# Patent

## Title
Antibiotic compound

## Abstract

Fermentation of a nutrient medium with a eubacterium Streptomyces sp. yields a novel antibacterial compound of structural formula (I).

## Background
This Application claims the benefit of U.S. Provisional Application 60/573,899, filed May 24, 2004.


BACKGROUND OF THE INVENTION
The present invention relates to a novel natural product that possesses antibacterial activity.
Infections caused by bacteria are a growing medical concern as many of these pathogens are resistant to various common antibiotics. Such microbes include Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, Actinobacter calcoaeticus, Escherichia coli and Stenotrophomonas maltophilia. The antibiotic of this invention comprises an important contribution to therapy for treating infections that are resistant to various known antibiotics. For a review see: F. D. Lowy The Journal of Clinical Investigation 2003, 111 (9), 1265.
In the present invention, a novel natural product isolated from the eubacterial fermentation of Streptomyces sp. is described. This compound displays antibacterial activity against various pathogens, many of which have demonstrated resistance to currently available antibiotics.
SUMMARY OF THE INVENTION
This invention describes the novel natural product shown in formula I and its use as an antibacterial agent:




or a pharmaceutically acceptable salt thereof which is effective in the treatment of bacterial infections.

The invention is also concerned with a process for the production of Compound I by fermentation with the eubacterium, Streptomyces sp. The invention is also concerned with a process for isolating the compounds of Formula I from fermentation broths.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a 13C NMR spectrum of Compound I in C5D5N.
FIG. 2 is a 1H NMR spectrum of Compound I in C5D5N.



DETAILED DESCRIPTION OF THE INVENTION
This invention describes the compound of structural formula I:




or a pharmaceutically acceptable salt thereof.

The pharmaceutically acceptable salts of the compound 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.
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.
Compound I of this invention displays antibiotic activity useful in the treatment of bacterial infections. It demonstrates antibacterial activity against various strains of S. aureus, S. pneumoniae, E. faecalis, E. faecium, B. subtilis and E. coli including species that are resistant to many known antibiotics such as methicillin-resistant S. aureus (MRSA), vancomycin-resistant Enterococcus sp. (VRE), multidrug-resistant E. faecium, macrolide-resistant S. aureus and S. epidermidis, and linezolide-resistant S. aureus and E. faecium. 
The compound of this invention can be formulated in pharmaceutical compositions by combining Compound I with a pharmaceutically acceptable carrier. Examples of such carriers are set forth below.
The compound may be employed in powder or crystalline form, in liquid solution, or in suspension. It may be administered by a variety of means; those of principal interest include topically, orally and parenterally by injection (intravenously or 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 (lyophillized or non-lyophillized) form for reconstitution at the time of delivery with a suitable vehicle, such as sterile water. In 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 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 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 treatment well known in the antibacterial arts.
The compositions for administration to humans per unit dosage, whether liquid or solid, may contain from about 0.01% 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 15 mg to about 2.5 g of Compound I, one embodiment of this range being from about 250 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.
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 Compound Ito the mammal in an amount effective to treat the infection.
One embodiment of the methods of administration of Compound I includes oral and parenteral methods, e.g., i.v. infusion, i.v. bolus and i.m. injection.
For adults, about 5-50 mg of Compound I per kg of body weight given one to four times daily is preferred. The preferred dosage is 250 mg to 1000 mg of the antibacterial given one to four times per day. More specifically, for mild infections a dose of about 250 mg two or three times daily is recommended. For moderate infections against highly susceptible gram positive organisms a dose of about 500 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 1000-2000 mg three to four times daily may be recommended.
For children, a dose of about 5-25 mg/kg of body weight given 2, 3, or 4 times per day is preferred; a dose of 10 mg/kg is typically recommended.
Another aspect of this invention is the process for producing Compound I, which comprises cultivating a Streptomyces sp. microorganism in a suitable nutrient medium and then recovering the compound of this invention from the fermentation broth. The Streptomyces sp. microorganism, ATCC# PTA-5942 identified as the eubacterium Streptomyces sp. with the following taxonomic studies is deposited in the Merck Culture Collection as MA7339.
The organism was subsequently placed on permanent deposit with the Amercian Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., 20852 and have been assigned accession number ATCC# PTA-5942 (Merck# MA7339).
Any restrictions relating to public access to the microorganism shall be irrevocably removed upon patent issuance. Although the use of these 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 compound of structural Formula I is produced by the aerobic fermentation of a suitable medium under controlled conditions via inoculation with a culture of the eubacterium, Streptomyces sp. The suitable medium is preferably aqueous and contains sources of assimilable carbon, nitrogen, and inorganic salts.
The medium employed for fermentation by the Streptomyces sp. is primarily the well-known Difco Tryptic Soy Broth, either alone or with added nutrients commonly used by those skilled in the art.
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.
The fermentation is conducted at temperatures ranging from about 10° C. to about 40° C.; however for optimum results it is preferred to conduct the fermentation at about 28° C. 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 invention, the invention is not limited to the use of the particular Streptomyces sp. with ATCC accession number, ATCC# PTA-5942 (Merck# MA7339). 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 Streptomyces genus insofar as they can produce the compound of this invention. The artificial production of mutant species or strains of Streptomyces from ATCC# PTA-5942 (Merck# MA7339) 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.
EXAMPLE 1
Production of Compound I—










SEED Media composition:










Component
g/L














Soluble starch
20



Dextrose
10



NZ Amine type E
5



Difco Beef Extract
3



Bacto (Difco) Peptone
5



Difco Yeast Extract
5



CaCO3
1







pH 7.0
















YME.TE (Production Medium, g/l)










Media Component
g/L














*Difco yeast extract
6



*Malt extract
15



*Dextrose
6



Trace Elements
5 ml



MOPS
20







pH = 7.0






A frozen suspension (1.3 mL) of a Streptomyces sp. ATCC# PTA-5942 (MA7339) was inoculated into a 250 mL flask containing 50 mL of seed medium. The flask was incubated at 28.0° C. with an agitation of 220 RPM for 48 hours. The second stage seed was developed by transferring 3% inoculum of the first stage seed into a 250 ml shake flask containing 50 mL of seed medium. The flask was incubated at 28.0° C. with an agitation of 220 RPM for 24 hours. A 5% inoculum of the second stage seed was transferred to 30ml of YME-TE in a 250ml and incubated at 32.0° C. with an agitation of 220 RPM for 12 days.

Isolation of Compound I:

To a two liter fermentation broth was added two liter of acetone and shaken on a shaker for two hours and filtered. The filtrate was concentrated under reduced pressure to remove most of the acetone and charged on to a 75 mL amberchrome (CG161s) column. The column was eluted with a gradient of 90% water to 100% methanol that eluted the compound in a borad zone which upon concentration and lyophilization afforded 170 mg of semi-purified fraction. A 80 mg portion of the fraction was purified by prep HPLC (Zorbax Rx C8 21.4×250 mm with a gradient of 20-98% aqueous acetonitrile containing 0.1% trifluoroacetic acid to produce 1.1 mg of compound I. The structure was elucidated by spectroscopic analysis (see below).

Physical Data of Compound I:








TABLE






















MW
425


MF
C24H27NO6


HEESIFTMS
Found: 426.1911; calcd for M + H: 426.1911


[α]23D
+2.1° (c 0.96, CH3OH)


UV (CH3OH) λmax
226 (ε 16,837) 296 (2,663) nm










1H and 13C NMR Assignment of Compound I at 500 MHz












#
C5D5N13C
Type
C5D5N1H
















 1
175.2
Co 




 2
32.2
CH2
 2.70, m



 3
31.8
CH2
 2.53, m






 1.92, m



 4
48.1
Co 




 5
204.1
Co 




 6
126.8
CH
 5.92, d, 10.0



 7
154.9
CH
 6.36, d, 10.0



 8
36.7
Co 




 9
40.4
CH
 2.00, t, 10.2



10
27.1
CH2
 1.60, m






 1.36, brt, 10.2



11
36.6
CH
 2.26, m



12
26.5
CH2
 1.78, m






 1.56, m



13
28.5
CH2
 1.56, t, 7.8






 1.80, m



14
44.9
CH
 1.93, d, 10.2






 2.15, d, 10.2



15
149.8
Co 




16
107.8
CH2
 4.71, brs






 4.87, brs



17
21.5
CH3
 1.09, s



 1′
175.2
Co 




 2′
107.6
Co 




 3′
158.8
Co 




 4′
115.8
Co 




 5′
158.3
Co 




 6′
110.4
CH
 6.88, d, 8.4



 7′
129.8
CH
 8.12, d, 8.4



 8′

NH
10.50, s



 3′-OH






 5′-OH










Characterization of Culture

General description of culture MA-7339 producer of compound I is described. 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).
Chemical composition of the cells was determined using the methods of Lechevalier and Lechevalier (1980).
Fatty acid composition was determined using a modified sample preparation (Sasser, 1990). 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×25 m). Individual fatty acids identification was determined by the Microbial Identification System software.
The complete 16S rDNA sequence was determined from the 1500 by PCR fragment obtained using primers 27f and 1525r (Lane, 1991). The PCR product was used as template in sequencing reactions using an ABI PRISMTM 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 Parsimony Analysis (PAUP) program version 4.0. (Swofford, 1993).
Source:
Strain MA7339 was obtained from a soil collected in Mallorca, Balearic Islands, Spain. The strain was isolated after pretreatment of the soil with 1% (w/v) chloramine T and plating on humic-acid based agar supplemented with 20 ug/ml nalidixic acid. After purification on Yeast Malt Extract agar, the isolate was detected active when tested as agar plug in the FabF_SPAR_C screen.
General Growth Characteristics.
Strain MA7339 grows well on a range of agar media such as Yeast Malt Extract, Oatmeal, Glycerol Asparagine, Inorganic Salts Starch and Trypticase Soy agars at 28° C. The gross colonial morphology is typical of streptomycetes and its growth characteristics, including spore-mass colour, substrate mycelial pigmentation and the production of different pigments were recorded in different agar media (Table 1).
Colony morphology (on Yeast Malt Extract Agar, ISP2): Substrate mycelium initially whitish yellow turns yellowish brown (5D7) after 21 days of incubation. The initial white aerial mycelium continues to develop after 21 days incubation turning whitish grey (5E1/5E2) with brownish wet exudate droplets.
Micromorphology: the spore-chain morphology was examined directly on the plates by light microscopy under 400× and 1000× magnification. Observations were made after 7, 14 and 21 days of cultivation on Yeast Malt Extract agar. The aerial mycelium arises from extensive branched substrate hyphae. Sparse branched aerial hyphae differentiate initially into short and irregularly tight coiled spore chain spirals. Sporophores are formed by less than 10-20 spores and with time tend to coalesce in a dark mucous mass of spores in older cultures. Similar morphologies were observed in most of the other test media but with different degrees of coalescence. On the contrary in the glycerol asparagine agar the strain grows as a sterile vegetative mycelium.
Chemotaxonomic Analysis.
The analysis of cell wall composition shows that strain MA7339 contains LL-A2pm in whole-organism hydrolysates, a characteristic of Streptomyces, and glucose and ribose as major cell wall sugars. The strain is rich in saturated straight-chain and iso- and anteiso- fatty acids and whole-cell methanolysates contain the predominant fatty acids 15:0 anteiso (12.43%) and 16:0 iso (17.94%), which are also typical of Streptomyces. Nevertheless the major component is the fatty acid species 15:0 iso (20.43%). A complete fatty acid composition is given in Table 2.
All these chemotaxonomic analyses indicate that the strain corresponds to a member of the genus Streptomyces. 
Physiological Properties.
Strain MA7339 presents the following carbon utilization pattern (Table 3):
good utilization of sucrose, D-xylose, D-fructose and raffinose; moderate utilization of D-glucose, I-inositol, and D-mannitol; and no utilization of L-arabinose, cellulose and rhamnose.
16S rDNA Sequence and Phylogenetic Analysis.
The complete 16S rDNA sequence has been determined for strain MA-7339 (FIG. 1). Sequence was aligned with Streptomyces nucleotide sequences from Genbank (AB045882) and the S. platensis strains MA7327 and MA7331. A phylogenetic tree based on these 16S rDNA sequences was built using the maximum parsimony method. Bootstrap replicates from each grouping was used as a measure of statistical confidence. A grouping found on 95% of bootstrap replicates was considered statistically significant.
The strain MA7339 is associated to the strain Streptomyces platensis ATCC 13865 and the strains MA7327 and MA7331. This close relationship is highly supported by the bootstrapping value (97%) and suggests that this isolate can be identified as another strain of the species Streptomyces platensis (FIG. 2).







TABLE 1







Cultural characteristics of Streptomyces sp. MA7339 (21 days, 28° C.)












Amount

Soluble
Substrate


Medium
of growth
Aerial Mycelium
pigments
Mycelium





Yeast Extract
Abundant
Whitish grey (5E1/5E2), dense
None
Yellowish brown


Malt Extract

growth, extensive aerial

(5D7)


(ISP2)

mycelium with short and tight




irregular spore chains forming




loops and coils. Sporophores




born in main and secondary aerial




branches, coalescence.


Oatmeal (ISP3)
Abundant
Grey (5E2/F2), extensive aerial
None
Olive brown




mycelium, coalescence of tight

(4E3)




coiled spore chains.


Inorganic Salts
Abundant
Greyish brown (5D3/F3),
None
Light yellow


Starch (ISP4)

extensive aerial mycelium,

(4A4)




coalescence of chains.


Glycerol
Sparse
none
None
Greyish


Asparagine



orange(5B4),


(ISP5)



sterile substrate






mycelium


Tyrosine
Abundant
Orange grey (5B2), extensive
none
Dark brown


Agar

aerial mycelium growth, short

(6F7)


(ISP7)

and tight spirals in aerial hyphae,




collapsing in coalescence and




knots
















TABLE 2







Major fatty acids found in strain MA7339.










Fatty acid
% of total fatty acids














14:0 iso
5.66



15:0 iso
20.43



15:0 anteiso
12.43



15:0 anteiso 2OH
8.00



15:0
3.48



16::0
2.52



16:0 iso
17.94



16:1 iso H
3.54



16:0 iso 20H
1.14



17:0 anteiso
3.58



17:0 cyclo
1.48



17:0 iso
3.36



17:1 iso C
2.28



17:1 anteiso C
2.28

















TABLE 3







Carbohydrate utilization patterns of strains MA7339.










Carbon source
Growth levels







D-glucose
2



L-arabinose
0



Sucrose
3



D-xylose
3



I-inositol
2



D-mannitol
2



D-fructose
3



Rhamnose
0



Raffinose
3



Cellulose
0







Growth on the following compounds as sole carbon sources; Observations were made at 7, 14 and 21 days, 28° C.; Growth levels: 3 = good utilization; 2 = moderate utilization; 1 = poor utilization; 0 = no utilization.






FIG. 1. 16S rDNA sequences of strain MA7339.









Strain MA7339 16S rDNA region (from: 1 to: 1445)










1
CCTCCTTCGG GAGGGGATTA GCTGGCGAAC GGGTGAGTAA CACGTGGGCA



 


51
ATCTGCCCTT CACTCTGGGA CAAGCCCTGG AAACGGGGTC TAATACCCGG


 


101
ATACGACACA CGACCGCATG GTCTGTGTGT GGAAAGCTCC GGCGGTGAAG


 


151
GATGAGCCCG CGGCCTATCA GCTTGTTGGT GGGGTGATGR CCTACCAAGG


 


201
CGACGACGGG TAGCCGGCCT GAGAGGGCGA CCGGCCACAC TGGGACTGAG


 


251
ACACGGCCCA GACTCCTACG GGAGGCAGCA GTGGGGAATA TTGCACAATG


 


301
GGCGAAAGCC TGATGCAGCG ACGCCGCGTG AGGGATGACG GCCTTCGGGT


 


351
TGTAAACCTC TTTCAGCAGG GAAGAAGCGA GAGTGACGGT ACCTGCAGAA


 


401
GAAGCGCCGG CTAACTACGT GCCAGCAGCC GCGGTAATAC GTAGGGCGCA


 


451
AGCGTTGTCC GGAATTATTG GGCGTAAAGA GCTCGTAGGC GGCTTGTCAC


 


501
GTCGGATGTG AAAGCCCGGG GCTTAACCCC GGGTCTGCAT TCGATACGGG


 


551
CAGGCTAGAG TTCGGTAGGG GAGATCGGAA TTCCTGGTGT AGCGGTGAAA


 


601
TGCGCAGATA TCAGGAGGAA CACCGGTGGC GAAGGCGGAT CTCTGGGCCG


 


651
ATACTGACGC TGAGGAGCGA AAGCGTGGGG AGCGAACAGG ATTAGATACC


 


701
CTGGTAGTCC ACGCCGTAAA CGTTGGGAAC TAGGTGTGGG CGACATTCCA


 


751
CGTCGTCCGT GCCGCAGCTA ACGCATTAAG TTCCCCGCCT GGGGAGTACG


 


801
GCCGCAAGGC TAAAACTCAA AGGAATTGAC GGGGGCCCGC ACAAGCAGCG


 


851
GAGCATGTGG CTTAATTCGA CGCAACGCCA AAGAACCTTA CCAAGGCTTG


 


901
ACATACACCG GAAACGTCTG GAGATCAGGC GCCCCCTTGT GTCCGGTGTA


 


951
TCATGGTGGT GCATGGCTGT CGTCAGCTCG TGTCGTGAGA TGTTGGGGTT


 


1001
TAAKTCCCCG CAACGAGCGC AACCCTTGTT CTGTGTTGCC AGCATGCCCT


 


1051
TCGGGGTGAT GGGGACTCAC AGGAGACTGC CGGGGTCAAC TCGGAGGAAG


 


1101
GTGGGGACGA CGTCAAGTCA TCATGCCCCT TATGTCTTGG GCTGCACACG


 


1151
TGCTACAATG GCCGGTACAA TGAGCTGCGA TACCGCGAGG TGGAGCGAAT


 


1201
CTCAAAAAGC CGGTCTCAGT TCGGATTGGG GTCTGCAACT CGACCCCATG


 


1251
AAGTCGGAGT TGCTAGTAAT CGCAGATCAG CATTGCTGCG GTGAATACGT


 


1301
TCCCGGGCCT TGTACACACC GCCGGTCACG TCACGAAAGT CGGTAACACC


 


1351
CGAAGCCGGT GGCCCAACCC CTTGTGGGAG GGAATCGTCG AAGGTGGGAC


 


1401
TGGCGATTGG GACGAAGTCG TAACAAGGTA GCCGTACCGG AAGGT






The protocols used to determine the antibacterial activity of Compound I are described below.
Materials:


    Cation-Adjusted Mueller Hinton Broth (MH; BBL)
    50% Lysed Horse Blood (LHB; BBL) (stored frozen)
    RPMI 1640 (BioWhittaker)
    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)
    Trypticase Soy+5% Sheep Blood Agar Plates (TSA; BBL)
    Sabouraud Dextrose Agar Plates (BBL)
    Chocolate Agar Plates (BBL)
    2× Skim Milk (Remel)
    Microbank Beads (Kramer Scientific)
    MIC 2000 Microtiter plate inoculator.
    2× Trypticase Soy Broth (TSB, BBL)+15% glycerol/50% horse serum.
    96-Well Microtiter plates, lids, inoculum trays (Dynex Laboratories)
    8-Channel Finn Multichannel pipettor, 0.5-10 μL volume

Methods:

Media Preparation



Cation-Adjusted Mueller Hinton Broth (BBL): Prepared according to manufacturer's 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: Defibrinated horse blood is diluted 1:1 with sterile distilled water; frozen, thawed and re-frozen (at least 7 times), then centrifuged. Stored frozen at −20° C.
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.
Cation-Adjusted Mueller Hinton+50% Human Serum: Aseptically add 50 mL Human Serum to 50 mL 2× 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 before use using a Corning 0.45 Tm cellulose acetate filter.
0.9% Sodium Chloride (Saline; Abbott Labs): Received prepared from manufacturer.
2× Skim Milk (Remel): Received prepared from manufacturer.
All agar plates are received prepared from manufacturer.










Conditions and Inoculum for Representative Strains
















Bacillus, Staphylococcus,
Incubation Conditions, 35° C.; MICs read at 18-22 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 influenzae:
Incubation Conditions, 35° C.; MICs read at 18-22



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 sol'n 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, 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 −80° C. in a.) Microbank beads; b.) 2× Skim Milk; or c.) in 2× Trypticase Soy Broth+15% glycerol/50% horse serum (Haemophilus and Streptococcus pneumoniae).
Inoculum Preparation
Selected isolates are subcultured 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 Agar (Candida) and incubated at 35° C. Haemophilus and Streptococcus pneumoniae are incubated in 5% CO2; all other isolates are incubated in ambient air. Isolates are subcultured 2× 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 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 CFU/well (TSB inoculum plated out 10−5, 10−6 onto either TSA II+5% SB or onto chocolate agar plates, incubated overnight, 35° C., CO2)
Plate Filling
All wells of 96-well microtiter plates (Dynex) are filled with 100 TL media. 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 col 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-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
The compounds are prepared on a weight basis. Compounds are prepared to 2 mg/mL in 100% DMSO, then diluted to 1 mg/mL in a 1:1 dilution of DMSO/2× 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 mg/mL).
Microbroth Dilution Assay
Using a Finn Automated Multichannel Pipette, (0.5-10 μL volume) 6.4 TLs of antimicrobial working solutions are added to wells of filled microtiter plates (concentration of antimicrobial in first well=64 μg/mL; concentration of DMSO=3.2%). 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.
Plate Inoculation
All wells of microtiter plates are inoculated with (saline-diluted) culture using the MIC 2000 System, an automated plate inoculating device which delivers an inoculum of 1.5 TL per well. Plates are incubated at 35° C. 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 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, S. pneumoniae, E. faecalis, E. faecium, B. subtilis and E. coli. Compound I also demonstrates antibacterial activity against various species that are resistant to many known antibiotics such as methicillin-resistant S. aureus (MRSA), 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 0.5 to 32 ug/mL. MICs are obtained in accordance to the NCCLS guidelines.

## Claims

1. An isolated compound of structural formula (I);




or a pharmaceutically acceptable salt thereof.

2. A process for the preparation of an isolated compound of structural formula (I):




which comprises the cultivation of a Streptomyce sp. with ATCC# PTA-5942 (MA7339) in a nutrient medium and isolating the Compound (I) from a fermentation broth.

3. The process of claim 2 wherein the fermentation is conducted at a temperature of about 10° C. to about 40° C.

4. The process of claim 3, wherein the fermentation is conducted at a temperature of about 28° C.

5. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a compound of structure (I) of claim 1.