EA040674B1 - POLY(ADP-RIBOSE)POLYMERASE (PARP) INHIBITORS - Google Patents

Description

Field of invention

This application relates to poly(ADP-ribose)polymerase (PARP) inhibitors, in particular PARP-1 inhibitors, and methods of using these inhibitors, such as the treatment or prevention of one or more PARP-associated diseases.

State of the art invention

The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP-1) is a member of the PARP family of enzymes. This growing family of enzymes consists of PARPs such as PARP-1, PARP-2, PARP-3 and Vault-PARP.

PARP is involved in the repair of DNA strand breaks, and therefore inhibition of this enzyme is a well-established approach in the treatment of cancer. PARP inhibition may be particularly effective in combination with DNA damaging treatments such as ionizing radiation or after treatment with DNA damaging agents such as methylating agents, topoisomerase I inhibitors and other chemotherapeutic agents such as cisplatin and bleomycin. Inhibition of PARP enzymatic activity will lead to increased sensitivity of tumor cells to DNA damaging treatment. PARP inhibitors have been reported to be effective in radiosensitizing (hypoxic) tumor cells and effective in preventing tumor cell regeneration following potentially lethal and sub-lethal DNA damage from radiotherapy, presumably due to their ability to prevent DNA break repair and to effects on some damage-initiated signaling pathways. DNA.

Inhibition of PARP-2 may confer protection against oxidative stress (see Szanto, et al., Cell Mol. Life Sci. 69:4079 (2012)). Accordingly, PARP inhibitors can be used to treat diseases characterized by oxidative stress (eg, ischemia-reperfusion injury, inflammatory diseases, burns, parkinsonism, Huntington's disease, Alzheimer's disease, and toxic strokes).

PARP-1 and PARP-2 are pro-inflammatory (see Rosado et al., Immunology 739:428 (2013)). Accordingly, their inhibition can be used to treat, for example, asthma, arthritis, colitis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), atherosclerosis, cardiac remodeling after myocardial infarction, sepsis, endotoxic shock, hemorrhagic shock, homologous disease , encephalomyelitis and autoimmune nephritis.

Inhibition of PARP can also provide protection against viral infections (see Atasheva et al., J. Virol. 55:2116 (2014); and Virag and Szabo Pharmacol. Rev., 54:375 (2002)), for example against infections caused by human immunodeficiency virus 1, Venezuelan equine encephalitis virus, herpes simplex virus, human hepatitis B virus and human cytomegalovirus (Virag and Szabo Pharmacol. Rev., 54:375 (2002)).

PARP enzymes are involved in the control of glucose homeostasis (see Bai and Canto Cell Metab, 16:290 (2012); Riffel et al., Nat. Rev. Drug Discovery, 11:923 (2012); and Yeh et al., Diabetes, 55:2476 (2009)). For example, PARP-1 inhibition improves glucose utilization and insulin sensitivity (see Bai and Canto Cell Metab., 16:290 (2012); and Pirinen et al., Cell Metab., 79:1034 (2014)). Accordingly, PARP inhibition can be used to treat diseases and conditions such as metabolic syndrome and type II diabetes and their subsequent complications such as diabetic neurological, renal and ophthalmic complications.

Accordingly, there is a need for new and improved PARP inhibitors that can be administered for the above and other therapeutic indications.

The essence of the invention

The present invention provides new compounds that are effective inhibitors of PARP (see examples 1-53). In particular, they have selective PARP-1 inhibitory activity over PARP-2 inhibition (see example 54). In addition, it has been shown that some of these PARP inhibitors can be used to increase the amount of NAD + in cells (see example 55).

In a first aspect, the present invention relates to a pharmaceutical composition for the treatment of a subject having a disease that can be attenuated by inhibition of poly(ADPribose)polymerase (PARP), comprising a pharmaceutically acceptable carrier or diluent and a compound represented by the following structural formula:

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or a pharmaceutically acceptable salt thereof, wherein ring A is phenyl optionally substituted with one or two groups selected from halogen and (C ₁ -C ₃ )αkyl, or a 5-6 membered heteroaryl optionally substituted with one or two groups selected from halo and (C1- _C3 ) alkyl;

ring B is phenyl, 5-6 membered heteroaryl, or 5-6 membered heterocyclyl, each of which optionally has one or more substituents represented by R ³ ;

----- absent or represents a link;

E is N or CH when ----- is absent, or E is C when ----- is a bond;

optionally substituted with (C1-C ₅ )alkyl or hydroxy(C1-C ₅ )alkyl;

each R3 is independently selected from the group consisting of -halogen, -CN, -C(=NR ^e )NR ^e R ^f , -C(=O)NR ^e R ^f and (C1-C ₅ )alkyl;

each Re ^is independently selected from the group consisting of -H and (C ₁ -C ₅ )αkyl;

each R ^f is independently selected from the group consisting of -H, (C1-C ₅ )alkyl, (C3-C ₆ )cycloalkyl optionally substituted with (C ₁ -C ₂ )alkyl, and 4-6 membered oxygenate heterocyclyl optionally substituted with (C ₁ -C ₂ )αkyl; or

-C(=NR ^e )NR ^e R ^f forms a 4-6 membered heterocyclyl optionally substituted with R ^e ; And

R ⁵ is -H or (C ₁ -C ₃ )alkyl;

where heteroaryl contains 1-4 heteroatoms selected from oxygen, nitrogen and sulfur, and heterocyclyl contains 1 or 2 heteroatoms selected from oxygen, nitrogen and sulfur.

In a preferred embodiment of the present invention, the compound in the above pharmaceutical composition is represented by a structural formula selected from the group consisting of

or a pharmaceutically acceptable salt thereof, wherein X ¹ , X ² , X3 and X ⁴ are each independently selected from the group consisting of N and CH, provided that no more than two of X ¹ , X ² , X3 and X ⁴ are N;

X ⁵ is NR ² , O or S;

ring B is phenyl, 5-6 membered heteroaryl, or 5-6 membered heterocyclyl, each of which optionally has one or more substituents represented by R3;

p N

N ₄ A % optionally has (C1-C3)-alkyl or hydroxy(C1-C3)alkyl as a substituent;

each R1 is independently selected from the group consisting of α-halogen and (C1-C3)alkyl;

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R ² is -H or (C ₁ -C ₃ )alkyl; and n is 0, 1, or 2.

In a more preferred embodiment of the present invention, the compound in the above pharmaceutical composition is represented by a structural formula selected from the group consisting of

or a pharmaceutically acceptable salt thereof, wherein ring B is phenyl, 5-6 membered heteroaryl, or 5-6 membered heterocyclyl, each of which optionally has one or more substituents represented by R ³ ; And

optionally substituted with (C1-C ₅ )alkyl or hydroxy(C1-C ₅ )alkyl;

each R ¹ is independently selected from the group consisting of halogen and (C ₁ -C ₃ )alkyl; and each R ³ is independently selected from the group consisting of -halogen, -CN, -C(=NR ^e )NR ^{e Rf} , -C(=O)NR ^e Rf and (C ₁ -C ₅ )αkyl.

In an even more preferred embodiment of the present invention, in the compound of the above pharmaceutical composition, ring B is selected from the group consisting of

each R ⁴ is -H or (C _g C ₅ )alkyl;

each p is independently 0 or 1;

each R ¹ is independently halogen or (C ₁ -C ₃ )alkyl;

each R ³ is independently selected from the group consisting of -halogen, -CN, -C(=NR ^e )NR ^e Rf, -C(=O)NR ^e Rf, and (C ₁ -C ₅ )αkyl;

each R ^e and each R ^f is independently selected from the group consisting of -H and methyl; or R ^e is -H and Rf is -(C ₃ -C ₆ )cycloalkyl or 4-6 membered oxygen-containing heterocyclyl, each of which is optionally substituted with (C ₁ -C ₂ )alkyl; and each m is 0, 1, or 2.

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In an even more preferred embodiment of the present invention, in the compound of the above pharmaceutical composition, each R ¹ is independently chlorine, fluorine or methyl;

each R ³ is independently selected from the group consisting of chlorine, fluorine, -CN, -C(=NR ^e )NR ^e R ^f ,

-C(=O)NR ^e R ^f and methyl;

group

optionally has methyl or hydroxymethyl as a substituent.

In an even more preferred embodiment of the present invention, in the compound of the above pharmaceutical composition, each R ³ is independently selected from the group consisting of chlorine,

fluorine, -CN, -C(O)NH(cyclopropyl), -C(O)NH2, -C(O)NH(CH3), -C(O)N(CH3>2,

'-C(=NH)NHCH ₃ and methyl.

-C(O)NH(cyclobutyl),

In another preferred embodiment of the present invention, in the compound of the above pharmaceutical composition, R ² is -H or methyl.

In a preferred embodiment of the present invention, the compound in the above pharmaceutical composition is 6-[(3S)-4-[3-(6-fluoro-4-oxo-3H-quinazolin-2yl)propanoyl]-3-methylpiperazin- 1-yl]pyridine-3-carbonitrile or a pharmaceutically acceptable salt thereof.

In a preferred embodiment of the present invention, the compound in the above pharmaceutical composition is 6-[(3S)-4-[3-(5-fluoro-4-oxo-3H-quinazolin-2yl)propanoyl]-3-methyl-piperazine- 1-yl]pyridine-3-carbonitrile or a pharmaceutically acceptable salt thereof.

In yet another aspect, the present invention is a compound of the following structural formula:

or a pharmaceutically acceptable salt thereof, wherein R ¹ is F or methyl and ην-^Λ.

JO

R ³ is -CN, -C(=NH)NHCH ₃ %· or methyl.

In a preferred embodiment of the above compound, R ¹ is F and R ³ is -CN.

In a preferred embodiment, the above compound is 6-[(3S)-4-[3-(6fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]-3-methyl-piperazin-1-yl]pyridine- 3-carbonitrile or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the above compound is 6-[(3S)-4-[3-(5fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]-3-methyl-piperazin-1-yl]pyridine- 3-carbonitrile or a pharmaceutically acceptable salt thereof.

In yet another aspect, the present invention relates to a method of treating a subject having a disease that can be attenuated by inhibition of poly(ADP-ribose)polymerase (PARP), which method includes administering to said subject an effective amount of the pharmaceutical composition as provided above or the compound as provided above, or its pharmaceutically acceptable salt.

In yet another aspect, the present invention relates to a method of treating a subject with acute kidney injury, wherein said method comprises administering to said subject an effective amount of a pharmaceutical composition as provided above, or a compound as provided above, or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention relates to a method of treating a subject suffering from cancer, which can be attenuated by inhibition of poly(ADP-ribose)polymerase (PARP), where

- 4 040674 said method comprises administering to said subject an effective amount of a pharmaceutical composition as provided above, or a compound as provided above, or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the disease that can be attenuated by PARP inhibition is selected from genetic lipodystrophy, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), ischemic/reperfusion kidney injury (IRI), Duchenne and Becker muscular dystrophy, diabetes (type I or type II), obesity, sarcopenia, Alpers disease, chronic progressive external ophthalmoplegia (CPEO), Kearns-Sayre syndrome (KSS), Leber hereditary optic neuropathy (LHON), MELAS - mitochondrial myopathy, encephalomyopathy, lactic acidosis and stroke-like episodes, MERRF - myoclonic epilepsy with torn muscle fibers, NARP - neurogenic muscle weakness, ataxia and retinitis pigmentosa, Pearson's syndrome, platinum chemotherapy-induced ototoxicity, Cockayne's syndrome, xeroderma pigmentosa A, Wallerian degeneration and HIV-induced lipodystrophy.

In a preferred embodiment, the pharmaceutical composition as provided above is for the treatment of a wound and/or burn in a subject.

In another preferred embodiment, the pharmaceutical composition provided above is for the treatment of acute kidney injury in a subject.

In yet another aspect, the present invention provides a method of treating a wound in a subject, wherein the method comprises administering to said subject an effective amount of a pharmaceutical composition as provided above, or a compound as provided above, or a pharmaceutically acceptable salt thereof.

In yet another aspect, the present invention provides a method of treating a burn in a subject, wherein the method comprises administering to said subject an effective amount of a pharmaceutical composition as provided above, or a compound as provided above, or a pharmaceutically acceptable salt thereof.

The term "pharmaceutically acceptable salt" refers to a pharmaceutical salt that has been medically evaluated as being suitable for use in contact with human and lower animal tissues, without excessive toxicity, without excessive irritation or allergic reaction, and with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al describe pharmacologically acceptable salts in J. Pharm. Sci., 1977, 66, 1-19.

The present invention includes pharmaceutically acceptable salts of the compounds described in this application. Compounds having basic groups can form pharmaceutically acceptable salts with pharmaceutically acceptable acid(s). Suitable pharmaceutically acceptable acid addition salts of the compounds described in this application include salts of inorganic acids (such as hydrochloric acid, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids) and organic acids (such as acetic acid, benzenesulfonic, benzoic, ethanesulfonic, methanesulfonic , succinic and trifluoroacetic acids). Compounds of the present invention with acidic groups, such as carboxylic acids, may form pharmaceutically acceptable salts with pharmaceutically acceptable base(s). Suitable pharmaceutically acceptable base salts include ammonium salts, alkali metal salts (such as sodium and potassium salts), and alkaline earth metal salts (such as magnesium and calcium salts).

Brief description of the drawings

Figure 1 shows that the PAYP1 inhibitors described in Example 29 and Example 30 reduce plasma creatinine and blood urea nitrogen (BUN) after 24 hours in an animal model of kidney injury.

Detailed description of the invention

Definitions.

The term halogen as used herein means a halogen atom and includes chlorine, fluorine, bromine and iodine.

The term alkyl, whether used alone or as part of the name of a larger group such as alkoxy or haloalkyl and the like, means a monovalent hydrocarbon radical with a saturated aliphatic straight or branched chain. Unless otherwise noted, an alkyl group typically contains 1-5 carbon atoms, i. e. is (C ₁ -C ₅ )alkyl. In the context of this description, the group (C1-C ₅ )alkyl means a radical containing from 1 to 5 carbon atoms in a normal or branched configuration. Examples include methyl, ethyl, n-propyl, iso-propyl, and the like.

The term alkoxy refers to an alkyl radical attached via a bonding oxygen atom, represented by -O-alkyl. For example, (C ₁ -C 4 )alkoxy includes methoxy, ethoxy, propoxy and butoxy.

The terms haloalkyl and haloalkoxy mean alkyl or alkoxy which may be substituted with one or more halogen atoms.

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The term cycloalkyl refers to a monocyclic saturated hydrocarbon ring system. For example, C ₃ . ₆ cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Unless otherwise noted, cycloalkyl contains three to six carbon atoms.

The term heteroaryl, heteroaromatic, heteroaryl ring, heteroaryl group, heteroaromatic ring, and heteroaromatic group, whether used alone or as part of the name of a larger moiety such as heteroaralkyl or heteroarylalkoxy, refers to monocyclic aromatic ring groups containing five or six ring atoms (i.e. . to 5-6-membered groups) selected from a carbon atom and at least one (usually 1-4, more often 1 or 2) heteroatoms (for example, an oxygen, nitrogen or sulfur atom).

Examples of monocyclic heteroaryl groups include furanyl (e.g. 2-furanyl, 3-furanyl), imidazolyl (e.g. N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), isoxazolyl (e.g. 3-isoxazolyl, 4-isoxazolyl , 5-isoxazolyl), oxadiazolyl (e.g. 2-oxadiazolyl, 5-oxadiazolyl), oxazolyl (e.g. 2-oxazolyl, 4-oxazolyl, 5-oxazolyl), pyrazolyl (e.g. 3-pyrazolyl, 4-pyrazolyl), pyrrolyl (e.g. 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), pyridyl (e.g. 2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (e.g. 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl), pyridazinyl (e.g. 3-pyridazinyl), thiazolyl (e.g. 2-thiazolyl, 4-thiazolyl, 5-thiazolyl), triazolyl (e.g. 2-triazolyl, 5-triazolyl), tetrazolyl (e.g. tetrazolyl), thienyl (e.g. 2-thienyl, 3-thienyl), pyrimidinyl, pyridinyl and pyridazinyl.

The term heterocyclyl refers to a monocyclic non-aromatic ring radical containing 4 to 6 ring atoms (ie, a 4-6 membered radical) selected from a carbon atom and 1 or 2 heteroatoms.

Each heteroatom is independently selected from a nitrogen atom, a quaternary nitrogen atom, an oxidized nitrogen atom (eg NO), an oxygen atom, and a sulfur atom, including a sulfoxide and a sulfone. Exemplary heterocyclyl groups include morpholinyl, thiomorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like. A substituted heterocyclyl group has a substituent on any one or more ring atoms (capable of having a substituent) that is a ring carbon atom or a ring nitrogen atom bonded to a hydrogen atom.

In this description, many groups (for example, alkyl, alkylene, cycloalkyl, cycloalkylene, aryl, arylene, heteroaryl, heteroarylene, heterocyclyl or heterocyclylene) are referred to as having a substituent or optionally having a substituent. When the name of a group is modified by one of these terms, it means, unless otherwise indicated, that any part of the group that is known to the person skilled in the art to be capable of having substituents may be substituted, i. have one or more substituents. If more than one substituent is present, each substituent may be selected independently of the other substituents. Such a substitution option is well known in the art and/or described in this invention. The optional substituents may be any substituents that are suitable for attachment to the moiety.

Suitable substituents are substituents that do not significantly adversely affect the ability of a compound of the invention to inhibit PARP. When suitable substituents are not directly listed, typical substituents include, without limitation, (C1-C ₅ )alkyl, (C1-C ₅ )hydroxyalkyl, (C1-C ₅ ) haloalkyl, (C1-C ₅ )alkoxy, (C1-C ₅ ) haloalkoxy, halogen atom, hydroxyl, cyano, amino, -CN, -NO ₂ , -OR ^c , -NR ^a R ^b , -S(O),R ^a , -NR ^a S(O),R ^b , -S (O),NR ^a R ^b , -C(=O)OR ^a , -OC(=O)OR ^a , -C(=S)OR ^a , -O(C=S)R ^a , -C(= O)NR ^a R ^b , -NR ^a C(=O)R ^b , -C(=S)NR ^a R ^b , -NR ^a C(=S)R ^b , -NR ^a (C=O)OR ^b , -O(C=O)NR ^a R ^b , -NR ^a (C=S)OR ^b , -O(C=S)NR ^a R ^b , -NR ^a (C=O)NR ^a R ^b , - NR ^a (C=S)NR ^a R ^b , -C(=S)R ^a , -C(=O)R ^a , phenyl or 5-6 membered heteroaryl. Each R ^a and each R ^b is independently selected from -H and (C1-C5)alkyl optionally substituted with hydroxyl or (C1-O3)alkoxy; R ^c is -H, (C1-C ₅ )haloalkyl or (C ₁ -C ₅ )alkyl, with (C ₁ -C ₅ )alkyl optionally substituted with hydroxyl or (C1-C ₃ )alkoxy.

Some of the compounds described in this application may exist in various stereoisomeric or tautomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. When the name or depicted structure of a compound of the invention does not indicate stereochemistry, the name or structure is intended to include all possible stereoisomers, geometric isomers, including substantially pure stereoisomers or geometric isomers, as well as combinations thereof.

In some cases, the compounds of the invention have tautomeric forms, such as the tautomeric structures shown below,

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Oh Oh

It is to be understood that when a compound in this application is represented by a structural formula or defined using a chemical name, all other tautomeric forms that may exist in that compound are included by that structural formula.

Some compounds of the invention may exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are pairs of stereoisomers that are non-superimposable mirror images, most commonly due to their having an asymmetric carbon atom that acts as a chiral center.

An enantiomer is one of paired molecules that are mirror images of each other that do not overlap in space. Diastereoisomers are stereoisomers that contain two or more asymmetric carbon atoms. Geometric isomers are stereoisomers that differ in the arrangement of substituent atoms relative to a carbon-carbon double bond, a carbocyclyl ring, or a bridged bicyclic system.

When a geometric isomer is indicated using a name or structural formula, it is to be understood that the isomeric purity of the named or depicted geometric isomer is at least 60, 70, 80, 90, 99, or 99.9% by weight. Isomeric purity of a geometric isomer is determined by dividing the weight of the named or depicted geometric isomer present in the mixture by the total weight of all geometric isomers present in the mixture.

When the stereochemistry of a compound of the invention is indicated in the name or shown in the structural formula, the purity of the named or depicted stereoisomer is at least 60, 70, 80, 90, 99, or 99.9% by weight relative to all other stereoisomers. Percent purity by weight relative to all other stereoisomers is the ratio of the mass of one stereoisomer to the mass of other stereoisomers. When a single enantiomer is specified using a name or structural formula, the optical purity (also referred to as enantiomeric purity) of the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight. Percent optical purity by weight is the ratio of the mass of a given enantiomer to the sum of the mass of a given enantiomer and the mass of its optical isomer.

When the stereochemistry of a compound of the invention is indicated in a name or displayed in a structural formula, and the named or depicted structure includes more than one stereoisomer (for example, as in the case of a diastereomeric pair), it is to be understood that one of said stereoisomers or any mixture of said stereoisomers is included. It is also to be understood that the stereoisomeric purity of the named or depicted stereoisomers is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight relative to all other stereoisomers. Stereoisomeric purity is here determined by dividing the total weight of stereoisomers corresponding to a given name or structure present in the mixture by the total weight of all stereoisomers present in the mixture.

When the stereochemistry of a compound of the invention is not indicated in the name or shown in the structural formula and the compound has a single chiral center, it is to be understood that the name or structure includes one enantiomer of the compound free from its corresponding optical isomer, a racemic mixture of the compound, and mixtures enriched in one enantiomer relative to its corresponding optical isomer.

When the stereochemistry of a compound of the invention is not indicated in the name or shown in the structural formula and, for example, the compound has at least two chiral centers, it is to be understood that the given name or structure includes one stereoisomer free from other stereoisomers, a mixture of stereoisomers, and a mixture of stereoisomers enriched in one or more stereoisomers relative to the other(s) stereoisomer(s). For example, the name or structure may include one stereoisomer free of other diastereomers, mixtures of stereoisomers, and mixtures of stereoisomers enriched in one or more diastereomers relative to other diastereomer(s).

Enantiomeric and diastereomeric mixtures can be separated into their constituent enantiomeric or stereoisomeric components using well known techniques such as chiral phase gas chromatography, chiral high performance liquid chromatography, crystallization of a compound as a chiral salt complex, or crystallization of a compound in chiral solvent. Enantiomers and diastereoisomers can also be prepared from diastereomeric or enantiomerically pure intermediates, reagents and catalysts using well known asymmetric synthesis techniques.

pharmaceutical compositions.

The compounds described in this application are PARP inhibitors (eg PARP1 inhibitors). The pharmaceutical composition of the present invention includes one or more PARP inhibitors (eg PARP-1 inhibitors), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.

A pharmaceutically acceptable carrier and a pharmaceutically acceptable diluent refer to a substance that facilitates the preparation of a formulation containing the active agent and/or the administration of the active agent to a subject and/or the absorption of the active agent by the subject, and the inclusion of which in the compositions of the present invention does not have a significant adverse toxic effect. on the subject. Non-limiting examples of pharmaceutically acceptable carriers and/or diluents include water, NaCl, saline solutions, lactated Ringer's solution, physiological sucrose levels, physiological glucose levels, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, saline solutions (such such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine and dyes, and the like. Such preparations may be sterilized and, if desired, mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffering agents, colorants and/or fragrances, and the like, which do not cause an adverse change or disruption in the activity of the compounds of the present invention. One of ordinary skill in the art will appreciate that other pharmaceutical excipients are also suitable for use with the compounds of the invention.

The pharmaceutical compositions of the present invention optionally include one or more pharmaceutically acceptable carriers and/or diluents such as lactose, starch, cellulose and dextrose. Other excipients such as flavoring agents, sweeteners and preservatives such as methyl, ethyl, propyl and butyl parabens may also be included. A more complete list of suitable excipients can be found in The Handbook of Pharmaceutical Excipients (5th ed., Pharmaceutical Press (2005)). One skilled in the art would be aware of how to prepare formulations suitable for various types of routes of administration. Standard procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003, 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19), 1999 edition. Carriers, diluents and/or excipients are acceptable in terms of their compatibility with other ingredients of the pharmaceutical composition and safety for the recipient.

Methods of treatment.

PARP enzymes are involved in a variety of cellular functions, including DNA repair, mitochondrial homeostasis, protection against oxidative stress, inflammation, metabolic regulation, circadian rhythms, differentiation, and aging. See, for example, Peter Bai, Molecular. Cell, 55:947 (2015). Accordingly, PARP inhibitors have the potential to treat a wide range of diseases, and several PARP inhibitors are already approved for use in the treatment of cancer.

The present invention provides a method of treating a subject having a disease that can be attenuated by PARP inhibition, comprising administering to the subject an effective amount of one or more compounds of the invention, or a pharmaceutically acceptable salt thereof, or an appropriate pharmaceutical composition.

The term subject refers to a mammal, preferably a human, but may also refer to an animal in need of veterinary treatment, such as pets (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses). etc.) and laboratory animals (eg rats, mice, guinea pigs, etc.).

Diseases that can be attenuated by PARP inhibition can be selected from genetic lipodystrophy, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), ischemic/reperfusion kidney injury (IRI), Duchenne and Becker muscular dystrophy, diabetes (type I or type II), obesity, sarcopenia, Alpers disease, chronic progressive external ophthalmoplegia (CPEO), Kearns-Sayre syndrome (KSS), Leber hereditary optic neuropathy (LHON), MELAS - mitochondrial myopathy, encephalomyopathy, lactic acidosis and stroke-like episodes, MERRF - myoclonic epilepsy with torn muscle fibers, NARP - neurogenic muscle weakness, ataxia and retinitis pigmentosa, Pearson's syndrome, platinum chemotherapy-induced ototoxicity, Cockayne's syndrome, xeroderma pigmentosa A, Wallerian degeneration and HIV-induced lipodystrophy.

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Methods of administration and dosage forms.

The precise amount of compound administered that is an effective amount for a given subject depends on the route of administration, the type and severity of the cancer, and on the subject's characteristics such as general health, age, sex, body weight, and drug tolerance. One skilled in the art will be able to determine appropriate dosages depending on these and other factors. When administered in combination with other therapeutic agents, for example when administered in combination with an anti-cancer agent, the effective amount of any additional therapeutic agent(s) depends on the type of drug used. Suitable doses of approved therapeutic agents are known and can be adjusted by one of skill in the art according to the condition of the subject, the type of condition(s) that(s) require(s) to be treated, and the amount of compound of the invention used per based, for example, on doses described in the literature and recommended in the Physician's Desk Reference (57th edition, 2003).

The term "effective amount" means an amount which, when administered to a subject, produces beneficial or desired results, including clinical results, such as inhibition, amelioration or reduction of the symptoms of the condition in the subject being treated, as compared to a control. For example, a therapeutically effective amount may be administered in unit dosage form (and is, for example, from 0.1 mg to about 50 g per day, alternatively from 1 mg to about 5 g per day, and otherwise from 10 mg to 1 g per day).

The terms enter, introduction (process), introduction, etc. in the context of this description refer to techniques that can be used to deliver compositions to the desired site of biological impact. These techniques include, without limitation, intra-articular (into the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, subcutaneous, oral, topical, intrathecal, inhalation, transdermal, rectal routes, and the like. Administration techniques that can be used with agents and techniques described herein can be found, for example, in Goodman and Gilman's The Pharmacological Basis of Therapeutics (current edition); Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa.

The particular route of administration and dosage regimen will generally be selected by the attending physician, taking into account the particularities of each individual case (eg, characteristics of the subject, disease, comorbid condition, particular treatment). Treatment may include daily, or multiple daily, or less frequent than daily (eg, weekly or monthly, etc.) doses administered over a period of several days to several months, or even years. However, one of ordinary skill in the art familiar with the dosages of approved compositions for the treatment of PARP-mediated disease would readily be able to determine appropriate and/or equivalent dosages using the data of the described PARP inhibitors (eg, PARP-1 inhibitors) for guidance.

The compounds or corresponding pharmaceutical compositions described herein may be administered to a patient in a variety of dosage forms, depending on the route of administration chosen, as will be understood by those skilled in the art. The compounds of the present invention may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, dispenser, or transdermal administration, and pharmaceutical compositions containing these compounds are presented in the appropriate form. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topical routes of administration. Parenteral administration may be by continuous infusion over a selected period of time.

The pharmaceutical composition of the present invention is in the form of a formulation compatible with its intended route of administration. According to one of the embodiments of the present invention, this composition is obtained in accordance with standard techniques in the form of a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to humans. According to preferred embodiments of the present invention, the pharmaceutical composition is in the form of a preparation intended for intravenous administration.

Generally, a compound of the present invention intended for oral therapeutic administration may be combined with an excipient and used in the form of swallowable tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, and the like.

Typically, parenteral solutions of a compound of the present invention can be prepared in water appropriately mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be obtained in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under standard conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

For injectable administration, sterile aqueous solutions or dispersions and sterile powders of the compound described in this application are usually suitable for the preparation of extemporaneous sterile injectable solutions or dispersions.

Examples

Abbreviations.

Me is methyl;

Et - ethyl;

Boc - tert-butyloxycarbonyl;

Ac - acetyl;

Ph - phenyl;

Tf - trifluoromethanesulfonyl;

DIPEA - diisopropylethylamine;

EDC - 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide;

HOBt - 1-hydroxybenzotriazole;

DCM - dichloromethane;

DMF - N,N-dimethylformamide;

DMSO - dimethyl sulfoxide;

TFA - trifluoroacetic acid;

THF - tetrahydrofuran;

TMS - trimethylsilane;

TMSOTf - trifluoromethanesulfonic acid trimethylsilyl ether, aq. water;

M is the concentration expressed in mol/l;

k.t. - room temperature;

TLC - thin layer chromatography;

HPLC - high performance liquid chromatography;

NMI - 1-methylimidazole;

LC-MS - liquid chromatography/mass spectrometry;

ESI+ - m/z values in mass spectroscopy (ESI ionization);

ESI- - m/z values in mass spectroscopy (ESI ionization);

¹ H NMR (DMSO-d6) δ (ppm) peak - ¹ H NMR in DMSO-L;

s - singlet (spectrum);

d - doublet (spectrum);

t - triplet (spectrum);

q - quartet (spectrum);

dd - double doublet (spectrum);

br - broadened line (spectrum);

m - multiplet (spectrum);

4-ANI - 4-amino-1,8-naphthalimide;

ADP - adenosine diphosphate;

CPM - number of pulses per minute;

DNA - deoxyribonucleic acid;

DTT - DL-dithiothreitol;

FB - flat bottom;

mg - milligram;

mM - millimolar;

NAD - nicotinamide adenine dinucleotide;

nM - nanomolar;

ng - nanogram;

PARP-1 - poly(ADP-ribose) polymerase 1;

SPA - scintillation proximity analysis;

mkCi - microcurie;

µl - microliter;

T3P - propylphosphonic anhydride;

NMM - 4-methylmorpholine;

CDI - 1,1'-carbonyldiimidazole;

EtOAc - ethyl acetate;

TEMPO - 2,2,6,6-tetramethyl-1-piperidinyloxy;

MTBE - tert-butyl methyl ether;

HATU - 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate;

IPA - isopropyl alcohol;

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DMA - Ν,N-dimethylacetamide;

BINAP - (1,1'-binaphthalene-2,2'-diyl)bis(diphenylphosphine);

NMP - 1-methyl-2-pyrrolidinone;

Dppf - 1,1'-bis(diphenylphosphino)ferrocene;

DMAP - 4-(dimethylamino)pyridine;

DIEA - Ν,N-diisopropylethylamine.

Example 1 Synthesis of 3-chloro-4-(4-(3-(8-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)propanoyl)piperazin1-yl)-N-cyclopropylbenzamide.

Stage 1

N32S2O4, H2O ____________________________ aq. NH ₃ , HC1

Stage 1

2

To a stirred solution of 3-chloro-2-nitrobenzoic acid (15 g, 0.074 mol) in water (105 ml) was added at room temperature 30% aqueous NH ₃ solution (6 ml) and an aqueous solution of sodium dithionite (52 g, 0.298 mol) and stirred for 1 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then acidified to pH 3 with concentrated HCl (30 ml), extracted with EtOAc (2x500 ml), washed with water (2x100 ml) and brine (150 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure to give the crude product, which was washed with Et ₂ O (50 ml) to give 2-amino-3-chlorobenzoic acid (9 g, 70%) as a yellowish solid.

LC-MS (m/z): 172.3 [M+H]+.

Stage 2

A stirred solution of 2-amino-3-chloro-benzoic acid (9 g, 0.052 mol) and CDI (9 g, 0.055 mol) in DMF (180 ml) was heated to 70°C for 1 h. Then a 30% aqueous solution was added NH3 (144 ml), maintaining the temperature at 70°C and stirring for 16 h (according to TLC analysis, the starting material was completely consumed). The temperature of the reaction mixture was brought to room temperature, the mixture was poured into ice water (1 l) and was extracted with EtOAc (2x250 ml). The combined organic extracts were washed with water (2x100 ml), brine (100 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude product, which was washed with Et ₂ O (2x30 ml) to give 2-amino-3- chlorobenzamide (5.4 g, 60%) as a yellowish solid.

LC-MS (m/z): 171.3 [M+H]+.

Stage 3

To a stirred solution of 2-amino-3-chloro-benzamide (2 g, 11.76 mmol) in pyridine (15 ml) in a sealed tube was added at 0 .64 mmol) in DCM (5 ml). This reaction mixture was heated to 100°C and stirred for 12 h (according to TLC analysis, the starting material was completely consumed). The temperature of the reaction mixture was brought to room temperature, the mixture was diluted with water (150 ml) and was extracted with EtOAc (2x150 ml). The combined organic extracts were washed with saturated aqueous NH4Cl (2x50 ml), brine (50 ml), dried over Na2SO4, concentrated under reduced pressure to give the crude desired compound, which was washed with toluene (20 ml), diethyl ether (2x10 ml) to give 8-chloro-2-(3-hydroxypropyl)-3H-quinazolin-4-one (600 mg, 21%) as a yellowish solid.

LC-MS (m/z): 239.4 [M+1]+.

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Stage 4

To a stirred solution of 8-chloro-2-(3-hydroxypropyl)-3H-quinazolin-4-one (500 mg, 2.10 mmol) in ACN (acetonitrile) (10 mL) was added TEMPO (65 mg, 0.414 mmol) and sodium phosphate buffer (8 ml, pH 6.5) and heated to 40°C. Sodium chlorite (3.75 g in 15 ml water) and sodium chlorite solution (4% in H2O, 15 ml) were then added portionwise at 40°C. The temperature of the reaction mixture was brought to room temperature, the mixture was basified to pH 8 by adding 1 N hydrochloric acid.

NaOH solution, poured into 1 N. Na ₂ S ₂ O ₃ solution (50 ml), washed with MTBE (2x25 ml). The aqueous layer was acidified to pH 1 by adding 1N. HCl and extracted with EtOAc (3x50 ml). The combined organic extracts were washed with brine (50 ml), dried over Na ₂ SO ₄ , concentrated under reduced pressure to give 3-(8-chloro-4-oxo-3H-quinazolin-2-yl)propanoic acid (250 mg, 47 %), which was used in the next stage of additional purification.

LC-MS (m/z): 253.3 [M+1]+.

Stage 5

To a stirred solution of 3-(8-chloro-4-oxo-3H-quinαzolin-2-yl)propanoic acid (285 mg, 1.13 mmol) and 3-chloro-N-cyclopropyl-4-piperazin-1-yl- benzamide (308 mg, 1.1 mmol) in DMF (2.6 ml) were added at room temperature EDC-HCl (432 mg, 2.26 mmol), HOBt (305 mg, 2.26 mmol) and DIPEA (0. 96 ml, 5.65 mmol) and stirred for 12 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was diluted with cold water (50 ml), was extracted with EtOAc (3x25 ml). The combined organic layers were washed with water (25 ml), brine (25 ml), dried over Na ₂ SO ₄ , concentrated under reduced pressure and the residue was purified by chromatography on Teledyne-ISCO Combiflash (5-7% MeOH-DCM, 4 g cartridge ) to give 3-chloro-4-(4-(3-(8-chloro4-oxo-3,4-dihydroquinazolin-2-yl)propanoyl)piperazin-1-yl)-N-cyclopropylbenzamide (100 mg, 85% according to LC-MS) which was further purified by preparative HPLC to yield the pure compound (25 mg, 5%) as a white solid.

¹ H NMR [300 MHz, DMSO-d6] δ 12.54 (br. s, 1H), 8.42 (d, J=3.9 Hz, 1H), 8.05-8.02 (m, 1H), 7.93-7.88 (m, 2H) , 7.76 (dd, J=8.1, 1.8 Hz, 1H), 7.42 (t, J=7.8 Hz, 1H), 7.16 (d, J=8.4 Hz, 1H), 3.70 (m, 2H), 3.61 (m, 2H), 3.08-2.92 (m, 2H), 2.8-2.79 (m, 7H), 0.71-0.62 (m, 2H), 0.60-0.52 (m, 2H).

LC-MS (m/z): 514.4 [M+H] ⁺ .

Example 2 Synthesis of 3-chloro-4-[4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]benzamide.

Stage 1

3-Chloro-4-fluoro-benzoic acid (2.0 g, 11.4 mmol), sulfuric acid (0.33 g, 3.4 mmol), MeOH (20 ml) was added to a round bottom flask and the mixture was refluxed within 16 h (according to TLC analysis, the starting material was completely consumed). Volatiles were removed under reduced pressure; the residue was diluted with water (15 ml) and was extracted with EtOAc (3x50 ml). The combined organic extracts were washed with brine (50 ml), dried over Na ₂ SO ₄ and concentrated in vacuo, the resulting crude residue was purified by column chromatography (100-200 silica gel, 40 g, 10% EtOAc-hexane) to yield methyl 3-chloro -4-fluoro-benzoate (1.5 g, 69%) as a light yellow oil.

1Н NMR [300 MHz, CDCl ₃ ] δ 8.08 (dd, J=6.9, 2.1 Hz, 1H), 7.94-7.89 (m, 1H), 7.18 (t, J=8.7 Hz, 1H), 3.90 (s, 3H ).

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Stage 2

To methyl 3-chloro-4-fluoro-benzoate (1.5 g, 7.9 mmol) in a sealed tube was added piperazine-1-carboxylic acid tert-butyl ester (1.48 g, 7.9 mmol ) and then K ₂ CO ₃ (3.29 g, 23 mmol) and DMSO (15 ml) and stirred for 10 h at 100° C. (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was poured into water cooled to 0°C (150 ml) and extracted with EtOAc (2x100 ml). The combined organic extracts were washed with brine (2x75 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure, the resulting crude residue was purified by column chromatography (100-200 silica gel, 30 g, 10% EtOAc-hexane) to yield t-butyl 4-(2-Chloro-4-methoxycarbonyl-phenyl)piperazine-1-carboxylate (2.1 g, 74%) as a white solid.

LC-MS (m/z): 355.4 [M+H]+.

Stage 3

To a stirred solution of mpem-butyl 4-(2-chloro-4-methoxycarbonyl-phenyl)piperazine-1-carboxylate (2 g, 5.6 mmol) in THF:MeOH:H ₂ O (10:1:1, 24 mL) , LiOH·H ₂ O (0.47 g, 11.2 mmol) was added, this mixture was heated for 12 h at 60° C. (according to TLC analysis, the starting material was completely consumed) and concentrated under reduced pressure. The residue was dissolved in water (30 ml), cooled to 0°C, acidified to pH 2-3 by adding 1N. HCl. The precipitated solid was collected by filtration and dried in vacuo to give 4-(4-tert-butoxycarbonylpiperazin-1-yl)-3-chloro-benzoic acid (1.4 g, 73%) as a white solid

va.

LC-MS (m/z): 341.4 [M+H] ⁺ .

Stage 4

EDC HC1, HOBt NH ₃ , DIPEA, THF, rt, 5 h

Stage 4

BocN^^ Cl ⁴ 5

EDC-HCl (0 .42 g, 2.2 mmol), HOBt-NH ₃ (0.33 g, 2.2 mmol) and DIPEA (0.75 ml, 4.4 mmol), and stirred for 5 h at room temperature (according to TLC analysis of the starting material was completely consumed). Volatiles were removed under reduced pressure and the residue was diluted with ice water (100 ml) and EtOAc (150 ml). The organic layer was separated, washed with brine (2x50 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure. This crude residue was washed with Et ₂ O (3x5 ml), pentane (3x5 ml), dried under high vacuum to give tert-butyl 4-(4-carbamoyl-2-chlorophenyl)piperazine-1-carboxylate (0.4 g, 80%) as a white solid.

LC-MS (m/z): 340.4 [M+H] ⁺ .

Stage 5

To a stirred solution of tert-butyl 4-(4-carbamoyl-2-chloro-phenyl)piperazine-1-carboxylate (0.4 g, 1.0 mmol) in DCM (4 ml) . HCl-dioxane (0.4 ml), and stirred for 3 h at room temperature (according to TLC analysis, the starting material was completely consumed). Volatiles were removed under reduced pressure and the residue was washed with Et ₂ O (3x10 ml), pentane (2x5 ml) and dried under high vacuum to give 3-chloro-4-13 040674 piperazin-1-yl-benzamide (300 mg, quantitative yield) as a white solid.

LC-MS (m/z): 240.4 [M+H]+.

Stage 6

To a stirred solution of 3-chloro-4-piperazin-1-yl-benzamide (0.1 g, 0.45 mmol) in DMF (1 mL) was added 3-(4-oxo-3H-quinazoline-2 -yl)propanoic acid (0.13 g, 0.50 mmol), HATU (0.26 g, 0.68 mmol) and DIPEA (0.23 ml, 1.37 mmol) and stirred for 8 h at room temperature (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then poured into water cooled to 0°C (10 ml), the precipitated solid was collected by filtration, washed with Et2O (3x5 ml), pentane (3x5 ml). MeOH (2x5 ml), dried under high vacuum to give 3-chloro-4-[4-[3-(4-oxo-3H-quinazolin-2yl)propanoyl]piperazin-1-yl]benzamide (0.04g , 20%) as a yellowish solid.

¹ H NMR [300 MHz, DMSO-de] δ 12.22 (br. s, 1H), 8.07 (dd, J=8.1, 1.2 Hz, 1H), 7.98 (br. s, 1H), 7.93 (d, J= 1.8 Hz, 1H), 7.83-7.78 (m, 2H), 7.76 (d, J=1.2 Hz, 1H), 7.74-7.48 (m, 1H), 7.47 (br. s, 1H), 7.44 (d, J =7.2 Hz, 1H), 3.69-3.61 (m, 4H), 3.07-2.97 (m, 4H), 2.89 (br. s, 4H).

LC-MS (m/z): 440.4 [M+H]+.

Example 3 Synthesis of 3-chloro-N-methyl-4-[4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1 yl]benzamide.

Stage 1

8

To a stirred solution of 4-(4-tert-butoxycarbonylpiperazin-1-yl)-3-chloro-benzoic acid (0.5 g, 1.4 mmol) in DMF (5 ml) was added CDI (0.35 g, 2. 2 mmol), cooled to 0°C, stirred for 10 min at room temperature and added a solution of methylamine (1 M solution in THF, 1.46 ml, 1.4 mmol). This reaction mixture was warmed to room temperature, stirred for 8 h (according to TLC analysis, the starting material was completely consumed), poured into water cooled to 0°C (50 ml) and extracted with EtOAc (2x50 ml). The combined organic extracts were washed with water (50 ml), brine (2x50 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure. This crude residue was washed with Et ₂ O (3x10 ml) and pentane (3x10 ml), dried under high vacuum to give tert-butyl 4-[2-chloro-4-(methylcarbamoyl)phenyl]piperazine-1-carboxylate (0.4 g , 77%) as a light brown solid.

1H NMR [300 MHz, DMSO-d6] δ 8.44 (br., 1H), 7.88 (d, J=2.1 Hz, 1H), 7.78-7.75 (m, 1H), 7.19 (d, J=8.4 Hz, 1H ), 3.47 (t, J=4.2 Hz, 4H), 2.98 (d, J=4.8 Hz, 4H), 2.75 (d, J=4.5 Hz, 3H), 1.42 (s, 9H).

LC-MS (m/z): 354.4 [M+H]+.

Stage 2

4 N . HCl-dioxane (1.1 ml), warmed to room temperature and stirred for 5 h (according to TLC analysis, the starting material was completely consumed). Volatile components were removed under reduced pressure and the residue was washed with Et ₂ O (2x5 ml) and pentane (2x5 ml) and dried under vacuum to obtain 3-chloro-N-methyl-4-piperazin-1-yl-benzamide (300 mg, 91 %) as a white solid.

LC-MS (m/z): 254.4 [M+H]+.

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Stage 3

ABOUT

O

To a stirred solution of 3-chloro-N-methyl-4-piperazin-1-yl-benzamide (0.1 g, 0.45 mmol) in DMF (1 ml) was added 3-(4-oxo-3H- quinazolin-2-yl)propanoic acid (0.14 g, 0.50 mmol), HATU (0.26 g, 0.68 mmol) and DIPEA (0.23 ml, 1.37 mmol) and stirred for 8 h at room temperature (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was poured into ice water and was extracted with EtOAc (2x50 ml). The combined organic extracts were separated, washed with water (30 ml), brine (50 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure. This crude residue was washed with Et2O (3x5 ml), pentane (3x5 ml), 10% MeOH-DCM (3x5 ml) and dried under vacuum to give 3-chloro-Nmethyl-4-[4-[3-(4-oxo- 3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]benzamide (0.04 g, 19%) as a yellowish solid.

¹ H NMR [300 MHz, DMSO-d6] δ 12.21 (br. s, 1H), 8.45-8.44 (br., 1H), 8.07 (dd, J=7.8, 1.2 Hz, 1H), 7.89 (d, J =2.1 Hz, 1H), 7.79-7.73 (m, 2H), 7.57 (d, J=7.8 Hz, 1H), 7.47-7.42 (m, 1H), 7.18 (d, J=8.4 Hz, 1H), 3.69 -3.59 (m, 4H), 3.07-2.97 (m, 4H), 2.89 (br. s, 4H), 2.76 (d, J=4.5 Hz, 3H).

LC-MS (m/z): 454.4 [M+H]+.

Example 4 Synthesis of 3-chloro-N-cyclopropyl-4-[4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin1-yl]benzamide.

Stage 1

To a stirred solution of 3-chloro-4-fluoro-benzoic acid (1 g, 5.73 mmol) and cyclopropanamine (0.47 ml, 6.76 mmol) in dry DMF (10 ml) was added at room temperature under argon EDC -HCl (1.64 g, 8.56 mmol), HOBt (1.2 g, 8.89 mmol) and NMM (3.1 ml, 28.24 mmol) and stirred for 4 h. After completion of the reaction ( according to TLC analysis) the reaction mixture was diluted with cold water (25 ml) and stirred for 15 min. The resulting solid was collected by filtration, washed with water (50 ml) and dried under vacuum to give 3-chloro-N-cyclopropyl-4-fluoro-benzamide (0.85 g, yield: 70%) as a white solid.

LC-MS (m/z): 214.3 [M+H]+.

Stage 2

To a stirred solution of 3-chloro-N-cyclopropyl-4-fluoro-benzamide (3.1 g, 14.55 mmol) in dry DMSO (25 ml) was added piperazine (6.26 g, 72. 77 mmol) and stirred for 30 h at 120°C (according to TLC analysis, the starting material was completely consumed). After cooling to room temperature, the reaction mixture was diluted with water (20 ml) and extracted with 10% IPA-DCM (5x100 ml). The combined organic extracts were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was washed with Et ₂ O (2x20 ml) to give 3-chloro-N-cyclopropyl-4-piperazin-1-yl-benzamide (3.9 g, yield: 96%) as a white solid.

LC-MS (m/z): 280.4 [M+H]+.

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Stage 3

To a stirred solution of 3-chloro-N-cyclopropyl-4-piperazin-1-yl-benzamide (100 mg, 0.36 mmol) and 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (94 mg , 0.43 mmol) in dry DMF (2 ml) was added at room temperature under argon EDC-HCl (103 mg, 0.54 mmol), HOBt (73 mg, 0.54 mmol) and DIPEA (0.2 ml , 1.15 mmol) and stirred for 4 hours. After completion of the reaction (according to TLC analysis), the reaction mixture was quenched with cold water (20 ml) and stirred for 15 minutes. The resulting solid was collected by filtration, washed with water (50 ml) and then Et ₂ O (2x5 ml) to obtain the compound 3-chloro-N-cyclopropyl-4-[4-[3-(4-oxo-3H-quinazoline- 2yl)propanoyl]piperazin-1-yl]benzamide (80 mg, yield: 47%) as a white solid.

1H NMR [300 MHz, DMSO-de] δ 12.31 (br. s, 1H), 8.41 (br. s, 1H), 8.08 (d, J=8.1 Hz, 1H), 7.89 (s, 1H), 7.79- 7.40 (m, 2H), 7.57 (d, J=8.1Hz, 1H), 7.45 (t, J=7.8Hz, 1H), 7.17 (d, J=8.4Hz, 1H), 3.69 (s, 2H), 3.62 (s, 2H), 3.07 (s, 2H), 2.97 (s, 2H), 2.88 (s, 4H), 2.85-2.81 (m, 1H), 0.71-0.65 (m, 2H), 0.56-0.55 ( m, 2H).

LC-MS (m/z): 480.5 [M+H]+.

Example 5 Synthesis of 3-chloro-N-cyclobutyl-4-[4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin1-yl]benzamide.

Stage 1

EDCHC1, HOBt 2 en \ | NMM, DMF, k.t., 4 h'

NOgS-^^S! CIH H ₂ N Stage 1 ² 3

To a stirred solution of 3-chloro-4-fluoro-benzoic acid (500 mg, 2.865 mmol) in DMF (5 ml) was added at room temperature cyclobutanamine hydrochloride (369 mg, 3.438 mmol), EDC-HCl (820 mg, 4.297 mmol) , HOBt (580 mg, 4.297 mmol) and NMM (1.6 ml, 14.325 mmol) and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was diluted with cold water (60 ml) and was extracted with EtOAc (3x60 ml). The combined organic extracts were washed with cold water (50 ml), dried over Na ₂ SO ₄ , concentrated under reduced pressure to give 3-chloro-N-cyclobutyl-4-fluoro-benzamide (550 mg, 84%), which was used in the next step without additional cleaning.

LC-MS (m/z): 228.22 [M+H]+.

Stage 2

To a stirred solution of 3-chloro-N-cyclobutyl-4-fluoro-benzamide (550 mg, 2.422 mmol) in DMSO (5.5 ml) was added piperazine (1.04 g, 12.114 mmol) at room temperature and heated for 16 h at 120°C (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then poured into water cooled to 0°C (40 ml), the precipitated solid was collected by filtration under argon to give crude 3-chloro-C-cyclobutyl-4-piperazin-1-yl-benzamide (410 mg, 58%). This crude material was used in the next step without further purification.

LC-MS (m/z): 294.39 [M+H]+.

Stage 3

To a stirred solution of 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (100 mg,

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0.458 mmol) in DMF (2 ml) was added at room temperature 3-chloro-N-cyclobutyl-4-piperazin1-yl-benzamide (134 mg, 0.458 mmol), EDC-HCl (131 mg, 0.687 mmol), HOBt (92 mg, 0.687 mmol) and DIPEA (0.16 ml, 0.916 mmol) and stirred for 16 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then poured into ice-cold water (20 ml), the precipitated solid was collected by filtration, washed with Et2O (20 ml) and dried under vacuum to give 3-chloro-N-cyclobutyl-4-[4-[3 -(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin1-yl]benzamide (90 mg, 40%) as a white solid.

¹ H NMR [300 MHz, DMSO-d ₆ ] δ 12.2 (s, 1H), 8.60 (d, J=7.2 Hz, 1H), 8.07 (d, J=7.7 Hz, 1H), 7.92 (s, 1H) , 7.80-7.73 (m, 2H), 7.56 (d, J=8.1 Hz, 1H), 7.45 (t, J=7.5 Hz, 1H), 7.17 (d, J=8.4 Hz, 1H), 4.42-4.37 ( m, 1H), 3.65 (d, J=22.5 Hz, 4Н), 3.07 (br. s, 2H), 2.97 (br. s, 2H), 2.89 (s, 4H), 2.18 (br. s, 2H) , 2.07-1.98 (m, 2H), 1.67-1.65 (m, 2H).

LC-MS (m/z): 494.70 [M+H]+.

Example 6 Synthesis of 3-chloro-N-(1-methylcyclopropyl)-4-[4-[3-(4-oxo-3H-quinαzolin-2yl)propanoyl]piperazin-1-yl]benzamide.

Stage 1

^^, CO ₂ H \ A

O L cihh ₂ n<T ^F J? EDC HCL, HOBt' F NMM, DMF, 4 h,

k.t.

Stage 1

To a stirred solution of 3-chloro-4-fluoro-benzoic acid (50 mg, 0.29 mmol) in DMF (2 ml) was added at room temperature 1-methylcyclopropanamine hydrochloride (36 mg, 0.34 mmol), EDC-HCl ( 82 mg, 0.429 mmol), HOBt (58 mg, 0.429 mmol) and NMM (0.16 ml, 1.43 mmol) and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then diluted with water (25 ml) and the precipitated solid was collected by filtration. This solid was washed with water (20 ml) and dried under high vacuum to give 3-chloro-4-fluoro-N-(1-methylcyclopropyl)benzamide (50 mg, 78%) as a yellowish

solid.

LC-MS (m/z): 228.19 [M+H]+.

Stage 2

A stirred solution of 3-chloro-4-fluoro-N-(1-methylcyclopropyl)benzamide (500 mg, 2.21 mmol) and piperazine (951 mg, 11.06 mmol) in DMSO (5 mL) was heated for 16 h at 120°C (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was diluted

EtOAc (200 ml), washed with water (1x100 ml) and brine (1x100 ml). The organic layer was separated, dried over Na ₂ SO ₄ , the volatiles were removed under reduced pressure to obtain crude 3-chloro-N-(1-methylcyclopropyl)-4-piperazin-1-yl-benzamide (800 mg), which was used in the next step without further purification.

LC-MS (m/z): 294.35 [M+H]+.

Stage 3

O

To a stirred solution of 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (300 mg, 1.37 mmol) in DMF (5 ml) was added at room temperature 3-chloro-N'-(1-methylcyclopropyl )-4-piperazin-1yl-benzamide (604 mg, 2.06 mmol), T3P (0.87 ml, 2.75 mmol, 50% solution in DMF) and DIPEA (0.75 ml, 4.12 mmol) and stirred for 18 h at room temperature (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then diluted with EtOAc (200 ml), washed with water (1x100 ml) and brine (1x100 ml). The organic layer was separated, dried over Na ₂ SO ₄ , the volatiles were evaporated under reduced pressure to give the desired crude compound, which was purified by preparative HPLC to yield 3-chloro-N-(1-17 040674 methylcyclopropyl)-4-[4-[ 3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]benzamide (140 mg,

20%) as a white solid.

1H NMR [400 MHz, DMSO-de] δ 12.20 (br. s, 1H), 8.64 (br. s, 1H), 8.07 (d, J=8.0 Hz, 1H), 7.87 (s, 1H),

7.76-7.75 (m, 2H), 7.57 (d, J=8.0 Hz, 1H), 7.45 (t, J=8.0 Hz, 1H), 7.15 (d, J=8.4 Hz, 1H), 3.69 (br. s , 2H), 3.61 (br. s, 2H), 3.06 (br. s, 2H), 2.96 (br. s, 2H), 2.89 (s, 4H), 1.34 (s, 3H), 0.71 (br. s , 2H), 0.60 (br. s, 2H).

LC-MS (m/z): 494.50 [M+H]+.

Example 7 Synthesis of 3-chloro-N-(3-methyloxetan-3-yl)-4-[4-[3-(4-oxo-3H-quinazolin-2yl)propanoyl]piperazin-1-yl]benzamide.

Stage 1

To a stirred solution of 3-chloro-4-fluoro-benzoic acid (500 mg, 2.86 mmol) in DMF (5 ml) was added at room temperature 3-methyloxetane-3-amine hydrochloride (420 mg, 3.43 mmol),

EDC-HCl (820 mg, 4.29 mmol), HOBt (580 mg, 4.29 mmol) and NMM (1.6 ml, 14.32 mmol) and stirred for 4 h (according to TLC analysis, the starting material was completely used up). Then the reaction mixture was diluted with EtOAc (200 ml), washed with water (1x100 ml), brine (1x100 ml). The organic layer was separated, dried over Na ₂ SO ₄ , the volatiles were evaporated under reduced pressure, and the residue was purified by column chromatography (100-200 silica gel, 7 g, 70% EtOAc-hexane) to give 3-chloro-4-fluoro-A3-methyloxetane- 3-yl)benzamide (610 mg, 87%) as a cream-colored solid.

LC-MS (m/z): 244.14 [M+H] ⁺ .

Stage 2

To a stirred solution of 3-chloro-4-fluoro-N-(3-methyloxetan-3-yl)benzamide (600 mg, 2.46 mmol) in DMSO (6 ml) was added piperazine (1 g, mmol) and heated for 16 h at 120°C (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was diluted with EtOAc (200 ml), washed with water (1x100 ml), brine (1x100 ml). The organic layer was separated, dried over Na ₂ SO ₄ , the volatiles were evaporated under reduced pressure to give crude 3-chloro-N-(3-methyloxetan-3-yl)-4-piperazin-1-yl-benzamide (900 mg), which was used in the next step without further purification.

LC-MS (m/z): 310.34 [M+H] ⁺ .

Stage 3

O

To a stirred solution of 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (200 mg, 0.917 mmol) in DMF (5 ml) was added at room temperature 3-chloro-N-(3-methyloxetan-3- yl)-4piperazin-1-yl-benzamide (425 mg, 1.37 mmol), T3P (0.58 ml, 1.83 mmol, 50% solution in DMF) and DIPEA (0.5 ml, 2.75 mmol ) and stirred for 18 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was diluted with EtOAc (100 ml), washed with water (1x100 ml), brine (1x100 ml). The organic layer was separated, dried over Na ₂ SO ₄ , the volatiles were evaporated under reduced pressure to give the desired crude compound, which was purified by preparative HPLC to give 3-chloro-N-(3-methyloxetan-3-yl)-4-[4 -[3-(4oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]benzamide (100 mg, 21%) as a white solid.

1H NMR [300 MHz, DMSO-de] δ 12.30 (br. s, 1H), 8.86 (br. s, 1H), 8.08 (dd, J=8.1, 1.2 Hz, 1H), 7.92 (d, J=1.8 Hz, 1H), 7.81-7.73 (m, 2H), 7.57 (d, J=7.8 Hz, 1H), 7.47-7.42 (m, 1H), 7.19 (d, J=8.4 Hz, 1H), 4.69 (d ,

- 18 040674

J=6.0 Hz, 2H), 4.36 (d, J=6.3 Hz, 2H), 3.70 (br. s, 2H), 3.62 (br. s, 2H), 3.08 (br. s, 2H), 2.98 (br. s, 2H), s, 2H),

2.90 (s, 4H), 1.58 (s, 3H).

LC-MS (m/z): 510.69 [M+H]+.

Example 8 Synthesis of 2-[3-oxo-3-(4-phenyl-1-piperidyl)propyl]-3H-quinazolin-4-one

Stage 1

Magnesium turnings (600 mg, 25 mmol) and dry THF (5 ml) were added to an oven-dried two-necked round-bottom flask with a volume of 100 ml at room temperature in an argon atmosphere. Iodine (20 mg) was added to this mixture, the mixture was heated to 70°C with vigorous stirring, and a solution of bromobenzene (1.57 g, 10 mmol) in dry THF (5 ml) was added, maintaining the temperature at 70°C, and the reaction was continued for 1 h in an argon atmosphere. The temperature of the reaction mixture was brought to room temperature and the mixture was added dropwise to a pre-cooled (-50°C) solution of tert-butyl 4-oxo-piperidine-1-carboxylate (1 g, 5.0 mmol) in dry THF (5 ml) under an atmosphere argon. The reaction mixture was allowed to warm to room temperature, stirred for 1 h (according to TLC analysis, the starting material was completely consumed), quenched with saturated aqueous NH ₄ Cl (10 ml) and was extracted with EtOAc (2x50 ml). The combined organic extracts were washed with water (10 ml), brine (25 ml) and dried over anhydrous Na ₂ SO ₄ . Volatiles were removed under reduced pressure, the resulting crude product was taken up in 18% HCl aqueous solution (15 ml) and heated for 3 h at 100°C (according to TLC analysis, the starting material was completely consumed). The solvent was removed under reduced pressure and the residue was washed with Et ₂ O (20 ml) and EtOAc (20 ml) to give 4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (700 mg, 71%) as a hygroscopic yellowish solid .

LC-MS (m/z): 160.3 [M+H] ⁺ .

Stage 2

10% Pd/C, H ₂ , MeOH, q.t., 4 h

Stage 4

8

To a stirred solution of 4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (300 mg, 1.53 mmol) in MeOH (10 mL) was added 10% Pd-C (100 mg) at room temperature under argon. This reaction mixture was purged with H ₂ (3 times) and stirred under H ₂ atmosphere (balloon) for 4 hours. After completion of the reaction (according to LC-MS analysis), the reaction mixture was filtered through a thin pad of diatomaceous earth (Celite) and washed with MeOH ( 5 ml). This filtrate was concentrated under reduced pressure and the residue was washed with dry diethyl ether (2x5 ml) to give 4-phenylpiperidine hydrochloride (300 mg, 99%) as a hygroscopic yellowish solid

substances.

LC-MS (m/z): 162.3 [M+H] ⁺ .

Stage 3

To a stirred solution of 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (200 mg,

0.917 mmol) and 4-phenylpiperidine hydrochloride (217 mg, 1.1 mmol) in dry DMF (1.5 ml) were added at room temperature under argon EDC-HCl (264 mg, 1.375 mmol), HOBt (187 mg, 1 .38 mmol) and Na ₂ CO ₃ (292 mg, 2.75 mmol). This reaction mixture was stirred for 18 h at room temperature (according to TLC analysis, the starting material was completely consumed), quenched by adding chilled to 0°C water (15 ml) and stirred for 30 min. The precipitate was collected by filtration, the resulting solid was washed with water (10 ml) and dried under vacuum. This crude product was purified by column chromatography (100-200 silica gel, 25 g, 5% MeOH-DCM) to give 2-[3-oxo-3-(4-phenyl-1-piperidyl)propyl]-3H-quinazoline-4 - 19 040674 she (30 mg, 25%) as a yellowish solid.

Ή NMR [300 MHz, DMSO-d6] δ 12.21 (s, 1H), 8.08 (d, J=6.6 Hz, 1H), 7.79-7.73 (m, 1H), 7.57-7.52 (m, 1H), 7.48- 7.43 (m, 1H), 7.38-7.22 (m, 5H), 4.51 (d, J=12.6 Hz, 1H), 4.07 (d, J=13.8 Hz, 1H), 3.14 (t, J=12.6 Hz, 4H ), 2.94-2.81 (m, 3H), 2.64-2.57 (m, 1H), 1.84-1.73 (m, 1H), 1.66-1.62 (m, 1H), 1.42-1.33 (m, 1H).

LC-MS (m/z): 362.5 [M+H]+.

Example 9 Synthesis of 2-[3-oxo-3-(4-phenylpiperazin-1-yl)propyl]-3H-quinazolin-4-one

Stage 1

To a stirred solution of 2-aminobenzamide (5 g, 36.76 mmol) in AcOH (acetic acid) (10 ml) was added at room temperature a solution of succinic anhydride (3.67 g, 36.76 mmol) in AcOH (10 ml). This reaction mixture was stirred for 2 h at room temperature (according to TLC analysis, the starting material was completely consumed), diluted with cold water (100 ml) and stirred for 15 min. The precipitate was collected by filtration, washed with cold water (30 ml) and dried under vacuum to give 4-(2-carbamoylanilino)-4-oxo-butanoic acid (8 g, 92%) as a white solid.

LC-MS (m/z): 237.4 [M+H] ⁺ .

Stage 2

A stirred suspension of 4-(2-carbamoylanilino)-4-oxo-butanoic acid (8 g, 33.86 mmol) and NaOAc (2.78 g, 33.86 mmol) in Ac ₂ O (10 mL) was heated for 1 h at 120°C (according to TLC analysis, the starting material was completely consumed). The temperature of the reaction mixture was brought to room temperature, the mixture was quenched with water (100 ml) and slowly added 1N. NaOH solution to pH 10. The resulting mixture was washed with EtOAc (30 ml), the aqueous layer was separated and acidified to pH 5 by adding AcOH, stirred for 1 h and filtered. The solid was washed with hexanes (3x20 ml) and dried under vacuum to give 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (5.0 g, 68%) which was used in the next step without further purification.

LC-MS (m/z): 219.3 [M+H] ⁺ .

Stage 3

To the stirred solution

3-(4-oxo-3H-quinazolin-2-ul) propanoic acid (100 mg,

0.458 mmol) and 1-phenylpiperazine (90 mg, 0.55 mmol) in dry DMF (1.5 ml) were added at room temperature under argon EDC-HCl (132 mg, 0.687 mmol), HOBt (93 mg, 0.688 mmol ) and Na ₂ CO ₃ (146 mg, 1.37 mmol). The reaction mixture was stirred for 18 h at room temperature under argon (the starting material was completely consumed according to TLC analysis), quenched by adding water (15 ml) cooled to 0°C, and stirred for 30 min. The precipitate was collected by filtration, washed with water (10 ml) and dried under vacuum. The resulting crude product was purified by column chromatography (100-200 silica gel, 50 g, 5% MeOH-DCM) to yield 2-[3-oxo-3-(4-phenylpiperazin-1-yl)propyl]-3H-quinazoline-4 -one (33 mg, 20%) as a yellowish solid.

1H NMR [300 MHz, DMSO^] δ 12.21 (s, 1H), 8.07 (dd, J=1.2, 8.1 Hz, 1H), 7.77-7.71 (m, 1H), 7.54 (d, J=7.8 Hz, 1H ), 7.47-7.42 (m, 1H), 7.23 (t, J=7.5 Hz, 2H), 6.92 (d, J=8.1 Hz, 2 H), 6.81 (t, J=7.2 Hz, 1H), 3.67- 3.57 (m, 4H), 3.20-3.06 (m, 4H), 2.89 (s, 4H).

LC-MS (m/z): 363.5 [M+H] ⁺ .

- 20 040674

Example 10 Synthesis of 2-[3-[4-(2-chlorophenyl)piperazin-1-yl]-3-oxo-propyl]-3H-quinazolin-4-one

Stage 1

Xantphos (34 mg, 0.0590 mmol), Pd ₂ (dba) ₃ (24 mg, 0.0262 mmol) and Cs ₂ CO ₃ (261 mg, 0.80 mmol). The resulting mixture was heated for 30 min at 100°C in a microwave oven (CEM) (according to TLC analysis, the starting material was completely consumed). The solvent was evaporated under reduced pressure, the residue was diluted with water (20 ml) and was extracted with EtOAc (3x20 ml). The combined organic extracts were washed with brine (20 ml), dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo. The residue was purified by column chromatography (100-200 silica gel, 10 g, 5% EtOAc-hexane) to give tert-butyl 4-(2-chlorophenyl)piperazine-1-carboxylate (100 mg, 63%) as a yellowish solid.

¹ H NMR [300 MHz, CDCy δ 7.37 (dd, J=7.8, 1.2 Hz, 1H), 7.22 (td, J=7.8, 1.2 Hz, 1H), 7.02-6.96 (m, 2H), 3.60 (t, J=5.1 Hz, 4H), 2.99 (t, J=5.1 Hz, 4H), 1.48 (s, 9H).

Stage 2

To a stirred solution of tert-butyl 4-(2-chlorophenyl)piperazine-1-carboxylate (250 mg, 0.844 mmol) in 1,4-dioxane (2 ml) was added dropwise at 0°C 4N. HCl in 1,4-dioxane (0.9 ml, 3.60 mmol). This reaction mixture was allowed to warm to room temperature and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). Volatiles were removed under reduced pressure to give the crude compound, which was washed with diethyl ether (2x20 ml) and dried under high vacuum to give 1-(2chlorophenyl)piperazine hydrochloride (165 mg, 84%) as a white solid.

Stage 3

To a stirred solution of 1-(2-chlorophenyl)piperazine hydrochloride (119 mg, 0.510 mmol) and 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (111 mg, 0.509 mmol) in dry DMF (2 ml ) was added at room temperature EDC-HCl (98 mg, 0.511 mmol), HOBt (69 mg, 0.510 mmol) and DIPEA (0.18 ml, 1.03 mmol) and stirred for 3 h (according to TLC analysis of starting material was completely used up). The reaction mixture was then quenched with cold water (20 ml) and stirred for 15 minutes. The precipitate formed was collected by filtration and the solid was washed with Et2O (2x5 ml) to give 2-[3-[4-(2-chlorophenyl)piperazin-1-yl]-3-oxo-propyl]-3H-quinazolin-4-one (90 mg, 44%) as a white solid.

1H NMR [300 MHz, DMSO-d ₆ ] δ 12.20 (s, 1H), 8.08 (dd, J=7.8, 1.2 Hz, 1H), 7.76-7.73 (m, 1H), 7.57 (d, J=8.1 Hz , 1H), 7.48-7.41 (m, 2H), 7.31-7.28 (m, 1H), 7.15-7.04 (m, 2H), 3.68 (s, 2H), 3.61 (s, 2H), 3.00 (s, 4H ), 2.89 (s, 4H).

LC-MS (m/z): 397.30 [M+H]+.

Example 11 Synthesis of 6-[4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]pyridine-3carbonitrile.

Stage 1

N-Boc-piperazine,

K ₂ CO ₃ , DMA, 60°C, II J

J T 6h__________ p Ν N

_{Cl_l} BocN^^

Stage 1

2

K2CO3 (3.4 g, 24.63 mmol), N -Boc-piperazine (2.7 g, 14.50 mmol) and stirred for 3 h. The reaction mixture was then heated for 3 h at

60°C (according to TLC analysis, the starting material was completely consumed) and poured into ice water (100 ml), the precipitated solid was collected by filtration, washed

Et ₂ O (3x10 ml), pentane (3x10 ml) and dried to give tert-butyl 4-(5-cyano-2-pyridyl)piperazine 1-carboxylate (3 g, 73%) which was used without further purification.

LC-MS (m/z): 289.3 [M+H]+.

Stage 2

To a stirred solution of tert-butyl 4-(5-cyano-2-pyridyl)piperazine-1-carboxylate (0.5 g, 1.736 mmol) in DCM was added dropwise 4 N sodium chloride. HCl in dioxane (0.5 ml) and stirred for 4 h at room temperature under argon (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was concentrated under reduced pressure, the residue was washed with Et ₂ O (2x5 ml), DCM (2x5 ml), pentane (2x5 ml) and dried under vacuum to obtain 6-piperazin-1-ylpyridine-3-carbonitrile hydrochloride (0. 3 g, yield: 93%), which was used in the next step without further purification.

1H NMR [300 MHz, DMSOCH] δ 9.58 (br. s, 2H), 8.54 (d, J=2.8 Hz, 1H), 7.91-7.33 (dd, J=3.2, 12.4 Hz, 1H), 7.02 (d, J=12.0 Hz, 1H), 3.91 (t, J=6.8 Hz, 4H), 3.14 (br. s, 4H).

Stage 3

ABOUT

To a stirred solution of 6-piperazin-1-ylpyridin-3-carbonitrile hydrochloride (0.2 g, 0.974 mmol) and 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (0.2 g, 0.913 mmol ) in DMF (2 ml) were added at room temperature EDC-HCl (0.35 g, 1.83 mmol), HOBt (0.24 g, 1.83 mmol) and DIPEA (0.8 ml, 4.59 mmol ) and stirred for 8 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then poured into water cooled to 0°C (30 ml) and stirred for 15 min, the precipitated solid was collected by filtration and purified by column chromatography (100-200 silica gel, 10 g, 5% MeOH in DCM) to give 6-[4-[3-(4-oxo-3H-quinazolin-2-ul)propanoyl]piperazin-1-yl]pyridine-3-carbonitrile (0.025 g, 20%) as white solid.

¹ H NMR [300 MHz, DMSO-d6] δ 12.02 (s, 1H), 8.51 (d, J=3.2 Hz, 1H), 8.08-8.05 (dd, J=1.6, 10.4 Hz, 1H), 7.90-7.86 (dd, J=3.2, 12.0 Hz, 1H), 7.77-7.71 (m, 1H), 7.54 (d, J=10.4 Hz, 1H), 7.47-7.41 (m, 1H), 6.94 (d, J=12.0 Hz, 1H), 3.77-3.75 (m, 2H), 3.64 (d, J=4.8 Hz, 4H), 3.57-3.56 (m, 2H), 2.89 (s, 4H).

LC-MS (m/z): 389.61 [M+H] ⁺ .

Example 12 Synthesis of 6-[4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]pyridine-3 carboxamide.

To a stirred solution of 6-[4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile (100 mg, 0.257 mmol) in toluene (1 mL) was added at room temperature under argon H2SO4 (127 mg, 1.285 mmol) and heated for 5 h at 80°C (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was concentrated under reduced pressure and subjected to distillation with toluene (3x5 ml), basified to pH 9 by adding 1N. NaOH solution, extracted using 10% MeOH-DCM (3x10 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure, the residue was purified by column chromatography (100-200 silica gel, 15 g, 10% MeOH in DCM) to obtain 6-[4-[3-(4-oxo3H-quinazolin-2-ul)propanoyl]piperazin-1-yl]pyridine-3-carboxamide (0.025 g, 20%) as white

- 22 040674 solids.

Ή NMR [300 MHz, DMSO-d6] δ 12.02 (s, 1H), 8.63 (d, J=3.2 Hz, 1H), 8.08-8.05 (dd, J=1.6,10.4 Hz, 1H), 8.00-7.96 ( dd, J=3.2,12.0 Hz, 1H), 7.79-7.71 (m, 2H), 7.54 (d, J=10.8 Hz, 1H), 7.44 (t, J=10.4 Hz, 1H), 7.17 (s, 1H ), 6.86 (d, J=12 Hz, 1H), 3.69-3.64 (m, 4H), 3.56 (s, 4H), 2.89 (s, 4H).

LC-MS (m/z): 407.5 [M+H]+.

Example 13 Synthesis of 6-[4-[3-(5-methyl-4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile.

Stage 1

To a stirred solution of 2-amino-6-methyl-benzoic acid (0.5 g, 3.31 mmol) in DMF (5 ml) at room temperature was added CDI (0.53 g, 3.31 mmol). This reaction mixture was heated for 2 h at 80°C, then an aqueous solution of ammonia (25%, 10 ml) was carefully added to the reaction mixture, maintaining the temperature at 80°C, and the reaction was continued for 4 h (according to TLC analysis, the initial substance was completely consumed). The temperature of the reaction mixture was slowly brought to room temperature, the mixture was diluted with water (30 ml) and was extracted with EtOAc (3x100 ml). The combined organic extracts were washed with water (2x50 ml), brine (40 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure, the resulting crude residue was washed with Et ₂ O (10 ml) and dried under high vacuum to obtain 2- amino-6-methylbenzamide (200 mg, 40%) as a white solid.

LC-MS (ESI+): m/z: 151.09 [M+H] ⁺ .

Stage 2

To a stirred solution of 2-amino-6-methyl-benzamide (0.2 g, 1.33 mmol) in AcOH (3 ml) was added succinic anhydride at room temperature and stirred for 4 h (according to TLC analysis, the starting material was completely used up). Then the reaction mixture was poured into water cooled to 0°C (5 ml) and stirred for 30 min, while a solid precipitated. This solid was collected by filtration, washed with water (20 ml), cold acetone (5 ml) and dried under high vacuum to give 4-(2-carbamoyl-3-methylanilino)-4-oxo-butanoic acid (250 mg, 75%) as a white solid.

LC-MS (ESI+) (m/z): 251.50 [M+H] ⁺ .

Stage 3

ABOUT

4-(2-Carbamoyl-3-methyl-anilino)-4-oxo-butanoic acid (0.25 g, 1.0 mmol) was added in 2N. aqueous solution of NaOH (5 ml) and stirred for 2 h at 100°C (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was slowly cooled to 0°C and acidified to pH 3-4 with 2N. aqueous HCl solution, whereupon a white solid precipitated. This suspension was stirred for 30 min at 0°C, filtered, washed with water (20 ml), cold acetone (2 ml) and dried under high vacuum to give 3-(5-methyl-4-oxo-3Hquinazoline-2- yl) propanoic acid (150 mg, 64%) as a yellowish solid.

LC-MS (ESI+) (m/z): 233.49 [M+H] ⁺ .

Stage 4

- 23 040674

To a stirred solution of 3-(5-methyl-4-oxo-3H-quinazolin-2-yl)propanoic acid (150 mg, 0.65 mmol) and 6-piperazin-1-ylpyridine-3-carbonitrile hydrochloride (145 mg, 0.77 mmol) in DMF (2 ml) was added at room temperature DIPEA (0.3 ml, 1.94 mmol) and T3P (50% solution in EtOAc, 0.4 ml, 1.29 mmol) and stirred for 8 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then quenched with water (10 ml) and extracted with EtOAc (3x40 ml). The combined organic extracts were washed with cold water (3x30 ml), brine (20 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure, the resulting crude residue was purified by column chromatography (100-200 silica gel, 10 g, 5% MeOH- DCM) yielding 6-[4-[3-(5-methyl-4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]pyridine-3-carbonitrile (60 mg, 27%) in form of a white solid.

¹ H NMR [300 MHz, DMSO-d ₆ ] δ 11.98 (br. s, 1H), 8.51 (d, J=2.1 Hz, 1H), 7.88 (dd, J=9.0, 2.1 Hz 1H), 7.55 (t , J=7.8 Hz, 1H), 7.34 (d, J=7.8 Hz, 1H), 7.16 (d, J=7.2 Hz, 1H), 6.93 (d, J=9.0 Hz, 1H), 3.77-3.76 (m , 2H), 3.64-3.62 (m, 4H), 3.57-3.55 (m, 2H), 2.88-2.83 (m, 4H), 2.75 (s, 3H).

LC-MS (ESI+) (m/z): 403.66 [M+H] ⁺ .

Example 14 Synthesis of 6-[4-[3-(6-methyl-4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile

Stage 1

To a stirred solution of 2-amino-5-methyl-benzoic acid (100 mg, 0.66 mmol) in THF (2 ml) was added at room temperature EDC-HCl (189 mg, 0.99 mmol), HOBt-NH ₃ ( 149 mg, 0.99 mmol) and DIPEA (0.35 ml, 1.99 mmol) and stirred for 1 h (TLC analysis indicated complete conversion of the starting material). The reaction mixture was then diluted with EtOAc (1x50 ml), washed with water (1x20 ml) and brine (1x20 ml). The organic layer was separated, dried over Na ₂ SO ₄ , the volatiles were evaporated under reduced pressure, the resulting crude residue was purified by chromatography on Combiflash R _f 200 Teledyne ISCO (100% EtOAc, 12 g cartridge) to yield 2-amino-5-methyl- benzamide (70 mg, 70%) as a white solid.

LC-MS (ESI+) (m/z): 151.13 [M+H] ⁺ .

Stage 2

To a stirred solution of 2-amino-5-methyl-benzamide (600 mg, 4.0 mmol) in AcOH (6 ml) was added succinic anhydride (480 mg, 4.80 mmol) at room temperature and stirred for 2 h (TLC analysis indicated complete conversion of the starting material). The reaction mixture was then diluted with water (1 x 50 ml) cooled to 0°C, stirred for 30 min, the precipitated solid was collected by filtration, washed with water (1 x 50 ml) and then with cold acetone (1 x 20 ml) and dried under conditions high vacuum to give 4-(2-carbamoyl-4-methylanilino)-4-oxo-butanoic acid (800 mg, 80%) as a white solid.

LC-MS (ESI+) (m/z): 273.56 [M+Na]+.

Stage 3

ABOUT

A stirred solution of 4-(2-carbamoyl-4-methyl-anilino)-4-oxo-butanoic acid (480 mg, 2.07 mmol) in 2N an aqueous solution of NaOH (15 ml) was heated for 4 h at 100°C (TLC analysis indicated complete conversion of compound 4). Then the reaction mixture was cooled to 0°C, acidified to pH 5 by adding AcOH, while a solid precipitated. This solid was collected by filtration, washed with water (1x80 ml) and then with cold acetone (1x20 ml) and dried under high vacuum to give 3-(6-methyl-4-oxo-3H-quinazolin-2-yl)propanoic acid (380 mg, 85%) as a yellowish solid.

LC-MS (ESI+) (m/z): 233.45 [M+H] ⁺ .

- 24 040674

Stage 4

To a stirred solution of 3-(6-methyl-4-oxo-3H-quinazolin-2-yl)propanoic acid (250 mg, 1.077 mmol) in DMF (5 mL) was added 6-piperazin-1-ylpyridine-3carbonitrile at room temperature hydrochloride (289 mg, 1.293 mmol), T3P (50% in DMF, 0.68 ml, 2.15 mmol) and DIPEA (0.57 ml, 3.23 mmol) and stirred for 6 h (according to TLC analysis the starting material was completely consumed). The reaction mixture was then diluted with water (1x80 ml), stirred for 5 minutes, the precipitated solid was collected by filtration. This solid was washed with water (1x70 ml) and dried under high vacuum to give 6-[4-[3(6-methyl-4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]pyridine -3-carbonitrile (170 mg, 39%) as a yellowish solid.

¹ H NMR [300 MHz, DMSO-de] δ 12.10 (br. s, 1H), 8.51 (s, 1H), 7.90-7.87 (m, 2H), 7.56 (d, J=8.1 Hz, 1H), 7.44 (d, J=8.4 Hz, 1H), 6.94 (d, J=9.3 Hz, 1H), 3.79-3.72 (m, 2H), 3.68-3.62 (m, 4H), 3.59-3.54 (m, 2H), 2.87 (s, 4H), 2.41 (s, 3H).

LC-MS (ESI+) (m/z): 403.66 [M+H] ⁺ .

Example 15 Synthesis of yl]pyridine-3-carbonitrile

Stage 1

6-[4-[3-(7-methyl-4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1EDCHC1

HOBtNH ₃ ,

DIPEA, THF,

ABOUT

Stage 1

To a stirred solution of 2-amino-4-methyl-benzoic acid (300 mg, 1.99 mmol) in THF (6 ml) was added at room temperature EDC-HCl (569 mg, 2.98 mmol), HOBt-NH ₃ ( 447 mg, 2.98 mmol) and DIPEA (1.06 ml, 5.96 mmol) and stirred for 1 h (the starting material was completely consumed according to TLC analysis). Then the reaction mixture was diluted with water (30 ml), was extracted with EtOAc (3x25 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure to give the crude desired compound, which was purified by flash column chromatography (100-200 silica gel, 5 g, 50% EtOAc-hexane) to yield 2-amino-4 -methyl-benzamide (190 mg, 64%) as a white solid.

¹ H NMR [300 MHz, DMSO-de] δ 7.62 (br. s, 1H), 7.42 (d, J=8.1 Hz, 1H), 6.93 (br. s, 1H), 6.54-6.46 (m, 3H) , 6.30–6.27 (m, 1H), 2.15 (s, 3H).

Stage 2

To a stirred solution of 2-amino-4-methyl-benzamide (190 mg, 1.27 mmol) in AcOH (5 ml) was added succinic anhydride (151 mg, 1.52 mmol) at room temperature and stirred for 2 h (according to TLC analysis of the starting material was completely consumed). The reaction mixture was then diluted with water (20 ml), the precipitated solid was collected by filtration and dried under vacuum to give the desired 4-(2-carbamoyl-5-methyl-anilino)-4-oxo-butanoic acid (250 mg, 89 %), which was used in the next step without further purification.

1H NMR [300 MHz, DMSO-de] δ 12.37 (br. s, 1H), 11.88 (s, 1H), 8.33 (s, 1H), 8.20 (s, 1H), 7.70 (d, J=8.1 Hz, 1H), 7.64 (s, 1H), 6.91 (d, J=7.8 Hz, 1H), 2.53-2.50 (m, 4H), 2.31 (s, 3H).

LC-MS (m/z): 273.50 [M+Na]+.

- 25 040674

Stage 3

Q

HE

4-(2-Carbamoyl-5-methyl-anilino)-4-oxo-butanoic acid (250 mg, 1 mmol) was added in 2N. NaOH solution (10 ml) and subjected to reflux for 3 h (according to TLC analysis, the starting material was completely consumed), cooled to 0°C and acidified to pH 4 by adding AcOH, while a solid precipitated. This solid was collected by filtration and dried under vacuum to give 3-(7-methyl-4-oxo-3H-quinazolin-2-yl)propanoic acid (220 mg, 95%) which was used in the next step without further purification.

LC-MS (m/z): 233.45 [M+H] ⁺ .

Stage 4

To a stirred solution of 3-(7-methyl-4-oxo-3H-quinazolin-2-yl)propanoic acid (150 mg, 0.65 mmol) and 6-piperazin-1-ylpyridin-3-carbonitrile hydrochloride (173 mg, 0.78 mmol) in DMF (4 ml) were added at room temperature EDC-HCl (185 mg, 0.97 mmol), HOBt (130 mg, 0.97 mmol) and DIPEA (0.46 ml, 2.58 mmol ) and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then poured into cold water (25 ml), stirred for 10 min, whereupon a solid precipitated. This solid was collected by filtration, dried under vacuum, washed with Et ₂ O (20 ml) and hexane (20 ml) to give 6-[4-[3-(7-methyl-4-oxo-3H-quinazoline-2- yl)propanoyl]piperazin-1-yl]pyridine-3-carbonitrile (75 mg, 29%) as a yellowish solid.

1H NMR [300 MHz, DMSO-dd δ 12.1 (s, 1H), 8.52 (s, 1H), 7.96-7.87 (m, 2H), 7.33 (s, 1H), 7.26 (d, J=8.1 Hz, 1H ), 6.94 (d, J=9.3 Hz, 1H), 3.78 (br. s, 2H), 3.65 (br. s, 4H), 3.57 (br. s, 2H), 2.87 (br. s, 4H), 2.39(s, 3H).

LC-MS (m/z): 403.69 [M+H] ⁺ .

Example 16 Synthesis of 6-[4-[3-(8-methyl-4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile

Stage 1

To a stirred solution of 2-amino-3-methyl-benzoic acid (0.5 g, 3.31 mmol) in THF (15 ml) was added at room temperature EDC-HCl (0.948 g, 4.97 mmol), HOBt-NH ₃ (0.745 g, 4.97 mmol) and DIPEA (1.76 ml, 9.93 mmol) and stirred for 6 h (TLC analysis indicated complete conversion of the starting material). Then the reaction mixture was diluted with water (30 ml) and was extracted with EtOAc (3x100 ml). The combined organic extracts were washed with water (2x50 ml), brine (40 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude residue. This crude material was purified by column chromatography (100-200 silica gel, 20 g, 50% EtOAc-hexane) to give 2-amino-3-methyl-benzamide (0.3 g, 60%) as a white solid.

LC-MS (m/z): 151.09 [M+H] ⁺ .

Stage 2

ABOUT

To a stirred solution of 2-amino-3-methyl-benzamide (0.3 g, 2.0 mmol) in AcOH (3 ml) was added succinic anhydride at room temperature and stirred for 4 h (according to TLC analysis, the starting material was completely used up). The reaction mixture was then poured into

- 26 040674 water cooled to 0°C (10 ml) and stirred for 30 min, while a solid precipitated. This solid was collected by filtration, washed with water (20 ml), cold acetone (5 ml) and dried under high vacuum to give 4-(2-carbamoyl-6-methylanilino)-4-oxo-butanoic acid (280 mg, 56%) as a white solid.

LC-MS (m/z): 251.48 [M+H]+.

Stage 3

4-(2-Carbamoyl-6-methyl-anilino)-4-oxo-butanoic acid (0.28 g, 1.12 mmol) in 2N. an aqueous solution of NaOH (5 ml) was stirred for 2 h at 100°C (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was cooled to 0°C and acidified to pH 3-4 with 2N. aqueous HCl solution, whereupon a white solid precipitated. This suspension was stirred for 30 min at 0°C, filtered, washed with water (20 ml), cold acetone (5 ml) and dried under high vacuum to give 3-(8-methyl-4-oxo-3H-quinazoline- 2yl) propanoic acid (180 mg, 69%) as a yellowish solid.

LC-MS (m/z): 233.45 [M+H] ⁺ .

Stage 4

ТЗР, DIPEA, CN DMF, k.t., 3 h

Stage 4

6

To a stirred solution of 3-(8-methyl-4-oxo-3H-quinazolin-2-yl)propanoic acid (100 mg, 0.43 mmol) and 6-piperazin-1-ylpyridine-3-carbonitrile hydrochloride (97 mg, 0.52 mmol) in DMF (3 ml) was added at room temperature DIPEA (0.23 ml, 1.29 mmol) and T3P (0.27 ml, 0.86 mmol) and stirred for 3 h (according to TLC- analysis, the starting material was completely consumed).

The reaction mixture was then quenched with water (10 ml) and extracted with EtOAc (3x60 ml). The combined organic extracts were washed with cold water (3x30 ml), brine (20 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude residue. This crude material was recrystallized from acetonitrile (5 ml) to give 6-[4-[3-(8methyl-4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]pyridine-3-carbonitrile (60 mg, 34%) as a white solid.

1H NMR [300 MHz, DMSO-d^ δ 12.20 (br. s, 1H), 8.51 (d, J=2.1 Hz, 1H), 7.92-7.86 (m, 2H), 7.60 (d, J=6.9 Hz, 1H), 7.31 (t, J=7.5 Hz, 1H), 6.93 (d, J=9.0 Hz, 1H), 3.77-3.75 (m, 2H), 3.66-3.65 (m, 4H), 3.59-3.57 (m , 2H), 2.90 (s, 4H), 2.46 (s, 3H).

LC-MS (m/z): 403.68 [M+H] ⁺ .

Example 17 Synthesis of 6-[4-[3-(5-fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]pyridine3-carbonitrile

To a stirred solution of 3-(5-fluoro-4-oxo-3H-quinazolin-2-yl)propanoic acid (250 mg, 1.054 mmol) in DMF (5 ml) was added 6-piperazin-1-ylpyridine-3carbonitrile at room temperature hydrochloride (283 mg, 1.265 mmol), EDC-HCl (302 mg, 1.582 mmol), HOBt (213 mg, 1.582 mmol) and DIPEA (0.56 ml, 3.164 mmol) and stirred for 7 h (according to TLC analysis the starting material was completely consumed). The reaction mixture was then diluted with water (1x80 ml), stirred for 5 min at room temperature, the precipitated solid was collected by filtration, washed with water (1x70 ml), dried under high vacuum to give 6-[4-[ 3-(5-fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile (120 mg, 23%) as a pale yellow solid.

1H NMR [300 MHz, DMSO-d ₆ ] δ 12.20 (br. s, 1H), 8.51 (d, J=2.1 Hz, 1H), 7.88 (dd, J=9.3, 2.4 Hz, 1H), 7.73-7.67 (m, 1H), 7.35 (d, J=8.1 Hz, 1H) 7.21-7.15 (m, 1H), 6.94 (d, J=9.3 Hz, 1H), 3.81-3.74 (m, 2H), 3.68-3.62 (m, 4H), 3.58-3.53 (m, 2H), 2.87 (s, 4H).

LC-MS (m/z): 407.60 [M+H] ⁺ .

- 27 040674

Example 18 Synthesis of 6-[4-[3-(6-fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]pyridine3-carbonitrile

To a stirred solution of 3-(6-fluoro-4-oxo-3H-quinazolin-2-yl)propanoic acid (100 mg, 0.42 mmol) and 6-piperazin-1-ylpyridine-3-carbonitrile hydrochloride (95 mg, 0.51 mmol) in DMF (2 ml) was added at room temperature DIPEA (0.2 ml, 1.27 mmol) and T3P (0.3 ml, 0.85 mmol) and stirred for 4 h (according to TLC- analysis, the starting material was completely consumed).

The reaction mixture was then quenched with water (10 ml) and extracted into EtOAc (3x40 ml). The combined organic extracts were washed with cold water (3x30 ml), brine (20 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude residue. This crude material was purified by column chromatography (100-200 silica gel, 10 g, 5% MeOH-DCM) to yield 6-[4-[3-(6-fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl ]piperazin-1-yl]pyridine3-carbonitrile (60 mg, 35%) as a white solid.

1H NMR [400 MHz, DMSO-de] δ 12.33 (br. s, 1H), 8.51 (d, J=2.1 Hz, 1H), 7.88 (dd, J=9.0, 2.1 Hz, 1H), 7.74 (d, J=8.1 Hz, 1H), 7.64-7.62 (m, 2H), 6.93 (d, J=9.3 Hz, 1H), 3.77-3.56 (m, 8H), 2.88 (s, 4H).

LC-MS (m/z): 407.61 [M+1]+.

Example 19 Synthesis of 6-[4-[3-(7-fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]pyridine3-carbonitrile

Stage 1

EDCHC1 _ HOBtNH ₃ , © p DIPEA, THF, A

Stage 1

To a stirred solution of 2-amino-4-fluoro-benzoic acid (300 mg, 1.93 mmol) in THF (6 ml) was added at room temperature EDC-HCl (553 mg, 2.90 mmol), HOBt-NH ₃ ( 435 mg, 2.90 mmol), DIPEA (1.03 ml, 5.79 mmol) and stirred for 1 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was diluted with water (25 ml) and was extracted with EtOAc (3x20 ml). The combined organic extracts were dried over Na2SO4, concentrated under reduced pressure, the resulting crude residue was purified by flash column chromatography (silica gel 100-200 mesh, 5 g, 50% EtOAc-hexane) to yield 2-amino-4-fluorobenzamide (195 mg, 65%) as a white solid.

1H NMR [300 MHz, DMSO-d ₆ ] δ 7.71 (br. s, 1H), 7.61–7.56 (m, 1H), 7.07 (br. s, 1H), 6.89 (s, 2H), 6.42 (dd, J=2.7, 12.0 Hz, 1H), 6.30-6.23 (m, 1H).

Stage 2

To a stirred solution of 2-amino-4-fluoro-benzamide (195 mg, 1.26 mmol) in AcOH (4 ml) was added succinic anhydride (151 mg, 1.51 mmol) at room temperature and stirred for 2 h (according to TLC analysis of the starting material was completely consumed). The reaction mixture was then diluted with water (20 ml) whereby a solid precipitated. This solid was collected by filtration, dried under vacuum to give 4-(2-carbamoyl-5-fluoro-anilino)-4-oxo-butanoic acid (260 mg, 81%), which was used in the next step without further purification.

1H NMR [300 MHz, DMSO-d ₆ ] δ 12.10 (s, 1H), 8.40–8.26 (m, 3H), 7.92–7.87 (m, 1H), 7.79 (br. s, 1H), 7.00–6.93 ( m, 1H), 2.56-2.49 (m, 4H).

- 28 040674

Stage 3

A stirred solution of 4-(2-carbamoyl-5-fluoro-anilino)-4-oxo-butanoic acid (250 mg, 0.98 mmol) in 2 N NaOH (10 ml) was subjected to reflux for 3 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was cooled to 0°C, acidified to pH 4 by adding AcOH, while a solid precipitated. This solid was collected by filtration and dried under vacuum to give 3-(7-fluoro-4-oxo-3H-quinazolin-2yl)propanoic acid (210 mg, 90%) which was used in the next step without further purification.

LC-MS (m/z): 237.43 [M+H]+.

Stage 4

To a stirred solution of 3-(7-fluoro-4-oxo-3H-quinazolin-2-yl)propanoic acid (150 mg, 0.63 mmol) and 6-piperazin-1-ylpyridine-3-carbonitrile hydrochloride (174 mg, 0.76 mmol) in DMF (4 ml) were added at room temperature EDC-HCl (181 mg, 0.95 mmol), HOBt (130 mg, 0.95 mmol) and DIPEA (0.45 ml, 2.54 mmol ) and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then poured into cold water (25 ml) whereby a solid precipitated. This solid was collected by filtration, dried under vacuum and washed with Et ₂ O (20 ml), hexane (20 ml), pentane (20 ml), EtOAc (15 ml) and again dried under vacuum to obtain 6-[4-[ 3-(7-Fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile (110 mg, 43%) as a yellowish solid.

1H NMR [300 MHz, DMSO-d ₆ ] δ 12.30 (s, 1H), 8.51 (d, J=2.1 Hz, 1H), 8.15-8.10 (m, 1H), 7.88 (dd, J=2.4, 9.6 Hz , 1H), 7.33-7.27 (m, 2H), 6.94 (d, J=9.0 Hz, 1H), 3.80-3.73 (m, 2H), 3.69-3.61 (m, 4H), 3.59-3.53 (m, 2H ), 2.89 (s, 4H).

LC-MS (m/z): 407.61 [M+H] ⁺ .

Example 20 Synthesis of 6-[4-[3-(8-fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]pyridine3-carbonitrile

Stage 1

EDCHC1

HOBt NH ₃ DIPEA ?

Stage 1 W<NH ₂ F 2

To a stirred solution of 2-amino-3-fluoro-benzoic acid (400 mg, 2.57 mmol) in THF (10 ml) was added at room temperature EDC-HCl (738 mg, 3.86 mmol), HOBt-NH ₃ ( 580 mg, 3.86 mmol) and DIPEA (1.38 ml, 7.73 mmol) and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was diluted with water (30 ml) and was extracted with EtOAc (3x40 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure to give the crude desired compound, which was purified by flash column chromatography (silica gel 100-200 mesh, 5 g, 50% EtOAc-hexane) to yield 2-amino- 3-fluoro-benzamide (250 mg, 63%) as a white solid.

LC-MS (m/z): 155.42 [M+H] ⁺ .

Stage 2

ABOUT

To a stirred solution of 2-amino-3-fluoro-benzamide (250 mg, 1.62 mmol) in AcOH (5 mL) to

- 29 040674 succinic anhydride (389 mg, 3.89 mmol) was added at room temperature and stirred for 1 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then diluted with water (20 ml) whereby a solid precipitated. This solid was collected by filtration and dried under vacuum to give 4-(2-carbamoyl-6-fluoro-anilino)-4-oxo-butanoic acid (400 mg, 97%) which was used in the next step without further purification.

LC-MS (m/z): 255.42 [M+H]+.

Stage 3

HE

4-(2-Carbamoyl-6-fluoro-anilino)-4-oxo-butanoic acid (400 mg, 1.57 mmol) was added in 2N. NaOH solution (10 ml) and heated for 3 h at 100°C (according to TLC analysis, the starting material was completely consumed). After completion of the reaction, the reaction mixture was cooled to 0°C, acidified to pH 4 by adding AcOH, while a solid precipitated. This solid was collected by filtration and dried under vacuum to give 3-(8-Fluoro-4-oxo-3Hquinazolin-2-yl)propanoic acid (250 mg, 67%), which was used in the next step without further purification.

LC-MS (m/z): 237.39 [M+H]+.

Stage 4

To a stirred solution of 3-(8-fluoro-4-oxo-3H-quinazolin-2-yl)propanoic acid (150 mg, 0.63 mmol) and 6-piperazin-1-ylpyridine-3-carbonitrile hydrochloride (170 mg, 0.76 mmol) in DMF (4 ml) was added at room temperature T3P (0.3 ml, 0.95 mmol) and DIPEA (0.45 ml, 2.54 mmol) and stirred for 4 h (according to TLC- analysis, the starting material was completely consumed). The reaction mixture was then poured into cold water (25 ml) whereby a solid precipitated. This solid was collected by filtration, dried under vacuum, washed with hexane (30 ml), chloroform (30 ml) and dried under vacuum again to give 6-[4-[3-(8-fluoro-4-oxo3H-quinazoline-2 -yl)propanoyl]piperazin-1-yl]pyridine-3-carbonitrile (80 mg, 23%) as a yellowish solid.

1H NMR [300 MHz, DMSO-d6] δ 8.51 (d,J=2.1 Hz, 1H), 7.90-7.87 (m, 2H), 7.65-7.59 (m, 1H), 7.46-7.39 (m, 1H), 6.93 (d, J=9.0 Hz, 1H), 3.77 (br. s, 2H), 3.64 (br. s, 4H), 3.57 (br. s, 2H), 2.90 (s, 4H).

LC-MS (m/z): 407.61 [M+H]+.

Example 21 Synthesis of 6-[4-[3-(4-oxo-3H-pyrido[3,2-d]pyrimidin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile

Stage 1

ABOUT

To a stirred solution of 3-aminopyridine-2-carboxamide (0.3 g, 2.19 mmol) in AcOH (3 ml) was added succinic anhydride (0.26 g, 2.63 mmol) at room temperature and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was poured into water cooled to 0°C (5 ml) and stirred for 30 min, while a solid precipitated. This solid was collected by filtration, washed with water (10 ml), cold acetone (5 ml), and dried under high vacuum to give 4-[(2-carbamoyl-3-pyridyl)amino]-4-oxo-butanoic acid ( 320 mg, 61%), which was used in the next step without further purification.

LC-MS (m/z): 238.41 [M+H]+.

- 30 040674

Stage 2

ABOUT

4-[(2-Carbamoyl-3-pyridyl)amino]-4-oxo-butanoic acid (300 mg, 1.27 mmol) was added in 2N. aqueous solution of NaOH (5 ml) and stirred for 2 h at 100°C (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was cooled to 0°C and acidified to pH 3-4 with 2 N. aqueous HCl solution, whereupon a white solid precipitated. The reaction mixture was stirred for 30 min at 0°C. The solid was collected by filtration, washed with water (10 ml), cold acetone (4 ml) and dried under high vacuum to give 3-(4-oxo-3H-pyrido[3,2-d]pyrimidin-2-yl) propanoic acid (200 mg, 72%), which was used in the next step without further purification.

LC-MS (m/z): 220.46 [M+H]+.

Stage 3

To a stirred solution of 3-(4-oxo-3H-pyrido[3,2-d]pyrimidin-2-yl)propanoic acid (100 mg, 0.46 mmol) and 6-piperazin-1-ylpyridine-3-carbonitrile hydrochloride (104 mg, 0.55 mmol) in DMF (3 ml) DIPEA (0.3 ml, 1.37 mmol) and T3P (0.23 ml, 0.92 mmol) were added at room temperature and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then quenched with water (10 ml) and extracted with EtOAc (3x40 ml). The combined organic extracts were washed with cold water (3x20 ml), brine (20 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude residue. This crude material was purified by column chromatography (100-200 silica gel, 10 g, 5% MeOH-DCM) to yield 6-[4-[3-(4-oxo-3H-pyrido[3,2-d]pyrimidin-2yl )propanoyl]piperazin-1-yl]pyridine-3-carbonitrile (60 mg, 36%) as a white solid.

¹ H NMR [400 MHz, DMSO-d6] δ 12.50 (br. s, 1H), 8.71 (dd, J=4.0, 1.2 Hz, 1H), 8.50 (d, J=2.0 Hz, 1H), 7.95 (dd , J=8.4, 1.6Hz, 1H), 7.88 (dd, J=9.2, 1.6Hz, 1H), 7.73 (dd, J=8.0, 4.0Hz, 1H), 6.93 (d, J=9.2Hz, 1H) ,

3.77-3.75 (m, 2H), 3.66-3.63 (m, 4H), 3.57-3.55 (m, 2H), 2.90 (s, 4H).

LC-MS (m/z): 390.69 [M+H] ⁺ .

Example 22 Synthesis of 6-[4-[3-(4-oxo-3H-pyrido[3,4-d]pyrimidin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile

Stage 1

To a stirred solution of 3-aminopyridine-4-carboxamide (300 mg, 2.18 mmol) in AcOH (6 ml) was added succinic anhydride (262 mg, 2.62 mmol) at room temperature and stirred for 22 h (according to TLC- analysis, the starting material was completely consumed). The reaction mixture was then diluted with water (20 ml) whereby a solid precipitated. This solid was collected by filtration and dried under vacuum to give 4-[(4-carbamoyl-3pyridyl)amino]-4-oxo-butanoic acid (430 mg, 83%) which was used in the next step without further purification.

LC-MS (m/z): 238.52 [M+H] ⁺ .

Stage 2

O

4-[(4-Carbamoyl-3-pyridyl)amino]-4-oxo-butanoic acid (430 mg, 1.81 mmol) was added in 2N.

- 31 040674 NaOH solution (8.6 ml) and subjected to reflux for 4 hours (according to TLC analysis, the starting material was completely consumed). After completion of the reaction, the reaction mixture was cooled to 0°C, acidified to pH 4 by adding AcOH, while a solid precipitated. This solid was collected by filtration and dried in vacuo to give 3-(4-oxo-3H-pyrido[3,4-d]pyrimidin-2-yl)propanoic acid (380 mg, 96%) which was used in the next step without additional cleaning.

LC-MS (m/z): 220.42 [M+H]+.

Stage 3

To a stirred solution of 3-(4-oxo-3H-pyrido[3,4-d]pyrimidin-2-yl)propanoic acid (200 mg, 0.91 mmol) and 6-piperazin-1-ylpyridine-3-carbonitrile hydrochloride (245 mg, 1.09 mmol) in DMF (5 ml) was added at room temperature T3P (0.87 ml, 1.36 mmol), DIPEA (0.64 ml, 3.65 mmol) and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was diluted (30 ml), was extracted with EtOAc (3x15 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure to give the crude desired compound, which was purified by column chromatography (silica gel 100-200 mesh, 4 g, 5% MeOH-DCM) to yield 6-[4-[3 -(4-oxo-3H-pyrido[3,4-d]pyrimidin-2yl)propanoyl]piperazin-1-yl]pyridine-3-carbonitrile (45 mg, 13%) as a white solid.

1H NMR [400 MHz, DMSO-d ₆ ] δ 12.55 (s, 1H), 8.94 (s, 1H), 8.59 (d, J=4.8 Hz, 1H), 8.51-8.50 (m, 1H), 7.91-7.87 (m, 2H), 6.94 (d, J=9.2 Hz, 1H), 3.77 (br. s, 2H), 3.65 (br. s, 4H), 3.56 (br. s, 2H), 2.92 (br. s , 4H).

LC-MS (m/z): 390.70 [M+H]+.

Example 23 Synthesis of 6-[4-[3-(4-oxo-3H-thieno[2,3-d]pyrimidin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile

Stage 1

2 3

To a stirred solution of 2-aminothiophene-3-carboxamide (300 mg, 2.11 mmol) in acetic acid (3 ml) was added succinic anhydride (253 mg, 2.53 mmol) at room temperature. This reaction mixture was stirred for 2 hours at room temperature (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then diluted with water (20 ml) and a precipitate formed. The mixture was filtered and the resulting solid was dried under vacuum to yield 4-[(3-carbamoyl-2-thienyl)amino]-4-oxo-butanoic acid (410 mg, 69%), which was used in the next step without further purification.

1H NMR [400 MHz, DMSO-d6] δ 12.20 (s, 1H), 12.14 (s, 1H), 7.89 (s, 1H), 7.50 (br. s, 1H), 7.39 (d, J=5.6 Hz, 1H), 6.93 (d, J=6.0 Hz, 1H), 2.67 (t, J=6.4 Hz, 2H), 2.56 (t, J=6.8 Hz, 2H).

LC-MS (m/z): 265.40 [M+Na]+.

Stage 2

4-[(3-Carbamoyl-2-thienyl)amino]-4-oxo-butanoic acid (350 mg, 1.44 mmol) was suspended in 2N. NaOH solution (7 ml) at room temperature. The resulting mixture was heated for 4 h at 100°C (according to TLC analysis, the starting material was completely consumed). After completion of the reaction, the reaction mixture was cooled to 0°C and acidified by adding AcOH (pH 4). The resulting solid was collected by filtration and dried under vacuum to give 3-(4-oxo-3H-thieno[2,3-d]pyrimidin-2-yl)propanoic acid (290 mg, 90%) which was used in the next step without additional cleaning.

LC-MS (m/z): 225.38 [M+H]+.

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Stage 3

To a stirred solution of 3-(4-oxo-3H-thieno[2,3-d]pyrimidin-2-yl)propanoic acid (200 mg, 0.89 mmol) and 6-piperazin-1-ylpyridine-3-carbonitrile hydrochloride (239 mg, 1.07 mmol) in DMF (5 ml) was added T3P (0.85 ml, 1.33 mmol), DIPEA (0.62 ml, 3.568 mmol) at room temperature. The reaction mixture was stirred for 4 h at room temperature (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was diluted (30 ml), was extracted with EtOAc (3x30 ml). The combined organic layers were dried over Na ₂ SO ₄ , the solvent was evaporated under reduced pressure to give the crude desired compound. The resulting crude compound was purified by column chromatography (100-200 silica gel, 4 g, 5% MeOH-DCM) to yield 6-[4-[3-(4-oxo-3H-thieno[2,3-d]pyrimidine-2 -yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile (80 mg, 22%) as a white solid.

1H NMR [400 MHz, DMSO-d6] δ 12.38 (s, 1H), 8.50 (d, J=2.0 Hz, 1H), 7.88 (dd, J=2.4, 9.2 Hz, 1H), 7.45 (d, J= 6.0 Hz, 1H), 7.32 (d, J=5.6 Hz, 1H), 6.93 (d, J=9.2 Hz, 1H), 3.77-3.74 (m, 2H), 3.65-3.62 (m, 4H), 3.57- 3.55 (m, 2H), 2.92-2.84 (m, 4H).

LC-MS (m/z): 395.62 [M+H]+.

Example 24 Synthesis of 6-[4-[3-(4-oxo-3H-thieno[3,2-d]pyrimidin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile

Stage 1

A stirred solution of methyl 3-aminothiophene-2-carboxylate (2 g, 12.72 mmol) in 1 M sodium hydroxide (14 ml, 14 mmol) was heated for 2 h at 100°C (according to TLC analysis, the starting material was completely consumed ). The temperature of the reaction mixture was brought to room temperature, the mixture was acidified to pH 2 by adding 1N. HCl and extracted with 50% THFEtOAc (2x100 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure to give the crude product, which was washed with n-pentane (2x20 ml) to yield 3-aminothiophene-2-carboxylic acid (1.4 g, 77%), which was used in the next step without further purification. LC-MS (ESI+) (m/z): 144.30 [M+H]+.

Stage 2

A solution of 3-aminothiophene-2-carboxylic acid (1 g, 6.99 mmol) and CDI (1.24 g, 7.69 mmol) in THF (20 ml) was heated for 1 h at 60°C, then 25 % aqueous ammonia solution (16 ml) and continued the reaction for 3 h at 60°C (according to TLC analysis, the starting material was completely consumed). The reaction mixture was brought to room temperature and the mixture was extracted with EtOAc (2x75 ml), the combined organic extracts were washed with brine (40 ml), dried over Na ₂ SO ₄ , concentrated under reduced pressure to give the crude residue, which was purified by column chromatography (100-200 silica gel, 20 g, 50-75% EtOAc-hexane) yielded 3-aminothiophene-2-carboxamide (400 mg, 40%) as a yellowish solid.

LC-MS (ESI+) (m/z): 143.33 [M+H]+.

Stage 3

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To a stirred suspension of 3-aminothiophene-2-carboxamide (390 mg, 2.74 mmol) in AcOH (10 ml) was added succinic anhydride (277 mg, 2.77 mmol) and stirred for 2 h at room temperature (according to TLC- analysis, the starting material was completely consumed). The reaction mixture was then quenched with water (40 ml), stirred for 15 min, the precipitate was collected by filtration, washed with water (10 ml) and dried to give 4-[(2-carbamoyl-3-thienyl)amino]-4-oxo- butanoic acid (585 mg, 88%) as a white solid.

LC-MS (ESI+) (m/z): 243.40 [M+H] ⁺ .

Stage 4

A solution of 4-[(2-carbamoyl-3-thienyl)amino]-4-oxo-butanoic acid (500 mg, 2.07 mmol) in 2 N. sodium hydroxide solution (16 ml, 32 mmol) was stirred for 2 h at 80°C (according to LC-MS analysis, the starting material was completely consumed). The temperature of the reaction mixture was brought to room temperature, the mixture was acidified to pH 5 by adding AcOH, the white precipitate was collected by filtration, washed with water (20 ml), dried under reduced pressure to obtain 3-(4-oxo-3H-thieno[3,2 -d]pyrimidin-2-yl)propanoic acid (330 g, 72%) as a white solid.

LC-MS (ESI+) (m/z): 225.42 [M+H] ⁺ .

Stage 5

To a stirred solution of 3-(4-oxo-3H-thieno[3,2-d]pyrimidin-2-yl)propanoic acid (200 mg, 0.89 mmol), 6-piperazin-1-ylpyridine-3-carbonitrile hydrochloride (240 mg, 1.07 mmol) and DIPEA (0.31 ml, 1.78 mmol) in DMF (10 ml) were added at room temperature 50% solution of T3P in EtOAc (0.85 ml, 1.34 mmol) and stirred for 3 h (according to LC-MS analysis, the starting material was completely consumed). The reaction mixture was then quenched with water (60 ml) and extracted with DCM (2x50 ml). The combined organic extracts were washed with brine (20 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude product, which was purified by column chromatography (100-200 silica gel, 20 g, 2-4% MeOH-DCM) with yield of 6-[4-[3-(4oxo-3H-thieno[3,2-d]pyrimidin-2-yl)propanoyl]piperαzin-1-yl]pyridin-3-carbonitrile (60 mg, 17%) as yellowish solid.

1H NMR [400 MHz, DMSO-d ₆ ] δ 12.37 (s, 1H), 8.50 (d, J=2.0 Hz, 1H), 8.12 (d, J=5.2 Hz, 1H), 7.88 (dd, J=5.6 , 9.2 Hz, 1H), 7.29 (d, J=5.2 Hz, 1H), 6.93 (d, J=9.2 Hz, 1H), 3.80-3.72 (m, 2H), 3.68-3.61 (m, 4H), 3.59 -3.55 (m, 2H), 2.92-2.85 (m, 4H).

LC-MS (ESI+) (m/z): 395.63 [M+H] ⁺ .

Example 25 Synthesis of 6-[(2S)-2-methyl-4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1 yl]pyridine-3-carbonitrile

Stage 1

To a stirred solution of tert-butyl (3S)-3-methylpiperazine-1-carboxylate (500 mg, 2.50 mmol) and 6-chloropyridine-3-carbonitrile (415 mg, 3.0 mmol) in DMSO (10 ml) was added K2CO3 (863 mg, 6.25 mmol) and Cu(MeCN) ₄ PF ₆ (18 mg, 0.05 mmol). This reaction mixture was heated for 2 h at 140°C (according to TLC analysis, the starting material was completely consumed). The temperature of the reaction mixture was brought to room temperature, the mixture was diluted with water (20 ml) and extracted with EtOAc (3x50 ml). The combined organic extracts were washed with water (2x30 ml) and brine (40 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to leave the crude material, which was purified by column chromatography (100-200 silica gel, 15 g, 30 % EtOAc-hexane) to yield tert-butyl (3S)-4-(5-cyano-2-pyridyl)-3-methyl-piperazine-1-carboxylate (260 mg, 34%) as a yellowish solid.

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LC-MS (m/z): 303.60 [M+H]+.

Stage 2

To a stirred solution of tert-butyl (3S)-4-(5-cyano-2-pyridyl)-3-methyl-piperazine-1-carboxylate (260 mg, 0.86 mmol) in DCM (5 ml) was added cooled to 0°C 4 n. HCl solution in dioxane (5 ml). The temperature of the reaction mixture was brought to room temperature and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then concentrated under reduced pressure to give the crude product, which was washed with Et ₂ O (5 ml) and dried under high vacuum to yield 6-[(2S)-2methylpiperazin-1-yl]pyridine-3-carbonitrile hydrochloride (120 mg, 68%) as a yellowish solid.

LC-MS (m/z): 203.45 [M+H] ⁺ .

Stage 3

To a stirred solution of 6-[(2S)-2-methylpiperazin-1-yl]pyridine-3-carbonitrile hydrochloride (100 mg, 0.46 mmol) and 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (111 mg, 0.55 mmol) in DMF (5 ml) cooled to 0°C, T3P (0.3 ml, 0.92 mmol, 50% solution in DMF) and DIPEA (0.25 ml, 1.38 mmol). This reaction mixture was warmed to room temperature and stirred for 8 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was quenched with water (20 ml) and extracted with EtOAc (3x60 ml). The combined organic extracts were washed with cold water (3x30 ml), brine (40 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude material, which was purified by column chromatography (100-200 silica gel, 8 g , 5% MeOH-EtOAc) to yield 6-[(2S)-2methyl-4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]pyridine-3-carbonitrile ( 40 mg, 21%) as a white solid.

1H NMR [300 MHz, DMSO-d ₆ ] δ 12.28 (s, 1H), 8.52 (d, J=1.8 Hz, 1H), 8.06 (d, J=7.5 Hz, 1H), 7.88 (dd, J=1.8 Hz, J=9.0 Hz, 1H), 7.76-7.17 (m, 1H), 7.51 (d, J=8.1 Hz, 1H), 7.44 (t, J=7.5 Hz, 1H), 6.90 (d, J=9.0 Hz, 1H), 4.66-4.58 (m, 1H), 4.25-4.14 (m, 2H), 4.05-3.87 (m, 1H), 3.47 (d, J=10.5 Hz, 1H), 3.12-2.99 (m, 2H), 2.91-2.84 (m, 4H), 1.20-.98 (m, 3H).

LC-MS (m/z): 403.66 [M+H] ⁺ .

Example 26 Synthesis of 6-[(2R)-2-methyl-4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile

Stage 1

To a stirred solution of tert-butyl (3R)-3-methylpiperazine-1-carboxylate (500 mg, 2.5 mmol), 6-chloropyridine-3-carbonitrile (415 mg, 3 mmol) in DMSO (10 ml) was added at room temperature potassium carbonate (690 mg, 5 mmol), Cu (MeCN) ₄ PF ₆ (18 mg, 0.05 mmol). This reaction mixture was heated for 4 h at 140°C (according to TLC analysis, the starting materials were completely consumed), the temperature of the reaction mixture was brought to room temperature, the mixture was diluted with water (30 ml) and was extracted with EtOAc (3x40 ml). The combined organic extracts were washed with water (30 ml), brine (40 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude desired compound. This crude material was purified by column chromatography (silica gel 100-200 mesh, 7 g, 30% EtOAc-hexane) to give tert-butyl (3R)-4-(5-cyano-2-pyridyl)-3-methyl-piperazine-1- carboxylate (415 mg, 55%) as a yellowish solid.

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1H NMR [300 MHz, CDCl ₃ ] δ 8.39 (d, J=2.4 Hz, 1H), 7.62-7.58 (m, 1H), 6.54 (d, J=9.0 Hz, 1H), 4.51 (br.

s, 1H), 4.13-3.89 (m, 3H), 3.27-3.13 (m, 2H), 3.00 (br. s, 1H), 1.45 (s, 9H), 1.23-1.16 (m, 3H).

LC-MS (m/z): 247.46 [M-tBu] ⁺ .

Stage 2

To a stirred solution of tert-butyl (3R)-4-(5-cyano-2-pyridyl)-3-methyl-piperazine-1-carboxylate (410 mg, 1.35 mmol) in 1,4-dioxane (4 ml), chilled to 0°C, 4 n. HCl solution in dioxane (1.35 ml, 5.43 mmol). The temperature of the reaction mixture was slowly brought to room temperature and the mixture was stirred for 3 h (according to TLC analysis, the starting material was completely consumed). The volatiles were removed under reduced pressure to give crude 6-[(2R)-2-methylpiperazin-1-yl]pyridine-3-carbonitrile (310 mg, 96%) which was used in the next step without further purification.

LC-MS (m/z): 203.41 [M+H] ⁺ .

Stage 3

EDC-HCl, HOBt, DIPEA, DMF ., 16 h

Stage 4

ON

To a stirred solution of 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (100 mg, 0.45 mmol), 6-[(2R)-2-methylpiperazin-1-yl]pyridine-3-carbonitrile (93 mg, 0.45 mmol) in DMF (2 ml) were added at room temperature EDC-HCl (131 mg, 0.68 mmol), HOBt (92 mg, 0.68 mmol) and DIPEA (0.16 ml, 0.91 mmol) and stirred for 16 h (according to TLC analysis, the starting materials were completely consumed). Then the reaction mixture was diluted with cold water (20 ml) and was extracted with EtOAc (3x30 ml). The combined organic extracts were washed with cold water (1x30 ml), brine (40 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude desired compound. This crude residue was purified by column chromatography (silica gel 100-200 mesh, 4 g, 3% MeOH-DCM) to give 6-[(2R)-2-methyl-4-[3-(4oxo-3H-quinazoline-2- yl)propanoyl]piperazin-1-yl]pyridine-3-carbonitrile (60 mg, 33%) as a pale pink solid.

1H NMR [300 MHz, DMSO-dd δ 12.30 (br. s, 1H), 8.52 (d, J=2.1 Hz, 1H), 8.06 (d, J=7.8 Hz, 1H), 7.88 (dd, J=9.0 , 2.1 Hz, 1H), 7.76-7.71 (m, 1H), 7.51 (d, J=8.1 Hz, 1H), 7.44 (t, J=7.5 Hz, 1H), 6.89 (d, J=9.0 Hz, 1H ), 4.66-4.58 (m, 1H), 4.24-4.14 (m, 2H), 4.05-3.87 (m, 1H), 3.34-3.31 (m, 1H), 3.05-2.72 (m, 6H), 1.20-0.98 (m, 3H).

LC-MS (m/z): 403.66 [M+H] ⁺ .

Example 27 Synthesis of 6-[(3R)-3-methyl-4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1 yl]pyridine-3-carbonitrile

Stage 1

To a stirred solution of 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (1 g, 4.58 mmol), tert-butyl (3R)-3-methylpiperazin-1-carboxylate (917 mg, 4.58 mmol) 58 mmol) in DMF (20 ml) was added

EDC-HCl (1.3 g, 6.88 mmol), HOBt (928 mg, 6.88 mmol) and DIPEA (1.6 ml, 9.17 mmol) and stirred for 16 h at room temperature (according to TLC analysis, the starting materials were completely consumed). Then the reaction mixture was diluted with cold water (40 ml) and was extracted with EtOAc (3x40 ml). The combined organic extracts were washed with cold water (50 ml), brine (40 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude desired compound. This crude compound was purified by column chromatography (silica gel 100-200 mesh, 10 g, 3% MeOH in DCM) to give t-butyl (3R)-3-methyl-4-[3-(4oxo-3H-quinazoline-2- yl)propanoyl]piperazine-1-carboxylate (410 mg, 22%) as a yellowish solid.

LC-MS (m/z): 401.67 [M+H] ⁺ .

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Stage 2

To a stirred solution of tert-butyl (3R)-3-methyl-4-[3-(4-oxo-3H-quinazolin-2yl)propanoyl]piperazine-1-carboxylate (410 mg, 1 mmol) in 1,4-dioxane (4 ml), cooled to 0°C, was added 4 n. HCl solution in dioxane (1.0 ml, 4 mmol). This reaction mixture was warmed to room temperature and stirred for 3 h (according to TLC analysis, the starting material was completely consumed). Volatiles were removed under reduced pressure to give crude 2-[3-[(2R)-2-methylpiperazin-1-yl]-3-oxo-propyl]-3H-quinazolin-4-one hydrochloride (310 mg, quantitative yield) , which was used in the next step without further purification.

LC-MS (m/z): 301.61 [M+H]+.

Stage 3

To a stirred solution of 2-[3-[(2R)-2-methylpiperazin-1-yl]-3-oxo-p-propyl]-3H-quinazolin-4-one hydrochloride (200 mg, 0.66 mmol), 6-chloropyridine-3 -carbonitrile (110 mg, 0.79 mmol) in DMSO (4 ml) was added at room temperature potassium carbonate (183 mg, 1.33 mmol), Cu(MeCN) ₄ PF6 (5 mg, 0.013 mmol). This reaction mixture was heated for 4 h at 140°C (according to TLC analysis, the starting materials were completely consumed), the temperature of the reaction mixture was slowly brought to room temperature, the mixture was diluted with cold water (30 ml), extracted with EtOAc (3x40 ml). The combined organic extracts were washed with cold water (30 ml), brine (40 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude desired compound. This crude material was purified by column chromatography (silica gel 100-200 mesh, 4 g, 5% MeOH-DCM) to give 6-[(3R)-3-methyl-4-[3-(4-oxo-3H-quinazoline- 2yl)propanoyl]piperazin-1-yl]pyridine-3-carbonitrile (65 mg, 24%) as a yellowish solid.

¹ H NMR [400 MHz, DMSO-de] δ 12.20 (s, 1H), 8.48 (s, 1H), 8.06 (d, J=7.6 Hz, 1H), 7.86 (d, J=7.6 Hz, 1H), 7.73 (br. s, 1H), 7.52 (m, 1H), 7.44 (t, J=2.0 Hz, 1H), 6.93 (br. s, 1H), 4.56 (br. s, 1H), 4.37-4.14 ( m, 3H), 3.52-3.31 (m, 2H), 2.98-2.88 (m, 5H), 1.20-1.00 (m, 3H).

LC-MS (m/z): 403.62 [M+H] ⁺ .

Example 28 Synthesis of 6-[(3S)-3-methyl-4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile

Stage 1

To a stirred solution of tert-butyl (2R)-2-methylpiperazin-1-carboxylate (0.55 g, 2.75 mmol) and 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (0.5 g, 2.29 mmol) in DMF (10 ml) cooled to 0°C, were added EDC-HCl (0.657 g, 3.44 mmol), HOBt (0.464 g, 3.44 mmol) and DIPEA (1.2 ml, 6.88 mmol). This reaction mixture was warmed to room temperature, stirred for 12 h (according to TLC analysis, the starting material was completely consumed), quenched with water (20 ml) and extracted with EtOAc (3x60 ml). The combined organic extracts were washed with cold water (3x30 ml), brine (40 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude desired compound. This crude material was purified by column chromatography (100-200 silica gel, 15 g, 5% MeOH-EtOAc) to give tert-butyl (3S)-3-methyl-4-[3-(4-oxo-3H-quinazoline-2 -yl)n-propanoyl]piperazine-1-carboxylate (0.3 g, 32%).

LC-MS (m/z): 401.67 [M+H]+.

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Stage 2

To a stirred solution of tert-butyl (3S)-3-methyl-4-[3-(4-oxo-3H-quinazolin-2yl)propanoyl]piperazine-1-carboxylate (0.2 g, 0.5 mmol) in DCM (5 ml), cooled to 0°C, was added 4 n. HCl solution in dioxane (5 ml). This reaction mixture was slowly warmed to room temperature and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then concentrated in vacuo to leave the crude material, which was washed with Et2O (5 mL) and dried under high vacuum to yield 2-[3-[(2S)-2-methylpiperazin-1-yl]-3-oxo -propyl]-3H-quinazolin-4-one hydrochloride (120 mg, 80%) as a yellowish solid. LC-MS (m/z): 301.61 [M+H]+. Stage 3

Stage 4

2-[3-[(2S)-2-methylpiperazin-1-yl]-3-oxo-propyl]-3H-quinazolin-4-one hydrochloride solution (100 mg, 0.33 mmol), 6-chloropyridin-3 -carbonitrile (55 mg, 0.39 mmol), K ₂ CO ₃ (138 mg, 0.10 mmol) and Cu(MeCN) ₄ PF ₆ (2 mg, 0.006 mmol) in DMSO (3 ml) was stirred for 4 h at 140°C (according to TLC analysis, the starting material was completely consumed). The temperature of the reaction mixture was brought to room temperature, the mixture was diluted with water (20 ml) and was extracted with EtOAc (3x50 ml). The combined organic extracts were washed with water (2x30 ml), brine (1x30 ml), dried over Na ₂ SO ₄ , and concentrated under reduced pressure to leave the crude material, which was purified by column chromatography (100-200 silica gel, 10 g, 5% MeOH-EtOAc) yielding 6-[(3S)-3-methyl-4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]pyridine-3carbonitrile (30 mg, 22%) as a white solid.

¹ H NMR [300 MHz, DMSO-d6] δ 12.24 (s, 1H), 8.49 (d, J=2.1, 1H), 8.06 (d, J=7.2 Hz, 1H), 7.87 (dd, J=9.0, 2.4 Hz, 1H), 7.78-7.70 (m, 1H), 7.55-7.42 (m, 2H), 7.01-6.91 (m, 1H), 4.61-4.51 (m, 1H), 4.38-4.15 (m, 2H) , 3.95-3.85 (d, J=12.9 Hz, 1H), 3.51-3.40 (m, 1H), 3.25-3.17 (m, 1H), 3.05-2.81 (m, 5H), 1.22-0.99 (m, 3H) .

LC-MS (m/z): 403.63 [M+H]+.

Example 29 Synthesis of 6-[(3S)-4-[3-(6-fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]-3-methyl-piperazin1-yl]pyridine-3-carbonitrile

Stage 1

To a stirred solution of 2-amino-5-fluoro-benzoic acid (15 g, 96.69 mmol) in THF (300 ml) was added at room temperature EDC-HCl (27.70 g, 145.03 mmol), HOBt-NH ₃ (21.75 g, 145.03 mmol) and DIPEA (51.0 ml, 290.07 mmol) and stirred for 6 hours (the starting material was completely consumed by TLC analysis). The reaction mixture was then concentrated under reduced pressure to leave the crude material, which was diluted with water (150 ml) and extracted with EtOAc (3x150 ml). The combined organic extracts were washed with water (2x100 ml), brine (1x100 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to leave the crude material. This crude material was purified by column chromatography (100-200 silica gel, 300 g, 40% EtOAc-hexane) to give 2-amino-5fluoro-benzamide (10.0 g, 67%) as a pale yellow solid.

LC-MS (m/z): 155.38 [M+H]+.

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Stage 2

To a stirred solution of 2-amino-5-fluoro-benzamide (7.0 g, 45.45 mmol) in AcOH (35 ml) was added succinic anhydride (5.45 g, 54.54 mmol) at room temperature and stirred for 3 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then poured into water cooled to 0°C (250 ml), the precipitated solid was stirred for 30 min at room temperature, collected by filtration, washed with water (50 ml), cold acetone (20 ml) and dried under high vacuum to give 4-(2-carbamoyl-4fluoro-anilino)-4-oxo-butanoic acid (9.0 g, 77%) as a white solid.

LC-MS (m/z): 255.57 [M+H]+.

Stage 3

A stirred solution of 4-(2-carbamoyl-4-fluoro-anilino)-4-oxo-butanoic acid (9.0 g, 35.43 mmol) in 2N an aqueous solution of NaOH (100 ml) was heated for 3 h at 100°C (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was cooled to 0°C and acidified to pH 4-5 with 2N. aqueous HCl solution, a white precipitate formed. The resulting suspension was stirred for 30 min at 0°C, filtered, washed with water (2x50 ml) and cold acetone (20 ml). The solid was dried under high vacuum to give 3-(6fluoro-4-oxo-3H-quinazolin-2-yl)propanoic acid (7.0 g, 83%) as a yellowish solid.

LC-MS (m/z): 237.43 [M+H] ⁺ .

Stage 4

To a stirred solution of 3-(6-fluoro-4-oxo-3H-quinazolin-2-yl)propanoic acid (5.0 g, 21.19 mmol) and tert-butyl (3S)-3-methylpiperazin-1-carboxylate (4.2 g, 21.19 mmol) in DMF (50 ml) cooled to 0°C was added EDC-HCl (6.06 g, 31.78 mmol), HOBt (4.29 g, 31.78 mmol) and DIPEA (11.2 ml, 63.56 mmol). This reaction mixture was warmed to room temperature, stirred for 12 h (according to TLC analysis, the starting material was completely consumed), quenched with water (100 ml) and extracted with EtOAc (3x200 ml). The combined organic extracts were washed with cold water (3x50 ml), brine (50 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to leave the crude material, which was purified by column chromatography (100-200 silica gel, 80 g, 5% MeOH-DCM) to yield tert-butyl (3S)-4-[3-(6-fluoro-4-oxo3H-quinazolin-2-yl)propanoyl]-3-methyl-piperazine-1-carboxylate (4, 0 g, 45%) as a yellowish solid.

LC-MS (m/z): 419.73 [M+H]+.

Stage 5

To a stirred solution of tert-butyl (3S)-4-[3-(6-fluoro-4-oxo-3H-quinazolin-2-yl)-propanoyl]3-methyl-piperazine-1-carboxylate (4.0 g, 9 .57 mmol) in 1,4-dioxane (20 ml) cooled to 0°C was added 4N. HCl solution in dioxane (10 ml, 40.0 mmol). The temperature of the reaction mixture was brought to room temperature and the mixture was stirred for 4 h (according to TLC analysis, the starting material was completely consumed). The volatiles were removed under reduced pressure to give

- 39 040674 in the residue of the crude material, which was washed with Et2O (10 ml), dried under high vacuum to yield 6-fluoro-2-[3-[(2S)-2-methylpiperazin-1-yl]-3-oxo- propyl]-3H-quinazolin-4-one hydrochloride (2.0 g, 65%), which was used in the next step without further purification.

LC-MS (m/z): 319.63 [M+H]+.

Stage 6

To a stirred solution of 6-fluoro-2-[3-[(2S)-2-methylpiperazin-1-yl]-3-oxo-propyl]-3Hquinazolin-4-one hydrochloride (2.5 g, 7.86 mmol) , 6-chloropyridine-3-carbonitrile (1.08 g, 7.86 mmol) and K ₂ CO ₃ (3.2 g, 23.58 mmol) in DMSO (20 ml) was added Cu(MeCN) ₄ PF ₆ ( 58 mg, 0.16 mmol) and stirred for 4 h at 140°C (according to TLC analysis, the starting material was completely consumed). The temperature of the reaction mixture was brought to room temperature, the mixture was diluted with water (50 ml) and was extracted with EtOAc (3x150 ml). The combined organic extracts were washed with water (2x100 ml), brine (1x10 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to leave the crude material. This crude product was purified by column chromatography (100-200 silica gel, 40 g, 5% MeOH-DCM) to give the desired compound, 6-[(3S)4-[3-(6-fluoro-4-oxo-3H-quinazoline -2-yl)propanoyl]-3-methyl-piperazin-1-yl]pyridine-3-carbonitrile (1.3 g, 40%) as a white solid.

¹ H NMR [400 MHz, DMSO-d6] δ 12.31 (s, 1H), 8.48 (d, J=2.4 Hz, 1H), 7.86 (dd, J=8.8, 2.0 Hz, 1H), 7.73 (dd, J =8.8, 2.8 Hz, 1H), 7.68-7.55 (m, 2H), 6.97-6.88 (s, 1H), 4.56-4.36 (m, 1H), 4.30-4.14 (m, 3H), 3.51-3.44 (m , 1H), 3.24-3.17 (m, 1H), 2.98 (s, 1H), 2.95-2.80 (s, 4H), 1.25-0.94 (m, 3H).

LC-MS (m/z): 421.72 [M+H] ⁺ .

Example 29a. Synthesis of 6-[(3R)-4-[3-(6-fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]-3-methylpiperazin-1-yl]pyridine-3-carbonitrile.

Stage 1

K2CO3 (12 .0 g, 0.09 mol). The resulting mixture was stirred for 2 h at 60°C (according to TLC analysis, the starting material was completely consumed). The temperature of the reaction mixture was brought to room temperature, the mixture was extinguished with water (150 ml) and was extracted with EtOAc (3x80 ml). The combined organic extracts were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by column chromatography (100-200 silica gel, 5% MeOH-DCM) to give 6-[(3R)-3-methyl-piperazin1-yl]-pyridine-3-carbonitrile (10.0 g, yield: 69% ).

Stage 2

To a stirred solution of 3-(6-fluoro-4-oxo-3H-quinazolin-2-yl)propanoic acid (5.0 g, 21.19 mmol, prepared in Example 29 Step 3) in dry DMF (40 mL, ~ 8 vol), was added at 10-15°C

6-[(3R)-3-methyl-piperazin-1-yl]-pyridine-3-carbonitrile (4.06 g, 20 mmol), EDCI-HCl (6.08 g, 31.6 mmol), HOBt ( 3.43 g, 25.4 mmol) and DIPEA (14.5 ml, 84.5 mol) and stirred for 22 h. The reaction mixture was then quenched with water cooled to 0°C (500 ml) and extracted with EtOAc ( 3x70 ml). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure to give the crude desired compound. This crude compound was stirred with EtOAc (50 ml) for 1 h at room temperature, filtered and dried to dryness. The resulting solid was resuspended in EtOAc. The solid was collected by filtration and washed with EtOAc (50 ml) to give (50 ml) 6-[(3R)-4-[3-(6-fluoro-4-oxo-3H-quinazolin-2yl)propanoyl]-3- methyl-piperazin-1-yl]pyridine-3-carbonitrile ((3.8 g, yield: 43%). Chiral HPLC was performed to confirm that this compound was the enantiomer of compound 29.

Column: Lux, 5 microns, cellulose-4 (250x4.6 mm, 5 microns), mobile phase: 50:50 n-hexane: (0.1%

HCOOH in ethanol:methanol (1:1)), flow rate: 1.0 ml/min, temperature: 25°C. Retention time of the R-enantiomer: 12.9 min; retention time of compound 29: 13.4 min.

Example 30 Synthesis of 6-[(3S)-4-[3-(5-fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]-3-methyl-piperazin1-yl]pyridine-3-carbonitrile.

Stage 1

To a stirred solution of 2-amino-6-fluoro-benzoic acid (15 g, 96.77 mmol) in THF (150 ml) was added at room temperature EDC-HCl (27 g, 145.16 mmol), HOBt-NH ₃ ( 21 g, 145.16 mmol) and DIPEA (52 ml, 290.32 mmol) and stirred for 6 h (TLC analysis indicated complete conversion of compound 1). The reaction mixture was then diluted with EtOAc (300 ml) and washed with water (3x100 ml), brine (1x150 ml). The organic layer was separated, dried over Na ₂ SO ₄ , the volatiles were evaporated under reduced pressure to give the crude product, which was washed using 10% EtOAc-hexanes (2x150 ml), dried under high vacuum to yield 2-amino-6fluoro-benzamide (12.2 g, 80%) as a pale yellow solid.

LC-MS (m/z): 155.42 [M+H]+.

Stage 2

To a stirred solution of 2-amino-6-fluoro-benzamide (12 g, 77.92 mmol) in AcOH (120 ml) was added succinic anhydride (9.3 g, 93.50 mmol) at room temperature and stirred for 4 h (TLC analysis indicated complete conversion of the starting material). Then the reaction mixture was diluted with water cooled to 0°C (1x200 ml), stirred for 30 min, while a solid precipitated. This solid was collected by filtration, washed with water (1x150 ml) and then with cold acetone (1x100 ml) and dried under high vacuum to give 4-(2-carbamoyl-3-fluoro-anilino)-4-oxo-butanoic acid ( 16 g, 81%) as a yellowish solid.

LC-MS (m/z): 277.46 [M+Na]+, 238.49 [M-NH ₂ ] ⁺ .

Stage 3

A stirred solution of 4-(2-carbamoyl-3-fluoro-anilino)-4-oxo-butanoic acid (15.5 g, 6.10 mmol) in 2N an aqueous solution of NaOH (160 ml) was heated for 4 h at 100°C (TLC analysis indicated complete conversion of the starting material). Then the reaction mixture was cooled to 0°C, acidified to pH 5 by adding AcOH, while a solid precipitated. This solid was collected by filtration, washed with water (250 ml) and then with cold acetone (1x100 ml) and dried under high vacuum to give 3-(5-fluoro-4-oxo-3H-quinazolin-2yl)propanoic acid (11 g, 80%) as a yellowish solid.

LC-MS (m/z): 237.47 [M+H] ⁺ .

Stage 4

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To a stirred solution of 3-(5-fluoro-4-oxo-3H-quinazolin-2-yl)propanoic acid (5.2 g, 22 mmol) in DMF (52 ml) was added tert-butyl 3-methylpiperazine-1-carboxylate (6.6 g, 33.05 mmol), EDC-HCl (6.2 g, 33.05 mmol), HOBt (4.4 g, 33.05 mmol) and DIPEA (12 ml, 66.10 mmol) and stirred for 16 h (TLC analysis indicated complete conversion of the starting material). The reaction mixture was then quenched with water (160 ml) cooled to 0°C, stirred for 30 min, while a solid precipitated, which was filtered off. The resulting filtrate was extracted with EtOAc (3x200 ml) and the combined organic extracts were washed with water (1x200 ml), brine (1x200 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to leave the crude substance, which was purified by column chromatography ( 100-200 silica gel, 60 g, 5% MeOH-DCM) yielding tert-butyl (3S)-4-[3-(5-fluoro-4-oxo-3Hquinazolin-2-yl)propanoyl]-3-methyl- piperazine-1-carboxylate (4 g, 43%) as a cream-colored solid.

LC-MS (m/z): 419.73 [M+H]+.

Stage 5

To a stirred solution of tert-butyl (3S)-4-[3-(5-fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]3-methyl-piperazine-1-carboxylate (6 g, 14.35 mmol) in DCM (60 ml) was added at room temperature 4N. HCl solution in dioxane (60 ml) and stirred for 3 h (TLC analysis indicated complete conversion of the starting material). The volatiles were evaporated under reduced pressure, the crude residue was distilled with toluene (2x100 ml) and dried under high vacuum to give 5-fluoro-2-[3-[(2S)-2-methylpiperazin-1-yl]-3 -oxo-propyl]-3H-quinazolin-4-one hydrochloride (4.7 g, 94%) as a white solid.

LC-MS (m/z): 319.59 [M+H] ⁺ .

Stage 6

To a stirred solution of 5-fluoro-2-[3-[(2S)-2-methylpiperazin-1-yl]-3-oxo-propyl]-3Hquinazolin-4-one hydrochloride (5.2 g, 14.73 mmol) K ₂ CO ₃ (4 g, 29.40 mmol), 6-chloropyridine-3carbonitrile (2 g, 14.73 mmol) and tetrakis(acetonitrile) were added in DMSO (52 ml) in a sealed tube at room temperature copper(I) hexafluorophosphate (109 mg, 0.29 mmol). This reaction mixture was degassed with argon for 5 min and stirred for 3 h at 110°C (LC-MS analysis indicated complete conversion of the starting material). The reaction mixture was then quenched with water (300 ml) and extracted with EtOAc (3x200 ml). The combined organic extracts were washed with cold water (1x200 ml), brine (1x200 ml), the organic layer was separated, dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude substance, which was purified by column chromatography (100-200 silica gel , 60 g, 5% MeOH-DCM) yielding a yellowish solid. This solid was washed with 5% MeOHETOAc to give 6-[(3S)-4-[3-(5-fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]-3-methyl-piperazin- 1 yl]pyridine-3-carbonitrile (1.4 g, 23%) as a white solid.

Ή NMR [400 MHz, DMSOCH] δ 12.22 (s, 1H), 8.47 (d, J=2.0 Hz, 1H), 7.85 (dd, J=9.2, 2.4 Hz, 1H), 7.73-7.65 (m, 1H) , 7.32 (t, J=8.4 Hz, 1H), 7.19-7.14 (m, 1H), 6.91(d, J=8.0 Hz, 1H), 4.55-4.35 (m, 1H), 4.29-4.11 (m, 3H ), 3.48 (t, J=9.2 Hz, 1H), 3.26-3.18 (m, 1H), 3.04-2.94 (m, 1H), 2.91-2.84 (m, 4H), 1.21-0.98 (m, 3H).

LC-MS (m/z): 421.72 [M+H] ⁺ .

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Example 31 Synthesis of 6-[3-(hydroxymethyl)-4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile

Stage 1

To a stirred solution of piperazin-2-ylmethanol dihydrochloride (100 mg, 0.53 mmol) in DMSO (3 ml) was added at room temperature 6-chloropyridine-3-carbonitrile (88 mg, 0.63 mmol), K ₂ CO ₃ ( 146 mg, 1.062 mmol) and Cu(MeCN) ₄ PF ₆ (3.9 mg, 0.01 mmol). This reaction mixture was heated for 12 h at 140°C (according to TLC analysis, the starting material was completely consumed), extinguished with water (10 ml) and was extracted with EtOAc (3x10 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure to leave the crude material, which was purified by column chromatography (100-200 silica gel, 5 g, 5% MeOH-DCM) to yield 6-[3-(hydroxymethyl )piperazin-1-yl]pyridine-3-carbonitrile hydrochloride (50 mg, 32%) as a white solid.

LC-MS (m/z): 219 [M+H]+.

Stage 2

To a stirred solution of 6-[3-(hydroxymethyl)piperazin-1-yl]pyridine-3-carbonitrile hydrochloride (120 mg, 0.55 mmol) and 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (120 mg, 0.55 mmol) in dry DMF (2 ml) were added at room temperature EDC-HCl (157 mg, 0.825 mmol), HOBt (113 mg, 0.825 mmol) and DIPEA (0.2 ml, 1.1 mmol) and stirred for 16 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then quenched with cold water (20 ml) and extracted with EtOAc (3x10 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure, the resulting residue was purified by preparative HPLC to yield 6-[3-(hydroxymethyl)-4-[3-(4-oxo-3H-quinazolin-2-yl) propanoyl]piperazin-1-yl]pyridine-3carbonitrile (26 mg, 11%) as a white solid.

¹ H NMR [400 MHz, DMSO-dd δ 12.17 (s, 1H), 8.49 (d, J=1.6 Hz, 1H), 8.08-8.06 (m, 1H), 7.88-7.85 (m, 1H), 7.74 ( t, J=8 Hz, 1H), 7.53 (d, J=8.4 Hz, 1H), 7.46-7.42 (m, 1H), 6.93-6.86 (m, 1H), 5.0-4.82 (m, 2H), 4.42 -3.88 (m, 4H), 3.56-3.47 (m, 2H), 2.99-2.89 (m, 6H).

LC-MS (m/z): 419.76 [M+H] ⁺ .

Example 32 Synthesis of 6-[4-[3-(6-fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]-3-(hydroxymethyl)piperazin1-yl]pyridine-3-carbonitrile.

To a stirred solution of 3-(6-fluoro-4-oxo-3H-quinazolin-2-yl)propanoic acid (300 mg, 1.27 mmol) and 6-[3-(hydroxymethyl)piperazin-1-yl]pyridin- 3-carbonitrile (277 mg, 1.27 mmol) in DMF (4 ml) was added at room temperature DIPEA (0.68 ml, 3.81 mmol), T3P (0.8 ml, 2.54 mmol). This reaction mixture was heated for 30 min at 80°C in a microwave oven (CEM) (according to TLC analysis, the starting material was completely consumed), the temperature of the reaction mixture was slowly brought to room temperature, the mixture was quenched with water (10 ml) and was extracted with EtOAc ( 3x60 ml). The combined organic extracts were washed with cold water (3x30 ml), brine (20 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude residue. This crude material was purified by column chromatography (100-200 silica gel, 15 g, 5-10% MeOH-DCM) to yield 6-[4-[3-(6-fluoro-4-oxo-3H-quinazolin-2yl)propanoyl ]-3-(hydroxymethyl)piperazin-1-yl]pyridine-3-carbonitrile (90% LC-MS, 300 mg), which was further purified by preparative HPLC to give the pure compound (154 mg, 27%) as a white solid substances.

1H NMR [400 MHz, DMSO-de] δ 12.31 (br. s, 1H), 8.48 (s, 1H), 7.85 (dd, J=9.2, 2.4 Hz, 1H), 7.74 (dd, J=8.8, 2.4 Hz, 1H), 7.66-7.61 (m, 2H), 6.92-6.86 (m, 1H), 5.07-4.84 (m, 2H), 4.44-4.37 (m, 1H), 4.33-4.16 (m,

- 43 040674

4H), 3.94-3.86 (m, 1H), 3.12-2.78 (m, 6H).

LC-MS (m/z): 437.75 [M+H]+.

Example 33 Synthesis of 6-[4-[3-(5-fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]-3-(hydroxymethyl)piperαzin1-yl]pyridine-3-carbonitrile.

To a stirred solution of 3-(5-fluoro-4-oxo-3H-quinazolin-2-yl)propanoic acid (400 mg, 1.69 mmol) and 6-[3-(hydroxymethyl)piperazin-1-yl]pyridin- 3-carbonitrile (369 mg, 1.69 mmol) in DMF (4 ml) DIPEA (0.88 ml, 5.08 mmol) was added at room temperature followed by T3P (1.0 ml, 3.39 mmol). This reaction mixture was heated for 30 min at 80°C in a microwave oven (CEM) (according to TLC analysis, the starting material was completely consumed), the temperature of the reaction mixture was slowly brought to room temperature, the mixture was quenched with water (10 ml) and was extracted with EtOAc ( 3x60 ml). The combined organic extracts were washed with cold water (3x30 ml), brine (20 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude residue. The residue was purified by column chromatography (100-200 silica gel, 15 g, 5-10% MeOH-DCM) to yield 6-[4-[3-(5-fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl ]-3(hydroxymethyl)piperazin-1-yl]pyridine-3-carbonitrile (90% LC-MS, 300 mg) which was purified by preparative HPLC to give the pure compound (160 mg, 21%) as a white solid .

1H NMR [400 MHz, DMSO-d6] δ 12.36 (br. s, 1H), 8.49 (s, 1H), 7.86 (dd, J=8.8, 2.0 Hz, 1H), 7.70 (q, J=8.0 Hz, 1H), 7.34 (d, J=8.4 Hz, 1H), 7.20-7.15 (m, 2H), 6.93-6.86 (m, 1H), 5.02-4.81 (m, 1H), 4.42-4.39 (m, 1H) , 4.33-3.88 (m, 3H), 3.53-3.41 (m, 2H), 2.95-2.87 (m, 6H).

LC-MS (m/z): 437.75 [M+H] ⁺ .

Example 34 Synthesis of 2-[3-oxo-3-[4-(2-pyridyl)piperazin-1-yl]propyl]-3H-quinazolin-4-one

Stage 1

K ₂ CO ₃ ,

.. Cu(MeCN) ₄ PF ₆ ,

J ₊ ^NBoc DMSO, 140°С, 12 h

HN- A ^x ^x ^N Stage 1 NN 1 ² 3 L^NBoc

To a stirred solution of 2-chloropyridine (750 mg, 6.60 mmol) in DMSO (10 mL) was added tert-butyl piperazine-1-carboxylate (1.4 g, 7.92 mmol), K2CO3 (1.8 g, 13.27 mmol) and Cu(MeCN) ₄ PF ₆ (49 mg, 0.132 mmol) and heated for 12 h at 140°C (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then quenched with water (40 ml) and extracted with EtOAc (3x60 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure to give the crude desired compound, which was purified by flash chromatography (100-200 silica gel, 8 g, 20% EtOAc-hexanes) to give t-butyl 4-( 2-pyridyl)piperazine-1-carboxylate (350 mg, 20%) as a yellow oil.

LC-MS (m/z): 264.55 [M+H] ⁺ .

Stage 2

To a stirred solution of tert-butyl 4-(2-pyridyl)piperazine-1-carboxylate (350 mg, 1.32 mmol) in 1,4-dioxane (4 ml) was added at 0°C under argon 4N. HCl solution in dioxane (1.3 ml, 5.30 mmol). This reaction mixture was slowly warmed to room temperature and stirred for 3 h (according to TLC analysis, the starting material was completely consumed). Volatiles were removed under reduced pressure to give the crude desired compound, which was washed with Et ₂ O (50 ml) to yield 1-(2-pyridyl)piperazine hydrochloride (210 mg, 96%) as a white solid.

LC-MS (m/z): 164.48 [M+H] ⁺ .

- 44 040674

Stage 3

To a stirred solution of 1-(2-pyridyl)piperazine hydrochloride (100 mg, 0.609 mmol) and 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (121 mg, 0.609 mmol) in dry DMF (2 ml ) was added at room temperature EDC-HCl (174 mg, 0.913 mmol), HOBt (123 mg, 0.913 mmol) and DIPEA (0.2 ml, 1.2 mmol) and stirred for 16 h (according to TLC analysis of starting material was completely used up). The reaction mixture was then quenched with cold water (20 ml), stirred for 15 min, a solid precipitated, which was collected by filtration, washed with Et ₂ O (2x5 ml) and dried under vacuum to obtain 2-[3-oxo-3 -[4-(2-pyridyl)piperazin-1-yl]propyl]3H-quinazolin-4-one (55 mg, 24%) as a white solid.

1H NMR [300 MHz, DMSO-d ₆ ] δ 12.20 (s, 1H), 8.13-8.11 (dd, J=4.8, 1.5 Hz, 1H), 8.08 (dd, J=7.8, 1.2 Hz, 1H), 7.76 -7.74 (m, 1H), 7.58-7.52 (m, 2H), 7.46-7.41 (m, 1H), 6.84 (d, J=8.7 Hz, 1H), 6.68-6.64 (m, 1H), 3.62 (s , 2H), 3.59–3.51 (m, 4H), 3.46–3.46 (m, 2H), 2.89 (br. s, 4H).

LC-MS (m/z): 364.62 [M+H]+.

Example 35 Synthesis of 2-[3-[4-(5-methyl-2-pyridyl)piperazin-1-yl]-3-oxo-propyl]-3H-quinazolin-4-one

Stage 1

To a stirred solution of 2-chloro-5-methyl-pyridine (500 mg, 2.68 mmol) in toluene (10 ml) was added at room temperature tert-butyl piperazine-1-carboxylate (409 mg, 3.22 mmol), Pd ₂ (dba) ₃ (122 mg, 0.13 mmol), BINAP (167 mg, 0.27 mmol) and tert-BuOK (903 mg, 8.06 mmol) and heated for 2 h at 100°C (according to TLC analysis of the starting material was completely consumed). The reaction mixture was then quenched with water (30 ml) and extracted with EtOAc (3x30 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure and the residue was purified by flash chromatography (100-200 silica gel, 8 g, 30% EtOAc-hexanes) to give tert-butyl 4-(5 methyl-2-pyridyl )piperazine-1-carboxylate (560 mg, 92%) as a yellow oil.

Stage 2

To a stirred solution of 1-(5-methyl-2-pyridyl)piperazine (560 mg, 1.80 mmol) in 1,4-dioxane (5 ml) cooled to 0° C. was added 4 N sodium hydroxide under argon. HCl solution in dioxane (1.8 ml, 7.22 mmol) and stirred for 3 h (according to TLC analysis, the starting material was completely consumed). Volatiles were removed under reduced pressure, the resulting residue was washed with Et2O (50 ml) and dried to yield 1-(5-methyl-2-pyridyl)piperazine hydrochloride (350 mg, 98%) as a white solid.

1H NMR [300 MHz, DMSO-de] δ 9.64 (br. s, 2H), 7.95 (s, 1H), 7.85 (d, J=8.7 Hz, 1H), 7.25 (d, J=8.7 Hz, 1H) , 3.90 (m, 4H), 3.22 (m, 4H), 2.22 (s, 3H).

Stage 3

To a stirred solution of 1-(5-methyl-2-pyridyl)piperazine hydrochloride (100 mg, 0.56 mmol) and 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (135 mg, 0.67 mmol) in dry DMF (2 ml) was added at room temperature EDC-HCl (161 mg, 0.84 mmol), HOBt (114 mg, 0.84 mmol) and DIPEA (0.2 ml, 1.1 mmol) and stirred for 16 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then quenched with water (10 ml) and extracted with DCM (3x10 ml).

- 45 040674

The combined organic extracts were dried over Na2SO4, concentrated under reduced pressure and the residue was purified by flash chromatography (100-200 silica gel, 4 g, 5% MeOH-DCM) to give compound 2-[3-[4-(5-methyl-2 -pyridyl)piperazin-1-yl]-3-oxo-propyl]-3H-quinazolin-4-one (50 mg, 23%) as a white solid.

1H NMR [400 MHz, DMSO-d6] δ 12.18 (s, 1H), 8.07 (dd, J=6, 0.9 Hz, 1H), 7.96 (s, 1H), 7.75-7.71 (m, 1H), 7.54 ( d, J=6 Hz, 1H), 7.46-7.38 (m, 2H), 6.78 (d, J=6.6 Hz, 1H), 3.62-3.61 (m, 2H), 3.59-3.51 (m, 4H), 3.39 -3.38 (m, 2H), 2.88 (br. s, 4H), 2.14 (br. s, 3H).

LC-MS (m/z): 378.37 [M+H]+.

Example 36 Synthesis of 2-[3-[4-(3-methyl-2-pyridyl)piperαzin-1-yl]-3-oxo-propyl]-3H-quinazolin-4-one

Stage 1

Pd ₂ (dba) ₃ , BINAP, , m/?ew-BuOK,

I ₊ i^^NBoc toluene, 100°C, 2 h ^N Step! NN η

2L NBoc

To a stirred solution of 2-chloro-3-methyl-pyridine (500 mg, 2.68 mmol) in toluene (10 ml) was added at room temperature tert-butyl piperazine-1-carboxylate (409 mg, 3.22 mmol), Pd (dba) ₃ (122 mg, 0.13 mmol), BINAP (167 mg, 0.26 mmol) and tert-BuOK (903 mg, 8.06 mmol) and heated for 2 h at 100°C (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then quenched with water (30 ml) and extracted with EtOAc (3x30 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure, the resulting residue was purified by flash chromatography (100-200 silica gel, 8 g, 30% EtOAc-hexane) to yield tert-butyl 4-(3-methyl-2 -pyridyl)piperazine-1-carboxylate (560 mg, 92%) as a yellow oil.

Stage 2

To a stirred solution of tert-butyl 4-(3-methyl-2-pyridyl)piperazine-1-carboxylate (560 mg, 1.80 mmol) in 1,4-dioxane (5 mL) cooled to 0°C was added argon atmosphere 4n. HCl solution in dioxane (1.8 ml, 7.2 mmol). This reaction mixture was slowly warmed to room temperature and stirred for 3 h (according to TLC analysis, the starting material was completely consumed). Volatile components were removed under reduced pressure and the residue was washed with Et ₂ O (50 ml) to obtain 1-(3-methyl-2-pyridyl)piperazine hydrochloride (350 mg, 98%), which was used in the next step without further purification.

Stage 3

To a stirred solution of 1-(3-methylpyridin-2-yl)piperazine hydrochloride (100 mg, 0.46 mmol) and 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (135 mg, 0.67 mmol) in dry DMF (2 ml) was added at room temperature EDC-HCl (161 mg, 0.84 mmol), HOBt (114 mg, 0.84 mmol) and DIPEA (0.2 ml, 1.1 mmol) and stirred for 16 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then quenched with water (10 ml) and extracted

EtOAc (3x10 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure and the resulting residue was purified by flash chromatography (100-200 silica gel, 3 g, 5% MeOH-DCM) to yield compound 2-[3-[4-(3 -methyl-2-pyridyl)piperazin-1-yl]-3-oxonpropyl]-3H-quinazolin-4-one (50 mg, 23%) as a white solid.

1H NMR [400 MHz, DMSO-d6] δ 12.18 (s, 1H), 8.12-8.06 (m, 2H), 7.77-7.73 (m, 1H), 7.59-7.57 (m, 1H), 7.53-7.51 (m , 1H), 7.46-7.42 (m, 1H), 6.96-6.93 (m, 1H), 3.66 (br. s, 2h), 3.59 (br. s, 2H), 3.11 (br. s, 2h), 2.98 (br. s, 2H), 2.89 (s, 4H), 2.26 (s, 3H).

LC-MS (m/z): 378.61 [M+H] ⁺ .

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Example 37 Synthesis of 2-[3-[4-(6-methyl-2-pyridyl)piperazin-1-yl]-3-oxo-propyl]-3H-quinazolin-4-one

Stage 1

To a stirred solution of 2-chloro-6-methyl-pyridine (500 mg, 2.68 mmol) in toluene (10 ml) was added at room temperature tert-butyl piperazine-1-carboxylate (409 mg, 3.22 mmol), Pd ₂ (dba) ₃ (122 mg, 0.13 mmol), BINAP (167 mg, 0.26 mmol), tert-BuOK (903 mg, 8.06 mmol). This reaction mixture was heated for 2 h at 100°C (according to TLC analysis, the starting material was completely consumed), diluted with water (20 ml), extracted with EtOAc (3x30 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure to give the crude desired compound, which was purified by flash chromatography (100-200 silica gel, 8 g, 30% EtOAc-hexane) to yield tert-butyl 4-(6 -methyl-2-pyridyl)piperazine-1-carboxylate (560 mg, 75%) as a yellow solid. LC-MS (m/z): 278.59 [M+H]+. Stage 2

To a stirred solution of tert-butyl 4-(6-methyl-2-pyridyl)piperazine-1-carboxylate (560 mg, 1.80 mmol) in 1,4-dioxane (5 ml) cooled to 0°C was added argon atmosphere 4 n. HCl solution in dioxane (1.8 ml, 7.22 mmol). The temperature of the reaction mixture was slowly brought to room temperature and the mixture was stirred for 3 h (according to TLC analysis, the starting material was completely consumed). Volatile components were removed under reduced pressure and the residue was washed with Et ₂ O (50 ml) and EtOAc (50 ml) to obtain 1-(6-methyl-2-pyridyl)piperazine hydrochloride (350 mg, 98%) as a yellowish solid.

LC-MS (m/z): 178.48 [M+H]+.

Stage 3

EDC-HCl (131 mg , 0.68 mmol), HOBt (92 mg, 0.68 mmol) and DIPEA (0.16 ml, 0.91 mmol). This reaction mixture was stirred for 6 h at room temperature (according to TLC analysis, the starting material was completely consumed), diluted with cold water (30 ml) and stirred for 10 min, while a solid precipitated. This solid was collected by filtration, washed with EtOAc (3x10 ml) and dried under vacuum to give 2-[3-[4-(6-methyl-2-pyridyl)piperazin-1-yl]-3-oxo-propyl]- 3Hquinazolin-4-one (62 mg, 36%) as a white solid.

¹ H NMR [400 MHz, DMSO-dd δ 12.19 (s, 1H), 8.07 (dd, J=7.6, 1.2 Hz, 1H), 7.76-7.72 (m, 1H), 7.545 (d, J=8.0 Hz, 1H), 7.44 (t, J=7.6 Hz, 2H), 6.62 (d, J=8.8 Hz, 1H), 6.53 (d, J=7.2 Hz, 1H), 3.63-3.61 (m, 2H), 3.57- 3.53 (m, 4H), 3.45-3.43 (m, 2H), 2.89 (s, 4H), 2.31 (s, 3H).

LC-MS (m/z): 378.57 [M+H]+.

Example 38 Synthesis of 2-[3-[4-(4-methyl-2-pyridyl)piperazin-1-yl]-3-oxo-propyl]-3H-quinazolin-4-one

Stage 1

To a stirred solution of 2-chloro-4-methyl-pyridine (200 mg, 1.07 mmol) in DMSO (3 ml) was added

- 47 040674 tert-butyl piperazine-1-carboxylate (161 mg, 1.29 mmol), Pd ₂ (dba) ₃ (49 mg, 0.05 mmol), BINAP (66 mg, 0.107 mmol) and t-BuOK (360 mg, 3.22 mmol). This reaction mixture was heated for 2 h at 100°C (according to TLC analysis, the starting material was completely consumed), extinguished with water (30 ml) and was extracted with EtOAc (3x30 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure and the residue was purified by flash chromatography (100-200 silica gel, 10 g, 30% EtOAc-hexane) to give tert-butyl 4-(4-methyl2-pyridyl) piperazine-1-carboxylate (240 mg, 80%) as a yellow oil.

Stage 2

To a stirred solution of tert-butyl 4-(4-methyl-2-pyridyl)piperazine-1-carboxylate (560 mg, 1.805 mmol) in 1,4-dioxane (5 ml) cooled to 0°C was added under argon 4 n. HCl solution in dioxane (1.8 ml, 7.220 mmol). This reaction mixture was warmed to room temperature and stirred for 3 h (according to TLC analysis, the starting material was completely consumed). The volatiles were removed under reduced pressure, the residue was washed with Et ₂ O (50 ml) and dried to give 1-(4-methyl-2-pyridyl)piperazine hydrochloride (350 mg, yield: 98%) as a white solid.

Stage 3

5

To a stirred solution of 1-(4-methyl-2-pyridyl)piperazine hydrochloride (100 mg, 0.564 mmol) and 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (135 mg, 0.677 mmol) in dry DMF (2 ml) was added at room temperature EDC-HCl (161 mg, 0.84 mmol), HOBt (114 mg, 0.84 mmol) and DIPEA (0.2 ml, 1.1 mmol) and stirred for 16 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then quenched with water (10 ml) and extracted with DCM (3x10 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure to leave the crude material, which was purified by flash chromatography (100-200 silica gel, 8 g, 5% MeOH-DCM) to yield 2-[3-[ 4-(4-methyl-2-pyridyl)piperazin-1-yl]-3-oxonpropyl]-3H-quinazolin-4-one (50 mg, 23%) as a white solid.

¹ H NMR [400 MHz, DMSO-d ₆ ] δ 12.18 (s, 1H), 8.08-8.06 (d, J=9.2 Hz, 1H), 7.99-7.97 (d, J=5.2 Hz, 1H), 7.77- 7.73 (m, 1H), 7.54 (d, J=8 Hz, 1H), 7.46-7.42 (m, 1H), 6.67 (s, 1H), 6.51 (d, J=5.2 Hz, 1H), 3.62-3.43 (m, 8H), 2.89 (s, 4H), 2.22 (s, 3H).

LC-MS (m/z): 378.63 [M+H]+.

Example 39 Synthesis of 5-[4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]pyrazine-2carbonitrile

Stage 1

Pd(dba) ₃ , BINAP, ¥^ΝΒοο K2CO3, NMP, sealed tube HN X, 200°C, 20 min

Stage 1

BocN

To a stirred solution of 5-bromopyrazine-2-carbonitrile (350 mg, 1.90 mmol) in NMP (4 ml) was added at room temperature tert-butyl piperazine-1-carboxylate (424 mg, 2.28 mmol), Pd ₂ ( dba) ₃ (35 mg, 0.04 mmol), X-Phos (72 mg, 0.152 mmol) and K ₂ CO ₃ (367 mg, 2.66 mmol). This reaction mixture was heated for 20 min at 200°C (according to TLC analysis, the starting material was completely consumed). After completion of the reaction, the reaction mixture was diluted with EtOAc (50 ml), washed with cold water (2x20 ml), the organic layer was dried over Na ₂ SO ₄ and concentrated under reduced pressure to obtain the crude desired compound. This crude material was purified by flash chromatography (100-200 silica gel, 5 g, 30% EtOAc-hexane) to give tert-butyl 4-(5-cyanopyrazin-2-yl)piperazin-1-carboxylate (320 mg, 58% ) as a white solid.

LC-MS (m/z): 290.61 [M+H] ⁺ .

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Stage 2

4 n. HCl solution in 1,4-dioxane (1.07 ml, 4.29 mmol). This reaction mixture was warmed to room temperature and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). Volatiles were removed in vacuo to give crude 5-piperazin-1-ylpyrazin-2-carbonitrile hydrochloride (210 mg, quantitative yield), which was used in the next step without further purification.

LC-MS (m/z): 190.46 [M+H]+.

Stage 3

To a stirred solution of 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (200 mg, 0.917 mmol) and 5-piperazin-1-ylpyrazin-2-carbonitrile hydrochloride (208 mg, 1.10 mmol) in DMF (4 mL) was added at room temperature with EDC-HCl (262 mg, 1.37 mmol), HOBt (185 mg, 1.37 mmol) and DIPEA (0.64 mL, 3.66 mmol) and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then diluted with cold water (40 ml) and extracted with EtOAc (3x40 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure to give the crude desired compound, which was purified by flash chromatography (100-200 silica gel, 5 g, 5% MeOH-DCM) to yield 5-[4-[ 3-(4-oxo-3Hquinazolin-2-yl)propanoyl]piperazin-1-yl]pyrazin-2-carbonitrile (70 mg, 20%) as yellowish

solid.

¹ H NMR [400 MHz, DMSO-d ₆ ] δ 12.19 (s, 1H), 8.58 (d, J=1.2 Hz, 1H), 8.43 (d, J=1.2 Hz, 1H), 8.07 (dd, J= 1.6, 8.0 Hz, 1H), 7.77-7.72 (m, 1H), 7.55 (d, J=8.0 Hz, 1H), 7.47-7.43 (m, 1H), 3.87-3.83 (m, 2H), 3.76-3.66 (m, 4H), 3.63-3.57 (m, 2H), 2.90 (s, 4H).

LC-MS (m/z): 390.68 [M+H] ⁺ .

Example 40 Synthesis of 2-[4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]thiazole-5carbonitrile

Stage 1

XX _|R Pd(OAc) ₂ , P(mp"w-Bu) ₃ HBF ₄ , ^Βθ Φ II ^oc K ₃ PO ₄ , PhMe, 80°C, 16 h CN + HN^> ------- ---------►

Stage 1

Stirred solution of 2-bromothiazole-5-carbonitrile (200 mg, 1.05 mmol), tert-butyl piperazine-1-carboxylate (800 mg, 4.3 mmol), potassium orthophosphate (260 mg, 1.22 mmol), acetate palladium (trimer) (40 mg, 0.06 mmol), tri-tert-butylphosphine tetrafluoroborate (20 mg, 0.06 mmol) in toluene (4 ml), in a microwave oven tube, was heated in an argon atmosphere with microwave radiation for 30 min at 80°C (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was diluted with water (10 ml) and was extracted with EtOAc (3x30 ml). The combined organic extracts were washed with brine (10 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure. The resulting crude product was purified by column chromatography (100-200 silica gel, 20 g, 10-30% EtOAc-hexane) to yield tert-butyl 4-(5-cyanothiazol-2yl)piperazine-1-carboxylate (200 mg, 64%) as a white solid.

LC-MS (ESI+) (m/z): 295.60 [M+H] ⁺ .

Stage 2

n. HC1 in

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To a stirred solution of tert-butyl 4-(5-cyanothiazol-2-yl)piperazine-1-carboxylate (200 g,

0.680 mmol) in dioxane (10 ml) was added at room temperature 4N. HCl solution in dioxane (5 ml) and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). Volatiles were removed under reduced pressure to give a crude product which was washed with EtOAc (2x15 ml) and then with diethyl ether (15 ml) to yield 2-piperazin1-ylthiazole-5-carbonitrile hydrochloride (155 mg, 98%) as a white solid .

LC-MS (ESI+) (m/z): 195.44 [M+H] ⁺ .

Stage 3

To a stirred solution of 2-piperazin-1-ylthiazol-5-carbonitrile hydrochloride (155 mg, 0.55 mmol) in anhydrous DMF was added DIPEA (0.3 ml, 1.74 mmol), 3-(4-oxo-3H- quinazolin-2yl)propanoic acid (120 mg, 0.67 mmol) and 50% solution of T3P in EtOAc (0.53 ml, 0.82 mmol), stirred for 16 h at room temperature (according to LC-MS analysis of the original substance was completely consumed). Then the reaction mixture was quenched with ice water (60 ml), extracted

DCM (2x50 ml), the combined organic extracts were washed with brine (25 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure. This crude residue was purified by column chromatography (100-200 silica gel, 200 g, 2-5% MeOH-DCM) to give 2-[4-[3-(4oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1 -yl]thiazole-5-carbonitrile (50 mg, 23%) as a pale yellow solid.

¹ H NMR [400 MHz, DMSO-de] δ 12.18 (br. s, 1H), 8.09-8.05 (m, 2Н), 7.77-7.72 (m, 1H), 7.55 (d, J=8.0 Hz, 1H) , 7.45 (t, J=7.2 Hz, 1H), 3.75-3.55 (m, 6H), 3.53-3.50 (m, 2H), 2.89 (s, 4H).

LC-MS (ESI+) (m/z): 395.62 [M+H] ⁺ .

Example 41 Synthesis of 6-[4-[3-(4-oxo-3H-pyrido[4,3-d]pyrimidin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile

Stage 1

O KOH, EtOH 9O

Mel, reflux, 1 h11

HN |1 ------------P T ⁺ P % Stage 1

Ν ।Ν ¹ (desired

K ^2a 2 product)

To a stirred solution of KOH (5.82 g, 0.104 mol) in EtOH (80 ml) was added 1H-pyrimidin-6-one (10 g, 0.104 mol) at room temperature followed by MeI (7.20 ml, 0.114 mol). This reaction mixture was subjected to reflux for 2 h (according to TLC analysis, 10-15% of unreacted starting material remained). Additional MeI (1.5 g, 0.01 mol) was added, the mixture was refluxed for 1 h (the starting material was completely consumed according to TLC analysis) and the temperature of the reaction mixture was slowly brought to room temperature. The reaction mixture was then filtered, washed with DCM (100 ml) and the filtrate was concentrated under reduced pressure to give the crude product, which was purified by column chromatography (100-200 silica gel, 200 g, 2-5% MeOH-DCM) to yield 3-methylpyrimidine- 4-one (4.5 g, 39%) as a yellowish solid.

LC-MS (ESI+) (m/z): 111.30 [M+H] ⁺ .

Stage 2

To sulfuric acid (50 ml) cooled to 10°C was added 3-methylpyrimidin-4-one (5.5 g, 50.00 mol) followed by fuming nitric acid (6.6 ml, 157.15 mol). The temperature of the reaction mixture was slowly brought to room temperature, the mixture was heated for 4 h at 100°C (according to TLC analysis, the starting material was completely consumed), the reaction mixture was again cooled to room temperature and poured onto crushed ice (500 g), then 50 % aqueous sodium hydroxide solution to pH 5. The reaction mixture was extracted with CHC13 (3x250 ml), the combined organic extracts were dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude desired compound, which was recrystallized from EtOH (15 ml) with the yield 3-methyl-5-nitro-pyrimidin-4-one (2 g, 26%) as a yellow solid.

- 50 040674

LC-MS (ESI+) (m/z): 156.36 [M+H] ⁺ .

Stage 3

O NO ₂ /% / ^s ° 2M ^e

NH ₄ OAc, MeOH 3 80°С, MVP, 2 h

Stage 3

To a Microwaveable Tube (CEM) was added 3-methyl-5-nitro-pyrimidin-4-one (300 mg, 1.94 mmol), methyl acetoacetate (2.7 g, 23.27 mmol), ammonium acetate (1 .79 g, 23.22 mmol) and MeOH (8.0 ml) and heated using microwave radiation for 2 h at 80°C (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then concentrated under reduced pressure to give the crude product, which was purified by column chromatography (100-200 silica gel, 40 g, 5-10% MeOH-DCM) to yield methyl 4-aminopyridine-3-carboxylate; received 6 batches of this reaction mixture (300 mg each), the resulting crude material after isolation was combined and purified to yield methyl 4-aminopyridine-3-carboxylate (900 mg, 51%) as a yellow solid.

LC-MS (ESI+) (m/z): 153.43 [M+H] ⁺ .

Stage 4

EH ₂ LiOH, THF-H ₂ O

D^CO ₂ Me 80°SDh____ 1 Stage 4

N 4

To a stirred solution of methyl 4-aminopyridine-3-carboxylate (2 g, 13.15 mmol) in EtOH-water (120 ml, 1:1) was added at room temperature LOH-^O (1.21 g, 28.80 mmol) and heated for 2 h at 80°C (according to TLC analysis, the starting material was completely consumed). Volatiles were removed under reduced pressure to give the crude desired compound, which was dissolved in water (30 ml), washed with EtOAc (2x25 ml) to remove non-polar impurities. The aqueous layer was acidified to pH 1 by adding 1N. HCl and extracted with EtOAc (2x10 ml). The combined organic extracts were concentrated under reduced pressure to give a crude residue which was crystallized in MeOH (20 ml) to give 4-aminopyridine-3-carboxylic acid (1 g, 55%) as a yellowish solid.

LC-MS (ESI+) (m/z): 139.31 [M+H] ⁺ .

Stage 5

SOC1 ₂ , THF, _o

Μ NH ₃ in MeOH II

k.t., 7 h______________

Stage 5 ^^^NH ₂

To a stirred solution of 4-aminopyridine-3-carboxylic acid (3 g, 0.013 mol) in THF (50 ml) was added at room temperature SOCl ₂ (3.6 ml, 0.045 mol) and a catalytic amount of DMF (30 μl) and stirred in for 3 h in an argon atmosphere (according to TLC analysis, the starting material was completely consumed). The volatiles were removed under reduced pressure to give a crude product which was dissolved in THF (30 ml), cooled to 0° C., and 7N hydrochloric acid was added. NH ₃ -methanol (22 ml). The reaction mixture was then slowly warmed to room temperature and stirred for 4 h, the precipitate was filtered off, the filtrate was concentrated under reduced pressure to give the crude desired compound, which was purified by column chromatography (100-200 silica gel, 60 g, 10% MeOH-10% NH ₄ OH-DCM) yielding 4-aminopyridine-3-carboxamide (475 mg, 27%), which was used in the next step without further purification.

LC-MS (ESI+) (m/z): 138.33 [M+H] ⁺ .

Stage 6

To a stirred solution of 4-aminopyridine-3-carboxamide (500 mg, 3.65 mmol), 4-tert-butoxy-4-oxo-butanoic acid (762 mg, 4.38 mmol), TEA (1 ml, 7.29 mmol) in DMF (10 ml) was added at room temperature a 50% solution of T3P in EtOAc (3.5 ml, 5.5 mmol) and stirred for 12 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then quenched with water (50 ml) and extracted with DCM (2x35 ml). The combined organic extracts were washed with brine (30 ml), dried over Na ₂ SO ₄ , concentrated under reduced pressure to give the crude product, which was purified by column chromatography (100-200 silica gel, 20 g, 10% MeOH-10% NH ₄ OH -DCM) to yield tert-butyl 4-[(3-carbamoyl-4pyridyl)amino]-4-oxo-butanoate (500 mg, 47%) as a yellow solid.

¹ H NMR [400 MHz, CDCy δ 11.74 (s, 1H), 9.07 (s, 1H), 8.69 (d, J=5.2 Hz, 1H), 8.52 (d, J=5.2 Hz, 1H), 7.11 (br s, 1H), 5.83 (br. s, 1H), 2.75–2.70 (m, 2H), 2.68–2.63 (m, 2H), 1.44 (s, 9H).

LC-MS (ESI+) (m/z): 316.59 [M+Na]+.

Stage 7

Water (0.9 ml ), LiOH·H ₂ O (645 mg, 15.35 mmol). The temperature of the reaction mixture was slowly brought to room temperature, the mixture was stirred for 2 h (according to TLC analysis, the starting material was completely consumed) and was extracted with EtOAc (2x30 ml). The combined organic extracts were washed with brine (30 ml), dried over Na ₂ SO ₄ , concentrated under reduced pressure to give the crude product, which was washed with pentane (2x15 ml) to yield tert-butyl 3-(4-oxo-3H-pyrido[4 ,3-d]pyrimidin-2yl)propanoate (700 mg, 83%) as a pale yellow solid.

LC-MS (ESI+) (m/z): 276.48 [M+H] ⁺ .

Stage 8

TFA (5 .7 ml, 74.50 mmol) and stirred for 4 h (according to LC-MS analysis, the starting material was completely consumed). Volatile components were removed under reduced pressure, the residue was subjected to distillation with toluene (2x10 ml) to obtain the crude product, which was triturated with pentane (2x20 ml) to yield 3-(4-oxo-3H-pyrido[4,3-d]pyrimidine- 2-yl) propanoic acid (351 mg, 63%) as a pale yellow solid.

LC-MS (ESI+) (m/z): 220.42 [M+H]+.

Stage 9

To a stirred solution of 3-(4-oxo-3H-pyrido[4,3-d]pyrimidin-2-yl)-propanoic acid (250 mg, 1.141 mmol), 6-piperazin-1-ylpyridine-3-carbonitrile hydrochloride (256 mg, 1.361 mmol) in DMF (2.5 mL) was added DIPEA (0.4 mL, 2.325 mmol) and 50% T3P in EtOAc (0.75 mL, 1.711 mmol). This reaction mixture was stirred for 4 h at room temperature (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then quenched with water (25 ml), extracted with DCM (3x30 ml), the combined organic extracts were washed with brine (10 ml) and dried over Na ₂ SO ₄ . The solvent was partially evaporated under reduced pressure to give a crude product which was purified by column chromatography (100-200 silica gel, 10 g, 10% MeOH-5% NH^H-DCM) to give 6-[4-[3-(4- oxo-3H-pyrido[4,3d]pyrimidin-2-yl)propanoyl]piperazin-1-yl]pyridine-3-carbonitrile (45 mg, 10%) as a yellowish solid.

¹ H NMR [400 MHz, DMSO-d ₆ ] δ 12.57 (br. s, 1H), 9.22 (s, 1H), 8.74 (d, J=5.6 Hz, 1H), 8.50 (d, J=2.0 Hz, 1H), 7.88 (dd, J=8.8, 2.0 Hz, 1H), 7.45 (d, J=5.6 Hz, 1H), 6.94 (d, J=9.2 Hz, 1H), 3.78-3.76 (m, 2H), 3.64 (t, J=4.8 Hz, 4H), 3.57-3.54 (m, 2H), 3.31 (s, 4H).

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LC-MS (ESI+) (m/z): 390.63 [M+H] ⁺ .

Example 42 Synthesis of 6-[4-[3-(4-oxo-3H-pyrido[2,3-d]pyrimidin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carbonitrile

Stage 1

LDA, THF, -78°С9 9 -90°С, 2 h 9

Ph^oA * _Stage| 'Ph^cA^O'Bu

3°

A solution of DIPEA (4.16 ml, 29.3 mmol) in dry THF (60 ml) was cooled to -78° C. in a dry ledacetone bath. n-BuLi in hexanes (1.6 M, 18.3 ml, 29.3 mmol) was added dropwise at -78°C and stirred for 30 minutes. Benzyl acetate (3.8 ml, 26.6 mmol) in dry THF (30 ml) was added dropwise to the reaction flask, keeping the temperature below -78°C. After the addition was complete, the flask was transferred to a dry ice-Et ₂ O bath and the reaction mixture was stirred until the temperature dropped below -90°C. To the resulting solution was added dropwise tert-butyl 2-bromoacetate (5.9 ml, 40.0 mmol) in THF (30 ml) and stirred for 1 h at -90°C. Then the reaction mixture was extinguished by slow addition of water (100 ml), warmed to room temperature and was extracted with Et ₂ O (2x150 ml). The combined organic extracts were dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to leave the crude material, which was purified by column chromatography (100-200 silica gel, 60 g, 5% EtOAc-hexane) to yield benzyl tert-butyl butanedioate (2.3 g, 27%) as a pale yellow oil.

LC-MS (m/z): 287.6 [M+Na]+.

Stage 2

To a stirred solution of benzyl tert-butyl butanedioate (2.3 g, 8.71 mmol) in dry THF (30 ml) was added 10% Pd-C (0.23 g) and stirred under H ₂ (balloon) for 16 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was filtered through diatomaceous earth (Celite) and washed with CHCl ₃ (100 ml). The filtrate was concentrated in vacuo, the resulting crude residue was purified by column chromatography (100-200 silica gel, 30 g, 35% EtOAc-hexane) to yield 4-tert-butoxy-4-oxo-butanoic acid (0.8 g, 52%) as a colorless oil.

¹ H NMR [400 MHz, CDCl3] δ 2.65–2.61 (m, 2H), 2.56–2.52 (m, 2H), 1.44 (s, 9H).

Stage 3

HOBt NH ₃ , О EDC-HCl, DIPEA, ° ^n^nh ₂ ^{Stage 3} 4 ^Λ νη ₂

6

To a stirred solution of 2-aminopyridine-3-carboxylic acid (0.9 g, 6.52 mmol) in THF (15 mL) was added EDC-HCl (1.86 g, 9.78 mmol), HOBt-NH ₃ ( 1.46 g, 9.78 mmol) and DIPEA (4.67 ml, 26.08 mmol) at room temperature. This reaction mixture was stirred for 5 h at room temperature (according to TLC analysis, the starting material was completely consumed), diluted with water (30 ml) and was extracted with EtOAc (3x75 ml). The combined organic extracts were washed with cold water (2x40 ml), brine (40 ml), separated, dried over Na2SO4 and concentrated under reduced pressure to give the crude material, which was purified by column chromatography (100-200 silica gel, 20 g, 60% EtOAc-hexane) yielding 2-aminopyridine-3-carboxamide (0.4 g, 45%) as a white solid.

LC-MS (m/z): 138.3 [M+H] ⁺ .

Stage 4

θ 4 7 O

To a stirred solution of 2-aminopyridine-3-carboxamide (0.35 g, 2.55 mmol) in DMF (15 ml) was added at room temperature 4-tert-butoxy-4-oxo-butanoic acid (0.66 g,

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3.83 mmol), HATU (1.45 g, 3.83 mmol) and Et ₃ N (0.7 ml, 5.04 mmol). The resulting reaction mixture was stirred for 24 h at 60°C (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was diluted with water (30 ml) and was extracted with EtOAc (3x50 ml). The combined organic extracts were washed with cold water (2x30 ml), brine (30 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to leave the crude material, which was purified by column chromatography (100-200 silica gel, 10 g, 2% MeOH-DCM) to yield tert-butyl 4-[(3-carbamoyl-2-pyridyl)amino]-4-oxo-butanoate (0.22 g, 26%) as a pale yellow solid.

LC-MS (m/z): 294.6 [M+H]+.

Stage 5

To a stirred solution of tert-butyl 4-[(3-carbamoyl-2-pyridyl)amino]-4-oxo-butanoate (0.22 g, 0.75 mmol) in a mixture of THF (8 ml) and water (0.3 ml) at 0°C was added LiOH·H ₂ O (0.16 g, 3.88 mmol). The temperature of the reaction mixture was brought to room temperature, the mixture was stirred for 6 h (according to TLC analysis, the starting material was completely consumed), diluted with water (10 ml) and was extracted with EtOAc (2x75 ml). The combined organic extracts were washed with brine (20 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give tert-butyl 3-(4-oxo-3H-pyrido[2,3-d]pyrimidin-2-ul)-propanoate ( 140 mg, 63%) as a pale yellow solid.

LC-MS (m/z): 276.4 [M+H] ⁺ .

Stage 6

To a stirred solution of tert-butyl 3-(4-oxo-3H-pyrido[2,3-d]pyrimidin-2-yl)-propanoate (0.14 g, 0.50 mmol) in DCM (5 ml) was added at room temperature TFA (1.1 ml, 15.0 mmol) and stirred for 3 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then concentrated under reduced pressure to give 3-(4-oxo-3H-pyrido[2,3-d]pyrimidin-2-ul)propanoic acid as the trifluoroacetic acid salt (0.14 g, 75%), which was used on the next stage without further purification, in the form of a pale yellow liquid.

LC-MS (m/z): 220.3 [M+H] ⁺ .

Stage 7

To a stirred solution of 3-(4-oxo-3H-pyrido[2,3-d]pyrimidin-2-yl)-propanoic acid as trifluoroacetic acid salt (0.087 g, 0.39 mmol) and 6-piperazin-1-ylpyridine -3-carbonitrile hydrochloride (0.133 g, 0.59 mmol) in DMF (4 mL) DIPEA (0.14 mL, 0.79 mmol) and 50% T3P in EtOAc (0.5 mL) were added at room temperature , 0.78 mmol) and stirred for 2 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then quenched with water (10 ml) and extracted with DCM (3x30 ml). The combined organic extracts were washed with brine (10 ml), dried over Na ₂ SO ₄ and concentrated to give the crude product (150 mg, 54% by LC-MS) which was subjected to preparative purification to yield 6-[4-[3-( 4-oxo-3H-pyrido[2,3-d]pyrimidin-2-yl)propanoyl]piperazin-1-yl]pyridine-3-carbonitrile (29 mg, 18%) as a white solid.

1H NMR [400 MHz, DMSO-d6] δ 12.49 (br. s, 1H), 8.86 (dd, J=4.0, 1.6 Hz, 1H), 8.50 (d, J=2.4 Hz, 1H), 8.45 (dd, J=7.6, 1.6Hz, 1H), 7.88 (dd, J=8.8, 2.0Hz, 1H), 7.47 (dd, J=8.0, 4.8Hz, 1H), 6.94 (d, J=9.2Hz, 1H), 3.83-3.77 (m, 2H), 3.69-3.62 (m, 4H), 3.58-3.53 (m, 2H), 2.93 (s, 4H).

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LC-MS (m/z): 390.67 [M+H]+.

Example 43 Synthesis of 6-[4-[3-(4-oxo-3H-quinazolin-2-yl)butanoyl]piperazin-1-yl]pyridine-3carbonitrile

Stage 1

i) KOH, VpBg, toluene, reflux, 5 h O ii) KOH, MeOHN ₂ O,

X-Λ reflux, 16 h g _n Q11

[ ABOUT

Stage 1,2I

Powdered KOH (6.0 g, 0.10 mol) was added to a solution of 3-methyltetrahydrofuran-2-one (2.0 g, 0.02 mol) and benzyl bromide (14.0 g, 0.08 mol) in toluene ( 36 ml). The resulting reaction mixture was stirred for 5 hours at 110° C. and the toluene was removed under vacuum to leave a crude product which was dissolved in MeOH (40 ml). KOH (2.0 g, 0.035 mol) and water (20 ml) were added to the resulting solution, and the reaction mixture was refluxed for 16 hours. The temperature of the reaction mixture was brought to room temperature, the mixture was washed with Et ₂ O (2x50 ml), aqueous the layer was acidified to pH 2-3 with concentrated HCl and the mixture was extracted with DCM (3x50 ml). The combined organic extracts were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo to give 4-benzyloxy-2-methyl-butanoic acid (3.4 g, 81%) as a pale yellow oil.

¹ H NMR [400 MHz, CDCh] δ 7.37-7.26 (m, 5H), 4.53-4.50 (m, 2H), 3.54 (t, J=6.4 Hz, 2H), 2.73-2.64 (m, 1H), 2.09 -2.00 (m, 1H), 1.76-1.68 (m, 1H), 1.21 (d, J=7.2 Hz, 3H).

Stage 2

To a stirred solution of 2-aminobenzamide (0.3 g, 2.20 mmol) in DMF (5 ml) was added at room temperature 4-benzyloxy-2-methyl-butanoic acid (0.59 g, 2.83 mmol), HATU (1.25 g, 3.28 mmol) and Et ₃ N (0.61 ml, 4.35 mmol). The resulting reaction mixture was stirred for 6 h at 80°C (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was diluted with water (30 ml) and was extracted with EtOAc (3x50 ml). The combined organic extracts were washed with cold water (2x30 ml), brine (30 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give a crude residue which was purified by column chromatography (100-200 silica gel, 20 g, 30% EtOAc-hexanes) yielding 2-[(4-benzyloxy-2-methyl-butanoyl)amino]benzamide (0.63 g, 79%) as a colorless liquid.

LC-MS (m/z): 327.6 [M+H] ⁺ .

Stage 3

2-[(4-Benzyloxy-2-methyl-butanoyl)amino]benzamide (0.63 g, 1.93 mmol) in 2N an aqueous solution of NaOH (12 ml) was stirred for 3 h at 100°C (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was cooled to 0°C, acidified to pH 3-4 with 2 N. aqueous HCl solution and was extracted with EtOAc (3x50 ml). The combined organic extracts were washed with brine (30 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give 2-(3-benzyloxy-1-methyl-propyl)-3H-quinazolin-4-one (450 mg, 67 %), which was used in the next step without further purification.

LC-MS (m/z): 309.5 [M+H] ⁺ .

Stage 4

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To a stirred solution of 2-(3-benzyloxy-1-methyl-propyl)-3H-quinazolin-4-one (0.55 g, 1.78 mmol) in THF (15 ml) was added 10% Pd-C as a catalyst (50% moisture, 1.0 g) and stirred under H ₂ atmosphere (balloon) for 5 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was filtered through diatomaceous earth (Celite) and washed with THF (30 ml). The filtrate was concentrated in vacuo to give 2-(3-hydroxy-1-methylpropyl)-3H-quinazolin-4-one (0.3 g, 78%) as a yellowish solid.

LC-MS (m/z): 219.49 [M+H]+.

Stage 5

DMP, DCM, 0°C-R. t., 5 h

OH Stage 6

7

Periodinan was added to a stirred solution of 2-(3-hydroxy-1-methyl-propyl)-3H-quinazolin-4-one (0.40 g, 1.83 mmol) in DCM (25 ml) cooled to 0°C. Dess-Martin (0.85 g, 2.00 mmol). This reaction mixture was slowly warmed to room temperature and stirred for 5 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was quenched with saturated aqueous Na ₂ S ₂ O ₃ and was extracted with EtOAc (2x30 ml). The combined organic extracts were washed with brine (25 ml), dried over Na ₂ SO ₄ and

concentrated in vacuo to give the crude residue, which was purified by silica gel column chromatography (100-200 silica gel, 10 g, 15% EtOAc-hexane) to yield 3-(4-oxo-3H-quinazolin-2-yl)butanal (220 mg, 56%) as a white solid.

LC-MS (m/z): 217.5 [M+H] ⁺ .

Stage 6

NaClO ₂ , NaH ₂ PO ₄ , 2-methyl-2-butene, otre / and-ViOH:H ₂ O (2: 1), THF, 0 ° C-k. t „ 12 h

Stage 7

8

tert-BuOH (5 ml), water (1.5 ml) and 2-methyl-2-butene (0.51 g, 7.28 mmol) and then

NaClO ₂ (0.25 g, 2.77 mmol) and NaH ₂ PO ₄ H ₂ O (0.43 g, 2.75 mmol). The temperature of the reaction mixture was slowly brought to room temperature, the mixture was stirred for 12 h, diluted with water (15 ml) and was extracted with EtOAc (2x30 ml). The combined organic extracts were washed with brine (25 ml), dried over Na ₂ SO ₄ and concentrated in vacuo to leave a crude material which was purified by column chromatography (100-200 silica gel, 10 g, 10% MeOH-DCM) to yield 3-(4-oxo-3H-quinazolin-2-yl)butanoic acid (150 mg, 52%), which was used in the next step without further purification.

LC-MS (m/z): 233.4 [M+H] ⁺ .

Stage 7

To a stirred solution of 3-(4-oxo-3H-quinazolin-2-yl)butanoic acid (0.15 g, 0.64 mmol) and 6-piperazin-1-ylpyridine-3-carbonitrile (0.15 g, 0 .71 mmol) in DMF (5 ml) was added under argon EDC-HCl (0.18 g, 0.96 mmol), HOBt (0.13 g, 0.96 mmol) and DIPEA (0.34 ml, 1 .93 mmol) and stirred for 8 h at room temperature (according to TLC analysis, the starting material was completely consumed). The volatiles were removed under reduced pressure and the residue was diluted with water (30 ml) and extracted with EtOAc (3x50 ml). The combined organic extracts were washed with cold water (2x25 ml), brine (20 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to leave the crude substance (100 mg, 71% LC-MS), which was subjected to preparative purification yielding 6-[4-[3-(4-oxo-3H-quinazolin-2-yl)butanoyl]piperazin-1yl]pyridine-3-carbonitrile (28 mg, 9%) as a white solid.

¹ H NMR [400 MHz, DMSO-dd δ 12.19 (br. s, 1H), 8.50 (d, J=2.4 Hz, 1H), 8.06 (d, J=7.6 Hz, 1H), 7.87 (dd, J= 8.8, 2.4 Hz, 1H), 7.75-7.70 (m, 1H), 7.53 (d, J=8.0 Hz, 1H), 7.43 (t, J=8.0 Hz, 1H), 6.92 (d, J=9.2 Hz, 1H), 3.76-3.66 (m, 4H), 3.62-3.51 (m, 4H), 3.25-3.08 (m, 2H), 2.64-2.59 (m, 1H), 1.26 (d, J=7.2 Hz, 3H) .

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LC-MS (m/z): 403.7 [M+H]+.

Example 44 Synthesis of 6-[(3S)-4-[3-(5,6-difluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]-3-methylpiperazin-1 -yl]pyridin-3 - carbonitrile.

Stage 1

Vos ₂ O, THF, c.t., 12h F

Stage 1

J ΓΊ2

2

To 3,4-difluoroaniline (5.5 g, 25.2 mmol) in dry THF (45 ml) was added di-tert-butyl dicarbonate (3.0 g, 23.2 mmol) and the resulting reaction mixture was stirred for 12 h at room temperature (according to TLC analysis, the starting material was completely consumed). Volatile components were removed under reduced pressure and the residue was washed with hexanes (15 ml). The resulting white solid was dried under high vacuum to yield tert-butyl N-(3,4-difluorophenyl)carbamate (5 g, 93%), which was used in the next step without further purification.

Ή NMR [400 MHz, CDCl ₃ ] δ 7.45–7.40 (m, 1H), 7.08–7.01 (m, 1H), 6.92–6.89 (m, 1H), 6.45 (br. s, 1H), 1.51 (s, 9H).

LC-MS (m/z): 174.4 [M+H-56] ⁺ .

Stage 2

To a stirred solution of tert-butyl N-(3,4-difluorophenyl)carbamate (1 g, 4.36 mmol) in THF (30 ml) at -78°C was added tert-BuLi (7.54 ml, 9. 82 mmol) and stirred for 3 h. Ethyl chloroformate (0.48 g, 5.1 mmol) was slowly added to the reaction mixture at -78°C and stirred for 1 h (according to TLC analysis, the starting material was completely consumed ). The temperature of the reaction mixture was brought to 0°C, the mixture was treated with saturated aqueous ammonium chloride solution (24 ml) for 10 min, then warmed to room temperature and diluted with EtOAc (100 ml) and water (50 ml). The layers were separated and the aqueous phase was extracted with EtOAc (2x30 ml). The combined organic extracts were washed with brine (50 ml), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to leave the crude material, which was purified by column chromatography (100-200 silica gel, 20 g, 2% EtOAc- hexane) to yield ethyl 6-(tert-butoxycarbonylamino)-2,3-difluoro-benzoate (0.5 g, 38%) as a white solid.

Ή NMR [400 MHz, CDCl3] δ 9.44 (br. s, 1H), 8.11-8.08 (m, 1H), 7.30-7.23 (m, 1H), 4.45 (q, J=6.8 Hz, 2H), 1.51 ( s, 9H), 1.41 (t, J=7.2 Hz, 3H).

LC-MS (m/z): 202.4 [M+H-100] ⁺ .

Stage 3

To a stirred solution of ethyl 6-(tert-butoxycarbonylamino)-2,3-difluoro-benzoate (0.5 g, 1.66 mmol) in DCM (14 ml) was added TFA (2.27 ml) dropwise at room temperature and stirred for 12 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then concentrated under reduced pressure to give ethyl 6-amino-2,3-difluorobenzoate (0.43 g, 89%), which was used in the next step without further purification.

LC-MS (m/z): 202.3 [M+H] ⁺ .

Stage 4

LiOHH ₂ O ₍ 0.46 g , 14.3 mmol). The resulting reaction mixture was stirred for 18 h at room temperature (according to TLC analysis, the starting material was

- 57 040674 completely consumed), acidified to pH 4-5 by adding 1 N. HCl and extracted with EtOAc (50 ml). The organic layer was separated, dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to give 6-amino-2,3-difluorobenzoic acid (0.2 g, 80%), which was used in the next step without further purification.

LC-MS (m/z): 174.39 [M+H]+.

Stage 5

To a stirred solution of 6-amino-2,3-difluoro-benzoic acid (0.7 g, 4.0 mmol) in THF (15 mL) was added EDC-HCl (1.15 g, 6.0 mmol) under argon. , HOBt-NH ₃ (0.91 g, 6.0 mmol) and DIPEA (2.17 ml, 12.0 mmol) and stirred for 6 h at room temperature (according to TLC analysis, the starting material was completely consumed). Volatiles were removed under reduced pressure and the residue was diluted with cold water (40 ml) and EtOAc (100 ml). The organic layer was separated, washed with cold water (2x20 ml), brine (2x20 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to leave the crude substance, which was purified by column chromatography (100-200 silica gel, 20 g , 25% EtOAc-hexane) to yield 6-amino-2,3-difluoro-benzamide (0.45 g, 65%) as a white solid.

LC-MS (m/z): 173.4 [M+H] ⁺ .

Stage 6

Succinic anhydride (0.31 g, 3.13 mmol) and stirred for 3 h at room temperature (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was poured into water (10 ml) cooled to 0°C and stirred for 30 min at room temperature. The precipitated solid was collected by filtration, washed with water (10 ml), cold acetone (5 ml) and dried under high vacuum to give 4-(2-carbamoyl-3,4-difluoroanilino)-4-oxo- butanoic acid (500 mg, 70%), which was used in the next step without further purification.

LC-MS (m/z): 273.5 [M+H] ⁺ .

Stage 7

F about

4-(2-Carbamoyl-3,4-difluoro-anilino)-4-oxo-butanoic acid (0.50 g, 1.83 mmol) in 2N. an aqueous solution of NaOH (5 ml) was stirred for 3 h at 100°C (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was cooled to 0°C and acidified to pH 3-4 with 2N. aqueous HCl solution, whereupon a white solid precipitated. This suspension was stirred for 30 min at 0°C, filtered, washed with water (10 ml), cold acetone (2 ml) and dried under high vacuum to give 3-(5,6-difluoro-4-oxo-3H- quinazolin-2yl)propanoic acid (300 mg, 65%), which was used in the next step without further purification.

LC-MS (m/z): 255.46 [M+H] ⁺ .

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Stage 8

To a stirred solution of (2S)-2-methylpiperazine (0.30 g, 2.1 mmol) in DMA (6 ml) was added 6-chloropyridine-3-carbonitrile (0.29 g, 2.3 mmol) and K ₂ CO ₃ . The resulting reaction mixture was heated to 60°C for 2 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was diluted with cold water (20 ml) and was extracted with EtOAc (3x25 ml). The combined organic extracts were washed with cold water (20 ml) and brine (2x20 ml). The organic layer was separated, dried over Na ₂ SO ₄ and concentrated under reduced pressure to leave the crude material, which was purified by column chromatography (100-200 silica gel, 10 g, 10% MeOH-DCM) to yield 6-[(3S) -3-methylpiperazin-1-yl]pyridine-3-carbonitrile (0.29 g, 67%) as a yellowish solid.

LC-MS (m/z): 203.4 [M+H]+.

Stage 9

To a stirred solution of 3-(5,6-difluoro-4-oxo-3H-quinazolin-2-yl)propanoic acid (0.25 g, 1.0 mmol) and 6-[(3,5)-3-methylpiperazine -1-yl]pyridine-3-carbonitrile (0.19 g, 1.0 mmol) in

DMF (5 ml) was added under argon EDC-HCl (0.28 g, 1.4 mmol), HOBt (0.22 g, 1.4 mmol) and DIPEA (0.5 ml, 2.9 mmol) and stirred for 24 h at room temperature (according to TLC analysis, the starting material was completely consumed). Volatiles were removed under reduced pressure and the residue was diluted with ice water (30 ml) and EtOAc (50 ml). The organic layer was separated, washed with ice water (2x15 ml), brine (2x15 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to leave the crude substance (200 mg, 47% according to LC-MS), which was subjected to preparative purification to give 6-[(3S)-4-[3-(5,6-difluoro-4oxo-3H-quinazolin-2-yl)propanoyl]-3-methyl-piperazin-1-yl]pyridin-3- carbonitrile (40 mg, 9%) as a white solid.

Ή NMR [400 MHz, CDCl ₃ ] δ 12.33 (br. s, 1H), 8.48 (d, J=2.4 Hz, 1H), 7.86 (dd, J=9.2, 2.4 Hz, 1H), 7.84-7.76 (m , 1H), 7.39-7.33 (m, 1H), 6.95-6.91 (m, 1H), 4.55-4.34 (m, 1H), 4.30-3.86 (m, 3H), 3.49-3.42 (m, 1H), 3.23 -3.15 (m, 1H), 3.05-2.94 (m, 1H), 2.89-2.78 (m, 4H), 1.20-0.98 (m, 3H). LC-MS (m/z): 439.1 [M+H] ⁺ .

Example 45 Synthesis of 2-[4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]ninerazin-1-yl]n-pyrimidin-5 carbonitrile

Stage 1

To a solution of 5-bromo-2-chloro-pyrimidine (0.5 g, 2.58 mmol) in 1,4-dioxane (20 ml) was added tert-butyl piperazine-1-carboxylate (0.722 g, 3.0 ml) at room temperature. 88 mmol) and K ₂ CO ₃ (0.713 g, 5.17 mmol). This reaction mixture was subjected to reflux for 4 h (according to TLC analysis, the starting material was completely consumed). The temperature of the reaction mixture was brought to room temperature, the mixture was diluted with water (20 ml) and was extracted with EtOAc (3x50 ml). The combined organic extracts were washed with water (2x40 ml), brine (1x40 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure. The resulting residue was further purified by column chromatography (100-200 silica gel, 15 g, 10% EtOAc-hexane) to yield t-butyl 4-(5-bromopyrimidin-2-yl)piperazine-1-carboxylate (0.7 g, 78 %) as a white solid.

1H NMR [400 MHz, CDCl ₃ ] δ 8.29 (s, 2H), 3.75 (t, J=4.8 Hz, 4H), 3.47 (t, J=5.2 Hz, 4H), 1.47 (s, 9H).

LC-MS (m/z): 287.44 [M-tBu] ⁺ .

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Stage 2

Zn(CN) ₂ (513 mg, 513 mg, 4.37 mmol) and X-phos (84 mg, 0.15 mmol). This reaction mixture was degassed using argon gas for 20 min, then Pd(PPh ₃ ) ₄ (168 mg, 0.15 mmol) was added and heated for 18 h at 100°C (according to TLC analysis, the starting material was completely consumed ). The temperature of the reaction mixture was slowly brought to room temperature, the mixture was diluted with water (20 ml) and was extracted with EtOAc (3x100 ml). The combined organic extracts were washed with water (2x40 ml), brine (1x40 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude material. This crude material was purified by column chromatography (100-200 silica gel, 12 g, 10% EtOAc-hexane) to give tert-butyl 4-(5-cyanopyrimidin-2-yl)piperazine-1-carboxylate (300 mg, 71%) as a white solid.

LC-MS (m/z): 290.49 [M+H]+.

Stage 3

To a stirred solution of tert-butyl 4-(5-cyanopyrimidin-2-yl)piperazine-1-carboxylate (300 mg, 0.5 mmol) in DCM (5 ml) at 0°C was added 4N. HCl solution in dioxane (5 ml). This reaction mixture was warmed to room temperature and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then concentrated under reduced pressure to leave the crude material, which was washed with Et ₂ O (5 ml) and dried under vacuum to yield 2-piperazin-1-ylpyrimidine-5-carbonitrile hydrochloride (200 mg, 80%) as white solid.

LC-MS (m/z): 190.46 [M+H] ⁺ .

Stage 4

To a stirred solution of 2-piperazin-1-ylpyrimidin-5-carbonitrile hydrochloride (130 mg, 0.69 mmol) and 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (100 mg, 0.46 mmol ) in DMF (2 ml) was added at room temperature with DIPEA (0.3 ml, 1.38 mmol) and T3P (291 mg, 0.92 mmol) and stirred for 8 h (according to TLC analysis, the starting material was completely consumed ). The reaction mixture was then quenched with water (10 ml) and extracted with EtOAc (3x40 ml). The combined organic extracts were washed with cold water (3x20 ml), brine (20 ml), dried over Na2SO4 and concentrated under reduced pressure to give a residue. This crude residue was purified by column chromatography (100-200 silica gel, 10 g, 5% MeOH-DCM) to give 2-[4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1 -yl]pyrimidine-5-carbonitrile (30 mg, 16%) as a white solid.

¹ H NMR [400 MHz, DMSO-d6] δ 12.20 (s, 1H), 8.79 (s, 2H), 8.06 (dd, J=7.6, 1.2 Hz, 1H), 7.78-7.73 (m, 1H), 7.57 (d, J=8.0 Hz, 1H), 7.47-7.43 (m, 1H), 3.93 (t, J=4.4 Hz, 2H), 3.81 (t, J=4.4 Hz, 2H), 3.66 (t, J= 4.8 Hz, 2H), 3.57 (t, J=5.6 Hz, 2H), 2.90 (s, 4H).

LC-MS (m/z): 390.67 [M+H] ⁺ .

Example 46 Synthesis of 2-methyl-5-[4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]pyrazole3-carbonitrile

Stage 1

Η

O^OMe > ^N 'NH2 ^h 2SO4oh ,, MeOH / H ₂ 0.70 ° C, 22 members - / and ------- ^me uG

Stage 1β

MeO^O

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To a solution of dimethyl but-2-indioate (8 g, 0.05 mmol) in MeOH (80 ml), H ₂ O (40 ml) was added Et3N (17.3 ml, 0.12 mol) and stirred for 30 min at room temperature. Methylhydrazine sulfate (8.92 g, 61.9 mol) was added and the reaction mixture was stirred for 22 h at 70°C.

The resulting solution was kept overnight at room temperature and the resulting solid was collected by filtration, dried under high vacuum to yield methyl

5-Hydroxy-2-methyl-pyrazole-3-carboxylate (3.5 g, 40%) as a pale brown solid. LC-MS (m/z): 157.3 [M+H]+.

Stage 2

To a stirred solution of methyl 5-hydroxy-2-methyl-pyrazole-3-carboxylate (0.3 g, 1.92 mmol) in DCM (30 ml) at 0°C was added pyridine (0.18 g, 2. 30 mmol) and then Tf ₂ O (0.59 g, 2.11 mmol). This reaction mixture was slowly warmed to room temperature and stirred for 2 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was slowly diluted with water (15 ml) and was extracted with DCM (2x30 ml). The combined organic extracts were washed with brine (25 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give methyl 2-methyl-5(trifluoromethylsulfonyloxy)pyrazole-3-carboxylate (580 mg, 93%), which was used in the next step without further purification.

LC-MS (m/z): 289.4 [M+H] ⁺ .

Stage 3

PdCl ₂ (dppf)gXMdppf,KOAc, ⁿ 9

Methyl 2-methyl-5-(trifluoromethylsulfonyloxy)pyrazole3-carboxylate (1.7 g, 5.90 mmol), bis(pinacolato)diboron (1.64 g, 6.48 mmol), KOAc (1 .73 g, 17.6 mmol) in 1,4-dioxane (40 ml) and this mixture was degassed under N ₂ for 15 minutes. Dppf (0.15 g, 0.29 mmol) and PdCl ₂ (dppf)-CH ₂ Cl ₂ (0.24 g, 0.29 mmol) were added and the mixture was again degassed under N ₂ for an additional 10 minutes. The resulting reaction mixture was stirred for 4 h at 90°C (according to TLC analysis, the starting material was completely consumed), diluted with toluene (30 ml), water (20 ml) and filtered through a pad of diatomaceous earth (Celite)®. A pad of diatomaceous earth (Celite) was washed with toluene (50 ml), the combined organic layers resulting from the washing were separated, dried over Na ₂ SO ₄ and concentrated under reduced pressure to obtain a residue of the crude substance, which was triturated with pentane (70 ml) and filtered. The pentane layer was concentrated under reduced pressure to give (5-methoxycarbonyl-1-methyl-pyrazol-3yl)boronic acid (1 g, 92%) as a pale yellow solid.

LC-MS (m/z): 185.5 [M+H] ⁺ .

Stage 4

To a stirred solution of (5-methoxycarbonyl-1-methyl-pyrazol-3-yl)boronic acid (1.05 g, 5.64 mmol) and N-Boc-piperazine (0.8 g, 4.34 mmol) in DCM (20 ml) at room temperature were added pyridine (1.03 g, 13.03 mmol), 4 A molecular sieves and Cu(OAc) ₂ (1.57 g, 8.67 mmol). The reaction mixture was stirred under O2 atmosphere (pressurized balloon) for 14 h at room temperature (the starting material was completely consumed by TLC analysis). Then the reaction mixture was diluted with water (10 ml) and was extracted with DCM (2x30 ml). The combined organic extracts were washed with brine (20 ml), dried over Na2SO4 and concentrated under reduced pressure to give the crude residue (41% purity), which was purified by column chromatography (100-200 silica gel, 30 g, 15% EtOAc-hexane ) yielding tert-butyl 4-(5-methoxycarbonyl-1-methyl-pyrazol-3-yl)piperazine-1-carboxylate (0.43 g, 60% by LC-MS) in

- 61 040674 as a white solid.

LC-MS (m/z): 325.7 [M+H]+.

Stage 5

To a stirred solution of tert-butyl 4-(5-methoxycarbonyl-1-methyl-pyrazol-3-yl)piperazine 1-carboxylate (0.43 g, 1.32 mmol) in THF:MeOH:H ₂ O (1:1 :1, 15 ml) was added at room temperature LiOH·H ₂ O (0.27 g, 6.63 mmol) and stirred for 5 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was acidified to pH 4-5 by adding 1N. HCl and extracted with EtOAc (2x30 ml). The combined organic extracts were washed with brine (20 ml), dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to give 5-(4-tert-butoxycarbonylpiperazin-1-yl)-2-methyl-pyrazole-3-carboxylic acid ( 0.24 g, 58%) as a white solid.

LC-MS (m/z): 311.4 [M+H]+.

Stage 6

stirred solution

5-(4-tert-Butoxycarbonylpiperazin-1-yl)-2-methyl-pyrazole-3 carboxylic acid (0.47 g, 1.51 mmol) in THF (20 ml) was added under argon EDC-HCl (0. 43 g, 2.27 mmol), HOBt-NH ₃ (0.34 g, 2.27 mmol) and DIPEA (0.8 ml, 4.55 mmol) and stirred for 6 h at room temperature (according to TLC- analysis, the starting material was completely consumed). Volatiles were removed under reduced pressure and the residue was diluted with water (40 ml) and EtOAc (100 ml). The organic layer was separated, washed with cold water (2x20 ml), brine (2x20 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to leave the crude substance, which was purified by column chromatography (100-200 silica gel, 20 g , 2.5% MeOH-DCM) to yield tert-butyl 4-(5-carbamoyl-1-methyl-pyrazol-3-yl)piperazine-1-carboxylate (0.40 g, 86%) as a white solid.

LC-MS (m/z): 310.7 [M+H]+.

Stage 7

To a stirred solution of tert-butyl 4-(5-carbamoyl-1-methyl-pyrazol-3-yl)piperazin-1-carboxylate (0.17 g, 0.55 mmol) in THF (5.0 ml) cooled to 0° C, Et ₃ N (0.19 ml, 1.37 mmol) was added followed by Tf ₂ O (0.27 ml, 1.90 mmol). The temperature of the reaction mixture was slowly brought to room temperature and the mixture was stirred for 1 h (according to TLC analysis, the starting material was completely consumed). Then the reaction mixture was diluted with water (10 ml) and was extracted with EtOAc (2x25 ml). The combined organic extracts were washed with saturated NaHCO ₃ solution (10 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give tert-butyl 4-(5-cyano-1-methyl-pyrazol-3-yl)piperazine-1-carboxylate ( 0.11 g, 68%) as a pale yellow solid.

LC-MS (m/z): 292.5 [M+H-100]+.

- 62 040674

Stage 8

To a stirred solution of tert-butyl 4-(5-cyano-1-methyl-pyrazol-3-yl)piperazin-1 carboxylate (0.11 g, 0.37 mmol) in 1,4-dioxane (5 ml), chilled to 0°C, 4 n. HCl solution in dioxane (3 ml). The temperature of the reaction mixture was brought to room temperature, the mixture was stirred for 5 h (according to TLC analysis, the starting material was completely consumed), concentrated under reduced pressure to obtain a residue of the crude material, which was washed with Et2O (5 ml) and dried under high vacuum yielding 2-methyl-5-piperazin-1-ylpyrazole-3-carbonitrile hydrochloride (70 mg, 73%) as a white solid.

LC-MS (m/z): 192.5 [M+H]+.

Stage 9

To a stirred solution of 2-methyl-5-piperazin-1-yl-pyrazol-3-carbonitrile hydrochloride (0.06 g, 0.28 mmol) and 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (0.075 g, 0.34 mmol) in DMF (2 ml) at room temperature was added 50% T3P in EtOAc (0.36 ml, 0.57 mmol) and DIPEA (0.1 ml, 0.57 mmol) and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then concentrated under high vacuum to give a crude residue which was diluted with 5% MeOH-DCM (75 ml) and washed with cold water (2x10 ml). The organic layer was separated, dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude compound (110 mg, 37% by LCMS) which was preparatively purified to yield 2-methyl-5-[4-[3-( 4-oxo-3H-quinazolin-2yl)propanoyl]piperazin-1-yl]pyrazole-3-carbonitrile (22 mg, 16%) as a white solid.

1H NMR [400 MHz, DMSO-de] δ 12.15 (br. s, 1H), 8.07 (d, J=6.8, 1.2 Hz, 1H), 7.76-7.72 (m, 1H), 7.53 (d, J=8.0 Hz, 1H), 7.44 (t, J=7.2 Hz, 1H), 6.62 (s, 1H), 3.83 (s, 3H), 3.61 (t, J=4.8 Hz, 2H), 3.54 (t, J=5.2 Hz, 2H), 3.18 (t, J=6.4 Hz, 2H), 3.06 (t, J=4.8 Hz, 2H), 2.87 (s, 4H).

LC-MS (m/z): 392.7 [M+H] ⁺ .

Example 47 Synthesis of 3-chloro-N,N-dimethyl-4-[4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin1-yl]benzamide

Stage 1

ABOUT

CDI, Me ₂ NH, /

THF, c. τ., 5 h ί| N

Stage 1 ^F I

Cl

Me ₂ NH (0.3 ml, 5.74 mmol, 1 M in THF) and CDI (0.7 g, 4.31 mmol), and stirred for 5 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then quenched with water (40 ml) and extracted with EtOAc (3x30 ml). The combined organic extracts were dried over Na2SO4, the volatiles were removed under reduced pressure, the resulting residue was washed with Et ₂ O and dried to yield 3-chloro-4-fluoro-N,N-dimethyl-benzamide (450 mg, 78%) as a white solid substances.

LC-MS (m/z): 202.40 [M+H] ⁺ .

Stage 2

ABOUT

CI

Solution of 3-chloro-4-fluoro-N,N-dimethyl-benzamide (0.4 g, 1.99 mmol) and piperazine (0.8 g, 9.38 mmol)

- 63 040674 in DMSO (5 ml) was stirred in an argon atmosphere for 16 h at 120°C (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then quenched with water (40 ml) and extracted with EtOAc (3x10 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure, and the resulting residue was washed with Et 2 O (10 ml) to yield 3-chloro-N,N-dimethyl-4-piperazin-1-yl-benzamide (550 mg, 65%) in form of a yellowish solid.

LC-MS (m/z): 268.56 [M+H]+.

Stage 3

To a stirred solution of 3-chloro-N,N-dimethyl-4-piperazin-1-yl-benzamide (150 mg, 0.55 mmol) and 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid ( 122 mg, 0.55 mmol) in dry DMF (2 ml) were added at room temperature EDC-HCl (160 mg, 0.83 mmol), HOBt (113 mg, 0.83 mmol) and DIPEA (0.2 ml , 1.16 mmol) and stirred for 16 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then quenched with water (20 ml) and extracted with EtOAc (3x10 ml). The combined organic extracts were dried over Na ₂ SO ₄ , concentrated under reduced pressure and the resulting residue was purified by flash chromatography (100-200 silica gel, 5 g, 5% MeOH-DCM) to yield 3-chloro-N,N-dimethyl-4 -[4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin1-yl]benzamide (80 mg, 43%) as a white solid.

¹ H NMR [400 MHz, DMSO-d6] δ 12.20 (s, 1H), 8.07 (dd, J=8, 1.2 Hz, 1H), 7.78-7.74 (m, 1H), 7.57 (d, J=8 Hz , 1H), 7.47-7.43 (m, 2H), 7.36 (dd, J=8.4, 2 Hz, 1H), 7.15 (d, J=8.4 Hz, 1H), 3.70 (br. s, 2H), 3.61 ( br. s, 2H), 3.05 (br. s, 2H), 2.94 (s, 8Н), 2.89 (s, 4H).

LC-MS (m/z): 468.73 [M+H]+.

Example 48 Synthesis of N-methyl-6-[4-[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1yl]pyridine-3-carboxamidine hydrochloride.

Stage 1

Xantphos, pd ₂ dba ₃ , /re/u-BuONa, PhMe, Βι sealed tube, 80°C, 4h ||

Stage ^1N

BocN. J_

Stirring solution of 2,5-dibromopyridine (2 g, 10.7 mmol), sodium t-butylate (1.6 g, 16.6 mmol), xantphos (400 mg, 0.7 mmol) and toluene (100 ml) in The hermetically sealed tube was purged with argon for 5 min. To this reaction mixture was added tert-butyl piperazine-1 carboxylate (3.4 g, 14.30 mmol) followed by Pd ₂ (dba) ₃ (200 mg, 0.21 mmol) and heated for 4 h at 80°C ( according to TLC analysis, the starting material was completely consumed). The reaction mixture was then diluted with EtOAc (200 ml), water (100 ml), filtered through a pad of diatomaceous earth (Celite) and washed with EtOAc (2x30 ml). The combined organic extracts were washed with brine (50 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude product, which was purified by column chromatography (100-200 silica gel, 50 g, 10-20% EtOAc hexanes) to yield t -butyl 4-(5-bromo-2-pyridyl)piperazine-1-carboxylate (3 g, 82%) as yellow

solid.

LC-MS (ESI+) (m/z): 342.57 [M+H]+.

Stage 2

To a stirred solution of tert-butyl 4-(5-bromo-2-pyridyl)piperazine-1-carboxylate (3 g, 8.77 mmol) in dioxane (30 ml) was added at room temperature 4N. HCl solution in dioxane (10 ml) and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). EtOAc (50 ml) was added to the reaction mixture, stirred for 30 min, the solid was collected by filtration, washed with diethyl ether (20 ml) and dried under reduced pressure to give 1-(5-bromo-2-pyridyl)piperazine hydrochloride ( 2.1 g, 93%) as a yellowish solid.

LC-MS (ESI+) (m/z): 242.43 [M+H] ⁺ .

Stage 3

To a stirred solution of 1-(5-bromo-2-pyridyl)piperazine hydrochloride (2.1 g, 8.12 mmol) in DMF (20 ml) was added DIPEA (3.8 ml, 22.01 mmol) at room temperature, 3-(4-oxo-3H-quinazolin-2-yl)propanoic acid (1.6 g, 7.34 mmol) followed by 50% T3P in EtOAc (7 ml, 11.00 mmol) and stirred for 12 h (according to LC-MS analysis, the starting material was completely consumed). The reaction mixture was then quenched with ice water (200 ml), stirred for 2 h, the resulting solid was collected by filtration, washed with water (50 ml), acetone (20 ml) and dried under vacuum to yield 2-[3-[4- (5-bromo-2-pyridyl)piperazin-1-yl]-3-oxo-propyl]3H-quinazolin-4-one (1.6 g, 50%) as a yellowish solid.

LC-MS (ESI+) (m/z): 442.59 [M+H] ⁺ .

Stage 4

To a stirred solution of 2-[3-[4-(5-bromo-2-pyridyl)piperazin-1-yl]-3-oxo-propyl]-3Hquinazolin-4-one (1.6 g, 3.62 mmol) potassium acetate (1.1 g, 11.21 mmol) and then bis(pinacolato)diboron (1.3 g, 5.12 mmol) and Pd (dppf)Cl ₂ (89 mg, 0.11 mmol). This reaction mixture was heated for 12 h at 80°C (according to LC-MS analysis, the starting material was completely consumed). The reaction mixture was then diluted with EtOAc (75 ml), filtered through a pad of diatomaceous earth (Celite)®, washed with EtOAc (2x50 ml); the combined organic extracts were washed with brine (25 ml), dried over sodium sulfate and concentrated under reduced pressure to give crude product, which was washed with pentane (2x10 ml) to give a mixture of 2-[3-oxo-3-[4-[5 (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]piperazin-1-yl]propyl]-3H-quinazolin-4-one and [6-[4 -[3-(4-oxo-3H-quinazolin-2-yl)propanoyl]piperazin-1-yl]-3-pyridyl]boronic acid (1.16 g), which was used in the next reaction without further purification. LC-MS (ESI+) (m/z): 408.69 [M+H]+ (boronic acid).

Stage 5

To a stirred solution of a mixture of 2-[3-oxo-3-[4-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2ul)-2-pyridyl]piperazin-1-yl ]propyl]-3H-quinazolin-4-one and [6-[4-[3-(4-oxo-3H-quinazolin-2yl)propanoyl]piperazin-1-yl]-3-pyridyl]boronic acid (1, 16 g), copper(I) thiophene-2-carboxylate (1.1 g, 5.77 mmol), tri-2-furylphosphine (140 mg, 0.60 mmol) in THF under argon was added Pd ₂ (dba ) ₃ (91 mg, 0.10 mmol). This reaction mixture was purged with argon for 5 min, then N,N'-bis(tert-butoxycarbonyl)-S-methylisothiourea (600 mg, 1.97 mmol) in THF (18 ml) was added under argon to give a cloudy homogeneous solution . This reaction mixture was heated for 18 h at 65°C (according to LC-MS analysis, 16% boronic acid remained), then the temperature of the reaction mixture was brought to room temperature, saturated aqueous NaHCO ₃ solution (20 ml) and EtOAc (30 ml) were added ). The organic layer was separated, washed with brine (25 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude product, which was purified by column chromatography (100-200 silica gel, 100 g, 2-5% MeOH-DCM) with exit

- 65 040674 tert-butyl N-[(E)-N-tert-butoxycarbonyl-C-[6-[4-[3-(4-oxo-3H-quinαzolin-2-yl)propanoyl]piperαzin-1yl]- 3-pyridyl]carbonimidoyl]-N-methyl-carbamate (240 mg, 12% by LC-MS) which was repurified by preparative HPLC to give the pure compound (13 mg) as a white solid.

LC-MS (ESI+) (m/z): 620.92 [M+H] ⁺ .

Stage 6

To a stirred solution of tert-butyl N-[(E)-N-tert-butoxycarbonyl-C-[6-[4-[3-(4-oxo-3Hquinazolin-2-yl)propanoyl]piperazin-1 -yl] - 3-pyridyl]carbonimidoyl]-N-methyl-carbamate (13 mg, 0.021 mmol) in dioxane (0.5 ml) was added 4N. HCl solution in dioxane (0.4 ml, 1.744 mmol) and stirred for 2 h at room temperature (according to LC-MS analysis, the starting material was completely consumed). The reaction mixture was then concentrated under reduced pressure to give the crude product, which was washed with EtOAc (2x5 ml) and then Et ₂ O (2x5 ml), and then lyophilized in a mixture of CH3CN (5 ml) and water (5 ml) to obtain N-methyl -6-[4-[3-(4-oxo-3Hquinazolin-2-yl)propanoyl]piperazin-1-yl]pyridine-3-carboxamidine hydrochloride (12 mg, 99%) in vi

de yellowish solid.

¹ H NMR [400 MHz, DMSO-d ₆ ] δ 8.58 (br. s, 1H), 8.14 (d, J=7.6 Hz, 2Н), 7.99 (br. s, 1H), 7.92-7.78 (m, 2H ), 7.63-7.57 (m, 1H), 7.35 (br. s, 1H), 7.13-7.08 (m, 1H), 4.26 (br. s, 5H), 3.81 (s, 2H), 3.68 (s, 4H ), 3.60 (s, 2H), 3.06 (s, 3H).

LC-MS (ESI+) (m/z): 420.70 [M+H] ⁺ .

Stage 7

Sun.

Mel, NaH, DMF, Ν

0°С-k. t., 3 hours

Stage 7

8'

60% sodium hydride ( 276 mg, 6.916 mmol) followed by MeI (0.32 ml, 5.162 mmol). This reaction mixture was warmed to room temperature and stirred for 3 h (according to TLC analysis, the starting material was completely consumed). The reaction mixture was then quenched with ice water (30 ml) and extracted with EtOAc (2x50 ml). The combined organic extracts were washed with brine (30 ml), dried over Na ₂ SO ₄ and concentrated under reduced pressure, the resulting residue was purified by column chromatography (100-200 silica gel, 20 g, 5-10% EtOAc-hexanes) to yield t- butyl N-(N-tert-butoxycarbonyl-C-methylsulfanyl-carbonimidoyl)-N-methyl-carbamate (650 mg, 63%) as a colorless oil.

LC-MS (ESI+) (m/z): 305.66 [M+H] ⁺ .

Example 49 Synthesis of 2-[3-[4-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-pyridyl]piperazin-1-yl]-3-oxopropyl]-3 H- quinazolin-4-one.

Stage 1

To a stirred solution of methyl 6-chloropyridine-3-carboxylate (500 mg, 2.91 mmol) in DMF (5 ml) was added at room temperature tert-butyl piperazine-1-carboxylate (720 mg, 3.87 mmol), K2CO3 ( 1.2 g, 8.74 mmol) and DMAP (35 mg, 0.29 mmol). This reaction mixture was heated in a microwave oven (CEM) for 30 min at 80°C (according to TLC analysis, the starting material was completely consumed) and diluted with water (20 ml), while a solid precipitated. This solid was collected by filtration and dissolved in EtOAc (50 ml), dried over Na ₂ SO ₄ , concentrated under reduced pressure to give the crude desired compound, which was purified by flash chromatography (100-200 silica gel, 5 g, 30% EtOAc -hexane) to yield tert-butyl 4-(5-methoxycarbonyl-2-pyridyl)piperazine-1-carboxylate (510 mg, 54%) as a white solid.

- 66 040674

LC-MS (m/z): 322.63 [M+H]+.

Stage 2

Ethylenediamine (0.25 ml, 3.73 mmol) was added to a stirred solution of trimethylaluminum (1.86 ml, 3.73 mmol, 2 M in toluene) in toluene (12 ml) cooled to 0°C. This reaction mixture was warmed to room temperature, stirred for 5 min and tert-butyl 4-(5-methoxycarbonyl-2-pyridyl)piperazine-1-carboxylate (200 mg, 0.62 mmol) in toluene (4 ml). The reaction mixture was refluxed for 3 h (according to TLC analysis, the starting material was completely consumed), the temperature of the reaction mixture was slowly brought to room temperature, the mixture was quenched with water (5 ml) and dissolved in MeOH (15 ml) and DCM (15 ml) . Then the reaction mixture was filtered through Na ₂ SO ₄ , the resulting filtrate was evaporated under vacuum to obtain the residue of the crude substance. The residue was dissolved in EtOAc (50 ml), heated for 5 min at 80°C, filtered through Na ₂ SO ₄ and concentrated under vacuum to obtain the crude desired compound. This crude residue was purified by column chromatography (100-200 silica gel, 4 g, 10% MeOH-80% DCM-10% aqueous NH3) to give tert-butyl 4-[5-(4,5dihydro-1H-imidazole-2- yl)-2-pyridyl]piperazine-1-carboxylate (160 mg, 78%) as a white solid.

LC-MS (m/z): 332.70 [M+H]+.

Stage 3

To a stirred solution of tert-butyl 4-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-pyridyl]piperazine-1-carboxylate (160 mg, 0.48 mmol) in 1,4-dioxane ( 3 ml), cooled to 0°C, was added 4 n. HCl solution in 1,4-dioxane (0.48 ml, 1.93 mmol). This reaction mixture was warmed to room temperature and stirred for 4 h (according to TLC analysis, the starting material was completely consumed). The volatiles were evaporated under reduced pressure to give 1-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-pyridyl]piperazine hydrochloride (130 mg, 100%), which was used in the next step without additional cleaning.

LC-MS (m/z): 232.56 [M+H]+.

Stage 4

1-[5-(4,5-dihydro-1H -imidazol-2yl)-2-pyridyl]piperazine hydrochloride (127 mg, 0.55 mmol), EDC-HCl (131 mg, 0.68 mmol), HOBt (93 mg, 0.68 mmol), and DIPEA (0. 32 ml, 1.83 mmol) and stirred for 16 h (according to TLC analysis, the starting material was completely consumed). The volatiles were removed under reduced pressure to leave a crude product which was diluted with cold water (30 ml) and a solid precipitated. This precipitate was collected by filtration, dried under vacuum to give the crude product (120 mg, 92% by LC-MS) which was purified by preparative HPLC yielding 2-[3-[4-[5-(4,5-dihydro- 1H-imidazol-2-yl)-2-pyridyl]piperazin-1yl]-3-oxo-propyl]-3H-quinazolin-4-one (55 mg, 28%) as a white solid.

1H NMR [400 MHz, DMSO-d ₆ ] δ 12.0 (s, 1H), 8.54 (d, J=2.0 Hz, 1H), 8.07 (dd, J=1.6, 8.0 Hz, 1H), 7.92 (dd, J =2.4, 9.2 Hz, 1H), 7.76-7.71 (m, 1H), 7.54 (d, J=8.0 Hz, 1H), 7.46-7.42 (m, 1H), 6.87(d, J=9.2 Hz, 1H) , 3.70–3.65 (m, 4H), 3.56 (br. s, 8H), 2.89 (br. s, 4H).

LC-MS (m/z): 432.73 [M+H]+.

Example 50 Synthesis of 8-chloro-2-(3-(4-(4-fluorophenyl)-3,6-dihydropyridin-1(2H)-yl)-3oxopropyl)quinazolin-4(3H)-one.

- 67 040674

Stage 1

2-Amino-3-chlorobenzoic acid (10 g, 58.3 mmol) was dissolved in DMF (30 ml) and then HOBt (10.2 g, 75.8 mmol), EDCI (13.4 g, 69, 9 mmol), ammonium chloride (12.5 g, 233 mmol) and DIEA (40.6 ml, 233 mmol). The resulting mixture was stirred for 20 hours at room temperature. The mixture was then partitioned between H ₂ O (200 ml) and EtOAc (200 ml) and the phases were separated. The aqueous layer was extracted with EtOAc (2x150 ml). The combined organic extracts were washed with 50% brine, dried (MgSO ₄ ) and filtered. The filtrate was concentrated to give a yellow/white solid residue. This solid residue was dissolved in DMF (10 ml). Addition of DCM (10 ml) resulted in the formation of a white precipitate which was collected by filtration. This process was repeated two more times to give 2-amino-3-chloro-benzamide (5.1 g) as a white fluffy solid.

LC-MS (m/z): 171.1 [M+H]+.

Stage 2

O

A suspension of 2-amino-3-chloro-benzamide (500 mg, 2.93 mmol) and succinic anhydride (293 mg, 2.93 mmol) in toluene (5 ml) was stirred at 110°C. The mixture became homogeneous after 1 hour. An additional 16 hours were required to form a precipitate. The mixture was cooled to room temperature and concentrated in vacuo. To the residue was added 1 N. NaOH solution (10 ml), and the resulting mixture was heated for 10 min at 110°C, then cooled to room temperature. Added 1 n. HCl solution until the pH reaches ~1. The resulting white precipitate was collected by filtration. This solid was washed with water (2x5 ml) and dried to give 3-(8-chloro-4-oxo-3H-quinazolin-2yl)propanoic acid (294 mg) as a white solid.

LC-MS (m/z): 253.0 [M+H] ⁺ .

Ή NMR (500 MHz, DMSO-d6) δ 12.45 (s, 1H), 12.20 (br. s, 1H), 8.04 (dd, J=7.9, 1.5 Hz, 1H), 7.92 (dd, J=7.8, 1.4 Hz, 1H), 7.43 (t, J=7.8 Hz, 1H), 2.89 (dd, J=10.6, 3.7 Hz, 2H), 2.78 (dd, J=10.6, 3.6 Hz, 2H).

Stage 3

To a solution of 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine hydrochloride (18.6 mg, 0.087 mmol) and 3-(8-chloro-4-oxo-3H-quinazolin-2-yl)propanoic acid (20.0 mg, 0.079 mmol) in DMF (3 mL) HATU (33.1 mg, 0.087 mmol) was added followed by DIEA (33.9 μL, 0.190 mmol). The resulting mixture was stirred for 3 h at room temperature, then diluted with EtOAc (40 ml) and washed with saturated aqueous NaHCO ₃ (10 ml), 50% brine (3x5 ml), dried (MgSO 4 ), filtered and concentrated under vacuum . The residue was purified by silica gel flash chromatography to give the crude product (13 mg) as a white solid which was 90% pure by LC-MS and NMR. This solid was triturated with EtOAc (3 ml), then the solid was collected by decanting the liquid phase to give 8-chloro-2-(3-(4-(4fluorophenyl)-3,6-dihydropyridin-1(2H)-yl) -3-oxopropyl)quinazolin-4(3H)-one (1.2 mg) as a white solid.

LC-MS (m/z): 412.1 [M+H] ⁺ .

Ή NMR (500 MHz, CDCl3) δ 11.08 (s, 1H), 8.18 (d, J=8.0 Hz, 1H), 7.79 (d, J=7.7 Hz, 1H), 7.38-7.28 (m, 3H), 7.03 (td, J=8.7, 2.0Hz, 2H), 5.98 (d, J=45.0Hz, 1H), 4.23 (d, J=77.1Hz, 2H), 3.80 (dt, J=100.6, 5.7Hz, 2H) , 3.17 (dd, J=13.7, 7.8 Hz, 2H), 3.02-2.84 (m, 2H), 2.65-2.48 (m, 2H).

Example 51 Synthesis of 2-(3-(4-(4-fluorophenyl)-3,6-dihydropyridin-1(2H)-yl)-3-oxopropyl)quinazoline-68 040674

This compound was synthesized according to the procedure for the preparation of 8-chloro-2-(3-(4-(4fluorophenyl)-3,6-dihydropyridin-1(2H)-yl)-3-oxopropyl)quinazolin-4(3H)-one (Example 50) using 4(3H)-one.

with a suitable starting material.

LC-MS (m/z): 378.4 [M+H]+.

1H NMR (500 MHz, DMSO-d ₆ ) δ 12.19 (s, 1H), 8.07 (d, J=7.9 Hz, 1H), 7.72 (ddd, J=8.5, 7.2, 1.6 Hz, 1H), 7.53 (d , J=8.1 Hz, 1H), 7.50-7.46 (m, 2H), 7.46-7.41 (m, 1H), 7.18 (t, J=8.8 Hz, 2H), 6.20-6.12 (m, 1H), 4.24 ( d, J=2.6 Hz, 1H), 4.09 (d, J=2.7 Hz, 1H), 3.72 (t, J=5.6 Hz, 1H), 3.66 (t, J=5.7 Hz, 1H), 2.98-2.85 ( m, 4H), 2.58 (s, 1H), 2.42 (s, 1H).

Example 52 Synthesis of 2-(3-(4-(4-fluorophenyl)piperazin-1-yl)-3-oxopropyl)quinazolin-4(3H)-one

ABOUT

This compound was synthesized according to the procedure for the preparation of 8-chloro-2-(3-(4-(4fluorophenyl)-3,6-dihydropyridin-1 (2H)-yl)-3-oxopropyl)quinazolin-4(3H)-one (Example 50) using the appropriate starting material.

LC-MS (m/z): 381.4 [M+H] ⁺ .

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 12.18 (s, 1H), 8.07 (dd, J=8.0, 1.2 Hz, 1H), 7.77-7.71 (m, 1H), 7.55 (d, J=8.0 Hz, 1H), 7.44 (ddd, J=8.1, 7.3, 1.1 Hz, 1H), 7.10-7.04 (m, 2H), 7.01-6.95 (m, 2H), 3.70-3.63 (m, 2H), 3.63- 3.54 (m, 2H), 3.15-3.10 (m, 2H), 3.04-2.98 (m, 2H), 2.89 (s, 4H).

Example 53 Synthesis of 2-(3-(4-(4-fluorophenyl)-3,6-dihydropyridin-1(2H)-yl)-3-oxopropyl)-7-methyl3,7-dihydro-4H-pyrrolo [2 ,3-d] pyrimidin-4-one.

ABOUT

This compound was synthesized according to the procedure for the preparation of 8-chloro-2-(3-(4-(4fluorophenyl)-3,6-dihydropyridin-1(2H)-yl)-3-oxopropyl)quinazolin-4(3H)-one (Example 50) using the appropriate starting material (2-amino-1-methyl-1H-pyrrole-32-carboxamide, CAS No. 1894093-24-3).

LC-MS (m/z): 381.4 [M+H] ⁺ .

1H NMR (500 MHz, DMSO) δ 11.70 (s, 1H), 7.48 (ddd, J=8.7, 5.6, 2.1 Hz, 2H), 7.21-7.14 (m, 2H), 6.98 (d, J=3.3 Hz, 1H), 6.37 (d, J=3.3Hz, 1H), 6.16 (t, J=3.3Hz, 1H), 4.23 (d, J=2.5Hz, 1H), 4.10 (d, J=2.6Hz, 1H) , 3.71 (t, J=5.6 Hz, 1H), 3.67 (t, J=5.7 Hz, 1H), 3.61 (d, J=6.5 Hz, 3H), 2.92-2.80 (m, 4H), 2.60-2.54 ( m, 1H), 2.46-2.40 (m, 1H).

Example 54 Human PARP Assay.

Poly(ADP-ribose) polymerase 1 (PARP-1) and poly(ADP-ribose) polymerase 2 (PARP-2) are two nuclear enzymes involved in many cellular processes, including DNA repair, that play a key role in maintaining DNA integrity. and chromatin structure. This assay is designed to evaluate the potential ability of a test substance to inhibit PARP-1 or PARP-2 activity using the Scintillation Proximity Assay (SPA) technique.

The Scintillation Proximity Assay (SPA) was developed to measure PARP activity using purified recombinant PARP-1 enzyme and is an ideal technique for high-throughput screening of small molecular inhibitors for drug discovery. In this case, recombinant human PARP-1 or PARP-2 enzyme was incubated with a substrate mixture (NAD, 3H-NAD and biotinylated NAD) and [ ³ H]- and biotin-labeled ADP-ribose polymers were immobilized using streptavidin conjugate SPA beads and horseradish peroxidase. In the absence of enzyme inhibition, a 100% signal was obtained. Inhibitors were identified by signal attenuation caused by a decrease in PARP-1 or PARP-2 mediated formation of poly(ADP-ribose) polymers.

- 69 040674

The chemicals and reagents used in this assay are listed below with their catalog number and source.

Order number Materials and Reagents Provider Catalog no. 1 recombinant PARP-1 enzyme human (high specific activity) Trevigen 4668-500-01 2 rh-PARP-2 enzyme, 10 µg/tube BPS Bioscience 80502 3 96-well microplate, white, FB Corning 3600 or CLS3600-100EA 4 96-well microplate, polypropylene, transparent Corning 3365 or P070 5 Activated calf thymus DNA Amersham Biosciences 27-4575 6 Nicotinamide adenine dinucleotide Sigma N1511-250MG 7 Biotinylated NAD (6biotin-17-NAD) Trevigen 4670-500-01 8 [Adenine-2,8-3N]-NAD, 250 µCi/tube Perkin Elmer NET443H250UC 9 Streptavidin- SPA-granules Perkin Elmer RPNQ 0007 10 DL-dithiotreitol Sigma 43815-1G AND Trizma® base Sigma T6791-100G 12 Magnesium chloride Sigma 449172-10G 13 Sperm Sigma 85590-5G 14 Potassium chloride Sigma 746436-500G 15 Substitute for NONIDET® R-40 Amresco M158 16 Dimethyl sulfoxide, ACS- reagent, >99.9% Sigma 472301-500 ml 17 4-Amino-1,8-naphthalimide (4-ANI) Alfa Aesar J64358 18 TOPSEAL-A 96 Perkin Elmer 6005185 19 Pipettes Eppendorf

Note: All chemicals were weighed using a balance accurate to 0.01 mg.

Gauges: Perkin Elmer; TopCount NXT.

Reagents and buffer preparations.

Buffer A 4X:

Tris pH 8: 100 mM; MgCl2: 4 mM; spermine: 4 mM; KCl: 200 mM; substitute for NonidetP-40: 0.04%.

Assay Mix A (per well).

Buffer A 4X: 12.5 µl;

DTT 100 mM: 0.5 µl;

PARP-1 enzyme: 1 unit/well, volume depends on the specific activity of a particular lot;

PARP-2 enzyme: 30 ng/well, volume dependent on specific activity of a particular lot;

H2O: up to 35 µl.

Assay Mix B (per well).

[Adenine-2,8-3H]-OVER 100 µCi/mL: 1 µL (0.1 µCi/well);

3H-OVER 100 µCi/mL: 2 µL (0.2 µCi/well);

OVER 1.5 mM: 0.05 µl;

biotinylated NAD 2501.iM: 0.03 µl;

activated calf thymus DNA: 50 mcg;

- 70 040674

H2O: up to 10 µl.

Mixture C for analysis.

streptavidin-SPA beads: 2.5 mg/ml in 200 mM EDTA pH 8.0 (for PARP-1 assay);

streptavidin-SPA beads: 2.5 mg/ml dH ₂ O (for PARP-2 assay).

Method of analysis.

mM A solution of the control compound, 4-amino-1,8-naphthalimide (4-ANI), was prepared using 100% DMSO. A 10 mM 4-ANI solution was diluted to a concentration of 2 mM and then diluted to a concentration of 200 μM using 100% DMSO. Serial 3-fold dilutions of a 200 μM 4-ANI solution in 100% DMSO were performed to give ten concentrations. 5 µl of each of the serial dilution samples were transferred to 95 µl of water to give 10-fold (10X) final concentrations used for analysis. The highest concentration of 4-ANI in this assay was 1 μM.

The previously prepared 2 mM 4-ANI solution was diluted with water to a concentration of 100 μM. 5 μl of this 100 μM 4-ANI solution was added to the negative control (NC) wells. The final concentration of 4-ANI in NC'-wells was 10 μM. NC wells were defined as wells giving the lowest signal.

mM A solution of the test compound was prepared using 100% DMSO. This 10 mM solution was diluted to 200-fold (200X) the desired final concentration used for analysis. Serial 3-fold dilutions of a 200X solution of the compound in 100% DMSO were performed to give ten concentrations.

Pre-Dilution Plate: 5 µl of each of the serial dilutions were transferred to wells of a polypropylene plate containing 95 µl of water to give 10-fold (10X) final concentrations used for analysis.

Conducting a PARP Reaction: Assay Plate.

Samples of 5 μl from the pre-dilution microplate were transferred to the wells of a 96-well white microplate (Corning 3600). 35 µl/well of Assay Mix A was added and incubated for 5 minutes at room temperature. To initiate the reaction, 10 μl/well of mixture B for analysis was added. The assay plate was incubated for 3 hours at room temperature.

Registration of signals read from the tablet for analysis.

Added 50 μl/well of mixture C for analysis. The plate was sealed using TOP SEAL-A 96. The assay plate was incubated for 15 minutes with gentle shaking. The assay plate was read on a TopCount using a protocol optimized for tritium and PVT-SPA beads.

analysis conditions.

TRIS pH 8: 25 mM;

MgCl ₂ : 1 mM;

Sperm: 1 mM;

KCl: 50 mM;

NonidetP-40: 0.01%;

DTT: 1 mM;

PARP-1: 1 unit/well;

PARP-2: 20-30 ng (depending on the specific activity of a particular batch);

Activated calf thymus DNA: 1 μg/ml;

Cold NAD: 1.5 µM;

Biotinylated NAD: 150 nM;

3H-OVER: 0.2 µCi;

Streptavidin-SPA-granules: 1.25 mg/ml.

Results and data analysis.

CRM was used as initial data. Four parameter non-linear regression was used to fit the concentration-response curve and calculate IC ₅₀ values. The duplicated CPM values for the NC and PC groups were averaged. The mean CPM for NC was subtracted from all baseline CPM values. Then, the CPM values obtained after subtracting the background were divided by the average CPM value of the positive control to obtain % activity. This % activity was subtracted from 100 to give % inhibition. These data were plotted and fitted to the following equation:

(MAX - MIN) % Inhibition = MIN + -----;——-----~;

। ^(Hill coefficient) +

[X]

Example 55 Cellular analysis of PARP-1.

In response to DNA damage, poly-(ADP-ribose) polymerase 1 (PARP-1), which is the main

- 71 040674 isoform of the PARP family, is rapidly activated by DNA strand breaks caused by environmental factors, cancer therapy, inflammation, ischemia-reperfusion injury, and neurodegeneration. After activation, the synthesis of a highly negatively charged poly-ADP-ribose (PAR) polymer begins, requiring the consumption of NAD+, this polymer is found on nuclear target proteins, including PARP-1 as the main acceptor. Due to PARP activation, extensive DNA damage can lead to depletion of NAD+ in the cell and cause cell necrosis. Therefore, PARP-1 is considered as a promising target for drug development that can be used in various treatment regimens for cancer, inflammation, ischemia, and neurodegenerative disease.

In this assay, to observe PARP activity inside cells, HeLa cells were treated with PARP-1 inhibitors and then DNA damage was induced using H2O2. The final activity of PARP-1 was assessed by measuring the level of NAD+ and NAD(H) in the collected cell lysates of treated and untreated cells.

Materials and reagents.

Order number Materials and Reagents Provider Catalog no. 1 HeLa cells ATCC ATCC® CCL-2™ 2 fetal calf serum, HI Invitrogen 10438-026 3 Penicillin-streptomycin (10,000 units/ml), 100 ml Invitrogen 15140-122 4 DMEM, High Glucose, Pyruvate, 6x 1000ml Invitrogen 11995-065 5 0.25% Trypsin-EDTA (IX), phenol red, 100 ml Invitrogen 25200-056 6 175 cm ² , Cell culture mat, 100/pack Corning CLS431306- 84EA 7 IX PBS (phosphate-buffered saline), pH 7.4, 500 ml Invitrogen 10010-023 8 96-well plate, white, flat bottom, sterile, 100/pack Corning CLS3917-100EA 9 96-well microplate, polypropylene, transparent Corning P070 10 Dimethyl sulfoxide, ACS reagent, >99.9% Sigma 472301-500 ml AND universal tips, transparent, 0.5-10 µl, 1000/bag Axygen T-300

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12 universal tips, clear, 1-200 µl, 1000/bag Axygen T-200-C 13 NAD/NADH-Glo Kit Promega G9072 14 Dodecyltrimethylammonium bromide Sigma D5047-5G 15 NaOH Sigma S8045-500G 16 sodium bicarbonate Sigma S5761-500G 17 sodium carbonate Sigma S7795-500G 18 Nicotinamide Sigma N5535-100G 19 TritonX-100 Sigma T9284-100 ml 20 Trizma® base Sigma 93362-250G 21 Hydrochloric acid Sigma H1758-100ml 22 Olaparib Medchem Express HY-10162 23 Envision Plate Reader Perkin Elmer 2104 24 CO ₂ Humidifier Incubator Thermo Scientific 25 Pipettes/serological pipettes Eppendorf / Corning

Measuring instruments. Registration: registration of luminescence in Envision Plate Reader/TopCount (Perkin Elmer).

Obtaining reagents and media.

Obtaining culture media.

DMEM media: 1X;

FBS (heat inactivated): 10%;

Penicillin-streptomycin (10,000 units/ml): 0.1 mg/ml;

L-Glutamine: 2 mM.

Obtaining a reagent for the registration of luciferin.

The reconstitution buffer was thawed. The temperature of the reconstitution buffer and the luciferin detection reagent was equilibrated to room temperature. The entire contents of the reconstitution buffer vial was transferred to a dark vial with lyophilized luciferin registration reagent. These two reagents were mixed by rotating or inverting the vial to obtain a homogeneous solution. No shaking. The reagent for registration of luciferin should easily pass into solution in less than 1 min.

Receipt of NAD/NADH-Glo™ reagent for registration.

An equal volume of NAD/NADH-Glo™ reagent was added to each sample containing NAD ⁺ or NAD(H) for registration.

The temperature of the re-dissolved reagent for registration of luciferin was balanced with room temperature. Reductase, reductase substrate, and NAD ⁺ cycling substrate were thawed at room temperature or on ice immediately prior to use. The NAD ⁺ cycling enzyme was redissolved by adding 275 µl of water. This mixture was stirred by gently swirling the tube and stored on ice. The required amount of NAD/NADH-Glo™ reagent for detection was obtained by adding 5 µl of reductase, 5 µl of reductase substrate, 5 µl of NAD ⁺ cycling enzyme, and 25 µl of NAD ⁺ cycling substrate to 1 ml of the reconstituted luciferin detection reagent. . This mixture was gently inverted five times.

Final conditions of the analysis.

Assay mix volume: 100 µl;

cell type: HeLa cells;

cell seeding density: 10,000 cells/well;

media: DMEM, 10% FBS, 0.1 mg/ml penicillin-streptomycin, 2 mM L-glutamine;

DMSO concentration: 0.5%;

assay plate: 96-well white, TC (with culture plate), sterile, with lid;

compound incubation time: 18 hours;

CO ₂ level: 5%;

- 73 040674 humidity: 95%;

temperature: 37°C;

H2O2 concentration: 200 μM.

Cell seeding and treatment with compounds.

In a 96-well cell culture microplate, HeLa cells were seeded at a density of 10,000 cells/well in a 90 μl culture medium. The plates were incubated for 4 hours at 37° C. under 5% CO2. 10 μl samples were added from 8 serial dilutions of 10X solutions of compounds in 5% DMSO (concentration range: 0.3-100 nM). Prepared plates were incubated for 18 hours at 37°C in 5% CO2. Added 5 μl of a solution of H2O2 in H2O (final concentration 200 μm) to induce DNA damage. Cells untreated with H ₂ O ₂ were stored in negative control wells. The plate was incubated for 5 min at 37°C and then carefully inverted to remove the medium. 50 μl of 1x PBS was added to all wells.

Determination of PARP activity: separate measurement of NAD+ and NADH levels.

This protocol is designed to analyze cells in 50 µl of PBS per well in a 96-well white luminometer plate. The contents of each well with cells were divided into two samples: one sample was treated with acid for subsequent quantitative determination of NAD+, and the other sample was treated with base for subsequent quantitative determination of NAD(H). At the stage of seeding cells into a plate, wells were reserved on the plate for subsequent division of samples. Alternatively, a second plate can be used to split samples.

To each well containing cells in 50 μl PBS was added 50 μl base solution with 1% DTAB (Dodecyltrimethylammonium bromide). The contents of the tablet was quickly mixed on a tablet shaker to ensure homogeneity and cell lysis. 50 μl of each sample was transferred to an empty well for subsequent acid treatment. To these samples were added 25 μl of 0.4 n. HCl per well; these wells contained samples treated with acid. The samples in the original wells were samples treated with base. The tablet was closed and incubated for 15 min at 60°C. The plate temperature was then equilibrated for 10 minutes to room temperature. To neutralize the acid, 25 μl of 0.5 M Tpizma® base was added to each well containing acid-treated cells. To each well containing the base-treated sample, 50 μl of HCl/Tpizma® solution was added. NAD/NADH-Glo™ registration reagent was prepared as described above. An equal volume of NAD/NADH-Glo™ detection reagent (eg, 100 µl) was added to each well. The plate was gently shaken to mix the samples. The tablet was incubated for 30-60 min at room temperature. Luminescence was recorded using a luminometer (Envision, PerkinElmer). Carried out the collection of data obtained during the registration of luminescence. A non-linear regression model was used to construct a dose-response curve and calculate IC ₅₀ values.

The table below shows the results of the analysis of the inhibitory effect of typical compounds of the present invention in relation to the activity of PARP1-1 and PARP-2. These results show that the compounds of the present invention selectively inhibit PARP1-1 over PARP-2 and can be used to increase the amount of NAD+ in cells.

Example No. Structure Mean IC ₅₀ PARP-1 (highest concentration 10 µM) (nM) Mean IC ₅₀ PARP-2 (highest concentration 10 µM) (nM) H ₂ O ₂ NAD ⁺ HeLa cell assay EC ₅₀ (nM) Prime R 1 ° |^ΑνΗ ^C| 27 181 909 Prime R 2 O ci O 7 69

- 74 040674

Prime R 3 O Cl O 16 196 1322 Prime R 4 2 jOr n Cl 26 1658 Prime R 5 0 JU? _l II _l JI JH L JL JL NJ Cl H 0 51 1096 Prime R 6 O . . O iCV^^ _l P _l JI 1 and Μ^Α^γ^Ι Cl ^H O 29 1271 Prime R 7 2 V7 O ii^r A II J h i^Y^NI^N— ^Λ^γΝ^ Cl ⁿ o thirty 2630 Prime R 8 10 (P=ψ^ΝΗ O 325 Prime R 9 i J0 .''Ύ^ΝΗ ABOUT 160 Prime R 10 .rcAyn O 180 Prime R eleven i jiY ^N [Pf'N H Μ^Α^γΝ^ι o 6 338 50 Prime R 12 o O |j ^Z ^f ^4NH 2 [ί^^Ζ^ΝΗ ι^Ν^Ν^ 25

- 75 040674

Prime R 13 1°ifT™ ___j about 54 Prime R 14 ,CN l GU Ν H ABOUT 23 974 Prime R 15 .CN ΑΑ _Ν Αχγ^ o 196 Prime R 16 uch .CN o gP ^Α _Ν Αχγ ^Ν Α 7 40 163 Prime R 17 ^f ° rT H ΧΊ\Α|\Γ ΙχΑιΑχγΰ___J o 8 215 16 Prime R 18 _F x rr“ UCh/Xn Xn— o 7 216 24 Prime R 19 ΛΓΥ pXN H<4---N' A I. A l N J ABOUT 83 Prime R 20 ° rr ^CN |ΛΛν h 12 288 Prime R 21 ^^. ^CN π ΐί^Υ^Ν H [^N'^N'J UnVynJ o 99 Prime R 22 χί rr ^CN H |^N----- O 395

- 76 040674

Prime R 23 O ^j^NH p^N^N^ 35 10294 552 Prime R 24 ° rr ^CN O eleven 1591 341 Prime R 25 ,Ν Xi IL ^JL JL n JL ABOUT 70 Prime R 26 ^N A jfj N H p^N^N^ U JL JL ___ nJ. ABOUT 47 770 Prime R 27 A jfj Un^nJ o · 94 Prime R 28 _S ΝHpW U JL JL nJ o- 10 910 138 Prime R 29 ο ί^Γ ^Ρ ·γ^Χ ^4ΝΗ p^N-^N^ xA _m A _h /'y ^y y1 12 611 156 Prime R thirty ^οΞ 12 624 110 Prime R 31 > ^Ν A Ρ'Υρ Ν Η ρ^Ν^Ν^ -ύ ^ν Χ ° Ύ)Η 6 42 440 Prime R 32 ° ry ^{CN f>} XV ^A4nh Ρ^νΑμ* ¹ ΧΑνΛ^υϋ 5 1232

- 77 040674

Prime R 33 ^F ° ° %n 7 2736 Prime R 34 [[^Αν Η UA^/J ABOUT 54 Prime R 35 o Μ-Α^ν ^Ν Α o 35 562 Prime R 36 (ΡΑνη ^νΌ ¹ Ιί,ΑΑ^^ΰ^χΙ o 120 Prime R 37 JOl about 359 Prime R 38 S2) M-A^A 129 Prime R 39 s x'A 4Α _Ν Αχ-γ^Ά O 14 468 754 Prime R 40 CN u K |^Ν ^Λ Ν MA/V^ o 53 1502 Prime R 41 .CN jjfy ϋ d. ¢1___ NJ ABOUT 97 Prime R 42 ° jfy ^CN O 58 12271

- 78 040674

Prime R 43 fl jfjl 42 3817 440 Prime R 44 ^F ° rv™ ^F Vir ^ΝΗ eleven 345 Prime R 45 Ο _Λ JI k JJ Η S Ν^ΓΤ o 92 Prime R 46 CN ABOUT Ν Η ^Ν^Ν ___ ABOUT 278 Prime R 47 ABOUT .2 .ΛL । Cl ABOUT 922 Prime R 48 NH° ^N - about thirty 7564 Prime R 49 HN^\ ο (^N^N^ ABOUT 36 22383 Prime R 50 ° ίΎ ^Ρ 255 Prime R 51 ζΛ ABOUT 83 339 Prime R 52 Ζ\Ανη 68 999 Prime R 53 ° ct ^f Οι Ср О 189

Example 56 Rat Model of Acute Kidney Injury (AKI).

Animals, surgery and doses. In these experiments, male Sprague-Dawley rats weighing approximately 300-350 g were used, which were provided with unlimited access to standard food and water. Rats were anesthetized with isoflurane and placed ventrally on a temperature-controlled heated surgical platform. A skin incision was made on the dorsal surface of the body, exposing both kidneys through lateral incisions. In the area of the gates of both kidneys, clamps were applied to clamp the vessels, occlusion lasted 45 minutes. After 45 minutes, the clamps were removed, the kidney reperfusion was verified to be successful, and then the surgical incisions were sutured. Animals in the surgical placebo group underwent a similar surgical procedure, except that no occlusive clamps were applied. Compound preparations were prepared on the day of administration as a clear solution of Example 29 or Example 30 in a mixture of N-methylpyrrolidone, PEG300, propylene glycol and saline (10:30:20:40). Compounds or vehicle were administered intravenously (intravenously) via the tail vein at a dose of 15 mg/kg (3 ml/kg) 4 hours after reperfusion on the day of surgery.

-

Claims (21)

On Day 1 (day post-surgery), animals were treated with vehicle or 15 mg/kg of Example 29 or Example 30 (3 ml/kg, iv) at the start of the light cycle. Animals in the surgical placebo group were similarly treated with vehicle. Plasma collection and biomarker quantitation. 24 hours after reperfusion, blood was taken from the retroorbital sinus in all groups into test tubes with the anticoagulant K ₂ EDTA, using isoflurane for light anesthesia of the animals. Plasma was separated by centrifugation at 3000 rpm for 10 min at 4°C. Plasma creatinine and blood urea nitrogen (BUN) analysis was performed using a fully automated clinical chemistry analyzer (Siemens Dimension® Xpand® Plus Integrated Chemistry System). Data analysis and statistical analysis. GraphPad Prism version 6.05 was used for plotting and statistical analysis. In all groups, the normality of the distribution of data on creatinine and BUN was tested using the D'Agostino-Pearson omnibus normality test or the Shapiro-Wilk normality test. Statistical significance (p<0.05) was determined using Student's t-test comparing the Placebo-Vehicle group with the IR-Vehicle group, or the IR-Vehicle group with the compound-treated group. ##p<0.01, ###p<0.001, ####p<0.0001 Placebo vs. IR-Carrier; *p<0.05, **p<0.01 IR-Carrier vs. the group receiving the compound (example 29 or example 30). Results. The PARP1 inhibitors described in Example 29 and Example 30, when administered i.v. after ischemia-reperfusion injury (IRI), reduced kidney damage. Both compounds significantly reduced plasma creatinine and BUN when administered at a dose of 15 mg/kg (see figure). Example 57 In vitro and in vivo micronucleus analysis. Some of the compounds of the present invention lacked any clastogenic activity according to the results of in vitro and/or in vivo micronucleus analysis. CLAIM 1. Pharmaceutical composition for the treatment of a subject having a disease that can be attenuated by inhibition of poly(ADP-ribose)polymerase (PARP), containing a pharmaceutically acceptable carrier or diluent and a compound represented by the following structural formula: or a pharmaceutically acceptable salt thereof, wherein ring A is phenyl optionally substituted with one or two groups selected from halogen and (C ₁ -C ₃ )alkyl, or 5-6 membered heteroaryl optionally substituted with one or two groups selected from halogen and (C ₁ -C ₃ )alkyl; ring B is phenyl, 5-6 membered heteroaryl, or 5-6 membered heterocyclyl, each of which optionally has one or more substituents represented by R ³ ; ----- absent or represents a link; E is N or CH when ----- is absent, or E is C when ----- is a bond; optionally substituted with (C ₁ -C ₅ )alkyl or hydroxy(C ₁ -C ₅ )alkyl; each R ³ is independently selected from the group consisting of -halogen, -CN, -C(=NR ^e )NR ^e R ^f , -C(=O)NR ^e R ^f and (C ₁ -C ₅ )alkyl; each R ^e is independently selected from the group consisting of -H and (C ₁ -C ₅ )alkyl; each R ^f is independently selected from the group consisting of -H, (C ₁ -C ₅ )alkyl, (C ₃ -C ₆ )cycloalkyl optionally substituted with (C ₁ -C ₂ )alkyl, and 4-6- membered oxygen-containing heterocyclyl optionally having (C ₁ -C ₂ )alkyl as a substituent; or -C(=NR ^e )NR ^e R ^f forms a 4-6 membered heterocyclyl optionally substituted with R ^e ; And R ⁵ is -H or (C ₁ -C ₃ )alkyl; and where heteroaryl contains 1-4 heteroatoms selected from oxygen, nitrogen and sulfur, and heterocyclyl contains 1 or 2 heteroatoms selected from oxygen, nitrogen and sulfur. - 80 040674 2. Pharmaceutical composition according to claim 1, wherein the compound is represented by a structural formula selected from the group consisting of or a pharmaceutically acceptable salt thereof, where each of X ¹ , X ² , X ³ and X ⁴ is independently selected from the group consisting of N and CH, provided that no more than two of X ¹ , X ² , X ³ and X ⁴ are N; X ⁵ is NR ² , O or S; ring B is phenyl, 5-6 membered heteroaryl, or 5-6 membered heterocyclyl, each of which optionally has one or more substituents represented by R ³ ; optionally substituted with (C ₁ -C ₅ )alkyl or hydroxy(C ₁ -C ₅ )alkyl; each R ¹ is independently selected from the group consisting of α-halogen and (C1-C ₃ )alkyl; R ² is -H or (C ₁ -C ₃ )alkyl; and n is 0, 1, or 2. 3. Pharmaceutical composition according to claim 2, wherein the compound is represented by a structural formula selected from the group consisting of or a pharmaceutically acceptable salt thereof, wherein ring B is phenyl, 5-6 membered heteroaryl, or 5-6 membered heterocyclyl, each of which optionally has one or more substituents represented by R ³ ; And optionally substituted with (C1-C5)alkyl or hydroxy( _C1 - _C5 )alkyl; each R1 is independently selected from the group consisting of halogen and (C ₁ -C ₃ )alkyl; and each R ³ is independently selected from the group consisting of -halogen, -CN, -C(=NR ^e )NR ^e R ^f , -C(=O)NR ^e R ^f and (C1-C5)alkyl. - 81 040674 4. Pharmaceutical composition according to claim 2 or 3, characterized in that ring B is selected from the group consisting of where each R ⁴ represents -H or (C ₁ -C ₅ )alkyl; each p is independently 0 or 1; each R ¹ is independently halogen or (C1-C ₃ )alkyl; each R ³ is independently selected from the group consisting of -halogen, -CN, -C(=NR ^e )NR ^e R ^f , -C(=O)NR ^e R ^f and (C1-C ₅ )alkyl; each R ^e and each R ^f is independently selected from the group consisting of -H and methyl; or R ^e is -H and R ^f is -(C ₃ -C ₆ )cycloalkyl or 4-6 membered oxygen-containing heterocyclyl, each of which is optionally substituted with (C ₁ -C ₂ )alkyl; and each m is 0, 1, or 2. 5. Pharmaceutical composition according to any one of paragraphs.2-4, characterized in that each R ¹ independently represents chlorine, fluorine or methyl; each R ³ is independently selected from the group consisting of chlorine, fluorine, -CN, -C(=NR ^e )NR ^e R ^f , C(=O)NR ^e R ^f and methyl; the e group optionally has a methyl or hydroxymethyl substituent. 6. Pharmaceutical composition according to claim 4 or 5, characterized in that each R ³ is independently selected from the group consisting of chlorine, fluorine, -CN, -C(O)NH(cyclopropyl), -C(O)NH ₂ , -C(O)NH(CH ₃ ), -C (O) N (CH ₃ ) 2, ⁿ '-C (O) KSH cyclobutyl), > -C(=NH)NHCH ₃ and methyl. 7. Pharmaceutical composition according to claim 2, characterized in that R ² is -H or methyl. 8. Pharmaceutical composition according to claim 1, characterized in that the compound is 6-[(3S)-4-[3-(6-fluoro-4-oxo-3H-quinazolin-2-yl)propanoyl]-3 -methyl-piperazin-1 -yl]pyridine-3-carbonitrile or a pharmaceutically acceptable salt thereof. 9. Pharmaceutical composition according to claim 1, characterized in that the compound is 6-[(3 S)-4-[3 -(5-fluoro-4-oxo-3 H-quinazolin-2-yl)propanoyl] - 3-methyl-piperazin-1-yl]pyridine-3-carbonitrile or a pharmaceutically acceptable salt thereof. 10. Compound of the following structural formula: or methyl. or a pharmaceutically acceptable salt thereof, wherein R ¹ is F or methyl; And R ³ is -CN, -C(=NH)NHCH3, 11. A compound or pharmaceutically acceptable salt thereof according to claim 10, wherein R ¹ is F; And R ³ is -CN. 12. The compound according to claim 10, which is 6-[(3S)-4-[3-(6-fluoro-4-oxo-3H-quinazolin-2yl)propanoyl]-3-methyl-piperazin-1-yl ]pyridine-3-carbonitrile or a pharmaceutically acceptable salt thereof. 13. The compound according to claim 10, which is 6-[(3S)-4-[3-(5-fluoro-4-oxo-3H-quinazolin-2yl)propanoyl]-3-methyl-piperazin-1-yl ]pyridine-3-carbonitrile or a pharmaceutically acceptable salt thereof. 14. A method of treating a subject having a disease that can be attenuated by inhibition of poly(ADP-ribose)polymerase (PARP), comprising administering to said subject an effective amount of a pharmaceutical composition according to any one of claims 1 to 9, or a compound, or a pharmaceutical thereof. - 82 040674 a maceutically acceptable salt according to any one of claims 10-13. 15. A method of treating a subject with acute kidney injury, comprising administering to said subject an effective amount of a pharmaceutical composition according to any one of claims 1-9, or a compound, or a pharmaceutically acceptable salt thereof, according to any one of claims 10-13. 16. A method of treating a subject suffering from cancer that can be attenuated by inhibition of poly(ADP-ribose) polymerase (PARP), comprising administering to said subject an effective amount of a pharmaceutical composition according to any one of claims 1 to 9, or a compound, or a pharmaceutically acceptable thereof salt according to any one of claims 10-13. 17. The method of claim 14, wherein the disease that can be attenuated by PARP inhibition is selected from genetic lipodystrophy, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), ischemic/reperfusion kidney injury (IRI), Duchenne and Becker muscular dystrophy, diabetes (type I or type II), obesity, sarcopenia, Alpers disease, chronic progressive external ophthalmoplegia (CPEO), Kearns-Sayre syndrome (KSS), Leber hereditary optic neuropathy (LHON), MELAS - mitochondrial myopathy , encephalomyopathy, lactic acidosis and stroke-like episodes, MERRF - myoclonic epilepsy with torn muscle fibers, NARP - neurogenic muscle weakness, ataxia and retinitis pigmentosa, Pearson's syndrome, platinum chemotherapy-induced ototoxicity, Cockayne's syndrome, xeroderma pigmentosa A, Wallerian degeneration and HIV-induced lipodystrophy . 18. Pharmaceutical composition according to any one of claims 1 to 9, intended for the treatment of a wound and/or burn in a subject. 19. Pharmaceutical composition according to any one of claims 1 to 9 for the treatment of acute kidney injury in a subject. 20. A method of treating a wound in a subject, comprising administering to said subject an effective amount of a pharmaceutical composition according to any one of claims 1-9, or a compound, or a pharmaceutically acceptable salt thereof, according to any one of claims 10-13. 21. A method of treating a burn in a subject, comprising administering to said subject an effective amount of a pharmaceutical composition according to any one of claims 1-9, or a compound, or a pharmaceutically acceptable salt thereof, according to any one of claims 10-13. -