Synthesis and biological evaluation of new N-substituted-N′-(3,5-di/ 1,3,5-trimethylpyrazole-4-yl)thiourea/urea derivatives

Article abstract of DOI:10.1016/j.ejps.2005.05.005

Several thiourea and urea derivatives were prepared by the reaction of 4-aminopyrazoles with substituted isothiocyanates or isocyanates. The novel compounds were tested anticonvulsant activity using by pentylenetetrazole- induced seizure (PTZ) and maximal electroshock seizure (MES) tests. Among the tested compounds, thiourea derivatives of 4b were afforded 90 and 100% protection in PTZ and MES tests at 50 mg/kg, respectively. Urea derivatives of 5a and 5b were afforded 82 and 100% protection both at 25 and 50 mg/kg. Also synthesized compounds were screened for antituberculosis activity against Mycobacterium tuberculosis H₃₇Rv at 6.25 μg/mL concentration but they were not found active at these concentration. In addition, some selected compounds were evaluated for in vitro anti-HIV activity and they were all negative.

Full text of DOI:10.1016/j.ejps.2005.05.005

European Journal of Pharmaceutical Sciences 26 (2005) 97–103
Synthesis and biological evaluation of new N-substituted-Nꢀ-(3,5-
di/1,3,5-trimethylpyrazole-4-yl)thiourea/urea derivatives
a
a,∗
b
b
˘
˘
¨
˘
˘
Bedia Koc¸yigit Kaymakc¸ıoglu , Sevim Rollas , Eylem Korcegez , Feyza Arıcıoglu
a
Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Haydarpas¸a, 81010 Istanbul, Turkey
b
Marmara University, Faculty of Pharmacy, Department of Pharmacology, Haydarpas¸a, 81010 Istanbul, Turkey
Received 6 May 2004; received in revised form 4 January 2005; accepted 9 May 2005
Available online 28 June 2005
Abstract
Several thiourea and urea derivatives were prepared by the reaction of 4-aminopyrazoles with substituted isothiocyanates or isocyanates.
The novel compounds were tested anticonvulsant activity using by pentylenetetrazole-induced seizure (PTZ) and maximal electroshock
seizure (MES) tests. Among the tested compounds, thiourea derivatives of 4b were afforded 90 and 100% protection in PTZ and MES tests
at 50 mg/kg, respectively. Urea derivatives of 5a and 5b were afforded 82 and 100% protection both at 25 and 50 mg/kg. Also synthesized
compounds were screened for antituberculosis activity against Mycobacterium tuberculosis H37Rv at 6.25 ␮g/mL concentration but they were
not found active at these concentration. In addition, some selected compounds were evaluated for in vitro anti-HIV activity and they were all
negative.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Anticonvulsant activity; 3,5-Dimethylpyrazole; Thiourea; Urea
1. Introduction
as structurally novel anticonvulsant. The anticonvulsant drug
design was based on the presumption (Pandeya et al., 1998)
that for the activity in maximal electroshock (MES) evalu-
ation at least one phenyl or similar aromatic group in close
proximity to two electron donor atoms and for activity in the
pentylenetetrazole (PTZ) evaluation an alkyl group substi-
tuted close to two electron donor atoms was required. It has
been hypothesized that, ureas and thioureas displaying anti-
convulsant activity interact at locations on the putative bind-
ing site designated as aryl binding site, a hydrogen bonding
domain and an auxiliary aryl or other hydrophobic binding
site (Dimmock et al., 2000). In view of these data, thiourea
moities have been attached to a pyrazole ring and to an aryl or
alkyl group. Thus, the aim of the present study was to prepare
a series of thiourea and ureas and evaluated for anticonvulsant
properties using MES and PTZ tests in mice.
Epilepsy is characterized by seizures, which are due to
abrupt discharge of a large population of neurons firing
synchronously. It is estimated that in developed countries
0.5–1% of the population suffer from epilepsy. However,
with the available antiepileptic drugs on the market, about
70% of the people with epilepsy achieve satisfactory seizure
control. During the 5 years, several new drugs have been
approved or in the process of being approved, e.g. lamotrig-
ine, gabapentin, tiagabine and milacemide. Although these
drugs have been shown to be effective in reducing seizures
in a number of patients their efficacy does not appear to be
superior to that of the drugs developed earlier. For that rea-
son, the need for more effective and less toxic antiepileptic
¨
drugs still exists (Loscher et al., 1991).
In recent years, urea and thioureas (Heinisch et al., 1997;
Masereel et al., 1997; Pandeya et al., 2001) have emerged
In addition to anticonvulsant activity, similar compounds
containing thiourea moiety have shown antituberculosis
¨ ¨
¨
(Kuc¸ukguzel et al., 2001) and anti-HIV (Bell et al., 1995;
Uc¸kun et al., 1999) activities. Therefore, all synthesized com-
pounds were screened for antituberculosis activity and some
∗
Corresponding author. Tel.: +90 216 414 2962; fax: +90 216 345 2952.
E-mail address: sevim@sevimrollas.com (S. Rollas).
0928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2005.05.005

98
B.K. Kaymakc¸ıog˘lu et al. / European Journal of Pharmaceutical Sciences 26 (2005) 97–103
2973, 1557–1440, 1332, 1297, 1249 cm−1 1H NMR,
.
selected compounds were evaluated for in vitro anti-HIV
activity.
δ ppm: 1.01 (t, 3H, CH3–CH2–), 1.98 (s, 6H, pyrazole
3,5-dimethyl), 3.43 (m, 2H, –CH2–CH3), 7.10 (s, 1H, NH),
8.50 (s, 1H, NH), 12.20 (s, 1H, pyrazole NH). Mass (EI) m/z:
198(M+), 153, 121, 120, 111, 110, 95, 88, 85, 70, 42(100%),
41, 39. Anal. calcd. for C8H14N4S: C, 48.46; N, 28.26; S,
16.17. Found: C, 48.77; N, 27.50; S, 15.17.
2. Materials and methods
2.1. Instrumentation and chemicals
Melting points were determined on a Buchi 530 melting
point apparatus and uncorrected. UV spectra were obtained
on a Shimadzu UV 2100 S spectrophotometer (1 mg/100 mL
in ethanol). Infrared (IR) spectra were run as potassium bro-
mide disk on Perkin-Elmer 1600 FTIR. 1H nuclear magnetic
resonance spectra were taken on a Bruker AVANC—DPX
400 spectrometer in DMSO. Chemical shifts were reported
in ppm related to the internal standard tetramethylsilane, the
following abbreviations were used: s, singlet; d, dublet; t,
triplet; m, multiplet. Mass spectra were measured on a Fisons
Instruments VG Platform II LC–MS Double focusing spec-
trometer. Elemental analyses were performed with Leco-932
(C, H, N, S-Elemental analyser) instrument at The Scien-
2.2.1.3. N-Propyl-Nꢀ-(3,5-dimethylpyrazole-4-yl)thiourea
(4c). Rt values in acetonitrile/water (60:40) 1.82 min.
White powder (45%); mp 227–228 ◦C; IR: 3194, 2979,
1554–1474, 1350, 1268, 1242 cm−1. 1H NMR, δ ppm: 0.93
(t, 3H, –CH3), 1.59 (q, 2H, –CH2–), 2.10 (s, 6H, pyrazole
3,5-dimethyl), 3.40 (s, 2H, –CH2–), 7.10 (s, 1H, NH), 8.40
(s, 1H, NH), 12.10 (s, 1H, pyrazole NH). Mass (EI) m/z:
212(M+), 179, 153, 121, 111, 95, 70, 42(100%), 41, 39.
Anal. calcd. for C9H16N4S: C, 50.91; N, 26.39; S, 15.10.
Found: C, 50.40; N, 25.88; S, 14.70.
2.2.1.4. N-Cylohexyl-Nꢀ-(3,5-dimethylpyrazole-4-
¨ ˙
tific and Technical Research Council of Turkey (TUBITAK)
yl)thiourea (4d). Rt values in acetonitrile/water (50:50)
1.78 min. White powder (52%); mp 251–253 ◦C; IR: 3260,
3186, 2937, 2848, 1594–1450, 1356, 1255 cm−1. 1H NMR,
δ ppm: 0.9–1.90 (m, 10H, cyclohexyl protons), 1.97 (s, 6H,
pyrazole 3,5-dimethyl), 4.10 (s, 1H, proton of cyclohexyl),
6.75 (s, 1H, NH), 8.50 (s, 1H, NH), 12.00 (s, 1H, pyrazole
NH). Mass (EI) m/z: 252(M+), 219, 153, 120, 111(100%),
110, 95, 42, 41, 39. Anal. calcd. for C12H20N4S: C, 57.11;
N, 22.20; S, 12.71. Found: C, 57.01; N, 21.72; S, 12.30.
Instrumentally Analysis Laboratory. HPLC analysis was per-
formed using a Waters Novopak C18 150 mm × 3.9 mm i.d.
column, eluting with 50:50 and 60:40% gradients of acetoni-
trile/water. All starting materials and reagents were commer-
cially available from Merck, Aldrich and Fluka.
2.2. Syntheses
2.2.1. General procedure for preparation of N-
substituted-Nꢀ-(3,5-di/1,3,5-trimethylpyrazole-4-
yl)thioureas (4a–m, 7a–b)
Starting compounds of 4-aminopyrazole derivatives 1 and
2 were prepared following reported procedures (Koc¸yigit
2.2.1.5. N-Benzyl-Nꢀ-(3,5-dimethylpyrazole-4-yl)thiourea
(4e). Rt values in acetonitrile/water (50:50) 1.61 min.
Creamy powder (64%); mp 248–250 ◦C; IR: 3200,
1589–1495, 1340, 1268–1240, 696 cm−1 1H NMR, δ
.
˘
˘
Kaymakc¸ıoglu and Rollas, 2002). A solution of appropriate
ppm: 2.00 (s, 6H, pyrazole 3,5-dimethyl), 4.60 (d, 2H,
–CH2–C6H5), 7.40 (m, 5H, Ar–H), 7.60 (s, 1H, NH), 8.75
(s, 1H, NH), 12.20 (s, 1H, pyrazole NH). Anal. calcd. for
C13H16N4S: C, 59.97; N, 21.52; S, 12.32. Found: C, 60.45;
N, 21.49; S, 12.41.
isothiocyanate (0.0085 mol) in anhydrous acetone (100 mL)
was added dropwise to a solution of the appropriate amine
(0.0085 mol) in anhydrous acetone (100 mL). Resulting mix-
ture was refluxed for 4–5 h and the solvent was evaporated
in vacuo to give the crude thioureas 4a–m and 7a–b, which
were purified by recrystallization from the ethanol.
2.2.1.6. N-Phenyl-Nꢀ-(3,5-dimethylpyrazole-4-yl)thiourea
(4f). Rt values in acetonitrile/water (60:40) 1.72 min. White
crystal (74%); mp 212–214 ◦C; IR: 3270–3172, 1653–1450,
1362, 1270, 1235, 693 cm−1. 1H NMR, δ ppm: 2.04 (s, 6H,
pyrazole 3,5-dimethyl), 7.10–7.60 (m, 5H, Ar–H), 8.80,
9.10, 9.80 (2H, NH–CS–NH), 12.20 (s, 1H, pyrazole NH).
Anal. calcd. for C12H14N4S: C, 58.51; N, 22.74; S, 13.02.
Found: C, 58.57; N, 22.27; S, 12.73.
2.2.1.1. N-Methyl-Nꢀ-(3,5-dimethylpyrazole-4-yl)thiourea
(4a). Rt values in acetonitrile/water (50:50) 1.25 min. White
powder (65%); mp 250–251 ◦C; IR: 3208,1530–1473,
1
1355, 1255 cm−1. H NMR, δ ppm: 1.67 (s, 6H, pyrazole
3,5-dimethyl), 2.67 (s, 3H, –CH3), 7.00 (s, 1H, NH), 8.70
(s, 1H, NH), 12.20 (s, 1H, pyrazole NH). Mass (EI) m/z:
184(M+), 154, 153, 135, 120, 111, 95, 70, 42(100%), 41, 39.
Anal. calcd. for C7H12N4S: C, 45.63; N, 30.41; S, 17.40.
Found: C, 46.07; N, 29.70; S, 17.63.
2.2.1.7. N-(2-Fluorophenyl)-Nꢀ-(3,5-dimethylpyrazole-4-
yl)thiourea (4g). Rt values in acetonitrile/water (60:40)
1.92 min. White powder (65%); mp 230 ◦C; IR: 3291, 3172,
2.2.1.2. N-Ethyl-Nꢀ-(3,5-dimethylpyrazole-4-yl)thiourea
(4b). Rt values in acetonitrile/water (50:50) 1.78 min.
Creamy powder (51%); mp 232–233 ◦C; IR (KBr): 3207,
2982, 1591–1453, 1362, 1284, 1240–1041, 864, 823 cm−1
.
1H NMR, δ ppm: 2.31 (s, 6H, pyrazole 3,5-dimethyl),
7.14–7.21 (m, 4H, Ar–H), 8.60, 8.85, 9.20, 9.50 (2H,

B.K. Kaymakc¸ıog˘lu et al. / European Journal of Pharmaceutical Sciences 26 (2005) 97–103
99
1
1073, 828 cm−1. H NMR, δ ppm: 2.05 (s, 6H, pyrazole
3,5-dimethyl), 7.40–7.80 (m, 4H, Ar–H), 9.15, 9.40, 10.05
(2H, NH–CS–NH), 12.20 (s, 1H, pyrazole NH). Mass (EI)
m/z: 314(M+), 282, 281, 220, 219(%100), 203, 153, 145,
121, 111, 95, 77, 73, 69, 51, 42, 41, 39, 32. Anal. calcd.
for C13H13F3N4S: C, 49.67; N, 17.82; S, 10.20. Found: C,
49.78; N, 17.47; S, 9.67.
NH–CS–NH), 12.19 (s, 1H, pyrazole NH). Anal. calcd. for
C12H13FN4S: C, 54.53; H, 4.96; N, 21.20; S, 12.13. Found:
C, 54.22, H, 4.72; N, 20.69; S, 11.95.
2.2.1.8. N-(3-Fluorophenyl)-Nꢀ-(3,5-dimethylpyrazole-4-
yl)thiourea (4h). Rt values in acetonitrile/water (50:50)
1.55 min. White powder (33%); mp 193–194 ◦C; IR: 3173,
1600–1488, 1243–1073, 1244, 1210, 851 cm−1. 1H NMR, δ
ppm: 2.20 (s, 6H, pyrazole 3,5-dimethyl), 6.88–7.65 (m, 4H,
Ar–H), 8.40, 8.65 (2H, NH–CS–NH), 11.60 (s, 1H, pyrazole
NH). Anal. calcd. for C12H13FN4S: C, 54.53; N, 21.20; S,
12.13. Found: C, 54.87; N, 21.12; S, 12.38.
2.2.1.14. N-Propyl-Nꢀ-(1,3,5-trimethylpyrazole-4-yl)thiou-
rea (7a). Rt values in acetonitrile/water (60:40) 1.93 min.
White powder (38%); mp 179–180 ◦C; IR: 3214, 2956,
2929, 2870, 1545–1480, 1345, 1260, 1230 cm−1. 1H NMR,
δ ppm: 0.80 (t, 3H, CH3), 1.50 (m, 2H, –CH2–), 1.93,2.00
(s, 6H, pyrazole 3,5-dimethyl), 3.35 (s, 2H, –CH2–NH),
3.62 (s, 3H, pyrazole N–CH3), 7.10 (s, 1H, NH), 8.50 (s,
1H, NH). Mass (EI) m/z: 226(M+), 193, 192, 167, 150, 125,
109, 84, 83, 82, 56(100%), 39. Anal. calcd. for C10H18N4S:
C, 53.06; N, 24.75; S, 14.17. Found: C, 53.51; N, 24.91; S,
13.82.
2.2.1.9. N-(4-Chlorophenyl)-Nꢀ-(3,5-dimethylpyrazole-4-
yl)thiourea (4i). Rt values in acetonitrile/water (60:40)
1.84 min. White powder (52%); mp 212 ◦C; IR: 3273, 3160,
1595–1492, 1404, 1266, 1235, 1089, 824 cm−1. H NMR,
1
δ ppm: 2.00 (s, 6H, pyrazole 3,5-dimethyl), 7.40–7.60 (m,
4H, Ar–H), 8.90, 9.10, 9.90 (2H, NH–CS–NH), 12.20 (s,
1H, pyrazole NH). Anal. calcd. for C12H13ClN4S: C, 51.33;
N, 19.95; S, 11.42. Found: C, 51.74; N, 19.90; S, 11.92.
2.2.1.15. N-Phenyl-Nꢀ-(1,3,5-trimethylpyrazole-4-yl)thiou-
rea (7b). Rt values in acetonitrile/water (60:40) 1.85 min.
White powder (77%); mp 220–222 ◦C; IR: 3170, 1592–1420,
1380, 1357, 1260, 1230, 691 cm−1. 1H NMR, δ ppm: 2.00,
2.08 (s, 6H, pyrazole 3,5-dimethyl), 3.28 (s, 3H, pyra-
zole N–CH3), 7.10–7.30 (m, 5H, Ar–H), 8.44 (s, 2H,
NH–CS–NH). Anal. calcd. for C13H16N4S: C, 59.97; N,
21.52; S, 12.32. Found: C, 60.22; N, 21.33; S, 12.43.
2.2.1.10. N-(4-Methylphenyl)-Nꢀ-(3,5-dimethylpyrazole-
4-yl)thiourea (4j). Rt values in acetonitrile/water (50:50)
2.16 min. White powder (65%); mp 210–211 ◦C; IR: 3256,
1
3166, 2980, 1590–1543, 1364, 1268, 1238, 816 cm−1. H
NMR, δ ppm: 2.00 (s, 6H, pyrazole 3,5-dimethyl), 2.30 (s,
3H, CH3–C6H5), 7.10–7.40 (m, 4H, Ar–H), 8.75, 9.00, 9.80
(2H, NH–CS–NH), 12.20 (s, 1H, pyrazole NH). Anal. calcd.
for C13H16N4S: C, 59.97; N, 21.52; S, 12.32. Found: C,
60.21; N, 21.20; S, 12.34.
2.2.2. General procedure for preparation of N-
substituted-Nꢀ-(3,5-di/1,3,5-trimethylpyrazole-4-yl)ureas
(5a–b, 8)
These compounds were prepared by treating 4-amino-
3,5-di/1,3,5-trimethylpyrazole with an equivalent amount of
appropriate isocyanate at 80 ◦C. Upon cooling the prod-
uct was washed petroleum ether and recrystallized from
ethanol.
2.2.1.11. N-(4-Methoxyphenyl)-Nꢀ-(3,5-dimethylpyrazole-
4-yl)thiourea (4k). Rt values in acetonitrile/water (50:50)
1.96 min. White powder (40%); mp 199–201 ◦C; IR: 3253,
3171, 3002, 1594–1512, 1370, 824 cm−1. 1H NMR, δ ppm:
2.05 (s, 6H, pyrazole 3,5-dimethyl), 3.73 (s, 3H, O–CH3),
6.90–7.25 (m, 4H, Ar–H), 8.80, 9.50 (2H, NH–CS–NH),
12.10 (s, 1H, pyrazole NH). Anal. calcd. for C13H16N4OS:
C, 56.50; N, 20.27; S, 11.60. Found: C, 56.51; N, 19.82; S,
11.47.
2.2.2.1. N-(2-Ethoxyphenyl)-Nꢀ-(3,5-dimethylpyrazole-4-
yl)urea (5a). Rt values in acetonitrile/water (50:50)
1.36 min. White powder (48%); mp 198–200 ◦C; IR: 3271,
2978–2925, 1652–1440, 1383, 1335, 830 cm−1. H NMR,
1
2.2.1.12. N-(4-Nitrophenyl)-Nꢀ-(3,5-dimethylpyrazole-4-
yl)thiourea (4l). Rt values in acetonitrile/water (50:50)
1.96 min. Yellow powder (57%); mp 210–211 ◦C; IR:
3278, 3154, 2988, 1653–1508, 1423, 1340, 1267–1237,
δ ppm: 1.34 (s, 3H, –CH2–CH3), 2.05 (s, 6H, pyrazole 3,5-
dimethyl), 4.06 (s, 2H, –CH2–), 6.54–8.07 (m, 4H, Ar–H),
8.10–8.07 (2H, NH–CO–NH), 12.00 (s, 1H, pyrazole NH).
Anal. calcd. for C14H18N4O2: C, 61.30; N, 20.42. Found: C,
60.60; N, 18.98.
850 cm−1 1H NMR, δ ppm: 2.04 (s, 6H, pyrazole 3,5-
.
dimethyl), 7.80–8.30 (m, 4H, Ar–H), 9.30, 9.40, 10.05 (2H,
NH–CS–NH), 12.20 (s, 1H, pyrazole NH). Anal. calcd. for
C12H13N5O2S: C, 49.47; N, 24.04; S, 11.01. Found: C,
49.73; N, 23.59; S, 10.93.
2.2.2.2. N-Phenyl-Nꢀ-(3,5-dimethylpyrazole-4-yl)urea (5b).
Rt values in acetonitrile/water (50:50) 4.40 min. White
powder (48%); mp > 250 ◦C; IR: 3290, 1640–1445, 1312,
2.2.1.13. N-(3-Trifluoromethylphenyl)-Nꢀ-(3,5-dimethylpy-
razole-4-yl)thiourea (4m). Rt values in acetonitrile/water
(50:50) 1.61 min. White powder (57%); mp 210–213 ◦C;
IR: 3260, 3170, 2987, 1597–1450, 1335, 1280, 1256, 1243,
731 cm−1 1H NMR, δ ppm: 2.00 (s, 6H, pyrazole 3,5-
.
dimethyl), 6.90–7.50 (m, 5H, Ar–H), 7.40, 8.50 (2H,
NH–CO–NH), 12.00 (s, 1H, pyrazole NH). Mass (EI) m/z:
230(M+), 212, 137, 111, 96, 95, 93, 80, 78, 77, 70, 69, 64,

100
B.K. Kaymakc¸ıog˘lu et al. / European Journal of Pharmaceutical Sciences 26 (2005) 97–103
Scheme 1. Conditions: (a) NaNO2, HCl, 0–5 ◦C; (b) acetylacetone, CH3COONa; (c) hydrazine hydrate, AcOH, reflux; (d) hydrazine hydrate, EtOH, 50 ◦C.
63, 43, 42(100%), 41, 39, 32. Anal. calcd. for C12H14N4O:
C, 62.59; N, 24.33. Found: C, 62.34; N, 23.78.
Sathyanarayana, 1984; Wawer and Koleva, 1993). As a result
of common fragmentation of thiourea compounds in EI–mass
spectrum, isothiocyanate and amines were observed due to
cleavege of NH–CS bond and the migration of hydrogen atom
on nitrogen to the other nitrogen atom (Baldwin et al., 1968;
Ueda et al., 1971). According to literature, thiourea com-
pounds having aryl group on nitrogen do not show molecular
2.2.2.3. N-(2-Ethoxyphenyl)-Nꢀ-(1,3,5-trimethylpyrazole-4-
yl)urea (8). Rt values in acetonitrile/water (60:40) 1.13 min.
White powder (31%); 185–187 ◦C; IR: 3316, 3065, 2980,
2934, 2888, 1666–1473, 1250, 1220, 748–685 cm−1 1H
.
¨ ¨
¨
ion peak in mass spectrum (Kuc¸ukguzel et al., 1994). There-
fore, mass spectrums of the compounds were not taken except
6h compound having aromatic ring on nitrogen.
NMR, δ ppm: 1.39 (m, 3H, –CH2–CH3 and 6H, pyrazole
3,5-dimethyl), 4.10 (m, 5H, –CH2– and pyrazole N–CH3),
6.90–7.20 (m, 4H, Ar–H), 8.00, 8.40 (2H, NH–CO–NH).
Mass (EI) m/z: 288(M+), 151, 137(%100), 111, 110, 109,
92, 77, 65, 63, 56, 55, 39, 32. Anal. calcd. for C15H20N4O2:
C, 62.48; N, 19.43. Found: C, 63.10; N, 18.60.
Compounds 5a and 5b displayed the resonance of urea
NH at 7.40–8.50 ppm in the same area of aromatic protons.
Different from the others the urea NH protons of 8 resonated
at 8.00 and 8.40 ppm as a two singlet (Pavia et al., 1990). The
main way of cleavage is the broken of NH CO bond as in
the thiourea derivatives in EI–mass spectrum of 5a and 8.
2.3. Structure determination
Starting compounds 4-amino-3,5-dimethylpyrazole 1
and 4-amino-1,3,5-trimethylpyrazole 2 were prepared in
four steps from aniline or p-amino ethylbenzoate following
2.4. Anticonvulsant activity
˘
˘
the reported procedure (Koc¸yigit Kaymakc¸ıoglu and Rollas,
2002). The intermediate products of 4-phenylazo-3,5-
dimethylpyrazole or 4-(4-ethoxycarbonylphenylazo)-1,3,5-
trimethylpyrazole were reduced with hydrazine hydrate
to give target 4-amino-3,5-di/1,3,5-trimethylpyrazole
(Scheme 1). The amine compounds 1 and 2 were reacted
with substituted isothiocyanates in dry acetone medium
and give some novel N-substituted-Nꢀ-(3,5-di/1,3,5-
trimethylpyrazole-4-yl)thioureas. Finally, urea compounds
(5a–b, 8) were prepared by treating 4-amino-3,5-di/1,3,5-
trimethylpyrazole with an equivalent amount of appropriate
isocyanate at 80 ◦C (Scheme 2). The purities of the
synthesized compounds were checked elemental analyses.
In the 1H NMR spectra of the thiourea derivatives the con-
taining alkyl groups attached to N1 showed N1–H and N2–H
at 6.75–7.10 and 8.40–8.70 ppm, respectively. The same pro-
tons of the compounds containing aryl group at N1 position
were observed as a wide spread peak at 8.75–10.05 ppm in
DMSO-d6 (except 4h in Aceton-d6) suggesting the existence
of two isomer (syn/anti) (Salman et al., 1991; Sudha and
Male and female adult Swiss albino mice weighing
20–30 g were used. The animals were housed in colongy
cages, under standart laboratory conditions, with free access
to food and tap water. All mice were maintanied at an ambient
temperature of 20 1 ◦C and on a natural dark cycle. After
the adaption period of 10 days, experimental groups were
chosen randomyl. Each mouse was used only once.
Scheme 2. Conditions: (a) RNCS, acetone, reflux; (b) RNCO, 80 ◦C.

B.K. Kaymakc¸ıog˘lu et al. / European Journal of Pharmaceutical Sciences 26 (2005) 97–103
101
2.4.1. PTZ test
2.7. Antituberculosis activity
The synthesized compounds suspended in a 5% aque-
ous suspension of gum acacia and were administered 10,
25 and 50 mg/kg intraperitoneally. Thirty minutes after the
administration of the test compounds, mice were injected
with PTZ 55–80 mg/kg intraperitoneally and observed for
15 min. Motor responses were graded 0–5 according to the
scale of Racine, where grade 1: no movements, grade 2: head
twitching and myoclonic jerks (MKJ), grade 3: clonic fore-
limb convulsions, grade 4: three plus change in posture, grade
5: falling back and generalized convulsions with tonic exten-
tion (Racine, 1972).
Primary screen was conducted at 6.25 ␮g/mL (or molar
equivalent of highest molecular weight compound in a series
of congeners) againist Mycobacterium tuberculosis H37Rv in
BACTEC 12B medium using the BACTEC 460 radiometric
system (Inderleid, 1991). Rifampicin was used as the standart
in the test. Compounds effecting <90% inhibition in the pri-
mary screen (MIC > 6.25 ␮g/mL) were not evaluated further.
3. Results and discussion
The anticonvulsant activity of the new compounds were
determined by using PTZ and MES tests. These rodent mod-
els are widely used as standard methods for predicting protec-
tion against generalized tonic-clonic and generalized absence
seizures in humans.
The synthesized compounds were administered 10, 25 and
50 mg/kg intraperitoneally. Thirty minutes after the adminis-
tration of the test compounds, mice were injected with PTZ
55–80 mg/kg intraperitoneally and observed for 15 min.
First of all ED50 is calculated for PTZ and 80 mg/kg is
applied. All compounds were administered 25 and 50 mg/kg
intraperitoneally and injected 30 min before MES. At the
beginning of the study, value of CC50 (convulsive current
50) of animals and its 95% fiducial limits were calculated by
the method of Litchfield and Wilcoxon (1949).
The animals of the control group received same volume of
saline and standart drugs were carbamazepine in PTZ tests
or phenytoin in MES. Statistical analysis was done using
“Chi-square” method. Locomotor activity were analysed by
one-way ANOVA followed by with Dunnett’s test for com-
parisons. ED50 values, as well as their statistical evaluation,
were estimated by computer probit analysis, according to
Litchfield and Wilcox. The anticonvulsant activity results
were shown in Table 1. The anticonvulsant potential of these
compounds were shown for both PTZ and MES models. In
Table 1, effect of all compounds in 25 and 50 mg/kg is pre-
sented. Beside percentage protection from grade 5 latency of
myoclonic jerk (MKJ) and percentage protection from death
are presented.
2.4.2. MES test
All compounds were administered 25 and 50 mg/kg
intraperitoneally and injected 30 min before MES. Each
mouse was used only once. The electroshocks were evoked
through a current transmitter producing 60 Hz square waves
(Ugo Basil and Arı Techincal ECT unit). Flow duration of
current and the duration of each square wave were fixed at
0.2 s, and at 0.4 m, respectively. During the shock, electrodes
were attached to each animal’s ears and the animals lay on
their backs, their tails being fixed. Thus, observation of the
tonic and clonic convulsions that appeared during the seizure
was ensured. In mice, maximal electroshock seizures consist
of a latency period lasting 1.6 s. Followed by a short initial
flexion period, then 13.2 s of tonic hindlimb extension and
7.6 s of terminal clonus. Total duration of seizure was 22.3 s.
The shock was applied in the afternoon between 2:00 and
5:00 p.m.
2.5. Locomotor activity
Locomotor activities were measured at by a wooden box
with a floor 35 cm × 50 cm and plexiglass wall 21 cm high.
The activity cage was divided into 12 equal areas, and there
were 12 equally spaced holes in the center of each area, 3 cm
diameter and 1 cm deep. Mice were placed in a corner and
locomotor activity was counted as cross of lines between
squares for 5 min (File and Wardill, 1975).
2.6. Anti-HIV activity
The results from PTZ model basically simulate petit mal
seizures. For PTZ model compounds 1, 4a, 4d, 4e, 4g, 4h, 4i,
4j, 4k, 4m, 5a and 5b did not show a dose-related effect. The
most active compound in suppressing MKJ was found as 7b
followed by 4f. In terms of protection from death 4a, 4h and
7b were found 100% effective in 25 mg/kg in PTZ-induced
convulsions. ED50 of the compounds were calculated for %
of protection for grade 5 PTZ-induced convulsions. ED50
values were found 22.76, 17.14, 20.62, 30.32, 22.01, 61.00
and 25.65 for 2, 4b, 4c, 4f, 4j, 4l and 7b, respectively. Among
these compounds, 4b was found the most active in percentage
protection of grade 5 in PTZ-induced convulsions and ED50
was 17.14.
The experimentation was performed at the National Can-
cer Institute, Bethesta, MD, USA, following the standart pro-
cedure in use by N.C.I. (Weislow et al., 1989). Some selected
thiourea compounds (4b, 4e, 4f, 4g, 4i, 4j, 4k, 4l, 4m and
7b) have been screened for their anti-AIDS (HIV-1) activity,
which involved susceptible human host cells (CEM cell line).
All tests are compared with at least one positive (e.g. AZT-
treated) control performed at the same time under identical
conditions, and with other appropriate controls (untreated
infected and untreated non-infected cells, drug containing
wells without cells, etc.) on the same plate. Data are reviewed
in comparison with other tests carried out at the same time
and determination of activity is performed.
The results from maximal electroshock (MES) model
basically simulate grand mal seizures. In MES-induced grade

102
B.K. Kaymakc¸ıog˘lu et al. / European Journal of Pharmaceutical Sciences 26 (2005) 97–103
Table 1
Anticonvulsant activity results of the compounds
Compound
R, R1
Dose (mg/kg)
PTZ testa (% of protection)
MES testa (% of protection)
MKJ
0
Grade 5
0
Exitus
0
Grade 5
0
Exitus
0
Control
1
–, H
25
50
20*
8
72***
77***
95***
77***
78***
80***
89***
100***
2
–, CH3
25
50
0
0
60**
80***
60**
100***
56**
78***
100***
100***
4a
4b
4c
Methyl, H
Ethyl, H
Propyl, H
25
50
30*
40**
100***
63**
67**
88***
100***
25
50
8
0
77***
90***
85***
90***
100***
100***
100***
100***
25
50
36*
0
64**
89***
90***
89***
29*
100***
57**
100***
4d
4e
Cylohexyl, H
Benzyl, H
25
0
82***
81***
100***
100***
25
50
0
27*
73***
64**
82***
73***
100***
100***
100***
100***
4f
Phenyl, H
25
50
70***
9
60**
64**
80***
82***
26
63**
87***
87***
4g
4h
4i
2-Fluorophenyl, H
3-Fluorophenyl, H
4-Chlorophenyl, H
4-Methylphenyl, H
4-Methoxyphenyl, H
4-Nitrophenyl, H
3-Trifluoromethylphenyl, H
2-Ethoxyphenyl, H
Phenyl, H
25
50
14
50
71***
42**
86***
58**
100***
100***
100***
100***
25
50
25*
0
78***
42**
100***
67**
100***
100***
100***
100***
25
50
9
0
64**
33
81***
55**
50**
63**
88***
63**
4j
25
50
0
10
64**
77***
64
78***
52**
64**
60**
85
4k
4l
25
50
9
9
55**
45
73***
55**
100***
100***
100***
100***
25
50
30*
40**
30*
40**
90***
100***
20*
100***
80***
100***
4m
5a
5b
7b
25
50
0
0
64**
20
73***
40**
100***
100***
100***
100***
25
50
9
0
82***
82***
82***
82***
100***
100***
100***
100***
25
50
9
0
80***
62**
70**
69**
75***
88***
88***
100***
Phenyl, CH3
25
50
100***
0
50**
80***
100***
80***
100***
100***
100***
100***
Carbamazepine/phenytoin
98***
100***
100***
100***
100***
Compounds were compared to control group and statistical significance is expressed.
a
Protected mice in the tests 30 min after intraperitoneal administration of 25 or 50 mg/kg of compound (n = 12–30).
*
**
p < 0.05.
p < 0.01.
p < 0.001.
***

B.K. Kaymakc¸ıog˘lu et al. / European Journal of Pharmaceutical Sciences 26 (2005) 97–103
103
5 seizures, compound 4b, 4d, 4e, 4g, 4h, 4k, 4m, 5a and 7b
were found 100% effective in 25 mg/kg. For MES model,
these compounds did not show a dose-related effect. ED50
values were found 21.44, 27.37, 28.33, 29.92, 35.57, 34.67,
31.72, 17.46 and 17.76 for 1, 2, 4a, 4c, 4f, 4i, 4l, 5a and 5b,
respectively.
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This study was supported by The Research Fund of
Marmara University (Project numbers 1999/45 and SAG-
˘
¨
¨
008/230902). We thank Dr. Joseph A. Maddry from the
Tuberculosis Antimicrobial Acquisition and Coordinating
Facility (TAACF), National Institute of Allergy and Infec-
tions Disease Southern Research Institute, GWL Hansen’s
Disease Center, Colorado State University, Birmingham,
Alabama, USA, for the in vitro evaluation of antimycobac-
terial activity using M. tuberculosis H37Rv. Also we are
grateful to Ph.D. Robert H. Shoemaker of the National Insti-
tute of Health, Bethesda, MD, USA, for anti-HIV screening.
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