Synthesis and antimicrobial study of novel heterocyclic compounds from hydroxybenzophenones

Article abstract of DOI:10.1016/j.ejmech.2005.04.005

The triazolothiadiazine analogues 6a-e were obtained via a multistep synthesis sequences beginning with the hydroxybenzophenones 1a-e. Hydroxybenzophenones on reaction with ethyl chloroacetate affords ethyl (2-aroylaryloxy)acetates 2a-e which on treatment

Full text of DOI:10.1016/j.ejmech.2005.04.005

European Journal of Medicinal Chemistry 40 (2005) 1156–1162
www.elsevier.com/locate/ejmech
Short Communication
Synthesis and antimicrobial study of novel heterocyclic compounds
from hydroxybenzophenones
Shaukath A. Khanum a, Sheena Shashikanth b,*, S. Umesha c, R. Kavitha c
a Department of Chemistry, Yuvaraja’s College, University of Mysore, Mysore 570 005, India
b Department of Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
c Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore 570 006, India
Received 3 May 2004; received in revised form 17 March 2005; accepted 12 April 2005
Available online 01 June 2005
Abstract
The triazolothiadiazine analogues 6a–e were obtained via a multistep synthesis sequences beginning with the hydroxybenzophenones
1a–e. Hydroxybenzophenones on reaction with ethyl chloroacetate affords ethyl (2-aroylaryloxy)acetates 2a–e which on treatment with
hydrazine hydrate yields 2-(2-aroylaryloxy)acetohydrazides 3a–e. Intramolecular cyclization of 3a–e with carbon disulfide affords 5-(2-
aroylaryloxy)methyl-1,3,4-oxadiazole-2-(3H)thiones 4a–e, which on treatment with hydrazine hydrate yields 4-amino-5-(2-aroyl
aryloxy)methyl-1,2,4-triazole-3-(2H)thiones 5a–e. Condensation of 5a–e with a-halocarbonyl compound results in 3-(2-aroylaryloxy)methyl-
6-phenyl-1,2,4-triazolo[3,4-b][1,3,4] thiadiazine 6a–e analogues. The compounds 4a–e, 5a–e and 6a–e were tested against variety of fungal
and bacterial strains in comparison to fluconazole and chloramphenicol, respectively.
© 2005 Elsevier SAS. All rights reserved.
Keywords: 1,3,4-Oxadiazole-2-(3H)thiones; 1,2,4-Triazole-3-(2H)thiones; 1,2,4-Triazolo thiadiazines; Synthesis; Antimicrobial activity
1. Introduction
commonly used antibiotics have become less and less effec-
tive against certain illnesses not only because many of them
produce toxic reactions but also due to emergence of drug
resistant bacteria. It is essential to investigate newer drugs
with lesser resistance [10].
Over the past several years the emergence of organisms
resistant to nearly all the class of antimicrobial agents has
become a serious public health concern [1,2]. In general, bac-
teria have the genetic ability to transmit and acquire resis-
tance to drugs, which are utilized as therapeutic agents. [3].
Since past two decades there has been significant increase
in the frequency of systematic fungal infection in man. The
first orally active antifungal agent that was effective against a
broad array of systematic and superficial fungal infections
was ketoconazole [4]. Further a number of azole antifungal
agents viz., itraconazole [5], fluconazole [6], voriconazole [7],
ravuconazole [8] etc., and glucan synthesis inhibitor caspo-
fungin [9] have been introduced to the clinic. Antibiotics are
one of our most important weapons in fighting bacterial infec-
tions and have greatly benefited the health-related quality of
human life since their introduction. However, over the past
few decades these health benefits are under threat as many
During the past years extensive evidences have been accu-
mulated to establish the efficiency of benzophenone ana-
logues as antimicrobial agent [11–14]. Benzophenone ana-
logue (garcinol) has been isolated from the stem bark of
Garcinia huillensis grown in Zaire and used in central-
African traditional medicine and this has been shown to
exhibit chemotherapeutical activity against gram-positive and
gram-negative cocci, mycobacteria and fungi [15]. Recently
Selvi et al have shown antifungal activity of benzophenone
analogues, at its lower concentration [16]. Besides chloro sub-
stituted benzophenones have exhibited more antifungal activ-
ity [17]. Moreover, a large number of oxadiazoles [18,19],
triazoles [20] and triazolothiadiazines [21] have been shown
to exhibit significant antimicrobial activity against S. aureus,
C. albicans, C. krusei, C. parapsilosis, T. paradoxa, E. Coli,
B. subtilis and P. aeruginosa. These initial reports, thereafter
stimulated us to integrate 1,3,4-oxadiazole-2-(3H)thione and
* Corresponding author. Tel.: +91 821 254 7279.
E-mail address: shashi56_2000@yahoo.com (S. Shashikanth).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.04.005

S.A. Khanum et al. / European Journal of Medicinal Chemistry 40 (2005) 1156–1162
1157
tively. The disappearance of amide C=O and NH2 stretching
bands of 3a–e and detection of strong C–O–C, C=S, and C=N
stretching bands at about 1130–1137, 1235–1245 and 1610–
1647 cm–1, respectively, are evidences for ring closure of
1,3,4-oxadiazoles-2(3H) thiones 4a–e. The disappearance of
C–O–C stretching bands of 4a–e and detection of strong NH2
bands at 3400–3412 cm–1 are evidences for conversion of
4a–e to 4-amino-5-(2-aroylaryloxy)methyl-1,2,4-triazole-3-
(2H)thiones 5a–e. Similarly disappearance of NH2 stretch-
ing bands of 5a–e and detection of strong C–S–C bands at
750–760 cm–1 are evidences for conversion of 5a–e to tria-
zolothiadiazine analogues 6a–e. In 1H NMR spectra, all pro-
tons were seen according to the expected chemical shift and
integral values. Aromatic methyl, phenoxymethyl and aro-
matic ring protons were seen at 2.0–2.32, 4.42–4.85 and 6.65–
7.8 ppm. The NH protons of 4a–e and 5a–e were seen at about
9.0–9.15 ppm, respectively, and the NH2 protons of 5a–e were
seen at 3.7–3.73 ppm. The S-CH2 protons of 6a–e were seen
at 3.2–3.23 ppm. The results of microanalyses were within
0.4% error.
The antimicrobial activities of compounds 4a–e, 5a–e and
6a–e were evaluated in vitro against some fungi such as
C. albicans, C. krusei, C. parapsilosis, A. flavus, A. ochra-
ceous, F. moniliforme and C. gloeosporioides. Bacteria such
as E. coli, P. solanacearum, P. fluorescens and B. subtilis
were evaluated by serial tube dilution technique and the results
are summarized in Tables 1 and 2. The antifungal screening
results have shown that the halo substituted compounds 4a,
4b, 5a, 5b, 5c, 6a, 6b and 6c exhibit in general growth inhibi-
tory activity more relevant than that of the reference flucona-
zole against F. moniliforme and C. gloeosporioides. It is worth
noting that when chloro group is at meta position in ring B as
in compounds 4a and 6a have shown more activity than flu-
conazole against A. ochraceous. Besides, when chloro group
is at para position in ring A as in compounds 4b, 5b and 6b
and at meta position in ring B as in compounds 4a, 5a and 6a,
also when bromo group is at meta position in ring A as in
compounds 5c and 6c, have shown more activity compared
to fluconazole against F. moniliforme. Nevertheless, with
A. flavus only bromo and chloro substituted compounds 5c
and 6b, respectively, have exhibited more activity than flu-
conazole. This is an example, which shows how the biologi-
cal properties are influenced by even minor structural modi-
fications. With C. albicans chloro substituted compound 5b
and with C. krusei chloro compounds 5a, 5b and 6b have
shown more activity compared to fluconazole. Correspond-
ingly, with C. gloeosporioides chloro compounds 4a, 4b, 5a,
5b, 6a, and 6b, bromo compounds 5c and 6c and methyl sub-
stituted compounds 5e and 6e have shown more activity com-
pared to standard drug. On the contrary, with C. parapsilosis
all compounds have shown less antifungal activity compared
to fluconazole.
Scheme 1
.
triazolothiadiazine moieties in benzophenone framework,
since these systems possess well documented antimicrobial
activity.
2. Chemistry
The synthesis of the hitherto unreported title compounds
is as outlined in Scheme 1 in 70% yield. Hydroxybenzophe-
nones 1a–e on reaction with ethyl chloroacetate affords ethyl
(2-aroylaryloxy)acetates 2a–e in excellent yield [22–25],
which on treatment with hydrazine hydrate yields correspond-
ing 2-(2-aroylaryloxy)acetohydrazides 3a–e [24,25]. Intramo-
lecular cyclization of 3a–e with carbon disulfide resulted 5-(2-
aroylaryloxy)methyl-1,3,4-oxadiazole-2-(3H)thiones 4a–e
[19,26]. Compounds 4a–e were further treated with hydra-
zine hydrate to obtain compounds 4-amino-5-(2-aroyla-
ryloxy)methyl-1,2,4-triazole-3-(2H)thiones 5a–e [23,25]. The
preparations of novel 3-(2-aroylaryloxy)methyl-6-phenyl-
1,2,4-triazolo[3,4-b][1,3,4] thiadiazine 6a–e were achieved
from 5a–e with phenacyl bromide [21].
3. Results and discussion
The structures of the compounds were elucidated by IR,
NMR and microanalyses. The IR spectra of 2-(2-aroyla-
ryloxy)acetohydrazides 3a–e have amide C=O and NH2
stretching bands at 1655–1670 and 3110–3223 cm–1, respec-
In case of antibacterial activity with few exceptional cases
all the compounds have shown higher activity compared to
chloramphenicol against E. coli, P. solanacearum, P. fluore-
scens and B. subtilis. It is worth noting that compound 5e

1158
S.A. Khanum et al. / European Journal of Medicinal Chemistry 40 (2005) 1156–1162
Table 1
The minimum inhibitory concentration (MIC)a of 4a–e, 5a–e and 6a–e for antifungal activity
Compounds
Tested fungi (MIC in mM)
C. albicans
7.0
C. krusei
6.0
C. parapsilosis
A. flavus
12.0
6.5
A. ochraceous
1.0
F. moniliforme
C. gloeosporioides
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
11.5
9.5
4.5
1.5
8.5
11.5
8.5
2.0
4.0
5.0
8.0
6.0
4.5
1.0
5.5
11.0
6.5
6.0
2.5
1.0
9.5
10.0
6.5
1.5
2.0
3.0
6.5
6.0
2.5
1.5
3.5
12.0
3.0
6.5
6.0
5.5
10.0
6.5
3.0
7.0
9.5
8.0
8.0
8.0
11.0
11.5
6.0
9.0
4.0
12.0
3.0
12.0
4.0
7.5
3.5
7.5
10.5
11.0
12.0
6.5
2.5
3.0
5.0
11.0
1.0
4.0
5.5
8.0
7.0
5.0
9.5
2.0
10.5
3.0
10.5
5.0
5.5
6.0
5.0
11.5
9.0
9.0
1.0
3.0
4.5
1.0
7.0
5.5
5.5
8.5
10.0
7.0
7.5
8.0
6.5
8.0
12.0
9.5
11.5
5.5
11.5
2.5
6.5
Fluconazole
3.0
5.0
2.0
2.0
a Average of at least three determinations.
Table 2
The MICa of 4a–e, 5a–e and 6a–e for antibacterial activity
(3H)thiones, 1,2,4-triazole-3-thiones and triazolothiadiaz-
ines linked benzophenones. It is interesting and significant to
note from the antifungal data in Table 1 that one or the other
chloro substituted compounds exhibit in general growth
inhibitory activity more relevant than that of the reference
compound against the strains. Besides, compounds with
methyl and methoxy groups in ring A and B, respectively, in
general exhibited lower growth inhibitory activity when com-
pared to reference compound against all the strains. It is inter-
esting to note from the antibacterial data in Table 2 that all
the compounds have shown higher activity compared to ref-
erence compound against all the strains with few exceptional
cases. In general, compound 5e with two methyl groups in
ringA and B has exhibit lower growth inhibitory activity com-
pared to reference compound against all the strains except
with P. fluorescens. One can conclude that these compounds
posses more antibacterial activity than antifungal activity. In
retrospect, there exists a bright prospect for the discovery of
many a new drug for the treatment of antibacterial and anti-
fungal activity.
Compounds
Tested bacteria (MIC in mM)
E. coli P. solanacearum P. fluorescens B. subtilis
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
2.0
4.5
3.0
4.0
5.0
6.0
3.0
0.5
4.5
11.5
0.5
2.5
5.5
4.0
2.0
12.0
2.5
5.5
3.5
4.0
2.5
7.5
6.5
3.0
12.0
4.0
1.0
11.0
3.5
1.0
7.0
8.0
2.0
5.5
6.0
6.5
2.5
1.5
11.5
3.5
1.0
1.0
8.0
3.5
3.5
1.0
10.0
2.5
1.5
4.5
6.5
5.5
0.5
2.5
11.0
0.5
8.0
3.0
3.0
4.0
1.0
3.5
5.0
Chloramphenicol 10.5
a Average of at least three determinations.
with two methyl groups at para position in ring A and B has
shown lesser activity compared to other substituents against
Escherichia coli. Whereas chloro substituted compounds 4a
and 5b, bromo substituted compound 6c and methyl substi-
tuted compound 5e, exhibit lesser activity compared to other
compounds against P. solanacearum. Nevertheless with
P. fluorescens strain bromo compound 5c has shown lesser
activity. Besides with B. subtilis compounds with two methyl
groups 4e and 5e, with methyl and methoxy groups 4d and
with bromo group 5c have shown lesser activity compared to
the other compounds. In general these compounds are found
to possess more antibacterial activity than antifungal activity.
5. Experimental
5.1. Chemistry
TLC was performed on aluminum-backed silica plated with
visualization by UV-light. IR spectra were determined with a
FT IR Shimadzu 8300 spectrophotometer using a potassium
1
bromide wafer. H and 13C NMR spectra were recorded in
CDCl3 at 300 and 100 MHz, respectively. Chemical shifts
are in ppm relative to internal TMS. Melting points were deter-
mined on a Thomas Hoover capillary melting point appara-
tus with a digital thermometer.
4. Conclusion
In conclusion our study shows a strong evidence for the
antimicrobial activity of halo substituted 1,3,4-oxadiazole-2-

S.A. Khanum et al. / European Journal of Medicinal Chemistry 40 (2005) 1156–1162
1159
5.1.1. Synthesis of ethyl [2-(3-chlorobenzoyl)-4-
methylphenoxy]acetate (2a)
171.01 (s), 187.02 (s). Anal. Calcd. for: C19H20O4: C, 73.07;
H, 6.41. Found: C, 73.04; H, 6.38%.
A mixture of 1a (5 g, 0.02 mol), ethyl chloroacetate (2.4 g,
0.02 mol) in dry acetone (60 ml) and anhydrous potassium
carbonate (2.8 g, 0.02 mol) was refluxed for 8 h then cooled
and the solvent removed under reduced pressure. The residual
mass was triturated with ice water to remove potassium car-
bonate and extracted with ether (3 × 50 ml) and the ether
layer was washed with 10% sodium hydroxide solution (3 ×
30 ml) followed by water (3 × 30 ml) and then dried over
anhydrous sodium sulfate and evaporated to dryness to get
crude solid, which on recrystallization with ethanol gave 2a
(5.39 g, 80%) as white flakes.
5.1.2. Synthesis of 2-[2-(3-chlorobenzoyl)-4-methylphe-
noxy]acetohydrazide (3a)
To 2a (2 g, 6 mmol) in methanol (10 ml), 80% hydrazine
hydrate (0.3 g, 6 mmol) was added in drops and stirred for
1 h at room temperature. A white solid separated, which on
recrystallization with ethanol gave 3a (1.43 g, 75%) as white
needles.
3a: M.p. 177–180 °C; IR (KBr): 1620 (C=O), 1655 (amide,
C=O), 3110–3215 cm–1 (NH–NH2); 1H NMR (CDCl3): d 2.3
(s, 3H, CH3), 3.7 (bs, 2H, NH2), 4.5 (s, 2H, CH2), 7.1–7.6
(m, 7H, Ar-H), 9.25 (bs, 1H, CONH); 13C NMR (CDCl3): d
20.91 (q), 78.0 (t), 113.7 (d), 123.3 (s), 128.21 (d), 129.62
(d), 129.7 (s), 130.5 (d), 131.8 (d), 132.6 (d), 133.51 (s), 133.9
(d), 139.2 (d), 160.6 (s), 170.3 (s), 187.0 (s). Anal. Calcd. for
C16H15ClN2O3 (318.5): C, 60.28; H, 4.70; Cl, 11.14; N, 8.79.
Found: C, 60.24; H, 4.67; Cl, 11.10; N, 8.75%.
3b: M.p. 181–183 °C; IR (KBr): 1630 (C=O), 1668 (amide,
C=O), 3120–3223 cm–1 (NH–NH2); 1H NMR (CDCl3): d 3.72
(bs, 2H, NH2), 4.55 (s, 2H, CH2), 7.15–7.75 (m, 8H, Ar-H),
9.35 (bs, 1H, CONH); 13C NMR (CDCl3): d 78.01 (t), 115.2
(d), 124.8 (s), 125.8 (s), 128.21 (d), 130.1 (d), 131.51 (d),
132.62 (d), 133.61 (d), 137.81 (s), 161.7 (s), 170.31 (s), 187.03
(s). Anal. Cal. for C15H13ClN2O3 (304.5): C, 59.11; H, 4.26;
Cl, 11.65; N, 9.19. Found: C, 59.12; H, 4.24; Cl, 11.67; N,
9.17%.
2a: M.p. 60–62 °C; IR (KBr): 1670 (C=O), 1735 cm–1
(ester, C=O); 1H NMR (CDCl3): d 1.2 (t, J = 7 Hz, 3H, CH3
of ester), 2.3 (s, 3H, CH3), 4.2 (q, J = 6 Hz, 2H, CH2 of ester),
4.45 (s, 2H, CH2), 7.2–7.6 (m, 7H,Ar-H); 13C NMR (CDCl3):
d 13.6 (q), 20.9 (q), 59.5 (t), 75.6 (t), 113.7 (d), 123.3 (s),
128.2 (d), 129.61 (d), 129.7 (s), 130.5 (d), 131.8 (d), 132.6
(d), 133.5 (s), 139.2 (s). 133.9 (d), 160.6 (s), 171.0 (s), 187.0
(s). Anal. Calcd. for C18H17O4Cl (332.5): C, 64.96; H, 5.11;
Cl, 10.67. Found: C, 64.94; H, 5.07; Cl, 10.64%.
2b: Oily product[23]; IR (KBr): 1672 (C=O), 1738 cm–1
(ester, C=O); 1H NMR (CDCl3): d 1.21 (t, J = 7 Hz, 3H, CH3
of ester), 4.23 (q, J = 6 Hz, 2H, CH2 of ester), 4.5 (s, 2H,
CH2), 7.2–7.75 (m, 8H, Ar-H); 13C NMR (CDCl3): d 13.62
(q), 59.52 (t), 75.61 (t), 115.2 (d), 124.8 (s), 125.8 (s), 128.22
(d), 130.1 (d), 131.5 (d), 132.21 (d), 133.6 (d), 137.8 (s), 161.7
(s), 171.8 (s), 187.03 (s).Anal. Calcd. for C17H15ClO4 (318.5):
C, 64.05; H, 4.70; Cl, 11.14. Found: C, 64.02; H, 4.67; Cl,
11.11%.
3c: M.p. 182–185 °C; IR (KBr): 1625 (C=O), 1670 (amide,
C=O), 3115–3220 cm–1 (NH–NH2); 1H NMR (CDCl3): d 3.72
(bs, 2H, NH2), 4.53 (s, 2H, CH2), 7.15–7.76 (m, 8H, Ar-H),
9.35 (bs, 1H, CONH); 13C NMR (CDCl3): d 78.01 (t), 117.1
(d), 122.4 (s), 123.8 (d), 127.8 (s), 128.2 (d), 130.1 (d), 132.2
(d), 133.3 (d), 137.8 (s), 165.8 (s), 170.3 (s), 187.03 (s).Anal.
Cal. for C15H13BrN2O3(349): C, 51.57; H, 3.72; Br, 23.92;
N, 8.02. Found: C, 51.54; H, 3.70; Br, 23.89; N, 8.0%.
3d: M.p. 175–177 °C; IR (KBr): 1610 (C=O), 1645 (amide,
C=O), 3100–3205 cm–1 (NH–NH2); 1H NMR (CDCl3): d 2.2
(s, 3H, CH3), 3.5 (bs, 2H, NH2), 3.9 (s, 3H, OCH3), 4.55 (s,
2c: M.p. 69–71 °C; IR (KBr): 1672 (C=O), 1736 cm–1
(ester, C=O); 1H NMR (CDCl3): d 1.2 (t, J = 7 Hz, 3H, CH3
of ester), 4.22 (q, J = 6 Hz, 2H, CH2 of ester), 4.45 (s, 2H,
CH2), 7.22–7.8 (m, 8H, Ar-H); 13C NMR (CDCl3): d 13.61
(q), 59.51 (t), 75.61 (t), 117.1 (d), 122.4(s), 123.8 (d), 127.8
(s), 128.21 (d), 130.1 (d), 132.2 (d), 133.3 (d), 137.8 (s), 165.8
(s), 171.0 (s), 187.03 (s). Anal. Calcd. for C17H15BrO4 (363):
C, 56.19; H, 4.13; Br, 22.03. Found: C, 56.17; H, 4.10; Br,
22.0%.
2d: M.p. 58–60 °C; IR (KBr): 1660 (C=O), 1730 cm–1
(ester, C=O); 1H NMR (CDCl3): d 1.2 (t, J = 7 Hz, 3H, CH3
of ester), 2.25 (s, 3H, CH3), 3.8 (s, 3H, OCH3), 4.2 (q, J
= 6 Hz, 2H, CH2 of ester), 4.42 (s, 2H, CH2), 7.0–7.6 (m, 7H,
Ar-H); 13C NMR (CDCl3): d 13.63 (q), 20.92 (q), 56.0 (q),
59.53 (t), 75.6 (t), 113.71 (d), 113.8 (d), 123.32 (s), 129.7 (s),
130.1 (s), 131.1 (d), 131.81 (d), 133.91 (d), 160.62 (s), 165.7
(s), 171.02 (s), 187.04 (s). Anal. Calcd. for C19H20O5 (328):
C, 69.51; H, 6.09. Found: C, 69.49; H, 6.05%.
2H, CH2), 7.2–7.9 (m, 7H, Ar-H), 9.4 (bs, 1H, CONH); 13
C
NMR (CDCl3): d 20.92 (q), 56.0 (q), 78.02 (t), 113.72 (d),
113.82 (d), 129.6 (d), 129.72 (s), 130.1 (s), 131.1 (d), 131.8
(d), 133.9 (d), 160.62 (s), 165.7 (s), 170.3 (s), 187.04 (s).
Anal. Cal. for C17H18N2O4(314): C, 64.96; H, 5.73; N, 8.91.
Found: C, 64.94; H, 5.70; N, 8.89%.
3e: M.p. 182–85 °C; IR (KBr): 1630 (C=O), 1670 (amide,
C=O), 3120–3220 cm–1 (NH–NH2); 1H NMR (CDCl3): d 2.2–
2.3 (d, J = 7 Hz, 6H, 2CH3), 3.55 (bs, 2H, NH2), 4.6 (s, 2H,
CH2), 7.2–7.8 (m, 7H,Ar-H), 9.35 (bs, 1H, CONH); 13C NMR
(CDCl3): d 20.91 (q), 78.01 (t), 113.71 (d), 123.3 (s), 128.9
(d), 129.7 (s), 130.0 (d), 131.8 (d), 133.9 (d), 134.8 (s), 141.4
(s), 160.61 (s), 170.31 (s), 187.01 (s). Anal. Cal. for
C17H18N2O3: C, 68.41; H, 6.0; N, 9.35. Found: C, 68.45; H,
6.04; N, 9.39%.
2e: M.p. 57–59 °C IR (KBr): 1740 (ester, C=O), 1665 cm–1
1
(C=O); H NMR (CDCl3): d 1.2 (t, J = 7 Hz, 3H, CH3 of
ester), 2.3–2.35 (d, J = 7 Hz, 6H, 2Ar-CH3), 4.25 (q, J = 6 Hz,
2H, CH2 of ester), 4.45 (s, 2H, OCH2), 7.2–7.8 (bm, 7H,
Ar-H); 13C NMR (CDCl3): d 13.61 (q), 20.92 (q), 59.51 (t),
75.61 (t), 113.71 (d), 123.31 (s), 128.9 (d), 129.7 (s), 130.0
(d), 131.8 (d), 133.9 (d), 134.8 (s), 141.4 (s), 160.61 (s),

1160
S.A. Khanum et al. / European Journal of Medicinal Chemistry 40 (2005) 1156–1162
5.1.3. Synthesis of 5-[2-(3-chlorobenzoyl)-4-methyl-phe-
noxymethyl] 1,3,4-oxadiazole-2- (3H)thione (4a)
Anal. Calcd. for C18H16N2O3S (340.4): C, 63.51; H, 4.74; N,
8.23; S, 9.42. Found: C, 63.53; H, 4.75; N, 8.21; S, 9.40%.
To a 0 °C solution of 3a (1 g, 3.13 mmol) and carbon dis-
ulfide (0.47 g, 6.27 mmol) in absolute ethanol (15 ml), potas-
sium hydroxide (0.20 g, 3.13 mmol) was added in one por-
tion. The resulting mixture was stirred and refluxed for 8 h.
The solvent was removed in vacuo and the residue was acidi-
fied with 2 M hydrochloric acid and extracted with ethyl
acetate (2 × 20 ml). Organic layers were washed with water
and dried with anhydrous sodium sulfate. Filtration and con-
centration in vacuo gave a solid, which was recrystallized from
ethanol to give 4a (0.75 g, 72%) as yellow solid.
4a: M.p. 112–114 °C; IR (KBr): 1130 (C–O–C linkage),
1235 (C=S), 1610 (C=N), 1640 (C=O), 3310 cm–1 (N–H);
1H NMR (CDCl3): d 2.31 (s, 3H, CH3), 4.51 (s, 2H, CH2),
6.8–7.5 (m, 7H, Ar-H), 9.0 (bs, 1H, NH); 13C NMR (CDCl3):
d 20.91 (q), 78.02 (t), 113.7 (d), 123.3 (s), 128.21 (d), 129.62
(d), 129.7 (s), 130.5 (d), 131.8 (d), 132.6 (d), 133.51 (s), 133.9
(d), 139.2 (s), 155.01 (s), 157.0 (s), 160.6 (s), 187.0 (s). Anal.
Calcd. for C17H13ClN2O3S (360.5): C, 56.59; H, 3.63; Cl,
9.83; N, 7.76; S, 8.89. Found: C, 56.57; H, 3.65; Cl, 9.85; N,
7.78; S, 8.88%.
4b: M.p. 117–119 °C; IR (KBr): 1133 (C–O–C linkage),
1238 (C=S), 1612 (C=N), 1643 (C=O), 3315 cm–1 (N–H);
1H NMR (CDCl3): d 4.53 (s, 2H, CH2), 6.95–7.7 (m, 8H,
Ar-H), 9.1 (bs, 1H, NH); 13C NMR (CDCl3): d 78.03 (t), 115.2
(d), 124.8 (s), 125.8 (s), 128.2 (d), 130.1 (d), 131.5 (d), 132.2
(d), 133.6 (d), 137.8 (s), 155 .02 (s), 157.02 (s), 161.7 (s),
187.01 (s).Anal. Calcd. for C16H11ClN2O3S (346.8): C, 55.41;
H, 3.20; Cl, 10.22; N, 8.08; S, 9.25. Found: C, 55.40; H, 3.22;
Cl, 10.25; N, 8.10; S, 9.22%.
4c: M.p. 122–124 °C; IR (KBr): 1132 (C–O–C linkage),
1237 (C=S), 1610 (C=N), 1641 (C=O), 3310 cm–1 (N–H);
1H NMR (CDCl3): d 4.51 (s, 2H, CH2), 6.85–7.76 (m, 8H,
Ar-H), 9.05 (bs, 1H, NH); 13C NMR (CDCl3): d 78.02 (t),
117.1 (d), 122.4 (s), 123.8 (d), 127.8 (s), 128.2 (d), 130.1 (d),
132.21 (d), 133.3 (d), 137.82 (s), 155 .02 (s), 157.02 (s), 165.8
(s), 187.02 (s).Anal. Calcd. for C16H11BrN2O3S (391.25): C,
49.12; H, 2.83; Br, 20.42; N, 7.16; S, 8.20. Found: C, 49.15;
H, 2.81; Br, 20.44; N, 7.18; S, 8.22%.
5.1.4. Synthesis of 4-amino-5-[2-(3-chlorobenzoyl)-4-meth-
ylphenoxy]methyl-1,2,4-triazole- 3-(2H)thiones (5a)
To a mixture of 4a (0.8 g, 2.51 mmol) in ethanol (10 ml),
0.23 ml of 24% hydrazine hydrate was added drop wise and
the mixture was refluxed for 5 h. After cooling water was
added and the mixture was acidified by excess of 3N HCl, the
separated solid was filtered off, washed with water and crys-
tallized from ethanol to give 5a (0.706 g, 75%) as white solid.
5a: M.p. 205–207 °C; IR (KBr): 1230 (C=S), 1625 (C=N),
1
1642 (C=O), 3330 (N–H), 3400 cm–1 (NH2); H NMR
(CDCl3): d 2.31 (s, 3H, CH3), 3.7 (bs, 2H, NH2), 4.51 (s, 2H,
CH2), 6.9–7.55 (m, 7H, Ar-H), 9.1 (bs, 1H, NH); 13C NMR
(CDCl3): d 20.92 (q), 74.1 (t), 113.71 (d), 123.31 (s), 128.2
(d), 129.61 (d), 129.71(s), 130.51 (d), 131.81 (d), 132.61 (d),
133.51 (s), 133.91 (d), 139.21 (s), 155.01 (s), 160.61 (s), 186.0
(s), 187.01 (s). Anal. Calcd. for C17H15ClN4O2S (374.8): C,
54.47; H, 4.03; Cl, 9.46; N, 14.95; S, 8.55. Found: C, 54.46;
H, 4.05; Cl, 9.45; N, 14.92; S, 8.53%.
5b: M.p. 210–212 °C; IR (KBr): 1233 (C=S), 1630 (C=N),
1
1647 (C=O), 3335 (N–H), 3408 cm–1 (NH2); H NMR
(CDCl3): d 3.71 (bs, 2H, NH2), 4.52 (s, 2H, CH2), 6.95–7.6
(m, 8H,Ar-H), 9.13 (bs, 1H, NH); 13C NMR (CDCl3): d 74.12
(t), 115.2 (d), 124.8 (s), 125.8 (s), 128.2 (d), 130.1 (d), 131.5
(d), 132.2 (d), 133.6 (d), 137.8 (s), 155.02 (s), 161.7 (s),
186.01 (s), 187.03 (s). Anal. Calcd. for C16H13ClN4O2S
(360.8): C, 53.26; H, 3.63; Cl, 9.83; N, 15.53; S, 8.89. Found:
C, 53.25; H, 3.65; Cl, 9.81; N, 15.50; S, 8.90%.
5c: M.p. 220–222 °C; IR (KBr): 1232 (C=S), 1622 (C=N),
1
1645 (C=O), 3332 (N–H), 3404 cm–1 (NH2); H NMR
(CDCl3): d 3.71 (bs, 2H, NH2), 4.51 (s, 2H, CH2), 6.9–7.7
(m, 8H,Ar-H), 9.12 (bs, 1H, NH); 13C NMR (CDCl3): d 74.11
(t), 117.1 (d), 122.4 (s), 123.8 (d), 127.8 (s), 128.2 (d), 130.1
(d), 132.21 (d), 133.3 (d), 137.81 (s), 155.01 (s), 165.8 (s),
186.02 (s), 187.04 (s). Anal. Calcd. for C16H13BrN4O2S
(405.2): C, 47.42; H, 3.23; Br, 19.72; N, 13.82; S, 7.91. Found:
C, 47.44; H, 3.21; Br, 19.75; N, 13.32; S, 7.93%.
5d: M.p. 225–227 °C; IR (KBr): 1230 (C=S), 1621 (C=O),
1
4d: M.p. 114–116 °C; IR (KBr): 1130 (C–O–C linkage),
1236 (C=S), 1610 (C=N), 1640 (C=O), 3312 cm–1 (N–H);
1H NMR (CDCl3): d 2.3 (s, 3H, CH3), 3.8 (s, 3H, OCH3), 4.5
1640 (C=N), 3331 (N–H), 3409 cm–1 (NH2); H NMR
(CDCl3): d 2.3 (s, 3H, CH3), 3.7 (bs, 2H, NH2), 3.85 (s, 3H,
OCH3), 4.51 (s, 2H, CH2), 6.8–7.65 (m, 7H, Ar-H), 9.12 (bs,
1H, NH); 13C NMR (CDCl3): d 21.2 (q), 56.01 (q), 74.12 (t),
113.8 (d), 114.5 (d), 120.4 (s), 121.2 (d), 130.1 (s), 131.01
(d), 131.1 (d), 142.4 (s), 155.01 (s), 163.5 (s), 165.7 (s), 186.03
(s), 187.04 (s). Anal. Calcd. for C18H18N4O3S (370.4): C,
58.36; H, 4.90; N, 15.12; S, 8.66. Found: C, 58.34; H, 4.92;
N, 15.15; S, 8.68%.
(s, 2H, CH2), 6.9–7.6 (m, 7H, Ar-H), 9.1 (bs, 1H, NH); 13
C
NMR (CDCl3): d 20.92 (q), 56.0 (q), 78.0 (t), 113.7 (d), 113.8
(d), 123.31 (s), 129.7 (s), 130.11 (s), 131.1 (d), 131.8 (d),
133.9 (d), 155.02 (s), 157.02 (s), 160.61 (s), 165.7 (s), 187.03
(s).Anal. Calcd. for C18H16N2O4S (356.4): C, 60.66; H, 4.53;
N, 7.86; S, 9.0. Found: C, 60.68; H, 4.55; N, 7.88; S, 9.03%.
4e: M.p. 125–127 °C; IR (KBr): 1139 (C–O–C linkage),
1245 (C=S), 1620 (C=N), 1645 (C=O), 3323 cm–1 (N–H);
1H NMR (CDCl3): d 2.3–2.35 (d, J = 7 Hz, 6H, CH3), 4.51
5e: M.p. 208–210 °C; IR (KBr): 1240 (C=S), 1638 (C=N),
1
1652 (C=O), 3340 (N–H), 3412 cm–1 (NH2); H NMR
(CDCl3): d 2.25–2.31 (d, J = 7 Hz, 6H, CH3), 3.73 (bs, 2H,
NH2), 4.52 (s, 2H, CH2), 7.0–7.78 (m, 7H, Ar-H), 9.15 (bs,
1H, NH); 13C NMR (CDCl3): d 20.91 (q), 21.21 (q), 74.11
(t), 114.5 (d), 120.41 (s), 121.2 (d), 128.9 (d), 130.01 (d),
131.01 (d), 134.8 (s), 141.4 (s), 142.41 (s), 155.02 (s), 163.51
(s, 2H, CH2), 7.0–7.8 (m, 7H, Ar-H), 9.13 (bs, 1H, NH); 13
C
NMR (CDCl3): d 20.91 (q), 78.01 (t), 113.71 (d), 123.32 (s),
128.9 (d), 129.7 (s) 130.01 (d), 131.8 (d), 133.91 (d), 134.8
(s), 141.4 (s), 155.01 (s), 157.02 (s), 160.62 (s), 187.02 (s).

S.A. Khanum et al. / European Journal of Medicinal Chemistry 40 (2005) 1156–1162
1161
(s), 186.02 (s), 187.03 (s). Anal. Calcd. for C18H18N4O2S
(354.4): C, 61.0; H, 5.12; N, 15.81; S, 9.05. Found: C, 61.03;
H, 5.15; N, 15.80; S, 9.03%.
6.8–7.72 (m, 12H, Ar-H); 13C NMR (CDCl3): d 20.91 (q),
36.32 (t), 63.02 (t), 113.71 (d), 123.33 (s), 128.6 (d), 128.9
(d), 129.03 (d), 129.7 (s), 130.0 (d), 130.82 (d), 131.22 (s),
131.83 (d), 133.9 (d), 134.8 (s), 141.42 (s), 147.02 (s), 160.62
(s), 162.03 (s), 164.63 (s), 187.04 (s). Anal. Calcd. for
C26H22N4O2S (454.5): C, 68.70; H, 4.88; N, 12.33; S, 7.05.
Found: C, 68.72; H, 4.86; N, 12.31; S, 7.03%.
5.1.5. Synthesis of 3-[2-(3-chlorobenzoyl)-4-methylphe-
noxy]methyl-1,2,4-triazolo[3,4-b] [1,3,4]thiadiazine (6a)
A mixture of 5a (0.5 g, 1.33 mmol) and phenacyl bromide
(0.19 g, 1.33 mmol) in anhydrous ethanol (10 ml) was refluxed
for 5 h. The solvent was removed under reduced pressure,
diethyl ether (15 ml) was added and the reaction mixture was
left at 0 °C overnight. The precipitated solid was filtered off,
dried ad recrystallized with ethanol to give 6a (0.44 g, 70%)
as white needles.
5.2. Antimicrobial activity
The antimicrobial activities of compounds 4a–e, 5a–e and
6a–e were evaluated in vitro by serial tube dilution technique
[27,28] at different concentrations (0.5, 1.0, 1.5,........12 mM).
Some fungi such as C. albicans, C. krusei and C. parapsilo-
sis, A. flavus, A. ochraceous, F. moniliforme and C. gloeospo-
rioides and bacteria such as E. coli, P. solanacearum, P. fluo-
rescens and B. subtilis were used. Fluconazole and
chloramphenicol were used as reference standard in antifun-
gal and antibacterial activity studies, respectively. The stock
solutions of the compounds were prepared in chloroform. To
the culture tubes containing 1.9 ml of media, 0.1 ml of test
solution was added at sterile conditions. To all the tubes
including standard and controls, the fresh inoculums was
added using Himedia flexiloop 4 calibrated to 0.001 ml.After
incubating all the tubes at 37 °C for 24 h, their absorbance
was recorded at 640 nm along with reference. Percentage of
inhibition was calculated using the following equation.
6a: M.p. 142–144 °C; IR (KBr): 750 (C–S–C), 1630
1
(C=N), 1645 cm–1 (C=O); H NMR (CDCl3): d 2.3 (s, 3H,
CH3), 3.2 (s, 2H, CH2), 4.8 (s, 2H, OCH2), 6.75–7.6 (m, 12H,
Ar-H); 13C NMR (CDCl3): d 20.91 (q), 36.3 (t), 63.0 (t), 113.7
(d), 123.31 (s), 128.2 (d), 128.61 (d), 129.0 (d), 129.6 (d),
129.7 (s), 130.5 (d), 130.8 (d), 131.2 (s), 131.8 (d), 132.6 (d),
133.5 (s), 133.9 (d), 139.2 (s), 147.0 (s), 160.61 (s), 162.01
(s), 164.6 (s), 187.01 (s). Anal. Calcd. for C25H19ClN4O2S
(474.9): C, 63.22; H, 4.03; Cl, 7.46; N, 11.80; S, 6.75. Found:
C, 63.24; H, 4.04; Cl, 7.44; N, 11.82; S, 6.73%.
6b: M.p. 151–153 °C; IR (KBr): 752 (C–S–C), 1633
(C=N), 1648 cm–1 (C=O); 1H NMR (CDCl3): d 3.22 (s, 2H,
CH2), 4.83 (s, 2H, OCH2), 6.8–7.65 (m, 13H, Ar-H); 13C
NMR (CDCl3): d 36.32 (t), 63.01 (t), 115.2 (d), 124.8 (s),
125.8 (s), 128.2 (d), 128.62 (d), 129.02 (d), 129.72 (s), 130.1
(d), 130.82 (d), 131.21 (s), 131.5 (d), 132.2 (d), 133.6 (d),
137.8 (s), 147.01 (s), 161.7 (s), 162.01 (s), 164.62 (s), 187.01
(s). Anal. Calcd. for C24H17ClN4O2S (460.9): C, 62.54; H,
4.72; Cl, 7.69; N, 12.16; S, 6.96. Found: C, 62.55; H, 4.74;
Cl, 7.66; N, 12.14; S, 6.94%.
6c: M.p. 157–159 °C; IR (KBr): 748 (C–S–C), 1632
(C=N), 1648 cm–1 (C=O); 1H NMR (CDCl3): d 3.21 (s, 2H,
CH2), 4.84 (s, 2H, OCH2), 6.85–7.7 (m, 13H, Ar-H); 13C
NMR (CDCl3): d 36.31 (t), 63.01 (t), 117.1 (d), 122.4 (s),
123.8 (d), 127.8 (s), 128.2 (d), 128.6 (d), 129.0 (d), 130.1 (d),
130.81 (d), 131.22 (s), 132.21 (d), 133.3 (d), 137.83 (s),
147.01 (s), 162.01 (s), 164.61 (s), 165.82 (s), 187.03 (s).Anal.
Calcd. for C24H17BrN4O2S (505.3): C, 57.04; H, 3.39; Br,
15.81; N, 11.09; S, 6.34. Found: C, 57.06; H, 3.37; Br, 15.83;
N, 11.09; S, 6.31%.
% Inhibition = 100 (m – n)/m
where m = absorbance without the test sample and n = absor-
bance with test sample.
Acknowledgments
We the authors express our sincere gratitude to University
of Mysore, Mysore for the laboratory facilities provided to
us. One of the authors S.A.K. is indebted to UGC for the
award of teacher’s fellowship.
References
[1] H.S. Gold, R.C. Moellering, N. Engl. J. Med. Chem. 335 (1996)
1445–1453.
[2] H. Vanden. Bossche, P. Marichal, F.C. Odds, Trends Microbiol. 10
(1994) 393–400.
[3] M.L. Cohen, Science 257 (1992) 1050–1055.
[4] R.C. Heel, R.N. Brogden, A. Carmine, P.A. Morley, T.M. Speight,
G.S. Avery, Drugs 23 (1982) 1–36.
[5] D.J. Sheehan, C.A. Hitchcock, C.M. Sibley, Clin. Microbiol. Rev. 12
(1999) 40–79.
6d: M.p. 162–164 °C; IR (KBr): 740 (C–S–C), 1630
(C=N), 1645 cm–1 (C=O); 1H NMR (CDCl3): d 2.31 (s, 3H,
CH3), 3.2 (s, 2H, CH2), 3.75 (s, 3H, OCH3), 4.75 (s, 2H,
OCH2), 6.8–7.65 (m, 12H,Ar-H); 13C NMR (CDCl3): d 20.92
(q), 36.33 (t), 56.04 (q), 63.02 (t), 113.71 (d), 113.8 (d), 123.33
(s), 128.6 (d), 129.02 (d), 129.72 (s), 130.1 (s), 130.83 (d),
131.1 (d), 131.2 (s), 131.8 (d), 133.9 (d), 147.02 (s), 160.61
(s), 162.03 (s), 164.6 (s), 165.7 (s), 187.03 (s). Anal. Calcd.
for C26H22N4O3S (470.5): C, 66.37; H, 4.71; N, 11.91; S,
6.81. Found: C, 66.35; H, 4.73; N, 11.93; S, 6.83%.
[6] C.A. Lyman, T.J. Walsh, Drugs 44 (1992) 9–35.
[7] C.J. Clancy, M.H. Nguyen, Eur. J. Clin. Microbiol. Infect. Dis. 17
(1998) 573–575.
6e: M.p. 171–173 °C; IR (KBr): 755 (C–S–C), 1645
(C=N), 1652 cm–1 (C=O); 1H NMR (CDCl3): d 2.25–2.3 (d,
J = 7 Hz, 6H, 2CH3), 3.23 (s, 2H, CH2), 4.83 (s, 2H, OCH2),
[8] J.C.E. Fung-Tomc, B. Huczko, D.P. Minassian, Bonner, Antimicrob.
Agents Chemother. 42 (1998) 313–318.
[9] A. Espinel-Ingroff, J. Clin. Microbiol. 36 (1998) 2950–2956.

1162
S.A. Khanum et al. / European Journal of Medicinal Chemistry 40 (2005) 1156–1162
[10] A. Sarkar, K.A. Kumar, N.K. Dutta, P. Chakraborty, S.G. Dastidar,
Indian J. Med. Microbiol. 21 (2003) 172–178.
[11] B. Trusheva, M. Popova, H. Naydenski, I. Tsvetkova, G.J. Rodriguez,
V. Bankova, Fitoterapia 75 (2004) 683–689.
[12] J. Lokvam, J.F. Braddock, P.B. Reichardt, T.P. Clausen, Phytochem-
istry 55 (2000) 29–34.
[18] S. Giri, H. Singh, L.D.S. Yadav, Agric. Biol. Chem. 40 (1976) 17–21.
[19] G. Sahin, E. Palaska, M. Ekizoglu, M. Ozalp, Farmaco 57 (2002)
539–542.
[20] B.S. Holla, K.N. Poojary, B. Kalluraya, Farmaco 51 (1996) 793–799.
[21] B.S. Holla, P.M. Akberali, M.K. Shivananda, Farmaco 56 (2001)
919–927.
[13] J. Curtze, C.H.G. Rudolph, L. Schroder, G. Albert, A.E.E. Rehnig,
E.G. Sieverding, U. Pat, Chem. Abstr. 129 (1998) 108898 (577366).
[14] L. Ke, Z. Wannian, Z. Wu, W. Xiaoyan, Chem. Abstr. 136 (2002)
167122 (Zhongguo Yiyao Gongye Zazhi 32 (2001) 115–117).
[15] P. Bakana, M. Claeys, J. Totte, L.A. Pieters, L.Van Hoof, D.A. Tamba-
Vemba, V.d. Berghe, A.J. Vlietinck J. Ethnopharmacol. 21 (1987)
75–84.
[22] M.R. Friedman, K.J. Toyne, J.W. Goodby, M. Hird, J. Mater. Chem.
11 (2001) 2759–2772.
[23] G.N. Walker, R.T. Smith, J. Org. Chem. 36 (1971) 305–308.
[24] G. Sahin, E. Palaska, P. Kelicen, R. Demirdamar, G. Altinok, Arz-
neim. Forsch, Drug Res. 51 (2001) 478–484.
[25] E. Palaska, G. Sahin, P. Kelicen, N.T. Durlu, G. Altinok, Farmaco 57
(2002) 101–107.
[16] A. Tamil Selvi, G.S. Joseph, G.K. Jayaprakasha, Food Microbiol. 20
(2003) 455–460.
[17] T. Grote, A. Gypser, J. Rheinheimer, I. Rose, P. Schaefer,
F. Schieweck, N. Goetz, K. Eicken, E. Ammermann, S. Strathmann,
G. Lorenz, R. Stierl, Chem. Abstr. 137 (2002) 232443 (WO Patent
(2002) 2002072523).
[26] M.D. Mullican, M.W. Wilson, D.T. Connor, C.R. Kostlan,
D.J. Schrier, R.D. Dyer, J. Med. Chem. 36 (1993) 1090–1099.
[27] C.H. Collins, P.M. Lyne, J.M. Grange, J.O. Falkinham, Collins and
Lyne’s Microbiological Methods, Butterworth, London, 1970.
[28] B.G. Mullen, R.T. Decory, T.J. Mitchell, D.S. Allen, C.R. Kinsolving
Vassil St, B. Georgiev, J. Med. Chem. 31 (1988) 2008–2014.