HK1185884A - Salts of isophosphoramide mustard and analogs thereof as anti-tumor agents - Google Patents

Description

Salts of isophosphoramide mustard and analogs thereof as antitumor agents

The application is a divisional application of a Chinese patent application No. 200580044803.6 with the same name of the invention, the original international application number is PCT/US2005/038523, and the international application date is 10 and 25 in 2005.

Statement of government rights

The invention was made with government support under fund No.5R44CA083552-03 awarded by the National Cancer Institute. The government has certain rights in this invention.

FIELD

The present specification relates to salts of isophosphoramide mustard (isophosphoramide mustard) and analogs thereof. Pharmaceutical compositions and methods of using such compositions in the treatment of hyperproliferative diseases are also disclosed.

Background

Autopsy of soldiers killed by mustard gas in world war i showed that dichloroethyl sulfide had an unbalanced effect on rapidly dividing cells and suggested that the dichloroethyl sulfide compound may have an anti-tumor effect. Indeed, early researchers attempted to treat cancer by direct injection of diclofenac into tumors. This study was limited by the extreme toxicity of the bischloroethyl sulfide compound, and nitrogen mustard analogs such as mechlorethamine were investigated as less toxic alternatives.

Bis (chloroethyl) sulfide

Generally, mustard gas compounds exert their cytotoxic effects by alkylating DNA, such as at the N-7 position of a guanine residue. The mechanism of alkylation of the mustard gas compound is explained in scheme 1. Referring to scheme 1, mustard gas compounds have an internal nucleophile that facilitates chlorine displacement, as shown by p-mechlorethamine, thereby forming propidiumAn intermediate. Because mechlorethamine carries two leaving groups, the nucleophilic substitution mechanism depicted in scheme 1 can be repeated, resulting in DNA or protein-DNA cross-linking.

Scheme 1

Mechlorethamine is very reactive and as a result non-selective. Thousands of alkylating agents have been designed and prepared using mechlorethamine as a model. However, few of these compounds show sufficient therapeutic advantage over mechlorethamine for clinical trials.

Due to the lack of selectivity of most mechlorethamine analogues, prodrugs, i.e., phosphoramide compounds, which can be activated by the high concentration of phosphoramidases present in tumor cells, have been investigated. Two phosphoramide alkylating agents, Cyclophosphamide (CPA) and its isomer compound, ifosfamide (Ifos), have proven particularly effective.

Cyclophosphamide (CPA) ifosfamide (Ifos)

The metabolic pathway of CPA is similar to that of Ifos (the metabolism of Ifos is explained in figure 1) and thus both compounds share a common deficiency. Perhaps most important is its dose-limiting toxicity due to hemorrhagic cystitis. Hemorrhagic cystitis is believed to be induced by acrolein production during CPA and Ifos activation. Acrolein is an active electrophile that reacts with thiols under physiological conditions, which may be responsible for its hepatotoxicity in the form of glutathione depletion. Finally, acrolein has been shown to be a teratogen and a potent mutagen, and this may lead to an association between CPA treatment and serious side effects such as bladder cancer and other malignancies.

Referring to figure 1, isophosphoramide mustard (IPM) is a common metabolite of CPA and Ifos. IPM is believed to produce anti-tumor activity exhibited at least in part by CPA and Ifos. Efforts to use IPM directly as an anticancer agent have not been successful due in part to the instability of the compound. IPM has been synthesized and preliminary biological evaluation of this compound has been performed, but unfortunately IPM is too unstable to be isolated and used for human therapy.

Summary of the disclosure

Disclosed herein are compounds of the formula:

wherein A is⁺Represents an ammonium species selected from: protonated (conjugate acid) or quaternary ammonium forms of aliphatic and aromatic amines, said amines comprisingIncluding basic amino acids, heterocyclic amines, substituted and unsubstituted pyridines, guanidines, and amidines; and X and Y independently represent a leaving group.

In one embodiment, pharmaceutical compositions comprising one or more of the above compounds are disclosed. In one aspect of this embodiment, the composition can include one or more therapeutic agents other than those described by the above formula for use in combination therapy.

In another embodiment, a method for treating a mammalian subject, such as a human subject, suffering from a hyperproliferative disease is disclosed. Such methods may employ one or more of the above-described compounds and compositions.

In another aspect, disclosed herein is a sterile pharmaceutical composition of a compound of the formula:

wherein X and Y independently represent a leaving group. Also disclosed are methods of preparing such compositions, including rendering the compositions sterile by using a sterile antimicrobial filter. In certain embodiments, such filtration may be performed with less than 10%, preferably less than 5%, 2%, or even less than 1% decomposition of the active component.

Also disclosed herein are methods for producing a lyophilizate comprising a compound of the above formula. In one embodiment, the method comprises contacting isophosphoramide mustard or an analog thereof with an amine base in the presence of water and freeze-drying the resulting mixture.

Brief Description of Drawings

FIG. 1 is a schematic diagram illustrating the metabolism of ifosfamide, including acrolein and ifosfamide mustard production.

FIG. 2 shows the temperature at 500MHzRecorded in D₂IPM in O (LYS)₂Is/are as follows¹H NMR spectrum.

FIG. 3 is an expanded portion of the spectrum of FIG. 2.

FIG. 4 shows IPM (LYS)₂Is/are as follows¹³C NMR spectrum.

Detailed Description

The following explanations of terms and examples are provided to better describe the compounds, compositions, and methods of the present invention and to guide those of ordinary skill in the art in the practice of the present invention. It is also to be understood that the terminology used in the description is for the purpose of describing particular embodiments and examples only, and is not intended to be limiting.

Ranges may be expressed herein as from one particular value of "about" and/or to another particular value of "about". When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

In this specification and in the claims which follow, reference will be made to a number of terms which shall be understood to have the following meanings:

"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The term "amino acid" means natural and unnatural amino acids, including α -amino acids, in the case of chiral amino acids in their D and L stereoisomeric forms. Examples of basic amino acid residues include those having a basic side chain, such as an amino or guanidino group. Basic amino acid residues include, but are not limited to, arginine, histidine, homoarginine, lysine, homolysine, and ornithine.

The term "antibody" means an immunoglobulin, whether natural or wholly or partially synthetically produced. Also included in this term are all derivatives thereof that maintain specific binding capacity. The term also encompasses any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources or produced partially or completely synthetically. The antibodies used herein may be monoclonal or polyclonal.

As used herein, "aliphatic amine" means a compound of the formula NR¹R²R³Wherein R is^1-3At least one of which is an aliphatic group.

The term "acyclic aliphatic amine" means an aliphatic amine as described above wherein at least one of the aliphatic groups is acyclic.

The term "heterocyclic amine" means a compound of the formula NR¹R²R³Wherein R is^1-3At least one of which is heterocyclyl or R¹、R²And/or R³Together with the nitrogen atom they share, form a ring.

I. Salts of IPM and IPM analogues

The compounds and compositions disclosed herein include IPM and IPM analogs formulated using one or more equivalents of a base. Because IPM and its analogs are acid sensitive and acidic, the presently disclosed compounds may provide greater stability and other advantages. The advantages of the disclosed formulations in terms of synthesis, stability and bioavailability will be apparent to those skilled in the art in view of the present disclosure.

In one embodiment, the disclosed compounds are salts of isophosphoramide mustard or isophosphoramide mustard analogs that include one or more cations. In one embodiment, the cation may be the conjugate acid of an amine base or may be a quaternary ammonium cation. Suitable counterions for isophosphoramide mustard and analogs thereof include the conjugate acid of a base (unless the context clearly indicates designation as a free amine, the term referring to an amine as used herein is to be understood to include the conjugate acid thereof), including basic amino acids, aliphatic amines, heterocyclic amines, aromatic amines, pyridines, guanidines, and amidines. Of the aliphatic amines, acyclic aliphatic amines and cyclic and acyclic di-and tri-alkyl amines are particularly suitable for use in the disclosed compounds. In addition, quaternary ammonium counterions are examples of suitable counterions that can be used.

Specific examples of suitable amine bases (and their corresponding ammonium ions) for use in the present invention include, but are not limited to, pyridine, N-dimethylaminopyridine, diazabicyclononane, diazabicycloundecene, N-methyl-N-ethylamine, diethylamine, triethylamine, diisopropylethylamine, mono-, di-or tri- (2-hydroxyethyl) amine, 2-hydroxy-tert-butylamine, tris (hydroxymethyl) methylamine, N-dimethyl-N- (2-hydroxyethyl) amine, tris- (2-hydroxyethyl) amine and N-methyl-D-glucamine.

In another embodiment, the above salts may include a second amine or ammonium group. In one embodiment, the compounds disclosed herein comprise more than one equivalent of an amine per equivalent of isophosphoramide mustard or isophosphoramide mustard analog. Such embodiments include those compounds having a non-integer ratio of amine to isophosphoramide mustard or an isophosphoramide mustard analog. In certain embodiments, the compound has a molar ratio of 2:1 or 3: 1 ratio of amine to isophosphoramide mustard or isophosphoramide mustard analog. In an effective embodiment, a salt is produced that contains two equivalents of amine base per equivalent of isophosphoramide mustard. In one embodiment, the amine base used to form the isophosphoramide mustard and isophosphoramide mustard analog salts includes more than one amino group; such bases may be referred to as "polybasic bases". More specifically, some examples of polybasic bases that can be used carry 2 amino groups; such compounds may be referred to as "dibasic bases". For example, one suitable dibasic molecule is N, N-dimethylaminopyridine, which includes two basic amino groups. In a specific embodiment of the compounds disclosed herein, the compound comprises isophosphoramide mustard or an isophosphoramide mustard analog and one equivalent of a dibasic amine.

In one embodiment, the disclosed compounds include one or more zwitterionic bases. Examples of such bases include basic amino acids, which are zwitterionic at physiological pH.

In one embodiment, the presently disclosed salts are more stable than isophosphoramide mustard and isophosphoramide mustard analogs. For example, isophosphoramide mustard decomposed by approximately 40% during storage at-20 ℃ for 3 months after lyophilization as the pure compound. In contrast, the lysine salt of IPM did not show any measurable decomposition, even after 10 months under similar storage conditions.

In certain embodiments, the disclosed compounds are stable isophosphoramide mustard salts or stable analogs of isophosphoramide salts, wherein the salts have a half-life at room temperature (e.g., about 23 ℃) in the presence of water that is greater than the half-life of isophosphoramide mustard under the same conditions in the presence of water. In certain preferred such embodiments, the isophosphoramide mustard salt has a half-life in the presence of water equal to or greater than 2 times as long as isophosphoramide mustard, more preferably equal to or greater than 5 times.

In certain embodiments, lyophilized products of the disclosed compounds are more stable than lyophilized products of isophosphoramide mustard. In certain preferred such embodiments, the lyophilized product of the disclosed compound has a longer shelf life than the lyophilized product of isophosphoramide mustard itself, preferably at least 2 times as long, more preferably at least 5 times as long.

In certain embodiments, a pharmaceutical composition of a pharmaceutically acceptable salt of IPM or an analog thereof, such as a compound of the general formula described above, is more stable under the same conditions than an otherwise identical composition of isophosphoramide mustard itself (i.e., a non-salt form). In certain preferred such embodiments, the disclosed compositions have a longer shelf life than the lyophilized preparation of isophosphoramide mustard, preferably at least 2 times as long, more preferably at least 5 times as long.

Certain isophosphoramide mustard and isophosphoramide mustard analog compounds disclosed herein include two leaving groups. Without being bound by theory, it is believed that these two leaving groups are displaced in vivo by biomolecule nucleophiles, such as nucleic acids and proteins, thereby crosslinking the biomolecules. The term "leaving group" means a group that can be displaced by a nucleophile. With respect to the presently disclosed compounds, a leaving group means a group that can be displaced to form an acridinium intermediate or can be directly displaced by a biomolecule nucleophile, such as a nucleic acid nucleophile, to form, for example, a 7-alkylated guanidinium species. Examples of suitable leaving groups include halogen and sulfonate (-SO)₂R). In one embodiment of the disclosed isophosphoramide analog salts, the compound is a "mixed" leaving group compound, comprising two different types of leaving groups, for example, a halogen and a sulfonate or two different halogens such as bromine and chlorine. A process for preparing such mixed leaving group compounds is taught in U.S. patent No. US 6,197,760 to Struck.

One embodiment disclosed herein relates to an anti-hyperproliferative agent of the formula:

referring to this formula, for each n, B can be an independently selected basic molecule. In one embodiment of this formula, B may be selected from basic amino acids, acyclic aliphatic amines, di-and trialkylamines, heterocyclic aliphatic amines, aromatic amines, substituted and unsubstituted pyridines, cyclic and acyclic guanidines, and cyclic and acyclic amidines. Generally, n is from 1 to about 3, such that the formula may include different basic molecules. Continuing with this formula, X and Y are leaving groups. It will be appreciated by those skilled in the art that the exemplified isophosphoramide mustard structure includes an acidic proton and as such exists primarily as its conjugate base at physiological pH and in the presence of a base such as B. Likewise, B, which is a basic group, exists primarily as its conjugate acid at physiological pH and in the presence of isophosphoramide mustard and isophosphoramide mustard analogs. Exemplary embodiments of the disclosed compounds are shown in table 1.

TABLE 1

In another embodiment, the disclosed compounds include a salt of isophosphoramide mustard. Some examples of such isophosphoramide mustard salts may be represented by the formula:

in the case of the above formula, B may be any basic group, in particular an amine. It will be appreciated that the above formula will exist primarily as the corresponding salt and thus may include compounds represented by the formula:

in the case of the above formula, such compounds may also include one or more additional equivalents of amine or ammonium species. Such compounds may be represented by the formula:

wherein G represents a second ammonium or amine species. In a specific example, G is a basic amino acid and BH⁺Represents the conjugate acid of the same or different basic amino acids.

In one embodiment, BHs⁺Is the conjugate acid of G. In this embodiment, the disclosed isophosphorsThe amidomustard salt may be represented by the formula:

wherein B is an amine and BH⁺Is the conjugate acid thereof.

In one embodiment, the compounds disclosed herein comprise a metal cation, such as an alkali metal cation. Examples of such cations include Li⁺、Na⁺、K⁺And Rb⁺And Cs⁺. In one aspect, such examples can be represented by the formula:

wherein M is⁺Represents an alkali metal cation and B is as defined above.

Compositions and methods

Another aspect of the present specification includes pharmaceutical compositions, preferably sterile pharmaceutical compositions, prepared for administration to a subject and which include an effective amount of one or more of the presently disclosed compounds. Such sterile compositions may be prepared by passing a solution of a salt of IPM or an analog thereof through a sterile antimicrobial filter. Such sterile compositions preferably contain the active ingredient of the invention in which the rate of decomposition is less than 10%, preferably less than 5%, 2%, or even less than 1% decomposition as measured by detecting the presence or absence of decomposition by-products such as phosphoric acid and its salts and substituted ethylamines.

The compounds disclosed herein can be administered orally, topically, transdermally, parenterally, by inhalation or spray and can be administered in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.

In general, it is preferred that the disclosed isophosphoramide mustard salts and analogs thereof be administered parenterally by injection. The inhibitor may be provided in a single dose or over a prolonged period of time, depending on the particular disease, the condition of the patient, the toxicity of the compound and as recognized by those skilled in the art.

The therapeutically effective amount of the compound or compounds administered may vary depending on the desired effect and the factors described above.

Pharmaceutical compositions for administration to a subject may include carriers, thickeners, diluents, buffers, preservatives, surfactants, and the like, as well as the selected molecule. The pharmaceutical composition may also include one or more additional active ingredients, such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutical formulation may include additional ingredients, such as carriers. Pharmaceutically acceptable carriers for these formulations are conventional. Compositions and formulations suitable for drug delivery of the compounds disclosed herein are described in Remington's Pharmaceutical Sciences, by e.w. mart in, Mack publishing co., Easton, PA,19th Edition (1995).

In general, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations typically contain injectable fluids, which include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like as vehicles. For solid compositions (e.g., powder, pill, tablet, or capsule dosage forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.

In one embodiment, the disclosed compounds are formulated for administration to a human subject. In aspects of this embodiment, the pharmaceutical composition comprises from about 0.1mg/mL to about 250mg/mL, such as from about 20 to about 100mg/mL, of the compound of isophosphoramide mustard salt or analog thereof.

In one aspect, certain embodiments of the pharmaceutical composition are formulated in a unit dosage form. For example, such unit dosage forms can contain from about 100mg to about 1500mg, such as from about 200mg to about 1500mg, of the disclosed isophosphoramide mustard salt or analog thereof per dosage unit.

Of particular interest in certain embodiments, the compounds of the invention are delivered by injection and/or implantation of a long-acting drug, e.g., comprising multivesicular liposomes such as DepoFoam (SkyePharma, Inc, San Diego, Calif.) (see, e.g., Chamberland et al Arch. neuro.1993,50, 261-264; Katri et al. J. Pharm. Sci.1998,87, 1341-1346; Ye et al, J. control Release 2000,64,155-166; and Howell, Cancer J.2001,7, 219-227).

Disclosed herein are methods for treating conditions characterized by aberrant or pathological proliferative activity or neoplasia by administering to a subject one or more of the disclosed compounds and compositions. By "neoplasia" is meant an abnormal and uncontrolled cellular growth process. Neoplasias are one example of proliferative diseases. The product of neoplasia is a neoplasm (tumor), which is an abnormal growth of tissue due to excessive cell division. Non-metastatic tumors are called "benign". Tumors that invade surrounding tissues and/or can metastasize are referred to as "malignant".

Diseases that can be treated according to the disclosed methods include those characterized by abnormal cell growth and/or differentiation, such as cancer and other neoplastic conditions. Typical examples of proliferative diseases that can be treated using the disclosed compounds and compositions are described below.

Examples of hematological tumors that can be treated using the compounds and compositions disclosed herein include: leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelogenous leukemia, and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia); chronic leukemias (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia); polycythemia vera; lymphoma; hodgkin's disease; non-hodgkin lymphoma (slow progression and high grade form); multiple myeloma; waldenstrom's macroglobulinemia; heavy chain disease; myelodysplastic syndrome; hairy cell leukemia; and myelodysplasia.

Additional examples of diseases that can be treated using the compounds and compositions disclosed herein include: solid tumors, such as sarcomas and carcinomas, including fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other sarcomas, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic carcinoma, breast carcinoma, lung carcinoma, ovarian carcinoma, prostate carcinoma, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver carcinoma, bile duct carcinoma, choriocarcinoma, wilms' tumor, cervical carcinoma, testicular tumor, bladder carcinoma, and CNS tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma (menengioma), melanoma, neuroblastoma, and retinoblastoma).

In one embodiment, the compounds disclosed herein grow better than CPA or Ifos alone against CPA resistant tumors. Accordingly, one aspect of the methods disclosed herein comprises treating a subject having a CPA resistant neoplastic condition with an isophosphoramide mustard salt or an analog thereof disclosed herein.

In one embodiment of the method, the subject is administered from about 0.2 mg/kg/day to about 20 mg/kg/day of the disclosed isophosphoramide mustard salt or analog thereof. For example, about 0.5 to about 10 mg/kg/day, such as about 1 to about 7.5 mg/kg/day of the disclosed compounds can be administered to a subject.

In another embodiment of the methodIn a subject, about 10 to about 700mg/m²A day, such as from about 20 to about 400mg/m²Per day or from about 100 to about 500mg/m²The day is. For example, from about 30 to about 100mg/m²A day, such as from about 40 to about 90mg/m²A day of the disclosed compound.

In one embodiment of the methods for treating a hyperproliferative disorder disclosed herein, a subject is administered the disclosed compounds according to a multiple daily dosing regimen. In such embodiments, the compound is administered on at least two days and up to 5 different days. In one aspect of a multiple daily dosing regimen, the compound is administered to the subject on consecutive days, such as 2-5 consecutive days.

In one embodiment of the method, one or more additional therapeutic agents are administered to the subject in addition to the presently disclosed compounds and compositions. For example, additional therapeutic agents that may be used include microtubule binding agents, DNA intercalating or crosslinking agents, DNA synthesis inhibitors, DNA and/or RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene modulators, and/or angiogenesis inhibitors.

By "microtubule binding agent" is meant an agent that interacts with tubulin to stabilize or destabilize microtubule formation, thereby inhibiting cell division. Examples of microtubule binding agents that may be used in combination with the presently disclosed isophosphoramide mustard salts and analogs thereof include, but are not limited to, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine), epothilones, colchicine, dolastatin 15, nocodazole, podophyllotoxin, and rhizoxin. Analogs and derivatives of such compounds may also be used and are well known to those skilled in the art. For example, suitable epothilones and epothilone analogs that incorporate the compounds of the present invention are described in international publication No. WO 2004/018478, which is incorporated herein by reference. Taxanes, such as paclitaxel and docetaxel, are currently believed to be particularly useful as therapeutic agents in the presently disclosed compounds. Other examples of useful taxanes, including taxol analogs, are taught in Holton, U.S. Pat. No. 6,610,860, Gurram et al, U.S. Pat. No. 3,5,530,020, and Wittman et al, U.S. Pat. No.5,912,264. Each of these patents is incorporated herein by reference.

Suitable DNA and/or RNA transcription modulators, including, but not limited to, actinomycin D, daunorubicin, doxorubicin, and derivatives and analogs thereof, are also suitable for use in combination with the presently disclosed compounds.

DNA intercalators and cross-linkers that can incorporate the disclosed compounds include, but are not limited to, cisplatin; carboplatin; oxaliplatin; mitomycins, such as mitomycin C; bleomycin; chlorambucil; cyclophosphamide; and derivatives and analogs thereof.

DNA synthesis inhibitors suitable for use as therapeutic agents include, but are not limited to, methotrexate, 5-fluoro-5' -deoxyuridine, 5-fluorouracil, and analogs thereof.

Examples of suitable enzyme inhibitors for use in combination with the presently disclosed compounds include, but are not limited to, camptothecin, etoposide, formestane, trichostatin, and derivatives and analogs thereof.

Suitable therapeutic agents for use with the presently disclosed compounds that affect gene regulation include agents that result in an increase or decrease in expression of one or more genes, such as, but not limited to, raloxifene, 5-azacytidine, 5-aza-2' -deoxycytidine, tamoxifen, 4-hydroxyttamoxifen, mifepristone, derivatives and analogs thereof.

The term "angiogenesis inhibitor" as used herein means molecules, including, but not limited to, biomolecules, such as peptides, proteins, enzymes, polysaccharides, oligonucleotides, DNA, RNA, recombinant vectors, and small molecules, which function to inhibit blood vessel growth. Angiogenesis is involved in certain pathological processes, such as those involved in diseases such as diabetic retinopathy, chronic inflammatory diseases, rheumatoid arthritis, dermatitis, psoriasis, gastric ulcers and most types of human solid tumors.

Angiogenesis inhibitors are known in the art and examples of suitable angiogenesis inhibitors include, but are not limited to, angiostatin K1-3, staurosporine, genistein, fumagillin, medroxyprogesterone, suramin, interferon- α, metalloproteinase inhibitors, platelet factor 4, somatostatin, thrombospondin, endostatin, thalidomide and derivatives and analogs thereof.

One or more other therapeutic agents, particularly antineoplastic agents, which may or may not fall within the above categories, are also suitable for administration in combination with the presently disclosed compounds. By way of example, such active agents include doxorubicin, apigenin, rapamycin, zebularine, cimetidine, and derivatives and analogs thereof.

Example III

The disclosure above is further illustrated by the following non-limiting examples.

Example 1

This example describes the synthesis of phenyl esters of IPM as follows.

2-chloroethylamine hydrochloride (116g;1.0mol) in 1200mL of dichloromethane was suspended in a 5L 3-necked round-bottomed flask equipped with a mechanical stirrer, 500mL dropping funnel and calcium chloride drying tube and stirred in an ice-water bath. When the temperature had dropped to 5 ℃ phenyl dichlorophosphonate (105.5g;0.5mol) (commercially available from ldrich, Milwaukee, Wis.) was added. Triethylamine (202g,2mol) was slowly added dropwise at 1 drop per second; the temperature does not exceed 5 ℃. The mixture was stirred overnight. On day 2, 200mL of concentrated HCl (12M) was mixed with 1800mL of water. To the reaction mixture was slowly added 200mL of the acid solution. The mixture became clear and was transferred to a 2L separatory funnel and the organic layer was separated from the aqueous layer. Using the acid solution 9X200mL, followed byWater 1x200mL extracted the organic layer. The organic layer was then separated and dried over sodium sulfate and filtered. The dichloromethane was then evaporated under reduced pressure and the oil residue was dissolved in 40mL ethyl acetate and 60mL hexane was added slowly with stirring; it was then covered with parafilm and kept overnight in a refrigerator at 5 ℃. The next day the white crystals were suction filtered and washed with 100mL of cold hexane and then air dried. The mother liquor was kept in a refrigerator for 9 hours and a second batch of crystals was formed and allowed to air dry. The mother liquor from the second batch was frozen overnight to form a third batch of crystals and allowed to air dry. The combined batches gave a yield of 117.3 g, 0.39 mol. The yield is 82 percent, m.p.53-55 percent, p.C₁₀H₁₅Cl₂N₂O₂Calculated values of P (F.W.297.13) analysis C,40.44%, H,5.09%, N,9.43%, found C,39.7%, H4.97%, N, 9.00%.

Example 2

This example describes the synthesis of IPM (M, N' -bis (2-chloroethyl) phosphorodiamidic acid (phosphorodiamidic acid)) from the IPMphenyl ester described in example 1.

The white solid ester from example 1 (0.39mol) was dissolved in 100mL95% ethanol and added to a Parr flask and 2.5g PtO was added₂. Hydrogenating the suspension at 50 PSI; after 2 hours the hydrogenation was terminated and 2.5g PtO were carefully added with stirring₂. Hydrogenation was continued at 50 PSI for 2 hours. It was then terminated, the system was brought to ambient pressure and heated on a hot plate with magnetic stirring. When the suspension boiled, it was immediately filtered by suction through a 5.5cm suction funnel using 2 layers of filter paper and the supernatant was stored at 5 ℃ for 2 hours; the catalyst was stored and added to the parr flask and stored overnight in a refrigerator. The white solid formed is filtered by suction through a 9cm suction funnel and stored in a pesticide-free bottleInternal; the mother liquor was added to a Parr flask and 1.25 grams of PtO were added₂The system was hydrogenated at 50 PSI for 2 hours. It was terminated, heated and filtered as before and the mother liquor was kept in a refrigerator overnight. The white crystals formed were suction filtered and the mother liquor was kept in a refrigerator overnight. The white crystals formed were suction filtered and combined with the first batch. The mother liquor was collected in a parr flask containing the used catalyst and 1.25 grams of PtO was added₂And hydrogenation was continued at 50 PSI for 2 hours. It was then terminated, heated and filtered to yield a third batch of material, which was combined with batches 1 and 2. The combined batch was stirred in 150mL acetone for 30 minutes, then stored at 5 ℃ for 2 hours and then filtered and stored in a vacuum desiccator for 2 hours. The yield is 38g, 0.17mol and 44 percent; mp (calibration) 112 and 114 for C₄H₁₁N₂O₂PCl₂(F.W.221.11) assay calculated C,21.73%, H,5.01%, N,12.67%, found C,22.12%, H5.02%, N, 12.23%.

Example 3

This example describes the preparation of IPM lysine salts from IPM produced according to example 2. L-lysine (26.4g) was weighed and water (6L) was metered in. L-lysine was added to water with stirring at 2-8 ℃. The bulk drug substance IPM (20g) was weighed and added slowly to the lysine solution with stirring at 2-8 ℃.

Once dissolved at 2-8 ℃, the solution was passed through a sterile antimicrobial filter (0.22 micron). The solution was maintained at 2-8 ℃ and dispersed into vials under sterile conditions.

The dissolved product was then lyophilized under the following conditions.

Alternatively, the dissolved product may be lyophilized under the following conditions

The vials were sealed under sterile conditions according to standard procedures. The lyophilized IPM lysine salt was packaged in an unprinted glass bottle with a corrugated rubber sealing plug. The container/closure system does not include a liner. Anion electrospray mass spectrometry revealed IPM (LYS)₂Characteristic peaks at M =219.0, 441.0 (dimer) and 662.7 mass units (trimer). In FIGS. 2-4D is provided₂IPM in O (LYS)₂1H NMR and¹³c NMR spectrum.

Cyclohexylamine and ammonium salts of IPM were prepared as described above for lysine salts. Each of these salts was isolated with a stoichiometry of 2:1 amine: IPM.

Example 4

This example describes the evaluation of IPM against several different cancer cell lines implanted in mice. Mice were well tolerated Intraperitoneal (IP) and Intravenous (IV) IPM treatment in each study; the only toxicity was the organ pathology associated with the induced tumor observed at necropsy.

First, IPM was evaluated on two L1210 variant L1210/0 and L1210/CPA cell lines implanted in mice, compared to Ifos. The dose of IPM was 50% of the dose of Ifos. ILS was observed for all three actives in the L1210/0 treatment group. However, for the L1210/CPA model, IPM treatment showed superiority over the other two groups (Ifos vs CPA). In CPA resistant tumor lines, IPM treated animals had a 2-fold increase in survival and a 7-fold decrease in tumor burden. For the L1210/0 tumor model, IPM activity was equal to CPA and Ifos, but dose was lower than them. This indicates that CPA resistant cells are not cross-resistant to IPM. The results of this study are reported in table 2 below.

TABLE 2

Treatment: IP, day 2 only, highest non-toxic dose (LD) over a range of doses₁₀Or LD₁₀Below).

IP;10⁶Individual cells in male CDF₁In mice.

The second study demonstrated the inhibitory effect of IPM on Lewis lung cancer in mice implanted with Lewis lung cancer tumors. Single day 2 IP administration of CPA, Ifos, PM and IPM to Lewis lung cancer bearing mice revealed that IPM produced 6/10 tumor-free survivors compared to 7/10 for Ifos and 5/10 for CPA at an equivalent toxic dose. A single dose regimen was used for each active agent and the activity (T-C) recorded was the same between all 4 active agents.

The results of this study, recorded in table 3, demonstrate that IPM is effective against Lewis lung cancer.

TABLE 3

Reaction of Lewis lung carcinoma to Isophosphoramide mustard

Implant size 20-30mg, implant site: subcutaneous, medication, IP

Tumor growth delay (T-C), where T = the median time (days) required for the treated group tumors to reach a predetermined weight (750mg), the above time (median of 120 each) for the C control group tumors. Tumor-free survivors were excluded from these calculations.

Control group: the number of days to death =29, the number of times to 750mg of tumor =10.4 days; there were no tumor-free survivors in 30 control mice.

Increased longevity, not including survivors.

Delta Log is calculated according to₁₀Cell killing (total): log kill = T-C value/(3.32T)₄). Wherein T is₄Tumor volume-doubling time determined as a best-fit straight line based on exponentially growing control tumors (100-400mg range). In this experiment, T for Lewis tumors₄=1.2。

The third study evaluated the efficacy of IPM in inhibiting the growth of B16 melanoma. A single dose administration of 150mg IPM revealed that IPM was slightly inferior to CPA but better than Ifos in this animal model of resistance. There were no statistical differences between% ILS responses between the three therapeutic agents. The results of this study, recorded in table 4, confirm the efficacy of IPM on melanoma.

TABLE 4

Comparison of subcutaneous B16 melanoma response to cyclophosphamide, ifosfamide mustard, phosphoramide mustard and ifosfamide

See footnotes of table 3 above. The predetermined weight was 750 mg.

The fourth study evaluated the role of IPM in inhibiting P388 leukemia in mice. In this animal model, as by>log₁₀Cell killingAs shown, IPM was equally effective against IP implanted P388 leukemia as CPA and Ifos, however, it produced fewer tumor-free survivors. However, for the P388/CPA tumor model, IPM was significantly improved in cell killing and% ILS compared to CPA and Ifos. The results of this study are reported in table 5. All data are statistically significant and demonstrate that IPM can be used against CPA resistant or treated tumors and in patients pre-treated with other active agents.

TABLE 5

Activity of Isophosphoramide mustard against P388/0 and P388/CPA leukemias (in # LD from dose-response study₁₀Optimum response at dose)

Treatment IP, day 1 only, highest non-toxic dose (LD) within a series of doses₁₀Or LD₁₀Below).

Implant IP 10⁶Individual cell in female CDF₁In mice.

The fifth study evaluated the inhibitory effect of IPM on implanted M5076 sarcoma in mice. IPM IP was injected into growing tumors at a dose of 18-40mg/kg daily for 5 consecutive days (the compound was injected daily IP on days 11-15). T-C is 40mg/kg for 6.1 days. These doses were well tolerated and the response was significantly improved. Mice are well tolerated with IP treatment; the only toxicity was the organ pathology associated with the induced tumor observed in autopsy. The results of this study, recorded in table 6, demonstrate that IPM is effective against sarcoma in a dose-dependent manner.

TABLE 6

Response of SC-implanted M5076 sarcoma to treatment with IPM

The sixth study evaluated the inhibition of mouse implanted 16/C breast tumors. Mice were implanted with 16/C breast tumors and treated with CPA, Ifos and IPM as separate active agents when tumors were accessible/measurable. CPA and Ifos were used as IPM controls. IP was administered at a dose of 30-60 mg/kg/day for 4 days starting on day 7 after tumor implantation. At all doses of the three agents, IPM had a statistical improvement in activity compared to CPA and Ifos. IPM is superior in 'days to reach 2 doublings' and 'days delayed (T-C)' when compared to Ifos and CPA against this aggressive murine mammary tumor at the same dose/day. All ratios are within the confidence limits. These data (recorded in table 7) confirm the efficacy of IPM against breast tumors and the 4-day administration further supports the superiority of IPM over multiple administrations.

TABLE 7

The seventh study evaluated the effect of IPM on IP implanted human lox-IMVI melanoma. Nude mice were IP implanted with human Lox melanoma and treated with CPA or IPM for 5 days. Both doses were 40mg/kg IV x5 days daily. The% ILS of CPA is +121 and the% ILS of IPM is + 52. However, an excellent response was observed and the dose was well tolerated. The reaction is within the confidence level range. The results of this study (recorded in table 8) confirm the efficacy of IV administration of IPM and further confirm the efficacy of IPM on human melanoma.

TABLE 8

The eighth study evaluated the inhibition of human MX-1 breast tumors using IPM. CPA, Ifos or IPM were administered on a daily X5 day schedule starting on day 12 (post-implantation) compared at 40-60mg/kg daily IP. The data recorded in table 9 demonstrate that IPM is active against human breast tumors. All ratios are within confidence limits.

TABLE 9

Response of SC MX-1 mammary tumors to treatment with CPA, IFOS and IPM

Example 5

This example compares IPM and IPM (LYS)₂And IPM (NH)₄)₂Efficacy of salt on various hyperproliferative cell lines.

From day 6 (post-implantation), IPM and IPM (LYS) were compared when the compound was administered by the IP route for 5 consecutive days at a dose of 20-125mg/kg daily x5 days₂Salts and IPM (NH)₄)₂Efficacy of salt on Lewis mouse lung tumors. IPM and its lysine salts are equally active at doses reflecting a 2-fold increase in the MTD (mg/kg/dose) of the salt over the parent drug. All ratios are within the confidence limits. Mice are well tolerant to IP administration of the salt; the only toxicity was the organ pathology associated with the induced tumor observed at necropsy. The results of this study (recorded in Table 10) confirm that IPM (LYS)₂Shows equivalent to IPM in resisting Lewis mouse lung tumor, and IPM (NH)₄)₂The salt is effective against Lewis mouse lung tumors.

Watch 10

Lewis mouse lung tumor

Implant 20-30mg of tumor fragments

Route of treatment intraperitoneal

Protocol q1d x5 day 6 Start

Despite statistical differences in T-C values (P =0.004), antitumor activity was comparable.

IPM, IPM (LYS) in terms of inhibition of MX-1 breast tumors₂Salts and IPM (NH)₄)₂Second comparison of the efficacy of salt. In this study, IPM and IPM (LYS) were compared starting on day 12 after implantation of MX-1 mammary tumors in mice when administered IP at a dose of 20-100 mg/kg/day x5 days₂The action of salt. IPM (LYS)₂The salts are 8-fold better than IPM at comparable dosages administered. The MTD of the lysine salt is also higher. All ratios are within confidence limits. Mice well tolerated use of IPM (LYS)₂And IPM (NH)₄)₂Salt IP therapy; the only toxicity was the organ pathology associated with the induced tumor observed at necropsy. This data (recorded in Table 11) demonstrates IPM (LYS)₂Salts and IPM (NH)₄)₂The salt is obviously superior to IPM in the aspect of resisting human breast tumor cells.

TABLE 11

MX-1 human mammary tumor

Implant 20-30mg of tumor fragments are subcutaneously implanted in mammary gland fat pad

Route of treatment intraperitoneal

Scheme q1d x5, starting on day 12

P-value =0.041

Example 6

This example describes the evaluation of acute toxicity of isophosphoramide mustard lysine salt after 3 days of daily intravenous (bolus) injection in mice. The study consisted of two phases.

The first phase, dose range-finding phase, consisted of 4 treatment groups (1 mouse/sex/group) which received the test preparation as a single daily dose for 3 consecutive days, each at a corresponding dose level of 100, 200, 400 and 600 mg/kg. Vehicle is 0.9% sodium chloride USP for injection, all doses being constant volume of 15 mL/kg. Animals were observed for 7 days after dosing. After the 7-day observation period, all survivors of the dose range-finding period at day 10 are listed in appendix F of this report. The dose levels selected for the main study period were 50, 75, 100, 200, 300, 500 and 600mg/kg based on the number of deaths noted in the dose range-finding period at 200, 400 and 600mg/kg (see below).

The second phase of the main study consisted of 8 treatment groups (5 mice/sex/group) which received a single daily dose of the test article for 3 consecutive days, each at a corresponding dose level of 50, 75, 100, 200, 300, 400, 500 and 600 mg/kg. An additional 1 group (5 mice/sex) served as a control group for the parent compound and received the isophosphoramide mustard parent compound at a dose level of 150mg/kg in the same manner. Vehicle is 0.9% sodium chloride USP for injection, all doses being constant volume of 15 mL/kg. Animals were observed for 11 days after the 3 day dosing period.

All animals were observed twice daily for mortality, morbidity, and food and water utilization. Observations of clinical signs were made daily during the course of the study (about 1 and 4 hours post-dose on days 1, 2 and 3, and 1 time per day on the non-dose days). Body weights of all surviving animals were measured and recorded on day 2 post-receiving, before randomization and on days-1 and 7. Body weights were also determined on day 14 for all surviving animals in the main study period. Visual assessment was performed on each of the primary study animals at necropsy (day 15).

Animal acquisition and environmental adaptation:

a total of 62 male and 61 female Crl: CD-l (lCR) BR mice (about 6 weeks old) were received from Charles River Laboratories, Portage, Michigan on 21 days 4/2003. During the 7-16 day acclimation period, the animal's sex was verified, weighed and observed twice daily for general health and any signs of disease. Upon receipt, animals were housed in 3-4 mice/cage to accommodate the automatic watering system. Animals were housed individually 3 days after receiving. All animals were given detailed clinical observations prior to study selection.

Randomization, study assignment and maintenance:

mice were weighed and examined for signs of disease and other physical abnormalities before being dispensed for study. Animals assigned for study had a body weight within 20% of the average body weight for each sex. Animals were entered into the treatment group using a simple randomization procedure. Additional animals obtained from this study were euthanized by carbon dioxide inhalation and discarded.

49 male and 49 female mice (weighing 24.8-29.1g and 21.5-24.2g, respectively, at randomization) were assigned to the treatment groups identified in table 12.

Each animal was assigned for Provantis^TMAnd a microchip with a unique identification number is implanted. Each animal number, implant number or study number constitutes an identifying feature that is unique to each animal. The cages were determined according to animal number, study number, group number and gender. As the data is recorded, animal identification characteristics were verified during the course of the study.

Animals were housed individually in suspended stainless steel wire mesh cages. Fluorescent illumination was provided for about 12 hours per day and controlled by an automatic timer. Temperature and humidity were monitored and recorded daily and maintained at 68-74 ° F and 30-68%, respectively.

The dose level for the dose range-finding period was selected based on data obtained from previous studies. The dose level for the main study period was set after reviewing the results from the dose range-finding period, but for the 150mg/kg parent compound control group, the dose level was selected based on data obtained from previous studies.

TABLE 12

Administration:

the 4 range-finding treatment groups (1 mouse/sex/group) received test article as a single daily dose by intravenous (bolus) injection for 3 consecutive days with corresponding dose levels of 100, 200, 400 and 600 mg/kg. All doses were 15mL/kg in volume and based on the most recent body weight.

The 8 main study treatment groups received the test article as a single daily dose by intravenous (bolus) injection for 3 consecutive days, with corresponding dose levels of 50, 75, 100, 200, 300, 400, 500 and 600 mg/kg. An additional 1 group (5 mice/sex) served as the parent compound control group and received the isophosphoramide mustard parent compound at a dose level of 150mg/kg in the same manner. All doses were 15mL/kg in volume and based on the most recent body weight.

When the animal is restrained, the drug delivery formulation is administered through a needle inserted into the tail vein and the presence of blood in the needle mount is observed to ensure proper placement of the needle in the vein. Each animal was then dosed in absolute dose volumes.

Observation and inspection:

all mice were observed twice daily for morbidity, mortality, injury and food and water utilization throughout the duration of the study.

Detailed clinical examinations were performed on each animal at 1 and 4 hours post-dose on days 1, 2 and 3 and 1 time per day on non-dose days. Observations include, but are not limited to, the evaluation of skin, fur, eyes, ears, nose, mouth, chest, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as saliva secretion; and nervous system effects including tremor, convulsions, responsiveness to manipulation, and abnormal behavior.

Body weights of all surviving animals were determined and recorded on day 2 post-receiving, before randomization and at days-1 and 7. Body weights were also determined on day 14 for all surviving animals in the main study period. Body weights recorded after receipt and before random selection were not reported but were saved in the study file.

On day 10, all dose range-finding surviving animals were euthanized and discarded. No necropsy was performed on any dose range-finding animals. All animals in the main study period received a full necropsy under the operating procedure approved by the veterinary pathologist. At termination of the study, surviving animals of all major study periods were euthanized by carbon dioxide inhalation and exsanguination via the abdominal vena cava.

Each animal was carefully examined for external abnormalities, including tumor mass. The skin was retraced from the ventral median incision and any subcutaneous abnormalities were identified and correlated with cases found antemortem. Abnormalities of the abdominal, thoracic and cranial cavities were examined and organs were removed and examined. All abnormal conditions are recorded. The tissue was not preserved and animal carcasses were discarded.

Statistics:

if appropriate, usingMedium Probit program (SAS institute, Inc.User's Guide, Version 6, Fourth Edition, Volume 2.Cary NC: SAS Institute;1989) calculates LD₅₀And LD₁₀And its 95% confidence limit (main study treatment group).

The computer systems used in conducting this study are listed in table 13.

Watch 13

As a result:

the following data are the results of the identified primary study period.

A summary of the mortality results is listed in table 14 below. Mortality results generally showed a typical dose-response effect, with IPM lysine salt toxicity being slightly higher in females than in males. The IPM parent compound control group showed the expected mortality and toxicity in females was higher than in males, which correlates with data from previous studies.

TABLE 14

Intravenous LD of IPM lysine salt in mice (pooled sex)₁₀Calculated as 133mg/kg (95% confidence limit 65-172), and intravenous LD₅₀The calculation was 220mg/kg (95% confidence limit 184-265 mg/kg).

LD of male and female₁₀The values were 140 and 179mg/kg (95% confidence limits for males 12-199 mg/kg; not calculated for females), respectively, while LD for males and females₅₀Values were 247 and 197mg/kg (95% confidence limit for males 187-.

No treatment-related visual findings were noted in either gender in the post-mortem observation.

And (4) conclusion:

mortality results generally showed typical dose-response effects, with IPM lysine salt being slightly more toxic in females than in males. No animal death at 50 or 75 mg/kg; at 100mg/kg, 1 out of 10 animals died; at 200mg/kg, 3 out of 10 animals died; at 300mg/kg, 9 out of 10 animals died; and at 400, 500 and 600mg/kg all animals died. The IPM parent compound control group showed the expected mortality (only 5 out of 10 animals) and the toxicity in female animals was higher than in male animals, which correlates with available data from previous studies. The onset of death at study time was slightly delayed, with the first death occurring at day 6 and the last at day 12. Clinical signs were observed in both sexes that roughly reflected the pre-mortem worsening status of the mice. These clinical signs include dying, decreased activity, increased activity, swelling (tail, nose/mouth and/or face), shortness of breath/slow/light/difficult/audible, tremor, cold skin to the touch, profound appearance, bent posture, impaired limb function, hair loss in the back and/or anogenital area, little/no feces, and reduced urination. By day 7, the mean weight gain reduction or in many cases weight loss associated with treatment was noted in the surviving animals, with at least partial recovery in those animals that survived to study termination by day 14. No visual findings associated with treatment were noted at necropsy.

Based on the conditions and findings of this study, intravenous LD of IPM 2Lys in mice (pooled sex)₁₀Calculated as 133mg/kg (95% confidence limit 65-172), and intravenous LD₅₀The calculation was 220mg/kg (95% confidence limit 184-265 mg/kg).

Example 7

This example summarizes extensive preclinical data results for IPM and its lysine salt. This data was used to design dosage regimens for use in human clinical trials.

Toxicity of IPM and its lysine salt has been studied by preclinical acute and subacute studies using mice, rats and dogs. Single dose oral, Intravenous (IV) and Intraperitoneal (IP) routes of administration of IPM have been studied in mice and rats. Multiple daily doses of-IV and IP were studied in mice and dogs. Sub-acute intravenous (3-day) administration in mice and dogs provides toxicology/pharmacokinetic information on toxicity and drug profiles used to design dosing and dosage regimens in humans. Sub-acute IV (3-day) dosing with IPM lysine salt was performed in mice.

Based on the results of the dose range finding study, IPM at doses higher than expected was required to produce mortality. Male oral LD for rats₅₀Values were calculated as 4443mg/kg, female 2786mg/kg and both sexes combined 3560 mg/kg. In each case, a 95% confidence limit may be calculated.

For mice, male oral LD₅₀Values were calculated as 1014mg/kg (95% confidence limit), females as 1962mg/kg (95% confidence limit 1523-2983mg/kg) and both sexes combined as 1432mg/kg (95% confidence limit 1128-1742 mg/kg).

Single dose intravenous LD in males for rats₅₀The value was calculated as 567mg/kg, female 400mg/kg and both sexes combined 428 mg/kg. In each case, a 95% confidence limit could not be calculated. In the case of mice, male intravenous LD₅₀The value was calculated as 929mg/kg (95% confidence limit), females were calculated as 484mg/kg (95% confidence limit 72-1364mg/kg), and both sexes were combined and calculated as 688mg/kg (95% confidence limit 398-1366 mg/kg).

Administration of IPM by the IV and IP routes did produce acute death in mice, rats and dogs. Mice were also evaluatedAnd rats orally and LD in these rodent species₅₀The values were determined to be in the range of 1.4-3.5 g/kg. Acute intravenous toxicity symptoms in mice, rats and dogs include reduced appetite, diarrhea, reduced activity and death.

The acceptable dose from the three (3) day dosing study is significantly different from the single dose regimen. The effect of the drug on bone marrow, spleen and renal tubular function was evaluated. The effect of IPM on these organs appears to contribute to the cause of death in both types of animals. This is summarized as follows.

IPM sub-acute IV studies in mice provide information about LD₁₀Values and information on possible toxicity in humans. Mortality results showed typical dose-response effects, with IPM being slightly more toxic in females than in males.

LD of intravenous IPM in mice (combined sex)₁₀The value was calculated as 119mg/kg (95% confidence limit 87-134mg/kg), and intravenous LD₅₀The value was calculated to be 149mg/kg (95% confidence limit of 132-169 mg/kg). LD of male and female₁₀Values of 168 and 125mg/kg, respectively, for LD in males and females₅₀The values are 176 and 132, respectively. In each case, a 95% confidence limit could not be calculated.

Sub-acute IPM lysine salt study included 40 male and 40 female mice in total (Crl: CD-1(1CR) BR) with body weights 24.8-29.1g and 21.5-24.2g at randomization, treated with 50-600mg/kg IV daily x3 days (Table 8.8).

Intravenous LD for 3-day mouse study with IPMLYS salts₁₀Calculated as 133mg/kg (95% confidence limit 65-172mg/kg) (combined sex), and intravenous LD₅₀The calculation was 220mg/kg (95% confidence limit 184-265mg/kg) (combined gender). LD of male and female₁₀The values were 140 and 179mg/kg respectively (95% confidence limits for males between 12 and 199 mg/kg; not calculated for females). LD of male and female₅₀Values were 247 and 179mg/kg, respectively (95% confidence limit for males 187-330 mg/kg; not calculated for females).

IPM lysine salts generally showed typical dose-response effects with slightly higher toxicity observed in females. No mice died at 50, 75 or 200 mg/kg; at 100mg/kg, 1 out of 10 animals died; at 300mg/kg, 9 out of 10 animals died; and at 400, 500 and 600mg/kg, all mice died. The maternal ipm control group showed the expected mortality and toxicity in females was higher than in males, which correlates with available data from previous studies. The onset of death at study time was slightly delayed, with the first death occurring at day 6 and the last at day 12. Clinical signs were observed in both sexes that roughly reflected the pre-mortem worsening status of the mice.

Based on microscopic findings, IPM administered daily for 3 consecutive days, either alone or as lysine salt IV, produced treatment-related bone marrow depletion, tubular necrosis, or a combination of both and was considered the cause of death. For IPM, there was severe bone marrow depletion in males at 178mg/kg and higher, and in females at 133mg/kg and higher. Tubular necrosis occurred in males at doses of 237mg/kg and higher, and tubular necrosis occurred in females at doses of 133mg/kg and higher. In addition, spleen lymphoid tissue depletion was noted in most male animals and all female animals that died during the course of the study. At 75mg/kg, no obvious treatment-related microscopic findings were noted in either gender. Clinical signs generally secondary to the worsening state of the mice before death were observed, but no significant evidence of body weight effects was observed in mice that survived to study termination.

Intravenous LD of Isophosphoramide mustard (IPM) and its lysine salt administered daily for 3 consecutive days₁₀Calculated as 119mg/kg and 133mg/kg, respectively, LD₅₀Calculated as 149mg/kg and 220mg/kg, respectively.

Acute and sub-acute toxicity studies were performed in rodents and dogs using IPM and its lysine salts. Will alsoThese studies were used to develop acceptable starting doses for human studies. A summary of toxicity data for rodents and dogs administered IV to IPM is recorded in Table 15 and IV is administered IPM (LYS)₂A summary of the mouse toxicity data of (a) is recorded in table 16.

Watch 15

TABLE 16

Intravenous IPM lysine salts

The dog's IPM MTD was 5 mg/kg/day x3 days and the corresponding initial dose was 100mg/m in humans²Three (3) consecutive days per day should be a safe starting point. Just IPM (LYS)₂In particular, LD for an intravenous three (3) day dose regimen in mice₁₀The calculation was 133 mg/kg/day x3 days. Consider IPM (LYS)₂Is the least toxic alkylating agent with a steep therapeutic range. The Mean Toxic Dose (MTD) of the lysine salt in humans was estimated as LD in mice based on mg/kg₁₀1/10 or 40mg/m²/d。

The estimated equivalent human IV doses are recorded in table 17.

TABLE 17

Estimated equivalent human intravenous dose

Medicine	Species (II)	Sub-acute IV LD10	Equivalent human IV dose
				IPM	Mouse	119mg/kg/d	30mg/m2/d
IPM	Dog	5mg/kg/d	100mg/m2/d
				IPM lysine salt	Mouse	133mg/kg/d	40mg/m2/d

Example 8

This example describes cancer treatment in human subjects with metastatic ovarian cancer.

IPM500mg/m for intravenous infusion daily for 3 consecutive days²Treating the subject. Serum electrolytes, such as phosphorus and chlorine, were corrected using supplemental electrolytes and discontinued after 7 days. BUN and creatinine were monitored in the normal range.

Example 9

This exampleThe use of IPM (LYS) is described₂Results of treatment of human subjects. Up to now, IPM (LYS) has been used₂Four (4) patients with advanced cancer were treated.

Initial dose of IPM lysine salt was 30mg/m²Daily by intravenous administration for three (3) consecutive days. 1 patient (group) was treated with increasing doses every 21-28 days in order to visualize toxicity. If there were no serious toxic events, the dose was increased by 40%. 4 patients have been treated-1 patient per dose-30, 42, 59 and 83mg/m²Daily administration by IV for 3 consecutive days without severe toxicity. By daily IV administration of 83mg/m²IPM·(LYS)₂After 3 days, the disease remained stable in 1 patient with rectal cancer.

Example 10

This example describes the treatment of non-small cell lung cancer that progresses to metastatic invasive moderately differentiated adenocarcinoma. The status of the disease can be confirmed by CAT scanning.

At 350mg/m per day²Isophosphoramide mechlorethamine lysine salt was administered intravenously for 3 consecutive days. After a 21-day rest period, the 3-day treatment regimen was repeated 1 time. Daily blood fluid chemistry and hematology studies were monitored during the treatment. Cancer status was monitored by CAT scan.

Example 11

This example demonstrates the effect of amine salt formation on compound stability.

Samples of lyophilized isophosphoramide mustard and its lysine salt were stored under different conditions and tested for purity. The results are listed in the following table:

compound (I)	0 month	1 month	3 months old	1 year
					IPM,-23℃	97%	88%	70%
IPM-LYS,-23℃	98%		98%	100%
					IPM-LYS, Environment	98%	98%	65%

In view of the above, the present invention also includes the following inventions.

1. A compound of the formula:

wherein A is⁺Represents a conjugated acid selected from quaternary ammonium, basic amino acids, aliphatic ammonium, heterocyclic ammonium, aromatic ammonium, substituted and unsubstituted pyridinesGuanidine (guanidine)And amidinesThe ammonium species of (a); and is

X and Y independently represent a leaving group.

2. A compound of item 1, wherein A⁺Represents BH⁺And B is an amine selected from the group consisting of basic amino acids, pyridine, N, NN-dimethylaminopyridine, diazabicyclononane, diazabicycloundecene, N-methyl-N-ethylamine, diethylamine, triethylamine, diisopropylethylamine, mono-, bis-or tris- (2-hydroxyethyl) amine, 2-hydroxy-tert-butylamine, tris (hydroxymethyl) methylamine, N-dimethyl-N- (2-hydroxyethyl) amine, tris- (2-hydroxyethyl) amine and N-methyl-D-glucamine.

3. A compound of item 2, wherein the compound has the formula:

and G represents a second ammonium or amine species.

4. The compound of item 2, wherein B is selected from the group consisting of lysine, homolysine, arginine, homoarginine, histidine, ornithine and combinations thereof.

5. The compound of item 2, wherein B is lysine.

6. A compound of item 2, wherein X and Y are independently selected from halogen and sulfonate.

7. A compound of item 2, wherein X and Y are halogen.

8. A compound of item 2, wherein X and Y are chloro.

9. A compound of item 3, wherein B and G are lysine and X and Y represent chloro.

10. A compound comprising an isophosphoramide mustard anion and at least one amine, ammonium cation, or both.

11. The compound of item 10, further comprising a second amine or ammonium cation.

12. The compound of item 10, further comprising an alkali metal cation or a quaternary ammonium cation.

13. The compound of item 10, wherein the ammonium cation is lysine.

14. A pharmaceutical composition comprising a compound of item 1 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition of item 14, wherein X and Y are independently selected from the group consisting of halogen and sulfonate.

16. The pharmaceutical composition of item 14, wherein B + is selected from the group consisting of lysine, homolysine, arginine, homoarginine, histidine, ornithine and combinations thereof.

17. The pharmaceutical composition of item 14, wherein X and Y are halogen.

18. The pharmaceutical composition of item 14, wherein X and Y are chloro.

19. The pharmaceutical composition of item 14, wherein the composition comprises a solution formulated for administration to a human subject.

20. The pharmaceutical composition of item 19, wherein said solution comprises about 0.1mg/mL to about 250mg/mL of said compound.

21. The pharmaceutical composition of item 20, wherein said solution comprises about 20 to about 100mg/mL of said compound.

22. The pharmaceutical composition of item 14, wherein the composition comprises about 200mg to about 1500mg of the compound per dosage unit.

23. A pharmaceutical composition comprising a compound of item 3 and a pharmaceutically acceptable carrier.

24. A pharmaceutical composition comprising a compound of item 9 and a pharmaceutically acceptable carrier.

25. A pharmaceutical composition comprising isophosphoramide mustard and an amine base, an ammonium counterion, or both; and a pharmaceutically acceptable carrier.

26. A pharmaceutical composition comprising a salt of isophosphoramide mustard and an amine base; and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition of clause 25, wherein the composition comprises two equivalents of the amine base, the ammonium counterion, or both per equivalent of isophosphoramide mustard.

28. The pharmaceutical composition of clause 25, wherein the amine base, ammonium counterion, or both are selected from the group consisting of basic amino acids, aliphatic amines, di-and trialkylamines, heterocyclic amines, aromatic amines, substituted and unsubstituted pyridines, guanidines, and amidines.

29. The pharmaceutical composition of clause 25, wherein the amine base, ammonium counterion, or both are selected from the group consisting of lysine, homolysine, arginine, homoarginine, histidine, ornithine, and combinations thereof.

30. The composition of clause 25, wherein the amine base is lysine.

31. A method for treating a subject having a hyperproliferative disease comprising administering to the subject a compound of item 1.

32. The method of item 31, comprising administering to the subject about 10mg/m²A day to about 700mg/m²The compound was given a daily dose.

33. The method of the item 31, wherein,comprises administering to the subject about 100mg/m²A day to about 500mg/m²The compound was given a daily dose.

34. The method of item 31, further comprising administering to the subject a second compound.

35. The method of item 34, wherein the second compound is selected from the group consisting of a microtubule binding agent, a DNA intercalating or crosslinking agent, a DNA synthesis inhibitor, a DNA and/or RNA transcription inhibitor, an enzyme inhibitor, a gene modulator, an enzyme, an antibody, and an angiogenesis inhibitor.

36. The method of item 34, wherein the second compound is selected from the group consisting of paclitaxel, docetaxel, daunorubicin, cisplatin, carboplatin, oxaliplatin, colchicine, dolastatin 15, nocodazole, podophyllotoxin, rhizomycin, vinblastine, vindesine, vinorelbine (navelbine), epothilone, mitomycin, bleomycin, chlorambucil, carmustine, melphalan, mitoxantrone, 5-fluoro-5 'deoxyuridine, camptothecin, topotecan, irinotecan, etoposide, teniposide, geldanamycin, methotrexate, doxorubicin, actinomycin D, mifepristone, raloxifene, 5-azacytidine, 5-aza-2' -deoxycytidine, zelaraine, tamoxifen, 4-hydroxytamoxifene, apigenin, rapamycin, angiostatin K1-3, L-asparaginase, staurosporine, genistein, fumagillin, endostatin, thalidomide and analogues thereof.

37. The method of clause 34, further comprising administering to the subject a third compound, wherein the third compound is selected from the group consisting of a microtubule binding agent, a DNA intercalating or crosslinking agent, a DNA synthesis inhibitor, a DNA and/or RNA transcription inhibitor, an enzyme inhibitor, a gene modulator, an enzyme, an antibody, and an angiogenesis inhibitor.

38. The method of item 31, wherein said hyperproliferative disease comprises breast cancer, bladder cancer, bone cancer, cervical cancer, colon cancer, central nervous system cancer, esophageal cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hodgkin's disease, non-hodgkin's lymphoma, laryngeal cancer, leukemia, lung cancer, melanoma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, retinoblastoma, gastric cancer, testicular cancer, or wilms ' tumor.

39. The method of item 31, wherein said hyperproliferative disease comprises an adenocarcinoma, a sarcoma, a leukemia, or a lymphoma.

40. The method of item 31, wherein said hyperproliferative disease comprises a skin tumor.

41. The method of item 40, wherein said tumor is metastatic.

42. The method of clause 40, wherein the hyperproliferative disease comprises melanoma, sarcoma, or both.

43. The method of clause 42, wherein the hyperproliferative disease comprises melanoma.

44. The method of item 31, wherein said hyperproliferative disease comprises a carcinoma of the ovary, breast, lung, prostate, or rectum.

45. The method of item 44, wherein said lung tumor is a non-small cell or small cell lung cancer tumor.

46. The method of clause 45, wherein said lung tumor is a non-small cell lung cancer tumor.

47. A method for treating a subject having a hyperproliferative disease comprising administering to the subject an anti-hyperproliferative agent having the general formula of item 10.

48. A process for producing a lyophilizate comprising a compound of item 1, comprising reacting a compound of the formula

Contacting in the presence of water at least one equivalent of an amine base selected from the group consisting of basic amino acids, aliphatic amines, heterocyclic amines, aromatic amines, substituted and unsubstituted pyridines, guanidines, and amidines, and

freeze-drying the resulting mixture, thereby forming said freeze-dried product.

49. The method of clause 48, wherein X and Y are chloro.

50. The method of clause 48, wherein contacting the compound comprises contacting the compound with at least two equivalents of the amine base.

51. The method of clause 48, wherein the amine base is lysine.

52. A lyophilizate produced by the method of item 48.

53. A compound of the formula:

wherein M is⁺Represents an alkali metal cation and B represents an amine base.

54. The compound of item 2, wherein the compound is an anti-hyperproliferative compound.

55. The compound of item 2, wherein the compound has intravenous LD in a mouse₅₀Intravenous LD greater than Isophosphoramide mechlorethamine₅₀。

56. The compound of item 2, wherein the compound has intravenous LD in a mouse₁₀Intravenous LD greater than Isophosphoramide mechlorethamine₁₀。

57. The compound of item 2, wherein the compound has an intravenous LD in the mouse of about 184 to about 265mg/kg₅₀。

58. The compound of item 2, wherein the compound has an intravenous LD in mouse of about 65 to about 172mg/kg₁₀。

59. The compound of item 2, wherein the compound has an intravenous LD in rats of about 400 to about 570mg/kg₅₀。

60. A stable isophosphoramide mustard salt, wherein the salt has a half-life in the presence of water that is greater than the half-life of isophosphoramide mustard in the presence of water.

61. The lyophilizate of item 52, wherein said compound is more stable than a lyophilized preparation of isophosphoramide mustard.

62. The compound of item 2, wherein the compound is more effective at CPA-resistant tumor growth than CPA, Ifos, or both.

63. A sterile composition comprising a compound of the formula:

wherein X and Y independently represent a leaving group.

64. The composition of clause 63, wherein less than 10% of the decomposition products are present in the compound relative to the compound itself.

65. The composition of clause 63, wherein the composition has a half-life at least twice as long as pure ifosfamide mustard.

66. A method for treating a subject having a hyperproliferative disease, comprising administering to the subject the composition of item 63.

67. A method of purifying a salt of a compound of the formula:

wherein X and Y independently represent a leaving group;

the method comprises filtering a solution of the compound through a sterile antimicrobial filter;

thus, the purified compound undergoes less than 10% decomposition during filtration.

68. The method of clause 67, wherein the purified compound undergoes less than 5% decomposition during filtration.

69. The method of clause 68, wherein the purified compound undergoes less than 1% decomposition during filtration.

70. The pharmaceutical composition produced by the method of clause 67.

71. A method for treating a subject having a hyperproliferative disease comprising administering to the subject the composition of item 70.

Claims (14)

1. A compound of the formula:

wherein A is⁺Represents a conjugated acid selected from quaternary ammonium, basic amino acids, aliphatic ammonium, heterocyclic ammonium, aromatic ammonium, substituted and unsubstituted pyridinesGuanidine (guanidine)And amidinesThe ammonium species of (a); and is

X and Y independently represent a leaving group.

2. The compound of claim 1, wherein a⁺Denotes acyclic aliphatic ammonium, for example tris (hydroxymethyl) methylamine.

3. The compound of claim 1, wherein a⁺Represents BH⁺And B is an amine selected from the group consisting of basic amino acids, pyridine, N-dimethylaminopyridine, diazabicyclononane, diazabicycloundecene, N-methyl-N-ethylamine, diethylamine, triethylamine, diisopropylethylamine, mono-, bis-or tris- (2-hydroxyethyl) amine, 2-hydroxy-tert-butylamine, tris (hydroxymethyl) methylamine, N-dimethyl-N- (2-hydroxyethyl) amine, tris- (2-hydroxyethyl) amine and N-methyl-D-glucamine.

4. The compound of claim 3, wherein the compound has the formula:

and G represents a second ammonium or amine species.

5. A compound according to any preceding claim, wherein X and Y are independently selected from halogen, for example chloro, and sulphonate.

6. A pharmaceutical composition comprising a compound according to any preceding claim and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, comprising a) a solution formulated for administration to a human subject, optionally, the solution comprises from about 0.1mg/mL to about 250mg/mL of the compound, such as from about 20mg/mL to about 100mg/mL of the compound, or the composition comprises from about 200mg to about 1500mg of the compound per dosage unit.

8. A compound or composition according to any preceding claim, for use in treating a subject suffering from a hyperproliferative disease, such as breast cancer, bladder cancer, bone cancer, cervical cancer, colon cancer, central nervous system cancer, esophageal cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hodgkin's disease, non-hodgkin's lymphoma, laryngeal cancer, leukemia, lung cancer, melanoma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, retinoblastoma, stomach cancer, testicular cancer, wilms ' tumor, adenocarcinoma, sarcoma, lymphoma, skin tumor, metastatic tumor, non-small cell lung cancer tumor, or small cell lung cancer tumor; optionally, wherein the compound is administered to the subject in an amount of about 10mg/m²A day to about 700mg/m²A day, e.g. 100mg/m²A day to about 500mg/m²The day is.

9. A compound or composition according to any one of claims 1 to 7 for use in the treatment of a hyperproliferative disease in combination with a second compound, for example a compound selected from: microtubule binding agents, DNA intercalating or crosslinking agents, inhibitors of DNA synthesis, inhibitors of DNA and/or RNA transcription, enzyme inhibitors, gene modulators, enzymes, antibodies and angiogenesis inhibitors, such as: paclitaxel, docetaxel, daunorubicin, cisplatin, carboplatin, oxaliplatin, colchicine, dolastatin 15, nocodazole, podophyllotoxin, rhizoxin, vinblastine, vindesine, vinorelbine (navelbine), epothilone, mitomycin, bleomycin, chlorambucil, carmustine, melphalan, mitoxantrone, 5-fluoro-5 'deoxyuridine, camptothecin, topotecan, irinotecan, etoposide, teniposide, geldanamycin, methotrexate, doxorubicin, actinomycin D, mifepristone, raloxifene, 5-azacytidine, 5-aza-2' -deoxycytidine, zebularine, tamoxifen, 4-hydroxytamoxifen, apigenin, rapamycin, angiostatin K1-3, L-asparaginase, staurosporine, genistein, doxycycline, vindesine, vinorelbine, and other salts thereof, Fumagillin, endostatin, thalidomide, or analogs thereof.

10. The compound or composition of claim 8 or 9, wherein the hyperproliferative disease comprises melanoma, sarcoma, or both.

11. A process for the production of a lyophilisate comprising a compound according to any of claims 1 to 5, comprising reacting a compound of formula

Contacting in the presence of water at least one equivalent of an amine base selected from the group consisting of basic amino acids, aliphatic amines, heterocyclic amines, aromatic amines, substituted and unsubstituted pyridines, guanidines, and amidines, and

freeze-drying the resulting mixture, thereby forming the freeze-dried product.

12. The method of claim 11, wherein contacting the compound comprises contacting the compound with at least two equivalents of the amine base, e.g., tris (hydroxymethyl) methylamine.

13. A lyophile produced by the method of claim 11, optionally wherein the compound is more stable than a lyophilized preparation of isophosphoramide mustard.

14. The compound of any one of claims 1-5, wherein the compound is an anti-hyperproliferative compound having intravenous LD in mouse₁₀Intravenous LD greater than Isophosphoramide mechlorethamine₁₀For example, the compounds have an intravenous LD in mice of about 184 to about 265mg/kg₅₀(ii) a Alternatively, the compound has intravenous LD in mice₁₀Intravenous LD greater than Isophosphoramide mechlorethamine₁₀For example, the compounds have an intravenous LD in mice of about 65 to about 172mg/kg₁₀(ii) a Alternatively, the compounds have an intravenous LD in rats of about 400 to about 570mg/kg₅₀(ii) a Optionally, wherein the compound is more effective against CPA-resistant tumor growth than CPA, Ifos, or both.