U.S. patent application number 14/454974 was filed with the patent office on 2014-11-27 for antibiotic compounds.
This patent application is currently assigned to PIRAMAL ENTERPRISES LIMITED. The applicant listed for this patent is PIRAMAL ENTERPRISES LIMITED. Invention is credited to GIRISH BADRINATH MAHAJAN, PRABHU DUTT MISHRA.
Application Number | 20140348919 14/454974 |
Document ID | / |
Family ID | 42990204 |
Filed Date | 2014-11-27 |
United States Patent
Application |
20140348919 |
Kind Code |
A1 |
MISHRA; PRABHU DUTT ; et
al. |
November 27, 2014 |
ANTIBIOTIC COMPOUNDS
Abstract
This invention relates to novel purified compounds of Formula I.
The invention includes all stereoisomeric forms and all tautomeric
forms of the compounds of Formula I and pharmaceutically acceptable
salts and derivatives. The present invention further relates to
processes for the production of the novel antibacterial compounds
by fermentation of the microorganism belonging to Streptomyces
species (PM0626271/MTCC 5447) and to pharmaceutical compositions
containing one or more of the novel compounds as active ingredient
and their use in medicines for treatment and prevention of diseases
caused by bacterial infections.
Inventors: |
MISHRA; PRABHU DUTT;
(Mumbai, IN) ; MAHAJAN; GIRISH BADRINATH; (Mumbai,
IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PIRAMAL ENTERPRISES LIMITED |
Mumbai |
|
IN |
|
|
Assignee: |
PIRAMAL ENTERPRISES LIMITED
Mumbai
IN
|
Family ID: |
42990204 |
Appl. No.: |
14/454974 |
Filed: |
August 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13393294 |
Feb 29, 2012 |
|
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|
PCT/IB2010/053897 |
Aug 31, 2010 |
|
|
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14454974 |
|
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61239186 |
Sep 2, 2009 |
|
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Current U.S.
Class: |
424/474 ;
514/2.4; 514/2.6; 514/2.7; 530/330 |
Current CPC
Class: |
C07D 513/18 20130101;
C07K 5/101 20130101; A61K 38/00 20130101; C07K 5/10 20130101; A61P
31/04 20180101 |
Class at
Publication: |
424/474 ;
530/330; 514/2.6; 514/2.7; 514/2.4 |
International
Class: |
C07K 5/10 20060101
C07K005/10 |
Claims
1-19. (canceled)
20. An isolated compound selected from the compound of Formula I(a)
or the compound of Formula I(b): ##STR00002## or a stereoisomer, or
a tautomer, or a pharmaceutically acceptable salt thereof.
21. The isolated compound as claimed in claim 20, wherein the
compound is isolated from fermented broth of a microorganism
belonging to Streptomyces species (PM0626271/MTCC 5447).
22. The isolated compound of Formula I(a) as claimed in claim 20,
wherein said compound is characterized by: (a) molecular weight of
1649.5, (b) molecular formula
C.sub.71H.sub.83N.sub.19O.sub.18S.sub.5, (c) .sup.1H NMR spectrum
as depicted in FIG. 1, and (d) .sup.13C NMR spectrum as depicted in
FIG. 2.
23. The isolated compound of Formula I(b) as claimed in claim 20,
wherein said compound is characterized by: (a) molecular weight of
1651.5, (b) molecular formula
C.sub.71H.sub.85N.sub.19O.sub.18S.sub.5, and (c) .sup.1H NMR
spectrum as depicted in FIG. 3.
24. A pharmaceutical composition comprising an effective amount of
the isolated compound of Formula I(a) or the isolated compound of
Formula I(b) as claimed in claim 20 and at least one
pharmaceutically acceptable excipient or carrier.
25. The pharmaceutical composition as claimed in claim 24, wherein
the pharmaceutical composition is in the form of a tablet, coated
tablet, capsule, granule, powder cream, ointment, gel, emulsion,
suspension, or solution for injection.
26. A method of treating or preventing a bacterial infection
comprising administering to a mammal in need thereof an effective
amount of the isolated compound of Formula I(a) or the isolated
compound of Formula I(b) as claimed in claim 20.
27. The method as claimed in claim 26, wherein the bacterial
infection is caused by bacteria belonging to Staphylococcus,
Streptococcus, Enterococci, Bacillus or Mycobacterium species.
28. The method as claimed in claim 27, wherein the bacteria
belonging to Staphylococcus species is methicillin-resistant, or
vancomycin-resistant or both.
29. The method as claimed in claim 27, wherein the bacteria
belonging to Enterococci species is vancomycin-resistant.
30. The method as claimed in claim 27, wherein the bacteria
belonging to Mycobacterium species is multi drug-resistant.
Description
CROSS REFERENCE APPLICATIONS
[0001] This is a divisional of copending application Ser. No.
13/393,294 filed on 29 Feb. 2012, which is a 371 of International
Application PCT/IB2010/053897 filed on 31 Aug. 2010, which
designated the U.S., claims the benefit thereof and incorporates
the same by reference. The nonprovisional application designated
above, namely application Ser. No. 13/393,294 filed 29 Feb. 2012,
claims the benefit of U.S. Provisional Application No. 61/239,186
filed 9 Sep. 2009 and incorporates the same by reference.
FIELD OF THE INVENTION
[0002] This invention relates to novel compounds of Formula I
having antibacterial activity. The compounds may be obtained by
fermentation of a microorganism belonging to Streptomyces species
(PM0626271/MTCC 5447). The invention also includes all
stereoisomeric forms and all tautomeric forms of compounds of
Formula I and pharmaceutically acceptable salts and derivatives
thereof. The present invention further relates to processes for the
production of the novel antibacterial compounds and to
pharmaceutical compositions containing one or more of the novel
compounds as an active ingredient and their use in medicines for
treatment and prevention of diseases caused by bacterial
infections.
BACKGROUND OF THE INVENTION
[0003] The dramatic rise in the prevalence of antibiotic resistance
among bacteria currently poses a serious threat to public health
worldwide. Of particular concern are infections caused by
methicillin-resistant Staphylococcus aureus (MRSA),
penicillin-resistant Streptococcus pneumoniae (PRSP),
vancomycin-resistant Enterococcus (VRE) (Clin. Microbiol. Infect.,
2005, 11, Supplement 3: 22-28) and multi drug resistant (MDR)
Mycobacterium tuberculosis (Eur. Respir. J., 2002, Supplement 36,
66S-77S).
[0004] Thiostrepton, an antibiotic isolated from Streptomyces
azureus, has been reported to be an effective anti-infective
medicine having the same general antibiotic spectrum as penicillin
and is used against gram-positive coccal infections (U.S. Pat. No.
2,982,689).
[0005] Siomycin, a sulfur-containing peptide antibiotic isolated
from Streptomyces sioyaensis, has been reported to be active
against gram-positive bacteria and mycobacteria with little or no
activity against gram-negative bacteria (The Journal Of
Antibiotics, 1969, 364-368).
[0006] There is a need to discover new compounds, which can be used
as drugs to treat patients who are at risk of infection or are
infected with bacteria, especially multi drug resistant bacteria
such as MRSA, VRE and Mycobacterium tuberculosis.
SUMMARY OF THE INVENTION
[0007] The present invention relates to novel compounds of Formula
I.
[0008] The present invention also relates to novel purified
compounds of Formula I, isolated from the fermented broth of the
microorganism belonging to Streptomyces species (PM0626271/MTCC
5447).
[0009] The invention also relates to all stereoisomeric forms and
all tautomeric forms of compounds of Formula I and pharmaceutically
acceptable salts and derivatives thereof.
[0010] The compounds of Formula I, and isomers, pharmaceutically
acceptable salts and derivatives thereof, have antibacterial
activity and are useful for the treatment or prevention of diseases
caused by bacteria, particularly multi drug resistant bacteria such
as MRSA, VRE and Mycobacterium tuberculosis.
[0011] The invention further relates to pharmaceutical compositions
comprising one or more of the novel compounds of Formula I, an
isomer, a pharmaceutically acceptable salt, or derivative thereof,
as an active ingredient for the treatment of diseases caused by
bacteria, particularly multi drug resistant bacteria such as MRSA,
VRE and Mycobacterium tuberculosis.
[0012] The present invention further relates to processes for the
production of the compounds of Formula I and/or their isomers from
the microorganism belonging to Streptomyces species (PM0626271/MTCC
5447).
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 Illustrates the .sup.1H NMR spectrum (500 MHz;
Instrument Bruker) of compound of Formula I(a) in
CDCl.sub.3:CD.sub.3OD (4:1).
[0014] FIG. 2 Illustrates the .sup.13C NMR spectrum (75 MHz;
Instrument Bruker) of compound of Formula I(a) in
CDCl.sub.3:CD.sub.3OD (4:1).
[0015] FIG. 3 Illustrates the .sup.1H NMR spectrum (500 MHz;
Instrument Bruker) of compound of Formula I(b) in
CDCl.sub.3:CD.sub.3OD (4:1).
DETAILED DESCRIPTION OF THE INVENTION
[0016] Before describing the present invention in detail, it has to
be understood that this invention is not limited to particular
embodiments. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting.
[0017] As used in the specification and claims, the singular forms
"a", "an" and "the" include plural references unless the context
clearly indicates otherwise.
[0018] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
the ordinary skill in the art to which the invention belongs.
[0019] As used herein, the term "derivative" refers to a compound
that is derived from a similar compound or a compound that can be
imagined to arise from another compound, if one atom is replaced
with another atom or group of atoms.
[0020] As used herein, the term "stereoisomer" refers to all
isomers of individual compounds that differ only in the orientation
of their atoms in space. The term stereoisomer includes mirror
image isomers (enantiomers), mixtures of mirror image isomers
(racemates, racemic mixtures), geometric (cis/trans or syn/anti or
E/Z) isomers, and isomers of compounds with more than one chiral
center that are not mirror images of one another
(diastereoisomers). The compounds of the present invention may have
asymmetric centers and occur as racemates, racemic mixtures,
individual diastereoisomers, or enantiomers, or may exist as
geometric isomers, with all isomeric forms of said compounds being
included in the present invention.
[0021] As used herein, the term "tautomer" refers to the
coexistence of two (or more) compounds that differ from each other
only in the position of one (or more) mobile atoms and in electron
distribution, for example, keto-enol and imine-enamine
tautomers.
[0022] As used herein, the term "fermented broth" refers to a
suspension of microbial culture in a nutrient medium containing
compounds produced by the microbes during its growth and also
having unconsumed nutrients.
[0023] As used herein, the term "mutant" refers to an organism or
cell carrying a mutation, which is an alternative phenotype to the
wild-type.
[0024] As used herein, the term "variant" refers to an individual
organism that is recognizably different from an arbitrary standard
type in that species.
[0025] The novel compounds of Formula I are structurally
represented by the following formula:
##STR00001##
[0026] The novel compound of Formula I(a) has the molecular formula
C.sub.71H.sub.83N.sub.19O.sub.18S.sub.5 (molecular weight 1649.5).
The novel compound of Formula I(b) has the molecular formula
C.sub.71H.sub.5N.sub.19O.sub.18S.sub.5 (molecular weight 1651.5).
The novel compounds of Formula I(a) and Formula I(b) may be
characterized by any one or more of their physico-chemical and
spectral properties, such as high performance liquid chromatography
(HPLC), mass spectrum (MS), infra red (IR) and nuclear magnetic
resonance (NMR) spectroscopic data as discussed herein below.
[0027] The structure of the novel compounds of Formula I(a) and
Formula I(b) has been elucidated and its complete characterization
is done by HPLC, high resolution MS (HRMS), IR and NMR
spectroscopic data. The compounds of Formula I(a) and Formula I(b)
are new antibiotics active against bacteria, particularly multi
drug resistant bacteria such as MRSA, VRE and Mycobacterium
tuberculosis. Compounds of Formula I(a) and Formula I(b) have
hitherto unreported structures.
[0028] The microorganism, which may be used for the production of
the compounds
of Formula I(a) and Formula I(b), is a strain of Streptomyces
species (PM0626271/MTCC 5447), herein after referred to as culture
no. PM0626271, isolated from a soil sample collected from
Schirmacher Oasis in Antarctic region.
[0029] The present invention further provides processes for the
production of the compounds of Formula I(a) and Formula I(b) from
culture no. PM0626271, comprising the steps of: cultivating the
culture no. PM0626271 under submerged aerobic conditions in a
nutrient medium containing one or more sources of carbon and one or
more sources of nitrogen and optionally nutrient inorganic salts
and/or trace elements; isolating the compounds of Formula I(a) and
Formula I(b) from the fermented broth; and purifying the compounds
of Formula I(a) and Formula I(b) using purification procedures
generally used in the related art.
[0030] Preliminary identification of culture no. PM0626271, which
is the producer of compounds of Formula I(a) and Formula I(b) was
performed by examination of its colony morphology, wet mount
observations and Gram stain reaction. Microscopic studies on the
strain of isolated culture no PM0626271 were carried out on
Actinomycete Isolation agar (AS-AIA; details given in Example
section) containing 1.5% agar and observations were made at 1, 2
and 3 days of incubation at 25.degree. C.
[0031] Growth on AS-AIA containing 1.5% agar develops as 1 mm
diameter colonies with white sporulation, scanty yellowish
substrate mycelia, slightly raised appearance, no diffusible
pigment and back side dark buff in color. Under phase contrast
light microscopy, wavy tangles of thin mycelia with sporulated tips
are observed at 400.times. magnification. They are Gram-positive.
The free spores are non-motile. The observed morphology classifies
this organism as a member of Streptomycetes family.
[0032] Culture no. PM0626271 has been deposited with Microbial Type
Culture Collection (MTCC), Institute of Microbial Technology,
Sector 39-A, Chandigarh--160 036, India, a World Intellectual
Property Organization (WIPO) recognized International Depository
Authority (IDA) and has been given the accession number MTCC
5447.
[0033] In addition to the specific microorganism described herein,
it should be understood that mutants, such as those produced by the
use of chemical or physical mutagens including X-rays, U.V. rays
etc. and organisms whose genetic makeup has been modified by
molecular biology techniques, may also be cultivated to produce the
compounds of Formula I(a) and Formula I(b).
[0034] The screening for suitable mutants and variants which can
produce the compound according to the invention can be confirmed by
HPLC and/or determination of biological activity of the active
compounds accumulated in the fermented broth, for example by
testing the compounds for antibacterial activity.
[0035] The medium and/or nutrient medium used for isolation and
cultivation of culture no. PM0626271, which produces the compounds
of Formula I(a) and Formula I(b), preferably contains sources of
carbon, nitrogen and nutrient inorganic salts. The carbon sources
are, for example, one or more of starch, glucose, sucrose, dextrin,
fructose, molasses, glycerol, lactose, or galactose. Preferred
carbon sources are soluble starch and glucose. The sources of
nitrogen are, for example, one or more of soybean meal, peanut
meal, yeast extract, beef extract, peptone, malt extract, corn
steep liquor, gelatin, or casamino acids. Preferred nitrogen
sources are peptone and yeast extract. The nutrient inorganic salts
are, for example, one or more of sodium chloride, potassium
chloride, calcium chloride, magnesium chloride, ferric chloride,
strontium chloride, cobalt chloride, potassium bromide, sodium
fluoride, sodium hydrogen phosphate, potassium hydrogen phosphate,
dipotassium hydrogen phosphate, magnesium phosphate, calcium
carbonate, sodium bicarbonate, sodium silicate, ammonium nitrate,
potassium nitrate, ferrous sulphate, sodium sulphate, ammonium
sulphate, magnesium sulphate, ferric citrate, boric acid or trace
salt solution. Calcium carbonate, sodium chloride, and magnesium
chloride are preferred.
[0036] The maintenance of culture no. PM0626271 may be carried out
at a temperature ranging from 22.degree. C. to 36.degree. C. and a
pH of about 7.5 to 8.0. Typically, culture no. PM0626271 is
maintained at 25.degree. C. to 27.degree. C. and a pH of about 7.4
to 7.8. The well-grown cultures may be preserved in the
refrigerator at 4.degree. C. to 8.degree. C.
[0037] Seed culture cultivation of culture no. PM0626271 may be
carried out at a temperature ranging from 25.degree. C. to
36.degree. C. and a pH of about 7.5 to 8.0 for 66 hours to 75 hours
at 200 rpm (revolutions per minute) to 280 rpm. Typically, culture
no. PM0626271 seed is cultivated at 29.degree. C. to 31.degree. C.
and a pH of about 7.4 to 7.8, for 72 hours at 230 rpm to 250
rpm.
[0038] The production of the compounds of Formula I(a) and Formula
I(b) may be carried out by cultivating culture no PM0626271 by
fermentation at a temperature ranging from 26.degree. C. to
36.degree. C. and a pH of about 6.5 to 8.5, for 24 hours to 96
hours at 60 rpm to 140 rpm and 100 lpm (liter per minute) to 200
lpm aeration. Typically, culture no. PM0626271 is cultivated at
30.degree. C. to 32.degree. C. and pH 7.4 to 7.8 for 40 hours to 96
hours at 90 rpm and 110 lpm aeration.
[0039] The production of the compounds of Formula I(a) and Formula
I(b) can be carried out by cultivating culture no. PM0626271 in a
suitable nutrient broth under conditions described herein,
preferably under submerged aerobic conditions, for example in shake
flasks, as well as in laboratory fermenters. The progress of
fermentation and production of the compounds of Formula I(a) and
Formula I(b) can be detected by high performance liquid
chromatography (HPLC) and by measuring the bioactivity of the
fermented broth against Staphylococci and/or Enterococci species by
the known microbial agar plate diffusion assay method. The
preferred culture is Staphylococcus aureus E710, which is a strain
resistant to methicillin, a .beta.-lactam antibiotic reported in
the literature, and Enterococcus faecium R2 (VRE) which is
resistant to vancomycin. In the resulting fermented broth, the
compounds of Formula I(a) and Formula I(b) are present in the
culture filtrate as well as in cell mass and can be isolated using
known separation techniques such as solvent extraction and column
chromatography. Thus, the compounds of Formula I(a) and Formula
I(b) can be recovered from the culture filtrate by extraction at a
pH of about 5 to 9 with a water immiscible solvent such as
petroleum ether, dichloromethane, chloroform, ethyl acetate,
diethyl ether or butanol, or by hydrophobic interaction
chromatography using polymeric resins such as "Diaion HP-20.RTM."
(Mitsubishi Chemical Industries Limited, Japan), "Amberlite XAD"
(Rohm and Haas Industries U.S.A.), activated charcoal, or by ion
exchange chromatography at pH 5 to 9. The active material can be
recovered from the cell mass by extraction with a water miscible
solvent such as methanol, acetone, acetonitrile, n-propanol, or
iso-propanol or with a water immiscible solvent such as petroleum
ether, dichloromethane, chloroform, ethyl acetate or butanol. One
other option is to extract the whole broth with a solvent selected
from petroleum ether, dichloromethane, chloroform, ethyl acetate,
methanol, acetone, acetonitrile, n-propanol, iso-propanol, or
butanol. Typically, the active material is extracted with ethyl
acetate from the whole broth. Concentration and lyophilization of
the extracts gives the active crude material.
[0040] The compounds of Formula I(a) and Formula I(b) of the
present invention can be recovered from the crude material by
fractionation using any of the following techniques: normal phase
chromatography (using alumina or silica gel as stationary phase;
eluents such as petroleum ether, ethyl acetate, dichloromethane,
acetone, chloroform, methanol, or combinations thereof); reverse
phase chromatography (using reverse phase silica gel such as
dimethyloctadecylsilyl silica gel, (RP-18) or dimethyloctylsilyl
silica gel (RP-8) as stationary phase; and eluents such as water,
buffers [for example, phosphate, acetate, citrate (pH 2 to 8)], and
organic solvents (for example, methanol, acetonitrile, acetone,
tetrahydrofuran, or combinations of these solvents); gel permeation
chromatography (using resins such as Sephadex LH-20.RTM. (Pharmacia
Chemical Industries, Sweden), TSKgel.RTM. Toyopearl HW (TosoHaas,
Tosoh Corporation, Japan) in solvents such as methanol, chloroform,
acetone, ethyl acetate, or their combinations, or Sephadex.RTM.
G-10 and G-25 in water); or by counter-current chromatography
(using a biphasic eluent system made up of two or more solvents
such as water, methanol, ethanol, iso-propanol, n-propanol,
tetrahydrofuran, acetone, acetonitrile, methylene chloride,
chloroform, ethyl acetate, petroleum ether, benzene, and toluene).
These techniques may be used repeatedly, alone or in combination. A
typical method is chromatography over normal phase using silica
gel.
[0041] The compounds of Formula I(a) and Formula I(b) and
stereoisomers thereof, can be converted into their pharmaceutically
acceptable salts and derivatives which are all contemplated by the
present invention.
[0042] Salts of the compounds can be prepared by standard
procedures known to one skilled in the art, for example, salts like
hydrochloride and sulphate salts, can be prepared by treating the
compounds of Formula I(a) and Formula I(b) and isomers thereof,
with a suitable acid, for example hydrochloric acid, sulphuric
acid.
[0043] The compounds of Formula I(a) and Formula I(b) have
antibacterial activity against a wide range of bacterial strains.
The compounds of Formula I(a) and Formula I(b) also have
antimycobacterial activity against MDR Mycobacterium tuberculosis
strains such as M. tuberculosis H37Rv; M. tuberculosis Clinical
isolate--S (Streptomycin), H (Isoniazid or Isonicotinyl hydrazine),
R (Rifampicin) and E (Ethambutol)--Resistant; and M. tuberculosis
Clinical isolate--S, H, R and E sensitive.
[0044] One or more of the compounds of Formula I(a) and Formula
I(b), stereoisomers and their pharmaceutically acceptable salts
thereof, alone or together, can be administered to animals, such as
mammals, including humans, as pharmaceuticals and in the form of
pharmaceutical compositions. One or more of the compounds of
Formula I(a) and Formula I(b), stereoisomers and their
pharmaceutically acceptable salts thereof, alone or together, can
be administered prophylatically to a patient who is at risk for
being infected by a bacterial infection. The patient may be someone
who may be exposed to the bacteria in a medical setting such as in
a hospital or in other setting where the bacteria may be
present.
[0045] Accordingly, the present invention also relates to the
compounds of Formula I(a) and Formula I(b), their stereoisomers and
their pharmaceutically acceptable salts for use as pharmaceuticals
and to the use of the compounds of Formula I(a) and Formula I(b),
stereoisomers and their pharmaceutically acceptable salts for the
manufacture of medicaments having antibacterial activity.
[0046] The present invention further relates to pharmaceutical
compositions, which contain an effective amount of one or more of
the compounds of Formula I(a) and Formula I(b) and/or stereoisomers
and/or one or more pharmaceutically acceptable salts and/or
derivatives thereof, together with at least one pharmaceutically
acceptable excipient or carrier useful for preventing or treating
bacterial infections.
[0047] The effective amount of the compounds of Formula I(a) and
Formula I(b), or its stereoisomers, or its pharmaceutically
acceptable salts or its derivatives as the active ingredient in the
pharmaceutical preparations normally is from about 0.01 mg to 1000
mg.
[0048] The present invention also relates to method of treating or
preventing a bacterial infection comprising administering to a
mammal in need thereof an effective amount of one or more of the
compounds of Formula I(a) and Formula I(b) and/or stereoisomers
and/or one or more pharmaceutically acceptable salts thereof.
[0049] The present invention also relates to a method for the
manufacture of a medicament containing one or more of the compounds
of Formula I(a) and Formula I(b) and/or stereoisomers and/or one or
more pharmaceutically acceptable salts thereof, for the treatment
or prevention of diseases caused by bacterial infections.
[0050] The compounds of the present invention are particularly
useful as anti-bacterial agents. The present invention accordingly
relates to the use of one or more of the compounds of Formula I(a)
and Formula I(b) and/or stereoisomers and/or one or more
pharmaceutically acceptable salts and/or derivatives thereof, for
the manufacture of a medicament for the prevention or treatment of
diseases caused by bacterial infections.
[0051] The bacterial infections for the treatment of which the
compounds of the present invention are used may be caused by
bacteria belonging to Staphylococcus, Streptococcus, Enterococcus,
Bacillus or Mycobacterium species. The bacteria belonging to
Staphylococcus species can be methicillin-resistant or vancomycin
resistant. The bacteria belonging to Enterococci species can be
vancomycin resistant. The bacteria belonging to Mycobacterium
species can be multi drug-resistant.
[0052] The term "Staphylococcus species" refers to Gram-positive
bacteria, which appear as grape-like clusters when viewed through a
microscope and as large, round, golden-yellow colonies, often with
.beta.-hemolysis, when grown on blood agar plates. Species of
Staphylococus include Staphylococcus aureus.
[0053] The term "Streptococcus species" refers to a genus of
spherical, Gram-positive bacteria, and a member of the phylum
Firmicutes. Streptococci are lactic acid bacteria. Streptococcus
species includes bacteria such as S. hemolyticus, S. mitis, S.
salivarius, S. pneumoniae. Streptococcus species are responsible
for infectious diseases such as meningitis, bacterial pneumonia,
endocarditis, erysipelas and necrotizing fasciitis (`flesh-eating`
bacterial infections).
[0054] The term "Enterococcus species" refers to a genus of lactic
acid bacteria of the phylum Firmicutes. They are Gram-positive
cocci which often occur in pairs (Diplococci for example
Diplococcus pneumoniae). Enterococci are facultative anaerobic
organisms.
[0055] The term "Bacillus species" refers to a large number of
diverse, rod-shaped Gram positive bacteria that are motile by
peritrichous flagella and are aerobic such as B. anthracis, B.
subtilis or anaerobic such as Clostridium spp. for example C.
difficile. These Bacilli belong to division Firmicutes.
[0056] The term "Mycobacterium species" refers to Gram-positive,
non-motile, pleomorphic rods related to the actinomyces.
Tuberculosis in humans is caused by Mycobacterium tuberculosis.
MDR-TB (multi-drug resistant tuberculosis) describes strains of
tuberculosis that are resistant to at least the two first-line TB
drugs, isoniazid and rifampicin.
[0057] The compounds of the present invention can be administered
orally, nasally, topically, parenterally such as subcutaneously,
intramuscularly, intravenously, or by other modes of
administration.
[0058] Pharmaceutical compositions which contain one or more of the
compounds of Formula I(a) and Formula I(b) or a stereoisomer or a
pharmaceutically acceptable salt or a derivative thereof,
optionally with other pharmaceutically acceptable excipient or
carrier, can be prepared by mixing the active compounds with one or
more pharmaceutically acceptable excipients and/or carriers such
as, wetting agents, solubilisers such as surfactants, vehicles,
tonicity agents, fillers, colorants, masking flavors, lubricants,
disintegrants, diluents, binders, plasticizers, emulsifiers,
ointment bases, emollients, thickening agents, polymers, lipids,
oils, cosolvents, complexation agents, or buffer substances, and
converting the mixture into a suitable pharmaceutical form such as,
for example, tablets, coated tablets, capsules, granules, powders,
creams, ointments, gels, syrup, emulsions, suspensions, or
solutions suitable for injection used for parenteral
administration.
[0059] Examples of excipients and/or carriers that may be mentioned
are cremophor, poloxamer, benzalkonium chloride, sodium lauryl
sulfate, dextrose, glycerin, magnesium stearate, polyethylene
glycol, starch, dextrin, lactose, cellulose, carboxymethylcellulose
sodium, talc, agar-agar, mineral oil, animal oil, vegetable oil,
organic and mineral waxes, paraffin, gels, propylene glycol, benzyl
alcohol, dimethylacetamide, ethanol, polyglycols, tween 80, solutol
HS 15, water and saline. It is also possible to administer the
active substances as such, without vehicles or diluents, in a
suitable form, for example, in capsules.
[0060] As is customary, the galenic formulation and the method of
administration as well as the dosage range which are suitable in a
specific case depend on the species to be treated and on the state
of the respective condition or disease, and can be optimized using
methods known in the art. On average, the daily dose of active
compound in a patient is 0.0005 mg to 50 mg per kg, typically 0.001
mg to 20 mg per kg.
[0061] The following are provided as illustrative examples of the
present invention and do not limit the scope thereof.
Example 1
Isolation of Culture No. PM0626271 from Soil Collected from
Antarctic Region
[0062] a) Composition of the isolation medium:
[0063] Modified artificial sea water agar: Peptone 1.5 g, yeast
extract 0.5 g, ferric chloride 0.007 g, 1.0 L water (750 mL
artificial sea water+250 mL demineralised water), agar powder 15.0
g, final pH (at 25.degree. C.) 7.4 to 7.8.
[0064] Composition of the artificial seawater: Sodium chloride 24.6
g, potassium chloride 0.67 g, calcium chloride.2H.sub.2O 1.36 g,
magnesium sulphate.7H.sub.2O 6.29 g, magnesium chloride.6H.sub.2O
4.66 g, sodium bicarbonate 0.18 g, demineralised water 1.0 L, final
pH (at 25.degree. C.) 7.8 to 8.2.
b) Procedure:
[0065] From Schirmacher Oasis region in Antarctica area, surface
level soil was collected and was stored at -20.degree. C.
throughout the journey to Piramal Life Sciences Limited, Goregaon,
Mumbai, India. The sample was stored at -20.degree. C. to
-22.degree. C. and later thawed to room temperature
(25.+-.2.degree. C.) for isolation of the microbes. The soil sample
(.about.1 g) was suspended in 25 mL of sterile 1% peptone water in
a 100 mL sterilized flask. The flask was vortexed for 30 seconds.
Serial dilutions up to 10.sup.-5 were prepared in sterile 1%
peptone water. 100 .mu.L of 10.sup.-5 dilution was surface spread
on modified artificial seawater agar. The plate was incubated at
room temperature (25.+-.2.degree. C.) till colonies were observed.
After incubation for one and a half month, the colony which
appeared on this medium was streaked on petri plates containing
actinomycete isolation agar [Hi Media] prepared in 75% artificial
sea water [Accumix.TM.](AS-AIA). The isolate was purified and was
provided culture ID number PM0626271. The culture no. PM0626271 was
thus isolated from amongst the growing microorganisms as single
isolate.
Example 2
Purification of Culture No. PM0626271
[0066] a) Composition of the purification medium (Actinomycete
Isolation Agar, agarified by 1.5% agar agar):
[0067] Glycerol 5.0 mL, sodium caseinate 2.0 g, L-asparagine 0.1 g,
sodium propionate 4.0 g, dipotassium phosphate 0.5 g, magnesium
sulphate 0.1 g, ferrous sulphate 0.001 g, 1.0 L water (750 mL
Artificial Sea Water+250 mL demineralised water), agar powder 15.0
g, final pH (at 25.degree. C.) 7.4 to 7.8.
[0068] Composition of the artificial seawater: Sodium chloride 24.6
g, potassium chloride 0.67 g, calcium chloride.2H.sub.2O, 1.36 g,
magnesium sulphate.7H.sub.2O 6.29 g, magnesium chloride.6H.sub.2O
4.66 g, sodium bicarbonate 0.18 g, demineralized water 1.0 L, final
pH (at 25.degree. C.) 7.8 to 8.2.
b) Procedure:
[0069] The culture no. PM0626271 was streaked on Actinomycete
Isolation Agar (containing 75% artificial sea water salts)
petriplate. The petriplate was incubated for 10 days at 25.degree.
C. One of the isolated colonies from the petriplate was transferred
to fresh slants of Actinomycete Isolation Agar prepared in 75%
artificial seawater. The slants were incubated for 10 days at
25.degree. C.
Example 3
Maintenance of Producer Strain--Culture No. PM0626271
[0070] a) Composition of the medium (Actinomycete Isolation
Agar):
[0071] Glycerol 5.0 mL, sodium caseinate 2.0 g, L-asparagine 0.1 g,
sodium propionate 4.0 g, dipotassium phosphate 0.5 g, magnesium
sulphate 0.1 g, ferrous sulphate 0.001 g, 1.0 L water (750 mL
artificial sea water+250 mL demineralised water), agar powder 15.0
g, final pH (at 25.degree. C.) 7.4 to 7.8.
[0072] Composition of the artificial sea water: Sodium chloride
24.6 g, potassium chloride 0.67 g, calcium chloride.2H.sub.2O 1.36
g, magnesium sulphate.7H.sub.2O 6.29 g, magnesium
chloride.6H.sub.2O 4.66 g, sodium bicarbonate 0.18 g, demineralized
water 1.0 L, final pH (at 25.degree. C.) 7.8 to 8.2.
b) After dissolving the ingredients thoroughly by heating, the
resultant solution was distributed in test tubes and sterilized at
121.degree. C. for 30 minutes. The test tubes were cooled and
allowed to solidify in a slanting position. The agar slants were
streaked with the growth of culture no. PM0626271 by a wire loop
and incubated at 27.degree. C. to 29.degree. C. until a good growth
was observed. The well-grown cultures were stored in the
refrigerator at 4.degree. C. to 8.degree. C.
Example 4
Fermentation of the Culture No. PM0626271 in Shake Flasks
[0073] a) Composition of seed medium [AS-274 (1)]:
[0074] Glucose 15 g, corn steep liquor 5 g, peptone 7.5 g, yeast
extract 7.5 g, calcium carbonate 2.0 g, sodium chloride 5.0 g,
volume made with 750 mL artificial sea water and 250 mL
demineralised water.
[0075] b) The above medium was distributed in 40 mL amounts in 500
mL capacity Erlenmeyer flasks and autoclaved at 121.degree. C. for
30 minutes. The flasks were cooled to room temperature
(25.+-.2.degree. C.) and each flask was inoculated with a loopful
of the well-grown producing strain (culture no. PM0626271) on the
slant and shaken on a rotary shaker for 72 hours at 230 rpm to 250
rpm at 30.degree. C. (.+-.1.degree. C.) to give seed culture.
c) Composition of the production medium [AS 36P (1)]:
[0076] Soluble Starch 20 g, glucose 15 g, yeast extract 2 g,
peptone 3 g, calcium carbonate 2 g, ammonium sulfate 0.5 g, corn
steep liquor 2 g, sodium chloride 2 g, magnesium phosphate 5 g,
cobalt chloride 1 mL/L from stock of 1 g/L, trace salt solution 1
mL/L, volume made to 1 L using with 75% artificial sea water and
25% demineralised water.
d) 40 mL of the production media in 500 mL capacity Erlenmeyer
flasks was autoclaved at 121.degree. C. for 30 minutes, cooled to
29.degree. C. to 30.degree. C. and seeded with 5% (v/v) of the seed
culture mentioned in Example 4b. e) Fermentation parameters:
[0077] The production flasks were incubated on shaker at 29.degree.
C. and 220 rpm for 96 hours. The production flasks were harvested
and the whole broth from each media flask was extracted with equal
volume of methanol under shaking condition for one hour at
29.degree. C. and centrifuged at 3500 rpm for half an hour. The
supernatant was used for antibacterial agar well diffusion assay
for monitoring of the activity.
[0078] The production of the compounds of Formula I(a) and Formula
I(b) in the fermentation broth was determined by testing the
bioactivity against S. aureus E710 (MRSA strain) and/or
Enterococcus faecium R2 (VRE) using the agar well diffusion method.
The harvest pH of the fermented broth was 7.0 to 8.0. The fermented
broth was harvested and the whole broth was used for isolation and
purification of the compounds of Formula I(a) and Formula I(b).
Example 5
Preparation of Seed Culture in Shake Flasks for Fermentation
[0079] a) Composition of the medium [AS-274 (1)]:
[0080] Glucose 15 g, corn steep liquor 5 g, peptone 7.5 g, yeast
extract 7.5 g, calcium carbonate 2.0 g, sodium chloride 5.0 g,
volume made with 750 mL Artificial Sea Water and 250 mL
demineralised water.
[0081] b) The above medium was distributed in 200 mL amounts in
1000 mL Erlenmeyer flasks and autoclaved at 121.degree. C. for 30
minutes. The flasks were cooled to room temperature
(25.+-.2.degree. C.) and each flask was inoculated with a loopful
of the well-grown producing strain (PM0626271) on the slant and
shaken on a rotary shaker for 70 hours to 74 hours at 230 rpm to
250 rpm at 29.degree. C. to 30.degree. C. to obtain the seed
culture.
Example 6
Cultivation of the Culture No PM0626271 in Fermenter
[0082] a) Composition of the production medium:
[0083] Artificial Sea Water (artificial sea water salt 28.32 g)
(75%), soluble starch 20 g, glucose 15 g, yeast extract 2 g,
peptone 3 g, calcium carbonate 2 g, ammonium sulphate 0.05 g, corn
steep liquor 2 g, sodium chloride 2 g, magnesium phosphate 5 g,
cobalt chloride (cobalt chloride 1 g demineralized water 1.0 L) 1
mL/L, trace salt solution (copper sulphate 7 g, ferrous sulphate 1
g, manganese chloride 8 g, zinc sulphate 2 g, demineralized water
1.0 L) 1 mL/L, demineralized water 1.0 L, pH 6.5 to 7.5 (before
sterilization).
b) 100 L of the production medium in 150 L fermenter along with 30
mL of desmophen as an antifoaming agent was sterilized in situ for
30 minutes at 121.degree. C., cooled to 29.degree. C. to 30.degree.
C. and seeded with 2.5 L to 3.5 L of the seed culture obtained
above (Example 5). c) Fermentation parameters: The fermentation was
carried out at temperature 29.degree. C. to 30.degree. C.,
agitation 100 rpm, aeration 60 lpm and harvested at 70 hours to 74
hours. The production of the compounds of Formula I(a) and Formula
I(b) in the fermentation broth was detected qualitatively by
testing the bioactivity against S. aureus E710 (MRSA strain) and/or
Enterococcus faecium R2 (VRE) using the agar well diffusion method.
The harvest pH of the fermented broth was 7.5 to 8.0. After the
harvest, whole broth was subjected to solvent extraction.
Example 7
Isolation and Purification of the Compounds of Formula I(a) and
Formula I(b)
[0084] The whole broth (10 L batch) was extracted using ethyl
acetate (1:1). The organic and aqueous layers were separated. The
organic layer was processed to evaporate the solvent to obtain
crude ethyl acetate extract (1.5 g). The crude extract was further
processed by flash chromatography (silica gel, 30 g, solvent:
methanol/chloroform step gradient, flow: 15 mL/minute). The active
compound eluted with 1% methanol to 5% methanol in chloroform,
which was concentrated to obtain the semipure compound (250 mg).
Further purification was carried out by repeated normal phase
preparative HPLC.
Preparative HPLC conditions: [0085] Column: Eurospher silica
(10.mu., 20.times.250 mm) [0086] Eluent: methanol:chloroform (5:95)
[0087] Flow rate: 20 mL/minute [0088] Detection (UV): 245 nm [0089]
Retention time: compound of Formula I(a) (5 to 6 minutes), [0090]
compound of Formula I(b) (8 to 10 minutes)
[0091] Purity of fractions was checked by bioassay against E.
faecium R2 and/or S. aureus 3066 and/or analytical HPLC. The
eluates containing the compounds of Formula I(a) and Formula I(b)
were pooled and concentrated under reduced pressure to remove the
solvent to obtain compound of Formula I(a) (40 mg), and compound of
Formula I(b) (3 mg).
Analytical HPLC conditions: [0092] Column: Eurospher RP-18, (3.mu.,
4.6.times.125 mm) [0093] Solvent system: Gradient (0% acetonitrile
to 100% in 15 minutes against water, followed by 100% acetonitrile
for 5 minutes) [0094] Flow rate: 1 mL/minute [0095] Detection (UV):
245 nm [0096] Retention time: compound of Formula I(a) (12 to 13
minutes), and compound of Formula I(b) (11 to 12 minutes)
A. Physical & Spectral Properties of the Compound of Formula
I(a):
[0096] [0097] Appearance: White powder [0098] Melting point:
240.degree. C. (decomposes) [0099] Solubility: Soluble in
chloroform, ethyl acetate, methanol and insoluble in water [0100]
MS [HR-ESI(+) MS)]m/z: 1650.4858 (M+H) [0101] Molecular weight:
1649.5 [0102] Molecular formula:
C.sub.71H.sub.63N.sub.19O.sub.18S.sub.5 [0103] IR (KBr): 3386,
2927, 1648, 1507, 1206, 756, 666 cm.sup.-1 [0104] .sup.1H NMR:
refer to Table 1 and FIG. 1 [0105] .sup.13C NMR: refer to Table 2
and FIG. 2
B. Physical & Spectral Properties of the Compound of Formula
I(b):
[0105] [0106] Appearance: White powder [0107] Solubility: Soluble
in chloroform, ethyl acetate, methanol and insoluble in water
[0108] MS [HR-ESI(+) MS)]m/z: 1674.4787 (M+Na) [0109] Molecular
weight: 1651.50 [0110] Molecular formula:
C.sub.71H.sub.85N.sub.19O.sub.18S.sub.5 [0111] .sup.1H NMR: refer
to Table 3 and FIG. 3
TABLE-US-00001 [0111] TABLE 1 .sup.1H NMR of the compound of
Formula I(a) in CDCl.sub.3:CD.sub.3OD (4:1) at 500 MHz Peak .delta.
1 0.7 (d, 3H) 2 0.74 (d, 3H) 3 0.95 (d, 3H) 4 1.04 (s, 3H) 5 1.08
(d, 3H) 6 1.2 (d, 3H) 7 1.28 (d, 3H) 8 1.34 (d, 3H) 9 1.37 (m, 1H)
10 1.5 (d, 3H) 11 1.6 (d, 3H) 12 2.1 (m, 1H) 13 2.2 (m, 1H) 14 2.2
(m, 1H) 3.99 (m, 1H) 15 2.8 (d, 1H) 16 3.05 (t, 1H) 3.5 (t, 1H) 17
3.49 (d, 2H) 18 3.67 (d, 1H) 19 3.7 (q, 1H) 20 4.33 (d, 1H) 21 4.33
(d, 1H) 22 4.62 ((q, 1H) 23 4.86 (dd, 1H) 24 5.19 (s, 1H) 25 5.19
(s, 1H), 5.67 (s, 1H) 26 5.2 (t, 1H) 27 5.6 (d, 1H) 28 5.62 (s,
1H), 6.44 (s, 1H) 29 5.65 (d, 2H) 30 5.72 (s, 1H), 6.61 (s, 1H) 31
6.1 (q, 1H) 32 6.25 (m, 1H) 33 6.28 (d, 2H) 34 6.8 (d, 1H) 35 6.91
(s, 1H) 36 6.94 (s, 1H) 37 7.2 (s, 1H) 38 7.43 (s, 1H) 39 7.45 (s,
1H) 40 7.65 (s, 1H) 41 7.87 (s, 1H) 42 8.05 (s, 1H) 43 8.17 (s, 1H)
44 8.2 (s, 1H) 45 8.5 (s, 1H) 46 8.67 (s, 1H) 47 8.99 (s, 2H) 48
9.72 (s, 1H) 49 9.8 (s, 1H)
TABLE-US-00002 TABLE 2 .sup.13C NMR of the compound of Formula I(a)
in CDCl.sub.3:CD.sub.3OD (4:1) at 75 MHz Signal .delta. 1 12.11 2
13.74 3 14.1 4 14.8 5 16.48 6 17.11 7 17.27 8 17.55 9 20.9 10 23.13
11 27.56 12 27.56 13 29.04 14 33.24 15 46.17 16 50.14 17 51.3 18
53.91 19 53.91 20 55.8 21 57.32 22 58.8 23 62.42 24 62.73 25 64.45
26 64.73 27 65.69 28 65.95 29 70.27 30 77.1 31 101.45 32 101.45 33
102.6 34 116.52 35 120.63 36 121.59 37 123.28 38 123.87 39 123.87
40 125.48 41 126.03 42 126.69 43 128.24 44 130.34 45 130.98 46
131.14 47 132.39 48 141.85 49 144.61 50 148.17 51 148.45 52 151.89
53 152.76 54 155.45 55 157.9 56 159.0 57 160.04 58 160.36 59 161.01
60 163.75 61 164.46 62 164.5 63 166.6 64 167.13 65 167.95 66 168.47
67 168.67 68 170.35 69 170.35 70 171.63 71 172.0
TABLE-US-00003 TABLE 3 .sup.1H NMR of the compound of Formula I(b)
in CDCl.sub.3:CD.sub.3OD (4:1) at 500 MHz Peak .delta. 1 0.88 (d,
3H) 2 0.95 (d, 3H) 3 0.99 (d, 3H) 4 1.0 (m, 1H) 5 1.16 (s, 3H) 6
1.18 (d, 3H) 7 1.32 (d, 3H) 8 1.35 (d, 3H) 9 1.43 (d, 3H) 10 1.5
(d, 3H) 11 1.61 (d, 3H) 12 1.72 (d, 3H) 13 2.02 (m, 1H) 14 2.28 (m,
1H) 15 2.8 (d, 1H) 16 3.17 (t, 1H) 3.69 (t, 1H) 17 3.43 (d, 1H) 18
3.46 (d, 1H) 19 3.58 (d, 1H) 20 3.8 (q, 2H) 21 4.06 (m, 1H) 22 4.45
((d, 1H) 23 4.59 (t, 1H) 24 4.61 (dd, 1H) 25 4.74 (t, 1H) 26 4.95
(t, 1H) 27 5.19 (s, 1H), 5.78 (s 1H) 28 5.19 (s, 1H) 29 5.3 (d, 1H)
30 5.52 (s, 1H), 6.65 (s, 1H) 31 5.65 (s, 1H) 32 5.72 (d, 1H) 33
5.8 (d, 1H) 34 6.1 (q, 1H) 35 6.34 (m, 1H) 36 6.36 (d, 1H) 37 6.73
(d, 1H) 38 6.91 (d, 1H) 39 6,94 (s, 1H) 40 7.14 (s, 1H) 41 7.3 (s,
1H) 42 7.46 (s, 1H) 43 7.54 (d 1H) 44 7.65 (d, 1H) 45 7.80 (s, 1H)
46 8.05 (s, 1H) 47 8.11 (s, 1H) 48 8.14 (s, 1H) 49 8.26 (s, 1H) 50
8.52 (t, 1H) 51 8.65 (d, 1H) 52 9.2 (s, 1H) 53 9.87 (s, 1H) 54 9.92
(s, 1H)
Biological Evaluation of the Compounds of Formula I(a) and Formula
I(b)
In-Vitro Assays
Example 8
[0112] The in-vitro potency was established by minimum inhibitory
concentration (MIC) determinations of the compounds of Formula I(a)
and Formula I(b) against bacterial strains, by using the
Macro-broth dilution method as per National Committee for Clinical
Laboratory Standards (2000) guidelines (Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria that Grow
Aerobically--Fifth Edition Approved Standard M7-A5. NCCLS, Wayne,
Pa., USA). Mueller-Hinton broth was used as nutrient medium for the
assay, unless stated otherwise. PM181104 (PCT publication no.
WO2007119201) was used as known standard in all in-vitro
experiments. For preparation of the stock solution the compounds of
Formula I(a) and Formula I(b) were dissolved in chloroform (5% of
the total required volume) and diluted using methanol (95% of the
total required volume).
Result:
[0113] The results obtained are shown in Table 4 and Table 5, and
demonstrate that the compounds of Formula I(a) and Formula I(b)
have utility in treating bacterial infections.
TABLE-US-00004 TABLE 4 MICS of the compound of Formula I(a) against
bacterial strains MIC Test Organism (.mu.g/ml) Test Organism MIC
(.mu.g/ml) S. aureus C1 MRSA 2 0.25 S. aureus E712, MRSA 0.25
Erythro.sup.R, 59 S. aureus C1 MRSA 3 0.25 S. aureus 503, MRSA, 62
>1 S. aureus C1 MRSA 5 0.25 S. aureus SG 511, MRSA, 63 1 S.
aureus C1 MRSA 7 0.125 S. aureus 789, MRSA, 64 >1 S. aureus C1
MRSA 8 0.25 S. aureus 209 P, MSSA 0.063 S. aureus C1 MRSA 9 0.25 S.
epidermidis 823, Teicho.sup.R, 230 0.25 S. aureus C1 MRSA 10 0.25
S. epidermidis 6098, Erythro.sup.R, 233 >1 S. aureus C1 MRSA 13
0.25 S. epidermidis 6729 II W, 0.125 Erythro.sup.R, 236 S. aureus
C1 MRSA 16 0.25 S. epidermidis 2361 W, 246 0.25 S. aureus C1 MRSA
17 0.125 S. epidermidis 4264 I (1) W, 0.125 247 S. aureus C1 MRSA
20 0.125 S. epidermidis Pat 01 IV, 251 0.25 S. aureus C1 MRSA 21
0.125 E. faecium C1 VRE 26 0.25 S. aureus C1 MRSA 22 0.25 E.
faecium C1 VRE 27 0.25 S. aureus C1 MRSA 23 0.016 E. faecium C1 VRE
28 0.125 S. aureus C1 MRSA 24 0.25 E. faecium C1 VRE 31 0.5 S.
aureus C1 MRSA 25 0.15 E. faecium C1 VRE 33 0.125 S. aureus KEM
MRSA 1 0.25 E. faecium C1 VRE 34 0.25 S. aureus KEM MRSA 2 0.25 E.
faecium KEM VRE 1 0.25 S. aureus KEM MRSA 3 0.25 E. faecium KEM VRE
2 0.5 S. aureus KEM MRSA 4 0.125 E. faecium KEM VRE 3 0.25 S.
aureus KEM MRSA 5 0.125 E. faecium KEM VRE 4 0.5 S. aureus MRSA 3
lilavati 0.125 E. faecium KEM VRE 5 0.25 S. aureus Misk MRSA 35
0.125 E. faecium R-2 (VRE), 0.25 S. aureus Misk MRSA 37 0.125
Enterococcus faecium, VSE 0.125 (322) S. aureus Misk MRSA 38 0.125
Bacillus cereus (121) 0.031 S. aureus E710, MRSA 0.125 Bacillus
subtilis ATCC 6633 0.031 (123) S. aureus ATCC 33591, 0.125 Bacillus
megaterium FH1127 0.125 MRSA (124)
TABLE-US-00005 TABLE 5 MICs of the compounds of Formula I(a) and
Formula I(b) against bacterial strains Minimal Inhibitory
Concentration (.mu.g/ml) Compound of Compound of Test culture
Formula I(a) Formula I(b) E. faecium, R-2 (VRE) 0.125 2 S. aureus
E710, (MRSA) 0.125 2 S. aureus 209P (MSSA) 0.064-0.125 0.25-0.5
[0114] Abbreviations used in Table 4 and Table 5 are--
S: Staphylococcus
E: Enterococci
Example 9
Evaluation of Antimycobacterial Activity
[0115] The assay was done as reported in J. Clin. Microbiol., 1999,
37, 1144.
[0116] 50 .mu.L of bacterial (as mentioned in Table 6) suspension,
equivalent to MacFarlands no. 2 standard (corresponding to
>5.times.10.sup.7 CFU/ml) (Remel, Lenexa, Kan.) was added to 400
.mu.L of G7H9 with and without compounds of Formula I(a) and
Formula I(b) (tested at 0.5 .mu.g/mL, 5 .mu.g/mL and 10 .mu.g/mL)
and incubated for 72 hours at 37.degree. C. After incubation 50
.mu.L of the high titer Luciferase reporter phage (phAE 129) and 40
.mu.L of 0.1M calcium chloride (CaCl.sub.2) were added to all the
vials and this set up was incubated for 4 hours at 37.degree. C.
After incubation 100 .mu.L of the mixture was taken from each vial
into a luminometer cuvette and equal amount of working D-luciferin
(0.3 mM in 0.05 M sodium citrate buffer, pH 4.5) solution was
added. The Relative Light Units (RLUs) were measured after 10
seconds of integration in the Luminometer (Monolight 2010).
[0117] Duplicate readings were recorded for each sample and the
mean was calculated. The percentage reduction in the RLU was
calculated for each test sample and compared with control. The
experiment was repeated when the mean RLU of the control was less
than 1000. The criterion for activity is antimycobacterial activity
indicated by fifty percent reduction in RLU in the presence of the
compound in comparison with compound free control.
TABLE-US-00006 TABLE 6 Antimycobacterial activity of the compounds
of Formula I(a) and Formula I(b) % reduction in RLU Strain
Compounds 0.5 .mu.g/mL 5 .mu.g/mL 10 .mu.g/mL M. tuberculosis
Compound of 36.36 77.17 83.76 H.sub.37Rv Formula I(a) Compound of
28.87 54.83 76.73 Formula I(b) Clinical isolate: Compound of 20.88
78.11 88.42 S, H, R and E Formula I(a) resistant Compound of 5.67
70.9 83.74 Formula I(b)
CONCLUSION
[0118] The compounds of Formula I(a) and Formula I(b) are active
against standard strain of TB (H.sub.37 RV) and MDR Mycobacterium
tuberculosis strains [resistant to 4 standard antibiotics: S
(Streptomycin), H (Isoniazid or Isonicotinyl hydrazine), R
(Rifampicin) and E (Ethambutol)].
* * * * *