Deoxy-cytidine or uridine derivatives for use in cancer therapies

Abstract

The present invention relates to a compound of Formula (I), or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof, wherein X, W.sub.1, W.sub.2, Y, Z, R.sub.1, R.sub.2 and R.sub.3 are as defined in the disclosure herein, for use in therapy, particularly for use in the treatment of cancer. The present invention also relates to methods of treating cancer comprising the administration of a compound of Formula (I) to a subject in need thereof, and to pharmaceutical compositions and kits comprising such compounds. ##STR00001##

Claims

1. A method of treating or preventing a cancer of the central nervous system in a subject in need thereof, which comprises administering to the subject a compound of Formula (IIa): ##STR00008## or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof, wherein: X is (CH.sub.2).sub.nX; n is an integer having a value of from 0 to 6; X is CHO, OH, OR, or OC(?O)R; R is methyl; R.sub.1 is OH or O(P(?O)(OH)O).sub.mH, where m is an integer having a value of from 1 to 3; and R.sub.2 is OH.

2. The method of claim 1, wherein the compound is a compound of Formula (IIIa) or (IIIc), or a stereoisomer, solvate, tautomer, or pharmaceutically acceptable salt thereof: ##STR00009## wherein X is as defined in claim 1.

3. The method of claim 1, wherein X is: a) CHO or CH.sub.2OH; or b) CH.sub.2OCH.sub.3 or CH.sub.2OC(?O)CH.sub.3.

4. The method of claim 1, wherein X is CHO or CH.sub.2OH.

5. The method of claim 1, wherein X is CH.sub.2OH.

6. The method of claim 1, wherein the compound is 5-formyl-2-deoxycytidine, 5-hydroxymethyl-2-deoxycytidine, 5-formyl-2-deoxycytidine-5-triphosphate or 5-hydroxymethyl-2-deoxycytidine-5-triphosphate, or a stereoisomer, solvate, tautomer, or pharmaceutically acceptable salt thereof.

7. The method of claim 6, wherein the compound is 5-formyl-2-deoxycytidine or 5-hydroxymethyl-2-deoxycytidine, or a stereoisomer, solvate, tautomer, or pharmaceutically acceptable salt thereof.

8. The method of claim 7, wherein the compound is 5-hydroxymethyl-2-deoxycytidine or a stereoisomer, solvate, tautomer, or pharmaceutically acceptable salt thereof.

9. The method of claim 1, wherein the cancer is a brain cancer.

10. The method of claim 1, wherein the cancer is a glioma.

11. The method of claim 1, wherein the cancer is resistant to treatment with gemcitabine, cytarabine, temozolomide, or 5-fluorouracil.

12. The method of claim 1, wherein the compound is administered at a dose of between 10 mg/kg and 405 mg/kg.

13. The method of claim 1, further comprising administering an additional anticancer agent.

14. The method of claim 2, wherein the compound is a compound of Formula (IIIa) or Formula (IIIc).

15. The method of claim 10, wherein the cancer is a glioblastoma.

16. The method of claim 13, wherein the additional anticancer agent is selected from the group consisting of: gemcitabine, cytarabine, temozolomide, 5-fluorouracil, and carmustine.

Description

(1) The invention will be further described with reference to the following non-limiting Examples in which:

(2) Figure 1 shows that 5-formyl-2-deoxycytidine and 5-hydroxymethylcytidine are well tolerated in mice and reduce human glioblastoma multiforme tumors in mouse xenograft models.

(3) FIG. 1 A: Single dose maximum tolerated dose protocol. Mice were dosed with a single intraperitoneal injection at the indicated dose. If all the mice in the relevant group tolerated the indicated dose, the dose was escalated as indicated.

(4) FIG. 1 B: Mice body weigh was measured at the indicated time point after mice were intraperitoneally injected with the indicated dose and indicated compound every three days for a total of five doses.

(5) FIGS. 1C-H: U87-MG cells were implanted in the flank of 32 immunodeficient mice. After tumors reached 129-131 mm.sup.3, mice were divided into four groups of 8 mice. The negative control group was treated with the vehicle, the positive control group was treated with 40 mg/kg temozolomide once a day for five days, the treatment groups were treated with 2000 mg/kg 5-formyl-2-deoxycytidine or 2000 mg/kg 5-hydroxymethyl-2-deoxycytidine once every three days for a total of five doses. Tumor volumes were measured every three days (FIG. 1C) and the mouse body weight was measured every three days (FIG. 1 D). At the completion of the study the percent tumor growth inhibition was computed (TGI(%)) (FIG. 1 E), tumors were resected, photographed (FIG. 1F), measured (FIG. 1G) and sectioned and stained with hematoxylin and eosin (FIG. 1H).

(6) FIG. 2 shows that 5-formyl-2-deoxycytidine and 5-hydroxymethyl-2-deoxycytidine kill glioblastoma multiforme by a mechanism unrelated to current nucleotide analogues:

(7) FIG. 2A: flow cytometry of 5-formyl-2-deoxycytidine and 5-hydroxymethyl-2-deoxycytidine treated cells, stained with Annexin V and 7AAD.

(8) FIG. 2B: Survival curves of HeLa cells treated with a titration of 5-formylcytosine, 5-formylcytidine, or 5-formyl-2-deoxycytidine.

(9) FIG. 2C: Survival curves of U87-MG cells treated with a titration 5-hydroxymethylcytosine, 5-hydroxymethylcytidine, or 5-hydroxymethyl-2-deoxycytidine.

(10) FIG. 3 shows 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 are shown not to be mutagenic.

(11) Figure 4 shows the level of cytotoxicity (% survival) of 5-formyl-2-deoxycytidine, 5-formylcytidine and 5-chloro-2-deoxycytidine against HeLa cells after three days of treatment.

(12) FIG. 5 shows the level of cytotoxicity (% survival) of 5-formyl-2-deoxycytidine (d5fC), 5-hydroxymethyl-2-deoxycytidine (d5hmC), 5-chloro-2-deoxycytidine (5CldC), 5-bromo-2-deoxycytidine (5BrdC), 5-lodo-2deoxycytidine (51dC), and Thymidine vs. U87-MG cells.

(13) FIG. 6 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.

(14) FIG. 7 shows the cytotoxic effects (% survival) of 5-formyl-2-deoxycytidine in combination with Temozolomide in U87-MG cells.

(15) FIG. 8 shows the cytotoxic effects (% survival) of 5-hydroxymethyl-2-deoxycytidine in combination with Temozolomide in U87-MG cells.

(16) FIG. 9 shows the cytotoxic effects of 5-methoxymethyl-2-deoxyuridine and 5-acetoxymethyl-2-deoxyuridine on U87-MG cells. Treatment for 72 hrs. Survival was quantified using an MTT assay.

(17) FIG. 10 shows % survival of various cells after treatment with d5fCTP or d5hmCTP

(18) FIG. 10A: % survival of HeLa after treatment with 5-formyl-2-deoxycytidine-5-triphosphate (d5fCTP) for 72 hrs. Survival was quantified using an MTT assay.

(19) FIG. 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.

(20) FIG. 11 shows the CDA expression levels in various cell lines. The values are normalized Log 2 CDA expression levels. The expression levels were determined using the Affymetrix Human Genome U133 Plus 2.0 Array platform and the Genevestigator database (https://genevestigator.com/gv/).

(21) FIG. 12 shows the linear CDA expression levels in various cell lines. The expression levels were determined using the Affymetrix Human Genome U133 Plus 2.0 Array Platform and the Genevestigator database (https://genevestigator.com/gv/).

(22) FIG. 13A and 13B show the CDA expression levels in various human brain tumours. The values are normalized Log 2 CDA expression levels. The expression levels were determined using the Affymetrix Human Genome U133 Plus 2.0 Array platform and the Genevestigator database (https://genevestigator.com/gv/).

(23) FIGS. 14A and 14B show the linear CDA expression levels in various human brain tumours. The expression levels were determined using the Affymetrix Human Genome U133 Plus 2.0 Array platform and the GENEVESTIGATOR? database (https://genevestigator.com/gv/).

(24) FIG. 15 shows the results of a PAMPA Assay demonstrating that 2d5hmC and 2d5fC can pass the blood-brain barrier.

EXAMPLES

(25) Materials and Methods

(26) Animals

(27) All aspects of this work, including housing, experimentation, and disposal of animals were performed in general accordance with the Guide for the Care and Use of Laboratory Animals: Eighth Edition (National Academy Press, Washington, D.C., 2011) in an AAALAC-accredited laboratory animal facility. The animal care and use protocol was reviewed and approved by the IACUC at Pharmacology Discovery Services Taiwan, Ltd.

(28) Cell Culture

(29) Primary glioma neural stem (GNS) cells (G7, G14, G144, G166) were cultured on poly-D-lysine (Merck Millipore, Cat. Nr. A-003-E) and laminin (R&D Systems, Cat.Nr. 3446-005-01) coated plates in neural stem cell medium (50% DMEM-F12 (Thermofisher, Cat. Nr. 21041025), 50% neurobasal medium (Thermofisher, Cat. Nr. 10888-022), N2 (Life Technologies, Cat. Nr. A-003-E) and B27 supplements (Life Technologies, Cat.Nr. 12587010), 1 mM sodium pyruvate (Life Technologies, Cat.Nr. 11360-039), 2 mM glutamax (Life Technologies, Cat. Nr. 35050038), 1 mM HEPES (Fisher Scientific, Cat. Nr. BP299-1), 0.1 mM p-mercaptoethanol (Life Technologies, Cat. Nr. 31350010), 1? nonessential amino acids (Life Technologies, Cat. Nr. 11140-035), 0,006% bovine serum albumin (Sigma, Cat. Nr. A8577-10ML),4 ?g/ml heparin (Sigma, Cat. Nr. H3149-25KU), 100 U/ml penicillin, 100 ?g/ml streptomycin, 20 ng/ml hEGF (R&D Systems, Cat. Nr. 236-EG-200), 10 ng/ml bFGF (Peprotech, Cat. Nr. 100-18B).

(30) HCoT16 were grown in McCoAs 5a Medium modified (Life technologies, Cat.Nr. 36600021) supplemented with 10% Fetal Bovine Serum and 100 U/mi penicillin 100 U/ml streptomycin.

(31) Arpe 19 were grown in DMEM:F12 medium (Life Technologies, 21331-020) supplemented with 10% Fetal Bovine Serum and 100 U/m penicillin 100 U/m5 streptomycin.

(32) HAP1 were grown in IMDM (Gibco, Cat. Nr. 12440-05) supplemented with supplemented with 10% Fetal Bovine Serum and 100 U/m penicillin 100 U/m5 streptomycin.

(33) Cell lines not mentioned above were grown in DMEM (Sigma, Cat.Nr. 06429) supplemented with 10% Fetal Bovine Serum and 100 U/mA penicillin 100 U/m5 streptomycin. All cells were maintained in a 5% 002, humidified, water-jacketed incubator at 37? C. Cells were passed between 70 and 90% confluence.

(34) Drugs

(35) Compounds used in the present Examples were obtained as follows (CAT#0 catalogue number).

(36) TABLE-US-00001 Compound CAS # Source CAT # 5-hydroxymethyl-2-deoxycytidine 7226-77-9 Berry and Associates PY 7588 5-formyl-2-deoxycytidine 137017-45-9 Berry and Associates PY 7589 5-carboxyl-2-deoxycytidine 1009808-62-1 Berry and Associates PY 7593 5-formylcytidine 148608-53-1 Berry and Associates PY 7599 5-formylcytosine 4425-59-6 Toronto Research F698975 Chemicals Temozolomide 85622-93-1 Sigma Aldrich T2577 5-flurouracil 51-21-8 Sigma Aldrich F6627 5-hydroxymethylcytidine 19235-17-7 Berry and Associates PY 7596 5-hydroxymethylcytosine 1123-95-1 Toronto Research H945870 Chemicals 5-chloro-2-deoxycytidine 32387-56-7 Carbosynth NC08279 5-bromo-2-deoxycytidine 1022-79-3 Carbosynth NB06450 5-iodo-2-deoxycytidine 611-53-0 Carbosynth ND05777 Thymidine 50-89-5 Sigma Aldrich T1895 5-methoxymethyl-2- 5116-22-3 Toronto Research M263610 deoxyuridine Chemicals 5-acetoxymethyl-2-deoxyuridine 148380-55-6 Toronto Research A167180 Chemicals 5-formyl-2-deoxycytidine-5- Trilink Biotech N-2064 triphosphate 5-Hydroxymethyl-2-deoxycytidine-5-Triphosphate Trilink Biotech N-2060
Survival Assay

(37) In a 96-well plate, 4000 cells in 100 ?l of corresponding medium were seeded. The following day, drugs, diluted in DMSO, were added in 8 concentrations in triplicate. Drugs were added as a 4-fold dilution series starting from 100 ?M. Cells were incubated with the drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, Cat. Nr. 30-1010K). Cell survival was normalized to the survival of cells treated with DMSO only. Experiment was performed three times, data represent mean of nine wells?SEM.

(38) MTD (Maximum Tolerated Dose)

(39) 5-hydroxymethyl-2-deoxycytidine and 5-formyl-2-deoxycytidine (Berry and Associates), were formulated in dimethyl sulfoxide (DMSO)/Solutol? R HS15/phosphate buffered saline (PBS) (5/5/90, v/v/v) at the concentration of 30 and 200 and 400 mg/mL for IP administration at the dosing volume of 5 mL/kg. A dosing volume at 10 or 20 mL/kg was applied.

(40) Male ICR mice weighing 23?3 g were provided by BioLasco Taiwan (under Charles River Laboratories Licensee). Animals were acclimated for 3 days prior to use and were confirmed with good health. All animals were maintained in a hygienic environment with controlled temperature (20-24? C.), humidity (30%-70%) and 12 hours light/dark cycles. Free access to sterilized standard lab diet [MFG (Oriental Yeast Co., Ltd., Japan)] and autoclaved tap water were granted.

(41) 5-hydroxymethyl-2-deoxycytidine and 5-formyl-2-deoxycytidine were administered IP to groups of three male ICR mice weighing 23?3 g. Animals received an initial dose of 300 mg/kg. If the animals survived for 72 hours, the dose for the next cohort was increased. If one or more animals died, the dose for the next cohort was decreased. The testing stopped when all animals survived at the upper bound, or when three dose levels had been tested or when the upper or lower bound had been reached. At each dose level, animals were observed for the presence of acute toxic symptoms (mortality, convulsions, tremors, muscle relaxation, sedation, etc.) and autonomic effects (diarrhea, salivation, lacrimation, vasodilation, piloerection, etc.) during the first 30 minutes, again at 1, 24, 48 and 72 hours. Body weights were recorded pre-dose and at 72 hours. The animals were observed and mortality noted daily after compound administration. Gross necropsy was performed on all animals without tissue collection, and the next dose level was determined based on the study design table.

(42) Multiple MTD

(43) 5-hydroxymethyl-2-deoxycytidine and 5-formyl-2-deoxycytidine (Berry and Associates) were formulated in dimethyl sulfoxide (DMSO)/Solutol? R HS15/phosphate buffered saline (PBS) (5/5/90, v/v/v) at the concentration of 15, 50, and 100 mg/mL for IP administration at the dosing volume of 20 mL/kg. The test compounds were dosed every three days for a total of 5 doses (q3d?5).

(44) Male ICR mice weighing 23?3 g were provided by BioLasco Taiwan (under Charles River Laboratories Licensee). Animals were acclimated for 3 days prior to use and were confirmed with good health. All animals were maintained in a hygienic environment with controlled temperature (20-24? C.), humidity (30%-70%) and 12 hours light/dark cycles. Free access to sterilized standard lab diet [MFG (Oriental Yeast Co., Ltd., Japan)] and autoclaved tap water were granted.

(45) Animals were observed for the presence of acute toxic 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). The mortality was noted at the same scheme. In addition, body weights were recorded before each treatment and at 24, 48 and 72 hours after the final administration. Gross necropsy was performed on all animals without tissue collection.

(46) Xenograft

(47) 5-hydroxymethyl-2-deoxycytidine and 5-formyl-2-deoxycytidine (Berry and Associates) dosing solutions were prepared fresh prior to each dose administration by first adding appropriate volume of DMSO to pre-weighed compound, then adding appropriate volumes of Solutol? and PBS (5% DMSO/5% Solutol?/90% PBS). Standard agent, Temozolomide, was provided by Oslo University Hospital in powder form and was formulated fresh prior to each dose by first adding appropriate volume of DMSO to pre-weighed compound, then adding 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. Standard agent, Temozolomide, was administered at a dose volume of 10 mL/kg.

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

(49) Female (nu/nu) nude mice aged 6-7 weeks obtained from BioLasco Taiwan (under Charles River Laboratories Licensee) were used. The animals were housed in individually ventilated cages (IVC, 36 Mini Isolator system). The allocation for 5 animals was 27?20?14 in cm. All animals were maintained in a hygienic environment under controlled temperature (20-24? C.) and humidity (30%-70%) with 12-hour light/dark cycle. Free access to standard lab diet [MFG (Oriental Yeast Co., Ltd., Japan)] and autoclaved tap water were granted.

(50) Viable U87-MG (ATCC HTB-14) cells were subcutaneously (SC) implanted (5?106 cells/mouse in PBS at 0.2 mL/mouse) into the right flank of female nu/nu mice.

(51) When group mean tumor volumes reached approximately 129 mm.sup.3 to 131 mm.sup.3, tumor implanted mice were divided into four treatment groups, each group containing eight animals, and dose administrations were initiated (denoted as Day 1).

(52) 5-hydroxymethyl-2-deoxycytidine, 5-formyl-2-deoxycytidine at 2000 mg/kg and corresponding vehicle (5% DMSO/5% Solutol?/90% PBS) were administered intraperitoneally (IP) once every three days for a total of five administrations. Temozolomide at 40 mg/kg was administered orally (PO) once daily for five total administrations.

(53) The tumor volume, body weight, mortality, and signs of overt toxicity were monitored and recorded twice weekly for 29 days. Tumor volume (mm.sup.3) was estimated according to the ellipsoid formula as: length?(width).sup.2?0.5. Tumor growth inhibition (T/C) was calculated by the following formula:

(54) $\%T/C=\left(\mathrm{Tn}/\mathrm{Cn}\right)?100\%$ Cn: Tumor weight measured on Day n in the control group Tn: Tumor weight measured on Day n in the treated group % T/C value?42% was considered significant antitumor activity (#).
Percent tumor growth inhibition (TGI) was also calculated by the following formula:

(55) $\%\mathrm{THI}=\left(1-\left(\mathrm{Tn}/\mathrm{Cn}\right)\right)?100\%$ % TGI value?58% was considered significant antitumor activity (#).

(56) Two-way ANOVA? followed by Bonferroni test was also used to ascertain the statistically significant difference compared to the negative control group during study; Day 1 through to Day 29. Differences are considered significant at p<0.05 (*).

(57) Upon study completion, tumors were excised from all animals on study and photographs were taken.

(58) HPRT Assay

(59) HPRT mutagenicity assay was performed in V79 cells. 50,000 V79 cells were treated with three different concentrations of either d5hmC or d5fC (1, 10, 100 ?M) for 24 hours in a 6 well plate. DMSO was used as a negative control. After the treatment, the cells were subcultured as needed in T75 flasks for 9 days to allow expression of HPRT-mutants. 10,000 cells were replated into 10 replica Petri dishes (100?15 mm) with selective media (2.5 ?g/ml of 6TG).

(60) Survival (relative plating efficiency) was determined by plating 200 cells into four replica Petri dishes (60?15 mm) without selective media. Colonies were fixed, Giemsa-stained and counted 7 days later. The mutant frequency is expressed as a total number of mutants counted on all plates divided by the number of cells seeded corrected by reseeding plating efficiency. Experiment was performed three times, data represent mean of triplicate?SEM.

(61) Apoptosis Flow Cytometry

(62) 100,000 cells were seeded in a 6-well plate and incubated with 100 ?M of Temozolomide, 2-Deoxy-5-hydroxymethylcytidine, 5-Formyl-2-deoxycytidine or DMSO for 72 hours. Detection of apoptotic cells was assessed using Annexin V-7-amino-actinomycin D (7-AAD) Apoptotic Detection Kit (Nordic Biosite AS, Cat. Nr. 640922) according to manufacturer's protocol.

(63) Fluorescence-activated cell sorting analysis was performed on LSR Fortessa (BD Biosciences) and data were analyzed on FlowJo software. All experiments were performed in triplicates.

(64) Western Blots

(65) Western blots were performed as previously described (Towbin et al., 1979. Biotechnology, 24, 145-149). Anti-CDA antiserum (Abcam, cat nr. Ab82346) was used according to the manufacturer's recommended concentration. Proteins were quantified by the signal generated by the oxidation of luminol by horse radish peroxidase conjugated to the secondary antibody.

Example 1: Cytotoxicity to Tumour Cells

(66) HeLa cells, grown as described above (Materials and Methods), were treated with 5-hydroxymethyl-2-deoxycytidine and 5-formyl-2-deoxycytidine. After three days of treatment with these compounds, cell survival was assessed as described above (Materials and Methods). While survival after treatment with 5-hydroxymethyl-2-deoxycytidine did not differ from DMSO treated cells, it was found, surprisingly, that 5-formyl-2-deoxycytidine was cytotoxic to HeLa cells.

(67) The cytotoxic effect of that 5-formyl-2-deoxycytidine was compared to two well-described cytotoxic compounds, 5-flurouracil and temozolamide. 5-formyl-2-deoxycytidine was determined to be more cytotoxic to HeLa (IC50=0.76 ?M) cells than both 5-flurouracil (IC50>25 ?M) and temozolomide (IC50>25 ?M).

(68) With the knowledge that 5-formyl-2-deoxycytidine is cytotoxic to cervical carcinoma (HeLa) cells, the study was expanded in two directions: (i) a further compound was assessed: 5-carboxyl-2-deoxycytidine; and (ii) their cytotoxic effects against a wide range of human cancer cell lines was assessed (Table 1).

(69) TABLE-US-00002 TABLE 1 Evaluation of 5-formyl-2-deoxycytidine (2d5fC), 5-hydroxymethyl-2-deoxycytidine (2d5hmc) and 5-carboxy- 2-deoxycytidine (2d5caC) in a range of human cancer cell lines, and as compared to Temozolomide and 5-flurouracil (5fU). IC.sub.50 is the concentration at which half of the cells are killed by the relevant compound. Cell Germ IC.sub.50 (?M) Line Disease Layer 2d5fC 2d5hmC 2d5caC Temozolomide 5fU U87-MG Glioma, Grade IV Ectoderm 0.3340 3.027 >25.00 >25.00 >25.00 HCT-116 Colon Carcinoma Endoderm >25.00 >25.00 >25.00 >25.00 3.545 A549 Lung Carcinoma Endoderm >25.00 >25.00 >25.00 1.018 1.853 22Rv1 Prostate Carcinoma Endoderm >25.00 >25.00 >25.00 >25.00 >25.00 NCI-N87 Gastric Carcinoma Endoderm 0.8057 >25.00 >25.00 >25.00 1.524 MIA PaCa-2 Pancreatic Endoderm 1.143 >25.00 >25.00 >25.00 8.096 A-498 Kidney Carcinoma Mesoderm >25.00 >25.00 >25.00 >25.00 2.367 U937 Lymophoma Mesoderm 0.9536 >25.00 >25.00 13.08 7.449 A375 Malignant Melanoma Mesoderm 3.188 >25.00 >25.00 >25.00 7.867 HL-60 Acute Promyelocytic Mesoderm 6.262 >25.00 >25.00 >25.00 >25.00 Leukemia SK-OV-3 Ovarian Mesoderm 9.690 >25.00 >25.00 >25.00 3.756 Adenocarcinoma MCF-7 Epithelial Mesoderm 0.8657 >25.00 >25.00 >25.00 >25.00 Adenocarcinoma (Breast) U2OS Bone Mesoderm 8.69 >25.00 >25.00 >25.00 2.889 HeLa Cervical Mesoderm 0.76 >25.00 >25.00 >25.00 >25.00 Adenocarcinoma HAP1 Chronic Mesoderm >25.00 6.53 n.d. n.d. n.d. Myelogenous Leukemia (CML) V79 Chinese Hamster Mesoderm >25.00 >25.00 n.d. n.d. n.d. Ovary H1437 Lung (metastatic Endoderm 5.65 n.d. n.d. n.d. n.d. non-small cell adenocarcinoma) H1573 Lung (metastatic Endoderm 7.72 n.d. n.d. n.d. n.d. adenocarcinoma)

(70) 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 is 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 has a narrower cytotoxicity profile; indeed this cytidine derivative was only cytotoxic to two cell lines evaluated: U87-MG, grade IV glioma cells (IC50>0.3340 ?M) and HAP1 Chronic Myelogenous Lukemia cells (IC50>3.027 ?M). This suggests that this compound may be well tolerated by patients. 5-formyl-2-deoxycytidine and 5-hydroxymethyl-2-deoxycytidine were observed as the most cytotoxic to glioblastoma multiforme cell (U87-MG) lines.

(71) Since the greatest cytotoxic effect of 5-formyl-2-deoxycytidine and 5-hydroxymethyl-2-deoxycytidine was observed in glioma grade IV (U87-MG) cells, the cytotoxic activity of these compounds was evaluated in a wider range of patient derived grade IV, glioma cells (Table 2). Due to lack of activity in previous assays, 5-carboxyl-2-deoxycytidine was not included in this more rigorous analysis.

(72) TABLE-US-00003 TABLE 2 Evaluation of cytotoxicity of 5-formyl-2-deoxycytidine (2d5fC) and 5-hydroxymethyl- 2-deoxycytidine (2d5hmc) against patient derived glioblastoma multiforme cell lines (Glioma, Grade IV), and as compared to Temozolomide and 5-flurouracil (5fU). IC.sub.50 is the concentration at which half of the cells are killed by the relevant compound. Cell Germ IC.sub.50 (?M) Line Disease Layer 2d5fC 2d5hmC Temozolomide 5fU U87-MG Glioma, Grade IV Ectoderm 0.3340 3.027 >25.00 >25.00 G7 Glioma, Grade IV Ectoderm 19.25 11.93 6.176 6.456 G14 Glioma, Grade IV Ectoderm >100 25.4 6.018 11.61 G26 Glioma, Grade IV Ectoderm 28.12 >100 7.338 7.125 G30 Glioma, Grade IV Ectoderm 18 28.13 21.6 23.33 G144 Glioma, Grade IV Ectoderm >100 >100 14.66 6.805 G166 Glioma, Grade IV Ectoderm >100 >100 >100 4.813 SF188 Glioma, Grade IV Ectoderm 28.24 8.975 >100 3.884 U3017 Glioma, Grade IV Ectoderm >100 14.12 >100 3.921 DIPG007 Glioma, Grade IV Ectoderm >100 >100 >100 4.302 U3013 Glioma, Grade IV Ectoderm >100 >100 >100 4.138 CB152 Glioma, Grade IV Ectoderm >100 >100 >100 6.496

(73) As shown in Table 2, after treatment with the relevant compound, 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 broad range of Gliomas. These results were benchmarked to both temozolomide, the current frontline treatment for Grade IV, Gliomas, and 5-flurouracil, a broad use anti-cancer drug. Temozolomide effectively killed 5 of 12 Glioma, Grade IV cell lines and 5-flurouracil killed 11 of 12 Glioma, Grade IV cell lines. Thus, the data demonstrates that 5-formyl-2-deoxycytidine is as effective as the current frontline treatment for Grade IV, Gliomas and that 5-hydroxymethyl-2-deoxycytidine is more effective than the current frontline treatment for Grade IV, Gliomas.

(74) 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 tumours. Alternatively, Table 2 indicates that temozolomide in combination with 5-formyl-2-deoxycytidine and/or 5-hydroxymethyl-2-deoxycytidine would be an optimal treatment.

Example 2: Cytotoxicity to Normal Cells

(75) As demonstrated in Example 1, 5-formyl-2-deoxycytidine and 5-hydroxymethyl-2-deoxycytidine are useful therapeutics for the treatment of cancers, particularly Grade IV gliomas, and particularly those that are resistant to temozolomide treatment.

(76) The present inventors further investigated the extent to which these compounds kill normal human cells; low cytotoxicity against normal human cells is advantageous property for anti-cancer agents. The cytotoxicity of these compounds in various normal human cell lines was therefore investigated (Table 3). Cells were grown and the survival assay was performed as described above (Materials and Methods).

(77) TABLE-US-00004 TABLE 3 Evaluation of cytotoxicity of 5-formyl-2-deoxycytidine (2d5fC) and 5-hydroxymethyl- 2-deoxycytidine (2d5hmC) to normal (non-cancerous) human cell lines,), and as compared to Temozolomide and 5-flurouracil (5fU). IC.sub.50 is the concentration at which half of the cells are killed by the relevant compound. Cell Germ IC.sub.50 (?M) Line Disease Layer 2d5fC 2d5hmC Temozolomide 5fU HEK293FT Normal Mesoderm, Kidney >25.00 >25.00 >25.00 Arpe19 Normal Ectoderm, ReMna >25.00 >25.00 >25.00 >25.00 HaCat Normal Mesoderm, 0.1024 >25.00 >25.00 0.5228 KeraMnocyte MRC5 Normal Endoderm, Lung >25.00 >25.00 >25.00 >25.00 Buffy A Normal Mesoderm, Bone >25.00 >25.00 n.d. >25.00 Marrow Buffy C Normal Mesoderm, Bone >25.00 >25.00 n.d. >25.00 Marrow Buffy M Normal Mesoderm, Bone >25.00 >25.00 n.d. >25.00 Marrow

(78) As shown in Table 3, 5-hydroxymethyl-2-dexycytidine was not cytotoxic to any of the normal cell lines evaluated. 5-formyl-2-deoxycytidine was cytotoxic only to one normal human cell line (HaCat, Keratinocytes). These results indicate that these compounds are not only effective cancer chemotherapeutics but also can be well tolerated by humans.

Example 3: Maximum Tolerated Dose

(79) Since 5-hydroxymethyl-2-dexycytidine and 5-formyl-2-deoxycytidine have limited effects on normal cells, the present inventors considered that the compounds could be given at relatively high doses without causing side effects normally associated with cancer chemotherapy. The maximum tolerated dose (MTD) of these compounds in mice was determined as described above (Materials and Methods). An MTD scheme was developed (FIG. 1A). While the end-point of the study was survival after 72 hours, the presence of acute toxic symptoms (mortality, convulsions, tremors, muscle relaxation, sedation, etc.) and autonomic effects (diarrhea, salivation, lacrimation, vasodilation, piloerection, etc.) in the animals was monitored during the first 30 minutes, and again at 1, 24, 48 and 72 hours. Body weights were recorded pre-dose and at 72 hours.

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

(81) The conversion between a mouse dose and a human dose is a factor of 0.081 (Nair et al., (2016) Basic Clin Pharm. 7(2): 27-31. The data therefore indicates that in humans, the maximum tolerated single dose of the compounds of Formula (I) is at least 162 mg/kg but less than 648 mg/kg.

(82) Cancer chemotherapeutic drugs that can be tolerated after multiple repetitive doses over many days are advantageous. Therefore, the present inventors conducted a repeated maximum tolerated dose evaluation for both 5-formyl-2-deoxycytidine and 5-hydroxymethyl-2-deoxycytidine as described above (Materials and Methods). As they were well tolerated as a single dose in both treatment groups, 300 mg/kg, 1000 mg/kg, and 2000 mg/kg doses were selected for repeated multiple tolerated dose evaluation.

(83) Mice were injected with the indicated compound at the indicated dose intraperitoneally once every three days for a total of 5 doses. While survival was an end-point of this study, the primary end-point of the study was body weight; the presence of acute toxic symptoms (mortality, convulsions, tremors, muscle relaxation, sedation, etc.) and autonomic effects (diarrhea, salivation, lacrimation, vasodilation, piloerection, etc.) in the animals was also monitored during the first 30 minutes, again at 1, 24, 48 and 72 hours. Body weights were recorded pre-dose and at 72 hours.

(84) Body weight in untreated animals was not statistically different from animals treated with either 5-formyl-2-deoxycytidine or 5-hydroxymethyl-2-deoxycytidine at any dose evaluated (FIG. 1B). These results indicate that these compounds are well tolerated at the indicated doses over extended periods of time.

Example 4: In Vivo Cytotoxicity

(85) 5-formyl-2deoxycytidine and 5-hydroxymethyl-2-deoxycytidine were evaluated in a Glioblastoma Multiforme mouse xenograft model as described above (Materials and Methods). U87-MG cells were injected subcutaneously into the flank of nude mice. Tumours were allowed to form as described above (Materials and Methods). After the tumours reached between 129 mm.sup.3 and 131 mm.sup.3 animals were divided into 4 groupstwo 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 one 2000 mg/kg dose of either 5-formyl-2-deoxycytidine or 5-hydroxymethyl-2-deoxycytidine every three days for a total of 5 doses. The negative control group was treated identically to the treatment group except the IP injection contained vehicle and no compound. The positive control group was treated with 5 daily doses of 40 mg/kg temozolomide.

(86) The tumour volume (FIG. 1C), body weight (FIG. 1D), mortality, and signs of overt toxicity were monitored and recorded twice weekly for 29 days. Tumour volume (mm.sup.3) was estimated according to the ellipsoid formula as: length?(width).sup.2?0.5. Percent tumour growth inhibition (% TGI) was determined using the following formula: % TGI=(1?[(Tn)/(Cn)])?100, where Tn=mean tumour volume of treated group on day n, and Cn=mean tumour volume of control group on day n. A % T/C value?42% or a percent TGI value?58% compared to that of the negative control group was considered significant anti-tumour activity. Two-way ANOVA? followed by Bonferroni test was also used to ascertain the statistically significant difference compared to the negative control group during the study; Day 1 through to Day 29 (*p<0.05).

(87) FIG. 1C indicates that treatment with 5-formyl-2-deoxycytidine or 5-hydroxymethyl-2-deoxycytidine resulted in marked decrease in tumour volume, comparable to that achieved with Temozolomide, over all time points.

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

(89) One mouse in the control group died during this experiment the tumour volume of this mouse was not reported on day 29; however, tumour volumes of this mouse are included for the prior days.

(90) Together, these surprising results demonstrate that 5-formyl-2-deoxycytidine and 5-hydroxymethyl-2-deoxycytidine efficiently kill Glioblastoma Multiforme cells (Glioma, Grade IV) and are not strongly cytotoxic to normal cells. Furthermore, these compounds are well tolerated in mice, and achieve marked reduction in tumour volume with minimal side effects in mice. The results demonstrate that the compounds of the invention can be used to treat human cancers, and they can be used at high doses to kill tumour cells without killing non-cancerous cells. Thus, there is a wide therapeutic window for the use of these compounds as anti-cancer drugs.

Example 5: Cytotoxic Vs. Cytostatic Effect

(91) The above cell line assays measure metabolic activity, and as such do not distinguish between inhibition of cell division and cell death. It was therefore evaluated whether 5-formyl-2deoxycytidine and 5-hydroxymethyl-2-deoxycytidine were killing sensitive cells or causing them to be delayed in the cell cycle. SF-188 grade IV glioma cells were treated either with DMSO, temozolomide, 5-formyl-2-deoxycytidine, or 5-hydroxymethyl-2-deoxycytidine at the indicated concentration. Cells were harvested and stained with Annexin V (detects apoptotic cells by its ability to bind to phosphatidylserine) and 7AAD (a DNA stain which does not readily pass through intact cell membranes; cells with compromised membranes will therefore be selectively stained) and analyzed by flow cytometery as described above (Materials and Methods). SF-188 cells treated with DMSO or temozolomide yielded similar cytometric profiles with a slight increase in the amount of dead cells in the temozolomide treated control. A majority of SF-188 cells treated with 5-formyl-2-deoxycytidine or 5-hydroxymethyl-2-deoxycytidine were dead according to the flow cytometry profile (FIG. 2A). It is clear that SF188 cells exposed to 5-formyl-2-deoxycytidine or 5-hydroxymethyl-2-deoxycytidine are dead and not arrested at a cell cycle checkpoint.

Example 6: Requirement for 2-Deoxy Sugar Derivatives

(92) 5-flurouracil, a widely used cancer chemotherapeutic drug, has multiple variants that are cytotoxic; these variants include the nucleoside (5-flurouridine and 5-fluro-2-deoxyuridine) and nucleobase 5-flurouracil. The present inventors evaluated whether the ribonucleoside and nucleobase variants of 5-formyl-2-deoxycytidine and 5-hydroxymethyl-2-deoxycytidine would also be cytotoxic.

(93) 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 survival assay as described above. Surprisingly, and in contrast to 5-fluorouridine and 5-fluorouracil, neither 5-formylcytidine nor 5-formylcytosine was cytotoxic to HeLa cells (FIG. 2B).

(94) Table I shows that 5-hydroxymethyl-2-deoxycytidine is cytotoxic to U87-MG cells. Cytotoxicity of the 5-hydroxymethylcytidine and 5-hydroxymethylcytosine in U87-MG cells was evaluated in a survival assay as described above. Surprisingly, and in contrast to 5-fluorouridine and 5-fluorouracil, 5-hydroxymethylcytidine and 5-hydroxymethylcytosine were shown not to be cytotoxic to U87-MG cells (FIG. 2C).

(95) These results indicate that the 2-deoxyribose sugar is necessary for cytotoxicity of the compounds of the present invention. In turn, this result indicates, surprisingly, that 5-formyl-2-deoxycytidine and 5-hydroxymethyl-2-deoxycytidine exploit a fundamentally different cellular pathway than fluropyrimidines.

Example 7: Non-Effect of Cytidine Deaminase

(96) Mutated nucleoside and nucleotide analogues are often removed from the nucleotide pool by cytidine deaminase (CDA). CDA inactivates gemcitabine and cytosine arabinosidetwo common nucleotide analogue anti-cancer agents. Anti-cancer agents that are not inactivated by CDA would be desirable. Since 5-formyl-2-deoxycytidine and 5-hydroxymethyl-2-deoxycytidine are cytidine derivatives, the present inventors hypothesized that cells expressing CDA would be resistant to treatment with 5-formyl-2-deoxycytidine and 5-hydroxymethyl-2-deoxycytidine.

(97) Surprisingly, however, there was in fact no correlation observed between CDA expression and resistance or sensitivity to either 5-formyl-2-deoxycytidine or 5-hydroxymethyl-2-deoxycytidine, as shown in Table 4A. The IC.sub.50 data in Table 4 is identical to that in Table 1. CDA expression levels in the cell lines specified 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. If more than one value was reported the lowest value was used.

(98) TABLE-US-00005 TABLE 4A No correlation between level of CDA expression and sensitivity to treatment with 5-formyl-2-deoxycytidine or 5-hydroxymethyl-2-deoxycytidine. CDA Cell Germ expression IC.sub.50 (?M) Line Disease Layer (TPM) 2d5fC 2d5hmC U87-MG Glioma, Grade IV Ectoderm 6 0.3340 3.027 HCT-116 Colon Carcinoma Endoderm 24 > >25.00 A549 Lung Carcinoma Endoderm 50 > >25.00 22Rv1 Prostate Carcinoma Endoderm 1 >25.00 >25.00 NCI-N87 Gastric Carcinoma Endoderm 46 0.8057 >25.00 MIA Pancreatic Carcinoma Endoderm 3 1.143 >25.00 A-498 Kidney Carcinoma Mesoderm 29 > >25.00 U937 Lymophoma Mesoderm 146 0.9536 >25.00 A375 Malignant Melanoma Mesoderm 0 3.188 >25.00 HL-60 Acute Promyelocytic Leukemia Mesoderm 10 6.262 >25.00 SK-OV-3 Ovarian Adenocarcinoma Mesoderm 295 9.690 >25.00 MCF-7 Epithelial Adenocarcinoma Mesoderm 0 0.8657 >25.00 U2OS Bone Osteoscarcoma Mesoderm 13.0 8.69 >25.00 HeLa Cervical Adenocarcinoma Mesoderm 42 0.76 >25.00 HAP1 Chronic Myelogenous Mesoderm N.A. >25.00 6.53 Leukemia (CML) V79 Chinese Hamster Ovary Mesoderm N.A. >25.00 >25.00 H1437 Lung (metastatic non-small Endoderm 49 5.65 n.d. cell adenocarcinoma) H1573 Lung (metastatic Endoderm 67 7.72 n.d. adenocarcinoma) MDA-MB- breast adenocarcinoma, Endoderm 153.0 231 HOP-92 non-small cell lung carcinoma Endoderm 321.0 Capan-2 pancreatic ductal Endoderm 248.0 adenocarcinoma

(99) A linear correlation between cytotoxicity and drug exposure showed a poor fit (Linear Model for 2d5fc, R.sub.z=0.00173; Linear Model for 2d5hmC, R.sub.z=0.02262). The data, taken together with the data from EMBL, suggested that CDA expression is not relevant to 5-formyl-2-deoxycytidine or 5-hydroxymethyl-2-deoxycytidine sensitivity or resistance.

(100) Additionally, the CDA expression level (TPM) of various glioma cell lines was determined, and is shown in Table 4B. CDA expression levels in the cell lines specified were identified using the Cancer Cell Line Encyclopedia (https://portals.broadinstitute.org/ccle), which is described in Barretina J et al. (2012) The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug s8 CDA expression is reported as RNA transcripts per million (TPM), as described in Wagner et al., (2012) Theory Biosci 131(4):281-285. If more than one value was reported the highest value was used.

(101) TABLE-US-00006 TABLE 4B Level of CDA expression in various glioma cell lines. Glioma Cell Line CDA expression (TPM) 42-MG-BA 1.0 8-MG-BA 3.0 A172 2.0 AM-38 7.0 CAS-1 0.2 DBTRG-05MG 0.7 DK-MG 0.1 GAMG 41.0 GB-1 0.5 GMS-10 24.0 GOS-3 0.5 KALS-1 24.0 KNS-42 0.4 KNS-60 30.0 KNS-81 0.6 KS-1 7.0 LN-18 11.0 LN-229 0.3 M059K 8.0 SF-295 3.0 SF126 16.0 SNB75 0.6 SNU-1105 7.0 SNU-201 0.3 SNU-466 0.1 SNU-489 3.0 SNU-626 0.2 T98G 3.0 U-87 MG 6.0 YH-13 4.0 YKG1 0.2

Example 8: Non-Mutagenic Effect

(102) A current frontline treatment for Glibolastoma MultiformeTemozolomideis potent mutagen. In fact, temozolomide exerts its cancer chemotherapeutic activity is by mutating tumour cell so severely that the tumour cells are killed. Temozolomide works by alkalyting DNA causing mutations. The expression of MGMTa protein responsible for removing alkalyated DNA damagemakes glioblastoma cells almost completely resistant to the cytotoxic effects of Temozolomide.

(103) Therefore, the present inventors investigated whether 5-formyl-2-deoxycytidine and 5-hydroxymethyl-2-deoxycytidine had a similar mechanism of action. The mutagenicity of these compounds was evaluated in an Hypoxanthine-guanine phosphoribosyltransferase (HPRT) assay as described above (Materials and Methods). As shown in the HPRT assay neither 5-hydroxymethyl-2-deoxycytidine nor 5-formyl-2-deoxycytidine were genotoxic to mammalian cells at any concentration evaluated (FIG. 3). These results indicate that the 5-hydroxymethyl-2-deoxycytidine and 5-formyl-2-deoxycytidine are not mutagens, i.e. that their cytotoxic effects are not due to mutagenic activity.

Example 9: Cytotoxicity of Other Compounds Vs. HeLa Cells

(104) HeLa cells were treated with either 5-formyl-2-deoxycytidine, 5-formylcytidine or 5-chloro-2-deoxycytidine. After three days of treatment with these compounds at a concentration of 100, 25, 6.25, 1.56, 0.39, 0.1, 0.02, or 0.006 ?M, cell survival was assessed by MTT assay as described above (Materials and Methods).

(105) As shown in FIG. 4. A markedly greater cytotoxic effect is observed following treatment with 5-formyl-2-deoxycytidine than following treatment with either 5-formylcytidine or 5-chloro-2-deoxycytidine.

Example 10: Cytotoxicity of Other Compounds Vs. Glioma Cells

(106) U87-MG cells (Glioma, Grade IV) were treated with either 5-formyl-2-deoxycytidine, 5-hydroxymethyl-2-deoxycytidine, 5-carboxy-2-deoxycytidine, Temozolomide, 5-flurouracil, 5-bromo-2-deoxycytidine, 5-iodo-2-deoxycytidineor 5-chloro-2-deoxycytidine at a concentration of 100, 25, 6.25, 1.56, 0.39, 0.1, 0.02, or 0.006 ?M. After three days of treatment with these compounds, cell survival was assessed by MTT assay as described above (Materials and Methods).

(107) Cell Culture

(108) U87-MG cell lines were grown in DMEM (Sigma, Cat.Nr. D6429) supplemented with 10% Fetal Bovine Serum. All cells were maintained in a 5% CO2, humidified, water-jacketed incubator at 37? C. Cells were passed between 70 and 90% confluence.

(109) Survival Assay

(110) In a 96-well plate, 4000 cells in 100 ?l of corresponding medium were seeded. The following day, drugs, diluted in DMSO, were added in triplicate at a concentration of 100, 25, 6.25, 1.56, 0.39, 0.1, 0.02, or 0.006 ?M. Cells were incubated with the drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, Cat. Nr. 30-1010K). Cell survival was normalized to the survival of cells treated with DMSO only. Experiment was performed three times, data represent mean of nine wells?SEM.

(111) As shown in Table 5, both 5-formyl-2-deoxycytidine and 5-hydroxymethyl-2-deoxycytidine performed surprisingly better in terms of cytotoxicity vs. U87-MG cells than the other compounds tested, including 5-bromo-2-deoxycytidine, 5-iodo-2-deoxycytidine and 5-chloro-2-deoxycytidine.

(112) TABLE-US-00007 TABLE 5 Evaluation of cytotoxicity of various compounds to U87-MG cells. IC.sub.50 is the concentration at which half of the cells are killed by the relevant compound. Cell Line: U87-MG Compound Tested IC.sub.50 (?M) 5-formyl-2-deoxycytidine 0.3340 5-hydroxymethyl-2-deoxycytidine 3.027 5-carboxy-2-deoxycytidine >25.00 Temozolomide >25.00 5-flurouracil >25.00 5-bromo-2-deoxycytidine >25.00 5-chloro-2-deoxycytidine >25.00 5-iodo-2-deoxycytidine >25.00

(113) Additionally, 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-lodo-2deoxycytidine (5IdC), and Thymidine. The results are shown in FIG. 5. d5hmC and d5fC are markedly more cytotoxic than 5CldC, 5BrdC, dldC, and thymidine to glioma cells.

(114) In a 96-well plate, 4000 cells in 100 ?l of corresponding medium were seeded. The following day, drugs, diluted in DMSO, were added in triplicate at a concentration of 100, 25, 6.25, 1.56, 0.39, 0.1, 0.02, or 0.006 ?M. Cells were incubated with the drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, Cat. Nr. 30-1010K). Cell survival was normalized to the survival of cells treated with DMSO only. Experiment was performed three times, data represent mean?SD.

Example 11: Effect not Mediated Via Thymidine Synthase

(115) Cell Culture

(116) U87-MG cell lines were grown in DMEM (Sigma, Cat.Nr. D6429) supplemented with 10% Fetal Bovine Serum. All cells were maintained in a 5% CO2, humidified, water-jacketed incubator at 37? C. Cells were passed between 70 and 90% confluence.

(117) Survival Assay

(118) In a 96-well plate, 4000 cells in 100 ?l of corresponding medium were seeded. The following day, drugs, diluted in DMSO, were added in 8 concentrations in triplicate. Drugs were added as a 4-fold dilution series starting from 100 ?M. Cells were incubated with the drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, Cat. Nr. 30-1010K). Cell survival was normalized to the survival of cells treated with DMSO only. Experiment was performed three times, data represent mean of three wells?SD.

(119) The results are shown in FIG. 6. Cytotoxicity by 5-formyl-2-deoxycytidine and 5-hydroxymethyl-2-deoxycytidine is not rescued by the addition of thymidine in U87-MG cells. This result indicates that 5-formyl-2-deoxycytidine and 5-hydroxymethyl-2-deoxycytidine do not act by inhibiting thymidine synthase.

Example 12: Combined Therapy with Temozolomide

(120) Cell Culture

(121) Glioblastoma multiforme cell lines (U87-MG) were grown in DMEM (Sigma, Cat.Nr. D6429) supplemented with 10% Fetal Bovine Serum. All cells were maintained in a 5% CO2, humidified, water-jacketed incubator at 37? C. Cells were passed between 70 and 90% confluence.

(122) Survival Assay

(123) In a 96-well plate, 4000 cells in 100 ?l of corresponding medium were seeded. The following day, drugs, diluted in DMSO, were added in 8 concentrations in triplicate. Drugs were added as a 4-fold dilution series starting from 100 ?M. Cells were incubated with the drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, Cat. Nr. 30-1010K). Cell survival was normalized to the survival of cells treated with DMSO only. Experiment was performed three times, data represent mean of these experiments.

(124) As shown in FIG. 7, a human glioblastoma multiforme cell line that is resistant to temozolomide was treated with temozolomide alone, 5-formyl-2-deoxycytidine (d5fC) or a combination of d5fC and temozolomide. The combination of temozolomide and d5fC is more effective at treating human glioblastoma multiforme than either chemical alone.

(125) As shown in FIG. 8, a human glioblastoma multiforme cell line that is resistant to temozolomide was treated with temozolomide alone, 5-hydroxymethyl-2-deoxycytidine (d5hmC) or a combination of d5hmC and temozolomide. The combination of temozolomide and d5hmC is more effective at treating human glioblastoma multiforme than either chemical alone.

(126) Thus, 5-hydroxymethyl-2-deoxycytidine and 5-formyl-2-deoxycytidine act synergistically with temozolomide.

Example 13: Uridine Analogues

(127) The cytotoxic effects of 5-methoxymethyl-2-deoxyuridine and 5-acetoxymethyl-2-deoxyuridine were evaluated.

(128) Cell Culture

(129) U87-MG cell lines were grown in DMEM (Sigma, Cat.Nr. D6429) supplemented with 10% Fetal Bovine Serum. All cells were maintained in a 5% CO2, humidified, water-jacketed incubator at 37? C. Cells were passed between 70 and 90% confluence.

(130) Survival Assay

(131) In a 96-well plate, 4000 cells in 100 ?l of corresponding medium were seeded. The following day, drugs, diluted in DMSO, were added in 8 concentrations in triplicate. Drugs were added as a 4-fold dilution series starting from 100 ?M. Cells were incubated with the drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, Cat. Nr. 30-1010K). Cell survival was normalized to the survival of cells treated with DMSO only. Experiment was performed three times, data represent mean of nine wells?SEM.

(132) As shown in FIG. 9, the U87-MG, glioblastoma multiforme, cell line is unable to survive a 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 anti-cancer agents, particularly against glioblastoma multiforme.

Example 14: Cytotoxicity of 5-Formyl-2-Deoxycytidine-5-Triphosphate (HeLa)

(133) Cell Culture

(134) HeLa cell lines were grown in DMEM (Sigma, Cat.Nr. D6429) supplemented with 10% Fetal Bovine Serum. All cells were maintained in a 5% CO.sub.2, humidified, water-jacketed incubator at 37? C. Cells were passed between 70 and 90% confluence.

(135) Survival Assay

(136) In a 96-well plate, 4000 cells in 100 ?l of corresponding medium were seeded. The following day, drugs, diluted in DMSO, were added in 8 concentrations in triplicate. Drugs were added as a 4-fold dilution series starting from 100 ?M. Cells were incubated with the drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, Cat. Nr. 30-1010K). Cell survival was normalized to the survival of cells treated with DMSO only. Experiment was performed three times, data represent mean of nine wells?SEM.

(137) As shown in FIG. 10A, HeLa cervical carcinoma cells are unable to survive a treatment with increasing concentrations of 5-formyl-2-deoxycytidine-5-triphosphate. This demonstrates 5-formyl-2-deoxycytidine-5-triphosphate's utility in the treatment of human cancers, for instance cervical carcinoma.

Example 15: Cytotoxicity of 5-Formyl-2-Deoxycytidine-5-Triphosphate and 5-Hydroxymethyl-2-Deoxycytidine-5-Triphosphate (Glioma)

(138) U87-MG cells (Glioma, Grade IV) were treated with either 5-formyl-2-deoxycytidine-5-triphosphate or 5-hydroxymethyl-2-deoxycytidine-5-triphosphate at a concentration of 100, 25, 6.25, 1.56, 0.39, 0.1, 0.02, or 0.006 ?M. After three days of treatment with these compounds, cell survival was assessed by MTT assay as described above (Materials and Methods).

(139) Cell Culture

(140) U87-MG cell lines were grown in DMEM (Sigma, Cat.Nr. D6429) supplemented with 10% Fetal Bovine Serum. All cells were maintained in a 5% CO2, humidified, water-jacketed incubator at 37? C. Cells were passed between 70 and 90% confluence.

(141) Survival Assay

(142) In a 96-well plate, 4000 cells in 100 ?l of corresponding medium were seeded. The following day, drugs, diluted in DMSO, were added in triplicate at a concentration of 100, 25, 6.25, 1.56, 0.39, 0.1, 0.02, or 0.006 ?M. Cells were incubated with the drugs for 72 hours. Cell proliferation was assessed by MTT assay according to the manufacturer's protocol (ATCC, Cat. Nr. 30-1010K). Cell survival was normalized to the survival of cells treated with DMSO only. Experiment was performed three times, data represent mean of at least 3 wells?SD.

(143) 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.

Example 16: CDA Expression

(144) The linear and logarithm base 2 transformed CDA expression level of various glioma cell lines was determined. CDA expression levels in the cell lines specified were identified using the GENEVESTIGATOR? database (https://genevestigator.com/gv/), by searching for CDA, organism: homosapiens, platform: Affymetrix Human Genome U133 2.0 Array. The results are shown in FIGS. 11 to 13 and Table 6.

(145) FIG. 11 shows the CDA normalized Log 2 CDA expression levels determined for various cancers. FIG. 12 shows the linear CDA expression levels determined for various cancers. FIGS. 13A and 13B show the CDA normalized Log 2 expression levels in various human brain tumours. FIGS. 14A and 14B show the linear CDA expression levels in various human brain tumours.

(146) Table 6 below shows the CDA expression levels of various cell lines and the IC.sub.50 values for 2d5hmC and/or 2d5fc in those cell lines. IC.sub.50 values were obtained as described above (Materials and Methods and Example 1).

(147) TABLE-US-00008 TABLE 6 CDA expression levels (Log2 normalized values) and 2d5hmC and/or 2d5fc IC.sub.50 values in various cell lines. CDA CDA Expression IC50 IC50 CDA Mean Expression Median Cell Line 2d5hmC 2d5fC Log2 SD Log2 Log2 U87-MG 3.027 0.334 9.84 0.42 9.94 HCT-116 25 25 11.11 0.62 10.93 A549 25 25 12 1.01 11.93 22Rv1 25 25 8.92 0.62 9.05 NCI-N87 25 0.8057 11.6 0.56 11.37 MIA PaCa-2 25 1.143 9.68 0.49 9.79 A-498 25 25 10.52 1.06 10.32 U937 25 0.9536 13.82 0.45 13.89 A375 25 3.188 9.36 0.62 9.56 HL-60 25 6.262 10.36 0.96 10.12 SK-OV-3 25 9.69 13.35 1.59 14.07 MCF-7 25 0.8657 9.26 0.39 9.2 U2OS 25 8.69 10.14 10.14 HeLa 25 0.76 13.15 0.92 13.33 HAP1 6.53 25 None HEK293 25 25 9.59 0.4 9.53 ARPE19 25 25 9.99 0.7 9.94 MRC5 25 25 9.87 0.29 9.82 H1437 5.65 13.02 1.12 12.44 H1573 7.72 13.63 0.18 13.67
Table 7 below shows the linear CDA expression levels of various cell lines and the IC.sub.50 values for 2d5hmC and/or 2d5fc in those cell lines. IC.sub.50 values were obtained as described above (Materials and Methods and Example 1).

(148) TABLE-US-00009 TABLE 7 Linear CDA expression levels and 2d5hmC and/or 2d5fc IC.sub.50 values in various cell lines. CDA CDA IC50 IC50 Linear CDA Linear Cell Line 2d5hmC 2d5fC Mean Linear SD Median U87-MG 3.027 0.334 951.65 276.02 989.44 HCT-116 25 25 2481.67 1559.27 1949.23 A549 25 25 5490.76 5549.26 3904.87 22Rv1 25 25 519.39 225.08 527.89 NCI-N87 25 0.8057 3327.03 1464.72 2654.16 MIA PaCa-2 25 1.143 860.01 261.17 881.38 A-498 25 25 1874.54 1443.37 1301.02 U937 25 0.9536 15148.69 4611.68 15231.25 A375 25 3.188 700.89 226.39 753.10 HL-60 25 6.262 1787.26 2129.78 1112.79 SK-OV-3 25 9.69 15500.02 11439.83 17236.82 MCF-7 25 0.8657 635.57 182.06 588.96 U2OS 25 8.69 1130.20 N/A 1130.20 HeLa 25 0.76 10915.38 6549.95 10272.63 HAP1 6.53 25 *** *** *** HEK293 25 25 800.57 225.25 738.45 ARPE19 25 25 1133.42 515.09 981.92 MRC5 25 25 950.82 212.00 901.07 H1437 5.65 11861.95 14192.00 5568.28 H1573 7.72 12779.53 1515.78 13045.67
Together these results demonstrate that many cancers, including all tested cancers of the central nervous system, do not over-express CDA; rather they 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

Example 17: Blood-Brain Barrier Permeability

(149) Blood brain barrier permeability was measured using a Parallel Artificial Membrane Permeability Assay Kit (PAMPA) from BioAssay systems. The manufacturer's instructions were followed to determine permeability.

(150) Manufacturer's instructions: 1. In separate centrifuge tubes, prepare 500 ?L of 500 ?M Test Compound: mix 25 ?L 10 mM Test Compound in DMSO+475 ?L PBS. If using the Permeability Controls, dilute them to 500 ?M as well: mix 25 ?L Permeability Control+475 ?L PBS. 2. In separate tubes, prepare 200 ?M Equilibrium Standards for each test compound and control: mix 80 ?L of 500 ?M Test Compound or Control with 120 ?L PBS. If the compound is able to permeabilize the membrane and fully reach equilibrium, 200 ?M will be the final concentration of solution in the Donor and Acceptor wells. Next, in a separate tube, mix 5 ?L DMSO+245 ?L PBS to prepare the Blank Control. Set aside the Equilibrium Standards and Blank Control for analysis the next day. 3. Add 300 ?L PBS to wells in the acceptor plate. 4. With the donor plate still in its tray, add 5 ?L 4% Lecithin in Dodecane directly to the well membranes of the donor plate. Be careful not to puncture the membranes with the pipette tip. 5. Add 200 ?L of each 500 ?M Test Compound and 500 ?M Permeability Controls to duplicate wells of the donor plate. Note: we recommend running all experimental variables in at least duplicate5. Carefully place the donor plate into the acceptor plate wells. Incubate at RT or 37? C. for 18 hours or the desired incubation time period (e.g. 16-24 hours) 6. Carefully remove donor plate and collect the liquid in acceptor plate wells for analysis. This will be referred to as Acceptor Solution 7. Add 100 ?L of Acceptor Solution and Equilibrium Standards for each Test Compound and Permeability Control. Also add 100 ?L Blank Control to wells of UV plate (Cat # P96UV). 8. Read Absorbance spectrum from 200 nm to 500 nm in 10 nm intervals to determine peak absorbance of test compounds. The Blank Control is to confirm peaks are due to the test compound and not the DMSO in the solution. Peak absorbance for High Permeability, Medium Permeability, and Low Permeability Controls are 280 nm, 270 nm, and 270 nm respectively.

(151) As shown in FIG. 15, 2d5hmC and 2d5fC pass the blood brain barrier.