GLYCO-PHOSPHORYLATED BIOLOGICALLY ACTIVE AGENTS

Field of Present Invention

The invention relates to glyco-phosphorylatcd therapeutic biologically active agents and their use for targeted and/or sustained release treatment regimens. Background of the Invention

Glyco-conjugation is a known art and it has advantages of increasing water solubility of the drugs, enhancing bioavailability, reducing liver metabolism, enhancing transport across the blood brain barrier, and promoting cancer cell adhesion and internalization due to glucose being the fuel for the cell's energy process.

However, glycosylation of organic molecules is cumbersome and not cost effective. Since glycosidation is substrate specific, there is no one unified method to bio- conjugate with glycosyl groups. Also, the choice of glycosyl donor, solvents and catalysts lead to mixtures of α and β anomers. The choice of glucosyl donors and reactaiits plays an important role to a high yielding and cleaner β-glucosides.

In view of the potential drawbacks to current approaches, there exists a need for more efficient and reliable method of glycosylation of biologically active agents.

Objects of the Invention

The main object of the present invention is to develop glyco-phosphorylated therapeutic biologically active agents.

Another object of the present invention is to develop a process for preparing glyco- phosphorylated therapeutic biologically active agents.

Yet another object of the present invention is develop methods of management of tumor, inflammation, infection and promoting passage across blood-brain barrier and related biological conditions. Statement of the Invention

The present invention relates to a compound of formula I:

O

Il DX-P-OA

I

°- (D wherein

DX is a radical of a biologically active agent comprising atom X;

X is selected from O and N; and

A is a radical of a saccharide, or an acid, salt, or ester thereof.

Further, the invention relates to a pharmaceutical composition comprising compound of formula I, a process of preparing the same and method of management of tumor, inflammation, infection and promoting passage across blood-brain barrier. Detailed description of the present Invention

The invention provides an efficient and reliable method for synthesizing glyco- phosphorylated therapeutics. Compounds of the invention feature a saccharide linked via a phosphodiester or phosphoroamidate linkage to a biologically active agent. The glyco-phosphorylated compounds can exhibit enhanced tumor specificity and/or blood brain barrier penetration in comparison to the aglycon "parent" drug. In a first aspect, the invention features a compound of formula I:

O

Il DX-P-OA

P- CQ ^, wherein DX is a radical of a biologically active agent comprising atom X; X is selected from O and N; and A is a radical of a saccharide, or an acid, salt, or ester thereof.

Tn the above aspect, the saccharide can be any saccharide described herein. Desirably, the saccharide is selected from D-glucose, D-mannose, D-xylose, D-galactose, D- glucuronic acid, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, sialyic acid, iduronic acid, L-fucose, D-ribose, D-arabinose, D-ribulose, fructose, sucrose, lactose, and maltose. The saccharide can also be a derivative, such as derivatives in which one fluorine or hydrogen atom replaces a hydroxyl group of the saccharide. The saccharide can also be deoxygenated saccharide (for eg deoxyglucose). In various embodiments of the above aspect, the compound is further described by any of formulas IT-VI:

In formulas II-VI, DX is a radical of a biologically active agent comprising atom X; Y is H ₅ OH or F; and X is selected from O and N, or an acid, salt, or ester thereof. In any of the above formulas, the biologically active agent is, desirably, a hormone (e.g., testosterone, dihydrotestesterone, estradiol, or estrone), a vitamin (e.g., vitamin E or vitamin D), an antiproliferative agent (e.g., irinotecan, topotecan, rubitecan, exatecan, lurtotecan, lO-hydroxy-9-ethyl-camptothecin, silatecan, afeletecan, gimatecan, or epipodophyllotoxin), an antiinflammatory agent (e.g., salicylic acid; acetaminophen; a cox-2 inhibitor, such as rofecoxib, celecoxib, valdecoxib, or lumiracoxib; or a corticosteroid, such as beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, corticosterone, cortisone, dexamethasone, hydrocortisone, meprednisone, paramethasone, prednisolone, or prednisone), or an antibiotic (e.g., daunorubicin or adriamycin). The biologically active agent can be any biologically active agent described herein.

Compounds of the invention include glycol-phosphorylated testosterone, glycol- phosphorylated estradiol, glycol-phosphorylated lO-hydroxy-9-ethyl-camptothecin, glycol-phosphorylated topotecan, glycol-phosphorylated irinotecan, glycol- phosphorylated epipodophyllotoxin, glycol-phosphorylated tocopherol, glycol- phosphorylated salicylic acid, and glycol-phosphorylated acetaminophen, among others.

In another embodiment of the above aspect, the compound of the invention is further described by formula VIII:

wherein the bond between Ci and C ₂ is a double or a single bond; Xi represents H or a halogen atom; X ₂ represents H, CH ₃ , or a halogen atom; X ₃ represents H or a halogen atom; Ri represents =O or -OH; R ₂ represents CH ₃ , SCH ₂ F, CH ₂ Cl, CH ₂ OH, CH ₂ O- P(O)(O ^" )* CH ₂ O-acyl, CH ₂ NHG ¹ , or CH ₂ OG ¹ ; R ₃ and R ₄ each independently represent H, C] _-4 alkyl, -OH, -0(CO)-Rg, -OR ₉ , or R ₃ and R ₄ combine to form a cyclic acetal described by formula IX:

in formula IX, n is a whole integer from O to 6; Wi represents H, CH ₃ , NRsG ¹ , or OG ¹ ; each of R5, R ₆ , R ₇ , Rs, and Rg is, independently, H or Ci _-4 alkyl; and G ¹ is described by formula X:

wherein, A is a radical of a saccharide; or an acid, salt, or ester thereof. In another aspect, the invention features pharmaceutical composition including a compound of the invention and a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition can be in any form described herein. Desirably, the pharmaceutical formulation is in the form of a capsule or a tablet. Alternatively, the pharmaceutical composition is formulated for topical administration (e.g., as a cream, lotion, spray, stick, or ointment).

In yet another aspect, the invention feature a method for promoting passage across the blood-brain barrier of a biologically active agent. The method includes the

glycophosphorylation of a hydroxyl oxygen atom or amino nitrogen atom of a biologically active agent to form a glyco-phosphorylated compound of the invention exhibiting enhanced passage across the blood-brain barrier in comparison to the unmodified biologically active agent (e.g., the aglycon). In still another aspect, the invention features a method for promoting tumor uptake of a biologically active agent. The method includes the glycophosphorylation of a hydroxyl oxygen atom or amino nitrogen atom of a biologically active agent to form a glyco- phosphorylated compound of the invention exhibiting enhanced tumor uptake in comparison to the unmodified biologically active agent (e.g., the aglycon). In another aspect, the invention features a method for treating a tumor in a patient by administering to the patient a glyco-phosphorylated antiproliferative agent in an amount sufficient to treat the tumor.

In yet another aspect, the invention features a method for treating inflammation in a patient by administering to the patient a glyco-phosphorylated antiinflammatory agent in an amount sufficient to treat the inflammation.

In still another aspect, the invention features a method of treating an infection in a patient by administering to the patient a glyco-phosphorylated antibiotic in an amount sufficient to treat the infection. By "corticosteroid" is meant any naturally occurring or synthetic compound characterized by a hydrogenated cyclopentanoperhydrophenanthrene ring system and having immunosuppressive and/or antinflammatory activity. Naturally occurring corticosteriods are generally produced by the adrenal cortex. Synthetic corticosteriods may be halogenated. Examples corticosteroids are provided herein. As used herein, the term "treating" refers to administering a pharmaceutical composition for prophylactic and/or therapeutic purposes. To "prevent disease" refers to prophylactic treatment of a patient who is not yet ill, but who is susceptible to, or otherwise at risk of, a particular disease. To "treat disease" or use for "therapeutic treatment" refers to administering treatment to a patient already suffering from a disease to improve the patient's condition. Thus, in the claims and embodiments, treating is the administration to a mammal either for therapeutic or prophylactic purposes.

The term "administration" or "administering" refers to a method of giving a dosage of a pharmaceutical composition to a mammal, wherein the corticosteroid conjugate is

administered by a route selected from, without limitation, inhalation, ocular administration, nasal instillation, parenteral administration, dermal administration, transdermal administration, buccal administration, rectal administration, sublingual administration, periungual administration, nasal administration, topical administration and oral administration. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, and intramuscular administration. The preferred method of administration can vary depending on various factors, e.g., the components of the pharmaceutical composition, site of the potential or actual disease and severity of disease. By "patient" is meant any mammal (e.g., a human).

By "promoting passage across the blood-brain barrier" for a compound of the invention is meant that the ratio of AUCbrain (area under the curve in brain tissue) to AUCblood (area under the curves in whole blood) is increaseed for the glycol-phosphorylated biologically active agent in comparison to the parent biologically active agent which is not glycol-phosphorylated (e.g., the aglycon) when administered under the same conditions.

By "promoting tumor uptake" for a compound of the invention is meant that the ratio of AUCtumor (area under the curve in tumor tissue) to AUCblood (area under the curves in whole blood) is increaseed for the glycol-phosphorylated biologically active agent in comparison to the parent biologically active agent which is not glycol-phosphorylated (e.g., the aglycon) when administered under the same conditions. As used herein, "an amount sufficient to treat the inflammation" is meant the amount of a compound, in a combination of the invention, required to treat or prevent an inflammatory disease. The effective amount of active compound used to practice the present invention for therapeutic treatment of conditions caused by or contributing to an inflammatory disease varies depending upon the manner of administration, the age, body weight, and general health of the patient. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an amount sufficient to treat inflammation. The terms "inflammation" and "inflammatory disease" encompass a variety of conditions, including autoimmune diseases such as rheumatoid arthritis, ulcerative colitis, Crohn's disease, stroke-induced brain cell death, septic shock syndrome, ankylosing spondylitis, fibromyalgia, asthma, multiple sclerosis, type I diabetes, systemic lupus erythematosus, scleroderma, systemic

sclerosis, inflammatory dermatoses, myasthenia gravis, and Sjogren's syndrome. Inflammatory dermatoses include, for example, psoriasis, acute febrile neutrophilic dermatosis, eczema (e.g., asteatotic eczema, dyshidrotic eczema, vesicular palmoplanar eczema), balanitis circumscripta plasmacellularis, balanoposthitis, Behcet disease, erythema annulare centrifugum, erythema dyschromicum perstans, erythema multiforme, granuloma annulare, lichen nitidus, lichen planus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, nummular dermatitis, pyoderma gangrenosum, sarcoidosis, subcorneal pustular dermatosis, urticaria, and transient acantholytic dermatosis. As used herein, "an amount sufficient to treat a tumor" is meant the amount of a compound, in a combination of the invention, required to treat a tumor (e.g., slow the growth of a tumor). The effective amount of active compound used to practice the present invention for therapeutic treatment of conditions caused by or contributing to tumor growth varies depending upon the manner of administration, the age, body weight, and general health of the patient. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an amount sufficient to treat a tumor.

As used herein, "an amount sufficient to treat the infection" is meant the amount of a compound, in a combination of the invention, required to treat or prevent (slow the spread of or kill the infectious agent) an infection of a host mammal by a parasitic microorganism (e.g., a fungus or bacterium). The effective amount of active compound used to practice the present invention for therapeutic treatment of conditions caused by or contributing to an infection varies depending upon the manner of administration, the age, body weight, and general health of the patient. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an amount sufficient to treat an infection.

In the generic descriptions of compounds of this invention, the number of atoms of a particular type in a substituent group is generally given as a range. For example, an alkyl group containing from 1 to 4 carbon atoms. Reference to such a range is intended to include specific references to groups having each of the integer number of atoms within the specified range. For example, an alkyl group from 1 to 4 carbon atoms includes each of Cl, C2, C3, and C4. Other numbers of atoms and other types of atoms are indicated in a similar manner.

By "CM alkyl" is meant a branched or unbranched saturated hydrocarbon group, desirably having from 1 to 4 carbon atoms. An alkyl may optionally include a monocyclic ring of three to four members. The alkyl group may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halogen, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. The invention provides an efficient and reliable method for synthesizing glyco- phosphorylated therapeutics. Compounds of the invention feature a saccharide linked via a phosphodiester or phosphoroamidate linkage to a biologically active agent. The glyco-phosphorylated compounds can exhibit enhanced tumor specificity and/or blood brain barrier penetration in comparison to the aglycon "parent" drug. Another advantage of attaching the glycose by utilizing a phosphate linker is that using this approach it is possible to prepare glyco-phosphorylated compounds on a commercial scale. The biologically active agent can be, for example, a hormone, vitamin, cancer drug, cardiovascular drug, antibiotic, or psychotropic drug.

Upon absorption, the glyco-phosphorylated agent can be cleaved in vivo by, for example, enzymatic action upon phosphate linker or upon the saccharide to yield the "parent" biologically active agent, or aglycon. Saccharides The invention features a biologically active agent attached to a phosphate, which in turn is attached to a saccharide at a synthetically available hydroxyl position. Suitable monosaccharides include, but are not limited to, any of several simple open or closed chain sugars (in the L or D configuration), typically having 5 or 6 carbons (a pentose monosaccharide or a hexose monosaccharide), as well as 7 carbons (heptose monosaccharide). Included are sugar derivatives in which the ring oxygen atom has been replaced by carbon, nitrogen or sulfur, amino sugars in which a hydroxyl substituent on the simple sugar is replaced with an amino group or sugars having a double bond between two adjacent carbon atoms, (e.g. glucosamine, 5-thio-D-glucose, nojirimycin, deoxynojirimycin, 1,5-anhydro-D-sorbitol, 2,5-anhydro-D-mannitol, 2- deoxy-D-galactose, 2-deoxy-D-glucose, 3-deoxy-D-glucose, allose, arabinose, arabinitol, fucitol, fucose, galactitol, glucitol, iditol, lyxose, mannitol, levo-rhamnitol, 2-deoxy-D-ribose, ribose, ribitol, ribulose, rhamnose, xylose, xylulose, allose, altrose, fructose, galactose, glucose, gulose, idose, levulose, mannose, psicose, sorbose,

tagatose, talose, galactal, glucal, fiical, rhamnal, arabinal, xylal, valienamine, validamine, valiolamine, valid, valiolon, valienol, valienone, glucuronic acid, galacturonic acid, N-acetylneuraminic acid, gluconic acid D-lactone, galaclonic acid .gamma.-lactone, galactonic acid .delta.-lactone, mannonic acid .gamma.-lactone, D- altro-heptulose, D-manno-heptulose, D-glycero-D-manno-heptose, D-glycero-D-gluco- heptose, D-allo-heptulose, D-altro-3-heptulose, D-glycero-D-manno-heptitoI, D- glycero-D-altro-heptit- ol and the like), The hydroxyl groups on monosaccharides may optionally be replaced with hydrogen (deoxyglycoses), alkoxy (e.g. 2-O-methyl-D- fructose), alkanoate or halogen groups (especially fluorine atoms). Included are sulfate and/or phosphate derivatives of monosaccharides as defined herein.

Suitable oligosaccharides include, but are not limited to, carbohydrates having from 2 to 10 or more monosaccharides linked together. The constituent monosaccharide unit may be, for example, a pentose monosaccharide, a hexose monosaccharide, or a pseudosugar (including a pseudoaminosugar). Oligosaccharides do not include bicyclic groups that are formed by fusing a monosaccharide to a benzene ring, a cyclohexane ring, or a heterocyclic ring.

Pseudosugars that may be used in the invention are members of the class of compounds wherein the ring oxygen atom of the cyclic monosaccharide is replaced by a methylene group. Pseudosugars are also known as "carba-sugars." It has been long known that fluorine atom is served as a dignostic tool in imaging. The fluorine atom can easily incorporated into the saccharide-phosphate-agent approach by utilizing the sugar moiety. Fluoro-glucose is often used for cancer diagnosis. Also phosphorous atom can be easily radio labeled or has a magnetic spin similar to fluorine and can be imaged to monitor the progress of the cure. Such a diagnosis & cure option within a single molecule is seen as the second generation drugs especially for brain and cancer cure. Fluorinated saccharides can be prepared using the methods described by, for example, Adam, M.J. Journal of Labeled Compounds & Radiopharmaceuticals 45:167 (2002); and Wong et al, Journal of Labeled Compounds & Radiopharmaceuticals 44: 385-394 (2001) Biologically Active Agents

The invention features a glyco-phosphorylated biologically active agent. The glyco- phospohryl group is attached to the biologically active agent at a synthetically available hydroxyl, amino, or phenolic position. Alternatively, a hydroxyl or amino functionality

can be made available by chemical modification of a biologically active agent. Biologically active agents that can be glyco-phosphorylated as described herein include therapeutic, diagnostic, and prophylactic agents. They can be naturally occurring compounds or synthetic organic compounds. Agents that can be modified as described herein include, but are not limited to, proteins, peptides, antibiotics, antiproliferative agents, rapamycin macrolides, analgesics, anesthetics, antiangiogenic agents, vasoactive agents, anticoagulants, immunomodulators, cytotoxic agents, tyrosinase inhibitors, antiviral agents, antithrombotic drugs, such as terbrogel and ramatrob, anantibodies, neurotransmitters and psychoactive drugs. Exemplary therapeutic agents include growth hormone, for example human growth hormone, calcitonin, granulocyte macrophage colony stimulating factor (GM-CSF), ciliary neurotrophic factor, and parathyroid hormone. Other specific therapeutic agents include parathyroid hormone-related peptide, somatostatin, testosterone, progesterone, estradiol, nicotine, fentanyl, norethisterone, clonidine, scopolomine, salicylate, salmeterol, formeterol, albeterol, Valium, heparin, dermatan, ferrochrome A, erythropoetins, diethylstilbestrol, lupron, estrogen estradiol, androgen halotestin, 6- thioguaniπe, 6-mercaptopurine, zolodex, taxol, lisinopril/zestril, streptokinase, aminobutytric acid, hemostatic aminocaproic acid, parlodel, tacrine, potaba, adipex, memboral, phenobarbital, insulin, gamma globulin, azathioprine, papein, acetaminophen, ibuprofen, acetylsalicylic acid, epinephrine, flucloronide, oxycodone percoset, dalgan, phreniline butabital, procaine, novocain, morphine, oxycodone, aloxiprin, brofenac, ketoprofen, ketorolac, hemin, vitamin B-12, folic acid, magnesium salts, vitamine D, vitamin C, vitamin E, vitamin A, Vitamin U, vitamin L, vitamin K, pantothenic acid, aminophenylbutyric acid, penicillin, acyclovir, oflaxacin, amoxicillin, tobramycin, retrovior, epivir, nevirapine, gentamycin, duracef, ablecet, butoxycaine, benoxinate, tropenzile, diponium salts, butaverine, apoatropine, feclemine, leiopyrrole, octamylamine, oxybutynin, albuterol, metaproterenol, beclomethasone dipropionate, triamcinolone acetamide, budesonide acetonide, ipratropium bromide, flunisolide, cromolyn sodium, ergotamine tartrate, and protein or peptide drugs such as TNF antagonists or interleukin antagonists. For example, the biologically active agent can be an antiinflammatory agent, such as an NSAID, corticosteriod, or COX-2 inhibitor, e.g., rofecoxib, celecoxib, valdecoxib, or lumiracoxib.

Corticosteroids

Corticosteroids which can be modified as described herein include, without limitation, hydrocortisone and compounds which are derived from hydrocortisone, such as 21 - acetoxypregnenolone, alclomerasone, algestone, amcinonide, beclomethasone, betamethasone, betamethasone valerate, budesonide, chloroprednisone, clobetasol, clobetasol propionate, clobetasone, clobetasone butyrate, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacon, desonide, desoximerasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flumethasone pivalate, flunisolide, flucinolone acetonide, fluocinonide, fluorocinolone acetonide, fluocortin butyl, fluocortolone, fluorocortolone hexanoate, diflucortolone valerate, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandenolide, formocortal, halcinonide, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone phosphate, hydrocortisone 21 -sodium succinate, hydrocortisone tebutate, mazipredone, medrysone, meprednisone, methylprednicolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 21-diedryaminoacetate, prednisolone sodium phosphate, prednisolone sodium succinate, prednisolone sodium 21-m-sulfobenzoate, prednisolone sodium 21- stearoglycolate, prednisolone tebutate, prednisolone 21 -trimethylacetate, prednisone, prednival, prednylidene, prednylidene 21-diethylaminoacetate, ^• tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide and triamcinolone hexacetonide. Structurally related corticosteroids having similar anti-inflammatory properties are also intended to be encompassed by this group. Rapamycin Macrolides Rapamycin (Sirolimus) is an immunosuppressive lactam macrolide that is produced by Streptomyces hygroscopicus. See, for example, McAlpine, J. B., et al., J. Antibiotics 44: 688 (1991); Schreiber, S. L., et al., J. Am. Chem. Soc. 1 13: 7433 (1991); and U.S. Patent No. 3,929,992, incorporated herein by reference. Exemplary rapamycin macrolides which can be used in the methods and compositions of the invention include, without limitation, rapamycin, CCI-779, Everolimus (also known as RADOOl), and ABT-578. CCI-779 is an ester of rapamycin (42 -ester with 3-hydroxy-2- hydroxymethyl-2-methylpropionic acid), disclosed in U.S. Patent No. 5,362,718. Everolimus is an alkylated rapamycin (40-O-(2-hydroxyethyl)-rapamycin, disclosed in

U.S. Patent No. 5,665,772. Antiproliferative Agents

Exemplary antiproliferative agents which can be used in the methods and compositions of the invention include, without limitation, mechlorethamine, cyclophosphamide, iosfamide, melphalan, chlorambucil, uracil mustard, estramustine, mitomycin C, AZQ, thiotepa, busulfan, hepsulfam, carmustine, lomustine, semustine, streptozocin, dacarbazine, cisplatin, carboplatin, procarbazine, methotrexate, trimetrexate, fluouracil, floxuridine, cytarabine, fludarabine, capecitabine, azacitidine, thioguanine, mercaptopurine, allopurine, cladribine, gemcitabine, pentostatin, vinblastine, vincristine, etoposide, teniposide, topotecan, irinotecan, 10-hydroxycamptothecin, 9- aminocamptothecin, lO-hydroxy-9-ethyl-camptothecin, paclitaxel, docetaxel, daunorubicin, doxorubicin, dactinomycin, idarubincin, plicamycin, mitomycin, amsacrine, bleomycin, aminoglutethimide, anastrozole, finasteride, ketoconazole, tamoxifen, flutamide, leuprolide, goserelin, GleevecTM (Novartis), leflunomide (Pharmacia), SU5416 (Pharmacia), SU6668 (Pharmacia), PTK787 (Novartis), IressaTM (AstraZeneca), TarcevaTM, (Oncogene Science), trastuzumab (Genentech), ErbituxTM (ImClone), PKIl 66 (Novartis), GW2016 (GlaxoSmithKline), EKB-509 (Wyeth), EKB-569 (Wyeth), MDX-H210 (Medarex),2C4 (Genentech), MDX-447 (Medarex), ABX-EGF (Abgenix), CI-1033 (Pfizer), AvastinTM (Genentech), IMC- ICl 1 (ImClone), ZD4190 (AstraZeneca), ZD6474 (AstraZeneca), CEP-701 (Cephalon), CEP-751 (Cephalon), MLN518 (Millenium), PKC412 (Novartis), 13-cis- retinoic acid, isotretinoin, retinyl palmitate, 4-(hydroxycarbophenyl) retinamide, misonidazole, nitracrine, mitoxantrone, hydroxyurea, L-asparaginase, interferon alfa, AP23573, Cerivastatin, Troglitazone, CRx-026DHA-paclitaxel, Taxoprexin, TPI-287, Sphingosine-based lipids, and mitotane. NSAIDs

Exemplary non-steroidal antiinflammatory drugs (NSAIDs) which can be used in the methods and compositions of the invention include, without limitation, naproxen sodium, diclofenac sodium, diclofenac potassium, aspirin, sulindac, diflunisal, piroxicam, indomethacin, ibuprofen, nabumetone, choline magnesium trisalicylate, sodium salicylate, salicylsalicylic acid (salsalate), fenoprofen, flurbiprofen, ketoprofen, meclofenamate sodium, meloxicam, oxaprozin, sulindac, and tolmetin.

Analgesics

Exemplary analgesics which can be used in the methods and compositions of the invention include, without limitation, morphine, codeine, heroin, ethylmorphine, 0- carboxymethylmorphine, O-acetylmorphine, hydrocodone, hydromorphone, oxymorphone, oxycodone, dihydrocodeine, thebaine, metopon, ethorphine, acetorphine, diprenorphine, buprenorphine, phenomorphan, levorphanol, ethoheptazine, ketobemidone, dihydroetorphine and dihydroacetorphine. Antimicrobials Exemplary antimicrobials which can be used in the methods and compositions of the invention include, without limitation, penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, azlocillin, temocillin, cepalothin, cephapirin, cephradine, cephaloridine, cefazolin, cefamandole, cefuroxime, cephalexin, cefprozil, cefaclor, loracarbef, cefoxitin, cefmatozole, cefotaxime, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefixime, cefpodoxime, ceftibuten, cefdinir, cefpirome, cefepime, BAL5788, BAL9141, imipenem, ertapenem, meropenem, astreonam, clavulanate, sulbactam, tazobactam, streptomycin, neomycin, kanamycin, paromycin, gentamicin, tobramycin, amikacin, netilmicin, spectinomycin, sisomicin, dibekalin, isepamicin, tetracycline, chlortetracycline, demeclocycline, minocycline, oxytetracycline, methacycline, doxycycline, erythromycin, azithromycin, clarithromycin, telithromycin, ABT-773, lincomycin, clindamycin, vancomycin, oritavancin, dalbavancin, teicoplanin, quinupristin and dalfopristin, sulphanilamide, para-aminobenzoic acid, sulfadiazine, sulfisoxazole, sulfamethoxazole, sulfathalidine, linezolid, nalidixic acid, oxolinic acid, norfloxacin, perfloxacin, enoxacin, ofloxacin, ciprofloxacin, temafloxacin, lomefloxacin, fleroxacin, grepafloxacin, sparfioxacin, trovafloxacin, clinafloxacin, gatifloxacin, moxifloxacin, gemifloxacin, sitafloxacin, metronidazole, daptomycin, garenoxacin, ramoplanin, faropenem, polymyxin, tigecycline, AZD2563, and trimethoprim. Synthesis The invention features compounds having a phosphate is attached to glycose at a synthetically available hydroxyl. Diacetone glucose or 1,2,5,6-diisopropylidenc-D- glucose, 2,3:5,6-Di-Oisopropylidene-α-D-mannofurose, l,2:3,4-Di-Oisopropylidene-α- D-galactopyranose or D-allofuranose are commercially available. One of the hydroxyl

groups is free and can be phosphorylated using reagents like phosphoryl chloride followed by reactive hydroxyl or amino group of the aglycon (parent drug). The above sequence of reactions can be performed in several high yielding steps using dibenzyl- N,Ndiisopropyl phosphoramidite or the H-Phosphonic acid method (i.e., a method often employed in nucleotide chemistry) using phosphorous trichloride coupling followed by oxidation. Conversely, the biologically active agent can be phosphorylated prior to protected glycose addition resulting in the same product. Preferential protection of the pyranose or the furanose of the hexoses is a known art and either the more reactive primary hydroxyl at the 6-position or readily hydrolysable anomeric position or synthetically freed 3-position are synthetically simple to accomplish. Either pentoses or hexoses (either as furanoside or pyronoside) can be used as the 'sugar' in the saccharide-phosphate-agent strategy. Exemplary starting materials are shown below.

Mannofuranose Allofuranose Galactopyranose

glucose glucofuranose fructofuranose

The usefulness of such phosphodiester compounds in medical applications, and the synthesis of such compounds, is well known. See, e.g., Desseaux et al., "Synthesis of Phosphodiester and Triester Derivatives of AZT with Tethered N-Methyl Piperazine and N,N,N'trimethylethylenediamine," Bioorg. & Med. Chem. Letters, vol. 3, no. 8, pp. 1547-50 (1993); PCT publication no. WO 96/27379. Recently, PCT publication no. WO 96/23526, incorporated herein by reference, describes phosphodiester compounds which are useful as contrast agents for diagnostic imaging.

A number of methods of making phosphodiester compounds, based on P(III) chemistry, are known. Typically, phosphorylation plays an important role in the synthesis of phosphodiester compounds.

One method for making phosphodiesters involves the use of phosphoramidite chemistry. See, e.g., Bannwarth et al., Helvetica Chimϊca Acta, 70:175 (1987); Bannwarth et al., Tetrahedron Letters, 30:4219 (1989); Moore et al., J. Org. Chem., 50:2019 (1985); Hebert et al., J. Org. Chem., 57:1777 (1992); Desseaux et al., Bioorg. & Med. Chem. Letters, 3:1547 (1993); and Pirrung et al., J. Org. Chem., 61 :2129 (1996). Methods involving the use of phosphodichloridates as the phosphorylating agent can also be used. See, e.g., Martin et al., J. Org. Chem., 59:4805 (1994); Martin et al., Tetrahedron Letters, 29:3631 (1988); Lammers et al., J. Royal Netherlands Chem. Soc, 98:243 (1979); and Martin et al., J. Org. Chem., 61:8016 (1996). Another method used for making phosphodiester compounds involves the use of PCI ₃ to generate hydfogen-phosphonate intermediates. See, e.g., Lindh et al., J. Org. Chem., 54:1338 (1989); Garcia et al., Tetrahedron, 47:10023 (1991); and Garigapati et al., Tetrahedron Letters, 34:769 (1993).

Any of the methods described above and those in the Examples can be used for the preparation of glyco-phosphorylated biologically active agents of the invention. Therapy

Compounds of the present invention may be administered by any appropriate route for treatment or prevention of a disease or condition. These may be administered to humans, domestic pets, livestock, or other animals with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form. Administration may be topical, parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, by suppositories, or oral administration. The compounds of the invention may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 0.01-95% by weight of the total weight of the composition. For example, the composition may be provided in a dosage form that is suitable for the oral, parenteral (e.g., intravenously, intramuscularly), rectal, cutaneous, nasal, vaginal, inhalant, skin (patch), or ocular administration route. Thus, the composition may be in the form of, e.g., tablets,

capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols. Methods well known in the art for making formulations are found, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., ed. A.R. Gennaro, 2000, Lippincott Williams & Wilkins). Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Nanoparticulate formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) may be used to control the biodistribution of the compounds. Other potentially useful parenteral delivery systems include ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel. The concentration of the compound in the formulation will vary depending upon a number of factors, including the dosage of the drug to be administered, and the route of administration.

The compound may be optionally administered as a pharmaceutically acceptable salt, such as a non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and the like. Administration of compounds in controlled release formulations is useful where the compound of formula I has (i) a narrow therapeutic index (e.g., the difference between the plasma concentration leading to harmful side effects or toxic reactions and the plasma concentration leading to a therapeutic effect is small; generally, the therapeutic index, TI, is defined as the ratio of median lethal dose (LD ₅₀ ) to median effective dose

(ED ₅₀ )); (ii) a narrow absorption window in the gastro-intestinal tract; or (iii) a short biological half-life, so that frequent dosing during a day is required in order to sustain the plasma level at a therapeutic level.

Many strategies can be pursued to obtain controlled release in which the rate of release outweighs the rate of metabolism of the therapeutic compound. For example, controlled release can be obtained by the appropriate selection of formulation parameters and ingredients, including, e.g., appropriate controlled release compositions and coatings. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes.

Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc).

Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium. Topical Formulations The compounds of the invention may be formulated for topical administration. Examples of skin care formulations which may be used include, without limitation, creams, lotions, sprays, sticks, ointments, hair care products; suncare products, or combinations thereof. Any conventional pharmacologically and cosmetically acceptable vehicles may be used. For example, the compounds may also be administered in liposomal formulations that allow compounds to enter the skin. Such liposomal formulations are described in U.S. Patnet Nos. 5,169,637; 5,000,958; 5,049,388; 4,975,282; 5,194,266; 5,023,087; 5,688,525; 5,874,104; 5,409,704; 5,552,155; 5,356,633; 5,032,582; 4,994,213; and PCT Publication No. WO 96/40061. Examples of other appropriate vehicles are described in U.S. Patent No. 4,877,805 and EP Publication No. 0586106A1. Suitable vehicles of the invention may also include mineral oil, petrolatum, polydecene, stearic acid, isopropyl myristate, polyoxyl 40 stearate, stearyl alcohol, or vegetable oil. The formulations can include various conventional colorants, fragrances, thickeners

(e.g., xanthan gum), preservatives, humectants, emollients (e.g., hydrocarbon oils, waxes, or silicones), demulcents, solubilizing excipienls, dispersants, penetration enhancers, plasticizing agents, preservatives, stabilizers, demulsifiers, wetting agents, sunscreens, emulsifiers, moisturizers, astringents, deodorants, and the like can be added to provide additional benefits and improve the feel and/or appearance of the topical preparation.

The formulations are typically used for the prophylaxis and/or treatment of the skin in the context of dermatological treatment. Accordingly, the formulations of the invention are desirably formulated as a cream, lotion, ointment, or spray when used for the treatment of skin disorders (e.g, a cream containing an glycol-phosphorylated antiinflammatory agent for the treatment of dermatitis).

The compositions of the invention are useful for the treatment of diseases. When administered to a human, the dosage of any of the compounds of the invention will depend on a variety of factors including the biologically active agent used, the disease being treating, the formulation used, and the route of administration. Typically, the dosage administered will be about that administered for the aglycol, e.g., the biologically active agent without glycol-phosphorylation. However, in some instances, the preferential delivery of the biologically active agent to the site of disease (e.g., a tumor) as a result of the modifications described herein will reduce the minimum efficacious dose, allowing the compositions of the invention to be administered at doses lower than that of the corresponding aglycol. Standard clinical trials maybe used to optimize the dose and dosing frequency for any particular compound. The following examples are to illustrate the invention. They are not meant to limit the invention in any way. Example 1: Preparation of protected glycosyl donors with one free hydroxyl group for phosphorylation:

IA. Preparation of 1-α or β-Hydroxy-2,3,4,6-tetra-0-acetyl β-D- glucopyranoside: Dimethlamine gas was generated from commercially available 30% aqueous solution by adding into sodium hydroxide pellets and was added to penta-acetyl glucose prepared as above. In a 2 necked round bottomed flask containing lOOg of NaOH pellets was added 200 ml of 30% dimethyl amine solution slowly through dropping

funnel. The liberated dimelhylamine gas was transferred to another 2L jacketed reactor cooled to -2O ⁰ C and dissolved in 800 mL of acetonitrile. After the acetonitrile solution attains PH of 9-11, glucose pentaacetate (65 g) was added and stirred for 15 minutes at -15°C. Completion of the reaction was confirmed by TLC monitoring. Acetonitrile and excess dimethylamine gas were removed quickly by rotory evaporator. The resulting paste was dissolved in 500 mL ethylacetate and washed once with 200 mL of water. The organic portion was dried over magnesium sulfate and evaporated. The mixture was pure enough for further use in phosphorylation reactions. IB. Preparation of 1- β-Hydroxy-2,3,4,6-tetra-0-acetyl-β-D-glucopyranoside: l-β-Acetyl-penta-O-acetyl-glucopyranoside was purchased from Sigma. A methanolic solution of 1-β-acetate was heated to 40°C with imidazole (1 molar equivalent) for 26 hours and TLC monitored for the completion of the anomeric deacetylation. Methanol was removed and after water addition and extraction with ethylacetate followed by 1 N hydrochloric acid wash gave exclusive beta hydroxyl anomer in almost quantitative manner.

1C. Preparation of l,2:5,6-Di-O-cyclohexylidene-α-D-glucofuranose: l OOOg (1050 mL, 10 mol) of redistilled cyclohexanone was added to a 3 L cooled to O ⁰ C. 62.5 mL of concentrated sulphuric acid was added slowly into the vigorously stirred cyclohexanone. 450 grams (2.5 mol) of finely powdered dried α-D-glucose was added slowly. The jacketed reactor was allowed to reach ambient temperature with continual stirring for over a period of 8 hours. The reaction mixture becomes progressively more viscous and finally sets into a solid off-white crystalline mass. The crystalline mass was agitated with 750 mL of heptane and a solution of 124 grams of sodium carbonate in 375mL of water at 70°C. The heptane layer was allowed to percolate by gravity into a Erlenmayer flask. Upon cooling the heptane layer, the crystals were filtered and the heptane was resubjected to the residue similarly. The purified l,2:5,6-di-O-cyclohexylidene-α-D-glucofuranose (recrystallized from heptane) has m.p. 131-132°C, [α]20D -2.2° (c 1.8 in EtOH); the yield is 380 grams (47%). ID. Preparation of l,2:4,5-Di-O-cydohexyIidene-D-fructopyranose: 200 grams (1.11 mol) of finely powdered dry D-fructose and 419 grams (440 mL, 4.49 mol) of ice-cooled cyclohexanone containing 30 mL of concentrated sulphuric acid in a jacketed reactor was stirred as in example 1C and the reaction mixture becomes solid

within 30 minutes. The mixture was left overnight at room temperature and the product was dissolved in 500 mL of chloroform. The organic layer was washed with dilute aqueous sodium hydroxide, dilute hydrochloric acid and water and finally dried and evaporated. The residue has m.p. 145-156°C, [α]20D -133.5°(c 1 in CHC13). The yield is 142 grams (37%).

IE. Preparation of l,2:5,6-Di-0-isopropylidene-α-D-glucofuranose: A suspension of 150 grams (0/83 mol) of dry D-glucose, 120 grains (0.83 mol) of anhydrous zinc chloride and 7.5 grams of phosphoric acid (88% v/v) in 1 L of dry acetone was stirred at ambient temperature for 30 hours. Unchanged glucose was removed by filtration, and inorganic salts were precipitated by the addition of a solution of 58 grams of sodium hydroxide in 85mL of water. The resulting suspension was filtered, the residue washed with acetone and the acetone layer evaporated. The mass was dissolved in 200 mL of water and extracted with five 100-mL portions of dichloromethane. The organic phase was dried and evaporated on rotary evaporator. Recrystallisation from light petroleum (b.p. 80-100°C) gave 70 grams of product, m.p. 109-110 ⁰ C, [α]20D -18.5° (c 5 in H ₂ O).

IF. Preparation of l,2:5,6-Di-0-isopropylidene-D-mannitol: To a solution of 60 grams of zinc chloride in 30OmL of acetone was added 10 grams of finely powdered D-mannitol. The mixture was stirred vigorously at 20°C until clear solution (2-3 hours) and was allowed to stand for further 16 hours. The reaction mixture was then poured into a solution of 70 grams of potassium carbonate in 70 mL of water and 300 mL of ether. The product was filtered and 100 mL of 1 :1 acetone- ether solution was used to wash the filtrate. The combined filtrates were evaporated to dryness on a rotary evaporator. The dry residue was successively extracted with five 250-mL portions of boiling light petroleum (b.p. 60-80 ⁰ C) and the combined filtrates cooled to give the product, 7.9 grams (55%), having m.p.l 19°C. IG. Preparation of l,2:4,5-0-Diisopropylidene-D-galactopyranose: To a suspension of acetone and cupric chloride (catalyst) was added galactose and the solution refluxed for 8 hours upon which time galactose was mostly dissolved. The reaction mixture was stirred for a further period of 10 hours at ambient temperature. Upon removal of acetone and extraction with ethylacetate and an aqueous wash afforded the diisopropylidene derivative in 80% yield. The product upon LC-MS examination gave molecular ion as sodium adduct 282 M ⁺ .

IH. Preparation of 1,2:4,5-O-Dicyclohexylidene- D-galactopyranose:

Instead of acetone cyclohexanone was used similar to example 7. The product was isolated after cyclohexanone removal (yield 72%) as above in example IG. Example 2. General Procedure for the glycophosphorylation of substrates: 2A. Preparation of H-Phosphonic acid derivatives of sugar moieties:

Protected sugar as in examples IA-H (10 mmol) was dissolved in dry toluene (50 mL) and freshly distilled phosphorous trichloride (25 mmol) was added at room temperature and the mixture was cooled to 10°C under inert atmosphere. A solution of triethylamine (20 mmol) in dry toluene (20 mL) was added over a period of 30 minutes to the cooled and stirred mixture. The mixture was stirred and monitored for the complete disappearance of the starting material. Precipitated triethylamine hydrochloride was removed by filtration and water (10 mL) was added and sufficient amount of sodium acetate was added when cold to neutralize the pH to about 5. The toluene layer was evaporated. Upon evaporation of the solvent, the product was obtained as a gummy solid and was used immediately for the next reaction without any purification. Direction injection using LC-MS (Applied Biosystems) gave a strong negative ion spectrum as expected. The TLC after work-up also showed most of the starting material was consumed and a more polar product same as the one monitored from the reaction mixture was obtained indicating that the dichloride can be hydrolyzed readily to the H-Phosphonic acid. The product was more water soluble with the diacetonides and the tetraacetates and thus ethyl acetate was substituted for the aqueous extraction. Thus dicyclohexylidene derivative obtained from fructose (from example ID) gave a gummy product in 85% isolated yield. LC-MS showed a strong negative molecular ion at 393 amu. 2B. Preparation of H-Phosphonic acid of agly cons.

General procedure: The aglycon (10 mmol) and phosphorous trichloride (25 mmol) in dry THF (75 mL) was stirred and cooled to 10°C and a solution of triethylamine (25 mmol) in dry THF (25 mL) was added over a period of 1 hour when cold. TLC monitoring of the reaction mixture maintained at room temperature showed a polar product. The reaction mixture was stirred for over 2 hours for the complete conversion. The product was isolated after addition of water and pH neutralization (5-6.5) using sodium bicarbonate, extraction with ethylacetate (3 times 50 mL). Organic portion was dried over magnesium sulfate and evaporated on a rotory evaporator to obtain the H-

Phosphonic acid derivative which was pure enough for subsequent conjugation with the free sugar hydroxyl group.

2C. Preparation of Tocopherol-H-Phosphonic acid:

Imidazole (11.2 grams; 7 equivalent), THF (100 mL) and PCl ₃ (12 niL) were mixed at O ⁰ C and stirred. Vitamin E (10 grams) in 60 mL THF containing triethylamine (8.2 grams) was added over 20 minutes. After workup with water, cthylacetate extraction (3 times 100 mL), sodium sulfate drying and evaporation gave 14 grams of tocopherol-H- phosphonic acid. The product was purified by silica gel column as described below in example 2D. 2D. Preparation of Tocopherol-H-Phosphonic acid (without imidazole):

In a 1 L flask 200 mL of THF under argon was cooled to O ⁰ C. 17.5 mL of PCl ₃ was added to the above stirred mixture. 20 grams of Vitamin E, 45 mL of triethylamine, and 50 mL of THF were combined and added slowly during 30 minutes to the tocopherol mixture. The reaction mass was warmed to 25 °C and stirred for 1 hour. The reaction mixture was added to 10% hydrochloric acid (50 mL) and stirred for 5 minutes. The THF layer was separated and the aqueous layer was further extracted with ethylacetate (3x200 mL). The organic portion was combined and evaporated to afford 22 grams of crude product. The product was purified by silica gel Column. Methanol-ethylacetate mixture gave pure product (16g; isolated; 69% yield). RF value: 0.25 in dichloromethane:methanol (85:15). LC-MS showed the molecular ion peak at m/z 493.

2E. Phosphorous oxychlorination of Vitamin E:

In a dried single-necked 100 mL round bottomed flask, dry THF (12.5 mL) was added and cooled to 0°C. Freshly distilled phosphorous oxychloride (1.0 mL) was added. A mixture of dried vitamin E (2.0 grams) and triethylamine(1.7 mL) in dry THF(12.5 mL) was added dropwise over a period of 15 minutes at 0°C. The reaction was stirred for 45 minutes at 0 ⁰ C, followed by 2 hours at room temperature. After the completion of reaction as monitored by TLC, the reaction was filtered to remove triethylammonium hydrochloride. The filtrate was then concentrated using high vacuum pump to remove the excess phosphorous oxychloride and solvents.

Example 3. Coupling Reactions:

3A. Coupling of H-Phosphonic acid sugar derivative with aglycon:

General procedure: H-Phosphonic acid derivative of the protected sugar was dried thoroughly by a high vacuum pump and 0.8 molar equivalent of the aglycon containing the free hydroxyl group was added in dry pyridine (10 niL per each millimole of sugar- H-Phosphonic acid). The resulting mixture was cooled to 0°C and 4 molar equivalents of pivolyl chloride was added. The mixture was stirred for a further period of 60 minutes at 0°C and 60 minutes at room temperature. To this mixture was added 1 equivalent of iodine dissolved in pyridine (or 3 equivalents of freshly prepared per- acetic acid). The mixture was stirred for 10 minutes at room temperature and partitioned between water and ethylacetate. The organic portion was evaporated and purified through silica gel to obtain the clean glyco-phosphorylated material. Depending upon the protecting group, the deprotection is performed. Ammonia gas was used for deprotecting the acetylated glycoses. Diketals were deprotected in refluxing 1 N hydrochloric acid/acetone mixture in almost quantitative yield.

3B. Coupling of Vitamin E H Phosphonate with 2,3,4,6-tetra-O-acetyl β-D- glucopyranoside:

In a 500 mL flame dried round bottomed Flask 23.0 grams of H-Phosphonate of Vitamin E and 24.35 grams of 2,3,4,6- tetra-O-acetate glucopyranoside were taken. 50 mL of toulene was added and connected to the high vacuum pump for 1.5 hours. 200 mL of pyridine was added and the mixture was cooled to 0°C. 23.7 mL of Pivaloyl chloride was added and after 10 minutes at 0°C, the reaction was warmed to room temperature and stirred for 1 hour. 21grams of I ₂ in 50ml of pyridine-water mixture (9:1) was added and stirred for 45 minutes. 15% sodium thiosulphate (300 mL) was added, the mixture was extracted with ethylacetate (3x300 mL), the organic portion dried, and concentrated to afford 56 grams of crude product. TLC monitoring showed an rf value of 0.35, CH ₂ Cl ₂ MeOH (85:15). LC-MS showed the molecular ions peak at m/z 839. 3C. Deacetylation of Vitamin E Glycophosphonate: In a 1 litre flask 56 grams of the above crude product was dissolved in 200 mL of MeOH and 150 mL of 25% NH ₃ solution were added. The mixture was heated to 65°C and maintained for about two hours during which time the TLC showed complete

deacetylation. The mixture was evaporated to dryness under vacuum. The desired product was partitioned was obtained after a silica gel column separation using methanol-ethylacetate mixture in about 78% yield (24 grams). LC-MS showed the molecular ions at m/z 671. HPLC examination showed that the product is a mixture of anomeric isomers.

3D. Preparation of l,2:5,6-Diisopropylidene-3-0-PhosphoryI-tocopherol:

The procedure was similar to as in example 3B. Instead of tetra-O-acetyl glucopyranose, diketalized glucofuranose was used.

3E. Dekatalization of l,2:5,6-Diisopropylidene-3-O-Phosphoryl-tocopheroI: l,2:5,6-Diisopropylidene-3-O-Phosphoryl-tocopherol was added to IN Hydrochloric acid (5 mL per mmol) in acetone and was refluxed until complete deprotection was achieved. Trifluroacetic acid can be replaced for hydrochloric acid for room temperature dekatalization. LC-MS showed the molecular ions at m/z 671 and the product was obtained as a crystalline solid. 3F. Coupling of dichlorophosphorylated vitamin E with 2,3,4,6-tetra-O-acetyI glucopyranose:

In a 100 mL round bottomed flask, dichlorophosphate of Vitamin E (prepared as described in example 2E) was dissolved in 12.5 mL of dry THF and the mixture was cooled externally using ice bath. A mixture of 2,3,4,6-tetra-o-acetyl-α-glucopyranoside (2.19 grams; thoroughly dried using , toluene) and triethylamine (1.7 mL) in THF( 12.5mL) was added slowly at 0°C over a period of 20 minutes. After addition, the ice bath was removed and mixture was allowed to warm to room temperature. The reaction was essentially complete within one hour after adding the sugar tetraacetate. The product was isolated after partitioning between water and dichloromethane. The organic layer was dried and evaporated to obtain a gummy residue. 3G. Isolation of TocopheroI-O-glycophosphate:

The gummy residue from above in acetone (20 mL) was hydrolyzed with IN hydrochloric acid at refluxing temperature. The reaction was checked by LC-MS/MS which gives a very strong negative ion peak at 840 and TLC using dichloromethane:methanol mixture. After the complete hydrolysis of the phosphoryl chloride under acidic conditions, the mixture was added to refluxing saturated methanol containing ammoniumhydroxide (20 mL of methanol and 7 mL of saturated ammonium hydroxide). The mixture was heated and stirred till the MS showed molecular ion peak

at 671 had total absence of + 42 amu peaks signifying that the deacetylation is complete. The product was extracted with ethylacetate and dried to afford crude vitamin E glycophosphate (2.95 grams). The mass spectrometer analysis showed expected molecular ion at 671 and very minor peak at 509 signifying that the coupling with the sugar is complete. The crude product showed mixture of alpha and beta anomers (M ⁺ at 671 amu) using reverse phase LC-MS/MS. The complete synthesis of tocopherol-O-glycophosphate is shown below in Scheme 1. Scheme 1

3H. Coupling of TBHQ with l-H-Phosphonic-2,3,4,6-tetra-0-acetyl-D- glucopyranose- acid: l-H~Phosphonic-2,3,4,6-tetra-O~acetyl~D- glucopyranose- acid was dried thoroughly with the TBHQ using toluene (2x20 mL) and then under high vacuum for 30 minutes. 30 mL of Pyridine was added and mixture cooled to 0 ⁰ C. Pivaloyl chloride (3.69 mL) was added slowly at 0 ⁰ C and the mixture was warmed and stirred for 1 hour at room temperature. 10 mL iodine solution (5.5 grams , 9:1 Pyridine: water) was added with stirring over a period of 30 minutes. The reaction was quenched with 15% Na ₂ S ₂ O ₃ solution (100 mL). The mixture was extracted with ethylacetate, dried over sodium sulfate and concentrated to afford 4-0-t-butyl hydroquinone-l '-O-α

& β-2',3',4',6'-tetra-O-acetyl-D-glucopyranose (6.2 grams).

31. Preparation of 4-O-t-butyl hydroquinone-l'-O-α & β-D-glucopyranose:

4-0-t-butyl hydroquinone-l'-O-α & β-2',3',4',6'-tetra-O-acelyl-D-glucopyranosc (6.2 g) was dissolved in methanol (50 niL) and ammonium hydroxide (30%; 40 mL) was added and the mixture heated to 60°C for about 2 hours during which time mass spectrometric analysis showed a complete absence of M ⁺ 42 ions and a sharp negative ion peak at 407 amu.. The product was isolated after neutralization using hydrochloric acid and evaporating the solvents. The product was isolated from the gum by acetone extraction (2.5 grams; 52%). The complete synthesis of 4-0-t-butyl hydroquinone-T- O-α & β-D-glucopyranose is shown below in Scheme 2.

Scheme 2

3 J. Coupling of Glycosyl-H-Phosphonic acid with 10-Hydroxy camptothecin: A solution of dried 10-hydroxycamptothecin (500 mg, 1.37 mmol) in dry pyridine (10 mL) was heated to 50°C until all of the solids dissolved. The mixture was cooled to O ⁰ C and 1-H-Phosphonate 2,3,4,6-tetra-O-acetyl-D-Glucose (853 mg, 1.5 equivalent, 2.06 mmol) was added. Pivoloyl chloride (0.7 mL) was added slowly over 1 hour and the mixture was stirred for 1 hour more at room temperature. A solution of

iodine (700 mg) in Pyridine:H ₂ O (9:1) was added and the mixture stirred for 30 minutes at room temperature. The mixture was worked up after addition of 40 mL of 15 % Na ₂ S ₂ O ₃ solution and extracted with chloroform (3x100 mL). The organic layer was washed once with 50 mL of saturated solution Of NaHCO ₃ . Removal of the solvent gave the desired tetraacetyl derivative (450 mg) after column chromatography over silicagel. The mass spectrum of the compound showed molecular ion at 773 (M-I; amu) in accordance with the structure. The complete synthesis of camptothecin-10-O- α & β-2',3',4',6'-tetra-0-acetyl-D-glucopyranose is shown below in Scheme 3. Scheme 3

Pyridine

Pivaloyl chloride iodine w ork up

The related compounds, shown below, can be prepared using similar methods.

a glyco-phosphorylated testosterone

a glyco-phosphorylated 3-epipodophyllotoxin

3-0-phospho-glycosylated estradiol 17-0-phospho-glycosylated estradiol

a glyco-phosphorylated-topotecan a glyco-phosphorylated-irinotecan

a phospho-glycosylated salicylic acid a phospho-glycosylatcd acetaminophen Other Embodiments

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims.