Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
  • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
  • C07D495/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the present invention relates to dithiolopyrrolone derivatives, their corresponding monoxides (xenomins) and dioxides (xenorxides) having antmeoplastic activities.
• the present invention also provides antineoplastic compositions comprising these compounds, the salts thereof, and methods of using the inventive compounds as antineoplastic agents.
• the present invention provides dithiolopyrrolone derivatives, their corresponding monoxides (xenomins) and dioxides (xenorxides) having antineoplastic activities.
• the present invention also provides antineoplastic compositions comprising these compounds, the salts thereof, and methods of using the inventive compounds as antineoplastic agents.
• Figure 1 Figure 2 and Figure 3 represent the structural formula of dithiolopyrrolones. their corresponding monoxides (xenomins) and dioxides (xenorxides), respectively.
• R ⁇ hydrogen, substituted or unsubstituted alkyl, cycloalkyl, aryl. aralkyl, or heterocyclyl group:
• R 2 hydrogen, substituted or unsubstituted alkyl. cycloalkyl. aryl. aralkyl, heterocyclyl or group acyl;
• R 3 hydrogen, alkyl, cycloalkyl. aralkyl. aryl or heterocyclyl group.
• Rj hydrogen, substituted or unsubstituted alkyl, cycloalkyl. aryl. aralkyl, or heterocyclyl group:
• R2 hydrogen, substituted or unsubstituted alkyl, cycloalkyl. aryl, aralkyl, heterocyclyl or group acyl;
• R3 hydrogen, alkyl. cycloalkyl. aralkyl. aryl or heterocyclyl group.
• Cancer is one of the major causes of death in humans and animals. Millions of people in the world are diagnosed every year as having cancer, and a large proportion of these people subsequently die of cancer. Although large effort has been made, cancers remain to be hard-to- treat diseases. There continues to be an urgent need for effective anticancer drugs.
• Soil organisms have been a traditional source of bioactive materials for the pharmaceutical and agrochemical industry.
• One of the recent developments has been the commercialization of a soil-living nematode-bacteria combination as biological control agents of insect pests.
• a crucial feature of this biocontrol agent is that the bacterial symbiont (Xenorhabdus spp. or Photorhabdus spp.) of the nematode produces a wide range of bioactive metabolites and some of these specific compounds have been isolated, identified and their structures elucidated (Forst and Nealson, 1996). Among these identified compounds, several are dithiolopyrrolone derivatives.
• Dithioiopyrrolones were initially isolated from Streptomyces in the 40s and have since then been isolated from other organisms. These compounds include aureothricin, thiolutin, holomycin and xenorhabdins (Hamao et al., 1948; Eisenman et al., 1953; Celmer and Solomons, 1955; von Daehne e al.. 1969; Mclnerney et al, 1991). These compounds possess antimicrobial activity against a wide range of micro-organisms. Recently, new antimicrobial substances, namely xenorxides (Webster et al., 1996), also were found from the bacterial cultures of Xenorhabdus spp.
• xenorxides As a result of part of the ongoing exploration of the potential usefulness of these metabolites of Xenorhabdus, the xenorxides. xenomins and the dithiolopyrrolones have been found to be highly active against animal and human cancer cells, and is the subject of this invention.
• thiolutin is the one that has been studied extensively. Thiolutin inhibits RNA polymerase synthesis (Jimenez et al.. 1973; Tipper, 1973) in yeast, has membrane stabilization activity and inhibit platelet aggregation (Yasuyuki et al., 1980) in rat.
• the compounds of the present invention may be synthesized chemically, the initial compounds were obtained from micro-organisms.
• the organisms used for this invention include Xenorhabdus bovienii, a symbiotic bacterium associated with entomopathogenic nematodes.
• X. bovienii and its nematode symbiont Steinernemafeltiae, used in this invention were collected from soil in British Columbia, Canada. Briefly, last instar larvae of the Greater Wax Moth, Galleria mellonella, were infected with infective juvenile (IJ) nematodes, carrying the bovienii A21 strain, at a rate of 25 Us/larvae.
• the dead insect larvae were surface disinfected by dipping them into 95% EtOH and igniting them.
• the cadavers were aseptically dissected, haemo lymph was streaked onto an agar culture medium and incubated in the dark at room temperature.
• the agar medium has the following composition in one litre of distilled water: beef extract 3 g peptone 5 g bromothymol blue 0.025 g
• X. bovienii Cultivation of the microorganism X. bovienii yields bioactive substances, xenomins, xenorxides and dithiolopyrrolones.
• X. bovienii may be cultivated (fermented), for example, at about 25 °C under submerged aerobic conditions in an aqueous, nutrient medium containing assimilable carbon (carbohydrate) and nitrogen sources to obtain xenomins, xenorxides and dithiolopyrrolones.
• the fermentation may be carried out for a time period such as approximately 48 to 96 hours, at the end of which time these compounds have been formed, and may be isolated from the fermentation medium and purified.
• the fermented broth may be filtered or centrifuged and the pH of the filtrate adjusted to about 7.0 by the addition of hydrochloric acid or kept as it is.
• the filtrate may then be extracted with a water immiscible, organic solvent, for example, with ethyl acetate or chloroform.
• the combined organic layers e.g. pooled ethyl acetate or chloroform extracts
• the oily residue may be mixed with a small amount of organic solvent and chromatographed on a silica gel column. After introduction of the sample, chloroform or other organic solvent may be applied to elute the bioactive fraction.
• the bioactive fraction may be purified further by high performance, liquid chromatographv (HPLC) with organic and/or aqueous solution.
• HPLC high performance, liquid chromatographv
• the major compounds produced by.K bovienii are dithiolopyrrolones while xenorxides and xenomins are present in relatively small amounts.
• dithiolopyrrolones may be produced from other micro-organisms, by chemical method(s) and/or a combination of both approaches.
• the monoxides and dioxides of the corresponding dithiolopyrrolones isolated fromZ bovienii, namely, xenorxides and xenomins, respectively may be transformed from their corresponding dithiolopyrrolone derivatives by biological or chemical means.
• biological means the culture broth of X. bovienii, with the corresponding dithiolopyrrolone derivatives present, may be filtered or centrifuged.
• the cell-free filtrate may be open to the air for extended periods from one week up to one month with or without stirring at room temperature or at other temperatures. This process may oxidize all or part of the corresponding dithiolopyrrolone derivatives to xenomins and xenorxides.
• Xenorxides and xenomins may be obtained by oxidation of the corresponding dithiolopyrrolone derivatives by chemical means.
• the pure dithiolopyrrolone derivatives or their mixture may be dissolved in a mixture of acetone and water, then oxidizing reagents such as potassium peroxymonosulfate and potassium bicarbonate may be added to the mixture.
• the mixture may be allowed to react for a period of several minutes up to more than one hour.
• the reaction mixture may be mixed with water, and extracted with organic solvent.
• the extracts may be combined, dried and purified by column chromatography or HPLC to obtain the corresponding xenorxides and/or xenomins.
• Additional monoxides and dioxides of the dithiolopyrrolones may be obtained from different microorganisms, synthesized chemically and/or produced by a combination of biolological and chemical methods, and several examples are disclosed (Takahashi, et al., 1995; Fujimoto, et al. 1996).
• the compounds of the present invention include dithiolopyrrolones, their corresponding monoxides such as xenomins, their corresponding dioxides such as xenorxides, and salts thereof.
• salts denotes acidic and/or basic salts, formed with inorganic and/or organic acids and bases. Suitable acids include, for example, hydrochloric, sulfuric, nitric, benzenesulfonic, acetic, maleic, tartaric and the like which are pharmaceutically acceptable. It is well known to one skilled in the art that an appropriate salt is chosen based on physical and chemical stability, flowability, hydroscopicity and solubility. While pharmaceutically acceptable salts are preferred, particularly when employing the compounds of the invention as medicaments, other salts find utility, for example, in processing these compounds, or where non-medicament- type uses are contemplated.
• the antineoplastic agents and use thereof are preferred.
• the present invention relates to pharmaceutical preparations which contain an active ingredient of these compounds or a pharmaceutically acceptable salt thereof.
• the dosage form and mode of administration. as well as the dosage amount, may be selected by the skilled artisan.
• Exemplary daily dosages for an adult human are those within the range of about 2.5 mg to about 2,000 mg/day.
• Administration to a mammalian host may, for example, be oral, parenteral. or topical.
• compositions of this invention can be formulated by known means.
• compositions include any solid (tablets, pills, capsules, granules, powder etc.) or liquid (solutions, suspensions or emulsions) compositions suitable for oral, topical or parenteral administration, and they may contain the pure compound or a salt thereof or in combination with any carrier or other pharmaceutically active compounds. These compositions ma ⁇ ' need to be sterile when administered parenterally.
• compositions of the present invention being employed as antineoplastic agents for treatment of animal and human illness can be easily prepared in such unit dosage form with the employment of pharmaceutical materials which themselves are available in the an and can be prepared by established procedures.
• the appropriate solid or liquid vehicle or diluent may be selected, and the compositions prepared, by methods known to the skilled artisan.
• the administration of any of the inventive compound and/or its pharmacologically active and physiologically compatible derivatives is useful for treating animals or humans having a neoplastic disease, for example, colon cancer, cevercal cancer, breast cancer, leukemia, lung cancer, ovarian cancer. CNS cancer, renal cancer, prostate cancer, etc. using the accepted protocols of the National Cancer Institute (NCI).
• NCI National Cancer Institute
• dosage administered will be dependent upon the identity of the neoplastic disease, the type of host involved including its age. health and weight, the kind of concurrent treatment, if any, and the frequency of treatment and therapeutic ratio.
• dosage levels of the administered active ingredients are intravenous, 0.1 to about 200 mg/kg; intramuscular. 1 to about 500 mg/kg and orally, 5 to about 1000 mg/kg of host body weight.
• an active ingredient can be present in the compositions of the present invention for localized use in a concentration from about 0.01 to about 50% w/w of the composition, preferably about 1 to about 20% w/w of the composition, and for parenteral use in a concentration of from about 0.
• Xenorxides Cultures o ⁇ X. bovienii were shaken at 180 rpm on an Eberbach gyrorotary shaker for 24 h at 25 °C . Bacterial fermentation was initiated by adding 100 ml of this bacterial culture to 900 ml of tryptic soy broth in a 2,000 ml flask. The flask was incubated in the dark at 25 °C on a gyrorotary shaker. After 96 h, the culture was immediately centrifuged (12,000 g, 20 minutes, 4 °C ) to separate the bacterial cells. The cell-free broth was then extracted with ethyl acetate four times.
• Xenomins For the purification of xenomins, the culture and extraction conditions were the same as for xenorxides. After the extraction, the concentrated crude extract was processed through a silica gel chromatographic column with ethyl acetate as the eluent. After the less polar bioactive material was eluted. the more polar bioactive fraction was obtained by eluting with methanol. The more polar bioactive fraction was concentrated under vacuum, and separated by a Cj chromatographic column first with water as eluent. then 25% methanol in water, 50% methanol, 75% methanol and finally, pure methanol. The most bioactive fraction was eluated with 75% methanol in water.
• the cell-free broth was obtained using the same method as described above, and was then stored at 4 °C to room temperature for 3 to 6 weeks. Then the aqueous broth was extracted with ethyl acetate, and the combined extracts were separated using the same process discussed above.
• XENORXIDE 1 was eluted at 33.6 min (2 mg/1)
• XENORXIDE 2 was eluted at 35.2 min (1.5 mg/1).
• XENORXIDE 1 (XO1): EIMS: 317(2), 316(M+, 13), 220(9), 219(9), 218(100). 186(23). 154(16). 99(40). 71(39); HRMS: 316.0555 (Calc. for C12H16N2O4S2: 316.0551, 20), 217.9824 (Calc. for C6H6N2O3S2: 217.9820, 100), 154.0197 (Calc. for C6H6N2OS: 154.0201. 16); IR (KBr): 3448. 3298.
• XENORXIDE 2 (XO2): EIMS: 330(M+, 10). 218(100); HRMS: 330.0707 (Calc. for C13H18N2O4S2: 330.0708, 18), 217.9829 (Calc. for C6H6N2O3S2: 217.9820. 100), 154.0213 (Calc. for C6H6N2OS: 154.0201, 16); IR (KBr): 3438, 3298, 1719, 1686, 1654, 1637, 1560, 1522, 1400. 1310, 1142, 551 cm-1; 1HNMR (CDCI3) ⁇ : 7.56 (1H, bs.CO-NH), 6.35 (1H, s. H-3), 3.20 (3H, s,
• Xenomin 2 (XM2): EIMS: 314(M + , 21), 218(15), 202(59), 187(18), 186(98). 185(73), 69(100); HRMS: 314.0759 (Calc. for C ⁇ 3 H, 8 N 2 O 3 S 2 : 314.0759, 12), 201.9871 (Calc.
• Example 3 Dithiolopyrrolones, the corresponding monoxides and dioxides as antineoplastic agents.
• the anticancer activity of a particular compound can be demonstrated by standard assays.
• the method normally used by the American National Cancer Institute (NCI) for the effectiveness of a compound is based on the LC 50 .
• the LC 5 0 is the compound concentration at which half of the cancer cell population is killed.
• the assay is commonly used by those skilled in the art and are accepted as indicative of anticancer activity in mammals.
• the test animal cancer cells are available from ATCC. NCI and other organizations.
• the anticancer activities of compounds of the present invention have been determined in cell cultures of various human cancer cells (see table below) using the standard NCI method with slight modifications. The specifications of the procedure was described by Skehan et al. (1990).
• cancer cells were grown in RPMI-1640 medium with glutamine and 10%) fetal calf serum, and were harvested from exponential-phase maintenance cultures.
• the harvested cells were counted and dispensed into replicate 96-well culture plates in 180 ⁇ l volumes for each well with a cell density of up to 2,500 cells/well.
• the cells were allowed to settle for about 4 hours at 37 °C.
• 20 ⁇ l of medium containing the test compound was added into each well, resulting in a suitable final test compound concentration.
• the test plates were then incubated at 37 °C. The test was terminated after incubation by adding to each well 50 ⁇ l of cold 50% trichloroacetic acid.
• the cells were fixed for an hour at 4 °C and then washed five times with tap water.
• the washed plates were air-dried and stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) which was dissolved in 1% > acetic acid.
• SRB sulforhodamine B
• the SRB was removed and plates were quickly rinsed five times with 1% acetic acid.
• the plates were air-dried and 100 ⁇ l of 10 mM tris base (pH 10.5) were added to each well to dissolve the dye bound to the cells.
• the plates were placed on a gyratory shaker and shaken (100 rpm) for 10 minutes. Finally, the plates were read in a microtiter plate reader at 570 nm. All the compounds tested exhibited very strong anticancer activity against these cancer cells (see table below).

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• 1997
  • 1997-09-05 CA CA002212237A patent/CA2212237A1/en not_active Abandoned
• 1998
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