Imidazo[2,1-b] [1,3,4]thiadiazoles with antiproliferative activity against primary and gemcitabine-resistant pancreatic cancer cells

Article abstract of DOI:10.1016/j.ejmech.2020.112088

A new series of eighteen imidazo [2,1-b] [1,3,4]thiadiazole derivatives was efficiently synthesized and screened for antiproliferative activity against the National Cancer Institute (NCI-60) cell lines panel. Two out of eighteen derivatives, compounds 12a and 12h, showed remarkably cytotoxic activity with the half maximal inhibitory concentration values (IC₅₀) ranging from 0.23 to 11.4 μM, and 0.29–12.2 μM, respectively. However, two additional compounds, 12b and 13g, displayed remarkable in vitro antiproliferative activity against pancreatic ductal adenocarcinoma (PDAC) cell lines, including immortalized (SUIT-2, Capan-1, Panc-1), primary (PDAC-3) and gemcitabine-resistant (Panc-1R), eliciting IC₅₀ values ranging from micromolar to sub-micromolar level, associated with significant reduction of cell-migration and spheroid shrinkage. These remarkable results might be explained by modulation of key regulators of epithelial-to-mesenchymal transition (EMT), including E-cadherin and vimentin, and inhibition of metalloproteinase-2/-9. High-throughput arrays revealed a significant inhibition of the phosphorylation of 45 tyrosine kinases substrates, whose visualization on Cytoscape highlighted PTK2/FAK as an important hub. Inhibition of phosphorylation of PTK2/FAK was validated as one of the possible mechanisms of action, using a specific ELISA. In conclusion, novel imidazothiadiazoles show potent antiproliferative activity, mediated by modulation of EMT and PTK2/FAK.

Full text of DOI:10.1016/j.ejmech.2020.112088

Journal Pre-proof
Imidazo[2,1-b] [1,3,4]thiadiazoles with antiproliferative activity against primary and
gemcitabine-resistant pancreatic cancer cells
Stella Cascioferro, Giovanna Li Petri, Barbara Parrino, Daniela Carbone, Niccola
Funel, Cecilia Bergonzini, Giulia Mantini, Henk Dekker, Daan Geerke, Godefridus J.
Peters, Girolamo Cirrincione, Elisa Giovannetti, Patrizia Diana
PII:
S0223-5234(20)30055-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112088
EJMECH 112088
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 October 2019
Revised Date: 16 January 2020
Accepted Date: 20 January 2020
Please cite this article as: S. Cascioferro, G.L. Petri, B. Parrino, D. Carbone, N. Funel, C. Bergonzini,
G. Mantini, H. Dekker, D. Geerke, G.J. Peters, G. Cirrincione, E. Giovannetti, P. Diana, Imidazo[2,1-b]
[1,3,4]thiadiazoles with antiproliferative activity against primary and gemcitabine-resistant pancreatic
cancer cells, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112088.
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Imidazo[2,1-b][1,3,4]thiadiazoles with antiproliferative activity against
primary and gemcitabine-resistant pancreatic cancer cells
1,a
1,2,a
1
1
Stella Cascioferro , Giovanna Li Petri , Barbara Parrino , Daniela Carbone , Niccola
3
2
2
2
4
Funel , Cecilia Bergonzini, Giulia Mantini, Henk Dekker, Daan Geerke, Godefridus J.
2
1
2,3,*
1,*
Peters , Girolamo Cirrincione , Elisa Giovannetti , and Patrizia Diana
1
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF),
Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy
2
Department of Medical Oncology, Amsterdam University Medical Center, VU University
Cancer Center Amsterdam, De Boelelaan 1117, 1081HV, Amsterdam, the Netherlands
3
Fondazione Pisana per la Scienza, via Via Ferruccio Giovannini 13, 56017 San Giuliano Terme,
Pisa, Italy
4
AIMMS Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical
Sciences, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1108, 1081 HZ
Amsterdam, The Netherlands
a
Equally contributed
*
Co-corresponding authors
KEYWORDS: imidazo[2,1-b][1,3,4]thiadiazole derivatives, pancreatic ductal adenocarcinoma,
antiproliferative activity, inhibition of migration, spheroids shrinkage, modulation of EMT,
PTK2/FAK
1

ABSTRACT: A new series of eighteen imidazo[2,1-b][1,3,4]thiadiazole derivatives was efficiently
synthesized and screened for antiproliferative activity against the National Cancer Institute (NCI-
60) cell lines panel. Two out of eighteen derivatives, compounds 12a and 12h, showed remarkably
cytotoxic activity with the half maximal inhibitory concentration values (IC ) ranging from 0.23 to
50
1
1.4 µM, and 0.29 to 12.2 µM, respectively. However, two additional compounds, 12b and 13g,
displayed remarkable in vitro antiproliferative activity against pancreatic ductal adenocarcinoma
PDAC) cell lines, including immortalized (SUIT-2, Capan-1, Panc-1), primary (PDAC-3) and
gemcitabine-resistant (Panc-1R), eliciting IC values ranging from micromolar to sub-micromolar
(
5
0
level, associated with significant reduction of cell-migration and spheroid shrinkage. These
remarkable results might be explained by modulation of key regulators of epithelial-to-
mesenchymal transition (EMT), including E-cadherin and vimentin, and inhibition of
metalloproteinase-2/-9. High-throughput arrays revealed a significant inhibition of the
phosphorylation of 45 tyrosine kinases substrates, whose visualization on Cytoscape highlighted
PTK2/FAK as an important hub. Inhibition of phosphorylation of PTK2/FAK was validated as one
of the possible mechanisms of action, using a specific ELISA. In conclusion, novel
imidazothiadiazoles show potent antiproliferative activity, mediated by modulation of EMT and
PTK2/FAK.
2

1
. Introduction
The synthesis of hybrid molecules, bearing two or more different biologically active scaffolds in
a single structure, is regarded as one of the most valuable approaches in drug development in
order to obtain new therapeutic strategies to treat oncological diseases [1–3].
The design of anticancer drugs characterized by two moieties with antitumor activity led to the
development of a number of molecules with improved biological potential compared to the
parent compounds. In particular, hybrid anticancer drugs showed improved specificity, a greater
ability to overcome drug-resistance mechanisms, better patient compliance and lower side effects
[
4,5].
The simultaneous presence of two pharmacophores often led to a synergism of the biological
activities and therefore to the capability to act towards more than one target. Many examples of
hybrid compounds with promising cytotoxic properties have been reported in the latest years.
Singla and collaborators described remarkable antiproliferative activity against the NCI-60 cell
lines panel of benzimidazole-triazine hybrids 1 (Fig. 1) which showed IC values from the low
5
0
micromolar to the nanomolar range [6]. The pyrimidine-triazole hybrids 2 (Fig. 1) exhibited
potent anticancer activity against the B16-F10 murine melanoma cancer cell line due to their
ability in reducing the pro-caspase3 level while increasing the p53 and active-caspase 3 levels
[
7]. Pyrazole-benzofuran hybrids 3 (Fig. 1) emerged as promising anticancer compounds against
human pancreatic (Panc-1 and PaCa-2 cells), lung (A549 and H-460), breast (MCF-7), colon
HT-29) and prostate (PC-3) cancer, with IC values in the range of 0.9-2.2 µM (Fig. 1) [8].
(
5
0
3

Fig. 1. Chemical structures of benzimidazole-triazine hybrids 1, pyrimidine-triazole hybrids 2,
pyrazole-benzofuran hybrids 3.
The imidazo[2,1-b][1,3,4]thiadiazole nucleus has been considered a privileged scaffold for the
development of molecules with various pharmacological activities, such as anticancer,[9]
analgesic [10], anti-leishmanial [11], antioxidant [12], antitubercular [13], anticonvulsant [14],
and antibacterial [15,16].
Concerning the antitumor activity, many imidazo[2,1-b][1,3,4]thiadiazole derivatives have been
described as potent anticancer molecules acting on several targets against different tumor
models. Compound 4 (Fig. 2) showed potent inhibitory activity (IC = 1.2 nM) against the
5
0
activin receptor-like kinase 5 (ALK5) proving to be selective toward the P38α kinase [17]. The
imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde 5 (Fig. 2) was three fold more potent than
melphalan, used as reference drug, against murine (L1210) and human (CEM) leukemia cells as
well as against immortalized cervical cancer (HeLa) cells, eliciting IC values of 0.89 µM, 0.75
5
0
µM and 0.90 µM, respectively [18].
4

O
O
COOH
N
O
O
N
S
N
S
S
N
S
N
N
N
F
S
H
Cl
5
4
N
N
N
N
H C
3
6
Fig. 2. Chemical structures of the anticancer compounds 4-6.
In the last decade the indole ring has emerged among the scaffolds recognized as privileged
pharmacophores for the development of new antitumor compounds [19–26].
The indole derivative 6 was described for its potent antiproliferative activity against diffuse
malignant peritoneal mesothelioma (DMPM) cell. Nortopsentin analogue 6 potently inhibited
CDK1 activity eliciting an IC value of 0.86 µM and consequently induced a marked cell cycle
5
0
arrest at the G2/M phase, which was paralleled by an increase in the apoptotic rate [27].
Therefore, on the basis of the interesting anticancer properties described for imidazo[2,1-
b][1,3,4]thiadiazole and indole scaffolds, we decided to evaluate the cytotoxic activity of a
library of thirty-six 3-(6-phenylimidazo[2,1-b][1,3,4]thiadiazol-2-yl)-1H-indole derivatives. In
particular derivative 7 (Fig. 3) was effective against all the tested cancer cell lines showing GI50
values ranging from 1.02 to 9.21 µM [28].
5

Fig 3. Hybrid compound 7 obtained by combining the two bioactive scaffolds indole and
imidazothiadiazole.
These preliminary results prompted further studies on nitrogen heterocyclic systems endowed
with antitumor activity [29–35] and we synthesized eighteen new 3-(imidazo[2,1-
b][1,3,4]thiadiazol-2-yl)-1H indole analogues in order to evaluate how structural modifications
on the indole nucleus, introduction of an aldehyde group at the position 5 of the
imidazothiadiazole scaffold or the replacement of the phenyl ring at the position 6 with a
thiophene ring could influence the anticancer activity of this class of compounds.
We decided to test our new compounds on clinically-relevant models of pancreatic ductal
adenocarcinoma (PDAC). This tumor is an extremely aggressive neoplasm, predicted to become
the second leading cause of cancer-related deaths before 2030 [36]. Cytotoxic chemotherapy
remains the mainstay of treatment for most PDAC patients. Treatment with 5-fluorouracil,
leucovorin, irinotecan and oxaliplatin (FOLFIRINOX) or with a combination of gemcitabine and
nab-paclitaxel, represent the standard-of-care for unresectable patients, and recent data support
the use of FOLFIRINOX as adjuvant therapy after surgical resection [37]. However, PDAC is
6

broadly chemoresistant, with a 5-year survival rate below 9%, and novel, more effective
therapeutics for PDAC remain an important unmet need [38–40].
2
. Chemistry
The new imidazothiadiazole derivatives 12-14 were efficiently synthesized following the
synthetic route described in Scheme 1.
The commercially available indole-3-carbonitrile 9a and the derivatives 9b-e, prepared by
reaction of the appropriate 1H-indole with chlorosulfonyl isocyanate (CSI), were subjected to a
methylation for obtaining the corresponding 1-methyl-1H-indole-3-carbonitriles 10a-e [16]. The
5-(1H-indol-3-yl)-1,3,4-thiadiazol-2-amines 11a-j were obtained in excellent yields (92-100%)
by treating the proper derivatives 9a-e or 10a-e with thiosemicarbazide.
The 1,3,4-thiadiazol-2-amines 11a-j underwent a reaction with the appropriate α-bromoacetyl
compounds in refluxing ethanol to give the hydrobromide derivatives 12a-r. Some of such
hydrobromides, 12a,b,d,e,f,h, were isolated as pure compounds (yields 55-68%) and were
characterized without further purifications. Instead, hydrobromides 12c,g,i,j were treated with
saturated aqueous NaHCO solution producing the corresponding free bases 13 which were
3
purified by column chromatography providing specimens with suitable analytical and spectral
data (yields 58-80%).
Finally, the free bases 13k-r, prepared through the treatment of the corresponding
hydrobromides 12 with saturated aqueous NaHCO solution, were subjected to a reaction of
3
formylation using standard Vilsmeier conditions to give the imidazo[2,1-b][1,3,4]thiadiazole
derivatives 14k-r (yields 70-90%).
Data on physicochemical properties of the compounds are reported in the Supplementary results
and Supplementary Tables 1-2.
7

R
CN
R
R
N
N
iii
i
NH2
N
S
N
1
N
8
b-e
R
H
1
1
R
9
1
a-e (
R = H)
8
b, R = Br; 8c, R = Cl; 8d, R = OCH
3
; 8e, R = F
ii
11a-j
1
0a-e (R = CH )
3
1
1
1
1a, R = H, R = H; 11b, R = H, R = CH ;
3
1
1
a, R = H; b, R = Br; c, R = Cl
11c, R = Br, R = H; 11d, R = Br, R = CH
;
3
1
1
d, R = OCH ; e, R = F
11e, R = Cl, R = H; 11f, R = Cl, R = CH
3
3
1
1
1
1
1g, R = OCH , R = H; 11h, R = OCH , R = CH3
3
3
1
1
1i, R = F, R = H; 11j, R = F, R = CH3
O
O
Br
Br
iv
3
R
or
O
N
H
S
R
R
2
2
R
R
N
N
N
vi
N
N
HBr
S
S
N
1
N
1
12a-r
R
R
1
4k-r
1
1
2c,g,i,j,k-r
3c,g,i,j,k-r (free bases)
v
Scheme 1. Synthesis of 3-(imidazo[2,1-b][1,3,4]thiadiazol-2-yl)-1H-indole derivatives 12-14.
Reagents and conditions: i) CH CN, CSI, 0 °C, 2 h, then DMF, 0 °C, 1.5 h (98-100%); ii) DMF,
3
(
CH O) CO, K CO , 130 °C, 3.5 h (98-100%); iii) trifluoroacetic acid, thiosemicarbazide, 60 °C,
3 2 2 3
3
.5 h (98-100%); iv) anhydrous ethanol, reflux, 24 h (42-80%); v) NaHCO saturated aqueous
3
solution (58-80 %); vi) POCl , DMF, 0 - 5 °C, then compound 13, DMF, 70 °C, 5 h.
3
Table 1. New 3-(imidazo[2,1-b][1,3,4]thiadiazol-2-yl)-1H-indole derivatives 12-14.
1
2
Comp
R
H
H
R
H
R
Yield
57%
55%
68%
63%
68%
58%
58%
80%
58%
70%
71%
91%
81%
60%
82%
70%
75%
90%
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2a
2b
2d
2e
2f
2h
3c
3g
3i
3j
4k
4l
4m
4n
4o
4p
4q
4r
tiophen-3-yl
tiophen-3-yl
tiophen-3-yl
tiophen-3-yl
tiophen-3-yl
tiophen-3-yl
tiophen-3-yl
tiophen-3-yl
tiophen-3-yl
tiophen-3-yl
CH
CH
H
CH
CH
H
3
3
Br
Cl
Cl
F
Br
F
OCH
OCH
H
H
H
H
H
3
3
H
H
3
3
CH
H
CH
H
3
3
C
C
H
H
6
5
6
5
4-F-C
3OCH -C H
3 6 4
2,5-OCH
2,5-OCH
4-NO
2,5-OCH
H
6 4
H
H
-C
-C
H
H
3
6
3
3
H
H
Br
CH
H
3
3
6
-C H
2 6 4
H
-C
6
H
3
3
8

3
. Results and discussion
3.1. Antiproliferative activity
The newly synthesized imidazo[2,1-b][1,3,4]thiadiazoles 12a,b,d,e,f,h, 13c,g,i,j and 14k,l,n,o,p
were submitted to the National Cancer Institute (NCI; Bethesda, MD) for the pharmacological
evaluation of their antitumor activity. They were initially pre-screened according to the NCI
protocol at one-dose of 10 µM on the full panel of 60 human cancer cell lines derived from 9
cancer cell types and grouped into disease subpanels including leukemia, non-small cell lung,
colon, central nervous system, melanoma, ovarian, renal, prostate, and breast cancers. The 12a
and 12h derivatives were selected for further screening at five concentrations at 10-fold dilution
-4
-8
(
10 -10 M) on the full panel. As shown in Table 2, both derivatives have interesting in vitro
anticancer activity with GI values ranging from micromolar to sub-micromolar level, i.e., 0.23-
5
0
11.4 µM, and 0.29-12.2 µM, respectively (Table 2).
In order to expand the NCI panel, we evaluated the in vitro antiproliferative activity of the
compounds 12a,b,d,e,f,h, 13c,g,i,j and 14k-r on a panel of PDAC cells, including SUIT-2,
Capan-1 and Panc-1, by Sulforhodamine-B (SRB) assay. PDAC is indeed broadly
chemoresistant tumor, with a 5-year survival rate below 9%, and novel, more effective
therapeutics for PDAC remain an important unmet need.
A pre-screening assay was initially performed at concentrations of 0.1, 1 and 16 µM. We then
expanded the cytotoxicity test, using at least 8 different concentrations (from 125 nM to 16 µM)
on the most promising compounds, in order to define more accurate half-maximal inhibitory
concentration (IC ) values. The compounds 12a,b,h and 13g exhibited remarkable
5
0
antiproliferative activity on all the preclinical models with IC values in the range from 0.85 to
5
0
4.86 µM (Table 3).
9

a
Table 2. GI and TGI of the compounds 12a and 12h.
5
0
1
2a
12h
Panel/Cell line
GI (µM)
TGI (µM)
GI (µM)
TGI (µM)
5
0
50
Leukemia
CCRF-CEM
HL-60(TB)
K-562
2.19
0.76
0.38
1.47
27.2
20.9
39.9
15.4
2.54
1.34
0.45
3.01
> 100
15.5
> 100
27
RPMI-8226
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
1.72
1.99
1.58
5.46
9.94
4.47
4.85
0.57
0.75
> 100
> 100
39.9
2.85
2.51
1.92
12.2
4.56
3.82
4.71
1.73
1.62
99.5
> 100
23.1
HOP-62
HOP-92
> 100
> 100
> 100
52.7
76.9
NCI-H226
NCI-H23
35.8
> 100
> 100
17.1
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
HCC-2998
HCT-116
> 100
> 100
24.5
2.99
0.63
0.49
0.41
0.50
0.43
26.7
70.6
15.6
10.2
10.5
> 100
5.64
2.04
0.61
0.48
1.04
0.48
33.9
26.4
HCT-15
> 100
14.8
HT29
KM12
> 100
> 100
SW-620
CNS Cancer
SF-268
3.84
1.74
1.49
2.56
0.45
1.21
> 100
6.44
4.60
> 100
8.09
17
6.58
2.24
2.18
3.44
1.29
2.76
> 100
9.39
SF-295
SF-539
7.54
SNB-19
> 100
6.45
SNB-75
U251
17.9
Melanoma
MALME-3M
M14
10.4
0.75
0.23
1.08
4.74
0.71
11.4
0.74
36.9
15.8
0.68
23.8
44.0
15.1
98.3
18.7
1.81
1.05
0.29
1.59
7.23
1.80
7.90
1.93
31.6
> 100
1.13
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
7.29
> 100
12.9
> 100
> 100
1.79
0.92
> 100
9.02
1.93
2.06
> 100
9.67
OVCAR-3
1
0

OVCAR-4
4.42
4.27
2.72
0.56
2.80
> 100
76.4
5.77
8.24
4.53
0.99
3.47
> 100
84.4
OVCAR-5
OVCAR-8
> 100
> 100
82.7
> 100
19.1
NCI/ADR-RES
SK-OV-3
70.9
Renal Cancer
7
86-0
3.06
5.97
3.23
1.00
2.05
1.15
5.21
1.71
> 100
23.6
7.62
4.09
3.39
2.42
1.59
4.71
4.92
1.72
52.3
50.0
86.8
57.4
5.73
> 100
19.4
> 100
A498
ACHN
52.5
CAKI-1
> 100
50.0
RXF 393
SN12C
> 100
33.5
TK-10
UO-31
> 100
Prostate Cancer
PC-3
2.07
2.86
> 100
> 100
3.19
3.67
> 100
31.6
DU-145
Breast Cancer
MCF7
1.17
1.08
2.65
2.87
2.92
20.2
7.88
> 100
31.7
27.8
0.83
3.23
2.34
5.54
1.44
30.0
30.6
13.4
77.4
5.47
MDA-MB-231/ATCC
HS 578T
BT-549
MDA-MB-468
[
a] Data obtained from the NCI in vitro disease-oriented human tumor cell line screen. [b] GI : concentration that
50
inhibit 50% net cell growth. [c] TGI total growth inhibition
PDAC is notoriously resistant to chemotherapy or radiotherapy. For several decades,
gemcitabine monotherapy has been used as a first-line treatment for metastatic PDAC and is still
a cornerstone of PDAC treatment in all stages of this disease. However, this drug has limited
clinical effects caused by primary PDAC resistance, as well as by the development of resistance
within a few weeks from treatment initiation [41]. Therefore, new therapeutic agents should be
tested for their ability to circumvent gemcitabine chemoresistance. For this reason, we assessed
the cytotoxic activity of the compounds 12a,b,h and 13g in the Panc-1R cells, a gemcitabine-
resistant sub-clone obtained by continuous incubation of Panc-1 with 1 µM of the drug [41].
Notably, all these compounds showed antiproliferative activity against Panc-1R, with IC50
ranging from 2.2 ± 0.37 µM (compound 12b) to 3.9 ± 0.25 µM (compound 13g) as reported in
the Fig. 4A and Table 4.
1
1

Table 3. Antiproliferative activity of compounds 12a,b,d,e,f,h, 13c,g,i,j and 14k-r on SUIT-2,
Capan-1 and Panc-1 cell lines.
a
b
IC50 (µM) ± SEM
Cell lines
Comp
SUIT-2
Capan-1
1.19 ± 0.06
1.35 ± 0.04
>16
>16
>16
1.93 ± 0.25
>16
4.52 ± 0.48
>16
>16
10.5 ± 0.21
>16
8.83 ± 0.17
>16
>16
5.73 ± 0.086
6.1 ± 0.019
8.24 ± 0.08
0.02 ± 0.001
0.47 ± 0.13
Panc-1
1.70 ± 0.20
1.69 ± 0.10
>16
>16
>16
2.37 ± 0.028
>16
4.86 ± 0.5
>16
>16
12.41 ± 0.16
>16
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2a
2b
2d
2e
2f
2h
3c
3g
3i
3j
4k
4l
4m
4n
4o
4p
4q
4r
0.85 ± 0.018
0.99 ± 0.078
>16
>16
>16
1.78 ± 0.017
>16
2.16 ± 0.039
>16
>16
9.56 ± 0.34
>16
7.93 ± 0.23
11.49 ± 0.36
13 ± 1.13
12.49 ± 0.18
5.32 ± 0.29
8.7 ± 0.20
0.01 ± 0.001
10.53 ± 037
>16
>16
>16
3.61 ± 0.4
10.56 ± 0.11
0.15 ± 0.01
4.3 ± 0.42
gemcitabine
5
-fluorouracil 0.91 ± 0.15
a
The values are means ± SEM of three separate experiments.
b
:
SEM Standard Error of the Mean.
Our previous studies showed different genetic and epigenetic modifications, including splicing
and phosphoproteomics aberrations [42,43], underlying the molecular mechanisms of
gemcitabine-resistance, and further studies will be carried out to identify how our new
compounds counteract these mechanisms. Moreover, the compounds 12a,b,h and 13g were
tested on a primary patient-derived PDAC cell culture, PDAC-3 (Fig. 4B-C). This cellular model
was chosen since our previous studies showed that its genetic and histological features were
similar to the original tumor [44]. In order to maintain the original characteristics of the primary
tumor, particularly from the genetic point of view, these cells have been kept in culture only for a
1
2

few passages. As shown in Fig. 4B the new imidazothiadiazoles maintained their
antiproliferative activity on PDAC-3 cells, with IC values slightly higher in comparison to the
50
values reported on the previously mentioned cancer cell lines. The phase contrast microscopy
images in the Fig. 4C highlighted the antiproliferative activity of compounds 12a and 12b
(central and right panel, respectively) compared to untreated cells (left picture) after 72 hours of
the treatment.
Because of their remarkable antiproliferative activity the compounds 12a,b,h and 13g were
selected for following mechanistic studies, in order to unravel the mechanisms underlying their
anticancer activity.
D
Cell line
PDAC-3 1.7 ± 0.39 1.7 ± 0.27 5.1 ± 0.37 3.9 ± 0.18
Panc-1R 2.7 ± 0.25 2.2 ± 0.37 2.8 ± 0.3 3.9 ± 0.8
12a
12b
12h
13g
Gemcitabine 5-fluorouracil
0.03 ± 0.01
0.56 ± 0.32
4.5 ± 0.54
>10
Fig. 4. Representative growth curves of Panc-1R (A) and PDAC-3 (B) cells treated with the
compounds 12a,b,h and 13g (from 0.125 to 16 µM). Points, mean values obtained from three
independent experiments; bars, SEM. (C) Representative pictures of PDAC-3 cells after 72 h
from the treatment at concentration of IC value. Left panel: untreated cells; central panel: cells
5
0
1
3

treated with compound 12a; right panel: cells treated with compound 12b. Original
magnification 20X. (D) IC values of compounds 12a,b,h and 13g on gemcitabine-resistant and
50
a
b
:
primary PDAC-3 cells ( The values are means of three separated experiments. SEM Standard
Error Mean).
Despite compound 7 has shown interesting GI values against all the tested cancer cell lines in
5
0
the NCI screening, preliminary biological evaluations of analogues 13k,l,n,p,r showed limited
antiproliferative activity [28]. We evaluated the citotoxicity of the compounds 13m, 13o and 13q
on SUIT-2, Capan-1 and Panc-1 but the IC values were above 16 µM.
5
0
Finally, we performed additional experiments to evaluate the in vitro cytotoxicity of the new
compounds 12a and 12b against the normal fibroblasts Hs27. The results of these experiments
allowed us to calculate the selectivity index (SI, IC non-tumor cell line/IC tumor cell line),
5
0
50
which was 4.5 and 7.2 for compounds 12a and 12b, respectively, and therefore our compounds
have SI similar to Gemcitabine and 5-fluorouracil and were regarded as highly cancer selective
compared to the primary pancreatic cells PDAC3 (Supplementary Table 3).
1
4

3.2. Volume reduction of PDAC-3-derived tumor spheres
Two-dimensional cytotoxicity assay obviously provide a useful method to screen libraries of
compounds with high-throughput efficiency, but they are not capable of resembling the complex
architecture and biology of solid tumors, which grow in three-dimensions (3D) [45]. For this
reason we evaluated two of our most promising compounds (emerging from monolayer assay) on
3D spheroids of PDAC-3 cells. These primary cultures are indeed able to form spheroids that are
more representative of the aggregation of tumor cells in vivo, as also reported in our previous
studies [46].
We treated spheroids with compounds 12a and 12b after 3 days of growth, at 5-times the IC ,
5
0
and we took a picture which represent the Day 1. Then the treatment was repeated every four
days (Day 5 and Day 8) and pictures were taken immediately before that (Fig. 5A).
Fig. 5. Size reduction of PDAC-3 spheroids treated with compounds 12a and 12b at 5-times the
IC (i.e. 8.5 µM). (A) Representative pictures of PDAC-3 spheroids exposed to 12a and 12b,
5
0
taken at day 1 of treatment, and after 5 and 8 days with an automated phase-contrast microscope.
B) Fold-change, corrected for control, of the spheroids size, at Day 1, Day 5 and Day 8. p-
values were determined by Two-way ANOVA followed by Tukey's multiple comparisons test,
*** = p<0.0001. The values were obtained taking into account the mean values of the areas of
at least ten different spheroids.
(
*
1
5

Reduction of the size of spheroids was calculated by measuring their area with ImageJ. As
shown in Fig. 5B, after five, but considerably more after eight days, both compounds clearly
showed their ability to hinder the spheroids formation. This reduction is shown as the fold-
change between treated spheroids compared to the controls and was statistically significant (p-
value < 0.0001). Therefore, these two compounds retained their activity in a 3D model.
3.3. Reduction of cell migration
Next to the lack of clinically relevant improvement in effective treatments, the high metastatic
potential of PDAC is one of the main causes for the poor outcome of this disease [36]. The
ability of the compounds 12a and 12b to inhibit the migratory behaviour of PDAC cells was
investigated by scratch wound-healing assays on SUIT-2, Capan-1, Panc-1, Panc-1R and PDAC-
3
cell lines.
4
Briefly, 5x10 cells/well were seeded into 96-well flat-bottom plates in a volume of 100 µL and
incubated for 24 h to create a monolayer. The scratches in the middle of the wells were created
by scraping with a specific tool with needles. The cells were then treated with the compounds
using 4x IC concentrations. These concentrations were chosen after preliminary experiments
5
0
demonstrating that the exposure for 24 h did not result in pro-apoptotic or necrotic effects. The
wound closure was monitored by phase-contrast microscopy and the pictures were captured
immediately after scratch (T = 0), and at 4, 8, 20 and 24 h from the treatment. As shown in the
Fig. 6A-D, the compounds 12a and 12b induced a remarkable reduction of cell migration rate in
2
Panc-1R and SUIT-2 cell lines. The scratch area (µm ) was already wider in the treated cells
compared to the untreated Panc-1R cells after 8 h of treatment. After 24 h from the beginning of
the treatment we observed a reduction of the cell migration with a fold-change value of
approximately 4 in both cell lines, SUIT-2 and Panc-1, treated with compounds 12a and 12b
1
6

(Fig. 6A,B). Statistical analyses revealed that these differences were significant, compared to the
respective controls (i.e., untreated cells) in both cell lines.
Fig. 6. Modulation of the migration rate in Panc-1R and SUIT-2 cells treated with the
compounds 12a and 12b at concentration of 4x IC . (A-B) Fold-changes in Panc-1R (A) and
50
SUIT-2 (B) cells were determined by taking into consideration at least four scratch areas. All the
P values were calculated with Student’s t-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.
(C-D) Percentages of migration monitored over time (0, 4, 8, 20 and 24 h) of Panc-1R (C) and
SUIT-2 (D) cells treated with compound 12a and 12b at concentration 4x IC . Points, mean
5
0
values obtained from the means of at least three different scratch areas. (E) Representative
images of the wounds closure captured with the microscope at 0, 8 and 20 h on Panc-1R cells.
Original magnification 5X.
1
7

Fig. 7. (A-D) Percentage of migration monitored over time (0, 4, 8, 20 and 24 h) of Capan-1 (A-
B) and Panc-1 (C) cells treated with the compounds 12a and 12b at concentrations of 4x IC .
5
0
Points, mean values obtained from the means of at least three different scratch areas. (D) (Table)
List of P values that were calculated with Student’s t-test. ****p<0.0001, ***p<0.001,
**p<0.01, *p<0.05, ns= not significant.
In particular, compared to untreated cells (set at 100%), the percentages of migration in cells
treated with the compounds 12a and 12b were of 33.3% and 32%, respectively, in Panc-1R (Fig.
6C), and 34.9% and 41%, respectively, in SUIT-2 (Fig. 6D) after 24 h from the start of the
treatment. The anti-migratory activity was also evident in Capan-1 and Panc-1 cells for which we
observed similar statistically significant results, with migration rates between 50% and 60% (Fig.
7A-C and Fig.-table 6D). Finally, as shown in the Supplementary data Fig. 1A, we detected
lower migration rates (64% and 71%, respectively) compared to the control (set at 100%) also in
1
8

the primary PDAC-3 cells treated with the compounds 12a and 12b. Overall, these data
highlighted the ability of our compounds to significantly reduce the rate of cell migration on all
the PDAC preclinical models.
3.4. Modulation of Epithelial Mesenchymal transition events as assessed by qRT-PCR, Western
blot and gelatine zymography
It is well known that epithelial-to-mesenchymal transition (EMT) events are essential for
embryonic development and other physiological events, such as the response to several injuries.
During EMT, epithelial cells undergo changes in their phenotypic tracts through the loss of
polarity, cell-cell adhesion and extracellular matrix, and they acquire mesenchymal features
including motility and invasiveness [47]. Several transcription factors within the cellular
microenvironment regulate the expression of epithelial and mesenchymal marker genes, among
which the most important include the zinc finger transcription factors SNAIL1 and SNAIL2,
potent epithelial repressors belonging to the SNAIL superfamily, E-cadherin (CDH1) and N-
cadherin (CDH12), calcium-dependent cell adhesion molecules, vimentin (VIM), type III
intermediate filament (IF) protein typically expressed in mesenchymal cells, and finally, the
matrix metalloproteinases (MMPs), enzymes involved in the breakdown of extracellular matrix
[
48]. Remarkably, their dysregulation induces a transition from the physiological function to the
pathological one, including mechanisms underlying the origin and progression of tumors and
tissue fibrosis [49]. The loss of CDH1 expression is considered the hallmark of EMTs in cancer
and recently, SNAIL1 and SNAIL2 have been identified as the major determinants for the
repression of its transcription through the direct binding to the E-cadherin E-box promoter [50–
52]. Simultaneously, the gain of mesenchymal markers, such as VIM, CDH12, MMPs and others
occur [53]. In PDAC, these genes contribute to a crucial network of signalling pathways that
contribute to the irreversible change of cell phenotype, both in metastasis and resistance to the
chemotherapy [54]. Because of their outstanding effects against PDAC cell migration, we
1
9

assessed the influence of the imidazothiadiazole derivatives 12a and 12b on these EMTs key
regulator expression, in SUIT-2, Capan-1 and Panc-1 cells, using RT-qPCR, Western blot and
gelatine zymography analyses. Capan-1 and SUIT-2 cells were selected for these experiments
because preliminary analyses of the housekeeping protein GAPDH showed that using lysates of
these cells the Western blot images were not “saturated” and were kept in the linear range (as
revealed by exposing blots to increasing times and drawing a plot of intensity and time
exposure). RT-qPCR reactions were performed in order to evaluate the modulation of the mRNA
expression of SNAIL1, SNAIL2, CDH1, CDH12. Briefly, SUIT-2 and Capan-1 cells
5
(
2.5×10 /well) were seeded into 6-well plates and incubated for 24 h to form a confluent
monolayer. Then they were treated with the compounds 12a and 12b at concentrations of 5x
IC . After 24 h, the cells were harvested using TRIzol, and we extracted the total RNA, which
5
0
was used to synthesize, by reverse transcription, the cDNA for the PCR reactions. The
expression levels were normalized to those of the glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) housekeeping gene, whose expression was constant in all cells, as described
previously [55]. As shown in Fig. 8A-B, the compounds 12a and 12b affected the genes leading
EMTs. In both cell lines the mRNA levels of SNAIL1 and SNAIL2 were increased from 1.5 to
approximately 1.9 fold compared to the GAPDH in the control cells, suggesting a low amount of
protein expression and consequently, due to a negative feedback mechanism, CDH1 protein
expression was upregulated, as reported in the Western blot analysis (Fig. 8C-D, Fig. 8G and
Supplementary data Fig. 2). Instead, the protein expression of VIM and MMP2 were
significantly reduced. Finally, CDH12 protein expression did not noticeably change compared to
the control.
2
0

Fig. 8. SNAIL1, SNAIL2, CDH1 and CDH12 mRNA expressions in SUIT-2 (A) and Capan-1
B) cells treated with the compounds 12a and 12b at 5x IC for 24 h. The expression levels were
(
5
0
determined by RT-qPCR and the results were obtained by the delta-delta Ct (cycle threshold)
analysis. The experiments were conducted in duplicates and the values are shown as means ±
SD. (C-D) SNAIL1, SNAIL2, CDH1, CDH12, VIM and MMP2 protein levels expression in
SUIT-2 (C) and Capan-1 (D) cells treated with compounds 12a and 12b. The protein levels were
determined after 24 h of treatment at concentration 5x IC value by densitometric analysis of the
5
0
Western Blot performed using ImageJ. All the P values were determined by Tukey's multiple
2
1

comparisons test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. (E-F) Gelatine zymography
analysis of media from SUIT-2 and Capan-1 cells incubated with serum-free medium for 24 h.
The enzymatic activity of MMP2 and MMP9 was determined by densitometric analysis. The
cells were treated with the compounds 12a and 12b at concentration 5x IC value for 24 h.
5
0
****p<0.0001, ***p<0.001. (G) Representative image of CDH1 expression determined by
Western blot analysis in Capan-1 cells treated with the compounds 12a and 12b at 5x IC50
concentrations after 24 h.
Furthermore, considering the pivotal role of MMPs in tumor invasion, through degradation of
extracellular matrix components, we evaluated the effect of the compounds 12a and 12b on the
proteolytic activity of MMP2 and MMP9 by using specific gelatine zymography assays. These
assays showed a significant decrease of the activity of MMP2 and MMP9 isolated from SUIT-2
and Capan-1 cells exposed to the compounds. In particular, the activities of the MMP2 and
MMP9 enzymes were decreased by about 50% after 24 h of treatment compared to the control
(Fig. 8E-F). However, as shown in the Supplementary data Fig. 3, the areas of lysis created by
the proteolytic activities of both enzymes isolated from Panc-1 cells treated with compounds 12a
and 12b were not significantly wider compared to the control.
3.5. Profiling of inhibition of kinase activity
To investigate the potential mechanism of action of our compounds, we performed a high-
throughput analysis with the Pamgene tyrosine kinase peptide substrate array (PamChip). The
PamChip consists of 4 identical arrays, each of which contains 144 peptide sequences
immobilized on a porous ceramic membrane (Fig. 9A). Each of these sequences harbours one or
more phosphorylation sites, derived from literature or computational predictions. Finally,
specific fluorescently labelled anti-phospho antibodies are used to detect the amount of
phosphorylated protein by tyrosine kinases from our samples.
2
2

Fig. 9. (A) Representative images of pictures taken at the end point of the control array (left) and
the treated array (right). (B) Network visualization obtained with Cytoscape of the significant
proteins containing differentially phosphorylated peptides, color legend indicates the
significance level of each protein. (C) Barplot of PTK2 phosphorylation in the control vs treated
with 5x IC of 12b.
5
0
The compound 12b significantly inhibited the phosphorylation of 45 peptide substrates in SUIT-
cells, and we visualized on Cytoscape a network highlighting the interactions between proteins
2
containing the phosphorylated peptides. Notably, PTK2/FAK emerged as an important hub
between those proteins (Fig. 9B). In particular, the phosphorylation of PTK2/FAK showed a
more than 2-fold inhibition after treatment with compound 12b, with FDR < 0.01 (Fig. 9C). This
result prompted us to validate the inhibition of phosphorylation of PTK2/FAK as one of the
possible mechanisms of action of our compound, using a specific ELISA assay, as detailed in the
following paragraphs.
2
3

3.6. Inhibition of PTK2/FAK as assessed by ELISA
The focal adhesion kinase (FAK), also known as protein tyrosine kinase 2 (PTK2), is a
downstream non-receptor tyrosine kinase able to mediate information from extracellular matrix
into the cytoplasmatic compartment, through a linker with intracellular tails of integrins [56].
FAK controls several cellular processes, including survival, proliferation and motility. However,
its overexpression is correlated with many aspects of the tumorigenesis. For instance, FAK
regulates the development of metastasis, driving adhesion, invasion and migration events.
Furthermore, the translocation of FAK in the nucleus induces the arrest of p53 activity and its
downstream gene transcription [57]. In PDAC, FAK coordinates several signalling pathways
involved in growth and metastasis processes [58]. Notably, a recent study reported the ability of
the indole-3-carbinol to affect EMTs genes and reduce FAK mRNA expression in MCF-7 cells
[
59]. Thereby, we conducted a quantitative analysis by the Enzyme-Linked Immunosorbent
Assay (ELISA) to investigate whether our imidazothiadiazole compounds could reduce FAK
phosphorylation at tyrosine residue 397 (FAK [pY397]), which is essential for the kinase activity
of this protein. This assay was carried out on lysates of SUIT-2, Capan-1 and Panc-1 cells treated
with compounds 12a and 12b at concentrations of 5x IC50s for 24 h. As shown in the Fig. 10,
these ELISA experiments showed a reduction of p-FAK in all the PDAC cell lines, with fold-
change values ranging from 0.4 to 0.5. Similar results were observed in the Western blot
analyses of the same compounds (Supplementary Fig. 4). These results suggest that FAK is a
target of our compounds and might explain how they can then suppress FAK-driven migration,
and growth in PDAC cells.
2
4

Fig. 10. Inhibition of FAK/PTK2 phosphorylation by compounds 12a and 12b. Modulation of
phosphorylated-FAK (pFAK) at tyrosine residue 397 by compounds 12a and 12b on Panc-1 (A),
Capan-1 (B) and SUIT-2 (C) cells. The amount of pFAK was measured in cell lysates after 24 h
from the treatment with compounds 12a and 12b at 5x IC . All the P values were calculated
5
0s
with Student’s t-test, *p<0.05.
4
. Conclusions
A new series of hybrid molecules, 12a,b,d,e,f,h, 13c,g,i,j and 14k-r compounds bearing in the
same structure the imidazo[2,1-b][1,3,4]thiadiazole and indole scaffolds were efficiently
synthesized and evaluated for their antiproliferative activity and mechanism of action on a panel
of PDAC cells, namely SUIT-2, Capan-1 and Panc-1.
Among the synthesized imidazothiadiazoles, compounds 12a,b,d,e,f,h, 13c,g,i,j and 14k,l,n,o,p
were screened by the NCI on the full panel of sixty human cancer cells at concentration of 10
µM. Notably, compounds 12a and 12h displayed relevant antiproliferative activity eliciting GI50
values in the range from micro- to sub-micromolar levels. In addition, compounds 12b and 13g
considerably reduced PDAC cell proliferation in SUIT-2, Capan-1, Panc-1, Panc-1R
(gemcitabine-resistant) and in the primary cells PDAC-3, growing as monolayers or as
spheroids. Noteworthy, compounds 12a,b,h and 13g are characterized by a thiophene ring at
position 6 of the imidazothiadiazole scaffold, suggesting the importance of this ring for the
pharmacological activity. However, in order to confirm the role of aldehyde for the
antiproliferative activity future studies in 14a-j compounds are warranted.
2
5

Through wound-healing assays we found remarkably reduction of cell migration in all the PDAC
preclinical models when treated with the most promising compounds 12a and 12b. These effects
might be explained by modulation of key regulators of EMT, including E-cadherin and vimentin,
as well as by the inhibition of MMP-2/-9 activities. Finally, high-throughput analysis with kinase
arrays revealed a significant inhibition of the phosphorylation of 45 tyrosine kinases substrates,
whose visualization on Cytoscape highlighted PTK2/FAK as an important hub between those
proteins. These results were validated using a specific ELISA assay, which demonstrated the
inhibition of phosphorylation of PTK2/FAK. Altogether these results support the high potential
of this type of compounds against EMT and PTK2/FAK, which play a key role in the
aggressiveness of pancreatic cancer [55]. In order to support our experimental findings we also
investigated the molecular structure of FAK. Unfortunately complete crystal structures of the
molecule are not available; e.g. the Tyr397 part is lacking in the published structures, which may
be due to the high flexibility of this region of the molecule. This means that proper molecular
docking is not yet feasible.
5
. Experimental section
5.1. Chemistry
All melting points were taken on a Büchi-Tottoly capillary apparatus and are uncorrected. IR
1
spectra were determined in bromoform with a Shimadzu FT/IR 8400S spectrophotometer. H and
1
3
C NMR spectra were measured at 200 and 50.0 MHz, respectively, in DMSO-d solution, using a
6
Bruker Avance II series 200 MHz spectrometer. Column chromatography was performed with
Merck silica gel 230-400 mesh ASTM or with Büchi Sepacor chromatography module (prepacked
cartridge system). Elemental analyses (C, H, N) were within ±0.4% of theoretical values and were
performed with a VARIO EL III elemental analyzer. The LC/HRMS have been obtained on a
Thermo Q-Exactive system equipped with a Dionex 3000 chromatographic system.
2
6

5.1.1. Synthesis of 1H-indole-3-carbonitriles (9b-e)
A solution of the suitable indole 8 (5.10 mmol) in anhydrous acetonitrile (4.5 mL) was treated
dropwise with chlorosulfonyl isocyanate (CSI) (0.44 mL, 5.10 mmol). The reaction mixture was
maintained at 0 °C under stirring for 2 h, then, anhydrous dimethylformamide (DMF) (2.8 mL,
3
6.39 mmol) was slowly added and the mixture was stirred at 0 °C for 1.5 h. The resulting
solution was poured into crushed ice. The solid obtained was filtered and dried (yields 98-
00%). Analytical and spectroscopic data for compounds 9b-e are in agreement with those
previously reported [60].
1
5.1.2 Synthesis of 1-methylindole-3-carbonitriles (10a-e)
To a solution of the suitable 3-cyanoindole 9 (7.03 mmol) in anhydrous DMF (10 mL) 3.61
mmol of K CO and dimethyl carbonate (1.8 mL, 21.4 mmol) were added and the mixture was
2
3
heated at 130 °C for 3.5 h. After cooling (0-5 °C), water and ice (25 mL) was slowly added
under stirring. The oily suspension obtained was extracted with diethyl ether (3x10 mL), the
organic phase was washed with water and brine, dried over Na SO and the solvent evaporated at
2
4
reduced pressure to obtain the 3-cyano-1-methylindoles 10 in excellent yields. Analytical and
spectroscopic data are in accordance to those reported in literature [16].
5.1.3. Synthesis of 5-(1H-indol-3-yl)-1,3,4-thiadiazol-2-amines (11a-j)
A mixture of the suitable indole-3-carbonitrile 9a-e or 10a-e (5 mmol), thiosemicarbazide (5
mmol) and trifluoroacetic acid (5 mL) was heated under stirring at 60 °C for 3.5 h. The reaction
mixture was then poured into ice and neutralized with NaHCO saturated solution. The solid
3
obtained was filtered off, washed with water, cyclohexane and diethyl ether to give 5-(1H-indol-
2
7

3-yl)-1,3,4-thiadiazol-2-amines 11a-j in excellent yields. Analytical and spectroscopic data for
the derivatives 11a-f are in accordance to those reported in literature [16].
5.1.3.1. 5-(5-Methoxy-1H-indol-3-yl)-1,3,4-thiadiazol-2-amine (11g)
-1 1
Light yellow solid. Yield: 98%, m.p. 216-217 °C IR: 3604 (NH), 3558 (NH ) cm ; HNMR
2
(
200MHz, DMSO-d ) δ: 3.79 (3H, s, CH ), 6.88 (1H, dd, J = 2.4, 8.8 Hz, Ar-H), 7.39 (1H, d, J =
6 3
8
.8 Hz, Ar-H), 7.53 (1H, d, J = 2.3 Hz, Ar-H), 8.02 (1H, s, Ar-H), 8.58 (2H, bs, NH ), 12.07 (1H,
2
1
3
bs, NH). C NMR (50 MHz, DMSO-d ) δ: 55.2 (q), 101.9 (d), 105.7 (s), 112.9 (d), 113.0 (d),
6
1
2
24.4 (s), 128.7 (d), 131.5 (s), 152.2 (s), 154.7 (s), 166.5 (s). Anal. Calcd for C H N OS (MW:
11 10 4
46.29): C, 53.64; H, 4.09; N, 22.75. Found: C, 53.72; H, 4.16; N, 22.98.
5.1.3.2. 5-(5-Methoxy-1-methyl-1H-indol-3-yl)-1,3,4-thiadiazol-2-amine (11h)
-
1
1
Light yellow solid. Yield: 99%, m.p. 205-206 °C IR: 3381 (NH ) cm ; HNMR (200MHz,
2
DMSO-d ) δ: 3.80 (6H, s, CH , OCH ), 6.90 (1H, dd, J = 2.5, 8.8 Hz, Ar-H), 7.13 (2H, s, NH ),
6
3
3
2
1
3
7
.41 (1H, d, J = 8.9 Hz, Ar-H), 7.61 (1H, d, J = 2.4 Hz, Ar-H), 7.81 (1H, s, Ar-H). C NMR (50
MHz, DMSO-d ) δ: 32.8 (q), 55.3 (q), 102.3 (d), 106.0 (s), 111.2 (d), 112.5 (d), 124.9 (s), 130.7
6
(
d), 132.0 (s), 152.2 (s), 154.6 (s), 165.4 (s). Anal. Calcd for C H N OS (MW: 260.31): C,
12 12 4
55.37; H, 4.65; N, 21.52. Found: C, 55.42; H, 4.80; N, 21.78.
5.1.3.3. 5-(5-Fluoro-1H-indol-3-yl)-1,3,4-thiadiazol-2-amine (11i)
-1
1
Light yellow solid. Yield: 98%, m.p. 257°C IR: 3609 (NH), 3461 (NH ) cm ; HNMR
2
(
200MHz, DMSO-d ) δ: 7.03-7.12 (1H, m, Ar-H), 7.33-750 (3H, m, Ar-H, NH ), 7.80 (1H, dd, J
6
2
1
3
=
2.5, 10.0 Hz, Ar-H), 7.95 (1H, s, Ar-H), 11.79 (1H, s, NH). C NMR (50 MHz, DMSO-d ) δ:
6
105.4 (d, J = 24 Hz), 107.4 (s, J = 4.5 Hz), 110.7 (d, J = 25.5 Hz), 113.1 (d, J = 10 Hz), 124.4 (s,
J = 11 Hz), 128.6 (d), 130.1 (s), 152.0 (s), 159.9 (s), 165.9 (s). Anal. Calcd for C H FN S (MW:
1
0
7
4
234.25): C, 51.27; H, 3.01; N, 23.92. Found: C, 51.38; H, 3.25; N, 24.12.
2
8

5.1.3.4. 5-(5-Fluoro-1-methyl-1H-indol-3-yl)-1,3,4-thiadiazol-2-amine (11j)
-1 1
Light orange solid. Yield: 98%, m.p. 183°C. IR: 3471 (NH ) cm ; HNMR (200MHz, DMSO-
2
d ) δ: 3.85 (3H, s, CH ), 7.12-7.21 (1H, m, Ar-H), 7.59 (1H, dd, J =4.4, 9.9 Hz, Ar-H), 7.76 (2H,
6
3
1
3
dd, J =2.5, 9.8 Hz, Ar-H), 8.10 (2H, s, NH ). C NMR (50 MHz, DMSO-d ) δ: 33.14 (q), 105.4
2
6
(s), 105.5 (d, J = 24 Hz), 110.9 (d, J = 26 Hz), 112.0 (d, J = 9.5 Hz), 124.3 (s), 124.5 (s), 133.1
(
d), 151.4 (s), 155.7 (s). Anal. Calcd for C H FN S (MW: 248.28): C, 53.21; H, 3.65; N, 22.57.
1
1
9
4
Found: C, 53.38; H, 3.88; N, 22.72.
5
.1.4. General procedure for the synthesis of 3-(6-phenylimidazo[2,1-b][1,3,4]thiadiazol-2-yl)-
H-indole derivatives (12 and 13)
1
A mixture of 5-(1H-indol-3-yl)-1,3,4-thiadiazol-2-amine 11a-j (0.92 mmol) and the suitable α-
bromoacetyl derivative (0.92 mmol) in 40 mL of anhydrous ethanol was stirred at reflux for 24
h. After cooling at room temperature the desired product 12 was filtered off and washed with
cold ethanol.
Derivatives 12a,b,d-f,h were isolated as pure compounds and were characterized without further
purifications. Whereas, compounds 12c,g,i,j were treated with saturated aqueous NaHCO3
solution to give the corresponding free bases 13 which were purified by silica gel column
chromatography eluting by petroleum ether:ethyl acetate, 1:1. Analytical and spectroscopic data
for the derivatives 12k-r are in accordance to those reported in literature [16].
5.1.4.1 3-[6-(Thiophen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl]-1H-indole hydrobromide
(12a)
-
1
1
White solid, yield: 57%, m.p. 288-289 °C, IR cm : 3630 (NH), 3458 (NH). HNMR (200 MHz,
DMSO-d ) δ: 7.27-7.34 (2H, m, 2xAr-H), 7.54-7.59 (2H, m, 2xAr-H), 7.68 (1H, dd, J = 2.9, 5.0
6
2
9