Synthesis and antiproliferative studies of 5-aryl-2-(3-thienylamino)-1,3,4- thiadiazoles

Article abstract of DOI:10.1016/j.bmcl.2014.04.043

A series of 5-aryl-2-(3-thienylamino)-1,3,4-thiadiazoles 3a-m were synthesized in good yields in two steps starting from thiophen-3- isothiocyanates. Those compounds as well as the thiosemicarbazide intermediates 2a-m were screened for their antiproliferative activity against a panel of six cancer cell lines. Among them, two 5-aryl-2-(3-thienylamino)-1,3,4-thiadiazoles (3f and 3i) have shown very interesting results with IC₅₀ <10 μM on three cell lines.

Full text of DOI:10.1016/j.bmcl.2014.04.043

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2724–2727
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiproliferative studies of
5-aryl-2-(3-thienylamino)-1,3,4-thiadiazoles
Germain Revelant a, Charlène Gadais a, Véronique Mathieu b, Gilbert Kirsch a, Stéphanie Hesse a,
⇑
a Université de Lorraine, Laboratoire de Synthèse et Réactivité des Systèmes Moléculaires Complexes UMR-SRSMC 7565, Equipe Hécrin, 1 boulevard Arago, 57070 Metz
Technopôle, France
b Laboratoire de toxicologie, Université Libre de Bruxelles, Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 January 2014
Revised 9 April 2014
Accepted 10 April 2014
Available online 19 April 2014
A series of 5-aryl-2-(3-thienylamino)-1,3,4-thiadiazoles 3a–m were synthesized in good yields in two
steps starting from thiophen-3-isothiocyanates. Those compounds as well as the thiosemicarbazide inter-
mediates 2a–m were screened for their antiproliferative activity against a panel of six cancer cell lines.
Among them, two 5-aryl-2-(3-thienylamino)-1,3,4-thiadiazoles (3f and 3i) have shown very interesting
results with IC50 <10 lM on three cell lines.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Thienylamino-1,3,4-thiadiazoles
Thienylthiosemicarbazides
Thienylisothiocyanates
Antiproliferative agents
MTT assay
1,3,4-Thiadiazoles are heterocyclic scaffolds incorporated in
many compounds presenting various pharmacological activities
such as antimicrobial,1 antitubercular,2 anti-oxydant,3 anti-leish-
manial,4 or anti-inflammatory5 agents. Especially, 1,3,4-thiadiazole
core is found in several marketed drugs such as acetazolamide, a
carbonic anhydrase inhibitor used in treatment of glaucoma or sul-
famethizole, an antibacterial agent. 1,3,4-Thiadiazole derivatives
have also been investigated for their antiproliferative properties.6
For example, 2-(4-fluorophenylamino)-5-(2,4-dihydroxy-phenyl)-
1,3,4-thiadiazole (FABT), a promising anticancer compound for
treating malignant tumors of the nervous system, decreased cell
division and inhibited cell migration.7 In the same way, Kumar
et al. have synthesized and evaluated a series of 5-(3-indolyl)-2-
substituted-1,3,4-thiadiazoles against six human cancer cell lines
(prostate (PC3, DU145 and LnCaP), breast (MCF7 and MDA-MB-
231) and pancreatic (PaCa2) cancer cell lines). Most of those com-
pounds showed cytotoxic effect and the authors have demonstrated
the importance of substituents at C-2 such as 3,4-dimethoxyphenyl
or 4-benzyloxy-3-methoxyphenyl which significantly increased
activity against all the cancer cell lines and especially against
MCF7.8 They have also studied a series of 2-arylamino-5-aryl-
1,3,4-thiadiazoles and screened them on the same six cell line
panel. Compounds with trimethoxyphenyl at C-5 position
displayed potent anticancer activity on MCF7 and PaCa2 cell lines
with IC50 4.3–9.2 l
M.9
A variety of synthetic methods for the preparation of 1,3,4-thiadiaz-
oles have been reported. Especially 2-amino-1,3,4-thiadiazoles have
been prepared via dithiocarbamate,10 thiosemicarbazone11 or thio-
semicarbazide12 intermediates. Some one-pot procedure was also
described and synthesis of 2-arylamino-5-aryl-1,3,4-thiadiazoles
was achieved by refluxing aryl aldehydes, hydrazine hydrate, and
aryl isothiocyanates in methanol followed by oxidative cyclization
with ferric ammonium sulfate.9 In continuation of our interest in
the synthesis of new 3-thienylisothiocyanates and their use in
organic transformations (syntheses of thienylimino-1,3-thiazoli-
din-4-ones13 and 2-aminothieno[3,2-d]thiazoles14), we focused
our attention on their use in the synthesis of 2-(3-thienylamino)-
1,3,4-thiadiazoles. Those compounds were indeed poorly described
in literature excepted three compounds related to 2-(3-benzothie-
nyl)-1,3,4-thiadiazoles.15 We first tried the one-pot procedure
described by Kumar;9 however, in our case, cyclization by FAS of
the thiosemicarbazone formed in situ required 24 h of reflux to
reach its completion. Moreover, we also observed formation of sec-
ondary products so that the expected compounds have to be purified
by column chromatography and yields were moderate (Scheme 1).
Condensation of 5-phenylthiophene-3-isothiocyanate with
benzaldehyde (or 4-fluorobenzaldehyde) and hydrazine gave the
expected thiadiazoles 1a–b after cyclization by FAS in 33% yield.
The same reaction conducted on 5-(4-methylphenyl)thiophene-
⇑
Corresponding author. Tel.: +33 3 87 54 71 97; fax: +33 3 87 31 58 01.
E-mail address: stephanie.hesse@univ-lorraine.fr (S. Hesse).
http://dx.doi.org/10.1016/j.bmcl.2014.04.043
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

G. Revelant et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2724–2727
2725
NCS
R1
N
H3PO4.18 The expected 5-aryl-2-(3-thienylamino)-1,3,4-thiadiaz-
oles 3a–m were obtained in good to excellent yields after 1 h at
room temperature in concentrated H2SO4 (Table in Supplementary
data section). This two-step procedure allowed formation of thi-
adiazoles19 in 39–93% global yield.
The synthesized thienylthiosemicarbazide intermediates 2a–m
and thiadiazoles 3a–m were screened in terms of antiproliferative
activity against a panel of six cancer cell lines20 (Table 1) which
included three cell lines that display sensitivity to pro-apoptotic
stimuli (human MCF-7 breast, human Hs683 anaplastic oligoden-
droglioma and mouse B16F10 melanoma cancer cell lines) and three
cell lines that display various level of resistance to pro-apoptotic
stimuli (human U373 glioblastoma, human SKMEL-28 melanoma
and human A549 non-small-cell-lung cancer cell lines) by means
of colorimetric MTT assay. Table 1 resumes IC50 concentrations
obtained, that is, the concentration that decreased by 50% the global
growth of the cell population after 72 h of exposure to the com-
pound. Antiproliferative activities of thienylthiosemicarbazide
R2
N
R2CHO
+
H2NNH2.H2O
a
+
R1
S
S
NH
S
1a R1 = R2 = Ph
1b R1 = Ph, R2= 4-FC6H4
1c R1 = 4-CH3C6H4, R2= Ph
1a-d
1d R1 = 4-CH3C6H4, R2= 4-FC6H4
Scheme 1. Synthesis of 5-aryl-2-(3-thienylamino)-1,3,4-thiadiazoles 1a–d.
Reagents and conditions: (a) (i) methanol, reflux, 2 h; (ii) FAS, reflux, 24 h.
O
R2
NH
NH
NCS
O
NH
H2N
R2
S
+
N
H
a
R1
R1
S
S
2a-m
intermediates 2a–m ranged between 5 to >100
the substituent and the cell line (Table 1). The most sensitive cell line
is B16F10 with four compounds having IC50 <10 M whereas almost
all compounds displayed weak cytotoxic effect on U373 cell line (ten
compounds with IC50 >40 M). Thiosemicarbazide 2g with a 5-(4-
lM depending on
R2
N
b
N
l
R1
S
NH
l
S
methylphenyl)thienyl substituent and a 4-methoxyphenyl substi-
tuent on the other side is the most potent one with a mean IC50 con-
3a-m
centration of 11
However, it should be noted that this compound is not selective as
it showed toxicity to normal fibroblast cell lines (IC50 = 22
on NHDF cell line and IC50 = 28 M on NHLF cell line). Thiosem-
icarbazide 2h with phenyl (R1) and 4-hydroxyphenyl (R2) substitu-
ents was the less active one (IC50 >50 M in five cell lines; mean
IC50 concentration of 74 M on the six cancer cell lines under study).
For most of thiosemicarbazides, the IC50 could be determined
with values <50 M in all six cell lines while the thiadiazoles
3a–m presented different dose–response curves as compared to
lM on the six cancer cell lines under study.
Scheme 2. Synthesis of 5-aryl-2-(3-thienylamino)-1,3,4-thiadiazoles 3a–m.
Reagents and conditions: (a) ethanol, reflux, 3 h; (b) concd H2SO4, rt, 1 h.
1 lM
3
l
3-isothiocyanate led to thiadiazoles 1c–d in respectively 46% and
25% yield.
l
We then turned our attention to a two-step process (Scheme 2)
and synthesized thienylthiosemicarbazide intermediates 2a–m by
condensation of isothiocyanates with several arylhydrazides
refluxing 3 h in ethanol. Most of the time, those compounds are
then cyclized in acidic media in concentrated H2SO4,16 PPA17 or
l
l
Table 1
In vitro cytotoxic data of thienylthiosemicarbazide intermediates 2a–m and 5-aryl-2-(3-thienylamino)-1,3,4-thiadiazoles 3a–m against a panel of six cancer cell lines IC50 (lM)
R1
R2
Compound
H683
U373
A549
MCF-7
SKMEL-28
22 11
B16F10
Mean
4-OMeC6H4
C6H5
4-MeC6H4
C6H5
4-MeC6H4
C6H5
4-MeC6H4
C6H5
4-MeC6H4
C6H5
4-MeC6H4
C6H5
4-MeC6H4
4-MeC6H4
4-MeC6H4
3,4,5-(OMe)3C6H2
3,4,5-(OMe)3C6H2
4-OMeC6H4
4-OMeC6H4
4-OHC6H4
2a
2b
2c
2d
2e
2f
2g
2h
2i
30
37
40
42
42
35
12
80
49
42
37
22
39
1
>100
62 17
35
40
37
39
39
36
11
75
44
39
32
20
36
3
40
30
29
43
31
32
5
2
2
1
1
1
1
7
1
>39 13
2
1
1
1
1
1
1
4
1
1
2
1
2
1
1
1
1
1
1
2
1
1
1
40
44
55
42
30
11
71
46
37
38
28
19
3
2
9
2
2
1
3
1
1
2
2
4
24
20
32
13
33
9
2
3
2
2
1
39
35
43
36
35
11
>74
49
38
31
27
27
5
4
3
5
2
2
38
44
47
41
19
1
1
1
2
1
1
1
>100
78
42
38
26
21
11
2
1
1
2
1
1
40
48
30
13
9
3
3
2
2
1
1
8
4-OHC6H4
64
40
41
59
47
3
3
2
4
7
7
3,5-(OMe)2C6H3
3,5-(OMe)2C6H3
3,4-(OMe)2C6H3
3,4-(OMe)2C6H3
2j
2k
2l
1
1
4
1
4-MeC6H4
2m
1
7
Mean
39
4
>54
7
37
4
33
5
37
4
22
4
4-OMeC6H4
C6H5
4-MeC6H4
C6H5
4-MeC6H4
C6H5
4-MeC6H4
4-MeC6H4
4-MeC6H4
3,4,5-(OMe)3C6H2
3,4,5-(OMe)3C6H2
4-OMeC6H4
3a
3b
3c
3d
3e
3f
ND (10)
ND (5)
ND (1)
ND (5)
ND (0.05)
>100
>100
ND (0.5)
>100
ND (5)
>100
>100
>100
>100
>100
ND (0.5)
>100
ND (1)
ND (53 21)
ND (64 19)
ND (34 21)
ND (68 21)
ND (2 2)
>45 18
ND (50)
ND (0.5)
ND (100)
ND (0.5)
34
3
4
1
ND (100)
>100
ND (0.5)
ND (1)
ND (0.5)
ND (0.5)
12
3
5
1
92
7
8
1
60
7
6
1
4-MeC6H4
C6H5
4-MeC6H4
C6H5
4-MeC6H4
C6H5
4-MeC6H4
4-OMeC6H4
4-OHC6H4
4-OHC6H4
3,5-(OMe)2C6H3
3,5-(OMe)2C6H3
3,4-(OMe)2C6H3
3,4-(OMe)2C6H3
3g
3h
3i
3j
3k
3l
ND (0.5)
ND (0.5)
ND (0.5)
ND (0.5)
ND (100)
ND (1)
ND (17 17)
26
17
1
2
58
8
92
2
4
15
1
24
1
57
14
2
1
8
6
1
1
31
9
1
6
1
8
1
10
2
ND (50)
ND (5)
ND (100)
ND (5)
ND (100)
ND (10)
>100
ND (100)
ND (5)
>100
>100
>100
>100
ND (50)
ND (1)
ND (0.5)
ND (5)
ND (80 11)
ND (22 16)
ND (80 18)
ND (43 19)
ND (10)
>100
>100
92
2
3m
ND (0.5)
ND (50)
Mean
ND (18 8)
ND (>59 13)
ND (>30 12)
ND (>52 12)
ND (>83 10)
ND (7 4)
In vitro growth inhibitory IC50 concentrations were determined using the MTT colorimetric assay conducted once in sextuplicate. Bold values show IC50 less than 10
not determined because of ‘plateau’ phase, values in bracket correspond to the first concentration for which there is less than 50% of viable cells.
lM. ND:

2726
G. Revelant et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2724–2727
thiadiazole series (compounds 3e and 3i). Moreover, thiosemicar-
bazides and their corresponding 5-aryl-2-(3-thienylamino)-1,3,4-
thiadiazoles might have different cellular targets as highlighted
by the different dose-response curve profiles.
Acknowledgments
We thank the ‘Ministère de l’Enseignement Supérieur et de la
Recherche’ for Ph.D. grants to G.R. and C.G. and the Belgian Brain
Tumor Support for its financial support. The authors also would
like to thank Robert Kiss for fruitful discussions.
.
Supplementary data
Figure 1. Dose–response curves of A549 NSCLC cells treated with 2a–m
compounds (black lines) or 3a–m compounds (gray dotted lines) as established
after MTT colorimetric assay.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
04.043. These data include MOL files and InChiKeys of the most
important compounds described in this article.
2a–m, with a ‘plateau’ phase (Fig. 1, dotted gray lines as compared
to black lines) indicating that increasing drug concentration, at
least till 100 lM, did not lead to any improvement of the activity.
References and notes
Generally, thiadiazoles were nevertheless able to decrease partially
the global growth of cancer cells. So, to evaluate the antiprolifera-
tive potential of thiadiazoles 3a–m, we reported nevertheless in
brackets in Table 1 the first tested concentration for which there
remains less than 50% of viable cells after treatment. Marked dif-
ferences of sensitivity between cell lines could be observed. Again
the most sensitive cell line is B16F10 (five compounds with IC50
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14. Revelant, G.; Hesse, S.; Kirsch, G. Tetrahedron 2011, 67, 9352.
15. Russo, F.; Santagati, M.; Caruso, A.; Trombadore, S.; Amico-Roxas, M. Farmaco
1987, 42, 437.
16. Mavrova, A. T.; Wesselinova, D.; Tsenov, Y. A.; Denkova, P. Eur. J. Med. Chem.
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17. Khan, I. M.; Ali, S.; Hameed, S.; Rama, N. H.; Hussain, M. T.; Wadood, A.; Uddin,
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19. Spectral data of some potent derivatives: 1-(4-Methoxyphenyl)-4-[5-(4-
methylphenyl)-3-thienyl]thiosemicarbazide (2g). Yield 96%; beige solid; mp
209 °C; 1H NMR (250 MHz, DMSO-d6) dH 2.30 (s, 3H, CH3), 3.82 (s, 3 H, OCH3),
7.03 (d, 2H, 2 Â CH, J = 7.5 Hz), 7.21 (d, 2H, 2 Â CH, J = 7.5 Hz), 7.46 (d, 2H,
2 Â CH, J = 7.5 Hz), 7.57 (s, 1H, CH), 7.66 (s, 1H, CH), 7.93 (d, 2H, 2 Â CH,
J = 7.5 Hz), 9.68 (s, 1H, NH), 9.88 (s, 1H, NH), 10.37 (s, 1H, NH); 13C NMR
(62,5 MHz, DMSO-d6) dC 20.7, 55.4, 96.8, 101.8, 113.5, 120.9, 124.6, 124.8,
129.7, 129.8, 130.8, 131.5, 137.1, 137.8, 162.1, 165.7; HRMS (APCI): m/z calcd
for [C20H17N3O2S+H]+: 364.1114; found: 364.1120. 2-(5-Phenyl-3-
thienylamino)-5-(4-methoxyphenyl)-1,3,4-thiadiazole (3f). Yield 88%; brown
solid; mp 264 °C; 1H NMR (250 MHz, DMSO-d6) dH 3.81 (s, 3H, OCH3), 7.05 (d,
2H, 2 Â CH, J = 10 Hz), 7.32 (t, 1H, CH, J = 7.5 Hz), 7.37 (d, 1H, CH, J = 1.5 Hz),
7.43 (t, 2H, 2 Â CH, J = 7.5 Hz), 7.54 (d, 1H, CH, J = 1.5 Hz), 7.63 (d, 2H, 2 Â CH,
J = 7.5 Hz), 7.78 (d, 2H, 2 Â CH, J = 10 Hz), 10.81 (s, 1H, NH); 13C NMR
(62,5 MHz, DMSO-d6) dC 55.4, 105.8, 114.6, 116.9, 122.9, 125.2, 127.9, 128.2,
129.2, 133.3, 138.8, 142.1, 157.5, 160.7, 163.1; HRMS (APCI): m/z calcd for
[C19H15N3OS2+H]+: 366.0729; found: 366.0730. 2-[5-(4-Methylphenyl)-3-
thienylamino]-5-(4-hydroxyphenyl)-1,3,4-thiadiazole (3i). Yield 98%; brown
solid; mp 240 °C; 1H NMR (250 MHz, DMSO-d6) dH 2.31 (s, 3H, CH3), 6.87 (d,
2H, 2 Â CH, J = 8.75 Hz), 7.23 (d, 2H, 2 Â CH, J = 8.75 Hz), 7.30 (s, 1H, CH), 7.48
(s, 1H, CH), 7.51 (d, 2H, 2 Â CH, J = 8.75 Hz), 7.66 (d, 2H, 2 Â CH, J = 8.75 Hz),
10.01 (s, 1H, OH), 10.72 (s, 1H, NH); 13C NMR (62,5 MHz, DMSO-d6) dC 20.7,
105.0, 115.9, 116.3, 121.4, 125.0, 128.3, 129.7, 131.5, 137.4, 138.8, 142.2, 157.9,
<12
pounds under study) whereas ten thiadiazoles displayed rather
no cytotoxic activity on SKMEL-28 cell line (IC50 >100 M; mean
IC50 concentration of >83 M considering all compounds under
lM; mean IC50 concentration of 7 lM considering all com-
l
l
study). Three thiadiazoles (3f, 3h and 3i) showed potent antiprolif-
erative activity on the six cell line panel with classical dose-
response curves. Thiadiazole 3f containing a 5-phenylthienylamino
substituent at C-2 and a 4-methoxyphenyl at C-5 exhibited growth
inhibition on five cancer cell lines with IC50 below 10
cell lines (Hs 683, IC50 = 5 M; A549, IC50 = 8 M; B16F10, IC50 = 6 -
M). In the same way, thiadiazole 3i containing a 5-(4-methyl-
lM for three
l
l
l
phenyl)thienylamino substituent at C-2 and a 4-hydroxyphenyl
at C-5 showed growth inhibition on all cancer cell lines with IC50
below 10
M; MCF-7, IC50 = 8
played a mean IC50 concentration of 31
lines under study but only one IC50 <10
l
M for four cell lines (U373, IC50 = 8
M; B16F10, IC50 = 6 M). Thiadiazole 3h dis-
M on the six cancer cell
M on B16F10 cell line.
lM; A549, IC50 = 6 -
l
l
l
l
l
Thiadiazoles 3e and 3g with 5-(4-methylphenyl)thienylamino sub-
stituent at C-2 and respectively a 3,4,5-trimethoxyphenyl and a 4-
methoxyphenyl at C-5 were also potent on almost all cancer cell
lines with respectively mean IC50 concentration of 2 and 17
on the six cancer cell lines under study.
lM
In conclusion, we have synthesized 26 new compounds and
evaluated their in vitro antiproliferative properties on a panel of
six cancer cell lines. Among them, two 5-aryl-2-(3-thienylami-
no)-1,3,4-thiadiazoles (3f and 3i) have shown very interesting
results with IC50 <10
lM on three cell lines and one thiosemicarba-
zide (2g) with a mean IC50 of 11
l
M on the six cancer cell line
panel. Presence of 5-(4-methylphenyl)thienylamino substituent
at C-2 instead of phenylthienylamino seems to enhance activity
in both series (mean IC50 concentrations on the six cancer cell line
panel are always lower for compounds with 5-(4-methyl-
phenyl)thienylamino substituent at C-2). Having a 4-methoxy-
phenyl group at C-5 gave potent compounds in both series
(compounds 2g and 3g) whereas 4-hydroxyphenyl and 3,4,5-tri-
methoxyphenyl moieties at C-5 increased activity only in

G. Revelant et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2724–2727
2727
159.3, 162.8; HRMS (APCI): m/z calcd for [C19H15N3OS2+H]+: 366.0729; found:
366.0737.
20. Determination of the IC50 growth inhibitory concentrations were determined
by means of the colorimetric MTT assay. Human cancer cell lines Hs683 and
U373 (glioma), A549 (NSCLC), MCF-7 (breast cancer), SKMEL-28 (melanoma)
and mouse B16F10 melanoma cells were purchased from the ATCC or ECACC
and were cultivated in RPMI culture medium supplemented with 10% heat
inactivated fetal bovine serum and antibiotics. Compounds were applied 72 h
on cells after 24 h of seeding. Reduction of MTT into formazan cristals by
succinate dehydrogenase is proportional to the number of viable cells and was
evaluated by measurement of the optical density at 570 nm after their
dissolution in DMSO.