Thymidine analogs and methods for their use in the treatments of cancer and other diseases

Abstract

Provided herein are methods of treating a cancer, comprising administering to a patient diagnosed with cancer an effective amount of a thymidine analog and a thymidylate synthase inhibitor, wherein the thymidine analog incorporates into DNA.

Description

THYMIDINE ANALOGS AND METHODS FOR THEIR USE IN THE TREATMENTS OF CANCER AND OTHER DISEASESRELATED APPLICATION

[0001] This application claims the benefit of US Provisional Application No. 63 / 653,287, filed May 30, 2024, which is incorporated by reference herein in its entirety for any purpose.FIELD

[0002] The present disclosure relates generally to compounds and methods for their use in the treatments of cancer and other diseases.BACKGROUND

[0003] Antimetabolite chemotherapy drugs have been used in cancer treatment since the 1950s. These synthetic molecules structurally resemble natural cellular metabolites and can disrupt essential biosynthetic pathways or get incorporated into macromolecules, such as DNA and RNA, and inhibit their normal function. Among antimetabolite drugs, pyrimidine analogs have been extensively used due to their ability’ to disrupt nucleotide metabolism, leading to DNA damage and RNA dysregulation. These compounds exert pleiotropic effects, including nucleic acid incorporation and inhibition of nucleotide handling enzymes. However, the extent to which each of these mechanisms contributes to overall therapeutic efficacy of many pyrimidine analogs remains unclear.

[0004] For example, 5-fluorouracil (5-FU) is utilized in the treatment of colorectal, pancreatic, gastric, and breast cancers. However, 5-FU is poorly tolerated, and has a short halflife in vivo.

[0005] Structurally, 5-FU is a heterocyclic aromatic organic compound and an analog of uracil, with a fluorine atom at the carbon 5 position in place of hydrogen. 5-FU is administered intravenously, and enters cells using the same facilitated transport mechanism as uracil. There are several postulated mechanisms by which 5-FU enters the nucleotide pool, yet the precise metabolic routes remain incompletely understood. One of these pathways involves 5-FU?s conversion to 5-fluorodeoxyuridine (5-FUdR, also known as floxuridine) by thymidine phosphorylase (TP, TYMP), followed by phosphory lation to fluorodeoxyuridine monophosphate (FdUMP). FdUMP forms a stable ternary7complex with thymidylate synthase (TS, TYMS), an enzyme that catalyzes the formation of deoxythymidine mono-phosphate (dTMP) from deoxyuridine mono-phosphate (dUMP) using 5’ 10-methylenetetrahydrofolate (5,10-mTHF) as a cofactor via the de novo pathway. dTMP is essential for DNA replication and repair, and itsdepletion therefore causes cytotoxicity. Thus, 5-FU is proposed to inhibit TS, which in turn inhibits dTMP production, resulting in impaired DNA synthesis, DNA damage, and ultimately cell death.

[0006] In addition to TS inhibition, 5-FU containing nucleotides have been speculated to be incorporated into RNA or DNA in place of uridine and thymidine, respectively. One proposed mechanism for RNA incorporation is that the enzyme orotate phosphoribosyl-transferase (OPRT) catalyzes the conversion of 5-FU to 5-FU monophosphate (5FUMP), which is subsequently converted to fluorouridine triphosphate (FUTP). FUTP can be incorporated into RNA in place of uridine triphosphate (UTP). For DNA incorporation, one proposed route involves the reduction of 5-FU to fluorodeoxyuridine diphosphate (FdUDP) by ribonucleotide reductase, followed by phosphorylation to fluorodeoxyuridine triphosphate (FdUTP), which could then be incorporated into DNA in place of dTTP, leading to DNA damage.

[0007] Trifluridine is another pyrimidine analog proposed to inhibit TS. Trifluridine has been used in the treatment of colorectal cancer, stomach adenocarcinoma, and gastroesophageal junction adenocarcinoma. Inside cells, trifluridine is rapidly degraded by TP. Trifluridine is coadministered with tipiracil, which is an inhibitor of TP.

[0008] Accordingly, there is a need for improved therapies involving thymidine analogs and rational combinations involving them.SUMMARY

[0009] Disclosed herein are compounds and methods for their use in the treatments of cancer and other diseases. The disclosed compounds can be used in combination w ith pemetrexed for cancer therapy. The disclosed approach enhances existing treatments for cancers, including nonsmall cell lung cancer, mesothelioma, pancreatic cancer, and colon cancer.

[0010] One embodiment provides a method of treating a cancer in a patient diagnosed with cancer, comprising administering to the patient an effective amount of a thymidine analog and a thymidylate synthase inhibitor, wherein the thymidine analog incorporates into DNA. Also provided is a combination comprising a thymidine analog and a thymidylate synthase inhibitor for use in the treatment of a cancer, wherein the thymidine analog incorporates into DNA. Also provided is use of a combination comprising a thymidine analog and a thymidylate synthase inhibitor in the treatment of a cancer, wherein the thymidine analog incorporates into DNA. Also provided is use of a combination comprising a thymidine analog and a thymidylate synthase inhibitor for the manufacture of a medicament for the treatment of a cancer, wherein the thymidine analog incorporates into DNA.

[0011] Another embodiment provides a thymidine analog for use in the treatment of a cancer in combination with a thymidylate synthase inhibitor, wherein the thy midine analog incorporates into DNA. Also provided is use of a thymidine analog in the treatment of a cancer in combination with a thymidylate synthase inhibitor, wherein the thymidine analog incorporates into DNA. Also provided is use of a thymidine analog in the manufacture of a medicament for treatment of a cancer, wherein the medicament is for use in combination with a thymidylate synthase inhibitor, and the thymidine analog incorporates into DNA.

[0012] Yet another embodiment provides a thymidylate synthase inhibitor for use in the treatment of a cancer in combination with a thymidine analog, wherein the thymidine analog incorporates into DNA. Also provided is use of a thymidylate synthase inhibitor in the treatment of a cancer in combination with a thymidine analog, wherein the thymidine analog incorporates into DNA. Also provided herein is use of a thymidylate synthase inhibitor in the manufacture of a medicament for treatment of a cancer, wherein the medicament is for use in combination with a thymidine analog, wherein the thymidine analog incorporates into DNA.

[0013] Another embodiment provides a pharmaceutical combination comprising a thymidine analog and a thymidylate synthase inhibitor, wherein the thymidine analog incorporates into DNA.

[0014] Also provided herein is a compound of the following structure:0127), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of either of the foregoing.

[0015] Also provided herein is a method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of HK-0127, or a pharmaceutically acceptable salt thereof, or pharmaceutical composition of either of the foregoing. Also provided is HK-0127, or a pharmaceutically acceptable salt thereof, or pharmaceutical composition of either of the foregoing, for use in treating cancer. Also provided is use of HK-0127, or a pharmaceutically acceptable salt thereof, or pharmaceutical composition of either of the foregoing, for treating cancer. Also provided is use of HK-0127, or a pharmaceutically acceptable salt thereof, or pharmaceutical composition of either of the foregoing, for the manufacture of a medicament for treating cancer.

[0016] Further nonlimiting embodiments include:

[0017] Embodiment 1. A thymidine analog, comprising a structure according to:where A is O or S; where R1is Ci-Csalkyl, Cs-Cscycloalkyl, halogen. Ci-Cshaloalkyl, C2-C3haloalkene, - CH2OH, -CH2OCH3, formyl, carboxylic acid, or -C=CR1A; where R1Ais H, halogen, Ci-Csalkyl, Ci-Cshaloalkyl, or Ci-Cshaloalkene, where X is a halogen; where R2and R3are independently H or halogen; where R4is -OH or R5; where R5is -O-P(O)(OH)2, where each H is independently optionally substituted with one or more alkyl or arylalkyl groups, and / or each -OH is independently optionally substituted with -NHR6; where R6is H, linear or branched Ci-Cioalkyl, Ci-Cioalkene, Cr-Cnalkylaryl, C?-Ci2alkenearyl, C7-Ci2heteroalkylaryl. or C7-Ci2heteroalkenearyl, where, when R4is OH, A is O, and R1is -C=CH, then at least one of R2and / or R3is a halogen, and where, when R4is OH and R1is Br, F, or CF3, then A is S and / or at least one of R2and / or R3is halogen.

[0018] Embodiment 2. A pharmaceutical composition, comprising: a thymidine analog of claim 1, and a pharmaceutically acceptable carrier.

[0019] Embodiment 3. The thymidine analog of claim 1 for use in treating cancer.

[0020] Embodiment 4. A method for treating cancer, comprising: providing an effective dose of a thymidine analog whose activity depends at least partly on DNA incorporation to a patient diagnosed with a cancer.

[0021] Embodiment 5. The method of Embodiment 4, wherein the thymidine analog is a thymidine analog of claim 1.

[0022] Embodiment 6. The method of Embodiment 4, wherein the cancer is a non-small cell lung cancer and / or a colon cancer.

[0023] Embodiment 7. The method of Embodiment 4, further comprising administering or coadministrating a mesothelioma or non-small cell lung cancer or colon cancer medication to the patient.

[0024] Embodiment 8. The method of Embodiment 7, wherein the mesothelioma or non- small cell lung cancer or colon cancer medication is pemetrexed.

[0025] Embodiment 9. The method of Embodiment 8, wherein pemetrexed inhibits thymidylate synthase, one or more tumor thymidine deoxyribonucleotide levels are lowered, and incorporate and efficacy of thymidine analog is enhanced.

[0026] Embodiment 10. The method of Embodiment 4, further comprising administering, or co-administering, a compound known to induce DNA damage repair machinery as its mode of activity.

[0027] Embodiment 11. A method for treating cancers, comprising administering or coadministering any compound disclosed herein and pemetrexed to a patient diagnosed with cancer.

[0028] The disclosed approach enhances existing treatments for non-small cell lung cancer and colon cancer. The disclosed data show that current thymidine analogs used for cancer treatment (5-FU and its formulations as well as trifluridine) work as thymidylate synthase inhibitors. The strategy' disclosed herein utilizes thymidine analogs whose activity depends at least partly on DNA incorporation. When pemetrexed inhibits thymidylate synthase, tumor thymidine levels are lowered and the incorporation and efficacy of these DNA incorporators are enhanced.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 shows intracellular metabolism of 5-FU.

[0030] FIG. 2A shows relative cell growth of HCT116 wild type (WT) cells in the presence ofHK-0141.

[0031] FIG. 2B shows relative cell growth of HCT116 sgTKl cells in the presence of HK-0141.

[0032] FIG. 2C shows relative cell growth of HCT116 WT cells in the presence of HK-0142.

[0033] FIG. 2D shows relative cell growth of HCT116 sgTKl cells in the presence of HK-0142.DETAILED DESCRIPTION

[0034] The features and other details of the disclosure will now be more particularly described. Certain terms employed in the specification, examples, and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and as understood by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.Definitions

[0035] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms '‘a,” “an”, and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and / or” unless stated otherwise.Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0036] Unless the context requires otherwise, throughout the present specification and claims, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.”

[0037] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size, or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the terms “about” and “approximately” mean ± 20%, ± 10%, ± 5%, or ± 1% of the indicated range, value, or structure, unless otherwise indicated.

[0038] “Alkyl” refers to an unbranched or branched, saturated hydrocarbon chain radical having from one to fifteen carbon atoms, e.g., from one to ten, from one to six. or from one to three carbon atoms. “(Ci-Cio)alkyl” refers to a hydrocarbon chain radical having from one to ten carbon atoms. Nonlimiting examples of alkyl include methyl, ethyl, / / -propyl. 1 -methylethyl (iso- propyl), n-butyl, n-pentyl, 1,1 -dimethylethyl ( / -butyl). 3-methylhexyl, 2-methylhexyl and the like.

[0039] “Alkenyl” and “alkene” refer to an unbranched or branched, unsaturated hydrocarbon chain radical, having one or more carbon-carbon double bonds and the indicated number of carbon atoms. Thus, for example, “(C2-Cio)alkenyl” refers to an unsaturated hydrocarbon chainradical, which contains one or more carbon-carbon double bonds and has from two to ten carbon atoms. Nonlimiting examples of alkenyl include ethenyl, prop-l-enyl, but-l-enyl, pent-l-enyl, penta-1, 4-dienyl, and the like.

[0040] “Aryl” refers to a carbocyclic, aromatic ring system radical comprising 6 to 18 carbon ring atoms (z.e., Ce-Cis aryl), preferably having 6 to 10 carbon ring atoms (z.e., Ce-Cio aryl) or 6 carbon ring atoms (z.e., Ce aryl). For purposes of embodiments of this disclosure, aryl is a monocyclic or polycyclic (e.g., bicyclic) ring system, which may include fused ring systems. Aryl includes, but is not limited to, radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene. s-indacene. indane, indene, naphthalene, phenalene, phenanthrene, phenyl, pleiadene, pyrene, and triphenylene. In some embodiments, ary l is phenyl.

[0041] “Alkenylaryl” and “alkenearyl” refer to an alkenyl radical attached to a molecule via a divalent ary l radical, wherein alkenyl and aryl are as described herein.

[0042] “Alkylaryl” refers to an alkyl radical attached to a molecule via a divalent ary l radical, wherein alkyl and aryl are as described herein.

[0043] “Arylalkyl” refers to an ary l radical attached to a molecule via a divalent alkyl radical, wherein aryl and alkyl are as described herein.

[0044] “Carboxylic acid” refers to -CO2H.

[0045] “Cycloalkyl” refers to a saturated, monocyclic or polycyclic, carbocyclic radical having the indicated number of ring carbon atoms. Thus, for example, “(C3-C5)cycloalkyl” refers to a carbocyclic radical having from three to five ring carbon atoms. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0046] “Formy l” refers to -C(=O)H.

[0047] “Halo” or “halogen” refers to bromo, chloro, fluoro, or iodo. In some embodiments, halo is fluoro, chloro, or bromo. In some embodiments, halo is fluoro or chloro. In some embodiments, halo is fluoro.

[0048] “Haloalkyl” refers to an alkyl, as defined above, that is substituted by one or more halo, as defined above. Each halo substituent may be the same or one or more of the halo substituents may be different from another halo substituent. Nonlimiting examples of haloalkyl include trifluoromethyl, difluoromethyl, tri chloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like. “Haloalkeny 1” and “haloalkene” refer to an alkenyl, as defined above, that is substituted by one or more halo, as defined above. Each halo substituent may be the same or one or more of the halo substituents may be different fromanother halo substituent. Nonlimiting examples of haloalkenyl include 1-fluoroethenyl, 1,1- difluoroethenyl, 1,1,2-trifluoropropenyl, 2-bromo- 1,1 -difluoropropenyl, and the like.

[0049] “Heteroalkyl” refers to a stable, unbranched or branched, saturated hydrocarbon chain radical having, wherein the chain atoms consist of carbon and one or more heteroatoms (e.g, selected from nitrogen, oxygen, and sulfur).

[0050] “Heteroalkenyl” and “heteroalkene” refer to a stable, unbranched or branched, unsaturated hydrocarbon chain radical having one or more double bonds, wherein the chain atoms consist of carbon and one or more heteroatoms (e.g., selected from nitrogen, oxygen, and sulfur).

[0051] “Heteroalkenylaryl” and “heteroalkenearyl” refer to a heteroalkenyl radical attached to a molecule via a divalent aryl radical, wherein alkenyl and aryl are as described herein and the heteroalkenylaryl has the indicated number of carbon atoms.

[0052] “Heteroalkylaryl” refers to a heteroalkyl radical attached to a molecule via a divalent aryl radical, wherein heteroalkyl and aryl are as described herein and the heteroalkylaryl has the indicated number of carbon atoms.

[0053] “Substituted,” as used herein, refers to the replacement of an atom (typically, hydrogen) with an indicated substituent group. The term “optionally substituted”, as used herein, means that substitution is optional and, therefore, it is possible for the atom or moiety designated as “optionally substituted” to be unsubstituted or substituted. In some embodiments, an optionally substituted group is unsubstituted. In some embodiments, an optionally substituted group is substituted. Unless otherwise indicated, e.g., as with the terms “substituted” or “optionally substituted,” a group designated herein is unsubstituted.

[0054] When a disclosed compound is depicted by a structure indicating stereochemistry, the stereochemistry indicates absolute configuration of the substituents around the one or more chiral centers.

[0055] “Effective amount” or “effective dose” of a compound or a composition refers to that amount of the compound or the composition that results in an intended result as desired based on the disclosure herein. Effective amounts can be determined by standard pharmaceutical procedures in cell cultures or experimental animals including, without limitation, by determining the EDso (the dose therapeutically effective in 50% of the population) and the LD50 (the dose lethal to 50% of the population). In some embodiments, an effective amount of a compound results in reduction or inhibition of symptoms or a prolongation of survival in a subject (z.e., a human patient). The results may require multiple doses of the compound.

[0056] “Treating” or “treatment” includes inhibiting a disease or arresting its development and / or ameliorating or causing regression of the disease. As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For the purposes of this disclosure, beneficial or desired results include, but are not limited to, one or more of the following: decreasing one or more symptoms resulting from cancer, diminishing the extent of the cancer, stabilizing the cancer (e.g. , preventing or delaying the worsening of the cancer), delaying the occurrence or recurrence of the cancer, delaying or slowing the progression of the cancer, ameliorating the cancer, providing a remission (whether partial or total) of the cancer, decreasing the dose of one or more other medications required to treat the cancer, enhancing the effect of another medication used to treat the cancer, delaying the progression of the cancerr, increasing the quality of life, and / or prolonging survival of a subject. Also encompassed by “treatment” is a reduction of pathological consequence of the disease or disorder.

[0057] As used herein, the terms “subjects )” and “patient(s)” mean any mammal. Examples include, but are not limited to, humans, domestic animals, such as laboratory7animals (e.g., dogs, monkeys, pigs, rats, mice, etc.), household pets (e g., cats, dogs, rabbits, etc.) and livestock (e.g., pigs, cattle, sheep, goats, horses, etc.), and non-domestic animals. In some aspects, a subject or patient is a human.

[0058] The terms “co-administration,” “administered in combination with,” and their grammatical equivalents, as used herein, encompass administration of two or more agents to an animal, including humans, so that both agents and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.

[0059] The phrase “pharmaceutically acceptable” means that the substance or composition the phrase modifies is. within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio.

[0060] As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences. 1977, 66, 1-19, the relevant teachings of which areincorporated herein by reference in their entirety. Pharmaceutically acceptable salts of the compounds described herein include salts derived from suitable inorganic and organic acids, and suitable inorganic and organic bases.

[0061] “Pharmaceutically acceptable carrier” refers to a non-toxic carrier or excipient that does not destroy the pharmacological activity of the agent with which it is formulated and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent. Nonlimiting examples of pharmaceutically acceptable carriers include any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.Compounds

[0062] Provided herein is a compound of Formula (I) or (II):or a pharmaceutically acceptable salt thereof, wherein:A is S or O;R1is Ci-Csalkyl, Cs-Cscycloalkyl, halogen, Ci-Cshaloalkyl, C2-C3haloalkenyl, -CH2OH, -CH2OCH3, -C(O)H, -CO2H, or -C=CR1A;R1Ais H, halogen, Ci-Csalkyl, Ci-C.ihaloalkyl, or Ci-Cshaloalkenyl;R2and R3are independently H or halogen;R4is -OH or R5;R5is -O-P(O)(OH)2, wherein each H is independently optionally substituted with alkyl or aiylalkyl and / or each -OH is independently optionally substituted with -NHR6;R6is H, linear or branched Ci-Cioalkyl, Ci-Cioalkenyl, Cv-Cnalkylaiyl, C?-Ci2alkenylaryl, C7-Ci2heteroalkylaryl, or C7-Ci2heteroalkenylaryl, provided that when R4is OH, A is O, and R1is -C=CH, then at least one of R2and / or R3is a halogen, and when R4is OH and R1is Br, F, or CF3, then A is S and / or at least one of R2and / or R3is halogen.

[0063] In some embodiments, provided is a compound in Table 1, or a pharmaceutically acceptable salt thereof.Table 1

[0064] In some embodiments, provided is a compound of the following structure:pharmaceutically acceptable salt thereof. In some embodiments, provided is a compound of the following structure:pharmaceutically acceptable salt thereof.

[0065] It is understood that in the present description, combinations of substituents and / or variables of the depicted formulae are permissible only if such combinations result in stable compounds.

[0066] Furthermore, all compounds which exist in free base or acid form can be converted to their pharmaceutically acceptable salts by treatment with the appropriate inorganic or organic base or acid by methods known to one skilled in the art. Salts of the compounds of Formula (I) or (II) can be converted to their free base or acid form by standard techniques.

[0067] The novel nucleoside analogs disclosed herein, including prodrugs thereof, can be designed and chemically synthesized using techniques known to those of skill in the art.Pharmaceutical Compositions and Formulations

[0068] Typically, for administration to a subject, a therapeutic agent (e.g. , a thymidine analog, a thymidylate synthase inhibitor) is formulated with one or more pharmaceutically acceptable carriers. The disclosure provides such compositions, including pharmaceutical compositions. Thus, one embodiment is a composition (e.g., pharmaceutical composition) comprising a thymidine analog and a pharmaceutically acceptable carrier. The compositions described herein can be used in the methods described herein, e.g., to supply the indicated compound.

[0069] Pharmaceutical compositions can be formulated in a conventional manner using techniques and excipients known in the art. Proper formulation is dependent upon the route of administration chosen. See, for example, Remington: The Science and Practice of Pharmacy.Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds.. Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems. Seventh Ed. (Lippincott Williams & Wilkins 1999). Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary, transmucosal, transdermal, vaginal, otic, nasal, and topical routes of administration. In addition, by way of example only, parenteral delivery includes intramuscular, subcutaneous, intravenous, and intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections.

[0070] In some embodiments, the concentration of therapeutic agent(s) (c.g. , thymidine analog) provided in the pharmaceutical compositions of the present disclosure is less than 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%. 0.0009%, 0.0008%, 0.0007%, 0.0006%. 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w / w. w / v or v / v; and / or greater than 90%. 80%. 70%. 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 1 %, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50%, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%. 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75%, 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125% , 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%. 0.007%, 0.006%, 0.005%, 0.004%, 0.003%. 0.002%, 0.001%. 0.0009%, 0.0008%, 0.0007%. 0.0006%. 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w / w, w / v, or v / v.

[0071] In some embodiments, the concentration of therapeutic agent(s) (e.g. , thymidine analog) provided in the pharmaceutical compositions ranges from approximately 0.0001% to approximately 50%, approximately 0.001% to approximately 40 %, approximately 0.01% to approximately 30%, approximately 0.02% to approximately 29%, approximately 0.03% to approximately 28%, approximately 0.04% to approximately 27%, approximately 0.05% to approximately 26%, approximately 0.06% to approximately 25%, approximately 0.07% to approximately 24%, approximately 0.08% to approximately 23%, approximately 0.09% toapproximately 22%, approximately 0.1% to approximately 21%, approximately 0.2% to approximately 20%, approximately 0.3% to approximately 19%, approximately 0.4% to approximately 18%, approximately 0.5% to approximately 17%, approximately 0.6% to approximately 16%. approximately 0.7% to approximately 15%, approximately 0.8% to approximately 14%, approximately 0.9% to approximately 12%, or approximately 1% to approximately 10% w / w, w / v or v / v.

[0072] The disclosure also provides combinations (e.g., pharmaceutical combinations) comprising a thymidine analog or a pharmaceutical composition thereof, and a thymidylate synthase inhibitor, or a pharmaceutical composition thereof.Methods of Use

[0073] The disclosed data show that current thymidine analogs used for cancer treatment (5- FU and its formulations or prodrugs including capecitabine as well as trifluridine) work as thymidylate synthase inhibitors. The strategy disclosed herein utilizes thymidine analogs whose activity depends at least partly on DNA incorporation, or that incorporate to a greater extent than 5-FU into DNA, or that show efficacy in combination with pemetrexed or synergize with pemetrexed, 5FU, capecitabine or other cancer therapies. When pemetrexed, 5FU, or capecitabine inhibit thymidylate synthase, tumor deoxythymidine nucleotide levels, such as deoxy thy mi dine triphosphate (dTTP), deoxythymidine diphosphate (dTDP), and deoxythymidine monophosphate (dTMP) are lowered and the incorporation and efficacy of these DNA incorporators are enhanced.

[0074] The novel nucleoside analogs disclosed herein can be used alone or in combination with pemetrexed, 5-FU, capecitabine, or other cancer therapies to treat cancer in a patient with primary or metastatic tumors, including of the lung, mesothelium, colon, rectum, pancreas, prostate, breast, head and neck, stomach, uterus, ovaries, endometrium, bladder, kidney, salivary- glands, and testes. The nucleoside analogs disclosed herein can further be used for hematological malignancies.

[0075] Embodiments of the present disclosure thus provide a method for treating a cancer in a patient diagnosed with cancer, the method comprising administering to the patient an effective amount of a thymidine analog (e.g. , a thymidine analog disclosed herein, such as a compound of Formula (I) or (II). or Table 1, or a pharmaceutically acceptable salt thereof). In some embodiments, the thymidine analog incorporates into DNA. In some embodiments, the thymidine analog incorporates into DNA to a greater extent than 5-FU. In some embodiments, the thymidine analog shows efficacy in combination with the thymidylate synthase inhibitor. In some embodiments, the thymidine analog synergizes with the thymidylate synthase inhibitor.

[0076] Thymidylate synthase inhibition and DNA incorporation can be assessed using assays known in the art and / or described herein, e.g., in the Examples. See, for example, Example Bib. In some embodiments, DNA incorporation of a thymidine analog is as detected (e.g.. measured) by liquid chromatography mass spectrometry (LC-MS) of nucleic acids following extraction of DNA from cells (e.g., cancer cells, such as cancer cells from the patient) contacted with the thymidine analog, and digestion. In some embodiments, DNA incorporation of a thymidine analog is as detected (e.g, measured) by LC-MS of nucleic acids following extraction of DNA from cells (e.g., cancer cells, such as cancer cells from the patient) contacted with the thymidine analog for about 15 minutes, about 2 hours, about 24 hours, about 48 hours, or about 72 hours, and digestion. In some embodiments, the cells are contacted with thymidine analog for about 8 hours. In some embodiments, the cells are contacted with the thymidine analog for about 24 hours.

[0077] In some embodiments, ratio of thymidine analog to deoxycytidine is 0.001 or greater. In some embodiments, ratio of thymidine analog to deoxy cytidine is 0.01 or greater. In some embodiments, ratio of thymidine analog to deoxycytidine is 0. 1 or greater. In some embodiments, ratio of thymidine analog to deoxy cytidine is greater than 0. 1. In some embodiments, ratio of thymidine analog to deoxy cytidine is from 0.001 to 0.01. In some embodiments, ratio of thymidine analog to deoxy cytidine is from 0.01 to 0.1 . In some embodiments, ratio of thymidine analog to deoxy cytidine is from greater than 0.01 to 0. 1. Ratio of thymidine analog to deoxy cytidine can be measured and calculated, for example, using the assay described in Example Bib herein. In some embodiments, ratio of thymidine analog to deoxy cytidine is as calculated from a mass spectrometry signal of thymidine analog from extracted and digested DNA to a mass spectrometry signal of deoxy cytidine from the extracted and digested DNA.

[0078] In some embodiments, DNA incorporation is 0.1% or greater. In some embodiments, DNA incorporation is 1% or greater. In some embodiments. DNA incorporation is 10% or greater. In some embodiments, DNA incorporation is greater than 10%. In some embodiments, DNA incorporation is from 0.1% to 1%. In some embodiments, DNA incorporation is from 1% to 10%. In some embodiments, DNA incorporation is from greater than 1% to 10%. Percent DNA incorporation can be measured and calculated, for example, using the assay described in Example Bib herein. In some embodiments, percent DNA incorporation is as calculated from the ratio of a mass spectrometry signal of thymidine analog from extracted and digested DNA to a mass spectrometry signal of deoxy cytidine from the extracted and digested DNA.

[0079] In some embodiments, the thymidine analog is antiproliferative. Antiproliferative activity can be assessed using assays known in the art and / or described herein, e.g, in the Examples.

[0080] Representative thymidine analogs include the compounds in Table 1. In some embodiments, the thymidine analog is a compound in Table 1, or a pharmaceutically acceptable salt thereof. In some embodiments, the thymidine analog is HK-0127, or a pharmaceutically acceptable salt thereof. In some embodiments, the thymidine analog is HK-0105. or a pharmaceutically acceptable salt thereof. In some embodiments, the thymidine analog is HK- 0141, or a pharmaceutically acceptable salt thereof.

[0081] The thymidine analogs described herein can also be administered in combination with one or more additional therapies. When administered "in combination” with such therapies, the thymidine analog can be administered before, after or concurrently with the other therapy (ies) (e.g., additional therapeutic agent(s)). When administered simultaneously (e.g., concurrently), the thymidine analog and additional therapy can be in separate formulations or the same formulation. Alternatively, the thymidine analog and additional therapy can be administered sequentially, either at approximately the same time or at different times, as separate compositions. When the thymidine analog and the additional therapy (e.g., therapeutic agent) are administered as separate formulations or compositions, the thymidine analog and the additional therapy can be administered by the same route of administration or by different routes of administration. A skilled clinician can determine appropriate timing for administration of each therapy being used in combination (e.g.. timing sufficient to allow an overlap of the pharmaceutical effects of the therapies). Typically, a combination therapy will provide beneficial effects of the drug combination in treating the diseases, conditions or disorders described herein.

[0082] In some embodiments, the method comprises administering to the patient an effective amount of a thymidine analog and a thymidylate synthase inhibitor. In some embodiments, the thymidylate synthase inhibitor is pemetrexed, 5-FU, capecitabine, or trifluridine. In some embodiments, the thymidylate synthase inhibitor is pemetrexed, 5-FU, or capecitabine. In some embodiments, the thymidylate synthase inhibitor is pemetrexed. It will be appreciated that in methods wherein a thymidine analog and a thymidylate synthase inhibitor are administered to a patient, the thymidine analog and thymidylate synthase inhibitor are different from one another. Thus, for example, when trifluridine, which is a thymidine analog and also has activity as a thymidylate synthase inhibitor, is administered to a patient in combination with a thymidine analog, the thymidine analog is not trifluridine.

[0083] The thymidine analogs described herein show promising activity in a variety of cancers, including primary and metastatic tumors and hematological malignancies. In some embodiments, the cancer is a primary or metastatic tumor. In some embodiments, the cancer is a primary tumor. In some embodiments, the cancer is a metastatic tumor. In some embodiments, the cancer is a hematological malignancy.

[0084] Specific examples of cancer treatable according to the methods described herein include Acute Lymphoblastic Leukemia (ALL); Acute Myeloid Leukemia (AML); Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS-Related Cancer (e.g., Kaposi Sarcoma, AIDS-Related Lymphoma, Primary CNS Lymphoma); Anal Cancer; Appendix Cancer; Astrocytomas, Childhood; Atypical Teratoid / Rhabdoid Tumor, Childhood, Central Nervous System; Basal Cell Carcinoma of the Skin; Bile Duct Cancer; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer (including Ewing Sarcoma, Osteosarcoma and Malignant Fibrous Histiocytoma); Brain Tumors / Cancer; Breast Cancer; Burkitt Lymphoma; Carcinoid Tumor (Gastrointestinal); Carcinoid Tumor, Childhood; Cardiac (Heart) Tumors, Childhood; Embry onal Tumors, Childhood; Germ Cell Tumor, Childhood; Primary' CNS Lymphoma; Cervical Cancer; Childhood Cervical Cancer; Cholangiocarcinoma; Chordoma, Childhood; Chronic Lymphocytic Leukemia (CLL); Chronic Myelogenous Leukemia (CML); Chronic Myeloproliferative Neoplasms; Colorectal Cancer; Childhood Colorectal Cancer;Craniopharyngioma, Childhood; Cutaneous T-Cell Lymphoma (e.g., Mycosis Fungoides and Sezary Syndrome); Ductal Carcinoma In Situ (DCIS); Embryonal Tumors, Central Nervous System, Childhood; Endometrial Cancer (Uterine Cancer); Ependymoma, Childhood; Esophageal Cancer; Childhood Esophageal Cancer; Esthesioneuroblastoma; Ewing Sarcoma; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Eye Cancer; Childhood Intraocular Melanoma; Intraocular Melanoma; Retinoblastoma; Fallopian Tube Cancer; Fibrous Histiocytoma of Bone, Malignant, and Osteosarcoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Childhood Gastric (Stomach) Cancer; Gastrointestinal Carcinoid Tumor; Gastrointestinal Stromal Tumors (GIST); Childhood Gastrointestinal Stromal Tumors; Germ Cell Tumors; Childhood Central Nervous System Germ Cell Tumors (e.g., Childhood Extracranial Germ Cell Tumors, Extragonadal Germ Cell Tumors, Ovarian Germ Cell Tumors, Testicular Cancer); Gestational Trophoblastic Disease; Hairy Cell Leukemia; Head and Neck Cancer; Heart Tumors, Childhood; Hepatocellular (Liver) Cancer; Histiocytosis, Langerhans Cell; Hodgkin Lymphoma; Hypophary ngeal Cancer; Intraocular Melanoma; Childhood Intraocular Melanoma: Islet Cell Tumors, Pancreatic Neuroendocrine Tumors; Kaposi Sarcoma; Kidney (Renal Cell) Cancer; Langerhans Cell Histiocytosis; Laryngeal Cancer; Leukemia; Lipand Oral Cavity Cancer; Liver Cancer; Lung Cancer (Non-Small Cell and Small Cell); Childhood Lung Cancer; Lymphoma; Male Breast Cancer; Malignant Fibrous Histiocytoma of Bone and Osteosarcoma; Melanoma; Childhood Melanoma; Melanoma, Intraocular (Eye); Childhood Intraocular Melanoma; Merkel Cell Carcinoma; Mesothelioma. Malignant; Childhood Mesothelioma; Metastatic Cancer; Metastatic Squamous Neck Cancer with Occult Primary: Midline Tract Carcinoma With NUT Gene Changes; Mouth Cancer; Multiple Endocrine Neoplasia Syndromes; Multiple Myeloma / Plasma Cell Neoplasms; Mycosis Fungoides; Myelodysplastic Syndromes, Myelodysplastic / Myeloproliferative Neoplasms; Myelogenous Leukemia, Chronic (CML); Myeloid Leukemia, Acute (AML); Myeloproliferative Neoplasms, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasophary ngeal Cancer; Neuroblastoma; Non-Hodgkin Lymphoma; Non-Small Cell Lung Cancer; Oral Cancer, Lip and Oral Cavity Cancer and Oropharyngeal Cancer; Osteosarcoma and Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer; Childhood Ovarian Cancer; Pancreatic Cancer; Childhood Pancreatic Cancer; Pancreatic Neuroendocrine Tumors; Papillomatosis (Childhood Laryngeal); Paraganglioma; Childhood Paraganglioma; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pharyngeal Cancer; Pheochromocytoma; Childhood Pheochromocytoma; Pituitary Tumor; Plasma Cell Neoplasm / Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Primary Central Nervous System (CNS) Lymphoma; Primary Peritoneal Cancer; Prostate Cancer; Rectal Cancer; Recurrent Cancer; Renal Cell (Kidney) Cancer; Retinoblastoma; Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Sarcoma (e.g., Childhood Rhabdomyosarcoma, Childhood Vascular Tumors, Ewing Sarcoma, Kaposi Sarcoma, Osteosarcoma (Bone Cancer), Soft Tissue Sarcoma, Uterine Sarcoma); Sezary Syndrome; Skin Cancer; Childhood Skin Cancer; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma; Squamous Cell Carcinoma of the Skin; Squamous Neck Cancer with Occult Primary. Metastatic; Stomach (Gastric) Cancer; Childhood Stomach (Gastric) Cancer; T-Cell Lymphoma, Cutaneous (e.g.. Mycosis Fungoides and Sezary Syndrome); Testicular Cancer; Childhood Testicular Cancer; Throat Cancer (e g.. Nasophary ngeal Cancer, Orophary ngeal Cancer, Hypophary ngeal Cancer); Thymoma and Thymic Carcinoma; Thyroid Cancer; Transitional Cell Cancer of the Renal Pelvis and Ureter; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Cancer, Endometrial; Uterine Sarcoma; Vaginal Cancer; Childhood Vaginal Cancer; Vascular Tumors; Vulvar Cancer; and Wilms Tumor and Other Childhood Kidney Tumors.

[0085] Examples of hematological malignancies include leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) such as FLT3 inhibitor-resistant AML or AMLwith high mTORC l expression and / or activity, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute myelogenous leukemia, chronic myelogenous leukemia, acute monocytic leukemia (AMoL)), lymphoma e.g.. non-Hodgkin's lymphoma, Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma), myeloma (e.g., multiple myeloma), myelodysplastic syndrome, or polycythemia vera. In certain embodiments, the cancer is a leukemia, preferably a T-cell leukemia, such as T-cell lymphoblastic leukemia. In certain embodiments, the cancer is a B-cell leukemia. In certain embodiments, the cancer is a lymphoma, preferably a T-cell lymphoma. In certain embodiments, the cancer is a B-cell lymphoma, for example, diffuse large B-cell lymphoma or a Burkitt lymphoma. In certain embodiments, the cancer is myeloma. In certain embodiments, the cancer is myelodysplastic syndrome. In certain embodiments, the cancer is polycythemia vera.

[0086] In some embodiments, the cancer is non-small cell lung cancer, mesothelioma, pancreatic cancer, colon cancer, breast cancer, gastric cancer, esophageal cancer, or rectal cancer. In some embodiments, the cancer is non-small cell lung cancer, mesothelioma, pancreatic cancer, or colon cancer. In some embodiments, the cancer is non-small cell lung cancer (e.g., non-squamous non-small cell lung cancer or squamous non-small cell lung cancer). In some embodiments, the cancer is non-squamous non-small cell lung cancer. In some embodiments, the cancer is small cell lung cancer, non-small cell lung cancer, melanoma, squamous cell skin cancer, or basal cell skin cancer.

[0087] In some embodiments, the cancer is a tumor of the lung, mesothelium, colon, rectum, pancreas, prostate, breast, head and neck, stomach, uterus, ovaries, endometrium, bladder, kidney, salivary glands, testes, cervix, nasopharynx, esophagus, rectum, anus, liver, skin, brain, or thyroid. In some embodiments, the cancer is a tumor of the lung, mesothelium, colon, rectum, pancreas, prostate, breast, head and neck, stomach, uterus, ovaries, endometrium, bladder, kidney, salivary’ glands, or testes.

[0088] In some embodiments, the cancer is cervical cancer, melanoma, ovarian cancer, pancreatic ductal adenocarcinoma, endometrial cancer, or colorectal cancer.

[0089] A compound of the disclosure or other therapeutic agent described herein can be administered via a variety of routes of administration, including, for example, oral, dietary, topical, transdermal, rectal, parenteral (e.g., intra-arterial, intravenous, intramuscular, subcutaneous injection, intradermal injection), intravenous infusion and inhalation (e.g, intrabronchial, intranasal or oral inhalation, intranasal drops) routes of administration, depending on the compound and the particular disease to be treated. Administration can be local or systemic.EXAMPLES

[0090] The following Examples are presented by way of illustration, not limitation. One skilled in the art can modify the procedures set forth in the illustrative examples to arrive at the desired products.

[0091] The following abbreviations may be relevant for the application:Example SI. Synthesis of HKNU-0127

[0092] Step 1: trimethylsilylformaldehyde

[0093] To a solution of oxalyl dichloride (4.57 g, 35.98 mmol, 3.15 mL, 1.5 eq) in DCM(125 mL) at -78°C was added dropwise to a solution of DMSO (3.09 g, 39.58 mmol, 3.09 mL, 1.65 eq) in DCM (65 mL) at -78°C over 20 min. After addition, the mixture was stirred at this temperature for 10 min, and trimethylsilylmethanol (2.5 g, 23.99 mmol, 23.99 mL, 1 eq) in DCM (200 mL) was added dropwise at -78 °C for 10 min, and TEA (8.74 g, 86.35 mmol, 12.02 mL, 3.6 eq) was then added dropwise. The resulting mixture was stirred at -78 °C for 20 min.The solution of trimethylsilylformaldehyde (4.09 g, crude) in DCM (388 mL) was used directly in next step.

[0094] Step 2: [(E)-2-(benzenesulfonyl)-2-fluoro-vinyl]-trimethyl-silane

[0095] To a solution of fluoromethylsulfonylbenzene (2.5 g, 14.35 mmol, 1 eq) in THF (50 mL) at -78°C was added LiHMDS (1 M, 25.95 mL, 1.81 eq) and [chloro(methoxy)phosphoryl] oxymethane (2.07 g, 14.35 mmol, 1.55 mL, 1 eq). The mixture was stirred at -78 °C for 1.5 h. The mixture was added dropwise in trimethylsilylformaldehyde (1.47 g, 14.35 mmol, 1 eq), and the resulting mixture was stirred at -78 °C for 1 h. warmed to 25 °C, and stirred for 1 h. The reaction mixture was quenched with saturated NH4CI (aq, 10 mL), and the mixture was extracted with EtOAc (10 mL*3). The combined organic layers were washed with brine (50 mL), dried over Na2SC>4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh. Petroleum ether / Ethyl acetate=8 / l to 5 / 1) to give [(E)-2-(benzenesulfonyl)-2-fluoro-vinyl]-trimethyl-silane (3 g, crude) as a white oil.

[0096] !H NMR: (400MHz, DMSO-de) 5 7.97-7.89 (m, 3H), 7.88-7.79 (m, 1H), 7.79-7.69 (m, 3H), 6.52-6.31 (m, 1H), 6.23-6.08 (m, 1H), 0.31 (s, 4H), 0.17 (s, 9H).

[0097] Step 3: [(E)-2-fluoro-2-tritert-butylstannyl-vinyl]-trimethyl-silane

[0098] To a solution of [(E)-2-(benzenesulfonyl)-2-fluoro-vinyl]-trimethyl-silane (1 g. 3.87 mmol, 1 eq) in Tol (20 mL) was added AIBN (31.78 mg, 193.51 pmol, 0.05 eq) and tributylstannane (3.05 g, 10.48 mmol, 2.78 mL, 2.71 eq). The mixture was stirred at 120 °C for 24 h. The reaction mixture was quenched with saturated KF (aq, 100 mL), and the mixture was stirred at 20 °C for 2 h. The mixture was extracted with DCM (30 mL*3). The combined organic layers were washed with brine (30 mL), dried over Na2SC>4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, n-hexane / Ethyl acetate=l / O) to give [(E)-2-fluoro-2-tritert-butylstannyl-vinyl]-trimethyl-silane (2.3 g, crude) as a white oil

[0099] Step 4: l-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl]-5- iodo-pyrimidine-2, 4-dione & l-[(2S,4S,5R)-4-hydroxy-5- (hydroxymethyl)tetrahydrothiophen-2-yl]-5-iodo-pyrimidine-2, 4-dione

[0100] A solution of l-[(4S,5R)-4-benzyloxy-5-(benzyloxymethyl)tetrahydrothiophen-2-yl]- 5-iodo-pyrimidine-2.4-dione (1.5 g, 2.73 mmol. 1 eq) in TFA (15 mL) was stirred at 80 °C for 12 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (neutral condition, column: WePure Biotech XP tC18 150*40*7pm; mobile phase: [EhOQOmM NH4HCO3)-ACN];gradient: l%-25% B) over 8.0 min to give l-[(2R,4S,5R)-4- hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl]-5-iodo-pyrimidine-2, 4-dione (130 mg, 351.20 pmol, 12.89% yield) as a white solid and l-[(2S,4S,5R)-4-hydroxy-5- (hydroxymethyl)tetrahydrothiophen-2-yl]-5-iodo-pyrimidine-2, 4-dione (190 mg, 513.29 pmol, 18.83% yield) as a white solid.

[0101] 'H NMR: (400MHz. DMSO-ds) 5 8.50-8.42 (m, 1H), 6.19 (t, J= 7.3 Hz, 1H), 5.40- 5.11 (m, 3H), 4.32 (br d. J = 3.1 Hz. 1H), 3.66-3.53 (m. 3H), 2.30-2.11 (m. 2H).

[0102] Step 5: 5-[(Z)-l-fluoro-2-trimethylsilyl-vinyl]-l-[(2R,4S,5R)-4-hydroxy-5- (hydroxymethyl)tetrahydrothiophen-2-yl]pyrimidine-2, 4-dione

[0103] To a solution of [(E)-2-fluoro-2-tritert-butylstannyl-vinyl ]-trimethyl-silane (110 mg, 270.09 pmol, 1 eq), l-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl]-5- iodo-pyrimidine-2, 4-dione (100 mg, 270.15 pmol, 1 eq) in DMF (2 mL) was added Pd(PPli3)4 (15.61 mg, 13.50 pmol, 0.05 eq). The mixture was stirred at 100 °C for 12 h. The reaction mixture was poured into H2O (10 mL). The mixture was extracted with EtOAc (5 mL*3). The combined organic layers were washed with brine (10 mL), dried over Na2SC>4, filtered, andconcentrated under reduced pressure. The residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate=l / l to 0 / 1) to give 5-[(Z)-l-fluoro-2-trimethylsilyl-vinyl]- l-[(2R.4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl]pyrimidine-2, 4-dione (55 mg crude) was as a brown solid.

[0104] Step 6: 5-(l-fhiorovinyl)-l-[(2R,4S,5R)-4-hydroxy-5- (hydroxymethyl)tetrahydrothiophen-2-yl]pyrimidine-2, 4-dione

[0105] A solution of 5-[(Z)-l-fluoro-2-trimethylsilyl-vinyl]-l-[(2R.4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl]pyrimidine-2, 4-dione (25.00 mg, 69.35 pmol, 1 eq) in TFA (0.01 mL) and DCM (0.5 mL) was stirred at 25 °C for 5 min. The reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (FA condition, column: Phenomenex Luna C18 100*30mm*3pm; mobile phase: [H2O(0.2% FA)-ACN] gradient: l%-25% B over 8.0 min) to give 5-(l-fluorovinyl)-l-[(2R,4S,5R)-4-hydroxy-5- (hydroxymethyl)tetrahydrothiophen-2-yl]pyrimidine-2,4-dione (1 mg, 3.23 pmol, 4.66% yield, 93.20% purity ) as a white solid.

[0106] 'H NMR: (400MHz, DMSO-de) 5 8.37 (s, 1H), 6.25 (t, 7.0 Hz, 1H), 5.71-5.46(m, 1H), 5.36-5.16 (m, 2H), 4.95-4.82 (m, 1H), 4.36-4.26 (m, 1H), 3.66-3.54 (m, 3H), 2.32-2.13 (m. 2H).

[0107] LCMS: (M+Na+): 311.0 @ 1.934 mm (0-60% ACN in H2O, 6 min)Example S2. Synthesis of HKNU-0142

[0108] Step 1: trimethyl- [5-(trifluoromethyl)-2-trimethylsilyloxy-pyrimidin-4-yl] oxysilane

[0109] To a solution of 5-(trifluoromethyl)-lH-pyrimidine-2, 4-dione (1 g, 5.55 mmol, 1 eq) in HMDS (10 mL) was added (NH^SCU (36.69 mg, 277.65 pmol, 20.73 pL, 0.05 eq). The mixture was stirred at 120 °C for 12 h. The resulting reaction mixture comprising trimethyl-[5-(trifluoromethyl)-2-trimethylsilyloxy-pyrimidin-4-yl] oxy-silane (1.5 g, crude) in HMDS was directly used in step 2 without purification.

[0110] Step 2: l-[(2R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)tetrahydrothiophen-2- yl]-5-(trifluoromethyl)pyrimidine-2, 4-dione[OHl] To a solution of (2R,3S)-3-benzyloxy-2-(benzyloxymethyl)-5-benzylsulfanyl- etrahydrothiophene (900 mg, 2.06 mmol, 1 eq) in MeCN (20 mL) was added 4A MS (500 mg) and trimethyl-[5-(trifluoromethyl)-2-trimethylsilyloxy-pyrimidin-4-yl]oxy-silane (1.4 g, 4.32 mmol, 2.09 eq), followed by NIS (1.16 g, 5.15 mmol. 2.5 eq). The mixture was stirred at 25 °C for 2 h. The reaction mixture was poured into H2O (30 mL). The mixture was extracted with DCM (15 mL*3). The combined organic layers were washed with brine (20 mL), dried over Na2SOr, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate=2 / lto 1 / 1) to give l-[(2R,4S,5R)-4- benzyloxy-5-(benzyloxymethyl) tetrahydrothiophen-2-yl]-5-(trifluoromethyl)pyrimidine-2,4- dione (250 mg crude) as a white solid.

[0112] Step 3: l-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl]-5-(trifluoromethyl)pyrimidine-2, 4-dione

[0113] A solution of l-[(2R.4S,5R)-4-benzyloxy-5-(benzyloxymethyl)tetrahydrothiophen-2-yl]-5-(trifluoromethyl)pyrimidine-2,4-dione (50 mg, 101 .52 pmol, 1 eq) in TFA (1 mL) was stirred at 80 °C for 12 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (neutral condition, column: Phenomenex Gemini-NX 80*40mm*3pm;mobile phase: [H2O(10mM NH4HCO3)-ACN];gradient: l%-25% B over 15.0 min) to give l-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2- yl] -5- (trifluoromethyl)pyrimidine-2, 4-dione (4.5 mg, 14.09 pmol, 13.88% yield, 97.80% purity) as a white solid.

[0114] 'H NMR: (400MHz. DMSO-ds) 5 8.76 (s. 1H), 6.17 (t, J = 6.7 Hz, 1H), 5.47-5.16 (m. 2H), 4.29 (q. J = 4.2 Hz. 1H), 3.69-3.64 (m. 1H), 3.62-3.57 (m. 1H), 3.29-3.27 (m. 1H), 2.29-2.16 (m, 2H).

[0115] LCMS: (M+Na+): 335.0 @ 1.772 mm (0-60% ACN in H2O, 6 min)

[0116] Step 4: benzyl(2S)-2-[[[(2R,3S,5R)-5-[2,4-dioxo-5-(trifhioromethyl)pyrimidin-l- yl]-3-hydroxy-tetrahydrothiophen-2-yl]methoxy-phenoxy-phosphoryl]amino]propanoate

[0117] A mixture of l-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl]-5- (trifluoromethyl)pyrimidine-2, 4-dione (95 mg, 304.23 pmol, 1 eq), benzyl (2S)-2- [[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl]amino]propanoate (152.52 mg, 304.23 pmol, 1 eq), DIEA (78.64 mg, 608.46 pmol. 105.98 pL. 2 eq), and MgCh (57.93 mg, 608.46pmol, 24.97 pL, 2 eq) in DMF (2 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 25 °C for 2 h under N2 atmosphere. The reaction mixture w as poured into H2O (10 mL). The mixture was extracted with EtOAc (5 mL*3). The combined organic layers were washed with brine (10 mL). dried over Na2SO4. filtered, and concentrated under reduced pressure. The residue w as purified by prep-HPLC (neutral condition, column: 2_Phenomenex Gemini C18 75*40mm*3pm;mobile phase: [H2O(10mM NH4HCO3)- ACN];gradient:25%-50% B over 8.0 min) to give benzyl (2S)-2-[[[(2R,3S,5R)-5-[2,4-dioxo-5- (trifluoromethyl)pyrimidin-l -yl]-3-hy droxy-tetrahydrothiophen-2-yl]methoxy -phenoxyphosphoryl] amino]propanoate (1.9 mg, 2.92 pmol, 0.96% yield, 96.80% purity) as a white solid.

[0118] !H NMR: (400MHz, DMSO-de) 5 11.87-11.51 (m, 1H), 8.29 (s, 1H), 7.39-7.29 (m, 7H), 7.22-7.13 (m, 3H), 6.21 (dd, J= 6.4, 8.4 Hz, 1H), 6.11 (dd, J= 10.2, 13.2 Hz, 1H), 5.48- 5.41 (m, 1H), 5.10 (d, J= 2.0 Hz, 2H), 4.39-4.22 (m, 2H), 4.03 (td, J= 6.7. 10.4 Hz, 1H), 3.96- 3.86 (m, 1H), 3.50-3.42 (m, 1H), 2.44-2.31 (m, 1H), 2.23 (ddd, J= 3.8, 6.3, 13.2 Hz, 1H), 1.30- 1.19 (m, 3H).

[0119] LCMS: (M+H~): 630.2 @ 3.960 min (0-60% ACN in H2O, 6 min)Example Bia. Cell growth assay of thymidine analogs, pemetrexed, or a combination thereof

[0120] Growth assays were conducted as follows. 5,000 HCT116 cells were seeded in 96- well plates with lOOpL DMEM + 10% dFBS + 1%P / S. For relative cell growth, the following day, 5()pL of 30pM thymidine analog and / or 3pM pemetrexed was added to each well for 72hr. The final concentration of thymidine analogs was lOpM and the final concentration of pemetrexed was IpM. Cell viability was quantified by the sulforhodamine B colorimetric assay. Day 0 values w ere subtracted from all samples, and Day 3 values for each drug were normalized to Day 3 vehicle as described in Vichai V. and Kirtikara K. 2006 Sulforhodamine B colorimetric assay for cytotoxicity’ screening Nat. Protoc. 1 1112-6.

[0121] For ICsos. the following day. 50pL of 3x thymidine analog was added to each well for 72hr. The final concentration of thymidine analog was lx, and typically ranged from lOnM to 30pM. Calculations of IC50 after treatment with each thymidine analog were performed using the SRB data and a 4-parameter logistic model in GraphPad Prism. IC50 curves included measurements from day 0 to assess baseline cell viability. This inclusion allowed determination of whether cells exhibited net proliferation or cell death, as values dropping below zero indicated a reduction in viable cell count relative to the initial population.

[0122] The data are reported in Table 2.Example Bib. TS inhibition and DNA incorporation assay

[0123] At time 0, and after 15 minutes, 2 hours, 8 hours, 24 hours, 48 hours, and 72 hours of treatment with thymidine analog (IpM for HK-0141, lOpM for all other analogs), intracellular metabolites (e.g., dUMP), and nucleic acids were extracted using 500 pL of ice-cold 80% methanol, followed by addition of 8.75 (iL neutralization buffer (15% NH4HCO3). Samples were stored at -80°C until LC-MS analysis. Once out of the freezer, samples were centrifuged at maximum speed at 4°C for 25 minutes. The pellet was retained for DNA and RNA incorporation analysis. From the supernatant, 200 L was transferred to new tubes and centrifuged again under the same conditions. The supernatant was used for LC-MS analysis. At each of the specified time points, culture media was also collected before metabolite extraction from the cells. The media underwent the same steps for sample preparation and mass spectrometry' as described for intracellular metabolites.

[0124] As mentioned above, following metabolomics sample preparation, the remaining pellet was processed for DNA and RNA extraction. Methanol was completely removed, and nucleic acids were extracted using the DNeasy Blood and Tissue Kits for DNA Isolation (Qiagen). After extraction, 17 pL of each sample was used for digestion using the (kit), according to the manufacturer's instructions. A 2: 1 mastermix of digestion buffer and digest enzymatic mix was prepared, and 3 pL of this mix was added to each sample, bringing the final reaction volume to 20 pL. Samples were mixed and incubated at 37°C for 1 hour. Following digestion, 180 pL of 100% acetonitrile was added to each sample and vortexed. 75 pL of each sample was loaded into mass spectrometry' tubes, along with blanks consisting of 100% acetonitrile.

[0125] Liquid chromatography separation was achieved using a Vanquish Horizon UHPLC System (Thermo Fisher Scientific) with a Waters XBridge BEH Amide XP column (150 mm (length) x 2.1 mm (i.d.); particle size, 2.5 mm). For the hydrophilic interaction chromatography (HILIC), solvent A was 20 mM ammonium acetate and 22.5 mM ammonium hydroxide in 95:5 (v / v) water: acetonitrile (pH 9.45) and solvent B was 100% acetonitrile. The gradient was 0-2 min, 90% B; 2-3 min, 90% B to 75% B; 3-7 min, 75% B; 7-8 min, 75% B to 70% B; 8-9 min, 70% B; 9-10 min, 70% B to 50% B; 10-12 min, 50% B; 12-13 mm, 50% B to 25% B; 13-14 min, 25% B; 14-16 min, 25% B to 0.5% B, 16-20.5 min, 0.5% B; 20.5-25 min, 90% B (75). The flow rate was 150 pl min-1. LC-MS data were collected on an Exploris 240 Orbitrap mass spectrometer (Thermo Fisher Scientific) operating in full scan mode with a scan range of m / z 70-1,000 and a resolving power of 160,000 at m / z 200. Other mass spectrometer parameters are as follows: sheath gas flow rate, 28 (arbitrary units); auxiliary gas flow rate, 10 (arbitrary' units); sweep gas flow rate, 1 (arbitrary units); spray voltage. 3.3 kV; capillary temperature, 320 °C; S-lens radiofrequency level, 65; automatic gain control (AGC) target, 3E6; and maximum injection time, 500 ms. LC-MS data acquisition was operated under dual scan positive and negative mode for all samples.

[0126] LC-MS raw data files (.raw) were converted to mzXML format using ProteoWizard (version 3.0.20315). El-MAVEN (version 0.12.1) was used to generate a peak table containing m / z, retention time, and intensity for the peaks. Parameters for peak picking were the defaults. The resulting peak table was exported as a .csv file. Ratio of thymidine analog to deoxy cytidine was calculated from the mass spectrometry signal of the analog from hydrolyzed DNA relative to the mass spectrometry signal of deoxy cytidine from the hydrolyzed DNA. Percent DNA incorporation was calculated from this ratio.

[0127] The data are reported in Table 2, wherein "yes" in the DNA Incorporation column indicates DNA incorporation and "no" in the DNA Incorporation column indicates DNA incorporation was less than the detection limit of 0.1% or 0.001 thymidine analog:deoxy cytidine.Table 2. Relative cell growth of HCT116 cells treated with a thymidine analog, pemetrexed, or a combination thereof* NT = not tested; inf = infiniteExample B2. Cell growth assays involving HmdU, pemetrexed, or a combination thereof in various cell lines

[0128] Growth assays for the initial compound screen were conducted as follows. 5,000 cells were plated in 24-well plates with 500pL DMEM + 10% dFBS + 1%P / S. The following day, 250pL of Hmdu alone, pemetrexed alone, or a combination of Hmdu and pemetrexed was added to each well for 72hr. Cell viability was quantified by the Sulforhodamine B colorimetric assay. Day 0 values were subtracted from all samples, and Day 3 values for each drug were normalized to Day 3 vehicle as described above. The data are reported in Table 3.Table 3. Relative cell growth of various cell lines treated with HmdU, pemetrexed, or a combination thereof

[0129] These data indicate that while pemetrexed and Hmdu both block cell growth, the combination is superior to each alone in a variety of cell lines.Example B3. Cell growth assays involving trifluridine or HK-0141 in multiple cell lines

[0130] Growth assays were conducted as follows. 5,000 cells were seeded in 96-well plates with lOOqL DMEM + 10% dFBS + 1 %P / S . The following day, 50pL of 3x HK-0141 was added to each well for 72hr. The final concentration of HK-0141 was lx, and ranged from lOnM to 30pM. Cell viability was quantified by the Sulforhodamine B colorimetric assay. Day 0 values were subtracted from all samples, and Day 3 values for each drug were normalized to Day 3 vehicle as described above. The calculated ICso values are reported in Table 4.Table 4. Relative cell growth of various cell lines treated with trifluridine or HK-0141

[0131] The data in Table 4 show that HK-0141 demonstrates similar efficacy to trifluridine in trifluridine-sensitive cells. HK-0141 demonstrates superior efficacy to trifluridine in cells with reduced sensitivity to trifluridine.Example B4. Cell growth assay in HCT116 WT and HCT116 sgTKl cell lines

[0132] Growth assays were conducted as follows. 5,000 cells (HCT116 WT or HCT116 sgTKl) were seeded in 96-well plates with lOOpL DMEM + 10% dFBS + 1 %P / S. The following day, 50pL of 3x HK-0142 was added to each well for 72hr. The final concentration of HK-0142 was lx, and ranged from lOnM to 30pM. Cell viability was quantified by the Sulforhodamine B colorimetric assay. Day 0 values were subtracted from all samples, and Day 3 values for each drug were normalized to Day 3 vehicle as described above. The results are shown in FIGs. 2A- 2D. TK1 knockout in HCT116 sgTKl cells were achieved by CRISPR CAS9 technology;

[0133] It was expected that protides would allow TK1 bypass. These data indicate that protide compound HK-0142 did not have efficacy in both WT and sgTKl cells, suggesting its metabolism is not independent of TK1, as expected.

[0134] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0135] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

CLAIMS1. A method of treating a cancer in a patient diagnosed with cancer, comprising administering to the patient an effective amount of a thymidine analog and a thymidylate synthase inhibitor, wherein the thymidine analog incorporates into DNA.

2. The method of claim 1, wherein the thymidylate synthase inhibitor is pemetrexed, 5- fluorouracil, or capecitabine.

3. The method of claim 2, wherein the thymidylate synthase inhibitor is pemetrexed.

4. The method of any one of claims 1-3, wherein the thymidine analog is a compound of one of the following structural formulae:or a pharmaceutically acceptable salt thereof, wherein:A is S or O;R1is Ci-Csalkyl, Cs-Cscycloalkyl, halogen, Ci-Cshaloalkyl. C2-Cshaloalkenyl, -CH2OH, -CH2OCH3, -C(O)H, -CO2H, or -C=CR1A;R1Ais H, halogen, Ci-Csalkyl, Ci-Cshaloalkyl, or Ci-Cshaloalkenyl;R2and R3are independently H or halogen;R4is -OH or R5;R5is -O-P(O)(OH)2, wherein each H is independently optionally substituted with alky l or arylalkyl and / or each -OH is independently optionally substituted with -NHR6;R6is H, linear or branched Ci-Cioalkyl, Ci-Cioalkenyl, Cr-Cnalkylaryl. C?-Ci2alkenylaryl, C7-Ci2heteroalkylaryl, or C7-Ci2heteroalkenylaryl. provided that w hen R4is OH, A is O, and R1is -C=CH, then at least one of R2and / or R3is a halogen, and when R4is OH and R1is Br, F. or CF3. then A is S and / or at least one of R2and / or R3is halogen.

5. The method of any one of claims 1-3, wherein the thymidine analog is a compound ofTable 1, or a pharmaceutically acceptable salt thereof.The method of claim 5, wherein the thymidine analog ipharmaceutically acceptable salt thereof.

7. The method of claim 5, wherein the thymidine analog is, or a pharmaceutically acceptable salt thereof.

8. The method of claim 5, wherein the thymidine analog ispharmaceutically acceptable salt thereof.

9. The method of claim 5, wherein the thymidine analog ipharmaceutically acceptable salt thereof.

10. The method of any one of claims 1-9, wherein the thymidine analog is antiproliferative.

11. The method of any one of claims 1-10, wherein the cancer is non-small cell lung cancer, mesothelioma, pancreatic cancer, colon cancer, breast cancer, gastric cancer, esophageal cancer, or rectal cancer.

12. The method of any one of claims 1-11. wherein the cancer is non-small cell lung cancer, mesothelioma, pancreatic cancer, or colon cancer.

13. The method of any one of claims 1-10, wherein the cancer is non-squamous non-small cell lung cancer.

14. The method of any one of claims 1-10, wherein the cancer is a tumor of the lung, mesothelium, colon, rectum, pancreas, prostate, breast, head and neck, stomach, uterus, ovaries, endometrium, bladder, kidney, salivary glands, testes, cervix, nasopharynx, esophagus, rectum, anus, liver, skin, brain, or thyroid.

15. The method of any one of claims 1-10 and 14, wherein the cancer is a tumor of the lung, mesothelium, colon, rectum, pancreas, prostate, breast, head and neck, stomach, uterus, ovaries, endometrium, bladder, kidney, salivary glands, or testes.

16. The method of any one of claims 1-10, wherein the cancer is cervical cancer, melanoma, ovarian cancer, pancreatic ductal adenocarcinoma, endometrial cancer, or colorectal cancer.

17. The method of any one of claims 1-10, wherein the cancer is a hematological malignancy.

18. The method of any one of claims 1-16, wherein the cancer is primary tumor.

19. The method of any one of claims 1-16, wherein the cancer is a metastatic tumor.

20. The method of any one of claims 1-19, wherein the patient is a human.

21. A thymidine analog for use in the treatment of a cancer in combination with a thymidylate synthase inhibitor, wherein the thymidine analog incorporates into DNA.

22. A thymidylate synthase inhibitor for use in the treatment of a cancer in combination with a thymidine analog, wherein the thymidine analog incorporates into DNA.

23. A pharmaceutical combination comprising a thymidine analog and a thymidylate synthase inhibitor, wherein the thymidine analog incorporates into DNA.

24. A compound of the following structure:pharmaceutically acceptable salt thereof.

25. A pharmaceutical composition comprising a compound of claim 24, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

26. A method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of the compound of claim 24, or a pharmaceutically acceptable salt thereof, or pharmaceutical composition of claim 25.

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