Combinations of deoxycytidine derivatives and PARP inhibitors for use in methods of treating HR-preserving cancers

Abstract

The present invention relates to a pharmaceutical combination for use in a method of treating HR-preserving cancer, comprising a PARP inhibitor and a compound of formula (I): The present invention relates to pharmaceutical combinations comprising JPEG2026522455000053.jpg17094 or its stereoisomers, solvates, tautomers, or pharmaceutically acceptable salts (wherein X, Y, Z, W1, W2, R1, R2, and R3 are as defined herein and in this disclosure). The present invention also relates to a method for treating HR-deficient cancer, comprising the step of administering a pharmaceutical combination to a subject in need thereof, as well as pharmaceutical compositions and kits comprising such pharmaceutical combinations.

Description

Technical Field

[0001] The present invention relates to a combination of a compound of formula (I) as defined herein and a PARP inhibitor in the treatment of cancer that retains its function in homologous recombination (HR).

Background Art

[0002] Cancer is a disease characterized by the loss of proper control of cell growth and proliferation. The American Cancer Society estimates that in 2022, there were over 1.9 million new cancer cases in the United States and approximately 600,000 deaths that year that were estimated to be caused by cancer. The World Health Organization estimates that cancer was the leading cause of death globally in 2010 and that the number of deaths caused by cancer will increase to 12 million annually by 2030.

[0003] A number of treatment methods are available, but additional cancer treatment methods are still needed. Resistance to known anticancer agents can be a problem in the success of cancer treatment in patients. Improved solutions to the problems of treating cancer and drug-resistant cancer are still needed in the art.

[0004] WO 2020 / 157335 discloses compounds for use in the treatment of cancer.

[0005] WO 2021 / 048235 discloses that the combination of a PARP inhibitor and hmdU sensitizes HR-deficient cancer cells to the PARP inhibitor.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

[0007] [Non-Patent Document 1] "Comprehensive Organic Synthesis" by B.M. Trost and I. Fleming, Pergamon Press, 1991. [Non-Patent Document 2] "Protective Groups in Organic Synthesis," 3rd edition, TW Greene and PGM Wutz, Wiley-Interscience (1999) [Non-Patent Document 3] Vyas and Change (2014) Nat Rev Cancer 14(7):502~509 [Non-Patent Document 4] Ame et al. (2004), Bioessays 26:882-893 [Non-Patent Document 5] Shen et al. (2013) Clin Cancer Res 19(18)5003-5015 [Non-Patent Document 6] The chapter "Cell Viability Assays" by editors Markossian S, Grossman A, Brimacombe K, et al., is available in the Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company [Non-Patent Document 7] National Center for Advancing Translational Sciences;2004-https: / / www.ncbi.nlm.nih.gov / books / NBK53196 / [Non-licensed document 8] Banasik J. Biol. Chem., 267:3, 1569-75, 1992

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

[0008] The inventors have discovered that combinations of selected class compounds with PARP inhibitors exhibit synergistic activity in the treatment of HR-preserving cancers, i.e., cancers that are not HR-deficient. Such combination therapies are generally useful in inhibiting the proliferation of HR-preserving cancer cells and are therefore useful in the treatment of HR-preserving cancers.

[0009] The combination of the present invention has been found to be more effective than the predicted additive sum of each component. This synergistic interaction makes the combination more effective against cancer. This synergy can result in higher activity and / or lower doses of the active ingredients. Beneficially, lower doses of the active ingredients can reduce side effects and save costs. Furthermore, drug-resistant cancers, particularly cancers resistant to any of the components as monotherapy, can be treated more effectively. [Means for solving the problem]

[0010] In a first aspect of the present invention, a pharmaceutical combination for use in a method of treating HR function-preserving cancer comprises a PARP inhibitor and a compound of formula (I):

[0011] [ka]

[0012] or its solvates, tautomers, or pharmaceutically acceptable salts. (In the formula, X is a group containing 1 to 20 nonhydrogen atoms and containing at least one functional group selected from aldehydes, alcohols, protected alcohols, ethers, anhydrides, esters, and carboxylic acids; W1 and W2 are independently O, S, or NH; Y is a group containing H or 1 to 15 non-hydrogen atoms; Z is -N(R x R y ) and here R x and R y It is independently a group containing H or 1 to 10 non-hydrogen atoms; R1 is a group containing H or 1 to 15 non-hydrogen atoms; R2 is H, -OH, -OPG, -F, -Cl, -Br, -I, or -N3; R3 is H, -F, -Cl, -Br, -I, or -N3; PG is an alcohol protecting group such as acetyl (Ac), benzyl (Bn), or benzoyl (Bz). A combination of pharmaceuticals including the above is provided.

[0013] Alternatively, a pharmaceutical combination is provided for use in treating cancers that are not HR-deficient, comprising a PARP inhibitor and a compound of formula (I).

[0014] Alternatively, PARP inhibitors and compounds of formula (I) are provided for use in methods of treating cancers that retain HR function. Alternatively, the cancer is not HR deficient.

[0015] Alternatively, the present invention provides a method for treating target HR function-preserving cancer, comprising the step of administering a pharmaceutical combination containing a PARP inhibitor and a compound of formula (I) to the target.

[0016] Alternatively, the present invention provides a method for treating target HR function-preserving cancer, comprising the step of administering a PARP inhibitor and a compound of formula (I) to the target.

[0017] Alternatively, the present invention provides a method for treating cancer that is not HR-deficient, comprising the step of administering a pharmaceutical combination comprising a PARP inhibitor and a compound of formula (I) to the target.

[0018] Alternatively, the present invention provides a method for treating cancers that are not HR-deficient, comprising the step of administering a PARP inhibitor and a compound of formula (I) to the target.

[0019] Alternatively, the use of a PARP inhibitor in the manufacture of a pharmaceutical product is provided for use in a method for treating target HR-preserving cancer, which includes the step of administering a pharmaceutical combination comprising a PARP inhibitor and a compound of formula (I) to the target. Alternatively, the cancer is not HR-deficient.

[0020] Alternatively, the use of a compound of formula (I) in the manufacture of a pharmaceutical for use in a method for treating target HR-preserving cancer, which includes the step of administering a pharmaceutical combination comprising a PARP inhibitor and a compound of formula (I) to the target. Alternatively, the cancer is not HR-deficient.

[0021] Alternatively, the use of compounds of formula (I) and PARP inhibitors (or drug combinations thereof) in the manufacture of pharmaceuticals for use in treating target HR-preserving cancers is provided. Alternatively, cancer is not HR-deficient.

[0022] In a further embodiment, a compound of formula (I) is provided for use in the treatment of HR-preserving cancer, wherein the compound of formula (I) is administered together with a PARP inhibitor. In other words, the cancer is not HR-deficient.

[0023] Alternatively, a method is provided for treating a target HR-preserving cancer, comprising the step of administering a compound of formula (I) to the target, wherein the target is receiving a PARP inhibitor. Alternatively, the cancer is not HR-deficient.

[0024] In a further embodiment, a PARP inhibitor is provided for use in the treatment of HR-preserving cancer, which is administered together with a compound of formula (I). In other words, the cancer is not HR-deficient.

[0025] Alternatively, a method is provided for treating a target HR-preserving cancer, comprising the step of administering a PARP inhibitor to the target, wherein the target is receiving a compound of formula (I). Alternatively, the cancer is not HR-deficient.

[0026] The administration of PARP inhibitors and the administration of compounds of formula (I) may be carried out separately, sequentially, in any order, in parallel, or simultaneously.

[0027] In a further embodiment, a PARP inhibitor is provided for use in a method of sensitizing a target HR-preserving cancer to treatment with a compound of formula (I). In other words, the cancer is not HR-deficient. Administration of the PARP inhibitor to the target sensitizes the HR-preserving cancer to treatment with a compound of formula (I).

[0028] Alternatively, the present invention provides a method for sensitizing a target HR-preserving cancer to treatment with a compound of formula (I), the method comprising the step of administering a PARP inhibitor to the target. Alternatively, the cancer is not HR-deficient. Administration of a PARP inhibitor to the target sensitizes the HR-preserving cancer to treatment with a compound of formula (I).

[0029] Alternatively, the use of PARP inhibitors in the manufacture of pharmaceuticals for use in a method of sensitizing target HR-deficient cancers to treatment with a compound of formula (I) is provided. Alternatively, the cancer is not HR-deficient.

[0030] In a further embodiment, a compound of formula (I) is provided for use in a method of sensitizing a target HR-preserving cancer to treatment with a PARP inhibitor. In other words, the cancer is not HR-deficient. Administration of the compound of formula (I) to the target sensitizes the HR-preserving cancer to treatment with a PARP inhibitor.

[0031] In a further embodiment, a method is provided for sensitizing a target HR-preserving cancer to treatment with a PARP inhibitor, the method comprising the step of administering a compound of formula (I) to the target. In other words, the cancer is not HR-deficient. Administration of a compound of formula (I) to the target sensitizes the HR-preserving cancer to treatment with a PARP inhibitor.

[0032] Alternatively, the use of a compound of formula (I) in the manufacture of a pharmaceutical for use in a method of sensitizing target HR-deficient cancers to treatment with PARP inhibitors is provided. Alternatively, the cancer is not HR-deficient.

[0033] A method for sensitizing HR-preserving cancer may, in the context of any aspect of the present invention, include administering to a subject a pharmaceutical combination comprising a PARP inhibitor and a compound of formula (I), i.e., a pharmaceutical combination comprising one as a sensitizer and the other as a drug to be sensitized. The administration of the PARP inhibitor and the compound of formula (I) may be carried out separately, sequentially, in any order, in parallel, or simultaneously. In embodiments, the sensitizer may be administered before, in parallel with, or simultaneously with the drug to be sensitized.

[0034] Simultaneous administration means the administration of two compounds of a single dosage form (i.e., two active ingredients / activators, i.e., a PARP inhibitor and a compound of formula (I)); parallel administration means the administration of two compounds of separate dosage forms at approximately the same time; and sequential administration means that one compound is administered, followed by the other. Sequential and / or separate administration can also take the form of simultaneous or parallel administration of two compounds, followed by discontinuation of simultaneous or parallel administration, and then continued administration of one of the two compounds alone. Separate administration should be understood as meaning that the two compounds are administered separately, for example, at different times of the day, or on different days, and according to different treatment regimens, while sequential administration means that the compounds are administered one after the other in any order.

[0035] In all embodiments, the compound of formula (I) and the PARP inhibitor can be co-formulated in a single composition. However, this is not necessary; they may be formulated separately and administered separately, sequentially, in parallel, or simultaneously in any order.

[0036] In a further embodiment, a pharmaceutical composition comprising a PARP inhibitor and a compound of formula (I) is provided.

[0037] In a further embodiment, a pharmaceutical composition is provided comprising a PARP inhibitor and a compound of formula (I) for use in a method of treating HR-preserving cancer (or, in other words, cancer that is not HR-deficient).

[0038] In a further embodiment, a method is provided for treating a target HR function-preserving cancer (or, in other words, a cancer that is not HR-deficient), comprising the step of administering a pharmaceutical composition comprising a PARP inhibitor and a compound of formula (I) to the target.

[0039] Depending on the circumstances, the composition may further include one or more pharmaceutically acceptable excipients.

[0040] In a further embodiment, a product, preferably a pharmaceutical composition, is provided comprising a PARP inhibitor and a compound of formula (I) as a combination preparation for separate, sequential, parallel, or concurrent use in the treatment of cancers that preserve HR function. In other words, cancer is not HR deficiency. The product may further comprise one or more pharmaceutically acceptable excipients.

[0041] Furthermore, products, particularly pharmaceuticals, comprising a PARP inhibitor co-formulated with a compound of formula (I) are provided. The product may further comprise one or more pharmaceutically acceptable excipients. The product is also provided for use in methods of treating HR-preserving cancers (or, in other words, cancers that are not HR-deficient).

[0042] In a further embodiment, a kit is provided comprising a PARP inhibitor and a compound of formula (I). Preferably, the kit is for treating HR-preserving cancer (in other words, cancer that is not HR-deficient). Preferably, the kit is for use in treating HR-preserving cancer (in other words, cancer that is not HR-deficient).

[0043] Each of the compounds of formula (I) and the PARP inhibitor in the kit of the present invention may be provided in separate compartments or containers. Where convenient and practical, a mixture of the components may be provided. The components may be provided in a dry form, e.g., a crystalline form, a freeze-dried form or a lyophilized form, or in a solution, typically such a liquid composition being aqueous and buffered with a standard buffer such as Tris or HEPES.

[0044] The kit may be for the separate, sequential, parallel, or simultaneous use of the compound of formula (I) and a PARP inhibitor in the treatment of HR-preserving cancer. The kit preferably includes instructions for the use of the components in the treatment of HR-preserving cancer.

[0045] In all aspects of the present invention, each of the compounds of formula (I) and the PARP inhibitors may be as described elsewhere in this specification, and preferred optional embodiments of the compounds described in one aspect of the present invention may be applied to any other aspect of the present invention with necessary modifications. [Modes for carrying out the invention]

[0046] X X is a group containing 1 to 20 non-hydrogen atoms and containing at least one functional group selected from aldehydes, alcohols, protected alcohols, ethers, anhydrides, esters, and carboxylic acids. Preferably, X is not -COOH. Preferably, X is not -OH. Preferably, X is neither -COOH nor -OH.

[0047] Preferably, X is a group containing 1 to 10 nonhydrogen atoms, more preferably 1 to 5 nonhydrogen atoms, even more preferably 1 to 3 nonhydrogen atoms, and most preferably 2 nonhydrogen atoms.

[0048] More preferably, X is a group containing at least two non-hydrogen atoms, i.e., a group containing 2 to 20 non-hydrogen atoms. Therefore, preferably, X is a group containing 2 to 10 non-hydrogen atoms, more preferably 2 to 5 non-hydrogen atoms, even more preferably 2 to 3 non-hydrogen atoms, and most preferably 2 non-hydrogen atoms.

[0049] Preferably, X contains at least one functional group selected from aldehydes, alcohols, protected alcohols, ethers, anhydrides, and esters. More preferably, X contains at least one functional group selected from aldehydes, alcohols, ethers, and esters. Most preferably, X contains at least one functional group selected from aldehydes and alcohols. For example, X preferably contains an aldehyde functional group. For example, X preferably contains an alcohol functional group.

[0050] Preferably, X contains only one functional group.

[0051] X is -LX' (In the formula, L is a bond, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, or haloalkoxy; X' is -CHO, -OH, -OPG, -COOH, -OR, -OC(=O)R, -C(=O)-OC(=O)-R, or -C(=O)OR, PG is an alcohol protecting group such as acetyl (Ac), benzyl (Bn), or benzoyl (Bz), and R is an alkyl group, preferably methyl. It can be defined as follows.

[0052] The term "alkyl" refers to linear and branched saturated aliphatic hydrocarbon chains. Preferably, alkyl is C 1~10Refers to alkyl. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl).

[0053] R can be any alkyl group such as those exemplified above. For example, R is -(CH2) n is H, where n can be 1 to 10, preferably 1 to 5, more preferably 1 to 3, and most preferably 1. When n is 1, R is CH3.

[0054] The term "alkenyl" refers to straight-chain and branched hydrocarbon chains having one or more, preferably one or two carbon-carbon double bonds. Preferably, alkenyl is C 2~10 Refers to alkenyl. Examples of alkenyl groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, and 4-methyl-3-pentenyl.

[0055] The term "alkynyl" refers to straight-chain and branched hydrocarbon chains having one or more, preferably one or two carbon-carbon triple bonds. Preferably, alkynyl is C 2~10 Refers to alkynyl. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, and propargyl.

[0056] The term "haloalkyl" refers to straight-chain and branched saturated aliphatic hydrocarbon chains substituted with one or more halogens (fluoro (F), chloro (Cl), bromo (Br), and iodo (I)). Preferably, haloalkyl is C 1~10This refers to haloalkyl groups. Examples of haloalkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2,2,2-trifluoroethyl, heptafluoropropyl, and heptachloropropyl.

[0057] The term "alkoxy" refers to an -O-alkyl group. Preferably, the alkoxy is C 1~10 This refers to alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), and t-butoxy.

[0058] The term "haloalkoxy" refers to the haloalkyl group defined above, bonded through oxygen crosslinking. Preferably, the haloalkoxy is C 1~10 This refers to haloalkoxy groups. Examples of haloalkoxy groups include, but are not limited to, trifluoromethoxy, 2,2,2-trifluoroethoxy, and pentafluoroethoxy.

[0059] If X is -L-X', the X group must still contain the required number of non-hydrogen atoms.

[0060] Preferably, L is a bond, alkyl, alkenyl or alkynyl, more preferably a bond or alkyl. For example, L is a bond or C 1~6 It may be an alkyl group. More preferably, L is a bond or C 1~4 It is alkyl. Most preferably, L is a bond or C1 alkyl(-CH2-).

[0061] X' is preferably -CHO, -OH, -OPG, -OR, -OC(=O)R, C(=O)-OC(=O)-R or -C(=O)OR, more preferably -CHO, -OH, -OR, -OC(=O)R, C(=O)-OC(=O)-R or -C(=O)OR, most preferably -CHO, -OH, -OR or -OC(=O)R.

[0062] Therefore, preferably, X is -(CH2) n -X', where n is 0 to 6, preferably 0 to 4, more preferably 0 or 1, and X' is as defined above, preferably -CHO, -OH, -OR, or -OC(=O)R, where R is as defined above.

[0063] Preferably, X is -(CH2) n -X', where n is 0 to 6, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is 0 to 6, and X' is -OH. Preferably, X is -(CH2) n It is -X', where n is 0 to 6, and X' is -CHO.

[0064] More preferably, X is -(CH2) n -X', where n is 0 to 4, and X' is -OH or -CHO. Preferably, X is -(CH2) n It is -X', where n is 0 to 4, and X' is -OH. Preferably, X is -(CH2) n It is -X', where n is 0 to 4, and X' is -CHO.

[0065] More preferably, X is -(CH2) n -X', where n is 0 to 2, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is between 0 and 2, and X' is -OH. Preferably, X is -(CH2). n It is -X', where n is 0 to 2, and X' is -CHO.

[0066] More preferably, X is -(CH2) n -X', where n is 0 or 1, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is 0 or 1, and X' is -OH. Preferably, X is -(CH2) n-X', where n is 0 or 1, and X' is -CHO.

[0067] More preferably, X is -CHO, -CH2OH, -CH2OCH3, or -CH2OC(=O)CH3.

[0068] More preferably, X is -CHO or -CH2OH.

[0069] In all of the above definitions of X, it is preferable that X is not -COOH or not -OH. And preferably, X is neither -COOH nor -OH. Therefore, if X' is -OH or -COOH, it is preferable that L is not a bond (i.e., n is not 0). In this case, n can be 1 to 6, preferably 1 to 4, more preferably 1 to 2, and most preferably 1.

[0070] W1 and W2 W1 and W2 are each independently O, S, or NH, preferably O or S, and more preferably O.

[0071] Therefore, preferably, W1 is O or S and W2 is O, S or NH; or W2 is O or S and W1 is O, S or NH.

[0072] More preferably, both W1 and W2 are O or S, and even more preferably, W1 is O and W2 is O or S; or W2 is O and W1 is O or S.

[0073] Most preferably, both W1 and W2 are O.

[0074] Y Y is a group containing H or 1 to 15 non-hydrogen atoms. Preferably, Y is a group containing H or 1 to 10 non-hydrogen atoms. More preferably, Y is a group containing H or 1 to 5 non-hydrogen atoms.

[0075] For example, Y can be H, -OH, -OPG, -F, -Cl, -Br, -I, -SH, or -N3, and PG is an alcohol protecting group such as acetyl, benzyl, or benzoyl.

[0076] If Y is a group containing H or 1 to 5 non-hydrogen atoms, Y may be H, -OH, -OAc, -F, -Cl, -Br, -I, -SH, or -N3.

[0077] Most preferably, Y is H.

[0078] Z Z is -N(R x R y ) and here R x and R y It is independently a group containing H or 1 to 10 non-hydrogen atoms.

[0079] R x and R y One or both of these may be amine protecting groups such as acetyl, benzyl, or benzoyl.

[0080] Preferably, R x and R y H or C 1~8 It is an ester. More preferably, R x and R y These are independently H or -C(O)O(CH2) n CH3, where n is 1 to 4, preferably 4.

[0081] Preferably, R x and R y At least one of them is H. For example, preferably R x H is R y These are independently H or -C(O)O(CH2) n CH3, where n is 1 to 4, preferably 4. More preferably R x and R y Both are H.

[0082] Therefore, Z is preferably -NH2.

[0083] R1 R1 is a group containing H or 1 to 15 non-hydrogen atoms, preferably a group containing H or 1 to 13 non-hydrogen atoms.

[0084] Preferably, R1 is H, -OH, -OPG, -F, -Cl, -Br, -I, -SH, -N3, or -A1(A2(=A3)(OH)O) n H is H, where n is 1 to 3, and in each case A1 is O, CH2 or NH, A2 is P or S, and A3 is O or S; PG is an alcohol protecting group such as acetyl, benzyl or benzoyl.

[0085] In some cases, A1 is O, A2 is P, and A3 is O; A1 is O, A2 is S, and A3 is O; A1 is O, A2 is P, and A3 is S; A1 is NH, A2 is P, and A3 is O; or A1 is CH2, A2 is P, and A3 is O.

[0086] Preferably, R1 is H, -OH, -F, -Cl, -Br, -I, -N3, or -A1(A2(=A3)(OH)O) n H (for example, -O(P(=O)(OH)O) n H) where n is 1 to 3, preferably 3. More preferably, R1 is H, -OH, or -A1(A2(=A3)(OH)O) n H (for example, -O(P(=O)(OH)O) n H) is where n is 1 to 3, preferably 3. More preferably R1 is -OH or -O(P(=O)(OH)O) n H is H, where n is 1 to 3, preferably 3. Most preferably, R1 is -OH.

[0087] R2 R2 is H, -OH, -OPG, -F, -Cl, -Br, -I, or -N3, and PG is an alcohol protecting group such as acetyl, benzyl, or benzoyl.

[0088] Preferably, R2 is H, -OH, -F, -Cl, -Br, -I, or -N3. More preferably, R2 is H or -OH, and most preferably, R2 is -OH.

[0089] R3 R3 is H, -F, -Cl, -Br, -I, or -N3, preferably H.

[0090] Most preferably, R1 is -OH or -O(P(=O)(OH)O) n R2 is H, where n is 1 to 3, preferably 3; R2 is -OH; and R3 is H.

[0091] Preferred Embodiment Preferably, the compound of formula (I) is the compound of formula (II), or its solvate, tautomer, or pharmaceutically acceptable salt:

[0092] [ka]

[0093] (In the formula, X, R1, and R2 are as defined above.) That is the case.

[0094] In this preferred embodiment, preferably X is -(CH2) n -X', where n is 0 to 6, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is 0 to 6, and X' is -OH. Preferably, X is -(CH2) n It is -X', where n is 0 to 6, and X' is -CHO.

[0095] More preferably, X is -(CH2) n -X', where n is 0 to 4, and X' is -OH or -CHO. Preferably, X is -(CH2) nIt is -X', where n is 0 to 4, and X' is -OH. Preferably, X is -(CH2) n It is -X', where n is 0 to 4, and X' is -CHO.

[0096] More preferably, X is -(CH2) n -X', where n is 0 to 2, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is between 0 and 2, and X' is -OH. Preferably, X is -(CH2). n It is -X', where n is 0 to 2, and X' is -CHO.

[0097] More preferably, X is -(CH2) n -X', where n is 0 or 1, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is 0 or 1, and X' is -OH. Preferably, X is -(CH2) n -X', where n is 0 or 1, and X' is -CHO.

[0098] More preferably, X is -CHO, -CH2OH, -CH2OCH3, or -CH2OC(=O)CH3. More preferably, X is -CHO or -CH2OH.

[0099] In all of the above definitions of X, it is preferable that X is not -COOH or not -OH. Preferably, X is neither -COOH nor -OH. Therefore, if X' is -OH or -COOH, it is preferable that n is not 0. In this case, n can be 1 to 6, preferably 1 to 4, more preferably 1 to 2, and most preferably 1.

[0100] In formula (II), preferably, R1 is -OH or -O(P(=O)(OH)O) n H is H, where n is 1 to 3, preferably 3; and R2 is -OH.

[0101] More preferably, the compound of formula (I) is a compound of formula (IIIa) or (IIIb), or a solvate, tautomer, or pharmaceutically acceptable salt thereof:

[0102] [ka]

[0103] (In the formula, X is as defined above.) That is the case.

[0104] More preferably, the compound of formula (I) is the compound of formula (IIIa), or a solvate, tautomer, or pharmaceutically acceptable salt thereof.

[0105] Formulas (IIIa) and (IIIb), in particular formula (IIIa), preferably X is -(CH2) n -X', where n is 0 to 6, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is 0 to 6, and X' is -OH. Preferably, X is -(CH2) n It is -X', where n is 0 to 6, and X' is -CHO.

[0106] More preferably, X is -(CH2) n -X', where n is 0 to 4, and X' is -OH or -CHO. Preferably, X is -(CH2) n It is -X', where n is 0 to 4, and X' is -OH. Preferably, X is -(CH2) n It is -X', where n is 0 to 4, and X' is -CHO.

[0107] More preferably, X is -(CH2) n -X', where n is 0 to 2, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is between 0 and 2, and X' is -OH. Preferably, X is -(CH2). nIt is -X', where n is 0 to 2, and X' is -CHO.

[0108] More preferably, X is -(CH2) n -X', where n is 0 or 1, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is 0 or 1, and X' is -OH. Preferably, X is -(CH2) n -X', where n is 0 or 1, and X' is -CHO.

[0109] More preferably, X is -CHO, -CH2OH, -CH2OCH3, or -CH2OC(=O)CH3. More preferably, X is -CHO or -CH2OH.

[0110] In all of the above definitions of X, it is preferable that X is not -COOH or not -OH. Preferably, X is neither -COOH nor -OH. Therefore, if X' is -OH or -COOH, it is preferable that n is not 0. In this case, n can be 1 to 6, preferably 1 to 4, more preferably 1 to 2, and most preferably 1.

[0111] Most preferably, the compound of formula (I) is a compound of formula (IVa), (IVb), (IVc), or (IVd), or a solvate, tautomer, or pharmaceutically acceptable salt thereof:

[0112] [ka]

[0113] That is the case.

[0114] Formula (IVa) is 5-formyl-2'-deoxycytidine (also known herein as 5f2dC, 5fdC, 2d5fC and d5fC). Formula (IVb) is 5-hydroxymethyl-2'-deoxycytidine (also known herein as 5hm2dC, 5hmdC, 2d5hmC and d5hmC). Formula (IVc) is 5-formyl-2'-deoxycytidine-5'-triphosphate. Formula (IVd) is 5-hydroxymethyl-2'-deoxycytidine-5'-triphosphate.

[0115] Therefore, preferably, compounds useful in the present invention are selected from 5-formyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, 5-formyl-2'-deoxycytidine-5'-triphosphate and 5-hydroxymethyl-2'-deoxycytidine-5'-triphosphate or their solvates, tautomers or pharmaceutically acceptable salts.

[0116] Most preferably, the compound is 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine or its solvates, tautomers, or pharmaceutically acceptable salts.

[0117] The compound of formula (I) of the present invention preferably has the stereochemistry shown below:

[0118] [ka]

[0119] (In the formula, X, Y, Z, W1, W2, R1, R2, and R3 are as defined above.) It holds.

[0120] Compounds of formula (I) useful in the present invention (i.e., compounds of formulas (I), (II), (IIIa), (IIIb), (IVa), (IVb), (IVc), and (IVd)) are commercially available, known in the literature, or can be obtained from available starting materials by conventional synthetic procedures in accordance with standard techniques, using appropriate reagents and reaction conditions. In this regard, those skilled in the art can refer, in particular, to "Comprehensive Organic Synthesis" by BM Trost and I. Fleming, Pergamon Press, 1991, and "Protective Groups in Organic Synthesis," 3rd edition, by TW Greene and PGM Wutz, Wiley-Interscience (1999). These compounds are also commercially available, for example, from Berry and Associates, Toronto Research Chemicals, Sigma Aldrich, Carbosynth, Trilink Biotech, and other well-known commercial suppliers.

[0121] PARP inhibitors As used herein, the term PARP refers to poly(ADP-ribose) polymerase (PARP), a family of related enzymes that share the ability to catalyze the transfer of ADP-ribose to target proteins. Among the PARP family, PARP-1 is the most well-studied target for cancer treatment, but all members of the family, including PARP-2 and PARP-5a, are potential cancer treatment targets (Vyas and Change (2014) Nat Rev Cancer 14(7):502-509). PARP inhibitors useful in the present invention may be inhibitors of any PARP, i.e., any member of the enzymes of the PARP family, as disclosed, for example, in Ame et al. (2004), Bioessays 26: 882-893.

[0122] Inhibitors of PARP1 (EC2.4.2.30, Genbank number: M32721; gene ID 142) are particularly preferred. PARP1 may have the reference amino acid sequence or a variant thereof of database accession number NP_001609.2, and may be encoded by the nucleotide sequence or a variant thereof of NM_001618.4.

[0123] Poly[ADP-ribose] polymerase 1 (PARP-1) is an enzyme encoded by the PARP-1 gene in humans. PARP-1 is thought to be involved in the repair of ssDNA through the base excision repair (BER) mechanism. PARP-1 inhibition is thought to lead to the accumulation of ssDNA lesions, which halt replication forks and ultimately lead to the accumulation of DNA double-strand breaks (DSBs). Repair of these DSBs is important for cell viability and may depend on processes such as non-homologous end joining (NHEJ), alternative form of NHEJ - alternative end joining (AEJ), and homologous recombination repair (HRR). PARP-1 may also be involved in AEJ, which functions as a backup for the NHEJ process.

[0124] Therefore, when used herein, the terms "PARP inhibitor" or "PARPi" preferably refer to PARP-1 inhibitors.

[0125] As used herein, the terms “PARP inhibitor” or “PARPi” refer to compounds that inhibit the expression level or biological activity of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP). A suitable PARP inhibitor may selectively inhibit PARP (preferably PARP-1) with an IC50 of less than 20 nM, less than 10 nM, less than 5 nM, or less than 2 nM in a cell-free assay (Shen et al. (2013) Clin Cancer Res 19(18)5003-5015). A suitable PARP inhibitor may selectively inhibit PARP (preferably PARP-1) in cancer cell lines, leading to cell death, with an IC50 value of less than 100 μM, less than 10 μM, less than 1 μM, less than 100 nM, less than 10 nM, less than 5 nM, or less than 2 nM in a cell viability assay. Such assays are widely known in the art, and any suitable assay may be used. For example, a cell-based assay used to determine cell death by IC50 value could be the MTT cell proliferation assay described in the Assay Guidance Manual [Internet], chapter "Cell Viability Assays" by editors Markossian S, Grossman A, Brimacombe K, et al., available from Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004, https: / / www.ncbi.nlm.nih.gov / books / NBK53196 / . Alternatively, a cell viability assay may include immunostaining of a proliferation marker (e.g., KI67) and / or DNA synthesis measurement (e.g., 3H-dT labeling) to assess cell viability, and such techniques are well known and routinely used in the art.

[0126] Suitable assays for measuring PARP inhibition, including fluorescence and chemiluminescence assays, are well known in the art. For example, PARP inhibition can be measured by determining the inhibition of PARP-mediated NAD+ depletion by linking NAD+ levels to a cycling assay involving alcohol dehydrogenase and diaphorases that produce fluorescent molecules such as resolphins (see, for example, the Fluorescence Homogeneous PARP Inhibition Assay Kit, catalog number 4690-096-K, Trevigen Inc., Maryland, USA).

[0127] PARP inhibition can induce the formation of multiple double-strand breaks. Cancer cells lacking HR are known to be unable to efficiently repair these double-strand breaks, leading to cell death. Normal non-cancerous cells do not replicate DNA as frequently as cancer cells and generally possess functional HR, so normal cells survive PARP inhibition. Surprisingly, the inventors have found that a combination of a PARP inhibitor and the compound of formula (I) has a synergistic effect in treating HR-preserving cancers and can efficiently repair double-strand breaks. Surprisingly, the inventors have also found that a combination of a PARP inhibitor and the compound of formula (I) does not have a synergistic effect in treating HR-deficient cancers.

[0128] Numerous examples of compounds that inhibit PARP are known and may be used as described herein, including: 1. Nicotinamides, such as 5-methylnicotinamide and O-(2-hydroxy-3-piperidinopropyl)-3-carboxylic acid amidoxime, as well as their analogues and derivatives. 2.3-substituted benzamides, such as 3-aminobenzamide, 3-hydroxybenzamide, 3-nitrosobenzamide, 3-methoxybenzamide, and 3-chloroprocainamide, and 4-aminobenzamide, as well as 1,5-di[(3-carbamoylphenyl)aminocarbonyloxy]pentane, and their analogs and derivatives. 3,2H-isoquinoline-1-one, 3H-quinazoline-4-one, 5-substituted dihydroisoquinolinone, e.g., 5-hydroxydihydroisoquinolinone, 5-methyldihydroisoquinolinone, and 5-hydroxyisoquinolinone, 5-aminoisoquinoline-1-one, 5-dihydroxyisoquinolinone, 3,4-dihydroisoquinoline-1(2H)-one, e.g., 3,4-dihydro-5-methoxyisoquinoline-1(2H)-one and 3,4-dihydro- 5-methyl-1(2H)isoquinolinone, isoquinoline-1(2H)-one, 4,5-dihydroimidazo[4,5,1-ij]quinoline-6-one, 1,6-naphthyridin-5(6H)-one, 1,8-naphthalimide, e.g., 4-amino-1,8-naphthalimide, isoquinolinone, 3,4-dihydro-5-[4-1(1-piperidinyl)butoxy]-1(2H)-isoquinolinone, 2,3-dihydrobenzo[de]isoquinoline-1-one, 1-11 Isoquinolinones and dihydroisoquinolinones containing β-dihydro-[2H]benzopyrano[4,3,2-de]isoquinolin-3-one, and tetracyclic lactams containing benzopyranoisoquinolinone, such as benzopyrano[4,3,2-de]isoquinolinone, as well as their analogs and derivatives. 4. Beriparib, niraparib, benzoxazole-4-carboxamide, benzimidazole-4-carboxamide, e.g., 2-substituted benzoxazole-4-carboxamide and 2-substituted benzimidazole-4-carboxamide, e.g., 2-(4-hydroxyphenyl)benzimidazole-4-carboxamide, 2-arylbenzimidazole-4-carboxamide and 2-cycloalkylbenzimidazole-4-carboxamide, quinoxaline carboxamide, imidazopyridine carboxamide, 2-phenylindole, 2-substituted benzoxazole, e.g., 2-phenylbenzoxazole and 2-(3-methoxyphenyl)benzimidazole, 2-substituted benzimidazole, e.g., 2-phenylbenzimidazole and 2-(3-methoxyphenyl)benzimidazole Benzimidazoles, indazoles and indoles, as well as their analogs and derivatives, comprising 1,3,4,5-tetrahydro-azepino[5,4,3-cd]indole-6-one, azepinindole and azepinoidrone, for example 1,5-dihydro-azepino[4,5,6-cd]indorin-6-one and dihydrodiazapinoidrinone, 3-substituted dihydrodiazapinoidrinone, for example 3-(4-trifluoromethylphenyl)-dihydrodiazepinoidrinone, tetrahydrodiazapinoidrinone and 5,6-dihydroimidazo[4,5,1-j,k][1,4]benzodiazopine-7(4H)-one, 2-phenyl-5,6-dihydroimidazo[4,5,1-jk][1,4]benzodiazepine-7(4H)-one and 2,3-dihydro-isoindole-1-one. 5. Phthalazine-1(2H)-one and quinazolinones, e.g., olaparib, 4-hydroxyquinazoline, phthalazinone, 5-methoxy-4-methyl-1(2)phthalazinone, 4-substituted phthalazinone, 4-(1-piperazinyl)-1(2H)-phthalazinone, tetracyclic benzopyrano[4,3,2-de]phthalazinone and tetracyclic indeno[1,2,3-de]phthalazinone and 2-substituted quinazolines, e.g., 8-hydroxy-2-methylquinazoline-4-(3H)-one, tricyclic phthalazinone and 2-aminophthalhydrazide, as well as their analogs and derivatives, and 1(2H)-phthalazinone and its derivatives as described in International Publication No. 02 / 36576. 6. Isoindolinone and its analogs and derivatives. 7. Phenanthridines and phenanthridinenes, e.g., 5[H]phenanthridine-6-one, substituted 5[H]phenanthridine-6-one, especially 2-,3-substituted 5[H]phenanthridine-6-one and sulfonamide / carbamide derivatives of 6(5H)phenanthridine, thieno[2,3-c]isoquinolone, e.g., 9-aminothieno[2,3-c]isoquinolone and 9-hydroxythieno[2,3-c]isoquinolone, 9-methoxythieno[2,3-c]isoquinolone, and N-(6-oxo-5,6-dihydrophenanthridine-2-yl)-2-(N,N-dimethylamino)acetamide, substituted 4,9-dihydrocyclopenta[lmn]phenanthridine-5-one, and their analogues and derivatives. 8. Benzopyrrones, such as 1,2-benzopyrone, 6-nitrosobenzopyrone, 6-nitroso1,2-benzopyrone, and 5-iodo-6-aminobenzopyrone, as well as their analogues and derivatives. 9. Unsaturated hydroxy acid derivatives, such as 0-(3-piperidino-2-hydroxy-1-propyl)nicotinamidexime, and its analogs and derivatives. 10. Pyridazines, including condensed pyridazines, and their analogues and derivatives. 11. Other compounds, such as caffeine, theophylline, and thymidine, as well as their analogs and derivatives.

[0129] Additional suitable PARP inhibitors include, for example, International Publication No. 14201972, International Publication No. 14201972, International Publication No. 12141990, International Publication No. 10091140, International Publication No. 9524379, International Publication No. 09155402, International Publication No. 009046205, International Publication No. 08146035, International Publication No. 08015429, International Publication No. 0191796, International Publication No. 0042040, US Patent Application Publication No. 2006004028, European Patent No. 2604610, European Patent No. 1802578, Chinese Patent Patent No. 104140426, Chinese Patent No. 104003979, US Patent No. 060229351, US Patent No. 7,041,675, International Publication No. 07041357, International Publication No. 2003057699, US Patent Application No. 06 / 444,676, US Patent Application Publication No. 20060229289, US Patent Application Publication No. 20060063926, International Publication No. 2006033006, International Publication No. 2006033007, International Publication No. 03051879, International Publication No. 2004108723, International Publication No. 20060661 Patent No. 72, International Publication No. 2006078503, U.S. Patent Application Publication No. 20070032489, International Publication No. 2005023246, International Publication No. 2005097750, International Publication No. 2005123687, International Publication No. 2005097750, U.S. Patent No. 7,087,637, U.S. Patent No. 6,903,101, International Publication No. 20070011962, U.S. Patent Application Publication No. 20070015814, International Publication No. 2006135873, U.S. Patent Application Publication No. 20070072912, International Publication No. 2006065 Patent No. 392, International Publication No. 2005012305, International Publication No. 2005012305, European Patent No. 412848, European Patent No. 453210, European Patent No. 454831, European Patent No. 879820, European Patent No. 879820, International Publication No. 030805, International Publication No. 03007959, U.S. Patent No. 6,989,388, U.S. Patent Application Publication No. 20060094746, European Patent No. 1212328, International Publication No. 2006078711, U.S. Patent Application No. 06 / 426,415, U.S. Patent Application No. 06 / 514,Patent No. 983, European Patent No. 1212328, U.S. Patent Application Publication No. 20040254372, U.S. Patent Application Publication No. 20050148575, U.S. Patent Application Publication No. 20060003987, U.S. Patent Application No. 06 / 635,642, International Publication No. 200116137, International Publication No. 2004105700, International Publication No. 03057145, International Publication No. 2006078711, International Publication No. 2002044157, U.S. Patent Application Publication No. 20056924284, International Publication No. 2005112935, U.S. Patent Application Publication No. 20046828319, International Publication No. 2005054201, International Publication No. 200505 Patent No. 4209, International Publication No. 2005054210, International Publication No. 2005058843, International Publication No. 2006003146, International Publication No. 2006003147, International Publication No. 2006003148, International Publication No. 2006003150, International Publication No. 2006003146, International Publication No. 2006003147, US Patent Application Publication No. 20070072842, US Patent Application No. 05 / 587,384, US Patent Application Publication No. 20060094743, International Publication No. 2002094790, International Publication No. 2004048339, European Patent No. 1582520, US Patent Application Publication No. 20060004028, International Publication No. 2005108400, U.S. Patent No. 6,964,960, International Publication No. 20050080096, International Publication No. 2006137510, U.S. Patent Application Publication No. 20070072841, International Publication No. 2004087713, International Publication No. 2006046035, International Publication No. 2006008119, International Publication No. 06008118, International Publication No. 20 U.S. Patent Publication No. 06042638, U.S. Patent Application Publication No. 20060229289, U.S. Patent Application Publication No. 20060229351, International Publication No. 2005023800, International Publication No. 1991007404, International Publication No. 2000042025, International Publication No. 2004096779, U.S. Patent No. 6,426,415, International Publication No. 2068407, U.S. Patent No. 6,476,International Publication No. 048, International Publication No. 2001090077, International Publication No. 2001085687, International Publication No. 2001085686, International Publication No. 2001079184, International Publication No. 2001057038, International Publication No. 2001023390, International Publication No. 01021615, International Publication No. 2001016136, International Publication No. 2001012199, International Publication No. 95024379, International Publication Publication No. 200236576, International Publication No. 2004080976, International Publication No. 2007149451, International Publication No. 2006110816, International Publication No. 2007113596, International Publication No. 2007138351, International Publication No. 2007144652, International Publication No. 2007144639, International Publication No. 2007138351, International Publication No. 2007144637, Banasik et al. (J. This is described in Biol. Chem., 267:3, 1569-75, 1992), Banasik et al. (Molec. Cell. Biochem, 138:185-97, 1994), Cosi et al. (Expert Opin. Ther. Patents 12(7), 2002), Southan and Szabo (Curr Med Chem, 10 321-340, 2003), Underhill C. et al. (Annals of Oncology, doi:10.1093 / annonc / mdq322, pp. 1-12, 2010), and Murai J. et al. (J. Pharmacol. Exp. Ther., 349:408-416, 2014).

[0130] Other examples of known PARP inhibitors include the hydrochloride of N-(-oxo-5,6-dihydrophenanthridine-2-yl)-N,N-dimethylacetamide and other analogues or similar compounds that exhibit PARP inhibition, such as INO-1001.

[0131] In some embodiments, the PARP inhibitor is a small molecule, an organic compound with a molecular weight of less than 900 daltons. In some embodiments, the PARP inhibitor is a polypeptide with a molecular weight of more than 900 daltons. In some embodiments, the PARP inhibitor is an antibody.

[0132] Preferred examples of PARP inhibitors that can be used in accordance with the present invention include olaparib (AZD2281; 1-(cyclopropylcarbonyl)-4-[5-[(3,4-dihydro-4-oxo-1-phthalanidyl)methyl]-2-fluorobenzoyl]piperazine; Pubchem CID 23725625), rucaparib (AG014699; 8-fluoro-2-{4-[(methylamino)methyl]phenyl}-1,3,4,5-tetrahydro-6H-azepino[5,4,3-cd]indole-6-one; Pubchem CID 9931954), and niraparib (MK4827; 2-{4-[(3S)-3-piperidinyl]phenyl}-2H-indazole-7-carboxamide; Pubchem CID: 24958200), thalazoparib (BMN-673; (8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazine-3-one; Pubchem CID 135565082), veliparib (ABT-888; 2-[(2R)-2-methyl-2-pyrrolidinyl]-1H-benzimidazole-4-carboxamide; Pubchem CID 11960529, veliparib), iniparib (BSI 201, Pubchem CID 9796068), pamiparib (BGB-290; (10aR)-2-fluoro-5,8,9,10,10a,11-hexahydro-10a-methyl-5,6,7a,11-tetraazacyclohepta[def]cyclopenta[a]fluoren-4(7H)-one; Pubchem CID: 135565554), CEP-9722 (11-methoxy-2-((4-methylpiperazine-1-yl)methyl)-4,5,6,7-tetrahydro-1H-cyclopenta[a]pyrrolo[3,4-c]carbazole-1,3(2H)-dione; Pubchem CID 24780387), E7016 (10-((4-hydroxypiperidine-1-yl)methyl)chromeno[4,3,2-de]phthalazine-3(2H)-one; Pubchem CID 11660296), Iobenguan (1-(3-iodobenzyl)guazinine; PubChem CID 60860), Cedilanib (AZD-2171;Recentin; 4-[(4-fluoro-2-methyl-1 / - / -indole-5-yl)oxy]-6-methoxy-7-[3-(pyrroridine-1-yl)propoxy]quinazoline; PubChem CID 9933475), SH33162, 2x 121-2X, Cellara Sertib (imino-methyl-[1-[6-[(3R)-3-methylmorpholine-4-yl]-2-(1 / - / -pyrrorol[2,3-b]pyridine-4-yl)pyrimidine-4-yl]cyclopropyl]-oxo-A; 6 -Sulfane; PubChem CID 54761306), JPI289 (Amelparib; 10-Ethoxy-8-(Morpholin-4-ylmethyl)-2,3,4,6-Tetrahydro-1 / - / -Benzo[h][1,6]naphthyridine-5-one; PubChem CID 58424881), JPI547, RBN2397, IDX1197 (NOV1401), IMP4297 (1-(4-Fluoro-3-(4-(Pyrimidine-2-yl)piperazine-1-carbonyl)benzyl)Quinazoline-2,4(1H,3H)-5-Fluorodione), SC10914, HWH340, SOMCL9112 (4-(4-Fluoro-3-(5-methyl-3-( Examples include (Lifluoromethyl)-5,6,7,8-4H-[1,2,4]triazolo[4,3-a]piperazine-7-carbonyl)benzyl)phthalazine-1(2H)-one), AZD9574(6-fluoro-5-[4-[(5-fluoro-2-methyl-3-oxo-4H-quinoxaline-6-yl)methyl]piperazine-1-yl]-N-methylpyridine-2-carboxamide; Pubchem CID 162524593); ABT767; WB1340; and STX-100s.

[0133] In preferred embodiments, the PARP inhibitor is talazoparib (BMN673), rucaparib (AG014699, PF-01367338), veliparib (ABT888), olaparib (AZD2281), pamiparib (BGB-290), or niraparib (MK4827), or any pharmaceutically acceptable salt, analogue, derivative, or mixture thereof.

[0134] The PARP inhibitor may be one or more of talazoparib, lucaparib, veliparib, olaparib, pamiparib, and niraparib, or any pharmaceutically acceptable salts, analogs, derivatives thereof, or mixtures thereof. In a preferred embodiment, the PARP inhibitor is veliparib, olaparib, talazoparib, lucaparib, pamiparib, or niraparib, or any pharmaceutically acceptable salt thereof. The PARP inhibitor may be one or more of veliparib, olaparib, talazoparib, lucaparib, and niraparib, or any pharmaceutically acceptable salt thereof. Preferably, the PARP inhibitor may be one or more of veliparib, olaparib, and niraparib, or any pharmaceutically acceptable salt thereof. In a preferred embodiment, the PARP inhibitor is veliparib or any pharmaceutically acceptable salt thereof. In a preferred embodiment, the PARP inhibitor is olaparib or any pharmaceutically acceptable salt thereof. In preferred embodiments, the PARP inhibitor is niraparib or any pharmaceutically acceptable salt thereof. In preferred embodiments, the PARP inhibitor is rucaparib or any pharmaceutically acceptable salt thereof. In preferred embodiments, the PARP inhibitor is talazoparib or any pharmaceutically acceptable salt thereof. In preferred embodiments, the PARP inhibitor is pamiparib or any pharmaceutically acceptable salt thereof.

[0135] PARP inhibitors useful in the present invention are commercially available, known in the literature, or can be obtained from available starting materials by conventional synthetic procedures in accordance with standard techniques, using appropriate reagents and reaction conditions. In this regard, those skilled in the art can refer, in particular, to "Comprehensive Organic Synthesis" by B.M. Trost and I. Fleming, Pergamon Press, 1991, and "Protective Groups in Organic Synthesis," 3rd edition, by T.W. Greene and P.M. Wutz, Wiley-Interscience (1999). PARP inhibitors are also commercially available from, for example, Selleckchem, Toronto Research Chemicals, Carbosynth, Biosynthesis, Merck, Sigma Aldrich, Fischer Scientific, MedChemExpress, and other well-known commercial suppliers.

[0136] The following are preferred drug combinations according to the present invention:

[0137] [Table 1A]

[0138] [Table 1B]

[0139] Combination I or combination J is particularly preferred.

[0140] When used herein in the context of treating a condition, the term "treatment" generally refers to treatments and therapies that achieve some desired therapeutic effect, such as inhibiting the progression of a condition, slowing the rate of progression, stopping the rate of progression, improving the condition, and curing the condition.

[0141] The treatment includes any treatment or therapy, whether in human or animal (for example, in veterinary use), in which some desired therapeutic effect is achieved, such as inhibiting or delaying the progression of a condition, reducing the rate of progression, stopping the rate of progression, improving the condition, curing or relieving the condition (whether partially or completely), preventing, delaying, reducing or stopping one or more symptoms and / or signs of the condition, or extending the survival of the subject or patient beyond the survival expected in the absence of the treatment.

[0142] "Treatment" is not limited to curative therapies (e.g., those resulting in the elimination of cancer cells or tumors or metastases from the patient), but includes any treatment that has a beneficial effect on cancer or the patient, such as tumor regression or reduction, reduction of metastatic potential, extension of overall survival, extension or prolongation of life or remission, induction of remission, slowing or reduction of disease progression or rate of disease progression, or tumor development, subjective improvement of quality of life, reduction of pain or other symptoms associated with the disease, improvement of appetite, reduction of nausea, or relief of any symptoms of cancer.

[0143] This also includes preventative measures (i.e., prevention). For example, individuals who are prone to or at risk of developing or recurring cancer may be treated as described herein. Such treatment may prevent or delay the development or recurrence of cancer in the individual. Thus, “treating” means treating or preventing.

[0144] In particular, treatment may include the prevention of cancer, the delay of its progression, or treatment of cancer. Treatment may include inhibiting cancer growth and / or inhibiting cancer metastasis, including complete remission of cancer. Cancer growth generally refers to one of several indicators that show changes within the cancer to a more developed form. Therefore, indicators for measuring inhibition of cancer growth include a decrease in cancer cell viability, a decrease in tumor volume or morphology (determined, for example, using computed tomography (CT), ultrasound, or other imaging methods), delayed tumor growth, destruction of the tumor vascular system, or improved performance in delayed-type hypersensitivity skin tests.

[0145] The treatment may include a decrease in the number of cancer cells or the elimination of cancer cells. The treatment is for a subject, i.e., a subject who needs it. Thus, the treatment may include a decrease in tumor size, or the prevention of tumor growth or further tumor growth, i.e., stabilization of tumor size.

[0146] Preferably, the combinations, compounds and compositions of the present invention have a direct effect on cancer / tumor cells. "Direct effect", as used herein, means that the compounds of formula (I) and the PARP inhibitor interact directly with cancer / tumor cells in order to exert their anti-cancer / anti-tumor effect. In other words, preferably, the compounds of the present invention, i.e., the compounds of formula (I) and the PARP inhibitor, are cytotoxic to cancer / tumor cells. Preferably, the compounds of the present invention are administered to a subject in order to exert a direct effect on cancer / tumor cells.

[0147] Cancer is characterized by the abnormal proliferation of malignant cancer cells compared to normal cells.

[0148] The cancers treated according to the present invention are cancers with intact homologous recombination function (HR-proficient cancers), i.e., they are not homologous recombination-deficient (HR-deficient) cancers. The terms "HR-proficient" and "HR-deficient" are clearly understood by those skilled in the art and are widely used in the field of oncology to distinguish cancers having normal or increased (HR-proficient), or decreased or inhibited (HR-deficient) HR ability, i.e., the ability to repair DNA double-strand breaks (DSBs) by HR. These same definitions are applied herein.

[0149] HR-deficient cancer is a cancer lacking in HR-dependent DNA DSB repair. HR-proficient cancer is a cancer that is proficient in HR-dependent DNA double-strand break (DSB) repair, i.e., the cancer cells are proficient in HR-dependent DNA DSB repair. Unless otherwise clear from the context, throughout this specification, references to "cancer" also refer to the "cancer cells" of said cancer, and vice versa.

[0150] Thus, HR-proficient cancer (or cancer cells) is capable of HR, i.e., HR-dependent DNA DSB repair.

[0151] The term "HR-proficient cancer" or "HR-proficient cancer cells" refers to a cancer (or cancer cells therein) having an HR ability (i.e., proficiency) that is approximately equal (e.g., equal) or increased compared to the HR ability of normal cells (also referred to herein as normal control cells). Such a cancer (or cancer cells) may have an increased HR ability as a result of increased levels or activities of one or more HR proteins. The increased HR ability may alleviate the increased replication stress induced by oncogenes.

[0152] HR-proficient cancer includes or consists of cancer cells having an ability to repair DNA DSBs by HR that is approximately equal (e.g., equal) or increased compared to the ability of normal cells to repair DNA DSBs by HR, i.e., HR is functional in the cancer cells and the HR-dependent DNA DSB repair activity is approximately equal (e.g., equal) or increased compared to the activity of said normal cells.

[0153] The terms proficiency, activity, function, ability, and capacity are used interchangeably herein with respect to HR, HR proteins, and HR genes. HR proficiency, ability, etc. mean the proficiency, ability, etc. of a cancer, or its cancer cells, to repair DNA double-strand breaks (DSBs) by HR.

[0154] The term "approximately equal" is synonymous with "similar," meaning slightly different, for example, slightly statistically different. HR-preserving cancers as used herein have HR-preserving (i.e., activity) similar to or greater than that of normal cells.

[0155] As used herein, the term “normal cells” refers to cells that possess normal homologous recombination function (i.e., activity, function, ability, or capacity). In other words, “normal cells” are HR function-preserving control cells. “Normal” cells may be cancerous or non-cancerous cells. Normal non-cancerous cells may be tissue-matched with the cancer cells involved, i.e., they may be of the same cell type or cell lineage, or originate from the same anatomical region. Alternatively, “normal cells” may be cancerous cells, such as cancer cell lines.

[0156] "Normal cells" (HR function-preserving control cells) are not limited to cells that possess normal homologous recombination function (i.e., activity or functionality). "Normal cells" may be any of several known wild-type mammalian cell lines (discussed further below) that do not have loss-of-function mutations in the HR gene. Loss-of-function mutations include, but are not limited to, point mutations, haploid defects, and complete deletions.

[0157] Specific examples of known mammalian cell lines that maintain normal homologous recombination function, i.e., are active, include, but are not limited to, human cell lines such as Nalm-6 (human precursor B-cell leukemia cell line), HT1080 (human fibrosarcoma cell line), U2OS (human osteosarcoma cell line), HeLa (human cervical cancer cell line), HCT116 (human colon adenocarcinoma cell line), MCF-7 (human mammary adenocarcinoma cell line), HAP1 (human chronic myeloid leukemia cell line), HEK293 (human fetal kidney cell line), TIG-7 (human lung cell line), TIG-3 (human lung cell line), iPS cells (human induced pluripotent stem cells; established from normal human cells), and ES cells (human embryonic stem cells).

[0158] HR-deficient cell lines, such as those containing loss-of-function or partial loss-of-function (i.e., low-phenotype) mutations in the HR gene, are unsuitable as normal cells (normal HR function-preserving control cells). For example, cell lines derived from human hereditary breast cancer or hereditary ovarian cancer typically have one or more of these mutations in the HR gene, either the BRCA1 or BRCA2 gene. Cell lines containing such mutations (and preferably not containing either loss-of-function or partial loss-of-function mutations in 53BP1) lack homologous recombination activity and are unsuitable as normal HR function-preserving control cells.

[0159] If cancer cells possess (i.e., exhibit or show) homologous recombination function that is approximately equal to (e.g., equal to) or increased compared to normal cells (normal HR function-retaining control cells), then the cancer is determined to be HR-retaining (i.e., not HR-deficient cancer).

[0160] "HR function-preserving cancer" may, for example, have HR function preservation (i.e., activity, ability, etc.) of at least 50%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% of normal cells.

[0161] "HR-deficient cancer" may, for example, retain less than 50%, preferably less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of HR function (i.e., activity, ability, etc.) of normal cells, or may not retain any HR function (i.e., activity, ability, etc.) of normal cells.

[0162] Proteins that mediate DNA DSB repair by HR ("HR proteins") are well-characterized in the art (see, for example, Wood et al. (2001) Science 291 1284-1289) and include BRCA1, BRCA2, MUS81, RAD52, RAD51, RAD50, ATM, BARD1, CHEK2, PALB2, NBS1, RBBP8 (CtIP), MRE11 (i.e., MRE11A), RAD51C, FANCD2, and RPA (i.e., one or more of RPA1, RPA2, RPA3, and RPA4, preferably all of them).

[0163] Preferably, the HR protein may further contain one or more of BLM, XRCC2, XRCC3, EXO1, and DNA2, preferably all of them.

[0164] In embodiments, the HR protein may include BRCA1, BRCA2, MUS81, RAD52, RAD51, RAD51C, RAD50, ATM / ATR, BARD1, BRIP1, CHEK1, CHEK2, FAM175A, NBN, PALB2, MRE11 (i.e., MRE11A), NBS1, RBBP8 (CtIP), RPA (i.e., one or more of RPA1, RPA2, RPA3 and RPA4, preferably all of them), MMR protein (preferably one or more of MLH-1, MSH-2, MSH-6 and PMS-2, preferably all of them), H2AX, EME1, TP53, and FANC Fanconi anemia (FA) protein (preferably one or more of FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG and FANCI, preferably all of them).

[0165] In a preferred embodiment, the HR protein may further comprise one or more, preferably all, of BLM, XRCC2, XRCC3, EXO1, and DNA2.

[0166] HR-preserving cancers include or consist of cancer cells that possess sufficient activity of the HR protein (e.g., collectively) to mediate HR-mediated DNA DSB repair, i.e., to provide HR-preserving properties. In cancer cells of HR-preserving cancers, the HR protein activity (e.g., collectively) is approximately equal to (e.g., equal to) or increased compared to the activity of the said protein in normal cells (normal HR-preserving control cells).

[0167] Preferably, in cancer treated according to the present invention (i.e., the cancer cells thereof), the activity of the HR proteins BRCA1, BRCA2, and MUS81 is approximately equal to (e.g., equal to) or increased compared to the activity of the said proteins in normal cells.

[0168] Preferably, in cancer treated according to the present invention (i.e., its cancer cells), the activity of BRCA1 is approximately equal to (e.g., equal to) or increased compared to the activity of the protein in normal cells; that is, preferably, the cancer does not contain loss-of-function mutations (or partial loss-of-function mutations) in BRCA1. Preferably, cancer cells have wild-type BRCA1, i.e., they contain a functional copy of BRCA1. Thus, preferred cancers are BRCA1-preserving (positive) (i.e., not BRCA1-deficient). In the context of BRCA1-preserving, “normal cells” are any cells that have a functional copy of wild-type BRCA1.

[0169] Genes encoding proteins that mediate the repair of DNA DSBs by HR are referred to as "HR genes" and may include BRCA1, BRCA2, MUS81, RAD52, RAD51, RAD50, ATM, BARD1, CHEK2, PALB2, NBS1, RBBP8 (CtIP), MRE11 (i.e., MRE11A), RAD51C, FANCD2, and RPA (i.e., one or more, preferably all, of RPA1, RPA2, RPA3, and RPA4).

[0170] Preferably, the HR gene may further include one or more, preferably all, of BLM, XRCC2, XRCC3, EXO1, and DNA2.

[0171] In an embodiment, the HR gene may include BRCA1, BRCA2, MUS81, RAD52, RAD51, RAD51C, RAD50, ATM / ATR, BARD1, BRIP1, CHEK1, CHEK2, FAM175A, NBN, PALB2, MRE11 (i.e., MRE11A), NBS1, RBBP8 (CtIP), RPA (i.e., one or more, preferably all, of RPA1, RPA2, RPA3, and RPA4), MMR genes (preferably one or more, preferably all, of MLH-1, MSH-2, MSH-6, and PMS-2), H2AX, EME1, TP53, and FANC Fanconi anemia (FA) genes (preferably one or more, preferably all, of FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, and FANCI).

[0172] In a preferred embodiment, the HR gene may further include one or more, preferably all, of BLM, XRCC2, XRCC3, EXO1, and DNA2.

[0173] HR-retaining cancers consist of cancer cells in which genes encoding the HR protein (i.e., collectively), known as HR genes, are sufficiently normally expressed or overexpressed to mediate HR-mediated DNA DSB repair, i.e., to provide HR-retaining cancer. In cancer cells of HR-retaining cancers, the expression level of the HR genes (i.e., collectively) is approximately equal to (e.g., equal to) or increased compared to the expression level of the said genes in normal cells (normal HR-retaining control cells). Cancers that are HR-retaining are positive for one or more of the aforementioned HR genes, preferably substantially all, more preferably all.

[0174] Preferably, in cancer treated according to the present invention (i.e., its cancer cells), the activity of protein 53BP1 (encoded by the TP53BP1 gene) is approximately equal to (e.g., equal to) or increased compared to the activity of the protein in normal cells; that is, the cancer cells do not contain loss-of-function mutations (or partial loss-of-function mutations) in 53BP1 / TP53BP1. Preferably, the cancer cells have wild-type TP53BP1, i.e., contain a functional copy of TP53BP1. Thus, preferred cancers are 53BP1-preserving (positive) (i.e., not 53BP1-deficient). In the context of 53BP1-preserving, “normal cells” are any cells that have a functional copy of wild-type TP53BP1.

[0175] Cancers treated according to the present invention are not homologous recombination-deficient (HR-deficient) cancers. In other words, the cancer (or the cancer cells within it) does not lack HR-dependent DNA DSB repair activity (i.e., ability, function retention, etc.).

[0176] Cancers treated according to the present invention do not have suppressed (i.e., eliminated) HR capacity, or preferably reduced HR capacity, compared to normal cells (also referred to herein as normal HR function-preserving control cells, as described above). In other words, HR is not dysfunctional in the cancer cells of the present invention.

[0177] Cancer treated according to the present invention does not have, preferably, a suppressed ability to repair DNA DSBs by HR, or a reduced ability to repair DNA DSBs by HR, compared to normal cells. In other words, the HR-dependent DNA DSB repair ability of cancer cells is neither eliminated nor preferably reduced compared to the ability of normal cells.

[0178] In HR-deficient cancer (i.e., in the cancer cells), the activity of one or more HR proteins may be reduced or absent (for example, compared to normal cells). Preferably, in cancer treated according to the present invention (i.e., in the cancer cells), the activity of HR proteins is neither reduced nor absent.

[0179] Preferably, the cancer (i.e., the cancer cells) treated according to the present invention does not contain one or more of the following HR proteins BRCA1, BRCA2, and MUS81, preferably any one of them with reduced or absent activity.

[0180] Preferably, the cancer (i.e., the cancer cells) treated according to the present invention does not contain reduced or abolished BRCA1 activity.

[0181] Preferably, cancer (i.e., cancer cells) treated according to the present invention does not contain any reduced or absent activity of any of the following HR proteins: BRCA1, BRCA2, MUS81, RAD52, RAD51, RAD50, ATM, BARD1, CHEK2, PALB2, NBS1, RBBP8 (CtIP), MRE11 (i.e., MRE11A), RAD51C, FANCD2, and RPA (i.e., one or more of RPA1, RPA2, RPA3, and RPA4, preferably all of them).

[0182] Preferably, the cancer (i.e., the cancer cells) treated according to the present invention also does not contain reduced or eliminated activity of one or more of BLM, XRCC2, XRCC3, EXO1, and DNA2, preferably any of them.

[0183] Preferably, the cancer (i.e., the cancer cells) treated according to the present invention are the following HR proteins: BRCA1, BRCA2, MUS81, RAD52, RAD51, RAD51C, RAD50, ATM / ATR, BARD1, BRIP1, CHEK1, CHEK2, FAM175A, NBN, PALB2, MRE11 (i.e., MRE11A), NBS1, RBBP8 (CtIP), RPA (i.e., one or more of RPA1, RPA2, RPA3 and RPA4, preferably) (or all of them), MMR proteins (preferably one or more of MLH-1, MSH-2, MSH-6, and PMS-2, preferably all of them), H2AX, EME1, TP53, and one or more of FANC Fanconi anemia (FA) proteins (preferably one or more of FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, and FANCI, preferably all of them), preferably none of which have reduced or absent activity.

[0184] In a preferred embodiment, the cancer treated according to the present invention (i.e., the cancer cells thereof) also does not contain any reduced or eliminated activity of one or more of BLM, XRCC2, XRCC3, EXO1, and DNA2, preferably any of them.

[0185] The cancer (i.e., the cancer cells) treated according to the present invention preferably does not contain any reduced or abolished activity of any HR protein.

[0186] In HR-deficient cancer (i.e., in the cancer cells), one or more HR genes may be mutated. Mutations in one or more HR genes may eliminate the expression or activity of the HR protein, thereby eliminating the HR activity of the cancer cells. Such mutations are referred to herein as “loss-of-function” mutations. Alternatively, mutations in one or more HR genes may reduce the expression or activity of the HR protein, thereby reducing the HR activity of the cancer cells. Such mutations are referred to herein as “hypophenotic” mutations.

[0187] The cancer (i.e., the cancer cells) treated according to the present invention preferably does not contain loss-of-function mutations in the HR gene and / or low-phenotypic mutations in the HR gene. Mutations that do not result in a decrease or elimination of HR protein expression or activity may be present in the HR gene of the cancer treated according to the present invention.

[0188] Preferably, the cancer (i.e., the cancer cells) treated according to the present invention does not contain loss-of-function mutations in one or more of BRCA1, BRCA2, or MUS81, preferably any one thereof, and / or low-phenotypic mutations. In other words, the cancer treated according to the present invention does not have a BRCA1-deficient, BRCA2-deficient, and / or MUS81-deficient phenotype, i.e., it is BRCA1-positive (i.e., functional), BRCA2-positive, and / or MUS81-positive (preferably BRCA1-positive, BRCA2-positive, and MUS81-positive).

[0189] Preferably, the cancer (i.e., the cancer cells) treated according to the present invention does not contain loss-of-function mutations in BRCA1 and / or low-phenotypic mutations in BRCA1, i.e., the cancer does not have a BRCA1 deficiency phenotype (the cancer is BRCA1 positive (i.e., functional)).

[0190] In other words, cancer treated according to the present invention includes one or more, preferably all, functional copies of BRCA1, BRCA2, and MUS81. Cancer treated according to the present invention may include wild-type BRCA1, BRCA2, and / or MUS81, preferably all of them.

[0191] A preferred cancer (i.e., its cancer cells) treated according to the present invention comprises a functional copy of BRCA1 and / or wild-type BRCA1.

[0192] It is known in the art that cancer cells with loss-of-function mutations in BRCA1 may acquire further mutations in BRCA1 that partially restore its functionality (i.e., activity). These are known as "revertant mutations" or "reversion mutations" and can result in altered sequences of the BRCA1 gene / protein (i.e., compared to the wild-type sequence of the BRCA1 gene / protein). Sequences of known BRCA1 revertant mutations and methods for detecting such mutations have been described in the art.

[0193] Preferably, the cancer (i.e., the cancer cells) treated according to the present invention does not contain one or more BRCA1 revertant (i.e., reversion) mutations; i.e., the cancer cells contain wild-type BRCA1.

[0194] Preferably, cancers treated according to the present invention are free from loss-of-function mutations and / or low-phenotypic mutations in one or more, preferably any of, of the following HR genes: BRCA1, BRCA2, MUS81, RAD52, RAD51, RAD50, ATM, BARD1, CHEK2, PALB2, NBS1, RBBP8 (CtIP), MRE11 (i.e., MRE11A), RAD51C, FANCD2, and RPA (i.e., one or more of RPA1, RPA2, RPA3, and RPA4, preferably all of them).

[0195] Preferably, cancers treated according to the present invention are further free from loss-of-function mutations and / or low-phenotypic mutations in one or more of BLM, XRCC2, XRCC3, EXO1, and DNA2, preferably any one of them.

[0196] In embodiments, cancers treated according to the present invention include the following HR genes: BRCA1, BRCA2, MUS81, RAD52, RAD51, RAD51C, RAD50, ATM / ATR, BARD1, BRIP1, CHEK1, CHEK2, FAM175A, NBN, PALB2, MRE11 (i.e., MRE11A), NBS1, RBBP8 (CtIP), RPA (i.e., one or more of RPA1, RPA2, RPA3, and RPA4, preferably all of them), MMR The genes (preferably one or more of MLH-1, MSH-2, MSH-6, and PMS-2, preferably all of them), H2AX, EME1, TP53, and FANC Fanconi anemia (FA) genes (preferably one or more of FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, and FANCI, preferably all of them), preferably one or more of any of them, are free from loss-of-function mutations and / or low-phenotypic mutations.

[0197] In a preferred embodiment, the cancer treated according to the present invention is further free from loss-of-function mutations and / or low-phenotypic mutations in one or more of BLM, XRCC2, XRCC3, EXO1, and DNA2, preferably any one of them.

[0198] In other words, cancers treated according to the present invention include one or more of the above-mentioned HR genes, preferably functional copies of each. Cancers treated according to the present invention may include wild-type HR genes, such as those listed above.

[0199] Cancers treated according to the present invention preferably do not contain loss-of-function mutations in any of the HR genes and / or hypophenotypic mutations in any of the HR genes. Mutations (or polymorphisms) in one or more genes encoding regulatory factors of one or more HR genes may also reduce or eliminate the expression or activity of the HR protein, thereby reducing or eliminating the HR activity of cancer cells. Such mutations (or polymorphisms) in such regulatory factors may be gain-of-function or loss-of-function mutations relating to the regulatory factor itself, but the phenotypic effect on cancer (or its cancer cells) is reduced or eliminated HR activity. Preferably, cancers treated according to the present invention also do not contain mutations (or polymorphisms) in genes encoding regulatory components that cause reduced or eliminated HR.

[0200] Cancers that retain HR function or are deficient in HR are well known and can be easily identified by those skilled in the art, for example, based on the presence, level, and / or activity of the HR protein and / or HR gene.

[0201] Methods / assays for simple and rapid detection of the presence or absence, or degree of HR activity, in cells, tissues, or organisms are also well known and widely available, as described, for example, in U.S. Patent Application Publication No. 20230151391 and European Patent No. 4130283, and any such method or assay may be used.

[0202] In some embodiments, the cancer of an individual may have been previously identified as HR-preserving. In other embodiments, the method described herein may include the step of identifying the cancer of an individual as HR-preserving. Appropriate methods for identifying HR-preserving cancers are well known in the Art.

[0203] Various methods and assays for determining HR function retention (i.e., activity, capacity, efficiency, etc.) have been described in the Art, and those skilled in the art can use any such method or assay to determine the HR function retention (i.e., activity, capacity, efficiency, etc.) of cancer (or its cancer cells), and thus identify the cancer of an individual as HR function retention. Such assays may include, for example, HR recombinant assays based on the transfection of one or more plasmids into cells, where HR function retention (i.e., activity, capacity, efficiency, etc.) can be measured or quantified by detecting or quantifying an "HR reporter" nucleic acid molecule produced only in the cells as a result of HR activity in the cells (i.e., as a result of HR between a first nucleotide sequence and a second nucleotide sequence in the one or more plasmids), and the HR reporter molecule is specifically detectable or quantifiable (e.g., by having a characteristic (i.e., unique) nucleotide sequence).

[0204] For example, the Norgen homologous recombination assay kit (Norgen Biotek Corporation, product number 35600) is one such assay based on the cotransfection of a first plasmid and a second plasmid into cells, where the HR between the nucleotide sequences of the first plasmid and the second plasmid produces an HR reporter nucleic acid molecule, which can be specifically detected and quantified (e.g., by quantitative PCR (qPCR)).

[0205] In such assays, HR function retention (i.e., activity, capacity, efficiency, etc.) can be identified by analyzing the presence or absence of HR reporter nucleic acid molecules, or by quantifying the amount or level of HR reporter nucleic acid molecules.

[0206] In quantitative PCR (qPCR), primers containing a detectable label (e.g., a fluorescent reporter dye, i.e., the primer is fluorescently labeled) may be used, and the primer is specific to the HR reporter nucleic acid molecule. The PCR reaction then produces a PCR product (an amplified HR reporter nucleic acid molecule), and the detectable label (e.g., a fluorescent label) can be detected (preferably quantified) to determine the presence or absence, or the amount or level of the PCR product.

[0207] Therefore, for example, when analyzed by quantitative PCR (qPCR), the fluorescence intensity of the PCR product (amplified HR reporter nucleic acid molecule) corresponds to the level, activity, or efficiency of HR between cotransfected plasmids (i.e., HR function retention) (and thus the cell's HR capability). If HR does not occur between the two plasmids, no HR reporter nucleic acid molecule is detected (e.g., the fluorescence intensity does not exceed a minimum threshold). Thus, the detection of the HR reporter nucleic acid molecule provides a direct readout of HR function retention (i.e., activity, capability, efficiency, etc.), and this detection can be used to identify cancer cells that retain HR function.

[0208] Therefore, a preferred assay for determining whether cancer is HR-preserving (i.e., for determining the HR-preserving state of cancer cells) is: i) a step of cotransfecting cancer cells (referred to as “Target Cancer Cells” elsewhere herein) with a first plasmid and a second plasmid, wherein the first plasmid comprises a first nucleotide sequence, the second plasmid comprises a second nucleotide sequence, and when HR occurs between the first and second nucleotide sequences, an “HR Reporter” nucleic acid molecule is produced, the HR Reporter nucleic acid molecule has a characteristic (i.e., unique) nucleotide sequence, and the HR Reporter nucleic acid molecule is not produced in the absence of HR; ii) Incubating the transfected cells for a period sufficient to allow HR to occur (if the cells are capable of HR (i.e., HR-retaining)), for example, 12 to 24 hours, and then isolating plasmid DNA from the cells (using a common DNA isolation technique known in the art); iii) A step of detecting or quantifying an HR reporter nucleic acid molecule by, for example, qPCR. Includes.

[0209] In such assays, the detection of HR reporter nucleic acid molecules indicates that the cancer is a cancer that retains HR function.

[0210] A particularly preferred assay is the Norgen homologous recombination assay kit (Norgen Biotek Corporation, product number 35600), which involves the following steps: i) a step of cotransfecting cancer cells (referred to as “target cancer cells” elsewhere herein) (e.g., seeded in a 24-well plate) with a first plasmid and a second plasmid (e.g., 0.5 μg each), wherein the first plasmid comprises a first sequence, the second plasmid comprises a second sequence, and when HR occurs between the first sequence and the second sequence, an “HR reporter” nucleic acid molecule is produced, the HR reporter nucleic acid molecule has a characteristic (i.e., unique) nucleotide sequence, and the HR reporter nucleic acid molecule is not produced in the absence of HR; ii) Incubating the transfected cells for a period sufficient to allow HR to occur (if the cells are capable of HR (i.e., HR-retaining)), for example, 12 to 24 hours, and then isolating plasmid DNA from the cells (using a common DNA isolation technique known in the art); iii) A step of detecting or quantifying an HR reporter nucleic acid molecule by, for example, qPCR. This assay includes the following:

[0211] In such assays, the detection of HR reporter nucleic acid molecules indicates that the cancer is a cancer that retains HR function.

[0212] In step iii), methods for detecting the target nucleic acid molecule (i.e., detecting the HR reporter nucleic acid molecule) are well known in the art, and any suitable method may be used. qPCR is a preferred method.

[0213] qPCR includes the steps of adding a primer pair specific to an HR reporter nucleic acid molecule to the isolated plasmid DNA obtained in step ii), wherein the primers are detectably labeled, for example, by fluorescent labeling (i.e., including a fluorescent reporter dye); and then carrying out a PCR reaction (preferably a qPCR reaction) to produce a PCR product (i.e., an amplified HR reporter nucleic acid molecule), and then detecting (preferably quantifying) the level of the PCR product.

[0214] qPCR reactions are widely known and can be carried out using standard qPCR procedures that are routine in the art. For example, a qPCR reaction preferably includes the following steps (i.e., PCR thermal cycler conditions): initial denaturation at 95°C for 3 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds, 61°C for 15 seconds, and 72°C for 15 seconds, followed by melting curve analysis.

[0215] Once the primers are fluorescently labeled, detection (preferably quantification) can then be performed by detecting (preferably quantifying) the amount of fluorescence intensity. The fluorescence intensity can be plotted against the number of cycles. Alternatively, or in addition, a cycle threshold (i.e., Ct value) can be determined, which is the number of cycles at which the qPCR reaction exceeds the fluorescence threshold.

[0216] Such assays may include obtaining internal calibration controls (positive and negative) by repeating the above steps with separate samples, i) a positive calibration control plasmid, or ii) a negative calibration control plasmid, to obtain positive and negative calibration controls, respectively, from the same cancer cells, but instead of the first and second plasmids. Since the negative calibration control plasmid can be either the first plasmid or the second plasmid alone (i.e., not the other), HR cannot occur between them as a result, and therefore, the HR reporter nucleic acid molecule cannot be produced. Since the positive calibration control plasmid can be a plasmid expressing the HR reporter nucleic acid molecule, the HR reporter nucleic acid molecule is present in the cell as a result without requiring HR-mediated production.

[0217] In a preferred embodiment, the HR function retention (i.e., activity, capacity, etc.) of cancer cells is determined in comparison to (i.e., by comparison with) positive or negative control cells. Positive control cells may be cells known in the Art to have normal HR function retention (e.g., as described elsewhere herein). Negative control cells may be HR-deficient cells known to be HR-deficient (e.g., cell lines derived from human hereditary breast cancer or hereditary ovarian cancer having one or more mutations in the BRCA1 or BRCA2 gene), or cells in which HR deficiency has been induced (e.g., by knockout or knockdown of one or more "HR genes" as described elsewhere herein).

[0218] In such embodiments, positive control cells (e.g., HR-retaining cell lines) or negative control cells (e.g., HR-deficient cell lines) are preferably tested for HR function retention (i.e., activity, capacity, efficiency, etc.) in parallel with the target cancer cells according to the assay described above.

[0219] Therefore, identifying cancer cells as HR-retaining can be done by comparing the level of the PCR product (i.e., amplified HR reporter nucleic acid molecule), such as the amount of fluorescence intensity or the cycle threshold (i.e., the Ct value, the number of cycles in which the qPCR reaction exceeds the fluorescence threshold), determined using the target cancer cells, with the level determined in positive control cells (e.g., HR-retaining cells) or negative control cells (e.g., HR-deficient cells or cells transfected with a negative control plasmid).

[0220] In some embodiments, cancer is identified as HR-preserving cancer by determining an increase (preferably a statistically significant increase) in the level of the PCR product (i.e., amplified HR reporter nucleic acid molecule) in the target cancer cells (or using the PCR product) compared to a level determined in (or using the PCR product) in negative control cells. Such an increase may be determined, for example, by determining an increase (preferably a statistically significant increase) in fluorescence intensity in the target cancer cells compared to a level determined in (or using the PCR product) in negative control cells, or by determining a decrease (preferably a statistically significant decrease) in Ct values.

[0221] In some embodiments, a cancer is identified as an HR-preserving cancer if the level of the PCR product (i.e., the amplified HR reporter nucleic acid molecule) determined in (or using) the target cancer cells is the same as (or equivalent to, or not statistically significantly different from) the level determined in (or using) the positive control cells. Similarly, the levels being compared may be, for example, the level of fluorescence intensity or the Ct value.

[0222] Alternatively, cancer may be identified as HR-preserving cancer by determining an increase (preferably a statistically significant increase) in the level of the PCR product (i.e., amplified HR reporter nucleic acid molecule) in the target cancer cells (or using the PCR product) compared to the level determined in (or using the PCR product) in positive control cells. Such an increase may be determined, for example, by determining an increase (preferably a statistically significant increase) in fluorescence intensity in the target cancer cells compared to the level determined in (or using the PCR product) in negative control cells, or by determining a decrease (preferably a statistically significant decrease) in Ct values.

[0223] A cancer may be identified as an "HR-retaining cancer" if it is determined, for example, that it (i.e., the target cancer cells) has at least 50%, at least 55%, at least 60%, at least 65%, preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the HR-retaining capacity (i.e., activity, ability, or efficiency) of positive control cells (i.e., HR-retaining cells), and preferably, the HR-retaining capacity is analyzed or quantified using a homologous recombination assay (i.e., as described above).

[0224] A cancer may be identified as an "HR-retaining cancer" if, for example, the level of the PCR product (i.e., the amplified HR reporter nucleic acid molecule) determined in (or using) the cancer (i.e., the target cancer cells) is at least 50%, at least 55%, at least 60%, at least 65%, preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the level determined in (or using) positive control cells, and preferably, HR-retaining cancer is analyzed or quantified using a homologous recombination assay (e.g., as described above).

[0225] Alternatively, online databases can be used to identify cancer cells that retain HR function, such as the Cancer Cell Line Encyclopedia found at https: / / sites.broadinstitute.org / ccle / , DepMap Portal found at https: / / depmap.org / portal / , cBioPortal found at https: / / www.cbioportal.org / , Catalogue of Somatic Mutations in Cancer (COSMIC) found at https: / / cancer.sanger.ac.uk / cosmic, The Network of Cancer Genes (NCG) found at http: / / network-cancer-genes.org, or the American Association for Cancer Research (AACR) Project GENIE found at https: / / www.aacr.org / professionals / research / aacr-project-genie / aacr-project-genie-data / , to determine the expression levels of the "HR gene" (as defined elsewhere herein) and / or the presence of functional mutations within it in cancer cell lines.

[0226] The HR function retention (i.e., activity, capacity, etc.) of cancer cells can be determined by comparing the expression levels of, for example, known "HR genes" (as defined elsewhere herein) to those of positive or negative control cells, which are also available in the online databases mentioned above. Positive control cells may be known cell lines with normal HR function retention (e.g., as described elsewhere herein). Negative control cells may be cells known in the art to be HR deficient (e.g., cell lines derived from human hereditary breast cancer or hereditary ovarian cancer with one or more mutations in the BRCA1 or BRCA2 gene).

[0227] Therefore, if the expression and / or function of the "HR gene" (as defined elsewhere herein) can be determined using an appropriate online database, and thus HR function retention can be identified, the above-described methods and assays for determining HR activity in cancer cells may not be required. However, after matching with the above-described online database, such methods and assays can be used to confirm the HR function retention status of cancer cells.

[0228] In some embodiments, the cancer (i.e., the cancer cells) treated according to the present invention are DNA damage response functional (i.e., not DNA damage response deficient). In preferred embodiments, the cancer (i.e., the cancer cells) treated according to the present invention are homologous recombination functional (HR functional) and retain function in one or more, preferably all, of the following DNA damage response pathways: base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), alt-NHEJ, microhomology-mediated end joining (MMEJ), and translesion synthesis (TLS). In other words, preferred cancers treated according to the present invention are free from homologous recombination deficiencies and free from deficiencies in one or more, preferably all, of the following DNA damage response pathways: base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), alternative NHEJ (alt-NHEJ), microhomology-mediated end joining (MMEJ), and damage-overcoming DNA synthesis (TLS).

[0229] In a preferred embodiment, the cancer (i.e., the cancer cells) treated according to the present invention retain homologous recombination function (HR function) and base excision repair function (BER function) (i.e., neither HR-deficient nor BER-deficient).

[0230] BER-retaining cancer is cancer in which BER-dependent DNA repair retains its function; in other words, cancer cells retain their function in BER-dependent DNA repair.

[0231] The terms "BER-preserving cancer" or "BER-preserving cancer cells" refer to cancer (or cancer cells within it) that have BER capacity (i.e., function preservation) that is approximately equal to (e.g., equal to) or increased compared to that of normal cells (also referred herein as normal control cells), that is, BER is functional in the cancer cells and BER-dependent DNA repair activity is approximately equal to (e.g., equal to) or increased compared to the activity in the normal cells.

[0232] In the context of BER function-preserving cancer, "normal cells" (BER function-preserving control cells) refer to cells that possess normal BER function preservation (i.e., activity, function, ability, or capacity), and are not limited to cells that possess normal BER function preservation (i.e., activity or functionality).

[0233] BER-deficient cell lines, such as those in which BER is inhibited, reduced, or suppressed (i.e., absent), are unsuitable as normal cells (control cells maintaining normal BER function). For example, cell lines containing loss-of-function mutations in XRCC1, or cell lines containing reduced or suppressed (i.e., absent) expression of XRCC1, are BER-deficient. Cell lines containing such mutations are unsuitable as control cells maintaining normal BER function.

[0234] As is evident from this disclosure, to avoid any doubt, preferred HR-retaining and BER-retaining cancers (preferably those that retain function in all of the aforementioned DNA damage response pathways) are BER-retaining (i.e., not BER-deficient) before the first addition or initial addition of the pharmaceutical combination according to the present invention (i.e., before the addition of the compound of formula (I) and the PARP inhibitor).

[0235] 2'-Deoxyribonucleoside 5'-phosphate N-hydrolase 1 (DNPH1; gene ID 10591) is a glycohydrolase that cleaves the N-glycosidic bond of deoxyribonucleoside 5'-phosphate. DNPH1 is a c-myc-stimulated transcription factor involved in the regulation of cell proliferation, differentiation, and apoptosis. DNPH1 has a reference amino acid sequence of NP_006434.1 or NP_954653.1 or its variants, and can be encoded by the nucleotide sequences of NM_006443.3 or NM_199184.2 or its variants.

[0236] Appropriate assays for measuring DNPH1 activity are known in the art. DNPH1 activity can be determined, for example, spectrophotometrically by incubating DNPH1 with dGMP and by tracking the production of 2-deoxyribose 5-phosphate (Dupouy et al. (2010) J. Biol. Chem. 285 53 41806~41814).

[0237] Cancers treated according to the present invention preferably do not have reduced or suppressed (absent) DNPH1 activity, for example, caused by reduced or suppressed (i.e., absent) expression levels of DNPH1. Preferably, cancers treated according to the present invention (i.e., their cancer cells) do not contain loss-of-function mutations or low-phenotypic mutations in DNPH1. In other words, cancers treated according to the present invention preferably do not have a DNPH1 deficiency phenotype, i.e., are DNPH1-positive (cells). In other words, cancers treated according to the present invention preferably contain a functional copy of DNPH1, i.e., contain normal DNPH1 activity (i.e., functionality). Cancers treated according to the present invention preferably contain wild-type DNPH1.

[0238] Preferably, the treatment according to the present invention does not include any steps that reduce DNPH1 activity. Therefore, preferably, the treatment according to the present invention does not include the administration of any agent that reduces DNPH1 activity, i.e., the administration of any DNPH1 inhibitor or antagonist. Preferably, the subject has not previously been treated with any agent that reduces DNPH1 activity, i.e., the treatment with any DNPH1 inhibitor or antagonist.

[0239] X-ray repair cross-complementation 1 (XRCC1; gene ID 7515) is a protein involved in DNA single-strand break repair (SSB repair) and base excision repair (BER). XRCC1 has a reference amino acid sequence of NP_006288.2 or a variant thereof and can be encoded by a nucleotide sequence of NM_006297.3 or a variant thereof.

[0240] Cancers treated according to the present invention preferably have, for example, reduced or suppressed (i.e., eliminated) XRCC1 expression levels, or reduced or suppressed (i.e., eliminated) XRCC1 activity caused by loss-of-function mutations or low-phenotypic mutations of XRCC1 resulting in reduced or suppressed (i.e., eliminated) XRCC1 activity, compared to normal cells (i.e., XRCC1-positive (i.e., function-retaining) cells, e.g., cells having a functional copy of XRCC1).

[0241] In other words, cancers treated according to the present invention preferably do not have an XRCC1 deficiency phenotype, i.e., they are XRCC1 positive (cells), i.e., they retain XRCC1 function (cells). In other words, cancers treated according to the present invention preferably contain a functional copy of XRCC1, i.e., they contain normal XRCC1 activity (i.e., functionality). Cancers treated according to the present invention preferably contain wild-type XRCC1.

[0242] Appropriate assays for measuring the expression level of XRCC1 are known in the art. For example, the expression levels of XRCC1 mRNA or protein can be determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) or Western blotting, respectively.

[0243] Preferably, the treatment according to the present invention does not include any steps that reduce XRCC1 activity. Therefore, preferably, the treatment according to the present invention does not include the administration of any agent that reduces the expression level of XRCC1 or any agent that reduces XRCC1 activity, i.e., the administration of any XRCC1 inhibitor or antagonist. Preferably, the subject has not previously been treated with any agent that reduces the expression level of XRCC1 or any agent that reduces XRCC1 activity, i.e., the treatment with any XRCC1 inhibitor or antagonist. Preferably, the cancer (i.e., the cancer cells) treated according to the present invention does not contain loss-of-function mutations and / or low-phenotypic mutations in any of BRCA1, BRCA2, MUS81, DNPH1 and XRCC1.

[0244] Therefore, a preferred cancer (i.e., its cancer cells) treated according to the present invention does not contain loss-of-function mutations and / or low-phenotypic mutations in one or more of BRCA1, BRCA2, MUS81, DNPH1, or XRCC1, preferably any one of them. In other words, a cancer treated according to the present invention does not have a BRCA1-deficient, BRCA2-deficient, MUS81-deficient, DNPH1-deficient, and / or XRCC1-deficient phenotype; that is, the cancer is one or more of BRCA1-positive (i.e., functionally retaining), BRCA2-positive, MUS81-positive, DNPH1-positive, and XRCC1-positive, preferably all of them. In other words, a cancer treated according to the present invention contains a functional copy of one or more of BRCA1, BRCA2, MUS81, DNPH1, and XRCC1, preferably all of them. A cancer treated according to the present invention may include wild-type BRCA1, BRCA2, MUS81, DNPH1, and / or XRCC1.

[0245] In some embodiments, the cancer (i.e., the cancer cells) treated according to the present invention are BRCA1-positive (i.e., functionally preserved), BRCA2-positive, MUS81-positive, and DNPH1-positive. In other embodiments, the cancer (i.e., the cancer cells) treated according to the present invention are BRCA1-positive (i.e., functionally preserved), BRCA2-positive, MUS81-positive, DNPH1-positive, and XRCC1-positive. In preferred embodiments, the cancer (i.e., the cancer cells) treated according to the present invention are MUS81-positive (i.e., functionally preserved) and DNPH1-positive, i.e., MUS81-positive and DNPH1-positive HR functionally preserved cancer.

[0246] Preferably, the cancer (i.e., its cancer cells) treated according to the present invention does not contain reduced or suppressed (i.e., absent) expression of the PARP-1 protein, and / or does not contain loss-of-function mutations in PARP-1. In other words, preferred cancers are PARP-1 positive (i.e., functionally stable), i.e., PARP-1 positive HR functionally stable cancers. In preferred embodiments, the cancer (i.e., its cancer cells) treated according to the present invention does not contain reduced or absent expression of the PARP-1 and PARP-2 proteins, and / or does not contain loss-of-function mutations in PARP-1 and PARP-2. Thus, preferred cancers (i.e., its cancer cells) treated according to the present invention are PARP-1 positive (i.e., functionally stable) and PARP-2 positive. In some embodiments, the cancer (i.e., the cancer cells) treated according to the present invention is also PARP-3 positive (i.e., functional) and / or PARP-16 positive, i.e., does not contain reduced or suppressed (i.e., absent) expression of the PARP-3 protein and / or PARP-16 protein, and / or does not contain loss-of-function mutations in PARP-3 and / or PARP-16.

[0247] Cancer treated according to the present invention (i.e., its cancer cells) may contain at least one, preferably at least two, wild-type alleles of the p53 gene. Cancer treated according to the present invention may also not contain loss-of-function mutations in at least one, preferably at least two, p53 alleles.

[0248] Any aspect of the present invention, particularly the aspect relating to sensitization, of HR-preserving cancer may be resistant (i.e., insensitive) to treatment with the compound of formula (I) described above. For example, HR-preserving cancer may have developed resistance (insensitivity) after treatment with the compound of formula (I), that is, the subject may have been previously treated with the compound of formula (I) and developed resistance to it.

[0249] Any aspect of the present invention, particularly the aspect relating to sensitization, of HR-preserving cancer may be resistant to PARP inhibition, i.e., treatment with PARP inhibitors, as described above. For example, HR-preserving cancer may have developed PARP inhibition resistance (i.e., insensitivity) after treatment with a PARP inhibitor; that is, the subject may have been previously treated with a PARP inhibitor and developed resistance to it.

[0250] Alternatively, the cancer may not be resistant to treatment with the compound of formula (I) (for example, it may not have developed resistance), or it may not be resistant to treatment with a PARP inhibitor, or it may not be resistant to either treatment. Preferably, the cancer is not resistant to the PARP inhibitor. In the treatment methods referred to herein, the HR-preserving cancer (or subjects suffering from it) is preferably not previously treated with a PARP inhibitor. In the treatment methods referred to herein, the HR-preserving cancer (or subjects suffering from it) is preferably not previously treated with the compound of formula I.

[0251] HR function-preserving cancers can occur in (or be present in) any tissue or organ of the body, or originate from any tissue or organ of the body. For example, the present invention can be used to treat or prevent any of the following cancers in a patient or subject:

[0252] HR-preserving cancers can be any solid tumor or any hematological cancer.

[0253] Preferably, HR function-preserving cancer is a hematological cancer. Examples of hematological cancers include leukemias such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL), as well as lymphomas such as Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma. Preferably, HR function-preserving cancer is not chronic myeloid leukemia. ALL, AML, CML, and lymphoma are preferred cancers to be treated according to the present invention. Examples of solid cancers include sarcomas, skin cancers, melanomas, bladder cancers, breast cancers, uterine cancers, oral cancers, ovarian cancers, prostate cancers, lung cancers, colorectal cancers, cervical cancers, liver cancers, head and neck cancers, esophageal cancers, kidney cancers, pancreatic cancers, adrenal cancers, gastric cancers, testicular cancers, gallbladder and biliary tract cancers, thyroid cancers, thymic cancers, bone cancers, brain cancers, and central nervous system cancers. Central nervous system cancers and cervical cancers are preferred cancers to be treated according to the present invention.

[0254] Preferably, HR function-preserving cancer is HR function-preserving human cancer (i.e., human HR function-preserving cancer).

[0255] Cancer can be familial or sporadic.

[0256] Human embryonic cells are arranged in distinct germ layers: the outer ectoderm, the inner endoderm, and the mesoderm, which develops between the ectoderm and endoderm. All organs of the body develop or differentiate in an orderly manner from these three major germ layers. In this invention, preferably, HR function-preserving cancer is cancer of tissue derived from the ectoderm, paraxial mesoderm, or lateral plate mesoderm, preferably the ectoderm. In some cases, HR function-preserving cancer does not originate from the bone marrow.

[0257] Preferably, the HR-preserving cancer is not chronic myeloid leukemia. Preferably, the HR-preserving cancer is not breast cancer. Preferably, the HR-preserving cancer is not lung cancer, such as non-small cell lung cancer (NSCLC) or squamous cell carcinoma of the lung. Preferably, the HR-preserving cancer is not colorectal cancer. In some cases, the cancer is not a glioma or is not brain cancer.

[0258] Preferably, the HR-preserving cancer is not chronic myeloid leukemia, breast cancer, lung cancer, or colorectal cancer. Preferably, the cancer is not any of these cancers.

[0259] Preferably, the HR function-preserving cancer is not chronic myeloid leukemia, breast cancer, or colorectal cancer. Preferably, the cancer is not any of these cancers. Preferably, the HR function-preserving cancer is a central nervous system cancer, preferably a brain cancer.

[0260] To avoid ambiguity, brain cancer is considered in the art and herein to be a central nervous system cancer. The central nervous system includes the brain and spinal cord. Therefore, preferably, the tumor is a central nervous system tumor, preferably a brain tumor. Preferably, CNS cancer / tumor is selected from the group consisting of CNS lymphoma, rhabdoid tumor, germ blastoma, germ cell tumor and chordoma, or brain cancer / tumor. Preferably, brain cancer / tumor is selected from the group consisting of glioma, acoustic neuroma, CNS lymphoma, craniopharyngioma, medulloblastoma, meningioma, metastatic brain tumor, pituitary tumor, primitive neuroectodermal tumor (PNET), schwannoma, pineal tumor, trilateral retinoblastoma and rhabdoid tumor.

[0261] Most preferably, the cancer / tumor is brain cancer / brain tumor, more preferably glioma. The glioma may be any type of glioma, e.g., astrocytoma, ependymal cell tumor, subependymal tumor, oligodendroglioma, brainstem glioma, optic glioma, or mixed glioma.

[0262] Preferably, the glioma is an astrocytoma. The astrocytoma may be a grade I astrocytoma (preferably a pilocytic astrocytoma or subependymal giant cell astrocytoma), a grade II astrocytoma (preferably a low-grade astrocytoma, pleomorphic xanthoastrocytoma, or mixed oligoastrocytoma), a grade III astrocytoma (anaplastic astrocytoma), or most preferably a grade IV glioblastoma.

[0263] Classification systems for central nervous system tumors are well known to those skilled in the art. Preferably, the World Health Organization (WHO) classification system is used. The WHO classification scheme is well known in the art and is based on certain characteristics that reflect the malignancy of tumors in terms of invasion and growth rate: the appearance of atypia, mitosis, endothelial proliferation, and necrosis.

[0264] Gliomas can also be classified according to whether they are located above or below the tentorium, the membrane that separates the cerebrum from the cerebellum. Supratentorial gliomas are found in the cerebrum above the tentorium, and infratentorial gliomas are found in the cerebellum below the tentorium. The glioma treated according to the present invention may be either a supratentorial glioma or an infratentorial glioma.

[0265] Therefore, in the context of the present invention, the cancer / tumor is particularly preferably a glioma, most preferably a glioma, grade IV, i.e., glioblastoma multiforme. Glioblastoma multiforme is a malignant astrocytoma and the most common primary brain tumor among adults. Glioblastoma multiforme is also known as glioma, grade IV, glioblastoma, and GBM.

[0266] For example, it is known in the art that achieving and maintaining high concentrations of PARP inhibitors in the brain is difficult due to insufficient permeability of PARP inhibitors across the blood-brain barrier and insufficient retention of PARP inhibitors in the brain. Therefore, the concentration of PARP inhibitors in the brain is usually too low to have the desired therapeutic effect of treating brain cancer.

[0267] The present invention demonstrates that co-administration of a compound of formula (I) sensitizes brain cancer to PARP inhibitors, and therefore reduces the concentration of PARP inhibitors required to achieve a therapeutic effect in these cancers. Thus, the pharmaceutical combination according to the present invention has the advantageous property of sensitizing HR-preserving brain cancer to PARP inhibitors, i.e., reducing or decreasing the therapeutically effective concentration of PARP inhibitors required to treat HR-preserving brain cancer.

[0268] Preferably, when the HR function-preserving cancer is a central nervous system cancer, preferably a brain cancer, the compound of formula (I) is 5-hydroxymethyl-2'-deoxycytidine or its solvate, tautomer, or pharmaceutically acceptable salt. When the HR function-preserving cancer is a central nervous system cancer, preferably a brain cancer, preferred pharmaceutical combinations according to the present invention are 5-hydroxymethyl-2'-deoxycytidine and veliparib, olaparib, thalazoparib, lucaparib, pamiparib, niraparib, or AZD9574.

[0269] Preferably, when the HR function-preserving cancer is a central nervous system cancer, preferably a brain cancer, the PARP inhibitor is pamiparib or AZD9574, preferably pamiparib. Therefore, in a particularly preferred embodiment, when the HR function-preserving cancer is a central nervous system cancer, preferably a brain cancer, the compound of formula (I) is 5-hydroxymethyl-2'-deoxycytidine (or its solvate, tautomer, or pharmaceutically acceptable salt), and the PARP inhibitor is pamiparib or AZD9574, preferably pamiparib.

[0270] In some embodiments, HR-preserving cancer may be a carcinoma, sarcoma, or germ cell tumor.

[0271] Preferably, the cancer is ovarian cancer, pancreatic cancer, skin cancer (preferably melanoma), gastric cancer, prostate cancer, colon cancer, colorectal cancer, kidney cancer, hematological cancer (preferably ALL, AML, CML, or lymphoma as defined above), cervical cancer, or central nervous system cancer (preferably glioma or glioblastoma as defined above).

[0272] More preferably, the cancer is skin cancer (preferably melanoma), colorectal cancer, kidney cancer, hematological cancer (preferably as defined above, preferably ALL, AML, CML, or lymphoma), cervical cancer, or central nervous system cancer (preferably as defined above, preferably glioma or glioblastoma).

[0273] Therefore, in preferred embodiments, HR-preserving cancers treated according to the present invention are glioma, glioblastoma, cervical cancer, ALL, renal cancer, colorectal cancer, melanoma, CML, lymphoma, and AML.

[0274] In other preferred embodiments, HR-preserving cancers treated according to the present invention include glioma, glioblastoma, cervical cancer, ALL, renal cancer, melanoma, lymphoma, and AML.

[0275] A HR-preserving cancer in any aspect of the present invention may be a cancer in which the human protein cytidine deaminase (CDA) is not overexpressed.

[0276] Preferably, the CDA expression level is 90% or less of the CDA expression level of a reference cancer cell line (determined using the same method under the same conditions), and the reference cancer cell line is MDA-MB-231.

[0277] Preferably, the CDA expression level, compared to the CDA expression level of a reference cancer cell line, is determined by referring to a database selected from the EMBL-EBI Expression Atlas Database (https: / / www.ebi.ac.uk / gxa / home), the GENEVESTIGATOR® database (https: / / genevestigator.com / gv / ), the Cancer Cell Line Encyclopaedia (https: / / portals.broadinstitute.org / ccle), and the Human Protein Atlas (https: / / www.proteinatlas.org / ).

[0278] The MDA-MB-231 cell line is an epithelial human breast cancer cell line established from the pleural fluid of a 51-year-old Caucasian woman with metastatic breast cancer, and is one of the most commonly used breast cancer cell lines in the Institute of Medicine. It can be obtained, for example, from the European Collection of Authenticated Cell Cultures (ECACC), catalog number 92020424. The expression level of CDA in the MDA-MB-231 cell line is 153 TPM.

[0279] Preferably, the CDA expression level is 80% or less, preferably 70% or less, preferably 60% or less, preferably 50% or less, preferably 40% or less, preferably 30% or less, and preferably 25% or less of the CDA expression level of a reference cancer cell line (determined using the same method under the same conditions), and the reference cancer cell line is MDA-MB-231.

[0280] Preferably, the cancer contains CDA RNA transcripts at a level of ≤140 TPM (less than or equal to 140 TPM). In other words, the preferred cancer of the present invention has a CDA expression level of ≤140 TPM. Therefore, the particularly preferred cancers treated according to the present invention are those that express CDA to a level of ≤140 TPM, more preferably ≤100 TPM, and more preferably ≤50 TPM.

[0281] The methods and conditions used to determine the CDA expression level can be any suitable methods and conditions. Those skilled in the art can easily determine the expression level of a target gene, such as CDA, in cancer cells. Such methods are part of common sense in the art, and any suitable method can be used in the context of this invention.

[0282] For example, expression levels can be measured at the protein level by methods such as Western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISPOT), radioimmunoassay (RIA), immunohistochemistry and immunoprecipitation, and fluorescence-activated cell sorting (FACS). However, preferably, gene expression levels, such as CDA expression levels, can be measured at the RNA level by methods such as microarrays, RT-PCR, quantitative real-time PCR, RNA sequencing, Northern blotting, primer extension, RNase protection, and RNA expression profiling. Preferably, the methods used are RNA-seq or microarrays.

[0283] The levels of RNA transcripts in relation to TPM can be obtained by those skilled in the art by routine methods such as quantitative real-time PCR or RNA sequencing. Such information is available from resources such as TCGA, EMBL-EBI Expression Atlas, GENEVESTIGATOR® database (https: / / genevestigator.com / gv / ), Cancer Cell Line Encyclopaedia (https: / / portals.broadinstitute.org / ccle), and Human Protein Atlas (https: / / www.proteinatlas.org / ), among others. Such methods are preferred herein. Methodologies for determining gene expression levels in TPM are described in the literature, e.g., Wagner et al., (2012) Theory Biosci 131(4):281-285 or Mortazavi A et al., (2008) "Mapping and quantifying mammalian transcriptomes by RNA-Seq." Nature Methods 5(7):621-8.

[0284] Preferably, HR-retaining cancer (i.e., its cancer cells) does not contain the Philadelphia chromosome. Preferably, HR-retaining cancer (i.e., its cancer cells) does not have BCR-ABL fusion.

[0285] A HR-preserving cancer in any embodiment of the present invention may be hormone-resistant, or it may be hormone-sensitive.

[0286] HR-preserving cancer can be at any stage (e.g., stage 0, stage 1, stage 2, stage 3, or stage 4) or any grade (e.g., grade 1, grade 2, grade 3, or grade 4).

[0287] In all aspects and embodiments of the present invention, the treatment may be for malignant tumors or benign tumors; treatment for malignant tumors is preferred.

[0288] A “subject” suitable for the treatment described herein is a subject suffering from the indicated condition, that is, a subject requiring the treatment (“subject requiring the treatment”).

[0289] Suitable subjects for the treatments described herein may be mammals, e.g., rodents (e.g., guinea pigs, hamsters, rats, mice), murids (e.g., mice), canids (e.g., dogs), felines (e.g., cats), equids (e.g., horses), primates, simians (e.g., monkeys or apes), monkeys (e.g., marmosets, baboons), apes (e.g., gorillas, chimpanzees, orangutans, gibbons), or humans. In some preferred embodiments, the subject is human.

[0290] In other preferred embodiments, the subjects are selected from non-human mammals, particularly mammals conventionally used as models to demonstrate therapeutic efficacy in humans (e.g., rodents, primates, pigs, canids, or rabbits).

[0291] Individuals with HR-preserving cancer may exhibit at least one identifiable sign, symptom, or laboratory finding sufficient to make a diagnosis of cancer according to clinical criteria known in the art. Examples of such clinical criteria can be found in medical textbooks such as Harrison's Principles of Internal Medicine, 15th edition, edited by Fauci AS et al., McGraw-Hill, New York, 2001. In some cases, the diagnosis of cancer in an individual may involve identifying a specific cell type (e.g., cancer cells) in a fluid or tissue sample obtained from the individual.

[0292] The combinations, compounds, and forms of pharmaceutical compositions, as well as the routes of administration, dosages, and regimens, naturally depend on the nature of the cancer being treated, the severity of the disease, the age, weight, and sex of the subject (e.g., the patient), or the desired duration of treatment. Determining the appropriate dosage form, route of administration, dosage, and regimen for a given subject is within the scope of the practitioner's competence.

[0293] Compounds of formula (I) and PARP inhibitors may be administered to a subject via any suitable route. The same applies to compositions or formulations containing compounds of formula (I). Compounds of formula (I) and PARP inhibitors may be administered to a subject via the same or different routes.

[0294] Compounds of formula (I), and PARP inhibitors, and therefore compounds containing them (e.g., pharmaceutical compositions) and formulations, may be provided in forms suitable for, for example, oral, nasal, parenteral, intravenous, topical, rectal, or intrathecal administration. Forms suitable for systemic (e.g., intravenous) administration are preferred.

[0295] Any common or standard mode of administration in the art, such as injection, infusion, topical administration, inhalation, or transdermal administration, may be used to both the internal and external body surfaces by any appropriate method known in the medical field. Therefore, possible modes of administration include oral, nasal, enteral, rectal, vaginal, transmucosal, topical, parenteral, or inhalation. Administration may also be direct to the tumor (intratumor administration).

[0296] Oral or parenteral administration is preferred. Preferred parenteral administration methods include intravenous, intramuscular, intraperitoneal, intracranial, and subcutaneous administration, as well as administration into the cerebrospinal fluid (intrathecal administration). More preferably, administration is intraperitoneal or intravenous, most preferably intravenous.

[0297] Preferably, administration is oral or intravenous. Intravenous administration may be by intravenous injection or intravenous infusion, most preferably by intravenous infusion (e.g., by an infusion pump).

[0298] Preferably, if X is -CHO, administration is intravenous. Preferably, if X is -CH2OH, administration is oral.

[0299] The combination of the compound of formula (I) and the anticancer agents described herein, such as PARP inhibitors, can be administered in a single dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. The most effective means of administration and methods for determining the dosage are well known to those skilled in the art and vary depending on the formulation used for treatment, the purpose of treatment, the target cells being treated, and the subject being treated. Single or multiple doses can be administered at dose levels and patterns selected by the treating physician.

[0300] As described above, the compound of formula (I) and the PARP inhibitor may be administered separately, sequentially, in parallel, or simultaneously. In some embodiments, the compound of formula (I) and the PARP inhibitor are administered sequentially, for example, at separate times, i.e., not together in the same composition. In alternative embodiments, the compound of formula (I) and the PARP inhibitor are administered simultaneously, for example, together in the same composition or in separate compositions. The timing of separate administration may be determined according to the specific compound of formula (I) or the specific PARP inhibitor, the formulation used, and / or the mode of administration. Thus, the compound of formula (I) may be administered before or after the PARP inhibitor.

[0301] For example, to time the delivery of the PARP inhibitor to the target site optimally, the PARP inhibitor may be administered first, followed by the compound of formula (I) at an appropriate time interval, and vice versa. Such decisions are entirely within the scope of routine clinician skills. Therefore, for example, the compound of formula (I) may be administered at least or up to 20, 30, 40, 50, 60, 70, or 90 minutes, or 2, 3, 4, 5, 6, 12, or 18 hours, or 1, 2, 3, 4, 5, 6, 7, or 14 days before or after the PARP inhibitor, preferably parenterally, more preferably intravenously or orally.

[0302] Dosage and dosage may be determined on a daily basis and may depend on the properties of the molecule, the purpose of the treatment, the patient's age, the mode of administration, etc. Any therapeutic agent of the present invention described above can be combined with pharmaceutically acceptable excipients to form a therapeutic composition. Dosage refers to a specified amount of the drug taken at one time; i.e., the terms "single dose" and "dosage" are used interchangeably. The course of treatment may include multiple administrations, i.e., multiple single doses, over a period of time. Dosage refers to a specified amount of the drug taken over a specific period of time.

[0303] In the method and use of the present invention, preferably, a therapeutically effective amount of the compound of formula (I) is administered, and a therapeutically effective amount of a PARP inhibitor is administered. In other words, the dose or amount preferably comprises a therapeutically effective amount of the compound of formula (I) and a therapeutically effective amount of a PARP inhibitor.

[0304] Therefore, the present invention provides a method for treating HR function-preserving cancers in which such treatment is required, comprising the step of administering a therapeutically effective amount of a compound of formula (I) and a therapeutically effective amount of a PARP inhibitor to the target.

[0305] In any aspect of the present invention, the combination is preferably a synergistic combination. Preferably, the compound of formula (I) and the PARP inhibitor are present in synergistic amounts. The terms “synergistic” or “synergistic” are used to mean that the result of a combination of two or more compounds (drugs) is greater than the sum of the individual drugs combined. The terms “synergistic” or “synergistic” also mean that there is an improvement in the disease condition or impairment being treated that exceeds the individual use of the two or more compounds (drugs). This improvement in the disease condition or impairment being treated is the “synergistic effect.” Since “synergistic” is defined herein, “synergistic amount” is the amount of a combination of two compounds (drugs) that produces a synergistic effect.

[0306] Once synergistic interactions between one or two compounds (drugs) are determined, the optimal range for efficacy and the respective absolute dose ranges for efficacy can be definitively measured by administering the compounds across different ratio ranges and doses to patients requiring treatment. However, observing synergistic effects in in vitro or in vivo models can predict efficacy in humans and other species, which can then be used to measure synergistic effects and, by applying pharmacokinetic / pharmacodynamic methods, predict effective dose and plasma concentration ratio ranges, as well as the absolute dose and plasma concentration required in humans and other species.

[0307] The compound of formula (I) may be administered in doses of ≤1000 mg / kg, preferably ≤750 mg / kg, ≤500 mg / kg, ≤400 mg / kg, ≤300 mg / kg, ≤250 mg / kg, ≤200 mg / kg, ≤150 mg / kg, or ≤100 mg / kg. The compound of formula (I) may be administered in doses of at least 10 mg / kg, more preferably at least 20 mg / kg, more preferably at least 30 mg / kg, more preferably at least 40 mg / kg, more preferably at least 50 mg / kg, and more preferably at least 100 mg / kg.

[0308] The compound of formula (I) is found in concentrations of approximately 10 mg / kg to 1000 mg / kg; approximately 50 mg / kg to 1000 mg / kg; approximately 100 mg / kg to 1000 mg / kg; approximately 50 mg / kg to 800 mg / kg; approximately 100 mg / kg to 800 mg / kg; approximately 50 mg / kg to 600 mg / kg; approximately 100 mg / kg to 600 mg / kg; approximately 50 mg / kg to It can be administered in doses of approximately 500 mg / kg; approximately 100 mg / kg to approximately 500 mg / kg; approximately 50 mg / kg to approximately 400 mg / kg; approximately 100 mg / kg to approximately 400 mg / kg; approximately 50 mg / kg to approximately 300 mg / kg; approximately 100 mg / kg to approximately 300 mg / kg; approximately 50 mg / kg to approximately 200 mg / kg; and approximately 100 mg / kg to approximately 200 mg / kg.

[0309] The compound of formula (I) may be administered in doses between approximately 100 mg / kg and approximately 500 mg / kg.

[0310] Appropriate dosages, administration regimens, and routes of administration for PARP inhibitors are those described in the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines).

[0311] PARP inhibitors can be administered in doses of approximately 10 mg / kg to 1000 mg / kg; approximately 50 mg / kg to 1000 mg / kg; approximately 100 mg / kg to 1000 mg / kg; approximately 50 mg / kg to 800 mg / kg; approximately 100 mg / kg to 800 mg / kg; approximately 50 mg / kg to 600 mg / kg; approximately 100 mg / kg to 600 mg / kg; approximately 50 mg / kg to 500 mg / kg; approximately 100 mg / kg to 500 mg / kg; approximately 50 mg / kg to 400 mg / kg; approximately 100 mg / kg to 400 mg / kg; approximately 50 mg / kg to 300 mg / kg; approximately 100 mg / kg to 300 mg / kg; approximately 50 mg / kg to 200 mg / kg; and approximately 100 mg / kg to 200 mg / kg.

[0312] PARP inhibitors can be administered in doses of 50-1000 mg, 50-800 mg, 50-700 mg, 50-600 mg, or 50-500 mg. PARP inhibitors can be administered in doses of 100-1000 mg, 100-800 mg, 100-700 mg, 100-600 mg, or 100-500 mg. PARP inhibitors can be administered in doses of approximately 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg.

[0313] PARP inhibitors may be administered once, twice, or three times a day, preferably once or twice a day.

[0314] PARP inhibitors can be administered once daily in doses of 100-800 mg or two or three times daily in doses of 50-400 mg. For example, PARP inhibitors can be administered once daily in doses of 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, or 800 mg, or two or three times daily in doses of 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, or 400 mg.

[0315] In some embodiments, the ratio of the compound of formula (I) to the PARP inhibitor is selected from any one of the following: 100000:1 to 1:100000, 10000:1 to 1:10000, 5000:1 to 1:5000, preferably 2500:1 to 1:2500, 2000:1 to 1:2000, 1000:1 to 1:1000, 500:1 to 1:500, 100:1 to 1:100, 50:1 to 1:50, 20:1 to 1:20, 10:1 to 1:10, 5:1 to 1:5, 2:1 to 1:2, 1:1.5 to 1.5:1, and 1:1. In some embodiments, the PARP inhibitor is present in a higher molar amount than the compound of formula (I). In some embodiments, the PARP inhibitor is present in a lower molar amount than the compound of formula (I). Preferably, the ratio is the ratio of the molar concentrations (M) of the two drugs.

[0316] Dosage and administration regimens may vary based on parameters such as the patient's age, weight, condition, and sex, the purpose of the treatment, the disease being treated, the patient's age and / or condition, and the mode of administration.

[0317] Appropriate dosages and regimens can be easily established. Appropriate dosage units can be easily prepared. Dosage regimens can be determined routinely.

[0318] Determining an appropriate dosage regimen and the relevant doses therein, based on the properties of the compound, the purpose of the treatment, the disease being treated, the patient's age and / or condition, the mode of administration, etc., is within the scope of the skills of a person skilled in the art.

[0319] The treatment may include a single dose of the compound of formula (I) and a single dose of a PARP inhibitor, or repeated doses of either or both of the drugs. The administration regimens of the compound of formula (I) and the PARP inhibitor do not need to be identical. The treatment may include a single dose of the compound of formula (I) and repeated doses of a PARP inhibitor, and vice versa.

[0320] In some embodiments, the compound of formula (I) and at least one PARP inhibitor (combination therapy agent) are administered using the same dosing regimen (dosage, frequency, and duration of treatment) as is typically employed when the agent is used as monotherapy to treat the same cancer. In other embodiments, the subject receives at least one of the combination therapy agents, e.g., a lower dose of the agent, a reduced frequency of administration, and / or a shorter duration of administration, than when the same agent is used as monotherapy. In some cases, dose levels lower than the lower limit of the above range may be more than sufficient, while in other cases, higher doses may be employed without causing any adverse side effects, provided that such higher doses are initially divided into several smaller doses for daily administration.

[0321] PARP inhibitors may be administered once daily in doses of 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, or 800 mg, or two or three times daily in doses of 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, or 400 mg.

[0322] Naturally, the specific initial and subsequent administration regimens for each patient will vary according to the nature and severity of the condition as determined by the diagnosing physician, the activity of the specific compound used, the patient's age and general condition, the time of administration, the route of administration, the rate of drug excretion, the drug combination, etc. The desired treatment mode and number of administrations for the compound of the present invention or its pharmaceutically acceptable salts, esters, or compositions can be determined by those skilled in the art using conventional treatment tests.

[0323] The dosages provided herein refer to the dose of the free base form of the PARP inhibitor, or are calculated as the free base equivalent of the administered salt form. Dosage regimens may be adjusted to produce an optimal therapeutic response. For example, doses may be proportionally reduced or increased as indicated by the emergency situation of the treatment.

[0324] In one embodiment, the PARP inhibitor is administered daily.

[0325] The compound of formula (I) may be administered once or twice a day, preferably twice a day, in doses of 1 to 4000 mg, 10 to 2000 mg, 50 to 1000 mg, or 100 to 500 mg.

[0326] In one embodiment, the compound of formula (I) is administered daily.

[0327] In any aspect of the present invention, a pharmaceutical combination comprising a compound of formula (I) and a PARP inhibitor may be used in combination with further, i.e., one or more further anticancer agents. The pharmaceutical combination may be administered in combination with one or more other treatments, such as cytotoxic chemotherapy or radiotherapy. The combinations, compositions, and kits of the present invention may comprise one or more further anticancer agents. In all aspects and embodiments of the present invention, the further anticancer agents may be any suitable anticancer agents known in the art. A wide range of different types of agents are known or proposed for use in the treatment of cancer, and any of these may be used regardless of their chemical properties or mode of action.

[0328] Therefore, anticancer agents included chemical molecules, whether naturally derived, synthetically derived, or prepared (e.g., small organic chemical molecules), as well as biomolecules such as proteins and peptides (e.g., immunotherapeutic agents). Thus, anticancer agents included chemotherapeutic agents or drugs that could be a wide range of different chemical or functional classes, as well as antibodies or antibody derivatives, and other biomolecules that act to stimulate, activate, or enhance various physiological processes or cells in the body, such as immune and / or anti-inflammatory responses or cells.

[0329] The anticancer agents may include kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, thyrophostine, protease inhibitors, herbimycin A, genistein, arbstatin, and lavendastine A. In one embodiment, the anticancer agents may be selected from, but are not limited to, one or a combination of the following classes: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, folic acid antimetabolites, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxins, hormone therapy, retinoids, agents for use in photosensitization or photodynamic therapy, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycin, bleomycin, anthracyclines, MDR inhibitors, and Ca2+ ATPase inhibitors.

[0330] Anticancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, monospecific, bispecific or multispecific antibodies, monobodies, or polybodies.

[0331] Alternative anticancer agents may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, as well as their growth factor mimics.

[0332] Examples of suitable anticancer agents include chemoactive agents, such as alkylating agents, such as temozolomide (Temodal® / Temodar®) (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide), cisplatin, mono(platinum), bis(platinum), trinuclear platinum complexes, oxaliplatin, and platinum complexes including carboplatin, thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates, such as busulfan, improsulfan and pigosulfan; aziridines, such as benzodopa, carbocon, meturedopa, and uredopa; and methylamelanamine, including ethyleneimine and altoretamine. lamines), triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustards, e.g., chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, novembichin, fenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas, e.g., carmustine (preferably gliadel® (carmustine wafer) (1,3-bis(2-chloroethyl)-1-nitrosourea)), chlorozotosine, fotemustine, lomustine, nimustine, ranimustine;Antibiotics, such as acrasinomycin, actinomycin, autoramycin, azaserin, bleomycin, cactinomycin, carabicin, caminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin (ma rcellomycin), mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, plicamycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, zolubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU), gemcitabine, fluorouracil, capecitabine, methotrexate Sodium toxin, larcitrexed, pemetrexed, tegafur, cytarabine (cytosine arabinoside), thioguanine, 5-azacitidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, pitavastatin, fludarabine phosphate, and cladribine; folic acid analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidines Synthetics, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenals, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folinic acid; acegraton; aldophosphamide glycoside;Aminolevulinic acid; Amsacrine; Bestrabucil; Bisanthren; Edatraxate; Defofamine; Demecolsin; Diadiquan; Elformithine; Erliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Ronidamin; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK; Lazoxane; Schizofuran; Spirogermanium; Tenuazonic acid; Triadicone; 2,2',2"-Trichlorotriethylamine; Urethane; Vindesine Decarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacytosine; Arabinoside (Ara-C); Taxoids, e.g., paclitaxel (TAXOL) and docetaxel (TAXOTERE); Chlorambucil; Gemcitabine; 6-Thiogunine; Mercaptopurine; Platinum analogs, e.g., cisplatin and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vinca alkaloids, e.g., vinblastine, vincristine, vinorelbine, and vindesine; Navelbine; Novantrone; Teniposide; Daunomycin; Aminopterin; Ibandronate; CPT11; Topoisomerase inhibitor RFS Examples include difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine (XELODA); topoisomerase inhibitors, such as doxorubicin HCl, daunorubicin triphosphate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), levamisol and irinotecan, hydroxyurea, cyclophosphamide, nitrosourea, camptothecin, bleomycin, L-asparaginase, leucovorin, imatinib mesylate, hexamethylenediamine, and any pharmaceutically acceptable salts, acids, or derivatives of the above.

[0333] Further anticancer agents may be selected from the group consisting of temozolomide, 5-fluorouracil, gemcitabine, cytarabine, doxorubicin, daunorubicin, cisplatin, and carmustine (preferably gliadel® (carmustine wafer) (1,3-bis(2-chloroethyl)-1-nitrosourea)).

[0334] In some embodiments, the further anticancer agent is an immunotherapy agent. Induction of an immune response to treat cancer is known as cancer "immunotherapy." Immunotherapy may involve, for example, cell-based therapy, antibody therapy, or cytokine therapy. In some cases, the further anticancer agent is an antibody, selected from the group consisting of alemtuzumab, bevacizumab, brentuximab vedotin, cetuximab, gemtuzumab ozogamicin, ibritumomab tiuxetan, ipilimumab, ofatumumab, panitumumab, rituximab, tocitumomab, and trastuzumab.

[0335] In some embodiments, additional anticancer agents are checkpoint inhibitors. Several checkpoint inhibitors are known and can be used in the present invention, such as those described in Creelan (2014) Cancer Control 21:80-89. Examples of checkpoint inhibitors include tremelimumab (CP-675,206); ipilimumab (MDX-010); nivolumab (BMS-936558); MK-3475 (formerly lambrolizumab); urelumab (BMS-663513); anti-LAG-3 monoclonal antibody (BMS-986016); and bavituximab (chimeric 3G4). All of these checkpoint inhibitors can be used in the present invention.

[0336] When a combination of drugs containing the compound of formula (I) and a PARP inhibitor (each a "cancer agent") is used in combination with one or more additional anticancer agents, the various drugs may be administered separately, sequentially, in parallel, or simultaneously by any convenient route. When an anticancer agent is used in combination with additional anticancer agents active against the same disease, the dose of each anticancer agent in the combination may differ from the dose of that agent when used alone. Appropriate doses will be readily understood by those skilled in the art. One or more additional anticancer agents may be administered by any convenient means.

[0337] The administration of a combination of the compound of formula (I) and an anticancer agent described herein, such as a PARP inhibitor, in combination with one or more additional anticancer agents, may be carried out in a single dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. The most effective means of administration and methods for determining the dosage are well known to those skilled in the art and vary depending on the formulation used for treatment, the purpose of treatment, the target cells being treated, and the subject being treated. Single or multiple doses may be administered at dose levels and patterns selected by the treating physician.

[0338] The pharmaceutical combinations of the present invention can be used in accordance with the present invention in the form of products, i.e., pharmaceutical compositions. As described above, the present invention provides products, in particular pharmaceutical compositions, comprising a compound of formula (I) and a PARP inhibitor, and optionally one or more pharmaceutically acceptable excipients.

[0339] In all aspects of the present invention, a pharmaceutical combination, product, or pharmaceutical composition may comprise one or more pharmaceutically acceptable excipients.

[0340] The combinations, compounds, and compositions provided (hereinafter simply referred to as “compositions”) can be formulated in any convenient manner according to techniques and procedures known in the pharmaceutical field, for example, using one or more pharmaceutically acceptable diluents, carriers, or excipients. Such formulations may be for medicinal or veterinary use. Suitable diluents, excipients, and carriers for use in such formulations are known to those skilled in the art.

[0341] As used herein, "pharmaceutically acceptable" refers to compatibility with other components of a combination, compound, and composition, and to components that are physiologically acceptable to the recipient. The properties, dosage, etc., of the composition and carrier or excipient materials may be selected in a routine manner according to the choice and desired route of administration, purpose of treatment, etc.

[0342] Therefore, "pharmaceutically acceptable" or "pharmaceutically acceptable" means, where necessary, molecular entities and compositions that, when administered to mammals, particularly humans, do not produce adverse reactions, allergic reactions, or other undesirable reactions. A pharmaceutically acceptable carrier or excipient means any kind of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid.

[0343] The combinations, compounds, and compositions may contain pharmaceutically acceptable vehicles for formulation. These may be dry, freeze-dried compositions that can be administered by adding isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, or mixtures of such salts), or optionally sterile water or physiological saline.

[0344] The combinations, compounds, and compositions offered may be provided in conventional pharmacological dosage forms such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, powders, capsules, or sustained-release formulations. Conventional pharmaceutical excipients and standard manufacturing methods may be employed in the preparation of these forms.

[0345] To prepare a pharmaceutical composition, an effective amount of the compound of formula (I) or further anticancer agents according to the present invention can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The composition may contain any known carrier, diluent or excipient. For example, formulations suitable for parenteral administration conveniently, preferably, generally contain a sterile aqueous solution and / or suspension of the active pharmaceutical ingredient made isotonic with the recipient's blood using sodium chloride, glycerin, glucose, mannitol, sorbitol, etc.

[0346] Excipients that may be included in any pharmaceutical composition include, among others, preservatives (e.g., p-hydroxybenzoate), chelating agents (e.g., EDTA), stabilizers, isotonic agents, antimicrobial agents, flocculants / suspending agents, wetting agents, solvents and solvent systems, antioxidants, and buffering agents. When formulating a pharmaceutical composition for a particular desired purpose, selecting and optimizing such excipients and their amounts is within the scope of the skills of those skilled in the art.

[0347] The composition is preferably in the form of an aqueous solution. Such a solution is prepared according to methods known in the art and then filled into injection vials or ampoules.

[0348] The present invention will be further described with reference to the following non-limiting embodiments. [Brief explanation of the drawing]

[0349] [Figure 1]This figure shows the cell viability of U-87 MG cells. After incubation for 96 hours in the presence or absence of 5hm2dC, olaparib, veliparib, niraparib, or a combination of 5hm2dC and any of olaparib, veliparib, or niraparib, metabolic activity was determined using the MTT cell proliferation assay. A: Treatment with any of 5hm2dC, olaparib, veliparib, or niraparib. B: Treatment with 5hm2dC, olaparib, or a combination of the two. C: Treatment with 5hm2dC, veliparib, or a combination of the two. D: Treatment with 5hm2dC, niraparib, or a combination of the two. The assay was performed in triplicate, and the data shown are averages. Concentrations refer to the total sum of both compounds. The highest concentrations of olaparib (200 μM) and 5hm2dC + olaparib (400 μM) were excluded from the dataset due to precipitation. [Figure 2] This figure shows the IC50 (μM) values ​​of U-87 MG cells after drug treatment. After incubation for 96 hours in the presence or absence of 5hm2dC, olaparib, veliparib, niraparib, or a combination of 5hm2dC and any of olaparib, veliparib, or niraparib, metabolic activity was determined using an MTT cell proliferation assay, and the IC50 values ​​were determined by 4-parameter logistic regression analysis. IC50: Half-opposition inhibitory concentration (μM). [Figure 3] This figure shows the cell viability of HeLa cells. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 96 hours and 35 minutes in the presence or absence of 5hm2dC, olaparib, veliparib, niraparib, or a combination of 5hm2dC and any of olaparib, veliparib, or niraparib. A: Treatment with either 5hm2dC or 5hm2dC combined with olaparib. B: Treatment with either 5hm2dC or 5hm2dC combined with veliparib. C: Treatment with either 5hm2dC or 5hm2dC combined with niraparib. The assay was performed in triplicate, and the data shown are mean values. The two reference lines on each graph show the effects of 1 μM and 10 μM PARP inhibitors on HeLa cells without 5hm2dC. [Figure 4]This figure shows the cell viability of HeLa cells after olaparib treatment. After incubation for 96 hours in the presence or absence of 5f2dC, or a combination of 5f2dC and either 0.1 μM or 1 μM olaparib, metabolic activity was determined using the MTT cell proliferation assay. A: Treatment with either 5f2dC or 5f2dC combined with 0.1 μM olaparib. B: Treatment with either 5f2dC or 5f2dC combined with 1 μM olaparib. The assay was performed in triplicate, and the data shown are mean values. The reference lines on each graph indicate the effects of 0.1 μM and 1 μM olaparib on HeLa cells without 5f2dC. The predicted additive effect is theoretical and based on the individual effects of 5f2dC and olaparib. [Figure 5] This figure shows the cell viability of HeLa cells after veliparib treatment. After incubation for 96 hours in the presence or absence of 5f2dC, or a combination of 5f2dC and either 5 μM or 10 μM veliparib, metabolic activity was determined using the MTT cell proliferation assay. A: Treatment with either 5f2dC or 5f2dC combined with 5 μM veliparib. B: Treatment with either 5f2dC or 5f2dC combined with 10 μM veliparib. The assay was performed in triplicate, and the data shown are mean values. The reference lines on each graph show the effects of 5 μM and 10 μM veliparib on HeLa cells without 5f2dC. The predicted additive effect is based on the individual effects of 5f2dC and veliparib. [Figure 6]This figure shows the cell viability of HeLa cells after niraparib treatment. After incubation for 96 hours in the presence or absence of 5f2dC, or a combination of 5f2dC with either 0.1 μM or 1 μM niraparib, metabolic activity was determined using the MTT cell proliferation assay. A: Treatment with either 5f2dC or 5f2dC combined with 0.1 μM niraparib. B: Treatment with either 5f2dC or 5f2dC combined with 1 μM niraparib. The assay was performed in triplicate, and the data shown are mean values. The reference lines on each graph indicate the effects of 0.1 μM and 1 μM niraparib on HeLa cells without 5f2dC. The predicted additive effect is based on the individual effects of 5f2dC and niraparib. [Figure 7] This figure shows the cell viability of Louisy cells after treatment with 5hm2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 95 hours in the presence or absence of 5hm2dC, or a combination of 5hm2dC with either 1 μM or 10 μM olaparib, veliparib, or niraparib. A: Treatment with either 5hm2dC or 5hm2dC combined with either 1 μM or 10 μM olaparib. B: Treatment with either 5hm2dC or 5hm2dC combined with either 1 μM or 10 μM veliparib. C: Treatment with either 5hm2dC or 5hm2dC combined with either 1 μM or 10 μM niraparib. The assay was performed in triplicate, and the data shown are mean values. The predicted additive effect is theoretical and based on the sum of the individual effects of 5hm2dC and PARP inhibitors on Louisy cells. [Figure 8]This figure shows the cell viability of Lucy cells after treatment with 5f2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 95 hours in the presence or absence of 5f2dC, or a combination of 5f2dC with either 1 μM or 10 μM olaparib, veliparib, or niraparib. A: Treatment with either 5f2dC or 5f2dC combined with either 1 μM or 10 μM olaparib. B: Treatment with either 5f2dC or 5f2dC combined with either 1 μM or 10 μM veliparib. C: Treatment with either 5f2dC or 5f2dC combined with either 1 μM or 10 μM niraparib. The assay was performed in triplicate, and the data shown are mean values. The predicted additive effect is theoretical and based on the sum of the individual effects of 5f2dC and PARP inhibitors on Lucy cells. [Figure 9] This figure shows the cell viability of DBTRG-05MG cells after treatment with 5hm2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 95 hours in the presence or absence of 5hm2dC, or a combination of 5hm2dC with either 1 μM or 10 μM olaparib, veliparib, or niraparib. A: Treatment with either 5hm2dC or 5hm2dC combined with either 1 μM or 10 μM olaparib. B: Treatment with either 5hm2dC or 5hm2dC combined with either 1 μM or 10 μM veliparib. C: Treatment with either 5hm2dC or 5hm2dC combined with either 1 μM or 10 μM niraparib. The assay was performed in triplicate, and the data shown are mean values. The predicted additive effect is theoretical and based on the sum of the individual effects of 5hm2dC and PARP inhibitors on DBTRG-05MG cells. [Figure 10]This figure shows the cell viability of DBTRG-05MG cells after treatment with 5f2dC and olaparib. Metabolic activity was determined using the MTT cell proliferation assay after 95 hours of incubation in the presence or absence of 5f2dC, or a combination of 5f2dC and either 1 μM or 10 μM olaparib. The assay was performed in triplicate, and the data shown are mean values. The predicted additive effect is theoretical and based on the sum of the individual effects of 5f2dC and olaparib on DBTRG-05MG cells. [Figure 11] This figure shows the cell viability of Caki-1 cells after treatment with 5hm2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 96 hours in the presence or absence of 5hm2dC, or a combination of 5hm2dC with either 1 μM or 10 μM olaparib, veliparib, or niraparib. A: Treatment with either 5hm2dC or 5hm2dC combined with either 1 μM or 10 μM olaparib. B: Treatment with either 5hm2dC or 5hm2dC combined with either 1 μM or 10 μM veliparib. C: Treatment with either 5hm2dC or 5hm2dC combined with either 1 μM or 10 μM niraparib. The assay was performed in triplicate, and the data shown are mean values. The predicted additive effect is theoretical and based on the sum of the individual effects of 5hm2dC and PARP inhibitors on Caki-1 cells. [Figure 12]This figure shows the cell viability of Caki-1 cells after treatment with 5f2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 96 hours in the presence or absence of 5f2dC, or a combination of 5f2dC with either 1 μM or 10 μM olaparib, veliparib, or niraparib. A: Treatment with either 5f2dC or 5f2dC combined with either 1 μM or 10 μM olaparib. B: Treatment with either 5f2dC or 5f2dC combined with either 1 μM or 10 μM veliparib. C: Treatment with either 5f2dC or 5f2dC combined with either 1 μM or 10 μM niraparib. The assay was performed in triplicate, and the data shown are mean values. The predicted additive effect is theoretical and based on the sum of the individual effects of 5f2dC and PARP inhibitors on Caki-1 cells. [Figure 13] This figure shows the cell viability of CCRF-CEM cells after treatment with 5hm2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 96 hours in the presence or absence of 5hm2dC, or a combination of 5hm2dC with either 1 μM or 10 μM olaparib, veliparib, or niraparib. A: Treatment with either 5hm2dC or 5hm2dC combined with either 1 μM or 10 μM olaparib. B: Treatment with either 5hm2dC or 5hm2dC combined with either 1 μM or 10 μM veliparib. C: Treatment with either 5hm2dC or 5hm2dC combined with either 1 μM or 10 μM niraparib. The assay was performed in triplicate, and the data shown are mean values. The predicted additive effect is theoretical and based on the sum of the individual effects of 5hm2dC and PARP inhibitors on CCRF-CEM cells. [Figure 14]This figure shows the cell viability of CCRF-CEM cells after treatment with 5f2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 96 hours in the presence or absence of 5f2dC, or a combination of 5f2dC with either 1 μM or 10 μM olaparib, veliparib, or niraparib. A: Treatment with either 5f2dC or 5f2dC combined with either 1 μM or 10 μM olaparib. B: Treatment with either 5f2dC or 5f2dC combined with either 1 μM or 10 μM veliparib. C: Treatment with either 5f2dC or 5f2dC combined with either 1 μM or 10 μM niraparib. The assay was performed in triplicate, and the data shown are mean values. The predicted additive effect is theoretical and based on the sum of the individual effects of 5f2dC and PARP inhibitors on CCRF-CEM cells. [Figure 15] This figure shows the cell viability of HT-29 cells after treatment with 5hm2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 96 hours in the presence or absence of 5hm2dC, or a combination of 5hm2dC with either 1 μM or 10 μM of olaparib, veliparib, or niraparib. A: Treatment with either 5hm2dC or 5hm2dC combined with either 1 μM or 10 μM of olaparib. B: Treatment with either 5hm2dC or 5hm2dC combined with either 1 μM or 10 μM of veliparib. C: Treatment with either 5hm2dC or 5hm2dC combined with either 1 μM or 10 μM of niraparib. The assay was performed in triplicate, and the data shown are mean values. The predicted additive effect is theoretical and based on the sum of the individual effects of 5hm2dC and PARP inhibitors on HT-29 cells. [Figure 16]This figure shows the cell viability of HT-29 cells after treatment with 5f2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 96 hours in the presence or absence of 5f2dC, or a combination of 5f2dC with either 1 μM or 10 μM olaparib, veliparib, or niraparib. A: Treatment with either 5f2dC or 5f2dC combined with either 1 μM or 10 μM olaparib. B: Treatment with either 5f2dC or 5f2dC combined with either 1 μM or 10 μM veliparib. C: Treatment with either 5f2dC or 5f2dC combined with either 1 μM or 10 μM niraparib. The assay was performed in triplicate, and the data shown are mean values. The predicted additive effect is theoretical and based on the sum of the individual effects of 5f2dC and PARP inhibitors on HT-29 cells. [Figure 17] This figure shows the cell viability of M14 cells after treatment with 5hm2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after 96 hours of incubation in the presence or absence of 5hm2dC, or 5hm2dC combined with various concentrations of rucaparib or talazoparib. A: Treatment with either 5hm2dC or 5hm2dC combined with 1 μM or 10 μM rucaparib. B: Treatment with either 5hm2dC or 5hm2dC combined with 0.01 μM or 0.1 μM talazoparib. The assay was performed in triplicate, and the data shown are averages. The concentration of 5hm2dC (μM) is plotted on the x-axis. The predicted additive effect is theoretical and based on the sum of the individual effects of 5hm2dC and PARP inhibitors on M14 cells. [Figure 18]This figure shows the cell viability of M14 cells after treatment with 5f2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after 96 hours of incubation in the presence or absence of 5f2dC, or 5f2dC combined with various concentrations of rucaparib or talazoparib. A: Treatment with either 5f2dC or 5f2dC combined with 1 μM or 10 μM rucaparib. B: Treatment with either 5f2dC or 5f2dC combined with 0.01 μM or 0.1 μM talazoparib. The assay was performed in triplicate, and the data shown are mean values. The concentration of 5f2dC (μM) is plotted on the x-axis. The predicted additive effect is theoretical and based on the sum of the individual effects of 5f2dC and PARP inhibitors on M14 cells. [Figure 19] This figure shows the cell viability of SN12C cells after treatment with 5hm2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after 96 hours of incubation in the presence or absence of 5hm2dC, or in combination with various concentrations of 5hm2dC and rucaparib or talazoparib. A: Treatment with either 5hm2dC or 5hm2dC combined with 1 μM or 10 μM rucaparib. B: Treatment with either 5hm2dC or 5hm2dC combined with 0.01 μM or 0.1 μM talazoparib. The assay was performed in triplicate, and the data shown are mean values. The concentration of 5hm2dC (μM) is plotted on the x-axis. The predicted additive effect is theoretical and based on the sum of the individual effects of 5hm2dC and PARP inhibitors on SN12C cells. [Figure 20]This figure shows the cell viability of SN12C cells after treatment with 5f2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after 96 hours of incubation in the presence or absence of 5f2dC, or 5f2dC combined with various concentrations of rucaparib or talazoparib. A: Treatment with either 5f2dC or 5f2dC combined with 1 μM or 10 μM rucaparib. B: Treatment with either 5f2dC or 5f2dC combined with 0.01 μM or 0.1 μM talazoparib. The assay was performed in triplicate, and the data shown are mean values. The concentration of 5f2dC (μM) is plotted on the x-axis. The predicted additive effect is theoretical and based on the sum of the individual effects of 5f2dC and PARP inhibitors on SN12C cells. [Figure 21] This figure shows the cell viability of HeLa cells after treatment with 5hm2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after 96 hours of incubation in the presence or absence of 5hm2dC, or in combination with various concentrations of 5hm2dC and rucaparib or talazoparib. A: Treatment with either 5hm2dC or 5hm2dC combined with 1 μM or 5 μM rucaparib. B: Treatment with either 5hm2dC or 5hm2dC combined with 0.01 μM or 0.1 μM talazoparib. The assay was performed in triplicate, and the data shown are mean values. The concentration of 5hm2dC (μM) is plotted on the x-axis. The predicted additive effect is theoretical and based on the sum of the individual effects of 5hm2dC and PARP inhibitors on HeLa cells. [Figure 22]This figure shows the cell viability of HeLa cells after treatment with 5f2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after 96 hours of incubation in the presence or absence of 5f2dC, or 5f2dC combined with various concentrations of rucaparib or talazoparib. A: Treatment with either 5f2dC or 5f2dC combined with 1 μM or 5 μM rucaparib. B: Treatment with either 5f2dC or 5f2dC combined with 0.01 μM or 0.1 μM talazoparib. The assay was performed in triplicate, and the data shown are mean values. The concentration of 5f2dC (μM) is plotted on the x-axis. The predicted additive effect is theoretical and based on the sum of the individual effects of 5f2dC and PARP inhibitors on HeLa cells. [Figure 23] This figure shows the cell viability of KCL-22 cells after treatment with 5hm2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 96 hours in the presence or absence of 5hm2dC, or 5hm2dC combined with various concentrations of olaparib, veliparib, or niraparib. A: Treatment with either 5hm2dC or 5hm2dC combined with 1 μM or 5 μM olaparib. B: Treatment with either 5hm2dC or 5hm2dC combined with 10 μM or 20 μM veliparib. C: Treatment with either 5hm2dC or 5hm2dC combined with 1 μM or 5 μM niraparib. The assay was performed in triplicate, and the data shown are averages. The concentration of 5hm2dC (μM) is plotted on the x-axis. The predicted additive effect is theoretical and based on the sum of the individual effects of 5hm2dC and PARP inhibitors on KCL-22 cells. [Figure 24]This figure shows the cell viability of KCL-22 cells after treatment with 5f2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 96 hours in the presence or absence of 5f2dC, or 5f2dC combined with various concentrations of olaparib, veliparib, or niraparib. A: Treatment with either 5f2dC or 5f2dC combined with 1 μM or 5 μM olaparib. B: Treatment with either 5f2dC or 5f2dC combined with 10 μM or 20 μM veliparib. C: Treatment with either 5f2dC or 5f2dC combined with 1 μM or 5 μM niraparib. The assay was performed in triplicate, and the data shown are mean values. The concentration of 5f2dC (μM) is plotted on the x-axis. The predicted additive effect is theoretical and based on the sum of the individual effects of 5f2dC and PARP inhibitors on KCL-22 cells. [Figure 25] This figure shows the cell viability of U937 cells after treatment with 5hm2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 96 hours in the presence or absence of 5hm2dC, or 5hm2dC combined with various concentrations of olaparib, veliparib, or niraparib. A: Treatment with either 5hm2dC or 5hm2dC combined with 1 μM or 5 μM olaparib. B: Treatment with either 5hm2dC or 5hm2dC combined with 10 μM or 20 μM veliparib. C: Treatment with either 5hm2dC or 5hm2dC combined with 10 μM or 20 μM niraparib. The assay was performed in triplicate, and the data shown are averages. The concentration of 5hm2dC (μM) is plotted on the x-axis. The predicted additive effect is theoretical and based on the sum of the individual effects of 5hm2dC and PARP inhibitors on U937 cells. [Figure 26]This figure shows the cell viability of U937 cells after treatment with 5f2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 96 hours in the presence or absence of 5f2dC, or 5f2dC combined with various concentrations of olaparib, veliparib, or niraparib. A: Treatment with either 5f2dC or 5f2dC combined with 1 μM or 5 μM olaparib. B: Treatment with either 5f2dC or 5f2dC combined with 10 μM or 20 μM veliparib. C: Treatment with either 5f2dC or 5f2dC combined with 10 μM or 20 μM niraparib. The assay was performed in triplicate, and the data shown are mean values. The concentration of 5f2dC (μM) is plotted on the x-axis. The predicted additive effect is theoretical and based on the sum of the individual effects of 5f2dC and PARP inhibitors on U937 cells. [Figure 27] This figure shows the cell viability of OCI-AML3 cells after treatment with 5hm2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 96 hours in the presence or absence of 5hm2dC, or 5hm2dC combined with various concentrations of olaparib, veliparib, or niraparib. A: Treatment with either 5hm2dC or 5hm2dC combined with 1 μM or 5 μM olaparib. B: Treatment with either 5hm2dC or 5hm2dC combined with 10 μM or 20 μM veliparib. C: Treatment with either 5hm2dC or 5hm2dC combined with 1 μM or 5 μM niraparib. The assay was performed in triplicate, and the data shown are mean values. The concentration of 5hm2dC (μM) is plotted on the x-axis. The predicted additive effect is theoretical and based on the sum of the individual effects of 5hm2dC and PARP inhibitors on OCI-AML3 cells. [Figure 28]This figure shows the cell viability of OCI-AML3 cells after treatment with 5f2dC and PARPi. Metabolic activity was determined using the MTT cell proliferation assay after incubation for 96 hours in the presence or absence of 5f2dC, or 5f2dC combined with various concentrations of olaparib, veliparib, or niraparib. A: Treatment with either 5f2dC or 5f2dC combined with 1 μM or 5 μM olaparib. B: Treatment with either 5f2dC or 5f2dC combined with 10 μM or 20 μM veliparib. C: Treatment with either 5f2dC or 5f2dC combined with 1 μM or 5 μM niraparib. The assay was performed in triplicate, and the data shown are mean values. The concentration of 5f2dC (μM) is plotted on the x-axis. The predicted additive effect is theoretical and based on the sum of the individual effects of 5f2dC and PARP inhibitors on OCI-AML3 cells. [Figure 29] This figure shows the cell viability of SNB19 cells after treatment with 5hm2dC, 5f2dC, and pamiparib. Metabolic activity was determined using the MTT cell proliferation assay after 96 hours of incubation in or without 5hm2dC, 5f2dC, or a combination of 5hm2dC or 5f2dC with either 5 μM or 20 μM pamiparib. A: Treatment with either 5hm2dC or 5hm2dC combined with either 5 μM or 20 μM pamiparib. B: Treatment with either 5f2dC or 5f2dC combined with either 5 μM or 20 μM pamiparib. The assay was performed in triplicate, and the data shown are mean values. The concentration (μM) of 5hm2dC or 5f2dC is plotted on the x-axis. The predicted additive effect is theoretical and based on the sum of the individual effects of 5hm2dC or 5f2dC and pamiparib on SNB19 cells. [Figure 30]This figure shows the cell viability of U-87 MG cells after treatment with 5hm2dC, 5f2dC, and pamiparib. Metabolic activity was determined using the MTT cell proliferation assay after 96 hours of incubation in or without 5hm2dC, 5f2dC, or a combination of 5hm2dC or 5f2dC with either 5 μM or 20 μM pamiparib. A: Treatment with either 5hm2dC or 5hm2dC combined with either 5 μM or 20 μM pamiparib. B: Treatment with either 5f2dC or 5f2dC combined with either 5 μM or 20 μM pamiparib. The assay was performed in triplicate, and the data shown are mean values. The concentration (μM) of 5hm2dC or 5f2dC is plotted on the x-axis. The predicted additive effect is theoretical and based on the sum of the individual effects of 5hm2dC or 5f2dC and pamiparib on U-87 MG cells. [Examples]

[0350] DMSO: Dimethyl sulfoxide 5hm2dC:5-hydroxymethyl-2'-deoxycytidine 5f2dC:5-Formyl-2'-Deoxycytidine MTT: 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide PBS: Phosphate-buffered saline SDS: Sodium dodecyl sulfate U: Unit

[0351] material Test items Name: 5hm2dC Chemical name: 5-hydroxymethyl-2'-deoxycytidine CAS number: 7226-77-9 Supplier examples 1, 2 and 4-6: Carbosynth Inc. Lot number: 45660 Supplier examples 7-9: WuXi Co., Ltd. Lot number: PC12804-146-FP-P Name: 5f2dC Chemical name: 5-formyl-2'-deoxycytidine CAS Number: 137017-45-9 Supplier: Carbosynth Inc. Lot Number: ND635561701 Name: Olaparib CAS Number: 763113-22-0 Supplier: Selleckchem Lot Number: S106024 Name: Veriparib CAS Number: 912444-00-9 Supplier: Selleckchem Lot Number: S100418 Name: Chiraparib CAS Number: 1038915-60-4 Supplier: Selleckchem Lot Number: S274105 Name: Lucaparib CAS Number: 283173-50-2 Supplier: Selleckchem Lot Number: S494803 Name: Thalazoparib CAS Number: 1207456-01-6 Supplier: Selleckchem Lot Number: S704810 Name: Pamiparib CAS Number: 1446261-44-4 Supplier: MedChemExpress Lot Number: 59119 Name: Dimethyl sulfoxide (DMSO) CAS number: 67-68-5 Supplier: PanReac AppliChem Lot number: 2G010433

[0352] All compounds were prepared as 100 mM solutions in 100% DMSO, further diluted with PBS, and a final DMSO concentration of 0.2% was achieved. The selected negative control was DMSO in PBS with a final concentration of 0.2% DMSO.

[0353] cell line Name: U-87 MG Tissue: Brain Supplier: ATCC Disease: Glioblastoma Mycoplasma: Not detected. Doubling time: 30-40 hours. After thawing the initial vial, the cells were subculturred 11 times before seeding, every 3-4 days or until a maximum of 90% confluence was reached.

[0354] Name: HeLa Tissue: Uterus Supplier: ATCC Disease: Adenocarcinoma Mycoplasma: Not detected. Doubling time: 20-24 hours. After thawing the initial vial, the cells were subculturred 12 times before seeding, every 3-4 days or until a maximum of 90% confluence was reached.

[0355] Name: Loucy Tissue: Peripheral Blood Supplier: ATCC CRL-2629 Disease: Acute lymphoblastic leukemia, T cell (T-ALL) Mycoplasma: Not detected. Doubling time: 50-60 hours. After thawing the first vial, pass the cells 10 times before seeding, every 3-4 days, or 2 × 10 6 The cells were passaged until the maximum confluence (cells / ml) was reached.

[0356] Name: DBTRG-05MG Organization: Brain Supplier: ATCC CRL-2020 Disease: Glioblastoma Mycoplasma: Not detected. Doubling time: 48 hours. After thawing the initial vial, the cells were subculturred three times before seeding, every 3-4 days or until a maximum of 90% confluence was reached.

[0357] Name: Caki-1 Tissue: Kidney Supplier: NCI Disease: Clear cell carcinoma Mycoplasma: Not detected. Doubling time: 40 hours. After thawing the initial vial, the cells were subculturred nine times before seeding, every 3-4 days or until 90% maximum confluence was reached.

[0358] Name: CCRF-CEM Organization: Peripheral Blood Supplier: NCI Disease: Acute lymphoblastic leukemia (ALL) Mycoplasma: Not detected. Doubling time: 26 hours. After thawing the first vial, pass the cells 9 times before seeding, every 3-4 days, or 2 × 10 6 The cells were passaged until the maximum confluence (cells / ml) was reached.

[0359] Name: HT-29 Tissue: Colon Supplier: NCI Disease: Adenocarcinoma Mycoplasma: Not detected. Doubling time: 20 hours. After thawing the initial vial, the cells were subculturred nine times before seeding, every 3-4 days or until 90% maximum confluence was reached.

[0360] Name: M14 Tissue: Skin Supplier: NCI Disease: Apigmented melanoma Mycoplasma: Not detected. Doubling time: 26 hours. After thawing the initial vial, the cells were subculturred four times before seeding, every 3-4 days or until 90% maximum confluence was reached.

[0361] Name: SN12C Tissue: Kidney Supplier: NCI Disease: Renal cell carcinoma Mycoplasma: Not detected. Doubling time: 26 hours. After thawing the initial vial, the cells were subculturred four times before seeding, every 3-4 days or until 90% maximum confluence was reached.

[0362] Name: KCL-22 Tissue: Blood Supplier: UCL Disease: Chronic myeloid leukemia Mycoplasma: Not detected. Doubling time: 24 hours. After thawing the first vial, pass the cells 11 times before seeding, every 3-4 days, or 2 × 10 6 The cells were passaged until the maximum confluence (cells / mL) was reached.

[0363] Name: U937 Tissue: Blood Supplier: UCL Disease: Histiocytic lymphoma Mycoplasma: Not detected. Doubling time: 30 hours. After thawing the first vial, pass the cells 11 times before seeding, every 3-4 days, or 2 × 10 6 The cells were passaged until the maximum confluence (cells / mL) was reached.

[0364] Name: OCI-AML3 Organization: Blood Supplier: UCL Disease: Acute myeloid leukemia Mycoplasma: Not detected. Doubling time: 30 hours. After thawing the first vial, pass the cells 11 times before seeding, every 3-4 days, or 2 × 10 6 The cells were passaged until the maximum confluence (cells / mL) was reached.

[0365] Name: SNB19 Organization: Brain Supplier: NCI Disease: Glioblastoma Mycoplasma: Not detected. Doubling time: 24 hours. After thawing the initial vial, the cells were subculturred 13 times before seeding, every 3-4 days or until 90% maximum confluence was reached.

[0366] (Example 1) The combined effect of 5hm2dC and a PARP inhibitor on the survival rate of cancer cells with preserved HR function (U-87 MG). The objective of the study was to determine whether 5hm2dC could be used in combination with the PARP inhibitors olaparib, veliparib, and niraparib to enhance the efficacy of 5hm2dC or PARP inhibitors as HR-preserving cancer treatments compared to their use individually.

[0367] HR-preserving glioblastoma cell line U-87 MG was cultured for 96 hours in the presence or absence of 5hm2dC in combination with olaparib, veliparib, niraparib, or any of olaparib, veliparib, or niraparib. Cell viability was determined by an MTT cell proliferation assay. The cells used were BRCA1-positive (i.e., functional), BRCA2-positive, MUS81-positive, DNPH1-positive, and XRCC1-positive.

[0368] Growth medium and conditions Cells were grown in GlutaMAX Dulbecco's modified Eagle medium (Gibco, catalog 31966047) supplemented with 10% final concentration fetal bovine serum (BioSera, catalog FB-1001 / 500), 100 U / mL penicillin, and 100 U / mL streptomycin. These were maintained at 37°C in a 5% CO2 humidified atmosphere.

[0369] Plate settings U-87 MG cells were harvested and seeded at 2000 cells / well in three 96-well plates. Each drug was diluted in an eight-point dilution series at a dilution ratio of 2.5. For wells with a single drug, the final concentrations were 200 μM, 80 μM, 32 μM, 12.8 μM, 5.12 μM, 2.05 μM, 0.82 μM, and 0.33 μM. For wells with two drugs, the total final concentrations were 400 μM, 160 μM, 64 μM, 25.6 μM, 10.24 μM, 4.10 μM, 1.64 μM, and 0.66 μM. The drugs were added in a triple series 4 hours after cell seeding, and the plates were maintained at 37°C in a 5% CO2 humidified atmosphere for 96 hours.

[0370] MTT assay After 96 hours, 10 μL of MTT reagent, prepared by filtration sterilization of a 5 mg / mL solution in PBS, was added to each well to achieve a final concentration of 0.45 mg / mL. After 3 hours, 100 μL of MTT solubilizing reagent (10% SDS in 0.01 M HCl) was added to each well, and the plate was maintained in the dark overnight. After shaking for 30 seconds, the absorbance at 570 nm was read.

[0371] Results and Discussion The results are shown in Figures 1 and 2, and Tables 1-3 (Tables 2-4).

[0372] [Table 2]

[0373] [Table 3]

[0374] [Table 4]

[0375] The combination of 5hm2dC with the PARP inhibitors olaparib, veliparib, and niraparib showed a synergistic effect on the viability of U-87 MG cells, with particularly noteworthy results at low concentrations (Figure 1, Table 2 (Table 3)). The combination effect also synergistically reduced the IC50 values ​​by orders of magnitude compared to the effects of the individual drugs (Table 3 (Table 4), Figure 2).

[0376] Data points corresponding to the highest concentrations of olaparib (200 μM) and the combination of 5hm2dC and olaparib (400 μM) were excluded from data analysis due to precipitation formation.

[0377] The use of 5hm2dC in combination with the PARP inhibitors olaparib, veliparib, and niraparib has a clear effect on the cell viability of HR-retaining U-87 MG cells, particularly improving drug efficacy at low drug concentrations.

[0378] (Example 2) Combination effect of 5hm2dC and PARP inhibitors on HR function-preserving cancer cell survival rate (HeLa). The objective of the study was to determine whether 5hm2dC could be used in combination with the PARP inhibitors olaparib, veliparib, and niraparib to enhance the efficacy of 5hm2dC or PARP inhibitors as HR function-preserving cancer therapies compared to their use individually. Based on previous studies, HeLa cells were specifically selected because they were i) HR function-preserving and ii) insensitive to 5hm2dC at concentrations up to 100 μM. The cells used were BRCA1-positive (i.e., function-preserving), BRCA2-positive, MUS81-positive, DNPH1-positive, and XRCC1-positive cells.

[0379] HeLa cells were cultured for 96 hours and 35 minutes in the presence or absence of 5hm2dC, olaparib, veliparib, niraparib, or any of olaparib, veliparib, or niraparib. Cell viability was determined by performing an MTT cell proliferation assay.

[0380] Growth medium and conditions Cells were grown in GlutaMAX Dulbecco's modified Eagle medium (Gibco, catalog 31966047) supplemented with 10% final concentration fetal bovine serum (BioSera, catalog FB-1001 / 500), 100 U / mL penicillin, and 100 U / mL streptomycin. These were maintained at 37°C in a 5% CO2 humidified atmosphere.

[0381] Plate settings HeLa cells were harvested and seeded at 2000 cells / well in three 96-well plates. 5hm2dC was diluted in a five-point dilution series at a dilution ratio of 2.5, with final concentrations of 10 μM, 2.5 μM, 0.63 μM, 0.16 μM, and 0.04 μM. PARP inhibitors were added to the 5hm2dC dilution series at either a concentration of 1 μM or 10 μM. The cell seeding volume was 90 μL, and the drug volume was 5 μL per drug addition. Wells to which only one drug was added contained an additional 5 μL of PBS solution with the same DMSO concentration as the added drug dilution to maintain a constant final volume of 100 μL. Drugs were added in a triple series 2 hours after cell seeding, and the plates were maintained at 37°C in a 5% CO2 humidified atmosphere for 96 hours and 35 minutes.

[0382] MTT assay After 96 hours and 35 minutes, 10 μL of MTT reagent, prepared by filtration sterilization of a 5 mg / mL solution in PBS, was added to each well to achieve a final concentration of 0.45 mg / mL. After 2 hours and 20 minutes, 100 μL of MTT solubilizing reagent (10% SDS in 0.01 M HCl) was added to each well. The plate was kept in the dark overnight, and after shaking for 30 seconds, the absorbance at 570 nm was read.

[0383] Results and Discussion The results are shown in Figure 3 and Tables 4-7 (Tables 5-8).

[0384] [Table 5]

[0385] [Table 6]

[0386] [Table 7]

[0387] [Table 8]

[0388] MTT assays confirmed that HeLa cells were insensitive to 5hm2dC up to the maximum concentration of 10 μM in this assay, as expected, and that no cell death occurred at any concentration (Tables 4, 5, 6 (Tables 5, 6, 7)). When 5hm2dC was combined with olaparib, veliparib, or niraparib at concentrations of 1 μM or 10 μM, HeLa cells became sensitive to 5hm2dC, even at the same PARP inhibitor concentration, and higher 5hm2dC concentrations corresponded to more cell death (Figure 3). These results demonstrate a synergistic effect between 5hm2dC and PARP inhibitors. The use of 5hm2dC in combination with PARP inhibitors has a clear efficacy as a combination therapy in HR-preserving cancers.

[0389] (Example 3) The combined effect of 5f2dC and PARP inhibitors on cancer cell survival rate (HeLa). The objective of the study was to determine whether 5f2dC could be used in combination with the PARP inhibitors (PARPi) olaparib, veliparib, and niraparib to enhance the efficacy of 5f2dC or PARP inhibitors as cancer treatments compared to their use individually.

[0390] HeLa cells were cultured for 96 hours in the presence or absence of 5f2dC, olaparib, veliparib, niraparib, or olaparib, veliparib, or niraparib in combination with 5f2dC. The cells used were BRCA1-positive (i.e., functional), BRCA2-positive, MUS81-positive, DNPH1-positive, and XRCC1-positive.

[0391] Growth medium and conditions Cells were grown in GlutaMAX Dulbecco's modified Eagle medium (Gibco, catalog 31966047) supplemented with 10% final concentration fetal bovine serum (BioSera, catalog FB-1001 / 500), 100 U / mL penicillin, and 100 U / mL streptomycin. These were maintained at 37°C in a 5% CO2 humidified atmosphere.

[0392] Plate settings HeLa cells were harvested and seeded at 1000 cells / well in three 96-well plates. 5f2dC was diluted in a five-point dilution series at a dilution factor of 4, with final concentrations of 1 μM, 0.25 μM, 0.063 μM, 0.016 μM, and 0.004 μM. Since HeLa cells have been previously shown to be highly sensitive to approximately 1.5 μM of 5f2dC, a starting concentration of 1 μM was chosen to avoid major cell death.

[0393] PARP inhibitors were added to a 5f2dC dilution series at final concentrations of 0.1 μM or 1 μM olaparib or niraparib, or 5 μM or 10 μM veliparib. The cell seeding volume was 90 μL, and the drug volume was 5 μL per drug addition. Wells to which only one drug was added contained an additional 5 μL of PBS solution with the same DMSO concentration as the added drug dilution to maintain a constant final volume of 100 μL. Drugs were added in triplicate 5 hours after cell seeding, and the plates were maintained at 37°C in a 5% CO2 humidified atmosphere for 96 hours.

[0394] MTT cell proliferation assay After 96 hours, 10 μL of MTT reagent, prepared by filtration sterilization of a 5 mg / mL solution in PBS, was added to each well to achieve a final concentration of 0.45 mg / mL. After 2 hours, 100 μL of MTT solubilizing reagent (10% SDS in 0.01 M HCl) was added to each well, and the plate was maintained in the dark overnight. After shaking for 30 seconds, the absorbance at 570 nm was read.

[0395] Results and Discussion The results are shown in Figures 4-6 and Tables 8-11 (Tables 9-12).

[0396] [Table 9]

[0397] [Table 10]

[0398] [Table 11]

[0399] [Table 12]

[0400] MTT cell proliferation assays were performed to determine cell viability, demonstrating a synergistic effect between 5f2dC and PARP inhibitors. Combining 5f2dC with either olaparib or niraparib at concentrations of 0.1 μM or 1 μM, or with veliparib at concentrations of 5 μM or 10 μM, resulted in HeLa cells becoming more sensitive to 5f2dC compared to the absence of a PARP inhibitor (Figures 4, 5, and 6). In particular, the combination of 5f2dC with either olaparib or niraparib resulted in significant cell death even at a low total drug concentration of 2 μM, with cell viability rates of 20.17% and 9.87%, respectively (Tables 8, 10 (9, 11)). Furthermore, the theoretical additive effect of each treatment predicted significantly less cell death than the actual combined effect (Figures 4, 5, and 6). The combination of 5f2dC with either 0.1 μM or 1 μM olaparib or niraparib, or either 5 μM or 10 μM veliparib, resulted in significantly higher cell death than the predicted additive effects of each individual drug and each combination. Since higher co-administrative effects can be achieved at lower total drug concentrations, these results demonstrate a synergistic effect between 5f2dC and PARPi, and highlight their usefulness as a combination therapy.

[0401] (Example 4) Combination effect of PARP inhibitors and 5hm2dC or 5f2dC on cancer cell survival rate (Loucy cells) The objective of the study was to determine whether 5hm2dC or 5f2dC could be used in combination with the PARP inhibitors olaparib, veliparib, and niraparib to enhance the efficacy of 5hm2dC, 5f2dC, or PARP inhibitors as cancer treatments in Luucy cells (acute lymphoblastic leukemia, T-cell (T-ALL) cell line) compared to their use individually. The cells used were BRCA1-positive (i.e., functional), BRCA2-positive, MUS81-positive, DNPH1-positive, and XRCC1-positive cells.

[0402] Loucy cells were cultured for 95 hours in or without 5hm2dC, 5f2dC, olaparib, veliparib, niraparib, or 5hm2dC combined with any of olaparib, veliparib, or niraparib, or 5f2dC combined with any of olaparib, veliparib, or niraparib. MTT cell proliferation assays were performed to determine cell viability, and it was shown that both 5hm2dC and PARP inhibitors, and 5f2dC and PARP inhibitors, had synergistic effects. All combinations of 5hm2dC or 5f2dC with each PARPi showed synergistic effects in Loucy cells.

[0403] Growth medium and conditions Loucy cells were harvested and seeded at 40,000 cells / well in six 96-well plates. 5hm2dC and 5f2dC were diluted in a five-point dilution series at a dilution ratio of 4, resulting in final concentrations of 1 μM, 0.25 μM, 0.063 μM, 0.016 μM, and 0.004 μM.

[0404] PARP inhibitors were added to 5hm2dC or 5f2dC dilution series at a final concentration of 1 μM or 10 μM of olaparib, veliparib, or niraparib. The cell seeding volume was 90 μL, and the drug volume was 5 μL per drug addition. Wells to which only one drug was added contained an additional 5 μL of PBS solution having the same DMSO concentration as the added drug dilution to maintain a constant final volume of 100 μL. Drugs were added in triplicate 5 hours after cell seeding, and the plates were maintained at 37°C in a 5% CO2 humidified atmosphere for 95 hours.

[0405] MTT assay After 95 hours, 10 μL of MTT reagent, prepared by filtration sterilization of a 5 mg / mL solution in PBS, was added to each well to achieve a final concentration of 0.45 mg / mL. After 2 hours, 100 μL of MTT solubilizing reagent (10% SDS in 0.01 M HCl) was added to each well, and the plate was maintained in the dark overnight. After shaking for 30 seconds, the absorbance at 570 nm was read.

[0406] 4.2 Results and Discussion The results are shown in Figures 7 and 8, and Tables 12-14 (Tables 13-15).

[0407] [Table 13]

[0408] [Table 14]

[0409] [Table 15]

[0410] The results demonstrate that 5hm2dC and 5f2dC exhibit synergistic effects with olaparib, veliparib, and niraparib in Lucy cells. Combinations of 5hm2dC or 5f2dC with PARP inhibitors resulted in greater cell death than predicted by the additive effect. For example, the combination of 1 μM 5hm2dC and 1 μM olaparib resulted in 52.1% cell death, while the theoretical additive effect predicted only 18.5% (Figure 7A). Similarly, the combination of 1 μM 5f2dC with 1 μM olaparib resulted in 47.4% cell death, while the theoretical additive effect predicted only 9.7% (Figure 8a).

[0411] (Example 5) Combination effect of PARP inhibitors and 5hm2dC or 5f2dC on cancer cell survival rate (DBTRG-05MG cells) The objective of the study was to determine whether 5hm2dC could be used in combination with the PARP inhibitors olaparib, veliparib, and niraparib, or 5f2dC in combination with the PARP inhibitor olaparib, to enhance the efficacy of 5hm2dC, 5f2dC, or PARP inhibitors as cancer treatments in DBTRG-05MG cells (glioblastoma cell line) compared to their use individually. The cells used were BRCA1-positive (i.e., functional), BRCA2-positive, MUS81-positive, DNPH1-positive, and XRCC1-positive cells.

[0412] DBTRG-05MG cells were cultured for 95 hours in or without 5hm2dC, 5f2dC, olaparib, veliparib, niraparib, or 5hm2dC combined with any of olaparib, veliparib, or niraparib, or 5f2dC combined with olaparib. Cell viability was determined by an MTT cell proliferation assay, which showed synergistic effects between 5hm2dC and PARP inhibitors and between 5f2dC and PARP inhibitors. All combinations of 5hm2dC with each PARPi, or 5f2dC with olaparib, showed synergistic effects in DBTRG-05MG cells.

[0413] Growth medium and conditions DBTRG-05MG cells were harvested and seeded at 1000 cells / well in six 96-well plates. 5hm2dC and 5f2dC were diluted in a five-point dilution series at a dilution ratio of 4, resulting in final concentrations of 1 μM, 0.25 μM, 0.063 μM, 0.016 μM, and 0.004 μM.

[0414] PARP inhibitors were added to the 5hm2dC or 5f2dC dilution series at a final concentration of 1 μM or 10 μM olaparib, veliparib, or niraparib for 5hm2dC, and at a final concentration of 1 μM or 10 μM olaparib for 5f2dC. The cell seeding volume was 90 μL, and the drug volume was 5 μL per drug addition. Wells to which only one drug was added contained an additional 5 μL of PBS solution with the same DMSO concentration as the added drug dilution to maintain a constant final volume of 100 μL. Drugs were added in triplicate 5 hours after cell seeding, and the plates were maintained at 37°C in a 5% CO2 humidified atmosphere for 95 hours.

[0415] MTT assay After 95 hours, 10 μL of MTT reagent, prepared by filtration sterilization of a 5 mg / mL solution in PBS, was added to each well to achieve a final concentration of 0.45 mg / mL. After 2 hours, 100 μL of MTT solubilizing reagent (10% SDS in 0.01 M HCl) was added to each well, and the plate was maintained in the dark overnight. After shaking for 30 seconds, the absorbance at 570 nm was read.

[0416] Results and Discussion The results are shown in Figures 9 and 10, and in Tables 15 (Table 16) and Table 16 (Table 17).

[0417] [Table 16]

[0418] [Table 17]

[0419] The results demonstrate that in DBTRG-05MG cells, 5hm2dC showed synergistic effects with olaparib, veliparib, and niraparib, and 5f2dC showed synergistic effects with olaparib. Combinations of 5hm2dC or 5f2dC with PARP inhibitors resulted in greater cell death than predicted by the additive effect. For example, the combination of 1 μM 5hm2dC and 1 μM olaparib resulted in 49.2% cell death, but the theoretical additive effect did not predict cell death (Figure 9A).

[0420] (Example 6) Combination effect of PARP inhibitors and 5hm2dC or 5f2dC on cancer cell survival rate (Caki-1, CCRF-CEM, and HT-29 cells) The objective of the study was to determine whether 5hm2dC or 5f2dC could be used in combination with the PARP inhibitors (PARPi) olaparib, veliparib, and niraparib to enhance the efficacy of 5hm2dC, 5f2dC, or PARP inhibitors as cancer treatments in Caki-1 cells (clear cell carcinoma cell line), CCRF-CEM cells (acute lymphoblastic leukemia (ALL) cell line), and HT-29 cells (adenocarcinoma cell line) compared to their use individually. The cells used were BRCA1-positive (i.e., functional), BRCA2-positive, MUS81-positive, DNPH1-positive, and XRCC1-positive cells.

[0421] Caki-1, CCRF-CEM, and HT-29 cells were cultured for 96 hours in or without 5hm2dC, 5f2dC, olaparib, veliparib, niraparib, or 5hm2dC combined with any of olaparib, veliparib, or niraparib, or 5f2dC combined with any of olaparib, veliparib, or niraparib. Cell viability was determined by an MTT cell proliferation assay, which showed that both 5hm2dC and PARP inhibitors, and 5f2dC and PARP inhibitors, had synergistic effects in these cell lines. All combinations of 5hm2dC or 5f2dC with each PARPi showed synergistic effects in Caki-1, CCRF-CEM, and HT-29 cells.

[0422] Growth medium and conditions Caki-1, CCRF-CEM, and HT-29 cells were harvested and seeded into six 96-well plates at 4000, 10000, and 3000 cells / well, respectively. 5hm2dC and 5f2dC were diluted in a five-point dilution series at a dilution ratio of 4, with final concentrations of 10 μM, 2.5 μM, 0.63 μM, 0.16 μM, and 0.04 μM, or 1 μM, 0.25 μM, 0.063 μM, 0.016 μM, and 0.004 μM, depending on the cell line. The CCRF-CEM dilution series had an initial concentration of 1 μM for both 5hm2dC and 5f2dC, Caki-1 had an initial concentration of 10 μM for 5hm2dC and 1 μM for 5f2dC, and HT-29 had an initial concentration of 10 μM for both 5hm2dC and 5f2dC.

[0423] PARP inhibitors were added to 5hm2dC or 5f2dC dilution series at a final concentration of 1 μM or 10 μM of olaparib, veliparib, or niraparib. The cell seeding volume was 90 μL, and the drug volume was 5 μL per drug addition. Wells to which only one drug was added contained an additional 5 μL of PBS solution having the same DMSO concentration as the added drug dilution to maintain a constant final volume of 100 μL. Drugs were added in triplicate 5 hours after cell seeding, and the plates were maintained at 37°C in a 5% CO2 humidified atmosphere for 96 hours.

[0424] MTT assay After 96 hours, 10 μL of MTT reagent, prepared by filtration sterilization of a 5 mg / mL solution in PBS, was added to each well to achieve a final concentration of 0.45 mg / mL. After 2-3 hours, 100 μL of MTT solubilizing reagent (10% SDS in 0.01 M HCl) was added to each well according to the cell line confluence. The plate was kept in the dark overnight, and after shaking for 30 seconds, the absorbance at 570 nm was read.

[0425] Results and Discussion The results are shown in Figures 11-16 and Tables 17-25 (Tables 18-26).

[0426] Table 18

[0427] Table 19

[0428] Table 20

[0429] Table 21

[0430] Table 22

[0431] Table 23

[0432] Table 24

[0433] Table 25

[0434] Table 26

[0435] The results demonstrate that 5hm2dC and 5f2dC exhibit synergistic effects with olaparib, veliparib, and niraparib in Caki-1, CCRF-CEM, and HT-29 cells. The combination of 5hm2dC and 5f2dC with PARP inhibitors resulted in greater cell death than predicted by the additive effect. For example, the combination of 1 μM 5hm2dC and 10 μM velip resulted in 81.6% cell death in CCRF-CEM cells, while the theoretical additive effect predicted only 11.3% cell death (Figure 13B).

[0436] (Example 7) Combination effect of PARP inhibitors with 5hm2dC or 5f2dC on cancer cell survival rate (M14, SN12C, and HeLa cells) The objective of the study was to determine whether 5hm2dC, 5f2dC, or 5hm2dC could be used in combination with the PARP inhibitors (PARPi) rucaparib and talazoparib to enhance the efficacy of 5hm2dC, 5f2dC, or PARP inhibitors as cancer treatments in M14 cells (achromatic melanoma cell line), SN12C cells (renal cell carcinoma cell line), and HeLa cells (adenocarcinoma cell line) compared to their use individually. The cells used were BRCA1-positive (i.e., functional), BRCA2-positive, MUS81-positive, DNPH1-positive, and XRCC1-positive cells.

[0437] M14, SN12C, and HeLa cells were cultured for 96 hours in or without 5hm2dC combined with either rucaparib or talazoparib, or 5f2dC combined with either rucaparib or talazoparib. Cell viability was determined by an MTT cell proliferation assay, which showed that both 5hm2dC and a PARP inhibitor, and 5f2dC and a PARP inhibitor, had synergistic effects in these cell lines. All combinations of 5hm2dC or 5f2dC with each PARPi showed synergistic effects in M14, SN12C, and HeLa cells.

[0438] Growth medium and conditions M14, SN12C, and HeLa cells were harvested and seeded at 3500, 3000, and 1000 cells / well in four 96-well plates per cell line, respectively. 5hm2dC and 5f2dC were diluted in a five-point dilution series at a dilution ratio of 4, resulting in final concentrations of 10 μM, 2.5 μM, 0.63 μM, 0.16 μM, and 0.04 μM, or 1 μM, 0.25 μM, 0.063 μM, 0.016 μM, and 0.004 μM, depending on the cell line.

[0439] Lucaparib was added to a 5hm2dC or 5f2dC dilution series at a final concentration of 1, 5, or 10 μM, and talazoparib was added to a 5hm2dC or 5f2dC dilution series at a final concentration of 0.01 or 0.1 μM. The cell seeding volume was 90 μL, and the drug volume was 5 μL per drug addition. Wells to which only one drug was added contained an additional 5 μL of PBS solution having the same DMSO concentration as the added drug dilution to maintain a constant final volume of 100 μL. Drugs were added in triplicate 2 hours after cell seeding, and the plates were maintained at 37°C in a 5% CO2 humidified atmosphere for 96 hours.

[0440] MTT assay After 96 hours, 10 μL of MTT reagent, prepared by filtration sterilization of a 5 mg / mL solution in PBS, was added to each well to achieve a final concentration of 0.45 mg / mL. After 2–4 hours, 100 μL of MTT solubilizing reagent (10% SDS in 0.01 M HCl) was added to each well according to the cell line confluence. The plate was kept in the dark overnight, and after shaking for 30 seconds, the absorbance at 570 nm was read.

[0441] Results and Discussion The results are shown in Figures 17-22 and Tables 26-31 (Tables 27-32).

[0442] [Table 27]

[0443] [Table 28]

[0444] [Table 29]

[0445] [Table 30]

[0446] [Table 31]

[0447] [Table 32]

[0448] The results demonstrate that 5hm2dC and 5f2dC showed synergistic effects with rucaparib and talazoparib in M14, SN12C, and HeLa cells.

[0449] When comparing the individual combination effects (predicted additive effect) of 5hm2dC / 5f2dC and rucaparib / talazoparib with the observed actual effects, lower cell viability is observed compared to what was expected. For example, the combination of 10 μM 5hm2dC and 0.01 μM talazoparib resulted in 73.1% cell death in M14 cells, but the theoretical additive effect predicted only 37.2% cell death (Figure 17B).

[0450] (Example 8) Combination effect of PARP inhibitors with 5hm2dC or 5f2dC on cancer cell survival rate (KCL-22, U937, and OCI-AML3 cells) The objective of the study was to determine whether 5hm2dC or 5f2dC could be used in combination with the PARP inhibitors (PARPi) olaparib, veliparib, and niraparib to enhance the efficacy of 5hm2dC, 5f2dC, or PARP inhibitors as cancer treatments in KCL-22 cells (chronic myeloid leukemia cell line), U937 cells (histiocytic lymphoma cell line), and OCI-AML3 cells (acute myeloid leukemia cell line) compared to their use individually. The cells used were BRCA1-positive (i.e., functional), BRCA2-positive, MUS81-positive, DNPH1-positive, and XRCC1-positive cells.

[0451] KCL-22, U937, and OCI-AML3 cells were cultured for 96 hours in or without 5hm2dC combined with olaparib, veliparib, or niraparib, or 5f2dC combined with olaparib, veliparib, or niraparib. Cell viability was determined by an MTT cell proliferation assay, which showed that both 5hm2dC and 5f2dC combined with PARP inhibitors had synergistic effects in these cell lines. All combinations of 5hm2dC or 5f2dC with each PARPi showed synergistic effects in KCL-22, U937, and OCI-AML3 cells.

[0452] Growth medium and conditions KCL-22, U937, and OCI-AML3 cells were harvested and seeded at 16,000, 20,000, and 20,000 cells / well in six 96-well plates per cell line, respectively. The sensitivity of each cell line to the drug was determined prior to this setup and used to optimize the final concentration. 5hm2dC and 5f2dC were diluted in a five-point dilution series at a dilution ratio of 4, resulting in final concentrations of 10 μM, 2.5 μM, 0.63 μM, 0.16 μM, and 0.04 μM for 5hm2dC, and 1 μM, 0.25 μM, 0.063 μM, 0.016 μM, and 0.004 μM for 5f2dC.

[0453] PARP inhibitors were added to a 5hm2dC or 5f2dC dilution series at final concentrations of 1, 5, 10, or 20 μM olaparib, veliparib, or niraparib. The cell seeding volume was 90 μL, and the drug volume was 5 μL per drug addition. Wells to which only one drug was added contained an additional 5 μL of PBS solution having the same DMSO concentration as the added drug dilution to maintain a constant final volume of 100 μL. Drugs were added in triplicate 1–2 hours after cell seeding, and the plates were maintained at 37°C in a 5% CO2 humidified atmosphere for 96 hours.

[0454] MTT assay After 96 hours, 10 μL of MTT reagent, prepared by filtration sterilization of a 5 mg / mL solution in PBS, was added to each well to achieve a final concentration of 0.45 mg / mL. After 2-3 hours, 100 μL of MTT solubilizing reagent (10% SDS in 0.01 M HCl) was added to each well according to the cell line confluence. The plate was kept in the dark overnight, and after shaking for 30 seconds, the absorbance at 570 nm was read.

[0455] Results and Discussion The results are shown in Figures 23-28 and Tables 32-40 (Tables 33-41).

[0456] [Table 33]

[0457] [Table 34]

[0458] [Table 35]

[0459] [Table 36]

[0460] [Table 37]

[0461] [Table 38]

[0462] [Table 39]

[0463] [Table 40]

[0464] [Table 41]

[0465] The results demonstrate that 5hm2dC and 5f2dC exhibit synergistic effects with olaparib, veliparib, and niraparib in KCL-22, U937, and OCI-AML3 cells. Combinations of 5hm2dC or 5f2dC with PARP inhibitors resulted in greater cell death than predicted by the additive effect. For example, the combination of 10 μM 5hm2dC and 1 μM niraparib resulted in 84.1% cell death in OCI-AML3 cells, but the theoretical additive effect did not predict any cell death (Figure 27C).

[0466] (Example 9) The combined effect of the PARP inhibitor pamiparib and 5hm2dC or 5f2dC on cancer cell survival rate (SNB19 and U-87 MG cells). The objective of the study was to determine whether 5hm2dC or 5f2dC could be used in combination with the blood-brain barrier (BBB) ​​permeable PARP inhibitor (PARPi) pamiparib to enhance the efficacy of 5hm2dC, 5f2dC, or pamiparib as cancer treatments in glioblastoma cell lines SNB19 and U-87 MG compared to their use individually. The cells used were BRCA1-positive (i.e., functional), BRCA2-positive, MUS81-positive, DNPH1-positive, and XRCC1-positive cells.

[0467] SNB19 and U-87 MG cells were cultured for 96 hours in or without 5hm2dC combined with pamiparib, or 5f2dC combined with pamiparib. Cell viability was determined by an MTT cell proliferation assay, which showed that both 5hm2dC and 5f2dC combined with pamiparib had a synergistic effect in these cell lines.

[0468] Growth medium and conditions SNB19 and U-87 MG cells were harvested and seeded in six 96-well plates at 3500 cells / well and 2000 cells / well per cell line. The sensitivity of each cell line to the drug was determined prior to this setup and used to optimize the final concentration. 5hm2dC and 5f2dC were diluted in a five-point dilution series at a dilution factor of 4, resulting in final concentrations of 10 μM, 2.5 μM, 0.63 μM, 0.16 μM, and 0.04 μM.

[0469] Pamiparib was added to 5hm2dC or 5f2dC dilution series at final concentrations of 5 μM and 20 μM. The cell seeding volume was 90 μL, and the drug volume was 5 μL per drug addition. Wells to which only one drug was added contained an additional 5 μL of PBS solution having the same DMSO concentration as the added drug dilution to maintain a constant final volume of 100 μL. Drugs were added in triplicate 1–2 hours after cell seeding, and the plates were maintained at 37°C in a 5% CO2 humidified atmosphere for 96 hours.

[0470] MTT assay After 96 hours, 10 μL of MTT reagent, prepared by filtration sterilization of a 5 mg / mL solution in PBS, was added to each well to achieve a final concentration of 0.45 mg / mL. After 2-3 hours, 100 μL of MTT solubilizing reagent (10% SDS in 0.01 M HCl) was added to each well according to the cell line confluence. The plate was kept in the dark overnight, and after shaking for 30 seconds, the absorbance at 570 nm was read.

[0471] Results and Discussion The results are shown in Figures 29 and 30, and in Tables 41 (Table 42) and 42 (Table 43).

[0472] [Table 42]

[0473] [Table 43]

[0474] The results demonstrate that 5hm2dC and 5f2dC exhibit synergistic effects with pamiparib in SNB19 and U-87 MG cells. The combination of 5hm2dC or 5f2dC with pamiparib resulted in greater cell death than predicted by the additive effect. For example, the combination of 0.63 μM 5hm2dC and 5 μM pamiparib resulted in 52.9% cell death in U-87 MG cells, while the theoretical additive effect predicted only 12.76% cell death (Figure 30A).

[0475] (Example 10) The combined effect of PARP inhibitors and 5hm2dC or 5f2dC on the survival rate of HR-deficient cancer cells. The objective of this study was to determine whether 5hm2dC or 5f2dC could be used in combination with the PARP inhibitors (PARPi) olaparib, veliparib, and niraparib to enhance the efficacy of 5hm2dC, 5f2dC, or PARP inhibitors in HR-deficient cell lines. The cells used were HT-29 cells (adenocarcinoma cell line), Caki-1 cells (clear cell carcinoma cell line), HeLa cells (adenocarcinoma cell line), and SN12C cells (renal cell carcinoma cell line), and HR deficiency was induced in these cells by knockdown of the HR gene BRCA1.

[0476] Next, HR-deficient HT-29, Caki-1, HeLa, and SN12C cells were cultured for 96 hours in or without 5hm2dC combined with olaparib, veliparib, or niraparib, or 5f2dC combined with olaparib, veliparib, or niraparib. Cell viability was determined by performing an MTT cell proliferation assay. The results indicate that the combination of 5hm2dC or 5f2dC with any of the PARP inhibitors did not produce a synergistic effect in HR-deficient HT-29, Caki-1, HeLa, and SN12C cells.

[0477] Growth medium and conditions Caki-1 and SN12C cells were harvested and seeded in 96-well plates at 4000 cells / well in a triple configuration. HT-29 and HeLa cells were seeded in 96-well plates at 3000 cells / well and 1000 cells / well, respectively, in a triple configuration.

[0478] Cells were transfected with a BRCA1 siRNA mixture (BRCA1 siRNA: s459, s457, and s458; Thermo Fisher Scientific; catalog numbers: 4390824_s459, 4390824_s457, and 4390824_s458), negative control siRNA (si negative control n.5, Thermo Fisher Scientific, catalog AM4642), or mock transfection to knock down the BRCA1 gene and induce HR deficiency. The transfection reagent RNAiMax (Lipofectamine RNAiMAX, Thermo Fisher Scientific, catalog 13778-150) was diluted in Opti-MEM medium (Gibco, catalog 31985062) according to the manufacturer's instructions. BRCA1 siRNA was also diluted in Opti-MEM medium in a separate tube. The diluted siRNA was added to the diluted lipofectamine RNAiMAX reagent in a 1:1 ratio. The final concentrations of BRCA1 siRNA and negative control siRNA were 1 pmol / well, and the total volume of the siRNA-lipid complex was 10 μL / well.

[0479] 5hm2dC and 5f2dC were diluted in a five-point dilution series at a dilution ratio of 4, with final concentrations of 10 μM, 2.5 μM, 0.63 μM, 0.16 μM, and 0.04 μM, or 1 μM, 0.25 μM, 0.063 μM, 0.016 μM, and 0.004 μM.

[0480] PARP inhibitors were added to 5hm2dC or 5f2dC dilution series at a final concentration of 1 μM or 10 μM of olaparib, veliparib, or niraparib. The cell seeding volume was 90 μL, and the drug volume was 5 μL per drug addition. Wells to which only one drug was added contained an additional 5 μL of PBS solution having the same DMSO concentration as the added drug dilution to maintain a constant final volume of 100 μL. Drugs were added in triplicate 24 hours after transfection, and the plates were maintained at 37°C in a 5% CO2 humidified atmosphere for 96 hours.

[0481] RNA extraction and gene expression Cells were harvested 48 hours after transfection to confirm BRCA1 knockdown. RNA was extracted using an RNA extraction kit (Monarch Total RNA Miniprep kit, New England Biolabs, catalog T2010S) according to the manufacturer's instructions. 1 μg of RNA was reverse transcribed to cDNA according to the manufacturer's instructions (High Capacity RNA-to-cDNA kit, Thermo Fisher Scientific, catalog 4387406). Quantitative PCR (qPCR) was performed using TaqMan technology by combining a cDNA template, TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, catalog 44445577), and a BRCA1 TaqMan assay probe (Thermo Fisher Scientific, catalog 4331182-Hs01556193_m1) according to the manufacturer's instructions.

[0482] MTT assay 96 hours after drug addition, 10 μL of MTT reagent, prepared by filtration sterilization of a 5 mg / mL solution in PBS, was added to each well to achieve a final concentration of 0.45 mg / mL. After 2-3 hours, depending on the cell line confluence, 100 μL of MTT solubilizing reagent (10% SDS in 0.01 M HCl) was added to each well. The plate was kept in the dark overnight, and after shaking for 30 seconds, the absorbance at 570 nm was read.

[0483] Results and Discussion Although the results are not shown, it was found that neither 5hm2dC and each PARP inhibitor nor 5f2dC and each PARP inhibitor produced a synergistic effect in HR-deficient HT-29, Caki-1, HeLa, and SN12C cells. In other words, the combination of 5hm2dC or 5f2dC with each PARPi did not result in greater cell death than the individual predicted additive effect of 5hm2dC or 5f2dC and the PARPi.

Claims

1. A combination of pharmaceuticals for use in treating HR-preserving cancers, comprising a PARP inhibitor and a compound of formula (II): 【Chemistry 1】 or its solvates, tautomers, or pharmaceutically acceptable salts. (In the formula, X is a group containing 1 to 20 nonhydrogen atoms and containing at least one functional group selected from aldehydes, alcohols, protected alcohols, ethers, anhydrides, esters, and carboxylic acids; R 1 This is a group containing H or 1 to 15 non-hydrogen atoms; R 2 is H, -OH, -OPG, -F, -Cl, -Br, -I, or -N 3 and; PG is an alcohol protecting group such as acetyl (Ac), benzyl (Bn), or benzoyl (Bz). A combination of medicines including [the specified ingredient].

2. X is -(CH 2 ) n -X', where n is 0 to 6, and X' is -CHO, -OH, -OR, or -OC(=O)R, where R is methyl; R1 is H, OH, OPG, F, Cl, Br, I, SH, N3, or A1(A2(=A3)(OH)O)nH, where n is 1 to 3, in each case A1 is O, CH2, or NH, A2 is P or S, A3 is O or S, and PG is an alcohol protecting group such as acetyl, benzyl, or benzoyl; R 2 is -OH, A pharmaceutical combination for use as described in claim 1.

3. R 1 is -OH or -O(P(=O)(OH)O) n H is such that n is between 1 and 3. A pharmaceutical combination for use according to claim 1 or 2.

4. The compound of formula (II) is a compound of formula (IIIa) or (IIIb): 【Chemistry 2】 or its solvates, tautomers, or pharmaceutically acceptable salts. (where X is -(CH 2 ) n -X', where n is from 0 to 6, and X' is -CHO, -OH, -OR or -OC(=O)R, where R is methyl) A pharmaceutical combination for use according to any one of claims 1 to 3.

5. X is CHO or -CH 2 A pharmaceutical combination for use according to any one of claims 1 to 4, wherein the combination is OH.

6. A pharmaceutical combination for use according to any one of claims 1 to 5, wherein X is -CHO.

7. X is CH 2 A pharmaceutical combination for use according to any one of claims 1 to 5, wherein the combination is OH.

8. The pharmaceutical combination for use according to any one of claims 1 to 5, wherein the compound is 5-formyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, 5-formyl-2'-deoxycytidine-5'-triphosphate or 5-hydroxymethyl-2'-deoxycytidine-5'-triphosphate, or a solvate, tautomer, or pharmaceutically acceptable salt thereof.

9. The pharmaceutical combination for use according to any one of claims 1 to 5, wherein the compound is 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine, or a stereoisomer, solvate, tautomer, or pharmaceutically acceptable salt thereof.

10. The pharmaceutical combination for use according to any one of claims 1 to 9, wherein the PARP inhibitor is talazoparib, lucaparib, veliparib, olaparib, pamiparib, or niraparib, or any pharmaceutically acceptable salt thereof.

11. A pharmaceutical combination for use according to any one of claims 1 to 10, wherein the HR function-preserving cancer has not been previously treated with a PARP inhibitor.

12. The pharmaceutical combination for use according to any one of claims 1 to 11, wherein the HR function-preserving cancer does not contain loss-of-function mutations in one or more HR genes.

13. The pharmaceutical combination for use according to any one of claims 1 to 12, wherein the HR function-preserving cancer is BRCA1-positive and BRCA2-positive.

14. The pharmaceutical combination for use according to any one of claims 1 to 13, wherein the HR function-preserving cancer is MUS81-positive and DNPH1-positive.

15. The pharmaceutical combination for use according to any one of claims 1 to 14, wherein the HR-preserving cancer is XRCC1 positive.

16. The pharmaceutical combination for use according to any one of claims 1 to 15, wherein the HR function-preserving cancer is a blood cancer, cervical cancer, kidney cancer, colorectal cancer, skin cancer, or central nervous system cancer.

17. The aforementioned hematological cancer is one or more of ALL, AML, CML, and lymphoma; and / or The skin cancer is melanoma; and / or The pharmaceutical combination for use according to claim 16, wherein the central nervous system cancer is one or more of gliomas and glioblastomas.

18. The pharmaceutical combination for use according to any one of claims 1 to 16, wherein the HR function-preserving cancer is ALL, AML, lymphoma, cervical cancer, renal cancer, skin cancer, or central nervous system cancer.

19. The pharmaceutical combination for use according to any one of claims 1 to 18, wherein the HR-preserving cancer is resistant to treatment with the compound of formula (II) alone.

20. A method for treating target HR function-preserving cancer, comprising the step of administering to the target a pharmaceutical combination comprising a PARP inhibitor and a compound of formula (II) as defined in any one of claims 1 to 9.

21. A PARP inhibitor for use in a method of sensitizing target HR function-preserving cancer to treatment with a compound of formula (II) as defined in any one of claims 1 to 9.

22. A compound of formula (II) as defined in any one of claims 1 to 9, for use in a method for sensitizing target HR function-preserving cancer to treatment with a PARP inhibitor.

23. The compound for use according to claim 22, wherein the HR function-preserving cancer is brain cancer.

24. A pharmaceutical composition comprising a PARP inhibitor and a compound of formula (II) as defined in any one of claims 1 to 9, and optionally further comprising one or more pharmaceutically acceptable excipients, wherein the PARP inhibitor and the compound of formula (II) are co-formulated.

25. A kit comprising a PARP inhibitor and a compound of formula (II) as defined in any one of claims 1 to 9, wherein the PARP inhibitor and the compound of formula (II) are provided in separate compartments within the kit.

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