MXPA97003484A - Macrocyclic lactone compounds and their product procedure - Google Patents

Abstract

This invention provides a process for producing a macrocyclic lactone compound, which comprises cultured Actinoplanes sp. FERM BP-3832, in the presence of L-proline, L-hydroxyproline or L-nipecotic acid, and then isolate macrocyclic lactone compound from the fermentation broth. The compounds produced by this process include a compound of the following formula: (See Formula). The present invention also relates to a pharmaceutical composition comprising said compound, which is useful as an immunosuppressant, antifungal, antitumor or similar agent.

Description

COMPOUNDS OF LYNCHRONIC LICENSES AND THEIR P OOLFORMANCE TF PRODUCTION TECHNICAL CñflPQ This invention refers to a new acrylonitrile lac + ona compound and in particular to a new lactone inacrocic lactone compound and the fermentation of a product called flet inopi e sp. , which has been doctored or FERM pP-p832. This invention also relates to a process for producing the acryocyclic lac-ona compounds and a pharmaceutical composition comprising said compounds, which is useful as a suppressive people, in a co-operative manner, a n + + uino ra 1 o si m 11 ar .. Ib PREVIOUS TECHNIQUE Fn 198 * 3, the United States Food and Drug Pr oduction and Drug Treatment and the use of ciclopopna in human beings as a result of rejection. The use of ciclospopna has revolutionized the field of organ transplant surgery. The drug acts as an inhibitor, in the immune system of the body, of the immobilization of its ample arsenal of natural protective agents to reject the foreign proteins of the transplants .. a that the ciclospop na is effective to combat the rejection a tr sl ten -.ufre lesventajas at causing kidney disorders, damage l '?? f > at? and ulcers that in many cases can be very serious .. Therefore, more safe drugs that have fewer side effects have been investigated. it was found that certain macrolide compounds, raparme i na and their analogues have immunosuppressive or similar activity (European Patent Publication No. 0184162, US Patents Nos. 3929992 and 3993749). European Patent Publication No. 0589703 (1 describes the 2 l - norrapain i ci na ut i j on the treatment or prevention of rejection of transplants, in the immune and im- mediate medals. In the Japanese Patent Application Laid-Open No. 292948/1993, there are disclosed some rapamycin analogs which have a novel composition. The object of the present invention is to provide a new compound of acroleic lactone which has an <- * - xce lens t .tivi i nmunosupresora, ant j mi cot i ca or ant + uinora 1, and a pharmaceutical composition quo contieno. Other objects of the present invention are , 'U provide a process for producing the new macroc lactones, pharmaceutical compositions containing them and methods for using them.

BRIEF DESCRIPTION OF THE INVENTION Accordingly, the present invention provides the chemical lactone compound idenified with 03-12.98, which has the following chemical formula: (i) Pdi citonally, this invention provides the lactic acid compounds known as CJ-13.502; C3 ~ 13.503 and 03 13.504 that have the characteristics described below. In addition, the present invention provides a process for producing the inazocyclic lactone compounds 01-12.798, 03-13.502, 03-13.503 and CJ-13.504, which • ..emprende - * ul +? Var a microorganism having the characteristics of the PctinoDlanes sr &i . FERM TJP-3832, or a mu + an + or rectangulent form thereof, in the presence of L-α-ol, L-hydroxy prolma or L-mpecot acid co. '-. In addition, the present invention provides a pharmaceutical composition for use in the treatment or prevention of rejection of transplants, autoimmune diseases, fungal diseases or tumors, which broke a compound selected from C3.12.798, C3-13.502, 0 03-13503 and 03-13504, and a pharmaceutically acceptable vehicle. BRFVE DESCRIPTION OF THE DRAWINGS Figure 1 is the H NMR spectrum of compound 5 03-12.798. Figure 2 is the mass spectrum I SI of the compound i J-l. 79B- Figure 3 is the mass spectral FST of the compound 03-13.502. 0 Figure 4 is the ESI mass spectrum of the compound 03-13.503.

Fig. 5 is the mass spectrum EST of compound 03 -13..504. Figure 6 is the UV spectrum of compound 03-12. 98. Figure 7 is the UV spectrum of compound 03-13502. The iig? Ra 8 is the UV spectrum of compound 03-13.503. Figure 9 is the UV spectrum of compound OJ13.504.

DETAILED DFSCRIPCTON OF THE INVENTION The microorganism that is used in this invention is a strain of Fc? InQPL, R? E <;, sp. which was deposited as Pcti opl anes sr ».

FFRN BP-3832 at the National Institute of B oscience and Human Technology, Pgency of Industrial Science and Technology (located at 1-3, Higashi 1-chome, Tsul-uba, Tbaraki, 305, Japan) under the Budapest Treaty on 13 Pbril 1992. The details of this strain, including its taxonomic properties, are described on the Patent application "Japanese open for public inspection No. 04 46/1993. In this invention, an inviting or recombinant form of FFRM BP-3832 can be used which has the ability to produce the lactone organic compounds 03-12,798, 03-13502, 03-13503 and 03-13504. I to rnufante reeornbinante form or may be obtained by spontaneous mutation n, + ificial mutation or radiation treatment or rum ultraviolet with a mutagen such horn or N-methyl N- t tro -tu rosogua d or methanesulfonate ethyl acetate, or a cellular technology method * such as cellular fusion, genomic or similar, according to well-known procedures, According to the present invention, the acyclophilic lactone compounds of the invention can be produced by lerrnon aerobic treatment of FERf DP-3832, or a mutant or The rewinder thereof, under conditions similar to those generally employed to produce bioactive compounds by fermentation (for example, as described in Japanese Patent Application Laid-Open No. 292948/1993), with the exception that L-proline, - 15 hydroxyprol or L-nipecotic acid is added to the fermentation broth. Y-l l Ulti o de Pe 11 no or ne < ---. FERM BP-3832, or a similar or recombinant form of the same, is normally carried out by submerged aerobic conditions and with stirring at a temperature of 20 to 0 ° C for 1 to 10 days, which can Mi v ri r - "*" 1 according to the fermentation conditions .. The cultivation of FERfl P-3832 to produce said macrocyclic lactone compounds preferably has a place in an aqueous nutrient medium in the presence of L- proline, L- h dr'ox i? r * oJ ma or Lm? ecot acid at a temperature of 25 a ? 5 ° C for 1 to 3 days, the -prolma or similar is added to the < fermentation at a concentration of 0.1 to 1.0% (weight / volume), preferably from "3.4 to 0.5% (that / vo lumen). The pH of the medium can be adjusted in the range between 4.0 and 9.0, preferably between 6.0 and 7.5. The nutrient medium 1 for fermentation includes a source of assimilable carbon such as sugars, starches and gly-rol; a source of nitrogen or organic such as casein, products of the enzymatic digestion of casein, soybean meal, cottonseed meal, peanut meal, wheat gluten, soybean meal, meat extract and fishmeal; and a source of nutrient substances such as mineral salts, sodium chloride and calcium carbonate; and oligoes such as iron, magnesium, copper, zinc, cobalt and manganese. If excessive sputtering occurs during fermentation, anti-foaming agents such as propylene glycols can be added or if they are added to the fermentation medium. The aeration of the fermentation medium for submerged growth is maintained from 3 to 200%, preferentially from 50 to 150% in volumes of sterile air per volume of medium and per minute, the rate of agitation depends on the type of agitator * used. A stirring flask is typically used at an agitation rate of 150 to 250 rpins, while a ferrner is normally used at a stirring speed of 300 to 2,000 rpm. Of course, aseptic conditions have to be maintained during the organism's response and throughout its growth. The thus produced inacrocyclic lactone compounds can be isolated by conventional techniques such as extraction and various chromatographic techniques. The four icos rnacrooícl lactone compounds, CJ- 12,798, OJ-13,502, CJ-13,503 and CJ-13,504 were isolated from the fermentation broth and examined by various techniques ospect roscopicas, as shown in FIGS. I to 9 and post analysis of HPLO. It is believed that OJ-12.798 has the following e t oreoo t structure In addition, compound CJ-12.798 has the characteristic SI spectrum spectrum shown in FIG. 2, with rn / z 908 (T : > * > + Na] 1-; the UV spec- trum shown in FIG. 6, with a rnáx. of UV at 267, 277 and 287 n in methane 1; and a retention time of 12.9 ínin in HPLO using a col Mna ODS Pegas l (trademark of Sonshu) (4.6 x 150 min) and eluting with rnet ano! -water (7: 3 to 10: 0) for 30 minutes at a flow rate of 0.7 mL / mm at 42 ° 0. The compound designated 03-13502 has the characteristic FST mass spectrum shown in FIG. 3, with n / z 908 rr + Na] +; the UV specimen shown in FIG. 7, with a max. of UV at 267, 277 and 288 nm in methanol; and a retention time of 12.0 rnnn in HPI C using an ODS Pegasil column (trademark of Senshu) (4.6 x 150 nm) and eluting with rnol anol-water (7.3 to 10: 0) for 30 minutes at a flow rate of 0.7 ml / rnin at 42 ° 0. Compound CJ-13.503 has the characteristic ESI mass spectrum shown in FIG. 4, with rn / z 896 Cfl + Nal- * in the FSI mass spectrum; the UV spec shown in the FTG. 8, with a rnax. from V to 267, 277, and 288 nm in methanol; and a retention time of 10.9 rn n in l-IPLC using an ODS Pegas il column (trademark of Senshu) (4.6 x 150 rn) and eluting with methanol-water (7: 3 to 10: 0 ) for 30 minutes at a flow rate of 0.7 rnl / rni at 42 ° 0 .. Fl composite CJ-13.504 has the characteristic ESI mass spec- trum shown in FIG. 5, with rn / z 910 ri * 1 + Nal +; the UV spectrum shown in FIG. 9, with an inax. of UV at 267, 277 and 208 nm in methanol; and with a retention time of 11.9 rnm in HPI C using an ODS Pegasil column (trademark do Senshu) (4.6 x J50mrn) and eluting with toluene-water (7: 3 to 10: 0) for 10 minutes a flow rate of 0.7 ml / inin at 4 ° C. The immunosuppressive properties of the acylcyclic laotone compound of formula (T) and of the other compounds produced by the process of this invention were measured by measuring their inhibitory activities of the mixed lymphocyte reaction. (MLR) human. The measurement of the human MLR activity of the acetone lactone compounds of this invention was carried out by conventional methods which are described in the literature (D. P. Dubey et al., In Manual of Clinical Laboratory Tnrnunology, 35 Ed., Pages 047-858, 1986). the cytotoxi- ties were measured by conventional procedures (T. flosmann, J », J Tinmuno 1. Methods, 5_: 55-63, 1983). Compounds 03-12.798, CJ-13.502 and CJ-J2.504 showed MLR inhibitory activities (values 0Isa) that were more than one hundred times stronger than their cytotoxic activities. Among these new macrocyclic lactones, compound OJ-12..798 showed a higher suprasuppressant activity *. The anti-fungal activities of the compounds of the present invention were determined by a paper disk method (8 nm, Pdvantec) (agar plate medium: Antibiotic Medium 11 (Di ugus); albica s). the macrocyclic lactone compounds CJ-12,798, OJ-J 3,502, CJ-13,503 and CJ-13,504 showed good anti-fungal activities, showing 03-12.798 the highest activity. To be used as an immunosuppressant agent, anti-mycobacterial agent in a mammal, especially in a human being, the inaccurate compounds of the present invention can be administered alone or with an inert vehicle in a pharmaceutical composition, in accordance with conventional pharmaceutical practice. The lactone acroleic compounds can be applied by oral or parenteral administration. The active ingredient can be included, for example, with the usual non-toxic pharmaceutically acceptable carriers, in tablets. Pills, capsules, suppositories, solutions, emulsions, suspensions and other forms suitable for use. The vehicles that can be used are water, glucose, lactose, goat arugula, gelatin, mannitol, starch paste, magnesium silicate tp, talc, corn starch, keratma, colloidal silica, potato starch, urea and other suitable vehicles for use in the manufacture of preparations. In addition, if necessary, adjuvants, stabilizers, color-before and perfumes can be used. In general, the macrocyclic lactone compounds of this invention can be present in said dosage forms at concentration levels ranging from 5 to 70% by weight, preferably from 10 to 50% by weight. The macrocyclic lactone compounds of this invention can be used in mammals as immunosuppressive, anticoagulant or antitumor agents at doses ranging between 0.01 and 20 mg / Lg. The dose that is used in a particular case will no longer vary according to numerous factors, such as the phase of the disease being treated, the potency of the individual compound being administered, the response of the particular subject and the route of administration. However, when a macrocyclic lactone compound of formula (I) is used in a human patient to treat or prevent rejections to transplants, oral or parenteral dose will be from 0.5 to 250 mg / kg and, preferably from 5 to 250 g / Kh of one to four times per day. EXAMPLES The present invention is illustrated by the following examples. However, it should be understood that the invention is not limited by the specific details of these examples. The UV spectrum was recorded in the methanol in a rototoinet.ro UV / V1S DASOO Ubest-30 spec. The NMR spectrum was measured in CDCla using a BruLer NMR spectrometer (AM-500) unless otherwise indicated and the positions of the expressed peaks < -n part per million (ppin) based on the internal standard of the peak of 0I) C1GJ at 7.25 ppm. The shapes of the peaks are named as indicated below; s, singlet, d, doublet; t, ripple; q, quadruple; , rnuJtiplete; br, wide. The mass spectra LSI (Secondary Ion in Liquid Phase) and ESI (Eletronization Electron) were measured by means of a Kratos mass spectrometer (model 1S) using a NaT matrix of di totereitol: di tioep t ritol (3: L) and a Sciex mass spectrometer (model PPT TIT) using an ammonium acetate matrix.

EXAMPLE 1 One hundred (100) i of Medium 1 (2% of glucose), 0.5% of Pol pepton, 0.3% of meat extract, 0.5% of wheat gluten, 5.0% of extract were inoculated. of yeast, 0.3% blood preparation and 0.4% of OaCO: -, pH 7.0-7, 7) in a 500 ml flask, with a vegetative cell suspension of a tilted culture of Pctinoplanes. sp. FERM BP-3032. The flask was stirred at 28 ° 0 for 3 days on a rotary shaker with a displacement of 7 and 220 rpm, until a first crop was obtained. A stirring medium containing medium was inoculated 1 (150 rnl) with 7.5 rnl of the first seminal culture. The flask was stirred at 28 ° 0 for 2 days on the rotary shaker until a second seed culture was obtained. The second use endpoint for inoculating a 6-liter fermentation vessel (1) containing 3 1 sterile medium (2: 2% glucose, 0.5% Polipepton, 0.3% ex. meat racto, 0.5% yeast extract and 0.4% CaCO.,? L-1 7.2-7.4). An aeration was carried out at 26o0 during 2 days with 1700 rpm at 3 1 per minute, until obtaining a third serum curtain. The third seed culture was centrifuged for 10 minutes at 3. UOO rprn in the fermentation vessel of 6 1 and was resuspended. in the original volume in a medium or tepl (Medium 9: 2.5% glucose, 2.5% MES, pH 7.2-7.4). Aeration was carried out at 26 ° C for 6 hours with 1,700 rprn 3 1 per minute. Fifteen grams of L-prolma (final concentration 0.5%) were added to the fermentation broth and an aeration was performed at 26"0 for 3 days with 1700 rprn at 3 a minute. (3 1) after the addition of 2 1 of MeOH, then the isolate was applied to a resin (Diaion HP20) (500 ml) and the hydrochloric lactones were eluted with 2 1 of acetone. The mixture was dried in an aqueous solution (11) and extracted three times with 1 l of ethyl acetate.The extract was dried over N ^ C and * and evaporated to an oily residue (10.4 g). (10 g) was applied to a column 50DS-UH Oherncosorb (trademark of Chernco) (20 x 250 inm) and eluted with inetanoi-water (8: 2) at a flow rate of 5 rnl / nin. by ahs? UV rhubarb at 305 nrn The eluted peak was collected to yield 03-12.798 (1.0 mg) The compound was detected by HPLC using an ODS Pegasil column (Trademark of Senshu ) (4.6 x 150 rn) and using methane! -Water (7: 3 to 10: 0) for 30 minutes at a flow rate of 0.7 l / inin at 42 ° C. The retention time of compound 03-12798 was 12.9 minutes (as compared to 15.2 minutes for rapani ciña). The defection is realized by UV at 280nrn. Adorno, the physicochemical properties of CJ-12.798 were determined as indicated below.

CJ-12.798 Appearance UVMnax white powder. (MoOH) 267, 277 288 Molecular weight 908 Molecular formula LSIMS / z 908, 5 LM- > Na] '1 H NMR (ppin) 3,14 (3 H, s, -OMe), 3.41 (3 H, s, -OMe) E3EHPLQ 2 A procedure similar to that of Example J was repeated, except that the amino acid feed of l.-proline was changed to L-hydroxyproline, and that Medium 2 was replaced by * Medium 2A (3% glucose, 1% corn starch, 0.5% Pharried soy, 0.5% Sungrowth, 0.75% inazo liquor, 0.0001% OoCl ... »fiH ^ oy 0, 4% CaOO-3, pH 7.2-7.4). As a result, eluted fumes were elicited to produce compounds 03-13503 (1.0 rng) and 03-13504 (1.5 rng). Compounds were detected by MPLC using ODS Pegasil column (trademark) of Senshu) (4.6 x L50 rnrn) and eluting with methanol-water (7: 3 to 10: 0) for 30 minutes at a flow rate of 0.7 ml / min at 42 ° C. The CJ-13.5Ü4 compound retention periods were 10.9 and 11.9 nm, respectively. The detection was performed by UV at 280 nrn. In addition, the physicochemical properties of OJ-13.503 se and 03-13.504 were determined as indicated below. C2-13.503 O_J.-13.504 Appearance White powder White powder UVXtnax. (MeOH) (nrn) 267, 277, 288 267, 277, 288 Molecular weight 908 910 FSTMS rn / z 908, 5 [Tl + Nal + 910.5 CM + Na] ".

Example 3 A procedure similar to that of Example 2 was repeated with the exception that the amino acid feed of L-hydroxyprolma was changed to L-rupecot co-acid. As a result, the elui or par-peak was collected to produce compound CJ-13.502 (1.0 rng). The compound was detected by HPLC using the ODS Pegasil column (trademark of Senshu) (4.6 x 150 m) and eluting with methanol-water (7: 3 10: 0) for 30 minutes at a flow rate of 0.7 in. / ín to 42 ° 0. TI retention time of compound CJ-13.502 was 12.8 minutes. The detection was carried out by UV at 200 nm. Also Jas physicochemical properties of 03-13.50? were determined as indicated below. • ___-- 13..502 Appearance UV white powder \? Nax. (MeOH) (n) 267, 277, 238 Molecular weight 900 FS1MS rn / z 908.5 TM + Na] ^

Claims (6)

1. The invention having been described as an antecedent, the contents of the following are claimed as property: CLAIMS 1. A compound of the microbial lactone selected from the group consisting of OJ-12.798; CJ13-502; CJ-13.503 and 0"J - 13.504, in which the said 0J- 12.798 has the following formula iini ca: (b) said CJ-13.502 has the characteristic mass speci of ESI modeled in the FTG. 3, with in / z 908 TM * Nal- »; the UV spectrum shown in FTG.7, with an ax. of UV at 267, 277 and 200 nm in ethanol; and a retention time of 12.8 min on HPLC using a solids ODS Pegasil column (4.6 x 150 nm) and operating with water-inertol (7: 3 to 10: 0) for 30 minutes at a flow rate of 0, 7 inl / tnin at 42 ° 0; (c) said OJ-13.503 has the characteristic EST spectrum shown in the FTG. 4, with m / z 896 CM + Na] + in the EST mass spectrum; the UV spectrum shown in FIG. 8, with an ax. of UV at 267, 277 and 288 nrn in methanol; and a retention time of 10.9 n. on HPLC using an ODS Pegas column f 4,6 x 150 in) and eluting with methanol-water (7: 3 to 10: 0) for 30 minutes at a flow rate of 0.7 ml / min at 42 ° C; and (d) said CJ-13.504 has the mass spec FST characteristic shown in the FTG. 5, m / z 910 ÍM < - Na] +; the UV spectrum shown in the FTG. 9, with a rnax. of UV at 267, 277 and 288 nrn in methanol; and a retention time of 11.9 min in IIPOL using an ODS Pegasil column (4.6 150 inrn) and eluting methane 1-water rum (7 :: 3 to J: 0) for 30 minutes at a flow rate of (1 , 7 l / min at 42 ° C.
2. 2. A rnacrocyclic lactone compound according to claim 1, which is 0J-12.978
3. 3. A process for producing an acrocyclic lactone compound according to claim 1, wherein it comprises culturing a microorganism having the identifying characteristics of Actinoola is sp.FERM BP-383? or a mutant or recoant form itself, in the presence of proline I, prolix L-hydrox or acid L- nor pecofi co.
4. 4. The process according to claim 3, further comprising the subsequent step of isolating said macroclic lactone compound from the fermentation broth.
5. 5. A process according to claim 3, wherein the cultivation is carried out in the presence of I-prolol.
6. 6. A pharmaceutical composition for use in the treatment or prevention of rejection of transplants, autoimmune diseases, diseases of my cotycase or tumors, which comprises a compound according to claim 1 and a pharmaceutically acceptable carrier. SUMMARY OF THE INVENTION The invention provides a reliable process for producing a macrocyclic lactone compound, comprising cultivating Pct and non I, sp. FERM BP-3832, in the presence of L-prolma, L-hi dr * ox i prolna or L-mpecot acid co, and then isolate * a lactone compound from the metabolic broth of the epithelium, the compounds produced by this procedure include a compound of the following formula: The present invention also relates to a pharmaceutical composition comprising said compound, which is useful as a suppressant, antifungal, oral antitumor or similar agent. PF / apn P97 / 370F