WO1992006992A2 - New process for biophosphorylating organic compounds - Google Patents

New process for biophosphorylating organic compounds Download PDF

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Publication number
WO1992006992A2
WO1992006992A2 PCT/US1991/006816 US9106816W WO9206992A2 WO 1992006992 A2 WO1992006992 A2 WO 1992006992A2 US 9106816 W US9106816 W US 9106816W WO 9206992 A2 WO9206992 A2 WO 9206992A2
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Prior art keywords
compound
phosphorylated
hydroxy
macrolide
hiv
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PCT/US1991/006816
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French (fr)
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WO1992006992A3 (en
Inventor
Shieh-Shung Tom Chen
Brian R. Petuch
Annjia T. Hsu
Byron H. Arison
Francis Dumont
Raymond F. White
David J. Mathre
Jane T. Wu
Lydia T. So
Robert A. Reamer
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Merck & Co., Inc.
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Priority claimed from US07/691,606 external-priority patent/US5198421A/en
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to JP4501234A priority Critical patent/JPH06502536A/en
Publication of WO1992006992A2 publication Critical patent/WO1992006992A2/en
Publication of WO1992006992A3 publication Critical patent/WO1992006992A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P9/00—Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/117—Esters of phosphoric acids with cycloaliphatic alcohols
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6527—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07F9/6533—Six-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
    • C07F9/65525—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a seven-(or more) membered ring
    • C07F9/65527—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a seven-(or more) membered ring condensed with carbocyclic rings or carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/01—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing oxygen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a new regio- specific biophosphorylation process for producing phosphorylated derivatives of "phosphate active" hydroxyl containing organic compounds utilizing the microorganism Rhizopus oryzae. ATCC No. 11145.
  • the process involves contacting the microorganism and an organic compound containing a free phosphate active hydroxyl group, under biotransformation conditions, which phosphorylate the hydroxyl group.
  • the process involves either resting Rhizopus cells or culturing the microorganism in the presence of the organic compound.
  • Rhizopus oryzae ATCC No. 11145 is known in the art primarily as a hydroxylating agent. It also degrades insect moulting hormones (J.C.S. Chem. Comm. 1974: 656-657, 1974); participates in hydroxylation of steroids (Can, J, Chem. 57:436-440 and 1585-1587, 1979; ibid., 59; 1651-1655, 1981; ibid., 63: 1127- 1131, 1985; H.J. Peppier, ed., Microbial Technology Reinhold, New York, p. 288-297, 1967; U.S.
  • Patent 2,646,370 is involved with transformations of sesquiterpene lactone costunolide (J.C.S, Perkin 1: 3022-3028, 1979); produces 16-hydroxyverrucarin A and B and 3'-hydroxyverrucarin A by transformation of verrucarins A and B (Appl. Environ, Microbiol, 46:480-483, 1983); and produces OH-products of sesquiterpene lactone costunolide (J.C.S, Perkin 1: 3022-3028, 1979); produces 16-hydroxyverrucarin A and B and 3'-hydroxyverrucarin A by transformation of verrucarins A and B (Appl. Environ, Microbiol, 46:480-483, 1983); and produces OH-products of sesquiterpene lactone costunolide (J.C.S, Perkin 1: 3022-3028, 1979); produces 16-hydroxyverrucarin A and B and 3'-hydroxyverrucarin A by transformation of verrucarins A and B (Appl.
  • Rhizopus is a genus of fungi which commonly occurs on mature fruits, grain and vegetables, as well as soil. Typically they are saprobes and facultative parasites, and form a branched, aseptate mycelium. Rhizopus spp are used commercially, e.g., in the preparation of carboxylic acids or steroids, or in the metabolism of hydrocarbons. Culture or fermentation of Rhizopus arrhizus ATCC 11145 is straightforward, conventional and well known. See, for example, U.S. 4,410,629. Adaptations, modifications and variations of the protocol are given in the Examples are within the routine skill of the fermentation microbiologist.
  • Regiospecific biophosphorylating agents are desired in the art since standard chemical phosphorylation using, e.g., POCI 3 or PCI 5 , are generally non-regiospecific and do not produce selectively phosphorylated compounds, which can lead to improved water solubility and pharmacokinetics.
  • a sample of MF 4974 has been deposited at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville MD 20852.
  • the culture access designation is ATCC No. 11145.
  • Figure 1 is an 1 H nuclear magnetic resonance (NMR) spectrum taken at 400 MHz of C-32 phosphorylated FR-900520 in CDCI 3 .
  • Figure 2 exhibits the assigned molecular structure for C-32 phosphorylated FR-900520.
  • Figure 3 is % nuclear magnetic resonance (NMR) spectrum taken at 400 MHz of the C-43
  • Figure 4 exhibits the assigned molecular structure for the C-43 phosphorylated rapamycin macrolide.
  • Figure 5 is 1 H nuclear magnetic resonance spectrum of echinocandin IIIA.
  • phosphate active hydroxyl containing organic compound as used herein is meant a compound containing a hydroxyl group which can stereo- specifically interact with the Rhizopus microorganism to undergo phosphorylation under biotransformation conditions .
  • a simple test with the organic compound under the biotransformation conditions described herein will determine, without undue experimentation, if the hydroxyl group in the organic molecule is phosphate active.
  • the process involves contacting resting
  • Rhizopus cells in a phosphate buffered medium containing e.g. glycerol, as a carbon nutrient, or by the fermentation of the microorganism Rhizopus oryzae, ATCC No. 11145 together, in the presence of the hydroxyl containing organic compound, e.g., an FK-506 type macrolide immunosuppressant i.e. FK-520, under submerged aerobic conditions in an aqueous carbohydrate medium, containing a nitrogen nutrient, said conditions being conducted at a pH of about 7 for a sufficient time, e.g. 24 hours at 27oC, to selectively biophosphorylate the phosphate active hydroxyl group. Either process can be utilized but preferred is the process using resting cells.
  • the hydroxyl containing organic compound e.g., an FK-506 type macrolide immunosuppressant i.e. FK-520
  • FK-506 type macrolide immunosuppressant i.e. FK-520
  • Rhizopus oryzae ATCC 11145 phosphorylation by Rhizopus oryzae ATCC 11145 is not limited to that particular strain. Rather, other Rhizopus oryzae strains can also be expected to be capable of acting to perform hydroxyl phosphorylation of hydroxy containing organic compounds.
  • a process for producing a biophosphorylated hydroxyl containing organic compound, wherein said hydroxyl group is phosphate reactive comprising the step of contacting a strain of Rhizopus oryzae ATCC No. 11145 microorganism together with the hydroxyl containing organic compound in an aqueous medium containing a carbon nutrient at ambient temperature for a sufficient time to produce the biophosphorylated hydroxyl containing organic compound.
  • the present invention involves a biotransformation process, which involves the contacting of resting cells, or the fermentation of, the microorganism, Rhizopus oryzae, together with an organic compound containing a free hydroxy group, to produce the phosphorylated derivative.
  • the microorganism is currently on deposit with the American Type Culture Collection, 12301 Parklawn Drive in Rockville,
  • phosphate reactive hydroxyl containing organic compounds includes a C-32 hydroxy-containing macrolide as described in Fujisawa's USP 4,894,366 of the formula:
  • R is H, C 1 -C 4 alkyl
  • R 2 is hydrogen, hydroxy or lower alkanoyloxy
  • R 3 is methyl, ethyl, propyl or allyl
  • n is an integer of 1 or 2, and the symbol of a line and dotted line is a single bond or a double bond, and a pharmaceutically acceptable basic salt thereof. Specifically included is where the compound FK-506 is where R is methyl, R 2 is hydroxy, R 3 is allyl, n is 2 and the double bond is absent; and the compound FK-520 is where R is methyl, R 2 is hydroxy, R 3 is ethyl, n is 2 and the double bond is absent.
  • phosphate reactive hydroxyl containing compounds including the following FK-506 type compounds (from USP 4,894,366 to Fujisawa): 7-Allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo- [22.3.1.0 4,9 ]octacos-18-ene-2,3,10,16-tetraone, 1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)- 1-methylvinyl]-23,25-dimethoxy-13,19,17,21,27-penta- methyl-11,28-dioxa-4-azatricyclo[22.3.1.0 4 ,9 ]octacos- 18-ene-2,3,10,16-tetraone, 16-Allyl-1
  • a) represent two vicinal hydrogen atoms, or b) form a second bond between the vicinal carbon atoms to which they are attached; in addition to it significance above,
  • R 2 can represent an C 1 -C 10 alkyl group
  • R 8 and R 9 independently represent H or OH
  • X represents 0, (H, OH), (H,H) or -CH 2 O-;
  • Y represents 0, (H, OH), (H,H), N-NR 11 R 12 or N-OR 13 wherein, R 11 and R 12 independently represent H, C 1 -C 10 alkyl, C 1 -C 10 aryl or tosyl, and
  • R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 22 and R 23 independently represent H or C 1 -C 10 alkyl;
  • R 20 independently represents 0, or it can independently represent (R 20 a,H); wherein R 20 a independently represents OH, O-C 1 -C 10 alkyl or OCH 2 OCH 2 CH 2 OCH 3 ; n is 1,2, or 3;
  • Y, R 10 and R 23 together with the carbon atoms to which they are attached, can represent a 5- or 6- membered N-, S- O- containing heterocyclic ring, which is saturated or unsaturated, and which can be substituted by one or more groups selected from C 1 -C 10 alkyl, hydroxyl, C 1 -C 10 alkyl substituted by one or more hydroxyl groups, O-C 1 -C 10 alkyl, benzyl and -CH 2 Se(C 6 H 5 );
  • R 9 represents OH
  • R 14 , R 15 , R 16 , R 17 , R 18 , R 19 and R 22 each represent methyl
  • R 20 a represents OCH 3
  • R 8 and R 23 each represent H
  • [R 3 and R 4 ] and [R 5 and R 6 ] each represent a carbon-carbon bond
  • R 2 , R 7 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R i7 , R 18 , R 19 , R 20 a, R 22 and R 23 comprise carbon-containing groups, those groups contain up to 10 carbon atoms, more preferably from 1 to 6, e.g., methyl or methoxyl.
  • each of R 14 , R 15 , R 16 , R 17 , R 18 , R 19 and R 22 represents methyl.
  • R 2 , R 7 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20a , R 22 and R 23 can comprise straight chain, branched and cyclic groups.
  • R 10 is allyl (i.e., allyl
  • prop-2-enyl propyl ethyl or methyl.
  • n 2;
  • R 7 is H or OH
  • R 1 and R 2 both represent H
  • X is preferably 0 or (H, OH);
  • R 20 a represents OH or OCH 3 ;
  • R 10 and R 23 together represent a N-,S- or O- containing heterocyclic ring, preferred is where that ring is five-membered, more preferably a pyrrole or tetrahydrofuran ring.
  • Preferred embodiments as products are C-32 phosphorylated FK-506, and C-32 phosphorylated FK-520,
  • the phosphorylated organic compound can be produced by either contacting resting cells of Rhizopus, in an aqueous buffered phosphate medium containing a carbon nutrient at ambient
  • aqueous nutrient medium containing sources of assimilable carbon and nitrogen, preferably under submerged aerobic conditions (e.g. shaking culture, submerged culture, etc.).
  • the aqueous medium is preferably maintained at a pH of about 7 at the initiation and termination (harvest) of the fermentation process. A higher pH leads to substantial and/or total loss of product.
  • the desired pH may be maintained by the use of a buffer such as morpholino- ethanesulfonic acid (MES), morpholinopropanesulfonic acid (MOPS), and the like, or by choice of nutrient materials which inherently possess buffering properties, such as production media described hereinbelow.
  • MES morpholino- ethanesulfonic acid
  • MOPS morpholinopropanesulfonic acid
  • the preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, xylose, galactose, glycerin, starch, dextrin, and the like.
  • Other sources which may be included are maltose, rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, and the like.
  • the preferred sources of nitrogen are yeast extract, meat extract, peptone, gluten meal, cottonseed meal, soybean meal and other vegetable meals (partially or totally defatted), casein hydrolysates, soybean hydrolysates and yeast hydrolysates, corn steep liquor, dried yeast, wheat germ, feather meal, peanut powder, distiller's solubles, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acids, and the like.
  • ammonium salts e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.
  • urea amino acids, and the like.
  • the carbon and nitrogen sources need not be used in their pure form, because less pure materials which contain traces of growth factors and considerable quantities of mineral nutrients, are also suitable for use.
  • the medium mineral salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, cobalt salts, and the like.
  • a defoaming agent such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone may be added.
  • the FK-520 starting material can be obtained by the fermentation of S. hygroscopicus var. ascomyceticus, ATCC No. 14891, as described in U.S. Patent 3,244,592, and by the fermentation of S. hygroscopicus subsp. yakushimaensis No. 7278, (to produce FR-900520, or "FK-520", and the other FK-506 type macrolides can be obtained by the processes) as described in EPO Publication No. 0184162 to Fujisawa, and PCT WO 89/05304 to Fisons, said above references hereby incorporated by reference for this particular purpose.
  • submerged aerobic culturel conditions are preferred therefor.
  • a shaking or surface culture in a flask or bottle is employed.
  • the vegetative form of the organism for inoculation in the production tanks in order to avoid growth lag in the process of production.
  • a vegetative inoculum of the organism by inoculating a relatively small quantity of culture medium with spores or mycelia of the organism produced in a "slant” and culturing said inoculated medium, also called the “seed medium”, and then to transfer the cultured vegetative inoculum aseptically to large tanks.
  • the fermentation medium in which the inoculum is produced, is substantially the same as or different from the medium utilized for the production of the phosphorylated organic compound and is generally autoclaved to sterilize the medium prior to inoculation.
  • the pH of the medium is generally adjusted to about 7.0 prior to the autoclaving step by suitable addition of an acid or base, preferably in the form of a buffering solution.
  • Agitation and aeration of the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor, by various pumping equipment or by the passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture.
  • the fermentation is usually conducted at a temperature between about 20oC and 40oC, preferably 25-35oC, for a period of about 10 hours to 24 hours, which may be varied according to fermentation conditions and scales.
  • the production cultures are incubated for about 24 hours at 27oC on a rotary shaker operating at 220 rpm, wherein the pH of the fermentation medium is maintained at 7.0 to harvest.
  • Preferred culturing/production media for carrying out the fermentation include the following media:
  • the produced phosphorylated organic compound can be recovered from the culture medium by conventional means which are commonly used for the recovery of other known biologically active substances.
  • the phosphorylated substance produced is found in the cultured mycelium and filtrate, and accordingly can be isolated and purified from the mycelium and the filtrate, which are obtained by filtering or centrifuging the cultured broth, by a conventional method such as concentration under reduced pressure, lyophilization, extraction with a conventional solvent, such as methanol and the like, pH adjustment, treatment with a conventional resin (e.g. anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g. activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), crystallization, recrystallization, and the like.
  • a preferred method is solvent extraction, particularly using methanol.
  • the phosphorylated organic compound obtained according to the resting cell or fermentation processes as explained above can be isolated and purified in a conventional manner, for example, extraction, precipitation, fractional crystallization, recrystallization, chromatography, and the like.
  • Suitable formulations of the material may also include conventional pharmaceutically acceptable biolabile esters of phosphorylated organic compound, formed via the hydroxy groups on the molecule, such as the acetate.
  • the phosphorylated organic compound, and particularly that of FK-520, of the present invention are water soluble possesses pharmacological activity such as immunosuppressive activity, antimicrobial activity, and the like, and therefore are useful for the treatment and prevention of the transplantation rejection of organs or tissues such as heart, kidney, liver, medulla ossium, skin, etc., graft-versus-host diseases by medulla ossium transplantation, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, and the like.
  • organs or tissues such as heart, kidney, liver, medulla ossium, skin, etc.
  • graft-versus-host diseases by medulla ossium transplantation autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematos
  • composition of this invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains the instant invention compounds, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications.
  • the active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use.
  • the carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form, and in addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used.
  • the active object compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of diseases.
  • a daily dose (calculated on the basis of a 70 kg man) of about 0.01-1000 mg, preferably 0.1-500 mg and more preferably 0.5-100 mg, of the active ingredient is generally given for treating diseases, and an average single dose of about 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250 mg and 500 mg is generally given for treating diseases, and an average single dose of about 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250 mg and 500 mg is generally
  • macrolide can be obtained via the present process by contacted resting Rhizopus cells in a phosphate buffered medium containing glycerol as a carbon nutrient, or by the fermentation of the microorganism Rhizopus oryzae, ATCC No. 11145 together, in the presence of the macrolide rapamycin, under submerged aerobic conditions in an aqueous carbohydrate medium, containing a nitrogen nutrient, said conditions being conducted at a pH of about 7 for a sufficient time, e.g. 24 hours at 27°C, to selectively C-43 phosphory- late the rapamycin type macrolide. Either process can be utilized but preferred is the process using resting cells.
  • the resultant C-43 phosphorylated macrolide exhibits immunosuppressant activity, similar to rapamycin i.e., inhibition of T-cell proliferation of mouse T lymphocytes stimulated with the combination of interleukin-2 plus PMA.
  • a positive sample in this assay will inhibit T-cell proliferation, as indicated by reduced tritiated thymidine uptake.
  • immunosuppressant identified as a phosphorylated macrolide comprising the step of contacting a strain of a Rhizopus microorganism capable of phosphorylating free hydroxy groups, e.g., Rhizopus oryzae, and specifically Rhizopus oryzae ATCC No. 11145, together with rapamycin macrolide (See U.S. Patent 3,929,992 for its preparartion), in an aqueous medium,
  • a new immunosuppressant being a phosphorylated macrolide which exhibits a proton nuclear magnetic resonance spectrum as identified in Figure 3, and a molecular weight of 993 as obtained by (FAB) mass spectrometry.
  • composition for the treatment of immunoregulatory disorders and diseases containing a therapeutically effective amount of the phosphorylated macrolide in combination with a pharmaceutically acceptable, substantially non-toxic carrier or excipient.
  • a method of use for treating a human host to prevent organ transplantation rejection i.e., heart, kidney, liver, lung, bone marrow, or for treating autoimmune diseases i.e., juvenile diabetes mellitus comprising administering to said host a therapeutically effective amount of the phosphorylated macrolide.
  • the present invention involves a biotransformation process, which involves the contacting of resting cells, or the fermentation of, the microorganism, Rhizopus oryzae, together with a rapamycin macrolide containing a free hydroxy group, to produce the phosphorylated macrolide derivative.
  • the microorganism is currently on deposit with the American Type Culture Collection, 12301 Parklawn Drive in
  • phosphorylated macrolide as used herein is meant the compound having a proton NMR spectrum as illustrated in Figure 3 (of the methylated derivative), a mass spectrum molecular ion of 993, and an assigned molecular structure of the formula:
  • the C-43 phosphorylated macrolide can be produced by either contacting resting cells, of Rhizopus in an aqueous buffered phosphate medium containing a carbon nutrient at ambient temperature, or by culturing (fermenting) the above-described microorganism, Rhizopus oryzae, in the presence of the known rapamycin macrolide, described in U.S. Patent 3,929,992, in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen, preferably under submerged aerobic conditions (e.g. shaking culture, submerged culture, etc.).
  • the aqueous medium is preferably maintained at a pH of about 7 at the initiation and termination (harvest) of the fermentation process. A higher pH leads to substantial and/ or total loss of product.
  • the desired pH may
  • MES morpholinoethanesulfonic acid
  • MOPS morpholino- propanesulfonic acid
  • the preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, xylose, galactose, glycerin, starch, dextrin, and the like.
  • Other sources which may be included are maltose, rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, and the like.
  • the preferred sources of nitrogen are yeast extract, meat extract, peptone, gluten meal, cottonseed meal, soybean meal and other vegetable meals (partially or totally defatted), casein hydrolysates, soybean hydrolysates and yeast hydrolysates, corn steep liquor, dried yeast, wheat germ, feather meal, peanut powder, distiller's solubles, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acids, and the like.
  • ammonium salts e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.
  • urea amino acids, and the like.
  • the carbon and nitrogen sources need not be used in their pure form, because less pure materials which contain traces of growth factors and considerable quantities of mineral nutrients, are also suitable for use.
  • the medium mineral salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, cobalt salts, and the like.
  • a defoaming agent such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone may be added.
  • the rapamycin starting material can be obtained by the known fermentation of S. hygroscopicus, NRRL No. 5491, as described in U.S. Patent 3,929,992.
  • submerged aerobic cultural conditions are preferred therefor.
  • a shaking or surface culture in a flask or bottle is employed.
  • the vegetative form of the organism for inoculation in the production tanks in order to avoid growth lag in the process of production.
  • a vegetative inoculum of the organism by inoculating a relatively small quantity of culture medium with spores or mycelia of the organism produced in a "slant” and culturing said inoculated medium, also called the “seed medium”, and then to transfer the cultured vegetative inoculum aseptically to large tanks.
  • the fermentation medium in which the inoculum is produced, is substantially the same as or different from the medium utilized for the production of the phosphorylated macrolide and is generally autoclaved to sterilize the medium prior to inoculation.
  • the pH of the medium is generally adjusted to about 7.0 prior to the autoclaving step by suitable addition of an acid or base, preferably in the form of a buffering solution.
  • Agitation and aeration of the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor, by various pumping equipment or by the passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture.
  • the fermentation is usually conducted at a temperature between about 20oC and 40oC, preferably 25-35oC, for a period of about 10 hours to 24 hours, which may be varied according to fermentation conditions and scales.
  • the production cultures are incubated for about 24 hours at 27oC on a rotary shaker operating at 220 rpm, wherein the pH of the fermentation medium is maintained at 7.0 to harvest.
  • Preferred culturing/production media for carrying out the fermentation include the following media: Seed and Transformation Medium g/L
  • the produced phosphorylated macrolide can be recovered from the culture medium by conventional means which are commonly used for the recovery of other known biologically active substances.
  • the phosphorylated macrolide produced is found in the cultured mycelium and filtrate, and accordingly can be isolated and purified from the mycelium and the filtrate, which are obtained by filtering or centrifuging the cultured broth, by a conventional method such as concentration under reduced pressure, lyophilization, extraction with a conventional solvent, such as methanol and the like, pH adjustment, treatment with a conventional resin (e.g.anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g. activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), crystallization, recrystallization, and the like.
  • a preferred method is solvent
  • the product phosphorylated macrolide from the fermentation exhibits positive immunosuppressive activity by the "T-cell proliferation assay” and possesses utility on this basis and exhibits the following physical characteristics:
  • the phosphorylated macrolide obtained according to the resting cell or fermentation processes as explained above can be isolated and purified in a conventional manner, for example, extraction, precipitation, fractional crystallization, recrystallization, chromatography, and the like.
  • Suitable formulations of the material may also include conventional pharmaceutically acceptable biolabile esters of the phosphorylated macrolide, formed via the hydroxy groups on the molecule, such as the acetate.
  • the phosphorylated macrolide of the present invention possesses pharmacological activity such as immunosuppressive activity, antimicrobial activity, and the like, and therefore are useful for the
  • organs or tissues such as heart, kidney, liver, medulla ossium, skin, etc.
  • graft-versus-host diseases by medulla ossium transplantation autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, and the like.
  • composition of this invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains the instant invention compounds, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications.
  • the active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use.
  • the carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form, and in addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used.
  • the active object compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of diseases.
  • a daily dose (calculated on the basis of a 70 kg man) of about 0.01-1000 mg, preferably 0.1-500 mg and more preferably 0.5-100 mg, of the active ingredient is generally given for treating diseases, and an average single dose of about 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250 mg and 500 mg is generally given for treating diseases, and an average single dose of about 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250 mg and 500 mg is generally
  • the present invention process also is applicable in a process for selectively
  • Echinocandins or echinocandin compounds are cyclohexapeptide compounds having a lipophilic side chain and having antifungal properties. Many are natural products but many compounds are
  • the present invention is especially directed to a compound having the formula (III):
  • R is - (OH) 2 or a cation salt thereof.
  • cation salt is meant a salt of Li, K, Mg, Na, Ca, and (C 1 -C 4 alkyl)ammonium.
  • R is - (OH) 2
  • the compound may be represented by formula (IIIA), and hereinafter referred to as Compound IIIA.
  • Fig. 5 is nuclear magnetic resonance spectrum of Compound III in which R is - (OH)(OK).
  • Compound IIIA has a molecular weight of 1144 by FAB-MS (observed (M + Na) + of 1167). NMR DATA
  • Compound IIIA is a white solid soluble in water and polar solvents such as lower alkanols and in dilute alkali metal, magnesium, calcium, and tetra (lower alkyl)ammonium bases. From the bases, salts in which R is a cation salt of phosphate may be obtained.
  • the compound of this invention has similar antibiotic properties as the non-phosphorylated compound and thus would be useful as an antibiotic for the control of parasites, especially Pneumocystis carinii, the causative agent of pneumocystis
  • Rhizopus arrhizus ATCC 11145 maintained in the Merck Culture Collection as MF 4974.
  • the culture was originally obtained from the American Type Culture Collection at 12301
  • Compound Z maybe produced by cultivating
  • Rhizopus arrhizus ATCC 11145 is also known as Rhizopus oryzae (J.J. Ellis, 1985, Mycologia 77: 243-247). The species has also been described under the names Rhizopus nodosus and Rhizopus tritici.
  • the strain MF 4974, ATCC 11145 exhibits all the essential features of R. arrhizus described by M.A.A. Schipper under the name R.
  • the strain is apparently heterothallic because zygospore spore formation was not observed. Colonies grow on most standard mycological media, but on cornmeal agar (Difco) are extremely fast-growing, reaching 35 mm in diameter in 36 hours at 20oC, reaching >90 mm in 36 hours at 37oC, with
  • Rhizoidal hyphae consisting of 3-10, thick, often contorted branches.
  • Compound IIIA may be produced by cultivating Rhizopus arrhizus ATCC 11145 in a suitable nutrient medium containing Compound Z under conditions
  • the sources of carbon include glycerol, sugars, sugar alcohols, starches and other carbohydrates, or carbohydrate deivatives such as dextran, cerelose, as well as complex nutrients such as oat flour, corn meal, millet, corn and the like.
  • carbohydrate deivatives such as dextran, cerelose, as well as complex nutrients such as oat flour, corn meal, millet, corn and the like.
  • the exact quantity of the carbon source which is utilized in the medium will depend, in part, upon the other ingredients in the medium, but it is usually found that an amount of carbohydrate between 0.5 and 40% by weight of the medium is satifactory.
  • These carbon sources can be used individually or several such carbon sources may be combined in the same medium.
  • the sources of nitrogen include amino acids such as glycine, arginine, threonine, methionine and the like, ammonium salt, and complex sources such as yeast hydrolysates, yeast autolysates, yeast cells, tomato paste, soybean meal, casein hydrolysates, yeast extracts, corn steep liquors, distillers
  • solubles, cottonseed meal, meat extract, and the like can be used alone or in combination in amounts ranging form 0.2 to 10 percent by weight of the medium.
  • the medium should contain a phosphate salt.
  • the phosphate salt should be at least about 10 percent by weight of the solid components. It is preferably from about 12 to about 15 percent.
  • a particularly suitable medium is soy-glucose medium of the following composition which may be employed both as a seed medium and a culture medium: Soy-Glucose Medium g/1
  • *Fidco yeast extract is a nitrogen source, product of Difco Laboratories, Detroit MI.
  • the fermentation may be carried out by first preparing a seed culture.
  • spores of Rhizopus arrhizus are obtained from oatmeal agar slants of MF 4974 maintained in the Merck Culture Collection and dispersed in water to obtain a spore suspension containing about 7 x 10 9 spores per milliliter.
  • the spore suspension of MF 4974 is inoculated into a seed flask containing the soy glucose broth and the inoculated suspension incubated on a rotary shaker in the temperature range of from about 15°C to about 30oC, preferably 25o to 28oC.
  • the agitation may be up to 400 rpm but generally about 220 rpm is preferred.
  • the incubation is carried out over a period of at least 24 hours to about two days.
  • the mycelia When growth is abundant, the mycelia are harvested by filtering through a nylon mesh. For biophosphorylation, the mycelia are suspended in a phosphate buffer containing 3 percent glycerol or some other simple carbon source. Compound Z then is added at a concentration of about 50 ⁇ g/ml in
  • DMSO dimethylsulfoxide
  • fractions may be assayed by HPLC.
  • the fractions determined to have the desired product as indicated by a retention time of 12.8 mins. are combined and concentrated under reduced pressure to obtain the product as residue.
  • the salts i.e., where R is a cationic salt of the phosphate, may be prepared by intimately contacting a base corresponding to the cation in an alcoholic or other polar solvent, then concentrating to initiate crystallization of the salt. Thereafter, the salt is recovered by filtration.
  • One method of preparing salts is to apply an aqueous solution of the acid onto a non-functionalized resin column.
  • Representative resins include "AMBERCHROM”-161 (divinylbenzene/polysyyrene resin, obtainable from TosoHaas; trademark name registered by Rohm and Haas), "DIAION” HP-20 and SP-207 (cross- linked styrene-divinylbenzene and brominated styrene- divinylbenzene, respectively, products of Mitsubishi Chemical).
  • the column is then washed with aqueous MH 2 PO 4 or M'(H 2 PO 4 ) 2 where M and M' are monovalent and divalent cations respectively, thereby converting the acid to a mono-cation salt form.
  • the column is washed with water to remove excess inorganic phosphate salt.
  • the product M or M' salt is then removed from the column by applying an aqueous eluant having greater than 50 percent organic content.
  • Useful eluants are 80 percent acetonitrile, 80 percent ethanol or 80 percent methanol.
  • the product is isolated by concentration to dryness and/or
  • This procedure also may be employed to prepare one salt from another.
  • the acid is dissolved in an aqueous mobile phase containing low amount of organic solvent such as acetonitrile and containing phosphate salt thereby forming a solution of the salt of the acid.
  • the solution is subjected to reduced pressure to remove the acetonitrile, then applied to a C-18 extraction column to retain the salt of the product on the column.
  • the salt of the product then may be removed as above described.
  • the phosphate is a compound which is active against certain yeast fungi such as C . albicans and C. tropicalis.
  • the activity may be seen in a microbroth dilution assay employing a Yeast Nitrogen Base (Difco) with 1% dextrose (YNBD).
  • YNBD Yeast Nitrogen Base
  • Compound IIIA was solubilized in 10 percent dimethyl sulfoxide (DMSO) and diluted to 2560 ⁇ g/ml.
  • DMSO dimethyl sulfoxide
  • the yeast cultures maintained on Sabouraud dextrose agar were transferred to YM broth (Difco) and incubated overnight at 35°C with shaking (250 rpm). After incubation, each culture was diluted in sterile water to yield a final concentration of 1-5 x 10 6 colony forming units (CFU)/ml.
  • 96-well microplates were inoculated using a MIC-2000 (Dynatech) which delivers 1.5 ⁇ l per well yielding a final inoculum per well of 1.5-7.5 x 10 3 cells.
  • the microplates were incubated at 35°C for 24 hours.
  • the minimum inhibitory concentrations (MICs) were recorded as the lowest concentrations of drug showing no visible growth.
  • the compounds of the present invention may be employed in inhibiting or alleviating Pneumocystis carinii infections.
  • Compound III/IIIA or a composition containing Compound III/IIIA may be administered in a therapeutically effective or
  • inhibitory amount to subjects infected with or
  • the suitability of the compounds of the present invention for therapeutic or anti-infective purposes may be determined in studies on immuno- suppressed rats when Sprague-Dawley rats (weighing approximately 200 grams) are immunosuppressed with dexasone in the drinking water (2.0 mg/L) and
  • a similar experiment may be carried out in which the rats are injected intraperitoneally (I.P.) twice daily for four days and then sacrificed, the lungs removed and processed, and the extent of disease determined by microscopic analysis of stained slides.
  • I.P. intraperitoneally
  • novel compositions contain at least a therapeutic antifungal or antipneumocystis amount of the active compound. Generally, the composition contains at least 1% by weight of Compound III.
  • Concentrate compositions suitable for dilutions prior to use may contain 90% or more by weight.
  • compositions include compositions suitable for rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), pulmonary (nasal or buccal inhalation), nasal administration, or insufflation.
  • the compositions may be prepacked by intimately mixing Compound III with the components suitable for the medium desired.
  • any method of administration may be used.
  • oral administration is frequently preferred.
  • oral administration is to be
  • the therapeutic agent is preferably formulated with water or aqueous compositions, but if desired, may be formulated with glycols, oils, alcohols, and the like.
  • solid preparations such as capsules and tablets, solid carriers such as starches, sugars, kaolin, ethyl cellulose, calcium and sodium carbonate, calcium phosphate, kaolin, talc, lactose, generally with lubricant such as calcium stearate, together with binders, disintegrating agents and the like.
  • tablets and capsules represent the most advantageous oral dosage form. It is especially advantageous to
  • compositions in unit dosage form for ease of administration and uniformity of dosage.
  • Composition in unit dosage form constitutes an aspect of the present invention.
  • the Compound III is preferably formulated in aqueous therapeutic compositions for intravenous or intraperitoneal injection or aerosol when use against Pneumocystis carinii is contemplated, and may be presented in unit dosage form in ampoules or in multidose containers, if necessary with an added preservative.
  • the compositions may also take such forms as solutions in aqueous vehicles such as 0.85 percent sodium chloride or 5 percent dextrose in water, and may contain formulating agents such as stabilizing and/or dispersing agents. Buffering agents as well as additives such as saline or glucose may be added to make the solutions isotonic.
  • the drug also may be solubilized in alcohol/propylene glycol or polyethylene glycol for drip intravenous administration.
  • the active ingredients may be in powder form for reconstituting with a suitable vehicle prior to administration.
  • unit dosage form refers to physically discrete units, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier.
  • unit dosage forms are tablets, capsules, pills, powder packets, wafers, measured units in ampoules or in multidose containers and the like.
  • a unit dosage of the present invention may contain from 100 to 1000 milligrams of one of the compounds.
  • the present invention is also concerned with a compound which inhibits the protease encoded by human immunodeficiency virus (HIV).
  • the compound, or pharmaceutically acceptable salt thereof is of value in the prevention of infection by HIV, the treatment of infection by HIV and the treatment of the resulting acquired immune deficiency syndrome (AIDS).
  • the present invention also relates to pharmaceutical compositions containing the compounds, and to a method of use of the present compounds with or without other agents for the treatment of AIDS &. viral infection by HIV.
  • a retrovirus designated human immunodeficiency virus (HIV) is the etiological agent of the complex disease that includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system.
  • This virus was previously known as LAV, HTLV-III, or ARV.
  • a common feature of retrovirus replication is the extensive post-trans- lational processing of precursor polyproteins by a virally encoded protease to generate mature viral proteins required for virus assembly and function. Interruption of this processing appears to prevent the production of normally infectious virus.
  • Crawford, S. et al ., J. Virol., 53 , 899, 1985 demonstrated that genetic deletion mutations of the protease in murine leukemia virus which prevent processing of precursor structural proteins result in non-infectious viral particles. Unprocessed structural proteins also have been observed in clones of non-infectious HIV strains isolated from human patients. These results suggest that inhibition of the HIV protease represents a viable method for the treatment of AIDS and the prevention or treatment of infection by HIV.
  • a biotransformed compound as herein defined is disclosed. This compound is useful in the inhibition of HIV protease, the prevention of infection by HIV, the treatment of infection by HIV and in the treatment of AIDS and/or ARC, either as a compound, pharmaceutically acceptable salt (when appropriate), pharmaceutical composition ingredient, whether or not as a prodrug or as a combination with other antivirals, anti-infectives, immunomodulators, antibiotics or vaccines. Methods of treating AIDS, methods of preventing infection by HIV, and methods of treating infection by HIV are also disclosed.
  • This invention is also concerned with the use of a compound given below, or pharmaceutically acceptable salts thereof, in the inhibition of HIV protease, the prevention or treatment of infection by HIV and in the treatment of the resulting acquired immune deficiency syndrome (AIDS).
  • the biotransformed compound is the product of the incubation of Rhizopus arrhizus (ATCC 11145) in the presence of L-702,083, an HIV protease inhibitor. It is defined as follows:
  • the pharmaceutically-acceptable salts of the compound of the present invention include the conventional non-toxic salts or the quaternary ammonium salts of this compound, which are formed, e.g., from inorganic or organic acids or bases.
  • acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,
  • Base salts include
  • alkali metal salts such as sodium and potassium salts
  • alkaline earth metal salts such as calcium and magnesium salts
  • salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth.
  • the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl
  • sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others.
  • Hydrates or esters are also encompassed by the present invention. Such hydrates or esters are those which would readily occur to the skilled artisan, and include, for example, C 1-4 alkyl esters.
  • the compound of the present invention is useful in the inhibition of HIV protease, the
  • HIV human immunodeficiency virus
  • Treating AIDS or preventing or treating infection by HIV is defined as including, but not limited to, treating a wide range of states of HIV infection:
  • the compound of this invention is useful in treating infection by HIV after suspected past exposure to HIV by e.g., blood transfusion, accidental needle stick, or exposure to patient blood during surgery.
  • the compound of the present invention may be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and vehicles.
  • a method of treating and a pharmaceutical composition for treating HIV infection and AIDS involves administering to a patient in need of such treatment a pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically-effective amount of the compound of the present invention.
  • compositions may be in the form of orally-administrable suspensions or tablets; nasal sprays; sterile injectable preparations, for example, as sterile injectable aqueous or oleagenous suspensions or suppositories.
  • compositions When administered orally as a suspension, these compositions are prepared according to
  • compositions for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweetners/flavoring agents known in the art.
  • these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.
  • compositions When administered by nasal aerosol or inhalation, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other
  • the injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • suitable non-toxic, parenterally-acceptable diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • these compositions When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug.
  • a suitable non-irritating excipient such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug.
  • Dosage levels of the order of 0.02 to 5.0 or 10.0 grams-per-day are useful in the treatment or prevention of the above-indicated conditions, with oral doses two-to-five times higher.
  • infection by HIV is effectively treated by the administration of from 10 to 50 milligrams of the compound per kilogram of body weight from one to three times per day.
  • the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age of the patient, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
  • the present invention is also directed to combinations of the HIV protease inhibitor compound with one or more agents useful in the treatment of AIDS.
  • the compound of this invention may be effectively administered, whether at periods of pre-exposure and/or post-exposure, in combination with effective amounts of other AIDS antivirals, immunomodulators, anti-infectives, or vaccines.
  • HIV Protease Inhibitor - II HIV Protease Inhibitor - II
  • the present invention is also further concerned with another compound which inhibits the protease encoded by human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • the compound, or pharmaceutically acceptable salt thereof, is of value in the prevention of infection by HIV, the treatment of infection by HIV and the treatment of the resulting acquired immune deficiency syndrome (AIDS).
  • AIDS acquired immune deficiency syndrome
  • the present invention also relates to pharmaceutical compositions
  • a retrovirus designated human immunodeficiency virus is the etiological agent of the complex disease that includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system.
  • This virus was previously known as LAV, HTLV-III, or ARV.
  • a common feature of retrovirus replication is the extensive post-translational processing of precursor polyproteins by a virally encoded protease to generate mature viral proteins required for virus assembly and function. Interruption of this processing appears to prevent the production of normally infectious virus. For example, Crawford, S. et al., J.
  • the compound of this invention is an inhibitor of HIV protease.
  • the compound of this invention provides a prodrug for the inhibition of HIV protease.
  • a biotransformed compound as herein defined is disclosed. This compound is useful in the inhibition of HIV protease, the prevention of infection by HIV, the treatment of infection by HIV and in the treatment of AIDS and/or ARC, either as a compound, pharmaceutically acceptable salt (when appropriate), pharmaceutical composition ingredient, whether or not as a prodrug or as a combination with other antivirals, anti-infectives, immunomodulators,
  • This invention is concerned with the use of a compound given below, or pharmaceutically acceptable salts thereof, in the inhibition of HIV protease, the prevention or treatment of infection by HIV and in the treatment of the resulting acquired immune deficiency syndrome (AIDS).
  • the biotransformed compound is the product of the incubation of Rhizopus arrhizus (ATCC 11145) in the presence of L-689,502, a HIV protease inhibitor. It is defined as follows:
  • the pharmaceutically-acceptable salts of the compound of the present invention include the conventional non-toxic salts or the quaternary ammonium salts of this compound, which are formed, e.g., from inorganic or organic acids or bases.
  • acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,
  • Base salts include
  • alkali metal salts such as sodium and potassium salts
  • alkaline earth metal salts such as calcium and magnesium salts
  • salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth.
  • the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl
  • sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others.
  • Hydrates or esters are also encompassed by the present invention. Such hydrates or esters are those which would readily occur to the skilled artisan, and include, for example, C 1-4 alkyl esters.
  • the compound of the present inventions is useful in the inhibition of HIV protease, the prevention or treatment of infection by the human immunodeficiency virus (HIV) and the treatment of consequent pathological conditions such as AIDS.
  • HIV human immunodeficiency virus
  • Treating AIDS or preventing or treating infection by HIV is defined as including, but not limited to, treating a wide range of states of HIV infection:
  • the compound of this invention is useful in treating infection by HIV after suspected past exposure to HIV by e.g., blood transfusion, accidental needle stick, or exposure to patient blood during surgery.
  • the compound of the present invention may be administered orally,
  • parenterally including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and vehicles.
  • a method of treating and a pharmaceutical composition for treating HIV infection and AIDS involves administering to a patient in need of such treatment a pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically-effective amount of the compound of the present invention.
  • compositions may be in the form of orally-administrable suspensions or tablets; nasal sprays; sterile injectable
  • compositions for example, as sterile injectable aqueous or oleagenous suspensions or suppositories.
  • these compositions When administered orally as a suspension, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweetners/flavoring agents known in the art.
  • these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.
  • compositions When administered by nasal aerosol or inhalation, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other
  • injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride
  • suitable dispersing or wetting and suspending agents such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • compositions When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating agent.
  • excipient such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidity and/or dissolve in the rectal cavity to release the drug.
  • Dosage levels of the order of 0.02 to 5.0 or 10.0 grams-per-day are useful in the treatment or prevention of the above-indicated conditions, with oral doses two-to-five times higher.
  • infection by HIV is effectively treated by the
  • the present invention is also directed to combinations of the HIV protease inhibitor compound with one or more agents useful in the treatment of AIDS.
  • the compound of this invention may be effectively administered, whether at periods of pre-exposure and/or post-exposure, in combination with effective amounts of other AIDS antivirals, immunomodulators, anti-infectives, or vaccines.
  • Simvastatin Analogs are also directed to combinations of the HIV protease inhibitor compound with one or more agents useful in the treatment of AIDS.
  • the compound of this invention may be effectively administered, whether at periods of pre-exposure and/or post-exposure, in combination with effective amounts of other AIDS antivirals, immunomodulators, anti-infectives, or vaccines.
  • simvastatin also included by the process of this invention are two phosphorylated derivatives of simvastatin, whose chemical name is 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-pyran- 2-one.
  • the above two compounds of this invention are useful as antihypercholesterolemic agents for the treatment of atherosclerosis, hyperlipemia and like diseases in humans. They may be administered orally or parenterally in the form of a capsule, a tablet, an injectable preparation or the like. It is usually desirable to use the oral route. Doses may be varied, depending on the age, severity, body weight and other conditions of human patients but daily dosage for adults is within a range of from about 2 mg to 2000 mg (preferably 10 to 100 mg) given in three or four divided doses. Higher doses may be favorably applied as required.
  • the compounds of rhis invention also have useful anti-fungal activities. For example, they may be used to control strains of Penicillium sp.,
  • the pharmaceutically acceptable salts of this invention include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium , zinc and tetramethylammonium as well as those salts formed from amines such as ammonia, ethylenediamine, N-methylglucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzyl- phenethylamine, 1-p-chlorobenzyl-2-pyrrolidine-1'- yl-methylbenzimidazole, diethylamine, piperazine, and tris(hydroxymethyl)aminomethane.
  • Zearalenone Zearalenone
  • biotransformation product alpha zearalenol 6'-phosphate:
  • the present invention also relates to the above new compound and an object of the present invention is to provide compounds which exhibit estrogenic activity or aid in increasing the rate of growth in meat-producing animals, e.g. cattle, lamb and swine.
  • the compound can be administered to animals by any suitable method including oral and parenteral administrations.
  • the compound can be blended with ordinary feed containing nutritional values in an amount sufficient to produce the desired rate of growth and can thus be fed directly to the animals, or the compound can be suspended in a suitable injection suspension medium such as peanut oil and injected parenterally.
  • a suitable injection suspension medium such as peanut oil and injected parenterally.
  • an animal feed composition may be prepared containing the usual nutritionally-balanced quantities of carbohydrates, proteins, vitamins and minerals, together with the compounds of the present invention.
  • Some of these usual dietary elements are grains, such as ground grain and grain by-products; animals protein substances, such as those found in fish meal and meat scraps; vegetable proteins like soybean oil meal or peanut oil meal; vitaminaceous materials, e.g. vitamin A and D complex members; and bone meal and limestone to provide minerals.
  • a type of conventional feed material for use with cattle includes alfalfa hay and ground corn cobs together with supplementary vitaminaceous substances is
  • ( ⁇ )-zearalenone a potent anabolic agent useful in the raising of meat-producing animals, originally was prepared by fermenting the microorganism, Gibberella zeae (Gordon), on a suitable nutrient medium
  • a spore sand culture containing Gibberella zeae (Gordon) NRRL-2830 was aseptically placed in a sterile tube containing 15 milliliters of Czapek's- Dox solution and a small amount of agar. This medium was then incubated for about 168 hours at approximately 25oC. At the end of the incubation period, the medium was washed with 5 milliliters of sterile deionized water and transferred to a sterile tube containing 45 milliliters of Czapek's-Dox solution. The contents of the tube were then incubated for about 96 hours at about 25oC after which the material was available for use in inoculation of a
  • the following example illustrates the fermentation of the organism Gibberella zeae (Gordon) NRRL-2830 to produce zearalenone.
  • the following example illustrates the recovery of the zearalenone from the fermentation medium.
  • a 300 gram portion of fermented material produced by the method of Example IIA was placed in 500 milliliters of deionized water and slurried.
  • the slurry was then heated for about 15 minutes at 75oC, 300 grams of filter aid were then added and the material was filtered.
  • the solid filtered material containing the anabolic substance was then air dried, and 333 grams of the dried cake were then extracted with 500 milliliters of ethanol. This procedure was repeated three more times.
  • the ethanol extract was evaporated to dryness under vacuum to give 6.84 grams of solid material.
  • This solid material was then dis- solved in 20 milliliters of chloroform and extracted with 30 milliliters of an aqueous solution containing 5% by weight of sodium carbonate having an adjusted pH of about 11.2.
  • the extraction process was repeated seven more times.
  • the pH of the sodiumcarbonate extract was then adjusted to 6.2 with hydrochloric acid, to yield an anabolic substance-containing precipitate.
  • the precipitate and the aqueous sodium carbonate extract were then each in turn extracted with 75 milliliters of ethyl ether. This procedure was repeated three more times to yield a light yellow ethereal solution, which was then evaporated to yield 116 milligrams of solid anabolic substance.
  • This material was then subjected to multiple transfer countercurrent distribution using 100 tubes and a solvent system consisting of two parts chloroform and two parts carbontetrachloride as the lower phase and four parts methanol and one part water as the upper phase, all parts by volume.
  • the solid material obtained from the multiple transfer countercurrent distribution was zearalenone.
  • each flask was harvested by centrifugation, washed once with sterile water, and resuspended in equal volume of 100 mM pH 7.0 PO 4 buffer containing 3% glycerol. FK-520 was added to achieve a final concentration of 0.2 mg/ml. The flasks were then incubated on a rotary shaker (220 rpm) at 27°C for 48 hours. Following incubation, the whole broth was extracted as described in the Isolation/Purification Section below.
  • the whole broth (400 ml) was adjusted to pH 3.5 and extracted three times with methylene chloride (3 x 400 ml). Methylene chloride extracts were combined and evaporated to dryness under reduced pressure at 30oC. The resulting oil was dissolved in methanol and subjected to HPLC purification. HPLC was carried out on Whatman Partisil 10 ODS-3, 9.4 mm x 25 cm at 50oC and monitored at 205 nm. The column was developed at 3 ml/minutes with a linear gradient from 35% acetonitrile in 0.1% phosphoric acid to 80% in 0.1% phosphoric acid in 60 minutes. The compound was collected during repeated injections of the above described extract.
  • Spleens from C57B1/6 mice were taken under sterile conditions and gently dissociated in ice-cold RPMI 1640 culture medium (GIBCO, Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal calf serum (GIBCO). Cells were pelleted by centrifugation at 1500 rpm for 8 minutes. Contaminating red cells were removed by treating the pellet with ammonium chloride lysing buffer (GIBCO) for 2 minutes at 4oC. Cold medium was added and cells were again centrifuged at 1500 rpm for 8 minutes.
  • RPMI 1640 culture medium Gib Island, N.Y.
  • GIBCO heat-inactivated fetal calf serum
  • T lymphocytes were then isolated by separation of the cell suspension on nylon wool columns as follows: Nylon wool columns were prepared by packing approximately 4 grams of washed and dried nylon wool into 20 ml plastic syringes. The columns were sterilized by autoclaving at 250oF for 30 minutes. Nylon wool columns were wetted with warm (37oC) culture medium and rinsed with the same medium. Washed spleen cells resuspended in warm medium were slowly applied to the nylon wool. The columns were then incubated in an upright position at 37oC for 1 hour. Non-adherent T lymphocytes were eluted from the columns with warm culture medium and the cell suspensions were spun as above.
  • Nylon wool columns were prepared by packing approximately 4 grams of washed and dried nylon wool into 20 ml plastic syringes. The columns were sterilized by autoclaving at 250oF for 30 minutes. Nylon wool columns were wetted with warm (37oC) culture medium and rinsed with the same medium. Washed spleen cells
  • T lymphocytes were resuspended at 2.5 x 10 5 cells/ml in complete culture medium composed of RPMI 1640 medium with 10% heat-inactivated fetal calf serum, 100 mM glutamine, 1 mM sodium pyruvate, 2 x 10 -5 M 2-mercaptoethanol and 50 ⁇ g/ml gentamycin. Ionomycin was added at 250 ng/ml and PMA at 10 ng/ml. The cell suspension was immediately distributed into 96 well flat-bottom microculture plates (Costar) at 200 ⁇ l/well. The control, being the medium without test drug, and various below-indicated dilutions of the above sample of purified C-32 phosphorylated
  • FK-520 to be tested were then added in triplicate wells at 20 ⁇ l/well .
  • FK-520 was used as a standard .
  • the culture plates were then incubated at 37oC in a humidified atmosphere of 3% CO 2 -95% air for 44 hours.
  • the proliferation of T lymphocytes was
  • tritiated thymidine NNN, Cambridge, MA
  • cultures were harvested on glass fiber filters using a multiple sample harvester. Radioactivity of filter discs corresponding to individual wells was measured by standard liquid scintillation counting methods (Betacounter). Mean counts per minute of replicate wells were calculated and the results expressed as percent inhibition of tritiated thymidine uptake (proliferation) as follows:
  • C-32-P FK-520 The results of % inhibition at various concentrations of C-32 phosphorylared FK-520 (C-32-P FK-520) are presented in the following table:
  • the mean IC 50 for C-32-P FK-520 was determined to be: 15.0 + 2.1 ng/ml (17.2 ⁇ 2.4 nM) in 3 independent experiments.
  • Rhizopus oryzae MF 4974 cultivated on oatmeal agar, were inoculated into 50 ml Soy-Glucose medium in a 250 ml Erlenmeyer flask and shaken at 25oC on a rotary shaker at 220 rpm for 24 hours.
  • the second stage flasks 50ml in a 250 ml Erlenmeyer flask
  • each flask was harvested by centrifugation, washed once with sterile water, and resuspended in equal volume of 100 mM P04 buffer containing 3% glycerol, FK506 was added to achieve a final concentration of 0.2 mg/ml.
  • the charged flasks were incubated on a rotary shaker (220 rpm) at 27oC for 24 hours. Following incubation, the whole broth was worked up as described below.
  • HPLC was carried out on Whatman Magnum 20 Partisil 1- ODS-3 Column (C 18 ,22.1 mm ID x 25 cm) at 50oC and monitored at 205 nm. The column was developed at 7 mL/min with linear gradient from 35% acetonitrile in 0.1% phosphoric acid to 80% acetonitrile in 0.1% phosphoric acid in 70 minutes.
  • the compound was collected during repeated injections of the above described extract. Fractions of retention time 50 minutes were pooled, adjusted to pH 3.0 and evaporated to remove acetonitrile. The compound was desalted using a C 18 Sep Pak (Water Associate) to yield 20 mg of product.
  • the compound was characterized by MS and confirmed by NMR as the C-32 phosphate ester
  • Example 3 The fermentation procedure of Example 3 was carried out substantially identical except that C-31 desmethyl FK-520 (available as for example, by the procedure in EPO Publication 0 349 061, published January 3, 1990) was used in place of FK-506.
  • FK-520 was characterized by mass spectrometry and proton nuclear magnetic resonance in which the obtained spectra was consistent with the assigned structure.
  • each flask was harvested by centrifugation, washed once with sterile water, and resuspended in equal volume of 100 mM pH 7.0 phosphate buffer containing 3% glycerol. Rapamycin macrolide was added to achieve a final concentration of 0.2 mg/ml. The flasks were then incubated on a rotary shaker (220 rpm) at 27 °C for 24 hours . Following incubation, the whole broth was extracted as described in the Isolation/Purification Section below.
  • the whole broth (500 ml) was maintained at pH 6.8 and centrifuged.
  • the mycelial cake was washed with water, then dicarded.
  • the clear filtrate and washings were pooled and passed through a Speed octadecyl cartridge (14% carbon load, Applied Separations) under vacuum.
  • the column was washed with 100 ml of water.
  • Column effluent and wash did not contain microbial transformation product when tested with HPLC.
  • the cartridge was eluted with 200 ml methanol. Methanol was evaporated to dryness under reduced pressure at 30°C. The resulting oil was dissolved in methanol and subjected to HPLC purification.
  • HPLC HPLC was carried out on Whatman Magnum 9 Partisil 10 ODS-3, 9.8 mm i.d. x 25 cm at 25oC and monitored at 225 nm.
  • the column was developed at 3 ml/minutes with a linear gradient from 35% to 80% acetonitrile in 0.1% phosphoric acid in 30 minutes.
  • the compound was collected during repeated injections of the above described extract.
  • the fractions of retention time 17.3 minutes, were pooled, adjusted to pH 3 and evaporated to remove acetonitrile.
  • the compound was further purified using C18 Sep Pak
  • the C-43 phosphorylated macrolide was characterized by FAB mass spectrometry and NMR spectrometry as the methyl phosphate ester derivative yielding the proton NMR spectrum of Figure 3, which confirms the assigned molecular structure in Figure 4. Methylation to produce the phosphate ester was necessary to minimize the considerable line broadening which characterized the proton NMR spectrum of the free acid.
  • the key features were the downfield displaced H-42 and H-43 signals (which are H-31 and H-32 in FK-506 nomenclature) at 3.87 ppm and 4.12 ppm, respectively, and the additional fine structure of H-43 resulting from coupling with the phosphorus.
  • Purified phosphorylated macrolide as prepared by HPLC above, was dissolved in absolute ethanol at 1 mg/ml and serially diluted in culture medium prior to addition to the cultures.
  • Nylon wool columns were wetted with warm (37oC) culture medium and rinsed with the same medium. Washed spleen cells resuspended in warm medium were slowly applied to the nylon wool. The columns were then incubated in an upright position at 37°C for 1 hour. Non-adherent T lymphocytes were eluted from the columns with warm culture medium and the cell suspensions were spun as above.
  • T lymphocytes were resuspended at 2.5 x 10 5 cells/ml in complete culture medium composed of RPMI 1640 medium with 10% heat-inactivated fetal calf serum, 100 mM glutamine, 1 mM sodium pyruvate, 2 x 10 -5 M 2-mercaptoethanol and 50 ⁇ g/ml gentamycin.
  • the cell suspension vas immediately
  • Proliferation was assessed at 48 hours of culture by tritiated thymidine uptake.
  • the mean IC 50 for the C-43 phosphory- lated rapamycin was determined to be: 7.6 ⁇ 0.2 ng/ml and 0.4 ⁇ 0.1 ng/ml for rapamycin in 3 independent experiments.
  • Step A Preparation of 4-tert-butyldimethylsilyloxy- phenylprop-2-en-1-yl bromide:
  • Step B Preparation of 5(S)-(1,1-dimethylethoxycarbonyl)amino-4(S)-(1',1'-dimethylethyl-1,1- dimethylsilyloxy)-6-phenyl-2(R)-(4-(1',1'- dimethylsilyloxy)phenylprop-2-en-yl)hex- anoic acid:
  • the resulting solution was aged at -78oC for 1 hour and the dropping funnel was charged with a solution of 4-tert-butyldimethylsilyloxyphenylprop- 2-en-1-yl bromide (17.53 g, 53.55 mmol) in 75 mL of dry THF.
  • the bromide solution was cooled to -78oC and was then added to the enolate solution dropwise over 45 minutes, keeping the temperature below
  • the DME was removed in vacuo and the aqueous residue was acidified with 10% aqueous citric acid.
  • This milky suspension was extracted with several portions of EtOAc and the combined extracts were washed with water and brine, dried (MgSO 4 ), filtered, and
  • Step C Preparation of N-(2(R)-hydroxy-1(S)-indanyl)- 5(S)-(1,1-dimethylethoxycarbonylamino)-4(S)- (1',1'-dimethylethyl-1,1-dimethylsilyloxy)-6- phenyl-2(R)-((4-(hydroxyphenyl)prop-2-en-1- yl)hexanamide:
  • Step D Preparation of N-(2(R)-hydroxy-1(S)-indanyl)- 5(S)-(1,1-dimethylethoxycarbonylamino)-4(S)- hydroxy-6-phenyl-2(R)-(4-(2-(4-morpholino)- ethoxy)phenylprop-2-en-1-yl)hexanamide:
  • Step A Preparation of (5S,1'S)-5-(1'-((1,1-di- methylethoxycarbonyl)amino)-2'-cyclo- hexylethyl)-4,5-dihydrofuran-2-(3H)-one:
  • Step B Steps B, C and D of Example 1 are repeated except that (5S, 1'S)-5-((1,1-dimethylethoxy- carbonyl)amino)-2-phenylethyl)dihydrofuran-2- (3H)-one in Step B is substituted with its cyclohexyl analog, (5S, 1'S)-5-((1-dimethyl- ethoxycarbonyl)amino)-2-cyclohexylethyl)- 4,5-dihydrofuran-2-(3H)-one.
  • the title compound, L-702,083 is obtained.
  • the neighboring methine (m) is slightly displaced downfield and is barely discernible as a shoulder at the base of the morpholine CH 2 O at 3.74 ppm. Also perturbed is the nearby H k which now appears underneath the lower field H j signal at 3.10 ppm. Its presence is revealed only by the additional area of the H j signal, compared with the H j double doublet in L-702,083.
  • step A The product of step A is reacted with monophenyl phosphorodichloridate according to the principles and practice of Chambers, R.W. and H . G . Khorana, J. Am. Chem. Soc. 80, 3749 (1958).
  • Another phosphorylating agent is dibenzyl phosphorochloridate.
  • HIV protease expressed in Escherichia coli with a tritiated peptide substrate [ 3 H]-acetyl- Val-Ser-Gln- Asn-(beta-napthyl-Ala)-Pro-Ile-Val-Gln- Gly-Arg-Arg-NH 2 (MW 1800).
  • the two arginine residues at the carboxyl terminus give this peptide an overall positive charge at acidic pH and enable it to bind to the H + form of DOWEX AG-50W-X8 resin and similar resins.
  • the HIV protease cleaves between the HIV protease expressed in Escherichia coli with a tritiated peptide substrate [ 3 H]-acetyl- Val-Ser-Gln- Asn-(beta-napthyl-Ala)-Pro-Ile-Val-Gln- Gly-Arg-Arg-NH 2 (MW 1800).
  • Step A Preparation of N-3(S)-[(1,1-Dimethylethoxy- carbonyl)amino]-2(RS)-hydroxy-4-phenyl-1-tri- methylsilyl butane:
  • Step A (22.8 g, 67.5 mmoL) in dry methylene chloride (400 mL) cooled in an ice bath and under nitrogen was added in a fine stream boron trifluoride etherate
  • Step C Preparation of N-3(S)-[(1,1-Dimethyl- ethpxycarbonyl)amino]-4-phenyl-1-butene:
  • Step D Preparation of 1(R)-[1'(S)-(1,1-Dimethyl- ethoxycarbonyl)amino-2-phenylethylloxirane: To a solution of the product of Step C (9.4 g, 38 mmol) in dry methylene chloride (100 mL) cooled in an ice bath and under nitrogen was added 3-chloroperoxybenzoic acid (technical grade, 80-85%; 41 g, 200 mmol). The mixture was stirred at 0oC for 18 hours and 25oC for 23 hours, then diluted with diethyl ether (300 mL), and poured in ice cold aqeous 10% sodium sulfite (1 L).
  • 3-chloroperoxybenzoic acid technical grade, 80-85%; 41 g, 200 mmol
  • Step F Preparation of (5S,1'S)-3-carboethoxy-3-(4- benzyloxyphenylmethyl)-5-[1-(1,1-dimethyl- ethoxycarbonyl)amino)-2-phenylethyl]dihydro- f ⁇ ran-2-(3H)-one
  • Step G Preparation of (3R,5S,1'S)-3-(4-benzyloxy- phenylmethyl)-5-(1((1,1-dimethylethoxy- carbonyl)amino)-2-phenylethyl)-dihydrofuran-
  • Step F The product of Step F, 13.6 g, was dissolved in 250 mL of 1,2-dimethoxyethane, and to it was added 117 mL of 1 M lithium hydroxide at room temperature. After stirring for 12 hours, the solvents were removed under reduced pressure, the residue suspended in 200 mL of 10% citric acid and extracted 3 X 500 mL of diethyl ether. The combined ether extracts were washed with 500 mL of brine, dried (MgSO 4 ) and the concentrated to dryness. The residue was dissolved in 250 mL of toluene, heated to reflux for 12 hours, then concentrated to dryness under reduced pressure. Purification by medium pressure chromatography over silica gel eluting with 15% ethyl acetate/hexanes gave 3.2 g of the 3R-lactone as a clear foam.
  • Step H Preparation of N'-(1,1-dimethylethoxy- carbonyl)-5(S)-amino-4(S)-(1',1'-dimethylethyl-1,1-dimethylsilyloxy)-6-phenyl-2(R)- (4-benzyloxyphenylmethyl-hexanoic acid.
  • Step G The product of Step G, 0.6 g, was dissolved in 30 mL of a 2:1 mixture of ethylene glycol dimethyl ether/water, and to it was added 5 mL of 1 M lithium hydroxide at room temperature. After stirring for 1 hour, the mixture was partitioned between 200 mL chloroform and 20 mL 10% citric acid. The layers were separated and the aqueous phase extracted with 3 X 20 mL chloroform. The combined organic layers were dried (Na 2 SO 4 ) and the solvent removed to yield 0.56 g of the crude hydroxy acid. This residue was dissolved in 5 mL of dry DMF and 0.845 g tert-butyl dimethylsilyl chloride and 0.725 g of imidazole were added.
  • Step J Preparation of N-(2(R)-hydroxy-1(S)-indanyl)- 5(S)-(1,1-dimethylethoxycarbonylamino)-4(S)- hydroxy-6-phenyl-2(R)-(4-benzyloxyphenyl- methyl) hexanamide
  • Step H The product from Step H, 0.12 g, was dissolved in 2 ml dry DMF and to it was added 40 mg of 1(S)-amino-2(R)-hydroxyindane, (Step I) 25 mg of 1-hydroxybenzotriazole hydrate and 70 mg of dimethyl- 3-(3-dimethyl aminopropyl)carbodiimide hydrochloride. Triethylamine was added to the stirred solution until the pH was 8.5 (32 mL).
  • Step K Preparation of N-(2(R)-hydroxy-1(S)-indanyl)- 5(S)-(1,1-dimethylethoxycarbonylamino)-4(S)- hydroxy-6-phenyl-2(R)-(4-hydroxyphenyl- methyl)hexanamide
  • Step J 85 mg was dissolved in 10 mL of methanol and 10 mL of THF, and to it was added 0.10 g of 10% palladium on carbon. The mixture was stirred under an atmosphere of hydrogen for 48 hours at room temperature, then filtered and concentrated to dryness. The residue was dissolved in 10 mL of hot ethanol and 20 mL water was added. On cooling the white solid precipitate was collected and dried under vacuum over P 2 O 5 . The yield was 72 mg (98% yield) of pure product: mp 218-219oC (effervesces, sinters at 215) elemental analysis, Calc'd for
  • a frozen vial (2.0 ml) of Rhizopus arrhizus MF4974 was used to inoculate a 250 ml baffled shake flask containing 50 ml of seed medium A.
  • the seed flask was incubated on a rotary shaker (220 rpm) at 27°C for 24 hours.
  • a 2.5 ml aliquot of the developed seed was used to inoculate a 250 ml non-baffled flask containing 50 ml of transformation medium B; L-689,502 in DMSO was added to the fermentation at 0 hour to achieve a final concentration of 0.05 mg/ml.
  • the shake flask contents were subsequently incubated at 27oC on a rotary shaker for 4 days.
  • the resultant whole broth was extracted as described in Section B.
  • HPLC high performance liquid chromatography
  • Phosphorus was identified and quantified by complexomatric and gravimetric methods. Phosphorylation at C 4 was established by NMR from the 0.5 ppm downfield displacement of the C 4 H relative to its chemical shift in the parent L-689,502.
  • the microbial transformation metabolite L-696,432 was determined to be a phosphate ester of L-689,502, a highly potent HIV protease inhibitor. To determine if the phosphate ester bond can be enzymatically cleaved, treatment with alkaline phosphatase was performed.
  • N-(cis-2(R)-Acetoxy-1(S)indanyl)-5(S)-[1,1- dimethylethoxycarbonylamino)-4(S)-hydroxy-6-phenyl- 2(R)-[(4-(2-(4-morpholinyl)ethoxy)phenyl]methyl]- hexanamide is prepared by the protocol of Example 1, except that 1(S)-amino-2(R)-acetoxyindane substitutes for 1(S)-amino-2(R)-hydroxyindane of step I.
  • step A The product of step A is reacted with monophenyl phosphorodichloridate according to the principles and practice of Chambers, R.W. and H.G. Khorana, J. Am. Chem. Soc. 80, 3749 (1958). Sub- sequent treatment with excess ammonia, followed by removal of the acetyl and phenyl groups under basic conditions, yields L-696,432.
  • Another phosphorylating agent is dibenzyl phosphorochloridate.
  • Rhizopus arrhizus MF 4974 from the Merck Culture Collection was inoculated in 50 milliliters of soy-glucose medium in a 250 milliliter flask and the flask incubated on a rotary shaker at 220 rpm at 27 °C for 24 hours to obtain the f irst stage seed culture. 2.5 milliliters of this seed culture was inoculated into each of 18 flasks
  • each flask was harvested by centrifugation, the mycelium washed twice with sterile saline and resuspended in pH 7.0 phosphate buffer containing 1 percent glycerol.
  • Compound Z was added to a final concentration of 58 ⁇ g/ml using 100 ⁇ l of (DMSO). The flasks were incubated on a rotary shaker at 220 rpm at 27oC for 48 hours.
  • the extracts of the mycelium and the extracts of the supernatant were pooled and evaporated to dryness at 30oC at reduced pressure to obtain an oil as residue.
  • the oil was dissolved in the mobile phase of 40/60 acetonitrile/water and further purified using ZORBAX C8 (9.6 mm x 25 cm) semi-preparative column.
  • the column was developed at 7.05 ml/min. using 40 percent aqueous acetonitrile containing 0.1 percent trifluoroacetic acid (TFA) at 45oC. Fractions having a retention time of 12.8 minutes were pooled, and the solvent evaporated to obtain 12 mg of Compound III in a yield which calculated to be 24%.
  • the product had the mass spectral data previously set forth.
  • a portion of the product was converted to the monopotassium salt. This was carried out by dissolving the biophosphorylation product in 70 percent aqueous acetonitrile containing 10 mM KH 2 PO 4 (pH 4.5). The mixture was subjected to reduced pressure to remove the acetonitrile and the aqueous residue loaded onto a water-equilibrated C-18 solid phase extraction column. The column was washed with water and then eluted with 70 percent aqueous
  • Rhizopus arrhizus were obtained from oatmeal agar slants of MF 4974 maintained in the Merck Culture Collection and employed to prepare a spore suspension in water of about 7 x 10 9 spores per milliliter for use in the preparation of seed culture.
  • Seed flasks each containing 500 milliliters of soy glucose broth of the composition previously given, were inoculated with 1 milliliter of spore suspension and incubated on a rotary shaker (220 rpm) at 27oC for 24 hours.
  • milliliters were pooled and filtered through a 10 micron nylon mesh.
  • the mycelial cake was slurried with 1000 milliliters of 50 percent aqueous methanol and filtered.
  • the mycelial cake was again extracted with aqueous methanol and the two aqueous methanol filtrates combined and diluted with 2000 milliliters of water.
  • the resulting aqueous solution was applied to a 15 mm x 300 mm column packed to a 220 mm bed height with water equilibrated "DIANION" HP20 resin.
  • the filtrate was pumped in a downflow mode at 15 ml/min. in 2000 ml aliquots.
  • the column was washed with 500 milliliters of water and the desired phosphorylated product eluted with 500 milliliters of 20 percent aqueous acetonitrile.
  • the remaining substrate and metabolite was eluted with 500 milliliters of 70 percent aqueous acetonitrile.
  • the diluted filtrates from the mycelial extracts were also pumped onto the- column, the column washed and then eluted and the eluate assayed by HPLC.
  • the assay condition was as described in
  • HPLC indicated the following mass balance for microbial phosphorylation and "DIAION" HP 20 isolation:
  • the eluate was concentrated under reduced pressure and the residue dissolved in 40 percent aqueous acetonitrile containing 10mM KH 2 PO 4 to obtain a monopotassium salt.
  • the aqueous solution was subjected to reduced pressure to remove the
  • the salt is converted to the acid by careful acidification.
  • the following salts are prepared by reacting an appropriate phosphate salt and Compound III in the manner described in Example 21 and concentrating under vacuum.
  • Calcium stearate 2.5 The finely powdered ingredients are mixed well and granulated with 10% starch paste. The granulation is dried and compressed into tablets.
  • a uniform mixture of the ingredients is prepared by blending and used to fill two-piece hard gelatin capsules.
  • 250 ml of an injectable solution are prepared by conventional procedures having the following formulation:
  • An aerosol composition may be prepared having the following formulation:
  • Compound Z the starting material, was prepared by inoculating 54 milliliters of P34-2 medium of the following composition per liter: corn steep liquor, 5 g; D-mannitol 25 g; glucose
  • a 25 liter sample of the resulting broth was then used to inoculate three fermenters each containing 475 liters of P34-2 medium containing 2 ml/L of P-2000 and cultivated for four days at 25oC, air flow of 250 L/min, pressure 0.7 kg/cm 2 gauge and 150 rpm.
  • the broth from the foregoing cultivation was first extracted with an equal volume of methanol.
  • the methanol-broth was clarified using a liquid-solid separator (centrifuge) to obtain clarified liquid as first extract and solid.
  • the extraction-clarification was repeated.
  • the extracts were combined and the water cont at adjusted to about 50 percent.
  • the resulting solution was passed through a "DIAION" SP-207 adsorption column to adsorb Compound III and the column washed with aqueous methanol. Thereafter Compound III was recovered with 100 percent methanol.
  • the water content of the methanol containing Compound III was adjusted to 50 percent and the aqueous methanol solution intimately mixed with an equal volume of 1:1 ethyl acetate/hexane and the two liquid phases thereafter separated.
  • the aqueous methanol layer was passed through a column of
  • the starting material also may be prepared by methods described in copending applications Serial Nos. 47/492,025 and 47/492,026.
  • N-methyl-N'-nitro-N-nitrosoguanidine (b) cultivating, thereafter (c) plating a portion of the growth on potato dextrose agar and (d) incubating for 14 days at 25oC to obtain spores then (e) harvesting the spores, (f) diluting the spores with sterile saline (g) plating on potato dextrose agar (h) incubating for 7 days for colony formation, (i) transferring separate colonies to slants of potato and (j) incubating for 14 days at 25oC.
  • Rhizopus oryzae MF 4974 cultivated on oatmeal agar, were inoculated in a
  • Soy-Glucose medium consisting of 20.0 g dextrose, 5.0 g Fidco yeast extract, 5.0 g NaCl, 5.0 g K 2 HPO 4 in a liter of distilled water.
  • the pH of the medium was adjusted to 7.0 before autoclaving.
  • the cultures were incubated at 27oC for 24 hours on a rotary shaker at 220 rpm.
  • a fresh Soy-Glucose medium was inoculated with the 24-hour seed culture at a ratio of 5% (v/v) and the fermentations were continued in the manner described above.
  • Cell free extracts were obtained by suspending the cells in a buffer containing 100 mM phosphate (pH 7.5) and 2 mM EDTA. The cells to buffer ratio was 2:1. Several procedures were then tried to break the cell membranes and release the cell contents, i.e., a high-pressure French press, sonication, grinding the frozen cells, and lysozyme treatment.
  • the enzyme reaction was carried out in 0.2 ml mixture containing 0.15 mM FK-520, 5 mM MgCl 2 , 5 ⁇ M ATP and various amount of enzyme.
  • the reaction mixture was incubated at different temperatures for 1 hour and terminated by the addition of 0.2 ml methanol.
  • the resulting solution was subjected to HPLC analysis on Whatman Partisil 10 ODS-3 at 55oC.
  • the column was developed at 1 ml/min with a linear gradient from 45% acetonitrile in 0.1% phosphoric acid to 807. in 0.1% phosphoric acid in 30 minutes.
  • FK-520 was 16,5 and 20 minutes, respectively.
  • the Km value for the substrate (FK-520) was about 0.5 mM and the Vmax was 2. In mole/min/mg protein. To determine metal ion requirement the crude enzyme was dialyzed thoroughly against 20 mM Tris-HCl buffer and 2 mM EDTA. The results show that the phosphorylating activity requires Mg +2 or Ca +2 as cofactor.
  • Example 3 produced two phosphorylated compounds, L-706, 546 and L-706, 527.
  • the preparation and properties of simvastatin are described in EP Publication No. 0033538 to Merck &. Co., Inc.
  • the compounds exhibit inhibition in the biosynthesis of cholesterol as also described for simvastatin in EP Publication No. 0 033 538.
  • Rhizopus arrhizus MF 4974
  • spore suspension harvested from oatmeal agar, wea incubated into 500 mL Soy-Glucose medium contained in a 2 liter Erlenmeyer flask and shaken a 27°C on a rotary shaker (220 rpm) for 24 hours.
  • the whole broth (1000 mL) was diluted with an equal volume of methanol, than
  • Preparative HPLC was carried out on Whatman Magnum 9 Partisil 10 ODS-3 column (C 18 , 9.8 mm ID x 25cm) at room temperature and monitored at 237 nm.
  • the column was
  • the buffer solution was incubated at 37oC for 4 hours. After incubation, 300 uL was removed and analysed by HPLC, using a Whatman Partisil 10 0DS-3 column (C 18 10 urn, 4.6 mm ID x 25 cm) at room temperature and monitored at 237 nm. The column was developed at 1 mL/min with a linear gradient gradient from 20%

Abstract

Described is a new microbial biophosphorylation process for regiospecifically phosphorylating the hydroxyl group, in a ''phosphate active'' hydroxyl containing organic compound, under biotransformation conditions utilizing the microorganism, Rhizopus oryzae ATCC No. 11145. The phosphorylated compounds produced by the process include e.g., macrolide FK-506 type immunosuppressants which are useful in preventing human host rejection of foreign organ transplants, e.g. bone marrow, liver, kidney, lung and heart transplants. Other hydroxyl containing compounds, applicable in this process include rapamycin, echinocandins, HIV protease inhibitors, simvastatin and zearalenone.

Description

TITLE OF THE INVENTION
NEW PROCESS FOR BIOPHOSPHORYLATING ORGANIC COMPOUNDS
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a combined continuation-in-part application of SN 594,500 (Case 18238) filed October 9, 1990; SN 594,214 (Case 18240) filed October 9, 1990; SN 595,894 (Case 18208) filed October 11, 1990; SN 691,606 (Case 18261) filed
April 26, 1991; SN 691,607 (Case 18382) filed
April 26, 1991; SN 701,387 (Case 18340) filed
May 16, 1991; and SN 735,963 (Case 18462) filed
July 25, 1991.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a new regio- specific biophosphorylation process for producing phosphorylated derivatives of "phosphate active" hydroxyl containing organic compounds utilizing the microorganism Rhizopus oryzae. ATCC No. 11145. The process involves contacting the microorganism and an organic compound containing a free phosphate active hydroxyl group, under biotransformation conditions, which phosphorylate the hydroxyl group. The process involves either resting Rhizopus cells or culturing the microorganism in the presence of the organic compound.
Rhizopus oryzae ATCC No. 11145 is known in the art primarily as a hydroxylating agent. It also degrades insect moulting hormones (J.C.S. Chem. Comm. 1974: 656-657, 1974); participates in hydroxylation of steroids (Can, J, Chem. 57:436-440 and 1585-1587, 1979; ibid., 59; 1651-1655, 1981; ibid., 63: 1127- 1131, 1985; H.J. Peppier, ed., Microbial Technology Reinhold, New York, p. 288-297, 1967; U.S. Patent 2,646,370); is involved with transformations of sesquiterpene lactone costunolide (J.C.S, Perkin 1: 3022-3028, 1979); produces 16-hydroxyverrucarin A and B and 3'-hydroxyverrucarin A by transformation of verrucarins A and B (Appl. Environ, Microbiol, 46:480-483, 1983); and produces OH-products of
imipramine (J. Pharmaceut. Sci. 70: 151-153, 1981). However, in the above references, there is no
description of its ability as a regiospecific
hydroxyl phosphorylating agent.
Rhizopus is a genus of fungi which commonly occurs on mature fruits, grain and vegetables, as well as soil. Typically they are saprobes and facultative parasites, and form a branched, aseptate mycelium. Rhizopus spp are used commercially, e.g., in the preparation of carboxylic acids or steroids, or in the metabolism of hydrocarbons. Culture or fermentation of Rhizopus arrhizus ATCC 11145 is straightforward, conventional and well known. See, for example, U.S. 4,410,629. Adaptations, modifications and variations of the protocol are given in the Examples are within the routine skill of the fermentation microbiologist.
Regiospecific biophosphorylating agents are desired in the art since standard chemical phosphorylation using, e.g., POCI3 or PCI5, are generally non-regiospecific and do not produce selectively phosphorylated compounds, which can lead to improved water solubility and pharmacokinetics.
ATCC PEPOSIT
A sample of MF 4974 has been deposited at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville MD 20852. The culture access designation is ATCC No. 11145. BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is an 1H nuclear magnetic resonance (NMR) spectrum taken at 400 MHz of C-32 phosphorylated FR-900520 in CDCI3 .
Figure 2 exhibits the assigned molecular structure for C-32 phosphorylated FR-900520.
Figure 3 is % nuclear magnetic resonance (NMR) spectrum taken at 400 MHz of the C-43
methylated phosphate ester of the phosphorylated rapamycin macrolide in MeOH.
Figure 4 exhibits the assigned molecular structure for the C-43 phosphorylated rapamycin macrolide. Figure 5 is 1H nuclear magnetic resonance spectrum of echinocandin IIIA.
SUMMARY OF THE INVENTION
We have discovered a new overall process for selectively biophosphorylating an organic compound containing "phosphate active" hydroxyl groups. The organic compound is suspended in an aqueous medium with a strain of Rhizopus oryzae ATCC No. 11145 for a sufficient time to produce the phosphorylated organic compound.
By the term "phosphate active hydroxyl containing organic compound" as used herein is meant a compound containing a hydroxyl group which can stereo- specifically interact with the Rhizopus microorganism to undergo phosphorylation under biotransformation conditions . A simple test with the organic compound under the biotransformation conditions described herein will determine, without undue experimentation, if the hydroxyl group in the organic molecule is phosphate active.
The process involves contacting resting
Rhizopus cells in a phosphate buffered medium containing e.g. glycerol, as a carbon nutrient, or by the fermentation of the microorganism Rhizopus oryzae, ATCC No. 11145 together, in the presence of the hydroxyl containing organic compound, e.g., an FK-506 type macrolide immunosuppressant i.e. FK-520, under submerged aerobic conditions in an aqueous carbohydrate medium, containing a nitrogen nutrient, said conditions being conducted at a pH of about 7 for a sufficient time, e.g. 24 hours at 27ºC, to selectively biophosphorylate the phosphate active hydroxyl group. Either process can be utilized but preferred is the process using resting cells.
It will be recognized by those skilled in the art that phosphorylation by Rhizopus oryzae ATCC 11145 is not limited to that particular strain. Rather, other Rhizopus oryzae strains can also be expected to be capable of acting to perform hydroxyl phosphorylation of hydroxy containing organic compounds.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
In accordance with this invention there is provided a process for producing a biophosphorylated hydroxyl containing organic compound, wherein said hydroxyl group is phosphate reactive, comprising the step of contacting a strain of Rhizopus oryzae ATCC No. 11145 microorganism together with the hydroxyl containing organic compound in an aqueous medium containing a carbon nutrient at ambient temperature for a sufficient time to produce the biophosphorylated hydroxyl containing organic compound.
The present invention involves a biotransformation process, which involves the contacting of resting cells, or the fermentation of, the microorganism, Rhizopus oryzae, together with an organic compound containing a free hydroxy group, to produce the phosphorylated derivative. The microorganism is currently on deposit with the American Type Culture Collection, 12301 Parklawn Drive in Rockville,
Maryland as ATCC No. 11145. The scope of the compounds included within the "phosphate reactive hydroxyl containing organic compounds" includes a C-32 hydroxy-containing macrolide as described in Fujisawa's USP 4,894,366 of the formula:
Figure imgf000008_0001
wherein
R is H, C1-C4 alkyl,
R2 is hydrogen, hydroxy or lower alkanoyloxy,
R3 is methyl, ethyl, propyl or allyl,
n is an integer of 1 or 2, and the symbol of a line and dotted line is a single bond or a double bond, and a pharmaceutically acceptable basic salt thereof. Specifically included is where the compound FK-506 is where R is methyl, R2 is hydroxy, R3 is allyl, n is 2 and the double bond is absent; and the compound FK-520 is where R is methyl, R2 is hydroxy, R3 is ethyl, n is 2 and the double bond is absent.
Also included are phosphate reactive hydroxyl containing compounds including the following FK-506 type compounds (from USP 4,894,366 to Fujisawa): 7-Allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo- [22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone, 1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)- 1-methylvinyl]-23,25-dimethoxy-13,19,17,21,27-penta- methyl-11,28-dioxa-4-azatricyclo[22.3.1.0 4 ,9]octacos- 18-ene-2,3,10,16-tetraone, 16-Allyl-1,13-dihydroxy-11-[-2-(4-hydroxy-3- methoxycyclohexyl)-1-methylvinyl]-22,24-dimethoxy- 12,18,20,26-tetramethyl-10,27-dioxa-4-azatricyclo- [21.3.1.04,8]heptacos-17-ene-2,3,9,15-tetraone. Further included within the term "FK-506 type macrolide" as used herein are specifically the compounds disclosed in Fisbn's EPO 0 323 042 of the formula:
Figure imgf000010_0001
wherein each vicinal pair of substituents [R1 and R2], [R3 and R4], [R5 and R6] independently:
a) represent two vicinal hydrogen atoms, or b) form a second bond between the vicinal carbon atoms to which they are attached; in addition to it significance above,
R2 can represent an C1-C10 alkyl group;
R7 represents H, OH or O-C1-C10 alkyl, or in conjunction with R1 it may represent =0;
R8 and R9 independently represent H or OH;
R10 represents H; C1-C10 alkyl, wherein said alkyl can be substituted by one or more hydroxyl groups; C1-C10 alkenyl, which can be substituted by one or more hydroxyl groups, or C1-C10 alkyl substituted by =0;
X represents 0, (H, OH), (H,H) or -CH2O-;
Y represents 0, (H, OH), (H,H), N-NR11R12 or N-OR13 wherein, R11 and R12 independently represent H, C1-C10alkyl, C1-C10aryl or tosyl, and
R13, R14, R15, R16, R17, R18, R19, R22 and R23 independently represent H or C1-C10 alkyl; R20 independently represents 0, or it can independently represent (R20a,H); wherein R20a independently represents OH, O-C1-C10 alkyl or OCH2OCH2CH2OCH3; n is 1,2, or 3;
in addition to their significances above, Y, R10 and R23, together with the carbon atoms to which they are attached, can represent a 5- or 6- membered N-, S- O- containing heterocyclic ring, which is saturated or unsaturated, and which can be substituted by one or more groups selected from C1-C10 alkyl, hydroxyl, C1-C10 alkyl substituted by one or more hydroxyl groups, O-C1-C10 alkyl, benzyl and -CH2Se(C6H5);
provided that when X and Y both represent 0 ; R9 represents OH; R14 , R15 , R16 , R17 , R18 , R19 and R22 each represent methyl; R20 a represents OCH3; R8 and R23 each represent H; [R3 and R4] and [R5 and R6] each represent a carbon-carbon bond;
and pharmaceutically acceptable salts thereof, which includes acid addition salts of any amine groups present. Preferably when R2, R7, R11, R12, R13 , R14, R15, R16, Ri7, R18, R19, R20a, R22 and R23 comprise carbon-containing groups, those groups contain up to 10 carbon atoms, more preferably from 1 to 6, e.g., methyl or methoxyl.
Also preferred is wherein each of R14, R15, R16, R17, R18, R19 and R22 represents methyl.
The alkyl groups: R2, R7, R11, R12, R13, R14, R15, R16, R17, R18, R19, R20a, R22 and R23 can comprise straight chain, branched and cyclic groups.
Alkyl groups substituted by =0, include R10 which can represent include ketone and aldehyde groups.
Preferably, R10 is allyl (i.e.,
prop-2-enyl), propyl ethyl or methyl.
Preferably, n is 2;
R7 is H or OH;
R1 and R2 both represent H;
X is preferably 0 or (H, OH);
R20a represents OH or OCH3; and
when Y, R10 and R23 together represent a N-,S- or O- containing heterocyclic ring, preferred is where that ring is five-membered, more preferably a pyrrole or tetrahydrofuran ring.
Preferred embodiments as products are C-32 phosphorylated FK-506,
Figure imgf000013_0001
and C-32 phosphorylated FK-520,
Figure imgf000014_0001
and C-32 phosphorylated FK-523;
Figure imgf000015_0001
and the corresponding C-32 phosphorylated C-31 desmethyl analogs, and also the following specific compounds from EPO 323,042 to Fisons:
1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)- 1-methylvinyl]-18-[(phenylseleno)methyl]-16,26,28- trimethoxy-13,22,24,30-tetramethyl-11,17,31-trioxa-4- azatetracyclo[25.3.1.04,9.016,20]hentriacont-21-ene- 2,3,10-trione,
17-Allyl-1,2,14-trihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo- [22.3.1.04,9]octacos-18-ene-3,10,16-trione, 17-Allyl-1,2,14,16-tetrahydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo- [22.3.1.04,9]octacos-18-ene-3,10-dione,
17-Propyl-1,14-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo- [22.3.1.04'9]octacosane-2,3,10,16-tetraone,
17-Propyl-1,14-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-l-methylethyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo- [22.3.1.04'9]octacos-18-ene-2,3,10,16-tetraone,
17-Propyl-1,14-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-1-methylethyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo- [22.3.1.04'9]octacosane-2,3,10,16-tetraone,
17-Propyl-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclo- hexyl)-1-methylvinyl-23,25-dimethoxy-13,19,21,27- tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]- octacosa-14,18-diene-2,3,10,16-tetraone,
17-Propyl-1-hydroxy-12-[2-(4-hydroxy-3-methoxycyclo- hexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27- tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04'9]- octacos]-18-ene-2,3,10,16-tetraone, 17-Allyl-1,14,20-trihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-
13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-
[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone,
17-(1-Hydroxyproρ-2-enyl)-1,14,20-trihydroxy-12- [2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl- 23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa- 4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16- tetraone,
17-Allyl-1,2-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo- [22.3.1.04,9]octacos]-18-ene-3,10,16-trione,
17-Allyl-1,16-dihydroxy-12-[2-(4-hydroxy-3-methoxy- cyclohexyl)-1-methylvinyl]-23,25-dimethoxy-
13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-
[22.3.1.04'9]octacosa]-14,18-diene-2,3,10-trione,
17-Allyl-1-hydroxy-12-[2-(4-hydroxy-3-methoxy- cyclohexyl)-1-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo- [22.3.1.04,9]octacos]-18-ene-2,3,10,16-tetraone,
17-(2,3-Dihydroxypropyl)-1,14-dihydroxy-12-[2-(4- hydroxy-3-methoxycyclohexyl)-1-methylvinyl-23,25-di- methoxy-13,19 21,27-tetramethyl- 1,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ent-2,3,10,16-tetraone, 17-Ethanalyl-1,14-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo- [22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone,
1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclo- hexyl)-1-methylvinyl]-26,28-dimethoxy-13,22,24,30- tetramethyl-11,31-dioxa-4,17-diazatetracyclo- [25.3.1.04,9.016,20]hentriaconta-16(20),18,21- triene-2,3,10-trione,
1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)- 1-methylvinyl]-26,28-dimethoxy-17-(2-hydroxyethyl)- 13,22,24,30-tetramethyl-11,31-dioxa-4,17-diazatetra- cyclo[25.3.1.0.4,9.016,20]hentriaconta-16(20),18,21- triene-2,3,10-trione,
1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)- 1-methylvinyl]-26,28-dimethoxy-13,22,24,30-tetra- methyl-17-phenylmethyl-11,31-dioxa-4,17-diazatetra- cyclo-[25.3.1.04,9.016,20]hentriaconta-16(20),18,21- triene-2,3,10-trione,
1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)- 1-methylvinyl]-26,28-dimethoxy-13,22,24,30-tetra- methyl-17-phenylmethyl-11,31-dioxa-4,17-diazatetra- cyclo[25.3.1.04,9.016'20]hentriaconta-16(20),18,21- triene-2,3,10-trione, 17-Allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxy- cyclohexyl)-1-methylvinyl]-23,25-dimethoxy-
13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-
[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone C16 oxime,
17-Allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxy- cyclohexyl)-1-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo- [22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone C16 oxime O-methyl ether,
17-Propyl-1-hydroxy-12-[2-(3,4-dihydroxycyclohexyl)-1- methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl- 11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene- 2,3,10,16-tetraone,
1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)- 1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetra- methyl-17-(2-oxopropyl)-11,28-dioxa-4-azatricylo- [22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone,
17-Allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-11,28-dioxa-4-aza-spiro-
[tricyclo[22.3.1.0.4,9]octacos-18-ene-2,2'-oxirane,- 3,10,16-trione,
17-Ethanalyl-1,2,14-trihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl-1-methylvinyl-23,25-dimethoxy- 13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo- [22.3.1.04,9]octacos-18-ene-3,10,16-trione, 1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)- 1-methylvinyl]-26,28-dimethoxy-18-[(phenylseleno)- methyl-13,22,24,30-tetramethyl-11,17,31-trioxa-4-azatetracyclo[25.3.1.0.4,90.16,20]hentriaconta-16(20),21- diene-2,3,10-trione,
4-Methyl-[17-Allyl-1,14-dihydroxy-12-[2-(4-hydroxy- 3-methoxycyclohexyl-1-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo- [22.3.1.04,9]octacos-18-ene-2,3,10-trione-16-ylidene]- hydrazide, or a pharmaceutically acceptable salt of any one thereof,
In general, the phosphorylated organic compound can be produced by either contacting resting cells of Rhizopus, in an aqueous buffered phosphate medium containing a carbon nutrient at ambient
temperature, or by culturing (fermenting) the above- described microorganism, Rhizopus oryzae, in the presence of the hydroxy containing organic compound in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen, preferably under submerged aerobic conditions (e.g. shaking culture, submerged culture, etc.). The aqueous medium is preferably maintained at a pH of about 7 at the initiation and termination (harvest) of the fermentation process. A higher pH leads to substantial and/or total loss of product. The desired pH may be maintained by the use of a buffer such as morpholino- ethanesulfonic acid (MES), morpholinopropanesulfonic acid (MOPS), and the like, or by choice of nutrient materials which inherently possess buffering properties, such as production media described hereinbelow.
The preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, xylose, galactose, glycerin, starch, dextrin, and the like. Other sources which may be included are maltose, rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, and the like.
The preferred sources of nitrogen are yeast extract, meat extract, peptone, gluten meal, cottonseed meal, soybean meal and other vegetable meals (partially or totally defatted), casein hydrolysates, soybean hydrolysates and yeast hydrolysates, corn steep liquor, dried yeast, wheat germ, feather meal, peanut powder, distiller's solubles, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acids, and the like.
The carbon and nitrogen sources, though advantageously employed in combination, need not be used in their pure form, because less pure materials which contain traces of growth factors and considerable quantities of mineral nutrients, are also suitable for use. When desired, there may be added to the medium mineral salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, cobalt salts, and the like. If necessary, especially when the culture medium foams seriously, a defoaming agent, such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone may be added. As one of the starting materials in
the process, the FK-520 starting material can be obtained by the fermentation of S. hygroscopicus var. ascomyceticus, ATCC No. 14891, as described in U.S. Patent 3,244,592, and by the fermentation of S. hygroscopicus subsp. yakushimaensis No. 7278, (to produce FR-900520, or "FK-520", and the other FK-506 type macrolides can be obtained by the processes) as described in EPO Publication No. 0184162 to Fujisawa, and PCT WO 89/05304 to Fisons, said above references hereby incorporated by reference for this particular purpose.
As to the conditions for the production of phosphorylated organic compound in massive amounts, submerged aerobic culturel conditions are preferred therefor. For the production in small amounts, a shaking or surface culture in a flask or bottle is employed. Furthermore, when the growth is carried out in large tanks, it is preferable to use the vegetative form of the organism for inoculation in the production tanks in order to avoid growth lag in the process of production. Accordingly, it is desirable first to produce a vegetative inoculum of the organism by inoculating a relatively small quantity of culture medium with spores or mycelia of the organism produced in a "slant" and culturing said inoculated medium, also called the "seed medium", and then to transfer the cultured vegetative inoculum aseptically to large tanks. The fermentation medium, in which the inoculum is produced, is substantially the same as or different from the medium utilized for the production of the phosphorylated organic compound and is generally autoclaved to sterilize the medium prior to inoculation. The pH of the medium is generally adjusted to about 7.0 prior to the autoclaving step by suitable addition of an acid or base, preferably in the form of a buffering solution.
Agitation and aeration of the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor, by various pumping equipment or by the passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture.
The fermentation is usually conducted at a temperature between about 20ºC and 40ºC, preferably 25-35ºC, for a period of about 10 hours to 24 hours, which may be varied according to fermentation conditions and scales. Preferably, the production cultures are incubated for about 24 hours at 27ºC on a rotary shaker operating at 220 rpm, wherein the pH of the fermentation medium is maintained at 7.0 to harvest.
Preferred culturing/production media for carrying out the fermentation include the following media:
Seed and Transformation Medium g/L
Glucose 20.0
Soy Flour (Sigma) 5.0
Yeast Extract (Fidco) 5.0 NaCl 5.0
K2HPO4 5.0
Adjust pH to 7.0 The produced phosphorylated organic compound can be recovered from the culture medium by conventional means which are commonly used for the recovery of other known biologically active substances. The phosphorylated substance produced is found in the cultured mycelium and filtrate, and accordingly can be isolated and purified from the mycelium and the filtrate, which are obtained by filtering or centrifuging the cultured broth, by a conventional method such as concentration under reduced pressure, lyophilization, extraction with a conventional solvent, such as methanol and the like, pH adjustment, treatment with a conventional resin (e.g. anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g. activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), crystallization, recrystallization, and the like. A preferred method is solvent extraction, particularly using methanol.
The phosphorylated organic compound obtained according to the resting cell or fermentation processes as explained above can be isolated and purified in a conventional manner, for example, extraction, precipitation, fractional crystallization, recrystallization, chromatography, and the like.
Suitable formulations of the material may also include conventional pharmaceutically acceptable biolabile esters of phosphorylated organic compound, formed via the hydroxy groups on the molecule, such as the acetate.
The phosphorylated organic compound, and particularly that of FK-520, of the present invention are water soluble possesses pharmacological activity such as immunosuppressive activity, antimicrobial activity, and the like, and therefore are useful for the treatment and prevention of the transplantation rejection of organs or tissues such as heart, kidney, liver, medulla ossium, skin, etc., graft-versus-host diseases by medulla ossium transplantation, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, and the like.
The pharmaceutical composition of this invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains the instant invention compounds, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications.
The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. The carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form, and in addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used. The active object compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of diseases.
For applying this composition to a human, it is preferable to apply if by parenteral or enteral administration. While the dosage of therapeutically effective amount of the C-32 phosphorylated FK-506, varies from, and also depends upon the age and condition of each individual patient to be treated, a daily dose (calculated on the basis of a 70 kg man) of about 0.01-1000 mg, preferably 0.1-500 mg and more preferably 0.5-100 mg, of the active ingredient is generally given for treating diseases, and an average single dose of about 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250 mg and 500 mg is generally
administered.
Rapamycin
It has been further found that a new immunosuppressant, a phosphorylated rapamycin
macrolide, can be obtained via the present process by contacted resting Rhizopus cells in a phosphate buffered medium containing glycerol as a carbon nutrient, or by the fermentation of the microorganism Rhizopus oryzae, ATCC No. 11145 together, in the presence of the macrolide rapamycin, under submerged aerobic conditions in an aqueous carbohydrate medium, containing a nitrogen nutrient, said conditions being conducted at a pH of about 7 for a sufficient time, e.g. 24 hours at 27°C, to selectively C-43 phosphory- late the rapamycin type macrolide. Either process can be utilized but preferred is the process using resting cells. The resultant C-43 phosphorylated macrolide exhibits immunosuppressant activity, similar to rapamycin i.e., inhibition of T-cell proliferation of mouse T lymphocytes stimulated with the combination of interleukin-2 plus PMA. A positive sample in this assay will inhibit T-cell proliferation, as indicated by reduced tritiated thymidine uptake.
Also, in accordance with this invention, there is provided a process for producing an
immunosuppressant, identified as a phosphorylated macrolide comprising the step of contacting a strain of a Rhizopus microorganism capable of phosphorylating free hydroxy groups, e.g., Rhizopus oryzae, and specifically Rhizopus oryzae ATCC No. 11145, together with rapamycin macrolide (See U.S. Patent 3,929,992 for its preparartion), in an aqueous medium,
containing a carbon nutrient, preferably at a pH below about 8.0, for a sufficient time to produce the C-43 phosphorylated macrolide.
Also provided is the unfiltered broth produced by the above process, which exhibits
positive inhibition of T-cell activation.
Also provided is a new immunosuppressant being a phosphorylated macrolide which exhibits a proton nuclear magnetic resonance spectrum as identified in Figure 3, and a molecular weight of 993 as obtained by (FAB) mass spectrometry.
Also provided is a pharmaceutical
composition for the treatment of immunoregulatory disorders and diseases containing a therapeutically effective amount of the phosphorylated macrolide in combination with a pharmaceutically acceptable, substantially non-toxic carrier or excipient. In addition, there is provided a method of use for treating a human host to prevent organ transplantation rejection, i.e., heart, kidney, liver, lung, bone marrow, or for treating autoimmune diseases i.e., juvenile diabetes mellitus comprising administering to said host a therapeutically effective amount of the phosphorylated macrolide.
The present invention involves a biotransformation process, which involves the contacting of resting cells, or the fermentation of, the microorganism, Rhizopus oryzae, together with a rapamycin macrolide containing a free hydroxy group, to produce the phosphorylated macrolide derivative. The microorganism is currently on deposit with the American Type Culture Collection, 12301 Parklawn Drive in
Rockville, Maryland as ATCC No. 11145.
By the term "phosphorylated macrolide" as used herein is meant the compound having a proton NMR spectrum as illustrated in Figure 3 (of the methylated derivative), a mass spectrum molecular ion of 993, and an assigned molecular structure of the formula:
Figure imgf000029_0001
where R= [HO]2-
Figure imgf000029_0002
In general, the C-43 phosphorylated macrolide can be produced by either contacting resting cells, of Rhizopus in an aqueous buffered phosphate medium containing a carbon nutrient at ambient temperature, or by culturing (fermenting) the above-described microorganism, Rhizopus oryzae, in the presence of the known rapamycin macrolide, described in U.S. Patent 3,929,992, in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen, preferably under submerged aerobic conditions (e.g. shaking culture, submerged culture, etc.). The aqueous medium is preferably maintained at a pH of about 7 at the initiation and termination (harvest) of the fermentation process. A higher pH leads to substantial and/ or total loss of product. The desired pH may
be maintained by the use of a buffer such as
morpholinoethanesulfonic acid (MES), morpholino- propanesulfonic acid (MOPS), and the like, or by choice of nutrient materials which inherently
possess buffering properties, such as production media described hereinbelow.
The preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, xylose, galactose, glycerin, starch, dextrin, and the like. Other sources which may be included are maltose, rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, and the like.
The preferred sources of nitrogen are yeast extract, meat extract, peptone, gluten meal, cottonseed meal, soybean meal and other vegetable meals (partially or totally defatted), casein hydrolysates, soybean hydrolysates and yeast hydrolysates, corn steep liquor, dried yeast, wheat germ, feather meal, peanut powder, distiller's solubles, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acids, and the like.
The carbon and nitrogen sources, though advantageously employed in combination, need not be used in their pure form, because less pure materials which contain traces of growth factors and considerable quantities of mineral nutrients, are also suitable for use. When desired, there may be added to the medium mineral salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, cobalt salts, and the like. If necessary, especially when the culture medium foams seriously, a defoaming agent, such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone may be added.
The rapamycin starting material can be obtained by the known fermentation of S. hygroscopicus, NRRL No. 5491, as described in U.S. Patent 3,929,992.
As to the conditions for the production of the phosphorylated macrolide in massive amounts, submerged aerobic cultural conditions are preferred therefor. For the production in small amounts, a shaking or surface culture in a flask or bottle is employed. Furthermore, when the growth is carried out in large tanks, it is preferable to use the vegetative form of the organism for inoculation in the production tanks in order to avoid growth lag in the process of production. Accordingly, it is desirable first to produce a vegetative inoculum of the organism by inoculating a relatively small quantity of culture medium with spores or mycelia of the organism produced in a "slant" and culturing said inoculated medium, also called the "seed medium", and then to transfer the cultured vegetative inoculum aseptically to large tanks. The fermentation medium, in which the inoculum is produced, is substantially the same as or different from the medium utilized for the production of the phosphorylated macrolide and is generally autoclaved to sterilize the medium prior to inoculation. The pH of the medium is generally adjusted to about 7.0 prior to the autoclaving step by suitable addition of an acid or base, preferably in the form of a buffering solution.
Agitation and aeration of the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor, by various pumping equipment or by the passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture.
The fermentation is usually conducted at a temperature between about 20ºC and 40ºC, preferably 25-35ºC, for a period of about 10 hours to 24 hours, which may be varied according to fermentation conditions and scales. Preferably, the production cultures are incubated for about 24 hours at 27ºC on a rotary shaker operating at 220 rpm, wherein the pH of the fermentation medium is maintained at 7.0 to harvest.
Preferred culturing/production media for carrying out the fermentation include the following media: Seed and Transformation Medium g/L
Dextrose 20.0 Soy Meal (Sigma) 5.0
Yeast Extract (Fidco) 5.0
NaCl 5.0 K2HPO4 5.0
Adjust pH to 7.0 The produced phosphorylated macrolide can be recovered from the culture medium by conventional means which are commonly used for the recovery of other known biologically active substances. The phosphorylated macrolide produced is found in the cultured mycelium and filtrate, and accordingly can be isolated and purified from the mycelium and the filtrate, which are obtained by filtering or centrifuging the cultured broth, by a conventional method such as concentration under reduced pressure, lyophilization, extraction with a conventional solvent, such as methanol and the like, pH adjustment, treatment with a conventional resin (e.g.anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g. activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), crystallization, recrystallization, and the like. A preferred method is solvent
extraction, particularly using methanol.
The product phosphorylated macrolide from the fermentation exhibits positive immunosuppressive activity by the "T-cell proliferation assay" and possesses utility on this basis and exhibits the following physical characteristics:
1. White amorphous powder
2. Solubility in methanol
3. Molecular weight of 993, as determined by
FAB mass spectroscopy and is consistent with the assigned molecular structure in Figure 3. The phosphorylated macrolide obtained according to the resting cell or fermentation processes as explained above can be isolated and purified in a conventional manner, for example, extraction, precipitation, fractional crystallization, recrystallization, chromatography, and the like.
Suitable formulations of the material may also include conventional pharmaceutically acceptable biolabile esters of the phosphorylated macrolide, formed via the hydroxy groups on the molecule, such as the acetate.
It is to be noted that in the aforementioned fermentation reactions and the post-treatment of the fermentation mixture therein, the tautomeric and conformational isomer(s) of the phosphorylated
macrolide, including those due to rearrangement of the hemiketal ring system are also included within the scope of the present invention.
The phosphorylated macrolide of the present invention possesses pharmacological activity such as immunosuppressive activity, antimicrobial activity, and the like, and therefore are useful for the
treatment and prevention of the transplantation rejection of organs or tissues such as heart, kidney, liver, medulla ossium, skin, etc., graft-versus-host diseases by medulla ossium transplantation, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, and the like.
The pharmaceutical composition of this invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains the instant invention compounds, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications.
The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. The carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form, and in addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used. The active object compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of diseases.
For applying this composition to a human, it is preferable to apply if by parenteral or enteral administration. While the dosage of therapeutically effective amount of the phosphorylated macrolide, varies from, and also depends upon the age and condition of each individual patient to be treated, a daily dose (calculated on the basis of a 70 kg man) of about 0.01-1000 mg, preferably 0.1-500 mg and more preferably 0.5-100 mg, of the active ingredient is generally given for treating diseases, and an average single dose of about 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250 mg and 500 mg is generally
administered.
Echinocandins
The present invention process also is applicable in a process for selectively
biophosphorylating a cyclic lipopeptide related to echinocandins and having a peptide skeleton bearing several hydroxy groups wherein in said phosphorylated cyclic lipopeptide, the phosphate group is attached to the hydroxy group of the 4-hydroxyproline
component of the lipopeptide.
Echinocandins or echinocandin compounds are cyclohexapeptide compounds having a lipophilic side chain and having antifungal properties. Many are natural products but many compounds are
semi-synthetic. The natural products are described in the literature by such names as echinocandins, aculeacins, mulundocandin, by number designations or by structure. Many have been known a long time and the structure and properties may be found summarized in the CRC Handbook of Antibiotic Compounds, Vol IV, Part I, pp 355-367, CRC Press, Inc., Boca Raton, Fla. 1980. Still others include a more recently
discovered compound such as that described in U.S. Patent 4,931,352.
The present invention is especially directed to a compound having the formula (III):
Figure imgf000037_0002
wherein R is - (OH)2 or a cation salt thereof.
By "cation salt" is meant a salt of Li, K, Mg, Na, Ca, and (C1-C4 alkyl)ammonium.
When R is -
Figure imgf000037_0004
(OH)2, the compound may be represented by formula (IIIA), and hereinafter referred to as Compound IIIA.
Figure imgf000037_0001
BRIEF DESCRIPTION OF THE DRAWING
Fig. 5 is nuclear magnetic resonance spectrum of Compound III in which R is -
Figure imgf000038_0001
(OH)(OK).
MASS SPECTRAL DATA
Compound IIIA has a molecular weight of 1144 by FAB-MS (observed (M + Na)+ of 1167). NMR DATA
1 H NMR Spectra of the compound isolated as a monopotassium salt in CD3OD at 400 MHz is seen in Fig. 5; and
13C NMR chemical shifts of the same isolate in CD3OD at 100 MHz are as follows:
δC 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 (doublet, JCP=4.0 Hertz), 74.3, 73.9, 70.6, 70.5, 69.7, 68.3, 62.7, 58.3, 56.3, 56.1, 55.6, 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.
On the basis of these and other data,
Compound IIIA is believed with considerable certainty to have the structure indicated.
Compound IIIA is a white solid soluble in water and polar solvents such as lower alkanols and in dilute alkali metal, magnesium, calcium, and tetra (lower alkyl)ammonium bases. From the bases, salts in which R is a cation salt of phosphate may be obtained. The compound of this invention has similar antibiotic properties as the non-phosphorylated compound and thus would be useful as an antibiotic for the control of parasites, especially Pneumocystis carinii, the causative agent of pneumocystis
pneumonia, a particular problem with immune
compromised patients, and for the control of fungi. Its antifungal properties are particularly useful against certain strains of yeast, such as Candida albicans and Candida tropicalis.
The compound in which R is -
Figure imgf000039_0002
(OH)2 is conveniently produced by incubating a compound having the formula (Z) (Compound Z)
Figure imgf000039_0001
with induced resting cells of Rhizopus arrhizus ATCC 11145 maintained in the Merck Culture Collection as MF 4974. The culture was originally obtained from the American Type Culture Collection at 12301
Parklawn Drive, Rockville, MD 20852.
Compound Z maybe produced by cultivating
Zalarion arboricola ATCC 74030 in nutrient medium as hereinafter described as well as by methods described in copending applications S.N. 492,025, filed
March 12, 1990 and S.N. 492,026, also filed
March 12, 1990.
The microorganism Rhizopus arrhizus ATCC 11145 is also known as Rhizopus oryzae (J.J. Ellis, 1985, Mycologia 77: 243-247). The species has also been described under the names Rhizopus nodosus and Rhizopus tritici. The strain MF 4974, ATCC 11145, exhibits all the essential features of R. arrhizus described by M.A.A. Schipper under the name R.
oryzae, CBS Studies in Mycology 25: 1-19 (1984).
During a recent regrowth of MF4974 ATCC 11145, the following diagnostic characteristics were observed.
The strain is apparently heterothallic because zygospore spore formation was not observed. Colonies grow on most standard mycological media, but on cornmeal agar (Difco) are extremely fast-growing, reaching 35 mm in diameter in 36 hours at 20ºC, reaching >90 mm in 36 hours at 37ºC, with
sporangiophores completely filling Petri dishes, hyaline at first but soon becoming pale yellowish gray to light gray, brownish gray, finally dark gray. Sporangiophores 200-1000 μm tall, 7.5-19 μm wide, aseptate, straight to curved at base, flared at apex, sometimes furcate, with walls slightly
thickened, with minutely granular surface, pale yellowish brown or yellowish gray, arising from rhizoidal hyphae. Rhizoidal hyphae consisting of 3-10, thick, often contorted branches.
Sporangia globose to subglobose, slightly flattened on underside, 100-230 μm in diameter, opaque, with surface spiny, dark gray to black.
Columellae 18-50 μm in diameter, hemispherical to subglobose, smooth, often collapsing, without adhering sporangia remnants. Sporangiospores 5-8 X 2.5-5 μm, subglobose to irregularly elliptical, or angular in sideview, with faint to prominent longitudinal striations, hyaline to pale yellowish brown.
Although the invention is discussed hereinbelow principally with respect to a specific strain, not only the strain described above, but varieties and mutants, whether obtained by natural selection, produced by the action of various mutating agents such as ionizing radiation or chemical agents such as nitrosoguanidine are contemplated within the scope of the present invention.
Compound IIIA may be produced by cultivating Rhizopus arrhizus ATCC 11145 in a suitable nutrient medium containing Compound Z under conditions
hereinafter described and thereafter recovering from the product medium by extracting the desired product from the fermentation medium with a suitable solvent, concentrating the component containing the desired compound, and then subjecting the concentrated material to chromatographic separation. The cultivation is carried out in a medium containing sources of carbon and nitrogen assimilable by the microorganism.
The sources of carbon include glycerol, sugars, sugar alcohols, starches and other carbohydrates, or carbohydrate deivatives such as dextran, cerelose, as well as complex nutrients such as oat flour, corn meal, millet, corn and the like. The exact quantity of the carbon source which is utilized in the medium will depend, in part, upon the other ingredients in the medium, but it is usually found that an amount of carbohydrate between 0.5 and 40% by weight of the medium is satifactory. These carbon sources can be used individually or several such carbon sources may be combined in the same medium.
The sources of nitrogen include amino acids such as glycine, arginine, threonine, methionine and the like, ammonium salt, and complex sources such as yeast hydrolysates, yeast autolysates, yeast cells, tomato paste, soybean meal, casein hydrolysates, yeast extracts, corn steep liquors, distillers
solubles, cottonseed meal, meat extract, and the like. The various sources of nitrogen can be used alone or in combination in amounts ranging form 0.2 to 10 percent by weight of the medium.
In addition, the medium should contain a phosphate salt. The phosphate salt should be at least about 10 percent by weight of the solid components. It is preferably from about 12 to about 15 percent. A particularly suitable medium is soy-glucose medium of the following composition which may be employed both as a seed medium and a culture medium: Soy-Glucose Medium g/1
Glucose 20.0
Soya meal 5.0
Fidco yeast extract* 5.0
NaCl 5.0
K2HPO4 5.0
Adjust pH to 5
--------------
*Fidco yeast extract is a nitrogen source, product of Difco Laboratories, Detroit MI.
The fermentation may be carried out by first preparing a seed culture. In preparing a seed culture, spores of Rhizopus arrhizus are obtained from oatmeal agar slants of MF 4974 maintained in the Merck Culture Collection and dispersed in water to obtain a spore suspension containing about 7 x 109 spores per milliliter.
The spore suspension of MF 4974 is inoculated into a seed flask containing the soy glucose broth and the inoculated suspension incubated on a rotary shaker in the temperature range of from about 15°C to about 30ºC, preferably 25º to 28ºC. The agitation may be up to 400 rpm but generally about 220 rpm is preferred. The incubation is carried out over a period of at least 24 hours to about two days.
When growth is abundant, the mycelia are harvested by filtering through a nylon mesh. For biophosphorylation, the mycelia are suspended in a phosphate buffer containing 3 percent glycerol or some other simple carbon source. Compound Z then is added at a concentration of about 50 μg/ml in
dimethylsulfoxide (DMSO). The pH of the production medium is important. The flasks are incubated, preferably with shaking at 220 rpm at 27ºC for 24 to 48 hours to produce Compound IIIA. It is critical that it be maintained in the range of about 6.0 to 6.3.
After completion of the incubation period the contents of all the flasks are pooled and
filtered through a nylon mesh filter. The mycelial cake on the filter is slurried with aqueous methanol and filtered. The procedure is repeated with the filter cake and the filtrate loaded onto a styrene/ divinylbenzene column. The column is then washed with water and the phosphorylated product eluted with 20 percent aqueous acetonitrile and the remaining metabolite and substrate eluted with 70 percent aqueous acetonitrile.
After elution, the fractions may be assayed by HPLC. The fractions determined to have the desired product as indicated by a retention time of 12.8 mins. are combined and concentrated under reduced pressure to obtain the product as residue.
The salts, i.e., where R is a cationic salt of the phosphate, may be prepared by intimately contacting a base corresponding to the cation in an alcoholic or other polar solvent, then concentrating to initiate crystallization of the salt. Thereafter, the salt is recovered by filtration.
One method of preparing salts is to apply an aqueous solution of the acid onto a non-functionalized resin column. Representative resins include "AMBERCHROM"-161 (divinylbenzene/polysyyrene resin, obtainable from TosoHaas; trademark name registered by Rohm and Haas), "DIAION" HP-20 and SP-207 (cross- linked styrene-divinylbenzene and brominated styrene- divinylbenzene, respectively, products of Mitsubishi Chemical). The column is then washed with aqueous MH2PO4 or M'(H2PO4)2 where M and M' are monovalent and divalent cations respectively, thereby converting the acid to a mono-cation salt form. The column is washed with water to remove excess inorganic phosphate salt. The product M or M' salt is then removed from the column by applying an aqueous eluant having greater than 50 percent organic content. Useful eluants are 80 percent acetonitrile, 80 percent ethanol or 80 percent methanol. The product is isolated by concentration to dryness and/or
lyophilization of the eluate.
This procedure also may be employed to prepare one salt from another.
Alternatively, the acid is dissolved in an aqueous mobile phase containing low amount of organic solvent such as acetonitrile and containing phosphate salt thereby forming a solution of the salt of the acid. The solution is subjected to reduced pressure to remove the acetonitrile, then applied to a C-18 extraction column to retain the salt of the product on the column. The salt of the product then may be removed as above described.
As previously noted, the phosphate is a compound which is active against certain yeast fungi such as C . albicans and C. tropicalis. The activity may be seen in a microbroth dilution assay employing a Yeast Nitrogen Base (Difco) with 1% dextrose (YNBD). In carrying out the assay, Compound IIIA was solubilized in 10 percent dimethyl sulfoxide (DMSO) and diluted to 2560 μg/ml. The compounds were
further diluted to 256 μg/ml in YNBD. Then 0.15 ml of the suspension was dispensed to the first row of a 96-well plate (each well containing 0.15 ml of YNDB) resulting in a drug concentration of 128 μg/ml. Twofold dilutions were then made to obtain final drug concentrations ranging from 128 to 0.06 μg/ml.
The yeast cultures, maintained on Sabouraud dextrose agar were transferred to YM broth (Difco) and incubated overnight at 35°C with shaking (250 rpm). After incubation, each culture was diluted in sterile water to yield a final concentration of 1-5 x 106 colony forming units (CFU)/ml.
96-well microplates were inoculated using a MIC-2000 (Dynatech) which delivers 1.5 μl per well yielding a final inoculum per well of 1.5-7.5 x 103 cells. The microplates were incubated at 35°C for 24 hours. The minimum inhibitory concentrations (MICs) were recorded as the lowest concentrations of drug showing no visible growth.
After recording the MIC, the plates were shaken to resuspend the cells. Thereafter, 1.5 μl samples from the wells in the 96-well microplate were transferred to a single well tray containing Sabouraud dextrose agar. The inoculated trays were incubated 24 hours at 28ºC and then read. The MFC is defined as the lowest concentration of drug showing no growth or less than 4 colonies per spot. Minimum Fungicidal Concentration Fungus (μg/ml)
Strain No. Compound IIIA
Candida albicans
MY 1055 64
MY 1028 64
Candida tropicalis
MY 1012 32
The foregoing illustrates particular suitability for treating mycotic infections.
The compounds of the present invention may be employed in inhibiting or alleviating Pneumocystis carinii infections. In such use, Compound III/IIIA or a composition containing Compound III/IIIA may be administered in a therapeutically effective or
inhibitory amount to subjects infected with or
susceptible to being infected with Pneumocystis carinii.
The suitability of the compounds of the present invention for therapeutic or anti-infective purposes may be determined in studies on immuno- suppressed rats when Sprague-Dawley rats (weighing approximately 200 grams) are immunosuppressed with dexasone in the drinking water (2.0 mg/L) and
maintained on a low protein diet for five weeks to induce the development of pneumocystis pneumonia from a latent infection. Before drug treatment, two rats are sacrificed to confirm the presence of Pneumocystis carinii pneumonia (PCP). Six rats then are inj ected twice daily for four days intravenously (I .V . ) via the tail vein with Compound III in 0.25 ml of vehicle (distilled water). A vehicle control is also carried out. All animals continue to receive dexasone in the drinking water and low protein diet during the treatment period. At the completion of the treatment, all animals are sacrificed, the lungs are removed and processed, and the extent of disease determined by microscopic analysis of stained slides.
A similar experiment may be carried out in which the rats are injected intraperitoneally (I.P.) twice daily for four days and then sacrificed, the lungs removed and processed, and the extent of disease determined by microscopic analysis of stained slides.
The outstanding properties are most effectively utilized when the compound is formulated into novel pharmaceutical compositions with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques.
The novel compositions contain at least a therapeutic antifungal or antipneumocystis amount of the active compound. Generally, the composition contains at least 1% by weight of Compound III.
Concentrate compositions suitable for dilutions prior to use may contain 90% or more by weight.
The compositions include compositions suitable for rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), pulmonary (nasal or buccal inhalation), nasal administration, or insufflation. The compositions may be prepacked by intimately mixing Compound III with the components suitable for the medium desired. When the compound is for antifungal use any method of administration may be used. For treating mycotic infection, oral administration is frequently preferred. When oral administration is to be
employed, it may be with a liquid composition
or a solid composition. For liquid preparations, the therapeutic agent is preferably formulated with water or aqueous compositions, but if desired, may be formulated with glycols, oils, alcohols, and the like. For solid preparations such as capsules and tablets, solid carriers such as starches, sugars, kaolin, ethyl cellulose, calcium and sodium carbonate, calcium phosphate, kaolin, talc, lactose, generally with lubricant such as calcium stearate, together with binders, disintegrating agents and the like.
Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage form. It is especially advantageous to
formulate the compositions in unit dosage form (as hereinafter defined) for ease of administration and uniformity of dosage. Composition in unit dosage form constitutes an aspect of the present invention.
The Compound III is preferably formulated in aqueous therapeutic compositions for intravenous or intraperitoneal injection or aerosol when use against Pneumocystis carinii is contemplated, and may be presented in unit dosage form in ampoules or in multidose containers, if necessary with an added preservative. The compositions may also take such forms as solutions in aqueous vehicles such as 0.85 percent sodium chloride or 5 percent dextrose in water, and may contain formulating agents such as stabilizing and/or dispersing agents. Buffering agents as well as additives such as saline or glucose may be added to make the solutions isotonic. The drug also may be solubilized in alcohol/propylene glycol or polyethylene glycol for drip intravenous administration. Alternatively, the active ingredients may be in powder form for reconstituting with a suitable vehicle prior to administration.
The term "unit dosage form" as used in the specification and claims refer to physically discrete units, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier. Examples of such unit dosage forms are tablets, capsules, pills, powder packets, wafers, measured units in ampoules or in multidose containers and the like. A unit dosage of the present invention may contain from 100 to 1000 milligrams of one of the compounds.
HIV Protease Inhibitors - I
The present invention is also concerned with a compound which inhibits the protease encoded by human immunodeficiency virus (HIV). The compound, or pharmaceutically acceptable salt thereof, is of value in the prevention of infection by HIV, the treatment of infection by HIV and the treatment of the resulting acquired immune deficiency syndrome (AIDS). The present invention also relates to pharmaceutical compositions containing the compounds, and to a method of use of the present compounds with or without other agents for the treatment of AIDS &. viral infection by HIV. A retrovirus designated human immunodeficiency virus (HIV) is the etiological agent of the complex disease that includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system. This virus was previously known as LAV, HTLV-III, or ARV. A common feature of retrovirus replication is the extensive post-trans- lational processing of precursor polyproteins by a virally encoded protease to generate mature viral proteins required for virus assembly and function. Interruption of this processing appears to prevent the production of normally infectious virus. For example, Crawford, S. et al ., J. Virol., 53 , 899, 1985, demonstrated that genetic deletion mutations of the protease in murine leukemia virus which prevent processing of precursor structural proteins result in non-infectious viral particles. Unprocessed structural proteins also have been observed in clones of non-infectious HIV strains isolated from human patients. These results suggest that inhibition of the HIV protease represents a viable method for the treatment of AIDS and the prevention or treatment of infection by HIV.
Nucleotide sequencing of HIV shows the presence of a pol gene in one open reading frame
[L. Ratner, et al., Nature, 313, 277(1985)]. Amino acid sequence homology provides evidence that the pol sequence encodes reverse transcriptase, an endonuclease and an HIV protease [H. Tol, e t al., EMBO J. 4, 1267 (1985); M. D. Power, et al., Science, 231, 1567 (1986); L. H. Pearl, et al., Nature 329, 351 (1987)]. Applicants demonstrate that the compound of this invention is an inhibitor of HIV protease. The compound of this invention provides a prodrug for the inhibition of HIV protease.
A biotransformed compound as herein defined is disclosed. This compound is useful in the inhibition of HIV protease, the prevention of infection by HIV, the treatment of infection by HIV and in the treatment of AIDS and/or ARC, either as a compound, pharmaceutically acceptable salt (when appropriate), pharmaceutical composition ingredient, whether or not as a prodrug or as a combination with other antivirals, anti-infectives, immunomodulators, antibiotics or vaccines. Methods of treating AIDS, methods of preventing infection by HIV, and methods of treating infection by HIV are also disclosed.
This invention is also concerned with the use of a compound given below, or pharmaceutically acceptable salts thereof, in the inhibition of HIV protease, the prevention or treatment of infection by HIV and in the treatment of the resulting acquired immune deficiency syndrome (AIDS). The biotransformed compound is the product of the incubation of Rhizopus arrhizus (ATCC 11145) in the presence of L-702,083, an HIV protease inhibitor. It is defined as follows:
Figure imgf000053_0001
or pharmaceutically acceptable salts, hydrates or esters thereof.
The pharmaceutically-acceptable salts of the compound of the present invention (in the form of water- or oil-soluble or dispersible products) include the conventional non-toxic salts or the quaternary ammonium salts of this compound, which are formed, e.g., from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,
fumarate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalene- sulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-ρropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate,
tosylate, and undecanoate. Base salts include
ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl
sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others. Hydrates or esters are also encompassed by the present invention. Such hydrates or esters are those which would readily occur to the skilled artisan, and include, for example, C1-4alkyl esters.
The compound of the present invention is useful in the inhibition of HIV protease, the
prevention or treatment of infection by the human immunodeficiency virus (HIV) and the treatment of consequent pathological conditions such as AIDS.
Treating AIDS or preventing or treating infection by HIV is defined as including, but not limited to, treating a wide range of states of HIV infection:
AIDS, ARC (AIDS related complex), both symptomatic and asymptomatic, and actual or potential exposure to HIV. For example, the compound of this invention is useful in treating infection by HIV after suspected past exposure to HIV by e.g., blood transfusion, accidental needle stick, or exposure to patient blood during surgery.
For these purposes, the compound of the present invention may be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and vehicles.
Thus, in accordance with the present invention there is further provided a method of treating and a pharmaceutical composition for treating HIV infection and AIDS. The treatment involves administering to a patient in need of such treatment a pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically-effective amount of the compound of the present invention.
These pharmaceutical compositions may be in the form of orally-administrable suspensions or tablets; nasal sprays; sterile injectable preparations, for example, as sterile injectable aqueous or oleagenous suspensions or suppositories.
When administered orally as a suspension, these compositions are prepared according to
techniques well-known in the art of pharmaceutical formulation and may contain microcrystalline
cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweetners/flavoring agents known in the art. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.
When administered by nasal aerosol or inhalation, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other
solubilizing or dispersing agents known in the art.
The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug.
Dosage levels of the order of 0.02 to 5.0 or 10.0 grams-per-day are useful in the treatment or prevention of the above-indicated conditions, with oral doses two-to-five times higher. For example, infection by HIV is effectively treated by the administration of from 10 to 50 milligrams of the compound per kilogram of body weight from one to three times per day. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age of the patient, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
The present invention is also directed to combinations of the HIV protease inhibitor compound with one or more agents useful in the treatment of AIDS. For example, the compound of this invention may be effectively administered, whether at periods of pre-exposure and/or post-exposure, in combination with effective amounts of other AIDS antivirals, immunomodulators, anti-infectives, or vaccines. HIV Protease Inhibitor - II
The present invention is also further concerned with another compound which inhibits the protease encoded by human immunodeficiency virus (HIV). The compound, or pharmaceutically acceptable salt thereof, is of value in the prevention of infection by HIV, the treatment of infection by HIV and the treatment of the resulting acquired immune deficiency syndrome (AIDS). The present invention also relates to pharmaceutical compositions
containing the compound, and to a method of use of the present compound with or without other agents for the treatment of AIDS & viral infection by HIV.
A retrovirus designated human immunodeficiency virus (HIV) is the etiological agent of the complex disease that includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system. This virus was previously known as LAV, HTLV-III, or ARV. A common feature of retrovirus replication is the extensive post-translational processing of precursor polyproteins by a virally encoded protease to generate mature viral proteins required for virus assembly and function. Interruption of this processing appears to prevent the production of normally infectious virus. For example, Crawford, S. et al., J. Virol., 53 , 899, 1985, demonstrated that genetic deletion mutations of the protease in murine leukemia virus which prevent processing of precursor structural proteins result in non-infectious viral particles. Unprocessed structural proteins also have been observed in clones of non-infectious HIV strains isolated from human patients. These results suggest that inhibition of the HIV protease represents a viable method for the treatment of AIDS and the prevention or treatment of infection by HIV.
Nucleotide sequencing of HIV shows the presence of a pol gene in one open reading frame
[Ratner, L. et al., Nature, 313 , 277(1985)]. Amino acid sequence homology provides evidence that the pol sequence encodes reverse transcriptase, an
endonuclease and an HIV protease [Toh, H. et al., EMBO J. 4, 1267 (1985); Power, M.D. et al ., Science,
231, 1567 (1986); Pearl, L.H. et al., Nature 329, 351 (1987)].
Applicants demonstrate that the compound of this invention is an inhibitor of HIV protease. The compound of this invention provides a prodrug for the inhibition of HIV protease.
A biotransformed compound as herein defined is disclosed. This compound is useful in the inhibition of HIV protease, the prevention of infection by HIV, the treatment of infection by HIV and in the treatment of AIDS and/or ARC, either as a compound, pharmaceutically acceptable salt (when appropriate), pharmaceutical composition ingredient, whether or not as a prodrug or as a combination with other antivirals, anti-infectives, immunomodulators,
antibiotics or vaccines. Methods of treating AIDS, methods of preventing infection by HIV, and methods of treating infection by HIV are also disclosed.
This invention is concerned with the use of a compound given below, or pharmaceutically acceptable salts thereof, in the inhibition of HIV protease, the prevention or treatment of infection by HIV and in the treatment of the resulting acquired immune deficiency syndrome (AIDS). The biotransformed compound is the product of the incubation of Rhizopus arrhizus (ATCC 11145) in the presence of L-689,502, a HIV protease inhibitor. It is defined as follows:
Figure imgf000060_0001
or pharmaceutically acceptable salts, hydrates or esters thereof.
The pharmaceutically-acceptable salts of the compound of the present invention (in the form of water- or oil-soluble or dispersible products) include the conventional non-toxic salts or the quaternary ammonium salts of this compound, which are formed, e.g., from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,
fumarate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalene- sulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate,
tosylate, and undecanoate. Base salts include
ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl
sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others. Hydrates or esters are also encompassed by the present invention. Such hydrates or esters are those which would readily occur to the skilled artisan, and include, for example, C1-4alkyl esters.
The compound of the present inventions is useful in the inhibition of HIV protease, the prevention or treatment of infection by the human immunodeficiency virus (HIV) and the treatment of consequent pathological conditions such as AIDS.
Treating AIDS or preventing or treating infection by HIV is defined as including, but not limited to, treating a wide range of states of HIV infection:
AIDS, ARC (AIDS related complex), both symptomatic and asymptomatic, and actual or potential exposure to HIV. For example, the compound of this invention is useful in treating infection by HIV after suspected past exposure to HIV by e.g., blood transfusion, accidental needle stick, or exposure to patient blood during surgery.
For these purposes, the compound of the present invention may be administered orally,
parenterally (including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and vehicles.
Thus, in accordance with the present invention there is further provided a method of treating and a pharmaceutical composition for treating HIV infection and AIDS. The treatment involves administering to a patient in need of such treatment a pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically-effective amount of the compound of the present invention.
These pharmaceutical compositions may be in the form of orally-administrable suspensions or tablets; nasal sprays; sterile injectable
preparations, for example, as sterile injectable aqueous or oleagenous suspensions or suppositories. When administered orally as a suspension, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweetners/flavoring agents known in the art. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.
When administered by nasal aerosol or inhalation, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other
solubilizing or dispersing agents known in the art.
The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride
solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating
excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidity and/or dissolve in the rectal cavity to release the drug.
Dosage levels of the order of 0.02 to 5.0 or 10.0 grams-per-day are useful in the treatment or prevention of the above-indicated conditions, with oral doses two-to-five times higher. For example, infection by HIV is effectively treated by the
administration of from 10 to 50 milligrams of the compound per kilogram of body weight from one to three times per day. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age of the patient, body weight, general health, sex, diet, mode and time of administration, rate of
excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
The present invention is also directed to combinations of the HIV protease inhibitor compound with one or more agents useful in the treatment of AIDS. For example, the compound of this invention may be effectively administered, whether at periods of pre-exposure and/or post-exposure, in combination with effective amounts of other AIDS antivirals, immunomodulators, anti-infectives, or vaccines. Simvastatin Analogs
Also included by the process of this invention are two phosphorylated derivatives of simvastatin, whose chemical name is 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-pyran- 2-one. See EPO Publication No. 0033 358 and The Merck Index, Eleventh Edition, for a description of the compound, its synthesis and utility as an anti-hypercholesterolemic agent in interfering with cholesterol biosynthesis.
Contacting simvastatin with the Rhizopus microorganisim under the conditions described herein results in two phosphorylated derivatives:
Figure imgf000066_0001
The above two compounds of this invention are useful as antihypercholesterolemic agents for the treatment of atherosclerosis, hyperlipemia and like diseases in humans. They may be administered orally or parenterally in the form of a capsule, a tablet, an injectable preparation or the like. It is usually desirable to use the oral route. Doses may be varied, depending on the age, severity, body weight and other conditions of human patients but daily dosage for adults is within a range of from about 2 mg to 2000 mg (preferably 10 to 100 mg) given in three or four divided doses. Higher doses may be favorably applied as required.
The compounds of rhis invention also have useful anti-fungal activities. For example, they may be used to control strains of Penicillium sp.,
Aspergillus niger, Cladosporium sp., Cochliobolus miyabeanns and Helminthosporium cynodnotis. For those utilities they are admixed with suitable
formulating agents, powders, emulsifying agents or solvents such as aqueous ethanol and sprayed or dusted on the plants to be protected.
The pharmaceutically acceptable salts of this invention include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium , zinc and tetramethylammonium as well as those salts formed from amines such as ammonia, ethylenediamine, N-methylglucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzyl- phenethylamine, 1-p-chlorobenzyl-2-pyrrolidine-1'- yl-methylbenzimidazole, diethylamine, piperazine, and tris(hydroxymethyl)aminomethane. Zearalenone
Also included by the process of this invention is the biotransformation product: alpha zearalenol 6'-phosphate:
Figure imgf000068_0001
α-Zearalenol-6' -phos phate
The present invention also relates to the above new compound and an object of the present invention is to provide compounds which exhibit estrogenic activity or aid in increasing the rate of growth in meat-producing animals, e.g. cattle, lamb and swine.
The compound can be administered to animals by any suitable method including oral and parenteral administrations. For example, the compound can be blended with ordinary feed containing nutritional values in an amount sufficient to produce the desired rate of growth and can thus be fed directly to the animals, or the compound can be suspended in a suitable injection suspension medium such as peanut oil and injected parenterally. The amount of compound fed to an animal, of course, varies depending upon the animal, desired rate of growth and the like.
When the new products are to administered in feeds, an animal feed composition may be prepared containing the usual nutritionally-balanced quantities of carbohydrates, proteins, vitamins and minerals, together with the compounds of the present invention. Some of these usual dietary elements are grains, such as ground grain and grain by-products; animals protein substances, such as those found in fish meal and meat scraps; vegetable proteins like soybean oil meal or peanut oil meal; vitaminaceous materials, e.g. vitamin A and D complex members; and bone meal and limestone to provide minerals. A type of conventional feed material for use with cattle includes alfalfa hay and ground corn cobs together with supplementary vitaminaceous substances is
desired.
The starting material racemic
(±)-zearalenone, a potent anabolic agent useful in the raising of meat-producing animals, originally was prepared by fermenting the microorganism, Gibberella zeae (Gordon), on a suitable nutrient medium
according to the techniques described in U.S. Patent No. 3,169,019. More recently there has been
described the total chemical synthesis of racemic (±)- zearalenone and the 2,4-dimethyl ether derivative thereof (Taub et al., Chemical Communications, 1967, p. 225). The process described in U.S. Patent No. 3,551,455 and in U.S. Patent No. 3,239,545 affords new and more direct routes to the total synthesis of (±)-zearalenones.
Example IA
A spore sand culture containing Gibberella zeae (Gordon) NRRL-2830 was aseptically placed in a sterile tube containing 15 milliliters of Czapek's- Dox solution and a small amount of agar. This medium was then incubated for about 168 hours at approximately 25ºC. At the end of the incubation period, the medium was washed with 5 milliliters of sterile deionized water and transferred to a sterile tube containing 45 milliliters of Czapek's-Dox solution. The contents of the tube were then incubated for about 96 hours at about 25ºC after which the material was available for use in inoculation of a
fermentation medium.
The following example illustrates the fermentation of the organism Gibberella zeae (Gordon) NRRL-2830 to produce zearalenone.
Example IIA
To a 2 liter flask were added 300 grams of finely divided corn. The flask and its contents were then sterilized and after sterilization 150 milliliters of sterile deionized water were added. To the mixture in the flask were then added 45 milliliters of the inoculum prepared by the process of Example IA and the material was thoroughly mixed The mixed material was then incubated for about 20 days at 25ºC in a dark room in a water-saturated atmosphere.
The following example illustrates the recovery of the zearalenone from the fermentation medium.
Example IIIA
A 300 gram portion of fermented material produced by the method of Example IIA was placed in 500 milliliters of deionized water and slurried.
The slurry was then heated for about 15 minutes at 75ºC, 300 grams of filter aid were then added and the material was filtered. The solid filtered material containing the anabolic substance was then air dried, and 333 grams of the dried cake were then extracted with 500 milliliters of ethanol. This procedure was repeated three more times. The ethanol extract was evaporated to dryness under vacuum to give 6.84 grams of solid material. This solid material was then dis- solved in 20 milliliters of chloroform and extracted with 30 milliliters of an aqueous solution containing 5% by weight of sodium carbonate having an adjusted pH of about 11.2. The extraction process was repeated seven more times. The pH of the sodiumcarbonate extract was then adjusted to 6.2 with hydrochloric acid, to yield an anabolic substance-containing precipitate. The precipitate and the aqueous sodium carbonate extract were then each in turn extracted with 75 milliliters of ethyl ether. This procedure was repeated three more times to yield a light yellow ethereal solution, which was then evaporated to yield 116 milligrams of solid anabolic substance. This material was then subjected to multiple transfer countercurrent distribution using 100 tubes and a solvent system consisting of two parts chloroform and two parts carbontetrachloride as the lower phase and four parts methanol and one part water as the upper phase, all parts by volume. The solid material obtained from the multiple transfer countercurrent distribution was zearalenone.
The following examples are given for the purpose of illustrating the present invention and should not be construed as being limitations on the scope or spirit of the instant invention.
EXAMPLE 1
C-32 Phosphorylated FK-520
Microorganism and Culture Conditions
Spores of Rhizopus oryzae MF4974 (ATCC
No. 11145) cultivated on an oatmeal agar flask were inoculated into 50 ml Soy-Glucose medium in a 250 ml Erlenmer flask and shaken at 27°C on a rotary shaker at 220 rpm for 24 hours. The second stage flask (50 ml in a 250 ml Erlenmeyer flask) were inoculated with 2.5 ml of seed culture and incubated on a rotary shaker (220 rpm) at 27ºC for 24 hours. Following incubation, each flask was harvested by centrifugation, washed once with sterile water, and resuspended in equal volume of 100 mM pH 7.0 PO4 buffer containing 3% glycerol. FK-520 was added to achieve a final concentration of 0.2 mg/ml. The flasks were then incubated on a rotary shaker (220 rpm) at 27°C for 48 hours. Following incubation, the whole broth was extracted as described in the Isolation/Purification Section below.
Media Soy Glucose Medium g/1
Dextrose 20.0
Soy Meal 5.0
Fido yeast extract 5.0
NaCl 5.0
K2HPO4 5.0
Adjust pH to 7.0
ISOLATION AND PURIFICATION
The whole broth (400 ml) was adjusted to pH 3.5 and extracted three times with methylene chloride (3 x 400 ml). Methylene chloride extracts were combined and evaporated to dryness under reduced pressure at 30ºC. The resulting oil was dissolved in methanol and subjected to HPLC purification. HPLC was carried out on Whatman Partisil 10 ODS-3, 9.4 mm x 25 cm at 50ºC and monitored at 205 nm. The column was developed at 3 ml/minutes with a linear gradient from 35% acetonitrile in 0.1% phosphoric acid to 80% in 0.1% phosphoric acid in 60 minutes. The compound was collected during repeated injections of the above described extract. The fractions at retention time, 32 minutes, were pooled, adjusted to pH 3 and evaporated to remove acetonitrile. The compound was further purified using C18 Sep Pak (Water Associates) and methanol-water elution solvent to yield 4.5 mg pf product.
Characterization
C-32 Phosphorylated FK-520 (FK-900520) was characterized by FAB mass spectrometry and FK-520 via NMR spectrometry yielding the proton NMR spectrum of Figure 1, which confirms the assigned molecular structure in Figure 2.
EXAMPLE 2
T-Cell Proliferation Assay
1. Sample Preparation
Purified C-32 phosphorylated FK-520, as prepared by HPLC above, was dissolved in absolute ethanol at 10 mg/ml.
2. Assay
Spleens from C57B1/6 mice were taken under sterile conditions and gently dissociated in ice-cold RPMI 1640 culture medium (GIBCO, Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal calf serum (GIBCO). Cells were pelleted by centrifugation at 1500 rpm for 8 minutes. Contaminating red cells were removed by treating the pellet with ammonium chloride lysing buffer (GIBCO) for 2 minutes at 4ºC. Cold medium was added and cells were again centrifuged at 1500 rpm for 8 minutes. T lymphocytes were then isolated by separation of the cell suspension on nylon wool columns as follows: Nylon wool columns were prepared by packing approximately 4 grams of washed and dried nylon wool into 20 ml plastic syringes. The columns were sterilized by autoclaving at 250ºF for 30 minutes. Nylon wool columns were wetted with warm (37ºC) culture medium and rinsed with the same medium. Washed spleen cells resuspended in warm medium were slowly applied to the nylon wool. The columns were then incubated in an upright position at 37ºC for 1 hour. Non-adherent T lymphocytes were eluted from the columns with warm culture medium and the cell suspensions were spun as above.
Purified T lymphocytes were resuspended at 2.5 x 105 cells/ml in complete culture medium composed of RPMI 1640 medium with 10% heat-inactivated fetal calf serum, 100 mM glutamine, 1 mM sodium pyruvate, 2 x 10-5 M 2-mercaptoethanol and 50 μg/ml gentamycin. Ionomycin was added at 250 ng/ml and PMA at 10 ng/ml. The cell suspension was immediately distributed into 96 well flat-bottom microculture plates (Costar) at 200 μl/well. The control, being the medium without test drug, and various below-indicated dilutions of the above sample of purified C-32 phosphorylated
FK-520 to be tested were then added in triplicate wells at 20 μl/well . FK-520 was used as a standard . The culture plates were then incubated at 37ºC in a humidified atmosphere of 3% CO2-95% air for 44 hours. The proliferation of T lymphocytes was
assessed by measurement of tritiated thymidine
incorporation. After 44 hours of culturing, the cells were pulse-labelled with 2 μCi/well of
tritiated thymidine (NEN, Cambridge, MA). After another 4 hours of incubation, cultures were harvested on glass fiber filters using a multiple sample harvester. Radioactivity of filter discs corresponding to individual wells was measured by standard liquid scintillation counting methods (Betacounter). Mean counts per minute of replicate wells were calculated and the results expressed as percent inhibition of tritiated thymidine uptake (proliferation) as follows:
Mean cpm sample tested
% Inhibition = 100 - Mean cpm control medium X 100
Figure imgf000076_0001
Figure imgf000076_0002
The results of % inhibition at various concentrations of C-32 phosphorylared FK-520 (C-32-P FK-520) are presented in the following table:
Figure imgf000076_0003
Notes: 1. Mouse T cell cultures were pulsed with 3H-thymidine for 4 hours prior to harvesting at 48 hours.
2. Standard FK-520 (4 ng/ml) gave 97%
inhibition.
3. The mean IC50 for C-32-P FK-520 was determined to be: 15.0 + 2.1 ng/ml (17.2 ± 2.4 nM) in 3 independent experiments.
4. Inhibition of T-Cell proliferation was reversed by the addition of 100
units/ml of recombinant human IL-2 at the initiation of culture.
EXAMPLE 3
C-32 Phosphorylated FK-506
Fermentation
Spores of Rhizopus oryzae MF 4974, cultivated on oatmeal agar, were inoculated into 50 ml Soy-Glucose medium in a 250 ml Erlenmeyer flask and shaken at 25ºC on a rotary shaker at 220 rpm for 24 hours. The second stage flasks (50ml in a 250 ml Erlenmeyer flask) were inoculated with 2.5 ml of seed culture and incubated on a rotary shaker (220 rpm) at 27ºC for 24 hours.
Following incubation, each flask was harvested by centrifugation, washed once with sterile water, and resuspended in equal volume of 100 mM P04 buffer containing 3% glycerol, FK506 was added to achieve a final concentration of 0.2 mg/ml. The charged flasks were incubated on a rotary shaker (220 rpm) at 27ºC for 24 hours. Following incubation, the whole broth was worked up as described below.
Media Soy Glucose Medium g/L
Dextrose 20.0
Soy meal 5.0
Fido yeast extract 5.0
NaCl 5.0
K2HPO4 5.0
Adjust pH to 7.0 ISOLATION AND PURIFICATION
The whole broth (200 ml) was maintained at pH 6.8 and centrifuged. The mycelial cake was washed with water, then discarded. The clear filtrate and washings were pooled and passed thru a Spe-ed
octadecyl cartridge (14% carbon load, Applied
Separations) under vaccum. The column was washed with 100 ml of water. Column effluent and wash did not contain microbial transformation product when tested with HPLC. The cartridge was eluted with 200 mL methanol. Methanol was evaporated to dryness under reduced pressure at 30ºC. The resulting oil was dissolved in methanol and subjected to HPLC purification.
HPLC was carried out on Whatman Magnum 20 Partisil 1- ODS-3 Column (C18,22.1 mm ID x 25 cm) at 50ºC and monitored at 205 nm. The column was developed at 7 mL/min with linear gradient from 35% acetonitrile in 0.1% phosphoric acid to 80% acetonitrile in 0.1% phosphoric acid in 70 minutes.
The compound was collected during repeated injections of the above described extract. Fractions of retention time 50 minutes were pooled, adjusted to pH 3.0 and evaporated to remove acetonitrile. The compound was desalted using a C18 Sep Pak (Water Associate) to yield 20 mg of product.
CHARACTERIZATION
The compound was characterized by MS and confirmed by NMR as the C-32 phosphate ester
derivative of FK 506.
EXAMPLE 4
C-32 Phosphorylated C-31-desmethyl FK-520
The fermentation procedure of Example 3 was carried out substantially identical except that C-31 desmethyl FK-520 (available as for example, by the procedure in EPO Publication 0 349 061, published January 3, 1990) was used in place of FK-506.
The isolation/purification procedures were virtually identical to those described in Example 3.
The C-32 phosphorylated C-31 desmethyl
FK-520 was characterized by mass spectrometry and proton nuclear magnetic resonance in which the obtained spectra was consistent with the assigned structure. EXAMPLE 5
Carrying out the above IL-2 assay described above in Example 2 for C-32 phosphorylated FK-506 (P-FK-506) and C-32 phosphorylated C-31 desmethyl FK-520 (P-31-desMe FK-520) yielded the following results:
Inhibition of T cell proliferation
by P-FK-506
Concentration % of
of P-FK-506 inhibition
(mg/ml)
50 98.5
25 97.7
12.5 93.3
6.2 80.3
3.1 42.9
IC50 = 3.4 ng/mL (3.9nM)
Inhibition of T cell proliferation
by P-31-desMe FK-520
Concentration % of
of P-31-desMe FK-520 inhibition
(mg/ml)
250 95.2
125 90.3
62.5 79.5
31.2 53.8
15.6 22.2
IC50 = 31.3 ng/mL (36.7 nM). EXAMPLE 6
Phosphorylated Rapamycin Macrolide
Microorganism and Culture Conditions
Spores of Rhizopus oryzae MF4974 (ATCC
No. 11145) cultivated on an oatmeal agar flask were inoculated into 50 ml Soy-Glucose medium in a 250 ml Erlenmer flask and shaken at 27°C on a rotary shaker at 220 rpm for 24 hours. The second stage flask (500 ml in a 1000 ml Erlenmeyer flask) were inoculated with 25 ml of seed culture and incubated on a rotary shaker (220 rpm) at 27ºC for 24 hours. Following incubation, each flask was harvested by centrifugation, washed once with sterile water, and resuspended in equal volume of 100 mM pH 7.0 phosphate buffer containing 3% glycerol. Rapamycin macrolide was added to achieve a final concentration of 0.2 mg/ml. The flasks were then incubated on a rotary shaker (220 rpm) at 27 °C for 24 hours . Following incubation, the whole broth was extracted as described in the Isolation/Purification Section below.
Media Soy Glucose Medium g/l
Dextrose 20.0
Soy Meal 5.0
Fido yeast extract 5.0
NaCl
5.0
K2HPO4 5.0
Adjust pH to 7.0 ISOLATION AND PURIFICATION
The whole broth (500 ml) was maintained at pH 6.8 and centrifuged. The mycelial cake was washed with water, then dicarded. The clear filtrate and washings were pooled and passed through a Speed octadecyl cartridge (14% carbon load, Applied Separations) under vacuum. The column was washed with 100 ml of water. Column effluent and wash did not contain microbial transformation product when tested with HPLC. The cartridge was eluted with 200 ml methanol. Methanol was evaporated to dryness under reduced pressure at 30°C. The resulting oil was dissolved in methanol and subjected to HPLC purification. HPLC was carried out on Whatman Magnum 9 Partisil 10 ODS-3, 9.8 mm i.d. x 25 cm at 25ºC and monitored at 225 nm. The column was developed at 3 ml/minutes with a linear gradient from 35% to 80% acetonitrile in 0.1% phosphoric acid in 30 minutes. The compound was collected during repeated injections of the above described extract. The fractions of retention time 17.3 minutes, were pooled, adjusted to pH 3 and evaporated to remove acetonitrile. The compound was further purified using C18 Sep Pak
(Water Associates) and methanol-water elution solvent to yield 14 mg pf product.
Characterization
The C-43 phosphorylated macrolide was characterized by FAB mass spectrometry and NMR spectrometry as the methyl phosphate ester derivative yielding the proton NMR spectrum of Figure 3, which confirms the assigned molecular structure in Figure 4. Methylation to produce the phosphate ester was necessary to minimize the considerable line broadening which characterized the proton NMR spectrum of the free acid. The key features were the downfield displaced H-42 and H-43 signals (which are H-31 and H-32 in FK-506 nomenclature) at 3.87 ppm and 4.12 ppm, respectively, and the additional fine structure of H-43 resulting from coupling with the phosphorus. The downfield shift of H-43 is a normal consequence of replacing the active hydrogen with an electron withdrawing substituent. H-42 is also displaced downfield by proximity to the new substituent. The obtained spectrum of the phosphorylated macrolides as the phosphate methyl ester with assignments of H-42, H-43, and the methyl ester peaks is shown in Figure 3,
EXAMPLE 7
T-Cell Proliferation Assay
1. Sample Preparation
Purified phosphorylated macrolide, as prepared by HPLC above, was dissolved in absolute ethanol at 1 mg/ml and serially diluted in culture medium prior to addition to the cultures.
2. Assay
The assay was carried out as described in J. Immunol. Vol. 144, pp.251 (1990) by F. Dumont, et al.
Spleens from C57B1/6 mice were taken under sterile conditions and gently dissociated in ice-cold RPMI 1640 culture medium (GIBCO, Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal calf serum (GIBCO). Cells were pelleted by centrifugation at 1500 rpm for 8 minutes. Contaminating red cells were removed by treating the pellet with ammonium chloride lysing buffer (GIBCO) for 2 minutes at 4ºC. Cold medium was added and cells were again centrifuged at 1500 rpm for 8 minutes. T lymphocytes were then isolated by separation of the cell suspension on nylon wool columns as follows : Nylon wool columns were prepared by packing approximately 4 grams of washed and dried nylon wool into 20 ml plastic
syringes. The columns were sterilized by autoclaving at 250ºF for 30 minutes. Nylon wool columns were wetted with warm (37ºC) culture medium and rinsed with the same medium. Washed spleen cells resuspended in warm medium were slowly applied to the nylon wool. The columns were then incubated in an upright position at 37°C for 1 hour. Non-adherent T lymphocytes were eluted from the columns with warm culture medium and the cell suspensions were spun as above.
Purified T lymphocytes were resuspended at 2.5 x 105 cells/ml in complete culture medium composed of RPMI 1640 medium with 10% heat-inactivated fetal calf serum, 100 mM glutamine, 1 mM sodium pyruvate, 2 x 10-5 M 2-mercaptoethanol and 50 μg/ml gentamycin. Human recombinant interleukin-2 (50 μg/ml) and PMA at 10 ng/ml. The cell suspension vas immediately
distributed into 96 well flat-bottom microculture plates (Costar) at 200 μl/well. The control, being the medium without test drug, and various below- indicated dilutions of the above sample of purified phosphorylated macrolide to be tested were then added in triplicate wells at 20 μl/well. Rapamycin (U.S. Patent 3,929,992) was used as a standard. The culture plates were then incubated at 37ºC in a humidified atmosphere of 5% CO2-95% air for 44 hours. The proliferation of T lymphocytes was assessed by measurement of tritiated thymidine incorporation. After 44 hours of culturing, the cells were pulselabelled with 2 μCi/well of tritiated thymidine (NEN, Cambridge, MA). After another 4 hours of incubation, cultures were harvested on glass fiber filters using a multiple sample harvester. Radioactivity of filter discs corresponding to individual wells was measured by standard liquid scintillation counting methods (Betacounter). Mean counts per minute of replicate wells were calculated and the results expressed as percent inhibition of tritiated thymidine uptake (proliferation) as follows: Mean cpm sample tested
% Inhibition = 100 - X 100
Mean cpm control medium
Figure imgf000085_0001
Figure imgf000085_0002
Proliferation was assessed at 48 hours of culture by tritiated thymidine uptake.
The results, expressed as IC50 values of three independent experiments, are presented in the following table:
Figure imgf000086_0001
Notes: 1. Mouse T cell cultures were pulsed with
3H-thymidine for 4 hours prior to harvesting at 48 hours.
2. The mean IC50 for the C-43 phosphory- lated rapamycin was determined to be: 7.6 ± 0.2 ng/ml and 0.4 ± 0.1 ng/ml for rapamycin in 3 independent experiments.
EXAMPLE 8
SYNTHESIS
The following preparation and synthesis follows, in general, U.S. Patent 4,661,473; Evans, B.E. et al, J. Org. Chem., 50, 4615, (1985) and
Evans, B.E. et al., "A Stereocontrolled Synthesis of Hydroxyethylene Dipeptide Isosteres," Proc. Am. Pept. Symp., 9, 743-6(1985), and Luly, J.R. et al, J. Org. Chem, 52, 1487 (1987).
N(2(R)-Hydroxy-1(S)indanyl)-5(S)-((1,1-dimethylethoxy- carbonyl)amino)-4(S)-hydroxy-6-ρhenyl-2(R)-3-(4-(2- (morpholino)ethoxy)phenyl)prop-2-en-1-yl)hexanamide:
Step A: Preparation of 4-tert-butyldimethylsilyloxy- phenylprop-2-en-1-yl bromide:
To a 1 L round bottomed flask with a stirring bar and an argon inlet was added 26.25 g (160 mmol) of p-hydroxycinnamic acid, 50.62 g (335 mmol) of tert- butyldimethylsilyl chloride, 32.68 g (480 mmol) of imidazole, and 250 mL of dry DMF. This mixture was stirred at room temperature for 24 hours. The DMF was removed in vacuo and the residue was partitioned between EtOAc and 10% aqeous citric acid. The layers were separated and the organic phase was washed with water (4x) and brine. Drying (MgSO4), filtration and removal of the solvent in vacuo gave 62.3 g of the bissilylether-ester. This ester was placed in an oven dried 2 L round bottomed, 3 necked flask with a mechanical stirrer, argon inlet, and addition funnel. Ether (460 mL) was added and the solution was cooled in an ice bath to 0ºC. To this solution was added a solution of Dibal-H 397 mL of a 1.0 M solution in hexanes), dropwise over 1 hour. The mixture was stirred for 1 hour and the reaction was then quenched by careful addition of 1 L of saturated aqueous sodium potassium tartrate solution. This viscous mixture was stirred for 20 hours at room temperature. The mixture was filtered through a celite pad, the filtrate layers were separated and the organic phase was extracted with 2 portions of EtOAc. The combined organic phases were washed with brine, dried (MgSO4), filtered and concentrated in vacuo to give the crude alcohol. This material was chromatographed on 500 g of silica gel using 20% EtOAc in hexanes as eluant. There was obtained 34.9 g of 4-tert-butyldimethylsilyloxycinnamyl alcohol as a viscous oil. This material crystallized on cooling to -15ºC. A portion of this alcohol (18.5 g, 69.96 mmol) was placed in a 300 mL round bottomed flask with a stirring bar and 125 mL of dry ether. This solution was cooled to 0°C and 20.83 g (76.95 mmol) of PBr3 was added dropwise with a syringe over 5 minutes. This solution was maintained at 0ºC for 20 minutes, diluted with hexanes (1L) and washed with aqueous NaHCO3. This solution was washed with brine, dried (MgSO4), filtered through a pad of silica gel, and concentrated in vacuo to give 17.53 g of 4-tert- butyldimethylsilyloxypenylprop-2-en-1-yl bromide as a colorless oil. Step B: Preparation of 5(S)-(1,1-dimethylethoxycarbonyl)amino-4(S)-(1',1'-dimethylethyl-1,1- dimethylsilyloxy)-6-phenyl-2(R)-(4-(1',1'- dimethylsilyloxy)phenylprop-2-en-yl)hex- anoic acid:
To a 500 mL, 3-necked, oven dried round bottomed flask with an argon inlet, stirring bar, low temperature thermometer, and a jacketed addition funnel was added 75 mL of dry THF and 15.39 mL (109.78 mmol) of diisopropylamine. This solution was cooled to -20ºC and n-butyllithium (42.84 mL, 107.11 mmol of a 2.5M solution in hexanes) was added slowly. The resulting solution was cooled to -78ºC and a solution of (5S,1'S)-5-((1,1-dimethylethoxycarbonyl)amino)- 2-phenylethyl)dihydrofuran-2-(3H)-one (16.36 g, 53.55 mmol) in 75 mL of dry THF was added at such a rate that the temperature of the solution did not rise above -70ºC (ca. 40 minutes required for this addition). The resulting solution was aged at -78ºC for 1 hour and the dropping funnel was charged with a solution of 4-tert-butyldimethylsilyloxyphenylprop- 2-en-1-yl bromide (17.53 g, 53.55 mmol) in 75 mL of dry THF. The bromide solution was cooled to -78ºC and was then added to the enolate solution dropwise over 45 minutes, keeping the temperature below
-70°C. When the addition was complete the solution was aged at -78ºC for 1 hour, warmed to -50ºC and quenched with a solution of NaHSO4 (33 g) in 250 mL of water. The mixture was diluted with EtOAc and the layers were separated. The organic phase was washed with NaHCO3 solution and brine. Drying (MgSO4), filtration, and removal of the solvent in vacuo provided the crude alkylation product as an oil.
This material was chromatographed on 1000 g of
silica gel using 20% EtOAc in hexanes as eluant.
There was obtained 23.6 g of pure (5S,3R,1'S)3- (4-(1',1'-dimethylethyl-1,1-dimethysilyloxy)phenyl- prop-2-en-1-yl)-5-(1-((1,1-dimethylethoxycarbonyl)- amino)-2-phenylethyl)dihydrofuran-2-(3H)-one as a colorless foam. This material was dissolved in 325 mL of DME and a solution of LiOH (6.99 g, 291.97 mmol) in H2O (325 mL) was added. This solution was stirred at room temperature for 24 hours. The DME was removed in vacuo and the aqueous residue was acidified with 10% aqueous citric acid. This milky suspension was extracted with several portions of EtOAc and the combined extracts were washed with water and brine, dried (MgSO4), filtered, and
concentrated in vacuo. To the crude α-hydroxy acid was added imidazole (56.79 g, 834 mmol), tert-butyl- dimethylsilyl chloride (62.87 g, 417 mmol) and dry DMF (325 mL). This mixture was stirred at room temperature for 24 hours. The mixture was then treated with methanol (325 mL) for 4 hours at room temperature. The solvents were removed in vacuo at 60ºC, 20 torr. The residue was dissolved in 1L of EtOAc and washed with 10% aqueous citric acid, water and brine. Drying (MgSO4), filtration and removal of the solvent gave 28.53 g of 5(S)-(1,1-dimethylethoxy- carbonylamino)-4(S)-(1',1'-dimethylethyl-1,1-dimethyl- silyloxy)-6-phenyl-2(R)-(4-(1',1'-dimethylethyl-1,1- dimethylsilyloxy)phenylprop-2-en-1-yl)hexanoic acid as a colorless foam. Step C: Preparation of N-(2(R)-hydroxy-1(S)-indanyl)- 5(S)-(1,1-dimethylethoxycarbonylamino)-4(S)- (1',1'-dimethylethyl-1,1-dimethylsilyloxy)-6- phenyl-2(R)-((4-(hydroxyphenyl)prop-2-en-1- yl)hexanamide:
To a 2L round bottomed flask with a stirring bar and an argon inlet was added 5(S)-(1,1-dimethyl- ethylethoxycarbonylamino)-4(S)-(1',1'-dimethylethyl- 1,1-dimethylsilyloxy)-6-phenyl-2(R)-(4(1',1'-dimethylethyl-1,1-dimethylsilyloxy)phenylprop-2-en-1-yl) hexanoic acid (28.53 g, 41.71 mmol) from Step B, 2(R)- hydroxy-1(S)-aminoindane (6.85 g, 45.88 mmol), 3-(N,N- dimethylaminopropyl)ethylcarbodiimide hydrochloride (8.79 g, 45.8 mmol), 1-hydroxybenztriazole hydrate (6.20 g, 45.88 mmol), and dry DMF (300 mL). When all of the solids had dissolved, triethylamine (12.79 mL, 91.76 mmol) was added and the mixture was stirred at room temperature for 18 hours. The mixture was partitioned between EtOAc (1500 mL) and 10% aqueous citric acid (1500 mL). The layers were separated and the organic phase was washed with water (3 x 1000 mL), and brine. Drying (MgSO4), filtration and removal of the solvent in vacuo gave 32 g of a yellow foam. 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 in vacuo. The resulting aqueous residue was extracted with EtOAc (1L). The EtOAc extract was dried (MgSO4), filtered, and concentrated in vacuo. This material was
chromatographed on 1 Kg of silica gel using 8L of 40% EtOAc in hexanes as eluant. There was obtained 20.07 g of N-(2(R)-hydroxy-1(S)-indanyl)-5(S)-(1,1-dimethyl- ethoxycarbonyl)amino-4(S)-(1',1'-dimethylethyl-1,1-dim ethylsilyloxy)-6-phenyl-2(R)-((4-hydroxyphenyl)prop-2- en-1-yl)hexanamide as a colorless foam.
Step D: Preparation of N-(2(R)-hydroxy-1(S)-indanyl)- 5(S)-(1,1-dimethylethoxycarbonylamino)-4(S)- hydroxy-6-phenyl-2(R)-(4-(2-(4-morpholino)- ethoxy)phenylprop-2-en-1-yl)hexanamide:
To a IL round bottomed flask with a stirring bar and an argon inlet was added N-(2(R)-hydroxy-1(S)- indanyl)-5(S)-(1,1-dimethylethoxycarbonyl)-4(S)-(1',- 1'-dimethylethyl-1,1-dimethylsilyloxy)-6-phenyl-2(R)- (4-(1',1'-dimethylethyl-1,1-dimethylsilyloxy)phenyl- prop-2-en-1-yl)hexanamide (19.49 g, 28.1 mmol) 1,4- dioxane (400 mL), 4-(2-chloroethyl)morpholine (12.61 g, 84.3 mmol), and powdered cesium carbonate (27.5 g, 84.3 mmol). This mixture was heated at 80ºC with vigorous stirring for 3 hours. The cooled reaction mixture was filtered through a celite pad and the 1,4-dioxane was removed in vacuo. To this residue was added a solution of tetrabutylammonium fluoride in THF (280 mL of a 1M solution, 280 mmol) and a stirring bar. This solution was stirred at room temperature under argon for 28 hours. The reaction mixture was poured into 3L of H2O, with stirring.
The white solid was collected by filtration and dried in vacuo overnight. The crude product was
chromatographed on silica gel using 5% MeOH in chloroform as eluant. The chromatographed material was recrystallized from boiling EtOAc-hexanes to give 13 g of analytically pure N-(2(R)-hydroxy-1(S)- indanyl)-5(S)-(1,1-dimethylethoxycarbonylamino)-4(S)- hydroxy-6-phenyl-2(R)-(4-(2-(4-morpholino)ethoxy)- phenylprop-2-en-1-yl)hexanamide as a white solid, mp:178-179ºC.
EXAMPLE 9
Preparation of N-(2R)-hydroxy-1(S)-indanyl)-5(S)- (1,1-dimethylethyloxycarbonylamino)-4(S)-hydroxy- 2(R)-(4-(2-(4-morpholino)ethoxy)phenyl)prop-2-en-1- yl-6-cyclohexylhexanamide, parent compound L-702, 083
Step A: Preparation of (5S,1'S)-5-(1'-((1,1-di- methylethoxycarbonyl)amino)-2'-cyclo- hexylethyl)-4,5-dihydrofuran-2-(3H)-one:
A solution of the (5S,1'S)-5-(1'-((1,1-di- methylethoxycarbonyl)amino)-2'-phenylethyl)-4,5- dihydrofuran-2-(3H)-one was dissolved in ethyl
acetate and rhodium on alumina was added. This mixture was shaken under a hydrogen atmosphere (50 psi) at 50°C overnight. Filtration and evaporation of the solvent afforded the title compound as a viscous oil, which solidified as a hard glass.
Step B: Steps B, C and D of Example 1 are repeated except that (5S, 1'S)-5-((1,1-dimethylethoxy- carbonyl)amino)-2-phenylethyl)dihydrofuran-2- (3H)-one in Step B is substituted with its cyclohexyl analog, (5S, 1'S)-5-((1-dimethyl- ethoxycarbonyl)amino)-2-cyclohexylethyl)- 4,5-dihydrofuran-2-(3H)-one. The title compound, L-702,083, is obtained. EXAMPLE 10 BIOTRANSFORMATION OF L-702, 083 Spores of Rhizopus arrhizus MF 4974, cultivated on oatmeal agar, were inoculated into 50 ml of seed medium [containing(in grams per liter) dextrose 20.0, soy meal 5.0, Fidco yeast extract 5.0, NaCl 5.0, K2HPO4 5.0, pH 7.0 before autoclaving] in a 250 ml 3-baffle Erlenmeyer flask. The flask was incubated for 24 hours on a rotary shaker (220 rpm) at 27°C. The 2.5 ml of the seed cultures was used to inoculate 50 ml of the same medium in a 250 ml
Erlenmeyer flask and incubated on a rotary shaker (220 rpm) at 27ºC for 24 hours. Following
incubation, each flask was harvested by
centrifugation, washed once with sterile saline solution, and resuspended in equal volume of 100 mM phosphate buffer (pH 7.0) containing 3% glycerol. L-702,083 in DMSO (20 mg/ml) was then added to achieve a final concentration of 0.1 mg/ml, and cultivation was continued at 27ºC on a rotary shaker at 220 rpm. After 12 hours incubation the whole broth was extracted as described in Example 16.
EXAMPLE 11 ISOLATION AND PURIFICATION
Two shake flasks were combined to yield 100 ml of broth. The broth was centrifuged. The
mycelial cake was washed with water and discarded. The clear filtrate and the wash were combined and passed thru a Spe-ed octadecyl 14% cartridge (Applied Separations) under vacuum. The column was washed with 100 ml of water and then eluted with 200 ml methanol. Methanol extract was concentrated in vacuo to a slightly oily residue. The residue was taken up in methanol and subjected to preparative HPLC
chromatography on a Whatman Magnum 20 Partisil 10 ODS-3,22.1 mm x 25 cm at room temperature at a flow rate of 7 ml/minute. The effluent stream was
monitored by U.V. absorption at 215 nm. Elution was achieved with a linear gradient from 30% acetonitrile in 0.1% phosphoric acid to 80% acetonitrile in 0.1% phosphoric acid in 80 minutes. The fractions at retention time of 35 minutes were combined, adjusted to pH 4.0 and evaporated to remove acetonitrile. The compound was then desalted using C-18 Sep Pak (Waters Associate) to afford 3 mg of product L-706, 579.
EXAMPLE 12
NMR CHARACTERIZATION
The product of Example 11, L-706, 579, was characterized by NMR as the phosphate ester
derivative at C-4 of L-702,083. The key observation was the 0.6 ppm downfield displacement of H1 in the Formula :
Figure imgf000096_0001
Compared to its chemical shift in the parent L-702,083, the neighboring methine (m) is slightly displaced downfield and is barely discernible as a shoulder at the base of the morpholine CH2O at 3.74 ppm. Also perturbed is the nearby Hk which now appears underneath the lower field Hj signal at 3.10 ppm. Its presence is revealed only by the additional area of the Hj signal, compared with the Hj double doublet in L-702,083.
EXAMPLE 13
ORGANIC SYNTHESIS OF L-706,579, PHOSPHATE ESTER
OF L-702,083
A. Acetylation of Indan
N-[(2R)-Acetoxy-1(S)-indanyl]-5(S)-(1,1-dimethyl- ethoxycarbonylamino)-4(S)-hydroxy-2(R)-[(4-(2-(4- morpholino)ethoxy)phenyl)prop-2-en-1-yl]-6-cyclo- hexanamide, L-702,083, is prepared by the protocol of Examples 1 and 2, except that 2(R)-hydroxy-1(S)- aminoindan is substituted with 2(R)-acetoxy-1(S)- aminoindan in Example 1, Step C.
B. L-706,579
The product of step A is reacted with monophenyl phosphorodichloridate according to the principles and practice of Chambers, R.W. and H . G . Khorana, J. Am. Chem. Soc. 80, 3749 (1958).
Subsequent treatment with excess ammonia, followed by removal of the acetyl groups under basic conditions, yields L-706, 579.
C Alternative Synthetic Routes
Another phosphorylating agent is dibenzyl phosphorochloridate. An extensive discussion of phosphorylation and phosphorylating agents can be found in Y. Mizuno, Studies in Org. Chem, 24, 171-175 (1986) and references cited therein.
EXAMPLE 14
Assay for Inhibition of Recombinant
HIV Protease
Inhibition studies were performed on the reaction of the HIV protease expressed in Escherichia coli with a tritiated peptide substrate [3H]-acetyl- Val-Ser-Gln- Asn-(beta-napthyl-Ala)-Pro-Ile-Val-Gln- Gly-Arg-Arg-NH2(MW 1800). The two arginine residues at the carboxyl terminus give this peptide an overall positive charge at acidic pH and enable it to bind to the H+ form of DOWEX AG-50W-X8 resin and similar resins. The HIV protease cleaves between the
β-napthyl-Ala and proline residues to yeild a product (3H-acetyl-val-ser-asn-(beta-napthyl-ala) that is either neutral or slightly negatively charged and does not bind to the cation exchange resin. It is therefore possible to conveniently separate the labelled product from the substrate.
Aliquots of 25 μl containing 6.0-8.0 nM HIV protease in assay buffer (100 mM sodium acetate, pH 5.5 and 0.1% BSA) are placed in assay tubes. The reaction is initiated by addition of 25 μl aliquots of 4.2 uM tritiated peptide substrate in 100mM sodium acetate, pH 5.5. After incubation for 60 min at 37ºC, the reaction is stopped with 100 μl of 5% H3PO4, then analysed by application of column chromatography. Results are as follows :
Conc. L-706,579(μM) %Inhibition
100 100
10 89
1 20
0.1 3
0.01 0
0.001
EXAMPLE 15
SYNTHESIS OF L-689,502
The preparation and synthesis follows, in general, U.S. Patent 4,661,473; Evans, B.E. et al, J. Org. Chem., 50, 4615, (1985) and Evans, B.E. et al., "A Stereocontrolled Synthesis of Hydroxyethylene Dipeptide Isosteres," Proc. Am. Pept. Symp., 9,
743-6(1985), and Luly, J.R. et al, J. Org. Chem, 52, 1487 (1987), all herein incorporated by reference. All temperatures are in degrees centigrade, unless indicated otherwise.
Preparation of N-(cis-2(R)-hydroxy-1(S)-indanyl)- 5(S)-(1,1-dimethylethoxycarbonylamino)-4(S)-hydroxy- 6-phenyl-2(R)-[(4-(2-(4-morpholinyl)ethoxy)phenyl) methyl]-hexanamide, L-689,502
Step A: Preparation of N-3(S)-[(1,1-Dimethylethoxy- carbonyl)amino]-2(RS)-hydroxy-4-phenyl-1-tri- methylsilyl butane:
To a stirred suspension of magnesium turnings (9.79 g, 403 mmol) in dry diethyl ether (200 mL) under nitrogen was added chloromethyltrimethylsilane (50 mL, 358 mmol). The reaction was initiated by gentle warming and then was cooled in an ice bath to maintain gentle reflux. After exotherm was complete the reaction was stirred at room temperature for 1 hour then cooled to -78ºC in a dry ice/acetone bath. To the solution of the Grignard was added dropwise with stirring a solution of N-2(S)-[(1,1-dimethyl- ethoxycarbonyl)amino]-3-phenyl propionaldehyde
(19.3 g, 77.4 mmol) in dry diethyl ether (250 mL) dropwise such that the temperature of the reaction remained below -55°C. The resultant gray suspension was allowed to warm to room temperature where it was stirred for 30 minutes then was quenched by pouring into a mixture of ice (500 g) and 10% citric acid (500 mL). The organic phase was collected and the aqueous phase was extracted with diethyl ether (3 X 300 mL). The combined organics were washed with
10% citric acid (1 X 300 mL) and brine (1 X 200 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to give crude N-3(S)-[(1,1-dimethyl- ethoxycarbonyl)amino]-2(RS)-hydroxy-4-phenyl-1-tri- methylsilyl butane (26.6 g, quantitative crude yield) as a yellow oil. An analytical sample was obtained by low pressure chromatography (silica gel, 230-400 mesh; diethyl ether: hexanes, 30%:70%) followed by recrystallization from heptane, mp = 91-95ºC;
elemental analysis. Calcd. for C18H31NO3Si (337.53):
C = 64.05, H = 9.26, N = 4.15;
Found: C = 64.05, H = 9.13, N = 4.22; [a]D20 = -40.0° Step B: Preparation of 3(S)-Amino-4-phenyl-1-butene.
To a stirred solution of the product of
Step A (22.8 g, 67.5 mmoL) in dry methylene chloride (400 mL) cooled in an ice bath and under nitrogen was added in a fine stream boron trifluoride etherate
(43 mL, 345 mmol). The solution was allowed to warm to room temperature where it was stirred for 4 days. Reaction was cooled in an ice bath and quenched by the dropwise addition of 10% sodium hydroxide
(400 mL). The organic phase was collected and the aqueous phase was extracted with methylene chloride (2 X 250 mL). The combined organics were washed with brine (1 X 200 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to give crude 3(S)-amino-4-phenyl-1-butene (14.2 g) as a yellow oil,
Step C: Preparation of N-3(S)-[(1,1-Dimethyl- ethpxycarbonyl)amino]-4-phenyl-1-butene:
A solution of the product of Step B (14.2 g) and di-tert-butyl dicarbonate (31.0 g, 142 mmoL) in dry methylene chloride (200 mL) was stirred at room temperature for 18 hours, washed with 10% citric acid (3 X 100 mL), water (1 X 100 mL), sat'd. sodium bicarbonate (3 X 125 mL), and brine (1 X 250 mL), dried over anhydrous magnesium sulfate, filtered and concentrated to yield crude N-3(S)-1[(1,1-dimethyl- ethoxycarbonyl)amino]-4-phenylbutene (34.6 g) as a yellow oil. Crude product was purified by low
pressure chromatography (silica gel, 230-400 mesh, 10 X 20 cm column; diethylether: hexanes, 20%: 80%) to yield N-3(S)-[(1,1-dimethylethoxylcarbonyl)amino]- 4-phenyl-1-butene (16.3 g, 97.6% yield) as a white solid. An analytical sample was obtained by
recrystallization from heptane, mp = 67.5-68.5ºC;
elemental analysis, Calcd. for C15H21NO2 (247.34):
C = 72.84, H = 8.56, N = 5.66.
Found: C = 72.78, H = 8.76, N = 5.64.
Step D: Preparation of 1(R)-[1'(S)-(1,1-Dimethyl- ethoxycarbonyl)amino-2-phenylethylloxirane: To a solution of the product of Step C (9.4 g, 38 mmol) in dry methylene chloride (100 mL) cooled in an ice bath and under nitrogen was added 3-chloroperoxybenzoic acid (technical grade, 80-85%; 41 g, 200 mmol). The mixture was stirred at 0ºC for 18 hours and 25ºC for 23 hours, then diluted with diethyl ether (300 mL), and poured in ice cold aqeous 10% sodium sulfite (1 L). The organic layer was collected and the aqueous layer was extracted with diethyl ether (3 X 100 mL). The combined organics were washed with 10% sodium sulfite (3 X 100 mL), satd. sodium bicarbonate (3 X 100 mL), and brine
(1 X 100 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give a white solid.
Crude product was purified by low pressure chromatography (silica gel 230 - 400 mesh, 8 X 15 cm
column; ethyl acetate: hexanes, 25%:75%) to yield 1(R)-[1'(S)-(1,1-dimethylethoxycarbonyl)amino-2- phenylethyl]oxirane (7.0 g, 70% yield) as a clear oil which crystallized upon standing. An analytical sample was obtained by recrystallization from heptane . mp = 51.5-52°C; elemental analysis, Calcd. for
C15H21NO2 (263.34):
C = 68.42, H = 8.04, N = 5.32.
Found: C = 68.22, H = 8.26, N = 5.29; [a]D 20 = 1.34° Step E: Preparation of(5S,1'S)-3-carboethoxy-
5-(1-((1',1'-dimethylethoxycarbonyl)amino)-2- phenylethyl)-dihydrpfuran-2-(3H)-one.
The product from Step D, 9.93 g, was dissolved in 100 mL of absolute ethanol and added to a solution of 2.6 g of sodium and 20.1 mL of diethyl malonate in 170 mL of absolute ethanol. After
stirring overnite, the reaction was acidified to pH 4 with 10% citric acid and extracted with 2 X 500 mL of ether. The combined organic extracts were washed 1 X 500 mL H2O, 1 X 500 mL sat'd NaHCO3, 1 X 500 mL sat'd brine and dried over MgSO4. The solvents were removed and the crude product purified by low pressure chromatography on silica gel eluting with 50% ether/hexanes (or EtOAc/hexanes). The yield of semi-solid product was 10.6 g. The later fractions contained 2.5 g of the undesired 5 R isomer as a white solid.
Step F: Preparation of (5S,1'S)-3-carboethoxy-3-(4- benzyloxyphenylmethyl)-5-[1-(1,1-dimethyl- ethoxycarbonyl)amino)-2-phenylethyl]dihydro- fυran-2-(3H)-one
To a stirred solution of (5S,1'S)-3-carbo- ethoxy-5-[1-((1',1'-dimethylethoxycarbonyl)amino)-7- phenylethyl)-dihydrofuran-2-(3H)-one (product of Step E), 2 g (5.3 mmol) in 25 mL of absolute ethanol was added a solution of 0.13 g of sodium in 2.2 mL of absolute ethanol followed by 1.30 g (5.5 mmol) of 4-benzyloxybenzyl chloride. The solution was heated to 50°C under nitrogen for 1 hour, then cooled in an ice bath and acidified with 20 mL of 10% citric acid and diluted with 200 mL of water. The mixture was extracted with 3 X 100 mL of ether and the combined ether extracts washed with 50 mL of water, 200 mL of sat'd NaHCO3 and dried over MgSO4. Removal of solvents under reduced pressure and purification by low pressure chromatography on silica gel, eluting with 40% ether in hexanes gave 1.56 g (51% yield) of a clear colorless glass essentially homogeneous by TLC (50% ether/hexanes).
Step G: Preparation of (3R,5S,1'S)-3-(4-benzyloxy- phenylmethyl)-5-(1((1,1-dimethylethoxy- carbonyl)amino)-2-phenylethyl)-dihydrofuran-
2-(3H)-one.
The product of Step F, 13.6 g, was dissolved in 250 mL of 1,2-dimethoxyethane, and to it was added 117 mL of 1 M lithium hydroxide at room temperature. After stirring for 12 hours, the solvents were removed under reduced pressure, the residue suspended in 200 mL of 10% citric acid and extracted 3 X 500 mL of diethyl ether. The combined ether extracts were washed with 500 mL of brine, dried (MgSO4) and the concentrated to dryness. The residue was dissolved in 250 mL of toluene, heated to reflux for 12 hours, then concentrated to dryness under reduced pressure. Purification by medium pressure chromatography over silica gel eluting with 15% ethyl acetate/hexanes gave 3.2 g of the 3R-lactone as a clear foam.
Further elution with the same solvents gave 6.15 g of the 3S-lactone as a white solid. Step H: Preparation of N'-(1,1-dimethylethoxy- carbonyl)-5(S)-amino-4(S)-(1',1'-dimethylethyl-1,1-dimethylsilyloxy)-6-phenyl-2(R)- (4-benzyloxyphenylmethyl-hexanoic acid.
The product of Step G, 0.6 g, was dissolved in 30 mL of a 2:1 mixture of ethylene glycol dimethyl ether/water, and to it was added 5 mL of 1 M lithium hydroxide at room temperature. After stirring for 1 hour, the mixture was partitioned between 200 mL chloroform and 20 mL 10% citric acid. The layers were separated and the aqueous phase extracted with 3 X 20 mL chloroform. The combined organic layers were dried (Na2SO4) and the solvent removed to yield 0.56 g of the crude hydroxy acid. This residue was dissolved in 5 mL of dry DMF and 0.845 g tert-butyl dimethylsilyl chloride and 0.725 g of imidazole were added. After stirring for 18 hours, the reaction was poured into 50 mL of water and extracted with 3 X 20 mL of ethyl acetate. The combined organic extracts were washed with 3 X 20 mL of 10% citric acid, 1 X 20 mL of water, 3 X 10 mL of saturated aqueous solution of Na2CO3, and 20 mL of brine. After drying (Na2SO4), the solvent was removed and the resulting residue dissolved in a mixture of 5 mL of THF, 5 mL of glacial acetic acid, and 2 mL of water. The mixture was stirred for 4 hours, then poured into 5v. mL of water and extracted with 3 X 20 mL of ether. The combined ether extracts were washed with 2 X 20 mL of water, brine, dried (Na2SO4), and the sclvent removed.
Purification by medium pressure chromatography over silica gel, eluting with MeOH/CHCl3 gave 0.60 g of the product as a white glassy solid. Step I: Resolution of 1-Amino-2-hydroxyindan
From the known racemic 1-amino-2- hydroxyindan, the resolution was carried out
as described for the 3-amino-1,2-dihydroxyindan in Example 7 below (Steps D and E). The (1S,2R)- 1-amino-2-hydroxyindan resulting from saponification of the higher Rf diastereomer was shown to have an aD of -58° (c = 1.0, CHCl3). The (1R, 2S)-1-amino- 2-hydroxyindan resulting from saponification of the lower Rf diastereomer was found to have an ap of
+62° (c = 1.0, CHCI3).
Step J: Preparation of N-(2(R)-hydroxy-1(S)-indanyl)- 5(S)-(1,1-dimethylethoxycarbonylamino)-4(S)- hydroxy-6-phenyl-2(R)-(4-benzyloxyphenyl- methyl) hexanamide
The product from Step H, 0.12 g, was dissolved in 2 ml dry DMF and to it was added 40 mg of 1(S)-amino-2(R)-hydroxyindane, (Step I) 25 mg of 1-hydroxybenzotriazole hydrate and 70 mg of dimethyl- 3-(3-dimethyl aminopropyl)carbodiimide hydrochloride. Triethylamine was added to the stirred solution until the pH was 8.5 (32 mL). After stirring for concentrated to dryness under reduced pressure, the residue was dissolved in 100 mL of chloroform and worked with 1 X 50 mL of 10% citric acid, 1 X 50 mL H2O, 1 X 50 mL sat'd NaHCO3, dried over MgSO4 and concentrated to dryness. The residue was dissolved in 1 mL of tetrahydrofuran and added to 2 mL of 1 M tetrabutyl- ammonium fluoride in THF. After stirring overnight at room temperature the reaction mixture was diluted with 10 mL of 10% citric acid and the white preci pitate collected by filtration. The product was purified by low pressure chromatography on silica gel eluting with 2% methanol/CH2Cl2 to give 85 mg of product which was essentially homogeneous by TLC (3% methanol/CH2Cl2).
Step K: Preparation of N-(2(R)-hydroxy-1(S)-indanyl)- 5(S)-(1,1-dimethylethoxycarbonylamino)-4(S)- hydroxy-6-phenyl-2(R)-(4-hydroxyphenyl- methyl)hexanamide
The product of Step J, 85 mg was dissolved in 10 mL of methanol and 10 mL of THF, and to it was added 0.10 g of 10% palladium on carbon. The mixture was stirred under an atmosphere of hydrogen for 48 hours at room temperature, then filtered and concentrated to dryness. The residue was dissolved in 10 mL of hot ethanol and 20 mL water was added. On cooling the white solid precipitate was collected and dried under vacuum over P2O5. The yield was 72 mg (98% yield) of pure product: mp 218-219ºC (effervesces, sinters at 215) elemental analysis, Calc'd for
C33H40N2O6: (560.696):
C, 70.69; H, 7.19; N, 5.00;
Found: C, 70.62; H, 7.39; N, 4.79.
Step L: Preparation of N-(cis-2(R)-hydroxy-
1(S)-indanyl)-5(S)-[1,1-dimethylethoxy- carbonylamino)-4(S)-hydroxy-6-phenyl-2(R)-
[(4-(2-(4-morpholinyl)ethoxy)phenyl]methyl]- hexanamide
A stirred mixture of Step K product, N-(2
(R)-hydroxy-1(S)-indanyl)-5(S)-[1,1-dimethylethoxy- carbonyl)amino]-4(S)-hydroxy-6-phenyl-2(R)-(4-hydroxy- phenylmethyl) hexanamide (0.50 g, 0.9 mmol), anhydrous cesium carbonate (1.0 g, 3 mmol) and N-(2-chloroethyl) morpholine, free base (2.35 g, 17 mmole) in 100 mL of anhydrous dioxane was heated to 80°C (internal temperature) for 3 hrs. After cooling to room
temperature the mixture was diluted with chloroform (50 mL) filtered, concentrated to dryness under reduced pressure, and the residue triturated with 50 ml of anhydrous ether and 10 mL of ethyl acetate. The white solid product was collected and dried under vacuum over P2O5. The yield was 0.54 g (89%) of pure product L-689,502: mp 195-7ºC. elemental analysis, Calc'd. for C3H51N3O: (673.856):
C, 69.52 H, 7.63; N, 6.23;
Found: C, 69.19 H, 7.45; N, 6.15. maleate hydrate: mp 112-113°C dec. elemental analysis,
Calc'd. for C39H51N3O7.C4H4O4.H2O: (807.946):
C, 63.92 H, 7.11; N, 5.20;
Found: C, 64.23 H, 6.94; N, 5.10.
EXAMPLE 16
A. Fermentation of L-689.502
A frozen vial (2.0 ml) of Rhizopus arrhizus MF4974 was used to inoculate a 250 ml baffled shake flask containing 50 ml of seed medium A. The seed flask was incubated on a rotary shaker (220 rpm) at 27°C for 24 hours. A 2.5 ml aliquot of the developed seed was used to inoculate a 250 ml non-baffled flask containing 50 ml of transformation medium B; L-689,502 in DMSO was added to the fermentation at 0 hour to achieve a final concentration of 0.05 mg/ml. The shake flask contents were subsequently incubated at 27ºC on a rotary shaker for 4 days. The resultant whole broth was extracted as described in Section B.
Media: Seed Medium A g/l
Dextrose 1.0
Dextrin 10.0
Beef Extract 3.0
Ardamine pH 5.0
NZ Amine Type E 5.0
MgSO4●7H2O 0.05
K2HPO4 0.3
Adjust pH to 7.1
Add to CaCO3 0.5 g/l
Transformation Medium B g/l
Glucose 10
Hycase SF 2
Beef Extract 1
Corn Steep Liquor 3
Adjust pH to 7.0
B. Isolation and Purification
The whole broth (400 ml) was extracted three times with 1-butanol (3 x 400 ml). The extracts were combined and concentrated under vacuum to an oily residue. The residue was dissolved in methanol and subjected to high performance liquid chromatography (HPLC). HPLC was carried out on Whatman Partisil 10 ODS-3,9.4 mm x 25 cm at room temperature and monitored at 215 mm. The column was developed at 3 ml/min with a linear gradient from 0.1% aqueous H3PO4-CH3CN, 80:20, to 0.17. aqueous H3PO4-CH3CN, 20:80, in 60 minutes. The compound was collected during
repeated injections of the above described extract. The fractions at retention time, 19.3 minutes, were pooled, adjusted to pH 6.5 and evaporated to remove acetonitrile. The compound was further purified using a C18 Sep-Pak (Waters Associates) and methanol- water elution solvent to yield 11 mg of product.
Anal. Calc'd. for C39H50O10PN3Na2:
C, 58.72 H, 6.27; N, 5.27; P, 3.89. Found: C, 59.78 H, 6.58; N, 4.97; P, 4.17.
NMR, elemental analysis, and MS showed the structure to be
Figure imgf000110_0001
Phosphorus was identified and quantified by complexomatric and gravimetric methods. Phosphorylation at C4 was established by NMR from the 0.5 ppm downfield displacement of the C4H relative to its chemical shift in the parent L-689,502.
EXAMPLE 17
Enzymatic Treatment of L-696,432
The microbial transformation metabolite L-696,432 was determined to be a phosphate ester of L-689,502, a highly potent HIV protease inhibitor. To determine if the phosphate ester bond can be enzymatically cleaved, treatment with alkaline phosphatase was performed.
To 0.5 ml of water containing 0.8 mg of L-696,432 was added 30 microliter of bacterial alkaline phosphatase (0.31 unit/microliter), and the reaction mixture was incubated at 37ºC and monitored by HPLC. The reaction was complete after four hours of incubation. The reaction product was purified by HPLC and subjected to FAB MS analysis. [M+H]+ion was observed at m/z 674. The fragmentation ions at m/z 574,556,469 and 425 confirmed the product is
L-689,502.
The results show that L-696,432 can be enzymatically hydrolyzed back to the parent compound L-689,502. EXAMPLE 18 Assay for Inhibition of Recombinant HIV Protease
Inhibition studies of the reaction of HIV protease expressed in Escherichia coli were carried out with a tritiated peptide substrate, [3H]-acetyl- Val-Ser-Gln-Asn-(beta-napthyl Ala)-Pro-Ile-Val-Gln- Gly-Arg-Arg-NH2 (MW=1800). The two arginine residues at the carboxyl terminus give this peptide an overall positive charge at acidic pH and enable it to bind to the H+ form of DOWEX AG-50W-X8 resin and similar resins. The HIV protease cleaves between the
β-napthyl-Ala and proline residues to yield a product [3H]-acetyl-val-ser-asn-(β-napthyl-ala) that is either neutral or slightly negatively charged and does not bind to the cation exchange resin. It is therefore possible to conveniently separate the labelled product from the substrate.
Aliquots of 25 μl containing 6.0-8.0 nM HIV protease in assay buffer (100 mM sodium acetate, pH 5.5 and 0.1% BSA) are placed in assay tubes. The reaction is initiated by addition of 25 μl aliquots of 4.2 μM tritated peptide substrate in 100 mM sodium acetate, pH 5.5. After incubation for 60 minutes at 37°C, the reaction is stopped with 100 μl of 5% H3PO4, then analysed by application of column chromatography.
L-696,432 demonstrated 32% inhibition at a concentration of 1 μg/ml in substantially purified form. EXAMPLE 19
Organic Synthesis of L-696,432,
phosphate ester of L-689,502
Organic Synthesis of L-696,432
A. Acetylation of Indan
N-(cis-2(R)-Acetoxy-1(S)indanyl)-5(S)-[1,1- dimethylethoxycarbonylamino)-4(S)-hydroxy-6-phenyl- 2(R)-[(4-(2-(4-morpholinyl)ethoxy)phenyl]methyl]- hexanamide is prepared by the protocol of Example 1, except that 1(S)-amino-2(R)-acetoxyindane substitutes for 1(S)-amino-2(R)-hydroxyindane of step I.
B. L-696,432
The product of step A is reacted with monophenyl phosphorodichloridate according to the principles and practice of Chambers, R.W. and H.G. Khorana, J. Am. Chem. Soc. 80, 3749 (1958). Sub- sequent treatment with excess ammonia, followed by removal of the acetyl and phenyl groups under basic conditions, yields L-696,432.
C. Alternative Synthetic Routes
Another phosphorylating agent is dibenzyl phosphorochloridate. An extensive discussion of phosphorylation and phosphorylating agents can be found in Y. Mizuno, Studies in Org. Chem, 24,
171-175 (1986) and references cited therein. EXAMPLE 20
A culture of Rhizopus arrhizus MF 4974 from the Merck Culture Collection was inoculated in 50 milliliters of soy-glucose medium in a 250 milliliter flask and the flask incubated on a rotary shaker at 220 rpm at 27 °C for 24 hours to obtain the f irst stage seed culture. 2.5 milliliters of this seed culture was inoculated into each of 18 flasks
containing 50 milliliters of soy-glucose medium which was also charged with Compound Z to a concentration of 5 μg/ml in 50 microliters of DMSO and the
resulting culture incubated on a rotary shaker at 220 rpm at 27ºC for 18 hours.
Following incubation, each flask was harvested by centrifugation, the mycelium washed twice with sterile saline and resuspended in pH 7.0 phosphate buffer containing 1 percent glycerol.
Compound Z was added to a final concentration of 58 μg/ml using 100 μl of (DMSO). The flasks were incubated on a rotary shaker at 220 rpm at 27ºC for 48 hours.
At the end of this period, the contents of eighteen flasks (900 milliliters) were pooled and the whole broth centrifuged. The mycelial cake was slurried with 100 milliliters of water, the pH adjusted to 3.5 and the slurry extracted twice with 100 milliliters of n-butanol. The supernatant was acidified to pH 3.5 and extracted twice with one-half the volume of n-butanol. Each organic extract was assayed by HPLC. The assay condition was as follows: Column: "ZORBAX" (DuPont) C8 Rx 4.5 x 250 mm
Mobile Phase: Acetonitrile + 10mM aq. KH2PO4,
gradient 20% to 80% over 20 minutes
Temperature: 45ºC
Flow rate: 1.5 ml/min.
Detection: 210 nm
Sample Vol: 50 μl
The extracts of the mycelium and the extracts of the supernatant were pooled and evaporated to dryness at 30ºC at reduced pressure to obtain an oil as residue.
The oil was dissolved in the mobile phase of 40/60 acetonitrile/water and further purified using ZORBAX C8 (9.6 mm x 25 cm) semi-preparative column. The column was developed at 7.05 ml/min. using 40 percent aqueous acetonitrile containing 0.1 percent trifluoroacetic acid (TFA) at 45ºC. Fractions having a retention time of 12.8 minutes were pooled, and the solvent evaporated to obtain 12 mg of Compound III in a yield which calculated to be 24%.
The product had the mass spectral data previously set forth.
A portion of the product was converted to the monopotassium salt. This was carried out by dissolving the biophosphorylation product in 70 percent aqueous acetonitrile containing 10 mM KH2PO4 (pH 4.5). The mixture was subjected to reduced pressure to remove the acetonitrile and the aqueous residue loaded onto a water-equilibrated C-18 solid phase extraction column. The column was washed with water and then eluted with 70 percent aqueous
acetonitrile and the eluate freeze-dried for
retention of the salt. EXAMPLE 21
Spores of Rhizopus arrhizus were obtained from oatmeal agar slants of MF 4974 maintained in the Merck Culture Collection and employed to prepare a spore suspension in water of about 7 x 109 spores per milliliter for use in the preparation of seed culture.
Seed flasks each containing 500 milliliters of soy glucose broth of the composition previously given, were inoculated with 1 milliliter of spore suspension and incubated on a rotary shaker (220 rpm) at 27ºC for 24 hours.
Following incubation, the mycelia from each flask were harvested by filtering through a 10 micron nylon mesh and then resuspended in an equal volume of a 100 mM pH 6.3 phosphate buffer containing 3 percent glycerol and added to the broth. Compound Z, was added to a concentration of about 50 μg/ml in
dimethylsulfoxide. The flasks were then incubated on a rotary shaker at 220 rpm at 27ºC for 24 hours.
After completion of the incubation period, the contents of sixteen flasks totaling 8000
milliliters were pooled and filtered through a 10 micron nylon mesh. The mycelial cake was slurried with 1000 milliliters of 50 percent aqueous methanol and filtered. The mycelial cake was again extracted with aqueous methanol and the two aqueous methanol filtrates combined and diluted with 2000 milliliters of water.
The resulting aqueous solution was applied to a 15 mm x 300 mm column packed to a 220 mm bed height with water equilibrated "DIANION" HP20 resin. The filtrate was pumped in a downflow mode at 15 ml/min. in 2000 ml aliquots. After loading, the column was washed with 500 milliliters of water and the desired phosphorylated product eluted with 500 milliliters of 20 percent aqueous acetonitrile. The remaining substrate and metabolite was eluted with 500 milliliters of 70 percent aqueous acetonitrile.
The diluted filtrates from the mycelial extracts were also pumped onto the- column, the column washed and then eluted and the eluate assayed by HPLC. The assay condition was as described in
Example 20.
HPLC indicated the following mass balance for microbial phosphorylation and "DIAION" HP 20 isolation:
Substrate Charged: 402.5 mg
Metabolite & Substrate Recovered: 383.8 mg
Total Recovery: 95.4%
Compound III 234.9. mg
Bioconversion yield: 61.2%
The eluate was concentrated under reduced pressure and the residue dissolved in 40 percent aqueous acetonitrile containing 10mM KH2PO4 to obtain a monopotassium salt. The aqueous solution was subjected to reduced pressure to remove the
acetonitrile and the aqueous residue loaded onto a water-equilibrated C-18 solid phase extraction column. The column was thereafter washed with water and then the monopotassium salt of Compound III eluted with 70 percent aqueous acetonitrile. The eluant was freeze dried to obtain the salt. The NMR spectrum of this salt is that previously detailed.
The salt is converted to the acid by careful acidification.
EXAMPLE 22
The following salts are prepared by reacting an appropriate phosphate salt and Compound III in the manner described in Example 21 and concentrating under vacuum.
Example No. R
IlIa PO(OH)(ONa)
Illb PO(OH)(OK)
IIIc PO(ONa)2
IIId PO(OLi)(OH)
IIIe PO(OH)(OMg)½
IIIf PO(OH)(ON(CH3)4)
EXAMPLE 23
1000 compressed tablets each containing 500 mg of Compound IIIA are prepared from the following formulation:
Compound Grams
Compound IIIA 500
Starch 750
Dibasic calcium phosphate hydrous 5000
Calcium stearate 2.5 The finely powdered ingredients are mixed well and granulated with 10% starch paste. The granulation is dried and compressed into tablets.
EXAMPLE 24
1000 hard gelatin capsules, each containing 500 mg of mono sodium salt of Compound III are prepared from the following formulation:
Compound Grams
Compound III, mono sodium salt 500
Starch 750
Dibasic calcium phosphate hydrous 5000
Calcium stearate 2.5
A uniform mixture of the ingredients is prepared by blending and used to fill two-piece hard gelatin capsules.
250 ml of an injectable solution are prepared by conventional procedures having the following formulation:
Dextrose 12.5 g
Water 250 mL
Compound III, mono potassium salt 400 mg
The ingredients are blended and thereafter sterilized for use. EXAMPLE 25
An aerosol composition may be prepared having the following formulation:
Per Canister
Compound IIIA 24 mg
Lecithin NF Liquid
Concentrated 1.2 mg
Trichlorofluoromethane, NF 4.026 g
Dichlorodifluoromethane, NF 12.15 g
Preparation of Starting Materials
Compound Z, the starting material, was prepared by inoculating 54 milliliters of P34-2 medium of the following composition per liter: corn steep liquor, 5 g; D-mannitol 25 g; glucose
monohydrate, 10 g; "PHARMAMEDIA," (nonhydrolyzed protein, Buckeye Oilseed Products, Memphis, Tenn.) 20 g KH2PO4, 9 g; FeSO4●7H2O, 10 mg MnSO4●4H2O 10 mg; CuCl2●2H2O, 0.25 mg; CaCl2●2H2O, 1 mg; H3BO3, 0.56 mg: (NH4)6 Mo7O24●H2O, 0.19 mg; ZnSO4●7H2O, 2 mg, with frozen vials of Zalerion arboricola MF5533 ATCC 74030 and the inoculated medium incubated with shaking at 220 rpm at 25ºC for four days. Twenty milliliters were used to inoculate four 2-liter flasks containing 500 milliliters of P34-2 medium and the inoculated medium incubated at 25ºC for four days at 220 rpm. The flask contents were pooled and used to inoculate into three fermenters each containing 180 liters of P34-2 medium and 2 ml/L of propylene glycol P-2000 (Dow Chemical) to reduce foaming and the inoculated medium cultivated for six days at 25ºC, an air flow of 90 L/min. a pressure of 0.7 kg/cm2 gauge, and an agitator speed of 200 rpm. A 25 liter sample of the resulting broth was then used to inoculate three fermenters each containing 475 liters of P34-2 medium containing 2 ml/L of P-2000 and cultivated for four days at 25ºC, air flow of 250 L/min, pressure 0.7 kg/cm2 gauge and 150 rpm.
425 liters of this seed broth was inoculated into each of three production fermenters containing 13,700 liters of TG106 medium of the following composition per liter: D-mannitol, 100 g; NZ-Amine type E (casein hydrolysate, Sheffield Products, Kraft Inc.) 33 g; Fidco 8005 yeast extract (Difco), 10 g; (NH4)2SO4, 5 g; KH2PO4, 9 g; P-2000, 2 ml, and the fermenters operated at a temperature of 27ºC, air flow of 2500 liters/minute, a pressure of 0.7 kg/cm2 gauge, and an agitator speed of 50 rpm. The pH was allowed to decrease from 6.0 to 5.5 and then
maintained at 5.5 + 3. After about 2 1/2 weeks the broth was harvested for product isolation.
The broth from the foregoing cultivation was first extracted with an equal volume of methanol.
The methanol-broth was clarified using a liquid-solid separator (centrifuge) to obtain clarified liquid as first extract and solid. The extraction-clarification was repeated. The extracts were combined and the water cont at adjusted to about 50 percent. The resulting solution was passed through a "DIAION" SP-207 adsorption column to adsorb Compound III and the column washed with aqueous methanol. Thereafter Compound III was recovered with 100 percent methanol. The water content of the methanol containing Compound III was adjusted to 50 percent and the aqueous methanol solution intimately mixed with an equal volume of 1:1 ethyl acetate/hexane and the two liquid phases thereafter separated. The aqueous methanol layer was passed through a column of
"DIAION" SP-207, the column washed with aqueous methanol, and Compound III eluted with 100 percent methanol. The eluant was vacuum concentrated to a minimum volume and the solvent composition adjusted to about 75:20:5 ethyl acetate/methanol/water.
The feed thus prepared was passed through a silica gel column and Compound III eluted with
85:10:5 ethyl acetate/methanol/water. The fractions showing 85 percent or greater area purity by HPLC were combined, vacuum concentrated to remove ethyl acetate and the concentrate adjusted to 50 percent aqueous methanol, the latter passed through "DIAION" HP-20 in the manner previously described concentrated and Compound III precipitated with acetronitrile and recovered by vaccum filtration and then dried.
The starting material also may be prepared by methods described in copending applications Serial Nos. 47/492,025 and 47/492,026.
Z. arboricola MF5533 ATCC 74030 is disclosed and claimed in copending application Serial No.
630,457, filed December 19, 1990. Briefly, it may be obtained by (a) inoculating a frozen vegatative mycelium of Z. arboricola ATCC 20957 (disclosed and claimed in copending application Serial No. 492,024) into KF seed medium of: corn steep liquor, 5 g/l; tomato; tomato paste 40 g/l, oat flour 10 g/l; glucose 10 g/l; FeSO4●7H2O 10 mg/l; MnSO4●4H2O 10 mg/l; CuCl2●2H2O, 0.25 mg/l; CaCl2●2H2O, 1 mg/l;
H3BO3, 0.56 mg/l; (NH4)6Mo7O24●H2O, 0.19 mg/l;
ZNSO4●7H2O, 2 mg/l; adding to the medium
N-methyl-N'-nitro-N-nitrosoguanidine (NTG) (b) cultivating, thereafter (c) plating a portion of the growth on potato dextrose agar and (d) incubating for 14 days at 25ºC to obtain spores then (e) harvesting the spores, (f) diluting the spores with sterile saline (g) plating on potato dextrose agar (h) incubating for 7 days for colony formation, (i) transferring separate colonies to slants of potato and (j) incubating for 14 days at 25ºC.
EXAMPLE 26
Biotransformation Enzymology-Isolation
Of Cell Free Activity
Spores of Rhizopus oryzae MF 4974, cultivated on oatmeal agar, were inoculated in a
Soy-Glucose medium consisting of 20.0 g dextrose, 5.0 g Fidco yeast extract, 5.0 g NaCl, 5.0 g K2HPO4 in a liter of distilled water. The pH of the medium was adjusted to 7.0 before autoclaving. The cultures were incubated at 27ºC for 24 hours on a rotary shaker at 220 rpm. A fresh Soy-Glucose medium was inoculated with the 24-hour seed culture at a ratio of 5% (v/v) and the fermentations were continued in the manner described above. The phosphorylating enzyme activity, as a function of time, was determined.
Cell free extracts were obtained by suspending the cells in a buffer containing 100 mM phosphate (pH 7.5) and 2 mM EDTA. The cells to buffer ratio was 2:1. Several procedures were then tried to break the cell membranes and release the cell contents, i.e., a high-pressure French press, sonication, grinding the frozen cells, and lysozyme treatment.
The enzyme reaction was carried out in 0.2 ml mixture containing 0.15 mM FK-520, 5 mM MgCl2, 5 μM ATP and various amount of enzyme. The reaction mixture was incubated at different temperatures for 1 hour and terminated by the addition of 0.2 ml methanol. The resulting solution was subjected to HPLC analysis on Whatman Partisil 10 ODS-3 at 55ºC. The column was developed at 1 ml/min with a linear gradient from 45% acetonitrile in 0.1% phosphoric acid to 807. in 0.1% phosphoric acid in 30 minutes. The retention time of phosphorylated FK-520 and
FK-520 was 16,5 and 20 minutes, respectively.
Protein concentration was determined by Pierce
BCA protein assay reagent using BSA as standard.
In a search for phosphorylating enzyme in cell free extracts of Rhizopus oryzae MF 4974 we have found phosphorylating activity: 1) is highest in the extracts of cells isolated after 24 hours incubation in Soy-Glucose medium; 2) both sonication and maceration of the cells under liquid nitrogen can be used to break the cell membranes for liberating the enzyme in soluble form. The pH optimum is 7.5; 4) the temperature optimum is 37ºC. 5) The phosphorylation of FK-520 was linear with respect to time and enzyme concentration for one hour and from 0.2 to 2.0 mg protein. The effect of the FK-520 concentration on the rate of phosphorylation have been studied using crude extract. The Km value for the substrate (FK-520) was about 0.5 mM and the Vmax was 2. In mole/min/mg protein. To determine metal ion requirement the crude enzyme was dialyzed thoroughly against 20 mM Tris-HCl buffer and 2 mM EDTA. The results show that the phosphorylating activity requires Mg+2 or Ca+2 as cofactor.
EXAMPLE 27
NMR analysis established that the biotransformation product obtained by incubating 24-deoxy FR-900520 with MF 4974 (Rhizopus arrhizus) is the 32 phosphorylated analog:
Figure imgf000125_0001
Contacting 24-deoxy FR 900520 made by the process described in Examples 28-29 with the Rhizopus microorganism as per the analogous procedure
described for 24-oxy FR 900520, yields the 32-phos- phorylated analog as determined by NMR spectroscopy. EXAMPLE 28
17-Ethyl-1-hydroxy-12-[2'-(3'',4''-dihydroxycyclo- hexyl)-1'-methylvinyl]-23,25-dimethoxy-13,19,21,27- tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]- octacos-14,18-diene-2,3,10,16-tetraone
To a stirred solution of 31-desmethyl
FK-900,520, 17-ethyl-1,14-dihydroxy-12-[2'-(3",4"- dihydroxycyclohexyl)-1'-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo- [22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone produced as described in EPO Publication. 0 349 061 (77 mg in 3 ml benzene) was added 6 mg of
p-toluenesulfonic acid and the mixture warmed to 60°C in an oil bath. After 25 minutes, the reaction mixture was cooled to room temperature, neutralized by the addition of a saturated aqueous NSLHCO3
solution and extracted with ethyl acetate (3 times). The combined organics were washed with saturated NaCl solution, dried over Na2SO4 and purified by flash chromatography (20% hexanes in ethyl acetate and 1% MeOH) to yield 40 mg of the title compound.
MASS: (FAB) 782 (m + Na).
Partial 1H NMR (200 mHz): δ 6.80 (dd, J1 = 16 Hz, J2 = 6 Hz), 6.16 (dd, J1 = 16 Hz, J2 = 1.5 Hz), 4.39 (broad d, J = 14 Hz), 4.26 (broad d, J = 5 Hz), 3.91 (dd, J1 = 8.8 Hz, J2 = 3 Hz). EXAMPLE 29
17-Ethyl-1-hydroxy-12-[2'-(3'',4''-dihydroxycyclohexyl)-1'-methylvinyl]-23,25-dimethoxy-13,19,21,27- tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]- octacos-18-ene-2,3,10,16-tetraone
To a solution of 17-ethyl-1-hydroxy-12- [2'-(3'',4''-dihydroxycyclohexyl)-1'-methylvinyl]- 23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4- azatricyclo[22.3.1.04,9]octacos-14,18-diene-2,3,10,16- tetraone (40 mg in 1.5 ml ethyl acetate) was added 3 mg of 57. Rh/Carbon catalyst. The reaction flask was fitted with a hydrogen balloon, evacuated and
recharged with hydrogen gas (3 times). After 45 minutes, the mixture was filtered over Celite, concentrated and purified by flash chromatography (CH2Cl2: MeOH: Hexane (10:1:2)). to yield 33 mg of the title compound.
MASS: (FAB) 768 (m + Li).
Partial 1H NMR (200 mHz): δ 4.55 (broad d, J = 5 Hz), 4.39 (broad d, J = 14 Hz), 3.86 (dd, J1 = 8.8 Hz, J2 = 3 Hz).
EXAMPLE 30
PHOSPHORYLATED ANTI-CHOLESTEROLEMIC
ISOLATES L-706,526 AND L-706,527
Contacting simvastatin, whose chemical name is: 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-pyran-2-one with the Rhizopus. microorganism under the analogous conditions
described in Example 3 produced two phosphorylated compounds, L-706, 546 and L-706, 527. The preparation and properties of simvastatin are described in EP Publication No. 0033538 to Merck &. Co., Inc.
NMR analysis of the biotransformation products obtained by incubating the above compound simvistatin with Rhizopus arrhizus confirms their assigned structures as illustrated:
Figure imgf000128_0001
The compounds exhibit inhibition in the biosynthesis of cholesterol as also described for simvastatin in EP Publication No. 0 033 538.
Key features in the NMR of L-706, 526 are the absence of a side chain methyl triplet and the presence of a novel CH3CH-O- moiety with signals at 1.10 ppm (CH3) and at 3.93 ppm (CH-O). Irradiating at 1.10 ppp collapsed the methine quintet to a doublet. Since the methine proton in the proposed structure has no vicinal neighbor the appearance as a doublet implies coupling with an unseen hetero atom, i.e., the phosphorus.
Phosphorylation at the H-6' sitea is suggested by the absence of a typical H-6' signal at 3.69 ppm and the presence of a broad , featureless s ignal at 4.26 ppm which is assigned to a displaced H-6'. Both the magnitude of the displacement and the loss of detail are reasonable consequences of phosphorylation.
EXAMPLE 31
Phosphorylation of Zearalenone
Figure imgf000129_0001
Chemical structure of
α-zearalenol-6'-phosphate
Incubation of zearalenone available from Sigma with resting cells of Rhizopus arrhizus MF 4974 for 24 hours results in two biotransformation
products. They have been identified as α-zearalenol and α-zearalenol-6'-phosphate. The fermentation, isolation, purification and enzymatic hydrolysis are described below. FERMENTATION
One Ml of a Rhizopus arrhizus (MF 4974) spore suspension, harvested from oatmeal agar, wea incubated into 500 mL Soy-Glucose medium contained in a 2 liter Erlenmeyer flask and shaken a 27°C on a rotary shaker (220 rpm) for 24 hours.
Following incubation, each flask was
harvested by filtration washed once with water, then resuspended in 500 mL or mM PO4 buffer (pH 7.0) containing 4% glycerol.
Zearalenone was added to a final
concentration of .05 mg/mL, using 0.8 mL DMSO. The charged flasks were incubated on a rotary shaker (220 rpm) at 27°C for 24 hours. Following incubation, the whole broth was worked up as described below.
Media Soy Glucose Medium g /L
Dextrose 20.0
Soy Meal 5.0
Fido yeast extract 5.0
NaCl 5.0
K2HPO4 5.0
Adjust pH to 7.0 ISOLATION AND PURIFICATION
The whole broth (1000 mL) was diluted with an equal volume of methanol, than
centrifuged. The mycelial cake was
discarded. The clear filtrate was applied to a 15mm X 30 cm column packed with HP 20 (Mitsubishi Chemical, 220 mm bed height water equilibrated). The filtrate was pumped onto the column in the downflow mode at 15 mL/min. After loading, the column was washed with 500 mL water. Metabolites or substrates were not detected in the spent broth or wash. The column was eluted with a step gradient of methanol:water. The most polar metabolite eluted between 10% and 30% methanol. The second metabolite and
substrate eluted in the column wash (100% methanol). Appropriate fractions were combined and evaporated to dryness under reduced pressure at 30ºC to yield yellow oils that was subjected to further
purification by preparative HPLC.
Preparative HPLC was carried out on Whatman Magnum 9 Partisil 10 ODS-3 column (C18, 9.8 mm ID x 25cm) at room temperature and monitored at 237 nm. The column was
developed at 4 mL/min with linear gradient from 20% acetonitrile in 0.1%phosphoric acid to 80% acetonitrile in 0.1% phosphoric acid in 40 minutes. The compounds were collected during repeated injections of the above described extract. Fractions of retention times 17.5 and 20.1 minutes were collected, pooled, diluted with three volumns of water and desalted using a Speed C18 column (Applied Separations) to yield 7 mg of 6-phosphate ester and 5 mg of α-zearalenol, respectively.
ENZYMATIC HYDROLYSIS
To prove that the 6'α-hydroxyl group is phosphorylated, enzymatic hydrolysis was examined using various sulfatases and alkaline phosphatases obtained from Sigma. In summary, a specific amount of enzyme was added to one mL of 100mM phosphate buffer containing ~50 ug/mL of zearalenol
phosphate ester. The buffer solution was incubated at 37ºC for 4 hours. After incubation, 300 uL was removed and analysed by HPLC, using a Whatman Partisil 10 0DS-3 column (C18 10 urn, 4.6 mm ID x 25 cm) at room temperature and monitored at 237 nm. The column was developed at 1 mL/min with a linear gradient gradient from 20%
acetonitrile in 0.1% phosphoric acid to 80% acetonitrile in 0.17. phosphoric acid in 20 minutes. Under these conditions,
α-zearalenol-6'-phosphate ester and
α-zearalenol have retention times of 13.93 and 18.17 minutes, respectively. The results of enzymatic hydrolysis are summarized below:
ENZYME pH UNITS ACTIVITY
P-4653
Alk. Phosphatase 7.0 1.0 100% α-OH @ lhr
P-5521
Alk. Phosphatase 7.0 100.0 100% α-OH @ lhr
S-8629
Sulfatase 5.0 10.0 0% α-OH @ lhr
S-1629
Sulfatase 7.0 1.0 100% α-OH @ lhr
P-8639
Alk. Phosphatase 7.0 10.0 6% α-OH @ lhr
P-1391
Alk. Phosphatase 7.0 10.0 100% α-OH @ lhr
S-9751
Sulfatase 5.0 100.0 52% α-OH @ lhr
CHARACTERIZATION
The compounds were identified by MS and NMR and confirmed by enzymatic hydrolysis as α-zearalenol-6'-phosphate and
α-zearalenol. Also, the presence of phosphorus was confirmed by an ICP elemental analysis.

Claims

WHAT IS CLAIMED IS:
1. A process for producing a biophosphorylated hydroxyl containing organic compound, wherein said hydroxyl group is phosphate reactive, comprising the step of contacting a strain of Rhizopus oryzae microorganism together with the hydroxy containing organic compound in an aqueous medium containing a carbon nutrient at ambient temperature for a sufficient time to produce the biophosphorylated hydroxyl containing organic compound.
2. The process of Claim 1 wherein said microorganism comprises resting cells of Rhizopus oryzae in a phosphate buffer containing glycerol as a carbon nutrient.
3. The process of Claim 1 wherein said Rhizopus oryzae is cultured with said hydroxyl containing organic compound under submerged aerobic fermentation conditions in an aqueous carbohydrate medium containing a nitrogen nutrient at a pH below about 8.0 at ambient temperature.
4. The process of claim 1 wherein said
Rhizopus oryzae strain is ATCC No. 11145.
5. A process for producing an immunos- suppressant, identified as a C-32 phosphorylated derivative of an FK-506 type macrolide comprising the step of contacting a strain of a Rhizopus oryzae microorganism together with a FK-506 type macrolide, containing a free C-32 hydroxy group, in an aqueous medium containing a carbon nutrient at ambient temperature for a sufficient time to produce the C-32 phosphorylated FK-506 type macrolide immunosuppressant.
6. A compound being the C-32 phosphorylated derivative of an FK-506 type macrolide.
7. The compound of Claim 1 of the formula:
Figure imgf000135_0001
wherein:
R is H, C1-C4 alkyl,
R1 is H2PO4,
R2 is hydrogen, hydroxy or lower alkanoyloxy, R3 is methyl, ethyl, propyl or allyl,
n is an integer of 1 or 2, and the symbol of a line and dotted line is a single bond or a double bond,
and a pharmaceutically acceptable salt thereof.
8. The compound of Claim 7 being the C-32 phsophorylated derivative of FK-506, FK-520, FK-523, FK-525, C-31 desmethyl FK-506, C-31 desmethyl FK-520,
9. The compound of Claim 7 being:
Figure imgf000136_0001
10. The compound of Claim 7 being:
Figure imgf000137_0001
11. The compound of Claim 7 being;
Figure imgf000138_0001
12. The compound of Claim 7 being:
Figure imgf000139_0001
13. The compound of Claim 7 being:
Figure imgf000140_0001
14. The compound of Claim 7 being:
Figure imgf000141_0001
15. The compound of Claim 7 being:
Figure imgf000142_0001
16. A pharmaceutical composition for the treatment of immunoregulatory disorders or diseases comprising a pharmaceutical carrier and a
therapeutically effective amount of compound of Claim 7.
17. A pharmaceutical composition for the topical treatment of inflammatory and
hyperproliferative skin diseases and or cutaneous manifestations of immunologically-mediated illnesses comprising a pharmaceutical carrier and a
therapeutically effective amount of compound of Claim 7.
18. A method for the treatment of immunoregulatory disorders or diseases comprising the administration to a mammalian species in need of such treatment an effective of a compound of Claim 7.
19. A process for producing an
immunosuppressant, identified as a phosphorylated rapamycin-type macrolide comprising the step of contacting a strain of a Rhizopus oryzae
microorganism together with rapamycin macrolide in an aqueous medium containing a carbon nutrient at ambient temperature for a sufficient time to produce the C-43 phosphorylated rapamycin macrolide
immunosuppressant.
20. The process of Claim 19 wherein said microorganism are resting cells of Rhizopus oryzae in a phosphate buffer containing glycerol as a carbon nutrient.
21. The process of Claim 19 wherein the resulting aqueous medium containing said phosphorylated macrolide exhibits positive inhibition of T-Cell activation.
22. The process of Claim 19 wherein said
Rhizopus oryzae is cultured with said rapamycin macrolide under submerged aerobic fermentation conditions in an aqueous carbohydrate medium containing a nitrogen nutrient at a pH below about
8.0 at ambient temperature.
23. A phosphorylated macrolide of the structural formula:
Figure imgf000144_0001
where R= [HO]2-
Figure imgf000144_0002
24. A phosphorylated macrolide exhibiting a characteristic proton NMR spectrum as illustrated in Figure 3 and exhibiting positive inhibition of T-cell proliferation, reversible by recombinant human IL-2, in a T-cell proliferation assay.
25. A phosphorylated macrolide having the structural formula as illustrated in Figure 4.
26. A method for producing a phosphorylated cyclic lipopeptide compound having the formula
Figure imgf000145_0001
wherein R is
Figure imgf000145_0002
(OH)2 which comprises cultivating
Rhizopus arrhizus ATCC 11145 in a nutrient medium containing a compound having the formula:
Figure imgf000146_0001
and a phosphate salt, said medium maintained in a pH range of from about 6.0 to 6.3.
27. A method according to Claim 26 in which the phosphate salt is in an amount of at least 10 percent by weight of the solid components.
28. A method according to Claim 26 in which the medium of the following composition in
grams/liter: glucose 20.0; soya meal 5.0; Fidco yeast extract 5.0: sodium chloride 5.0; and potassium hydrogen phosphate 5.0; at pH 5.
29. A method according to Claim 26 in which the cultivation is carried out in the temperature range of from about 15ºC to about 30ºC with agitation in the range of 220 rpm to 400 rpm for a period of from 24 hours to about two days.
30. A method for selectively
phosphorylating a compound having the formula
Figure imgf000147_0001
at the 4-hydroxyproline hydroxyl which comprises cultivating Rhizopus arrhizus ATCC 11145 in a soy glucose medium containing said compound in an amount of 40-60 μg/ml, wherein said soy glucose medium is of the following composition in grams/liter: glucose 20.0; soya meal 5.0; Fidco yeast extract 5.0; sodium chloride 5.0; and potassium hydrogen phosphate 5.0; at pH 5.
31. A phosphorylated cyclic lipopeptide compound obtained by biophosphorylating with Rhizopus arrhizus ATCC 11145, a cyclic lipopeptide related to echinocandins and having a peptide skeleton bearing several hydroxy groups wherein in said phosphorylated cyclic lipopeptide, the phosphate group is attached to the hydroxy group of the 4-hydroxyproline
component of the lipopeptide.
32. A phosphorylated cyclic lipopeptide compound having the formula:
Figure imgf000148_0001
wherein R is
Figure imgf000148_0002
(OH)2 or a cation salt thereof ,
33. A compound according to Claim 32 wherein
R is - (OH)2,
Figure imgf000148_0003
34. A compound according to Claim 32 wherein
R is - (OH)(OK)
Figure imgf000149_0001
35. A compound according to Claim 32 wherein R is - (OH)(ONa).
Figure imgf000149_0002
36. A composition comprising the compound of Claim 32 in admixture with a pharmaceutically acceptable carrier.
37. A method for controlling the growth of fungi comprising administering an antifungally
effective amount of the compound of Claim 32.
38. A method for the treatment of or for the prevention of Pneumocystis carinii infections in mammals which comprises administering to mammals an anti-infective or therapeutic amount of the compound of Claim 32.
39. A compound of the Formula:
Figure imgf000150_0001
or pharmaceutically acceptable salt, hydrate, or ester thereof.
40. A method of preparing the compound of Claim 39, comprising the steps of
(a) providing a quantity of N-(2(R)-hydroxy- 1(S)-indanyl)-5(S)-(1,1-dimethylethoxy- carbonylamino)-4(S)-hydroxy-2(R)-[(4-(2- (4-morpholino)ethoxy)phenyl)prop-2-en-1- yl]-6-cyclohexyl-hexanamide; (2) incubating the compound of step (a) in a microbial culture of Rhizopus
arrhizus MF4974; and
(3) isolating the compound of Claim 39.
41. A pharmaceutical composition useful for inhibiting HIV protease, comprising an effective amount of a compound of Claim 39, and a
pharmaceutically acceptable carrier.
42. A pharamceutical composition useful for preventing or treating infection of HIV or for
treating AIDS or ARC, comprising an effective amount of a compound of Claim 39, and a pharmaceutically acceptable carrier.
43. A method of inhibiting HIV protease, comprising administering to a suitable mammal in need of such treatment an effective amount of a compound of Claim 39.
44. A method of preventing infection of HIV, or of treating infection by HIV or of treating AIDS or ARC, comprising administering to a suitable mammal in need of such treatment an effective amount of a compound of Claim 39.
45. A compound of the Formula:
Figure imgf000152_0001
or pharmaceutically acceptable salt, hydrate, or ester thereof.
46. A method of preparing the compound of Claim 45, comprising the steps of
(1) providing a quantity of N-(2(R)-hydroxy- 1(S)-indanyl)-5(S)-(1,1-dimethylethoxy- carbonylamino)-4(S)-hydroxy-6-phenyl- 2(R)-[(4-(2-(4-morpholinyl)ethoxy)- phenyl)methyl]-hexanamide; (2) incubation the compound of step (1) in a microbial culture of Rhizopus
arrhizυs MF4974; and
(3) isolating the compound of Claim 45.
47. A pharmaceutical composition useful for inhibiting HIV protease, comprising an effective amount of a compound of Claim 45, and a
pharmaceutically acceptable carrier.
48. A pharamceutical composition useful for preventing or treating infection of HIV or for treating AIDS or ARC, comprising an effective amount of a compound of Claim 45, and a pharmaceutically acceptable carrier.
49. A method of inhibiting HIV protease, comprising administering to a mammal an effective amount of a compound of Claim 45.
50. A method of preventing infection of HIV, or of treating infection by HIV or of treating AIDS or ARC, comprising administering to a mammal an effective amount of a compound of Claim 45.
51. A phosphorylated simvastatin derivative of the following structure:
Figure imgf000154_0001
52. A phosphorylated zearalenol compound of the following structure:
Figure imgf000154_0002
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