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WO2025209499A1 - INHIBITEUR DE POLθ ET COMPOSITION PHARMACEUTIQUE DE CELUI-CI ET UTILISATION - Google Patents

INHIBITEUR DE POLθ ET COMPOSITION PHARMACEUTIQUE DE CELUI-CI ET UTILISATION

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Publication number
WO2025209499A1
WO2025209499A1 PCT/CN2025/086748 CN2025086748W WO2025209499A1 WO 2025209499 A1 WO2025209499 A1 WO 2025209499A1 CN 2025086748 W CN2025086748 W CN 2025086748W WO 2025209499 A1 WO2025209499 A1 WO 2025209499A1
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Prior art keywords
mmol
methoxy
reaction
tetrahydrobenzo
thiazol
Prior art date
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Pending
Application number
PCT/CN2025/086748
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English (en)
Chinese (zh)
Inventor
陆平波
杨佳乐
祝辉
王敏超
邓民杰
王知
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Alicorn Pharmaceutical Co Ltd
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Alicorn Pharmaceutical Co Ltd
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Publication of WO2025209499A1 publication Critical patent/WO2025209499A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/438—The ring being spiro-condensed with carbocyclic or heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/502—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with carbocyclic ring systems, e.g. cinnoline, phthalazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C07D277/68—Benzothiazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
    • C07D277/82—Nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the present invention belongs to the field of medicinal chemistry, and specifically relates to a Pol ⁇ inhibitor or its stereoisomers, tautomers, and pharmaceutically acceptable salts, as well as to a pharmaceutical composition of the compound and its use in treating cancer.
  • DNA damage repair pathways include base excision repair (BER), nucleotide excision repair (NER), DNA mismatch repair (MMR), and DNA double-strand break (DSB) repair.
  • Double-strand DNA damage repair pathways mainly include homologous recombination (HR), non-homologous end joining (NHEJ), and microhomology-mediated end joining (alt-EJ or MMEJ).
  • Homologous recombination mainly relies on homologous chromosomes to guide the correction of damaged DNA, while non-homologous end joining directly connects the two broken DNA chains together to achieve the purpose of repair.
  • BRCA1 and BRCA2 the key proteins in homologous recombination, are two important tumor suppressor factors.
  • NHEJ non-homologous end joining
  • DNA polymerase ⁇ (Pol ⁇ or POLQ) is a synthetic lethal target of homologous recombination (HR) defects and plays an important role in the DNA damage response (DDR) pathway for double-strand breaks (DSBs).
  • HR DNA damage response
  • DSBs double-strand breaks
  • BRCA2 BRCA2 not only recruits the recombinase RAD51 to the DSB to promote HR but also inhibits repair pathways such as alt-NHEJ.
  • homologous recombination-mediated repair is impaired (HR deficiency), as in the case of BRCA1 or BRCA2 mutations, Pol ⁇ is highly expressed and directs DSB repair toward alt-NHEJ, initiating the DNA repair process of microhomology-mediated end joining (MMEJ).
  • MMEJ microhomology-mediated end joining
  • Pol ⁇ is expressed in only a few tissue types but is highly expressed in many cancer cells. Pol ⁇ can confer resistance to cancer therapies and promote the survival of abnormal cells lacking DNA damage repair pathways.
  • the primary function of DNA polymerase ⁇ (Pol ⁇ ) is to act as a reverse transcriptase. In healthy cells, Pol ⁇ acts as an RNA-mediated DNA repair enzyme. In cancer cells, Pol ⁇ is highly expressed and promotes cancer cell growth and drug resistance.
  • Documents such as CN114127062A, CN114667167A, WO2020160213, WO2022118210, WO2021123785, and WO2023134708 disclose DNA polymerase Theta (Pol ⁇ ) inhibitors.
  • the heterocyclic compounds provided by the present invention exhibit excellent Pol ⁇ inhibitory effects.
  • the present invention provides a compound represented by formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are each independently selected from H, deuterium, halogen, amino, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy;
  • R 8 is selected from H, hydroxy, C 1 -C 6 alkoxy, NR a R b ;
  • R 9 is phenyl, heteroaryl, heterocyclyl, -O(CH 2 ) r -cycloalkyl, -O(CH 2 ) r -heterocycloalkyl, -O(CH 2 ) r -heteroaryl, and the phenyl, heteroaryl, heterocyclyl, -O(CH 2 ) r -cycloalkyl, -O(CH 2 ) r -heterocycloalkyl, -O(CH 2 ) r -heteroaryl may be optionally substituted with one or more R n ;
  • R n is selected from H, halogen, cyano, NR a R b , hydroxy, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 alkylamino, C 1 -C 6 haloalkoxy, -(CH 2 ) r -OC 1 -C 6 alkyl, -C(O)NR a R b , -S(O) 2 NR a R b , -NH(CH 2 ) r C 1 -C 6 haloalkyl, -NH(CH 2 ) r OH, -(CH 2 ) r OC 1 -C 6 haloalkyl, C 1 -C 6 haloalkyl;
  • R a and R b are each independently selected from absent, hydrogen, and C 1 -C 6 alkyl;
  • r is an integer from 0 to 3.
  • R 4 is selected from methoxy.
  • R 8 is selected from hydrogen, hydroxy, methoxy, amino, and aminomethyl.
  • the compounds of the present invention possess asymmetric centers. Compounds of the present invention containing asymmetrically substituted atoms can be separated into optically active or racemic forms. Those skilled in the art will appreciate how to prepare optically active forms, such as by racemate resolution or synthesis from optically active starting materials. Unless otherwise indicated with respect to specific stereochemistry or isomeric forms, the present invention encompasses all chiral, diastereoisomer, and racemic forms. Methods for preparing the compounds of the present invention and intermediates thereto are also intended to be included in the present invention. All tautomers of the compounds of the present invention are also intended to be included in the present invention.
  • the term "optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.
  • “optionally substituted alkyl” refers to events or circumstances where the alkyl group may be substituted as well as instances where the alkyl group is not substituted.
  • substituted refers to a moiety having a substituent that replaces a hydrogen on one or more carbons of the main chain. It should be understood that “substituted” or “substituted by" includes implicit conditions, i.e., such substitution is consistent with the allowed valence of the substituted atom and the substituent, and the substitution produces a stable compound, for example, it will not spontaneously undergo transformations such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is intended to include all permissible substituents of an organic compound.
  • permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound.
  • the permissible substituents can be one or more and the same or different.
  • a heteroatom such as nitrogen can have a hydrogen substituent and/or any permissible substituent of an organic compound as described herein that satisfies the valence of the heteroatom.
  • Substituents can include any substituent described herein, such as halogen, hydroxyl, alkyl, alkoxy, amino, cyano, heteroaryl, heterocyclic, etc. It will be understood by those skilled in the art that substituents may themselves be substituted, if appropriate.
  • halogen refers to fluorine, chlorine, bromine, or iodine, and particularly to fluorine, chlorine, and bromine, with fluorine and chlorine being particularly preferred.
  • cyano refers to a group in which a carbon atom is triple-bonded to a nitrogen atom.
  • alkoxy is a customary expression and refers to an alkyl group attached to the rest of the molecule via an oxygen atom, and refers to an alkyl group containing an oxygen atom. Examples of such groups include methoxy, ethoxy, or propoxy.
  • alkyl refers to an alkyl group having a specified number of carbon atoms, which is a linear or branched alkyl group, and which may include subgroups thereof.
  • C1 - C6 alkyl group it may also include sub-ranges of groups represented by C1 - C4 alkyl, C1 - C3 alkyl, C2 - C6 alkyl, C2 - C4 alkyl, etc., as well as specific groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, etc.
  • haloalkyl refers to an alkyl group as defined above that is attached to the rest of the molecule via a halo linkage.
  • a C 1-6 haloalkyl group refers to an alkyl group having 1 to 6 carbon atoms or 1 to 3 carbon atoms that is attached to the rest of the molecule via a halo linkage.
  • Preferred haloalkyl groups include, but are not limited to, -CH 2 Cl, -CHCl 2 , -CF 3 , and the like.
  • hydroxy or "hydroxyl,” by itself or in combination with other terms, means -OH.
  • Nitrogen and sulfur atoms may be optionally oxidized, and nitrogen atoms may be optionally quaternized.
  • the fused rings completing the bicyclic and tricyclic groups may contain only carbon atoms and may be saturated, partially saturated or fully unsaturated.
  • Heterocyclic groups may be attached to any available nitrogen or carbon atom.
  • the heterocyclic radicals include heterocycloalkyl and the like.
  • heteroaryl refers to substituted and unsubstituted aromatic 5- or 6-membered monocyclic groups, 9- or 10-membered bicyclic groups, and 11- to 14-membered tricyclic groups having at least one heteroatom (O, S, or N) in at least one ring, the heteroatom-containing ring preferably having 1, 2, or 3 heteroatoms selected from O, S, and N.
  • Each ring of the heteroaryl containing heteroatoms can contain one or two oxygen or sulfur atoms and/or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less, and each ring has at least one carbon atom.
  • the fused rings completing the bicyclic and tricyclic groups can contain only carbon atoms and can be saturated, partially saturated, or unsaturated.
  • the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atom may optionally be quaternized.
  • a bicyclic or tricyclic heteroaryl must include at least one fully aromatic ring, but the other one or more fused rings may be aromatic or non-aromatic.
  • heteroaryl examples include, but are not limited to, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, 1H-tetrazolyl, oxadiazolyl, triazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzofuranyl, benzothienyl, benzotriazinyl, phthalazinyl, thianthracene, dibenzofuranyl, benzothiophenyl ...
  • Step 6 Synthesis of 2'-chloro-N-[6-(6-fluoro-4-methylpyridin-3-yl)-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl]-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide
  • 6-(6-Fluoro-4-methylpyridin-3-yl)-4,5,6,7-tetrahydrobenzo[d]thiazol-2-amine (90.0 mg, 0.34 mmol, 1.0 eq) and 2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxylic acid (95.1 mg, 0.34 mmol, 1.0 eq) were dissolved in ultra-dry N,N-dimethylformamide (2 mL), and N-methylimidazole (84.2 mg, 1.03 mmol, 3.0 eq) and N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (105.8 mg, 0.41 mmol, 1.2 eq) were added, followed by reaction at room temperature for 2.0 hours.
  • Step 5 Synthesis of 2'-chloro-N- ⁇ 6-[2-fluoro-4-(trifluoromethyl)phenyl]-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl ⁇ -5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide
  • N-methylimidazole (88.23 mg, 1.08 mmol, 3.0 eq) and N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (150.7 mg, 0.54 mmol, 1.5 eq) were then added and reacted at room temperature for 2 hours. After the reaction, preparative separation and purification were performed to obtain 2'-chloro-N- ⁇ 6-[2-fluoro-4-(trifluoromethyl)phenyl]-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl ⁇ -5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide (42.7 mg, 20.62% yield) as a white solid.
  • 1,4-Dioxyaspirin[4.5]dec-7-en-8-yl trifluoromethanesulfonate (8.19 g, 28.4 mmol, 1.0 eq) and (2,4,5-trifluorophenyl)boronic acid (5.0 g, 28.4 mmol, 1.0 eq) were added to 1,4-dioxane (80 mL), followed by cesium carbonate (27.78 g, 85.3 mmol, 3.0 eq) and water (20 mL). Finally, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (831 mg, 1.1 mmol, 0.04 eq) was added.
  • Step 6 Synthesis of 2'-chloro-5'-methoxy-6-methyl-N-[6-(2,4,5-trifluorophenyl)-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl]-[4,4'-bipyridine]-3-carboxamide
  • 1,4-Dioxyaspirino[4.5]dec-7-en-8-yl trifluoromethanesulfonate (2.0 g, 6.94 mmol, 1.0 eq) was dissolved in 1,4-dioxane (10 mL).
  • (4-Cyano-3-fluorophenyl)boronic acid (1.01 g, 6.94 mol, 1.0 eq)
  • cesium carbonate (6.79 g, 20.8 mmol, 3.0 eq)
  • deionized water 2 mL
  • [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (253.8 mg, 0.35 mmol, 0.05 eq) were added.
  • Step 5 Synthesis of 2'-chloro-N-(6-(4-cyano-3-fluorophenyl)-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide
  • Step 3 Synthesis of phenyl 4-[2-(2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridyl]-3-carboxamido)-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl]acetate
  • Step 4 Synthesis of 2'-chloro-N-[6-(4-hydroxyphenyl)-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl]-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide
  • Step 5 Synthesis of 2'-chloro-5'-methoxy-6-methyl-N-[6-(4-methylthiophen-2-yl)-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl]-[4,4'-bipyridine]-3-carboxamide
  • Methyl 2'-(difluoromethyl)-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxylate (550 mg, 1.78 mmol, 1.0 eq) was dissolved in methanol (6 mL) and lithium hydroxide monohydrate (112.5 mg, 2.67 mmol, 1.5 eq) was added. The mixture was reacted at 60°C for 1 hour. After completion of the reaction, the reaction solution was cooled to room temperature, the pH was adjusted to neutral, and the mixture was extracted three times with ethyl acetate.
  • Step 3 Synthesis of 2'-(difluoromethyl)-5'-methoxy-6-methyl-N-(6-phenyl-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)-[4,4'-bipyridine]-3-carboxamide
  • 6-phenyl-4,5,6,7-tetrahydrobenzo[d]thiazol-2-amine 25 mg, 0.108 mmol, 1.0 eq
  • N-methylimidazole (26.6 mg, 0.324 mmol, 3.0 eq)
  • N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (45.7 mg, 0.162 mmol, 1.5 eq) were added in sequence and reacted at room temperature for 2 hours.
  • 1,4-Dioxyaspirino[4.5]dec-7-en-8-yl trifluoromethanesulfonate (4.0 g, 13.87 mmol, 1.0 eq) and 4-fluorophenylboronic acid (2.64 g, 13.87 mmol, 1.0 eq) were dissolved in ultra-dry 1,4-dioxane (30.0 mL).
  • Deionized water (7.5 mL), cesium carbonate (13.56 g, 41.62 mmol, 3.0 eq), and dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium (0.456 g, 0.624 mmol, 0.41 eq) were then added.
  • Step 5 Synthesis of 2'-chloro-N-[6-(4-fluorophenyl)-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl]-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide
  • Step 2 Synthesis of 5'-methoxy-2',6'-dimethyl-N-(6-phenyl-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)-[4,4'-bipyridine]-3-carboxamide
  • 1,4-Dioxyaspirino[4.5]dec-7-en-8-yl trifluoromethanesulfonate (3.0 g, 10.4 mmol, 1.0 eq) and 4-(difluoromethyl)phenylboronic acid (2.64 g, 13.87 mmol, 1.0 eq) were dissolved in ultra-dry 1,4-dioxane (16.0 mL), and deionized water (4.0 mL), cesium carbonate (10.17 g, 31.2 mmol, 3.0 eq) and dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium (228 mg, 0.312 mmol, 0.03 eq) were added.
  • Step 5 Synthesis of 2'-chloro-N- ⁇ 6-[4-(difluoromethyl)phenyl]-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl ⁇ -5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide
  • Step 1 Synthesis of tert-butyl [4-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)phenyl]carbamate
  • 1,4-Dioxaspiro[4.5]dec-7-en-8-yl trifluoromethanesulfonate (4 g, 13.9 mmol, 1.0 eq), ⁇ 4-[(tert-butoxycarbonyl)amino]phenyl ⁇ boronic acid (3.62 g, 15.2 mmol, 1.1 eq), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (1.02 g, 1.39 mmol, 0.1 eq) and cesium carbonate (9.03 g, 27.8 mmol, 2.0 eq) were dissolved in 1,4-dioxane (40 mL) and water (4 mL) and reacted at 80 ° C under nitrogen protection for 5 hours.
  • reaction solution was filtered, concentrated, and stirred, then purified by normal phase column chromatography (ethyl acetate/petroleum ether, 30% ethyl acetate) to obtain tert-butyl [4-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)phenyl]carbamate (2.13 g, 46% yield) as a yellow solid.
  • Step 2 Synthesis of tert-butyl [4-(1,4-dioxaspiro[4.5]decan-8-yl)phenyl]carbamate
  • Step 3 Synthesis of tert-butyl [4-(4-oxocyclohexyl)phenyl]carbamate
  • Step 4 Synthesis of tert-butyl [4-(3-bromo-4-oxocyclohexyl)phenyl]carbamate
  • Step 6 Synthesis of tert-butyl ⁇ 4-[2-(2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridyl]-3-carboxamido)-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl]phenyl ⁇ carbamate
  • 6-Morpholinyl-4,5,6,7-tetrahydrobenzo[d]thiazol-2-amine (256 mg, 1.07 mmol, 1.0 eq) was dissolved in acetonitrile (5 mL), and 2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxylic acid (298 mg, 1.07 mmol, 1.0 eq), tetramethylchlorouronium hexafluorophosphate (359 mg, 1.21 mmol, 1.2 eq) and N-methylimidazole (263 mg, 3.21 mmol, 3.0 eq) were added. The reaction was carried out at room temperature under nitrogen protection for 12 hours.
  • 1,4-Dioxaspiro[4.5]decane-7-ene-8-trifluoromethanesulfonate (3.4 g, 10.6 mmol, 1.2 eq)
  • tert-butyl[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl]carbamate (2.55 g, 8.85 mmol, 1.0 eq)
  • Step 3 Synthesis of [5-(1,4-dioxaspiro[4.5]n-decane-8-yl)pyridin-2-yl]decane
  • 1,4-Dioxaspirino[4.5]decan-8-one (6.5 g, 41.67 mmol, 1.0 eq) was dissolved in ultra-dry tetrahydrofuran (40.0 mL). Under nitrogen, a solution of (4-cyanophenyl)magnesium bromide in tetrahydrofuran (75.0 mL, 1.2 eq) was added dropwise at 0°C. The mixture was allowed to react at room temperature for 3 hours. After completion, the reaction was quenched with saturated aqueous ammonium chloride in an ice bath. The mixture was then extracted with water and ethyl acetate.
  • Step 5 Synthesis of 2'-chloro-N-[6-(4-cyanophenyl)-6-hydroxy-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl]-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide
  • N-methylimidazole (121 mg, 1.48 mmol, 4.0 eq) was added, followed by N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (155 mg, 0.55 mmol, 1.5 eq) in an ice bath.
  • the mixture was reacted at room temperature for 16 hours.
  • preparative separation and purification were performed to obtain 2'-chloro-N-[6-(4-cyanophenyl)-6-hydroxy-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl]-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide (41 mg, yield 20.92%).
  • Step 5 Synthesis of 2'-chloro-N-[6-(4-cyano-2-methoxyphenyl)-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl]-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide
  • Step 1 Synthesis of 3-methoxy-4-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)benzaldehyde
  • Step 2 Synthesis of 8-[4-(difluoromethyl)-2-methoxyphenyl]-1,4-dioxaspiro[4.5]dec-7-ene
  • Step 3 Synthesis of 8-[4-(difluoromethyl)-2-methoxyphenyl]-1,4-dioxaspiro[4.5]decane
  • Step 5 Synthesis of 6-[4-(difluoromethyl)-2-methoxyphenyl]-4,5,6,7-tetrahydrobenzo[d]thiazol-2-amine
  • Step 6 Synthesis of 2'-chloro-N- ⁇ 6-[4-(difluoromethyl)-2-methoxyphenyl]-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl ⁇ -5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide
  • Step 6 Synthesis of 2'-chloro-N-[6-(4-cyano-3-methoxyphenyl)-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl]-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide
  • Step 1 Synthesis of 3-methoxy-4-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)benzaldehyde
  • Step 2 Synthesis of 8-[4-(difluoromethyl)-3-methoxyphenyl]-1,4-dioxaspiro[4.5]dec-7-ene
  • Step 3 Synthesis of 8-[4-(difluoromethyl)-3-methoxyphenyl]-1,4-dioxaspiro[4.5]decane
  • Step 5 Synthesis of 6-[4-(difluoromethyl)-3-methoxyphenyl]-4,5,6,7-tetrahydrobenzo[d]thiazol-2-amine
  • Step 6 Synthesis of 2'-chloro-N- ⁇ 6-[4-(difluoromethyl)-3-methoxyphenyl]-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl ⁇ -5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide
  • Step 1 Synthesis of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-(2,2,2-trifluoroethyl)pyrimidin-2-amine
  • Step 4 Synthesis of 4-[4-(2,2,2-trifluoroethyl)amino]phenylcyclohexane-1-one
  • Step 5 Synthesis of 6- ⁇ 4-[(2,2,2-trifluoroethyl)amino]phenyl ⁇ -4,5,6,7-tetrahydrobenzo[d]thiazol-2-amine
  • Step 6 Synthesis of 2'-chloro-5'-methoxy-6-methyl-N- ⁇ 6-[4-(2,2,2-trifluoroethyl)amino]phenyl ⁇ -4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl-[4,4'-bipyridine]-3-carboxamide
  • N-methyl-4-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)benzenesulfonamide (2.4 g, 6.4 mmol, 1.0 eq) and palladium on carbon (480 mg) were dissolved in methanol (5 mL). The mixture was replaced with hydrogen three times and allowed to react at room temperature for 16 hours. After the reaction was complete, the mixture was filtered and the filtrate was concentrated to obtain N-methyl-4-(1,4-dioxaspiro[4.5]dec-8-yl)benzenesulfonamide (2.4 g) as a white solid, which was used directly in the next reaction.
  • N-Methyl-4-(1,4-dioxaspiro[4.5]dec-8-yl)benzenesulfonamide (800 g, 2.57 mmol, 1.0 eq) and p-toluenesulfonic acid hydrate (977 mg, 5.1 mmol, 2.0 eq) were dissolved in acetone (9 mL) and water (3 mL) and reacted at 60°C for 16 hours. After the reaction was complete, the mixture was quenched with sodium bicarbonate solution and extracted with ethyl acetate and water.
  • N,N-Dimethyl-4- ⁇ 1,4-dioxaspiro[4.5]dec-8-yl ⁇ benzenesulfonamide 600 mg, 1.85 mmol, 1.0 eq
  • p-toluenesulfonic acid hydrate 527 mg, 2.77 mmol, 1.5 eq
  • acetone 9 mL
  • water 3 mL
  • the mixture was quenched with sodium bicarbonate solution and extracted with ethyl acetate and water.
  • N,N-Dimethyl-4-(4-oxocyclohexyl)benzenesulfonamide 150 mg, 0.534 mmol, 1.0 eq
  • thiourea 81.3 mg, 1.07 mmol, 2.0 eq
  • iodine 163 mg, 0.641 mmol, 1.2 eq
  • Step 4 Synthesis of 2'-chloro-N- ⁇ 6-[4-(N,N-dimethylsulfamoyl)phenyl]-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl ⁇ -5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide
  • Step 6 Synthesis of 6- ⁇ 6-[(2,2,2-trifluoroethyl)amino]pyridin-3-yl ⁇ -4,5,6,7-tetrahydrobenzo[d]thiazol-2-amine
  • Step 7 Synthesis of 2'-chloro-5'-methoxy-6-methyl-N-(6-((2,2,2-trifluoroethyl)amino)pyridin-3-yl)-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)-[4,4'-bipyridine]-3-carboxamide
  • N-ethyl-4-(4-oxocyclohexyl)benzenesulfonamide (9.1 g, 32.38 mmol, 1.0 eq), thiourea (4.9 g, 64.79 mmol, 2.0 eq), and iodine (12.3 g, 48.57 mmol, 1.5 eq) were dissolved in ethanol (200 mL) and reacted at 80°C for 2 hours. After the reaction was complete, the reaction mixture was concentrated and then quenched with saturated sodium bicarbonate solution and sodium thiosulfate solution.
  • Step 6 Synthesis of 2'-chloro-5'-methoxy-6-methyl-N- ⁇ 6-[4-(N-ethylaminosulfonyl)phenyl]-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl ⁇ -[4,4'-bipyridine]-3-carboxamide
  • Step 1 Synthesis of tert-butyl 4-(2,2,2-trifluoroethyl)amino)piperidine-1-carboxylate
  • Step 3 Synthesis of tert-butyl (6-(4-(2,2,2-trifluoroethyl)amino)piperidin-1-yl)-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)carbamate
  • tert-butyl (6-(4-(2,2,2-trifluoroethyl)amino)piperidin-1-yl)-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)carbamate (600 mg, 1.38 mmol, 1.0 eq) in dichloromethane (8 mL). Slowly add trifluoroacetic acid (1 mL) and react at room temperature for 30 minutes. After the reaction is complete, extract with water and ethyl acetate (100 mL x 3).
  • Step 3 Synthesis of 2'-chloro-5'-methoxy-6-methyl-N- ⁇ 6-[(1-methylazetidin-3-yl)methoxy]-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl ⁇ -[4,4'-bipyridine]-3-carboxamide
  • 6-Bromo-N-(2,2,2-trifluoroethyl)pyridin-3-amine (427 mg, 1.67 mmol, 1.0 eq), 4,4,5,5-tetramethyl-2-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)-1,3,2-dioxaborolane (489 mg, 1.84 mmol, 1.1 eq), 1,1-bis(diphenylphosphino)diborane iron palladium dichloride (122 mg, 0.167 mmol, 0.1 eq), and potassium carbonate (577 mg, 4.18 mmol, 2.5 eq) were added to 1,4-dioxane (10 ml) and water (2 ml).
  • 6-(1,4-Dioxyaspirino[4.5]dec-7-en-8-yl)-N-(2,2,2-trifluoroethyl)pyridin-3-amine 328 mg, 1.04 mmol, 1.0 eq
  • palladium on carbon 66 mg
  • the mixture was filtered through celite and the filtrate was concentrated to obtain 6-(1,4-Dioxyaspirino[4.5]dec-8-yl)-N-(2,2,2-trifluoroethyl)pyridin-3-amine (290 mg, 87.9% yield) as a brown oil.
  • 6-(1,4-Dioxyaspirin[4.5]dec-8-yl)-N-(2,2,2-trifluoroethyl)pyridin-3-amine 290 mg, 0.92 mmol, 1.0 eq
  • p-toluenesulfonic acid monohydrate 174 mg, 0.92 mmol, 1.0 eq
  • acetone 9 ml
  • water 3 ml
  • ethyl acetate (20 mL) and water (15 mL) were added for extraction.
  • Step 6 Synthesis of 2'-chloro-5'-methoxy-6-methyl-N- ⁇ 6-[5-(2,2,2-trifluoroethyl)amino]pyridin-2-yl ⁇ -4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl-[4,4'-bipyridine]-3-carboxamide
  • 6-Chloropyridine-3-sulfonyl chloride (6 g, 28.29 mol, 1.0 eq), methylamine hydrochloride (2.3 g, 0.034 mol, 1.2 eq), and triethylamine (8.6 g, 0.085 mol, 3.0 eq) were added sequentially to dichloromethane (20 ml) and allowed to react at room temperature for 2 hours.
  • H2O 100 mL
  • ethyl acetate 100 ml x 3
  • the organic phase was concentrated to afford 6-chloro-N-methylpyridine-3-sulfonamide (2.5 g, 43.3% yield) as a brown oil.
  • N-Methyl-6-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)pyridine-3-sulfonamide 980 mg, 3.16 mmol, 1.0 eq
  • palladium/carbon 67 mg, 0.63 mmol, 0.2 eq
  • Filtration and concentration afforded a crude yellow solid of N-methyl-6-(1,4-dioxaspiro[4.5]dec-8-yl)pyridine-3-sulfonamide (1.0 g), which was used directly in the next reaction.
  • N-Methyl-6-(1,4-dioxaspiro[4.5]dec-8-yl)pyridine-3-sulfonamide (1.0 g, 3.2 mmol, 1.0 eq) and toluenesulfonic acid monohydrate (183 mg, 0.96 mmol, 0.3 eq) were added to acetone/water (6 mL/3 mL) and reacted at 60°C for 2 hours. The pH was adjusted to neutral by adding saturated aqueous sodium bicarbonate. The mixture was extracted with ethyl acetate (15 mL x 2).
  • N-Methyl-6-(4-oxocyclohexyl)pyridine-3-sulfonamide 200 mg, 0.75 mmol, 1.0 eq
  • ethanol 5 ml
  • Iodine (227 mg, 0.89 mmol, 1.2 eq)
  • thiourea 113 mg, 1.49 mmol, 2.0 eq
  • the mixture was allowed to react at 80°C for 2 hours. After completion, the reaction was quenched by addition of saturated sodium thiosulfate solution.
  • Step 7 Synthesis of 2'-chloro-5'-methoxy-6-methyl-N- ⁇ 6-[5-(N-methylsulfamoyl)pyridin-2-yl]-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl ⁇ -[4,4'-bipyridine]-3-carboxamide
  • 6-(2-Amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-N-methylpyridine-3-sulfonamide 60 mg, 0.18 mmol, 1.0 eq
  • 2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxylic acid 51 mg, 0.18 mmol, 1.0 eq
  • N,N-dimethylformamide 3 mL
  • N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate 78 mg, 0.27 mmol, 1.5 eq
  • N-methylimidazole 46 mg, 0.54 mmol, 3.0 eq
  • Step 2 Synthesis of N-(8-(4-chlorophenyl)-1,4-dioxaspiro[4.5]decan-8-yl)-2-methylpropane-2-sulfonamide
  • Step 3 Synthesis of 2-methyl-N- ⁇ 8-[4-(2,2,2-trifluoroethyl)amino]phenyl ⁇ -1,4-dioxaspiro[4.5]decan-8-yl)propane-2-sulfonamide
  • N-(8-(4-chlorophenyl)-1,4-dioxaspiro[4.5]decan-8-yl)-2-methylpropane-2-sulfonamide (1.00 g, 2.7 mmol, 1.0 eq) was dissolved in 1,4-dioxane (10 mL), followed by the addition of sodium tert-butoxide (777 mg, 8.09 mmol, 3.0 eq) and methanesulfonic acid (2-di-tert-butylphosphino-3.6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II) (230 mg, 0.27 mmol, 0.1 eq).
  • Step 5 Synthesis of 6- ⁇ 4-[(2,2,2-trifluoroethyl)amino]phenyl ⁇ -4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine
  • Step 6 Synthesis of N- ⁇ 6-amino-6-[4-(2,2,2-trifluoroethyl)amino]phenyl ⁇ -4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)-2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide
  • the purpose of this test example is to test the effect of compounds on the proliferation of HCT116 BRCA2 -/- cells.
  • DNA polymerase theta is a synthetic lethal target with homologous recombination deficiency and plays an important role in the DNA damage response pathway for double-strand breaks (DSBs).
  • DSBs double-strand breaks
  • HR deficiency homologous recombination-mediated repair
  • MMEJ microhomology-mediated end joining
  • inhibition of Pol ⁇ leads to cell death through the accumulation of toxic RAD51 intermediates and inhibition of the alt-EJ repair pathway. Therefore, by measuring the inhibitory effect of compounds on the proliferation of HCT116 BRCA2 -/- cells, it can be used to screen for Pol ⁇ protein inhibitors.
  • HCT116 BRCA2 knockout cells and screen for single clones.
  • HCT116 BRCA2 -/- and WT cells in logarithmic growth phase were seeded into 96-well plates, with 1000 cells/well (90 ⁇ L) per well, and incubated overnight at 37°C. The next day, 10 ⁇ L of compound was added at various concentrations, with a maximum concentration of 100 ⁇ M.
  • 10 ⁇ L of compound was added at various concentrations, with a maximum concentration of 100 ⁇ M.
  • Nine 10-fold dilutions were performed (final DMSO concentration was 1%), and the cells were incubated at 37°C for 6 days.
  • the old culture medium was aspirated and 110 ⁇ L of culture medium (culture medium to CCK8 at a ratio of 100:10) was added.
  • the cells were incubated at 37°C for 1-4 hours.
  • the absorbance was measured at 450 nM, and the IC50 value was calculated using GraphPad software. Compounds were screened by
  • the compounds of the present invention can inhibit the proliferation of HCT116 BRCA2-/- cells, with IC50 values reaching the nM level.
  • the activity of some compounds is surprisingly significantly better than that of the positive control. This strong inhibitory effect has important therapeutic significance for the treatment of symptoms or diseases related to Pol ⁇ inhibition; while the inhibitory effect on HCT116 WT cells is weak, with excellent selectivity.
  • the purpose of this test case is to test the effect of compounds on the proliferation of DLD1 BRCA2 -/- cells.
  • DLD1 BRCA2 knockout cells were constructed and single clones were screened.
  • DLD1 BRCA2 -/- and WT cells in logarithmic growth phase were seeded into 96-well plates, with 1000 cells/well (90 ⁇ L) per well, and incubated overnight at 37°C. The next day, 10 ⁇ L of compound was added at various concentrations, with a maximum concentration of 100 ⁇ M.
  • 10 ⁇ L of compound was added at various concentrations, with a maximum concentration of 100 ⁇ M.
  • Nine 10-fold dilutions were performed (final DMSO concentration was 1%), and the cells were incubated at 37°C for 6 days.
  • the old culture medium was aspirated and 110 ⁇ L of culture medium (culture medium to CCK8 ratio of 100:10) was added.
  • the cells were incubated at 37°C for 1-4 hours.
  • the absorbance was measured at 450 nM, and the IC50 was calculated using GraphPad software. Compounds were screened by
  • Pol ⁇ consists of a C-terminal family A DNA polymerase and an N-terminal superfamily 2 (SF2) DNA helicase, separated by a long, poorly conserved central domain of unknown function.
  • ART558 binds to an allosteric site within the Pol ⁇ polymerase catalytic domain and inhibits MMEJ in a dose-dependent manner. Therefore, compounds that inhibit Pol ⁇ polymerase function can be used to screen for Pol ⁇ inhibitors.
  • POL ⁇ -polymerase domain protein expression The POL ⁇ -polymerase domain sequence (residues 1819-2590) was cloned into the pET24N vector containing an N-terminal (His) 6x tag and a tobacco etch virus (TEV) protease cleavage site.
  • the constructed vector was transformed into Escherichia coli Rosetta (DE3) and incubated at an OD of 0.6. 1 mM IPTG was added and cultured at 12°C for 16 h. The cells were harvested, ultrasonically disrupted, and the supernatant was centrifuged. The protein was purified by Ni-NTA and dialyzed for determination of protein concentration and purity.
  • Purified POL ⁇ -polymerase domain protein was added to assay buffer (20 mM Tris, pH 7.80, 50 mM KCl, 10 mM MgCl, 1 mM DTT, 0.01% BSA, 0.01% Tween 20) at a protein concentration of 4 nM. Test compounds (an 11-point dilution series) were then added. The enzyme and test compound inhibitor mixture were incubated at room temperature for 15 minutes.
  • the compounds of the present invention have very strong inhibitory effects on the polymerase function of the Pol ⁇ -polymerase domain protein, with IC50 values reaching the nanomolar range. Some compounds have inhibitory effects comparable to or superior to the positive control drug. This strong inhibitory effect has important therapeutic implications for the treatment of conditions or diseases associated with Pol ⁇ inhibition.
  • the compound of the present invention and control compound A were administered by gavage (50 mg/kg), PO solvent: 40% PEG300 + 15% Tween 80 + 45% water, prepared on the day of administration.
  • Blood was collected from the jugular vein 0h before administration and 0.25h, 0.5h, 1h, 2h, 4h, 7h, 24h after administration (gavage), and about 0.02mL of each sample was collected.
  • K2-EDTA was used for anticoagulation and the samples were placed on ice after collection.
  • Plasma sample processing After blood collection, the blood samples were placed on ice and centrifuged within 1 hour to separate the plasma (centrifugation conditions: 4000g, 5 minutes, 2-8°C). Plasma samples were stored in a -80°C refrigerator before analysis.
  • Phoenix WinNonlin7.0 was used to calculate the pharmacokinetic parameters based on the blood drug concentration data at different time points. The experimental results are shown in Table 4 below:
  • mice The pharmacokinetic data of the compounds of the present invention in mice showed that the oral exposure of the tested representative compounds in mice was unexpectedly good compared with the control compound A.

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Abstract

La présente invention concerne un composé hétérocyclique pour inhiber l'activité d'une ADN polymérase Theta(Polθ), un stéréoisomère, un tautomère, ou un sel pharmaceutiquement acceptable du composé et une utilisation de celui-ci dans la préparation d'un médicament pour le traitement ou la prévention du cancer. Plus particulièrement, l'invention concerne un composé tel que représenté dans la formule I ou une forme stéréoisomère et un sel pharmaceutiquement acceptable de celui-ci, la définition de chaque groupe étant telle que décrite dans la description. Le composé a une activité inhibitrice de Polθ relativement élevée, de telle sorte que le composé peut être utilisé pour traiter des maladies liées à la surexpression de Polθ.
PCT/CN2025/086748 2024-04-03 2025-04-02 INHIBITEUR DE POLθ ET COMPOSITION PHARMACEUTIQUE DE CELUI-CI ET UTILISATION Pending WO2025209499A1 (fr)

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CN202410398889 2024-04-03
CN202410398889.7 2024-04-03
CN202410601956 2024-05-14
CN202410601956.0 2024-05-14
CN202411073608.7 2024-08-06
CN202411073608 2024-08-06
CN202411238132.8 2024-09-04
CN202411238132 2024-09-04
CN202510088603 2025-01-20
CN202510088603.X 2025-01-20

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