JPH06502536A - Novel biological phosphorylation method for organic compounds - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] name of invention Background of the invention of a novel biological phosphorylation method for organic compounds 1. Industrial applications The present invention uses microorganisms Rh1zous or zae.

Contains “phosphate active” hydroxyl using ATCC No. 11145 Novel site-specific biological phosphates for producing phosphorylated derivatives of organic compounds related to the conversion method. The method involves phosphorylating hydroxyl groups under biological conversion conditions. A method that brings microorganisms into contact with an organic compound containing free phosphate-active hydroxyl groups. be. The method utilizes dormant and erected cells or the presence of organic compounds. Cultivate microorganisms in the presence of

Rh1zo us or zae ATCC No.

11145 is primarily known to those skilled in the art as a hydroxylating agent. The microorganism is Decomposition of insect molting hormone J.

C, S, Chem, Comm, 1974: 656-657.1974), Ste. Can, J. Chem, 57:436-4. 40 and 1585-1587.1979. Ibid., 59: 1651-1655 ．． 1981; supra, 63: 1127-1131゜1985; H, J, Pe Edited by ppier, Mtcrobial Technology, Re1nhold , New York, p. 288-297. 1967; U.S. Patent No. 2.6 46,370), involved in the conversion of sesquiterbenlactone costanolite J , C, S, Perkin 1: 3022-3028.1979), Belcarin Conversion of A and B yields 16-hydroxykaline A and B and 3°-hydroxy produces silcarin A (Appl, Environ, Microbiol, 46 :480-483. 1983), producing the ○H product of imipramine (J, P harmaceut, Sci, 70:151-153.1981), L Kashinaga et al., the above-mentioned literature describes its ability as a site-specific hydroxyl phosphorylating agent. is not listed.

Motan 1 left and Eyu 1 are fungi commonly found in ripe fruits, grains and vegetables, as well as in soil. It is a genus of the same species. Typically, this microorganism is a saprotrophic fungus and a facultative parasite; Forms sterile mycelium. For example, Rh1z tachitanyu species contain carboxylic acids or It is used commercially in the production of hydrocarbons or in the metabolism of hydrocarbons.

The cultivation or fermentation of 145 is simple and generally well known8, e.g., as described in U.S. Pat. See No. 4,410.629 for adaptation of the protocols described in the Examples. Adaptations, modifications and changes are within the ordinary skill of the fermentation microbiologist.

Standard chemical phosphorylation using e.g. POC13 or PCls is generally Selectively phosphorylated compounds that are position specific and can improve water solubility and pharmacokinetics site-specific biological phosphorylating agents are desired in the industry. Ru.

AT Standing I The sample of MF4974 is 12301 Parklawn Drive, Roc American Type Cu1 located in kville MD 20852 It has been deposited at the ture Collection (ATCC).

The culture access designation number is ATCC No. 11145.

・　F　工　H Figure 1 shows C-32 phosphorylated FR-900520 in CDCl at 400MHz. +H nuclear magnetic resonance (NMR) spectrum.

Figure 2 shows the assigned molecular structure of C-32 phosphorylated FR-900520.

Figure 3 shows C-43 methylated phosphorus of phosphorylated lavamycin macrolide in MeOH. 1H nuclear magnetic resonance (NMR) spectrum at 400MHz of acid ester .

Figure 4 shows the assigned molecular structure of C-43 phosphorylated lavamycin macrolide.

FIG. 5 is a 1H nuclear magnetic resonance spectrum of echinocandin IIIA.

i milk seat 1 sword The present inventors have selectively analyzed organic compounds containing "phosphate-active" hydroxyl groups in biological studies. We have discovered a new comprehensive method for target phosphorylation. produces phosphorylated organic compounds Rh1zo us or zaeATCCNo. , 11145 in an aqueous medium.

As used herein, the term "phosphate-active hydroxyl-containing organic compound" refers to Rh1Zo us microorganisms and stereospecificity to be phosphorylated under biological conversion conditions 0 refers to a compound containing a hydroxyl group capable of reacting with Simple tests with organic compounds under conditions of physical transformation can eliminate unwarranted practices. Determines whether a hydroxyl in an organic molecule is phosphate active without the need for experimentation. Ru.

The method of the present invention uses phosphoric acid [1 medium] containing, for example, glycerol as a carbon nutrient source. Aqueous charcoal containing a nitrogen nutrient source can be used to contact resting Rh and erect cells in the body. Hydroxyl-containing organic compounds (e.g. PK- Microorganism Rh1z in the presence of macrolide immunosuppressant type 506 (FK-520) o us or zae.

It is carried out by fermenting ATCC No. 11145, and the material is phosphoric acid. Sufficient time to selectively biologically phosphorylate active hydroxyl groups (e.g. 24 hours at 27° C.) and a pH of about 7. You can use either method , methods using resting cells are preferred.

Phosphorylation by Rh1zo us or zae ATCC11145 is responsible for this characteristic. It will be understood by those skilled in the art that it is not limited to fixed strains, but rather hydroxyl-containing Other Rh1z to perform hydroxyl phosphorylation of organic compounds.

It can be expected that the Ldan J Or Zae stock can also be used.

Bino-t・ According to the invention, containing biological phosphorylated hydroxyl groups that are phosphorylated A method of making an organic compound is provided, the method comprising a biologically phosphorylated hydroxyl. in an aqueous medium containing a carbon nutrient source for a sufficient time to produce organic compounds. Incubate Rh1zo us or zae ATCC No. 11145 microorganisms at room temperature. contacting with a droxyl-containing organic compound.

The present invention aims to transform resting cells of the microorganism Rh1zous or zae into free hydroxyl by contacting with or fermenting in the presence of an organic compound containing a group, Oxidation Induction 2301 Parklawn Drive, Rockville, M AmericanType Culture Co11 located in aryland It has been deposited with ATCC No. 11145 at

The range of compounds included in “phosphoric acid-reactive hydroxyl-containing organic compounds” is defined by the formula : engineering (wherein R is 1(, C, -C, alkyl, R2 is hydrogen, hydroxy or lower alkanoyloxy, and R3 is methyl, ethyl, propyl or allyl. , n is an integer of 1 or 2, and the parallel parts of the solid line and broken line indicate single bonds or double bonds. U.S. Patent No. 4,894.36 in the name of Fujisawa, which is a double bond) C-32 hydroxy-containing macrolides and pharmaceuticals thereof as described in No. 6 Contains acceptable basic salts.

Specifically, R is methyl, R2 is hydroxy, and R3 is allyl. , +1 is 2 and there is no double bond, such as compound FK-506, and R is methyl , R2 is hydroxy, R3 is ethyl, n is 2, and double Mention may be made of the compound FK-520 in which binding is absent.

Furthermore, the following FK-506 type compounds (U.S. Patent Nos. 4 and 8 in the name of Fujisawa) No. 94,366). Wear.

7-allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methane) Toxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13 ,19,21゜27-tetramethyl-11,28-dioxa-4-azatricic [22,3,1,0”]]octas-18-ene-2,310,16-tet Raon, 1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxy) cyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,1 9,17,21,27-bentamethyl-11,28-dioxa-4-azatolysi Chlo[22,3,1,0"lioctacosu-18-en-2,3,10,16-te Traon, 16-allyl-1,13-dihydroxy-11-[-2-(4-dihydroxy-3 -methoxycyclohexyl)-1-methylvinyl]-22,24-dimethoxy- 12,18゜20.26-tetramethyl-10,2fusioxer 4-azatri To Cyclo [21,3,1,0”] Butassu-1 Funin-2.3,915-te Traon.

Furthermore, as used herein, the term "rFK-506 type macrolide" specifically includes ,formula: (wherein each adjacent substituent pair [R+ and R2], [R3 and R4 co, [R! and R6 ] each independently a) represent two adjacent hydrogen atoms, or b) these substituents A second bond is formed between the bonded adjacent carbon atoms, and in addition to the above meaning, R2 is C It may represent a 1-C1° alkyl group, and R7 is H-OH or OC+C+. Archi or may represent =0 together with R1, and R@ and R9 each independently represents H or OH, R'' is H1 optionally substituted by one or more hydroxyl groups C+C+o alkyl, optionally substituted by one or more hydroxyl groups C, -C, substituted by C+C+o alkenyl or =0. represent alkyl , X represents 01(H,OH), (H,H) or -CH20-, and Y represents 0, (H , OH), (H, H), N-NR” represents R12 or N-0R13, and R” and and R'' are independently H, C, -C, .alkyl, C, -C, .aryl or tosyl. represents R13, R14, l:tIs, Rth, R1f, Rth, R'', R22 and and R2 are independently H or C, -C, represents alkyl, R” is independently 0 or independently (R”a, H) (R”a is independently 0H1OC+ CI. alkyl or OCH20CH2CH20CH3), where n is 1, 2 or 3, In addition to the above meanings, Y, R'' and R2' are the carbon atoms to which these groups are bonded. In addition, C+C+ in some cases. alkyl, hydroxyl, one or more hydroxy CI-〇, substituted with a ru group. Alkyl, 0-CI-C,. alkyl, ben saturated or unsaturated substituted by Zyl and -CH2S　e　(Cs　Hs　) May represent a 5-membered ring or a 6-membered ring I'l-1S- or 〇-containing heterocycle, provided that and Y both represent 0, R9 represents OH, R", R", R", R+', R 1”, Rlg and R22 each represent methyl, R”a represents OCH3, R@ and and R” each represent H, and [Rj and R4] and [RS and R6] each represent carbon-carbon. European Patent No. EPOO 323042 in the name of Fison (representing an elementary bond) The disclosed compounds and their pharmaceutically acceptable compounds, including any acid addition salts of amine groups. Contains salt.

Preferably, R2, R RI I, R+2, R1, R”, R1 5゜When R", R1?, R", R", R"a, R" and R2 contain a carbon-containing group, and these groups contain up to 10, more preferably 1 to 6 carbon atoms (e.g. methyl or methoxyl).

R11, RIS, R! @, RI7. each of R", R" and R" is methyl Also suitable are compounds representing

Alkyl group R”, R’, RIl, R”, R”, R”, R15゜R”, R”, R” , R'', R20a, R22 and R'' may include straight chain, branched and cyclic groups.

Examples of the alkyl group substituted by 10 included in R'' include ketones and aldehydes. Examples include groups.

Preferably R'' is allyl (i.e. prob-2-enyl), propyl, ethyl or It is methyl.

Preferably n is 2, R7 is H or OH, and R1 and R2 are both I ], and X is preferably 0 or (I(.

OH), R2°a represents OH or OCH, and Y, R'0 and R'' are When represents an N-, S- or 〇-containing heterocycle, the ring is a 5-membered ring, more preferably is preferably a pyrrole or tetrahydrofuran ring.

A preferred embodiment of the product is C-32 phosphorylated FK-506: Dioxa-4-azatricyclo[22,3,1,0'']]octasus-18-e -3,1o dione, 17-brobyl-1,14-dihydroxy-12-[2- (4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23 ,25-dimethoxy-13,19゜21.27-tetramethyl-11,28-di Oxa-4-azatricyclo[22,3,1,0”]]octacosan-23,1 0.16-chitraone, 17-broby-1,14-dihydroxy-12-[2-(4~hydroxy~3 -methoxycyclohexyl)-1-methylethyl]-23,25-dimethoxy- 13,19゜21.27-tetramethyl-11,28-dioxa-4-azatri cyclo[22,3,1,0”]]octas-18-ene-2,310,16- Chitraone, 17-broby-1,14-dihydroxydi-12-[2-(4-hyperoxidase) droxyl 3-methoxycyclohexyl)-1~methylethylco23.25- dimethoxy-13,19゜2], 27-tetramethyl-11,28-dioxa -4-Azatricyclo[22,3,1,0'lioctacosan-2,3,10,1 6-Chitraone, 17-broby-1-hydroxy-12-[2-(4-hydroxy-3-methoxy) cyclohexyl)-1-methylvinyl-23,25-dimethoxy-13,19 ,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[2 2,3,1,0'']]octacocer14.18-diene2.3,10.16-thi Throne, 17-broby-1-droxy-11[2-(1-hydroxy-3 -methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy- 13,19,21゜27-tetramethyl-11,28-dioxa-4-azatri cyclo[22,3,1,0”cooctacos]-18-ene-2,3,10,16 -titraone, 17-allyl-1,14,20-trihydroxy-12-[2- (4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23 ,25-dimethoxy-13゜19.21.27-tetramethyl-11,28-di Oxa-4-azatricyclo[22,3,1,0”]]octas-18-ene -2,310,16-titraone, 17-(1-hydroxyprob-2-enyl )-1,14,20-)dihydroxydi-12-[2-(4-hydroxy-3-methane) Toxycyclohexylton-1-methylvinyl-23,25-dimethoxy-13 ．． 19,21.27-tetramethyl-11.28-dioxa-4-azatricic B [22, 3.1.0” Kooctacos = 18-en-2.3.

10,16-chitraone, 17-allyl-1,2-dihydroxy-12-[2-(4-hydroxy-3-methyl Toxycyclohexyl)-1-methylvinyl]-23.25-dimethoxy-13 ．． 19,21.

27-tetramethyl-11.28-dioxa-4-azatricyclo[22.3. 1.0” cooctacos]-18-ene-3.10.16-trione, 17-allyl-1,16-dihydroxydi1 2-[2-(4-hydroxy- 3-Methoxycyclohexyl>-i-methylvinyl]-23.25-dimethoxy -13.19.

21,27-tetramethyl-11.28-dioxa-4-azatol) cyclo[2 2.3.1.01g Kooctakosakho 14,18-diene-2.3.10-1- dione, 17-allyl-1-hydroxy-1 2-[2-(4-hydroxy- 3-Methoxycyclohexyl)-1-methylvinylco23.25-dimethoxy -13.19,21.27-tetramethyl-11.28-dioxa-4-azato Recyclo [22.3.1.0”]]Octacos]-18-en-2.310.1 6-thitraone, 17-(2,3-dihydroxypropyl)-1,14-dihyde Roxy-1 2- [2- (4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl]-23.25-dimethoxy-13.19.21.27-thi Tramethyl-11,28-dioxa-4-azatricyclo[22.3.

1,04lioctacos-18-ene-2.3,10.16-titraone, 17-ethanaryl-1,14-dihydroxy-12-[2-(4-hydroxy- 3-Methoxycyclohexyl)-1-methylvinylco23.25-dimethoxy -13.19.

21,27-tetramethyl-11.28-dioxa-4=azatricyclo[22 ．． 3.1.0″9 octacos-18-ene-2.3,10.16-titraone , 1,14-dihydroxy-1 2-[2'-(4-hydroxy).

(3-methoxycyclohexyl)-1-methylvinylco26.28-dimeth xy-13.22,24.30-tetramethyl-11.31-dioxa-4,1 7-Diazatetracyclo[25,3,1,0's,O""]]hentriaco 16 (20), 18.21-triene-2,3°10-trione, 1.14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclo hexyl)-1-methylvinyl]-26.28-dimethoxy-17-<2-hydro (oxyethyl)-13,22,24,30-tetramethyl-11,31-dioxy Cir-4,17-diazatetracyclo[25,3゜1.0''', O'''']] (contour 1620).

18.21-1-Lien-2.3.10-)dione, 1.14-dihydroxy -12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methyl and Nylco26.28-dimethoxy-13,22,24,30-tetramethyl- 17-phenylmethyl-11,31-dioxa-4,17-diazatetracyclo [25,3,1,0'%, Q 1G, 20] Hentria contour 16 (20) , 18.21-triene-2,3,10-)dione, 1.14-dihydroxy- 12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylbi Nirro 26.28-dimethoxy-13,22,24,30-tetramethyl-1 7-phenylmethyl-11,31-dioxa-4,17-diazatetracyclo[ 25,3,1,0'9, Q l@, 20] Hentria Contour 16 (20), 18.21-triene-2,3,10-1-dione, 17-allyl-1,14- Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl]-23,25-dimethoxy-13,19゜21.27-te tramethyl-11,28-dioxa-4-azatricyclo[22,3,1,0” ] ] Octasus-18-ene-2,310,16-tetraone C16 oxime, 17-allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-1 3,19゜21.27-tetramethyl-11,28-dioxa-4-azatolysi Kuro[22,3,1,0''']octacos-18-en-2,3,10,16- Tetraone C16 oxime O-methyl ether, 17-broby-1-hydroxy-12-[2-(3゜4-dihydroxycyclo hexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21 ,27-tetramethyl-11,28-dioxa-4-azatricyclo[22,3 ,1,O''-octaros-18-ene-2゜3.10.16-titraone, 1.14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclo hexyl)-1-methyl and nylco-23.25-dimethoxy-13,19,21 ,27-titramethyl-17-(2-oxopropyl)-11,28-dioxa -4-Azatricyclo[22,3,1,0'lioctacos-18-ene-2,3 ,10,16-chitraone, 17-allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-1 3,19゜21.27-tetramethyl-11,28-dioxa-4-aza-spi b[tricyclo[22,3,1,0'lioctacos-18-ene-2,2'-o xylan-3,10°16-trione, 17-ethanaryl-1.2.14-)dihydroxy-12-[2-(4-hydro xy-3-methoxycyclohexyl-1-methylvinyl]-23,25-dimeth xy-13゜19.21.27-tetramethyl-11,28-dioxa-4-a zatricyclo[22,3,1,0'' cooctacosu-18-ene-3,10,16 -) dione, 1,14-dihydroxy-12-[2-(4-hydroxy-3-methane) Toxycyclohexyl)-1-methylvinylrho26.28-dimethoxy-18 -[(phenylseleno)methylco13.22,24.30-tetramethyl-1 1゜17.31-) Lyoxer 4-azatetracyclo[25゜3, 1, 0 4.9. Q 18.20] Hentriaconta-16(20)221-diene- 2,3,10-)dione, 4-methyl-[17-allyl-1,14-dihydroxy C-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methy rubinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl -11,28-dioxa-4-azatricyclo[22,3,1,0”]octa Sus-18-ene-2,310-)linen-16-ylidene]hydrazide, or a pharmaceutically acceptable salt of any one of the above compounds.

Phosphorylated organic compounds are generally oxidized in an aqueous buffered phosphate medium containing a carbon nutrient source at room temperature. Once the resting cells are brought into contact with each other, preferably under submerged aerobic conditions. hydroxy-containing organic compounds in an aqueous nutrient medium containing assimilable carbon and nitrogen sources. Cultivate (ferment) the above microbial milk IL in the presence of (e.g. (for example, shaking culture, submerged culture, etc.). Aqueous medium is preferred or maintained at p) 1 of about 7 at the beginning and end (harvesting) of the fermentation process. pH is More than 1 morpholite results in substantial and/or complete loss of product. Noethanesulfonic acid (MES), morpholinopropanesulfonic acid (MOPS), etc. or by using a wL buffer such as the production medium described below. Maintain desired pH by selecting nutritional materials that inherently have MWi properties can do.

Preferred carbon sources in the nutrient medium are glucose, xylose, galactose, and glycerin. carbohydrates such as , starch, dextrin, etc. Other carbon sources include malt -Rhamnose, Raffinose, Arabinose, Mannose, Salicin, Koha Examples include sodium citrate.

Suitable nitrogen sources include yeast extract, meat extract, peptone, gluten flour, cottonseed meal, and soybeans. and other vegetable flour (partially or completely defatted), thawed casein, thawed soybean melted yeast, cornstarch liquor, dried yeast, wheat malt, feather flour, beer Nut powder, distiller solubles, etc., as well as e.g. ammonium salts (e.g. ammonium nitrate). monium, ammonium sulfate, ammonium phosphate, etc.), urea, amino acids, etc. It is an inorganic and organic nitrogen compound.

Although it is advantageous to use a combination of carbon and nitrogen sources, trace amounts of growth factors and Not necessarily in pure form, as low-purity materials containing large amounts of inorganic nutrients are also suitable for use. There is no need to use it. Sodium carbonate, calcium carbonate, phosphoric acid as needed Sodium, potassium phosphate, sodium chloride, potassium chloride, sodium iodide Inorganic salts such as aluminum, potassium iodide, magnesium salts, copper salts, cobalt salts, etc. If necessary, a liquid package may be added to the body, especially if the culture medium is quite foamy. Defoamers such as raffin, fatty oils, vegetable oils, mineral oils or silicones may be added. stomach.

As one type of starting material in the process, the PK-,520 starting material is described in U.S. Pat. S, h rosco 1cus va as described in No. 244,592 Fermentation of r, ascom ceticus, ATCC No. 14891 and S, h Rosco 1cus 5ubs.

Obtained by fermentation of P. akushimaensis No. 7278 (FR-90 0520 or FK-520), the other FK-506 type macrolide is European Patent No. EPO’0184162 in the name of ujisawa and Fi According to the method described in PCT WO89105304 in the name of 5ons These references are hereby incorporated by reference for this specific purpose. Add to part of.

For mass production of phosphorylated organic compounds, submerged aerobic culture conditions are suitable. Ru. For small-scale production, shaking or surface culture in flasks or bottles is used. Furthermore , when growing in large tanks, to avoid delays in growth during the manufacturing process. It is preferred to use the vegetative form of the organism for inoculating the tank. Therefore , the spores or mycelium of organisms produced by “tilt culture” are exposed to a relatively small amount of medium. First, by culturing the inoculated medium, also called ``seed medium''. to produce a vegetative inoculum of the organism, and then sterilely transfer the cultured vegetative inoculum into large tanks. The fermentation medium producing the inoculum, which is preferably transferred in a state in which phosphorylated organic compounds The culture medium may be substantially the same or different from the medium used for the production of the Autoclaved to destroy the ground. The pH of the medium is generally preferred. before the autoclaving step by appropriate addition of acid or base in the form of MWI solution. It is adjusted to about 7.0.

Agitation and aeration of the culture mixture can be carried out in various ways.

Agitation may be achieved by using a propeller or similar mechanical agitation device, or by rotating the fermenter. by shaking, using various pump devices, or by passing sterile air through the medium. This is done by Aeration can be carried out by passing sterile air through the fermentation mixture .

Fermentation is typically carried out at temperatures of about 20-40°C, preferably 25-30°C, depending on fermentation conditions and scale. It is carried out at a temperature of 5° C. for about 10 hours to 24 hours. Preferably, the production culture is 22 The fermentation was incubated at 27°C for approximately 24 hours on a rotary shaker operated at Orpm. The pH of the medium is maintained at 7.0 for harvest.

Suitable culture/production media for carrying out the fermentation include the following media:

Ko IU Trouble” 1 Kameka ■Z top Glucose 20.0 Soybean meal (Sigma) 5.0 Yeast extract (Fidco) 5.0 NaC15.0 K2HPO,' 5.0 Adjust p'H to 7.0.

The phosphorylated organic compounds produced are of interest in the recovery of other known biologically active substances. It can be recovered from the culture medium by conventional means commonly used. generated phosphorus Oxidized substances are found in the cultured mycelia and P fluid, and are therefore concentrated under reduced pressure and freeze-dried. , extraction with conventional solvents such as methanol etc., p11 modulation, conventional resins (e.g. treatment with anion or cation exchange resins, nonionic adsorption resins, etc.), conventional absorption Treatment with adhesives (e.g. activated carbon, silicic acid, silica gel, cellulose, alumina, etc.) Filter or centrifuge the culture broth by conventional methods such as crystallization, crystallization, recrystallization, etc. It can be isolated or purified from the mycelium and P solution obtained by. The preferred method is In particular, solvent extraction using methanol.

Phosphorylated organic compounds obtained by resting cells or fermentation methods as described above such as extraction, precipitation, fractional crystallization, recrystallization, chromatography, etc. It can be isolated and purified using conventional methods.

Suitable compositions of the substance may also be formed through the hydroxyl group on the molecule, such as acetate. Conventional pharmaceutically acceptable piolevil esters of phosphorylated organic compounds prepared may include.

The phosphorylated organic compounds of the present invention, particularly that of FK-520, are water-soluble and immunostimulatory. It has pharmacological activities such as inhibitory activity, antibacterial activity, etc., and therefore has a strong effect on the heart, kidneys, Rejection of transplantation of organs or tissues such as liver, bone marrow, skin, etc., GVH disease due to bone marrow transplantation autoimmune diseases (e.g. rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's disease) , multiple sclerosis, myasthenia gravis, type II diabetes, vaginitis, etc.). Useful.

The pharmaceutical composition of the invention may be prepared in an organic or inorganic carrier suitable for external, enteral or parenteral application. For example, a solid containing a compound of the invention as an active ingredient together with carriers or excipients. It can be used in semi-solid or liquid form formulations. The active ingredient is e.g. tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions and any other suitable can be formulated with non-toxic pharmaceutically acceptable carriers conventionally used in the desired use form. .

Possible carriers include those used in the preparation of solid, semi-solid or liquid form preparations. suitable water, glucose, lactose, gum arabic, gelatin, manganese Nitol, starch paste, magnesium trigaate, talc, corn starch, Includes keratin, colloidal silica, potato starch, urea and other carriers In addition, auxiliaries, stabilizers, thickeners, colorants and fragrances may be used. The active compounds of the invention are present in an amount sufficient to provide the desired effect on a disease process or condition. is incorporated into a pharmaceutical composition.

In order to apply this composition to the human body, it can be administered parenterally or through the small intestine. The therapeutically effective dosage of C-32 phosphorylated FK-506 is preferably one. For the treatment of disease, the The daily dose (calculated for a man weighing 70 kg) is generally 0.01~ 1000mg, preferably 0.1-500mg, more preferably 0.5-100mg mg, and the average unit dose is generally about 0.5 mg, 1 mg, 5 mg, 10 mg , 50mg, 100mg, 250mg and 500mg.

Lavamycin Phosphoric acid containing glycerol as a carbon nutrient source was suspended in medium. o Method of contacting 1J cells or selectively contacting lavamycin-type macrolide with C -43 phosphorylation at a pH of approximately 7 (e.g., 24 hours at 27°C) macrolides under submerged aerobic conditions in an aqueous hydrocarbon medium containing a nitrogen nutrient source. Microorganisms Rh1zo us or zae, ATCC No. in the presence of lavamycin , 11145, a novel immunosuppressant according to the method of the present invention. It has further been found that certain phosphorylated lavamycin macrolides are obtained. Which The method using resting cells can also be used, but the method using resting cells is preferred.

The C-43 phosphorylated macrolide formed has immunosuppressive activity similar to lavamycin. Mice stimulated with interleukin-2+PMA combination Positive samples in this assay are Suppresses T cell proliferation as shown by decreased 3H thymidine incorporation.

Further according to the invention, the production of immunosuppressive agents identified as phosphorylated macrolides A method is provided, the method comprising: in an aqueous medium containing a carbon nutrient source for a period of time, preferably at a pH of about 8.0 or less. , Rh1zous microorganisms capable of phosphorylating free hydroxyl groups (e.g. If you stand on the first floor, you should stop.

9 "Nishi Noe l" 工, more specifically dan "Shi 1" Nyu" - Raw 5oLlzae A TCC No. 11145) and lavamycin macrolide (its manufacturing method is available in the US). 3,929°992)).

Also provided is a non-rhyperbroth produced by the above method and which exhibits positive inhibition of T cell activation. It will be done.

Proton nuclear magnetic resonance spectrum and (FAB) mass spectrometry as shown in Figure 3. A novel immunosuppressant, a phosphorylated macrolide with a molecular weight of 993, obtained by Medications are also provided.

Useful as a therapeutic agent with a pharmaceutically acceptable substantially non-toxic carrier or excipient Pharmaceutical composition for the treatment of immunoregulatory disorders and diseases containing an effective amount of phosphorylated macrolides Compositions are also provided.

Additionally, we treat recipients to prevent rejection of organ transplants such as heart, kidneys, liver, lungs, and bone marrow. or used to treat autoimmune diseases (e.g. juvenile diabetes). Also provided is a method comprising pre-preparing a therapeutically effective amount of a phosphorylated macrolide. It consists of administering to a recipient.

The present invention provides lavamycin macrolide containing free hydroxyl groups with microbial 11 Contact the resting cells of the main two, or the presence of the macrolide. Fermenting the microorganism in the presence of a phosphorylated macrolide derivative to produce a phosphorylated macrolide derivative. This includes biological conversion methods. Microorganisms are currently 12301 Parklaw n Drive.

American Type located in Rockville, Maryland Deposited at Culture Co., Ltd. as ATCC No. 11145. has been done.

As used herein, the term "phosphorylated macrolide" is shown in Figure 3 (Metal ) Proton NMR spectrum of chilled derivative, mass spectrum molecular ion of 993 means a compound having the formula:

Generally, C-43 phosphorylated macrolides are prepared in an aqueous buffer containing a carbon nutrient source at room temperature. Contact resting cells of Rh1zous in phosphoric acid medium or preferably Assimilable carbon and nitrogen under aerobic conditions (e.g. shaking culture, submerged culture, etc.) In an aqueous nutrient medium containing the known In the presence of lavamycin macrolide, the above microorganism Rh1zo us 1 bifurcated 1 It can be produced by fermentation of E. The aqueous medium is preferably fermented. A pH of approximately 7 is maintained at the beginning and end of the process (recovery). pH is higher than this This results in substantial and/or complete loss of product. The desired pH is morpholinoe Such as tansulfonic acid (MES), morpholinopropanesulfonic acid (MOPS), etc. By using buffers such as can be maintained by selecting nutritional materials with M-stimulating properties.

Preferred carbon sources in the nutrient medium are glucose, xylose, galactose, and glycerin. carbohydrates such as , starch, dextrin, etc. Other carbon sources include malt s, rhamnose, raffinose, arabinose, mannose, salicin, amber Examples include sodium acid.

Suitable nitrogen sources include yeast extract, meat extract, peptone, gluten flour, cottonseed meal, and soybeans. Other vegetable flour (partially or completely defatted), thawed casein, thawed soybean and yeast thaw, cornstarch liquor, dried yeast, wheat malt, feather flour, beer Nut powder, distiller solubles, etc., as well as ammonium salts (e.g. ammonium nitrate) , ammonium sulfate, ammonium phosphate, etc.), urea, amino acids, etc. and organic nitrogen compounds.

Although carbon and nitrogen sources are advantageous when used in combination, trace amounts of growth factors and Low-purity materials containing large amounts of inorganic nutrients can also be used, so they do not necessarily need to be used in pure form. No need to use sodium carbonate, calcium carbonate, sodium phosphate as needed potassium phosphate, sodium chloride, potassium chloride, sodium iodide, Inorganic salts such as potassium iodide, magnesium salts, copper salts, cobalt salts, etc. are used as media. Liquid parallax may be added if necessary, especially if the culture medium is quite foamy. Defoaming sides such as fins, fatty oils, vegetable oils, mineral oils or silicones may be added. .

The lavamycin starting material is as described in U.S. Pat. No. 3,929,992. Known fermentation of eel S, h rosco 1cus, NRRL No. 5491 More can be obtained.

Regarding the conditions for mass production of phosphorylated macrolides, see submerged aerobic culture conditions. The conditions are suitable. For small-scale production, shaking or surface cultivation in flasks or bottles is recommended. used.

Furthermore, when propagating in large tanks, delays in propagation during the production process can be avoided. Therefore, it is preferable to use the vegetative form of the organism for inoculating production tanks. Therefore, a relatively small amount of spores or mycelium of the organism produced by "tilt culture" to inoculate a culture medium and cultivate the inoculated medium, also called a "seed medium". First, a vegetative inoculum of an organism is produced, and then the cultured vegetative inoculum is The fermentation medium producing the inoculum, which should be transferred under aseptic conditions to a large tank, is phosphorized. may be substantially the same or different from the medium used to produce the oxidized macrolide; The medium is generally autoclaved to sterilize it before inoculation. The pH of the medium is In general, autoclaving can be achieved by appropriate addition of an acid or base, preferably in the form of a buffer solution. It is adjusted to about 7.0 before the processing step.

Agitation and aeration of the culture mixture can be carried out in various ways.

Agitation may be achieved by using a propeller or similar mechanical agitation device, or by rotating the fermenter. by shaking, using various pump devices, or by passing sterile air through the medium. It can be implemented by Aeration is carried out by passing sterile air through the fermentation mixture. obtain.

Fermentation is typically carried out at a temperature of about 20-40°C, preferably 25-35°C, depending on fermentation conditions and scale. It is carried out for about 10 to 24 hours at a temperature of .degree. Preferably, the production culture is 22Orp The fermentation medium was incubated at 27°C for approximately 24 hours on a rotary shaker operated at pH is maintained at 7.0 for collection.

Fermentation/production media suitable for carrying out the fermentation include the following media:

LZ on the north side Dextrose 20.0 Soybean meal (Sigma) 5. Q Yeast extract (Fidco) 5.0 NaC15.0 K2HP0. 5. O The pH was adjusted to 7.0.

The phosphorylated macrolides produced can be used for the recovery of other known biologically active substances. It can be recovered from the culture medium by conventional means commonly used. The generated resource Oxidized macrolides are found in cultured mycelium and P fluid, and therefore can be concentrated under reduced pressure. , freeze drying, extraction with conventional solvents like methanol etc., pH adjustment, conventional resins. treatment with (e.g. anion or cation exchange resins, nonionic adsorption resins, etc.), Conventional adsorbents (e.g. activated carbon, silica gel, cellulose, alumina, etc.) The culture broth may be strained or separated by conventional methods such as treatment with It can be isolated or purified from the mycelium or r-fluid obtained by heart separation. suitable The method is a solvent extraction, especially using methanol.

Phosphorylated macrolides, which are fermentation products, have been shown to have positive immunity in T cell proliferation assays. Shows inhibitory activity and is therefore useful and has the following physical characteristics: 1. White amorphous powder 2. Methanol soluble 3, the molecular weight of 993 determined by FAB mass spectroscopy and shown in Figure 3. Assigned molecular structure shows.

Phosphorylated organic compounds obtained by resting cells or fermentation methods as described above such as extraction, precipitation, fractional crystallization, recrystallization, chromatography, etc. It can be isolated and purified using conventional methods.

Suitable compositions of the substance may also be formed through the hydroxyl group on the molecule, such as acetate. Conventional pharmaceutically acceptable pyolevir esters of phosphorylated macrolides prepared may contain files.

In the above fermentation reaction and post-treatment of the fermentation mixture, rearrangement of the hemiketal ring system is used. Tautomers and conformational isomers of phosphorylated macrolides are also within the scope of the present invention. Please note that this is included in

The phosphorylated macrolide of the present invention has pharmacological activities such as immunosuppressive activity, antibacterial activity, etc. Therefore, organs or systems such as the heart, kidneys, liver, bone marrow, skin, etc. Rejection of tissue transplantation, GVH disease due to bone marrow transplantation, autoimmune diseases (e.g. Paraphritis, systemic lupus erythematosus, Hashimoto's disease, multiple sclerosis, myasthenia gravis, type I sugar It is useful for the treatment and prevention of urinary diseases, uveal length, etc.).

The pharmaceutical composition of the invention may be prepared in an organic or inorganic carrier suitable for external, enteral or parenteral application. For example, a solid containing a compound of the invention as an active ingredient together with carriers or excipients. It can be used in semi-solid or liquid form formulations. The active ingredient is e.g. tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions and any other suitable can be formulated with non-toxic pharmaceutically acceptable carriers conventionally used in the desired use form. .

Possible carriers include those used in the preparation of solid, semi-solid or liquid form preparations. suitable water, glucose, lactose, gum arabic, gelatin, manganese Nithole, starch paste, magnesium trisilicate, talc, corn starch, Mention gelatin, colloidal silica, potato starch, urea and other carriers In addition, auxiliaries, stabilizers, thickeners, colorants and fragrances may be used. The active compounds of the invention are present in an amount sufficient to provide the desired effect on a disease process or condition. is incorporated into a pharmaceutical composition.

In order to apply this composition to the human body, it can be administered parenterally or through the small intestine. is preferable. The effective dosage of phosphorylated macrolides as therapeutic agents is not fixed. , depending on the age and condition of each individual patient treated, for the treatment of disease. The dosage (calculated for a man weighing 70 kg) is generally 0.01 to 1000 of the active ingredient. mg, preferably 0.1 to 500 mg, more preferably 0.5 to 100 mg. The average unit dose is generally about 0.5 mg, 1 mg, 5 mg, 10 mg, 50 m g, 100mg, 250mg and 500mg.

Enter A standing position The method of the invention further comprises a peptide skeleton carrying several hydroxy groups. Selective biological phosphorylation of cyclic ribopeptides related to echinocandins It is also applicable to the method for The group binds to the hydroxy group of the 4-hydroxyproline component of bobebutide.

Echinocandins or echinocandin compounds have lipophilic side chains and antibacterial properties. It is a cyclohexapeptide compound. Most are natural products, but there are also many semi-synthetic products. , natural products have names like echinocandin, gum arabic, marandocandin, Described in the literature by number designation or structure. Many compounds have remained unknown for many years. The structure and properties are based on the CRC Handbook of Antibiotics. Compounds, Vol IV, Part I.

PP 355-367, CRC Press, Inc.

, Boca Raton, FIa, 19804. Other transformation Examples of compounds include recently developed compounds such as those described in U.S. Pat. No. 4,931.352. There are compounds that have been observed.

The present invention particularly relates to formula (III): It concerns the cationic salt.

The term "r cation salt" refers to L'i, Mg, Na, Ca and (CIC4 a ammonium salt.

and is referred to as compound IIIA in the following text.

Kokucho 91114 Group This is a nuclear magnetic resonance spectrum.

smooth router Compound IIIA has a molecular weight of 1144 according to FAB-MS ((M+N a)” observed value 1167).

Concave” Figure 5 shows the compound isolated as monopotassium salt in CD, OD at 400 MHz. 'HNMR spectrum is shown, CD at 100 MHz, same in OD. The 13c NMR conversion of one isolate, Schiff l-, is as follows.

δe　177.2. 175.9. 174.3. 173.2. 172.9 ．． 172.58. 172.57, 169.0. 158.4. 133.1 ．． 129.6. 116.2. 77.0. 75.8. 75゜2 (double line , Jc,=4.0Hz>,74.3. 73°9, 70.6. 70.5.　 69.7.　68.3゜62.7. 58.3, 56.3. 56.1. 5 5.6. 51.2. 47.0. 45.9.　39゜4, 38.1. 37 ,6. 36.7.　35.0゜34.6. 32.9. 31.24. 31 ．． 20°30.8. 30.6. 30.3. 28.1. 27.0. 20 ．． 7. 20.2. 19.8.　11゜6゜ Based on the above and other data, compound IIIA has the title structure. Estimated with accuracy.

Compound fiI Rimetallic, magnesium, calcium and tetra(lower alkyl)ammonium salts It is a white solid that is soluble in base. From these bases, R is a cationic salt of phosphoric acid You can get salt like this.

The compounds of the invention have similar antibiotic properties to non-phosphorylated compounds and therefore , a parasite that is the causative agent of pneumodystis pneumonia, which is a particular problem in patients with immune disorders. (especially Pneumoc 5tiscar "L soil" 2) protection and fungal defense Useful as an antibiotic for Its antibacterial properties are based on Candida albi cans and cand i da- It is particularly useful against certain yeast strains.

Iture Co11ection is M% as MF4974. Lord! and 1-Tachieki ± Menguni derivatives of ATCC11145, together with the formula (Z): Preferably produced by incubating a compound (compound Z) having will be built. Cultures were initially grown at 12301 Parklawn Drive, Roc. American Type C located in kvi I Ie, MD 20852 Obtained from u1ture Co11ection.

Compound Z is added to Z a l a r ion arb in a nutrient medium as described below. oricola ATCC74030 and co-pending 199 U.S. Patent Application No. 492,025 dated March 12, 1990; 492.026.

Microorganism Rh1zo us arrhizus ATCC11145 is Rh1zo Also known as us or zae (J. J. EIlis, 1985 ．． Mycologia 77: 243 247>, this species is tanhizo u It is also described under the names S. nodosus and Rh1zo ustritici. There is. Strains MF4974, ATCC11145 are M, A, A, 5chipper By R, or zae, CBS 5 studiesin Mycolog’y 25:1-19 (1984). During the recent repopulation of MF4974 ATCC11145, which exhibits all of the following symptoms: The cutting characteristics described below were observed.

The strain appears to be heterothallic as no zygospore formation was observed.

Colonies grow on most standard mycological media, but on cornmeal agar (Dir. co) grows extremely rapidly and reaches a diameter of 35 mm within 36 hours at 20°C; At 7°C, the sporangium reaches >90 mm within 36 hours and the sporangium completely fills the vetri dish. , initially clear, but quickly turning pale yellowish-gray to light gray to brownish. The color turned gray, and finally dark gray. The sporangium stalk is 200-1000 μm long and 7. 5 to 19 μm, non-diaphragm, linear to curved at the base, and toward the top. once wide, sometimes bifurcated, with rather thick walls and a finely grained surface. It is pale yellowish brown or yellowish gray in color and is derived from rhizoid hyphae. tentative The root hyphae are composed of 3 to 10 thick, often curved branches.

The sporangia are spherical to subglobular, with a slightly flattened lower part, and a diameter of 100 to 230 μm. It is opaque, has a prickly surface, and is dark gray to black in color. The diameter of the column axis is 18 ~50 μm hemispherical to subspherical, smooth, often bent, adhesive cysts There is no ascus remnant. The sporangial stalk is 5-8 x 25-5 μm, subglobose to irregularly oval. Circular or angular on the sides, with inconspicuous to countersignal longitudinal striations. It is transparent to yellowish brown in color.

In the following, the present invention will be explained mainly in terms of specific aspects, but it is applicable not only to the above-mentioned strains but also to natural selection. various mutagens such as ionizing radiation or nitrosoguanidine Variants and mutant strains, whether produced by the action of chemicals such as Within the scope of the present invention.

Compound IIIA was added to Rh1z in a suitable nutrient medium containing Compound Z under the following conditions: o us arrhizus ATCC11145, and then fermentation medium The desired product is extracted with a suitable solvent and the components containing the desired compound are concentrated. , the concentrated material can be recovered from the product medium by chromatographic separation. can be generated by

Cultivation is carried out in a medium containing carbon and nitrogen sources assimilable by the microorganisms.

Carbon sources can be glycerol, sugars, sugar alcohols, starches and other carbohydrates, or carbohydrates chemical derivatives such as dextran, celerose), and complex nutrients (oat flour, corn meal, millet, corn, etc.). Strict carbon source used in culture medium The amount depends in part on the other components in the medium, but is usually between 0.5 and 40% by weight of the medium. % carbohydrate content is sufficient. These carbon sources can also be used individually or , several such carbon sources may be combined in the same medium.

Nitrogen sources include amino VI (e.g. glycine, arginine, threonine, methionine, etc.) ), ammonium salts and complex sources (e.g. yeast ice melt, yeast autolysate, yeast cells) , tomato paste, soybean meal, casein thaw, yeast extract, corn staple (liquor, distiller solubles, cottonseed waste, meat extract, etc.). Various nitrogen sources can be added to the culture medium. Can be used alone or in combination in an amount equivalent to 0.2 to 10% by weight .

Additionally, the medium should contain phosphate. Phosphate has a solid content of at least about 1 It should be 0% by weight. Preferably it is about 12 to about 15%. Particularly suitable culture The earth has the following composition: Fidco yeast extract *5. O p + (adjusted to 5.0.

(*Fidco yeast extract is available from Difco Lab located in Detroit, MI. is a nitrogen source that is a product of oratories. ) It is a soybean-glucose medium with a can do.

Fermentation can be carried out by first preparing a seed culture. seed culture When preparing Rh1zous arrhizus spores, Merck Cu Approximately 7 x 109 pieces 7/m1 stored in 1ture Co11ection Automated MF4974 dispersed in water to obtain a spore suspension containing spores of obtained from agar agar slant cultures.

A spore suspension of MF4974 was applied to a seed flask containing soybean glucose broth. The seeded and inoculated suspension is heated to a temperature range of about 15 to about 30°C, preferably 25 to 28°C. Incubate on a rotating shaker. Stirring is possible up to 400 rpm, but Generally preferred is about 220 rpm; incubation is for at least 24 hours. The event will be held for two days.

When the mycelium has grown sufficiently, the mycelium is recovered by applying P with a nylon mesh. Living For physical phosphorylation, 3% glycerol or some other simple carbon source Suspend the mycelium in a phosphate buffer containing . Then dimethyl sulfoxide ( DMSO) Compound Z is added to a concentration of approximately 50 μg/m+. production The pH of the live medium is important. The flask is preferably heated at 27°C for 24-48 hours. Incubate with shaking at 2 Orpm to produce compound IIIA. pH It is important to maintain this in the range of about 6.0 to 6.3.

After the incubation period is complete, pool the contents of all flasks and cover with a nylon membrane. Pass it through a TSUSH filter. Clean the mycelial cake on the filter with aqueous methanol. It becomes a rally and P-entitlement. Repeat this procedure for filter cake, 7f4 Pack the liquid into a styrene/divinylbenzene column. 8. Next, wash the column with water and rinsing it. The oxidation products were eluted with 20% acetonitrile in water, and the remaining metabolites and substrates were removed. Elute with 70% acetonitrile in water.

After elution, each fraction is assayed by HPLC.

Fraction determined to contain desired product by retention time of 128 minutes are combined and concentrated under reduced pressure to obtain the product as a residue.

Salts in which R is a cationic salt of phosphate are cationic salts in alcohol or other polar solvents. in close contact with the corresponding base and then condensation to initiate crystallization of the salt. It can be prepared by The salt is then recovered by filtration.

One method to prepare the salt is to add an aqueous solution of the acid to a non-functionalized resin column. 0 Representative resins include “AMB ER, CHROM”-161 (Toso Divinylbenzene/polystyrene resin available from Haas, Rohman (registered trademark of d Ha as), “DIAION” HP-20 and 5P-20 7 (crosslinked styrene, each a product of Mitsubishi Chemical) divinylbenzene and styrene bromide-divinylbenzene). . The column is then heated to MH, PO, or M' (H, PO, >2, where M and M ゛ are monovalent and divalent cations, respectively). Convert to state. Wash the column with water to remove excess inorganic phosphate. After that, 50 The product M or M by applying an aqueous eluent with an organic content of more than % 'Remove the salt from the column. Useful eluents are 80% acetonitrile, 80% ethyl alcohol or 80% methanol. Concentrate and dry the product and/or freeze the molten product. Isolate by drying.

This procedure can also be used to prepare certain salts from other salts.

Alternatively, an aqueous solution containing a small amount of an organic solvent such as acetonitrile and a phosphate Dissolve the acid in the mobile phase to form an acid salt solution. Remove solution from acetonitrile under reduced pressure. is added to a C-18 extraction column, retaining the product salts on the column.

The product salts are then removed as described above.

As has already been noted, phosphates are It is a compound that is active against certain yeast fungi such as ``Dandanyu''. The activity is 1 Using Yeast Nitrogen Base (Dirco) (YNBC) containing % Dextrose can be demonstrated in a microbroth dilution assay.

To perform the assay, Compound IIIA was added to 10% dimethylsulfoxy It was solubilized in DMSO and diluted to 2560 μg/ml. more compounds It was diluted to 256 μg/ml with YNBD. Then add 0.15ml of suspension. 96-well plate (each well accommodates 0.15 ml of 'r'NDDB) Distributed into the first column, and the drug concentration was 128 μg / ml. Dilutions were made to give final drug concentrations of 128-0.06 μg, .m+.

Yeast cultures maintained on 5 abouraud dextrose agar were transferred to YM broth ( Difco) and incubate overnight at 35°C with shaking (250 rpm). I did it. After incubation, each culture was diluted with sterile water and divided into 1-5 x 10 cells. The final concentration of roni-forming units (CFU)/ml is to inoculate 96-deletion microplates using a final inoculum volume of 1.5-7.5X 10' cells/well. Incubate the microplate at 35°C for 24 hours. Incubated. The minimum inhibitory concentration (MI C) was recorded.

After recording the MIC, the plate was shaken and the cells resuspended.

Thereafter, 1.5 μl samples from wells in 96-defect microplates 1 to 5 Transferred to single tray containing abouraud dextrose agar. inoculated Trays were incubated for 24 hours at 28°C and then counted. MFC shows proliferation or the lowest drug concentration such that the number of colonies per spot is less than 4. It is defined as

M Y I O2864 Candida tro icalis MY1012 32 The above shows that it is particularly suitable for treating fungal infections.

In the case of 2 compounds I I I/I I IA or compound I I I/I IIA Pneumoc 5tis carinii infected with the composition containing the or are suspected of having the drug, administer only a therapeutically effective or suppressive dose.

The suitability of the compounds of the invention for therapeutic or anti-infection purposes is determined by the Sprague-D awley rats (200 g body weight) were treated with dexason (2,0 mg/L) in their drinking water. ) and fed a paper protein diet for 5 weeks to prevent latent infection and pneumodysstasis. can be determined in a test that induces pneumonia. Two rats were killed before drug treatment. and confirm the presence of Pneumodystis carinii pneumonia (PCP). Next, Compound III in 0.25 ml of distilled water was administered into the tail vein of 6 rats for 4 days. Inject intravenously (1, V,) twice a day. Vehicle controls are performed similarly. all Animals remain on dexason in the drinking water and low protein diet throughout the treatment period.

After completion of treatment, all animals were sacrificed, lungs were removed and processed, and stained slides were analyzed for microscopic analysis. The extent of the disease is determined by analysis.

After injecting rats intraperitoneally (1,P,) twice a day for 4 days, the animals were sacrificed and the lungs were removed. A similar procedure is performed in which the extent of disease is determined by microscopic analysis of the stained slides. Experiments can also be carried out.

Compounds with pharmaceutically acceptable carriers according to conventional drug compounding techniques The above properties are most effectively exploited when formulated into pharmaceutical compositions.

The novel compositions provide at least a therapeutically effective antifungal or antipneumodystis amount. Contains ingredients. Generally, the composition will contain at least 1% by weight of Compound III. Ru. Concentrated compositions suitable for use when diluted before use may contain 90% or more by weight. 0 compositions are suitable for rectal, topical, extraintestinal (including subcutaneous, intramuscular and intravenous), pulmonary (nasal) intravenous or lateral inhalation), nasal administration or insufflation. The composition comprises compound III in a desired vehicle. It can be prepackaged by intimately mixing with quality appropriate ingredients.

Any method of administration may be used when the compound is used as an antifungal. Oral administration is often preferred for treating fungal infections. oral administration If it is to be used, it can be a liquid or solid composition. For liquid formulations , the therapeutic agent is preferably formulated with water or an aqueous composition, but optionally with glycol. It may be mixed with oil, alcohol, etc. Solids such as capsules and tablets For formulations, lubricants such as calcium stearate, binders, and disintegrating Along with agents, starch, sugar, kaolin, ethylcellulose, calcium carbonate, sodium carbonate, etc. Solid carriers such as thorium, calcium phosphate, kaolin, talc, and lactose Preferably, it is mixed with Riya. Tablets and caps are available for ease of administration. Cells are the most advantageous oral dosage form. 5 In terms of ease of administration and uniformity of dosage It is particularly advantageous to formulate the composition in unit dosage form (as defined below). . Compositions in unit dosage form are part of the invention! ! Configure the following.

Compound mIII is administered intravenously when used as an anti-Pneumodystis carinii. or as an aqueous therapeutic composition for intraperitoneal injection or aerosol. Prepared in unit dosage form in ampoules or multi-dose containers, with preservatives as required. can be done. The composition further includes 0.85% sodium chloride or 5% dextrose in water. It may take the form of a solution in an aqueous vehicle, such as a solution, and stabilizers and It may also contain formulation agents such as dispersants. saline to make the solution isotonic Or buffers and additives such as glucose may be added. For intravenous drip administration To avoid this, mix the drug with alcohol/propylene glycol or polyethylene glycol. It may be solubilized. Alternatively, the active ingredient may be powdered and mixed with a suitable vehicle prior to administration. You can also reconfigure it using a file.

As used herein and in the claims, the term "unit dosage form" refers to the presence of a predetermined amount calculated to give the desired therapeutic effect together with the carrier; Refers to physically distinct units each containing an active ingredient. Unit dosage forms such as Examples are tablets, capsules, pills, powder packets, cachets, ampoules or multiple use It is a measured unit, etc. in a volume container. The unit dose of the invention is between 100 and 1000 mg. may contain one type of compound.

HIV proase inhibitor-1 The present invention utilizes a protease encoded by the human immunodeficiency virus (HIV). It also relates to inhibiting compounds. The compound or a pharmaceutically acceptable salt thereof may be used to treat HIV infection. Useful for prevention of HI, treatment of HI infection, and treatment of acquired immunodeficiency syndrome (AIDS) 6 The present invention further provides pharmaceutical compositions containing the compound and AIDS and Hr. The use of compounds of the invention with or without other therapeutic agents for viral infections of V. It also relates to how it is used.

A retrovirus named Human Immunodeficiency Virus ()IIV) is a A complex disease (acquired immunodeficiency) that involves progressive destruction of the central and peripheral nervous systems and degeneration of the central and peripheral nervous systems. It is the causative agent of the total syndrome AIDS). This virus was previously known as LAV, HTLV -III or ARV. A common feature of retrovirus replication is that to produce mature viral proteins necessary for virus assembly and function. Extensive production of precursor polyproteins by virus-encoded proteases This is similar to post-translation processing. If you interfere with this processing, it will feel normal. It is thought to be able to prevent the production of infectious viruses. For example, Crawford, S. .

et, al., J. Virol, 53. 899°1985 is a precursor structure tag. of proteases in murine leukemia virus that interfere with protein processing. We demonstrated that gene deletion mutations form non-infectious virus particles. unprocessed Structural proteins were also isolated from human patients □ In clones of non-infectious HIV strains observed. These results demonstrate that inhibition of HIV protease may be useful in the treatment of AIDS and This suggests that it is an effective method for preventing or treating HIV infection.

According to nucleotide sequencing of HIV, one unique leading frame The existence of the gene "P Niji" was found in [L, Ratner, et at,,N ature.

ISL↓ sequence is reversed due to amino acid sequence homology. Proven to encode transcriptase, endonuclease and HIV protease [H.

Tol, et al., EMBOJ, 4. 1267 (1985); M, D, Power, et al.

5science, 231. 1567 (1986); L, H, Pearl, et al. al., Nature 329, 351 (1987)].

The inventors have demonstrated that the compounds of the invention are inhibitors of HIV protease. The compounds of the present invention provide prodrugs for the inhibition of HIV protease. do.

The present invention relates to biologically transformed metals as defined herein. this compound may be used as a prodrug or with other antiviral, antiinfective, immunomodulatory, or antiviral agents. The compound itself, whether in combination with a biological agent or a vaccine, HIV protea, optionally as a pharmaceutically acceptable salt or as a component of a pharmaceutical composition. inhibition of HIV infection, prevention of HIV infection, treatment of HIV infection, and AIDS and/or 1 The present invention is useful for the treatment of ARC, and is a method for treating AIDs, H■vr! i1 dyed Prevention methods and HIVr! ! It also relates to a treatment method for A staining.

The invention further provides methods for inhibiting HIV protease, preventing or treating HIV infection, and post-inhibition of HIV protease. The following compounds or their pharmaceutically acceptable compounds for the treatment of innate immunodeficiency syndrome (AIDS) Concerning the possible use of salt. The bioconversion compound is an HIV protease inhibitor. In the presence of L-702,083, which is 11145) It is. This compound has the following formula: or a pharmaceutically acceptable salt, hydrate or ester thereof.

Pharmaceutically acceptable salts of the compounds of the invention (water-soluble, oil-soluble, water-dispersible or oil-dispersible) products) include conventional non-toxic salts or quaternary ammonium salts of this compound, e.g. For example, they are formed from inorganic or organic acids or bases. Examples of such acid addition salts are , acetate, adipate, alginate, aspartate, benzoate, ben Zensulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonic acid salt, cyclopentane propionate, digluconate, dodecyl KM salt, ethane Sulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate salt, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2 -Hydroxyethanesulfonate, lactate, maleate, methanesulfonate , 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectic acid salt, persulfate, 3-phenylpropionate, picrate, pinocrate, Ropionate, succinate, tartrate, thiocyanate, tosylate and unde Canonicate is mentioned. Examples of base salts include ammonium salts and alkali metal salts. (e.g. sodium and potassium salts), alkaline earth metal salts (e.g. calcium and potassium salts), salts with organic bases (e.g. dicyclohexylamine salts), salts with organic bases (e.g. dicyclohexylamine salts), , N-methyl-D-glucamine) and amino acids (e.g. arginine, lysine, etc.) Examples include salt. Additionally, basic nitrogen-containing groups can be replaced with lower alkyl halides (e.g. For example, methyl chloride, ethyl chloride, propyl chloride, butyl chloride, methyl bromide, ethyl bromide. Chyl, propyl bromide, butyl bromide, methyl iodide, ethyl iodide, propyl iodide butyl iodide), dialkyl sulfates (e.g. dimethyl sulfate, diethyl sulfate, dibutyl sulfate and cyamyl sulfate), long halides (e.g. decyl chloride, cyamyl chloride) Lauryl, myristyl chloride, stearyl chloride, decyl bromide, lauryl bromide, bromide myristyl, stearyl bromide, decyl iodide, lauryl iodide, myristyl iodide aralkyl halides (e.g. benzyl bromide and It may be quaternized with a substance such as phenethyl bromide, etc.). Hydrates and esters are also included in the present invention. Such hydrates or esters can be easily conceived by those skilled in the art. For example, C1□ alkyl esters can be aged.

The compounds of the present invention are useful for inhibiting HIV protease, for human immunodeficiency virus (H1) Useful for the prevention or treatment of infections caused by IV) and the treatment of pathological conditions such as AIDS It is. The treatment of AIDS or the prevention or treatment of HIV infection is not limited to However, AIDS, ARC (AIDS-related syndrome), which is a widespread HIV infection state, shall include the treatment of symptomatic or non-symptomatic actual or potential HIV exposure. For example, the compounds of the invention may be used for example in blood transfusions, accidental punctures or surgical procedures. To treat suspected HIV infection after exposure to HIV during surgery, etc. Useful.

For these purposes, the compounds of the invention can be used in conventional non-toxic pharmaceutically acceptable compounds. Oral, parenteral ( subcutaneous injection, intravenous injection, intramuscular injection, intrasternal injection or infusion), inhalation spray It may be administered by the intravenous or rectal route.

Therefore, according to the present invention, methods and pharmaceutical compositions for treating HIV infection and AIDS More things will be provided. Treatment includes providing medical care to patients in need of such treatment. and a therapeutically effective amount of a compound of the invention. This will be implemented by

These pharmaceutical compositions are available as orally administrable suspensions or tablets, a-cavity sprays, In the form of bacterial injectable preparations (eg, sterile injectable aqueous or oily suspensions or suppositories).

When administered orally as suspensions, these compositions can be prepared using techniques well known in the pharmaceutical art. Bulking agents (microcrystalline cellulose), suspending agents (algium acid or sodium alginate), viscosity increaser (methylcellulose), sweetness/flavor May contain flavoring agents. As direct release tablets, these compositions are well known to those skilled in the art. microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and and lactose and/or other excipients, binders, extenders, disintegrants, diluents and a lubricant.

When administered by nasal aerosol or inhalation, these compositions are well known in the pharmaceutical field. prepared according to known techniques and containing benzyl alcohol or other suitable Preservatives, absorption enhancers to enhance bioavailability, fluorocarbons and/or may be prepared as a saline solution using other solubilizing or dispersing agents.

A suitable non-toxic parenterally acceptable diluent or solvent (e.g. mannitol, 1.3-butanediol, water, Ringer's solution or isosomic sodium chloride solution), or suitable dispersing or wetting and suspending agents including synthetic mono- or diglycerides. according to known techniques using sterile, non-irritating fatty oils and fatty acids, including oleic acid. Injectable solutions or suspensions may also be prepared.

When administered by the rectal route in suppository form, these compositions are solid at room temperature; , cocoa butter, synthetic glycerin which liquefies and/or dissolves in the rectal cavity to release the drug. Drugs with suitable non-irritating excipients such as lidoesters or polyethylene glycols can be prepared by mixing.

Dosage levels of about 0.02 to 5.0 or 10.0 g/day for the treatment or prevention of the above conditions. For example, HIV infection is useful when administered orally. By administering 10 to 50 mg/kg body weight of the compound 1 to 3 times a day. is effectively treated. However, specific dosing levels and dosing times for particular patients may The number depends on the activity of the particular compound used, the metabolic stability and duration of action of the compound, and the patient. age, weight, general health, gender, regular diet, administration method and time, excretion rate, drug combination and may vary depending on a variety of factors, including the severity of the particular condition and the condition of the recipient. be understood.

The invention further provides HIV protease inhibitor compounds useful for the treatment of AIDS. For example, the compounds of the present invention may be used in combination with one or more substances such as Regardless of whether the virus is closed at 11:00 a.m., an effective amount of other AIDS antiviral drugs, immunotherapy It can be effectively administered in conjunction with regulatory agents, anti-infectives or vaccines.

The present invention further provides treatment for human immunodeficiency diseases. Another compound that inhibits the protease encoded by H.I.V. It depends. The compound or a pharmaceutically acceptable salt thereof can be used for the prevention of HIV infection, The present invention is effective for the treatment of AIDS and acquired immunodeficiency syndrome (AIDS). and pharmaceutical compositions containing the compounds, as well as for the treatment of AIDS and HIV virus infections. The present invention relates to methods of using the compounds of the present invention with or without therapeutic agents.

The I-lovirus, named Human Immunodeficiency Virus (HIV), is a virus that affects the immune system. A complex disease that causes progressive destruction and degeneration of the central and peripheral nervous system (acquired immunodeficiency syndrome) It is the causative agent of the syndrome AIDS. This virus was previously known as LAV, HTL\r- It was known as xr or ARV. A common feature of retrovirus replication is that virus assembly and function to produce the mature viral proteins necessary for virus assembly and function. Large-scale production of the precursor polyprotein by a protease encoded by This is post-translation processing. If this processing is interfered with, the infection is usually For example, Crawford, S.

et al., J. Virol, 5th edition, 899° 1985. A protease residue in murine leukemia virus that interferes with protein processing. We demonstrated that gene deletion mutations form non-infectious virus particles. Non-processing structure Synthetic proteins have also been observed in clones of non-infectious HIV strains isolated from human patients. It was. These results demonstrate that inhibition of HIV protease may be useful in the treatment of AIDS and HIV. This suggests that it is an effective method for preventing or treating contagious infections.

According to nucleotide sequencing of HIV, one open reading frame The existence of the Ldan gene was found in [Ratner, L. et al., Nat. ure.

y top 3. 277 (1985)], the L earthwork sequence is reversed due to amino acid sequence homology. Proven to encode transcriptase, endonuclease and HIV protease [T.

h, H, et al, EMBOJ, 4. 1267 (1985); Pow er, M, D, et al.

5science, 231. 1567 (1986); Pearl, L.H.e. tal, Nature±λ, 351 (1987)].

The inventors have demonstrated that the compounds of the invention are inhibitors of HIV protease. do. Compounds of the invention provide prodrugs for inhibition of HIV protease do.

The present invention relates to biological conversion compounds as defined herein. This compound is as a drug or other antiviral, antiviral, antiinfective, immunomodulatory, or antibiotic agent. The compound itself, whether in combination with a substance or vaccine (if as a pharmaceutically acceptable salt or as a component of a pharmaceutical composition. inhibition, prevention of HIV infection, HIV! ! ! treatment of infections and AIDS and/or Useful in the treatment of ARC. The present invention provides methods for treating AIDS, prevention of HIV infection, The present invention also relates to methods and methods of treating HIV infection.

The present invention further provides inhibition of HIV protease, prevention or treatment of HIV infection and The following compounds or their pharmaceutical approval for the treatment of acquired immunodeficiency syndrome (AIDS) Concerning the use of acceptable salt. Biotransformation compounds are HIV protease inhibitors Rh1z in the presence of L-689,502.

arrhizus (ATCC11145). This is the incubation product obtained. This compound has the following formula: or a pharmaceutically acceptable salt, hydrate or ester thereof.

Pharmaceutically acceptable salts of the compounds of the invention (water-soluble, oil-soluble, water-dispersible or oil-dispersible) products) include conventional non-toxic salts or quaternary ammonium salts of this compound, e.g. For example, they are formed from inorganic or organic acids or bases. Such acids are examples of salt addition. , acetate, adipate, alginate, aspartate, benzoate, ben Zensulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonic acid salt, cyclopentane propionate, digluconate, dodecyl sulfate, ethane Sulfonate, fumarate, glucoheptanoate, glyceptanophosphate, hemisulfate salt, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2 -Hydroxyethanesulfonate, lactate, maleate, methanesulfonate , 2-naphthalenesulfonate, nicotinate, oxalate, pamoic acid, pectate , persulfate, 3-phenylpropionate, picrate, pivalate, propylene onate, succinate, tartrate, thiocyanate, tosylate and undecane Examples include acid salts. Examples of base salts include ammonium salts, alkali metal salts (e.g. alkaline earth metal salts (e.g. sodium and potassium salts), alkaline earth metal salts (e.g. calcium salts) and magnesium salts), salts with organic bases (e.g. dicyclohexylamine salts, N -methyl-D-glucamine) and amino acids (e.g. arginine, lysine, etc.) Salt is an example. Additionally, basic nitrogen-containing groups can be replaced with lower alkyl halides (e.g. Methyl chloride, ethyl chloride, propyl chloride, butyl chloride, methyl bromide, ethyl bromide , propyl bromide, butyl bromide, methyl iodide, ethyl iodide, propyl iodide and butyl iodide), dialkyl sulfate (e.g. dimethyl sulfate, diethyl sulfate, sulfate dibutyl and cyamyl sulfate), long chain halogenated! 1115 (e.g. decyl chloride, lauryl chloride, myristyl chloride, stearyl chloride, decyl bromide, lauryl bromide, Myristyl bromide, stearyl bromide, decyl iodide, lauryl iodide, myristyl iodide styl, stearyl iodide), aralkyl halides (e.g. benzyl bromide), hydrates and esters, which may be quaternized with substances such as phenethyl bromide and phenethyl bromide, The present invention also includes these types. Such hydrates or esters can be easily imagined by those skilled in the art. Examples include C1□ alkyl esters.

Compounds of the invention are useful for inhibiting HIV protease, human immunodeficiency virus (HI) V) and in the treatment of pathological conditions such as AIDS. Ru. Treatment of AIDS or prevention or treatment of HIV infection includes, but is not limited to, AIDS, ARC (AIDS-related syndrome), symptomatic and treatment of non-symptomatic actual or potential HIV exposure. For example, the present invention For example, compounds may be exposed to patient blood during blood transfusions, accidental punctures, or surgical procedures. Useful in treating suspected HIV infection following exposure to HIV.

For these purposes, the compounds of the invention can be used in conventional non-toxic pharmaceutically acceptable compounds. Oral, parenteral ( subcutaneous injection, intravenous injection, intramuscular injection, intrasternal injection or infusion), inhalation spray It may be administered by the intravenous or rectal route.

Therefore, according to the present invention, methods and pharmaceutical compositions for treating HIV infection and AIDS are provided. A composition is further provided. Treatment includes providing medical care to patients who require such treatment. and a therapeutically effective amount of a compound of the invention. This will be implemented by

These pharmaceutical compositions are available as orally administrable suspensions or tablets, nasal sprays, disposable In the form of bacterial injectable preparations (eg, sterile injectable aqueous or oily suspensions or suppositories).

When administered orally as suspensions, these compositions can be prepared using techniques well known in the pharmaceutical art. Bulking agents (microcrystalline cellulose), suspending agents (algium acid or sodium alginate), viscosity increaser (methylcellulose), sweetness/flavor May contain flavoring agents. As direct release tablets, these compositions are well known to those skilled in the art. microcrystalline cellulose, phosphorous-12 calcium, starch, magnesium stearate and and lactose and/or other excipients, binders, extenders, disintegrating agents, diluents and a lubricant.

When administered by nasal aerosol or inhalation, these compositions are well known in the pharmaceutical field. prepared according to known techniques and containing benzyl alcohol or other suitable Preservatives, absorption enhancers to enhance bioavailability, fluorocarbons and/or may be prepared as a saline solution using other solubilizing or dispersing agents.

A suitable non-toxic parenterally acceptable diluent or solvent (e.g. mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution), or suitable dispersing or wetting and suspending agents including synthetic mono- or diglycerides. according to known techniques using sterile, non-irritating fatty oils and fatty acids, including oleic acid. Injectable solutions or suspensions may also be prepared.

When administered by the rectal route in suppository form, these compositions are solid at room temperature; , cocoa butter, synthetic glycerin which liquefies and/or dissolves in the rectal cavity to release the drug. Suitable non-irritating excipients and agents such as lidoesters or polyethylene glycols can be prepared by mixing.

Dosage levels of about 0.02 to 5.0 or 10, Og/day for treatment or prevention of the above symptoms. For example, HI V angry staining is useful for oral administration. , effective by administering 10 to 50 mg/kg body weight of the compound 1 to 3 times a day. be treated. However, the specific dosage level and number of doses for a particular patient may vary depending on the usage. the activity of the particular compound used, the metabolic stability and duration of action of the compound, the age of the patient, Weight, general health, gender, regular diet, administration method and time, excretion rate, drug combination, special It is understood that this will vary depending on a variety of factors, including the severity of the condition and the condition of the recipient. Good morning.

The present invention further provides HI V protease inhibitor compounds and their use in the treatment of AIDS. For example, the compounds of the invention may be used in combination with one or more substances for and/or an effective amount of other AIDS antiviral agents, immunization, regardless of time after assault. It can be effectively administered in conjunction with regulatory agents, anti-infectives or vaccines.

Simba Stan Chemical name 6 (R)-[:2-(8'-(S)-2",2"-dimethylbutano) yloxy-2° (S), 6' (R>-dimethyl-1°, 2', 6°, 7 ’, 8’, 8°a(R)-hexahydronaphthyl-1’(S)-ethyl] -4(R)-Hydroxy-3.4,5.6-titrahydro-2H-bilan-2 Two phosphorylated derivatives of dimvastatin with -on were also produced by the method of the present invention. will be built. anti-hypercholesterol compound, which interferes with its synthesis and cholesterol biosynthesis For a description of its usefulness as a liquid agent, see European Patent EPO No. 0033 3. No. 58 and The Merck Index, 11th edition.

Rh1z simvastatin under the conditions described herein.

When contacted with L and U microorganisms, two types of phosphorylated derivatives shown below are formed.

L-706,526 L-706,527 The above two compounds of the present invention can be used to treat diseases such as atherosclerosis and hyperlipidemia in the human body. It is useful as an anti-hypercholesterol agent in the treatment of diseases. These compounds are in capsules It can be administered orally or parenterally as tablets, injection preparations, etc. Usually by oral route It is preferable to use However, the daily dose for adults is approximately 2 mg to 2000 mg (preferably 10 to 1 mg). 00 mg) in 3 or 4 divided doses. For higher doses if necessary. Gargling may be a good idea.

The compounds of the invention further have useful antifungal activity in strains of 6, e.g. Penicillium sp. Coch, which is Aser 1llus1uee, Cladosporium sp. 　l　i　obolus once worked Udan eanus and He1m1nth.

It can also be used to protect against sorium c nodnotis. this When used for such purposes, mix it with an appropriate medium and spray or spray it on the plants to be protected. do.

The pharmaceutically acceptable salts of the present invention include sodium, potassium, aluminum, calcium, Calcium such as aluminum, lithium, magnesium, zinc and tetramethylammonium Salts formed from thiones, as well as ammonia, ethylenediamine, N-methyl Lucamine, lysine, arginine, ornithine, choline, N,N'-dibenzyle Tilene diamine, chloropropylene, jetanolamine, propylene, N-benzene dilphenethylamine, 1-p-chlorobenzyl-2-pyrrolidine-1'-y -Methylbenzimidazole, diethylamine, piperazine and tris(hydrochloride) (oxymethyl)aminomethane.

α-zearalenol 6′-phosphate α-zearaleno, a biological conversion product l-6'-phosphate is also produced by the method of the present invention.

The present invention further relates to the above novel compound, and an object of the present invention is to exhibit estrogenic activity. Compounds that help increase the growth rate of meat animals (e.g. cattle, sheep and pigs) It is to provide.

The compounds may be administered to animals by any suitable method, including oral and parenteral administration. For example, a compound containing a sufficient amount of nutritional value to obtain a desired growth rate. It can be blended with normal feed and fed directly to animals, or the compound can be added to peanut oil. may be suspended in a suitable injectable suspension medium and administered parenterally to the animal. The amount of compound used will, of course, vary depending on the animal, desired growth rate, etc.

If the new product is administered in feed, the compound of the invention may be combined with normal nutrition. Animal feed composition containing balanced amounts of carbohydrates, proteins, vitamins and minerals prepare something These common feed ingredients include powdered feed (e.g. ground powdered feed and powdered feed). by-products), animal proteins (e.g. fishmeal and meatmeal), vegetable proteins (e.g. bean oil cake or peanut oil cake), vitamin minerals (e.g. vitamin A and D complex agent) and bone meal and lime for providing minerals. with a cow One example of a conventional feed used is alfalfa hay, ground corn It is desirable to use cob in conjunction with supplementary vitamin substances.

The starting material racemic (±)-ase is a powerful anabolic agent useful in raising meat animals. Aralenone can be prepared in a suitable manner according to the method described in U.S. Patent No. 3,169.019. Fermenting the microorganism Gibberella zeae (Gordon) on a nutrient medium It was first prepared by More recently, racemic (±)-zearalenone and A complete chemical synthesis of its 2,4-dimethyl ether derivative has been described ( Taub et al, Chemical Communications, 1967, p. 225), U.S. Pat. No. 3,551. '455 and the same No. 3, 23 The method described in No. 9,545 is a novel method for the complete synthesis of (±)-zearalenone. Provides a regular and more direct route.

fireworks Collect 15ml of CzaPek’s-Dox solution and a small amount of agar. Gibberella zeae (Gordon) NRRL The spore sand culture containing 2830 was placed under sterilization. This medium was heated for about 168 hours. Incubated at 25°C. Sterilize and deionize the medium after the incubation period. Wash with 5 ml of water and place in a sterile tube containing 45 ml of Czapek' 5-Dox solution. Moved. After incubating the contents of the tube for about 96 hours at about 25°C, the material is fermented. It was used to inoculate the culture medium.

The following example describes the biochemical transformation of 1hberella to produce zearalenone. Fermentation of eae (Gordon) NRRL-2830 will be described.

Support! 300 g of finely ground corn was added to a 2 liter flask. frass The container and its contents were sterilized, and after sterilization, 150 ml of sterile deionized water was added. frass Add 45 ml of the inoculum prepared according to Example IA to the mixture in the pot and mix thoroughly with the material. It was mixed. The mixed materials were then incubated in a dark room under a water-saturated atmosphere at 25°C for approximately 20 days. Cubated.

The following example describes the recovery of zearalenone from this fermentation medium.

Dai 1LLL Yunoyo 300 g of fermented material prepared by the method of Example 11A was added to 500 ml of deionized water. In addition, it was made into a slurry. The slurry is then heated to 75°C for approximately 15 minutes and filtered. 300 g of auxiliary agent was added and the material was filtered. Following solid filtration materials containing assimilable substances 333 g of the dried cake was extracted with 500 ml of ethanol. This step Repeated three more times. The ethanol extract was evaporated to dryness and the solid material 6,8 4g was obtained. This solid material was then dissolved in 20 ml of chloroform to bring the pH to approx. Extract with 30ml of an aqueous solution containing 1% sodium carbonate 5wt adjusted to 11.2. Ta. The extraction process was repeated 7 more times with pHt hydrochloric acid of the sodium carbonate extract. 6.2 to obtain a precipitate containing assimilable substances. Precipitation and aqueous sodium carbonate extraction Each product was extracted with 75 ml of ethyl ether. Repeat this step three more times and A yellow ethereal solution was obtained which was evaporated to give 116 mg of solid assimilable material. . This material was then combined with 100 tubes and 2 volumes of chloroform and 1 part of tetrasalt as the lower phase. Composed of 1 part by volume of carbon dioxide, and 4 parts by volume of methanol and 1 part by volume of water as the upper phase. Multiple transport countercurrent distribution was performed using a solvent system. obtained from multiple transport countercurrent distribution The solid material used was zearalenone.

The following examples are for the purpose of illustrating the invention and are intended to limit the scope and spirit of the invention. It's not something you can do.

11k C-1 FK- ■ Rhi2ZO2JLs or zae cultured on oatmeal agar flasks MF4974 (ATCC No. 11145) spores in a 250ml Erlenmeyer flask Inoculate 50 ml of soybean-glucose medium in a colander, and use a rotary shaker for 220 r at 27°C. Shake at pm for 24 hours. Second stage flask (5 in a 250 ml Erlenmeyer flask) 0ml> was inoculated with 2.5ml of the seed culture, and placed in a rotary shaker (220 rpm) at 27°C. m) for 24 hours. After incubation, in each flask. The container was collected by centrifugation and washed once with sterile water containing 3% glycerol. Resuspended in an equal volume of 100mM pH 7.0 POt solution. FK-520 The flask was shaken rotatably to give a final concentration of 0.2 mg/m+.　 (22Orpm) and incubated at 27°C for 48 hours. incubation After lysis, the whole broth was extracted as described in the Isolation/Purification section below.

iI Lucose lt LLL Dextrose 20.0 Soybean meal 5.0 Fido yeast extract 5.0 NaC15.0 K2HPO,5・O Adjust the pH to 7.0.

111L1 [ The total broth (400 ml) was adjusted to pH 3,5 and diluted with methylene chloride (3x40 0ml) was extracted. The combined methylene chloride extracts were evaporated to dryness under reduced pressure at 30°C. Ta. The obtained oil was dissolved in methanol and purified by HPLC. HPLC Whatman Partisil 100DS-3,9,4mmX25c m at 50° C. and monitored at 205 nm. 35% acetoni in 0.1% phosphoric acid 6 at 3ml/min using a linear gradient from tolyl to 80% in 0.1% phosphoric acid. The column was developed for 0 minutes. Compounds were absorbed during repeated injections of the above extract. Protection Fractions with a retention time of 32 minutes were pooled, PI adjusted to pH 3, and evaporated to acetate. Nitriles were removed. C18Sep　Pak(Water　As5ociate The compound was further purified using methanol-water eluent and product 4.5 mg was obtained.

11 纺L c-32 phosphorylated FK-520 (PK-9 00520> and further analyzed the proton NM in Figure 1 by NMR spectrometry. An R spectrum was obtained and the assigned molecular structure of FIG. 2 was confirmed.

Support 11L T...Se 1. The purified C-32 phosphorylated FK-520 prepared by the above HPLC was dissolved in anhydrous ethanol. The spleens of C57B1/6 mice were taken under sterile conditions. ice-cold RPM supplemented with 10% heat-inactivated fetal bovine serum (GIBCO) I 1640 culture medium (GIBCO, Grand 15land, N, Y,) Quietly dissociated. Cells were pelleted by centrifugation at 1500 rpm for 8 min. Ta. The pellet was soaked in Mtll solution for dissolving ammonium chloride (GIBCO) for 2 minutes at 4°C. Contaminant red blood cells were removed by treatment with Add cold medium and incubate cells again. Centrifuge for 8 min at 500 rpm and then transfer onto a nylon wool column as follows. T lymphocytes were isolated by separating the cell suspension. nylon wool collar Approximately 4g of washed and dried nylon wool is placed in a 20ml plastic syringe. Prepared by filling. Autoclave the column at 250″F for 30 minutes. Sterilize by washing. Wet the nylon wool column with warm (37°C) culture medium. Gently incubate the washed tile cells, resuspended in warm medium and rinsed with the same medium. The column was incubated upright at 37°C for 1 hour. . Nonadherent T lymphocytes were eluted from the column with warm culture medium, and the cell suspension was purified as described above. Agitated culture was carried out as follows.

Purified T lymphocytes were mixed with 10% heat-inactivated fetal bovine serum, 100mM glutamine, 1mM sodium pyruvate, 2XIO-'M 2-mercaptoethanol and Composed of l' (PMI 1640 medium supplemented with 50 μg/ml gentamicin) 05 cells/m+ in complete culture medium. Io 250 ng/ml of Nomycin and 10 ng/ml of PMA were added. Thin Transfer 200 μl of the cell suspension directly into a 96-section flat bottom microculture plate (Costar). /Distributed according to the gaps. Next is a control, which is a medium without the test drug, and a Various dilutions of the above sample of purified C-32 phosphorylated FK-520 as described below. The solution was added to three sets of wells at a rate of 20 μl/'. Marked FK-520 It was used as a standard. The culture plates were then incubated at 37°C in an atmosphere of 5% CO2-95% air. T Lymphocyte proliferation was examined. After 44 hours of culture, cells were treated with 3H thymidine (NEN, Ca (Mbridge, MA) pulse-labeled with 2 μCi/dose. Incubate for another 4 hours After fermentation, transfer the culture onto a glass fiber filter using a multiplex sample collector. It was recovered. The radioactivity of the filter disk corresponding to each individual well was measured using a standard liquid system. Measured by anti-chillation counting method (Betaco 11nter) Ta. Calculate the average counts per minute for wells under the same conditions and determine 1H thymidine incorporation (proliferation). Percent inhibition of: % inhibition = 100 - (test sample average cpm/control medium average cpm) x 100 The results are expressed as

Various concentrations of C-32 phosphorylated FK-520 (C-32-P FK-520) The results of % inhibition rate are shown in the table below.

: C-32-P FK-520 This T CC-32-P-FK-520 (n /ml) 26L"1l-3qmi"1-2589,6 12,571,3 6,228,7 3,10,0 Note: 1. Pulse mouse T cells with 3H-thymidine for 4 hours before harvesting after 48 hours. Ta.

2. I[Semi-FK-520 (4 ng,/ml) had an inhibition rate of 97%.

3. Average IC of C-32-P FK-520. 15 in three independent experiments. 0±2. log/ml (172±2.4 nM).

・1. By adding 100 units/ml of recombinant human IL-2 at the beginning of the culture. Reversed inhibition of T cell proliferation.

Kusotatsu 1 C-21n FK-06 Exemption 1 Danhizo us o knee vzae MF497 cultured on oatmeal agar 4 spores were inoculated into 50 ml of soybean-glucose medium in a 250 ml Erlenmeyer flask. The mixture was shaken on a rotary shaker at 25° C. and 220 rpm for 24 hours. 2nd stage flask (50 ml in a 250 ml Erlenmeyer flask) was inoculated with 2.5 ml of the seed culture, It was incubated for 24 hours at 27°C on a rotary shaker (220rp+n).

After incubation, the contents of each flask were collected by centrifugation and diluted with sterile water. Wash twice and resuspend in an equal volume of 100 mM PO containing 3% glycerol. Then, FK-506 was added to a final concentration of 0.2 mg/ml.

The filled flask was incubated at 27°C for 24 hours on a rotary shaker (22Orpm). I started. After incubation, remove the whole blot as described in the Isolation/Purification section below. extracted.

ul"Se, Lucose ■Tate name 2 Z 2" 4 dextrose 20.0 Soybean meal 5.0 Fido yeast extract 5.0 NaC15.0 K2HP0. 5. O The pH was adjusted to 7.0.

1LL11 The whole broth (200ml) was maintained at pH 6.8 and centrifuged. mycelium cake After washing with water, it was discarded. The clear P solution and washing solution were pooled and heated under vacuum at 5 speeds. Kutadecyl cartridge (14% carbon filling, Applied 5eparati ons). The column was washed with 100ml of water.

Column effluent and washes were tested by HPLC to ensure that they did not contain microbial transformation products. It was. The cartridge was eluted with 200 mL of methanol. Methanol under reduced pressure The mixture was evaporated to dryness at 30°C. The obtained oil was dissolved in methanol and purified by HPLC. Manufactured.

HP L C is Whatman Magnum 20 Partisil 1- It was carried out at 50 °C on an ODS-3 column (C4, 22, 1 mm ID x 25 cm), and 2 Monitored at 0.05 nm. 0°1% phosphorus from 35% acetonitrile in 0.1% phosphoric acid Color for 70 min at 7 ml/min using a linear gradient to 80% acetonitrile in acid. The system was expanded.

Compounds were collected during repeated injections of the above extract. Fraction with retention time of 50 minutes The samples were pooled, adjusted to pH 3.0 and evaporated to remove acetonitrile. C , 5epPak (Water As5ociates) was used to remove the compound. Salting gave 20 mg of product.

L1L The compound was analyzed by MS, and the C-32 phosphate ester of FK506 was further analyzed by NMR. It was confirmed that it was a stell derivative.

Person "1st ward" - C-21 C-31--C-3 instead of Sumeru FK-520PK-506 1 desmethyl FK-520 (e.g. European patent publication dated January 3, 1990) Example 3. Substantially the same fermentation procedure was carried out.

The isolation/purification procedure was nearly identical to that described in Example 3.

C-32 phosphorylated C-31 desmethyl FK-520 was analyzed by mass spectrometry and and proton nuclear magnetic resonance, and the spectrum was found to be consistent with the assigned structure. Ta.

K East fLL C-32 phosphorylated FK-506 (P-FK-506) and C-32 phosphorylated C- Conducted on 31-desmethyl FK-520 (P-31-desMeFK-520) The above IL-2 assay described in Example 2 was performed with the following results.

I Cs o = 3, 4 n g / m L (3, 9 n M) P-3 Inhibition of T by 1-DesMeFK-520P-31--SMeFK-520 'nml'/25095, 2 125 90, 3 62, 5 79. 5 31, 2 53. 8 15, 6 22. 2 ICso=31. 3ng/mL (36.7nM).

1-Paminsuma Low ■Standing Rh1zo us or zae MF cultured on oatmeal agar flasks 4974 (ATCC No. 11145) in a 250 ml Erlenmeyer flask. Inoculate 50ml of soybean-glucose medium and shake! ill at 27°C and 220 rpm The mixture was shaken at m for 24 hours. Second stage flask (1000ml Erlenmeyer flask contains 5 00ml) with 25ml of seed culture and shake with rotation! l (220rpm ) at 27°C for 24 hours. After incubation, each flask The contents were collected by centrifugation, washed once with sterile water, and contained 3% glycerol. The cells were resuspended in an equal volume of 100mM pH 7.0 phosphorus HMm. Lavamycin Macrolides were added to a final concentration of 0.2 mg/ml.

The flask was then incubated for 24 hours at 27°C on a rotary shaker (22Orpm). did. After incubation, drain the entire broth as described in the Isolation/Purification section below. Extracted.

11 Lucose l LLk Dextrose 20.0 Soybean meal 5.0 Fido yeast extract 5.0 NaC15.0 K2HP 04 5, O The pH was adjusted to 7.0.

``Single'' Shibun Muko [1m The whole broth (500 ml) was maintained at pH 6.8 and centrifuged. water mycelium cake After washing, it was discarded. Pool the clear R solution and wash solution and heat at 5 octadecy under vacuum. Cartridge (14% carbon filling, Applied 5 separations) passed through. The column was washed with 100ml of water. HPLC column effluent and washings When tested, it did not contain any microbial conversion products. Cartridge with methanol Eluted with 200 rnI. The methanol was evaporated to dryness under reduced pressure at 30°C.

The resulting oil was dissolved in methanol and purified by HPLC. HPLC is W a t m a n M a g n u m9 Partisil 10 0DS-3,9,8mm i, d, x25cm at 25℃, 225nm I watched it. A linear gradient of 35% → 80% acetonitrile in 0.1% phosphoric acid was used. The column was developed for 30 minutes at 3ml/min. Add the above extract during the delayed injection. The compound was charged with electricity.

Fractions with a retention time of 17.3 minutes were pooled, adjusted to pH 3, and evaporated to Setonitrile was removed. Cl8Sep Pak (Water As5ocia The compound was further purified using methanol-water eluent and product 1 4 mg was obtained.

C-43 phosphorylated macrolides were analyzed by FAB mass spectrometry and NMR spectroscopy. (FK-900520) was identified as a phosphoric acid methyl ester derivative by chromometry. I was caught.

The proton NMR spectrum of [2I3 supports the molecular structure of [g4.

To minimize the pronounced line broadening that characterizes proton NMR spectra of free acids. It was necessary to generate a phosphate ester by methylation. Main features H-42 and H-42 of 3.87PPm and 4.12ppm, respectively, moved to the lower magnetic field side. H-43 jig + le (H-31 and H-32 in the name of FK-506) and phosphorus. The additional fine structure of H-43 was derived from the coupling of . H-43 low magnetism A field shift is the normal result when an active hydrogen is replaced with an electron-withdrawing substituent. similar H-42 has moved downfield due to its proximity to the new substituent. H-' Phosphoric acid methyl ester with assignment of 42.8-43 and methyl ester beak Figure 3 shows the spectrum of the phosphorylated macrolide obtained.

picture[ T...Se 1. 2 otsu ffl The purified phosphorylated macrolide prepared by HPLC above was added to absolute ethanol 1. Dissolve at a ratio of mg/ml and serially dilute with culture medium before addition of culture. Ta.

2 Assay The assay was performed in J. Immunol, Vol. 144. PP, 251 <1990 ) was performed as described by F. Dumont et al.

Spleens were harvested from C57B1/6 mice under sterile conditions and heat inactivated. Ice-cold RPMI 1640 culture medium supplemented with fetal bovine serum (GIBCO) ( GIBCO, Grand 15land, N, Y) was used for gentle dissociation. cell was pelleted by centrifugation at 1500 rpm for 8 minutes. Pellets with ammonium chloride By treating with MWRn for dissolving monium (GI BCO) for 2 minutes at 4°C. Contaminant red blood cells were removed. Add cold medium and incubate cells again at 1500 rpm for 8 minutes. Centrifuge for 0.000 min and then separate the cell suspension on a nylon wool column as follows: T lymphocytes were isolated by. Nylon wool column washed and dried By filling approximately 4g of nylon wool into a 20ml plastic syringe. It was prepared as follows. By autoclaving the column at 250″F for 30 minutes Sterilized. Moisten a nylon wool collar with warm (37°C) culture medium and add the same culture medium. Rinse the washed cells resuspended in warm medium and slowly add them to the nylon wool. The column was then incubated upright for 1 hour at 37°C. non-adhesive T Lymphocytes were eluted from the column with warm culture medium, and the cell suspension was transferred to a stirred medium as described above. fed.

Purified T lymphocytes were mixed with 10% heat-inactivated fetal bovine serum, 100mM glutamine, 1mM sodium pyruvate, 2XIO-'M 2-mercaptoethanol and Composed of RPMI 1640 medium supplemented with 50 μg/ml gentamicin. The cells were resuspended in complete culture medium at a rate of 2.5 x 10' cells/ml. human recombinant in Tarleukin-2 (50μg)%ml and PMA 10ng/m Added l. Transfer the cell suspension directly to a 96-cut flat-bottom microculture plate (Costar ) at a ratio of 200 μm/dose, which is a control medium containing no test drug. and a variety of the above mentioned samples of purified phosphorylated macrolides to be tested. dilutions were added to three sets of wells at a ratio of 20 μm/hole. Lavamycin ( A zero-order culture plate using U.S. Pat. No. 3,929,992 as standard. Incubated for 44 hours at 37°C in a humidified atmosphere of 5% CO and -95% air. . T lymphocyte proliferation was examined by measuring 3H thymidine incorporation.

After 4 hours of culture, cells were treated with 3H thymidine (NEN, Cambridge, MA). ) Pulse labeling was performed with 2 μCi/dose.

After an additional 4 hours of incubation, the cultures were garnished using a multiplex sample collector. of the filter disc corresponding to each well collected on the fiber filter. Radioactivity was measured by standard liquid scintillation counting method (Betacounter). Established. Calculate the average counts per minute for wells under the same conditions and calculate the % inhibition rate = 400 - (test sample average cpm/control medium average Results were expressed as average cpm)×100.

Proliferation was examined after 48 hours of culture by 3H-thymidine incorporation.

The results, expressed as IC5° values, of three independent experiments are shown in the table below.

Table: IC of T.　n　m　l Note: 1. Incubate mouse T cell cultures with 3H-thymidine for 4 hours before harvesting after 48 hours. I lost it.

2. Average IC of 3 independent experiments. is C-43 phosphorylated lavamycin 7.6 ±0.2 ng/ml, and lavamycin 0.4 ± 0.1 ng/ml.

E11 ratio 1e The following preparations and syntheses are generally described in U.S. Pat. No. 4,661.473, Evans, B. , E, et al., J, Or, Chem, 50. 4615. (1985), Ev ans.

B, E, et al. “A 5tereocontrolled Synthesis” f Hydroxyethylene Dipeptide l5ostere s, ″Proc, Am, Pept, SymP, +9,743-6 (+985 ) and Lu Iy, J R, et al., T, Org, Cb c rr+, Jiang, 1487 (1987).

WR-Hydro-C I S innyl-5S), -11-dimerue 1- carbonyl aminouni”Cro-sidoro-cy 6-phenyl-2R--4 -2-morpho-1-el-cyphenyl pro-2-en-1-yl → Namidonis 1/Pu A: /1-t, e r t, -Blue Dime Sill Schiff) x Nilprop-2-7-1-ylpromide: Stirring bar and Argo II-capacity round-bottomed flask with an inlet for P-hydroxycinnamic acid 2(,25 g<160 mmol), t-butyldimethylsilyl chloride 50. 62g (335m o I), imidazole 32, 68g (4 80 m m()1) and 250 mL of dry DMF were added. Mix this mixture at room temperature. Stirred for 24 hours. The DMF was removed under vacuum and the residue was dissolved in EL OA C and 10 % citric acid aqueous solution.

The layers were separated and the organic phase was washed with water (/ly:) and brine.

De+(MgSO+>-filtration and remove solvent under vacuum, Bif, silyl ether) D. Self-stirring machine, argon inlet and addition funnel to obtain 2.3g of Stell G A 2L round-bottomed 3-necked flask containing I put the ru. Ether (460 mL) was added and the solution was cooled to 0° C. in a water bath. child Add 397 mL of Dibal-T solution (1°0M hexane solution) to the solution for 1 hour. It dripped.

After stirring the mixture for 1 hour, a saturated aqueous sodium potassium tartrate solution IL was carefully added. The reaction was stopped by adding

This viscous mixture was stirred for 20 hours at room temperature. Filter the mixture through a Celite band; - The liquid layers were separated and the organic phase was extracted twice with EtOAC. Bry the organic phase together. Washed with water, deflated (MgSO4), passed through P, and concentrated in vacuo to obtain crude alcohol. . on 500 g of silica gel using 20% EtOAc in hexane as eluent. This material was purified by Chroma 1 to Graphie. 4-L e as a viscous oil 34.9 g of rt-butyldimethylsilyloxycinnamyl alcohol was obtained.

This material was cooled to -15'C to form a crystallizer in a 300 mL round bottom flask equipped with a stir bar. A portion of this alcohol (18°5 g, 69.96 mmol) and the dry alcohol were placed in a lasco. 125 mL of ether was added. This solution was cooled to 0°C, PBr, 20.83 g (76,95mmo I> was added dropwise with a syringe over 5 minutes. This solution was heated to 0°C. Maintain for 20 min, dilute with hexane (IL), and wash with aqueous N a HCO2 solution. did. This solution was washed with brine, dehydrated (MgSO4), and washed with a silica gel pad. Filter and concentrate in vacuo to 4-tert-butyldimethylsilyloxyphenylpropylene. 17.53 g of p-2-en-1-ylbromide were obtained as a colorless oil.

Sue B: 5 S--Jime Rue Sil 1, Nil Mino-4S-'1' - Dime Ruel 1 - Dime Luci 1 Rui -6-phenyl-2R-4-1 ”-dimerylsilyl phenylpropyl-enylhen-niargo 500 mL with inlet, stir bar, low temperature thermometer, and addition funnel with shaker tip. Oven-dry a 3-neck round-bottomed flask and add 75 mL of dry THF and 15.39 mL (109.78 mmol) of diisopropylamine was added. this The solution was cooled to -20°C and added with n-butyllithium (42.84 mL, 2.5 M 107.11 mmol of xane solution) was slowly added. The obtained solution is 178 Cool to ℃ and add (5S, 1'5)-5-((1, 1-dimethylethoxycarbonyl)amino)-2-phenylethyl)dihydrof Dissolve ran-2-(3H)-one (16.36 g, 53.55 mmo+). The solution was added at such a rate that the temperature of the solution did not rise above 170°C (this addition (required for approximately 40 minutes), the resulting solution was aged at -78°C for 1 hour, and then diluted with dry iTHF. Add 4-tert-butyldimethylsilyloxyphenylprop-2-ethyl to 75 mL. A solution prepared by dissolving ton-1-ylpromide (17.53 g, 53°55 mmol) The liquid was filled into a dropping funnel.

After cooling the bromide solution to -78°C, the enolate was added while maintaining the temperature below -70°C. It was added dropwise to the solution over a period of 45 minutes. Once the addition is complete, age the solution at -78°C for 1 hour. The temperature was raised to -50°C and reacted with a solution of NaH9O, (33 g) in 250 mL of water. stopped responding. The mixture was diluted with EtOAc and the layers were separated. Organic phase is Na Washed with HCOs solution and brine. Dehydration (MgSO<), filtration and under vacuum The solvent was removed to give the crude alkylated product as an oil. 20% E in hexane This material was chromatographed on 1000 g of silica gel using tOAc as eluent. Graphically purified. Thus pure (5S, 3R, 1°5)3-(4-(1 °.

1'-dimethylethyl-1,1-dimethylsilyloxy)phenylprop-2- en-1-yl)-5-(1-11,1-dimethylethoxycarbonyl)amino )-2-phenylethyl)dihydrofuran-2-(3H)-one (23°6 g) Obtained as a colored foam. This material was dissolved in 325 m of DME and Li0H (6, A solution of 99 g, 291.97 mmol> in H2O (325 mL) was added. This melt The solution was stirred at room temperature for 24 hours. The DME was removed under vacuum and the aqueous residue was quenched with 10% Acidified with an aqueous solution of phosphoric acid. This milky suspension was extracted several times with EtOAc and the extract was Combined, washed with water and brine, dehydrated (M g S 04), filtered, Concentrated under vacuum. Imidazole (56°79g, 83 4mmo+), tert-butyldimethylcy”) Lua 0lide (62,87g , 417 mmol) and dry DMF (325 mL) were added. Pour this mixture into the chamber Stir at room temperature for 24 hours. The mixture was then treated with methanol (325 mL) for 4 hours at room temperature. Processed. The solvent was removed under vacuum at 60° C. and 20 torr. The residue was dissolved in EtOAc I Washed with 10% aqueous citric acid, water and brine. Dehydration (Mg sO,), l filtrate and remove solvent, 5(S)-(1,1-dimethylethoxycarbonate rubonylamino)-4(S)-(1°,1'-dimethylethyl-1,1-dimethyl lucilyloxy)-6-phenyl-2(R)-(4-<1', 1'-dimethyl Ethyl-1,1-dimethylsilyloxy)phenylprop-2-en-1-yl ) 28.53 g of hexanoic acid were obtained as a colorless foam.

Lou -; Mail: lle ζ - two - R - -en-1-le he ≧ de-2 In a 2 L round bottom flask equipped with a stirrer and argon inlet, add 5 (5) from step B. S)-(1,1-dimethylethylethoxycarbonylamino)-4(S)-(1 ', 1°-dimethylethyl-1,1-dimethylsilyloxy)-6-phiniru 2(R)-(4(1',1'-dimethylethyl-1゜1-dimethylsilyl) oxy)phenylpro-1-2-en-1-yl)hexanoic acid (28,53g, 4 1.71 mmof), 2(R)-hydroxy-1(S)-aminoindan (6, 85g, 45.88mmol), 3-(N,N-dimethylaminopropyl)ethyl Lupodiimide hydrochloride (8.79g, 45.8mmol), 1-hydrochloride Roxybenztriazole hydrate (6.20g, 45°88mmo 1) and Dry DMF (<300 mL) was added.

Dissolve all solids and add triethylamine (12.79 mL).

91.76 mmo1) was added and the mixture was stirred at room temperature for 18 hours. mixture Between EtOAc (1500 mL) and 10% citric acid aqueous solution (1500 mL) Separate the 0 layer and wash the organic phase with water (3x 1000 mL) and brine. It was. Dehydrate (MgSO<), pass through F and remove solvent under vacuum, leaving a yellow foam 32 I got g. This material was dissolved in 500 mL of methanol, and LiOH (4.99 g , 208.55 mmol) was added. This solution was stirred at room temperature for 1 hour.

The solution was acidified with aqueous citric acid and the methanol was removed under vacuum. obtained water The residue was extracted with EtOAc ('LL). The EtOAc extract was dehydrated (MgSO ), filtered and concentrated under vacuum. Elute with 8 L of 40% EtOAc in hexane. This material was chromatographically purified on 1 Kg of silica gel using . Thus N-(2(R)-hydroxy-1(S)-indanyl)-5(S) -(1,1=dimethylethoxycarbonyl)amino-4(S) -(1°, 1 °-dimethylethyl-1,1-dimethylsilyloxy)-6-phenyl-2(R )-((4-(hydroxyphenyl)prop-2-en-1-yl)hexanami 20.07 g of chloride was obtained as a colorless foam.

Soup D: N-2R-Hero Sea S-11-D MELUE SIL 5, NILUA ≧NO -4S -HI'MOUTH 2--Phenyl-2R- 4--4-morphoethyl phenylpropylene Into an IL round-bottomed flask equipped with a stir bar and argon inlet, add N-(2(R) -hydroxy-1(s)-indanyl)-5(S)-(1,1-dimethylethoxy cyclocarbonylamino)-4(S)-(1', 1°-dimethylethyl-1,1- dimethylsilyloxy)-6-phenyl-2(R)-(4-(1°, 1°-dimethyl Tylethyl-1,1-dimethylsilyloxy)phenylprop-2-ene-1- il) hexanamide (19.49 g, 28.1 mmol), 1,4-dioxane (400 mL), 4 (2-chloroethyl)morpholine (12.61 g, 84. 3 mmol) and cesium carbonate powder <27.5 g, 84.3 mmol) were added. .

The mixture was heated to 80° C. with vigorous stirring for 3 hours.

The cooled reaction mixture was filtered through a pad of Celite and the 1,4-dioxane was removed under vacuum. Removed. A THF solution (LM solution) of tetrabutylammonium fluoride was added to this residue. 280mL.

280 mmol) was added and stirred with a stirring bar. This solution was added under an argon atmosphere. The mixture was stirred at room temperature for 28 hours. The reaction mixture was poured into H2C3L under stirring and filtered. The whiter solid was collected and dried under vacuum overnight with 5% MeOI (5% MeOI as eluent). Chromatographic purification of the crude product on silica gel using chloroform solution did. The chromatographically purified material was reconsolidated in boiling EtOAc-hexane. Crystallized and analytically pure N-<2(R)-hydroxy-1(S)-indanyl' I-5(S)-(1,1-dimethylethoxycarbonylamino)-4(S)- Hydroxy-6-phenyl-2(R)-(4-(2-(4-morpholino)ethoxy) c) 13g of phenylprop-2-en-1-yl)hexanamide as a white solid. I got it. m, p, 178-179°C0 and 11 N-2R--Hydro C I S-Indanyl-5S-11-dimerue dirubonylamino)-4(S-hydroxy-2(R)-(4-(2 Go to -4 -45-dihydrofuran-2-3H2J"ren0"l [: (5S, 1' S>-5-(1°-((1,1-dimethylethoxycarbonyl) amino)-2°-phenylethyl)-4,5-dihydrofuran-2-(3H)- ion was dissolved in ethyl acetate and rhodium supported on alumina was added.

The mixture was shaken under a hydrogen atmosphere (50 ps i) at 50° C. overnight. Filtration and Evaporation of the solvent and the title compound was obtained as a viscous oil, which on standing turned into a hard glass. It solidified.

Left LLL shadow: (5S, 1' 5)-5-((1,1-jime) of Example 8 Step B thylethoxycarbonyl)amine)-2-phenylethyl)dihydrofuran-2 -(3)()-one is its cyclohexyl analogue (5S, 1'5)-5 -<　(1-dimethylethoxycarbonyl)amino)-2-cyclohexylethyl Example except that the substitution was made with 1)-4,5-dihydrofuran-2-3H)-one 8, steps B, C and D. 1 compound L-702゜083 Obtained.

Fire m”- L-702083 p″1 Rh1zo us arrhizus MF49 cultured on oatmeal agar 74 spores were placed in a seed medium of 50 ml in a 250 ml Erlenmeyer flask with 3 baffles. m1 [dextrose 20.0g/L, soybean meal 5.0g/L, Fidco yeast Contains extract 5.0g/L, NaC 15.0g/L, K, HPo, 5.0g/L, [pH 7.0] before autoclaving]. Place the flask on a rotary shaker (22O rpm) for 24 hours at 27°C. This seed culture Th2.5ml Inoculate 50 ml of the same medium in a 250 ml Erlenmeyer flask and shake once. The cells were incubated at 27° C. for 24 hours in a machine (22 rpm). incubation After washing, the contents of each flask were collected by centrifugation, washed once with sterile saline solution, and Resuspend in an equal volume of 100 mM phosphate buffer (pH 7.0) containing % glycerol. Made it muddy. Next, add a DMSO solution (20 mg/ml) of L-702,083 to the final concentration. Add the solution to a concentration of 0.1 mg/ml and heat it at 27°C on a rotary shaker (22Orpm). Cultivation continued. After 12 hours of incubation, whole broth was added as described below. was extracted.

ANDILLLL 11 branches m The contents of the two shake flasks were combined to obtain 100 ml of broth. Centrifuge the broth I let go. After washing the yarn cake with water, it was discarded. Combine the clear P solution and washing solution and place under vacuum. 5peed octadecyl 14% cartridge (Applied 5epara tions). After washing the column with 100ml of water, 200ml of methanol It was eluted with . The methanol extract was concentrated under vacuum to give a slightly oily residue.

Take the residue in methanol and m 20Par and 1si1100DS-3, 22, Intm:' 25 c m The mixture was subjected to preparative HPLC chromatography with flow 1 of 7 ml/min at room temperature.

Effluent flow was monitored by UV absorption at 215 nm. 30% acetate in 0.1% phosphoric acid Elute for 80 minutes using a linear gradient from setonitrile to 80% in 0.1% phosphoric acid. I let go. Separate the fractions with a retention time of 35 minutes, adjust the pH to 4.0, and evaporate. to remove acetonitrile. C-18Sep　Pak(WatersAs 3111 g of product L-706 ,579 was obtained.

Fire 1” t13- xyp'' U1 passed away. The product of Example 11, L-706,579, at C-4 of L-702°083 It was analyzed by NMR as a phosphate ester derivative. The main feature is the formula: There was a 0.6 ppm shift of Hl to the downfield side.

Compared to the chemical shift 1~ in the parent L-702,083, the vicinal methine (m) J slightly moves to the lower magnetic field side, and at 3.74 ppm, it becomes the shoulder of the base of morpholine CH2O. barely recognizable. H nearby. also moved, and the low magnetic field of HJ decreased to 3.10 ppm. It is perceived as a signal below the field side. Its existence is L-702,083 Compared to Hl, which is a doublet, it is an additional region of H signal. That's it.

E11L1 L-700 Inn Sul 7L-706579-8 A. Inn's Asel The 2(R)-hydroxy-1(S)aminoindan of Example 8, Step C was converted to 2( Example 8 and R)-acetoxy-1(S)-aminoindan except that 9, N-[(2(R)-acetoxy-1(S)-indanyl]- 5(S)-(1,1-dimethylethoxycarbonylamino)-4(S)-hydro Roxy-2(R)-[(4-(2-(4-morpholino)ethoxy)phenyl) Prop-2-en-1-yl]-6-cyclohexanamide, L-702°083 was prepared.

B-Sini Nishi±65ヱ”- Chambe rs, R, W, and H, G, Kh o r a na, J , Am, Chem, Soc, 80. According to the principles and methods of 3749 (1958) The product of step A was then reacted with monophenylphosphorodichloridate. . It is then treated with excess ammonia to remove the acetyl group under basic conditions, and L I got -706,579.

C-艮1i1 [k month- Another phosphorylating agent is dibenzylposborochloridate. phosphorylation and phosphoric acid For details of the curing agent, see Y. Mizuno.

5tudies in Org, Chem, and.

171-175 (1986) and its cited references.

XJ1Kohong ) IIV Brose's Tusei HIV protease expressed in E. coli and sH peptide group! ['H]-acetyl -Va 1-3e r-G l n-Asn-(β-naphthyl-Ala)-Pr o-I 1e-Val-G I n-G I y-Arg-Arg-Nl2(M An inhibition test was carried out by reacting with W1800). Two arginines at the amino terminus The residues give this peptide an overall positive charge with acidic P) (DOWEX AG This peptide can be attached to the H-form of -50W-X8 resin and similar resins. can. HIV protease cleaves between β-naphthyl-Ala and proline residues Therefore, it is neutrally or slightly negatively charged, and Vat-Ser-Gln-Asn-(β -naphthyl-Ala)) is produced. Therefore, the labeled product should be appropriately separated from the substrate. It is possible to

Assay 11m solution (100mM sodium acetate, pH 5.5 and 0.1% BSA ) in 6.0-8. 25μm aliquot containing OnM HIV protease was put into a test tube. 4.2 μM in 100 mM sodium acetate, pH 5.5. The reaction was started by adding a 25 μl aliquot of 3H peptide substrate. 6 After incubation at 37°C for 0 min, react with 100 μl of 5% HsPO4. It was stopped and analyzed by application of column chromatography.

The results are shown below.

L-706,579'-M) Inhibition rate % dog m above j- Input of L-689502 Preparation and synthesis are described in U.S. Pat. No. 4, which is hereby incorporated by reference. , No. 661, 473, Evan S.

B, E, et al., J, Or, Chem, 50. 4615 (1985), Ev ans, B, E, et al. “A Stereo controlled 5ynthes iso f Hy d r o x y e t h y I e n e D i p e p tide l5osteres,” Proc, Am, Pe pt, Symp, 9,743-6 (1985) and Luy, J. R., et al. J. Org. Chem. 52° 1487 (1987). Total temperature Degrees are in degrees Celsius unless otherwise specified.

N-cis-2R-Hiwa Sea I S-Nil-5S-11-Dimerue Seal 1, fluoramino-48-hydroxy-6-phenyl-2R under nitrogen atmosphere Magnesium turnings (9,79 g, 40 g in diethyl ether (200 mL) dried with chloromethyltrimethylsilane (50 mL) with stirring. , 358 mmol) was added. The reaction is started by gently raising the temperature, and After the end of zero exotherm, the reaction was cooled in a water bath to maintain a constant reflux for 1 hour at room temperature. The mixture was stirred for a while and then cooled to -78°C in a dry ice/acetone solution. Grini N-2(S)-[(1,1-dimethylethoxycarbonyl)amino Drying of co-3-phenylpropionaldehyde (19.3g, 77.4mmo+) A solution of dry diethyl ether (250 mL) was added dropwise with stirring, and the reaction temperature was increased to -55°C. Maintained below. The resulting gray suspension was warmed to room temperature and stirred at room temperature for 30 minutes. After stirring, pour into a mixture of ice (500 g) and 10% citric acid (500 mL). The reaction was stopped. The organic phase was collected and the aqueous phase was diluted with diethyl ether (3X300m Extracted with L). Combine the organic phases and add 10% citric* (IX 300 mL) and Wash with 200 mL of chlorine (IX), dehydrate with anhydrous magnesium oxide, filter, and concentrate. Condensation to give crude N-3(S)-[(1,1-dimethylethoxycarbonyl)amine]- 2(R3)-hydroxy-4-phenyl-1-trimethylsilylbutane (26, 6 g, quantitative crude yield) was obtained as a yellow oil.

Low pressure chromatography purification (silica gel, 230-4°O mesh, diethyl Ether; hexane = 30% nia.

%) and then recrystallized from heptane to obtain a sample for analysis.

m, P, 91-95°C0 elemental analysis: CIIH31NO! Si (337, Calculated value of 53): C=64.05. H=9.26°N=4.15. Actual measurement value C =64.05. H=9.13, N=4.22. [(ZID”=40.0” .

Step B: 3(S-amino-4-phenyl-1-buty'L!!2JLLni) The product of Step A (22.8 g, 67.5 mmol) was dissolved in dry methylene chloride (4 00 mL) solution was cooled in a water bath under a nitrogen atmosphere, and fluorotrifluoride was added to the solution with stirring. Uron etherate (43 mL, 345 mmo+) was added as a fine stream. solution The temperature was raised to room temperature and stirred at room temperature for 4 days.

Cool the reaction in a water bath and add 10% sodium hydroxide (400 mL) dropwise. The reaction was stopped. Combine the organic phase and dilute the aqueous phase with methylene chloride (2 x 250 mL). Extracted with.

The combined organic phases were washed with brine (IX 200 mL) and washed with anhydrous magnesium sulfate. Dry, filter, and concentrate to give the crude product 3(S)-amino-4-phenyl-1-butene. (14.2 g) was obtained as a yellow oil.

Sue Bu C: N-3--Jime Rue carbonyl ζ-4-phenyl-1-boon; Product of Step B (14,2 g) and di-tert-butyl bicarbonate (31,0 g, 142 mmo+) in dry methylene chloride (200 mL) at room temperature for 18 hours. Stir and add 1096 citric acid (3X100mL), water (IXloommL), saturated Wash with sodium carbonate (3X125nL) and brine (IX250mL); Dry over anhydrous magnesium, filter and concentrate to give crude N-3(S)-[(1,1 -dimethylethoxycarbonyl)aminoco-4-phenylbutene (34.6g) was obtained as a yellow oil. The crude product was purified by low pressure chromatography (silica gel, 230-400 mesh, 1010X2O column, diethyl ether:hexane =20%=80%) and purified by N-3(S)-[(1,1-dimethylethoxy cyclocarbonyl)amine]-4-phenyl-1-butene (16.3 g, 97.6% Yield) was obtained as a white solid. Analytical samples by recrystallization from heptane I got Le. mp=67.5~68.5℃ 1 element eIFr: Cl5H21NO! ( Calculated value of 247,34): C near 2.84. l-1=8.56. N=5.6 6. Actual value: C=72.78. H=8.76, N=5.64゜Suitable D: I　R-1'　S　-1-Jimele　Shino,・Nil　≧　--２l　え　えし　 Yo and Zlzll-ni Product of Step C (9.4 g, 38 mmol >77) Dry methylene chloride (1 00 mL) solution was cooled in a water bath under nitrogen atmosphere, and 3-chloroperoxide was added to this solution. Take xybenzoic acid (industrial use, 80-85%; 41 g, 200 mmol) Ta. After stirring the mixture at 0°C for 18 hours and at 25°C for 23 hours, diethyl ether (3 00 mL) and poured into a water-cooled 10% aqueous sodium sulfite solution (IL). The organic layers were combined and the aqueous layer was extracted with diethyl ether (3x 100 mL).

Combine the organic layers and add 10% sodium sulfite (3X 100 mL), saturated sodium bicarbonate. Wash with thorium (3 x 100 mL) and brine (IX room mL), anhydrous sulfuric acid. Dried over sodium, filtered and concentrated to give a white solid. Low pressure chromatography of crude product torography (silica gel 230-400 mesh, 8x15cm column, acetic acid Ethyl: Hexa 2225% Nia 5%), purified by 1(R)-[1°(S) -(1,1-dimethylethoxycarbonyl)amino-2-phenylethyl]oxy The silane (7.0 g, 70% yield) was obtained as a clear oil, which after standing crystallized into. Analytical samples were obtained by recrystallization from heptane. mp =51.5~52℃ Single element analysis: Calculation of Cl5H21NO2 (263,34) Value: C=68.42. H=8.04°N=5.32; Actual value C=68.22 ．． 8=8.26. N=5.29; [α]. ”=1.34°.

Soup E: S1'S--le 1, Eto C-1-1 　Syl　1, Nyl≧--Phenylethyl-dihydrofuran--3H-- d Dissolve 9.93 g of product 97 in step D in 100 mL of absolute ethanol, and 2.6 g of thorium and 20.1 mL of diethyl malonate were added to 170 ml of absolute ethanol. It was added to a solution prepared by dissolving L. - After stirring overnight, the reaction was diluted with 10% citric acid p)4 4 and extracted with 2×500 mL of ether.

The organic extracts were combined at Hz OI 　X　500. mL, washed with 500 mL of saturated brine IX, and desorbed with Mg5O< I watered it. The solvent was removed and the crude product was subjected to low pressure chromatography (silica gel, eluent Purified with 50% ether/hexane (or EtOAc/hexane) as agent did. The yield of semi-solid product was 10.6 g. Subsequent fractions are desirable It contained 2.5 g of the 5R isomer as a white solid.

Swoop F: 5S 1°S -3-Lepoet Sea 3-(4-Penjiruoji) Phenylmethyl -5-1-11-dimethylsilbonyl ≧) -2-ph phenyleyl-dihydrofuran-2-3H-one alpha disorder 1: (53,1' 5)-3-carboethoxy-5-[1-((1',1'-dimethy ethoxycarbonyl)amine)-7-phenylethyl)-dihydrofuran-2 -(3H)-one (product of step E>2 g (5,3 mmo 1)) in anhydrous Add 0.13 g of sodium to a stirred solution of 25 mL of ethanol in anhydrous ethanol. A solution prepared by dissolving 2.2 mL of alcohol, then 4-benzyloxybenzylchloro 1.30 g of lido (5.5 mmol I) was added. The solution was incubated for 1 hour under nitrogen atmosphere. After heating to 50°C for a while, it was cooled in a water bath and acidified with 20ml of 10% citric H. , diluted with 200 mL of water. The mixture was extracted with 100 mL of ether 3X, The combined tel extracts were washed with 50 mL of water and 200 mL of saturated NaHCOz, and the Mg Dehydrated with SO4. The solvent was removed under reduced pressure, and the Purified by low pressure chromatography using % ether as eluent and purified by TLC (50% % ether/hexane), almost homogeneous, colorless transparent glass 1.56 g (yield 51%) was obtained.

Suit 1G: 3R5S 1'S -3-4-benzyl siphenyl mail - 5-111-dimelethylbonyl a≧ノ-2-phenyleth Rusig 1.2- Dissolve in 250 mL of dimethoxyethane and add 1M lithium hydroxide to this solution at room temperature. 117rnL was added. After stirring for 12 hours, the solvent was removed under reduced pressure and the residue was reduced to 10% Suspend in 200 mL of Quen D and extract with 3 x 500 mL of diethyl ether did. The ether extracts were combined, washed with 500 mL of brine, and dehydrated. ) L, concentrated and dried. Dissolve the residue in 250 mL of toluene and heat under reflux for 12 hours. After that, the mixture was dried under reduced pressure. 15% ethyl acetate, 'hexane on silica gel was purified by medium pressure chromatography using as eluent to obtain 3.2 g of 3R-lactone. was obtained as a transparent foam. Further elution with the same solvent yielded 6.15 g of 3S-lactone. Obtained as a white solid.

Step H: N'-1-dimethylcarbonyl-5S-aceno4 S -1° 1°-Zimele Roux -Zimele Luci 1 Le・Si -6-Phenyl -2 (R-4-Pendyloxyphenylmer 1 Yue)” Obituary I-2 0.6 g of the product tm of step G was added to 2 ml of ethylene glycol dimethyl ether/water. :1 mixture in 30 mL, and add 5 mL of IM lithium hydroxide to this solution in the room. Added warm. After stirring for 1 hour, the mixture was dissolved in 200 mL of chloroform and 10% citric acid. One layer was separated between 20 ml of vi Extracted with L. The organic layers were combined and dehydrated (Nazso-), the solvent was removed and the crude 0.56 g of hydroxy acid was obtained. This residue was dissolved in 5 mL of dry DMF and tc 0.845 g of rt-butyldimethylsilyl chloride and 0.725 g of imidazole g was added. After stirring for 18 hours, the reaction was poured into 50 mL of water and 3×20 ethyl acetate was added. Extracted with mL. Combine the organic extracts and add 10% citric acid 3 x 20 mL, water I 20 mL, washed with 3 x 10 mL of a saturated aqueous solution of NaCO and 20 mL of brine. Ta. After dehydration (N a 2 S O 4), the solvent was removed and the resulting residue was dissolved in THF 5 mL, 5 mL of glacial acetic acid, and 2 mL of water. Stir the mixture for 4 hours. After stirring, the mixture was poured into 50 mL of water and extracted with 3×20 mL of ether. Ether drawing Combine the extracts, wash with 2 x 20 mL of water, brine, and dehydrate (Na! S O4 ') L, solvent was removed. Dissolve MeOH/CHCl3 on silica gel Purification was carried out by medium pressure chromatography as a releasing agent, and 0.60 g of the product was purified using white gas. Obtained as a lath-like solid.

Soup ■: -: Known racemic 1-amino- of -2-herosine From 2-hydroxyindane, 3-amino The resolution was carried out as described for -1,2-dihydroxyindan. High R, (Is, 2R)-1-amino-2-hydroxy derived from saponification of diastereomers Siindan is α. = -58° (c = 1, 0, CHCI y) It turned out that. Low R, derived from saponification of the diastereomer (IR,2S)- 1-amino-2-hydroxyindan is α. =+62° (c=1.0.CH C13).

Suitable J: N-2R-Hydro Sheen S-Nyl-S-1-Dimethyl EC Carbonyl Mino-4S-Hydro C 6- Enyl 2 R-4- Benzyl Diphenyl Hekayu” Hosei Takumi I-ni Dissolve 0.12 g of the product from step H in 2 ml of dry DMF and add 1 ml to this solution. (S)-Amino-2(R)-hydroxyindan (Step I) 40 mg, 1 -25 mg of hydroxybenzotriazole hydrate and dimethyl-3-(3-dimethyl 70 mg of (thylaminopropyl) carbodiimide hydrochloride was added. pH is Trimethylamine was added to the stirred solution until 8.5 (32 mL). under reduced pressure After stirring to concentrate and dry, the residue was dissolved in 100 mL of chloroform and diluted with 10% Citric [I The extract was extracted with 50 mL of X, dehydrated with Mg5O1, and concentrated to dryness. Tetra the residue IM tetrabutylammonium fluoride in THF was dissolved in 1 mL of hydrofuran. It was added to 2 mL of Orido. - After stirring overnight at room temperature, the reaction mixture was mixed with 10% citric acid (10ml) The white precipitate was collected by filtration. The product was diluted with 2% meth on silica gel. Purified by low pressure chromatography using Nor/CH2CI2 as eluent. , almost homogeneous product 8 by TLC (3% methanol/CHz Cl2) 5 mg was obtained.

To Suitup: N-2 R-Hero C I S-Danil 15S-11-D methylethylbonylamino)-48-hydroxy-6-phenyl-2R- To 4-hydrocyphenyl mel ≧1' to key I-2 Generation of step J? 585 mg in 10 mL of methanol and THFloml 10% palladium on carbon was dissolved in this solution. Added Log. Mixed The mixture was stirred at room temperature under a hydrogen atmosphere for 48 hours, then filtered and concentrated to dryness. heat the residue It was dissolved in 10 mL of ethanol, and 20 mL of water was added.

After cooling, the white solid precipitate was collected and dried under vacuum with P2O.

The yield was 72 mg (98% yield) of pure product. mp218-219℃ (21 Boiling and sintering at 5℃). Elemental analysis: C33H4ON+06 (560, 6961) Calculated value: C70,69, H7,19, H5,00 + Actual value C70,62゜shi The product N-(2( R)-hydroxy-1(S)-indanyl)-5(S)-[(1,1-dimethyl ethoxycarbonyl)amino]-4(S)-hydroxy-6=phenyl-2(R )-(4-hydroxyphenylmethyl)hexanamide (0.50 g, 0.9 mm ol), anhydrous cesium carbonate (1,0 g, 3 mmo+) and N-(2-chloroethyl ) Morpholine free base (2.35 g, 17 mmol) in anhydrous dioxane 10 The stirred mixture in 0 mL was heated to 80° C. (internal temperature) for 3 hours. After cooling to room temperature, mix The mixture was diluted with chloroform (50 mL), filtered, and concentrated to dryness under reduced pressure. was triturated with 50 ml of anhydrous ether and 10 ml of ethyl acetate. white solid formation The material was collected and dried under vacuum at P 20 s. Pure product L-689,502 Yield was 0.54 g (89%), mp 195-7°C.

Elemental analysis: C, IH5IN30? < 673, 856) calculated value C69 , 52, H7, 63, H6, 23; Actual value C69, 19, H7, 45, H6, 15゜Maleic acid hydrate: mpH2-113℃ (decomposition) 0 Elemental analysis: C3gH s+N　xo　t・C,H,○,・H20(807,946) calculation value C63, 92, H7, 11, H5, 20; Actual value C64, 23, H6, 94, H5, 1 0゜X1ヱ1-( A frozen vial (2,0 ml) is used to contain 250 ml of seed medium A. A 1-seed flask inoculated into a 1 m capacity shake flask with a baffle plate was placed in a rotary shaker (22 Orpm) for 24 hours at 27°C. 2.5ml of propagated seeds Use an aliquot to create a 250 ml volume block containing 8 50 ml of conversion medium. A plateless flask was inoculated and L-689,502 in DMSO was added to the fermentation product for 0 hours. was added to give a final concentration of 0.05 mg/ml. Then the shake flask The contents were incubated at 27°C on a rotary shaker for 4 days. Whole broth formed was extracted as described in Section B.

ArdamLne pH5,O NZ amine type E 5.0 M g S O4・7HiOO,05KtHPO,Ol3 pH adjusted to 7.1 Added to Cacos 0.5g/l The pH was adjusted to 7.0.

B, 13121 Ward” l [ The whole broth (400ml) was extracted three times (3x400ml) with 1-butanol.

The combined extracts were concentrated under vacuum to an oily residue. Dissolve the residue in methanol HPLC was subjected to high performance liquid chromatography (HPLC). atman Partisil 100DS-3゜9.4mmX25cm chamber It was carried out at room temperature and monitored at 215 nm. )1ffPO, 0.1% aqueous solution: C1-1 , CN=80:20 to 0.1% aqueous solution H=　P　O=　:　CHs　CN　 The column was developed for 60 minutes at 3 ml/min with a linear gradient of 20:80.

One sample was collected during repeated injections of the extract. E19.3 minute hula when held The solutions were pooled, adjusted to pH 6.5 and evaporated to remove acetonitrile. Ta. C+5SeP--Pak (Waters As5ociates) and meta The compound was further purified using alcohol-aqueous M solvent to yield 11 mg of product.

Elemental analysis: CstHs. Calculated value of o + oP N sN a 2: C58゜7 2, H6,27, H5,27, r'3.89; Actual value C5978, H6,58 ,H4,97,P4゜17゜ As a result of NMR1 elemental analysis and MS, it was found to have the following tlI structure.

L-696,432 Phosphorus was identified and quantified by titration and heavy electric analysis.

According to NMR, C and H are 0 relative to the chemical shift in parent L-689,502. ．． The phosphorylation of 04 was verified from the fact that it shifted to the lower magnetic field side by 5 ppm.

Embarrassing day-L Shi-94 Microbially transformed metabolite L-696,432 is an extremely potent HIV protease inhibitor. It turned out to be a phosphoric acid ester of bitter L-689,502. Rin To determine whether acid ester bonds can be cleaved enzymatically, alkaline phosphorus Fatase treatment was performed.

Bacterial alkaline phosphatide was added to 0.5 ml of water containing 0.8 mg of L-696,432. Add 30μ+ (0.31 units/μm) of atase and incubate the reaction mixture at 37°C. and monitored by HPLC. After 4 hours incubation, The completed reaction product was purified by HPLC and subjected to FAB MS analysis. . [M+1-1]+ ion was observed at m/z 674. m/z574, The product from the fragmentation ions of 556.469 and 425 -68 It was confirmed that the number was 9,502.

As a result, L-696,432 was converted to the parent compound ThL-689 by enzymatic hydrolysis. It was shown that it can be reconverted to 502.

Large” 113 yu HIV Prose Assay 'H peptide substrate [CoH]-]acetyl-Val-Ser-GinAsn -(β-naphthylAla)-Pr.

-I Ie-Val-Gln-Gly-Arg-Arg-N+1. (MW=18 00) was used to conduct an inhibition test on the reaction of HIV protease expressed in E. coli. provided. The two arginine residues at the amine terminus give this peptide an acidic I) 11 Gives a positive charge to the whole, DOWEX AG-50Vl-X8 resin and similar resins can be attached to the H+ form of .

HI V protease cleaves between β-naphthyl-Ala and proline residues. , the product is neutral or slightly negatively charged and is not bound to the cation crosslinking resin [' H]-acetyl-Val-3er-GIr+-Asn-(β-naphthyl-AIa ) is generated. Therefore, it is possible to appropriately separate the labeled product from the substrate.

Assay buffer (100mM sodium acetate, pH 5.5 and 0.1% BSA) Inside 6. C1-8, 25 μl aliquot containing OnM l-I IV protease. The recoat was placed in a test tube. 4.2μ in 100mM sodium acetate pH 5.5 The reaction was started by adding a 25 μl aliquot of M'H peptide substrate.

After incubation for 60 minutes at 37°C, 5% H, P0. React with 100μl It was stopped and then analyzed by application of column chromatography.

L-696,432 exhibits 32% inhibition in substantially purified form at a concentration of 1 μg/l. The damage rate was shown.

Support 11LL L-68902's 1(S)-Ami of 7L-696 Step I 1(S)-amino-2(R)-a instead of no-2(R)-hydroxyindan N-(c i s-2(R)-acetoxy-1(S)indanyl)-5(S)-[1,1 -dimethylethoxycarbonylamino)-4(S)-hydroxy-6-phenyl -2(R)-[(4-(2-(4-morpholinyl)ethoxy)phenyl)methy ]hexanamide was prepared.

B, L-6642 Chambe rs, R, W, and H, G, Kh o r a na, J , Am, Chem, Soc, 80. According to the principles and methods of 3749 (1958) The product of step A was reacted with monophenylphosphorodichloridate. The acetyl and phenyl groups are then removed under basic conditions after treatment with excess ammonia. was removed to obtain L-696,432.

C, 1st class 1st class 5- Another phosphorylating agent is dibenzyl phosphorochloridate. phosphorylation and phosphoric acid For details on the curing agent, see Y, Mizuno, 5tudies in Org, Ch. em.

L et al., 171-175 (1986) and its cited references.

叉"Kyoichidan"- Rh1zo obtained from Merck Culture Co11ection arrhizus MF4974 in a 250 ml flask. 50 ml of bean-glucose medium was inoculated and the flask was placed on a rotary shaker at 220 rpm. After incubation at 27°C for 24 hours, a first stage seed culture was obtained. 50μl D Soybean-glucose similarly loaded with compound Z to a concentration of 5 μg/ml in MSO Add 2.5 ml of this seed culture to each of 18 flasks containing 50 ml of medium. inoculate and incubate the resulting culture for 18 hours at 27°C on a rotary shaker at 220 rpm. It was incubated.

After incubation, the contents of each flask are collected by centrifugation to kill the mycelium. Wash twice with bacterial saline and p) I'7.0 phosphate buffer containing 1% glycerol. resuspended in.

Compound Z was added to a final concentration of 58 μg/ml using 100 μ m DMSO. added. Incubate flasks on a rotary shaker at 220 rpm for 48 hours at 27°C. I did it.

After two incubation periods, the contents of the 18 flasks (900 ml) were plated. The whole broth was then centrifuged.

Slurry the mycelium with 100ml of water, adjust the pH to 3.5, and make the slurry. Extracted twice with 100 ml of n-butanol. Acidify the supernatant to pH 3.5 for 2 min. Extracted twice with 1 volume of 0-butanol. Each organic extract was analyzed by HPLC. Said. Assay conditions are as follows.

Column: “ZORBAX” (DuPont) C8Rx 4.5x250mm Mobile phase Niacetonium) IJ Lu + 10ml 1’l KH2PO4 aqueous solution 20%~ 20 minutes at 80% gradient Temperature: 45℃ Flow 1: 1.5ml/min Detection: 210n theory Sample volume: 50μl.

The mycelium extract and supernatant extract were pooled and evaporated to dryness under reduced pressure at 30°C. An oil was obtained as a liquid.

The oil was dissolved in a 40/60 acetonitrile/water mobile phase and added to ZORBAX C Further purification was performed using a 3 (9.6 mm x 25 cm) semi-preparative column. 0.1% 4 using a 40% acetonitrile aqueous solution containing lifluoroacetic acid (TFA). The column was developed at 7.05 ml/min at 5°C.

Fractions with a retention time of 128 min were pooled, the solvent was evaporated, and compound I 12 mg (yield 24%) of II was obtained.

The product had mass spectral data as described above.

A portion of the product was converted to monopotassium salt. This is 10m　M　K　Hz　P Biological solution was added to a 70% acetonitrile aqueous solution containing O + (P This was done by dissolving the oxidation products. Reduce the mixture to acetonitrile under reduced pressure. and the aqueous residue was loaded onto a water equilibrated C-18 solid phase extraction column. column Wash with water and elute with 70% acetonitrile aqueous solution, keeping the eluate in a salt state. Lyophilized.

Dai 101 11”- MF 4 stored in Merck Culture Co11ection Rh1zo us arrhizus from oatmeal agar slant cultures of 974 spores were obtained and used to prepare approximately 7X10g spores/ A spore suspension in water containing m1 spores was prepared.

The spores were placed in seed flasks each containing 500 ml of soybean glucose broth of the above composition. Inoculate 1 ml of the suspension and incubate at 27°C for 24 hours in a 5-revolution shaker (22 Orpm). Incubated.

After incubation, mycelia from each flask were separated with a 10 μm nylon mesh. Collected by filtration and made an equal volume of 100mM containing 3% glycerol. Resuspend in pH 6.3 phosphorus u% solution and add to broth. Compound Z with dimethyl sulfo It was added at a concentration of approximately 50 μg/m + oxidation width.

The flask was then inked for 24 hours at 27°C on a rotary shaker at 220 rp + n. It was incubated.

After the end of the incubation period, the contents of the 16 flasks totaled 8000 m 1 were pooled and passed through a 10 μm nylon mesh. Mycelium cake with 50% water The slurry was made into a slurry with 10,100 liters of methanol and filtered. Aqueous mycelium cake again Extract with methanol, combine the two aqueous methanol P solutions, and dilute with 2000ml of water. I interpreted it.

220mm bed height The obtained aqueous solution was added to a 15 mm x 300 mm column packed in a column. liquid was added. Divide the r liquid into 2000ml each and pump it at a rate of 15ml/min using the forward flow method. I entered. After packing, the column was washed with 500 ml of water to remove 20% of the desired phosphorylated product. Elution was carried out with 500 ml of aqueous acetonitrile. Remove residual substrates and metabolites with 70% aqueous aqueous solution. Elution was performed with 500 ml of setonitrile.

The diluted solution from the mycelial extract was also pumped into the column, and after washing the column, the solution layer was , the eluate) was assayed by IPLC. A/Say conditions are as described in Example 2o. I obeyed.

HPLC is used for microbial phosphorylation and “DIAION” HP20 isolation, and the following The material balance is shown below.

Substrate loading 1: 402.5mg Metabolite and substrate recovery amount: 383.8mg total recovery rate, 95.4% Kajimono III: 234.9mg Biological conversion yield, 61.2%.

The eluate is concentrated under reduced pressure and contains 10 mM KH, PO. 40? aqueous The residue was dissolved in acetonitrile to obtain the monopotassium salt. Acetate the aqueous solution under reduced pressure. The tonitrile was removed and the aqueous residue was loaded onto a C-18 solid phase extraction column equilibrated with water. Ta. After that, the column was washed with water, and the monopotassium salt of compound III was dissolved in 70-6 acetonate. Elution was carried out with aqueous tolyl solution. The eluate was lyophilized to obtain the salt. NMR spectrum of this salt Kutle is a Shino that has already been detailed.

The salt was converted to the acid by careful acidification.

111 support Reacting the appropriate phosphate with compound III as described in Example 21, The following salts were prepared by concentrating:

X Kyousen〇L1　L rlJa　PO(OR>(ONa) rllb PO (0) 1) (OK> 11 re PO (ONg) 2 111d PO (OLi) (0) 1) Ice PO(0)1)(OMg)+72111f PO(OH)(ON(CH , N4).

Ten J eyes 1" 2" 1,000,111 compressed tabs each containing 500 mg of qgzrA were prepared below. Prepared from the composition below.

Bakago L thing-1 Compound 11 IA 500 Starch 750 Dibasic hydrated calcium phosphate 5000 Calcium stearate 2.5゜ Rry) The crushed ingredients were thoroughly mixed and made into p-granules with 10% starch paste. Granulation The material was dried and compressed into tablets.

mL top 1000 pieces of hard gelatin each containing 500 mg of the monosodium salt of the compound TTI Capsules were prepared from the following composition.

LiJ! L　L Compound III, monosodium salt 500 Starch 750 Dibasic hydrated calcium phosphate 5000 Calcium stearate 2.5゜ A homogeneous mixture of the ingredients is prepared and used to form two-part hard gelatin capsules. Filled the cell.

Composition of: Dextrose 12.5g Water 250mL Compound fill I, 1 Potassium salt 250 ml of injection solution containing 400 mg of conventional Prepared according to the procedure.

After blending the ingredients, it was sterilized and used.

! An aerosol composition can be prepared having the following composition.

m jin 1 to 1 Compound IT IA 24mg Lecithin NP concentrate 1.2mg Trichlorofluoromethane, NF 4.026g Dichlorodifluoromethane, N F12. L5gL11111 per liter cornstarch liquor 5g, D-mannitol 25g, glue 10g of course engineered hydrate, “PHARMAMEDIA” (non-hydrolyzable protein, Buckeye 0ilseed Products, Memphis, Te nn, >20g, Kl (zPO+ 9g, Fe50.7H+0 10mg, M n5O<・4020 10mg, CuCI2・2H200, 25mg, CaCI z・2HzO1mg, H3BO>0. 56mg, (NH<)sM oto 2*・I20 0.19mg, Zn5O<7Hz0 2mg group Zaleri into 54 ml of PF3-2 medium with

Inoculate a frozen vial of P. narboricola MF5533ATCC74030 Compound Z, which is the starting material, was prepared by The cells were incubated at 25'C with shaking for 40 minutes. using 20ntl and attached to a 4III 2-hole flask containing 500 ml of PF3-2 medium. The seeded and inoculated media were incubated at 22 Orpm for 40 minutes at 25°C. Hula Pool the Sco contents and add 180 liters of P3/l-2 medium to reduce foaming. 2 ml/L of propylene glycol P-2000 (Dow Che+ ++1 cal) were inoculated into three fermenters each containing , 25℃, airflow Ji 90L, z minutes, pressure 0.7kg 7'cm2 gauge 2 of the 0th order obtained broth incubated with and stirrer speed 20 Q r 1: + m Contains 2 ml,,'L of P-2000 using 5 Lindor Sun 7"/shi Inoculate three fermenters each containing 475 liters of PF3-2 medium; 4 days, 25℃, air flow rate 25OL, -'min, pressure 0.7kg/cm'' gauge and cultured at 150 rpm.

D-mannitol 100g, NZ-amicitib E (casein Ice melt, 5hefffield Products, Kraft, Inc.) 33g, Fi(l CO8005 Yeast Extract (Difco>1.0g, (N1 14) T G with a composition of 2SO45g-KH2PO19g, P20002ml 106 culture tl! 3111 production fermenters each containing 13,700 liters was inoculated with 425 liters of this seed broth and maintained at a temperature of 27°C and an air flow of 12,500 liters. liter 7'min, pressure Q, 7kg/cm2 gauge and stirrer ('l' speed 5orp) The fermenter was operated at m. After lowering the pH from 6.0 to 5.5, 5.5±3 maintained. After approximately 2.5 weeks, the broth was collected and the product isolated.

The broth from the above culture was extracted with an equal volume of methanol. Solid-liquid separator (centrifugal Clarify the methanol-broth using a separator to remove the first extract and solids. A clear liquid was obtained. The extraction-clarification process was repeated. Combine the extracts to reduce the water content to approx. 11 adjusted to 50%. The resulting solution was applied to a “DIAION” 5P-207 adsorption column. Compound III was adsorbed through the column and the column was washed with aqueous methanol. after that , Compound III was recovered with 100% methanol.

The water content of methanol containing the compound ThI I was adjusted to 50%, and the aqueous meth t&, the ethanol solution was intimately mixed with an equal volume of 1:1 ethyl acetate/hexane, and two The liquid phase was separated. Transfer the aqueous methanol layer to the column of ``DIAION'' 5P-207. Wash the column with aqueous methanol and remove compound III with 100% methanol. eluted. The eluate was concentrated in vacuo to a minimum volume and the solvent composition was adjusted to approximately 75:20:5 ethyl acetate. Adjusted to chill/methanol/water.

The feed thus prepared was passed through a silica gel column and compound III was added at 85:1 Elute with 0:5 ethyl acetate/methanol/water. More than 85% by HPLC Fractions showing areal purity were combined, concentrated in vacuo to remove ethyl acetate, and concentrated. The solution was adjusted to 50% aqueous methanol and added to “DIAION”H as described above. P-20, concentrated and compound elIII was precipitated with acetonitrile and vacuum- It was collected by filtration and then dried.

This starting material is similar to co-pending U.S. patent application Ser. Nos. 47/492.025 and 47 It can also be prepared by the method described in No./492,026.

Z, arboricola MF5533 ATCC74030 is pending at the same time No. 630.457, dated December 19, 1990, is claimed in U.S. Patent Application No. 630.457. ing.

In summary, this microorganism (a) (co-pending U.S. Patent Application No. 492.024) Z, arboricola ATCC 20957 cold The frozen mycelium was mixed with 5 g/l of co-stay liquor, 40 g/'l of tomato paste, Oat flour 10 g/l, glucose Log/1. Fe5o<・7Hz O10rng/l, Mn5On-482010mg/1-CuC1g・282 0 0.25mg/l, CaClz・2H201mg/I, HzBOz 0 ．． 56mg Kawa, (NH4) sMoto2<・) 1200, 19mg/l , inoculated into a KF seed medium with a composition of 202 mg of ZnSO47H/kawa, and inoculated into this medium. Add N-methyl-No-nitro-N-nitrosoguanidine (NTG), (b) (C) A portion of the enrichment was plated on potato dextrose agar. (d) incubate at 25°C for 14 days to obtain spores, then ( e) collecting the spores; (f) diluting the spores with sterile saline; and (g) collecting potato dextrose. Plate on Trose agar and (h) incubate for 7 days to develop colonies. (i) transfer distinct colonies to potato slants, and (j) incubate for 14 days 2 Obtained by incubating at 5°C.

Dai 1ba1su J- On the brink, six μ-fuki no ゛ Rh1zo us orν zac MF 49 cultured on oatmeal agar 74 spores in distilled + 20.0 g dextrose in 1 bottle, Fidco Consists of 5.0g of yeast extract, 5.0g of NaCl, and 5.0g of HPo. The soybean-glucose medium was inoculated. Before autoclaving, the pH of the medium was adjusted to 7. Adjusted to 0. The culture was incubated at 27°C for 24 hours on a rotary shaker (22Orpm). It was incubated. 24-hour seed culture at 5% (V/ The activity of 6-phosphorylated enzyme was inoculated at the ratio of V) and fermentation was continued as above. determined as a function.

Pour the cells into a buffer containing 100mM phosphate (p)17.5> and 2mM EDTA. A cell-free extract was obtained by suspending the cells. The ratio of cells to buffer is 2: It was 1.

The cell membrane is then ruptured and the cell contents released using several methods (i.e. high-pressure heating). Chipress, sonication, frozen cell crushing, and lysozyme treatment) were attempted.

Enzyme reaction: 0.15mM PK-520, 5mM MgC1□, 5gM ATP and in 02 ml of mixtures containing various amounts of enzyme. Various reaction mixtures Incubate for 1 hour at a temperature of The response was completed. The obtained solution was heated to 55°C using a Whatman Partisi+1 HPLC analysis was performed on a 00DS-3. 45% acetonitrile in 0.1% phosphoric acid 1 ml/ml using a linear gradient from 0.1% to 80% acetonitrile in 0.1% phosphoric acid. The column was developed for 30 minutes.

The retention times of phosphorylated FK-520 and FK-520 were 16.5 and 20 minutes, respectively. there were. Pierce BCA protein assay using BSA as standard Protein concentration was determined by reagent.

Phosphorase in cell-free extract of W foot izo us or zae MF4974 Upon investigating the base, the present inventors found that phosphorylation activity was 1) 2 in soybean-glucose medium. highest in cell extracts isolated after 4 h incubation; 2) enzyme Sound cells under a liquid nitrogen atmosphere to disrupt cell membranes to liberate soluble forms Both wave treatment and isolation can be used; 3) the optimum pH value is 75; 1) The optimum temperature is 37°C, 5) Phosphorylation of FK-520 is carried out for 1 hour at 02-2 ．． It was found to be linear with time and enzyme concentration at 0 mg protein.

■The effect of FK-52071 degree on the phosphorylation rate was investigated using the extract. . The Km value of the group [(FK-520) is about 0.5mM, and the Vmax is 2.1n It contained 7 mg protein. Determine metal ion requirements In order to The offspring were dialyzed with TA. As a result, the phosphorylation activity is enhanced with Mg'' or Ca as cofactors. ” was found to be necessary.

Support [ As a result of NMR analysis, 24-deoxy E, FR-900520 was converted into MF 4974 ( Rhizopus art-l+1zus). The resulting biological transformation product is a 32-phosphorylated analog tree: It has been proven that.

Example 28~・Same as described for 24-oxy FR900520 in two 24-deoxy FR900520 was added to Rh1zo usli & raw oxidized analogues by NMR spectroscopy. I got something that was judged to be true.

Tomoyoshishi Haruka 17-Eru 1-Hydro Sea 12-2°-3'11"-Jihi 20 Shishi Mouth He Jill -1°-Melvinyl -2325-Jimeshi 13 21 27- -Tone Roux 1128-Ji-4-1 Shiro 22.3.1.0'' Kosu 14 18-diene-231016--)-en 31 manufactured according to the procedure described in European Patent Publication No. 0 349 061 -desmethyl PK-900,520, i.e. 17-ethyl-1,14-dihydroxy C-12-[2’-(3”,4”-dihydroxycyclohexyl)-1°-methy rubinyl]-23,25-dimethoxy-13゜19.21.27-tetramethyl -11,28-dioxa-4-azatricyclo[22,3,1,0”]octa Stir in 8M (3 ml benzene) of After 25 minutes, the reaction mixture was cooled to room temperature. , saturated NaHCO, neutralized by adding aqueous solution and extracted with ethyl acetate (3 times). I put it out. The combined organic phases were washed with saturated NaCl solution, dried over Nalso, Flash chromatography (20% hexane and 1% MeOH in ethyl acetate) ) to obtain 40 mg of the title compound, MASS: (FAB) 782 ( nt+Na).

Partial 'H NMR (200mHz): δ6. 80 (dd, J+ = 1 6 Hz, J 2 = 6 Hz), 6, 1 6 ( dd, JI=16Hz, J2=1.5Hz), 4.39(broad d, J = 14Hz), 4.26 (bro a d d, J = 5H Z) , 3, 9 1 (d d, J + = 8, 8 Hz, J 2 = 3 Hz) - + m 17-E Lu 1-H'ro C--°-"4"-Dihydro Dicyclo Sill -1°-Metal Vinyl-2-Jime Sea 319127-Tone Ru 11 28-Di---Si17 Nithi-1-Hydroxy-1 2- [2’-(3”).

4″-dihydroxycyclohexyl)-1°-methylvinyl]-23.25-di Methoxy-13.19,21.27-tetramethyl-11,28-dioxa-4 -Azatricyclo[22,3,1,0''']octacos-14,18-diene- A solution of 2,3,10,16-titraone (40 mg in 1.5 ml of ethyl acetate) A hydrogen balloon was attached to a reaction flask containing 3 + n g of 5% Rh/carbon catalyst. Evacuate and refill with hydrogen gas (3 times). After 45 minutes, filter hot through Celite. , concentrated, flash chromatography (CH2Cl2:MeOH :hexane (10:1:2)) to obtain 33 mg of the title compound.

MASS: (FAB) 768 (m1Li).

Partial 'HNMR (200mHz): δ4.55 (broad d, J=5 ) [z), 4. 39 (broadd, J=14Hz), 3.86 (d d, J1=8°811Z, J2=3H2)- and 111th Phosphorus cocysterol L-706526L~706527 Simvastatin (chemical name + 6 (R) - [2-<8' (S) ~ 2'', 2''- Dimethylbutanoyloxy-2' (S).

6'(R)-dimethyl-1', 2', 6', 7', 8'.

8'a (R)-hexahydronaphthyl-1°(S)-ethyl]-4(R )-hydroxy-3,4,5,6-tetrahydro-2H-bilan-2-one) When contacted with the microorganism Rh1zous under conditions similar to those described in Example 3, Two phosphorylated compounds L-706,546 and L-706°527 were produced.

For the manufacturing method and properties of simvastatin, please refer to Merck & Co., Inc. European Patent Publication No. 0033538 of the same name.

The above compound simvastatin was added to the ink together with Rh1zo us arrhizus. As a result of NMR analysis of the biological conversion product obtained by cubating, the compound It was confirmed that the substance had the following structure.

L-706,526 and 706.52 These compounds are described in European Patent Publication No. 0 033 538 as simvastatin. Inhibition of cocysterol biosynthesis as described for.

Lord of NMR4:J:6L-706,526! 'Fe sign is rl! M Methyl Mie The line disappears, i, topp mouth + (CH)) and 3.93ppm (CH-0 ) with a new signal in CI, the presence of a portion of CH-0. 1. lOp When illuminated with pm q1, the methine quintet was reduced to a doublet. Methine proton in the proposed structure The fact that it is a doublet because it has no adjacent atoms has never been shown before. (i.e., phosphorus).

++ Phosphorylation at the -6' position is 3.69[, +pm typical) l-6° The characteristic of 11.26ppm attributed to 11-6° where the signal disappeared and moved. This is indicated by the presence of no broad signal. The amount of movement and loss of microstructure is This is appropriate as the result of oxidation.

SI L1LL There-Lennon's 1nl o u s a r r r I i z u s M Two biological transformation products were obtained. these products The substance is the same as α-zearalenol and α-zearalenol-6′-phosphate. The determined zero fermentation, isolation, purification and enzymatic hydrolysis are described below.

Exemption 1 ■! L-e and knee rhiz 1ml of us (MF 4974) spore suspension was added to 50ml of soybean-glucose medium. A 2 liter Erlenmeyer flask containing 0 mL was inoculated and placed on a rotary shaker (2 liters) at 27°C. The mixture was shaken at 20 I-pm for 24 hours.

Incubation r! , f! t Collect the contents of the flask by filtration and dilute once with water. Wash, then 500 mL or mM PO < It was resuspended in EX FR solution (pH 7,0). Use 0.8mL DMSO to Aralenone was added to a final concentration of 0.05 mg/mL. filled flask The cells were incubated for 24 hours at 27°C with rotary shaking (>220 rpm). After incubation, the whole broth was extracted as described below.

11 Lucose l standing 521” Yu Dextrose 20.0 Soybean meal 5.0 Fido yeast extract 5.0 NaCI 5.0 2) (Po, 5.0 The pH is 7.0! Adjustment.

uILa-bi' L number- After diluting the whole broth (1000+nL) with an equal volume of methanol, centrifuge it. did. The mycelium cake was discarded. HP20 (Mitsubtshi Chemi ca+, 220mm bed height, 15mm x filled with water) Clear R solution was added to the 30 cm column. Pour the r solution in the downward direction at 15 mL/min. A pump was installed in the ram. After loading, the column was washed with 500 mL of water. Consumed blocks Loss or wash? No metabolites or substrates were detected in the 'p product. Column with methanol Le: eluted with a step gradient of water. The most polar metabolite is 10% to 30% methanol. It was eluted with The second metabolite and substrate were column washes (100% methanol). It eluted. The appropriate fractions were combined and evaporated to dryness under reduced pressure at 30°C to give a yellow An oil was obtained which was further purified by preparative HPLC. What is preparative HPLC? man Magnum 9 Partisillo Ds-3 column (C+a, 9.8 mm IDX 25 cm) at room temperature and monitored at 237 nm. 0゜1 % 20% acetonitrile in phosphoric acid to 80% acetonitrile in 0.1% phosphoric acid The column was developed for 40 minutes at 4 mL/min using a linear gradient.

Compounds were collected during repeated injections of the extract. Retention time 17.5 minutes and 20 ．． Collect 1 minute fractions, pool, dilute with 3 volumes of water, 5 peed Desalt using a C+a column (Applied 5 separations), 7 mg of 6-phosphate ester and 5 mg of α-zearalenol were obtained, respectively.

11 summer source 1 To prove that the 6° α-hydroxyl group is phosphorylated, S i Using various sulfatase and alkaline phosphatase obtained from GMA Enzymatic hydrolysis was tested. In summary, ~50 μg 7’ mL of Zearale Specified for 1ml of 100mM phosphate buffer containing norphosphate amount of enzyme was added. The MWi solution was incubated at 37°C for 4 hours. incu After vation, take out 300 μL and add Whatman Partisil 1 Use 00DS-3 column (C+s 10μm, 4.6mm ID''-25cm). Analyzed by HPLC at room temperature and monitored at 237 nm. 0.1% phosphorus Linear gradient of 20% acetonitrile in acid - 80% acetonitrile in 0,1% phosphoric acid The column was developed for 20 minutes at 1 mL/min using α-zea under staining above Larenol-6'-phosphate ester and α-zearalenol were each 13.93 minutes and a retention time of 18.17 minutes.

The enzymatic hydrolysis results are summarized below.

LL　L±　jLL　11 Alkaline phos77tase 7.0 1.0100$a-OH@1 hour F'-55 21 Alkaline phosphatase 7.0 100.0100$α-0) 1@1 hour Fatase 7.0 1.0100$a-OH@1 hour Alkaline Phosphatase 7.0 10.0 6$a-OH@1 hour alkaline phosphatase 7.0 10.0 100$a-OH@1 hour sulfatase 5.0 100.0 5 2$a-OH @ 1 hour service 4 new- The above-mentioned compound was identified by MS and NMR, and enzymatic hydrolysis was performed. α-zearalenol-6°-phosphate and α-zearalenol I was sure that. Furthermore, the presence of phosphorus was confirmed by ICP elemental analysis.

IGvu2 international search report international search report Continuation of front page (31) Priority claim number: 595,894 (32) Priority date: October 1, 1990 1st (33) Priority claim country United States (US) (31) Priority claim number: 691,606 (32) Priority date: April 26, 1991 Japan (33) Priority claim country United States (US) (31) Priority claim number 691, 607 (32) Priority date April 26, 1991 (33) Priority claim country United States (U.S. S) (31) Priority claim number 701,387 (32) Priority date May 16, 1991 Japan (33) Priority claim country United States (US) (31) Priority claim number 735,963 (32) Priority date July 25, 1991 Japan (33) Priority claim country United States (US) (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, PR, GB, GR, IT, LU, NL, SE), C A, JP, US (72) Inventor: Pichutsch, Pryor R. United States, New Sci. Jersey 08518, Florence, Carriage Stop 4l-2 (72) Inventor Shu, Anthea Tee, USA, Ohio 4322 0, 4180 Landmore Court, Columbus (72) Inventor: Allison, Va. Elon A, New Chassis 07060, Watu-chun, USA. Century Lane 88 (72) Inventor Damon, Francis West Chi, Rahway, New Chassis 07065, United States erry street si (72) Inventor: White, Raymond F. New York, United States of America Sea 07726, English Town, Belakerat Road 12 (72) Inventor: Mother, Dipid G.A., New York, USA Sea 08817, Edison, Reading Road 19 Sea (72) Inventor Riemer, Robert A. New Chassis, USA - 114 Maoris Avenue, Bloomfield, 07003 Continuation of front page (72) Inventor Wu, Jian T. New Chassis, USA 5 Black Birch Road, Scotch Plains 07076 (72) Inventor Thor, Reidea T. New Chassis, United States of America Prospect Ave, Maylarado, 07607

Claims (52)

[Claims] 1. Biologically phosphorylated hydroxyl groups whose hydroxyl groups are phosphate-reactive A method for producing an organic compound containing a sil group, the method comprising biologically phosphorylated hydrogen. a carbon nutrient source for a sufficient period of time to produce organic compounds containing droxyl groups. Rhizous oryzae microorganism strain at room temperature in an aqueous medium containing A method comprising the step of contacting with a roxy-containing organic compound. 2. The microorganism is in a phosphate buffer containing glycerol as a carbon nutrient source. Claim 1, characterized in that it consists of resting cells of Rhizous oryzae. Method described. 3. At room temperature under submerged aerobic fermentation conditions in an aqueous carbohydrate medium containing a nitrogen nutrient source. the Rhiz in the presence of the hydroxyl-containing organic compound at a pH of less than about 8.0. The method according to claim 1, characterized in that P. ous oryzae is cultured. 4. The Rhizous oryzae strain is ATCC No. This is 11145 The method according to claim 1, characterized in that: 5. Immunology identified as a C-32 phosphorylated derivative of FK-506 type macrolide A method for producing a C-32 phosphorylated FK-506 type macrolide immunosuppressant. room temperature in an aqueous medium containing a carbon nutrient source for a sufficient time to produce an inhibitory agent. Rhizous oryzae microbial strain containing a free C-32 hydroxy group in A method characterized in that the method comprises the step of contacting with a FK-506 type macrolide. . 6. It is characterized by being a C-32 phosphorylated derivative of FK-506 type macrolide. Compounds that do. 7. formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼I (In the formula, R is H, C1-C4 alkyl, R1 is H2PO4, and R2 is hydrogen, hydroxy or lower alkanoyloxy; R3 is methyl, ethyl, Propyl or allyl, n is an integer of 1 or 2, seed cotton and the parallel part of the broken line 2 represents a single bond or a double bond) and its pharmaceutically acceptable salts . 8. FK-506, FK-520, FK-523, FK-525, C-31 death Methyl FK-506, C-32 phosphorylation induction of C-31 desmethyl FK-520 8. A compound according to claim 7, characterized in that it is a compound. 9. formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ The compound according to claim 7, characterized in that it is represented by: 10. formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ The compound according to claim 7, characterized in that it is represented by: 11. formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ The compound according to claim 7, characterized in that it is represented by: 12. formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ The compound according to claim 7, characterized in that it is represented by: 13. formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ The compound according to claim 7, characterized in that it is represented by: 14. formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ The compound according to claim 7, characterized in that it is represented by: 15. formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ The compound according to claim 7, characterized in that it is represented by: 16. a pharmaceutical carrier and a therapeutically effective amount of a compound according to claim 7. A pharmaceutical composition for the treatment of an immunomodulatory disorder or disease. 17. a pharmaceutical carrier and a therapeutically effective amount of a compound according to claim 7. Topical treatment of skin manifestations of inflammatory and hyperproliferative skin diseases or immune-mediated diseases with Pharmaceutical composition for use. 18. Administering an effective amount of a compound according to claim 7 to a mammalian species in need of treatment. A method for treating an immunomodulatory disorder or disease, characterized by: 19. Production of immunosuppressants identified as phosphorylated lavamycin-type macrolides A method for producing a C-43 phosphorylated lavamycin macrolide immunosuppressant. Rhizopus at room temperature in an aqueous medium containing a carbon nutrient source for sufficient time to contacting the S. soryzae symptomatic strain with lavamycin macrolide. A method characterized by: 20. The organisms are placed in a phosphate buffer containing glycerol as a carbon nutrient source. Claim 19, characterized in that the cells are resting cells of Rhizopus oryzae. The method described in. 21. The aqueous medium containing the phosphorylated macrolide positively inhibits T cell activation. 20. A method according to claim 19, characterized in that it shows: 22. At room temperature under submerged aerobic fermentation conditions in an aqueous carbohydrate medium containing a nitrogen nutrient source. the Rhiz in the presence of the lavamycin macrolide at a pH of about 8.0 or less. The method according to claim 19, characterized in that Opus oryzae is cultured. 23. Structural formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R=[HO]2▲There are mathematical formulas, chemical formulas, tables, etc.▼) Phosphorylated macrolides. 24. In addition to showing the characteristic proton NMR spectrum as shown in Figure 3, T cell proliferation Phosphate showing reversible positive inhibition of T cell proliferation by recombinant human IL-2 in assay cation macrolide. 25. A phosphorylated macrolide having a structural formula as shown in FIG. 26. formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ A phosphorylated cyclic ribopeptide compound having the formula (wherein R is -P(OH)2) It is a manufacturing method, and there are formulas: ▲mathematical formulas, chemical formulas, tables, etc.▼ and a phosphate and maintained at a pH range of about 6.0 to 6.3. Rhizopusarrhizus ATCC 11145 was cultured in a nutrient medium containing A method characterized by nurturing. 27. characterized in that the content of phosphate is at least 10% by weight of solids 27. The method of claim 26. 28. The composition of the medium is glucose 20.0g/1, soybean meal 5.0g/1, Fid. co yeast extract 5.0g/1, sodium chloride 5.0g/1 and potassium hydrogen phosphate 27. The method according to claim 26, wherein the pH is 5.0 g/1. 29. 220 rpm to 40 for 24 hours to about 2 days at a temperature range of about 15°C to about 30°C 27. The culture according to claim 26, characterized in that the culture is carried out with stirring in a range of 0 rpm. Method. 30. formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ selectively phosphorylates compounds with 4-hydroxyproline hydroxyl position. 2. A method for producing a compound containing 40 to 60 μg/ml of the compound and Lucose 20.0g/1, soybean meal 5.0g/1, Fidco yeast extract 5.0 g/1, sodium chloride 5.0 g/1 and potassium hydrogen phosphate 5.0 g/1, p Rhizous arrhizus in soybean glucose medium with the composition of H5 A method characterized by culturing ATCC 11145. 31. Peptide skeleton related to echinocandins and carrying several hydroxy groups Rhizopus arrhizus ATCC1 Phosphorylated cyclic ribopeptides obtained by biological phosphorylation with 1145 a compound in which the phosphate group in the phosphorylated cyclic ribopeptide is It is characterized by being bonded to the hydroxyl group of the 4-hydroxyproline component of Tido. Compounds that do. 32. formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R is ▲a mathematical formula, chemical formula, table, etc.▼or its cation salt) A phosphorylated cyclic ribopeptide compound with 33. Claim 3 characterized in that R is ▲There are mathematical formulas, chemical formulas, tables, etc.▼ 2. The compound described in 2. 34. Claim 3 characterized in that R is ▲There are mathematical formulas, chemical formulas, tables, etc.▼ 2. The compound described in 2. 35. Claim 3 characterized in that R is ▲There are mathematical formulas, chemical formulas, tables, etc.▼ 2. The compound described in 2. 36. containing a compound according to claim 32 together with a pharmaceutically acceptable carrier. Composition. 37. Administering an antifungal effective amount of a compound according to claim 32. A method to protect against fungal growth. 38. administering to a mammal an anti-infective or therapeutic amount of a compound according to claim 32; Pneumocystis carinii in mammals, characterized by Methods for treating or preventing infection. 39. formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ A compound represented by: or a pharmaceutically acceptable salt, hydrate or ester thereof. 40. (a) a certain amount of N-(2(R)-hydroxy-1(S)-indanyl)- 5(S)-(1,1-dimethylethoxycarbonylamino)-4(S)-hydro xy-2(R)-[(4-(2-(4-morpholino)ethoxy)phenyl)pro preparing 2-en-1-yl]-6-cyclohexylhexanamide; , (b) in a microbial culture of Rhizous arrhizus MF4974. incubating the compound of step (a); and (c) as claimed in claim 39. and isolating the compound according to claim 39. manufacturing method. 41. comprising an effective amount of a compound according to claim 39 and a pharmaceutically acceptable carrier. A pharmaceutical composition useful for inhibiting HIV protease, comprising: 42. comprising an effective amount of a compound according to claim 39 and a pharmaceutically acceptable carrier. have. For prevention or treatment of HIV infection or treatment of AIDS or ARC Useful pharmaceutical compositions. 43. administering an effective amount of a compound according to claim 39 to a suitable mammal in need of treatment. A method for inhibiting HIV protease, which method comprises administering. 44. administering an effective amount of a compound according to claim 39 to a suitable mammal in need of treatment. Prevention or treatment of HIV infection or AIDS or is a treatment method for ARC. 45. formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ A compound represented by: or a pharmaceutically acceptable salt, hydrate or ester thereof. 46. (a) a certain amount of N-(2(R)-hydroxy-1(S)-indanyl)- 5(S)-(1,1-dimethylethoxycarbonylamino)-4(S)-hydro xy-6-phenyl-2(R)-[(4-(2-(4-morpholino)ethoxy) (b) preparing Rhizopus phenyl)methyl]hexanamide; arrhizus MF4974 in a microbial culture of step (a). (c) isolating the compound of claim 45. 46. A method for producing the compound according to claim 45, comprising: 47. comprising an effective amount of a compound according to claim 45 and a pharmaceutically acceptable carrier. A pharmaceutical composition useful for inhibiting HIV protease, comprising: 48. comprising an effective amount of a compound according to claim 45 and a pharmaceutically acceptable carrier. For the prevention or treatment of HIV infection or the treatment of AIDS or ARC, Useful pharmaceutical compositions. 49. characterized in that an effective amount of the compound according to claim 45 is administered to a mammal. A method for inhibiting HIV protease. 50. characterized in that an effective amount of the compound according to claim 45 is administered to a mammal. A method for preventing or treating HIV infection or treating AIDS or ARC. 51. Structure below: ▲There are mathematical formulas, chemical formulas, tables, etc.▼L-706,526or ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ Phosphorylated shim represented by L-706,527 Vastatin derivative. 52. Structure below: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ A phosphorylated zearalenol compound represented by