HK1058005B - Modified prodrug forms of ap/amp - Google Patents

Description

Improved prodrug forms of AP/AMP

Background

The reductive conversion of nucleotides to deoxynucleotides by Ribonucleotide Reductase (RR) is a key rate-controlling step in the DNA biosynthetic pathway (Cory, J.g. "inhibitors of nucleotide diphosphate reductase activity", International Encyclopedia of Pharmacology and Therapeutics, Cory, J.G.; Cory, A.H. ed.; Pergamon Press; New York, (1989); section 128, pages 1-16). Since the levels of deoxynucleotides in mammalian cells are very low, a good correlation between tumor growth rate and specific activity of ribonucleotide reductase has been shown (Elford, et al, j.biol.chem. (1970), 245, 5228). Mammalian nucleotide reductases are composed of two distinct proteins, R1, which binds to a nucleotide substrate, and R2, which contains non-heme iron and free tyrosyl groups (Reichard P. Ehrenberg, A. science, (1983), 221, 514). Both R1 and R2 have an effect on enzyme activity.

Currently, there are two major classes of RR inhibitors. The first class includes nucleoside analogs, whose mechanism of action involves binding to the R subunit of the enzyme; some of these inhibitors are entering clinical development. Among these inhibitors, 2 ', 2 ' -difluoro-2 ' -deoxycytidine (Gemcitabine, trade name: Gemcitabine, Eli lilly) has recently been approved by the U.S. food and drug administration for the treatment of pancreatic cancer (Baker, et al, J.Med.Chem. (1991), 34, 1879), and 2 ' -fluoromethylene-2 ' -deoxycytidine are under evaluation in clinical trials concerning the treatment of various tumors (McCarthy, J.R., and Sunkara, design, synthesis, and anticancer activity of nucleotide reductase inhibitors of P.S., Weiner, D.B.; Williams, W.V. eds.; CRC Press, Boca Raton, (1994), 681364). A second class of RR inhibitors includes N-hydroxyurea (Reichard and Ehrenberg, Science, (1983), 221, 514 and Wright et al, Cell Biol. (1990), 68, 1364) and HCTs [ N-heterocyclic aldehyde thiosemicarbazones ], which act by destroying the free radical of the R2 subunit. HCTs have been shown to be the most potent inhibitor enzymes of ribonucleotide reductases, 80-5000 fold stronger than N-hydroxyurea in vitro (see Liu et al, J.Med.chem. (1992), 35, 3672 and J.Med.chem.science, (1995)38, 4234).

It is now recognized that HCTs may exert their enzyme inhibitory effects through their high affinity for iron on the R2 subunit, since iron is an essential element at the active site of ribonucleotide reductase. Phase I clinical evaluation of the lead compound 5-HP in this system was performed many years ago (decnti, et al, Cancer Res. (1972), 32, 1455 and Moore, et al, Cancer Res. (1971), 31, 235), and the results indicated that this compound was well active in animal models but not in patients with solid tumors, presumably in connection with its rapid metabolism in humans. 5-HP has been modified by the introduction of steric hindrance and the substitution of hydroxyl groups with amino moieties to provide a range of 3-amino-containing compounds (e.g., 1A (3-AP) and 1B (3-AMP) (see below)). Of these inhibitors, 3-AP showed excellent anticancer activity (Liu et al, j.med.chem. (1992), 35, 3672) and significantly reduced clearance (clearance rate). It is currently in phase 1 clinical trials. A single dose clinical trial can be discontinued when the drug reaches the pharmacokinetic endpoint without showing any drug-related toxicity. Additional phase 1 studies on long-term (extended) dosing regimens (5 times daily, 96 hour continuous infusion) are ongoing.

1A R＝H 3-AP(Triapine^TM)

1B R＝CH₃ 3-AMP

Although 3-AP shows in vivo activity, the therapeutic potential of this compound may be limited due to its poor aqueous solubility. Thus, to improve their water solubility and therapeutic index, two phosphate-containing water-soluble prodrugs 2 (para 3-AP prodrug) and 3 (ortho 3-AP prodrug) were developed. The phosphate-containing prodrug is intended to impart excellent water solubility to the compound at neutral pH and to improve its bioavailability.

Preliminary in vitro evaluations of 3-AP prodrugs showed that they were rapidly enzymatically converted to 3-AP by alkaline phosphatase. However, in vivo PK studies in Beagle dogs showed that the half-life of 3-AP released from the ortho-phosphate containing 3 was 14.2 hours, whereas the half-life of the para-prodrug 2 was only 1.5 hours. Also, prodrugs 2 and 3 were evaluated in mice bearing M-109 solid tumors (compared to 3-AP and cyclophosphamide). The results of these experiments indicate that the ortho-prodrug 3 is more potent in reducing toxicity than the parent 3-AP, and that its activity is comparable to that of cyclophosphamide (cytoxan). To further improve the biological and pharmaceutical properties of 3-AP prodrugs and to optimize their therapeutic applications, a series of ortho-phosphate containing prodrugs were designed.

2-para-3-AP prodrug 3-ortho-3-AP prodrug

Object of the Invention

In one aspect of the invention, one object of the invention is a compound, pharmaceutical composition and method for treating neoplasma (including cancer) patients.

In another aspect of the invention, it is an object of the present invention to provide a method for treating neoplasia with specific compositions that exhibit good and enhanced properties with respect to activity, pharmacokinetics, bioavailability and reduced toxicity.

It is another object of the present invention to provide compositions and methods for treating cancer that is resistant to treatment with traditional chemotherapeutic agents.

One or more of these and other objects of the present invention will be readily apparent from the following description of the invention.

Summary of The Invention

The present invention relates to compounds having the structure:

wherein R is H or CH₃；

R₂Is in the form of phosphoric acid or a salt thereof;

R₃is H, F, Cl, Br, I, OCH₃、OCF₃、CF₃Or C₁-C₃An alkyl group;

R₄is H, F, Cl, Br, I, OCH₃、OCF₃、CF₃、NO₂、CN、SO₂CF₃、COOCH₃、SF₅、SO₂CH₃、COCH₃、NH₂、N(CH₃)₂、SCH₃OH; and

R₅and R₆Independently of one another, H, F, Cl, Br, I, OCH₃、OCF₃、CF₃、NO₂、CN、SO₂CF₃、COOCH₃、SF₅、SO₂CH₃、COCH₃、NH₂、N(CH₃)₂、SCH₃Or an OH group, or a mixture of OH,

the conditions are as follows: r₃、R₄、R₅Or R₆Is not H, and when R is₃、R₄、R₅Or R₆When any 2 of them are not H, R₃、R₄、R₅Or R₆The other 2 of (a) is H.

In the above-mentioned compounds of the present inventionPreferably, when R is₃、R₄、R₅Or R₆When any 2 of them are not H, R₃、R₄、R₅Or R₆The other 2 of (a) is H.

Among the above-mentioned compounds of the present invention, preferably, wherein R is₃、R₄、R₅Or R₆Two of which are not H, but are selected from F, Cl, Br or I.

In particularly preferred compounds of the invention, when R is₃、R₅、R₆When is H, R₄Is Cl, F or Br (preferably Cl). In other preferred compounds of the invention, when R is₃、R₄、R₆When is H, R₅Is F, Cl, OCH₃Or OCF₃(preferably F). In other preferred compounds of the invention, when R₃、R₄、R₅Or R₆When 2 of them are selected from F, Cl, Br, or I (preferably, both substituents are the same, more preferably, both substituents are Cl), R is₃、R₄、R₅Or R₆The other 2 of (a) is H. In other preferred compounds of the invention, when R is₄And R₅Or R₅And R₆When both are F or Cl (preferably both are Cl), the other R₃And R₆Or R₃And R₄Are all H. The compounds of the invention and particularly preferred compositions described herein are compounds that are highly effective in the treatment of neoplasia, including cancer, with at least one or more of significantly enhanced anti-neoplastic activity, significantly improved Maximum Tolerated Dose (MTD) and reduced toxicity, as well as extended half-life and favorable pharmacokinetics, as compared to para or ortho 3-AP prodrugs 2 and 3. These results are unexpected. In this way, higher doses of preferred compounds of the invention can be administered, thereby achieving greater anti-neoplastic (including cancerous) efficacy, and exhibiting prolonged blood flow half-life and reduced toxicity.

The compounds of the present invention are useful in pharmaceutical compositions having biological/pharmacological activity useful, for example, in the treatment of neoplasia (cancer) and many other conditions and/or disease states, as intermediates in the synthesis of biologically active compounds, and as standards for determining the biological activity of the compounds of the present invention as well as other biologically active substances. In certain applications, the compounds of the present invention are useful for treating microbial infections, including in particular viral infections. These compositions comprise an effective amount of any one or more of the above disclosed compounds, optionally in combination with a pharmaceutically acceptable additive, carrier or excipient.

Another aspect of the invention relates to the treatment of neoplasia, including cancer, comprising administering to a patient in need of such treatment an effective amount of a compound described above, optionally in combination with a pharmaceutically acceptable additive, carrier or excipient. The present invention also relates to methods for inhibiting the growth of neoplasia, including malignant tumors or cancers, comprising contacting the neoplasia with an inhibitory or therapeutically effective amount or concentration of at least one of the compounds described above. These methods are useful therapeutically for treating neoplasia, including cancer, and also for comparative testing, such as assays for determining the activity of related analogs, and assays for determining the sensitivity of a patient's cancer to one or more of the present invention. The main application of the compound is in the treatment of neoplasia, including carcinomas, including inter alia lung, breast and prostate carcinomas, and the like.

The invention also relates to the use of a compound of the invention for the preparation of a medicament for the treatment of neoplasia.

The present invention also provides a method of treating neoplasia comprising administering to a patient in need of such treatment an effective amount of a compound of the present invention in combination with a DNA-damaging anticancer agent.

The invention also relates to the use of the compounds of the invention and DNA damaging anticancer agents for the preparation of a medicament for the treatment of neoplasia.

Brief Description of Drawings

Figure 1 represents certain chemicals of the present invention.

FIGS. 2-3 represent chemical synthetic routes for compounds of the invention.

Figures 4-15 are experimental results of efficacy, pharmacokinetics, bioavailability, combination chemotherapy and toxicity of compounds according to preferred embodiments of the present invention.

Detailed Description

The following terms are used in the context of the present invention.

In this specification, the term "patient" refers to an animal (including mammals and preferably humans) treated (including prophylactic treatment) with a composition of the invention. In treating an infection, disorder or disease state that is patient-specific for an animal (e.g., a human), the term "patient" refers to that particular animal.

In this specification, the term "effective amount" refers to a concentration or amount of a compound of the invention sufficient to effect a beneficial change in the disease or condition being treated. Depending on the treatment or condition, the changes include: remission, reduction in cancer or tumor growth or size, favorable physiological consequences, and reduction in growth or breakdown (metabolism) of microorganisms, among others.

In the present specification, the term "alkyl" refers to an alkyl group containing 1 to 3 carbon units. The alkyl group in the present invention includes linear or branched groups such as methyl, ethyl, propyl and isopropyl.

The term "salt" refers to any salt that is used in relation to the compounds of the present invention. Where the compounds are used for pharmaceutical indications, including the treatment of neoplasia, including cancer, the term "salt" denotes a pharmaceutically acceptable salt of the compound which is useful as a pharmaceutical.

The term "neoplasia" refers to a pathological process that results in the formation and growth of neoplasms, i.e., abnormal tissue growth resulting from cell proliferation that grows faster than normal tissue and continues to grow after the stimulus that caused the neoplasm ceases. Neoplasias, which are characterized by a partial or complete lack of structural organization and functional coordination with normal tissue, often form a distinct tissue mass that may be either benign (benign tumor) or malignant (carcinoma). The term "cancer" is a generic term used to describe any type of malignancy, most of which invade surrounding tissues, can metastasize to several sites, and may recur after seeking resection, and if not adequately treated, can lead to patient death. The term cancer as used herein is included within "neoplasia".

A preferred therapeutic aspect of the invention relates to a method for neoplasia (including benign and malignant tumors and carcinomas) in an animal or human patient, which carcinoma has developed resistance, such as multi-drug resistant breast cancer, in a preferred embodiment, which method comprises administering a therapeutically effective amount or concentration of one or more compounds to inhibit the growth or spread of, or to actually shrink, the neoplasia in the animal or human patient being treated.

For example, cancers that may be treated with the compositions of the present invention include: stomach, colon, rectum, liver, pancreas, lung, breast, cervix, uterus, ovary, prostate, testis, bladder, kidney, brain/central nervous system, head and neck, throat, hodgkin's disease, non-hodgkin's leukemia, multiple myeloma leukemia, skin melanoma, acute lymphocytic leukemia, acute granulocytic leukemia, ewing's sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, nephroblastoma, neuroblastoma, hairy cell leukemia, mouth/pharynx, esophagus, larynx, melanoma, kidney, and lymphoma, and the like. The compounds of the invention are particularly useful in the treatment of lung, breast and prostate cancer.

In the methods of the invention, in certain preferred embodiments, it has been found that the combination with at least one additional antineoplastic agent is advantageous for the treatment of neoplasia, including cancer. In these aspects of the invention, an effective amount of one or more compounds of the invention may be combined with an effective amount of at least one additional anti-neoplastic/anti-cancer agent, such as: cyclophosphamide (cyclophosphamide), mitomycin C and etoposide, and the like, including topo I and topo II inhibitors such as doxorubicin, topotecan and irinotecan (irinotecan), other drugs such as gemcitabine (gemcitabine), camptothecin (campothecin) and camptothecin-based drugs, and cisplatin, and the like, alkylating agents include chlorambucil (chlorambucil) and melphalan (melphalan). It has been unexpectedly found that the compounds of the present invention (i.e., R) function by reducing or preventing the mechanisms of DNA repair₃、R₄、R₅And R₆A compound that is H) may act synergistically with the DNA damaging compound. Thus, the compounds of the invention can be advantageously used in combination with any compound that acts by damaging DNA, the latter including, in particular, alkylating agents and platting agents. By "in combination with" is meant that the compound of the invention and the compound of the combination are found in the bloodstream of the patient simultaneously, regardless of whether the compounds of the combination are administered simultaneously. In many cases, it is surprising that the combination of a compound of the invention with a conventional anti-cancer agent results in a synergistic effect (i.e., greater than an additive effect).

Pharmaceutical compositions based on the novel compounds of the present invention are useful for the treatment of conditions or diseases such as neoplasia (including cancer) comprising a therapeutically effective amount of the above compounds optionally in combination with a pharmaceutically acceptable additive, carrier or excipient.

Many compounds in pharmaceutical dosage forms are useful as prophylactics for the prevention of certain diseases or conditions.

The compounds of the present invention or derivatives thereof may be provided in the form of pharmaceutically acceptable salts thereof. The term "pharmaceutically acceptable salts or complexes" as used herein refers to suitable salts or complexes (complexs) of the active compounds of the present invention which retain the desired biological activity of the parent compound and exhibit limited toxicological effects on normal cells. Non-limiting examples of such salts include sodium and potassium phosphates and the like. Modifications to the active compound can alter the solubility, bioavailability and rate of metabolism of the chemical, which can allow control over chemical delivery. In addition, modifications may affect the anti-cancer activity of the compound (sometimes more so than the parent compound). The derivatives can be readily prepared and tested for evaluation of their anti-cancer activity by one skilled in the art using routine methods.

The compounds of the invention may be incorporated into formulations suitable for administration by all routes, including, for example: oral and parenteral, including intravenous, intramuscular, intraperitoneal, buccal, transdermal and suppository forms. Among them, parenteral administration is preferred, and intravenous or intramuscular administration is particularly preferred.

Pharmaceutical compositions based on these novel compounds comprise a therapeutically effective amount of the above-described compounds for the treatment of neoplasia, cancer and other diseases and conditions, optionally in combination with pharmaceutically acceptable additives, carriers and/or excipients. It will be appreciated by those skilled in the art that the therapeutically effective amount of one or more compounds of the invention may be adjusted depending on the infection or disorder to be treated, the treatment regimen employed, the pharmacokinetics of the drug employed and the patient (animal or human) being treated.

In the pharmaceutical aspect of the present invention, it is preferably formulated in a form in which the compound of the present invention is mixed with a pharmaceutically acceptable carrier. Generally, it is preferred to administer the pharmaceutical composition parenterally, particularly preferably in an intravenous intramuscular dosage form, but many formulations can also be administered by other parenteral routes, for example transdermal, buccal, subcutaneous, suppository or other routes including oral administration. Intravenous and intramuscular formulations are preferably administered as sterile saline. Of course, those skilled in the art, given the teachings of this invention, may modify the formulation to provide a variety of formulations for a particular route of administration without compromising the stability of the compositions of the invention and without compromising their therapeutic activity. In particular, modifications to the compounds of the invention, such as to improve their solubility in water or other carriers, can be readily accomplished, for example, using minor modifications (e.g., salt formation, etc.) well known to those of ordinary skill in the art. In order to control the pharmacokinetics of the compounds of the present invention to produce the most beneficial effect in a patient, one skilled in the art can modify the route of administration and the dosage regimen of a particular compound.

One skilled in the art can utilize the advantageous pharmacokinetic parameters of the prodrug form compounds of the invention to advantageously deliver the compounds of the invention to a target site in the host organism or patient to maximize the desired effect of the compound.

The amount of the compound in the therapeutically active formulation of the present invention is an effective amount for treating an infection or disorder. In a preferred embodiment, the compounds of the invention, especially when R₄Is Cl or R₅Is F, Cl, OCH₃Or OCF₃And the remaining substituents on the phenyl ring (other than the phosphate and urethane moieties) are H, the compounds are preferably used to treat neoplasia and especially cancer (including in some cases drug resistant cancers). Generally, a therapeutically effective amount of a preferred compound of the invention in a dosage form is from about 0.025mg/kg to about 2.5mg/kg, preferably from about 2.5mg/kg to about 5mg/kg to about 100mg/kg of patient body weight, more preferably from about 20mg/kg to about 50mg/kg, more preferably about 25mg/kg, and these dosage ranges are possible for use in the present invention, but should be adjusted depending on the particular compound used, the condition or infection being treated, and the route of administration. As regards the preferred compositions of the invention mentioned above, namely R₄Is Cl or R₅Is F, Cl, OCH₃Or OCF₃And the remaining substituents on the benzene (not the phosphate and urethane moieties) are H, these compounds can be administered at levels 3-10 times higher than tripine 1a and have significantly lower toxicity due to their enhanced anticancer activity, combined with reduced overall toxicity to non-cancerous host cells and higher bioavailability. At these dosage levels, the AUC (area under the curve) of tripine released from the prodrug form is about 5-25 times that of tripine used in the non-prodrug form. Thus, the compounds of the present invention show unexpected results and are excellent drugs for the treatment of neoplasias, especially cancer. Generally, the above-recited dosage ranges will result in effective blood levels of the active agent, i.e., concentrations in the patient's blood of less than about 0.04-400 μ g/cc or higher. With the prior art Triapine^TMCompared to (on a molar basis), the compounds of the invention have more favorable bioavailability characteristics, reduced toxicity and greater activity, and are useful as therapeutics for neoplasmsForm unexpectedly advantageous compounds, including cancers.

The active substances of the invention can be administered continuously (i.v. instillation), including by bolus injection (i.v. infusion), intravenously or intramuscularly less often than once to several times per day, including by topical, parenteral, intramuscular, intravenous, subcutaneous, transdermal (including penetration enhancers), buccal and suppository administration, and in some cases by other routes of administration including oral administration.

To prepare the pharmaceutical compositions of the present invention, a therapeutically effective amount of one or more compounds of the present invention is intimately admixed with a pharmaceutically acceptable carrier, preferably using conventional pharmaceutical compounding techniques, to prepare a formulation. Various carriers can be selected depending on the intended form of administration (e.g., intravenous or intramuscular) of the formulation. In preparing the pharmaceutical compositions in suitable dosage form, any of the conventional pharmaceutical media may be employed. For parenteral formulations, the carrier will typically comprise sterile water or an aqueous sodium chloride solution, and may also include other ingredients to aid dispersion. Of course, the compositions and carriers must be sterilized despite the use of sterile water and aseptic procedures. Liquid carrier, suspending agent, etc. can also be used to prepare injection suspension.

The compounds of the present invention are useful for treating animal patients and particularly mammals including humans. Thus, an effective amount of one or more of the compounds of the present invention, or derivatives or pharmaceutically acceptable salts thereof, can be administered to treat humans, horses (equines), dogs, cows and other animals, and especially mammals, suffering from tumors and especially cancers or other diseases disclosed herein, optionally in admixture with a pharmaceutically acceptable or diluent, either alone or in combination with other known agents depending on the disease being treated. These treatments may also be combined with other conventional cancer therapies such as radiation therapy or surgery.

The active substance in a pharmaceutically acceptable carrier or diluent is employed in an amount sufficient to deliver to the patient a therapeutically effective amount of the drug required for the intended indication without causing serious toxic effects to the patient being treated.

The compounds may be conveniently administered in any suitable unit dosage form including, but not limited to, dosage forms containing 1 to 3000mg, preferably 5 to 500mg, of active ingredient per unit amount.

The concentration of the active agent in the pharmaceutical composition will depend on the absorption, distribution, inactivation and excretion rates of the drug, as well as those factors well known to those skilled in the art. It should be noted that the dosage should vary as the condition is alleviated. It will be understood that any particular subject, at the discretion of the individual need and the professional responsible for administering the administration instructions for administering the composition, will have to adjust the specific dosage regimen at any time, and that the concentration ranges described herein are exemplary only and are not limiting as to the scope or application of the claimed composition. The active ingredient may be administered immediately, or divided into a plurality of smaller doses administered at different intervals.

The active substances of the invention can also be combined with other active substances which do not impair the desired effect of the active substances of the invention, or with active substances which enhance the desired effect (for example other anticancer agents), in some cases, depending on the desired therapy or target, with antibiotics, antifungal agents, anti-inflammatory agents or antiviral compounds, etc.

Solutions or suspensions for parenteral, intradermal, subcutaneous, or topical administration may be employed that include the following components: sterile diluents such as water for injection, saline solution, fixed oils (fixed oils), polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates and isotonicity adjusting agents such as sodium chloride or dextrose. Formulations for parenteral administration may be presented in ampoules, disposable syringes or glass or plastic multi-dose vials. If intended for intravenous administration, preferred carriers include, for example, physiological saline or Phosphate Buffered Saline (PBS).

In one embodiment, the active agent may be formulated with a carrier that prevents rapid elimination of the compound from the body, for example, as a controlled release formulation including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers may be used, such as ethylene-vinyl acetate copolymers, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods of preparing such formulations will be apparent to those skilled in the art.

Liposomal suspensions may also be used as pharmaceutical carriers. These formulations may be formulated according to methods well known to those skilled in the art. For example, liposome formulations can be prepared by dissolving the appropriate lipid in an inorganic solvent and then evaporating the solvent to leave a dry lipid film on the surface of the container. An aqueous solution of the active substance is then introduced into the container. The container is then manually swirled to peel the lipid material from the container walls and disperse the material into lipid aggregates, thereby forming a liposomal suspension. Other methods of preparation known to those of ordinary skill in the art may also be used in these aspects of the invention.

The anti-cancer activity of a compound can be assessed using various biological assays that have been used and accepted by those skilled in the art. Any of these methods can be used to evaluate the activity of the compounds disclosed herein.

One conventional method for assessing activity is to employ a panel of cancer cell lines. These assays allow the in vitro evaluation of the anti-cancer activity of a particular compound in cancer cell lines and provide reference data for the in vivo use of the test compound. Other assays include in vivo evaluation of the effect of the compounds on humans or appropriate animal models, for example using mouse tumor cells implanted or transplanted into mice or other appropriate animal models.

Chemical synthesis

Preliminary in vitro evaluations of 3-AP prodrugs showed that they were rapidly enzymatically converted to 3-AP by alkaline phosphatase. In contrast, in vivo PK studies in Beagle dogs showed that the half-life of 3-AP released from the ortho-phosphate containing prodrug was only 14.2 hours, while the half-life of the para-prodrug was only 1.5 hours. Also, prodrugs 2 and 3 were evaluated in mice bearing M-109 solid tumors (compared to anti-3-AP and cyclophosphamide). The results of these experiments indicate that the ortho-prodrug 3 is more potent in reducing toxicity than the parent 3-AP (1A) and that its activity is comparable to cyclophosphamide. To further improve the biological and pharmaceutical properties of 3-AP prodrugs and to optimize their therapeutic applications, a series of ortho-phosphate based prodrugs were designed using the following principles.

The principle used to design new prodrugs is: the 3-AP phosphate linked prodrug is dephosphorylated to release 3-AP to give 4, and subsequently cleaved with a benzyl group (fragmentation) to give quinone methide 5 which can be used as a biological alkylating agent.

Without wishing to be bound by theory, it is speculated that the rate-controlling step in the prodrug activation process may be a P — O bond cleavage step (catalyzed by alkaline phosphatase). The subsequent fragmentation step is generally faster. This may be due to the long circulating half-life of the 3-AP prodrug, which acts as a 3-AP reservoir, or due to the distribution of the prodrug, which is different from the parent drug, which may give it the desired properties. This is achieved by slowing down the dephosphorylation step, which is the rate-limiting step in the biological activation of 3-AP-containing phosphate prodrugs by introducing bulky substituents into the phosphate group at the alpha position. These alkyl groups create steric hindrance by approaching the P-O bond cleavage site, slowing down enzymatic dephosphorylation. Another approach introduces electron-releasing or electron-withdrawing groups in the phenyl ring that can affect the rate of P-O bond cleavage. Likewise, substitution with electron-releasing and electron-withdrawing groups at other positions may affect subsequent fragmentation steps.

Based on these considerations, a number of phosphate prodrugs (FIG. 1) were readily synthesized in large quantities and evaluated. The disodium salts of these prodrugs are very soluble in water.

The synthesis of 5-chloro prodrug compound 6 is shown in figure 2. Thus, for example, adoptThe acid 20 is prepared from methyl 2-chloro-3-nicotinate 18 or a derivative in two steps including a Heck reaction (see Jeffery, Tetrahedron (1996), 52, 10113 and Dieck and Heck, j.org.chem. (1975), 40, 1083) and NaOH-promoted ester hydrolysis. Chloro ortho phosphate linker 21 was prepared by oxidative coupling between bis-TMSE phosphite and 2-hydroxybenzyl alcohol (McCombie et al, j. chem. soc. (1945), 381). The large-scale preparation of linker 21 encounters a problem in that it is decomposed during purification, which lowers the yield. With Et₃N is a buffer to neutralize the acidity of the silica gel, and the conditions are adjusted so that the linker (88%) can be obtained in large quantities. The reaction mixture containing acid 20, linker 21, triethylamine and diphenylphosphoryl azide was heated under Curtius rearrangement conditions (Shipps et al, j.bioorg.med.chevm. (1996), 4, 655) to provide the desired carbamate 22 (58%), which was then converted to the aldehyde 23 (72%) and the corresponding thiosemicarbazone 24 (63% yield). The 2-Trimethylsilylethyl (TMSE) group in 24 was removed with TFA (Chao et al, J.org.chem. (1994), 59, 6687) to provide 3-AP prodrug free acid 6, which was then treated with saturated sodium bicarbonate solution and purified with a reverse phase column to convert to disodium salt 25.

Another substituted ortho prodrug is synthesized by essentially the same route using an appropriate phosphate-containing substituted benzyl linker, e.g., 21. These linkers are coupled to 25 and then treated with functional groups to give prodrugs (FIG. 3). This synthesis demonstrates that the prodrugs of the invention can be readily converted to their corresponding phosphates. These phosphate compounds have excellent water solubility (significantly higher than their non-prodrug forms). The parent 3-AP has a solubility in aqueous solution of less than 0.1mg/ml, while the prodrug has a solubility of 16-35 mg/ml.

Following the general description above, the following examples are intended to illustrate preferred and other embodiments and comparisons. These examples should not be construed as limiting the scope of the invention as defined by the foregoing and the appended claims. Other compounds not specifically exemplified in the examples section of this application may be readily synthesized using similar methodologies and/or known synthetic methods readily available in the art. All compounds described can be readily synthesized by one of ordinary skill in the art by either using the synthetic methods described herein directly or by modifying/modifying the synthetic methods using techniques known in the art.

Examples

All reagents were purchased as commercial grade products without further purification, and the solvents were dried and/or distilled before use if necessary. All NMR spectra (¹H、¹³C and³¹p) were all determined using a BruckerrAC 300 spectrometer. Chemical shifts (relative to tetramethylsilane) are in parts per million (ppm). Coupling constants are reported in hertz (Hz). Flash Column Chromatography (FCC) was performed using Merck silica gel 60 (230-. Reverse Phase Column Chromatography (RPCC) was packed with CAT gel (Waters, preparative column C18125 , 55-105 μm) and eluted with milli-Q grade deionized water.

General procedure for the preparation of nicotinic acid (20) examples 1-3

Example 1

Preparation of methyl 2-chloronicotinate (18)

To a mixture of 2-chloronicotinic acid (Aldrich, 100.0g, 0.63mol) in 1, 4-dioxane (500ml) was added thionyl chloride (70ml, 0.96 mol). The suspension was heated at reflux for 22 hours and the hydrogen chloride gas was absorbed by a gas separator. After evaporation of the solvent, the residue was dissolved in methanol (300 ml). Triethylamine (TEA, 120ml, 1.26mol) was added dropwise to the solution at 0 ℃ over 2 hours. The solvent was evaporated and the residue was suspended in ethyl acetate. The precipitate was removed by filtration. The filtrate was concentrated to give ester 18 as an oil (92.3g, 86%):

R_f(1: 5v/v ethyl acetate-hexane) 0.38.

¹H NMR(300MHz，CDCl₃) Δ 8.53(dd, 4.8Hz, 1H), 8.19(dd, 7.6Hz, 1H), 7.37(dd, 7.7Hz, 1H) and 3.97(s, 3H).

¹³C NMR(75MHz，CDCl₃) δ 164.5, 151.6, 149.6, 140.0, 126.4, 121.9 and 52.5.

Example 2

Preparation of 2-styryl nicotinic acid methyl ester (19)

To a solution of ester 18(48.8g, 0.28mol) in DMF (450ml) was added styrene (165ml, 1.42mol), palladium acetate (6.5g, 30mmol), sodium acetate (47g, 0.57mol) and triphenylphosphine (30g, 0.11 mol). The mixture was heated at reflux for 22 hours. The palladium catalyst was removed by filtration through a pad of Celite. The filtrate was concentrated under reduced pressure and the residue was dissolved in a minimum amount of ethyl acetate. Hexane was added to the above solution. After removing the precipitate by filtration, the filtrate was concentrated. The crude product was purified by FCC (1: 1v/v ethyl acetate-hexanes) to give ester 19(55.0g, 81%) as a pale yellow oil:

rf (1: 5v/v ethyl acetate-hexane) 0.41.

¹H NMR(300MHz，CDCl₃) δ 8.70(dd, 1H), 8.10(dd, 1H), 8.16(d, 1H), 7.94(d, 1H), 7.64(d, 2H), 7.4-7.3(m, 3H), 7.18(dd, 1H) and 3.94(s, 3H).

¹³C NMR(75MHz，CDCl₃) δ 166.7, 155.3, 152.0, 138.6, 136.7, 135.9, 128.6, 128.5, 127.5, 24.8, 123.8, 121.3 and 52.4.

Example 3

Preparation of 2-styrylnicotinic acid (20)

A solution of ester 19(55.0g, 0.23mol) in THF (100ml) was treated with a 3N NaOH solution (110ml, 0.25mol) for 21 hours at ambient temperature. After removal of the solvent, the residue was taken up with water and diethyl ether. The phases were separated and the aqueous phase was washed with ether (2 ×). The resulting aqueous phase was neutralized with 2N HCl solution and the precipitate was collected by filtration to give acid 20 as a cream solid (50.2g, 97%):

¹H NMR(300MHz，DMSO-d6)δ8.72(dd，1H)8.19(dd 1H), 8.10(d, 1H), 7.86(d, 1H), 7.62(d, 2H) and 7.4-7.3(m, 4H).

¹³C NMR (75MHz, DMSO-d6) delta 167.9, 153.7, 151.8, 138.6, 136.4, 134.5, 128.9, 128.7, 127.2, 125.3 and 122.1.

Examples 4-5 general procedure for preparation of phosphate linkers (21, 26a-k)

Example 4

Preparation of bis (2-trimethylsilylethyl) phosphite (TMSE-phosphite)

To a solution of 2-trimethylsilane) ethanol (Aldrich, 25.0g, 0.21mol) in ether (200ml) with pyridine (11.4ml, 0.14mol) was added phosphorus trichloride (6.2ml, 70mmol) in one portion at-78 ℃. The reaction mixture was kept under stirring for 5 minutes and then diluted with diethyl ether (500 ml). After warming to ambient temperature, the mixture was stirred continuously for 18 hours, the precipitate was removed by filtration, and the filtrate was bubbled with ammonia gas for 10 minutes. The precipitate was removed by filtration through a pad of Celite and the filtrate was concentrated to give TMSE-phosphite (20.7g, 99%) as a colorless oil:

¹H NMR(300MHz，CDCl₃) δ 6.76(d, 1H), 4.13(m, 4H), 1.07(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 64.0(d), 19.6(d) and-1.6 (d).

³¹P NMR(121MHz，CDCl₃)δ18.5。

Example 5

Preparation of 2- (TMSE-Phosphonoxy) benzyl alcohol (21, 26a-k)

General procedureTo a corresponding solution of 2-hydroxybenzyl alcohol (10mmol) in acetonitrile (40ml) were added N, N' -diisopropylethylamine (DIEA, 11mmol), 4-dimethylaminopyridine (DMAP, 1mmol) and carbon tetrachloride (50 mmol). Bis (2-trimethylsilane) was added to the solution immediately with stirring at-30 deg.CEthyl) phosphite (refrigerator storage, 11 mmol). After warming to ambient temperature, the reaction mixture was stirred for 3 hours. The solvent was evaporated under reduced pressure and the residual product was purified by FCC (1: 1v/v ethyl acetate-hexanes) to afford the corresponding TMSE-protected phosphate linker (21, 26 a-k).

2-bis (2-trimethylsilylethyl) phosphonoxybenzyl alcohol (H in phenyl position 5, 21a)

The oily ortho phosphate linker 2-bis (2-trimethylsilylethyl) phosphonoxybenzyl alcohol (39.78g, 81%) was obtained via 2-hydroxybenzyl alcohol (15.0g, 0.12mmol) as described above:

¹h NMR (300 MHz, CDCl)₃) δ 7.43(d, J ═ 8.5Hz, 1h), 7.27-7.16(m, 3h), 4.63(s, 2h), 4.29-4.19(m, 4h), 4.12(m, 4h), 1.14-1.08(m, 4h), and 0.00(s, 18 h);

¹³c NMR (75MHz, CDCl)₃)148.4(d, J ═ 8.84Hz), 133.0(d, J ═ 4.59Hz), 130.9, 129.1, 25.8, 121.0(d, J ═ 4.47Hz), 67.5(d, J ═ 6.93), 60.1, 9.5(d, J ═ 5.76Hz), and-1.563.

³¹P NMR(121MHz，CDCl₃)δ6.4.

2-bis (2-trimethylsilylethyl) phosphonoxy-5-chlorobenzyl alcohol (21)

Following the above procedure, over 5-chloro-2-hydroxybenzyl alcohol (5.0g, 32mmol), 21 was obtained as an oil (12.2g, 88%):

¹H NMR(300MHz，CDCl₃) δ 7.33(d, 1H), 7.11(m, 1H), 7.02(m, 1H), 4.49(s, 2H), 4.12(m, 4H), 1.00(m, 4H and-0.07 (s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 146.5(d), 134.7(d), 130.8, 130.2, 128.6, 122.0(d), 67.7(d), 59.4, 19.5(d) and-1.6.

³¹P NMR(121MHz，CDCl₃)δ6.1.

2-bis (2-trimethylsilylethyl) phosphonoxy-5-fluorobenzyl alcohol (26a)

Following the above procedure, 5-fluoro-2-hydroxybenzyl alcohol (17.0g, 119mmol) gave 26a (31.7g, 62%) as an oil:

¹H NMR(300MHz，CDCl₃) δ 7.2-7.1(m, 1H), 7.0-6.9(m, 1H), 4.63(s, 1H), 4.3-4.1(m, 4H), 1.2-1.1(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 159.8(d), 143.7(dd), 135.2(dd), 121.8(dd), 116.4(d), 115.0(d), 67.6(d), 59.4, 19.5(d) and-1.6.

³¹P NMR(121MHz，CDCl₃)δ6.4.

¹⁹F NMR(282MHz，CDCl₃)δ-59.8.

2-bis (2-trimethylsilylethyl) phosphonoxy-5-nitrobenzyl alcohol (26b)

Following the above procedure, 2-hydroxy-5-nitrobenzyl alcohol (4.5g, 27mmol) gave 26b (6.4g, 53%) as an oil:

¹H NMR(300MHz，CDCl₃) δ 8.14(m, 1H), 7.81(m, 1H), 7.14(m, 1H), 4.48(s, 2H), 4.06(m, 4H), 0.90(m, 4H) and-0.20 (m, 18H).

¹³C NMR(75MHz，CDCl₃) δ 152.0(d), 144.6, 134.7(d), 123.7, 123.4, 119.8, 67.9(d), 58.4, 19.3(d) and-1.6.

³¹P NMR(121MHz，CDCl₃)δ4.4.

2-bis (2-trimethylsilylethyl) phosphonoxy-5-methoxybenzyl alcohol (26c)

Following the above procedure, 2-hydroxy-5-methoxybenzyl alcohol (11.0g, 25mmol) gave 26c (7.7g, 70%) as an oil:

¹H NMR(300MHz，CDCl₃) δ 7.06(dd, 1H), 6.94(d, 1H), 6.74(dd, 1H), 4.57(s, 2H), 4.34.1(m, 4H), 3.74(s, 3H), 1.1-1.0(m, 4H) and 0.0(m, 18H).

¹³C NMR(75MHz，CDCl₃) δ 157.1, 141.7(d), 134.0(d), 121.9(d), 125.3, 114.5, 67.5(d), 60.2, 55.6, 19.6(d) and-1.6.

³¹P NMR(121MHz，CDCl₃)δ6.9.

2-bis (2-trimethylsilylethyl) phosphonoxy-5-trifluoromethoxybenzyl alcohol (26d)

Following the above procedure, 2-hydroxy-5-trifluoromethoxybenzyl alcohol (1.9g, 9.1mmol) gave 26d (3.3g, 62%) as an oil:

¹H NMR(300MHz，CDCl₃) δ 7.20(d, 1H), 7.19(dd, 1H), 7.09(dd, 1H), 4.61(s, 2H), 4.24(m, 4H), 1.08(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 146.4(dd), 135.0(d), 123.0, 122.1, 121.4, 67.8(d), 59.6, 19.6(d) and-1.6.

³¹P NMR(121MHz，CDCl₃)δ6.2.

¹⁹F NMR(282MHz，CDCl₃)δ-58.7.

2-bis (2-trimethylsilylethyl) phosphonoxy-5-trifluoromethylbenzyl alcohol (26e)

Following the above procedure, 2-hydroxy-5-trifluoromethylbenzyl alcohol (4.1g, 22mmol) gave 26e (7.9g, 77%) as an oil:

¹H NMR(300MHz，CDCl₃) δ 7.72(br s, 1H), 7.51(dd, 1H), 7.29(d, 1H), 4.66(s, 2H), 4.23(m, 4H), 1.09(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 150.6(d), 133.8(d), 127.7(d), 126.1, 121.3(d), 68.0(d), 59.6, 19.6(d) and-1.6.

³¹P NMR(121MHz，CDCl₃)δ5.8.

¹⁹F NMR(282MHz，CDCl₃)δ-62.9.

2-bis (2-trimethylsilylethyl) phosphonoxy-3, 5-dichlorobenzyl alcohol (26f)

Following the above procedure, 3, 5-dichloro-2-hydroxybenzyl alcohol (4.6g, 24mmol) gave 26f (7.2g, 63%) as an oil:

¹H NMR(300MHz，CDCl₃) δ 7.34(d, 1H), 7.32(dd, 1H), 4.56(s, 2H), 4.25(m, 4H), 1.08(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 149.9, 143.3(d), 136.9(d), 131.2(d), 129.9, 129: 5,127.6 (d), 68.3(d), 59.8, 19.5(d) and-1.6.

³¹P NMR(121MHz，CDCl₃)δ5.7.

2-bis (2-trimethylsilylethyl) phosphonoxy-4, 5-dichlorobenzyl alcohol (26g)

Following the above procedure, over 4, 5-dichloro-2-hydroxybenzyl alcohol (3.6g, 18mmol), 26g (5.2g, 59%) of oil was obtained:

¹H NMR(300MHz，CDCl₃) δ 7.53(s, 1H), 7.28(s, 1H), 4.55(s, 2H), 4.21(m, 4H), 1.08(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 146.6(d), 133.5(d), 131.9(d), 131.6, 129.5(d), 123.0(d), 68.1(d), 59.1, 19.6(d) and-1.5.

³¹P NMR(121MHz，CDCl₃)δ6.0.

2-bis (2-trimethylsilylethyl) phosphonoxy-5, 6-dichlorobenzyl alcohol (26h)

Following the above procedure, 5, 6-dichloro-2-hydroxybenzyl alcohol (4.8g, 25mmol) gave 26h (8.6g, 73%) as an oil:

¹H NMR(300MHz，CDCl₃) δ 7.35(d, 1H), 7.04(dd, 1H), 4.76(s, 2H), 4.22(m, 4H), 1.08(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 147.6(d), 134.7, 133.5(d), 130.6(d), 129.9, 120.8(d), 68.1(d), 57.3, 19.6(d) and-1.6.

³¹P NMR(121MHz，CDCl₃)δ6.4.

2-bis (2-trimethylsilylethyl) phosphonoxy-3-methylbenzyl alcohol (26i)

Following the above procedure, 2-hydroxy-3-methylbenzyl alcohol (2.0g, 14mmol) gave 26i (1.7g, 88%) as an oil:

¹H NMR(300MHz，CDCl₃) δ 7.16(m, 1H), 7.0-6.9(m, 2H), 4.48(s, 2H), 4.13(m, 4H), 2.22(s, 3H), 0.97(m, 4H) and-0.09 (s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 146.9(d), 133.5(d), 131.0, 130.4(d), 129.4, 125.6(d), 67.6(d), 60.1, 19.5(d), 16.8 and-1.6.

³¹P NMR(121MHz，CDCl₃)δ6.9.

2-bis (2-trimethylsilylethyl) phosphonoxy-4-chlorobenzyl alcohol (26j)

Following the above procedure, over 4-chloro-2-hydroxybenzyl alcohol (4.2g, 26mmol), 26j (9.6g, 84%) was obtained as an oil:

rf (4: 1v/v ethyl acetate-hexane) 0.67.

¹H NMR(300MHz，CDCl₃) δ 7.27(d, 1H), 7.2-7.1(m, 2H), 4.55(s, 2H), 4.21(m, 4H), 1.07(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 148.5(d), 133.9, 131.7(d), 131.5, 126.0, 121.4(d), 67.9(d), 59.4, 19.5(d) and-1.6.

³¹P NMR(121MHz，CDCl₃)δ5.8.

2-bis (2-trimethylsilylethyl) phosphonoxy-4-methoxybenzyl alcohol (26k)

Following the above procedure, over 2-hydroxy-4-methoxybenzyl alcohol (2.7g, 17mmol), 26k (2.5g, 33%) was obtained as an oil:

rf (4: 1v/v ethyl acetate-hexane) 0.70.

¹H NMR(300MHz，CDCl₃) δ 7.29(d, 1H), 6.8-6.7(m, 2H), 4.53(s, 2H), 4.22(m, 4H), 3.75(s, 3H), 1.09(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 160.1(d), 149.1(d), 131.9, 125.3(d), 111.2, 107.3(d), 67.6(d), 59.7, 55.5, 19.6(d) and-1.6.

³¹P NMR(121MHz，CDCl₃)δ6.4.

EXAMPLE 6 general preparation of 103-AP prodrug (25, 30a-k)

Example 6

Preparation of 2- (TMSE-Phosphonoxy) benzyl (2-styrylpyridin-3-yl) carbamate (22, 27a-k) (Curtius rearrangement)

General procedureTo a solution of 2-styrylnicotinic acid (20, 20mmol) in benzene (100ml) containing triethylamine (TEA, 32mmol) was added diphenylphosphorylazide (32 mmol). The solution was heated to reflux for 10 minutes and then the corresponding TMSE protection prepared as described above was addedThe phosphate linker of (21 or 26a-k, 20 mmol). The reaction mixture was kept at reflux for 3 hours. Then, the solvent was evaporated under reduced pressure. The residual product was purified by FCC (1: 4v/v ethyl acetate-hexane) to give the corresponding carbamate (22 or 27 a-k).

(2-styrylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonooxy-benzyl ester (22a)

The crude carbamate salt was obtained from 2-bis (2-trimethylsilylethyl) phosphonoxybenzyl alcohol (21a) (29.72g, 0.073mol) according to the above procedure and was used for another reaction without purification.

(2-styrylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-5-chlorobenzyl ester (22)

Following the above procedure, via 21(9.4g, 21mmol), 22 was obtained as an oil (10.6g, 58%):

¹H NMR(300MHz，CDCl₃) δ 8.23(d, 1H), 7.92(br s, 1H), 7.56(d, 1H), 7.4-7.0(m, 11H), 5.11(s, 2H), 4.10(m, 4H), 0.94(m, 4H) and-0.14 (s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 153.7, 151.9, 147.3(d), 146.8, 145.2, 136.5, 134.8, 131.4, 130.2, 129.5, 12, 129.2, 128.5, 128.3, 127.2, 122.3, 121.3, 121.2, 67.4(d), 61.6, 19.4(d) and-1.7.

³¹P NMR(121MHz，CDCl₃)δ5.1.

C₃₁H₄₂ClN₂O₆PSi₂Mass calculated value: 661.277, respectively; measured value: 661.2

(2-styrylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-5-fluorobenzyl ester (27a)

The crude carbamate salt 27a was obtained from 26a (31.0g, 73mmol) according to the above procedure and was used directly for another reaction without purification.

(2-styrylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-5-nitrobenzyl ester (27b)

Following the above procedure, over 26b (4.3g, 9.6mmol), 27b was obtained as an oil (2.3g, 35%):

¹H NMR(300MHz，CDCl₃) δ 8.3-7.0(m, 14H), 5.21(s, 2H), 4.16(m, 4H), 0.99(m, 4H) and-0.12 (m, 18H).

¹³C NMR(75MHz，CDCl₃) δ 153.5, 153.2(d), 149.9, 145.6, 144.3, 136.4, 135.1, 131.1, 129.3, 129.0(d), 128.5, 128.4, 127.2, 124.9, 124.8, 122.4, 120.9, 120.3, 68.0(d), 62.1, 19.5(d), 17.1 and-1.6.

³¹P NMR(121MHz，CDCl₃)δ4.5.

(2-styrylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-5-methoxybenzyl ester (27c)

The crude carbamate salt 27c was obtained as above via 26c (6.0g, 26mmol) and was used directly for another reaction without purification.

(2-styrylpyridin-3-yl) carbamic acid 2-carbamic acid (2-trimethylsilylethyl) phosphonoxy-5-trifluoromethoxybenzyl ester (27d)

Following the above procedure, over 26d (1.9g, 8.5mmol), 27d was obtained as an oil (3.4g, 83%):

¹H NMR(300MHz，CDCl₃) δ 8.39(dd, 1H), 8.13(br s, 1H), 7.74(d, 1H), 7.60(m, 2H), 7.41(dd, 1H), 7.4-7.1(m, 7H), 5.30(s, 2H), 4.27(m, 4H), 1, 10(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃)δ153.4，147.3(d)，145.7，145.4，136.6，135.4，131.2，129.8，129.7，129.2(d), 128.6, 128.5, 127.4, 127.0, 125.2, 122.7, 122.5, 122.2, 121.5, 120.8, 120.0(d), 118.6, 67.6(d), 62.0, 19.6(d) and-1.6.

³¹P NMR(121MHz，CDCl₃)δ5.3.

¹⁹F NMR(282MHz，CDCl₃)δ-58.7.

(2-styrylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-5-trifluoromethylbenzyl ester (27e)

Following the above procedure, over 26e (5.1g, 11mmol), 27e (5.3g, 71%) was obtained as an oil:

¹H NMR(300MHz，CDCl₃) δ 8.40(dd, 1H), 8.14(br s, 1H), 7.74(d, 2H), 7.6-7.5(m, 4H), 7.4-7.1(m, 8H), 5.29(s, 2H), 4.29(m, 4H), 1.11(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 153.4, 145.4(m), 136.5, 135.4, 131.1, 129.7, 128.6, 128.5, 128.2, 128.1, 127.4, 127.1(d), 122.5, 120.8, 120.5, 120.0(d), 67.7(d), 61.9, 19.6(d) and 1.6.

³¹P NMR(121MHz，CDCl₃)δ5.0.

¹⁹F NMR(282MHz，CDCl₃)δ-62.7.

(2-styrylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-3, 5-dichlorobenzyl ester (27f)

Following the above procedure, over 26f (6.3g, 13mmol), 27f (7.1g, 76%) was obtained as an oil:

¹H NMR(300MHz，CDCl₃) δ 8.39(dd, 1H), 8.11(br s, 1H), 7.74(d, 1H), 7.59(br d, 2H), 7.4-7.2(m, 8H), 7.18(dd, 2H), 5.35(s, 2H), 4.29(m, 4H), 1.11(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 153.3, 145.4(d), 136.5, 135.4, 131.9(d), 131.1, 131.0, 130.2, 129.8, 129.7, 128.7, 128.6, 128.3, 128.0(d), 127.4, 122.5, 120.8, 67.9(d), 62.2, 19.5(d) and-1.6.

³¹P NMR(121MHz，CDCl₃)δ5.0.

(2-styrylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-4, 5-dichlorobenzyl ester (27g)

Following the above procedure, 26g (11.3g, 50mmol) gave 27g (17.4g, 75%) of an oil:

¹H NMR(300MHz，CDCl₃) δ 8.38(dd, 1H), 8.12(br s, 1H), 7.74(d, 1H), 7.60(dd, 2H), 7.53(s, 1H), 7.51(d, 1H), 7.4-7.2(m, 6H), 7.17(dd, 2H), 5.23(s, 2H), 4.27(m, 4H), 1.10(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 153.4, 147.7(d), 145.4, 136.6, 135.4, 133.1, 131.3, 131.2, 128.9, 128.6, 128.5, 127.7(d), 127.4, 122.5, 120.8, 67.8(d), 61.5, 19.6(d) and-1.6.

³¹p NMR(121MHz，CDCl₃)δ5.2.

(2-styrylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-5, 6-dichlorobenzyl ester (27h)

Following the above procedure, over 26h (6.2g, 28mmol), 27h (9.6g, 75%) was obtained as an oil:

¹H NMR(300MHz，CDCl₃) δ 8.38(dd, 1H), 8.20(br s, 1H), 7.73(d, 1H), 7.60(br d, 2H), 7.48(d, 1H), 7.4-7.2(m, 7H), 7.18(dd, 2H), 5.48(s, 2H), 4.28(m, 4H), 1.10(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 153.4, 149.2(d), 145.2, 136.5, 135.4, 134.7, 131.3, 131.0, 129.8, 129.4, 128.6, 128.5, 127.4, 127.3(d), 122.5, 120.7, 119.6(d), 67.7(d), 59.9, 19.6(d) and-1.6.

³¹P NMR(121MHz，CDCl₃)δ5.0.

(2-styrylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-3-methylbenzyl ester (27i)

Following the above procedure, over 26i (1.4g, 6.2mmol), 27i was obtained as an orange oil (1.5g, 53%):

¹H NMR(300MHz，CDCl₃) δ 8.23(dd, 1H), 7.97(br s, 1H), 7.66(m, 1H), 7.58(d, 1H), 7.46.9 (m, 10H), 5.27(s, 2H), 4.11(m, 4H), 2.27(s, 3H), 0.94(m, 4H) and-0.11 (s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 153.9, 147.6(d), 146.4, 145.1, 136.6, 134.7, 131.7, 131.6, 131.0, 130.8(d), 128.5, 128.4(d), 128.3, 127.6, 127.3, 125.3, 122.3, 121.2, 67.1(d), 62.9, 19.5(d), 17.1 and-1.6.

³¹P NMR(121MHz，CDCl₃)δ5.6.

C₃₂H₄₅N₂O₆PSi₂Mass calculated value: 640.859, respectively; measured value: 640.2

(2-styrylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-4-chlorobenzyl ester (27j)

Following the above procedure, 26j (9.3g, 21mmol) gave 27j (12.6g, 87%) as an oil:

rf (1: 1v/v ethyl acetate-hexane) 0.82.

¹H NMR(300MHz，CDCl₃)δ8.28(dd，1H)，815(br s, 1H), 7.72(d, 1H), 7.60(d, 2H), 7.47.3(m, 7H), 7.2-7.1(m, 2H), 5.26(s, 2H), 4.28(m, 4H), 1.11(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 153.6, 149.7(d), 145.2, 136.6, 135.3, 135.1, 131.4, 131.3, 128.6, 128.5, 127.4, 125.9(d), 125.4, 122.4, 120.9(d), 120.8, 67.6(d), 62.1, 19.5(d) and-1.6.

³¹P NMR(121MHz，CDCl₃)δ5.1.

(2-styrylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-4-methoxybenzyl ester (27k)

Following the above procedure, over 26k (2.8g, 6.5mmol), 27k (3.7g, 86%) was obtained as an oil: rf (1: 1v/v ethyl acetate-hexane) 0.50.

¹H NMR(300MHz，CDCl₃) δ 8.36(dd, 1H), 8.18(br s, 1H), 7.72(d, 1H), 7.4-7.3(m, 6H), 7.62(d, 2H), 7.2-7.1(m, 1H), 6.97(m, 1H), 6.71(dd, 1H), 5.24(s, 2H), 4.29(m, 4H), 3.80(s, 3H), 1.11(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 160.5, 153.9, 150.0(d), 145.0, 136.7, 135.1, 131.9, 131.6, 130.0, 128.6, 128.4, 127.4, 122.4, 120.9, 119.2(d), 110.4, 67.3(d), 62.4, 55.5, 19.6(d) and 1.6.

³¹P NMR(121MHz，CDCl₃)δ5.2.

Example 7

Preparation of 2- (TMSE-Phosphonoxy) benzyl (2-formylpyridin-3-yl) carbamate (23, 28a-k) (ozonolysis method)

General procedureThe corresponding 2-styrylpyridine (22 or 27a-k, 10mmol) was dissolved in dichloromethane (50ml) and ethanol (40 ml). Treating the pale yellow solution with ozone at-50 deg.CLiquid until the solution turns light blue. The solution was bubbled through nitrogen for 30 minutes to expel excess ozone. Then, dimethyl sulfide (5ml) was added to the solution, and the resulting compound was stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure and the residual product was purified by FCC (1: 9v/v ethyl acetate-hexane) to give the corresponding pyridine-2-aldehyde (carboxaldehydes) (23 or 28 a-k).

(2-formylpyridin-3-yl) carbamic acid 2- (trimethylsilylethyl-phosphonooxy) benzyl ester (23a)

Crude 22a prepared as described above gave 23a as an oil (29.81g, 73%) as follows:

¹H NMR(300MHz，CDCl₃) δ 10.49(s, 1H), 10.06(s, 1H), 8.84(d, J ═ 8.36Hz, 1H), 8.43(d, J ═ 5.36Hz, 1H), 7.49-7.16(m, 5H), 5.34(s, 2H), 4.32-4.24(m, 4H), 1.11(dd, J ═ 8.59Hz, 6.57Hz, 4H) and 0.0 (18H).

¹³C NMR(75MHz，CDCl₃)δ197.0，153.2，149.1(d，J＝6.45Hz)，143.7，138.5，136.7，130.1，129.8，128.6(d，J＝6.68Hz)，126.3，124.9，120.0，67.2(d，J＝5.39Hz，2C)，62.5，19.5(d，J＝6.58Hz，2C)，-1.6.

³¹P NMR(121MHz，CDCl₃)δ5.2.

(2-formylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-5-chlorobenzyl ester (23)

Following the above procedure, 22(2.4g, 3.7mmol) gave 23(1.6g, 72%) as an oil:

¹H NMR(300MHz，CDCl₃) δ 10.38(br s, 1H), 9.90(s, 1H), 8.67(d, 1H), 8.28(dd, 1H), 7.47.3(m, 2H), 7.23(dd, 1H), 7.13(dd, 1H), 5.14(s, 2H), 4.12(m, 4H), 0.97(m, 4H) and-0.14 (m, 18H).

¹³C NMR(75MHz，CDCl₃) δ 197.0, 152.9, 147.3(d), 143.7, 138.3, 136.7, 130.1, 129.6, 129.5, 128.6, 128.5, 126.2, 121.3, 67.4(d), 61.6, 19.4(d) and-1.7.

³¹P NMR(121MHz，CDCl₃)δ5.1.

(2-formylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-5-fluorobenzyl ester (28a)

Following the above procedure, crude 27a (31.0g, 73mmol) gave 28a (26.9g, 64%) as an oil:

¹H NMR(300MHz，CDCl₃) δ 10.58(s, 1H), 10.10(s, 1H), 8.86(d, 1H), 8.48(dd, 1H), 7.52(m, 1H), 7.4-7.3(m, 1H), 7.21(dd, 1H), 7.1-6.9(m, 1H), 5.30(s, 2H), 4.4-4.2(m, 4H), 1.21.0(m, 4H) and 0.0(s, 18H).

¹³C NMR (75MHz, CDCl3) delta 197.1, 159.3(d), 152.9, 144.5(dd), 143.7, 143.5, 138.4, 136.8, 121.4(dd), 116.2(d), 115.9(d), 67.3(d), 61.8, 19.5(d) and-1.6.

³¹P NMR(121MHz，CDCl₃)δ5.5.

¹⁹F NMR(282MHz，CDCl₃)δ-59.3.

(2-formylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-5-nitrobenzyl ester (28b)

Following the above procedure, over 27b (4.2g, 9.4mmol), 28b was obtained as an oil (2.8g, 50%):

¹H NMR(300MHz，CDCl₃) δ 10.42(br s, 1H), 9.89(s, 1H), 8.64(d, 1H), 8.28(dd, 1H), 8.21(d, 1H), 8.05(dd, 1H), 7 × 47(d, 1H), 7.33(dd, 1H), 5.21(s, 2H), 4.17(m, 4H), 0.98(m, 4H) and-0.13 (m, 18H).

¹³C NMR(75MHz，CDCl₃) δ 197.0, 153.5(d), 152.7, 144.3, 143.9, 138.1, 136.8, 128.6, 128.3(d), 126.2, 125.4, 125.2, 120.3, 67.9(d), 61.4, 19.5(d), and-1.7.

³¹P NMR(121MHz，CDCl₃)δ4.5.

(2-formylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-5-methoxybenzyl ester (28e)

Following the above procedure, crude 27c (7.5g, 17mmol) gave 28e (9.8g, 73%) as an oil:

¹H NMR(300MHz，CDCl₃) δ 10.37(s, 1H), 9.93(s, 1H), 8.71(d, 1H), 8.30(d, 1H), 7.34(dd, 1H), 7.19(d, 1H), 6.85(d, 1H), 6.70(d, 1H), 5.18(s, 2H), 4.2-4.0(m, 4H), 3.66(s, 3H), 1.1-0.9(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 196.9, 156.4, 153.1, 143.6, 142.4(d), 138.5, 136.7, 128.6, 127.7(d), 126.2, 120.9, 115.1, 114.3, 67.1(d), 62.3, 55.5, 19.4(d), and-1.7.

³¹P NMR(121MHz，CDCl₃)δ5.8.

(2-formylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-5-trifluoromethoxybenzyl ester (28d)

Following the above procedure, over 27d (4.7g, 6.6mmol), 28d (3.2g, 75%) was obtained as an oil:

¹H NMR(300MHz，CDCl₃) δ 10.54(br s, 1H), 10.06(s, 1H), 8.82(d, 1H), 8.44(dd, 1H), 7.48(dd, 1H), 7.44(dd, 1H), 7.32(d, 1H), 7.25(s, 1H), 7.2-7.1(m, 1H), 5.30(s, 2H), 4.26(m, 4H), 1.10(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃)δ197.2，153.0，147.1(d)，145.7，143.9，138.4，136.9, 129.8, 128.8, 128.7, 126.4, 122.6, 122.2, 121.3, 120.0, 119.9, 67.6(d), 61.8, 19.5(d), and-1.6.

³¹P NMR(121MHz，CDCl₃)δ5.3.

¹⁹F NMR(282MHz，CDCl₃)δ-58.8.

(2-formylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-5-trifluoromethylbenzyl ester (28e)

Following the above procedure, over 27e (12.2g, 18mmol), 28e was obtained as an oil (6.9g, 63%):

¹H NMR(300MHz，CDCl₃) δ 10.54(br s, 1H), 10.06(d, 1H), 8.82(br d, 1H), 8.44(dd, 1H), 7.72(br s, 1H), 7.6-7.5(m, 2H), 7.48(dd, 1H), 7.3-7.1(m, 2H), 5.33(s, 2H), 4.27(m, 4H), 1.10(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 197.1, 153.0, 151.5(d), 143.9, 138.4, 136.9, 129.8, 129.7, 128.7, 127.7, 127.6, 127.3(d), 127.1, 127.0, 126.4, 126.3, 125.2, 120.3(d), 120.0(d), 67.7(d), 61.8, 19.6(d), and-1.6.

³¹P NMR(121MHz，CDCl₃)δ4.9.

¹⁹F NMR(282MHz，CDCl₃)δ-62.7.

(2-formylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonooxy-3, 5-dichlorobenzyl ester (28f)

Following the above procedure, over 27f (8.0g, 12mmol), 28f (5.1g, 71%) was obtained as an oil:

¹H NMR(300MHz，CDCl₃)δ10.54(br s，1H)，10.05(s，1H)，8.79(d，1H)，8.42(dd，1H)，7.46(dd，1H)，7.35(dd，2H)，7.24(s，1H)，5.38(s，2H)，4.27(m, 4H), 1.12(m, 4H) and 0.0(m, 18H).

¹³C NMR(75MHz，CDCl₃) δ 197.2, 152.8, 149.9, 143.9, 143.6(d), 138.4, 136.8, 131.8(d), 131.0(d), 130.1, 128.7, 128.0(d), 127.8(d), 126.3, 120.0(d), 67.8(d), 62.0, 19.6(d), and 1.6.

³¹P NMR(121MHz，CDCl₃)δ5.1.

(2-formylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonooxy-4, 5-dichlorobenzyl ester (28g)

Following the above procedure, 27g (17.4g, 25mmol) gave 28g (13.4g, 86%) of oil:

¹H NMR(300MHz，CDCl₃) δ 10.51(br s, 1H), 10.05(s, 1H), 8.79(d, 1H), 8.42(dd, 1H), 7.53(dd, 1H), 7.5-7.4(m, 1H), 7.24(s, 1H), 6.96(dd, 1H), 5.23(s, 2H), 4.28(m, 4H), 1.10(m, 4H) and 0.0(m, 18H).

¹³C NMR(75MHz，CDCl₃) δ 197.2, 152.9, 147.4(d), 144.0, 138.3, 136.9, 133.1, 131.0, 128.8(d), 128.7, 127.2(d), 126.3, 122.2, 122.0, 67.8(d), 61.3, 19.6(d), and-1.6.

³¹P NMR(121MHz，CDCl₃)δ5.1.

(2-formylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonooxy-5, 6-dichlorobenzyl ester (28h)

Following the above procedure, over 27h (9.5g, 14mmol), 28h (6.5g, 77%) was obtained as an oil:

¹H NMR(300MHz，CDCl₃) δ 10.44(br s, 1H), 10.05(s, 1H), 8.86(d, 1H), 8.44(dd, 1H), 7.50(d, 1H), 7.47(d, 1H), 7.41(d, 1H), 7.38(m, 1H), 5.46(s, 2H), 4.25(m, 4H), 1.10(m, 4H) and 0.0(m, 18H).

¹³C NMR(75MHz，CDCl₃) δ 197.0, 153.0, 149.9, 149.2(d), 143.8, 138.5, 136.8, 135.0, 131.2, 129.8, 129.6, 128.7, 126.6(d), 126.3, 119.5(d), 67.7(d), 59.9, 19.5(d), and-1.6.

³¹P NMR(121MHz，CDCl₃)δ4.9.

(2-formylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-3-methylbenzyl ester (28i)

Following the above procedure, over 27i (1.5g, 2.2mmol), 28i (1.1g, 86%) was obtained as an oil:

¹H NMR(300MHz，CDCl₃) δ 10.35(br s, 1H), 9.92(s, 1H), 8.71(d, 1H), 8.28(dd, 1H), 7.32(dd, 1H), 7.16(d, 1H), 7.05(d, 1H), 6.96(dd, 1H), 5.3.1(s, 2H), 4.12(m, 4H), 2.28(s, 3H), 0.97(m, 4H) and-0.12 (m, 18H).

¹³C NMR(75MHz，CDCl₃) δ 196.9, 153.2, 147.2(d), 143.6, 138.6, 136.6, 131.6, 130.9(d), 128.6, 128.0(d), 127.4, 126.2, 125.3, 67.1(d), 62.9, 19.4(d), 17.0, and-1.7.

³¹P NMR(121MHz，CDCl₃)δ5.8.

(2-formylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-4-chlorobenzyl ester (28j)

Following the above procedure, 27j (12.2g, 19mmol) gave 28j (8.9g, 80%) as an oil:

rf (1: 1v/v ethyl acetate-hexane) 0.66.

¹H NMR(300MHz，CDCl₃) δ 10.49(br s, 1H), 10.04(s, 1H), 8.80(d, 1H), 8.42(dd, 1H), 7.5-7.4(m, 3H), 7.16(dd, 1H), 5.26(s, 2H), 4.27(m, 4H), 1.11(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 197.1, 153.1, 149.3(d), 143.8, 138.5, 136.8, 135.0, 131.0, 128.7, 126.3, 125.3(d), 125.2, 120.5(d), 67.5(d), 61.9, 19.5(d), and-1.6.

³¹P NMR(121MHz，CDCl₃)δ5.0.

(2-formylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-4-methoxybenzyl ester (28k)

Following the above procedure, over 27k (5.1g, 8.0mmol), 28k (2.7g, 58%) was obtained as an oil:

rf (1: 1v/v ethyl acetate-hexane) 0.44.

¹H NMR(300MHz，CDCl₃) δ 10.50(br s, 1H), 10.05(s, 1H), 8.84(d, 1H), 8.42(dd, 1H), 7.46(dd, 1H), 7.36(dd, 1H), 7.01(s, 1H), 6.71(dd, 1H), 5.24(s, 2H), 4.27(m, 4H), 3.80(s, 3H), 1.10(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，CDCl₃) δ 197.0, 160.9, 153.4, 150.2(d), 143.6, 138.7, 136.7, 131.7, 128.7, 126.3, 118.5(d), 110.6, 106.1(d), 67.3(d), 62.4, 55.5, 19.5(d), and-1.6.

³¹P NMR(121MHz，CDCl₃)δ5.0.

Example 8

Preparation of pyridine-2-carboxaldehydethiosemicarbazone (24, 29a-k)

General procedureThe corresponding pyridine-2-carbaldehyde (23, 23a or 28a-k, 10mmol) was dissolved in ethanol-water (2: 1v/v, 150 ml). To the solution was added thiosemicarbazide (11 mmol). The solution was stirred at ambient temperature for 30 minutes. After addition of water (50ml), the reaction mixture was stirred vigorously for 2 hours. The yellow precipitate was collected by filtration, washed with ethanol-water (1: 4v/v) and vacuum filteredDrying gave the corresponding pyridine-2-carboxaldehyde thiosemicarbazone (24, 24a, 29 a-k).

(2-Aldolaminothiosemicarbazone methylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-5-chlorobenzyl ester (24) (2-thiosemicarbazone methyl pyridine-3-yl) carbamic acid 2-bis (2-trimethylsilyl ethyl) phosphoryloxy-5-chloro benzyl ester)

Following the above procedure, 23(6.9g, 12mmol) gave 24(4.9g, 63%) as a yellow solid:

¹H NMR(300MHz，DMSO-d₆) δ 11.77(br s, 1H), 10.03(br s, 1H), 8.40(dd, 1H), 8.28(br s, 1H), 8.26(s, 1H), 7.94(br s, 1H), 7.5-7.4(m, 4H), 5.20(s, 2H), 4.06(m, 4H), 0.98(m, 4H) and-0.03 (m, 18H).

¹³C NMR(75MHz，DMSO-d₆) δ 178.5, 153.4, 148.0(d), 144.2, 142.9, 141.0, 133.9, 129.5(d), 128.9, 128.4, 128.0, 124.4, 121.6, 65.1(d), 61.2, 18.9(d) and-1.5.

³¹P NMR(121MHz，DMSO-d6)δ9.8.

For C₂₅H₃₉ClN₅O₆PSSi₂Mass calculated value: 660.267, respectively; measured value: 660.

2- (2-Aldolaminothiosemicarbazone-methylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonooxybenzyl ester (24a)

Following the above procedure, over 23a (29.74g, 0.54mol), a yellow solid 24a (33.67g, 90%)):

¹H NMR(DMSO-d₆，300MHz)：d 11.76(s，1H)，10.04(s，1H)，8.38(d，J＝4.32Hz，1H)，8.29(s，2H)，7.86-7.26(m，5H)，5.27(s，2H)，4.264.18(m，4H)，1.05(dd，J＝9.08Hz，7.64Hz，4H)，0.0(s，18H)；

¹³C NMR(DMSO-d6，75MHz)：d 178.9，153.3，148.0(d，J＝6.42Hz)，144.6，144.3，140.6，133.9，129.5，127.2(d，J＝5.54Hz)，125.2，124.1，119.8(d，J＝4.32Hz)，119.6，66.7(d，J＝7.8Hz，2C)，61.3，18.9(d，J＝6.5Hz，2C)，-1.6；

³¹P NMR(DMSO-d₆，121MHz)：d 9.7。

2-bis (2-trimethylsilylethyl) phosphonoxy-5-fluorobenzyl (2-carboxaldehyde thiosemicarbazone-methylpyridin-3-yl) carbamate (29a)

Following the above procedure, via 28a (14.3g, 25mmol), gave 29a (12.9g, 80%) as a yellow solid:

¹H NMR(300MHz，DMSO-d₆) δ 11.85(s, 1H), 10.13(s, 1H), 8.45(d, 1H), 8.26(s, 1H), 7.5-7.1(m, 5H), 5.21(s, 2H), 4.2-4.0(m, 4H), 1.0-0.9(m, 4H) and 0.0(s, 18H).

¹³C NMR(75MHz，DMSO-d₆) δ 178.6, 159.8, 156.0, 153.4, 145.1(d), 143.5, 141.5, 140.4, 134.2, 129.5, 124.5, 121.4(d), 115.5, 64.4(d), 61.3, 18.9(d) and-1.6.

³¹P NMR(121MHz，DMSO-d6)δ10.5.

¹⁹F NMR(282MHz，DMSO-d₆)δ-62.5.

C₂₅H₃₉FNSO₆PSSi₂Mass calculated value: 643.813 found: 644.2

(2-Aldolaminothiosemicarbazone-methylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-5-nitrobenzyl ester (29b)

Following the above procedure, over 28b (1.6g, 2.7mmol), yellow solid 29b (1.3g, 77%) was obtained:

¹H NMR(300MHz，DMSO-d₆)δ11.86(br s, 1H), 10.14(br s, 1H), 8.4-8.2(m, 4H), 7.87(br s, 2H), 7.64(m, 1H), 7.52(m, 1H), 5.26(s, 2H), 4.05(m, 4H), 0.97(m, 4H) and-0.03 (m, 18H).

¹³C NMR(75MHz，DMSO-d₆) δ 178.6, 155.3(d), 153.4, 143.7, 142.7, 140.7, 134.1, 128.1(d), 125.2, 124.6, 124.5, 120.1, 64.8(d), 61.2, 18.9(d) and-1.5.

³¹P NMR(121MHz，DMSO-d₆)δ9.2.

(2-Aldolaminothiosemicarbazone-methylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-5-methoxybenzyl ester (29c)

Following the above procedure, over 28c (5.0g, 8.8mmol), 29c (4.4g, 77%) was obtained as a yellow solid:

¹H NMR(300MHz，DMSO-d₆) δ 11.85(s, 1H), 10.08(s, 1H), 8.41(d, 1H), 8.35(d, 1H), 8.30(s, 1H), 8.03(s, 2H), 7.51(dd, 1H), 7.26(d, 1H), 7.01(d, 1H), 6.90(dd, 1H), 5.26(s, 2H), 4.2-4.0(m, 4H), 3.75(s, 3H), 1.1-0.11(m, 4H) and 0.0(s, 18H).

¹³C NMR (75MHz, DMSO-d6) delta 178.7, 155.7, 153.6, 143.9, 142.6(d), 134.2, 129.4, 128.4(d), 124.5, 121.1, 114.1, 113.9, 64.9(d), 61.9, 55.6, 19.1(d), and-1.4.

³¹P NMR(121MHz，DMSO-d6)δ10.3.

C₂₆H₄₂NSO₇PSSi₂Mass calculated value: 655.848, respectively; measured value: 656.2

(2-Aldolaminothiosemicarbazone-methylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonooxy-5-trifluoromethoxybenzyl ester (29d)

Following the above procedure, over 28d (2.5g, 3.9mmol), 29d (1.9g, 68%) was obtained as a yellow solid:

¹H NMR(300MHz，DMSO-d₆) δ 11.81(s, 1H), 10.04(br s, 1H), 8.42(d, 1H), 8.26(s, 1H), 7.95(br s, 1H), 7.5-7.2(m, 4H), 5.24(s, 2H), 4.07(m, 4H), 0.96(m, 4H) and-0.04 (m, 18H).

¹³C NMR(75MHz，DMSO-d₆) δ 178.4, 153.4, 147.8(d), 144.1, 144.0, 133.9, 129.4(d), 124.4, 121.9, 121.7, 121.6, 121.4, 118.3, 65.1(d), 61.2, 18.9(d), and-1.6.

³¹P NMR(121MHz，DMSO-d₆)δ9.8.

¹⁹F NMR(282MHz，DMSO-d₆)δ-53.0.

(2-Aldolaminothiosemicarbazone-methylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonooxy-5-trifluoromethylbenzyl ester (29e)

Following the above procedure, over 28e (5.3g, 8.5mmol), 29e (3.6g, 61%) was obtained as a yellow solid:

¹H NMR(300MHz，DMSO-d₆) δ 11.81(s, 1H), 10.03(br s, 1H), 8.42(d, 1H), 8.4-8.3(m, 2H), 8.26(s, 1H), 7.89(br s, 1H), 7.79(s, 1H), 7.75(d, 2H), 7.56(d, 2H), 7.49(dd, 1H), 5.27(s, 2H), 4.06(m, 4H), 0.97(m, 4H) and-0.04 (m, 18H).

³¹P NMR(121MHz，DMSO-d₆)δ9.5.

¹⁹F NMR(282MHz，DMSO-d₆)δ-56.2.

(2-Aldolamide Methylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-3, 5-dichlorobenzyl ester (29f)

Following the above procedure, over 28f (4.8g, 7.7mmol), 29f (4.6g, 85%) was obtained as a yellow solid:

¹H NMR(300MHz，DMSO-d₆) δ 11.75(s, 1H), 10.04(s, 1H), 8.39(d, 1H), 8.30(d, 1H), 8.26(s, 1H), 7.91(br s, 1H), 7.69(dd, 1H), 7.50(d, 1H), 7.43(dd, 1H), 5.29(s, 2H), 4.24(m, 4H), 1.03(m, 4H) and-0.01 (m, 18H).

³¹P NMR(121MHz，DMSO-d₆)δ10.3.

(2-Aldolaminothiosemicarbazone-methylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonooxy-4, 5-dichlorobenzyl ester (29g)

Following the above procedure, 28g (2.0g, 3.2mmol) gave 29g (1.5g, 70%) of a yellow solid:

¹H NMR(300MHz，DMSO-d₆) δ 11.85(s, 1H), 10.08(s, 1H), 8.42(d, 1H), 8.31(d, 1H), 8.26(s, 1H), 7.89(m, 1H), 7.70(s, 1H), 7.59(s, 1H), 7.5-7.2(m, 2H), 5.18(s, 2H), 4.02(m, 4H), 0.95(m, 4H) and 0.0(m, 18H).

¹³C NMR(75MHz，DMSO-d₆) δ 178.6, 153.3, 148.8, 143.8, 142.4, 140.6, 134.0, 130.8, 130.2(d), 129.5, 128.3(d), 125.8(d), 124.4, 121.5, 119.9, 64.9(d), 60.8, 18.9(d), and-1.5.

³¹P NMR(121MHz，DMSO-d₆)δ9.7.

(2-Aldolaminothiosemicarbazone-methylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonooxy-5, 6-dichlorobenzyl ester (29h)

Following the above procedure, over 28h (5.9g, 9.5mmol), a yellow solid was obtained for 29h (3.6g, 55%):

¹H NMR(300MHz，DMSO-d6)δ11.82(s，1H)，9.97(br s，1H)，8.50(m，1H)，8.41(d，1H)，8.27(d，1H)，8.23(s，1H)，7.72(m，1H)，7.67(d，1H)，7.48(m，1H)，7.41(d，1H)，7.3-7.1(m，1H)，5.32(s，2H)，4.03(m，4H)，0.96(m, 4H) and-0.06 (m, 18H).

¹³C NMR(75MHz，DMSO-d₆) δ 178.3, 153.3, 150.6(d), 143.8, 140.8, 134.0, 133.8, 133.3, 131.2, 130.3, 129.4, 126.7(d), 124.4, 120.1, 119.9(d), 64.8(d), 59.4, 18.9(d) and-1.6.

³¹P NMR(121MHz，DMSO-d₆)δ9.6.

(2-Aldolaminothiosemicarbazone-methylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonooxy-3-methylbenzyl ester (29i)

Following the above procedure, over 28i (1.1g, 1.9mmol), 29i (0.7g, 57%) was obtained as a yellow solid:

¹H NMR(300MHz，DMSO-d₆) δ 11.76(br s, 1H), 9.99(br s, 1H), 8.42(br s, 1H), 8.37(m, 1H), 8.27(br s, 1H), 8.24(m, 1H), 8.03(br s, 1H), 7.45(dd, 1H), 7.25(d, 1H), 7.19(d, 1H), 7.11(dd, 1H), 5.30(s, 2H), 4.08(m, 4H), 2.29(s, 3H), 1.01(m, 4H) and-0.03 (m, 18H).

¹³C NMR(75MHz，DMSO-d₆) δ 178.7, 153.8, 147.5(d), 144.4, 143.2, 141.2, 134.3, 131.1, 130.7(d), 128.9(d), 126.4, 124.9, 124.6, 65.4(d), 62.4, 19.2(d), 16.9 and-1.4.

³¹P NMR(121MHz，DMSO-d₆)δ10.4.

(2-Aldolaminothiosemicarbazone-methylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonooxy-4-chlorobenzyl ester (29j)

Following the above procedure, 28j (10.5g, 18.5mmol) gave 29j (11.8g, 97%) as a yellow solid:

¹H NMR(300MHz，DMSO-d₆)δ11.74(s，1H)，10.05(br s，1H)，8.4-8.3(m，3H)，7.85(br s，1H)，7.56(d，1H)，7.5-7.4(m，3H)，5.21(s, 2H), 4.22(m, 4H), 1.04(m, 4H) and-0.01 (s, 18H).

¹³C NMR(75MHz，DMSO-d₆) δ 153.2, 148.5, 148.4, 144.8, 144.5, 133.8, 133.0, 130.8, 126.5, 126.4, 125.4, 124.1, 119.7, 67.2(d), 60.8, 18.9(d), and-1.6.

³¹P NMR(121MHz，DMSO-d₆)δ9.5.

(2-Aldolaminothiosemicarbazone-methylpyridin-3-yl) carbamic acid 2-bis (2-trimethylsilylethyl) phosphonoxy-4-methoxybenzyl (29k)

Following the above procedure, over 28k (2.6g, 4.5mmol), 29k (2.7g, 91%) was obtained as a yellow solid:

¹H NMR(300MHz，DMSO-d₆) δ 11.74(d, 1H), 9.98(br s, 1H), 8.4-8.3(m, 3H), 7.82(br s, 1H), 7.44(m, 1H), 6.87(m, 3H), 5.16(s, 2H), 4.19(m, 4H), 3.77(s, 3H), 1.03(m, 4H) and 0.01(s, 18H).

¹³C NMR(75MHz，DMSO-d₆)178.4, 160.0, 153.2, 150.2, 148.4, 144.8, 144.5, 133.8, 133.0, 130.8, 124.1, 119.0, 110.4, 106.0, 66.8(d), 61.4, 55.3, 18.9(d), and-1.6.

³¹P NMR(121MHz，DMSO-d₆)δ9.6.

Example 9

Preparation of free phosphonic acid (6-17)

General procedureTrifluoroacetic acid (TFA, 20-50ml) was added to a solution of the corresponding TMSE-protected phosphate (24, 24a or 29a-k, 10mmol) in dichloromethane (300-500ml) at 0 ℃. The reaction mixture was stirred vigorously in an ice bath for 2 hours. The precipitate was collected by filtration, washed with cold dichloromethane and then dried in vacuo. The solvent is usually evaporated off and the resulting residual mixture is then dried in vacuo. The corresponding free phosphonic acid obtained (6-17 and 6a not shown) is yellowA colored solid or a glassy solid.

Example 10

Preparation of disodium salt of phosphonic acid (25, 30a-k)

General procedureWith saturated sodium bicarbonate (NaHCO)₃) The aqueous solution (50-100ml) neutralized the corresponding free phosphonic acid (6-17, 10 mmol). The suspension was stirred at ambient temperature for 2 hours, then a minimum amount of water was added to homogenize it. The aqueous solution was purified by reverse phase column chromatography (eluting with deionized water). By using³¹Fractions were monitored by P NMR and pooled. After drying by low pressure sublimation, the corresponding disodium salt (25 or 30a-k) was obtained as a pale yellow powder.

(2-Aldolamide Thiouromethylpyridin-3-yl) carbamic acid 2- (Phosphonoyloxydisodium) -5-chlorobenzyl ester (25)

Following the above procedure, over 24(1.1g, 1.7mmol), gave 25(0.4g, 49%) as a pale yellow powder:

¹H NMR(300MHz，D₂o) delta 7.94(br s, 2H), 7.72(s, 1H), 7.2-7.0(m, 3H) and 4.98(s, 2H).

¹³C NMR(75MHz，D₂O) δ 179.6, 157.0, 153.2, 147.2(d), 145.2, 136.8, 131.4, 130.5, 128.8, 127.8, 123.6 and 65.4.

³¹P NMR(121MHz，D₂O)δ14.3.

(2-Aldolamide Thiouromethylpyridin-3-yl) carbamic acid 2- (Phosphonoyloxydisodium) -5-fluorobenzyl ester (30a)

From 29a (10.5g, 16mmol) 7(6.2g, 86%) was obtained as described above via NaHCO₃Work-up gave a pale yellow powder 30a (4.0g, 59%):

¹H NMR(300MHz，D₂o) Δ 8.2(br s, 1H), 7.8(br m, 1H), 7.57(br s, 1H), 7.15(m, 1H), 6.93(m, 1H), 6.81(m, 1H), 6.78(m, 1H) and 4.93(s，2H)。

¹³C NMR(75MHz，D₂O) δ 179.4, 161.5, 158.4, 156.5, 150.3, 147.3, 146.5, 136.7, 130.8, 130.4, 127.7, 123.5, 117.5, 117.2 and 65.2.

³¹P NMR(121MHz，D₂O)δ14.5.

¹⁹F NMR(282MHz，D₂O)δ-57.4.

2- (Phosphonoyloxydisodium) -5-nitrobenzyl (2-carboxaldehyde thiosemicarbazone-methylpyridin-3-yl) carbamate (30b)

Following the above procedure, over 29b (2.1g, 3.0mmol), a dark yellow powder 30b (1.0g, 73%) was obtained:

¹H NMR(300MHz，D₂o) Δ 8.0-7.8(m, 4H), 7.40(m, 1H), 7.17(m, 1H) and 5.06(s, 2H).

³¹P NMR(121MHz，D₂O)δ13.8.

2- (Phosphonoyloxydisodium) -5- (methoxybenzyl) carbamic acid 2- (3-yl-2-carboxaldehyde thiosemicarbazone methylpyridin-3-yl ester (30c)

The above procedure was followed through 29c (4.3g, 16mmol) to give 9(2.9g, 98%) over NHCO₃Work-up gave 30c (1.6g, 43%) as a pale yellow powder:

¹H NMR(300MHz，D₂o) δ 7.96(br s, 1H), 7.70(br s, 1H), 7.21(br s, 1H), 7.08(br s, 1H), 6.73(s, 2H), 5.05(s, 2H) and 3.65(s, 3H).

¹³C NMR(75MHz，D₂O) δ 174.5, 151.8, 151.1, 143.4, 142.1, 141.7, 135.9, 131.6, 126.4, 124.9, 122.6, 118.4, 111.7, 60.8 and 53.2.

³¹P NMR(121MHz，D₂O)δ14.6.

(2-Aldolamide Thiouromethylpyridin-3-yl) carbamic acid 2- (Phosphonoyloxydisodium) -5-trifluoromethoxybenzyl ester (30d)

Following the above procedure, over 29d (1.9g, 2.6mmol), gave 30d (0.5g, 31%) as a pale yellow powder:

¹H NMR(300MHz，D₂o) δ 7.93(br s, 1H), 7.86(br d, 1H), 7.71(s, 1H), 7.25(d, 1H), 7.02(m, 4H) and 5.01(s, 2H).

¹³C NMR(75MHz，D₂O)δ179.5，173.5，157.1，153.3，147.1，146.8，145.5，141.2(m)，136.5，132.4(m)，130.2(d)，127.7(d)，124.5，124.0，123.1，122.6，121.0-65.4.

³¹P NMR(121MHz，D₂O)δ14.3.

¹⁹F NMR(282MHz，D₂O)δ-56.3.

(2-Aldolamide Thiouromethylpyridin-3-yl) carbamic acid 2- (Phosphonoyloxydisodium) -5-trifluoromethylbenzyl ester (30e)

Following the above procedure, over 29e (3.6g, 5.2mmol), gave 30e (1.3g, 45%) as a pale yellow powder:

¹H NMR(300MHz，D₂o) δ 7.98(br s, 1H), 7.89(d, 1H), 7.77(s, 1H), 7.4-7.3(m, 3H), 7.08(m, 1H) and 5.04(s, 2H).

³¹p NMR(121MHz，D₂O)δ14.0.

¹⁹F NMR(282MHz，D₂O)δ-59.4.

(2-Aldolanzothiourea methylpyridin-3-yl) carbamic acid 2- (phosphonooxy disodium) -3, 5-dichlorobenzyl ester (30f)

Following the above procedure, over 29f (4.5g, 6.5mmol), gave 30f (0.8g, 24%) as a pale yellow powder:

¹H NMR(300MHz，D₂o) Δ 8.31(br s, 1H), 7.88(br d, 2H), 7.6-7.5(m, 2H), 7.2-6.8(m, 5H) and 5.07(s, 2H).

¹³C NMR(75MHz，D₂O) δ 179.6, 156.6(d), 149.4(d), 147.4, 146.8(d), 136.6, 134.2, 131.5, 131.1, 130.7(d), 130.1, 128.5, 127.7(d) and 65.6.

³¹P NMR(121MHz，D₂O)δ14.4.

2- (Phosphonoyloxydisodium) -4, 5-dichlorobenzyl (2-Aldcarbodimide Thiouromethylpyridin-3-yl) carbamate (30g)

Following the above procedure, 29g (2.5g, 3.0mmol) gave 30g (0.4g, 23%) of a pale yellow powder:

¹H NMR(300MHz，D₂o) Δ 8.07(s, 1H), 7.99(m, 1H), 7.85(s, 1H), 7.39(s, 1H), 7.19(m, 2H) and 4.99(s, 2H).

³¹p NMR(121MHz，D₂O)δ14.3.

(2-Aldolanzothiouronomethylpyridin-3-yl) carbamic acid 2- (phosphonooxy disodium) -5, 6-dichlorobenzyl ester (30h)

Following the above procedure, over 29h (4.6g, 6.6mmol), a pale yellow powder was obtained for 30h (2.3g, 64%):

¹H NMR(300MHz，D₂o) Δ 8.01(s, 1H), 7.91(br s, 1H), 7.73(s, 1H), 7.23(dd, 2H), 7.12(m, 1H) and 5.18(s, 2H).

³¹P NMR(121MHz，D₂O)δ14.2.

(2-Aldolamide Thiouromethylpyridin-3-yl) carbamic acid 2- (Phosphonoyloxydisodium) -3-methylbenzyl ester (30i)

Following the above procedure, over 29i (1.2g, 1.8mmol), a yellow powder 30i (0.5g, 57%) was obtained:

¹H NMR(300MHz，D₂o) Δ 8.11(br s, 2H), 7.91(m, 2H), 7.71(m, 1H), 7.00(m, 2H), 6.84(m, 1H), 5.22(s, 2H) and 2.14(s, 3H).

¹³C NMR(75MHz，D₂O) δ 146.1, 134.6, 133.6, 131.1, 128.8, 127.9, 125.7, 66.6 and 19.1.

³¹P NMR(121MHz，D20)δ14.2.

(2-Aldolamide Thiouromethylpyridin-3-yl) carbamic acid 2- (Phosphonoyloxydisodium) -4-chlorobenzyl ester (30j)

Following the above procedure, over 29j (4.2g, 6.6mmol), gave 30j (1.6g, 48%) as a pale yellow powder:

¹H NMR(300MHz，D₂o) δ 7.98(s, 1H), 7.90(m, 1H), 7.74(s, 1H), 7.31(s, 1H), 7.09(m, 3H), 6.85(m, 1H) and 5.00(s, 2H).

¹³C NMR(75MHz，D₂O) δ 180.0, 157.7, 155.9, 147.6, 147.4, 142.3, 137.1, 136.8, 133.2, 132.9, 128.3, 127.9, 124.9 and 65.9.

³¹P NMR(121MHz，D₂O)δ14.3.

(2-Aldolamide Thiouromethylpyridin-3-yl) carbamic acid 2- (Phosphonoyloxydisodium) -4-methoxybenzyl ester (30k)

Following the above procedure, over 29k (2.9g, 4.4mmol), we obtained 30k (1.2g, 54%) as a pale yellow powder:

¹H NMR(300MHz，D₂o) Δ 8.06(s, 1H), 7.94(s, 1H), 7.64(s, 1H), 7.13(m, 1H), 7.0-6.8(m, 3H), 6.43(m, 1H), 4.06(s, 2H) and 3.58(s, 3H).

¹³C NMR(75MHz，D₂O) δ 161.5, 161.3, 155.1, 133.1, 127.3, 127.0, 111.4, 108.3 and 57.9.

³¹P NMR(121MHz，D₂O)δ14.3.

Biological assay/data

Alkaline phosphatase catalyzed bioconversion of prodrugs to 3-AP

Using 4.65X 10^-5The biological activation of the dimethyl para-prodrug and phosphate prodrug subtypes was studied in unit phosphatase enzyme solution (type VII-SA from Bovine IntestrialMucose, Sigma). During incubation with phosphatase, all prodrugs are converted to the parent drug 3-AP. Under this experimental condition, the biological activation of the ortho phosphate prodrug did not increase its half-life (T/2) compared to the unsubstituted ortho prodrug (Table 1 below). The prodrug is placed in human serum and is subjected to heat preservation at 37 ℃ for human serum stability study. This study showed that 4-chlorophosphate prodrug 16 is a slow release prodrug with a half-life 1.5 times that of the ortho prodrug.

Table 1: enzymatic bioconversion and serum stability of 3-AP phosphate prodrugs

Half life
				Prodrugs	Alkaline phosphatase 37 deg.C	Human serum 37 deg.C	Buffered saline pH7.6, 37 deg.C
Ortho- (2)	16.3min	2.7hr	Not hydrolyzed
				Para- (3)	9.2min	1.2hr	5.5hr
5-Cl-(6)	30.5min	3.4hr	162hr
				5-F-(7)	Not tested	3.8hr	Not hydrolyzed
5-CH3O(9)	22.1min	3.2hr	151hr
				4-Cl-(16)	29.9min	4.0hr	Not hydrolyzed
4-CH3O(17)	Not tested	13.3hr	15.7hr

In vivo PK Studies on prodrugs containing ortho-phosphate 3-AP

A single dose of 7.2-8.5mg/kg of the 3-AP phosphate prodrug (equivalent to 3mg/kg of 3-AP) was administered to Beagle intravenous dogs to characterize the pharmacokinetic profile of the 3-AP prodrug. Each prodrug is administered once per week. After each dose, a washout period (washout period) of at least 6 days is maintained before the next dose is administered. The concentrations of 3-AP (Triapine) and prodrug in the serum were determined by HPLC, from which various PK parameters were calculated. These PK parameters were compared to 3-AP (equimolar dose) parameters from another independent study. Mean blood concentration data over time were analyzed using compartmental and non-compartmental models. Calculating AUC, Total clearance (Cl), Steady State volume of distribution (Vd) for 3-AP and prodrug_ss) Terminal half life (T)_1/2)C_maxAnd T_max。

The pharmacokinetic parameters for equimolar doses of the prodrug are shown in table 2 below. When incubated with alkaline phosphatase in vitro, the ortho-phosphate prodrug appeared to undergo premature rapid biotransformation to 3-AP. However, this transformation was rather slow in vivo, indicating that the effect of alkaline phosphatase on the drug in vivo (off-rate) was delayed. No intermediate phenol (i.e. the expected cleavage product) was ever detected. The serum half-life of the partial ortho-prodrug is relatively extended compared to the half-life of 3-AP in dogs (about 1.5 hours), which is equivalent to Triapine^TM(3-AP) half-life in humans. The AUCs and half-lives of the 4-chloro (16), 5-methoxy (9) and 5-fluoro (7) analogs are also predictive of this.

Table 2: PK values in dogs for 3-AP prodrugs containing ortho-phosphates

Prodrugs	Cmax(μg/mL)	AUC(μg·min/L)	T1/2	Cl(mL/min/kg)	Vss(L/kg)
						Ortho- (3)	125	63309	5.9hr	0.11	0.14
5-Cl-(6)	136	24263	2.1hr	0.32	0.08
							139	27939	2.3hr	0.28	0.09
5-F-(7)	114	44829	4.5hr	0.47	0.11
						5-NO2-(8)	126	3396	19min	2.33	0.12
5-CH3O-(9)	126	51460	4.7hr	0.15	0.12
						5-CF3O-(10)	144	17584	1.4hr	0.48	0.09
5-CF3-(11)	156	9579	43min	0.86	0.08
						3, 5-bis-Cl- (12)	140	6939	34min	1.2	0.08
4, 5-bis-Cl- (13)	220	21499	1.1hr	0.39	0.15
						5, 6-bis-Cl- (14)	202	34211	2.0hr	0.24	0.12
3-CH3-(15)	147	66756	5.3hr	0.11	0.08
						4-Cl-(16)	120	46321	4.5hr	0.17	0.10

The pharmacokinetic parameters for 3-AP after i.v. administration of equimolar doses of prodrug are shown in table 3 below. The results of the study demonstrate that the prodrug ortho (3), 5-fluoro (7) and 4-chloro (16) prolong the release of the parent 3-AP, which allows a sustained concentration of 3-AP in the serum equivalent to the other prodrug under study. These compounds exhibit improved stability in aqueous solutions.

Table 3: PK values of 3-AP in dogs

Prodrugs	Cmax(μg/mL)	AUC(μg·min/L)	Tmax(min)	T1/2(hour)	V/F(L/kg)
						Para- (2)	1.6	74.5	---	1.5	2.80
Ortho- (3)	0.6	698	8.6	14.2	1.77
						5-Cl-(6)	2.2	456	16.7	2.2	0.42
	2.1	395	---	2.5	3.67
						5-F-(7)	0.5	619	1.2	13.2	1.85
5-NO2-(8)	1.9	84	---	0.5	4.27
						5-CH3O-(9)	1.5	987	7.9	7.7	0.67
5-CF3O-(10)	9.2	709	---	0.9	0.92
						5-CF3-(11)	3.0	280	---	1.1	2.74
3, 4-bis-Cl- (12)	2.1	163	---	0.9	3.99
						4, 5-bis-Cl- (13)	2.7	392	---	1.7	3.14
5, 6-bis-Cl- (14)	3.6			1.6	2.32
						3-CH3-(15)	1.5	998	7.8	7.8	5.18
4-Cl-(16)	0.8	888	9.0	12.4	1.21

Initially, a single dose of two prodrugs, each 7.5-7.7mg/kg (equivalent to 3mg/kg Triapine), was studied. Based on this result, dose-escalating pharmacokinetic and toxicological studies were performed on 5-fluoro-prodrugs (20, 40, and 80mg/kg) and 4-chloro-prodrugs (20 and 30 mg/kg).

The PK parameters for Triapine and prodrug are shown in tables 4 and 5 and compared to the results of an independent study with 3mg/kg Triapine administered (equivalent to about 7.5mg/kg prodrug). Dogs receiving both 4-chloro and 5-fluoro prodrugs (equimolar dose) showed improved Triapine contact time (expressed as AUC) compared to dogs treated with Triapine. Studies with escalating doses show that the peak plasma concentrations of tripine and AUCs are linearly related to prodrug doses.

TABLE 4 comparative pharmacokinetics of Triapine phosphate prodrugs in dogs

Prodrugs	Dosage (mg/kg)	Cmax(μg/mL)	AUC(mg·min/L)	Tmax(min)	T1/2(hour)	V/F(L/kg)
							5-fluoro- (7)	7.5	0.5	619	1.2	13.2	1.85
20	6.8	490	---	0.8	2.90
						40		12.4	1153	---	1.1	3.22
80	32.0	2713	---	1.0	2.50
						4-chloro- (16)		7.7	0.8	888	9.0	12.4	1.21
20	13.0	2592	---	2.3	1.53
							30	31.9	5905	---	2.1	0.94
Triapine	3	2.3	124	---	1.8								3.57

TABLE 5 comparative pharmacokinetics of Triapine phosphate prodrugs in dogs

Prodrugs	Dosage (mg/kg)	Cmax(μg/mL)	AUC(mg·min/L)	T1/2(hour)	Cl(mL/min/kg)	Vss(L/kg)
							5-fluoro- (7)	7.5	114	44829	4.5hr	0.47	0.11
20	299.6	35877	1.4hr	0.56	0.11
						40		412.0	56679	1.6hr	0.35	0.07
80	377.4	43863	1.3hr	0.46	0.06
						4-chloro- (16)		7.7	120	46321	4.5hr	0.17	0.10
20	464	63080	1.6hr	0.32	0.07
							30	556	90291	1.9hr	0.33	0.15
Triapine	3	2.3	124	---	1.8								3.57

On the other hand, the peak serum concentrations of the prodrug and the AUCs are not linearly related to the dose, and appear to saturate at the dose studied. The plasma concentration-time profile of the prodrug is shown in figures 4 and 5. These data indicate that at IV dose levels greater than or equal to 20mg/kg, both prodrugs exhibit prolonged biotransformation to the parent Triapine, with levels above 1 μ M (0.2 μ g/ml) being maintained for 24 hours. Sustained serum Triapine levels may be due to higher prodrug serum levels and their lower overall clearance. The pharmacokinetics of Triapine of the 4-chloro prodrug is shown in FIG. 6. Higher blood levels (> 1. mu.M) of Triapine were observed at 24 hours and dogs were better tolerated at this dose level.

And (3) clinical observation: early death did not occur in dogs receiving the 4-chloro-prodrug. Treatment-related clinical observations of dogs were recorded. On days 2 and 3 (day 1 ═ day of dosing), male dogs treated with 20mg/kg developed loose stools. One female dog treated with 40mg/kg showed vomiting, diarrhea, yellow mucus in the stool, decreased mobility and cyanosis (grey mouth) 1 day after administration. On day 2, the above phenomenon was not observed. On day 2, the only adverse clinical sign was absence of stool. The clinical signs of male dogs treated with 30mg/kg were similar to female dogs treated with 40 mg/kg. Thus, the MTD of the 4-chloro-prodrug was determined to be between 30-40 mg/kg.

Early death did not occur in dogs receiving the 5-fluoro-prodrug. No adverse signs were observed in dogs treated with 20 (female) and 40mg/kg (male). Treatment-related clinical observations were recorded for 80mg/kg treated females. The female dogs vomited during and after infusion. In addition, the dogs were observed to develop discoloration (pale skin), diarrhea, and yellow mucus in the stool. All of the above observations occurred on day 1 and recovered on day 2. Based on these results, the MTD of the 5-chloro-prodrug was determined to be 80 mg/kg.

Notably, at MTDs levels (30 mg/kg for the 4-chloro prodrug and 80mg/kg for the 5-fluoro prodrug), both prodrugs reached a peak plasma level of Triapine (approximately 32 μ g/ml), suggesting that toxicity observed in dogs was due to Triapine, not to the prodrug itself.

In vivo antitumor efficacy

The efficacy and toxicity of the twelve (12)3-AP prodrug was evaluated in Balb/c mice using the M109 murine lung cancer model. The experimental method is as follows: female Balb/c mice (about 20g) aged 8 weeks were injected subcutaneously on day 0 in the right flank with 5X 10⁵Mouse M109 murine Lung cancer cells. The mice were then randomly grouped, each group consisting of 8-10 mice. Treatment was started on day 3 or 5 according to the schedule shown in table 6. 3-AP and Triapine^TMThe formulations are used together with all of the 3-AP prodrug dissolved or suspended in sterile deionized water. Body weight and tumor volume of mice were measured twice weekly until tumor necrosis in control group or tumor necrosis in control groupAt least one animal died.

The results obtained are shown in Table 6 below. Based on the stable pharmacokinetics and activity of these drugs, it is clear that the 5-fluoro and 4-chloro analogs (7 and 16, respectively) exhibit the greatest activity as anticancer agents. In addition, 5-trifluoromethoxy derivative (10), 4, 5-dichloro derivative (13) and 5, 6-dichloro derivative (14) also exhibited unexpectedly superior activities.

Table 6: activity in M109 Lung cancer model

Prodrugs	Dosage (mpk, ip)	Time-meter (Tian)	Inhibition (%)	Body weight loss (%)	Relative vigor
						3-AP(1A)	5.5(bid)	3-7，10-14	60	8.09	＜CTX
Ortho- (3)	48QD	5-9，12-16	70	12.6	＜CTX
						5-Cl-(6)	60QD	5-9，12-16	67	5.3	＝CTX
5-Fl-(7)	60QD	5-9，12-16	75	4.7	＝CTX
						5-NO2-(8)	60QD	3-7，10-14，17-21	33	9.7	＜＜CTX
5-CH3O-(9)	48QD	5-9，12-16	73	13.5	ND
						5-CF3O-(10)	100QD	3-7，10-14，17-21	81	8.7	＝CTX
5-CF3-(11)	100QD	3-7，10-14，17-21	77	9.1	＜CTX
						3, 5-bis-Cl- (12)	60QD	3-7，10-14，17-21	58	9.0	＜CTX
4, 5-bis-Cl- (13)	60QD	3-7，10-14，17-21	57	10.0	＜CTX
						5, 6-bis-Cl- (14)	60QD	3-7，10-14，17-21	59	7.0	＜CTX
3-CH3-(15)	60QD	3-7，10-14	64	7.9	＜CTX
						4-Cl-(16)	60QD	5-9，12-16	74	12.6	＝CTX

CTX ═ cyclophosphamide

Typically, prodrugs of 3-AP can be administered at a dose 8 times (molar) higher than the MTDs of 3-AP. These drugs may also be administered on a daily 1-5 day/week (QD1-5) schedule for a period of time during which mice do not experience excessive mortality. Treatment of M109 lung cancer with compounds 3, 6, 7, 9, 10-16 gave better efficacy compared to 3-AP precursor at MTD levels. Subsequent studies of M109 lung cancer with prodrugs 7 and 16 according to the schedule shown in FIG. 7c have shown that they are significantly effective against M109 lung cancer. In mice, the effects of these drugs (prodrugs 7 and 16) on other cancer cell lines such as HTB human lung carcinoma, B16-F10 melanoma, DLD-1 colon carcinoma were also tested and shown to be significantly more potent than cyclophosphamide (see FIGS. 7D, E and F).

Further studies (e.g., optimal dosages and dosage regimens, different routes of administration, and combinations with chemotherapy) were performed on 5-chloro (6), 5-fluoro (7), and 4-chloro (16) and the like. The results of these experiments are shown in the accompanying figures (FIGS. 4-10). The results, which are readily derived from the figures, show that the prodrugs of the invention are better combined with the DNA damaging agents cyclophosphamide and mitomycin C (FIGS. 8A-D and 9A-C). Likewise, the compounds of the invention were also more effective against human colon cancer and human Lovo colon cancer (fig. 10A and B) in combination with etoposide (fig. 9D) and cisplatin.

Efficacy of 3-AP prodrug on M109 lung carcinoma in Balb/c mice

Materials: m109 lung cancer cells; BALB/c mice (female, 9 weeks old, 18-20 g); cyclophosphamide (Sigma); 3-AP prodrug (ortho 3-AP prodrug (3), 5-methoxy 3-AP prodrug (9), 5-chloro 3-AP prodrug (6), 4-chloro 3-AP prodrug (16), 5-fluoro 3-AP prodrug (7), and 4-fluoro 3-AP prodrug (12).

The 110 Balb/c mice were randomly divided into 12 groups:

grouping: mouse

1. 0.2ml of carrier, and 10 Qd (5-9 days; 12-16; 19-23)

2.200 mpk Cyclophosphamide, I.P., l/w 10

3.48 mpk ortho 3-AP prodrug, I.P., Qd (5-9 days; 12-16; 19-23) 10

4.60 mpk ortho 3-AP prodrug, I.P., Qd (5-9 days; 12-16; 19-23) 10

5.48 mpk 5-methoxy 3-AP prodrug, I.P., Qd (5-9 days; 12-16; 19-23) 10

6.55 mpk 5-methoxy 3-AP prodrug, I.P., Qd (5-9 days; 12-16; 19-23) 10

7.48 mpk 5-chloro 3-AP prodrug, I.P., Qd (5-9 days; 12-16; 19-23) 10

8.60 mpk 5-chloro 3-AP prodrug, I.P., Qd (5-9 days; 12-16; 19-23) 10

9.48 mpk 4-chloro 3-AP prodrug, I.P., Qd (5-9 days; 12-16; 19-23) 10

10.60 mpk 4-chloro 3-AP prodrug, I.P., Qd (5-9 days; 12-16; 19-23) 10

11.48 mpk 5-fluoro 3-AP prodrug, I.P., Qd (5-9 days; 12-16; 19-23) 7

12.48 mpk 4-fluoro 3-AP prodrug, I.P., Qd (5-9 days; 12-16; 19-23) 8

(Note: W/week; I.P./intraperitoneal injection; Qd/daily)

Formulation of 3-AP prodrug: prior to injection, the prodrug was dissolved in deionized sterile water to make a stock solution (10.0mg/ml) of each 3-AP prodrug. The 3-AP prodrug stock solution was further diluted with water to formulate each of the following prodrugs.

Volume of test tube stock solution water prodrug concentration

1. 1.8ml 1.2ml 6.0mg/ml 3ml

2.1.65 ml 1.35ml 5.5mg/ml (methoxy 3-AP) 3ml

3. 1.44ml 1.56ml 4.8mg/ml 3ml

By trypsin treatmentM109 cells were harvested in the logarithmic growth phase, washed with PBS and reconstituted to 2.5X 10⁶Cells/ml PBS. On day 0, M109 suspension (0.2ml, 5X 10) was implanted subcutaneously in the right flank⁵Cell/mouse). Mice were randomly regrouped as described above. Drug treatment was started on day 5 according to the schedule described above. Mice were placed in a clean, thermostated laboratory. The feeding bedding is changed at least twice a week. Provide sufficient biological and drinking water for the mice. The drinking water is processed by hot pressing before use. Treatment with the ortho-phosphate 3-AP prodrug (3) and the 5-methoxy 3-AP prodrug (9) was stopped before the end of the experiment due to severe toxic reactions or death to the mice. Body weight and tumor were measured twice weekly before the end of the experiment. Mice were observed daily for mortality and appearance.

FIGS. 7A, 7B and 7C show the potency of 5-fluoro 3-AP prodrug (7) and 4-chloro 3-AP prodrug (16) relative to 200mpk cyclophosphamide (compared to control). The 3-AP prodrug showed excellent tumor-shrinking efficacy comparable to cyclophosphamide without death.

Efficacy of 3-AP prodrug/cyclophosphamide combination chemotherapy on M109 lung cancer in Balb/c mice

Materials: m109 lung cancer cells; BALB/c mice (female, 9 weeks old, 19-21 g); cyclophosphamide (Sigma corporation); mitomycin C; 5-chloro 3-AP prodrug (6), and 5-fluoro 3-AP prodrug (7).

120 Balb/c mice were randomly divided into 15 groups, each group consisting of 8 mice.

Grouping: mouse

1. 0.2ml of carrier, Qd (3-7 days; 10-14) 8

2.200 mpk Cyclophosphamide, I.P., 1/Wx 3 (starting on day 3) 8

3.3 mpk mitomycin C, I.V., Qd (days 3 and 17) 8

4.45 mpk 5-chloro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +100mpk cyclophosphate 8

Amide, I.P., 1/W (starting on day 4)

5.45 mpk 5-chloro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +150mpk cyclophosphate 8

Amide, I.P., 1/W (starting on day 4)

6.45 mpk 5-chloro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +200mpk cyclophosphate 8

Amide, I.P., 1/W (starting on day 4)

7.60 mpk 5-chloro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +100mpk cyclophosphate 8

Amide, I.P., 1/W (starting on day 4)

8.60 mpk 5-chloro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +150mpk cyclophosphate 8

Amide, I.P., 1/W (starting on day 4)

9.60 mpk 5-chloro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +200mpk cyclophosphate 8

Amide, I.P., 1/W (starting on day 4)

10.45 mpk 5-fluoro 3-AP prodrug, I.P./daily (3-7 days; 10-14) +100mpk Ring 8

Phosphoramide, I.P., 1/W (starting on day 4)

11.45 mpk 5-fluoro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +150mpk Ring 8

Phosphoramide, I.P., 1/W (starting on day 4)

12.45 mpk 5-fluoro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +200mpk Ring 8

Phosphoramide, I.P., 1/W (starting on day 4)

13.60 mpk 5-fluoro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +100mpk Ring 8

Phosphoramide, I.P., 1/W (starting on day 4)

14.60 mpk 5-fluoro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +150mpk Ring 8

Phosphoramide, I.P., 1/W (starting on day 4)

15.60 mpk 5-fluoro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +200mpk Ring 8

Phosphoramide, I.P., 1/W (starting on day 4)

(Note: I.V./intravenous injection)

Preparing a 3-AP prodrug stock solution: prior to injection, the prodrug was dissolved in deionized sterile water to make up a stock solution (10.0mg/ml) of each 3-AP prodrug. The 3-AP prodrug stock solution was diluted with water to prepare each of the following prodrugs.

Volume of test tube stock solution water prodrug concentration

1. 1.8ml 1.2ml 6.0mg/ml 3ml

2. 1.35ml 1.65ml 4.5mg/ml 3ml

M109 cells were collected at logarithmic growth phase by trypsinization, washed with PBS and reconstituted to 2.5X 10⁶Cells/ml PBS. On day 0, M109 suspension (0.2ml, 5X 10) was implanted subcutaneously in the right flank⁵Cell/mouse). Mice were randomly regrouped as described above. Drug treatment was started on day 3 according to the schedule described above. Mice were placed in a clean, thermostated laboratory. The feeding bedding is changed at least twice a week. Provide sufficient food and drinking water for the mice. For drinking waterAnd (6) performing hot pressing treatment. Body weight and tumor were measured twice weekly before the end of the experiment. Mice were observed daily for mortality and appearance. Figure 7C is another comparative experiment with cyclophosphamide and prodrug (3).

FIGS. 7D-F show the activity of 5-fluoro 3-AP prodrug (7) and 4-chloro prodrug (16) against mouse HTB177 human lung carcinoma B16-F10 melanoma and DLD-1 colon carcinoma under the conditions specified.

Figures 8A-D show the efficacy of 5-chloro 3-AP prodrug (6) and 5-fluoro 3-AP prodrug (7) in combination with chemotherapy (in combination with cyclophosphamide) against M109 lung cancer relative to 200mpk cyclophosphamide (compared to control). The 3-AP prodrug in combination with cyclophosphamide exhibits superior synergistic tumor reduction efficacy compared to cyclophosphamide alone. Notably, no animals died during this experiment.

Combination chemotherapy pair based on 5-fluoro 3-AP prodrug/cyclophosphamide or mitomycin Efficacy of M109 Lung cancer in Balb/C mice

Materials: m109 lung cancer cells; BALB/c mice (female, 9 weeks old, 19-21 g); cyclophosphamide (Sigma corporation); mitomycin C and 5-fluoro ortho-3-AP prodrug (7).

121Balb/c mice were randomly divided into 12 groups, each group consisting of 10 mice.

Grouping: mouse

1. 0.2ml of carrier, Qd (3-7 days; 10-14) 11

2.200 mpk Cyclophosphamide, I.P., 1/Wx 3 (starting on day 3) 10

3.3 mpk mitomycin C, I.V., QD (3 and 13 days) 10

4.45 mpk 5-fluoro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +150mpk cyclophosphate 10

Amide, I.P., 1/W (starting on day 4)

5.45 mpk 5-fluoro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +200mpk cyclophosphate 10

Amide, I.P., 1/W (starting on day 4)

6.60 mpk 5-fluoro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +150mpk cyclophosphate 10

Amide, I.P., 1/W (starting on day 4)

7.60 mpk 5-fluoro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +200mpk cyclophosphate 10

Amide, I.P., 1/W (starting on day 4)

8.45 mpk 5-fluoro 3-AP prodrug, I.P./Qd (3-7 days; 10-14) +2mpk mitomycin 10

Element C, i.v., QD (days 3 and 13)

9.60 mpk 5-fluoro 3-Ap prodrug, I.P./Qd (3-7 days; 10-14) +2mpk mitomycin 10

Element C, i.v., QD (days 3 and 13)

10.120 mpk 5-fluoro 3-AP, S.C. qd (3-7 days)^* 10

11.150 mpk 5-fluoro 3-AP, S.C. qd (3-7 days; 10-14)^** 10

12.200 mpk 5-fluoro 3-AP, S.C. qd (3-6 days)^*** 10

(Note:^*only one dose regimen was given due to death of the mice;^**after administration of the two dose regimen, due to weight lossAnd stopping the processing;^***treatment was stopped after 4 days because of death of the mice; S.C./subcutaneous injection)

Formulation of 3-AP prodrug: prior to injection, the prodrug was dissolved in deionized sterile water to prepare a stock solution (20.0mg/ml) of the 5-fluoro-3-AP prodrug (7). The 3-AP prodrug stock solution was further diluted with water to formulate each of the following prodrugs.

Volume of test tube stock solution water prodrug concentration

1. 3.0ml 0ml 20.0mg/ml 3ml

2. 2.25ml 0.75ml 15.0mg/ml 3ml

3. 1.8ml 1.2ml 12.0mg/ml 3ml

4. 1.5ml 1.5ml 10.0mg/ml 3ml

2. 0.9ml 2.1ml 6.0mg/ml 3ml

2. 0.68ml 2.32ml 4.5mg/ml 3ml

M109 lung and cancer cells (1X 10) stored in liquid nitrogen⁶Cells/ml × 1ml) were thawed rapidly at 37 ℃ and placed in 25ml DMEM medium (containing 10% FCS, 37 ℃ C., 5% CO)₂In (1). After passage 2, the cells were washed twice with PBS (pH7.2), trypsinized and re-cultured in flasks containing 50ml of medium. Finally, M109 cells in the logarithmic growth phase (about 90-95% saturation) were collected by trypsinization, washed with PBS and reconstituted to 5X 10⁶Cells/ml PBS for tumor implantation. On day 0, M109 suspension (0.2ml, 5X 10) was implanted subcutaneously in the right flank⁵Cell/mouse). Mice were randomly regrouped as described above. According to the above-mentioned time scheduleDrug treatment was started on day 3. Mice were placed in a clean, thermostated laboratory. The feeding bedding is changed at least twice a week. Provide sufficient food and drinking water for the mice. The drinking water is processed by hot pressing before use. Body weight and tumor were measured twice weekly before the end of the experiment. Mice were observed daily for mortality and appearance.

FIGS. 9A-C show the efficacy of 5-fluoro 3-AP prodrug (7) and 5-chloro 3-AP prodrug (6) in combination with chemotherapy (mitomycin C) versus 200mpk cyclophosphamide, and comparison to control. The combination of 3-AP prodrug with cyclophosphamide and mitomycin showed a favorable synergistic tumor reduction efficacy compared to cyclophosphamide or mitomycin C alone. Figure D shows the results of an experiment comparing the efficacy of 5-chloro 3-AP prodrug (6) in combination with etoposide versus etoposide alone or a control. The experiment shows that the drug combination shows the activity of resisting M109 lung cancer synergistically.

FIGS. 10A and 10B show the efficacy of 4-chloro 3-AP (16) in combination with cisplatin against DLD-1 human colon cancer (FIG. 10A) and human LoVo colon cancer (FIG. 10B). Combination therapy in these two experiments showed that the combination therapy had synergistic activity against the tested tumors.

LD of 5-fluoro 3-AP prodrug on C57BL/6J mice₅₀

Materials: c57BL/6J mice (female, 8 weeks old); 5-fluoro 3-AP prodrug (7).

55C57BL/6J mice were randomly divided into 11 groups, each group consisting of 5 mice:

grouping: mouse

1. Vehicle 0.2ml sterile deionized water, i.p.qd 5

2.100 mpk 5-fluoro 3-AP prodrug, I.P.qd 5

3.125 mpk 5-fluoro 3-AP prodrug, I.P.qd 5

4.150 mpk 5-fluoro 3-AP prodrug, I.P. qd 5

5.175 mpk 5-fluoro 3-AP prodrug, I.P.qd 5

6.200 mpk 5-fluoro 3-AP prodrug, I.P. qd 5

7.175 mpk 5-fluoro 3-AP prodrug, S.C. qd 5

8.200 mpk 5-fluoro 3-AP prodrug, S.C. qd 5

9.225 mpk 5-fluoro 3-AP prodrug, S.C. qd 5

10.250 mpk 5-fluoro 3-AP prodrug, S.C. qd 5

11.300 mpk 5-fluoro 3-AP prodrug, S.C. qd 5

Formulation of 3-AP prodrug: the 5-fluoro-3-AP prodrug (7) was dissolved in sterile deionized water to prepare a stock solution (30 mg/ml). The 3-AP prodrug stock solution was further diluted with water to formulate each of the following prodrugs.

Volume of test tube stock solution water prodrug concentration

1. 3.0ml 0ml 30.0mg/ml 3ml

2. 2.5ml 0.5ml 25.0mg/ml 3ml

3. 2.25ml 0.75 22.5mg/ml 3ml

4. 2ml 1ml 20mg/ml 3ml

5. 1.75ml 1.25ml 17.5mg/ml 3ml

6. 1.5ml 1.5ml 15mg/ml 3ml

7. 1.25ml 1.75ml 12.5mg/ml 3ml

8. 1ml 2ml 10mg/ml 3ml

Treatment was started on day 0 according to the schedule described above. The animals were recorded daily for mortality. Body weights were measured twice weekly and mice were observed daily for appearance and behavior. FIG. 11 shows the determination of LD for 5-fluoroaP prodrugs₅₀(approximately 160 mpk).

Pharmacokinetics study

The pharmacokinetics of the 3-AP (1A) ortho phosphate prodrug (2), and the 5-F ortho phosphate prodrug (7, 30a, FIG. 3) in Beagle dogs (Canis familiaris) were determined.

The dogs received the following doses: 20mg/kg, 30mg/kg and 40mg/kg of the 3-AP and ortho phosphate prodrug, and 20mg/kg, 40mg/kg and 80mg/kg of the 5-fluoro ortho phosphate prodrug. The dosing regimen for each compound was determined based on the maximum tolerated dose for each prodrug, with the 5-fluoro ortho phosphate prodrug being higher than the 3-AP or ortho phosphate prodrug (3). (notably, even at 80mg/kg doses of 5-fluoro ortho phosphate did not produce toxicity in animals, whereas 3-AP and ortho phosphate prodrug (3) produced toxicity at 30-40mg/kg levels.)

The levels of drug in the animals were measured at intervals shown in FIGS. 12-15. These figures show that the ortho phosphate prodrug has a significant effect on the bioavailability of 3-AP, and that the 5-fluorophosphate prodrug allows 3-AP to be sustained with significantly greater bioavailability and high concentrations. The pharmacokinetic data for 3-AP and ortho-phosphate prodrug (3) are shown in FIG. 12.

With respect to the 5-fluorophosphate prodrug (7), the data shown in FIGS. 13-15 indicate that the 5-fluorophosphate derivatives can provide greater bioavailability of the prodrug compound itself, as well as greater bioavailability of 3-AP (resulting from degradation of the prodrug). In addition. The high level of 3-AP duration (see 13) of the 5-fluorophosphate prodrug is longer than that of the 3-AP or ortho-phosphate prodrug.

The above studies indicate that high levels of 5-fluorophosphate prodrug (7) are better tolerated than 3-AP or ortho-phosphate prodrug (3) and that initially higher plasma concentrations of 3-AP can be delivered and maintained for longer periods of time than either the ortho-phosphate prodrug form or the 3-AP drug itself.

It will be understood by those skilled in the art that the foregoing description and examples are illustrative only of the practice of the invention, and are not intended to limit the scope of the invention in any way. Modifications and variations may be made to the invention as herein detailed without departing from the scope of the invention as defined in the following claims.

Claims (59)

1. A compound having the structure:

wherein R is H or CH₃；

R₂Is in the form of phosphoric acid or a salt thereof;

R₃is H, F, Cl, Br, I, OCH₃、OCF₃、CF₃Or C₁-C₃An alkyl group;

R₄h, F, Cl is,Br、I、OCH₃、OCF₃Or CF₃(ii) a And

R₅and R₆Independently of one another, H, F, Cl, Br, I, OCH₃、OCF₃Or CF₃，

The conditions are as follows: r₃、R₄、R₅And R₆Is not H, and when R is₃、R₄、R₅And R₆When any 2 of them are not H, R₃、R₄、R₅And R₆The other 2 of (a) is H.

2. A compound according to claim 1, wherein when R is₃、R₅And R₆When is H, R₄Is Cl, F or Br.

3. A compound according to claim 2, wherein R₄Is Cl.

4. A compound according to claim 1, wherein when R is₃、R₄And R₆When is H, R₅Is F, Cl, OCH₃Or OCF₃。

5. A compound according to claim 4, wherein R₅Is F or Cl.

6. A compound according to claim 5, wherein R₅Is F.

7. A compound according to claim 5, wherein R₅Is Cl.

8. A compound according to claim 1, wherein R₃、R₄、R₅And R₆Two of which are not H, but are selected from F, Cl, Br or I.

9. A compound according to claim 8, wherein R₃、R₄、R₅And R₆Two of which are F or Cl.

10. A compound according to claim 8, wherein R₄And R₅Is F or Cl.

11. A compound according to claim 8, wherein R₅And R₆Is F or Cl.

12. A compound according to claim 10, wherein R₄And R₅Is Cl.

13. A compound according to claim 11, wherein R₅And R₆Is Cl.

14. A pharmaceutical composition comprising an effective amount of a compound for treating neoplasia, the compound having the structure:

wherein R is H or CH₃；

R₂Is in the form of phosphoric acid or a salt thereof;

R₃is H, F, Cl, Br, I, OCH₃、OCF₃、CF₃Or C₁-C₃An alkyl group;

R₄is H, F, Cl, Br, I, OCH₃、OCF₃Or CF₃(ii) a And

R₅and R₆Independently of one another, H, F, Cl, Br, I, OCH₃、OCF₃Or CF₃，

The conditions are as follows: r₃、R₄、R₅And R₆Is not H, and when R is₃、R₄、R₅And R₆When any 2 of them are not H, R₃、R₄、R₅And R₆Another 2 of (a) is H, said compoundThe compound is combined with pharmaceutically acceptable additives, carriers or excipients.

15. The composition according to claim 14, wherein when R is₃、R₅And R₆When is H, R₄Is Cl, F or Br.

16. The composition according to claim 15, wherein R₄Is Cl.

17. The composition according to claim 14, wherein when R is₃、R₄And R₆When is H, R₅Is F, Cl, OCH₃Or OCF₃。

18. The composition according to claim 17, wherein R₅Is F or Cl.

19. The composition according to claim 18, wherein R₅Is F.

20. The composition according to claim 18, wherein R₅Is Cl.

21. The composition according to claim 14, wherein R₃、R₄、R₅And R₆Two of which are not H, but are selected from F, Cl, Br or I.

22. The composition according to claim 21, wherein R₃、R₄、R₅And R₆Two of which are F or Cl.

23. The composition according to claim 21, wherein R₄And R₅Is F or Cl.

24. The composition according to claim 21, wherein R₅And R₆Is F or Cl.

25. The composition according to claim 23, wherein R₄And R₅Is Cl.

26. The composition according to claim 24, wherein R₅And R₆Is Cl.

27. The composition according to claim 14, wherein the neoplasm is cancer.

28. Use of a compound of the formula:

wherein R is H or CH₃；

R₂Is in the form of phosphoric acid or a salt thereof;

R₃is H, F, Cl, Br, I, OCH₃、OCF₃、CF₃Or C₁-C₃An alkyl group;

R₄is H, F, Cl, Br, I, OCH₃、OCF₃Or CF₃(ii) a And

R₅and R₆Independently of one another, H, F, Cl, Br, I, OCH₃、OCF₃Or CF₃，

The conditions are as follows: r₃、R₄、R₅And R₆Is not H, and when R is₃、R₄、R₅And R₆When any 2 of them are not H, R₃、R₄、R₅And R₆The other 2 of (a) is H, in combination with a pharmaceutically acceptable additive, carrier or excipient.

29. The use according to claim 28, wherein when R is₃、R₅And R₆When is H, R₄Is Cl, F or Br.

30. Use according to claim 29, wherein R₄Is Cl.

31. The use according to claim 28, wherein when R is₃、R₄And R₆When is H, R₅Is F, Cl, OCH₃Or OCF₃。

32. Use according to claim 31, wherein R₅Is F or Cl.

33. Use according to claim 32, wherein R₅Is F.

34. Use according to claim 32, wherein R₅Is Cl.

35. Use according to claim 28, wherein R₃、R₄、R₅And R₆Two of which are not H, but are selected from F, Cl, Br or I.

36. Use according to claim 35, wherein R₃、R₄、R₅And R₆Two of which are F or Cl.

37. Use according to claim 35, wherein R₄And R₅Is F or Cl.

38. Use according to claim 35, wherein R₅And R₆Is F or Cl.

39. Use according to claim 37, wherein R₄And R₅Is Cl.

40. Use according to claim 38, wherein R₅And R₆Is Cl。

41. The use according to claim 28, wherein the neoplasm is cancer.

42. The use according to claim 41, wherein the cancer is gastric, colon, rectal, liver, pancreatic, lung, breast, cervical, endometrial, ovarian, prostate, testicular, bladder, kidney, brain/central nervous system, head and neck, throat, Hodgkin's disease, non-Hodgkin's leukemia, multiple myeloma leukemia, skin melanoma, acute lymphocytic leukemia, acute myeloid leukemia, Ewing's sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, nephroblastoma, neuroblastoma, hairy cell leukemia, mouth/throat, esophageal, throat, melanoma, renal cancer or lymphoma.

43. The use according to claim 41, wherein said cancer is lung, breast or prostate cancer.

44. Use of a compound of the formula and a DNA damaging anticancer agent in the manufacture of a medicament for the treatment of neoplasia:

wherein R is H or CH₃；

R₂Is in the form of phosphoric acid or a salt thereof;

R₃is H, F, Cl, Br, I, OCH₃、OCF₃、CF₃Or C₁-C₃An alkyl group;

R₄is H, F, Cl, Br, I, OCH₃、OCF₃Or CF₃(ii) a And

R₅and R₆Independently of one another, H, F, Cl, Br, I, OCH₃、OCF₃Or CF₃，

The conditions are as follows:R₃、R₄、R₅and R₆Is not H, and when R is₃、R₄、R₅And R₆When any 2 of them are not H, R₃、R₄、R₅And R₆The other 2 of (a) is H, in combination with a pharmaceutically acceptable additive, carrier or excipient.

45. The use according to claim 44, wherein when R is₃、R₅And R₆When is H, R₄Is Cl, F or Br.

46. The use according to claim 45, wherein R₄Is Cl.

47. The use according to claim 44, wherein when R is₃、R₄And R₆When is H, R₅Is F, Cl, OCH₃Or OCF₃。

48. The use according to claim 47, wherein R₅Is F or Cl.

49. The use according to claim 48, wherein R₅Is F.

50. The use according to claim 48, wherein R₅Is Cl.

51. The use according to claim 44, wherein R₃、R₄、R₅And R₆Two of which are not H, but are selected from F, Cl, Br or I.

52. Use according to claim 51, wherein R₃、R₄、R₅And R₆Two of which are F or Cl.

53. According to claim 52, wherein R₄And R₅Is F or Cl.

54. The use according to claim 52, wherein R₅And R₆Is F or Cl.

55. The use according to claim 44, wherein the neoplasm is cancer.

56. The use according to claim 55, wherein the cancer is gastric, colon, rectal, liver, pancreatic, lung, breast, cervical, uterine corpus, ovarian, prostate, testicular, bladder, kidney, brain/central nervous system, head and neck, throat, Hodgkin's disease, non-Hodgkin's leukemia, multiple myeloma leukemia, skin melanoma, acute lymphocytic leukemia, acute myeloid leukemia, Ewing's sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, nephroblastoma, neuroblastoma, hairy cell leukemia, mouth/throat, esophageal, throat, melanoma, renal or lymphoma.

57. The use according to claim 56, wherein said DNA damaging anticancer agent is selected from the group consisting of: cyclophosphamide, mitomycin C, etoposide, doxorubicin, topotecan, irinotecan, gemcitabine, camptothecin, cisplatin, chlorambucil, melphalan, or mixtures thereof.

58. The use according to claim 56, wherein R₃、R₄And R₆Is H, R₅Is F or Cl, and said DNA damaging anticancer drug is selected from cyclophosphamide or mitomycin C.

59. The use according to claim 58, wherein the cancer is lung cancer, prostate cancer, colon cancer, melanoma or breast cancer.