NOVEL COMPOUNDS AS ANTI-TUBERCULAR AGENTS
FIELD OF THE INVENTION
The present invention relates to novel compounds useful as anti-tubercular agents. More
particularly, the present invention relates to substituted derivatives, methods for their
preparation, pharmaceutical compositions containing these compounds and uses of these
compounds in the treatment of tuberculosis.
BACKGROUND OF THE INVENTION
Tuberculosis causes nearly two million deaths annually. Recent years have witnessed a
resurgence of efforts directed at tuberculosis (TB) drug research and development. An
analysis of the chronology of anti-tuberculosis therapy suggests a renewed interest in
tuberculosis research. Since the 1960s and 70s same generation of antibiotics or their
derivatives are being used against Mycobacterium tuberculosis even though there is
resistance against the drugs with the development of drug resistant strains such as Multidrug
resistance TB (MDR-TB)which are resistant to at least isoniazid (INH) and rifampicin
(RMP), the two most powerful first-line treatment anti-TB drugs; extensively drug resistance
TB (XDR-TB)which are resistant to isoniazid and rifampin, plus any fluoroquinolone and at
least one of three injectable second-line drugs such as amikacin, kanamycin, or capreomycin
and totally drug resistance TB (TDR-TB) which are resistant to all first and second line drugs
tested such as isoniazid, rifampicin, streptomycin, ethambutol, pyrazinamide, ethionamide,
para-aminosalicylic acid, cycloserine, ofloxacin, amikacin, ciprofloxacin, capreomycin,
kanamycin.
India has the world's one of the highest burden of tuberculosis, wherein MDR tuberculosis
accounts for nearly 5 lakhs cases annually which have been increased by more than 6% in the
past years.
It is said that M. tuberculosis develops resistance to drugs by induced or spontaneous
mutation of its genome. It is also hypothesized that in case of certain drugs the bacterial cell
wall, does not permit adequate permeability, thereby resulting in inadequate/minor amount of
drug entering the bacteria. Such amount is insufficient to kill the bacteria, but the presence of
minor amount, induces the bacteria to become resistant to the same. It is also hypothesized
that M. tuberculosis acquires resistance by modification of its enzyme by unknown
mechanisms. Traditional antitubercular drugs administered via oral route, act by inhibiting
the synthesis of mycolic acid and/or by inhibiting the mycobacterial arabinosyltransferase, an
essential component of mycobacterial cell wall. Few systemic antitubercular drugs also act by
crossing the lipid bilayer and bind to one of the ribosome sub-unit, inhibiting the protein
synthesis. Based on mechanism of action of these drugs, these drugs have a high probability
of inducing mutation and/or have problems of permeability, increasing the chances of drug
resistant strains.
G-protein coupled receptors such as GPR109A also plays an important role in tuberculosis.
GPR109A receptor is located on the cell surface and inhibits adenylyl cyclase along with
consequent suppression of PKA-signaling resulting in reduced triglyceride turnover which is
further responsible for accumulation of lipid body inside the cell. Increase in the
concentration of lipid body inside the cell favors the growth of M. tuberculosis and prevents
respiratory burst. A GPR109A inhibitor prevents the formation of lipid body by creating a
hostile environment for the tuberculosis and thus induces respiratory burst.Host macrophages
infected by M. tuberculosis acquire foamy phenotype characterized by the intracellular
accumulation of lipid bodies induced by pathogen through modulation of lipolysis of neutral
lipids. This dysregulation influences the lipolysis by modulating the cAMP dependent
signaling pathway.
Current resistant tuberculosis treatment, has greater risk of side effects and lasts for 18-24
months and target processes or enzymes within M. tuberculosis but suffers from the risk of
generating newer variants exhibiting drug resistance. Since, M. tuberculosis survives within
the human macrophage through modulation of a range of host cellular processes hence, a
pharmacological target within the host that has been co-opted by M. tuberculosis for its
survival should be a breakthrough approach for therapy. Additionally such an approach
should be insensitive to whether the infecting strain was drug sensitive or drug resistant and
preclude the development of resistance.
OBJECT OF THE INVENTION
An object of the invention is to provide compounds useful as anti-tubercular agents.
SUMMARY OF THE INVENTION
The present invention provides novel ula 1:
Formula 1
wherein,
X is selected from O, NH or N(alkyl);
Ri and R2are independently selected from hydrogen, deuterium, hydroxyl, Cj-io straight chain
or branched chain alkyl, 3-7 membered cycloalkyl, Ci.6alkoxy, aryl, amino, NH(alkyl),
N(alkyl)2 OCOR5, heteroaryl containing 1-3 heteroatoms selected from the group comprising
O, N or S; or Ri and R2 may combined to form an aryl or a heteroaryl ring containing 1-3
heteroatoms selected from the group comprising O, N or S;
R3isselected from hydrogen, hydroxyl, Ci-6 straight chain or branched chain alkyl, 3-7
membered cycloalkyl, Ci^alkoxy, aryl, aromatic or non-aromatic heterocyclic ring or fused
heterocyclic rings elected from:
X may in conjunction with R3 form a 5-7 membered heterocyclic ring comprising 1-3
heteroatoms selected from group comprising N, O or S. The heterocyclic ring be further
substituted with one or more lower alkyl groups, halogens, amino, NH(alkyl), N(alkyl)2, NHaralkyl;-
R4 is selected from a hydrogen, lower straight chain or branched alkyl, halogen, deuterium,
CI-6 alkoxy, amino, NH(alkyl), N(alkyl)2, -COOR8, CONR8R9;
R5 is hydrogen, hydroxy, Cl-6alkyl, Cl-6alkoxy, amino, NH(alkyl), N(alkyl)2;
R6 and R7 are independently selected from the group comprising hydrogen, CI-10 alkyl, -
COR8, -CH20COR8, -CH20CONHR8R9, -COOR8, -CONR8R9, -S02R8, aryl, aralkyl;
R8 and R9 are independently selected from the group comprising hydrogen, or CI-6 straight
chain or branched chain alkyl;
n is 1,2, or 3.and salts, hydrates and stereoisomers thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1, depicts the effects of the compounds of the present invention in MYC-431 infected
THP-1 macrophages
Figure 2, depicts the effects of the compounds of the present invention on Lipid Bodies
Figure 3 depicts the potency of compound 1085 of the present invention in Cellular M.tb
Figure 4, represents the effect of compound 1085 on adenylyl cyclase (c-AMP) in THP-1
macrophages
Figure 5a and 5b, represents the Co-localization of M. tuberculosis with degradative
vesicles: acidified lysosomes and autophagosomes in the presence of compound 1085
Figure 6, represents the effect of compound 1085 on cellular lipid body
Figure 7, represents the effect of compound 1085 on various mycobacterial strains
Figure 8, represents the effect of compound 1085 on the mycobacterial growth alone and in
combination with other pharmaceutical agents
Figure 9a and 9b, represents the pharmacokinetic parameters of compound 1085
administered via intraperitoneal route
Figure 10, represents the combination treatment of compound 1085 with Antituberculer
therapy (ATT)
DETAILED DESCRIPTION OF THE INVENTION
A. Compounds of the present invention:
Accordingly, the present invention provides novel compound of Formula 1 as antitubercular
agents.
The present invention relates to novel compounds of Formula ( 1) :
Formula 1
wherein,
X is selected from O or NH;
and R are independently selected from hydrogen, deuterium, hydroxyl, C straight
chain or branched chain alkyl, 3-7 membered cycloalkyl, Ci- alkoxy, aryl, amino, NH(alkyl),
N(alkyl)2 OCOR5, heteroaryl containing 1-3 heteroatoms selected from the group comprising
O, N or S; or Ri and R2 may combined to form an aryl or a heteroaryl ring containing 1-3
heteroatoms selected from the group comprising 0 , N or S;
R3 is selected from hydrogen, hydroxyl, Ci-6 straight chain or branched chain alkyl, 3-7
membered cycloalkyl, Ci- alkoxy, aryl, aromatic or non-aromatic heterocyclic ring or fused
heterocyclic ring selected from:
X may in conjunction with R3 form a 5-7 membered heterocyclic ring comprising 1-3
heteroatoms selected from group comprising N, 0 or S. The heterocyclic ring be further
substituted with one or more lower alkyl groups, halogens, amino, NH(alkyl), N(alkyl)2, NHaralkyl;
R4 is selected from a hydrogen, lower straight chain or branched alkyl, halogen, deuterium,
CI-6 alkoxy, amino, NH(alkyl), N(alkyl)2, -COOR8, CONR8R9;
R5 is hydrogen, hydroxy, Cl-6alkyl, Cl-6alkoxy, amino, NH(alkyl), N(alkyl)2;
R6 and R7 are independently selected from the group comprising hydrogen, CI-10 alkyl, -
COR8, -CH20COR8, -CH20CONHR8R9, -COOR8, -CONR8R9, -S02R8, aryl, aralkyl;
R8 and R9 are independently selected from the group comprising hydrogen, or CI-6 straight
chain or branched chain alkyl;
n is 1,2, or 3;
and salts, hydrates and stereoisomers thereof.
Further, the present invention relates to novel compounds of Formula (1):
Formula 1
wherein
X is NH;
Ri and R are selected from hydrogen, hydroxyl, C Oalkyl, C1-6alkoxy;
R 3 is selected from substituted aromatic or non-aromatic heterocyclic ring or fused
heterocyclic ring selected fro
R 4 is selected from hydrogen, deuterium, halogen, Ci-6 straight chain or branched chain alkyl,
Ci-6 alkoxy, amino, NH(alkyl), N(alkyl) 2;
R and R7 are independently selected from hydrogen, Ci-10 alkyl, -COR8, -CH2OCOR8, -
CH2OCONHR8R9, -COOR , -CONR R9, -S0 2R , phenyl, benzyl;
R8 and R9 are independently selected from hydrogen or C1-6 straight chain or branched chain
alkyl;
n is 1,2 or 3.
and salts, hydrates and stereoisomers thereof.
The term "alkyl" refers to a linear or branched saturated monovalent hydrocarbon, wherein
the alkylene may optionally be substituted as described herein. The term "alkyl" also
encompasses both linear and branched alkyl, unless otherwise specified. In certain
embodiments, the alkyl is a linear saturated monovalent hydrocarbon that has the specified
number of carbon atoms, or branched saturated monovalent hydrocarbon of specified number
of carbon atoms. As used herein, linear CI- C6 and branched C3- C6 alkyl groups are also
referred as "lower alkyl. "Examples of alkyl groups include, but are not limited to, methyl,
ethyl, propyl (including allisomeric forms), n-propyl, isopropyl, butyl (including all isomeric
forms), rc-butyl, isobutyl, sec-butyl, t-buly\, pentyl (including all isomeric forms), and hexyl
(including all isomeric forms). For example, CI - C6 alkyl refers to a linear saturated
monovalent hydrocarbon of 1 to 6 carbon atoms or a branched saturated monovalent
hydrocarbon of 3 to 6 carbon atoms.
The term "aryl" refers to a monocyclic aromatic group and/or multicyclic monovalent
aromatic group that contain at least one aromatic hydrocarbon ring. In certain embodiments,
the aryl has from 6 to 20 (C6 - C20), from 6 to 15 (C6 - CI 5), or from 6 to 10 (C6 - CIO) ring
atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl,
azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl. Aryl also refers to bicyclic
or tricyclic carbon rings, where one of the rings is aromatic and the others of which may be
saturated, partially unsaturated, or aromatic, for example, dihydronaphthyl, indenyl, indanyl,
or tetrahydronaphthyl (tetralinyl). In certain embodiments, aryl may be optionally substituted
as described herein.
The term "alkoxy" refers to the group R'0~ wherein R' is alkyl. The term "lower alkoxy"
refers to alkoxy groups having from 1 to 3 carbon atoms; examples include methoxy, ethoxy,
isopropoxy, and the like.
The term "cycloalkyl" as used herein refers to a saturated or partially unsaturated,
monocyclic, fused bicyclic or bridged polycyclic ring assembly containing from 3 to 12 ring
atoms, or the number of atoms indicated. Cycloalkyl can include any number of carbons,
such as C3-6 , C -6, C5-6 , C3-8 , C4-8 , C5-8 , and C6 8. Saturated monocyclic cycloalkyl rings
include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl.
Saturated bicyclic and polycyclic cycloalkyl rings include, for example, norbornane,
[2.2.2]bicyclooctane, decahydronaphthalene and adamantane. Cycloalkyl groups can also be
partially unsaturated, having one or more double bonds in the ring.
Representative cycloalkyl groups that are partially unsaturated include, but are not limited to,
cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers),
cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers),
norbornene, and norbornadiene. Unless otherwise stated, cycloalkyl groups are unsubstituted.
A "substituted cycloalkyl" group can be substituted with one or more moieties selected from
halo, hydroxy, amino, alkylamino, nitro, cyano, and alkoxy.
The term "aralkyl" or "aryl-alkyl" refers to a monovalent alkyl group substituted with aryl.
Incertain embodiments, the alkyl and aryl moieties are optionally substituted as described
herein.
The term "heteroaryl" refers to a monocyclic aromatic group and/or multicyclic aromatic
group that contain at least one aromatic ring, wherein at least one aromatic ring contains one
or more heteroatoms independently selected from 0, S, and N. Each ring of a heteroaryl group
may contain one or two O atoms, one or two S atoms, and/or one to four N atoms, provided
that the total number of heteroatoms in each ring is four or less and each ring contains at least
one carbon atom. In certain embodiments, the heteroaryl has from 5 to 20, from 5 to 15, or
from 5 to 10 ring atoms. Examples of monocyclic heteroaryl groups include, but are not
limited to, furanyl,imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxadiazolyl, oxazolyl,
pyrazinyl, pyrazolyl,pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, thiadiazolyl, thiazolyl,
thienyl, tetrazolyl, triazinyl,and triazolyl. Examples of bicyclic heteroaryl groups include, but
are not limited to,benzofuranyl, benzimidazolyl, benzoisoxazolyl, benzopyranyl,
benzothiadiazolyl, benzothiazolyl, benzothienyl, benzothiophenyl, benzotriazolyl,
benzoxazolyl, furopyridyl, imidazopyridinyl, imidazothiazolyl, indolizinyl, indolyl,
indazolyl, isobenzofuranyl, isobenzothienyl, isoindolyl, isoquinolinyl, isothiazolyl,
naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridyl,
pyrrolopyridyl, quinolinyl, quinoxalinyl, quinazolinyl, thiadiazolopyrimidyl, and
thienopyridyl. Examples of tricyclic heteroaryl groupsinclude, but are not limited to,
acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, perimidinyl, phenanthrolinyl,
phenanthridinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and xanthenyl. In
certain embodiments, heteroaryl may also be optionally substituted as described herein.
The term heteroaralkyl refers to an aralkyl group as defined above, in which one or more
(preferably 1, 2, 3 or 4) carbon atoms have been replaced by an oxygen, nitrogen, silicon,
selenium, phosphorus, boron or sulphur atom (preferably oxygen, sulphur or nitrogen), that is
to say groups that in accordance with the above definitions contain both aryl or heteroaryl
and alkyl, alkenyl, alkynyl and/or heteroalkyl and/or cycloalkyl and/or heterocycloalkyl
groups. A hetero-aralkyl group preferably contains one or two aromatic ring systems ( 1 or 2
rings) with from 5 or 6 to 10 carbon atoms and one or two alkyl, alkenyl and/or alkynyl
groups having from 1 or 2 to 6 carbon atoms and/or a cycloalkyl group having 5 or 6 ring
carbon atoms, with 1, 2, 3 or 4 of those carbon atoms having been replaced by oxygen,
sulphur or nitrogen atoms. Examples are aryl-heteroalkyl, aryl-heterocycloalkyl, arylheterocycloalkenyl,
arylalkyl-heterocycloalkyl, arylalkenyl-heterocycloalkyl, arylalkynylheterocycloalkyl,
arylalkyl-heterocycloalkenyl, heteroarylalkyl, heteroarylalkenyl,
heteroarylalkynyl, heteroaryl-heteroalkyl, heteroarylcyclo-alkyl, heteroarylcycloalkenyl,
heteroaryl-heterocycloalkyl, heteroaryl-heterocycloalkenyl, heteroarylalkylcycloalkyl, _ g _
heteroarylalkyl-heterocycloalkenyl, heteroaryl-heteroalkyl-cycloalkyl, heteroarylheteroalkylcycloalkenyl
and hetero-aryl-heteroalkyl-heterocycloalkyl groups, the cyclic
groups being saturated or mono-, di- or tri-unsaturated. Specific examples are the
tetrahydroisoquinolinyl, benzoyl, 2- or 3-ethylindolyl, 4-methylpyridino, 2-, 3- or 4-
methoxyphenyl, 4-ethoxyphenyl and 2-, 3- or 4-carboxylphenylalkyl groups.
The term "heterocyclyl" or "heterocyclic" refers to a monocyclic non-aromatic ring system
and/or multicyclic ring system that contains at least one non-aromatic ring, wherein one or
more of the non-aromatic ring atoms are heteroatoms independently selected from 0 , S, or N;
and the remaining ring atoms are carbon atoms. In certain embodiments, the heterocyclyl or
heterocyclic group has from 3 to 20, from 3 to 15, from 3 to 10, from 3 to 8, from 4 to 7, or
from 5 to 6 ring atoms. In certain embodiments, the heterocyclyl is a monocyclic, bicyclic,
tricyclic, or tetracyclic ring system, which may include a fused or bridged ring system, and in
which thenitrogen or sulfur atoms may be optionally oxidized, the nitrogen atoms may be
optionally quaternized, and some rings may be partially or fully saturated, or aromatic. The
heterocyclyl may be attached to the main structure at any heteroatom or carbon atom which
results in thecreation of a stable compound. Examples of such heterocyclic compounds
include, but are not limited to, azepinyl, benzodioxanyl, benzodioxolyl, benzofuranonyl,
benzopyranonyl, benzopyranyl, benzotetrahydrofuranyl, benzotetrahydrothienyl,
benzothiopyranyl, benzoxazinyl, p-carbolinyl, chromanyl, chromonyl, cinnolinyl,
coumarinyl, decahydroisoquinolinyl, dihydrobenzisothiazinyl, dihydrobenzisoxazinyl,
dihydrofuryl, dihydroisoindolyl, dihydropyranyl, dihydropyrazolyl, dihydropyrazinyl,
dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl, 1,4-dithianyl, furanonyl,
imidazolidinyl, imidazolinyl, indolinyl, isobenzotetrahydrofuranyl,
isobenzotetrahydrothienyl, isochromanyl, isocoumarinyl, isoindolinyl, isothiazolidinyl,
isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazolidinonyl,
oxazolidinyl, oxiranyl, piperazinyl, piperidinyl, 4-piperidonyl, pyrazolidinyl, pyrazolinyl,
pyrrolidinyl, pyrrolinyl, quinuclidinyl, tetrahydrofuryl, tetrahydroisoquinolinyl,
tetrahydropyranyl, tetrahydrothienyl, thiamorpholinyl, thiazolidinyl, tetrahydroquinolinyl,
and 1,3,5-trithianyl. In certain embodiments, heterocyclic may also be optionally substituted
as described herein.
The term "halogen", "halide" or "halo" refers to fluorine, chlorine, bromine, and iodine.
The term "heteroatom" as used herein means an atom of any element other than carbon or
hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur, phosphorus and selenium.
The term "aromatic", as used herein, refers to an unsatured cyclic moiety that has an aromatic
character. The term is intended to encompass both hydrocarbon aromatic compounds and
heteroaromatic compounds. The terms "hydrocarbon aromatic ring" or "hydrocarbon
aromatic compound" refer to an aromatic ring or compound in which the aromatic moieties
have only carbon and hydrogen atoms. The terms "heteroaromatic ring" or "heteroaromatic
compound' refer to an aromatic ring or compound wherein in at least one aromatic moiety
one or more of the carbon atoms within the cyclic group has been replaced by another atom,
such as nitrogen, oxygen, sulfur, or the like.
The term "non-aromatic", as used herein, refers to a cyclic moiety, that may be unsaturated,
but that does not have an aromatic character.
The term "substituted" refers to where hydrogen radical on a molecule has been replaced by
another atom radical, a functional group radical or a moiety radical; these radicals being
generally referred to as "substituents."
The term "optionally substituted" is intended to mean that a group, such as an alkyl, alkylene,
alkenyl, alkenylene, alkynyl, alkynylene, alkoxy, alkylamino, dialkylamino, carboxamido,
cycloalkyl, cycloalkylene, aryl, arylene, heteroaryl, heteroarylene, heterocyclyl, or
heterocyclylene, may be substituted with one or more substituents independently selected
from,e.g., (a) CI - C6 alkyl, C2 - C6 alkenyl, C2 - C6 alkynyl, C3 - C7 cycloalkyl, C6 - C14
aryl, C7 - C15aralkyl, heteroaryl, and heterocyclyl, each optionally substituted with one or
more substituents; and (b) halo, cyano (-CN), nitro (-N02),-C(O)R3 , -C(0)OR3 , -
C(0)NRbRC , -C(NR3)NR)RC , -OR3 , -OC(0)R3 , -OC(0)OR3, -OC(0)NRbRC, -
OC(=NR3)NR)RC, -OS(0)R3, -OS(0)2R3, -OS(0)NRbRC, -OS(0)2NRbRc, -NRbRc,-
NR3C(0)Rd„ -NR3C(0)ORd, -NR3C(0)NRbRC ,-NR3C(=NRd)NRbRC , -NR3S(0)Rd, -
NR3S(0)2Rd, -NR3S(0)NRbRC, -NR3S(0)NRbRc, -SR3, -S(0)R3, -S(0)2R3, -
S(0)NRbRC, and -S(0)2NRbRC, wherein each R3, Rb, Re, and Rd isindependently (i)
hydrogen; (ii) C - C6 alkyl, C2 - C6 alkenyl, C2 - C6 alkynyl, C3 - C7cycloalkyl, C6 - C14
aryl, C7 - C15 aralkyl, heteroaryl, or heterocyclyl, each optionally substituted with one or
more substituents; or (iii) Rb and Re together with the N atom to which they are attached
form heteroaryl or heterocyclyl, optionally substituted with one or more, in one embodiment,
one, two, three, or four, substituents. As used herein, all groups that may be substituted are
"optionally substituted," unless otherwise specified.
The use of terms "a" and "an" and "the" and similar references in the context of describing
the invention (especially in the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein or clearly contraindicated
by context.
The term "salt(s)", as employed herein, denotes acidic and/or basic salts formed with
inorganic and/or organic acids and bases. Zwitterions (internal or inner salts) are included
within the term "salt(s)" as used herein (and may be formed, for example, where the R
substituents comprise a basic moiety such as an amino group). Also included herein are
quaternary ammonium salts such as alkyl ammonium salts. Pharmaceutically acceptable (i.e.,
non-toxic, physiologically acceptable) salts are preferred.
The term "pharmaceutically acceptable salts" refers to the acid addition salt compound
formed with a suitable acid selected from an inorganic acid such as hydrochloric acid,
hydrobromic acid; or an organic acid such as benzene sulfonic acid, maleic acid, oxalic acid,
fumaric acid, succinic acid, p-toluenesulfonic acid and malic acid.
The term "hydrate" as used herein designates a crystalline molecular compound in which
water molecules are incorporated into the crystal lattice. Generally speaking, a hydrate thus
designates a crystalline form of a molecular compound, whereby the only further molecules
incorporated into the crystal lattice are water molecules.
The term "stereoisomer' s" refers to at least two compounds having the same molecular
formula and connectivity of atoms, but having a different arrangement of atoms in a threedimensional
space. In view of the present disclosure, a stereoisomer can be, for example, an
enantiomer, a diastereomer, or a meso compound.
The term "prophylactic" as used herein refers variously to medicaments, amounts or
quantities, methods, uses and effects, etc., that prevent and/or aid in preventing infections.
The term "therapeutic" as used herein refers to preventing, ameliorating, treating, improving,
or curing a disease or condition.
The term "Directly Observed Treatment Short-Course" (DOTS) refers to treatment regimen
consisting of a four-drug such as Rifampicin, Isoniazid, Ethambutol and Pyrazinamide)
intensive phase of two months, followed by a two-drug (Rifampicin and Isoniazid)
continuation phase of four months.
The term "Multidrug resistance Tuberculosis" (MDR-TB) as used herein refers to
tuberculosis which are resistant to atleast isoniazid (INH) and rifampicin (RMP), the two
most powerful first-line treatment anti-Tuberculosis drugs.
The term "Extensively drug resistance Tuberculosis" (XDR-TB) as used herein refers to the
tuberculosis which are resistant to atleast three of the six classes of second line anti¬
tuberculosis drugs as well as isoniazid and rifampicin, plus any fluoroquinolone and at least
one of three injectable second-line drugs such asamikacin, kanamycin, or capreomycin.
The term "Totally Drug Resistance Tuberculosis" (TDR-TB) as used herein refers to the
tuberculosis which are resistant to all first and second line drugs tested such as isoniazid,
rifampicin, streptomycin, ethambutol, pyrazinamide, ethionamide, para-aminosalicylic acid,
cycloserine, ofloxacin, amikacin, ciprofloxacin, capreomycin, kanamycin.
The term"H37Rv" as used herein refers to a pathogenic M. tuberculosis strain that has been
sequenced.
The term "JAL2287" as used herein refers to the MDR (multi drug resistant) strainof
tuberculosis
The term "MYC43 1" as used herein refers to the XDR (extensive drug resistant) strain of M.
tuberculosis
The term "GPR109A" as used herein refers to the G-protein coupled receptor GPR109A.
The present invention provides compound represented by formula (1) that inhibits
mycobacterium colony growth alone or in combination with DOTS (Directly Observed
Treatment Short Course) therapy.
The compounds of the present invention may be illustrated but not limited to the examples as
provided at Table 1.
Table 1: Illustrative compounds of present invention

The present invention also provides for compounds of formula (1) as below:
i . 1-methylpiperidin-3-yl-2-hydroxy-2,2-diphenylacetate;
ii. 3-(2-hydroxy-2,2-diphenylacetoxy)-l-(((isopropylcarbamoyl)oxy)methyl)-lmethylpiperidin-
1-ium;
iii. piperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
iv. l-benzylpiperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
v. 1-(methylsulfonyl)piperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
vi. 1-(dimethylcarbamoyl)piperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
vii. 1-benzylpiperidin-4-yl 2-hydroxy-2,2-diphenylacetate;
viii. 3-(2-hydroxy-2,2-diphenylacetoxy)-l-methylquinuclidin-l-ium;
ix. l-benzylpyrrolidin-3-yl 2-hydroxy-2,2-diphenylacetate;
x. l-ethylpiperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xi. 1-(isopropylcarbamoyl)piperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xii. l-propylpiperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xiii. 1-methylpiperidin-4-yl 2-hydroxy-2,2-diphenylacetate;
xiv. l-benzylazetidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xv. l-acetylpiperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xvi. 2-hydroxy-2,2-diphenyl-N-(piperidin-3-yl)acetamide;
xvii . N-( 1-benzylpiperidin-4-yl)-2-hydroxy-2,2-diphenylacetamide;
xviii. l-methylpyrrolidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xix. quinuclidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xx. 2-hydroxy-N-(l-methylpiperidin-3-yl)-2,2-diphenylacetamide;
xxi. N-( 1-benzylpiperidin-4-yl)-2-hydroxy-2,2-diphenylacetamide;
xxii . N-( 1-benzylazetidin-3 -yl)-2-hydroxy-2,2-diphenylacetamide;
xxiii. azetidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xxiv. (S)-l-benzylpiperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xxv. (R)-l -benzylpiperidin-3 -yl 2-hydroxy-2,2-diphenylacetate;
xxvi. (S)-l-methylpiperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xxvii. (R)-l-methylpiperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xxviii. (R)-N-( 1-benzylpiperidin-3-yl)-2-hydroxy-2,2-diphenylacetamide;
xxix. (R)-N-(l-benzylpiperidin-3-yl)-2-hydroxy-2,2-diphenylacetamide;
xxx. 2-hydroxy-2,2-diphenyl-N-(piperidin-4-yl)acetamide;
xxxi. (S)-2-hydroxy-N-(l-methylpiperidin-3-yl)-2,2-diphenylacetamide;
xxxii. (R)-2-hydroxy-N-(l-methylpiperidin-3-yl)-2,2-diphenylacetamide;
xxxiii. l-benzylazetidin-3-yl 2-hydroxy-2-phenylpropanoate;
xxxiv. (R)-N-( 1-benzylpiperidin-3 -yl)-2,2-diphenylacetamide;
xxxv. N-((S)- 1-benzylpiperidin-3 -yl)-2-hydroxy-2-phenylacetamide;
xxxvi. N-((R)- 1-benzylpiperidin-3 -yl)-2-hydroxy-2-phenylpropanamide;
xxxvii. (R)-(R)- 1-methylpiperidin-3-yl 2-hydroxy-2-phenylacetate;
xxxviii. (R)-(S)- 1-methylpiperidin-3-yl 2-hydroxy-2-phenylacetate;
xxxix. (S)-(R)-l-methylpiperidin-3-yl 2-hydroxy-2-phenylacetate;
xl. (S)-(S)-l-methylpiperidin-3-yl 2-hydroxy-2-phenylacetate;
xli. (R)-l-methylpiperidin-4-yl 2-hydroxy-2-phenylacetate;
xlii. (S)-2-hydroxy-N-( 1-methylpiperidin-4-yl)-2-phenylacetamide;
xliii. (R)-2-hydroxy-N-(l-methylpiperidin-4-yl)-2-phenylacetamide;
xliv. (R)-2-hydroxy-N-((R)- 1-methylpiperidin-3-yl)-2-phenylacetamide;
xlv. (R)-2-hydroxy-N-((S)-l-methylpiperidin-3-yl)-2-phenylacetamide;
xlvi. (S)-2-hydroxy-2-phenyl-N-(pyridin-3-yl)acetamide;
xlvii. (S)-l-methylpiperidin-4-yl 2-hydroxy-2-phenylacetate;
xlviii. (S)-l-benzylazetidin-3-yl 2-hydroxy-2-phenylacetate;
xlix. (S)-(R)-quinuclidin-3-yl 2-hydroxy-2-phenylacetate;
1. (S)-(S)-quinuclidin-3-yl 2-hydroxy-2-phenylacetate;
li. (R)-N-(l-benzylpiperidin-4-yl)-2-hydroxy-2-phenylacetamide;
i . (S)-N-(l-benzylpiperidin-4-yl)-2-hydroxy-2-phenylacetamide;
liii. (R)-3-((R)-2-hydroxy-2-phenylacetoxy)- 1,1-dimethylpiperidin- 1-ium;
liv. (S)-3 -((R)-2-hydroxy-2-phenylacetoxy)- 1,1-dimethylpiperidin- 1-ium;
lv. (R)-2-hydroxy-2-phenyl-N-(pyridin-3-yl)acetamide;
lvi. (R)-(S)-l-methylpiperidin-3-yl 2-acetoxy-2-phenylacetate;
lvii. (R)-(R)- 1-methylpyrrolidin-3-yl 2-hydroxy-2-phenylacetate;
lviii. (R)-(S)-l-methylpyrrolidin-3-yl 2-hydroxy-2-phenylacetate;
lix. (S)-(R)-l-methylpiperidin-3-yl 2-methoxy-2-phenylacetate;
lx. 2-methoxy-2-phenylacetic acid;
lxi. (R)-2-hydroxy-2-phenyl-N-(piperidin-4-yl)acetamide;
lxii. (R)-piperidin-4-yl 2-hydroxy-2-phenylacetate;
lxiii. (R)-3 -(2-hydroxy-2,2-diphenylacetoxy)- 1,1-dimethylpiperidin- 1-ium;
lxiv. (R)-3-(2-hydroxy-2,2-diphenylacetoxy)- 1,1-dimethylpiperidin- 1-ium;
lxv. (R)-3-((R)-2-hydroxy-2-phenylacetoxy)- 1,1-dimethylpiperidin- 1-ium;
lxvi. (R)-3-((R)-2-hydroxy-2-phenylacetoxy)- 1,1-dimethylpiperidin- 1-ium;
lxvii . (S)-(R)- 1-methylpiperidin-3 -yl 2-methoxy-2-phenylacetate;
lxviii. (S)-(S)-l-methylpiperidin-3-yl 2-methoxy-2-phenylacetate;
lxix. (S)-2-hydroxy-N-((R)-l-methylpiperidin-3-yl)-2-phenylacetamide;
lxx. (S)-2-hydroxy-N-((S)-l-methylpiperidin-3-yl)-2-phenylacetamide;
lxxi . 3-(2,2-diphenylacetamido)- 1,1-dimethylpiperidin- 1-ium;
lxxii . (2S)-1 -benzyl-2-((2,2-diphenylacetamido)methyl)- 1-methylpyrrolidin- 1-ium;
lxxiii. 3-(2-hydroxy-2,2-diphenylacetamido)- 1,1 -dimethylpiperidin- 1-ium;
lxxiv. (R)-2-methoxy-l-((S)-3-methylmorpholino)-2-phenylethanone;
lxxv. (R)-2-methoxy- 1-((R)-3 -methylmo holino)-2-phenylethanone;
lxxvi. (R)-l-((2R,3S)-2,3-dimethylmo holino)-2-methoxy-2-phenylethanone;
lxxvii. (S)-2-methoxy-l-((S)-3-methylmorpholino)-2-phenylethanone;
lxxviii. (1R,3R)- 1-benzyl-3-(2,2-diphenylacetamido)- 1-methylpiperidin- -ium;
lxxix. (lS,3R)-l-benzyl-3-(2,2-diphenylacetamido)-l-methylpiperidin-l-ium;
lxxx. (lS,3R)-l-benzyl-3-(2,2-diphenylacetamido)-l-methylpiperidin-l-ium;
lxxxi. (1R,3R)- 1-benzyl-3-(2,2-diphenylacetamido)- 1-methylpiperidin- 1-ium;
lxxxii. l-(3-(benzylamino)piperidin-l-yl)-2,2-diphenylethanone;
lxxxiii. N-((l-benzylpyrrolidin-2-yl)methyl)-2,2-diphenylacetamide;
lxxxiv. 1-benzyl-2-((2,2-diphenylacetamido)methyl)- 1-methylpyrrolidin- 1-ium;
lxxxv. (R)-2-methoxy-2-phenyl-N-(piperidin-4-yl)acetamide;
lxxxvi. . 2-hydroxy-2-phenyl-N-(piperidin-4-yl)acetamide;
lxxxvii. (S)-2-hydroxy-2-phenyl-N-(piperidin-4-yl)acetamide;
lxxxviii. (S)-2-methoxy-2-phenyl-N-(piperidin-4-yl)acetamide;
lxxxix. 2-hydroxy-2-phenyl-N-(piperidin-4-yl)propanamide;
xc. 2-(3-bromo-2,6-difluorophenyl)-2-hydroxy-N-(piperidin-4-yl)acetamide;
xci. (R)-2-methoxy-N-methyl-2-phenyl-N-(piperidin-4-yl)acetamide;
xcii. (R)-N-(l-benzylpiperidin-4-yl)-2-methoxy-N-methyl-2-phenylacetamide;
xciii. (S)-l-benzylpiperidin-4-yl 2-methoxy-2-phenylacetate;
xciv. (R)-ethyl 4-(2-methoxy-2-phenylacetamido)piperidine-l -carboxylate;
B. Salts and Isomers and Counter Ions
The present invention includes within its scope the salts and isomers. Compounds of the
present invention after being novel may in some cases form salts which are also within the
scope of this invention.
All stereoisomer' s of the present compounds, such as those which may exist due to
asymmetric carbons on the R substituents of the compound, including enantiomeric and
diastereomeric forms, are contemplated within the scope of this invention.
The present invention may also optionally envisage within its scope the effect of selection of
suitable counter ions. The present invention includes in its scope, the modification of
deuterated compounds. Deuterated compounds are those wherein the compounds have
selective incorporation of deuterium in place of hydrogen.
The Compounds of the present invention may be present in their enantiomeric pure forms or
their mixtures.
C. Synthesis of the Compounds of the Present Invention
The compounds of the present invention may be synthesized by the any of the synthetic
schemes as shown below:
General Synthetic Scheme
The compounds of present invention may be synthesized by coupling A with B in the
presence of (i) as depicted in the scheme above.
X is selected from O, NH or N(alkyl);
R and R2are independently selected from hydrogen, deuterium, hydroxyl, C O straight chain
or branched chain alkyl, 3-7 membered cycloalkyl, C - alkoxy, aryl, amino, NH(alkyl),
N(alkyl) OCOR , heteroaryl containing 1-3 heteroatoms selected from the group comprising
O, N or S; or R and R2 may combined to form an aryl or a heteroaryl ring containing 1-3
heteroatoms selected from the group comprising O, N or S;
R3isselected from hydrogen, hydroxyl, Ci-6 straight chain or branched chain alkyl, 3-7
membered aryl, aromatic or non-aromatic heterocyclic ring or fused
heterocyclic ring selected from:
X may in conjunction with R3 form a 5-7 membered heterocyclic ring comprising 1-3
heteroatoms selected from group comprising N, O or S. The heterocyclic ring be further
substituted with one or more lower alkyl groups, halogens, amino, NH(alkyl), N(alkyl)2, NHaralkyl;
R4 is selected from a hydrogen, lower straight chain or branched alkyl, halogen, deuterium,
CI-6 alkoxy, amino, NH(alkyl), N(alkyl)2, -COOR8, CONR8R9;
R5 is hydrogen, hydroxy, Cl-6alkyl, Cl-6alkoxy, amino, NH(alkyl), N(alkyl)2;
R6 and R7 are independently selected from the group comprising hydrogen, CI-10 alkyl, -
COR8, -CH20COR8, -CH20CONHR8R9, -COOR8, -CONR8R9, -S02R8, aryl, aralkyl;
R8 and R9 are independently selected from the group comprising hydrogen, or CI -6 straight
chain or branched chain alkyl;
n is 1,2, or 3. nd salts, hydrates and stereoisomers thereof.
A general procedure involves the coupling reactions of acid with an amine or alcohol (R X).
In one method, an acid (A) is used as a starting material which can be activated by converting
it to corresponding acid chloride using reagent such thionyl chloride or oxalyl chloride in a
solvent such as DCM with or without a small amount of DMF at temperature ranging from
room temperature to reflux and then by reaction of this acid chloride with a desired amine or
alcohol (R3X)to arrive at other members of the series.
Another method involves the use of an acid coupling reagent is such as carbodiimide in an
organic solvent such as DMF with or without an organic base such DMAP. Alternatively,
acid can be activated by converting to corresponding ester using alcohol such as methanol
first before reacting it with an amine or alcohol in a solvent such as benzene with or without a
base such as sodium methoxide to arrive at other members of the series.
D. Methods of Use and Pharmaceutical Composition containing the Novel Entities
of the Invention
The invention thus provides the use of the novel compounds as defined herein for use in
human or veterinary medicine. The compounds of the present invention may be used in
the treatment and prevention of diseases caused by mycobacteria such as tuberculosis. The
said tuberculosis may be caused by mycobacterial species consisting of Mycobacterium
tuberculosis, M. bovis, M africanum, M. canettior M. microti. Particularly, the compounds of
the present invention are effective in inhibiting the growth of the M. tuberculosis.
Tuberculosis as mentioned herein comprises active tuberculosis or latent tuberculosis. The
active tuberculosis comprises drug sensitive, mono-drug resistant, multi-drug-resistant
tuberculosis (MDR), extensively drug- resistant tuberculosis (XDR) or totally drug resistant
tuberculosis.
In an aspect, the compounds of the present invention may be used in the treatment of
tuberculosis including MDR, XDR and TDR tuberculosis.
In an aspect, the compounds of the present invention may be used either alone or in
combination with cycloserine, amikacin, linezolid, ethambutol, rifampicin, isoniazid,
ethionamide, moxyfloxacin, clarithromycin, PAS, clofazamine, streptomycin, capreomycin
and kanamycin and DOTS ("Directly Observed Treatment, Short-course") therapy.
In another aspect, the compounds of the present invention may also be used prophylactically
in preventing the occurrence of tuberculosis in healthcare providers, health workers,
community members and the alike who are directly in contact with tuberculosis patients.
The compounds of the present invention may be used for treatment of the infection caused by
resistant and non-resistant Mycobacterium tuberculosis as defined above. The compounds of
the present invention are effective against tuberculosis and said tuberculosis includes drugsensitive,
mono-drug resistance, multi drug-resistant (MDR), extensively drug-resistance
(XDR) and totally drug resistance (TDR) caused by a strain of Mycobacterium tuberculosis
Mycobacterium strains. The compounds of the present invention may be used for the
treatment of multi-drug-resistant (MDR), extensively drug-resistance (XDR) and totally drug
resistance (TDR) tuberculosis caused by a strain Mycobacterium tuberculosis and for
treatment of the infection caused by resistant and non-resistant Mycobacterium tuberculosis
as defined above by inhibition of GPR109A inhibition by an agent.
The compound for use as a pharmaceutical may be presented as a pharmaceutical
composition. The invention therefore provides in a further aspect a pharmaceutical
composition comprising the novel compounds of the invention along with pharmaceutically
acceptable excipients/carriers thereof and optionally other therapeutic and/or prophylactic
ingredients. The excipients/carriers must be "acceptable" in the sense of being compatible
with the other ingredients of the composition and not deleterious to the recipient thereof.
Suitably the pharmaceutical composition will be in an appropriate formulation.
The pharmaceutical formulations may be any formulation and include those suitable for oral,
intranasal, or parenteral (including intramuscular and intravenous) administration. The
formulations may, where appropriate, be conveniently presented in discrete dosage units and
may be prepared by any of the methods well known in the art of pharmacy. All methods
include the step of bringing into association the active compound with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the product into the desired
formulation.
For these purposes the compounds of the present invention may be administered orally,
topically, intranasally, , parenterally, by inhalation spray or rectally in dosage unit
formulations containing conventional non-toxic pharmaceutically acceptable carriers,
adjuvants and vehicles. The term parenteral as used herein includes subcutaneous injections,
intravenous, intramuscular, intrasteral injection or infusion techniques. In addition to the
treatment of warm-blooded animals such as mice, rats, horses, dogs, cats, etc. The
compounds of the present invention are effective in the treatment of humans.
In an aspect, compound of the present invention may be administered in a dose ranging from
0.1 to 100 mg/kg body weight per day. The compounds of the present invention are useful for
the prevention and treatment of tuberculosis and may be used as GPR109A inhibitors.
E. Experimental
The compounds of present invention may be prepared by the schemes as here below:
Synthetic Scheme 1:
Step 1:
To a solution of OH (21.0 gm) in water (42.0 ml), ethanol (54.0 ml) and the compound [1]
(25.Og, 19mmol) was added and the resulting solution was refluxed for 30 minutes and
poured into a glass plate and left overnight at RT. The semisolid obtained was dissolved in
water (400 ml) and washed with ethyl acetate. The pH of the aqueous layer was adjusted to
acidic with 50% HCI, and extracted with ethyl acetate. The ethyl acetate layer was dried over
anhydrous Na2S0 4 and concentrated to give 2-hydroxy-2,2-diphenylacetic acid,(12.0 g,
45%). Analytical Data: [2] ESIMS: 229 [M++l]
Step 2
To a solution of the compound obtained in the above step (12g, 52.6mmol) in Methanol
(100.0 ml) at 0°C, thionyl chloride (5.0 ml) was added and the resulting solution was refluxed
for 4 hr. Ethanol was concentrated under vacuum and the residue was purified by column
chromatography using 20% ethyl acetate in hexane to give ethyl 2-hydroxy-2,2-
diphenylacetateas liquid (10.08g, 76%).Analytical Data: [3] ESIMS: 257 [M++l]
Step 3:
To a solution of [3](1.00 g, 5.23 mmol) in benzene (90 mL),sodium ( 10 mg) was added.
After refluxing for 2.5 h, a solution of methylbenzilate (1.27 g, 5.23 mmol) was added and
the reaction refluxed overnight. The benzene was evaporated in vacuo and the residue
purified by flash chromatography (eluted first with 20% EtOAc/Hexane, then
5%MeOH/CH2Cl2) to give [1O04] as transparent liquid (981 mg,46%).Analytical Data:
[1004] ESIMS: 402[M++1]
Step 4:
To a stirred solution of [1004] (0. 5 g, 1.24mmol) in a mixture of ethyl acetate and methanol
(1;1, 5 ml), was added a slurry of 10% Pd/C (0.02 g) at room temperature. Hydrogen balloon
pressure was applied and the reaction mixture was stirred for 3hr at RT. Reaction was
monitored using TLC. The reaction mass was filtered over celite and excess of solvent was
removed under vacuum to afford light brown sticky material, which was further purified
using silica gel column and 2% methanol in dichloromethane as eluent to afford [1003] as an
off-white sticky material(0.22g, 57%).Analytical Data: [1003] ESIMS: 312 [M++l]
Step 5 :
To a stirred solution of [1003] (0.1 2g, 0.38 mmol) in DMF, anhydrous K2C0 3 (0.05g, 0.41
mmol) was added at 0°C under nitrogen atmosphere. After an additional stirring for 15
minutes at same temperature, Methyl iodide (0.02 ml, 0.41 mmol) was added drop-wise. The
reaction temperature was allowed to increase up to 25°C and stirring was continued for 3 h.
Consumption of [1003] was monitored by TLC. After complete consumption of [1003], water
(50 ml) was added and organic layer was extracted with ethyl acetate (2x 50 ml). The
combined organic layers were washed with water, brine and dried over sodium sulphate. The
organic layer was concentrated to afford light brown sticky material which was further
purified using silica gel column chromatography using 5% ethyl acetate/hexane as eluent to
afford [1001] as white solid (0.08g, 65%,). Analytical Data: [1001] ESIMS: 326 [M++l].
Step 6:
To a stirred solution of [1003] (0.05g, 0.16 mmol) in DCM, triethylamine (0.03ml, 0.24
mmol) was added at 0°C under nitrogen atmosphere. After an additional stirring for 5 minutes
at same temperature, Dimethylcarbamoyl chloride (0.02 ml, 0.24 mmol) was added dropwise.
The reaction temperature was allowed to increase up to 25°C and stirring was continued
for 1 h. Consumption of [1003] was monitored by TLC. After complete consumption of
[1003], water (50 ml) was added and organic layer was extracted with ethyl acetate (2 x 25
ml). The combined organic layers were washed with water, brine and dried over sodium
sulphate. The organic layer was concentrated to afford light brown sticky material which was
washed further with DCM and pentane to afford [1006] (0.04g, 65%).Analytical
Data:[1006]ESIMS: 385 [M++l].
Synthesis of [1010], [1012], [1008], [1013], [1018], [1007], [1009], [1014], [1019] and
[1023] were carried out by procedure described for [1004].
Synthesis of [1005], [1011] and [1015] were carried out by procedure described for [1006].
Synthetic Scheme 2 :
[1024] [1025]
Step 1:
To a stirred solution of [5] (2.0g, 10.47mmol), vinyl acetate (10.0ml, lOO.Ommol) was added
at room temperature. After an additional stirring for 5 minutes at same temperature, PS-IM
(0.20g, 10%) was added. The reaction temperature was allowed to increase up to 40°C and
stirring was continued for 16 h. Reaction mixture was passed through the bed of celite and
evaporated to dryness which was further purified using silica gel column chromatography
using 10% ethyl acetate/hexane as eluent to afford [6](0.9g, 45%)and [7] (0.8g, 40%) as
transparent sticky material. Analytical Data: [6] ESIMS: 192 [M++ 1][7]ESIMS: 234 [M++l].
Step 2 :
To a solution of [6] (0.45 g, 1.8mmol) in benzene (45 L) was added sodium (0.03g,
1.6mmol). After refluxing for 2.5 h, a solution of ethyl benzilate (0.35 g, 1.8mmol) was
added and the reaction refluxed overnight. The benzene was evaporated in vacuo and the
residue purified by flash chromatography (eluted first with 20% EtOAc/Hexane, then
5%MeOH/CH2Cl ) to give [1024] as transparent liquid (0.35g,48%).
Analytical Data: [1024] ESIMS: 402[M++1]
Step 3 :
To a solution of [7] (0.60g, 2.5mmol) in a mixture of THF/MeOH/H 20 (9ml) was added
Lithium hydroxide (0.20g, 5.0mmol). The reaction mixture was stirred at room temperature
for 4h. TLC showed complete consumption of starting material. Reaction mixture was
evaporated to dryness. Water was added and extracted with ethylacetate (2x50ml) to afford
light transparent sticky material [8] (0.35g, 73%).Analytical Data: [8] ESIMS: 192[M++1]
Step 4:
[1025] was synthesized as described in step 2.Analytical Data:[1025]ESIMS: 402[M++1]
Synthesis of [1026] and [1027] were carried out by procedure described for [1024]
Synthetic Scheme 3:
[1020] [1016]
Step 1:
To a stirred solution of [5] (l.Og, 5.2mmol) in DCM (20.0ml) was added TEA (1.9g,
15.2mmol) at room temperature. After an additional stirring for 5 minutes at same
temperature, Mesyl chloride (0.55ml, 7.8mmol) was added. The reaction temperature was
allowed to stirred at this temperature for 3h. TLC showed complete consumption of starting
material. Water (100 ml) was added and organic layer was extracted with ethyl acetate (2 x
100 ml). The combined organic layers were washed with water, brine and dried over sodium
sulphate. The organic layer was concentrated to afford light brown sticky material [9] which
was used further without any purification (l.lg, 78%).Analytical Data: [9]ESIMS: 270
[M++l].
Step 2 :
To a stirred solution of [9] (0.5g, 1.8 mmol) in DMF (10.0ml) was added sodium azide
(0.46g, 7.2mmol) at room temperature. The temperature of reaction mixture was raised to
100°C and allowed to heat at this temperature for 2 hr. TLC showed complete consumption
of starting material. Water (100 ml) was added and organic layer was extracted with ethyl
acetate (2 x 100 ml). The combined organic layers were washed with water, brine and dried
over sodium sulphate. The organic layer was concentrated to afford light brown sticky
material [10] which was used further without any purification (0.3g, 77%).Analytical
Data:[10] ESIMS: 217 [M++l].
Step 3 :
To a stirred solution of [10] (0.3g, 1.3mmol) in THF (10.0ml) was added triphenylphosphine
(0.5g, 1.9mmol) at room temperature. After an additional stirring for 5 minutes at same
temperature, water (0.2ml, 9.1mmol) was added. The reaction temperature was allowed to
reflux at 70°C for 2h. TLC showed complete consumption of starting material. Reaction
mixture was evaporated and water (100 ml) was added and organic layer was extracted with
ethyl acetate (2 x 100 ml). The combined organic layers were washed with water, brine and
dried over sodium sulphate. The organic layer was concentrated to afford light brown sticky
which was subjected to column chromatography with 2% MeOH/DCM as eluent to afford
light brown sticky material [11] (0.2g, 80%).Analytical Data: [11]ESIMS: 191 [M++l]
Step 4 :
To a stirred solution of [12] (0.2g, 0.87mmol) in DMF (5.0ml) was added EDC (0.25g,
lJmmol), HOBT (0.1 7g, 1.3mmol) at room temperature. After an additional stirring for 5
minutes at same temperature, [ll](0 '.18g, 0.91mmol) and NMM (0.37ml, 2.61mmol) was
added. The reaction temperature was allowed to stirred at room temperature for overnight.
TLC showed complete consumption of starting material. Water (100 ml) was added and
organic layer was extracted with ethyl acetate (2 x 100 ml). The combined organic layers
were washed with water, brine and dried over sodium sulphate. The organic layer was
concentrated to afford light brown sticky which was subjected to column chromatography
with 1% MeOH/DCM as eluent to afford light brown solid material[13] (0.25g,
76%).Analytical Data: [13JESIMS: 400 [M++l]
Step 5:
To a stirred solution of [13] (0.25 g, 0.62mmol) in a mixture of ethyl acetate and methanol
(1;1, 5 ml), was added a slurry of 10% Pd/C (0.02 g) at room temperature. Hydrogen balloon
pressure was applied and the reaction mixture was stirred for 3hr at RT. Reaction was
monitored using TLC. The reaction mass was filtered over celite and excess of solvent was
removed under vacuum to afford light brown sticky material, which was further purified
using silica gel column and 2% methanol in dichloromethane as eluent to afford [1016] as off
white sticky material (0.1 1 g, 57%).Analytical Data: [1016]ESIMS: 3 11 [IvT+1]
Step 6:
To a stirred solution of [1016] (0.1 Og, 0.32 mmol) in DMF, anhydrous K2C0 (0.06g, 0.48
mmol) was added at 0°C under nitrogen atmosphere. After an additional stirring for 15
minutes at same temperature, Methyl iodide (0.03 ml, 0.48 mmol) was added drop-wise. The
reaction temperature was allowed to increase up to 25°C and stirring was continued for 3 h.
Consumption of [1016] was monitored by TLC. After complete consumption of [1016], water
(50 ml) was added and organic layer was extracted with ethyl acetate (2x 50 ml). The
combined organic layers were washed with water, brine and dried over sodium sulphate. The
organic layer was concentrated to afford light brown sticky material which was further
purified using silica gel column chromatography using 10 % ethyl acetate/hexane as eluent to
afford [1020] as white solid (0.05g, 50%,).Analytical Data: [1020]ESIMS: 325 [M++l]
Synthesis of [1017], [1021], [1022], [1028], [1029], [1030], [1031] and [1032] were carried
out by procedure described for [1020]
Synthetic Scheme 4 :
Step 1:
To a stirred solution of [14] (0.50g, 4.34 mmol) in benzene (30ml), PTSA (0.74g, 4.34 mmol)
was added at room temperature under nitrogen atmosphere. After an additional stirring for 5
minutes at same temperature, [15](0.65g, 4.34mmol) was added. The reaction temperature
was allowed to increase up to 90°C and stirring was continued for 5 h. Consumption of [15]
was monitored by TLC. After complete consumption of [15], water (50 ml) was added and
organic layer was extracted with ethyl acetate (3 x 50 ml). The combined organic layers were
washed with water, brine and dried over sodium sulphate. The organic layer was concentrated
to afford light brown sticky material which was further purified using silica gel column
chromatography using 30 % ethyl acetate/hexane as eluent to afford mixture of [1037] and
[1038] which was further separated by prep-HPLC to afford [1037] (0.1 Og, 9% ) and [1038]
(0.08g, 8%) as transparent sticky material. Analytical Data: [1037 andl038 ]ESIMS: 250
[M++l].
Step 2 :
To a stirred solution of [1037] (0.05g, 0.20 mmol) in Acetonitrile (2ml), methyl iodide
(0.01ml, 0.20 mmol) was added at room temperature under nitrogen atmosphere. The reaction
temperature was allowed to stirred at this temperature for overnight. After complete
consumption of [1037], reaction mixture was evaporated to dryness to afford light yellow
solid which was further purified by washing with Dichloromethane and ether to afford pure
product [1065] (0.04g,51%). Analytical Data: [1065]ESIMS: 364 [M+].
Step 3 :[1064] was synthesized similar to [1065] as described in step 2.Analytical Data:
[1064JESIMS: 364[M+]
Synthesis of [1039], [1040], [1041], [1042], [1043], [1044], [1045], [1046], [1047], [1048],
[1049], [1050], [1051], [1052], [1055], [1057], [1058], [1059], [1053], [1054], [1063],
[1067], [1068], [1069], [1070], [1071], [1073], [1061], [1062], [1074], [1075], [1076],
[1077]and [1093] were carried out by procedure described for [1064] and [1065].
Synthetic Scheme 5:
Step 1:
To a stirred solution of [17] (0.08g, 0.42mmol) in DMF (5.0ml) was added EDC (0.12g, 0.63
mmol), HOBT (0.08g, 0.63mmol) at room temperature. After an additional stirring for 5
minutes at same temperature, [16](0.08g, 0.42mmol) and NMM (0.14ml, 2.3mmol) was
added. The reaction temperature was allowed to stir at room temperature for overnight. TLC
showed complete consumption of starting material. Water (100 ml) was added and organic
layer was extracted with ethyl acetate (2 x 100 ml). The combined organic layers were
washed with water, brine and dried over sodium sulphate. The organic layer was concentrated
to afford light brown sticky which was subjected to column chromatography with 1%
MeOH/DCM as eluent to afford off white solid material [18] (0.1 lg, 68%). Analytical Data:
[18] ESIMS: 385 [M++l].
Step 2:
To a stirred solution of [18] (0.1 lg, 0.28 mmol) in Acetonitrile (2ml), methyl iodide (0.02ml,
0.28 mmol) was added at room temperature under nitrogen atmosphere. The reaction
temperature was allowed to stirred at this temperature for overnight. After complete
consumption of [18], reaction mixture was evaporated to dryness to afford light yellow solid
as a mixture of [ 1 1081] and [ 1082] which was further purified by prep-HPLC to afford
[1078] (0.03g, 20%) and [1079] (0.4g, 27%) as light yellow solid material. Analytical Data:
[1078 and 1079] ESIMS: 399 [M .
Synthesis of [1080], [1081], [1082], [1083], [1084] and [1034] were carried out by procedure
described for [1078] and [1079]
Synthetic Scheme 6 :
Step 1 :
To a stirred solution of [19] (0.80g, 4.81mmol) in DMF (5.0ml) was added EDC (1.40g, 7.2
mmol), HOBT (0.97g, 7.2mmol) at room temperature. After an additional stirring for 5
minutes at same temperature, [20]( l .l lml, 5.30mmol) and NMM (1.6ml, 14.43mmol) was
added. The reaction temperature was allowed to stirred at room temperature for overnight.
TLC showed complete consumption of starting material. Water (100 ml) was added and
organic layer was extracted with ethyl acetate (2 x 100 ml). The combined organic layers
were washed with water, brine and dried over sodium sulphate. The organic layer was
concentrated to afford light yellow powder which was triturated with pentane to afford off
white solid material [21] (1.20g, 75%).Analytical Data: [21] ESIMS: 339 [M++l].
Step 2 :
To a stirred solution of [21] (0.5 g, 1.4mmol) in methanol (10ml), was added a slurry of 10%
Pd/C (0.05 g) at room temperature. Hydrogen balloon pressure was applied and the reaction
mixture was stirred for 3hr at RT. Reaction was monitored using TLC. The reaction mass was
filtered over celite and excess of solvent was removed under vacuum to afford off white
powder [1085] (0.3 g, 86%).Analytical Data: [1085] ESIMS: 249 [M++l].
Step 3 :
To a stirred solution of [1085 Hydrochloride] (0.05g, 0.20mmol) in DCM, triethylamine
(0.07ml, 0.5mmol) was added at 0°C under nitrogen atmosphere. After an additional stirring
for 5 minutes at same temperature, Ethyl chloroformate (0.02 ml, 0.22mmol) was added
drop-wise. The reaction temperature was allowed to increase up to 25°C and stirring was
continued for 1 h. Consumption of [1085] was monitored by TLC. After complete
consumption of [1085], water (50 ml) was added and organic layer was extracted with ethyl
acetate (2 x 25 ml). The combined organic layers were washed with water, brine and dried
over sodium sulphate. The organic layer was concentrated to afford light brown sticky
material which was washed further with DCM and pentane to afford [1094] (0.03g,
64%).Analytical Data: [1094] ESIMS: 321 [M++l].
Synthesis of [1086], [1087], [1088], [1089], [1090], [1091], [1092], [1035] and [1036] were
carried out by procedure described for [1085].
F. Biological Testing of the Compounds of Present Invention
Example 1: Inhibition of mycobacterium colony growth in macrophage by compounds
of the present invention.
THP1 cells were seeded in a tissue culture plate in complete RPMI + 10% FCS and were
allowed to differentiate to macrophages by addition of PMA with incubation at 37°C with 5%
CO . After 16-20 hr of PMA differentiation, cells were washed and replenished with
complete RPMI. PMA-differentiated THP-1 cell were infected with tuberculosis bacteria
H37Rv at an MOI of 10. Infection was performed in antibiotic free RPMI supplemented with
10% FCS. After adding bacteria, culture plates were centrifuged prior to incubation at 37°C
with 5% C0 2. After 4hr, infected cells were washed twice with warm RPMI and replenished
with complete RPMI containing Amikacin to remove any remaining extra-cellular bacteria.
Compound was added at concentrations of 5 to 500nM at 16hr and medium containing the
appropriate dose of compound was refreshed every 24hrupto the 64hr time point. At the end
of the assay at 90hr, cells were lysed with lysis buffer (7H9+ .06% SDS) and the residual
bacterial loads determined as CFU counts. The results are graphically depicted in Table 2
which demonstrates that growth of Mycobacterial colony stood inhibited upon use of these
compounds.
Table 2 : Screening of compounds for H37Rv% inhibition at lOOnM and 500nM
Compound No. H37Rv %Inhibition at 500 nM
1001 73
1002 NT
1003 22
1004 33
1005 55
1006 66
1007 46
1008 42
1009 46
1010 46
101 1 5 1
1012 5 1
1013 55
1014 77
1015 73
1016 65
1017 75
1018 66
1019 60
1020 64
1021 74
1022 75
1023 50
1024 72
1025 79
1026 7 1
1027 85
1028 77
1029 6 1
1030 52
1031 74
1032 72
1033 78
1034 64
1035 62
1036 58
1037 40
1038 56
1039 6 1
1040 79
1041 7 1
1042 73
1043 72
1044 65
1045 66
1046 56
1047 68
1048 79
1049 56
1050 42
1051 59
1052 73
1053 78
1054 74.
1055 52
1056 78
1057 76
1058 55
1059 74
1060 78
1061 NT
1062 NT
1063 NT
1064 NT
1065 NT
1066 NT
1067 NT
1068 NT
1069 NT
1070 NT
1071 NT
1072 NT
1073 NT
1074 24
1075 46
1076 50
1077 52
1085 93
NT= Not Tested
Example 3: In vitro infection of human PBMC derived macrophages, compound
addition and cfu determination.
Heparinised human blood diluted 1:1 with RPMI 1640 was layered onto equal volume of
Ficoll-Paque followed by centrifugation at 1600 rpm for 30 min. The PBMC layer formed at
the interface was collected carefully and washed twice with RPMI. The cells were diluted in
RMPI medium (without serum) to a concentration 2 l 06/ml and 10 ml of diluted cells were
put into a 75-cm 2 tissue culture flask, and incubated for 2 hrs in a humidified 37°C incubator.
The non-adherent cells were removed by aspiration, followed by two washes with RPMI.
Complete media (with 10% FCS) was added and the cells were allowed to spontaneously
differentiate into macrophages for 4 days in a humidified 37°C, 5% C0 2 incubator. Bacteria
were grown in Middlebrooke 7H9 broth supplemented with 10% ADC, 0.4% Glycerol and
0.05% Tween-80 until the mid-log phase. The bacteria were then harvested, washed with
RPMI and re-suspended in the same media. The suspension was dispersed by aspiration
twelve times each with a 23- and then a 26-gauge needle, followed by an additional
dispersion for 3 times through a 30-gauge needle. This suspension was allowed to stand for 5
min. The upper half of the suspension was then used for the experiments. Bacteria were
quantified by measuring the absorbance at a wavelength 600nm (0.6 O.D. corresponds to
100 X 106 bacteria).
Human PBMC derived macrophages were infected with tuberculosis bacteria at a MOI of 10
(i.e., 10 bacteria per cell). Infection was performed in antibiotic free RPMI supplemented
with 10% FCS. After adding bacteria, culture plates were centrifuged at 700 rpm for 5 min
prior to incubation at 37°C with 5% C0 2. After 4h, infected cells were washed twice with
warm RPMI and replenished with complete RPMI containing 00mg/mL amikacin for 2h to
remove any remaining extra-cellular bacteria. Subsequently, cells were washed and then
maintained in complete RPMI for the rest of the experiment. Inhibitor addition was
performed at 16hrs post infection (p.i) and the medium containing the appropriate dose of
inhibitor was replenished at 40hrs and 64hrs p.i. At 90h post infection, the cells were lysed in
50 mΐ of 0.06% SDS for 10 min at room temperature. Lysate dilutions of 1:10 were plated in
duplicate sets on 7H1 1 agar plates. Square plates (12 X 12 cm) were used for the plating, by
the track dilution method in which 10 mΐ of each dilution was spotted on one side of square
plate. The plate was then tipped onto its side (at a 45°- 90° angle), and the spots were allowed
to gently flow in parallel tracks along the agar surface. The plates were then allowed to dry
and subsequently incubated in a humidified incubator at 37°C. Colonies were counted on the
14th day and converted into cfu/well. The effect of one of the compounds of the present
invention in reducing mycobacterial growth in human PBMC derived macrophages is shown
as means of illustration at Table 3.
Table 3 : Demonstration of reduction of colony count on use of the compound 1085
Example 4: In vitro infection of THP-1 macrophages, compound addition and cfu
determination.
The human monocyte/ macrophage cell line THP-1 was cultured in RPMI 1640
supplemented with 10% FCS and were maintained between 2 and 10 X 105 cells per ml at
37°C in a humidified, 5% C0 2 atmosphere. Before infection, cells were plated in 96 well
plates at 1X 104 cells per well and differentiated with PMA (30 ng/ml) for a period of 48 hrs.
Bacteria were grown in Middlebrooke 7H9 broth supplemented with 10% ADC, 0.4%
Glycerol and 0.05% Tween 80 until the mid-log phase. The bacteria were then harvested,
washed with RPMI and re-suspended in the same media. The suspension was dispersed by
aspiration twelve times each with a 23- and then a 26-gauge needle, followed by an additional
dispersion for 3 times through a 30-gauge needle. This suspension was allowed to stand for 5
min. The upper half of the suspension was then used for the experiments. Bacteria were
quantified by measuring the absorbance at a wavelength 600nm (0.6 O.D. corresponds to ~
100 X 106 bacteria).
PMA-differentiated THP-1 cells were infected with tuberculosis bacteria at a MOI of 10 (i.e.,
10 bacteria per cell). Infection was performed in antibiotic free RPMI supplemented with
10% FCS. After adding bacteria, culture plates were centrifuged at 700 rpm for 5 min prior to
incubation at 37°C with 5%C0 . After 4h, infected cells were washed twice with warm
RPMI and replenished with complete RPMI containing 200 mL amikacin for 2h to remove
any remaining extra-cellular bacteria. Subsequently, cells were washed and then maintained
in complete RPMI for the rest of the experiment. Inhibitor addition was performed at 16hrs
post infection (p.i) and the medium containing the appropriate dose of inhibitor was
replenished at 40hrs and 64hrs p.i. At 90h post infection, the cells were lysed in 50 m of
0.06% SDS for 10 min at room temperature. Lysate dilutions of 1:10 were plated in duplicate
sets on 7H1 1 agar plates. Square plates (12 X 12 cm) were used for the plating, by the track
dilution method in which 10 m of each dilution was spotted on one side of square plate. The
plate was then tipped onto its side (at a 45°- 90° angle), and the spots were allowed to gently
How in parallel tracks along the agar surface. The plates were then allowed to dry and
subsequently incubated in a humidified incubator at 37°C. Colonies were counted on the 14th
day and converted into cfu/well. The results are represented at Figure 1. From Figure 1, it
may be inferred that compounds of the present invention produces dose dependent reduction
in mycobacterial cfu's in MYC 431 infected macrophages.
Example 5: Determination of the effect of the compounds of the present invention on
lipid bodies.
THP-1 cells were seeded onto # 1 thickness, 12 mm diameter glass cover-slips in 24-well
tissue culture plates at a density of 0.3x106 cells per cover slip. These cells were then
infected M. tb (H37Rv) at a MOI of 10 and incubated for 4h at 370 C in 5% C02.
Extracellular bacteria were removed by washing and subsequently by supplementing the
medium with 200 mg/ amikacin for 2h. SPR1 13 was added in increasing concentrations
immediately after the amikacin treatment, and the medium containing the appropriate
inhibitor was replenished at 16h and 40h post infection. At 48hrs post infection cells were
fixed with 3.7% para-formaldehyde and washed with PBS. HCS LipidTox Red neutral lipid
stain, diluted 1:1000 in PBS was added to the cells for 30 min. The cell nuclei were stained
using 300nM DAPI solution (in H20) for 5 min and then washed. Stained cells were
observed with a Nikon EclipseTi-E laser scanning confocal microscope equipped with
60X/1 .4NA Plan Apochromat DIC objective lens. DAPI and Lipid Tox were excited at 408
nm and 543 nm with a blue diode and a Helium-Neon laser respectively. The emissions were
recorded through emission filters set at 450 and 605/75 nm. Images were acquired with a
scanning mode format of 512 x 512 pixels. The transmission and detector gains were set to
achieve best signal to noise ratios and the laser powers were tuned to limit bleaching
fluorescence. All images were quantified using Image-Pro Plus version 6.0, a commercially
available software package from Media Cybernetics. The results are represented herebelow at
Figure 2. Figure 2 shows dose dependent reduction in cellular lipid bodies in the presence of
the compounds of the present invention.
Example 6: Effect of compounds against different mycobacterial strains.
THP-1 cells independently infected with r M.tb strains [JAL2287, JAL2261, XDR]. Addition
of the media containing compound was performed at 16 hr and the medium containing the
appropriate dose of compound was refreshed every 24 hr up to the 64 hr time point. At the
end of the total culture period of 90 hr, cells were lysed and CFUs were determined and
results are as shown graphically in Figure 3.The cellular Potency of compound 1085 was
measured in MTB infected THP1 and EC50 of H37Rv was 0.5nM and MYC431 and
JAL2287 were 0.1 nM. The graph shows that compound inhibited all the mycobacterial
strains at concentration of 0.01 to 500nM.
Example 7: Effect of compounds on c-AMP levels.
THP-1 macrophages were incubated with compd. 1085 at the indicated concentrations for 30
min, following which 3-hydroxybutyric acid. (3HBA, lOuM), a GPR109A agonist, was
added to cells for 90 min. In the absence of compound 1085, 3HBA reduces intracellular
cAMP levels while compound 1085 inhibits the activity in a dose dependent manner. The
results are represented at Figure 4.
Example 8 : Co-localization of M. tuberculosis with degradative vesicles: acidified
lysosomes and autophagosomes in the presence of compound 1085
THP-1 cells were seeded onto glass cover-slips in 24-well tissue culture plates at a density of
0.3x1 06 cells per cover slip. These cells were then infected GFP- tagged M. tb (H37Rv-GFP)
at a MOI of 10 and incubated for 4h at 37° C in 5% C0 2. Extracellular bacteria were removed
by washing and subsequently by supplementing the medium with 200 mg/ml Amikacin for
2hr. compound 1085 was added at 100 nM concentration immediately after the amikacin
treatment, and the medium containing the compound was replenished at 16h and 40h post
infection. At 48h cells were incubated with IOOhM of the acidotropic dye for 60 min
followed by fixation of cells with 3.7% para-formaldehyde for 20 min, and washed. The dye
stained cells were permeabilized with 0.2%(v/v) Triton X-100 for 20 min, washed with PBS
and blocking buffer 3% (w/v)BSAwas added for 60 min. The cells were washed and LC3B
rabbit Ab (Cell Signaling Technology), at a 1:200 dilution, was added for 60 min at room
temperature. Cells were washed with PBST (once) and PBS (twice). Alexa Fluor 405 goat
anti-rabbit Ab at 1:200 dilution was added for 45 min at room temperature. Cells were
washed with PBST (once) and PBS (twice). The coverslips were mounted on slides with an
antifade reagent. Stained cells were observed with a laser scanning confocal microscope. GFP
and dyes were excited at 488 nm, 408 nm and 543 nm with an argon ion, blue diode and a
Helium-Neon laser respectively. The emissions were recorded through emission filters set at
515/30; 450 and 605/75 nm. Serial confocal sections (0.5 m h thick) within a z-stack spanning
a total thickness of 10 mhi were taken in individual channels green, blue and red using the
motor drive focusing system. Images were acquired with a scanning mode format of 512 x
512 pixels. The transmission and detector gains were set to achieve best signal to noise ratios
and the laser powers were tuned to limit bleaching fluorescence. All images were quantified.
The merged confocal images were deconvolved and subjected to co-localization analysis to
determine the "Overlap Coefficient" as previously described (Manders et al, 1993). The
results are presented at Figures 5a and 5b. Autophagosomes and acidified lysosomes are
degradative vesicles that function in maintaining cellular homeostasis and are also an
important component of the antimicrobial responses mounted by a macrophage to clear off
intracellular infection. M. Tuberculosis modulates both these degradative pathways in order
to ensure its continued survival within the macrophage. Interventions which lead to activation
of these pathways in infected macrophages will lead to targeted killing of intracellular M.
Tuberculosis and thus reduce mycobacterial load. The significance of the experiments done
with compound 1085 is that treatment of infected macrophages with this compound leads to
an increased co localization of mycobacteria with these degradative vesicles which is
reflected by the increased value of the co localization coefficient (higher value of co
localization coefficient indicates a higher percentage of bacteria present inside these
degradative vesicles). The bacteria present inside these vesicles upon compound 1085
treatment are thus primed to be subsequently killed which in turn is reflected in the lower in
vitro cfu's obtained with compound 1085.
Example 9: GPR109A specific effect of compound no. 1085 on cellular lipid bodies
THP-1 cells were seeded onto glass cover-slips in 24-well tissue culture plates at a density of
0.3xl0 6 cells per cover slip. Ligand for GPR109A, 3-HB (10 uM) was added to the cells and
in parallel sets. Compd. 1085 was added in increasing concentrations. At 48h post 3HB
addition cells were fixed with 3.7% para-formaldehyde and washed with PBS. HCS LipidTox
Red neutral lipid stain, diluted 1:1000 in PBS was added to the cells for 30 min. The cell
nuclei were stained using 300nM DAPI solution (in H20 ) for 5 min and then washed. Stained
cells were observed with a laser scanning confocal microscope equipped with Apochromat
DIC objective lens. DAPI and Lipid Tox were excited at 408 nra and 543 nm with a blue
diode and a Helium-Neon laser respectively. The emissions were recorded through emission
filters set at 450 and 605/75 nm. Images were acquired with a scanning mode format of 512 x
512 pixels. The transmission and detector gains were set to achieve best signal to noise ratios
and the laser powers were tuned to limit bleaching fluorescence. All images were quantified
and the results are graphically depicted in Figure 6 which demonstrates that significant effect
on average fluorescent intensity was observed in presence of compound 1085 representing
decrease in the cellular lipid body.
Example 10: Effect of compound 1085 on intracellular mycobacterial load in THP-1
macrophages infected individually with 8 different strains of M. tuberculosis
The human monocyte/ macrophage cell line THP-1 was cultured in RPMI 1640
supplemented with 10% FCS and were maintained between 2 and 10 X 105 cells per ml at
37°C in a humidified, 5% C0 2 atmosphere. Before infection, cells were plated in 96 well
plates at 1 X 10 cells per well and differentiated with PMA (30 ng/ml) for a period of 48 hrs.
Bacteria were grown in Middlebrooke 7H9 broth supplemented with 10% ADC (Becton
Dickinson), 0.4% Glycerol and 0.05% Tween 80 until the mid-log phase. The bacteria were
then harvested, washed with RPMI and re-suspended in the same media. The suspension was
dispersed by aspiration twelve times each with a 23- and then a 26-gauge needle, followed by
an additional dispersion for 3 times through a 30-gauge needle. This suspension was allowed
to stand for 5 min. The upper half of the suspension was then used for the experiments.
Bacteria were quantified by measuring the absorbance at a wavelength 600nm (0.6 O.D.
corresponds to ~ 100 X 106 bacteria).
PMA-differentiated THP-1 cells were infected with tuberculosis bacteria at a MOI of 10 (i.e.,
10 bacteria per cell). Infection was performed in antibiotic free RPMI supplemented with
10% FCS. After adding bacteria, culture plates were centrifuged at 700 rpm for 5 min prior to
incubation at 37°C with 5% C0 2. After 4h, infected cells were washed twice with warm
RPMI and replenished with complete RPMI containing 200 g/mL amikacin for 2h to remove
any remaining extra-cellular bacteria. Subsequently, cells were washed and then maintained
in complete RPMI for the rest of the experiment. Inhibitor addition was performed at 16hrs
post infection (p.i) and the medium containing the appropriate dose of inhibitor was
replenished at 40hrs and 64hrs p.i. At 90hr post infection, the cells were lysed in 50 mΐ of
0.06% SDS for 10 min at room temperature. Lysate dilutions of 1:10 were plated in duplicate
sets on 7H1 1 agar plates. Square plates (12 X 12 cm) were used for the plating, by the track
dilution method in which 10 m of each dilution was spotted on one side of square plate. The
plate was then tipped onto its side (at a 45°- 90° angle), and the spots were allowed to gently
flow in parallel tracks along the agar surface. The plates were then allowed to dry and
subsequently incubated in a humidified incubator at 37°C. Colonies were counted on the 14th
day and converted into cm/well. The results are graphically depicted in Figure 7 representing
steady decrease in mycobacteria] load with increase in concentration of compound 1085 for
all 8 strains of M. tuberculosis.
Example 11: Effect of combinations of compound 1085 with known anti-mycobacterial
antibiotics on intracellular mycobacterial load in THP-1 macrophages infected with M.
tuberculosis H37Rv.
The human monocyte/ macrophage cell line THP-1 was cultured in RPMI 1640 (Gibco
Laboratories) supplemented with 10% FCS (Hyclone) and were maintained between 2 and 10
X 10 5 cells per ml at 37°C in a humidified, 5% C0 atmosphere. Before infection, cells were
plated in 96 well plates at 1 X 104 cells per well and differentiated with PMA (30 ng/ml) for a
period of 48 hrs. Bacteria were grown in Middlebrooke 7H9 broth (Difco) supplemented with
10% ADC (Becton Dickinson), 0.4% Glycerol and 0.05% Tween 80 until the mid-log phase.
The bacteria were then harvested, washed with RPMI and resuspended in the same media.
The suspension was dispersed by aspiration twelve times each with a 23- and then a 26-gauge
needle, followed by an additional dispersion for 3 times through a 30-gauge needle. This
suspension was allowed to stand for 5 min. The upper half of the suspension was then used
for the experiments. Bacteria were quantified by measuring the absorbance at a wavelength
600nm (0.6 O.D. corresponds to ~ 100 X 106 bacteria).
PMA-differentiated THP-1 cells were infected with tuberculosis bacteria at a MOI of 10 (i.e.,
10 bacteria per cell). Infection was performed in antibiotic free RPMI supplemented with
10% FCS. After adding bacteria, culture plates were centrifuged at 700 rpm for 5 min prior to
incubation at 37°C with 5% C0 2. After 4h, infected cells were washed twice with warm
RPMI and replenished with complete RPMI containing 200mg/mL amikacin for 2h to remove
any remaining extra-cellular bacteria. Subsequently, cells were washed and then maintained
in complete RPMI for the rest of the experiment. Inhibitor addition was performed at 16hrs
post infection (p.i) and the medium containing the appropriate dose of inhibitor was
replenished at 40hrs and 64hrs p.i. At 90h post infection, the cells were lysed in 50 m of
0.06% SDS for 10 min at room temperature. Lysate dilutions of 1:10 were plated in duplicate
sets on 7H1 1 agar plates. Square plates (12 X 12 cm) were used for the plating, by the track
dilution method in which 10 mΐ of each dilution was spotted on one side of square plate. The
plate was then tipped onto its side (at a 45°- 90° angle), and the spots were allowed to gently
flow in parallel tracks along the agar surface. The plates were then allowed to dry and
subsequently incubated in a humidified incubator at 37°C. Colonies were counted on the 14th
day and converted into cfu/well. The Figure 8 shows that the compound 1085 does not
negatively affect the activities of the known anti TB antibiotics and in some cases there might
be additive/synergistic effects.
Example 12: Compound 1085 treatment in mice (oral dosing)
Groups of naive mice (female BALB/c mice 4-6 wk of age at 5/group) were infected with
MDR- M. tb (JAL2287) through the aerosol route by delivering between 150-200 bacteria per
lung during 30 min of exposure. One group of mice was sacrificed 24 h later and lung
homogenates were plated onto 7H1 1 agar plates for confirming infection. At fifteen days post
infection, compoundl085 treatment was initiated by delivering the compound orally.
Individual groups of mice were treated at the following doses: 100, 30, and 10 mg/kg body
weight/ day (1085 was solubilized in PEG400). At fourteen days following treatment, mice
were sacrificed and lung and spleen enriched using a homogenizer. An aliquot (100 mΐ) of the
serially diluted homogenate (10-2 & 10-3) was plated onto 7H1 1 agar plates for determining
the mycobacterial load. The cfu's were counted at 18 days post plating on the agar plates. The
results of pharmacokinetic parameters via intraperitoneal route are represented graphically at
Figures 9a and 9b. The pharmacokinetic parameter via oral route is presented herebelow at
Table 3 and 4. From the figures and tables it may be inferred that compounds of the present
invention, illustrated by an exemplary compound 1085 were found to be effective at all
experimental doses. The pharmacokinetic (PK) data of compound 1085 is represented in
Table 5 and 6 demonstrating that compound 1085 has very good bioavailability in both
mouse and dog.
Table 3 : Reduction of CFU in lung (Oral Dosing)
Table 4: Reduction of CFU in spleen (Oral Dosing)
Compd. 1085 (mg/kg/day) CFU/spleen (xlO ) % Reduction p value
0 11.8
10 0.4 96.6 0.0001
30 0.2 98.3 0.0001
100 0.2 98.3 0.0001
Table 5: P data of Compound 1085 in Mouse
Dog Bioavailability: 57%
Example 13: Compound 1085 + ATT combination treatment in mice (oral dosing)
Groups of naive mice (female BALB/c mice 4-6 wk of age at 5/group) were infected with
drug susceptible M. tb (H37Rv) through the aerosol route by delivering between 150-200
bacteria per lung during 30 min of exposure. One group of mice was sacrificed 24 h later and
lung homogenates were plated onto 7H1 1 agar plates for confirming infection. At fifteen days
post infection, oral drug treatment was initiated with a single dose per day. One group
received Anti-TB therapy, ATT (Isoniazid: 1.5mg/kg + Rifampicin: lmg/kg + Pyrazinamide:
1.5mg/kg + Ethambutol : 1.5mg/kg), one group received compd. 1085 (lOmg/kg) and a
parallel group received a combination of comd. 1085 and ATT (Isoniazid: 1.5mg/kg +
Rifampicin: lmg/kg + Pyrazinamide: 1.5mg/kg + Ethambutol : 1.5mg/kg + Compound 1085:
lOmg/kg). At 1 week, 2 weeks and 4 weeks post treatment mice were sacrificed along with a
control (mocka treated) group and lungs enriched using a homogenizer. An aliquot (100 mΐ)
of the serially diluted homogenate (10" & 10 ) was plated onto 7H1 1 agar plates for
determining the mycobacterial load. The CFU's were counted at 18 days post plating on the
agar plates. Combination treatment of compd. 1085 with ATT demonstrates significant
reduction in lung CFU as depicted in Figure 10. The data is represented in below Table7.
Table 7: Combination treatment of compd. 1085 with ATT
GROUP CFU/Lung (xlO5) SD
Untreated 19.54 2.09
Compd. 1085 (lOmg/kg) 4.76 0.76
ATT
• Isoniazid: 1.5mg/kg
• Rifampicin: lmg/kg 1.76 0.42
• Pyrazinamide: 1.5mg/kg
• Ethambutol: 1.5mg/kg
Compd. 1085 + ATT 0.36 0.1 1
Claims:
1. A compound of structural Formula (1), including its salts, hydrates and stereoisomers:
Formula 1
wherein
X is selected from O, NH or N(alkyl);
Ri and R2are independently selected from hydrogen, deuterium, hydroxyl, C straight
chain or branched chain alkyl, 3-7 membered cycloalkyl, Ci-6alkoxy,aryl, amino,
NH(alkyl), N(alkyl) 2 OCOR , heteroaryl containing 1-3 heteroatoms selected from the
group comprising O, N or S; or Ri and R2 may combined to form an aryl or a heteroaryl
ring containing 1-3 heteroatoms selected from the group comprising O, N or S;
R3isselected from hydrogen, hydroxyl, C i- straight chain or branched chain alkyl, 3-7
membered cycloalkyl,Ci -6alkoxy, aryl, aromatic or non-aromatic heterocyclic ring or
fused heterocyclic ring selected from:
X may in conjunction with Reform a5-7 membered heterocyclic ring comprisingl-3
heteroatoms selected from group comprising N, O or S. The heterocyclic ring be further
substituted with one or more lower alkyl groups, halogens, amino, NH(alkyl), N(alkyl) 2,
NH-aralkyl;
4 is selected from a hydrogen, lower straight chain or branched alkyl, halogen,
deuterium, C - alkoxy, amino, NH(alkyl), N(alkyl) 2, -COORg, CONR R ;
R5 is hydrogen, hydroxy, Ci^alkyl, Ci^alkoxy, amino, NH(alkyl), N(alkyl) 2;
R and R are in dependently selected from the group comprising hydrogen, CJ.JO alkyl, -
COR8, -CH2OCOR , -CH2OCONHR 8R , -COOR , -CONR 8R , -S0 2R , aryl, aralkyl;
R8 and R areindependently selected from the group comprising hydrogen, or Ci.6 straight
chain or branched chain alkyl;
n is 1,2, or 3.
2. The compound of structural Formula (1) as claimed in claim 1, including its salts,
hydrates and stereoisomers:
Formula
wherein
X is NH;
Ri and R2 are independently selected from hydrogen, hydroxyl, Ci.io alkyl,Ci - alkoxy;
R is selected from substituted aromatic or non-aromatic heterocyclic ring or fused
heterocyclic ring selected from:
4 is selected from hydrogen, deuterium,halogen,Ci - straight chain or branched chain
alkyl, Ci. 6 alkoxy, amino, NH(alkyl), N(alkyl) 2;
and R7 are independently selected from hydrogen, C Oalkyl, -COR , -CH 2OCOR , -
CH OCONHR R , -COOR , -CONR R , -S0 2R8, phenyl, benzyl;
R and R9are independently selected from hydrogen or Cj_6 straight chain or branched
chain alkyl;
n is 1,2 or 3.
3 . A compounds of Formula (1), including its salts, hydrates and stereoisomer' s thereof
wherein said compound is selected from the group comprising:
i. 1-Methylpiperidin-3-yl-2-hydroxy-2,2-diphenylacetate;
ii. 3-(2-Hydroxy-2,2-diphenylacetoxy)-l-(((isopropylcarbamoyl)oxy)methyl)-lmethylpiperidin-
1 -ium;
iii. Piperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
iv. l-Benzylpiperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
v. 1-(Methylsulfonyl)piperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
vi. l-(Dimethylcarbamoyl) piperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
vii. 1-Benzylpiperidin-4-yl 2-hydroxy-2,2-diphenylacetate;
viii. 3-(2-Hydroxy-2,2-diphenylacetoxy)-l-methylquinuclidin-l-ium;
ix. l-Benzylpyrrolidin-3-yl 2-hydroxy-2,2-diphenylacetate;
x. l-Ethylpiperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xi. 1-(Isopropylcarbamoyl) piperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xii. l-Propylpiperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xiii. 1-Methylpiperidin-4-yl 2-hydroxy-2,2-diphenylacetate;
xiv. l-Benzylazetidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xv. l-Acetylpiperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xvi. 2-Hydroxy-2,2-diphenyl-N-(piperidin-3-yl)acetamide;
xvii. N-( 1-Benzylpiperidin-4-yl)-2-hydroxy-2,2-diphenylacetamide;
xviii. l-Methylpyrrolidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xix. Quinuclidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xx. 2-Hydroxy-N-(l-methylpiperidin-3-yl)-2,2-diphenylacetamide;
xxi. N-(l-Benzylpiperidin-4-yl)-2-hydroxy-2,2-diphenylacetamide;
xxii. N-(l-Benzylazetidin-3-yl)-2-hydroxy-2,2-diphenylacetamide;
xxiii. Azetidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xxiv. (S)-l-Benzylpiperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xxv. (R)-l -Benzylpiperidin-3 -yl 2-hydroxy-2,2-diphenylacetate;
xxvi. (S)-l-Methylpiperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xxvii. (R)-l-Methylpiperidin-3-yl 2-hydroxy-2,2-diphenylacetate;
xxviii . (R)-N-( 1-Benzylpiperidin-3 -yl)-2-hydroxy-2,2-diphenylacetamide;
xxix. (R)-N-( 1-Benzylpiperidin-3-yl)-2-hydroxy-2,2-diphenylacetamide;
xxx. 2-Hydroxy-2,2-diphenyl-N-(piperidin-4-yl)acetamide;
xxxi . (S)-2-Hydroxy-N-( 1-methylpiperidin-3 -yl)-2,2-dipheny lacetamide ;
xxxii. (R)-2-Hydroxy-N-(l-methylpiperidin-3-yl)-2,2-diphenylacetamide;
xxxiii. l-Benzylazetidin-3-yl 2-hydroxy-2-phenylpropanoate;
xxxiv. (R)-N-(l -Benzylpiperidin-3 -yl)-2,2-diphenylacetamide;
xxxv. N-((S)- 1-Benzylpiperidin-3-yl)-2-hydroxy-2-phenylacetamide;
xxxvi. N-((R)- 1-Benzylpiperidin-3 -yl)-2-hydroxy-2-pheny Ipropanamide ;
xxxvii. (R)-(R)-1 -Methylpiperidin-3 -yl 2-hydroxy-2-phenylacetate;
xxxviii . (R)-(S)- 1-Methylpiperidin-3 -yl 2-hydroxy-2-phenylacetate;
xxxix. (S)-(R)-l-Methylpiperidin-3-yl 2-hydroxy-2-phenylacetate;
xl. (S)-(S)-l-Methylpiperidin-3-yl 2-hydroxy-2-phenylacetate;
xli. (R)- 1-Methylpiperidin-4-yl 2-hydroxy-2-phenylacetate;
xlii. (S)-2-Hydroxy-N-(l-methylpiperidin-4-yl)-2-phenylacetamide;
xliii . (R)-2-Hydroxy-N-( 1-methylpiperidin-4-yl)-2-phenylacetamide ;
xliv. (R)-2-Hydroxy-N-((R)-l -methylpiperidin-3 -yl)-2-phenylacetamide;
xlv. (R)-2-Hydroxy-N-((S)-l-methylpiperidin-3-yl)-2-phenylacetamide;
xlvi. (S)-2-Hydroxy-2-phenyl-N-(pyridin-3-yl)acetamide;
xlvii . (S)- -Methylpiperidin-4-yl 2-hydroxy-2-phenylacetate;
xlviii. (S)-l-Benzylazetidin-3-yl 2-hydroxy-2-phenylacetate;
xlix. (S)-(R)-Quinuclidin-3-yl 2-hydroxy-2-phenylacetate;
1. (S)-(S)-Quinuclidin-3-yl 2-hydroxy-2-phenylacetate;
li. (R)-N-(l-Benzylpiperidin-4-yl)-2-hydroxy-2-phenylacetamide;
i . (S)-N-( 1-Benzylpiperidin-4-yl)-2-hydroxy-2-phenylacetamide;
liii . (R)-3 -((R)-2-Hydroxy-2-phenylacetoxy)- 1,1-dimethylpiperidin- 1-ium;
liv. (S)-3 -((R)-2-Hydroxy-2-phenylacetoxy)- 1,1-dimethylpiperidin- 1-ium;
lv. (R)-2-Hydroxy-2-phenyl-N-(pyridin-3-yl)acetamide;
lvi. (R)-(S)-l-Methylpiperidin-3-yl 2-acetoxy-2-phenylacetate;
lvii. (R)-(R)- 1-Methylpyrrolidin-3-yl 2-hydroxy-2-phenylacetate;
lviii. (R)-(S)- -Methylpyrrolidin-3-yl 2-hydroxy-2-phenylacetate;
lix. (S)-(R)-l-Methylpiperidin-3-yl 2-methoxy-2-phenylacetate;
lx. 2-Methoxy-2-phenylacetic acid;
lxi. (R)-2-Hydroxy-2-phenyl-N-(piperidin-4-yl)acetamide;
lxii. (R)-Piperidin-4-yl 2-hydroxy-2-phenylacetate;
lxiii. (R)-3-(2-Hydroxy-2,2-diphenylacetoxy)- 1,1-dimethylpiperidin- 1-ium;
lxiv. (R)-3-(2-Hydroxy-2,2-diphenylacetoxy)- 1,1 -dimethylpiperidin- 1-ium;
lxv. (R)-3 -((R)-2-Hydroxy-2-phenylacetoxy)- 1,1-dimethylpiperidin- 1-ium;
lxvi. (R)-3 -((R)-2-Hydroxy-2-phenylacetoxy)- 1,1-dimethylpiperidin- 1-ium;
lxvii. (S)-(R)-l-Methylpiperidin-3-yl 2-methoxy-2-phenylacetate;
lxviii. (S)-(S)-l-Methylpiperidin-3-yl 2-methoxy-2-phenylacetate;
lxix. (S)-2-Hydroxy-N-((R)-l-methylpiperidin-3-yl)-2-phenylacetamide;
lxx. (S)-2-Hydroxy-N-((S)- 1-methylpiperidin-3-yl)-2-phenylacetamide;
lxxi. 3-(2,2-Diphenylacetamido)- 1,1-dimethylpiperidin- 1-ium;
lxxii. (2S)-l-Benzyl-2-((2,2-diphenylacetamido)methyl)-l-methylpyrrolidin-l-ium;
lxxiii. 3-(2-Hydroxy-2,2-diphenylacetamido)- 1,1-dimethylpiperidin- 1-ium;
lxxiv. (R)-2-Methoxy-l-((S)-3-methylmorpholino)-2-phenylethanone;
lxxv. (R)-2-Methoxy- 1-((R)-3-methylmorpholino)-2-phenylethanone;
lxxvi. (R)-l-((2R,3S)-2,3-Dimethylmorpholino) -2-methoxy-2-phenylethanone;
lxxvii. (S)-2-Methoxy-l-((S)-3-methylmorpholino)-2-phenylethanone;
lxxviii. (1R,3 R)- 1-Benzyl-3 -(2,2-diphenylacetamido)- 1-methylpiperidin- -ium;
lxxix. (1S,3 R)- 1-Benzyl-3 -(2,2-diphenylacetamido)- 1-methylpiperidin- 1-ium;
lxxx. (1S,3 R)- 1-Benzyl-3 -(2,2-diphenylacetamido)- 1-methylpiperidin- 1-ium;
lxxxi. (1R,3R)-1 -Benzyl-3-(2,2-diphenylacetamido)- 1-methylpiperidin- 1-ium;
lxxxii . 1-(3 -(Benzylamino)piperidin- 1-yl)-2,2-diphenylethanone;
lxxxiii. N-(( 1-Benzylpyrrolidin-2-yl)methyl)-2,2-diphenylacetamide;
lxxxiv. 1-Benzyl-2-((2,2-diphenylacetamido)methyl)- 1-methylpyrrolidin- 1-ium;
lxxxv. (R)-2-Methoxy-2-phenyl-N-(piperidin-4-yl)acetamide;
lxxxvi. 2-Hydroxy-2-phenyl-N-(piperidin-4-yl)acetamide;
lxxxvii. (S)-2-Hydroxy-2-phenyl-N-(piperidin-4-yl)acetamide;
lxxxviii. (S)-2-Methoxy-2-phenyl-N-(piperidin-4-yl)acetamide;
lxxxi . 2-Hydroxy-2-pheny l-N-(piperidin-4-yl)propanamide;
xc. 2-(3-Bromo-2,6-difluorophenyl)-2-hydroxy-N-(piperidin-4-yl)acetamide;
xci. (R)-2-Methoxy-N-methyl-2-phenyl-N-(piperidin-4-yl)acetamide;
xcii. (R)-N-(l-Benzylpiperidin-4-yl)-2-methoxy-N-methyl-2-phenylacetamide;
xciii. (S)-l-Benzylpiperidin-4-yl 2-methoxy-2-phenylacetate;
xciv. (R)-Ethyl 4-(2-methoxy-2-phenylacetamido)piperidine- 1-carboxylate;
A compounds having Formula (1), including its salts, hydrates and stereoisomer' s
wherein said compound is selected from the group comprising:
i . (R)-2-Methoxy-2-phenyl-N-(piperidin-4-yl)acetamide;
ii. 2-Hydroxy-2-phenyl-N-(piperidin-4-yl)acetamide;
iii . (S)-2-Hydroxy-2-phenyl-N-(piperidin-4-yl)acetamide;
iv. (S)-2-Methoxy-2-phenyl-N-(piperidin-4-yl)acetamide;
v. 2-Hydroxy-2-phenyl-N-(piperidin-4-yl)propanamide;
vi. 2-(3-Bromo-2,6-difluorophenyl)-2-hydroxy-N-(piperidin-4-yl)acetamide;
(R)-2-Hydroxy-2-phenyl-N-(piperidin-4-yl)acetamide;
vii . (R)-3-(2-Hydroxy-2,2-diphenylacetoxy)- 1,1-dimethylpiperidin- 1-ium;
5. A process for preparing the compounds as claimed in claim 1 having Formula (1),
comprising:
coupling of compound A with that of compound B in presence of coupling agent (i) and
an organic solvent;
wherein:
X is selected from O, NH or N(alkyl);
Ri and R2are independently selected from hydrogen, deuterium, hydroxyl, Cj.io straight
chain or branched chain alkyl, 3-7 membered cycloalkyl, Ci^alkoxy, aryl, amino,
NH(alkyl), N(alkyl) 2 COR , heteroaryl containing 1-3 heteroatoms selected from the
group comprising O, N or S; or and R2 may combined to form an aryl or a heteroaryl
ring containing 1-3 heteroatoms selected from the group comprising O, N or S;
R is selected from hydrogen, hydroxyl, C i- straight chain or branched chain alkyl, 3-7
membered cycloalkyl,C i-6alkoxy, aryl, aromatic or non-aromatic heterocyclic ring or
fused heterocyclic rings elected from:
X may in conjunction with R3 form a 5-7 membered heterocyclic ring comprising 1-3
heteroatoms selected from group comprising N, O or S. The heterocyclic ring be further
substituted with one or more lower alkyl groups, halogens, amino, NH(alkyl), N(alkyl)2,
NH-aralkyl;
R4 is selected from a hydrogen, lower straight chain or branched alkyl, halogen,
deuterium, CI-6 alkoxy, amino, NH(alkyl), N(alkyl)2, -COOR8, CONR8R9;
R5 is hydrogen, hydroxy, Cl-6alkyl, Cl-6alkoxy, amino, NH(alkyl), N(alkyl)2;
R6 and R7 are independently selected from the group comprising hydrogen, Cl-10 alkyl,
-COR8, -CH20COR8, -CH20CONHR8R9, -COOR8, -CONR8R9, -S02R8, aryl,
aralkyl;
R8 and R9 are independently selected from the group comprising hydrogen, or C -6
straight chain or branched chain alkyl;
n is 1,2, or 3.and salts, hydrates and stereoisomers thereof;
6 . The compounds as claimed in claim 1 to 4 for use in treatment of the infection caused by
resistant and non-resistant Mycobacterium tuberculosis.
7. The compounds as claimed in claim 1 when combined with cycloserine, amikacin,
linezolid, ethambutol, rifampicin, isoniazid, ethionamide, moxyfloxacin, clarithromycin,
PAS, clofazamine, streptomycin, capreomycin and kanamycin and other anti-tubercular
agents for treatment of the infection caused by resistant and non-resistant Mycobacterium
tuberculosis.
8. The pharmaceutical composition comprising a compound of formula (1) as claimed in
Claim 1 to 4 along with pharmaceutically acceptable excipients.
9. The pharmaceutical composition as claimed in Claim 8, further to be used either alone or
in combination with atleast one additional pharmaceutical compound for treatment of the
infection caused by resistant and non-resistant Mycobacterium tuberculosis as defined
above.
10. The pharmaceutical composition as claimed in Claim 8, wherein the said pharmaceutical
compound is selected from the group consisting of cycloserine, amikacin, linezolid,
ethambutol, rifampicin, isoniazid, ethionamide, moxyfloxacin, clarithromycin, PAS,
clofazamine, streptomycin, capreomycin and kanamycin.
1 . The use as claimed in claim 11, wherein the said tuberculosis includes drug-sensitive,
mono-drug resistance, multi drug-resistant (MDR), extensively drug-resistance (XDR)
and totally drug resistance (TDR) caused by a strain of Mycobacterium tuberculosis
12. The use as claimed in claim 11, wherein a compound of formula (1) to be used for the
treatment of multi-drug-resistant (MDR), extensively drug-resistance (XDR) and totally
drug resistance (TDR) tuberculosis caused by a strain Mycobacterium tuberculosis
13. The use as claimed in claim 11 for treatment of the infection caused by resistant and nonresistant
Mycobacterium tuberculosis as defined above by inhibition of GPR109A by an
agent.