Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

• the treatment of tumor can be approached by several modes of therapy, including surgery, radiation, chemotherapy, or any combination of any of these treatments.
• chemotherapy is indispensable for inoperable or metastatic forms of cancer.
• NCI National Cancer Institute
• This in vitro screening system is based on human tumor cell line panels consisting of approximately 60 cell lines of major human tumors (e.g., leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, or breast cancer), and serves as a tool for identifying compounds that possess anti-tumor activities.
• major human tumors e.g., leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, or breast cancer
• Each of Ri and R 2 is H, C(O)R d , C(O)OR d , C(O)NR d R e , or SO 2 R d ; and each of R a , R b , R c , R d , and R e , independently, is H, alkyl, aryl, heteroaryl, cyclyl, or heterocyclyl.
• R a and R b taken together are cyclyl or heterocyclyl; and, also optionally, R ⁇ and R or Ri and R b taken together are cyclyl or heterocyclyl.
• a subset of the piperazinedione compounds of this invention is featured by that both ⁇ ⁇ - and ZJ Z are double bonds.
• Ar is pyridyl linked to B at position 2
• R c is H
• R O is arylalkoxy linked to position 5 of pyridyl
• both Ri and R 2 are H
• one of R a and R b is aryl or heteroaryl
• the other of R a and R b is H.
• Another subset of the piperazinedione compounds of this invention is featured by that both ⁇ and ⁇ 1 are single bonds.
• Ar is pyridyl linked to B at position 2
• R° is H
• R 3 O is arylalkoxy linked to position 5 of pyridyl
• both Ri and R 2 are H
• one of R and R b is H, aryl, or heteroaryl
• the other of R and R b is H.
• Alkyl, aryl, heteroaryl, cyclyl, and heterocyclyl mentioned herein include both substituted and unsubstituted moieties.
• substituted refers to one or more substituents (which may be the same or different), each replacing a hydrogen atom.
• substituents include, but are not limited to, halogen, hydroxyl, amino, alkylamino, arylamino, dialkylamino, diarylamino, cyano, nitro, mercapto, carbonyl, carbamido, carbamyl, carboxyl, thioureido, thiocyanato, sulfoamido, C ⁇ C 6 alkyl, C !
• heteroaryl refers to a hydrocarbon ring system having at least one aromatic ring which contains at least one heteroatom such as O, N, or S.
• heteroaryl moieties include, but are not limited to, furyl, fluorenyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyridinyl, pyrimidinyl, quinazolinyl, and indolyl.
• Another aspect of the present invention relates to a pharmaceutical composition that contains a pharmaceutically acceptable carrier and an effective amount of at least one of the piperazinedione compounds described above.
• Z is CH(R c R d ) when Z 1 ⁇ is a single bond, or C(R°R d ) when ⁇ is a double bond;
• each of R] and R 2 independently, is H, C(O)R e , C(O)OR e , C(O)NR e R f , or SO 2 R e ; and each of R a , R b , R°, R d , R e , and R , independently, is H, alkyl, aryl, heteroaryl, cyclyl, or heterocyclyl, provided that one of R° and R d is aryl or heteroaryl.
• R a and R b taken together are cyclyl or heterocyclyl; and, also optionally, Ri and R a or R ⁇ and R b taken together are cyclyl or heterocyclyl.
• R° and R d is 2- pyridyl
• the other of R° and R is H
• both Ri and R 2 are H
• one of R a and R is H, aryl, or heteroaryl
• the other of R a and R b is H.
• the piperazinedione compounds described above include the compounds themselves, as well as their salts and their prodrugs, if applicable.
• Such salts can be formed between a positively charged substituent (e.g., amino) on a piperazinedione compound and an anion.
• Suitable anions include, but are not limited to, chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, and acetate.
• a negatively charged substituent (e.g., carboxylate) on a piperazinedione compound can form a salt with a cation.
• Suitable cations include, but are not limited to, sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as teteramethylammonium ion.
• prodrugs include esters and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing piperazinedione compounds described above.
• a composition containing one or more of the piperazinedione compounds described above for use in treating tumor and the use of such a composition for the manufacture of a medicament for the just-described use.
• the piperazinedione compounds described above can be prepared by methods well known in the art, as well as by the synthetic routes disclosed herein. For example, one can react a piperazine-2,5-dione compound with a heteroaryl formaldehyde to produce an intermediate heteroaryl-methylidene-piperazine-2,5-dione. The intermediate can then be reduced to heteroaryl-methyl-piperazine-2,5-dione (a compound of this invention), or reacted with a ketone or another formaldehyde, followed by a base treatment, to produce a mixture of piperazinedione isomers, which are cis- or trans- or E- or Z- double bond isomeric forms.
• the desired isomeric product can be separated from others by high pressure liquid chromatography (HPLC). If preferred, proper functional groups can be introduced into the heteroaryl ring by further modifications. Alternatively, a desired reduced product can be obtained by reacting the product with a reducing agent.
• HPLC high pressure liquid chromatography
• the piperazinedione compounds contain at least two double bonds, and may further contain one or more asymmetric centers. Thus, they can occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans- or E- or Z- double bond isomeric forms. All such isomeric forms are contemplated.
• a pharmaceutical composition that contains an effective amount of at least one piperazinedione compound of the present invention and a pharmaceutically acceptable carrier. Further, this invention covers a method of administering an effective amount of one or more of the piperazinedione compounds described in the "Summary" section above to a subject in need of tumor treatment.
• the piperazinedione compounds can function via one or more of the above described action mechanisms, or via any other mechanism.
• “An effective amount” refers to the amount of the compound which is required to confer a therapeutic effect on the treated subject.
• the interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described in Freireich et al, (1966) Cancer Chemother Rep 50: 219.
• Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537.
• An effective amount of the piperazinedione compounds can range from about 0.1 mg/Kg to about 50 mg/Kg. Effective doses will also vary, as recognized by those skilled in the art, depending on the types of tumors treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments such as use of other anti-tumor agents or radiation therapy.
• a piperazinedione compound- containing composition can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
• parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
• a sterile injectable composition for example, a sterile injectable aqueous or oleaginous suspension, can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.
• the sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
• suitable vehicles and solvents that can be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides).
• Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
• These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents.
• Other commonly used surfactants such as Tweens or Spans or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.
• a composition for oral administration can be any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.
• carriers which are commonly used include lactose and corn starch.
• Lubricating agents, such as magnesium stearate, are also typically added.
• useful diluents include lactose and dried corn starch.
• the carrier in the pharmaceutical composition must be "acceptable” in the sense of being compatible with the active ingredient of the formulation (and preferably, capable of stabilizing it) and not deleterious to the subject to be treated.
• solubilizing agents such as cyclodextrins, which form specific, more soluble complexes with the piperazinedione compounds, or one or more solubilizing agents, can be utilized as pharmaceutical excipients for delivery of the piperazinedione compounds.
• examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow # 10.
• the piperazinedione compounds can be preliminarily screened for their efficacy in treating cancer by one or more of the following in vitro assays.
• One assay is based on the NCI screening system, which consists of approximately 60 cell lines of major human tumors. See Monks, et al. (1991) JNCI, J Natl. Cancer Inst. 83: 757-766; Alley, et al. (1988) Cancer Res. 48: 589-601; Shoemaker, et al. (1988) Prog. Clin. Biol. Res. 276: 265-286; and Stinson, et al. (1989) Proc. Am. Assoc. Cancer Res. 30: 613.
• a cell suspension that is diluted according to the particular cell type and the expected target cell density (5,000-40,000 cells per well based on cell growth characteristics) is added (100 ⁇ L) into a 96-well microtiter plate.
• a pre-incubation is preformed at 37° C for 24 hr.
• Dilutions at twice of an intended test concentration are added at time zero in 100 ⁇ L aliquots to each well of the microtiter plate.
• a test compound is evaluated at five 10-fold dilutions. In a routine testing, the highest concentration of the test compound is 10 "4 M. Incubations are performed for 48 hr in 5% CO 2 atmosphere and 100% humidity.
• the cells are assayed by using the sulforhodamine B assay described by Rubinstein, et al. (1990, J/VCY, J Natl. Cancer Inst. 82: 1113-1118) and Skehan, et al. (1990, MCI, J. Natl. Cancer Inst. 82: 1107-1112).
• a plate reader is used to read the optical densities and a microcomputer processes the optical densities into the special concentration parameters.
• the NCI has renamed an IC 5 o value, the concentration that causes 50% growth inhibition, a GI 50 value to emphasize the correction for the cell counted at time zero; thus, the GI 50 measures the growth inhibitory power of the test compound. See Boyd, et al. (1992) In Cytotoxic Anticancer
• a piperazinedione compound is tested for its cytotoxicity on PC-3 cells (a prostate cancer cell line). More specifically, cells are incubated with a test compound in a serum-free medium for 24 hr.
• the cytotoxic effect can be determined using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay method described in Boyd (In Principle of Practice of Oncology Devita, J.T., Hellman, S., and Rosenberg, S.A. (Eds.) Vol. 3, PPO Update, No. 10, 1989).
• Another in vitro assay can be used to evaluate the efficiency of a piperazinedione compound in arresting the cell cycle progression. More specifically, a test piperazinedione compound is added to PC-3 cells in a concentration-dependent manner using propidium iodide-stained flow cytometric assessment. The cell population of sub-Grj/Gi, Go/Gi, S, and G 2 /M phase is then determined. In addition, the effect of a piperazinedione compound on the Ras activity can be examined to determine its regulation of Ras protein-dependent signal transduction pathway. The anti-tumor activity of a piperazinedione compound can be further assessed by an in vivo animal model.
• Example 1 Synthesis of 3-[(5-benzyoxypyridin-2-yl)methylidene]-6-phenylmethylidene piperazine-2,5-dione (Compound 1).
• l,4-Diacetyl-piperazine-2,5-dione (8.6 g) was added to a solution of 5- benzyoxypyridin-2-yl-formaldehyde (4.0 g) in 5.6 mL of triethylamine and 40 mL of dimethylformamide. The mixture was stirred at room temperature for 16 hr and then cooled at ice bath to produce a yellow precipitate.
• Example 8 Synthesis of 3-[(5-benzyoxypyridin-2-yl)methylidene]-6-[(thien-2-yl) methylidene]piperazine-2,5-dione (Compound 8).
• Compound A (2.8 g), obtained from Example 1, was added to a 40 mL of dimethylformamide solution containing 2 mL of thiophene-2-carbaldehyde and 4 equivalents of triethylamine. The solution was refluxed at 60°C for 2 days and cooled at ice bath to produce a yellow precipitate.
• Example 11 Synthesis of 3-[(5-phenylmethoxypyridin-2-yl)methylidene]-6-(2-oxo- 3- indolylidenepiperazine-2,5-dione (Compound 11).
• Example 14 Synthesis of 3-[(5-acetoxypyridin-2-yl)methylidene]-6-(benzylmethylidene) piperazine-2,5-dione (Compound 14). 3-[(5-benzyloxypyridin-2-yl)methylidene]-6-(benzylmethylidene)piperazine-2,5- dione (Compound 1, 0.5 g, 1.26 mmol) and NaOH (0.5 g, 12.5 mmol) were dissolved in 100 mL of methanol. The mixture was hydrogenated with 0.5 g palladium/charcoal under 1 atmospheric pressure.
• Example 16 Synthesis of 3-[(5-(4-toluenesulfonyl)pyridin-2-yl)methylidene]-6- (benzylmethylidene) piperazine-2,5-dione (Compound 16).
• Example 17 Synthesis of 3-[(5-(4-chlorophenylcarbamic)pyridin-2-yl)methylidene]-6- (benzylmethylidene) piperazine-2,5-dione (Compound 17).
• Example 18 Screening for anti-tumor activities (NCI cell lines).
• the most potent compound exhibited GI 50 values ⁇ 10 "4 M for all 60 cell lines, with GI50 values ⁇ 10 "8 M for 9 cell lines.
• Example 19 Screening for anti-tumor activities (a prostate cell line). The cytotoxic activities of a number of piperazinedione compounds and taxol (a well- known anti-tumor agent) were tested on PC-3 cells. Cells were incubated in the presence of each compound in a serum-free medium for 24 hr. The cytotoxic activities were determined by the MTT assay. All test compounds are active. Unexpectively, the most potent piperazinedione compound has an EC 50 value around 0.3 microM, >30 times more potent than taxol.
• PC-3 cells were incubated in the presence of a piperazinedione compound in a serum- free medium and harvested, fixed, and stained with propidium iodide at the 6 th , 12 th , 18 th , and 24 th hr, respectively.
• the stage of cell cycles was determined based on flow cytometric measurements.
• the test compound induced an arrest of the cell cycle as entranced by a large number of cells at G /M phase.
• a piperazinedione compound had a marked effect on the regulation of Ras activity
• Example 21 In vitro assay (disturbance of tubulin/microtubulin assembly). Tubulin/microtubulin was incubated in the presence of a piperazinedione compound at different concentrations in a solution (0.1 M MES, 1 mM EGTA, 0.5 mM MgCl 2 , 0.1 mM EDTA, and 2.5 M glycerol) at 37°C. Then, GTP was added to induce polymerization of tubulin/microtubulin. Optical density (OD) was measured at 350 nm at various time points to determine the degree of the polymerization. The test compound inhibited the polymerization at 10 '6 - 10 "5 M.
• a piperazinedione compound at different concentrations in a solution (0.1 M MES, 1 mM EGTA, 0.5 mM MgCl 2 , 0.1 mM EDTA, and 2.5 M glycerol) at 37°C. Then, GTP was added to induce
• Example 22 In vivo assay (inhibition of tumor enlargement)
• SCID mice subcutaneously injected into PC-3 cells, developed a tumor more than 800 mm 3 in volume.
• a piperazinedione compound significantly diminished the tumor volume after a 14-28 days treatment.
• Example 23 In vivo assay (regulation of angiogenesis activity)

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