Cyclization of 1-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]-acetyl} thiosemicarbazides to 1,2,4-triazole and 1,3,4-thiadiazole derivatives and their pharmacological properties

Article abstract of DOI:10.1135/cccc20050051

By the reaction of 4-methyl-4H-1,2,4-triazole-3-thiol with ethyl bromoacetate, ethyl [(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetate (1) was obtained. This compound was converted to [(4-methyl-4H-1,2,4-triazol-3-yl) sulfanyl]acetohydrazide (2). In the reaction of 2 with isothiocyanates, new thiosemicarbazides 3a-3g were obtained. The cyclization of 3a-3g in 2% aqueous solution of sodium hydroxide led to the formation of 4H-1,2,4-triazole-3(2H)- thione derivatives 4a-4g, whereas the cyclization in acid media led to the formation of 2-amino-1,3,4-thiadiazole derivatives 5a-5g. Molecular structure was confirmed by X-ray structure analysis of 3a, 4g, 5a and 5g. Compounds 4a, 4b and 4g were investigated pharmacologically to determine their effect on the central nervous system (CNS) in mice.

Full text of DOI:10.1135/cccc20050051

Cyclization and Pharmacological Properties of Thiosemicarbazides
51
CYCLIZATION OF 1-{[(4-METHYL-4H-1,2,4-TRIAZOL-3-YL)SULFANYL]-
ACETYL}THIOSEMICARBAZIDES TO 1,2,4-TRIAZOLE AND
1,3,4-THIADIAZOLE DERIVATIVES AND THEIR PHARMACOLOGICAL
PROPERTIES
a
a2
Alicja MALISZEWSKA-GUZ , Monika WUJECa1, Monika PITUCHA
,
Maria DOBOSZ *, Anna CHODKOWSKAb1, Ewa JAGIEŁŁO-WÓJTOWICZ
Liliana MAZUR and Anna E. KOZIOŁ
,
a,
b2
c1
c2
a
Department of Organic Chemistry, Faculty of Pharmacy, Medical University,
6 Staszica Str., 20-081 Lublin, Poland; e-mail: wujec@panaceum.am.lublin.pl,
pitucha@panaceum.am.lublin.pl
Department of Toxicology, Medical University,
8 Chodzki Str., 20-093 Lublin, Poland; e-mail: ewafarm@asklepios.am.lublin.pl,
paulina@asklepios.am.lublin.pl
Department of Crystallography, Faculty of Chemistry, Maria Curie-Sklodowska University,
20-031 Lublin, Poland; e-mail: lmazur2@op.pl, akoziol@hermes.umcs.lublin.pl
1
2
b
c
1
2
1
2
Received July 13, 2004
Accepted October 6, 2004
By th e reaction of 4-m eth yl-4H-1,2,4-triazole-3-th iol with eth yl brom oacetate, eth yl
[(4-m eth yl-4H-1,2,4-triazol-3-yl)sulfan yl]acetate (1) was obtain ed. Th is com poun d was con -
verted to [(4-m eth yl-4H-1,2,4-triazol-3-yl)sulfan yl]acetoh ydrazide (2). In th e reaction of 2
with isoth iocyan ates, n ew th iosem icarbazides 3a–3g were obtain ed. Th e cyclization of 3a–3g
in 2% aqueous solution of sodium h ydroxide led to th e form ation of 4H-1,2,4-triazole-
3(2H)-th ion e derivatives 4a–4g, wh ereas th e cyclization in acid m edia led to th e form ation
of 2-am in o-1,3,4-th iadiazole derivatives 5a–5g. Molecular structure was con firm ed by X-ray
structure an alysis of 3a, 4g, 5a an d 5g. Com poun ds 4a, 4b an d 4g were in vestigated ph ar-
m acologically to determ in e th eir effect on th e cen tral n ervous system (CNS) in m ice.
Keyw ord s: 1,2,4-Triazoles; 1,3,4-Th iadiazoles; Th iosem icarbazides; Cyclization s; X-ray dif-
fraction ; Crystal structure determ in ation ; Ph arm acological screen in g; An tidepressives.
4-Meth yl-4H-1,2,4-triazole-3-th iol can exist in two tautom eric form s sh own
in Sch em e 1. Nucleoph ilic substitution reaction s, depen din g on th e con di-
tion s used, can led eith er to th e S- or N-derivatives. In th is paper th e substi-
tution reaction with brom oacetate ester was in vestigated. Th e S-derivative,
eth yl [(4-m eth yl-4H-1,2,4-triazol-3-yl)sulfan yl]acetate (1) was obtain ed.
Th is com poun d was con verted to th iosem icarbazide derivative 3 an d, after
cyclization in alkalin e m edia, a group of n ew 5-{[(4-m eth yl-4H-1,2,4-triazol-
Collect. Czech. Chem. Commun. (Vol. 70) (2005)
doi:10.1135/cccc20050051

52
Maliszewska-Guz et al.:
3-yl)sulfan yl]m eth yl}-4H-1,2,4-triazole-3(2H)-th ion es 4 was obtain ed, wh ile
th e cyclization in acidic m edia gave n ew 2-(alkyl(aryl)am in o)-5-{[(4-m eth yl-
4H-1,2,4-triazol-3-yl)sulfan yl]m eth yl}-1,3,4-th iadiazoles 5.
Depen din g of th e n ature of substituen ts, th e 1,2,4-triazole an d 1,3,4-th ia-
diazole derivatives can sh ow various ph arm acological activity. New deriva-
tives of 1,2,4-triazole, wh ich were presen ted in previous papers, sh ow
strictly defin ed ph arm acological activity, e.g. poten tial action on th e cen -
tral n ervous system 1 an d an tich olin ergic2, an tiseptic3 an d an algesic4 action .
Th e 1,3,4-th iadiazole derivatives sh ow also som e ph arm acological activity,
e.g. an ticon vulsion 5–7, an tiviral an d an tibacterial8,9, ch em oth erapeutic10 as
well as an th elm in tic11 action .
Th e 1,2,4-triazole an d 1,3,4-th iadiazole derivatives could be prepared via
th e cyclization of acyl derivatives of th iosem icarbazide. It h as been foun d
th at cyclization in alkalin e m edia led to th e 1,2,4-triazole system 12–34 wh ile
in acidic m edia th e 1,3,4-th iadiazole system was obtain ed16,17,26,27,29,30
.
Th is paper is a con tin uation of th e system atic in vestigation s on th e
cyclization of n ew acyl derivatives of th iosem icarbazide. Th e reaction s are
presen ted in Sch em e 1. Som e of th e obtain ed com poun ds (4a, 4b, 4g) were
tested in vivo.
N
N
N
O
N
NH
N
+
BrCH2
O
OC2H5
N
N
H3C
N
H3C
SCH2C
S
SH
H3C
NH2NH2·H2O
OC2H5
1
N
N
N
N
N
R
NCS
O
N
H3C
SCH2CONHNHCNHR
SCH2C
H3C
NHNH2
S
2
3a-3g
2% NaOH
conc. H2SO4
R
a
b
c
d
e
f
Ph
4-CH3C6H4
4-OCH3C6H4
4-BrC6H4
2-FC6H4
Me
N
N
N
N
N
NH
N
N
S
CH2
S
CH2
S
NHR
N
N
N
S
CH3
CH3
R
4a-4g
5a-5g
g
Et
SCHEME 1
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Cyclization and Pharmacological Properties of Thiosemicarbazides
53
RESULTS AND DISCUSSION
Chemistry
4-Meth yl-4H-1,2,4-triazole-3-th iol is a startin g m aterial for th e syn th esis of
n ew derivatives of 1,2,4-triazole an d 1,3,4-th iadiazole. In th e reaction with
eth yl brom oacetate, eth yl [(4-m eth yl-4H-1,2,4-triazol-3-yl)sulfan yl]acetate
(1) was obtain ed. Th is com poun d was treated with 100% h ydrazin e h ydrate
to give [(4-m eth yl-4H-1,2,4-triazol-3-yl)sulfan yl]acetoh ydrazide (2). New
th iosem icarbazide derivatives 3 were obtain ed by reaction of 2 with
isoth iocyan ates. Th eir structure was con firm ed by X-ray an alysis of 3a
(Fig. 1a). Th e reaction was carried out in an h ydrous m eth an ol at room tem -
perature. Th iosem icarbazides 3 were cyclized in 2% aqueous solution of
NaOH to give derivatives of 1,2,4-triazole-3(2H)-th ion e 4a–4g, wh ereas th e
cyclization in acidic m edia led to th e form ation of 2-(alkyl(aryl)am in o)-
1,3,4-th iadiazole derivatives 5a–5g.
Th e obtain ed products were ch aracterized by th eir elem en tal an alysis, IR
1
an d H NMR spectra. Th e structures of selected com poun ds (3a, 4g, 5a, 5g)
were con firm ed by X-ray crystallograph y.
C5p
C4p
C3
S2
N2
N2
C6p
N4
N1
C5
N12
N11
C3
N4
C3p
C2p
C12
N1
C4
C1p
C5
N13
C41
C42
N13
C12
S1
S1
C2
C1
N11
C4
C1
C2
S2
N12
O1
a
b
C42
C4
N4
S1
C41
C3p
N11
C2p
C4
N4
N12
C1p
S2
S1
C1
C2
C4p
C5p
C3
C12
N12
C5
N1
N13
C1
C3
C2
C12
C5
N1
N13
N2
S2
C6p
N11
N2
c
d
FIG. 1
Molecular structure an d atom ic n um berin g sch em e for: a 3a, b 4g, c 5g, d 5a
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

54
Maliszewska-Guz et al.:
In th e IR spectra of th iosem icarbazide derivatives 3, th e followin g ch arac-
teristic absorption ban ds were observed: 1670–1681 cm –1 correspon din g to
C=O group an d 3120–3137 cm –1 correspon din g to NH group. In th e com -
poun ds 4 con tain in g th e 1,2,4-triazole system , th e absorption ban ds of th e
C–N group appeared at about 1520 cm –1 an d th ose for th e C=N group at
1610 cm –1. For 1,3,4-th iadiazole 5, th e absorption ban ds of th e NH group
were observed at 3220–3246 cm –1 an d of th e C–S–C group of th iadiazole
at 707–719 cm –1
.
1H NMR spectra of th e th iosem icarbazide derivatives 3 sh ow th ree-
proton sign als typical of th e NH group in th e δ 8.40–10.46 ppm ran ge.
In 1H NMR spectra of 1,2,4-triazole derivatives 4, proton sign als of th e NH
group were observed in th e δ 13.58–13.96 ppm ran ges. Th e above observa-
tion s suggest th at th e com poun ds exist m ain ly in th e th ion e form , both
in th e solid state an d in solution . X-ray an alysis con firm ed th at com poun d
4g exists in th e th ion e form (Fig. 1b). Th e 1H NMR spectra of 1,3,4-th ia-
diazole derivatives 5 sh ow th e proton sign als typical of th e NH group in
th e δ ran ge 7.62–10.30 ppm . X-ray an alysis of com poun ds 5a an d 5g con -
firm ed th at th e syn th esized com poun ds con tain th e 1,3,4-th iadiazole sys-
tem . Th e n um berin g sch em e an d gen eral view of th e m olecules are sh own
in Figs 1c an d 1d.
Pharmacology
Prelim in ary ph arm acological studies in dicated th at com poun ds 4a, 4b an d
4g exh ibit a weak an tidepressive activity in m ice. All th e derivatives of
1,2,4-triazole-3(2H)-th ion e given in a dose 100 m g/kg i.p. sign ifican tly pro-
lon ged th e sleep tim e in duced by th iopen tal (Table I), as well as an algesic
activity (Table II). On ly on e com poun d, 4a, given in a dose 50 m g/kg
sign ifican tly decreased th e pain reactivity of m ice (ca. 38% with respect to
con trol group). In th e rem ain in g tests, th e n ew com poun ds did n ot pro-
duce an y sign ifican t activity on th e CNS of m ice.
EXPERIMENTAL
Ch em istry
Meltin g poin ts were determ in ed in a Fish er–Joh n s block an d are n ot corrected. IR spectra
(ν, cm –1) were recorded in KBr usin g a Specord IR-75 spectroph otom eter. 1H NMR spectra
were recorded on a Tesla BS-567 A spectrom eter (100 MHz) in DMSO-d6 with TMS as an
in tern al stan dard. Ch em ical sh ifts are given in ppm (δ-scale), couplin g con stan ts (J) in Hz.
Ch em icals were purch ased from Merck Co. or Lan caster an d used with out furth er purifi-
cation .
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Cyclization and Pharmacological Properties of Thiosemicarbazides
55
TABLE I
Th e in fluen ce of in vestigated com poun ds on th e th iopen tal-in duced sleep (N = 10)
Sleepin g tim e
Dose
Com poun d
m g/kg. i.p.
m in ± SEM
19.4 ± 5.5
%
Con trol
–
100.0 ± 28.4
4a
50
100
50
10.4 ± 4.9
53.6 ± 25.3
230.4 ± 92.3a
67.0 ± 5.7
44.7 ± 17.9a
13.0 ± 1.1
4b
4g
100
50
71.4 ± 14.2a
30.4 ± 14.6
55.9 ± 15.8a
368 ± 73.2a
156.7 ± 74.4
288.1 ± 81.4a
100
a
p < 0.001 vs th e con trol group.
TABLE II
An algesic activity of com poun ds of “writh in g syn drom e” test in m ice (N = 10)
Dose
m g/kg i.p.
Mean writh in g
n um ber
In h ibition a
%
Com poun d
Con trol
–
15.6 ± 2.5
0
4a
25
50
16.0 ± 1.7
10.3 ± 2.1b
8.3 ± 1.9b
13.7 ± 2.7
4.9 ± 1.5b
13.8 ± 2.1
8.4 ± 1.2b
0.7
38b
47b
12
100
50
4b
4g
100
50
68b
12.5
46b
100
a
b
% of in h ibition obtain ed by com parison with con trol groups. p < 0.001 vs th e con trol
group.
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

56
Maliszewska-Guz et al.:
Eth yl [(4-Meth yl-4H-1,2,4-triazol-3-yl)sulfan yl]acetate (1)
A m ixture of 4-m eth yl-4H-1,2,4-triazole-3-th iol (1.15 g, 10 m m ol), an h ydrous potassium car-
bon ate (2 g) an d eth yl brom oacetate (1.84 g, 11 m m ol) in an h ydrous aceton e (20 m l) was
refluxed for 15 h . After coolin g, th e solid was filtered off, an d th e solven t was evaporated
un der reduced pressure. Th e residue was extracted with dieth yl eth er an d th e residue after
evaporation was purified by crystallization from eth an ol. Yield 1.7 g (85%), m .p. 48–49 °C.
For C7H11N3O2S (201.2) calculated: 41.78% C, 5.51% H, 20.88% N; foun d: 41.67% C,
5.46% H, 20.78% N. IR (KBr): 3060 (CHar); 2972, 1445 (CHal); 1710 (C=O); 1550 (C=N).
1H NMR (DMSO-d6): 1.16 t, 3 H, J = 7 (CH3); 3.61 s, 3 H (CH3); 3.98 s, 2 H (CH2); 4.11 q,
2 H, J = 7 (CH2); 8.55 s, 1 H (CH).
[(4-Meth yl-4H-1,2,4-triazol-3-yl)sulfan yl]acetoh ydrazide (2)
Hydrazin e h ydrate 100% (1 m l) was added to a solution of 1 (2.01 g, 10 m m ol) in an h y-
drous eth an ol (5 m l) an d th e m ixture was kept at room tem perature for 24 h . Precipitated
h ydrazide was th en filtered off an d dried. Yield 1.6 g (85%), m .p. 169–170 °C. For C5H9N5OS
(187.2) calculated: 32.07% C, 4.84% H, 37.41% N; foun d: 31.92% C, 4.64% H, 37.62% N.
IR (KBr): 3300 (NH); 3064 (CHar); 2970, 1445 (CHal); 1710 (C=O); 1556 (C=N). 1H NMR
(DMSO-d6): 3.64 s, 3 H (CH3); 3.80 s, 2 H (CH2); 4.34 s, 2 H (NH2); 8.60 s, 1 H (CH); 9.33 s,
1 H (NH).
1-{[(4-Meth yl-4H-1,2,4-triazol-3-yl)sulfan yl]acetyl}th iosem icarbazides 3a–3g.
Gen eral Procedure
A m ixture of h ydrazide 2 (1.87 g, 10 m m ol) an d th e correspon din g isoth iocyan ate (11 m m ol)
in an h ydrous m eth an ol (25 m l) was kept at room tem perature for 4 days. Th e form ed prod-
uct was th en filtered off, wash ed with dieth yl eth er an d crystallized from m eth an ol.
1-{[(4-Methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}-4-phenylthiosemicarbazide (3a ). Yield
2.96 g (92%), m .p. 166–167 °C. For C12H14N6OS2 (322.4) calculated: 44.70% C, 4.37% H,
26.07% N; foun d: 44.50% C, 4.35% H, 26.00% N. IR (KBr): 3130 (NH); 3050 (CHar); 2930,
1450 (CHal); 1680 (C=O); 1530, 1360 (C=S). 1H NMR (DMSO-d6): 3.67 s, 3 H (CH3); 3.90 s,
2 H (CH2); 7.19–7.58 m , 5 H (5 × CHar); 8.63 s, 1 H (CHar); 9.78, 9.87, 10.39 3 s, 3 H (3 ×
NH).
1-{[(4-Methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}-4-(p-tolyl)thiosemicarbazide (3b ). Yield
3.16 g (94%), m .p. 162–163 °C. For C13H16N6OS2 (336.4) calculated: 46.41% C, 4.79% H,
24.98% N; foun d: 46.34% C, 4.68% H, 24.79% N. IR (KBr): 3120 (NH); 3048 (CHar); 2934,
1452 (CHal); 1679 (C=O); 1528, 1364 (C=S). 1H NMR (DMSO-d6): 2.34 s, 3 H (CH3); 3.66 s,
3 H (CH3); 3.90 s, 2 H (CH2); 7.16–7.43 m , 4 H (4 × CHar.); 8.62 s, 1 H (CHar); 9.71, 9.79,
10.37 3 s, 3 H (3 × NH).
4-(4-Methoxyphenyl)-1-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}thiosemicarbazide (3c).
Yield 3.27 g (93%), m .p. 163–164 °C. For C13H16N6O2S2 (352.4) calculated: 44.31% C,
4.58% H, 23.85% N; foun d: 44.11% C, 4.47% H, 23.65% N. IR (KBr): 3133 (NH); 3045
(CHar); 2931, 1452 (CHal); 1678 (C=O); 1536, 1361 (C=S). 1H NMR (DMSO-d6): 3.66 s, 3 H
(CH3); 3.80 s, 3 H (CH3); 3.90 s, 2 H (CH2); 6.92–6.95 d, 2 H, J = 9 (2 × CHar); 7.38–7.40 d,
2 H, J = 9 (2 × CHar); 8.62 s, 1 H (CHar); 9.67, 9.76, 10.35 3 s, 3 H (3 × NH). 13C NMR: 30.90
(CH3); 35.20 (CH2); 55.30 (CH3); 113.20, 114.60, 127.30, 129.60 (4 × CHar); 131.90, 156.80
(2 × Car); 147.00 (CH); 149.10 (C-S); 167.20 (C=S); 181.10 (C=O).
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Cyclization and Pharmacological Properties of Thiosemicarbazides
57
4-(4-Bromophenyl)-1-{[(4-Methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}thiosemicarbazide (3d ).
Yield 3.69 g (92%), m .p. 176–177 °C. For C12H13BrN6OS2 (401.3) calculated: 35.91% C,
3.26% H, 20.94% N; foun d: 35.76% C, 3.31% H, 20.97% N. IR (KBr): 3121 (NH); 3041
(CHar); 2927, 1449 (CHal); 1680 (C=O); 1547, 1355 (C=S). 1H NMR (DMSO-d6): 3.61 s, 3 H
(CH3); 3.83 s, 2 H (CH2); 7.50–7.52 m , 4 H (4 × CHar); 8.58 s, 1 H (CHar); 9.86, 9.92, 10.38
3 s, 3 H (3 × NH).
4-(2-Fluorophenyl)-1-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}thiosemicarbazide (3e).
Yield 3.20 g (94%), m .p. 182–183 °C. For C12H13FN6OS2 (340.4) calculated: 42.34% C,
3.85% H, 24.69% N; foun d: 42.46% C, 3.72% H, 24.52% N. IR (KBr): 3133 (NH); 3061
(CHar); 2934, 1447 (CHal); 1679 (C=O); 1540, 1362 (C=S); 1 247 (C–O–C). 1H NMR
(DMSO-d6): 3.59 s, 3 H (CH3); 3.88 s, 2 H (CH2); 7.13–7.30 m , 4 H (4 × CHar); 8.51 s, 1 H
(CHar); 9.72, 9.89, 10.46 3 s, 3 H (3 × NH). 13C NMR: 30.80 (CH3); 35.00 (CH2); 115.60,
115.90, 124.00, 127.10 (4 × CHar); 130.70, 160.80 (2 × Car); 146.20 (CH); 149.10 (C-S);
167.10 (C=S); 182.30 (C=O).
4-Methyl-1-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}thiosemicarbazide (3f). Yield 2.49 g
(96%), m .p. 160–161 °C. For C7H12N6OS2 (260.3) calculated: 32.30% C, 4.65% H, 32.28% N;
foun d: 32.15% C, 4.48% H, 32.34% N. IR (KBr): 3127 (NH); 3070 (CHar); 2931, 1445 (CHal);
1681 (C=O); 1539, 1366 (C=S). 1H NMR (DMSO-d6): 2.93 s, 3 H (CH3); 3.59 s, 3 H (CH3);
3.83 s, 2 H (CH2); 8.59 s, 1 H (CHar); 8.40, 9.37, 10.21 3 s, 3 H (3 × NH). 13C NMR: 30.90
(CH3); 31.00 (CH3); 34.20 (CH2); 146.30 (CH); 149.40 (C-S); 167.00 (C=S); 182.20 (C=O).
4-Ethyl-1-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}thiosemicarbazide (3g). Yield 2.60 g
(95%), m .p. 168–169 °C. For C8H14N6OS2 (274.4) calculated: 35.02% C, 5.14% H, 30.63% N;
foun d: 35.17% C, 5.31% H, 30.74% N. IR (KBr): 3137 (NH); 3055 (CHar); 2937, 1452 (CHal);
1670 (C=O); 1533, 1359 (C=S). 1H NMR (DMSO-d6): 1.13 t, 3 H, J = 7 (CH3); 3.58 q, 2 H,
J = 7 (CH2); 3.65 s, 3 H (CH3); 3.86 s, 2 H (CH2); 8.62 s, 1 H (CHar); 8.43, 9.33, 10.23 3 s,
3 H (3 × NH).
5-{[(4-Meth yl-4H-1,2,4-triazol-3-yl)sulfan yl]m eth yl}-4H-1,2,4-triazole-3(2H)-th ion e
Derivatives 4a–4g. Gen eral Procedure
A m ixture of th iosem icarbazide 3a–3g (10 m m ol) an d 2% aqueous solution of sodium
h ydroxide (15 m l) was refluxed for 2 h (3a–3e) or kept at room tem perature for 5 days (3f,
3g). Th en , th e solution was n eutralized with acetic acid an d th e form ed precipitate was fil-
tered off an d crystallized from m eth an ol.
5-{[(4-Methyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-4-phenyl-4H-1,2,4-triazole-3(2H)-thione
(4a). Yield 2.62 g (86%), m .p. 193–194 °C. For C12H12N6S2 (304.4) calculated: 47.34% C,
3.97% H, 27.61% N; foun d: 47.26% C, 3.76% H, 27.47% N. IR (KBr): 3101 (CHar); 2922,
1462 (CHal); 1610 (C=N); 1520 (C–N). 1H NMR (DMSO-d6): 3.54 s, 3 H (CH3); 4.21 s, 2 H
(CH2); 7.50–7.64 m , 5 H (5 × CHar); 8.60 s, 1 H (CHar); 13.89 s, 1 H (NH).
5-{[(4-Methyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-4-(p-tolyl)-4H-1,2,4-triazole-3(2H)-thione
(4b). Yield 2.80 g (88%), m .p. 213–214 °C. For C13H14N6S2 (318.4) calculated: 49.03% C,
4.43% H, 26.39% N; foun d: 49.22% C, 4.64% H, 26.52% N. IR (KBr): 3098 (CHar); 2919,
1455 (CHal); 1608 (C=N); 1524 (C–N). 1H NMR (DMSO-d6): 2.40 s, 3 H (CH3); 3.49 s, 3 H
(CH3); 4.15 s, 2 H (CH2); 7.30–7.38 m , 4 H (4 × CHar); 8.52 s, 1 H (CHar); 13.81 s, 1 H (NH).
4-(4-Methoxyphenyl)-5-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-4H-1,2,4-triazole-
3(2H)-thione (4c). Yield 2.97 g (89%), m .p. 205–206 °C. For C13H14N6OS2 (334.4) calculated:
46.69% C, 4.22% H, 25.13% N; foun d: 46.48% C, 4.13% H, 25.34% N. IR (KBr): 3099 (CHar);
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

58
Maliszewska-Guz et al.:
2931, 1459 (CHal); 1608 (C=N); 1526 (C–N); 1280 (C–O–C). 1H NMR (DMSO-d6): 3.50 s, 3 H
(CH3); 3.84 s, 3 H (CH3); 4.14 s, 2 H (CH2); 7.08–7.37 m , 4 H (4 × CHar); 8.56 s, 1 H (CHar);
13.78 s, 1 H (NH). 13C NMR: 27.70 (CH2); 30.90 (CH3); 55.50 (CH3); 114.60, 129.60 (4 ×
CHar); 125.70 (C-N); 146.60, 159.80 (2 × Car); 146.90 (CH); 148.60 (C-S); 168.50 (C=S).
4-(4-Bromophenyl)-5-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-4H-1,2,4-triazole-
3(2H)-thione (4d ). Yield 3.25 g (85%), m .p. 223–224 °C. For C12H11BrN6S2 (383.2) calculated:
37.61% C, 2.89% H, 21.93% N; foun d: 37.50% C, 2.78% H, 21.73% N. IR (KBr): 3091 (CHar);
2925, 1459 (CHal); 1 620 (C=N); 1 530 (C–N). 1H NMR (DMSO-d6): 3.51 s, 3 H (CH3); 4.19 s,
2 H (CH2); 7.45 d, 2 H, J = 9 (2 × CHar); 7.78 d, 2 H, J = 9 (2 × CHar); 8.56 s, 1 H (CHar);
13.86 s, 1 H (NH).
4-(2-Fluorophenyl)-5-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-4H-1,2,4-triazole-
3(2H)-thione (4e). Yield 2.64 g (82%), m .p. 199–200 °C. For C12H11FN6S2 (322.4) calculated:
44.71% C, 3.44% H, 26.07% N; foun d: 44.58% C, 3.19% H, 26.23% N. IR (KBr): 3075 (CHar);
2933, 1457 (CHal); 1619 (C=N); 1522 (C–N). 1H NMR (DMSO-d6): 3.49 s, 3 H (CH3); 4.18 d,
2 H, J = 5 (CH2); 7.38–7.65 m , 4 H (4 × CHar); 8.55 s, 1 H (CHar); 13.96 s, 1 H (NH).
13C NMR: 27.40 (CH2); 30.80 (CH3); 116.70, 120.70, 130.70, 132.60 (4 × CHar); 125.40
(C-N); 146.60, 158.80 (2 × Car); 146.80 (CH); 148.40 (C-S); 168.70 (C=S).
4-Methyl-5-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione
(4f). Yield 2.18 g (90%), m .p. 174–175 °C. For C7H10N6S2 (242.3) calculated: 34.69% C,
4.16% H, 34.68% N; foun d: 34.61% C, 4.21% H, 34.49% N. IR (KBr): 3078 (CHar); 2930,
1466 (CHal); 1603 (C=N); 1519 (C–N). 1H NMR (DMSO-d6): 3.57 s, 3 H (CH3); 3.77 s, 3 H
(CH3); 4.37 s, 2 H (CH2); 8.59 s, 1 H (CHar); 13.58 s, 1 H (NH). 13C NMR: 28.50 (CH2); 31.00
(CH3); 31.80 (CH3); 125.50 (C-N); 147.70 (CH); 149.50 (C-S); 168.20 (C=S).
4-Ethyl-5-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-4H-1,2,4-triazole-3(2H)-thione (4g).
Yield 2.38 g (93%), m .p. 175–176 °C. For C8H12N6S2 (256.3) calculated: 37.48% C, 4.72% H,
32.78% N; foun d: 37.37% C, 4.63% H, 32.53% N. IR (KBr): 3098 (CHar); 2 927, 1462 (CHal);
1616 (C=N); 1523 (C–N). 1H NMR (DMSO-d6): 1.28 t, 3 H, J = 7 (CH3); 3.57 s, 3 H (CH3);
4.04 q, 2 H, J = 7 (CH2); 4.41 s, 2 H (CH2); 8.60 s, 1 H (CHar); 13.59 s, 1 H (NH).
2-(Alkyl(aryl)am in o)-5{[(4-m eth yl-4H-1,2,4-triazol-3-yl)sulfan yl]m eth yl}-1,3,4-th iadiazole
Derivatives 5a–5g. Gen eral Procedure
Startin g com poun d 3 (10 m m ol) was dissolved in con cen trated sulfuric acid (5 m l) an d th e
solution was kept at room tem perature for 10 days. Th e correspon din g product 5 was ob-
tain ed after n eutralization of th e solution with dilute am m on ium h ydroxide. Th e isolated
product was filtered off, dried an d crystallized from m eth an ol.
2-Anilino-5-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-1,3,4-thiadiazole (5a ). Yield
2.46 g (81%), m .p. 189–190 °C. For C12H12N6S2 (304.4) calculated: 47.34% C, 3.97% H,
27.61% N; foun d: 47.16% C, 3.68% H, 27.48% N. IR (KBr): 3220 (NH); 3051 (CHar); 2938,
1449 (CHal); 710 (C–S–C). 1H NMR (DMSO-d6): 3.55 s, 3 H (CH3); 4.64 s, 2 H (CH2);
6.96–7.59 m , 5 H (5 × CHar); 8.61 s, 1 H (CHar); 10.30 s, 1 H (NH).
5-{[(4-Methyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-2-(p-tolylamino)-1,3,4-thiadiazole (5b ).
Yield 2.64 g (83%), m .p. 191–192 °C. For C13H14N6S2 (318.4) calculated: 49.03% C, 4.43% H,
26.39% N; foun d: 48.90% C, 4.18% H, 26.18% N. IR (KBr): 3240 (NH); 3056 (CHar); 2938,
1455 (CHal); 710 (C–S–C). 1H NMR (DMSO-d6): 2.35 s, 3 H (CH3); 3.56 s, 3 H (CH3); 4.64 s,
2 H (CH2); 6.92–7.43 m , 4 H (4 × CHar); 8.61 s, 1 H (CHar); 10.28 s, 1 H (NH).
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Cyclization and Pharmacological Properties of Thiosemicarbazides
59
2-(4-Methoxyanilino)-5-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-1,3,4-thiadiazole (5c).
Yield 2.81 g (84%), m .p. 167–168 °C. For C13H14N6OS2 (334.4) calculated: 46.69% C,
4.22% H, 25.13% N; foun d: 46.50% C, 4.12% H, 25.16% N. IR (KBr): 3242 (NH); 3043
(CHar); 2931, 1449 (CHal); 1260 (C–O–C); 709 (C–S–C). 1H NMR (DMSO-d6): 3.56 s, 3 H
(CH3); 3.73 s, 3 H (CH3); 4.60 s, 2 H (CH2); 6.93–7.48 m , 4 H (4 × CHar); 8.60 s, 1 H (CHar);
10.09 s, 1 H (NH). 13C NMR: 30.90 (CH3); 31.50 (CH2); 55.20 (CH3); 114.30, 119.20 (4 ×
CHar); 146.60 (CH); 147.90, 153.70, 165.90 (3 × C-S); 154.60, 155.20 (2 × Car).
2-(4-Bromoanilino)-5-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-1,3,4-thiadiazole (5d ).
Yield 3.06 g (80%), m .p. 197–198 °C. For C12H11BrN6S2 (383.2) calculated: 37.61% C,
2.89% H, 21.93% N; foun d: 37.48% C, 2.65% H, 21.69% N. IR (KBr): 3241 (NH); 3048
(CHar); 2931, 1449 (CHal); 716 (C–S–C). 1H NMR (DMSO-d6): 3.55 s, 3 H (CH3); 4.65 s, 2 H
(CH2); 7.48–7.59 m , 4 H (4 × CHar); 8.61 s, 1 H (CHar); 9.83 s, 1 H (NH).
2-(2-Fluoroanilino)-5-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-1,3,4-thiadiazole (5e).
Yield 2.77 g (86%), m .p. 158–159 °C. For C12H11FN6S2 (322.4) calculated: 44.71% C,
3.44% H, 26.07% N; foun d: 44.69% C, 3.50% H, 26.02% N. IR (KBr): 3246 (NH); 3055
(CHar); 2934, 1446 (CHal); 719 (C–S–C). 1H NMR (DMSO-d6): 3.56 s, 3 H (CH3); 4.64 s, 2 H
(CH2); 7.03–8.35 m , 4 H (4 × CHar); 8.61 s, 1 H (CHar); 10.13 s, 1 H (NH). 13C NMR: 30.90
(CH3); 31.40 (CH2); 115.30, 120.50, 123.00, 124.70 (4 × CHar); 146.70 (CH); 147.80, 153.50,
165.40 (3 × C-S); 150.20, 157.30 (2 × Car).
2-(Methylamino)-5-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-1,3,4-thiadiazole (5f).
Yield 1.94 g (80%), m .p. 135–136 °C. For C7H10N6S2 (242.3) calculated: 34.69% C, 4.16% H,
34.68% N; foun d: 34.47% C, 4.34% H, 34.75% N. IR (KBr): 3244 (NH); 3050 (CHar); 2931,
1446 (CHal); 707 (C–S–C). 1H NMR (DMSO-d6): 2.83 d, 3 H, J = 5 (CH3); 3.54 s, 3 H (CH3);
4.54 s, 2 H (CH2); 7.62 d, 1 H, J = 5 (NH); 8.59 s, 1 H (CHar). 13C NMR: 30.80 (CH3); 31.10
(CH3); 31.70 (CH2); 146.60 (CH); 147.80, 153.80, 170.40 (3 × C-S).
2-(Ethylamino)-5-{[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]methyl}-1,3,4-thiadiazole (5g).
Yield 2.18 g (85%), m .p. 160–161 °C. For C8H12N6S2 (256.3) calculated: 37.48% C, 4.72% H,
32.78% N; foun d: 37.19% C, 4.57% H, 32.59% N. IR (KBr): 3237 (NH); 2927, 1450 (CHal);
713 (C–S–C). 1H NMR (DMSO-d6): 1.14 t, 3 H, J = 7 (CH3); 3.19–3.28 m , 2 H (CH2); 3.53 s,
3 H (CH3); 4.53 s, 2 H (CH2); 7.66 t, 1 H, J = 5 (NH); 8.59 s, 1 H (CHar).
Crystallograph y
Sin gle-crystal X-ray diffraction data for com poun ds 3a, 4g, 5a an d 5g were m easured at
room tem perature in th e ω/2θ scan m ode on a KM4 four-circle diffractom eter usin g CuKα
radiation . Details of th e crystal data, X-ray data collection an d refin em en t are given in
35
Table III. Crystal structures were solved by direct m eth ods usin g th e SHELXS97
an d refin ed by th e full-m atrix least-squares m eth od on F2 usin g th e SHELXL97
program
program .
36
Th e n on -h ydrogen atom s were refin ed with an isotropic displacem en t param eters. H-atom
position s were located from th e geom etry an d isotropic factors 1.2Ueq of th e bon ded
C-atom s were given ; for th e C–H bon d, “ridin g m odel” was used in th e refin em en t.
CCDC 229220, 229221, 229222 an d 229223 (for com poun ds 3a, 4g, 5a an d 5g) con tain
th e supplem en tary crystallograph ic data for th is paper. Th ese data can be obtain ed free of
ch arge via www.ccdc.cam .ac.uk/con ts/retrievin g.h tm l (or from th e Cam bridge Crystallo-
graph ic Data Cen tre, 12, Un ion Road, Cam bridge, CB2 1EZ, UK; fax: +44 1223 336033; or
deposit@ccdc.cam .ac.uk).
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

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Maliszewska-Guz et al.:
Ph arm acology
Th e experim en ts were carried out on m ale Albin o Swiss m ice (20–25 g). Th e an im als were
h oused in colon y cages with free access to tap water an d food (stan dard laboratory pellets,
Bacutil, Motycz, Polan d) an d m ain tain ed in th e 12/12 h ligh t-dark cycle (ligh t on from
7 a.m . to 7 p.m .). Experim en tal an d con trol groups con sistin g of 10 an im als. Th e experi-
m en ts were perform ed between 8 a.m . an d 3 p.m . Th e in vestigated com poun ds 4a, 4b an d
4g were adm in istered in traperiton eally (i.p.) in doses of 25, 50 an d 100 m g/kg (equivalen t to
0.025, 0.05 an d 0.1 of th eir LD50) as suspen sion s in a 1% Tween 80 in th e con stan t volum e
10 m l/kg. Con trol an im als received th e equivalen t volum e of solven t. Effects of n ew com -
poun ds on th e CNS of m ice were studied in eigh t tests:
1. Chimney test. Th e effect of com poun ds 4a, 4b an d 4g in a dose of 100 m g/kg i.p. on
m otoric im pairm en t was quan tified with th e ch im n ey test37. Briefly, m ice h ad to clim b up
backwards in a plastic tube (3 cm in n er diam eter, 25 cm lon g). Mice un able to perform th e
task with in 60 s were con sidered to display m otor im pairm en t. Motoric im pairm en t was
quan tified as th e percen tage of an im als th at failed to com plete th e test.
2. Body temperature. Th e rectal body tem perature in m ice (m easured with an Ellab th er-
m om eter) was recorded 15, 30, 45, 60, 90 an d 120 m in after th e adm in istration of in vesti-
gated com poun ds in a dose 100 m g/kg i.p.
TABLE III
Crystallograph ic data an d details of th e structure refin em en t
Param eter
3a
4g
5g
5a
Crystal system
Space group
a, Å
m on oclin ic
P21/c
m on oclin ic
P21/n
m on oclin ic
P21/c
orth orh om bic
Pbca
8.184(1)
20.954(4)
8.551(1)
90
8.699(1)
12.328(2)
11.013(2)
90
15.235(3)
7.267(1)
10.441(2)
90
13.988(3)
8.665(1)
22.360(4)
90
b, Å
c, Å
α, °
β, °
100.15(1)
90
97.41(1)
90
101.44(3)
90
90
γ, °
90
Volum e, Å3
1443.4(4)
4.22–80.23
3135
1171.2(3)
5.41–76.15
2383
1133.0(3)
2.96–76.16
2222
2710.2(8)
5.06–76.17
2780
θ ran ge, °
No. of un ique reflection s
No. of param eters
Fin al R1 in dices
wR2, [I > 2σ(I)]
∆ρm ax; ∆ρm in , e Å–3
192
146
147
182
0.0576
0.0517
0.0572
0.0514
0.1031
0.31; –0.47
0.0985
0.1340
0.1527
0.31; –0.42
0.44; –0.45
0.67; –0.49
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Cyclization and Pharmacological Properties of Thiosemicarbazides
61
3. Four-plate test38. An xiolytic activity was assessed 30 m in after in jection of th e com -
poun d in a dose of 100 m g/kg i.p. Th e n um ber of pun ish ed crossin gs was coun ted for
1 m in .
4. Forced swimming test39. Th e in vestigated com poun ds in a dose of 100 m g/kg were
adm in istered 30 m in before th e test. Mice were in dividually placed an d forced to swim in
a glass cylin der (27 × 16 cm ) con tain in g 15 cm of water (25 °C). A m ouse was con sidered
im m obile wh en it floated in th e water, in uprigh t position , an d sh owed on ly sm all m ove-
m en ts to keep its h ead above water. Th e total im m obility tim e of m ice was m easured durin g
th e last 4 m in of th e 6-m in test.
5. Thiopental-induced sleep. Th iopen tal (60 m g/kg i.p.) was given 30 m in after adm in istra-
tion of th e tested derivatives in doses of 50 an d 100 m g/kg i.p. Th e sleepin g tim e (from loss
to recovery of righ tin g reflex) was m easured.
6. “W rithing syndrome” test40. Com poun ds were adm in istered in doses of 25, 50 or
100 m g/kg i.p., an d after 30 m in th e an im als were in jected 0.6% acetic acid i.p. Th e n um ber
of writh in g episodes was coun ted for 30 m in .
7. Pentetrazole-induced convulsions. Pen tetrazole (100 m g/kg sc) was given 30 m in after
adm in istration of com poun ds 4a, 4b an d 4g in a dose of 100 m g/kg i.p. Th e observation
(durin g 30 m in ) of in dividual an im als, to record occurren ce of ton ic con vulsion s as well as
m ortality of m ice, started im m ediately after in jection of pen tetrazole.
8. Head twitches. Th e in vestigated com poun ds were given in a dose of 100 m g/kg i.p.
30 m in before L-5-h ydroxytryptoph an (L-5-HTP, 180 m g/kg i.p.). Th e n um ber of h ead twitch
episodes of m ice was coun ted durin g 60 m in after th e in jection of L-5-HTP.
Th e obtain ed results were evaluated statistically with th e Studen t’s t-test.
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