Synthesis and preliminary evaluation of some pyrazine containing thiazolines and thiazolidinones as antimicrobial agents

Article abstract of DOI:10.1016/j.bmc.2004.02.024

A series of N^′-[3,4-disubstituted-1,3-thiazol-2(3H)- ylidene]-2-(pyrazin-2-yloxy)acetohydrazide 11-66 and N^′-[(2Z)- 3-(4-bromophenyl)-4-oxo-1,3-thiazolidin-2-ylidene]-2-(pyrazin-2-yloxy) acetohydrazide 68-74 were synthesized using appropriate synthetic route. The entire test compounds 11-66 and 68-74 were assayed in vitro for antibacterial activity against two different strains of Gram-negative (E. coli and S. typhi), Gram-positive (S. aureus and B. subtilis) bacteria and the antimycobacterial activity was evaluated against H₃₇Rv strain of Mycobacterium tuberculosis. The minimum inhibitory concentration (MIC) was determined for test compounds and for reference standards. The test compounds showed significant antibacterial and antimycobacterial activity against the microbial strains used, when tested in vitro. In general, pyrazine ring and substituted thiazoline ring are essential for antimicrobial activity. Among the compounds tested, compounds 11, 12 and 40 were found to be most potent. The toxicity of most potent compounds 11, 12 and 40 were determined using hemolytic assay and minimal hemolytic concentration (MHCs) were determined. The test compounds were found to be nontoxic up to a dose level of 250μg/mL.

Full text of DOI:10.1016/j.bmc.2004.02.024

Bioorganic & Medicinal Chemistry 12 (2004) 2151–2161
Synthesis and preliminary evaluation of some pyrazine containing
thiazolines and thiazolidinones as antimicrobial agents
Chandrakant G. Bonde* and Naresh J. Gaikwad
Department of Pharmaceutical Sciences, Nagpur University Campus, Nagpur University, Amravati Road, Nagpur 440033, India
Received 6 November 2003; revised 17 February 2004; accepted 23 February 2004
Abstract—A series of N0-[3,4-disubstituted-1,3-thiazol-2(3H)-ylidene]-2-(pyrazin-2-yloxy)acetohydrazide 11–66 and N0-[(2Z)-3-(4-
bromophenyl)-4-oxo-1,3-thiazolidin-2-ylidene]-2-(pyrazin-2-yloxy)acetohydrazide 68–74 were synthesized using appropriate syn-
thetic route. The entire test compounds 11–66 and 68–74 were assayed in vitro for antibacterial activity against two different strains
of Gram-negative (E. coli and S. typhi), Gram-positive (S. aureus and B. subtilis) bacteria and the antimycobacterial activity was
evaluated against H37Rv strain of Mycobacterium tuberculosis. The minimum inhibitory concentration (MIC) was determined for
test compounds and for reference standards. The test compounds showed significant antibacterial and antimycobacterial activity
against the microbial strains used, when tested in vitro. In general, pyrazine ring and substituted thiazoline ring are essential for
antimicrobial activity. Among the compounds tested, compounds 11, 12 and 40 were found to be most potent. The toxicity of most
potent compounds 11, 12 and 40 were determined using hemolytic assay and minimal hemolytic concentration (MHCs) were
determined. The test compounds were found to be nontoxic up to a dose level of 250 lg/mL.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
probability of resistance formation.10–12 The biochemical
basis of both intrinsic and acquired resistance are now
known13–15 and has contributed significantly towards the
design of new entities by rational strategies that can be
used to counteract the resistance. The development of
new potential drugs, which will be devoid of side-effect
profile of currently available drugs, will be one of the
possible solutions to treat various infectious diseases
with multi-drug treatment over a long period of time.
Moreover, in immunocompromized patients, tubular
pathology is often accompanied by bacterial infections;
therefore there is a need to develop the new entities,
which will act both as antibacterial and antimycobac-
terials.
The deterioration of human population due to the
enhance prevalence of infectious diseases is becoming a
worldwide problem. Over the last few years, tuberculosis
is retrieving its place among theses infectious diseases
and today, nearly one-third of the worldÕs population is
infected with Mycobacterium tuberculosis with approxi-
mately three million patients deceasing every year.1 The
contemporary treatment of these infectious diseases
involves administration of multi-drug regimen over a
long period of time,2;3 which lead to patient noncom-
pliance and rapid emergence of multi-drug resistance
strain.4–7 The resistance problem demands to seek anti-
microbial agents effective against pathogenic microor-
ganisms resistant to current treatment.8 Furthermore,
treatment of infectious diseases is more difficult in
immunodeficient patients such as those infected with
human immunodeficiency virus (HIV).9 Recent studies
showed that the application of appropriate dosage reg-
imen with highly potent antimicrobial agents not only
eradicates bacterial growth but also minimizes the
Literature survey reveals that pyrazine ring is important
for antimycobacterial activity.16 In addition, many
thiazoline derivatives exhibit a wide variety of biological
activities such as antimicrobial,17 antiinflammatory,18
antihistaminic,19 antihypertensive,20 hypnotic21 and
anticonvulsant,22 etc. In view of the fact that pyrazine
ring possess antimycobacterial activity and as a part of
our ongoing studies in the area of antibacterial and
antimycobacterial agents,16 we have synthesized some
novel N0-[3,4-disubstituted-1,3-thiazol-2(3H)-ylidene]-
2-(pyrazin-2-yloxy)acetohydrazide and N0-[(2Z)-3-(4-
bromophenyl)-4-oxo-1,3-thiazolidin-2-ylidene]-2-(pyra-
zin-2-yloxy)acetohydrazide with the aim of obtaining
Keywords: Antimicrobial; Thiazoline; Thiazolidinones; Hemolytic
assay.
* Corresponding author. Tel.: +91-712-2500324; fax: +91-712-25003-
55; e-mail: chandubonde@yahoo.co.in
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.02.024

2152
C. G. Bonde, N. J. Gaikwad / Bioorg. Med. Chem. 12 (2004) 2151–2161
the new broad spectrum antimicrobial agents, which will
devoid of side effects associated with current therapy.
ylidene]-2-(pyrazin-2-yloxy)acetohydrazide 11–66 and/
or N-[(2Z)-2-[(aryl/alkyl substituted)imino]-4-(aryl/alkyl
substituted)-1,3-thiazol-3(2H)-yl]-2-(pyrazin-2-yloxy)acet-
amide 67. In fact, only one product was obtained as
confirmed by TLC. The structure of the products 11–66
and 67 was based on previous discussion of the struc-
tures of similar compounds.23 The structures of the
reaction products were confirmed by elemental analysis,
IR, 1H NMR and FABMS analyses. IR spectra revealed
Thus in the present investigation, 56 different derivatives
of N0-[3,4-disubstituted-1,3-thiazol-2(3H)-ylidene]-2-(pyr-
azin-2-yloxy)acetohydrazide and 8 different derivatives of
N0-[(2Z)-3-(4-bromophenyl)-4-oxo-1,3-thiazolidin-2-ylid-
ene]-2-(pyrazin-2-yloxy)acetohydrazide were synthesized
and evaluated for their antibacterial and antimycobac-
terial activity. The most potent compounds of each
series were further evaluated for their toxicity using
hemolytic assay.
that the disappearance of NH band at 3215–3230 cmꢀ1
.
1
The H NMR spectra also lacked the NH signals and
showed new singlet signal at d 5.8–6.1 attributed to C5–
H of thiazoline ring. The reaction of 4–10 with chloro-
acetic acid in boiling ethanol containing fused sodium
acetate afforded the corresponding N0-[(2Z)-3-(4-alkyl/
aryl substituted)-4-oxo-1,3-thiazolidin-2-ylidene]-2-(pyr-
azin-2-yloxy)acetohydrazide. Compounds 68–74 and
not N-{(2Z)-2-[(4-alkyl/aryl substituted)imino]-4-oxo-
1,3-thiazolidin-3-yl}-2-(pyrazin-2-yloxy)acetamide as ex-
pected from our previous discussion.23
2. Chemistry
The synthesis of the intermediate and target compounds
were performed by the reaction illustrated in Figure 1.
Compound 2 namely ethyl(pyrazin-2-yloxy)acetate was
synthesized in an excellent yield by electrophilic substi-
tution on 2-hydroxy pyrazine by ethyl chloroacetate
under reflux condition. Compound 2 on amination with
hydrazine hydrate afforded 2-(pyrazin-2-yloxy)aceto-
hydrazide 3. Reaction of 3 with alkyl/aryl isothiocynate
in ethanol gives compounds 4–10. The structures of the
compounds 4–10 were confirmed on the basis of ele-
mental analysis and spectral data. The IR spectra
showed NH and CS stretching bands at 3215–3230 and
1309–1348 cmꢀ1, respectively. The 1H NMR showed
downfield signal at d 11.6–14.23 attributed to 3-substi-
tuted NH and NH of 2-methoxy amido appeared as a
singlet signal at d 10.25–10.8. These two signals disap-
peared with D2O. The other protons appeared at the
expected chemical shifts. Condensation of 4–10 with
appropriate phenacyl bromide in boiling ethanol
containing anhydrous sodium acetate may lead to
formation of N0-[3,4-disubstituted-1,3-thiazol-2(3H)-
3. Result and discussion
In the present investigation, different derivatives of
N0-[3,4-disubstituted-1,3-thiazol-2(3H)-ylidene]-2-(pyra-
zin-2-yloxy)acetohydrazide 11–66 and N0-[(2Z)-3-(4-al-
kyl/aryl substituted)-4-oxo-1,3-thiazolidin-2-ylidene]-2-
(pyrazin-2-yloxy)acetohydrazide. Compounds 68–74
were synthesized and evaluated for their physical, ana-
lytical and spectral data (Tables 1 and 2).
The MIC values of the test compounds are summarized
in Table 3. For comparison, the MICs of compounds
2, 3 and 4–10 are included in Table 3. The results
revealed that the test compounds 11–66 exhibit
remarkable antimycobacterial activity against H37 Rv
strain of M. tuberculosis. The MIC values are in the
N
OH
N
OCH2COOC2H5
N
OCH2CONHNH2
ClCH2COOC2H5
NH2NH2.H2O
N
1
N
2
N
3
N
N
OCH2CONHN
RNCS
S
N
R
N
N
OCH2CONHNH
S
R'COCH2Br
11- 66
R
NH
4-10
R'
ClCH2COOH
NR
S
N
N
OCH2CONH
N
N
OCH2CONHN
N
R
N
S
O
R'
68-74
67
Figure 1. General scheme for synthesis of compounds 11–74 (R ¼ 4-chloro-2-nitrophenyl, 4-chlorophenyl, 2,4-dichlorophenyl, 4-flurophenyl,
n-butyl, t-butyl, R0 ¼ 4-bromo, 4-chloro, 4-fluro, 4-methoxy, 4-hydroxy, 2,4-dichloro methyl).

C. G. Bonde, N. J. Gaikwad / Bioorg. Med. Chem. 12 (2004) 2151–2161
Table 1. Physical and analytical data of compounds
2153
Compound
R
R
Molecular formulaa
MP
Yieldb
Mass [Mþ]
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
4-Chloro-2-nitrophenyl
4-Chloro-2-nitrophenyl
4-Chloro-2-nitrophenyl
4-Chloro-2-nitrophenyl
4-Chloro-2-nitrophenyl
4-Chloro-2-nitrophenyl
4-Chloro-2-nitrophenyl
4-Chloro-2-nitrophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
4-Ethoxyphenyl
4-Ethoxyphenyl
4-Ethoxyphenyl
4-Ethoxyphenyl
4-Ethoxyphenyl
4-Ethoxyphenyl
4-Ethoxyphenyl
4-Ethoxyphenyl
n-Butyl
4-Bromo
4-Chloro
4-Fluro
C21H14N6O4SClBr
C21H14N6O4SCl2
C21H14N6O4SClF
C22H17N6O5SCl
C21H15N6O5SCl
C21H13N6O4SCl3
C21H15N6O4SCl
C22H17N6O4SCl
C21H15N5O2SClBr
C21H15N5O2SCl2
C21H15N5O2SClF
C22H18N5O3SCl
C21H16N5O3SCl
C21H14N5O2SCl3
C21H16N5O2SCl
C22H18N5O2SCl
C21H14N5O2SCl2Br
C21H14N5O2SCl3
C21H14N5O2SCl2F
C22H17N5O3SCl2
C21H15N5O3SCl2
C21H13N5O2SCl4
C21H15N5O2SCl2
C22H17N5O2SCl2
C23H20N5O3SBr
C23H20N5O3SCl
C23H20N5O3SF
C24H23N5O3S
169–170
172–175
185–186
212–213
218–219
217–218
156–157
169–170
159–160
165–166
178–179
170–172
195–196
201–202
221–222
226–227
235–236
198–199
201–202
209–210
199–200
182–183
191–191
256–257
248–249
243–244
256–257
196–197
209–210
258–259
186–187
249–250
267–268
135–136
168–169
233–234
265–266
241–242
215–216
222–223
189–190
169–170
158–159
178–179
171–172
163–164
204–205
256–257
198–199
166–167
185–186
143–144
168–169
184–185
196–197
212–213
62
65
72
59
58
56
67
63
49
59
63
47
58
59
46
65
68
73
75
65
58
52
63
69
71
46
59
58
64
52
63
68
59
76
78
54
63
75
68
59
81
55
56
67
53
68
56
75
68
79
54
68
56
54
81
76
561c
517c
504c
512c
498c
551c
482c
497c
516c
472c
459c
467c
453c
506c
437c
451c
551c
506c
494c
502c
488c
541c
472
4-Methoxy
4-Hydroxy
2,4-Dichloro
––
4-Methyl
4-Bromo
4-Chloro
4-Fluro
4-Methoxy
4-Hydroxy
2,4-Dichloro
––
4-Methyl
4-Bromo
4-Chloro
4-Fluro
4-Methoxy
4-Hydroxy
2,4-Dichloro
––
4-Methyl
4-Bromo
4-Chloro
4-Fluro
486c
526c
481c
469c
477
4-Methoxy
4-Hydroxy
2,4-Dichloro
––
C23H21N5O4S
C
463
23H19N5O3SCl
C23H21N5O3S
C24H23N5O3S
516c
447
4-Methyl
4-Bromo
4-Chloro
4-Fluro
461
C
19H20N5O2SBr
462c
417c
405c
413
n-Butyl
n-Butyl
C19H20N5O2SCl
C19H20N5O2SF
C20H23N5O3S
C19H21N5O3S
n-Butyl
n-Butyl
4-Methoxy
4-Hydroxy
2,4-Dichloro
––
399
n-Butyl
n-Butyl
C
19H19N5O2SCl2
452c
383
C19H21N5O3S
C20H23N5O2S
C18H18N5O2SBr
C18H18N5O2SCl
C18H18N5O2SF
C19H21N5O3S
C18H19N5O3S
n-Butyl
Isopropyl
4-Methyl
4-Bromo
4-Chloro
4-Fluro
397
448c
403c
391c
399
Isopropyl
Isopropyl
Isopropyl
Isopropyl
4-Methoxy
4-Hydroxy
2,4-Dichloro
––
385
Isopropyl
Isopropyl
C
18H17N5O2SCl2
438c
369
C18H19N5O2S
C19H21N5O2S
C19H21N5O2SBr
C19H21N5O2SCl
C19H21N5O2SF
C20H23N5O3S
C19H21N5O3S
Isopropyl
t-Butyl
4-Methyl
4-Bromo
4-Chloro
4-Fluro
383
462c
417c
405c
413
t-Butyl
t-Butyl
t-Butyl
t-Butyl
4-Methoxy
4-Hydroxy
2,4-Dichloro
––
399
t-Butyl
t-Butyl
t-Butyl
C
19H19N5O2SCl2
452c
383
C19H21N5O2S
C20H23N5O2S
4-Methyl
397
a CHN analyses were found to be within the limit of ꢁ0.4%.
b All the compounds were recrystallized from ethanol.
c Values represent [Mþ2] due to appearance of an isotopic peak.
range of 1.5–12.5 while the antibacterial activity against
the Gram-positive and Gram-negative stains of bacteria
is moderate, also compound no 11, 12 and 40 showed
good antibacterial activity against both the Gram-
positive and Gram-negative stains of bacteria.
The structure–activity relationship revealed that thiazo-
line ring is essential for antibacterial activity as com-
pounds 2, 3 and 4–10 showed comparatively less activity
than 11–66. Amide linkage plays an important role in
imparting the antimycobacterial activity, as compound 2

2154
C. G. Bonde, N. J. Gaikwad / Bioorg. Med. Chem. 12 (2004) 2151–2161
Table 2. Physical and analytical data of compounds
Compound
R
Molecular formulaa
MP
Yieldb
Mass [M+]
4
4-Chloro-2-nitrophenyl
4-Chlorophenyl
2,4-Dichlorophenyl
4-Ethoxyphenyl
n-Butyl
C13H11N6O4SCl
C13H12N5O2SCl
C13H11N5O2SCl2
C15H17N5O3S
245–246
220–221
215–216
201–202
152–154
233–234
174–175
256–257
247–248
218–219
253–254
206–207
225–226
241–242
86
84
84
89
91
75
81
76
66
71
84
76
64
69
382.78c
337.78c
372.22c
347.39
269.32
283.34
269.32
422.80c
377.80c
412.25c
387.41
323.37
309.34
323.37
5
6
7
8
C
11H17N5O2S
10H15N5O2S
C11H17N5O2S
9
Isopropyl
t-Butyl
C
10
68
69
70
71
72
73
74
4-Chloro-2-nitrophenyl
4-Chlorophenyl
2,4-Dichlorophenyl
4-Ethoxyphenyl
n-Butyl
C15H11N6O5SCl
C15H12N5O3SCl
C15H11N5O3SCl2
C17H17N5O4S
C
13H17N5O3S
12H15N5O3S
C13H17N5O3S
Isopropyl
t-Butyl
C
a CHN analyses were found to be within the limit of ꢁ0.4%.
b All the compounds were recrystallized from ethanol.
c Values represent [Mþ2] due to appearance of an isotopic peak.
is less active than compound 3. Amide linkage and
thiazoline ring contributes significantly towards anti-
microbial activity. The different substituent in com-
pounds 11–66 over the side chain at 3 and 4 position of
thiazoline ring exerts significant influence on biological
activity. In general, aromatic substituted compounds at
3-position were found to be more active than aliphatic
substituents. Further, the presence of electron-with-
drawing groups (both halogen and nitro substituents) in
11–17 showed maximum antimycobacterial activity.
Literature survey reveals that electrons-withdrawing or
donating groups amend the lipophilicity of the test
compounds, which in turn alters permeability across the
bacterial cell membrane. Further the MIC values of 11–
66 were found to be comparatively less after 21 days
than 14 days against mycobacterium strain and 24 h
against bacterial strains. These values were also found to
be higher than MIC values of 11–66, which indicated
that the test compounds were metabolizing with time in
the biological environment. In order to confirm this fact
in vitro, the most potent compound 12 was incubated in
phosphate buffer solution (pH 7.4) at 37 ꢁC and after
every 12 h, 20 lL of aliquot was analyzed in Analytical
RP-HPLC (reverse phase high performance column
chromatography) over a period of 15 days. Result of the
study showed that after 14 days, the compound 12
(parent peak, retention time: 14.63 min) get metabolized
(appearance of second peak at 9.05 min, Fig. 3). Among
the compounds tested, 11, 12 and 40 were found to be
most potent. Again in comparison to thiazoline ring,
thiazolidine ring showed less activity 68–74. Thus
thiazolidine ring is not essential for imparting the anti-
bacterial activity to the compounds containing pyrazine
ring. Further, the toxicity of most potent compounds
(11, 12 and 40) were assessed using hemolytic assay and
minimal hemolytic concentration (MHC) and LD50
values (Log concentration of test compound required
for 50% hemolysis) were determined from the graph of
% lyses against Log concentration (Fig. 2). The most
potent compounds (11, 12 and 40) were found to be
nontoxic up to a dose level of 250 lg/mL (MHC) and
their corresponding LD50 values were found to be within
the range of 560–770 lg/mL (11, 770; 12, 650; 40, 560;
lg/mL). Thus, in comparison with antibiotics commonly
used in therapy, our most potent compounds showed
similar or slightly less in vitro antimicrobial activity
against various bacterial (Gram-positive and Gram-
negative) and mycobacterial strains with marked
reduction in toxicity (hemolytic activity), suggested that
this class of compounds could be used as potent broad-
spectrum antimicrobial agent to treat various clinical
conditions associated with multiple infectious diseases.
Further studies are in progress to optimize these lead
compounds and to characterize the mode of action.
3.1. Antimicrobial activity
All the test compounds were assayed in vitro for anti-
bacterial activity against two different strains of Gram-
negative (E. coli and S. typhi) and Gram-positive
(S. aureus and B. subtilis) bacteria and the antimyco-
bacterial activity was evaluated against H37 Rv strain of
M. tuberculosis strain using standard protocol.24 The
minimum inhibitory concentration (MIC) was deter-
mined by the test tube dilution technique using Mueller–
Hinton nutrient broth (for antibacterial) and modified
KirchnerÕs culture medium containing 0.5% sterilized
horse serum for antimycobacterial activity. The MIC
values were also tested for standard antibiotics (Ampi-
cillin, Penicillin-G and Chloramphenicol) to compare
the antibacterial activity of our test compounds.
Rifampin and Isoniazid (INH) were used as stan-
dard for antimycobacterial activity. Further, the toxicity
of most potent compounds 11, 12 and 40 were
assessed using hemolytic assay and minimal hemolytic
concentration (MHCs) and LD50 values were deter-
mined.25
4. Experimental
Melting points were determined in open capillaries using
microprocessor-based melting point apparatus, Model

C. G. Bonde, N. J. Gaikwad / Bioorg. Med. Chem. 12 (2004) 2151–2161
Table 3. In vitro antibacterial and antimycobacterial activity of test compounds
2155
Compound
Antibacterial activitya
Antimycobacterial activityb
M. tuberculosis
21d
Gram positive
Gram negative
S. typhi
S. aureus
B. subtilis
E. coli
14c
e
e
e
e
e
e
e
e
e
e
e
e
2
3
4
157
168
187
158
123
165
109
3
185
195
169
142
158
158
136
5
175
125
174
156
147
141
125
8
>200
139
125
121
159
123
158
4
52
62
61
74
65
92
56
63
49
6
5
6
57
85
7
8
45
56
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
51
1.0
0.3
6.1
2.7
4.0
3.6
5.2
4.5
10.7
11.5
12.7
11.6
12.4
10.8
12.5
12.1
10.5
11.9
12.2
9.6
10.8
10.6
12.4
11.5
8.9
8.7
10.2
8.7
10.4
1.5
3.5
3.1
7.4
7.6
7.6
6.6
7.9
7.2
9.2
8.7
8.1
8.7
8.5
7.5
8.4
8.2
10
6
1
2
9
4
19
17
15
19
12
12
19
37
14
12
16
21
32
23
41
9
15
14
12
11
25
15
13
36
19
16
21
29
18
48
26
11
9
17
17
32
23
63
25
25
42
25
21
29
32
39
56
48
15
14
13
36
59
54
28
84
54
68
85
23
2
15
13
19
15
16
23
14
25
36
39
24
12
25
89
39
12
18
19
57
58
62
69
56
63
74
56
69
3
9
8
7
5
9
12
16
23
17
16
24
13
26
52
22
16
11
15
21
15
23
21
17
19
22
14
15
8
12
7
9
27
23
42
17
25
26
59
26
15
1
35
45
39
16
36
52
63
39
25
7
25
26
26
69
26
59
56
28
49
68
28
14
57
24
27
56
56
63
85
46
95
56
69
69
28
95
58
25
48
57
63
56
59
68
28
58
26
65
85
26
58
76
28
25
38
37
58
39
56
45
27
63
49
67
57
57
54
64
29
54
45
54
128
25
35
95
26
39
35
49
52
26
16
16
47
26
69
26
58
17
37
27
95
47
25
64
69
25
23
36
25
26
15
12
19
13
15
18
16
13
21
20
15
16
21
22
11
14
18
13
18
19
13
17
12
9.5
6.3
7.2
6.9
5.8
7.1
(continued on next page)

2156
C. G. Bonde, N. J. Gaikwad / Bioorg. Med. Chem. 12 (2004) 2151–2161
Table 3 (continued)
Compound
Antibacterial activitya
Antimycobacterial activityb
M. tuberculosis
21d
Gram positive
B. subtilis
Gram negative
S. typhi
S. aureus
E. coli
14c
64
65
36
56
98
85
56
65
63
25
6.6
8.7
15
18
66
68
45
95
85
46
73
23
41
8.2
85
13
89
104
125
158
109
186
174
124
12.5
0.01
5
69
70
189
175
135
163
163
189
2.6
NA
86
79
96
102
148
169
156
191
128
NA
NA
NA
0.2
105
154
175
159
196
135
NA
NA
NA
0.22
71
72
168
NA
156
NA
25
125
NA
185
175
12.5
0.016
7
73
74
Std1f
Std2g
Std3h
Std4i
Std5j
0.09
7
0.015
6
51
1
Inactive
0.9
Inactive
57
Inactive
Inactive
0.005
0.008
a DMF has no antimicrobial activity at the concentration used to dissolve the test compounds.
b MIC: minimum inhibitory concentration.
c Antimycobacterial activity was measured after 14 days of incubation.
d Antimycobacterial activity was measured after 21 days of incubation.
e Compound 2 was found to be inactive at 200 lg/mL. NA: indicates activity not measured.
f Std1: Ampicillin.
g Std2: Penicillin-G.
h Std3: Chloramphenicol.
i Std4: Rifampin.
j Std5: Isoniazid (INH).
4.1. Syntheses
120
100
80
60
40
20
0
Specific examples presented below illustrate general
synthetic procedures.
Synthesis of 2: To a mixture of TEA (6.58 g, 0.065 mol),
2-hydroxy pyrazine (5 g, 0.0575 mol), was added drop-
wise a solution of ethyl chloroacetate (10.87 mL,
0.065 mol) in 1:4 dioxane (50 mL). The temperature was
maintained at 90 ꢁC for 1 h and then the reaction mix-
ture was stirred for 7–8 h. The excess solvent was
removed under reduced pressure to obtain sticky mass,
which was poured into ice-cold water and extracted
from chloroform. The chloroform was removed under
vacuum and solid product obtained was recrystallized
from chloroform. Yield 72%, Rf: 0.56 (acetoni-
trile:methanol, 1:1); mp 68–69 ꢁC, IR: 1730 (C@O
Comp. No. 13
Comp. No 14
Comp. No 50
0
1
2
3
4
Log Concentration
Figure 2. Graph of % lyses against Log concentration for compounds
11, 12 and 40.
stretching), 2985, 2989 (CH2CH3 stretching) cmꢀ1; H
1
NMR (CDCl3): 1.37 (t, 3H, CH3), 4.34–4.61 (q, 2H,
CH2), 4.70 (s, 2H, OCH2), 8.76 (t, 1H, pyrazine C5-H),
8.89 (d, 1H, pyrazine C6-H), 9.254 (d, 1H, pyrazine C2-
H), FABMS (m=z, 100%): 182.15 [Mþ, 100%); Anal.
found: C, 52.74; H, 5.53; N, 15.37. For C8H10N2O3
(182.17): C, 52.73; H, 5.59; N, 15.32.
PMP-DM (Mumbai, India) and are uncorrected. Purity
of the compounds was checked by precoated TLC plates
(E. Merck Kieselgel 60 F25, Mumbai, India). IR spectra
were recorded using KBr pellets on Perkin–Elmer 337
spectrophotometer from Perkin–Elmer International
Incorporation, Rorkreuz, Switzerland (Vmax in cmꢀ1 1H
NMR spectra on a Varian 300 MHz instrument at
RSIC, IIT, Powai. Elemental analyses were carried out
using FLASH EA 1112 CHN analyzer from Thermo
Finnigen, Italy. Mass spectra (FAB-MS) were recorded
on 70 eV on Jeol D-300 spectrometer (Jeol Ltd., Tokyo,
Japan).
Synthesis of 3: A mixture of 2 (3.75 g, 0.02 mol), 85%
hydrazine hydrate (4.12 mL, 0.08 mol) in ethanol
(40 mL) was refluxed for 12 h. The excess solvent was
removed under reduced pressure and the reaction mix-
ture was cooled at 4–5 ꢁC. The solid crystals separated
were filtered, washed with cold water, dried and
recrystalized from ethanol. Yield 62%, Rf: 0.62 (sceto-

C. G. Bonde, N. J. Gaikwad / Bioorg. Med. Chem. 12 (2004) 2151–2161
2157
nitrile:methanol, 1:1); mp 170–171 ꢁC, IR: 3382, 3355
(NHNH2), 1733 (C@O stretching), cmꢀ1 1H NMR
stretching), 1315 (C@S stretching) cmꢀ1
;
1H NMR
;
(CDCl3): d 0.94 (t, 3H, butyl CH3) 1.39 (m, 2H, butyl
CH2), 1.53 (m, 2H, butyl CH2), 3.62 (m, 2H, butyl CH2),
4.71 (s, 2H, OCH2), 7.02–7.61 (m, 4H, ArH), 7.73 (s,
1H, CONH), 8.74 (t, 1H, pyrazine C5-H), 8.81 (d, 1H,
pyrazine C6-H), 9.22 (d, 1H, pyrazine C2-H), 10.44 (s,
1H, NH), 11.81 (s, 1H, NHAr), FABMS (m=z, 100%):
283.33 ([Mþ], 100%); Anal. found: C, 46.61; H, 6.06; N,
24.74. For C11H17N5O2S (283.35): C, 46.63; H, 6.05; N,
24.72.
(CDCl3): d 4.40 (s, 2H, NH2), 4.70 (s, 2H, OCH2), 7.78
(s, 1H, CONH) 8.74 (t, 1H, pyrazine C5-H), 8.85 (d, 1H,
pyrazine C6-H), 9.24 (d, 1H, pyrazine C2-H), FABMS
(m=z, 100%): 168.13 [Mþ, 100%); Anal. found: C, 42.86;
H, 4.80; N, 33.32. For C6H8N4O2 (168.15): C, 42.83; H,
4.80; N, 33.31.
Synthesis of 4: To a solution of 3 (0.01 mol) in ethanol
(50 mL), various aliphatic/aromatic isothiocynates
(0.01 mol) were added and the reaction mixture was
refluxed for 12 h. Excess solvent was removed under
vacuum. The residue was washed with diethyl ether and
recrystallized using methanol.
9. Yield 83%, Rf: 0.56 (acetonitrile:methanol, 1:1); mp
233–234 ꢁC, IR: 3213, 3229 (NH stretching), 2986, 2990
(CH(CH3)2 stretching), 1732 (C@O stretching), 1315
1
(C@S stretching) cmꢀ1; H NMR (CDCl3): d 1.2 (dd,
6H, isopropyl CH3), 4.00 (m, 1H, isopropyl CH), 4.71 (s,
2H, OCH2), 7.74 (s, 1H, CONH) 8.73 (t, 1H, pyrazine
C5-H), 8.82 (d, 1H, pyrazine C6-H), 9.21 (d, 1H, pyr-
azine C2-H), 10.44 (s, 1H, NH), 11.82 (s, 1H, NHAr),
FABMS (m=z, 100%): 269.35 ([Mþ], 100%); Anal.
found: C, 44.64; H, 5.62; N, 26.05. For C10H15N5O2S
(269.32): C, 44.60; H, 5.61; N, 26.00.
Yield 85%, Rf: 0.68 (acetonitrile:methanol, 1:1); mp
245–246 ꢁC, IR: 3215, 3230 (NH), 1731 (C@O stretch-
1
ing), 1315 (C@S stretching) cmꢀ1; H NMR (CDCl3):
4.70 (s, 2H, OCH2), 7.24–7.52 (m, 3H, ArH), 7.78 (s,
1H, CONH), 8.74 (t, 1H, pyrazine C5-H), 8.85 (d, 1H,
pyrazine C6-H), 9.24 (d, 1H, pyrazine C2-H), 10.40 (s,
1H, NH), 11.81 (s, 1H, NHAr), FABMS (m=z, 100%):
384.75 ([Mþþ2], 100%); Anal. found: C, 40.78; H, 2.92;
N, 21.98. For C13H11N6O4SCl (382.78): C, 40.79; H,
2.90; N, 21.96.
10. Yield 86%, Rf: 0.60 (acetonitrile:methanol, 1:1); mp
174–175 ꢁC, IR: 3213, 3224 (NH stretching), 2981, 2986,
2990, (C(CH3)3 stretching) 1731 (C@O stretching), 1316
1
(C@S stretching) cmꢀ1; H NMR (CDCl3): d 1.2 (dd,
5. Yield 81%, Rf: 0.65 (acetonitrile:methanol, 1:1); mp
220–221 ꢁC, IR: 3217, 3232 (NH), 1738 (C@O stretch-
9H, t-butyl CH3), 4.72 (s, 2H, OCH2), 7.73 (s, 1H,
CONH), 8.71 (t, 1H, pyrazine C5-H), 8.84 (d, 1H, pyr-
azine C6-H), 9.23 (d, 1H, pyrazine C2-H), 10.45 (s, 1H,
NH), 11.83 (s, 1H, NHAr), FABMS (m=z, 100%): 283.32
([Mþ], 100%); Anal. found: C, 46.62; H, 6.05; N, 24.73.
For C11H17N5O2S (283.35): C, 46.63; H, 6.05; N, 24.72.
1
ing), 1313 (C@S stretching) cmꢀ1; H NMR (CDCl3): d
4.71 (s, 2H, OCH2), 7.39–7.62 (m, 4H, ArH), 7.75 (s,
1H, CONH), 8.73 (t, 1H, pyrazine C5-H), 8.82 (d, 1H,
pyrazine C6-H), 9.21 (d, 1H, pyrazine C2-H), 10.44 (s,
1H, NH), 11.83 (s, 1H, NHAr), FABMS (m=z, 100%):
339.76 ([Mþþ2], 100%); Anal. found: C, 46.20; H, 3.56;
N, 20.76. For C13H12N5O2SCl (337.78): C, 46.22; H,
3.58; N, 20.73.
Synthesis of 11: The mixture of the thiosemicarbazide
(0.01 mol), appropriate phenacyl bromide (0.01 mol) and
sodium acetate (0.2 mol) in ethanol (50 mL) was refluxed
for 7 h. The mixture was cooled, diluted with enough
water to develop turbidity and left overnight to obtain
the product. The product was filtered, dried and
recrystallized using aqueous ethanol. The yields and
physical constants are summarized in Table 1.
6. Yield 78%, Rf: 0.62 (acetonitrile:methanol, 1:1); mp
215–216 ꢁC, IR: 3212, 3235 (NH), 1734 (C@O stretch-
1
ing), 1317 (C@S stretching) cmꢀ1; H NMR (CDCl3): d
4.72 (s, 2H, OCH2), 7.21–7.54 (m, 3H, ArH), 7.74 (s,
1H, CONH), 8.73 (t, 1H, pyrazine C5-H), 8.82 (d, 1H,
pyrazine C6-H), 9.21 (d, 1H, pyrazine C2-H), 10.43 (s,
1H, NH), 11.82 (s, 1H, NHAr), FABMS (m=z, 100%):
374.21 ([Mþþ2], 100%); Anal. found: C, 41.92; H, 2.91;
N, 18.85. For C13H11N5O2SCl2 (372.23): C, 41.95; H,
2.98; N, 18.81.
Rf: 0.68 (acetonitrile:methanol, 1:1), IR: 3225 (NH),
1731 (C@O stretching), 1576, 1480, 1051 (thiazoline),
1
3001 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 4.71
(s, 2H, OCH2), 6.02 (s, 1H, thiazoline), 7.14–7.62 (m,
7H, ArH), 7.77 (s, 1H, CONH), 8.75 (t, 1H, pyrazine
C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.23 (d, 1H,
pyrazine C2-H), FABMS (m=z, 100%): 563.81
([Mþþ2], 100%); Anal. found: C, 44.94; H, 2.52; N,
14.98. For C21H14N6O4SClBr (561.80): C, 44.90; H,
2.51; N, 14.96.
7. Yield 86%, Rf: 0.59 (acetonitrile:methanol, 1:1); mp
201–202 ꢁC, IR: 3214, 3236 (NH), 1733 (C@O stretch-
1
ing), 1318 (C@S stretching) cmꢀ1; H NMR (CDCl3): d
1.22, (t, 3H, CH2 CH3), 3.61(q, 2H, CH2 CH3), 4.72 (s,
2H, OCH2), 7.01–7.49 (m, 4H, ArH), 7.74 (s, 1H,
CONH), 8.73 (t, 1H, pyrazine C5-H), 8.82 (d, 1H, pyr-
azine C6-H), 9.21 (d, 1H, pyrazine C2-H), 10.43 (s, 1H,
NH), 11.82 (s, 1H, NHAr), FABMS (m=z, 100%): 347.39
([Mþ], 100%); Anal. found: C, 51.84; H, 4.94; N, 20.19.
For C15H17N5O3S (372.23): C, 51.86; H, 4.93; N, 20.16.
12. Rf: 0.61 (acetonitrile:methanol, 1:1), IR: 3226 (NH),
1732 (C@O stretching), 1577, 1481, 1050 (thiazoline),
1
3002 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 4.73
(s, 2H, OCH2), 6.04 (s, 1H, thiazoline), 7.09–7.72 (m,
7H, ArH), 7.78 (s, 1H, CONH), 8.75 (t, 1H, pyrazine
C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.23 (d, 1H, pyr-
azine C2-H), FABMS (m=z, 100%): 517.37 ([Mþþ2],
100%); Anal. found: C, 48.72; H, 2.74; N, 16.29. For
C21H14N6O4SCl2 (517.35): C, 48.76; H, 2.73; N, 16.25.
8. Yield 78%, Rf: 0.62 (acetonitrile:methanol, 1:1); mp
152–153 ꢁC, IR: 3209, 3232 (NH stretching), 2984, 2989,
2991 (CH2CH2CH2CH3 stretching) 1733 (C@O

2158
C. G. Bonde, N. J. Gaikwad / Bioorg. Med. Chem. 12 (2004) 2151–2161
13. Rf: 0.72 (acetonitrile:methanol, 1:1), IR: 3225 (NH),
1733 (C@O stretching), 1576, 1481, 1051 (thiazoline),
19. Rf: 0.58 (acetonitrile:methanol, 1:1), IR: 3223 (NH),
1732 (C@O stretching), 1574, 1482, 1052 (thiazoline),
1
1
3003 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 4.72
3004 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 4.71
(s, 2H, OCH2), 5.99 (s, 1H, thiazoline), 7.19–7.63 (m,
7H, ArH), 7.79 (s, 1H, CONH), 8.75 (t, 1H, pyrazine
C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.23 (d, 1H, pyr-
azine C2-H), FABMS (m=z, 100%): 500.87 ([Mþþ2],
100%); Anal. found: C, 50.3 8; H, 2.84; N, 16.76. For
C21H14N6O4SCl2 (500.89): C, 50.36; H, 2.82; N, 16.78.
(s, 2H, OCH2), 6.05 (s, 1H, thiazoline), 7.14–7.52 (m,
8H, ArH), 7.77 (s, 1H, CONH), 8.75 (t, 1H, pyrazine
C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.23 (d, 1H,
pyrazine C2-H), FABMS (m=z, 100%): 518.82
([Mþþ2], 100%); Anal. found: C, 48.83; H, 2.92; N,
16.94. For C21H15N5O2SClBr (516.80): C, 48.81; H,
2.93; N, 16.91.
14. Rf: 0.58 (acetonitrile:methanol, 1:1), IR: 3225 (NH),
1738 (C@O stretching), 1572, 1486, 1057 (thiazoline),
3005 (ArH stretching) cmꢀ1; 1H NMR (CDCl3): d 1.2 (t,
3H, OCH3–C6H4), 4.72 (s, 2H, OCH2), 5.97 (s, 1H,
thiazoline), 7.18–7.65 (m, 7H, ArH), 7.76 (s, 1H,
CONH), 8.75 (t, 1H, pyrazine C5-H), 8.86 (d, 1H, pyr-
azine C6-H), 9.23 (d, 1H, pyrazine C2-H), FABMS
(m=z, 100%): 514.95 ([Mþþ2], 100%); Anal. found: C,
51.56; H, 3.33; N, 16.36. For C22H17N6O5SCl (512.93):
C, 51.52; H, 3.34; N, 16.38.
23. Rf: 0.59 (acetonitrile:methanol, 1:1), IR: 3226 (NH),
1733 (C@O stretching), 1572, 1484, 1057 (thiazoline),
1
3003 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 4.72
(s, 2H, OCH2), 5.95 (s, 1H, thiazoline), 7.16–7.64 (m,
8H, ArH), 7.75 (s, 1H, CONH), 8.75 (t, 1H, pyrazine
C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.20 (s, 1H,
C6H4OH) 9.23 (d, 1H, pyrazine C2-H), FABMS (m=z,
100%): 500.8 ([Mþþ2], 100%); Anal. found: C, 55.54; H,
3.55; N, 15.46. For C21H16N5O3SCl (453.90): C, 55.57;
H, 3.56; N, 15.43.
15. Rf: 0.62 (acetonitrile:methanol, 1:1), IR: 3228 (NH),
1734 (C@O stretching), 1573, 1485, 1056 (thiazoline),
26. Rf: 0.65 (acetonitrile:methanol, 1:1), IR: 3226 (NH),
1734 (C@O stretching), 1575, 1484, 1056 (thiazoline),
3004 (ArH stretching) cmꢀ1; 1H NMR (CDCl3): d 1.3 (t,
3H, CH3–C6H4), 4.72 (s, 2H, OCH2), 5.98 (s, 1H,
thiazoline), 7.18–7.65 (m, 8H, ArH), 7.75 (s, 1H,
CONH), 8.75 (t, 1H, pyrazine C5-H), 8.86 (d, 1H, pyr-
azine C6-H), 9.23 (d, 1H, pyrazine C2-H), FABMS
(m=z, 100%): 453.95 ([Mþþ2], 100%); Anal. C, 58.49; H,
4.05; N, 15.53. For C22H18N5O2SCl (451.93): C, 58.47;
H, 4.04; N, 15.50.
1
3004 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 4.72
(s, 2H, OCH2), 5.96 (s, 1H, thiazoline), 7.12–7.69 (m,
7H, ArH), 7.78 (s, 1H, CONH), 8.75 (t, 1H, pyrazine
C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.19 (s, 1H,
C6H4OH), 9.23 (d, 1H, pyrazine C2-H), FABMS (m=z,
100%): 500.8 ([Mþþ2], 100%); Anal. found: C, 51.56; H,
3.33; N, 16.36. For C21H15N6O5SCl (498.9): C, 50.56; H,
3.03; N, 16.85.
16. Rf: 0.63 (acetonitrile:methanol, 1:1), IR: 3225 (NH),
1733 (C@O stretching), 1578, 1482, 1052 (thiazoline),
30. Rf: 0.77 (acetonitrile:methanol, 1:1), IR: 3224 (NH),
1739 (C@O stretching), 1573, 1485, 1056 (thiazoline),
1
3005 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 4.73
1
(s, 2H, OCH2), 6.07 (s, 1H, thiazoline), 7.09–7.72 (m,
6H, ArH), 7.78 (s, 1H, CONH), 8.75 (t, 1H, pyrazine
C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.23 (d, 1H,
pyrazine C2-H), FABMS (m=z, 100%): 553.76
([Mþþ2], 100%); Anal. found: C, 45.73; H, 2.39; N,
15.25. For C21H13N6O4SCl3 (551.79): C, 45.71; H, 2.38;
N, 15.23.
3003 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 1.26
(t, 3H, OCH3–C6H4), 4.72 (s, 2H, OCH2), 5.97 (s, 1H,
thiazoline), 7.09–7.71 (m, 7H, ArH), 7.76 (s, 1H,
CONH) 8.75 (t, 1H, pyrazine C5-H), 8.86 (d, 1H, pyr-
azine C6-H), 9.23 (d, 1H, pyrazine C2-H), FABMS
(m=z, 100%): 504.35 ([Mþþ2], 100%); Anal. found: C,
52.63; H, 3.42; N, 13.97. For C22H17N5O3SCl2 (502.37):
C, 52.60; H, 3.41; N, 13.94.
17. Rf: 0.56 (acetonitrile:methanol, 1:1), IR: 3226 (NH),
1732 (C@O stretching), 1579, 1484, 1052 (thiazoline),
32. Rf: 0.67 (acetonitrile:methanol, 1:1), IR: 3224 (NH),
1735 (C@O stretching), 1579, 1483, 1053 (thiazoline),
1
3005 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 4.73
1
(s, 2H, OCH2), 6.05 (s, 1H, thiazoline), 7.02–7.73 (m,
9H, ArH), 7.77 (s, 1H, CONH), 8.75 (t, 1H, pyrazine
C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.23 (d, 1H, pyr-
azine C2-H), FABMS (m=z, 100%): 553.76 ([Mþþ2],
100%); Anal. C, 52.25; H, 3.12; N, 17.44. For
C21H15N6O4SCl (482.90): C, 52.23; H, 3.13; N, 17.40.
3004 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 4.73
(s, 2H, OCH2), 6.07 (s, 1H, thiazoline), 7.18–7.70 (m,
6H, ArH), 7.78 (s, 1H, CONH), 8.75 (t, 1H, pyrazine
C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.23 (d, 1H, pyr-
azine C2-H), FABMS (m=z, 100%): 543.25 ([Mþþ2],
100%); Anal. found: C, 46.62; H, 2.44; N, 12.97. For
C21H13N5O2SCl4 (541.24): C, 46.60; H, 2.42; N, 12.94.
18. Rf: 0.65 (acetonitrile:methanol, 1:1), IR: 3226 (NH),
1737 (C@O stretching), 1573, 1484, 1055 (thiazoline),
3003 (ArH stretching) cmꢀ1; 1H NMR (CDCl3): d 1.2 (t,
3H, CH3–C6H4), 4.72 (s, 2H, OCH2), 5.97 (s, 1H,
thiazoline), 7.18–7.65 (m, 7H, ArH), 7.76 (s, 1H,
CONH), 8.75 (t, 1H, pyrazine C5-H), 8.86 (d, 1H, pyr-
azine C6-H), 9.23 (d, 1H, pyrazine C2-H), FABMS
(m=z, 100%): 498.94 ([Mþþ2], 100%); Anal. found: C,
53.15; H, 3.43; N, 16.95. For C22H17N6O4SCl (496.93):
C, 53.18; H, 3.45; N, 16.91.
33. Rf: 0.56 (acetonitrile:methanol, 1:1), IR: 3225 (NH),
1731 (C@O stretching), 1575, 1481, 1055 (thiazoline),
1
3002 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 4.72
(s, 2H, OCH2), 6.04 (s, 1H, thiazoline), 7.09–7.71 (m,
9H, ArH), 7.77 (s, 1H, CONH), 8.75 (t, 1H, pyrazine
C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.23 (d, 1H, pyr-
azine C2-H), FABMS (m=z, 100%): 474.36 ([Mþþ2],
100%); Anal. C, 53.42; H, 3.22; N, 14.84. For
C21H15N6O4SCl (472.35): C, 53.40; H, 3.20; N, 14.83.

C. G. Bonde, N. J. Gaikwad / Bioorg. Med. Chem. 12 (2004) 2151–2161
2159
37. Rf: 0.71 (acetonitrile:methanol, 1:1), IR: 3223 (NH),
1734 (C@O stretching), 1575, 1482, 1053 (thiazoline),
CH2), 4.72 (s, 2H, OCH2), 5.98 (s, 1H, thiazoline),
7.15–7.61 (m, 4H, ArH), 7.76 (s, 1H, CONH), 8.75 (t,
1H, pyrazine C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.23
(d, 1H, pyrazine C2-H), FABMS (m=z, 100%): 397.53
([Mþ], 100%); Anal. found: C, 60.45; H, 5.85; N,
17.64.For C20H23N5O2S (397.50): C, 60.43; H, 5.83; N,
17.62.
1
3002 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 4.72
(s, 2H, OCH2), 6.01 (s, 1H, thiazoline), 7.08–7.61 (m,
8H, ArH), 7.79 (s, 1H, CONH), 8.75 (t, 1H, pyrazine
C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.23 (d, 1H, pyr-
azine C2-H), FABMS (m=z, 100%): 467.48 ([Mþþ2],
100%); Anal. found: C, 59.31; H, 4.35; N, 15.09. For
C23H20N5O3FS (465.50): C, 59.35; H, 4.33; N, 15.05.
52. Rf: 0.51 (acetonitrile:methanol, 1:1), IR: 3224 (NH),
1732 (C@O stretching), 1573, 1488, 1056 (thiazoline),
1
39. Rf: 0.67 (acetonitrile:methanol, 1:1), IR: 3224 (NH),
1733 (C@O stretching), 1571, 1484, 1055 (thiazoline),
3002 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 1.23
(dd, 6H, isopropyl CH3), 4.03 (m, 1H, isopropyl CH),
4.72 (s, 2H, OCH2), 6.06 (s, 1H, thiazoline), 7.08–7.76
(m, 4H, ArH), 7.79 (s, 1H, CONH), 8.75 (t, 1H,
pyrazine C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.23 (d,
1H, pyrazine C2-H), FABMS (m=z, 100%): 405.87
([Mþþ2], 100%); Anal. found: C, 48.72; H, 2.74; N,
16.29. For C18H18N5O2SCl (403.89): C, 48.76; H, 2.73;
N, 16.25.
1
3003 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 1.22,
(t, 3H, CH2CH3), 3.61 (q, 2H, CH2CH3), 4.72 (s, 2H,
OCH2), 6.07 (s, 1H, thiazoline), 7.03–7.67 (m, 8H,
ArH), 7.78 (s, 1H, CONH), 8.75 (t, 1H, pyrazine C5-H),
8.86 (d, 1H, pyrazine C6-H), 9.19 (s, 1H, C6H4OH)
9.23 (d, 1H, pyrazine C2-H), FABMS (m=z, 100%):
463.51 ([Mþ], 100%); Anal. found: C, 59.65; H, 4.55; N,
15.13. For C21H15N5O4S (498.9): C, 59.60; H, 4.57; N,
15.11.
55. Rf: 0.49 (acetonitrile:methanol, 1:1), IR: 3223 (NH),
1733 (C@O stretching), 1575, 1490, 1060 (thiazoline),
1
41. Rf: 0.72 (acetonitrile:methanol, 1:1), IR: 3224 (NH),
1735 (C@O stretching), 1575, 1482, 1052 (thiazoline),
3005 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 1.22
(dd, 6H, isopropyl CH3), 4.05 (m, 1H, isopropyl CH),
4.73 (s, 2H, OCH2), 6.05 (s, 1H, thiazoline), 7.05–7.66
(m, 4H, ArH), 7.79 (s, 1H, CONH), 8.75 (t, 1H, pyr-
azine C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.18 (s, 1H,
C6H4OH), 9.23 (d, 1H, pyrazine C2-H), FABMS (m=z,
100%): 385.46 ([Mþ], 100%); Anal. found: C, 56.05; H,
4.94; N, 18.12. For C18H19N5O3S (385.44): C, 56.09; H,
4.96; N, 18.17.
1
3004 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 4.72
(s, 2H, OCH2), 5.99 (s, 1H, thiazoline), 7.05–7.49 (m,
9H, ArH), 7.78 (s, 1H, CONH), 8.75 (t, 1H,
pyrazine C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.23 (d,
1H, pyrazine C2-H), FABMS (m=z, 100%): 447.53
([Mþ], 100%); Anal. found: C, 61.73; H, 4.73; N,
15.65. For C23H21N5O3S (447.51): C, 61.73; H, 4.73; N,
15.65.
57. Rf: 0.73 (acetonitrile:methanol, 1:1), IR: 3225 (NH),
1734 (C@O stretching), 1578, 1485, 1054 (thiazoline),
43. Rf: 0.53 (acetonitrile:methanol, 1:1), IR: 3226 (NH),
1732 (C@O stretching), 1575, 1481, 1053 (thiazoline),
1
3003 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 4.73
1
3002 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 0.94
(s, 2H, OCH2), 5.58 (s, 1H, thiazoline), 7.03–7.69 (m,
9H, ArH), 7.78 (s, 1H, CONH), 8.75 (t, 1H, pyrazine
C5-H), 8.86 (d, 1H, pyrazine C6-H), 9.23 (d, 1H, pyr-
azine C2-H), FABMS (m=z, 100%): 369.46 ([Mþ],
100%); Anal. C, 58.54; H, 5.19; N, 18.99. For
C18H19N5O2S (369.44): C, 58.52; H, 5.18; N, 18.96.
(t, 3H, butyl CH3), 1.39 (m, 2H, butyl CH2), 1.53 (m,
2H, butyl CH2), 3.62 (m, 2H, butyl CH2), 4.71 (s, 2H,
OCH2), 5.99 (s, 1H, thiazoline), 7.16–7.68 (m, 4H, ArH),
7.77 (s, 1H, CONH), 8.75 (t, 1H, pyrazine C5-H), 8.86
(d, 1H, pyrazine C6-H), 9.23 (d, 1H, pyrazine C2-H),
FABMS (m=z, 100%): 464.38 ([Mþþ2], 100%); Anal.
found: C, 49.33; H, 4.32; N, 15.17. For C19H20N5O2SBr
(462.36): C, 49.36; H, 4.30; N, 15.15.
59. Rf: 0.59 (acetonitrile:methanol, 1:1), IR: 3226 (NH),
1732 (C@O stretching), 1575, 1481, 1053 (thiazoline),
1
3002 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 1.39
47. Rf: 0.62 (acetonitrile:methanol, 1:1), IR: 3225 (NH),
1734 (C@O stretching), 1571, 1487, 1056 (thiazoline),
(s, 9H, t-butyl), 4.72 (s, 2H, OCH2), 6.01 (s, 1H, thiaz-
oline), 7.06–7.69 (m, 4H, ArH), 7.76 (s, 1H, CONH),
8.75 (t, 1H, pyrazine C5-H), 8.86 (d, 1H, pyrazine C6-
H), 9.23 (d, 1H, pyrazine C2-H), FABMS (m=z, 100%):
464.35 ([Mþþ2], 100%); Anal. found: C, 49.34; H, 4.31;
N, 15.18. For C19H20N5O2SBr (462.36): C, 49.36; H,
4.30; N, 15.15.
1
3001 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 0.95
(t, 3H, butyl CH3), 1.38 (m, 2H, butyl CH2), 1.54 (m,
2H, butyl CH2), 3.63 (m, 2H, butyl CH2) d 4.72 (s, 2H,
OCH2), 5.94 (s, 1H, thiazoline), 7.19–7.56 (m, 4H, ArH),
7.75 (s, 1H, CONH), 8.75 (t, 1H, pyrazine C5-H), 8.86
(d, 1H, pyrazine C6-H), 9.20 (s, 1H, C6H4OH) 9.23 (d,
1H, pyrazine C2-H), FABMS (m=z, 100%): 399.49
([Mþþ2], 100%); Anal. found: C, 57.19; H, 5.31; N,
17.33. For C19H21N5O3S (399.47): C, 57.13; H, 5.30; N,
17.32.
62. Rf: 0.64 (acetonitrile:methanol, 1:1), IR: 3225 (NH),
1733 (C@O stretching), 1576, 1484, 1057 (thiazoline),
1
3003 (ArH stretching) cmꢀ1; H NMR (CDCl3): d 1.21
(t, 3H, OCH3–C6H4), 1.40 (s, 9H, t-butyl), 4.73 (s, 2H,
OCH2), 6.03 (s, 1H, thiazoline), 7.06–7.69 (m, 4H, ArH),
7.76 (s, 1H, CONH), 8.75 (t, 1H, pyrazine C5-H), 8.86
(d, 1H, pyrazine C6-H), 9.23 (d, 1H, pyrazine C2-H),
FABMS (m=z, 100%): 413.48 ([Mþ], 100%); Anal.
found: C, 57.16; H, 5.32; N, 17.59. For C20H23N5O3S
(413.49): C, 57.12; H, 5.30; N, 17.54.
50. Rf: 0.52 (acetonitrile:methanol, 1:1), IR: 3225 (NH),
1736 (C@O stretching), 1573, 1482, 1059 (thiazoline),
3004 (ArH stretching) cmꢀ1; 1H NMR (CDCl3): 0.96 (t,
3H, butyl CH3), 1.34 (t, 3H, CH3–C6H4), 1.39 (m, 2H,
butyl CH2), 1.56 (m, 2H, butyl CH2), 3.65 (m, 2H, butyl

2160
C. G. Bonde, N. J. Gaikwad / Bioorg. Med. Chem. 12 (2004) 2151–2161
66. Rf: 0.65 (acetonitrile:methanol, 1:1), IR: 3225 (NH),
1734 (C@O stretching), 1573, 1486, 1054 (thiazoline),
3005 (ArH stretching) cmꢀ1; 1H NMR (CDCl3): 1.34 (t,
3H, CH3–C6H4), 1.40 (s, 9H, t-butyl), 4.72 (s, 2H,
OCH2), 5.97 (s, 1H, thiazoline), 7.15–7.61 (m, 4H, ArH),
7.76 (s, 1H, CONH), 8.75 (t, 1H, pyrazine C5-H), 8.86
(d, 1H, pyrazine C6-H), 9.23 (d, 1H, pyrazine C2-H),
FABMS (m=z, 100%): 397.51 ([Mþ], 100%); Anal.
found: C, 60.46; H, 5.84; N, 17.61. For C20H23N5O2S
(397.50): C, 60.43; H, 5.83; N, 17.62.
activity.24 The MIC values were also tested for three
well-known antibiotics (penicillin-G, ampicillin and
chloramphenicol) to compare the antibacterial activity
of our test compounds with the antibiotics, which are
currently in therapy. Rifampcin and isoniazid (INH)
were used as reference standard for antimycobacterial
activity. The stock solution (2–4 lg/mL) of test com-
pounds was prepared in a mixture of sterile water and
dimethylformamide (8:2) solvent. The stock solution
was sterilized by passing through a 0.2 mm polycar-
bonate sterile membrane (Nuclepore) filters. Further,
the serial dilution of test compounds was carried out
and the following concentration was used: 1000, 500,
250, 125, 62, 32, 16, 8, 4 and 1 lg/mL. Test compounds
at various concentrations were added to culture medium
in a sterilized borosilicate test tube and different bacte-
rial strains were inoculated at 106 bacilli/mL concen-
tration. The tubes were incubated at 37 ꢁC for 24 h for
antibacterial activity and 14 and 21 days for antimyco-
bacterial activity and then examined for the presence or
absence of growth of the test organisms. All experiments
were performed in triplicate. The MIC values were
obtained from the lowest concentration of the test
compound where the tubes remained clear, indicated
that the bacterial growth was completely inhibited at
this concentration. The MIC values were expressed in
lg/mL and the results are shown in Table 3.
Synthesis of 68: A mixture of the thiosemicarbazide
(0.01 mol), chloroacetic acid (0.01 mol) and sodium
acetate (0.2 mol) in ethanol (60 mL) was refluxed for 10
h. The mixture was cooled, diluted with enough water to
develop turbidity and left overnight to obtain the
product. The product was filtered, dried and recrystal-
lized using aqueous ethanol. The yields and physical
constants are summarized in Table 2. Yield 71%, Rf:
0.56 (acetonitrile:methanol, 1:1); mp 256–257 ꢁC, IR:
3224 (NH), 1732 (C@O stretching) cmꢀ1 1H NMR
;
(CDCl3): d 3.76 (s, 2H, thiazolidine CH2), 4.70 (s, 2H,
OCH2), 7.15–7.61 (m, 3H, ArH), 7.75 (s, 1H, CONH),
8.73 (t, 1H, pyrazine C5-H), 8.84 (d, 1H, pyrazine C6-
H), 9.23 (d, 1H, pyrazine C2-H), FABMS (m=z, 100%):
424.82 ([Mþþ2], 100%); Anal. found: C, 42.68; H, 2.65;
N, 19.91. For C15H11N6O5SCl (382.78): C, 42.61; H,
2.63; N, 19.88.
70. Yield 71%, Rf: 0.61 (acetonitrile:methanol, 1:1); mp
218–219 ꢁC, IR: 3214 (NH), 1731 (C@O stretching)
4.3. Hemolytic assay
cmꢀ1 1H NMR (CDCl3): d 3.98 (s, 2H, thiazolidine
;
CH2), 4.72 (s, 2H, OCH2), 7.15–7.62 (m, 3H, ArH), 7.75
(s, 1H, CONH), 8.73 (t, 1H, pyrazine C5-H), 8.82 (d,
1H, pyrazine C6-H), 9.21 (d, 1H, pyrazine C2-H),
FABMS (m=z, 100%): 414.26 ([Mþþ2], 100%); Anal.
found: C, 43.71; H, 2.71; N, 17.01. For C15H11N5O3SCl2
(372.23): C, 43.70; H, 2.69; N, 16.99.
Fresh human blood (5 mL) was centrifuged at 700g
(3500 rpm) for 10 min and the erythrocytes (RBCs) were
collected. The erythrocytes were washed and centrifuged
three times with phosphate buffered saline (PBS, pH 7.4,
50 mL) and the supernatant was decanted carefully. The
erythrocyte suspension was diluted two times with saline
solution and total erythrocytes were counted using
haemocytometer. Finally necessary dilution was carried
out with saline solution to get total count to
6.4 · 108 cells/mL. The stock solution of the test com-
pounds was made at an initial concentration of 4 mg/mL
using the same saline solution. Assay was carried out in
1 mL eppendorf tubes. In an eppendorf, varying vol-
umes (900–650 lL) of the saline solution was added. To
this varying volume of the stock solution of test com-
pounds (1–250 lL) was added followed by 100 lL of
RBC suspension. For the positive control that is, total
cell lyses, 10 lL of 20% w/v Triton was added instead of
the test compounds and the plain saline solution was
used as control. The eppendorf tubes were then incu-
bated at 37 ꢁC for 30 min, tubes were centrifuged at
4000 rpm for 10 min and carefully 200 lL of the super-
natant was transferred to 96-well microtritre plates with
U-shaped wells. The absorbance of the samples was read
at 404 nm with Spectra Max-190 microplate spectro-
photometer. The percentage lyses was determined as
(Atest compounds ꢀ AblankÞ=ðApositive control ꢀ AblankÞ ꢂ 100. Fur-
ther the % lyses was plotted against Log concentration
of test compounds used (Fig. 3) and from the graph
MHC and LD50 (concentration required for 50% lyses)
was determined.25
74. Yield 69%, Rf: 0.65 (acetonitrile:methanol, 1:1); mp
241–242 ꢁC, IR: 3212 (NH stretching), 2982, 2987, 2989,
1
(C(CH3)3 stretching) 1732 (C@O stretching), cmꢀ1; H
NMR (CDCl3): d 1.2 (dd, 9H, t-butyl CH3), 3.01 (s, 2H,
thiazolidine CH2), 4.72 (s, 2H, OCH2), 7.74 (s, 1H,
CONH), 8.71 (t, 1H, pyrazine C5-H), 8.84 (d, 1H, pyr-
azine C6-H), 9.23 (d, 1H, pyrazine C2-H), FABMS
(m=z, 100%): 323.37 ([Mþ], 100%); Anal. found: C,
48.30; H, 5.32; N, 21.71. For C13H17N5O2S (283.35): C,
48.29; H, 5.30; N, 21.66.
4.2. Microbiology
All the test compounds were assayed in vitro for anti-
bacterial activity against two different strains of Gram-
negative (E. coli ATCC3750 and S. typhi NCTC 786)
and Gram-positive (S. aureus ATCC 3750, B. subtilis
6633) bacteria and the antimycobacterial activity was
evaluated against H37Rv strain of M. tuberculosis. The
MIC was determined by the test tube dilution technique
using Mueller–Hinton nutrient broth (for antibacterial)
and modified KirchnerÕs culture medium containing
0.5% sterilized horse serum for antimycobacterial

C. G. Bonde, N. J. Gaikwad / Bioorg. Med. Chem. 12 (2004) 2151–2161
2161
Training Research and Testing, Parel, Mumbai 12 for
his assistance during the antimicrobial testing and
enzymatic assay. Thanks to Dr. Ravi Chambare, Nov-
artis Enterprises Limited, Navi Mumbai for technical
support.
References and notes
1. Rouhi, A. M. Chem. Eng. News 1999, 17, 52.
2. Clarck, C.; Jacobs, M.; Appelbaum, P. J. Clin. Microbiol.
1998, 36, 3579.
3. Davidson, P. T.; Le, H. Q. Drugs 1992, 43, 651.
4. Vincent, T. A. Int. J. Antimicrob. Agents 2000, 16, 317.
5. Tan, Y. T.; Tillett, D. J.; Mckay, I. A. Mol. Med. Today
2000, 6, 309.
6. MacManus, M. C. Am. J. Health-Syst. Pharm. 1997, 54,
1420.
7. Heinemann, J. A. Drug Discov. Today 1999, 4, 72.
8. Chopra, I.; Hodgson, J.; Metcalf, B.; Poste, G. Antimic-
rob. Agents Chemother. 1997, 41, 497.
9. Sander, P.; Bottger, E. C. Chemotherapy 1999, 45, 95.
10. Chan, W. C.; Li, R. C.; Ling, J. M. J. Antimicrob.
Chemother. 1999, 43, 55.
Figure 3. Analytical reverse phase high performance liquid chro-
matogram of compound 12 and its metabolite. RP-HPLC conditions:
Beckman Gold Nouveau System equipped with a 168 photodiode-
ꢀ
array detector. Column; Vydac 218TP53 (C18, 300 A, 5 mm 3.2 mm
i.d. · 250 mm). 0.5 mL/min, Buffer A¼0.1% TFA in water. Buffer
B¼90% ACN (acetonitrile) containing 10% buffer A. Linear gradient
from 0% B to 90% B over 10 min, total run time 20 min. Retention time
(Rt) for parent peak 9.05 min, metabolite Rt¼14.63 min.
11. Bush, K. Curr. Opin. Chem. Biol. 1997, 1, 169.
12. Stapleton, P.; Shannon, K.; Phillips, I. J. Antimicrob.
Chemother. 1995, 36, 483.
5. Conclusion
13. Chopra, I. J. Antimicrob. Chemother. 1992, 30, 737.
14. Coleman, K.; Athalye, M.; Clancey, A.; Davison, M.;
Payne, D. J.; Perry, C. R.; Chopra, I. J. Antimicrob.
Chemother. 1994, 33, 1091.
15. Sykes, R. B.; Bonner, D. P. Br. Med. Bull. 1984, 40, 96.
16. Bonde, C. G.; Nadkarni, B. A.; Khadse, B. G. Indian J.
Heter. Chem. 2001, 10, 271.
We have described the synthesis, antimicrobial activity
and Hemolytic studies of pyrazine containing thiazoline
and thiazolidine derivatives. The number of compounds
showed good potency in in vitro assays. Out of which
compounds 11, 12 and 40 showed good potency against
Gram-positive, Gram-negative bacteria and M. tuber-
culosis. Again compounds 28 and 30 showed good
potency against Gram-positive organisms. The anti-
mycobacterial activity of all the compounds are less in
21 days than in 14 days, means the test drug is meta-
bolizing with time in biological environment. As some
compounds show good antibacterial and antimycobac-
terial activity, such compounds would represent a
fruitful matrix for the development of a new class of
dual antibacterial–antimycobacterial agents that would
deserve further investigation and derivatization.
17. Sihgh, S. P.; Parmar, S. S.; Raman, K.; Stenherb, V. I.
Chem. Pharm. Bull. 1981, 81, 197.
18. Patel, P. B.; Trivedi, J. T. Ind. Chem. Soc. 1987, 54, 765.
19. Vittoria, D. M.; Mazzoni, O.; Piscopo, E.; Caligmano, A.;
Bolognese, A. J. Med. Chem. 1992, 35, 2910.
20. Omar, A. M.; Eshba, N. H. J. Pharm. Sci. 1984, 73, 1166.
21. Chaudhary, M.; Parmar, S. S.; Chaudhari, S. K.; Rama-
sastry, B. V. J. Pharm. Sci. 1976, 65, 443.
22. Ragab, F. A.; Hussain, m. M.; Hanna, M. m.; Hassan, G.
S. J. Pharm. Sci. 1993, 34, 387.
23. Hassan, I. Y.; Koussi, A. A.; Farghaly, Z. S. Chem.
Pharm. Bull. 1998, 46(5), 863.
24. A Manual of Clinical Microbiology; Sahm, D. F., Wash-
ington, J. A., Ballows, A., Housler, W. J., Herrmann, K.
L., Isenberg, H. D., Shadomy, H. J., Eds.; fifth ed.; ASM:
Washington, DC, 1991; p 1105.
Acknowledgements
25. Kikuchi, K. E.; Bernard, M.; Sadownik, A.; Regen, S. L.;
Armstrong, D. Antimicrob. Agents Chemother. 1997, 41,
1433.
The authors are thankful to Dr. A. S. Bobade,
Department of Chemotherapy, Haffkine Institute for