WO2008024285A2

WO2008024285A2 - Antibiotic compound

Info

Publication number: WO2008024285A2
Application number: PCT/US2007/018288
Authority: WO
Inventors: Angela Basilio Carnicer; Olga Genilloud; Sheo B. Singh; Francisca Vicente; Chaowei Zhang; Deborah L. Zink
Original assignee: Merck & Co., Inc.; Merck Sharp & Dohme De Espana, S.A.
Priority date: 2006-08-23
Filing date: 2007-08-17
Publication date: 2008-02-28
Also published as: WO2008024285A3

Abstract

Fermentation of a nutrient medium with a Actinoplanes sp. yields a novel broad spectrum antibiotic compound of structural formula (I), or a pharmaceutically acceptable salt, ester, enantiomer, diastereomer or mixture.

Description

TITLE OF THE INVENTION ANTIBIOTIC COMPOUND

This application claims the benefit of U.S. Provisional Application No.

60/839,497, filed August 23, 2006, the disclosure of which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to broad spectrum thiazolyl-peptide antibiotic compounds that are useful in treating bacterial infections.

Infections caused by bacteria are a growing medical concern as many of these bacteria are resistant to various antibiotics. Such microbes include Staphylococcus aureus, Staphylococcus hemolyticus, Pediococcus spp., and Streptococcus pyogenes, Streptococcus pneumoniae, Pseudomonas aeruginosa, Vibrio cholerae, Vibrio parahemolyticus, Actinobacter calcoaeticus, Stenotrophomonas maltophilia. The antibiotic of this invention, thus comprises an important contribution to therapy for treating infections which are resistant to various known antibiotics.

Known compounds that are also thiazolyl-peptide containing, and which are used for treating bacterial infections are thiostrepton, GE2270A, nocathiacins, glycothiohexide, and nosiheptide. hi the present invention thiazolyl-peptide antibiotics are produced from an Actinoplanes sp. fermentation and possess antibacterial activity against bacterial infections that are sensitive and resistance to currently available antibiotics.

2205 IY

SUMMARY OF THE INVENTION

This invention is concerned with a novel thiazolyl-peptide antibiotic of the formula I:

5 or a pharmaceutically acceptable salt, ester, enantiomer, diastereomer or mixture thereof,

wherein R represents:

The invention is also concerned with a process for the production of the 10 compounds of formula I by fermentation with a Actinoplanes sp. The invention is also concerned with a process for isolating the compound of formula I from the fermentation broth. 2205 IY

DETAILED DESCRIPTION OF THE INVENTION

This invention is concerned with the compounds of formula I in particular compounds of structural formula Ia:

2205 IY

and structural formula Ib:

The pharmaceutically acceptable salts of the compounds of this invention include the conventional non-toxic salts, as formed from non-toxic inorganic or organic bases. For example, such conventional non-toxic salts include those derived from inorganic bases such as an alkali or alkaline earth metal hydroxide, e.g., potassium, sodium, lithium, calcium, or magnesium, and the like: and the salts prepared from organic bases such as an amine, e.g., dibenzylethylene-diamine, trimethylamine, piperidine, pyrrolidine, benzylamine and the like, or a quaternary ammonium hydroxide such as tetramethylammonium hydroxide and the like.

10 The pharmaceutically acceptable salts can be synthesized from the compounds of this invention by conventional chemical methods. Generally, the salts are prepared by reacting the free acid with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic base in a suitable solvent or various combinations of solvents.

The compounds of this invention are broad spectrum antibiotics useful in the

15 treatment of bacterial infections. They demonstrate antibacterial activity primarily against S. aureus, E.faecalis, E.faecium, S. pneumoniae, B. subtilus including species that are resistant to 2205 IY

many known antibiotics. The minimum inhibitory concentration (MlC) values range from 0.0001 to less than 200 ug/mL for test strains such as Staphylococcus aureus, Staphylococcus hemolyticus, Streptococcus pyogenes, Streptococcus pneumoniae, and E. faecalis. The compounds of the invention can be formulated in pharmaceutical compositions by combining the 5 compounds with a pharmaceutically acceptable carrier. Examples of such carriers are set forth below.

The compounds may be employed in powder or crystalline form, in liquid solution, or in suspension. They may be administered by a variety of means; those of principal interest include: topically, orally and parenterally by injection (intravenously or

10 intramuscularly) .

Compositions for injection, one route of delivery, may be prepared in unit dosage form in ampules, or in multidose containers. The injectable compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain various formulating agents. Alternatively, the active ingredient may be in powder

15 (lyophilized or non-lyophilized) form for reconstitution at the time of delivery with a suitable vehicle, such as sterile water, hi injectable compositions, the carrier is typically comprised of sterile water, saline or another injectable liquid, e.g., peanut oil for intramuscular injections. Also, various buffering agents, preservatives and the like can be included.

Topical applications may be formulated in carriers such as hydrophobic or

20 hydrophilic bases to form ointments, creams, lotions, in aqueous, oleaginous or alcoholic liquids to form paints or in dry diluents to form powders.

Oral compositions may take such forms as tablets, capsules, oral suspensions and oral solutions. The oral compositions may utilize carriers such as conventional formulating agents, and may include sustained release properties as well as rapid delivery

25 forms.

The dosage to be administered depends to a large extent upon the condition and size of the subject being treated, the route and frequency of administration, the sensitivity of the pathogen to the Compound, the virulence of the infection and other factors. Such matters, however, are left to the routine discretion of the physician according to principles of

30 treatment well known in the antibacterial arts.

The compositions for administration to humans per unit dosage, whether liquid or solid, may contain from about 0.001% to as high as about 99% of Compound I, one embodiment of the range being from about 10-60%. The composition will generally contain from about 2 mg to about 2.5 g of Compound I, one embodiment of this range being from

35 about 2 mg to 1000 mg. In parenteral administration, the unit dosage will typically include pure Compound I in sterile water solution or in the form of a soluble powder intended for solution, which can be adjusted to neutral pH and isotonicity. 2205 IY

The invention described herein also includes a method of treating a bacterial infection in a mammal in need of such, treatment comprising the administration of the compound of formula I to the mammal in an amount effective to treat the infection.

One embodiment of the methods of administration of a compound of formula I 5 includes oral and parenteral methods, e.g., i.v. infusion, i.v. bolus and i.m. injection.

For adults, about 0.1-50 mg of a compound of formula I per kg of body weight given one to four times daily is preferred. The preferred dosage is 2 mg to 1000 mg of the antibacterial given one to four times per day. More specifically, for mild infections a dose of about 5-200 mg two or three times daily is recommended. For moderate infections against

10 highly susceptible gram positive organisms a dose of about 20-1000 mg three or four times daily is recommended. For severe, life-threatening infections against organisms at the upper limits of sensitivity to the antibiotic, a dose of about 100-2000 mg three to four times daily may be recommended. For children, a dose of about 0.1-50 mg of a compound of formula I per kg of body weight given one to four times daily is typically recommended. Another

15 aspect of this invention is realized when the dosage is 2 mg to 1000 mg of the antibacterial given one to four times per day.

Another aspect of this invention is the process for producing the compounds of formula I which comprises cultivating a Actinoplanes sp. microorganism (identified herein as either MA 7347/P-000503667 or ATCC PTA 7551) in a suitable nutrient medium and then

20 recovering the compound of this invention from the fermentation broth. The organism in question was deposited on April 12, 2006 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852 under the terms of the Budapest Treat for Deposit of Microorganisms and registered with accession ATCC PTA 7551. The microorganism is also deposited in the Merck culture collection with an accession number of MA 7347/P-

25 000503667. Although the use of this particular species is described in connection with this invention, there may be other species and mutants of the above organism capable of producing Compound I, and their use is contemplated in carrying out the process of this invention.

The compounds of structural formula I are produced by the aerobic fermentation of a suitable medium under controlled conditions via inoculation with a culture of the MA

30 7347/P-000503667 or ATCC PTA 7551. The suitable medium is preferably aqueous and contains sources of assimilable carbon, nitrogen, and inorganic salts.

The medium employed for fermentation non-streptomyces actinomycete is primarily the well-known Difco Tryptic Soy Broth, either alone or with added nutrients, which is commonly used by those skilled in the art.

35 It should be noted that the nutrient media described herein are merely illustrative of the wide variety of media which may be employed and are not intended to limit the scope of this invention in any way. 2205 IY

The fermentation is conducted at temperatures ranging from about 10⁰C to about 40⁰C; however for optimum results it is preferred to conduct the fermentation at about 26-30⁰C. The pH of the nutrient medium during the fermentation can be about 5.5 to about 7.5.

It is to be understood that for the fermentative production of the compound of this 5 invention, the invention is not limited to the use of the particular Actinoplanes sp. with ATCC PTA 7551. It is especially desired and intended that there be included in the scope of this invention the use of other natural or artificial mutants produced or derived from the described cultures, or other variants or species of the Actinoplanes genus insofar as they can produce the compound of this invention. The artificial production of mutant species or strains of 10 Actinoplanes from ATCC PTA 7551 may be achieved by conventional, physical or chemical mutagens, for example, ultraviolet irradiation of the described culture, or nitrosoguanidine treatment and the like. Recombinant DNA techniques such as protoplast fusion, plasmid incorporation, chromosome fragment incorporation and the like also may prove useful.

Abbreviations employed herein include the following: ACN = acetonitrile;

15 CAMHB = cation adjusted Mueller-Hinton broth; DMSO = dimethyl sulfoxide; HRFTMS = high resolution fourier transform mass spectrometry; i.m. = intramuscular; i.v. = intravenous; MIC = minimum inhibitory concentration; MOPS = 3-(N-morpholino) propanesulfonic acid; NCCLS = National Committee on Clinical Laboratory Standards; RPMI-1640 = Roswell Park Memorial Hospital 1640 culture medium; ID NMR = one-dimensional NMR; 2D NMR = two-dimensional 20 NMR; TFA = trifluoroacetic acid.

EXAMPLE 1

Fermentation for Production of Compound Ia/Ib by Actinoplanes sv. P-000503667 (ATCC 25 PTA 7551)

A. Media Composition:

Seed Medium g/L

Soluble Starch 20.0

30 Dextrose 10.0

NZ Amine Type E 5.0

Beef Extract 3.0

Yeast Extract 5.0

Peptone 5.0 35 (pH adjust to 7.0)

Calcium Carbonate 1.0 2205 IY

MPG (Production Medium)

Glucose 10.0

Millet 20.0

Pharmamedia 20.0

5 MOPS 20.0

B. Seed Culture

A first seed culture of the strain was prepared by inoculating 10 ml of -seed medium in a 50 ml tube with frozen agar plugs of the producing strain and incubating the tube at 28 ⁰C with shaking 10 220 rpm for about 96 hours. A second seed culture was prepared by inoculating 50 mL of seed medium in a 250 mL flask with 2ml of the first seed.

C. Production Culture

4 ml of seed culture was transferred to 500 ml flask containing 100 ml of production medium and 15 the flask was incubated at 28 ⁰C with 220 rpm agitation for 13 days.

Isolation and purification of Compound Ia/Ib from Actinoylanes sy. (ATCC PTA 7551)

The IL broth was extracted with IL Acetone by shaking for 2 hours. 50% Acetone extract was filtered in a sintered glass funnel packed with celite. The filtrate was 20 concentrated to 15% acetone in H2O, which was subjected to Amberchrome chromatography

(50ml column, 10 ml/min, 5-100% aqueous MeOH over lOOmin, hold 100% MeOH 25min, 5min/fraction, finally, washed column with 200ml acetone.). Five 100% MeOH fractions plus acetone wash fractions were combined to give combined fraction weighing 201.8mg.

A portion of combined fraction (50mg) was subjected to prep-HPLC (Zorbax C8,

25 21.2x250mm, lOml/min, 10-95% ACN with 0.1% TFA over 40min). Fractions with biological activity were combined to give 5.0mg material, which was subjected to second prep-HPLC run (Zorbax C8, 21.2x250mm, lOml/min, 38% ACN with 0.1% TFA over 38min, 38-95% ACN for 5min). Fractions eluting at 39min were lyophilized to give Compound Ib (1.5mg) as amorphous powder. The structure was elucidated based on analysis of HRFTMS, ID and 2D NMR spectra.

30 Remainder of the combined fraction was subjected to prep-HPLC (Zorbax C8,

21.2x250mm, 10ml/min, 35-57% ACN without 0.1% TFA over 40min). Fractions eluting at 28min were lyophilized to give Compound Ia (9.4 mg) as amorphous powder. The structure was elucidated based on analysis of HRFTMS, ID and 2D NMR spectra. 2205 IY

Compound Ib Compound Ia C_6IH₆₁N₁₃O₁₉S₆ MW 1471 C₇₄H₈₃N₁₃O₂₅S₆ MW 1745

Phvsiochemical Properties of Compounds Ia and Ib

The structure of Compound I was determined by the use of mass spectroscopy, lH NMR and 13C NMR.

Compound Ia

Molecular weight: 1745,

Molecular formula: 074HgSN₁₃O₂₅Se,

UV (methanol+THF, 1:1) W 259(ε 38855), 306(ε 26407), 350(ε 13960),

10 IR (ZnSe) v_max 3328, 2937, 1650, 1530, 1482, 1424, 1247, 1192, 1140, 1119, 1068, 985, 926, 837cm^"\

HRFTMS: 1746.4074 (observed for M+H), 1746.4026 (calcd for M+H),

¹H NMR (CDC13, 500MHz) δ 10.41 (IH, s), 9.70 (IH, s), 8.42 (IH, s), 8.25 (IH, s), 8.07 (IH, d, 9.6Hz), 7.90 (IH, d, 7.5Hz), 7.90 (IH, s), 7.86 (IH, d, 8.5Hz), 7.66 (IH, s), 7.60 (IH, s), 7.40

15 (IH, d, 10.4), 7.32 (IH, dd, 8.5Hz, 7.2Hz), 7.13 (IH, d, 7.0Hz), 6.53 (IH₃ bs), 5.90 (IH, dd,

10.9Hz, 4.2Hz), 5.59 (IH, bs), 5.50 (IH, d, 11.4Hz), 5.41 (IH, d₅ 11.4Hz), 5.41 (IH, d, 1.5Hz), 5.01 (IH, dd, 9.2Hz, 7.3Hz), 4.84 (IH, d, 10.9Hz), 4.83 (IH, d, 10.9Hz), 4.74 (IH, bs), 4.62 (IH, d, 8.8Hz), 4.50 (IH, dd, 7.1Hz, 4.0Hz), 4.02 (IH, dd, 14.1Hz, 4.7Hz), 4.00 (IH₅ dd, 3.2Hz, 2.0Hz), 3.95 (IH, m), 3.91 (IH, bs), 3.80 (3H, s), 3.63 (IH, m), 3.62 (IH, m), 3.61 (IH, m), 3.60

20 (IH, m), 3.59 (IH, m), 3.50 (3H, s), 3.47 (IH, m), 3.43 (3H, s), 3.38 (3H, s), 3.37 (IH, t, 9.6Hz), 2205 IY

3.25 (IH, m), 3.22 (IH₃ dd, 9.3Hz, 3.4Hz), 3.10 (3H₅ s), 3.08 (IH, dt, 4.6Hz, 9.4Hz), 2.52 (IH, d, 12.8Hz)₅ 2.34 (IH₅ dd, 13.6Hz₃ 12.1Hz)₅ 2.02 (IH₃ m), 1.95 (3H₅ s)₃ 1.81 (IH₅ bd, 9.8Hz)₃ 1.44 (IH₅ m), 1.42 (IH₃ m), 1.26 (3H₅ d, 5.3Hz)₅ 1.18 (3H₅ d, 6.1Hz)₃ 1.12 (3H₅ d₃ 5.9Hz), 1.04 (3H₅ d₃ 6.3Hz)₃

5 ¹³C NMR (CDCD₃ 125MHz) δ 180.7, 170.2, 169.8, 169.4, 167.9, 165.9, 164.5, 162.1, 161.3, 161.2, 160.6, 160.3, 159.9, 158.4, 155.1, 154.5, 151.2, 150.0, 149.5, 145.7, 143.7, 137.3, 134.I₅ 132.7, 131.3 (2C)₃ 130.4, 129.4, 127.O₅ 126.2, 125.5, 125.3, 124.7, 124.3, 124.0, 120.3, 117.6, 114.4, 110.0, 104.0, 101.9, 101.8, 101.5, 80.6, 80.O₃ 79.8₃ 76.7, 75.8, 74.O₅ 71.1, 71.0, 69.8, 68.3, 66.8, 66.6 (2C), 64.3, 59.1, 57.6, 57.4, 56.7, 56.6, 55.6, 53.7, 49.5, 39.9, 30.4, 29.4, 29.3, 17.8,

10 17.6, 17.3, 17.0, 13.4,

¹³C NMR (DMSO-d₆₃ 125MHz) δ 182.7, 169.8, 169.3, 168.9, 167.1, 165.1, 163.8, 162.3, 160.9, 160.1, 160.0, 159.7, 159.6, 158.4, 156.4, 153.2, 150.8, 150.1, 149.8, 146.2, 142.5, 137.4, 135.2, 134.4, 132.4, 131.5, 130.0, 128.9, 127.5, 127.4, 126.4, 125.2, 124.6, 124.4, 123.3, 120.0, 117.8, 114.9, 109.8, 103.8, 102.2, 102.0, 101.6, 80.5, 79.7, 79.0, 76.6, 75.1, 73.6, 70.6, 69.2, 68.8 (2C),

15 67.3, 66.5, 66.2, 64.1, 58.6, 57.1, 56.3, 56.2, 56.0 (2C)₃ 54.0, 48.6, 40.8, 30.6 (2C)₃ 29.2, 18.7, 18.1, 17.7 (2C)₅ 13.0.

Compound Ib

Molecular weight: 1471,

20 Molecular formula: CβiHβiNBOigSβ,

UV (methanol+THF, 1:1) W 245(ε 17603), 298(ε 11866), 350(ε 6864),

IR (ZnSe) v_max 3257₃ 2959, 289O₃ 1673, 1536, 1440, 1368, 1344, 1191, 1121, 1061, 1036, 961,

924₃ 840cm^"1,

HRFTMS: 1472.2593 (observed for M+H), 1472.2609 (calcd for M+H),

25 ¹H NMR (CDC13, 500MHz) δ 8.44 (IH, s), 8.29 (IH, s)₃ 8.16 (IH₃ d, 9.5Hz), 8.00 (IH, d,

7.4Hz), 7.93 (IH₃ s), 7.90 (IH₅ d, 8.4Hz)₃ 7.69 (IH, s), 7.64 (IH₃ s)₃ 7.45 (IH₃ d, 10.4Hz)₅ 7.36 (IH, dd, 8.4Hz, 7.0Hz)₃ 7.18 (IH, d₃ 7.0Hz)₃ 6.59 (IH₃ bs), 5.93 (IH₅ bdd, 4.5Hz, 2.0Hz)₅ 5.60 (IH₅ bs)₃ 5.53 (IH₃ d, 11.5Hz), 5.50 (IH₅ d, 11.5Hz)₃ 5.46 (IH, d, 1.6Hz)₅ 5.05 (IH, bd, 6.5Hz), 4.86 (2H, bs), 4.59 (IH, bd, 3.9Hz), 4.07 (IH, dd, 13.8Hz, 4.4Hz)₃ 4.05 (IH₃ dd, 3.1Hz₅ 1.7Hz),

30 3.95 (IH, ddd, 11.6Hz₃ 6.6Hz₃ 2.1Hz)₃ 3.84 (3H₃ s), 3.67 (IH, dq, 8.8Hz₃ 6.2Hz), 3.64 (IH₃ dd, 14.3Hz₃ 1.8Hz)₃ 3.58 (IH₅ dq₃ 3.8Hz₃ 6.2Hz), 3.56 (IH₃ dd, 9.0Hz₃ 3.0Hz)₃ 3.54 (IH₅ 1, 9.0Hz), 3.54 (3H, s)₅ 3.49 (3H, s), 3.11 (3H₅ s), 2.54 (IH, dd, 13.8Hz, 2.0Hz)₅ 2.39 (IH₅ dd, 13.7Hz, 11.5Hz), 1.98 (3H, s)₅ 1.30 (3H₃ d, 6.2Hz)₃ 1.09 (3H₃ d, 6.1Hz). 1³C NMR (CDCB₃ 125MHz) δ 180.9, 170.4, 169.8, 169.5, 168.0, 165.2, 164.6, 162.2, 161.4,

35 161.2, 160.5, 160.0, 159.O₅ 154.9, 154.6, 151.3, 150.3, 149.7, 145.8, 143.9, 137.4, 134.4, 132.7, 131.4, 130.4, 129.6, 127.1, 126.3, 125.5, 125.4, 124.7, 124.4, 124.1, 120.4, 117.7, 114.5, 110.2, 2205 IY

104.0, 102.2, 80.0, 75.4, 71.5, 70.9 (2C), 66.7, 66.6, 64.3, 59.4, 57.8, 57.4, 56.7, 55.8, 53.6, 49.1, 40.0, 29.4, 20.4, 17.6, 17.2.

General description of strain Actinoulanes SO. (P-000503667, ATCC PTA 7551)

5 Observations of growth, general cultural characteristics and carbon source utilization were made in accordance with the methods of Shirling and Gottlieb (Int. J. Syst. Bacteriol. (1966) 16: 313-340). Coloration of the cultures was determined by comparison with color standards contained in the Methuen Handbook of Colour (A. Kornerup and J.H. Wauscher, Third Edition, 1978).

10

Chemical composition of the cells was determined using the methods of Lechevalier and Lechevalier (Lechevalier, 1980, The chemotaxonomy of actinomycetes in Actinomycete Taxonomy, A.Dietz and D.W. Thayer, Ed. Society for Industrial Microbiology) Fatty acid composition was determined using a modified sample preparation (Sasser 1990.

15 Identification of bacteria by gas chromatography of cellular fatty acids. Tech. Note 101. MIDI, Newark, DE.). Analysis of fatty acid methyl esters (FAMEs) was carried out by capillary gas chromatography using a Hewlett Packard Model 6890N gas chromatograph/Microbial Identification System software (MIDI, Inc., Newark, Del) equipped with a phenyl methyl silicone column (0.2 mm x 25 m). Individual fatty acids identification was determined by the Microbial

20 Identification System software.

The complete 16S rDNA sequence was determined from the 1500 bp PCR fragment obtained using primers 27f and 1525r (Lane, 1991, Nucleic acid techniques in bacterial systematics. E. Stackebrandt and M. Goodfellow (Eds). The PCR product was used as template

25 in sequencing reactions using an ABI PRISM™ DYE Terminator Cycle sequencing Kit (Perkin Elmer). Partial sequences were assembled using the GCG Fragment Assembly System (Wisconsin Package, version 8) and sequences were aligned with the program CLUSTALW (Intelligenetics, Inc.). The phylogenetic analysis of the aligned sequences was performed using the maximum-parsimony analysis with the branch-and-bound algorithm of the Phylogeny Using

30 Parsimony Analysis (PAUP) program version 4.0. (Swofford D. L. (1993). PA UP: Phylogenetic analysis using parsimony, version 3.1.1. Washington D.C.: Laboratory of Molecular Systematics, Smithsonian Institute).

Isolation conditions

35 The strain was obtained from a soil collected in Namaqualand, South Africa and associated to the rhizosphere of a specimen of Dinteranthus microspermus. The strain was isolated after high 2205 IY

centrifiigation speed enrichment method and plating on NZ amine A based agar supplemented with 20 ug/ml nalidixic acid.

General growth characteristics

5 The strain grows well on a range of agar media such as Yeast Malt Extract, Oatmeal, Glycerol Asparagine, Inorganic Salts Starch and Tyrosine agars at 28⁰C. The gross colonial morphology is typical of Micromonosporaceae and its growth characteristics, sporulation structures, substrate mycelial pigmentation and the production of different pigments were recorded in different agar media (Table 1).

10 - Colony morphology (on Yeast Malt Extract Agar, ISP2): flat colonies with well developed light orange (5A5) substrate mycelium, sterile even after 21 days of incubation, no aerial mycelium is present.

- Micromorphology: mycelial development and morphology were examined directly on the plates by light microscopy under 40Ox and IOOOX magnification. Observations were made after

15 7, 14 and 21 days of cultivation on Yeast Malt Extract, Oatmeal, Glycerol Asparagine, Inorganic Salts Starch and Tyrosine agars. The extensive branched substrate mycelium was sterile in all media excepting on Inorganic Salts Starch agar where globose vegetative sporangia develop quite early, at the end of sporophores formed on substrate hyphae after 7 days incubations.

20 Chemotaxonomic analysis

The analysis of cell wall composition shows that strain P-000503667 contains meso-diamino pimelic acid in whole-cell hydrolysates, a characteristic of Micromonosporaceae, and galactose as major cell wall sugar.

The strain cultivated in Trypticase Soy Agar (TSBA) for 13 days at 28°C is rich in saturated and 25 unsaturated fatty acids and whole-cell methanoly sates contain the predominant fatty acids 15:0 iso (9.78%),15:0 (10.61%), 16:0 iso (22.76%), 16:1 cis9 (6.71%) and 17:1 cis9 (30.84%). A complete fatty acid composition is given in Table 2.

Physiological properties

30 Growth on the following compounds as sole carbon sources was determined after observations made at 7, 14 and 21 days. Strain P-000503667 presents the following carbon utilization pattern: good utilization of D-glucose^", D-mannitol, rhamnose, L-arabinose, D-xylose, D-fructose, I- inositol and sucrose; no utilization of cellulose and rafϊinose.

35 16S rDNA Sequence and Phylogenetic Analysis

Sequence was aligned with Micromonosporaceae nucleotide sequences from Genbank. See Sequence below a phylogenetic tree based on these 16S rDNA sequences was built using the 2205 IY

maximum parsimony method. Bootstrap replicates from each grouping was used as a measure of statistical confidence.

The 16s rRNA sequence analysis indicates that strain P-000503667 belongs to the genus Actinoplanes. The general description, both colony morphology and micro morphology is also consistent with the genus Actinoplanes. Based on the data presented herein, P-000503667 should be classified and identified as a species in the genus Actinoplanes.

Furthermore the strain P-000503667 is associated in the phylogenetic tree to the strain Actinoplanes philippinensis DSM 43019. This close relationship is highly supported by the

10 bootstrapping value (88%) and suggests that this isolate could be identified as another strain of the species Actinoplanes philippinensis.

Table 1. Cultural characteristics of Actinoplanes P-000503667 (21 days, 28⁰C)

2205 IY

Table 2: Major fatty acids found in strain P-000503667.

5 16S rDNA sequence of strain P-000503667 (ATCC# PTA 7551).

(SEQ ID NO:1) Length: 1474

1 GACGAACGCT GGCGGCGTGC TTAACACATG CAAGTCGAGC GGAAAGGCCC

10

51 TTCGGGGTAC TCGAGCGGCG AACGGGTGAG TAACACGTGA GTAACCTGCC ioi CTGGACTTTG GGATAACCCT CGGAAACGGG GGCTAATACC GAATACGACC

15 151 TTGGACCGCA TGGTGTCTTG GTGGAAAGTT TTTTCGGTTT GGGATGGACT

201 CGCGGCCTAT CAGCTTGTTG GTGGGGTAAT GGCCTACCAA GGCGACGACG

251 GGTAGCCGGC CTGAGAGGGC GACCGGCCAC ACTGGGACTG AGACACGGCC

20

301 CAGACTCCTA CGGGAGGCAG CAGTGGGGAA TATTGCACAA TGGGCGGAAG 2205 IY

351 CCTGATGCAG CGACGCCGCG TGAGGGATGA CGGCCTTCGG GTTGTAAACC

401 TCTTTCAGCA GGGACGAAGC GCAAGTGACG GTACCTGCAG AAGAAGCGCC 5

451 GGCCAACTAC GTGCCAGCAG CCGCGGTAAG ACGTAGGGCG CGAGCGTTGT

501 CCGGATCCAA TGGGCGTaAA GAGCTCGTAg GCgGCTTGTC GCGTCGAATG

10 551 TGAAAACTTG GGGCTCAACT CCAAGCTTGC ATTCGATACG GGCAGGCTAG

601 AGTTCGGTAG GGGAGACTGG AATTCCTGGT GTAGCGGTGA AATGCGCAGA

651 TATCAGGAGG AACACCGGTG GCGAAGGCGG GTCTCTGGGC CGATACTGAC 15

701 GCTGAGGAGC GAAAGCGTGG GGAGCGAACA GGATTAGATA CCCTGGTAGT

751 CCACGCTGTA AACGTTGGGC GCTAGGTGTG GGGACCCTCT CCGGGTTTCT

20 801 GCGCCGCAGC TAACGCATTA AGCGCCCCGC CTGGGGAGTA CGGCCGCAAG

851 GCTAAAACTC AAAGGAATTG ACGGGGGCCC GCACAAGCGG CGGAGCATGC

901 GGATTAATTC GATGCAACGC GAAGAACCTT ACCTGGGTTT GACATGCACG 25

951 GAAATCCTCC AGAGATGGGG GGTCCTTCGG GGTCGTGCAC AGGTGGTGCA

1001 TGGCTGTCGT CAGCTCGTGT CGTGAGATGT TGGGTTAAGT CCCGCAACGA

30 1051 GCGCAACCCT CGTTCGATGT TGCCAGCGCG TTATGGCGGG GACTCATCGA

1101 AGACTGCCGG GGTCAACTCG GAGGAAGGTG GGGATGACGT CAAGTCATCA

1151 TGCCCCTTAT GTCCAGGGCT TCACGCATGC TACAATGGCC GGTACAAAGG 35

1201 GCTGCGAGAC CGTGAGGTTG AGCGAATCCC AAAAAGCCGG TCTCAGTTCG 2205 IY

1251 GATCGGGGTC TGCAACTCGA CCCCGTGAAG TCGGAGTCGC TAGTAATCGC

1301 AGATCAGCAA CGCTGCGGTG AATACGTTCC CGGGCCTTGT ACACACCGCC

5 1351 CGTCACGTCA CGAAAGTCGG CAACACCCGA AGCCGGTGGC CTAACCCCTT

1401 GTGGGAGGGA GCCGTCGAAG GTGGGGCTGG CGATTGGGAC GAAGTCGTAA

1451 CAAGGTAGCC GTACCGGAAG GTGC 10

Phylogenetic tree of MA7347 (=P-000503667)

THE PROTOCOLS USED TO DETERMINE THE ANTIBACTERIAL ACTIVITY OF THE

COMPOUNDS OF FORMULA I ARE DESCRIBED BELOW. 15

MATERIALS:

Cation-Adjusted Mueller Hinton Broth (MH; BBL)

50% Lysed Horse Blood (LHB; BBL) (stored frozen)

RPMI 1640 (BioWMttaker) 20 Human Serum (Pel-Freez)

RPMI 1640 (BioWhittaker)

Haemophilus Test Medium (HTM, Remel)

Trypticase Soy Broth (TSB, 5 mL/tube; BBL)

0.9% Sodium Chloride (Saline; Baxter) 25 Trypticase Soy + 5% Sheep Blood Agar Plates (TSA; BBL)

Sabouraud Dextrose Agar Plates (BBL)

Chocolate Agar Plates (BBL)

2X Skim Milk (Remel)

Microbank Beads (Kramer Scientific) 30 MIC 2000 Microtiter plate inoculator.

2X Trypticase Soy Broth (TSB₅ BBL) + 15% glycerol/50% horse serum.

96- Well Microtiter plates, lids, inoculum trays (Dynex Laboratories)

8-Channel Finn Multichannel pipettor, 0.5-10 uL volume 2205 IY

METHODS:

MEDIA PREPARATION

Cation- Adjusted Mueller Hinton Broth (BBL): Prepared according to manufacturer's 5 instructions (22 gms dissolved in 1000'mL water; autoclaved 22 minutes). Stored refrigerated. Filter-sterilized before use using a Corning 0.45 Tm cellulose acetate filter.

50% Lysed Horse Blood: Defϊbrinated horse blood is diluted 1 : 1 with sterile distilled water; frozen, thawed and re-frozen (at least 7 times), then centrifuged. Stored frozen at -2O⁰C. 10

Cation- Adjusted Mueller Hinton + 2.5% Lysed Horse Blood: Aseptically add 5 mL 50% lysed horse blood to 100 mL Cation- Adjusted Mueller Hinton Broth. Filter-sterilize before use using a Corning 0.45 Tm cellulose acetate filter.

15 Cation- Adjusted Mueller Hinton + 50% Human Serum: Aseptically add 50 mL Human Serum to 50 mL 2X Cation-Adjusted Mueller Hinton Broth. Filter-sterilize before use using a Corning 0.45 Tm cellulose acetate filter.

Haemophilus Test Medium (Remel): Received prepared from manufacturer. Filter-sterilized 20 before use using a Corning 0.45 Tm cellulose acetate filter.

0.9% Sodium Chloride (Saline; Abbott Labs): Received prepared from manufacturer.

2X Skim Milk (Remel): Received prepared from manufacturer. 25

All agar plates are received prepared from manufacturer.

2205 IY

CONDITIONS AND FOR REPRESENTATIVE STRAINS

INOCULUM

BACILLUS, INCUBATION CONDITIONS, 35°C; MICS READ AT 18-22

STAPHYLOCOCCUS, HOURS;

ENTEROCOCCUS:

ESCHERICHI: CATION-ADJUSTED MUELLER HINTON (CAMHB; BBL);

INOCULUM = 10⁵ CFU/ML

STREP. PNEUMONIAE: INCUBATION CONDITIONS, 35°C; MICS READ AT 22-24

HOURS;

CATION-ADJUSTED MUELLER HINTON+ 2.5% LYSED

HORSE BLOOD (LHB); INOCULUM = 10⁵ CFU/ML

HAEMOPHILUS INCUBATION CONDITIONS, 35°C; MICS READ AT 18-22

INFLUENZAE: HOURS;

HAEMOPHILUS TEST MEDIUM (HTM; REMEL); INOCULUM

= 10⁵ CFU/ML

CANDIDA: INCUBATION CONDITIONS, 35°C; MICS READ AT 24

HOURS; RPMI 1640 MEDIUM (BIOWHITTAKER)

INOCULUM = 10³ CFU/ML

HIGHEST CONCENTRATION OF ANTIBIOTIC TESTED = 64 μG/ML (WHEN STARTING

FROM A 1 MG/ML SOL¹N IN 50% DMSO)

FINAL CONCENTRATION OF DMSO PER WELL = 3.2%

SELECTION AND MAINTENANCE OF ISOLATES

The strains used are isolates from either the Merck Culture Collection, the Merck Clinical Culture Collection or from Clinical Trials. The strain of Haemophilus influenzae is a mouse pathogen used for in vivo testing at Merck. The Escherichia coli strain is a cell wall permeable strain. The Candida albicans strain is used as a control. These culture are maintained as frozen stocks at -8O⁰C in a) Microbank beads; b) 2X Skim Milk; or c) in 2X Trypticase Soy Broth + 15% glycerol/50% horse serum (Haemophilus and Streptococcus pneumoniae).

10

INOCULUM PREPARATION

Selected isolates are sub-cultured onto either Chocolate Agar Plates (Haemophilus influenzae), onto Trypticase Soy + 5% Sheep Blood Agar Plates (Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Enterococcus, Bacillus) or onto Sabouraud Dextrose

15 Agar (Candida) and incubated at 35°C. Haemophilus and Streptococcus pneumoniae are 2205 IY

incubated in 5% CO₂; all other isolates are incubated in ambient air. Isolates are sub-cultured 2X before assay.

Colonies are selected from plates and used to prepare an inoculum equivalent to a 0.5 McFarland standard in Trypticase Soy Broth. An inoculum with a density equivalent to a 1.0 5 McFarland standard is prepared for Streptococcus pneumoniae. The inoculum density for all cultures is ~10⁸ CFU/mL in TSB. This TSB inoculum is diluted 1 : 10 in sterile saline (4 mL inoculum + 36 mL saline; equivalent to ~10⁷ CFU/mL) and kept on ice until used to inoculate microtiter plates.

Colony counts are performed on randomly-selected isolates to confirm

10 CFU/well (TSB inoculum plated out 10^~5, 10^"6 onto either TSA II + 5% SB or onto chocolate agar plates, incubated overnight, 35⁰C, CO₂)

PLATE FILLING

All wells of 96-well microtiter plates (Dynex) are filled with 100 TL media.

15 Haemophilus test media plates are prepared to test Haemophilus influenzae; Cation- Adjusted Mueller Hinton + 5% Lysed Horse Blood plates are prepared to test Streptococcus pneumoniae; Cation-Adjusted Mueller Hinton Broth plates are prepared to test Enterococcus, Staphylococcus aureus, Escherichia coli and Bacillus subtilis. RPMI 1640 is used to test Candida. The MICs against S. aureus Smith are determined in Cation-adjusted Mueller Hinton and in Cation-

20 Adjusted Mueller Hinton + 50% Human Serum, to determine if the compound is inactivated by some component in serum (indicated by an increase in the MIC). Filled plates are wrapped in plastic bags (to minimize evaporation), stored frozen and thawed before use.

PREPARATION OF COMPOUNDS 25 The compounds are prepared on a weight basis. Compounds are prepared to 2-10 mg/rnL in 100% DMSO, then diluted to lmg/mL in a 1 :1 dilution of DMSO/2x CAMHB (final concentration=50%DMSO/50% CAMHB). Compounds are serially diluted 1:1 in 50%

DMSO/50% CAMHB in BD Biosciences Deep Well Polypropylene 96 well plates (starting concentration 1-5 mg/mL). 30

MICROBROTH DILUTION ASSAY

Using a Finn Automated Multichannel Pipette, (0.5-10 μL volume) 6.4 mLs of antimicrobial working solutions are added to wells of filled microtiter plates (concentration of antimicrobial in first well = 512-64 microg/mL; concentration of DMSO = 3.2%). 35 Antimicrobials are added in this manner to keep constant the amount of DMSO in each well (to keep compounds solubilized and to account for the possibility of non-specific killing by the

DMSO. The last row contains a growth control of 3.2% DMSO. With each assay, controls are run. They are Penicillin G and chloramphenicol, prepared in the same manner as the compounds. Ertapenem is included as a control for the serum protein binding assay.

5 PLATE INOCULATION

All wells of microliter plates are inoculated with (saline-diluted) culture using the MIC 2000 System, an automated plate inoculating device which delivers an inoculum of 1.5 mL per well. Plates are incubated at 35⁰C in ambient air. An uninoculated plate is also incubated as a sterility check. Results are recorded after 22-24-hours' incubation. Plates were read to no 10 growth. The MIC is defined as the lowest antimicrobial level which resulted in no growth after 22-24-hours' incubation.

Compound I demonstrates antibacterial activity against various strains of S. aureus, E. faecalis, E. faecium, B. subtilus and S. pneumoniae. Compounds Ia and Ib also

15 demonstrate antibacterial activity against various species that are resistant to many known antibiotics such as methicillin-resistant S. aureus (MRSA)₅ vancomycin-resistant Enterococcus sp. (VRE), multidrug-resistant E. faecium, macrolide-resistant S. aureus and S. epidermidis, and linezolid-resistant S. aureus and E. faecium. The minimum inhibitory concentration (MIC) values for these test strains range from 0.0001 to 200 μg/mL. MICs are obtained in accordance to the

20 NCCLS guidelines.

- 20 -

Claims

2205 IYWHAT IS CLAIMED IS:

1. A compound of structural formula I:

or a pharmaceutically acceptable salt, ester, enantiomer, diastereomer or mixture thereof, wherein R represents:

2. A compound which is:

or a pharmaceutically acceptable salt, ester, enantiomer, diastereomer or mixture thereof.

- 22 - 2205 IY

3. A compound which is:

5 4. A process for the preparation of the compound of structural formula I of claim I₅ which comprises the cultivation of a Actinoplanes sp. ATCC PTA 7551 or a natural or artificial mutant thereof in a nutrient medium and recovering the compound of structural formula I from the fermentation broth.

10 5. The process of Claim 4 wherein the fermentation is conducted at a temperature of about 10⁰C to about 40⁰C.

6. The process of Claim 5, wherein the fermentation is conducted at a temperature of about 26-30⁰C. 15 2205 IY

7. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a compound of structural formula I of claim 1.

8. Use of a compound of formula I of claim 1 for the manufacture of a 5 medicament for treating a bacterial infection in a host in need of such treatment.

9. A method of treating a bacterial infection in a host in need of such treatment comprising the administration of an effective amount of the compound of formula I.

10 10. Actinoplanes sp. with ATCC accession number ATCC PTA 7551 (MA

7347) or a natural or artificial mutant thereof.