Synthesis of novel indole hydrazone derivatives and evaluation of their antiplatelet aggregation activity

Article abstract of DOI:10.1248/cpb.c12-00597

Based on the existing reports regarding the antiplatelet aggregation activity of hydrazone derivatives, a series of indole hydrazone derivatives were considered as potential antiplatelet agents and synthesized. The structures of the synthesized compounds

Full text of DOI:10.1248/cpb.c12-00597

144
Regular Article
Chem. Pharm. Bull. 61(2) 144–150 (2013)
Vol. 61, No. 2
Synthesis of Novel Indole Hydrazone Derivatives and Evaluation of Their
Antiplatelet Aggregation Activity
Vida Mashayekhi,a Kamaleddin Haj Mohammad Ebrahim Tehrani,a Salimeh Amidi,a and
,a,b
Farzad Kobarfard*
a Department of Medicinal Chemistry, School of Pharmacy, Shahid Beheshti University of Medical Sciences; and
b Phytochemistry Research Centre, Shahid Beheshti University of Medical Sciences; Vali Asr Ave. Niayesh Junction,
Tehran, P.O. Box 14155–6153, Iran.
Received July 8, 2012; accepted October 2, 2012; advance publication released online November 16, 2012
Based on the existing reports regarding the antiplatelet aggregation activity of hydrazone derivatives,
a series of indole hydrazone derivatives were considered as potential antiplatelet agents and synthesized.
The structures of the synthesized compounds were confirmed by spectral data and elemental analysis. The
new indole hydrazone derivatives were evaluated for their ability to inhibit platelet aggregation induced by
adenosine diphosphate (ADP) and arachidonic acid (AA). Compounds 1h and 3h exhibited remarkable activi-
ty against arachidonic acid induced platelet aggregation with IC50 values comparable to that of indomethacin
and compound 1i efficiently inhibited platelet aggregation induced by both ADP and AA.
Key words antiplatelet aggregation; indole; hydrazone
Thromboembolic disorders including cardiovascular and of antiplatelet derivatives which act in the arachidonic acid
cerebrovascular events continue to be the major causes of pathway probably by inhibition of platelet COX-1 enzyme.
death in the industrial countries and worldwide.1) In the Jordao described the synthesis of N-substituted-phenyl-1,2,3-
pathogenesis of thromboembolic disorders, platelets play the triazole-4-acylhydrazones with antiplatelet activity. These
major role. Activation of the platelets by a complex of bio- compounds inhibited arachidonic acid-induced platelet ag-
chemical pathways and multitude of mediators is the primary gregation in rabbit and human citrated platelet rich plasma.10)
step in the progress of these diseases.2) Of the physiological Studies also showed that hydrazone group may play an impor-
agonists of platelet aggregation with medical importance in- tant role for the antithrombotic activity.9,11–13)
clude arachidonic acid (AA) that is metabolized by platelet
Indole ring is another structural moiety which has been
cyclooxygenase-1 (COX-1) to different prostaglandins with reported to have antiplatelet aggregation activity. Indole-3-car-
inflammatory and platelet-aggregation properties; adenosine binol, a natural compound found in cruciferous vegetables,
diphosphate (ADP) that acts on purinergic receptors on the is known to have antiplatelet and antithrombotic activities in
platelet—known as P2Y receptors—and thrombin, a potent vitro and in vivo. Indole-3-carbinol has been shown to inhibit
agonist that forms stable platelet aggregates.2,3)
collagen-induced platelet aggregation in human platelet rich
By targeting the pathways in which the above mentioned plasma (PRP) in a dose-dependent manner. This compound
mediators are involved, antiplatelet drugs have been developed has also suppressed the death of mice with pulmonary throm-
to control the pathogenic activation of the platelets. Acetyl bosis induced by intravenous injection of collagen and epi-
salicylic acid (a nonselective irreversible COX inhibitor) and nephrine.14)
clopidogrel (an irreversible antagonist of P2Y12) are the most
Considering this background, in the present study, by
commonly prescribed oral antiplatelet drugs to date. However, molecular hybridization between indole and hydrazone moi-
use of these drugs is associated with a high risk of bleeding.4) eties, a series of indole hydrazone compounds have been
Also undesired complications of neutropenia and thrombotic designed and synthesized as potential antiplatelet aggregation
thrombocytopenic purpura (TTP) pose limitation on the use derivatives. In compounds of series 1 and 2, various hydra-
of clopidogrel.4,5) Besides, resistance to both drugs makes the zone-containing moieties are attached to C-3 of the indole or
antiplatelet therapy more complicated.6) Hence, research for 5-bromoindole ring respectively. In order to investigate the
novel antiplatelet drugs with improved safety and efficacy potential correlation between activity and positional isomer-
profiles is necessary.
ism in the derivatives of series 3, hydrazone-containing moi-
Hydrazones are an important class of compounds which eties are linked to C-2 of indole ring system in another series
have been demonstrated to possess antimicrobial, analgesic, of compounds. In addition to indole derivatives of series 1 to
antiinflamatory, anticonvulsant, antitubercular, antitumoral 3, in the search for new structure backbones with potential
and antiplatelet activities.7) Many evidences suggest that the antiplatelet aggregation activity some other new derivatives
hydrazone moiety possess a pharmacophoric character for the
inhibition of COX and thus antiplatelet aggregation activity.
Fraga et al., have reported a novel group of heterocyclic ac-
ylhydrazone derivatives with antiplatelet aggragation activity
on rabbit platelet-rich plasma.8) They also reported a group of
phenothiazine-1-acylhydrazone derivatives9) as new prototype
The authors declare no conflict of interest.
Fig. 1. Indole-3-carbinol
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: kobarfard@sbmu.ac.ir

February 2013
145
Reaction condition: reflux in ethanol and few drops of acetic acid.
Chart 1. Synthesis of Compounds of Series 1–4
have been synthesized in which a diphenylmethyl moiety has
replaced the indole moiety and is link to the hydrazone frag-
ment as in compounds of series 4.
Chemistry The general synthetic route is shown in
Chart 1. Final compounds were obtained by a Schiff base
formation reaction between selected aldehydes and hydrazine
derivatives. The aldehydes were commercially available and
purchased from Merck Chemicals or Sigma-Aldrich chemical
companies. 4-amino-1,2,4-triazole-5-one15) was prepared by
cyclization of carbohydrazide16) in triethyl orthoformate and
4-amino-1,2,4-triazole-5-thione17) was prepared by refluxing
thiocarbohydrazide18) in formic acid.
Fig. 2. The Structure Suggested for Compound 5
The Schiff base formation reactions were performed in eth- NH, two thioamide hydrogens and NH2 hydrogens in each
1
anol with a few drops of acetic acid. This reaction in the ma- case. The H- and 13C-NMR of 5, however, showed a different
1
jority of the cases was straight forward with acceptable yields. pattern. In the H-NMR spectrum of this compound a quartet
However, the reaction between 5-bromoindole-3-carbaldehyde exists in 4.5ppm that turns to doublet by addition of D2O,
and phenylhydrazine in ethanol was not productive and the while the singlet assigned to imine hydrogen in compounds
1
product apparently was tending to decompose so fast that the 1c, 2c and 3c is absent in the H-NMR of compound 5. In ad-
desired hydrazone was not obtainable. Similar results were dition, in 13C-NMR spectrum of this compound, one peak ex-
obtained by changing the solvent to acetic acid alone.
ists in 68.50ppm which is also absent in 13C-NMR spectrum
The derivatives 1c, 2c, 3c and 5 were obtained by the reac- of 1c, 2c and 3c. These data together suggest that the product
1
tion of thiocarbohydrazide with the desired aldehydes. The H- of the reaction of thiocarbohydrazide with diphenylacetalde-
and 13C-NMR data of compounds 1c, 2c and 3c show that the hyde could have a tetrazine structure as shown in Fig. 2. The
1
1
hydrazone derivatives are obtained. In the H-NMR spectrum quartet in 4.5ppm in H-NMR spectrum of this compound
of these derivatives, the existence of singlets at 8.0 to 8.4ppm can be assigned to C6–H and its multiplicity can be caused by
were assigned to imine hydrogen and existence of five D2O- coupling with N1,5–H and (ph)2C–H. Coupling of C6–H with
exchangeable hydrogens represents the corresponding indole N1,5–H is in line with the observation that addition of D2O

146
Vol. 61, No. 2
changes to quartet into a doublet. The peak in 68.50ppm in
13C-NMR confirms the existence of the aliphatic C-6.
In the case of the reactions between 4-amino-1,2,4-triazole-
5-one(thione) and the aldehydes, the reaction proceeded just
partly in ethanol and catalytic amount of acetic acid and ef-
forts to remove water from the reaction mixture to force the
reaction to completion, such as addition of sodium sulfate or
distilling off a portion of ethanol and adding a new portion
of absolute ethanol were unsuccessful. In another effort, the
solvent was switched to acetic acid which gave satisfactory
results and the products were obtained in excellent yields by
heating the starting materials in acetic acid for about 20min.
Chart 2. Preparation of Thiadiazole Derivatives 1i, 2i and 5i
essentiality of both hydrazone and indole moieties for exerting
The monothiocarbohydrazone derivatives (1c, 2c, 3c) and antiplatelet aggregation activity in the synthesized compounds
compound 5 were cyclized by heating the mentioned de- in the present study.
rivatives in triethyl orthoformate (Chart 2) which lead to the
Compounds 1i and 2i exhibited promising inhibitory activ-
formation of 1i, 2i and 4i. These three derivatives were suc- ity against both platelet aggregation agonists of ADP and AA.
cessfully prepared and their structures were ascertained by This can be considered as a beneficial property for the com-
1H- and 13C-NMR.
pounds designed as therapeutic agents for antiplatelet therapy.
In the 13C-NMR spectra of these three derivatives the C-2 It is worth to mention that most of the medications currently
of thiadiazole ring resonates at 170ppm which is characteristic used for antiplatelet therapy suffer from limited mode of ac-
for C-2 of N-substituted 2-aminothiadiazoles.19,20)
tion and efficacy, which translates to longer time course of
Biological Activity Evaluation In vitro platelet aggrega- drug therapy and use of regimens consisting of multiple drugs.
tion was evaluated by turbidimetric method reported by Born
The two most potent compounds, i.e. 1h and 3h, both have
and Cross21) using APACT 4004 aggregometer. All the synthe- phenylhydrazone moiety on the indole ring. Therefore it could
sized compounds were tested for their ability to inhibit platelet be speculated that the interaction with this aromatic unit may
aggregation of human platelet-rich plasma (PRP) with AA or play an important role in improving the anti platelet activity
ADP as potent aggregation inducers, and indomethacin and perhaps by molecular recognition through a π-stacking with
aspirin were used as positive controls.
the planar phenyl ring.
Replacing the indole ring with a diphenylmethyl unit (se-
ries 4) had been detrimental to anti platelet activity except
Results and Discussion
Antiplatelet aggregation activity of the synthesized com-
=44µm) in which the phenylhydrazone
for compound 4f (IC50
pounds is listed in Table 1. All the derivatives were initially moiety has been switched to a 4-pyridoylhydrazone. Further
tested at 250µm and IC50 was measured for those with more studies on this type of compounds may lead to a new group of
than 50% platelet aggregation inhibitory activity in the initial anti platelet agents.
concentration of 250µm.
The most potent antiplatelet aggregation derivatives in the
Among the tested derivatives, phenyl hydrazones of indole- present study i.e. compounds 1h, 3h and 1i have an excellent
2-carbaldehyde and indole-3-carbaldehyde (compounds 1h, potential to be developed as lead compounds to achieve anti-
3h) were the most potent derivatives, inhibiting platelet ag- platelet derivatives with optimized pharmacological and phar-
gregation induced by AA with IC50 values of 10µm and 3µm maceutical properties and further studies on these derivatives
respectively. These IC50 values are comparable to that of indo- are ongoing in our laboratory.
methacin (3µm), a well known nonselective inhibitor of cyclo-
oxygenase. Compounds 1i (IC50=34µm) and 1b (IC50=66µm)
exhibited the highest inhibitory activity against platelet aggre-
gation induced by ADP.
Conclusion
In the present study, some hydrazone derivatives of indole
were synthesized and their ability to inhibit platelet aggrega-
Among the four series of derivatives, hydrazone deriva- tion induced by ADP and AA were evaluated. Among the
tives of indole-3-carbaldehyde (series 1) were the most ac- tested compounds hydrazone derivatives of indole-3-carbal-
tive compounds. Replacement of H-5 of indole with Br (as dehyde and indole-2-carbaldehyde efficiently inhibited platelet
in compounds of series 2) was accompanied by a significant aggregation induced by AA. Compound 1i was successful in
fall in the antiplatelet aggregation activity. This may be due inhibition of platelet aggregation induced by both ADP and
to steric, lipophilic or positional effect of Br and in order to AA. In general, compounds of series 1 which are hydrazone
clarify which one of the mentioned effects is responsible for derivatives of indole-3-carbaldehyde were the most active
the decrease in the activity further complementary studies on derivatives with antiplatelet aggregation potentials. Further
the new derivatives of indole is being undertaken in our lab. studies based on the results of the current study are ongoing
Considering compound 3h as an exception, hydrazone deriva- in our laboratory to obtain more efficient antiplatelet aggrega-
tives of indole-2-carbaldehyde (series 3) exhibited weaker an- tion derivatives with the potential to be a lead compound for
tiplatelet aggreration activity in comparison to the derivatives antiplatelet drug development.
of series 1. This finding contains primary clues to the effect of
the steric relationship between hydrazone and indole moieties
on the antiplatelet activity of the tested derivatives. In addi-
Experimental
General Compounds 1a,22) 1b,23) 1h,24) 1f,25) 4a,26) 3b,23)
tion, replacing indole with diphenyl moiety leads to significant 3f, 3g, 3h27) and 4h28) are known compounds and synthesis
decreased antiplatelet aggregation activity. This suggests the and spectral data of them are available in the literature. Melt-

February 2013
147
Table 1. Antiplatelet Aggregation Activity of the Synthesized Derivatives
ADP
AA
Derivative
Inhibition %a) in 250µm
IC50 (µm)a)
Inhibition %a) in 250µm
IC50 (µm)a)
1a
1b
1c
1d
1e
1f
1g
1h
1i
2a
2b
2c
2d
2e
2f
2g
2i
3b
3c
3e
3f
3g
3h
4a
4b
4c
4f
4g
4h
74 2.3
70 0.9
41 1.0
11 0.9
17 1.4
154 3.7
66 2.1
>250
>250
>250
>250
>250
250
93 1.4
90 2.3
92 1.0
194 5.8
140 3.9
192 7.1
>250
>250
220 5.8
8
5
1.1
1.0
4
1.1
83 2.3
0.7
0
6
>250
50 0.6
95 3.3
30 3.3
58 1.0
27 0.6
0
80 0.4
99 0.0
89 2.1
91 1.4
0
10 0.6
52 1.7
158 4.0
174 4.6
>250
>250
>250
>250
>250
96 2.5
>250
>250
>250
>250
>250
34 2.4
>250
135 3.4
>250
>250
>250
>250
>250
166 5.1
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
959 7.2
0
8
0.8
49 0.8
1.1
8
1.5
20 0.8
1.0
8
4
60 1.2
19 0.7
39 1.0
0
81 2.3
27 0.4
7
0.7
0
9
0.3
10 0.4
40 1.3
93 4.2
0
28 0.9
0
3
0.21
3
5
8
0.7
0.6
0.9
>250
>250
>250
44 2.3
>250
14 0.7
41 2.6
33 1.4
20 1.4
21 1.6
16 0.5
42 1.1
21 0.6
—
92 1.2
11 0.8
82 1.0
181 9.2
>250
4i
8
1.7
Indomethacin
Aspirin
Indole-3-carbinolb)
100
100
—
3
0.2
30 2.6
>1000
a) Values are presented as mean S.E. of three separate determination. b) IC50 against collagen was 827 12µm.
ing points were determined using an Electrothermal 9100 (1H, s, D2O exchangeable, NH), 11.19 (1H, s, D2O exchange-
1
model apparatus. H-NMR spectra were taken on a Bruker able, NH), 11.60 (1H, s, D2O exchangeable, NH); 13C-NMR
400MHz in DMSO-d6 with tetramethylsilane (TMS) as in- (DMSO-d6, 100MHz) δ: 111.42, 112.24, 121.19, 122.45,
ternal standard. 13C-NMR spectra were taken with the same 123.27, 124.52, 131.10, 137.21, 141.66, 175.86; IR (KBr): 3383,
spectrometer at 100MHz. IR spectra were obtained with 3289, 3171, 1620, 1553, 1127, 998, 753cm−1; Anal. Calcd for
Perkin-Elmer 843 spectrophotometer employing KBr as dilu- C10H11N5S (233.29): C, 51.48; H, 4.75; N, 30.02; S, 13.74.
ent. Electrospray ionization (ESI)-MS spectra were obtained Found: C, 51.54; H, 4.74; N, 29.94; S, 13.70.
by Agilent 6410 Triple Quad. LC-MS. All the compounds
were analyzed for C, H, N and S on a Costech model 4010 White powder, yield (72%) mp 288–290°C. H-NMR (DMSO-
Indole-3-carbaldehyde p-Hydroxybenzoylhydrazone (1g):
1
and agreed with the proposed structures within 0.4% of the d6, 400MHz) δ: 6.88 (2H, d, J=8.4Hz, phenyl H-3,5),
theoretical values.
7.14–7.23 (2H, m, indole H-5,6), 7.45 (1H, d, J=7.6Hz, indole
General Procedure for Synthesis of Hydrazones Deriva- H-7), 7.81 (1H, s, indole H-2), 7.84 (2H, d, J=8.4Hz, phenyl
tives In a 50mL flask equipped with a magnetic stirrer, hy- H-2,6), 8.32 (1H, d, J=7.2Hz, indole H-4), 8.62 (1H, s, azome-
drazide derivatives (2mmol) and aldehydes (2mmol) were dis- thine H), 10.07 (1H, s, D2O exchangeable), 11.34 (1H, s, D2O
solved in ethanol (20mL) and acetic acid (0.4mL). The mixure exchangeable), 11.57 (1H, s, D2O exchangeable); 13C-NMR
was heated under reflux for 4h, then cooled and filtered. The (DMSO-d6, 100MHz) δ: 112.13, 112.26, 115,45, 120.96,
resulting solid was recrystallized from ethanol to yield title 122.40, 123.21, 124.70, 124.74, 129.91, 130.29, 137.24, 145.00,
compounds.
160.61, 163.05; IR (KBr): 3321, 1615, 1589, 1268, 739cm−1;
Indole-3-carbaldehyde Thiocarbohydrazone (1c): Pale yel- MS (ESI) m/z: 280 (M+H+), 302 (M+Na+); Anal. Calcd for
1
low powder, yield (91%) mp 205–208°C. H-NMR (DMSO-d6, C16H13N3O2 (279.29): C, 68.81; H, 4.69; N, 15.05. Found: C,
400MHz) δ: 4.85 (2H, s, D2O exchangeable, NH2), 7.15 (1H, 68.99; H, 4.70; N, 15.01.
t, J=7.2Hz, indole H-6), 7.21 (1H, t, J=7.4Hz, indole H-5),
5-Bromoindole-3-carbaldehyde Semicarbazone (2a): White
7.44 (1H, d, J=8Hz, indole H-7), 7.77 (1H, s, indole H-2), 8.23 powder, yield (32%) mp 220–223°C; 1H-NMR (DMSO-d6,
(1H, d, J=8Hz, indole H-4), 8.26 (1H, s, azomethine H), 9.09 400MHz) δ: 6.30 (2H, s, D2O exchangeable, NH2), 7.31 (1H,

148
Vol. 61, No. 2
d, J=8.8Hz, indole H-6), 7.40 (1H, d, J=8.4Hz, indole H-7), (M+Na+); Anal. Calcd for C16H12BrN3O2 (358.19): C, 53.65; H,
7.78 (1H, d, J=2Hz, indole H-4), 8.08 (1H, s, azomethine H), 3.38; N, 11.73. Found: C, 53.80; H, 3.38; N, 11.70.
8.27 (1H, s, indole H-2), 9.99 (1H, s, D2O exchangeable, NH),
Indole-2-carbaldehyde Thiocarbohydrazone (3c): Yellow
11.66 (1H, s, D2O exchangeable, NH); 13C-NMR (DMSO-d6, powder, yield (37%) mp 214–217°C. 1H-NMR (DMSO-d6,
100 MHz) δ: 111.57, 113.53, 114.28, 123.90, 125.60, 126.05, 400MHz) δ: 4.90 (2H, s, D2O exchangeable, NH2), 6.77 (1H,
130.69, 135.94, 138.31, 157.52; IR (KBr): 3430, 3379, 3332, s, indole H-3), 7.02 (1H, t, J=7.6Hz, indole H-5), 7.19 (1H,
1642, 1609, 1407, 1100, 806cm−1; MS (ESI) m/z: 281 (100%), t, J=7.6Hz, indole H-6), 7.37 (1H, d, J=8Hz, indole H-4),
283 (100%) (M+H+); Anal. Calcd for C10H9BrN4O (281.11): C, 7.56 (1H, d, J=8Hz, indole H-7), 8.00 (1H, s, azomethine
42.73; H, 3.23; N, 19.93. Found: C, 42.82; H, 3.22; N, 19.98.
H), 9.90 (1H, s, D2O exchangeable, NH), 11.47 (1H, s, D2O
5-Bromoindole-3-carbaldehyde Thiosemicarbazone (2b): exchangeable, NH), 11.65 (1H, s, D2O exchangeable, indole
Pale yellow powder, yield (88%) mp 240–243°C. 1H-NMR NH); 13C-NMR (DMSO-d6, 100MHz) δ: 106.33, 111.53,
(DMSO-d6, 400MHz) δ: 7.35 (1H, dd, J=8.6, 2Hz, indole 119.92, 121.39, 123.92, 128.47, 133.41, 134.42, 137.55, 177.09;
H-6), 7.42 (1H, d, J=8.6Hz, indole H-7), 7.56 (1H, s, D2O ex- IR (KBr): 3400, 3278, 3171, 1597, 1509, 1006, 806cm−1; Anal.
changeable, NH2), 7.89 (1H, d, J=2Hz, indole H-4), 8.05 (1H, Calcd for C10H11N5S (233.07): C, 51.48; H, 4.75; N, 30.02; S,
s, D2O exchangeable, NH2), 8.29 (1H, s, azomethine H), 8.33 13.74. Found: C, 51.33; H, 4.76; N, 30.12; S, 13.77.
(1H, d, J=1.2Hz, indole H-2), 11.21 (1H, s, D2O exchange-
Diphenylacetaldehyde Thiosemicarbazone (4b): White
able, NH), 11.81 (1H, s, D2O exchangeable, NH); 13C-NMR powder, yield (89%) mp 90–93°C. 1H-NMR (DMSO-d6,
(DMSO-d6, 100MHz) δ: 111.07, 113.96, 114.35, 124.15, 125.90, 400MHz) δ: 4.93 (1H, d, J=7.6Hz, aliphatic H), 7.38–7.22
125.93, 132.51, 136.04, 141.30, 176.68; IR (KBr): 3472, 3342, (10H, m, phenyl H), 7.48 (1H, s, D2O exchangeable, NH), 7.91
3218, 1630, 1592, 1127, 808cm−1; Anal. Calcd for C10H9BrN4S (1H, d, J=7.6Hz, azomethine H), 8.15 (1H, s, D2O exchange-
(297.17): C, 40.42; H, 3.05; N, 18.85; S, 10.79. Found: C, 40.54; able, NH), 11.26 (1H, s, D2O exchangeable, NH); 13C-NMR
H, 3.06; N, 18.81; S, 10.76.
(DMSO-d6, 100MHz) δ: 53.34, 127.41, 128.58, 129.28, 141.40,
5-Bromoindole-3-carbaldehyde Thiocarbohydrazone (2c): 148.00, 178.11; IR (KBr): 3252, 3150, 1594, 753, 696cm−1; MS
Pale yellow powder, yield (64%) mp 215–218°C. 1H-NMR (ESI) m/z: 279 (M+H+); Anal. Calcd for C15H15N3S (269.36):
(DMSO-d6, 400MHz) δ: 4.87 (2H, s, D2O exchangeable, NH2), C, 66.88; H, 5.61; N, 15.60; S, 11.90. Found: C, 66.74; H, 5.62;
7.32 (1H, d, J=8.8Hz, indole H-6), 7.40 (1H, d, J=8.4Hz, in- N, 15.62; S, 11.93.
dole H-7), 7.89 (1H, s, indole H-2), 8.25 (1H, s, indole H-4),
6-Benzhydryl-1,2,4,5-tetrazinane-3-thione (5): White pow-
8.44 (1H, s, azomethine H), 9.40 (1H, s, D2O exchangeable, der, yield (88%) mp 196–199°C. 1H-NMR (DMSO-d6,
NH), 11.18 (1H, s, D2O exchangeable, NH), 11.79 (1H, s, D2O 400MHz) δ: 3.85 (1H, d, J=10Hz, benzylic H), 4.52 (1H,
exchangeable, indole NH); 13C-NMR (DMSO-d6, 100MHz) δ: quart, J=10Hz, tetrazine H-6), 4.75 (2H, d, J=10Hz, D2O
111.38, 113.81, 114.16, 124.64, 125.75, 126.02, 132.55, 136.22, exchangeable, NH) 7.26 (10H, m, phenyl H), 9.19 (2H, s,
140.87, 176.29; IR (KBr): 3398, 3149, 1604, 1546, 1258, D2O exchangeable, thioamide NH); 13C-NMR (DMSO-d6,
794cm−1; MS (ESI) m/z: 312(95%), 314(100%) (M+H+), 334 100 MHz) δ: 52.41, 68.50, 126.79, 128.53, 128.80, 142.26,
(100%), 336 (100%) (M+Na+); Anal. Calcd for C10H10BrN5S 172.45; IR (KBr): 3302, 3164, 1545, 1407, 906, 751, 706cm−1;
(312.19): C, 38.47; H, 3.23; N, 22.43; S, 10.27. Found: C, 38.58; Anal. Calcd for C15H16N4S (284.38): C, 63.35; H, 5.67; N,
H, 3.22; N, 22.39; S, 10.30.
19.70; S, 11.28. Found: C, 63.30; H, 5.68; N, 19.72; S, 11.29.
5-Bromoindole-3-carbaldehyde Isonicotinoylhydrazone (2f):
Diphenylacetaldehyde Isonicotinoylhydrazide (4f): White
Yellow powder, yield (74%) mp 304°C (dec.). 1H-NMR powder, yield (88%) mp 140–143°C. 1H-NMR (DMSO-d6,
(DMSO-d6, 400MHz) δ: 7.36 (1H, d, J=8.2Hz, indole H-6), 400MHz) δ: 5.07 (1H, d, J=7.4Hz, aliphatic H), 7.27–7.41
7.45 (1H, d, J=8.2Hz, indole H-7), 7.85 (2H, d, J=5.4Hz, (10H, m, phenyl H), 7.79 (2H, d, J=5Hz, pyr H-3,5), 8.30 (1H,
pyr H-3,5), 7.96 (1H, s, indole H-4), 8.47 (1H, s, indole d, J=7.4Hz, azomethine H), 8.77 (2H, d, J=5Hz, pyr H-2,6),
H-2), 8.62 (1H, s, azomethine H), 8.80 (2H, d, J=5.4Hz, pyr 11.86 (1H, s, D2O exchangeable, NH); 13C-NMR (DMSO-d6,
H-2,6), 11.80 (1H, s, D2O exchangeable, NH), 11.84 (1H, s, 100 MHz) δ: 53.60, 122.01, 127.51, 128.59, 129.33, 140.76,
D2O exchangeable, NH); 13C-NMR (DMSO-d6, 100MHz) δ: 141.28, 150.59, 154.80, 162.46; IR (KBr): 3221, 1663, 1547,
111.44, 113.83, 114.47, 122.00, 124.50, 125.86, 126.30, 132.50, 772, 695cm−1; MS (ESI) m/z: 316 (M+H+), 354 (M+K+);
136.05, 141.35, 146.38, 150.66, 161.62; IR (KBr): 3137, 1665, Anal. Calcd for C19H17N3 (287.36): C, 79.41; H, 5.96; N, 14.62.
1618, 1413, 792cm−1; MS (ESI) m/z: 323 (100%), 325 (100%) Found: C, 79.25; H, 5.97; N, 14.65.
(M+H+); Anal. Calcd for C15H11BrN4O (343.18): C, 52.50; H,
3.23; N, 16.33. Found: C, 52.63; H, 3.22; N, 16.29.
Diphenylacetaldehyde p-Hydroxybenzoylhydrazone (4g):
White powder, yield (98%) mp 244–247°C. H-NMR (DMSO-
1
5-Bromoindole-3-carbaldehyde p-Hydroxybenzoylhydrazone d6, 400MHz) δ: 5.01 (1H, d, J=7.4Hz, aliphatic H), 6.85 (1H,
1
(2g): White powder, yield (54%) mp 240°C (dec.). H-NMR d, J=8.4Hz, phenyl H-3′,5′), 7.39–7.25 (10H, m, phenyl H),
(DMSO-d6, 400MHz) δ: 6.90 (2H, d, J=8.4Hz, phenyl H-3,5), 7.77 (1H, d, J=8.4Hz, phenyl H-2′,6′), 8.26 (1H, d, J=7.4Hz,
7.34 (1H, d, J=8.8Hz, indole H-6), 7.43 (1H, d, J=8.8Hz, azomethine H), 10.12 (1H, s, D2O exchangeable), 11.42 (1H, s,
indole H-7), 7.85 (2H, d, J=8.4Hz, phenyl H-2,6), 7.88 (1H, D2O exchangeable); 13C-NMR (DMSO-d6, 100MHz) δ: 53.58,
s, indole H-4), 8.51 (1H, s, azomethine H), 8.62 (1H, s, in- 115.44, 124.07, 127.33, 128.64, 129.26, 130.03, 141.78, 152.26,
dole H-2), 10.01 (1H, s, D2O exchangeable), 11.43 (1H, s, D2O 160.93, 163.31; IR (KBr): 3257, 1634, 1611, 741, 691cm−1; MS
exchangeable), 11.77 (1H, s, D2O exchangeable); 13C-NMR (ESI) m/z: 331 (M+H+); Anal. Calcd for C21H18N2O2 (330.38):
(DMSO-d6, 100MHz) δ: 111.83, 113.64, 114.31, 115.48, 124.55, C, 76.34; H, 5.49; N, 8.48. Found: C, 76.15; H, 5.51; N, 8.50.
124.64, 125.70, 126.37, 129.94, 131.58, 136.01, 144.46, 160.71,
General Procedure for Preparation of Schiff Bases
163.07; IR (KBr): 3325, 1605, 1279, 805cm−1; MS (ESI) m/z: of 4-Amino-1H-1,2,4-triazole-5(4H)-thione Derivatives
358 (100%), 360 (90%) (M+H+), 380 (100%), 382 (100%) 4-Amino-1,2,4-triazole-5(4H)-thione (1mmol) and appropriate

February 2013
149
aldehydes were added to acetic acid (2mL) and the mixture s, D2O exchangeable, NH); 13C-NMR (DMSO-d6, 100MHz) δ:
was heated under reflux for 30min. Then mixture was cooled, 112.41, 112.93, 120.58, 122.19, 125.56, 127.76, 131.20, 139.02,
filtered and washed with cold water.
139.12, 154.28, 162.98; IR (KBr): 3300, 1607, 1558, 953,
4-((1H-Indol-3-yl)methyleneamino)-1H-1,2,4-triazol-5(4H)- 771 cm−1; MS (ESI) m/z: 236 (M+H+), 266 (M+Na+); Anal.
one (1d): Pale yellow powder, yield (66%). mp 314–317°C. Calcd for C11H9N5S (243.29): C, 54.31; H, 3.73; N, 28.79; S,
1H-NMR (DMSO-d6, 400MHz) δ: 7.24 (2H, m, indole H-5,6), 13.18. Found: C, 54.19; H, 3.72; N, 28.87; S, 13.14.
7.50 (1H, d, J=8Hz, indole H-7), 8.02 (1H, s, indole H-2), 8.21
General Procedure for Cyclization of Monothiocarbohy-
(1H, d, J=7.6Hz, indole H-4), 8.35 (1H, s, azomethine H), drazones Monothiocarbohydrazones (2mmol) were heated
9.53 (1H, s, triazole H), 11.85 (1H, s, D2O exchangeable, NH), under reflux in triethylorthoformate (15mL) for 3h and then
11.97 (1H, s, D2O exchangeable, NH); 13C-NMR (DMSO-d6, cooled in an ice bath and filtered. The crude was recrystal-
100 MHz) δ: 111.08, 112.64, 121.49, 122.39, 123.48, 124.46, lized from ethanol to afford the desired products.
133.85, 136.78, 137.69, 151.68, 153.01; IR (KBr): 3270, 1713,
2-(2-((1H-Indol-3-yl)methylene)hydrazinyl)-1,3,4-thiadiazole
1670, 1572, 930, 764cm−1; MS (ESI) m/z: 228 (M+H+), 250 (1i): Yellow powder, yield (85%) mp 250–253°C (dec.).
(M+Na+); Anal. Calcd for C11H9N5O (227.22): C, 58.14; H, 1H-NMR (DMSO-d6) δ: 7.16–7.24 (2H, m, indole H-5,6), 7.46
3.99; N, 30.82. Found: C, 58.32; H, 3.98; N, 30.89.
(1H, d, J=7.6Hz, indole H-7), 7.80 (1H, s, imine H-1), 8.16
4-((1H-Indol-3-yl)methyleneamino)-1H-1,2,4-triazole-5(4H)- (1H, d, J=7.6Hz, indole H-4), 8.32 (1H, s, indole H-2), 8.77
thione (1e): White powder, Yield (70%) mp 250–253°C. (1H, s, thiadiazole H), 11.58 (1H, s, NH), 12.03 (1H, s, NH);
1H-NMR (DMSO-d6, 400MHz) δ: 7.22–7.31 (2H, m, in- 13C-NMR (DMSO-d6) δ: 111.73, 112.44, 121.15, 121.86, 123.27,
dole H-5,6), 7.53 (1H, d, J=7.6Hz, indole H-7), 8.10 (1H, d, 124.31, 130.12, 137.33, 142.16, 143.99, 170.49. IR (KBr): 3183,
J=2.8Hz, indole H-1), 8.27 (1H, d, J=7.6Hz, indole H-4), 1581, 1418, 1106, 747cm−1; MS (ESI) m/z: 244 (M+H+); Anal.
8.88 (1H, s, azomethine H), 9.38 (1H, s, triazole H), 12.03 Calcd for C11H9N5S: C, 54.31; H, 3.73; N, 28.79; S, 13.18.
(1H, s, D2O exchangeable, NH), 13.84 (1H, s, D2O exchange- Found: C, 54.21; H, 3.72; N, 28.87; S, 13.17.
able, NH); 13C-NMR (DMSO-d6, 100MHz) δ: 110.45, 112.81,
2-(2-((5-Bromo-1H-indol-3-yl)methylene)hydrazinyl)-1,3,4-
121.93, 122.61, 123.78, 124.62, 135.38, 137.76, 139.06, 159.28, thiadiazole (2i): Purple powder, yield (85%) mp 273°C
162.69; IR (KBr): 3103, 1600, 1549, 1110, 947, 765cm−1; (dec.). 1H-NMR (DMSO-d6) δ: 7.35 (1H, dd, J=8.8, 2Hz,
MS (ESI) m/z: 244 (M+H+), 266 (M+Na+); Anal. Calcd indole H-6), 7.44 (1H, d, J=8.8Hz, indole H-7), 7.86 (1H,
for C11H9N5S (243.29): C, 54.31; H, 3.73; N, 28.79; S, 13.18. d, J=2.4Hz, imine H), 8.29 (1H, s, indole H-4), 8.32 (1H, s,
Found: C, 54.20; H, 3.73; N, 28.85; S, 13.17.
indole H-2), 8.80 (1H, s, thiadiazole H), 11.74 (1H, s, NH),
4-((5-Bromo-1H-indol-3-yl)methyleneamino)-1H-1,2,4- 12.11 (1H, s, NH); 13C-NMR (DMSO-d6) δ: 111.44, 113.57,
triazol-5(4H)-one (2d): White powder, yield (83%) mp 315°C 11447, 124.28, 125.55, 126.11, 131.57, 136.23, 141.38, 143.76,
(dec). 1H-NMR (DMSO-d6, 400MHz) δ: 7.38 (1H, d, J=8.4Hz, 170.25. IR (KBr): 3203, 1611, 1538, 1276, 1116, 811cm−1; MS
indole H-6), 7.48 (1H, d, J=8.4Hz, indole H-7), 8.08 (1H, s, (ESI) m/z: 322 (97%), 324 (100%) (M+H+). Anal. Calcd for
azomethine H), 8.34 (1H, s, indole H-2), 8.41 (1H, s, indole C11H8BrN5S: C, 41.01; H, 2.50; N, 21.74; S, 9.95. Found: C,
H-4), 9.52 (1H, s, triazole H), 11.99 (1H, s, D2O exchange- 41.13; H, 2.50; N, 21.70; S, 9.97.
able, NH), 12.03 (1H, s, D2O exchangeable, NH); 13C-NMR
2-(2-(2,2-Diphenylethylidene)hydrazinyl)-1,3,4-thiadiazole
1
(DMSO-d6, 100MHz) δ: 110.45, 114.28, 114.72, 124.32, 125.99, (5i): Yellow powder, yield (51%) mp 207°C (dec.). H-NMR
126.16, 134.81, 136.15, 136.20, 151.48, 152.82; IR (KBr): 3219, (DMSO-d6, 400MHz) δ: 4.99 (d, J=7.2Hz, 1H, aliphatic H),
1704, 1598, 1442, 937, 716cm−1; MS (ESI) m/z: 306 (100%), 7.42–7.25 (m, 10H, phenyl H), 7.89 (s, 1H, thiadiazole H), 8.76
308 (100%) (M+H+); Anal. Calcd for C11H8BrN5O (306.12): C, (d, J=7.2Hz, 1H, azomethine H), 12.07 (s, 1H, D2O exchange-
43.16; H, 2.63; N, 22.88. Found: C, 43.30; H, 2.62; N, 22.81.
able, NH); 13C-NMR (DMSO-d6, 100MHz) δ: 53.27, 127.29,
4-((5-Bromo-1H-indol-3-yl)methyleneamino)-1H-1,2,4- 128.73, 129.19, 141.70, 144.49, 148.65, 170.25; IR (KBr): 3068,
triazole-5(4H)-thione (2e): White powder, Yield (81%) mp 1591, 1452, 1119, 757, 699cm−1; MS (ESI) m/z: 295 (M+H+);
1
282–285°C. H-NMR (DMSO-d6, 400MHz) δ: 7.42 (1H, dd, Anal. Calcd for C16H14N4S (294.37): C, 65.28; H, 4.79; N,
J=8.8, 1.8Hz, indole H-6), 7.51 (1H, d, J=8.8Hz, indole H-7), 19.03; S, 10.89. Found: C, 65.20; H, 4.80; N, 19.06; S, 10.91.
8.16 (1H, d, J=2.8Hz, indole H-2), 8.43 (1H, d, J=1.8Hz,
Procedure for in Vitro Evaluation of Antiplatelet Ag-
indole H-4), 8.92 (1H, s, azomethine H), 9.38 (1H, s, triazole gregation Activity Whole blood was obtained from healthy
H), 12.20 (1H, s, D2O exchangeable, NH), 13.86 (1H, s, D2O volunteers that had not been taking any drugs at least for 14d
exchangeable NH); 13C-NMR (DMSO-d6, 100MHz) δ: 109.98, before the test. The blood was gently mixed with trisodium
114.62, 114.90, 124.71, 126.34, 136.34, 136.50, 138.92, 158.67, citrate dihydrate 3.8% (1 part citrate, 9 part blood) and cen-
162.77, 186.41; IR (KBr): 3273, 1589, 1290, 937, 793cm−1; MS trifuged at 1000rpm for 8min to obtain PRP. The remained
(ESI) m/z: 322 (98%), 324 (100%) (M+H+), 344 (100%), 346 blood was centrifuged at 3000rpm for 15min and PPP was
(100%) (M+Na+); Anal. Calcd for C11H8BrN5S (322.18): C, removed which was used as the blank. The platelet count was
41.01; H, 2.50; N, 21.74; S, 9.95. Found: C, 41.12; H, 2.51; N, adjusted to 250000plts/mL. Then 1µL of the compounds dis-
21.69; S, 9.97.
solved in dimethylsulfoxide (DMSO) was added to 200µL of
4-((1H-Indol-2-yl)methyleneamino)-1H-1,2,4-triazole-5(4H)- PRP and incubated for 5min. Aggregation was induced by
thione (3e): White powder, Yield (66%) mp 238°C (dec.). addition of ADP (5µm) or arachidonic acid (1.25mg/mL) and
1H-NMR (DMSO-d6, 400MHz) δ: 7.08 (1H, t, J=7.6Hz, aggregation was measured with aggregometer (APACT 4004)
indole H-5), 7.17 (1H, s, indole H-3), 7.28 (1H, t, J=7.6Hz, for 5min. DMSO (0.5% v/v) was used as negative control
indole H-6), 7.51 (1H, d, J=8.4Hz, indole H-7), 7.68 (1H, d, and indomethacin and aspirin were used as standard drugs.
J=8Hz, indole H-4), 8.91 (1H, s, azomethine H), 9.45 (1H, s, The platelet aggregation inhibitory activity was expressed
triazole H), 11.92 (1H, s, D2O exchangeable, NH), 13.98 (1H, as percent inhibition by comparison with that measured for

150
Vol. 61, No. 2
P., Castro H. C., Zingali R. B., Fraga C. A. M., de Souza M. C. B.
V., Ferreira V. F., Barreiro E. J., Bioorg. Med. Chem., 11, 2051–2059
(2003).
the vehicle (DMSO) alone and IC50 was calculated from
log(concentration)−inhibition (%) diagram and was defined as
the concentration of the test compound that inhibits the plate-
let aggregation by 50%. Data were presented as mean S.E.M.
of three independent experiments performed in triplicate.
12) Lima L. M., Frattani F. S., Dos Santos J. L., Castro H. C., Fraga
C. A. M., Zingali R. B., Barreiro E. J., Eur. J. Med. Chem., 43,
348–356 (2008).
13) Silva A. G., Zapata-Sudo G., Kummerle A. E., Fraga C. A. M., Bar-
reiro E. J., Sudo R. T., Bioorg. Med. Chem., 13, 3431–3437 (2005).
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S. H., Phytother. Res., 22, 58–64 (2008).
15) Jianguo Z., Tonglai Z., Kaibei Y., Chemistry of Heterocyclic Com-
pounds, 39, 461–466 (2003).
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Acknowledgment The authors would like to thank Dr.
Marjan Esfahani Zadeh and Kimia Tabib for their kind assis-
tance to evaluate the IC50s of indole-3-carbinol against ADP,
AA and collagen.
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