Design, synthesis and biological evaluation of novel 1,3,4-thiadiazole derivatives as anti-glioblastoma agents targeting the AKT pathway

Article abstract of DOI:10.1016/j.bioorg.2020.104362

In spite of progress in understanding biology of glioblastoma (GBM), this tumor remains incurable with a median survival rate of 15 months. Previous studies have shown that 2-(4-fluorophenyloamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole (FPDT) and 2-(3

Full text of DOI:10.1016/j.bioorg.2020.104362

Bioorganic Chemistry 105 (2020) 104362
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of novel 1,3,4-thiadiazole
derivatives as anti-glioblastoma agents targeting the AKT pathway
,
*
b
c
b
Monika Szeligaa , Monika Karpinska , Radosław Rola , Andrzej Niewiadomy
´
a Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego Str, 02-106 Warsaw, Poland
b Łukasiewicz Research Network – Institute of Industrial Organic Chemistry, 6 Annopol Str., 03-236 Warsaw, Poland
c Department of Neurosurgery and Paediatric Neurosurgery of the Lublin Medical University, 8 Jaczewskiego Str, 20-090 Lublin, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords:
In spite of progress in understanding biology of glioblastoma (GBM), this tumor remains incurable with a median
survival rate of 15 months. Previous studies have shown that 2-(4-fluorophenyloamino)-5-(2,4-dihydrox-
yphenyl)-1,3,4-thiadiazole (FPDT) and 2-(3-chlorophenyloamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole
(CPDT) diminished viability of cancer cell lines of different origin. In the current study, we have examined
activity of these compounds in several GBM cell lines and patient-derived GBM cells. We have also designed,
Glioblastoma
Anti-glioblastoma compound
Thiadiazole derivative
Viability
Proliferation
–
synthesized and evaluated anti-GBM activity of novel 1,3,4-thiadiazole derivatives containing additional Cl or
AKT pathway
–
CH2CH3 substitute at C5-position of 2,4-dihydroxyphenyl. The tested compounds presented a considerable
cytotoxicity against all GBM cell lines examined as well as patient-derived GBM cells. They were 15–110 times
more potent than temozolomide, the first-line chemotherapeutic agent for GBM. Notably, in anticancer con-
centrations three of the derivatives were not toxic to human astrocytes. FPDT appeared to be the most promising
compound with IC50 values between 45 μM and 68 μM for GBM cells and >100 μM for astrocytes. It augmented
activity of temozolomide and inhibited proliferation migration and invasion of GBM cells. Treatment with FPDT
diminished phosphorylation level of GSK3β and AKT. Pretreatment with PDGF-BB, an AKT activator, partially
protected cells from death caused by FPDT, indicating that FPDT-mediated decrease in cell viability is causatively
related to the inhibition of the AKT pathway.
1. Introduction
with GBM remains still poor: median survival time is approximately one
year [2]. Diffuse growth and as well as inter- and intratumor heteroge-
Glioblastoma (GBM, WHO IV) is the most frequent malignant primary
brain tumor in adults. The standard of care for newly diagnosed GBM is
maximal safe surgical resection, radiotherapy and concomitant and
adjuvant chemotherapy with alkylating agent temozolomide (TMZ)
(Fig. 1A) [1]. Despite the progress in understanding of biology of glio-
magenesis made during the past decade, the clinical outcome of patients
neity are the most important hallmarks of GBM, which significantly
contribute to therapeutic failure [3]. Moreover, the blood-brain barrier
(BBB) and blood-tumor barrier (BTB) represent major obstacles to effec-
tive drug delivery [4]. Furthermore, side effects of each treatment cause
significantdecreaseinqualityoflife. Therefore, continuouseffortsshould
be made to find new molecular targets and effective therapies of GBM.
Abbreviations: ATCC, American Type Culture Collection; BBB, blood-brain barrier; BrdU, bromodeoxyuridine; BTB, blood-tumor barrier; CPCDT, 2-(3-chlor-
ophenylamino)-5-(5-chloro-2,4-dihydroxyphenyl)-1,3,4-thiadiazole; CPDT, 2-(3-chlorophenyloamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole; CPEDT, 2-(3-
chlorophenylamino)-5-(5-ethyl-2,4-dihydroxyphenyl)-1,3,4-thiadiazole; DMEM, Dulbecco’s Modified Eagle’s Medium; DMSO, dimethyl sulfoxide; EMEM, Eagle’s
Minimum Essential Medium Eagle; FBS, fetal bovine serum; FCS, fetal calf serum; FPCDT, 2-(4-fluorophenylamino)-5-(5-chloro-2,4-dihydroxyphenyl)-1,3,4-thia-
diazole; FPDT, 2-(4-fluorophenyloamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole; FPEDT, 2-(4-Fluorophenylamino)-5-(5-ethyl-2,4-dihydroxyphenyl)-1,3,4-thia-
diazole; GBM, glioblastoma; HA, human astrocytes; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; 2-(3-chlorophenyloamino)-5-(2, 4-
dihydroxyphenyl)-1,3,4-thiadiazole; PBS, phosphate buffer saline; PDGF-BB, platelet-derived growth factor-BB; SClTB, sulfinylbis(5-chloro-2,4-dihydrox-
ythiobenzoyl); SETB, sulfinylbis((5-ethyl-2,4-dihydroxyphenyl)methanethione); STB, sulfinylbis(2,4-dihydroxythiobenzoyl); TMZ, temozolomide.
* Corresponding author.
´
E-mail addresses: mszeliga@imdik.pan.pl (M. Szeliga), monika_karpinska2@wp.pl (M. Karpinska), rola.radoslaw@gmail.com (R. Rola), andrzej.niewiadomy@up.
lublin.pl (A. Niewiadomy).
https://doi.org/10.1016/j.bioorg.2020.104362
Received 13 July 2020; Received in revised form 3 September 2020; Accepted 6 October 2020
Available online 9 October 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.

M. Szeliga et al.
Bioorganic Chemistry 105 (2020) 104362
on the viability of normal rat astrocytes and neurons [18,19]. The aim of
the present study was to evaluate the efficacy of FPDT and CPDT in
human GBM cell lines and cells isolated from human GBM tissues. The
key premise is that potential anti-glioma drug should display anti-
proliferative activity in tumor cells without affecting normal tissues,
therefore we also examined the influence of FPDT and CPDT on normal
human primary astrocytes (HA). Moreover, the analysis was extended to
four 1,3,4,-thiadiazole derivatives not tested so far. The newly tested
–
–
CH2CH3 substitute at C5-
compounds contain additional
Cl or
position of 2,4-dihydroxyphenyl. The choice of these modifications
was guided by the literature data indicating that the hydrophobic sub-
stituent (π > 0) in the resorcinol ring can improve antiproliferative
potency against cancer cells. It has been reported that the presence of a
chlorine atom or ethyl (methyl, isopropyl) substituents in position 5 of
resorcinol moiety of resorcylazoles can enhance their anticancer prop-
erties [20–22]. This finding was confirmed in the groups of 1H-benz-
imidazoles, 1,3-thiazolo[5,4-b]pyridines and 4H-3,1-benzothiazines
substituted with 2,4-dihydroxyphenyl substituent [23–25]. Herein, both
FPDT and CPDT notably decreased survival of GBM cells and FPDT did
not display cytotoxic activity against non-transformed HA. The addition
– –
of either Cl or CH2CH3 substituent enhanced anti-GBM properties of
the tested derivatives, but also significantly enhanced their toxic effects
on HA. Hence, FPDT was subsequently subjected to further analysis to
gain insight into possible biological mechanism of this compound.
2. Materials and methods
2.1. Synthesis of thiadiazole derivatives
The synthesis procedure of CPDT and FPDT was described previously
[26]. Four other thiadiazole derivatives analyzed in this study were: 2-
(4-Fluorophenylamino)-5-(5-chloro-2,4-dihydroxyphenyl)-1,3,4-thia-
diazole (FPCDT); 2-(4-Fluorophenylamino)-5-(5-ethyl-2,4-dihydrox-
yphenyl)-1,3,4-thiadiazole (FPEDT); 2-(3-Chlorophenylamino)-5-(5-
chloro-2,4-dihydroxyphenyl)-1,3,4-thiadiazole (CPCDT); 2-(3-Chlor-
ophenylamino)-5-(5-ethyl-2,4-dihydroxyphenyl)-1,3,4-thiadiazole
(CPEDT). The chemical structures of the tested compounds are depicted
in Fig. 2.
All derivatives were prepared by the reaction of sulfinylbis(2,4-
dihydroxythiobenzoyl) (STB), sulfinylbis(5-chloro-2,4-dihydroxythi
obenzoyl) (SClTB) or sulfinylbis((5-ethyl-2,4-dihydroxyphenyl)meth
anethione) (SETB) with commercially available thiosemicarbazides in
the endocyclization reaction. Briefly, STB, SClTB or SETB was mixed
with either 4-(4-fluorophenyl)-3-thiosemicarbazide or 4-(3-chlor-
ophenyl)-3-thiosemicarbazide (Alfa Aesar) in methanol and refluxed for
3 h. The reaction mixture was hot filtered and the product was crys-
tallized from methanol. Subsequently, the compounds were subjected to
in-depth analytical studies. The results of these analyses are following:
2-(4-Fluorophenylamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadia-
Fig. 1. Chemical structure of the first-line chemotherapeutic for GBM, temo-
zolomide (A) and two thiadiazole based clinically used drugs, acetazolamide
(B) and methazolamide (C).
Thiadiazoles occurrs in nature in four isomeric forms 1,2,3-thiadia-
zole; 1,2,4-thiadiazole; 1,2,5-thiadiazole and 1,3,4,-thiadiazole. They
exhibit a wide variety of biological activities, including antimicrobial,
antifungal, antiviral, anti-inflammatory or anticonvulsant properties
andincreasingnumberofthiadiazolebaseddrugsarecurrentlyavailable
in the market [5]. Antineoplastic activity of different thiadiazole de-
rivatives has been documented in a wide range of tumor cell lines [6–10]
and in vivo [11,12]. The molecular mechanism underlying this activity
most likely depends on the type of modification of thiadiazole ring. N-(5-
sulfamoyl-1,3,4-thiadiazol-2-yl)acetamide (acetazolamide) (Fig. 1B)
and N-(3-Methyl-5-sulfamoyl-3H-1,3,4-thiadiazol-2-ylidene) ethana-
mide(methazolamide)(Fig. 1C)areclinicallyusedinhibitorsofcarbonic
anhydrase [13]. Bis-2-(5 phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl
sulfide (BPTES) is an allosteric inhibitor of kidney type glutaminase
isoform C [14], and similar mode of action was ascribed to a series of
BPTES analogs [11,15,16]. A very recent study showed that 5-(3,5-
dinitrophenyl)-1,3,4-thiadiazolederivativesincorporatedwithdifferent
heterocyclic systems inhibits activity of dihydrofolate reductase [17].
Two thiadiazole derivatives: 2-(4-fluorophenyloamino)-5-(2,4-dihy-
droxyphenyl)-1,3,4-thiadiazole (FPDT) and 2-(3-chlorophenyloamino)-
5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole (CPDT) presented anti-
proliferative activity in several tumor cell lines derived from cancers of
different origins including rat glioma C6 cell line, but had no influence
◦
zole (FPDT): Yield: 69%, mp: 279–280 C. EI-MS (m/z, %): 303 (M+,
100), 168 (39), 135 (11), 136 (14), 121 (4), 109 (3), 110 (8), 95 (5), 94
(12), 83 (6), 66 (6). 1H NMR (500 MHz, DMSO‑d6, δ): 10,85 (s, 1H, C2-
OH), 10,27 (s, 1H, C4-OH), 9,92 (s, 1H, NH), 7,82–7,84 (d, 1H, C6-H),
7,61–7,70 (m, 2H, C3′,5′-H), 7,12–7,26 (m, 2H, C2′,6′-H), 7,00–7,08
(m, 1H, C4′-H), 6,44–6,45 (s, 1H, C3-H), 6,42–6,43 (d, 1H, C-5). 13C
NMR (125 MHz, DMSO‑d6, δ): 102,40; 108,74; 108,45; 115,50; 118,83;
128,55; 137,36; 154,78; 155,66; 157,94; 160,23; 163,57. IR (KBr,
cmꢀ 1): 3421 (NH), 3400, 3259, 3216 (OH, NH), 1629 (C N), 1590
–
–
–
–
–
–
–
– –
C S
(C C), 1231 (C OH), 1134 (C F), 1052 (N
C
–
N), 677
–
–
– –
(C
S C). Anal. Calcd for C14H10FN3O2S (303,32): C, 55,44; H, 3,22;
N, 13,85. Found: C, 55,61; H, 3,23; N, 13,79.
2-(4-Fluorophenylamino)-5-(5-chloro-2,4-dihydroxyphenyl)-1,3,4-
thiadiazole (FPCDT): Yield: 75%, mp: 241–243 ◦C. EI-MS (m/z, %): 337
(M+, 100), 303 (6), 187 (11), 168 (39), 154 (6), 141 (10), 136 (12), 128
(10), 110 (9), 95 (14), 83 (9), 69 (8), 63 (2), 51 (4). 1H NMR (500 MHz,
DMSO‑d6, δ): 11,05 (s, 1H, C2-OH); 10,65 (s, 1H, C4-OH); 10,28 (s, 1H,
2

M. Szeliga et al.
Bioorganic Chemistry 105 (2020) 104362
Fig. 2. Chemical structure of the 1,3,4-thiadiazole derivatives tested in this study.
3

M. Szeliga et al.
Bioorganic Chemistry 105 (2020) 104362
–
–
–
NH); 7,93 (s, 1H, CAr-H); 7,67 (q, 2H, CAr-H); 7,19 (t, 2H, CAr-H); 6,68
(s, 1H, CAr-H). 13C NMR (125 MHz, DMSO‑d6, δ): 164,44; 158,08;
156,19; 155,29; 153,94; 152,75; 137,36; 127,37; 119,05; 115,66;
115,48; 111,65; 109,88; 103,73. IR (KBr, cmꢀ 1): 3642 (NH), 3196 (OH),
(C C), 1513 (C C), 1482, 1453, 1390, 1318, 1278, 1261, 1225, 1187,
–
1136, 1094, 993, 926, 876, 819, 786, 771, 722, 671. Anal. Calcd for
C16H14N3O2SCl (348,47): C, 55,37; H, 4,02; N, 12,05. Found: C, 55,47;
H, 3,99; N, 12,11.
–
–
–
3153 (NH), 2897 (CH), 2834 (CH), 1622 (C N); 1566 (C C), 1509
–
–
–
(C C), 1484, 1447, 1391, 1295, 1260 (C OH), 1219, 1189, 1158,
–
2.2. Reagents
1105, 1052, 1011, 973, 866, 833, 811, 732. Anal. Calcd for
C14H9N3O2SFCl (338,35): C, 49,90; H, 2,66; N, 12,41. Found: C, 49,81;
H, 2,68; N, 12,47.
The tested thiadiazole derivatives were dissolved in DMSO at a
concentration of 5 mM. These working solutions were stored at room
temperature (RT). TMZ was purchased from Sigma-Aldrich and a 100
mM working solution in dimethyl sulfoxide (DMSO) (Sigma-Aldrich)
was prepared prior to storage at ꢀ 20 ◦C. For all experiments, final
concentration of DMSO was 0.1% (v/v). Minimum Essential Medium
Eagle (MEME) was obtained from Sigma-Aldrich and Eagle’s Minimum
Essential Medium Eagle (EMEM) from ATCC. Dulbecco’s Modified Ea-
gle’s Medium (DMEM), fetal bovine serum (FBS), fetal calf serum (FCS),
non-essential amino acids penicillin and streptomycin were purchased
from Gibco. Platelet-derived growth factor-BB (PDGF-BB) was pur-
2-(4-Fluorophenylamino)-5-(5-ethyl-2,4-dihydroxyphenyl)-1,3,4-
thiadiazole (FPEDT):
Yield: 73%, mp: 235–236 ◦C. EI-MS (EI, m/z, B): 331 (M+, 100), 316
(M-Me, 72), 181 (8), 168 (11), 148 (9), 110 (6), 95 (5), 69 (10), 65 (3).
1H NMR (500 MHz, DMSO‑d6, δ): 10,61 (s, 1H, C2-OH); 10,26 (s, 1H,
C4-OH); 9,83 (s, 1H, NH); 7,67 (q, 3H, CAr-H); 7,19 (t, 2H, CAr-H); 6,55
(s, 1H, CAr-H); 2,51 (k, 2H, CH2Me); 1,14 (t, 3H, CH2Me). 13C NMR
(125 MHz, DMSO‑d6, δ): 163,52; 158,02; 156,12; 155,11; 153,72;
137,41; 127,33; 122,52, 118,96; 115,62; 115,44; 107,87; 102,27; 20,07
(2C); 14,32 (3C). IR (KBr, cmꢀ 1): 3379 (NH), 3257 (OH), 3212 (OH),
chased from Cell Signaling Technology. A 5 μg/ml working solution in
20 mM citrate, pH 3.0 was prepared prior to storage at ꢀ 20 ◦C.
Phosphate-buffered saline (PBS) and thiazolyl blue tetrazolium bromide
(MTT) were obtained from Sigma-Aldrich.
–
–
–
–
3165 (OH), 2959 (CH), 2832 (CH), 1625 (C N); 1586 (C C), 1510
–
–
(C C), 1446, 1392, 1380, 1316, 1260, 1232 (C OH), 1186, 1160,
–
1140, 1126, 1058, 1012, 973, 876, 824, 766, 744, 671. Anal. Calcd for
C16H14N3O2SF (332,02): C, 58,12; H, 4,22; N, 12,65. Found: C, 58,03;
H, 4,25; N, 12,73.
2.3. Cell lines
2-(3-Chlorophenylamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadia-
zole (CPDT):
Three human GBM cell lines were used in this study: T98G, U87MG
Yield: 77%, mp: 265–266 ◦C. EI-MS (m/z, %): 319 (M+, 100), 184
(36), 167 (5), 152 (5), 153 (10), 149 (16), 136 (8), 121 (5), 111 (6), 94
(10), 66 (5), 52 (3), 39 (4). 1H NMR (200 MHz, DMSO‑d6, δ): 9,80–11,02
(3H, C2,4-OH, NH), 7,92 (s, 1H, CAr-H), 7,81–7,84 (d, 1H, C6-H),
7,31–7,55 (m, 2H, CAr-H), 6,97–7,05 (m, 1H, CAr-H), 6,52 (s, 1H, C3-
H), 6,42 (s, 1H, C5-H). 13C NMR (125 MHz, DMSO‑d6, δ): 102,42;
108,15; 108,43; 115,65; 116,57; 120,94; 128,50; 130,55; 133,44;
142,16; 155,09; 155,66; 160,40; 163,10. IR (KBr, cmꢀ 1): 3245 (OH,
and LN229. T98G cell line purchased from ATCC (CRL-1690™) was
maintained in MEME supplemented with 50 U/mL penicillin, 50 μg/mL
streptomycin, 1% non-essential amino acids and 10% FBS. U87MG cell
line purchased from Sigma-Aldrich (89081402) was maintained in
EMEM supplemented with 50 U/mL penicillin, 50 μg/mL streptomycin
and 15% FBS. LN229 cell line (kindly provided by Rafał Krętowski, PhD,
Department of Pharmaceutical Biochemistry, Medical University of
Białystok, Poland) was maintained in DMEM supplemented with 50 U/
–
–
–
–
–
–
NH), 1626 (C N), 1598 (C C), 1225 (C OH), 1110 (C Cl), 677
mL penicillin, 50 μg/mL streptomycin, 10% FBS and glucose (4.5 g/l).
– –
(C
S C). Anal. Calcd for C14H10ClN3O2S (319,77): C, 52,59; H, 3,15;
Cells were routinely tested for mycoplasma contamination using My-
coplasma Detection Kit-Quick Test (Biotool). The used passage numbers
were below 25. All cell lines were authenticated by ATCC using human
short tandem repeat (STR) analysis. Normal human astrocytes (HA)
were purchased from Lonza (CC-2565) and cultured in the AGM Astro-
cyte Growth Medium BulletKit containing Basal Medium supplemented
with AGM SingleQuots Supplements according to the manufacturer’s
instruction. Cells were incubated at 37 ◦C, 5% CO2 and 95% humidity.
N, 13,14. Found: C, 52,39; H, 3,13; N, 13,19.
2-(3-Chlorophenylamino)-5-(5-chloro-2,4-dihydroxyphenyl)-1,3,4-
thiadiazole (CPCDT):
Yield: 74%, mp: 264–266 ◦C. EI-MS (m/z, %): 353 (M+, 100), 319
(7), 200 (3), 186 (15), 184 (41), 169 (13), 149 (17), 128 (11), 111 (12),
100 (6), 90 (4), 75 (9), 69 (8), 63 (5), 51 (5). 1H NMR (500 MHz,
DMSO‑d6, δ): 11,11 (s, 1H, C2-OH); 10,69 (s, 1H, C4-OH); 10,54 (s, 1H,
NH); 7,96 (t, 2H, CAr-H); 7,48 (q, 1H, CAr-H); 7,38 (t, 1H, CAr-H); 7,04
(m, 1H, CAr-H); 6,71 (s, 1H, CAr-H). 13C NMR (125 MHz, DMSO‑d6, δ):
161,83; 155,37; 153,92; 153,08; 142,13; 133,43; 130,53; 127,22;
120,97; 116,62; 115,69; 111,61; 109,75; 101,67. IR (KBr, cmꢀ 1): 3421
(NH), 3242 (OH), 3100 (OH), 2966 (CH), 2943 (CH), 2825 (CH), 1619
2.4. Primary GBM culture
Clinical samples from patients with WHO grade IV GBM were
collected from the Department of Neurosurgery and Pediatric Neuro-
surgery of the Medical University in Lublin, Poland. Prior written
informed consent was obtained from patients, and the local Ethics
Committee approved all procedures. Tumor samples were frozen vitally
in freezing medium (FCS containing 10% DMSO) at ꢀ 80 ◦C. Until
–
–
–
–
–
–
–
(C N), 1599 (C C), 1565 (C C), 1510 (C C), 1480, 1454, 1415,
–
–
1397, 1298 (C OH), 1223, 1181, 1168, 1137, 1097, 992, 970, 925,
868, 845, 812, 771, 734, 721, 672, 615. Anal. Calcd for
C14H9N3O2SCl2 (354,81): C, 47,59; H, 2,54; N, 11,84. Found: C, 47,68;
H, 2,51; N, 11,89.
◦
unthawing, samples were kept at ꢀ 80 C for a maximum of 6 weeks.
2-(3-Chlorophenylamino)-5-(5-ethyl-2,4-dihydroxyphenyl)-1,3,4-
thiadiazole (CPEDT):
Patient-derived GBM cell lines were established according to the pro-
tocol by Mullins et al. [27]. Briefly, frozen tumor samples were thawed
at 37 ◦C, minced by scalpels in DMEM/HAM’s F12 medium supple-
mented with 10% FCS, 2 mM L-glutamine, penicillin-streptomycin and
passed through cell strainer to obtain a single cell suspension. Cells were
washed with PBS and seeded in 6-well plates coated with collagen.
Outgrowing cells were detached with trypsin and expanded for subse-
quent analyses.
Yield: 78%, mp: 236–239 ◦C. EI-MS (m/z, %): 347 (M+, 100), 332
(M-Me, 84), 318 (3), 212 (3), 181 (12), 165 (10), 148 (14), 122 (6), 111
(8), 107 (6), 99 (3), 77 (6), 69 (15), 65 (5), 39 (4). 1H NMR (500 MHz,
DMSO‑d6, δ): 10,64 (s, 1H, C2-OH); 10,43 (s, 1H, C4-OH); 9,84 (s, 1H,
NH); 7,94 (d, 1H, CAr-H); 7,69 (s, 1H, CAr-H); 7,45 (q, 1H, CAr-H); 7,35
(t, 1H, CAr-H); 7,01 (q, 1H, CAr-H); 6,50 (s, 1H, CAr-H); 2,51 (k, 2H,
CH2Me); 1,44 (t, 3H, CH2Me). 13C NMR (125 MHz, DMSO‑d6, δ):
163,01; 158,08; 155,46; 153,70; 142,23; 133,51; 130,53; 127,26;
122,55; 120,94; 116,63; 115,65; 107,92; 102,26; 22,08 (2C); 14,30
(3C). IR (KBr, cmꢀ 1): 3367 (NH), 3252 (OH), 3187 (OH), 3126 (OH),
2.5. Cell viability assay
For the determination of cell viability, cells were seeded into 96-well
plates at a density of 4 × 104 cells/well and incubated overnight at
–
–
–
3058 (OH), 2961 (CH), 2829 (CH), 1621 (C N), 1598 (C N), 1555
–
4

M. Szeliga et al.
Bioorganic Chemistry 105 (2020) 104362
37 ◦C. After this time, the cells were treated with the tested compounds
or TMZ at serial concentrations for 48 h. Next, the medium was
removed, the cells were washed with PBS and incubated in the culture
medium with MTT solution at the final concentration of 0.5 mg/ml for 2
h at 37 ◦C. Then the medium was replaced with DMSO and absorbance
was read at 570 nm using Elisa Bio-Rad Microplate Reader. Each com-
pound in each concentration was tested in triplicate in a single experi-
ment and five independent experiments were performed. On the basis of
obtained results, the half maximal inhibitory concentration (IC50) was
calculated using the software GraphPad Prism 7.
Table 1
Evaluation of the cytotoxic properties (mean IC50 ± SD [
μ
M])a of the 1,3,4-thia-
diazole derivatives in GBM cells and human astrocytes HA.
Compound
T98Gb
U87MGb
LN229b
LUB04c
LUB07c
HAd
FPDT
53.53
± 4.77
53.41
± 4.94
80.83
± 9.85
48.16
± 9.93
30.78
± 9.54
28.75
± 9.94
3426
44.98 ±
9.71
55.20 ±
9.07
56.37 ±
7.18
67.73 ±
3.00
> 100
FPCDT
FPEDT
CPDT
48.21 ±
9.99
89.96 ±
8.64
62.84 ±
6.33
78.85 ±
7.33
97.27
± 3.42
27.22
± 8.77
90.33
± 9.30
32.42
± 8.37
21.17
± 6.53
nd
50 ±
51.35 ±
14.28
58.43 ±
6.81
47.61 ±
7.85
8.17
49.19 ±
9.73
63.15 ±
9.83
55.08 ±
9.84
73.22 ±
5.70
CPCDT
CPEDT
TMZ
19.53 ±
9.03
35.60 ±
9.44
37.49 ±
5.09
34.89 ±
5.12
2.6. Cell proliferation and colony formation assay
28.60 ±
4.92
30.00 ±
9.77
30.46 ±
2.39
38.76 ±
2.46
DNA synthesis as a marker for cellular proliferation was measured by
bromodeoxyuridine (BrdU) incorporation using the colorimetric Cell
Proliferation Elisa BrdU assay (Roche) according to the manufacturer’s
instruction. Briefly, cells (2x103 cells/well) were seeded in 96-well
plates and incubated for 24 h. After this time, the culture medium was
changed and the cells were treated with serial dilutions of FPDT or
DMSO for 48 h. Next, BrdU was added for 2 h, and then cells were fixed
for 30 min and incubated with BrdU antibody for 90 min. The absor-
bance values were measured at 415 nm using Elisa Bio-Rad Microplate
Reader. Both FPDT in each concentration and DMSO was tested in
triplicate in a single experiment and five independent experiments were
performed.
892 ±
37.20
978 ±
67.00
2826 ±
240
3597 ±
267
± 270
nd - not determined.
a
The concentration of compound that inhibit 50% of cell viability after 48 h of
drug exposure measured by MTT assay.
b
c
Human GBM cell line.
Human patient-derived GBM cells.
d
Human astrocytes.
supplemented with cocktails of protease and phosphatase inhibitors
and sodium fluoride (Sigma-Aldrich). The lysates were centrifuged at
12,000g for 10 min at 4 ◦C and the protein concentration in the super-
natants was determined using the BCA Protein Assay Kit (Pierce). Thirty
To examine long-term toxicity of the tested compounds, a colony
formation assay was performed. Cells were seeded in 6-well plates (100
cells/well). After 24 h the culture medium was changed and the cells
were treated with serial dilutions of FPDT or DMSO for 2 weeks. Next,
cell culture plates containing colonies were gently washed with PBS and
fixed with 4% formaldehyde for 10 min. Wells were rinsed once again
with PBS, colonies were stained with 0.5% crystal violet solution in 25%
methanol for 10 min and the grossly visible colonies were counted. Four
independent experiments were performed.
μ
g of proteins were separated on 12% SDS-PAGE and transferred to
nitrocellulose membranes. The membranes were blocked in 5% bovine
serum albumin (BSA) in TBST (20 mM TRIS-HCl pH 7.5, 150 mM NaCl,
0.1% Tween 20) for 1 h at RT and next incubated with a primary anti-
body overnight at 4 ◦C. The following antibodies obtained from Cell
Signaling (pAKT S473, #4060; AKT, #4691; pGSK3β S9, #5558; GSK3β,
#9315) were used according to the manufacturer’s instruction. After
washing in TBST, the membranes were incubated with a secondary anti-
rabbit antibody (Sigma-Aldrich) conjugated to horseradish peroxidase
(HRP) for 1 h at RT. The blots were visualized on X-ray film using a
SuperSignal West Pico Chemiluminescent Substrate (Pierce). Next, the
membranes were stripped in 0.1 M glycine pH 2.9 and reused with a
HRP-conjugated monoclonal antibody against glyceraldehyde 3-phos-
phate dehydrogenase (GAPDH) (Proteintech, #HRP-60004). The
densitometric analysis was performed using G:Box system and Gene-
Tools software (Syngene).
2.7. Cell migration assay
The cells were grown to confluence in 6-well plates grown to
confluence. The cells were scratched using P200 pipette tip, washed
with PBS and incubated in the serum free medium containing 25 μM
FPDT or DMSO. The scratch was photographed under JuLI Smart Cell
Analyzer and its width was measured at 0 and 24 h after scratching. Four
independent experiments were performed.
2.8. Transwell chamber invasion assay
2.10. Human phospho-kinase array
The invasion of GBM cells was evaluated using the transwell inserts
with PET membrane (8 mm pore size) coated with matrigel (Corning) as
a mimic of the extracellular matrix. After rehydration of the matrigel, 5
The human phospho-kinase array (R&D Systems, #ARY003) was
performed according to the manufacturer’s instructions, with a total
protein amount of 400 μg per assay. Cells treated with DMSO and FPDT
× 104 cells in medium with 0.5% FBS containing 25
μM FPDT or DMSO
were handled and analyzed in parallel throughout the entire procedure.
Exposure and data collection of each set of arrays was also performed in
parallel. For data quantification, densitometric analysis of spots was
performed using G:Box system and GeneTools software (Syngene). In-
tensity of the kinases’ spots was normalized to the assay-internal posi-
tive controls after local background subtraction.
were seeded in the upper compartment of the insert. The lower
compartment was filled with medium containing either 10% (for T98G
and LN229 cells) or 15% FBS (for U87MG cells). Following 24-hour
incubation, the upper surface of the membrane was wiped with a cot-
ton swab to remove the remaining cells. Cells that invaded to the bottom
side of the membrane were fixed with methanol and stained with 0.5%
crystal violet solution in methanol. Membranes were placed on a glass
slide, and three fields from each membrane were counted using an op-
tical microscope. Three independent experiments were performed.
2.11. Statistical analysis
Western blot analysis and phospho-kinase array were performed
three times. All other experiments were repeated at least four times.
Results are reported as means and standard deviations. Statistical sig-
nificance of comparisons was based on one-way ANOVA (multiple
comparisons) or by paired t-test (comparison of two groups) in the
software Graphpad Prism 7. Differences were considered statistically
significant when P < 0.05.
2.9. Protein isolation and western blot
Twenty-four hours after seeding, T98G cells were exposed either to
DMSO or FPDT at IC50. After 48 h of treatment, the cells were washed
with PBS, harvested and sonicated in an ice cold lysis buffer (50 mM
TRIS-HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40)
5

M. Szeliga et al.
Bioorganic Chemistry 105 (2020) 104362
3. Results and discussion
3.1. Influence of thiadiazole derivatives on cell viability
As mentioned in the Introduction section, previous studies revealed
cytotoxic activity of FPDT and CPDT against several cancer cell lines
including rat glioma C6 cell line [18,19], but their activity against
human GBM cells has not been examined so far. Therefore, the primary
aim of this study was to evaluate cytotoxicity of FPDT, CPDT, and their
derivatives in GBM cells and commercially available HA. The IC50 (μM)
values of the examined compounds and TMZ, the first-line chemother-
apeutic for GBM, are listed in Table 1.
Fig. 3. Treatment with FPDT enhances an inhibitory effect of TMZ on viability
of GBM cells. Graphs showing viability of cells treated with FPDT, TMZ or both
agents after 48 h of continuous exposure at IC50. Data are presented as mean ±
SD (n = 5) expressed as percentage of control (DMSO-treated cells); *P < 0.05
(one-way ANOVA).
Our results clearly indicate that FPDT and CPDT inhibit viability of
all three human GBM cell lines (T98G, U87MG and LN229) used in this
study in a dose-dependent manner. These two compounds displayed a
comparable cytotoxic activity against GBM cells ranging between 44.98
± 9.71
μ
M and 55.20 ± 9.01
μ
M in case of FPDT or between 48.16 ±
9.93 M and 63.15 ± 9.83
μ
μM in case of CPDT. Cell lines do not fully
and 67.73 μM without affecting HA at 100 μM concentration, we sub-
recapitulate GBM heterogeneity which significantly contributes to
therapeutic failure. Therefore, we have also tested the compounds’ ac-
tivity in the patient-derived GBM cells (LUB04 and LUB07) in which
both FPDT and CPDT displayed considerable potency ranging from
sequently selected this compound for further studies.
3.2. Effect of FPDT on TMZ activity in GBM cells
56.37 ± 7.18
± 5.70
times more potent than TMZ. It should be emphasized that FPDT pre-
sented IC50 > 100 M for HA, suggesting its selective toxicity against
GBM cells. CPDT appeared to be slightly toxic towards HA with IC50
value around 90 M.
A growing body of evidence suggests that the hydrophobic substit-
uent ( > 0) in the resorcinol ring of the compound may increase its
potency against cancer cells [20–25]. Here we evaluated the cytotoxicity
μM to 67.73 ± 3.00 μM and from 55.08 ± 9.84 μM to 73.22
In order to determine whether FPDT can augment inhibitory effect of
TMZ on cell viability, T98G, U87MG, LN229, LUB04 and LUB07 cells
were incubated with FPDT, TMZ or both compounds at IC50. Combined
treatment with FPDT and TMZ resulted in enhanced inhibition of cell
viability when compared to the single agent treatment (Fig. 3).
μ
M, respectively. Either of compounds appeared to be 18–70
μ
μ
π
3.3. Influence of FPDT on proliferation of GBM cells
–
–
of derivatives of FPDT and CPDT containing either Cl or CH2CH3
substituent at C5-position of 2,4-dihydroxyphenyl. In case of compounds
containing the chlorine substituent at C3-position of the phenyl group,
our results confirmed the initial hypothesis that introduction of either
chlorine or ethyl substituent at C5-position of the resorcinol ring leads to
an increased activity of the 1,3,4-thiadiazole derivatives. CPCDT con-
taining chlorine atom at C5-position of 2,4-dihydroxyphenyl showed
increased cytotoxic activity against both GBM cells (IC50 between 19.53
Previously, the effect of FPDT on tumor cells was attributed to
decreased DNA synthesis [19,29]. Herein, we first measured the level of
DNA synthesis by analysis of BrdU incorporation into DNA. In all three
cell lines and patient-derived GBM cells exposed for 48 h to increasing
concentrations of FPDT, a dose-dependent inhibition of BrdU incorpo-
ration was observed (Fig. 4A). Treatment with the highest, 100 μM,
concentration of FPDT reduced DNA synthesis to 13, 13, 18, 9 or 14%
respectively in T98G, U87MG, LN229, LUB04 or LUB07 cells as
compared to the dimethyl sulfoxide (DMSO)-treated cells. We further
investigated the antiproliferative effect of FPDT by colony forming
assay. After 2 weeks of incubation under continuous drug exposure, a
dose-dependent decrease of colony number was observed in all three cell
± 9.03
μ
M and 37.49 ± 5.09
μM). Similarly, the presence of the ethyl
substituent resulted in enhancement of cytotoxicity as CPEDT displayed
IC50 values between 28.60 ± 4.92
μ
M and 38.76 ± 2.46 μM on GBM
cells. Of note, the IC50 values of each of CPCDT and CPEDT against GBM
cells appeared to be 30–110 times lower compared to that of TMZ.
However, this increased activity was also observed in HA (IC50 of 32.42
lines and patient-derived cells (Fig. 4B). Treatment with 50
completely inhibited the ability of these cells to form colonies.
μM
± 8.37
μM and 21.17 ± 6.53 μM, respectively), disqualifying CPCDT and
CPEDT from further studies.
In case of compounds containing the fluoro substituent at C4-
position of the phenyl group, our initial hypothesis was not confirmed
as neither chlorine, nor ethyl substituent at C5-position of the resorcinol
ring significantly improved anti-GBM activity. FPCDT carrying chlorine
atom at C5-position of 2,4-dihydroxyphenyl displayed IC50 from 48.21
3.4. Influence of FPDT on migration and invasion of GBM cells
Diffuse infiltrative growth of GBM is one of the major factors in
therapeutic failure, therefore new therapeutic strategies should inhibit
not only proliferation, but also migration of GBM cells [28]. Here the
effect of FPDT on ability of GBM cells to migrate was analyzed by a
scratch assay. This analysis was conducted in T98G, U98MG and LN229
cell lines, as it was impossible to obtain a confluent monolayer of
patient-derived GBM cells required to perform a scratch assay. In each
± 9.99 μM to 89.96 ± 9.77 μM towards GBM cells and 97.27 ± 3.42 μM
towards HA. The presence of the ethyl substituent did not enhanced the
anticancer activity as FPEDT showed IC50 values between 47.61 ± 7.85
μ
M and 80.83 ± 9.85 μM for GBM cells. However, such a modification
markedly increased cytotoxicity against HA (IC50 of 27.22 ± 8.77 μM).
cell line examined, markedly fewer cells treated for 24 h with 25 μM
Based on current knowledge, it is impossible to point out the reasons
of these findings. It has previously been postulated that the changes in
the activity of 2-amino-1,3,4-thiadiazole derivatives may result from i).
modifications of electronic properties important for interactions be-
tween compound and its molecular target; ii). effects of both intra- and
inter-molecular hydrogen bonds [26]. Whether any of these elements
influences the activity of derivatives examined in this study remains an
open question.
FPDT migrated into the scratched area compared to the control cells.
Therefore, the width of the scratch gap in the FPDT-treated cells was
significantly higher compared to the DMSO-treated cells (Fig. 5A, B).
These results indicate that FPDT inhibits ability of T98G, U87MG and
LN229 cells to migrate. We further assessed the influence of FPDT on
invasive properties of GBM cells in Matrigel-coated Transwell inserts. In
each cell line examined, significantly lower number of cells treated for
24 h with 25
μM FPDT invaded through the insert membranes compared
Since FPDT exhibited anti-GBM activity ranging between 44.98 μM
to the DMSO-treated cells (Fig. 5C, D).
6

M. Szeliga et al.
Bioorganic Chemistry 105 (2020) 104362
Fig. 4. FPDT decreases proliferation and ability to form colonies of GBM cells. A The cells were treated with serial dilutions of FPDT or DMSO for 48 h and next cell
proliferation was assessed by BrdU assay. Data are presented as mean ± SD (n = 5) expressed as percentage of control (DMSO-treated cells); *P < 0.05 (one-way
ANOVA). B The cells were treated with serial dilutions of FPDT or DMSO for 2 weeks and subsequently the colonies formed were fixed with paraformaldehyde,
stained with crystal violet and counted. Data are presented as mean ± SD (n = 4) expressed as percentage of control (DMSO-treated cells); *P < 0.05 (one-
way ANOVA).
Fig. 5. Effect of FPDT on migration and invasion of GBM cells. The cells were scratch-wounded with a P200 tip and incubated in the serum free medium containing
25 μM FPDT or DMSO for 24 h. The width of the gap was measured at 0 and 24 h after scratching. A Relative scratch gap was calculated as the ratio of the remaining
scratch gap at the given time point and the original gap at 0 h. Data are presented as mean ± SD (n = 4); *P < 0.05 (t-test). B Representative images of the scratch
assay. C Quantitative analysis of cell invasion across Matrigel-coated Transwell inserts. Data are presented as mean ± SD (n = 3); *P < 0.05 (t-test). D Representative
images of the invasion assay.
3.5. Effect of FPDT on the AKT pathway
phospho-kinase array in T98G cells to identify the molecules that could
be potentially responsible for the antiproliferative and antimigratory
effect of FPDT. This analysis showed a significant downregulation of p-
Our previous study showed that FPDT inhibited phosphorylation of
MEK, ERK, RSK90 kinases as well as CREB protein in non-small lung
carcinoma cells [29]. Downregulation of several other kinase pathways,
such as c-Met [10], FAK [30] or AKT [31], was observed in cancer cells
treated with distinct 1,3,4-thiadiazole derivatives. Here we performed a
GSK3α/β (S21/S9) and p-AKT (S473) in the cells treated with FPDT
compared to the cells treated with DMSO (Fig. 6A, B). This down-
regulation was confirmed by Western blot analysis (Fig. 6C, D). The level
of total AKT and GSK3β remained unchanged upon FPDT treatment
7

M. Szeliga et al.
Bioorganic Chemistry 105 (2020) 104362
Fig. 6. Treatment with FPDT leads to downregulation of p-GSK3β and p-AKT which partially contributes to the diminished cell viability. A T98G cells were treated
with DMSO or FPDT for 48 h prior to collecting the lysates. The lysates were incubated with the membranes of the human phospho-kinase array kit (R&D Systems)
and bound phospho-proteins were detected according to the manufacturer’s instructions. Each membrane contains antibody against particular kinase or positive
control (P) spotted in duplicate. The membranes shown are representative for three independent experiments. B Densitometric analysis of spots 1 (p-GSK3α/β S21/
S9), 2 (p-p53 S392), 3 (p-AKT S473), 4 (p-p53 S46), 5 (CREB), 6 (p-p53 S15) and 7 (HSP60) was performed using G:Box system and GeneTools software (Syngene).
Each bar represents the mean ± SD of duplicate spots from three independent experiments; *P < 0.05 (t-test). C The level of selected proteins in T98G cells treated
with DMSO or FPDT for 48 h was analyzed by Western blot. GAPDH was used as a loading control. D The graph shows quantification of protein bands intensity after
normalization to GAPDH. Data are presented as mean ± SD (n = 3); *P < 0.05 vs DMSO (t-test).
8

M. Szeliga et al.
Bioorganic Chemistry 105 (2020) 104362
approximately 303 g/mol, FPDT seems to meet the first of these re-
quirements. Further studies are necessary to conclude whether the sec-
ond requirement is also fulfilled.
In summary, six 1,3,4-thiadiazole derivatives containing or not an
– –
additional Cl or CH2CH3 substituent at C5-position of 2,4-dihydrox-
yphenyl displayed anticancer activity in both human GBM cell lines and
patient-derived GBM cells. The tested compounds presented the IC50
values 15–110 times lower compared to that of TMZ, the first-line
chemotherapeutic agent for GBM. Introduction of either chlorine or
ethyl substituent at C5-position of the resorcinol ring increased cyto-
toxicity of the 1,3,4-thiadiazole derivatives containing the chlorine
substituent at C3-position of the phenyl group. Such a modifications did
not improve anti-GBM activity of the 1,3,4-thiadiazole derivatives
containing the fluoro substituent at C4-position of the phenyl group, but
increased their toxicity to HA. FPDT appeared to be the most promising
of the tested compounds, as it has shown IC50 values between 45
μ
M and
68
μM for GBM cells and >100 μM for HA. FPDT significantly
augmented activity of TMZ and inhibited proliferation and ability to
migrate of GBM cells. It also diminished phosphorylation level of GSK3β
and AKT, which partially contributed to inhibition of cell viability. Our
findings warrant further research on activity of FPDT against GBM.
Declaration of Competing Interest
Fig. 7. The viability of T98G cells cultured in medium with or without PDGF-
BB (50 ng/ml) for 24 h prior the exposure to FPDT at IC50 for 48 h. Data are
presented as mean ± SD (n = 5); *P < 0.05 (t-test).
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
which implies that this compound decreases phosphorylation, but not
expression of AKT and GSK3β.
Acknowledgements
As phosphorylation of GSK3β may be mediated by several kinases
[32], its decreased phosphorylation upon FPDT treatment may not be a
direct consequence of diminished AKT phosphorylation. The activity of
AKT is tightly regulated at many levels in a context-dependent manner
[33]. Therefore, further experimental work is needed in order to clarify
the precise molecular mechanism underlying inhibition of AKT and
GSK3β phosphorylation upon FPDT treatment.
The authors wish to thank the patients for providing biological
´
specimens for this study. We are grateful to Małgorzata Bogacinska-
´
Karas (MMRC PAS) for assistance in cell culturing. This work was
financially supported by the National Science Centre of Poland, grant
no: 2013/11/D/NZ7/00925 (to MS).
References
Both AKT and GSK3β are involved in several cellular processes such
as proliferation, cell cycle, growth, motility. Deregulated activity of each
of these molecules as well as their targets, often observed in GBM,
contributes to tumor growth and invasiveness. Hence, AKT and GSK3β
signaling pathways constitute attractive targets for GBM therapy [34].
We therefore speculated that decreased phosphorylation of AKT and
GSK3β observed in FPDT-treated cells might contribute to the reduced
viability of these cells. Indeed, our results confirmed this hypothesis, as
pretreatment with PDGF-BB, an activator of AKT phosphorylation [35],
resulted in a significantly increased viability of FPDT-treated cells
compared to the not pretreated with PDGF-BB counterparts (Fig. 7).
Our finding that FPDH influences the AKT/GSK3 β pathway in GBM,
together with the previous notion that it modulates the ERK pathway in
lung carcinoma cells [29], suggests that the direct molecular target of
FPDT regulates action of several kinase-pathways, probably in a cell
type-dependent manner. It is tempting to speculate that the mechanism
underlying FPDT’s activity is linked to some upstream signal molecules
of the AKT and ERK pathways. For instance, several receptor tyrosine
kinases (RTK) have been shown to activate RAS proteins, which in turn
modulate both AKT and ERK pathways [36].
[1] M. Weller, T. Cloughesy, J.R. Perry, W. Wick, Standards of care for treatment of
recurrent glioblastoma – are we there yet? Neuro Oncol. 15 (2013) 24–27, https://
doi.org/10.1093/neuonc/nos273.
[2] D. Gramatzki, P. Roth, E.J. Rushing, J. Weller, N. Andratschke, S. Hofer, et al.,
Bevacizumab may improve quality of life, but not overall survival in glioblastoma:
an epidemiological study, Ann. Oncol. 29 (2018) 1431–1436, https://doi.org/
10.1093/annonc/mdy106.
[3] H.G. Wirsching, M. Weller, The Role of Molecular Diagnostics in the management
of patients with gliomas, Curr. Treat. Options. Oncol. 17 (51) (2016), https://doi.
org/10.1007/s11864-016-0430-4.
[4] S.A. Sprowls, T.A. Arsiwala, J.R. Bumgarner, N. Shah, S.S. Lateef, B.N. Kielkowski,
et al., Improving CNS delivery to brain metastases by blood-tumor barrier
disruption, Trends, Cancer. 5 (2019) 495–505, https://doi.org/10.1016/j.
trecan.2019.06.003.
[5] S. Haider, M.S. Alam, H. Hamid, 1,3,4-Thiadiazoles: a potent multi targeted
pharmacological scaffold, Eur. J. Med. Chem. 92 (2015) 156–177, https://doi.org/
10.1016/j.ejmech.2014.12.035.
[6] S. Abu-Melha, M.M. Edrees, H.H. Salem, N.A. Kheder, S.M. Gomha, M.R. Abdelaziz,
Synthesis and biological evaluation of some novel thiazole-based heterocycles as
potential anticancer and antimicrobial agents, Molecules 24 (2019) 539, https://
doi.org/10.3390/molecules24030539.
[7] S. Cascioferro, G.L. Petri, B. Parrino, D. Carbone, N. Funel, C. Bergonzini, et al.,
Imidazo[2,1-b] [1,3,4]thiadiazoles with antiproliferative activity against primary
and gemcitabine-resistant pancreatic cancer cells, Eur. J. Med. Chem. 189 (2020),
112088, https://doi.org/10.1016/j.ejmech.2020.112088.
Aside from the question of the molecular target of FPDT, there is a
question concerning its activity in vivo. Although discrepancy between
the drug efficacy in vitro and in vivo is not a rule, it is relatively frequent
phenomenon. In case of compounds that could serve as drugs against
brain tumors, there is also a question about their ability to cross the BBB.
The general assumption is that the molecule crosses the BBB if its mo-
lecular mass is less than 400–500 Da and it forms less than 8–10
hydrogen bonds with solvent water [37]. With a molecular mass of
[8] K.M. Dawood, T.M. Eldebss, H.S. El-Zahabi, M.H. Yousef, P. Metz, Synthesis of
some new pyrazole-based 1,3-thiazoles and 1,3,4-thiadiazoles as anticancer agents,
Eur. J. Med. Chem. 70 (2013) 740–749, https://doi.org/10.1016/j.
ejmech.2013.10.042. Epub 2013 Oct 30.
[9] D. Kumar, N. Maruthi Kumar, K.H. Chang, K. Shah, Synthesis and anticancer
activity of 5-(3-indolyl)-1,3,4-thiadiazoles, Eur. J. Med. Chem. 45 (2010)
464–4668, https://doi.org/10.1016/j.ejmech.2010.07.023.
[10] L. Zhang, J. Zhao, B. Zhang, T. Lu, Y. Chen, Discovery of [1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole derivatives as novel, potent and selective c-Met kinase
inhibitors: Synthesis, SAR study, and biological activity, Eur. J. Med. Chem. 150
(2018) 809–816, https://doi.org/10.1016/j.ejmech.2018.03.049.
9

M. Szeliga et al.
Bioorganic Chemistry 105 (2020) 104362
´
[11] Z. Chen, D. Li, N. Xu, J. Fang, Y. Yu, W. Hou, et al., Novel 1,3,4-selenadiazole-
containing kidney-type glutaminase inhibitors showed improved cellular uptake
and antitumor activity, J. Med. Chem. 62 (2019) 589–603, https://doi.org/
10.1021/acs.jmedchem.8b01198.
[24] J. Matysiak, M.M. Karpinska, A. Niewiadomy, J. Wietrzyk, D. Kłopotowska, One-
pot synthesis of new (1,3-thiazolo[5,4-b]pyridin-2-yl)benzenediols and their
antiproliferative activities against human cancer cell lines, Chem. Biodivers. 9
(2012) 48–57.
´
[12] V. Raj, A.S. Bhadauria, A.K. Singh, U. Kumar, A. Rai, A.K. Keshari, et al., Novel
1,3,4-thiadiazoles inhibit colorectal cancer via blockade of IL-6/COX-2 mediated
JAK2/STAT3 signals as evidenced through data-based mathematical modeling,
Cytokine 118 (2019) 144–159, https://doi.org/10.1016/j.cyto.2018.03.026.
[13] S. Lindskog, Structure and mechanism of carbonic anhydrase, Pharmacol. Ther. 74
(1997) 1–20.
[25] A. Niewiadomy, J. Matysiak, M.M. Karpinska, Synthesis and anticancer activity of
new 2-aryl-4h-3,1-benzothiazines, Arch. Pharm. (Weinheim) 344 (2011) 224–230.
[26] J. Matysiak, A. Opolski, Synthesis and antiproliferative activity of N-substituted 2-
amino-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazoles, Bioorg. Med. Chem. 14 (2006)
4483–4489.
[27] C.S. Mullins, B. Schneider, F. Stockhammer, M. Krohn, C.F. Classen, M.
Linnebacher, Establishment and characterization of primary glioblastoma cell lines
from fresh and frozen material: a detailed comparison, PLoS One. (2013) 8(8):
e71070. doi: 10.1371/journal.pone.0071070. eCollection 2013.
[14] K. Thangavelu, C.Q. Pan, T. Karlberg, G. Balaji, M. Uttamchandani, V. Suresh, et
al., Structural basis for the allosteric inhibitory mechanism of human kidney-type
glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell
metabolism, Proc. Natl. Acad. Sci. USA 109 (2012) 7705–7710, https://doi.org/
10.1073/pnas.1116573109.
[28] F. Lefranc, E. Le Rhun, R. Kiss, M. Weller, Glioblastoma quo vadis: Will migration
and invasiveness reemerge as therapeutic targets? Cancer. Treat. Rev. 68 (2018)
145–154, https://doi.org/10.1016/j.ctrv.2018.06.017.
[15] L.A. McDermott, P. Iyer, L. Vernetti, S. Rimer, J. Sun, M. Boby, et al., Design and
evaluation of novel glutaminase inhibitors, Bioorg. Med. Chem. 24 (2016)
1819–1839, https://doi.org/10.1016/j.bmc.2016.03.009.
˙
[29] M. Juszczak, J. Matysiak, M. Szeliga, P. Pozarowski, A. Niewiadomy, J. Albrecht, et
al., 2-Amino-1,3,4-thiadiazole derivative (FABT) inhibits the extracellular signal-
regulated kinase pathway and induces cell cycle arrest in human non-small lung
carcinoma cells, Bioorg. Med. Chem. Lett. 22 (2012) 5466–5469, https://doi.org/
10.1016/j.bmcl.2012.07.036.
[16] K. Shukla, D.V. Ferraris, A.G. Thomas, M. Stathis, B. Duvall, G. Delahanty, et al.,
Design, synthesis, and pharmacological evaluation of bis-2-(5-phenylacetamido-
1,2,4-thiadiazol-2-yl)ethyl sulfide 3 (BPTES) analogs as glutaminase inhibitors,
J. Med. Chem. 55 (2012) 10551–10563, https://doi.org/10.1021/jm301191p.
[17] M. El-Naggar, H.A. Sallam, S.S. Shaban, S.S. Abdel-Wahab, A.E.E. Amr, M.E. Azab,
et al., Design, synthesis, and molecular docking study of novel heterocycles
incorporating 1,3,4-thiadiazole moiety as potential antimicrobial and anticancer
agents, Molecules 24 (2019) pii: E1066, https://doi.org/10.3390/
molecules24061066.
[30] J. Sun, Y.S. Yang, W. Li, Y.B. Zhang, X.L. Wang, J.F. Tang, et al., Synthesis,
biological evaluation and molecular docking studies of 1,3,4-thiadiazole
derivatives containing 1,4-benzodioxan as potential antitumor agents, Bioorg.
Med. Chem. Lett. 21 (2011) 6116–6121, https://doi.org/10.1016/j.
bmcl.2011.08.039.
[31] J.Y. Chou, S.Y. Lai, S.L. Pan, G.M. Jow, J.W. Chern, J.H. Guh, Investigation of
anticancer mechanism of thiadiazole-based compound in human non-small cell
lung cancer A549 cells, Biochem. Pharmacol. 66 (2003) 15–24.
[32] E. Beurel, S.F. Grieco, R.S. Jope, Glycogen synthase kinase-3 (GSK3): regulation,
actions, and diseases, Pharmacol. Ther. 148 (2015) 114–131, https://doi.org/
10.1016/j.pharmthera.2014.11.016.
[18] M. Juszczak, J. Matysiak, A. Niewiadomy, W. Rzeski, The activity of a new 2-
amino-1,3,4-thiadiazole derivative 4ClABT in cancer and normal cells, Folia
Histochem. Cytobiol. 49 (2011) 436–444.
´
[19] W. Rzeski, J. Matysiak, M. Kandefer-Szerszen, Anticancer, neuroprotective
activities and computational studies of 2-amino-1,3,4-thiadiazole based
compound, Bioorg. Med. Chem. 15 (2007) 3201–3207.
[33] J.R. Hart, P.K. Vogt, Phosphorylation of AKT: a mutational analysis, Oncotarget 2
(2011) 467–476, https://doi.org/10.18632/oncotarget.293.
[20] P.A. Brough, X. Barril, M. Beswick, B.W. Dymock, M.J. Drysdale, L. Wright, et al.,
3-(5-Chloro-2,4-dihydroxyphenyl)-pyrazole-4-carboxamides as inhibitors of the
Hsp90 molecular chaperone, Bioorg. Med. Chem. Lett. 15 (2005) 5197–5201.
[21] P.A. Brough, W. Aherne, X. Barril, J. Borgognoni, K. Boxall, J.E. Mansfield, et al.,
4,5-diarylisoxazole Hsp90 chaperone inhibitors: potential therapeutic agents for
the treatment of cancer, J. Med. Chem. 51 (2008) 196–218.
[34] E. Majewska, M. Szeliga, AKT/GSK3β Signaling in Glioblastoma, Neurochem. Res.
42 (2017) 918–924, https://doi.org/10.1007/s11064-016-2044-4.
¨
[35] B.H. Rauch, A. Weber, M. Braun, N. Zimmermann, K. Schror, PDGF-induced Akt
phosphorylation does not activate NF-kappa B in human vascular smooth muscle
cells and fibroblasts, FEBS Lett. 481 (2000) 3–7.
[22] S.Y. Sharp, K. Boxall, M. Rowlands, C. Prodromou, S.M. Roe, A. Maloney, et al., In
vitro biological characterization of a novel, synthetic diaryl pyrazole resorcinol
class of heat shock protein 90 inhibitors, Cancer Res 67 (2007) 2206–2216.
[36] D.K. Simanshu, D.V. Nissley, F. McCormick, RAS proteins and their regulators in
human disease, Cell 170 (2017) 17–33, https://doi.org/10.1016/j.
cell.2017.06.009.
´
[23] M.M. Karpinka, J. Matysiak, A. Niewiadomy, Synthesis of novel 4-(1H-
[37] W.M. Pardridge, The blood-brain barrier: bottleneck in brain drug development,
NeuroRx 2 (2005) 3–14, https://doi.org/10.1602/neurorx.2.1.3.
benzimidazol-2-yl)benzene-1,3-diols and their cytotoxic activity against human
cancer cell lines, Arch. Pharm. Res. 34 (2011) 1639–1647.
10