American Journal of Infectious Diseases 7 (3): 61-66, 2011

ISSN 1553-6203
2011 Science Publications

Design, Synthesis, Biological Screening and Structure Activity Relationship

Study of 4, 6-Dimethyl-2-(Substituted) Mercapto-3-(Substituted)
Pyridines as Anti Tubercular Agents
1

Mehul M. Patel and 2Mahesh T. Chhabriya

Department of Pharmaceutical Chemistry, Ramanbhai Patel College of Pharmacy,
Charotar University of Science and Technology, Changa 388421, India
2
Department of Pharmaceutical Chemistry, L.M. College of Pharmacy,
Gujarat University, India

Abstract: Problem statement: Tuberculosis is a leading global mortality factor which has not been
effectively controlled, with 1.7 million deaths per year and 8.9 million new cases. Aerobic microbe
Mycobacterium tuberculosis H37Rv (MTB) is the causative agent of tuberculosis. Although many
active antitubercular agents have since been developed, drug resistance will continue to be a problem.
Therefore, there is a clear need for the discovery of new derivatives with antitubercular activity for the
management of tuberculosis. It was observed from the literature that pyridine nucleus have significant
antitubercular activity. On the basis of that we have synthesized some new derivatives of pyridines and
investigated their antimycobacterial properties. Approach: Two series of 3-cyano-4, 6-dimethyl-2(Alkyl/Aryllthio) pyridine and 4, 6-dimethyl-2-(Alkyl/Aryllthio) nicotinamide have been designed and
synthesized from malanonitrile. The title compounds were evaluated at 12.5, 25 and 100 g mL1
concentrations for their anti tubercular activity against Mycobacterium tuberculosis H37 Rv using
Lowenstein Jensen method (proportion method). Results: Many of the synthesized compounds
exhibit significant anti tubercular activity in comparison to Isoniazide while other compounds have
shown promising ant tubercular activity. All the synthesized compounds screened for
antimycobacterial activity were found significantly active against M. tuberculosis at the concentration
12.5 g mL1. Conclusion: The antituberculosis screening data revealed that all the tested compounds
3a-3d and 4a-4d showed moderate to very good inhibitory activity. The compounds 4a-4d showed very
good antituberculosis activity. The good activity is attributed to the presence of substituted alkyl group
at position-2 and amide group at position-3 of pyridine ring. Structure Activity Relationship (SAR)
study reveals that with the increase in the chain length of alkyl group at position-2 has tremendously
increased the activity of the title molecules.
Key words: Substituted pyridines, lowenstein jensen method, antitubercular activity, synthesized
compounds, Mycobacterium tuberculosis, Acid Fast Bacillus (AFB)
patients throughout the world. Although many active
antitubercular agents have since been developed, a
disturbing co-occurrence with the use of present drugs
as single agent has developed drug resistance
(Swaminathan, 2002; Patel et al., 2007; Williams et al.,
2002; Attaby et al., 1999; Rajni and Meena, 2011). The
development of this resistance can be forestalled
through the use of combination regimens, it is clear that
drug resistance will continue to be a problem.
Therefore, there is a clear need for the discovery of new
derivatives with antitubercular activity for the
management of tuberculosis. It was observed from the

INTRODUCTION
Tuberculosis is a characterized as a chronic
bacterial infection caused by Mycobacterium
tuberculosis, an aerobic Acid Fast Bacillus (AFB). TB
is contagious and spreads through the air; if not treated
properly, each person infects average 10-15 people
every year. 2 billion people, equal to one third of the
worlds total population, are infected with TB bacilli.
Two of every five person-more than 400 million have
latent tuberculosis infection. Tuberculosis is currently
the leading killer of the youth, women and AIDS

Corresponding Author: Mehul M. Patel, Pharmaceutical Chemistry Department, Ramanbhai Patel College of Pharmacy,
Charotar University of Science and Technology, At and po: Changa-388 421,
Dist-Anand, Gujarat, India Tel: 91-9879247492 Fax: 02697-247100

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Am. J. Infect. Dis., 7 (3): 61-66, 2011

literature that certain six member heterocyclic
compounds possess interesting biological activity.
Among them the compounds bearing pyridine nucleus
have wide applications in medicinal chemistry. These
compounds also have been reported to have significant
antitubercular activity (Agrawal et al., 2007).
In view of these facts and in continuation of our
studies on the synthesis of biologically active substituted
pyridines, it was considered of interest to synthesize
2,3,4,6 tetra substituted pyridine compounds. For SAR
study, alkyl substituent has been introduced at positions 2
to increase the lipophilicity. On the other hand, 3-cyano
group has been replaced with the amide group. The title
compounds thus synthesized were evaluated for their ant
tubercular properties.

was added. The mixture was continuous bubbled with

H2S gas for 3-4 h. with occasional shaking. Two crops
of crystals were isolated. Recrystallized from ethanol,
to yield 7.5 g (75% w/w) of crystalline product. M.P.
115-116C (116-117C).
Synthesis
of
3-Cyano-4,6-dimethyl-2mercaptopyridine (2) (Schmidth and Kubitzek,
1960): To a suspension of thiocyanoacetamide (10 g, 0.1
mol) in ethanol (100 mL), a mixture of acetylacetone (10
g, 0.1 mol) and triethylamine (1 mL) was added
dropwise with constant shaking. The mixture was
allowed to stand at room temperature for 1 h. Solid
obtained was filtered under suction, dried and
recrystallized from ethanol, to yield 12.49 g (76.21%w/w)
of crystalline product M.P. 260-261C (264C).

MATERIALS AND METHODS

General procedure for the synthesis of 3-cyano-4,6dimethyl-2-alkylthiopyridine (3a-3d): 3-cyano-4,6dimethyl-2-alkylthiopyridines (3a-3d) were synthesized
by alkylation of thiolate anion obtained by reaction of
3-cyano-4,6-dimethyl-2-mercaptopyridine (2) with
sodium hydroxide as per Fig. 2.
To a cool solution of 20%w/v NaOH (15 mL), 3cyano-4, 6-dimethyl-2-mercaptopyridine (1.64 g, 0.01
mol) was added. The mixture was stirred at 5-10C.
Tetrabutyl ammonium bromide was added as a phase
transfer catalyst. Then Alkyl halide in ethanol (0.1mol
in 10 mL ethanol) was added dropwise. After complete
addition of, Alkyl halide, mixture was further stirred for
4-5 h and then kept overnight at room temperature.

Chemistry: The synthesis of 3-cyano-4, 6-dimethyl-2alkylthiopyridines and 4, 6-dimethyl-2-(alkylthio)

nicotinamide from malanonitrile was performed as
shown in Fig. 1. In the initial step, H2S gas pass
through Malanonitrile In the presence of triethylamine
gave thiocyanocetamide which was further react with
acetylacetone to gave Cyano-4, 6-dimethyl-2mercaptopyridine (2). The compounds 3a-3d were
synthesized by reacting Cyano-4, 6-dimethyl-2mercaptopyridine (2) with alkyl/aryl halide.
Compounds 4a-4d was synthesized by acid catalyzed
hydrolysis of compounds 3a-3d. The purity of the
synthesized compounds was controlled by TLC.
Spectral data like IR, 1H NMR and mass of all the
newly synthesized compounds were in full agreement
with the proposed structures.
General: All the melting points were determined in
open capillaries and are uncorrected. Thin layer
chromatography was performed on microscopic slides
(27.5 cm sec) coated with Silica-Gel-G and spots were
visualized by exposure to iodine vapor. UV spectra were
recorded in UV-VIS 160A Shimadzu spectrophotometer.
IR spectra of all compounds were recorded in KBr on
FT-IR 8400S Shimadzu spectrophotometer using KBr.
Mass spectra were obtained using 2010EV LCMS
Shimadzu instrument. 1H and 13C NMR spectra were
obtained in CDCl3 on BRUKER Advance-II 400 MHZ
instrument and chemical shift were measured as parts
per million downfield from Tetramethylsilane (TMS)
as internal standard.

Fig. 1: (a) H2S,ethanol ,triethylamine (b) Acetyl

acetone, ethanol ,triethylamine; (c) NaOH, 0 10 C (d) Alkyl halide (R-X),
R =
C3H7,C4H9,C6H13,CH2C6H5, X = Cl or Br,
TBAB(Tetra Butyl Ammonium Bromide); (e)
H2SO4(50%), reflux, 1-2h Thiocyanoacetamide
(1), 3-Cyano-4,6-dimethyl-2-mercaptopyridine
(2), 3-cyano-4,6-dimethyl-2-alkylthiopyridine
(3a-3d), R=C3H7(3a), C4H9(3b), C6H13(3c),
CH2C6H5(3d)
and
4,6-dimethyl-2(alkylthio)nicotinamide (4a-4d) R = C3H7(4a),
C4H9(4b), C6H13(4c), CH2C6H5(4d)

Experimental section:
Synthesis of thiocyanoacetamide (1) (Schmidth and
Kubitzek, 1960): To a solution of malanonitrile (6.6 g,
0.1 moL) in ethanol (20 mL), 0.5 mL of triethylamine
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Am. J. Infect. Dis., 7 (3): 61-66, 2011

Table 1: Physical characteristics of 3-cyano-4,6-dimethyl-2-alkylthiopyridine (3a-3d)

--------------------------------------------------------------------------------------------------------------------------------------------------------------------------1
Molecular
Mol.wt
Yield
max
H NMR
I.R.
Comp. No. R
formulae
g/mol B.P.(C) M.P.(C) (%w/w) (nm)
Mass (m/e) ( ppm, CDCl3)
(cm1, KBr)
3a
C3H7
C11H14N2S
206
>300
--61.16
311.5
207(M+1) --2964, 2922(C-H)
270
163(M-43)
2216(CN)
1579(C-N)
879(C-H.pyr)
3b
C4H9
C12H16N2S
220
>300
--81.81
312
1.45(sextet,2H,S
2956, 2929(C-H)
270
(CH2)2 CH2 CH3),
1.70(quintet,2H,SCH2
2217 (CN)
CH2CH2CH3),
2.42(s,3H,-6CH
1579(C-N)
3-pyridine),
2.49(s,3H,
873(C-H pyr)
-4CH3-pyridine),
3.25(t,2H,SCH2CH2CH2CH3),
6.75(s,1H,pyridine proton)
C14H20N2S
248
>300
--81.81
254
249
--2952, 2929(C-H)
3c
C6H13
(M+1)
2216.06(C-CN)
163.2
1579(C-N)
(M-85)
873(C-H pyr)
3d
CH2C6H5 C15H14N2S
254
--87-89
55
244
255(M+1) --2979, 2929(C-H)
2216.06(C-CN)
1587(C-N)
873(C-H pyr)

mercaptopyridine (1.64 g, 0.01 mol) was added. The

mixture was stirred at 5-10C. Tetrabutyl ammonium
bromide was added as a phase transfer catalyst. Then
Propyl bromide in ethanol (0.1mol in 10 mL ethanol)
was added drop wise. After complete addition, mixture
was further stirred for 4-5 h and then kept overnight at
room temperature. Two layers were separated out, upper
organic layered was collected and washed with water.

Fig. 2: Synthesis
of
3-cyano-4,
alkylthiopyridine (3a-3d)

Synthesis
of
2-(butylthio)-4,6dimethylnicotinonitrile(3b): To a cool solution of
20%w/v NaOH (15 mL), 3-cyano-4,6-dimethyl-2mercaptopyridine (1.64 g, 0.01 mol) was added. The
mixture was stirred at 5-10C. Tetrabutyl ammonium
bromide was added as a phase transfer catalyst. Then
butyl bromide in ethanol (1.0736 mL in 10 mL ethanol)
was added drop wise. After complete addition of butyl
bromide, mixture was further stirred for 4-5 h and then
kept it overnight at room temperature. Two layers were
separated out, upper organic layer was collected and
washed with water, to yield 1.80 g (81.81%w/w)
product B.P.>300C.

6-dimethyl-2-

Two layers were separated out, upper organic

layered was collected and washed with water.
Physical characteristics data of 3-cyano-4, 6dimethyl-2-alkylthiopyridine (3a-3d) were given in
Table 1.

Synthesis
of
2-(hexylthio)-4,6dimethylnicotinonitrile(3c): To a cool solution of
20%w/v NaOH (15 mL), 3-cyano-4,6-dimethyl-2mercaptopyridine (1.64 g, 0.01 mol) was added. The
mixture was stirred at 5-10C. Tetrabutyl ammonium
bromide was added as a phase transfer catalyst.

Synthesis
of
2-(propylthio)-4,6dimethylnicotinonitrile(3a): To a cool solution of
20%w/v NaOH (15 mL), 3-cyano-4,6-dimethyl-263

Am. J. Infect. Dis., 7 (3): 61-66, 2011

Table 2: Physical characteristics of 4, 6-dimethyl-2-(alkylthio) nicotinamide (4a-4d)

--------------------------------------------------------------------------------------------------------------------------------------------------------------Molecular
Mol.wt
Yield
max
Mass
1H NMR
I.R.
(cm1, KBr)
Comp. No. R
formulae
g/mol
M.P. (C) (%w/w)
(nm)
(m/e)
( ppm, CDCl3)
4a
C3H7
C11H16N2OS 224
146-148
70.55
293.5
1.01(t, 3H, SCH2CH2CH3)
3419.59(N-H)
358
1.70(sextei, 2H, SCH2CH2CH3) 2929, 2956(C-H)
2.29(s, 3H,-4CH3-pyridine)
1639 (C = O)
2.44(s, 3H,-6CH3-pyridine)
1585.38 (C-N pyr)
3.17(t, 2H, SCH2CH2CH3)
873(C-H pyri)
5.85(s, 1H, C=NH)
6.13(s, 1H,C-OH)
6.70(s, 1H, pyridine proton)
4b
C4H9
C12H18N2OS 238
140-142
73.11
293.5
239
3409.91(N-H),
358
(M+1)
2929, 2954(C-H),
221.9
1635(C = O),
(M-16)
1585(C-N pyr.),
873(C-H pyri.)
4c
C6H13
C14H22N2OS 266
112-114
80.64
254
267
3417(N-H)
(M+1)
2920, 2956(C-H)
250
1643.24(C = O)
(M-16)
1587.31(C-N pyr)
873.6(C-H pyri)
4d
CH2C6H5 C15H16N2OS 272
132-134
75.07
244
2.28(s,3H,-4CH3-pyridine)
3369.41(N-H)
2.49(s,3H,-6CH3-pyridine)
2923, 2974(C-H)
4.44(s,2H,SCH2C6H5)
1639.38(C=O)
5.58(s,1H,C=NH)
1581(C-N pyr.
5.77(s,1H,C-OH)
871.7(C-H pyri.)
6.74(s, 1H, pyridine proton)
7.25(5H, Ar-H)

Then hexyl bromide in ethanol (1.40 mL in 10 mL

ethanol) was added dropwise. After complete addition
of hexyl bromide, mixture was further stirred for 4-5 h
and then kept it overnight at room temperature. Two
layers were separated out, upper organic layer was
collected and washed with water, to yield 1.35 g
(81.81%w/w) product B.P. >300C.

dimethyl-2-(alkylthio)nicotinamide
(4a-ad).
Acid
catalyzed hydrolysis gave promising yield in 2thioalkyl derivatives as per Fig. 3.
Figure
2
Synthesis
of
(alkylthio)nicotinamide (4a-4d):

4,6-dimethyl-2-

R = C3H7 (3a), C4H9 (3b), C6H13 (3c), CH2C6H5 (3d)

Synthesis
of
2-(benzylthio)-4,6dimethylnicotinonitrile (3d): To a cool solution of
20%w/v NaOH (15 ml), 3-cyano-4,6-dimethyl-2mercaptopyridine (1.64 g, 0.01 mol) was added. The
mixture was stirred at 5-10C. Tetrabutyl ammonium
bromide was added as a phase transfer catalyst. Then
benzyl chloride in ethanol (0.01mole in 10 mL ethanol)
was added drop wise. After complete addition of benzyl
chloride, mixture was further stirred for 4-5 h and then
kept it overnight at room temperature. Solid obtained
was collected and washed with water, to yield 1.394 g
(55%w/w) product. M.P. 87-89C (87C).

3-Cyano-4, 6-dimethyl-2-(alkyllthio)pyridine (1.96

g, 0.0095 mol) was added to 12 ml of 50% v/v H2SO4
and reflux for 1-2 h. The reaction mixture was brought
to room temperature and neutralized by adding 20%
w/v NaOH solution till basic to litmus. The solid
obtained was filtered under suction, dried at room
temperature and recrystallized from benzene. Physical
characteristics
data
of
4,
6-dimethyl-2(alkylthio)nicotinamide (4a-4d) were given in Table 2.

General procedure for the synthesis of 4,6-dimethyl2-(alkylthio)nicotinamide (4a-4d): Acid catalyzed

hydrolysis of 4,6-dimethyl-2-(alkylthio)nicotinonitrile
(3a-3d) under controlled temperature yielded 4,6-

Synthesis of 2-(propylthio)-4,6-dimethylnicotinamide
(4a): 2-(propylthio)-4,6-dimethylnicotinonitrile (1.96 g,
0.0089 mol) was added to 12 mL of 50%
v/v H2SO4 and reflux
for 1-2 h at 100C.

R = C3H7 (4a), C4H9 (4b), C6H13 (4c), CH2C6H5 (4d)

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Am. J. Infect. Dis., 7 (3): 61-66, 2011

Table 3: Comparision of screened compound for activity against M.Tuberculosis with reference to isoniazide
Compound
Concentration (12.5 g mL1)
Concentration (25 g mL1)
1
Isoniazide (1g mL )
++++
3a
++
+++
3b
++
+++
3c
+++
++++
3d
+++
+++
4a
+++
+++
4b
+++
+++
4c
++++
++++
4d
+++
++++

Concentration (100 g mL1)

++++
++++
++++
++++
++++
+++
++++
++++

Tetrabutyl ammonium bromide was added as a phase

transfer catalyst. Then benzyl chloride in ethanol
(1.153 ml in 10 mL ethanol) was added drop wise.
After complete addition of benzyl chloride, mixture
was further stirred for 4-5 h and then kept it overnight
at room temperature. Solid obtained was collected and
washed with water, to yield 1.394 g (55%w/w)
product. M.P. 87-89C (87C).

Fig. 3: Synthesis
of
4,6-dimethyl-2(alkylthio)nicotinamide (4a-4d)
The reaction mixture was brought to room temperature
and neutralized by adding 20% w/v NaOH solution till
basic to litmus. The precipitate obtained was filtered
under suction, dried at room temperature and
recrystallized from benzene, to yield 1.55 g
(73.11%w/w) of crystalline product M.P. 140-142C.

Antituberculosis activity by Lowenstein Jensen

assay method: M. tuberculosis H37Rv was grown in
Lowenstein Jensen media. The culture was diluted to
McFarland 1 standard with the same medium. From
this, 50 mL of this culture were added to 15 mL of
fresh medium in McCartney tubes. Stock solutions (1
mg mL1) of the test compounds were prepared in
dimethyl sulphoxide (DMSO). The compounds were
tested at 12.5, 25 and 100 g mL1 concentrations.
Control wells had the same volumes of DMSO without
any compound. Isoniazid (1g mL1) served as positive
control. After incubation at 37C for 7th week, it was
found that all the McCartney tubes containing different
dilutions of the test compounds did not show any type
of growth of the mycobacteria as compared to control
(isoniazide 1 g mL1). This was further confirmed by
the Ziehl-Neilson staining of all sample, which shown
absence of mycobacteria.

Synthesis of 2-(butylthio)-4,6-dimethylnicotinamide
(4b): 2-(butylthio)-4,6-dimethylnicotinonitrile (1.96
g, 0.0089 mol) was added to 12 ml of 50% v/v
H2SO4 and reflux for 1-2 h at 100C. The reaction
mixture was brought to room temperature and
neutralized by adding 20% w/v NaOH solution till
basic to litmus. The precipitate obtained was filtered
under suction, dried at room temperature and
recrystallized from benzene, to yield 1.55 g
(73.11%w/w) of crystalline product M.P. 140142C.
Synthesis
of
2-(hexylthio)-4,6dimethylnicotinamide(4c):
2-(hexylthio)-4,6dimethylnicotinonitrile (1.96 g, 0.0079 moL) was
added to 12 mL of 50% v/v H2SO4 and reflux for 1-2 h.
The reaction mixture was brought to room temperature
and neutralized by adding 20% w/v NaOH solution till
basic to litmus. The precipitate obtained was filtered
under suction, dried at room temperature and
recrystallized from benzene, to yield 2.54 g
(80.64%w/w) of crystalline product. M.P.112-114C

RESULTS
Pharmacolgy:
Antitubercular activity: The encouraging results from
the antibacterial studies impelled us to go for the
preliminary screening of the title compounds for their
in vitro antituberculosis activity. The compounds were
evaluated against Mycobacterium tuberculosis H37Rv
ATCC 27294 strain using Lowenstein Jensen method;
the observed MICs are presented in Table 3. Isoniazid
(INH) (1g mL1) was used as standard drugs. The
antituberculosis screening data revealed that all the
tested compounds 3a-3d and 4a-4d showed moderate to
very good inhibitory activity. The compounds 4a-4d
showed very good antituberculosis activity.

Synthesis
of
2-(benzylthio)-4,6dimethylnicotinonitrile (4d) (Zawisza and Malinka,
1986): To a cool solution of 20%w/v NaOH (15 mL),
3-cyano-4,6-dimethyl-2-mercaptopyridine (1.64 g, 0.01
mol) was added. The mixture was stirred at 5-10C.
65

Am. J. Infect. Dis., 7 (3): 61-66, 2011

DISCUSSION

REFERENCES

The good activity is attributed to the presence of

substituted alkyl group at position-2 and amide group
at position -3 of pyridine ring. Structure Activity
Relationship (SAR) study reveals that with the increase
in the chain length of alkyl group at position-2 has
tremendously increased the activity of the title
molecules.
3-cyano-4,6-dimethyl-2-alkylthiopyridine (3a-3d),
R = C3H7(3a), C4H9(3b), C6H13(3c), CH2C6H5(3d). 4,6dimethyl-2-(alkylthio)nicotinamide (4a-4d), R =
C3H7(4a), C4H9(4b), C6H13(4c), CH2C6H5(4d).

Attaby, F.A., M.A. Elneairy and M.S. Elsayed, 1999.

Synthesis and antimicrobial evaluation of new
pyridine, thienopyridine and pyridothienopyrazole
derivatives. Arch. Pharm. Res., 22: 194-201.
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Patel, R.J., G.C. Patel, M.M. Patel and N.J. Patel,
2007. HIV Infection and Tuberculosis. Indian J.
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Rajni and L.S. Meena, 2011. Unique characteristic
features of Mycobacterium tuberculosis in relation
to immune system. Am. J. Immunol., 7: 1-8. DOI:
10.3844/ajisp.2011.1.8
Schmidth, U. and H. Kubitzek, 1960. Synthesen mit
den Thioamiden der Malonsure, II. Thiopyridone
aus Cyan-thioacetamid. Chem. Ber., 93: 15591565. DOI: 10.1002/cber.19600930716
Swaminathan, S., 2002. Clinical presentation and
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Williams, D.A., W.O. Foye, T.L. Lemke, 2002.
Foye's Principles of Medicinal Chemistry. 5th
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Philadelphia, ISBN: 9780683307375, pp: 1114.
Zawisza, T. and W. Malinka, 1986. Synthesis and
properties of some derivatives of 2H-4,6dimethylpyrido[3,2-d]isothiazolin-3-one-1,1dioxide. Farmaco Sci., 41: 676-683. PMID:
3491004

CONCLUSION
The present study reports the successful synthesis,
antibacterial and antituberculosis studies of a new class
of
4,
6-dimethyl-2-(substituted)
mercapto-3(substituted) pyridines carrying biologically active
groups. Their screening results revealed that all the
compounds showed moderate to very good activity
against pathogenic strains. Study of structure-activity
relationship showed that the presence of lipophilic group
at position-2 and the existence of amide at position-3 are
responsible for increased antituberculosis activity of the
newly synthesizedcompounds. It can be concluded that
2-(alkyl) mercapto-3-(amido) pyridine derivatives has
antituberculosis effect. All the compounds are found to
be active against Mycobacterium tuberculosis at 12.5 g
mL1 concentration.
ACKNOWLEDGEMENT
The researchers are thankful to authorities of L.
M. College of Pharmacy, Ahmedabad for providing
necessary facilities and especially thankful to Dr.
(Mrs.) Madhulika A. Mistry, Pathologist and
Bacteriologist. K. J. Mehta of T. B. Hospital,
Amargadh,
Bhavnagar
for
antimycobacterial
evaluations. Authors are also thankful to Punjab
University, Chandigarh and Oxygen Healthcare Ltd.,
Ahmedabad for providing 1H NMR and GC-MS
spectral analyses of the compounds.

66