Pepsin inhibitors and preparation thereof
United States Patent [1 1 Murao et a1.  
 [4 1 Apr. 15, 1975 [54] PEPSIN INHIBITORS AND PREPARATION THEREOF [76] lnventors: Sawao Murao, 8-12, 2-cho,  
 Horiagemidori-cho, Osaka; Shuzo Satoi, 1 1-5, ichome, Sumiyoshi- Kamokgahara cho, Kobe; Noriaki Kuwana, 74, Higashiohizumi-cho, Tokyo; Masayuki Fukumura, 2447-1 Hiratsuka Hirarsukushi, Hiratsuka; Tamio Kawamura, 12-8, 5 chome, Koishikawa, Tokyo, all of Japan [22] Filed: Apr. 28, 1971 [21] Appl. No.: 138,083  
 [30] Foreign Application Priority Data Apr. 28, 1970 Japan 45-35900 [52] US. Cl. 260/1125; 195/29; 424/177 [51] Int. Cl..... C07c 103/52; C07g 7/00; C08h 1/00 [58] Field of Search 195/80 R; 260/1125 [56] References Cited UNITED STATES PATENTS 6/1973 Umezawa et al 195/80 R 10/1974 Umezawa et a1. 260/1125 3,840,516 10/1974 Umezawa et al. 260/1 12.5  
 OTHER PUBLICATIONS Umezawa et al., J. of Antibiotics, 23, 259 (1970). Morishima et al., J. of Antibiotics, 23, 263 (1970).  
 L. Fieser and M. Fieser, Reagents for Organic Synthesis, Wiley and Sons, NY. (1967), pp. 192-193.  
 Primary Examiner-Lewis Gotts Assistant ExaminerRegina1d J. Suyat Attorney, Agent, or FirmCooper, Dunham, Clark, Griffin &amp; Moran [57] ABSTRACT 2 Claims, 1 Drawing Figure PATENTEDAPRI&#39;SIBTS 3.878.185  
  FIG. I I00 4000&#39; 3000&#39; 2000 f &#39;16&#39;00&#39; I400 I200 I000 P &#39;680 FIG. 2  
 4000&#39; 3000&#39; 2000 6 &#39;l8&#39;00&#39; I400 I200 I000 F A OA /AK/ KUWA/VA TAM/0 KAWAMUAA INVENTORS 1 2 PEPSIN INHIBITORS AND PREPARATION The first object of the present invention is to provide THEREOF new pepsin inhibitors, namely, the substance S-Pl and the substance Me S-Pl which have no undesirable side- This invention relates to the new pepsin inhibitors effect such as that presented by the known sulfuric acid hereinafter called &#34;substance S-Pl&#34; and substance Me 5 esters of polysaccharide. S-Pl, the methyl ester of the former, and a process for the preparation thereof. These substances are characterized by their enhanced inhibiting effect against enzymatic activity of acid protease and in particular pepsin.  
  The substance 8-H and the substance Me S-Pl ac- 10 The third object ofthe present invention is to provide cording to the present invention are represented by the a process for the production of the substance Me S-Pl The second object of the present invention is to provide a process for the production of the said substance S-Pl by fermentation of a new Actimomycetes.  
 formula by chemical treatment of the substance S-Pl.  
  CH CH CH CH 3 3 CH CH CH CH CH 3 3 3 3 I I OH CH CH CH CH l l OH l 9 CH CONH-CH-CO-NH-CH-CO-NH-CH-CH-CH -CO-NH-CH-CO-NH-CH-CH-CH -COOR wherein R is hydrogen or methyl. and hen R i y As the result of extensive search over wide range of gen. the ormula represents hc SubSta C S-PI. microorganisms, we have succeeded in finding out a whereas hen R s methyl. he formula represents the strain belonging to the genus Streptomyces which is casubstance Me S-Pl. pable of producing a new pepsin inhibitor as a metabo- According to the present invention, the substance lite of said strain. The present invention indeed relys S-Pl is obtained by fermenting thc Streptomyces naniwupon the discovery and utilization of said microorganaensis (EF 44-201) in a suitable nutrient medium and ism for the production of said new pepsin inhibitor,  
 &#39;pecially acid protease is formed as a metabolite of mi- 45 recovering the said substance as a metabolite from the namely, the substance S-Pl.  
 fermentation broth. The other substance, that is, the Surprisingly we have found that the substance substance MC the w hand obwmcfj by exhibits a remarkable activity as pepsin inhibitor, its further treatment of the 531d substance pepsin inhibitory activity amounting to approximate l0 methyluting flgc thousand times of that presented by the known sulfuric It was ell known that Substances having Inhibitory acid esters of polysaccharide. The substance S-Pl is an effect against enzymatic action of trypsin and microbial ongopeptide having the chemical composition Prom&#34;lSCS are Widely distributed in natural Sourcessented by the aforementioned formula, which is quite Such as beans Cereals, P cgg&#39;whiteblood and 40 different from that of the known sulfuric acid esters of the like P- y P g 128 and 213 polysaccharide, and is free from undesirable side-effect (1960), reported by P. D. Boyer et 21].). such as prevention of blood-coagulation.  
 lt was, however, hitherto scarcely known that a sub- Th b t M S- stance having inhibitory effect agamst protease and es- 6 Su S e P] also Shows pepsm inhibitor) effect almost equivalent to that of the substance S-Pl.  
 The both substances are therefore equally utilizable for treatment in safety of patient suffering from pepticulcer.  
 croorganism.  
  K. Matsuhima et al reported that they had succeeded to isolate a protease inhibitor from extract of fermentation of the Penicilium cyclopium [comp. Agricultural The Stfam uflllzcd l out the Process of and gi i Chemistry (J 53 pages 544 and present invention has been isolated by the present In- 549 (1969)]. The authors in the article mentionthat venlors &#39;Q in Japan and named n&#39; p l&#39; the said protease inhibitor does not nevertheless show namwaensls (EF 44301). any inhibition against pepsin which is a typical acid Specimens of said strain have been deposited to the protease.  
  55 Fermentation Research Institute of Japan under Recently, in accompany w th a remarkable advance FERM P N0 278. and also to the American p the l P Studles regard ulcera&#39; ture Collection under the assigned Number ATCC non of digestive organs, many attempts are directed to 21689.  
 the aspect of enzymatic action of pepsin in order to solve the causes of ulceration as well as developments h vegetative li f h Strain forms many of ulcersbranched mycelia having diameter of about 1 micron lt was known that sulfuric acid esters of polysacchad forms b d ri l elia,  
 ride are effective anti-ulcer agent. The pharmacological effects of these substances are supported mainly by Well grown aerial mycelium of the strain is fragtheir pepsin inhibitory activity. Unfortunately, it is, mem d b s mti t fo oph but n r ohowever, known that the substances, when adminisrangium. The vegetative mycelium of the strain does trated, are accompanied with injurious side-effect such not septate. From the fact above mentioned, the strain as prevention of blood-coagulation. is identified to be Streptomyces (Genus).  
  Further bacteriological characteristics of said strain are as follows: A. Morphorogical characteristics 1. Aerial mycelium:  
 Aerial mycelium is abundant. Sporphores are long loose spirals and irregularly branch. 2. Spore:  
 Oval or eliptical, irregular in sizes of from 0.5 to  
  0.8 X 0.9 to 1.5 microns inclusive. Surfaces of spore are covered with long spines. B. Growth of the strain in various nutrient media at In the above. the sign denotes the negative utilization; the sign the positive utilization, and the sign (i) the uncertain utilization. respectively.  
  Fermentation of Streptomyces naniwacnsis (EF 44-201) for the production of the substance S-Pl according to the present invention may be conducted in accordance with conventional arts and knowledges hitherto known with respect to the fermentation of Aetinomycetes. in preparation of a suitable nutrient medium, there may be employed as essential ingredients, a nitrogen source such as soybean meal. corn steep liquor, peptone. yeast extract, meat extract, dry yeast,  
 27C. for 14 days is tabulated in the following Table: 15 inorganic nitrate. ammonium salt and so on: and a car- Table 1 Nutrient medium Status of growth Aerial mycelium Soluble pigment triptone-yeast extract agar slightly grown starch-casein well grown: agar colorless to dark brown Starch-peptonewell grown: meat extract agar pale yellowish brown egg-albumin agar slightly grown:  
 colorless potato-glucose well grown; agar colorless to brown potato plug well grown;  
 black arginine-glucose well grown;  
 none  
 light brownish gray slightly produced: grayish white slightly produced: light brownish gray none none  
 light brownish gray light brownish gray slightly produced: white powder slightly produced: light brownish gray light brownish gray light purple to brownish purple none slightly pale brown none none  
 none  
 none  
 brownish purple to dark brown light amber none brownish purple dark brown to black brownish purple agar colorless to browmsh purple C) Physiological properties: I C hromogenic action 2) Cellulose decomposition 3) Thyrosinase activity 4) Hydrogen sulfide formation 5) Reduction of nitrate 55 6) Hydrolysis of starch 7) C atalase activity 8) Coagulation of milk with strong peptonization 9) Liquefaction of gelation I D) Utilization of carbohydrate 60 l arahinose i 2) raffinose i 3) xylose 4) galactose 5) rhamnose 6) mannose 7) lactose 65 8) maltose 9) fructose l0 inositol l l salicin 12) glucose bon source such as starch hydrolyzate, glucose. molasses and the like. If necessary, there may be used any other suitable inorganic salts such as K HPO MgSO FeSO MnSO, etc. and suitable additives such as antifoaming agent and the like.  
  The substance S-Pl aimed at in the present invention accumulates in the broth obtained by fermentation of the Streptomyces naniwaensis (EF 44-201) in a nutrient medium in accordance with a conventional procedure such as a submerged culture, a stationary culture or a surface culture at a temperature of from about 20C. to about 35C. for about 10-96 hours.  
  The resulting substance S-Pl may be recovered from the fermented broth advantageously based on the benefit of its chemical and physical properties in accordance with the usual procedure known in the art. As for example, the fermented broth may immediately be evaporated to dryness; or the broth may first be treated with active carbon to take up the substance S-Pl. In the latter case. the recovered active carbon is then treated with a water-missible solvent such as methanol to elute the desired substance. From the eluate. the substance S-Pl is recovered by evaporation to dryness.  
  The active carbon which carries the substance S-Pl may otherwise be washed with an aqueous alkali solution previous to the step of the aforementioned treatment with the water-missible solvent.  
  In some cases. it has been found desirable to repeat the treatment of the recovered substance S-Pl with active carbon in order to eliminate any colored materials. Alternatively, the crude substance S-Pl may be recovered from the fermented broth by means of precipitation with ammonium sulfate.  
  The crude substance S-Pl. if necessary. may be further purified by conventional column chromatography on SEPHADEX LH-(the registered trade mark). silica gel, ion exchange resins and the like.  
  In addition to the above, a purified substance S-Pl may be obtained as the precipitate by acidifying the alkaline solution of the crude substance at a high concentration with a mineral acid.  
  The production of the substance Me S-Pl that constitutes the other important aspect of the present invention can be effected by treating the aforementioned crude, semi-purified or purified substance S-PI with a known methyl esterifying agent such as diazomethane or methanol in the presence of thionyl chloride or dry hydrogen chloride. The resulting substance Me S-Pl may be recovered by a conventional procedure in a form of white powder. Like the free substance S-Pl, the substance Me S-PI thus obtained exhibits a marked inhibiting effect against pepsin and other acid proteases.  
  Followings are the physical properties of the substance S-Pl and the substance Me S-Pl:  
 1. Properties of the substance S-Pl The substance S-Pl begins coloration at about 215C, followed by decomposition at about 227-230C. It gives no sharp melting point.  
  The substance is sparingly soluble in water of from acid to neutral pH range but soluble in alkaline pH range; soluble in methanol and acetic acid; less soluble in ethanol and butanol; and practically insoluble in benzene, ether, petroleum ether, chloroform, carbon tetrachloride, hexane and ethyl acetate. In consideration of the result of paper electrophoresis and others, it is recognized that the substance S-Pl is an acidic substance.  
  ln ninhydrine reaction, the substance per se is negative while its hydrolyzate is positive.  
  Rydon-Smith reaction of the substance [H. N. Rydon and P. W. G. Smith; Nature 169, 922 1952)] is positive.  
  Thin layer chromatography of the substance on silica gel GF- (the registered trade mark, sold by Merk A. G. of Germany) gave the following R; values respectively:  
 0.40-0.50 in methanol-benzene (l l);  
 0.1-0.2 in methanol-chIoroform-benzene (l l l);  
 and  
 0.28-0.32 in benzene-methanol-acetic acid 20 2. Properties of the substance Me S-Pl The substance Me S-Pl has a melting point of 247-249C.  
  It is sparingly soluble in water throughout the entire sphere of pH range; soluble in methanol and acetic acid; sparingly soluble in ethanol and butanol. ln benzene, ether, petroleum ether and chloroform, the substance is practically insoluble.  
  In view of the result of paper electrophoresis and others. it is recognized that the substance is a neutral substance.  
  Within the region of ultraviolet to visible rays, the substance does not show any particular photoabsorption.  
  lnfra-red spectrum of the compound in potassium bromide tablet is graphically shown in accompanying FIG. 2, wherein the wave lengths in cm unit are given on the abscissa and the percentage absorptions on the ordinate. In the graph, marked absorptions are observed at the following wave lengths;  
  1370, 1295, 1263, 1225, 1175, 1153, 1072, 990, 927, 892, 865, 850, 820, 795, 755 and 715.  
  Specific rotation of the substance is from -9l to 93 (6 01 in methanol).  
  ln ninhydrine reaction, the substance per se is negative while its hydrolyzate is positive.  
  Rydon-Smith reaction of the substance [H. N. Rydon and P. W. G. Smith; Nature169, 922 (1952)] is positive.  
  Thin layer chromatography of the substance on silica gel GF- (the registered trade mark, sold by Merk A. G. of Germany) gave the following R values respectively:  
 0.80-0.90 in methanol-benzene (l l);  
 0.85-0.95 in methanol-chloroform-benzene (l l l and 0.35-0.40 in benzene-methanol-acetic aicd (80 20 As the results of instrumental analyses such as amino acid analysis. gas chromatographic analysis, IR and NMR and high resolution mass spectrometry, we confirmed that the substance S-Pl has the following structural formula:  
 Pcpsin inhibitory effect represented by the substance- S-Pl and the substance Me S-Pl were demonstrated by the following test:  
  To each 2.5 ml of 1% casein aqueous solution (pH 2.5), there was added respectively 0.2 ml of an aqueous solution of the substance S-Pl or 0.2 mol of an aqueous methanol solution of the substance Me S-Pl. the concentrations of the substances in the respective solutions varying from one another.  
  To each of the resulting mixtures kept at the temperature of 37C., was added 0.5 ml ofa aqueous solution of pepsin and the whole were incubated at 37C. for minutes. 2 ml of a 10% aqueous solution of trichloroacetic acid was added to each of the solutions and the mixtures were allowed to stand at 37C. for 30 minutes.  
 Precipitated excess casein was removed by filtration.  
  To each 1 ml aliquot of the recovered filtrates was added 5 ml ofa 0.44 molar aqueous solution of sodium carbonate and 1 ml of a phenol reagent (Folins reagent), and the whole were shaken well. After 60 minutes, photo-absorption at 660 my. wave length of each of the resulting mixtures was inspected. At the same time, two tests for control were carried out under the same conditions as those aforementioned with exception that the substance S-Pl and the substance Me S-Pl were eliminated.  
 In comparison with the control tests, the individual :amount of the substance 8-H and the substance Me S-Pl required for a 50% diminution of the pepsin activity were determined, which are then represented by the term [Pl] as the measure of pepsin inhibitory effect thereof.  
 The results thus obtained are:  
 [P11 1 1.2-1.8 ug for the substance 8-H, and  
 [Pl] 1.2-1.8 p.g for the substance Me S-Pl By the aforementioned chemical and physical as well as biological characteristics of the substance S-Pl and the substance Me S-Pl, they are distinguished from the hiterto known protease inhibitors and are considered the new compounds.  
  The following examples serve to illustrate embodiment of the invention. Example 1 To 14 liters of an aqueous nutrient medium, which contains 4% of glucose, 2% of meat extract, 2% of polypeptone and 0.2% of sodium chloride, there was added 1 liter of fermented liquor previously prepared by incubating the Streptomyces naniwaensis (EF 44-201 (w/v) of active carbon were added to the filtrate and the mixture was stirred for about 30 minutes to accomplish adsorption of the resultant substance S-Pl on the carbon. The active carbon was recovered by filtration and extracted three times each with 4 liters of methanol. The combined methanol extracts were subjected to distillation under reduced pressure to remove the solvent.  
 The residue was dissolved in 1.1 liters of water. The  
  solution was again treated with active carbon. The lat- I The methanol solution was poured onto a column of 3 cm diameter and 45 cm height filled with SEPHADEX LH-20 and eluted with methanol. 150-210 ml of the fractions of the eluate were collected and the solvent was removed therefrom by distillation.  
 There were obtained 1.58 grs. of the substance S-Pl as white amorphous powder.  
 under shaking at 27C. for 24 hours in an aqueous nu- Example 2 The Streptomyces naniwaensis (EF 44-201) was inoculated to ml ofa nutrient medium containing 5% of peptone, 0.1% of sodium chloride, 0.1% of dipotassium phosphate, 0.05% of magnesium sulfate, 0.001% of ferrous sulfate, 0.000l% of manganese sulfate, 0.0001% of copper sulfate and 0.0001% of zinc sulfate.  
  The whole was incubated at 27C. for 48 hours under shaking. After the period of that time, accumulation of 1.2 mg/ml of the substance S-PI was found in the fermented broth.  
  The broth after filtration was further worked up successively by fractionation with ammonium sulfate, adsorption with active carbon, and column chromatography with SEPHADEX LH-20 and silica gel.  
  There was obtained 13 mg of the purified substance S-Pl Elementary analysis of sodium salt of the substance S-Pl gave:  
 C H N O Na (Molecular weight 665.8)  
  500 mg of substance S-Pl obtained in Example 1 were dissolved in 10 ml of methanol. To the solution.  
 C H N Calculated (/1) 58.42 9.03 10.64 Found (71) 58.59 9.04 l().80  
 Example 4 500 mg of the substance S-Pl obtained in EXAMPLE I where dissolved in 50 ml of absolute methanol. The resulting solution was saturated with dry hydrogen chloride. The solvent was then removed from the solution by distillation under reduced pressure. The residue was dissolved in a small amount (about 5 ml) of methanol and followed by concentration under reduced pres- I sure. The treatments with methanol were further repeated three times. The resulting residue was dissolved in a small amound (about 5 ml) of methanol, and any solid materials were removed by filtration. The filtrate was subjected to chromatography on silica gel and then on SEPHADEX LH-ZO. There were obtained 300 mg of the substance Me S-Pl in a form of white amorphous powder.  
  The product in thin layer chromatography gave a single spot.  
 What is claimed is:  
  1. A pepsin inhibitor S-Pl which is acidic; soluble in water and aqueous alkali; insoluble in benzene. ether. petroleum. ether, chloroform. carbon tetrachloride. hexane and ethyl acetate; reacting negatively in the ninhydrin test, but positively after hydrolysis; convertible to a methyl ester by reaction with diazomethane in the presence of hydrogen chloride. convertible to a methyl ester by reaction with diazomethane in the presence of hydrogen chloride; and exhibiting an infrared cur&#39;ve substantially as shown in FIG. 1 hereof in a potassium bromide tablet.  
 2. A pepsin inhibitor Me S-Pl which is neutral in electrophoresis; soluble in methanol; insoluble in benzene.  
 ether. petroleum ether and chloroform; and exhibiting an infrared curve substantially as shown in FIG. 2 hereof in a potassium bromide tablet.