RUBRADIRIN DERIVATIVES FOR TREATMENT OF HIV INFECTION

Abstract: The compounds rubradirin, rubradirin B, rubradirin aglycone and the novel compounds protorubradirin, protorubradirin D, rubradirin D or pharmaceutically acceptable salts thereof can be used to treat humans infected with one or more than one strain of human immunodeficiency virus.

Description: RUBRADIRIN DERIVATIVES FOR TREATMENT OF HIV INFECTION Field of the Invention This invention is the novel compounds protorubradirin, protorubradirin D, rubradirin D and a novel treatment of patients infected with a human immunodeficiency virus. Background of the Invention An estimated one to one and one-half million people in the United States are infected with a human retrovirus, the human immunodeficiency virus type I, HIV-1, which is the etiological agent of acquired immunodeficiency syndrome, AIDS, Norman, C., Science, 661662 (1986). Of those infected, an estimated two hundred and fifty thousands people will develop AIDS in the next five years, Curran, J.W., et al., Science, 1352-1357 (1985). On March 20, 1987, the FDA approved the use of the compound, zidovudine (AZT), to treat AIDS patients with a recent initial episode of pneumocystis carinii pneumonia, AIDS patients with conditions other than pneumocystis carinii pneumonia or patients infected with the virus with an absolute CD4 lymphocyte count of less than 200/mm3 in the peripheral blood. AZT is a known inhibitor of viral reverse transcriptase, an enzyme necessary for human immunodeficiency virus replication. Information Disclosure Rubradirin, rubradirin B, rubradirin aglycone and pharmaceutically active salts are known compounds and are described in United States Patents 3,335,057, 4,107,295, and 4,171,437. The production, isolation and purification of rubradirin from Streptomyces achromogenes v. rubradiris is described in United States Patent 3,335,057. Rubradirin has also been isolated from the fermentation beers of Micromonospara sartamica sp. nov. Routien, ATCC 31178, using procedures described in United States Patent 4,032,631. The production, isolation and purification of rubradirin B is disclosed in United States Patent 4,107,295. The production, isolation and purification of rubradirin aglycone is disclosed in United States Patent 4,171,437. Rubradirin is known to inhibit protein synthesis apparently by blocking the initiation of polypeptide synthesis on the ribosome, Reusser, F., Antibiotics VI ed. F.E. Hahn, 187-198 (1983). Summary of the Invention This invention is a method for treating a human infected with one or more than one strain of a human immunodeficiency virus which comprises administering an effective amount of a compound selected from a group consisting of rubradirin, rubradirin B, rubradirin aglycone, protorubradirin, protorubradirin D, rubradirin D or pharmaceutically acceptable salts thereof, to inhibit viral replication in the infected human; a novel compound selected from the group consisting of protorubradirin, protorubradirin D, rubradirin D or pharmaceutically acceptable salts thereof; and the use of compound selected from the group consisting of rubradirin, rubradirin B, rubradirin aglycone, protorubradirin, protorubradirin D, rubradirin D or pharmaceutically acceptable salts thereof, to prepare a medicament to treat humans infected with one or more than one strain of a human immunodeficiency virus. Detailed Description of the Invention In this document, the term human immunodeficiency virus means human immunodeficiency virus type I, human immunodeficiency virus type II, or strains, apparent to one skilled in the art, which belong to the same viral family and which create similar physiological effects in humans as human immunodeficiency virus types I or II. Rubradirin, rubradirin B, rubradirin aglycone, protorubradirin, protorubradirin D, or rubradirin D can be used and administered as the free acid or as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts are derivatives of the compounds used to practice this invention which one skilled in the art would recognize as having substantially the same pharmaceutical and pharmacological properties as the free acid. Examples of pharmaceutically acceptable salts include sodium, potassium and calcium salts of the compounds used to practice this invention. Those skilled in the art would know how to formulate the compounds used to practice 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. When the compounds used to practice this invention are administered orally, an effective amount is sufficient to prevent the occurrence of symptomatic acquired immune deficiency defining infections in the infected human. Specifically, an effective amount ia from about 1 to 100 mg per kg per day. A typical unit dose for a 70 kg human would be from about 200 mg to 1000 mg taken one to four times per day. Either solid or fluid dosage forms can be prepared for oral administration. Solid compositions are prepared by mixing the compounds used to practice 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 used to practice 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 used to practice this invention with an acceptable inert oil such as vegetable oil or light liquid petrolatum. Syrups are prepared by dissolving the compounds used to practice this invention in an aqueous vehicle and adding sugar, aromatic flavoring agents and preservatives. 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 used to practice this invention are administered parenterally, it can be given by injection or by intravenous infusion. An effective amount is from about 1 to 100 mg per kg per day. Parenteral solutions are prepared by dissolving the compounds used to practice this invention in an aqueous solution buffered to physiological pH and sterilizing the solution by known methods 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 used to practice this invention are sterilized with ethylene oxide or suitable gas before it is suspended in the vehicle. The exact route of administration, dose and frequency of administration would be readily determined by those skill in the art and is dependant on the age, weight, general physical condition, or other clinical symptoms specific to the patient to be treated. Patients to be treated would be those individuals: 1) infected with one or more than one strain of a human immunodeficiency virus as determined by the presence of either measurable viral antibody or antigen in the serum and 2) having either a symptomatic AIDS defining infection such as i) disseminated histoplasmosis, ii) isoporiasis,iii) bronchial and pulmonary candidiasis including pneumocystis pneumonia iv) non-Hodgkin's lymphoma or v) Kaposi's sarcoma and being less than sixty years old; or having an absolute CD4 lymphocyte count of less than 200/mm3 in the peripheral blood. Treatment would consist of maintaining an inhibitory level of the compounds used to practice this invention in the patient at all times and would continue until the occurrence of a second symptomatic AIDS defining infection indicates alternate therapy is needed. The novel compounds protorubradirin, protorubradirin D, and rubradirin D are isolated from fermentation beers from a mutant strain of Streptomyces achromogenes v. rubradiris. A culture of this living organism was deposited on 4 May 1988, in compliance with the terms of the Budapest Treaty at the Agricultural Research Patent Culture Collection, Microbial Properties Research, Northern Regional Research Center, Peoria, IL., United States, deposit number NRRL 18366. Primary seed flasks, 300 ml closed systems, are inoculated with the sample and incubated for seventy two hours on a rotary shaker at 28 "C. The seed media is Pharmamedia, 25 g/l; glucose, 25 g/l; and polypropylene glycol, 0.2 ml/l; pH 7.2. Secondary seed vessels contain 250 1 of the above medium and are inoculated with a single closed system flask. Growing conditions are: temperature, 28"C; agitation rate, 280 rpm; aeration rate, 200 slm; back pressure, 7 psi; and incubation time, forty eight hours. For compound preparation, a 250 1 fermentor containing media of the following composition: distillers solubles (Solulac, Grain Processing Corp.), 10 g/l; cornstarch, 10 g/l; corn steep liquor, 20 g/l; sodium nitrate, 3 g/l; pH adjusted to 7.2 with sodium hydroxide then add calcium carbonate, 5 g/l; soybean oil, 10 ml/l; polypropylene glycol, 0.2 mull, is inoculated with 12.5 1 of secondary seed culture and incubated for about ninety hours at 28"C. Growing conditions are: agitation rate, 280 rpm; aeration rate, 200 slm; back pressure, 7 psi; and pH is controlled at 7.5 with sulfuric acid. Non-ionic resins, 60 ml/l, such as HP-21 (Mitsubishi) or XAD-16- (Rohm and Haas), are added as separate sterile additions at about twenty to about twenty-three hours of incubation. The resins are filtered from the bulk fermentation media and the excess water is removed by suction using a sintered glass funnel. Fatty material is removed from the resin by washing with a 4:1::cy- clohexane:methylene chloride solution. The compounds are removed from the resin and isolated by elution with ethyl acetate, removal of the solvent, redissolution in a small volume of ethyl acetate, dilution with Skellysolve B, and filtration. The resulting mixture is dried and separated by high performance liquid chromatography using two C-18 columns connected in series. The mixture is loaded onto the C-18 columns in tetrahydrofuran and eluted with 1:T::0.05 M ammonium acetate adjusted to pH 4.0 with glacial acetic acid:tetrahydrofuran. The fractions are collected, the organic solvents are removed, and the residue is filtered. The residue is washed with water, dissolved in chloroform, dried over sodium sulfate, precipitated by the addition of four volumes of Skellysolve B, filtered and dried. Further separation of the components of the mixture by high performance liquid chromatography using 65:35::acetonitrile:ammonium acetate pH 4.0 allows separation of protorubradirin and rubradirin. Without - further elaboration, those skilled in the art can practice the present invention to its fullest extent. The following detailed examples further describe how to use rubradirin, rubradirin B, rubradirin aglycone, protorubradirin, protorubradirin D, rubradirin D or pharmaceutically acceptable salts thereof to treat humans infected with one or more than one strain of a human immunodeficiency virus. These examples are merely illustrative and are not limitations of the preceding disclosure. Those skilled in the art will promptly recognize appropriate variations from the examples. In each example, rubradirin could be replaced with rubradirin B, rubradirin aglycone, protorubradirin, protorubradirin D, rubradirin D or a pharmaceutically acceptable salt thereof. Example 1 - Hard Gelatin Capsules One thousand two-piece hard gelatin capsules for oral use, each capsule containing 50 mg of rubradirin, are prepared from the following: Rubradirin 50 gm Lactose 100 gm Cornstarch 20 gm Talc 20 gm Magnesium Stearate 2 gm The rubradirin is added to the other ingredients, mixed and encapsulated in the usual manner. Example 2 Tablets One thousand tablets, each containing 50 mg of rubradirin, are prepared from the following: Rubradirin 50 gm Lactose 75 gm Cornstarch 50 gm Magnesium Stearate 4 gm Light liquid petrolatum 5 gm The rubradirin is added to the other ingredients, mixed and slugged. The slugs are broken down by forcing through a number sixteen screen. The resulting granules are then compressed into tablets. ExamDle 3 Parenteral solution A sterile aqueous solution for parenteral intravenous injection containing 150 mg of rubradirin sodium salt in one liter of buffered solution is prepared from the following: Rubradirin, sodium salt 150 mg Water for injection, qs 1000 mg buffed to physiological pH The rubradirin sodium salt is sterilized, added to the sterile buffered water, filled into sterile containers and sealed. Example 4 Preparation of protorubradirin A crude rubradirin mixture in tetrahydrofuran (20 mL) is placed on two PrepPak 500 C18 columns in series on the Waters Prep 500 instrument, and eluted using l:l::tetrahydrofuran 0.05 M ammonium acetate adjusted to pH 4.0 with acetic acid. Fractions are analyzed on an analytical Brownlee RP-18 Spheri-lO cartridge, 10 cm x 4/6 mm I.D., monitoring at 330 nm. Fractions showing only peaks of rubradirin-protorubradirin in the mobile phase acetonitrile--pH 4.0 buffer in a gradient from 40 to 60% of acetonitrile, are combined, and the majority of acetonitrile is removed on the rotating evaporator. This material is extracted with chloroform, dried over sodium sulfate concentrated, and the mixture is precipitated by the addition of 4 volumes of Skellysolve B. A mixture of rubradirin and protorubradirin (4.3 g) is dissolved in tetrahydrofuran (20 ml), diluted with acetonitrile (20 ml) and placed on the two PrepPak 500-C18 columns in series, which had been equilibrated with 65:35::acetonitrile:pH 4.0 ammonium acetate. Fractions are analyzed on the Brownlee C-18 analytical column using the same acetonitrile:pH 4.0 ammonium acetate solution. Fractions showing peaks corresponding to mixtures of the two components are stripped on a rotating evaporator, extracted into chloroform, dried over sodium sulfate and the solvent is removed. Fractions showing only the peak of protorubradirin on this analytical column are worked up similarly except that the concentrated chloroform solution was diluted with 4 volumes of cyclohexane to give protorubradirin. Physical characteristics are: Elemental Analysis: Found: C, 56.79; H, 4.73; N, 5.61. (M.W. not yet estab lished). pKa 6.4 and 8.2 (in dimethylformamate - 60% aqueous ethanol, 1:1). Optical Rotation: (a1 (c 0.2735 in acetone +921 . IR., mineral oil mull, cm-l 3610.7; 3556.7; 3361.9; 3278.0; 2940.5; 2868.1; 2854.6; 2855.6; 2724.4; 2668.5; 2418.7; 2185.3; 2035.9; 1726.3; 1690.6; 1670.4; 1641.4; 1616.3; 1573.9; 1544.0; 1598.3; 1462.0; 1378.1; 1325.1; 1282.7; 1246.0; 1232.5; 1197.8; 1186.2; 1163.1; 1145.7; 1122.6; 1104.2; 1086.9; 1048.3; 1033.8; 1008.8; 982.7; 964.4; 940.3; 924.9; 891.1; 871.8; 854.5; 840.0; 814.0; 804.3; 793.7; 782.1; 758.0; 734.9; 718.5; 691.5; 683.8; 658.7; 609.5. U V. 1. in 0.001N sodium hydroxide in 85% methanol. Amax(nm) 211; 236; 304; 347; 412; 425; 585, 2. in 0.001N sulfuric acid in 85% methanol. Amax(nm) 226; 268; 281; 314; 321; 519. 13C nmr chemical shifts in p.p.m. in d6-acetone: 11.611; 16.723; 16.897;, 19.459; 21.675; 25.226; 35.556; 49.344; 56.285; 62.545; 63.092; 68.538; 71.047; 78.322; 79.857; 91.122; 96.501; 100.892; 110.249; 112.745; 113.559; 122.355; 124.557; 125.812; 13Q.283; 132.472; 133.086; 134.301; 148.302; 153.134; 157.725; 158.620; 163.865; 176.185. Examole 5 Preparation of Rubradirin D HP-21 resin (about 1.5 L) is placed in a sintered glass funnel (4 L), and excess water was removed under suction. Fatty material is removed from the resin by washing with 4:1::cyclohexane:methylene chloride (3x2 1) initially under gravity with each batch and then under suction. These washes are discarded. Similar elution with ethyl acetate (total 10 1), removal of the solvent, redissolution of the residue in a small volume of ethyl acetate, and dilution with 4 volumes of Skellysolve B gives a mixture of compounds. The mixture is dissolved in tetrahydrofuran (20 mL) and transferred to two C-18 columns connected in series, prep-equilibrated with mobile phase (0.05 M aqueous ammonium acetate adjusted to pH 4.0 by the addition of glacial acetic acid-tetrahydrofuran, 1:1. Elution is commenced using the same (1:1) system isocratically, at a flow rate of 150 mL/min., monitoring at 365 nm. Appropriate fractions are combined, and volatile material is removed on the rotating evaporator. This solid is removed by filtration under suction on a sintered glass funnel, washed with water, and dissolved in chloroform. After drying over sodium sulfate, a four-fold volume of Skellysolve B is added with magnetic stirring, the precipitate is collected under suction on a sintered glass filter funnel, and dried in a vacuum desiccator. Elemental Analysis: Found: C, 57.89; H, 4.93; N, 5.09, C49H48N4020 requires: C, 58.09; H, 4.79; N, 5.53. pKa 5.6 and 8.1 (in dimethylformamide - 60% aqueous ethanol, 1:1). Optical Rotation: [a]D + 487 (c 0.0303 in acetone). Mass spectrum: mle 1012, 825. I.R., mineral oil mull, cm-l 3616.5; 3553.8; 3502.7; 3375.4; 2963.6; 2859.5; 2852.7; 2723.5; 2670.4; 2630.9; 2417.8; 2182.5; 2031.0; 1822.7; 1735.0; 1699.3; 1665.5; 1639.5; 1616.3; 1574.9; 1543.0; 1508.3; 1463.0; 1426.4; 1378.1; 1329.0; 1302.9; 1271.1; 1246.0; 1232.5; 1198.8; 1163.1; 1115.8; 1105.2; 1093.6; 1080.1; 1059.9; 1049.3; 1002.0; 945.1; 922.9; 868.9; 846.7; 835.2; 812.0; 803.3; 788.9; 781.2; 758.0; 73Q.9; 689.6; 658.7; 620.1. UV 1. in 0.001N sodium hydroxide in 85% methanol. Amax 212 (77,270), 241 (38,050), 257 (32,530), 286 (27,080), 304 (33,380), 342 (17,500), 404 (12,040), 420 (10,580), 585 (921) nm. 2. in 0.001N sulfuric acid in 85% methanol. Amax 216 (23,480), 285 (10,450), 307 (11,520), 317 (11,590) nm. 13C nmr chemical shifts in p.p.m. in d6-acetone: 11.613; 16.727; 16.874; 19.438; 21.694; 25.247; 36.542; 45.891; 49.350; 56.293; 62.529; 63.090; 68.538; 71.035; 78.313; 79.889; 91.132; 96.514; 100.894; 110.254; 112.765; 113.553; 122.340; 125.811; 130.271; 130.378; 130.752; 132.488; 134.277; 136.374; 144.680; 148.312; 153.132; 157.739; 158.607; 176.180; 179.492. Example 6 Preparation of protorubradirin D. Material isolated from crude rubradirin complex by reversedphase chromatography in the tetrahydrofuran-pH 4.0 aqueous ammonium acetate system as described for Rubradirin D and which had been sheltered from light gives essentially a single peak, not distinguished from rubradirin D at the Brownlee analytical column in the tetrahydrofuran buffer gradient. Chromatography of this material over the two Preppak-500/C18 columns in series, eluting with an isocratic mixture of acetonitrile and pH 4.0 buffer (53:47 v/v), monitoring the chromatogram at 340 nm, and examining fractions on the p-Bendapak column using the same mobile phase results in the isolation of mixtures of rubradirin D and protorubradirin D followed by pure protorubradirin D. Removal of the volatile solvent gives an aqueous suspension which is extracted into chloroform, dried over sodium sulfate, and the solvent is removed to give a residue. The residue is redissolved in a small volume of chloroform and diluted with cyclohexane (4 volumes), filtered and washed with cyclohexane. Elemental Analysis: Found: C, 57.23; H, 5.02; N, 5.37. (molecular weight un known). pKa 5.6 and 8.0 (in dimethylformamide - 60% aqueous ethanol, 1:1). Optical Rotation: [a]D + 551 (c 0.02960 in acetone). I.R., mineral oil mull, cm 1 3613.6; 3548.0; 3493.1; 3466.1; 3383.1; 3295.4; 3082.2; 2909.6; 2868.1; 2854.6; 2722.5; 2672.4; 2181.5; 2037.8; 1735.0; 1697.4; 1664.6; 1640.5; 1617.3; 1574.9; 1541.1; 1508.3; 1463.0; 1378.1; 1329.0; 1302.9; 1262.4; 1246.0; 1231.5; 1198.8; 1188.1; 1163.1; 1114.9; 1105.2; 1093.6; 1079.2; 1050.2; 1002.0; 943.2; 922.0; 875.7; 836.1; 812.0; 803.3; 788.9; 781.2; 758.0; 732.9; 692.4; 660.6; 621.1. UV 1. in 0.OOlN sodium hydroxide in 85% methanol. Amax 212; 241; 250; 286; 305; 407; 424; 585. 2. in 0.001N sulfuric acid in 85% methanol. Amax 218; 288; 310; 320; 348; 515. 13C nmr chemical shifts in p.p.m. in d6 acetone: 11.636; 16.724; 17.096; 18.478; 19.483; 21.721; 34.650; 45.908; 49.332; 56.245; 62.444; 62.780; 68.558; 72.489; 78.258; 79.566; 79.925; 96.279; 100.621; 104.667; 110.140; 112.583; 113.487; 122.344; 124.475; 125.735; 130.268; 132.430; 133.106; 134.327; 143.516; 144.433; 148.273; 152.992; 157.698; 158.660; 162.913; 163.145; 163.756; 176.153; 179.463. The utility of this invention is demonstrated by the ability of the compounds used to practice this invention to inhibit viral reverse-transcriptase, an enzyme essential for human immunodeficiency virus replication. This enzyme has characteristics which differentiate it from other known cellular polymerases and it is a unique enzyme which is not found in uninfected cells. Viral reverse transcriptase is found in extracts from bacterial clones prepared according to the procedure described by Goff, S. P., et al., Journal of Virology, 743-745 (1986). Inhibition of this enzyme is determined in a cell free assay which measures the level of radioactive precursors incorporated into DNA. Extracts prepared according to the procedure of Kleid, D. G., et al., Science, 1125-1129 (1981) are incubated in a mixture of inhibitor, 20 mM dithiothreitol, 60 mM sodium chloride, 0.05% NP-40, 10 mM magnesium chloride, 50 mM Tris pH 8.3, 10 AM [35S-labeled deoxynuleoside-5'-triphosphate, 10 pg/ml RNA template (poly rC or poly rG) and 5 Ag/ml DNA primer (oligo dA or oligo dT) for 30 minutes at 37"C. Incorporation of radio-labeled precursor is determined by spotting aliquots of the reaction mixture on DE81 paper, washing the papers to remove unincorporated precursor, drying and determining counts. Chart 1 contains the results of the assay for rubradirin, rubradirin aglycone, protorubradirin, protorubradirin D, rubradirin D and rubradirin B. Chart 1 Sample Concentration (mM) % Inhibition Reverse Transcristase rubradirin 0.10 40 0'05 20 rubradirin 0.10 99 aglycone 0.05 91 protorubradirin 0.10 34 protorubradirin D 0.10 41 rubradirin D 0.10 84, 77, 97, 93 rubradirin B 0.10 29

Claims: CLAIMS 1. A method for treating a human infected with one or more than one strain of a human immunodeficiency virus which comprises administering an effective amount of a compound selected from a group consisting of rubradirin, rubradirin B, rubradirin aglycone, protorubradirin, protorubradirin D, rubradirin D or pharmaceutically acceptable salts thereof, to inhibit viral replication in the infected human. 2. A compound selected from the group consisting of protorubradirin, protorubradirin D, rubradirin D or pharmaceutically acceptable salts thereof. 3. Use of a compound selected from the group consisting of rubradirin, rubradirin B, rubradirin aglycone, protorubradirin, protorubradirin D, rubradirin D or pharmaceutically acceptable salts thereof, to prepare a medicament to treat humans infected with one or more than one strain of a human immunodeficiency virus. 4. A method according to claim 1 where the human is infected with human immunodeficiency virus type I. 5. A method according to claim 1 where the human is infected with human immunodeficiency virus type II. 6. A method according to.claim 1 where the administration is oral. 7. A method according to claim 1 where the effective amount is from about 1 to 100 mg per kg per day. 8. A method according to claim 1 where the administration is parenteral. 9. A method according to claim 1 wherein said effective amount of compound is sufficient to prevent the occurrence of symptomatic acquired immune deficiency defining infections in the infected human.

Inventors: ALTHAUS IRENE W, BANNISTER BRIAN, REUSSER FRITZ, TARPLEY WILLIAM GARY, ZAPOTOCKY BARBARA, ALTHAUS, IRENE, W., BANNISTER, BRIAN, REUSSER, FRITZ, TARPLEY, WILLIAM, GARY, ZAPOTOCKY, BARBARA
Applicants: UPJOHN CO, THE UPJOHN COMPANY
Application year: 1988
Main classification: A61K31
IPC codes: A61K31, A61K35, A61P31, C07D498, C07H17, C12P17, C12R1