Patent Grant
7737146
The present invention relates to the field of treatment of various diseases caused by pancreatic lipase activity. Particularly the present invention relates to isolation of a new compound useful for inhibiting pancreatic lipase activity. More particularly the present invention relates to a novel compound, Nonadeca-6-enoic acid-3-(hexadecyloxy-hydroxy-thiophosphoryloxy)quinoxalin-2-yl ester, designated as streptolipin, useful for pancreatic lipase inhibition, isolated from the culture of Streptomyces vayuensis strain N 2 having a molecular formula C.sub.43H.sub.73 N sub 2 PS O.sub.5 and a process for the preparation thereof. The present invention also relates to a method of inhibiting lipase activity.
Lipase catalyzes the hydrolysis of triglycerides to fatty acids and glycerol. Pancreatic lipase is the principle enzyme for digestion of dietary triglycerides and there fore pays a key role in absorption of fat from the small intestine. In conjunction with cofactors co lipase, pancreatic lipase hydrolyzes medium and long chain triglycerides to oil water interface to fatty acids and 2 mono glycerides. These fatty acids get accumulated in the body as a reserve source of energy. Excess accumulation of fat in the body leads to obesity. Inhibitor against this enzyme can be used as an antiobesity drug.
M. K. Meir, J. Tricari and Sullivan. Studies on the antiobesity activity of tetrahydrolipstatin, A potent and selective inhibitor of pancreatic lipase. International Journal of Obesity. (1987) 11, 35-42.
Obesity and hyperlipidaemia are medical conditions associated with a series of risk factors such as insulin resistance, impaired glucose tolerance, hypertension, hart diseases and stroke leading to an increased rate of mortality. Inhibitors against these enzymes thus have a potential application in medical sector.
Comparison of galenic formulations of orlistat (Tetrahydrolipstatin) A pharmacological approach. Drug Invest: 5(1): 44-50 (1993)
Weibel et al (1987) have reported that therapeutically active compound such as lipstatin as pancreatic lipase inhibitor (Weibel E K, Hadvary P, Hochuli E, Kupfer E and Lengsfeld H. The Journal of Antibiotics XL (1987) 1081-10191.
Panclicins, as novel pancreatic lipase inhibitors have been reported. Masae Mutoh, Naoki Nakada, Shoka Matsukuma, Shoichi Ohshima, Kiyoshi Yoshinari, Junko Watanabe and Mikio Arisawa. The Journal of Antibiotics vol 47, 1369-1375. Wherein the authors have discussed the use of various pancreatic lipase inhibitors in treatment of obesity.
The main object of the present invention is to provide A novel compound, Nonadeca-6-enoic acid-3-(hexadecyloxy-hydroxy-thiophosphoryloxy)-quinoxalin-2-yl ester, designated as streptolipin, useful for pancreatic lipase inhibition, isolated from the culture of Streptomyces vayuensis strain N2 having molecular formula C43H73N2 PSO5 and structural formula as hereunder:
An another object of the present invention is to provide a process for the isolation of said compound.
Yet another object of the present invention is to provide a method of treatment to inhibit lipase enzyme and for the prevention of obesity and treatment of disorders of the central nervous system; damage to the central nervous system; cardiovascular disorders; gastrointestinal disorders; diabetes, sleep apnea.
To meet the above objectives, the present invention provides a novel compound, Nonadeca-6-enoic acid-3-(hexadecyloxy-hydroxy-thiophosphoryloxy)-quinoxalin-2-yl ester, designated as streptolipin, useful for pancreatic lipase inhibition, isolated from the culture of Streptomyces vayuensis strain N2 having molecular formula C.sub.43H.sub.73 N sub 2 PS O.sub.5. The present invention also provides a process for the isolation/preparation of said compound from Streptomyces sp and a method treatment in inhibiting lipase inhibitor, for the treatment of obesity and treatment of disorders of the central nervous system; damage to the central nervous system; cardiovascular disorders; gastrointestinal disorders; diabetes, sleep apnea.
Accordingly, the present invention provides a novel compound, Nonadeca-6-enoic acid-3-(hexadecyloxy-hydroxy-thiophosphoryloxy)-quinoxalin-2-yl ester, designated as streptolipin, useful for pancreatic lipase inhibition, isolated from the culture of Streptomyces vayuensis strain N2, said compound having a molecular formula C43H73N2 PSO5 and the structural formula as hereunder:
wherein the said compound having a basic skeleton of quinoxaline moiety having substituents carbonyl ester and phosphotidyl groups. Said compound is soluble in an organic solvent selected from the group consisting of chloroform, acetone and dimethyl sulphoxide. The detailed solubility character of the said compound is as hereunder:
wherein the said compound is insoluble in phosphate buffer (pH 7.4, 8.0), acetate buffer (pH 5.0, 6.0), tris buffer (pH 8.0, 9.0), 5% sodium hydroxide, 5% acetic acid, 5% sodium bicarbona and the said compound having the physical characteristics as given below:
Lipase inhibition activity: IC.sub.50 value of the compound against purified pancreatic lipase inhibitory activity is 49 nM; Off white solid. Melting Point: 184.degree. C. λ max (chloroform): 210, 260; IR: 1434, 1312, 1046, 954, 761 for quinoxaline, 3436, 2913, 1771, 1714, 1659, 1236, 667 cm−1. Molecular formula: C.sub.43H.sub.73 N sub. 2 PS O.sub.5 EI-MS m/z: 761 (M+), 763 [M+2]+, [M−X]+468.4, [M−Y]+427, [M−Z]+584.4, [M-C18H33] 514.4, [M-C12H24] 595.4, PO3S—C16H34 337[Y], O2C19H35 295 [X], C13H25 181[Z], C15H15O2N2 255, PO3HS 112, CH2—(CH2)14—CH3 225, C9H4N2 128. 1H NMR (500 MHz, CDCl3) δ: 7.82-8.15 (4H, m, Ar—H—) 0.87-0.93 (3H, t, CH.sub.3) 1.26-1.38 (2H, m, CH sub 2) 1.6-1.7 (1H, t, ═CH.sub.2) 5.86 (1H, dd, C—CH═C) 2.04-2.12 (2H, t, O═C—CH sub 2) 2.36-2.39 (2H, t, O—CH sub 2), 5.37 (1H, s, —OH), 1.92-1.96 (2H, q, CH2—CH2). 13C NMR spectra (δ ppm): quinoxaline-N═C2—O 171.19, quinoxaline-N═C—OP 155.3, quanoxaline-CH2 CH2═CH2 130.41, quanoxaline-CH2═CH2—CH2— 128.422, quanoxaline-CH2—CH2═CH2— 128.28, quanoxaline-CH2CH2—CH2— 130.12, quanoxaline-CH2—CH2—NH— 138.84, quanoxaline-CH2—CH2—NH— 140.268, CH═CH 147.833, CH═CH 122.425, O—C═O 179.7, —CH3 14.4, —CH2 23.03, ═CH2 32.3, C—CH═C 39.6, CH═CH—C 30.6, O—CH2— 24.32, —CH2—C 29.45.
Yet another embodiment of the present invention is to provide a pharmaceutical composition, useful for effecting lipase inhibition in subjects mammals, comprising the said novel compound optionally along with pharmaceutically accepted salts, diluents and other excipients selected from the group consisting of carriers, colorants, flow modifiers and stabilizers. The said pharmaceutical composition is useful particularly for prevention of obesity and treatment of disorders of the central nervous system; damage to the central nervous system; cardiovascular disorders; gastrointestinal disorders; diabetes and sleep apnea. The said pharmaceutical composition is used in the form of oral, parental, buccal and ocular administration.
Yet another objective of the present invention is to provide a process for the preparation a novel compound, Nonadeca-6-enoic acid-3-(hexadecyloxy-hydroxy-thiophosphoryloxy)-quinoxalin-2-yl ester designated as streptolipin, useful for pancreatic lipase inhibition, which comprises the steps of:
The following examples are given by a way of illustrating the present invention and therefore shall not be construed to limit scope of the present invention.
The Isolation of the Streptomyces vayuensis Strain N2
Fermentation
Strain N2 is propagated on Glycerol Aspargine Agar slant composed of glycerol 1%, L-aspargine 0.1%, K2HPO4 0.1% and trace salt solution 0.1%. After incubation for 7 days at 30.degree. C., a portion of the mature agar slant is inoculated into 100 ml of a seed liquid medium of the same medium composition in a 500-ml Erlenmeyer flask and incubated at 30.degree. C. on a rotary shaker at 250 rpm.
A 10-ml of the seed culture is transferred into 500-ml Erlenmeyer flasks each containing 90 ml of soybean meal 1 g, dextrose 2 g, corn steep liquor 0.5 g, calcium carbonate 0.5 g for 100 ml. The inoculated flasks are incubated for 7 days at 30.degree. C.
Isolating the strain N2 of Streptomyces sp from manure rich soil and propagating the strain obtained from step on a Glycerol Aspargine Agar medium and incubating for 7 days at 30.degree. C. Inoculating with a slant of step into seed liquid medium contained in Erlenmeyer flask, incubating the liquid medium in Erlenmeyer flask at 28.degree. C. on a rotary shaker at 250 rpm to obtain the seed culture; transferring the culture of step into Erlenmeyer flasks containing soya bean meal, soluble starch, dextrose, corn steep liquor, calcium carbonate and incubated for 7 days at 30.degree. C. to obtain fermented broth. Biomass was separated by centrifugation and treating the fermented broth with an organic solvent for two hours to obtain an organic solvent extract; separating the organic solvent extract of step. the organic layer was separated by separating funnel; drying the organic layer of step over anhydrous sodium sulfate and concentrating under reduced pressure to obtain a solid; the compound is loaded on to silica gel (#60-120) column chromatography and eluted with hexane, hexane:ethylacetate (9:1) where it elutes as a colorless fraction in hexane ethylacetate (9:1), concentrated under reduced pressure chromatographed on sephadex LH-20 with methanol, checked for the active fraction on TLC and further purified on preparative TLC using benzene:ethyl acetate (7:3) and benzene:methanol (9:1). The silica gel was extracted with diethyl ether to obtain an off white solid. The silica gel was extracted with diethyl ether to obtain an off white solid. The biomass was dried at 40. degree. C and the dried biomass was extracted with ethyl acetate and further purification of the compound was carried by steps g, h, i, j, k which were carried out for broth extraction.
The composition of glycerol asparagine media: (g/) Asparagine 1, glycerol 10, K2HPO4 1; 1 ml trace salt solution (from stock of 1 Litre) CuSO4 64 mg, FeSO4 11 mg, MnCl2 79 mg, ZnSO4 15 mg, PH 7 and The seed liquid medium composition (g/L): glucose 50, yeast extract 50 NaCl 2.5, K2HPO4 0.5, ZnSO4 0.1, CaCO3 0.4, PH 7 The other ingredients occurring elsewhere in the patent Starch is 10 g/l, soybean meal 50 g/L.
For separating the biomass the centrifugation speed used is 5000 rpm for 20 minutes.
The compound is off white solid. It is soluble in acetone, chloroform and dimethyl sulphoxide, slightly soluble in ethylacetate, methanol, acetonitrile, diethyl ether, but insoluble in water, phosphate buffer (pH 7.4, 8.0), acetate buffer (pH 5.0, 6.0), trisbuffer (pH 8.0, 9.0), 5% sodium hydroxide, 5% acetic acid, 5% sodium bicarbonate. The EI-MS spectra of the compound showed the molecular ions at m/z 761.
The novel compounds of the invention can be used in a variety of pharmaceutical dosage forms. Thus, oral, buccal, ocular and other forms can be used. When such forms are formulated they will include pharmaceutically acceptable excipients such as colorants, carriers, perfumes, stabilizers, flow modifiers and the like in suitable amounts (i.e., from 0.001 to 0.99 wt %).
The compound of the invention is useful in methods of inhibiting the effects of pancreatic lipase. The compound is also used to treat a host, preferable a mammal, which is suffering from a disorder associated with a metabolism of disorders of the central nervous system; damage to the central nervous system; cardiovascular disorders; gastrointestinal disorders; diabetes, sleep apnea.
A biologically pure culture of Streptomyces sp N2, from which the compound of investigation is derived, has been deposited with the Microbial Type Culture Collection (MTCC), The Institute of Microbial Technology (IMTECH), India. The assigned MTCC Number is 5219
Taxonomy
Morphology The actinomycetes mycelium on different ISP medium agar had abundant spores more than 50, spiral, spore chains are long, non motile gray colored spores when matured.
Cultural and Physiological Characteristics
The growth characteristics of N2 on different media is given in table 2. The aerial mycelium at maturity formed long chains of spores of gray color, which are spiral, with warty surface and are non motile. Good growth on most of the media. Optimum temperature is between 10 to 45° C. There is no distinctive substrate mycelial pigments, no pigmentation of substrate mycelium, no production of diffusible pigments, no sensitivity of substrate pigment to pH, no sensitivity of diffusible pigment to pH, melanin production is negative. There are no sporulation on substrate mycelium, no sclerotia formation, and no fragmentation of mycelium. It doesn't show any antagonist activity against any organism. It degrades guanine, hypoxanthine, adenine, Xanthine, starch, casein, urea, gelatin. It is not resistant to all antibiotics, it grows at pH between 5 and 13. The sodium chloride tolerance is up to 5%. diffusible pigment is not produced. The color of the colonies is medium dependent (Table-2). Diagnostic amino acid of petidoglycan is meso-diaminopimelic acid. Whole cell hydrolysates contain glucose and small quantity of xylose, galactose and arabinose. The phosphotidylethanolamine is the diagnostic phospholipid. Predominant fatty acids are anreso-15:0, iso-16:0 and cyclo 17:0.
In Table 2 illustrating the growth on yeast extract and malt extract is good, colour of the aerial mycelium is blackish gray and substrate mycelium is black. Oat meal the growth is good, aerial mycelium is gray, substrate mycelium is dark brown. Inorganic salt starch agar, the growth is good, grayish white aerial mycelium, reddish brown substrate mycelium. The growth on glycerol aspargine agar, the growth is good, the colour of aerial mycelium is grayish white, substrate mycelium is blackish. Peptone yeast extract iron agar, the is moderate, colour of aerial mycelium is grayish black, gray colour substrate mycelium.
Table 5 illustrating the growth of Streptomyces sp N2 in the presence of various carbon and nitrogen sources. It had very good growth on DL-α-amino butyric acid, potassium nitrate, L-cysteine, L-threonine, L-serine, L-lysine, L-methionine, L-histidine, L-hydroxy proline, tryptophan, glutamic acid, tyrosine, ornithine mono hydrochloride, glycine, L-leucine, dopa, alanine, L-arabinose, sorbitol, starch, D-xylose, meso-inositol, mannitol, D-fructose, D-glucose, L-rhamnosse, maltose, D-mannose, D-lactose, trehalose, D-melibiose, D-galactose, cellobiose, moderate growth on L-valine, cellulose, sodium acetate, sodium citrate, xylitol, sodium pyruvate, sodium propionate and no growth on sodium malonate, insulin, sucrose aspartic acid.
Taxonomic Position
The strain Actinomycete N2 is isolated from rich manure soil sample from local region. The characteristics indicated that the strain belongs to Streptomyces group. According to the descriptions of Bergeys Manual of Bacteriology the strain is related to Streptomyces sp.
The taxonomic relationships between the strains and five other cultures Streptomyces erumpens, Streptomyces albduncus, Streptomyces olivvaceiscleroticus, Streptomyces viridodiastaticus, Streptomyces viridiviolaceus that did not give the bioactive molecules during screening are compared and given in Table 2.
Comparative Study:
In Table 11 illustrating Comparison between the strains N2 and five other related species which did not give the compound I. The spore surface of N2 was warty surface as the NRRL cultures of the above five strains are smooth surface and it also differs with the standard strains in physiological and biochemical.
The activity of pancreatic lipase against P-nitrophenyl butyrate was measured in 0.15 M of sodium chloride, 0.1M sodium phosphate buffer pH 7.4 at 25.degree.C. in a glass cuvette with a 1-cm light path. The substrate concentration 0.05 mM (from 10 mM stock solution of acetonitrile). The total volume three ml and the assay mixture enzyme concentration was x ml. The release of P-nitrophenol was monitored using spectrophotometer at 400 nm, and the reaction rate is determined from the slope of the straight-line portion of the curve. The activity was calculated from the initial rate of the reaction after the spontaneous hydrolysis of the substrate had been subtracted.
Results
The inhibitor compound having molecular formula C.sub.43H.sub.73 N sub 2 PS O.sub.5 is discovered in the fermented broth of a species of Streptomyces sp N2. The compound is successfully purified to homogeneity. The IC.sub.50 value of the compound against purified pancreatic lipase inhibitory activity is determined to be 349 nM.
Novelty
The compound, Nonadeca-6-enoic acid-3-(hexadecyloxy-hydroxy-thiophosphoryloxy)-quinoxalin-2-yl ester, designated as streptolipin, useful for pancreatic lipase inhibition, isolated from the culture of Streptomyces vayuensis strain N2, per se is novel. The process is novel because the culture ‘Streptomyces vayuensis strain N2’ and the product Nonadeca-6-enoic acid-3-(hexadecyloxy-hydroxy-thiophosphoryloxy)-quinoxalin-2-yl ester obtained in the process are novel.
Bacillus subtilis NCIB 3610
Pseudomonas fluorescens NCIB 9046
Escherichia coli NCIB 9132
Micrococcus luteus NCIB 196
Candida albicans CBS 562
Saccharomyces cerevisiaae CBS
Streptomyces murinus ISP 5091
Aspergillus niger LIV 131
Streptomyces
Streptomyces
Streptomyces
Streptomyces
Streptomyces
erumpens
albduncus
olivvaceiscleroticus
viridodiastaticus
viridiviolaceus
Streptomyces
Streptomyces
Streptomyces
Streptomyces
Streptomyces
erumpens
albduncus
olivvaceiscleroticus
viridodiastaticus
viridiviolaceus
Bacillus subtilis
Pseudomonas
fluorescens NCIB
Escherichia coli
Micrococcus luteus
Candida albicans
Saccharomyces
cerevisiaae CBS
Streptomyces
murinus ISP 5091
Aspergillus niger
Streptomyces
Streptomyces
Streptomyces
Streptomyces
Streptomyces
erumpens
albduncus
olivvaceiscleroticus
viridodiastaticus
viridiviolaceus
| Number | Date | Country | Kind |
|---|---|---|---|
| 1896/DEL/2005 | Oct 2005 | IN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2006/002964 | 10/23/2006 | WO | 00 | 10/9/2008 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2007/049119 | 5/3/2007 | WO | A |
| Number | Date | Country |
|---|---|---|
| 0040569 | Jul 2000 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20090163443 A1 | Jun 2009 | US |
