Deoxycytidine derivative or deoxyuridine derivative to be used in cancer treatment

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 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 treatment with known anti-cancer agents. Thus, the present invention relates to the use of the compounds of the present invention, both alone and in combination with one or more additional or further anti-cancer agents, in the treatment or prevention of cancer. The present invention also relates to methods of treating or preventing cancer using the compounds of the present invention, either alone or in combination with one or more further anti-cancer agents. [Background technology]

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

[0003] Although there are numerous therapies available, resistance to known anti-cancer drugs can be a problem in successfully treating cancer in patients. Additional cancer therapies remain needed.

[0004] In some 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 dividing normal, healthy cells. 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, i.e., 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 aggressive cancer of the central nervous system. It is the second most common cancer in children. Adults diagnosed with glioblastoma multiforme represent some of the largest unmet needs in oncology. With currently available treatments, the average survival from diagnosis is 14.6 months. Fewer than 5% of patients diagnosed with glioblastoma multiforme survive longer than 5 years. Long-term survival after a glioblastoma multiforme diagnosis is rare and is more common in children.

[0007] Current therapies are largely palliative and designed to improve quality of life. After diagnosis, current glioblastoma multiforme treatments follow a similar course: surgical removal of the affected brain area, with the patient expected to survive the surgery, followed by radiation and chemotherapy. Glioblastoma multiforme forms tentacle-like structures within the affected brain, thus making complete surgical removal difficult and often impossible, even when indicated. Besides surgical intervention and radiation therapy, three drugs have been approved for the treatment of glioblastoma multiforme: 1) Avastin® (bevacizumab)—an angiogenesis inhibitor with multiple oncology indications. Avastin® is believed to inhibit tumor growth by blocking the development of new blood vessels within tumors, effectively starving the tumor. While Avastin® is approved in the United States and Japan, Avastin® 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 studies, the patient benefit is often minimal, and no benefit is provided by Avastin® treatment in patients with recurrent 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)—a mutagenic DNA alkylating agent; generic formulations are also available. Temozolomide is thought to inhibit glioblastoma multiforme growth by significantly mutating tumor DNA, which induces cell death. Due to its mechanism of action, cells expressing the DNA repair protein MGMT are almost universally 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® (carmustine wafer) (1,3-bis(2-chloroethyl)-1-nitrosourea)—a cytotoxic nitrogen mustard. It is delivered as a biodegradable disc implanted directly into the brain after surgical resection. Randomized trials have demonstrated that gliadel® improves median survival by 2.1 months. Patients treated with gliadel® report fewer and less severe side effects than temozolomide-treated patients, but overall survival is 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 ed., T.W. Greene and P.G.M. 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 enables predictive modeling of anticancer drug sensitivity. Nature. 483:603~7 Summary of the Invention [Problem to be solved by the invention]

[0009] There remains a need for additional anti-cancer drugs for the treatment of glioblastoma multiforme and other cancers, particularly anti-cancer drugs that effectively treat cancers that are resistant to treatment with temozolomide.

[0010] The human protein cytidine deaminase (CDA) catalyzes the hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Some known anticancer drugs are nucleoside / nucleotide analogs, such as gemcitabine (2,2-difluorodeoxycytidine) and cytarabine (Ara-C, cytosine arabinoside). CDA inactivates such anticancer drugs, including gemcitabine and cytarabine, in a problematic manner. There remains a need for additional anticancer drugs that are not inactivated by CDA. [Means for solving the problem]

[0011] The present inventors have discovered a select class of compounds that exhibit activity in treating glioblastoma multiforme and a wide range of other cancers. Such compounds are suitable for inhibiting tumor cell proliferation in general, and those associated with brain cancers in particular, glioblastoma multiforme.

[0012] In a first aspect, the present invention provides a compound of formula (I):

[0013] [ka]

[0014] [In the formula, X is a group containing 1 to 20 non-hydrogen atoms containing at least one functional group selected from an aldehyde, an alcohol, a protected alcohol, an ether, an ester, and a carboxylic acid, with the proviso that X is not -COOH; W1 and W2 are each independently O, S, or NH; Y is H or a group containing 1 to 15 non-hydrogen atoms; Z is -OPG, -OR z or -N(R x R y ), where R x , R y and R zare independently H or a group containing 1 to 10 non-hydrogen atoms; R1 is H or a group containing 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). or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof to provide.

[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):

[0016] [ka]

[0017] [In the formula, X is a group containing 1 to 20 non-hydrogen atoms containing at least one functional group selected from an aldehyde, an alcohol, a protected alcohol, an ether, an ester, and a carboxylic acid, with the proviso that X is not -COOH; W1 and W2 are each independently O, S, or NH; Y is H or a group containing 1 to 15 non-hydrogen atoms; Z is -OPG, -OR z or -N(R x R y ), where R x , R y and R z are independently H or a group containing 1 to 10 non-hydrogen atoms; R1 is H or a group containing 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). or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof to provide.

[0018] The present invention is applicable to any cancer. Cancer is broadly defined herein to include any neoplastic condition, particularly including malignant or pre-malignant conditions. Cancer can cause, result in, or arise from solid tumors, but is not limited to these, and also includes cancers of the hematopoietic system. Throughout, the terms "cancer" and "cancer cells" are used interchangeably. Throughout, the terms "tumor" and "tumor cells" are used interchangeably. Benign tumors and malignant tumors are also included in the term cancer as used herein, i.e., the terms cancer and tumor are used interchangeably. Treatment of malignant tumors is preferred.

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

[0020] In an alternative aspect, the present invention provides a compound of formula (I) for use as an anti-cancer agent. In an alternative aspect, the present invention provides the use of a compound of formula (I) for treating or preventing cancer.

[0021] In an alternative aspect, the present invention provides a compound of formula (I) for use as an antitumor agent. In an alternative aspect, 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 invention provides for the use of a compound of formula (I) in the manufacture of an anti-cancer therapeutic product (i.e. a preparation or medicament, such as a pharmaceutical composition, formulation, combination product, co-formulated product or kit), or alternatively, for the manufacture of a medicament for use as an anti-cancer agent or in the treatment or prevention of cancer.

[0023] In an alternative view, this aspect of the invention provides for the use of a compound of formula (I) in the manufacture of an anti-tumour therapeutic product (i.e. a preparation or medicament, such as a pharmaceutical composition, formulation, combination product, co-formulated product or kit), or alternatively for the use of a compound of formula (I) for the manufacture of a medicament for use as an anti-tumour agent or in the treatment or prevention of tumours.

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

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

[0026] In some preferred embodiments of the present invention, a compound of formula (I) is used as the sole active agent (the only active agent in a treatment regimen). Thus, 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). Thus, in some preferred embodiments, a compound of formula (I) is used alone. "Sole active agent" (or sole active ingredient) means the only agent or component that is therapeutically active (or biologically active). Thus, ingredients such as preservatives or excipients or agents unrelated to the disease being treated are not considered to be active agents.

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

[0028] The compounds of formula (I) may be used in any method of treating or preventing cancer (ie, tumors) in a subject according to the present invention, either alone or in combination with an additional anti-cancer agent.

[0029] As demonstrated in the Examples, the use of compounds of formula (I) in combination with one or more additional anti-cancer agents results in a synergistic cytotoxic effect.

[0030] Thus, in a further aspect, the present invention provides a compound of formula (I) together with a further anti-cancer agent for use in treating or preventing cancer (i.e., tumours), or alternatively provides a compound of formula (I) for use together with a further anti-cancer agent for treating or preventing cancer (i.e., tumours).

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

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

[0033] The compound of formula (I) and the additional anti-cancer agent may be co-formulated into a single composition. However, this is not required. The medicament may be a combined preparation, composition, kit, etc., and in any of the aspects of the present invention, it is not required that the compound of formula (I) and the additional anti-cancer agent be co-formulated into a single composition—they may be formulated separately and administered separately, including sequentially or simultaneously.

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

[0035] More particularly, the present invention provides products (especially pharmaceutical products) comprising a compound of formula (I) and a further (i.e. one or more further or second) anti-cancer agent(s) as a combined preparation for separate, sequential or simultaneous use in the treatment or prevention of cancer (i.e. tumours).

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

[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 an additional anti-cancer agent.

[0038] For all aspects of the invention, the compounds of the invention are compounds of formula (I) as described elsewhere herein, and preferred and optional embodiments for compounds described with respect to one aspect of the invention apply mutatis mutandis to any and all other aspects of the invention.

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

[0040] X X is a group containing from 1 to 20 non-hydrogen atoms containing at least one functional group selected from aldehydes, alcohols, protected alcohols, ethers, esters, and carboxylic acids, with the proviso that X is not —COOH.

[0041] Preferably, X is a group containing from 1 to 10 non-hydrogen atoms, more preferably from 1 to 5 non-hydrogen atoms, even more preferably from 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 from 2 to 20 non-hydrogen atoms. Thus, preferably, X is a group containing from 2 to 10 non-hydrogen atoms, even more preferably from 2 to 5 non-hydrogen atoms, even more preferably from 2 to 3 non-hydrogen atoms, and most preferably 2 non-hydrogen atoms.

[0043] Preferably, X contains at least one functional group selected from aldehyde, alcohol, protected alcohol, ether, and ester. More preferably, X contains at least one functional group selected from aldehyde, alcohol, ether, and ester. Most preferably, X contains at least one functional group selected from aldehyde and alcohol. 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, with the proviso that X is not -COOH or -OH.

[0046] X may 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 where R is an alkyl group, preferably methyl.

[0047] The term "alkyl" refers to straight and branched saturated aliphatic hydrocarbon chains. Preferably, alkyl is a C 1~10 refers to alkyl. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl).

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

[0049] The term "alkenyl" refers to straight and branched hydrocarbon chains having one or more, preferably one or two, carbon-carbon double bonds. Preferably, alkenyl is C 2~10 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 straight and branched hydrocarbon chains having one or more, preferably one or two, carbon-carbon triple bonds. Preferably, alkynyl is C 2~10 Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, and propargyl.

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

[0023] refers to haloalkyl. 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, alkoxy is a C 1~10 refers to alkoxy. 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 attached through an oxygen bridge. Preferably, haloalkoxy is C 1~10

[0023] refers to haloalkoxy. Examples of haloalkoxy groups include, but are not limited to, trifluoromethoxy, 2,2,2-trifluoroethoxy, and pentafluorothoxy.

[0054] When 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 More preferably, L is a bond or C 1~4 Most preferably, L is a bond or C alkyl (—CH—).

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

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

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

[0060] More preferably, X is -(CH2) n -X', where n is 0 to 2, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is 0 to 2, and X' is -OH. Preferably, X is -(CH2) n-X', where n is 0 to 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, —CH 2 OH, —CH 2 OCH 3 or —CH 2 OC(═O)CH 3 .

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

[0064] More preferably, X is —CHO or —CH 2 OH, most preferably CH 2 OH.

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

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

[0067] Thus, 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, W1 and W2 are both O.

[0070] Y Y is H or a group containing 1 to 15 non-hydrogen atoms. Preferably, Y is H or a group containing 1 to 10 non-hydrogen atoms. More preferably, Y is H or a group containing 1 to 5 non-hydrogen atoms.

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

[0072] When Y is H or a group containing 1 to 5 non-hydrogen atoms, 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 ), where R x , R y and R z are independently H or a group containing 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 zis H or a group containing 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 are independently H or C 1~8 More preferably, R x and R y are independently H or -C(O)O(CH2) n CH3, where n is 1 to 4, preferably 4.

[0078] Preferably, R x and R y At least one of R is H. For example, preferably, x is H and R y are independently H or -C(O)O(CH2) n CH3, where n is 1 to 4, preferably 4. More preferably, R x and R y are both H.

[0079] Thus, Z is preferably -NH or -OH. More preferably, when X is -CHO or -CHOH, most preferably -CHOH, Z is -NH; when X is -CHOCH or -CHOC(=O)CH, Z is -OH.

[0080] When Z is —OH, the compounds of formula (I) may be depicted in tautomeric forms as shown below.

[0081] [ka]

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

[0083] In this case, R z 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 ) where PG, R x and R y is as defined above.

[0085] Most preferably, Z is —N(R x R y ), where R x and R y is as defined above.

[0086] Thus, Z is preferably -NH2.

[0087] R1 R1 is H or a group containing 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, where n is 1 to 3, and where 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 1 to 3, preferably 3. More preferably, R1 is H, -OH or -O(P(=O)(OH)O). nH, where n is 1 to 3, preferably 3. Even more preferably, R1 is -OH or -O(P(=O)(OH)O) n H, where n is 1 to 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 H, where n is 1 to 3, preferably 3; R2 is —OH; and R3 is H. [Brief explanation of the drawings]

[0094] [Figure 1A] Figure 1 shows that 5-formyl-2'-deoxycytidine and 5-hydroxymethylcytidine are well tolerated in mice and reduce human glioblastoma multiforme tumors in a mouse xenograft model. Single-dose maximum tolerated dose protocol. Mice were dosed at the indicated dose by a single intraperitoneal injection. If all mice in the relevant group tolerated the indicated dose, the dose was escalated as indicated. [Figure 1B]5 shows that 5-formyl-2'-deoxycytidine and 5-hydroxymethylcytidine are well tolerated in mice and reduce human glioblastoma multiforme tumors in a mouse xenograft model. Mice were injected intraperitoneally with the indicated doses and compounds every three days for a total of five doses, after which mice were weighed at the indicated time points. [Figure 1C] ~ [Figure 1H] Figure 1 shows that 5-formyl-2'-deoxycytidine and 5-hydroxymethylcytidine are well tolerated in mice and reduce human glioblastoma multiforme tumors in a mouse xenograft model. U87-MG cells were implanted into the flanks of 32 immunocompromised mice. After tumors reached 129-131 mm3, the mice were divided into four groups of eight mice each. The negative control group was treated with vehicle, the positive control group was treated with 40 mg / kg temozolomide once daily for 5 days, and the treatment groups were 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 volumes were measured every 3 days (Figure 1C), and mouse weights were measured every 3 days (Figure 1D). At the completion of the study, percent tumor growth inhibition (TGI (%)) was calculated (Figure 1E), and tumors were excised, photographed (Figure 1F), measured (Figure 1G), sectioned, and stained with hematoxylin and eosin (Figure 1H). [Figure 2] Figure 2 shows that 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine kill glioblastoma multiforme by a mechanism independent of current nucleotide analogs. Figure 2A: Flow cytometry of 5-formyl-2'-deoxycytidine and 5'-hydroxymethyl-2'-deoxycytidine-treated cells stained with Annexin V and 7AAD. Figure 2B: Survival curves of HeLa cells treated with titrations of 5-formylcytosine, 5-formylcytidine, or 5-formyl-2'-deoxycytidine. Figure 2C: Survival curves of U87-MG cells treated with increasing doses of 5-hydroxymethylcytosine, 5-hydroxymethylcytidine, or 5-hydroxymethyl-2'-deoxycytidine. [Figure 3] Quantification of mutations in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene induced by 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine in mammalian cells. 5-Formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine have been shown to be non-mutagenic. [Figure 4] FIG. 1 shows the levels of cytotoxicity (% survival) of 5-formyl-2′-deoxycytidine, 5-formylcytidine and 5-chloro-2′-deoxycytidine on HeLa cells after 3 days of treatment. [Figure 5] FIG. 1 shows the levels of cytotoxicity (% survival) of U87-MG cells 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. [Figure 6] FIG. 1 shows that the cytotoxic effects (% survival) of 5-formyl-2′-deoxycytidine and 5-hydroxymethyl-2′-deoxycytidine are not rescued by the addition of thymidine in U87-MG cells, indicating that 5-formyl-2′-deoxycytidine and 5-hydroxymethyl-2′-deoxycytidine do not act by inhibiting thymidine synthase. [Figure 7] FIG. 1 shows the cytotoxic effect (% survival) of 5-formyl-2′-deoxycytidine in combination with temozolomide in U87-MG cells. [Figure 8] FIG. 1 shows the cytotoxic effect (% survival) of 5-hydroxymethyl-2′-deoxycytidine in combination with temozolomide in U87-MG cells. [Figure 9]Figure 1 shows the cytotoxic effect of 5-methoxymethyl-2'-deoxyuridine and 5-acetoxymethyl-2'-deoxyuridine on U87-MG cells. Treatment for 72 hours. Survival was quantified using the MTT assay. [Figure 10] Figure 10 shows the % survival of various cells after treatment with d5fCTP or d5hmCTP. Figure 10A: % survival of HeLa cells after treatment with 5-formyl-2'-deoxycytidine-5'-triphosphate (d5fCTP) for 72 hours. Survival was quantified using an MTT assay. Figure 10B: % survival of U87-MG cells (glioma, grade IV) after treatment with either 5-formyl-2'-deoxycytidine-5'-triphosphate or 5-hydroxymethyl-2'-deoxycytidine-5'-triphosphate for 72 hours. Survival was quantified using an MTT assay. [Figure 11] Figure 1 shows CDA expression levels in various cell lines. 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]

[0023] Figure 1 shows 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]

[0023] Figure 1 shows CDA expression levels in various human brain tumors. 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]

[0023] Figure 1 shows CDA expression levels in various human brain tumors. 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]

[0023] Figure 1 shows 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]

[0023] Figure 1 shows 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] FIG. 1 shows the results of a PAMPA assay demonstrating that 2d5hmC and 2d5fC can cross the blood-brain barrier. DETAILED DESCRIPTION OF THE INVENTION

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

[0096] [ka]

[0097] wherein X, R1 and R2 are as defined above. or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof.

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

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

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

[0101] More preferably, X is -(CH2) n -X', where n is 0 to 2, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is 0 to 2, and X' is -OH. Preferably, X is -(CH2) n -X', where n is 0 to 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), X is preferably —CHO or —CHOH, most preferably —CHOH. In formula (IIb), X is preferably —CHOCH or —CHOC(═O)CH.

[0105] In all of these preferred embodiments, X is preferably not -OH. Thus, when X' is -OH, n is preferably not 0. In this case, n can be 1 to 6, preferably 1 to 4, more preferably 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 1 to 3, preferably 3; and R2 is -OH.

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

[0108] [ka]

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

[0110] 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 formulae (IIIa), (IIIb), (IIIc) and (IIId), especially in formulae (IIIa) and (IIIc), preferably X is -(CH2) n -X', where n is 0 to 6, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is 0 to 6, and X' is -OH. Preferably, X is -(CH2) n -X', where n is 0 to 6, and X' is -CHO.

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

[0114] More preferably, X is -(CH2) n -X', where n is 0 to 2, and X' is -OH or -CHO. Preferably, X is -(CH2) n -X', where n is 0 to 2, and X' is -OH. Preferably, X is -(CH2) n -X', where n is 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 formula (IIIa) and formula (IIIc), X is preferably -CHO or -CHOH, most preferably -CHOH. In formula (IIIb) and formula (IIId), X is preferably -CHOCH or -CHOC(=O)CH.

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

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

[0120] [ka]

[0121] or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof.

[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, the compound used in the present invention is preferably 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 a stereoisomer, solvate, tautomer, or pharmaceutically acceptable salt thereof. More preferably, the compound is a) 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine, or a stereoisomer, solvate, tautomer, or pharmaceutically acceptable salt thereof; or b) 5-methoxymethyl-2'-deoxyuridine or 5-acetoxymethyl-2'-deoxyuridine, or a stereoisomer, solvate, tautomer, or pharmaceutically acceptable salt thereof.

[0124] Alternatively, the compound for use in the present invention is 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 a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof.

[0125] Most preferably, the compound is 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof, most preferably 5-hydroxymethyl-2'-deoxycytidine or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof.

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

[0127] Thus, as used herein, "treatment" can refer to reducing, alleviating, ameliorating, or eliminating the cancer being treated or one or more symptoms thereof compared to the cancer or symptoms before treatment. Treatment can include reducing or eliminating cancer cells, for example, in a tumor, for example, in a solid tumor. Treatment is treatment of a subject, i.e., a subject in need thereof. Thus, treatment can include reducing tumor size, or preventing tumor growth or further tumor growth, i.e., stabilizing tumor size.

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

[0129] Preferably, the compounds of the present invention have a direct effect on cancer / tumor cells. As used herein, "direct effect" means that the compounds of the present invention directly interact with cancer / tumor cells to exert their anti-cancer / anti-tumor effects. In other words, preferably, the compounds of the present invention, i.e., compounds of formula (I), are cytotoxic to cancer / tumor cells. Preferably, the compounds of the present invention are administered to a subject to exert a direct effect on cancer / tumor cells.

[0130] Preferably, the method of the present invention does not include the administration of the compound of the present invention to deplete the cell population administered as part of cell-based therapy.Cell-based therapy is well known as the therapy that cell population is administered to subject to bring about specific therapeutic effect.Well-known cell-based therapy includes T cell therapy, for example, CAR T cell therapy.

[0131] Preferably, the methods of the present invention do not include administration of a compound of the present invention after administration of a cell population administered as part of a cell-based therapy.

[0132] Preferably, the methods of the present invention do not involve CAR T cell therapy. Preferably, the methods of the present invention do not involve T cell therapy. Preferably, the methods of the present invention do not involve cell-based therapy.

[0133] Preferably, the methods of the invention comprise administering a compound of the invention to a subject who has not undergone CAR T cell therapy, preferably T cell therapy, preferably cell-based therapy, In other words, preferably, the subject has not received and is not scheduled to receive CAR-T cell therapy, preferably T cell therapy, preferably cell-based therapy, as part of their treatment.

[0134] As referred to herein, a 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. Thus, preferably, the cancer referred to herein is a human cancer, and the tumor referred to herein is preferably present in a human subject.

[0135] In some embodiments, treatment according to the present invention may be used in subjects at risk of cancer relapse, recurrence, or metastasis. Thus, in an alternative view, 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 or at risk of developing cancer.

[0137] Alternatively or additionally, the present invention encompasses assessing or monitoring the effect of administration of compounds of formula (I) and / or other anti-cancer agents on a subject, or more particularly on cancer (tumor), or on the development or progression of cancer (tumor). Procedures and means for assessing and / or monitoring anti-cancer effects, for example, 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 any cancer. Cancer is broadly defined herein to include any neoplastic condition, particularly including malignant or premalignant conditions. Cancer can cause, result in, or arise from solid tumors, but is not limited thereto, and also includes cancers of the hematopoietic system. Benign tumors and malignant tumors are also included in the term cancer as used herein, i.e., 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; appendix 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's lymphoma; Carcinoid tumors; Cardiac (heart) tumors; Cervical cancer (cervical adenocarcinoma); Chordoma; Acute promyelocytic leukemia; Chronic lymphocytic leukemia (CLL); Chronic myelogenous 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 carcinoma; 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; Cardiac cancer; Hepatocellular (liver) cancer; Histiocytosis; Langerhans cell; Hodgkin's lymphoma; Hypopharyngeal cancer; Intraocular melanoma; Islet cell tumors; Pancreatic neuroendocrine tumors; Kaposi's sarcoma; Kidney cancer (including renal cell and Wilms' tumor); Langerhans cell histiocytosis; Laryngeal cancer; Leukemia (including acute lymphoblastic (ALL); acute myeloid (AML); chronic lymphocytic (CLL); chronic myeloid (CML)); Lip and oral cavity cancer; Liver cancer (primary); Lobular carcinoma in situ (LCIS); Lung cancer; Lymphoma; Macroglobulinemia; Waldenstrom's disease; Melanoma (malignant melanoma); Merkel cell carcinoma; Mesothelioma; Metastatic cervical squamous cell carcinoma of unknown primary; Midline duct carcinoma involving the NUT gene Tract Carcinoma Involving NUT Gene); Mouth Cancer; Multiple Endocrine Neoplasia Syndrome; Children; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndrome; Myelodysplastic / Myeloproliferative Neoplasm; Multiple Myeloma; Myeloproliferative Disorders; Nasal Cavity and Sinus Cancer; Nasopharyngeal Carcinoma; Neuroblastoma; Non-Hodgkin's Lymphoma; Non-Small Cell Lung Cancer; Mouth Cancer; Oral Cavity Cancer; Oropharyngeal Cancer; Osteosarcoma; Ovarian Cancer (Ovarian Adenocarcinoma); Pancreatic Cancer;Pancreatic neuroendocrine tumors (islet cell tumors); papillomatosis; paraganglioma; paranasal sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; epithelial adenocarcinoma; plasma cell neoplasm / 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; squamous cell carcinoma of the cervix of unknown primary; metastatic; gastric 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; Waldenstrom's macroglobulinemia; and Wilms' tumor.

[0141] The cells of the human embryo are arranged 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, the cancer / tumor is preferably a cancer / tumor of tissue derived from the ectoderm, paraxial mesoderm or lateral plate mesoderm, preferably from the ectoderm.

[0143] Preferably, the cancer is a cancer of the central nervous system, preferably brain cancer. For the avoidance of doubt, brain cancer is considered to be a cancer of the central nervous system in the art and herein. The central nervous system includes the brain and spinal cord. Therefore, preferably, the tumor is a tumor of the central nervous system, preferably a brain tumor. Preferably, the CNS cancer / tumor is selected from the group consisting of CNS lymphoma, rhabdoid tumor, embryonal tumor, germ cell tumor, and chordoma, or is a brain cancer / tumor. Preferably, the brain cancer / tumor is selected from the group consisting of glioma, acoustic neuroma, CNS lymphoma, craniopharyngioma, medulloblastoma, meningioma, metastatic brain tumor, pituitary tumor, primitive neuroectodermal tumor (PNET), schwannoma, pineal tumor, trilateral retinoblastoma, and rhabdoid tumor.

[0144] Most preferably, the cancer / tumor is a brain cancer / tumor, more preferably a glioma. The glioma can be any type of glioma, for example, an astrocytoma, an ependymoma, a subependymoma, an oligodendroglioma, a brainstem glioma, an optic glioma, or a mixed glioma.

[0145] Preferably, the glioma is an astrocytoma, which may be grade I astrocytoma (preferably pilocytic astrocytoma or subependymal giant cell astrocytoma), grade II (preferably low-grade astrocytoma, pleomorphic xanthoastrocytoma, or mixed oligoastrocytoma), grade III (anaplastic astrocytoma), or most preferably 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, mitoses, endothelial proliferation, and necrosis, which reflect the aggressiveness 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 cerebella tentorium, the membrane that separates the cerebrum from the cerebellum. Supratentorial gliomas are found above the cerebella tentorium in the cerebrum, while infratentorial gliomas are found below the cerebella tentorium in the cerebellum. The gliomas treated according to the present invention can be supratentorial gliomas or infratentorial gliomas.

[0148] Therefore, in the context of the present invention, the cancer / tumor is particularly preferably a glioma, most preferably a grade IV glioma, i.e., a glioblastoma multiforme. A glioblastoma multiforme is a malignant astrocytoma and is the most common primary brain tumor among adults. A glioblastoma multiforme is also known as a grade IV glioma, 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 (as defined above, preferably glioma, more preferably glioblastoma), skin cancer and breast cancer, more preferably brain cancer (as defined above, preferably glioma, more preferably glioblastoma) and skin cancer.

[0153] In these embodiments, preferred compounds of the invention are those in which X contains an aldehyde functionality, preferably where X is -(CH) n -X', where X' is -CHO and n is 0 to 6, more preferably 0 to 4, more preferably 0 to 2, more preferably 0 or 1, and most preferably where X is -CHO and 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 and limited cytotoxicity against non-cancerous cells.

[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, preferred compounds of the invention are those in which X contains an alcohol functional group, preferably wherein X is -(CH2) n -X', where X' is -OH and n is 0 to 6, more preferably 0 to 4, more preferably 0 to 2, more preferably 0 or 1, and most preferably where X is -CHOH and Z is -NH. As demonstrated in the examples of the present invention, such compounds of the present invention have advantageously specific cytotoxicity against these preferred cancer types with limited cytotoxicity against off-target cancerous and non-cancerous cells.

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

[0156] However, preferably the cancer is not lung cancer or breast cancer. Preferably the cancer is not pancreatic cancer, stomach cancer, testicular cancer or vaginal cancer. Preferably the cancer is not kidney cancer or 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 also not chronic myeloid leukemia, and in this case, preferred compounds of the present invention (i.e., compounds of formula (I), (IIa), (IIb), (IIIa), (IIIb), (IIIc), (IIId), (IVa), (IVb), (IVc), (IVd), (IVe) and (IVf)) are compounds wherein X contains an aldehyde functional group, preferably wherein X is -(CH) n -X', where X' is -CHO and n is 0 to 6, more preferably 0 to 4, more preferably 0 to 2, more preferably 0 or 1, most preferably where X is -CHO and preferably Z is -NH2.

[0160] Further preferably, the cancer is i) is not breast cancer, preferably is not pancreatic, gastric, testicular or vaginal cancer, more preferably is not intestinal cancer; and / or ii) Not having liver cancer, breast cancer, ovarian cancer or cervical cancer.

[0161] Preferably, the cancer is not a cancer 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, gastric cancer, testicular cancer, vaginal cancer and intestinal cancer. Preferably, the cancer is not any of these cancers.

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

[0163] Breast cancers that are preferably not treated according to the present invention are preferably: i) adenocarcinoma, and / or ii) are triple negative, i.e., do not express estrogen receptor (ER-), progesterone receptor (PR-) or HER2 (HER2-), and / or iii) expresses a mutant variant of p53, and / or iv) homozygous for p53 Only breast cancer.

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

[0165] Thus, in a preferred embodiment, the cancer / tumor is one in which CDA is expressed, preferably one in which CDA is expressed: i) is overexpressed; ii) not overexpressed; iii) are underexpressed; iv) not underexpressed; v) is overexpressed or underexpressed, or vi) not over- or under-expressed It is something.

[0166] Each of the above cancer / tumor types i) to vi) represents a preferred embodiment of the present invention. In particularly preferred embodiments, 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, i.e., increased / higher or decreased / lower levels, respectively.

[0168] Overexpression (or increased or high level) or underexpression (or decreased or lower level) can be as determined in comparison to any suitable control (e.g., a control level or control sample or biopsy). For example, a control level can be the level in a sample (e.g., a blood or serum sample or a 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 one of skill in the art. An appropriate control "value" can also be easily determined without running a control "sample" with every test, for example, by reference to a range for healthy subjects.

[0169] Preferably, the terms overexpressed and underexpressed mean that the level of an RNA transcript from the gene in question in a cancerous cell is higher (overexpressed) or lower (underexpressed) than in a non-cancerous cell from the same tissue as the cancerous cell, when assessed using the same methods and conditions in both cases. Preferred methods and conditions for assessing the level of gene expression, e.g., CDA expression, are as disclosed elsewhere herein.

[0170] Preferably, the 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 by chance alone. 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). The p-value is a measure of the probability that a difference between groups occurred by chance during an experiment. For example, a p-value of 0.01 means that there is a 1 in 100 chance that the result occurred by chance. The lower the p-value, the more likely the difference between groups is caused by the treatment. Preferably, the probability value is less than 0.05 or less than 0.01.

[0171] In some embodiments, increased levels (i.e., overexpression) can be at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, or at least a 50% increase (e.g., compared to a control level). In some embodiments, decreased levels (i.e., underexpression) can be at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, or at least a 50% decrease (e.g., compared to a control level).

[0172] Preferably, the level of gene expression in a cell of interest is determined by referring to a database containing such information. Information sources such as The Cancer Genome Atlas (TCGA, https: / / cancergenome.nih.gov / ), EMBL-EBI Expression Atlas (https: / / www.ebi.ac.uk / gxa / home), Human Protein Atlas (https: / / www.proteinatlas.org / ), GENEVESTIGATOR® (https: / / genevestigator.com / gv / ), 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 cancers, 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 derived 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 over- or underexpression of a gene of interest can be easily identified. It is within the ability of one skilled in the art to utilize such sources for this purpose.

[0173] Thus, preferably, the cancer / tumor is as defined anywhere herein, and wherein CDA expression within said cancer / tumor is determined by reference 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, the cancer / tumor does not express CDA to a level greater than the expression level in a non-cancerous cell derived from the same tissue as the cancer / tumor, wherein said overexpression is determined by reference 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 when determined using the same method under the same conditions, wherein said reference cancer cell line is MDA-MB-231.

[0176] MDA-MB-231 is a well-characterized cell line that is widely available commercially. The MDA-MB-231 cell line is an epithelial human breast cancer cell line established from the pleural effusion of a 51-year-old Caucasian woman with metastatic breast adenocarcinoma and is one of the most commonly used breast cancer cell lines in medical laboratories. It is available, for example, from the European Cell Culture Collection (ECACC) under catalog number 92020424. As shown in Table 4A, the expression level of CDA in the MDA-MB-231 cell line is 153 TPM.

[0177] MDA-MB-231 is a triple-negative breast cancer (TNBC) cell line that is highly aggressive, invasive, and poorly differentiated due to the lack of estrogen receptor (ER) and progesterone receptor (PR) expression and HER2 (human epidermal growth factor receptor 2) amplification. The cell line is recognized as belonging to the claudin-low subtype because it exhibits features associated with breast cancer stem cells (CSCs), such as downregulation of claudin-3 and claudin-4, low expression of the Ki-67 proliferation marker, enrichment for markers associated with epithelial-mesenchymal transition, and a CD44+CD24- / low phenotype. In three-dimensional culture, the cell line exhibits an endothelial-like morphology and is distinguished by its invasive phenotype, often with stellate processes bridging multiple cell colonies. Standard conditions for culturing this cell line are well known. Preferred culture conditions are growth at 37°C in Leibovitz's L-15 medium supplemented with 2 mM glutamine and 15% fetal bovine serum (FBS). This medium supports cell growth in an environment without CO2 equilibration. MDA-MB-231 cells are preferably grown at 1-3 x 10 4 cells / cm 2 They are seeded at a density between 100 and 1500 and subcultured when they are 70-80% confluent.

[0178] Preferably, the cancer / tumor has a CDA expression level that is not more than 80%, preferably not more than 70%, preferably not more than 60%, preferably not more than 50%, preferably not more than 40%, preferably not more than 30%, preferably not more than 25% of the CDA expression level in a reference cancer cell line when determined using the same method under the same conditions, wherein said reference cancer cell line is MDA-MB-231.

[0179] Preferably, the cancer / tumor has a CDA expression level that is at least 2-fold, preferably at least 2.5-fold, preferably at least 3-fold, preferably at least 3.5-fold, preferably at least 4-fold, preferably at least 4.5-fold lower than the CDA expression level in a reference cancer cell line when determined using the same method under the same conditions, wherein said reference cancer cell line is MDA-MB-231. "At least X-fold lower" in this context means that the maximum CDA expression level in the cancer / tumor is exactly X-fold lower than the expression level in the reference cancer cell line.

[0180] The methods and conditions used to determine the CDA expression level in a cancer / tumor and in a reference cancer cell line may be any suitable methods and conditions, provided that the same methods and conditions used to determine the CDA expression level in a reference cancer cell line are used to determine the CDA expression level in a cancer / tumor. The reference cancer cell line is therefore a control. Those skilled in the art can easily determine the expression level of a gene of interest, such as CDA, in cancerous and non-cancerous cells as well. Such methods are part of common general knowledge in the art, and any suitable method can 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 the protein, such as an antibody, and then determining the protein expression level based on the level of the detected reagent, preferably after normalization. Examples of methods that generally involve the use of antibodies include, but are not limited to, Western blot, immunoblot, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT), radioimmunoassay (RIA), immunohistochemistry, and immunoprecipitation. Other methods suitable for measuring protein expression levels may be used, which do not necessarily involve the use of antibodies, including, but not limited to, fluorescence activated cell sorting (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 level, for example CDA expression level, can be measured at RNA level.Methods for measuring RNA level are well known in the art, and any suitable method can be used in the context of the present invention.For example, microarray, RT-PCR, quantitative real-time PCR, RNA sequencing, Northern blot, primer extension, RNase protection and RNA expression profiling can be used.Preferably, the method used is RNA-seq or microarray.RNA-seq is a well-known method, which uses next-generation sequencing (NGS) to determine the presence and quantity of RNA in biological samples at a given moment.

[0183] Preferably, the method used to determine the CDA expression level in a cancer / tumor of interest compared to the CDA expression level in a reference cancer cell line (wherein the reference cancer cell line is MDA-MB-231) is the "microarray method A" referred to below.

[0184] Microarray method A includes the following steps. 1) extracting RNA from cancer / tumor cells of interest, said extraction preferably being performed using the Norgen Total RNA Purification Kit (Norgen Biotek Catalog No. 17200); 2) preparing poly(A)+ RNA from the extracted RNA, preferably using the MEGA Pure kit according to the manufacturer's instructions (Ambion); 3) preparing complementary DNA (cDNA) from poly(A)+RNA by treatment with reverse transcriptase, i.e., performing reverse transcription; 4) fragmenting the cDNA using TdT (terminal deoxynucleotidyl transferase); 5) Biotinylating the cDNA fragments using the GeneChipWT End Labeling Kit (Affymetrix); 6) repeating steps 1 to 5 using a reference cell line, wherein said reference cell line is MDA-MB-231; 7) hybridizing 5.5 μg of the biotinylated cDNA fragments obtained in step 5 to a first DNA microarray at 45° C. for 16 hours and 5.5 μg of the biotinylated cDNA fragments obtained in step 6 to a DNA microarray at 45° C. for 16 hours, preferably wherein the microarray is an Affymetrix GeneChip Human Gene 2.0ST array (Applied Biosystems); 8) washing and staining the hybridized microarray, preferably in an Affymetrix GeneChip Fluidics Station 450 (Applied Biosystems); 9) scanning the stained microarray using an Affymetrix GeneChip Scanner 3000 7G utilizing Affymetrix GeneChip Command Console® software to generate raw data signal values ​​in the form of CEL files; 10) normalizing the CEL files to generate gene-level expression values ​​using the Robust Multiarray Average (RMA) implementation in the Affymetrix software package (version 1.36.1), preferably as described in R.A. Irizarry et al., Exploration, normalization, and summaries of high-density oligonucleotide array probe level data. Biostatistics 4, 249-264 (2003); 11) Assessing the quality of the arrays by calculating the relative logarithmic expression (RLE) and normalized unscaled standard error (NUSE) using the affyPLM package (version 1.34.0). The output from the affyPLM package is a boxplot of both the RLE and NUSE distributions. Arrays for which the RLE boxplot is not centered around 0 and the NUSE boxplot is not centered around 1 are flagged as low quality and excluded from further analysis. If an array is excluded, the previous steps of the method are repeated. 12) Performing principal component analysis (PCA) using the Prcomp R function with expression values ​​normalized 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 high dimensionality of gene expression datasets while retaining most of the variation in the data. Thus, the performance of PCA achieves a lower dimensional representation of the data for downstream analysis and visualization. 13) assessing differential expression of CDA in the cancer / tumor cells of interest and in the reference cell line MDA-MB-231 using a moderated (empirical Bayes) t-test as 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 for statistical computing (version 2.15.1).

[0185] Preferably, the CDA expression level in the cancer / tumor of interest compared to the CDA expression level in a reference cancer cell line, wherein said reference cancer cell line is MDA-MB-231, is determined by reference 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] The expression level of a gene is typically presented in terms of the relative amount of RNA transcripts for that gene compared to the total amount of RNA transcripts in the relevant cell / tissue. The expression level is typically presented in units of transcripts per million (TPM), i.e., how many [x] of the gene of interest are produced per million RNA molecules in the cell / tissue of interest. Again, the level of RNA transcripts 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 sources such as 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 / ), among others. 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 that overexpress CDA contain CDA RNA transcripts at levels greater than 140 TPM. Conversely, cancerous cells / tumors that do not overexpress CDA preferably contain CDA RNA transcripts at levels of 140 TPM or less (less than or equal to 140 TPM). Alternatively, preferred cancerous cells / tumors of the present invention have CDA expression levels of 140 TPM or less. Thus, particularly preferred cancers / tumors to be treated according to the present invention are those that express CDA to 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 cancer / tumor of interest in units of TPM is obtained by consulting 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 cancer / tumor of interest, in units of TPM, is determined by quantitative real-time PCR or RNA sequencing (RNA-seq) and quantification using cells derived from said cancer / tumor. Preferably, the method used is RNA sequencing. Preferably, the method used is the "RNA-seq method A" referred to below.

[0190] The expression level of a gene in TPM units can be determined using any known method, but is preferably determined using RNA-seq. RNA-seq methods are well known in the art and widely available commercially. Any suitable RNA-seq method can be used to determine the CDA expression level in TPM units in a cancer of interest. The following method, referred to as RNA-seq method A, is preferred, and includes the following steps: 1. Extracting total RNA from the cancer / tumor cells of interest, which can be performed using a standard RNA extraction kit, preferably the QIAGEN AllPrep DNA / RNA Mini Kit (Hilden, Germany), according to the manufacturer's instructions. 2. Optionally quantifying and assessing the extracted RNA for purity, preferably by 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 / protein), and provides an "RNA Integrity Number" (RIN) that quantifies fragmentation of the RNA sample. Preferably, samples are only used further in the process if they have an RIN of at least 7, preferably at least 8. 3. 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 500-1000 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. Treating the extracted RNA to deplete bacterial and eukaryotic ribosomal RNA, preferably using the Ribo-Zero rRNA removal kit (Epicentre, Madison, WI, USA) according to the manufacturer's instructions. 3b. Treating the remaining RNA with reverse transcriptase and random hexamers to generate single-stranded cDNA fragments of 100-150 bases in length, or preferably 200-300 bases in length. 3c. treating the single-stranded cDNA fragment with DNA polymerase I and RNase H to generate double-stranded complementary DNA fragments (cDNA); and 3d. Ligate RNA-seq adapters to the ends of the cDNA fragments to generate a library of adapter-cDNA sequences that can be analyzed by RNA-seq. RNA-seq adapters are well known in the art, and any suitable adapter can be used. The adapters contain functional elements that enable sequencing, such as an amplification element and a primary sequencing site. Preferably, the adapters are Illumina index adapters. An adapter can be ligated to one end of each cDNA fragment to generate a single-end library (which will result in one "read" per fragment upon sequencing), in which case the cDNA fragments in step 3b are 100-150 bases long. However, preferably, adapters are ligated to both ends of each cDNA fragment to generate a paired-end library (which will result in two "reads" per fragment upon sequencing, so-called "mate reads" or "paired reads"), in which case the cDNA fragments in step 3b are 200-300 bases long. 4. Optionally amplifying the adapter-cDNA sequence by PCR. This step can be performed if the amount of DNA is insufficient to perform 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 loaded into the flow cell of the sequencer in step 5). Methods for assessing the amount of DNA in a sample are routine, and any suitable method can be used. Preferably, the method used is fluorometry, preferably using a Qubit® fluorometer. 5. Sequencing the adapter-cDNA sequences, 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-end library is generated in step 3, or more preferably using a sequencing cycle of 200-300 base pairs if a paired-end library is generated in step 3. Preferably, 70-135 μl, more preferably 125 μl, of a 20 pM solution of DNA is loaded into the flow cell. The resulting raw data set will provide a unique sequence identifier for each fragment analyzed, its sequence (a so-called "read"), and an indication of the confidence in the sequencer's determination of the sequence at each base position within the read (a so-called Phred quality score). 6. Preparing a quality-filtered dataset of reads by removing the following from the dataset of determined sequences: i) bases in the read with a Phred quality score of less than 10; ii) a base in a read that is downstream (i.e., following the direction of sequencing) of a base in the same read that has a Phred quality score of less than 10; iii) reads containing any undetermined ("TBD") base ("N"); iv) reads that map to a contaminant reference genome, wherein the contaminant reference genome is an Escherichia coli (E. coli) genome; and v) If the RNA-seq library used is a paired-end library, the reads whose "mate reads" are discarded in one of steps 6(iii) to 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 human reference genome from the Ensembl database version 98, preferably Genome Human GRCh38 and the Ensembl Gene Reference Traits database (version Ensembl Genome 45, GENCODE 32). Suitable alignment tools for this step are well known and widely available, and any suitable alignment tool can be used. Preferably, the alignment tool is TopHat2 (version 2.1.1) [Trapnell et al., (2010) Nature Biotechnology 28, 511-515]. 8. Determine the number of read counts for CDA genes, and optionally any other genes of interest. Bioinformatics tools for this step are well known and widely available, and any suitable tool can be used. Preferably, the read count per gene is determined using the HTSeq package (htseq count). This step thereby provides raw read count data. 9. 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. Determining TPM values ​​involves normalizing the raw data for i) gene length and ii) sequencing depth, so providing count data in units of TPM allows for meaningful assessment of gene expression levels. i) Regarding gene length normalization in raw data, higher read counts may be observed for longer genes simply because the genes are longer and therefore more fragments align to that gene. Gene length normalization involves dividing the read counts for each gene by the gene length (in kilobases). ii) Sequencing depth is a sample-to-sample effect that alters the comparison of read counts between the same gene in different samples. To normalize this, the read counts per kilobase obtained in step 9i) are divided by a "per million scaling factor," which is itself obtained by dividing the total number of reads in the sample by 1 million.

[0191] Gene expression data obtained using RNA-seq can be presented in units of RPKM (reads per kilobase per million reads). i. Dividing the total number of reads in the sample by 1,000,000 to provide a "per million scaling factor"; ii. Dividing the read count per gene by a "per million" scaling factor that normalizes for sequencing depth, thereby providing units of reads per million (RPM); and iii. Divide the RPM value by the length of the gene in kilobases to normalize for gene length, thereby providing units of RPKM It is calculated by:

[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 every read corresponds to a single sequenced fragment. FPKM is applicable to paired-end RNA-seq, where two reads can correspond to a single fragment, or one read in a pair can correspond to a single fragment if one read does not map. The only difference between RPKM and FPKM is that FPKM takes into account that two reads can map to one fragment (and therefore does not count this fragment twice).

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

[0194] The only difference when calculating TPM compared to RPKM or FPKM is that when calculating TPM, normalization for gene length is performed first. However, the result is that when using units of TPM, the sum of all TPMs in each sample is the same 1 million. This allows for a more meaningful comparison of the proportion of reads that mapped to genes in each sample.

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

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

[0197] The gene expression level determined by microarray can be expressed in any scale, for example, linear scale.Data from microarray can be preferably transformed by logarithmic base 2 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 that is up-regulated by a factor of 2 has a log2-transformed value of 1.

[0198] Preferably, the cancer / tumor is one that has a log base 2 transformed CDA expression level of less than 11.75. Such cancer / tumor is described as not overexpressing CDA. Preferably, the cancer / tumor is one that has a log base 2 transformed CDA expression level of 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 cancer / tumor is described as not overexpressing 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 base 2 transformed expression level of CDA in the cancer / tumor of interest is obtained by reference 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 are aware that there may be variations in the expression level of some genes between tumors of the same cancer type.For example, some breast cancers may overexpress CDA, while others may not overexpress CDA.Therefore, preferably, the cancers / tumors described as preferred elsewhere in this specification are preferably cancers / tumors of the type in which CDA is expressed, and preferably, CDA is i) is overexpressed; ii) not overexpressed; iii) are underexpressed; iv) not underexpressed; v) is overexpressed or underexpressed, or vi) not over- or under-expressed It is something.

[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 elsewhere herein as not preferred are preferably those types of cancer / tumours only, preferably those in which CDA: i) is overexpressed; ii) not overexpressed; iii) are underexpressed; iv) not underexpressed; v) is overexpressed or underexpressed, or vi) not over- or under-expressed It is something.

[0204] Preferably, the cancer / tumor is one in which CDA is overexpressed, CDA overexpression being as defined above.

[0205] In a preferred embodiment, the cancer / tumor is resistant to gemcitabine and / or cytarabine treatment. Thus, a cancer / tumor described as preferred elsewhere herein 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 a brain cancer, preferably a glioma, more preferably glioblastoma multiforme.

[0206] The human protein O-6-methylguanine-DNA methyltransferase (MGMT) removes alkylating DNA lesions. MGMT expression renders cancer cells, such as glioblastoma cells, almost completely resistant to the cytotoxic effects of temozolomide, which exerts its cancer chemotherapy activity by severely mutating tumor cells so that they die. Temozolomide acts by alkylating DNA, thereby causing mutations.

[0207] As shown in the examples of the present invention, the compound of formula (I) is not mutagenic in HPRT assay.Therefore, the compound of formula (I) is of particular use in treating cancers in which MGMT is expressed, preferably overexpressed.Therefore, the cancers / tumors described as preferred elsewhere in this specification are preferably cancers / tumors 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 a first-line treatment for glioblastoma multiforme and is also used to treat 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 a preferred embodiment, the cancer / tumor is a temozolomide-resistant cancer / tumor.

[0209] Thus, cancers / tumors described as preferred elsewhere herein are preferably types of cancers / tumors that are resistant to temozolomide treatment. Preferably, the temozolomide-resistant cancer / tumor is a brain cancer, preferably a glioma, 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 a preferred embodiment, the cancer is a fluoropyrimidine-resistant cancer.Therefore, the 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, 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 the cancer / tumor does not respond positively to treatment with the relevant anti-cancer drug, i.e., the treatment with the anti-cancer drug does not reduce, alleviate, ameliorate, or eliminate the cancer or one or more symptoms thereof, or does not reduce or eliminate cancer cells within the tumor compared to the cancer, tumor, or symptoms before treatment. The cancer / tumor may be resistant at the beginning of treatment or may become resistant during treatment.

[0213] As mentioned above, in the treatment or prevention of cancer, the compound of formula (I) can be used alone or, optionally, in combination with further, i.e., one or more further 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 may be used, regardless of their chemical nature or mode of action.

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

[0215] Representative examples of anticancer drugs within the "chemotherapy" class include fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin, and oxaliplatin, mitomycin, dacarbazine, procarbazine, etoposide, teniposide, campatecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, and mitoxanthin. These include, but are not limited to, thiazolinone, L-asparaginase, epimbicin, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustard, BCNU, nitrosoureas such as carmustine and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, or topotecan.

[0216] Anti-cancer agents may include kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, herbimycin A, genistein, erbstatin, and lavendustin 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 agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, antifolates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, epipodophyllotoxins, hormone therapy, retinoids, photosensitizers or 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] The additional anti-cancer agent may be selected from, but is not limited to, a cytokine, a chemokine, a growth factor, a growth inhibitor, a hormone, a soluble receptor, a decoy receptor, a monoclonal or polyclonal antibody, a monospecific, bispecific or multispecific antibody, a monobody, a polybody.

[0219] Alternative anti-cancer agents may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.

[0220] In one exemplary embodiment, the drug is a small molecule, more particularly a small molecule chemotherapeutic agent. Small molecule agents may be defined as having a molecular weight of less than 2000 Da, more particularly less than 1800, 1500, 1200, 1000, 900, 800, or 700 Da, and typically less than 1000 Da. For example, small molecule agents may have a size in the range of 100 to 1000 Da, e.g., 100 to 800 Da or 300 to 700 Da.

[0221] In an alternative embodiment, the additional anti-cancer agent is an immunotherapeutic agent. Inducing an immune response to treat cancer is known as cancer "immunotherapy." Immunotherapy can involve, for example, cell-based therapy, antibody therapy, or cytokine therapy. All three approaches take advantage of the fact that cancer cells often have distinct cell surface markers, or cancer antigens, that can be detected by the immune system. These antigens are most commonly proteins, but can also include other molecules, such as carbohydrates. Another example of immunotherapy is through checkpoint inhibition, whereby checkpoint proteins are inhibited, as discussed further below.

[0222] Thus, immunotherapy is used to stimulate the immune system to attack cancer cells, and as discussed further below, various molecules can be targeted by immunotherapy-based approaches. For example, targets for immunotherapeutic intervention in cancer include "CD" ("cluster of differentiation") 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 activation 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 targeted for immunotherapeutic intervention in cancer.

[0223] The immunotherapeutic agent may be a peptide, polypeptide, or protein. The immunotherapeutic agent may be selected from an antibody, a cytokine, and a checkpoint inhibitor. As noted above, therapeutic anti-cancer antibodies may have a range of targets, including checkpoint proteins. Thus, the antibody may be a checkpoint inhibitor.

[0224] Thus, in a first example, the immunotherapeutic agent is an antibody. The antibody may be selected from monoclonal or polyclonal antibodies, monospecific, bispecific or multispecific antibodies, monobodies and polybodies, or indeed from any of the many antibody-like or antibody derivative molecules currently known in the art. The term "antibody" is therefore used broadly herein to include 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] Thus, the antibody may be: (a) any of the various classes or subclasses of immunoglobulins, such as IgG, IgA, IgM, IgD or IgE, derived from any animal, such as any of the conventionally used animals, such as sheep, rabbit, goat or mouse, or from egg yolk; (b) a monoclonal or polyclonal antibody; (c) Intact antibodies or fragments of monoclonal or polyclonal antibodies, which contain fragments lacking the binding region of the antibody, e.g., the Fc portion (e.g., Fab, Fab', F(ab')2, Fv), so-called "half molecule" fragments obtained by reductive cleavage of the disulfide bonds connecting the heavy chain components in the intact antibody. Fv can be defined as a fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two 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 altered antibody-like structures.

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

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

[0228] In many cases, monoclonal antibodies are unmodified antibodies, and most currently used therapeutic antibodies fall into this category. However, in one embodiment of the present invention, an antibody, e.g., a monoclonal antibody, is conjugated or fused to an additional molecule, e.g., a toxic or radioactive substance. Thus, a conjugated or fused antibody is attached 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 and directs the toxin or radiation to the tumor.

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

[0230] In a second example, the immunotherapeutic agent is a cytokine. Cytokines include immune modifiers such as interleukins (IL) and interferons (IFN), as well as stimulatory factors, tumor necrosis factors (TNF), and other regulatory molecules. Cytokines have been classified as lymphokines, interleukins, and chemokines based on their function, secreting cells, or targets of action. Each cytokine has a corresponding cell surface receptor that initiates a cascade of intracellular signaling that alters cell 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 encompassed for use in accordance with the present invention. Thus, in one embodiment, the immunotherapeutic 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 (IL) are 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 that are normally involved in antiviral responses, but also have applications in the treatment of cancer. There are three classes of interferons (IFNs): type I (IFNα and IFNβ), type 2 (IFNγ), and the relatively newly discovered type III (IFNλ).

[0233] All known forms of the cytokines discussed above, including functionally equivalent variants, derivatives, and fragments thereof, may be used in the present invention. Thus, the term "cytokine," as used herein, includes amino acid sequence variants of known cytokine polypeptides, and fragments of cytokine polypeptides, or derivatives thereof, so long 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, Inc. (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 to the immune system which cells are healthy and which cells 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, the cell 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 speculated that checkpoint proteins contribute to the failure of some cancer immunotherapies.

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

[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 (chimeric 3G4). All of these checkpoint inhibitors may be used in the present invention.

[0237] An alternative option for immunotherapy relates to immune cell therapy, and the present invention can also be used in combination with such therapy, for example, adoptive cell transfer. Several T cell-based therapies have been developed to treat cancer, and these treatments, 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 periphery or tumor site. These cells are expanded ex vivo and reinfused back into the patient.

[0238] Two alternative therapies are available, which involve modifying the patient's own T cells with receptors capable of recognizing tumors. In one option, TcRs active against cancer antigens can be isolated and characterized, and the genes encoding the TcRs can be inserted into T cells and reinfused into the patient. While this therapy has been shown to shrink solid tumors in some patients, it is associated with a significant drawback: the TcRs used must match the patient's immune type. Therefore, as an alternative to the use of TcRs, therapies involving the expression of chimeric antigen receptors (CARs) on T cells have also been suggested. CARs are fusion proteins containing an antibody linked to the signaling domain of the TcR complex; once a suitable antibody is selected, they can be used to target T cells against tumors. 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 optionally be modified to express a CAR.

[0240] Thus, according to the present invention, the immunotherapeutic agent may be a cell, particularly an immune cell such as a lymphocyte, in particular a T cell or an NK cell as described above, for example, a T cell may be a TIL or may be modified to express a TcR or a CAR, or an NK may be modified to express a 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 that 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 its poly(A) tail, or inefficient translation of the mRNA into protein. miRNAs are similar to the siRNAs mentioned above, except that miRNAs are derived from regions of RNA transcripts that fold together to form short hairpins, whereas siRNAs are derived from longer regions of double-stranded RNA. Many miRNAs have been found to be linked to various types of cancer and are therefore sometimes referred to as "oncomia."

[0242] MicroRNA can be used in microRNA-based oncology therapeutics in the treatment of cancer.The rationale for developing miRNA therapeutics is based on the premise that aberrantly expressed miRNAs play a key role in the development of cancer, and correcting these miRNA defects by either antagonizing or restoring miRNA function, for example, by miRNA replacement therapy, can provide therapeutic benefits.

[0243] Any suitable miRNA can be used as an additional anti-cancer agent according to the present invention.MiRNA can be in free form, i.e., not bound to other molecules.Alternatively, miRNA can be conjugated or bound to other molecules, such as antibodies as discussed herein.

[0244] Most preferably, the additional anticancer drug is selected from the group consisting of temozolomide, 5-fluorouracil, gemcitabine, cytarabine, and gliadel®. Preferably, the additional anticancer drug is temozolomide. Preferably, the additional anticancer drug is 5-fluorouracil. Preferably, the additional anticancer drug is gemcitabine. Preferably, the additional anticancer drug is cytarabine. Preferably, the additional anticancer drug is gliadel®.

[0245] The compound of formula (I) and the additional anti-cancer agent may be used according to the present invention in the form of a composition, i.e., a pharmaceutical composition. The present invention provides a pharmaceutical composition comprising a compound of formula (I) and one or more pharmaceutically acceptable excipients, and optionally further comprising an additional anti-cancer agent.

[0246] A product (especially a pharmaceutical product) comprising a compound of formula (I) and a further (i.e. one or more further or second) anti-cancer agents may be a combined preparation for separate, sequential or simultaneous use in the treatment or prevention of cancer (i.e. tumors), or may be a product in which the compound of formula (I) is co-formulated with the further anti-cancer agent.

[0247] Thus, the compound of formula (I) and the additional anti-cancer agent may be formulated together in a single composition or in separate compositions for separate administration, which will depend on the nature of the additional anti-cancer agent and its selected or required mode of administration.

[0248] Compositions for use in the present invention can be formulated in any convenient manner, for example, using one or more pharmaceutically acceptable excipients, carriers, or additives, according to techniques and procedures known in the pharmaceutical arts. 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] "Pharmaceutically acceptable," as referred to herein, refers to a component that is compatible with the other components of the composition and physiologically acceptable to the recipient. The nature, dosage, and the like of the composition and carrier or additive materials can be selected in a routine manner according to the selected and desired route of administration, purpose of treatment, and the like.

[0250] Thus, "pharmaceutical" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid, or liquid filler, excipient, encapsulating material, or formulation aid of any type.

[0251] The pharmaceutical compositions contain pharmaceutically acceptable vehicles for formulation, which may in particular be isotonic sterile saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc. or mixtures of such salts) or dry, freeze-dried compositions that, on addition of sterile water or saline, as the case may be, allow the administration of the solution.

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

[0253] To prepare a pharmaceutical composition, an effective amount of a compound of formula (I) or an additional anticancer drug according to the present invention can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The composition can contain any known carrier, excipient, or additive. For example, suitable formulations for parenteral administration conveniently comprise sterile aqueous solutions and / or suspensions of the pharmaceutically active ingredient, preferably made isotonic with the recipient's blood using sodium chloride, glycerin, glucose, mannitol, sorbitol, or the like.

[0254] Additives that may be included in any pharmaceutical composition include, among others, preservatives (such as p-hydroxybenzoates), chelating agents (such as EDTA), stabilizers, tonicity adjusters, antimicrobial agents, flocculating / suspending agents, wetting agents, solvents and solvent systems, antioxidants, and buffers. It is within the ability of one skilled in the art to select and optimize such additives and their amounts when formulating a pharmaceutical composition for a particular desired purpose.

[0255] The composition is preferably in the form of an aqueous solution, which 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 will necessarily depend on the nature of the cancer to be treated, the severity of the disease, the age, weight and sex of the patient, etc., or alternatively the desired duration of treatment.

[0257] Treatment involves the administration of a compound of formula (I), optionally together with an additional anti-cancer agent.

[0258] The compounds of formula (I) for use according to the present invention may be administered to a subject via any suitable route. The same applies to compositions or formulations comprising compounds of formula (I).

[0259] The compounds of formula (I), and thus compositions and formulations containing them, may be presented in a form suitable for, for example, oral, nasal, parenteral, intravenous, topical, rectal or intrathecal administration. Preferably, the compounds are presented in a form suitable for systemic (e.g., intravenous) administration.

[0260] Any mode of administration common or standard in the art may be used, such as injection, infusion, topical administration, inhalation, transdermal administration, etc., to both internal and external surfaces of the body by any suitable method known in the pharmaceutical arts. Thus, modes of administration include oral, nasal, enteral, rectal, vaginal, transmucosal, topical or parenteral administration, or by inhalation. Administration may be directly to the tumor (intratumoral administration).

[0261] Oral or parenteral administration is preferred. Preferred parenteral administration means are intravenous, intramuscular, intraperitoneal, intracranial, and subcutaneous administration, as well as administration into the cerebrospinal fluid (intrathecal administration). More preferably, administration is intraperitoneal or intravenous, most preferably intravenous.

[0262] Preferably, administration is oral or intravenous.

[0263] Intravenous administration can be by intravenous injection or intravenous infusion, most preferably by intravenous infusion (eg, via an infusion pump).

[0264] The compound of formula (I) and the additional anti-cancer agent may be administered by the same or different routes.

[0265] As noted above, the compound of formula (I) and the additional anticancer drug can be administered simultaneously, separately, or sequentially. In a preferred embodiment, the compound of formula (I) and the additional anticancer drug are administered sequentially, for example, at different times, i.e., not together in the same composition. In an alternative embodiment, the compound of formula (I) and the additional anticancer drug are administered together at the same time, for example, in the same composition or in separate compositions. The timing of separate administration can be determined according to the specific compound of formula (I) or the specific additional anticancer drug, formulation, and / or administration mode used. Thus, the compound of formula (I) can be administered before or after the additional anticancer drug.

[0266] For example, the additional anticancer drug may be administered first, and the compound of formula (I) may be administered later at a suitable time interval to optimize the delivery time of the additional anticancer drug to the target site, or vice versa. Such a determination is entirely within the routine skill of a clinician. Thus, for example, the compound of formula (I) may be administered parenterally, more preferably intravenously, at least or at most 20, 30, 40, 50, 60, 70, or 90 minutes, or 2, 3, 4, 5, or 6 hours before or after the additional anticancer drug.

[0267] Doses and dosages may be determined in a routine manner and may depend on the nature of the molecule, the purpose of treatment, the age of the patient, the mode of administration, etc. Any of the therapeutic agents of the present invention, as described above, can be combined with pharmaceutically acceptable excipients to form a therapeutic composition. A dose refers to a specified amount of drug taken at one time, i.e., the terms "single dose" and dose are used interchangeably. A course of treatment may include multiple doses, i.e., multiple single doses, over a period of time.

[0268] In the methods and uses of the present invention, preferably an effective amount of the compound of formula (I) and, if present, any further anti-cancer agent is administered. In other words, the dose preferably comprises an effective amount of the compound of formula (I) and, if present, any further anti-cancer agent.

[0269] As shown in the examples, the compound of formula (I) is resistant to high doses compared to the dose required to kill tumors.This property is different from many chemotherapy compounds, and in fact, most chemotherapy compounds have substantial side effects at the dose required to kill cancer.The examples of the present invention indicate that the compound of formula (I) can be advantageously administered at a dose that far exceeds the dose required to kill tumors.

[0270] As shown in the Examples, mice tolerated single doses of 300 mg / kg and 2000 mg / kg of the compound of formula (I), but not a single dose of 8000 mg / kg. These data suggest that the maximum tolerated dose of the compound of formula (I) in mice is at least 2000 mg / kg but less than 8000 mg / kg. The conversion coefficient 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 tolerated dose of the compound of formula (I) in humans is at least 162 mg / kg but less than 648 mg / kg.

[0271] Thus, preferably, the compound of formula (I) is administered at a dose 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, more preferably 24.3 mg / kg or less. Preferably, the compound of formula (I) is administered at a dose 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, more preferably at least 100 mg / kg.

[0272] Preferably, the compound of formula (I) is administered at a dose 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, more preferably between 40 mg / kg and 81 mg / kg.

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

[0274] The dosage and dosage regimen may vary based on parameters such as the age, weight, condition and sex of the subject, the purpose of treatment, the disease being treated, the age and / or condition of the patient, the mode of administration, and the like.

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

[0276] Treatment may involve a single administration of a compound of formula (I), optionally together with an additional anti-cancer agent, or may involve repeated administration of a compound of formula (I), optionally together with an additional anti-cancer agent. The dosing regimens of the compound of formula (I) and the additional anti-cancer agent, if present, need not be the same. Alternatively, treatment may involve a single administration of a compound of formula (I) and repeated administration of the additional anti-cancer agent, or vice versa.

[0277] Preferably, the compound of formula (I) is administered, preferably at any one of the doses described above, 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 administrations, more preferably 3 to 8 administrations, more preferably 4 to 6 administrations, more preferably 5 administrations.

[0278] However, it will be within the ability of one skilled in the art to determine an appropriate dosing regimen and relevant doses therein based on the nature of the compound, the purpose of treatment, the disease being treated, the age and / or condition of the patient, the mode of administration, etc.

[0279] The present invention also provides a product or kit comprising a compound of formula (I) and an additional anti-cancer agent. The kit or product can be used in any of the uses or methods described herein, i.e., for use in treating or preventing cancer. In particular, the kit or product is for simultaneous, separate, or sequential use. Preferably, the compound of formula (I), and optionally the additional anti-cancer agent, are formulated for parenteral administration, preferably intravenous administration.

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

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

[0282] The compounds of the present invention (i.e., compounds of formulae (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 from available starting materials according to standard techniques using appropriate reagents and reaction conditions. In this respect, those skilled in the art may refer, inter alia, to "Comprehensive Organic Synthesis", BM Trost and I. Fleming, Pergamon Press, 1991 and "Protective Groups in Organic Synthesis", 3rd edition, TW Greene and PGM Wutz, Wiley-Interscience (1999).

[0283] Compounds of the 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 invention will now be further described with reference to the following non-limiting examples. [Example]

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

[0286] cell culture Primary glioma neural stem (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), 1x non-essential amino acids (Life Technologies, Catalog No. 11360-039), 1x ATP ... Cells were cultured on poly-D-lysine (Merck Millipore, Catalog No. A-003-E) and laminin (R&D Systems, Catalog No. 3446-005-01) coated plates in a medium containing 100 μg / ml of erythrocyte monolayer (Sigma, Catalog No. A8577-10ML), 4 μg / ml of heparin (Sigma, Catalog No. H3149-25KU), 100 U / ml of penicillin, 100 μg / ml of streptomycin, 20 ng / ml of human epidermal growth factor (hEGF) (R&D Systems, Catalog No. 236-EG-200), and 10 ng / ml of bFGF (Peprotech, Catalog No. 100-18B).

[0287] HCT116 were grown in McCoy's modified 5a medium (Life Technologies, catalog number 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 was grown in IMDM (Gibco, catalog no. 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, Cat. No. 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 between 70 and 90% confluence.

[0291] drugs The compounds used in the examples of the present invention were obtained as follows (CAT No. = catalogue number):

[0292] [Table 1]

[0293] Viability assay 4000 cells were seeded in 100 μl of the corresponding medium in a 96-well plate. The next day, drugs diluted in DMSO were added in triplicate at eight concentrations. Drugs were added in a four-fold dilution series starting from 100 μM. Cells were incubated with the drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, Cat. No. 30-1010K). Cell survival was normalized to that of cells treated with DMSO alone. Experiments were performed in triplicate, and data represent the mean ± SEM of nine wells.

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

[0295] Male ICR mice weighing 23±3 g were provided by BioLasco Taiwan (a licensee of Charles River Laboratories). Animals were acclimated for 3 days prior to use and confirmed to be in good health. All animals were maintained in a hygienic environment with controlled temperature (20-24°C), humidity (30%-70%), and a 12-h light / dark cycle. They were given free access to sterilized standard laboratory diet [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±3 g. Animals received an initial dose of 300 mg / kg. If 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 when all animals survived at the upper limit, when three dose levels had been tested, or when the upper or lower limit was reached. At each dose level, animals were observed for the first 30 minutes and again at 1, 24, 48, and 72 hours for the presence of acute toxicity symptoms (mortality, convulsions, tremors, muscle relaxation, sedation, etc.) and autonomic effects (diarrhea, salivation, lacrimation, vasodilation, piloerection, etc.). Body weights were recorded before dosing and at 72 hours. Animals were observed, and mortality was noted daily after compound administration. Gross necropsies were performed on all animals without tissue collection, and the next dose level was determined based on the study design table.

[0297] Multiple MTD 5-Hydroxymethyl-2'-deoxycytidine and 5-formyl-2'-deoxycytidine (Berry and Associates) were formulated in dimethyl sulfoxide (DMSO) / Solutol® HS15 / phosphate buffered saline (PBS) (5 / 5 / 90, v / v / v) at concentrations of 15, 50, and 100 mg / mL for intraperitoneal administration in a dose volume of 20 mL / kg. Test compounds were dosed every three days for a total of five doses (q3d x 5).

[0298] Male ICR mice weighing 23±3 g were provided by BioLasco Taiwan (a licensee of Charles River Laboratories). Animals were acclimated for 3 days prior to use and confirmed to be in good health. All animals were maintained in a hygienic environment with controlled temperature (20-24°C), humidity (30%-70%), and a 12-h light / dark cycle. They were given free access to sterilized standard laboratory diet [MFG (Oriental Yeast Co., Ltd., Japan)] and autoclaved tap water.

[0299] Animals were observed for the presence of acute toxicity symptoms (mortality, convulsions, tremors, muscle relaxation, sedation, etc.) and autonomic effects (diarrhea, salivation, lacrimation, vasodilation, piloerection, etc.) during 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 weights were recorded before each treatment and at 24, 48, and 72 hours after the final dose. Gross necropsies were performed on all animals without tissue collection.

[0300] xenograft 5-Hydroxymethyl-2'-deoxycytidine and 5-formyl-2'-deoxycytidine (Berry and Associates) dosing solutions were prepared freshly before each dose by first adding the appropriate volume of DMSO to the pre-weighed compound, followed by the appropriate volumes of Solutol® and PBS (5% DMSO / 5% Solutol® / 90% PBS). The standard agent, temozolomide, was provided in powder form by Oslo University Hospital and was formulated freshly before each dose by first adding the appropriate volume of DMSO to the pre-weighed compound, followed by the appropriate volumes 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 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). Cells were cultured in minimal essential medium containing 5% fetal bovine serum (FBS) at 37°C in an incubator with 5% CO.

[0302] Six- to seven-week-old female (nu / nu) nude mice obtained from BioLasco Taiwan (a licensee of Charles River Laboratories) were used. Animals were individually housed in ventilated cages (IVC, 36-cm mini-isolator system). Each cage consisted of five animals, each measuring 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 chow [MFG (Oriental Yeast Co., Ltd., Japan)] and autoclaved tap water.

[0303] Viable U87-MG (ATCC HTB-14) cells were implanted subcutaneously (SC) into the right flank of female nu / nu mice (5 × 10 cells / mouse in 0.2 mL / mouse of PBS). Group mean tumor volumes were approximately 129 mm. 3 from 131mm 3 Once the tumor-implanted mice reached day 1, they were divided into four treatment groups, each containing eight animals, and dosing began (denoted 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 overt toxicity were monitored and recorded twice weekly for 29 days. 3 ) into the formula for an ellipsoid: length x (width) 2 The tumor growth inhibition (T / C) was calculated according to 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 ≦42% was considered as significant antitumor activity (#).

[0306] Percent tumor growth inhibition (TGI) was also calculated by the following formula: %TGI = (1-(Tn / Cn)) x 100% A %TGI value ≥ 58% was considered as significant antitumor activity (#).

[0307] Two-way ANOVA® followed by Bonferroni's test was also used to determine statistically significant differences compared to the negative control group during the study; days 1 to 29. Differences were considered significant at p<0.05 (*).

[0308] At the completion of the study, tumors were excised and photographed from all animals in the experiment.

[0309] HPRT assay HPRT mutagenicity assays were performed in V79 cells. 50,000 V79 cells were treated in six-well plates with three different concentrations of either d5hmC or d5fC (1, 10, or 100 μM) for 24 hours. DMSO was used as a negative control. After treatment, cells were subcultured in T75 flasks for 9 days as needed to allow expression of HPRT mutants. 10,000 cells were replated onto 10 replica Petri dishes (100 × 15 mm) in selective medium (2.5 μg / ml 6TG).

[0310] Survival (relative plating efficiency) was determined by plating 200 cells onto four replica Petri dishes (60 x 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 cells plated, corrected for replating efficiency. Experiments were performed in triplicate, and data represent the mean ± SEM of triplicates.

[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 on an LSR Fortessa (BD Biosciences) and data were analyzed with FlowJo software. All experiments were performed in triplicate.

[0313] Western blot Western blots were performed as previously described (Towbin et al., 1979. Biotechnology, 24, 145-149). Anti-CDA antiserum (Abcam, catalog no. Ab82346) was used according to the manufacturer's recommended concentration. Protein was quantified by the signal generated 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 assessed as described above (Materials and Methods). Surprisingly, 5-formyl-2'-deoxycytidine was found to be cytotoxic to HeLa cells, although survival after treatment with 5-hydroxymethyl-2'-deoxycytidine was not different from that of DMSO-treated cells.

[0315] The cytotoxic effects of 5-formyl-2'-deoxycytidine were compared with two well-described 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] With the knowledge that 5-formyl-2'-deoxycytidine is cytotoxic to cervical cancer (HeLa) cells, the study was extended in two directions: (i) an additional compound was evaluated: 5-carboxyl-2'-deoxycytidine; and (ii) their cytotoxic effects against a wide range of human cancer cell lines were evaluated (Table 1).

[0317] [Table 2]

[0318] As shown in Table 1, 5-carboxyl-2'-deoxycytidine had no 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 wide range of cancers. 5-Hydroxymethyl-2'-deoxycytidine had a narrower cytotoxicity profile; in fact, this cytidine derivative was cytotoxic to only 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 well tolerated by patients. 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine were observed to be the most cytotoxic against the glioblastoma multiforme cell line (U87-MG).

[0319] Because the greatest cytotoxic effects of 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine were observed in glioma grade IV (U87-MG) cells, the cytotoxic activity of these compounds was evaluated in a broader range of patient-derived grade IV glioma cells (Table 2). Due to 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 related compounds, 5 of 12 grade IV glioma cell lines were killed by 5-formyl-2'-deoxycytidine, and 6 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 of 12 grade IV glioma cell lines, and 5-fluorouracil killed 11 of 12 grade IV glioma cell lines. Thus, 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 demonstrates that both 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine are cytotoxic to cell lines that are resistant to the current frontline treatment (temozolomide). This provides evidence that these compounds may perform better than current treatments for such temozolomide-resistant tumors. Alternatively, Table 2 indicates that temozolomide in combination with 5-formyl-2'-deoxycytidine and / or 5-hydroxymethyl-2'-deoxycytidine may be the optimal treatment.

[0323] Example 2 Cytotoxicity to 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, particularly those that are resistant to temozolomide treatment.

[0324] We further investigated the extent to which these compounds kill normal human cells; low cytotoxicity to normal human cells is an advantageous property for anticancer drugs. Therefore, we investigated the cytotoxicity of these compounds in various normal human cell lines (Table 3). Cells were grown and survival assays were performed as described above (Materials and Methods).

[0325] [Table 4]

[0326] As shown in Table 3, 5-hydroxymethyl-2'-deoxycytidine (dexycytidine) 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 indicate that these compounds are not only effective cancer chemotherapy agents, but may also be well tolerated by humans.

[0327] Example 3 maximum capacity Because 5-hydroxymethyl-2'-deoxycytidine and 5-formyl-2'-deoxycytidine have limited effects on normal cells, we reasoned that the compounds could be administered at relatively high doses without causing the side effects typically associated with cancer chemotherapy. The maximum tolerated doses (MTDs) of these compounds in mice were 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 animals were monitored for the presence of acute toxicity symptoms (mortality, convulsions, tremors, muscle relaxation, sedation, etc.) and autonomic effects (diarrhea, salivation, lacrimation, vasodilation, piloerection, etc.) within the first 30 minutes and again at 1, 24, 48, and 72 hours. Body weights were recorded before dosing 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 were unable to tolerate a single dose of 8000 mg / kg of 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine (not shown). 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 between mouse dose and human dose is a factor of 0.081 (Nair et al., (2016) Basic Clin Pharm. 7(2): 27-31). Thus, the data indicate that in humans, the maximum tolerated 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 many days are advantageous. Therefore, we performed repeated maximum tolerated dose assessments of both 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine as described above (Materials and Methods). Doses of 300 mg / kg, 1000 mg / kg, and 2000 mg / kg were selected for repeated multiple tolerated dose assessments because they were well tolerated as single doses in both treatment groups.

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

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

[0333] Example 4 In vivo cytotoxicity 5-Formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine were evaluated in a glioblastoma multiforme mouse xenograft model as previously described (Materials and Methods). U87-MG cells were injected subcutaneously into the flanks of nude mice. Tumors were allowed to form as previously described (Materials and Methods). Tumors grew to 129 mm 3 from 131mm 3After reaching the age of 18, 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 a single 2000 mg / kg dose of either 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine 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 vehicle and no compound. The positive control group was treated with five daily doses of 40 mg / kg temozolomide.

[0334] Tumor volume (Fig. 1C), body weight (Fig. 1D), mortality, and signs of overt toxicity were monitored and recorded twice weekly for 29 days. 3 ) into the formula for an ellipsoid: length x (width) 2 The tumor growth inhibition percentage (%TGI) was estimated according to a 2×0.5 ratio. 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 with that of the negative control group, a %T / C value of ≦42% or a TGI percentage value of ≧58% was considered to indicate significant antitumor activity. Two-way ANOVA® followed by Bonferroni's test was also used to elucidate statistically significant differences compared with the negative control group from day 1 to day 29 of the study (*p<0.05).

[0335] FIG. 1C indicates 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 demonstrated significant antitumor activity, with TGIs of 83% and 93%, respectively (Figure 1E). The positive control group treated with temozolomide demonstrated 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 the study, tumors were excised from the mice, photographed (Figure 1F), and measured (Figure 1G). Significant cytotoxic effects were observed in both treatment groups.

[0337] One mouse in the control group died during the experiment and the tumor volume for this mouse was not reported on day 29, but the tumor volumes for this mouse are included for the previous days.

[0338] Taken together, these surprising results demonstrate that 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine efficiently kill glioblastoma multiforme cells (glioma, grade IV) without being highly cytotoxic to normal cells. Furthermore, these compounds are well tolerated in mice, achieving a significant reduction in tumor volume with minimal side effects in mice. The results demonstrate that the compounds of the present invention can be used to treat human cancers, and that the compounds can be used at high doses that kill tumor cells without killing non-cancerous cells. Therefore, the use of these compounds as anti-cancer drugs has a wide therapeutic window.

[0339] Example 5 Cytotoxicity vs. cytostatic effect The cell line assay described above measures metabolic activity and therefore does not distinguish between inhibition of cell division and cell death. Therefore, we evaluated whether 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine kill sensitive cells or delay 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 phosphatidylserine) and 7AAD (a DNA stain that does not readily cross intact cell membranes; therefore, cells with compromised 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 cytometric profiles, with a slight increase in the amount of dead cells compared with temozolomide-treated controls. Flow cytometric profiles showed that the majority of SF-188 cells treated with 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine died (Figure 2A). SF188 cells exposed to 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine died and clearly did not arrest at cell cycle checkpoints.

[0340] Example 6 Requirement for 2'-deoxy sugar derivatives 5-Fluorouracil, a widely used cancer chemotherapy drug, has multiple cytotoxic variants, including the nucleoside (5-fluorouridine and 5-fluoro-2'-deoxyuridine) and the nucleobase 5-fluorouracil. We evaluated whether the ribonucleoside and nucleobase variants of 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine are also cytotoxic.

[0341] As shown in Table 1, 5-formyl-2'-deoxycytidine is cytotoxic to HeLa cells. The cytotoxicity of 5-formylcytidine and 5-formylcytosine in HeLa cells was evaluated in a viability assay as 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 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 in a viability assay as described above. Surprisingly, in contrast to 5-fluorouridine and 5-fluorouracil, 5-hydroxymethylcytidine and 5-hydroxymethylcytosine were shown not to be cytotoxic to U87-MG cells (Figure 2C).

[0343] These results indicate that the 2'-deoxyribose sugar is required for the cytotoxicity of the compounds of the present invention, which in turn surprisingly indicates that 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine utilize fundamentally different cellular pathways than fluoropyrimidines.

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

[0345] However, surprisingly, in fact, no correlation was observed between CDA expression and resistance or sensitivity to either 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine, as shown in Table 4A. 50 Data are identical to those in Table 1. CDA expression levels in the indicated cell lines were identified using the EMBL Expression Atlas (https: / / www.ebi.ac.uk / gxa / home, using the search term CDA). CDA expression is reported as RNA transcripts per million (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. When more than one value was reported, the lowest value was used.

[0346] [Table 5]

[0347] The linear correlation between cytotoxicity and drug exposure was poorly fitted (linear model for 2d5fc, Rz =0.00173; Linear model for 2d5hmC, R z = 0.02262). The data, together with 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, CDA expression levels (TPM) of various glioma cell lines were determined and are shown in Table 4B. CDA expression levels in the designated cell lines were identified using the Cancer Cell Line Encyclopedia (https: / / portals.broadinstitute.org / ccle) as described in Barretina, J et al. (2012) The Cancer Cell Line Encyclopedia enables predictive modeling of anticancer drug sensitivity. Nature. 483:603-7. CDA expression is reported as RNA transcripts per million (TPM) as described in Wagner et al. (2012) Theory Biosci 131(4):281-285. When more than one value was reported, the highest value was used.

[0349] [Table 6]

[0350] Example 8 Lack of mutagenic activity Temozolomide, the current frontline treatment for glioblastoma multiforme (Glibolastoma), is a potent mutagen. In fact, temozolomide exerts its cancer chemotherapy activity so intensely by mutating tumor cells that they die. Temozolomide acts by alkylating DNA, causing mutations. Expression of MGMT, a protein responsible for removing alkylated DNA damage, renders glioblastoma cells almost completely resistant to the cytotoxic effects of temozolomide.

[0351] Therefore, we investigated whether 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine have a similar mechanism of action. The mutagenicity of these compounds was assessed 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 was 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, i.e., their cytotoxic effects are not due to mutagenic activity.

[0352] Example 9 Cytotoxicity of other compounds vs. 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 assessed 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 Cytotoxicity of other compounds versus glioma cells U87-MG cells (glioma, grade IV) were treated with 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 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 assessed by MTT assay as described previously (Materials and Methods).

[0355] cell culture The U87-MG cell line was grown in DMEM (Sigma, Cat. No. 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 between 70 and 90% confluence.

[0356] Viability assay 4000 cells were seeded in 100 μl of the corresponding medium in a 96-well plate. The next day, drugs diluted in DMSO were 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 drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, Cat. No. 30-1010K). Cell survival was normalized to that of cells treated with DMSO alone. Experiments were performed in triplicate, and data represent the mean ± SEM of nine wells.

[0357] As shown in Table 5, both 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine performed surprisingly better than other compounds tested, including 5-bromo-2'-deoxycytidine, 5-iodo-2'-deoxycytidine, and 5-chloro-2'-deoxycytidine, in terms of cytotoxicity versus U87-MG cells.

[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 are 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 next day, drugs diluted in DMSO were 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 drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, Cat. No. 30-1010K). Cell survival was normalized to that of cells treated with DMSO alone. Experiments were performed in triplicate, and data represent the mean ± SD.

[0361] Example 11 Effects not mediated through thymidine synthase cell culture The U87-MG cell line was grown in DMEM (Sigma, Cat. No. 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 between 70 and 90% confluence.

[0362] Viability assay 4000 cells were seeded in 100 μl of the corresponding medium in a 96-well plate. The next day, drugs diluted in DMSO were added in triplicate at eight concentrations. Drugs were added as a 4-fold dilution series starting from 100 μM. Cells were incubated with drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, Cat. No. 30-1010K). Cell survival was normalized to the survival of cells treated with DMSO alone. Experiments were performed in triplicate, and data represent the mean ± SD of triplicate wells.

[0363] The results are shown in Figure 6. The cytotoxicity caused by 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine was 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 multiforme cell line (U87-MG) was grown in DMEM (Sigma, Cat. No. 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 between 70 and 90% confluence.

[0365] Viability assay 4000 cells were seeded in 100 μl of the corresponding medium in a 96-well plate. The next day, drugs diluted in DMSO were added in triplicate at eight concentrations. Drugs were added in a four-fold dilution series starting from 100 μM. Cells were incubated with the drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, Cat. No. 30-1010K). Cell survival was normalized to the survival of cells treated with DMSO alone. Experiments were performed in triplicate, and the data represent the average of these experiments.

[0366] As shown in Figure 7, human glioblastoma multiforme 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 in treating human glioblastoma multiforme than either chemical alone.

[0367] As shown in Figure 8, human glioblastoma multiforme cell lines that are 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 in treating human glioblastoma multiforme than either chemical alone.

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

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

[0370] cell culture The U87-MG cell line was grown in DMEM (Sigma, Cat. No. 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 between 70 and 90% confluence.

[0371] Viability assay 4000 cells were seeded in 100 μl of the corresponding medium in a 96-well plate. The next day, drugs diluted in DMSO were added in triplicate at eight concentrations. Drugs were added in a four-fold dilution series starting from 100 μM. Cells were incubated with the drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, Cat. No. 30-1010K). Cell survival was normalized to that of cells treated with DMSO alone. Experiments were performed in triplicate, and data represent the mean ± SEM of nine wells.

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

[0373] Example 14 Cytotoxicity of 5-formyl-2'-deoxycytidine-5'-triphosphate (HeLa) cell culture HeLa cell lines were grown in DMEM (Sigma, Cat. No. 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 between 70 and 90% confluence.

[0374] Viability assay 4000 cells were seeded in 100 μl of the corresponding medium in a 96-well plate. The next day, drugs diluted in DMSO were added in triplicate at eight concentrations. Drugs were added in a four-fold dilution series starting from 100 μM. Cells were incubated with the drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, Cat. No. 30-1010K). Cell survival was normalized to that of cells treated with DMSO alone. Experiments were performed in triplicate, and data represent the mean ± SEM of nine wells.

[0375] As shown in Figure 10A, HeLa cervical cancer cells cannot survive treatment with increasing concentrations of 5-formyl-2'-deoxycytidine-5'-triphosphate, demonstrating the utility of 5-formyl-2'-deoxycytidine-5'-triphosphate in treating 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 assessed by MTT assay as described above (Materials and Methods).

[0377] cell culture The U87-MG cell line was grown in DMEM (Sigma, Cat. No. 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 between 70 and 90% confluence.

[0378] Viability assay 4000 cells were seeded in 100 μl of the corresponding medium in a 96-well plate. The next day, drugs diluted in DMSO were 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 drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, catalog number 30-1010K). Cell survival was normalized to the survival of cells treated with DMSO alone. Experiments were performed in triplicate, and data represent the mean ± SD of at least three wells.

[0379] As shown in FIG. 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-base 2 transformed CDA expression levels were determined for various glioma cell lines. CDA expression levels in the indicated cell lines were identified using the GENEVESTIGATOR® database (https: / / genevestigator.com / gv / ) by searching CDA, Organism: Homo sapiens, Platform: Affymetrix Human Genome U133 2.0 array. Results are shown in Figures 11-13 and Table 6.

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

[0383] [Table 8]

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

[0385] [Table 9]

[0386] Taken together, these results demonstrate that many cancers, including all tested cancers of the central nervous system, do not overexpress CDA but rather express low levels of CDA. 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, Inc. Permeability was determined according to the manufacturer's instructions.

[0388] Manufacturer's instructions: 1. In a separate centrifuge tube, prepare 500 μL of 500 μM test compound: Mix 25 μL of 10 mM test compound in DMSO + 475 μL of PBS. If permeability controls are used, also dilute them to 500 μM: Mix 25 μL of permeability control + 475 μL of PBS. 2. In a separate tube, prepare 200 μM equilibrium standards for each test compound and control: Mix 80 μL of 500 μM test compound or control with 120 μL of PBS. If the compound is able to permeabilize the membrane and reach complete equilibrium, 200 μM will be the final concentration of the solution in the donor and acceptor wells. Next, prepare a blank control by mixing 5 μL of DMSO + 245 μL of PBS in a separate tube. Save the equilibrium standards and blank controls for analysis the next 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 donor plate well membrane. Be careful 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 duplicate wells of the donor plate. Note: We recommend running all experimental variables in at least duplicate. 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 equilibrated standards for each test compound and permeability control. 100 μL of blank control is also added to a well of the UV plate (Cat. No. P96UV). 8. Determine the peak absorbance of the test compound by reading the absorbance spectrum from 200 nm to 500 nm at 10 nm intervals. The blank control is to ensure that the peak is due to the test compound and not due to DMSO in the solution. The peak absorbances for the high, medium, 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. A compound of formula (I): 【Chemical 1】 [In the formula, X is a group containing 1 to 20 non-hydrogen atoms containing at least one functional group selected from an aldehyde, an alcohol, a protected alcohol, an ether, an ester, and a carboxylic acid, with the proviso that X is not -COOH; W 1 and W 2 are each independently O, S, or NH; Y is H or a group containing 1 to 15 non-hydrogen atoms; Z is -OPG, -OR z or -N(R x R y ), where R x , R y and R z are independently H or a group containing 1 to 10 non-hydrogen atoms; R 1 is H or a group containing 1 to 15 non-hydrogen atoms, R 2 is H, -OH, -OPG, -F, -Cl, -Br, -I or -N 3 and R 3 is H, -F, -Cl, -Br, -I or -N 3 and where PG is an alcohol protecting group such as acetyl (Ac), benzyl (Bn), or benzoyl (Bz). or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof.

2. 1. A compound of formula (I): 【Chemistry 2】 [In the formula, X is a group containing 1 to 20 non-hydrogen atoms containing at least one functional group selected from an aldehyde, an alcohol, a protected alcohol, an ether, an ester, and a carboxylic acid, with the proviso that X is not -COOH; W 1 and W 2 are each independently O, S, or NH; Y is H or a group containing 1 to 15 non-hydrogen atoms; Z is -OPG, -OR z or -N(R x R y ), where R x , R y and R z are independently H or a group containing 1 to 10 non-hydrogen atoms; R 1 is H or a group containing 1 to 15 non-hydrogen atoms, R 2 is H, -OH, -OPG, -F, -Cl, -Br, -I or -N 3 and R 3 is H, -F, -Cl, -Br, -I or -N 3 and where PG is an alcohol protecting group such as acetyl (Ac), benzyl (Bn), or benzoyl (Bz). or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof.

3. Formula (IIa) or Formula (IIb), preferably Formula (IIa): 【Chemistry 3】 [where X, R 1 and R 2 is as defined in claim 1 or claim 2] or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof.

4. Formula (IIIa), (IIIb), (IIIc) or (IIId), preferably formula (IIIa) or (IIIc): 【Chemistry 4】 wherein X is as defined in claim 1 or claim 2.

4. The compound for use according to any one of claims 1 to 3, which is a compound of the formula: or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof.

5. X is -(CH 2 ) n 5. The compound for use according to any one of claims 1 to 4, wherein n is from 0 to 6, and X' is -CHO, -OH, -OR or -OC(=O)R, wherein R is methyl.

6. 6. The compound for use according to any one of claims 1 to 5, wherein X is a group containing from 2 to 20 non-hydrogen atoms.

7. 6. The compound for use according to any one of claims 1 to 5, wherein X is not -COOH or -OH.

8. X is, a) -CHO or -CH 2 OH, or b)-CH 2 OCH 3 or -CH 2 OC(=O)CH 3 8. The compound for use according to any one of claims 1 to 7, wherein

9. X is -CHO or -CH 2 9. The compound for use according to any one of claims 1 to 8, wherein said compound is OH.

10. X is CH 2 10. The compound for use according to any one of claims 1 to 9, wherein said compound is OH.

11. Z is -NH 2 11. The compound for use according to any one of claims 1 to 10, wherein

12. 9. The compound for use according to any one of claims 1 to 8, which is 5-formyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, 5-methoxymethyl-2'-deoxyuridine, 5-acetoxymethyl-2'-deoxyuridine, 5-formyl-2'-deoxycytidine-5'-triphosphate or 5-hydroxymethyl-2'-deoxycytidine-5'-triphosphate, or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof.

13. 13. The compound for use according to claim 12, which is 5-formyl-2'-deoxycytidine or 5-hydroxymethyl-2'-deoxycytidine, or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof.

14. 14. The compound for use according to claim 13, which is 5-hydroxymethyl-2'-deoxycytidine or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof.

15. 15. The compound for use according to any one of claims 2 to 14, wherein the cancer is a cancer of tissue derived from the ectoderm, paraxial mesoderm or lateral plate mesoderm, preferably from the ectoderm.

16. 16. The compound for use according to any one of claims 2 to 15, wherein the cancer is a cancer of the central nervous system, preferably a brain cancer.

17. 17. The compound for use according to any one of claims 2 to 16, wherein the cancer is a glioma, preferably a glioblastoma.

18. 18. The compound for use according to any one of claims 2 to 17, wherein the cancer is a cancer in which CDA is not overexpressed, preferably wherein said cancer expresses CDA at a level of 140 transcripts per million (TPM) or less.

19. 19. The compound for use according to any one of claims 2 to 18, wherein the CDA expression level in the cancer is less than or equal to 90% of the CDA expression level in a reference cancer cell line when determined under the same conditions and using the same method, wherein said reference cancer cell line is MDA-MB-231.

20. 20. The compound for use according to any one of claims 2 to 19, wherein the cancer is resistant to treatment with gemcitabine, cytarabine, temozolomide or 5-fluorouracil.

21. 21. The compound for use according to any one of claims 2 to 20, wherein the treatment comprises administering the compound at a dose of between 10 mg / kg and 405 mg / kg.

22. 22. The compound for use according to any one of claims 2 to 21, wherein the treatment further comprises the administration of a further anti-cancer agent.

23. 15. A kit comprising a compound according to any one of claims 1 to 14 and a further anti-cancer agent for use in the treatment or prevention of cancer.

24. 15. A pharmaceutical composition comprising a compound according to any one of claims 1 to 14, and optionally a further anti-cancer agent, together with one or more pharmaceutically acceptable excipients.

25. 25. The compound for use according to claim 22, the kit according to claim 23, or the pharmaceutical composition according to claim 24, wherein the further anticancer agent is selected from the group consisting of gemcitabine, cytarabine, temozolomide, 5-fluorouracil, and gliadel (registered trademark).

26. 19. A method of treating or preventing cancer in a subject, comprising administering to the subject a compound of any one of claims 1 to 14, wherein the cancer is as defined in any one of claims 2 or 15 to 17, and the treatment is as defined in any one of claims 2, 21, 22 or 25.

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