Deoxycytidine derivatives or deoxyuridine derivatives for use in cancer therapy

Abstract

To provide a further anticancer agent for treating glioblastoma multiforme and other cancers, particularly, an anticancer agent for effectively treating cancer which is resistive to treatment by temozolomide.SOLUTION: A pharmaceutical for treating or preventing chronic myeloid leukemia in a subject which consists of a compound of formula (IIa) or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compounds for use in therapy, particularly in the treatment or prevention of cancer. The present invention also relates to pharmaceutical formulations containing such compounds, and to the use of such pharmaceutical formulations in therapy, particularly in the treatment or prevention of cancer. The present invention also relates to such compounds for combination therapy with known anticancer agents. Thus, the present invention relates to the use of the compounds of the present invention in the treatment or prevention of cancer, both alone and in combination with one or more additional or further anticancer agents. The present invention also relates to methods for treating or preventing cancer by using the compounds of the present invention either alone or in combination with one or more further anticancer agents. [Background technology]

[0002] Cancer is a disease characterized by a loss of proper control over cell growth and proliferation. The American Cancer Society estimates that there were more than 1.5 million new cases of cancer in the United States in 2010, and that approximately 570,000 deaths that year were attributed to cancer. The World Health Organization estimates that cancer was the leading cause of death globally in 2010, and that by 2030, cancer will cause 12 million deaths annually.

[0003] Although numerous therapies are available, resistance to known anticancer drugs can pose a challenge to the success of cancer treatment in patients. Further cancer therapies are still needed.

[0004] In certain contexts, anticancer drugs that can kill a wide range of cancer cell types are desirable. Fluorouracil (5-FU) is one such anticancer drug that is routinely administered for the treatment of a wide range of cancers.

[0005] However, many anticancer drugs that can kill a wide range of cancer cell types also kill non-cancerous cells, such as normal, healthy cells that are dividing. This is problematic in some contexts, and therefore, there is a need for broadly cytotoxic anticancer drugs that are not cytotoxic to non-cancerous cells. In addition, there is a need for anticancer drugs that have more specific cytotoxicity, that is, drugs that kill a narrower range of cancer cell types. Such more specific anticancer drugs are less likely to have undesirable off-target effects.

[0006] Glioblastoma multiforme is the most common and invasive cancer of the central nervous system. It is the second most common cancer in children. Adults diagnosed with glioblastoma multiforme have several of the most unmet needs in oncology. With currently available treatments, the mean survival from diagnosis is 14.6 months. Less than 5% of patients diagnosed with glioblastoma multiforme survive for more than 5 years. Long-term survival after a glioblastoma multiforme diagnosis is rare and more common in children.

[0007] Current therapies are largely palliative and designed to improve quality of life. Following diagnosis, current treatment for glioblastoma multiforme follows a similar course: surgical resection of the affected brain region, in which the patient is expected to survive the surgery, followed by radiation and chemotherapy. Glioblastoma multiforme forms tentacle-like structures within the affected brain, and therefore, complete surgical resection is difficult and often impossible, even when directed. In addition to surgical intervention and radiation therapy, three drugs are approved for the treatment of glioblastoma multiforme: 1) Avastin (bevacizumab) - An angiogenesis inhibitor with multiple oncological indications. Avastin is thought to inhibit tumor growth by inhibiting the development of new blood vessels within tumors and effectively starving the tumor. Avastin is approved in the United States and Japan, but it is not approved for the treatment of GBM in the European Union. In fact, a Phase II trial indicated that Avastin does not provide an overall survival benefit (NCI 06-C-0064E). While Avastin slightly improves progression-free survival in some trials, the patient benefit is often minimal, and there is no benefit to Avastin treatment in patients with relapsed glioblastoma multiforme. 2) Temozolomide (Temodal® / Temodar®) (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide) - mutagenic DNA alkylating agent; generic formulations are also available. Temozolomide is thought to inhibit glioblastoma multiforme growth by significantly mutating tumor DNA that induces cell death. Due to its mechanism of action, cells expressing the DNA repair protein MGMT are almost without exception resistant to temozolomide treatment. Temozolomide treatment improves overall survival by only 2.5 months. At the same time, temozolomide treatment often causes significant side effects. Temozolomide is the first-line treatment for glioblastoma multiforme. 3) Gliadel (registered trademark) (carmustine wafer) (1,3-bis(2-chloroethyl)-1-nitrosourea)-cytotoxic nitrogen mustard. This is delivered as a biodegradable disc directly implanted in the brain after surgical resection. A randomized trial demonstrated that Gliadel improved median survival by only 2.1 months. Patients treated with Gliadel reported fewer and less severe side effects than patients treated with temozolomide, but overall survival was reduced compared to temozolomide. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] RA Irizarry et al., Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249~264(2003) [Non-Patent Document 2] Wagner et al., (2012) Theory Biosci 131(4):281~285 [Non-Patent Document 3] Mortazavi A et al. (2008) “Mapping and quantifying mammalian transcriptomes by RNA-Seq.” Nature methods 5(7):621~8 [Non-Patent Document 4] Trapnell et al. (2010) Nature Biotechnology 28, 511–515 [Non-Patent Document 5] Creelan (2014) Cancer Control 21:80~89 [Non-Patent Document 6] Nair et al., (2016) Basic Clin Pharm. 7(2): 27~31 [Non-Patent Document 7] "Comprehensive Organic Synthesis", BM Trost and I. Fleming, Pergamon Press, 1991 [Non-Patent Document 8] "Protective Groups in Organic Synthesis," 3rd edition, TW Greene and PGM Wutz, Wiley-Interscience (1999) [Non-Patent Document 9] Guide for the Care and Use of Laboratory Animals: 8th edition (National Academy Press, Washington, DC, 2011) [Non-Patent Document 10] Towbin et al., 1979. Biotechnology, 24, 145-149. [Non-Patent Document 11] Barretina, J et al. (2012) The Cancer Cell Line Encyclopedia predictive enables modeling of anticancer drug sensitivity. Nature. 483:603~7 [Overview of the project] [Problems that the invention aims to solve]

[0009] Further anticancer drugs are still needed to treat glioblastoma multiforme and other cancers. In particular, there is still a need for anticancer drugs that can effectively treat cancers that are resistant to temozolomide treatment.

[0010] Human protein cytidine deaminase (CDA) catalyzes the hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Some known anticancer agents are nucleoside / nucleotide analogs, e.g., gemcitabine (2,2-difluorodeoxycytidine) and cytarabine (Ara-C, cytosine arabinoside). CDA inactivates such anticancer agents, including gemcitabine and cytarabine, in problematic ways. Further anticancer agents that are not inactivated by CDA are still needed. [Means for solving the problem]

[0011] The inventors have identified a selected class of compounds that exhibit activity when treating glioblastoma multiforme and a wide range of other cancers. Such compounds are generally suitable for inhibiting the proliferation of tumor cells, and in particular those associated with brain cancers, especially glioblastoma multiforme.

[0012] In a first aspect, the present invention relates to a formula (I) for use in therapy:

[0013] [ka]

[0014] [In the formula, X is a group containing 1 to 20 nonhydrogen atoms, having at least one functional group selected from aldehydes, alcohols, protected alcohols, ethers, esters, and carboxylic acids, provided that X is not -COOH. W1 and W2 are independently O, S, or NH. Y is a group containing H or 1 to 15 non-hydrogen atoms. Z is -OPG, -OR z or -N(R x R y ) and here, R x , R y and R zis, independently, H, or a group containing from 1 to 10 non-hydrogen atoms, R1 is H, or a group containing from 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, where PG is an alcohol protecting group such as acetyl (Ac), benzyl (Bn) or benzoyl (Bz), a compound of, or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof is provided.

[0015] The compounds of the present invention are particularly for use in the treatment or prevention of cancer. Thus, in a further aspect, the present invention provides a compound of formula (I) for use in the treatment or prevention of cancer:

[0016]

Chemical formula

[0017] [wherein, X is a group containing from 1 to 20 non-hydrogen atoms and containing at least one functional group selected from aldehyde, alcohol, protected alcohol, ether, ester and carboxylic acid, provided that X is not -COOH, W1 and W2 are each independently O, S or NH, Y is H, or a group containing from 1 to 15 non-hydrogen atoms, Z is -OPG, -OR [[ID=​​​​​​​​​​​​​​R2 is H, -OH, -OPG, -F, -Cl, -Br, -I, or -N3. R3 is H, -F, -Cl, -Br, -I, or -N3. Here, PG is an alcohol protecting group such as acetyl (Ac), benzyl (Bn), or benzoyl (Bz). Compounds thereof, or their stereoisomers, solvates, tautomers, or pharmaceutically acceptable salts. To provide.

[0018] This invention is applicable to all cancers. Cancer is defined herein broadly to include all neoplastic conditions, particularly malignant or premalignant conditions. Cancer can cause, result in, or arise from solid tumors, but is not limited to solid tumors, and also includes hematopoietic cancers. Throughout this text, the terms “cancer” and “cancer cells” are used interchangeably. Throughout this text, the terms “tumor” and “tumor cells” are used interchangeably. Benign tumors and malignant tumors are also included in the term cancer when used herein; that is, the terms cancer and tumor are used interchangeably. Treatment of malignant tumors is preferred.

[0019] Therefore, in an alternative view, the present invention provides a compound of formula (I) for use in the treatment or prevention of tumors.

[0020] Alternatively, the present invention provides a compound of formula (I) for use as an anticancer agent. Alternatively, the present invention provides the use of a compound of formula (I) for treating or preventing cancer.

[0021] Alternatively, the present invention provides a compound of formula (I) for use as an antitumor agent. Alternatively, the present invention provides the use of a compound of formula (I) for treating or preventing tumors.

[0022] In an alternative view, this aspect of the present invention provides the use of a compound of formula (I) in the manufacture of an anti-cancer therapeutic product (i.e., preparation or pharmaceutical, e.g., pharmaceutical composition, formulation, complex product, co-formulated product or kit), or alternatively, the use of a compound of formula (I) in the manufacture of a pharmaceutical for use as an anti-cancer agent or in the treatment or prevention of cancer.

[0023] In an alternative view, this aspect of the present invention provides the use of a compound of formula (I) in the manufacture of an antitumor therapeutic product (i.e., preparation or pharmaceutical, e.g., pharmaceutical composition, formulation, complex product, co-formulated product or kit), or alternatively, the use of a compound of formula (I) in the manufacture of a pharmaceutical for use as an antitumor agent or in the treatment or prevention of tumors.

[0024] In a further embodiment, the present invention also provides a method for treating or preventing cancer in a subject, comprising the step of administering a compound of formula (I) to the subject.

[0025] In a further embodiment, the present invention also provides a method for treating or preventing a tumor in a subject, comprising the step of administering a compound of formula (I) to the subject.

[0026] In some preferred embodiments of the present invention, the compound of formula (I) is used as the sole activator (the sole activator in the treatment regimen). Therefore, in some preferred embodiments, the treatment is monotherapy. Monotherapy refers to the use of a single drug to treat a disease or condition in this cancer (i.e., tumor). Therefore, in some preferred embodiments, the compound of formula (I) is used alone. "Sole activator" (or sole active ingredient) means the sole active substance or ingredient that is therapeutically active (or biologically active). Therefore, ingredients such as preservatives, excipients, or active substances unrelated to the disease being treated are not considered activators.

[0027] In the treatment or prevention of cancer (i.e., tumors), the compounds of formula (I) may be used alone or, as may be, in combination with further, i.e., one or more other anticancer agents (i.e., additional or second anticancer agents).

[0028] The compound of formula (I) may be used alone or in combination with further anticancer agents in any method of treating or preventing cancer (i.e., tumors) in a subject, according to the present invention.

[0029] As demonstrated in the examples, the use of the compound of formula (I) in combination with one or more additional anticancer agents results in a synergistic cytotoxic effect.

[0030] Accordingly, in a further embodiment, the present invention provides a compound of formula (I) in combination with a further anticancer agent for use in treating or preventing cancer (i.e., tumors), or alternatively, a compound of formula (I) for use in combination with a further anticancer agent for treating or preventing cancer (i.e., tumors).

[0031] In an alternative view, this aspect of the present invention provides the use of a compound of formula (I) in the manufacture of an anti-cancer (i.e., antitumor) therapeutic product (i.e., preparation or pharmaceutical, e.g., pharmaceutical composition, formulation, complex product or kit), or alternatively, the use of a compound of formula (I) as an anticancer agent (i.e., antitumor agent) or for the manufacture of a pharmaceutical for use in the treatment or prevention of cancer (i.e., tumor), wherein the treatment further comprises the administration of a further anticancer agent.

[0032] In a further embodiment, the present invention also provides a method for treating or preventing cancer (i.e., a tumor) in a subject, comprising the step of administering a compound of formula (I), optionally together with a further (i.e., a second) anticancer agent, to the subject. In particular, in this embodiment, the method comprises the step of administering an effective amount of the compound of formula (I) and an optional further anticancer agent.

[0033] The compound of formula (I) and further anticancer agents may be co-formulated into a single composition. However, this is not necessary. The pharmaceutical product may be a combination preparation, composition or kit, and in any aspect of the present invention, it is not necessary for the compound of formula (I) and further anticancer agents to be co-formulated into a single composition—they may be formulated separately and administered separately, including sequentially or simultaneously.

[0034] Accordingly, the present invention also provides a kit comprising a compound of formula (I) and further (i.e., one or more further or second) anticancer agents for use in the treatment or prevention of cancer (i.e., tumors).

[0035] More specifically, the present invention provides products (particularly pharmaceutical products) comprising a compound of formula (I) and further (i.e., one or more further or second) anticancer agents as combination preparations for use separately, sequentially, or simultaneously in the treatment or prevention of cancer (i.e., tumors).

[0036] In addition, the present invention provides products (particularly pharmaceutical products) comprising a compound of formula (I) co-formulated with further (i.e., one or more further or second) anticancer agents.

[0037] The present invention provides a pharmaceutical composition comprising a compound of formula (I) and one or more pharmaceutically acceptable excipients, and optionally further comprising a further anticancer agent.

[0038] In all aspects of the present invention, the compounds of the present invention are compounds of formula (I) as described elsewhere in this specification, and preferred and optional embodiments of the compounds described in one aspect of the present invention shall apply mutatis mutandis to all other aspects of the present invention.

[0039] In all aspects and embodiments of the present invention, treatment of malignant tumors is preferred.

[0040] X X is a group containing 1 to 20 nonhydrogen atoms, which contains at least one functional group selected from aldehydes, alcohols, protected alcohols, ethers, esters, and carboxylic acids, provided that X is not a -COOH group.

[0041] Preferably, X is a group containing 1 to 10 non-hydrogen atoms, more preferably 1 to 5 non-hydrogen atoms, even more preferably 1 to 3 non-hydrogen atoms, and most preferably 2 non-hydrogen atoms.

[0042] 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, even more preferably 2 to 5 non-hydrogen atoms, even more preferably 2 to 3 non-hydrogen atoms, and most preferably 2 non-hydrogen atoms.

[0043] Preferably, X contains at least one functional group selected from aldehydes, alcohols, protected alcohols, ethers, 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.

[0044] Preferably, X contains only one functional group.

[0045] Preferably, X is a group as defined herein, except that X is neither -COOH nor -OH.

[0046] X can be defined as -L-X', where, L is a bond, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, or haloalkoxy. X' is -CHO, -OH, -OPG, -COOH, -OR, -OC(=O)R, or -C(=O)OR, where PG is an alcohol protecting group such as acetyl (Ac), benzyl (Bn), or benzoyl (Bz), and R is an alkyl group, preferably methyl.

[0047] The term "alkyl" refers to linear and branched saturated aliphatic hydrocarbon chains. Preferably, alkyl is C 1~10 This refers to alkyl groups. 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).

[0048] R can be any alkyl group as exemplified above. For example, R could be -(CH2) n H may be H, where n is from 1 to 10, preferably from 1 to 5, more preferably from 1 to 3, and most preferably 1. When n is 1, R is CH3.

[0049] The term "alkenyl" refers to a linear and branched hydrocarbon chain having one or more, preferably one or two, carbon-carbon double bonds. Preferably, the alkenyl is C 2~10 This refers to alkenyl groups. 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.

[0050] The term "alkynyl" refers to a linear and branched hydrocarbon chain having one or more, preferably one or two, carbon-carbon triple bonds. Preferably, the alkynyl is C 2~10 This refers to alkynyl groups. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, and propargyl.

[0051] The term "haloalkyl" refers to linear and branched saturated aliphatic hydrocarbon chains substituted with one or more halogens (fluoro(F), chloro(Cl), bromo(Br), and iod(I)). Preferably, the haloalkyl is C 1~10 This 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.

[0052] 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.

[0053] The term "haloalkoxy" refers to a haloalkyl group as defined above, bonded via an oxygen bridge. 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 pentafluorothoxy.

[0054] If X is -L-X', the X group must still contain the required number of non-hydrogen atoms.

[0055] 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 alkyl. More preferably, L is a bond or C 1~4 It is alkyl. Most preferably, L is a bond or C1 alkyl(-CH2-).

[0056] X' is preferably -CHO, -OH, -OPG, -OR, -OC(=O)R or -C(=O)OR, more preferably -CHO, -OH, -OR, -OC(=O)R or -C(=O)OR, and most preferably -CHO, -OH, -OR or -OC(=O)R.

[0057] Therefore, preferably, X is -(CH2) n -X', where n is from 0 to 6, preferably from 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.

[0058] Preferably, X is -(CH2) n -X', where n is from 0 to 6, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is from 0 to 6, and X' is -OH. Preferably, X is -(CH2) n -X', where n is between 0 and 6, and X' is -CHO.

[0059] More preferably, X is -(CH2) n -X', where n is from 0 to 4, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is from 0 to 4, and X' is -OH. Preferably, X is -(CH2) n -X', where n is between 0 and 4, and X' is -CHO.

[0060] More preferably, X is -(CH2) n -X', where n is between 0 and 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-X', where n is between 0 and 2, and X' is -CHO.

[0061] 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.

[0062] More preferably, X is -CHO, -CH2OH, -CH2OCH3, or -CH2OC(=O)CH3.

[0063] More preferably, X is a) -CHO or -CH2OH; or b) -CH2OCH3 or -CH2OC(=O)CH3.

[0064] More preferably, X is -CHO or -CH2OH, most preferably CH2OH.

[0065] In all of the above definitions of X, it is preferable that X is not -OH. Therefore, if X' is -OH, it is preferable that L is not a bond (i.e., n is not 0). In this case, n can be from 1 to 6, preferably from 1 to 4, more preferably from 1 to 2, and most preferably 1.

[0066] W1 and W2 W1 and W2 are each independently O, S, or NH, preferably O or S, and more preferably O.

[0067] 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.

[0068] More preferably, W1 and W2 are both 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.

[0069] Most preferably, both W1 and W2 are O.

[0070] 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.

[0071] For example, Y may be H, -OH, -OPG, -F, -Cl, -Br, -I, or -N3, where PG is an alcohol protecting group such as acetyl, benzyl, or benzoyl.

[0072] If Y is a group containing H or 1 to 5 non-hydrogen atoms, then Y can be H, -OH, -OAc, -F, -Cl, -Br, -I, or -N3.

[0073] Most preferably, Y is H.

[0074] Z Z is -OPG, -OR z or -N(R x R y ) and here, R x , R y and R z This is independently a group containing H or 1 to 10 non-hydrogen atoms, where PG is an alcohol protecting group such as acetyl, benzyl, or benzoyl.

[0075] Preferably, Z is -OR z or -N(R x R y )

[0076] Preferably, R zThis is a group containing H or 1 to 5 non-hydrogen atoms, more preferably H or a group containing 1 to 3 non-hydrogen atoms, most preferably H.

[0077] 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 between 1 and 4, preferably 4.

[0078] 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 from 1 to 4, preferably 4. More preferably R x and R y All of these are H.

[0079] Therefore, Z is preferably -NH2 or -OH. More preferably, if X is -CHO or -CH2OH, most preferably -CH2OH, then Z is -NH2; if X is -CH2OCH3 or -CH2OC(=O)CH3, then Z is -OH.

[0080] When Z is -OH, the compound of formula (I) can be depicted in tautomerized forms as shown below.

[0081] [ka]

[0082] Alternatively, Z is preferably -OPG, -OR z or -N(R x R y ) and here, PG, R x and Ry As defined above, R z This is a group containing 1 to 10 non-hydrogen atoms.

[0083] In this case, R z This is preferably a group containing 1 to 5 non-hydrogen atoms, more preferably a group containing 1 to 3 non-hydrogen atoms.

[0084] In this case, Z is preferably -OPG or -N(R x R y ) and here, PG, R x and R y This is as defined above.

[0085] Most preferably, Z is -N(R x R y ) and here, R x and R y This is as defined above.

[0086] Therefore, Z is preferably -NH2.

[0087] R1 R1 is a group containing H or 1 to 15 non-hydrogen atoms, preferably H or a group containing 1 to 13 non-hydrogen atoms.

[0088] Preferably, R1 is H, -OH, -OPG, -F, -Cl, -Br, -I, -N3, or -O(P(=O)(OH)O) n H is the alcohol protecting group, where n is between 1 and 3, and PG is an alcohol protecting group such as acetyl, benzyl, or benzoyl.

[0089] Preferably, R1 is H, -OH, -F, -Cl, -Br, -I, -N3, or -O(P(=O)(OH)O) n H, where n is between 1 and 3, preferably 3. More preferably, R1 is H, -OH, or -O(P(=O)(OH)O) nH is H, where n is between 1 and 3, preferably 3. More preferably, R1 is -OH or -O(P(=O)(OH)O) n H is H, where n is between 1 and 3, preferably 3. Most preferably, R1 is -OH.

[0090] R2 R2 is H, -OH, -OPG, -F, -Cl, -Br, -I, or -N3, where PG is an alcohol protecting group such as acetyl, benzyl, or benzoyl.

[0091] Preferably, R2 is H, -OH, -F, -Cl, -Br, -I, or -N3. More preferably, R2 is H or -OH, and most preferably, R2 is -OH.

[0092] R3 R3 is H, -F, -Cl, -Br, -I, or -N3, preferably H.

[0093] Most preferably, R1 is -OH or -O(P(=O)(OH)O) n R2 is H, where n is between 1 and 3, preferably 3; R2 is -OH; and R3 is H. [Brief explanation of the drawing]

[0094] [Figure 1A] This figure shows that 5-formyl-2'-deoxycytidine and 5-hydroxymethylcytidine are well tolerated in mice and reduce human glioblastoma pleomorphism tumors in a mouse xenograft model. Single-dose maximum tolerated dose protocol. Mice were administered the prescribed dose by a single intraperitoneal injection. If all mice in the relevant group showed tolerance to the prescribed dose, the dose was escalated as instructed. [Figure 1B]This figure shows that 5-formyl-2'-deoxycytidine and 5-hydroxymethylcytidine are well-tolerated in mice and reduce human glioblastoma pleomorphism tumors in a mouse xenograft model. Mice were intraperitoneally injected with the indicated dose and compound every 3 days for a total of 5 doses, and their weight was measured at the indicated time. [Figure 1C] ~ [Figure 1H] This figure shows that 5-formyl-2'-deoxycytidine and 5-hydroxymethylcytidine are well-tolerated in mice and reduce human glioblastoma pleomorphism tumors in a mouse xenograft model. U87-MG cells were transplanted into the flanks of 32 immunodeficient mice. After the tumors reached 129-131 mm3, the mice were divided into four groups of eight. The negative control group was treated with a vehicle, the positive control group was treated with 40 mg / kg temozolomide once daily for 5 days, and the treatment group was treated with 2000 mg / kg 5-formyl-2'-deoxycytidine or 2000 mg / kg 5-hydroxymethyl-2'-deoxycytidine once every 3 days for a total of 5 doses. Tumor volume was measured every 3 days (Figure 1C), and mouse body weight was measured every 3 days (Figure 1D). At the completion of the study, the tumor growth inhibition percentage (TGI(%)) was calculated (Figure 1E), the tumors were excised, photographed (Figure 1F), measured (Figure 1G), incised, and stained with hematoxylin and eosin (Figure 1H). [Figure 2] This figure shows that 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine kill glioblastoma multiforme through a mechanism unrelated to current nucleotide analogs. Figure 2A: Flow cytometry of cells treated with 5-formyl-2'-deoxycytidine and 5'-hydroxymethyl-2'-deoxycytidine, stained with Annexin V and 7AAD. Figure 2B: Survival curves of HeLa cells treated by titration with 5-formylcytosine, 5-formylcytidine, or 5-formyl-2'-deoxycytidine. Figure 2C: Survival curves of U87-MG cells treated with escalating 5-hydroxymethylcytosine, 5-hydroxymethylcytidine, or 5-hydroxymethyl-2'-deoxycytidine. [Figure 3] This figure shows the quantification of mutations in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene induced by 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine in mammalian cells. It has been shown that 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine are not mutagenic. [Figure 4] This figure shows the levels of cytotoxicity (survival %) of 5-formyl-2'-deoxycytidine, 5-formylcytidine, and 5-chloro-2'-deoxycytidine in HeLa cells 3 days after treatment. [Figure 5] This figure shows the levels of cytotoxicity (survival %) of U87-MG cells against 5-formyl-2'-deoxycytidine (d5fC), 5-hydroxymethyl-2'-deoxycytidine (d5hmC), 5-chloro-2'-deoxycytidine (5CldC), 5-bromo-2'-deoxycytidine (5BrdC), 5-iodo-2'-deoxycytidine (5IdC), and thymidine. [Figure 6] This figure shows that the cytotoxic effects (survival percentage) of 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine are not rescued by the addition of thymidine in U87-MG cells, and that 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine do not act by inhibiting thymidine synthase. [Figure 7] This figure shows the cytotoxic effect (survival percentage) of 5-formyl-2'-deoxycytidine combined with temozolomide in U87-MG cells. [Figure 8] This figure shows the cytotoxic effect (survival percentage) of 5-hydroxymethyl-2'-deoxycytidine combined with temozolomide in U87-MG cells. [Figure 9]This figure shows the cytotoxic effects of 5-methoxymethyl-2'-deoxyuridine and 5-acetoxymethyl-2'-deoxyuridine on U87-MG cells over a 72-hour treatment period. Survival was quantified using the MTT assay. [Figure 10] This figure shows the survival percentages of various cells after treatment with d5fCTP or d5hmCTP. Figure 10A: HeLa survival percentage after 72 hours of treatment with 5-formyl-2'-deoxycytidine-5'-triphosphate (d5fCTP). Survival was quantified using an MTT assay. Figure 10B: U87-MG cells (glioma, grade IV) survival percentage after 72 hours of treatment with either 5-formyl-2'-deoxycytidine-5'-triphosphate or 5-hydroxymethyl-2'-deoxycytidine-5'-triphosphate. Survival was quantified using an MTT assay. [Figure 11] This figure shows CDA expression levels in various cell lines. The values ​​are normalized Log2 CDA expression levels. Expression levels were determined using the Affymetrix Human Genome U133 Plus 2.0 array platform and the Genevestigator database (https: / / genevestigator.com / gv / ). [Figure 12] This figure shows the linear CDA expression levels in various cell lines. Expression levels were determined using the Affymetrix Human Genome U133 Plus 2.0 array platform and the Genevestigator database (https: / / genevestigator.com / gv / ). [Figure 13A] This figure shows CDA expression levels in various human brain tumors. The values ​​are normalized Log2 CDA expression levels. Expression levels were determined using the Affymetrix Human Genome U133 Plus 2.0 array platform and the Genevestigator database (https: / / genevestigator.com / gv / ). [Figure 13B]This figure shows CDA expression levels in various human brain tumors. The values ​​are normalized Log2 CDA expression levels. Expression levels were determined using the Affymetrix Human Genome U133 Plus 2.0 array platform and the Genevestigator database (https: / / genevestigator.com / gv / ). [Figure 14A] This figure shows the linear CDA expression levels in various human brain tumors. Expression levels were determined using the Affymetrix Human Genome U133 Plus 2.0 array platform and the GENEVESTIGATOR® database (https: / / genevestigator.com / gv / ). [Figure 14B] This figure shows the linear CDA expression levels in various human brain tumors. Expression levels were determined using the Affymetrix Human Genome U133 Plus 2.0 array platform and the GENEVESTIGATOR® database (https: / / genevestigator.com / gv / ). [Figure 15] This figure shows the results of a PAMPA assay demonstrating that 2d5hmC and 2d5fC can cross the blood-brain barrier. [Modes for carrying out the invention]

[0095] Preferably, the compound of formula (I) is of formula (IIa) or formula (IIb):

[0096] [ka]

[0097] [In the formula, X, R1, and R2 are as defined above.] The compound, or its stereoisomer, solvate, tautomer, or pharmaceutically acceptable salt.

[0098] More preferably, the compound of formula (I) is the compound of formula (IIa), or a stereoisomer, solvate, tautomer, or pharmaceutically acceptable salt thereof.

[0099] In these preferred embodiments, in particular, in formula (IIa), X is preferably -(CH2) n -X', where n is from 0 to 6, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is from 0 to 6, and X' is -OH. Preferably, X is -(CH2) n -X', where n is between 0 and 6, and X' is -CHO.

[0100] More preferably, X is -(CH2) n -X', where n is from 0 to 4, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is from 0 to 4, and X' is -OH. Preferably, X is -(CH2) n -X', where n is between 0 and 4, and X' is -CHO.

[0101] More preferably, X is -(CH2) n -X', where n is between 0 and 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 -X', where n is between 0 and 2, and X' is -CHO.

[0102] 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.

[0103] More preferably, X is -CHO, -CH2OH, -CH2OCH3 or -CH2OC(=O)CH3. More preferably, X is a) -CHO or -CH2OH; or b) -CH2OCH3 or -CH2OC(=O)CH3. More preferably, X is -CHO or -CH2OH, most preferably CH2OH.

[0104] In formula (IIa), preferably, X is -CHO or -CH2OH, most preferably -CH2OH. In formula (IIb), preferably, X is -CH2OCH3 or -CH2OC(=O)CH3.

[0105] In all of these preferred embodiments, it is preferred that X is not -OH. Therefore, when X' is -OH, it is preferred that n is not 0. In this case, n can be from 1 to 6, preferably from 1 to 4, more preferably from 1 to 2, and most preferably 1.

[0106] In formulas (IIa) and (IIb), preferably, R1 is -OH or -O(P(=O)(OH)O) n H, where n is from 1 to 3, preferably 3; and R2 is -OH.

[0107] More preferably, the compound of formula (I) is of formula (IIIa), (IIIb), (IIIc) or (IIId):

[0108] [[ID= 25]]

Chemical formula

[0109] [where X is as defined above] or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof.

[0110] [[ID= 39]] More preferably, the compound of formula (I) is a compound of formula (IIIa), (IIIb) or (IIIc) or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof.

[0111] Even more preferably, the compound of formula (I) is a compound of formula (IIIa) or (IIIc) or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof.

[0112] In formulas (IIIa), (IIIb), (IIIc) and (IIId), particularly in formulas (IIIa) and (IIIc), preferably, X is -(CH2) n -X', where n is from 0 to 6 and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is from 0 to 6 and X' is -OH. Preferably, X is -(CH2) n -X', where n is from 0 to 6 and X' is -CHO.

[0113] More preferably, X is -(CH2) n -X', where n is from 0 to 4 and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is from 0 to 4 and X' is -OH. Preferably, X is -(CH2) n -X', where n is from 0 to 4 and X' is -CHO.

[0114] More preferably, X is -(CH2) n -X', where n is from 0 to 2 and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is from 0 to 2 and X' is -OH. Preferably, X is -(CH2) n -X', where n is from 0 to 2 and X' is -CHO.

[0115] 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.

[0116] More preferably, X is -CHO, -CH2OH, -CH2OCH3, or -CH2OC(=O)CH3. More preferably, X is a) -CHO or -CH2OH; or b) -CH2OCH3 or -CH2OC(=O)CH3. More preferably, X is -CHO or -CH2OH, most preferably CH2OH.

[0117] In formulas (IIIa) and (IIIc), X is preferably -CHO or -CH2OH, most preferably -CH2OH. In formulas (IIIb) and (IIId), X is preferably -CH2OCH3 or -CH2OC(=O)CH3.

[0118] In all of these preferred embodiments, it is preferable that X is not -OH. Therefore, if X' is -OH, it is preferable that n is not 0. In this case, n can be from 1 to 6, preferably from 1 to 4, more preferably from 1 to 2, and most preferably 1.

[0119] Most preferably, the compound of formula (I) is formula (IVa), (IVb), (IVc), (IVd), (IVe), or (IVf):

[0120] [ka]

[0121] The compound, or its stereoisomer, solvate, tautomer, or pharmaceutically acceptable salt.

[0122] Formula (IVa) is 5-formyl-2'-deoxycytidine (also referred to herein as 5f2dC, 5fdC, 2d5fC, and d5fC). Formula (IVb) is 5-hydroxymethyl-2'-deoxycytidine (also referred to herein as 5hm2dC, 5hmdC, 2d5hmC, and d5hmC). Formula (IVc) is 5-methoxymethyl-2'-deoxyuridine. Formula (IVd) is 5-acetoxymethyl-2'-deoxyuridine. Formula (IVe) is 5-formyl-2'-deoxycytidine-5'-triphosphate. Formula (IVf) is 5-hydroxymethyl-2'-deoxycytidine-5'-triphosphate.

[0123] Therefore, preferably, the compounds used in the present invention are selected from 5-formyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, 5-methoxymethyl-2'-deoxyuridine, 5-acetoxymethyl-2'-deoxyuridine, 5-formyl-2'-deoxycytidine-5'-triphosphate and 5-hydroxymethyl-2'-deoxycytidine-5'-triphosphate or their stereoisomers, solvates, tautomers or pharmaceutically acceptable salts. More preferably, the compounds are a) 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine or their stereoisomers, solvates, tautomers or pharmaceutically acceptable salts; or b) 5-methoxymethyl-2'-deoxyuridine or 5-acetoxymethyl-2'-deoxyuridine or their stereoisomers, solvates, tautomers or pharmaceutically acceptable salts.

[0124] Alternatively, the compounds used in the present invention are preferably 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 stereoisomers, solvates, tautomers or pharmaceutically acceptable salts.

[0125] Most preferably, the compound is 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine or its stereoisomers, solvates, tautomers, or pharmaceutically acceptable salts, most preferably 5-hydroxymethyl-2'-deoxycytidine or its stereoisomers, solvates, tautomers, or pharmaceutically acceptable salts.

[0126] The terms “treatment” or “therapy” include any treatment or therapy that results in improvement of the patient’s health or condition, or of the symptoms of the cancer the patient has. “Treatment” is not limited to curative treatment (e.g., resulting in the elimination of cancer cells or tumors or metastases from the patient), but includes any therapy that has a beneficial effect on the patient’s cancer, such as tumor regression or reduction, reduction of metastatic potential, increase in overall survival, extension or prolongation of life or remission, induction of remission, slowing or reducing the rate of disease progression or tumor development, subjective improvement in quality of life, reduction of pain or other symptoms related to the disease, improvement of appetite, reduction of nausea, or alleviation of any symptoms of cancer.

[0127] Therefore, as used herein, “treatment” may mean reducing, alleviating, relieving, or eliminating the treated cancer or one or more of its symptoms compared to the cancer or symptoms before treatment. Treatment may include, for example, reduction or elimination of cancer cells in a tumor, for example, in a solid tumor. Treatment is treatment of a subject, i.e., a subject that requires it. Therefore, treatment may include reduction of tumor size, or prevention of tumor growth or further tumor growth, i.e., stabilization of tumor size.

[0128] "Prevention" refers, for example, to delaying or preventing the onset of cancer symptoms in the development of a tumor.

[0129] Preferably, the compounds of the present invention have a direct effect on cancer / tumor cells. “Direct effect,” as used herein, means that the compounds of the present invention interact directly with cancer / tumor cells in order to exert their anti-cancer / anti-tumor effects. In other words, preferably, the compounds of the present invention, i.e., the compounds of formula (I), 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.

[0130] Preferably, the method of the present invention does not involve the administration of the compound of the present invention to deplete a cell population administered as part of a cell-based therapy. Cell-based therapies are well known as therapies in which a cell population is administered to a target to elicit a specific therapeutic effect. Well-known cell-based therapies include T-cell therapies, such as CAR T-cell therapy.

[0131] Preferably, the method of the present invention does not involve the administration of the compound of the present invention after the administration of a cell population administered as part of a cell-based therapy.

[0132] Preferably, the method of the present invention does not include CAR T cell therapy. Preferably, the method of the present invention does not include T cell therapy. Preferably, the method of the present invention does not include cell-based therapy.

[0133] Preferably, the method of the present invention involves administering the compound of the present invention to a subject who has not experienced CAR T-cell therapy, preferably T-cell therapy, or preferably cell-based therapy. In other words, preferably, the subject has never received, and does not plan to receive, CAR-T-cell therapy, preferably T-cell therapy, or preferably cell-based therapy as part of their treatment.

[0134] Where referred to herein, the subject may be any human or non-human animal, preferably a mammal, such as a cow, horse, sheep, pig, goat, rabbit, cat, dog, and especially preferably a human. Therefore, preferably, cancer referred to herein is human cancer, and tumor referred to herein is preferably present in a human subject.

[0135] In some embodiments, treatments according to the present invention may be used in subjects at risk of cancer relapse, recurrence, or metastasis. Therefore, in alternative views, compounds of formula (I) may be used in the prevention of cancer relapse, recurrence, or metastasis.

[0136] In certain embodiments, the present invention may involve first identifying or determining that the subject to be treated has cancer (i.e., a tumor) or is susceptible to cancer or at risk of developing it.

[0137] Alternatively or additionally, the present invention encompasses evaluating or monitoring the effects of administering compounds of formula (I) and / or other anticancer agents on a subject, or more specifically on cancer (tumors), or on the development or progression of cancer (tumors). Procedures and means for evaluating and / or monitoring anticancer effects by determining or monitoring symptoms, clinical status, tumor size or extent (e.g., by imaging techniques) or other cancer or tumor indicators, such as cancer / tumor markers, are well known in the art.

[0138] As mentioned above, the present invention is applicable to all cancers. Cancer is broadly defined herein to include all neoplastic conditions, particularly malignant or premalignant conditions. Cancer can cause, result in, or arise from solid tumors, but is not limited to solid tumors, and also includes cancers of the hematopoietic system. Benign and malignant tumors are also included in the term cancer as used herein; that is, the terms cancer and tumor are used interchangeably. Treatment of malignant tumors is preferred.

[0139] Cancer can appear in any tissue or organ of the body. For example, the present invention may be used in the treatment or prevention of any of the following cancers in a patient or subject.

[0140] Cancers of the central nervous system, preferably brain cancer, preferably glioma; acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); adrenocortical carcinoma; AIDS-related cancers (e.g., Kaposi's sarcoma and lymphoma); anal cancer; appendiceal cancer; basal cell carcinoma; bile duct cancer; extrahepatic bladder cancer Cancer; bone cancer (e.g., Ewing's sarcoma; osteosarcoma and malignant fibrous histiocytoma); breast cancer; bronchial tumors; Burkitt lymphoma; carcinoid tumors; cardiac tumors; cervical cancer (cervical adenocarcinoma); chordoma; acute promyelocytic leukemia; chronic lymphocytic leukemia (CLL); chronic myeloid leukemia (CML); chronic myeloproliferative disorders; colon cancer; colorectal cancer; cutaneous T-cell lymphoma; bile duct cancer; extrahepatic bile duct carcinoma in situ (DCIS); embryonal tumors; endometrial cancer; esophageal cancer; nasal neuroblastoma; Ewing's sarcoma; extracranial germ cell tumors; extragonadal germ cell tumors; extrahepatic bile duct cancer; eye cancer (including intraocular melanoma and retinoblastoma); fibrous histiocytoma of bone; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumors; gastrointestinal stromal tumors (GIS) T); germ cell tumors; gestational trophoblastic disease; hairy cell leukemia; head and neck cancer; heart cancer; hepatocellular carcinoma (liver cancer); histiocytosis; Langerhans cell tumors; Hodgkin lymphoma; hypopharyngeal cancer; intraocular melanoma; islet cell tumors; pancreatic neuroendocrine tumors; Kaposi's sarcoma; kidney cancer (including renal cell and Wilms tumors); Langerhans cell histiocytosis; laryngeal cancer; leukemia (including acute lymphoblastic (ALL); acute myeloid (AML); chronic lymphocytic (CLL); chronic myeloid (CML)); lip and oral cancer; liver cancer (primary); lobular carcinoma in situ (LCIS); lung cancer; lymphoma; macroglobulinemia; Waldenström's disease; melanoma (malignant melanoma); Merkel cell carcinoma; mesothelioma; metastatic cervical squamous cell carcinoma of unknown primary origin; median ductal carcinoma (Midline) involving the NUT gene Tract Carcinoma Involving NUT Gene); Oral cancer; Multiple endocrine neoplasia; Pediatric cancer; Multiple myeloma / plasmacytic neoplasm; Mycosis fungoides; Myelodysplastic syndrome; Myelodysplasia / myeloproliferative neoplasm; Multiple myeloma; Myeloproliferative disorders; Nasal and paranasal sinus cancer; Nasopharyngeal cancer; Neuroblastoma; Non-Hodgkin lymphoma; Non-small cell lung cancer; Oral cancer; Oral cancer; Oropharyngeal cancer; Osteosarcoma; Ovarian cancer (ovarian adenocarcinoma); Pancreatic cancer;Pancreatic neuroendocrine tumors (islet cell tumors); papillomatosis; paraganglioma; sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; epithelial adenocarcinoma; plasma cell neoplasms / multiple myeloma; pleuropulmonary blastoma; pregnancy and breast cancer; prostate cancer; rectal cancer; renal cell (kidney) cancer; renal pelvis and ureter; transitional cell carcinoma; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; sarcoma; Sézary syndrome; skin cancer; small cell lung cancer; Small intestine cancer; soft tissue sarcoma; squamous cell carcinoma; cervical squamous cell carcinoma of unknown primary origin; metastatic; stomach cancer; T-cell lymphoma; testicular cancer; throat cancer; thymoma and thymic carcinoma; thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; urethral cancer; uterine cancer; endometrium; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenström macroglobulinemia; and Wilms' tumor.

[0141] The cells of a human embryo are located in different 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 primary germ layers.

[0142] In the present invention, cancer / tumors are preferably cancers / tumors of tissue derived from the ectoderm, paraxial mesoderm, or lateral plate mesoderm, preferably the ectoderm.

[0143] Preferably, cancer is a cancer of the central nervous system, preferably brain cancer. To avoid doubt, brain cancer is considered in the art and herein to be a cancer of the central nervous system. The central nervous system includes the brain and spinal cord. Therefore, preferably, tumor is a tumor of the central nervous system, preferably brain tumor. Preferably, cancer / tumor of the CNS is selected from the group consisting of CNS lymphoma, rhabdoid tumor, embryonic tumor, 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 neuroectoderm (PNET), schwannoma, pineal tumor, trilateral retinoblastoma and rhabdoid tumor.

[0144] Most preferably, the cancer / tumor is brain cancer / brain tumor, more preferably glioma. The glioma may be any type of glioma, e.g., astrocytoma, ependymoma, subependymoma, oligodendroglioma, brainstem glioma, optic glioma, or mixed glioma.

[0145] 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 xanthoblastoma, or mixed oligoastrocytoma), a grade III astrocytoma (anaplastic astrocytoma), or most preferably a grade IV glioblastoma.

[0146] Grading systems for classifying tumors of the central nervous system are well known to those skilled in the art. Preferably, the World Health Organization (WHO) grading system is used. The WHO grading scheme is well known in the art and is based on the appearance of certain features: atypia, mitosis, endothelial proliferation, and necrosis, which reflect the malignancy of the tumor in terms of invasion and growth rate.

[0147] Gliomas can also be classified according to whether they are located above or below the tentorium cerebelli, the membrane that separates the cerebellum from the cerebrum. Supratentorium gliomas are found above the tentorium cerebrum, while infratentorium gliomas are found below the tentorium cerebelli. The gliomas treated according to the present invention may be either supratentorial gliomas or infratentorium gliomas.

[0148] 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.

[0149] Preferably, the cancer / tumor is selected from the group consisting of brain cancer (preferably glioma, more preferably glioblastoma, as defined above), gastric cancer, pancreatic cancer, lymphoma, lung cancer, skin cancer, acute promyelocytic leukemia, ovarian cancer, breast cancer, bone cancer, and cervical cancer.

[0150] More preferably, the cancer / tumor is selected from the group consisting of brain cancer (preferably glioma, more preferably glioblastoma, as defined above), gastric cancer, lymphoma, breast cancer, and cervical cancer.

[0151] Preferably, the cancer / tumor is selected from the group consisting of brain cancer (preferably glioma, more preferably glioblastoma, as defined above), gastric cancer, pancreatic cancer, skin cancer, acute promyelocytic leukemia, breast cancer, and cervical cancer.

[0152] Preferably, the cancer / tumor is selected from the group consisting of brain cancer (preferably glioma, more preferably glioblastoma, as defined above), skin cancer, and breast cancer, more preferably brain cancer (preferably glioma, more preferably glioblastoma, as defined above), and skin cancer.

[0153] In these embodiments, the preferred compounds of the present invention contain an aldehyde functional group X, preferably where X is -(CH2) n The compound is -X', where X' is -CHO, n is from 0 to 6, more preferably from 0 to 4, more preferably from 0 to 2, more preferably 0 or 1, most preferably X is -CHO, and preferably Z is -NH2. As demonstrated in the examples of the present invention, such compounds of the present invention have advantageously broad cytotoxicity against a wide range of cancer types, while cytotoxicity against non-cancerous cells is limited.

[0154] Alternatively, preferably, the cancer / tumor is selected from the group consisting of brain cancer (preferably glioma, more preferably glioblastoma, as defined above) and chronic myeloid leukemia. In these embodiments, the preferred compounds of the present invention are such that X contains an alcohol functional group, preferably where X is -(CH2) n The compound is -X', where X' is -OH, n is from 0 to 6, more preferably from 0 to 4, more preferably from 0 to 2, more preferably 0 or 1, most preferably X is -CH2OH, and preferably Z is -NH2. As demonstrated in the examples of the present invention, such compounds of the present invention have advantageously specific cytotoxicity against these preferred oncological types, and cytotoxicity against off-target cancerous and non-cancerous cells is limited.

[0155] Preferably, gastric tumors are gastric cancer. Preferably, pancreatic tumors are pancreatic cancer. Preferably, skin cancers are malignant melanoma. Preferably, ovarian cancers are ovarian adenocarcinoma. Preferably, breast cancers are epithelial adenocarcinoma. Preferably, bone cancers are osteosarcoma. Preferably, lung cancers are metastatic adenocarcinoma, preferably metastatic non-small cell adenocarcinoma. Preferably, cervical cancers are cervical adenocarcinoma.

[0156] However, preferably, the cancer is neither lung cancer nor breast cancer. Preferably, the cancer is neither pancreatic cancer, stomach cancer, testicular cancer, nor vaginal cancer. Preferably, the cancer is neither kidney cancer nor intestinal cancer.

[0157] Preferably, the cancer is not lung cancer. Preferably, the cancer is not prostate cancer, kidney cancer, liver cancer, breast cancer, colon cancer, ovarian cancer, or cervical cancer.

[0158] Preferably, the cancer / tumor is not colon cancer, lung cancer, prostate cancer, or kidney cancer. Preferably, the cancer / tumor is not pancreatic cancer.

[0159] Preferably, the cancer / tumor is not chronic myeloid leukemia, and in this case, the preferred compounds of the present invention (i.e., compounds of formulas (I), (IIa), (IIb), (IIIa), (IIIb), (IIIc), (IIId), (Iva), (IVb), (IVc), (IVd), (IVe), and (IVf)) contain an aldehyde functional group X, preferably where X is -(CH2) n -X', where X' is -CHO, and n is from 0 to 6, more preferably from 0 to 4, more preferably from 0 to 2, more preferably 0 or 1, most preferably X is -CHO and preferably Z is -NH2.

[0160] Furthermore, preferably, cancer i) Not breast cancer, preferably not pancreatic cancer, stomach cancer, testicular cancer or vaginal cancer, more preferably not intestinal cancer; and / or ii) Not liver cancer, breast cancer, ovarian cancer, or cervical cancer.

[0161] Preferably, the cancer is not selected from the group consisting of lung cancer, prostate cancer, kidney cancer, liver cancer, breast cancer, colon cancer, ovarian cancer, cervical cancer, chronic myeloid leukemia, pancreatic cancer, stomach cancer, testicular cancer, vaginal cancer, and intestinal cancer. Preferably, the cancer is none of these cancers.

[0162] Preferably, breast cancer treated according to the present invention is i) Invasive ductal carcinoma, and / or ii) Expressing wild-type p53 and / or iii) Not triple-negative, i.e., expressing one or more of the estrogen receptor (ER+), progesterone receptor (PR+), and HER2 (HER2+), preferably ER+ and PR+, and / or iv) It is heterozygous for p53.

[0163] Breast cancers that are not preferably treated according to the present invention are preferably, i) It is an adenocarcinoma and / or ii) Triple negative, i.e., does not express estrogen receptor (ER-), progesterone receptor (PR-), or HER2 (HER2-), and / or iii) Expressing a mutant variant of p53, and / or iv) Homozygous for p53 It is only breast cancer.

[0164] Human protein cytidine deaminase (CDA) catalyzes the hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Some known anticancer agents are nucleoside / nucleotide analogs, e.g., gemcitabine (2,2-difluorodeoxycytidine) and cytarabine (Ara-C, cytosine arabinoside). CDA inactivates such anticancer agents, including gemcitabine and cytarabine, in problematic ways. As demonstrated in the examples of the present invention, the compounds of the present invention exert their cytotoxic effects independently of CDA expression.

[0165] Therefore, in a preferred embodiment, cancer / tumor is one in which CDA is expressed, preferably CDA is expressed i) Overexpressed, ii) Not overexpressed, iii) Underexpressed, iv) Not underexpressed, v) Overexpressed or underexpressed, vi) Neither overexpressed nor underexpressed It is.

[0166] Each of the above cancer / tumor types i) through vi) represents a preferred embodiment of the present invention. In a particularly preferred embodiment, the cancer / tumor treated according to the present invention is one in which CDA is not overexpressed.

[0167] In the context of cancer / tumor cell gene expression, the terms "overexpressed" and "underexpressed" have clear and widely understood meanings, namely increased / higher levels and decreased / lower levels, respectively.

[0168] Overexpression (or increased or higher levels) or underexpression (or decreased or lower levels) may be determined by comparison with any appropriate control (e.g., control level or control sample or biopsy). For example, the control level may be the level in a sample (e.g., blood or serum sample or tissue sample or biopsy) from a healthy subject (e.g., a subject without cancer). An appropriate control level (or control sample or value) can be readily selected by those skilled in the art. An appropriate control "value" can also be readily determined without performing a control "sample" in every test, for example, by referring to a range for healthy subjects.

[0169] Preferably, the terms overexpressed and underexpressed mean that the level of RNA transcript derived from the gene in question in cancer cells is higher (overexpressed) or lower (underexpressed) than the level in non-cancerous cells from the same tissue as the cancer cells, when evaluated using the same methods and conditions in both cases. Preferred methods and conditions for evaluating the level of gene expression, such as CDA expression, are disclosed elsewhere in this specification.

[0170] Preferably, overexpression or underexpression is significant. Significantly overexpressed / underexpressed, i.e., significantly higher / lower, means statistically significantly over / underexpressed (statistically significantly higher / lower). Statistically significant means that the observed increase or decrease in level is greater than what might be expected to occur randomly on its own. Statistical significance can be determined by any method known in the art. For example, statistical significance can be determined by a probability value (p-value). A p-value is a measure of the probability that the difference between groups occurred randomly during the experiment. For example, a p-value of 0.01 means that there is a 1 in 100 chance that the result occurred randomly. The lower the p-value, the greater the probability that the difference between groups was caused by the treatment. Preferably, the probability value is less than 0.05 or less than 0.01.

[0171] In some embodiments, an increase in level (i.e., overexpression) may be an increase of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% (compared to, for example, a control level). In some embodiments, a decrease in level (i.e., underexpression) may be a decrease of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% (compared to, for example, a control level).

[0172] Preferably, the level of gene expression in the target cells is determined by referring to databases containing such information. Sources such as the Cancer Genome Atlas (TCGA, https: / / cancergenome.nih.gov / ), the EMBL-EBI Expression Atlas (https: / / www.ebi.ac.uk / gxa / home), the Human Protein Atlas (https: / / www.proteinatlas.org / ), GENEVESTIGATOR® (https: / / genevestigator.com / gv / ), the Cancer Cell Line Encyclopedia (https: / / portals.broadinstitute.org / ccle), and others contain information on gene expression levels in a wide range of cell types, including cancer, and also provide statistical data on whether the level of gene expression in a particular cancer type is significantly higher or lower than that in non-cancerous cells from the same tissue (i.e., whether the gene is overexpressed or underexpressed in that cancer type). These databases are preferred. Therefore, cancer types with statistically significant overexpression or underexpression of the target gene can be easily identified. Using such information sources for this purpose is within the capabilities of those skilled in the art.

[0173] Therefore, preferably, cancer / tumor is as defined in any part of this specification, where CDA expression within the cancer / tumor 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 Encyclopedia (https: / / portals.broadinstitute.org / ccle), and the Human Protein Atlas (https: / / www.proteinatlas.org / ).

[0174] Preferably, cancer / tumors do not express CDA to a level higher than that of non-cancerous cells derived from the same tissue as the cancer / tumor, where the overexpression 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 Encyclopedia (https: / / portals.broadinstitute.org / ccle), and the Human Protein Atlas (https: / / www.proteinatlas.org / ).

[0175] Preferably, the cancer / tumor has a CDA expression level that does not exceed 90% of the CDA expression level in a reference cancer cell line determined using the same method under the same conditions, where the reference cancer cell line is MDA-MB-231.

[0176] MDA-MB-231 is a widely available, well-characterized cell line. 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 medical laboratories. It can be obtained, for example, from the European Certified Cell Culture and Cell Culture Preservation Service (ECACC) under catalog number 92020424. As shown in Table 4A (Table 5), the expression level of CDA in the MDA-MB-231 cell line is 153 TPM.

[0177] MDA-MB-231 is a highly invasive, invasive, and poorly differentiated triple-negative breast cancer (TNBC) cell line lacking estrogen receptor (ER) and progesterone receptor (PR) expression, as well as HER2 (human epidermal growth factor receptor 2) amplification. The cell line is recognized as belonging to the claudin-low molecular weight subtype due to its downward regulation of claudin-3 and claudin-4, low expression of the Ki-67 proliferation marker, enrichment for markers related to epithelial-mesenchymal transition, and expression of breast cancer stem cell (CSC) characteristics such as CD44+CD24- / low phenotype. In 3D culture, the cell line exhibits an endothelial-like morphology and is distinguished by its invasive phenotype, often possessing stellate processes that bridge multiple cell colonies. Standard conditions for culturing this cell line are well known. The preferred culture conditions are growth at 37°C in Leibowitz L-15 medium supplemented with 2 mM glutamine and 15% fetal bovine serum (FBS). This medium supports cell growth in an environment without CO2 equilibrium. MDA-MB-231 cells are preferably 1-3 × 10⁶ 4 cells / cm 2 Seeds are sown at densities between 70% and 80%, and subcultured when the density reaches 70-80%.

[0178] Preferably, the cancer / tumor has a CDA expression level that does not exceed 80%, preferably not exceed 70%, preferably not exceed 60%, preferably not exceed 50%, preferably not exceed 40%, preferably not exceed 30%, and preferably not exceed 25%, where the reference cancer cell line is MDA-MB-231.

[0179] Preferably, the cancer / tumor has a CDA expression level that is at least half, preferably at least 1 / 2.5, preferably at least 1 / 3, preferably at least 1 / 3.5, preferably at least 1 / 4, and preferably at least 1 / 4.5 of the CDA expression level in a reference cancer cell line determined using the same method under the same conditions, where the reference cancer cell line is MDA-MB-231. In this context, "at least 1 / X" means that the maximum CDA expression level in the cancer / tumor is exactly 1 / X of the expression level in the reference cancer cell line.

[0180] The methods and conditions used to determine CDA expression levels in cancer / tumors and in reference cancer cell lines may be any preferred methods and conditions, provided that the same methods and conditions used to determine CDA expression levels in reference cancer cell lines are used to determine CDA expression levels in cancer / tumors. The reference cancer cell lines are therefore controls. Those skilled in the art can similarly and readily determine the expression levels of a gene of interest, such as CDA, in cancerous and non-cancerous cells. Such methods are part of the general knowledge in the art, and any preferred method may be used in the context of the present invention.

[0181] For example, expression levels can be measured at the protein level. Methods for measuring protein expression levels are well known in the art. These methods generally involve contacting a biological sample of interest with one or more detectable reagents suitable for measuring the expression level of a protein, such as an antibody, and then, preferably after normalization, determining the protein expression level based on the detected reagent levels. Examples of methods generally involving the use of antibodies include, but are not limited to, Western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISPOT), radioimmunoassay (RIA), immunohistochemistry, and immunoprecipitation. Other suitable methods for measuring protein expression levels, not necessarily involving the use of antibodies, may be used, but are not limited to, fluorescence-activated cell classification (FACS), microscopy such as atomic force microscopy, flow cytometry, microcytometry, protein binding assays, ligand binding assays, microarrays, polyacrylamide gel electrophoresis such as SDS-PAGE, surface plasmon resonance (SPR), Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), chemiluminescence, fluorescence polarization, phosphorescence, mass spectrometry such as liquid chromatography-mass spectrometry (LC-MS) or liquid chromatography-mass spectrometry / mass spectrometry (LC-MS-MS), matrix-assisted laser desorption / ionization time of flight (MALDI-TOF), surface-enhanced laser desorption / ionization time of flight (SELDI-TOF), and magnetic resonance imaging (MRI).

[0182] However, preferably, gene expression levels, such as CDA expression levels, can be measured at the RNA level. Methods for measuring RNA levels are well known in the art, and any suitable method can be used in the context of the present invention. For example, microarrays, RT-PCR, quantitative real-time PCR, RNA sequencing, Northern blotting, primer extension, RNase protection, and RNA expression profiling methods can be used. Preferably, the method used is RNA-seq or microarrays. RNA-seq is a well known method that uses next-generation sequencing (NGS) to determine the presence and amount of RNA in a biological sample at a given moment.

[0183] Preferably, the method used to determine the CDA expression level in the target cancer / tumor by comparing it with the CDA expression level in a reference cancer cell line (where the reference cancer cell line is MDA-MB-231) is the "microarray method A" as referred to below.

[0184] Microarray method A includes the following steps: 1) A step of extracting RNA from target cancer / tumor cells, wherein the extraction is preferably carried out using the Norgen Total RNA Purification Kit (Norgen Biotek catalog number 17200). 2) Preferably, the MEGA Pure Kit is used in accordance with the manufacturer's instructions (Ambion) to prepare poly(A)+ RNA from the extracted RNA. 3) The step of preparing complementary DNA (cDNA) from poly(A)+RNA by processing with reverse transcriptase, i.e., the step of performing reverse transcription. 4) A step of fragmenting cDNA using TdT (terminal deoxyribonucleotide transferase), 5) A step of biotinylating cDNA fragments using the GeneChipWT end labeling kit (Affymetrix). 6) A step in which steps 1 to 5 are repeated using a reference cell line, wherein the reference cell line is MDA-MB-231. 7) A step comprising hybridizing 5.5 μg of biotinylated cDNA fragment obtained in step 5 to a first DNA microarray at 45°C for 16 hours, and hybridizing 5.5 μg of biotinylated cDNA fragment obtained in step 6 to a DNA microarray at 45°C for 16 hours, wherein the microarray is preferably the Affymetrix GeneChip Human Gene 2.0ST Array (Applied Biosystems). 8) The hybridized microarray is washed and stained, preferably in an Affymetrix GeneChip Fluidics Station 450 (Applied Biosystems). 9) The stained microarray is scanned using an Affymetrix GeneChip scanner 3000 7G with Affymetrix GeneChip Command Console® software to generate raw data signal values ​​in the form of a CEL file. 10) Using the robust multi-array averaging (RMA) implementation in the Affymetrix software package (version 1.36.1), preferably as described in RA Irizarry et al., Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249~264 (2003), the CEL file is normalized to generate gene-level expression values. 11) A step to evaluate the quality of the array by calculating the relative log-expression (RLE) and normalized unscaled standard error (NUSE) using the affyPLM package (version 1.34.0). The output from the affyPLM package is box plots for both the RLE and NUSE distributions. Arrays whose RLE box plot does not unfold around 0 and whose NUSE box plot does not unfold around 1 are flagged as low quality and excluded from further analysis. If an array is excluded, the previous step of the method is repeated. 12) Perform principal component analysis (PCA) using the Prcomp R function, which normalizes expression values ​​across all samples to a mean of zero and a standard deviation of one. PCA is used as a dimensionality reduction method to reduce the dimensionality of gene expression datasets while preserving most of the variability in the data. Therefore, the performance of PCA enables a lower-dimensional representation of the data for downstream analysis and visualization. 13) A step to evaluate differential expression of CDA in target cancer / tumor cells and in the reference cell line MDA-MB-231 using a moderated (empirical Bayesian) t-test implemented in the limma package (version 3.14.4, http: / / bioinf.wehi.edu.au / limma), Here, microarray analysis steps 10 to 13 are performed using the R environment (version 2.15.1) for statistical calculations.

[0185] Preferably, the CDA expression level in the target cancer / tumor compared to the CDA expression level in a reference cancer cell line (where the reference cancer cell line is MDA-MB-231) 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 Encyclopedia (https: / / portals.broadinstitute.org / ccle), and the Human Protein Atlas (https: / / www.proteinatlas.org / ).

[0186] Gene expression levels are typically presented in terms of the relative amount of RNA transcript for the gene in question compared to the total amount of RNA transcript in the relevant cells / tissues. Expression levels are typically presented in units of transcripts per million (TPM), i.e., how many [x] transcripts [x] of the gene of interest were produced per million RNA molecules in the cell / tissue of interest. Here again, the level of RNA transcript in terms of TPM can be obtained by those skilled in the art by routine methods such as quantitative real-time PCR or RNA sequencing, and such information is available from a number of sources, including TCGA, EMBL-EBI Expression Atlas, GENEVESTIGATOR® database (https: / / genevestigator.com / gv / ), Cancer Cell Line Encyclopedia (https: / / portals.broadinstitute.org / ccle), and Human Protein Atlas (https: / / www.proteinatlas.org / ). Such methods are preferred herein. Methodologies for determining gene expression levels in TPM are described in the literature, for example, 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.

[0187] Preferably, cancerous cells / tumors overexpressing CDA contain CDA RNA transcripts at levels greater than 140 TPM. Conversely, cancerous cells / tumors not overexpressing CDA preferably contain CDA RNA transcripts at levels of 140 TPM or less (less than or equal to 140 TPM). In alternative view, preferred cancerous cells / tumors of the present invention have CDA expression levels of 140 TPM or less. Therefore, particularly preferred cancers / tumors treated according to the present invention express CDA at levels of 140 TPM or less, more preferably 100 TPM or less, and more preferably 50 TPM or less.

[0188] Preferably, the expression level of CDA in the target cancer / tumor, in units of TPM, is obtained 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 Encyclopedia (https: / / portals.broadinstitute.org / ccle), and the Human Protein Atlas (https: / / www.proteinatlas.org / ).

[0189] Preferably, the level of CDA in the target cancer / tumor, in units of TPM, is determined using cells derived from the cancer / tumor by quantitative real-time PCR or RNA sequencing (RNA-seq) and quantification. Preferably, the method used is RNA sequencing. Preferably, the method used is "RNA-seq method A" as referred to below.

[0190] The gene expression level at the TPM level can be determined using any known method, but is preferably determined using RNA-seq. RNA-seq methods are well known in the art and are widely available commercially. Any suitable RNA-seq method can be used to determine the CDA expression level in the target cancer at the TPM level. The following method, referred to as RNA-seq method A, is preferred and includes the following steps. 1. The step of extracting total RNA from the target cancer / tumor cells. This can be carried out using a standard RNA extraction kit, preferably the QIAGEN AllPrep DNA / RNA Mini kit (Hilden, Germany), according to the manufacturer's instructions. 2. The extracted RNA is optionally quantified and evaluated for purity, preferably using an automated electrophoresis tool, preferably a 2100 Bioanalyzer (Agilent Technologies). The Agilent Bioanalyzer is a microfluidic platform used for sizing, quantification, and quality control of RNA (and DNA / proteins), and provides an "RNA integrity number" (RIN) that quantifies the fragmentation of the RNA sample. Preferably, the sample is used further in the process only if it has at least 7, preferably at least 8 RINs. 3. A step of preparing an RNA sequencing library, preferably using the Illumina TruSeq® RNA sample preparation kit (Illumina, San Diego, CA, USA) and associated protocols. In this process, preferably 500 to 1000 μg of total RNA is used per sample. This kit and protocol are well known in the art. This step includes the following steps: 3a. Preferably, the step of depleting bacterial and eukaryotic ribosomal RNA by processing the extracted RNA using the Ribo-Zero rRNA removal kit (Epicentre, Madison, WI, USA) in accordance with the manufacturer's instructions. 3b. The remaining RNA is treated with reverse transcriptase and random hexamer to produce single-stranded cDNA fragments of 100-150 bases in length, or preferably 200-300 bases in length. 3c. A step of treating a single-stranded cDNA fragment with DNA polymerase I and RNase H to generate a double-stranded complementary DNA fragment (cDNA), and 3d. A step of generating an adapter-cDNA sequence library that can be analyzed by RNA-seq by ligating an RNA-seq adapter to the ends of cDNA fragments. RNA-seq adapters are well known in the art, and any suitable adapter can be used. The adapter contains functional elements that enable sequencing, such as amplification elements and primary sequencing sites. Preferably, the adapter is an Illumina index adapter. The adapter can be ligated to one end of each cDNA fragment to generate a single-ended library (which will result in one "read" per fragment when sequenced), in which case the cDNA fragment length in step 3b is 100-150 bases. However, preferably, the adapter is ligated to both ends of each cDNA fragment to generate a paired-ended library (which will result in two "reads" per fragment, so-called "mate reads" or "paired reads" when sequenced), in which case the cDNA fragment length in step 3b is 200-300 bases. 4. A step to optionally amplify the adapter-cDNA sequence by PCR. This step may be performed if the amount of DNA is insufficient to carry out the sequence analysis step 5. The amount of DNA required for sequence analysis is well known to those skilled in the art (preferably 70-135 μl, more preferably 125 μl, of a 20 pM solution of DNA is packed into the sequencer flow cell in step 5). Methods for evaluating the amount of DNA in the sample are routine, and any suitable method may be used. Preferably, the method used is fluorescence spectroscopy, and preferably a Qubit® fluorometer is used. 5. The adapter-cDNA sequence is sequenced, preferably using an Illumina flow cell sequencer, preferably an Illumina flow cell HiSeq 2500 sequencer or an Illumina flow cell NovaSeq 6000 sequencer, preferably using a sequencing cycle of 100-150 base pairs if a single-ended library is generated in step 3, and more preferably using a sequencing cycle of 200-300 base pairs if a paired-ended library is generated in step 3. Preferably 70-135 μl, more preferably 125 μl, of a 20 pM solution of DNA is packed into the flow cell. The raw dataset obtained will provide a unique sequence identifier for each analyzed fragment, its sequence (so-called "read"), and an indication of the reliability of the sequencer's determination of the sequence at each base position within the read (so-called Fred quality score). 6. Step 1: Prepare a read quality-filtered dataset by removing the following from the determined sequence dataset: i) Bases in reads with a Fred quality score of less than 10, ii) Bases in the read that are downstream (i.e., continue in the direction of sequencing) of bases in the same read that have a Fred quality score of less than 10, iii) Reads containing any undetermined ("undetermined") base ("N") iv) Reads that map to a contaminant reference genome, wherein the contaminant reference genome is the Escherichia coli genome, and v) If the RNA-seq library used is a paired-end library, the "mate read" is a read that is discarded in one of steps 6(iii) through 6(iv). 7. Aligning the quality-filtered reads to the genome of the target species, preferably the human genome. Preferably, the quality-filtered reads are aligned to the Ensembl database version 98, preferably the human GRCh38 genome, and to a human reference genome derived from the Ensembl gene reference feature database (version Ensembl genome 45, GENCODE 32). Suitable alignment tools for this step are well known and widely available, and any suitable alignment tool may be used. Preferably, the alignment tool is TopHat2 (version 2.1.1) [Trapnell et al., (2010) Nature Biotechnology 28, 511-515]. 8. A step to determine the number of read counts for the CDA gene and, optionally, any other gene of interest. Bioinformatics tools for this step are well known and widely available, and any suitable tool may be used. Preferably, the read count per gene is determined using an HTSeq package (htseq count). This step provides raw read count data. 9. The step of converting the raw read count data obtained in step 8 into units of transcripts per million (TPM). This is a standard mathematical operation routinely used in the art. Since determining the TPM value involves normalizing the raw data for i) gene length and ii) sequencing depth, providing the count data in units of TPM allows for a meaningful assessment of gene expression levels. i) Regarding gene length normalization in raw data, longer genes may have higher read counts simply because longer genes have more fragments aligned with them. Gene length normalization involves dividing the read count for each gene by the gene length (in kilobases). ii) Sequencing depth is an inter-sample effect that modifies the comparison of read counts between the same gene in different samples. To normalize this, the read count per kilobase obtained in step 9i) is divided by the "scaling factor per million," which itself is obtained by dividing the total number of reads in the sample by 1 million.

[0191] Gene expression data obtained using RNA-seq may be presented in units of RPKM (reads per kilobase per million reads). i. Divide the total number of reads in the sample by 1,000,000 to obtain the "scaling factor per million". ii.1 Divide the read count per gene by a scaling factor "per million" that normalizes with respect to sequencing depth, thereby providing units of reads per million (RPM), and iii. Divide the RPM value by the gene length in kilobases, which is normalized with respect to gene length, thereby providing the RPKM unit. It is calculated by [this method].

[0192] Another unit of gene expression is fragments per kilobase per million reads (FPKM), which is very similar to RPKM. RPKM is applicable when single-end RNA-seq is used, where all reads correspond to a single sequenced fragment. FPKM is applicable when paired-end RNA-seq, where two reads may correspond to a single fragment, or one read may correspond to a single fragment if one read in the pair did not map. The only difference between RPKM and FPKM is that FPKM takes into account that two reads can map to one fragment (and thus does not count this fragment twice).

[0193] TPM is very similar to RPKM and FPKM. The only difference is the order of operations (i) to (iii) above. Thus, TPM is determined as follows. i. Divide the read count per gene by the length of the gene in kilobases that normalizes for gene length, thereby providing a unit of reads per kilobase (RPK). ii. Divide the total RPK value in the sample by 1,000,000 to provide a "scaling factor per million", and iii. Divide the RPK value obtained in step (i) by the "per million" scaling factor obtained in step (ii) that normalizes for sequencing depth, thereby providing a unit of TPM.

[0194] The only difference when calculating TPM compared to RPKM or FPKM is that normalization for gene length is performed first when calculating TPM. However, the result is that when using the unit of TPM, the sum of all TPMs in each sample is the same million. This allows for a more meaningful comparison of the proportion of reads mapped to genes in each sample.

[0195] Using the above-described RNA-seq method A that determines the expression level of CDA in units of TPM, it is also possible to determine the expression level of CDA in the target cancer / tumor in units of TPM compared to a control as defined above, i.e., a control cell or a reference cell line, where the reference cell line is MDA-MB-231. In these embodiments, the RNA-seq method A may be performed using the target cancer / tumor cells and may be repeated using the control or reference cells, where the determined TPM values are then compared. Alternatively, the RNA-seq method A may be performed using the target cancer / tumor cells, and the CDA expression level determined in units of TPM is compared to the value for the CDA expression level in units of TPM previously obtained using the same method using the control or reference cell line.

[0196] Alternatively, the level of gene expression can be determined via a microarray, which is a standard technique in the art. Any suitable microarray technology can be used in the context of the present invention. Microarrays enable the simultaneous detection of the expression of thousands of genes.

[0197] The level of gene expression determined by the microarray can be expressed on any scale, such as a linear scale. The data from the microarray may preferably be transformed by a base-2 logarithm transformation, which has the advantage of generating a continuous spectrum of values and treating up- and down-regulated genes in a similar manner. A gene up-regulated by a factor of 2 has a log2 transformation value of 1.

[0198] Preferably, the cancer / tumor is one in which the base-2 logarithm-transformed CDA expression level is less than 11.75. Such a cancer / tumor is described as having non-overexpressed CDA. Preferably, the cancer / tumor is one in which the base-2 logarithm-transformed CDA expression level is less than 11.5, more preferably less than 11, more preferably less than 10.5, more preferably less than 10, more preferably less than 9.5.

[0199] Preferably, the cancer / tumor is one in which the linear CDA expression level is less than 6500. Such a cancer / tumor is described as having non-overexpressed CDA. Preferably, the cancer / tumor is one in which the linear CDA expression level is less than 6000, more preferably less than 5000, more preferably less than 4000, more preferably less than 3000, more preferably less than 2000, more preferably less than 1500.

[0200] Preferably, the linear or log-to-cardinal-2 converted expression levels of CDA in the target cancer / tumor are obtained 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 Encyclopedia (https: / / portals.broadinstitute.org / ccle), and the Human Protein Atlas (https: / / www.proteinatlas.org / ).

[0201] Those skilled in the art will be aware that even among tumors of the same cancer type, there can be variations in the expression levels of some genes. For example, some breast cancers may overexpress CDA, while others may not. Therefore, preferably, cancers / tumors described as preferred elsewhere in this specification are preferably of the type of cancer / tumor in which CDA is expressed, and preferably CDA is expressed i) Overexpressed, ii) Not overexpressed, iii) Underexpressed, iv) Not underexpressed, v) Overexpressed or underexpressed, vi) Neither overexpressed nor underexpressed It is.

[0202] Preferably, the cancer / tumor is one in which CDA is not overexpressed. Preferably, the cancer / tumor is one in which CDA is overexpressed. CDA overexpression is as defined above.

[0203] Conversely, cancers described as undesirable elsewhere in this specification are preferably only those types of cancer / tumors, preferably CDA. i) Overexpressed, ii) Not overexpressed, iii) Underexpressed, iv) Not underexpressed, v) Overexpressed or underexpressed, vi) Neither overexpressed nor underexpressed It is.

[0204] Preferably, cancer / tumors are those in which CDA is overexpressed. CDA overexpression is as defined above.

[0205] In preferred embodiments, the cancer / tumor is resistant to gemcitabine and / or cytarabine treatment. Therefore, any cancer / tumor described as preferred elsewhere in this specification is preferably a type of cancer / tumor that is resistant to gemcitabine and / or cytarabine treatment. Preferably, the gemcitabine and / or cytarabine-resistant cancer / tumor is brain cancer, preferably glioma, more preferably glioblastoma multiforme.

[0206] The human protein O-6-methylguanine-DNA methyltransferase (MGMT) removes alkylated DNA damage. MGMT expression makes cancer cells, such as glioblastoma cells, almost completely resistant to the cytotoxic effects of temozolomide. This exerts its cancer chemotherapy activity by violently mutating tumor cells, leading to their death. Temozolomide acts by alkylating DNA, thereby inducing mutations.

[0207] As shown in the examples of the present invention, the compound of formula (I) is not mutagenic in HPRT assays. Therefore, the compound of formula (I) is of particular use when treating cancers in which MGMT is expressed, preferably overexpressed. Thus, any cancer / tumor described as preferred elsewhere in this specification is preferably a type of cancer / tumor in which MGMT is expressed, preferably overexpressed.

[0208] Temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide) is the first-line treatment for glioblastoma multiforme and is also used in the treatment of some other brain cancers. However, as shown in the examples of the present invention, temozolomide effectively killed less than half of the glioblastoma cell lines evaluated, and temozolomide-resistant cell lines were effectively killed by the compounds of the present invention. Therefore, in preferred embodiments, the cancer / tumor is a temozolomide-resistant cancer / tumor.

[0209] Therefore, any cancer / tumor described as preferred elsewhere in this specification is preferably a type of cancer / tumor that is resistant to temozolomide treatment. Preferably, temozolomide-resistant cancers / tumors are brain cancers, preferably gliomas, and more preferably glioblastoma multiforme.

[0210] As shown in the examples of the present invention, 5-fluorouracil-resistant cell lines are effectively killed by the compounds of the present invention. Therefore, in preferred embodiments, the cancer is a fluoropyrimidine-resistant cancer. Thus, any cancer / tumor described as preferred elsewhere in this specification is preferably a type of cancer / tumor that is resistant to fluoropyrimidine treatment. Preferably, the fluoropyrimidine-resistant cancer / tumor is brain cancer, preferably glioma, and more preferably glioblastoma multiforme. Preferably, the fluoropyrimidine is 5-fluorouracil.

[0211] Preferably, the cancer / tumor is resistant to both temozolomide and fluoropyrimidine treatment.

[0212] In the context of cancer / tumor therapy, the term "resistance" has a clear and well - understood meaning in the art. "Resistance" means that a cancer / tumor does not respond positively to treatment with a relevant anti - cancer agent, i.e., treatment with an anti - cancer agent does not reduce, alleviate, remit or eliminate the cancer or one or more of its symptoms, or does not reduce or eliminate cancer cells within the tumor compared to the cancer, tumor or symptoms prior to treatment. A cancer / tumor may be resistant at the start of treatment or may become resistant during treatment.

[0213] As mentioned above, in the treatment or prevention of cancer, the compounds of formula (I) can be used alone or optionally in combination with one or more further, i.e., additional, anti - cancer agents. In all aspects and embodiments of the present invention, the further anti - cancer agent can be any suitable anti - cancer agent 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 can be used regardless of their chemical nature or mode of action.

[0214] Thus, anti - cancer agents include chemical molecules (e.g., small organic chemical molecules), whether naturally or synthetically derived or prepared, as well as biological molecules such as proteins and peptides (e.g., immunotherapeutic agents as discussed below). Thus, anti - cancer drugs include chemotherapeutic agents or drugs, which can be of a wide range of different chemical or functional classes, and antibodies or antibody derivatives, and other biological molecules that act to stimulate, activate or enhance various physiological processes or cells in the body, e.g., immune and / or anti - inflammatory responses or cells.

[0215] Representative examples of anticancer drugs in the "chemotherapy" class include fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbazine, etoposide, teniposide, campatecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, and mitoxant. This includes, but is not limited to, lon, L-asparaginase, epimbicin, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamizole, irinotecan, estramustine, etoposide, nitrogen mustard, BCNU, carmustine and lomustine, nitrosoureas, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatinib mesylate, hexamethylmelamine, or topotecan.

[0216] Anticancer agents may include kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrofostine, protease inhibitors, herbimycin A, genistein, erbustatin, and lavendastine A.

[0217] In one embodiment, the anticancer agent may be selected from, but is not limited to, one or a combination of the following classes of active agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, antifolic acid agents, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, epipodophyllotoxins, hormone therapy, retinoids, photosensitizers or active agents for use in photodynamic therapy, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycin, bleomycin, anthracyclines, MDR inhibitors, and Ca2+ATPase inhibitors.

[0218] Further 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, and polybodies.

[0219] Alternative anticancer agents may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and their growth factor mimics.

[0220] In one typical embodiment, the drug is a small molecule, more specifically, a small molecule chemotherapeutic agent. A small molecule agent can be defined as having a molecular weight of less than 2000 Da, more specifically less than 1800, 1500, 1200, 1000, 900, 800, or 700 Da, typically less than 1000 Da. For example, a small molecule agent may have a size in the range of 100 to 1000 Da, for example, 100 to 800 Da or 300 to 700 Da.

[0221] In alternative embodiments, further anticancer agents are immunotherapies. Inducing an immune response to treat cancer is known as cancer "immunotherapy." Immunotherapy may involve, for example, cell-based therapies, antibody therapies, or cytokine therapies. All three approaches take advantage of the fact that cancer cells often have different cell surface markers or cancer antigens that are detectable by the immune system. These antigens are most commonly proteins, but can also include other molecules such as carbohydrates. Another example of immunotherapy is checkpoint inhibition, which inhibits checkpoint proteins. This will be discussed further below.

[0222] Therefore, immunotherapy is used to stimulate the immune system to attack cancer cells, and as will be discussed further below, various molecules can be targets for immunotherapy-based approaches. For example, targets for immunotherapy intervention in cancer include "CD" ("differentiation antigens") proteins such as CD52, CD30, CD33, CD20, CD152 (also known as CTLA4) and CD279 (also known as programmed cell death 1 protein PD-1); growth factors such as vascular endothelial growth factor (VEGF); growth factor receptors such as epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor 2 (HER2); lymphocyte activator gene 3 (LAG3); and B7 family proteins such as B7-H3 and B7-H4. However, these are only representative examples, and other molecules can be targets for immunotherapy intervention in cancer.

[0223] Immunotherapy agents can be peptides, polypeptides, or proteins. Immunotherapy agents can be selected from antibodies, cytokines, and checkpoint inhibitors. As noted above, therapeutic anti-cancer antibodies may have a range of targets, including checkpoint proteins. Therefore, antibodies can also be checkpoint inhibitors.

[0224] Therefore, in the first example, the immunotherapy agent is an antibody. Antibodies can be selected from monoclonal or polyclonal antibodies, monospecific, bispecific or multispecific antibodies, monobodies and polybodies, or from any of the many antibody-like or antibody derivative molecules currently known in the art. Accordingly, the term “antibody” is used broadly herein and includes any such antibody and any antibody fragment, derivative or variant as known in the art. The antibody may be of any convenient or desired species, class or subtype. Furthermore, the antibody may be natural, derivatized or synthetic.

[0225] Therefore, the antibody could be one of the following: (a) Any animal, e.g., an animal commonly used, e.g., any of sheep, rabbits, goats or mice, or any of egg yolks, e.g., any of IgG, IgA, IgM, IgD or IgE (b) Monoclonal or polyclonal antibodies, (c) Intact antibodies, or fragments of monoclonal or polyclonal antibodies, the fragments being fragments lacking the antibody binding region, e.g., the Fc region (e.g., Fab, Fab', F(ab')2, Fv), and so-called "half-fragments" obtained by reductive cleavage of the disulfide bond connecting the heavy chain components in an intact antibody. Fv may be defined as a fragment containing a variable region of the light chain and a variable region of the heavy chain expressed as two separate chains. (d) Antibodies produced or modified by recombinant DNA or other synthetic techniques, including monoclonal antibodies, antibody fragments, humanized antibodies, chimeric antibodies, or synthetically produced or modified antibody-like structures.

[0226] This also includes functional derivatives or "equivalents" of antibodies, such as single-chain antibodies. A single-chain antibody can be defined as a genetically engineered molecule containing a variable light chain region and a variable heavy chain region, linked by a polypeptide linker suitable as a fused single-chain molecule. Methods for producing antibodies, antibody fragments, and derivatives are well known in the art.

[0227] In a preferred embodiment, the antibody is a monoclonal antibody.

[0228] In most cases, monoclonal antibodies are unmodified antibodies, and most therapeutic antibodies currently in use fall into this category. However, in one embodiment of the present invention, an antibody, such as a monoclonal antibody, is conjugated or fused with an additional molecule, such as a toxic substance or a radioactive substance. Thus, the conjugated or fused antibody is bound to another molecule, which is either toxic to cells (e.g., a drug) or radioactive. The antibody binds to a specific antigen on the surface of cancer cells, directing the toxin or radiation towards the tumor.

[0229] Known and approved antibodies include alemtuzumab, bevacizumab, brentuximab vedotin, cetuximab, gemtuzumab ozogamicin, ibritumomab tiuxetan, ipilimumab, ofatumumab, panitumumab, rituximab, tocitumomab, and trastuzumab.

[0230] In a second example, the immunotherapy agent is a cytokine. Cytokines include immunomodulators such as interleukins (ILs) and interferons (IFNs), as well as stimulants, tumor necrosis factor (TNF), and other regulatory molecules. Cytokines have been classified as lymphokines, interleukins, and chemokines based on their function, the cells they secrete, or the targets of their action. Each cytokine has a suitable cell surface receptor that initiates a cascade of intracellular signaling that modifies cellular function. In the context of cancer, cytokines are produced by many cell types present within tumors. Cytokines are well known in the art, and all such cytokines are covered for use according to the present invention. Therefore, in one embodiment, the immunotherapy agent is a cytokine. In a preferred embodiment, the cytokine is an interleukin or an interferon.

[0231] Interleukins are a group of cytokines that have a wide range of effects on the immune system. Examples of interleukins (ILs) include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, and IL-17.

[0232] Interferons are cytokines produced by the immune system, which is normally involved in antiviral responses, but they also have applications in cancer treatment. Interferons (IFNs) are classified into three groups: type I (IFNα and IFNβ), type II (IFNγ), and the relatively recently discovered type III (IFNλ).

[0233] All known forms of the cytokines discussed above, including functionally equivalent variants, derivatives, and fragments, may also be used in the present invention. Therefore, the term “cytokine,” as used herein, includes amino acid sequence variants of known cytokine polypeptides, and fragments or derivatives of cytokine polypeptides, insofar as such fragments, variants, or derivatives are active or “functional,” i.e., retain at least one function or activity (e.g., biological activity) of the relevant cytokine. Cytokines may be recombinant polypeptides, synthetic polypeptides, or isolated from natural sources. Suitable cytokines are commercially available and will be known to those skilled in the art; for example, human cytokines are available from GenScript (Piscataway, NJ, USA).

[0234] In a third example, immunotherapeutic agents are agents that target immune checkpoints, i.e., checkpoint inhibitors. Checkpoint proteins suppress the immune system by indicating which cells are healthy and which should be destroyed. Checkpoint proteins act as a "brake" on the immune system by preventing T cell activation. If a cell does not have enough checkpoint proteins on its surface, it can be destroyed by the immune system. In the case of cancer cells, there may be molecules that signal that the cell is cancerous, but if there are enough checkpoint proteins on the cell surface, the cell can evade the immune response, and it has been hypothesized that checkpoint proteins contribute to the failure of some cancer immunotherapies.

[0235] Several checkpoint inhibitors, such as those described in Creelan (2014) Cancer Control 21:80-89, are known and can be used in the present invention.

[0236] 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 (chimera 3G4). All of these checkpoint inhibitors can be used in the present invention.

[0237] Alternative options for immunotherapy, relating to immunocell therapy, can also be used in combination with such therapies, such as adoptive cell transfer. A number of T-cell-based therapies have been developed to treat cancer, and these procedures, known as adoptive cell transfer (ACT), have become increasingly attractive in recent years. Three main ACT strategies have been utilized to date. The first of these, and the most developed, involves the isolation of the patient's own tumor-reactive T cells (known as tumor-infiltrating lymphocytes (TILs)) from the peripheral or tumor site. These cells are expanded ex vivo and reinjected into the patient.

[0238] Two alternative therapies are available, both involving modification of the patient's own T cells with receptors capable of recognizing tumors. One option involves isolating and characterizing TcRs that are active towards cancer antigens, inserting the gene encoding the TcR into T cells, and reinjecting them into the patient. While this therapy has been shown to shrink solid tumors in some patients, it is associated with a significant drawback: the TcR used must be matched to the patient's immune type. Therefore, as an alternative to the use of TcRs, therapies involving the expression of chimeric antigen receptors (CARs) in T cells have also been suggested. CARs are fusion proteins containing antibodies linked to the signaling domain of the TcR complex, and when a suitable antibody is selected, they can be used to direct T cells towards the tumor. Unlike TCRs, CARs do not need to be MHC matched with the recipient.

[0239] Alternatively, the cells may be natural killer (NK) cells, which may be modified to express CARs.

[0240] Therefore, according to the present invention, the immunotherapy agent may be a cell, particularly an immune cell such as a lymphocyte, particularly a T cell or NK cell as described above. For example, a T cell may be a TIL, or may be modified to express TcR or CAR. An NK cell may be modified to express CAR.

[0241] An alternative option for further anticancer drugs is microRNA (miRNA). MicroRNAs are small, non-coding RNA molecules (containing approximately 22 nucleotides) found in plants, animals, and some viruses, and they function in RNA silencing and post-transcriptional regulation of gene expression. miRNAs function through base pairing with complementary sequences within mRNA molecules. As a result, these mRNA molecules are silenced by cleavage of the mRNA strand into two pieces, destabilization of the mRNA by shortening of its poly(A) tail, or inefficient translation of the mRNA into protein. miRNAs are similar to siRNAs mentioned above, except that miRNAs derive from regions of RNA transcripts that fold to form short hairpins, whereas siRNAs derive from longer regions of double-stranded RNA. Many miRNAs have been found to have associations with various types of cancer and are therefore sometimes referred to as "oncomirs."

[0242] MicroRNAs can be used in microRNA-based oncological therapies for cancer treatment. The rationale for developing miRNA therapies is based on the premise that abnormally expressed miRNAs play a major role in cancer development, and that correcting these miRNA deficiencies by either antagonistizing or repairing miRNA function, for example through miRNA replacement therapy, may provide therapeutic benefits.

[0243] Any suitable miRNA may be used as a further anticancer agent according to the present invention. The miRNA may be in a free form, i.e., not bound to another molecule. Alternatively, the miRNA may be conjugated or bound to another molecule, such as an antibody as discussed herein.

[0244] Most preferably, the further anticancer agent is selected from the group consisting of temozolomide, 5-fluorouracil, gemcitabine, cytarabine, and gliadel (registered trademark). Preferably, the further anticancer agent is temozolomide. Preferably, the further anticancer agent is 5-fluorouracil. Preferably, the further anticancer agent is gemcitabine. Preferably, the further anticancer agent is cytarabine. Preferably, the further anticancer agent is gliadel (registered trademark).

[0245] The compound of formula (I) and further anticancer agents can be used in accordance with the present invention in the form of a composition, i.e., a pharmaceutical composition. The present invention provides a pharmaceutical composition comprising the compound of formula (I) and one or more pharmaceutically acceptable excipients, and optionally further comprising further anticancer agents.

[0246] Products (especially pharmaceutical products) comprising a compound of formula (I) and further (i.e., one or more further or second) anticancer agents may be combination preparations for use separately, sequentially or concurrently in the treatment or prevention of cancer (i.e., tumors), or products in which the compound of formula (I) is co-formulated with further anticancer agents.

[0247] Therefore, the compound of formula (I) and further anticancer agents may be formulated together in a single composition or in separate compositions for separate administration. This will depend on the properties of the further anticancer agents and the selected or required mode of administration.

[0248] The compositions for use in the present invention may be formulated in any convenient manner according to techniques and procedures known in the pharmaceutical field, for example, using one or more pharmaceutically acceptable excipients, carriers, or additives. Such formulations may be for pharmaceutical or veterinary use. Suitable excipients, additives, and carriers for use in such formulations are known to those skilled in the art.

[0249] Where used herein, "pharmaceutically acceptable" refers to a component that is compatible with the other components of the composition and is physiologically acceptable to the recipient. The properties of the composition and the carrier or excipient materials, dosage, etc., may be selected in a routine manner according to the choice and desired route of administration, purpose of treatment, etc.

[0250] Therefore, “pharmaceutically acceptable” or “pharmaceutically acceptable” means molecular entities and compositions that, when administered appropriately to mammals, particularly humans, do not produce harmful, allergic, or other adverse reactions. A pharmaceutically acceptable carrier or excipient means any kind of non-toxic solid, semi-solid, or liquid filler, excipient, encapsulating material, or formulation aid.

[0251] The pharmaceutical composition contains a pharmaceutically acceptable vehicle for formulation. These may be a dry, freeze-dried composition that allows for the administration of the solution, particularly with the addition of an isotonic sterile saline solution (monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or a mixture of such salts), or optionally with sterile water or saline solution.

[0252] The compounds of the present invention may be presented in conventional pharmacological dosage forms, such as tablets, coated tablets, nasal sprays, liquids, emulsions, liposomes, powders, capsules, or sustained-release formulations. Conventional pharmaceutical excipients and typical production methods can be used to prepare these forms.

[0253] 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, excipient or additive. For example, formulations suitable for parenteral administration conveniently generally contain a sterile aqueous solution and / or suspension of the pharmaceutically active ingredient, preferably isotonic with the recipient's blood using sodium chloride, glycerin, glucose, mannitol, sorbitol, etc.

[0254] Additives that may be included in any pharmaceutical composition include, among many others, preservatives (such as p-hydroxybenzoate), chelating agents (such as EDTA), stabilizers, tonicity modifiers, antimicrobial agents, flocculants / suspending agents, wetting agents, solvents and solvent systems, antioxidants, and buffering agents. When formulating a pharmaceutical composition for a specific required purpose, selecting and optimizing such additives and their amounts is within the capabilities of those skilled in the art.

[0255] 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.

[0256] The form of the pharmaceutical composition, the route of administration, the dosage, and the regimen are necessarily determined by the nature of the cancer being treated, the severity of the disease, the patient's age, weight, and sex, or, alternatively, the desired duration of treatment.

[0257] The treatment may involve administering the compound of formula (I), along with additional anticancer drugs, if necessary.

[0258] Compounds of formula (I) for use in accordance with the present invention can be administered to a subject via any suitable route. The same applies to compositions or formulations containing compounds of formula (I).

[0259] Compounds of formula (I), and therefore compositions and formulations containing them, may be presented in forms suitable for, for example, oral, nasal, parenteral, intravenous, topical, rectal, or subarachnoid administration. Preferably, the compound is presented in a form suitable for systemic (e.g., intravenous) administration.

[0260] 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 surfaces of the body by any preferred method known in the pharmaceutical field. Therefore, modes of administration include oral, nasal, intestinal, rectal, vaginal, transmucosal, topical, or parenteral administration, or inhalation. Administration may be direct to the tumor (intratumor administration).

[0261] 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 (subarachnoid administration). More preferably, administration is intraperitoneal or intravenous, most preferably intravenous.

[0262] Preferably, administration is by oral or intravenous means.

[0263] Intravenous administration may be by intravenous injection or intravenous infusion, most preferably by intravenous infusion (e.g., by an infusion pump).

[0264] The compound of formula (I) and further anticancer agents may be administered by the same or different routes.

[0265] As noted above, the compound of formula (I) and further anticancer agents may be administered simultaneously, separately, or sequentially. In preferred embodiments, the compound of formula (I) and further anticancer agents 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 further anticancer agents are administered together at the same time, for example, 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 further anticancer agent used, formulation and / or mode of administration. Thus, the compound of formula (I) may be administered before or after further anticancer agents.

[0266] For example, an additional anticancer agent may be administered first, and the compound of formula (I) may be administered later at a suitable time interval to align the optimal time for delivery of the additional anticancer agent to the target site, or vice versa. Such decisions are entirely within the routine skill of the clinician. 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, or 6 hours before or after the additional anticancer agent, preferably parenterally, and more preferably intravenously.

[0267] Dosage and dosage may be determined in a routine manner 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 as described above can be combined with pharmaceutically acceptable additives to form a therapeutic composition. Dosage refers to a specified amount of the drug taken at one time; that is, the terms "single dose" and "dose" are used interchangeably. The course of treatment may include multiple doses, i.e., multiple single doses, over a period of time.

[0268] In the method and use of the present invention, preferably an effective amount of the compound of formula (I) and, if present, an optional further anticancer agent are administered. In other words, the dose preferably comprises an effective amount of the compound of formula (I) and, if present, an optional further anticancer agent.

[0269] As shown in the examples, the compound of formula (I) is tolerant to doses higher than those required to kill tumors. This property differs from many chemotherapeutic compounds, most of which have substantial side effects at doses required to kill cancer cells. The examples of the present invention advantageously indicate that the compound of formula (I) can be administered at doses far exceeding those required to kill tumors.

[0270] As shown in the examples, mice showed tolerance to single doses of the compound of formula (I) at 300 mg / kg and 2000 mg / kg, but not to a single dose of 8000 mg / kg. These data suggest that the maximum tolerable dose of the compound of formula (I) in mice is at least 2000 mg / kg but less than 8000 mg / kg. The conversion factor between mouse doses and human doses is 0.081 (Nair et al., (2016) Basic Clin Pharm. 7(2): 27~31). Therefore, the data in the examples indicate that the maximum tolerable dose of the compound of formula (I) in humans is at least 162 mg / kg but less than 648 mg / kg.

[0271] Therefore, preferably, the compound of formula (I) is administered in doses of 405 mg / kg or less, preferably 324 mg / kg or less, more preferably 243 mg / kg or less, more preferably 162 mg / kg or less, more preferably 81 mg / kg or less, more preferably 40.5 mg / kg or less, and more preferably 24.3 mg / kg or less. Preferably, the compound of formula (I) is 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 500 mg / kg, and more preferably at least 100 mg / kg.

[0272] Preferably, the compound of formula (I) is administered in doses between 10 mg / kg and 405 mg / kg, preferably between 20 mg / kg and 324 mg / kg, more preferably between 20 mg / kg and 243 mg / kg, more preferably between 30 mg / kg and 162 mg / kg, and more preferably between 40 mg / kg and 81 mg / kg.

[0273] These dosages are preferred doses for human subjects.

[0274] Dosage and dosage 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.

[0275] Appropriate dosages and regimens can be easily established. Appropriate dosage units can be easily prepared. Dosage regimens can be determined in a routine manner.

[0276] The treatment may include a single dose of the compound of formula (I) along with, if applicable, further anticancer agents, or it may include repeated doses of the compound of formula (I) along with, if applicable, further anticancer agents. The drug regimens of the compound of formula (I) and any further anticancer agents do not need to be identical. Alternatively, the treatment may include a single dose of the compound of formula (I) and repeated doses of further anticancer agents, or vice versa.

[0277] Preferably, the compound of formula (I) is administered in any of the above-described doses, every 1, 2, 3, 4, 5, or 6 days, more preferably every 2, 3, or 4 days, more preferably every 3 days, for a total of 2 to 10 doses, more preferably 3 to 8 doses, more preferably 4 to 6 doses, and more preferably 5 doses.

[0278] However, determining an appropriate drug 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., would be within the capabilities of a person skilled in the art.

[0279] The present invention also provides products or kits comprising the compound of formula (I) and further anticancer agents. The kits or products may be used in any of the uses or methods described herein, i.e., for use in treating or preventing cancer. In particular, the kits or products may be used simultaneously, separately, or sequentially. Preferably, the compound of formula (I), and optionally further anticancer agents, are formulated for parenteral administration, preferably intravenous administration.

[0280] Each component of the kit of the present invention (i.e., each anticancer agent) may be provided in a separate compartment or vessel. Where convenient and practical, a mixture of components may be provided. The components may be provided in a dry form, e.g., crystallized, freeze-dried or lyophilized, or in solution, typically such liquid compositions would be aqueous and buffered with a standard buffer such as Tris or HEPES.

[0281] Preferably, the kit is intended for use in treating cancer, for example, in the method or use of the present invention as described herein.

[0282] The compounds of the present invention (i.e., compounds of formulas (I), (IIa), (IIb), (IIIa), (IIIb), (IIIc), (IIId), (Iva), (IVb), (IVc), (IVd), (IVe), and (IVf)) are either commercially available, known in the literature, or can be obtained by conventional synthetic procedures, following standard techniques, from available starting materials, using appropriate reagents and reaction conditions. In this regard, those skilled in the art may 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).

[0283] The compounds of the present invention are commercially available from, for example, Berry and Associates, Toronto Research Chemicals, Sigma Aldrich, Carbosynth, Trilink Biotech, and other well-known commercial suppliers.

[0284] The present invention will be further described with reference to the following non-limiting examples. [Examples]

[0285] Materials and methods animal All aspects of this work, including animal housing, experimentation, and disposal, were carried out within an AAALAC-accredited laboratory animal facility, generally in accordance with the Guide for the Care and Use of Laboratory Animals: 8th Edition (National Academy Press, Washington, DC, 2011). The animal care and use protocols were reviewed and approved by IACUC at Pharmacology Discovery Services Taiwan, Ltd.

[0286] cell culture Primary glioma nerve trunk (GNS) cells (G7, G14, G144, G166) were cultured in neural stem cell medium (50% DMEM-F12 (Thermofisher, catalog no. 21041025), 50% Neurobasal medium (Thermofisher, catalog no. 10888-022), N2 (Life Technologies, catalog no. A-003-E) and B27 supplement (Life Technologies, catalog no. 12587010), 1 mM sodium pyruvate (Life Technologies, catalog no. 11360-039), 2 mM glutamax (Life Technologies, catalog no. 35050038), 1 mM HEPES (Fisher Scientific, catalog no. BP299-1), 0.1 mM β-mercaptoethanol (Life Technologies, catalog no. 31350010), 1 × non-essential amino acids (Life The cells were cultured on plates coated with poly-D-lysine (Merck Millipore, catalog number A-003-E) and laminin (R&D Systems, catalog number 3446-005-01) in 0.006% bovine serum albumin (Sigma, catalog number A8577-10ML), 4 μg / ml heparin (Sigma, catalog number H3149-25KU), 100 U / ml penicillin, 100 μg / ml streptomycin, 20 ng / ml hEGF (R&D Systems, catalog number 236-EG-200), and 10 ng / ml bFGF (Peprotech, catalog number 100-18B).

[0287] HCT116 was grown in McCoy 5a modified medium (Life Technologies, catalog no. 36600021) supplemented with 10% fetal bovine serum and 100 U / ml penicillin and 100 U / ml streptomycin.

[0288] Arpe 19 was grown in DMEM:F12 medium (Life Technologies, 21331-020) supplemented with 10% fetal bovine serum and 100 U / ml penicillin and 100 U / ml streptomycin.

[0289] HAP1 cells were grown in IMDM (Gibco, catalog number 12440-05) supplemented with 10% fetal bovine serum and 100 U / ml penicillin and 100 U / ml streptomycin.

[0290] Cell lines not mentioned above were grown in DMEM (Sigma, catalog number D6429) supplemented with 10% fetal bovine serum and 100 U / ml penicillin and 100 U / ml streptomycin. All cells were maintained at 37°C in a humidified water-jacketed incubator with 5% CO2. Cells were passaged at a density between 70 and 90%.

[0291] drugs The compounds used in the examples of the present invention were obtained as follows (CAT number = catalog number).

[0292] [Table 1]

[0293] Survival assay 4000 cells were seeded in 100 μl of corresponding medium in a 96-well plate. The following day, the drug, diluted in DMSO, was added in a triple series at eight different concentrations. The drug was added as a 4-fold dilution series, starting from 100 μM. Cells were incubated with the drug for 72 hours. Cell proliferation was evaluated by MTT assay according to the manufacturer's protocol (ATCC, catalog no. 30-1010K). Cell viability was normalized to the viability of cells treated with DMSO alone. The experiment was performed three times, and the data represent the mean ± SEM of the nine wells.

[0294] MTD (maximum tolerance) 5-Hydroxymethyl-2'-deoxycytidine and 5-formyl-2'-deoxycytidine (Berry and Associates) were formulated for intraperitoneal administration at concentrations of 30, 200, and 400 mg / mL in dimethyl sulfoxide (DMSO) / Solutol® HS15 / phosphate buffer solution (PBS) (5 / 5 / 90, v / v / v) at a dosage volume of 5 mL / kg. Dosage volumes of 10 or 20 mL / kg were also applied.

[0295] Male ICR mice weighing 23±3g were provided by BioLasco Taiwan (under license from Charles River Laboratories). The animals were acclimatized for three days prior to use to ensure good health. All animals were maintained in a hygienic environment with controlled temperature (20-24°C), humidity (30-70%), and a 12-hour light / dark cycle. They were given free access to sterile standard laboratory food [MFG (Oriental Yeast Co., Ltd., Japan)] and autoclaved tap water.

[0296] 5-Hydroxymethyl-2'-deoxycytidine and 5-formyl-2'-deoxycytidine were administered intraperitoneally to groups of three male ICR mice weighing 23±3g. The animals received an initial dose of 300 mg / kg. If the animals survived for 72 hours, the dose for the next cohort was increased. If one or more animals died, the dose for the next cohort was decreased. The study was stopped if all animals survived up to the upper limit, or if all three dose levels had been tested, or if the upper or lower limit was reached. At each dose level, the animals were observed for acute toxicity symptoms (mortality, convulsions, tremors, muscle relaxation, sedation, etc.) and autonomic effects (diarrhea, salivation, lacrimation, vasodilation, piloerection, etc.) for the first 30 minutes, and again at 1, 24, 48, and 72 hours. Body weight was recorded before administration and at 72 hours. The animals were observed, and the mortality rate was noted daily after compound administration. Macroscopic autopsies were performed on all animals without tissue collection, and the following dose levels were determined based on the study design.

[0297] Multiple MTDs 5-Hydroxymethyl-2'-deoxycytidine and 5-formyl-2'-deoxycytidine (Berry and Associates) were formulated for intraperitoneal administration at concentrations of 15, 50, and 100 mg / mL in dimethyl sulfoxide (DMSO) / Solutol® HS15 / phosphate buffer solution (PBS) (5 / 5 / 90, v / v / v) at a dosage volume of 20 mL / kg. The test compounds were administered in a total of 5 doses every 3 days (q3d × 5).

[0298] Male ICR mice weighing 23±3g were provided by BioLasco Taiwan (under license from Charles River Laboratories). The animals were acclimatized for three days prior to use to ensure good health. All animals were maintained in a hygienic environment with controlled temperature (20-24°C), humidity (30-70%), and a 12-hour light / dark cycle. They were given free access to sterile standard laboratory food [MFG (Oriental Yeast Co., Ltd., Japan)] and autoclaved tap water.

[0299] Animals were observed for acute toxic symptoms (mortality, convulsions, tremors, muscle relaxation, sedation, etc.) and autonomic nervous system effects (diarrhea, salivation, lacrimation, vasodilation, piloerection, etc.) for the first 30 minutes after each treatment (days 1, 4, 7, 10, and 13), and again at 1, 24, 48, and 72 hours after the final dose (day 13). Mortality was noted using the same scheme. In addition, body weight was recorded before each treatment and at 24, 48, and 72 hours after the final dose. Gross necropsy was performed on all animals without tissue collection.

[0300] xenograft The 5-hydroxymethyl-2'-deoxycytidine and 5-formyl-2'-deoxycytidine (Berry and Associates) administration solution was freshly prepared before each dose by first adding an appropriate volume of DMSO to a pre-weighed compound, and then adding an appropriate volume of Solutol® and PBS (5% DMSO / 5% Solutol® / 90% PBS). The standard active ingredient, temozolomide, was provided in powder form from the University Hospital of Oslo and was freshly formulated before each dose by first adding an appropriate volume of DMSO to a pre-weighed compound, and then adding an appropriate volume of Solutol® and PBS (5% DMSO / 5% Solutol® / 90% PBS). 5-hydroxymethyl-2'-deoxycytidine and 5-formyl-2'-deoxycytidine were administered at a dose volume of 20 mL / kg. The standard active agent, temozolomide, was administered at a dose volume of 10 mL / kg.

[0301] The human brain malignant glioma cell line, U87-MG (ATCC HTB-14, epithelial glioblastoma), was obtained from the American Type Culture Collection (ATCC). The cells were cultured in an incubator in minimal essential medium containing 5% fetal bovine serum (FBS) at 37°C and 5% CO2.

[0302] Six-to-seven-week-old female (nu / nu) nude mice obtained from BioLasco Taiwan (under license from Charles River Laboratories) were used. The animals were individually housed in ventilated cages (IVC, 36 mini-isolator system). The allocation for five animals was 27 × 20 × 14 cm. All animals were maintained in a hygienic environment with controlled temperature (20-24°C), humidity (30-70%), and a 12-hour light / dark cycle. They were given free access to standard laboratory food [MFG (Oriental Yeast Co., Ltd., Japan)] and autoclaved tap water.

[0303] Viable U87-MG (ATCC HTB-14) cells were subcutaneously (SC) transplanted into the right flank of female nu / nu mice (5 × 10⁶ cells / mouse in 0.2 mL / mouse PBS). The average tumor volume per group was approximately 129 mm². 3 From 131mm 3 Once this stage was reached, the tumor-transplanted mice were divided into four treatment groups, each containing eight animals, and dose administration was initiated (shown as Day 1).

[0304] 2000 mg / kg of 5-hydroxymethyl-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, and the corresponding vehicle (5% DMSO / 5% Solutol® / 90% PBS) were administered intraperitoneally (IP) once every three days for a total of five doses. 40 mg / kg of temozolomide was administered orally (PO) once daily for a total of five doses.

[0305] Tumor volume, body weight, mortality, and signs of obvious toxicity were monitored and recorded twice a week for 29 days. Tumor volume (mm 3 ) is the formula for an ellipsoid: length × (width) 2 It was estimated according to a multiplier of 0.5. Tumor growth inhibition (T / C) was calculated using the following formula: %T / C=(Tn / Cn)×100% Cn: Tumor mass measured on day n in the control group. Tn: Tumor mass measured on day n in the treatment group. A %T / C value of ≤42% was considered to indicate significant antitumor activity (#).

[0306] The tumor growth inhibition percentage (TGI) was also calculated using the following formula: %TGI=(1-(Tn / Cn))×100% A %TGI value of ≥58% was considered to indicate significant antitumor activity (#).

[0307] Using a two-way ANOVA®, followed by the Bonferroni test, we identified statistically significant differences between the study group and the negative control group from day 1 to day 29. Differences were considered statistically significant if p<0.05(*).

[0308] Upon completion of the study, tumors were removed from all animals used in the experiment and photographs were taken.

[0309] HPRT assay HPRT mutagenicity assays were performed in V79 cells. 50,000 V79 cells were treated in a 6-well plate with one of three different concentrations of d5hmC or d5fC (1, 10, 100 μM) for 24 hours. DMSO was used as a negative control. After treatment, cells were secondary cultured in T75 flasks for 9 days as needed to enable HPRT mutant expression. 10,000 cells were re-cultured in 10 replica petri dishes (100 × 15 mm) supplemented with selective medium (2.5 μg / ml 6 TG).

[0310] Survival (relative plate efficiency) was determined by plate culture of 200 cells in four replica petri dishes (60 × 15 mm) without selective medium. Colonies were fixed, Giemsa stained, and counted after 7 days. Mutation frequency is expressed as the total number of mutants counted on all plates divided by the number of seeded cells corrected for plate efficiency by reseeding. The experiment was performed three times, and the data represent the mean ± SEM of the triplicate.

[0311] Apoptosis flow cytometry 100,000 cells were seeded in 6-well plates and incubated with 100 μM temozolomide, 2'-deoxy-5-hydroxymethylcytidine, 5-formyl-2'-deoxycytidine, or DMSO for 72 hours. Apoptotic cells were detected using the Annexin V-7-amino-actinomycin D (7-AAD) Apoptosis Detection Kit (Nordic Biosite AS, catalog no. 640922) according to the manufacturer's protocol.

[0312] Fluorescence-activated cell sorting analysis was performed using an LSR Fortessa (BD Biosciences), and the data was analyzed using FlowJo software. All experiments were performed in triplicate.

[0313] Western blot Western blotting was performed as described above (Towbin et al., 1979. Biotechnology, 24, 145-149). Anti-CDA antiserum (Abcam, catalog number Ab82346) was used at the manufacturer's recommended concentration. Protein quantification was performed by the signal produced by oxidation of luminol by horseradish peroxidase conjugated to the secondary antibody.

[0314] (Example 1) Cytotoxicity against tumor cells HeLa cells grown as described above (materials and methods) were treated with 5-hydroxymethyl-2'-deoxycytidine and 5-formyl-2'-deoxycytidine. After 3 days of treatment with these compounds, cell viability was evaluated as described above (materials and methods). Surprisingly, cell viability after treatment with 5-hydroxymethyl-2'-deoxycytidine was not different from that of DMSO-treated cells, but 5-formyl-2'-deoxycytidine was found to be cytotoxic to HeLa cells.

[0315] The cytotoxic effects of 5-formyl-2'-deoxycytidine were compared to those of two well-documented cytotoxic compounds, 5-fluorouracil and temozolomide. 5-formyl-2'-deoxycytidine was determined to be more cytotoxic to HeLa cells (IC50 = 0.76 μM) than both 5-fluorouracil (IC50 > 25 μM) and temozolomide (IC50 > 25 μM).

[0316] Knowing that 5-formyl-2'-deoxycytidine is cytotoxic to cervical cancer (HeLa) cells, we expanded our research in two directions: (i) we evaluated further compounds: 5-carboxyl-2'-deoxycytidine; and (ii) we evaluated their cytotoxic effects against a wide range of human cancer cell lines (Table 1 (Table 2)).

[0317] [Table 2]

[0318] As shown in Table 1 (Table 2), 5-carboxyl-2'-deoxycytidine did not exhibit cytotoxic properties in any of the cancer cell lines evaluated. Interestingly, 5-formyl-2'-deoxycytidine was cytotoxic to a wide range of human cancer cells, indicating its potential use in the treatment of a broad range of cancers. 5-hydroxymethyl-2'-deoxycytidine had a narrower cytotoxicity profile, and in fact, this cytidine derivative was cytotoxic only to the two cell lines evaluated: U87-MG, grade IV glioma cells (IC50 > 0.3340 μM) and HAP1 chronic myeloid leukemia (Lukemia) cells (IC50 > 3.027 μM). This suggests that this compound may be more tolerant in patients. 5-Formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine were observed to be the most cytotoxic to glioblastoma pleomorphism (U87-MG) cell line.

[0319] The greatest cytotoxic effects of 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine were observed in glioma grade IV (U87-MG) cells; therefore, the cytotoxic activity of these compounds was evaluated in a wider range of patient-derived grade IV glioma cells (Table 2 (Table 3)). Due to the lack of activity in the previous assay, 5-carboxyl-2'-deoxycytidine was not included in this more rigorous analysis.

[0320] [Table 3]

[0321] As shown in Table 2 (Table 3), after treatment with the relevant compounds, 5 out of 12 grade IV glioma cell lines were killed by 5-formyl-2'-deoxycytidine, and 6 out of 12 grade IV glioma cell lines were killed by 5-hydroxymethyl-2'-deoxycytidine. This indicates the ability of these compounds against a wide range of gliomas. These results were benchmarked against both temozolomide, the current frontline treatment for grade IV gliomas, and 5-fluorouracil, a widely used anticancer drug. Temozolomide effectively killed 5 out of 12 grade IV glioma cell lines, while 5-fluorouracil killed 11 out of 12 grade IV glioma cell lines. Therefore, the data demonstrate that 5-formyl-2'-deoxycytidine is as effective as the current frontline treatment for grade IV glioma, and that 5-hydroxymethyl-2'-deoxycytidine is more effective than the current frontline treatment for grade IV glioma.

[0322] Furthermore, Table 2 (Table 3) demonstrates that both 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine are cytotoxic to cell lines resistant to the current frontline treatment (temozolomide). This provides evidence that these compounds may perform better than the current treatment for such temozolomide-resistant tumors. Alternatively, Table 2 (Table 3) indicates that temozolomide combined with 5-formyl-2'-deoxycytidine and / or 5-hydroxymethyl-2'-deoxycytidine would be the optimal treatment.

[0323] (Example 2) Cytotoxicity against normal cells As demonstrated in Example 1, 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine are useful therapeutic agents for the treatment of cancer, particularly grade IV gliomas, and are resistant to temozolomide treatment.

[0324] The inventors further investigated the extent to which these compounds kill normal human cells; low cytotoxicity to normal human cells is an advantageous characteristic for anticancer drugs. Therefore, the cytotoxicity of these compounds in various normal human cell lines was investigated (Table 3 (Table 4)). Cells were grown, and survival assays were performed as described above (materials and methods).

[0325] [Table 4]

[0326] As shown in Table 3 (Table 4), 5-hydroxymethyl-2'-deoxycytidine was not cytotoxic to any of the normal cell lines evaluated. 5-formyl-2'-deoxycytidine was cytotoxic to only one normal human cell line (HaCat, keratinocytes). These results suggest that these compounds are not only effective cancer chemotherapy agents but may also be well-tolerated by humans.

[0327] (Example 3) maximum capacity Since 5-hydroxymethyl-2'-deoxycytidine and 5-formyl-2'-deoxycytidine have limited effects on normal cells, the inventors hypothesized that the compounds could be administered at relatively high doses without causing the side effects typically associated with cancer chemotherapy. The maximum tolerated dose (MTD) of these compounds in mice was determined as described above (Materials and Methods). An MTD scheme was developed (Figure 1A). The endpoint of the study was survival after 72 hours, but the presence of acute toxicity symptoms (mortality, convulsions, tremors, muscle relaxation, sedation, etc.) and autonomic effects (diarrhea, salivation, lacrimation, vasodilation, piloerection, etc.) in the animals was monitored for the first 30 minutes, and again at 1, 24, 48, and 72 hours. Body weight was recorded before administration and at 72 hours.

[0328] The results indicated that mice could tolerate single doses of 300 mg / kg and 2000 mg / kg of 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine. Mice could not tolerate (or were not shown to tolerate) a single dose of 8000 mg / kg of 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine. These results suggest that the maximum tolerated single dose of both 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine in mice is at least 2000 mg / kg but less than 8000 mg / kg.

[0329] The conversion factor between mouse doses and human doses is 0.081 (Nair et al., (2016) Basic Clin Pharm. 7(2): 27~31). Therefore, the data indicate that in humans, the maximum tolerable single dose of the compound of formula (I) is at least 162 mg / kg but less than 648 mg / kg.

[0330] Cancer chemotherapy drugs that can be tolerated after multiple repeated administrations over several days are advantageous. Therefore, the inventors performed repeated maximum tolerated dose evaluations for both 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine as described above (materials and methods). Since both treatment groups showed good tolerance as single doses, doses of 300 mg / kg, 1000 mg / kg, and 2000 mg / kg were selected for repeated multiple tolerated dose evaluations.

[0331] Mice were intraperitoneally injected with the indicated compound at the indicated dose every three days for a total of five doses. Survival was the endpoint of this study, and body weight was the primary endpoint. However, the presence of acute toxicity symptoms (mortality, convulsions, tremors, muscle relaxation, sedation, etc.) and autonomic effects (diarrhea, salivation, lacrimation, vasodilation, piloerection, etc.) in the animals was also monitored during the first 30 minutes and again at 1, 24, 48, and 72 hours. Body weight was recorded before administration and at 72 hours.

[0332] Body weight in untreated animals was not statistically different from that of animals treated with either 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine at either of the evaluated doses (Figure 1B). These results indicate that these compounds are well-tolerated over long periods at the indicated doses.

[0333] (Example 4) In vivo cytotoxicity 5-Formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine were evaluated in a mouse xenograft model of glioblastoma pleomorphism as described above (materials and methods). U87-MG cells were subcutaneously injected into the flank of nude mice. Tumors were formed as described above (materials and methods). The tumor was 129 mm. 3 From 131mm 3After reaching a certain stage, the animals were divided into four groups: two treatment groups, a negative control group, and a positive control group. The two treatment groups were 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine. Mice in the treatment groups received either 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine at a dose of 2000 mg / kg every three days for a total of five doses. The negative control group was treated identically to the treatment groups, except that the IP injection contained a vehicle but not the compound. The positive control group was treated with temozolomide at a daily dose of 40 mg / kg for five days.

[0334] Tumor volume (Figure 1C), body weight (Figure 1D), mortality rate, and signs of obvious toxicity were monitored and recorded twice a week for 29 days. Tumor volume (mm 3 ) is the formula for an ellipsoid: length × (width) 2 Tumor growth inhibition percentage (%TGI) was estimated according to a multiplier of 0.5. The tumor growth inhibition percentage (%TGI) was determined using the following formula: %TGI = (1 - [(Tn) / (Cn)]) × 100, where Tn = the mean tumor volume of the treatment group on day "n" and Cn = the mean tumor volume of the control group on day "n". Compared to the negative control group, a %T / C value ≤ 42% or a TGI percentage value ≥ 58% was considered to indicate significant antitumor activity. Two-way ANOVA®, followed by the Bonferroni test, was used to identify statistically significant differences compared to the negative control group from day 1 to day 29 during the study (*p<0.05).

[0335] Figure 1C shows that treatment with 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine resulted in a significant reduction in tumor volume at all time points, comparable to that achieved with temozolomide.

[0336] At the end of the study, both 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine showed significant antitumor activity, with TGI of 83% and 93%, respectively (Figure 1E). The positive control group treated with temozolomide showed a 94% TGI. All compounds were well tolerated by the mice, and no significant changes in body weight were observed as a result of treatment (Figure 1D). At the completion of this study, tumors were dissected from the mice, photographed (Figure 1F), and measured (Figure 1G), and significant cytotoxic effects were observed in both treatment groups.

[0337] One mouse in the control group died during this experiment, and its tumor volume was not reported on day 29, but its tumor volume is included for the days prior to that.

[0338] In summary, these remarkable results demonstrate that 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine efficiently kill glioblastoma multiforme cells (glioma, grade IV) without strong cytotoxicity to normal cells. Furthermore, these compounds exhibit good tolerance in mice, resulting in a significant reduction in tumor volume with minimal side effects. The results demonstrate that human cancers can be treated using the compounds of this invention, and that these compounds can be used at high doses to kill tumor cells without killing non-cancerous cells. Therefore, these compounds have a broad therapeutic scope for use as anticancer drugs.

[0339] (Example 5) Cytotoxicity and anti-cell quiescent effects The cell line assays described above measure metabolic activity and therefore do not distinguish between inhibition of cell division and inhibition of cell death. Accordingly, we evaluated whether 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine either killed sensitive cells or delayed their cell cycle. SF-188 grade IV glioma cells were treated with the indicated concentrations of either DMSO, temozolomide, 5-formyl-2'-deoxycytidine, or 5-hydroxymethyl-2'-deoxycytidine. Cells were harvested, stained with Annexin V (which detects apoptotic cells by its ability to bind to phosphatidylserine) and 7AAD (a DNA stain that does not readily pass through intact cell membranes; therefore, cells with damaged membranes are selectively stained), and analyzed by flow cytometry as described above (Materials and Methods). SF-188 cells treated with DMSO or temozolomide produced similar cytometry profiles, with a slightly increased amount of dead cells compared to the temozolomide-treated control. Flow cytometry profiles showed that the majority of SF-188 cells treated with 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine were killed (Figure 2A). It is clear that SF-188 cells exposed to 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine were killed and did not stop at the cell cycle checkpoint.

[0340] (Example 6) Requirements for 2'-deoxy sugar derivatives 5-fluorouracil, a widely used cancer chemotherapy drug, has several cytotoxic variants, including nucleosides (5-fluorouridine (flurouridine) and 5-fluoro(fluro)-2'-deoxyuridine) and the nucleic acid base 5-fluorouracil. The inventors also evaluated whether the ribonucleoside and nucleic acid base variants of 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine are also cytotoxic.

[0341] As shown in Table 1 (Table 2), 5-formyl-2'-deoxycytidine is cytotoxic to HeLa cells. The cytotoxicity of 5-formylcytidine and 5-formylcytosine in HeLa cells was evaluated using the survival assay described above. Surprisingly, in contrast to 5-fluorouridine and 5-fluorouracil, neither 5-formylcytidine nor 5-formylcytosine was cytotoxic to HeLa cells (Figure 2B).

[0342] Table 1 (Table 2) shows that 5-hydroxymethyl-2'-deoxycytidine is cytotoxic to U87-MG cells. The cytotoxicity of 5-hydroxymethylcytidine and 5-hydroxymethylcytosine in U87-MG cells was evaluated using the survival assay described above. Surprisingly, in contrast to 5-fluorouridine and 5-fluorouracil, 5-hydroxymethylcytidine and 5-hydroxymethylcytosine were shown to be non-cytotoxic to U87-MG cells (Figure 2C).

[0343] These results indicate that 2'-deoxyribose sugar is required for the cytotoxicity of the compounds of the present invention. Surprisingly, these results now indicate that 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine utilize cellular pathways fundamentally different from those of fluoropyrimidines.

[0344] (Example 7) Ineffectiveness of cytidine deaminase Mutant nucleosides and nucleotide analogs are often removed from the nucleotide pool by cytidine deaminase (CDA). CDA inactivates two common nucleotide analog anticancer agents, gemcitabine and cytosine arabinoside. Anticancer agents that are not inactivated by CDA would be desirable. Since 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine are cytidine derivatives, we hypothesized that cells expressing CDA would be resistant to treatment with 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine.

[0345] However, surprisingly, as shown in Table 4A (Table 5), no correlation was observed between CDA expression and resistance or sensitivity to either 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine. IC in Table 4 (Tables 5 and 6) 50 The data are identical to those in Table 1 (Table 2). CDA expression levels in the specified cell lines were identified using the EMBL Expression Atlas (https: / / www.ebi.ac.uk / gxa / home, search term CDA). CDA expression is reported as RNA transcripts per million cells (TPM), as described in Wagner et al., (2012) Theory Biosci 131(4):281~285 and Mortazavi A et al., (2008) "Mapping and quantifying mammalian transcriptomes by RNA-Seq.", Nature methods 5(7):621~8. If more than one value was reported, the lowest value was used.

[0346] [Table 5]

[0347] The linear correlation between cytotoxicity and drug exposure shows an insufficient fit (linear model for 2d5fc, Rz Linear model for =0.00173;2d5hmC, R z The result was 0.02262). The data, along with the data from EMBL, suggest that CDA expression is not related to either sensitivity or resistance to 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine.

[0348] In addition, the CDA expression levels (TPM) of various glioma cell lines were determined and are shown in Table 4B (Table 6). CDA expression levels in specified cell lines were identified using the Cancer Cell Line Encyclopedia (https: / / portals.broadinstitute.org / ccle) described in Barretina, J et al., (2012) The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature. 483:603~7. CDA expression is reported as RNA transcripts per million cells (TPM), as described in Wagner et al., (2012) Theory Biosci 131(4):281~285. If more than one value was reported, the highest value was used.

[0349] [Table 6]

[0350] (Example 8) Lack of mutagenicity Temozolomide, the current frontline treatment for glioblastoma pleomorphism, is a potent mutagen. In fact, temozolomide exerts its cancer chemotherapy activity so intensely on mutated tumor cells that the tumor cells die. Temozolomide works by alkylating DNA, inducing mutations. The expression of MGMT—a protein responsible for removing alkylated DNA damage—makes glioblastoma cells almost completely resistant to the cytotoxic effects of temozolomide.

[0351] Therefore, the inventors investigated whether 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine have similar mechanisms of action. The mutagenicity of these compounds was evaluated in a hypoxanthine-guanine phosphoribosyltransferase (HPRT) assay as described above (materials and methods). As shown in the HPRT assay, neither 5-hydroxymethyl-2'-deoxycytidine nor 5-formyl-2'-deoxycytidine were genotoxic to mammalian cells at any of the concentrations evaluated (Figure 3). These results indicate that 5-hydroxymethyl-2'-deoxycytidine and 5-formyl-2'-deoxycytidine are not mutagens, that is, their cytotoxic effects are not due to mutagenic activity.

[0352] (Example 9) Cytotoxicity of other compounds versus HeLa cells HeLa cells were treated with either 5-formyl-2'-deoxycytidine, 5-formylcytidine, or 5-chloro-2'-deoxycytidine. After 3 days of treatment with these compounds at concentrations of 100, 25, 6.25, 1.56, 0.39, 0.1, 0.02, or 0.006 μM, cell viability was evaluated by MTT assay as described above (materials and methods).

[0353] As shown in Figure 4, a significantly greater cytotoxic effect is observed after treatment with 5-formyl-2'-deoxycytidine than after treatment with either 5-formylcytidine or 5-chloro-2'-deoxycytidine.

[0354] (Example 10) Other compound cytotoxic counter glioma cells U87-MG cells (glioma, grade IV) were treated with one of the following at concentrations of 100, 25, 6.25, 1.56, 0.39, 0.1, 0.02, or 0.006 μM: 5-formyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, temozolomide, 5-fluorouracil, 5-bromo-2'-deoxycytidine, 5-iodo-2'-deoxycytidine, or 5-chloro-2'-deoxycytidine. After 3 days of treatment with these compounds, cell viability was evaluated by MTT assay as described above (materials and methods).

[0355] cell culture U87-MG cell lines were grown in DMEM (Sigma, catalog number D6429) supplemented with 10% fetal bovine serum. All cells were maintained at 37°C in a humidified water-jacketed incubator with 5% CO2. Cells were passaged at a density between 70 and 90%.

[0356] Survival assay 4000 cells were seeded in 100 μl of the corresponding medium in a 96-well plate. The following day, the drug, diluted in DMSO, was added in triplicate at concentrations of 100, 25, 6.25, 1.56, 0.39, 0.1, 0.02, or 0.006 μM. Cells were incubated with the drug for 72 hours. Cell proliferation was evaluated by MTT assay according to the manufacturer's protocol (ATCC, catalog no. 30-1010K). Cell viability was normalized to the viability of cells treated with DMSO alone. The experiment was performed three times, and the data represent the mean ± SEM of the nine wells.

[0357] As shown in Table 5 (Table 7), both 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine performed remarkably well in terms of cytotoxicity against U87-MG cells compared to other compounds tested, including 5-bromo-2'-deoxycytidine, 5-iodo-2'-deoxycytidine, and 5-chloro-2'-deoxycytidine.

[0358] [Table 7]

[0359] In addition, U87-MG cells were treated with 5-formyl-2'-deoxycytidine (d5fC), 5-hydroxymethyl-2'-deoxycytidine (d5hmC), 5-chloro-2'-deoxycytidine (5CldC), 5-bromo-2'-deoxycytidine (5BrdC), 5-iodo-2'-deoxycytidine (5IdC), and thymidine. The results are shown in Figure 5. d5hmC and d5fC were significantly more cytotoxic to glioma cells than 5CldC, 5BrdC, 5IdC, and thymidine.

[0360] 4000 cells were seeded in 100 μl of the corresponding medium in a 96-well plate. The following day, the drug, diluted in DMSO, was added in triplicate at concentrations of 100, 25, 6.25, 1.56, 0.39, 0.1, 0.02, or 0.006 μM. Cells were incubated with the drug for 72 hours. Cell proliferation was evaluated by MTT assay according to the manufacturer's protocol (ATCC, catalog no. 30-1010K). Cell viability was normalized to the viability of cells treated with DMSO alone. The experiment was performed three times, and data are expressed as mean ± SD.

[0361] (Example 11) Effects not mediated by thymidine synthase cell culture U87-MG cell lines were grown in DMEM (Sigma, catalog number D6429) supplemented with 10% fetal bovine serum. All cells were maintained at 37°C in a humidified water-jacketed incubator with 5% CO2. Cells were passaged at a density between 70 and 90%.

[0362] Survival assay 4000 cells were seeded in 100 μl of the corresponding medium in a 96-well plate. The following day, the drug, diluted in DMSO, was added in a triple series at eight different concentrations. The drug was added as a 4-fold dilution series, starting from 100 μM. Cells were incubated with the drug for 72 hours. Cell proliferation was evaluated by MTT assay according to the manufacturer's protocol (ATCC, catalog no. 30-1010K). Cell viability was normalized to the viability of cells treated with DMSO alone. The experiment was performed three times, and the data represent the mean ± SD of the three wells.

[0363] The results are shown in Figure 6. Cytotoxicity caused by 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine is not rescued by the addition of thymidine in U87-MG cells. This result indicates that 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine do not act by inhibiting thymidine synthase.

[0364] (Example 12) Combination therapy with temozolomide cell culture Glioblastoma pleomorphism cell line (U87-MG) was grown in DMEM (Sigma, catalog number D6429) supplemented with 10% fetal bovine serum. All cells were maintained at 37°C in a humidified water-jacketed incubator with 5% CO2. Cells were passaged at a density between 70 and 90%.

[0365] Survival assay 4000 cells were seeded in 100 μl of the corresponding medium in a 96-well plate. The following day, the drug, diluted in DMSO, was added in a triple series at eight different concentrations. The drug was added in a 4-fold dilution series, starting at 100 μM. Cells were incubated with the drug for 72 hours. Cell proliferation was evaluated by the MTT assay according to the manufacturer's protocol (ATCC, catalog no. 30-1010K). Cell viability was normalized to the viability of cells treated with DMSO alone. The experiment was performed three times, and the data represent the mean of these experiments.

[0366] As shown in Figure 7, human glioblastoma pleomorphism cell lines resistant to temozolomide were treated with temozolomide alone, 5-formyl-2'-deoxycytidine (d5fC), or a combination of d5fC and temozolomide. The combination of temozolomide and d5fC was more effective than either chemical alone in treating human glioblastoma pleomorphism.

[0367] As shown in Figure 8, human glioblastoma pleomorphism cell lines resistant to temozolomide were treated with temozolomide alone, 5-hydroxymethyl-2'-deoxycytidine (d5hmC), or a combination of d5hmC and temozolomide. The combination of temozolomide and d5hmC was more effective than either chemical alone in treating human glioblastoma pleomorphism.

[0368] Therefore, 5-hydroxymethyl-2'-deoxycytidine and 5-formyl-2'-deoxycytidine act synergistically with temozolomide.

[0369] (Example 13) Uridine analog The cytotoxic effects of 5-methoxymethyl-2'-deoxyuridine and 5-acetoxymethyl-2'-deoxyuridine were evaluated.

[0370] cell culture U87-MG cell lines were grown in DMEM (Sigma, catalog number D6429) supplemented with 10% fetal bovine serum. All cells were maintained at 37°C in a humidified water-jacketed incubator with 5% CO2. Cells were passaged at a density between 70 and 90%.

[0371] Survival assay 4000 cells were seeded in 100 μl of corresponding medium in a 96-well plate. The following day, the drug, diluted in DMSO, was added in a triple series at eight different concentrations. The drug was added as a 4-fold dilution series, starting from 100 μM. Cells were incubated with the drug for 72 hours. Cell proliferation was evaluated by MTT assay according to the manufacturer's protocol (ATCC, catalog no. 30-1010K). Cell viability was normalized to the viability of cells treated with DMSO alone. The experiment was performed three times, and the data represent the mean ± SEM of the nine wells.

[0372] As shown in Figure 9, the U87-MG glioblastoma pleomorphism cell line cannot withstand treatment with gradually increasing concentrations of 5-methoxymethyl-2-deoxyuridine or 5-acetoxymethyl-2'-deoxyuridine. Therefore, 5-methoxymethyl-2-deoxyuridine and 5-acetoxymethyl-2'-deoxyuridine are particularly effective anticancer agents against glioblastoma pleomorphism.

[0373] (Example 14) Cytotoxicity of 5-formyl-2'-deoxycytidine-5'-triphosphate (HeLa) cell culture HeLa cell lines were grown in DMEM (Sigma, catalog number D6429) supplemented with 10% fetal bovine serum. All cells were maintained at 37°C in a humidified water-jacketed incubator with 5% CO2. Cells were subculturised at a density between 70 and 90%.

[0374] Survival assay 4000 cells were seeded in 100 μl of corresponding medium in a 96-well plate. The following day, the drug, diluted in DMSO, was added in a triple series at eight different concentrations. The drug was added as a 4-fold dilution series, starting from 100 μM. Cells were incubated with the drug for 72 hours. Cell proliferation was evaluated by MTT assay according to the manufacturer's protocol (ATCC, catalog no. 30-1010K). Cell viability was normalized to the viability of cells treated with DMSO alone. The experiment was performed three times, and the data represent the mean ± SEM of the nine wells.

[0375] As shown in Figure 10A, HeLa cervical cancer cells cannot withstand treatment with gradually increasing concentrations of 5-formyl-2'-deoxycytidine-5'-triphosphate. This demonstrates the usefulness of 5-formyl-2'-deoxycytidine-5'-triphosphate in the treatment of human cancers, such as cervical cancer.

[0376] (Example 15) Cytotoxicity of 5-formyl-2'-deoxycytidine-5'-triphosphate and 5-hydroxymethyl-2'-deoxycytidine-5'-triphosphate (glioma) U87-MG cells (glioma, grade IV) were treated with either 5-formyl-2'-deoxycytidine-5'-triphosphate or 5-hydroxymethyl-2'-deoxycytidine-5'-triphosphate at concentrations of 100, 25, 6.25, 1.56, 0.39, 0.1, 0.02, or 0.006 μM. After 3 days of treatment with these compounds, cell viability was evaluated by MTT assay as described above (materials and methods).

[0377] cell culture U87-MG cell lines were grown in DMEM (Sigma, catalog number D6429) supplemented with 10% fetal bovine serum. All cells were maintained at 37°C in a humidified water-jacketed incubator with 5% CO2. Cells were passaged at a density between 70 and 90%.

[0378] Survival assay 4000 cells were seeded in 100 μl of the corresponding medium in a 96-well plate. The following day, the drug, diluted in DMSO, was added in triplicate at concentrations of 100, 25, 6.25, 1.56, 0.39, 0.1, 0.02, or 0.006 μM. Cells were incubated with the drug for 72 hours. Cell proliferation was evaluated by MTT assay according to the manufacturer's protocol (ATCC, catalog no. 30-1010K). Cell viability was normalized to the viability of cells treated with DMSO alone. The experiment was performed three times, and data represent the mean ± SD of at least three wells.

[0379] As shown in Figure 10B, both 5-formyl-2'-deoxycytidine-5'-triphosphate and 5-hydroxymethyl-2'-deoxycytidine-5'-triphosphate were cytotoxic to U87-MG glioma cells.

[0380] (Example 16) CDA expression Linear and log-to-cardinal-2 converted CDA expression levels were determined in various glioma cell lines. CDA expression levels in specified cell lines were identified by searching the GENEVESTIGATOR® database (https: / / genevestigator.com / gv / ) for CDA, organism: Homo sapiens, and platform: Affymetrix Human Genome U133 2.0 array. The results are shown in Figures 11 to 13 and Table 6 (Table 8).

[0381] Figure 11 shows the CDA-normalized Log2 CDA expression levels determined for various cancers. Figure 12 shows the linear CDA expression levels determined for various cancers. Figures 13A and 13B show the CDA-normalized Log2 expression levels in various human brain tumors. Figures 14A and 14B show the linear CDA expression levels in various human brain tumors.

[0382] Table 6 (Table 8) below shows the CDA expression levels of various cell lines and the ICs for 2d5hmC and / or 2d5fc in those cell lines. 50 The value is shown. IC 50 The values ​​were obtained as described above (Materials and Methods and Example 1).

[0383] [Table 8]

[0384] Table 7 (Table 9) below shows the linear CDA expression levels of various cell lines and the ICs for 2d5hmC and / or 2d5fc in those cell lines. 50 The value is shown. IC 50 The values ​​were obtained as described above (Materials and Methods and Example 1).

[0385] [Table 9]

[0386] In summary, these results demonstrate that many cancers, including all central nervous system cancers tested, do not overexpress CDA, but rather express it at low levels. The results also demonstrate that there is no correlation between CDA expression and sensitivity to either 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine.

[0387] (Example 17) blood-brain barrier permeability Blood-brain barrier permeability was measured using the Parallel Artificial Membrane Permeability Assay Kit (PAMPA) from BioAssay Systems. Permeability was determined according to the manufacturer's instructions.

[0388] Manufacturer's instructions: 1. Prepare 500 μL of 500 μM test compound in a separate centrifuge tube: Mix 10 mM test compound in 25 μL of DMSO with 475 μL of PBS. If using permeable controls, dilute them to 500 μM as well: Mix 25 μL of permeable control with 475 μL of PBS. 2. Prepare 200 μM equilibrium standards for each test compound and control in separate tubes: Mix 80 μL of 500 μM test compound or control with 120 μL of PBS. 200 μM is the final concentration of the donor and acceptor well solutions if the compound can permeate the membrane and reach complete equilibrium. Next, prepare a blank control by mixing 5 μL of DMSO with 245 μL of PBS in a separate tube. Set aside the equilibrium standards and blank controls for analysis the following day. 3. Add 300 μL of PBS to the wells in the acceptor plate. 4. With the donor plate still in its tray, add 5 μL of 4% lecithin in dodecane directly to the well membrane of the donor plate. Take care not to puncture the membrane with the pipette tip. 5. Add 200 μL of each 500 μM test compound and 500 μM permeability control to the double wells of the donor plate. Note: The inventors recommend performing all experimental variables in at least double wells. Carefully place the donor plate into the acceptor plate wells. Incubate at room temperature or 37°C for 18 hours or the desired incubation period (e.g., 16–24 hours). 6. Carefully remove the donor plate and collect the liquid in the acceptor plate wells for analysis. This is referred to as the acceptor solution. 7. Add 100 μL of acceptor solution and equilibrium standard to each test compound and permeability control. 100 μL of blank control is also added to the well of the UV plate (catalog number P96UV). 8. The absorbance spectrum from 200 nm to 500 nm is read at 10 nm intervals to determine the peak absorbance of the test compound. The blank control is used to confirm that the peak is due to the test compound and not to DMSO in the solution. The peak absorbances for the high-transmittance, medium-transmittance, and low-transmittance controls are 280 nm, 270 nm, and 270 nm, respectively.

[0389] As shown in Figure 15, 2d5hmC and 2d5fC cross the blood-brain barrier.

Claims

1. Formula (IIa): 【Chemistry 1】 [In the formula, X is -(CH 2 ) n -X', where n is between 0 and 6, and X' is -OH; R1 is -OH or O(P(=O)(OH)O) n H, where n is 1 to 3; and R2 is -OH] A compound of the same, or a pharmaceutically acceptable salt thereof, for the treatment or prophylaxis of chronic myeloid leukemia in a subject.

2. The compound is of formula (IIIa) or (IIIc): 【Chemistry 2】 The agent according to claim 1, wherein X is a compound of [wherein X is as defined in claim 1] or a pharmaceutically acceptable salt thereof.

3. X is -CH 2 The agent according to claim 1 or 2, wherein it is an OH group.

4. The agent according to any one of claims 1 to 3, wherein the compound is 5-hydroxymethyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine-5'-triphosphate, or a pharmaceutically acceptable salt thereof.

5. The agent according to claim 4, wherein the compound is 5-hydroxymethyl-2'-deoxycytidine or a pharmaceutically acceptable salt thereof.

6. The agent according to any one of claims 1 to 5, wherein chronic myeloid leukemia is resistant to treatment with gemcitabine, cytarabine, temozolomide, or 5-fluorouracil.

7. The agent according to any one of claims 1 to 6, wherein the treatment comprises administering the compound in a dose of 10 mg / kg to 405 mg / kg.

8. The agent according to any one of claims 1 to 7, wherein the treatment further comprises the administration of a further anticancer agent.

9. The agent according to claim 8, wherein the further anticancer agent is selected from the group consisting of gemcitabine, cytarabine, temozolomide, 5-fluorouracil, and carmustine wafer.

10. A kit comprising an agent defined in any one of claims 1 to 5 and a further anticancer agent for use in the treatment or prevention of chronic myeloid leukemia.

11. The kit according to claim 10, wherein the further anticancer agent is selected from the group consisting of gemcitabine, cytarabine, temozolomide, 5-fluorouracil, and carmustine wafers.

12. Formula (IIa): 【Transformation 3】 [Here, X is -(CH 2)n -X', where n is 0 to 6 and X' is -OH; R1 is -OH or O(P(=O)(OH)O) n H, where n is 1 to 3; and R2 is -OH] The use of the compound, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prevention of chronic myeloid leukemia in a subject.

13. The compound is of formula (IIIa) or (IIIc): 【Chemistry 4】 The use according to claim 12, wherein X is a compound of [wherein X is as defined in claim 1] or a pharmaceutically acceptable salt thereof.

14. X is -CH 2 The use according to claim 12 or 13, wherein it is OH.

15. The use according to any one of claims 12 to 14, wherein the compound is 5-hydroxymethyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine-5'-triphosphate, or a pharmaceutically acceptable salt thereof.

16. The use according to claim 15, wherein the compound is 5-hydroxymethyl-2'-deoxycytidine or a pharmaceutically acceptable salt thereof.

17. The use according to any one of claims 12 to 16, wherein chronic myeloid leukemia is resistant to treatment with gemcitabine, cytarabine, temozolomide, or 5-fluorouracil.

18. The use according to any one of claims 12 to 17, wherein the treatment comprises administering the compound in a dose of 10 mg / kg to 405 mg / kg.

19. The use according to any one of claims 12 to 18, wherein the treatment further comprises the administration of a further anticancer agent.

20. The use according to claim 19, wherein the further anticancer agent is selected from the group consisting of gemcitabine, cytarabine, temozolomide, 5-fluorouracil, and carmustine wafers.

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