GB2271571A - Aminoalkyl ether of cyclohexapeptidylamine compounds - Google Patents

Abstract

Aminoalkyl others of cyclohexapeptidylamine compounds are prepared which have antibiotic properties.

Description

TITLE OF THE INVENTION AMINOALKYL ETHER OF CYCLOHEXAPEPTIDYLAMINE COMPOUNDS -The present invention is directed to certain aminoalkyl ethers of cyclohexapeptidylamine compounds or their quaternary ammonium salts.

The compounds of the present invention are compounds in which one amino group is directly on the ring through a continuous carbon chain but in which the second amino group is on a substituent which forms a derivative of a functional group on the ring. The compounds include (a) amines represented by the formula wherein R is an amino alkyl group:

(IA) Seq ID No 1 and their acid addition salts, and (b) quaternary ammonium salts represented by the formula wherein R is an amino alkyl group:

Also included within the scope of this invention are compounds in which the quarternary nitrogen is part of the amino-alkyl ether group represented by the formula where R1 represents the group having a quaternary ammonium nitrogen:

CIC) Seq ID No 1 and their acid addition salts.

A fourth group of compounds are those in which both amino functions are quaternary nitrogen:

(ID) Seq ID No 1 In the foregoing and succeeding formulas, R is CnH2nNRVRvI R' is CnH2n#NRVRVIRVIIIY# R1 is C9-C21 alkyl, C9-C21 alkenyl or C1-C10 alkoxyphenyl, or C1-C10 alkoxynaphthyl R11 is H, C1-C4 alkyl or benzyl RIII is H, C1-C4 alkyl or benzyl or RII and RIII together is -(CH2)4- or -(CH2)5-- RIV is C1-C4 alkyl RV is H, C1-C4 alkyl or benzyl RVI is H, C1-C4 alkyl or benzyl.

or RV and RVI together is -(CH2)4- or -(CH2)5- RVII is H or C1-C4 alkyl Q is O or S Y is an anion of a pharmaceutically acceptable salt Z is an anion of a pharmaceutically acceptable salt, and n is an integer from 2 to 4, inclusive.

Where the expression "alkyl", "alkenyl" or "alkoxy" is employed, it is intended to include branched as well as straight chain radicals.

Pharmaceutically acceptable salts suitable as acid addition salts as well as salts providing the anion of the quaternary salt are those from acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, maleic, citric, acetic, tartaric, succinic, oxalic, malic, glutamic, trifluoroacetic acetic and the like, and include other acids related to the pharmaceutically acceptable salts listed in Journal of Pharmaceutical Science, 66, 2 (1977).

The sequence IDs of the starting and intermediate materials are as follows: Compound (A): Seq ID No 2; Compound (B): Sequence ID No 3 and Compound (C): Sequence ID No 4. Since the nucleus amino acid does not change with the substituent R on the ornithine nitrogen, all compounds which differ only in R will have the same sequence ID. Also, further substitution on the amine in compound (C) will not change the sequence ID.

Since all compounds are variations in the derivative group and the cyclopeptidyl ring does not change, the products all have Seq. ID No 1.

The amine compounds are soluble in lower alcohols and polar aprotic solvents such as dimethylformamide (DMF) and pyridine. They are insoluble in solvents such as ether and acetonitrile.

The compounds of the present invention are useful as an antibiotic, especially as an antifungal agent or as an antiprotozoal agent. As antifungal agents they are useful for the control of both filamentous fungi and yeasts. They are especially adaptable to be employed for the treatment of mycotic infections in mammals, especially those caused by Candida species such as C; albicans, C. tropicalis and C. pseudotropicalis, and Aspergillus species such as A.

fumigatus, A. flavus and A. niger. They are also useful for the treatment and/or prevention of Pneumocvstis carinii pneumonia to which immune compromised patients are especially susceptible as hereinafter described.

A compound which is of particular interest for the control of mycotic infections, as hereinafter detailed is a compound represented by the following formula IA-1 (Seq ID 1)

Seq ID Nol The compounds of the present invention may be obtained from natural products or derivatives of natural products through a sequence of reactions seen in the following flow diagram. (It is to be noted that alternate routes are also possible but the noted one is one of the preferred routes.)

Step D

reductive alkylation seq ID No 1

Seq ID No 1 The starting material (Compound A) for the.

preparation maybe a natural product in which R is 9,11-dimethyltridecyl or a derivative of a natural product obtained as hereinafter described.- In carrying out the preparation, Compound A is dehydrated (Step A) at the carboxamide group which is part of the glutamine component of the cyclopeptide and which extends from the cyclopeptide ring to obtain a novel compound (B) which has a nitrile in said position.The nitrile compound is then subjected to reduction (Step B) to obtain a novel amine compound (C) which may be alkylated by reductive alkylation (Step D) with an appropriate aldehyde and a reducing agent such as sodium cyanoborohydride to obtain (IB). The amine compound (C) or alkylated derivative (IB) may be quaternized (Step F' or F respectively) by causing the amine to react with an excess alkylating agent such as an alkyl halide or alkyl sulfate in the presence of a mild base such as diisopropylethylamine in an inert solvent to obtain a mono quaternary compound (ID).

This amine compound (C, IB and ID) may be selectively etherified at the 5-hydroxy position of the ornithine component (Steps C, C', C" respectively) with an amino-alcohol or aminoalkylthiol resulting in bis-amine compounds, IA, IC, IE respectively, as their acid addition salts.

The first dehydration step is carried out by intimately contacting the cyclohexapeptide starting material with a dehydrating agent. The reaction is preferably carried out under nitrogen with cyanuric chloride in a solvent in the presence or-absence of molecular sieves.

.Suitable reagents which may be employed in place of cyanuric chloride are anhydrides such as acetic anhydride, trifluoroacetic anhydride and phosphorus pentoxide; acid chlorides such as oxalyl chloride, phosphorus oxychloride, thionyl chloride, p-toluenesulfonyl chloride and chlorosulfonyl isocyanate; phosphonium reagents such as phosphorus pentachloride, triphenylphosphine/carbon tetrachloride, triphenylphosphonium ditriflate and triphenylphosphonium dichloride; carbodiimides such as dicyclohexylcarbodiimide; other dehydrating agents such as aluminum chloride, titanium tetrachloride, (methoxycarbonylsulfamoyl)triethylammonium hydroxide inner salt.

Suitable solvents include dimethylformamide (DMF) or weakly basic solvents such as pyridine, collidine and the like.

Molecular sieves may be in the size range 3A to 5A.

The relative amounts of Compound A (Seq. ID No. 2) and reagents vary, but in general the dehydrating agent is used in excess. From about 1.5 to 15 equivalents of the dehydrating agent are employed.

When employed, the molecular sieves are used in amounts of at least tenfold by weight.

In carrying out the reaction, a suspension of molecular sieves in a rigorously dried solvent is first prepared, and while stirring under an atmosphere of nitrogen, there is added, cyanuric chloride or other dehydrating agent and thoroughly mixed. To the resulting mixture while stirring under an atmosphere of nitrogen is added the starting material Compound A and the stirring continued for about 12 to 24 hours or until HPLC analysis of the reaction mixture indicates substantial completion of the reaction with the formation of the nitrile. When the HPLC analysis shows substantial completion of the reaction, the sieves are removed by filtration, preferably on a sintered glass funnel, and the filtrate concentrated and purified by preparative HPLC. The mobile phase used in the purification are varying ratios of a water/acetonitrile composition containing 0.1 percent trifluoroacetic acid.For most part, water/acetonitrile compositions range from 40/60 to 60/40. The exact mobile phase used for HPLC as says and the mobile phase used in preparative HPLCs may differ not only from each other but also from compound to.compound, but can be determined by the skilled artisan without difficulty.

In carrying out the reaction in the absence of sieves, solid cyanuric chloride is added in a single portion to a solution of Compound A in an aprotic solvent and stirred rapidly for a short time and the reaction mixture then quenched by adding aqueous sodium acetate directly to the reaction mixture. The volatiles are then removed in vacuo to obtain a solid residue which may be purified as above described.

The reduction of the nitrile to the amine may be carried out employing either chemical or catalytic reduction. Sodium borohydride with cobaltous chloride in alcoholic solvent has been found to be particularly useful. When this combination of reagents is used, from about 5 to 50 molar equivalent of sodium borohydride and from 2 to 10 molar equivalents of cobaltous chloride are used for each molar amount of the nitrile.

Other hydride reducing agents such as sodium cyanoborohydride, aluminum hydride, diborane, diisobutyl aluminum hydride and the like also may be used.

Frequently these reducing agents are used in combination with a Lewis acid such as cobaltous chloride or aluminum chloride as in the present combination of sodium borohydride and cobaltous chloride.

Catalytic hydrogenation also may be carried out over a variety of catalysts including palladium on carbon, platinum oxide, or rhodium on alumina.

Typical solvents depending on the reagent include alcohols, especially methanol and ethanol, dimethylformamide, pyridine, tetrahydrofuran (THF) or other ethers.

When the reduction of the nitrile to the amine is carried out using the preferred chemical procedure, the reaction may be carried out by adding the chemical reducing agent to the nitrile in an alcoholic solution under an atmosphere of nitrogen, and stirring until HPLC analysis using detection by ultraviolet absorption at 210 nm shows substantial completion of the reaction. When sodium borohydride is used in combination with cobaltous chloride, cobaltous chloride is added while stirring to a solution in methanol, or other solvent, of the nitrile, prepared as above described, at ambient. temperature, followed by portionwise addition of the sodium borohydride which is accompanied by gas evolution. Stirring is continued for from 12 to 24 hours. The reaction mixture may be quenched 2N hydrochloric acid at this time.Then the mixture is diluted with a highly aqueous mobile phase, 70/30 H20/CH3CN, filtered and purified by chromatography. The eluate fractions are lyophilized to obtain the amine intermediate.

To prepare the aminoalkyl ethers of the amine intermediate, i.e., the bisamine product Compound IA, camphorsulfonic acid or hydrochloric acid is added to a solution containing the cyclohexapeptidylamine compound (Compound C), and the appropriate aminoalkanol or anfinoalkylthiol. The mixture is allowed to stir at room temperature for one to seven days. The progress of the reaction is conveniently monitored by HPLC as subsequently described using water/acetonitrile containing 0.1% TFA as the eluting agent. After the reaction is substantially complete, the reaction mixture is diluted with water and the resulting solution applied to a reverse phase flash silica gel column and eluted with an appropriate mixture of acetonitrile and water to obtain Compound IA.

A large excess of the aminoalkanol or thiol is employed, preferably on the order of one-hundred molar equivalents. The amount of camphorsulfonic acid or hydrochloric acid is one equivalent for every mole of the cyclohexapeptidyl amine. The reaction medium is a suitable aprotic solvent such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF) or dioxane, or combinations thereof.

For monitoring the progress of the reaction, an analytical "ZORBAX" (DuPont) column with 10 to 50 percent aqueous acetonitrile containing 0.1 percent trifluoroacetic acid (TFA) or acetic acid is suitable.

For preparative purification, a reverse phase column such as "LICHROPREP" C18 of particle size 40-63 microns with 5-15 percent aqueous acetonitrile to remove solvent and 10 to 50 percent acetonitrile (containing 0.1% TFA or acetic acid) to elute the product may be employed.

When it is desired to introduce an alkyl group on the cyclohexapeptidyl amine (c), the group may be introduced by formylating, followed by reduction of the hydroxymethyl group with sodium cyanoborohydride or other reducing agent. When the desired alkyl group on the nitrogen is a higher alkyl, a prefered procedure is a reductive alkylation of N-benzyl derivative with an aldehyde and a reducing agent such as sodium cyanoborohydride, and purifying the product with reverse phase chromatography to obtain a benzyl and a higher alkyl substituted tertiary amine. The benzyl group may be removed by hydrogenation using palladium on carbon or other suitable catalyst.

When a quaternary ammonium salt (ID) or (IE) is to be prepared, the appropriate amine is caused to react with an alkylating agent such as alkyl iodide, other alkyl halide, or alkyl sulfate in the presence of diisopropylethylamine in an inert solvent. The alkylating agent is used in large molar excess. About six to tenfold molar excess. A slight molar excess of base is employed.

When it is desired to prepare a quaternary ammonium salt of mixed alkyl composition it is desirable to introduce the groups on the nitrogen prior to the final quaternization step using procedures above described since alkylation using analkylating agent is difficult to control.

The compounds of the present invention are antimicrobial agents active against many fungi and particularly against Candida, Aspergillus and Cryptococcus species and also antiparasital agents for the control of organisms infecting mammals such as Pneumocvstis carinii.

The usefulness for the control of fungi causing mycotic infections may be determined, for example, in a microbroth dilution assay employing as medium a Yeast Nitrogen Base (Difco) with 1% dextrose (YNBD). In such assay, Compound I is solubilized in 10 percent dimethyl sulfoxide (DMSO) and diluted to 2560 Fg/ml. The compounds are then diluted to 256 Fg/ml in YNBD. 0.15 ml portions of the suspension are dispensed to the top row of a 96-well plate (each well containing 0;15 ml of YNDB) resulting in a drug concentration of 128 Fg/ml.

The yeast cultures, maintained on Sabouraud dextrose agar are transferred to TM broth (Difco) and incubated overnight at 35"C with shaking (250 rpm).

After incubation, each culture is diluted in sterile water to yield a final concentration of 1-5 x 106 colony forming units (CFU)/ml.

96-well micro-plates are inoculated using a MIC-2000 (Dynatech) which delivers 1.5 1 per well yielding a final inoculum per well of 1.5-7.5 x 103 cells. The microplates are incubated at 35"C for 24 hours and read. The minimum inhibitory concentration of the drug is that showing no visible growth.

After recording the MIC, the plates are shaken to resuspend the cells. Thereafter, 1.5 p1 samples from the wells in the 96-well microplate are transferred to a single well tray containing Sabouraud dextrose agar. The inoculated trays are incubated 24 hours at 28"C and then read. The MEC is defined as the lowest concentration of drug showing no growth or less than 4 colonies per spot.

The compounds also show activity in the inhibiting 1,3-glucan synthase which indicates anti pneumocystis activity. The l,3-i3-glucan synthesis inhibition assay may be carried out using protoplasts of Candida albicans in 80 1 of a mixture of 125 mM trisHCl (pH 7.0), 0.25 mM dithiothreitol, 0.15 mM phenylmethylsulfonyl fluoride, 0.40 M glycerol, 0.75 mM EDTA, 1.0 percent bovine serum albumin, 40.0 nM guanosine 5 '-(a-thio)-trisphosphate (tetralithium salt) and determining IC50 as more fully described in Proc. Natl. Acad. Sci. USA 87 (1990), p 5950.

In addition to the use of the compounds as therapeutic agents in the treatment of mycotic infections in mammals, they may be used wherever growth of fungi is desired to be controlled, e.g. consumer goods, plants and plant parts, plant products, wood and lumber, pulp and paper, and the like.

When employed for controlling mycotic infections, a therapeutically effective amount'is administered but the actual dosage may be varied according to the particular compound employed, the physical condition of the subject being treated, and the severity and nature of the infection. Therapy is usually started at a low dosage and increased to achieve the desired effect.

The compounds may be administered parenterally, orally, topically, by inhalation, by insufflation or by other means for drug administration.

The outstanding properties are most effectively utilized when the compounds are formulated into novel pharmaceutical compositions with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques.

The novel compositions contain at least a therapeutically effective amount of Compound I, usually at least 1 percent although concentrate compositions may contain up to 90 percent by weight. In preparing the compositions, Compound I is intimately admixed with any of the usual pharmaceutical media.

The compositions may be prepared in oral dosage form. For liquid preparations, Compound I is formulated with liquid carriers such as water, glycols, oils, alcohols, and the like; and for solid preparations such as capsules and tablets, Compound I is formulated with solid carriers such as starches, sugars, kaolin, ethyl cellulose, calcium and sodium carbonate, calcium phosphate, kaolin, talc, lactose, generally also with lubricant such as calcium stearate, together with binders, disintegrating agents and the like. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage form. It is especially advantageous to formulate the compositions in unit dosage form for ease of administration and uniformity of dosage.

Compound I may be formulated in compositions for injection and may be presented in unit dosage form in ampoules or in multidose containers, if necessary with an added preservative. The compositions may also take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles such as 0.85 percent sodium chloride or 5 percent dextrose in water, and may conta.in formulating agents such as suspending, stabilizing and/or dispersing agents. Buffering agents as well as additives such as saline our glucose may be added to make the solutions isotonic. Alternatively, the active ingredients may be in powder form for reconstituting with a suitable vehicle prior to administration.

The administration by inhalation, the compounds of the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or nebulizers. The compounds also may be delivered as powders which may be formulated and the powder composition may be inha-led with the aid of an insufflation powder inhaler device. The preferred delivery system for inhalation is a metered dose inhalation (MDI) aerosol, which may be formulated as a suspension or solution of Compound I in suitable propellants, such as fluorocarbons or hydrocarbons.

The term "unit dosage form" refer to physically discrete units, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier. Examples of such unit dosage forms are tablets, capsules, pills, powder packets, wafers, measured units in ampoules or in multidose containers and the like. A unit dosage form for antifungal application may contain from 10-200 milligrams of one of the compounds.

If the application is to be topical, the compound may be formulated in conventional creams and ointments such as white petrolatum, anhydrous lanolin, cetyl alcohol, cold cream, glyceryl monostearate, rose water and the like. Usually a 1 to 2 percent cream or solution is prepared and applied to the area to be treated.

The following examples illustrate the invention but are not to be construed as limiting: EXAMPLE I

seq. ID No. 1 A. Preparation of the Nitrile Intermediate (B) Seq ID No. 3.

To a solution of 250 milligrams (0.232 mmol) of Compound A in 3.0 mL of DMF was added 64 milligrams (0.348 mmol) of cyanuric chloride. After allowing the reaction to take place over 5.5 minutes, the reaction was quenched by the addition of 0.55 milliliter of 2M sodium acetate. The reaction mixture was then diluted with methanol and injected onto two "ZORBAX' C18 columns (25 mm x 25 cm) in series and eluted with 50/50 water/acetonitrile (both containing 0.1% trifluoroacetic acid) at 6.0 mL/min.Pure fractions as determined by HPLC (eluting with 45/55 E20/CH3CN (both containing 0.1% TFA), 4.5 nm x 25 cm "ZORBAX" C18 column, flow = 1.5 mL/min, temperature =40"C, k=210 mm) were combined and lyophilized to obtain 45 milligrams (18 percent yield) of nitrile product Compound B ( > 98% by HPLC: 4.6 mm x 25 cm "ZORBAX" C18; isocratic elution with 45/55 H20/CH3CN both 0.1 % TFA; flow rate = 1.5 mL/min; temperature = 40"C; X = 210 mm EPLC retention time = 5.0/min)) MS (FAB) 1054 (M+Li) B. Preparation of the Amine Intermediate (C) Seq ID No.

4.

To a solution of 38.5 mg (0.036 mmol) of nitrile in 2 milliliters of methanol was added 32 milligrams (0.25 mmol) of CoC12X6H20. When all the cobaltous chloride had dissolved, 46 milligrams (1.22 mmol) of NaBH4 was cautiously added in four portions.

After three hours, the reaction was judged complete by HPLC (45/55 H20/CH3CN (both 0.1% TFA), 4.5-mm x 25 cm "ZORBAX" C18 column, flow = 1..5 mL/min, temperature = 40"C; X = 210mm). The reaction mixture was quenched by the addition of 0.68 milliliter of 2N HC1. The resulting solution was filtered and injected onto a "ZORBAX" C18 column (25 mm x 25 cm) and eluted with 50/50 H20/CH3CN (both 0.1% TFA) at 6.0 mL/min. Pure fractions as determined by HPLC were combined and lyophilized to obtain 13 milligrams (33.7 percent) of amine product of > 98% purity by HPLC (HPLC retention time = 5.5 min).

MS (FAB) 1058 (M+Li) C. Preparation of Bisamine Product (Compound IA-l) To a solution of 222 milligrams (200 eq) of ethanolamine in a mixture of 0.6 milliliters of DMSO and 0.1 milliliter of dimethylformamide was added 12.8 milligrams of the amine (prepared as described in Part B), followed by 2.7 milligrams (1 equivalent) of camphorsulfonic acid. The reaction was allowed to proceed for one week and the mixture then injected onto a "ZORBAX" C18 column (25 mm x 25 cm). The mixture was eluted with 45/55 water/acetonitrile (both 0.1% TFA) at 6.0 mL/min.Pure fractions as determined by HPLC were combined and lyophilized to obtain 45 milligrams (18%) of aminoethyl ether product of > 98% purity by HPLC (4.6 mm x 25 cm "ZORBAX" C18, isocratic elution 66/4.0 H20/CH3CN both 0.1% TFA; flow rate = 1.5 mL/min; temp = 40"C; X = 210 nm; retention time = 4.96 min).

MS (FAB) 1100 (M+Li) 1H NMR (40.0 MHz, CD30D) 6 7.12 (d, 2H), 6.75 (d, 2H), 5.18 (d, 1H), 4.97 (d, 1H), 4.21 (dd, 1H), 4.15 (td, 1H), 3.96 (dd, 1H), 3.87 (dd, 1H), 3.80 (d, 1H), 3.02 (t, 1H), 2.42 (m, 1H), 2.21 (t, 2H), 1.82 (m, 1H), 1.18 (d, 3H).

EXAMPIIE II

To a solution of 44 milligrams (42 mmol) of Compound C (prepared as described in Example I) and 100 equivalent of hydroxyethyltrimethyl ammonium chloride is added 20 milligrams (2 eq) of camphorsulfonic acid in about 200 L DMSO and the resulting mixture stirred at room temperature until HPLC analysis indicated conversion of the starting material. The reaction mixture is then injected directly onto a "ZORBAX" (25 mm x 25 cm) C18 column and eluted with 50/50 water/acetonitrile (0.lZ TFA) at 8.0 mL/min. Pure fractions as determined by HPLC are pooled and lyophilized to the desired product Compound (IC-l) (Seq ID No. 1), M.W.=1172.9 as the monochloride.

EXAMPLE III

A. Preparation of Amine Intermediate To a solution of 44 milligrams (42 mol) of Compound C (pre.pared as described in Example I) in 1.0 milliliter of sieve dried DMF (13X, 3A molecular sieves) is added 4.5 milligrams (53 Fool) of sodium bicarbonate, followed by 250 milligrams of 4A sieves and finally 26 milliliters (417 Fmol, 10 eq) of methyl iodide and the resulting solution is stirred at room temperature for 5 hours. At this time, 2.5 mg (30 mol) of sodium bicarbonate and 26 milliliters (417 Fmol) of methyl iodide are added and the resulting mixture stirred overnight at room temperature. The reaction mixture is then applied directly to a preparative HPLC column and eluted with 55/45 water/acetonitrile (both 0.1Z TFA) at 8.0 ml/min. Pure fractions as determined by HPLC are pooled and lyophilized to obtain Compound (IB).

B. Preparation of ID To a solution of 17 milligrams (17 Fool) of (IB) (prepared as above described) and 100 equivalents of 2-N,N-dimethylaminoethanol hydrochloride in DMSO is added to milligrams (2 eq) of camphorsulfonic acid and the resulting mixture is stirred at room temperature until HPLC analysis indicated conversion of the starting material. The reaction mixture is then injected directly onto a "ZORBAX" (25 mm x 23 cm) C18 column and eluted with 50/50 water/acetonitrile (both 0.1% TFA) at 8.0 mL/min. Pure fractions as determined by HPLC are pooled and lyophilized to the desired product Compound ID-1. MW = 1200.92 (as the monochloride).

EXAMPLE IV In a manner similar to that carried out in Example I, the following compounds are prepared: (1) A compound of MW = 1095 and having the formula

(2) A compound of MW= 1138 and having the formula

EXAMPLE V In operations carried out in a manner similar to that described in Examples I and II, the following compounds are prepared.

Example R3 R1 R11 RIII V-A OCH2CH2N(CH3)CH2CH5 DMTD* CH3 CH3 V-B OCH2CH2N#(CH3)2CH2C6H5I# DMTD CH3 CH3 V-C OCH2CH2N(CH3)(C4H9-n) DMTD H H V-D 0CH2CH2CH2NHCH2C6H5 DMTD H H V-E OCH2CH2NH2 DMTD H H V-F OCH2CH2N(CH3)2 DMTD H H V-G oCH2CH2N@(CH3)3 le DMTD H H *DMTD = 9,11-dimethyltridecyl Compounds V-A through V-D are of Seq ID No. 1; Compounds V-E through V-G are of Seq ID No. 2.

EXAMPLE VI In operations carried out in a similar matter, compounds of the formulas may be prepared:

Seq. ID No. 1

(Seq ID No 1) EXAMPLE III 1000 compressed tablets each containing 500 mg of Compound of Example.I (Seq ID No 1) are prepared from the following formulation: Component Grams Compound of Example I 500 Starch 750 Dibasic calcium phosphate, hydrous 5000 Calcium stearate 2.5 The finely powdered ingredients are mixed well and granulated with 10 percent starch paste. The granulation is dried and compressed into tablets.

EXAMPLE VIII 1000 hard gelatin capsules, each containing 500 mg of Compound of Example II are prepared from the following formulation: Component Grams Compound of Example II 500 Starch 250 Lactose 750 Talc 250 Calcium stearate 10 A uniform mixture of the ingredients is prepared by blending .and used to fill two-piece hard gelatin capsules.

EXAMPLE IX An aerosol composition may be prepared having the following formulation: Per Canister Compound of Example III 24 mg Lecithin NF Liquid Concentrated 1.2 mg Trichlorofluoromethane, NF 4.026 g Dichlorodifluoromethane, NF 12.15 g EXAMPLE 250 milliliters of an injectible solution may be prepared by conventional procedures having the following formulation: Dextrose 12.5 g 26% aqueous polyethylene glycol 300 250 ml Compound IA of Example IV (1) 400 mg The ingredients are blended and thereafter sterilized for use.

Preparation of Starting Materials The starting material, Compound (A), Seq. ID.

No. 2., in which R is 9, ll-dimethyltridecyl may be produced by cultivating Zalerion arboricola ATCC 20868 in a nutrient medium enriched in mannitol as the primary source of carbon as described in U.S. Patent No. 5,021,341, June 4, 1991.

Compound (A) in which RI is other than 9, ll-dimethyltridecyl may be produced from Compound (A) by subjecting the latter in an aqueous buffered nutrient medium to which a deacylating enzyme obtained from or found present in intact.cells of microorganisms of the family Pseudomondaceae or Actinoplanaceae until deacylation is complete, isolating the nucleus compound from the fermentation broth by centrifuging the broth, and subjecting the supernatant to chromatographic purification steps and recovering the deacylated cyclopeptide, and thereafter acylating the deacylated cyclopeptide by mixing together with and appropriate active ester RCOZ where Z is halogen, pentachlorophenoxy, pentafluorophenoxy, p-nitrophenoxy and the like.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: HAMMOND, MILTON L. AND ZAMBIAS, ROBERT A.

(ii) TITLE OF INVENTION: (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: MERCK & CO., INC.

(B) STREET: P.O. BOX 200, EAST LINCOLN AVE.

(C) CITY: RAIlWAY (D) STATE: NEW JERSEY (E) COUNTRY: USA (F) ZIP: 07065 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette - 5.25 inch, 360Kb (B) COMPUTER: WANG PC 381 (C) OPERATING SYSTEM: MD-DOS 3.30.10 (D) SOFTWARE: Wang Integrated Word Processing (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: ~~~~~~~~~~~~~~~~~~ (B) FILING DATE: ~ (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: ALICE O.ROBERTSON (B) REGISTRATION NUMBER: 18,525 (C) REFERENCE/DOCKET NUMBER: 18870 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 908-594-4372 (B) TELEFAX: 908-594-4720 (C) TELEX: (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 (B) TYPE: AMINO ACID (C) STRANDEDNESS: NA (D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE: (A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 Xaa Thr Xaa Xaa Xaa Xaa 1 5 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 (B) TYPE: AMINO ACID (C) STRANDEDNESS: NA (D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE: (A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 Xaa Thr Xaa Xaa Xaa Xaa 1 5 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 (B) TYPE: AMINO ACID (C) STRANDEDNESS: NA (D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE: (A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 Xaa Ser Thr Xaa Xaa Xaa 1 5 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 (B) TYPE: AMINO ACID (C) STRANDEDNESS: NA (D) TOPOLOGY: CIRCULAR (ii) MOLECULE TYPE: (A) DESCRIPTION: PEPTIDE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 Xaa Thr Xaa Xaa Xaa Xaa 1 5

Claims (4)

WHAT IS CLAIMED IS

1. A compound selected from the group consisting of (a) a compound having the formula

and its acid addition salt, (b) a compound having the formula

and its acid addition salt, (c) a compound having the formula

and its acid addition salt, and (d) a compound having the formula

wherein R is CnH2nRVRVI, R' is CnH2n#NRVIRVIIIY# RI is C9-C21 alkyl, C9-C21 alkenyl or Cl-ClO alkoxyphenyl, or C1-C10 alkoxynaphthyl RII is H, C1-C4 alkyl or benzyl RIII is H, C1-C4 alkyl or benzyl or RII and RIII together is -(CH2)4- or -(CH2)5 RIV is C1-C4 alkyl RV is H, C1-C4 alkyl or benzyl RVI is H, C1-C4 alkyl or benzyl or RV and RVI together is -(CH2)4- or -tCH2)5- RVII is H or C1-C4 alkyl Q is O or S Y is an anion of a pharmaceutically acceptable salt Z is an anion of a pharmaceutically acceptable salt n is an integer from 2 to 4, inclusive.

2. An antibiotic composition comprising an amount of a compound of Claim 1 in a pharmaceutically acceptable carrier.

3. A composition according to Claim 2 in unit dosage form wherein the compound of Claim 1 is present in an amount of about 10 milligrams to 200 milligrams.

4. A compound according to Claim 1 having the formula:

Seq ID Nol