WO2023046060A1 - 治疗kras突变的癌症患者 - Google Patents

治疗kras突变的癌症患者 Download PDF

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WO2023046060A1
WO2023046060A1 PCT/CN2022/120817 CN2022120817W WO2023046060A1 WO 2023046060 A1 WO2023046060 A1 WO 2023046060A1 CN 2022120817 W CN2022120817 W CN 2022120817W WO 2023046060 A1 WO2023046060 A1 WO 2023046060A1
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cancer
kras
mutation
tumor
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WO2023046060A9 (zh
WO2023046060A8 (zh
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段建新
李安蓉
孟繁英
齐天阳
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深圳艾欣达伟医药科技有限公司
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Priority to CN202280060627.9A priority patent/CN117956998A/zh
Publication of WO2023046060A1 publication Critical patent/WO2023046060A1/zh
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Definitions

  • the present invention relates to a treatment method for cancer, especially a treatment method for cancer patients with KRAS mutation.
  • the full name of the KRAS gene is Kirsten ratsarcoma viral oncogene homolog, translated into Chinese as "Kirsten rat sarcoma virus oncogene homolog".
  • the protein encoded by the KRAS gene is a small GTPase (smallGTPase), which belongs to the RAS superprotein family.
  • the RAS gene includes KRAS, NRAS and HRAS.
  • the proteins encoded by these genes are GTPases, which are in the pathway of regulating cell proliferation and survival function as a molecular switch. Based on current COSMIC data, KRAS was the most frequently mutated gene among the three (22%), followed by NRAS (8%), and lastly HRAS (3%).
  • KRAS gene has a great influence on human cancer, about 30% of cancer patients have KRAS mutation, including 90% of pancreatic cancer, 50% of colon cancer and 25% of lung cancer.
  • KRAS gene mutation accounts for 20-30%, mostly in lung adenocarcinoma, but rare in lung squamous cell carcinoma.
  • KRAS gene mutations are mainly concentrated at the 12th, 13th and 61st codon positions, and the mutations at the 12th codon position account for more than 80%, including G12A, G12C, G12D, G12R, G12S and G12V.
  • KRAS-G12C mutations The most common way of KRAS gene activation is point mutation, and 95% of KRAS mutations mainly occur in codon 12 (>80%) and codon 13 of exon 2.
  • KRAS-G12C mutations 93% of all KRAS mutations
  • Loong HHF, Du N Cheng C, Lin H, Guo J, Lin G, Li M, Jiang T, Shi Z, Cui Y, Jin X, Yao J, Xing Y, Yao M, Wang K, Mok TSK, Liu L.
  • KRAS G12C mutations in Asia A landscape analysis of 11, 951 Chinese tumor samples. Transl Lung Cancer Res 2020. doi: 10.21037/tlcr-20-455).
  • the G12C mutation of the KRAS gene whose mutant has a cysteine residue (glycine at position 12 to cysteine), has been used to design covalent inhibitors with preclinical activity.
  • AMG-510 the first KRAS-G12C inhibitor in clinical development
  • MRTX849 have shown strong anti-tumor activity.
  • Sotorasib (AMG-510, Lumakras), known in the industry as a "revolutionary anticancer drug" effective against KRAS mutations, was approved by the FDA for the treatment of non-small cell lung cancer patients with KRAS-G12C mutations , these patients had received at least one prior systemic therapy.
  • AMG510 is specifically targeting the mutant subtype of KRAS-G12C, with high selectivity, and can specifically bind to, lock and inactivate KRAS-G12C in more than 6,000 proteins.
  • Adagrasib Adagrasib, MRTX849 Breakthrough Therapy Designation for the treatment of non-small cell lung cancer with KRAS-G12C mutations who have previously received systemic therapy (NSCLC) patients.
  • the drug is a specific and optimized oral inhibitor of the KRAS-G12C mutant.
  • MRTX849 is effective in treating non-small cells carrying KRAS-G12C gene mutations It has shown promising safety and anticancer activity in lung cancer (NSCLC) and colorectal cancer (CRC) and other solid tumors.
  • AST-3424 is a first-in-class DNA alkylation cancer therapy targeting overexpressed aldoketone reductase 1C3 (AKR1C3), which can selectively target cancers overexpressing AKR1C3 , and selectively releases a potent DNA alkylating agent in the presence of the AKR1C3 enzyme.
  • This selective mode of activation distinguishes AST-3424 from conventional alkylating agents such as cyclophosphamide and ifosfamide.
  • AKR 1C3 overexpression is present in a variety of treatment-resistant and refractory cancers, including HCC, castration-resistant prostate cancer (CRPC), and T-cell acute lymphoblastic leukemia (T-ALL).
  • AKR1C3 is highly expressed in as many as 15 solid and hematologic tumors.
  • the drug is undergoing phase II clinical trials in China and the United States (US OBI-3424-NCT03592264-II, castrated prostate cancer and liver cancer; US OBI-3424-NCT04315324-II, T-lymphocyte acute leukemia T-ALL; China AST-3424-CTR20191399-II phase, solid tumor; China AST-3424-CTR20201915-II phase, T lymphocyte acute leukemia T-ALL and B lymphocyte acute leukemia B-ALL).
  • AST-3424 Discovery of AST-3424 and compounds in in vivo animal model studies of this drug and similar drugs (hereinafter referred to as AST) has a good therapeutic effect on the KRAS mutant G12D subtype tumor model that highly expresses AKR1C3.
  • the application provides the following methods for treating cancer and pharmaceutical uses of the compounds.
  • a treatment method which uses a drug containing a DNA alkylating agent prodrug compound activated by AKR1C3 as a single drug or in combination with other therapeutic drugs to treat cancer and tumor patients with KRAS mutations.
  • the pharmaceutical application of the DNA alkylating agent prodrug compound activated by AKR1C3, the compound is used for the preparation of a single drug or in combination with other therapeutic drugs to treat cancer and tumor patients with KRAS mutation.
  • the AKR1C3-activated DNA alkylating agent prodrug compound means that the compound is a prodrug, and the prodrug molecule reacts with the AKR1C3 enzyme to release a cytotoxic DNA alkylating agent after the reaction.
  • AST-3424 is taken as an example. These compounds, as specific substrates of aldehyde and ketone reductase AKR1C3, can be quickly and effectively reduced only in cancer cells with high expression of AKR1C3, thereby releasing the cytotoxin-DNA alkylating agent AST-2660, AST-2660 cross-links DNA causing cancer cells to:
  • Therapeutic method uses the drug single drug containing the DNA alkylating agent prodrug compound of AKR1C3 activation or in combination with other therapeutic drugs to treat cancer and tumor patients with KRAS mutation, and the compound is selected from structural formula 1/2 and its salts, esters, solvents compounds, isotopomers:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 8 , R 9 , and R 10 are as described in the claims of patent application PCT/CN2020/089692, publication number WO2020228685A1. details as follows:
  • R 1 is C 6 -C 10 aryl or Z substituted aryl, 4-15 membered heterocycle or Z substituted heterocycle, 5-15 membered heteroaryl or Z substituted heteroaryl, 7-15 membered condensed ring Or Z replaces the fused ring;
  • R2 is hydrogen, halogen atom, cyano or isocyano, hydroxyl, mercapto, amino, OTs, OMS, C 1 -C 6 alkyl or Z substituted alkyl, C 2 -C 6 alkenyl or Z substituted alkenyl C 2 -C 6 alkynyl or Z substituted alkynyl, C 3 -C 8 cycloalkyl or Z substituted cycloalkyl, C 6 -C 10 aryl or Z substituted aryl, 4-15 membered heterocycle or Z-substituted heterocycle, 5-15 membered heteroaryl or Z-substituted heteroaryl, ether with 1-6 carbon atoms or Z-substituted alkoxy with 1-6 carbon atoms, -CONR 6 R 7 , -SO 2NR6R7 , -SO2R6 , -OCOO - R6 , -COOR6 , -NR6COR7 ,
  • R3 is hydrogen, halogen, cyano or isocyano, hydroxyl, mercapto, amino, OTs, OLCMS, C 1 -C 6 alkyl or Z substituted alkyl, C 2 -C 6 alkenyl or Z substituted alkenyl , C 2 -C 6 alkynyl or Z substituted alkynyl, C 3 -C 8 cycloalkyl or Z substituted cycloalkyl, C 6 -C 10 aryl or Z substituted aryl, 4-15 membered heterocycle or Z Substituted heterocycle, 5-15 membered heteroaryl or Z substituted heteroaryl, C 1 -C 6 alkoxy or Z substituted C 1 -C 6 alkoxy, -CONR 6 R 7 , -SO 2 NR 6 R 7 , -SO 2 R 6 , -OCO-R 6 , -OCOO-R 6 , -COOR 6 , -NR 6 COR 7 ,
  • R 4 and R 5 are each independently hydrogen, halogen atom, cyano or isocyano, hydroxyl, mercapto, amino, OTs, OLCMS, C 1 -C 6 alkyl or Z-substituted alkyl, C 2 -C 6 Alkenyl or Z substituted alkenyl, C 2 -C 6 alkynyl or Z substituted alkynyl, C 3 -C 8 cycloalkyl or Z substituted cycloalkyl, C 6 -C 10 aryl or Z substituted aryl, 4 -15-membered heterocycle or Z-substituted heterocycle, 5-15-membered heteroaryl or Z-substituted heteroaryl, C 1 -C 6 alkoxy or Z-substituted C 1 -C 6 alkoxy, -CONR 6 R 7 , -SO 2 NR 6 R 7 , -SO 2 R 6 , -OCOO-R 6 , -COOR
  • R 6 and R 7 are each independently hydrogen, cyano or isocyano, C 1 -C 6 alkyl or Z substituted alkyl, C 2 -C 6 alkenyl or Z substituted alkenyl, C 2 -C 6 alkyne or Z-substituted alkynyl, C 3 -C 8 cycloalkyl or Z-substituted cycloalkyl, C 6 -C 10 aryl or Z-substituted aryl, 4-15 membered heterocycle or Z-substituted heterocycle, 5-15 Elementary heteroaryl or Z-substituted heteroaryl, C 1 -C 6 alkoxy or Z substituted C 1 -C 6 alkoxy, or R 6 , R 7 groups form a 5- 7-membered heterocyclic group or Z-substituted 5-7-membered heterocyclic group;
  • R 8 and R 10 are each independently hydrogen, deuterium, aryl or Z substituted aryl, C 1 -C 6 alkyl or Z substituted alkyl, C 2 -C 6 alkenyl or Z substituted alkenyl, C 2 - C 6 alkynyl or Z substituted alkynyl, C 3 -C 8 cycloalkyl or Z substituted cycloalkyl, and one of them must be hydrogen or deuterium;
  • R 9 is a substituted C 6 -C 10 aryl group having at least one fluorine atom or a nitro group, a substituted 4-15 membered heterocycle having at least one fluorine atom or a nitro group, a substituted 4-15 membered heterocycle having at least one fluorine atom or a nitro group Substitute 5-15 membered heteroaryl.
  • Z substituent is halogen atom, cyano or isocyano, hydroxyl, mercapto, amino, OTs, OMS, C 1 -C 3 alkyl or substituted alkyl, C 1 -C 3 alkoxy or substituted alkoxy , C 2 -C 3 alkenyl or substituted alkenyl, C 2 -C 3 alkynyl or substituted alkynyl, C 3 -C 8 cycloalkyl or substituted cycloalkyl, aromatic ring, heterocyclic ring, heteroaryl ring and fused Ring or substituted aromatic rings, heterocyclic rings, heteroaryl rings and condensed rings, the substitution mode is mono-substitution or gem-di-substitution;
  • Substituted C 6 -C 10 aryl, substituted 4-15 membered heterocyclic, substituted 5-15 membered heteroaryl in R 9 are halogen atom, nitro, cyano or isocyano, hydroxyl, amino , C 1 -C 3 alkyl or alkoxy, alkenyl, alkynyl, cycloalkyl or benzene ring, substituted benzene ring, C 1 -C 3 alkoxy or halogen atom substituted alkoxy.
  • the compound of formula (1) (2) is selected from:
  • the compound of structural formula 1/2 is the same as AST-3424 and AST, both of which are prodrugs of AST-2660, which will be activated under the action of AKR1C3 enzyme to produce AST-2660 (a DNA alkylating agent) to exert anticancer effect :
  • Therapeutic method uses the drug single drug containing the DNA alkylating agent prodrug compound of AKR1C3 activation or in combination with other therapeutic drugs to treat cancer and tumor patients with KRAS mutation, the compound is selected from structural formula 3 and its salts, esters, solvates , Isotopic isomers:
  • A is H, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, CFH 2 , CF 2 H, CF 3 , F, Cl, Br, I, OCF 3 , COR or CON(R) 2 ;
  • E is SO or SO2 ;
  • X is Cl, Br, I or OSO 2 R;
  • Y is Cl, Br, I or OSO 2 R;
  • Each R is independently H or C1-C6 alkyl
  • G is a free radical selected from the group comprising formulas (B)-(AA):
  • R1 is H, C1-C6 alkyl, CH2 ( CH2 )nOH, CH2CH (OH) CH2OH , phenyl, pyridyl, benzyl or pyridylmethyl, provided that when R1 is benzene
  • R 2 and R 3 are each independently H, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, OR 6 , N(R 6 )(R 7 ), CFH 2 , CF 2 H, CF 3 , F, Cl, Br, I, OCF 3 , COR 6 , CON(R 6 )(R 7 ), SOR 6 , SON(R 6 )(R 7 ), SO2R 6 , SO2N(R 6 )(R 7 ), CN or NO 2 ;
  • R 4 is N(R 6 )(R 7 ), OH, OCH 2 (CH 2 )nN(R 6 )(R 7 ) or CH 2 (CH 2 )nN(R 6 )(R 7 );
  • R 5 is H or C1-C6 alkyl group
  • R 6 and R 7 are each independently H or C1-6 alkyl, or R 6 and R 7 together form a substituted or unsubstituted 5-membered or 6-membered heterocyclic ring:
  • Z is CH or N
  • W is CH2 , O, S, SO or SO2 ;
  • n 0 to 6;
  • the compound of formula (3) is selected from:
  • the compound of structural formula 3 is similar to AST-3424 and AST in principle, and they are all nitrogen mustard analogs Prodrugs of , which are activated by the enzyme AKR1C3 to produce nitrogen mustard analogs (a DNA alkylating agent) exerts anticancer effect:
  • Therapeutic method uses the drug containing the DNA alkylating agent prodrug compound containing AKR1C3 activation to treat cancer and tumor patients with KRAS mutation as a single drug or in combination with other therapeutic drugs.
  • the compound is selected from structural formula 4 and its salts, esters, and solvates Compounds, isotopomers:
  • R 1 is H, C 1-6 alkyl, C 3-6 cycloalkyl, 4-6 membered heterocycloalkyl, 5-6 membered heteroaryl or phenyl, wherein said C 1-6 alkyl, C 3-6 cycloalkyl, 4-6 membered heterocycloalkyl, 5-6 membered heteroaryl and phenyl are optionally substituted by 1, 2 or 3 R a ;
  • Each R is independently H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy or C 1-3 alkyl;
  • R 2 is H or C 1-6 alkyl
  • R1 and R2 are joined together to form a 4-6 membered heterocycloalkyl group together with the N atom to which they are attached, wherein the 4-6 membered heterocycloalkyl group is optionally substituted by 1, 2 or 3 R b ;
  • each R is independently H, F, Cl, Br, I, -CN, -OH, -NH2 , -OCH3 , -OCH2CH3 , -CH3 , or -CH2CH3 ;
  • R 3 is H, F, Cl, Br, I, -OH, -NH 2 , C 1-3 alkoxy or C 1-3 alkyl;
  • T 1 is -(CR c R d ) m -or -(CR c R d ) n -O-;
  • n 1, 2 or 3;
  • n 1 or 2;
  • T2 is N or CH
  • R c and R d are each independently H, F, C 1-3 alkyl or C 1-3 alkoxy;
  • R 4 , R 5 and R 6 are each independently H, F, Cl, Br, I, C 1-3 alkyl or C 1-3 alkoxy;
  • T is N or CH
  • R 7 and R 8 are each independently H, F, Cl, Br or I;
  • R 9 and R 10 are each independently H, F, Cl, Br, I, -CN or
  • the 4-6 membered heterocycloalkyl and 5-6 membered heteroaryl each contain 1, 2, 3 or 4 heteroatoms independently selected from N, -O- and -S-.
  • the compound of formula (4) is selected from:
  • the compound of structural formula 3 is the same as AST-3424 and AST, both of which are prodrugs of AST-2660, which will be activated under the action of AKR1C3 enzyme to produce AST-2660 (a DNA alkylating agent) to exert anticancer effect:
  • Therapeutic method uses the drug containing the DNA alkylating agent prodrug compound containing AKR1C3 activation as a single drug or in combination with other therapeutic drugs to treat cancer and tumor patients with KRAS mutation, the compound is selected from structural formula 5 and its salts, esters, solvates Compounds, isotopomers:
  • X 10 is O, S, SO or SO 2 ;
  • R 1 and R 2 are each independently hydrogen, C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 or -NR 13 COR 14 ;
  • X, Y and Z are each independently hydrogen, CN, halo, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 or -NR 13 COR 14 ;
  • R is hydrogen, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 4-15 membered hetero Ring, ether, -CONR 13 R 14 or -NR 13 COR 14 ;
  • R 13 and R 14 are each independently hydrogen, C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 4-15 membered heterocycle or ether
  • T comprises a phosphoramidate alkylating agent comprising one or more Z5- X5 - Y5 moieties bonded to an -OP( Z1 ) moiety, wherein Z5 is a heteroatom containing nitrogen, sulfur or oxygen, X5 is substituted or unsubstituted ethylidene, Y5 is halo or another leaving group, or Z5 - X5 - Y5 together form an aziridinyl (NCH 2 CH 2 ) moiety and Z 1 is O or S; and
  • alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, heteroaryl, ether groups are substituted or unsubstituted.
  • the compound of formula (5) is selected from:
  • the compound of structural formula 5 is similar to AST-3424 and AST, and is a prodrug of phosphoramidate alkylating agent, which will be activated under the action of AKR1C3 enzyme to produce T (a phosphoramidate alkylating agent, AST- 2660 is a phosphoramidate alkylating agent) to exert anti-cancer effects:
  • Therapeutic method uses the drug containing the DNA alkylating agent prodrug compound containing AKR1C3 activation as a single drug or in combination with other therapeutic drugs to treat cancer and tumor patients with KRAS mutation, the compound is selected from structural formula 6 and its salts, esters, solvates Compounds, isotopomers:
  • R 100 , R 101 and R 102 are each independently hydrogen, C1-C8 alkyl, C6-C12 aryl; or R 101 and R 102 form a 5-7 membered heterocyclic ring together with the nitrogen atom to which they are attached;
  • alkyl group and the aryl group are each substituted by 1-3 halo groups or 1-3 C1-C6 alkyl groups;
  • R 1 and R 2 are each independently phenyl or methyl
  • X, Y, and Z are each independently hydrogen or halo
  • R is hydrogen or C1-C6 alkyl or halogen-substituted alkyl.
  • Compound also of course includes the compound itself as well as the solvate, salt, ester or isotope of the compound and the like.
  • Cx-Cy or " Cxy " before a group refers to the range of the number of carbon atoms present in the group.
  • C1-C6 alkyl refers to an alkyl group having at least 1 and at most 6 carbon atoms.
  • Alkyl means a monovalent saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms, and in some embodiments 1 to 6 carbon atoms.
  • Cy-y alkyl refers to an alkyl group having x to y carbon atoms.
  • the term includes, for example, straight chain and branched chain hydrocarbon groups such as methyl ( CH3- ), ethyl ( CH3CH2- ), n- propyl ( CH3CH2CH2- ), isopropyl group ((CH 3 ) 2 CH-), n-butyl group (CH 3 CH 2 CH 2 CH 2 -), isobutyl group ((CH 3 ) 2 CHCH 2 -), sec-butyl group ((CH 3 )(CH 3 CH 2 )CH-), tert-butyl ((CH 3 ) 3 C-), n-pentyl (CH 3 CH 2 CH 2 CH 2 CH 2 - ) and neopentyl ((CH 3 ) 3 CCH 2 - ).
  • straight chain and branched chain hydrocarbon groups such as methyl ( CH3- ), ethyl ( CH3CH2- ), n- propyl ( CH3CH2CH2- ), isopropyl group ((CH 3 ) 2 CH
  • Aryl means an aromatic group having from 6 to 14 carbon atoms and containing no ring heteroatoms and having a single ring (for example, phenyl) or multiple condensed (fused) rings (for example, naphthyl or anthracenyl). group.
  • aryl is used when the point of attachment is at an aromatic carbon atom or "Ar” is suitable (eg, 5,6,7,8 tetrahydronaphthalen-2-yl is aryl because its point of attachment is at the 2 position of the aromatic phenyl ring).
  • Arylenyl refers to a divalent aryl group having an appropriate hydrogen content.
  • Cycloalkyl means a saturated or partially saturated cyclic group having from 3 to 14 carbon atoms and no ring heteroatoms and having a single ring or multiple rings including fused, bridged and spiro ring systems.
  • cycloalkyl applies when the point of attachment is at a non-aromatic carbon atom (e.g. 5, 6, 7, 8-tetrahydronaphthalen-5-yl).
  • cycloalkyl includes cycloalkenyl groups.
  • cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and cyclohexenyl.
  • Cycloalkylene refers to a divalent cycloalkyl group having an appropriate hydrogen content.
  • Halo refers to one or more of fluorine, chlorine, bromine and iodine.
  • Heteroaryl means an aromatic group having 1 to 14 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur and includes monocyclic rings such as imidazolyl-2-yl and imidazol-5-yl) and polycyclic systems (eg imidazopyridinyl, benzotriazolyl, benzimidazol-2-yl and benzimidazol-6-yl).
  • monocyclic rings such as imidazolyl-2-yl and imidazol-5-yl
  • polycyclic systems eg imidazopyridinyl, benzotriazolyl, benzimidazol-2-yl and benzimidazol-6-yl.
  • heteroaryl such as 1,2,3,4-tetrahydroquinolin-6-yl and 5,6,7,8-tetrahydroquinolin-3-yl.
  • the nitrogen and/or sulfur ring atoms of the heteroaryl are optionally oxidized to provide an N-oxide (N ⁇ O), sulfinyl or sulfonyl moiety.
  • heteroaryl includes, but is not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuryl, benzothiofuryl, benzothiophenyl, benzoxa Azolyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzothienyl, benzimidazolyl, carbazolyl , NH-carbazolyl, carbolinyl, chromanyl (chromanyl), benzopyranyl (chromenyl), cinnolinyl (cinnolinyl), dithiazinyl, furyl, furyl, Imidazolidinyl
  • Heterocyclic or “heterocycle” or “heterocycloalkyl” or “heterocyclyl” means a heteroatom having 1 to 14 carbon atoms and 1 to 6 selected from the group consisting of nitrogen, sulfur or oxygen and include monocyclic and polycyclic ring systems including fused, bridged and spiro ring systems.
  • heterocyclic For polycyclic ring systems having aromatic and/or non-aromatic rings, the terms “heterocyclic”, “heterocyclic” when at least one ring heteroatom is present and the point of attachment is at an atom of the non-aromatic ring , “heterocycloalkyl” or “heterocyclyl” are suitable (such as 1,2,3,4-tetrahydroquinolin-3-yl, 5,6,7,8-tetrahydroquinolin-6-yl and decahydroquinolin-6-yl).
  • the heterocyclyl group herein is a 3-15 membered, 4-14 membered, 5-13 membered, 7-12 or 5-7 membered heterocyclic ring.
  • the heterocycle contains 4 heteroatoms. In some other embodiments, the heterocycle contains 3 heteroatoms. In another embodiment, the heterocycle contains up to 2 heteroatoms. In some embodiments, the nitrogen and/or sulfur atoms of the heterocyclyl are optionally oxidized to provide N-oxide, sulfinyl, sulfonyl moieties.
  • Heterocyclic groups include, but are not limited to, tetrahydropyranyl, hexahydropyridinyl, N-methylhexahydropyridin-3-yl, hexahydropyrazinyl, N-methylpyrrolidin-3-yl, 3 -pyrrolidinyl, 2-pyrrolidinon-1-yl, morpholinyl and pyrrolidinyl.
  • a prefix indicating the number of carbon atoms (eg, C3-10) refers to the total number of carbon atoms in the heterocyclyl moiety excluding the number of heteroatoms.
  • Divalent heterocyclic groups will have suitably adjusted hydrogen content.
  • Biaryl refers to a structure in which two aromatic rings are connected through a C-C single bond, such as biphenyl, bipyridine, etc.
  • a group may be substituted with one or more substituents (eg 1 , 2, 3, 4 or 5 substituents).
  • the substituent is selected from the group consisting of chlorine, fluorine, -OCH 3 , methyl, ethyl, isopropyl, cyclopropyl, -CO 2 H and its salts, and C 1 -C 6 alkyl Esters, CONMe 2 , CONHMe, CONH 2 , -SO 2 Me, -SO 2 NH 2 , -SO 2 NMe 2 , -SO 2 NHMe, -NHSO 2 Me, -NHSO 2 CF 3 , -NHSO 2 CH 2 Cl, -NH 2 , -OCF 3 , -F 3 and -OCHF 2 .
  • the compound of formula (6) is selected from:
  • the compound of structural formula 6 is similar to AST-3424 and AST, and is a prodrug of AST-2660, which will be activated under the action of AKR1C3 enzyme to produce AST-2660 to exert anticancer effect:
  • AST-3424 (OBI-3424), (hereinafter code-named AST) and The synthesis method and spectral data of the patent application: PCT/US2016/021581, publication number WO2016145092A1, corresponding to Chinese application number 2016800150788, publication number CN107530556A; PCT/US2016/062114, publication number WO2017087428A1, corresponding to Chinese application number 2016800200101A, publication PCT/CN2020/089692, disclosed by publication number WO2020228686; related preparations concentrated injections, and related prescriptions, preparation methods, clinical compatibility, and administration methods are described and disclosed in detail by related patents: WO2021008520A1, WO2021043275A1, and the present invention combines the above The full text of the application text is incorporated.
  • Monotherapy that is, monotherapy.
  • Combined use that is, combined drug therapy.
  • Monotherapy refers to the use of only one anticancer drug in a course of treatment.
  • Combination therapy refers to the simultaneous or successive use of two or more anticancer drugs in one course of treatment.
  • combination therapy needs to explore different dosages and administration cycles according to the characteristics of the disease and the types of drugs used in combination. Only based on the above conditions can the combination drug treatment plan obtained through exploration be able to achieve better therapeutic effects than single drug therapy.
  • the drug dosage and dosage cycle of single drug and combined therapy need to be obtained through clinical trials with reference to the dosage and dosage of AST-3424 and its similar compounds and other drugs mentioned above.
  • the dosage of a single drug can be determined by referring to the animal experiment doses of WO2019062919A1, WO2016145092A1, and WO2017087428A1.
  • the KRAS mutation is selected from KRAS-G12D mutation, KRAS-G12V mutation and KRAS-G12C mutation.
  • Any one or two gene mutations in the genes corresponding to KRAS can be detected and diagnosed by commercially available (accompanied) diagnostic kits, such as the diagnostic kits approved in China:
  • NGS sequencing (YS 450 gene NGS large panel) can also be used to determine the specific KRAS mutation subtype.
  • the KRAS mutation is selected from the KRAS-G12D mutation.
  • the TMB (Tumor Mutation Burden) level of the gene mutation is moderate.
  • TMB Tumor mutation load (burden), that is, tumor gene mutation load
  • burden Tumor mutation load
  • Mb represents every million bases
  • CheckMate-032 This is a Phase II clinical trial of 401 advanced lung cancer patients who failed first-line treatment, receiving PD-1 inhibitors alone or in combination with Yipimu.
  • TMB Tumor mutation load
  • the effective rates of the three groups are 62%, 20%, and 23%, respectively, and the effective rate of the high TMB group is 3 times; and the median overall survival of the three groups were: 22.0 months, 3.6 months, 3.4 months - 22.0 months and 3.4 months, a difference of 6 times!
  • prodrug compound is preferably selected from:
  • the aforementioned cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, liver cancer, colon cancer, rectal cancer, lung cancer, bladder cancer.
  • Other therapeutic drugs are selected from KRAS inhibitors, immunotherapeutic drugs (immune checkpoint inhibitors).
  • KRAS inhibitors are substances that inhibit the enzymatic activity of KRAS.
  • KRAS inhibitors please refer to the review literature Goebel, Lisa&Müller, Matthias&Goody, Roger&Rauh, Daniel.(2020).KRasG12C inhibitors in clinical trials: A short historical perspective.RSC Medicinal Chemistry.11.10.1039/D0MD00096E.
  • KRAS inhibitors that have entered clinical development or are already on the market include Sotorasib (AMG510) developed by Amgen, adagrasib (MRTX849) developed by Mirati Therapeutics, GDC6036 developed by Roche, LY3499446 developed by Eli Lilly, JNJ74699157 (ARS3248) jointly developed by Araxes and Janssen ), D-1553 developed by Inventio, JAB-3312 developed by JACOS, JAB-3068 developed by JACOS, GH35 developed by Gen House, BPI-421286 developed by Betta Pharmaceuticals, BI17016963 developed by Boehringer Ingelheim, mRNA-5671 developed by Moderna, and AZD-4785 jointly developed by AstraZeneca and Ionis.
  • Sotorasib AMG510
  • MRTX849 adagrasib
  • Mirati Therapeutics GDC6036 developed by Roche
  • LY3499446 developed by Eli Lilly
  • the KRAS inhibitor is selected from Sotorasib (AMG510), adagrasib (MRTX849), GDC6036, LY3499446, JNJ74699157 (ARS3248), D-1553;
  • Immune checkpoint molecules are inhibitory regulatory molecules in the immune system that are critical for maintaining self-tolerance, preventing autoimmune responses, and minimizing tissue damage by controlling the timing and intensity of immune responses .
  • Immune checkpoint molecules are expressed on immune cells, which will inhibit the function of immune cells, so that the body cannot produce effective anti-tumor immune responses, and tumors form immune escape.
  • the tumor-related immune checkpoint molecules mainly include: PD1, PD-L1, CTLA4, Tim3, and LAG3, etc.
  • PD1, PD-L1, and CTLA4 are the most studied.
  • Immune checkpoint inhibitors are some monoclonal antibody drugs developed for the corresponding immune checkpoints. inhibition. Immunotherapy drugs are selected from PD-1 monoclonal antibody and PD-L1 monoclonal antibody.
  • the cancer and tumor are preferably selected from gastric cancer, pancreatic cancer, and lung cancer.
  • the above-mentioned medicines should also add pharmaceutically acceptable auxiliary materials or excipients according to the specificity of medicines, medicines and preparations.
  • the medicine can be in any dosage form for clinical application, such as tablet, suppository, dispersible tablet, enteric-coated tablet, chewable tablet, orally disintegrating tablet, capsule, sugar-coated agent, granule, dry powder, oral solution, small needle for injection , Freeze-dried powder for injection or large infusion.
  • the pharmaceutically acceptable adjuvant or excipient in the medicine may include one or more of the following: diluent, solubilizer, disintegrant, suspending agent, lubricant, viscose Mixtures, fillers, flavoring agents, sweeteners, antioxidants, surfactants, preservatives, coating agents, and pigments, etc.
  • Figure 1 is Growth curves of tumor volume in each group of mice in the gastric cancer GA6201 model
  • Figure 2 is Relative tumor inhibition rate curves of mice in each group in the gastric cancer GA6201 model
  • Figure 3 is Body weight curves of mice in each group in the gastric cancer GA6201 model
  • Figure 4 is Body weight change percentage curves of mice in each group in the gastric cancer GA6201 model
  • Figure 5 is Growth curves of tumor volume in each group of mice in the pancreatic cancer PA1222 model
  • Figure 6 is The relative tumor inhibition rate of mice in each group in the pancreatic cancer PA1222 model
  • Figure 7 is Body weight curves of mice in each group in the pancreatic cancer PA1222 model
  • Figure 8 is Body weight change percentage curves of mice in each group in the pancreatic cancer PA1222 model
  • Figure 9 is The growth curves of the tumor volume of mice in each group in the lung cancer LU11693 model.
  • Figure 10 is Relative tumor inhibition rate curves of mice in each group in the lung cancer LU11693 model
  • Figure 11 is Body weight curves of mice in each group in the lung cancer LU11693 model
  • Figure 12 is Body weight change percentage curves of mice in each group in the lung cancer LU11693 model
  • Figure 13 is the tumor volume growth curves of mice in each group in the subcutaneous xenograft model of human pancreatic cancer HPAF-II;
  • Fig. 14 is the percentage change curve of mouse body weight in each group in the subcutaneous xenograft model of human-derived pancreatic cancer HPAF-II;
  • Figure 15 is Tumor volume growth curves of mice in each group in the subcutaneous model of lung cancer LU5161;
  • Figure 16 is Body weight change percentage curves of mice in each group in the lung cancer LU5161 subcutaneous model
  • Figure 17 is Tumor volume growth curves of mice in each group in the colon cancer CR3820 subcutaneous model
  • Figure 18 is Body weight change percentage curves of mice in each group in the intestinal cancer CR3820 subcutaneous model
  • Figure 19 is Tumor volume growth curves of mice in each group in the subcutaneous model of pancreatic cancer PA2637;
  • Figure 20 is Body weight change percentage curves of mice in each group in the pancreatic cancer PA2637 subcutaneous model
  • Figure 21 is Tumor volume growth curves of mice in each group in the subcutaneous model of lung cancer LU11873;
  • Figure 22 is Body weight change percentage curves of mice in each group in the lung cancer LU11873 subcutaneous model
  • Figure 23 is Tumor volume growth curves of mice in each group in the subcutaneous model of pancreatic cancer PA1383;
  • Figure 24 is Body weight change percentage curves of mice in each group in the pancreatic cancer PA1383 subcutaneous model
  • Figure 25 is a photo of the IHC staining results of GA6201, LU11693, PA1222 models and the control group.
  • administering or “administration of” a drug (and grammatical equivalents of this phrase) to a patient means direct administration (which may be administered to the patient by a medical professional or may be self-administered) and/or indirect administration, It is the act of prescribing a drug.
  • direct administration which may be administered to the patient by a medical professional or may be self-administered
  • indirect administration It is the act of prescribing a drug.
  • a physician who instructs a patient to self-administer a drug and/or provides a prescription for the drug to the patient is administering the drug to the patient.
  • Cancer refers to leukemias, lymphomas, carcinomas and other malignancies (including solid tumors) of potentially unrestricted growth that can spread locally by invasion and systemically by metastasis.
  • cancers include, but are not limited to, adrenal, bone, brain, breast, bronchi, colon and/or rectum, gallbladder, head and neck, kidney, larynx, liver, lung, nervous tissue, pancreas, prostate, parathyroid, Cancers of the skin, stomach and thyroid.
  • cancers include acute and chronic lymphocytic and granulocytic neoplasms, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and carcinoma in situ, Ewing's sarcoma, epidermoid carcinoma, giant Cytoma, glioblastoma multiforme, hair cell tumor, enteric ganglioneuroma, hyperplastic corneal neuroma, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemia, lymphoma, Malignant carcinoid tumor, malignant melanoma, malignant hypercalcemia, marfanoid habitus tumor, medullary epithelial carcinoma, metastatic skin cancer, mucosal neuroma, myeloma, mycosis fungoides, neuroderm Cytoma, osteosarcoma, osteogenic and other sarcomas, ovarian tumors, pheochromocytoma,
  • a patient and “individual” are used interchangeably and refer to a mammal in need of cancer treatment.
  • the patient is a human.
  • the patient is a human being diagnosed with cancer.
  • a "patient” or “individual” may refer to a non-human mammal, such as a non-human primate, dog, cat, rabbit, pig, mouse, for screening, characterization and evaluation of drugs and therapies or rats.
  • Solid tumor refers to solid tumors including, but not limited to, metastatic tumors in bone, brain, liver, lung, lymph nodes, pancreas, prostate, skin, and soft tissue (sarcomas).
  • a "therapeutically effective amount" of a drug is that amount of a drug that, when administered to a patient with cancer, will have the desired therapeutic effect (eg, alleviation, amelioration, remission, or elimination of the clinical manifestations of one or more cancers in the patient) .
  • a therapeutic effect does not necessarily occur through administration of one dose, and may only occur after administration of a series of doses. Thus, a therapeutically effective amount can be administered in one or more administrations.
  • Treating means taking steps to obtain a beneficial or desired result (including a clinical result).
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer; reduction in extent of disease; delay or slowing of disease progression; amelioration, remission or stabilization of disease state; or other beneficial outcomes.
  • treatment of cancer can result in a partial response or stable disease.
  • Tumor cell refers to a tumor cell of any appropriate species (eg, a mammal such as murine, canine, feline, equine or human).
  • a patient and “individual” are used interchangeably and refer to a mammal in need of cancer treatment.
  • the patient is a human.
  • the patient is a human being diagnosed with cancer.
  • a "patient” or “individual” may refer to a non-human mammal, such as a non-human primate, dog, cat, rabbit, pig, mouse, for screening, characterization and evaluation of drugs and therapies or rats.
  • Treating or “treating a patient” refers to administering, using or administering to a patient a therapeutically effective amount of a drug relevant to the present invention.
  • administering or “administering” "using" a drug to a patient refers to direct administration or administration (which may be administered or administered to the patient by a medical professional or may be self-administered or administered) and/or indirect administration or administration, which may prescribe the drug the behavior of.
  • direct administration or administration which may be administered or administered to the patient by a medical professional or may be self-administered or administered
  • indirect administration or administration which may prescribe the drug the behavior of.
  • a physician who instructs a patient to self-administer or administer a drug and/or provides a prescription for the drug to the patient is administering or administering the drug to the patient.
  • Treatment of a condition or patient refers to steps taken to obtain beneficial or desired results (including clinical results).
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer; reduction in extent of disease; delay or slowing of disease progression; amelioration, remission or stabilization of disease state; or other beneficial outcomes.
  • treatment of cancer can result in a partial response or stable disease.
  • test substances AST, AST-3424 and Ifosfamide were in Pharmacodynamic evaluation in subcutaneous xenograft model of gastric cancer GA6201
  • Gastric cancer GA6201 PDX model is a model of KRAS pathogenic mutation (KRAS-G12D) with G12D amino acid mutation.
  • BALB/c nude mice were inoculated subcutaneously
  • the model GA6201 tumor mass was used to establish a subcutaneous transplanted tumor model of human gastric cancer.
  • the test was divided into test drug Ifosfamide (ifosfamide) 60mg/kg group, test drug AST-3424 5mg/kg group, AST 2.5mg/kg and 5mg/kg groups and normal saline (pH 7.0-7.6) vehicle control group, A total of 5 groups, 5 mice in each group.
  • the normal saline (pH 7.0-7.6) vehicle control group, the test drug AST-3424 5mg/kg, AST 2.5mg/kg and 5mg/kg groups were given by tail vein injection, administered once a week, and administered three times in total. Week, look around.
  • the Ifosfamide 60mg/kg group was administered by intraperitoneal injection, five consecutive days a week and two days off, for a total of two weeks of administration, and five weeks of observation.
  • the curative effect is evaluated according to the relative tumor inhibition rate TGI (%), and the safety evaluation is carried out according to the animal body weight change and death.
  • Table 1 Different doses of test drugs in Experimental design of antitumor effect in gastric cancer GA6201 tumor model
  • mice The tumor volumes of mice in different groups were measured on different days, and the average values were obtained. The results are shown in Table 2 below.
  • Table 2 In Changes of tumor volume of mice in each group with treatment time in gastric cancer GA6201 model
  • T/C% is the percentage value of the relative tumor volume or tumor weight between the treatment group and the control group at a certain time point. Calculated as follows:
  • T and C are the average relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively.
  • Table 4 The relative tumor inhibition rate of tumors in each group in the gastric cancer GA6201 model
  • Figure 2 can be obtained by making the above table 4 as a graph.
  • mice The body weights of mice in different groups were measured on different days, and the average values were obtained. The results are shown in Table 5 below.
  • Table 6 The percentage of body weight change of mice with different inoculation days in gastric cancer GA6201 model (%Group Mean
  • Group01, Group02, Group03, Group04, and Group05 in Table 4/5/6 are the above-mentioned Group 1, Group 2, Group 3, Group 4, Group 5, and Group 6.
  • 0/1/2/3/4/7/8/9/10/11/14/15/16/17/18/21/24/28/31/35/38 is the number of days after vaccination.
  • test drug AST-3424 was at a dose of 5 mg/kg, and the test drug AST was at a dose of 2.5 mg/kg and 5 mg/kg.
  • Gastric cancer GA6201 has a significant inhibitory effect on tumor growth, and there are statistically significant differences compared with the control group.
  • Test drug Ifosfamide at a dose of 60mg/kg, on Gastric cancer GA6201 has a certain effect of inhibiting tumor growth, but there is no statistically significant difference compared with the control group.
  • test substances AST and Gemcitabine were in Pharmacodynamics and safety evaluation in subcutaneous xenograft model of pancreatic cancer PA1222
  • the PA1222 PDX model of pancreatic cancer is a model of a KRAS pathogenic mutation with a G12D amino acid mutation (KRAS-G12D).
  • BALB/c nude mice were inoculated subcutaneously
  • the model PA1222 tumor mass was used to establish a subcutaneous transplanted tumor model of human pancreatic cancer.
  • the test was divided into test drug Gemcitabine (gemcitabine) 120mg/kg group, test drug AST 10mg/kg and 7.5% absolute ethanol + 7.5% polyoxyethylene (35) castor oil + 85% glucose injection D5W (pH7.4) vehicle Control group, 3 groups in total, 5 mice in each group.
  • test drug AST 10mg/kg group are tail vein injection administration, every week Administration once, continuous administration for three weeks.
  • the test drug Gemcitabine 120mg/kg group was administered by intraperitoneal injection once a week for 3 consecutive weeks.
  • the curative effect is evaluated according to the relative tumor inhibition rate TGI (%), and the safety evaluation is carried out according to the animal body weight change and death.
  • the administration volume is 10 ⁇ l/g
  • mice The tumor volumes of mice in different groups were measured on different days, and the average values were obtained. The results are shown in Table 8 below.
  • Table 8 Changes of tumor volume of mice in each group with treatment time in PA1222 model of pancreatic cancer
  • Table 9 Drug efficacy analysis table of each group in gastric cancer GA6201 model
  • T/C% Relative tumor proliferation rate
  • T and C are the average relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively.
  • Table 10 The relative tumor inhibition rate of each group of tumors in the pancreatic cancer PA1222 model
  • mice The body weights of mice in different groups were measured on different days, and the average values were obtained. The results are shown in Table 11 below.
  • Table 11 Body weight of mice with different inoculation days in PA1222 model of pancreatic cancer
  • Test drug Gemcitabine at 120mg/kg (Group 2) dose, on Pancreatic cancer PA1222 has a certain inhibitory effect on tumor growth, and there is a statistically significant difference compared with the control group.
  • Test drug AST at 10mg/kg (Group 3) dose, on Pancreatic cancer PA1222 has a significant tumor inhibitory effect, which is statistically significantly different from that in the control group, and two mice in this group were cured, with a cure rate of 40%.
  • mice in the test drug Gemcitabine (120mg/kg, Group 2) treatment group, AST 10mg/kg (Group 3) treatment group, and control group (Group 1) did not have any significant weight loss. Well tolerated.
  • test substances AST and Cisplatin are in Pharmacodynamics and safety evaluation in subcutaneous xenograft model of lung cancer LU11693
  • the lung cancer LU11693 PDX model is a model of KRAS pathogenic mutations with G12C amino acid mutations.
  • BALB/c Nude nude mice subcutaneously inoculated
  • the model LU11693 tumor mass was used to establish a subcutaneous transplanted tumor model of human lung cancer.
  • the test was divided into test drug Cisplatin 4mg/kg group, test drug AST 10mg/kg group and 7.5% absolute ethanol + 7.5% polyoxyethylene (35) castor oil + 85% glucose injection D5W (pH7.4) vehicle control group , a total of 3 groups, 6 mice in each group, administered by tail vein injection, administered once a week, for 3 consecutive weeks.
  • the curative effect is evaluated according to the relative tumor inhibition rate TGI (%), and the safety evaluation is carried out according to the animal body weight change and death.
  • Table 13 Different doses of test drugs in Experimental design of antitumor effect in lung cancer LU11693 PDX tumor model
  • the administration volume is 10 ⁇ l/g
  • mice The tumor volumes of mice in different groups were measured on different days, and the average values were obtained. The results are shown in Table 14 below.
  • Table 14 Changes of tumor volume of mice in each group with treatment time in lung cancer LU11693 model
  • Table 15 Drug efficacy analysis table of each group in lung cancer LU11693 model
  • the relative tumor proliferation rate, T/C% is the percentage value of the relative tumor volume or tumor weight between the treatment group and the control group at a certain time point. Calculated as follows:
  • T and C are the average relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively.
  • Table 16 The relative tumor inhibition rate of each group of tumors in the lung cancer LU11693 model
  • mice The body weights of mice in different groups were measured on different days, and the average values were obtained. The results are shown in Table 17 below.
  • Table 17 Body weight of mice with different inoculation days in lung cancer LU11693 model
  • the rate TGI (%) was 23.98%.
  • test drug AST (10mg/kg) treatment group showed a certain tumor inhibitory effect on the 28th day (Day 28) after the first administration, and there was a statistically significant difference compared with the control group (p ⁇ 0.001).
  • the inhibition rate TGI (%) is 54.64%, TGI is less than 60%, and there is no obvious tumor inhibitory effect.
  • mice in the test drug AST (10 mg/kg) and Cisplatin (4 mg/kg) treatment groups experienced severe weight loss, which may be related to the potential toxicity of high-dose drugs.
  • AST-3424 and AST have significant efficacy in KRAS pathogenic mutation models with G12D amino acid mutation: gastric cancer GA6021 and pancreatic cancer PA1222 PDX models, with TGI% greater than 90%;
  • AST in the KRAS pathogenic mutation model with G12C amino acid mutation the tumor inhibitory effect in lung cancer LU11693 is not obvious, and the TGI% is less than 60%;
  • AST-3424 and AST have generally significant curative effects on various cancer indications.
  • the curative effect of AST is not only related to the cancer type, but also related to other factors.
  • the human pancreatic cancer HPAF-II subcutaneous xenograft model is a CDX model with a KRAS G12D pathogenic mutation.
  • mice were subcutaneously inoculated with human pancreatic cancer HPAF-II cells to establish a subcutaneous transplantation model of human pancreatic cancer.
  • the test is divided into test drug treatment groups: Ifosfamide 60mg/kg single drug group (Group 2) is intraperitoneally administered once a day for 5 consecutive days, rested for 2 days, and then administered once a day for continuous administration 5 days; AST 4mg/kg single-drug group (Group 3) was given tail vein administration once a day for 5 consecutive days, rested for 2 days, rested for another 2 weeks, and administered once a day for continuous administration AST-3424 1 mg/kg single drug group (Group 4) was administered via tail vein once a week for 3 weeks in total; AST-3424 1 mg/kg single drug group (Group 5) was administered via tail vein Administration, administration once a day, continuous administration for 5 days, rest for 2 days, rest for 2 weeks, administration once a day, continuous administration for 5 days; and glucose injection (pH7.7-8.0
  • Table 19 Administration routes, doses and regimens in human-derived pancreatic cancer HPAF-II animal models
  • the abbreviations related to the route of administration i.v. means tail vein injection, i.p. , QD*5" means administer once a day for 5 consecutive days, rest for 2 days, rest for 2 weeks, and then administer once a day for 5 consecutive days.
  • the tumor growth of each treatment group and control group was recorded on different days of the experiment, as shown in Table 20, and the corresponding growth curves of the tumor volumes of mice in each group are shown in FIG. 13 .
  • the curative effect was evaluated according to the relative tumor proliferation rate and the relative tumor inhibition rate, and the drug efficacy analysis of each group is shown in Table 21.
  • the results of the mouse body weight changes are shown in Table 22.
  • the change curve is shown in Figure 14.
  • Table 20 Changes in tumor volume of mice in each group with treatment time in the subcutaneous model of human pancreatic cancer HPAF-II (mm 3 )
  • Table 21 Drug efficacy analysis table of each group in human pancreatic cancer HPAF-II subcutaneous model
  • the average tumor volume of the mice in the vehicle control group was 2212.64 mm 3 on the 31st day (Day 31) after the start of administration.
  • the average tumor volume of the test drug Ifosfamide in the 60mg/kg dose treatment group (group 2) on Day 31 was 2678.00mm 3 , and the relative tumor inhibition rate TGI (%) was -14.65%, which was statistically insignificant compared with the control group (p>0.05).
  • the test drug AST was in the 4mg/kg (QD ⁇ 5, 2 days off, 2 weeks off, QD ⁇ 5) and 8mg/kg (QW ⁇ 3) dose treatment groups (groups 3 and 4), and AST-3424 In the 1mg/kg (QD ⁇ 5, 2 days off, 2weeks off, QD ⁇ 5) dose group (group 5), the average tumor volumes on Day 31 were 457.66, 170.65 and 685.85mm 3 , compared with the control group, the statistically There was a significant difference (p ⁇ 0.05), and the relative tumor inhibition rates TGI (%) were 79.55%, 92.64% and 69.46%, respectively.
  • AST-3424 and AST in the model with KRAS G12D pathogenic mutation AST at 8mg/kg (QW ⁇ 3), 4mg/kg (QD ⁇ 5, 2 days off, 2 weeks off, QD ⁇ 5)
  • AST-3424 has a significant anti-tumor effect on the human pancreatic cancer HPAF-II subcutaneous model
  • the mice in each test drug treatment group did not lose weight during the treatment, and the mice were well tolerated.
  • the lung cancer LU5161 subcutaneous model is a PDX model with a KRAS G12D pathogenic mutation.
  • mice Balb/nude female mice were inoculated subcutaneously
  • the tumor mass of lung cancer LU5161 was used to establish the subcutaneous transplantation tumor model of human lung cancer.
  • the test is divided into the test drug Ifosfamide 60mg/kg single drug group (Group 2), administered once a day for 5 consecutive days, resting for 2 days, and then administered once a day for 5 consecutive days; AST 4mg/kg monotherapy Drug group (Group 3) and AST 8mg/kg single drug group (Group 4), both administered once a week for a total of 3 weeks; AST 4mg/kg single drug group (Group 5) and AST-34241mg/kg
  • the single-drug group (Group 6) was administered once a day for 5 consecutive days, rested for 2 days, rested for 2 weeks, and then administered once a day for 5 consecutive days, as well as glucose injection (pH7.
  • Vehicle control group (Group 1), administered once a day for 5 consecutive days, rested for 2 days, rested for another 2 weeks, and then administered once a day for 5 consecutive days. There were 6 groups in this study, 6 mice in each group.
  • the test drug Ifosfamide was administered intraperitoneally, and the vehicle control group, AST, and AST-3424 were all administered by tail vein injection. See Table 23 for the route of administration, dosage and regimen of the experimental design.
  • Table 23 Administration route, dose and regimen in subcutaneous model of lung cancer LU5161
  • the tumor growth of each treatment group and control group was recorded on different days of the experiment, as shown in Table 24, and the corresponding growth curves of the tumor volumes of the mice in each group are shown in FIG. 15 .
  • the curative effect was evaluated according to the relative tumor proliferation rate and the relative tumor inhibition rate, and the efficacy analysis of each group is shown in Table 25.
  • Table 24 Changes in tumor volume of mice in each group with treatment time in the subcutaneous model of lung cancer LU5161 (mm 3 )
  • Table 25 Drug efficacy analysis table of each group in the subcutaneous model of lung cancer LU5161
  • mice in the vehicle control group were 2238.97 mm 3 on the 29th day (Day 28) after the start of administration.
  • the average tumor volume of the test drug Ifosfamide in the 60mg/kg dose treatment group (Group 2) on Day 28 was 1636.39mm 3 , and the relative tumor inhibition rate TGI (%) was 27.39%. There was no statistically significant difference compared with the control group ( p>0.05).
  • AST-3424 and AST in the model with KRAS G12D pathogenic mutation show that AST-3424 and AST in the model with KRAS G12D pathogenic mutation, AST 4mg/kg (QW ⁇ 3), 8mg/kg (QW ⁇ 3), 4mg/kg (QD ⁇ 5, 2 days off, 2 weeks off, QD ⁇ 5) dose treatment groups (Group 3, Group 4 and Group 5), and AST-3424 at 1 mg/kg (QD ⁇ 5, 2 days off, 2 weeks off, QD ⁇ 5) dose Group (Group 6) under the test dosage and dosing frequency of this study, to The subcutaneous model of lung cancer LU5161 has significant anti-tumor effect. Ifosfamide administration group did not produce anti-tumor effect. The mice in each test drug treatment group tolerated well during the treatment period.
  • test substance AST and Ifosfamide monotherapy in Antitumor effect and safety evaluation of intestinal cancer CR3820 subcutaneous model
  • the colon cancer CR3820 subcutaneous model is a PDX model with a KRAS G12D pathogenic mutation.
  • NOD.SCID female mice were inoculated subcutaneously Intestinal cancer CR3820 tumor mass was used to establish a subcutaneous transplanted tumor model of human intestinal cancer.
  • the test was divided into test drug Ifosfamide, 60mg/kg monotherapy group (QD ⁇ 5/week ⁇ 2 weeks, Group 2), intraperitoneal administration, once a day, for 5 consecutive days, rest for 2 days, and then give daily AST 8 mg/kg single drug group (QW ⁇ 3, Group 3), administered via tail vein once a week for a total of 3 weeks; AST 4 mg/kg single drug Group (QD ⁇ 5, 2 days off, 2 weeks off, QD ⁇ 5, Group 4) and the vehicle control group glucose injection (pH7.7-8.0, Group 1), both were administered through the tail vein, and the administration cycle was It was administered once a day for 5 consecutive days, rested for 2 days, rested for 2 weeks, and then administered once a day for 5 consecutive days; the experiment consisted of 4 groups, 6 mice in each group. See Table 27 for the
  • Table 27 Administration route, dose and regimen in subcutaneous model of intestinal cancer CR3820
  • the tumor growth of each treatment group and control group was recorded on different days of the experiment, as shown in Table 28, and the growth curves of the tumor volumes of mice in each group are shown in FIG. 17 .
  • the curative effect was evaluated according to the relative tumor proliferation rate and the relative tumor inhibition rate, and the drug efficacy analysis of each group is shown in Table 29.
  • Record the body weight changes of the treatment group and the control group after administration, and study The safety of each group in the colon cancer CR3820 subcutaneous xenograft model, and the results of mouse body weight changes are shown in Table 30, and the curves of body weight changes over time for each treatment group are shown in Figure 18 .
  • Table 28 Changes in tumor volume of mice in each group with treatment time in the subcutaneous model of colon cancer CR3820 (mm 3 )
  • Table 29 Drug efficacy analysis table of each group in the colon cancer CR3820 subcutaneous model
  • mice in the vehicle control group were 1792.37mm 3 on the 25th day (Day 24) after the start of administration.
  • the average tumor volume of the test drug Ifosfamide in the 60mg/kg dose treatment group (QD ⁇ 5/week ⁇ 2 weeks, Group 2) on Day 24 was 1199.09mm3, and the relative tumor inhibition rate TGI (%) was 37.73%, compared with the control There was no statistically significant difference between groups (p>0.05).
  • test drug AST was dosed at 8mg/kg (QW ⁇ 3, Group 3), 4mg/kg (QD ⁇ 5, 2 days off, 2 weeks off, QD ⁇ 5, Group 4).
  • the tumor volumes were 110.59 mm 3 and 146.58 mm 3 , and the relative tumor inhibition rates TGI (%) were 94.08% and 92.44%, respectively, which were statistically significantly different from the control group (p ⁇ 0.05).
  • the test drugs Ifosfamide and AST were well tolerated at the doses tested in this study.
  • the PA2637 subcutaneous model of pancreatic cancer is a PDX model with a KRAS G12D pathogenic mutation.
  • NOD.SCID female mice were inoculated subcutaneously Pancreatic cancer PA2637 tumor mass was used to establish a subcutaneous transplanted tumor model of human pancreatic cancer.
  • the test was divided into test drug Ifosfamide, 60mg/kg monotherapy group (QD ⁇ 5/week ⁇ 2 weeks, Group 2), intraperitoneal administration, once a day, for 5 consecutive days, rest for 2 days, and then give daily The drug was administered once for 5 consecutive days; AST 8mg/kg single drug group (QW ⁇ 3, Group 3) was administered via tail vein once a week for a total of 3 weeks; AST 4mg/kg single drug group (QD ⁇ 5, 2 days off, 2 weeks off, QD ⁇ 5, Group 4), AST-3424 1mg/kg single drug group (QD ⁇ 5, 2 days off, 2 weeks off, QD ⁇ 5, Group 5) As well as the vehicle control group glucose injection (pH7.7-8.0, Group 1), both were administered through the tail vein, and the administration cycle was administered once a day for
  • Table 31 Administration route, dosage and regimen in the subcutaneous model of pancreatic cancer PA2637
  • the tumor growth of each treatment group and control group was recorded on different days of the experiment, as shown in Table 32, and the growth curves of the tumor volumes of mice in each group are shown in FIG. 19 .
  • the curative effect was evaluated according to the relative tumor proliferation rate and the relative tumor inhibition rate, and the drug efficacy analysis of each group is shown in Table 33.
  • Table 32 Changes of tumor volume of mice in each group with treatment time in PA2637 subcutaneous model of pancreatic cancer (mm 3 )
  • Table 33 Drug efficacy analysis table of each group in the subcutaneous model of pancreatic cancer PA2637
  • mice in the vehicle control group were 977.46 mm 3 on day 36 (Day 35) after the start of administration.
  • the test drug Ifosfamide in the 60mg/kg dose treatment group (Group 2) had an average tumor volume of 938.33mm3 on Day 35, and the relative tumor inhibition rate TGI (%) was 2.74%. There was no statistically significant difference compared with the control group (p >0.05).
  • the average tumor volume of the dose treatment group on Day 35 was 123.47mm 3 , 141.48mm 3 and 186.08mm 3 , and the relative tumor inhibition rate TGI (%) They were 87.28%, 85.46% and 80.78%, respectively, which were statistically significantly different from the control group (p ⁇ 0.001).
  • the lung cancer LU11873 subcutaneous model is a PDX model with a KRAS G12C pathogenic mutation.
  • NOD.SCID female mice were inoculated subcutaneously
  • the tumor mass of lung cancer LU11873 was used to establish a subcutaneous transplanted tumor model of human lung cancer.
  • the test is divided into the test drug Ifosfamide 60mg/kg single drug group (Group 2), administered once a day for 5 consecutive days, rested for 2 days, and then administered once a day for 5 consecutive days; AST 4mg /kg single drug group (Group 5), administered once a day for 5 consecutive days, rested for 2 days, rested for 2 weeks, and then administered once a day for 5 consecutive days; and glucose injection ( pH7.7-8.0) vehicle control group (Group 1), administered once a day for 5 consecutive days, rested for 2 days, rested for 2 weeks, and then administered once a day for 5 consecutive days; A total of 3 groups of 6 mice were studied.
  • the test drug Ifosfamide was administered intraperitoneally.
  • the vehicle control group and the AST groups were administered by tail vein injection. See Table 35 for the route of
  • Table 35 Administration route, dosage and regimen in subcutaneous model of lung cancer LU11873
  • Table 36 Changes in tumor volume of mice in each group with treatment time in the subcutaneous model of lung cancer LU11873 (mm 3 )
  • Table 37 Drug efficacy analysis table of each group in the subcutaneous model of lung cancer LU11873
  • the average tumor volume of the mice in the vehicle control group was 1677.89 mm 3 on the 31st day (Day 31) after the start of administration.
  • the average tumor volume on Day 31 of the test drug Ifosfamide in the 60mg/kg dose treatment group (Group 2) was 1866.37mm 3 , and the relative tumor inhibition rate TGI (%) was -10.08%, which was not statistically significant compared with the control group Difference (p>0.05).
  • the average tumor volume of the test drug AST at 4mg/kg (QD ⁇ 5, 2 days off, 2 weeks off, QD ⁇ 5) in the treatment group (Group 5) was 406.40mm 3 on Day 31, which was statistically higher than that in the control group. There was a significant difference (p ⁇ 0.05), and the relative tumor inhibition rate TGI (%) was 75.16%.
  • Example 3 the tumor inhibitory effect of AST in the KRAS pathogenic mutation model lung cancer LU11693 with G12C amino acid mutation was not obvious, and the TGI% was 54.64% at a dose of 10 mg/kg; while in this example Among them, AST had a significant anti-tumor effect in the lung cancer LU11873 model with KRAS G12C pathogenic mutation, and the TGI% was 75.16% at a dose of 4mg/kg.
  • LU11693 was derived from a 58-year-old female patient who clinically showed cachexia and mild ulcers;
  • LU11873 was derived from a 51-year-old male patient who clinically showed mild weight loss and mild ulceration.
  • test substance AST and Ifosfamide monotherapy in Antitumor effect and safety evaluation of PA1383 subcutaneous model of pancreatic cancer
  • the PA1383 subcutaneous model of pancreatic cancer is a PDX model with a KRAS G12C pathogenic mutation.
  • mice Balb/nude female mice were inoculated subcutaneously Pancreatic cancer PA1383 tumor mass was used to establish a subcutaneous transplanted tumor model of human pancreatic cancer.
  • the test is divided into the test drug Ifosfamide 60mg/kg single drug group (Group 2), administered once a day for 5 consecutive days, resting for 2 days, and then administered once a day for 5 consecutive days; AST 8mg/kg monotherapy
  • the drug group (Group 4) was administered once a week for a total of 3 weeks;
  • the AST 4mg/kg single drug group (Group 5) was administered once a day for 5 consecutive days, rested for 2 days, and then rested for 2 days.
  • Table 39 Administration route, dose and regimen in the subcutaneous model of pancreatic cancer PA1383
  • the tumor growth of each treatment group and control group was recorded on different days of the experiment, as shown in Table 40, and the growth curves of the tumor volumes of mice in each group are shown in FIG. 23 .
  • the curative effect was evaluated according to the relative tumor proliferation rate and the relative tumor inhibition rate, and the drug efficacy analysis of each group is shown in Table 41.
  • Table 40 Changes of tumor volume of mice in each group with treatment time in PA1383 subcutaneous model of pancreatic cancer (mm 3 )
  • Table 41 Drug efficacy analysis table of each group in the subcutaneous model of pancreatic cancer PA1383
  • mice in the vehicle control group were 1536.48 mm 3 on the 31st day (Day 31) after the start of administration.
  • the average tumor volume on Day 31 of the test drug Ifosfamide in the 60mg/kg treatment group (Group 2) was 1202.01mm 3 , and the relative tumor inhibition rate TGI (%) was 21.84%. Compared with the control group, there was no statistically significant difference (p>0.05).
  • the average tumor volume of the test drug AST at 8 mg/kg (QW ⁇ 3) and 4 mg/kg (QD ⁇ 5, 2 days off, 2 weeks off, QD ⁇ 5) in the treatment groups (Group 4 and Group 5) on Day 31 were respectively They were 18.57mm 3 and 39.94mm 3 , which were statistically significantly different from the control group (p ⁇ 0.05), and the relative tumor inhibition rates TGI (%) were 98.72% and 97.46%, respectively, and each had 2 small The tumors of the mice were completely cleared, with a clearance rate of 33.3%.
  • the AKR1C3LOG2 (FPKM) of GA6201 was 6.78, LU11693 was 11.14, PA1222 was 7.39, HPAF-II was 8.31, LU5161 was 11.56, CR3820 was 8.34, PA2637 was 9.12, LU11873 was 10.26, and PA1383 was 9.57, see Table 43.
  • the AKR1C3 protein content of these three tissues was determined (commercial IHC reagents were used, the primary antibody was rabbit antibody Rabbit IgG mAb from Abcam Company, and the secondary antibody was optimized polymer from Leica Company Detection system Bond Polymer Refine Detection, staining conditions: antigen retrieval 100°C, pH 9.0 EDTA buffer 20min, dilution ratio: 1:800), and H-SCORE scoring of the staining results: immunohistochemical staining intensity will be divided into 0 (negative), 1+ (weak staining), 2+ (medium staining), 3+ (strong staining), manually set the thresholds of weak staining, medium staining, and strong staining on the scoring instrument, and then use image processing software to perform staining The sample photos are color-identified, and the staining photos of all samples are scored according to a unified standard, and the staining situation corresponding
  • H-Score was calculated as the IHC result score of each sample by the following formula. The H-score will be between 0 and 300, and the higher the score, the higher the expression level of the target (AKR1C3 enzyme protein) corresponding to the antibody in the sample. Calculated as follows:
  • H-Score (%at 0) ⁇ 0+(%at 1) ⁇ 1+(%at 2) ⁇ 2+(%at 3) ⁇ 3
  • Table 44 IHC results and H-SCORE scores of GA6201, LU11693, PA1222 models and control groups
  • AST-3424 and AST have a positive effect on cancers with high expression of AKR1C3 and KRAS pathogenic mutations with G12D amino acid mutations.
  • AST may have a significant therapeutic effect on cancers with high expression of AKR1C3 and KRAS pathogenic mutations with G12C amino acid mutations.
  • AKR1C3 in some tumors may be associated with KARS (pathogenic) mutation subtypes, that is, the high expression or overexpression of AKR1C3 in some tumors is often associated with certain subtypes of KARS (pathogenic) mutations co-existence, and this phenomenon would lead to tumor models with these characteristics being more sensitive to AST-3424 or AST.
  • PCT/NZ2019/050030 publication number WO2019190331A1 (corresponding to Chinese application number 2019800234236, publication number CN111918864A);
  • AKR1C3-activated DNA alkylating agent prodrug is useful for the treatment of cancer and tumor patients with KRAS mutation, especially KRAS-G12D subtype mutation. patients had a significant therapeutic effect.

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Abstract

治疗KRAS突变的癌症、肿瘤患者的方法及制药用途。

Description

治疗KRAS突变的癌症患者 技术领域
本发明涉及癌症的治疗方法，特别是KRAS突变的癌症患者的治疗方法。
背景技术
KRAS基因的全名叫Kirsten ratsarcoma viral oncogene homolog，翻成中文是“Kirsten大鼠肉瘤病毒癌基因同源物”。KRAS基因编码的蛋白是一种小GTP酶(smallGTPase)，它属于RAS超蛋白家族，RAS基因包括KRAS，NRAS和HRAS三种，这些基因编码的蛋白是GTPases，在调控细胞增殖和存活的通路中起到分子开关的作用。基于目前的COSMIC数据显示，KRAS是三者中最常见的突变基因(22％)，其次是NRAS(8％)，最后是HRAS(3％)。KRAS基因对人类癌症影响很大，大约有30％的癌症患者都存在KRAS突变，其中包括90％的胰腺癌，50％的结肠癌和25％的肺癌。在非小细胞肺癌中，KRAS基因突变占20～30％，多存在于肺腺癌中，肺鳞癌中比较罕见。KRAS基因突变，主要集中在第12，13及61号密码子位置，其中，第12号密码子位置的突变占到80％以上，包括G12A，G12C，G12D，G12R，G12S及G12V。KRAS基因被激活最常见的方式是点突变，95％的KRAS突变主要发生在2号外显子的第12号密码子(＞80％)和13号密码子上。在常见的突变形式有KRAS-G12C突变(占所有KRAS突变的13％)，KRAS-G12V(20％)和KRAS-G1 2D(29％)突变(Loong HHF，Du N，Cheng C，Lin H，Guo J，Lin G，Li M，Jiang T，S hi Z，Cui Y，Jin X，Yao J，Xing Y，Yao M，Wang K，Mok TSK，Liu L.KRAS G12C mutations in Asia：A landscape analysis of 11，951 Chinese tumor samples.Transl Lung Ca ncer Res 2020.doi：10.21037/tlcr-20-455)。
KRAS基因G12C突变，其突变体具有一个半胱氨酸残基(第12位的甘氨酸变成半胱氨酸)，已被用于设计具有前期临床活性的共价抑制剂。比如AMG-510(临床开发的第一个KRAS-G12C抑制剂)和MRTX849，已表现出强抗肿瘤活性。
2021年5月29日，针对KRAS突变有效，被业界称为“革命性抗癌药”Sotorasib(AMG-510，Lumakras)获得FDA批准，用于治疗患有KRAS-G12C突变的非小细胞肺癌患者，这些患者至少接受过一种前期全身性治疗。AMG510是专门针对KRAS-G12C这种突变亚型的，具有很高的选择性，能与6,000多种蛋白质中的KRAS-G12C特异性结合，锁定并令其失活。
2021年6月24日，美国食品药品监督管理局(FDA)授予Adagrasib(阿达格拉西布，MRTX849)突破性疗法认定，用于治疗先前接受过全身治疗的携带KRAS-G12C突变的非小细胞肺癌(NSCLC)患者。该药是一款针对KRAS-G12C突变体的特异性优化口服抑制剂。通过在非活性状态下与KRAS-G12C不可逆转地选择性结合，阻止其发送细胞生长信号并导致癌细胞死亡，经过初步的人体临床试验证实，MRTX849在治疗携带KRAS-G12C基因突变的非小细胞肺癌(NSCLC)和结直肠癌(CRC)及其他实体肿瘤时表现出可喜的安全性和抗癌活性。
然而针对KRAS突变中的G12D这个更广泛的突变亚型却没有药物上市。
发明内容
AST-3424(OBI-3424)是一款first-in-class的以过表达醛酮还原酶1C3(AKR1C3)为标靶的DNA烷化癌症治疗药物，它可以选择性靶向过度表达AKR1C3的癌症，并在AKR1C3酶存在的情况下，选择性释放强效的DNA烷化剂。这种选择性激活模式将AST-3424与传统的烷化剂(如环磷酰胺cyclophosphamide与异环磷酰胺ifosfamide)区分开来。AKR 1C3过度表达在多种治疗抵抗性和难治性癌症中存在，包括HCC、去势抵抗性前列腺癌(CRPC)和T细胞急性淋巴性白血病(T-ALL)。AKR1C3在多达15种实体瘤和血液肿瘤中均有高度表达。目前，该药物在中国和美国进行II期临床试验(美国OBI-3424-NCT03592264-II期，去势前列腺癌和肝癌；美国OBI-3424-NCT04315324-II期，T淋巴细胞急性白血病T-ALL；中国AST-3424-CTR20191399-II期，实体瘤；中国AST-3424-CTR20201915-II期，T淋巴细胞急性白血病T-ALL和B淋巴细胞急性白血病B-ALL)。
在对该药物及类似药物的动物体内模型研究中发现AST-3424及化合物
Figure PCTCN2022120817-appb-000001
(以下简称AST)对高表达AKR1C3的KRAS突变G12D亚型肿瘤模型有较好的治疗效果。
基于实验结果，本申请提供以下的治疗癌症方法和化合物的制药用途。
治疗方法，其使用含有AKR1C3活化的DNA烷化剂前药化合物的药物单药或联用其他治疗药物治疗KRAS突变的癌症、肿瘤患者。
AKR1C3活化的DNA烷化剂前药化合物的制药用途，该化合物用于制备单药或联用其他治疗药物治疗KRAS突变的癌症、肿瘤患者的药物。
AKR1C3活化的DNA烷化剂前药化合物是指该化合物为前药，该前药分子与AKR1C3酶反应，反应后释放出具有细胞毒性的DNA烷化剂。
具体的以AST-3424为例，这些化合物作为醛酮还原酶AKR1C3特异性底物，可仅在AKR1C3高表达的癌细胞内快速有效地还原，从而释放细胞毒素-DNA烷化剂AST-2660，AST-2660与DNA交联而使得癌细胞：
Figure PCTCN2022120817-appb-000002
治疗方法，其使用含有AKR1C3活化的DNA烷化剂前药化合物的药物单药或联用其他治疗药物治疗KRAS突变的癌症、肿瘤患者，该化合物选自结构式1/2及其盐、酯、溶剂合物、同位素异构体：
Figure PCTCN2022120817-appb-000003
AKR1C3活化的DNA烷化剂前药化合物的制药用途，该化合物用于制备单药或联用其 他治疗药物治疗KRAS突变的癌症、肿瘤患者的药物，该化合物选自结构式1/2及其盐、酯、溶剂合物、同位素异构体。
其中，R 1、R 2、R 3、R 4、R 5、R 8、R 9、R 10的定义如专利申请PCT/CN2020/089692，公开号WO2020228685A1中的权利要求书所记载。具体如下：
其中，
R 1是C 6-C 10芳基或Z取代的芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、7-15元的稠环或Z取代稠环；
R 2是氢、卤素原子、氰基或异氰基、羟基、巯基、胺基、OTs、OMS、C 1-C 6烷基或Z取代烷基、C 2-C 6烯基或Z取代烯基、C 2-C 6炔基或Z取代炔基、C 3-C 8环烷基或Z取代环烷基、C 6-C 10芳基或Z取代芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、1-6个碳原子的醚或Z取代的1-6个碳原子的烷氧基、-CONR 6R 7、-SO 2NR 6R 7、-SO 2R 6、-OCOO-R 6、-COOR 6、-NR 6COR 7、-OCOR 6、-NR 6SO 2R 7、-NR 6SO 2NR 6R 7或者R 2和与其所键结的R 1基团上的原子一起形成7-15元的稠环或Z取代稠环；
R 3是氢、卤素、氰基或异氰基、羟基、巯基、胺基、OTs、OLCMS、C 1-C 6烷基或Z取代烷基、C 2-C 6烯基或Z取代烯基、C 2-C 6炔基或Z取代炔基、C 3-C 8环烷基或Z取代环烷基、C 6-C 10芳基或Z取代芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、C 1-C 6烷氧基或Z取代的C 1-C 6烷氧基、-CONR 6R 7、-SO 2NR 6R 7、-SO 2R 6、-OCO-R 6、-OCOO-R 6、-COOR 6、-NR 6COR 7，-OCOR 6、-NR 6SO 2R 7；
R 4、R 5各自独立地是氢、卤素原子、氰基或异氰基、羟基、巯基、胺基、OTs、OLCMS、C 1-C 6烷基或Z取代烷基、C 2-C 6烯基或Z取代烯基、C 2-C 6炔基或Z取代炔基、C 3-C 8环烷基或Z取代环烷基、C 6-C 10芳基或Z取代芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、C 1-C 6烷氧基或Z取代的C 1-C 6烷氧基、-CONR 6R 7、-SO 2NR 6R 7、-SO 2R 6、-OCOO-R 6、-COOR 6、-NR 6COR 6、-OCOR 6、-NR 6SO 2R 7或者R 4、R 5和与其所键结的苯环上的原子一起形成7-15元的稠环或Z取代稠环；
R 6和R 7各自独立地是氢、氰基或异氰基、C 1-C 6烷基或Z取代烷基、C 2-C 6烯基或Z取代烯基、C 2-C 6炔基或Z取代炔基、C 3-C 8环烷基或Z取代环烷基、C 6-C 10芳基或Z取代芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、C 1-C 6烷氧基或Z取代的C 1-C 6烷氧基，或者R 6、R 7基团与其所键结的原子一起形成5-7元杂环基或Z取代5-7元杂环基；
R 8、R 10各自独立地为氢、氘、芳基或Z取代芳基、C 1-C 6烷基或Z取代烷基、C 2-C 6烯基或Z取代烯基、C 2-C 6炔基或Z取代炔基、C 3-C 8环烷基或Z取代环烷基且必有一个为氢、氘；
R 9为至少具有一个氟原子或硝基取代的取代C 6-C 10芳基、至少具有一个氟原子或硝基取代的取代4-15元杂环、至少具有一个氟原子或硝基取代的取代5-15元杂芳基。
Z取代基为卤素原子、氰基或异氰基、羟基、巯基、胺基、OTs、OMS、C 1-C 3烷基或取代烷基、C 1-C 3烷氧基或取代烷氧基、C 2-C 3烯基或取代烯基、C 2-C 3炔基或取代炔基、C 3-C 8环烷基或取代环烷基、芳环、杂环、杂芳环和稠环或取代芳环、杂环、杂芳环和稠环，取代的方式为单取代或偕二取代；
R 9中的取代C 6-C 10芳基、取代4-15元杂环、取代5-15元杂芳基的取代基为卤素原子、硝基、氰基或异氰基、羟基、胺基、C 1-C 3烷基或烷氧基、烯基、炔基、环烷基或苯环、取代苯环、C 1-C 3烷氧基或卤原子取代烷氧基。
具体的，式(1)(2)的化合物选自：
Figure PCTCN2022120817-appb-000004
Figure PCTCN2022120817-appb-000005
Figure PCTCN2022120817-appb-000006
具体定义和含义以及制备方法、波谱数据参见专利申请PCT/CN2020/089692，公开号WO2020228685A1，在此将该申请全文引入。
显然结构式1/2的化合物与AST-3424、AST相同，都是AST-2660的前药，其会在AKR1C3酶的作用下活化而产生AST-2660(一种DNA烷化剂)发挥抗癌效果：
Figure PCTCN2022120817-appb-000007
治疗方法，其使用含有AKR1C3活化的DNA烷化剂前药化合物的药物单药或联用其他治疗药物治疗KRAS突变的癌症、肿瘤患者，该化合物选自结构式3及其盐、酯、溶剂合物、同位素异构体：
Figure PCTCN2022120817-appb-000008
AKR1C3活化的DNA烷化剂前药化合物的制药用途，该化合物用于制备单药或联用其他治疗药物治疗KRAS突变的癌症、肿瘤患者的药物，该化合物选自结构式3及其盐、酯、溶剂合物、同位素异构体：
Figure PCTCN2022120817-appb-000009
其中，A、E、G、X、Y的定义如专利申请PCT/NZ2019/050030，公开号WO2019190331A1(对应中国申请号2019800234236，公开号CN111918864A)中的权利要求书所记载。具体如下：
其中：
A为H、C1-C6烷基、C1-C6烯基、C1-C6炔基、CFH 2、CF 2H、CF 3、F、Cl、Br、I、OCF 3、COR或CON(R) 2；
E为SO或SO 2；
X为Cl、Br、I或OSO 2R；
Y为Cl、Br、I或OSO 2R；
每个R独立地为H或C1-C6烷基；
G是选自包括式(B)-(AA)的组中的自由基：
Figure PCTCN2022120817-appb-000010
其中：
R 1为H、C1-C6烷基、CH 2(CH 2)nOH、CH 2CH(OH)CH 2OH、苯基、吡啶基、苄基或吡啶基甲基，条件是当R 1为苯基、吡啶基、苄基或吡啶基甲基时，R 1任选地在任何可用位置处被C1-C6烷基、C1-C6烯基、C1-C6炔基、OR 6、N(R 6)(R 7)、CFH 2、CF 2H、CF 3、F、Cl、Br、I、OCF 3、COR 6、CON(R 6)(R 7)、SOR 6、SON(R 6)(R 7)、SO 2R 6、SO 2N(R 6)(R 7)、CN或NO 2取代；
R 2和R 3各自独立地为H、C1-C6烷基、C1-C6烯基、C1-C6炔基、OR 6、N(R 6)(R 7)、CFH 2、CF 2H、CF 3、F、Cl、Br、I、OCF 3、COR 6、CON(R 6)(R 7)、SOR 6、SON(R 6)(R 7)、SO2R 6、SO2N(R 6)(R 7)、CN或NO 2；
R 4为N(R 6)(R 7)、OH、OCH 2(CH 2)nN(R 6)(R 7)或CH 2(CH 2)nN(R 6)(R 7)；
R 5为H或C1-C6烷基基团；
R 6和R 7各自独立地为H或C1-6烷基，或者R 6和R 7一起形成取代或未被取代的5元或6元杂环：
Z为CH或N；
W为CH 2、O、S、SO或SO 2；
n为0至6；
*表示与式(I)的连接点。
具体的，式(3)的化合物选自：
Figure PCTCN2022120817-appb-000011
Figure PCTCN2022120817-appb-000012
Figure PCTCN2022120817-appb-000013
Figure PCTCN2022120817-appb-000014
Figure PCTCN2022120817-appb-000015
Figure PCTCN2022120817-appb-000016
Figure PCTCN2022120817-appb-000017
Figure PCTCN2022120817-appb-000018
Figure PCTCN2022120817-appb-000019
Figure PCTCN2022120817-appb-000020
Figure PCTCN2022120817-appb-000021
Figure PCTCN2022120817-appb-000022
具体定义和含义以及制备方法、波谱数据参见专专利申请PCT/NZ2019/050030，公开号WO2019190331A1(对应中国申请号2019800234236，公开号CN111918864A)，在此将该申请全文引入。
结构式3的化合物与AST-3424、AST原理类似，都是氮芥类似物
Figure PCTCN2022120817-appb-000023
的前药， 其会在AKR1C3酶的作用下活化而产生氮芥类似物
Figure PCTCN2022120817-appb-000024
(一种DNA烷化剂)发挥抗癌效果：
Figure PCTCN2022120817-appb-000025
治疗方法，其使用含有含有AKR1C3活化的DNA烷化剂前药化合物的药物单药或联用其他治疗药物治疗KRAS突变的癌症、肿瘤患者，该化合物选自结构式4及其盐、酯、溶剂合物、同位素异构体：
Figure PCTCN2022120817-appb-000026
AKR1C3活化的DNA烷化剂前药化合物的制药用途，该化合物用于制备单药或联用其他治疗药物治疗KRAS突变的癌症、肿瘤患者的药物，该化合物选自结构式4及其盐、酯、溶剂合物、同位素异构体：
Figure PCTCN2022120817-appb-000027
其中，Rw的定义如专利申请PCT/CN2020/120281，公开号WO2021068952A1中的权利要求书所记载。具体如下：
Rw为
Figure PCTCN2022120817-appb-000028
R 1为H、C 1-6烷基、C 3-6环烷基、4-6元杂环烷基、5-6元杂芳基或苯基，其中所述C 1-6烷 基、C 3-6环烷基、4-6元杂环烷基、5-6元杂芳基和苯基任选被1、2或3个R a所取代；
各R a独立地为H、F、Cl、Br、I、-CN、-OH、C 1-3烷氧基或C 1-3烷基；
R 2为H或C 1-6烷基；
或者R 1和R 2连接在一起，与其相连的N原子一起形成4-6元杂环烷基，其中所述4-6元杂环烷基任选被1、2或3个R b所取代；
各R b独立地为H、F、Cl、Br、I、-CN、-OH、-NH 2、-OCH 3、-OCH 2CH 3、-CH 3或-CH 2CH 3；
R 3为H、F、Cl、Br、I、-OH、-NH 2、C 1-3烷氧基或C 1-3烷基；
或者R 2和R 3连接在一起使结构单元
Figure PCTCN2022120817-appb-000029
为
Figure PCTCN2022120817-appb-000030
Figure PCTCN2022120817-appb-000031
T 1为-(CR cR d) m-或-(CR cR d) n-O-；
m为1、2或3；
n为1或2；
T 2为N或CH；
R c和R d各自独立地为H、F、C 1-3烷基或C 1-3烷氧基；
R 4、R 5和R 6各自独立地为H、F、Cl、Br、I、C 1-3烷基或C 1-3烷氧基；
T为N或CH；
R 7和R 8各自独立地为H、F、Cl、Br或I；
R 9和R 10各自独立地为H、F、Cl、Br、I、-CN或
所述4-6元杂环烷基和5-6元杂芳基各自包含1、2、3或4个独立选自N、-O-和-S-的杂原子。
具体的，式(4)的化合物选自：
Figure PCTCN2022120817-appb-000032
具体定义和含义以及制备方法、波谱数据参见专利申请PCT/CN2020/120281，公开号WO2021068952A1，在此将该申请全文引入。
显然结构式3的化合物与AST-3424、AST相同，都是AST-2660的前药，其会在AKR1C3酶的作用下活化而产生AST-2660(一种DNA烷化剂)发挥抗癌效果：
Figure PCTCN2022120817-appb-000033
治疗方法，其使用含有含有AKR1C3活化的DNA烷化剂前药化合物的药物单药或联用其他治疗药物治疗KRAS突变的癌症、肿瘤患者，该化合物选自结构式5及其盐、酯、溶剂合物、同位素异构体：
Figure PCTCN2022120817-appb-000034
AKR1C3活化的DNA烷化剂前药化合物的制药用途，该化合物用于制备单药或联用其他治疗药物治疗KRAS突变的癌症、肿瘤患者的药物，该化合物选自结构式5及其盐、酯、溶剂合物、同位素异构体：
Figure PCTCN2022120817-appb-000035
其中，X、Y、Z、R、T、A以及X 10的定义如专利申请PCT/US2016/062114，公开号WO2017087428A1(对应中国申请号2016800200132，公开号CN108136214A)中的权利要求书所记载。具体如下：
X 10是O、S、SO或SO 2；
A是C 6-C 10芳基、5-15元杂芳基或-N＝CR 1R 2；
R 1及R 2各自独立是氢、C 1-C 6烷基、C 3-C 8环烷基、C 6-C 10芳基、4-15元杂环、醚、-CONR 13R 14或-NR 13COR 14；
X、Y及Z各自独立是氢、CN、卤基、C 1-C 6烷基、C 2-C 6烯基、C 2-C 6炔基、C 3-C 8环烷基、C 6-C 10芳基、4-15元杂环、醚、-CONR 13R 14或-NR 13COR 14；
R是氢、C 1-C 6烷基、C 2-C 6烯基、C 2-C 6炔基、C 3-C 8环烷基、C 6-C 10芳基、4-15元杂环、醚、-CONR 13R 14或-NR 13COR 14；
R 13及R 14各自独立是氢、C 1-C 6烷基、C 3-C 8环烷基、C 6-C 10芳基、4-15元杂环或醚
T包含胺基磷酸酯烷化剂，所述胺基磷酸酯烷化剂包含一或多个键结至-O-P(Z 1)部分的Z 5-X 5-Y 5部分的烷化剂，其中Z 5是包含氮、硫或氧的杂原子，X 5是经取代或未经取代的伸乙基，Y 5是卤基或另一离去基，或Z 5-X 5-Y 5一起形成氮丙啶基(NCH 2CH 2)部分且Z 1是O或S；且
其中这些烷基、烯基、炔基、环烷基、芳基、杂环、杂芳基、醚基经取代或未经取代。
具体的，式(5)的化合物选自：
Figure PCTCN2022120817-appb-000036
Figure PCTCN2022120817-appb-000037
Figure PCTCN2022120817-appb-000038
Figure PCTCN2022120817-appb-000039
Figure PCTCN2022120817-appb-000040
Figure PCTCN2022120817-appb-000041
Figure PCTCN2022120817-appb-000042
Figure PCTCN2022120817-appb-000043
Figure PCTCN2022120817-appb-000044
具体定义和含义以及制备方法、波谱数据参见专利申请PCT/US2016/062114，公开号WO2017087428A1(对应中国申请号2016800200132，公开号CN108136214A)，在此将该申请全文引入。
显然结构式5的化合物与AST-3424、AST类似，是胺基磷酸酯烷化剂的前药，其会在AKR1C3酶的作用下活化而产生T(一种胺基磷酸酯烷化剂，AST-2660就是一种胺基磷酸酯烷化剂)发挥抗癌效果：
Figure PCTCN2022120817-appb-000045
治疗方法，其使用含有含有AKR1C3活化的DNA烷化剂前药化合物的药物单药或联用其他治疗药物治疗KRAS突变的癌症、肿瘤患者，该化合物选自结构式6及其盐、酯、溶剂合物、同位素异构体：
Figure PCTCN2022120817-appb-000046
AKR1C3活化的DNA烷化剂前药化合物的制药用途，该化合物用于制备单药或联用其他治疗药物治疗KRAS突变的癌症、肿瘤患者的药物，该化合物选自结构式6及其盐、酯、 溶剂合物、同位素异构体：
Figure PCTCN2022120817-appb-000047
其中：
A是取代或未经取代的C6-C10的芳基、联芳基或取代的联芳基、5-15元的杂芳基或-N＝CR 1R 2，其中取代时的取代基选自由以下组成的群：卤基、-CN、-NO 2、-O-(CH 2)-O-、-CO 2H及其盐、-OR 100、-CO 2R 100、-CONR 101R 102、-NR 101R 102、-NR 100SO 2R 100、-SO 2R 100、-SO 2NR 101R 1 02、C1-C6烷基、C3-C10杂环基；
其中，R 100、R 101及R 102各自独立是氢、C1-C8烷基、C6-C12芳基；或R 101及R 102与其附接至的氮原子一起形成5-7元杂环；
其中烷基及芳基各自是经1-3个卤基或1-3个C1-C6烷基取代；
R 1及R 2各自独立是苯基或甲基；
X、Y及Z各自独立是氢或卤基；
R是氢或C1-C6烷基或卤素取代烷基。
“化合物”也当然包括化合物本身以及该化合物的溶剂合物、盐、酯或同位素异构体等。
基团前的“Cx-Cy”或“C x-y”是指存在于该基团中的碳原子数目的范围。举例而言，C1-C6烷基是指具有至少1个且最多6个碳原子的烷基。
“烷基”是指具有1至10个碳原子且在一些实施例中具有1至6个碳原子的单价饱和脂肪族烃基。“Cx-y烷基”是指具有x至y个碳原子的烷基。此术语包括(举例而言)直链及具支链烃基，例如甲基(CH 3-)、乙基(CH 3CH 2-)、正丙基(CH 3CH 2CH 2-)、异丙基((CH 3) 2CH-)、正丁基(CH 3CH 2CH 2CH 2-)、异丁基((CH 3) 2CHCH 2-)、仲丁基((CH 3)(CH 3CH 2)CH-)、叔丁基((CH 3) 3C-)、正戊基(CH 3CH 2CH 2CH 2CH 2-)及新戊基((CH 3) 3CCH 2-)。
“芳基”是指具有6至14个碳原子且不含环杂原子且具有单环(例如，苯基)或多个缩合(稠合)环(例如，萘基或蒽基)的芳香族基团。对于包括不具有环杂原子的具有芳香族环及非芳香族环的稠合、桥连及螺环系统的多环系统而言，当附接点位于芳香族碳原子处时，术语“芳基”或“Ar”适用(例如，5，6，7，8四氢萘-2-基是芳基，此乃因其附接点是位于芳香族苯基环的2位处)。“伸芳基”是指具有适当氢含量的二价芳基。
“环烷基”是指具有3至14碳原子且没有环杂原子且具有单环或包括稠合、桥连及螺环系统的多环的饱和或部分饱和环状基团。对于不具有环杂原子的具有芳香族及非芳香族环的多环系统而言，当附接点是位于非芳香族碳原子处时，术语“环烷基”适用(例如5，6，7，8-四氢萘-5-基)。术语“环烷基”包括环烯基。环烷基的实例包括(例如)金刚烷基、环丙基、环丁基、环戊基、环辛基及环己烯基。“伸环烷基”是指具有适当氢含量的二价环烷基。
“卤基”是指氟、氯、溴及碘中的一或多者。
“杂芳基”是指具有1至14个碳原子及1至6个选自由氧、氮及硫组成的群的杂原子的芳香族基团且包括单环(例如咪唑基-2-基及咪唑-5-基)及多环系统(例如咪唑并吡啶基、苯并三唑基、苯并咪唑-2-基及苯并咪唑-6-基)。对于包括具有芳香族及非芳香族环的稠合、桥连及螺环系统的多环系统而言，若存在至少一个环杂原子且附接点是位于芳香族环的原子处，则应用 术语“杂芳基”(例如1，2，3，4-四氢喹啉-6-基及5，6，7，8-四氢喹啉-3-基)。在一些实施例中，杂芳基的氮及/或硫环原子视情况经氧化以提供N-氧化物(N→O)、亚磺酰基或磺酰基部分。术语杂芳基包括(但不限于)吖啶基、吖辛因基(azocinyl)、苯并咪唑基、苯并呋喃基、苯并硫代呋喃基、苯并噻吩基(benzothiophenyl)、苯并恶唑基、苯并噻唑基、苯并三唑基、苯并四唑基、苯并异恶唑基、苯并异噻唑基、苯并噻吩基(benzothienyl)、苯并咪唑啉基、咔唑基、NH-咔唑基、咔啉基、苯并二氢吡喃基(chromanyl)、苯并吡喃基(chromenyl)、噌啉基(cinnolinyl)、二噻嗪基、呋喃基、呋咕基、咪唑啶基、咪唑啉基、咪唑并吡啶基、咪唑基、吲唑基、二氢吲哚基(indolenyl)、吲哚啉基、吲嗪基、吲哚基、异苯并呋喃基、异苯并二氢吡喃基(isochromanyl)、异吲唑基、异吲哚啉基、异吲哚基、异喹啉基(isoquinolinyl)、异喹啉基(isoquinolyl)、异噻唑基、异恶唑基、萘啶基、八氢异喹啉基、恶二唑基、恶唑啶基、恶唑基、嘧啶基、啡啶基、啡啉基、吩嗪基、吩噻嗪基、吩恶噻基、吩恶嗪基、酞嗪基、六氢吡嗪基、蝶啶基、嘌呤基、吡喃基、吡嗪基、吡唑啶基、吡唑啉基、吡唑基、嗒嗪基、吡啶并恶唑基、吡啶并咪唑基、吡啶并噻唑基、吡啶基(pyridinyl)、吡啶基(pyridyl)、嘧啶基、吡咯基、喹唑啉基、喹啉基、喹喏啉基、奎宁环基、四氢异喹啉基、四氢喹啉基、四唑基、噻二嗪基、噻二唑基、噻蒽基、噻唑基、噻吩基(thienyl)、噻吩并噻唑基、噻吩并恶唑基、噻吩并咪唑基、噻吩基(thiophenyl)、三嗪基及咕吨基。“伸杂芳基”是指具有适当氢含量的二价杂芳基。
“杂环状”或“杂环”或“杂环烷基”或“杂环基”是指具有1至14个碳原子及1至6个选自由氮、硫或氧组成的群的杂原子的饱和或部分饱和环状基团且包括单环及包括稠合、桥连及螺环系统的多环系统。对于具有芳香族及/或非芳香族环的多环系统而言，当存在至少一个环杂原子且附接点是位于非芳香族环的原子处时，术语“杂环状”、“杂环”、“杂环烷基”或“杂环基”适用(例如1，2，3，4-四氢喹啉-3-基、5，6，7，8-四氢喹啉-6-基及十氢喹啉-6-基)。在一些实施例中，此处杂环基是3-15元、4-14元、5-13元、7-12或5-7元杂环。在一些其他实施例中，杂环含有4个杂原子。在一些其他实施例中，杂环含有3个杂原子。在另一实施例中，杂环含有最多2个杂原子。在一些实施例中，杂环基的氮及/或硫原子视情况经氧化以提供N-氧化物、亚磺酰基、磺酰基部分。杂环基包括(但不限于)四氢吡喃基、六氢吡啶基、N-甲基六氢吡啶-3-基、六氢吡嗪基、N-甲基吡咯啶-3-基、3-吡咯啶基、2-吡咯啶酮-1-基、吗啉基及吡咯啶基。指示碳原子数的前缀(例如，C3-10)是指杂环基部分中除杂原子数之外的总碳原子数。二价杂环基将具有适当调整的氢含量。
“联芳基”是指两个芳环通过C-C单键相联的结构，如联苯、联吡啶等。
术语“视情况经取代”是指经取代或未经取代的基团。基团可经一或多个取代基(例如1、2、3、4或5个取代基)取代。较佳地，取代基选自由以下组成的群：侧氧基、卤基、-CN、NO 2、-N 2+、-CO 2R 100、-OR 100、-SR 100、-SOR 100、-SO 2R 100、-NR 100SO 2R 100、-NR 101R 102、-CONR 101R 102、-SO 2NR 101R 102、C 1-C 6烷基、C 1-C 6烷氧基、-CR 100＝C(R 100) 2、-CCR 100、C 3-C 10环烷基、C 3-C 10杂环基、C 6-C 12芳基及C 2-C 12杂芳基或诸如-O-(CH 2)-O-、-O-(CH 2) 2-O-及其14个甲基经取代的形式等二价取代基，其中R 100、R 101及R 102各自独立是氢或C 1-C 8烷基；C 3-C 12环烷基；C 3-C 10杂环基；C 6-C 12芳基；或C 2-C 12杂芳基；或R 101及R 102与其附接至的氮原子一起形成5-7元杂环；其中烷基、环烷基、杂环基、芳基或杂芳基各自视情况经1-3个卤基、1-3个C 1-C 6烷基、1-3个C 1-C 6卤烷基或1-3个C 1-C 6烷氧基取代。较佳地，取代基选自由以下组成的群：氯、氟、-OCH 3、甲基、乙基、异丙基、环丙基、-CO 2H及其盐及C 1-C 6烷基酯、CONMe 2、CONHMe、CONH 2、-SO 2Me、-SO 2NH 2、-SO 2NMe 2、-SO 2NHMe、-NHSO 2Me、-NHSO 2CF 3、-NHSO 2CH 2Cl、-NH 2、-OCF 3、-F 3及-OCHF 2。
具体的，式(6)的化合物选自：
Figure PCTCN2022120817-appb-000048
Figure PCTCN2022120817-appb-000049
Figure PCTCN2022120817-appb-000050
Figure PCTCN2022120817-appb-000051
Figure PCTCN2022120817-appb-000052
Figure PCTCN2022120817-appb-000053
Figure PCTCN2022120817-appb-000054
Figure PCTCN2022120817-appb-000055
Figure PCTCN2022120817-appb-000056
Figure PCTCN2022120817-appb-000057
具体定义和含义以及制备方法、波谱数据参见PCT/US2016/021581，公开号WO2016145092A1(对应中国申请号2016800150788，公开号CN107530556A)；PCT/US2016/062114，公开号W02017087428A1(对应中国申请号2016800200132，公开号CN108136214A)；PCT/CN2020/089692，公开号WO2020228686所公开，在此将这些申请全文引入。
显然结构式6的化合物与AST-3424、AST类似，是AST-2660的前药，其会在AKR1C3酶的作用下活化而产生AST-2660发挥抗癌效果：
Figure PCTCN2022120817-appb-000058
AST-3424(OBI-3424)、
Figure PCTCN2022120817-appb-000059
(以下代号为AST)以及
Figure PCTCN2022120817-appb-000060
的合成方法、波谱数据被专利申请：PCT/US2016/021581，公开号WO2016145092A1，对应中国申请号2016800150788，公开号CN107530556A；PCT/US2016/062114，公开号WO2017087428A1，对应中国申请号2016800200132，公开号CN108136214A；PCT/CN2020/089692，公开号WO2020228686所公开；相关的制剂浓缩注射液，并且相关处方、制备方法和临床配伍、施用方法被相关专利：WO2021008520A1、WO2021043275A1所详细说明并公开，在此本发明将上述申请文本的全文引入。
单药，即单药治疗。联用，即联合用药治疗。单药治疗是指在一个疗程中仅使用一种抗癌药物。联合治疗是指在一个疗程中同时或先后使用两种或两种以上的抗癌药物。
一般而言，联合治疗需要根据病情特点、联用药物种类探索不同的给药剂量、给药周期，只有根据上述情况，探索得到的联合用药治疗方案才可能取得较单一用药治疗好的治疗效果。
单药和联用治疗方案的药物给药剂量、给药周期均需要在参考上述AST-3424及其类似化合物和其他药物的剂量、给药方案通过临床试验探索得到。
单药可以参考WO2019062919A1以及WO2016145092A1、WO2017087428A1的动物实验剂量来确定给药剂量。
进一步，KRAS突变选自KRAS-G12D突变、KRAS-G12V突变和KRAS-G12C突变。
KRAS对应的基因中的任意一个基因突变或两个基因突变可以通过市售的(伴随)诊断试剂盒进行检测诊断，比如在中国获批的诊断试剂盒：
厦门艾德生物Amoy Dx，国械注准20153401126，人类KRAS基因突变检测试剂盒(荧光PCR法)
上海透景生命，国械注准20163401341，人K-RAS基因7种突变检测试剂盒(PCR荧光法)。
当然也可以使用NGS测序(YS 450基因NGS大panel)来判定具体的KRAS突变亚型。
更优选的，KRAS突变选自KRAS-G12D突变。
所述的基因突变的TMB(肿瘤基因突变负荷)水平为中。
由于不同瘤种之间TMB(Tumor mutation load(burden)即肿瘤基因突变负荷)高低不同：一般认为：TMB超过20个突变/Mb(Mb代表的就是每百万个碱基)，就是高；低于10个突变/Mb，就是低，处于中间的就是中。2017年世界肺癌大会上，施贵宝公司公布过一项名为CheckMate-032的临床试验结果。这是一项纳入了401名一线治疗失败的晚期肺癌患者的II期临床试验，接受PD-1抑制剂单独或联合伊匹木治疗。按照TMB高低划分成TMB高、TMB中、TMB低三类病人，那么在接受联合治疗的人群中，三组的有效率分别为62％、20％、23％，TMB高的人群有效率高3倍；而三组的中位总生存期，分别为：22.0个月、3.6个月、3.4个月——22.0个月与3.4个月，相差6倍！该试验证明，对于不同的癌症治疗药物，不同的TMB水平对于药物的疗效有很大的影响。
进一步，前药化合物优选选自：
Figure PCTCN2022120817-appb-000061
上述癌症选自卵巢癌、乳腺癌、胰腺癌、输卵管癌、原发性腹膜癌、胃癌、前列腺癌、肝癌、结肠癌、直肠癌、肺癌、膀胱癌。
其他治疗药物选自KRAS抑制剂、免疫治疗药物(免疫检查点抑制剂)。
KRAS抑制剂即抑制KRAS酶活性的物质。具体可以参考综述文献Goebel，Lisa&Müller， Matthias&Goody，Roger&Rauh，Daniel.(2020).KRasG12C inhibitors in clinical trials：A short historical perspective.RSC Medicinal Chemistry.11.10.1039/D0MD00096E.
进入临床开发或已经上市的KRAS抑制剂包括美国安进开发的Sotorasib(AMG510)、Mirati Therapeutics开发的adagrasib(MRTX849)、罗氏开发的GDC6036、礼来开发的LY3499446、Araxes和Janssen联合开发的JNJ74699157(ARS3248)、益方生物开发的D-1553、加科思开发的JAB-3312、加科思开发的JAB-3068、勤浩医药(Gen House)开发的GH35、贝达药业开发的BPI-421286、勃林格殷格翰开发的BI17016963、Moderna开发的mRNA-5671、阿斯利康与Ionis公司合作开发的AZD-4785。
Figure PCTCN2022120817-appb-000062
其中，KRAS抑制剂选自Sotorasib(AMG510)、adagrasib(MRTX849)、GDC6036、LY3499446、JNJ74699157(ARS3248)、D-1553；
免疫检查点分子(immune cheeckpoint)是免疫系统中起抑制作用的调节分子，其对于维持自身耐受、防止自身免疫反应、以及通过控制免疫应答的时间和强度而使组织损伤最小化等至关重要。免疫检查点分子表达于免疫细胞上，将抑制免疫细胞功能，使机体无法产生有效的抗肿瘤免疫应答，肿瘤形成免疫逃逸。与肿瘤相关的免疫检查点分子主要有：PD1、PD-L1、CTLA4、Tim3和LAG3等，目前研究较多的为PD1、PD-L1、CTLA4。免疫检查点抑制剂就是针对相应的免疫检查点研发的一些单抗类药物，其主要作用为阻断表达免疫检查点的肿瘤细胞与免疫细胞之间的作用，从而阻断肿瘤细胞对免疫细胞的抑制作用。免疫治疗药物选自PD-1单抗、PD-L1单抗。
所述癌症、肿瘤优选选自胃癌、胰腺癌、肺癌。
上述药物除含有对应的前药化合物外，还应根据药品、药物、制剂的特定，添加药学上可接受的辅料或赋形剂。所述药物可以为临床施用的任何剂型，例如片剂、栓剂、分散片、肠溶片、咀嚼片、口崩片、胶囊、糖衣剂、颗粒剂、干粉剂、口服溶液剂、注射用小针、注射用冻干粉针或大输液。根据具体剂型和施用方式，所述药物中的药学上可接受的辅料或赋形剂可以包括下述的一种或多种：稀释剂、增溶剂、崩解剂、悬浮剂、润滑剂、粘合剂、填充剂、矫味剂、甜味剂、抗氧化剂、表面活性剂、防腐剂、包裹剂、和色素等。
附图说明
图1为
Figure PCTCN2022120817-appb-000063
胃癌GA6201模型中各组小鼠肿瘤体积的生长曲线；
图2为
Figure PCTCN2022120817-appb-000064
胃癌GA6201模型中各组小鼠相对肿瘤抑制率曲线；
图3为
Figure PCTCN2022120817-appb-000065
胃癌GA6201模型中各组小鼠体重曲线；
图4为
Figure PCTCN2022120817-appb-000066
胃癌GA6201模型中各组小鼠体重变化百分比曲线；
图5为
Figure PCTCN2022120817-appb-000067
胰腺癌PA1222模型中各组小鼠肿瘤体积的生长曲线；
图6为
Figure PCTCN2022120817-appb-000068
胰腺癌PA1222模型中各组小鼠相对肿瘤抑制率；
图7为
Figure PCTCN2022120817-appb-000069
胰腺癌PA1222模型中各组小鼠体重曲线；
图8为
Figure PCTCN2022120817-appb-000070
胰腺癌PA1222模型中各组小鼠体重变化百分比曲线；
图9为
Figure PCTCN2022120817-appb-000071
肺癌LU11693模型中各组小鼠肿瘤体积的生长曲线；
图10为
Figure PCTCN2022120817-appb-000072
肺癌LU11693模型中各组小鼠相对肿瘤抑制率曲线；
图11为
Figure PCTCN2022120817-appb-000073
肺癌LU11693模型中各组小鼠体重曲线；
图12为
Figure PCTCN2022120817-appb-000074
肺癌LU11693模型中各组小鼠体重变化百分比曲线；
图13为人源胰腺癌HPAF-II皮下异种移植模型中各组小鼠肿瘤体积生长曲线；
图14为人源胰腺癌HPAF-II皮下异种移植模型中各组小鼠体重变化百分比曲线；
图15为
Figure PCTCN2022120817-appb-000075
肺癌LU5161皮下模型中各组小鼠肿瘤体积生长曲线；
图16为
Figure PCTCN2022120817-appb-000076
肺癌LU5161皮下模型中各组小鼠体重变化百分比曲线；
图17为
Figure PCTCN2022120817-appb-000077
肠癌CR3820皮下模型中各组小鼠肿瘤体积生长曲线；
图18为
Figure PCTCN2022120817-appb-000078
肠癌CR3820皮下模型中各组小鼠体重变化百分比曲线；
图19为
Figure PCTCN2022120817-appb-000079
胰腺癌PA2637皮下模型中各组小鼠肿瘤体积生长曲线；
图20为
Figure PCTCN2022120817-appb-000080
胰腺癌PA2637皮下模型中各组小鼠体重变化百分比曲线；
图21为
Figure PCTCN2022120817-appb-000081
肺癌LU11873皮下模型中各组小鼠肿瘤体积生长曲线；
图22为
Figure PCTCN2022120817-appb-000082
肺癌LU11873皮下模型中各组小鼠体重变化百分比曲线；
图23为
Figure PCTCN2022120817-appb-000083
胰腺癌PA1383皮下模型中各组小鼠肿瘤体积生长曲线；
图24为
Figure PCTCN2022120817-appb-000084
胰腺癌PA1383皮下模型中各组小鼠体重变化百分比曲线；
图25为GA6201、LU11693、PA1222模型及对照组的IHC染色结果照片。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
向患者“投与”(“administering”或“administration of”)药物(及此词组的语法等效形式)是指直接投与(其可由医学专业人士向患者投与或可自投与)及/或间接投与，其可是开处药物的行为。举例而言，指示患者自投与药物及/或将药物的处方提供给患者的医师是向患者投与药物。
“癌症”是指可通过侵袭而局部扩展且通过转移而全身扩展的潜在无限制生长的白血病、淋巴瘤、癌及其他恶性肿瘤(包括实体肿瘤)。癌症的实例包括(但不限于)肾上腺、骨、脑、乳房、支气管、结肠及/或直肠、胆囊、头及颈、肾、喉、肝、肺、神经组织、胰脏、前列腺、副甲状腺、皮肤、胃及甲状腺的癌症。癌症的某些其他实例包括急性及慢性淋巴细胞及粒细胞肿瘤、腺癌、腺瘤、基底细胞癌、子宫颈上皮分化不良及原位癌、尤文氏肉瘤(Ewing’s sarcoma)、表皮样癌、巨细胞瘤、多型性神经胶母细胞瘤、毛细胞肿瘤、肠神经节细胞瘤、增生性角膜神经肿瘤、胰岛细胞癌、卡波西肉瘤(Kaposi’s sarcoma)、平滑肌瘤、白血病、淋巴瘤、恶性类癌瘤、恶性黑色素瘤、恶性高钙血症、马方样体型肿瘤(marfanoid habitus tumor)、髓样上皮癌、转移性皮肤癌、黏膜神经瘤、骨髓瘤、蕈状肉芽肿、神经胚细胞瘤、骨肉瘤、骨原性及其他肉瘤、卵巢瘤、嗜铬细胞瘤、真性红血球增多症、原发性脑瘤、小细胞肺癌、溃疡型及乳头型二者的鳞状细胞癌、增生、精原细胞瘤、软组织肉瘤、视网膜母细胞瘤、横纹肌肉瘤、肾细胞肿瘤、局部皮肤病灶、网状细胞肉瘤及威尔姆氏肿瘤(Wilm’s tumor)。
“患者”及“个体”可互换使用，是指需要癌症治疗的哺乳动物。通常，患者是人类。通常，患者是诊断患有癌症的人类。在某些实施例中，“患者”或“个体”可指用于筛选、表征及评估药物及疗法的非人类哺乳动物，例如非人类灵长类动物、狗、猫、兔、猪、小鼠或大鼠。
“实体肿瘤”是指包括(但不限于)骨、脑、肝、肺、淋巴结、胰脏、前列腺、皮肤及软组织(肉瘤)中的转移肿瘤的实体肿瘤。
药物的“治疗有效量”是指当向患有癌症的患者投与时，将具有预期的治疗效应(例如患者中一或多种癌症的临床表现的缓和、改善、缓解或消除)的药物的量。治疗效应不必通过投 与一个剂量而出现，且可仅在投与一系列剂量后出现。因此，治疗有效量可以一或多次投与来投与。
病况或患者的“治疗”(“Treating”、“treatment of”或“therapy of”)是指采取步骤以获得有益或期望结果(包括临床结果)。出于本发明的目的，有益或期望临床结果包括(但不限于)一或多种癌症症状的缓和或改善；疾病程度的减弱；疾病进展的延迟或减缓；疾病状态的改善、缓解或稳定；或其他有益结果。在一些情形下，癌症的治疗可使得部分反应或稳定疾病。
“肿瘤细胞”是指任何适当物种(例如，哺乳动物，例如鼠类、犬、猫、马或人类)的肿瘤细胞。
“患者”及“个体”可互换使用，是指需要癌症治疗的哺乳动物。通常，患者是人类。通常，患者是诊断患有癌症的人类。在某些实施例中，“患者”或“个体”可指用于筛选、表征及评估药物及疗法的非人类哺乳动物，例如非人类灵长类动物、狗、猫、兔、猪、小鼠或大鼠。
“治疗”或“治疗患者”是指向患者投与、使用或施用本发明相关的治疗有效量的药物。
向患者“投与”或“施用”“使用”药物是指直接投与或施用(其可由医学专业人士向患者投与或施用或者可自投与或施用)及/或间接投与或施用，其可是开处药物的行为。举例而言，指示患者自投与或施用药物及/或将药物的处方提供给患者的医师是向患者投与或施用药物。
病况或患者的“治疗”是指采取步骤以获得有益或期望结果(包括临床结果)。出于本发明的目的，有益或期望临床结果包括(但不限于)一或多种癌症症状的缓和或改善；疾病程度的减弱；疾病进展的延迟或减缓；疾病状态的改善、缓解或稳定；或其他有益结果。在一些情形下，癌症的治疗可使得部分反应或稳定疾病。
下述实施例中的实验方法，如无特殊说明，均为常规方法。所用的药材原料、试剂材料等，如无特殊说明，均为市售购买产品。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。
以下提供本发明的具体实验。
1、受试物AST，AST-3424和Ifosfamide在
Figure PCTCN2022120817-appb-000085
胃癌GA6201皮下异种移植模型中的药效学评价
Figure PCTCN2022120817-appb-000086
胃癌GA6201 PDX模型是带有G12D氨基酸突变的KRAS致病突变(KRAS-G12D)的模型。BALB/c裸小鼠皮下接种
Figure PCTCN2022120817-appb-000087
模型GA6201瘤块，建立人胃癌皮下移植肿瘤模型。试验分为测试药Ifosfamide(异环磷酰胺)60mg/kg组、测试药AST-3424 5mg/kg组、AST 2.5mg/kg和5mg/kg组及生理盐水(pH 7.0-7.6)溶媒对照组，共5组，每组5只小鼠。其中，生理盐水(pH 7.0-7.6)溶媒对照组、测试药AST-3424 5mg/kg、AST 2.5mg/kg和5mg/kg组是尾静脉注射给药，每周给药一次，共给药三周，观察四周。Ifosfamide 60mg/kg组腹腔注射给药，每周连续给五天停两天，共给药两周，观察五周。根据相对肿瘤抑制率TGI(％)进行疗效评价，根据动物体重变化和死亡情况进行安全性评价。
具体每一组的该药方案下表1所示。
表1：不同剂量的测试药在
Figure PCTCN2022120817-appb-000088
胃癌GA6201肿瘤模型中的抗肿瘤作用实验设计
Figure PCTCN2022120817-appb-000089
Figure PCTCN2022120817-appb-000090
分别在不同天数对不同组别的小鼠的肿瘤体积进行测量，并取得平均值，其结果如下表2所示。
表2：在
Figure PCTCN2022120817-appb-000091
胃癌GA6201模型中各组小鼠肿瘤体积随治疗时间的变化
Figure PCTCN2022120817-appb-000092
根据表2的数据制作体现各治疗组和对照组肿瘤生长情况的图2。
根据表2的数据制作药效评估分析数据，具体数据见表3。
表3：
Figure PCTCN2022120817-appb-000093
胃癌GA6201模型中各组药效分析表
Figure PCTCN2022120817-appb-000094
Figure PCTCN2022120817-appb-000095
表3中相对肿瘤增殖率，T/C％，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。计算公式如下：
T/C％＝TRTV/CRTV×100％(TRTV：治疗组平均RTV；CRTV：溶媒对照组平均RTV；RTV＝Vt/V0，V0为分组时该动物的瘤体积，Vt为治疗后该动物的瘤体积)；
相对肿瘤抑制率，TGI(％)，计算公式如下：TGI％＝(1-T/C)×100％。(T和C分别为治疗组和对照组在某一特定时间点的平均相对肿瘤体积(RTV)。
表4：
Figure PCTCN2022120817-appb-000096
胃癌GA6201模型中各组肿瘤的相对肿瘤抑制率
Figure PCTCN2022120817-appb-000097
将上表4制作为曲线图，可以得到图2。
分别在不同天数对不同组别的小鼠的体重进行测量，并取得平均值，其结果如下表5所示。
表5：
Figure PCTCN2022120817-appb-000098
胃癌GA6201模型中不同接种天数小鼠的体重
Group 0 1 2 3 4 7 8 9 10 11 14 15 16 17 18 21 24 28 31 35 38
Group 01 25.3     25.4   25.3     24.4   25.4     24.6   23.8 24.3 23.7 24.0 24.4 25.1
Group 02 25.8 25.5 25.0 25.5 24.4 24.4 24.5 24.1 24.4 24.4 24.3     25.1   26.3 26.1 25.5 25.2 26.0 26.2
Group 03 24.8     25.1   25.1     24.5   25.4     25.0   25.5 25.6 25.3 25.4 25.7 26.0
Group 04 25.0     24.4   24.8     24.5   25.2     24.9   25.4 25.1 24.6 25.2 25.6 25.7
Group 05 25.3     25.1   25.0     25.0   25.2     24.9   25.4 25.8 25.0 25.4 25.6 25.4
将上表制作为曲线图，可以得到图3，即
Figure PCTCN2022120817-appb-000099
胃癌GA6201模型中各组小鼠体重曲线。
同理对表5的数据进行处理可以得到下表6。
表6：
Figure PCTCN2022120817-appb-000100
胃癌GA6201模型中不同接种天数小鼠的体重变化百分比(％Group Mean
Change＝mean((T-T0)/T0)*100，T表示current value，T0表示initial value)
Group 0 1 2 3 4 7 8 9 10 11 14 15 16 17 18 21 24 28 31 35 38
Group 01 0.00％     0.72％   -0.09％     -3.34％   0.31％     -2.58％   -4.69％ -2.40％ -4.98％ -3.68％ -2.33％ 0.69％
Group 02 0.00％ -1.02％ -2.93％ -1.20％ -5.32％ -5.50％ -5.07％ -6.48％ -5.33％ -5.32％ -5.80％     -2.46％   1.96％ 1.12％ -0.90％ -2.41％ 0.86％ 1.53％
Group 03 0.00％     1.21％   1.46％     -1.11％   2.52％     0.87％   3.03％ 3.31％ 2.27％ 2.52％ 3.58％ 4.87％
Group 04 0.00％     -2.44％   -0.77％     -2.14％   0.65％     -0.46％   1.57％ 0.53％ -1.72％ 0.85％ 2.51％ 2.73％
Group 05 0.00％     -0.70％   -1.21％     -1.22％   -0.28％     -1.77％   0.50％ 1.97％ -1.37％ 0.38％ 1.23％ 0.43％
上表4/5/6中的Group01、Group02、Group03、Group04、Group05即上述的第1组、第2组、第3组、第4组、第5组、第6组。0/1/2/3/4/7/8/9/10/11/14/15/16/17/18/21/24/28/31/35/38是接种后的天数。
将上表制作为曲线图，可以得到图4。
分析实验数据可知，疗效方面：
测试药AST-3424在5mg/kg剂量下，测试药AST在2.5mg/kg和5mg/kg剂量下，对H
Figure PCTCN2022120817-appb-000101
胃癌GA6201具有显著抑制肿瘤生长的作用，相较对照组统计学上均有显著性差异。测试药Ifosfamide在60mg/kg剂量下，对
Figure PCTCN2022120817-appb-000102
胃癌GA6201具有一定的抑制肿瘤生长的作用，但相较对照组统计学上均无显著性差异。
分析实验数据可知，荷瘤小鼠对Ifosfamide、AST-3424、AST在测试剂量下耐受良好。
2、受试物AST和Gemcitabine在
Figure PCTCN2022120817-appb-000103
胰腺癌PA1222皮下异种移植模型中的药效学及安全性评价
Figure PCTCN2022120817-appb-000104
胰腺癌PA1222 PDX模型是带有G12D氨基酸突变的KRAS致病突变(KRAS-G12D)的模型。BALB/c裸小鼠皮下接种
Figure PCTCN2022120817-appb-000105
模型PA1222瘤块，建立人胰腺癌皮下移植肿瘤模型。试验分为测试药Gemcitabine(吉西他滨)120mg/kg组、测试药AST 10mg/kg及7.5％无水乙醇+7.5％聚氧乙烯(35)蓖麻油+85％葡萄糖注射液D5W(pH7.4)溶媒对照组，共3组，每组5只小鼠。其中，7.5％无水乙醇+7.5％聚氧乙烯(35)蓖麻油+85％葡萄糖注射液D5W(pH7.4)溶媒对照组、测试药AST 10mg/kg组是尾静脉注射给药，每周给药一次，连续给药三周。测试药Gemcitabine 120mg/kg组腹腔注射给药，每周给药一次，连续给药3周。根据相对肿瘤抑制率TGI(％)进行疗效评价，根据动物体重变化和死亡情况进行安全性评价。
具体每一组的该药方案下表7所示。
表7：不同剂量的测试药在
Figure PCTCN2022120817-appb-000106
胰腺癌PA1222 PDX肿瘤模型中的抗肿瘤作用实验设计
Figure PCTCN2022120817-appb-000107
注：1.给药体积为10μl/g
2.QW×3；每周给药一次，给药三周
分别在不同天数对不同组别的小鼠的肿瘤体积进行测量，并取得平均值，其结果如下表8所示。
表8：
Figure PCTCN2022120817-appb-000108
胰腺癌PA1222模型中各组小鼠肿瘤体积随治疗时间的变化
Figure PCTCN2022120817-appb-000109
各治疗组和对照组肿瘤生长情况见表8和图5，药效评估见表9。
表9：
Figure PCTCN2022120817-appb-000110
胃癌GA6201模型中各组药效分析表
Figure PCTCN2022120817-appb-000111
表9：相对肿瘤增殖率，T/C％，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。计算公式如下：
T/C％＝TRTV/CRTV×100％(TRTV：治疗组平均RTV；CRTV：溶媒对照组平均RTV；RTV＝Vt/V0，V0为分组时该动物的瘤体积，Vt为治疗后该动物的瘤体积)；
相对肿瘤抑制率，TGI(％)，计算公式如下：TGI％＝(1-T/C)×100％。(T和C分别为治疗组和对照组在某一特定时间点的平均相对肿瘤体积(RTV)。
表10：
Figure PCTCN2022120817-appb-000112
胰腺癌PA1222模型中各组肿瘤的相对肿瘤抑制率
Figure PCTCN2022120817-appb-000113
将上表制作为曲线图，可以得到图6。
分别在不同天数对不同组别的小鼠的体重进行测量，并取得平均值，其结果如下表11所示。
表11：
Figure PCTCN2022120817-appb-000114
胰腺癌PA1222模型中不同接种天数小鼠的体重
Group 0 3 7 10 14 17 21 24 28
Group 01 24.0 24.1 24.6 24.6 24.4 24.3 25.1 24.8 25.0
Group 02 24.1 23.9 24.4 24.3 24.6 24.2 25.1 25.1 25.4
Group 03 24.0 23.5 24.0 23.6 23.6 23.6 24.0 24.0 24.1
将上表制作为曲线图，可以得到图7，即
Figure PCTCN2022120817-appb-000115
胰腺癌PA1222模型中各组小鼠体重曲线。
同理对表11的数据进行处理可以得到下表12。
表12：
Figure PCTCN2022120817-appb-000116
胰腺癌PA1222模型中不同接种天数小鼠的体重变化百分比(％Group mean Change＝mean((T-T0)/T0)*100，T表示current value，T0表示initial value)
Figure PCTCN2022120817-appb-000117
将上表制作为曲线图，可以得到图8。
分析实验数据可知，疗效方面：
测试药Gemcitabine在120mg/kg(Group 2)剂量下，对
Figure PCTCN2022120817-appb-000118
胰腺癌PA1222具有一定的抑制肿瘤生长的作用，相较对照组统计学上有显著性差异。测试药AST在10mg/kg(Group 3)剂量下，对
Figure PCTCN2022120817-appb-000119
胰腺癌PA1222有显著的抑瘤作用，相较对照组统计学上有显著性差异，并且该组有两只小鼠肿瘤被治愈，治愈率均为40％。测试药AST 10mg/kg(Group 3)的抑瘤效果显著优于测试药Gemcitabine(120mg/kg，Group 2)(p＝0.000778)。
分析实验数据可知，测试药Gemcitabine(120mg/kg，Group 2)治疗组、AST 10mg/kg(Group 3)治疗组以及对照组(Group 1)的小鼠没有任何明显的体重下降的现象，治疗期间耐受良好。
3、受试物AST和Cisplatin在
Figure PCTCN2022120817-appb-000120
肺癌LU11693皮下异种移植模型中的药效学及安全性评价
Figure PCTCN2022120817-appb-000121
肺癌LU11693 PDX模型是带有G12C氨基酸突变的KRAS致病突变的模型。BALB/c Nude裸小鼠皮下接种
Figure PCTCN2022120817-appb-000122
模型LU11693瘤块，建立人肺癌皮下移植肿瘤模型。试验分为测试药Cisplatin 4mg/kg组、测试药AST 10mg/kg组及7.5％无水乙醇+7.5％聚氧乙烯(35)蓖麻油+85％葡萄糖注射液D5W(pH7.4)溶媒对照组，共3组，每组6只小鼠，尾静脉注射给药，每周给药一次，连续给3周。根据相对肿瘤抑制率TGI(％)进行疗效评价，根据动物体重变化和死亡情况进行安全性评价。
具体每一组的该药方案下表13所示。
表13：不同剂量的测试药在
Figure PCTCN2022120817-appb-000123
肺癌LU11693 PDX肿瘤模型中的抗肿瘤作用实验设计
Figure PCTCN2022120817-appb-000124
注：1.给药体积为10μl/g
2.QW×3；每周给药一次，给药三周
分别在不同天数对不同组别的小鼠的肿瘤体积进行测量，并取得平均值，其结果如下表14所示。
表14：
Figure PCTCN2022120817-appb-000125
肺癌LU11693模型中各组小鼠肿瘤体积随治疗时间的变化
Figure PCTCN2022120817-appb-000126
Figure PCTCN2022120817-appb-000127
各治疗组和对照组肿瘤生长情况见表14和图9，药效评估见表15。
表15：
Figure PCTCN2022120817-appb-000128
肺癌LU11693模型中各组药效分析表
Figure PCTCN2022120817-appb-000129
表15中，相对肿瘤增殖率，T/C％，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。计算公式如下：
T/C％＝TRTV/CRTV×100％(TRTV：治疗组平均RTV；CRTV：溶媒对照组平均RTV；RTV＝Vt/V0，V0为分组时该动物的瘤体积，Vt为治疗后该动物的瘤体积)；
相对肿瘤抑制率，TGI(％)，计算公式如下：TGI％＝(1-T/C)×100％。(T和C分别为治疗组和对照组在某一特定时间点的平均相对肿瘤体积(RTV)。
表16：
Figure PCTCN2022120817-appb-000130
肺癌LU11693模型中各组肿瘤的相对肿瘤抑制率
Figure PCTCN2022120817-appb-000131
将上表制作为曲线图，可以得到图10。
分别在不同天数对不同组别的小鼠的体重进行测量，并取得平均值，其结果如下表17所示。
表17：
Figure PCTCN2022120817-appb-000132
肺癌LU11693模型中不同接种天数小鼠的体重
Group 0 4 7 11 14 18 21 25 28
Group 01 24.5 24.6 24.5 24.5 24.9 24.9 24.6 24.3 24.4
Group 02 24.3 23.6 23.9 22.8 24.0 22.4 23.0 23.4 23.6
Group 03 24.3 24.3 23.9 23.3 23.6 23.3 23.0 22.4 22.8
将上表制作为曲线图，可以得到图11，即
Figure PCTCN2022120817-appb-000133
肺癌LU11693模型中各组小鼠体重曲线。
同理对表17的数据进行处理可以得到下表18。
表18：
Figure PCTCN2022120817-appb-000134
肺癌LU11693模型中不同接种天数小鼠的体重变化百分比(％Group Mean Change＝mean((T-T0)/T0)*100，T表示current value，T0表示initial value)
Group 0 4 7 11 14 18 21 25 28
Group 01 0.00％ 0.53％ 0.07％ 0.20％ 1.54％ 1.81％ 0.63％ -0.54％ -0.18％
Group 02 0.00％ -2.95％ -1.68％ -6.23％ -1.32％ -8.04％ -5.33％ -3.86％ -2.74％
Group 03 0.00％ 0.15％ -1.86％ -4.24％ -3.09％ -4.15％ -5.47％ -7.78％ -6.38％
将上表制作为曲线图，可以得到图12。
分析实验数据可知，疗效方面：
测试药Cisplatin(4mg/kg)治疗组在首次给药后的第28天(Day28)表现出一定的抑瘤作用，相较对照组统计学上有显著性差异(p＝0.0152)，相对肿瘤抑制率TGI(％)为23.98％。
测试药AST(10mg/kg)治疗组在首次给药后的第28天(Day 28)表现出一定的抑瘤作用，相较对照组统计学上有显著性差异(p＜0.001)，相对肿瘤抑制率TGI(％)为54.64％，TGI小于60％，没有明显的抑瘤效果。
分析实验数据可知，测试药AST(10mg/kg)和Cisplatin(4mg/kg)治疗组有个别小鼠出现体重严重下降的现象，可能与高剂量药物潜在的毒性有关。
由上述三组实验数据我们可以得出以下结论：
1.AST-3424和AST在带有G12D氨基酸突变的KRAS致病突变模型：胃癌GA6021和胰腺癌PA1222 PDX模型中均具有显著的药效，TGI％均大于90％；
2.AST在带有G12C氨基酸突变的KRAS致病突变模型：肺癌LU11693中的抑瘤效果不明显，TGI％小于60％；
3.AST-3424和AST在各模型中均具有比较好的耐受性。
发明人进一步通过研究发现，在带有G12D氨基酸突变的KRAS致病突变模型中，AST-3424和AST对多种癌症适应症具有普遍的显著疗效。而在带有G12C氨基酸突变的KRAS致病突变模型中，AST的疗效不仅与癌种有关，还与其他因素有关。
4、受试物AST、AST-3424和Ifosfamide在人源胰腺癌HPAF-II皮下异种移植模型中的抗肿瘤作用及安全性评价
人源胰腺癌HPAF-II皮下异种移植模型是带有KRAS G12D致病突变的CDX模型。
Balb/c nude雌性小鼠皮下接种人源胰腺癌HPAF-II细胞，建立人源胰腺癌皮下移植模型。试验分为的测试药物治疗组Ifosfamide 60mg/kg单药组(Group 2)为腹腔给药，每天给药1次，连续给药5天，休息2天，再每天给药1次，连续给药5天；AST 4mg/kg单药组(Group 3)为尾静脉给药，每天给药1次，连续给药5天，休息2天，再休息2周，再每天给药1次，连续给药5天；AST 8mg/kg单药组(Group 4)为尾静脉给药，每周给药1次，共给药3周；AST-3424 1mg/kg单药组(Group 5)为尾静脉给药，每天给药1次，连续给药5天，休息2天，再休息2周，再每天给药1次，连续给药5天；以及葡萄糖注射液(pH7.7-8.0)溶媒对照组(Group 1)为尾静脉给药，每天给药1次，连续给药5天，休息2天，再休息2周，再每天给药1次，连续给药5天；该研究共5组，每组6只小鼠。实验设计的给药途径、剂量及方案见表19。
表19：人源胰腺癌HPAF-II动物模型中的给药途径、剂量及方案
Figure PCTCN2022120817-appb-000135
Figure PCTCN2022120817-appb-000136
本申请中，给药途径相关缩写，i.v.表示尾静脉注射，i.p.表示腹腔注射；给药周期相关缩写，QW表示每周一次，QD表示每天一次，“QD*5，2 days off，2 weeks off，QD*5”表示每天给药一次，连续给5天，休息2天，再休息2周，再每天给药一次，连续给5天。
在试验不同天数记录各治疗组和对照组肿瘤生长情况，如表20所示，相应地各组小鼠肿瘤体积的生长曲线如图13所示。根据相对肿瘤增殖率和相对肿瘤抑制率对疗效进行评价，各组药效分析如表21所示。记录治疗组和对照组给药后体重变化，研究人源胰腺癌HPAF-II皮下异种移植模型中各组的安全性，小鼠体重变化结果如表22所示，各治疗组体重变化百分比随时间变化曲线图如图14所示。
表20：人源胰腺癌HPAF-II皮下模型中各组小鼠肿瘤体积随治疗时间的变化(mm 3)
Figure PCTCN2022120817-appb-000137
表21：人源胰腺癌HPAF-II皮下模型中各组药效分析表
Figure PCTCN2022120817-appb-000138
Figure PCTCN2022120817-appb-000139
Figure PCTCN2022120817-appb-000140
溶媒对照组小鼠在开始给药后的第31天(Day 31)平均肿瘤体积为2212.64mm 3。测试药Ifosfamide在60mg/kg剂量治疗组(第2组)在Day 31平均肿瘤体积为2678.00mm 3，相对肿瘤抑制率TGI(％)为-14.65％，相较对照组统计学上没有显著性差异(p＞0.05)。
测试药AST在4mg/kg(QD×5，2 days off，2 weeks off，QD×5)和8mg/kg(QW×3)剂量治疗组(第3组和第4组)，以及AST-3424在1mg/kg(QD×5，2 days off，2weeks off，QD×5)剂量组(第5组)在Day 31平均肿瘤体积分别为457.66，170.65和685.85mm 3，相较对照组统计学上有显著性差异(p＜0.05)，相对肿瘤抑制率TGI(％)分别为79.55％，92.64％和69.46％。
上述实验结果表明，AST-3424和AST在带有KRAS G12D致病突变模型中，AST在8mg/kg(QW×3)，4mg/kg(QD×5，2 days off，2 weeks off，QD×5)剂量下，以及AST-3424在1mg/kg(QD×5，2 days off，2 weeks off，QD×5)剂量下，对人源胰腺癌HPAF-II皮下模型有显著的抗肿瘤作用，且各测试药治疗组小鼠在治疗期间小鼠体重均没有下降，耐受良好。
5、受试物AST、AST-3424和Ifosfamide单药在
Figure PCTCN2022120817-appb-000141
肺癌LU5161皮下模型中的抗肿瘤作用及安全性评价
Figure PCTCN2022120817-appb-000142
肺癌LU5161皮下模型是带有KRAS G12D致病突变的PDX模型。
Balb/nude雌性小鼠皮下接种
Figure PCTCN2022120817-appb-000143
肺癌LU5161瘤块，建立人肺癌皮下移植肿瘤模型。试验分为的测试药Ifosfamide 60mg/kg单药组(Group 2)，每天给药1次，连续给5天，休息2天，再每天给药1次，连续给5天；AST 4mg/kg单药组(Group 3)和AST 8mg/kg单药组(Group 4)，均每周给药1次，共计给药3周；AST 4mg/kg单药组(Group 5)和AST-34241mg/kg单药组(Group 6)，每天给药1次，连续给药5天，休息2天，再休息2周，然后再每天给药1次，连续给药5天，以及葡萄糖注射液(pH7.7-8.0)溶媒对照组(Group 1)，每天给药1次，连续给药5天，休息2天，再休息2周，然后再每天给药1次，连续给药5天。该研究共6组，每组6只小鼠。其中测试药Ifosfamide为腹腔给药，溶媒对照组，AST，和AST-3424均为尾静脉注射给药。实验设计的给药途径、剂量及方案见表23。
表23：
Figure PCTCN2022120817-appb-000144
肺癌LU5161皮下模型中的给药途径、剂量及方案
Figure PCTCN2022120817-appb-000145
在试验不同天数记录各治疗组和对照组肿瘤生长情况，如表24所示，相应地各组小鼠肿瘤体积的生长曲线如图15所示。根据相对肿瘤增殖率和相对肿瘤抑制率对疗效进行评价，各组药效分析如表25所示。记录治疗组和对照组给药后体重变化，研究
Figure PCTCN2022120817-appb-000146
肺癌LU5161皮下异种移植模型中各组的安全性，小鼠体重变化结果如表26所示，各治疗组体重变化百分比随时间变化曲线图如图16所示。
表24：
Figure PCTCN2022120817-appb-000147
肺癌LU5161皮下模型中各组小鼠肿瘤体积随治疗时间的变化(mm 3)
Figure PCTCN2022120817-appb-000148
表25：
Figure PCTCN2022120817-appb-000149
肺癌LU5161皮下模型中各组药效分析表
Figure PCTCN2022120817-appb-000150
Figure PCTCN2022120817-appb-000151
溶媒对照组小鼠在开始给药后的第29天(Day 28)平均肿瘤体积为2238.97mm 3。测试药Ifosfamide在60mg/kg剂量治疗组(Group 2)在Day 28平均肿瘤体积分别为1636.39mm 3，相对肿瘤抑制率TGI(％)为27.39％，相较对照组统计学上没有显著性差异(p＞0.05)。
测试药AST在4mg/kg(QW×3，Group 3)，8mg/kg(QW×3，Group 4)，4mg/kg(QD×5，2 days off，2 weeks off，QD×5，Group 5)剂量治疗组，以及AST-3424在1mg/kg(QD×5，2 days off，2 weeks off，QD×5)剂量组(Group 6)在Day 28平均肿瘤体积分别为71.72mm 3，68.18mm 3，37.14mm 3和94.42mm 3，相较对照组统计学上有显著性差异(p＜0.001)，相对肿瘤抑制率TGI(％)分别为96.92％，97.05％，98.38％和95.62％。测试药AST和AST-3424的所有治疗组均各有1只小鼠肿瘤被清除，清除率为16.7％。
上述实验结果表明，AST-3424和AST在带有KRAS G12D致病突变模型中，AST 4mg/kg(QW×3)，8mg/kg(QW×3)，4mg/kg(QD×5，2 days off，2 weeks off，QD×5)剂量治疗组(Group 3，Group 4和Group 5)，以及AST-3424在1mg/kg(QD×5，2 days off，2 weeks off，QD×5)剂量组(Group 6)在本研究的测试剂量和给药频率下，对
Figure PCTCN2022120817-appb-000152
Figure PCTCN2022120817-appb-000153
肺癌LU5161皮下模型均有显著的抗肿瘤作用。Ifosfamide给药组未产生抑瘤作用。各测试药治疗组小鼠在治疗期间小鼠耐受良好。
6、受试物AST和Ifosfamide单药在
Figure PCTCN2022120817-appb-000154
肠癌CR3820皮下模型中的抗肿瘤作用及安全性评价
Figure PCTCN2022120817-appb-000155
肠癌CR3820皮下模型是带有KRAS G12D致病突变的PDX模型。
NOD.SCID雌性小鼠皮下接种
Figure PCTCN2022120817-appb-000156
肠癌CR3820瘤块，建立人肠癌皮下移植肿瘤模型。试验分为测试药物Ifosfamide，60mg/kg单药治疗组(QD×5/week×2 weeks，Group 2)，腹腔给药，每天给药1次，连续给5天，休息2天，再每天给药1次，连续给5天；AST 8mg/kg单药组(QW×3，Group 3)，尾静脉给药，每周给药1次，共计给药3周；AST 4m g/kg单药组(QD×5，2 days off，2 weeks off，QD×5，Group 4)以及溶媒对照组葡萄糖注射液(pH7.7-8.0，Group 1)，均为尾静脉给药，给药周期均为每天给药1次，连续给药5天，休息2天，再休息2周，然后再每天给药1次，连续给药5天；该实验共4组，每组6只小鼠。实验设计的给药途径、剂量及方案见表27。
表27：
Figure PCTCN2022120817-appb-000157
肠癌CR3820皮下模型中的给药途径、剂量及方案
Figure PCTCN2022120817-appb-000158
在试验不同天数记录各治疗组和对照组肿瘤生长情况，如表28所示，相应地各组小鼠肿瘤体积的生长曲线如图17所示。根据相对肿瘤增殖率和相对肿瘤抑制率对疗效进行评价，各组药效分析如表29所示。记录治疗组和对照组给药后体重变化，研究
Figure PCTCN2022120817-appb-000159
肠癌CR3820皮下异种移植模型中各组的安全性，小鼠体重变化结果如表30所示，相应地各治疗组体重变化百分比随时间变化曲线图如图18所示。
表28：
Figure PCTCN2022120817-appb-000160
肠癌CR3820皮下模型中各组小鼠肿瘤体积随治疗时间的变化(mm 3)
Figure PCTCN2022120817-appb-000161
表29：
Figure PCTCN2022120817-appb-000162
肠癌CR3820皮下模型中各组药效分析表
Figure PCTCN2022120817-appb-000163
Figure PCTCN2022120817-appb-000164
溶媒对照组小鼠在开始给药后的第25天(Day 24)平均肿瘤体积为1792.37mm 3。测试药Ifosfamide在60mg/kg剂量治疗组(QD×5/week×2 weeks，Group 2)在Day 24平均肿瘤体积分别为1199.09mm3，相对肿瘤抑制率TGI(％)分别为37.73％，相较对照组统计学上没有显著性差异(p＞0.05)。
在Day 24，测试药AST在8mg/kg(QW×3，Group 3)，4mg/kg(QD×5，2 days off，2 weeks off，QD×5，Group 4)剂量治疗组在Day 24平均肿瘤体积为110.59mm 3和146.58mm 3，相对肿瘤抑制率TGI(％)分别为94.08％和92.44％，相较对照组统计学上有显著性差异(p＜0.05)。
上述实验结果表明，AST在带有KRAS G12D致病突变模型中，测试药AST 8mg/kg(QW×3，Group 3)，4mg/kg(QD×5，2 days off，2 weeks off，QD×5，Group 4)在该实验中的测试剂量以及给药频率下，对
Figure PCTCN2022120817-appb-000165
肠癌CR3820皮下模型中具有统计学意义的显著抗肿瘤作用。测试药Ifosfamide和AST在本研究的测试剂量下均有良好耐受。
7、受试物AST、AST-3424和Ifosfamide单药在
Figure PCTCN2022120817-appb-000166
胰腺癌PA2637皮下模型中的抗肿瘤作用及安全性评价
Figure PCTCN2022120817-appb-000167
胰腺癌PA2637皮下模型是带有KRAS G12D致病突变的PDX模型。
NOD.SCID雌性小鼠皮下接种
Figure PCTCN2022120817-appb-000168
胰腺癌PA2637瘤块，建立人胰腺癌皮下移植肿瘤模型。试验分为测试药物Ifosfamide，60mg/kg单药治疗组(QD×5/week×2 weeks，Group 2)，腹腔给药，每天给药1次，连续给5天，休息2天，再每天给药1次，连续给5天；AST 8mg/kg单药组(QW×3，Group 3)，尾静脉给药，每周给药1次，共计给药3周；AST 4mg/kg单药组(QD×5，2 days off，2 weeks off，QD×5，Group 4)、AST-3424 1mg/kg单药组(QD×5，2 days off，2 weeks off，QD×5，Group 5)以及溶媒对照组葡萄糖注射液(pH7.7-8.0，Group 1)，均为尾静脉给药，给药周期均为每天给药1次，连续给药5天，休息2天，再休息2周，然后再每天给药1次，连续给药5天。该实验共5组，每组6只小鼠。实验设计的给药途径、剂量及方案见表31。
表31：
Figure PCTCN2022120817-appb-000169
胰腺癌PA2637皮下模型中的给药途径、剂量及方案
Figure PCTCN2022120817-appb-000170
在试验不同天数记录各治疗组和对照组肿瘤生长情况，如表32所示，相应地各组小鼠肿瘤体积的生长曲线如图19所示。根据相对肿瘤增殖率和相对肿瘤抑制率对疗效进行评价，各组药效分析如表33所示。记录治疗组和对照组给药后体重变化，研究
Figure PCTCN2022120817-appb-000171
胰腺癌PA2637皮下异种移植模型中各组的安全性，小鼠体重变化结果如表34所示，相应地各治疗组体重变化百分比随时间变化曲线图如图20所示。
表32：
Figure PCTCN2022120817-appb-000172
胰腺癌PA2637皮下模型中各组小鼠肿瘤体积随治疗时间的变化(mm 3)
Figure PCTCN2022120817-appb-000173
表33：
Figure PCTCN2022120817-appb-000174
胰腺癌PA2637皮下模型中各组药效分析表
Figure PCTCN2022120817-appb-000175
Figure PCTCN2022120817-appb-000176
溶媒对照组小鼠在开始给药后的第36天(Day 35)平均肿瘤体积为977.46mm 3。测试药Ifosfamide在60mg/kg剂量治疗组(Group 2)在Day 35平均肿瘤体积分别为938.33mm3，相对肿瘤抑制率TGI(％)为2.74％，相较对照组统计学上没有显著性差异(p＞0.05)。
测试药AST在8mg/kg(QW×3，Group 3)，4mg/kg(QD×5，2 days off，2 weeks of f，QD×5，Group 4)，测试药AST-3424在1mg/kg(QD×5，2 days off，2 weeks off，QD×5，Group 5)剂量治疗组在Day 35平均肿瘤体积为123.47mm 3，141.48mm 3和186.08mm 3，相对肿瘤抑制率TGI(％)分别为87.28％，85.46％和80.78％，相较对照组统计学上有显著性差异(p＜0.001)。
上述实验结果表明，AST和AST-3424在带有KRAS G12D致病突变模型中，测试药AST 8mg/kg(QW×3，Group 3)，4mg/kg(QD×5，2 days off，2 weeks off，QD×5，Group 4)和测试药AST-34241mg/kg(QD×5，2 days off，2 weeks off，QD×5，Group 5)在该实验中的测试剂量以及给药频率下，对
Figure PCTCN2022120817-appb-000177
胰腺癌PA2637皮下模型中均具有统计学意义的显著抗肿瘤作用。在实验过程中，各测试药治疗组小鼠在治疗期间均耐受良好。
8、受试物AST和Ifosfamide单药在
Figure PCTCN2022120817-appb-000178
肺癌LU11873皮下模型中的抗肿瘤作用及安全性评价
Figure PCTCN2022120817-appb-000179
肺癌LU11873皮下模型是带有KRAS G12C致病突变的PDX模型。
NOD.SCID雌性小鼠皮下接种
Figure PCTCN2022120817-appb-000180
肺癌LU11873瘤块，建立人肺癌皮下移植肿瘤模型。试验分为的测试药Ifosfamide 60mg/kg单药组(Group 2)，每天给药1次，连续给药5天，休息2天，然后再每天给药1次，连续给药5天；AST 4mg/kg单药组(Group 5)，每天给药1次，连续给药5天，休息2天，再休息2周，然后再每天给药1次，连续给药5天；以及葡萄糖注射液(pH7.7-8.0)溶媒对照组(Group 1)，每天给药1次，连续给药5天，休息2天，再休息2周，然后再每天给药1次，连续给药5天；该研究总共3组，每组6只小鼠。测试药Ifosfamide为腹腔给药。溶媒对照组，AST各组均为尾静脉注射给药。实验设计的给药途径、剂量及方案见表35。
表35：
Figure PCTCN2022120817-appb-000181
肺癌LU11873皮下模型中的给药途径、剂量及方案
Figure PCTCN2022120817-appb-000182
在试验不同天数记录各治疗组和对照组肿瘤生长情况，如表36所示，相应地各组小鼠肿瘤体积的生长曲线如图21所示。根据相对肿瘤增殖率和相对肿瘤抑制率对疗效进行评价，各组药效分析如表37所示。记录治疗组和对照组给药后体重变化，研究
Figure PCTCN2022120817-appb-000183
肺癌LU11873皮下异种移植模型中各组的安全性，小鼠体重变化结果如表38所示，相应地各治疗组体重变化百分比随时间变化曲线图如图22所示。
表36：
Figure PCTCN2022120817-appb-000184
肺癌LU11873皮下模型中各组小鼠肿瘤体积随治疗时间的变化(mm 3)
Figure PCTCN2022120817-appb-000185
Figure PCTCN2022120817-appb-000186
表37：
Figure PCTCN2022120817-appb-000187
肺癌LU11873皮下模型中各组药效分析表
Figure PCTCN2022120817-appb-000188
Figure PCTCN2022120817-appb-000189
溶媒对照组小鼠在开始给药后的第31天(Day 31)平均肿瘤体积为1677.89mm 3。测试药Ifosfamide在60mg/kg剂量治疗组(Group 2)剂量下Day 31平均肿瘤体积分别为1866.37mm 3，相对肿瘤抑制率TGI(％)为-10.08％，相较对照组统计学上没有显著性差异(p＞0.05)。测试药AST在4mg/kg(QD×5，2 days off，2 weeks off，QD×5)剂量治疗组(Group 5)在Day 31平均肿瘤体积为406.40mm 3，相较对照组统计学上有显著性差异(p＜0.05)，相对肿瘤抑制率TGI(％)为75.16％。
上述实验结果表明，AST在带有KRAS G12C致病突变模型中，测试药AST 4mg/kg(QD×5，2 days off，2 weeks off，QD×5)剂量治疗组(Group 5)在本研究的测试剂量和给药频率下，对
Figure PCTCN2022120817-appb-000190
肺癌LU11873皮下模型中的有显著的抗肿瘤作用，Ifosfamide给药组未产生抑瘤作用。各测试药治疗组小鼠在治疗期间小鼠体重均没有下降，耐受良好。
特别地，申请人发现在实施例3中AST在带有G12C氨基酸突变的KRAS致病突变模型肺癌LU11693中的抑瘤效果不明显，10mg/kg剂量下TGI％为54.64％；而在本实施例中，AST在带有KRAS G12C致病突变的肺癌LU11873模型中有显著的抗肿瘤作用，4mg/kg剂量下TGI％为75.16％，两者差别明显，显示这两个PDX模型可能存在某种差别，从模型的来源资料来看：
LU11693来源于58岁的女性患者，其在临床中显示有恶病质，轻微溃疡；
LU11873来源于51岁的男性患者，其在临床中显示有轻微的体重减少，轻微溃疡。
9、受试物AST和Ifosfamide单药在
Figure PCTCN2022120817-appb-000191
胰腺癌PA1383皮下模型中的抗肿瘤作用及安全性评价
Figure PCTCN2022120817-appb-000192
胰腺癌PA1383皮下模型是带有KRAS G12C致病突变的PDX模型。
Balb/nude雌性小鼠皮下接种
Figure PCTCN2022120817-appb-000193
胰腺癌PA1383瘤块，建立人胰腺癌皮下移植肿瘤模型。试验分为的测试药Ifosfamide 60mg/kg单药组(Group 2)，每天给药1次，连续给5天，休息2天，再每天给药1次，连续给5天；AST 8mg/kg单药组(Group 4)，每周给药1次，共计给药3周；AST 4mg/kg单药组(Group 5)每天给药1次，连续给药5天，休息2天，再休息2周，然后再每天给药1次，连续给药5天；以及葡萄糖注射液(pH7.7-8.0)溶媒对照组(Group 1)，每天给药1次，连续给药5天，休息2天，再休息2周，然后再每天给药1次，连续给药5天。该研究共4组，每组6只小鼠。其中测试药Ifosfamide为腹腔给药，溶媒对照组，AST为尾静脉注射给药。实验设计的给药途径、剂量及方案见表39。
表39：
Figure PCTCN2022120817-appb-000194
胰腺癌PA1383皮下模型中的给药途径、剂量及方案
Figure PCTCN2022120817-appb-000195
在试验不同天数记录各治疗组和对照组肿瘤生长情况，如表40所示，相应地各组小鼠肿瘤体积的生长曲线如图23所示。根据相对肿瘤增殖率和相对肿瘤抑制率对疗效进行评价，各组药效分析如表41所示。记录治疗组和对照组给药后体重变化，研究
Figure PCTCN2022120817-appb-000196
胰腺癌PA13 83皮下异种移植模型中各组的安全性，小鼠体重变化结果如表42所示，相应地各治疗组体重变化百分比随时间变化曲线图如图24所示。
表40：
Figure PCTCN2022120817-appb-000197
胰腺癌PA1383皮下模型中各组小鼠肿瘤体积随治疗时间的变化(mm 3)
Figure PCTCN2022120817-appb-000198
表41：
Figure PCTCN2022120817-appb-000199
胰腺癌PA1383皮下模型中各组药效分析表
Figure PCTCN2022120817-appb-000200
Figure PCTCN2022120817-appb-000201
溶媒对照组小鼠在开始给药后的第31天(Day 31)平均肿瘤体积为1536.48mm 3。测试药Ifosfamide在60mg/kg剂量治疗组(Group 2)剂量下Day 31平均肿瘤体积为1202.01mm 3，相对肿瘤抑制率TGI(％)为21.84％，相较对照组统计学上均没有显著性差异(p＞0.05)。
测试药AST在8mg/kg(QW×3)以及4mg/kg(QD×5，2 days off，2 weeks off，QD×5)剂量治疗组(Group 4和Group 5)在Day 31平均肿瘤体积分别为18.57mm 3和39.94mm 3，相较对照组统计学上均有显著性差异(p＜0.05)，相对肿瘤抑制率TGI(％)分别为98.72％和97.46％，且均各有2只小鼠的肿瘤被完全清除，清除率为33.3％。
上述实验结果表明，AST在带有KRAS G12C致病突变模型中，AST 8mg/kg(QW×3)以及4mg/kg(QD×5，2 days off，2 weeks off，QD×5)剂量治疗组(Group 4和Group 5)在本研究的测试剂量和给药频率下，对
Figure PCTCN2022120817-appb-000202
胰腺癌PA1383皮下模型有显著的抗肿瘤作用。Ifosfamide给药组未产生抑瘤作用。各测试药治疗组小鼠在治疗期间小鼠体重均没有下降，耐受良好。
10、组织中的AKR1C3的RNA表达水平及酶含量的检测
A、AKR1C3 RNA表达水平FPKM检测
根据文献(Meng，F.，Li，W.F.，Jung，D.，Wang，C.C.，Qi，T.，Shia，C.S.，Hsu，R.Y.，Hsieh，Y.C.，&Duan，J.(2021).A novel selective AKR1C3-activated prodrug AST-3424/OBI-3424 exhibits broad anti-tumor activity.American journal of cancer research，11(7)，3645-3659.)的记载方法，使用RNA-Seq分析了上述胃癌GA6021、胰腺癌PA1222、肺癌LU11693组织中的AKR1C3的RNA表达水平(AKR1C3 RNA expression level，并使用Log2 FPKM进行定量)，结果如下：
GA6201检测得到AKR1C3LOG2(FPKM)为6.78，LU11693为11.14，PA1222为7.39，HPAF-II为8.31，LU5161为11.56，CR3820为8.34，PA2637为9.12，LU11873为10.26，PA1383为9.57，参见表43。
根据上述文献，可知这九个肿瘤组织的AKR1C3 RNA均为高表达。
B、AKR1C3蛋白含量的IHC方法检测及H-SCORE打分
根据常用的IHC(免疫组织化学)染色法对这三个组织的AKR1C3蛋白含量进行测定(使用商用的IHC试剂，一抗为Abcam公司的兔抗体Rabbit IgG mAb，二抗为Leica公司的聚合物优化检测系统Bond Polymer Refine Detection，染色条件：抗原修复100℃，pH9.0 EDTA缓冲液20min，稀释比：1∶800)，并对染色结果进行H-SCORE打分：免疫组化染色强度将被分为0(阴性)，1+(弱染色)，2+(中染色)，3+(强染色)，人工在打分仪器上设置弱染色、中染色、强染色的阈值，然后通过图像处理软件对染色样本照片进行色彩识别，所有样品的染色照片按照统一的标准，由评分软件对某个细胞对应的染色情况进行评分：0/1/2/3。并统计不同染色强度的阳性细胞数量占切片中总细胞数量的百分数。通过下面的公式计算H-Score作为每个样品的IHC结果评分。H-score得分将在0～300之间，评分越高则表示该抗体对应的靶点(AKR1C3酶蛋白)在该样品中表达量越高。计算公式如下：
H-Score＝(％at 0)×0+(％at 1)×1+(％at 2)×2+(％at 3)×3
GA6201、LU11693、PA1222及两个对照组的染色结果如图25，打分结果如下表43：
表43：九个模型及对照组的IHC和RNA分析检测结果
Figure PCTCN2022120817-appb-000203
Figure PCTCN2022120817-appb-000204
GA6201、LU11693、PA1222模型及对照组的具体染色统计结果如下表44：
表44：GA6201、LU11693、PA1222模型及对照组的IHC结果及H-SCORE打分
Figure PCTCN2022120817-appb-000205
上述染色结果中阳性和阴性对照结果在控制范围内即表明本次IHC染色剂H-SCORE打分结果是可信的。
根据上述结果可知，这九个组织对应的AKR1C3蛋白均为高表达。
综合以上九个模型药效实验结果以及这九个模型使用的组织均为人AKR1C3高表达的肿瘤组织可知：AST-3424和AST对高表达AKR1C3且带有G12D氨基酸突变的KRAS致病突变的癌症具有普遍的显著的治疗效果；AST对于高表达AKR1C3且带有G12C氨基酸突变的KRAS致病突变的癌症可能具有显著的治疗效果。这意味着AKR1C3在某些肿瘤中高表达可能与KARS(致病)突变亚型是伴生的，即在某些肿瘤中AKR1C3高表达或过表达常常与KARS(致病)突变的某些亚型是同时存在的，而这种现象会导致具有这些特征的肿瘤模型对AST-3424或AST更敏感。
根据文献(Meng，F.，Li，W.F.，Jung，D.，Wang，C.C.，Qi，T.，Shia，C.S.，Hsu，R.Y.，Hsieh，Y.C.，&Duan，J.(2021).A novel selective AKR1C3-activated prodrug AST-3424/OBI-3424 exhibits broad anti-tumor activity.American journal of cancer research，11(7)，3645-3659；Evans，K.，Duan，J.，Pritchard，T.，Jones，C.D.，McDermott，L.，Gu，Z.，Toscan，C.E.，E1-Zein，N.，Mayoh，C.，Erickson，S.W.，Guo，Y.，Meng，F.，Jung，D.，Rathi，K.S.，Roberts，K.G.，Mullighan，C.G.，Shia，C.S.，Pearce，T.，Teicher，B.A.，Smith，M.A.，…Lock，R.B.(2019).OBI-3424，a Novel AKR1C3-Activated Prodrug，Exhibits Potent Efficacy against Preclinical Models of T-ALL.Clinical cancer research：an official journal of the American Association for Cancer Research，25(14)，4493-4503；Yanlan Wang，Yue Liu，Changhua Zhou，Chunnian Wang，Ning Zhang，Donglin Cao，Qing Li&Zhong Wang(2020)An AKR1C3-specific prodrug with potent anti-tumor activities against T-ALL，Leukemia&Lymphoma，61(7)，1660-1668。)以及对应的专利申请：
PCT/US2016/021581，公开号WO2016145092A1(对应中国申请号2016800150788，公开号CN107530556A)，
PCT/US2016/062114，公开号WO2017087428A1(对应中国申请号2016800200132，公开号CN108136214A)，
PCT/CN2020/089692，公开号WO2020228685A1；
PCT/NZ2019/050030，公开号WO2019190331A1(对应中国申请号2019800234236，公开号CN111918864A)；
PCT/CN2020/120281，公开号WO2021068952A1，
所公开，这些专利中的化合物与AST-3424、AST化合物类似，都是AKR1C3活化的抗癌前药，在AKR1C3活化后裂解出DNA烷化剂
Figure PCTCN2022120817-appb-000206
或氮芥结构结构。
因此，结合上述AST-3424、AST的实验结果，可以推测AKR1C3活化的DNA烷化剂前药对于单药或联用其他治疗药物治疗KRAS突变的癌症、肿瘤患者，特别是KRAS-G12D亚型突变的患者具有显著的治疗效果。

Claims (13)

  1. 治疗方法，其使用含有AKR1C3活化的DNA烷化剂前药化合物的药物单药或联用其他治疗药物治疗KRAS突变的癌症、肿瘤患者。
  2. 根据权利要求1所述的治疗方法，其中，所述化合物选自结构式1/2/3/4/5/6及其盐、酯、溶剂合物、同位素异构体：
    Figure PCTCN2022120817-appb-100001
    其中，R 1、R 2、R 3、R 4、R 5、R 8、R 9、R 10的定义如专利申请PCT/CN2020/089692，公开号WO2020228685A1中的权利要求书所记载；
    Figure PCTCN2022120817-appb-100002
    其中，A、E、G、X、Y的定义如专利申请PCT/NZ2019/050030，公开号WO2019190331A1(对应中国申请号2019800234236，公开号CN111918864A)中的权利要求书所记载；
    Figure PCTCN2022120817-appb-100003
    其中，Rw的定义如专利申请PCT/CN2020/120281，公开号WO2021068952A1中的权利要求书所记载；
    Figure PCTCN2022120817-appb-100004
    其中，X、Y、Z、R、T、A以及X 10的定义如专利申请PCT/US2016/062114，公开号WO201708 7428A1(对应中国申请号2016800200132，公开号CN108136214A)中的权利要求书所记载；
    Figure PCTCN2022120817-appb-100005
    其中：
    A是取代或未经取代的C6-C10的芳基、联芳基或取代的联芳基、5-15元的杂芳基或-N＝CR 1R 2，其中取代时的取代基选自由以下组成的群：卤基、-CN、-NO 2、–O-(CH 2)-O-、-CO 2H及其盐、-OR 100、-CO 2R 100、-CONR 101R 102、-NR 101R 102、-NR 100SO 2R 100、-SO 2R 100、-SO 2NR 101R 1 02、C1-C6烷基、C3-C10杂环基；
    其中，R 100、R 101及R 102各自独立是氢、C1-C8烷基、C6-C12芳基；或R 101及R 102与其附接至的氮原子一起形成5-7元杂环；
    其中烷基及芳基各自是经1-3个卤基或1-3个C1-C6烷基取代；
    R 1及R 2各自独立是苯基或甲基；
    X、Y及Z各自独立是氢或卤基；
    R是氢或C1-C6烷基或卤素取代烷基。
  3. 根据权利要求1所述的治疗方法，其中，所述癌症选自卵巢癌、乳腺癌、胰腺癌、输卵管癌、原发性腹膜癌、胃癌、前列腺癌、肝癌、结肠癌、直肠癌、肺癌、膀胱癌。
  4. 根据权利要求1或2所述的治疗方法，其中，KRAS突变选自KRAS-G12D突变、KRAS-G12V和KRAS-G12C突变，优选选自KRAS-G12D突变。
  5. 根据权利要求4所述的治疗方法，其中，所述突变的TMB(肿瘤基因突变负荷)水平为中。
  6. 根据权利要求1或2所述的治疗方法，其中，其他治疗药物选自KRAS抑制剂、免疫治疗药物，KRAS抑制剂选自Sotorasib(AMG510)、adagrasib(MRTX849)、GDC6036、LY3499446、JNJ74699157(ARS3248)、D-1553，免疫治疗药物选自PD-1单抗、PD-L1单抗。
  7. 根据权利要求2所述的治疗方法，其中，式(1)(2)的化合物选自：
    Figure PCTCN2022120817-appb-100006
    Figure PCTCN2022120817-appb-100007
    Figure PCTCN2022120817-appb-100008
    式(3)的化合物选自：
    Figure PCTCN2022120817-appb-100009
    Figure PCTCN2022120817-appb-100010
    Figure PCTCN2022120817-appb-100011
    Figure PCTCN2022120817-appb-100012
    Figure PCTCN2022120817-appb-100013
    Figure PCTCN2022120817-appb-100014
    Figure PCTCN2022120817-appb-100015
    Figure PCTCN2022120817-appb-100016
    Figure PCTCN2022120817-appb-100017
    Figure PCTCN2022120817-appb-100018
    Figure PCTCN2022120817-appb-100019
    Figure PCTCN2022120817-appb-100020
    式(4)的化合物选自：
    Figure PCTCN2022120817-appb-100021
    式(5)的化合物选自：
    Figure PCTCN2022120817-appb-100022
    Figure PCTCN2022120817-appb-100023
    Figure PCTCN2022120817-appb-100024
    Figure PCTCN2022120817-appb-100025
    Figure PCTCN2022120817-appb-100026
    Figure PCTCN2022120817-appb-100027
    Figure PCTCN2022120817-appb-100028
    Figure PCTCN2022120817-appb-100029
    Figure PCTCN2022120817-appb-100030
    及
    Figure PCTCN2022120817-appb-100031
    式(6)的化合物选自：
    Figure PCTCN2022120817-appb-100032
    Figure PCTCN2022120817-appb-100033
    Figure PCTCN2022120817-appb-100034
    以及
    Figure PCTCN2022120817-appb-100035
    Figure PCTCN2022120817-appb-100036
    Figure PCTCN2022120817-appb-100037
    Figure PCTCN2022120817-appb-100038
    Figure PCTCN2022120817-appb-100039
    Figure PCTCN2022120817-appb-100040
    Figure PCTCN2022120817-appb-100041
    Figure PCTCN2022120817-appb-100042
    Figure PCTCN2022120817-appb-100043
    Figure PCTCN2022120817-appb-100044
    及
    Figure PCTCN2022120817-appb-100045
  8. AKR1C3活化的DNA烷化剂前药化合物的制药用途，该化合物用于制备单药或联用其他治疗药物治疗KRAS突变的癌症、肿瘤患者的药物。
  9. 根据权利要求8所述的制药用途，其中，所述化合物选自结构式1/2/3/4/5/6及其盐、酯、溶剂合物、同位素异构体：
    Figure PCTCN2022120817-appb-100046
    其中，R 1、R 2、R 3、R 4、R 5、R 8、R 9、R 10的定义如专利申请PCT/CN2020/089692，公开号WO2020228685A1中的权利要求书所记载；
    Figure PCTCN2022120817-appb-100047
    其中，A、E、G、X、Y的定义如专利申请PCT/NZ2019/050030，公开号WO2019190331A1(对应中国申请号2019800234236，公开号CN111918864A)中的权利要求书所记载；
    Figure PCTCN2022120817-appb-100048
    其中，Rw的定义如专利申请PCT/CN2020/120281，公开号WO2021068952A1中的权利要求书所记载；
    Figure PCTCN2022120817-appb-100049
    其中，X、Y、Z、R、T、A以及X 10的定义如专利申请PCT/US2016/062114，公开号WO2017087428A1(对应中国申请号2016800200132，公开号CN108136214A)中的权利要求书所记载；
    Figure PCTCN2022120817-appb-100050
    其中：
    A是取代或未经取代的C6-C10的芳基、联芳基或取代的联芳基、5-15元的杂芳基或-N＝CR 1R 2，其中取代时的取代基选自由以下组成的群：卤基、-CN、-NO 2、–O-(CH 2)-O-、-CO 2H及其盐、-OR 100、-CO 2R 100、-CONR 101R 102、-NR 101R 102、-NR 100SO 2R 100、-SO 2R 100、-SO 2NR 101R 1 02、C1-C6烷基、C3-C10杂环基；
    其中，R 100、R 101及R 102各自独立是氢、C1-C8烷基、C6-C12芳基；或R 101及R 102与其附接至的氮原子一起形成5-7元杂环；
    其中烷基及芳基各自是经1-3个卤基或1-3个C1-C6烷基取代；
    R 1及R 2各自独立是苯基或甲基；
    X、Y及Z各自独立是氢或卤基；
    R是氢或C1-C6烷基或卤素取代烷基。
  10. 根据权利要求8所述的制药用途，其中，所述癌症选自卵巢癌、乳腺癌、胰腺癌、输卵管癌、原发性腹膜癌、胃癌、前列腺癌、肝癌、结肠癌、直肠癌、肺癌、膀胱癌。
  11. 根据权利要求8或9所述的制药用途，其中，KRAS突变选自KRAS-G12D突变、KRAS-G12V和KRAS-G12C突变，优选选自KRAS-G12D突变。
  12. 根据权利要求11所述的制药用途，其中，所述突变的TMB(肿瘤基因突变负荷)水平为中。
  13. 根据权利要求8或9所述的制药用途，其中，其他治疗药物选自KRAS抑制剂、免疫治疗药物，KRAS抑制剂选自Sotorasib(AMG510)、adagrasib(MRTX849)、GDC6036、LY3499446、JNJ74699157(ARS3248)、D-1553，免疫治疗药物选自PD-1单抗、PD-L1单抗。
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