panitumumab, panobinostat, pantoprazole, pazopanib, pegaspargase, PEG-epoetin beta (methoxy PEG-epoetin beta), pembrolizumab, pegfilgrastim, peginterferon alfa-2b, pembrolizumab, pemetrexed, pentazocine, pentostatin, peplomycin, Perflubutane, perfosfamide, Pertuzumab, picibanil, pilocarpine, pirarubicin, pixantrone, plerixafor, plicamycin, poliglusam, polyestradiol phosphate, polyvinylpyrrolidone + sodium hyaluronate, polysaccharide-K, pomalidomide, ponatinib, porfimer sodium, pralatrexate, prednimustine, prednisone, procarbazine, procodazole, propranolol, quinagolide, rabeprazole, racotumomab, radium-223 chloride, radotinib, raloxifene, raltitrexed, ramosetron, ramucirumab, ranimustine, rasburicase, razoxane, refametinib, regorafenib, ribociclib, risedronic acid, rhenium- 186 etidronate, rituximab, rolapitant, romidepsin, romiplostim, romurtide, rucaparib, samarium (153Sm) lexidronam, sargramostim, sarilumab, satumomab, secretin, siltuximab, sipuleucel-T, sizofiran, sobuzoxane, sodium glycididazole, sonidegib, sorafenib, stanozolol, streptozocin, sunitinib, talaporfin, talimogene laherparepvec, tamibarotene, tamoxifen, tapentadol, tasonermin, teceleukin, technetium (99mTc) nofetumomab merpentan, 99mTc-HYNIC-[Tyr3]-octreotide, BHC233018 - FC - 151 / 152 - tegafur, tegafur + gimeracil + oteracil, temoporfin, temozolomide, temsirolimus, teniposide, testosterone, tetrofosmin, thalidomide, thiotepa, thymalfasin, thyrotropin alfa, tioguanine, tisagenlecleucel, tislelizumab, tocilizumab, topotecan, toremifene, tositumomab, trabectedin, trametinib, tramadol, trastuzumab, trastuzumab emtansine, treosulfan, tretinoin, trifluridine + tipiracil, trilostane, triptorelin, trametinib, trofosfamide, thrombopoietin, tryptophan, ubenimex, valatinib, valrubicin, vandetanib, vapreotide, vemurafenib, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, vismodegib, vorinostat, vorozole, yttrium-90 glass microspheres, zinostatin, zinostatin stimalamer, zoledronic acid, zorubicin. 8. Use of a compound of general formula (I) according to any one of claims 1 or 2 for the treatment or prophylaxis of a disease. 9. Use of a compound of general formula (I) according to any one of claims 1 or 2 for the preparation of a medicament for the treatment or prophylaxis of a disease. 10. Use according to claim 7, wherein the disease is a neoplastic disorder, such as cancer or conditions with dysregulated immune responses or other disorders associated with aberrant KRAS signaling, for example. 11. Method for controlling neoplastic diseases in humans and animals by administering an anti- neoplastic effective amount of at least one compound as defined in one of claims 1 or 2.
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