CA2212237A1 - Novel antineoplastic agents - Google Patents
Novel antineoplastic agentsInfo
- Publication number
- CA2212237A1 CA2212237A1 CA002212237A CA2212237A CA2212237A1 CA 2212237 A1 CA2212237 A1 CA 2212237A1 CA 002212237 A CA002212237 A CA 002212237A CA 2212237 A CA2212237 A CA 2212237A CA 2212237 A1 CA2212237 A1 CA 2212237A1
- Authority
- CA
- Canada
- Prior art keywords
- hydrogen
- aralkyl
- cycloalkyl
- aryl
- compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Compounds, isolated from the bacteria Xenorhabdus bovienii, having the formula (see fig. I) Or (see fig. II) wherein n=0,1, or 2;R1 = 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 or the salts thereof have antineoplastic activity. The invention provides pharmaceutical compositions containing the compounds and the methods for employing them as medicaments, particularly in the treatment of human and animal cancers.
Description
SUMMARY OF THE INVENTION
The present invention provides dithiolopyrrolone derivatives, their corresponding monoxides (xenomins) and dioxides (xenorxides) having antineoplastic activities. The present CA 02212237 1997-09-0~
Page 2 of 22 Webster et aL Novel antineoplastic agents invention also provides antineoplastic compositions comprising these compounds, the salts thereof, and methods of using the inventive compounds as antineoplastic agents.
BACKGROUND
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 org~ni~m~ 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. Dithiolopyrrolones were initially isolated from Streptomyces in the 40s and have since then been isolated from other org~ni~m~. These compounds include aureothricin, thiolutin, holomycin and xenorhabdins (Hamao et al., 1948; Eisenman et al., 1953;
Celmer and Solomons, 1955; von Daehne et al., 1969; McInerney et al., 1991). These compounds possess antimicrobial activity against a wide range of micro-org~nism.~. Recently, new antimicrobial substances, namely xenorxides (Webster et al., 1996), also were found from the bacterial cultures of Xenorhabdus spp. As a result of part of the ongoing exploration of the potential usefulness of these metabolites of Xenorhabdus, the xenorxides, xenomins and the CA 02212237 1997-09-0~
Page 3 of 22 Webster et al. Novel antineoplastic agents dithiolopyrrolones have been found to be highly active against animal and human cancer cells, and is the subject of this invention.
Animal cells normally undergo various enzymatic and biochemical changes when influenced by various chemical factors. For example, when animal cells were challenged by carcinogens many enzymatic and biochemical activities can be promoted and some of the promoted activities can be experimentally detected. Among dithiolopyrrolones and their derivatives, 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. It has been reported (Menta and Moon, 1991; Sharma et al., 1994; Arnold et al., 1995) that thiolutin inhibited the promotion of several such activities that are possibly related to carcinogenesis in m~mm~ry cells that have been exposed to carcinogens. This suggested that thiolutin might be involved in the initiation of cell transformation or the progression of prem~lign:lnt cells becoming m~lign~nt cancer cells. However, this activity has not experimentally established. Although the study of dithiolopyrrolones has been on-going for about 50 years, the anticancer activity of thiolutin and other dithiolopyrrolones against m~lign~nt m~mm~ry cells has never been evaluated and reported until now. Xenorxides and xenomins have been discovered very recently, and there is no report in the prior art on their anticancer activity.
DESCRIPTION OF THE INVENTION
Although the compounds of the present invention may be synthesized chemically, the initial compounds were obtained from micro-org~nisms. The org~ni.sms used for this invention include Xenorhabdus bovienii, a symbiotic bacterium associated with entomopathogenic CA 02212237 1997-09-0~
Page 4 of 22 Websfer et al. Novel antineoplastic agents nematodes. X bovienii and its nematode symbiont Sfeinernema feltiae, 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 X bovienii A21 strain, at a rate of 25 IJs/larvae. After 24 to 48 h the dead insect larvae were surface disinfected by dipping them into 95% EtOH and igniting them. The cadavers were aseptically dissected, haemolymph 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 2,3,5-triphenyltetrazolium 0.04 g Agar 15 g Sterilized at 121 ~C for 15 minutes.
The resulting primary form of X bovienii was maintained and subcultured at 14 d intervals. For consistency, 14% sucrose lyophilized powder of the bacteria stored at -20~C was frequently used as the starting material for cultures. Cultures of X bovienii A21 strain from which the inventive compounds are obtained exhibit the characteristics listed in Table 1 and Table 2:
Table 1. Biochemical properties of Xenorhabdus bovienii A21 strain.
CA 02212237 1997-09-0~
Page 5 of 22 Webster et al. Novel antineoplastic agents Gram reaction -*
Cell size (,um) 5.3x2.2 Mobility +
Cell peritrichous +
Pigmentation yellow Catalase Oxidase Urease Lecithinase +
Lipase(Tween 80) +
* + positive; - negative.
Table 2. Acid production and utilization of carbon sources by Xenorhabdus bovienii A2 1 strain.
Acid production* Utilization of carbon sources D-Arabinose +/- Asparagine +
Esculine - Cystine D-Fructose + Glysine D-Galactose - Tyrosine +
D-Glucose + Nicotinic acid Inositol +/- Ethanol Inulin - Methanol D-Lactose - Inositol +/-D-Maltose + Mannose +
D-Mannitol - D-Galatose D-Mannose + D-Glucose +
D-Raffinose - D-Lactose D-Sorbitol +/- D-Mannitol L-Sorbose - D-Sorbitol D-xylose - Ribose +
* + positive; +/-: weakly positive; - negative.
These characteristics are in agreement with those described for X bovienii by Akhurst, R.
J. and N. E. Boemare (1988), and, therefore, establishes the identity of the organism as X
bovienii. The bacterial strain from which the compounds of this invention were isolated was deposited under the Budapest Treaty in the American Type Culture Collection (ATCC), Rochville, MD with a deposition number of ATCC 55743.
CA 02212237 1997-09-0~
Page 6 of 22 Webster et al. Novel antineoplastic agents 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.
After the fermentation has been completed, 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) may be concentrated under vacuum (e.g., at about 30 ~C ) to an oily residue. 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 chromatography (HPLC) with organic and/or aqueous solution.
In a conventional culture, the major compounds produced by X bovienii are dithiolopyrrolones while xenorxides and xenomins are present in relatively small amounts.
Alternatively, dithiolopyrrolones may be produced from other micro-org~ni~m.~, by chemical method(s) and/or a combination of both approaches.
CA 02212237 1997-09-0~
Page 7 of 22 Webster et al. Novel antineoplastic agents The monoxides and dioxides of the corresponding dithiolopyrrolones isolated from X
bovienii, namely, xenorxides and xenomins, respectively may be transformed from their corresponding dithiolopyrrolone derivatives by biological or chemical means. By 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. Several, additional monoxides and dioxides of the dithiolopyrrolones may obtained from different microorg~nisms, synthesized chemically and/or produced by a combination of both methods as well disclosed (T~k~h:~chi, 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.
The term "salts", as used herein, 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 CA 02212237 1997-09-0~
Page 8 of 22 Webster et al. Novel antineoplastic agents well known to one skilled in the art that an ~pl.~pliate 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.
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 a~1mini~tration, 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 m~mm~ n host may, for example, be oral, parenteral, or topical.
When these compounds or the salts thereof are used as therapeutics, they can be a~lmini~trated alone or in a pharmaceutically suitable formulation containing, in addition to the active ingredient, one or more conventional carriers. Depending on the nature of the disease and/or route of ~lmini~tration, the composition of this invention can be formulated by known means.
Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granules, powder etc.~ or liquid (solutions, suspensions or emulsions) compositions suitable for oral, topical or parenteral a(lmini~tration, and they may contain the pure compound or a salt thereof or in combination with any carrier or other pharmaceutically active compounds. These compositions may need to be sterile when a~lmini.~tered parenterally.
CA 02212237 1997-09-0~
Page 9 of 22 Webster et aL Novel antineoplastic agents The therapeutic 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 ph~ ceutical materials which themselves are available in the art and can be prepared by established procedures. The ~ l;ate solid or liquid vehicle or diluent may be selected, and the compositions prepared, by methods known to the skilled artisan. The a(lministration 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). The dosage ~lministered 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. Illustratively, dosage levels ofthe a(lministered 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. Expressed in terms of concentration, an active ingredient can be present in the compositions ofthe 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. 05 to about 50% w/v of the composition and preferably from about 5 to about 20% w/v. The active ingredients to be employed as antineoplastic agents, can be easily prepared in such unit dosage form with the employment of pharmaceutical materials which themselves are available in the art and can be prepared by established procedures.
CA 02212237 1997-09-0~
Page 10 of 22 Webster et al. Novel antineoplastic agents EXAMPLE 1. preparation of xenorxides, xenomins and the dithiolopyrrolones A. Preparation of selected dithiolopyrrolones.
Several dithiolopyrrolones have been reported so far, these include aureothricin, thiolutin, holomycin, xenorhabdins and thiomarinol. Aureothricin was initially reported by Umezawa et al. (1948), the structure were fully disclosed by Celmer and Solomons (1955).
Thiolutin is produced by several bacterial species including Streptoverticillium album, which is available from the American Type Culture Collection(ATCC), Rockville, MD
with an ATCC number of 33049. The production of thiolutin and holomycin by chemical synthesis was published by Schmidt and Geiger (1962), Hagio, K. and Yoneda, N. (1974) and Stachel, H.D., et al. (1992). Production of xenorhabdins was reported and fully reported by McInerney et al. (1991) and by Li et al.(1995). The preparation of additional different compounds with the dithiolopyrrolone ring was recently disclosed by Baggaley et al. (1994a, b) and by T~k~h~hi et al (1994). The dithiolopyrrolones used for the subject invention are prepared by the methods described in the cited references, and the structure of each dithiolopyrrolone derivative confirmed by its NMR spectroscopy. Skilled chemists will be able to use procedures as disclosed herein, and others, to obtain these dithiolopyrrolones from available stock substances. In carrying out such operations, any suitable filtration, chromatographic, and other purification techniques might be employed by those skilled in the art. It will be apparent to those skilled in the art that materials and reagents to carry out such operations are commercially available from chemical companies, so no details are given respecting them.
CA 02212237 1997-09-0~
Page 11 of 22 Webster et aL Novel antineoplastic agents B. Preparation of the corresponding dioxides from dithiolopyrrolones by microbial fermentation.
Xenorxides: Cultures of 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. The combined extracts were dried with anhydrous sodium sulfate and then filtered through filter paper. The filtrate was concentrated on a rotary evaporator below 30 ~C, under vacuum to yield a brown oil. After the above experiment was repeated 10 times, approximately 3 g of the oil was obtained. The crude extracts were then loaded onto a silica gel (200 g silica gel 60, 40 cm x 5 cm, EM Science, Darmstadt, Germany) chromatographic column.
The yellow bioactive fraction was eluted out with ether or ethyl acetate. This bioactive fraction was then subject to HPLC on a C,8preparative column (Spherisorb 10 (ODS(l)), 250 X 10 mm, 10 micro, Phenomenex, Torrance, CA) with a program (isocratic at 10% acetonitrile in water for 5 min, then gradually increasing to 85% acetonitrile in 35 min, isocratic for 5 min, then decreasing back to 10% in 2 min ) at 2.5 ml/min. The eluate was monitored at 254 nm.
XENORXIDE 1 (about 0.3 mg per liter of the culture broth) was eluted at 33.6 min, and this was followed by XENORXIDE 2 (0.2 mg/l) which was eluted at 35.2 min.
C. Preparation of the corresponding monoxides from dithiolopyrrolones by microbial fermentation.
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 CA 02212237 1997-09-0~
Page 12 of 22 Webster et al. Novel antineoplastic agents 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 C,8 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. This bioactive fraction was then concentrated and separated by HPLC on a C,8 preparative column (Spherisorb 10 (ODS(l)), 250 x 10 mm, 10 micro, Phenomenex, Torrance, CA) with a program (30% MeCN in H2O for 1 min and gradually increasing to 70% MeCN in H2O in 24 min, isocratic for 5 min) at 2.0 ml/min. The eluate was monitored at 254 nm. Active peak 1 (25.4 min) and peak 2 (25.8 min) were collected. Active peak 1 was concentrated, and further separated by preparative silica gel TLC with 60% ethyl acetate in dichloromethane as the eluent to give xenomin 1 (Rf = 0.32). Active peak 2 was concentrated, and further separated by preparative silica gel TLC with 60% ethyl acetate in dichloromethane as the eluent to give xenomin 2 (Rf = 0.31).
D. Preparation of xenorxides from their corresponding dithiolopyrrolone derivatives by biological transformation.
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/l), XENORXIDE 2 was eluted at 35.2 min (1.5 mg/l).
E. Preparation of the corresponding dioxides from dithiolopyrrolones by chemical oxidation.
CA 02212237 1997-09-0~
Page 13 of 22 Webster et aL Novel antineoplastic agents One hundred and fifty two mg of 6-(hexanamido)-4-methyl-4,5-dihydro-1.2-dithiolo[4,3-b] pyrrol-5-one (XN1) were dissolved in a mixture of 20 ml of acetone and 15 ml of water, and then cooled to 0 ~C with ice. Potassium peroxymonosulfate (510 mg) was added to the solution at 0 ~C . The reaction mixture was stirred for one hour at 0 ~C. Then 5 ml of saturated solution of potassium bicarbonate was added to the reaction mixture with continuous stirring for 0.5 h at 0~C. The reaction mixture, after water was added, was extracted with ethyl acetate three times.
The extracts were combined, dried over Na2 SO4 and evaporated to give the crude product, which was then purified by silica gel column chromatography with hexane: ethyl acetate (2:1) to give pure xenorxide 1 (78 mg, 51% yield). Similarly, xenorxide 3 was produced from 6-(5'-methylhexanamido)-4,5-dihydro-1,2-dithiolo[4,3-b] pyrrol-5One with 50% yield.
Example 2. Identification of the active components NMR spectra were recorded on a Bruker WM400 spectrometer in CDCl3, using residual CDCl3 (~7.25) as internal standard. Low resolution mass spectra were obtained on a Hewlett-Packard 5985B GC/MS system operating at 70 eV using a direct probe. High resolution MS
spectra were recorded on a Kratos MS80 instrument. IR spectra were recorded as neat film on NaCl using a Perkin-Elmer 599B spectrometer. (Abbreviations used as follows: EI = Electron Impact, M = Molecular Ion, t = triplet, J = coupling constant, Hz = Hertz, d = doublet, m =
multiplet, bs = broad singlet).
6-(Acetamido)-4-methyl-4,5-dihydro-1.2-dithiolo[4,3-b] pyrrol-5-one (Thiolutin) (XN0): lHNMR (DMSO-d6) ~ 2.05 (3H, s), 3.92 (3H, s), 7.19 (lH, s) and 9.85 (lH, broad s); EIMS m/e 228 (M+), 186.
CA 02212237 1997-09-0~
Page 14 of 22 Webster et al. Novel antineoplastic agents 6-(Hexanamido)-4-methyl-4,5-dihydro-1.2-dithiolo[4,3-b] pyrrol-5-one (XNl):
lHNMR (CDCl3) ~ 0.90 (3H, t, J=6.9Hz), 1.35 (4H, m), 1.70 (2H, m), 2.35 (2H, t, J=7.4Hz), 3.35 (3H, s), 6.63 (lH, s) and 7.43 (lH, broad s); EIMS m/e 284 (M+), 186.
6-(5'-Methylhexanamido)-4,5-dihydro-1,2-dithiolo[4,3-b] pyrrol-5One (XN3): IHNMR(CDCl3) ~ 0.89 (6H, d, J=6.6Hz), 1.24 (3H, m), 1.70 (2H, m), 2.32 (2H, t, J=7.6Hz), 6.74 (lH, s), 7.44 (lH, broad s) and 7.94 (lH, broad s); EIMS m/e 284 (M+), 172.
XENORXIDE 1 (XOl): 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, 3275, 1720, 1686, 1654, 1637, 1560, 1522, 1310, 1139, 551 cm-l; lHNMR
(CDCl3) ~: 7.56 (lH, bs, CO-NH), 6.35 (lH, s, H-3), 3.20 (3H, s, N-Me), 2.38 (2H, t, CO-CH2, J=7.4 Hz), 1.67 (2H, m, CH2), 1.32 (4H, m, CH2CH2), 0.89 (3H, t, J=7.0 Hz); l3CNMR
(CDCl3) ~: 171.6(s, CON), 164.7 (s, CO), 145.4(s, C7), 121.3(s, C6), 116.2(s, C8), 109.2(d, C3), 36.4, 31.2, 27.8, 24.6, 22.3, 13.8.
XENORXIDE 2 (X02): EIMS: 330(M+, 10), 218(100); HRMS: 330.0707 (Calc. for C13HlgN2O4S2: 330.0708, 18), 217.9829 (Calc. for C6H6N2O3S2: 217.9820, 100), 154.0213 (Calc. for C6H6N20S: 154.0201, 16);; IR (KBr): 3438, 3298, 1719, 1686, 1654, 1637, 1560, 1522, 1400, 1310, 1142, 551 cm-l; lHNMR (CDCl3) ~: 7.56 (lH, bs,CO-NH), 6.35 (lH, s, H-3), 3.20 (3H, s, N-Me), 2.36 (2H, t, CO-CH2, J=7.4 Hz), 1.67 (2H, m, CH2), 1.2-1.6 (lH, m, CH), 1.22 (2H, m, CH2), 0.89 (6H, d, J=6.6Hz); Different NOE experiment showed the NOE effect between the peak at 6.35 ppm and 3.20 ppm; l3CNMR (CDCl3) ~: 171.6(s, CON), 164.7 (s, Page 15 of 22 Webster et aL Novel antineoplastic agents CO), 145.4(s, C7), 121.3(s, C6), 116.2(s, Cg), 109.2(d, C3), 38.2(t, CH2), 36.7(t, CH2), 28.0(q, CH3), 27.8(d, CH), 22.8(t, CH2), 22.4(q, CH3).
XENORXIDE 3 (X03): EIMS: 316(M+, 7.5), 218(9), 204(65) 185(7), 172(15), 105 (25), 95(95), 69(100), 43 (70); lHNMR (CDC13) ~: 10.10 (lH, bs), 9.32 (lH, bs), 6.85 (IH, s, H-3), 2.53 (2H, t, CO-CH2, J=7.5 Hz), 1.66 (2H, m, CH2), 1.58 (lH, m), 1.21 (2H, m, ), 0.87 (6H, d, J=6.6 Hz).
Xenomin l(XMl): EIMS: 300(M+, 13), 202(36), 186(44), 185(41), 85(62), 69(100);
HRMS: 300.0605 (Calc. for C,2HI6N203S2: 300.0602, 12), 201.9871 (Calc. for C6H6N2O2S2:
201.9871, 30); IHNMR (CDCl3) d: 7.52 (lH, bs,CO-NH), 6.46 (IH, s, H-3), 3.30 (3H, s, N-Me), 2.49 (2H, t, CO-CH2, J=7.4 Hz), 1.79 (2H, m, CH2), 1.41 (4H, m, CH2-CH2), 0.90 (3H, t, J=6.9).
Xenomin 2 (XM2): EIMS: 314(M+, 21), 218(15), 202(59), 187(18), 186(98), 185(73), 69(100); HRMS: 314.0759 (Calc. for Cl3HI8N2O3S2: 314.0759, 12), 201.9871 (Calc. for C6H6N2O2S2: 201.9871, 45); ~HNMR (CDCl3) d: 7.43 (lH, bs,CO-NH), 6.46 ('H, s, H-3), 3.30 (3H, s, N-Me), 2.48 (2H, t, CO-CH2, J=7.6 Hz), 1.71 (2H, m, CH2), 1.29 (3H, m, CH and CH2), 0.88 (6H, d, J=7.0).
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 LC50. The LC50 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 m~mm~l.s. The test animal cancer cells are CA 02212237 1997-09-0~
Page 16 of 22 Webster et al. Novel antineoplastic agents 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). Briefly, 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 ~11 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. Then 20 ~l of medium cont~ining 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 termin:~te~l after incubation by adding to each well 50 111 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. At the end of the staining period, the SRB was removed and plates were quickly rinsed five times with 1% acetic acid. After being rinsed, 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).
CA 02212237 1997-09-0~
Page 17 of 22 Webster et a/. Novel antineoplastic agents Antitneoplastic activity:
LC50 (10-6 M) Cancer cells XN0 XN1 XN3 XO1 X02 XM1 XM2 HT29 0.13 0.11 0.11 0.22 0.85 0.47 0.11 MCF-7 0.35 0.56 0.39 0.89 0.95 0.47 0.55 Hela 0.31 0.46 0.32 0.16 0.13 0.80 0.21 P388 --* -- -- 0.66 0.28 NCI-H460 -- -- 0.03 0.28 0.22 Sk-Mel-28 -- -- 0.24 0.19 0.73 LNCap -- -- -- 0.54 0.19 DU-145 (Prostate cancer) -- -- 0.14 1.80 1.60 UO-31 (Renal cancer) -- -- 0.18 1.85 6.0 SF-295 (CNS cancer) -- -- 0.04 1.58 2.12 * not tested.
From the foregoing embodiments and examples, it is apparent that an invention has been herein described and illustrated. While our above description contains many specificities, these should not be construed as limitations on the scope of the invention, but rather as examples of preferred embodiments. Accordingly, the scope of the invention should not be determined by the embodiments presented, but by the appended claims and their legal equivalents.
CA 02212237 1997-09-0~
Page 21 of 22 Webster et aL Novel antineoplastic agents REFERENCES CITED
Akhurst, R. J. and N. E. Boemare, 1988. .J: Gen. Microbiol. Vol. 134, 1835- 1845 Arnold, J. T. et al., 1995. Cancer Research 55:537-543.
Baggaley, K.H. et al., 1994a.WO 94/26750.
Baggaley, K.H. et al., 1994b. WO 94/28001.
Celmer, W. D. and I. A. Solomons 1955. J. Amer. Chem. Soc. 77:2861-2865.
Eisenman, W. et al., 1953. Antibiotics and Chemotherapy 3: 385-392.
Forst, S. and K. Nealson, 1996. Microbiol. Rev. 60:21-43.
Fujimoto, K. et al., 1996. WO 96/23795.
Hagio, K. and Yoneda, N. 1974. Bull. Chem. Soc. Japan. 47:1484-1489.
Jimenez, A. et al., 1973. Antimicrob. Ag Chemother. 729-738.
Li, et al., 1995. Appl. Environ. Microbiol. 61 :4329-4333.
Li, J. et al., 1995. J Nat. Prod. 58:1081-1085.
McInerney, B. V. et al., 1991. J Nat. Prod. 54:774-784.
Menta R. G. and R. C. Moon, 1991. Anticancer Research 11 :593-596.
Monks et al., 1991. J: Natl. Cancer Inst. 83 :757-766.
Ninomiya, Y. T. et al., 1980. Chem. Pharm. Bull. 28 :3157-3162.
Sharma, S. et al., 1994. Cancer Research 54:5848-5855.
Shroeder et al., 1981. J. Med. Chem. 24: 1078-1083.
Skehan, P. et al., 1990. J Natl. Cancer Inst 82: 1107- 1118.
Stachel, H.D. et al. 1992. Liebigs Ann. Chem. 473-480.
T~k~h~hi S. et al., 1994. US patent. 5,292,892.
Page 22 of 22 Webster et aL Novel antineoplastic agents Tz~k~h~shi S. et al., 1995. US patent. 5,399,711.
Tipper D. J. 1973. J. Bacteriol. 116:245-256.
Umezawa, H. et al. 1948. Jap. Med. J. 1 :512-517.
Webster et al. WO: 96/32396.
von Daehne, W. et al., 1969. J. Antibiotics 22:233-235.
* * *
The present invention provides dithiolopyrrolone derivatives, their corresponding monoxides (xenomins) and dioxides (xenorxides) having antineoplastic activities. The present CA 02212237 1997-09-0~
Page 2 of 22 Webster et aL Novel antineoplastic agents invention also provides antineoplastic compositions comprising these compounds, the salts thereof, and methods of using the inventive compounds as antineoplastic agents.
BACKGROUND
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 org~ni~m~ 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. Dithiolopyrrolones were initially isolated from Streptomyces in the 40s and have since then been isolated from other org~ni~m~. These compounds include aureothricin, thiolutin, holomycin and xenorhabdins (Hamao et al., 1948; Eisenman et al., 1953;
Celmer and Solomons, 1955; von Daehne et al., 1969; McInerney et al., 1991). These compounds possess antimicrobial activity against a wide range of micro-org~nism.~. Recently, new antimicrobial substances, namely xenorxides (Webster et al., 1996), also were found from the bacterial cultures of Xenorhabdus spp. As a result of part of the ongoing exploration of the potential usefulness of these metabolites of Xenorhabdus, the xenorxides, xenomins and the CA 02212237 1997-09-0~
Page 3 of 22 Webster et al. Novel antineoplastic agents dithiolopyrrolones have been found to be highly active against animal and human cancer cells, and is the subject of this invention.
Animal cells normally undergo various enzymatic and biochemical changes when influenced by various chemical factors. For example, when animal cells were challenged by carcinogens many enzymatic and biochemical activities can be promoted and some of the promoted activities can be experimentally detected. Among dithiolopyrrolones and their derivatives, 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. It has been reported (Menta and Moon, 1991; Sharma et al., 1994; Arnold et al., 1995) that thiolutin inhibited the promotion of several such activities that are possibly related to carcinogenesis in m~mm~ry cells that have been exposed to carcinogens. This suggested that thiolutin might be involved in the initiation of cell transformation or the progression of prem~lign:lnt cells becoming m~lign~nt cancer cells. However, this activity has not experimentally established. Although the study of dithiolopyrrolones has been on-going for about 50 years, the anticancer activity of thiolutin and other dithiolopyrrolones against m~lign~nt m~mm~ry cells has never been evaluated and reported until now. Xenorxides and xenomins have been discovered very recently, and there is no report in the prior art on their anticancer activity.
DESCRIPTION OF THE INVENTION
Although the compounds of the present invention may be synthesized chemically, the initial compounds were obtained from micro-org~nisms. The org~ni.sms used for this invention include Xenorhabdus bovienii, a symbiotic bacterium associated with entomopathogenic CA 02212237 1997-09-0~
Page 4 of 22 Websfer et al. Novel antineoplastic agents nematodes. X bovienii and its nematode symbiont Sfeinernema feltiae, 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 X bovienii A21 strain, at a rate of 25 IJs/larvae. After 24 to 48 h the dead insect larvae were surface disinfected by dipping them into 95% EtOH and igniting them. The cadavers were aseptically dissected, haemolymph 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 2,3,5-triphenyltetrazolium 0.04 g Agar 15 g Sterilized at 121 ~C for 15 minutes.
The resulting primary form of X bovienii was maintained and subcultured at 14 d intervals. For consistency, 14% sucrose lyophilized powder of the bacteria stored at -20~C was frequently used as the starting material for cultures. Cultures of X bovienii A21 strain from which the inventive compounds are obtained exhibit the characteristics listed in Table 1 and Table 2:
Table 1. Biochemical properties of Xenorhabdus bovienii A21 strain.
CA 02212237 1997-09-0~
Page 5 of 22 Webster et al. Novel antineoplastic agents Gram reaction -*
Cell size (,um) 5.3x2.2 Mobility +
Cell peritrichous +
Pigmentation yellow Catalase Oxidase Urease Lecithinase +
Lipase(Tween 80) +
* + positive; - negative.
Table 2. Acid production and utilization of carbon sources by Xenorhabdus bovienii A2 1 strain.
Acid production* Utilization of carbon sources D-Arabinose +/- Asparagine +
Esculine - Cystine D-Fructose + Glysine D-Galactose - Tyrosine +
D-Glucose + Nicotinic acid Inositol +/- Ethanol Inulin - Methanol D-Lactose - Inositol +/-D-Maltose + Mannose +
D-Mannitol - D-Galatose D-Mannose + D-Glucose +
D-Raffinose - D-Lactose D-Sorbitol +/- D-Mannitol L-Sorbose - D-Sorbitol D-xylose - Ribose +
* + positive; +/-: weakly positive; - negative.
These characteristics are in agreement with those described for X bovienii by Akhurst, R.
J. and N. E. Boemare (1988), and, therefore, establishes the identity of the organism as X
bovienii. The bacterial strain from which the compounds of this invention were isolated was deposited under the Budapest Treaty in the American Type Culture Collection (ATCC), Rochville, MD with a deposition number of ATCC 55743.
CA 02212237 1997-09-0~
Page 6 of 22 Webster et al. Novel antineoplastic agents 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.
After the fermentation has been completed, 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) may be concentrated under vacuum (e.g., at about 30 ~C ) to an oily residue. 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 chromatography (HPLC) with organic and/or aqueous solution.
In a conventional culture, the major compounds produced by X bovienii are dithiolopyrrolones while xenorxides and xenomins are present in relatively small amounts.
Alternatively, dithiolopyrrolones may be produced from other micro-org~ni~m.~, by chemical method(s) and/or a combination of both approaches.
CA 02212237 1997-09-0~
Page 7 of 22 Webster et al. Novel antineoplastic agents The monoxides and dioxides of the corresponding dithiolopyrrolones isolated from X
bovienii, namely, xenorxides and xenomins, respectively may be transformed from their corresponding dithiolopyrrolone derivatives by biological or chemical means. By 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. Several, additional monoxides and dioxides of the dithiolopyrrolones may obtained from different microorg~nisms, synthesized chemically and/or produced by a combination of both methods as well disclosed (T~k~h:~chi, 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.
The term "salts", as used herein, 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 CA 02212237 1997-09-0~
Page 8 of 22 Webster et al. Novel antineoplastic agents well known to one skilled in the art that an ~pl.~pliate 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.
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 a~1mini~tration, 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 m~mm~ n host may, for example, be oral, parenteral, or topical.
When these compounds or the salts thereof are used as therapeutics, they can be a~lmini~trated alone or in a pharmaceutically suitable formulation containing, in addition to the active ingredient, one or more conventional carriers. Depending on the nature of the disease and/or route of ~lmini~tration, the composition of this invention can be formulated by known means.
Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granules, powder etc.~ or liquid (solutions, suspensions or emulsions) compositions suitable for oral, topical or parenteral a(lmini~tration, and they may contain the pure compound or a salt thereof or in combination with any carrier or other pharmaceutically active compounds. These compositions may need to be sterile when a~lmini.~tered parenterally.
CA 02212237 1997-09-0~
Page 9 of 22 Webster et aL Novel antineoplastic agents The therapeutic 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 ph~ ceutical materials which themselves are available in the art and can be prepared by established procedures. The ~ l;ate solid or liquid vehicle or diluent may be selected, and the compositions prepared, by methods known to the skilled artisan. The a(lministration 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). The dosage ~lministered 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. Illustratively, dosage levels ofthe a(lministered 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. Expressed in terms of concentration, an active ingredient can be present in the compositions ofthe 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. 05 to about 50% w/v of the composition and preferably from about 5 to about 20% w/v. The active ingredients to be employed as antineoplastic agents, can be easily prepared in such unit dosage form with the employment of pharmaceutical materials which themselves are available in the art and can be prepared by established procedures.
CA 02212237 1997-09-0~
Page 10 of 22 Webster et al. Novel antineoplastic agents EXAMPLE 1. preparation of xenorxides, xenomins and the dithiolopyrrolones A. Preparation of selected dithiolopyrrolones.
Several dithiolopyrrolones have been reported so far, these include aureothricin, thiolutin, holomycin, xenorhabdins and thiomarinol. Aureothricin was initially reported by Umezawa et al. (1948), the structure were fully disclosed by Celmer and Solomons (1955).
Thiolutin is produced by several bacterial species including Streptoverticillium album, which is available from the American Type Culture Collection(ATCC), Rockville, MD
with an ATCC number of 33049. The production of thiolutin and holomycin by chemical synthesis was published by Schmidt and Geiger (1962), Hagio, K. and Yoneda, N. (1974) and Stachel, H.D., et al. (1992). Production of xenorhabdins was reported and fully reported by McInerney et al. (1991) and by Li et al.(1995). The preparation of additional different compounds with the dithiolopyrrolone ring was recently disclosed by Baggaley et al. (1994a, b) and by T~k~h~hi et al (1994). The dithiolopyrrolones used for the subject invention are prepared by the methods described in the cited references, and the structure of each dithiolopyrrolone derivative confirmed by its NMR spectroscopy. Skilled chemists will be able to use procedures as disclosed herein, and others, to obtain these dithiolopyrrolones from available stock substances. In carrying out such operations, any suitable filtration, chromatographic, and other purification techniques might be employed by those skilled in the art. It will be apparent to those skilled in the art that materials and reagents to carry out such operations are commercially available from chemical companies, so no details are given respecting them.
CA 02212237 1997-09-0~
Page 11 of 22 Webster et aL Novel antineoplastic agents B. Preparation of the corresponding dioxides from dithiolopyrrolones by microbial fermentation.
Xenorxides: Cultures of 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. The combined extracts were dried with anhydrous sodium sulfate and then filtered through filter paper. The filtrate was concentrated on a rotary evaporator below 30 ~C, under vacuum to yield a brown oil. After the above experiment was repeated 10 times, approximately 3 g of the oil was obtained. The crude extracts were then loaded onto a silica gel (200 g silica gel 60, 40 cm x 5 cm, EM Science, Darmstadt, Germany) chromatographic column.
The yellow bioactive fraction was eluted out with ether or ethyl acetate. This bioactive fraction was then subject to HPLC on a C,8preparative column (Spherisorb 10 (ODS(l)), 250 X 10 mm, 10 micro, Phenomenex, Torrance, CA) with a program (isocratic at 10% acetonitrile in water for 5 min, then gradually increasing to 85% acetonitrile in 35 min, isocratic for 5 min, then decreasing back to 10% in 2 min ) at 2.5 ml/min. The eluate was monitored at 254 nm.
XENORXIDE 1 (about 0.3 mg per liter of the culture broth) was eluted at 33.6 min, and this was followed by XENORXIDE 2 (0.2 mg/l) which was eluted at 35.2 min.
C. Preparation of the corresponding monoxides from dithiolopyrrolones by microbial fermentation.
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 CA 02212237 1997-09-0~
Page 12 of 22 Webster et al. Novel antineoplastic agents 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 C,8 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. This bioactive fraction was then concentrated and separated by HPLC on a C,8 preparative column (Spherisorb 10 (ODS(l)), 250 x 10 mm, 10 micro, Phenomenex, Torrance, CA) with a program (30% MeCN in H2O for 1 min and gradually increasing to 70% MeCN in H2O in 24 min, isocratic for 5 min) at 2.0 ml/min. The eluate was monitored at 254 nm. Active peak 1 (25.4 min) and peak 2 (25.8 min) were collected. Active peak 1 was concentrated, and further separated by preparative silica gel TLC with 60% ethyl acetate in dichloromethane as the eluent to give xenomin 1 (Rf = 0.32). Active peak 2 was concentrated, and further separated by preparative silica gel TLC with 60% ethyl acetate in dichloromethane as the eluent to give xenomin 2 (Rf = 0.31).
D. Preparation of xenorxides from their corresponding dithiolopyrrolone derivatives by biological transformation.
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/l), XENORXIDE 2 was eluted at 35.2 min (1.5 mg/l).
E. Preparation of the corresponding dioxides from dithiolopyrrolones by chemical oxidation.
CA 02212237 1997-09-0~
Page 13 of 22 Webster et aL Novel antineoplastic agents One hundred and fifty two mg of 6-(hexanamido)-4-methyl-4,5-dihydro-1.2-dithiolo[4,3-b] pyrrol-5-one (XN1) were dissolved in a mixture of 20 ml of acetone and 15 ml of water, and then cooled to 0 ~C with ice. Potassium peroxymonosulfate (510 mg) was added to the solution at 0 ~C . The reaction mixture was stirred for one hour at 0 ~C. Then 5 ml of saturated solution of potassium bicarbonate was added to the reaction mixture with continuous stirring for 0.5 h at 0~C. The reaction mixture, after water was added, was extracted with ethyl acetate three times.
The extracts were combined, dried over Na2 SO4 and evaporated to give the crude product, which was then purified by silica gel column chromatography with hexane: ethyl acetate (2:1) to give pure xenorxide 1 (78 mg, 51% yield). Similarly, xenorxide 3 was produced from 6-(5'-methylhexanamido)-4,5-dihydro-1,2-dithiolo[4,3-b] pyrrol-5One with 50% yield.
Example 2. Identification of the active components NMR spectra were recorded on a Bruker WM400 spectrometer in CDCl3, using residual CDCl3 (~7.25) as internal standard. Low resolution mass spectra were obtained on a Hewlett-Packard 5985B GC/MS system operating at 70 eV using a direct probe. High resolution MS
spectra were recorded on a Kratos MS80 instrument. IR spectra were recorded as neat film on NaCl using a Perkin-Elmer 599B spectrometer. (Abbreviations used as follows: EI = Electron Impact, M = Molecular Ion, t = triplet, J = coupling constant, Hz = Hertz, d = doublet, m =
multiplet, bs = broad singlet).
6-(Acetamido)-4-methyl-4,5-dihydro-1.2-dithiolo[4,3-b] pyrrol-5-one (Thiolutin) (XN0): lHNMR (DMSO-d6) ~ 2.05 (3H, s), 3.92 (3H, s), 7.19 (lH, s) and 9.85 (lH, broad s); EIMS m/e 228 (M+), 186.
CA 02212237 1997-09-0~
Page 14 of 22 Webster et al. Novel antineoplastic agents 6-(Hexanamido)-4-methyl-4,5-dihydro-1.2-dithiolo[4,3-b] pyrrol-5-one (XNl):
lHNMR (CDCl3) ~ 0.90 (3H, t, J=6.9Hz), 1.35 (4H, m), 1.70 (2H, m), 2.35 (2H, t, J=7.4Hz), 3.35 (3H, s), 6.63 (lH, s) and 7.43 (lH, broad s); EIMS m/e 284 (M+), 186.
6-(5'-Methylhexanamido)-4,5-dihydro-1,2-dithiolo[4,3-b] pyrrol-5One (XN3): IHNMR(CDCl3) ~ 0.89 (6H, d, J=6.6Hz), 1.24 (3H, m), 1.70 (2H, m), 2.32 (2H, t, J=7.6Hz), 6.74 (lH, s), 7.44 (lH, broad s) and 7.94 (lH, broad s); EIMS m/e 284 (M+), 172.
XENORXIDE 1 (XOl): 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, 3275, 1720, 1686, 1654, 1637, 1560, 1522, 1310, 1139, 551 cm-l; lHNMR
(CDCl3) ~: 7.56 (lH, bs, CO-NH), 6.35 (lH, s, H-3), 3.20 (3H, s, N-Me), 2.38 (2H, t, CO-CH2, J=7.4 Hz), 1.67 (2H, m, CH2), 1.32 (4H, m, CH2CH2), 0.89 (3H, t, J=7.0 Hz); l3CNMR
(CDCl3) ~: 171.6(s, CON), 164.7 (s, CO), 145.4(s, C7), 121.3(s, C6), 116.2(s, C8), 109.2(d, C3), 36.4, 31.2, 27.8, 24.6, 22.3, 13.8.
XENORXIDE 2 (X02): EIMS: 330(M+, 10), 218(100); HRMS: 330.0707 (Calc. for C13HlgN2O4S2: 330.0708, 18), 217.9829 (Calc. for C6H6N2O3S2: 217.9820, 100), 154.0213 (Calc. for C6H6N20S: 154.0201, 16);; IR (KBr): 3438, 3298, 1719, 1686, 1654, 1637, 1560, 1522, 1400, 1310, 1142, 551 cm-l; lHNMR (CDCl3) ~: 7.56 (lH, bs,CO-NH), 6.35 (lH, s, H-3), 3.20 (3H, s, N-Me), 2.36 (2H, t, CO-CH2, J=7.4 Hz), 1.67 (2H, m, CH2), 1.2-1.6 (lH, m, CH), 1.22 (2H, m, CH2), 0.89 (6H, d, J=6.6Hz); Different NOE experiment showed the NOE effect between the peak at 6.35 ppm and 3.20 ppm; l3CNMR (CDCl3) ~: 171.6(s, CON), 164.7 (s, Page 15 of 22 Webster et aL Novel antineoplastic agents CO), 145.4(s, C7), 121.3(s, C6), 116.2(s, Cg), 109.2(d, C3), 38.2(t, CH2), 36.7(t, CH2), 28.0(q, CH3), 27.8(d, CH), 22.8(t, CH2), 22.4(q, CH3).
XENORXIDE 3 (X03): EIMS: 316(M+, 7.5), 218(9), 204(65) 185(7), 172(15), 105 (25), 95(95), 69(100), 43 (70); lHNMR (CDC13) ~: 10.10 (lH, bs), 9.32 (lH, bs), 6.85 (IH, s, H-3), 2.53 (2H, t, CO-CH2, J=7.5 Hz), 1.66 (2H, m, CH2), 1.58 (lH, m), 1.21 (2H, m, ), 0.87 (6H, d, J=6.6 Hz).
Xenomin l(XMl): EIMS: 300(M+, 13), 202(36), 186(44), 185(41), 85(62), 69(100);
HRMS: 300.0605 (Calc. for C,2HI6N203S2: 300.0602, 12), 201.9871 (Calc. for C6H6N2O2S2:
201.9871, 30); IHNMR (CDCl3) d: 7.52 (lH, bs,CO-NH), 6.46 (IH, s, H-3), 3.30 (3H, s, N-Me), 2.49 (2H, t, CO-CH2, J=7.4 Hz), 1.79 (2H, m, CH2), 1.41 (4H, m, CH2-CH2), 0.90 (3H, t, J=6.9).
Xenomin 2 (XM2): EIMS: 314(M+, 21), 218(15), 202(59), 187(18), 186(98), 185(73), 69(100); HRMS: 314.0759 (Calc. for Cl3HI8N2O3S2: 314.0759, 12), 201.9871 (Calc. for C6H6N2O2S2: 201.9871, 45); ~HNMR (CDCl3) d: 7.43 (lH, bs,CO-NH), 6.46 ('H, s, H-3), 3.30 (3H, s, N-Me), 2.48 (2H, t, CO-CH2, J=7.6 Hz), 1.71 (2H, m, CH2), 1.29 (3H, m, CH and CH2), 0.88 (6H, d, J=7.0).
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 LC50. The LC50 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 m~mm~l.s. The test animal cancer cells are CA 02212237 1997-09-0~
Page 16 of 22 Webster et al. Novel antineoplastic agents 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). Briefly, 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 ~11 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. Then 20 ~l of medium cont~ining 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 termin:~te~l after incubation by adding to each well 50 111 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. At the end of the staining period, the SRB was removed and plates were quickly rinsed five times with 1% acetic acid. After being rinsed, 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).
CA 02212237 1997-09-0~
Page 17 of 22 Webster et a/. Novel antineoplastic agents Antitneoplastic activity:
LC50 (10-6 M) Cancer cells XN0 XN1 XN3 XO1 X02 XM1 XM2 HT29 0.13 0.11 0.11 0.22 0.85 0.47 0.11 MCF-7 0.35 0.56 0.39 0.89 0.95 0.47 0.55 Hela 0.31 0.46 0.32 0.16 0.13 0.80 0.21 P388 --* -- -- 0.66 0.28 NCI-H460 -- -- 0.03 0.28 0.22 Sk-Mel-28 -- -- 0.24 0.19 0.73 LNCap -- -- -- 0.54 0.19 DU-145 (Prostate cancer) -- -- 0.14 1.80 1.60 UO-31 (Renal cancer) -- -- 0.18 1.85 6.0 SF-295 (CNS cancer) -- -- 0.04 1.58 2.12 * not tested.
From the foregoing embodiments and examples, it is apparent that an invention has been herein described and illustrated. While our above description contains many specificities, these should not be construed as limitations on the scope of the invention, but rather as examples of preferred embodiments. Accordingly, the scope of the invention should not be determined by the embodiments presented, but by the appended claims and their legal equivalents.
CA 02212237 1997-09-0~
Page 21 of 22 Webster et aL Novel antineoplastic agents REFERENCES CITED
Akhurst, R. J. and N. E. Boemare, 1988. .J: Gen. Microbiol. Vol. 134, 1835- 1845 Arnold, J. T. et al., 1995. Cancer Research 55:537-543.
Baggaley, K.H. et al., 1994a.WO 94/26750.
Baggaley, K.H. et al., 1994b. WO 94/28001.
Celmer, W. D. and I. A. Solomons 1955. J. Amer. Chem. Soc. 77:2861-2865.
Eisenman, W. et al., 1953. Antibiotics and Chemotherapy 3: 385-392.
Forst, S. and K. Nealson, 1996. Microbiol. Rev. 60:21-43.
Fujimoto, K. et al., 1996. WO 96/23795.
Hagio, K. and Yoneda, N. 1974. Bull. Chem. Soc. Japan. 47:1484-1489.
Jimenez, A. et al., 1973. Antimicrob. Ag Chemother. 729-738.
Li, et al., 1995. Appl. Environ. Microbiol. 61 :4329-4333.
Li, J. et al., 1995. J Nat. Prod. 58:1081-1085.
McInerney, B. V. et al., 1991. J Nat. Prod. 54:774-784.
Menta R. G. and R. C. Moon, 1991. Anticancer Research 11 :593-596.
Monks et al., 1991. J: Natl. Cancer Inst. 83 :757-766.
Ninomiya, Y. T. et al., 1980. Chem. Pharm. Bull. 28 :3157-3162.
Sharma, S. et al., 1994. Cancer Research 54:5848-5855.
Shroeder et al., 1981. J. Med. Chem. 24: 1078-1083.
Skehan, P. et al., 1990. J Natl. Cancer Inst 82: 1107- 1118.
Stachel, H.D. et al. 1992. Liebigs Ann. Chem. 473-480.
T~k~h~hi S. et al., 1994. US patent. 5,292,892.
Page 22 of 22 Webster et aL Novel antineoplastic agents Tz~k~h~shi S. et al., 1995. US patent. 5,399,711.
Tipper D. J. 1973. J. Bacteriol. 116:245-256.
Umezawa, H. et al. 1948. Jap. Med. J. 1 :512-517.
Webster et al. WO: 96/32396.
von Daehne, W. et al., 1969. J. Antibiotics 22:233-235.
* * *
Claims (6)
1. A pharmaceutical composition for treating neoplastic diseases comprising a compound having the following structure formula Or wherein n = 0, 1 or 2; R1= hydrogen, substituted or unsubstituted alkyl, cycloalkyl, aryl, aralkyl, or heterocyclyl group; R2= = hydrogen, substituted or unsubstituted alkyl, cycloalkyl, aryl, aralkyl, or heterocyclyl, acyl group; R3 = hydrogen, alkyl, cycloalkyl, aralkyl, aryl or heterocyclyl group, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutical carrier or dilution.
2. A pharmaceutical composition for treating neoplastic diseases comprising a compound of claim 1, wherein n = 0, 1 or 2; R1 = hydrogen; R2= acyl; R3 = hydrogen, methyl; or a pharmaceutically acceptable salt thereof, in association with a pharmaceutical carrier or dilution.
3. A pharmaceutical composition for treating neoplastic diseases comprising a compound of claim 1, wherein n = 0, 1 or 2; R1= hydrogen, R2= acyl group with a straight or branched one Page 19 of 22 to ten carbon chain; R3= hydrogen, or methyl, or a pharmaceutically acceptable salt thereof in association with a pharmaceutical carrier or dilution.
4. A method of inhibiting the growth of mammalian tumors, comprising administrating to a subject in need of such treatment, an effective antineoplastic amount of a compound of the structures shown below, Or wherein n = 0, 1 or 2; R1= hydrogen, substituted or unsubstituted alkyl, cycloalkyl, aryl, aralkyl, or heterocyclyl group; R2= = hydrogen, substituted or unsubstituted alkyl, cycloalkyl, aryl, aralkyl, or heterocyclyl, acyl group; R3 = hydrogen, alkyl, cycloalkyl, aralkyl, aryl or heterocyclyl group, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutical carrier or dilution.
5. A method of inhibiting the growth of mammalian tumors, comprising administrating to a subject in need of such treatment, an effective antineoplastic amount of a compound of claim 4, wherein n = 0, 1 or 2; R1 = hydrogen; R2= acyl; R3 = hydrogen, methyl; or a pharmaceutically acceptable salt thereof, in association with a pharmaceutical carrier or dilution.
Page 20 of 22
Page 20 of 22
6. A method of inhibiting the growth of mammalian tumors, comprising administrating to a subject in need of such treatment, an effective antineoplastic amount of a compound of claim 4, wherein n = 0, 1 or 2; R1= hydrogen, R2= acyl group with a straight or branched one to ten carbon chain; R3= hydrogen, or methyl, or a pharmaceutically acceptable salt thereof in association with a pharmaceutical carrier or dilution.
Priority Applications (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002212237A CA2212237A1 (en) | 1997-09-05 | 1997-09-05 | Novel antineoplastic agents |
| CN98810328A CN1276723A (en) | 1997-09-05 | 1998-09-03 | Dithiolopyrrolones and their corresponding monoxides and dioxides as antineoplastic agent |
| AU90570/98A AU759990B2 (en) | 1997-09-05 | 1998-09-03 | Dithiolopyrrolones and their corresponding monoxides and dioxides as antineoplastic agents |
| JP2000510440A JP2001515861A (en) | 1997-09-05 | 1998-09-03 | Dithiolopyrrolones and their corresponding monooxides and dioxides as antitumor agents |
| PCT/CA1998/000841 WO1999012543A1 (en) | 1997-09-05 | 1998-09-03 | Dithiolopyrrolones and their corresponding monoxides and dioxides as antineoplastic agents |
| EP98942414A EP1009401A1 (en) | 1997-09-05 | 1998-09-03 | Dithiolopyrrolones and their corresponding monoxides and dioxides as antineoplastic agents |
| CN01131401A CN1360891A (en) | 1997-09-05 | 2001-09-06 | Dithiolane amylene hepyrroleketone and its relative monoxide and dioxide using as antitumour agent |
| HK02107990.3A HK1046373A1 (en) | 1997-09-05 | 2002-11-02 | Dithiolopyrrolones and their corresponding monoxides and dioxides as antineoplastic |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002212237A CA2212237A1 (en) | 1997-09-05 | 1997-09-05 | Novel antineoplastic agents |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2212237A1 true CA2212237A1 (en) | 1999-03-05 |
Family
ID=4161181
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002212237A Abandoned CA2212237A1 (en) | 1997-09-05 | 1997-09-05 | Novel antineoplastic agents |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP1009401A1 (en) |
| JP (1) | JP2001515861A (en) |
| CN (2) | CN1276723A (en) |
| AU (1) | AU759990B2 (en) |
| CA (1) | CA2212237A1 (en) |
| HK (1) | HK1046373A1 (en) |
| WO (1) | WO1999012543A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1238668A1 (en) * | 2001-03-09 | 2002-09-11 | Aventis Pharma Deutschland GmbH | Use of thiolutin dioxide and its derivatives in the manufacture of a medicament for the treatment of CNS disorders and a process for the preparation thereof |
| KR100839214B1 (en) | 2002-03-26 | 2008-06-17 | 웰리켐 바이오 테크 인크. | Novel dithiolopyrrolones with therapeutic activity |
| CN101522688B (en) * | 2006-09-29 | 2011-09-14 | 北京文丰天济医药科技有限公司 | Novel dithiolopyrrolones and their therapeutical applications |
| CN101381371B (en) | 2007-09-05 | 2011-05-18 | 上海医药工业研究院 | Dithiolopyrrolones compounds and preparation method and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1214130A (en) * | 1982-10-26 | 1986-11-18 | Stuart H. Rhodes | Xenorhabdin antibiotics |
| DE19513040A1 (en) * | 1995-03-29 | 1996-10-02 | Schering Ag | New borne esters, processes for their preparation and their pharmaceutical use |
| CA2217957A1 (en) * | 1995-04-11 | 1996-10-17 | Genhui Chen | Xenorxides with antibacterial and antimycotic properties |
-
1997
- 1997-09-05 CA CA002212237A patent/CA2212237A1/en not_active Abandoned
-
1998
- 1998-09-03 WO PCT/CA1998/000841 patent/WO1999012543A1/en active IP Right Grant
- 1998-09-03 EP EP98942414A patent/EP1009401A1/en not_active Withdrawn
- 1998-09-03 AU AU90570/98A patent/AU759990B2/en not_active Ceased
- 1998-09-03 JP JP2000510440A patent/JP2001515861A/en active Pending
- 1998-09-03 CN CN98810328A patent/CN1276723A/en active Pending
-
2001
- 2001-09-06 CN CN01131401A patent/CN1360891A/en active Pending
-
2002
- 2002-11-02 HK HK02107990.3A patent/HK1046373A1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| HK1046373A1 (en) | 2003-01-10 |
| AU759990B2 (en) | 2003-05-01 |
| CN1360891A (en) | 2002-07-31 |
| JP2001515861A (en) | 2001-09-25 |
| CN1276723A (en) | 2000-12-13 |
| EP1009401A1 (en) | 2000-06-21 |
| AU9057098A (en) | 1999-03-29 |
| WO1999012543A1 (en) | 1999-03-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5010099A (en) | Discodermolide compounds, compositions containing same and method of preparation and use | |
| US5583239A (en) | Antimicrobial sterol conjugates | |
| EP0486565B1 (en) | Discodermolide compounds, compositions containing same and methods of preparation and use | |
| OKAMOTO et al. | Studies of platelet activating factor (PAF) antagonists from microbial products. I.: bisdethiobis (methylthio) gliotoxin and its derivatives | |
| US4268676A (en) | Mitomycin analogs | |
| JP2003522166A (en) | Compound having antitumor activity: method for producing the same and pharmaceutical composition containing the same | |
| US7166634B2 (en) | Bis-indole pyrroles useful as antimicrobials agents | |
| Sakai et al. | Xanthocillins as thrombopoietin mimetic small molecules | |
| AU759990B2 (en) | Dithiolopyrrolones and their corresponding monoxides and dioxides as antineoplastic agents | |
| US6583171B1 (en) | Antineoplastic agents | |
| LI et al. | Sommaire du brevet 2212237 | |
| US5298523A (en) | Method for treating transplant patients using mycalamide compounds | |
| Reddy et al. | Chemical investigation of Mycale mytilorum and a study on toxicity and antidiabetic activity of 5-octadecylpyrrole-2-carboxaldehyde | |
| LI et al. | Patent 2212237 Summary | |
| LI et al. | Patent 2212237 Summary | |
| US4460599A (en) | Mitomycin analogs | |
| JPWO2019189331A1 (en) | New K95-5901-1 substance and its manufacturing method | |
| KR960005150B1 (en) | Novel pyrazinoxide compound of nf-1616-904 | |
| WO1999032115A1 (en) | Modulators of ryanodine receptors comprising 2-(aryl)-4,7-dioxobenzothiazoles and analogues thereof | |
| CA2217957A1 (en) | Xenorxides with antibacterial and antimycotic properties | |
| US4514330A (en) | Carbon-7-substituted atinomycin D analogue | |
| WO2007010307A1 (en) | Antineoplastic compounds | |
| IE832491L (en) | Sparsomycin (sc-rs) derivatives. | |
| JP5175742B2 (en) | Novel macrolide compounds with antibacterial and antitumor properties | |
| US5910509A (en) | Antimicrobial agent for agriculture |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Dead |