HK1089368B - Water-soluble shps as novel alkylating agents - Google Patents

Description

Water-soluble sulfonyl hydrazide prodrugs as novel alkylating agents

Technical Field

The present invention relates to metabolically activated Sulfonyl Hydrazide Prodrugs (SHPs) having anti-mammalian tumor activity. Another aspect of the invention is a method of treating tumors, particularly cancers, using the compounds of the invention.

The invention is government supported, and issued by the ministry of health and public health as permit number 1R 43CA 92968-01. The government therefore has certain rights in the invention.

Background

Alkylating agents are one of the most effective therapeutic agents for the treatment of various malignant tumors at present, and are widely used in clinical settings (katzeng,Basic & Clinical Pharmacology7 th edition 1998 Appleton&Lange, Stamford, 881). Its high cytotoxicity is attributed to its ability to induce cross-linking between DNA strands, thereby inhibiting the level of replication (Rajski and Williams,Chem Reviews1998, 98: 2723). Among alkylating agents, the CNU (chloroethyl nitrosoureas) series are widely used clinically for the treatment of brain tumors, colon cancer and lymphoma (DeVita et al.Cancer Res.1965, 25: 1876; and Nissen et al.Cancer1979, 43: 31) however, its clinical use has been limited due to delayed and cumulative myelosuppressive effects and hepatotoxicity (Panasci et al).Cancer Res.1977, 37: 2615; and a combination of Gibson and Hickman,Biochem Pharmacol.1982，31：2795)。

a series of 1, 2-bis (sulfonyl) hydrazine prodrugs (SHPs) from CNU have recently been developed that have the ability to generate chloroethylated and carbamylated groups but lack hydroxyethylated and vinylated groups (Sartorlli et al, see U.S. Pat. No. 6,040,338; U.S. Pat. No. 5,637,619; U.S. Pat. No. 5,256,820; U.S. Pat. No. 5,214,068; U.S. Patents)No. 5,101,072; U.S. patent nos. 4,849,563; and U.S. patent No. 4,684,747). Its antitumor activity is thought to be caused by chloroethylation followed by crosslinking with DNA (Kohn,Recent Results in Cancer Researchcarter et al, 1981, Springer, Berlin, vol.76: 141, a solvent; and Sheary et al,J Med Chem.1984, 27: 664). Carbamoylated groups (i.e., isocyanates) can react with thiol and amine functional groups on proteins, thereby inhibiting DNA polymerase (Baril et al).Cancer Res.1975, 35: 1) repair of DNA strand breaks (Kann et al.Cancer Res.1974, 34: 398) and RNA synthesis and processing (Kann et al.Cancer Res.1974, 34: 1982). However, DNA hydroxyethylation will lead to carcinogenic and/or mutagenic consequences (Swenson et al.J Natl Cancer Inst.1979，63：1469)。

1, 2-bis (methylsulfonyl) -1- (2-chloroethyl) -2- (methylaminocarbonyl) hydrazine (VNP40101M) is a lead compound in the current SHP series, which has low toxicity to the host and better antitumor activity than Chloroethylnitrosourea (CNU) derivatives and other SHP analogues (Shyam et al) against L1210 murine leukemia, L1210/BCNU, L1210/CTX, L1210/MEL (1, 3-bis (2-chloroethyl) -1-nitrosourea, cyclophosphamide and the melphalan resistant sublines), P388 leukemia, M109 lung carcinoma, B16 melanoma, C26 colon carcinoma and U251 glioma.J Med Chem.1999, 42: 941). In addition, VNP40101M can effectively cross the Blood Brain Barrier (BBB), thereby completely killing implanted intracranial leukocytes (> 6.54 log cell lethal dose), activating the potential of BCNU (Finch et al).Cancer Biochem Biophys.2001，61：3033)。

VNP40101M has antitumor activity probably due to the release of 90CE and methyl isocyanate. Further cleavage of 90CE gave methyl 2-chloroethyldiazosulfone (1) FIG. 1, a compound having a relative relationshipSpecific O⁶A guanine chloroethylating agent which may be present in the N of guanine⁷Slight alkylation at the-position (Penkenth et al.J Med Chem.1994, 37: 2912; and Penkenth et al.Biochem Pharmacol.2000, 59: 283). Methyl isocyanate released by VNP40101M has the effect of inhibiting various DNA repair enzymes including O⁶-alkylguanine-DNA alkyltransferase ability to induce O in DNA⁶Stabilization of the alkylguanine monoalkyl group gives a higher percentage of interchain crosslinking (Baril et al.Cancer Res.1975，35：1)。

VNP40101M is currently in clinical trials on patients with solid tumors and hematological malignancies. VNP40101M does not dissolve well in aqueous solutions; polyethylene glycol (PEG) and ethanol are added into the solvent for preparing the product to improve the solubility. Both PEG and ethanol are acceptable vehicles for human use, but as found in animal trials, they may also cause side effects such as hemolysis and phlebitis at high concentrations. If PEG and ethanol can be eliminated from the vehicle, they can be administered at higher doses due to the excellent tolerability of VNP40101M in humans. This theoretically yields better efficacy. Therefore, our aim was to synthesize a series of SHPs: (a) the water solubility and stability of the water-soluble chitosan can be improved in an aqueous solution with the pH of 3-9; (b) capable of forming a chloroethylated group; (c) no hydroxyethylation activity; (d) capable of forming methyl isocyanate; and (e) able to improve the type of drug metabolism (e.g., longer half-life in vivo).

The present inventors considered that SHPs (I) activated by water-soluble enzymes can satisfy the above conditions. An example of such an SHP may be a phosphate-containing derivative as shown in figure 2 for the following reasons.

(a) In general, analogs with phosphate, including their salt forms, may have good water solubility and stability at neutral pH;

(b) it is believed that compounds of formula I form a phenol intermediate by cleavage of the oxygen-phosphorus bond by Alkaline Phosphatase (AP), which is subsequently cleaved to form a chloroethylated or methylated product and a carbamylation agent without formation of a hydroxyethylation agent, as shown in figures I and 2.

(c) Compound I biotransformation can also produce quinone methides, which themselves can destroy DNA and thus exhibit an inhibitory effect on cell replication (Lin et al).J Med Chem.1986，29：84)。

(d) Compound I can be considered as a prodrug of VNP40101M, which has been demonstrated to be a broad spectrum anti-cancer alkylating agent against tumor pathologies including, for example, various solid tumors. Thus, compound I can produce substances with the same activity as VNP 40101M.

Other examples of biologically active prodrugs are shown in figures 3 and 4. Nitro analogs as shown in figure 3 are examples of compounds that are both soluble in water and selectively oxidizable under anoxic conditions. Release of VNP401 40101M is only possible after reduction of the nitro group under anoxic conditions. Compound II can be reduced to the corresponding amino analog by Nitroreductase (NR), which is then cleaved to form VNP40101M and quinone-imine methide. NR is an enzyme isolated from E.coli (E.coli) or Bacillus sp (Bacillus spp.) and widely used for ADEPT (antibody-directed enzyme prodrug therapy) or GDEPT (gene-directed enzyme prodrug therapy) in cancer therapy (Anlezark et al, WO93/08288, 1993).

Figure 4 depicts the use of peptidases to generate VNP40101M intratumorally. It has been previously described that compounds resulting from the combination of glutamyl residues with suitably substituted phenols and aromatic amines can be cleaved by carboxypeptidases such as carboxypeptidase G2(CPG2) and carboxypeptidase A (CPA). CPG2 is an enzyme isolated from Pseudomonas (Pseudomonas) that removes glutamate residues from folate and methotrexate. It has been used in ADEPT or GDEPT systems to activate prodrugs containing glutamate residues (Bagshawe et al, WO88/07378, 1988; Springer et al, US6,025,340, 2000; Springer et al, US6,004,550, 1999). CPA from bovine pancreas can be conveniently cleaved to pharmaceutical alpha-peptides (in which derivatives the alpha-carboxyl of the amino acid and glutamate moietiesGroups are linked). It is also used in ADEPT (Wolfe et al.Bioconjug Chem.1999，10：38；Huennekens，Adv Enzyme Regul.1997, 37: 77; and vitals et al.Cancer Res.1995, 55: 478). As shown in FIG. 4, compound III and compound IV can be cleaved by the corresponding CPG2 or CPA to form VNP40101M and quinone methide or quinone-imine methide, CPG2 or CPA can be introduced either as antibody conjugates or as transgenes (Pawelek et al, U.S. Pat. No. 6,190,657, 2001).

Object of the Invention

In one aspect, the present invention aims to provide compounds, pharmaceutical compositions and methods for the treatment of neoplasia, including mammalian and human cancers.

In another aspect, the present invention aims to provide a method for treating neoplasia with a class of pharmaceutical compositions that have good and high activity, pharmacokinetics, bioavailability and low toxicity.

It is yet another object of the present invention to provide compositions and methods for treating cancers that are resistant to traditional chemotherapeutic agents.

One or more of the above objects and/or other objects of the present invention will become apparent from the following description of the invention.

Summary of The Invention

The present invention relates to compounds according to structure (I):

wherein R is-CH₃or-CH₂CH₂Cl；

R' is C₁-C₇Alkyl or-CH₂CH₂Cl；

R₂Or R₄One but not all selected from OPO₃H₂、NO₂OCO (Glu), NHCO (Glu) and NHR₇Another unspecified R₂Or R₄And R₃、R₅And R₆Independently selected from H, F, Cl, Br, I, OH, OPO₃H₂、OCH₃、CF₃、OCF₃、NO₂、CN、SO₂CH₃、SO₂CF₃、COCH₃、COOCH₃、SCH₃、SF₅、NHR₈、N(R₉)₂、OPO₃H₂And C₁-C₇Alkyl with the proviso that R₂、R₃、R₄、R₅And R₆Is H;

R₇is H, glutamyl, preferably alpha-glutamyl (-COCH (NH)₂)CH₂CH₂CO₂H) Or a polyglutamic polypeptide residue (-COCH (NHR) having 1-50 peptide bonds, preferably having 2-10 peptide bonds_7a)CH₂CH₂CO₂H, wherein R_7aIs a glutamyl (preferably alpha-glutamyl) or polyglutamic polypeptide residue);

R₈is H or C₁-C₇An alkyl group; and

R₉is CH₃Or CH₂CH₃. The phosphate and/or glutamate substituents may be in the free acid form or a pharmaceutically acceptable salt thereof.

In certain preferred aspects of the invention, the preferred agent in compound I is a series carrying a ortho phosphate, wherein R is-CH₂CH₂Cl; r' is-CH₃；R₂Is a phosphate group which may be in the form of the free acid or a pharmaceutically acceptable salt thereof, preferably Na. In a particularly preferred aspect of the SHPs carrying ortho-phosphates, when R₃、R₅And R₆When is H, R₄Is Cl, F or Br (preferably Cl). In another preferred aspect of the SHPs carrying ortho-phosphates, when R₃、R₄And R₆When is H, R₅Is Cl, F or Br (preferably Cl, F). In a further preferred aspect of the SHPs carrying ortho-phosphates, R₃、R₄、R₅And R₆Two of which are selected from F, Cl, Br or I (preferably the two substituents are the same, more preferably both substituents are Cl), R₃、R₄、R₅And R₆The other two of (a) are H.

Preferred agents in compound II are nitro-containing analogs of SHPs carrying meta-phosphate. The phosphate group may be the free acid or a pharmaceutically acceptable salt thereof (preferably Na). In a particularly preferred aspect of the nitro-containing SHPs, when R₄When is H, R₂Is NO₂. In another preferred method of using nitro-containing SHPs, when R is₂When is H, R₄Is NO₂。

Preferred agents in compounds III and IV are conjugated analogs of the glutamyl residues of SHPs. In a particularly preferred aspect of both SHPs, the terminal acid may be the free acid or a pharmaceutically acceptable salt thereof (preferably Na). In still another preferred aspect of Compound IV, R₇May be H or a polyglutamic polypeptide residue.

The compounds according to the invention as defined above and in particular the preferred compositions according to the invention are particularly effective compounds for the treatment of neoplasias. They have at least one or more improved properties relative to VNP40101M, such as increased antitumor activity, reduced toxicity response, higher water solubility, or a more satisfactory pharmacokinetic profile. Thus, preferred compounds of the invention may have a higher therapeutic index (i.e. better therapeutic efficacy/risk ratio) than VNP 40101M.

The compounds according to the invention are useful in pharmaceutical compositions for the treatment of cancer, as well as a variety of other diseases and/or disorders. Embodiments of the invention may be used as intermediates in the synthesis of other compounds having biological activity and as standards for measuring the biological activity of the compounds of the invention. In certain applications, the compounds of the present invention are useful for treating microbial infections, including in particular viral, bacterial and fungal infections. These compounds contain an effective amount of any one or more of the compounds as disclosed above, optionally in combination with pharmaceutically acceptable additives, carriers, or excipients.

Another aspect of the invention relates to a method of treating cancer comprising administering to a patient in need of such treatment an effective amount of a compound as disclosed above, optionally in combination with a pharmaceutically acceptable additive, carrier, or excipient. The present invention also relates to a method of treating neoplasia in a mammal comprising administering to a patient suffering from cancer an effective amount of a compound as disclosed above. A preferred embodiment of the present invention is a method of treating solid malignancies, leukocytosis, and lymphomas comprising administering to a patient an antitumor effective amount of one or more of the above agents. The compounds of the present invention are also effective for the treatment of various other related diseases. The methods of the invention can be used in a variety of comparative assays, such as assays for measuring the activity of related analogs, and assays for measuring the sensitivity of a patient's cancer to one or more compounds of the invention.

Brief description of the figures and tables

FIG. 1 shows the proposed mechanism of action for VNP 40101M.

FIGS. 2, 3 and 4 are diagrams illustrating certain compound embodiments of the present invention and their mechanism of action.

FIGS. 5-9 are chemical reaction schemes showing the synthesis of compounds of the present invention.

Figures 10-15 show the results of tests relating to the therapeutic efficacy and toxicity response of certain preferred embodiments of the present invention in this application.

Detailed Description

The following terms are used throughout the specification to describe the present invention.

The term "patient" as used in the specification means an animal including a mammal, particularly preferably a human, treated, including prophylactic treatment, with a composition of the invention. For the treatment of an infection, disorder or disease state directed to a particular animal, e.g., a human, the term patient refers to that particular animal. Preferably, in a main aspect of the invention, the patient is a human.

The term "effective amount" as used in the specification refers to the concentration or amount of a compound of the invention that produces the desired effect, which effect as used herein and in the context of the present application generally refers to a favorable change in the disease or condition being treated (whether the change is a reduction in the growth or size of the cancer or tumor), a favorable physiological effect, and a reduction or elimination in the growth of the tumor, cancerous tissue, etc., depending on the disease or condition being treated.

The term "neoplasia" as used in the specification refers to the pathological process of formation and growth of a tumor, i.e., abnormal tissue that grows faster than normal tissue by cell proliferation and continues to grow after the appearance of a stimulus that causes new growth to cease. Neoplasia may be a distinct tissue mass, which may be benign (benign tumor) or malignant (carcinoma). The term neoplasia as used herein refers to all of the various cancerous diseases, including or involving pathological processes associated with malignant hematopoiesis, ascites, and solid tumors. The term "cancer" and the term "tumor" as used in this application are used interchangeably with the term "neoplasia".

Cancers that may be treated using the compositions of the present invention include, for example, stomach, colon, rectal, liver, pancreatic, lung, breast, cervical, uterine corpus, ovarian, prostate, testicular, bladder, renal (renal), brain/CNS, head and neck, throat, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, melanoma, acute lymphocytic leukemia, acute myelogenous leukemia, Ewing's sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, wilms' tumor, neuroblastoma, hairy cell leukemia, mouth/pharynx, esophageal, laryngeal, renal (kidney), lymphoma, and the like. In a preferred embodiment of the invention, the tumor treatment comprises administering to the patient an anti-tumor effective amount of one or more of these agents.

The term "alkyl" as used in the specification refers to a fully saturated hydrocarbon group containing 1 to 7 carbon units. The alkyl group used in the present invention includes a linear, branched or cyclic group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, cyclohexyl, methylcyclopropyl and methylcyclohexyl.

The term "salt" as used in the specification refers to any salt that is compatible with the use of the compounds of the present invention. Where the compounds are used for pharmaceutical indications including the treatment of cancer, the term "salt" may denote a pharmaceutically acceptable salt which is compatible with the use of the compound as a medicament.

The term "Glu" or "Glu", as used in the formulae herein, refers to a glutamic acid residue or derivative which can be bound at its amino group to another carboxyl group (e.g., RCOOH) or at one of its carboxyl groups to a hydroxyl or amino group. Glutamic acid (HOOCCH (NH)₂)CH₂CH₂COOH) has two reactive carboxylic acid groups and one reactive amino group, which can be used to form glutamyl groups in the compounds of the invention. As shown in the chemical structure, glutamic acid may be bonded to other amino group (RNH) at one of its two carboxyl groups₂) Or hydroxy groups (ROH) to form the corresponding α -glutamyl or γ -glutamine or ester compounds. Alternatively, glutamate can form an N-glutamine derivative with a carboxyl group at its amino group. Single glutamyl residues, as well as polyglutamyl polypeptide residues (which are polypeptides formed from two or more glutamate amino acids) may be present in the compounds of the invention. The term OCO (Glu) denotes a glutamyl residue (alpha-or gamma-) which combines with a free hydroxyl group to form an alpha-or gamma-carboxylic acid (preferably alpha of glutamic acid)-carboxylic acid groups). The term nhco (glu) denotes a glutamyl residue (α -or γ -) which binds to a free amino group to form an amide with an α -or γ -carboxylic acid group, preferably the α -carboxylic acid group of glutamic acid. The term "glutamyl" refers to glutamate amino acids that are derivatized as esters or amides. Alpha-glutamyl refers to glutamate derivatives formed on the alpha-carboxyl group of glutamic acid (ester or amide). Gamma-glutamyl refers to the glutamate derivative formed on the gamma-carboxyl group of glutamic acid (ester or amide). N-glutamyl refers to the glutamate derivative formed on the amino group of glutamic acid (amide). A polyglutamic polypeptide residue refers to a polypeptide residue that contains more than one glutamic acid and preferably only glutamic acid.

The present invention relates to compounds according to structure (I):

wherein R is-CH₃or-CH₂CH₂Cl；

R' is C₁-C₇Alkyl or-CH₂CH₂Cl；

R₂Or R₄One but not all selected from OPO₃H₂、NO₂OCO (Glu), NHCO (Glu) and NHR₇And another unspecified R₂Or R₄And R₃、R₅And R₆Independently selected from H, F, Cl, Br, I, OH, OPO₃H₂、OCH₃、CF₃、OCF₃、NO₂、CN、SO₂CH₃、SO₂CF₃、COCH₃、COOCH₃、SCH₃、SF₅、NHR₈、N(R₉)₂、OPO₃H₂And C₁-C₇Alkyl with the proviso that R₂、R₃、R₄、R₅And R₆ToTwo less are H;

R₇is H, glutamyl, preferably alpha-glutamyl (-COCH (NH)₂)CH₂CH₂CO₂H) Or a polyglutamic polypeptide residue-COCH (NHR)_7a)CH₂CH₂CO₂H wherein R_7aIs a glutamyl (preferably alpha-glutamyl) or polyglutamic polypeptide residue having 1-50 peptide bonds, preferably having 2-10 peptide bonds;

R₈is H or C₁-C₇An alkyl group; and

R₉is CH₃Or CH₂CH₃. Phosphoric acid and glutamic acid (glutamyl) can be in the free acid form or pharmaceutically acceptable salts thereof.

The compounds of the present invention represent prodrug forms of certain intermediates, as shown in FIGS. 1-4, which are believed to exhibit their activity via chloroethylation, methylation, and/or carbamylation mechanisms. The rationale for the design of this new prodrug is that an enzyme-activated prodrug can be converted to the active alkylating group 1 and methyl isocyanate through a series of enzyme activations and rapid cleavage. For compound I, dephosphorylation is accomplished by AP enzyme activation to give intermediate 2 or 3, followed by benzyl cleavage to generate alkylated and carbamylated species, as shown in figure 2. For compound II, as shown in fig. 3, the dephosphorylation by AP activation and the reduction by NR activation can give intermediates 6 or 7, which can generate alkylated and carbamylated species by rapid cleavage. For compounds III and IV, as shown in fig. 4, cleavage of the compound can be catalyzed by CPG2 or CPA enzymes to yield the appropriately substituted phenol 10 and aromatic amine 11, which are then rapidly cleaved to yield alkylated and carbamylated species.

While not wishing to be bound by any theory, it is theorized that the rate-controlling step in the prodrug activation process may be a P-O bond cleavage step, which is catalyzed by the AP enzyme. The subsequent breaking step is usually faster. This may be due to the fact that the phosphate linked prodrug has a longer circulating half-life, so that it acts as a reservoir for the active alkylating group; or the prodrug may have desirable properties due to its different distribution from VNP 40101M. One way to achieve this is to slow down the dephosphorylation step, the rate limiting step, in the process of bioactivation of phosphate-bearing SHPs by introducing bulky substituents in the alpha position of the phosphate group. These alkyl groups are sterically hindered by close proximity to the P — O bond cleavage site, thereby slowing enzymatic dephosphorylation. Another approach is to introduce electron-releasing or electron-withdrawing groups on the phenyl ring that can affect the rate of cleavage of the P-O bond. In addition, substitution of the benzene ring with electron-releasing or electron-withdrawing groups may also affect the subsequent cleavage step. Based on these considerations, various phosphate-carrying SHPs can be conveniently synthesized in large quantities and evaluated. The disodium salts of these prodrugs are very soluble in water.

The compounds according to the invention are useful primarily because of their anti-tumor activity, including anti-solid tumor activity. In addition, these compositions are useful as intermediates in the chemical synthesis of other useful antineoplastic agents, which are useful as therapeutic agents or for other purposes.

In a preferred compound I of the invention, R is-CH₂CH₂Cl; r' is-CH₃；R₂Are phosphate groups which may be in the free acid or salt (preferably Na) form. In a particularly preferred aspect of the SHPs carrying ortho-phosphates, when R₃、R₅And R₆When is H, R₄Is Cl, F or Br (preferably Cl). In other preferred aspects of the SHPs carrying ortho-phosphates, when R₃、R₄And R₆When is H, R₅Is Cl, F or Br (preferably Cl, F). In other preferred aspects of the SHPs carrying ortho-phosphates, R₃、R₄、R₅And R₆Two of which are selected from F, Cl, Br or I (preferably the two substituents are the same and more preferably both are Cl), R₃、R₄、R₅And R₆The other two of (a) are H.

In the preferred compounds II of the invention, they are carriersAnalogs of SHPs with meta-phosphate and nitro groups. The phosphate group may be a free acid or salt (preferably sodium). In a particularly preferred aspect of SHPs containing nitro groups, when R is₄When is H, R₂Is NO₂. In another preferred aspect of SHPs containing nitro groups, when R is₂When is H, R₄Is NO₂。

In preferred compounds III and IV of the invention, they are conjugated analogs of the glutamyl residues of SHPs. In a particularly preferred aspect of both SHPs, the terminal acid may be a free acid or a salt (preferably Na). In still another preferred aspect of Compound IV, R₇May be H or polyglutamyl.

The compounds of the present invention can be synthesized and derived from 90CE by methods well known in the art. The synthesis of 90CE is shown in FIG. 5 (see Sartorelli et al, U.S. Pat. No. 4,684,747, 1987, relevant portions incorporated herein by reference).

As shown in fig. 6, compound I2-aminocarbonyl-1, 2-bis (methylsulfonyl) -1- (substituted) hydrazine (19 and 20, R ═ CH) was synthesized by reacting 90CE with phosgene or its equivalent, e.g., triphosgene or trichloromethyl chloroformate, respectively₂CH₂Cl) (see Majer et al.J Org Chem.1994, 59: 1937; and Pridden et al.J Org Chem.1989, 54: 3231) and then further reacted with a suitable N-alkyl-N-benzylamine (15 or 16, wherein R' is-CH)₃；R₂Or R₄Is a phosphate group, such as diethylphosphonoxy; and R₂、R₃、R₄、R₅And R₆Each of the unspecified groups in (a) is independently the structure shown or a related alkyl group, with the proviso that when the group R is not specified₂、R₃、R₄、R₅Or R₆When any two of them are other than H, the other two unspecified radicals R₂、R₃、R₄、R₅Or R₆Is H) in situ condensation. The coupling reaction can be carried out in high yield under the following conditions: using N, N-Diisopropylethylamine (DIEA) as a base, maintaining the reaction at 0 deg.C, dissolving in anhydrous acetonitrile-dichloromethaneThe formulation was overnight. Deprotection with trimethylsilylbromide (TMSBr) at 17 or 18 (Matulic-Adamic et al).J Org Chem.1995, 60: 2563) thereafter, the phosphate free acid form 19 or 20 was treated with saturated sodium bicarbonate (NaHCO)₃) Solution treatment gave the corresponding disodium salt 21 or 22, respectively. The above water-soluble SHPs (19-22) can be purified by reverse phase column chromatography.

As shown in FIG. 7, N-benzyl-N-methylamines (15 and 16) can be prepared from the corresponding salicylaldehyde, salicylic acid, or 4-substituted phenol. Commercially available salicylaldehyde was reacted with diethyl chlorophosphate under mild conditions to give its corresponding diethylphosphonoxy-benzaldehyde (23 or 24). The corresponding 23 or 24 was reductively aminated with methylamine using sodium borocyanide as a reducing agent to give the corresponding N-benzylmethylamine (15 or 16). Commercially available salicylic acid was first reduced to the corresponding salicyl alcohol (25) using Lithium Aluminium Hydride (LAH) as reducing agent at reflux. Selective phosphorylation of phenol 25 was achieved using diethyl phosphite, carbon tetrachloride, DIEA and a catalytic amount of 4-Dimethylaminopyridine (DMAP) (Silverberg et al.Tetrahedron Lett.1996, 37: 771) to give benzyl alcohol (26). Pyridine chlorochromate (PCC) -oxidation reaction was used (Kasmai et al.J Org Chem.1995, 60: 2267) the benzyl alcohol (26) is converted to benzaldehyde (24). Under the dalfof formylation conditions, Hexamethylenetetramine (HMTA) was used in refluxing trifluoroacetic acid (TFA) (Lindoy et al,Synthesis1998, 1029) from a commercially available 4-substituted phenol to give the corresponding salicylaldehyde (27 or 28) which is then converted to 24 and 16 by phosphorylation and reductive amination steps similar to those described above. The synthesis of suitable N-alkyl-N-benzylamine derivatives for use in the above reaction schemes is well known in the art and standard chemical methods may also be employed.

As shown in FIG. 8, the synthesis of compounds II containing SHPs containing nitro groups can be carried out by similar methods. Reaction with diethyl chlorophosphate under mild conditions using commercially available nitrobenzaldehyde as starting material gave its corresponding diethylphosphonoxy-benzaldehyde (29 or 30). Using sodium borocyanide as a reducing agent, corresponding 29 or 30 formazanReductive amination of the amine gives the corresponding N-benzylmethylamine (31 or 32) containing nitro groups. The use of phosgene or its equivalent as the carbonyl coupling agent and DIEA as the base allows the completion of the 31 or 32 coupling reaction using 90CE to be achieved in high yield. After deprotection of 33 or 34TMSBr, phosphate free acid form 35 or 36 is reacted with saturated NaHCO₃The solution was reacted to give the corresponding disodium salt 37 or 38, respectively. The above water-soluble compound II (35-38) can be purified by reverse phase column chromatography.

The preparation of glutamic acid substituted phenols of compound III is outlined in figure 9. Reacting di-tert-butyl glutamate (39) with phosgene or its equivalent, and then condensing with 4-hydroxy-benzaldehyde in situ at 0 ℃ overnight to obtain benzaldehyde 40 carrying glutamate with high yield. 40 to obtain the secondary amine 41 in a good yield after reductive amination. Compound 41 was reacted with phosgene at 0 ℃ followed by in situ condensation with 90CE to give 42. According to the prior art method (Mann et al.Tetrahedron1990, 46: 5377) deprotection of 42 to give the free acid 43 is conveniently achieved by treatment with formic acid. 43 was further treated with saturated NaHCO₃The corresponding water soluble glutamate 44, such as the disodium salt, triethanolamine salt, triethylamine salt, or lutidine salt, can be obtained from the solution or suitable amine treatment.

The synthesis of glutamyl substituted aromatic amino analogues (compound IV) is depicted in figure 10. According to the relevant literature methods (Jones et al.Bio-org Med Chem Lett.2000, 10: 1987) commercially available N-Boc-glutamic acid 5-tert-butyl ester (45) was reacted with 4-aminobenzyl alcohol under mild conditions using 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 1-Hydroxybenzotriazole (HOBT) as promoters to give amide 46 in high yield. As an alternative to the preparation of N-benzyl-N-methylamine by PCC-oxidation and reductive amination, the disclosed one-pot conversion of benzyl alcohol 46 to secondary amine 47 using magnesium dioxide in sodium borocyanide (Kanno et al) can be used.Tetrahedron Lett.2002, 43: 7337) the following reaction is carried out. Amine 47 was reacted with phosgene at 0 ℃ and condensed in situ with 90CE using DIEA as a promoter to give 48. The 48 can be conveniently removed by treatment with dilute hydrochloric acidProtection to obtain free acid 49. Finally, saturated NaHCO was further used₃The solution is processed to form a pharmaceutically acceptable salt, such as sodium salt 50.

After synthesis, the crude product is usually purified by reverse phase column chromatography and lyophilization. The above syntheses demonstrate that the SHPs of the invention can be conveniently converted to their corresponding phosphates. The synthetic methods disclosed above may be readily modified by one of ordinary skill in the art in order to obtain alternative synthetic routes for the compounds of the present invention.

Pharmaceutical compositions based on the novel compounds of the present invention contain an amount of the above-described compounds effective to treat a condition or disease such as cancer, optionally in combination with pharmaceutically acceptable additives, carriers or excipients.

Certain pharmaceutical dosage forms of certain compounds 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. The term "pharmaceutically acceptable salt or complex" as used herein refers to suitable salts or complexes of the active compounds of the present invention which retain the desired physiological activity of the parent compound. Non-limiting examples of such salts include the sodium and potassium salts of phosphoric acid and glutamic acid, others such as the triethanolamine, triethylamine, lutidine, or other pharmaceutically acceptable salts known in the art. Modification of the active compound can affect the solubility, pharmacokinetic parameters and metabolic rate of the active compound, thereby achieving control over the delivery of the active compound. In addition, the modification may affect the anti-cancer activity of the compound, and in some cases may increase the activity of the parent compound. Such modifications can be readily assessed by preparing derivatives according to methods known to those of ordinary skill in the art and then testing for anti-cancer activity.

The compounds of the present invention may be incorporated into formulations suitable for various routes of administration, including, for example, oral and parenteral administration, including intravenous, intramuscular, intraperitoneal, buccal, transdermal and suppository forms. Parenteral administration is preferred, in particular intravenous or intramuscular administration.

Pharmaceutical compositions based on these novel compounds comprise the above-mentioned compounds in an amount effective for the treatment of cancer as well as other diseases and conditions already described above, optionally in combination with pharmaceutically acceptable additives, carriers and/or excipients. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount of a compound of the invention will depend on the infection or disorder being treated, the severity, the treatment regimen being used, the pharmacokinetics of the agent being used, and the patient (animal or human) being treated.

In the pharmaceutical aspect of the present invention, the compounds of the present invention are preferably formulated in admixture with a pharmaceutically acceptable carrier. In general, the pharmaceutical compositions are preferably administered parenterally, particularly in intravenous or intramuscular dosage forms, but many formulations may be administered by other parenteral routes, such as transdermal, buccal, subcutaneous, suppository or other routes, including oral administration. Intravenous and intramuscular formulations are preferably administered in the form of sterile saline. Of course, one of ordinary skill in the art, given the teachings of this specification, can modify these formulations to obtain different formulations appropriate for a particular route of administration, provided that the compositions of the invention do not become unstable or otherwise interfere with their therapeutic activity. In particular, modification of the compounds of the present invention to render them more soluble in water or other vehicles may be conveniently accomplished by slight modifications (e.g., salt formation, etc.) known to those of ordinary skill in the art. It is also well known to those skilled in the art that modifications may be made to the route and regimen of administration of a particular compound 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 take full advantage of the favorable pharmacokinetic parameters of the prodrug forms of the invention to maximize the desired effect of the compounds when delivering the compounds of the invention to a target site in a host organism or patient.

The active compounds of the present invention may be administered continuously (i.v. instillation), including bolus, intravenous or intramuscular administration, once to several times daily administration, and may also include topical, parenteral, intravenous, intramuscular, subcutaneous, transdermal (which may include a permeation enhancer), buccal and suppository administration, and in some cases, other routes of administration include 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 preferably intimately admixed with a pharmaceutically acceptable carrier to prepare a dosage unit using conventional pharmaceutical compounding techniques. Various types of carriers can be selected depending on the desired form of administration (e.g., intravenous or intramuscular) preparation. In preparing pharmaceutical compositions in suitable dosage form, any of the usual pharmaceutical media may be employed. For parenteral formulations, the carrier may comprise sterile water or aqueous sodium chloride solution, together with other ingredients to aid dispersion, such as ethanol and other pharmaceutically acceptable solvents, including DMSO and the like. Of course, even if the solution is used and stored under sterile conditions, the composition and the carrier must be sterilized. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed.

Solutions or suspensions for parenteral, intradermal, subcutaneous, or topical use may include the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, 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. The parenteral formulations may be presented in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Preferred carriers, if used for intravenous administration, include, for example, physiological saline or Phosphate Buffered Saline (PBS).

In preparing pharmaceutical compositions in oral dosage form, any one or more of the usual pharmaceutical media may be employed. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives can be employed including water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like. For solid oral formulations such as powders, tablets, capsules, and solid formulations such as suppositories, suitable carriers and additives may be used including starches, sugar carriers such as dextrose, mannitol, lactose and related carriers, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. Tablets or capsules may be provided with enteric coatings or sustained release formulations, if desired, by conventional techniques.

In one embodiment, the active compound is prepared with a carrier that prevents rapid elimination of the compound from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers may be used, such as ethylene vinyl acetate, 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 pharmaceutically acceptable carriers. They can be prepared according to methods known to the person skilled in the art. For example, a liposomal formulation 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. The active compound is then introduced into the container. The liposome suspension is then formed by manually rotating the container to cause the lipid material to peel from the container walls thereby dispersing the lipid aggregates. Other methods of preparation known to those of ordinary skill in the art may also be used in this aspect of the invention.

The compounds of the invention are useful for treating animal patients, particularly mammals including humans. Thus, humans, horses, dogs, cattle and other animals, particularly mammals, suffering from tumors, particularly cancer, or other diseases described herein may be treated by administering to the patient an effective amount of one or more compounds of the present invention or derivatives or pharmaceutically acceptable salts thereof, optionally in admixture with a pharmaceutically acceptable carrier or diluent, either alone or in combination with other known agents, depending on the disease being treated. The above treatment may also be combined with other conventional cancer therapies, such as radiation therapy or surgery. In a preferred aspect, the compositions of the invention are also useful for treating tumors or cancers that are resistant, particularly those that are resistant to traditional cancer drugs.

The amount of active compound contained in a pharmaceutically acceptable carrier or diluent should be sufficient to deliver to the patient a therapeutically effective amount of the drug as required for the intended indication, while not causing serious toxic effects in the treated patient.

The compounds of the present invention are prodrug forms of active intermediates. In certain pharmaceutical dosage forms, the compounds of the present invention may be modified into other prodrug forms to suit the particular route of administration of the active compound. One of ordinary skill in the art will understand how to readily modify the compounds of the present invention into additional prodrug forms to facilitate delivery of the active compound to a target site within a patient. One of ordinary skill in the art can also take full advantage of the favorable pharmacokinetic parameters of the prodrug form when delivering the compounds of the invention to a target site in a patient, thereby maximizing the anti-neoplastic effect of the compound.

The therapeutically active formulation of the present invention comprises the compound in an amount effective to treat cancer. Generally, a therapeutically effective amount of a compound of the invention in a dosage form will generally be from less than about 0.05mg/kg to about 500mg/kg of the body weight of the patient being treated, or more specifically, will depend upon the compound used, the type of tumor being treated, the ability of the active compound to localize in the tissue being treated, the route of administration, and the pharmacokinetics of the compound in the patient. In the case of treating cancer, the compound is preferably administered at a single dose of about 0.05mg/kg to about 250mg/kg or more. The above dosage ranges generally produce effective blood levels of about 0.01 to about 500 micrograms of active compound per milliliter of blood of the patient being treated. The duration of treatment may be one or more days, or for months or even longer (years), depending on the disease state being treated. In a more preferred embodiment, the compound is administered to the patient at a dose of 0.1mg/kg to 100mg/kg, twice daily or once every 14 days for a period of 1 week to 52 weeks.

The concentration of the active compound in the patient depends on the absorption, distribution, inactivation, and excretion rate of the drug, as well as other factors known to those skilled in the art. It should be noted that the dosage at which the patient takes the drug should also vary with the degree of remission. It is further understood that the specific dosage regimen should be adjusted at any time for any particular patient, according to the individual needs and the professional judgment of the professional in charge of or supervising the administration of the composition, and that the concentration ranges set forth herein are exemplary only and are not meant to limit the scope or application of the claimed composition. The active ingredient may be administered immediately or in multiple smaller doses taken at different time intervals.

The active compounds of the present invention may also be mixed with other active materials which do not affect the desired activity, or with materials which enhance the desired effect, such as other anti-cancer agents, and in some cases antibiotics, antifungal agents, anti-inflammatory agents, or antiviral compounds, etc., which are selected according to the desired therapy or target.

The compounds of the present invention may be administered alone or in combination with other agents, especially other compounds encompassed by the present invention. In this aspect of the invention, an effective amount of one or more compounds of the invention are administered in combination with an effective amount of at least one other anti-tumor/anticancer agent, such as an antimetabolite, cytarabine, etoposide, doxorubicin, paclitaxel, hydroxyurea, vincristine, cyclophosphamide, or mitomycin C, others including topoisomerase I and topoisomerase II inhibitors, such as doxorubicin, topotecan, camptothecin, and irisoneTikon, other agents such as gemcitabine and camptothecin and cisplatin based agents. Theoretically, a compound of the invention that acts by disrupting a DNA mechanism may have a synergistic effect with a compound that acts by reducing or hindering a DNA repair mechanism. Thus, the compounds of the present invention may be advantageously used in combination with any compound that acts by reducing or hindering DNA repair mechanisms, including, inter alia, enzyme-catalyzed DNA repair inhibitors, such as Ribonucleotide Reductase (RR) inhibitors and O⁶-alkylguanine-DNA Alkyltransferase (AGT) inhibitors. By "co-administered" is meant that the compound of the invention and the combined compounds are both detectable in the bloodstream of the patient at the same time, regardless of when the compounds are administered (including at the same time), after the patient has been administered the compound of the invention. In many cases, it is surprising that the administration of the compounds of the present invention in combination with conventional anti-cancer agents results in a synergistic effect (i.e., greater than a simple addition). In another embodiment, the compounds of the invention are administered simultaneously or sequentially with each antibody (conjugated or non-conjugated), virus, or bacterium. The antibody, virus, or bacterium may carry an enzyme or a gene-encoded enzyme that activates the compound of the present invention. These enzymes are not limited to NR, CPG2 and CPA.

In another aspect of the invention, the compositions of the invention may be used to treat tumors and/or cancers that are resistant to one or more conventional anti-tumor/anti-cancer agents, such as those already described above. In this aspect of the invention, an effective amount of the composition is administered to a patient having a drug-resistant tumor or cancer to treat the tumor or cancer. In this aspect of the invention, the compositions of the invention may be administered alone or in combination with other effective anti-tumor/anti-cancer agents.

Without wishing to be bound by any theory, it is believed that the compounds of the present invention, by having the combined function of a chloroethylating agent and a carbamylating agent, mainly induce a therapeutic effect for the treatment of malignant tumors.

After the invention has been summarized, preferred embodiments of the invention are further described below, along with other embodiments and comparative examples, with reference to various specific examples. The included embodiments are not to be construed as limiting the scope of the invention, which is defined in the foregoing and the appended claims as broadly as possible. Other compounds not specifically mentioned in the examples section of this application can be conveniently synthesized according to analogous methods and/or alternative synthetic means known and existing in the art. All of the defined and described compounds can be readily synthesized by one of ordinary skill in the art by simply employing these particular synthetic methods directly or by making variations/modifications to these synthetic methods using techniques well known in the art, without undue experimentation.

Examples

All reagents were purchased on a commercial scale and used without further purification, if necessary, the solvent was dried and/or distilled prior to use. All NMR spectra (¹H，¹³C and³¹p) were measured on a bruker ac300 spectrometer. Chemical shifts are measured in parts per million (ppm) relative to tetramethylsilane. Coupling constants are expressed in hertz (Hz). Flash Column Chromatography (FCC) was performed using Merck silica gel 60(230- & 400 mesh), Reverse Phase Column Chromatography (RPCC) was performed using CAT gel (Water, preparative C-18,55-105 μm) and eluted with milli-Q deionized water. Electrospray mass (ESMS) analysis was performed by Keck Laboratory of Yale university on Q-Tof manufactured by Micromass (Manchester, UK) with mass precision of < 0.02%.

Examples 1 to 3

Preparation of salicylaldehyde (27a, 27b, 28b) by formylation of 4-substituted phenol (Daffy formylation)

A general method. To a solution of the appropriately substituted phenol (10.0g) in TFA (100mL) was added a small amount of HMTA (1.1 equiv.). The reaction solution was heated at reflux overnight. After cooling, the solution was taken up in 50% H₂SO₄The solution (40mL) was treated at room temperature for 4h and then extracted with ether (3X 100 mL). The combined ether phases were washed with 5M HCl solution and water and then with anhydrous MgSO₄And (5) drying. After filtration, the filtrate was evaporated and purified.

2-hydroxy-5-trifluoromethyl-benzaldehyde (27 a). According to the general procedure, FCC purification was performed with 30% ethyl acetate-hexane to give 2-hydroxy-5-trifluoromethyl-benzaldehyde 27(3.9g, 34%) as a pink solid from 4-trifluoromethylphenol (10.0g, 61.7 mmol).

Rf (20% ethyl acetate-hexane): 0.47.

¹H NMR(300MHz，CDCl₃) δ 11.31(s, 1H, OH), 9.96(s, 1H, CHO), 7.87(d, J ═ 1.6Hz, 1H, C6-H (ph)), 7.76(dd, J ═ 2.0 and 8.5Hz, 1H, C4-H (ph)), and 7.11(d, J ═ 8.8Hz, 1H, C3-H (ph)).

¹³C NMR(75MHz，CDCl₃) δ 195.8, 163.8, 133.4(d), 131.0(d), 125.3, 122.1(m), 119.8 and 118.6.

4, 5-dichloro-2-hydroxy-benzaldehyde (27b) and 5, 6-dichloro-2-hydroxy-benzaldehyde (28). According to the general procedure, FCC purification using 5-10% ethyl acetate-hexane afforded 5, 6-dichloro-2-hydroxy-benzaldehyde 28(2.2g, 19%) as a pale yellow solid from 3, 4-dichlorophenol (10.0g, 61.1 mmol).

Rf (40% ethyl acetate-hexane): 0.63.

¹H NMR(300MHz，CDCl₃) δ 11.98(s, 1H, OH), 10.44(s, 1H, CHO), 7.55(d, J ═ 9.4Hz, 1H, C4-H (ph)) and 6.89(d, J ═ 9.3Hz, 1H, C3-H (ph)).

¹³C NMR(75MHz，CDCl₃) δ 195.4, 162.4, 137.8(2C), 135.6, 123.8 and 118.1。

According to the same reaction, FCC purification was successively performed with 10% ethyl acetate-hexane to obtain 4, 5-dichloro-2-hydroxy-benzaldehyde 27b (1.8g, 15%) as a pale yellow solid.

Rf (40% ethyl acetate-hexane): 0.47.

¹H NMR(300MHz，CDCl₃) Δ 10.97(s, 1H, OH), 9.84(s, 1H, CHO), 7.64(s, 1H, C3-H (Ph)) and 7.15(s, 1H, C6-H (Ph)).

¹³C NMR(75MHz，CDCl₃) δ 194.7, 160.0, 149.9, 141.5, 134.0, 123.6 and 119.9.

Examples 4 to 6

Preparation of Diethylphosphonyloxy-benzaldehyde from phenol Formaldehyde (23, 24a-b)

A general method. To a stirred ice-cold solution of the appropriately substituted aldehyde (10.0g) in acetonitrile (120mL) was added diethyl chlorophosphate (1.1 equiv.) and TEA (1.1 equiv.). The reaction mixture was left at room temperature overnight. After removing the precipitate by filtration, the filtrate was evaporated and dried. The crude diethylphosphonoxy-benzaldehyde can be used as such without further purification.

4-diethylphosphonoxy-benzaldehyde (23). According to the general procedure 4-hydroxybenzaldehyde (9.0g, 73.8mmol) was converted to 4-diethylphosphonoxy-benzaldehyde 23(18.9g, 99%) which was isolated as a pale yellow oil.

¹H NMR(300MHz，CDCl₃)δ9.98(s，1H，CHO)，7.91(d，J＝9.0Hz，2H，C3-H(Ph))，7.39(d，J＝8.4Hz，2H，C2-H(Ph))，4.26(m，4H，CH₂) And 1.38(t, J ═ 6.9Hz, 6H, CH₃)。

¹³C NMR(75MHz，CDCl₃) δ 190.5, 155.2(d), 133.0, 131.4, 120.3(d), 64.7(d) and 15.8 (d).

³¹P NMR(121MHz，CDCl₃)δ4.6.

TOF ESMS calculated for (M + H) 259.07, observed 259.10.

2-diethylphosphonoxy-benzaldehyde (24 a). Following the general procedure, salicylaldehyde (9.0g, 73.8mmol) gave 24a (18.9g, 99%) as a colorless oil.

¹H NMR(300MHz，CDCl₃)δ10.42(s，1H，CHO)，7.90(d，J＝7.7Hz，1H，C3-H(Ph))，7.61(t，J＝8.0Hz，1H，C5-H(Ph))，7.48(d，J＝8.7Hz，1H，C6-H(Ph))，7.31(t，J＝7.1Hz，1H，C4-H(Ph))，4.27(m，4H，CH₂) And 1.37(t, J ═ 7.1Hz, 6H, CH₃)。

¹³C NMR(75MHz，CDCl₃) δ 188.2, 152.5(d), 135.5, 128.5, 127.1(d), 125.2, 120.9(d), 64.9(d) and 15.8 (d).

³¹P NMR(121MHz，CDCl₃)δ5.0。

TOF ESMS calculated for (M + H) 259.07, observed 259.07.

5-chloro-2-diethylphosphonoxy-benzaldehyde (24 b). Following the general procedure, 5-chlorosalicylaldehyde (10.0g, 73.8mmol) gave 24b (19.4g, 90%) as a colorless oil.

¹H NMR(300MHz，CDCl₃) δ 10.34(s, 1H, CHO), 7.84(d, J ═ 2.5Hz, 1H, C3-H (ph)), 7.55(dd, J ═ 8.8 and 2.5Hz, 1H, C5-H (ph)), 7.45(d, J ═ 8.8Hz, 1H, C6-H (ph)), 4.27(m, 4H, CH 6-H (ph)), and combinations thereof₂) And 1.38(t, J ═ 6.8Hz, 6H, CH₃)。

¹³C NMR(75MHz，CDCl₃) δ 187.0, 151.1(d), 135.2, 131.2, 128.2(d), 128.1, 122.6(d), 65.2(d) and 16.0 (d).

³¹P NMR(121MHz，CDCl₃)δ5.1。

TOF ESMS calculated for (M + H) 293.03, observed 293.04.

Examples 7 to 9

Preparation of 4-chloro-2-diethylphosphonoxy-benzaldehyde (24c) from 4-chlorosalicylic acid

5-chloro-2-hydroxymethyl-phenol (25). A solution of 4-chlorosalicylic acid (10.0g, 58.0mmol) in THF (150mL) was treated with LAH (1.5 equiv.) at reflux for 2 hours. After cooling to ambient temperature, the reaction solution was quenched with 1N NaHSO₄The solution (200mL) was quenched and then extracted with ether (300 mL). After separation, the organic layer was separated with anhydrous MgSO₄Dried, filtered and concentrated. The crude product 25 was obtained as a grey solid (7.5g, 81%) which was pure enough to be used without further purification.

¹H NMR (300MHz, DMSO-d6) δ 9.85(s, 1H, Ph-OH), 7.27(d, J ═ 8.2Hz, 1H, C3-H (Ph)), 6.81(d, J ═ 8.2Hz, 1H, C4-H (Ph)), 6.78(s, 1H, C6-H (Ph)), 5.03(m, 1H, OH) and 4.41(d, J ═ 4.1Hz, 2H, PhCH)₂)。

¹³C NMR (75MHz, DMSO-d6) delta 155.0, 131.0, 128.6, 128.0, 118.5, 114.2 and 57.7.

4-chloro-2-diethylphosphonoxy-benzyl alcohol (26). A solution of 25(8.6g, 54.8mmol), DIEA (2.1 equiv.), and DMAP (0.1 equiv.) in acetonitrile (200mL) was placed in a-20 ℃ bath. Adding CCl to the above cooled solution₄(5.0 equiv.) and diethyl phosphite (1.1 equiv.). The reaction solution was left at room temperature for 2 hours. The solvent was removed by rotary evaporation and the crude oil was purified by FCC, eluting with 60% ethyl acetate-hexanes to give 26 as a light yellow oil (10.9g, 68%).

Rf (80% ethyl acetate-hexane): 0.34.

¹H NMR(300MHz，CDCl₃)δ7.40(d，J＝8.2Hz，1H，C3-H(Ph))，7.23(s，1H，C6-H(Ph))，7.19(d，J＝8.5Hz，1H，C4-H(Ph))，4.62(s，2H，PhCH₂)，4.24(m，4H，CH₂) And 1.37(t, J ═ 7.4Hz, 6H, CH₃)。

¹³C NMR(75MHz，CDCl₃) δ 148.2(d), 133.6(d), 131.6(d), 131.0, 125.8, 120.9(d), 65.1(d), 59.1 and 15.9 (d).

³¹P NMR(121MHz，CDCl₃)δ6.0。

4-chloro-2-diethylphosphonoxy-benzaldehyde (24 c). To a stirred solution of 26(10.7g, 36.3mmol) in dichloromethane (600mL) was added a small amount of PCC over 30 minutes at room temperature. The reaction was monitored by TLC. The reaction mixture was then filtered through a celite filter pad and the filtrate was rotary evaporated. The residual oil was purified by pad of silica gel eluting with ethyl acetate to give 24d as a green oil (9.5g, 90%).

¹H NMR(300MHz，CDCl₃)δ10.34(s，1H，CHO)，7.84(s，1H，C3-H(Ph))，7.52(s，1H，C6-H(Ph))，7.29(s，1H，C4-H(Ph))，4.29(m，4H，CH₂) And 1.39(s, 6H, CH)₃)。

¹³C NMR(75MHz，CDCl₃) δ 186.8, 152.4(d), 140.9, 129.3, 125.4(d), 125.3, 121.1, 64.9(d) and 15.6 (d).

³¹P NMR(121MHz，CDCl₃)δ4.8。

TOF ESMS calculated for (M + H) 293.03, observed 293.06.

Examples 10 to 13

Preparation of N- (diethylphosphonooxybenzyl) -N-methylamine (15, 16a-c)

A general method. To a solution of the corresponding diethylphosphonoxy-benzaldehyde (23 or 24a-c, 10mmol) in dichloromethane (10mL) was added methylamine (2N in THF, 2.0 equiv.). The reaction solution was left at room temperature overnight, filtered through a pad of silica gel and the filtrate was rotary evaporated and dried in vacuo. The crude oil obtained is dissolved in methanol(50 mL). Adding a small amount of NaBH into the solution at 0 DEG C₄(2.0 equiv.) the solution was stirred for 4 hours. After evaporation, the residue was distributed between water (50mL) and dichloromethane (50 mL). The aqueous phase was separated and extracted once with dichloromethane (50 mL). The combined organic phases were dried over anhydrous MgSO₄Dried, filtered and evaporated. The crude N- (diethylphosphonooxybenzyl) -N-methylamine (15 or 16a-c) was pure enough to be used without further purification.

N- (4-diethylphosphonooxybenzyl) -N-methylamine (15). According to the general procedure, 23(29.9g, 116mmol) gave 15(22.3g, 71%) as a yellow oil.

¹H NMR(300MHz，CDCl₃)δ7.31(d，J＝8.0Hz，2H，C3-H(Ph))，7.17(d，J＝8.5Hz，2H，C2-H(Ph))，4.21(m，4H，CH₂)，3.73(s，2H，PhCH₂)，2.42(s，3H，NCH₃) And 1.34(t, J ═ 6.9Hz, 6H, CH₃)。

¹³C NMR(75MHz，CDCl₃) δ 149.4(d), 135.6, 129.3, 119.5(d), 64.2(d), 54.5, 35.1 and 15.7 (d).

³¹P NMR(121MHz，CDCl₃)δ5.3。

TOF ESMS calculated for (M + H) 274.11, observed 274.11.

N- (2-diethylphosphonooxybenzyl) -N-methylamine (16 a). According to the general procedure, 24a (19.0g, 73.6mmol) gave 16a (16.4g, 82%) as a pale yellow oil.

¹H NMR(300MHz，CDCl₃)δ7.37(d，J＝7.4Hz，1H，C3-H(Ph))，7.33(d，J＝7.9Hz，1H，C6-H(Ph))，7.24(t，J＝7.2Hz，1H，C5-H(Ph))，7.14(t，J＝7.3Hz，1H，C4-H(Ph))，4.22(m，4H，CH₂)，3.82(s，2H，PhCH₂)，2.44(s，3H，NCH₃) And 1.35(t, J ═ 7.0Hz, 6H, CH₃)。

¹³C NMR(75MHz，CDCl₃) δ 148.6(d), 130.5(d), 130.2, 128.1, 124.8, 119.7, 64.4(d), 49.9, 35.5 and 15.8 (d).

³¹P NMR(121MHz，CDCl₃)δ5.6。

TOF ESMS calculated for (M + H) 274.11, observed 274.13.

N- (4-chloro-2-diethylphosphonooxybenzyl) -N-methylamine (16 b). Following the general procedure, from 24b (21.9g, 74.9mmol) 16b was obtained as a pale yellow oil (18.3g, 80%).

¹H NMR(300MHz，CDCl₃) δ 7.39(d, J ═ 2.2Hz, 1H, C3-H (ph)), 7.27(d, J ═ 7.9Hz, 1H, C6-H (ph)), 7.20(dd, J ═ 8.3 and 2.6Hz, 1H, C5-H (ph)), 4.23(m, 4H, CH, ph ═ 8.3, ph) (ph)), and 4.23(m, 4H, CH, ph₂)，3.78(s，2H，PhCH₂)，2.45(s，3H，NCH₃) And 1.36(t, J ═ 7.0Hz, 6H, CH₃)。

¹³C NMR(75MHz，CDCl₃) δ 147.1(d), 132.8(d), 130.1(d), 129.7, 127.7, 121.1, 64.6(d), 49.6, 35.6 and 15.9 (d).

³¹P NMR(121MHz，CDCl₃)δ5.6。

TOF ESMS calculated for (M + H) 308.07, observed 308.08.

N- (5-chloro-2-diethylphosphonooxybenzyl) -N-methylamine (16 c). According to the general procedure, 24c (23.9g, 81.7mmol) gave 16c (18.9g, 76%) as a pale yellow oil.

¹H NMR(300MHz，CDCl₃)δ7.35(s，1H，C6-H(Ph))，7.30(d，J＝10.4Hz，1H，C3-H(Ph))，7.14(d，J＝8.4Hz，1H，C4-H(Ph))，4.24(m，4H，CH₂)，3.78(s，2H，PhCH₂)，2.43(s，3H，NCH₃) And 1.37(t, J ═ 6.8Hz, 6H, CH₃)。

¹³C NMR(75MHz，CDCl₃) δ 140.9(d), 133.0, 131.0, 129.4(d), 125.1, 120.3(d), 64.7(d), 49.4, 35.5 and 15.9 (d).

³¹P NMR(121MHz，CDCl₃)δ5.4。

TOF ESMS calculated for (M + H) 308.07, observed 308.08.

Examples 14 to 15

Preparation of free phosphoric acid (19, 20a-c)

A general method. To a cooled stirred solution of 90CE (10mmol) in acetonitrile (40mL) was added phosgene (20% in toluene, 1.0 eq.) and DIEA (1.0 eq.). The reaction solution was left at 0 ℃ for 20 minutes. To the above solution was then added the corresponding N- (diethylphosphonooxy-benzyl) -N-methylamine (15 or 16a-c, 10mmol) in dichloromethane (5mL) and DIEA (plus 1.0 equiv.). The resulting reaction solution was left at 5 ℃ overnight. After evaporation, the residue was distributed over water (80mL) and dichloromethane (80 mL). The aqueous phase was separated and extracted twice with dichloromethane (80 mL). The combined organic phases were dried over anhydrous MgSO₄Dried, filtered and evaporated. The crude protected phosphate (17 or 18a-c) was obtained as an oil.

A solution of the corresponding diethyl-protected phosphate (17 or 18a-c, 10mmol) in dichloromethane (60mL) was treated with excess TMSBr (40mL) at 5 ℃ overnight. Evaporation followed by vacuum drying gave the crude free phosphoric acid (19 or 20a-c) as a glassy solid.

To the crude compound (19 or 20a, 10mmol) was added water (approximately 30 mL). The suspension was stirred at ambient temperature for 2 hours, then a small amount of water was added to dissolve completely. The aqueous solution was purified by RPCC and eluted with deionized water. Fraction passing³¹p NMR was monitored and collected. After lyophilization, purified free phosphoric acid (19 or 20a) was obtained as a white powder.

1, 2-bis (methanesulfonyl) -1- (2-chloroethyl) -hydrazine (90 CE). According to the prior art (Shyam et al.J Med Chem.1987,30: 2157) reaction of 2-hydroxyethyl-hydrazine and methanesulfonyl chloride in the presence of pyridine base gave deferoxamine (mesylate), which was subsequently reacted with lithium chloride to give 90 CE. After purification by FCC, eluting with 5% methanol in dichloromethane, 90CE was obtained as a white solid.

Rf (50% ethyl acetate-hexane): 0.30.

¹H NMR(300MHz，CDCl₃)δ6.82(s，1H，NH)，3.99(t，J＝6.8Hz，2H，ClCH₂)，3.86(t，J＝5.6Hz，2H，NCH₂)，3.19(s，3H，SCH₃) And 3.13(s, 3H, SCH)₃)。

¹³C NMR(75MHz，CDCl₃) δ 54.0, 41.0, 40.2 and 38.4.

Phosphoric acid mono- {4- { N- [1, 2-bis (methanesulfonyl) -2- (2-chloroethyl) -hydrazinocarbonyl ] -N-methylaminomethyl } -phenyl } ester (19). Following general procedure, 4- { N- [1, 2-bis (methanesulfonyl) -2- (2-chloroethyl) -hydrazinocarbonyl ] -N-methylaminomethyl } -phenyl ester diethyl ester (17, 25.2g, 80%) phosphate was obtained from 15(15.4g, 56.8mmol) and 90CE (14.2g, 1.0 eq). Compound 17(11.0g, 20.1mmol) was converted to 19(3.8g, 38%) as a white powder.

¹H NMR(300MHz，D₂O)δ7.11(d，J＝8.1Hz，2H，C3-H(Ph))，6.95(d，J＝7.5Hz，2H，C2-H(Ph))，4.36(m，2H，PhCH₂)，3.89(m，2H，ClCH₂)，3.67(m，2H，NCH₂)，3.23(s，3H，NCH₃)，2.92(s，3H，SCH₃) And 2.87(s, 3H, SCH)₃)。

¹³C NMR(75MHz，D₂O) δ 156.6, 153.8(d), 133.4, 132.0, 123.0(d), 57.3, 55.7, 43.6, 42.0, 40.4 and 39.1.

³¹P NMR(121MHz，D₂O)δ9.9。

TOF ESMS calculated for (M + H) 494.01, observed 493.98.

Phosphoric acid mono- {2- { N- [1, 2-bis (methanesulfonyl) -2- (2-chloroethyl) -hydrazinocarbonyl ] -N-methylaminomethyl } -phenyl } ester (20 a). Following the general procedure, 2- { N- [1, 2-bis (methanesulfonyl) -2- (2-chloroethyl) -hydrazinocarbonyl ] -N-methylaminomethyl } -phenyl ester diethyl ester (18a, 29.7g, 89%) phosphate was obtained from 16a (16.4g, 60.5mmol) and 90CE (15.1g, 1.0 eq). Compound 18a (10.5g, 19.2mmol) gave 20a (2.6g, 27%) as a white powder.

¹H NMR(300MHz，D₂O) delta 7.1-7.2(m, 3H, C3-H, C5-H and C6-H (Ph)), 6.94(m, 1H, C4-H (Ph)), 4.45(m, 2H, PhCH₂)，3.84(m，2H，ClCH₂)，3.64(m，2H，NCH₂)，3.20(s，3H，NCH₃)，2.93(s，3H，SCH₃) And 2.92(s, 3H, SCH)₃)。

¹³C NMR(75MHz，D₂O) δ 156.8, 152.5(d), 131.8, 131.7, 128.7(d), 126.7, 122.5, 57.2, 51.4, 43.5, 42.1, 40.5 and 39.9.

³¹P NMR(121MHz，D₂O)δ9.8。

TOF ESMS calculated for (M + H) 494.01, observed 494.00.

Examples 16 to 19

Preparation of disodium salt (21, 22a-c)

A general method. The corresponding crude phosphoric acid (19 or 20a-c, 10mmol) was treated with saturated sodium bicarbonate (NaHCO)₃) Aqueous solution (100mL) was neutralized. The suspension was stirred at ambient temperature for 2 hours and then added to a small amount of water to homogenize. The aqueous solution was passed through RPCC with deionized water. Purification fraction by³¹PNMR was monitored and collected. After lyophilization, the corresponding disodium salt (21 or 22a-c) was obtained as a white powder.

Phosphoric acid 4- { N- [1, 2-bis (methanesulfonyl) -2- (2-chloroethyl) -hydrazinocarbonyl ] -N-methylaminomethyl } -phenyl ester disodium salt (21). According to the general procedure, 21(5.6g, 54%) was obtained as a white powder from crude product 19(9.5g, 19.3 mmol).

¹H NMR(300MHz，D₂O)δ7.08(d，J＝8.3Hz，2H，C3-H(Ph))，6.97(d，J＝8.1Hz，2H，C2-H(Ph))，4.38(m，2H，PhCH₂)，3.92(m，2H，ClCH₂)，3.71(m，2H，NCH₂)，3.26(s，3H，NCH₃)，2.95(s，3H，SCH₃) And 2.89(s, 3H, SCH)₃)。

¹³C NMR(75MHz，D₂O) δ 156.5, 156.1(d), 131.7, 131.4, 122.9(d), 57.4, 55.8, 43.7, 42.0, 40.5 and 39.0.

³¹P NMR(121MHz，D₂O)δ14.2。

TOF ESMS calculated for (M-H) 492.01, observed 492.10.

Phosphoric acid 2- { N- [1, 2-bis (methanesulfonyl) -2- (2-chloroethyl) -hydrazinocarbonyl ] -N-methylaminomethyl } -phenyl ester disodium salt (22 a). According to the general procedure, crude product 20a (8.8g, 17.8mmol) gave 22a (5.7g, 59%) as a white powder.

¹H NMR(300MHz，D₂O)δ7.21(d，J＝8.4Hz，1H，C3-H(Ph))，7.0-7.2(m，2H，C5-H and C6-H(Ph))，6.86(t，J＝7.2Hz，1H，C4-H(Ph))，4.52(m，2H，PhCH₂)，3.90(m，2H，ClCH₂)，3.68(m，2H，NCH₂)，3.27(s，3H，NCH₃)，2.97(s，3H，SCH₃) And 2.93(s, 3H, SCH)₃)。

¹³C NMR(75MHz，D₂O) δ 156.7, 154.6(d), 131.3, 130.9, 128.3(d), 124.7, 122.4, 57.3, 51.4, 43.6, 42.0, 40.5 and 39.9.

³¹P NMR(121MHz，D₂O)δ14.1。

TOF ESMS calculated for (M-H) 492.01, observed 492.05.

Phosphoric acid 2- { N- [1, 2-bis (methanesulfonyl) -2- (2-chloroethyl) -hydrazino-carbonyl ] -N-methylaminomethyl } -4-chloro-phenyl ester disodium salt (22 b). From crude product 20b (14.5g, 27.6mmol) 22b was obtained as a white powder (8.3g, 53%) according to the general procedure.

¹H NMR(300MHz，D₂O)δ7.26(s，1H，C3-H(Ph))，7.17(m，1H，C5-H(Ph))，7.08(d，J＝7.2Hz，1H，C6-H(Ph))，4.48(m，2H，PhCH₂)，3.93(m，2H，ClCH₂)，3.72(m，2H，NCH₂)，3.30(s，3H，NCH₃)，3.03(s，3H，SCH₃) And 3.01(s, 3H, SCH)₃)。

¹³C NMR(75MHz，D₂O) δ 156.8, 153.3(d), 134.6, 130.8, 128.9, 126.7, 123.6, 57.3, 51.3, 43.6, 42.1, 40.6 and 40.1.

³¹P NMR(121MHz，D₂O)δ14.2。

TOF ESMS calculated for (M-H) 525.96, observed 525.93.

2- { N- [1, 2-bis (methanesulfonyl) -2- (2-chloroethyl) -hydrazino-carbonyl ] -N-methylaminomethyl } -5-chloro-phenyl ester disodium phosphate (22 c). According to the general procedure, crude product 20c (16.2g, 30.8mmol) gave 22c (8.9g, 51%) as a white powder.

¹H NMR(300MHz，D₂O)δ7.28(s，1H，C6-H(Ph))，7.03(d，J＝8.4Hz，1H，C3-H(Ph))，6.87(d，J＝8.2Hz，1H，C4-H(Ph))，4.47(m，2H，PhCH₂)，3.91(m，2H，ClCH₂)，3.70(m，2H，NCH₂)，3.26(s，3H，NCH₃)，3.00(s，3H，SCH₃) And 2.96(s, 3H, SCH)₃)。

¹³C NMR(75MHz，D₂O) δ 156.7, 155.3(d), 135.6, 132.0, 127.0(d), 124.5, 122.4, 57.3, 51.0, 43.7, 42.0, 40.5 and 40.0.

³¹p NMR(121MHz，D₂O)δ14.1。

TOF ESMS calculated for (M-H) 525.96, observed 526.01.

Example 20

Preparation of Carbamate (40)

4-Formylphenoxycarbonyl-glutamic acid di-tert-butyl ester (40). To a cold stirred solution of 4-hydroxybenzyl alcohol (2.0g, 16.9mmol) in acetonitrile (50mL) and dichloromethane (50mL) was added phosgene (20% in toluene, 1.0 eq.) and DIEA (1.0 eq.). The reaction solution was left at 0 ℃ for 30 minutes. To the above solution was then added a solution of DIEA (2.0 equivalents) in di-tert-butyl glutamate 39(1.0 equivalent) in dichloromethane (50 mL). The reaction mixture was left at 0 ℃ overnight. The mixture was then diluted with 0.5N KHSO₄Solution (50 mL). After separation, the organic phase was washed with brine (80mL), anhydrous MgSO₄Drying, rotary evaporation and vacuum drying. The crude carbamate 40(6.7g, 97%) was obtained as a pale yellow semi-solid.

¹H NMR(300MHz，CDCl₃)δ9.97(s，1H，CHO)，7.89(d，J＝8.1Hz，2H，C3-H(Ph))，7.32(d，J＝8.7Hz，2H，C2-H(Ph))，5.91(d，J＝7.8Hz，1H，NH)，4.32(m，1H，C¹H)，2.35(m，2H，C²H)，2.00(m，2H，C³H) 1.50 and 1.46(s, 2X 9H, CH)₃)。

¹³C NMR(75MHz，CDCl₃) δ 191.0, 172.1, 170.6, 155.6, 153.1, 133.4, 131.1, 121.9, 82.7, 80.9, 54.1, 31.4, 28.0, 27.9 and 27.5.

TOF ESMS calculated for (M + H) 408.20, observed 408.19.

Example 21

Preparation of N-benzyl-N-methylamine (41)

4- (methylaminomethyl) phenoxycarbonyl-cerealDi-tert-butyl ester of amino acid (41). A stirred solution of 40(6.1g, 14.9mmol) in dichloromethane (50mL) was treated with a solution of 2N methylamine in THF (10mL) at 0 deg.C overnight. After removal of the solvent, the residual oil was dissolved in methanol (80mL) while being placed in an ice bath. To the above solution was added a small amount of sodium borohydride over 30 minutes. The reaction solution was left at 0 ℃ for 1 hour, and then the solvent was removed by evaporation. The residue was treated with brine and dichloromethane. After separation, the organic phase is separated with anhydrous MgSO₄Drying, rotary evaporation and vacuum drying. The crude amine 41 was obtained as a pale yellow glassy solid (5.4g, 78%).

¹H NMR(300MHz，CDCl₃)δ7.04(d，J＝8.3Hz，2H，C3-H(Ph))，6.78(d，J＝8.1Hz，2H，C2-H(Ph))，5.36(d，J＝7.4Hz，1H，CONH)，4.39(m，1H，C¹H)，2.86(d，J＝4.6Hz，3H，NCH₃)，2.74(d，J＝4.8Hz，2H，NCH₂)，2.34(m，2H，C²H)，1.92(m，2H，C³H) 1.49 and 1.42(s, 2X 9H, CH)₃)。

¹³C NMR(75MHz，CDCl₃) δ 172.6, 172.4, 157.9, 156.1, 128.6, 128.3, 115.6, 82.0, 80.7, 53.9, 51.5, 33.8, 31.6, 28.0, 27.9 and 27.6.

TOF ESMS calculated for (M + H) 423.25, observed 423.24.

Example 22

Preparation of N-benzyl-N-methylaminocarbonyl-hydrazine (42)

4- { N- (1, 2-bis (methylsulfonyl) -1- (2-chloroethyl) hydrazino-2-yl-carbonyl) -N-methylaminomethyl } phenoxycarbonyl-glutamic acid di-tert-butyl ester (42). To a cold stirred solution of 90CE (1.5g, 5.9mmol) in acetonitrile (30mL) was added phosgene (20% in toluene, 1.0 eq.) and DIEA (1.0 eq.). The reaction solution was left at 0 ℃ for 30 minutes. To the above solution was then added a solution of DIEA (1.0 eq) in 41(1.0 eq) acetonitrile (30 mL). The reaction mixture was left at 0 ℃ overnight. After removal of the solvent by evaporation, the residue obtained is taken up in water and dichloromethaneAnd (6) processing. After separation, the organic phase is separated with anhydrous MgSO₄Drying, rotary evaporation and vacuum drying. The crude product N-benzyl-N-methylaminocarbonyl-hydrazine 42(3.6g, 87%) was obtained as a pale yellow glassy solid.

¹H NMR(300MHz，CDCl₃)δ7.31(d，J＝8.0Hz，2H，C3-H(Ph))，7.21(d，J＝8.2Hz，2H，C2-H(Ph))，5.38(d，J＝7.5Hz，1H，CONH)，4.52(bs，1H，C¹H)，3.82(m，2H，ClCH₂)，3.67(m，2H，NCH₂)，3.55(s，3H，NCH₃) 3.23 and 3.14(s, 2X 3H, SO)₂CH₃)，2.87(s，2H，NCH₂Ph)，2.35(m，2H，C²H)，1.98(m，2H，C³H) 1.47 and 1.43(s, 2X 9H, CH)₃)。

¹³C NMR(75MHz，CDCl₃) δ 172.7, 172.1, 157.6, 148.8, 137.0, 128.9, 128.7, 121.0, 81.7, 80.5, 53.8, 53.4, 51.2, 41.9, 41.7, 41.3, 40.4, 31.5, 27.9, 27.8 and 27.4.

TOF ESMS calculated for (M + H) 699.21, observed 699.18.

Example 23

Preparation of glutamic acid (43) and its disodium salt (44)

4- { N- (1, 2-bis (methylsulfonyl) -1- (2-chloroethyl) hydrazino-2-yl-carbonyl) -N-methylaminomethyl } phenoxycarbonyl-glutamic acid (42) and its disodium salt (44). The crude glassy solid 42(4.4g, 6.4mmol) was treated with formic acid (200mL) at 5 ℃ overnight. After freezing at-78 ℃, the desired glutamic acid 43 was obtained as a viscous white solid by lyophilization.

Without further purification, the crude product 43 was purified with saturated NaHCO₃The solution (200mL) was treated at room temperature for 2 hours. The resulting viscous mixture was purified by RPFCC and eluted with deionized water. Fractions were monitored by HPLC and collected. After lyophilization, disodium salt 44 was obtained as a white powder (0.78g, 20%).

¹H NMR(300MHz，D₂O)δ7.15(d，J＝8.0Hz，2H，C3-H(Ph))，7.08(d，J＝7.8Hz，2H，C2-H(Ph))，4.31(bs，1H，C¹H)，3.83(m，2H，ClCH₂)，3.67(m，2H，NCH₂)，3.46(s，3H，NCH₃)，3.16(s，3H，SO₂CH₃)，2.95(m，2H，NCH₂Ph)，2.70(s，3H，SO₂CH₃)，1.99(m，2H，C²H) And 1.75(m, 2H, C)³H)。

¹³C NMR(75MHz，D₂O) δ 185.0, 182.7, 162.0, 154.4, 150.8, 140.0, 131.0, 123.6, 59.1, 55.1, 53.5, 43.7, 42.9, 36.7, 36.4 and 31.2.

TOF ESMS calculated for (M + H) 631.05 observed 631.04, (M + Na) 653.03 observed 653.03.

Example 24

Determination of solubility and stability in aqueous solution

VNP40101M has a solubility in water of 0.66mg/mL at room temperature (Krishna et al.AAPS PharmsciTech2001,2: article 14). As shown in Table 1, the newly synthesized SHPs (19, 20a, 21, and 22a) of the present invention have higher solubility than VNP 40101M.

For free phosphoric acid 19 and 20a, the excess drug was placed in a glass vial containing 2.0mL of water. The vials were placed on a Glas Col rotating apparatus at room temperature and shaken for 24 hours. Centrifuging the suspension containing the undissolved drug; the supernatant was carefully separated and analyzed by HPLC for the concentration of the drug. The solubility of 19 and 20a was measured to be 293 and 46mg/mL, respectively. 19 and 20a are colorless. Similarly, the solubility of the two drugs was measured by incremental addition of disodium salts 21 and 22a to a glass vial containing 2.0mL of water. The vial was shaken on a Glas Col rotary device at room temperature until the drug was completely dissolved. A defined amount of drug was added and the vial shaken until complete dissolution. The above procedure was repeated until no more drug was dissolved. Compounds 21 and 22a are very soluble in water. Equilibrium solubility cannot be measured due to limited drug supply. Solubility of 21 and 22a was measured to be > 0.98 and > 1.35g/mL, respectively.

TABLE 1 aqueous solubility of designated SHPs at room temperature

Compound (I)	Water solubility, mg/mL
		VNP40101M	0.66
PAP-101M(19)	293
		OAP-101M(20a)	46
PAP-Na-101M(21)	＞980
		OAP-Na-101M(22a)	＞1350

The stability of PAP-101M (19), OAP-101M (20a), and VNP40101M was measured at room temperature (22-25 ℃) in potassium phosphate buffers (50mM) at pH 3, 5, 7, and 9. One sample was prepared for each drug at each pH; the initial drug concentration in each sample was 50. mu.g/mL. Each sample was analyzed by HPLC at different time points to determine the concentration of the corresponding drug. The first order pharmacokinetic half-life of each drug was calculated. As shown in table 2 below, the results clearly show that the phosphate-carrying prodrugs (19 and 20a) are quite stable relative to VNP 40101M.

TABLE 2 Water stability of designated SHPs

Example 25

In vitro bioconversion and stability measurements

Table 3 shows the biotransformation of compounds 19 and 20a in the presence of AP (from bovine intestinal mucosa, Sigma) or in human plasma. The final concentration of approximately 50. mu.g/mL drug was incubated in 50mM Tris buffered saline (pH7.6) containing approximately 0.055 units/mL phosphatase at 37 ℃.

TABLE 3 in vitro enzymatic bioconversion and human plasma stability

Control samples of each drug were also incubated at 37 ℃ with a final concentration of 50. mu.g/mL in 50mM Tris buffered saline (pH7.6) without AP. Periodically withdrawing aliquots of each solution; the test drug was measured by HPLC until no more appeared.

The stability of compounds 19, 20a and VNP40101M was evaluated in 100% human plasma (BioChemed) at a final concentration of 50. mu.g/mL. Will be provided withEach drug (19 or 20a) was incubated in human plasma at 37 ℃ for up to two hours. Aliquots of the incubation mixture were removed at different time points and extracted with acetonitrile. The extracts were centrifuged and analyzed directly by HPLC. In a similar manner, VNP40101M was incubated in human plasma at 37 ℃ for up to 1 hour. At various time points, aliquots of the incubation mixture were removed and incubated with 0.5% H₃PO₄Extracting the solution of acetonitrile. The extracts were centrifuged and analyzed directly by HPLC. For control purposes, the stability of each drug incubated in 50mM Tris buffered saline (pH7.6) instead of 100% human plasma was also measured.

It is clear that (a) the designated prodrugs 19 and 20a are more stable in buffered saline and human plasma than VNP 40101M; and (b) they can be activated rapidly by alkaline phosphatase. OAP-101M (20a) was shown to have a longer half-life than PAP-101M (19).

Example 26

Pharmacokinetic studies in rats

The pharmacokinetic profile of prodrugs 19 and 20a was initially studied in female Sprague-Dawley rats (10 weeks old, 250g, Charles River). Each prodrug was administered by intravenous jugular injection at a dose of 50mg per kg (mpk) body weight. Blood samples were collected at the following time points after dosing: pre-dose and approximately 2, 10, 30 minutes, 1, 2, and 24 hours post-dose. At each time point, approximately 0.2mL of blood was collected in a tube containing anticoagulant (heparin), which was immediately acidified by the addition of 0.005mL of 2.0M citric acid solution. The tube was then inverted 4-6 times and immediately placed in ice. The blood samples were centrifuged at 3,000rpm for 10-20 minutes at 2-8 ℃ within 30 minutes after collection and the plasma fractions were transferred to a labeled Nunc cryo-al. Plasma samples were immediately frozen on dry ice and stored at-20 ℃ until HPLC-UV analysis. Animals were allowed to inhale CO after the test₂And (6) killing the dead with the dead.

The above prodrugs were quantified in rat plasma using bioanalytical methods using 220nm or 230nm HPLC-UV. Each plasma sample (0.1mL) was extracted with 0.2mL acetonitrile. The extracts were centrifuged and then directly analyzed by HPLC-UV. HPLC calibration standards were prepared in control rat plasma according to the method described above. The standard curve has a straight line ranging from 1.0 to 50. mu.g/mL.

Since 19 had been transformed or cleared very rapidly in the rat circulation, measurements thereof could not be completed. As shown in Table 4, various pharmacokinetic parameters (area under the concentration-time curve-AUC, Total body clearance-Cl, volume of distribution equilibrium-Vss, maximum concentration-Cmax, and end-point half-life-T were calculated_1/2). The plasma half-life of 20a is approximately 14 minutes, longer than 19. Control with VNP101M (10 mpk of radioactive VNP101M was used in the previous experiment) was difficult because of the difference in dose used in the two studies.

Tables 4.19 and 20a pharmacokinetic parameters in rats

Medicine	AUC (min. ug/mL)	Cl (mL/min/kg)	Vss(mL/kg)	Cmax(μg/mL)	T(minutes)
						19	---	---	---	---	---
20a	1312.0	37.8	145.6	238.6	14.0
						VNP40101M	325.8±113.8	2.0±0.6	0.91±0.16	11.3±2.1	20.9±8.7

^*The plasma VNP40101M concentration (10. mu.g/mL) is administered¹⁴C]Peak at 2 min after VNP 40101M. VNP40101M concentration decreased with half-life of-20 minutes. After the first 2 hours, no VNP401 40101M was detectable in plasma (see Almassian et al.Proceedings AACR，2001，42：326，article 1756)。

Example 27

Evaluation of antitumor Activity in vivo

The anti-tumor effects of VNP40101M and prodrugs including PAP-101M (19), OAP-101M (20a), PAP-Na-101M (21), and OAP-Na-101M (22a) were evaluated in B16-F10 murine melanoma and HTB177 human lung cancer models. B16-F10 melanoma cells were implanted subcutaneously (5X 10)⁵Cells) in C57BL/6 miceThey were randomly divided into 10 groups immediately after tumor cell implantation (day 0). On day 2, mice were injected intraperitoneally with 0.1mL PBS or drug injections. The above treatments were performed weekly for four consecutive weeks. The spatial size of the tumor was measured every other week according to the formula L x H x W/2, where L, H, and W represent length, height, and width, respectively. As shown in FIG. 11, B16-F10 tumors in the PBS control group grew exponentially and reached sizes approaching 4,000mm on day 24³. VNP40101M and the test prodrug effectively inhibited the growth of B16-F10 melanoma. On day 24, 81% of the tumor growth was inhibited in mice by treatment with 80mg/kg VNP40101M, and 75-91% of the tumor growth was inhibited in mice by treatment with equimolar doses of the prodrug. Of all prodrugs, OAP-101M (20a) and OAP-Na-101M (22a) are most potent; the tumor growth inhibition rates were 91% and 89.5%, respectively. The inhibition was much higher (p < 0.05) than in the other groups. Tumor-bearing mice dosed with OAP-101M (20a) survived longer than mice dosed with other drugs (FIG. 12). As shown in fig. 13, the toxic response of these drugs in mice was evaluated by weight loss and animal appearance. As shown in FIGS. 14 and 15, the antitumor effects of the above drugs were also evaluated in nu/nu CD-1 mice implanted with HTB177 human lung cancer cells, and the results showed that OAP-101M (20a) and OAP-Na-101M (22a) were still superior.

Briefly, we found that water-soluble SHP prodrugs, OAP-101M (20a) and OAP-Na-101M (22a), had anti-tumor activity against B16-F10 murine melanoma and HTB177 human lung cancer, with effects as good as or better than VNP 40101M.

Summary of the invention

Briefly, phosphate-bearing SHPs OAP-101M (20a) and OAP-Na-101M (22a) have the following properties: (a) has excellent water solubility and stability in an aqueous solution with a pH of 3-9; (b) its conversion can be catalyzed by Alkaline Phosphatase (AP); (c) has a longer half-life in saline and human plasma than PAP-101M (19) and VNP 40101M; (d) has a better in vivo PK profile than PAP-101M; (e) has better anti-tumor activity against B16-F10 murine melanoma and HTB177 human lung cancer than PAP-101M (19), PAP-Na-101M (21) and VNP 40101M; and (f) its sodium salt (22a) has higher water solubility and similar anti-tumor activity than the free acid 20 a.

It is to be understood by persons skilled in the art that the foregoing description and examples are illustrative of the present invention only and are not intended to be in any way limiting. Various modifications may be made in the details set forth without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (33)

1. A compound of the formula:

wherein R is-CH₃or-CH₂CH₂Cl；

R' is C₁-C₇Alkyl or-CH₂CH₂Cl；

R₂Or R₄At the same timeAre not all selected from OPO₃H₂、NO₂OCO (Glu), NHCO (Glu) and NHR₇Another unspecified R₂Or R₄And R₃、R₅And R₆Independently selected from H, F, Cl, Br, I, OH, OPO₃H₂、OCH₃、CF₃、OCF₃、NO₂、CN、SO₂CH₃、SO₂CF₃、COCH₃、COOCH₃、SCH₃、SF₅、NHR₈、N(R₉)₂And C₁-C₇Alkyl with the proviso that R₂、R₃、R₄、R₅And R₆Is H;

R₇is H, glutamyl or polyglutamic polypeptide residue-COCH (NHR)_7a)CH₂CH₂CO₂H, wherein R_7aIs glutamyl, or a polyglutamic polypeptide residue having 1-50 peptide bonds;

R₈is H or C₁-C₇An alkyl group; and

R₉is CH₃Or CH₂CH₃。

2. The compound according to claim 1, wherein R is-CH₂CH₂Cl。

3. The compound according to claim 1 or 2, wherein said R' is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, isohexyl or substituted hexyl.

4. A compound according to claim 3, wherein R' is methyl.

5. A compound according to claim 1 or 2, wherein R₂Is OPO₃H₂A group or a pharmaceutically acceptable salt thereof.

6. A compound according to claim 1 or 2, wherein when R is₃、R₅And R₆Each is H, R₄Is F, Cl or OCH₃。

7. A compound according to claim 1 or 2, wherein when R is₃、R₄And R₆Each is H, R₅Is F, Cl, OCH₃Or OCF₃。

8. A compound according to claim 1 or 2, wherein R₃、R₄、R₅Or R₆Are independently F or Cl.

9. A compound according to claim 8, wherein R₄And R₅Independently F or Cl.

10. A compound according to claim 8, wherein R₅And R₆Independently F or Cl.

11. A compound according to claim 9, wherein R₄And R₅Is Cl.

12. A compound according to claim 10, wherein R₅And R₆Is Cl.

13. A compound according to claim 1 or 2, wherein R₅Is OPO₃H₂A group or a pharmaceutically acceptable salt thereof.

14. A compound according to claim 1 or 2, wherein R₂Is NO₂And R is₃、R₄And R₆Each is H.

15. A compound according to claim 1 or 2, wherein R₄Is NO₂And R is₂、R₃And R₆Each is H.

16. A compound according to claim 1 or 2, wherein R₄Is OCO (Glu), and R₂、R₃、R₅And R₆Each is H.

17. A compound according to claim 16, wherein oco (glu) is in the form of a pharmaceutically acceptable salt.

18. A compound according to claim 1 or 2, wherein R₄Is NHCO (Glu), and R₂、R₃、R₅And R₆Each is H.

19. The compound according to claim 18, wherein nhco (glu) is in the form of a pharmaceutically acceptable salt.

20. A compound according to claim 1 or 2, wherein R₄Is NHR₇And R is₂、R₃、R₅And R₆Each is H.

21. A compound according to claim 20, wherein R₇Is H, alpha-glutamyl or a pharmaceutically acceptable salt thereof or a polyglutamic acid polypeptide residue having 1-50 peptide bonds or a pharmaceutically acceptable salt thereof.

22. A compound according to claim 21, wherein R₇Is alpha-glutamyl or a pharmaceutically acceptable salt thereof or a polyglutamic polypeptide residue having 2-10 peptide bonds or a pharmaceutically acceptable salt thereof.

23. A pharmaceutical composition comprising an effective amount of a compound according to any one of claims 1-22, or a pharmaceutically acceptable salt thereof, optionally in combination with a pharmaceutically acceptable additive, carrier or excipient.

24. Use of an effective amount of a compound according to any one of claims 1-22, or a pharmaceutically acceptable salt thereof, optionally in combination with a pharmaceutically acceptable additive, carrier, or excipient, in the manufacture of a medicament for treating cancer in a patient in need of such treatment.

25. The use according to claim 24, wherein the cancer is selected from the group consisting of gastric, colon, rectal, liver, pancreatic, lung, breast, cervical, uterine corpus, ovarian, prostate, testicular, bladder, kidney, brain/CNS, head and neck, throat, multiple myeloma, melanoma, acute lymphocytic leukemia, acute myelogenous leukemia, ewings 'sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, wilms' tumor, neuroblastoma, hairy cell leukemia, mouth/pharynx, esophageal, laryngeal, renal, or lymphoma.

26. The use according to claim 25, wherein the lymphoma is hodgkin's disease or non-hodgkin's lymphoma.

27. Use of an effective amount of a compound according to any one of claims 1-22 in the manufacture of a medicament for treating drug-resistant cancer in a patient in need of such treatment.

28. Use of an effective amount of a compound according to any one of claims 1-22 in combination with at least one other anti-cancer agent in the manufacture of a medicament for treating cancer in a patient in need of such treatment.

29. Use of an effective amount of a compound according to any one of claims 1 to 22 in the manufacture of a medicament for the treatment of cancer in a patient in need of such treatment, in combination with at least one additional anti-cancer agent selected from the group consisting of antimetabolites, cytarabine, etoposide, doxorubicin, paclitaxel, hydroxyurea, vincristine, cyclophosphamide, mitomycin C, doxorubicin, topotecan, camptothecin, irinotecan, gemcitabine, camptothecin, and cisplatin.

30. Use of an effective amount of a compound according to any one of claims 1-22, or a pharmaceutically acceptable salt thereof, optionally in combination with a pharmaceutically acceptable additive, carrier, or excipient, in the manufacture of a medicament for treating neoplasia in a patient in need of such treatment.

31. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 22 in combination with at least one other anti-cancer agent, optionally in combination with pharmaceutically acceptable additives, carriers or excipients.

32. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 22 in combination with at least one additional anti-cancer agent selected from the group consisting of antimetabolites, cytarabine, etoposide, doxorubicin, paclitaxel, hydroxyurea, vincristine, cyclophosphamide, mitomycin C, doxorubicin, topotecan, camptothecin, irinotecan, gemcitabine, camptothecin and cisplatin.

33. Use of a compound according to any one of claims 1-22 in the manufacture of an anti-cancer medicament containing an effective amount of said compound, optionally in combination with a pharmaceutically acceptable additive, carrier or excipient.