BACKGROUND OF THE INVENTION Cancer is a disease of abnormal cell growth often leading to death. Cancer is treated by three principal means; surgical removal of the tumor, therapeutic radiation, and treatment with anti-tumor chemical compounds. Treatment with chemical compounds, termed chemotherapy, is often hindered by the inherent toxicity of the chemicals to the patient and resistance of the tumor to the chemical treatment. Therefore the identification of less toxic anti-tumor agents capable of inhibiting growth of resistant tumors is of great importance.

Nakano et al. disclose that the dithiolanone oxide antiobiotic Leinamycin has anti- tumor activity, supposedly via its DNA cleaving properties [J. Antibiot., 42,1768 (1989)].

Gates et al discloses that simple dithiolanone oxides analogous to a portion of Leinamycin are thiol-activated DNA cleaving agents, either via production of electrophilic intermediates that cause DNA cleavage directly, or via formation of DNA damaging oxygen radicals [Biochemistry, 35,1768 (1996)]. Dithiolane containing calcium channel blockers have been shown to enhance the growth inhibitory effects of conventional cancer chemotherapeutic agents such as doxorubicin and vincristine in otherwise chemotherapeutically resistant tumors, presumably by affecting the multidrug resistance pathway [Yin et al, Cancer Research, 49,4729 (1989) and Eliason et al, Euro. J. of Cancer, 31A, 2354, (1995)].

However, nucleosides linked covalently to dithiolane at the 5 position of the base had very poor anti-tumor activity (J. Med. Chem., 25,522 [1982]).

OBJECTS OF THE INVENTION In one aspect of the invention, an object of the present invention is to provide compunds and methods for the treatment of cancer, and in particular, chemotherapeutically resistant cancer, in mammals.

In another aspect of the invention, an objective of the present invention is to provide methods which relate to the use of 3H-1,2-Dithiolo [4,3-d] pyrimidin-5,7-diones and 4H-1,3- Dithiino [5,4-d] pyrimidin-8-one-7S-oxides and the respective nucleosides and nucleotides in the management of cancer.

In a further aspect of the invention, an objective of the present invention is to provide methods for the use of 3H-1,2-Dithiolo and 4H-1,3-Dithiino [5,4- d] pyrimidin-8-one-7S-oxides and the respective nucleosides and nucleotides in the management of breast cancer.

In other aspects of the invention, objects of the present invention provide compounds and methods for the treatment of neoplasia, hyperproliferative cell growth, psoriasis, chronic inflammatory diseases including rheumatoid and osteoarthritis and microbial infections including viral infections.

These and/or other objects of the present invention may be readily gleaned from the description of the present invention which follows.

DESCRIPTION OF THE INVENTION The present invention is concerned with 3H-1,2-Dithiolo [4,3-d] pyrimidin-5,7-diones and 4H-1,3-Dithiino [5,4-d] pyrimidin-8-one-7S-oxides and their respective nucleosides and nucleotides. These compounds have been shown to have growth inhibiting properties against several forms of human cancer grown in culture.

The method of the present invention involves the use of compounds to treat neoplasia and other diseases and conditions of animals, including humans encompassed by the following formulas: R = H, Cl-C8 (preferably, Cl-C3) alkyl, Cl-C8 (preferably, Cl-C3) hydroxyl alkyl, ribose, deoxyribose and arabinose including a 2'halo-substituted arabinose IX and IXa Where M is Zn, Ca, Mg, or Mn R = H, Cl-C8 (preferably, Cl-C3) alkyl, Cl-C8 (preferably, Cl-C3) hydroxyl alkyl, ribose, deoxyribose, arabinose (including a 2'-halo arabinose such as F, Cl, Br, I).

The compounds of the present invention are used to treat benign and malignant neoplasia, including various cancers such as, stomach, colon, rectal, liver, pancreatic, lung, breast, cervix uteri, corpus uteri, ovary, prostate, testis, bladder, renal, brain/cns, head and neck, throat, Hodgkins disease, non-Hodgkins leukemia, multiple myeloma leukemias, skin melanoma, acute lymphocytic leukemia, acute mylogenous leukemia, Ewings Sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, Wilms Tumor, neuroblastoma, hairy cell leukemia, mouth/pharynx, oesophagus, larynx, melanoma, kidney, lymphoma, among others. Compounds according to the present invention are particularly useful in the treatment of breast cancer, including breast cancer which is of a multiple drug resistant phenotype.

A method of treating hyperproliferative cell growth and psoriasis and related conditions using one or more of the disclosed compositions are other inventive aspects of the present invention.

Further inventive aspects of the present invention relate to the use of the present compositions in the treatment of arthritis and chronic inflammatory diseases, including rheumatoid arthritis and osteoarthritis, among others.

Compounds according to the present invention may also be used as anti-microbials, in particular antivirals for use in the treatment of retroviral infections and other infections including HIV, HSV (herpes simplex virus), heptatitis viruses (A, B and C) and other viruses.

The present invention also relates to methods for inhibiting the growth of neoplasia, including a malignant tumor or cancer comprising exposing the neoplasia to an inhibitory or therapeutically effective amount or concentration of at least one of the disclosed compounds.

This method may be used therapeutically, in the treatment of neoplasia, including cancer or in comparison tests such as assays for determining the activities of related analogs as well as for determining the susceptibility of a patient's cancer to one or more of the compounds according to the present invention.

Methods for treating abnormal cell proliferation or growth of non-transformed cells, including the treatment of psoriasis and for treating chronic inflammatory disease, including arthritis, comprising administering a therapeutically effective amount of one or more of the disclosed compounds for treating the condition or disease are also contemplated within the scope of the present invention.

Detailed Description of the Invention The following terms shall be used throughout the specification to describe the present invention.

The term"patient"is used throughout the specification to describe an animal, preferably a human, to whom treatment, including prophylactic treatment, with the compositions according to the present invention is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal.

The term"effective amount"is used throughout the specification to describe concentrations or amounts of compounds according to the present invention which may be used to produce a favorable change in the disease or condition treated, whether that change is a remission, a decrease in growth or size of cancer or a tumor, a favorable physiological result, a reduction in the growth or elaboration of a microbe, or the like, depending upon the disease or condition treated.

The term"alkyl"is used throughout the specification to describe a hydrocarbon radical containing between one and eight carbon units. Alkyl groups for use in the present invention include linear or branched-chain groups. There term"hydroxyalkyl"is used throughout the specification to describe alcohol-substituted alkyl groups which contain primary, secondary or tertiary alcohol groups anywhere on the alkyl chain.

The term"ribose"is used to describe a five-carbon sugar synthon having a hydroxyl group at the 2'position of the sugar which is traditionally found in naturally occurring nucleoside compounds attached to a base, usually at a nitrogen position of such base. An example of a ribose group appears in Figure 2 (compound 3 a).

The term"deoxyribose"or 2'-deoxyribose is used to describe a five-carbon sugar synthon having a hydrogen group at the 2'position of the sugar which is traditionally found in naturally occurring nucleoside compounds attached to a base, usually at a nitrogen position of such base. An example of a deoxyribose group appears in Figure 2 (compound 3 b).

The term"arabinose"is used to describe a five-carbon sugar synthon having a hydroxyl group at the 2'position of the sugar which is traditionally found in many synthetic nucleoside compounds attached to a base, usually at a nitrogen position of such base. An example of an arabinose group appears in Figure 2 (compound 6a, where R is hydrogen).

The term"halogen-substituted arabinose"or"2'-halogen-substituted arabinose"is a halogen- substituted five carbon sugar synthon exemplified by the chemical structure which appears in Figure 2 (compound 6b, where R is hydrogen).

The term"neoplasia"is used to describe the pathological process that results in the formation and growth of a neoplasm, i. e., an abnormal tissue that grows by cellular proliferation more rapidly than normal tissue and continues to grow after the stimuli that initated the new growth cease. Neoplasia exhibits partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue which may be benign (benign tumor) or malignant (carcinoma). The term "cancers used as a general term to describe any of various types of malignant neoplasms, most of which invade surrounding tissues, may metastasize to several sites and are likely to recur after attempted removal and to cause death of the patient unless adequately treated. As used herein, the term cancer is subsumed under the term neoplasia.

A preferred therapeutic aspect according to the present invention relates to methods for treating neoplasia, including benign and malignant tumors and cancer in animal or human patients, and in preferred embodiments, cancers which have developed drug resistance, such as multiple drug resistant breast cancer comprising administering therapeutically effective amounts or concentrations of one or more of the compounds according to the present invention to inhibit the growth or spread of or to actually shrink the neoplasia in the animal or human patient being treated.

Pharmaceutical compositions based upon these novel chemical compounds comprise the above-described compounds in a therapeutically effective amount for the treatment of a condition or disease such as neoplasia, including cancer, hyperplastic cell growth (the abnormal cell growth of a non-transformed cell) or a related condition or disease optionally in combination with a pharmaceutically acceptable additive, carrier or excipient.

Certain of the compounds, in pharmaceutical dosage form, may be used as prophylactic agents for preventing a disease or condition from manifesting itself. In certain pharmaceutical dosage forms, the pro-drug form of the compounds according to the present invention may be preferred. In particular, prodrug forms which rely on Cl to C2o ester groups or amide groups (preferably on the 5'OH position of ribose, deoxyribose or arabinose sugars or other free hydroxyl groups or alternatively, one or more nitrogen groups which may be formed into an amide group, or monophosphate groups, diphosphate groups, triphosphate groups or phosphodiester groups on the sugar synthons) may be particularly useful in this context.

The present compounds or their derivatives, including prodrug forms of these agents, can be provided in the form of pharmaceutically acceptable salts. As used herein, the term pharmaceutically acceptable salts or complexes refers to appropriate salts or. complexes of the active compounds according to the present invention which retain the desired biological activity of the parent compound and exhibit limited toxicological effects to normal cells.

Nonlimiting examples of such salts are (a) acid addition salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, and polyglutamic acid, among others; (b) base addition salts formed with metal cations such as zinc, calcium, sodium, potassium, and the like, among numerous others.

Modifications of the active compound can affect the solubility, bioavailability and rate of metabolism of the active species, thus providing control over the delivery of the active species. Further, the modifications can affect the anticancer activity of the compound, in some cases increasing. the activity over the parent compound. This can easily be assessed by preparing the derivative and testing its anticancer activity according to known methods well within the routineer's skill in the art.

Compounds according to the present invention may include the respective nucleoside and nucleotide analogs. As used herein, the term"nucleoside"is used as is well known in the art, i. e., as a compound containing a base (e. g., any number of related pyrimidine or purine bases or compounds which are derivatives of these bases) to which is attached a ribose or deoxyribose sugar. The term"nucleotide"refers to a nucleoside compound containing a phosphate group at the 5'OH position of sugar synthon. Monophosphates, diphosphates, triphosphates and phosphodiesters are all subsumed under the term"nucleotide"as used herein.

The compounds of this invention may be incorporated into formulations for all routes of administration including for example, oral and parenteral including intravenous, intramuscular, intraperitoneal, intrabuccal, transdermal and in suppository form.

Pharmaceutical compositions based upon these novel chemical compounds comprise the above-described compounds in a therapeutically effective amount for treating neoplasia, cancer and other diseases and conditions which have been described herein, including viral diseases, psoriasis, arthritis and chronic inflammatory diseases, optionally in combination with a pharmaceutically acceptable additive, carrier and/or excipient. One of ordinary skill in the art will recognize that a therapeutically effective amount of one of more compounds according to the present invention will vary with the infection or condition to be treated, its severity, the treatment regimen to be employed, the pharmacokinetics of the agent used, as well as the patient (animal or human) treated.

In the pharmaceutical aspect according to the present invention, the compound according to the present invention is formulated preferably in admixture with a pharmaceutically acceptable carrier. In general, it is preferable to administer the pharmaceutical composition in orally-administrable form, but a number of formulations may be administered via a parenteral, intravenous, intramuscular, transdermal, buccal, subcutaneous, suppository or other route. Intravenous and intramuscular formulations are preferably administered in sterile saline. Of course, one of ordinary skill in the art may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration without rendering the compositions of the present invention unstable or compromising their therapeutic activity. In particular, the modification of the present compounds to render them more soluble in water or other vehicle, for example, may be easily accomplished by minor modifications (salt formulation, esterification, etc.) which are well within the ordinary skill in the art. It is also well within the routineer's skill to modify the route of administration and dosage regimen of a particular compound in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect to the patient.

In certain pharmaceceutical dosage forms, the pro-drug form of the compounds may be preferred. One of ordinary skill in the art will recognize how to readily modify the present compounds to pro-drug forms to facilitate delivery of active compounds to a targeted site within the host organism or patient. The routineer also will take advantage of favorable pharmacokinetic parameters of the pro-drug forms, where applicable, in delivering the present compounds to a targeted site within the host organism or patient to maximize the intended effect of the compound.

The amount of compound included within therapeutically active formulations according to the present invention is an effective amount for treating the infection or condition. In its most preferred embodiment, the present compounds, and in particular, compound I (where R=H), are used for treating multiple drug resistant breast cancer. In general, a therapeutically effective amount of the present preferred compound in dosage form usually ranges from slightly less than about 0.025mg./kg. to about 2.5 g./kg., preferably about 2.5-5 mg/kg to about 100 mg/kg of the patient or considerably more, even more preferably about 20-50 mg/kg, more preferably about 25 mg/kg, depending upon the compound used, the condition or infection treated and the route of administration, although exceptions to this dosage range may be contemplated by the present invention. In the case of breast cancer, the compound I (R=H) is preferably administered in amounts ranging from about 1 mg/kg to about 100 mg/kg. This dosage range generally produces effective blood level concentrations of active compound ranging from less than about 0.04 to about 400 micrograms/cc or more of blood in the patient.

Administration of the active compound may range from continuous (intravenous drip) to several oral administrations per day (for example, Q. I. D.) and may include oral, topical, parenteral, intramuscular, intravenous, sub-cutaneous, transdermal (which may include a penetration enhancement agent), buccal and suppository administration, among other routes of administration.

To prepare the pharmaceutical compositions according to the present invention, a therapeutically effective amount of one or more of the compounds according to the present invention is preferably intimately admixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques to produce a dose. A carrier may take a wide variety of forms depending on the form of preparation desired for administration, e. g., oral or parenteral. In preparing pharmaceutical compositions in oral dosage form, any of the usual pharmaceutical media may be used. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives including water, glycols, oils, alcohols, flavouring agents, preservatives, colouring agents and the like may be used. For solid oral preparations such as powders, tablets, capsules, and for solid preparations such as suppositories, suitable carriers and additives including starches, sugar carriers, such as dextrose, mannitol, lactose and related carriers, diluents, granulating agents, lubricants, binders, disintegrating agents and the like may be used. If desired, the tablets or capsules may be enteric-coated or sustained release by standard techniques.

For parenteral formulations, the carrier will usually comprise sterile water or aqueous sodium chloride solution, though other ingredients including those which aid dispersion may be included. Of course, where sterile water is to be used and maintained as sterile, the compositions and carriers must also be sterilized. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.

The present compounds may be used to treat animals, and in particular, mammals, including humans, as patients. Thus, humans, equines, canines, bovines and other animals, and in particular, mammals, suffering from tumors, and in particular, cancer, or other diseases as disclosed herein, can be treated by administering to the patient an effective amount of one or more of the compounds according to the present invention or its derivative or a pharmaceutically acceptable salt thereof optionally in a pharmaceutically acceptable carrier or diluent, either alone, or in combination with other known pharmaceutical agents, depending upon the disease to be treated). This treatment can also be administered in conjunction with other conventional cancer therapies, such as radiation treatment or surgery.

The active compound is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount for the desired indication, without causing serious toxic effects in the patient treated.

The compound is conveniently administered in any suitable unit dosage form, including but not limited to one containing 1 to 3000 mg, preferably 5 to 500 mg of active ingredient per unit dosage form. An oral dosage of 25-250 mg is usually convenient.

The concentration of active compound in the drug composition will depend on absorption, distribution, inactivation, and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. The active ingredient may be administered at once, or may be divided into a . number of smaller doses to be administered at varying intervals of time.

Oral compositions will generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound or its prodrug derivative can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.

The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a dispersing agent such as alginic acid or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it can contain, in addition to material-of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents.

The active compound or pharmaceutically acceptable salt thereof may also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like.

A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

The active compound or pharmaceutically acceptable salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as other anticancer agents, and in certain instances depending upon the desired therapy or target, antibiotics, antifungals, antinflammatories, or antiviral compounds.

Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include. the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. If administered intravenously, preferred carriers include, for example, physiological saline or phosphate buffered saline (PBS).

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

Liposomal suspensions may also be pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. For example, liposome formulations may be prepared by dissolving appropriate lipid (s) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container.

An aqueous solution of the active compound are then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension. Other methods of preparation well known by those of ordinary skill may also be used in this aspect of the present invention.

A wide variety of biological assays have been used and are accepted by those skilled in the art to assess anti-cancer activity of compounds. Any of these methods can be used to evaluate the activity of the compounds disclosed herein.

One common method of assessing activity is through the use of test panels of cancer cell lines. These tests evaluate the in vitro anti-cancer activity of particular compounds in cancer cell lines, and provide predictive data with respect to the use of tested compounds in vivo. Other assays include in vivo evaluations of the compound's effect on human or in an appropriate animal model, for example, using mouse tumor cells implanted into or grafted onto mice or in other appropriate animal models.

Chemical Synthesis Several compounds of this invention are synthesized by oxidative cleavage of the corresponding p-anisaldehyde dithioacetal. The corresponding nucleosides and nucleotides are synthesized by traditional methods, methods which are well known in the art. All compounds may be prepared by analogy using the methods taught herein as well as readily available methods used in the art. See, for example, Kishi, et al., J. Am. Chem Soc. 1973, 95,6490-6492; Kishi, et al., J. Am. Chem Soc.. 1973,95,6492-6493; Kishi, et al., J. Am Chem Soc.. 1973,95,6493-6495; Kishi, et al., J. Am. Chem Soc. 1973,95,6493-6495, Fukuyama, et al., J. Am. Chem. Soc., 1976,98,6723-6724; Kanda, et al., J. Am. Chem. Soc., 1993,115,8451-8452; Vorbruggen, et al. J. Org. Chem., 1974,39,3654,3660,3668,3672; Vorbruggen, et al., Chem. Ber. Johnson, et al. Nucleosides & Nucleotides Jung, et al., J. Org. Chem., Kishi, et al., J. Am. Chem. Soc.

Watanabe, et al., J. Med. Chem., 1979,22,21; Reichman, et al., J. Carbohyd. Res., 1975,42,233.

The following provides a schematic general synthesis of the individual compounds of the present invention.

Compound I (3H-1,2-dithiolo [4,3-d] pyrimidin-5,7 dione) As set forth in the attached scheme in Figure 4, the preparation of I is accomplished by the acid catalyzed condensation of p-anisaldehyde with 2-mercaptoacetate, forming the corresponding bis-thioacetal. The latter is cyclized in sodium ethoxide to 2- (p- methoxyphenyl)-5-hydroxy-6-ethoxycarbonyl-4H-1,3-dithiin (I-1). Reaction of I-1 with 2- methyl-2-thio-pseudouronium iodide and potassium hydroxide produces the dithiino [5,4-d]- pyrimidine (I-2). Hydrolysis of the methylthio group with HC1 in ethanol produces the 1,3- dithiinouracil (I-3). Treatment of I-3 with concentrated sulfuric acid, or with metachlorobenzoic acid (MCPBA) followed by BC13 gives the desired compound I.. Note that this synthesis is very similar to the synthesis which is described in Figure 1 of the present application.

Compounds II (the N-1-alkyl cytosine analogs of I) As set forth in attached Figure 5 (II), I-2 (above) is chlorinated with POCL3 in the presence of dimethylaniline to give the 4-chloro derivative (11-1). Hydrolysis of the 2- methylthio group of 11-1 gives the 2-oxo compound (11-2) which is converted by treatment with MCPBA and BC13 to the cyclic disulfide 11-3. Silylation with hexamethylenedisilizane followed by treatment with R-X (where R = alkyl, hydroxyalkyl, ribosyl, or 2'-deoxyribosyl, and X = chlorine, bromine), in the presence of triethyl amine gives the N-alkyl derivative II- 4, which upon treatment with ammonia in ethanol gives the desired N-1-alkyl (or N-1- glucosyl) cytosine analog, II.

Compound III As set forth in attached Figure 5 (III), the 2-methylthio group of I-2 is reacted with concentrated HC1 or HF to give the corresponding 2-chloro or 2-fluoro derivatives (III-1), which then is deprotected as in the previous examples either, using conc. sulfuric acid or MCPBA followed by BC13 to give the halogen analogs (III).

Compound IV As set forth in attached Figure 5 (IV), compound I is silylated and reacted with RX, as in the synthesis of II. Removal of the silyl group gives IV.

Compound V As set forth in attached Figure 5 (V), starting with the thymine derivate V-1 (eg, appropriately protected 4-thiothymidine), the methyl group is selectively brominated using, for example, NBS, to give V-2. V-2 is then reacted with sodium sulfhydride to give the 5- thiomethyl derivative V-3. Oxidation with air at high dilution results in formation of the cyclic disulfide, V.

Compound VI As set forth in attached Figure 6 (VI), the parent compound I is silylated (as described for compound II above), and subsequently lithiated using n-butyl lithium and tert- butylpotassium oxide to give VI-1. VI-1 is then reacted with the appropriate alkyl halide, RX (where R = alkyl, hydroxyalkyl, ribosyl or 2'-deoxyribosyl) and triethylamine. Desilylation with acid/water gives directly VI. Alternatively, the synthesis can be carried out by first brominating I with NBS and then coupling it with the lithium derivative of the appropriate alkyl group (Rli).

Compound VII As set forth in attached Figure 6 (VII), the ethoxy carbonyl derivative I-1 is reacted with guanidine in sodium ethoxide to give the 2-amino pyrimidine dithiane (VII-1). VII-1 was converted to the dithiino derivative in the usual manner (using MCPBA followed by BC13) to give the 2-amino substitued derivate, VII-2 (III C). VII-2 is silylated followed by lithiation to VII-3, which then was reacted with RX followed by desilylation with acid/water to give VII.

Compound VIII As set forth in attached Figure 6 (VIII), the 2-chloro substituent of III-A is removed by catalytic hydrogenation to give VIII-1 which then is silylated and subsequently lithiated to give VIII-2. VIII-2 is coupled with RX as defined above and then desilylated to give compounds VIII.

Compounds IV, IX and X.

As set forth in attached Figure 6 (IX), compound IV is reacted with MCPBA to give IX and X.. IX can be readily chelated to produce IXa.

Compound XI As set forth in attached Figure 7 (XI), compound IX is oxidized with potassium permanganate to give the sulfone, XI.

Compound XII As set forth in attached Figure 7 (XII), compound I is reduced with DTT and reacted with a mixture of formic acid and acetic anhydride resulting in formylation at S-5 followed by ring closure to give the 2-hydroxy 1,3-dithiin, XII.

Compound XIII and XIV As set forth in attached Figure 7 (XIII and XIV), compound IV is reduced with DTT to 5-mercapto-6-thiomethyluracil derivative XIII-1. The latter is reacted with formaldehyde, then MCPBA to give XIII and XIV.

Compound XVa Compound XIV is oxidized with permanganate to give the sulfone, XVA.

Compound XVb As set forth in attached Figure 7, compound XIII-1 is methylated with methyl iodide, followed by oxidation to the sulfoxide formation with MCPBA, and to the sulfone with potassium permanganate.

Compound XVI Compound XIII is oxidized with potassium permanganate to give XVI.

The introduction of the cyclic dithiane attached at the 5,6 position of the pyrimidine moiety surprisingly confers a significantly higher potency to the compounds of the present invention. In addition, this substitution also surprisingly confers a decrease in resistance of the tumors. The introduction of sugar substituents at the 1 position of the pyrimidine moiety can be accomplished according to the methods routinely used in the art or as otherwise disclosed in the present specification.. See, for example, Kotik, et al., J. Org. Chem.. 1969, 34,3806.

Structural analogy with leinamycin and other dithiolanone oxide derivatives shown to have DNA cleaving ability suggests that M-47 (Structure 1, where R = H) may act by one of two proposed mechanisms: 1) nucleophilic attack of thiols on the dithiolanone ring producing an electrophilic intermediate that causes DNA cleavage, or 2) thiol-mediated oxygen-radical formation. Alternatively, based on the powerful chelating affinity of 5- mercaptouracil and M-47 toward zinc, as well as on analogy with the proposed mode of action of dithianes (such as NSC 624151) against the zinc fingers of retroviral nucleocapsid proteins, the mechanism of M-47 may involve binding to the zinc fingers of replication proteins, thereby inhibiting ssDNA function and repair.

EXAMPLES The following examples illustrate the invention.

EXAMPLE 1 Synthesis of 3H-1,2-Dithiolo [4,3-d] pyrimidin-5,7-diones and 4H-1,3-Dithiino [5,4- d] pyrimidin-8-one-7S-oxides. a) Methods of preparation of intermediates and final products (see Figure 1): M47-A: Diethyl 4 (p-anisyl)-3,5-dithiopimelate Prepared in 86% yield by the condensation of p-anisaldehyde with 2 equivalents of ethyl 2-mercaptoacetate (also known as ethyl thioglycolate), using p-toluenesulfonic acid as a catalyst. The product was characterized by NMR and IR.

M47-B: 2- (p-Anisyl)-6-carbethoxy-5-hydroxy-4H-1,3-dithiine.

Sodium (7.0 g, 0.304 eq) was dissolved in 160 mL of absolute ethanol under inert atmosphere at 0°C, and to this solution was added (slowly) M47-A (90 g, 0.251 mole). The reaction was allowed to warm to ambient temperature and was stirred under inert atmosphere for 16 hr. Diethyl ether (300 mL) was added to precipitate a sodium salt of M47-B. The precipitated yellow salt was filtered and washed with diethyl ether. The salt was then suspended in 150 mL of water, the pH adjusted to 2 with dilute hydrochloric acid, and the solution extracted with dichloromethane. After water washing of the dichloromethane extract and thorough drying with a drying agent such as anhydrous sodium sulfate, the extract was concentrated at reduced pressure to yield 60 g (85% yield) of M47-B as a yellow oil which could be used without further purification in the next step, or could be crystallized from absolute ethanol to give a solid of mp 61-63°C. This material was characterized by NMR, IR, UV and elemental analysis (see below).

M47-C: 2- (p-Anisyl)-6- (methylthio)-4H-1, 3-dithiino [5,4-d] pyrimidin-8-one.

M47-B (58.0 g, 0.207 mole), 2-methyl-2-thiopseudourea sulfate (40.0 g, 0.144 mole) and potassium hydroxide (13.0 g, 0.23 mole) were combined in 600 mL of ethanol and refluxed for 15 minutes, then stirred for 16 hr at ambient temperatures. The precipitate was collected by filtration, and the filtrates were diluted with 500 mL water, inducing precipitation of a second crop of crystals. The combined portions of solid precipitates were washed with water, cold ethanol, and diethyl ether to give M47-C as a yellow solid (16.2 g, 23.1 % yield), mp 236-238°C.. The product was characterized by NMR, IR, UV and elemental analysis.

M47-D: 2- (p-Anisyl)-4H-1, 3-dithiino [5,4-d] pyrimidin-6,8-dione.

To a suspension of M47-C (6.0 g, 0.018 mole) in 1.4 L of ethanol was introduced 40 mL of 20% aqueous hydrogen chloride and the mixture was refluxed for 16 hr. The resulting clear yellow solution was concentrated at reduced pressure to remove the ethanol, and the resulting precipitate was filtered and washed with water, cold ethanol and diethyl ether until the ether filtrates were colorless. A further washing with acetone and diethyl ether gave 4.5 g (82.3% yield) M47-D, mp 255-260°C (dec.). The product was characterized by NMR, IR, UV and elemental analysis.

M47-E: 3H-1,2-Dithiolo [4,3-d] pyrimidin-5,7-dione.

M47-D (2.1 g, 0.0068 mole) was oxidized with m-chloroperbenzoic acid (1.2 g, 0.007 mole in ethanol at ambient temperature over an 18 hr period. The resulting precipitate was filtered and washed with aqueous sodium bicarbonate, giving 1.7 g (77.0% yield) of 2- (p- anisyl)-4H-1,3-dithiino [5,4-d] pyrimidin-6,8-dione-1-(or 3-)-S-oxide. The crude product exhibited the sulfoxide IR absorption peak at 1055 cm-1. While the extreme insolubility of this S-oxide prevented further purification, the analogous 5,7-dichloro derivative, which had the same IR peak, was fully purified and characterized. M-47-D (0.52 g, 0.0016 mole) was stirred in 7 mL of concentrated sulfuric acid at ambient temperature for 5 hr. The reaction solution was then quenched with icewater and the resulting precipitate filtered and washed with water and acetone, to yield 0.21 g (0.0011 mole, 69.6% yield) of M47-E, mp 248-253°C (slow dec.). This compound was characterized by NMR, IR, UV and Mass Spectroscopy.

While the elemental analysis was unsatisfactory, the sulfur/nitrogen ratio was the correct calculated ratio. b) Spectroscopic Data for M47-A through M47-E: M47-A: NM47R (CDC13): _ 1.12 (t, J=7.0 Hz, 6H), 3.11 (d, J=15.5 Hz, 2H), 3.46 (d, J=15.5 Hz, 2H), 3.76 (s, 3H), 4.15 (q, J=7.0 Hz, 4H), 5.32 (s, 1H), 6.81 (d, J=9 Hz, 2H), 7. 37 (d, J=9 Hz); IR (neat): 1725 cm-1 (C=O, ester).

M47-B: NMR (CDC13): _ 1.27 (t, J=7 Hz, 3 H), 3.62 (s, 2H), 3.81 (s, 3H), 4.27 (q, J=7 Hz, 2H), 5.35 (s, 1H), 6.86 (d, J=8.5 Hz, 2H), 7.44 (d, J=8.5 Hz, 2H), 12.7 (s, 1H); IR (KBr): 3360,3600 cm-1 (broad, OH), 1645 cm-1 (C=O, ester); UV (CHC13): max 310 nm (3.59 x 103), 278 nm (sh, 3.71 x 103), 242 nm (6.68 x 103), min 294 nm (2.89 x 103); ANAL.: Calc. for C14H16S204: C, 53.83; H, 5.16; S, 20.53.

Found: C, 53.90; H, 5.26; S, 20.46.

M47-C: NMR (DMSO-d6): 2.43 (s, 3H), 3.56 (d, J=7.0 Hz, 1H), 4.16 (d, J=7.0 Hz, 1H), 5.63 (s, 1H), 6.90 (d, J=8.5 Hz, 2H), 7.36 (d, J=8.5 Hz, 2H); IR (KBr): 1635 cm-1 (C=O, amide); UV (95% ethanol): max 322 nm (1.02 x 104), 266 nm (5.2 x 103), min 290 nm (1.09 x 103); ANAL.: Calc. for C14H14N202S3: C, 49.68; H, 4.17; N, 8.28; S, 28.42.

Found: C, 49.83; H, 4.19; N, 8.17; S, 28.28.

M47-D: NMR (DMSO-d6): 3.46 (d, J=17.0 Hz, 1H), 3.70 (s, 3H), 3.99 (d, J=17.0 Hz, 1 H), 5.54 (s, 1H), 6.80 (d, J=8.5 Hz, 2H), 7.27 (d, J=8.5 Hz, 2H), 11.1 and 11.3 (broad,- NH); IR (KBr): 1730,1635 cm-1 (C=O, amides); UV (95% ethanol): max 310 nm (4.8x 103), 279 nm) 3.20x 103); ANAL.: Calc. for C13H1203N2S2: C, 50.62; H, 3.92; N, 9.08; S, 20.80.

Found: C, 50.54; H, 3.98; N, 9.03; S, 20.83.

M47-E: NMR (DMSO-d6): 4.49 (s), 11.58 (broad, exchangeable with D20); IR (KBr): 1700,1655 cm-1 (C=O, amides); UV (95% ethanol): max 279 nm (1.18 x 104), 224 nm (2.0 x 104), min 252 nm (3.62 x 104),; after dithiothreitol (DTT) was added max 340 nm (2.88 x 104); ANAL.: Calc. for C5H4N202S2: C, 31.91; H, 2.14; N, 14.88, S, 34.04 Found: C, 30.63; H, 3.12; N, 12.89, S, 29.37. b) Methods of preparation of nucleosides and nucleotides of base units As shown in attached figure 2, base units according to the present invention may be derivatized to the corresponding nucleoside and nuceotide compounds by well known methods in the art. As set forth in attached figure 2, the appropriately protected (acyl, etc.) ribose or deoxyribose acetal derivative 1 as the acetyl or chloro derivative may be coupled to silylated 3H-1,2-Dithiolo [4,3-4] pyrimidin-5,7-dione (M-47E) using the method as described <BR> <BR> <BR> <BR> in Vorbruggen, J. Org. Chem., 3660,3668,3672 and Chem. Ber. 1981,114, 1234 to produce derivative 2, containing ester blocking groups at the hydroxyl positions of the sugar, which can be removed with sodium cyanide in methanol to produce the corresponding nucleoside (ribofuranosyl 3a) or deoxyribonucleoside (2'-deoxyribofuranosyl 3b). See, Johnson, et al. Nucleosides & Nucleotides Note that coupling of the the riboside derivative corresponding to la provides a single p-anomer (putatively beccause of steric hindrance about the a-position, coupling of the deoxyribo derivative lb results in a mixture of a and ß derivatives, which may be separated using standard chromatographic techniques.

The arabinofuranosyl derivatives may prepared by an analogous approach. Similar coupling of benzylated arabinofuranosyl chloride 4a or a bromide derivative of its 2-deoxy-2- fluoro analog (see, Watanabe, at al., J. Med. Chem., 1979,22,21 and Reichman, et al. and Carbohvd. Res., gives the corresponding anomeric mixtures of protected nucleosides 5. Separation of these anomeric mixtures of nucleosides will provide the ß- anomers of 5 which can be debenzylated by treatment with iodotrimethylsilane (see, Jung, et al., J. Org. Chem., to give the target nucleosides 6a and 6b, respectively.

Alternatively, if difficulties are encountered, the debenzylation step can be effected by treatment with BC13 which is compatible with the epidithio functionality of 6. See Kishi, et al., J. Am. Chem. Soc. 1973,95,6490.

Biology a) Cell Growth Inhibition. The antiproliferative potency of M-47 was determined by the sulforhodamine B assay, which is based on the colorimetric determination of total cell protein remaining after drug treatment (Invest. New Drugs, 8,347 [1990], J. Natl. Cancer Inst., 82,1107, [1990]). Human colon, ovarian, lung and both"wild-type"and"multi- drug resistant"breast tumor cells were plated at a density of 400 cells/well in 96-well plates and were allowed to attach overnight. M-47 was dissolved in dimethylsulfoxide (DMSO) and further diluted with growth medium (RPMI 1640). Triplicate wells were exposed to various treatments. After 96 h of incubation, 100) ice-cold 50% trichloroacetic acid were added to each well and incubated for 1 h at 4°C. Plates were then washed five times with water to remove trichloroacetic acid and serum proteins, and 50p1 of 0.4% sulforhodamine B were added to each well. Following the 5-min. incubation, plates were rinsed five times with 0.1 % acetic acid and air dried. The dye was then solubilized with 10mm Tris base (pH 10.5) for 5 min. on a rotational shaker.

Absorbance was measured at 570 nm. Experiments were repeated at least twice.

Cytotoxicity assays were also repeated with M-47 using a long (72 hours) and short (4 hours) exposure period on the"wild-type"and"multi-drug-resistant"breast cancer cells (Figure 2).

Results : The ICso values determinedfollowing a 72 hour exposure to M-47 were 2.5, uM and 3.1 Ifor the"wild-type"and"multi-drug resistant"human breast cells respectively. The <BR> <BR> <BR> <BR> ICso values determinedfrom this assay were 18AMand exposure respectively. Similar results were obtainedfor"wild-type"colon, ovarian and lung tumors. b) Effect of M-47 directly on DNA. The ability of M-47 to cause strand breaks directly on cell-free DNA in the absence and presence of a chemical reducing agent was determined.

Plasmid, pUC118, was incubated in 12 p1 buffer (0.01M NaP04/0. 145M NaCI) containing M-47 alone or M-47 plus dithiothreitol (DTT). M-47 was added at 1 M or 2pM and DTT was at 1 ßM. A positive control incubation included 0.1 mM CuSO4 and 1.0 mM H202. Plasmid in buffer alone served as the negative control. After a 30 min. incubation at room temperature, the plasmid was precipitated and analyzed on a 0.9% agarose gel with ethidium bromide straining. The amount of plasmid in supercoiled and relxed forms was determined by densitometry using a laser densitometer to scan the photographic negative of the stained gel. The amount of nicked plasmid and average nicks per plasmid were then calculated as follows.

Nicked fraction = [ (density, relaxed form)/1.6] [density, supercoiled form + (density, relaxed form/1.6) 1-nicked = unnicked fraction Average nicks per plasmid =-ln (unnicked fraction) The results in Table I demonstrate that M-47 had no direct strand breaking activity under the conditions usedfor this experiment.

Table 1. Results from the forms analysis of pUC118 exposed to M-47.

Treatment Nicked Fraction Average Nicks per Plasmid Control 0.19 0.21 H202/CuSO4 0.62 0.97 M-47, lnM 0.14 0.15 M-47,2 0.07 0.07 M-47, lpM/DTT, lgM 0.21 0.24 M-47,2M/DTT, lllM 0.18 0.20 C) Effect of M-47 on DNA within cells. Two approaches were used to evaluate the effect of M-47 on DNA within cells. The fraction of duplex DNA in cells after treatment was determined using a fluorescence enhancement assay (3) (fea), and the amount of DNA damage in individual cells was evaluated by the"comet assay" (4). LCC6 cells were plated at IX106/flask in 10ml medium, grown for 2 days and then treated with M-47. After the incubation period, cells were washed with PBS and resuspended in 1.5-2.0 ml PBS. For FEA, cells were divided into three tubes and treated as follws: Tube A: Cells plus 0.1M NaOH, 0. 1M HC1 and dye solution, followed by 15 seconds sonication. The DNA in this tube will be all duplex form.

Tube B: Cells plus 0.1M NaOH incubated undisturbed for 30 minutes in the dark, then 0.1M HC1 and dye solution followed by 15 seconds sonication. The DNA in this tube will be partially duplex form based on the extent of DNA damage.

Tube C: Cells plus 0.1M NaOH sonicated 5 seconds and incubated 30 minutes, then 0.1M HC1 and dye solution followed by 15 seconds sonication. The DNA in this tube will be completely single stranded (no duplex form).

The fluorescence in each tube was read and an F value (the amount of DNA in duplex form after partial alkaline denaturation) was determined by: F= (B-C)/ (A-C) The lower the F value the more DNA damage induced by the agent Table 2. The extent of DNA damage induced by M-47 in LCC6 cells.

Treatment Time (hours) F % of control Control 1 0.93 100.0 4 0.54 100.0 5pM 1 0.73 78.5 4 0.50 92.5 10tm 1 0.68 73.1 4 0.10 18.5 30pM 1 0.59 63.0 4 0.13 24.0 lOOpM 1 ND- 4 0.0 0. 0 For the comet assay, cells treated as above were embedded in low melting point agarose on slides that had been precoated with high melting point agarose. After a period of cell lysis and alkaline denaturation, the slides were subjected to electrophoresis in alkaline buffer for 15-25 minutes. Cells were rinsed in Tris buffer to neutralize the alkali and were stained with ethidium bromide. Stained cells were viewed by fluorescence microscopy to evaluate the formation of the comets indicating damaged DNA leaving the nucleus during electrophoresis.

The results from Table 2 demonstrate there was a dose dependent increase in DNA damage <BR> <BR> <BR> <BR> <BR> in cells treated with M-47. This is in agreement with the results of the comet assay, where a dose dependent increase in cometformation was also observed. This indicated that M-47 was damaging DNA directly via single strand breaks or by theformation of alkali labile adducts that caused strand breaks during the alkaline denaturation step and subsequent electrophoresis.