WO2026029682A1 - Prodrugs of alkylthio-dadme-immucillin a compounds and their use as anticancer therapeutic agents - Google Patents

Abstract

Amide prodrugs of alkylthio-DADMe-Immucillin A compounds as inhibitors of MTAP (5- methylthioadenosine phosphorylase) and their use as therapeutic agents against various cancers. The invention relates to compounds, pharmaceutical compositions, methods for inhibiting MTAP, and methods for treating cancer. The inhibitors are compounds of the general formula: (I).

Description

PRODRUGS OF ALKYLTHIO-DADME-IMMUCILLIN A COMPOUNDS AND THEIR USE AS ANTICANCER THERAPEUTIC AGENTS

TECHNICAL FIELD

This invention relates generally to novel compounds and their use as prodrugs for the treatment of various cancers. In particular, the invention relates to certain compounds that are prodrugs of selected alkylthio-DADMe-Immucillin-A compounds and to their use as therapeutic agents against various cancers.

BACKGROUND OF THE INVENTION

Methylthio-DADMe-Immucillin-A (MTDIA) is an orally available transition state analogue inhibitor of 5-methylthioadenosine phosphorylase (MTAP). MTAP is a key enzyme in mammalian cells and is critical for cellular growth. MTAP is a component of the polyamine biosynthesis pathway, and also contributes to purine recycling, S-adenosylmethionine synthesis, methylation of histones and regulatory proteins via protein arginine methyltransferase (PRMT5), and regulation of gene expression. Inhibition of MTAP leads to a build-up of its substrate, 5'-methylthioadenosine (MTA). Elevated levels of MTA produce a semi-toxic effect on tumour cells. MTDIA has been shown to lead to the inhibition of growth in several human cancers in mouse models.

MTDIA MTA

Approximately 15% of human cancers have genetic deletion of MTAP, often co-deleted with the adjacent CDK2 region. Since MTAP is synthetic-lethal with inhibition of methionine adenosyltransferase 2A (MAT2A) and/or PRMT5, some MAT2A and PRMT5 inhibitors have been developed to treat patients with MTAP-deficient tumours. This approach excludes the 85% of cancers with normal CDK2-MTAP genetics.

MTDIA renders MTAP+ cancer cells susceptible to MAT2A and/or PRMT5 inhibitors. However, one problem with MTDIA is that it has low orally bioavailability and sub-optimal pharmacologic and biological activity. There is therefore a need to find analogues of MTDIA having increased oral bioavailability, pharmacologic and biological activity. Nucleoside analogues are compounds that are structurally similar to natural nucleosides. Many nucleoside analogues have anti-cancer properties. However, like MTDIA, many suffer from poor oral bioavailability due to their high polarity and low intestinal permeability. The use of prodrugs of nucleoside analogues is a well-known strategy for improving oral absorption. See, for example, Li et ah, J. Pharma. Sei., 2008, 97(3), 1109- 1133.

Li et al. consider a range of prodrug design strategies including ester, carbamate and amide prodrugs which can be hydrolysed by carboxyesterases to release the active drug molecule, Notably, Li et al. point out that N-acylation of amines in prodrug design has limited use because amides tend to have high stability in vivo.

Amide prodrugs of the antiviral drug Remdesivir have been shown to have decreased antiviral activity. The reason postulated is that the biostable amide group obstructs hydrogen bond formation necessary for activity. See Cao et al., Sci. Transl. Med. (2022).

A review on nucleoside prodrugs identified examples of amide prodrugs of pyrimidine nucleoside analogues, but not of adenosine analogues. See Zhang et al. Asian J. Pharma. Sci., 2014, 9, 65-74.

The applicants initially chose to synthesise ester prodrugs of MTDIA by esterifying the hydroxyl group on the pyrrolidine ring. However, these were found to be highly unstable. The applicants then chose to target the primary amino group and synthesise carbamate prodrugs of MTDIA. They again found these compounds to be highly unstable. Then, despite the above reports indicating that amide prodrugs are too stable to be effective, the applicants surprisingly found that amide prodrugs of MTDIA were sufficiently stable, but not too stable, to show increased oral availability relative to MTDIA therefore making them potential candidates as anti-cancer agents. The applicants also found that prodrugs of ethylthio-DADMe- Immucillin-A (ETDIA) are potential candidates as anti-cancer agents. Accordingly, the compounds of the invention are prodrugs of alkylthio-DADMe-Immucillin-A compounds.

It is therefore an object of the invention to provide novel amide prodrugs of alkylthio- DADMe-Immucillin-A compounds, particularly prodrugs of MTDIA and ETDIA.

SUMMARY OF THE INVENTION

In a first aspect of the invention there is provided a compound of formula (I): wherein:

R¹ is methyl, ethyl, n-propyl or n-butyl; and

R² is Ci-6 alkyl, C2-6 alkenyl or C2-6 alkynyl each of which may be substituted by one or more phenyl groups, amino groups or amide groups, or R² is Ph, or a pharmaceutically acceptable salt thereof.

In some embodiments of the invention, R¹ is methyl or R¹ is ethyl.

In some embodiments of the invention, R² is C1-6 alkyl, for example, -(CH2)4CH3, - CH(CH₃)2, or -CH(CH₂CH₃)2.

In some embodiments, R² is -CH(Ph)2.

In some embodiments, R² is Ph.

In some embodiments, R² is C1-6 alkyl substituted by a phenyl group, or two amino groups or an amide group.

In some embodiments, R² is C1-6 alkyl substituted by a phenyl group and by an amide group.

Preferably the amide group is NHAc.

The compound of formula (I) may be, but is not limited to, any one of the following compounds:

In another aspect, the invention provides a pharmaceutical composition comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a method for inhibiting MTAP (5- methylthioadenosine phosphorylase) in a subject which comprises administering to a subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a method of treating cancer which comprises administering to a subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cancer.

In another aspect, the invention provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of cancer.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph showing the correlation of in vitro drug activation and glCso in MTAP^+/+ HT-29 cells treated with MTDIA prodrugs in combination with 1 mM AG-270.

DETAILED DESCRIPTION

The invention provides amide prodrugs of selected alkylthio-DADMe-Immucillin A compounds as inhibitors of MTAP and their use for the treatment of cancer. The invention is based on the finding that amide prodrug compounds of general formula (I) are effective inhibitors of MTAP. Once a prodrug compound is in the body of a subject, the amide bond is hydrolysed, primarily by enzyme activity, to provide the active amine compound. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the inventions belong. Although any assays, methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, various assays, methods, devices and materials are now described.

It is intended that reference to a range of numbers disclosed herein (for example 1 to 10) also incorporates reference to all related numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

As used in this specification, the words "comprises", "comprising", and similar words, are not to be interpreted in an exclusive or exhaustive sense. In other words, they are intended to mean "including, but not limited to".

The term "alkyl" means any saturated hydrocarbon radical and is intended to include both straight- and branched-chain alkyl groups. The term "Ci-Ce alkyl" means any saturated hydrocarbon radical having up to 6 carbon atoms. Examples include, but are not limited to methyl, ethyl, n-propyl, /so-propyl, n-butyl, /so-butyl, sec-butyl, t-butyl, n-pentyl, 1,1- dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-ethylpropyl, n-hexyl and l-methyl-2-ethylpropyl.

The term "alkenyl" means any hydrocarbon radical having at least one double bond and is intended to include both straight- and branched-chain alkenyl groups. Examples of alkenyl groups include, but are not limited to, ethenyl, n-propenyl, /so-propenyl, n-butenyl, iso- butenyl, sec-butenyl, n-pentenyl, 1,1-dimethylpropenyl, 1,2-dimethylpropenyl, 1- ethylpropenyl, 2-ethylpropenyl, n-hexenyl and l-methyl-2-ethylpropenyl.

The term "alkynyl" means any hydrocarbon radical having at least one carbon-carbon triple bond and is intended to include both straight- and branched-chain alkynyl groups. Examples of alkynyl groups include, but are not limited to, ethynyl, n-propynyl and n-butynyl.

The term "prodrug" as used herein refers to a compound that, when administered to a subject, undergoes chemical or enzymatic transformation to yield a compound that is therapeutically active.

The term "pharmaceutical composition" as used herein refers to a mixture of one or more of the compounds of formula (I), or pharmaceutically acceptable salts, or hydrates thereof, with other chemical components, such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Non-limiting examples of such pharmaceutical carriers include liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carrier may also be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and colouring agents may be used. Other examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, herein incorporated by reference.

The term "pharmaceutically acceptable salt" as used herein refers to any salt of a compound provided herein which retains its biological properties and which is not toxic or otherwise undesirable for pharmaceutical use and is intended to include salts derived from inorganic or organic acids including, for example hydrochloric, hydrobromic, sulfuric, nitric, perchloric, phosphoric, formic, acetic, lactic, maleic, fumaric, succinic, tartaric, glycolic, salicylic, citric, methanesulfonic, benzenesulfonic, benzoic, malonic, trifluoroacetic, trichloroacetic, naphthalene-2 sulfonic and other acids. Pharmaceutically acceptable salt forms may also include forms wherein the ratio of molecules comprising the salt is not 1: 1. For example, the salt may comprise more than one inorganic or organic acid molecule per molecule of base, such as two hydrochloric acid molecules per molecule of compound of formula (I).

The terms "treatment", "treating" and the like include the alleviation of one or more symptoms, or improvement of a state associated with the disease or disorder, for example, improvement in cognition, improvement in memory function.

The terms "preventing", "prevention" and the like include the prevention of one or more symptoms associated with the disease or disorder.

The compounds of the invention are useful in both free base form and in the form of salts and/or solvates.

Although the invention is described by way of example, it should be appreciated the variations or modifications may be made without departing from the scope of the invention. Furthermore, when known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification.

Compounds of the invention

The compounds of the invention have the general formula (I): wherein:

Ri is methyl, ethyl, n-propyl or n-butyl; and

R2 is C1-6 alkyl, C2-6 alkenyl or C2-6 alkynyl each of which may be substituted by one or more phenyl groups or amide groups, or R2 is Ph; or a pharmaceutically acceptable salt thereof.

In some preferred embodiments of the invention, R¹ is methyl or R¹ is ethyl.

In some embodiments of the invention, R² is C1-6 alkyl, or R² is -CH(Ph)2, or R² is Ph, or R² is C1-6 alkyl substituted by a phenyl group, one or two amino groups, or an amide group, or R² is C1-6 alkyl substituted by a phenyl group and by an amide group, Preferably the amide group is NHAc.

Some specific compounds of the invention include the following:

Preparation of compounds

The compounds of the invention may be prepared according to the methods described in the Examples. It will be appreciated that the methods used for preparing the compounds of the Examples may also be used for other compounds of the invention together with standard techniques and procedures as would be understood by those skilled in the art.

5'-Methylthioadenosine phosphorylase (MTAP)

The compounds of this invention are designed to inhibit the metabolic enzyme 5'- methylthioadenosine phosphorylase (MTAP, EC:2.4.2.28). Loss of MTAP is the most frequent metabolic gene deletion in human cancers due to its close (100 kb) sequence proximity to the CDKN2A/B locus. Genetic knockdown screening libraries revealed that in the 15% of cancers with MTAP loss, enhanced dependence is conferred on MAT2A (EC:2.5.1.6) and PRMT5 (EC:2.1.1.320). These synthetic lethal interactions between MTAP loss and MAT2A or PRMT5 knock-down have driven the development of MAT2A and PRMT5 inhibitors that are now in clinical testing for treatment of malignancies with the MTAP-/- genotype. See Bedard et al., J. Biol. Chem. (2024), 300(1), 105492.

The MTAP, MAT2A, and PRMT5 gene products are functionally linked through their involvement in the methionine/S-adenosyl-L-methionine (SAM) cycle. MAT2A catalyses the synthesis of SAM, an essential metabolite in polyamine biosynthesis and methylation reactions. Spermine production from SAM produces two equivalents of 5'-methylthioadenosine (MTA), which requires MTAP for recycling to methionine. SAM is also used as a cofactor for methyltransferases, including PRMT5, that mediate regulatory posttranslational and epigenetic methylations on proteins and DNA. SAM-consuming methyltransferases contain adenosyl- recognition motifs that can be inhibited by adenosyl metabolites including SAH or MTA. Regulation of inhibitory metabolites is critical for maintaining cellular homeostasis. Therefore, methyl-metabolite pool disruption can have tumour suppressing activity if alterations affect the activity of oncodriving methyltransferases.

PRMT5 is a transformation-driving type II arginine methyltransferase responsible for the majority of protein symmetric dimethylarginine (SDMA, also Rme2s) post-translational modifications. PRMT5 catalyses the vast majority of SDMA posttranslational modifications on histones, splicing factors, transcription factors and enhancer-binding proteins, metabolic enzymes, and cell signalling proteins implicating its role in cancer progression and survival. PRMT5 is overexpressed and negatively correlated with survival in patients with colorectal cancer and other cancers. PRMT5 is unique among methyltransferases in that its affinity for SAM is 40-times weaker than its affinity for MTA (SAM KM = 10.3 pM; MTA Ki = 0.26 pM) making PRMT5 uniquely sensitive to metabolite pool sizes of MTA and SAM. Consequently, in MTAP-/- tumours the synthetic lethal inhibition of MAT2A ultimately converges on PRMT5 inhibition that reduces cancer growth and induces cell death.

MTDIA binds tightly to MTAP, causing MTA accumulation in blood and tissues of treated mice. MTDIA induces apoptosis in xenografts of head and neck squamous carcinomas, reduces growth and metastases in mice bearing subcutaneous lung carcinoma xenografts, and extends the lifespan of a familial adenomatous polyposis mouse model by significantly inhibiting tumour growth. MTDIA binds 58,000 times tighter than MTA to the MTAP active site to drive the accumulation of MTA. MTDIA shows low toxicity, and high specificity for MTAP inhibition in the mouse. Therefore, MTDIA is effective in mimicking the metabolic phenotype of MTAP-/- cancers.

Pharmacokinetic and Pharmacodynamics

Example 18 describes an experiment where 30 mg/kg doses by intraperitoneal (IP) or oral gavage (PO) were given to mice and tail vein blood samples taken at timed intervals for each compound. Extraction of prodrug compounds, drugs, and MTA from whole blood allowed LC-MS quantification and generation of an area under the curve (AUC) for each compound of interest. The results are summarised in Table 1. In the mouse, MTDIA has a half-life (ti/2) of approximately 380 minutes with a time of maximum concentration (tMAx) at 30 minutes for IP injection and about 1 hour for PO. After MTDIA injection, serum MTA levels rise from low nanomolar to micromolar levels (1000-fold increase) with a tMAx at around 4-8 hours and a ti/2 >24 hours. Compared to IP administration, MTDIA reaches plasma levels at only 5% of the value when given PO, and total MTA exposure is only 19.5% of the response when given PO compared to IP.

Prodrug compounds of MTDIA and ETDIA demonstrated increased bioavailability up to 58%, such as for the hexanoyl compound 2. For many prodrug compounds, considerable active compound was detected in the blood, indicating that amidases are activating the prodrugs. The prodrug efficacy, measured by MTA exposure over 24 hours after a single dose, is a combination of its bioavailability and its rate of conversion to active form. Table 2 demonstrates that conjugation of MTDIA and ETDIA to lipophilic side chains increases the bioavailability of the prodrug compounds from 5% to as much as 58%. The best prodrug compound, MTDIA prodrug 2, had an IP dose AUC approximately 80% of the total AUC from an equivalent dose of MTDIA, indicating that the prodrug compound was efficiently converted to active compound. Because of its increased bioavailability and efficient conversion to active compound, prodrug compound 2 delivered five times the amount of MTDIA when given orally and caused a doubling of the MTA plasma exposure (Table 1). The nature of the lipophilic group for other prodrug compounds had variable effects on the total oral MTDIA or ETDIA delivered, likely due to their different membrane penetrations and activities on amidases. However, because the prodrug compounds themselves are weak inhibitors of MTAP, some degree of MTA accumulation was seen for all compounds tested.

Prodrug conversion to active compound

Example 20 shows the conversion of prodrug to active compound in blood. Incubation of MTAP inhibitor prodrugs with erythrocyte lysates demonstrates significant MTAP and prodrug-activating enzyme activity in the blood. Incubation of prodrugs with intact and lysed human erythrocytes tests for cellular permeability and for the action of enzymatic activities capable of converting specific prodrugs to their active forms. Examples are provided by prodrugs 2, 4 and 6. Incubation of prodrugs (250 pM) with lysed human blood (at 25% protein level of whole blood) gave prodrug-to-drug conversion for every prodrug tested. Specifically, incubation with lysed erythrocytes led to product formation at rates of 0.37, 1.29, and 10.5 pM per hour for compounds 4, 2 and 6, respectively, demonstrating catalytic conversion to the active MTDIA from cellular amidases (Table 3). In whole blood, the rates for these compounds were 0.35, 1.25, and 1.42 pM per hour suggesting that the lipophilicity of the side chain impacts its membrane permeance.

Prodrugs are weak inhibitors of human MTAP

The inhibitory potential of MTDIA prodrugs for recombinant human MTAP was tested with purified enzyme inhibition assays as described in Example 21. For this comparison, the values were compared to MTDIA, as none of the prodrug forms exhibited the slow-onset inhibition seen for MTDIA, giving a slow-onset * value of 86 pM. The addition of N-6 lipophilic amides to MTDIA decreased the initial rate binding affinity for MTAP by factors of 90 to 4,400-fold compared to the affinity of MTDIA (Table 4). Despite being orders of magnitude weaker inhibitors than MTDIA, some of the inhibition constants are in the nanomolar range. It was considered possible that small amounts of the prodrugs might be converted to MTDIA during experimental procedures, prompting the evaluation of compound purity.

The possibility that the observed inhibition by prodrugs was from small impurities of the tight-binding MTDIA was tested by incubating solutions of prodrugs with MTAP at 5 to 10% of the molar fraction of the prodrug concentration. Under these conditions, the enzyme would sequester any small amount of MTDIA generated in solution immediately prior to assay readout. Ultrafiltration was used to isolate the treated prodrugs from the MTAP fraction. The purified filtrate prodrugs were then retested and gave similar values as untreated prodrugs (Table 4). Thus, the prodrugs are stable under the assay conditions and are weak inhibitors of MTAP.

Prodrug synergy with MAT2A inhibitors in cell-based assays MTAP inhibitors have anti-cancer actions linked to the accumulation of MTA in cells. Inhibition of the protein arginine methyltransferase 5 (PRMT5) via the competition of MTA for substrate SAM leads to decreased cell cycling, alternative transcript splicing, and cell death resulting in tumor growth inhibition. This effect is enhanced by depleting the substrate SAM via MAT2A inhibition, thereby providing a synthetic-lethal strategy for anti-cancer therapeutics. The applicants sought to determine if prodrugs of MTDIA had anti-cancer activity in combination with MAT2A inhibitor AG-270 in HT-29 CRC cells. See Example 22. MTDIA in combination with 1 pM of AG-270 had an IC50 value of 52 nM, similar to previously reported values. Prodrugs of MTDIA varied significantly in anticancer efficiency, ranging from 30 nM to 5.5 pM, with compound 3 providing growth suppression of HT-29 cells superior to MTDIA (see Example 19, Table 2).

The relationship between growth IC50 (glCso) observed in the cellular model and the bioavailability observed in the animal model was explored by incubating prodrugs with HT-29 colorectal carcinoma cells for 8 hr to assess amidase conversion of the prodrugs. Prodrug conversion to active drug ranged from 0.2% (compound 2) to near quantitative conversion. There was good agreement between the prodrug activation by endogenous amidases and drug glCso (see Example 22 and Figure 1). Prodrugs with increases in glCso of 10-fold had approximately 10-fold less activation. Thus, prodrugs of MTDIA can be effective anti-cancer agents in combination with MAT2A inhibitors when activated by endogenous amidases.

Formulations and administration

The compounds of the invention may be administered to a patient by a variety of routes, including orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally or via an implanted reservoir. For parenteral administration, injections may be given intravenously, intra-arterially, intramuscularly or subcutaneously.

The amount of a compound of the invention to be administered to a patient will vary widely according to the nature of the patient and the nature and extent of the disorder to be treated. Typically the dosage for an adult human will be in the range of about 0.01 pg/kg to about 1 g/kg, and preferably about 0.01 mg/kg to about 100 mg/kg. The specific dosage required for any particular patient will depend upon a variety of factors, such as the patient's age, body weight, general health, gender and diet. Optimal doses will depend on other factors such as mode of administration and level of progression of the disease or disorder. Doses may be given once daily, or two or more doses may be required per day. For example, a dosage regime for a malaria patient might require one dose in the morning and one in the evening. Alternatively, a dosage regime for such a patient might require four hourly doses.

For oral administration the compounds can be formulated into solid or liquid preparations, for example tablets, capsules, granules, powders, solutions, suspensions, syrups, elixirs and dispersions. Such preparations are well known in the art as are other oral dosage regimes not listed here.

For parenteral administration, compounds of the invention can be formulated into sterile solutions, emulsions and suspension.

Compounds of the invention may be mixed with suitable vehicle and then compressed into the desired shape and size. The compounds may be tableted with conventional tablet bases such as lactose, sucrose and corn starch, together with a binder, a disintegration agent and a lubricant. The binder may be, for example, corn starch or gelatin, the disintegrating agent may be potato starch or alginic acid, and the lubricant may be magnesium stearate. For oral administration in the form of capsules, diluents such as lactose and dried cornstarch may be employed. Other components such as colourings, sweeteners or flavourings may be added. Tablets, capsules or powders for oral administration may contain up to about 99% of a compound of the invention.

When liquid preparations are required for oral use, a compound of the invention may be combined with a pharmaceutically acceptable carriers such as water, an organic solvent such as ethanol, or a mixture of both, and optionally other additives such as emulsifying agents, suspending agents, buffers, preservatives, and/or surfactants may be used. Colourings, sweeteners or flavourings may also be added.

The compounds may also be administered by injection in a pharmaceutically acceptable diluent such as water or saline. The diluent may comprise one or more other ingredients such as ethanol, propylene glycol, an oil or a pharmaceutically acceptable surfactant.

The compounds of the invention may also be administered topically. Carriers for topical administration of the compounds include mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. The compounds may be present as ingredients in lotions or creams, for topical administration to skin or mucous membranes. Such creams may contain the active compounds suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

The compounds of the invention may further be administered by means of sustained release systems. For example, they may be incorporated into a slowly dissolving tablet or capsule. EXAMPLES

The following examples further illustrate the invention. It is to be appreciated that the invention is not limited to the examples.

Example 1: General synthesis of 6-N-acyl-9-deazaadenine compounds

A suspension of 9-deazaadenine in dry pyridine (2 mL per mmol) was stirred at 0 °C and the acyl chloride (3-5 eq) was added. The reaction mixture was stirred at RT. When tic analysis showed the 9-deazaadenine was consumed, the reaction was quenched with water. Ethyl acetate was added (20 mL per mmol) and the solution was washed with water (x3) and brine and concentrated to dryness. The crude product, which was a mixture of mono- di- and tri-acylated compounds, was dissolved in methanol (3 mL per mmol) and dichloromethane (1.5 mL per mmol). 7N NH3 in methanol was carefully added and the solution was monitored by thin layer chromatography on silica gel (tic) until the over-acylated material was consumed leaving the 6-/V-acyl-9-deazaadenine. The solution was concentrated to dryness. Chromatography on silica gel eluted with ethyl acetate and hexanes mixtures afforded the 6-N- acyl-9-deazaadine compound.

Example 2: Synthesis of 6-N-benzoyl-9-deazaadenine

The synthesis was carried out according to the procedure reported in Mason, Jennifer M.; Yuan, Hongling; Evans, Gary B.; Tyler, Peter C.; Du, Quan; Schramm, Vern L., European Journal of Medicinal Chemistry (2017), 127, 793-809).

Example 3: Synthesis of 6-N-isobutyryl-9-deazaadenine

The synthesis was carried out according to the procedure of Example 1 to provide 6-/V- Isobutyryl-9-deazaadenine. ^XH NMR (400 MHz, CDCI3) 6 11.08 (s, 1H), 10.15 (s, 1H), 8.62 (s, 1H), 7.57 (t, J = 2.7 Hz, 1H), 6.72 (dd, J = 3.2, 1.4 Hz, 1H), 2.83 (hept, J = 6.8 Hz, 1H), 1.29 (d, J = 6.9 Hz, 6H). ¹³C NMR (101 MHz, CDCI3) 6 177.6, 152.3, 149.2, 142.8, 130.7, 115.8, 102.8, 36.0, 19.5. Example 4: Synthesis of 6-N-(2-ethylbutyrl)-9-deazaadenine

The synthesis was carried out according to the procedure of Example 1 to provide 6-/V-(2- ethylbutyrl)-9-deazaadenine. ^XH NMR (400 MHz, CDCI3) 6 11.05 (s, 1H), 8.66 (s, 1H), 7.59 (t, J = 3.0 Hz, 1H), 6.74 (dd, J = 3.2, 2.1 Hz, 1H), 2.36 (tt, J = 8.8, 5.2 Hz, 1H), 1.84 - 1.68 (m, 2H), 1.65-1.55 (m, 2H), 0.95 (t, J = 7.5 Hz, 6H). ¹³C NMR (101 MHz, CDCI3) 6 176.6, 152.5, 149.4, 142.5, 130.8, 116.1, 102.9, 51.3, 25.7, 11.9.

Example 5: Synthesis of 6-N-hexanoyl-9-deazaadenine

The synthesis was carried out according to the procedure of Example 1 to provide 6-/V- hexanoyl-9-deazaadenine. ^XH NMR (400 MHz, CDCI3) 6 11.06 (s, 1H), 10.62 (s, 1H), 8.67 (s, 1H), 7.58 (t, J = 2.8 Hz, 1H), 6.73 (dd, J = 3.2, 1.5 Hz, 1H), 2.60 - 2.51 (m, 2H), 1.81 - 1.69 (m, 2H), 1.40 - 1.30 (m, 4H), 0.86 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, CDCI3) 6 173.7, 152.4, 149.2, 142.8, 130.8, 116.0, 102.8, 37.0, 31.2, 25.0, 22.3, 13.8.

Example 6: Synthesis of 6-N-diphenylacetyl-9-deazaadenine

The synthesis was carried out according to the procedure of Example 1 to provide 6-/V- diphenylacetyl-9-deazaadenine. ^XH NMR (400 MHz, CDCI3) 6 10.96 (s, 1H), 10.51 (s, 1H), 8.50 (s, 1H), 7.49 (dd, J = 3.3, 1.5 Hz, 1H), 7.28 - 7.20 (m, 10H), 6.70 (d, J = 3.1 Hz, 1H), 5.20 (s, 1H). ¹³C NMR (101 MHz, CDCI3) 6 172.6, 152.6, 149.0, 142.5, 137.9, 131.0, 128.9, 128.8, 128.7, 127.7, 115.8, 103.0, 58.9. Example 7: 6-N-[(2S)-2-acetamido-3-phenylpropanoyl]-9-deazaadenine

A solution of 9-deazaadenine (0.466 g, 3.48 mmol) and (2S)-2-acetamido-3- phenylpropanoic acid (0.515 g, 2.48 mmol) in dry DMF (10 mL) was stirred at 0 °C while triethylamine (1.05 mL, 7.45 mmol) and then HBTU (1.71 g, 4.47 mmol) were added. After Ih the solution was concentrated to dryness under high vacuum. The residue was dissolved in chloroform and the solution was washed with sat. aq sodium bicarbonate and concentrated to dryness. Chromatography (1-10% MeOH/CHCh) gave title compound (0.55 g, 1.70 mmol, 68%). ^XH NMR (400 MHz, CDCI3) 6 10.86 (s, IH), 10.40 (s, IH), 8.51 (s, IH), 7.54 - 7.46 (m, 2H), 7.12 (s, 5H), 6.69 (d, J = 3.1 Hz, IH), 5.19 (q, J = 7.3 Hz, IH), 3.25 (dd, J = 13.9, 6.7 Hz, IH), 3.08 (dd, J = 13.9, 7.6 Hz, IH), 1.97 (s, 3H). ¹³C NMR (101 MHz, CDCI3) 6 172.1, 171.4, 152.4, 149.5, 141.9, 135.7, 131.0, 129.1, 128.8, 128.7, 127.2, 116.0, 102.6, 55.5, 37.8, 22.8.

Example 8: General synthesis of compounds of the invention by Mannich reaction of a (3R,4S)-4-((alkylthio)methyl)pyrrolidin-3-ol with a 6-N-acyl-9-deazaadenine

A mixture of the thioalkylpyrrolidinol as the HCI salt (J. Med. Chem. 2005, 48, 14, 4679-4689) with ethanol (3 mL per mmol), the 6-/V-acyl-9-deazaadenine (2.0 eq), water (0.3 mL per mmol), sodium acetate (3.0 eq) and 37% aq formaldehyde (2.0 eq) was stirred at 50- 70 °C until tic analysis showed the reaction was complete. The solution was concentrated to dryness, chloroform was added to the residue and the mixture was filtered, and the filtrate was concentrated to dryness. Chromatography on silica gel eluted with mixtures of methanol and chloroform afforded the product.

Example 9: Synthesis of 2-ethyl-N-(7-(((3R,4S)-3-hydroxy-4-((methylthio)methyl)- pyrrolidin-1 -yl)methyl)-5H-pyrrolo[3,2-d]pyrimidin-4-yl)butanamide (1)

The synthesis was carried out according to the procedure of Example 8 to provide 2- ethyl- N-(7-(((3R,4S)-3-hydroxy-4-((methylthio)methyl)pyrrolidin-l-yl)methyl)-5H- pyrrolo[3,2-d]pyrimidin-4-yl)butanamide. ^XH NMR (400 MHz, CDCI3) 6 11.12 (s, 1H), 10.35 - 10.30 (m, 1H), 8.62 (s, 1H), 7.72 (s, 1H), 7.55 (s, 1H), 4.15 - 4.01 (m, 3H), 3.32 (p, J = 5.8 Hz, 1H), 2.99 - 2.91 (m, 2H), 2.73 - 2.64 (m, 1H), 2.54 - 2.35 (m, 4H), 2.08 (s, 3H), 1.83 - 1.68 (m, 2H), 1.67 - 1.52 (m, 2H), 0.95 (t, J = 7.5 Hz, 6H). ¹³C NMR (101 MHz, CDCI3) 6 176.7, 150.9, 149.1, 142.7, 131.4, 116.1, 110.6, 75.7, 61.0, 57.6, 51.0, 47.8, 47.1, 37.0, 25.6, 15.6, 11.9.

Example 10: Synthesis of N-(7-(((3R,4S)-3-hydroxy-4-((methylthio)methyl)- pyrrolidin-1 -yl )methyl)-5H-pyrrolo[3,2-d]pyrimidin-4-yl )hexanamide (2)

The synthesis was carried out according to the procedure of Example 8 to provide N-(7- (((3R,4S)-3-hydroxy-4-((methylthio)methyl)pyrrolidin-l-yl)methyl)-5H-pyrrolo[3,2- d]pyrimidin-4-yl)hexanamide. 1H NMR (400 MHz, CDCI3) 6 11.03 (s, 1H), 8.57 (s, 1H), 7.67 (s, 1H), 4.10 (dd, J = 5.9, 2.9 Hz, 1H), 4.06 (d, J = 13.7 Hz, 1H), 4.00 (d, J = 13.4 Hz, 1H), 3.36 - 3.25 (m, 1H), 2.98 (dd, J = 10.8, 2.8 Hz, 1H), 2.90 (dd, J = 10.8, 6.0 Hz, 1H), 2.67 (dd, J = 12.2, 5.7 Hz, 1H), 2.60 - 2.36 (m, 5H), 2.07 (s, 3H), 2.04 (s, 1H, partial acetic acid), 1.33 (m, 4H), 0.94 - 0.83 (m, 3H). 13C NMR (101 MHz, CDCI3) 6 173.6, 150.8, 149.1, 142.7, 131.1, 115.9, 110.8, 75.8, 61.1, 57.8, 47.9, 47.2, 37.1, 36.9, 31.2, 24.9, 22.3, 15.6, 13.8.

Example 11: Synthesis of N-(7-(((3R,4S)-3-hydroxy-4-((methylthio)methyl)- pyrrolidin-l-yl)methyl)-5H-pyrrolo[3,2-d]pyrimidin-4-yl)-2,2-diphenylacetamide (3)

The synthesis was carried out according to the procedure of Example 8 to provide N-(7- (((3R,4S)-3-hydroxy-4-((methylthio)methyl)pyrrolidin-l-yl)methyl)-5H-pyrrolo[3,2- d]pyrimidin-4-yl)-2,2-diphenylacetamide. ^XH NMR (400 MHz, CDCI3) 6 10.81 (s, 1H), 10.57 - 10.21 (br s, 1H), 8.52 (s, 1H), 7.47 (s, 1H), 7.28 - 7.13 (m, 10H), 5.15 (s, 1H), 4.31 (br s, 1H), 4.01 (dt, J = 6.4, 3.2 Hz, 1H), 3.91 (d, J = 13.4 Hz, 1H), 3.84 (d, J = 13.4 Hz, 1H), 3.13 (dd, J = 9.4, 7.4 Hz, 1H), 2.79 (dd, J = 10.2, 3.1 Hz, 1H), 2.71 (dd, J = 10.3, 5.9 Hz, 1H), 2.60 (dd, J = 12.7, 7.2 Hz, 1H), 2.45 (dd, J = 12.7, 7.9 Hz, 1H), 2.36 - 2.23 (m, 1H), 2.02 (s, 3H). ¹³C NMR (101 MHz, CDCI3) 6 172.4, 151.1, 148.9, 142.4, 138.0, 130.7, 128.8, 128.8, 128.7, 127.7, 116.1, 112.7, 76.4, 61.8, 58.8, 58.5, 48.3, 47.4, 37.5, 15.6.

Example 12: Synthesis of N-(7-(((3R,4S)-3-hydroxy-4-((methylthio)methyl)- pyrrolidin-l-yl)methyl)-5H-pyrrolo[3,2-d]pyrimidin-4-yl)isobutyramide (4)

The synthesis was carried out according to the procedure of Example 8 to provide N-(7- (((3R,4S)-3-hydroxy-4-((methylthio)methyl)pyrrolidin-l-yl)methyl)-5H-pyrrolo[3,2- d]pyrimidin-4-yl)isobutyramide. ^XH NMR (400 MHz, CDCI3) 6 8.55 (s, 1H), 5.47 (br s, 2H), 4.18 - 4.04 (m, 3H), 3.39 - 3.28 (m, 1H), 3.03 (dd, J = 11.1, 2.8 Hz, 1H), 2.95 (dd, J = 11.0, 6.0 Hz, 1H), 2.80 (m, 1H), 2.72 - 2.61 (m, 1H), 2.53 - 2.39 (m, 3H), 2.06 (s, 3H), 1.28 (d, J = 6.9 Hz, 6H). ¹³C NMR (101 MHz, CDCI3) 6 177.5, 150.6, 149.3, 142.8, 131.5, 115.9, 109.9, 75.5, 60.8, 57.6, 47.9, 47.0, 36.8, 36.0, 23.2, 19.4, 15.6.

Example 13: Synthesis of N-(7-(((3R,4S)-3-hydroxy-4-((methylthio)methyl)- pyrrolidin-1 -yl )methyl)-5H-pyrrolo[3,2-d]pyrimidin-4-yl )benzamide (5)

The synthesis was carried out according to the procedure of Example 8 to provide N-(7- (((3R,4S)-3-hydroxy-4-((methylthio)methyl)pyrrolidin-l-yl)methyl)-5H-pyrrolo[3,2- d]pyrimidin-4-yl)benzamide. ^XH NMR (500 MHz, CDCI3) 6 11.01 (s, 1H), 8.48 (s, 1H), 8.04 - 7.97 (m, 2H), 7.66 - 7.58 (m, 2H), 7.55 - 7.46 (m, 2H), 4.09 (dt, J = 6.2, 3.2 Hz, 1H), 3.98 (d, J = 13.4 Hz, 1H), 3.91 (d, J = 13.4 Hz, 1H), 3.21 (dd, J = 9.5, 7.4 Hz, 1H), 2.90 (dd, J = 10.5, 2.9 Hz, 1H), 2.80 (dd, J = 10.5, 5.9 Hz, 1H), 2.64 (dd, J = 12.8, 7.4 Hz, 1H), 2.51 (dd, J = 12.8, 7.8 Hz, 1H), 2.40 - 2.27 (m, 2H), 2.09 (s, 3H). ¹³C NMR (126 MHz, CDCI3) 6 166.6, 151.0, 149.3, 142.4, 133.2, 132.7, 130.8, 129.0, 127.8, 115.8, 112.1, 76.4, 61.5, 58.4, 48.2, 47.2, 37.3, 15.6.

Example 14: Synthesis of (S)-2-acetamido-N-(7-(((3R,4S)-3-hydroxy-4- ( ( methylthio)methyl)-pyrrolidin-l -yl )methyl)-5H-pyrrolo[3,2-d]pyrimidin-4-yl)-3- phenylpropanamide (6)

The synthesis was carried out according to the procedure of Example 8 to provide (S)- 2-acetamido-N-(7-(((3R,4S)-3-hydroxy-4-((methylthio)methyl)pyrrolidin-l-yl)methyl)-5H- pyrrolo[3,2-d]pyrimidin-4-yl)-3-phenylpropanamide. ^XH NMR (400 MHz, CDCI3) 6 10.86 (s, 1H), 8.44 (s, 1H), 7.66 - 7.55 (m, 1H), 7.53 (s, 1H), 7.15 (d, J = 4.1 Hz, 5H), 5.12 (q, J = 7.3 Hz, 1H), 4.06 (dt, J = 5.6, 2.9 Hz, 1H), 3.99 - 3.85 (m, 2H), 3.26 (q, J = 6.7 Hz, 2H), 3.08 (dd, J = 14.2, 7.9 Hz, 1H), 2.89 (dd, J = 11.3, 5.8 Hz, 1H), 2.80 (q, J = 5.8 Hz, 1H), 2.63 (dd, J = 12.6, 5.7 Hz, 1H), 2.50 - 2.26 (m, 3H), 2.04 (s, 3H), 1.97 (s, 3H). ¹³C NMR (101 MHz, CDCI3) 6 172.1, 171.5, 150.7, 149.3, 142.1, 136.0, 131.2, 129.1, 128.6, 127.1, 116.0, 116.0, 111.0, 75.9, 61.3, 58.2, 58.1, 55.5, 48.1, 47.3, 47.2, 37.6, 37.1, 22.9, 15.6.

Example 15: Synthesis of 2-ethyl-N-(7-(((3R,4S)-3-hydroxy-4-((ethylthio)methyl)- pyrrolidin-l-yl)methyl)-5H-pyrrolo[3,2-d]pyrimidin-4-yl)butanamide (7)

The synthesis was carried out according to the procedure of Example 8 to provide of 2- ethyl-N-(7-(((3R,4S)-3-hydroxy-4-((ethylthio)methyl)pyrrolidin-l-yl)methyl)-5H-pyrrolo[3,2- d]pyrimidin-4-yl)butanamide. ^XH NMR (500 MHz, CDCI3) 6 11.05 (s, 1H), 9.63 (s, 1H), 8.61 (s, 1H), 7.73 (br s, 1H), 4.17 - 4.02 (m, 2H), 3.30 (dd, J = 10.0, 7.7 Hz, 1H), 3.00 - 2.90 (m, 2H), 2.70 (dd, J = 12.8, 7.3 Hz, 1H), 2.59 - 2.49 (m, 3H), 2.46 (dd, J = 10.0, 7.3 Hz, 1H), 2.41-2.33 (m, 2H), 1.75 (ddq, J = 14.6, 8.7, 7.4 Hz, 2H), 1.62 (dqd, J = 14.8, 7.4, 5.2 Hz, 2H),1.22 (t, J = 7.4 Hz, 3H), 0.96 (t, J = 7.4 Hz, 6H). ¹³C NMR (126 MHz, CDCI3) 6 176.4, 150.9, 149.3, 142.5, 131.2, 116.0, 110.9, 76.0, 61.1, 58.0, 51.4, 48.2, 47.4, 34.4, 26.1, 25.7, 14.7, 11.9.

Example 16: Synthesis of N-(7-(((3R,4S)-3-hydroxy-4-((ethylthio)methyl)pyrrolidin- 1 -yl)methyl)-5H-pyrrolo[3,2-d]pyrimidin-4-yl)-2,2-diphenylacetamide (8)

The synthesis was carried out according to the procedure of Example 8 to provide N-(7- (((3R,4S)-3-hydroxy-4-((ethylthio)methyl)pyrrolidin-l-yl)methyl)-5H-pyrrolo[3,2-d]pyrimidin- 4-yl)-2,2-diphenylacetamide. ^XH NMR (500 MHz, CDCI3) 6 10.81 (s, 1H), 8.48 (s, 1H), 7.46 (s, 1H), 7.32 - 7.23 (m, 10H), 5.27 (s, 1H), 4.08 (dt, J = 6.2, 2.8 Hz, 1H), 4.02 (d, J = 13.4 Hz,

1H), 3.95 (d, J = 13.4 Hz, 1H), 3.29 (dd, J = 9.5, 7.3 Hz, 1H), 2.95 (dd, J = 10.9, 2.9 Hz,

1H), 2.87 (dd, J = 10.9, 5.9 Hz, 1H), 2.67 (dd, J = 12.7, 7.1 Hz, 1H), 2.56 - 2.46 (m, 3H),

2.43 - 2.31 (m, 1H), 1.20 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCI3) 6 172.3, 150.9,

149.2, 142.2, 138.0, 131.2, 129.0, 129.0, 128.9, 128.9, 128.9, 128.8, 128.8, 128.8, 128.3,

128.2, 127.8, 115.8, 76.1, 61.2, 58.9, 58.2, 48.2, 47.5, 34.5, 26.1, 14.7.

Example 17: Synthesis of N-(7-(((3R,4S)-3-hydroxy-4-((ethylthio)methyl)pyrrolidin- 1 -yl)methyl)-5H-pyrrolo[3,2-d]pyrimidin-4-yl)benzamide (9)

The synthesis was carried out according to the procedure of Example 8 to provide N-(7- (((3R,4S)-3-hydroxy-4-((ethylthio)methyl)pyrrolidin-l-yl)methyl)-5H-pyrrolo[3,2-d]pyrimidin- 4-yl)benzamide. ^XH NMR (500 MHz, CDCI3) 6 11.07 (s, 1H), 8.47 (s, 1H), 8.03 - 7.97 (m, 2H), 7.73 (s, 1H), 7.65 - 7.58 (m, 1H), 7.51 (t, J = 7.8 Hz, 2H), 4.14 (dt, J = 6.1, 3.2 Hz, 1H), 4.09 (d, J = 13.4 Hz, 1H), 4.02 (d, J = 13.4 Hz, 1H), 3.31 (dd, J = 9.9, 7.6 Hz, 1H), 3.00 (dd, J = 10.9, 3.0 Hz, 1H), 2.92 (dd, J = 10.8, 5.8 Hz, 1H), 2.69 (dd, J = 12.7, 7.2 Hz, 1H), 2.60 - 2.31 (m, 5H), 1.22 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCI3) 6 166.7, 151.0, 149.4, 142.5, 133.2, 132.7, 131.4, 129.0, 129.0, 127.8, 127.8, 115.8, 110.7, 75.9, 61.1, 58.1, 48.2, 47.4, 34.4, 26.1, 14.7. Example 18: Pharmacokinetics, Pharmacodynamics and Animal Bioavailability

Prodrug compound solutions were made up to 7.5 mg/mL in either sterile lx PBS or DMSO. Three male BALB/c mice weighing approximately 25 grams each received 0.1 mL of the prodrug compound either by oral gavage or IP injection for a total dose of 30 mg/kg. Blood was drawn at timed intervals via tail vein clippings for 24 hours (0, 0.5, 1, 2, 4, 8, 24 hrs), after which the animals were sacrificed by CO2 asphyxiation and cervical dislocation in accordance with IAUCUC protocol. 25 pL of whole blood was mixed with 5 pL heparin to prevent clotting, then metabolites and prodrug compound were extracted by mixing with 150 pL of extraction buffer (40% acetonitrile, 40% methanol, 20% water, 0.1% formic acid) containing internal standard (SAM, methyl-ds). The extract was dried by SpeedVac evaporator and reconstituted with 40 pL water. Samples were injected into an Agilent 6490 triple quadrupole mass 148 spectrometer (LC-MS) to obtain normalised peak intensities for prodrugs, activated drugs, and MTA.

Normalised peak areas for each species (prodrug compound, active drug, and MTA) were plotted as a function of time, and the area under the curve (AUC) determined for IP- injected and orally-gavaged animals. AUCs are shown for compound 2 in Table 1. Bioavailability for the prodrug and the active drug and MTA Exposure were determined via the following equations:

Bioavailability (%) = AUC Oral Gavage I AUC IP injection * 100%

MTA Plasma Exposure (%) = AUC Oral Gavage I AUC IP injection * 100%

The amount of active drug (MTDIA or ETDIA) in the plasma after prodrug dose compared to injection of active drug alone was determined by taking the AUC of MTDIA from animals injected with the prodrug and dividing by the AUC of MTDIA from animals injected with the active drug alone and is expressed as a percentage by route of administration (IP injection or oral gavage). The amount of MTA in the plasma after prodrug dose compared to injection of active drug alone was determined by taking the AUC of MTA from animals injected with the prodrug and dividing by the AUC of MTA from animals injected with the active drug alone and is expressed as a percentage by route of administration (IP injection or oral gavage). Assays were performed with n=3 mice per condition. The results are shown in Table 1. Table 1: MTAP prodrug pharmacokinetic and pharmacodynamic parameters in mice administration, both at 30 mg/kg. b Concentration of MTDIA or ETDIA appearing in blood as AUC over 24 hr following oral administration of 30 mg/kg test agent. c Concentration of MTA appearing in blood as AuC over 24 hr following oral administration of 30 mg/kg test agent.

*Value estimated from ratio of concentration at tcMax.

Example 19: Prodrug conversion to MTDIA by HT-29 cells

To a 6-well plate containing IxlO⁶ HT-29 colorectal carcinoma cells was added 10 pM of purified prodrug compound. Cells were incubated for 8 hours, then were washed with lx cold PBS and the metabolites extracted with cold extraction solution (40% acetonitrile, 40% methanol, 20% H2O, 0.1% formic acid) containing an internal standard of SAM (methyl-d3). The extract was transferred to Eppendorf tubes, the cellular debris removed by centrifugation and the supernatant dried in a SpeedVac Evaporator. Dried samples were resuspended in 40 pL H2O and injected into an Agilent 6490 triple quadrupole mass 148 spectrometer (LC-MS). Normalised peak intensities were converted to molar quantities using standard curves for each prodrug compound, and the fractional conversion to active drug was determined as an 8-hour endpoint. The results are shown in Table 2. Table 2: HT-29 colorectal carcinoma cell growth inhibition by MTDIA prodrugs in combination with MAT2A inhibitor AG-270 a Conversion detected after incubation for 8 hours. b Assay performed in the presence of 1 pM MAT2a inhibitor AG-270. c N/A = not applicable.

Example 20: Conversion of MTDIA prodrugs in blood

Standard curves for MTDIA were generated by injection of 250 pL samples from 9.4 to 150 pM in 2-fold dilutions over HPLC (Waters ARC system). Materials were separated by a pBondapak C18 10 pm 125 A 3.9 x 300 mm column with a gradient of: solvent A - 50 mM ammonium formate, pH 4.0, and solvent B - 100% acetonitrile with 0.1% formic acid; 0 - 5 min 100% solvent A, 5 - 22.5 min gradient to 100% solvent B, 22.5 - 25 minutes return to 100% solvent A, and 25 - 30 minutes hold 100% solvent A. Expected elution time for MTDIA was 11.6 minutes. Deidentified human blood samples in heparin anticoagulant tubes were used for conversion of MTDIA prodrugs in both whole or lysed samples to measure the rate of conversion to MTDIA. Whole blood was diluted 1 : 1 in 50 mM K2HPO4 (pH 7.4) followed by a second 1 : 1 (v/v) mix with prodrug to a final concentration of 250 pM prodrug and 25% blood. Prodrugs were incubated with gentle shaking at 37 °C for 0, 1, 3, 9, and 24 hours. The filtrate was collected from an Amicon Ultra 3K centrifugal filter and a 250 pL sample applied to HPLC (Waters ARC system). MTDIA and prodrugs were separated by HPLC as described above.

Lysed blood was prepared by a 1: 1 dilution into 0.1% triton X-100 in 50 mM K2HPO4 (pH 7.4) and incubated on ice for 30 minutes prior to being mixed in a 1: 1 (v/v) ratio with prodrugs to final concentrations of 250 pM and 25% original blood volume. Samples were incubated with gentle shaking at 37 °C for 0, 1, 3, 9, and 24 hours. Metabolites were extracted by use of an Amicon Ultra 3K centrifugal filter and analysed as described above.

Table 3: MTDIA formation from prodrug compounds in whole and lysed human blood a All values are from multiple sampling of a single biological replicate. Standard errors are taken from the error on fitting rate curves.

Example 21: Enzyme inhibition assays

Human MTAP was expressed and purified from E. coli as previously described (Singh, V., et al., Biochemistry, 2005, 44, 11647-11659). Prodrug compounds were purified by HPLC prior to assay. MTAP was assayed in a 96-well plate format by coupling the release of adenine to the generation of hydrogen peroxide by coupling with adenine deaminase from Plasmodium falciparum (pfADA) and xanthine oxidase (XanOx). Detection of peroxide was performed continuously using Amplex red and horse radish peroxidase (HRP) to detect resorufin fluorescence from 510 nm excitation and 590 nm emission. In 96-well plate format, near saturating MTA (500 pM, Km = 4 pM) was added to wells that were pre-incubated with 10 nM MTAP. Reactions contained varying inhibitor concentrations, buffer solution (100 mM phosphate pH 7.4 and 50 mM KCI), and coupled enzymes with Amplex red. Initial rates were determined over 30 min. The inhibition constant K was estimated using the Cheng-Prussof equation: = ICso/ (1 + [S] I K_m) where IC50 is the concentration at which the inhibitor causes 50% inhibition, [S] and Km are the concentration of the substrate in the assay and the Michaelis constant of the substrate, respectively. Assays were performed in triplicate.

Trace amounts of MTDIA in the prodrugs could cause erroneous inhibition, therefore, a second purification was performed by using MTAP protein to remove any tight-binding inhibitor. Prodrug solutions (300-700 pM) were incubated with MTAP (10 pM) in 100 mM phosphate buffer pH 7.4 containing 50 mM KCI. The enzyme-prodrug mixture was allowed to pre-incubate for 30 min to bind any MTDIA. The mixture was eluted through a lOkDa molecular weight retention filter, calibrated by spectral analysis and immediately used in MTAP inhibition assays. Both HPLC- or MTAP-purification showed similar K values (Table 4). Enzyme and cell assays were therefore performed with HPLC-purified drugs.

Table 4: MTDIA and prodrug inhibition of MTAP in vitro and in erythrocytes a Assay performed at 500 pM MTA. K estimated using the Cheng-Prusoff equation using the K_m of 4 pM for human MTAP. b No pre-incubation of enzyme and inhibitor. c Apparent K represents combined inhibition of prodrug and activated compound. Preincubated for 1 hour prior. d Decreased apparent K represents slow-onset inhibition (MTDIA) during the assay and/or erythrocytic conversion of prodrug to MTDIA during a 1 hr preincubation.

Example 22: HT-29 cell growth with MAT2A and MTDIA prodrug intervention

Growth inhibition assays were performed by plating 10³ HT-29 cells to each well of a 96 well plate. Cells were treated with 1 pM AG-270 (MAT2A inhibitor) and varying concentrations of prodrugs ranging from 1 nM to 10 pM. After 5 days of treatment, the media was aspirated and replaced with 110 pL of fresh growth media containing 10% WST-1 prepared growth reagent (Millipore-Sigma). Plates were incubated at 37 °C for 2 hours for WST-1 colour development before transferring 90 pL to a fresh 96-well plate for analysis (25 °C at 440 nm). Corrected absorbances were plotted as a function of concentration and calculated growth IC50 values interpolated from five replicates (Figure 1).

Claims

1. A compound of formula (I) : wherein :

Ri is methyl, ethyl, n-propyl or n-butyl; and

R2 is C1-6 alkyl, C2-6 alkenyl or C2-6 alkynyl each of which may be substituted by one or more phenyl groups or amide groups, or R2 is Ph; or a pharmaceutically acceptable salt thereof.

2. A compound as claimed in claim 1, wherein Ri is methyl or Ri is ethyl.

3. A compound as claimed in claim 1 or claim 2, wherein R2 is C1-6 alkyl.

4. A compound as claimed in any one of claims 1 to 3 wherein, R2 is -(CH2)4CH3, -CH(CH₃)2, or -CH(CH₂CH₃)2.

5. A compound as claimed in any one of claims 1 to 4, wherein R2 is -CH(Ph)20r Ph.

6. A compound as claimed in any one of claims 1 to 4, wherein R2 is C1-6 alkyl substituted by a phenyl group, or one or two amino groups, or an amide group.

7. A compound as claimed in any one of claims 1 to 4, wherein R2 is C1-6 alkyl substituted by a phenyl group and by an amide group.

8. A compound as claimed in claim 6 or claim 7, where in the amide group is NHAc.

9. A compound selected from the group comprising :

10. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

11. A method for inhibiting MTAP (5-methylthioadenosine phosphorylase) in a subject which comprises administering to a subject a therapeutically effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof.

12. A method of treating cancer which comprises administering to a subject a therapeutically effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof.

13. The use of a compound of claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer.

14. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof for use in the treatment of cancer.