AU702122B2 - Microbial synthesis of HIV protease inhibitors - Google Patents

Microbial synthesis of HIV protease inhibitors Download PDF

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AU702122B2
AU702122B2 AU39663/95A AU3966395A AU702122B2 AU 702122 B2 AU702122 B2 AU 702122B2 AU 39663/95 A AU39663/95 A AU 39663/95A AU 3966395 A AU3966395 A AU 3966395A AU 702122 B2 AU702122 B2 AU 702122B2
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compound
hiv
aids
combination
treating
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Byron H. Arison
Shieh-Shung T. Chen
George M Garrity
Brian Heimbuch
Randall R Miller
Ali Shafiee
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Merck and Co Inc
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    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D241/00Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/10Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D241/14Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D241/24Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00Medicinal preparations containing organic active ingredients
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • A61P37/04Immunostimulants
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    • C07DHETEROCYCLIC COMPOUNDS
    • C07D241/00Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/04Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
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    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D401/00Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D413/00Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/06Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

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Description

1- TITLE OF THE INVENTION MICROBIAL SYNTHESIS OF HIV PROTEASE INHIBITORS BACKGROUND OF THE INVENTION The present invention is concerned with a novel process for synthesizing compounds that inhibit the protease encoded by human immunodeficiency virus (HIV), and in particular certain oligopeptide analogs, such as derivatives of Compound J in the Examples below.
These compounds are 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). These compounds are also useful for inhibiting renin and other proteases.
15 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. Inhibition of this processing prevents the production of normally infectious virus. For example, Kohl, N. E. er al., Proc. Nat'l Acad. Sci., 85, 4686 (1988) demonstrated that genetic inactivation of the HIV encoded protease resulted in the production of immature, non-infectious virus particles. These results indicate that inhibition of the HIV protease represents a viable method for the treatment of AIDS and the prevention or treatment of infection by HIV.
The nucleotide sequence of HIV shows the presence of a pol gene in one open reading frame [Ratner, L. et al., Nature, 313, 277 (1985)J. Amino acid sequence homology provides evidence that the pol sequence encodes reverse transcriptase, an endonuclease and an HIV protease [Toh, H. et al., EMBO 4, 1267 (1985); Power, M. D. et al., WO 96/12492 PCT/US95/13628 -2- Science, 231, 1567 (1986); Pearl, L. H. et al., Nature, 329, 351 (1987)].
Compounds, including certain oligopeptide analogs that can be made from the novel processes of this invention are inhibitors of HIV protease. See EPO 541,168, which published on May 12, 1993. See also, for example, Compound J therein.
Previously, the synthesis of Compound J and related compounds was accomplished via a 12-step procedure. This procedure is described in EPO 541,168. The extreme length of this route (12 steps), renders this process time consuming and labor intensive, and it requires the use of many presently expensive reagents and a presently expensive starting material. A route requiring fewer reaction steps and reagents would provide desirable economical and time-saving benefits.
Applicants have identified and synthesized a variety of derivatives of Compound J, by incubating Compound J with a selected microbial system, MA7065. The new compounds are active and potent inhibitors of HIV protease.
SUMMARY OF THE INVENTION Biotransformation products of a fermentation with culture MA7065 are potent HIV protease inhibitors. These products are useful in the prevention or treatment of infection by HIV and in the treatment of AIDS, either as compounds, pharmaceutically acceptable salts, pharmaceutical composition ingredients, whether or not in combination with other antivirals, immunomodulators, antibiotics or vaccines.
Methods of treating AIDS and methods of preventing or treating infection by HIV are also described.
DETAILED DESCRIPTION OF THE INVENTION A method is disclosed for synthesizing biotransformation products of the Compound J, having the structure: WO 96/12492 PCT/US95/13628 -3-
OH
NH 0O or salt or hydrate thereof, comprising the steps of providing a culture of MA7065; incubating said culture with Compound J; isolating biotransformation products comprising: NH 0 Compound A
OH
Compound C1 WO 96/12492 PCT/US95/13628 -4-
OH
NH
OH
Compound C2 N
N
NH 0
OH
Compound D
OH
S NH O Compound E or salt or hydrate thereof. A preferred compound is compound A.
Also covered are the purified biotransformation products of this method, as well as the corresponding chemical compounds.
mwww 0
B
4 4e 4 tee..
B
4 'be.
4044
BC
6* 04 0 ATCC Deposit Before the U.S. filing date of the present application, a sample of the microorganism (Merck Culture Collection MA7065) was deposited at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 on 17 August 1994. The culture access designation is ATCC 55604. This deposit will be maintained in the ATCC for at least 30 years and will be made available to the public upon the grant of a patent disclosing it. It should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.
General Characteristics of ATCC The physical characteristics and taxonomy, including morphological, cultural, biological and physiological characteristics are briefly described herein below.
On the basis of the taxonomic analysis performed thus far, the culture has been assigned to the order Streptomyces.
15 The following is a general description of Streptomyces sp. MA7065 (AS2023, CRA- 3-4 ATCC 55604). This culture produces analogs of Compound J, an inhibitor of HIV protease, by biotransformation. Observations of growth, general cultural characteristics and carbon source utilisation were made in accordance with the methods of Shirling and Gottleib (Internat. J. System. Bacteriol. 16,313-340). Chemical composition of the cells was determined using the methods of Lechevalier and Lechevalier (in Actinomycete Taxonomy, A. Dietz and D. W. Thayer, Ed., Society for Industrial Microbiology, 1980).
Whole cell fatty acids were derivatised and analysed as methyl esters (FAMEs) by gas chromatography by the procedure of Miller and Berger using a MIDI Microbial Identification System (Microbial Identification Systems, Newark, Delaware). Coloration of the culture was determined by comparision with colour standards contained in the Inter-Society Colour Council-National Bureau 00 *4
C
c [n:\libc]02067:MEF WO 96/12492 PCT/US95/13628 -6of Standards Centroid Color Charts (US Dept. of Commerce National Bureau of Standards supplement to NBS Circular 553, 1985).
Source Culture MA7065 was isolated from a soil sample collected in a fire-break beside a fallow field that had undergone controlled burning 48h prior to sampling. The field was located in Santa Rosa Park, Guanacaste PR, Costa Rica.
Chemotaxonomic characteristics The peptidoglycan of MA7065 contains LL-diaminopimelic acid. Major whole cell fatty acids are listed in Table 1.
TABLE 1 Major Whole Cell Fatty Acids Found in MA7065 Fatty acid age 14:0 iso 6.04 15:0 iso 10.56 15:0 anteiso 9.61 15:0 2.23 16:0 iso H 7.77 16:0 iso 28.50 15:0 iso 2 OH 4.81 15:0 anteiso 2 OH 3.55 16:0 3.55 17:0 anteiso C 4.01 17:0 iso 4.09 17:0 anteiso 4.71 16:0 iso 20H 3.31 18:1 iso H 1.60 General growth characteristics Good to excellent growth was observed on yeast-extract malt-extract agar, glycerol-asparagine, WO 96/12492 PCT/US95/13628 -7inorganic salts-starch agar, Czapek's agar, Sabouraud Maltose agar, oatmeal agar and trypticase soy broth agar. Fair growth was observed on tap water agar. Growth occurred at 27° and 37 0 C. See Table 2.
TABLE 2 Cultural Characteristics of Actinomycete sp. MA7065 Medium Amount of Aerial Soluble Substrate growth mycelium pigments mycelium Yeast Extract Malt Extract Glucose Aspargine Inorganic Salts-Starch Oatmeal Czapek Tap Water Excellent Good Good Excellent Good Fair H2S negative, Melanin negative Light gray (264 l.Gy) Short, loosely coiled spirals on pseudoverticils Light gray (264 1.Gy) Short, loosely coiled spirals on pseudoverticils Black (267 Black) Short, loosely coiled spirals on pseudoverticils, extensive coalescence Light gray (264 l.Gy) Short, loosely coiled spirals on pseudoverticils White (263 White) Flexous filaments, few spirals Light gray (264 1.Gy) Short, loosely coiled spirals on pseudoverticils, highly fragmented None None None None None None Dark orange yellow (72d.
OY)
Yellowish white (92 y.
White) Grayish yellow (90 gy.Y) Grayish yellow (90 gy.Y) Colorless arK yellow brown (75 d y.Br) Peptone Ion
I
WO 96/12492 PCTIUS95/13628 -8- Colony morphology (On yeast-malt agar at 21d) Substrate mycelium is a dark yellow brown. Aerial spore mass is abundant, cottony and yellowish gray in color. Colonies are opaque, raised, with an entire edge and matte surface. The colonies are rubbery in texture.
Micromorphology Aerial mycelia (0.57 tm) arise from substrate mycelia in a verticilate fashion. In mature cultures (7 28d the aerial mycelium terminates in chains of spores that occur as short, loosely coiled spirals. Sporulation occurs on yeast extract malt extract agar, inorganic salts-starch agar, oatmeal, glycerol asparagine agar, Czapek's agar and tap-water agar. The aerial spore mass coalesces on inorganic salts-starch agar.
Miscellaneous physiological reactions Culture does not produce H2S in peptone-iron agar. Melanoid pigments were not formed in either peptone-iron agar or tryptone-yeast extract broth. Starch was weakly hydrolyzed. Carbon source utilization pattern is as follows: good utilization of D-arabinose, L-arabinose, D-fructose, a-D-glucose, inositol, a-D-lactose, p-D-lactose, D-maltose, D-mannitol, D-mannose, L-rhamnose, and sucrose; moderate utilization of D-raffinose and D-xylose. See Table 3.
WO 96/12492 PCT/US95/13628 -9- TABLE 3 Carbon Source Utilization Pattern of Strains MA7065 Carbon source Growth D-arabinose 2 L-arabinose 2 D-fructose 3 inositol 3 a-D-lactose 3 (-D-lactose 3 D-maltose 3 D-mannitol 3 D-mannose 3 D-raffinose 3 L-rhamnose 2 sucrose 3 D-xylose 2 a-D-glucose (control) 3 3=good utilization, 2=moderate utilization, l=poor utilization, 0=no utilization Diagnosis Cell wall analysis reveals that MA7065 has a type I cell wall. Morphological studies reveal that the culture produces short chains of spores on spiral sporophores, arranged in pseudoverticils which arise from the aerial mycelium. These are characteristics that are typical for some strains of Streptomyces spp. A comparison of the .phenotypic data for MA7065 with that of the validly published species of Streptomyces in the taxonomic literature shows that this strain bears some resemblance to Streptomyces albogriseolus, Stmy. lydicus, Stmy.
parvulus and Stmy. rocheii. Of these species, only Stmy. lydicus has been reported to exhibit coalescence of the aerial spore mass. However, the sporophores of Stmy. lydicus are not arranged in pseudoverticles.
WO 96/12492 PCTIUS95/13628 Comparison of the fatty acid profile of MA7065 against the MIDI actinomycete library (Version 3.7) showed a distant match to Stmy.
lydicus.
A cluster analysis was done by furthest neighbor analysis using Euclidean distance as the metric. The results of this analysis showed MA7065 to be more similar to Stmy. lydicus than the other reference strains. These findings are consistent with morphological and physiological data. Based upon these findings, MA7065 is a novel strain of Stmy. lydicus.
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 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 compounds of this invention can also be obtained by synthetic organic procedures by a skilled artisan.
As to the conditions for the production of MA7065 in massive amounts, submerged aerobic cultural conditions are preferred. For the production in small amounts, a shaking or WO 96/12492 PCT/US95/13628 -11surface 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 forms 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 generally autoclaved to sterilize the medium prior to inoculation.
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 20'C and 40'C, preferably 25-35°C, for a period of about 10 hours to 64 hours, which may be varied according to fermentation conditions and scales. Preferably, the production cultures are incubated for about 48 hours at 28 0 C on a rotary shaker operating at 220 rpm, wherein the pH of the fermentation medium is maintained at 4.85 to harvest.
Preferred culturing/production media for carrying out the fermentation include the following media: Seed medium (KE medium) consisted of: 0.1% dextrose; I% dextrin; 0.3% beef extract; 0.5% ardamine pH; 0.5% NZ amine type E; 0.005% MgSO4o7H20, and 0.037% K2HPO4 with pH adjusted to 7.1 with 0.05% CaCO3. Biotransformation medium (soy-glucose) contained: 2% glucose; 0.5% soya meal; 0.5% yeast extract; NaCl; 0.98% MES with pH adjusted to WO 96/12492 PCT/US95/13628 -12- The products can be recovered from the culture medium by conventional means which are commonly used for the recovery of other known substances. The substances produced 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 methylene chloride or methanol and the like, pH adjustment, treatment with a conventional resin anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), crystallization, recrystallization, and the like.
The compounds of this invention are useful in the preparation and execution of screening assays for antiviral compounds.
For example, the compounds of this invention are useful for isolating enzyme mutants, which are excellent screening tools for more powerful antiviral compounds. Furthermore, the compounds of this invention are useful in establishing or determining the binding site of other antivirals to HIV protease, by competitive inhibition. Thus the compounds of this invention are commercial products to be sold for these purposes.
The compounds of the present invention are 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 compounds of this invention are useful in treating infection by HIV after suspected past exposure to HIV by, blood transfusion, organ transplant, exchange of body fluids, bites, accidental needle stick, or exposure to patient blood during surgery.
For these purposes, the compounds of the present invention may be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intrastemal injection or infusion WO 96/12492 PCT/US95/13628 13 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 a compound of the present invention, or a pharmaceutically acceptable salt thereof.
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, parenterallyacceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable WO 96/12492 PCT/US95/13628 -14dispersing 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 gramsper-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 to 50 milligrams of the compound per kilogram of body weight from one to four times per day. In one preferred regimen, dosages of 100- 400 mg every six hours are administered orally to each patient. 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, 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 inhibitory compounds with one or more agents useful in the treatment of AIDS. For example, the compounds of this invention may be effectively administered, whether at periods of preexposure and/or post-exposure, in combination with effective amounts of the AIDS antivirals, immunomodulators, anti-infectives, or vaccines known to those of ordinary skill in the art.
PCTIUS95/13628 WO 96/12492 15 TABLE C
ANTIVIRALS
Drug Name Manufacturer Indication AL-721 Recombinant Human Interferon Beta Acemannan Ethigen (Los Angeles, CA) Triton Biosciences (Almeda, CA) Carrington Labs (Irving, TX) ARC, PGL HIV positive, AIDS AIDS, Kaposi's sarcoma, ARC
ARC
(See also immunomodulators) sight threatening CMV Cytovene Syntex Ganciclovir d4T Didehydrodeoxythymidine ddl Dideoxyinosine (Palo Alto, CA) Bristol-Myers (New York, NY) Bristol-Myers (New York, NY) Elan Corp, PLC (Gainesville, GA) peripheral CMV retinitis AIDS, ARC AIDS, ARC HIV infection (See also immunomodulators) WO 96/12492 PCT/US95/13628 16- Drug Name Trisodium Phosphonoformate Dideoxycytidine; ddC Novapren Manufacturer Astra Pharm.
Products, Inc (Westborough, MA) Hoffman-La Roche (Nutley, NJ) Novaferon Labs, Inc.
(Akron, OH) Diapren, Inc.
(Roseville, MN, marketer) Indication CMV retinitis, HIV infection, other CMV infections AIDS, ARC HIV inhibitor Peptide T Octapeptide Sequence Peninsula Labs (Belmont, CA)
AIDS
Zidovudine; AZT AIDS, adv, ARC Burroughs Wellcome (Rsch. Triangle Park,
NC)
AIDS, adv, ARC pediatric AIDS, Kaposi's sarcoma, asymptomatic HIV infection, less severe HIV disease, neurological involvement, in combination with other therapies.
Ansamycin LM 427 Adria Laboratories (Dublin, OH) Erbamont (Stamford, CT)
ARC
WO 96/12492 PCT/US95/13628 17- Drug Name Dextran Sulfate Virazole Ribavirin Alpha Interferon Acyclovir Antibody which neutralizes pH labile alpha aberrant Interferon in an immuno-adsorption column Manufacturer Ueno Fine Chem.
Ind. Ltd.
(Osaka, Japan) Viratek/ICN (Costa Mesa, CA) Burroughs Wellcome (Rsch. Triangle Park, NC) Burroughs Wellcome Advanced Biotherapy Concepts (Rockville, MD) Indication AIDS, ARC, HIV positive asymptomatic asymptomatic HIV positive, LAS, ARC Kaposi's sarcoma, HIV in combination w/Retrovir AIDS, ARC, asymptomatic HIV positive, in combination with
AZT.
AIDS, ARC Merck (Rahway, NJ) AIDS, ARC, asymptomatic HIV positive, also in combination with
AZT.
WO 9612492PCTIUS95/13628 WO 96/12492 18 Drui! Name Manufacturer Merck (Rahway, NJ) Indication AIDS, ARC, asymptomnatic HIV positive, also in combination with
AZT.
AIDS, ARC, asymptomatic HIV positive, also in combination with
AZT.
Nevirapine B oehringer Ingeiheim WO 96/12492 PCT/US95/13628 19-
IMMUNO-MODULATORS
Drug Name Manufacturer Indication AS-101 Bropirimine Acemannan CL246,738 Wyeth-Ayerst Labs.
(Philadelphia, PA) Upjohn (Kalamazoo, MI) Carrington Labs, Inc.
(Irving, TX) American Cyanamid (Pearl River, NY) Lederle Labs (Wayne, NJ) Elan Corp, PLC (Gainesville, GA) advanced AIDS AIDS, ARC (See also anti-virals) AIDS, Kaposi's sarcoma
AIDS
HIV infection (See also antivirals) Gamma Interferon Genentech San Francisco,
CA)
ARC, in combination w/TNF (tumor necrosis factor) PCT/US95/13628 WO 96/12492 Drug Name Manufacturer Indication Granulocyte Macrophage Colony Stimulating Factor Granulocyte Macrophage Colony Stimulating Factor Granulocyte Macrophage Colony Stimulating Factor Genetics Institute (Cambridge, MA) Sandoz (East Hanover, NJ) Hoeschst-Roussel (Somerville, NJ) Immunex (Seattle, WA) Schering-Plough (Madison, NJ)
AIDS
AIDS
AIDS
HIV Core Particle Immunostimulant IL-2 Interleukin-2 IL-2 Interleukin-2 Immune Globulin Intravenous (human) Rorer (Ft. Washington, PA) Cetus (Emeryville, CA) Hoffman-La Roche (Nutley, NJ) Immunex Cutter Biological (Berkeley, CA) AIDS, in combination w/AZT seropositive HIV AIDS, in combination w/AZT AIDS, ARC, HIV, in combination w/AZT pediatric AIDS, in combination w/AZT WO 96/12492 PCTIUS95/13628 -21 Drug Name IMREG-1 IMREG-2 Imuthiol Diethyl Dithio Carbamate Alpha-2 Interferon Methionine- Enkephalin
MTP-PE
Muramyl- Tripeptide Granulocyte Colony Stimulating Factor rCD4 Recombinant Soluble Human CD4 Manufacturer Imreg (New Orleans, LA) Imreg (New Orleans, LA) Merieux Institute (Miami, FL) Schering Plough (Madison, NJ) TNI Pharmaceutical (Chicago, IL) Ciba-Geigy Corp.
(Summit, NJ) Amgen (Thousand Oaks, CA) Genentech San Francisco,
CA)
Indication AIDS, Kaposi's sarcoma, ARC, PGL AIDS, Kaposi's sarcoma, ARC, PGL AIDS, ARC Kaposi's sarcoma w/AZT: AIDS AIDS, ARC Kaposi's sarcoma AIDS, in combination w/AZT AIDS, ARC rCD4-IgG AIDS, ARC hybrids WO 96/12492 PCT/US95/13628 -22- Drug Name Recombinant Soluble Human CD4 Interferon Alfa 2a SK&F106528 Soluble T4 Thymopentin Tumor Necrosis Factor; TNF Manufacturer Biogen (Cambridge, MA) Hoffman-La Roche (Nutley, NJ) Smith, Kline French Laboratories (Philadelphia, PA) Immunobiology Research Institute (Annandale, NJ) Genentech San Francisco,
CA)
ANTI-INFECTIVES
Indication AIDS, ARC Kaposi's sarcoma AIDS, ARC, in combination w/AZT HIV infection HIV infection ARC, in combination w/gamma Interferon Drug Name Clindamycin with Primaquine Manufacturer Upjohn (Kalamazoo, MI) Pfizer (New York, NY)
PCP
Indication Fluconazole cryptococcal meningitis, candidiasis WO 96/12492 WO 9612492PCTIUS95/13628 23 Dru Name Pastille Nystatin Pastille Manufacturer Squibb Corp.
(Princeton, NJ) Merrell Dow (Cincinnati, OH) LyphoMed (Rosemont, IL) Indication, prevention of oral candidiiasis Ornidyl Eflomnithine
PCP
Pentamnidine Isethionate (IM IV) PCP treatment Trimethoprim antibacterial antibacterial Trimethoprim/sulfa Piritrexim Pentamidine isethionate for inhalation Spiramycin Intraconazole- R51211 Burroughs Wellcome (Rsch. Triangle Park, NC) Fisons Corporation (Bedford, MA) Rhone-Poulenc Pharmaceuticals (Princeton, NJ) Janssen Pharm.
(Piscataway, NJ) PCP treatment PCP prophylaxis cryptosporidial diarrhea histoplasmosis; cryptoco ccal meningitis Trimetrexate TrimetexateWarner-Lambert PC PCP WO 96/12492 PCT/US95/13628 -24-
OTHER
Drug Name Manufacturer Indication Recombinant Human Ortho Pharm. Corp. severe anemia Erythropoietin (Raritan, NJ) assoc. with AZT therapy Megestrol Acetate Bristol-Myers treatment of (New York, NY) anorexia assoc.
w/AIDS Total Enteral Norwich Eaton diarrhea and Nutrition Pharmaceuticals malabsorption (Norwich, NY) related to AIDS It will be understood that the scope of combinations of the compounds of this invention with AIDS antivirals, immunomodulators, anti-infectives or vaccines is not limited to the list in the above Table, but includes in principle any combination with any pharmaceutical composition useful for the treatment of AIDS.
Certain compounds of Table C are the following: Compound B is 6-chloro-4-(S)-cyclopropyl-3,4-dihydro- 4 2 pyridyl)ethynyl)quinazolin- 2 (lH)-one; Compound C 6-chloro-4(S)-trifluoromethyl-1,2-dihydro-4(H)-3,1benzoxazin-2-one; nevirapine is 11-cyclopropyl-5,1 -dihydro-4-methyl- 6H-dipyrido[3,2-b:2',3'-e][l,4]diazepin-6-one. Compounds B and C are synthesized by the methods of EP 0,569,083, herein incorporated by reference for this purpose. Nevirapine is synthesized by Klunder, J. M.
et al., J. Med. Chem. 35, 1887 (1992); Hargrave, K. D. et al., J. Med Chem. 34, 2231 (1991); Cohen, K. A. et al.,J. Biol. Chem. 266, 14670 (1991), all three references herein incorporated by reference.
Preferred combinations are simultaneous or alternating treatments of an inhibitor of HIV protease and a non-nucleoside WO 96/12492 PCT/US95/13628 inhibitor of HIV reverse transcriptase. An optional third component in the combination is a nucleoside inhibitor of HIV reverse transcriptase, such as AZT, ddC or ddl. A preferred inhibitor of HIV protease is Compound A. Preferred non-nucleoside inhibitors of HIV reverse transcriptase include Compound B, Compound C or nevirapine. These combinations may have synergistic effects on limiting the spread of HIV. Preferred combinations include the following Compound A, with a preferred non-nucleoside inhibitor of HIV reverse transcriptase, and, optionally, AZT or ddl or ddC; Compound A, and any of AZT or ddl or ddC.
Assay for Inhibition of Microbial Expressed HIV Protease Inhibition studies of the reaction of the protease expressed in Eschericia coli with a peptide substrate [Val-Ser-Gln-Asn- (betanapthyl)Ala-Pro-Ile-Val, 0.5 mg/mL at the time the reaction is initiated] were in 50 mM Na acetate, pH 5.5, at 30 0 C for 1 hour.
Various concentrations of inhibitor in 1.0 tl DMSO were added to p.l of the peptide solution in water. The reaction is initiated by the addition of 15 ptl of 0.33 nM protease (0.11 ng) in a solution of 0.133 M Na acetate pH 5.5 and 0.1% bovine serum albumin. The reaction was quenched with 160 ptl of 5% phosphoric acid. Products of the reaction were separated by HPLC (VYDAC wide pore 5 cm C-18 reverse phase, acetonitrile gradient, 0.1% phosphoric acid). The extent of inhibition of the reaction was determined as IC50 from the peak heights of the products. HPLC of the products, independently synthesized, proved quantitation standards and confirmation of the product composition.
CELL SPREAD ASSAY Inhibition of the spread of HIV in cell culture was measured according to Nunberg, J. H. et al., J. Virol. 65, 4887 (1991).
In this assay, MT-4 T-lymphoid cells were infected with HIV-1 (wildtype, unless otherwise indicated) by using a predetermined inoculum, and cultures were incubated for 24 h. At this time, <1 of the cells WO 96/12492 PCT/US95/13628 -26were positive by indirect immunofluorescence. Cells were then extensively washed and distributed into 96-well culture dishes. Serial twofold dilutions of inhibitor were added to the wells, and cultures were continued for 3 additional days. At 4 days postinfection, 100% of the cells in control cultures were infected. HIV-1 p24 accumulation was directly correlated with virus spread. The cell culture inhibitory concentration was defined as the inhibitor concentration in nanomoles/liter which reduced the spread of infection by at least or INHIBITION OF VIRUS SPREAD A. Preparation of HIV-infected MT-4 cell Suspension.
MT cells were infected at Day 0 at a concentration of 250,000 per ml with a 1:1000 dilution of HIV-1 strain IIIb stock (final 125 pg p24/ml; sufficient to yield infected cells on day 1 and 100% on day Cells were infected and grown in the following medium: RPMI 1640 (Whittaker BioProducts), 10% inactivated fetal bovine serum, 4 mM glutamine (Gibco Labs) and 1:100 Penicillin- Streptomycin (Gibco Labs).
The mixture was incubated overnight at 37 0 C in 5% C02 atmosphere.
B. Treatment with Inhibitors A matrix of nanomolar range concentrations of the pairwise combinations is prepared. At Day 1, aliquots of 125 ul of inhibitors are added to equal volumes of HIV-infected MT-4 cells (50,000 per well) in a 96-well microtiter cell culture plate. Incubation is continued for 3 days at 37°C in 5% C02 atmosphere.
C. Measurement of Virus Spread Using a multichannel pipettor, the settled cells are resuspended and 125 [l harvested into a separate microtiter plate. The supernatant is assayed for HIV p2 4 antigen.
WO 96/12492 PCT/US95/13628 -27 The concentration of HIV p24 antigen is measured by an enzyme immunoassay, described as follows. Aliquots of p2 4 antigen to be measured are added to microwells coated with a monoclonal antibody specific for HIV core antigen. The microwells are washed at this point, and at other appropriate steps that follow. Biotinylated HIV-specific antibody is then added, followed by conjugated streptavidinhorseradish peroxidase. A color reaction occurs from the added hydrogen peroxide and tetramethylbenzidine substrate. Color intensity is proportional to the concentration of HIV p24 antigen.
Calculation of Degree of Svnergv or Enhanced Inhibition When there is synergy pairwise combinations of inhibitors are found to exhibit markedly enhanced inhibition of virus spread, in comparison to each inhibitor alone, or in comparison to merely additive inhibition of each inhibitor.
The data is processed as follows: fractional inhibitory concentration ratios (FIC) are calculated according to Elion, et al., J.
Biol. Chem., 208, 477 (1954). The minimum sum of FICS, which is the maximum synergy, is determined for various pairwise combinations.
The smaller the number, the greater the synergy.
EXAMPLE 1 A. Culture Preparation Culture MA7065 was grown in seed and biotransformation media, respectively. Seed medium (KE medium) consisted of: 0.1 dextrose; 1% dextrin; 0.3% beef extract; 0.5% ardamine pH; 0.5% NZ amine type E; 0.005% MgSO4*7H20, and 0.037% K2HPO4 with pH adjusted to 7.1 with 0.05% CaCO3. Biotransformation medium (soyglucose) contained: 2% glucose; 0.5% soya meal; 0.5% yeast extract; NaCl; 0.98% MES with pH adjusted to WO 96/12492 PCT/US95/13628 -28 B. Biotransformation Screening MA7065 was grown in KE seed medium. After overnight incubation at 27 0 C with gyratory shake (220 rpm) two milliliters of each culture was transferred into 250 ml baffled flasks containing 50 ml of soy-glucose bioconversion medium. At zero time, 5 mg of Compound J. dissolved in 0.5 ml DMSO, was added to each flask and incubation continued as for seed culture. At various time intervals, a sample of the biotransformation culture was examined by HPLC. At the time of maximal conversion of the substrate, as determined by HPLC, the cultures were harvested and subjected to isolation, purification and characterization.
C. Isolation and Characterization of the Biotransformation Products The content of ten flasks initially containing 25 mg of the HIV-protease inhibitor, Compound J, were pooled and centrifuged. The supernatant was recovered and applied on the top of an activated column containing 14% octadecyl support. The column was washed with water and then eluted with a gradient of an aqueous methanol from 20 to Each eluted fraction was examined by HPLC on an analytical column.
This column was developed with a gradient solvent system consisting of reservoir A and B. Reservoir A contained 10% ammonium acetate 0.1% formic acid and reservoir B contained 67% acetonitrile 33% methanol 0.1% formic acid. The gradient was run from 30 to solvent B in 30 min. Based on the result from the analytical column, fractions containing desired metabolites were eluted with 60 to aqueous methanol from the activated column. This fraction was therefore further purified on a semi-prep. Four resolved fractions from the analytical column was each isolated and after further purification were submitted for NMR, FAB-MS analysis and biological evaluation.
WO 96/12492 PCT/US95/13628 -29- EXAMPLE 2 Four samples isolated from an incubation of the HIV protein inhibitor Compound J with bacterial culture MA7065 were characterized by mass spectral analysis. As shown below, Compounds A, Cl, C2, D and E were characterized. Oxidation of the indanyl, phenyl, and pyridine rings was observed.
Mass spectra and daughter ion spectra were obtained by LC/MS/MS on a mass spectrometer using the ionspray interface.
Samples were analyzed by direct injection in a mobile phase that consisted of 50% CH3CN/50% 10 mM NH40Ac/0.1% TFA. Positive ion detection was used.
N OH N OH O NH Compound J The mass spectrum of J gave an 614, which indicated a molecular weight of 613 Da. Four key fragment ions in the daughter ion spectrum of J at m/z 513, 465, 421, and 338, made it possible to determine the general site of metabolism for these samples.
The following table summarizes the results of the analysis of these spectra and includes samples of MA7065 biotransformation products (68051-A, 68051-9C, 68051-12 FI and 68051-12 F3) as well as products from other microorganisms.
WO 96/12492 PCTIUS95/13628
TABLE
Sample Compound Mol. Weight Addition of: Site of Addition 68051-5 X1 68051-5 X2 68051-5 X3 645 629 627 two hydroxyls phenyl, indanyl 68051-9 A* (E) 629 629 68051-9 B 68051-9 C 68051-12 F1 (Cl C2) 68051-12 F2 (D) 68051-12 F3 (E)
(A)
Sample was determined to compounds 629 629 629 629 629 hydroxyl ketone oxygen hydroxyl hydroxyl hydroxyl hydroxyl hydroxyl oxygen hydroxyl indanyl indanyl pyridine indanyl indanyl indanyl indane phenyl pyridine indanyl be a mixture of two oxygenated The structures of these compounds were conformed by NMR spectroscopic analysis.
EXAMPLE 3 HIV Protease Inhibitory Activity of Compound J Bioconversion Products Seven Compound J bioconversion products were assayed for in vitro HIV protease inhibitory by the assay for inhibition of microbial expressed HIV Protease, protocol given above. Four of the bioconversion products were isolated from MA7065 incubations, Compounds A, C1, C2, D and E. The IC50 of the parent Compound J WO 96/12492 PCT/US95/13628 31 was divided by the IC50 of each test compound and multiplied by 100 in order to calculate the percent potency of each compound relative to J.
In some cases, the dilution of the test compound was based on nominal weight. In other cases, especially when the weight was less than 250 jtg, the concentration was checked by HPLC using J as standard.
Potency Relative to Compound J Compound Wt. Rec'd Based On Nominal Wt.
27% Based on HPLC a 750 ag (2,3-Trans-OH)
B
(2,3-Cis-OH)
C
or 6-OH-Indane) 240 tpg 115% 50 gg 8.6% 86%
D
(p-OH-Phenyl)
E
(N-Oxide)
F
(3-Ketone) 50 igg 3.1% 31% 250 igg 34% 1000 igg 16% aNot calculated WO 96/12492 PCT/US95/13628 -32- EXAMPLE 4 Preparation of Amide 1 0 1) Y OCOCI O NH2 O 00H 3 1 r 0 OCHEt3 2) O H A solution of (-)-cis-l-aminoindan-2-ol (884 g, 5.93 mol) in 17.8 L of dry THF (KF 55 mg/mL) (KF stands for Karl Fisher titration for water) and triethylamine (868 mL, 6.22 mol) in a 50 L round bottom flask equipped with a thermocouple probe, mechanical stirrer, and a nitrogen inlet adapter and bubbler, was cooled to Then, 3-phenylpropionyl chloride (1000 g, 5.93 mol) was added over minutes, while the internal temperature was kept between 14-24°C with an ice-water cooling batch. After addition, the mixture was aged at 18 to 20 0 C for 30 minutes and checked by HPLC analysis for the disappearance of (-)-cis-l-aminoindan-2-ol.
Progress of the reaction is monitored by high performance liquid chromatography (HPLC) analysis: 25 cm Dupont C8-RX column, 60:40 acetonitrile/10 mM (KH2PO4/K2HPO4), 1.0 mL/min, injection volume 20 mL, detection 200 nm, sample preparation 500 X dilution. Approximate retention times: retention time (min) identity 6.3 cis-aminoindanol The reaction was treated with pyridinium p-toluenesulfonate (241 g, 0.96 mol, 0.16 equiv.) and stirred for 10 minutes (the pH of the mixture after diluting 1 mL sample with an equal volume of water is between Then, 2-methoxypropene (1.27 L, 13.24 mol, 2.2 equiv.) was added and reaction was heated to 38-40°C for 2 h.
WO 96/12492 PCT/US95/13628 33 The reaction mixture was cooled to 20 0 C and partitioned with ethyl acetate (12 L) and 5% aqueous NaHCO3 (10 The mixture was agitated and the layers were separated. The ethyl acetate extract was washed with 5% aqueous NaHCO3 (10 L) and water (4 The ethyl acetate extract was dried by atmospheric distillation and solvent switched to cyclohexane (total volume of -30L). At the end of the distillation and concentration (20 volume of ethyl acetate extraction volume), the hot cyclohexane solution was allowed to slowly cool to 0 C to crystallize the product. The resulting slurry was further cooled to 10 0 C and aged for 1 h. The product was isolated by filtration and the wet cake was washed with cold (10 0 C) cyclohexane (2 X 800 mL). The washed cake was dried under vacuum (26" of Hg) at 40 0 C to afford 1.65 kg of acetonide 1 98 area% by HPLC), IH NMR (300.13 MHz, CDC13, major rotamer) 6 7.36-7.14 9 5.03 J=4.4, 1 4.66 1 H) 3.15 2 3.06 (br s, 2 2.97 2 1.62 3 H), 1.37 3 13C NMR (75.5 MHz, CDC13, major rotamer) 8c 168.8, 140.9, 140.8, 140.6, 128.6, 128.5, 128.4, 127.1, 126.3, 125.8, 124.1, 96.5, 78.6, 65.9, 38.4. 36.2, 31.9, 26.5, 24.1. Anal. Calcd for C21H23NO2: C, 78.47; H, 7.21; N, 4.36. Found: C, 78.65; H, 7.24; N, 4.40.
EXAMPLE Preparation of Epoxide 3 0 ~i SX >I OTs \N O O H 2 H0 1 Base 0 WO 96/12492 PCT/US95/13628 -34- A solution of acetonide 1 (1000 g, 3.11 mol) and 2(S)glycidyl tosylate 2 (853 g, 3.74 mol, 1.2 equiv.) in 15.6 L of THF (KF 22 mg/mL) in a 50 L 4-neck round bottom flask, equipped with a thermocouple, mechanical stirrer, addition funnel and nitrogen inlet adapter was degassed 3 times via vacuum-nitrogen purge and cooled to -56 0 C. Then, lithium hexamethyldisilazide (LiN[(CH3)3Si]2)(2.6 L, 1.38 M, 1.15 equiv.) was added over 2 h, while keeping the internal temperature between -50 to -45 0 C. The reaction mixture was stirred at to -40 0 C for 1 h and then allowed to warm to -25 0 C over 1 h. The mixture is stirred between -25 to -22 0 C for 4 h (or until the starting acetonide is 3.0 area Progress of the reaction is monitored by HPLC analysis: cm X 4.6 nm Zorbax Silica column, 20% ethyl acetate in hexane, mL/min, injection volume 20 mL, detection 254 nm, sample preparation 100 X dilution. Approximate retention times: retention time (min). identity amide 1 glycidyl tosylate 2 13.5 epoxide 3 The reaction mixture was quenched with DI water (6.7 L) at -15°C and partitioned with ethyl acetate (10 The mixture was agitated and the layers were separated. The ethyl acetate extract was washed with a mixture of 1% aqueous NaHCO3 (5 L) and saturated NaCI (0.5 The ethyl acetate extract (28.3 L) was concentrated by vacuum distillation (28" of Hg) and additional ethyl acetate was added to complete the solvent switch to ethyl acetate (final volume 11.7 L).
The ethyl acetate concentrate was further solvent switched to MeOH to crystallize the product and concentrated to a final volume of 3.2 L. The residual ethyl acetate solvent was removed by charging 10 L of methanol and collecting 10 L of distillate. The resulting slurry was stirred at 22°C for 1 h, then cooled to 5 0 C and aged for 0.5 h. The product was isolated by filtration and the wet cake was washed with cold WO 96/12492 PCT/US95/13628 methanol (2 X 250 mL). The washed cake was dried under vacuum (26" of Hg) at 25 0 C to afford 727 g of epoxide 3 98.7 area of the major epoxide by HPLC): 13C NMR (75.5 MHz, CDC13) 8 171.1, 140.6, 140.5, 139.6, 129.6, 128.8, 128.2, 127.2, 126.8, 125.6, 124.1, 96.8, 79.2, 65.8, 50.0, 48.0, 44.8, 39.2, 37.4, 36.2, 26.6, 24.1.
EXAMPLE 6 Preparation of penultimate 6 1) BocN
LNH
t-Bu-NH
O
BocN tBN, t-Bu-NH 0 2) aq. HCI
ON
HN
OH
N NH OH t-Bu-NH 0 6 6- WO 96/12492 PCT/US95/13628 -36 CI N OH O H N N
NHI
t-Bu-NH O
J
A slurry of the 2(S)-t-butylcarboxamide-4-N-Bocpiperazine 4 (1950 g, 6.83 mol, >99.5% ee) (ee enantiomeric excess) and the epoxide 3 (2456 g, 97.5:2.5 mixture of 4S/R epoxides, 6.51 mol) in isopropanol (2-propanol, 18.6 L) in a 72 L round bottom flask with four inlets, equipped with a mechanical stirrer, reflux condenser, steam bath, Teflon coated thermocouple and nitrogen inlet, was heated to reflux (internal temperature was 84-85 0 After 40 min, a homogeneous solution was obtained. The mixture was heated at reflux for 28 h.
The internal temperature during reflux was 84-85 0
C.
Progress of the reaction was monitored by HPLC analysis: 25 cm Dupont C8-RX column, 60:40 acetonitrile/10 mM (KH2PO4/K2HPO4), 1.0 mL/min, detection 220 nm, sample preparation 2 pL, reaction mixture diluted to 1 mL in acetonitrile. Approximate retention times: retention time (min) identity 4.8 piperazine 4 8.9 epoxide 3 15.2 coupled product After 28 h, the remaining epoxide 3 and coupled product (by HPLC analysis) were 1.5 area and 91-93 area respectively.
The mixture was cooled to 0 to 5 0 C and 20.9 L of 6 N HCI was added while keeping the temperature below 15°C. After the addition was WO 96/12492 PCTIUS95/13628 -37 complete, the mixture was warmed to 22 0 C. Evolution of gas is noted at this point (isobutylene). The mixture was aged at 20 to 22 0 C for 6 h.
Progress of the reaction was monitored by HPLC analysis: same conditions as above. Approximate retention times: retention time (min) identity cis-aminoindanol 11.9 penultimate 6 15.1 coupled product The mixture was cooled to 0°C and 7.5 L of 50% NaOH was slowly added to adjust the pH of the mixture to pH=l 1.6, while keeping the temperature less than 25 0 C during the addition. The mixture was partitioned with ethyl acetate (40 L) and water (3 The mixture was agitated and the layers were separated. The organic phase L) was concentrated under reduced pressure (29" of Hg) and solvent switched to DMF and concentrated to a final volume of 10.5 L (KF 1.8 mg/mL). The HPLC assay yield of 6 in ethyl acetate was 86.5%.
The penultimate compound 6 in DMF was directly used in the next step without further purification. For isolated 6: 13C NMR (75.4 MHz, CDC13) 8 175.2, 170.5, 140.8, 140.5, 139.9, 129.1, 128.5, 127.9, 126.8, 126.5, 125.2, 124.2, 73.0, 66.0, 64.8, 62.2, 57.5, 49.5, 47.9, 46.4, 45.3, 39.6, 39.3, 38.2, 28.9.
To 10.0 g (0.019 mol) of N-(2(R)-hydroxy-l(S)-indanyl)- 2(R)-phenylmethyl-4(S)-hydroxy)-5-( (-2(S)-N-(t-butylcarbamoyl)piperazinyl)-pentaneamide 6 and 3.45 g (0.021 mol) of 3-picolyl chloride dissolved in 40 mL of DMF was added 5.85 mL (0.042 mol) of triethylamine. After 3 hours an additional 0.313 g of 3-picolyl chloride was added. After an additional 2 hours the reaction was diluted with 400 mL of EtOAc and washed with water (3 x 75 mL), brine (1 x 100 mL), dried over MgSO4 and concentrated. The residue was triturated with 30 mL of EtOAc and the resulting white precipitate was collected.
Further recrystallization from EtOAc provided the product Compound J 167.5-168°C).
WO 96/12492 PCT/US95/13628 -38 EXAMPLE 7 Pvrazine-2-tert-butyl carboxamide 9 N
CN
N COOH N CONHt-Bu 8 9 2-Pyrazinecarboxylic acid 3.35 kg (27 mol) Oxalyl chloride 3.46 kg (27.2 mol) tert-Butylamine (KF 460 gg/ml) 9.36 L (89 mol) EtOAc (KF 56 ig/ml) 27 L DMF 120 mL 1-Propanol 30 L The carboxylic acid 8 was suspended in 27 L of EtOAc and 120 mL of DMF in a 72 L 3-neck flask with mechanical stirring under N2 and the suspension was cooled to 2 0 C. The oxalyl chloride was added, maintaining the temperature between 5 and 8°C.
The addition was completed in 5 h. During the exothermic addition CO and C02 were evolved. The HCI that was formed remained largely in solution. A precipitate was present which is probably the HCL salt of the pyrazine acid chloride. Assay of the acid chloride formation was carried out by quenching an anhydrous sample of the reaction with t-butylamine. At completion of acid 8 remained.
The assay for completion of the acid chloride formation is important because incomplete reaction leads to formation of a bis-tertbutyl oxamide impurity.
The reaction can be monitored by HPLC: 25 cm Dupont Zorbax RXC8 column with 1 mL/min flow and detection at 250 nm; linear gradient from 98% of 0.1 aqueous H3P04 and 2% CH3CN to WO 96/12492 PCT/US95/13628 39 aqueous H3P04 and 50% CH3CN at 30 min. Retention times: acid 8 10.7 min, amide 9 28.1 min.
The reaction mixture was aged at 5°C for 1 h. The resulting slurry was cooled to 0°C and the tert-butylamine was added at such a rate as to keep the internal temperature below 20 0
C.
The addition required 6 h, as the reaction was very exothermic. A small portion of the generated tert-butylammonium hydrochloride was swept out of the reaction as a fluffy white solid.
The mixture was aged at 18 0 C for an additional 30 min.
The precipitated ammonium salts were removed by filtration. The filter cake was washed with 12 L of EtOAc. The combined organic phases were washed with 6 L of a 3% NaHCO3 and 2 X 2 L of saturated aq. NaC1. The organic phase was treated with 200 g of Darco carbon and filtered through Solka Flok and the cake was washed with 4 L of EtOAc.
Carbon treatment efficiently removed some purple color in the product.
The EtOAc solution of 9 was concentrated at 10 mbar to of the original volume. 30 L of 1-propanol were added, and the distillation was continued until a final volume of 20 L was reached.
At this point, the EtOAc was below the limit of detection in the 1H NMR The internal temperature in this solvent change was 30 0 C. A 1-propanol/EtOAC solution of 3 was stable to reflux atatmospheric pressure for several days.
Evaporation of an aliquot gave a tan solid m.p. 87-88 0 C. 13C NMR MHz, CDC13, ppm) 161.8, 146.8, 145.0, 143.8, 142.1, 51.0, 28.5.
WO 96/12492 PCT/US95/13628 EXAMPLE 8 rac-2-tert-Butvl-carboxamide-piperazine N H 2 /Pd(OH) 2
N
N CONHt-Bu N CONHt-Bu 9
H
Materials Pyrazine-2-tert-butylcarboxamide 9 2.4 kg (13.4 mol) in 1- Propanol solution 12 L 20% Pd(OH)2/C 16 wt.% water 144 g.
The pyrazine-2-tert-butylcarboxamide 9/1-propanol solution was placed into the 5 gal autoclave. The catalyst was added and the mixture was hydrogenated at 65 0 C at 40 psi (3 atm) of H2.
After 24 h the reaction had taken up the theoretical amount of hydrogen and GC indicated of 9. The mixture was cooled, purged with N2 and the catalyst was removed by filtration through Solka Floc. The catalyst was washed with 2 L of warm 1-propanol.
It was found that the use of warm 1-propanol during washing of the filter cake improved filtration and lowered the losses of product on the filter cake.
The reaction was monitored by GC: 30 m Megabore column, from 100°C to 160C at 10 0 C/min, hold 5 min, then at 0 C/min to 250 0 C, retention times: 9 7.0 min, 10 9.4 min. The reaction could also be monitored by TLC with EtOAc/MeOH (50:50) as solvent and Ninhydrin as developing agent.
Evaporation of an aliquot indicated that the yield over amidation and hydrogenation is 88% and that the concentration of 10 is 133g/L.
Evaporation of an aliquot gave 10 as a white solid m.p.
150-151°C; 13C NMR (75 MHz, D20, ppm) 173.5, 59.8, 52.0, 48.7, 45.0, 44.8, 28.7.
WO 96/12492 PCT/US95/13628 -41 EXAMPLE 9 (S)-2-tert-Butyl-carboxamide-piperazine bis (S)-Camphorsulfonic acid salt (S)-i1 H
H
N 2 N 2 (+)-CSA N CONHt-Bu N CONHt-Bu H
H
11 Materials rac-2-tert-Butyl-carboxamide-piperazine 10 in 1-Propanol Solution acid 1-Propanol Acetonitrile Water 4.10 kg (22.12 mol) in 25.5 Kg solvent 10.0 Kg (43.2 mol) 12 L 39 L 2.4 L The solution of amine 10 in 1-propanol was charged to a 100 L flask with an attached batch concentrator. The solution was concentrated at 10 mbar and a temperature 25°C to a volume of ca 12
L.
At this point the product had precipitated from the solution, but went back into a solution when the mixture was heated to 50 0
C.
Analysis of a homogeneous aliquot indicated that the concentration of 10 was 341 g/L. The concentration was determined by HPLC: 25 cm Dupont Zorbax RXC8 column with 1.5 mL/min flow and detection at 210 nm, isocratic (98/2) CH3CN/0.1 aqueous H3P04.
Retention time of 10 2.5 min.
Acetonitrile (39 L) and water (2.4 L) were added to give a clear, slightly brown solution.
Determination of the water content by KF titration and CH3CN/1-propanol ratio by 1H NMR integration showed that the WO 96/12492 PCTIUS95/13628 -42- CH3CN/1-propanol/H20 ratio was 26/8/1.6. The concentration in the solution was 72.2 g/ L.
The (S)-10-camphorsulfonic acid was charged over 30 min in 4 portions at 20°C. The temperature rose to 40 0 C after the CSA was added. After a few minutes a thick white precipitate formed. The white slurry was heated to 76 0 C to dissolve all the solids, the slightly brown solution was then allowed to cool to 21 C over 8 h.
The product precipitated at 62 0 C. The product was filtered without aging at 21 0 C, and the filter cake was washed with 5 L of the CH3CN 1-propanol H20 26/8/1.6 solvent mixture. It was dried at in the vacuum oven with N2 bleed to give 5.6 Kg of 11 as a white crystalline solid m.p. 288-290°C (with decomp.) [a]D 2 5 18.9° (c 0.37, H20). 13C NMR (75 MHz, D20, ppm) 222.0, 164.0, 59.3, 54.9, 53.3, 49.0, 48.1, 43.6, 43.5, 43.1, 40.6, 40.4, 28.5, 27.2, 25.4, 19.9, 19.8.
The ee of the material was 95% according to the following chiral HPLC assay: an aliquot of 11 (33 mg) was suspended in 4 mL of EtOH and 1 mL of Et3N. Boc20 (11 mg) was added and the reaction mixture was allowed to age for 1 h. The solvent was completely removed in vacuo, and the residue was dissolved in ca. 1 mL of EtOAc and filtered through a Pasteur pipet with SiO2, using EtOAc as eluent.
The evaporated product fractions were redissolved in hexanes at ca. 1 mg/mL. The enantiomers were separated on a Daicel Chiracell AS column with a hexane/IPA (97:3) solvent system at a flow rate of 1 mL/min and detection at 228 nm. Retention times: S antipode 7.4 min, R 9.7 min.
WO 96/12492 PCTIUS95/13628 -43- EXAMPLE (S)-2-tert-Butylcarboxamide-4-tert-butoxycarbonyl-piperazine 4 from salt 11
H
N 2 (Boc) 2 0 N CONHt-Bu
H
11 Materials (S)-2-tert-Butyl-carboxamide-piperazine Bis CSA salt 11, 95% ee Di-tert-butyl dicarbonate Et3N EtOH Punctilious 200 proof EtOAc Boc
NS
N CONHt-Bu
H
4 5.54 Kg (8.53 mol) 1.86 Kg (8.53 mol) 5.95L (42.6 mol) 55 L 2L To the (S)-CSA salt 11 in a 100 L 3-neck flask with an addition funnel under N2 was added EtOH, followed by triethylamine at 0 C. The solid dissolved readily on the addition of the Et3N. The was dissolved in EtOAc and charged to the addition funnel. The solution of Boc20 in EtOAc was added at such a rate as to keep the temperature below 25'C. The addition took 3 h. The reaction mixture was aged for 1 h after completion of the addition of the Boc20 solution.
The reaction can be monitored by HPLC: 25 cm Dupont Zorbax RXC8 column with 1 mL/min flow and detection at 228 nm, isocratic (50/50) CH3CN/0.1 M KH2PO4 adjusted to pH=6.8 with NaOH. Retention time of 4 7.2 min. The chiral assay was carried out using the same system as in the previous step. The reaction could also be monitored by TLC with a 100% EtOAc as the solvent. (Rf=0.7) The solution was then concentrated to ca. 10 L at an internal temperature of <20 0 C in a batch-type concentrator under mbar vacuum. The solvent switch was completed by slowly bleeding in WO 96/12492 PCT/US95/13628 -44 L of EtOAc and reconcentrating to ca 10 L. The reaction mixture was washed into an extractor with 60 L of EtOAc. The organic phase was washed with 16 L of 5% aqueous Na2CO3 solution, 2 X 10 L Di water and 2 X 6 L of saturated aqueous sodium chloride. The combined aqueous washes were back extracted with 20 L of EtOAc and the organic phase was washed with 2 X 3 L water and 2 X 4 L of saturated aqueous sodium chloride. The combined EtOAc extracts were concentrated under 10 mbar vacuum with an internal temperature of 0 C in a 100 L batch-type concentrator to ca. 8 L. The solvent switch to cyclohexane was achieved by slowly bleeding in ca. 20 L of cyclohexane, and reconcentrating to ca. 8 L. To the slurry was added L of cyclohexane and 280 mL of EtOAc and the mixture was heated to reflux, when everything went into solution. The solution was cooled and seed (10 g) was added at 58 0 C. The slurry was cooled to 22'C in 4 h and the product was isolated by filtration after a 1 h age at 22 0 C. The filter cake was washed with 1.8 L of cyclohexane and dried in the vacuum oven at 35 0 C under N2 bleed to give 1.87 Kg >99.9 area by HPLC, R-isomer below level of detection) of 4 as a slightly tan powder. [a]D 2 5 22.00 (c 0.20, MeOH), m.p. 107 0 C; 13C NMR MHz, CDC13, ppm) 170.1, 154.5, 79.8, 58.7, 50.6, 46.6, 43.6, 43.4, 28.6, 28.3.
While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptations, or modifications, as come within the scope of the following claims and its equivalents.

Claims (11)

1. A method of synthesizing biotransformation products of the Compound J having the structure: OH NH 0 or salt or hydrate thereof, comprising the steps of providing a culture of MA7065; incubating said culture with Compound J; isolating biotransformation products comprising: NH O PCT[US95/13628 WO 96/12492 PTU9/32 46 OH NH 0 N NH 0 OH ,OH OH NH 0 PCT/US95/13628 WO 96/12492 -47 OH I N O NH/ or salt or hydrate thereof.
2. A purified biotransformation product of the method of Claim 1.
3. The compound, which is NH O NH O HO NH 0 NH O or salt or hydrate thereof. A method of synthesising biotransformation products useful in treating and O NH O O :1 or salt or hydrate thereof.
4. A purified biotransformation product, substantially as hereinbefore described with reference to any one of compounds A, C 1 C 2 D, or E of Example 2.
5. A method of synthesising biotransformation products useful in treating and Sdelaying the onset of AIDS, the method being substantially as hereinbefore described with reference to the Examples.
6. A pharmaceutical composition comprising the compound of any one of claims 2 to 4, and a pharmaceutically acceptable carrier.
7. A combination of compounds, which is a compound of any one of claims 2 to 4, and any one of AZT or ddi or ddC.
8. A method of treating and delaying the onset of AIDS, the method comprising administering to a mammal, an effective amount of a compound of any one of claims 2 to R 4 or of the composition of claim 6 or of the combination of claim 7. [N:\LIBZZ]00051:tab 49
9. A method of preventing infection by HIV, the method comprising administering to a mammal, an effective amount of a compound of any one of claims 2 to 4 or of the composition of claim 6 or of the combination of claim 7. A method of treating infection by HIV, the method comprising administering to a mammal in need of such treatment an effective amount of a compound of any one of claims 2 to 4 or of the composition of claim 6 or of the combination of claim 7.
11. A method of inhibiting HIV protease, the method comprising administering to a mammal in need of such treatment an effective amount of a compound of any one of claims 2 to 4 or of the composition of claim 6 or of the combination of claim 7.
12. The use of a compound of any one of claims 2 to 4 or of the composition of claim 6 or of the combination of claim 7 for the manufacture of a medicament for a purpose selected from the group consisting of treating and delaying the onset of AIDS, preventing infection by HIV, treating infection by HIV, inhibiting HIV protease and synthesising biotransformation products. 4 15 13. A compound of any one of claims 2 to 4 or the composition of claim 6 or the S combination of claim 7 when used for a purpose selected from the group consisting of treating and delaying the onset of AIDS, preventing infection by HIV, treating infection by HIV, inhibiting HIV protease and synthesising biotransformation products. Dated 8 December, 1998 Merck Co., Inc. S* Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 0O 0 [N:\LIBZZ]0005 I:NJC
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US5612217A (en) * 1994-10-25 1997-03-18 Merck & Co., Inc. Streptomyces sp. MA 7074 (ATCC 55605) used for microbial synthesis of HIV protease inhibitors
EP0817789B1 (en) * 1996-01-26 2002-01-02 Roche Diagnostics Corporation Bis-maleimido cross-linking agents
US5763196A (en) * 1996-01-26 1998-06-09 Boehringer Mannheim Corporation Assays using cross-linked polypeptide fragments of β-galactosidase
US5976857A (en) * 1996-01-26 1999-11-02 Boehringer Mannheim Corporation Cross-linked polypeptide fragments of β-galactosidase
JP2004115510A (en) * 2002-09-05 2004-04-15 Toray Fine Chemicals Co Ltd Method for producing piperazine derivative
AU2003264365A1 (en) * 2002-09-05 2004-03-29 Toray Fine Chemicals Co, . Ltd. Process for producing oxycarbonyl-substituted piperazine derivative
CN101497608B (en) * 2009-03-05 2011-11-09 中国科学院广州生物医药与健康研究院 HIV protease inhibitor derivative, preparation thereof and use in antineoplastic medicament preparation
WO2024171717A1 (en) * 2023-02-17 2024-08-22 株式会社トクヤマ Manufacturing method for selexipag and purifying method therefor

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US3160560A (en) * 1957-08-23 1964-12-08 Upjohn Co Streptolydigin and production thereof
US4497797A (en) * 1980-10-27 1985-02-05 Shionogi & Co., Ltd. β-Galactosidase inhibitor GT-2558 and its derivatives

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CA2081970C (en) * 1991-11-08 1997-07-08 Joseph P. Vacca Hiv protease inhibitors useful for the treatment of aids
CA2110827A1 (en) * 1992-12-14 1994-06-15 Maki Nishio Antiviral antibiotic bu-4724v and preparation thereof
SK279471B6 (en) * 1993-03-31 1998-11-04 Merck & Co. Compound composition, pharmaceutical agents containing thereof and their use
US5612217A (en) * 1994-10-25 1997-03-18 Merck & Co., Inc. Streptomyces sp. MA 7074 (ATCC 55605) used for microbial synthesis of HIV protease inhibitors

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US3160560A (en) * 1957-08-23 1964-12-08 Upjohn Co Streptolydigin and production thereof
US4497797A (en) * 1980-10-27 1985-02-05 Shionogi & Co., Ltd. β-Galactosidase inhibitor GT-2558 and its derivatives

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