WO1988007540A1 - Hydrogen sulfide adducts of paulomycin - Google Patents

Abstract

Novel hydrogen sulfide adducts of paulomycin compounds are disclosed and claimed. Also provided are methods for preparing these compounds.

Description

HYDROGEN SULFIDE ADDUCTS OF PAULOMYCIN FIELD OF INVENTION

The present invention relates to novel compositions of matter, and methods for producing them. The present invention particularly provides new hydrogen sulfide adducts of paulomycin and derivatives of paulomycins including esters and salts thereof. BACKGROUND OF THE INVENTION

Antibiotics A₂ , C, D, E, F, 273a₁, 273a₁α, 273a₁β, 273a₂,

273a2α, 273a₂β, and O-demethyl paulomycins A and B have been dis covered in the fermentation broth of Streptomyces paulus which also produces the known antibiotics paulomycin A and paulomycin B. The structures of these paulomycins are shown in Chart A.

Paulomycin A- and B-hydrogen sulfide adducts are two new an tibiotics produced by Streptomyces paulus. The two compounds, which are formed by addition of H₂S to paulomycins A and B respectively. The paulomycin A- and B-H₂S adducts are present as minor components in fermentations of S. paulus. They were first detected in purified preparations obtained from mother liquors from crystallization of paulomycins A and B. These compounds have the properties of adversely affecting the growth of Gram-positive bacteria, for example, Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes and Streptococcus faecalis. Thus, they can be used alone or in combination with other antibacterial agents to prevent the growth of, or reduce the number of such microorganisms present in various environments. INFORMATION DISCLOSURE

Antibiotics paulomycin A and paulomycin B are disclosed in United States Patent 4 , 335 , 108 . They are prepared by fermentation using Streptomyces paulus. strain 273 NRRL 12251. Essentially pure crystalline preparations of paulomycins A and B are disclosed in Examples 2 and 3 thereof, respectively. Antibiotics 273a₁α and 273a₁β are disclosed in United States patent 4,505,895. Novel methods of preparing antibiotics 273a₁α and 273a₁β and derivatives thereof are disclosed in copending application S.N. 812,178, filed 23 December 1985.

A.D. Argoudelis, et al., "Paulomycins A and B Isolation and Characterization", J. Antibiotics, 35, pp. 285-94 (1982), refers to the isolation and characterization of paulomycins A and B. Paulo mycin C is mentioned therein. Specifically, this publication describes the thin layer chromatography and HPLC behavior of paulomycin C. No isolation procedure or chemical or biological properties of the compound are reported. We have also learned that the spot identified therein as paulomycin C also contains O-demethyl paulomycin B.

P.F. Wiley, et al., "The Structure and Chemistry of Paulomycin", J. Org. Chem., 51, pp. 2493-99 (1986), refer to the gross structure and absolute stereochemistry of paulomycins A and B and some degradation products thereof.

All of the foregoing references are incorporated herein by reference. SUMMARY OF THE INVENTION

The present invention particularly provides an antibacterially active compound of the Formula II, or a pharmacologically acceptable salt thereof I. wherein R₁ is OCH₃ and R₂ is

A. CH₃-CH₂CH(CH₃)-COOCH(CH₃)-,

B. CH₃CH(CH₃)-COOCH(CH₃)-, C. (CH₃)₂CHCH₂COOCH(CH₃)-,

D . CH₃CH₂-COOCH(CH₃) - ,

E. CH₃COOCH(CH₃)-,

F. CH₃C(=O)-, or

G. CH₃CH(OH)-; or II. wherein R₁ is H and R₂ is

A. CH₃-CH₂CH(CH₃) -COOCH (CH₃) - , or

B . CH₃CH(CH₃)-COOCH(CH₃)-; or a pharmacologically acceptable salt thereof.

Pharmacologically acceptable cations include pharmacologically acceptable metal cations, ammonium, amine cations, or quarternary ammonium cations.

Especially preferred metal cations are those derived from the alkali metals, e.g., lithium, sodium, and potassium, and from the alkaline earth metals, e.g., aluminum, zinc, and iron which are within the scope of this invention.

Pharmacologically acceptable amine cations are those derived from primary, secondary, or tertiary amines. Examples of suitable amines are methylamine, dimethylamine, trimethylamine, ethvlamin,, dibutylamine, triisopropylamine, M-methylhexylamine, decylamine, dodecylamine, allylamine, crotylamine, cyclopentylamine, dicyclohexylamine, benzylamine, dibenzylamine, a-phenylethylamine, b- phenylethylamine, ethylenediamine, diethylehetriamine, and the like, aliphatic, cycloaliphatic, araliphatic amines containing up to and including about 18 carbon atoms, as well as heterocyclic amines, e.g., piperidine, morpholine, pyrrolidine, piperazine, and lower- alkyl derivatives thereof, e.g., 1-methylpiperidine, 4-ethylmorpholine, 1-isopropylpyrrolidine, 2-methylpyrrolidine., 1,4-dimethylpiperazine, 2-methylpiperidine, and the like, as well as amines containing water-solubilizing or hydrophilic groups, e.g., ono- , di-, and triethanolamine, ethyldiethanolamine, N-butylethanolamine, 2- amino-1-butanol, 2-amino-2-ethyl-1,3-propanediol, 2-amino-2-methyl- 1-propanol, tris(hydroxymethyl)aminomethane, N-phenylethanolamine, N-(p-tert-amylphenyl)-diethanolamine, N-methylglycamine, N-methylglucosamine, ephedrine, phenylephrine, epinephrine, procaine, and the like. Further useful amine salts are the basic amino salts, e.g., lysine and arginine.

Examples of suitable pharmacologically acceptable quaternary ammonium cations are tetramethylammonium, tetraethylainmonium, benzyltrimethylammonium, phenyltriethylammonium, and the like. DETAILED DESCRIPTION OF THE INVENTION

The present invention is disclosed more fully in the following examples. Example 1 Microbial Production of Paulomycin A- and B-H₂S

Adducts

A. Assay and Testing Procedures

Antibiotic production and purification was measured by a microbiological disc-plate assay procedure with Micrococcus luteus as the assay organism.

B. Thin-Layer Chromatographic Procedures

The production of paulomycins and other related metabolites was followed by thin-layer chromatography on silica gel G using chloroform-ethanol-water (25:30:5, v/v) or chloroform-methanol (90:10, v/v) as the solvent system. Paulomycins were separated by TLC using Brinkman's cellulose-coated plates and pH 7.0 phosphate buffer as the solvent system. The antibiotics present in the fermentation or in preparations obtained during purification were detected by bioauto graphy on M. luteus seeded trays.

C. Spectroscopic Methods

Proton magnetic resonance (PMR) spectra were recorded on a Varian XL-200 spectrometer operating at 200 MHz. Solutions (ca. 0.4 ml, ca. 0.25 M) of the compounds in dimethylsulfoxide-d₆ or acetoned₆ were used. Carbon magnetic resonance (CMR) spectra were recorded on a Varian CFT-20 spectrometer operating at 20.0 MHz. PMR and CMR chemical shifts are reported as ppm relative to tetramethylsllane. High resolution mass spectra were obtained on a ZAB-2F high resolution mass spectrometer using a fast atom bombardment (FAB) source.

D. Analytical-High Performance Liquid Chromatography (HPLC)

All HPLC chromatography was carried out with a Varian Model 5560 (Varian Instruments, Sugar Land, Texas) instrument equipped with a LKB Rapid Spectral Detector (LKB, Broma, Sweden). A Zorbax C-8 25 cm 4.6 mm stainless steel column packed with C8 (6μ) reverse phase silica was used with a mobile phase composed of 60% water, 20% acetonitrile, and 20% tetrahydrofuran. The solvent also contained 0.2% glacial acetic acid. The flow rate was 1.5 ml/min. Injection volume was 25 μl. E. Fermentation Conditions

The fermentation conditions were in general identical to the conditions described in U.S. Patent 4,335,108 which is incorporated herein by reference. The only exception was that Amberlite XAD-2 resin, ca. 150 liters, was added per 5000 liters of fermentation beer. Under these conditions, all paulomycins produced are adsorbed on the resin. F. Isolation Procedures

1. Isolation of Antibiotics Produced by Streptomyces paulus a. General Procedure The whole beer (ca. 5000 liters) was adjusted to pH

4.5 (using aqueous sulfuric acid) and then passed through a large vibrating screen in order to remove the Amberlite XAD-2 resin. The resin was slurried In 100 liters of water and screened off on the vibrating screen. The washed resin was packed in a column, washed with 500 liters of cyclohexane-methylene chloride (4:1, v/v) mixture, and then eluted with 1400 liters of ethyl acetate. The ethyl acetate eluate was concentrated to dryness. The dry solid was triturated twice with heptane and then crystallized from methylene chloride. The crude crystalline preparation obtained was found to contain, in addition to paulomycin A and B, several other bioactive components. Further purification was obtained by the procedures described below. b. Summary of Crude Crystalline Preparations Obtained by Extraction

Weight of Fermentation Crude Crystals (g) Preparation I + II 3820 A

III 1230 B Preparations A and B were used in the recrystallization studies described below.

2. Recrystallization a. General Procedure

The crude crystals obtained as described above were dissolved in ethyl acetate (5 ml of ethyl acetate per g of crystals). This solution was mixed with filter aid and Darco G-60 (50 mg of each per g of crude crystals) and stirred for 1 hr. It was then filtered over filter aid and the filtrate was mixed with hexane (5 ml per g of crude crystals). The mixture was allowed to stand at 5°C for 24 hours. The precipitated crystals of paulomycins A and B were separated by filtration. The filtrate (mother liquors) was concentrated to dryness to give a residue enriched in bioactive materials other than paulomycins A and B. This material was used for the isolation of the paulomycin A- and B-H₂S adducts as described below. b. Summary of Recrystallization

Preparation A was recrystallized from ethyl acetate as described above. The following materials were obtained: 1) Preparation C , 3050 g (paulomycin A and B) ; 2) Preparation D , 360 g, containing several bioactive materials.

Similarly preparation B, by recrystallization, yielded 990 g of paulomycins A and B (preparation E) and preparation F (188 g).

Preparations D and F were used as the starting material for the work which led to the isolation of the paulomycin A- and B-H₂S adducts.

3. Isolation of "Polar Paulomycins" Including Paulomycin A- and B-H₂S Adducts. HPLC Chromatography Instrument: Waters Prep. 500A Support: Waters C-18 Reverse Phase Silica Packed Columns Starting Material: Preparation F, 40 g

Mobile Phase: Acetonitrile--0.01 M pH 5.5 phosphate buffer

(1:1 v/v) The starting material was dissolved in 400 ml of acetonitrile - pH 5.5 phsophate buffer (1:1 v/v) and was injected into the column. The chromatography was followed by UV and/or refractive index detectors. In addition, selected fractions were analyzed by

TLC using KC-18 reverse phase silica plates and acetonitrile-methanol - pH 5.5, 0.01 M phosphate buffer (1:1:1 v/v) as the mobile phase.

Fractions containing the "polar paulomycins" (paulomycins C, D, E, O-demethylpaulomycin A, O-demethylpaulomycIn B, and the paulomycin A- and B-H₂S adducts were combined and this solution was extracted twice with methylene chloride. The methylene chloride extracts were concentrated to dryness to yield material which was used for further purification as described below.

A total of 12 runs were made, using the conditions described above, and preparations D and F as starting materials. The final preparations containing all "polar paulomycins" were kept as preparation G, 108.5 g.

4. Separation of "Polar Paulomycins." HPLC Chromatography Instrument: Waters Prep 500A

Support: Waters C-18 Reverse Phase Silica Packed Columns Starting Material: Preparation G, 10.0 g Mobile Phase : Acetonitrile - pH 5.5, 0.01 M phosphate buffer (45:55 v/v) The starting material was dissolved in 400 ml of acetonitrile - pH 5.5, 0.01 M phosphate buffer and the solution was Introduced into the column. The chromatography was followed by UV and/or refractive Index detectors and by TLC using the system described above. Three fractions were collected.

Fraction 1 contained materials with retention times close to those of paulomycin C and was designated "C-Fraction."

Fraction 2 contained materials with retention times close to those of paulomycin D and was designated "D-Fraction."

Fraction 3 contained materials with retention times close to those of paulomycin E and was designated "E-Fraction."

A total of 10 runs were made using preparation G as the starting material. Similar fractions from all runs were combined and extracted twice with methylene chloride. The combined methylene chloride extracts from the C-Fraction yielded preparation H, 13.5 g, by concentration to dryness. The methylene chloride extracts from the D-Fractions yielded preparation I, 38.1 g. Finally, the methyl ene chloride extracts from the E-Fractions yielded preparation J, 28.8, g.

HPLC analysis of preparation I, obtained from the D- Fraction indicated the presence of the paulomycin A-H₂S adduct among other paulomycin-related materials. Similarly, analysis of preparation J, obtained from the E-Fraction indicated the presence of the paulomycin B-H₂S adduct in this material. These two preparations were purified further as indicated below:

5. Isolation of the Paulomycin A-H₂S Adduct a. Purification of Preparation I obtained from the D- Fraction. HPLC Chromatography. Instrument: Waters Prep 500A

Support: Waters C-18 Reverse Phase Silica Packed Columns Starting Material: Preparation I, 13.0 g

Mobile Phase: Acetonitrile - 0.01 M pH 5.5 Phosphate

Buffer (40:60 v/v)

Preparation I was dissolved in ca. 400 ml of the mobile phase and the solution was introduced into the column. The chromatography was followed by UV and/or refractive index detectors.

Three fractions were collected. Fractions 1 and 3 did not contain the paulomycin A-H₂S adduct and were not purified any further.

Fraction 2, which contained the paulomycin A-H₂S adduct was extracted twice with methylene chloride. The methylene chloride extract was concentrated to dryness to yield a residue which was purified further as shown below.

A total of three runs were made under conditions identical to those described above. Similar fractions were treated as described above. Preparation K, 28.1 g, obtained by combining the residues from the methylene chloride extracts of Fraction 2 from each of the three runs was used as the starting material in the chromatography described below. b. Isolation of Pure Paulomycin A-H₂S Adduct. HPLC Chromatography. Instrument: Waters Prep 500A

Support: Waters C-18 Reverse Phase Silica Packed Columns Starting Material: Preparation K, 2.0 g

Mobile Phase: Acetonitrile - pH 5.5, 0.1 M phosphate buffer (40:60 v/v) Flow Rate: 200 ml/minute

The starting material was dissolved in 12 ml of acetonitrile and 12 ml of water and injected into the column. The chromatography was followed by UV detector (at 254 and 280 nm) and by analysis of selected fractions, by analytical HPLC. Fractions containing the paulomycin A-H₂S adduct only were combined. The combined solution was extracted once with 15% of its volume of methylene chloride. The extract was dried over anhydrous sodium sulfate and the dried solution was concentrated in vacuo to a residue. The residue (preparation L) was dissolved In 7 ml of acetone and this solution was mixed with 400 ml of cyclohexane-ether (325:75 v/v). The mixture was stirred for 10 minutes. The precipitated pure (by HPLC) paulomycin A-H₂S adduct was isolated by filtration and dried (preparation M, 1.6 g).

Characterization of this material follows in the characterization section.

6. Isolation of the Paulomycin B-H₂S Adduct a. Purification of Preparation J Obtained from the E- Fraction (see F-4 above). HPLC Chromatography. Instrument: Waters Prep 500A

Support: Waters C-18 Reverse Phase Silica Packed Columns Starting Material: Preparation J, 14.4 g

Mobile Phase: Acetonitrile - pH 5.5, 0.01 M Phosphate

Buffer (45:55 v/v)

Preparation J was dissolved in ca. 400 ml of the mobile phase and the solution was introduced Into the column. The chromatography was followed by UV and/or refractive index detectors.

Fractions containing material with retention times similar to those of paulomycin E were combined.

A total of two runs were made using the above starting material and conditions identical to those described above. The "paulomycin E-like" fractions were combined and the solution was extracted twice with methylene chloride. The extract was dried over sodium sulfate and concentrated to dryness in vacuo to yield preparation N, 22.7 g. Purification of this material which contained the paulomycin B-H₂S adduct which was continued as described below. b. Purification of "Paulomycin E-Like" Material. HPLC Chromatography.

Instrument: Waters Prep 500A Support: Waters C-18 Reverse Phase Silica Packed

Columns Starting Material: Preparation N, 22.7 g Mobile Phase: Acetonitrile - pH 5.5, 0.01 M Phosphate Buffer (40:60 v/v) Preparation N was dissolved in 400 ml of the mobile phase and the solution was introduced into the column. The chromato graphy was followed by UV and/or refractive Index detectors. Fractions containing paulomycin E and the paulomycin B-H₂S adduct were combined and this solution was extracted with methylene chlor ide. The extract was concentrated to dryness to yield preparation 0, 15.7 g. Preparation 0 was purified once more as described below. c. Isolation of Purified Paulomycin B-H₂S Adduct. HPLC Chromatography.

Instrument: Waters Prep 500A Support: Waters C-18 Reverse Phase Silica Packed

Columns Starting Material: Preparation 0, 15.7 g Mobile Phase: Gradient from acetonitrile -0.02 M pH 7.0 ammonium acetate (30:70 v/v) to acetonitrile -0.02 M pH 7.0 ammonium acetate (70:30 v/v). Preparation 0 was dissolved in 400 ml of acetonitrilebuffer (30:70 v/v) and this solution was introduced into the column. The chromatography was followed by UV and/or refractive index detectors.

Fractions containing only paulomycin E and paulomycin B-H₂S adduct were combined, dried over sodium sulfate and concentrated to dryness to yield preparation P, 8.9 g, which was purified as described below. d. Isolation of Pure paulomycin B-H₂S adduct. HPLC Chromatography.

Instrument: Waters Prep 500A Support: Waters C-18 Reversed Phase Silica Packed

Columns Starting Material: Preparation P, 2.0 g Mobile Phase: Gradient from Solvent A (100%) to Solvent B (100%) within 1 hours. Solvent A: Acetonitrile - 0.05 M

Phosphate Buffer, pH 5.5 (35:65 v/v) Solvent B: Acetonitrile - Phosphate Buffer, pH 5.5 (50:50 v/v)

Flow Rate: 100 ml/minute

Preparation P was dissolved in 50 ml of acetonitrile-water (1:1 v/v) and this solution was introduced Into the column. The chromatography was followed by UV detection at 254 and 320 nm. Fractions containing (by analytical HPLC) only paulomycin B-H₂S adduct were combined. The combined solution was extracted with 50% of its volume of methylene chloride. The extract was dried over sodium sulfate and then was concentrated to dryness to yield preparation Q which contained only paulomycin B-H₂S adduct when examined by analytical HPLC.

In another series of experiments, 2 g of preparation P was purified by HPLC using an isocratic mobile phase consisting of acetonitrile, 0.05 M pH 5.5 phosphate buffer (40:60 v/v). However, no separation between the paulomycin B-H₂S adduct and paulomycin E was obtained. Fractions containing paulomycin E and/or the paulomycin B-H₂S adduct were combined and extracted with methylene chloride. The extract gave, by concentration to dryness, preparation R, 1.13 g, which was purified by HPLC as described above with the exception that the mobile phase consisted of acetonitrile - 0.05 M pH 5.5 phosphate buffer (35:65 v/v). Fractions containing only paulomycin B-H₂S adduct were combined and extracted with methylene chloride. The extract was dried over sodium sulfate and concentrated to dryness. This residue, which contained, by analytical HPLC, only paulomvcin B- H₂S adduct was combined with preparation Q. The conbined preparation was dissolved in acetone, ca. 5 ml, and this solution was poured into 400 ml of a mixture of cyclohexane-ether (3:1 v/v). The precipitated material, which contained pure paulomycin B-H₂S adduct, was kept as preparation S. Characterization of paulomycin B-H₂S adduct (prepara tion S) is described in the characterization section.

CHARACTERIZATION 1. Characterization of Paulomycin A-H₂S Adduct a. Appearance: Colorless amorphous material b. Solubility: Soluble in lower alcohols, ketones, ethyl acetate, chloroform, methylene chloride; less soluble in ether; insoluble in saturat ed hydrocarbon solvents. c. Molecular Composition: C₃₄H₄₈N₂O₁₇S₂ Calcd. Mol. Weight: 820.2394

Found (HR-FAB/MS): 820.2385 d. Analytical Data: Calcd for C₃₄H₄₈N₂O₁₇S₂ : C, 49.75; H,

5.85; N, 3.41; S, 7.81; 0, 33.17 e. [α]_D ²⁵: -12° (C, 1.04, methanol) +5° (C, 0.74, chloroform) f. IR Spectrum: Tabulation of the IR bands follows. g. UV Spectrum: The tabulated UV spectrum of the paulomycin

A-H₂S adduct follows : λ_max(nm) a ε 245 14.08 11,545

278 15.10 12,382

322 8.92 7,314 h. C-13 Nuclear Magnetic Resonance Spectrum

A list of the absorptions in the ¹³C NMR spectrum of the paulomycin A-H₂S adduct in dg-acetone is presented in Table 1. i. Mass Spectrum: Tabulation of ions observed in the FAB/mass spectrum of the paulomycin A-H₂S .adduct follows: Most Intense Ions:

820 (9999) 58 (9638) 195 (8674) 176 (8026) 196 (7061) 183 (5678) 821 (4715) 184 (4072) 151 (3311) 101 (3143) j. Antimicrobial In Vitro Testing: The bioactivity of paulomycin A-H₂S adduct is shown in Table 2. 2. Characterization of Paulomycin B-H₂S Adduct a. Appearance: Colorless amorphous material b. Solubility: Soluble in lower alcohols, ketones, ethyl acetate, chloroform, methylene chloride; less soluble in ether; insoluble in saturated hydrocarbon solvents. c. Molecular Composition: C₃₃H₄₆N₂O₁₇S₂ Calcd Mol. Weight: 806.2238

Found (HR-FAB/MS): 806.2231 d. Analytical Data: Calcd for C₃₃H₄₆N₂O₁₇S₂: C, 49.13; H, 5.71; N, 3.47; S, 7.94; O, 33.75 e. [α]_D ²⁵: -22° (C, 0.695, methanol) +6° (C, 0.862, chloroform) f. IR Spectrum: Tabulation of the IR bands follows. g. UV Spectrum: The tabulated UV spectrum of the paulomycin B-H₂S adduct follows: λ_max(nm) a ε

244 17.05 13,744 278 17.15 13,827

322 10.45 8,424 h. C-13 Nuclear Magnetic Resonance Spectrum: A list of the absorptions in the ¹³C NMR spectrum of the paulomycin B-H₂S adduct is presented in Table 3. i. Mass Spectrum: Tabulation of ions observed In the FAB/Mass spectrum of paulomycin B-H₂S adduct follows: Most Intense Ions:

806 (9999) 58 (7161) 195 (6860) 176 (6138) 196 (5453)

183 (4273) 807 (4221) 184 (3111) 151 (2350) 808 (2036) j. Antimicrobial In Vitro Testing: The bioactivity of paulomycin B-H₂S adduct is shown in Table 4. The Structure of the paulomycin A and B-H₂S Adducts

The structure of paulomycins A and B is shown in Chart A.

Consideration of the mass spectra of paulomycins A, B and the paulomycin A and B-H₂S adducts and comparison of the proton and carbon-13 nuclear magnetic resonance spectra indicated that the structure of the paulomycin A and B-H₂S adducts Is as set forth in

Chart A.

Inspection of Chart A indicates that the paulomycin A- and B-H₂S adducts are produced in fermentations of Streptomyces paulus by an addition of one molecule of hydrogen sulfide to paulomycins A and B respectively. The addition of H₂S results in the formation of two additional asymmetric centers at "C-2" and "C-3" of the paulic acid of the paulomycin A or B molecule.

TABLE 1

Absorptions Observed in the ¹³C NMR^a Spectrum, of the

Paulomycin A H₂S Adduct

Chemical Shift Multiplicity_b Assignment

(δ)

200.41 S-C-N

S

198 .81 S C-4

188 .82 S C-7

175.81 S C-1'''

171 .08 S C-1''

169 .89 S S-1''''

169 .10 S C-1

159 .76 S C-3

100.42 S C-2

99 .74 D C-1'

78 .72 S C-6

78 .51 D C-8

76, .58 D C-10

74.89 D C-3'

74. .03 S C-4'

72. ,74 D C-12

71. 06 D C-11

70. 82 D C-7'

70.49 D C-9

69. 77 D C-2''

68. 05 D C-5'

62. 80 T C-13

57. 10 Q -OC_H3

48.47 T C-5

48. 27 D C-3''

41. 98 D C-2'''

31. 18 T C-2' 26 . 91 T C-3''' 22.41 Q C-4'' 20. 60 Q C-2'''' 17.29 Q C-4'" 15 .94 Q

C-6' or C-8'

15.76 Q

11.92 Q C-5''' ad₆-acetone was used as solvent. bMultiplicity observed in the off-resonance spectrum. cFor carbon assignments, see structure of the paulomycin A-H₂S adduct in Chart B.

TABLE 2 Antimicrobial In Vitro Testing of the Paulomycin A-H₂S Adduct Minimum Inhibitory Concentration - MCG per M1

Paulomycin A Organism Name UC^® No. H₂S Adduct Paulomycin A pH 6.0 pH 6.0

Staphylococcus Aureus 6675 16 0.125 Staphylococcus Aureus 9218 8 0.125

Staphylococcus Aureus 3665 16 0.125

Staphylococcus Aureus 6685 16 0.125

Staphylococcus Aureus 9213 16 0.125

Staphylococcus Epidermidis 30031* 8 0.125 Streptococcus Pneumoniae 41 4 0.125

Streptococcus Pyogenes 152 2 0.06

Streptococcus Faecalis 694 64 0.5

Streptococcus Faecalis 9217 32 0.25

Escherichia Coll 9451 >64 >64 Klebsiella Pneumoniae 58 >64 >64

Pseudomonas Aeruginosa 9191 >64 >64

Salmonella Schottmuellerl 126 >64 >64

Proteus Vulgaris 9679 >64 64

*Non-UC^® culture Band Tabulation of the Infrared Spectrum of the Paulomycin A-H₂S Adduct

Band Band

Freq. Inten. Type Freq. Inten Type

3477.6 51 SH 1192.0 17 AVG

3409.1 42 SH 1137.0 15 AVG

3354.2 39 BRD 1118.7 15 AVG

3270.2 37 BRD 1097.4 18 AVG

3233.6 37 BRD 1053.1 14 AVG

3101.5 67 SH 1027.0 17 AVG

2920.2 0 BRD M 997.1 19 AVG

2870.0 4 AVG M 953.7 61 SH

2853.6 1 AVG M 937.4 60 SH

2723.4 75 BRD M 932.5 60 AVG

2660.7 77 AVG 910.3 45 AVG

2616.4 81 SH 893.0 54 AVG

2045.5 88 BRD 867.0 61 AVG

1995.3 92 SH 835.1 67 AVG

1735.9 5 AVG 815.8 57 SHP

1700.2 18 AVG 784.0 67 AVG

1625.9 32 AVG 748.3 59 SH

1576.8 20 AVG 731.0 53 SH

1458.1 4 AVG M 726.1 53 SH M

1377.1 10 AVG M 690.5 51 AVG

1340.5 34 AVG 665.4 44 SH

1297.1 18 AVG 637.4 39 AVG

1259.5 17 SH 603.7 33 AVG

1244.0 13 AVG

Band Freq. : Band frequencies in wavenumbers (cm-1)

Inten.: Intensity in percent transmittance (%T)

Data type in local peak region: BRD = Broad I AVG = Average

SHP = Sharp SH = Shoulder

M: Possible interference from mineral oil 25 Strongest Peaks

%T Freq.

0 2920.1

1 2853.5

4 2870 . 0 4 1458.0

5 1735.8

10 1377.0

13 1244.0

14 1053.0

15 1137.0

15 1118.6

17 1259.5

17 1192.0

17 1027.0

18 1700.1

18 1297.0

18 1097.3

19 997.0

20 1576.7

32 1625.8

33 603.6

34 1340.5

37 3270.1

37 3233.5

39 3354.1

39 637.3

Prep: Mineral Oil Mull Max %T: 94 @ 1927.8 %T at 3800 (CM-1): 89 Density (CM-1/Pt) : 0.964

TABLE 3 Absorptions Observed in the ¹³C NMR^a Spectrum of the Paulomycin B-H₂S Adduct Chemical Shift Multiplicity_b Assignment^c (δ ) 200.27 S -S-C-N-

S

198.74 S C-4 188.81 S C-7 176.30 S C-1'" 170.92 S C-1'' 169.80 S C-1'''' 169.69 S C-1 159.71 S C-3 100.33 S C-2 99.65 D C-1' 78.63 S C-6 78.54 D C-8 76.27 D C-10 74.85 D C-3' 74.06 S C-4' 72.58 D C-12 71.04 D C-11 70.63 D C-7' 70.46 D C-9 69.67 D C-2'' 67.88 D C-5' 62.71 T C-13 56.95 Q OCH₃ 48.32 T C-5 48.14 D C-3'' 34.54 D C-2''' 31.06 T C-2' 22.39 Q C-4'' 20.45 Q C-2'''' 19.35 Q

C-3''' OR C-4'''

19.20 Q 15.90 Q

15.63 Q C-6' OR C-8' ^ad₆-Acetone was used as solvent. bMultiplicity observed in the off-resoannce spectrum. cFor carbon assignments, see structure of the paulomycin B-H₂S adduct in Chart B. TABLE 4 Antimicrobial In Vitro Testing of the Paulomycin B-H₂S Adduct Minimum Inhibitory Concentration - MCG per M1 Paulomycin B

Organism Name UC^® No. H₂S Adduct Paulomycin A pH 6.0 pH 6.0 Staphylococcus Aureus 9218 16 0.25 Staphylococcus Aureus 3665 32 0.25 Staphylococcus Aureus 6685 16 0.25 Staphylococcus Aureus 9218 32 0.5 Staphylococcus Epidermidis 30031* 8 0.125 Staphylococcus Pneumoniae 41 4 0.125 Streptococcus Pyogenes 152 2 0.125 CItrobacter Freundij 3507 >128 >128 Enterobacter Cloacae 9381 >128 >128 Enterobacter Cloacae 9382 >128 >128 Eschericia Coli 9379 >128 >128 Eschericia Coli 9380 >128 >128 Eschericia Coli 9451 >128 >128

Klebsiella Oxytoca 9383 >128 >128 Klebsiella Oxytoca 9384 >128 >128 Klebsiella Pneumoniae 58 >128 >128 Proteus Vulgaris 9679 >128 64 Sebratia Marcescens 6886 >128 >128

Pseudomonas Aeruginosa 231 >128 >128 Pseudomonas Aeruginosa 9191 >128 >128 *Non-UC^® culture

Band Tabulation of the Infrared Spectrum of the Paulomycin B-H₂S Adduct

Band Band

Freq. Inten Type Freq. Inten. Type

3609.7 83 SH 1244.0 22 AVG

3475.7 60 SH 1195.8 27 AVG 3359.9 49 BRD 1157.2 35 AVG

3272.2 49 BRD 1136.0 24 AVG

3235.5 49 BRD 1117.7 23 AVG

2955 . 9 0 AVG M 1099.4 25 AVG 2931.7 0 BRD M 1053.1 22 AVG

2868.1 2 SH M 1027.0 26 AVG

2856.5 1 AVG M 999.1 28 AVG

2727.3 77 AVG M 929.6 62 AVG

2670.4 80 BRD M 910.3 53 SHP

2040.6 87 BRD 893.0 60 AVG

2000.1 91 SH 854.4 66 AVG

1735.9 10 AVG 840.9 69 SH

1700.2 26 AVG 815.8 61 SHP

1625.0 41 AVG 780.2 70 SH

1577.7 29 AVG 748.3 61 SH

1461.0 5 AVG M 722.3 53 AVG M

1377.1 13 AVG M 689.5 54 AVG

1342.4 40 AVG 659.6 46 SH

1298.0 26 AVG 634.5 41 SH

Band Freq.: Band frequencies ; in wavenumbers (cm-1)

Inten.: Intensity in percent : transmittance (%T)

Data type in local peak region: BRD = Broad AVG = Average

SHP = Sharp SH = Shoulder

M: Possible interference from mineral oil

25 Strongest Peaks

%T Freq.

0 2955.8

0 2931.6

1 2856.5

2 2868.0

5 1461.0

10 1735.8

13 1377.0

22 1244.0

22 1053.0

23 1117 . 6

24 1136.0

25 1099.3

26 1700.1

26 1298.0

26 1027.0

27 1195.7 28 999.0

29 1577.6

35 1157.1

40 1342.3

41 1625.0

41 634.5

46 659.5

49 3359.8

49 3272.1 Prep: Mineral Oil Mull

Max %T: 94 @ 1938.4

%T at 3800 (CM-1): 92

Density (CM-1/Pt) : 0.964

Example 2 Production of Paulomycin-H₂S Adducts by Chemical Synthesis

This example describes the production of the paulomycin A-H₂S adduct by a chemical reaction between paulomycin A and H₂S. The procedures used for the production of paulomycin A-H₂S adduct can be used with other paulomycins, e.g., paulomycin A₂, B, C, D, E, F, O-demethylpaulomycin A and O-demethylpaulomycin B to produce the corresponding adducts shown in Chart B. As set forth in Chart B, in all cases R₁ is the same. The compounds vary in R₂ and R₃.

A. Assay and Testing Procedures See Example 1. B. Thin-Layer Chromatographic Procedures See Example 1.

C. Spectroscopic Methods See Example 1.

D. Analytical-High Performance Liquid Chromatography (HPLC) See Example 1.

Preparation of Paulomycin A-H₂S Adduct

A solution of 0.2 g of paulomycin A in 20 ml of absolute methanol was kept under stirring. A stream of nitrogen and hydrogen sulfide was bubbled into the solution. The reaction was monitored by HPLC. All paulomycin was transformed to paulomycin A-H₂S adduct within one hour at which time the solution was concentrated to dryness. The residue was purified by precipitation from acetonecyclohexane to give preparation A which was characterized described below.

Characterization of Synthetically Prepared Paulomycin A-H₂S Adduct A. Appearance: Colorless amorphous material B. Solubility: Soluble in lower alcohols, ketones, ethyl acetate, chloroform, methylene chloride; less soluble in ether; insoluble in saturated hydro carbon solvents.

C. Molecular Composition: C₃₄H₄₈N₂O₁₇S₂ Calcd. Mol. Weight: 820.2394

Found (HR-FAB/MS): 820.2381

D. Analytical Data: Calcd for C₃₄H4₈N₂O_17S2 : C, 49.75; H, 5.85;

N, 3.41; S, 7.81; O, 33.17

E. [α]_D ²⁵: -24° (C, 0.31, methanol) +3° (C, 0.51, chloroform)

F. IR Spectrum: Tabulation of the IR bands follows

G. UV Spectrum: The UV spectrum of this paulomycin A-H₂S adduct follows: λ_max(nm) δ ε 246 14.51 11,900

277 13.96 11,450

322 8.92 7,314

H. C-13 Nuclear Magnetic Resonance Spectrum

A list of absorptions is presented in Table 5. I. Mass Spectrum: Fast atom bombardment (FAB) spectrum follows:

Most Intense Ions:

198 (9999) 196 (6137) 183 (4272) 58 (3062) 823 (3509)

178 (3549) 151 (3493) 104 (2765) 101 (2279) 138 (2108)

TABLE 5 Absorptions Observed in the ¹³C NMR^a Spectrum of

Synthetically-Produced Paulomycin A-H₂S Adduct

Chemical Shift Multiplicity_b Assignment

(δ)

200.52 S S-C-N

S

198.98 S C-4 189.15 S C-7 175.96 S c-1'''

171. 17 S C-1''

171. 01 S C-1''''

169. 23 S C-1

159. 87 S C-3

100. 55 S C-2

99. 91 D C-1'

78. 75 S C-6

77. 12 D C-8

76. 66 D C-10

75 .06 D C-3'

74.18 S C-4'

72.89 D C-12

71. .23 D C-11

70 .99 D C-7'

70 .65 D C-9

69 .91 D C-2''

68 .19 D C-5'

62 .94 T C-13

57 .25 Q -OCH₃

48 .55 T C-5

48 .38 D C-3''

42 .15 D C-2'''

31 .31 T C-2'

27 .29 T C-3'''

22 .63 Q C-4''

20 .71 Q C-2''''

17 .43 Q C-4'''

16 .07 Q

C-6' or C-8'

15 .91 Q

12 .02 Q C-5''' ad₆-acetone was used as solvent. bMultiplicity observed in the off-resonance spectrum. cFor carbon assignments, see the structure of the paulomycin A-H₂S adduct in Chart B. Band Tabulation of the Infrared Spectrum

Band Band

Freq. Inten. Type Freq. Inten. Type

3468.9 53 SH 1259.5 34 SH

3361.9 47 BRD 1244.0 30 AVG

3270.2 46 BRD 1192.9 37 AVG

3234.6 47 BRD 1137.9 31 AVG

3103.4 57 BRD 1117.7 31 AVG

2963.6 0 BRD M 1098.4 33 AVG

2857.5 0 BRD M 1054.0 33 AVG

2724.4 68 BRD M 1027.0 34 AVG

2672.3 70 BRD M 998.1 39 AVG

2633.7 73 SH 933.5 65 AVG

1736.8 18 AVG 910.3 58 AVG

1702.1 37 AVG 893.0 64 AVG

1625.0 48 AVG 867.9 69 AVG

1577.7 38 AVG 857.3 70 SH

1462.0 2 AVG M 836.1 73 SH

1377.1 8 AVG M 815.8 67 SHP

1368.4 22 SH M 780.2 73 SH

1342.4 41 AVG 768.6 71 SH

1298.0 32 AVG 722.3 53 AVG M

Band Freq.: Band frequencies in wavenumbers (cm-1) Inten.: Intensity in percent transmittance (%T) Data type in local peak region: BRD = Broad AVG = Average

SHP = Sharp SH = Shoulder M.: Possible interference from mineral oil

25 Strongest Peaks

%T Freq.

0 2963.5 0 2857.5 2 1462.0

1377.0

18 1736.7 22 1368.3 30 1244.0 31 1137.8 31 1117.6 32 1298.0

33 1098.3

33 1054.0

34 1259.5

34 1027.0

37 1702.0

37 1192.8

38 1577.6

39 998.0

41 1342.3

46 3270.1

47 3361.8

47 3234.5

48 1625.0

53 3468.8

53 722.2

Prep: Mineral Oil Mull Max %T: 94 @ 1974.1 %T at 3800 (CM-1): 71 Density (CM-1/Pt) : 0.964

The antibacterially-active compounds of the present invention can be used for the same antibacterial purposes as paulomycins A and B. For example, the compounds of the invention can be used alone or in combination with other antibiotic agents to prevent the growth of, or reduce the number of the above described susceptible bacteria in many environments. For example, they can be used as disinfectants on various dental and medical equipment contaminated with Staphylococcus aureus. Further, they are useful in wash solutions for sanitation purposes, as in the washing of hands and in the cleaning of equipment, floors, or furnishings or contaminated rooms or laboratories; they are also useful as an industrial preservative, for example, as a bacterlostatic rinse for laundered clothes and for impregnating papers and fabrics; and they are useful for suppressing the growth of sensitive organisms in plate assays and other microbiological media. They also can be used as feed supplements to promote the growth of animals, for example, mammals, birds, fish, and reptiles.

The compounds of the subject invention are useful as antibacterial agents in suitable compositions. These compositions are preferably presented for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the active compound in the form of the free base, or its pharmacologically acceptable salts.

The tribasic salts are particularly desirable compounds because of their long term stability.

For oral administration, either solid or fluid unit dosage forms can be prepared. For preparing solid compositions such as tablets, the principal active ingredient is mixed with conventional ingredients such as talc, magnesium, stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, acacia, methylcellulose, and functionally similar materials as pharmaceutical diluents or carriers. The tablets can be laminated or otherwise compounded to provide a dosage form affording the advantage of prolonged or delayed action or predetermined successive action of the enclosed medication. For example, the tablet can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist distintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids or mixture of polymeric acids with such materials as shellac, cetyl alcohol, cellulose acetate phthalate, styrene maleic acid copolymer and the like. Alternatively, the two component system can be utilized for preparing tablets containing two or more incompatible active ingredients. Wafers are prepared in the same manner as tablets, differing only in shape and the inclusion of sucrose or other sweetener and flavor. In their simplest embodiment, capsules, like tablets, are prepared by mixing the compound of the formulation with an inert pharmaceutical diluent and filling the mixture into a hard capsule of appropriate size. In another embodiment, capsules are prepared by filling hard gelatin capsules with polymeric acid coated beads containing the active compound. Soft gelatin capsules are prepared by machine encapsulation of a slurry of the active compound with an acceptable vegetable oil, light liquid petrolatum or other inert oil.

Fluid unit dosage forms for oral administration such as syrups, elixirs, and suspensions can be prepared. The water-soluble forms of the active compound can be dissolved in an aqueous vehicle together with sugar, aromatic flavoring agents and preservatives to form a syrup. An elixir is prepared by using a hydro-alcoholic (ethanol) vehicle with suitable sweeteners such as sucrose together with an aromatic flavoring agent. Suspensions can be prepared of the insoluble forms with a syrup vehicle with the aid of a suspending agent such as acacia, tragacanth, methylcellulose and the like.

Topical ointments can be prepared by dispersing the active compound in a suitable ointment base such as petrolatum, lanolin, polyethylene glycols, mixtures thereof, and the like. Advantageously, the compound is finely divided by means of a colloid mill utilizing light liquid petrolatum as a levitating agent prior to dispersing in the ointment base. Topical creams and lotions are prepared by dispersing the compound in the oil phase prior to the emulsification of the oil phase in water.

For parenteral administration, fluid unit dosage forms are prepared utilizing the active compound and a sterile vehicle, water being preferred. The active compound, depending on the form and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions, a water-soluble form of the active compound can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampule and sealing. Advantageously adjuvants such as a local anesthetic, preservative and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection is supplied to reconstitute the powder prior to use. Parenteral suspensions are prepared in substantially the same manner except that the active compound is suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration. The active compound can be sterilized by exposure to ethylene oxide before suspending the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active compound. The term unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for human subjects and animals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical diluent, carrier or vehicle. The specifications for the novel unit dosage forms of this invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for therapeutic use in humans and animals, as disclosed in detail in this specification, these being features of the present invention. Examples of suitable unit dosage forms in accord with this invention are tablets, capsules, pills, troches, suppositories, powder packets, granules, wafers, cachets, teaspoonfuls, tablespoonfuls, dropperfuls, ampuls, vials, segregated multiples of any of the foregoing, and other forms as herein described.

In addition to the administration of the active compound as the principal active ingredient of compositions for the treatment of the conditions described herein, the said compound can be included with other types of compounds to obtain advantageous combinations of properties. Such combinations include the active compound with antibiotics such as spectinomycins, chloramphenicol, novobiocin, dihydronovobiocin, tetracyclines (e.g., tetracycline, oxytetracycline and chlortetracycline), penicillins, erythromycin, kanamycin, streptomycin, neomycin, polymyxin, bacitracin, nystatin, filipin, fumagillin and endomycin to broaden the bacterial spectrum of the composition and for synergistic action against particular bacteria; steroids having anti-inflammatory activity such as hydrocortisone, prednisolone, 6α-methylprednisolone, 6α-fluoroprednisolone and the like; analgesics such as aspirin, sodium salicylate (acetylsalicyclic acid)-anhydride, N-acetyl-p-aminophenyl and salicylamide; antihistamines, such as chlorpheniramine maleate, diphenylhydramine, promethazine, pyrathiazine, and the like; sulfas, such as sulf adiazine, sulfamethazine, sulfamerazine sulfacetamide, sulfadimethyloxazole, sulfamethizole, and the like; antifungals, such as undecylenic acid, sodium propionate, salicylanilide, sodium caprylate, and hexetidine; and the vitamins. The dosage of the active compound for treatment depends on route of administration; the age, weight, and condition of the patient; and the particular disease to be treated. A dosage schedule of from about 15 to 500 mg., 1 to 4 times daily (every six hours), embraces the effective range for the tratment of most conditions for which the compositions are effective. For children, the dosage is calculated on the basis of 15 to 30 mg/kg/day to be administered every six hours. The selection of suitable patients and dosages for the treatment of humans and animals with the compounds of this invention is readily undertaken by an ordinarily skilled physician or veterinarian.

The active compound is compounded with a suitable pharmaceutical carrier in unit dosage form for convenient and effective administration. In the preferred embodiments of this invention, the dosage units contain the compound in: 15, 30, 50, 125, 250, and 500 mg amounts for systemic treatment; in 0.25, 0.5, 1, 2 and 5% amounts for topical or localized treatment; and 5 to 65% w/v for parenteral treatment. The dosage of compositions containing the active compound and one or more other active ingredients is to be determined with reference to the usual dosage of each such ingredient.

CHART A The structures of paulomycins A, B, A₂, C, D, E, F, O-demethyl paulomycins A and B and the paulomycin A- and B-H₂S adducts are represented by Formula I as follows:

wherein R₁ is

wherein R₄ is hydrogen; wherein R₆ is CH₃; wherein R is :

1. Paulomycin A: CH₃-CH₂CH(CH₃) -COOCH(CH₃) - 2. Paulomycin B: CH₃CH(CH₃)-COOCH(CH₃)- 3. Paulomycin A₂: (CH₃)₂CHCH₂COOCH(CH₃)- 4. Paulomycin C: CH₃CH₂-COOCH(CH₃)- 5. Paulomycin D: CH₃COOCH(CH₃)- 6. Paulomycin E: CH₃C(=O)- 7. Paulomycin F: CH₃CH(OH)- wherein R6 is H;

8. O-Demethyl paulomycin A: (otherwise same as paulomycin A) 9. O-Demethyl paulomycin B: (otherwise same as paulomycin B) wherein R₁ is

wherein R₆ is CH₃; wherein R is : 10. Paulomycin A-H₂S Adduct: CH₃-CH₂CH(CH₃)-COOCH(CH₃)- 11. Paulomycin B-H₂S Adduct: CH₃CH(CH₃)-COOCH(CH₃)-

Claims

CLAIMS 1. A compound of the formula II:

or a pharmacologically acceptable salt thereof;

I. wherein R₁ is OCH₃ and R₂ is

A. CH₃-CH₂CH(CH₃)-COOCH(CH₃)-,

B . CH₃CH(CH₃)-COOCH(CH₃)-,

C . (CH₃)₂CHCH₂COOCH(CH₃)-,

D . CH₃CH₂-COOCH(CH₃)-,

E. CH₃COOCH(CH₃)-,

F. CH₃C(=O)-, or

G. CH₃CH(OH)-; or

II. wherein R₁ is H and R₂ is

A. CH₃-CH₂CH(CH₃)-COOCH(CH₃)-, or

B. CH₃CH(CH₃)-COOCH(CH₃)-; or a pharmacologically acceptable salt thereof.

2. A compound of claim 1 I. wherein R₁ is OCH₃ and R₂ is

A. CH₃-CH₂CH(CH₃)-COOCH(CH₃)-, or

B. CH₃CH(CH₃)-COOCH(CH₃)-; or a pharmacologically acceptable salt thereof .

3 . A compound of Claim 2 wherein R₂ is CH₃-CH₂CH(CH₃) -COOCH(CH₃) - .

4. A compound of Claim 2 wherein R₂ is CH₃CH(CH₃) -COOCH(CH₃) -.