Synthesis and biological evaluation of new 3-substituted indole derivatives as potential anti-inflammatory and analgesic agents

Article abstract of DOI:10.1016/j.bmc.2007.03.024

Treatment of 3-cyanoacetyl indole 1 with the diazonium salts of 3-phenyl-5-aminopyrazole and 2-aminobenzimidazole afforded the corresponding hydrazones 4 and 5. 3-Cyanoacetyl indole reacted with phenylisothiocyanate to give the corresponding thioacetanili

Full text of DOI:10.1016/j.bmc.2007.03.024

Bioorganic & Medicinal Chemistry 15 (2007) 3832–3841
Synthesis and biological evaluation of new 3-substituted
indole derivatives as potential anti-inflammatory
and analgesic agents
Mohamed A. A. Radwan,a,* Eman A. Ragab,b
Nermien M. Sabrya and Siham M. El-Shenawyc
aApplied Organic Chemistry Department, National Research Centre, Dokki, Cairo, Egypt
bChemistry Department, Faculty of Science, Cairo University, Giza, Egypt
cPharmacology Department, National Research Centre, Dokki, Cairo, Egypt
Received 30 December 2006; revised 5 March 2007; accepted 8 March 2007
Available online 13 March 2007
Abstract—Treatment of 3-cyanoacetyl indole 1 with the diazonium salts of 3-phenyl-5-aminopyrazole and 2-aminobenzimidazole
afforded the corresponding hydrazones 4 and 5. 3-Cyanoacetyl indole reacted with phenylisothiocyanate to give the corresponding
thioacetanilide derivative 7. Treatment of 7 with hydrazonoyl chlorides afforded the corresponding 1,3,4-thiadiazole derivatives
8a–f and 9. Also, the thioacetanilide reacted with a-haloketones to afford thiophene derivatives 10a,b (tenidap analogues), or
thiazolidin-4-one derivative 11. The newly synthesized compounds were found to possess potential anti-inflammatory and analgesic
activities.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
substitution of heterocyclic moiety at 3-position of the
indole ring obviously influenced the anti-inflammatory
activity.21 Besides these, pyrazole derivatives were found
to possess anti-inflammatory22 and antimicrobial23
activities. Also, 1,3,4-thiadiazoles were reported as
highly anti-inflammatory,24 antimicrobial25 and anti-
convulsant26 agents. Encouraged by the above observa-
tions and considering the interesting pharmacological
profile of tenidap, we synthesized new 3-substituted
indoles incorporating an extra heterocyclic ring: pyrazole;
benzimidazole; 1,3,4-thiadiazoles; thiophenes (tenidap
analogues) and thiazolidinone as promising anti-inflam-
matory and analgesic agents.
Indomethacin and tenidap are non-steroidal anti-inflam-
matory drugs (NSAIDs) (Fig. 1) and have been shown
to exert anti-inflammatory effects.1,2 Tenidap is an
inhibitor of prostaglandin3 interleukin-14 production
in the body used for the treatment of rheumatoid arthri-
tis and osteoarthritis. It also inhibits both enzymes
cyclooxygenase and 5-lipoxygenase,5 which convert ara-
chidonic acid into prostaglandin and leukotrienes3 and
exhibit superior activity compared to indomethacin.
Synthetic approaches based on NSAIDs have been
taken with the aim of improving their profile6–8 where
the action of NSAID is in lowering the prostaglandin
production through inhibition of cyclooxygenase
(COX). Indole, the potent basic pharmacodynamic
nucleus, has been reported to possess a wide variety of
biological properties viz., anti-inflammatory,9–11 anti-
convulsant,12 cardiovascular13 and antibacterial.14 In
addition, the indole nucleus is incorporated in various
natural products such as alkaloids.15–20 Furthermore,
The biological activity and structure–activity relationship
(SAR) of the newly synthesized compounds were evalu-
ated compared to indomethacin and were found to pos-
sess potent anti-inflammatory and analgesic activities.
2. Results and discussion
2.1. Chemistry
Keywords: Indole; 1,3,4-Thiadiazoles; Thiophenes; Anti-inflammatory
and tenidap.
In continuation of the previous work on the synthesis
of indole alkaloid derivatives,27 we report here
*
Corresponding author. Tel.: +20 2 7201588; fax: +20 2 3370931;
e-mail: m1radwan@yahoo.com
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.024

M. A. A. Radwan et al. / Bioorg. Med. Chem. 15 (2007) 3832–3841
3833
O
OH
O
O
S
Cl
N
O
O
N
O
H2N
Cl
Indomethacin
Tenidap
Figure 1.
synthesis of some new 3-substituted indole derivatives.
The reactivity of 3-cyanoacetyl indole 1 towards some
heterocyclic diazonium salts such as 3-phenyl-5-ami-
no-1H-pyrazole diazonium salt 2 and 2-amino-1H-
benzimidazole diazonium salt 3 was investigated. Thus,
3-cyanoacetyl indole 1 was found to couple smoothly
with the diazonium salts 2 and 3 to afford the corre-
sponding hydrazones 4 and 5, respectively (Scheme 1).
The structure of compound 4 was established on the
basis of its elemental analysis and spectral data. Its
1H NMR spectrum revealed singlet signal at d 7.5 char-
acteristic of the pyrazole proton (H-4) which was con-
firmed by 13C NMR and HSQC experiments at d
107.18. The structure of compound 5 was established
on the basis of its elemental analysis and spectral data.
1H NMR spectrum of compound 5 revealed a singlet
signals at d 12.01 and 12.71 characteristic for NH
groups. In addition, the mass spectrum revealed the
peak at m/z 328 corresponding to its molecular ion
(see Section 4).
which was confirmed by 13C NMR and HSQC experi-
ments at d 13.86 and 63.12, respectively. In addition to
AA0 XX0 system signals at d 7.68 and 7.78 correspond-
ing to p-substituted phenyl (129.34 and 129.80), respec-
tively, and two C@O at d 165.10 and 179.17 in 13C
NMR spectra (Fig. 2).
Moreover, the structure of compounds 8a–f was further
supported by an independent synthesis from the
treatment of the potassium salt intermediate 6 with
1-chloro-1-(2-phenylhydrazono)propan-2-one and/or ethyl
2-chloro-(2-phenylhydrazone)acetate 12a–f at room
temperature (Scheme 2).
Also, compound 7 reacted similarly with 1-[chloro-
(phenyl)methylene]-2-phenylhydrazine 13 to afford
3-(1H-indole-3-yl)-2-(3,5diphenyl-1,3,4-thiadiazol-2(3H)-
ylidene)-3-oxopropanenitrile 9 based on its elemental
analysis and spectral data. It has characteristic absorption
peaks in IR spectrum at 3223, 2191 and 1569 cmꢀ1 due to
NH, CN and carbonyl groups, respectively. In addition,
the mass spectrum revealed a peak at m/z 420 correspond-
ing to its molecular ion (see Section 4).
All attempts to perform intramolecular cyclization of
hydrazones 4 and 5 to the closed form by prolonged
heating in pyridine were unsuccessful and the starting
materials 4 and 5 were recovered unaffected (Scheme 1).
Next, compound 7 reacted with phenacyl bromide and
with a-chloroacetone in refluxing ethanol and in the
presence of a catalytic amount of triethylamine to give
the corresponding 3-(3-indolyl)thiophene derivatives
10a,b which were considered as analogues of tenidap,
as depicted in Scheme 3.
Treatment of compound 1 with phenylisothiocyanate
and potassium hydroxide in dimethylformamide at
room temperature afforded the potassium salt interme-
diate 6 which was converted into the corresponding
thioacetanilide derivative 7 upon treatment with 10%
hydrochloric acid. The thioacetanilide 7 reacted with
hydrazonoyl chlorides 12a–f in refluxing ethanol and
in the presence of triethylamine to afford, in each case,
only 1,3,4-thiadiazole isolable product. Elemental anal-
yses and spectral data of the reaction products were in
complete agreement with those of the corresponding
1,3,4-thiadiazole structures 8a–f (Scheme 2). For exam-
Compound 10a showed characteristic peaks at 3223,
2190 and 1704 cmꢀ1 due to NH, CN and C@O groups,
respectively. IR spectrum and its 1H NMR spectrum re-
vealed a singlet signal at d 1.90 characteristic of the
COCH3 protons and singlet signal at d 7.01 characteristic
of the NHPh group. As well a peak at m/z 357 corre-
sponds to its molecular ion in the mass spectrum. Simi-
larly compound 10b showed characteristic peaks at
3277, 2201 and 1687 cmꢀ1 due to NH, CN and C@O
groups, respectively (in its IR spectrum), and its 1H
1
ple, the H NMR spectrum of compound 8f revealed a
triplet and quartet signals at d 1.35 and 4.43 due to
methyl and methylene of the ester group, respectively,

3834
M. A. A. Radwan et al. / Bioorg. Med. Chem. 15 (2007) 3832–3841
CN
O
N
H
1
-
+
N2Cl
+
-
N
N2 HSO4
NH
N
H
N
Ph
2
3
CN
N
O
CN
N
O
H
N
NH
N
NH
N
NH
N
N
H
H
5
4
Ph
Scheme 1.
NMR spectrum revealed singlet at d 7.76 characteristic
of the NHPh group. In addition, the peak at m/z 419 cor-
responds to its molecular ion in its mass spectrum.
zone 4, which comes from the diazonium salt of
3-phenyl-5-aminopyrazole 2. As shown in Table 1, the
3-(3-indolyl)thiophene derivative 10a was the most
potent anti-inflammatory compound which was consid-
ered as analogue of tenidap. The thioacetanilide 7
showed good activity in comparison with the other
tested compounds (28%). Also, 1,3,4-thiadiazole deriva-
tive 8b was found to be more potent than indomethacin.
However, thiazolidin-4-one derivative 11 showed the
least inhibitory effect (7.7%).
While, thioacetanilide derivative 7 reacted similarly
with ethyl chloroacetate under the same reaction condi-
tions to give the corresponding thiazolidin-4-one deriv-
ative 11. The structure of compound 11 was established
on the basis of its elemental analysis and spectral data.
Its IR spectrum showed the presence of absorption
peaks at 3304, 2198, 1717 and 1616 cmꢀ1 due to NH,
CN and two C@O groups, respectively, and its 1H
NMR spectrum revealed a singlet signal at d 4.01 char-
acteristic of the methylene protons of thiazolidinone
ring. In addition, the mass spectrum revealed a
peak at m/z 359 corresponding to its molecular ion
(see Section 4).
From the structure–activity relationship (SAR) view-
point, the anti-inflammatory activity of the 5-acetyl 3-
(3-indolyl)thiophene derivative 10a was found to be
the highest one. 5-Acetyl-1,3,4-thiadiazole derivative of
substituted p-methylphenyl 8b was more potent anti-
inflammatory compound than p-chlorophenyl derivative
8c (CH3 > Cl). The anti-inflammation effect of the thiaz-
olidine ester derivatives 8e,f is less than that of its acetyl
derivatives. In addition, the chlorinated ester derivative
of 1,3,4-thiadiazole system 8f was found to be slightly
more effective than its non-chlorinated one 8e. Also
1,3,4-thiadiazole with biphenyl groups 9 showed good
activity (31%).
2.2. Pharmacology
2.2.1. Anti-inflammatory activity. The activity of the
newly synthesized compounds compared to indometha-
cin as a reference was measured before and 4 h after car-
rageenan injection. Percent of the oedema inhibition was
calculated as regards saline control group and potency
was calculated as regards the percentage of the change
of the indomethacin and tested compounds, as depicted
in Table 1a and Table 1b. All the tested compounds
showed a reasonable inhibition of oedema size ranging
between 5.5% for compound 8d and 44.4% for com-
pound 10a and 29.9% for indomethacin. Hydrazone 5,
which comes from the diazonium salt of 2-aminobenz-
imidazole 3, was found to be more potent than hydra-
2.2.2. Analgesic activity. Next, the analgesic activity
(antinociceptive) of the synthesized 3-indole derivatives
was also investigated. It was assessed by two different
models: the acetic acid-induced writhing test and the
hot-plate test. Chemical models of nociception, by using
acetic acid-induced writhing (visceral pain), and thermal
model by using hot-plate test (thermal pain). Collier
et al.36 proposed that acetic acid acts indirectly by

M. A. A. Radwan et al. / Bioorg. Med. Chem. 15 (2007) 3832–3841
CN
3835
O
-
+
S K
DMF
HN
1
Ph
N
PhNCS/ KOH
H
6
O
N
Ar
R
N
HCl
H
Cl
12a-f
O
CN
SH
Ph
O
N
Ar
R
N
CN
O
H
Cl
12a-f
R
O
S
HN
N
EtOH/ TEA
HN
Ph
H
N
N
H
7
NH
Ar
-PhNH2
O
Ph
N
Ph
EtOH/ TEA
N
H
Cl
CN
13
Ar
N
N
CN
S
O
N
H
COR
S
N
8a-f
Ph
HN
Ph
N
H
NH
R
Ar
8,12
Ph
-PhNH2
a
C6H5
CH3
CH3
CN
O
Ph
b
4-MeC6H4
4-ClC6H4
C6H5
N
N
S
CH3
c
d
N
H
Ph
OC2H5
9
e
f
4-MeC6H4
4-ClC6H4
OC2H5
OC2H5
Scheme 2.
inducing the release of endogenous mediators which stim-
ulate the nociceptive neurons sensitive to non-steroidal
anti-inflammatory drugs (NSAIDS) and opioids. While
the hot-plate model of analgesia measures nociception
induced by central mechanisms. Some of the 3-indole
derivatives exhibited analgesic effects as shown in Table 2
and Table 3. Compared with the control, the potency
of the tested compounds 11 and 8c was found to be
the highest (Table 3). Hydrazone 5, which comes from
the diazonium salt of 2-aminobenzimidazole 3, was
found to be less potent than hydrazone 4, which comes
from the diazonium salt of 3-phenyl-5-aminopyrazole 2.
According to the structure–activity relationship (SAR) it
is clear that the thiazolidin-4-one ring system is more ac-
tive than both the 1,3,4-thiadiazole ring and hydrazone
moiety. The reactivity of PhCO > CH3CO group in 3-(3-
indolyl)thiophene derivatives 10a,b. Among the same
ring system (1,3,4-thiadiazole derivatives), we noticed

3836
M. A. A. Radwan et al. / Bioorg. Med. Chem. 15 (2007) 3832–3841
O
that the chlorinated ketone derivatives are more effective
than their non-chlorinated analogues (Table 3).
121.64
117.04
122.86
NC
179.17
126.65
112.10
Cl
74.96
131.36
121.81
3. Conclusions
157.98
N
129.80
113.32
135.66
137.25
In conclusion, simple and convenient methods for the
synthesis of the new anti-inflammatory 3-indole hetero-
cyclic derivatives starting from 3-cyanoacetyl indole
were demonstrated. In this approach cyanoacetyl group
at the 3-position of indole is used as a precursor to con-
struct the appropriately substituted hydrazones, 1,3,4-
thiadiazole derivatives, thiophene derivatives as tenidap
analogues, and thiazolidin-4-one derivative. The anti-
inflammatory, analgesic activities and structure–activity
relationship (SAR) of the newly synthesized compounds
were provided. 3-(3-Indolyl)thiophene derivative 10a,
135.61
129.34
N
S
H
N
149.56
O
63.12
165.1
O
13.86
Figure 2. Complete 13C assignment of compound 8f.
CN
O
O
SH
Ph
O
Cl
HN
O
R
N
H
Cl
15
14
7
CN
O
CN
O
O
S
O
S
HN
O
HN
R
Ph
N
H
Ph
N
H
-EtOH
-H2O
CN
O
Ph
N
NHPh
S
H2N
O
S
NHPh
O
NC
N
H
S
COR
11
COR
N
N
H
H
10a,b
R
10, 14
a
CH3
C6H5
b
Scheme 3.

M. A. A. Radwan et al. / Bioorg. Med. Chem. 15 (2007) 3832–3841
3837
Table 1a. Inhibitory effect of some new 3-indole derivatives on carrageenan-induced oedema of the hind paw in mice
Compound Dose (mg/kg)
Oedema
Oedema (%) (X SE) Oedema inhibition (%) Potency
Zero min (basal) 4 h oedema (cm) (% increase)
Control
1 mL saline
0.23 0.006
0.26 0.03
0.21 0.003
0.20 0.002
0.22 0.002
0.22 0.004
0.22 0.001
0.22 0.009
0.20 0.004
0.20 0.09
0.20 0.004
0.46 0.01
0.39 0.03
0.41 0.2
109.1 6.3
76.5 3.6*
94.8 5.3
81.3 4.8*
78.3 4.1*
73.4 4.4*
92.7 5.1*
96.4 3.9
88.3 3.2*
60.7 4.9*
100.7 4.1
—
—
1
IND
4
35
70
70
70
70
70
70
70
70
70
ꢀ29.9
ꢀ13.1
ꢀ25.48
ꢀ28
0.4
0.9
0.9
1.1
0.5
0.4
0.6
1.5
0.3
5
0.37 0.01
0.38 0.03
0.37 0.02
0.42 0.01
0.43 0.01
0.38 0.009
0.32 0.004
0.41 0.01
7
8b
8c
8e
8f
ꢀ32.7
ꢀ15
ꢀ11.5
ꢀ19.1
ꢀ44.4
ꢀ7.7
10a
11
Data represent mean values SE of six mice per group and the percent changes versus basal (zero min) values and 4 h post-carrageenan injection.
Data were analyzed using one-way ANOVA and Duncan’s multiple comparison test *P < 0.05.
Percent oedema inhibition was calculated as regards saline control group.
Potency was calculated as regards the percentage change of the indomethacin treated group.
IND, indomethacin.
Table 1b. Inhibitory effect of some new 3-indole derivatives on carrageenan-induced oedema of the hind paw in mice
Compound Dose (mg/kg)
Oedema
Oedema (%) (X SE) Oedema inhibition (%) Potency
Zero min (basal) 4 h oedema (cm) (% increase)
Control
IND
8a
1 mL saline
0.21 0.006
0.23 0.02
0.23 0.03
0.22 0.01
0.24 0.002
0.22 0.009
0.43 0.01
0.37 0.04
0.41 0.03
0.44 0.02
0.40 0.01
0.42 0.03
103.8 4.6
65.2 2.8*
81.1 5.1*
98.07 4.2
71.58 2.4*
89.02 2.9*
—
—
1
35
70
70
70
70
ꢀ36.79
ꢀ21.87
ꢀ5.5
0.6
0.2
0.9
0.4
8d
9
10b
ꢀ31.04
ꢀ14.24
Data represent mean values SE of six mice per group and the percent changes versus basal (zero min) values and 4 h post-carrageenan injection.
Data were analyzed using one-way ANOVA and Duncan’s multiple comparison test *P < 0.05.
Percent oedema inhibition was calculated as regards saline control group.
Potency was calculated as regards the percentage change of the indomethacin treated group.
IND, indomethacin.
Table 2. Analgesic effect of oral administration of some new 3-indole derivatives on visceral pain by using writhing test in mice
Compound
Dose (mg/kg)
Number of writhing/30 min (X SE)
Change (%)
Potency
Saline
Indomethacin
1 mL
35
70
70
70
70
70
70
70
70
70
70
70
70
70
83.5 3.6
16 1.2*
31.5 2.3*
30.75 1.8*
30 2.1*
23.75 1.5*
35.2 2.9*
45.3 3.5*
32 1.5*
18.75 1.3*
20 1.4*
—
—
1
ꢀ80.7
ꢀ62.5
ꢀ63.2
ꢀ64.1
ꢀ71.6
ꢀ57.8
ꢀ45.7
ꢀ61.7
ꢀ77.5
ꢀ76
4
0.77
0.78
0.79
0.88
0.7
5
7
8a
8b
8c
8d
8e
8f
9
0.57
0.76
0.96
0.9
54 3.5*
ꢀ35.3
ꢀ68.6
ꢀ66.8
ꢀ60.9
0.4
10a
10b
11
26.25 2.2*
27.75 1.3*
32.6 2.8*
0.85
0.8
0.75
Data represent mean values SE of six mice per group and percentage inhibition of number of writhing/30 min. Statistical comparison of the
difference between saline control group and treated groups was done by one-way ANOVA and Duncan’s multiple comparison test *P < 0.05.
Potency was calculated as regards the percentage change of the indomethacine.
analogue of tenidap, was the most potent anti-inflam-
matory compound. According to this study, we could
point out that the 3-substituted indole derivatives played
a very important role as anti-inflammatory and analge-
sic agents and considering the interesting pharmacologi-
cal profile of tenidap. These observations may provide
some predictions in order to design further promising
3-indole compounds.

3838
M. A. A. Radwan et al. / Bioorg. Med. Chem. 15 (2007) 3832–3841
Table 3. Analgesic effect of oral administration of some new 3-indole derivatives on thermal pain by using hot-plat test in mice
Compound
Dose (mg/kg)
Latency (S)
Change (%)
Potency
Basal (X SE)
1 h (X SE)
Saline
Indomethacin
1 mL
35
70
70
70
70
70
70
70
70
70
70
70
70
70
11.3 0.8
11.5 0.6
11.3 1.1
12.8 0.9
12 0.7
12 0.9
14.1 1*
6.1
22.6
28.3
8.6
—
1
4
14.5 1.2*
13.9 0.8
14.9 1.1*
12.5 0.8
14.4 0.9*
21.2 1.5*
13.7 0.7
12.7 0.9
1.3
0.4
1.1
0.1
0.9
3.1
0.4
0.5
0.4
1.2
0.5
1.9
3.4
5
7
24.2
2.4
8a
8b
8c
8d
8e
8f
9
12.2 0.6
11.9 0.5
12.5 0.9
12.5 1.1
11.4 0.5
11.1 0.7
11 0.5
21
69.6
9.6
11.4
9.9
12.2
1
14 0.6*
13.9 1.1
15.7 1.3*
20.3 1.6*
27.3
11.2
42.7
76.5
10a
10b
11
12.5 1.1
11 0.6
11.5 0.9
Data represent mean values SE of six mice per group, shown at the basal (zero time) and 1 value for each group (saline, indomethacin and tested
compounds). Statistical comparisons between basal (pre-drug values) and post-drug values.
Data were analyzed using one-way ANOVA and Duncan’s multiple comparison test *P < 0.05.
Percentage change was calculated from basal (pre-drug) values and post-drug values.
Potency was calculated as regards the percentage change of the indomethacin.
4. Experimental
(s, 1H, NH), 7.50 (s, 1H, H-4, pyrazole), 7.54 and 8.12
(m, 5H, Ph), 7.65 (d, 1H, H-7), 7.77 (d, 1H, H-4), 8.1
(s, 1H, NH), 8.67 (s, 1H, H-2), 12.65 (br s, 1H, NH);
13C NMR: d 107.18, 109.90, 122.86, 127.25, 130.98,
133.11, 135.07, 136.77, 139.15, 140.54, 144.54, 146.66,
147.86, 160.58, 167.82. EIMS m/z (%) 336 (M+ꢀ18,
100), 310 (21), 166 (43), 139 (25), 116 (11), 77 (54), 51
(48). Anal. Calcd for C20H14N6O: C, 67.79; H, 3.89;
N, 23.72; O, 4.87. Found: C, 67.69; H, 3.93; N, 23.68;
O, 4.97.
4.1. Chemistry
4.1.1. General. Melting points were determined on a
Gallenkamp melting point apparatus and the values
are uncorrected. IR spectra were recorded on Shimadzu
1
FT-IR 8101 PC infrared spectrophotometer. H NMR
spectra were recorded on a Jeol EX-270 MHz spectrom-
eter using DMSO-d6 as solvent and TMS as the internal
standard, 13C NMR and 2D NMR spectra were
recorded on a varian Mercury VX 300 NMR using
DMSO-d6 as solvent and TMS as an internal standard.
Mass spectra were recorded on a Finnigan mat.
SSQ-7000 GC-MS spectrometer. Microanalyses were
performed at the Microanalytical Center of Cairo
University. 3-Cyanoacetyl indole 1,28 hydrazonoyl chlo-
rides 12a–c,29 12d–f30 and 1331 and phenacyl bromides
14b32 were prepared as reported in the literature.
4.1.2.2. 2-(2-(1H-Benzo[d]imidazol-2-yl)hydrazono)-3-
(1H-indole-3-yl)-3-oxopro-panenitrile (5). In 0.51 g (78%)
yield; mp > 300 °C; IR (KBr) mmax 3448 (NH), 3226
(NH), 2221 (CN), 1704 (C@O), 1626 (C@N),
1
1228 cmꢀ1; H NMR: d 7.19 (m, 2H, H-5, H-6), 7.42
(m, 4H, benzoimidazol), 7.5 (d, 1H, H-7), 8.32 (d, 1H,
H-4), 8.56 (s, 1H, H-2), 12.01 (s, 1H, NH), 12.71 (br s,
1H, NH); 13C NMR: d 111.19, 111.92, 113.02, 113.77,
121.50, 121.65, 122.70, 122.98, 123.98, 123.22, 126.68,
130.64, 135.91, 136.27, 157.39, 181.02. EIMS m/z (%)
328 (M+, 85), 311 (45), 164 (18), 144 (100), 116 (75),
89 (65), 63 (25). Anal. Calcd for C18H12N6O: C, 65.85;
H, 3.68; N, 25.60; O, 4.87. Found: C, 65.73; H, 3.88;
N, 25.59; O, 4.91.
4.1.2. Reaction of the 3-cyanoacetyl indole (1) with
diazonium salts of 3-phenyl-5-aminopyrazole (2) and 2-
aminobenzimidazole (3): general procedure. To a stirred
cold solution of 3-cyanoacetyl indole 1 (0.37 g, 2 mmol)
in pyridine (25 mL) was added the diazonium salt 2 or 3
(2 mmol) portionwise over a period of 30 min. The reac-
tion mixture was kept in an icebox overnight and then
diluted with water. The solid that precipitated was fil-
tered off, washed with water and dried. Recrystallization
from DMF gave the corresponding hydrazones 4 or 5,
respectively.
4.1.3. Synthesis of the thioacetanilide derivative (7). To a
stirred solution of potassium hydroxide (0.6 g, 10 mmol)
in dimethylformamide (50 mL), 3-cyanoacetyl indole 1
(1.8 g, 10 mmol) was added. After stirring for 30 min,
phenylisothiocyanate (1.4 g, 10 mmol) was added to
the resulting mixture. The stirring was continued for fur-
ther 6 h then poured over crushed ice containing hydro-
chloric acid. The solid product was filtered off, washed
with water, dried and finally recrystallized from DMF/
H2O to afford 3-(1H-indole-3-yl)-2-(mercapto(phenyl-
amino)methylene)-3-oxopropanenitrile 7 as a orange
4.1.2.1. 2-(2-(3-Phenyl-1H-pyrazol-5-yl)hydrazono)-3-
(1H-indole-3-yl)-3-oxopro-panenitrile (4). In 0.58 g (82%)
yield; mp > 300 °C; IR (KBr) mmax 3178 (NH), 3172
(NH), 2226 (CN), 1658 (C@O), 1596 (C@N),
1228 cmꢀ1; H NMR: d 7.34 (m, 2H, H-5, H-6), 7.49
1

M. A. A. Radwan et al. / Bioorg. Med. Chem. 15 (2007) 3832–3841
3839
powder in 2.74 g (86%) yield; mp 190–193 °C; IR (KBr)
mmax 3329 (NH), 3245 (NH), 2196 (CN), 1704 (C@O),
IR (KBr) mmax 3229 (NH), 2195 (CN), 1684 (C@O),
1571 (C@N) cmꢀ1; 1H NMR: d 1.36 (t, 3H, CH3,
J = 7.2 Hz), 4.45 (q, 2H, CH2, J = 7.2 Hz), 7.19 (m,
5H, Ph), 7.23 (m, 2H, H-5, H-6), 7.81 (d, 1H, H-7),
8.32 (d, 1H, H-4), 8.46 (s, 1H, H-2), 11.87 (s, 1H,
NH); MS m/z (%) 416 (M+, 51), 323 (19), 273 (25), 144
(100). Anal. Calcd for C22H16N4O3S: C, 63.45; H, 3.87;
N, 13.45; O, 11.53; S, 7.70. Found: C, 63.49; H, 3.66;
N, 13.39; O, 11.58; S, 7.64.
1
1626 (C@N) cmꢀ1; H NMR: d 7.16 (m, 5H, Ph), 7.26
(m, 2H, H-5, H-6), 7.75 (d, 1H, H-7), 8.17 (d, 1H, H-
4), 8.64 (s, 1H, H-2), 11.88 (s, 1H, NH), 14.25 (s, 1H,
SH); MS m/z (%) 319 (M+, 34), 166 (49), 144 (100),
139 (15), 116 (19), 77 (67). Anal. Calcd for C18H13N3OS:
C, 67.69; H, 4.10; N, 13.16; O, 5.01; S, 10.04. Found: C,
67.23; H, 4.05; N, 13.34; S, 10.15.
4.1.4. Reaction of the thioacetanilide derivative 7 with
hydrazonoyl chlorides. To a solution of the thioacetani-
lide derivative 7 (0.64 g, 2 mmol) in absolute ethanol
(20 mL), the appropriate hydrazonoyl chlorides 12a–f
or 13 (2 mmol) were added, in the presence of triethyl-
amine (0.3 mL). The reaction mixture was refluxed for
1 h and then allowed to cool. The formed solid product
was filtered off, washed with ethanol and recrystallized
from EtOH/DMF to afford the corresponding thiadiaz-
ole derivatives 8a–f and 9.
4.1.4.5. Ethyl 5-(1-cyano-2-(1H-indol-3-yl)-2-oxoethy-
lidene)-4,5-dihydro-p-tolyl-1,3,4-thiadiazole-2-carboxylate
(8e). Yellow-orange (0.61 g, 71%); mp 246–248 °C; IR
(KBr) mmax 3389 (NH), 2196 (CN), 1708 (C@O), 1571
(C@N) cmꢀ1; 1H NMR:
d 1.35 (t, 3H, CH3,
J = 7.2 Hz), 2.43 (s, 3H, p-CH3–Ph), 4.43 (q, 2H, CH2,
J = 7.2 Hz), 7.21 (m, 2H, H-5, H-6), 7.34 (d, 2H,
J = 7.8 Hz), 7.52 (d, 2H, J = 7.8 Hz), 7.78 (d, 1H, H-
7), 8.25 (d, 1H, H-4), 8.33 (s, 1H, H-2), 11.89 (s, 1H,
NH); MS m/z (%) 430 (M+, 21), 339 (39), 266 (65),
144 (100). Anal. Calcd for C23H18N4O3S: C, 64.17; H,
4.21; N, 13.01; O, 11.15; S, 7.45. Found: C, 64.39; H,
3.99; N, 13.11; O, 11.68; S, 7.54.
4.1.4.1. 2-(5-Acetyl-3-phenyl-1,3,4-thiadiazol-2(3H)-
lidene)3-(1H-indole-3-yl)-3-oxopropa-nenitrile (8a). Or-
ange-yellow (0.53 g, 69%); mp 270–273 °C; IR (KBr)
mmax 3220 (NH), 2198 (CN), 1687 (C@O), 1574 (C@N)
4.1.4.6. Ethyl 5-(1-cyano-2-(1H-indol-3-yl)-2-oxoethy-
lidene)-4,5-dihydro-4-chlorophenyl-1,3,4-thiadiazole-2-car-
boxylate (8f). Yellow (0.67 g, 74%); mp 289–291 °C; IR
(KBr) mmax 3272 (NH), 2195 (CN), 1735 (C@O), 1570
1
cmꢀ1; H NMR: d 2.3 (s, 3H, COCH3) 7.19 (m, 5H,
Ph), 7.28 (m, 2H, H-5, H-6), 7.86 (d, 1H, H-7), 8.35
(d, 1H, H-4), 8.61 (s, 1H, H-2), 12.02 (s, 1H, NH); MS
m/z (%) 386 (M+, 38), 343 (49), 309 (25), 266 (15), 144
(100), 77 (67). Anal. Calcd for C21H14N4O2S: C, 65.27;
H, 3.65; N, 14.50; O, 8.28; S, 8.30. Found: C, 65.12;
H, 3.54; N, 14.56; O, 8.31; S, 8.35.
(C@N) cmꢀ1; 1H NMR:
d 1.35 (t, 3H, CH3,
J = 7.2 Hz), 4.43 (q, 2H, CH2, J = 7.2 Hz), 7.20 (m,
2H, H-5, H-6), 7.68 (d, 2H, J = 7.8 Hz), 7.78 (d, 2H,
J = 7.8 Hz), 7.48 (d, 1H, H-7), 8.26 (d, 1H, H-4), 8.35
(s, 1H, H-2), 11.93 (s, 1H, NH); 13C NMR: d 13.86,
63.12, 74.95, 112.11, 113.32, 117.04, 121.64, 121.80,
122.86, 126.70, 129.34, 129.80, 131.36, 135.61, 135.66,
137.25, 149.56, 157.98, 165.1, 179.17; MS m/z (%) 450
(M+, 21), 338 (55), 265 (65), 144 (100). Anal. Calcd for
C22H15ClN4O3S: C, 58.60; H, 3.35; Cl, 7.86; N, 12.43;
O, 10.65; S, 7.11. Found: C, 58.44; H, 3.38; Cl, 7.86;
N, 12.53; O, 10.71; S, 7.07.
4.1.4.2. 2-(5-Acetyl-3-p-tolyl-1,3,4-thiadiazol-2(3H)-
lidene)3-(1H-indole-3-yl)-3-oxopropa-nenitrile (8b). Yel-
low needles (0.61 g, 76%); mp > 300 °C; IR (KBr) mmax
3222 (NH), 2197 (CN), 1676 (C@O), 1567 (C@N)
1
cmꢀ1; H NMR: d 2.44 (s, 3H, COCH3), 2.5 (s, 3H, p-
CH3–Ph), 7.20 (m, 2H, H-5, H-6), 7.44 (d, 2H,
J = 7.5 Hz), 7.60 (d, 2H, J = 7.5 Hz), 7.50 (d, 1H, H-
7), 8.25 (d, 1H, H-4), 8.33 (s, 1H, H-2), 11.88 (s, 1H,
NH); MS m/z (%) 400 (M+, 54), 357 (35), 309 (71),
268 (15), 144 (100), 77 (67). Anal. Calcd for
C22H16N4O2S: C, 65.98; H, 4.03; N, 13.99; O, 7.99; S,
8.01. Found: C, 65.76; H, 4.11; N, 13.87; O, 8.05; S,
8.12.
4.1.4.7. 3-(1H-Indole-3-yl)-2-(3.5-diphenyl-1,3,4-thia-
diazol-2(3H)- lidene)-3-oxopropane-nitrile (9). Orange-
yellow (0.53 g, 69%); mp 286–289 °C; IR (KBr) mmax
3223 (NH), 2191 (CN), 1589 (C@O), 1564 (C@N)
1
cmꢀ1; H NMR: d 7.19–7.27 (m, 10H, 2Ph), 7.31 (m,
2H, H-5, H-6), 7.76 (d, 1H, H-7), 8.31 (d, 1H, H-4),
8.44 (s, 1H, H-2), 11.92 (s, 1H, NH); MS m/z (%) 420
(M+, 28), 343 (41), 266 (19), 144 (100), 77 (67). Anal.
Calcd for C25H16N4OS: C, 71.41; H, 3.84; N, 13.32; O,
3.80; S, 7.63. Found: C, 71.36; H, 3.75; N, 13.34; O,
3.76; S, 7.61.
4.1.4.3. 2-(5-Acetyl-3-4-chlorophenyl-1,3,4-thiadiazol-
2(3H)-lidene)3-(1H-indole-3-yl)-3-oxopropanenitrile (8c).
Yellow (0.65 g, 77%); mp 283 °C; IR (KBr) mmax 3271
1
(NH), 2192 (CN), 1679 (C@O), 1570 (C@N) cmꢀ1; H
NMR: d 2.47 (s, 3H, COCH3), 7.19 (m, 2H, H-5, H-
6), 7.41 (d, 2H, J = 7.8 Hz), 7.63 (d, 2H, J = 7.8 Hz),
7.68 (d, 1H, H-7), 8.35 (d, 1H, H-4), 8.61 (s, 1H, H-2),
11.93 (s, 1H, NH); MS m/z (%) 420 (M+, 34), 384 (12),
377 (41), 268 (55), 144 (100), 77 (37). Anal. Calcd for
C21H13ClN4O2S: C, 59.93; H, 3.11; Cl, 8.42; N, 13.31;
O, 7.60; S, 7.62. Found: C, 59.79; H, 3.17; Cl, 8.33; N,
13.39; O, 7.67; S, 7.56.
4.1.5. Reaction of thioacetanilide derivative 7 with a-
haloketones: general procedure. To a solution of the thio-
acetanilide derivative 7 (0.64 g, 2 mmol) in ethanol
(20 mL), the appropriate a-haloketone, chloroacetone
14a, phenacyl bromide 14b or ethyl chloroacetate 15
(2 mmol) were added. Triethylamine (0.2 mL) was
added dropwise and the reaction mixture was refluxed
for 1 h then allowed to cool. The formed solid product
was filtered off, washed with ethanol and recrystallized
from EtOH/DMF to afford the corresponding thiophene
4.1.4.4. Ethyl 5-(1-cyano-2-(1H-indol-3-yl)-2-oxoethy-
lidene)-4,5-dihydro-4-phenyl-1,3,4-thiadiazole-2-carboxyl-
ate (8d). Yellow needles (0.62 g, 75%); mp 260–261 °C;

3840
M. A. A. Radwan et al. / Bioorg. Med. Chem. 15 (2007) 3832–3841
derivatives 10a,b and thiazolidin-4-one derivative 11,
respectively.
thickness was measured before and 4 h after carra-
geenan injection. The oedema was expressed as a per-
centage of change from the control group (pre-drug)
values. The dose of the tested compounds and indo-
methacin was based on the rat dose converted to that
of mice according to Paget and Barnes.39
4.1.5.1. 5-Acetyl-4-(1H-indole-3-yl)-2-(phynylamino)thio-
phene-3-carbonitrile (10a). Yellow (0.53 g, 74%);
mp > 300 °C; IR (KBr) mmax 3223 (NH), 2190 (CN),
1
1704 (C@O), 1574 (C@N) cmꢀ1; H NMR: d 1.90 (s,
3H, COCH3), 7.01 (s, 1H, NH), 7.18 (m, 2H, H-5, H-
6), 7.46 (d, 1H, H-7), 7.42-7.52 (m, 5H, Ph), 8.23 (d,
1H, H-4), 8.26 (s, 1H, H-2), 11.60 (s, 1H, NH); MS m/z
(%) 357 (M+, 14), 280 (46), 265 (56), 239 (25), 224
(100), 77 (67). Anal. Calcd for C21H15N3OS: C, 70.57;
H, 4.23; N, 11.76; O, 4.48; S, 8.97. Found: C, 70.43;
H, 4.27; N, 11.68; O, 4.49; S, 8.86.
In anti-inflammatory study, the percentage of oedema
inhibition was calculated from the mean effect in the
control and treated animals according to the following
equation:
% Oedema inhibition
¼ f½ð% oedema formation of control group
ꢀ % oedema formation of treated groupÞꢁ
=½% oedema formation of control groupꢁ ꢂ 100g
4.1.5.2. 5-Benzoyl-4-(1H-indole-3-yl)-2-(phynylamino)-
thiophene-3-carbonitrile (10b). Yellow (0.55 g, 66%);
mp > 300 °C; IR (KBr) mmax 3277 (NH), 2201 (CN),
While the potency was calculated as regards indometha-
cin treated group according to the following equation:
1
1687 (C@O), 1574 (C@N) cmꢀ1; H NMR: d 7.23 (m,
2H, H-5, H-6), 7.40–7.62 (m, 10H, 2Ph), 7.76 (s, 1H,
NH), 7.80 (d, 1H, H-7), 8.30 (d, 1H, H-4), 8.56 (s, 1H,
H-2), 12.02 (s, 1H, NH); MS m/z (%) 419 (M+, 14),
280 (46), 265 (56), 239 (25), 224 (100), 77 (67). Anal.
Calcd for C26H17N3OS: C, 74.44; H, 4.08; N, 10.02; O,
3.81; S, 7.64. Found: C, 74.49; H, 4.03; N, 10.11; O,
3.75; S, 7.61.
Potency
¼ f% oedema inhibition of treated groupg
=f% oedema inhibition of indomethacin
treated groupg
4.2.3. Antinociceptive activity. This activity was deter-
mined by measuring the responses of animals to the
Koster test and hot-plate test.
4.1.5.3. 3-(1H-Indole-3-yl)-2-(4-oxo-3-phenylthiazoli-
din-2-ylidene)-3-oxopropanenitrile (11). Yellow (0.55 g,
66%); mp >300 °C; IR (KBr) mmax 3304 (NH), 2198
(CN), 1717 (C@O), 1616 (C@N) cmꢀ1; 1H NMR: d 4.01
(s, 2H, CH2) 7.20 (m, 2H, H-5, H-6), 7.46-7.52 (m, 5H,
Ph), 7.54 (d, 1H, H-7), 8.20 (d, 1H, H-4), 8.25 (s, 1H, H-
2), 11.90 (s, 1H, NH); MS m/z (%) 359 (M+, 24), 266
(12), 144 (100), 77 (16). Anal. Calcd for C20H13N3O2S:
C, 66.84; H, 3.65; N, 11.69; O, 8.90; S, 8.92. Found: C,
74.49; H, 4.03; N, 10.11; O, 3.75; S, 7.61.
4.2.3.1. Koster test. Acetic acid-induced writhing in
mice was performed according to the Koster test.35,36
The mice were divided into 15 groups and received all
the tested compounds at dose of 70 mg/kg and indometh-
acin at dose of 35 mg/kg. After 60 min interval, the mice
received 0.6% acetic acid ip (0.2 mL/mice). The number
of writhings in 30 min period was counted and evaluated.
4.2. Pharmacology
4.2.3.2. Hot-plate test. Hot-plate test was conducted
according to Eddy and Leimback37 using an electroni-
cally controlled hot-plate (Ugo Basile, Italy) adjusted
at 52 0.1 °C and the cut-off time was 60 s. Fifteen
groups of mice each of six were used. The mice were di-
vided and received the same doses of tested compounds
and indomethacin as mentioned before. The time
elapsed until either paw licking or jumping occurs is re-
corded before and 1 h after oral administration.
4.2.1. Animals. Swiss mice (weighing 20–30 g) purchased
from the animal house of National Research Centre
were used. The animals were housed in standard metal
cages in an air-conditioned room at 22 3 °C, 55 5%
humidity and provided with standard laboratory diet
and water ad libitum. Experiments were performed be-
tween 9 am and 3 pm. Group of six mice was used for
each experiment. All experimental procedures were con-
ducted in accordance with the Guide for Care and Use
of Laboratory Animals and in accordance with the Lo-
cal Animal Care and Use Committee. The distilled water
was used as a vehicle for all tested compounds and 5%
sodium bicarbonate for indomethacin.
4.2.4. Statistical analysis. Data are expressed as
means SE. In anti-inflammatory study data are
expressed as means SE. The results of carrageenan-in-
duced paw oedema are expressed as percentage of
change to the control (pre-drug) values. Differences be-
tween vehicle control and treatment groups were tested
using one-way ANOVA followed by multiple compari-
sons by the Duncan’s multiple range test. A probability
value <0.05 was considered statistically significant.
4.2.2. Anti-inflammatory activity. Anti-inflammatory
activity in acute model was carried out according to
Winter et al.33 and Obkowicz et al.34 Mice were divided
into 15 groups each of six. They received orally saline as
control, tested compounds (70 mg/kg) and indometha-
cin (35 mg/kg), according to Rani et al. and Souza
et al.38,40 after induction of oedema by subplanter injec-
tion of 50 lL of 1% carrageenan (Sigma, USA) in saline
into the pad of right paw. The difference in hind footpad
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