Related Applications This application claims the benefit of provisional application 60/466,696 filed April 30, 2003, entitled"Antitumor 2-aminocarbonyl-1, 2-bis (methylsulfonyl)-1-(2-chloroethyl) hydrazines and methods of treating tumors", which is incorporated by reference herein in its entirety.

Background of the Invention Cyclophosphamide represents the antitumor alkylating agent with the least toxicity to the bone marrow and gastrointestinal tract and, in certain instances, represents an agent of choice.

See, Chabner, et al. ,"Antineoplastic Agents", in: The Pharmacological Basis of Therapeutics, Hardman, et al. , (eds. ), Ninth edition, McGraw-Hill, New York, pp. 1233-1287 (1996); Colvin, et al. ,"Alkylating Agents", in: Cancer Chemotherapy : Principles and Practice, Chabner and Collins, (eds. ), J. B. Lippincott Co. , Philadelphia, pp. 276-313 (1990); and Russo. , et al.,"The Role of Aldehyde Dehydrogenase Isoenzymes in Cellular Resistance to Alkylating Agent Cyclophosphamide", in: Enzymology and Molecular Biology of Carbonyl Metabolism, vol. 2, Liss, A. R. (ed. ), New York, pp. 65-79 (1989). This is the result of the conversion of cyclophosphamide's metabolites, hydroxycyclophosphamide and aldophosphamide, to the non- toxic 4-ketocyclophosphamide and carboxycyclophosphamide, respectively, by the relatively high aldehyde dehydrogenase activity present in these tissues. See Chabner, et al. , ibid.

The search for alkylating agents which exhibit enhanced antineoplastic activity in combination with reduced toxicity is an ongoing concern. The focus of the present invention is to combine the aldehyde dehydrogenase-reduced toxicity exhibited by cyclophosphamide with the DNA O6-guanine directed alkylating activity associated with aminocarbonylhydrazines to produce less toxic aminocarbonylhydrazines exhibiting enhanced antineoplastic activity.

Objects of the Invention It is an object of the invention to provide antineoplastic agents effective in the treatment of numerous neoplastic and cancerous conditions, including solid tumors in animals and humans.

It is another aspect of the invention to provide novel aminocarbonylhydrazines exhibiting antineoplastic activity combined with reduced toxicity.

It is an additional object of the invention to provide pharmaceutical compositions based upon the use of these novel antineoplastic agents.

It is still another object of the invention to provide methods of treating neoplasia, including solid tumors, in animals and humans.

These and/or other objects of the invention may be readily gleaned from the description of the invention which follows.

Brief Description of the Figures Figure 1 shows a chemical scheme for producing compounds according to the present invention.

Figure 2 shows a postulated mechanism of activation for a representative compound (R is a morpholine group) as described in more detail in the present specification.

Summary of the Invention The present invention relates to novel aminocarbonylhydrazine compounds of the formula: Where R is a group according to the chemical structure: and Rl, R R3 and R4 are independently selected from H or a Cl-C3 alkyl group; and where applicable, pharmaceutically acceptable salts thereof.

In preferred aspects of the present invention, Rl, R2 and R4 are preferably the same and are preferably selected from H or CH3, more preferably H. R3 is preferably CH3. In cases where R3 or R4 is H or CH3, the compound is preferably in the form of its pharmaceutically acceptable salt form. R is preferably The compounds according to the present invention are produced by synthetic methods which are readily known to those of ordinary skill in the art and include the disclosed chemical synthetic methods.

The present invention also relates to pharmaceutical compositions comprising an effective amount of a compound according to the present invention, including a pharmaceutically acceptable salt thereof, as set forth above. Pharmaceutical compositions according to the present invention preferably also include a pharmaceutically acceptable additive, carrier or excipient.

The present invention also relates to a method for treating neoplasia in mammals comprising administering an antineoplastic effective amount of a compound according to the present invention as otherwise described above to a patient suffering from neoplasia. The treatment of solid malignant tumors comprising administering to a patient an antitumor effective amount of one or more of these agents is a preferred embodiment of the present invention. The treatment of various cancers such as stomach, colon, rectal, liver, pancreatic, lung, breast, cervix uteri, corpus uteri, ovary, prostate, testis, bladder, 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, oesophagus, larynx, kidney, lymphoma, among others. Treatment of various other related disease states may also be effected using the compounds of the present invention.

The treatment of cancerous tumors is particularly preferred.

Compounds according to the present invention are designed to be less toxic to the bone marrow and the intestinal mucosa than aminocarbonyl derivatives of the prior art. Thus, the compounds according to the present invention are expected to show selective toxicity and more importantly, reduced toxicity to the bone marrow and the intestinal mucosa, and enhanced activity to cancer cells. It is expected that these compounds are converted to non-toxic products by aldehyde dehydrogenase after hydroxylation. Aldehyde dehydrogenase is present in relatively high concentrations in the bone marrow and intestinal mucosa, while the hydroxylated species is converted to cytotoxic species in other parts of the body, where the enzyme is in reduced supply. This may help explain the selective toxicity exhibited by compounds according to the present invention. Accordingly, the compounds of the present invention represent a clear advance in the art of treating cancer.

Detailed Description of the Invention The term"patient"is used throughout the specification to describe an animal, preferably a human, to whom treatment with the compositions according to the present invention is provided.

For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal.

The term"neoplasia"is used throughout the specification to refer to the pathological process that results in the formation and growth of a cancerous or malignant neoplasm, i. e., abnormal tissue that grows by cellular proliferation, often more rapidly than normal and continues to grow after the stimuli that initiated the new growth cease. Malignant neoplasms show partial or complete lack of structural organization and functional coordination with the normal tissue and most invade surrounding tissues, metastasize to several sites, and are likely to recur after attempted removal and to cause the death of the patient unless adequately treated. As used herein, the term neoplasia is used to describe all cancerous disease states and embraces or encompasses the pathological process associated with malignant hematogenous, ascitic and solid tumors. Representative cancers include, for example, stomach, colon, rectal, liver, pancreatic, lung, breast, cervix uteri, corpus uteri, ovary, prostate, testis, bladder, 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, oesophagus, larynx, kidney, lymphoma, among others, which may be treated by one or more compounds according to the present invention.

The term"effective amount"is used throughout the specification to describe an amount of the present compound or composition which is used to effect an intended result. In most aspects of the present invention the term effective amount is used in conjunction with the treatment of a patient suffering from neoplasia, in preferred embodiments, a cancerous tumor to prevent the further growth of the neoplasms-to bring that growth under control or even kill cancerous cells and preferably, produce a remission of the tumor.

The term effective amount with respect to the presently described compounds and compositions is used throughout the specification to describe that amount of the compound according to the present invention which is administered to a mammalian patient, especially including a human patient, suffering from cancer, to reduce or inhibit the growth or spread of the cancer, and in particular, a hematogenous, ascitic or solid tumor. Preferably, treatment with the compounds described in the present invention will result in a remission of the malignant hematogenous, ascitic or solid tumor. In the case of solid tumors, in certain preferred aspects, the compounds according to the present invention will inhibit the further growth of the tumor tissue and shrink the existing tumor.

The term coadministered is used to describe the administration of a compound according to the present invention in combination with another drug, generally an anticancer agent, to a patient, regardless of the time of administration, such that effective amounts of the present compounds and the coadministered drugs are present in the patient at the same time during therapeutic treatment.

The present invention is directed to 2-aminocarbonyl-1, 2-bis (methylsulfonyl)-1- (2- chloroethyl) hydrazine compounds of the formula: Where R is a group according to the chemical structure: And R', R2, R3 and R4 are independently selected from H or a C,-C3 alkyl group; or a pharmaceutically acceptable salt thereof.

In preferred aspects of the present invention, R', R2, R3 and R4 are preferably the same and are preferably selected from H or CH3, more preferably H. In cases where R3 or R4 is H or CH3, the compound is preferably in the form of its pharmaceutically acceptable salt form. R is preferably The compounds tested, exhibit pronounced activity against the L1210 leukemia and the B16 melanoma solid tumors.

The present compounds represent prodrug forms of intermediates which are believed to exhibit their activity through chloroethylation and/or carbamoylation mechanisms in cancer cells, but have been designed to exhibit reduced toxicity to bone marrow cells and to cells of the gastrointestinal tract of the patient.

The compounds according to the present invention are primarily useful for their antineoplastic activity, including their activity against solid tumors. In addition, these compositions may also find use as intermediates in the chemical synthesis of other useful antineoplastic agents which are, in turn, useful as therapeutic agents or for other purposes.

Compounds according to the present invention are synthesized by the adaptation of techniques which are well known in the art. For example, as set forth in Figure 1, compounds according to the present invention are synthesized by first reacting 1, 2-bis (methylsulfonyl)-1-(2- chloroethyl) hydrazine (1) with triphosgene in the presence of a tertiary amine such as N, N- diisopropylethylamine (DIEA) to produce 1, 2-bis (methylsulfonyl)-2-chlorocarbonyl-1-(2- chloroethyl) hydrazine (2). Compound 1 may be prepared by following the procedure which is set forth by Shyam, et al., J. Med. Chem., 33,2259-2264 (1990). Compound 2 is then reacted with an appropriate secondary amine in methylene chloride or acetonitrile in the presence of DIEA to produce a compound according to the present invention by displacement of the chloro group in the chlorocarbonyl moiety of compound 2 with an optionally substituted amine as indicated to form the appropriate compound according to the present invention.

After synthesis, the residue may be triturated, washed with dilute acid or water, dried, triturated again and recrystallized from an appropriate solvent, for example, ethanol or ethanol/petroleum ether. Modification of the disclosed chemical synthetic methods may be readily made by those of ordinary skill in the art in order to provide alternative synthetic pathways to the present compounds.

The present invention also relates to pharmaceutical compositions comprising a therapeutically effective amount of a 2-aminocarbonyl-1, 2-bis (methylsulfonyl)-1-(2- chloroethyl) hydrazine compound as set forth above. A therapeutically effective amount of one or more of these compounds is that amount which may be used to treat patients suffering from cancer. These pharmaceutical compositions preferably also include a pharmaceutically acceptable additive, carrier or excipient. In pharmaceutical compositions according to the present invention, which relate to the treatment of malignant solid tumors, those compositions comprise an amount of one or more 2-aminocarbonyl-1, 2-bis (methylsulfonyl)-1- (2- chloroethyl) hydrazine compounds as set forth above effective to inhibit the growth of the treated tumor and, in certain cases, to actually shrink the treated tumor.

One of ordinary skill in the art will recognize that a therapeutically effective amount of the compounds according to the present invention to be used to treat malignant tumors will vary with the disease state or condition to be treated, its severity, the treatment regimen to be employed, the result desired (remission, shrinkage of tumor in combination with surgical techniques or radiation), the type of administration used to deliver the compounds, the pharmacokinetics of the compounds used, as well as the patient (animal or human) treated.

In the pharmaceutical aspect according to the present invention, one or more compounds according to the present invention are formulated preferably in admixture with a pharmaceutically acceptable additive, carrier or excipient. In general, it is preferable to administer the pharmaceutical composition in parenteral-administrable form (preferably, intravenous), but consideration should be given to other formulations administered via oral or other route. Of course, one of ordinary skill in the art may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration without rendering the compositions of the present invention unstable or compromising the therapeutic activity.

For example, modifying the present compounds to render them more soluble in water or other vehicle may be easily accomplished by minor modifications (salt formulation, esterification, etc. ) which are well within the ordinary skill in the art. It is also within ordinary skill to modify the route of administration and dosage regimen of a particular compound in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect in the patient to be treated. Sustained and/or controlled release forms of the pharmaceutical compositions are also contemplated by the present invention.

The present compounds are prodrug forms of reactive intermediates. In certain pharmaceutical dosage forms, the present compounds may be modified to other prodrug forms to take advantage of a particular route of administration of the active compounds. One of ordinary skill in the art will recognize how to readily modify the present compounds to alternative prodrug forms to facilitate delivery of active compounds to a targeted site within the patient.

The individual of ordinary skill also will take advantage of favorable pharmacokinetic parameters of the prodrug forms, where applicable, in delivering the present compounds to a targeted site within the patient to maximize the intended antineoplastic effect of the compound.

The amount of compound included within the therapeutically active formulations according to the present invention is an effective amount for treating the malignant tumor. In general, a therapeutically effective amount of the compound according to the present invention in dosage form usually ranges from less than about 0.05 mg/kg to about 500 mg/kg of body weight of the patient to be treated, or considerably more, depending upon the compound used, the tumor type to be treated, the ability of the active compound to localize in the tissue to be treated, the route of administration and the pharmacokinetics of the compound in the patient. In the case of treating solid tumors, the compound is preferably administered in amounts ranging from about 0.05 mg/kg to about 250 mg/kg or more at one time. This dosage range generally produces effective blood level concentrations of active compound ranging from about 0.01 to about 500 micrograms per ml of blood in the patient to be treated. The duration of treatment may be for one or more days or may last for several months or considerably longer (years) depending upon the disease state treated.

Administration of the active compound may range from continuous (intravenous drip) to several oral administrations per day (for example, Q. I. D. ) and may include parenteral as a preferred route of administration, including intravenous and oral, among other routes of administration.

To prepare the pharmaceutical compositions according to the present invention, a therapeutically effective amount of one or more of the compounds according to the present invention is preferably intimately admixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques to produce a dose. A carrier may take a wide variety of forms depending on the form of preparation desired for administration, e. g., parenteral or oral.

For parenteral formulations, the carrier may comprise sterile water or aqueous sodium chloride solution in combination with other ingredients which aid dispersion, such as ethanol and other pharmaceutically acceptable solvents, including propylene glycol, among others. Of course, where solutions are to be used and maintained as sterile, the compositions and carriers must also be sterilized. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.

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

The present compounds or their derivatives, can be provided in the form of pharmaceutically acceptable salts. As used herein, the term"pharmaceutically acceptable salts" refers to appropriate salts or complexes of the active compounds according to the present invention which retain the desired biological activity of the parent compound and exhibit limited toxicological effects to normal cells. Nonlimiting examples of such salts are (a) acid addition salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, isethionic acid and polyglutamic acid, among others; (b) base addition salts formed with metal cations such as zinc, magnesium calcium, sodium, potassium, and the like, among numerous others.

The compounds and compositions according to the present invention are used to treat cancer in mammals, including humans. Generally, to treat malignant tumors, the compositions will be administered in parenteral, preferably intravenous dosage form in amounts ranging from about 25 micrograms up to about 500 mg or more one to four times per day. The present compounds are preferably administered parenterally, but they also may be administered in an alternative manner, for example, orally.

Compounds according to the present invention may be administered alone or in combination with other agents, especially including other compounds of the present invention.

In addition, the administration of one or more compounds according to the present invention with other antineoplastic agents, in combination chemotherapy, such as antimetabolites, etoposide, doxorubicin, taxol, vincristine, cyclophosphamide, mitomycin C, or other aminocarbonylhydrazine compounds of the prior art, among numerous others, is contemplated by the present invention.

Mechanism of Action The postulated mechanism of activation for a representative compound (R is a morpholine group) is shown in attached Figure 2. Ring oxidation of ld, possibly by the hepatic cytochrome P450 enzyme system (see Foley, et al, Proc. Am. Assoc. Cancer Res., 3,11, 1960 and Cohen, et al. , J. Pharmacol. Exp. Ther., 174,206-210, 1970), gives the hydroxy metabolite 3, which would exist in a steady-state equilibrium with the acylic aldehyde 4. While 4 would be prone to base-catalyzed activation to form compound 1, an agent with known antitumor activity and an isocyanate 6, the cyclic tautomer 3 would be oxidized to the corresponding non-toxic carbonyl derivative 5 by the hepatic aldehyde oxidase system, thereby minimizing hepatic damage and/or aldehyde dehydrogenase in the bone marrow and gastrointestinal tract, thereby minimizing the toxic effects of this agent to these two drug sensitive tissues, in a manner analogous to cyclophosphamide, delivering the alkylation stress preferentially to malignant tissues, while minimizing damage to the bone marrow and the gastrointestinal tract.

The present invention is now described, purely by way of illustration, in the following examples. It will be understood by one of ordinary skill in the art that these examples are in no way limiting and that variations of detail can be made without departing from the spirit and scope of the present invention.

EXAMPLES Chemical Synthesis Melting points were determined on a Thomas-Hoover melting point apparatus and are uncorrected. Elemental analyses were performed by the Baron Consulting Co. , Orange, Connecticut.

Synthesis of 1 2-Bis (methylsulfonyl)-l- (2-chloroethyl)-2- (N, N-diethvlaminocarbonyl) hydrazine To an ice-cold stirred solution of 1, 2-bis (methylsulfonyl)-1- (2-chloroethyl) hydrazine (1.25 g) and triphosgene (0.62 g) in anhydrous dioxane (10 ml) was added a solution of N, N- diisopropylethylamine (1.5 g) in anhydrous dioxane (10 ml) over a period of 40 minutes. The ice-bath was removed and the reaction mixture stirred for 6 hr. The reaction mixture was evaporated to dryness on a rotary evaporator. The residue was taken up in dry acetonitrile (15 ml) and treated with N, N-diethylamine (1.4 g). The reaction mixture was stirred for 16 hr and evaporated to dryness in vacuo. The residue was taken up in ethyl acetate (150 ml) and washed with 10% w/v hydrochloric acid (2 x 20 ml). The organic layer was dried over anhydrous sodium sulfate, filtered and the filtrate evaporated to dryness to give an oil. This oil was triturated with absolute ethanol to give a solid which was recrystallized twice from ethanol to give 0.32 g of the title compound. m. p. 113-114°C.

Anal. Calc.: C, 30.90 ; H, 5.76 ; N, 12.01.

Found: C, 30.88 ; H, 5.64 ; N, 12.03.

Synthesis of 1, 2-Bis (methylsulfonyl)-1- (2-chloroethyll-2- (1-pyrrolidinocarbonyl ydrazine To an ice-cold stirred solution of 1, 2-bis (methylsulfonyl)-1- (2-chloroethyl) hydrazine (1.25 g) and triphosgene (0.50 g) in anhydrous dioxane (10 ml) was added a solution of N, N- diisopropylethylamine (1.3 g) in anhydrous dioxane (10 ml) over a period of 1.5 hr. The ice-bath was removed and the stirring continued for an additional 3.5 hr. The reaction mixture was evaporated to dryness in vacuo. The residue was taken up in dry acetonitrile (15 ml) and treated with pyrrolidine (0.36 g), followed by N, N-diisopropylethylamine (0.65 g) and stirred for 16 hr.

After removing the solvent and the volatiles in vacuo the residue was taken up in ethyl acetate (150 ml), washed with 10% w/v hydrochloric acid (2 x 15 ml) and the organic layer dried over anhydrous magnesium sulfate. The ethyl acetate solution filtered and the filtrate evaporated to dryness. The residue was chromatographed on silica gel (preparative TLC; 2000 microns; chloroform) and the appropriate band was extracted with acetone. The solvent was removed and the residue recrystallized from ethanol to give 0.42 g of the target molecule. m. p. 135-137°C.

Anal. Calc.: C, 31.08 ; H, 5.22 ; N, 12.08.

Found: C, 31.30 ; H, 5.25 ; N, 11.95.

Synthesis of 1, 2-Bis (methylsulfonyl)-1- (2-chloroethvl)-2- (4-morpholinocarbonyl) hydrazine To an ice-cold stirred solution of 1, 2-bis (methylsulfonyl)-1-(2-chloroethyl) hydrazine (2.50 g) and triphosgene (1.00 g) in anhydrous dioxane (20 ml) was added a solution of N, N- diisopropylethylamine (2.6 g) in anhydrous dioxane (20 ml) over a period of 1 hr. The ice-bath was removed and the stirring continued for an additional 4 hr. The reaction mixture was evaporated to dryness in vacuo. The residue was taken up in dry acetonitrile (30 ml) and treated with morpholine (0.87 g), followed by N, N-diisopropylethylamine (1.3 g) and stirred for 16 hr.

After removing the solvent and the volatiles in vacuo the residue was taken up in ethyl acetate (150 ml), washed with 10% w/v hydrochloric acid (2 x 25 ml) and the organic layer dried over anhydrous sodium sulfate. The ethyl acetate solution was filtered and the filtrate evaporated to dryness. The residue was chromatographed on silica gel (preparative TLC; 2000 microns; chloroform) and the appropriate band was extracted with acetone. The solvent was removed and the residue recrystallized from ethanol to give 0.55 g of the target molecule. m. p. 139-141°C.

Anal. Calc.: C, 29.71 ; H, 4.99 ; N, 11.55.

Found: C, 30.00 ; H, 4.72 ; N, 11.41.

Synthesis of 1, 2-Bis (methylsulfonYl-(2-chloroethyl)-2-[l-(2s5 dimethyyrrolidino) carbonyl] hYdrazine To an ice-cold stirred solution of 1, 2-bis (methylsulfonyl)-1-(2-chloroethyl) hydrazine (1.25 g) and triphosgene (0.50 g) in anhydrous dioxane (10 ml) was added a solution of N, N- diisopropylethylamine (1.3 g) in anhydrous dioxane (10 ml) over a period of 45 min. The ice- bath was removed after 30 min and the stirring continued for an additional 3.75 hr. The reaction mixture was evaporated to dryness in vacuo. The residue was taken up in dry acetonitrile (10 ml), cooled in an ice-bath and treated with a solution of 2, 5-dimethylpyrrolidine (0.5 g) and N, N- diisopropylethylamine (0.65 g) in acetonitrile (10 ml) and the reaction mixture was stirred for 16 hr. After removing the solvent and the volatiles in vacuo the residue was taken up in ethyl acetate (150 ml), washed with 10% w/v hydrochloric acid (2 x 10 ml) and the organic layer dried over anhydrous sodium sulfate. The ethyl acetate solution was filtered and the filtrate evaporated to dryness. The residue was chromatographed on silica gel (preparative TLC; 2000 microns; chloroform) and the appropriate band was extracted with acetone. The solvent was removed and the residue recrystallized from ethanol to give 0.28 g of the target molecule. m. p. 145-147°C.

Anal. Calc.: C, 35.15 ; H, 5.90 ; N, 11.18.

Found: C, 35.40 ; H, 5.76 ; N, 11.08.

Synthesis of 1, 2-Bis (methelsulfonyl !-l-(2-chloroethyl)-2-[4-meth piperazinocarbonyl] hydrazine To an ice-cold stirred solution of 1, 2-bis (methylsulfonyl)-1-(2-chloroethyl) hydrazine (1.25 g) and triphosgene (0.50 g) in anhydrous methylene chloride (15 ml) was added a solution of N, N-diisopropylethylamine (1.3 g) in anhydrous methylene chloride (15 ml) over a period of 2 hr. The stirring was continued in the cold for an additional 4 hr. To this mixture was added 1- methylpiperazine (0.5 g) and N, N-diisopropylethylamine (1.3 g), and the reaction mixture was stirred for 16 hr. After removing the solvent and the volatiles in vacuo the residue was taken up in ethyl acetate (150 ml), washed with water (2 x 25 ml) and the organic layer dried over anhydrous sodium sulfate. The ethyl acetate solution was filtered and the filtrate evaporated to dryness. The residue was chromatographed on silica gel (preparative TLC; 2000 microns; chloroform) and the appropriate band was extracted with acetone. The solvent was removed and the residue recrystallized from ethanol to give 0.27 g of the target molecule. m. p. 131-132°C.

Anal. Calc. : C, 31.87 ; H, 5.62 ; N, 14.87.

Found: C, 32.10 ; H, 5.46 ; N, 14.77.

Biological Activity The compounds according to the present invention were evaluated for antineoplastic activity against the L1210 leukemia in mice according to the procedure set forth below. The data are summarized in Table 1, below."Cures"of the mice bearing the L1210 tumor were obtained with compounds where R is a group selected from the series consisting of diethylamino, morpholino and pyrrolidino. The compound having the morpholino substituent gave the best results, producing several"cures"of tumor-bearing mice at single intraperitoneal doses of 80 and 160 mg/kg.

Leukemia L1210 cells were obtained from the Frederick Cancer Research Facility, Division of Cancer Treatment Tumor Repository of the National Cancer Institute, and were maintained by serial passage in tissue culture. Every 8 weeks, tumor cells were injected intraperitoneally into five donor CD2F1 mice 8-to 10-weeks of age and were allowed to grow for 7 days. The peritoneal fluid was withdrawn and the suspension was centrifuged for 5 min at 1600g. The supernatant was decanted and 105 cells/mL were seeded into 10 mL of RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% L-glutamine, and once again maintained in culture. To assay for antineoplastic activity, 0.1 mL of the cell suspension containing 105 L1210 leukemia cells was injected ip into each recipient mouse. Test compounds were administered as a single dose over a range of dosage levels, beginning 24 h after tumor implantation. All drugs were administered ip as a solution in 100% dimethylsulfoxide (DMSO), in a volume not exceeding 0.025 mL. In each experiment, animals were distributed into groups of five mice of comparable weight and maintained throughout the course of the experiment on Purina Laboratory Chow pellets and water ad libitum. Control tumor-bearing animals given comparable volumes of vehicle were included in each experiment. Mice were weighed during the course of the experiments, and the percentage change in body weight from onset to termination of therapy was used as an indication of drug toxicity. Determination of the sensitivity of neoplasms to these agents was based upon the prolongation of survival time afforded by the drug treatments.

Results of L1210 Testing The tumor-inhibitory properties of the compounds were determined in initial tests by measuring their effects on the survival time of mice bearing the intraperitoneally (ip) implanted L1210 leukemia; the results of these tests are summarized in Table 1, below. A number of "cures"were seen (defined as tumor-free animals 60 days post-tumor implant) in 60 to 100 % of mice bearing the L1210 leukemia at one or more of the dosage levels examined following ip administration.

The compounds tested were those of the general chemical structure: where R is as indicated in Table 1, below.

Table 1 Compound R Dose,a mg/kg % T/Cb LUTS' 101DE N, N-Diethylamino 20 150--- 40 224 --- 80 632 4/5 160 100--- 101 PYR Pyrrolidino 20 129--- 40 161--- 80 189--- 160 --- 5/5 101 DMP 2,5-Dimethylpyrrolidino 20 108--- 40 137 --- 80 155--- 160 174 --- 101 MOR Morpholino 20 139--- 40 176 --- <BR> <BR> <BR> 80---5/5<BR> <BR> <BR> <BR> <BR> <BR> 160 296 3/5 a Female CD2Fl mice were inoculated i. p. with 1 X 10 leukemia cells. A single i. p. treatment was administered 24 hours later. b% T/C = mean life span of treated (T) mice/mean life span of control (C) mice x 100. T/C calculations do not include long-term survivors.

CLTS= Long-term survivors, mice alive and tumor-free 60 days after inoculation.