WO2006037090A2 - Drug-phosphate conjugates as prodrugs - Google Patents

Abstract

Phosphate-substituted therapeutic compounds and analogs thereof that may serve as prodrugs are described. The prodrugs may include phosphorus-containing compounds, drug-phosphate conjugates, and analogs of drug-phosphate conjugates. Also described are analogs of drugs, including phosphorylated drugs, where the linker connecting the phosphate fragment to the drug fragment is other than oxygen, such as but not limited to linkers forming phosphoramidates, phosphonates, difluorophosphonates, oxophosphonates, and the like.

Description

PRODRUGS OF DRUG-PHOSPHATE CONJUGATES AND ANALOGS

THEREOF

CROSSREFERENCETORELATEDAPPLICATION This application claims the benefit under 35 U.S.C. § 119(e) of U.S.

Provisional Application Serial No. 60/614,338, filed September 28, 2004, and U.S. Provisional Application Serial No. 60/615,134, filed September 30, 2004, the disclosures of which are hereby incorporated by reference.

FIELD OF INVENTION

This invention relates to intracellular delivery of phosphate-substituted therapeutic compounds and analogs thereof. More particularly, this invention relates to prodrugs of phosphorus containing compounds, and prodrugs of drug-phosphate conjugates and analogs of drug-phosphate conjugates.

BACKGROUND OF THE INVENTION

Significant research efforts have been directed toward developing systems for enhancing the intracellular delivery of phosphate-substituted compounds, such as biologically active peptides and nucleotides. These phosphate-substituted compounds possess a relatively high localized negative charge that may lead to low cellular permeability.

For example, there is interest in the intracellular delivery of tyrosine phosphate peptides and peptidomimetics for use in blocking the uncontrolled proliferation of tumor cells. In this regard, protein tyrosine kinases are important regulators of cell cycle progression, and represent attractive targets for the rational design of novel anticancer agents. Protein tyrosine kinases are activated upon phosphorylation of specific tyrosine residues of the kinases and the phosphotyrosine residue along with the three adjacent amino acids (p Y-E-E-I) serve as a binding site for Src homology 2 (SH2) domains of intracellular signaling proteins. Interaction between an SH2 domain of an intracellular signaling molecule and an activated protein tyrosine kinase is essential for effective cell cycle progression. Thus, molecules that mimic the phosphotyrosine moiety of protein tyrosine kinases and block the binding of protein tyrosine kinases to SH2 domains represent promising antiproliferative agents for the treatment of cancers.

The phosphate dianion on tyrosine-phosphorylated protein tyrosine kinases is implicated in the successful kinase-SH2 domain interaction. The crystal structures of a number of ligand-SH2 domains have been reported, the interactions have been modeled, and many different inhibitors have been synthesized. In all of these phosphotyrosine peptides and peptidomimetic inhibitors, the phosphotyrosine residue is involved in a complex network of hydrogen bonding and charge-charge interactions that involve extensive interaction of the SH2 domain with the phosphate dianion of the tyrosine phosphate moiety. The importance of this dianionic interaction is illustrated by the fact that substitution of the phosphate group with a wide variety of other anionic and neutral hydrogen-bonding substituents has lead to a significant loss in binding affinity. Thus, the intracellular delivery of tyrosine phosphate peptides and peptidomimetics where the phosphate is in the form of the dianion has been viewed as advantageous.

Nucleotide analogs represent another class of promising agents for use in treating disease states caused by uncontrolled viral or cellular replication. Like tyrosine phosphate peptidomimetics, the phosphate dianion of nucleotide analogs is implicated in the capacity of these compounds to block viral or cellular replication. General approaches for the use of nucleotide analogs as therapeutic agents involve delivering the nucleoside analog as a prodrug across the cell membrane and to rely on the cellular machinery to link a phosphate group to the sugar portion of the purine or pyrimidine base-containing nucleoside to form the corresponding nucleotide analog. However, many nucleosides with therapeutic promise are not converted to the corresponding nucleotide by existing cellular pathways. Furthermore, tumor cells or virus-infected cells may become resistant to treatment by down-regulating the activity of nucleoside kinases, and, for many previously developed nucleotide prodrugs, the intracellular activation process may be so inefficient that effective nucleotide concentrations do not accumulate within the cell.

SUMMARY OF THE INVENTION

The invention described herein is directed to drugs or drug conjugates that include one or more phosphate fragments or analogs of phosphate fragments. In the case of analogs of phosphate fragments, various linkers other than oxygen connecting the phosphate fragment to the remaining portion of the drug are included, such as but not limited to linkers forming phosphoramidates, phosphonates, difluorophosphonates, oxophosphonates, and the like. The invention described herein is also directed to drugs that are phosphorylated after delivery. It is appreciated that those drugs that are phosphorylated after delivery may only become active as drugs following phosphorylation. The invention described herein is also directed to analogs of drugs that are phosphorylated after delivery. Analogs of drugs that are phosphorylated after delivery include phosphorylated drugs where the linker connecting the phosphate fragment to the drug fragment is other than oxygen, such as but not limited to linkers forming phosphoramidates, phosphonates, difluorophosphonates, oxophosphonates, and the like.

The formation of prodrugs of each of these drugs, drug-phosphate conjugates, and phosphate analogs thereof may enhance their efficacy and specificity. In one embodiment, prodrugs of drugs that include a phosphate fragment or phosphate fragment analog are described, hi another embodiment, analogs of prodrugs of drugs that include a phosphate fragment or phosphate fragment analog are described, hi this latter embodiment, the phosphate frgament or phosphate fragment analog that is generally associated with the drug is replaced with a phosphate fragment analog or another phosphate fragment analog, respectively. In another embodiment, prodrugs of drugs that are phosphorylated after delivery are described. In this latter embodiment of prodrugs of drugs that are phosphorylated after delivery, the prodrugs may be derived from phosphorylated versions of the drug and/or from analogs of phosphorylated versions of the drug, including but not limited to phosphoramidates, phosphonates, difluorophosphonates, oxophosphonates, and the like. Also in this latter embodiment of prodrugs of drugs that are phosphorylated after delivery, delivery is to be understood to include any in vitro, in vivo, or other in situ situation, condition, or set of conditions where the drug is introduced to the situation, condition, set of conditions, or reaction condition under which it is phosphorylated. As used throughout, the term "drug" from which prodrugs as described herein are derived will refer both to drugs that include a phosphate moiety or analog thereof, and to drugs that do not include a phosphate moiety or analog thereof but are phosphorylated after delivery such as in vivo by an endogenous process. It will be understood by the context whether the term drug is referring to a drug that does not include a phosphate moiety or analog thereof or referring to a drug that ordinarily includes a phosphate moiety or analog thereof, but where that phosphate moiety or analog thereof has been replaced by a phosphate analog described herein. It is also to be understoon that the term "drug" includes derivatives of drugs and drug fragments described above. Such derivatives may be prepared to include additional atoms or functional groups that are used for attaching the phosphorus-containing portion of the prodrug.

The general strategy for the intracellular delivery of phosphate- containing compounds utilized in the present invention is outlined in Scheme 1.

O DRUG O DRUG O DRUG

DELIVERY— P-Q *- DELIVERY— P-Q⁷ *- O— P— Q

MASK O - O^"

Scheme 1

In Scheme 1, Delivery is a delivery group, Mask is a masking group, Drag is the drug or drug fragment, and Q is an optional linker forming the drug-phosphate conjugate. Briefly, the phosphate-substituted compound to be delivered intracellularly is synthesized to include a delivery group and a masking group. In one embodiment, the masking group is illustratively a (substituted alkyl)amino or bis(substituted alkyl) amino moiety that may be modified intracellularly. It is appreciated in this embodiment that such (substituted alkyl)amines may be secondary or tertiary amines, i.e., there may be remaining hydrogen on the amine, or it may be further substituted with alkyl as descrbied herein, hi one aspect, modification of the masking group occurs while retaining the delivery group. In another aspect, the delivery group is removed before modification of the masking group occurs. In another aspect, the masking group undergoes intramolecular cyclization and subsequent cleavage by hydrolysis. In another aspect, intramolecular cyclization occurs contemporaneously, or even simultaneously in a concerted or anchimerically-assisted process, with hydrolysis of the masking group. The masking group, either before or after intracellular modification, is generally stable as long as the delivery group remains attached to the phosphorus atom. The delivery group is generally a group that is subject to intracellular hydrolysis, such as a biologically labile ester-forming group, or an ester that is readily hydrolyzed intracellularly. Illustratively, the delivery group includes nitrofuryl groups, perhydrooxazines, optionally substituted benzyl groups, and the like.

In one illustrative embodiment, a mechanism of prodrug release of drug-phosphate, optionally substituted drug-phosphoramidate, or optionally substituted drug-phosphonate conjugates described herein is outlined in Scheme 2.

Scheme 2 In Scheme 2, the delivery group is illustratively a nitrofuryhriethyloxy group (X = O) that has been bioreduced, and the masking group is illustratively a chlorobutylmethylamine. Upon intracellular activation of the delivery group by bioreduction, and subsequent removal, the masking group undergoes intramolecular cyclization and P-N bond cleavage (i.e., spontaneous or via hydrolysis) in the intracellular environment, thus releasing the desired phosphate-containing compound, or phosphate analog thereof. It is appreciated that the sequence of steps leading to release of the drug or drug-conjugate free of both the masking and delivery groups may occur in a different order. For example, removal of the delivery group may precede any intracellular modification of the masking group, as shown above. Alternatively, cyclization of the masking group may be followed by immediate P-N bond cleavage prior to removal of the delivery group. It is to be understood that further variation of the mechanisms of release of the drug or drug-conjugate are contemplated.

In one aspect, the (substituted alkyl)amine or bis(substituted alkyl)amine is a substituted ethylamine or bis(substituted ethyl)amine, such as an ethyl substituted with a leaving group including halo, alkoxy, acyloxy, optionally substituted alkylsulfonyloxy, optionally substituted arylsulfonyloxy, and the like. The leaving group is understood to be any electrophilic group capable of being nucleophilically displaced from the carbon atom to which it is bound, either intermolecularly or intramolecularly. It is appreciated that such nucleophilic lability of the leaving group can be exploited in a controlled fashion to facilitate the unmasking of the prodrug and ultimate release of the drug phosphate conjugate or analog thereof. It is appreciated that the substituted ethylamine or bis(substituted ethyl)amine variants of the masking group, including the haloethylamine or bis(haloethyl)amine derivatives, may also lead to irreversible adducts such as formation of cross-linked structures with nucleic acids like DNA and RNA, enzymes, and the like. In another aspect, the (substituted alkyl)amine or bis(substituted alkyl)amine includes a longer chain (substituted alkyl)amine, such as a substituted butyl and/or substituted pentyl group. Similarly, the substitutions include leaving groups such as halo, alkoxy, acyloxy, optionally substituted alkylsulfonyloxy, optionally substituted arylsulfonyloxy, and the like. It is appreciated that in each of the illustrative examples, the alkyl may have branching substituents, including additional alkyl groups, spiro-ring fusions, halo groups, such as fluoro, and the like. In one illustrative aspect, the branching substituents are not also leaving groups.

The prodrugs described herein may be formed from a wide variety of drugs or drug fragments that include a phosphate or phosphate analog, or drugs that are phosphorylated, and analogs thereof. Illustratively, prodrugs of nucleotide analogs, phosphotyrosine peptides and peptidomimetics, and other pharmaceutically significant compounds containing phosphate moieties are described. Further, prodrugs of phosphotyrosine peptides and peptidomimetics may be synthesized from protected phosphotyrosine precursor phosphoramidates as described herein. Thus, the invention is also directed to protected phosphotyrosine precursors and related protected peptides, for example, with N-Boc (tert-butoxycarbonyl) and N-Fmoc (fluorenylmethoxycarbonyl) groups. In addition, prodrugs of phosphoserine and phosphothreonine analogs are described herein. Synthesis of these variants may be accomplished by conventional methods, and similarly to the syntheses described herein for phosphotyrosine analogs, which may be correspondingly adapted.

In another illustrative embodiment, the prodrugs described herein are formed from drugs that are phosphorylated in vivo after delivery or administration to a patient being treated for a disease state for which the drug is useful. In one aspect, the drug is more active, or activated only, after phosphorylation by a biological pathway in the patient. It is appreciated that the efficacy of the drug may be hampered by the dependence upon that additional endogenous functionalization pathway. Further, in some disease states, the necessary pathway is compromised, further minimizing the efficacy of the drug. The prodrugs described herein include the necessary or desirable phosphorus-containing fragment at dosing to the patient being treated, and therefore do not require additional in vivo modification or activation. In addition, the prodrugs described herein are illustratively prepared to mask the negative charge, or double negative charge of the phosphorus-containing fragment. It is appreciated that highly charged molecules often suffer from poor absorption and/or transport to the relevant tissues of cells that are desirably treated. hi another illustrative embodiment, the prodrugs described herein are resistant to phosphatases, including intracellular phosphatases that may cleave the phosphorus-containing fragment from the prodrug conjugate or conjugate after the prodrug is converted into the phosphorus-containing drug or drug-conjugate analog. As stated herein, the efficacy of many drugs are aided by or dependent upon phosphorylation. Thus, prodrugs described herein include embodiments that are resistant to metabolic degradation and therefore have increased half-lives in the patient being treated. It is appreciated that phosphatases may be particularly abundant and their presence may hamper treatment efforts. hi another illustrative embodiment, the prodrugs described herein are formed from drugs containing phosphate or drug-phosphate conjugates, and are resistant to degradation by phosphatases or other enzymes that cleave phosphorus- oxygen bonds, including nucleotidases, endonucleases, exonucleases, and the like. Such degradation resistance may be accomplished by the replacement of the oxygen atom linker that connects the phosphorus fragment with the rest of the drug or drug- phosphate conjugate with a one or more carbon atoms, each of which may be substituted, such as with alkyl, fluorine, and oxo substituents, and the like. In another illustrative embodiment, a compound of formula

is described, where R¹ is alkyl, including C₁-C₆ alkyl, or -(CR₂)_nX; n is an integer from 2 to about 5; R² is -(CR₂)_mX; m is an integer from 2 to about 5; R is in each instance an independently selected substituent, including hydrogen, alkyl, fluoro, and the like; or any geminal pair of such groups R can be taken together with the attached carbon to form a ring, including a carbocyclic ring, or heterocyclic ring; X is a nucleophilically labile group, including halo, alkoxy, phenoxy, acyloxy, sulfonyloxy, and the like; R³ is a biologically labile ester forming group; Drug is a biologically active compound, the activity of which is increased upon phosphorylation after delivery, or a fragment of a biologically active compound-phosphate conjugate; and A and B are each independently selected substituents, including hydrogen, alkyl, fluoro, and the like; or A and B are taken together with the attached carbon to form a carbonyl group or a carbocyclic ring.

In another illustrative embodiment, a compound of formula

is described, where R¹ is alkyl, including C₁-C₆ alkyl, or -(CR₂)_nX; n is an integer from 2 to about 5; R² is -(CR₂)_mX; m is an integer from 2 to about 5; R is in each instance an independently selected substituent, including hydrogen, alkyl, fluoro, and the like; or any geminal pair of such groups R can be taken together with the attached carbon to form a ring, including a carbocyclic ring, or heterocyclic ring; X is a nucleophilically labile group, including halo, alkoxy, phenoxy, acyloxy, sulfonyloxy, and the like; R³ is a biologically labile ester forming group; Drug is a biologically active compound, the activity of which is increased upon phosphorylation after delivery, or a fragment of a biologically active compound-phosphate conjugate; and R⁴ is hydrogen, alkyl, acyl, and the like.

In another illustrative embodiment, a compound of the formula

is described, where R is alkyl, including C₁-C₆ alkyl, or -(CR₂)_nX; n is an integer from 2 to about 5; R² is -(CR₂)_mX; m is an integer from 2 to about 5; R is in each instance an independently selected substituent, including hydrogen, alkyl, fluoro, and the like; or any geminal pair of such groups R can be taken together with the attached carbon to form a ring, including a carbocyclic ring, or heterocyclic ring; X is a nucleophilically labile group, including halo, alkoxy, phenoxy, acyloxy, sulfonyloxy, and the like; R³ is a biologically labile ester forming group; and Drug is a biologically active compound, the activity of which is increased upon phosphorylation after delivery, or a fragment of a biologically active compound-phosphate conjugate. It is appreciated that in every prodrug described herein, the drug may be modified as is necessary to provide a suitable point of attachment or provide a suitable linking atom or atoms to allow conjugation of the drug with the phosphorus- containing portion of the prodrug conjugate to be prepared. For example, the phosphorus may be attached directly to a drug through a linker, such as methylene, difluoromethylene, carbonyl, and the like, on an aromatic portion of the drug. Alternatively, the drug may be derivatized with, for example, a suitable heteroatom, such as optionally substituted amino, and the drug derivative is then attached to the phosphorus through an optional linker. In the case of an optionally substituted amino, that nitrogen may be directly attached to the phosphorus to form a prodrug including a phosphordiamidate.

In one illustrative aspect of these embodiments, the integers n and m are each 2. hi another illustrative aspect, the integers n and m are each 4 or 5. In another illustrative aspect, each R is hydrogen. In another illustrative aspect, X is halogen, such as chloro. In another illustrative aspect, both A and B are hydrogen or both A and B are fluoro. hi another illustrative aspect, R³ is heteroarylalkyloxy, optionally substituted with an electron withdrawing group, such as nitro, alkylsulfonyl, cyano, halo, and the like, hi another illustrative aspect, R³ is optionally substituted arylalkyloxy, such as optionally substituted benzyl, hi another illustrative aspect where R³ is optionally substituted benzyl, that compound may be prepared as an intermediate in the synthesis of the prodrugs described herein where R³ is converted into heteroarylalkyloxy, optionally substituted with an electron withdrawing group. In another illustrative aspect, R³ is optionally substituted 1,3- heterocycloalkyl, such as an optionally substituted tetrahydro-l,3-oxazine alkyl. hi another illustrative aspect, Drug is a phosphotyrosine peptide or peptidomimetic, a nucleoside or nucleoside analog, and the like.

In one aspect where R³ is optionally substituted 1,3-heterocycloalkyl, R³ is 4₅4,6-trimethyl-3,4,5,6-tetrahydro-l,3-[2H]-oxazin-2-ylethyl. Prodrugs described herein that include R³ as 4,4,6-trimethyl-3,4,5,6-tetrahydro-l,3-[2H]- oxazin-2-ylethyl may be prepared by conventional methods, including those described by Borch et al. in U.S. Patent No. 5,233,031, the synthetic methods of which are incorporated herein by reference. The prodrugs described herein may be applied to increase cell permeability of the drug or drug-phosphate conjugate or analog thereof, by selective activation. In the illustrative aspect where R³ is optionally substituted heteroarylalkyloxy, one variation includes nitrofuryhnethyloxy. It is appreciated that the prodrug activation may be initiated by bioreductive elimination of the delivery group followed by spontaneous liberation of the masking group, as generally described by Meyers, C.L.F.; Hong, L.; Joswig, C; and Borch, R.F. in J. Med. Chem. 43:4313-4318 (2000), the disclosure of which is incorporated herein by reference. The presence of a reducing environment in solid tumors has been established by Borch, R.F.; Liu, J.; Schmidt, J.P.; Marakovits, J.T.; Joswig, C; Gipp, JJ.; Mulcahy, R.T. in J. Med. Chem. 43:2258-22658 (2000), the disclosure of which is incorporated herein by reference, and allows a bioreductive activation prodrug strategy to achieve selectivity in tumor cells, as described herein.

In one aspect, the prodrugs described herein include a phosphonate linkage between the phosphorus and the drug or drug-conjugate fragment. In another aspect, the prodrugs described herein include a difluorophosphonate linkage between the phosphorus and the drug or drug-conjugate fragment. In another aspect, the prodrugs described herein include a carbonyl linkage between the phosphorus and the drug or drug-conjugate fragment. Illustrative examples of the delivered component include one or more of the following

DRUG Y₁ ,DRUG O _/DRUG

HK O— P— N O— P— N

13 where Drug is virtually any biologically active compound that may be phosphorylated after delivery or a fragment of any biologically active compound that would generally be delivered as a drug-phosphate conjugate, or analog thereof. Illustrative biologically active compounds include enzyme inhibitors, receptor antagonists, receptor agonists, and the like. DETAILED DESCRIPTION OF THE INVENTION

In one illustrative embodiment, pharmaceutical formulations comprising an effective amount of prodrug compounds for treatment of cancers are described. As used herein, an effective amount of the prodrug compound with respect to the treatment of cancer refers to an amount of the compound which, upon administration to a patient, inhibits growth or proliferation of tumor cells, kills malignant cells, reduces the volume or size of the tumors, or eliminates tumors in the treated patient.

The effective amount to be administered to a patient may be based on body surface area, patient weight, and patient condition. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich, E.J., et al., Cancer Chemother. Rep., 50 (4): 219 (1966). Body surface area may be approximately determined from patient height and weight (see e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, New York, pages 537-538 (1970)). An effective amount of the prodrug compounds for use in treating cancer, can range from about 0.05 mg/kg to about 100 mg/kg, about 0.25 mg/kg to about 50 mg/kg, and most typically about 0.1 to about 10 mg/kg per dose. Effective doses may also vary dependent on route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments, including other chemotherapeutic agents, and radiation therapy.

Pharmaceutical formulations of the prodrugs described herein may be administered via any route, including a parenteral route, including subcutaneously, intraperitoneally, intramuscularly and intravenously. Examples of parenteral dosage forms include aqueous solutions or suspensions of the active agent in isotonic saline, 5% glucose or other well-known pharmaceutically acceptable liquid carrier, hi one aspect of the pharmaceutical compositions described herein, the compound is dissolved in a saline solution containing 5% of dimethyl sulfoxide and about 10% Cremphor EL (Sigma Chemical Company). Additional solubilizing agents such as cyclodextrins, which can form more soluble complexes with the present compounds, or other solubilizing agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the present compounds for cancer therapy. Altematively, the present compounds can be formulated into dosage forms for other routes of administration utilizing conventional methods. The pharmaceutical compositions can be formulated, for example, in dosage forms for oral administration in a capsule, a gel seal or a tablet. Capsules may comprise any well- known pharmaceutically acceptable material such as gelatin or cellulose derivatives. Tablets may be formulated in accordance with conventional procedure by compressing mixtures of the active prodrugs and solid carriers and lubricants well- known to those familiar with the art. Examples of solid carriers include starch, sugar, bentonite. The compounds described herein can also be administered in a form of a hard shell tablet or capsule containing, for example, lactose or mannitol as a binder and conventional fillers and tableting agents.

With regard to illustrative formulae:

where R¹, R², R³, R⁴, A, B, and Drug are as defined herein, the electrophilic group X is illustratively halo, such as chloro, bromo, or iodo. However, in another aspect, the nature of group X is flexible and encompasses a wide variety of electrophilic groups, provided that the group X can serve as a good leaving group to enable cyclization and concomitant quaternization of the phosphorous-bound nitrogen atom in vivo following administration of the phosphoramidite prodrugs to a patient. Other electrophilic leaving groups such as acetoxy, haloacetoxy, alkyl and arylsulfonyloxy, each of which may be optionally substituted, such as methane sulfonyloxy, trifluoromethane sulfonyloxy, tosyloxy, brosyloxy, nosyloxy, and the like may also be used. The term "biologically labile ester forming group" as used herein refers to those ester forming groups derived from alcohols that form ester derivatives that are stable under drug manufacture and storage conditions but are subject to hydrolysis when exposed to biological conditions in vivo. Illustratively, the ester forming groups used herein exhibit minimal susceptibility to hydrolysis in the body fluids in the extracellular space but exhibit susceptibility to hydrolysis in the intracellular space where ester-degrading reductive conditions are prominent. Thus, in one embodiment the biologically labile ester forming group on the prodrugs described herein are those ester forming groups that are susceptible to hydrolysis under mild reductive conditions which include those groups such as nitroaryl, including nitrofuryl, nitrothienyl, nitropyrroyl, nitroimidazoyl, and the like, indanyl, napthoquinolyl, perhydrooxazinyl, and the like. The nature of the ester forming group R³ is not critical provided that the group exhibits susceptibility to hydrolysis under intracellular conditions, typically biological conditions that exhibit reductive potential.

The therapeutic compounds that can be derivatized as described herein to form prodrugs include amino acids, illustratively in the form of their doubly protected (both carboxyl protected and amino protected) form, peptides, proteins, peptidomimetics, nucleotide analogs, and other drug compounds that can exhibit biological activity as their corresponding phosphates, optionally substituted phosphoramidates, or optionally substituted phosphonate analogs thereof. It is understood that for drugs that already include a phosphate radical, fragment, or analog thereof, such as a phosphonate analog of a phosphate, the drug fragment excluding the phosphate radical or analog thereof is used as described herein as the drug in preparing prodrugs. Illustratively, the drug that is converted to a prodrug as described herein is a nucleotide or nucleoside analog, such as fluorodeoxyuridine, which exhibits anti-tumor activity as its corresponding monophosphate derivative, hi another embodiment the drug is an amino acid, a peptide or a peptidomimetic.

Drugs that are phosphorylated after delivery and therefore become biologically active or more biologically active include drugs such as antitumor nucleosides, phosphotyrosine peptides and peptidomimetics, and others. In general, any drag that is phosphorylated after delivery may be prepared as a prodrug described herein, as a drug-phosphate conjugate analog described herein, or as a prodrug of a drug-phosphate conjugate analog described herein.

In another illustrative embodiment, the drug, or analog or derivative thereof is a substituted aromatic compound. In one aspect, the phosphate analog is a carbonyl phosphonate. Such compounds may be prepared according to the synthetic route shown in Scheme 3.

Scheme 3. (a) P(OR^a)₃; (b) TMS-halide, (COCl)₂; or SOCl₂; (c) Cl(CH₂)_nNMe, or an acid addition salt thereof, base; (d) Ar-CH₂-OH, base.

Referring to Scheme 3, L is a leaving group, such as acyloxy, sulfonyloxy, halo, and the like; R^a is alkyl, aryl, or arylalkyl; n is an integer from 1 to about 5, and is illustratively 4; Ar is an optionally substituted aryl group, such as phenyl, furyl, thiophenyl, and the like, and R represents the balance of the drug, or analog or derivative thereof, including ring fusions, and one or more substitutents such as an amino acid, or peptide, and the like. Referring to step (a), the reaction may be performed without solvent, or with solvent, such as dipolar solvents and polar aprotic solvents such as DMSO, DMF, CH₂Cl₂, (CH₂Cl)₂, and the like. Referring to step (b), suitable solvents include dipolar solvents and polar aprotic solvents such as DMSO, DMF, CH₂Cl₂, (CH₂Cl)₂, and the like. Referring to step (c), suitable solvents include dipolar solvents such as CH₂Cl₂, (CH₂Cl)₂, and the like; and suitable bases include amine bases such as Et₃N, (i-Pr)₂NEt, pyridine, collidine, lutidine, and the like. Suitable solvents for step (d) include ether, THF, DME, and the like; and suitable bases include organolithium bases such as n-BuLi, LiHMDS, sec-BuLi, and the like.

In another aspect, the phosphate analog is a difluorophosphonate. Such compounds may be prepared according to the synthetic route shown in Scheme 4.

Scheme 4. (a) BrCdCF₂P(O)(OR^a)₂, CuCl; (b) TMS-halide, (COCl)₂; or SOCl₂; (c)

Cl(CH₂)_nNMe, or an acid addition salt thereof, base; (d) Ar-CH₂-OH, base. Referring to Scheme 4, L is a leaving group, such as acyloxy, sulfonyloxy, halo, and the like; R^a is alkyl, aryl, or arylalkyl; n is an integer from 1 to about 5, and is illustratively 4; Ar is an optionally substituted aryl group, such as phenyl, furyl, thiophenyl, and the like, and R represents the balance of the drug, or analog or derivative thereof, including ring fusions, and one or more substitutents such as an amino acid, or peptide, and the like. Referring to step (a), the reagent BrCdCF₂P(O)(OR^a)₂ is prepared according to the method of Qabar, et. al, Tetraheron, 53:11171-11178 (1997), the synthetic disclosure of which is incorporated herein by reference; and suitable solvents include dipolar solvents and polar aprotic solvents such as DMSO, DMF, CH₂Cl₂, (CH₂Cl)₂, and the like. Referring to step (b), suitable solvents include dipolar solvents and polar aprotic solvents such as DMSO, DMF, CH₂Cl₂, (CH₂Cl)₂, and the like. Referring to step (c), suitable solvents include dipolar solvents such as CH₂Cl₂, (CH₂Cl)₂, and the like; and suitable bases include amine bases such as Et₃N, (i-Pr)₂NEt, pyridine, collidine, lutidine, and the like. Suitable solvents for step (d) include ether, THF, DME, and the like; and suitable bases include organolithium bases such as n-BuLi, LiHMDS, sec-BuLi, and the like. Alternatively, the difluorophosphonate functional group may be prepared according to Scheme 5.

Scheme 5. (a) (diethylamino)sulfur trifluoride (DAST). Referring to Scheme 5, L, R^a, and R are as defined herein, an step (a) may be performed without solvent or with solvent, such as dipolar solvents including CH₂Cl₂, (CH₂Cl)₂, and the like.

It is appreciated that in aspects where the group R includes an ester, acid, or amide, such functional groups may be interconverted prior to or after variations of the steps shown in Schemes 3-5. Illustratively, the group R is a carboxylic acid, or analog or derivative thereof or an amino acid, or analog or derivative thereof, having any of the formulae shown in Scheme 6.

Scheme 6.

Ester-acid, acid-ester, and acid-amide interconversion may be performed on any of the above formulae using conventional procedures. See generally, Larock in "Comprehensive Organic Transformations," VCH Publishers, Inc. (New York, 1989); Greene & Wuts in "Protective Groups in Organic Synthesis," 2d Ed., John Wiley & Sons (New York, 1991), the synthetic disclosures of which are incorporated herein by reference. Referring to Scheme 6, L and R^a are as defined in Schemes 1-3; R^b is hydroxy, alkoxy, aryloxy, arylalkoxy, or amino, each of which is optionally substituted or protected with a protecting group; Rc is hydrogen, or alkyl, aryl, arylalkyl, or amino, each of which may be optionally substituted or protected with a protecting group, or the balance of a peptide; and m is an integer from 0 to about 3. Illustratively, the starting compound is an acid, ester, or amide, where R^b is hydroxy, alkoxy, aryloxy, arylalkoxy, or amino, each of which is optionally substituted or protected with a protecting group, and the product is a different acid, ester, or amide.

Illustratively, esters and amides may be formed from the corresponding acid using coupling reactions, such as carbodiimide, activated acid derivative, and like coupling reactions. Activated acid derivatives include acid halides, pentafiorophenyl esters, N-hydroxybenzotriazole (HOBt) esters, N- hydroxysuccinimide (NOS) esters, and the like. Other esters such as fluorenylmethyl esters may be prepared with conventional procedures, such as by reacting with Fmoc- Cl and DMAP in the presence of a base, such as an amine base like Et₃N, (i-Pr)₂NEt, pyridine, collidine, lutidine, and the like, in an appropriate solvent, such as a dipolar solvent like CH₂Cl₂ or (CH₂Cl)₂. In addition, acids may be prepared from the corresponding esters by hydrolysis, such as with LiOH/THF/H₂O, and the like. Fluorenylmethyl esters may be hydrolyzed to the corresponding acids with conventional procedures, such as by treatment with bases including DBU, and the like, in an appropriate solvent, such as a dipolar solvent like CH₂Cl₂ or (CH₂Cl)₂. Illustratively, the compounds shown in Tables 1-2 maybe prepared according to Schemes 3-5.

Table 1

Table 2

In another illustrative embodiment, the drug is a peptidomimetic of the formula:

where q and k are independently 1 or O, and B is H, amino, protected amino, or C₁-C₄ alkanoylamino, and the (*) carbon is attached to phosphorus or the optional linker Q, including an optionally substituted phosphonate carbon. In another illustrative embodiment, the prodrugs are prepared from drug and derivatives of amino acids and amino acid esters of the formula

wherein (*), q, k, and B are as defined above and R is hydrogen or a carboxy protecting group, particularly an ester forming group. Such tyrosine or tyrosine analogs are useful for the preparation of peptides or peptidomimetic compounds capable of facile transmembrane delivery as an intracellular corresponding source of the peptide phosphate or peptidomimetic phosphate. Alternatively such compounds can themselves provide an intracellular source of the corresponding amino acid or amino acid analog. In one aspect, the drug fragment is a compound of formula

where R⁶ is hydrogen, alkyl, acyl, an amide protecting group, or an amino acid, peptide, or peptidomimetic; and the (*) carbon is attached to phosphorus, or the optional linker Q, such as a substituted phosphonate carbon.

In one illustrative embodiment where the drug is a phosphotyrosine peptidomimetic, the prodrug is a compound of formulae I, II, or III group

II

group

III where R⁷ is in each case hydrogen, or one or more optional substituents, each of which is independently selected, including halo, alkyl, alkoxy, cycloalkoxy, cycloalkylalkoxy, and the like; and R⁸ is in each case hydroxy, an hydroxy protecting group, such as -O-Fmoc, alkoxy, such as C₁-C₆ alkoxy, alkenyloxy, such as C₂-C₆ alkenyloxy, unsubstituted or substituted amino, and the like. Further, analogs of the compounds of formulae I, II, and III are described wherein R⁸ is monosubstituted amino such as an optionally substituted benzylamino. It is appreciated that in each of these illustrative embodiments, one or more chiral centers may be included, and that both possible optical isomers at each chiral center are contemplated to be included in the invention described herein. Further, it is also to be understood that various mixtures, including racemic mixtures, or other diastereomeric mixtures of the various optical isomers may be used in one or more embodiments described herein.

Compounds of formulae I, II, and III are prepared from the corresponding dialkyl phosphonates by conversion to the nitrofurfuryl N-methyl-N-chlorobutyl phosphoramidates.

The prodrug activation chemistry was verified for these difluoromethyl phosphonate analogs by hydrogenolysis of a model compound to the corresponding phosphonamidic acid, as shown in Scheme 7 where R⁷ and R⁸ are as defined above in formulae I, II, and III.

Scheme 7 The intermediate phosphoramide-half acid was then dissolved in buffer and its conversion to the phosphonic acid monitored by P nmr; a half life of 44 minutes (37⁰C , pH = 7.4) was observed for this conversion, confirming the feasibility of this prodrug approach for the delivery of difluoromethyl phosphonates.

In another embodiment, an illustrative intermediate V in the bioreduction of the prodrug IV is prepared, as shown in Scheme 8.

Scheme 8

Referring to Scheme 8, reduction of VI via catalytic hydrogenolysis also provided V which underwent the process shown in Scheme 8. A kinetic experiment using ³¹P NMR to monitor the conversion of V to VIII, in an aqueous buffer at pH 7.4, showed a half life of 4 minutes at 37°C.

In another embodiment, prodrugs described herein are prepared by the synthesis shown in Scheme 9, and exemplified for compound XIb which is a phosphatase resistant analog of SH2 domain inhibitor XIa (Z = O) with IC₅₀ = 11 ± 2 μM in a cell-based assay and its negative control compound X.

X Z = CF2

IX

PyBOP, DIEA, DCM

74 % XIb Z = CF2

Scheme 9

It is appreciated that compound XIb includes a chiral center, and that both possible optical isomers are contemplated to be included in the invention described herein. Further, it is also to be understood that various mixtures, including a racemic mixture, of the two optical isomers may be used in one or more embodiments.

Additional illustrative prodrugs are described where the Drugs are compounds of the following formulae:

where Base refers to the nucleoside bases present on guanine, cytosine, uracil, adenine, thymine, inosine, and analogs and derivatives thereof, such as the uracil analogs described herein; and phosphorus is attached to the (*) carbon, or attached to the (*) carbon through the optional linker Q, such as a linker like -C(AB)-, where A and B are each independently selected from hydrogen, alkyl, fluoro, and the like; or A and B are taken together with the attached carbon to from a carbocycle or a carbonyl group or analog thereof. Additional analogs of bases useful in nucleoside analogs as described herein include substituted imidazoles, substituted triazoles, and the like. Substitutions include nitro, optionally substituted amino, carboxylic acid derivatives, including acids, esters, and optionally substituted amides, cyano, alkyl, alkenyl, and alkynyl, including ethenyl.

In another illustrative embodiment, prodrugs described herein may include more than one prodrug feature. Such multiple prodrug features may be included in any prodrug that may be derived from a drug or drug fragment, or analog or derivative thereof, the biological activity or properties of which might be improved by having multiple phosphate or phosphate analogs conjugated thereto. For example, drugs that are phosphorylated after delivery at more than one location are contemplated to be advantageously modified with more than one prodrug features capable of delivering a phosphate conjugate or phosphate conjugate analog. In another illustrative embodiment, the prodrugs described herein are useful in methods to treat various disease states. Many of these disease state will involve the phosphorylated substrates of enzymes, such as kinases, nucleases, phosphorylases, nucleotidases, and the like. In general, any disease state that is responsive to changes in the biological pathways leading to the formation or cleavage of phosphorus-oxygen bonds in substrates may be treated using the prodrugs described herein. It is to be understood that disease states arising from the dysfunction of enzymes that use ATP as a substrate or co-factor and account for that disease state may be treated with the prodrugs described herein. For example, phosphorylation of tyrosine residues is regulated by protein-tyrosine kinases (PTKs), and such protein kinases have been implicated in solid tumor treatment, hi addition, phosphorylated tyrosine leads to interaction with SH2 domain-containing proteins to trigger a signal response in tumors. In one aspect, the prodrugs described herein may be useful in for delivering HIV drugs, such as ribavirin, acyclovir, didanosine, ganciclovir, foscarnet zalcitabine, gemcitabine, EICAR, NAIR, and nucleoside reverse transcriptase inhibitors. It is appreciated that many such HIV drugs are phosphoryled in vivo by nucleoside kinases, such as thymidine kinases. In another aspect, prodrugs are formed from zidovudine, stavudine, and abacavir, all of which are phosphorylated in vivo, such as by peripheral blood mononuclear cells. In another embodiment, the prodrugs described herein may be used to treat cancer. Evaluation of the prodrugs described herein useful for treating cancer may involve in vitro studies that measure cytotoxicicity of the prodrugs against various cancer cell lines, hi one illustrative aspect, the cancer cell is a B 16 melanoma cell, hi another illustrative aspect, the cancer cell is an L1210 leukemia cell. EXAMPLES

The following examples further illustrate the compounds described herein as prodrugs, and should not be understood to limit the invention in any way. Illustratively, modifications of the following examples are contemplated, including modifications of the drug or drug analog, or fragment of a drug-phosphate conjugate or analog thereof. Such other drugs may be prepared as pro-drugs using the phosphonate resistant conjugates described herein, such as drugs useful for treating bone diseases or bone metabolism and/or bone remodeling dysfunction. EXAMPLE 1. Biological Activity. A. Cytotoxicity against B16 Melanoma Cells. B 16 cells in exponential growth (2-? ^" 10⁶ cells in 10 ml of serum- free MEM medium) are treated with prodrug for 2 hr. The cells are separated, washed, and resuspended in MEM medium supplemented with 10% fetal bovine serum. The cells are plated in 60-mm culture dishes at a density of 50-50,000 cells/plate (depending upon the drug concentration to be used initially) and then incubated for 8 days in a CO₂ incubator at 37 ⁰C. The colonies are fixed and stained with 0.5% crystal violet in ethanol and counted.

EXAMPLE 2. Biological Activity. B. Cyctotoxicity Against L1210 Leukemia Cells. In addition to the B16 melanoma cell assay, compounds described herein may be evaluated for growth inhibitory activity against L1210 leukemia cells. Stock solutions of the compounds are prepared in 95% ethanol, and serial dilutions of drug are prepared in ethanol such that 50 μ\ of drug solution added to 10 ml of cell suspension give the desired final concentration. L1210 cells in exponential growth are suspended in Fischer's medium supplemented with 10% horse serum, 1% glutamine, and 1% antibiotic-antimycotic solution to give 10-ml volumes of cell suspension at a final density of 3-6 x 10⁴ /ml. Appropriate volumes of the solutions of each compound are transferred to the cell suspensions, and incubation is continued for 2, 8, 24, or 48 hr. The cells are spun down, resuspended in fresh drug-free medium, and returned to the incubator, then counted with a Coulter Counter 48 hr after treatment with the compound.

EXAMPLE 3. Biological Activity. C. Growth Inhibition in L1210 Mouse Leukemia Cells. Stock solutions of drugs are prepared in absolute ethanol, and serial dilutions of drug are prepared in ethanol. L1210 cells are suspended in Fisher's medium supplemented with 10% horse serum, 1% glutamine, and 1% antibiotic-antimycotic solution to give 10 mL volumes of cell suspension at a final density of 3-6 X 10⁴ cells/mL. Appropriate volumes of the drug solution are transferred to the cell suspensions, and incubation is continued for 2, 8, 24, or 48 hours. The cells are spun down, resuspended in fresh drug-free medium and returned to the incubator. Final cell counts are determined 48 hours after the start of drug treatment. The data are analyzed by sigmoidal curve fit of cell count vs. Log (drug concentration) and the results expressed as the ICs₀ (the drug concentration that inhibits cell growth to 50% of control value). EXAMPLE 4. Biological Activity. D. Luciferase inhibition on transfected J77 cells. J77 cells (2 X 10⁷ cells in 500 niL of FBS-free RPMI media) are transfected with 15 mg of NF-AT-Luciferase plasmid. Cells are transferred to a flask with 10 mL of complete RPMI media and incubated for 24 hours. After this time cells are spun down, resuspended in 12 mL of complete RPMI media and divided into a 6-well plate (2 mL/well). Drug stock solutions in DMSO are made and each well is treated with a different concentration and incubated for 2 hours. Each well is then treated with 10 mL of a mixture of 1.25 mg of PMA and 9 mg of Ionomycin in 115 mL of FBS-free RPMI, followed by the addition of 2 mg of Anti-CD3. Cells are incubated for 6 hours, spun down, washed with PBS, lysed for 15 min and centrifuged. Supernantant is collected and stored at -78 ⁰C overnight. Luciferase activity is measured using a luminometer with a mixture of 50 mL of luciferase substrate and 10 ml of supernatant.

EXAMPLE 5. Biological Activity. E. Tritium Assay in L1210. LM and LM(TK-) Cells. Stock solutions of drugs are prepared in absolute ethanol. L1210 cells are suspended in Fisher's medium, LM and LM (TK-) cells are suspended in minimum essential medium to give a final density of 11.24 X lO⁶ cells/mL. Aliquots (445 mL) of the cell suspension are placed in ependorf tubes, followed by addition of 5 mL of drug solution and incubated at 37°C for 2 hours. Tritium release reaction is started by the addition of 50 mL of serum free media containing 1 mCi [5-³H]dCyt in a 0.5 mM dCyt solution. Allowed reaction to incubate for 60 min in a shaking water bath at 37°C. Reaction is terminated by transferring 100 mL of the reaction mixture to ependorf tubes containing 100 mL of a 20% mixture of activated charcoal suspension in 4% aqueous perchloric acid. Tubes are vigorously vortexed, then centrifuged in a microfuge. Radioactivity of 100 mL of the supernatant is measured using a scintillation spectrometer. The data are analyzed by sigmoidal curve fit of CMP vs. Log (drug concentration) and the results expressed as the IC₅₀ (the drug concentration that inhibits cell growth to 50% of control value). EXAMPLE 6. Illustrative Pharmaceutical Formulations. The following parenteral formulations may be prepared for use in cancer therapy

where the compound is of the formula

and DRUG is illustratively selected from one or more of the following:

* <\ /> (CH₂)_q[CHB]_kCO₂R

where (*), q, B, k, and Base are as described herein.

Additional examples of the synthesis of illustrative prodrugs of the invention are also described. Additional examples of chemical transformations that may be used in the synthesis of additional illustrative prodrugs of the invention are also described. Li each of the following examples, the citations are incorporated herein by reference for all they describe regarding the chemical transformation that is the subject of the example.

EXAMPLE 7. Diethyl benzoylphosphonate. This Example was prepared according to the general method of Terauchi & Sakurai, Bull. Chem. Soc. Jpn. 43:883-90 (1970), the synthetic disclosure of which is incorporated herein by reference, hi particular, benzoyl chloride (3.65 mL, 4.42 g, 31.4 mmol) was added dropwise to stirring P(OEt)₃ (7.0 mL, 6.8 g, 41 mmol) at 0 °C under an Ar atmosphere. The reaction mixture was warmed to ambient temperature over 1 h, and the title compound was distilled from the reaction mixture (147-172 °C, in vacuo) to give 4.85 g (64%).

EXAMPLE 8. Diethyl difluorobenzylphosphonate. This Example was prepared according to the general method of Burke et al, Tetrahedron Letters 34:4125-4128 (1993), the synthetic disclosure of which is incorporated herein by reference. Li particular, diethyl benzoylphosphonate (EXAMPLE 7, 2.85g, 10.65 mmol) and (diethylamino)sulfur trifluoride (DAST) (6.0 mL, 45.41 mL, 4.26 eq) were stirred together at O⁰C under an Ar atmosphere. The reaction was warmed to ambient temperature over 1 h and continued to stir for 20 h. The reaction mixture was then cooled again to O⁰C and it was added to a 25% solution OfNaHCO₃ in H₂O (100 mL) cooled to O⁰C. The suspension was stirred for 15 min, 150 mL of H₂O was added and the aqueous phase was extracted with four-50 mL portions of CHCl₃. The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated to a dark oil. The crude material was purified by flash column chromatography using 10% EtOAc/hexane. The product was isolated as an amber oil 1.18g (42%).

EXAMPLE 9. This Example was prepared according to the general method of Qabar, et. al, Tetraheron, 53:11171-11178 (1997), the synthetic disclosure of which is incorporated herein by reference, hi particular, cadmium powder (3.7g, 33.31 mmol, 3.2 eq) was stirred in dry DMF (36 mL) under an atmosphere of Ar. Chlorotrimethylsilane (0.13 mL, 1.04 mmol, 0.1 eq) was added dropwise to activate the metal. After 5 min, diethyl bromodifluoromethylphosphonate (5.7 niL, 32.27 mmol, 3.1 eq) was added dropwise from an addition funnel. The reaction mixture became warm and cloudy. After stirring for 3h at ambient temperature, formation of BrCdCF₂P(O)(OEt)₂ was deemed complete as indicated by the absence of starting material by TLC (15% EtOAc/hexane). N-Fmoc-L-4-iodophenylalanine methyl ester (5.49g, 10.41 mmol, 1 eq) in 8 mL DMF was treated with CuCl (4.12g, 41.64 mmol, 4.0 eq) and was stirred at ambient temperature under Ar for 10 min. The organocadmium solution was transferred to the solution with CuCl by cannula. After 2 days the reaction was quenched with 40 mL saturated aqueous NH₄Cl and was filtered through a pad of celite. Phases of the filtrate were separated. The aqueous phase was extracted with Et₂O (3 x 4OmL). Combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated to a gold oil. The oil was subjected to Kugelrohr distillation (80 ⁰C, 1.55 torr) to remove volatile side products. The remaining oil was further purified by flash column chromatography (gradient elution: 0%, 20%, 50% EtOAc/hexane) to provide the product as a yellow oil (4.92g, 80%).

EXAMPLE 1OA. This example was prepared according to the general method of Landry, et al, JAm Chem Soc 118:5881-5890 (1996), the synthetic disclosure of which is incorporated herein by reference. In particular, diethyl phosphonate from EXAMPLE 9 (3.06g, 5.21 mmol) in 50 mL dry CH₂Cl₂ was treated with bromotrimethylsilane (2.40 mL, 15.6 mmol 3 eq) and the mixture was stirred under a drying tube for 18h. Solvent was removed under reduced pressure and the residue was co-evaporated twice with CH₂Cl₂ (30 mL) to remove excess bromotrimethylsilane. The resulting oil was taken up in CH₂Cl₂ (50 mL) and oxalyl chloride (1.37 mL, 15.6 mmol, 3 eq) was added followed by 2 drops of dry DMF. The reaction mixture was stirred under an Ar atmosphere for 1.5h, solvent was removed under reduced pressure and the residue was co-evaporated two times with CH₂Cl₂ to remove excess oxalyl chloride. The resulting oil was taken up in CH₂Cl₂ (50 mL) and was cooled to -2O⁰C with stirring under an Ar atmosphere iV-methyl-JV- (2-chlorobutyl)amine hydrochloride (0.91g, 5.7 mmol, 1.1 eq) was added followed by dropwise addition of diisopropylethylamine (2.00 mL, 11.5. mmol. 3.0 eq). The reaction mixture was kept at -2O⁰C for 2Oh. Solvent was removed under reduced pressure and the residue was purified by flash column chromatography using 3% Et₂O/CH₂Cl₂ as eluent providing 2.89g (85%).

EXAMPLES 1OB, 1OC, 1OD, 1OE and 1OF listed below were also prepared by the method described for EXAMPLE 1OA.

EXAMPLE 11. Diethyl difluorobenzylphosphonate (EXAMPLE 8) (0.25g, 0.95 mmol) was dissolved in thionyl chloride (5 mL) and the mixture was refluxed under an atmosphere of Ar for 5 days. Thionyl chloride was removed under reduced pressure and the residue was taken up in dry CH₂Cl₂ (12 mL) and was cooled to -78⁰C with stirring under an Ar atmosphere. N-methyl-N-(2-chlorobutyl)amine hydrochloride (0.13g, 0.88 mmol, 0.93 eq) was added followed by the addition of triethylamine (0.27 mL, 1.94 mmol, 2.2 eq). The reaction was kept at -78⁰C for 2Oh. Solvent was removed under reduced pressure and the residue was purified by flash column chromatography using 10%EtOAc/hexane as eluent to provide a pale yellow oil 0.17g (57%).

EXAMPLE 12E. Nitrofurfuryl alcohol (0.26g, 1.83 mmol, 2 eq) along with 4-(phenylazo)diphenylamine (3 mg) in THF (10 niL) was cooled to -78 ⁰C with stirring under an Ar atmosphere. A 1.0M solution of lithium hexamethyldisilazide was added dropwise via syringe until the color of the reaction mixture changed from orange to violet. This required 2.0 niL (2 mmol, 2.17 eq) of lithium base. This mixture stirred at -78 ⁰C for 15 min while a solution of chlorophosphonamidate from EXAMPLE 1OA (0.6Og, 0.92 mmol) in THF (6.0 mL) under an Ar atmosphere was prepared and cooled to -78 ⁰C. The alkoxide solution was transferred rapidly by cannula to the chlorophosphamidate and the resulting mixture was kept at -78 ⁰C for 1Oh. The reaction mixture was quenched with 10 mL saturated NH₄Cl solution and the aqueous phase was extracted three times with Et₂O (15 mL each). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated to a dark, amber oil. The crude material was purified by flash column chromatography using gradient elution (25%, 50% EtOAc/hexane) to provide a light, amber oil 0.41g (58%).

EXAMPLES 12A, 12B, 12C, 12D, 12F₅ 12G and 12H listed below were also prepared by the method described for EXAMPLE 12E. Analytical Data for EXAMPLES 12A - 12H are as follows:

EXAMPLE 12A. ¹H NMR (CDCl₃): δ 7.55 (m, 2H); 7.42 (m, 3H); 7.20 (d, IH, J =3.62Hz); 6.53 (d, IH, J = 3.62 Hz); 5.09 (m, 2H); 3.58 (m, 2H); 3.05 (m, 2H); 2.65 (d, 3H, J = 8.96 Hz); 1.63 (m, 4H); ³¹P NMR (CDCl₃): δ -10.40 (dd, J =115.53 Hz).

EXAMPLE 12B. ¹HNMR (CDCl₃): δ 7.59 (m, 2H); 7.35 (m, 8H); 5.03 (d, 2H, J = 7.63 Hz); 3.47 (m, 2H); 2.99 (m, 2H); 2.62 (d, 3H, J = 8.81 Hz); 1.57 (m, 4H); ³¹P NMR (CDCl₃): δ -11.32 ( dd, J = 113.84 Hz).

EXAMPLE 12C. ¹H NMR (CDCl₃): δ 7.40 (m, 13H); 6.54 (m, IH); 5.30 (m, IH); 4.98 (m, 2H); 4.72 (m, 3H); 4.39 (m, 2H); 4.21 (m, IH); 3.54 (m, 2H); 3.18 (m, 2H); 2.63 (d, 3H, J = 9 Hz); 2.56 (s, IH); 1.72 (m, 4H); ³¹P NMR (CDCl₃): δ -10.45 (dd, J = IlOHz). EXAMPLE 12D. ¹H NMR (CDCl₃): δ 7.73 (d, 2H, J = 7.52 Hz); 7.36

(m, HH); 6.49 (d, 1 H, J = 2.29 Hz); 5.26 (m, IH); 4.99 (m, 2H); 4.43 (d, 2H, J = 5.83 Hz); 4.15 (m, 1H3.70 (s, 2H), 3.5 (m, 2H); 3.05 (m, 2H); 2.74 (d, 3H, J = 8.95 Hz); 1.62 (m, 4H); ³¹P NMR (CDCl₃): δ -10.44 (dd, J = 116.45 Hz)

EXAMPLE 12E. ¹H NMR (CDCl₃): δ 7.76 (m, 2H,); 7.39 (m, 9H); 6.54 (m, IH); 5.27 (m, IH); 5.04 (m, 2H); 4.68 (m, IH); 4.41 (m, 2H); 4.22 (m, IH); 3.74 (s, 3H); 3.53 (m, 2H); 3.09 (m, 4H); 2.69 (d, 3H, J = 8.74 Hz); 1.69 (m, 4H); ³¹P NMR (CDCl₃): δ -11.68 (dd, J = 116.44 Hz).

EXAMPLE 12F. ¹H NMR (CDCl₃): δ 7.79 (m, 2H₅); 7.58 (m, 2H); 7.38 (m, 13H); 7.12 (m, IH); 6.51(m, IH); 5.30 (m, IH); 4.98 (m, 2H); 4.72 (m, IH); 4.38 (m, 2H); 4.18 (m, IH); 3.50 (m, 2H); 3.07 (m, 4H); 2.65 (d, 3H, J = 8.33 Hz); 1.67 (m, 4H); ³¹P NMR (CDCl₃): δ -10.72 (dd, J = 115.35 Hz).

EXAMPLE 12G. ¹H NMR (CDCl₃): δ 7.53 (d, 2H, J = 7.81 Hz); 7.31 (m, 7H); 7.22 (d, IH, J = 3.63 Hz); 6.53 (IH, d, J = 3.58 Hz); 5.01 (m, 2H); 3.75 (s, 2H); 3.56 (m, 2H); 3.05 (m, 2H); 2.67 (d, 3H, J = 8.92 Hz); 1.65 (m, 4H); ³¹P NMR (CDCl₃): δ -10.78 (dd, J = 118.34 Hz).

EXAMPLE 12H. ¹H NMR (CDCl₃): δ 7.55 (d, 2H, J = 7.84 Hz); 7.40 (d, 2H, 8.01 Hz); 7.25 (d, IH, J = 3.6 Hz); 6.54 (d, IH, J = 3.6 Hz); 5.08 (m, 2H); 3.72 (s, 3H); 3.68 (s, 2H); 3.52 (m, 2H); 3.11 (m, 2H); 2.74 (d, 3H, J = 9.00 Hz); 1.67 (m, 4H); ³¹P NMR (CDCl₃): δ -10.54 (t, J = 112.00 Hz).

Λ^J NHFmoc ' EXAMPLE 13. N-Fluorenylmethyloxycarbonyl-4-iodophenylalanine (5.08g, 9.89mmol) was suspended in MeOH (16 niL) under a drying tube. Thionyl chloride (1.4 mL, 19.79 mmol, 2 eq) was added dropwise by syringe. The reaction stirred at ambient temperature and became a solid mass over two days. The solid mass was treated with 100 mL EtOAc and was sonicated until a solution was obtained. The solution was washed with saturated NaHCO₃ (50 mL), water (5OmL), and brine (5OmL). The organic phase was dried over Na₂SO₄ and was concentrated to give a white solid which was used without purification. 5.21g, (99%).

A^J NHFmoc EXAMPLE 14. N-Fluorenylmethyloxycarbonyl-4-iodophenylalanine

(0.94g, 1.82 mmol), benzyl alcohol (1.3 mL, 12.76 mmol, 7eq) and dimethylaminopyridine (O.Olg, 0.09 mmol, 0.05 eq) were stirred together under an Ar atmosphere in CH₂Cl₂ (9 mL). The mixture was cooled to 0 ⁰C and diisopropylcarbodiimide (0.28 mL, 1.82 mmol, 1 eq) was added by syringe. The reaction came to ambient temperature over Ih and stirred at ambient temperature for 4 days. SiO₂ (2g) was added and solvent was removed under reduced pressure. The compound adhered to silica gel was purified by flash column chromatography using gradient elution (0%, 10%, 20%, 30% EtOAc/hexane) to provide the product as a white solid 0.69g (63%).

EXAMPLE 15. Diethyl difluoromethyl phosphonate (EXAMPLE 16, 0.42g, 0.73 mmol, 1 eq) in dry CH₂Cl₂ (6.0 mL) was stirred under an Ar atmosphere and was treated with propargyl alcohol (0.21 mL, 3.64 mmol, 5 eq) and dimethylaminopyridine (0.004g, 0.03 mmol, 0.05 eq). The reaction mixture was cooled to 0 ⁰C, diisopropylcarbodiimide (0.11 mL, 0.73 mmol, 1 eq) was added, and stirring continued for 20 h. SiO₂ (Ig) was added and solvent was removed under reduced pressure. The compound adhered to silica gel was purified by flash column chromatography using gradient elution (0%, 20%, 40% EtOAc/hexane) to provide the product as an oil 0.38g (85%).

EXAMPLE 16. Methyl ester prepared as described in EXAMPLE 9 (1.67g, 2.84 mmol, 1 eq) in THF (40 mL) was cooled to 0 ⁰C. LiOH (0.24g, 5.68mmol, 2 eq) in H₂O (35 mL) was added and the reaction mixture was stirred at 0 ⁰C. After 15 min the reaction mixture was poured into 250 mL EtOAc/lM HCl (1/1). The phases were separated and the aqueous phase was washed with 10OmL EtOAc. The combined organic phases were washed with brine and dried over Na₂SO₄ to give a viscous oil which was purified by flash column chromatography using 3%MeOH, 1%ACOH/CH₂C1₂ as eluent to provide the product 1.03g (64%).

EXAMPLE 17. Process A. The corresponding Fmoc ester (0.2Og, 0.30mmol, 1 eq) in CH₂Cl₂ (4.0 mL) was stirred under an Ar atmosphere and was cooled to 0 ⁰C. A 0.17 M solution of DBLVCH₂Cl₂ (1.96 mL, 0.33 mmol, 1.1 eq) was added dropwise. After 25 min the reaction was quenched with saturated NH₄Cl solution. The phases were separated and the aqueous phase was extracted four times with EtOAc (5 mL each). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated to an orange oil which was purified by flash column chromatography using 2% MeOH, 1% ACOHZCH₂CI₂ as eluent to provide the product as a gold solid 0.1 Og (67%). Process B. The corresponding methyl ester prepared as described in

EXAMPLE 12H (1.09g, 2.13 mmol, 1 eq) in THF (32 mL) and H₂O (3.4 mL) was stirred at 0 ⁰C. A solution of 0.3M LiOH/H₂O (14.2 mL, 4.26 mmol, 2 equiv) was added dropwise in the following increments: 7.10 mL, 2.4 mL, 2.4 mL and 2.4 mL. The reaction was monitored by TLC after each addition. After the final addition, the reaction was quenched with 10% aqueous citric acid, and the aqueous phase was extracted four times with EtOAc (40 mL each). The combined organic phases were washed with brine, dried over Na₂SO₄ and concentrated to a dark oil which was purified by flash column chromatography using gradient elution (50%, 100% -- EtO Ac/hex, then 1% AcOH/ EtOAc 0.77g (73%). ^

EXAMPLE 18. Methyl ester from EXAMPLE 12E (0.58g, 0.76 mmol, 1 eq) in THF (11 mL) and H₂O (2 mL) was stirred at O ⁰C. A solution of 0.3M LiOHTH₂O (5.1 mL, 1.52 mmol, 2 equiv) was added dropwise in the following increments: 2.55 mL, 0.85 mL, 0.85 mL and 0.85 mL. The reaction was monitored by TLC after each addition. After the final addition the reaction was quenched with 10% aqueous citric acid, and the aqueous phase was extracted four times with EtOAc (1O mL each). The combined organic phases were washed with brine, dried over

Na₂SO₄ and concentrated to a foam which was used without further purification 0.46g (81%).

EXAMPLE 19. Carboxylic acid from EXAMPLE 18 (0.07g, 0.10 mmol, 1 eq) in THF (ImL) was stirred at 0 ⁰C under an Ar atmosphere and was treated with N-hydroxysuccinimide (0.0 Ig, 0.10 mmol, 1 eq). Diisopropylcarbodiimide (0.02 mL, 0.14 mmol, 1.5 eq) was added. After stirring at 0 ⁰C for 2h a IM solution OfNH₄HCO₃ (0.48 mL. 0.48 mmol, 5 eq) was added and the reaction mixture allowed to reach ambient temperature as it stirred for 2Oh. The reaction mixture was diluted with CH₂Cl₂ (3 mL) and the organic phase was washed successively with 5 mL each of IM HCl, saturated NaHCO₃ solution and brine. Solvent was removed under reduced pressure and the crude material was purified by flash column chromatography using gradient elution (50%, 75% EtOAc/hexane) to give the product as an oil 0.05g (71%).

EXAMPLE 20. 4-Iodophenylacetic acid (2.Og, 7.6 mmol, 1 eq) in CH₂Cl₂ (30 mL) was treated with diisopropylethylamine (1.32 mL, 7.6 mmol, 1 eq) and the mixture was cooled to 0 ⁰C with stirring under an atmosphere of Ar. 9- Fluorenylmethyl chloroformate (2.16g, 8.36 mmol, 1.1 eq) dissolved in CH₂Cl₂ (7.6 mL) was added followed by the addition of dimethylaminopyridine (0.14g, 1.14 mmol, 0.1 eq). The reaction was stirred at ambient temperature for 30 min and was then diluted with CH₂Cl₂ (15 mL) and washed successively with 30 mL portions of IM HCl, saturated NaHCO₃ solution and brine. The organic phase was dried over Na₂SO₄ and concentrated under reduced pressure. The product was used without further purification 3.34g (99%).

Claims

CLAIMS:

1. A prodrug of the formula:

wherein

R¹ is alkyl or -(CR₂)_nX; n is an integer from 2 to about 5; R² is alkyl or -(CR₂)_mX; m is an integer from 2 to about 5; R is in each instance an independently selected substituent selected from the group consisting of hydrogen, alkyl, and fluoro; or any two groups R covalently attached to the same carbon atom can be optionally taken together with the attached carbon to form a carbocyclic ring or heterocyclic ring;

X is in each instance an independently selected nucleophilically labile group;

R³ is a biologically labile ester forming group; Drug is a biologically active compound, the activity of which is increased upon phosphorylation after delivery, or a fragment of a biologically active compound-phosphate conjugate; and A and B are each independently selected from the group consisting of hydrogen, alkyl, and fluoro; or A and B are taken together to form a double bonded oxygen.

2. A prodrug of the formula:

wherein

R¹ is alkyl or -(CR₂)_nX; n is an integer from 2 to about 5;

R² is alkyl or -(CR₂)_mX; m is an integer from 2 to about 5; R is in each instance an independently selected substituent selected from the group consisting of hydrogen, alkyl, and fluoro; or any two groups R covalently attached to the same carbon atom can be optionally taken together with the attached carbon to form a carbocyclic ring or heterocyclic ring; X is in each instance an independently selected nucleophilically labile group;

R³ is a biologically labile ester forming group;

Drug is a biologically active compound, the activity of which is increased upon phosphorylation after delivery, or a fragment of a biologically active compound-phosphate conjugate; and

R⁴ is hydrogen, alkyl, or acyl.

3. A prodrug of the formula:

wherein R¹ is alkyl or -(CR₂)_nX; n is an integer from 2 to about 5;

R² is alkyl or -(CR₂)_mX; m is an integer from 2 to about 5;

R is in each instance an independently selected substituent selected from the group consisting of hydrogen, alkyl, and fluoro; or any two groups R covalently attached to the same carbon atom can be optionally taken together with the attached carbon to form a carbocyclic ring or heterocyclic ring;

X is in each instance an independently selected nucleophilically labile group; R³ is a biologically labile ester forming group; and

Drug is a biologically active compound, the activity of which is increased upon phosphorylation after delivery, or a fragment of a biologically active compound-phosphate conjugate.

4. The prodrug of any one of claims 1, 2, or 3 wherein R¹ is Ci-C₆ alkyl.

5. The prodrug of any one of claims 1 , 2, or 3 wherein X is halo, alkoxy, phenoxy, acyloxy, or sulfonyloxy.

6. The prodrug of any one of claims 1 , 2, or 3 wherein the integer n is 2.

7. The prodrug of any one of claims 1, 2, or 3 wherein the integer n is 4 or 5.

8. The prodrug of any one of claims 1, 2, or 3 wherein each R is hydrogen.

9. The prodrug of any one of claims 1 , 2, or 3 wherein each X is a halo group.

10. The prodrug of claim 1 wherein both A and B are hydrogen.

11. The prodrug of claim 1 wherein both A and B are fluoro.

12. The prodrug of any one of claims 1, 2, or 3 wherein R³ is heteroarylalkyloxy optionally substituted with an electron withdrawing group.

13. The prodrug of any one of claims 1 , 2, or 3 wherein Drug is a phosphotyrosine peptide, a phosphotyrosine peptidomimetic, a nucleoside, or a nucleoside analog.

14. Use of the prodrug of any one of claims 1, 2, or 3 in the manufacture of a medicament for treating a dysfunction of an enzyme in a phosphate- dependent biological process in a mammal.

15. The use of claim 14 wherein the enzyme is a nuclease.

16. The use of claim 14 wherein the enzyme is a ligase.

17. The use of claim 14 wherein the enzyme is a polymerase.

18. A pharmaceutical composition comprising the prodrug of any one of claims 1, 2, or 3, and a pharmaceutically acceptable carrier, excipient, or diluent therefor.

19. A method for treating a disease state arising from a dysfunction of an enzyme in a phosphate-dependent biological process in a mammal, the method comprising the step of administering to the mammal an effective amount of the prodrug of any one of claims 1 , 2, or 3.

20. The method of claim 19 wherein the enzyme is a tyrosine kinase.

21. The method of claim 19 wherein the enzyme is a nuclease.

22. The method of claim 19 wherein the enzyme is a ligase.

23. The method of claim 19 wherein the enzyme is a polymerase.