One pot synthesis and biological activity evaluation of novel schiff bases derived from 2-hydrazinyl-1,3,4-thiadiazole

Article abstract of DOI:10.1248/cpb.c12-00651

Considering the structural features of a group of known potent inhibitors of human platelet aggregation containing hydrazone structural backbone, a series of novel hydrazone derivatives of 2-hydrazinyl-1,3,4- thiadiazole were synthesized using a one-pot process and tested for their inhibitory activity against platelet aggregation induced by arachidonic acid and ADP. Among the derivatives, compounds 3l, 3o and 3p exhibited the highest antiplatelet aggregation activity. The derivatives were also screened for their potential antimycobacterial activity and compounds 3g, 3k, 3p and 3q were among the most active compounds.

Full text of DOI:10.1248/cpb.c12-00651

160
Regular Article
Chem. Pharm. Bull. 61(2) 160–166 (2013)
Vol. 61, No. 2
One Pot Synthesis and Biological Activity Evaluation of Novel Schiff
Bases Derived from 2-Hydrazinyl-1,3,4-thiadiazole
Kamaleddin Haj Mohammad Ebrahim Tehrani,a Soroush Sardari,b Vida Mashayekhi,a
,a,c
Marjan Esfahani Zadeh,a Parisa Azerang,b and Farzad Kobarfard*
a Department of Medicinal Chemistry, School of Pharmacy, Shahid Beheshti University of Medical Sciences;
c Phytochemistry Research Center, Shahid Beheshti University of Medical Sciences; Vali Asr Ave. Niayesh
b
Junction, Tehran P.O. Box 14155–6153, Iran: and Drug Design and Bioinformatics Unit, Department of Medical
Biotechnology, Biotechnology Research Center, Pasteur Institute; Tehran 13164, Iran.
Received July 24, 2012; accepted October 10, 2012; advance publication released online November 27, 2012
Considering the structural features of a group of known potent inhibitors of human platelet aggregation
containing hydrazone structural backbone, a series of novel hydrazone derivatives of 2-hydrazinyl-1,3,4-
thiadiazole were synthesized using a one-pot process and tested for their inhibitory activity against platelet
aggregation induced by arachidonic acid and ADP. Among the derivatives, compounds 3l, 3o and 3p exhib-
ited the highest antiplatelet aggregation activity. The derivatives were also screened for their potential anti-
mycobacterial activity and compounds 3g, 3k, 3p and 3q were among the most active compounds.
Key words 1,3,4-thiadiazole-2-yl hydrazone; antiplatelet aggregation; thiosemicarbazone; antimycobacterial
Platelet activation starts the initial steps in development of new hydrazones which effectively inhibited platelet aggrega-
thromboembolic disorders including cardiovascular and cere- tion with selective inhibitory activity toward platelet aggrega-
brovascular events; the major causes of death in the industrial tion induced by arachidonic acid. They found active group
countries and also worldwide.1,2) Based on the role of platelets of hydrazones with arylsulfonate acylhydrazone,8) phenothi-
in initiation and progression of these diseases, some medica- azine-1-acylhydrazone,9) N-substituted-phenyl-1,2,3-triazole-
tions have been developed that target the biochemical path- 4-acylhydrazone10) and pyrazolylhydrazone11) structures.
ways triggering or mediating platelet activation, self-affinity Although numerous derivatives have been found to exhibit
and aggregation.
antiplatelet activity in these studies, they share a strongly pre-
The medications of clinical importance include: acetyl- served structural backbone that is two (hetero)aromatic ring
salicylic acid (a nonselective irreversible inhibitor of the en- systems linked by a hydrazone bond (Fig. 1).
zyme cyclooxygenase) which prevents the transformation of
Many derivatizations have been made in both Ar1 and Ar2
arachidonic acid (AA) to its potent proaggregatory metabolite positions and introduction of both carbocyclic and heterocyclic
thromboxane A2 and clopidogrel (an irreversible antagonist aromatic rings in each position led to active derivatives.
of P2Y12) which blocks the activation of platelet purinergic
Considering this background, in the present study by an
recepteors of P2Y family (mainly P2Y12) by adenosine diphos- isosteric substitution strategy, some hydrazone analogues
phate (ADP), another potent agonist of platelet aggregation.2,3) have been designed and prepared as potential antiplatelet
However, some undesired effects are associated with the derivatives by introducing a thiadiazole ring in Ar1 position
use of these agents. Bleeding is the major side effect of both and various carbocyclic and heterocyclic aromatic rings in Ar2
drugs.4) Undesired complications such as neutropenia and position (Fig. 2).
thrombotic thrombocytopenic purpura (TTP) pose limitation
This paper reports the preparation and inhibitory activity of
on the antiplatelet therapies using clopidogrel.4,5) Resistance the above mentioned derivatives against platelet aggregation
to both drugs is another problem that further complicates the induced by AA and ADP.
anti-platelet therapy.6) Considering the above mentioned limi-
tations of antiplatelet drugs, research aiming at introducing
new therapeutic agents with unique modes of action, high ef-
ficacy and improved safety is strongly demanding.
Hydrazone derivatives are an interesting group of com-
pounds with various reported biological properties. They
also hold a place in the area of antiplatelet drug discovery
and many interesting studies have been performed focused
on the development of hydrazone derivatives as inhibitors of
platelet aggregation. Coquelet et al. have reported some ar-
ylhydrazone derivatives of aryl methyl ketones as inhibitors
of platelet cyclooxygenase (COX) and lipoxygenase (LOX),
blocking the transformation of arachidonic acid to its proag-
gregatory metabolites.7) In light of the finding of this study,
Barreiro et al. in a series of studies managed to develop some
Fig. 1. Schematic Representation of the General Hydrazone Structural
Backbone with Antiplatelet Activity
Coquelet et al. reported the most active derivatives with R=CH3 and NH2, while
Barreiro et al. have mainly focused on the derivatives with R=H.
The authors declare no conflict of interest.
Fig. 2. General Structure of the Newly Designed Derivatives
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: kobarfard@sbmu.ac.ir

February 2013
161
Fig. 3. Some Thiosemicarbazones Known as Potent Antitubercular Compounds
(a) SRI-224,15) (b) SRI-286,15) (c) the drug thiacetazone, (d) KBF611, a potent analogue of thiacetazone.16)
Chart 1. Total Synthesis of Final Derivatives 3a–r from Thiocarbohy-
drazide (1)
Fig. 4. The New Hydrazone Derivatives as Cyclic Analogues of Thio-
semicarbazones
three decades of negligence, there is now renewed interest in
the development of new drugs for the treatment and preven-
tion of tuberculosis.
Considering the structural features for the designed com-
pounds in the present study, anti mycobacterial activity is also
expected for them beside anti platelet activity.
Therefore, in addition to antiplatelet activity, the antimy-
cobacterial activity of the new derivatives was also assessed
Fig. 5. Monothiocarbohydrazone Intermediates
By reviewing the studies focused on antimycobacterial drug against Mycobacterium bovis BCG and the results have been
discovery and based on our previous experiences in this area, discussed in this paper.
we found that the structural features of the newly designed
An inherent issue associated to this type of approach is the
hydrazone derivatives, would give them the possibility to be fact that these compounds can show promiscuous activity for
considered as suitable candidates to be tested for their poten- both platelet and mycobacterium. Therefore, selectivity con-
tial antimycobacterial activity. The evidence comes from the siderations have to be addressed very early after the prelimi-
available background of hydrazone derivatives and specially nary activities are assessed.
(thio)semicarbazones in antitubercular drug discovery and
Chemistry The synthetic procedure is illustrated in Chart
drug therapy. Some thiosemicarbazone derivatives known 1. To prepare the desired derivatives, a one-pot procedure was
to possess antitubercular activity are shown in Fig. 3. The employed: the selected aldehydes were added to thiocarbohy-
mechanism of action of these compounds has not been firmly drazide (I) in glacial acetic acid as the solvent and within a
determined, but studies on thiacetazone suggests that this time interval triethyl orthoformate was added and the final
compound undergoes metabolic activation in Mycobacterium desired compounds were obtained upon heating the mixture
tuberculosis, producing reactive intermediate(s) that interfere for three hours.
with the synthesis of mycolic acid and hence integrity of the
In this synthetic procedure as shown in Chart 1, two main
steps are distinguishable: the first step is the formation of
monothiocarbohydrazone intermediates (2) resulted by react-
ing thiocarbohydrazide with equimolar amounts of desired
aldehydes; and the second step is the cyclization of monothio-
carbohydrazones in the presence of triethyl orthoformate lead-
ing to the compounds 3a–r.
cell wall.12–14)
In a general view, these derivatives are distinguished by
a thiosemicarbazone moiety linked via an imine bond to an
aromatic ring system. In this regard, the newly designed hy-
drazone derivatives could be considered as a cyclic version of
thiosemicarbazones with the thioamide moiety incorporated in
a thiadiazole ring (Fig. 4).
A new chemical entity, regardless of the rationale behind
its design and synthesis, could be considered as an unexplored
subject in medicinal chemistry that unanticipated biological
activities may be observed for it when tested against different
To ascertain the validity of the proposed intermediate 2,
in the case of compounds 3q and 3r, the primary reaction
mixture was added to water before the addition of triethyl or-
thoformate and the solid mass thus obtained was analyzed by
1H-NMR. In the NMR
spectroscopic methods such as IR and
biotargets. This is particularly beneficial in the early stages of spectra of these derivatives, the presence of D2O exchangeable
hit identification for those groups of diseases whose statuses peaks related to thiocarbohydrazone N–H protons and indole
are on WHO alert, such as Tuberculosis. Following nearly N–H and also the peaks assigned to imine and indole C–H

162
Vol. 61, No. 2
Fig. 7. (a) Compounds 3O (X=O) and 3P (X=S), (b) 2-Acetylthio-
Fig. 6. 1H-NMR Assignment of the Intermediate 2q Reported as Chem-
ical Shift (Multiplicity)
phene-2-thiazolylhydrazone,7) (c) LASSBio-2,11) (d) LASSBio-31611)
Table 1. Antiplatelet Aggregation Acvitiy of the Synthesized Derivatives
ADP
AA
Derivative
Ar
Inhibition (%)a,b)
IC50 (µm)
Inhibition (%)b)
IC50 (µm)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
Phenyl
2-Fluorophenyl
3-Fluorophenyl
4-Fluorophenyl
2-Chlorophenyl
3-Chlorophenyl
4-Chlorophenyl
2-Bromophenyl
3-Bromophenyl
4-Bromophenyl
2-Nitrophenyl
3-Nitrophenyl
4-Nitrophenyl
3-Pyridyl
32.1
45.8
38.4
46.6
ND
60.1
24.9
48.9
67.6
49.7
1.9
>100
>100
>100
>100
ND
81.92
>100
>100
95.23
>100
>100
>100
>100
18.1
>100
>100
34.2
>100
>100
>100
99.8
99.9
100.3
100.0
100.0
99.7
100.0
99.9
99.8
100.5
99.1
100.0
99.9
103.0
100.1
100.0
85.0
84.9
18.3
21.4
79.8
5.8
7.3
63.4
15.5
7.8
8.3
18.1
2.6
7.8
21.1
2.2
29.7
3.1
81.3
34.8
33.4
70.6
38.3
42.2c)
21.4c)
2-Furyl
2-Thienyl
3-Indolyl
3.8
51.9
96.2
3.0
3r
5-Bromo-3-indolyl
53.0
100.0
99.8
Indomethacin
Aspirin
30.3
a) All the values have been reported as mean. b) The derivatives were tested at 100µm concentration. c) Tested at 250µm.
protons confirms the formation of monothiocarbohydrazones 1H-NMR spectra, the singlet at 12.03–12.80ppm was assigned
of series 2 which were not isolated in the synthetic procedure to N–H proton, which was confirmed by D2O exchange exper-
of the other final derivatives (Fig. 6). Synthesis of the men- iments. Two singlet peaks were distinguishable in the spectra,
tioned monothiocarbohydrazones was also tried in ethanol/ one resonating at 8.76–8.87ppm and the other at 7.96–8.45.
water solution and few drops of glacial acetic acid and similar These two were assigned to thiadiazole H and imine H respec-
results were obtained, but preparation of the derivatives by the tively. The assignment of the remaining peaks of the spectra
described one-pot procedure proved to be more convenient appearing in the aromatic zone was straightforward because
and accompanied by higher yields ranging from 49 to 85%.
of their splitting patterns and coupling constants. In the
The structures of the final hydrazone derivatives 3a–r were 13C-NMR spectra the peaks at ca. 170ppm and ca. 144ppm
confirmed by different spectroscopic methods. IR, NMR and are characteristic for C-2 and C-5 of thiadiazole ring, since
MASS data and CHNS elemental analysis are in agreement these two peaks are observed in 2-amino-1,3,4-thiadiazole
with the proposed structures. In the IR spectra the N–H spectra (171ppm for C-2 and 144ppm for C-5).17)
stretch bond appeared as weak, vanishing and broad band in
Biological Activity Evaluation The in vitro antiplatelet
the range of 3350–3500cm−1 or was not observed at all. In the aggregation activity was evaluated using turbidimetric method
region between 1605–1620cm−1 the medium intensity bands reported by Born and Cross18) on an APACT 4004 aggregom-
were observed which could be assigned to the stretching of eter.
C=N bonds present in the synthesized derivatives. In the
The tests were performed using human platelet-rich plasma

February 2013
163
(PRP) and aggregation was induced by AA or ADP. Indo- ClogP and MIC values suggests an optimal ClogP for the
methacin and aspirin were used as standard drugs in this test. antitubercular activity of the studied derivatives, since the
The antimycobacterial activity of the compounds was eval- ClogP values calculated for the most potent derivatives range
uated by the broth microtiter dilution method19) against My- between approximately 3 to 4. This is in agreement with the
cobacterium bovis BCG (1173P2) and ethambutol and thiacet- observation that bromophenyl containing derivatives 3h–j
azone were used as standard drugs. The minimal inhibition (ClogP=4.0) and 5-bromoindolyl substituted 3r (ClogP=4.17)
concentration (MIC) was defined as the lowest concentration with higher lipophilic characters and compounds of the other
which could inhibit the bacterial growth.
extremity including 3n (ClogP=2.23) and 3o (ClogP=2.41)
fail to exert a satisfactory anti-tubercular activity. Analysis of
the other physicochemical parameters including surface area,
Results and Discussion
The antiplatelet aggregation activity of the derivatives is molecular volume, refractivity and polarizability did not lead
listed in Table 1. All the tested derivatives effectively inhib- to extracting a firm correlation between MICs and the men-
ited platelet aggregation higher than 50% at 100µm concentra- tioned parameters. This could be because of the complexity of
tion. The majority of the compounds showed lower IC50 values the action mechanism of the studied derivatives or the com-
than that of aspirin and among them, compounds 3l, 3o and plexity of the relationship between physicochemical param-
3p exhibited comparable IC50 values to that of indometha- eters and the observed activities, so that several parameters
cin, a potent inhibitor of the enzyme COX. The results show
that the tested compounds have a similar activity profile to
those of previously studied hydrazone derivatives, that is they
inhibited AA-induced platelet aggregation more effectively
than once ADP was used as inducer. However, compound 3n
showed satisfying activity with IC50 value of 18.1µm against
ADP-induced platelet aggregation; notably that this derivatives
proved to be effective to inhibit platelet aggregation induced
by both ADP and AA.
with different orders of strength may affect the biological
activity of the derivatives.
The synthesized derivatives showed promising antiplatelet
aggregation activity and also antimycobacterial activity. The
results obtained in this study gave us valuable clues to con-
duct our further works in antiplatelet and antimycobacterial
drug research, some of which are already in progress by our
group.
Of the most potent derivatives, compounds 3o and 3p with
IC50 values of 2.2µm and 3.8µm both contain five membered
Conclusion
This paper describes a class of novel aromatic aldehyde
electron-rich heterocyclic rings and this would suggest that 2-thiadiazolyl hydrazones designed by considering the struc-
the existence of these ring systems can be an important factor tural features of the previously reported hydrazone derivatives
affecting the antiplatelet activity. This has been confirmed by proved to be active inhibitors of platelet aggregation. The de-
the previous studies, as Coquelet et al. in their study found rivatives were prepared by a one-pot procedure and evaluated
2-acetylthiophene-2-thiazolylhydrazone as the most potent for their antiplatelet aggregation activity using AA and ADP
derivative exerting dual inhibition of COX/LOX.7) Compar- as aggregation inducers. The derivatives effectively inhibited
ing the structures of this compound and compound 3p shows platelet aggregation at 100µm concentration and some of them
that these two have a high extent of similarity; suggesting
that they also may share common mechanisms of action and
structure–activity relationships (SARs) (Fig. 7). This is also
in line with the findings of Barreiro’s group to discover some
derivatives with furan substituent as potent inhibitors of AA-
induced platelet aggregation.11)
Table 2. Inhibitory Activity and ClogP Values of the Synthesized De-
rivatives against Mycobacterium bovis BCG
MIC (µg/mL)
Derivatives
number
ClogPa)
24h
48h
Of the synthesized derivatives, compound 3n with dual
inhibition of ADP/AA-induced platelet aggregation and com-
pounds 3l, 3o and 3p as the most active inhibitors of AA-
induced platelet aggregation have molecular weights ranging
from 194 to 249 and ClogP values ranging from 2.23 to
3.18. Therefore the mentioned derivatives have ideal physi-
cochemical characteristics to be considered as starting points
for further lead optimization studies in the area of antiplatelet
aggregation drug research.
The anti-mycobacterial activitis and ClogP values of the
derivatives are listed in Table 2. The most potent derivatives
against Mycobacterium bovis BCG include 3g with 4-chloro-
phenyl, 3k with 2-nitrophenyl, 3p with 2-thienyl and 3q with
3-indolyl substituents, all with MIC value of 7.8µg/mL. The
derivatives containing 3-fluorophenyl 3c (MIC=15.62 µg/mL),
phenyl 3a (31.25 µg/mL) and 3-pyridyl 3n (46.87µg/mL) fall
into the lower levels with moderate activity. Among the most
potent derivatives, compounds 3p and 3q contain electron
rich heterocyclic rings in their structures while compounds 3g
and 3k contain carbocyclic rings with electron-withdrawing
substituents. Investigating the possible relationship between
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3.3
31.25
62.5
15.62
62.5
62.5
62.5
7.8
62.5
62.5
62.5
7.81
31.25
62.5
31.25
62.5
3.36
3.36
3.36
3.92
3.92
3.92
4.0
93.75
125
7.8
125
62.5
62.5
7.81
125
125
62.5
62.5
11.7
7.8
4.0
4.0
3.18
3.18
3.18
2.23
2.41
3.15
3.47
4.17
0.14
0.99
125
93.75
46.87
62.5
7.8
7.8
3r
250
0.75
<1.9
6.5% v/v
250
0.75
>1.9
Ethambutol
Thiacetazone
DMSO
6.5% v/v
a) Calculated from organic-chemistry.org.

164
Vol. 61, No. 2
exhibited inhibitory activities comparable to those of aspirin
and indomethacin.
Synthesis and study of anti platelet activity of 5-substituted tals (0.29g, 67%) mp 220–223°C. H-NMR (DMSO-d6) δ: 7.22
thiadiazole derivatives will provide valuable information about (1H, m, Ar H-5), 7.48 (3H, m, Ar H-2, 4, 6), 8.08 (1H, s, imine
the SAR of this group of compounds.
The derivatives were also tested for their antimycobacterial (DMSO-d6) δ: 112.1, 112.5, 116.0, 116.3, 122.6, 122.7, 130.7,
2-(2-(3-Fluorobenzylidene)hydrazinyl)-1,3,4-thiadiazole
(3c): Recrystallized from tetrahydrofuran (THF). White crys-
1
H), 8.83 (1H, s, thiadiazole H), 12.52 (s, 1H, N–H). 13C-NMR
activity and some showed activity with satisfying MICs.
130.9, 136.5, 136.7, 142.2, 144.3, 160.4, 164.3, 169.8. IR (KBr)
cm−1: 3219, 3112, 1620, 1596, 1461, 1433, 1274, 1147, 972,
879, 805, 694. ESI-MS m/z: 223 (M+H+). Anal. Calcd for
Experimental
General All the solvents and reagents were freshly used. C9H7FN4S: C, 48.64; H, 3.17; N, 25.21; S, 14.43. Found: C,
The reactions were monitored using TLC silica gel 60 F254 48.60; H, 3.16; N, 25.29; S, 14.40.
plates (Merck) with chloroform–methanol (20:1) as mobile
2-(2-(4-Fluorobenzylidene)hydrazinyl)-1,3,4-thiadiazole
phase. Melting points were obtained by an Electrothermal (3d): Recrystallized from THF. White crystals (0.25g,
9100 apparatus and are uncorrected. The Infrared spectra 57%) mp 245–248°C. 1H-NMR (DMSO-d6) δ: 7.27 (2H, t,
were recorded by a Perkin-Elmer 843 spectrometer with KBr J=8.80Hz, Ar H-2, 6), 7.70 (2H, dd, J=8.80Hz, J=5.64Hz,
as diluent. Proton nuclear magnetic resonance (1H-NMR) and Ar H-3, 5), 8.08 (1H, s, imine H), 8.82 (1H, s, thiadiazole H),
carbon nuclear magnetic resonance (13C-NMR) spectra were 12.40 (1H, s, N–H). 13C-NMR (DMSO-d6) δ: 115.6, 115.8,
obtained on a Bruker Avance DRX 500MHz spectrometer. 128.4, 130.6, 142.6, 143.9, 161.7, 163.6, 169.7. IR (KBr) cm−1:
The chemical shift values are reported as δ (ppm) in DMSO- 3196, 3127, 1622, 1596, 1457, 1301, 1147, 1036, 775. ESI-MS
d6 solution and tetramethylsilane (0.05% v/v) as internal m/z: 223 (M+H+). Anal. Calcd for C9H7FN4S: C, 48.64; H,
standard. Electron spray ionization mass spectra (ESI-MS) 3.17; N, 25.21; S, 14.43. Found: C, 48.52; H, 3.17; N, 25.25; S,
were obtained using Agilent 6410 Triple Quad. LC/MS. The 14.39.
elemental analysis for C, H, N and S was performed by a
2-(2-(2-Chlorobenzylidene)hydrazinyl)-1,3,4-thiadiazole
Costech model 4010 and the percentage values agreed with (3e): Recrystallized from ethyl acetate. White crystals (0.36g,
1
the proposed structures within
0.4% of the theoretical 75%) mp 223–226°C. H-NMR (DMSO-d6) δ: 7.41 (2H, m, Ar
values. The ClogP values were calculated from the server H-4, 5), 7.50 (1H, m, Ar H-3), 7.89 (1H, m, Ar H-6), 8.44 (1H,
tool available online at (www.organic-chemistry.org). Other s, imine H), 8.86 (1H, s, thiadiazole H), 12.61 (1H, s, N–H).
physicochemical parameters including surface area, molecular 13C-NMR (DMSO-d6) δ: 126.3, 127.5, 129.8, 130.9, 131.1,
volume, refractivity and polarizability were calculated by Hy- 132.3, 139.5, 144.7, 169.6. IR (KBr) cm−1: 3217, 1606, 1583,
perchem 8.0 software. Thiocarbohydrazide (1) was prepared 1451, 1430, 1150, 1031, 762. ESI-MS m/z: 239, 241 (M+H+).
by heating a mixture of carbon disulfide and aqueous solution Anal. Calcd for C9H7ClN4S: C, 45.29; H, 2.96; N, 23.47; S,
of hydrazine hydrate according to the known method.20)
13.43. Found: C, 45.14; H, 2.95; N, 23.48; S, 13.42.
General Procedure for the Synthesis of Hydrazones
2-(2-(3-Chlorobenzylidene)hydrazinyl)-1,3,4-thiadiazole
(3a–r) A mixture of thiocarbohydrazide (0.21g, 0.002mol), (3f): Recrystallized from ethyl acetate. White crystals (0.32g,
1
the appropriate aldehydes (0.002mol) and glacial acetic acid 68%) mp 199–202°C. H-NMR (DMSO-d6) δ: 7.44 (2H, m, Ar
(1mL) was heated for 20min, then triethyl orthoformate H-4, 5), 7.61 (1H, d, J=6.60Hz, Ar H-6), 7.68 (1H, s, Ar H-2),
(5mL) was added and the mixture was heated under reflux 8.07 (1H, s, imine H), 8.83 (1H, s, thiadiazole H), 12.54 (1H,
for 3h. The mixture was then cooled in an ice bath and the s, N–H). 13C-NMR (DMSO-d6) δ: 125.0, 125.6, 129.0, 130.6,
precipitates were filtered by vacuum. The solid thus obtained 133.6, 136.3, 142.0, 144.3, 169.8. IR (KBr) cm−1: 3186, 1607,
was recrystallized from an appropriate solvent to afford the 1589, 1451, 1428, 1149, 1042, 799, 690. ESI-MS m/z: 239, 241
title compounds.
2-(2-Benzylidenehydrazinyl)-1,3,4-thiadiazole (3a): Recrys- 23.47; S, 13.43. Found: C, 45.30; H, 2.96; N, 23.42; S, 13.45.
(M+H+). Anal. Calcd for C9H7ClN4S: C, 45.29; H, 2.96; N,
tallized from ethanol. White crystals (0.26g, 64%) mp
2-(2-(4-Chlorobenzylidene)hydrazinyl)-1,3,4-thiadiazole
209–212°C. IR (KBr) cm−1: 3179, 1605, 1574, 1449, 1439, 1422, (3g): Recrystallized from ethyl acetate. White crystals (0.28g,
1
1127, 1032, 774, 693. H-NMR (DMSO-d6) δ: 7.43 (3H, m, Ar 60%) mp 224-227°C. 1H-NMR (DMSO-d6) δ: 7.49 (2H, d,
H), 7.65 (2H, d, J=8.4Hz, Ar H), 8.09 (1H, s, imine H), 8.82 J=8.53Hz, Ar H-3, 5), 7.67 (2H, d, J=8.53Hz, Ar H-2, 6),
(1H, s, thiadiazole H), 12.39 (1H, s, N–H). 13C-NMR (DMSO- 8.08 (1H, s, imine H), 8.83 (1H, s, thiadiazole H), 12.48 (1H,
d6) δ: 126.3, 128.8, 129.5, 134.0, 143.6, 144.1, 169.8. ESI-MS s, N–H). 13C-NMR (DMSO-d6) δ: 127.9, 128.2, 128.8, 132.9,
m/z: 205 (M+H+). Anal. Calcd for C9H8N4S: C, 52.92; H, 3.95; 133.8, 142.3, 144.1, 169.7. IR (KBr) cm−1: 3204, 3113, 1615,
N, 27.43; S, 15.70. Found: C, 53.01; H, 3.95; N, 27.38; S, 15.72. 1602, 1493, 1460, 1150, 1090, 888. ESI-MS m/z: 239, 241 (M+
2-(2-(2-Fluorobenzylidene)hydrazinyl)-1,3,4-thiadiazole H+). Anal. Calcd for C9H7ClN4S: C, 45.29; H, 2.96; N, 23.47;
(3b): Recrystallized from ethanol. White crystals (0.25g, S, 13.43. Found: C, 45.18; H, 2.95; N, 23.40; S, 13.50.
1
57%) mp 215–218°C. H-NMR (DMSO-d6) δ: 7.27 (2H, 2nd
2-(2-(2-Bromobenzylidene)hydrazinyl)-1,3,4-thiadiazole
order triplet, Ar H-5, 6), 7.45 (1H, m, Ar H-4), 7.82 (1H, (3h): Recrystallized from ethyl acetate. White crystals (0.34g,
1
t, J=7.54Hz, Ar H-3), 8.09 (1H, s, imine H), 8.82 (1H, s, 60%) mp 220–223°C. H-NMR (DMSO-d6) δ: 7.33 (1H, dt,
thiadiazole H), 12.59 (1H, s, N–H). 13C-NMR (DMSO-d6) J=7.87Hz, J=1.7Hz, Ar H-4), 7.45 (1H, t, J=7.42Hz, Ar H-5),
δ: 115.8, 116.1, 121.5, 121.7, 124.8, 124.9, 125.8, 125.9, 131.2, 7.67 (1H, d, J=8.00Hz, Ar H-3), 7.87 (1H, dd, J=7.87Hz,
131.3, 136.1, 144.4, 158.1, 162.0, 169.7. IR (KBr) cm−1: 3187, J=1.7Hz, Ar H-6), 8.04 (1H, s, imine H), 8.86 (1H, s, thiadia-
1615, 1592, 1446, 1427, 1292, 1149, 1027, 753. ESI-MS m/z: zole H), 12.63 (1H, s, N–H). 13C-NMR (DMSO-d6) δ: 122.6,
223 (M+H+). Anal. Calcd for C9H7FN4S: C, 48.64; H, 3.17; N, 126.6, 128.0, 131.0, 132.7, 133.0, 141.7, 144.4, 169.6. IR (KBr)
25.21; S, 14.43. Found: C, 48.75; H, 3.16; N, 25.18; S, 14.45.
cm−1: 3164, 1592, 1566, 1422, 1148, 1025, 767. ESI-MS m/z:

February 2013
165
283, 285 (M+H+). Anal. Calcd for C9H7BrN4S: C, 38.18; H, d, J=4.65Hz, Pyr H-4), 8.80 (1H, s, Pyr H-2), 8.84 (1H, s,
2.49; N, 19.79; S, 11.32. Found: C, 38.11; H, 2.49; N, 19.81; S, thiadiazole H), 12.58 (1H, s, N–H). 13C-NMR (DMSO-d6) δ:
11.30.
123.9, 130.0, 132.8, 140.8, 144.4, 148.0, 150.1, 169.8. IR (KBr)
2-(2-(3-Bromobenzylidene)hydrazinyl)-1,3,4-thiadiazole cm−1: 3220, 1613, 1595, 1454, 1143, 1035, 814, 706. ESI-MS
(3i): Recrystallized from ethyl acetate. White crystals (0.38g, m/z: 206 (M+H+). Anal. Calcd for C8H7N5S: C, 46.82; H, 3.44;
67%) mp 213–216°C. 1H-NMR (DMSO-d6) δ: 7.39 (1H, t, N, 34.12; S, 15.62. Found: C, 46.74; H, 3.45; N, 34.08; S, 15.59.
J=7.87Hz, Ar H-5), 7.57 (1H, d, J=8.01Hz, Ar H-4), 7.65 (1H,
2-(2-(Furan-2-ylmethylene)hydrazinyl)-1,3,4-thiadiazole
d, J=7.78Hz, Ar H-6), 7.82 (1H, s, Ar H-2), 8.06 (1H, s, imine (3o): Recrystallized from aqueous ethanol. Yellow crystals
H), 8.83 (1H, s, thiadiazole H), 12.54 (1H, s, N–H). 13C-NMR (0.21g, 54%) mp 197–200°C (dec.). 1H-NMR (DMSO-d6)
(DMSO-d6) δ: 122.1, 125.3, 128.5, 130.8, 131.9, 136.5, 142.0, δ: 6.60 (1H, dd, J=3.40, 1.65Hz, furan H-4), 6.82 (1H, d,
144.2, 169.7. IR (KBr) cm−1: 3175, 1595, 1578, 1447, 1418, J=3.40Hz, furan H-3), 7.80 (1H, d, J=1.65Hz, furan H-5),
1137, 791, 682. ESI-MS m/z: 283, 285 (M+H+). Anal. Calcd 7.96 (1H, s, imine H), 8.79 (1H, s, thiadiazole H), 12.34 (1H,
for C9H7BrN4S: C, 38.18; H, 2.49; N, 19.79; S, 11.32. Found: C, s, N–H). 13C-NMR (DMSO-d6) δ: 112.1, 112.8, 133.9, 144.0,
38.20; H, 2.48; N, 19.73; S, 11.29.
144.7, 149.0, 169.6. IR (KBr) cm−1: 3184, 1619, 1590, 1446,
2-(2-(4-Bromobenzylidene)hydrazinyl)-1,3,4-thiadiazole 1034, 1020, 765, 682. ESI-MS m/z: 195 (M+H+). Anal. Calcd
(3j): Recrystallized from ethyl acetate. White crystals (0.28g, for C7H6N4OS: C, 43.29; H, 3.11; N, 28.85; S, 16.51. Found: C,
1
49%) mp 221–224°C. H-NMR (DMSO-d6) δ: 7.6 (2H, 2nd 43.19; H, 3.10; N, 28.86; S, 16.48.
order dd, Ar H-2, 3, 5, 6), 8.06 (1H, s, imine H), 8.83 (1H, s,
2-(2-(Thiophen-2-ylmethylene)hydrazinyl)-1,3,4-thiadiazole
thiadiazole H), 12.48 (1H, s, N–H). 13C-NMR (DMSO-d6) δ: (3p): Recrystallized from ethanol. Yellow crystals (0.32g,
122.6, 128.2, 131.7, 133.3, 142.4, 144.2, 169.7. IR (KBr) cm−1: 77%) mp 211–214°C (dec.). H-NMR (DMSO-d6) δ: 7.10 (1H,
1
3210, 1612, 1597, 1451, 1396, 1140, 1034, 800. ESI-MS m/z: dd, J=5.02, 3.60Hz, thiophene H-4), 7.38 (1H, d, J=3.60Hz,
283, 285 (M+H+). Anal. Calcd for C9H7BrN4S: C, 38.18; H, thiophene H-3), 7.60 (1H, d, J=5.02Hz, thiophene H-5), 8.27
2.49; N, 19.79; S, 11.32. Found: C, 38.09; H, 2.49; N, 19.75; S, (1H, s, imine H), 8.76 (1H, s, thiadiazole H), 12.37 (1H, s,
11.33.
N–H). 13C-NMR (DMSO-d6) δ: 127.8, 127.9, 129.5, 139.2,
2-(2-(2-Nitrobenzylidene)hydrazinyl)-1,3,4-thiadiazole (3k): 139.4, 143.7, 169.3. IR (KBr) cm−1: 3198, 1598, 1452, 1424,
Recrystallized from ethanol. Yellow crystals (0.38g, 77%) mp 1141, 1031, 720. ESI-MS m/z: 211 (M+H+). Anal. Calcd for
214–217°C. H-NMR (DMSO-d6) δ: 7.62 (1H, t, J=7.76Hz, Ar C7H6N4S2: C, 39.98; H, 2.88; N, 26.64; S, 30.50. Found: C,
H-4), 7.78 (1H, t, J=7.53Hz, Ar H-5), 7.99 (1H, d, J=7.88Hz, 39.86; H, 2.88; N, 26.66; S, 30.39.
1
Ar H-6), 8.03 (1H, d, J=8.17Hz, Ar H-3), 8.45 (1H, s, imine
2-(2-((1H-Indol-3-yl)methylene)hydrazinyl)-1,3,4-thiadiazole
H), 8.86 (1H, s, thiadiazole H), 12.74 (1H, s, N–H). 13C-NMR (3q): Recrystallized from ethanol. Yellow crystals (0.41g,
1
(DMSO-d6) δ: 124.6, 127.7, 128.2, 130.0, 133.5, 138.9, 144.8, 85%) mp 250–253°C (dec.). H-NMR (DMSO-d6) δ: 7.16–7.24
147.5, 169.7. IR (KBr) cm−1: 3202, 1611, 1593, 1575, 1519, 1421, (2H, m, indole H-5, 6), 7.46 (1H, d, J=7.6Hz, indole H-7),
1338, 1265, 1134, 750. ESI-MS m/z: 250 (M+H+). Anal. Calcd 7.80 (1H, s, imine H-1), 8.16 (1H, d, J=7.6Hz, indole H-4),
for C9H7N5O2S: C, 43.37; H, 2.83; N, 28.10; S, 12.86. Found: 8.32 (1H, s, indole H-2), 8.77 (1H, s, thiadiazole H), 11.58 (1H,
C, 43.40; H, 2.83; N, 28.11; S, 12.87.
s, NH), 12.03 (1H, s, NH). 13C-NMR (DMSO-d6) δ: 111.73,
2-(2-(3-Nitrobenzylidene)hydrazinyl)-1,3,4-thiadiazole (3l): 112.44, 121.15, 121.86, 123.27, 124.31, 130.12, 137.33, 142.16,
Recrystallized from 2-propanol. Yellow crystals (0.35g, 143.99, 170.49. IR (KBr) cm−1: 3183, 1581, 1418, 1106, 747.
70%) mp 221–224°C. 1H-NMR (DMSO-d6) δ: 7.72 (1H, t, ESI-MS m/z: 244 (M+H+). Anal. Calcd for C11H9N5S: C, 54.31;
J=7.98Hz, Ar H-5), 8.08 (1H, d, J=7.8Hz, Ar H-4), 8.20 (2H, H, 3.73; N, 28.79; S, 13.18. Found: C, 54.21; H, 3.72; N, 28.87;
d, J=8.40Hz, Ar H-6 and imine H), 8.45 (1H, s, Ar H-2), 8.84 S, 13.17.
(1H, s, thiadiazole H), 12.69 (1H, s, N–H). 13C-NMR (DMSO-
2-(2-((5-Bromo-1H-indol-3-yl)methylene)hydrazinyl)-1,3,4-
d6) δ: 120.4, 123.5, 130.3, 132.3, 135.9, 141.4, 144.4, 148.1, thiadiazole (3r): Recrystallized from ethanol. Purple crystals
1
169.8. IR (KBr) cm−1: 3108, 1585, 1522, 1447, 1350, 1149, (0.55g, 85%) mp 273°C (dec.). H-NMR (DMSO-d6) δ: 7.35
1032, 815, 745, 677. ESI-MS m/z: 250 (M+H+). Anal. Calcd for (1H, dd, J=8.8, 2Hz, indole H-6), 7.44 (1H, d, J=8.8Hz, in-
C9H7N5O2S: C, 43.37; H, 2.83; N, 28.10; S, 12.86. Found: C, dole H-7), 7.86 (1H, d, J=2.4Hz, imine H), 8.29 (1H, s, indole
43.28; H, 2.82; N, 28.01; S, 12.83.
H-4), 8.32 (1H, s, indole H-2), 8.80 (1H, s, thiadiazole H),
2-(2-(4-Nitrobenzylidene)hydrazinyl)-1,3,4-thiadiazole (3m): 11.74 (1H, s, NH), 12.11 (1H, s, NH). 13C-NMR (DMSO-d6)
Recrystallized from ethyl acetate. Yellow crystals (0.28g, δ: 111.44, 113.57, 11447, 124.28, 125.55, 126.11, 131.57, 136.23,
57%) mp 234°C (dec.). 1H-NMR (DMSO-d6) δ: 7.89 (2H, 141.38, 143.76, 170.25. IR (KBr) cm−1: 3203, 1611, 1538, 1276,
d, J=8.86Hz, Ar H-2, 6), 8.18 (1H, s, imine H), 8.27 (2H, 1116, 811. ESI-MS m/z: 322(97%), 324(100%) (M+H+). Anal.
d, J=8.86Hz, Ar H-3, 5), 8.87 (1H, s, thiadiazole H), 12.80 Calcd for C11H8BrN5S: C, 41.01; H, 2.50; N, 21.74; S, 9.95.
(1H, s, N–H). 13C-NMR (DMSO-d6) δ: 124.0, 127.1, 140.4, Found: C, 41.13; H, 2.50; N, 21.70; S, 9.97.
141.2, 144.7, 147.2, 169.8. IR (KBr) cm−1: 3094, 1607, 1570,
In Vitro Evaluation of Antiplatelet Aggregation Activity
1338, 1147, 851. ESI-MS m/z: 250 (M+H+). Anal. Calcd for The blood samples were obtained from healthy volunteers
C9H7N5O2S: C, 43.37; H, 2.83; N, 28.10; S, 12.86. Found: C, with negative history of smoking or taking any medications
43.31; H, 2.82; N, 28.05; S, 12.89.
since 14d prior to blood collection. To blood samples was
2-(2-(Pyridin-3-ylmethylene)hydrazinyl)-1,3,4-thiadiazole added trisodium citrate dihydrate 3.8% (1 part citrate, 9 part
(3n): Recrystallized twice from 2-propanol. Yellow crystals blood) and centrifuged at 1000rpm for 8min to obtain PRP.
(0.24g, 59%) mp 236–239°C (dec.). 1H-NMR (DMSO-d6) The remaining was centrifuged at 3000rpm for 15min and
δ: 7.45 (1H, dd, J=7.95, 4.65Hz, Pyr H-5), 8.05 (1H, td, PPP was collected from the above layer which was used as the
J=7.95, 1.9Hz, Pyr H-6), 8.12 (1H, s, imine H), 8.56 (1H, test blank. The platelet count was adjusted to 250000plts/mL

166
Vol. 61, No. 2
by diluting PRP with appropriate amount of PPP once needed.
Acknowledgment This work was partially supported by
Of the test compounds previously dissolved in dimethyl sulf- Shahid Beheshti University of Medical Sciences, Deputy of
oxide (DMSO), 1µL was added to 200µL of PRP and incubat- Research.
ed at 37°C for 5min. To induce platelet aggregation, a solution
of ADP (5µm) or arachidonic acid (1.25mg/mL) was added
to the samples and aggregation was measured using APACT
4004 aggregometer for a 5-minute period. DMSO (0.5% v/v)
was used as negative control and indomethacin and aspirin as
standard drugs. Platelet aggregation inhibition (%) was calcu-
lated by the following formula:
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