Pyrrolidine derivative and application thereof in medicine

Abstract

The invention relates to a pyrrolidine derivative and application thereof in medicine, in particular to a pyrrolidine derivative shown in a general formula (I) or a stereoisomer, a solvate, a prodrug, a metabolite, a deuterated substance, pharmaceutically acceptable salt or eutectic of the pyrrolidine derivative, and a pharmaceutical composition containing the pyrrolidine derivative. The invention also discloses application of the pyrrolidine derivative or the pharmaceutical composition in preparation of antitumor drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a pyrrolidine derivative and its application in medicine. Background Art

[0002] Mammalian cells primarily repair DNA double-strand breaks (DSBs) through non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ or alt-EJ) pathways to ensure genomic stability. DNA polymerase theta (Polθ or POLQ) is a key component of the MMEJ pathway and is involved in DNA double-strand break repair.

[0003] Polθ is one of 15 DNA polymerases in the human genome, consisting of a C-terminal family A DNA polymerase and an N-terminal superfamily 2 (SF2) type DNA helicase, separated by a long and poorly conserved central domain of unknown function.

[0004] Pol θ is barely expressed in normal tissues but is highly expressed in a variety of tumor types, such as breast cancer, ovarian cancer, HNSCC, and lung cancer. When DNA end resection occurs, in the presence of BRCA2, BRCA2 not only recruits the recombinase RAD51 to the DSB to promote HR, but also inhibits repair pathways such as MMEJ. When homologous recombination-mediated repair is impaired (HR deficiency), such as in BRCA1 or BRCA2 mutations, Pol θ is highly expressed and directs DSB repair toward alt-EJ, initiating the DNA repair process of MMEJ. In the case of HR deficiency, inhibition of Pol θ leads to cell death through the accumulation of toxic RAD51 intermediates and inhibition of the alt-EJ repair pathway (Jia Zhou, et al. A first-in-class polymerase theta inhibitor selectively targets homologous-recombination-deficient tumors. Nature Cancer, 2021: 598–610).

[0005] Polθ is therefore an attractive novel synthetic lethality therapeutic target in cancers with DNA repair defects. Summary of the Invention

[0006] The present invention aims to provide a new pyrrolidine derivative having an inhibitory effect on Polθ and its application in preparing antitumor drugs.

[0007] One or more embodiments of the present invention provide a compound represented by general formula (I) or all stereoisomers, solvates, prodrugs, metabolites, deuterated substances, pharmaceutically acceptable salts or cocrystals thereof:

[0008]

[0009] in:

[0010] A is selected from 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl, wherein the 3-8 membered heterocycloalkyl contains 1 to 4 heteroatoms selected from N, O or S;

[0011] B is selected from 5-12 membered heteroaryl, wherein the 5-12 membered heteroaryl contains 1 to 4 heteroatoms selected from N, O or S;

[0012] C is selected from 5-12 membered aryl or 5-12 membered heteroaryl, wherein the 5-12 membered heteroaryl contains 1 to 4 heteroatoms selected from N, O or S;

[0013] R a Can be the same or different, R a Selected from H, C 1-6 Alkyl or OH, the C 1-6 The alkyl group is optionally further substituted with one or more substituents selected from halogen;

[0014] R2, R3 are H, or R2, R3 and the carbon atom to which they are connected form C=O;

[0015] R b Can be the same or different, R b Selected from H, C 1-6 Alkyl or CN, the C 1-6 The alkyl group is optionally further substituted with one or more substituents selected from halogen;

[0016] R1 is C 1-3 alkyl;

[0017] R c Can be the same or different, R c selected from halogen;

[0018] a is 0, 1, 2, or 3;

[0019] b is 1, 2, or 3;

[0020] c is 1, 2, or 3.

[0021] In one or more embodiments of the present invention, the compound or all stereoisomers, solvates, prodrugs, metabolites, deuterated substances, pharmaceutically acceptable salts or cocrystals thereof,

[0022] in:

[0023] A is selected from 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl, wherein the 3-8 membered heterocycloalkyl contains 1 to 4 heteroatoms selected from N, O or S;

[0024] B is selected from 5-12 membered heteroaryl, wherein the 5-12 membered heteroaryl contains 1 to 4 heteroatoms selected from N, O or S;

[0025] C is selected from 5-12 membered aryl;

[0026] R a Can be the same or different, R a Selected from H, C 1-6 Alkyl or OH, the C 1-6 The alkyl group is optionally further substituted with one or more substituents selected from halogen;

[0027] R2, R3 are H, or R2, R3 and the carbon atom to which they are connected form C=O;

[0028] R b Can be the same or different, R b Selected from H, C 1-6 Alkyl or CN, the C 1-6 The alkyl group is optionally further substituted with one or more substituents selected from halogen;

[0029] R1 is C 1-3 alkyl;

[0030] R c Can be the same or different, R c selected from halogen;

[0031] a is 0, 1, 2, or 3;

[0032] b is 1, 2, or 3;

[0033] c is 1, 2, or 3.

[0034] In one or more embodiments of the present invention, the compound, or its stereoisomer, solvate, prodrug, metabolite, deuterated substance, pharmaceutically acceptable salt or cocrystal is selected from:

[0035]

[0036] One or more embodiments of the present invention provide a pharmaceutical composition comprising:

[0037] (1) a compound of the present invention or a stereoisomer, solvate, prodrug, metabolite, deuterated form, pharmaceutically acceptable salt, or cocrystal thereof;

[0038] (2) optionally one or more other active ingredients; and

[0039] (3) Pharmaceutically acceptable carriers and / or excipients.

[0040] One or more embodiments of the present invention provide the use of the pharmaceutical composition of the present invention or the compound or its stereoisomer, solvate, prodrug, metabolite, deuterated substance, pharmaceutically acceptable salt or cocrystal in the preparation of an anti-tumor drug.

[0041] Unless stated otherwise, the terms used in the specification and claims have the following meanings.

[0042] The carbon, hydrogen, oxygen, sulfur, nitrogen or F, Cl, Br, I involved in the groups and compounds of the present invention include their isotopes, and the carbon, hydrogen, oxygen, sulfur or nitrogen involved in the groups and compounds of the present invention are optionally further replaced by one or more of their corresponding isotopes, wherein the isotopes of carbon include 12 C. 13 C and 14 C, hydrogen isotopes include protium (H), deuterium (D, also called heavy hydrogen), tritium (T, also called super tritium), oxygen isotopes include 16 O. 17 O and 18 O, sulfur isotopes include 32 S. 33 S. 34 S and 36 S, nitrogen isotopes include 14 N and 15 N, fluorine isotopes include 17 F and 19 F, chlorine isotopes include 35 Cl and 37 Isotopes of Cl, bromine include 79 Br and 81 Br.

[0043] "Alkyl" refers to a linear or branched saturated aliphatic hydrocarbon group of 1 to 20 carbon atoms, preferably an alkyl group of 1 to 8 carbon atoms, more preferably an alkyl group of 1 to 6 carbon atoms, and even more preferably an alkyl group of 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, neobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and various branched isomers thereof. When an alkyl group is substituted, it may optionally be further substituted with one or more substituents.

[0044] "Cycloalkyl" refers to a saturated cyclic hydrocarbon group, which may be a 3- to 10-membered monocyclic ring, a 4- to 12-membered bicyclic ring, or a 10- to 20-membered polycyclic ring system, preferably having 3 to 10 carbon atoms, more preferably 3 to 8 carbon atoms. Non-limiting examples of "cycloalkyl" include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, 1,5-cyclooctadienyl, 1,4-cyclohexadienyl, and cycloheptatrienyl. When substituted, it may be further substituted by zero or more substituents.

[0045] "Heterocycloalkyl" refers to a substituted or unsubstituted saturated non-aromatic ring group, which may be a 3- to 8-membered monocyclic ring, a 4- to 12-membered bicyclic ring, or a 10- to 15-membered tricyclic ring system, and contains 1 to 3 heteroatoms selected from N, O, or S, preferably a 3- to 8-membered heterocyclic ring. The N and S atoms optionally substituted in the heterocycloalkyl ring may be oxidized to various oxidation states; the heterocycloalkyl group may be attached to a heteroatom or a carbon atom; and the heterocycloalkyl group may be a bridged ring or a spirocyclic ring. Non-limiting examples of “heterocycloalkyl” include oxiranyl, aziridinyl, oxetanyl, azetidinyl, 1,3-dioxolanyl, 1,4-dioxolanyl, 1,3-dioxanyl, azepanyl, piperidinyl, piperidinyl, morpholinyl, thiomorpholinyl, 1,3-dithianyl, tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydropyranyl, azabicyclo[3.2.1]octanyl, azabicyclo[5.2.0]nonanyl, oxatricyclo[5.3.1.1]dodecyl, azaadamantyl, and oxaspiro[3.3]heptanyl.

[0046] "Aryl" refers to a substituted or unsubstituted aromatic ring, which can be a 5-8 membered (e.g., 5, 6, 7, 8 membered) monocyclic ring, a 5-12 membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12 membered) bicyclic ring, or a 10-15 membered (e.g., 10, 11, 12, 13, 14, 15 membered) tricyclic ring system, which can be a bridged ring or a spirocyclic ring, non-limiting examples of which include phenyl and naphthyl. The aryl group may optionally be further substituted with one or more substituents.

[0047] "Heteroaryl" refers to a substituted or unsubstituted aromatic ring, which can be a 3-8 membered (e.g., 3, 4, 5, 6, 7, 8 membered) monocyclic ring, a 5-12 membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12 membered) bicyclic ring or a 10-15 membered (e.g., 10, 11, 12, 13, 14, 15 membered) tricyclic ring system, and contains 1 to 6 (e.g., 1, 2, 3, 4, 5, 6) heteroatoms selected from N, O or S, preferably a 5- to 8-membered heteroaryl group, and the 1 to 4 (e.g., 1, 2, 3, 4) N and S optionally substituted in the heteroaryl ring can be oxidized to various oxidation states. The heteroaryl group may be attached to a heteroatom or carbon atom, and may be a cyclic, bridged, or spirocyclic ring. Non-limiting examples include cyclic pyridyl, furyl, thienyl, pyranyl, pyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, piperidinylbenzimidazolyl, benzopyridinyl, and pyrrolopyridinyl. The heteroaryl group may be further substituted with one or more substituents. When the "alkyl," "cycloalkyl," "heterocycloalkyl," "aryl," or "heteroaryl" described above is substituted, it may be further substituted with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents selected from F, Cl, Br, I, hydroxyl, sulfhydryl, nitro, cyano, amino, C 1-6 Alkylamino, =O, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, -NR q4 R q5 、=NR q6 、-C(=O)OC 1-6 Alkyl, -OC(=O)C 1-6 Alkyl, -C(=O)NR q4 R q5 、C 3-8 Cycloalkyl, C 3-8 Heterocycloalkyl, C 6-10 Aryl, C 5-10 Heteroaryl, -C(=O)OC 6-10 Aryl, -OC(=O)C 6-10 Aryl, -OC(=O)C 5-10 Heteroaryl, -C(=O)OC 5-10 Heteroaryl, -OC(=O)C 3-8 Heterocycloalkyl, -C(=O)OC 3-8 Heterocycloalkyl, -OC(=O)C 3-8 Cycloalkyl, -C(=O)OC 3-8 Cycloalkyl, -NHC(=O)C 3-8 Heterocycloalkyl, -NHC(=O)C 6-10 Aryl, -NHC(=O)C 5-10Heteroaryl, -NHC(=O)C 3-8 Cycloalkyl, -NHC(=O)C 3-8 Heterocycloalkyl, -NHC(=O)C 2-6 Alkenyl or -NHC(=O)C 2-6 substituted by a substituent of an alkynyl group, wherein the substituent C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl, C 3-8 Heterocycloalkyl, C 6-10 Aryl, C 5-10 Heteroaryl, -NHC(=O)C 6-10 Aryl, -NHC(=O)C 5-10 Heteroaryl, -NHC(=O)C 3-8 Heterocycloalkyl or -NHC(=O)C 3-8 The cycloalkyl group is optionally further substituted by 1 to 3 groups selected from OH, F, Cl, Br, I, C 1-6 Alkyl, C 1-6 Alkoxy, -NR q4 R q5 Or substituted by a substituent of =O; R q1 Selected from C 1-6 Alkyl, C 1-6 Alkoxy or C 6-10 Aryl; R q2 、R q3 Selected from H or C 1-6 Alkyl; R q4 、R q5 Selected from H, C 1-6 Alkyl, -NH(C=NR q1 )NR q2 R q3 、-S(=O)2NR q2 R q3 、-C(=O)R q1 or -C(=O)NR q2 R q3 , wherein the C 1-6 The alkyl group is optionally further substituted by one or more groups selected from OH, F, Cl, Br, I, C 1-6 Alkyl, C 1-6 Alkoxy, C 6-10 Aryl, C 5-10 Heteroaryl, C 3-8 Cycloalkyl or C 3-8 is substituted by a substituent of a heterocycloalkyl group; or R q4 With R q5and the N atom form a 3- to 8-membered heterocyclic ring, which may contain one or more heteroatoms selected from N, O or S.

[0048] Halogens include F, Cl, Br and I.

[0049] "Pharmaceutically acceptable salt" or "pharmaceutically acceptable salt thereof" refers to a salt of the compound of the present invention that retains the biological effectiveness and properties of the free acid or free base, and the free acid is obtained by reacting with a non-toxic inorganic base or organic base, or the free base is obtained by reacting with a non-toxic inorganic acid or organic acid.

[0050] "Pharmaceutical composition" refers to a mixture of one or more compounds of the present invention, their pharmaceutically acceptable salts or prodrugs and other chemical components, wherein "other chemical components" refers to pharmaceutically acceptable carriers, excipients and / or one or more other therapeutic agents.

[0051] "Carrier" refers to a material that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

[0052] "Excipient" refers to an inert substance added to a pharmaceutical composition to facilitate administration of a compound. Non-limiting examples include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives (including microcrystalline cellulose), gelatin, vegetable oils, polyethylene glycols, diluents, granulating agents, lubricants, binders, and disintegrants.

[0053] "Prodrugs" refer to compounds of the present invention that can be converted into biologically active compounds through in vivo metabolism. Prodrugs of the present invention are prepared by modifying amino or carboxyl groups in compounds of the present invention. These modifications can be removed by conventional manipulation or in vivo to yield the parent compound. When the prodrugs of the present invention are administered to a mammalian subject, the prodrugs are cleaved to form free amino or carboxyl groups.

[0054] A "cocrystal" is a crystal formed by the active pharmaceutical ingredient (API) and cocrystal former (CCF) bound together by hydrogen bonds or other non-covalent bonds. Both the API and CCF are solid in their pure form at room temperature, and the components exist in a fixed stoichiometric ratio. A cocrystal is a multi-component crystal, encompassing both binary cocrystals formed between two neutral solids and multi-component cocrystals formed between a neutral solid and a salt or solvate.

[0055] "Stereoisomers" refer to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, and conformational isomers.

[0056] "Optional" or "optionally" or "selectively" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "heterocyclyl optionally substituted with alkyl" means that the alkyl group may but need not be present, and that the description includes instances where the heterocyclyl group is substituted with alkyl and instances where the heterocyclyl group is not substituted with alkyl. DETAILED DESCRIPTION

[0057] The following examples illustrate the technical solutions of the present invention in detail, but the protection scope of the present invention includes but is not limited to them.

[0058] The structures of the compounds were confirmed by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) are given in units of 10-6 (ppm). NMR measurements were performed using a Bruker Avance III 400 and Bruker Avance 300 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS).

[0059] MS was determined using (Agilent 6120B (ESI) and Agilent 6120B (APCI));

[0060] HPLC determination was performed using an Agilent 1260DAD high-pressure liquid chromatograph (Zorbax SB-C18 100 × 4.6 mm, 3.5 μM);

[0061] Thin layer chromatography silica gel plates used were Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The specifications of the silica gel plates used for thin layer chromatography (TLC) were 0.15 mm to 0.20 mm, and the specifications used for thin layer chromatography separation and purification products were 0.4 mm to 0.5 mm.

[0062] Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier;

[0063] The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from companies such as Titan Technology, Anage Chemical, Shanghai Demer, Chengdu Kelon Chemical, Shaoyuan Chemical Technology, Bailingwei Technology, and Jiangsu Aikon Biopharmaceutical Research and Development.

[0064] Example 1

[0065] (1S,3aR,6aS)-N-(3-chloro-4-fluorophenyl)-N-methyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide compound 1-1

[0066] (1S,3aR,6aS)-N-(3-chloro-4-fluorophenyl)-N-methyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide

[0067] (1R,3aS,6aR)-N-(3-chloro-4-fluorophenyl)-N-methyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide compound 1-2

[0068] (1R,3aS,6aR)-N-(3-chloro-4-fluorophenyl)-N-methyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide

[0069]

[0070] first step:

[0071] 2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopentyl[c]pyrrole-1-carboxylic acid 1b

[0072] 2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrol e-1-carboxylic acid

[0073] Methyl 2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxylate (1.0 g, 3.0 mmol) was dissolved in methanol (10.0 mL), followed by the addition of lithium hydroxide monohydrate (240 mg, 5.8 mmol). The mixture was stirred at 25°C for 5 h. LCMS monitoring confirmed the completion of the reaction. The reaction solution was adjusted to pH 6, extracted with ethyl acetate, dried, and concentrated to afford the target compound 1b, 2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxylic acid (950 mg, yellow solid, 96% yield).

[0074] LCMS m / z (ESI) = 329.10 [M+1].

[0075] Step 2:

[0076] N-(3-chloro-4-fluorophenyl)-N-methyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide compound 1

[0077] N-(3-chloro-4-fluorophenyl)-N-methyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide

[0078] 2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxylic acid 1b (450 mg, 1.2 mmol) was dissolved in pyridine (10 g, 13 mmol), followed by the addition of 1-n-propylphosphoric acid cyclic anhydride (3.80 g, 13 mmol), and then 3-chloro-4-fluoro-N-methylaniline (240 mg, 1.3 mmol). The mixture was reacted at room temperature for 4 h, and the reaction solution was directly spin-dried and purified by reverse phase column chromatography to obtain N-(3-chloro-4-fluorophenyl)-N-methyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide compound 1 (250 mg, white solid, 44% yield).

[0079] LC-MS m / z (ESI) = 470.29 [M+1].

[0080] 1 H NMR(400MHz,Chloroform-d)δ8.66(s,1H),7.64(s,1H),7.37(s,1H),7.31(t,1H),7.08(s,1H),4.73(d,1H),4.34–4.31(m,1H),3.29( s,3H),2.59(s,3H),2.57–2.56(m,1H),2.10–2.09(m,1H),1.95–1.84(m,1H),1.76–1.73(m,2H),1.61–1.52(m,1H),1.44–1.41(m,1H) .

[0081] Step 3:

[0082] (1S,3aR,6aS)-N-(3-chloro-4-fluorophenyl)-N-methyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide compound 1-1

[0083] (1S,3aR,6aS)-N-(3-chloro-4-fluorophenyl)-N-methyl-2-(6-methyl-4-(trifluoromet hyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide

[0084] (1R,3aS,6aR)-N-(3-chloro-4-fluorophenyl)-N-methyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide compound 1-2

[0085] (1R,3aS,6aR)-N-(3-chloro-4-fluorophenyl)-N-methyl-2-(6-methyl-4-(trifluoromet hyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide

[0086] Compound 1-1 and compound 1-2 were separated by chiral prep-HPLC. Analytical method: C3 IC MB 20% 1 mL 10 min 1 cm column, ethanol as the mobile phase, flow rate 1 mL / min, retention time of compound 1-1 was 2.104 min, and retention time of compound 1-2 was 4.287 min.

[0087] Compound 1-1 (106 mg, white solid):

[0088] LC-MS m / z (ESI) = 470.29 [M+1].

[0089] 1H NMR(400MHz,Chloroform-d)δ8.66(s,1H),7.64(s,1H),7.37(s,1H),7.31(t,1H),7.08(s,1H),4.73(d,1H),4.34–4.31(m,1H),3.29( s,3H),2.59(s,3H),2.57–2.56(m,1H),2.10–2.09(m,1H),1.95–1.84(m,1H),1.76–1.73(m,2H),1.61–1.52(m,1H),1.44–1.41(m,1H).

[0090] Compound 1-2 (106 mg, white solid):

[0091] LC-MS m / z (ESI) = 470.29 [M+1].

[0092] 1 H NMR(400MHz,Chloroform-d)δ8.66(s,1H),7.64(s,1H),7.37(s,1H),7.31(t,1H),7.08(s,1H),4.73(d,1H),4.34–4.31(m,1H),3.29( s,3H),2.59(s,3H),2.57–2.56(m,1H),2.10–2.09(m,1H),1.95–1.84(m,1H),1.76–1.73(m,2H),1.61–1.52(m,1H),1.44–1.41(m,1H).

[0093] Example 2

[0094] N-methyl-N-(1-methyl-1H-benzo[d]imidazol-6-yl)-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide compound 2

[0095] N-methyl-N-(1-methyl-1H-benzo[d]imidazol-6-yl)-2-(6-methyl-4-(trifluorometh yl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide

[0096]

[0097] first step:

[0098] N-(1-methyl-1H-benzo[d]imidazol-6-yl)-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide 2a

[0099] N-(1-methyl-1H-benzo[d]imidazol-6-yl)-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide

[0100] 2-(6-Methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxylic acid 1b (500 mg, 1.5 mmol) was dissolved in 10 mL of pyridine, followed by the addition of 1-methyl-1H-benzimidazol-6-amine (450 mg, 3 mmol) and 1-n-propylphosphonic anhydride (10 g, 16.5 mmol). The mixture was reacted at 25°C for 2 h. The reaction was monitored by LCMS for completion. The target compound, N-(1-methyl-1H-benzo[d]imidazol-6-yl)-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide 2a, was obtained by direct reverse phase purification as a white solid (600 mg).

[0101] LC-MS m / z (ESI) = 458.10 [M+H] + .

[0102] Step 2:

[0103] N-methyl-N-(1-methyl-1H-benzo[d]imidazol-6-yl)-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide compound 2

[0104] N-methyl-N-(1-methyl-1H-benzo[d]imidazol-6-yl)-2-(6-methyl-4-(trifluorometh yl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide

[0105] N-(1-methyl-1H-benzo[d]imidazol-6-yl)-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide 2a (400 mg, 0.87 mmol) was dissolved in 5 mL THF and 1 mL DMSO. F, nitrogen was replaced, the temperature was lowered to 0°C, and sodium hydride (60 mg, 1.3 mmol) was subsequently added. The mixture was stirred at room temperature for 0.5 h, then lowered to 0°C, and iodomethane (250 mg, 1.74 mmol) was slowly added dropwise. The reaction was allowed to react at room temperature for 1 h. The reaction was monitored by LCMS. Saturated aqueous NH4Cl solution was slowly added dropwise to the reaction solution at 0°C, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and then spin-dried. The reaction was purified by reverse phase purification to obtain the target compound N-methyl-N-(1-methyl-1H-benzo[d]imidazol-6-yl)-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide compound 2 (white solid, 285 mg).

[0106] LC-MS m / z (ESI) = 472.10 [M+H] + .

[0107] 1 H NMR(400MHz,Chloroform-d)δ8.66(s,1H),8.04(s,1H),7.92(d,1H),7.56(s,1H),7.26(s,1H),7.08(d,1H),4.78(d,1H),3.92(s,3H ),3.38(s,3H),3.30(m,1H),2.65(s,4H),2.10–2.03(m,1H),1.85(m,1H),1.59(m,1H),1.52–1.42(m,1H),1.32(m,1H),0.84(m,1H).

[0108] Example 3

[0109] N-(5-chloro-2,4-difluorophenyl)-N-(methyl-d3)-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopentyl[c]pyrrole-1-carboxamide compound 3

[0110] N-(5-chloro-2,4-difluorophenyl)-N-(methyl-d3)-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopenta[c]pyrrole-1-carboxamide

[0111]

[0112]

[0113] 2-(6-Methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopentyl[c]pyrrole-1-carboxylic acid 1b (300 mg, 0.9 mmol) and 5-chloro-2,4-difluoro-N-(methyl-d3)aniline (221 mg, 1.2 mmol) were dissolved in pyridine (1.5 mL), cooled to 0 ° C, and then 1-n-propylphosphoric anhydride (1.5 mL, 20 mmol) was added and nitrogen was used. After protection, the reaction was carried out at 25°C for 4 hours, and the reaction solution was directly dried, and water and ethyl acetate were added for separation. The organic phase was dried, concentrated, and purified by reverse phase column chromatography to obtain N-(5-chloro-2,4-difluorophenyl)-N-(methyl-d3)-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxooctahydrocyclopentyl[c]pyrrole-1-carboxamide compound 3 (60 mg, yellow solid, 15% yield).

[0114] LC-MS m / z (ESI) = 491.10 [M+1].

[0115] 1 H NMR(400MHz,Chloroform-d)δ8.68(s,1H),7.92–7.88(m,1H),7.19–7.14(m,1H),7.10(s,1H),4.72–4.71(m,1H),3.32–3.26(m,1H) ),2.63(s,3H),2.57–2.56(m,1H),2.10–2.09(m,1H),1.95–1.84(m,1H),1.76–1.73(m,2H),1.61–1.52(m,1H),1.44–1.41(m,1H).

[0116] Example 4

[0117] N,3a-dimethyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxo-N-(m-tolyl)octahydrocyclopentyl[c]pyrrole-1-carboxamide compound 4

[0118] N,3a-dimethyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxo-N-(m-tolyl)oc tahydrocyclopenta[c]pyrrole-1-carboxamide

[0119]

[0120]

[0121] N-Methyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxo-N-(m-tolyl)octahydrocyclopenta[c]pyrrole-1-carboxamide 4a (130 mg, 0.26 mmol) was dissolved in tetrahydrofuran (1.0 mL), cooled to -78°C, and then n-BuLi (1 mL, 2.5 mmol) was added. Under nitrogen protection, the mixture was reacted at -78°C for 2 h, and then iodomethane (25 mg, 0.3 mmol) was added and the reaction was continued at -78°C for 2 h. The reaction solution was added dropwise to an aqueous ammonium chloride solution to quench excess n-BuLi, and then extracted with ethyl acetate. The organic phase was concentrated and dried, and then purified by column chromatography to obtain the target compound N,3a-dimethyl-2-(6-methyl-4-(trifluoromethyl)pyridin-2-yl)-3-oxo-N-(m-tolyl)octahydrocyclopentyl[c]pyrrole-1-carboxamide compound 4 (50 mg, white solid, 48% yield).

[0122] LC-MS m / z (ESI) = 474.10 [M+1].

[0123] 1 H NMR(400MHz,Chloroform-d)δ8.68(s,1H),7.39(t,1H),7.32–7.27(m,2H),7.24(d,1H),7.07(s,1H),4.69(d,1H), 3.30(s,3H),2.64–2.60(m,1H),2.61(s,3H),2.44(s,3H),2.23–2.20(m,2H),2.20–2.11(m,2H),1.83–1.70(m,2H).

[0124] Biological test cases

[0125] 1. Polθ enzyme activity test

[0126] In this experiment, through The inhibitory ability of the compound on Polθ activity was determined using a dsDNA dye (Thermo Fisher, P7581). The specific operation process is as follows:

[0127] Compounds were prepared as 10 mM stocks in DMSO and serially diluted with 1% DMSO (v / v) (starting at 10 μM, 1:3 dilutions, 10 steps). 0.1 μL of each diluted compound was transferred to a 384-well plate (PerkinElmer, 6008260); two replicates were performed for each concentration. 5 μL of 4 nM Polθ enzyme solution (containing 25 mM Tris-HCl (pH 7.5), 12.5 mM NaCl, 0.5 mM MgCl2, 5% glycerol, 0.01% Triton X-100, 0.01% BGG, and 1 mM DTT) was added to each well, and the plates were centrifuged at 1000 rpm for 1 min at 4°C and incubated at room temperature (25°C) for 10 min. The reaction was initiated by adding 5 μL of substrate working solution (containing 40 μM dNTPs and 100 nM dsDNA) and incubated at room temperature (25°C) for 60 min. Add 5 μL of PicoGreen dye and incubate at room temperature (25°C) for 90 min. Use a microplate reader to read the fluorescence signals at 485 nm and 520 nm, calculate the inhibition rate of each well, and use GraphPad Prism to perform curve fitting and IC 50 Value calculation.

[0128] The results showed that the compounds of the present invention had significant biological inhibitory activity against Polθ.

[0129] 2. Clone formation experiment

[0130] In this experiment, the effect of compounds on DLD1 was evaluated by the number of cell clones formed. BRCA- / - The specific operation process is as follows:

[0131] DLD1 in the logarithmic growth phase BRCA- / -Cells were digested with trypsin, resuspended, and counted. Cells (200 cells / well) were seeded into a transparent 12-well plate (Corning, 3460) and cultured overnight in a 37°C incubator (5% CO2). The compound was prepared into a 10mM stock solution with DMSO, and the stock solution was serially diluted (starting concentration 10μM, 1:3 dilution, 5 gradients) using 1% DMSO (v / v) 1640 medium (containing 10% FBS, 1% Penicillin-Streptomycin). Subsequently, 1mL of the diluted medium containing the test compound was added to each well and cultured in a 37°C incubator (5% CO2) for 10 days, with the medium changed every 5 days. Discard the culture medium and wash the cells twice with PBS; add 500 μL of paraformaldehyde solution to each well for fixation and place at room temperature for 30 minutes; discard the solution and wash the cells twice with PBS; add 500 μL of crystal violet stain to each well and stain at room temperature for 30 minutes; discard the dye and wash the cells with PBS for 5 minutes each time until the background is clear. Dry the culture dish at room temperature, then count the number of colonies in the well, calculate the inhibition rate of each well, and use GraphPad Prism to perform curve fitting and IC 50 Value calculation.

[0132] The results showed that the compounds of the present invention had an effect on DLD1 BRCA- / - Cell colony formation has a significant biological inhibitory effect.

[0133] 3. MMEJ pathway inhibition experiment

[0134] The test compound was diluted with DMSO and transferred to a 384-well plate. The reaction system was diluted three-fold over nine concentrations, starting at a final concentration of 10 μM, to a final DMSO concentration of 0.1%. HEK293T cells were transfected with MMEJ dsDNA substrate (ICE Bioscience) using the Neon transfection system. Transfected cells were seeded at a density of 4,000 cells / well in a 384-well plate (25 μL / well). After incubation of the compound and cells at 37°C, 5% CO₂ in a humidified incubator for 24 hours, 40 μL / well of the Nano-Glo Luciferase Assay kit (Promega, N1120) was added. The cells were incubated at 300 rpm in the dark for 3 minutes, and fluorescence was measured using a BMG multi-function microplate reader.

[0135] MMEJ = (fluorescence value of compound well - fluorescence value of Yangshen) / (fluorescence value of DMSO well - fluorescence value of Yangshen), then use the compound concentration as the horizontal axis and MMEJ as the vertical axis to fit the IC value of the compound using Graphpad software. 50 The results are shown in the following table.

[0136]

[0137] Note: A stands for IC 50 ≦100nM; B represents 100nM>IC 50 ≦500nM; C represents 500nM>IC 50 ≦1000nM.

[0138] The results showed that the compound of the present invention had a significant inhibitory effect on the cellular MMEJ pathway.

[0139] The specification of the present invention describes the specific implementation scheme in detail. Those skilled in the art should recognize that the above implementation scheme is exemplary and cannot be understood as limiting the present invention. For those skilled in the art, without departing from the principles of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A compound represented by general formula (I) or any stereoisomer, solvate, prodrug, metabolite, deuterated substance, pharmaceutically acceptable salt or cocrystal thereof: in: A is selected from 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl, wherein the 3-8 membered heterocycloalkyl contains 1 to 4 heteroatoms selected from N, O or S; B is selected from 5-12 membered heteroaryl, wherein the 5-12 membered heteroaryl contains 1 to 4 heteroatoms selected from N, O or S; C is selected from 5-12 membered aryl or 5-12 membered heteroaryl, wherein the 5-12 membered heteroaryl contains 1 to 4 heteroatoms selected from N, O or S; R a Can be the same or different, R a Selected from H, C 1-6 Alkyl or OH, the C 1-6 The alkyl group is optionally further substituted with one or more substituents selected from halogen; R2, R3 are H, or R2, R3 and the carbon atom to which they are connected form C=O; R b Can be the same or different, R b Selected from H, C 1-6 Alkyl or CN, the C 1-6 The alkyl group is optionally further substituted with one or more substituents selected from halogen; R1 is C 1-3 alkyl; R c Can be the same or different, R c selected from halogen; a is 0, 1, 2, or 3; b is 1, 2, or 3; c is 1, 2, or 3.

2. The compound according to claim 1 or any stereoisomer, solvate, prodrug, metabolite, deuterated form, pharmaceutically acceptable salt or cocrystal thereof, in: A is selected from 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl, wherein the 3-8 membered heterocycloalkyl contains 1 to 4 heteroatoms selected from N, O or S; B is selected from 5-12 membered heteroaryl, wherein the 5-12 membered heteroaryl contains 1 to 4 heteroatoms selected from N, O or S; C is selected from 5-12 membered aryl; R a Can be the same or different, R a Selected from H, C 1-6 Alkyl or OH, the C 1-6 The alkyl group is optionally further substituted with one or more substituents selected from halogen; R2, R3 are H, or R2, R3 and the carbon atom to which they are connected form C=O; R b Can be the same or different, R b Selected from H, C 1-6 Alkyl or CN, the C 1-6 The alkyl group is optionally further substituted with one or more substituents selected from halogen; R1 is C 1-3 alkyl; R c Can be the same or different, R c selected from halogen; a is 0, 1, 2, or 3; b is 1, 2, or 3; c is 1, 2, or 3.

3. The compound according to claim 1 or 2, or any stereoisomers, solvates, prodrugs, metabolites, deuterated substances, pharmaceutically acceptable salts or cocrystals thereof, wherein the compound is selected from:

4. A pharmaceutical composition comprising: (1) The compound of claims 1 to 3 or its stereoisomers, solvates, prodrugs, metabolites, deuterated substances, pharmaceutically acceptable salts or cocrystals; (2) one or more other active ingredients; as well as (3) Pharmaceutically acceptable carriers and / or excipients.

5. Use of the compound of claims 1 to 3 or its stereoisomers, solvates, prodrugs, metabolites, deuterated substances, pharmaceutically acceptable salts or cocrystals, or the pharmaceutical composition of claim 4 in the preparation of antitumor drugs.

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