PAULOMYCIN RELATED COMPOUNDS BACKGROUND OF THE INVENTION

The present invention relates to novel compositions of matter, and novel synthetic methods. The present invention particularly provides new paulomycin C and O-demethylpaulomycins A and B, antibac¬ terially active paulomycin related compounds, which can be considered derivatives of paulomycins, including: esters and salts thereof. Also provided are methods of preparing these compounds.

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. These derivatives of paulomycins, except for the esters, are, advantageously, more soluble in aqueous solutions than paulomycin thereby facilitating the formulation of the antibiotic. Paulomycin is relatively insoluble in water. 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 273a ₁ α and 273a _χJ 8 are disclosed in United States patent 4,505,895. Novel methods of preparing antibiotics and derivatives thereof are disclosed in United States patent 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. Paulomy¬ cin 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-demethylpaulomycin 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 degrada¬ tion products thereof. SUMMARY OF THE INVENTION

The present invention particularly provides an antibacterially active compound of the Formula I, or a pharmacologically acceptable salt thereof; wherein R is

(a) R'-CH ₂ CH(CH ₃ )-CO0CH(CH ₃ )-, or (b) CH ₃ CH ₂ -COOCH(CH ₃ )-; wherein R' is hydrogen or methyl; wherein R _x is selected from formulas 1-4; wherein X _x is

(a) hydrogen,

(b) C _x to C ₁₂ alkyl, branched or straight chain, or (c) a pharmacologically acceptable cation; wherein R ₂ and R _g are the same or different and are selected from the group

(a) -CH ₂ C00X ₂ ,

(b) formulas 5-8, (c) -CH ₂ CH(NHR ₅ )C00X ₂ ,

(d) -CH ₂ CH ₂ CH(NHR ₅ )C00X ₂ ,

(e) -CH ₂ CH0HCH ₂ 0H,

(f) -CH ₂ [CH0H] _n CH ₂ 0H,

(g) -CH ₂ CH ₂ 0H, and (h) -CH ₂ CH(NHC0CH ₃ )C00X ₁ ; wherein n is 3 or 4; and wherein R _A is hydrogen or a pharmacologically acceptable cation; wherein R ₅ is hydrogen or acetyl; wherein R ₆ is hydrogen or methyl; and wherein X ₂ is hydrogen or a pharmacologically acceptable cation; with the proviso that when R is R'-CH ₂ CH(CH ₃ )COOCH(CH ₃ )- , then R ₆ is hydrogen.

The carbon atom content of various hydrocarbon-containing moieties is indicated by a prefix designating the minimum and maximum number of carbon atoms in the moiety, i.e., the prefix (C^-C _j indicates a moiety of the integer "i" to the integer "j" carbon atoms, inclusive. Thus (C _j^ -C ₈ alkyl refers to alkyl of one to 3 carbon atoms, inclusive, or methyl, ethyl, propyl, and isopropyl.

Examples of alkyl of one to 12 carbon atoms, inclusive, are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, and isomeric forms thereof.

Pharmacologically acceptable cations within the scope of X _χ include pharmacologically acceptable metal cations, ammonium, amine cations, or quaternary 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 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, dimeth lamine, trimethylamine, ethylamine, dibutylamine, triisopropylamine, M-methylhexylamine, decylamine, dodecylamine, allylamine, crotylamine, cyclopentylamine, dicyclo- hexylamine, benzylamine, dibenzylamine, a-phenylethylamine, b- phenylethy1amine, ethylenediamine, diethylenetriamine, 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-ethylmor- pholine, 1-isopropylpyrrolidine, 2-methylpyrrolidine, 1,4-dimethyl- piperazine, 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-l,3-propanediol, 2-amino-2-methyl- 1-propanol, tris(hydroxymethyl)aminomethane, N-phenylethanolamine, N-(p-tert-amylphenyl)-diethanolamine, glactamine, 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, tetraethylammonium, benzyl- trimethyla monium, phenyltriethyla monium, and the like. Paulomycins A^. , C, ύ , E; antibiotics 273a ₂ , 273a ₂ , 273a ₂ 0, 273a _χ , 273a _χ α and 17 & _χ β and O-demethylpaulomycins A and B have been discovered 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 I. DETAILED DESCRIPTION OF THE INVENTION

The present invention is disclosed more fully in the following examples.

"Paulomycin[s]" as used herein refers generically to all paulomycin-related compounds. Fermentation Procedures

The fermentation procedure described in U.S. Patent 4,335,108, entitled "Paulomycins A and B and Preparation Thereof", except for some differences described below, may be used to prepare the paulo¬ mycins of the present invention. The disclosure of U.S. Patent 4,335,108 is hereby expressly incorporated by reference.

Many of the processes used for preparing synthetic derivatives of paulomycin C and O-demethylpaulomycins A and B, for example the bis- and monoadducts, are set forth in detail in European patent application Nos. 85302616.9 and 85301535.2 which are incorporated herein by reference and referred to hereafter as "the European applications". Assay and Testing Procedures

Antibiotic production and purification is measured by a micro¬ biological disc-plate assay procedure with Micrococcus luteus as the assay organism.

Thin-layer Chromatographic Procedures The reaction of paulomycin with N-acetyl-L-cysteine is followed by thin-layer chromatography on silica gel G using chloroform- ethanol-water (25:30:5 v/v) or on cellulose using pH 7.0 phosphate buffer as the solvent system. The antibiotics present in reaction mixtures in preparations obtained during purification are detected by bioautography on M. luteus--seeded trays. Spectroscopic Methods

Proton magnetic resonance spectra are recorded on a Varian XL- 200 spectrometer operating at 200 MHz. Solutions (ca. 0.4 ml, ca. 0.25 M) of the compounds in d ₆ - imethylsulfoxide or d ₆ -acetone are used. Carbon magnetic resonance (CMR) spectra are recorded on a Varian CFT-80 spectrometer operating at 20.0 MHz. Proton magnetic resonance (PMR) and CMR chemical shifts are reported as ppm relative to tetramethylsilane. Mass spectra are obtained on a ZAB-2F high

resolution mass spectrometer using a fast atom bombardment (FAB) source.

High-Performance Liquid Chromatography (HPLC)

HPLC is done on a Hewlett-Packard Model 1084B (Hewlett-Packard, Avondale, California) instrument equipped with an HP model 79875A variable wave length detector and operating in the dual pump mode. A Brownlee 10 cm x 4.6 mm stainless steel column packed with C ₁₈ (lOμ) reverse phase is used. Mobile phases are prepared using Burdick and Jackson distilled in glass solvents. All samples and aqueous phases are filtered through a 0.45 micron filter. The mobile phases used consists of acetonitrile - pH 5.5, 0.1 M phosphate buffer. Samples are prepared as 1 mg/ml solutions in the initial mobile phase. Injection volume is 50 μl .

The following examples are illustrative of the processes and products of the invention, but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. Example 1 Preparation of Antibiotic 273a _χ α

N-Acetyl-L-cysteine, 4.2 g, was dissolved in 100 ml of 0.1 M pH 7.85 phosphate buffer. The solution was adjusted to pH 8.7 with IN aqueous potassium hydroxide. Paulomycin A, 1.0 g, was added to this solution and dissolved under stirring. After standing at room temperature for 1 hour, the solution was adjusted to pH 3.0 with 2N aqueous hydrochloric acid and extracted four times with 100 ml portions of ethyl acetate. The ethyl acetate extracts were combined, dried over sodium sulfate and concentrated to dryness to give prep. - 144.1. Preparation - 144.1 was found highly active vs. M. luteus, ca. 300 bu/mg, and contained antibiotic 273a _j α as the only bioactive component. Prep. -144.1 was dissolved in 15 ml of acetone; this solution was added under stirring to 200 ml of ether. The precipitated material was isolated by filtration, dried on the filter, and redissolved in 15 ml of acetone. The new solution was also poured into 200 ml of ether under stirring. The precipitate formed was isolated by filtration and dried (prep. -145.1, 910 mg) . The two filtrates (filtrate I and filtrate II) from the above precipitations gave, by concentration to dryness, preparations -145.2 and -145.3, respectively.

Preparation -145.1 contained, by TLC and HPLC, antibiotic 273a ₁ o. Preparation -145.2 contained N-acetyl-L-cysteine. Prepara¬ tion -145.3 contained N-acetyl-L-cysteine, small amounts of anti¬ biotic 273a _χ α and traces of a polar unidentified material. Example 2 Preparation of Antibiotic 273a _χ )3

N-Acetyl-L-cysteine, 4.2 g, was dissolved in 100 ml of 0.1 M pH 7.85 phosphate buffer. The solution was adjusted to pH 8.7 with 1 N aqueous potassium hydroxide. Paulomycin B, 1.0 g, was added to this solution and dissolved under stirring. After standing at room temperature for 1 hour, the solution was adjusted to pH 3.0 with 2N aqueous hydrochloric acid and extracted four times with 100 ml portions of ethyl acetate. The ethyl acetate extracts were combined, dried over sodium sulfate and concentrated to dryness to give prep. -146.1. Preparation -146.1 was found highly active vs. M. lu- teus, ca. 300 bu/mg, and contained antibiotic 273a _χ ^ as the only bioactive component.

Prep. -144.1 was dissolved in 15 ml of acetone; this solution was added under stirring to 200 ml of ether. The precipitated material was isolated by filtration, dried on the filter, and redissolved in 15 ml of acetone. The new solution was also poured into 200 ml of ether under stirring. The precipitate formed was isolated by filtration and dried (prep. -147.1, 111 g) . The two filtrates (filtrate I and filtrate II) from the above precipitations gave, by concentration to dryness, preparations -147.2 and -147.3, respectively.

Preparation -147.1 contained, by TLC and HPLC, _. antibiotic 273a _χ 3. Preparation -147.2 contained N-acetyl-L-cysteine. Prepara¬ tion -147.3 contained N-acetyl-L-cysteine, small amounts of anti¬ biotic 273a _χ ^ and an unidentified polar compound. Utilizing a procedure similar to that described in Examples 1 and 2, but substituting paulomycin C, or O-demethylpaulomycin A or B for paulomycins A and B, there are obtained the antibiotic analogs of paulomycin C and O-demethylpaulomycins A and B that correspond to antibiotics 273a _χ α and 2TSa _χ β. Example 3 Reaction of Paulomycins A, A ₂ , B, C, D, E and 0- demethylpaulomycins A and B with Esters of N-Acetyl-L- Cysteine

1. Preparation of N-Acetyl-L-Cysteine Methyl, Ethyl, Butyl, and Octyl Esters.

N-Acetyl-L-cysteine (1 equivalent) is dissolved in excess methyl, ethyl, butyl, or octyl alcohol. Thionyl chloride (1.1 to 1.5 equivalents) is then added to the N-acetyl-L-cysteine-alcohol mixture and this mixture is allowed to stand at room temperature for 1-3 hours. The reaction mixture is then concentrated to dryness. Crystallization of the desired product is obtained from ether-heptane mixture. 2. Reaction of Paulomycins (A, A ₂ , B, C, D, E and O-demethyl- paulo ycins A and B) with N-Ace yl-L-Cysteine Methyl, Ethyl, Butyl and Octyl Esters

Paulomycin (1 equivalent) is dissolved in tetrahydrofuran containing catalytic amounts of triethylamine. Ten equivalents of the corresponding N-acetyl-L-cysteine (methyl, ethyl, butyl, or octyl ester) is then added under stirring. After 30 minutes at room temperature the reaction mixture is concentrated to dryness. The residue is then dissolved in methylene chloride and this solution is mixed with Skellysolve B. The paulomycin-N-acetyl-L-cysteine ester adducts precipitate and are isolated by filtration and dried. Characterization is obtained by fast atom bombardment mass spectrome- try.

3. Purification. Counter Double Current Distribution The solvent consists of cyclohexane-ethyl acetate-acetone-water (1:1:1:1 v/v) . The residue obtained as described above is dissolved in both phases of the solvent system and added in one tube (center tube) of an all-glass counter double current distribution apparatus (100 tubes). After one hundred transfers, fractions are analyzed by thin layer chromatography. Fractions containing the "mono" adduct are concentrated to dryness. The residue is purified by precipita¬ tion from acetone-Skellysolve mixtures. Characterization is obtained by fast atom bombardment.

Example 4 Reaction of Paulomycins (A, A ₂ , B, C, D, E and 0- demethylpaulomycins A and B) with Mercaptoacetic Acid (Compound Is, Chart II)

Mercaptoacetic acid is obtained from commercial suppliers such as Sigma Co., Aldrich Co., and Alfa Co.

Reaction of Paulomycins with Mercaptoacetic Acid

Mercaptoacetic acid (20 eq) is dissolved in pH 8.5 phosphate buffer. The pH is then adjusted to 8.7 with IN KOH and 1 equivalent of paulomycin A, A _j , B, C, D, E or O-deme h lpaulomycin A or B is added under stirring. After 2 hours at room temperature under stirring the solution is adjusted to pH 3.0 and the paulomycin- mercaptoacetic acid addition compounds are isolated by extraction with methylene chloride or ethyl acetate. The extracts are dried over sodium sulfate and concentrated to dryness. The residues obtained are then purified by precipitations from methylene chloride- heptane combinations and the precipitate is purified by counter double current distribution and characterized by fast atom bombard¬ ment mass spectrometry.

Example 5 Reaction of Paulomycin (A, A^ , B, C, D, E or 0- demethylpaulomycin A or B) with Mercaptopropionic Acid

(compound 2s, Chart II) and Thiomalic Acid (compound

3s, Chart II)

Mercaptopropionic acid and thiomalic acid are commercially available. The procedure described in Example 4 is followed and specifically the ratio of the acids to paulomycin is about 20:1 equivalents. The precipitates obtained by the methylene chloride precipitation are analyzed by TLC, HPLC, and fast atom bombardment mass spectroscopy.

Example 6 Reaction of Paulomycins A, AJJ , B, C, D, E and 0- demethylpaulomycins A and B with Cysteine (compound

4s, Chart II), Homocysteine (compound 5s, Chart II) and Glutathione (compound 6s, Chart II) Compounds 4s, 5s, and 6s are obtained from commercial suppliers of chemicals such as Sigma Co., Aldrich Co., and Alfa Co. 1. Reaction with Cysteine

Cysteine hydrochloride (439 mg, 2.5 mmoles) was dissolved in 20 ml of pH 7.85 0.1 M phosphate buffer. The pH of the solution was adjusted to 8.7 with IN potassium hydroxide. Paulomycin (a mixture of paulomycins A and B) (200 mg, 0.25 mmoles), was added under stirring. The reaction was followed by thin layer chromatography. The same procedure can be utilized substituting paulomycins A, A ₂ , B, C, D, E or O-demethylpaulomycin A or B for the mixture of paulomycins A and B.

2. Reaction with Homocysteine

The procedure described above was followed using 337.5 mg of homocysteine (2.5 mmoles) and 200 mg of paulomycin (mixture of paulomycins A and B). Likewise, the procedure can be utilized substituting paulomycins A, A _j , B, C, D, E or O-demethylpaulomycin A or B for the mixture of paulomycins A and B.

3. Reaction with Glutathione

The procedure described above was followed using 767.5 mg of glutathione (2.5 mmoles) and 200 mg of paulomycin (mixture of paulomycins A and B) . Paulomycins A, ^ , B, C, D, E or O-demethyl¬ paulomycin A or B can be substituted for the mixture of paulomycins A and B. . -

4. Purification of Reaction Products

The products of the reactions of paulomycins A, A^ , B, C, D, E and O-demethylpaulomycins A and B with cysteine, homocysteine and glutathione can be extracted by butanol at pH 6.0. The butanolic extract is concentrated to dryness and the residue is then purified by chromatography over silica gel using chloroform-methanol mixtures as the mobile phase. Alternatively, the residue obtained from the butanolic extract can be purified by reverse phase chromatography using C-18 or C-8 silica and acetonitrile, 1 M phosphate buffer pH 5.5 mixtures.

The products of the reactions of paulomycin with cysteine, homo¬ cysteine, and glutathione were characterized by fast atom bombardment mass spectrometry.

Example 7 Reaction of Paulomycins A, A _j , B, C, D, E and 0- demethylpaulomycins A and B with Thioglucose (compound 7s, Chart II) or Thioglycerol (compound 8s, Chart II) Thioglucose or thioglycerol (20 eq) were dissolved in pH 7.85 phosphate buffer. The pH was then adjusted to 8.7 with IN KOH solution and 1 equivalent of paulomycin (A and B as mixture) was added under stirring. After 1 hour at room temperature the solution was adjusted to pH 5.5 and passed over Amberlite XAD-4. The paulo¬ mycin-thioglucose or -thioglycerol reaction products were absorbed on the resin and eluted with acetone. The acetone solution was con¬ centrated to dryness. The residue was dissolved in acetone or methylene chloride and this solution was mixed with an ether-hexane mixture. The precipitated paulomycin-thioglucose or paulomycin-

thioglycerol product was isolated by filtration and dried. Purifica¬ tion of the "mono" adducts is obtained by counter double current distribution. Characterization of these materials was obtained by fast atom bombardment mass spectroscopy. Likewise paulomycins A, A ₂ , B, C, D, E and O-demethylpaulomycins A and B can be reacted under essentially the same conditions to give the corresponding A, A ₂ , B, C, D, E and O-demethylpaulomycin A or B analogs. Example 8 Reaction of Paulomycins A, A _j , B, C, D, E and 0- demethylpaulomycins A and B with a 1-Deoxy-l-Thiopen- titol or with a 1-Deoxy-l-thiohexitol The 1-deoxy-l-thio-pentitol—or- -hexitol. (20. eq) ..is dissolved in pH 7.85 phosphate buffer. The pH is then adjusted to 8.7 with IN OH solution and 1 equivalent of paulomycin (A, ₂ , B, C, D, E or 0- demethylpaulomycin A or B) is added under stirring. After 1 hour at room temperature the solution is adjusted to pH 5.5 and passed over Amberlite XAD-4. The paulomycin-thio-pentitol or -hexitol reaction products are absorbed on the resin and eluted with acetone. The acetone solution is concentrated to dryness. The residue is dis- solved in acetone or methylene chloride and this solution is mixed with ether-hexane mixture. The precipitated paulomycin-thio-pentitol or paulomycin-thiohexitol product is isolated by filtration and dried. Characterization of these materials is obtained by fast atom bombardment mass spectroscopy. Mono Adducts

Mono adducts, compounds of Formula I wherein R _χ is formula 4, are prepared by reacting the paulomycin with limited amounts of mercapto-containing compounds of the type, e.g., R- _j SH wherein ^ is the same as described above (see Chart II) . "Limited amounts" means about 1.5 equivalents.

The reaction conditions are as follows: The mercapto-compound is dissolved in phosphate buffer. The pH is adjusted to approxi¬ mately 9.0; paulomycins A or B are then added under stirring (ratio of mercapto-compound to paulomycins is approximately 1.5:1). The reaction is stopped at about 30 minutes. At this time the resulting addition products are extracted from the reaction mixture with ethyl acetate or 1-butanol at the appropriate pH's (usually about 4.0). The residue obtained after removal of the solvent is a mixture of the

corresponding mono and bis adducts. Purification and separation of the desired mono adduct is obtained by chromatography over silica gel using methanol-chloroform mixtures or by reverse phase chromatography in C-18 or C-8 silica gel using acetonitrile-pH 5.5 phosphate buffer mixtures. Counter double current distribution using cyclohexane- ethyl acetate-acetone-water (1:1:1:1) can be used for the purifica¬ tion of the mono adducts. The products of the reaction can be characterized by fast atom bombardment mass spectrometry. The compounds thus produced by addition of 1 molecule of mercapto compound to paulomycins A or B have biological properties similar to those of antibiotics 273a ₂ , i.e. they are active against gram- positive organisms -including Staphylococcus aϋreus ^" resistant to methicillin, lincosaminide and macrolide antibiotics. Mixed Adducts Mixed adducts of Formula I, wherein R _x is formula 2, are formed by reacting a bis-adduct of paulomycin, obtained by reacting a thiol in aqueous medium at pH 8.7, with a second thiol under the same conditions, with the replacement of one of the initial thio-sub- stituents by the second, yielding a mixed bis-adduct. The group displaced is that of the dithiocarbamate (i.e., from the original isothiocyanate group). Such mixed bis-adducts, which show high antibacterial activity both in vitro and in vivo, were inaccessible previously. The Structure of the Mixed Adducts In the reaction between the bis(2-mercapto-ethanol) adduct of Paulomycin A (R _j - ca. 16 min under the HPLC conditions on a reversed phase C ₁₈ column and N-acetyl-L-cysteine, the major new peak (R _j -1 ca. 12 min) is intermediate in value between the starting material and the bis(N-acetyl-L-cysteine) adduct (Antibiotic 273a ₁ α) (R^i ca. 10 min) , and this intermediate value is consistent with the replacement of one -SCH ₂ CH ₂ 0H group in the starting material by the more polar -SCH ₂ CH(NHCOCH ₃ )C00H group.

Similarly, in the reaction between the bis(N-acetyl-L-cysteine) adduct (Antibiotic 273a ₁ α) (R _j . - ca. 10 min) and 2-mercapto-ethanol, the major naw peak (R^ i- ca. 12.5 min) is intermediate in value between the starting material and the bis(2-mercapto-ethanol) adduct (R _τ - 16 min); and this intermediate value is consistent with the

replacement of one -SCH ₂ CH(NHC0CH ₃ )C00H group in the starting material by the less polar -SCH ₂ CH ₂ 0H group.

Proof that the two products described above though having very similar R _j ± values, were indeed distinct entities, lay in the separation of two poorly resolved peaks by HPLC on coinjection. By negative ion FAB-MS., each compound as its mono-sodium salt, showed a molecular ion (M- ) at m/z 1049 a.m.u. , the value calculated for (Paulomycin A + 2-mercapto-ethanol + N-acetyl-L-cysteine, monosodium salt, C ₁ H ₆₀ N ₃ 0 ₂₁ S ₃ Na) ; furthermore, each showed low-mass ions at m/z 77 (H0CH ₂ CH ₂ S. ^} and 162 (H00CCH(NHC0CH ₃ )CH ₂ S ) a.m.u., additional evidence that each contained the 2-mercapto-ethanol and N-acetyl-L- cysteine fragments, but no fragment ion revealed the environment of either substituent. The U.V. spectrum of each showed λ _maχ 250, 274, and 328 nm characteristic of adducts of the Paulomycins of both bis- and mono- (i.e., dithiocarbamates retaining the conjugated double bond of the paulic acid ester) adducts. Similarly, the IR and N.M.R. (both 'H and ¹³ C) could be interpreted only as evidence of the absence of the -N-C-S and conjugated ester double bond, and gave no indication of the relative positions of the two thio-substituents. Under the same HPLC conditions, Antibiotic 273a ₂ α has an R _τ value of 11.6 min. On reaction with 2-mercapto-ethanol, the peak corresponding to the 273a ₂ α disappeared very rapidly, with the generation of a major new doublet peak of RT ca. 12 min together with a minor peak of RT ca. 16 min. After 2 1/2 hours, the 16 min peak was the major product, the 12 min peak having diminished markedly. The 16 min peak was coincident with that of the bis(2-mercapto- ethanol) adduct on coinjection of the two materials. The generation of this bis(2-mercapto-ethanol) product from 273a ₂ α demonstrates that the thio-component of the dithiocarbamate group of an adduct is displaced readily by a second thiolate anion.

The doublet peak of ca R _τ 12 min, seen at the earlier time of assay and at which point the starting 273a ₂ α has disappeared, is a mixture of components of RT 12.12 and 12.35 min. The former is not distinguished from the mixed bis-adduct derived from the reaction between the bis(2-mercapto-ethanol) adduct and N-acetyl-L-cysteine, and must be derived from the addition of 2-mercapto-ethanol to the conjugated double bond of the ester of 273a ₂ α. The second inter¬ mediate, of RT 12.35 min, must be that of displacement of the N-

acetyl-L-cysteine anion by the 2-mercapto-ethanol with retention of the conjugated double bond of 273a ₂ α (11.6 min).

Example 9 Reaction of Paulomycins with 2-mercapto-ethanol

2-Mercapto-ethanol (992 mg, 0.89 ml, 10 equiv) is dissolved in aqueous phosphate buffer (0.05 M, pH 7.85, 100 ml), and the solution is adjusted to pH 8.7 by the addition of aqueous sodium hydroxide (N) . Paulomycin A (1.0 g, 1 equiv. solid) is added to this vigorous¬ ly stirred solution and dissolves completely within 10 min. Analyti¬ cal HPLC shows the presence of only one product, and the complete disappearance of Paulomycin A.

The stirred reaction solution is acidified with aqueous hydro¬ chloric acid (N) of pH 5.5 ^" , causing the precipitation of the product,— which is collected by filtration and is washed with water. HPLC analysis shows the product to be free of excess 2-mercapto-ethanol. A solution of the solid in ethyl acetate is dried using sodium sulfate followed by removal of the solvent in vacuo. The residue is dissolved in acetone and this solution, under stirring, is diluted with ether and Skellysolve B, giving a colorless, amorphous precipi¬ tate which is dried in vacuo at room temperature. This bis-adduct is characterized by fast atom bombardment mass spectrometry.

Utilizing a procedure similar to that described in Example 9, but substituting Paulomycins A ₂ , B, C, D and E and O-demethyl¬ paulomycins A and B for Paulomycin A there are obtained the cor- responding bis-adducts.

Example 10 Reaction of the bis(2-mercapto-ethanol) adduct of Paulomycins with N-acetyl-L-cysteine - the mixed (2- mercaptoethanol)-N-acetyl-L-cysteine adduct The bis-(2-mercapto-ethanol) adduct of Paulomycin A (900 mg, 1 equiv, solid) is added to a vigorously stirred solution of N-acetyl- L-cysteine (1.56 g, 10 equiv.) in aqueous pH 7.85 phosphate buffer (0.05 M, 100 ml), which has been adjusted to pH 8.7 by the addition of aqueous sodium hydroxide (N) ; all of the solid dissolved within 15 min. Analytical HPLC examination of an aliquot of the reaction solution after 1 1/2 hours showed starting material (minor, RT ca. 16 min) and product (major, RT ca. 12 min); after 24 hours, very little starting material remains, and a minor zone, R _j 4- ca. 10 min, is present.

On acidification of the reaction solution with aqueous hydro¬ chloric acid (2N) to pH 3.0, a colorless flocculent precipitate is formed. Extraction with ethyl acetate, drying with sodium sulf te _^ and removal of the solvent in vacuo gives an amorphous solid, which is dissolved in acetonitrile (5 ml), injected onto a partisil 40 ODS-3 C ₁₈ reversed phase column, and eluted with a mixture of aqueous phosphate buffer (pH 5.5) and acetonitrile using a gradient of 15% acetonitrile to 40%. Elution is monitored at 220 nm. Fractions corresponding to the peaks are collected, resulting in pools which are assayed by analytical HPLC. These fractions are acidified separately to pH 3 with aqueous hydrochloric acid (N), extracted with ethyl acetate, dried with sodium sulfate _^ and.the solvent, removed in_ vacuo, giving colorless amorphous residues.

Fraction A, RT ca. 10 min, separates from acetone solution on the addition of ether as a colorless, amorphous solid, identified by HPLC, U.V. spectrum, and FAB-MS as antibiotic 273a _χ .

Fraction B, RT4 ca 12 min, is dissolved in ethyl acetate, and a solution of sodium 2-ethylhexanoate in the same solvent is added with stirring, producing a precipitate of the mono-sodium salt of the mixed 2-mercapto-ethanol-N-acetyl-L-cysteine adduct, characterized by FAB-MS.

Utilizing a procedure similar to that described in Example 10 but substituting the corresponding bis-adducts of Paulomycins A, A _j , B, C, D and E or O-demethylpaulomycin A or B for the bis-adduct of Paulomycin A there is obtained the corresponding mixed adducts. Example 11 Isolation and Characterization of Paulomycin C

A. Assay and Testing Procedures

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

B. Thin-Layer Chromatographic Procedures

The production of paulomycin was followed by thin-layer chroma¬ tography on silica gel G using chloroform-ethanol-water (25:30:5, v/v) or chlorofor -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 bioautography on M. luteus seeded trays.

C. Spectroscopic Methods

Proton magnetic resonance 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-d ₆ or acetone-d ₆ were used. Carbon magnetic resonance 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 tetramethylsilane. High resolution mass spectra were obtained on a Varian MAT-731 double-focusing high resolution mass spectrometer using the field desorption probe for in beam introduction of the- -sample in electron impact mode.

D. Analytical-High Performance Liquid Chromatography (HPLC) HPLC chromatography was carried out on a Hewlett-Packard Model 1084B (Hewlett-Packard, Avondale, CA) instrument equipped with an HP model 79875A variable wave length detector and operating in the dual pump mode. A Brownlee 10 cm x 4.6 mm stainless steel column packed with C-18 (10 μ) reverse phase was used with a mobile phase composed of 38% acetonitrile (Burdick & Jackson, Muskegon, MI) and 62% 0.5 M pH 7 potassium phosphate buffer. The aqueous buffer was filtered through a 0.45 μ filter prior to use. The following instrumental conditions were established:

Flow -2.00 Oven temperature 30

% % BB 3 388..00 Wavl S:R 320:430

S-Temperature A 30 Chart Speed 0.30

S-Temperature B 30 Attention 2t

E. Fermentation Conditions

The fermentation conditions were in general identical to the conditions described in US Patent 4,335,108, supra, except that

Amberlite XAD-2 resin, ca. 150 liters, was added per 5000 liters of fermentation. Under these conditions all paulomycins produced are adsorbed on the resin.

F. Isolation Procedures 1. Isolation of Antibiotics Produced by Streptomyces paulus

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.

2. Recrystallization The crude crystals obtained as described above were dissolved in ethyl acetate (5 ml of ethyl acetate per g of crys¬ tals). 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 paulomycin A and B were separated by filtration. The filtrate (mother liquors) was con¬ centrated to dryness to give a residue (Preparation A) enriched in bioactive materials other than paulomycins A and B. This material was used for the isolation of paulomycin C as described below.

3. Isolation of Paulomycin C by High Performance Liquid Chromatography

Support: 1.5 Kg of Dupont Zorbax RP-18 Starting Material: 17 g of preparation A Mobile Phase: Acetonitrile-water (40:60 v/v) containing 10 ml of tetrahydrofuran and 5 ml of acetic acid per liter.

The starting material was dissolved in 100 ml of mobile phase and the solution was introduced into the column (flow rate 15 ml/minute). Fractions were analyzed for bioactivity and by TLC.

Fractions containing paulomycin C were combined and the solution was kept as preparation B, 150 ml. Preparation B was concentrated to dryness to give preparation C.

Preparation C was dissolved in 5 ml of chloroform. This solution was mixed with 50 ml of ether; the precipitated material was removed by filtration. The filtrate was diluted with 5 ml of chloroform and the solution was mixed with 200 ml of n-hexane.

Amorphous paulomycin C was precipitated and isolated by filtration.

The precipitate was dried to give preparation D, 750 mg. Charac¬ terization of preparation D (paulomycin C) follows in the charac¬ terization section.

CHARACTERIZATION OF PAULOMYCIN C 1. Appearance: Colorless amorphous material

2. Solubility: Soluble in lower alcohols, ketones, ethyl acetate, chloroform, methylene chloride; less soluble in ether; insoluble in saturated hydrocarbon solvents.

3. Molecular Composition: C ₃₂ H ₄₂ N ₂ 0 ₁₇ S Calculated Mol. Weight: 758

4. IR Spectrum: Tabulation of the IR bands follows.

5. UV Spectrum: The UV spectrum of paulomycin C in methanol is as follows:

Λmax (nm) 7 £

237 19. .35 14650

277 13. .66 10350

321 10, .35 7850

C-13 NMR (5) chloroform solvent:

187.44 (S), 187.78 (S) , 170.77 (S) , 169.33 (S) , 169.24 (S) , 160.27 (S), 158.07 (S), 143.10 (S) , 136.56 (D) , 123.06 (S) , 100.09 (S) ,

100.02 (D), 78.74 (S), 78.63 (D) , 77.67 (D) , 76.40 (D) , 73.36 (S),

72.45 (D), 71.18 (D) , 69.64 (D) , 69.09 (D) , 68.85 (D) , 61.77 (T) ,

57.76 (Q), 47.81 (T) , 30.54 (T) , 27.96 (T) , 20.71 (Q) , 15.72 (Q) ,

15.56 (Q), 15.17 (Q) , 14.70 (Q) . 6. Antimicrobial In Vitro Testing: The bioactivity of paulomycin C is shown as follows:

Antimicrobial In Vitro Testing Minimum Inhibitory Concentration -MCG Per Ml-

Organism Name UC # Paulomycin C Paulomycin A pH 6 pH 6

Enterobacter cloacae 9381 >64 >64

Enterobacter cloacae 9382 >64 >64

Klebsiella Oxytoca 9383 >64 >64

Klebsiella Oxytoca 9384 >64 >64

Escherichia coli 9379 >64 >64

Escherichia coli 9380 >64 >64

Escherichia coli 311 >64 >64

Staphylococcus aureus 6675 0.5 0.12

Staphylococcus aureus 3665 0.25 0.12 Staphylococcus aureus 6685 0.25 0.12 Streptococcus faecalis 694 1 0.25 Klebsiella pneumoniae 58 >64 >64 Pseudomonas aeruginosa 9191 >64 64 Pseudomonas aeruginosa 6432 >64 >64 Serratia marcescens 6888 >64 >64 Citrobacter preundii 3507 >64 >64 Proteus vulgaris 30264 64 16

Note: "UC" is a registered trademark of The Upjohn Company

BAND TABULATION OF THE INFRARED SPECTRUM OF PAULOMYCIN C

Band Band

Frequencv Intensity Type Frequency Intensity Type

3467.0 50 SH 2042.6 29 AVG

3376.3 40 BRD 1885.4 92 BRD

3272.2 46 BRD 1735.9 2 AVG

3233.6 47 BRD 1699.2 15 AVG

2898.0 0 BRD M 1639.4 29 AVG

2851.7 0 AVG M 1625.9 27 AVG

2726.3 71 AVG M 1575.8 19 AVG

2671.4 74 BRD M 1461.0 2 AVGM

2634.7 77 SH 1377.1 6 AVGM

2245.1 90 BRD 1343.4 27 AVG

2176.6 81 SH 1297.1 18 AVG

2117.8 52 SH 1263.3 9 AVG

Band Band

Frequencv Intensity T pe Frequency Intensity Type

1245.0 14 SH 866.0 60 AVG

1190.0 27 AVG 824.5 65 AVG

1154.3 21 AVG 815.8 60 SHP

1137.0 16 AVG 783.0 63 AVG

1117.7 17 AVG 752.2 46 SHP

1098.4 19 AVG 723.3 51 AVGM

1055.0 19 AVG 690.5 57 AVG

1027.0 13 AVG 663.5 54 SH

992.3 23 AVG 636.5 53 SH

931.6 60 BRD 619 .1 50 SH 909.4 41 AVG 602.7 45 AVG 894.9 50 SH

Band Freq. : Band frequencies in wave numbers (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 Frequency

0 2898.0

0 2851.6

2 1735.8 2 1461.0

6 1377.0

9 1263.2

13 1027.0

14 1245.0 15 1699.1

16 1137.0

17 1117.6

18 1297.0

19 1575.7 19 1098.3

19 1055.0

21 1154.2

23 992.2

27 1625.8 27 1343.3

27 1190.0

29 2042.5

29 1639.3

40 3376.2 41 909.3

45 602.6

Prep: Mineral Oil Mull Tape: 122 File: 106

Max %T: 95 #3745.7 %T at 3800 (CM ^-1 ): 95 Density (CM ^_1 /PT): 0.964

Example 12 Isolation and Characterization of O-demeth lpaulo- mycins A and B

Assay and testing procedures, TLC procedures, spectroscopic methods, analytical HPLC, and fermentation conditions were as described in Example 11. A. Isolation Procedures 1. Isolation of Antibiotics Produced by Streptomyces paulus a. General Procedure

See Example 11. - - .. b. Summary of Crude Crystalline Preparations Obtained by Extraction Weight of

Fermentation Crude Crystals fq". Preparation 830148 685 A

830261 398 B

830289 785 C 830366 240 D

830501 1324 E

830383 371 F

830429 885 G

830473 589 H The above crude crystalline preparations were used in the recrystallization studies described below. 2. Recrystallization a. General Procedure See Example 11. b. Summary of Recrystallization

Preparations A, B and C were combined and recrystal- lized from ethyl acetate as described above. The following materials were obtained: 1) Prep H, 1158 g (paulomycin A and B) ; 2) Prep I, ca. 154 g (containing several bioactive materials) . Similarly combination of preparations D and E and recrystallization yielded 855 g of paulomycins A and B (Preparation J) and Preparation K (ca. 225 g) .

Finally, combination of preparations F, G and H and recrystallization yielded 1418 g of paulomycins A and B (Preparation L) and Preparation M (ca. 206 g) .

Preparations K and M were combined to yield prepara- tion N, 431 g, which was used as the starting material for the work which led to the isolation of purified preparations of O-demethyl¬ paulomycins A and B.

3. Isolation of O-Demethylpaulomycins A and B a. High Performance Liquid Chromatography, Isolation of "Polar Paulomycins"

Instrument: Waters Prep 500A

Support: _. Waters C-18 Reverse- Phase Silica Packed columns

Starting Material: Preparation N. Mobile Phase: Acetonitrile - 0.1 M, pH 5.5 phosphate buffer (1:1, v/v) Flow Rate: 200 ml/minute

The starting material was dissolved in 400 ml of acetonitrile-pH 5.5 phosphate buffer (1:1, v/v) and was injected into the column. The chromatography was followed by UV and refractive index detectors. In addition, selected fractions were analyzed by TLC using KC-18 reverse phase silica plates and acetonitrile-metha- nol-pH 5.5, 0.01 M phosphate buffer (1:1:1, v/v) as the mobile phase. Seven fractions were collected and characterized as follows: Fraction 1 - Polar Paulomycins (including O-Demethyl¬ paulomycins A and B) Fraction 2 - Paulomycin B Fraction 3 - Paulomycin B, A Fraction 4 - Paulomycin A Fraction 5 - Paulomycin A (tail-Fraction I)

Fraction 6 - Paulomycin A (tail-Fraction II) Fraction 7 - Methanolic wash

A total of 12 runs were made using a total of 250 g of preparation N. Similar fractions from all 12 runs were combined and extracted twice with methylene chloride. The combined methylene chloride extracts from Fraction 1 were concentrated to dryness to give preparation 0. This material was used for isolation of purified O-Demethylpaulomycin A and B as described below.

b. Isolation of Purified O-Demethylpaulomycins A and B, HPLC Chromatography

Instrument: Waters Prep 500A

Support: Waters C-18 Reverse Phase Silica Packed columns Starting Material: Preparation 0

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 ca. 400 ml of aceto- nitrile-pH 5.5, 0.1 M phosphate buffer (1:1, v/v) and the solution was introduced into the column. The chromatography was followed by

UV and refractive index..detectors and by TLC. Seven fractions were collected and designated as follows:

Fraction 1 - Non-designated materials Fraction 2 — Non-designated materials

Fraction 3 — Non-designated materials Fraction 4 - Non-designated materials, Paulomycin E Fraction 5 - Paulomycin E

Fraction 6 - Paulomycin E, D, some paulomycin C Fraction 7 - Paulomycin D, C

A total of 4 runs were run using all of prepara¬ tion 0. Similar fractions from all 4 runs were combined and ex¬ tracted twice with methylene chloride. The combined methylene chloride extracts from Fraction 6 (paulomcyins E, D, and some C) were concentrated to dryness to give preparation P, 4.98 g. This prepara¬ tion contained mainly paulomycin D and some paulomycin C, and was purified further as described below.

Instrument: Waters Prep 500A

Support: Waters C-18 Reverse Phase Silica Packed columns

Starting Material: Preparation P, 2.5 g Mobile Phase: Acetonitrile-pH 5.5, 0.1 M, phosphate buffer (40:60, v/v) Flow Rate: 90-100 ml/minute The starting material was dissolved in ca. 400 ml of acetonitrile-pH 5.5, 0.1 M phosphate buffer (1:1, v/v) and the solution was introduced into the column. The chromatography was followed by UV and refractive index detectors and by TLC.

Fractions containing, by TLC, paulomycin D or paulomy¬ cin D-like material were extracted with methylene chloride. The extract was dried over sodium sulfate and concentrated to dryness. The obtained preparation was kept as Preparation Q. Preparation Q contained paulomycin D and O-demethylpaulomycin B.

O-Demethylpaulomycin B: Analysis of Preparation Q by FAB/MS (negative ion probe) indicated the presence of two components, paulomycin D (molecular weight 744) and an equal amount of another compound with molecular weight identical to that of paulomycin C (molecular weight 758) . Bio-TLC of Preparation Q indicated the presence of paulomycin D only. This finding suggested that the "unknown," paulomycin C-like component was either bioinactive or it was bioactive with polarity identical to that of paulomycin D.

Preparative HPLC separation was repeated and after bio-TLC analysis three tubes (leading, middle and tail of the paulomycin D fraction) were analyzed by FAB/MS. Results are tabulated below:

Ratio of Paulomycin D (MW 744) Unknown (MW 758) Tube No. 18 >10 Tube No. 25 1.3

Tube No. 31 0.4

FAB/MS (positive ion mode) of the material in Tube 18 (mainly paulomycin D) showed fragment ions at m/z 231 and 199 assigned to 1 and la (Chart III) . Fragment la results from 1 by elimination of methanol.

FAB/MS of the material in Tube 31 (mainly the paulomycin C-like unknown) showed fragment ions at m/z 245 and 227 assigned to 2 and 2a (Chart III) . This indicated that the unknown paulomycin-C like compound is indeed O-demethylpaulomycin B, molecular weight 758. The lack of the methoxy group increases the polarity of the molecule so that O-demethylpaulomycin B behaves like paulomycin D in TLC.

Fractions containing, by TLC, paulomycin C or paulo¬ mycin C-like material were extracted with methylene chloride. The extract was dried over sodium sulfate and concentrated to dryness. The isolated preparation was kept as Preparation R. Preparation R contained paulomycins C and O-demethylpaulomycin A.

O-Demethylpaulomycin A: FAB/MS (in the negative ion probe) of preparation R indicated this material to be a compound of similar

molecular weight to paulomycin B (molecular weight 772) . However bio-TLC indicated this material to behave like paulomycin C. Positive ion FAB/MS showed the presence of fragments 259 and 241 assigned to 3 and 3a (Chart III) . This indicated that the unknown paulomycin B-like compound is indeed O-demethylpaulomycin A. 0- Demeth lpaulomycin A, because of the lack of the methoxy group, behaves in TLC like paulomycin C.

Both O-demethylpaulomycins A and B appear to have identical antibacterial spectrum as the other paulomycins, i.e. they are active mainly vs. gram-positive organisms.

ADDUCTS OF PAULOMYCIN C AND O-DEMETHYLPAULOMYCINS A AND B A. Bis-Adducts ^~

Referring generally to formula I and Chart I, the structures of paulomycin C and O-demethylpaulomycins A and B are presented in Chart I. Paulomycin C and O-demethylpaulomycins A and B are related to paulomycins A and B, the structures of which are also presented in

Chart I.

The reactions of paulomycins A and B with N-acetyl-L-cysteine and other mercapto-containing compounds, to yield the corresponding bis-adducts, are described, supra, and in the European applications. Like paulomycins A and B, paulomycin C and O-demethylpaulomycins A and B with large excess (5-10 equivalents) of N-acetyl-L-cysteine yield the corresponding bis-adducts, wherein R _x is formula 1 and X _χ is hydrogen. Similarly paulomycin C and O-demethylpaulomycins A and B react with methyl, ethyl, butyl or octyl esters of N-acetyl-L-cysteine to give the corresponding bis-adducts.

Referring now to Chart II, paulomycin C and O-demethylpaulo¬ mycins A and B react with a variety of compounds of the general formula R^H like mercaptoacetic acid (Is), mercaptopropionic acid (2s) , thiomalic acid (3s) , cysteine (4s) , homocysteine (5s) , gluta¬ thione (6s), thioglucose (7s), thioglycerol (8s) and thioalditols (9s, 10s).

Reactions of paulomycin C and O-demethylpaulomycin A and B with excess of compounds Is-lOs yield the corresponding bis-adducts wherein R _λ is formula 2 and R^ and R _g are the same.

The conditions for the reaction of paulomycin C and O-demethyl¬ paulomycin A and B with compounds Is-10s are identical to those

described for the production of antibiotics 273a _χ o and 273a _χ j8 disclosed in the European applications. An excess of the mercapto- compound (i.e. Is-10s) is dissolved in phosphate buffer. The pH is adjusted to ca. 9.0; paulomycin C or O-demethylpaulomycin A or B is then added under stirring. The reaction is complete within one hour. At this time the resulting addition product is extracted from the reaction mixture with ethyl acetate or butanol at the appropriate pH's (usually ca. 4.0). The residue obtained by removal of the solvent is then purified by chromatography over silica gel using methanol-chloroform mixtures or by reverse phase chromatography in C- 18 or C-8 silica gel using acetonitrile-pH 5.5 phosphate buffer mixtures. The products of the reactions are characterized by fast . atom bombardment mass spectrometry. The products resulting by addition of 2 molecules of Is-10s and O-demethylpaulomycin A or B have biological properties similar to those of antibiotics 273a _j , i.e. they are active against gram-positive organisms including Staphylococcus aureus resistant to methicillin, lincosaminide and macrolide antibiotics. B. Mono-Adducts of Paulomycin C and O-Demethylpaulomycins A and B As described in the European applications, reaction of paulo¬ mycins A and B with limited amounts of mercapto-containing compounds (ca. 1.5 equivalents) results in the production of mono-adducts. Similarly paulomycin C and O-demethylpaulomycin A and B react with limited amounts of compounds of the general formula R ₂ SH like N- acetyl-L-cysteine, N-acetyl-L-cysteine methyl ester, N-acetyl-L- cysteine ethyl ester, N-acetyl-L-cysteine butyl ester, N-acetyl-L- cysteine octyl ester, mercapto acetic acid, mercaptopropionic acid, thiomalic acid, cysteine, homocysteine, glutathione, thioglucose, thioglycerol and thioalditols. Reaction of paulomycin C or O-demethylpaulomycin A or B with limited amounts of compounds of the general formula ^SH results in the production of bis-adducts as above and the mono-adducts wherein R _x is formula 4.

The ratio of molar equivalents of reactants paulomycin C, or 0- demethylpaulomycin A or B and compounds of the general formula ^SH effect the products of the reaction. Large excess of the thiol- containing compounds (paulomycin to R^H ratio of ca. 10-20 results in the exclusive production of the bis-adducts. At low paulomycin to

^SH ratio (1:1 or 1:2) both the bis-adducts and the mono-adducts are produced. Separation of the mono- from the bis-adduct is obtained by a variety of methods including counter double current distribution and several chromatographic procedures. The conditions for the reaction of paulomycin C, or O-demethyl¬ paulomycin A or B with the thiol containing compounds for the production of the mono-adducts are identical to those described for the production of antibiotics 273a ₂ α and 273a ₂ 9 in the European applications. The mercapto-compound is dissolved in phosphate buffer. The pH is adjusted to ca. 9.0; paulomycin C or O-demethyl¬ paulomycin A or B is added under stirring (ratio of mercapto-compound to paulomycin ca. 1.5:1). The- -reaction- is stopped at cav 30 mi¬ nutes. At this time the resulting addition products are extracted from the reaction mixture with ethyl acetate or 1-butanol at the appropriate pH's (usually ca. 4.0). The residue obtained after removal of the solvent is a mixture of the corresponding mono- and bis-adducts. Purification and separation of the desired mono-adduct is obtained by chromatography over silica gel using methanol-chloro¬ form mixtures or by reverse phase chromatography in C-18 or C-8 silica gel using acetonitrile-pH 5.5 phosphate buffer mixtures. Counter double current distribution using cyclohexane-ethyl acetate- acetone-water (1:1:1:1) can be used for the purification of the mono- adducts. The products of the reaction can be characterized by fast atom bombardment mass spectrometry. The mono-adducts produced by addition of 1 molecule of the thiol-compounds on paulomycin C or 0- demethylpaulomycin A or B have biological properties similar to those of antibiotics 273a ₂ , i.e. they are active against gram-positive organisms including Staphylococcus aureus resistant to methicillin, lincosaminide and macrolide antibiotics. The antibacterially-active compounds of the present invention can be used for the same antibacterial purposes as paulomycin 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 equip-

ment, floors, or furnishings or contaminated rooms or laboratories; they are also useful as an industrial preservative, for example, as a bacteriostatic 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 antibac¬ terial 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 phar- macologically acceptable salts.

The tribasic and tri acid 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 ingre¬ dients 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 an 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 disintegration 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 embodi¬ ment, 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. Advanta¬ geously, 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 ster¬ ilized before filling into a suitable vial or ampul 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. Advanta¬ geously, 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 de¬ scribed.

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; anti- histamines, such as chlorpheniramine maleate, diphenylhydramine, pro- methazine, pyrathiazine, and the like; sulfas, such as sulfadiazine, 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 treatment of most conditions for which the compositions are effective. For children, the dosage is calcu¬ lated 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 veteri- narian.

The active compound is compounded with a suitable pharmaceutical carrier in unit dosage form for convenient and effective administra¬ tion. 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.

FORMULAS

(1) CH,CH CH C0O-

³ I I

S NH

I I

CH2 OS

CH3CO-NH-CH S '•

I I

COO i CH _jj

CH-NHCOCHg

COOX _j

(3) CH ₃ CH-C-C00- N-OS

(4) CHgCH-CCOO-

NH

I

OS i

S a

FORHULAS (continued)

(5) CHJCHCOOXJ

(9)

CHART I The structxires of paulomycins A, B, A _j , C, D, E, antibiotics 273a ₁ α, 273a ₁ ^ _> 273a _j α and 273a ₂ 0, and O-demeth l paulomycins A and B are represented by Formula I as follows:

13

ORe wherein R _j is formula 3; wherein ₄ is hydrogen; wherein R _g is CH ₃ ; wherein R is:

1. Paulomycin A: CH ₃ -O^OUCHa ) -C00CH CH ₃ ) - 2. Paulomycin B: CH ₃ CH(CH ₃ ) -COOOKCH _g ) - 3. Paulomycin A _j : (CH ₃ ) ₂ CHC^ C00CH(CH ₃ ) - 4. Paulomycin C CHaCI^-COOClKCHa)- 5. Paulomycin D CHg COOClUCH _g ) - 6. Paulomycin E CH _g C J)-

wherein R6 is H;

7. O-Demethylpaulomycin A: (otherwise sane as paulomycin A) 8. O-Demethylpaulomycin B: (otherwise same as paulomycin B)

wherein R ₁ is formula 1; wherein X ₁ is H; wherein R _g is CH ₃ ; wherein R is:

9. ntibiotic 273a _χ o: CH ₃ -O_ ₂ CT(OT ₃ )-C00CH(CH ₃ )- 10. Antibiotic 273a^: CH ₃ CH(CH ₃ )-COOCH(CH ₃ )-

CHART I (continued) wherein R _χ is formula 4; wherein R ₂ is -CH ₂ CH(NHR ₅ )C00X ₂ ; wherein X- is H; and wherein R ₅ is acetyl; wherein R _g is CH ₃ ; wherein R is:

11. Antibiotic 273a ₂ α: CH ₃ -CH ₂ CH(CH ₃ )- COOCH (CH ₃ ) -

12. Antibiotic 273a ₂ /3: CH ₃ CH(CH ₃ )- COOCH (CH ₃ )-

CHART II R _j SH s. R _j - -CH ₂ COOH (mercaptoacetic acid)

s. R _j - CH ₃ CHCOOH (mercaptopropionic acid)

s. R _j - (thiomalic acid)

s. 1^ - -CH ₂ CHCOOH (cysteine) NHa

s. R2 - -CH ₂ CH ₂ CHC00H (homocysteine) N1Ϊ2

s. R2 - H _jj N-CH-Ca^CH _j CONHCHCONHCH _j COOH (glutathione) COOH CH ₂ -

s. R2 - (3-2- CHOH

CH ₂ 0H

s. R2 - CH - 10s. IL _j - Cl-2- 11s. R2— CH2CH ₂ 0H

CHOH CHOH

I I

CHOH CHOH

I I

CHOH CHOH

I I

CH ₂ 0H CHOH

I

CI^OH

CHART III

: (199) : Ci3H2lθ5(345) -R2θH 3a C12H19O4 (237) R ₇ «CH3CH R ₆ -H R ₇ *CH3CH- CH3 OH3

: Cl3H23θ5(3S9) -R2θH 3a: C13H21O4 (341) R ₇ =CH3CH2CH-;R ₆ =H _L-=CH3CH2CH- CH3 CH3