WO1993004055A2

WO1993004055A2 - Compounds used for the inhibition of hiv-protease

Info

Publication number: WO1993004055A2
Application number: PCT/US1992/005250
Authority: WO
Inventors: Lester Arthur Dolak; Eric Peter Seest; Joyce Irma Cialdella; Grace P. Li; Michael John Bohanon
Original assignee: The Upjohn Company
Priority date: 1991-08-27
Filing date: 1992-06-29
Publication date: 1993-03-04
Also published as: WO1993004055A3; AU2267992A

Abstract

This invention relates to new compounds, known compounds and new uses for those compounds. The compounds of this invention are comprised of tetronic acid type compounds, panosialin type compounds and virustomycin type compounds that are inhibitors of HIV protease. The invention further comprises the extraction, isolation, and purification of the compounds plus a method of treating a human cell system, including a patient possibly infected with a human retrovirus (HRV), to prevent or retard the further replication of the HRV in that human cell system or patient.

Description

COMPOUNDS USED FOR THE INHIBITION OF HIV-PROTEASE FIELD OF THE INVENTION

This invention relates to both novel and known chemical compounds, their production, extraction, isolation, and purification; and a new method of treating a human cell system, including a patient, possibly infected with a human retrovirus (HRV), with a novel chemical compound and with known compounds, to prevent or retard the further replication of the HRV in that human cell system or patient. BACKGROUND OF THE INVENTION AIDS is a disease that is characterized by a severe immune deficiency primarily caused by a decreased cell-mediated immune response. Gottlieb, et al., N. Engl. J. Med., 305: 1425- 1431 (1981); Masur, et al, N. Engl. J. Med., 305: 1431-1438 (1981). The immunodeficient state is characterized by a decrease in TA lymphocytes, also known as helper T cells, a reversal of the normal T4< + > :78< + > cell ratio, lymphopenia, and opportunistic infections often caused by Pneumocystis carinii. Some patients also develop lymphoma or Kaposi's sarcoma at increased incidence. The disease is usually fatal.

Since the first description of the malady in the early part of this decade AIDS and its devastating consequences have been subjects of continuous and intense coverage in both the lay and scientific press. Full editions of both Science and Scientific American were devoted entirely to AIDS. Science, 239: No. 4840 (1988); Scientific American, 259: No. 4 (1988). The literature on the disease and the virus is already so vast as to defy thorough citation. Unfortunately, scientific understanding of the disease is still very incomplete.

The virus that the majority of scientists believes causes AIDS, first identified in 1983, has been described by several names. It is the third known human T-lymphotropic virus (HTLV-III) and has the capacity to replicate within cells of the immune system and thereby lead to a profound destruction of TA< + > J-cells (or CD4< + > cells). See, e.g., Gallo, et al., Science, 224: 500-503 (1984), and Popovic, et al, Science, 224: 497-500 (1984). This retrovirus has been known as lymphadenopathy-associated virus (LAV) or AIDS-related virus (ARV) and, more recently, as human immunodeficiency virus (HIV). Two distinct AIDS viruses, HIV-1 and HIV-2, have been described. HIV-1 is the virus originally identified in 1983 by Montagnier and co-workers at the Pasteur Institute in Paris. Montagnier, et al., Ann. de Virologie, 135 E: No. 1, 119-134 (1984), while HIV-2 was more recently isolated by Montagnier and his coworkers in 1986. Guyader, Nature, 326: 662-669 (1987). Additional distinct AIDS viruses may exist. As used herein, HIV is meant to refer to all of these viruses in a generic sense.

Retroviruses are enveloped RNA tumor viruses. See, Hayward and Neel, Curr. Top. Microbiol. Immunol., 91: 217-276 (1981). The virus particle consists of a ribonucleoprotein core enclosed by an outer membrane envelope derived from the host cell plasma membrane. Viral envelope glycoproteins protrude from the outer envelope. The viral genome consists of two identical single-stranded RNA molecules. Haseltine and Wong-Stall, Scientific American, 259: 52-62 (1988).

Sequence analysis of the complete genomes from several infective and non-infective HIV-isolates has shed considerable light on the make-up of the virus and the types of molecules that are essential for its replication and maturation to an infective species.

U.S. Patent 4,724,232 claims a method of treating humans having AIDS utilizing 3- azido-3-deoxythymidine. On March 20, 1987, the FDA approved the use of this compound, zidovudine (AZT), to treat AIDS patients with a recent initial episode of pneumocystis carinii pneumonia and for treatment of patients infected with the virus with an absolute CD4 lymphocyte count of less than 200/mπr in the peripheral blood. AZT is a known inhibitor of viral reverse transcriptase. Reverse transcriptase (RT) is an enzyme unique to retroviruses that catalyzes the conversion of viral RNA into double stranded DNA. Blockage at any point during the tran¬ scription process, by AZT or any other aberrant deoxynucleoside triphosphate incapable of elongation, is postulated to have dramatic consequences relative to viral replication, although no such therapy has yet been perfected. The principal receptor on the 74 cell for HIV is the so-called CD4 molecule. This molecule, a nonpolymorphic surface glycoprotein, has been targeted as an intervention point in AIDS therapy. Fisher, et al., Nature, 331: 76-78 (1988); Hussey, et al, Nature, 331: 78-81 (1988); and Deen, et al., Nature, 331: 82-84 (1988).

The present invention concerns another therapeutic target in AIDS, the inhibition of the viral protease (or proteinase) that is essential for processing HIV-fusion polypeptide precursors. In HIV and several other retroviruses, the proteolytic maturation of the gag (group specific antigen) and gag/pol (poly merase) fusion polypeptides (a process indispensable for generation of infective viral particles) has been shown to be mediated by a protease that is, itself, encoded by i epol region of the viral genome. Yoshinaka, et al, Proc. Natl. Acad. Sci., USA, 82: 1618-1622 (1985); Yoshinaka, et al., J. Virol., 55: 870-873 (1985); Yoshinaka, et al, J. Virol., 57: 826-832 (1986).

The protease (or proteina_e)enzymes, consisting of only 99 amino acids, are among the smallest enzymes known. Nutt, et al., Proc. Natl. Acad. Sci., USA, 85: 7129-7133 (1988). Pearl and Taylor, Nature, 329: 351-354 (1987). Its demonstrated homology to aspartyl proteases such as pepsin and renin led to inferences regarding the three-dimensional structure and mechanism of the enzyme that have since been borne out experimentally. Pearl and Taylor, Nature, 329: 351-354 (1987). Active HIV protease has been expressed in bacteria (e.g., Darke, et al, J. Biol. Chem., 264: 2307-2312 (1989)) and chemically synthesized. Schneider and Kent, Cell, 54: 363-368 (1988); and Nutt, et al, Proc. Natl. Acad. Sci., USA, 85: 7129-7133 (1988). To date, an effective means of inhibiting retroviruses in a human hosting such a virus, and thereby effectively treating a disease such as AIDS, has not been found. INFORMAΗON DISCLOSURE

There are three different types of compounds disclosed in this application. A. One type is a tetronic acid. B. The second type is a panosialin. C. The third type is a virustomycin. The panosialin and virustomycin type compounds show structural similarities to publications describing panosialin and virustomycin.

A. Tetronic Acid Family. The following information disclosures applies to the tetronic acid compounds.

A broad class of two tetronic acid compounds intended to be used as protease inhibitors for the promotion of "healing," is disclosed by the Takeda Chemical Company in a Japanese patent application, JO 1308-277-A, filed 13 May 1989 (Derwent No. 90-028213/04). Additionally, Kato discloses the synthesis of 2-O-Alkylascorbic Acids, and their possible use as scavengers against active oxygen. Kato, et al., J. Med. Chem, 31: 793-798 (1988).

In addition to the above documents another ascorbic acid derivative, also made by the Takeda Chemical Company, is disclosed by a European Patent. We are claiming a method of use of that known compound in this application. This known ascorbic acid derivative is described by European Patent Number, EP 202589-B, issued 28 February 1990, first disclosed in European Patent Application, Publication Number 0 202 589, Filed on 14 May 1986, Application Number 86106521.7. Other compounds useful as protease inhibitors that have no structural similarity to the tetronic acid compound claimed in this invention have been disclosed. General aspartyl proteinase inhibitors such as Pepstatin A have been evaluated for inhibition of HIV-1 protease. Seelmeier, et al, Proc. Natl. Acad. Sci., USA, 85: 6612-6616 (1986). Other substrate derived inhibitors of HIV-1 protease have also been disclosed. See, Richards, et al, FEBS Letters, 247: 113-117 (1989); Moore, et ah, Biochem. Biophys, Res. Commun., 159: 420 (1989); Billich, et al., J. Biol. Chem., 263: 17905-17908 (1988).

B. Panosialin Family. The following information disclosures applies to the panosialin type compounds.

T. Aoyagi, M. Yagisawa, M. Kumagai, M. Hamada, Y.Okami, T. Takeuchi, & H. Umezawa. An Enzyme Inhibitor, Panosialin, Produced by Streptomyces - 1. Biological Activity, Isolation, and Characterization of Panosialin. Journal of Antibiotics 24(12):860-869 (1971).

M. Kumagai, Y. Suhara, T. Aoyagi, & H. Umezawa. An Enzyme Inhibitor Panosialin, Produced by Streptomyces - 2. Chemistry of Panosialin, 5-Alkylbenzene-l,3- Disulfates. Journal of Antibiotics 24(12):870-875(1971). C. Virustomycin Family. The following information disclosures applies to the virustomycin type compounds.

S. Omura, N. Imamura, K.Hinotozawa, K.Otoguro, G. Lukacs, R. Faghih, R. Tolmann, B.H. Arison & J.L. Smith, Journal of Antibiotics, Vol. 36, 1783 (1983).

Y. Hayakawa, K. Takaku, K. Furihata, K.Nagai & H. Seto. Journal of Antibiotics, Vol. 44, p 1294 (1991).

JO 3290-193-A, Abstract from Japanese Application, by Takeda Chemical Ind. KK (23.01.90-JP-013022 and 27.11.90-JP-327778). SUMMARY OF THE INVENTION

A) Tetronic Add Family. Immediately below is a summary of the tetronic acid compounds. The tetronic acid aspect of the present invention relates to a compound of formula A-1, except as existing or occurring in nature, represented by the following formula:

₅ CH₂CH₂CH₂C_₃

(FORMULA A-1)

the methyl is attached to one of five carbon atoms as indicated or a pharmacologically acceptable salt thereof. Additionally, the present invention provides: 1) The compound of formula A-1 is claimed in an essentially pure form; 2) A pharmaceutical composition of the compound of formula one adapted for administration to obtain an antiviral effect; 3) A method of retarding or preventing the replication of human retrovirus (HRV) in a cell system or a human patient exhibiting or susceptible to said HRV, comprising the systematic administration of an amount of the compound of formula A-1, or a pharmacologically acceptable salt thereof, or the compound of formula A-2, represented by the following formula:

₁₆CH₃ (FORMULA A-2)

or a pharmacologically acceptable salt thereof, effective to prevent or retard the further replication of the HRV in that human cell system or patient; 4) A process for producing a compound of formula A-1 and A-2 that comprises: cultivating a subculture of the organism, deposited as NRRL 18839, in an aqueous nutrient medium containing a source of assimilable carbohydrate and assimilable nitrogen under aerobic conditions; 5) A process of cultivating a subculture of the organism, wherein the compound is isolated by filtering the fermentation medium and subjecting the medium to purification procedures; and 6) A biologically pure culture of the microorganism characterized as being essentially the strain identified as NRRL 18839.

B) Panosialin Family. Below is a summary of the panosialin type compounds. The panosialin aspect of the present invention relates to a compound of formula B-1, except as existing or occurring in nature, represented by the following formula:

(FORMULA B-1)

or a pharmacologically acceptable salt thereof. Additionally, the present invention provides: 1) The compound of formula B-1 is claimed in an essentially pure form; 2) A pharmaceutical composition of the compound of formula one adapted for administration to obtain an antiviral effect; 3) A method of retarding or preventing the replication of human retrovirus (HRV) in a cell system or a human patient exhibiting or susceptible to said HRV, comprising the systematic administration of an amount of the compound of formula B-1, effective to prevent or retard the further replication of the HRV in that human cell system or patient; 4) A process for producing a compound of formula B-1 that comprises: cultivating a subculture of the organism, deposited as NRRL 18942, in an aqueous nutrient medium containing a source of assimilable carbohydrate and assimilable nitrogen under aerobic conditions; 5) A process of cultivating a subculture of the organism, wherein the compound is isolated by filtering the fermentation medium and subjecting the medium to purification procedures; and 6) A biologically pure culture of the microorganism characterized as being essentially the strain identified as NRRL 18942.

C) Virustomycin Family. Below is a summary of the virustomycin type compounds. The virustomycin aspect of the present invention relates to a compound of formula C-l, except as existing or occurring in nature, represented by the following formula:

(FORMULA C-l)

or a pharmacologically acceptable salt thereof. Additionally, the present invention provides: 1) The compound of formula C-l is claimed in an essentially pure form; 2) A pharmaceutical composition of the compound of formula one adapted for administration to obtain an antiviral effect; 3) A method of retarding or preventing the replication of human retrovirus (HRV) in a cell system or a human patient exhibiting or susceptible to said HRV, comprising the systematic administration of an amount of the compound of formula C-l, effective to prevent or retard the further replication of the HRV in that human cell system or patient; 4) A process for producing a compound of formula C-l that comprises: cultivating a subculture of the organism, deposited as NRRL 18943, in an aqueous nutrient medium containing a source of assimilable carbohydrate and assimilable nitrogen under aerobic conditions; 5) A process of cultivating a subculture of the organism, wherein the compound is isolated by filtering the fermentation medium and subjecting the medium to purification procedures; and 6) A biologically pure culture of the microorganism characterized as being essentially the strain identified as NRRL 18943.

DETAILED DESCRTPTTON This invention relates to new compounds, to known compounds and to new uses for those compounds.

A) Tetronic acid Family. The compounds of the tetronic acid type are comprised of tetronic acids that are inhibitors of HIV protease. The tetronic acid type compounds are identified by the structures provided in formula A-1, the new compound, and in formula A-2. Formula A-1 and A-2 are provided above, in the Summary of Invention. The invention further comprises the extraction, isolation, and purification of the new compound plus a method of treating a human cell system, including a patient possibly infected with a human retrovirus (HRV), to prevent or retard the further replication of the HRV in that human cell system or patient. These compounds are designated as a tetronic acids.

The compound of formula A-1 is prepared, in an essentially pure form, by extraction from fermentations of an organism isolated from a soil sample collected in Pakistan. A subculture of the organism was deposited on June 25, 1991, under the provisions of the Budapest Treaty in the permanent collection of the Agricultural Research Culture Collection (NRRL) International Depositary Authority also known as the Midwest Area National Center for Agriculture Utilization Research, formerly called the Northern Regional Research Center, Agricultural Research Service; U.S. Dept. of Agriculture; Peoria, Illinois, USA. The accession number given to the microorganism by the International Depositary Authority is NRRL 18839. The morphological and cultural characteristics of the culture suggest that the organism is an actinomycete that belongs to the group of Streptomyces that produces pseudo-verticils.

Production of the compound is accomplished by fermentation of the organism followed by isolation and purification of the active metabolites. The fermentation, isolation and purification of the compounds can be accomplished by one skilled in the art. Specific examples and preferred procedures are provided below. The compound is designated by the structure provided in formula A-1. The arrow in the formula indicates attachment of the methyl group to one of the carbon atoms within the parenthesis. The compound is characterized and uniquely identified by the data and information provided in Tables Al - A4. Glossary, abbreviations, trade terms and other symbols are provided in Table Glossary.

B) Panosialin Family. The compounds of the panosialin type compounds are comprised of 5-alkylber__ene-l,3-disulfates that are inhibitors of HIV protease. These panosialin type compounds are identified by the structures provided in formula B-1. Formula B-1 is shown above, in the Summary of Invention. The invention further comprises the extraction, isolation, and purification of the compound plus a method of treating a human cell system, including a patient possibly infected with a human retrovirus (HRV), to prevent or retard the further replication of the HRV in that human cell system or patient. These compounds are designated as 5-alkylbenzene-l,3- disulfates.

The compound of formula B-1 is prepared, in an essentially pure form, by extraction from fermentations of an organism isolated from a soil sample collected in Texas. A subculture of the organism was deposited on January 23, 1992, under the provisions of the Budapest Treaty in the permanent collection of the Agricultural Research Culture Collection (NRRL) International Depositary Authority, also known as the Midwest Area National Center for Agriculture Utilization Research, formerly called the Northern Regional Research Center, Agricultural Research Service; U.S. Dept. of Agriculture; Peoria, Ηlinois, USA. The accession number given to the microorganism by the International Depositary Authority is NRRL 18942.

The morphological and cultural characteristics of the culture suggest that the organism belongs to the group Streptomyces .

Production of the compound is accomplished by fermentation of the organism followed by isolation and purification of the active metabolites. The fermentation, isolation and purification of the compounds can be accomplished by one skilled in the art. Specific examples and preferred procedures are provided below.

The compound is designated by the structure provided in formula B-1. The compound is characterized and uniquely identified by the data and information provided in Tables Bl - B5. Glossary, abbreviations, trade terms and other symbols are provided in Table Glossary.

C) Virustomycin Family. The compounds of the virustomycin type compounds are comprised of the compounds of formula C-l. The structure of formula C-l is provided above, in the Summary of Invention. The invention further comprises the extraction, isolation, and purification of the new compound plus a method of treating a human cell system, including a patient possibly infected with a human retrovirus (HRV), to prevent or retard the further replication of the HRV in that human cell system or patient. These compounds are designated as virustomycin type compounds.

The compound of formula C-l is prepared, in an essentially pure form, by extraction from fermentations of an organism isolated from a soil sample collected in Kansas. A subculture of the organism was deposited on January 23, 1992, under the provisions of the Budapest Treaty in the permanent collection of the Agricultural Research Culture Collection (NRRL) International Depositary Authority, also known as the Midwest Area National Center for Agriculture Utilization Research, formerly called the Northern Regional Research Center, Agricultural Research Service; U.S. Dept. of Agriculture; Peoria, Illinois, USA. The accession number given to the microorganism by the International Depositary Authority is NRRL 18943. The morphological and cultural characteristics of the culture suggest that the organism belongs to the group of Streptomyces.

The compound is designated by the structure provided in formula C-l. The arrow in the formula indicates attachment of the methyl group to one of the carbon atoms within the parenthesis. The compound is characterized and uniquely identified by the data and information provided in Tables Cl - C3. Glossary, abbreviations, trade terms and other symbols are provided in Table Glossary.

D) The HTV protease assay. Surprisingly and unexpectedly, the compounds of the present invention are effective and potent inhibitors of HIV protease. The HIV protease assay is described below. Because the compounds of the present invention inhibit retroviral proteases they are expected to inhibit the replication of the HIV virus. The compounds are thus useful for treating human patients infected with a human retrovirus, such as human immunodeficiency virus (strains of HIV-1 or HIV-2) or human T-cell leukemia viruses (HTLV-I or HTLV-II) that results in acquired immunodeficiency syndrome (AIDS) and/or related diseases. The term human retrovirus (HRV) includes human immunodeficiency virus type I, human immunodeficiency virus type II, or strains thereof, as well as human T cell leukemia virus 1 and 2 (HTLV-1 and HTLV-2) or strains apparent to one skilled in the art, that belong to the same or related viral families and that create similar physiological effects in humans as various human retroviruses. Patients to be treated are those individuals: 1) infected with one or more strains of a human retrovirus as determined by the presence of either measurable viral antibody or antigen in the serum and 2) in the case of HIV, having either a symptomatic AIDS defining infection such as i) disseminated histoplasmosis, ii) isopsoriasis, iii) bronchial and pulmonary candidiasis including pneumocystic pneumonia, iv) non-Hodgkin's lymphoma or v) Kaposi's sarcoma. Treatment consists of maintaining an inhibitory level of the compound used according to this in¬ vention in the patient at all times and would continue until the occurrence of a second symptomatic AIDS defining infection indicates alternate therapy is needed.

More specifically, an example of one such human retrovirus is the human immunodeficiency virus (HIV, also known as HTLV-III or LAV) that has been recognized as the causative agent in human acquired immunodeficiency disease syndrome (AIDS), Gallo, et al., Science, 224: 500-503 (1984). HIV contains a retro viral encoded protease, HIV-I protease, that cleaves the fusion polypeptides into the functional proteins of the mature virus particle. Lillehoj, et al., J. Virology, 62: 3053-3058 (1988); Debouck, et al., Proc. Natl. Acad. Sci., USA, 84: 8903-8906 (1987). This enzyme, HIV-I protease, has been classified as an aspartyl protease and has a demonstrated homology to other aspartyl proteases such as renin. Pearl and Taylor, Nature, 329: 351-354 (1987); Katoh, et al, Nature, 329: 654-656 (1987). Inhibition of HIV-I protease blocks the replication of HIV and thus is useful in the treatment of human AIDS. Clercq, J. Med. Chem., 29: 1561-1569 (1986). Inhibitors of HIV-I protease are useful in the treatment of AIDS.

The utility of the compounds of this invention is demonstrated in the HIV-1 protease assay. The HIV protease assay measures the cleavage of the HIV gag protein, that takes place at the Tyr-Pro bond. A synthetic octapeptide, H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-OH (GSP), that corresponds to the sequence from residue 128 to 135 in the HIV gag protein serves as a substrate for the measurement of HIV-1 protease activity. Tomasselli, et al, Biochemistry, 29: 264-269 (1990). Conditions for the assay and inhibition results for the compound of formula A-1, A-2, B-1 and C-l are provided in the specific examples and preferred procedures below. As is apparent to those of ordinary skill in the art, the compounds of the present invention can occur in several diastereomeric forms, depending on the configuration around the asymmetric carbon atoms. All such diastereomeric forms are included within the scope of the present invention. The compounds of the present invention are prepared as described in the specific examples and preferred procedures below, or are prepared by methods analogous thereto, are readily known and available to one of ordinary skill in the art of chemical synthesis.

The present invention provides for compounds of formula A-1, A-2, B-1, and C-l or pharmacologically acceptable salts and/or hydrates thereof. Pharmacologically acceptable salts refers to those salts that would be readily apparent to a manufacturing pharmaceutical chemist to be equivalent to the parent compound in properties such as formulation, stability, patient acceptance and bioavailability.

The acetyltetronic acids form base addition salts when reacted with bases of sufficient strength. The pharmaceutically acceptable salts include both inorganic and organic bases. The pharmaceutically acceptable salts are preferred over the free acids since they produce compounds that are more water soluble and more crystalline. The preferred pharmaceutically acceptable salts include, but are not limited to, salts of the mono and divalent metals such as: calcium, lithium, magnesium, potassium, or sodium; and salts formed with organic bases, such as: hydroxide, tro-methamine (TΗAM), 2-amino-2-(hydroxymethyl)-l,3-propanediol, and other salts as would be apparent to one skilled in the art.

Those skilled in the art would know how to formulate all three types of the compounds of this invention into appropriate pharmaceutical dosage forms. Examples of the dosage forms include oral formulations, such as tablets or capsules, or parenteral formulations, such as sterile solutions.

The compounds of the present invention are useful for treating patients infected with human immunodeficiency virus (HIV) that results in acquired immunodeficiency syndrome (AIDS) and related diseases. For this indication, the compounds of formula A-1, A-2, B-1 and C-l are administered by oral, nasal, transdermal and parenteral (including i.m. and i.v.) routes in doses of 1 μg to 100 mg/kg of body weight.

When the compounds in this invention are administered orally, an effective amount is from about 1 μg to 100 mg per kg per day. Either solid or fluid dosage forms can be prepared for oral administration. Solid compositions are prepared by mixing the compounds of this invention with conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, acacia, methyl cellulose, or functionally similar pharmaceutical diluents and carriers. Capsules are prepared by mixing the compounds of this invention with an inert pharmaceutical diluent and placing the mixture into an appropriately sized hard gelatin capsule. Soft gelatin capsules are prepared by machine encapsulation of a slurry of the compounds of this invention with an acceptable inert oil such as vegetable oil or light liquid petrolatum. Syrups are prepared by dissolving the compounds of this invention in an aqueous vehicle and adding sugar, aromatic flavoring agents and preserva- tives. Elixirs are prepared using a hydroalcoholic vehicle such as ethanol, suitable sweeteners such as sugar or saccharin and an aromatic flavoring agent. Suspensions are prepared with an aqueous vehicle and a suspending agent such as acacia, tragacanth, or methyl cellulose.

When the compounds of this invention are administered parenterally, they can be given by injection or by intravenous infusion. An effective amount is from about 1 μg to 100 mg per kg per day. Parenteral solutions are prepared by dissolving the compounds of this invention in water and filter sterilizing the solution before placing in a suitable sealable vial or ampule. Parenteral suspensions are prepared in substantially the same way except a sterile suspension vehicle is used and the compounds of this invention are sterilized with ethylene oxide or suitable gas before it is suspended in the vehicle. The exact route of administration, dose, or frequency of administration would be readily determined by those skilled in the art and is dependant on the age, weight, general physical condition, or other clinical symptoms specific to the patient to be treated. DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is more fully described in the three sections below. This PREFERRED EMBODIMENTS section is in 3 parts corresponding to the 3 types of compounds in this invention. The tetronic acid compounds and uses are described first, in part A. Panosialin related compounds and uses are described second, in part B. The virustomycin type compounds are described last, in part C.

A. TETRONIC ACID TYPE COMPOUNDS

1. THE ORGANISM PRODUCING THE COMPOUND REPRESENTED BY FORMULA A-1 A subculture of the organism was deposited on June 25, 1991 under the provisions of the Budapest Treaty in the permanent collection of the Agricultural Research Culture Collection (NRRL) International Depositary Authority, also know as the Midwest Area National Center for Agricultural Utilization Research, formerly called the Northern Regional Research Center, Agricultural Research Service; U.S. Dept. of Agriculture; Peoria, Illinois, USA. The accession number for the microorganism as given by the International Depositary Authority is NRRL 18839.

The isolate produces thick and dark gray colonies on ISP media 2, 3 and 4. The reverse side of the colony is dark brown on all three media. It produces spiral spore chains that often arise in verticils from long axial hyphae suggesting monoverticillate morphology. Spirals are medium-length and often open and wavy; hooked spore chains are also present. The spore surface is smooth. Melanoid pigment is produced on ISP media 1, 2, 3, 6 and 7. Pink to red pigment is also formed in ISP media 1 and 4. The strain grows in the temperature range of 10-55°C. It utilizes a wide range of sugars. 2. PRODUCTION Frozen agar plugs containing spores of the organism are inoculated directly into 100 mL of a sterile seed medium contained in a 500 mL wide-mouth flask and are incubated at 28 °C on a rotary shaker at 250 rpm for 3 days. The seed medium contains in each liter of tap water: 25 g glucose and 25 g Phaπnamedia. The pH of the mixture is adjusted to 7.2 prior to sterilization. The mature seed culture is transferred (5%) to the production medium (100 mL/500 mL flask).

This production medium contains, per liter of tap water: 10 g glucose, 25 g corn starch, 4 g corn gluten meal, 5 g Brewer's yeast and 4 g of calcium carbonate (mixture adjusted to pH 7.2 prior to sterilization). Fermentation is carried out at 28°C on a rotary shaker at 250 rpm. Days 3-6 of the fermentation produce metabolites that give strong inhibition in the HIV protease assay. Multiple flasks are prepared to produce 2 L of fermentation broth for use in the isolation of metabolites. 3. ISOLATION

The 2 L pool (obtained after four days of fermentation), from the production step above, is filtered at harvest pH over a bed of Celite filter aid in a 3 L sintered glass funnel using house vacuum with a filter flask. The resulting mycelial cake is washed with 1.5 L of deionized water, the wash water is discarded, the mycelial cake is then extracted with 3 L of acetone. The 3 L acetone extract is concentrated on a rotary evaporator to about 500 mL of an aqueous residue. The aqueous residue is extracted with chloroform, the chloroform layer dried with magnesium sulfate, this is filtered and concentrated to a solid residue. The aqueous is lyophilized to get additional solids. 4. PURIFICATION

The solid residue from the isolation above is suspended in methanol and filtered. The methanol solution is injected onto a Rainin 1 inch octadecyl (C-l 8) HPLC column and eluted with a gradient of 80-100% MeOH in water over 20 min followed by 15 min of 100% MeOH. The column is eluted at 12 mL/min and the eluate is monitored at 275 nm. The compound elutes at 22 min. as a partially-resolved pair of peaks just separated from a "hump." Portions of the sample from above, 50 to 100 mg, are then fractionated with countercurrent chromatography in a Ito Multi-Layer Coil Separator Extractor, model number 1. A system containing: cyclohexane: ethyl acetate:95% ethano water with 0.1% trifluoroacetic acid gave nearly pure product. Ratios of the four solvents that provided good separation were: 1:1:1:1, 1:2:2:1, 1:2:2:2 and 2:1:1:1 (V/V) systems (each with 0.1% TFA). The lower phase is used as the stationary phase and the upper phase is pumped at 2.5 mL/min, collecting 5.0 mL/fraction. The last solvent ratio is preferred.

Pooling of the fractions judged to be free of the yellow impurity and rich in the compound (275 nm) followed by addition of water and ethyl acetate to the pools allows one to obtain the compound free of water. After each run the orgamc phase is dried and the solvent stripped on a rotary evaporator. The white residue dissolves in warm methanol and the solution turns cloudy on addition of water. On cooling, small white crystals appear.

5. ACΉVΠΎ

The activity of the compounds of formula A-1 were measured in the HIV-1 protease assay. The HIV protease assay measures the cleavage of the HIV gag protein, that takes place at the Tyr-Pro bond. A synthetic peptide, H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-OH (GSP), that corresponds to the sequence from residue 128 to 135 in the HIV gag protein serves as a substrate for the measurement of HIV-1 protease activity. Tomasselli, et al, Biochemistry, 29: 264-269 (1990). HIV-1 protease activity is measured at 30°C in 50 mM sodium acetate, pH 5.5, containing 10% glycerol, 5% ethylene glycol, and 0.1% Nonidet P-40 in a total volume of 50 μL. After 10 and 20 min. of incubation, 20 μL samples are withdrawn into a vial containing 30 μL of 1% tri-fluoroacetic acid (TFA) and subjected to HPLC analysis. HPLC is carried out with a Vydac C column (0.46 x 15 cm), eluting with a linear gradient of 0-30% acetonitrile over a period of 25 min. at a flow rate of 1.0 mL/min. The K_j of the compound of formula A- 1 was found to be approximately 130 μM using the synthetic GSP octapeptide as the substrate and monitoring by HPLC.

6. STRUCTURE ELUCIDATION

The compound of the invention is identified by its novel properties and spectra. Tables A-1 through A-4 identify the compound of the invention. Table A-1 lists some properties of the compound that define it as a novel compound. Table A-2 shows the infrared spectrum of the compound (digital). Table A-3 shows the Proton NMR spectrum, in dg-DMSO solution, of the compound (digital). Table A-4 shows the Carbon-13 NMR spectrum in d^-pyridine (digital). These tables and spectra identify one compound represented by formula A-1.

7. PREPARATION OF THE KNOWN COMPOUND REPRESENTED BY FORMULA A-2 The compound represented by formula A-2 can be synthesized according to the following procedures: To a rapidly stirred suspension of ascorbic acid (1; 300 g, 2 mol) in acetone (3 L) add acetyl chloride (25mL, 0.1 Mol), stir the mixture at ambient temperature for 18 hours. Collect the precipitate by filtration, wash with EtOAc, and dry in vacuo to afford 5,6- -Isopropylideneascorbic Acid. (310 g 84%): m.p. 202-204 °C; NMR (d₆-DMSO) δ 1.33 (6 H,s), 3.90^.45 (4 H,m), 4.61 (lH,d, J = 3 Hz). Anal. (C₉H₁₂0₆) C, H.

The hydroxyl group at the 3-carbon is protected with a benzyl group with the following procedure: 5,6-O-Isopropylideneascorbic Acid, from above, is stirred in solution (108 g, 0.5 mol) in THF (300 mL) and DMF (100 mL) to this solution is added potassium carbonate (70 g, 0.5 mol). Stir the mixture for 10 min., then add benzyl bromide (0.5 mol) to the mixture while the temperature is maintained below 30°C. Stir at room temperature for 4 hours then dilute the reaction mixture with water (300mL), neutralize with 2 N HCL, and extract with EtOAc. The organic layer is washed with water, dried, and evaporated. The residue is chromatographed on silica gel with EPE/EtOAc (1:1) as eluent. Recrystallization from IPE gives 5,6-0-Isopropylidene-3-_⁾-benzylascorbic Acid. Alkylation of this compound with alkyl halides in the presence of potassium carbonate gives the corresponding dialkylated derivative. The alkylation is accomplished by the following procedure: To a solution of 5,6-0-Isopropylidene-3-0-benzylascorbic Acid (0.1 mol), from above, and a (CH^Me halide (0.11 mol) in THF (80 mL) and DMSO (90 mL) add potassium carbonate (16 g, 0.11 mol), and vigorously stir the reaction mixture at 50 °C for 3 hours. Dilute the reaction mixture with water (300 mL), neutralize with 2 N HCL, and extract with EtOAc. The organic layer is washed with water, dried, and evaporated. The residue is chromatographed on silica gel with IPE as eluent to produce 2-_>-alkyl-3-0-benzyl-5,6-O- isopropylideneascorbic acid. The protective group at the 3 position is then removed.

The protective benzyl group is subjected to acid hydrolysis followed by catalytic hydrogenation to give the corresponding 2- 0-alkylascorbic acid, according to the following procedure: A solution of 2-0-alkyl-3-_⁾-benzyl-5,6-O-isopropyl_deneascorbic acid (0.023 mol), from above, in THF (20 mL), methanol (20 mL), and 2 N HCL (20 mL) is stirred at room temperature and extracted with EtOAc. The organic layer is washed with water, dried, and evaporated. The resulting 2-0-alkyl-3-0-benzylascorbic acid is dissolved in ethanol (20 mL) and hydrogenated with 5% palladium charcoal (0.5 g) for 18 hours at atmospheric pressure. The catalyst is removed by filtration, and the filtrate is concentrated. The residue is recrystallized from IPE/EtOAc to afford the compound of formula A-2. Typical NMR spectrum for the compound of formula A-2 (d₆-DMSO) δ 0.85 (3 H,m), 1.26 (32 H, m), 3.45 (2 H, m), 3.86 (3 H, m), 4.70 (1H, d, J = 1 Hz). These procedures are described in Kato, et al., Studies on Scavengers of Active Oxygen Species. 1. Synthesis and Biological Activity of 2- O-Alkylascorbic Acids, J. Med. Chem. 31: 793-798 (1988) and provided in European Patent Application, Publication Number 0 202 589, filed on 14 May, 1986, Application Number 86106521.7. issued on 28 February 1990 as European Patent No., EP-202589-B.

B. PANOSIALIN TYPE COMPOUNDS 1. THE ORGANISM PRODUCING THE COMPOUND REPRESENTED BY FORMULA B-1 A subculture of the organism was deposited on January 23, 1992 under the provisions of the Budapest Treaty in the permanent collection of the Agricultural Research Culture Collection (NRRL) International Depositary Authority, also known as the Midwest Area National Center for Agricultural Utilization Research, formerly called the Northern Regional Research Center, Agricultural Research Service; U.S. Dept. of Agriculture; Peoria, Illinois, USA. The accession number for the microorganism as given by the International Depositary Authority is NRRL 18942.

The isolate is a Streptomyces species, given the reference name Streptomyces violaceochromogenes. It was isolated from a soil sample collected in Texas. Morphological Characterization: The mature spore chains produced are generally short with 5 to 15, or occasionally more than 15, spores per chain. The short spore chains form incomplete or imperfect spirals, hooks, and flexuous chains. Some longer chains have terminal spirals suggesting RA (Retinaculiaperti) morphology, as defined by International Streptomyces Project (ISP). The spore surfaces are smooth. Vegetative hyphae show no evidence of fragmentation. Cultural and Physiological Characterization: The culture grows well on most media. The production of sporulating aerial hyphae, however, is poor or absent on ISP media 2, 5, 6, and 7 and most of the confirmatory media. The production of sporulation mass is abundant on ISP media 3 and 4. These aerial hyphae are white first, changing to medium to dark gray upon sporulation. The reverse side of the colony (substrate mycelia) is yellow brown or yellow gray to brown or dark brown on most media. On ISP medium 2, however, the substrate mycelia are light purple first gradually changing to yellow brown, that may be due to the pH change of the medium since this pigment was found to be pH-sensitive. The growth pellets in tryptone-yeast extract broth (ISP medium 1) also often contain blue pigments. Melanoid pigments are formed in tryptone-yeast extract broth and peptone-yeast-iron agar (ISP medium 6) but not in tyrosine agar (ISP medium 7). Brown pigments are also formed in most confirmatory agar media. Trace of yellow brown pigments are formed in ISP media 2 and 3. 2. PRODUCTION

Frozen agar plugs containing spores of the organism are inoculated directly into 100 mL of a sterile seed medium contained in a 500 mL wide-mouth flask and are incubated at 28 °C on a rotary shaker at 250 rpm for 3 days. The seed medium contains in each liter of tap water: 25 g glucose and 25 g Pharmamedia. The mixture is adjusted to pH 7.2 with aqueous 29% NH₄OH prior to sterilization. The mature seed culture is transferred (5%) to the production medium (100 mL/500 mL flask).

The production medium ∞ntains, per liter of tap water: glucose, 10 g; lactose, 10 g; cottonseed meal, 10 g; brewers yeast, 10 g; MgS0₄Η₂0, 5 g; K₂HP0₄, 2 g per L of tap water. The broth is adjusted to pH 7.2 with aqueous 45% KOH (w/v) prior to sterilization. Fermentation is carried out at 28 °C on a rotary shaker at 250 rpm. Using these conditions, production of panosialin begins on day 1 of the fermentation and is consistently high on days 2- 4. On days 5 and 6 the compound appears to convert to its less active resorcinol analogs.

Production of panosialin can also be achieved in 10 L tanks. For these fermentations, a two stage seed is utilized. The primary seed flask (grown as previously described) is used as inoculum (5%) for multiple secondary seeds. The secondary seeds are grown under the same conditions for 24 hours (instead of three days) prior to inoculation (5%) into the tank. CBS-38 is used as the production medium. Tank fermentations are performed at 28 °C with agitation at 250 rpm and aeration at 7-8 slm. Production of panosialin in tanks is equivalent to shake flask fermentations on day 2 but titers began to drop on day 3, this is earlier than that observed in shake flasks. 3. ISOLATION

The 2 L pool (obtained after four days of fermentation), from the production step above, is filtered at harvest pH over a bed of Celatom FW-40 filter aid in a 3 L sintered glass funnel using house vacuum with a filter flask. The resulting mycelial cake is washed with deionized water until the filtrate is clear, the wash water is discarded, the mycelial cake is then extracted with 1 L of acetone. The 1 L acetone extract is dried on a rotary evaporator. Most of the activity is in the acetone cake extract. Similar processing of pools from 10 L pools on day 4, or from 2 L pools from day 6 of fermentation yield inactive 5-alky lbenzene-l,3-diols. A more efficient method of isolation is possible. A 2 L shake flask pool obtained after two days of fermentation is filtered as before and the filtrate and water cake wash are saved and pooled, 2.5 L combined. The cake is extracted with 1 L methanol followed by 1 L acetone. These extracts are combined and concentrated to an aqueous by rotary evaporation. Aqueous 1 % KC1 (w/v) is added to the cake extract aqueous in order to form the scarcely water soluble, K⁺ salt. The cake extract aqueous is extracted three times with 100 ml n-butanol. The organic layers are combined and washed with aqueous 2% KC1 (w/v) followed by rotary evaporation to a yellowish powder. Further washing of sample with methylene chloride and methanol followed by vacuum filtration yields a white powder. 4. PURIFICATION

The concentrated acetone cake extract from the 2 L beer harvested on day 4, from above, is purified by repetitive injection onto a Rainin C-l 8 HPLC column (21.4 mm ID x 25 cm; 83-221-C) using an automated Rainin Rabbit HPX system. A 20 to 100% methanol/water gradient over 20 minutes is used at a flow rate of 12 ml/min. The runs are monitored using a Knauer Variable Wavelength Monitor at 210 nm. Compounds of interest emerged at 19.4 minutes and 20.3 minutes. Portions of the sample from above are further purified by Ito Coil Extracter countercurrent chromatography using a 1:1:1:1 cyclohexane/ethyl acetate/95% ethanol/water system with the upper phase being mobile. A 3 ml/min flow rate is used with 52 two minute fractions being collected. The majority of the material (weight and activity both) remained in the coil. The residue is dried by rotary evaporation. 5. ACTIVITY

The activity of the compounds of formula B-1 were measured in the HIV-1 protease assay. The HIV protease assay measures the cleavage of the HIV gag protein, that takes place at the Tyr-Pro bond. A synthetic peptide, H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-OH (GSP), that corresponds to the sequence from residue 128 to 135 in the HIV gag protein serves as a substrate for the measurement of HIV-1 protease activity. Tomasselli, et al, Biochemistry, 29: 264-269 (1990).

HIV-1 protease activity is measured at 30°C in 50 mM sodium acetate, pH 5.5, containing 10% glycerol, 5% ethylene glycol, and 0.1% Nonidet P-40 in a total volume of 50 μL. After 10 and 20 minutes of incubation, 20 μL samples are withdrawn into a vial containing 30 μL of 1% tri-fluoroacetic acid (TFA) and subjected to HPLC analysis. HPLC is carried out with a Vydac C₄ column (0.46 x 15 cm), eluting with a linear gradient of 0-30% acetonitrile over a period of 25 min. at a flow rate of 1.0 mL/min. The results of the HIV-1 protease assay are shown in Table B-5. 6. STRUCTURE ELUCIDATION

The compound of the invention is identified by its novel properties and spectra. Tables B-1 through B-4 identify the compound of die invention. Table B-1 lists some properties of the compound that define it. Table B-2 shows the infrared spectrum of the compound (digital). Table B-3 shows the Proton NMR spectrum, in d^-DMSO solution, of the compound (digital). Table B-4 shows the Carbon-13 NMR spectrum in dg-DMSO solution (digital). These tables and spectra identify one compound represented by formula B-1. C. VIRUSTOMYCIN TYPE COMPOUNDS

1. THE ORGANISM PRODUCING THE COMPOUND REPRESENTED BY FORMULA C-l

A subculture of the organism was deposited on January 23, 1992 under the provisions of the Budapest Treaty in the permanent collection of the Agricultural Research Culture Collection (NRRL) International Depositary Authority, also known as the Midwest Area National Center for Agricultural Utilization Research, formerly called the Northern Regional Research Center, Agricultural Research Service; U.S. Dept. of Agriculture; Peoria, Illinois, USA. The accession number for the microorganism as given by the International Depositary Authority is NRRL 18943.

The producing organism was isolated from a soil sample collected in Kansas. On ISP media (Difco) 2, 3, and 4, the organism grows well and produces white aerial hyphae that turn gray when sporulation occurs. The reverse side of the colony is yellow-brown on ISP medium 2 and red-orange to red-brown on ISP media 3 and 4. The organism produces tight spiral spore chains with spiny spore surface. Melanoid pigment is produced on ISP media 1, 6 and 7. The optimum growth temperature range is 28-35°. The strain contains LL-diaminopimelic acid in whole cell hydrolysates and no diagnostic sugars. The morphological characteristics and cell wall type place the strain in the genus Streptomyces.

The culture grows well on most media. The production of sporulating aerial hyphae however, is poor on agar plates of ISP media 2, 3, and 5 and absent on agar slants of ISP media 6 and 7. On the agar plate of ISP medium 7, the culture produces abundant aerial hyphae and sporulates well after three weeks incubation. The production of aerial hyphae are generally abundant on ISP medium 9, carbon utilization medium, added with various carbohydrate sources. The aerial hyphae, when first produced, are white but turn to light to medium gray or pink gray when sporulation occurs. The sporulation is the best on ISP medium 4 and is generally good on ISP medium 9 plus various sugars. The reverse side of the colony is red orange to red brown on ISP media 2-5 and dark brown on ISP media 6 and 7. It is dark red on ISP medium 9 added with glucose, mannitol, fructose, or rhamnose. The reverse mycelium pigment is not a pH indicator. Melanoid pigments are formed in tryptone-yeast extract broth (ISP medium 1), peptone-yeast-iron agar (ISP medium 6), and tyrosine agar (ISP medium 7). Light red brown pigments are also produced in ISP media 3 and 4 and in many of the carbon utilization media. These pigments are not pH indicators. The culture utilizes glucose, arabinose, sucrose, xylose, inositol, mannitol, fructose, rhamnose, and raffinose. It does not utilize cellulose. The culture strongly solubilizes calcium malate and weakly solubilizes tyrosine and xanthine; it does not solubilize casein. The culture grows in the temperature range of 24-40°C. No growth occurs at 10°C or 45°C.

2. PRODUCTION Frozen agar plugs of the organism are inoculated into a 500 mL wide-mouth flask containing 100 mL of a sterile seed medium (GS-7). The culture is then incubated at 28° on a rotary shaker, 250 rpm, for three days. The seed medium is composed of glucose, 25 g and Pharmamedia (Traders Protein, Inc.) 25 g/L of tap water. The medium is adjusted to pH 7.2 with NH₄OH prior to sterilization. The mature seed culture is transferred (5%) to the production medium (100 mL/500 mL wide-mouth flask). The production medium is formulated as follows: glucose, 2 g; dextrin, 20 g; molasses (Brer Rabbit), 10 g; soybean meal, 15 g; Trypticase Soy Broth (BBL), 5 g; yeast extract, 3 g; cottonseed oil, 10 mL; K₂Cr0₄, 0.5 g; CaC0₃, 3 g/L of tap water. The mixture is adjusted to pH 7.2 with KOH slurry prior to sterilization. Fermentation is carried out at 28° on a rotary shaker at 250 rpm. Using these conditions, production of Inhibitor U-95314 begins on day 2 of the fermentation with maximum titers attained on days 3-4. The levels of compound gradually decline on latter days of fermentation. Adequate aeration is critical to production. The compound is not detectable in the fermentation when agitation on the rotary shakers was less than 200 rpm.

3. ISOLATION AND PARTIAL PURIFICATION A total of 9L of shake flask-grown beer is collected and frozen in 3L portions from each of Days 2-4. The pool for each day is filtered separately over a bed of 2L of Celatom- FW40 filter aid in a Buchner funnel/vacuum filter flask assembly. The filter cake is washed with 1L of deionized water and combined that with the filtered beers. The filtered beers plus washes are discarded. The filter cake for each day is extracted with 2.0L of acetone. The filtrates are contracted separately to an aqueous on a rotary evaporator. The aqueous residues are frozen with Dry Ice-acetone and lyophilized to black, tarry oils.

Each oily residue is partitioned between 1.0 L of cyclohexane and 500 mL each of ethyl acetate, 95% ethanol and water. The solids from the upper phase are discarded. The lower phase from this partition was extracted with 1.8 L of fresh ethyl acetate, giving new upper phases and new lower phases for Days 2-4 respectively. Solids from the latter, lower phase, are discarded.

The samples for the upper phases, for days 3 and 4, were combined. The appearance of these samples was, day 3, 4.57 gm, dark oil, and day 4, 0.88 gm, tan solid. The combined samples were placed a CCCD apparatus. Sample was placed in the first two tubes (10 mL/phase) of a CCCD apparatus using the 2:2:2:1 cyclohexane:ethyl acetate:95% ethanol:water system.

After 200 transfers, the solvent is withdrawn in every 10th tube into a 25 mL tube and it is blown down to a aqueous under a stream of nitrogen while in a 40° water bath (a Model 10 N-Evap apparatus, Organomation Assoc, Worcester, Massachusetts. 5.0 mL of 95% ethanol is added to each tube and the tube is agitated on a "wiggle bug" (Vortexer 2, VWR Scientific) to homogenize the solutions. A dilution of this solution, 1:10, is placed in a UV cell with a 1 cm path. The absorbance is read at 280 nm. On the basis of the UV absorbances and enzyme assays (not shown) the tubes are pooled as follows: CCCD Tubes Pool Sample 16-35 Group A

85-125 Group B

13-15 Group C

11-12 Group D

8-9 Group E 6-7 Group F

The above samples are chromatographed separately on a Rainin 1" C-l 8 HPLC System wim detection at 280 nm and a flow rate of 12 mL/min. The gradient progressed from 25 to 100% ethanol in water over 20 minutes, holding at 100% ethanol for ten minutes.

Samples from Group B, when chromatographed, gives a peak near 17.0 minutes. This sample contains the free acid form of the compound of figure C-l.

Samples from Groups A, C, D, E, and F give major peaks near 15 minutes with increasing amounts of materials eluting near the void volume of the column as the origin of the samples approached tube zero of the CCCD apparatus. Sample A gives 13.3 mg of a tan solid. Samples C, D, E and F give white solids from evaporation of the Rt = 15 minute peak. Samples C, D, E and F are combined to give 278.8 mg of a white solid. This sample gives one tic spot at Rf 0.60 using 1:1 ethyl acetate:95% ethanol as the mobile phase (Whatman LK6DF plates, channeled for densitometry and with a preadsorbant band). It melted at 145- 146°. This sample is pure and the NMR data referred to in this application was made with this sample using pyridine as the solvent. 5. ACΉVΠΎ

The activity of the compounds of formula C-l were measured in the HIV-1 protease assay. The HIV protease assay measures the cleavage of the HIV gag protein, that takes place at the Tyr-Pro bond. A synthetic peptide, H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-OH (GSP), that corresponds to the sequence from residue 128 to 135 in the HIV gag protein serves as a substrate for the measurement of HIV-1 protease activity. Tomasselli, et al, Biochemistry, 29: 264-269 (1990).

HIV-1 protease activity is measured at 30°C in 50 mM sodium acetate, pH 5.5, containing 10% glycerol, 5% ethylene glycol, and 0.1% Nonidet P-40 in a total volume of 50 μL. After 10 and 20 min. of incubation, 20 μL samples are withdrawn into a vial containing 30 μL of 1% tri-fluoroacetic acid (TFA) and subjected to HPLC analysis. HPLC is carried out with a Vydac C₄ column (0.46 x 15 cm), eluting with a linear gradient of 0-30% acetonitrile over a period of 25 min. at a flow rate of 1.0 mL/min. The IC50 of the compound of formula C-l was found to be approximately 14 μM using the synthetic GSP octapeptide as the substrate and monitoring by HPLC. 6. STRUCTURE ELUCIDATION The compound of the invention is identified by its novel properties and spectra. Tables

C-l through C-3 identify the compound of the invention. Table C-l lists some properties of the compound that define it as a novel compound. Table C-2 shows the infrared spectrum of the compound (digital). Table C-3 shows the Carbon-13 NMR spectrum in d^-pyridine (digital) and the Proton NMR spectrum, (digital), in a Carbon-Proton Connectivity table. These tables and spectra identify one compound represented by formula C-l.

Table A-1 Characterization of Compound

Appearance: White, crystalline solid

Melting point: 101-102° (methanol/water)

Mass Spectra: FAB [M+H]⁺ at m/z 369.2637; (Calculated for C₂₁H₃₇0₅: 369.2641) EI M⁺ at m z 368.2594 (Calculated for C₂₁H₃₆0₅: 368.2563)

Elemental Analysis: C, 68.44; H, 9.89 (Found) (C, 68.47; H, 9.78 calculated for C^gO_j)

UV Spectra: Ethanol, e₂₂β = 10,600; _₂ = 11,900 Ethanol + HCl, e₂₀₀ = 18,000; e₂₆₅ = 11,100 Hexane, e₂₆₈ = 6,100

FTIR Spectrum: 2914, 2847, 1749, 1663, 1607, 1469, 1049 cm^"1

Optical Rotation: [α]_D = +52° (95% ethanol, c = 9)

Table A-3 Proton NMR Spectrum in dg-DMSO (digital) Chemical Shift measured in ppm* 6.5 (2H, broad, exchangeable) 4.75 (1H, broad, S)

3.75 (2H DQ)

2.76 (2H, T) 1.51 (2H, M) 1.4 (21H, M)

0.85 (6H, D plus T)

*Multiplicities; D = Doublet; DQ = Doublet of Quartet; M = Multiplet; S = Singlet;

T = Triplet; Q = Quartet. H is Hydrogen

*Multiplicity; S = Singlet; D = Doublet; T = Triplet; Q = Quartet

Table B-1 Characterization of Compound (Characterization of the dipotassium salt.) Appearance: White Powder

Melting point: 245° (dec.)

Elemental Analysis: C, 45.30; H, 6.16; K, 14.04; S, 11.52 calculated for C₂₁H3₄0gS₂K₂ C, 45.79; H, 6.20; N, 0.24; S, 7.90; ash, 13.59 found

UV Spectra: 262 nm (e=396), water; no shift in acid or base FTffi. Spectrum: 2917, 2849, 1611, 1594, 1470, 1282, 1257 (mineral oil) 1227, 1129, 1065, 1052, 987, 884, 795, 755, 684, 670 cm^'1

Table B-3 Proton NMR Spectrum in d_β-DMSO (digital) (Chemical Shift measured in ppm, multiplicities in parenthesis*) pom # H. M*

6.79 1H, (S)

6.73 2H, (S)

2.50 4H, (M)

1.51 4H, (M) 1.24 17H, (M)

0.83 6H, (D + T)

*Multiplicities: S = Singlet; D = Doublet; DQ = Doublet of Quartet T = Triplet; M = Multiplet, H is Hydrogen, ; M = Multiplet; Q = Quartet.

Table B-4

Carbon-13 NMR spectrum in dg-DMSO (digital)

(Chemical Shift measured in ppm, multiplicities in parenthesis*)

Table B-5 HIV-1 Protease Assay Results

Sample Percent Inhibition at the following concentrations (u /mD 500 50 25 10 5 2.5

Formula B-1 100 105 66 37 0

Formula B-1 potassium salt 95 95 - - 23 -

Table C-l Characterization of Compound

Appearance: White solid

Melting point: 145-146°

Mass Spectra: FAB [M+H]⁺ at m/z 369.2637; (Calculated for C₂₁H₃₇0₅: 369.2641) EI M⁺ at m/z 368.2594 (Calculated for C₂₁H₃₆0₅: 368.2563)

Elemental Analysis: C, 58.54; H, 8.31; N, 0.26 (Found)

UV Spectra: ^A243 ⁼ 0-5604; A₂₈₁ = 0.2510 ( no change with addition of IN HCl or IN NH₄OH; 0.01 mg/mL in 95% EtOH)

FΗR Spectra: 3427, 3373, 2979, 2966, 2933, 2896, 2878, (Fig. 2) 1711, 1689,

1637, 1584, 1259, 1103, 971 cm^"1

TLC: Rf = 0.60, 1:1 EtOAc:EtOH, silica gel HPLC: Rt = 15 min, C-l 8, 25-100% EtOH (20 min)

c

3871 3816 3734 3427 3373 2979 2966 2933 2896 2878 2831 2362 2342 1711 1689 1637 1584 1450 1387 1363 1309

Table C-3

Carbon - Proton Connectivities

(Chemical shift in ppm, solution is d -pyridine)

Table C-3 (continued)

^Multiplicities: S = Singlet; D = Doublet; DQ = Doublet of Quartet T = Triplet; M = Multiplet, H is Hydrogen, ; M = Multiplet; Q = Quartet.

Table - Glossary Glossary, abbreviations, trade terms and symbols used. Chemical Terms

C is Carbon H is Hydrogen

O is Oxygen OH is hydroxy Asn is Asparagine Gin is Glutamine lie is Isoleucine

Pro is Proline Ser is Serine Tyr is Tyrosine Val is Valine Chromatographic Terms

HPLC is high pressure liquid chromatography.

Ito Multi-Layer Coil Separator Extractor (Ito Coil), Model 1, is a liquid-liquid counter current chromatography system manufactured by P.C. Inc., Potomac, Maryland. Spectroscopic Terms Mass Spectroscopy

MS is Mass Spectroscopy. FAB is Fast Atom Bombardment. EI is Electron Ionization. Infrared Spectroscopy FTIR is Fourier Transform Infrared Spectroscopy cm[-l] is the unit of measure for wave number, it is the inverse of the wavelength in centimeters or the number of waves per centimeter. %T is Percent Transmittance. Nuclear Magnetic Resonance Spectroscopy NMR (nmr) is Nuclear Magnetic Resonance. Table Glossary - Continued Trade Names

ISP media is "International Streptomycin Project" media described by Shirling & Gottlieb Int. J. Syst. Bacteriology, 16: 313-340 (1966). Celite is a diatomite filter aid made by Celite Corp., in Lompoc, California.

CCCD apparatus is a Craig Counter Current Distribution apparatus. Rainin is manufactured by Rainin Instrument Co. Inc., Woburn, MA. Ito Multi-Layer Coil Separator Extractor (Ito Coil), Model No. 1, is manufactured by P.C. Inc. P.C. Inc. is located in Potomac, Maryland. Pharmamedia is a culture media produced from cotton seed meal, it is available from

Proctor and Gamble Oil Seeds Product Company, Memphis, Tennessee. Vydac columns are available from the Separations Group, Inc., Hesperia, California. Nonidet P-40 is a nonionic detergent, it is an octylphenol-ethylene oxide condensate containing an average of 9 moles ethylene oxide per mole of phenol. Units of Measure

°C is degree centigrade, g is gram.

K_j is Equilibria constant for the inhibitor. L is Liter. M or mol is mole, mg is milligram, min is minute. mL is milliliter. mM is milliMole. m z is mass per unit charge, nm is nanometers, rpm is revolutions per minute, slm is standard liters per minute. μL is microliter.

Claims

CLABVISWhat is claimed is:

1. A compound of formula A-1, except as existing or occurring in nature, represented by the following formula:

₅ CH₂CH₂CH₂CH₃

(FORMULA A-1)

wherein the methyl is attached to one of five carbon atoms as indicated or a pharmacologically acceptable salt thereof.

2. The compound of claim 1 in an essentially pure form.

3. A pharmaceutical composition of the compound of claim 1 adapted for administration to obtain an antiviral effect.

4. Use of a substance or composition of formula A-1,

₅ CH₂CH₂CH₂CH₃

(FORMULA A-1)

or formula A-2,

(FORMULA A-2)

for the manufacture of a medicament for the retarding or preventing the replication of human retrovirus (HRV) in a cell system or a human patient exhibiting or susceptible to said HRV.

5. A process for producing a compound of formula A-1 that comprises: cultivating a subculture of the organism, deposited as NRRL 18839, in an aqueous nutrient medium containing a source of assimilable carbohydrate and assimilable nitrogen under aerobic conditions.

6. A process of claim 5, wherein the compound is isolated by filtering the fermentation medium and subjecting the medium to purification procedures.

7. A biologically pure culture of the microorganism characterized as being essentially the strain identified as NRRL 18839.

8. A compound of formula B-1, except as existing or occurring in nature, represented by the following formula:

(FORMULA B-1)

or a pharmacologically acceptable salt thereof.

9. The compound of claim 8 in an essentially pure form.

10. A pharmaceutical composition of the compound of claim 8 adapted for administration to obtain an antiviral effect.

11. Use of a substance or composition of formula B-1, represented by the structure below,

(FORMULA B-1)

for the manufacture of a medicament for retarding or preventing the replication of human retrovirus (HRV) in a cell system or a human patient exhibiting or susceptible to said HRV.

12. A process for producing a compound of formula B-1 that comprises: cultivating a subculture of the organism, deposited as NRRL 18942, in an aqueous nutrient medium containing a source of assimilable carbohydrate and assimilable nitrogen under aerobic conditions.

13. A process of claim 12, wherein the compound is isolated by filtering the fermentation medium and subjecting the medium to purification procedures.

14. A biologically pure culture of the microorganism characterized as being essentially the strain identified as NRRL 18942.

15. A compound of formula C-l, except as existing or occurring in nature, represented by the following formula:

(FORMULA C-l) or a pharmacologically acceptable salt thereof.

16. The compound of claim 15 in an essentially pure form.

17. A pharmaceutical composition of the compound of claim 15 adapted for administration to obtain an antiviral effect.

18. Use of a substance or composition of formula C-l, represented by the structure below,

(FORMULA C-l)

19. A process for producing a compound of formula C-l that comprises: cultivating a subculture of the organism, deposited as NRRL 18943, in an aqueous nutrient medium containing a source of assimilable carbohydrate and assimilable nitrogen under aerobic conditions.

20. A process of claim 19, wherein the compound is isolated by filtering the fermentation medium and subjecting the medium to purification procedures.

21. A biologically pure culture of the microorganism characterized as being essentially the strain identified as NRRL 18943.