New heparanase-inhibiting triazolo-thiadiazoles attenuate primary tumor growth and metastasis

Article abstract of DOI:10.3390/cancers13122959

Compelling evidence ties heparanase, an endoglycosidase that cleaves heparan sulfate side (HS) chains of proteoglycans, with all steps of tumor development, including tumor initiation, angiogenesis, growth, metastasis, and chemoresistance. Moreover, heparanase levels correlate with shorter postoperative survival of cancer patients, encouraging the development of heparanase inhibitors as anti-cancer drugs. Heparanase-inhibiting heparin/heparan sulfate-mimicking compounds and neutralizing antibodies are highly effective in animal models of cancer progression, yet none of the compounds reached the stage of approval for clinical use. The present study focused on newly synthesized triazolo–thiadiazoles, of which compound 4-iodo-2-(3-(p-tolyl)-[1,2,4]triazolo[3,4b][1,3,4]thiadiazol-6-yl)phenol (4-MMI) was identified as a potent inhibitor of heparanase enzymatic activity, cell invasion, experimental metastasis, and tumor growth in mouse models. To the best of our knowledge, this is the first report showing a marked decrease in primary tumor growth in mice treated with small molecules that inhibit heparanase enzymatic activity. This result encourages the optimization of 4-MMI for preclinical and clinical studies primarily in cancer but also other indications (i.e., colitis, pancreatitis, diabetic nephropathy, tissue fibrosis) involving heparanase, including viral infection and COVID-19.

Full text of DOI:10.3390/cancers13122959

cancers
Article
New Heparanase-Inhibiting Triazolo-Thiadiazoles Attenuate
Primary Tumor Growth and Metastasis
Uri Barash 1,† , Shobith Rangappa 2,†, Chakrabhavi Dhananjaya Mohan 3, Divakar Vishwanath 4
Ilanit Boyango 1, Basappa Basappa 4 , Israel Vlodavsky 1,* and Kanchugarakoppal S. Rangappa 5,
,
*
1
Technion Integrated Cancer Center (TICC), the Rappaport Faculty of Medicine, Technion,
Haifa 31096, Israel; ubarash@yahoo.com (U.B.); bilanit@rambam.health.gov.il (I.B.)
Adichunchanagiri Institute for Molecular Medicine, BG Nagara, Nagamangala Taluk 571448, India;
shobithrangappa@gmail.com
Department of Studies in Molecular Biology, University of Mysore, Manasagangotri, Mysore 570006, India;
cd.mohan@yahoo.com
Laboratory of Chemical Biology, Department of Studies in Organic Chemistry, University of Mysore,
Manasagangotri, Mysore 570006, India; divakardivi166@gmail.com (D.V.); salundibasappa@gmail.com (B.B.)
Institution of Excellence, Vijnana Bhavan, University of Mysore, Manasagangotri, Mysore 570006, India
Correspondence: vlodavsk@mail.huji.ac.il (I.V.); rangappaks@gmail.com (K.S.R.)
Equal contribution.
2
3
4
5
*
†
Simple Summary: Heparanase is an endoglycosidase that plays a critical role in tumor progression
and metastasis. The expression of heparanase in the tumor microenvironment is positively correlated
with the aggressiveness of the tumor and is associated with poor prognosis. In this study, we have
demonstrated that a new triazole–thiadiazole-bearing small molecule showed good heparanase
inhibition along with attenuation of tumor growth and metastasis. To the best of our knowledge, this
is the first report showing a marked decrease in primary tumor growth in mice treated with a small
molecule that inhibits heparanase enzymatic activity. Given these encouraging results, studies are
underway to better elucidate the mode of action and clinical significance of triazolo–thiadiazoles.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Barash, U.; Rangappa, S.;
Mohan, C.D.; Vishwanath, D.;
Boyango, I.; Basappa, B.; Vlodavsky,
I.; Rangappa, K.S. New
Heparanase-Inhibiting
Triazolo-Thiadiazoles Attenuate
Primary Tumor Growth and
Metastasis. Cancers 2021, 13, 2959.
https://doi.org/10.3390/cancers
13122959
Abstract: Compelling evidence ties heparanase, an endoglycosidase that cleaves heparan sulfate
side (HS) chains of proteoglycans, with all steps of tumor development, including tumor initiation,
angiogenesis, growth, metastasis, and chemoresistance. Moreover, heparanase levels correlate with
shorter postoperative survival of cancer patients, encouraging the development of heparanase in-
hibitors as anti-cancer drugs. Heparanase-inhibiting heparin/heparan sulfate-mimicking compounds
and neutralizing antibodies are highly effective in animal models of cancer progression, yet none
of the compounds reached the stage of approval for clinical use. The present study focused on
newly synthesized triazolo–thiadiazoles, of which compound 4-iodo-2-(3-(p-tolyl)-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazol-6-yl)phenol (4-MMI) was identified as a potent inhibitor of heparanase enzymatic
activity, cell invasion, experimental metastasis, and tumor growth in mouse models. To the best
of our knowledge, this is the first report showing a marked decrease in primary tumor growth in
mice treated with small molecules that inhibit heparanase enzymatic activity. This result encourages
the optimization of 4-MMI for preclinical and clinical studies primarily in cancer but also other
indications (i.e., colitis, pancreatitis, diabetic nephropathy, tissue fibrosis) involving heparanase,
including viral infection and COVID-19.
Academic Editor: Ian R. Hardcastle
Received: 27 April 2021
Accepted: 22 May 2021
Published: 13 June 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Keywords: triazolo–thiadiazoles; small molecules; heparanase-inhibiting compounds; primary
tumor growth; cell invasion; metastasis
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
The extracellular matrix (ECM) is a supramolecular network of macromolecules (i.e.,
collagens, glycoproteins proteoglycans) that provide structural and biochemical support to
Cancers 2021, 13, 2959. https://doi.org/10.3390/cancers13122959
https://www.mdpi.com/journal/cancers

Cancers 2021, 13, 2959
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the cellular compartment of tissues [
1,2
]. Heparan sulfate (HS) is a linear glycosaminogly-
can polysaccharide made up of tandem disaccharide units of glucuronic acid or iduronic
acid and D-glucosamine, which are covalently attached to a core protein (i.e., syndecan,
glypican, perlecan) together forming heparan sulfate proteoglycans (HSPG) [
there are only two repeating sugar units, they exhibit broad diversity in structure generated
by a complex pattern of deacetylation, sulfation, and epimerization [ ]. Importantly, the
sulfated regions of HS serve as docking sites for ECM components, extrinsic proteins,
growth-promoting factors (i.e., VEGF, FGF, HGF, TGF ), chemokines, and cytokines [ ],
indicating that HSPG is crucial in maintaining tissue/cell architecture, growth, and dif-
ferentiation [ ]. Heparanase is the only -endoglycosidase that partially degrades the
HS saccharide side chains of HSPG to generate fragments of 4–7 kDa in length [ ]. The
3,4]. Although
3
β
4,5
3
,6
β
7–9
degradation products of HS and, even more so, the liberated biologically active molecules,
serve as a bioavailable source of HS-bound proteins that activate signal transduction and
promote tissue remodeling, repair, neovascularization, and growth [8,9]. Compelling ev-
idence gathered during the last two decades tie heparanase levels not only with tumor
metastasis but with all steps of tumor development, including tumor initiation, angiogene-
sis, growth, metastasis, and chemoresistance [
with shorter postoperative survival of cancer patients [10
8
,
9
]. Moreover, heparanase levels correlate
13] altogether indicating that
–
heparanase is causally involved in cancer progression, encouraging the development of
heparanase inhibitors as anti-cancer drugs. Heparanase-inhibiting heparin/HS-mimicking
compounds, small molecules, and antibodies are being developed [14
eral saccharide-based compounds were and/or are being examined in cancer clinical
trials [17 18], yet none of the compounds reached the stage of approval for clinical use. The
–16], of which sev-
,
present study is a continuation of our previously reported in vitro screening of a library of
small molecules characterized by a variety of scaffolds [19]. It focuses on newly developed
triazolo–thiadiazole small molecules selected out of nearly 150 compounds [19], including
10 newly synthesized triazole amino thiol derivatives of substituted [1,2,4]triazolo[3,4-
b][1,3,4]thiadiazoles. Our compound of choice {4-iodo-2-(3-(p-tolyl)-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazol-6-yl)phenol = 4-MMI} [19] was found to efficiently inhibit heparanase
enzymatic activity, cell invasion, experimental metastasis, and tumor growth in mouse
models. To the best of our knowledge, this is the first report showing a marked decrease
in primary tumor growth in mice treated with small molecules that inhibit heparanase
enzymatic activity.
2. Materials and Methods
All chemicals and solvents were purchased from Sigma-Aldrich (Bangalore, India).
The completion of the reaction was monitored by pre-coated silica gel TLC plates. An Agi-
lent mass spectrophotometer was used to record the mass of the synthesized compounds.
1H and 13C NMR were recorded on Agilent and Jeol NMR spectrophotometers (400 MHz).
TMS was used as an internal standard, and DMSO was used as a solvent. Chemical shifts
are expressed as ppm.
2.1. General Procedure for the Synthesis of 4-amino-5-substituted phenyl-4H-1,2,4-
triazole-3-thiol (4a–e)
A mixture of ethyl benzoate (1 mmol) in 15 mL of ethanol, as well as hydrazine hydrate
(1 mmol), was added and refluxed for 5 h. Completion of the reaction was monitored by
TLC followed by a water wash to remove traces of hydrazine hydrate, and acid hydrazide
was obtained as a white solid. The acid hydrazide (1 mmol) was added to a stirred solution
of ethanol (15 mL) containing KOH (2 mmol), and CS2 (1.3 mmol) was added and refluxed
for 10 h. After completion of the reaction as monitored by TLC, diethyl ether was added
and stirred for 1 h. The potassium salt of hydrazine carbothioate was obtained as a solid,
filtered, and washed with diethyl ether. The above potassium salt (1 mmol) was dissolved
in 15 mL of water, and hydrazine hydrate (2 mmol) was added and refluxed at 100 ◦C for
4 h, during which H2S evolved and the color of the reaction mass turned to green. Then,
the reaction mass was cooled and acidified with concentrated HCl. The solid obtained

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was recrystallized in ethanol to obtain pure triazole. The reaction progress was monitored
using TLC.
2.2. General Procedure for the Synthesis of 6-substituted-(3-substituted
phenyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole)
To a mixture of 4-amino-5-substituted phenyl-4H-1,2,4-triazole-3-thiol (1 mmol) and
◦
substituted salicylic acid (1 mmol), 6 mL of POCl3 was added and refluxed at 80 C for 8 h.
The completion of the reaction was monitored by TLC. The reaction mass was quenched
with crushed ice and neutralized by potassium carbonate to pH 8. The solid obtained
was filtered, washed with water, and recrystallized with an appropriate solvent to obtain
6-substituted-(3-substituted-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole).
2.3. 2,4-diiodo-6-(3-(4-nitrophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)phenol (4-NDI)
1
Yellow solid. Yield: 70%. H NMR (DMSO, 400 MHz):
δ
9.04 (s, 1H), 8.73 (s, 1H),
164.14, 163.23,
8.54–8.36 (m, 2H), 8.19 (s, 1H), 7.96 (s, 1H). 13C NMR (DMSO, 100 MHz):
δ
150.27, 148.23, 138.30, 131.76, 131.15, 126.79, 125.24, 124.71, 120.15, 95.80, 90.81. LCMS:
590.8359 (m/z), 591.7344 [M+1]+.
2.4. 2-(3-(3-bromophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)-4,6-diiodophenol (3-BDI)
1
Brown solid. Yield: 81%. H NMR (DMSO, 400 MHz):
δ 8.41(s, 1H), 8.33 (d, J = 8 Hz,
1H), 8.09 (d, J = 1.6 Hz, 1H), 7.85 (d, J = 1.6 Hz, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.64–7.50 (m, 1H).
13C NMR (DMSO, 100 MHz):
δ 166.49, 165.79, 150.41, 147.60 (2C), 134.22, 132.74, 131.99,
129.09, 128.33, 124.95, 122.76, 116.01, 98.33, 70.09. LCMS: 623.7613 (m/z), 624.5974 [M+1]+.
2.5. 2-(3-(3-chlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)-4,6-diiodophenol (3-CDI)
1
Pale yellow solid. Yield: 75%. H NMR (DMSO, 400 MHz):
δ 8.27(s, 2H), 7.76 (s, 1H),
7.68 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H). 13C NMR (DMSO,
100 MHz):
δ 168.66, 167.62, 159.54, 151.40, 149.52, 136.42, 134.36, 131.86, 131.75, 127.45,
126.80, 126.35, 116.0, 90.50, 82.69. LCMS: 579.8118 (m/z), 580.6468 [M+1]+.
2.6. 2,4-diiodo-6-(3-(4-chlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)phenol (4-CDI)
Yellow solid. Yield: 78%. 1H NMR (DMSO, 400 MHz):
δ
8.30 (d, J = 8.4 Hz, 2H),
163.50,
8.23 (s, 1H), 8.06 (s, 1H), 7.68 (d, J = 8 Hz, 2H). 13C NMR (DMSO, 100 MHz):
δ
160.67, 148.17 (2C), 134.38 (2C), 129.24, 127.53, 127.32, 124.78, 117.98, 95.15, 80.83. LCMS:
579.8118 (m/z), 580.6678 [M+1]+.
2.7. 2,4-diiodo-6-(3-(p-tolyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)phenol (4-MDI)
1
Yellow solid. Yield: 80%. H NMR (DMSO, 400 MHz):
δ 8.18 (d, J = 8 Hz, 2H), 8.08
(s, 1H), 7.81 (s, 1H), 7.39 (d, J = 7.2 Hz, 2H), 2.36 (s, 3H). 13C NMR (DMSO, 100 MHz):
δ
166.94, 165.46, 147.37, 139.75, 134.09, 130.20, 128.11, 126.43, 126.11, 124.28, 115.97, 98.61,
69.22, 21.65. LCMS: 559.8665 (m/z), 558.8661 [M-1]+.
2.8. 4-iodo-2-(3-(4-nitrophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)phenol (4-NMI)
Off-white solid. Yield: 76%. 1H NMR (DMSO, 400 MHz):
δ 9.00 (s, 1H), 8.62 (d,
J = 7.6 Hz, 1H), 8.39–8.25 (m, 2H), 7.96–7.82 (m, 2H), 7.67 (d, J = 7.2 Hz, 1H). 13C NMR
(DMSO, 100 MHz):
δ 161.88, 161.08, 156.03, 148.04, 141.86, 134.76, 131.41, 130.97, 126.95,
124.30, 119.85, 119.26, 82.00. LCMS: 464.9393 (m/z), 465.9448 [M+1]+.
2.9. 2-(3-(3-bromophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)-4-iodophenol (3-BMI)
1
Off-white solid. Yield: 84%. H NMR (DMSO, 400 MHz):
δ 8.30 (d, J = 8.8 Hz, 2H), 8.20
(d, J = 7.6 Hz, 1H), 7.85 (d, J = 8 Hz, 1H), 7.70 (d, J = 8 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.61–
7.47 (m, 1H). 13C NMR (DMSO, 100 MHz):
δ
160.85, 156.12, 150.53, 141.74, 135.21, 132.72,
131.24, 127.92, 127.47, 124.49, 122.31, 122.19, 121.12, 119.36, 87.18. LCMS: 497.8647 (m/z),
498.8651 [M+1]+.

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2.10. 2-(3-(3-chlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)-4-iodophenol (3-CMI)
Yellow solid. Yield: 81%. 1H NMR (DMSO, 400 MHz):
8.38–8.23 (m, 2H), 8.09
(d, J = 2.4 Hz, 1H), 7.75–7.59 (m, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.34–7.22 (m, 1H), 6.35 (d,
J = 8.8 Hz, 1H). 13C NMR (DMSO, 100 MHz):
168.55, 167.57, 159.48, 151.38, 149.47, 136.31,
δ
δ
134.32, 131.87, 131.75, 130.28, 127.41, 126.69, 126.31, 115.85, 90.63. LCMS: 453.9152 (m/z),
454.9253 [M+1]+.
2.11. 2-(3-(4-chlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)-4-iodophenol (4-CMI)
1
Yellow solid. Yield: 80%. H NMR (DMSO, 400 MHz):
δ 8.27 (d, J = 8.4 Hz, 1H), 7.98
(d, J = 2.4 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.75–7.63 (m, 2H), 6.83 (d, J = 8.8 Hz, 1H), 7.55
(d, J = 8.4 Hz, 1H). 13C NMR (DMSO, 100 MHz):
δ 168.43, 164.73, 157.79, 150.08, 142.76,
138.69, 136.34, 129.66, 128.07, 124.24, 120.28, 81.56. LCMS: 453.9152 (m/z), 454.9253 [M+1]+.
2.12. 4-iodo-2-(3-(p-tolyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)phenol (4-MMI)
1
Yellow solid. Yield: 84%. H NMR (DMSO, 400 MHz):
δ 8.22 (s, 1H), 8.19–8.00 (m, 2H),
7.71 (d, J = 9.2 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.46–7.29 (m, 2H), 2.35 (s, 3H). 13C NMR
(DMSO, 100 MHz):
δ 161.14, 158.33, 150.56, 148.22, 140.24, 135.20, 130.15, 130.05, 126.26,
126.19, 123.28, 121.42, 84.01, 21.51. LCMS: 433.9698 (m/z), 434.8964 [M+1]+.
2.13. Cells and Cell Culture
U87 human glioma cells were purchased from the American Type Culture Collection.
(ATCC). MPC-11 mouse myeloma cells were kindly provided by Dr. Ralph Sanderson
(the University of Alabama at Birmingham, Birmingham, AL, USA) [20] and luciferase-
labeled 4T1 mouse breast carcinoma cells were kindly provided by Dr. Yuval Shaked (TICC,
Technion, Haifa, Israel) [21]. U87 and 4T1cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) and the MPC-11 cells in RPMI medium, supplemented with glutamine,
pyruvate, antibiotics, and 10% fetal calf serum (Biological Industries, Beit Haemek, Israel)
in a humidified atmosphere containing 5% CO2 at 37 ◦C.
2.14. Colorimetric Heparanase Assay
The assay was carried out in a 96-well plate in which the formation of the disaccharide
product upon cleavage of fondaparinux (Arixtra, GlaxoSmithKline, Brentford, UK) was
estimated using WST-1 tetrazolium salt as described earlier [22
,23]. In brief, the reaction
system (100 L) comprised sodium acetate buffer (pH 5.0, 40 mM) and fondaparinux
µ
(100 mM) with or without different doses of chemicals under investigation. The assay was
initiated by the addition of recombinant heparanase to make the final concentration of
◦
140 pM. The reaction vessels were placed at 37 C for 18 h, followed by the addition of stop
solution (100 µL). WST-1 prepared in 0.1 M NaOH served as a stop solution. Thereafter,
the plates were incubated at 60 ◦C for 60 min, and absorbance was measured at 584 nm.
N-(4-([4-(1H-Benzoimidazol-2-yl)-arylamino]-methyl)-phenyl)-benzamide (compound #98)
was used as a reference compound. The title compounds were examined for their inhibitory
efficacy towards heparanase at 10 and 50 µg/mL. Compounds that yielded inhibition at
these concentrations were further tested at lower concentrations to determine their IC50.
2.15. ECM Degradation Heparanase Assay
ECM degradation heparanase assay was carried out as reported earlier [24–26]. Briefly,
ECM labeled with sulfate [35S] [25] and coating the surface of 35-mm cultures plates was
treated with heparanase (200 ng/mL) with or without compounds under investigation.
The medium was collected to examine the cleavage of HS proteoglycans and subjected
to molecular sieving on Sepharose 6B columns. The radioactivity was measured in the
eluted fractions (0.2 mL). Blue dextran and phenol red were used to mark the excluded
volume (Vo) and included volume (Vt), respectively. The cleaved HS chains were eluted at
0.5 < Kav < 0.8 (fractions 16–32), and the nearly intact proteoglycan was eluted with the

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void volume [24]. Each experiment was carried out thrice and variation in elution positions
did not exceed ±15%.
2.16. Cell Invasion
Invasion assay was performed using Boyden chambers as reported previously [27
,28].
Briefly, U87 cells (5
×
105) were seeded into the upper chamber of the invasion chamber in
a medium devoid of serum, and the lower chamber contained media supplemented with
serum. The cell invasion on treatment with title compounds (4-MMI, 4-CMI; 10 mg/mL)
was analyzed by staining with 0.5% crystal violet (Sigma-Aldrich) and photographed.
2.17. Experimental Metastasis
4TI mouse breast cancer cells tagged with luciferase (1.5
mouse) were inoculated intravenously [21 29]. Test compounds (i.p., 500
×
105/0.1 mL/BALB/c
g/mouse) or
,
µ
DMSO (0.1 mL/mouse) were administered 20 min before cell injection. The mice were
analyzed using IVIS bioluminescent on day 7 and day 13 post-inoculation. These cells
metastasize primarily to the lungs.
2.18. IVIS Imaging
The bioluminescence imaging of tumors generated from luciferase-expressing can-
cer cells was carried out using a highly sensitive, cooled charge-coupled device camera
assembled in a specimen box (IVIS; Xenogen Corp, Hopkinton, MA, USA). The imaging
has advantages such as real-time capture, non-invasive, and quantitative values. For this,
the tumor-bearing mice were intraperitoneally administered with a luciferase substrate
D-luciferin at a dose of 150 mg/kg body weight. The experimental animal was anesthetized
and kept in a camera box followed by continuous exposure to isoflurane (EZAnesthesia,
Palmer, PA, USA). The bioluminescence from the tumor mass was detected by the IVIS
system, and, subsequently, the tumor burden was quantified using Living Image software
(Xenogen) [30,31].
2.19. MPC-11 Tumor Growth
The mouse myeloma (MPC-11) cells were gently washed with PBS and the cell number
was adjusted to 5
were subcutaneously injected with washed MPC-11 cells (5
mental animals were treated with either DMSO alone (0.1 mL/mouse) or 4-MMI (daily
200 g/mouse, i.p.) starting from day 2 after the implantation of tumor cells, for 12 days.
The tumor size was measured using calipers on day 8 and day 12. The tumor volume (V)
was determined using the equation V = L 0.52, where L is the length and W is the
W2
×
106 cells/mL. Thereafter, six-week-old female BALB/c mice (n = 6)
×
105/0.1 mL). The experi-
µ
×
×
width of the tumor. Lastly, the mice were euthanized and xenograft tumors were removed,
weighed, and fixed using formalin for further studies [32].
2.20. Statistics
Data are presented as means
±
SE. Statistical significance was analyzed by the 2-
tailed Student’s t-test. Values of p < 0.05 were considered significant. Data sets passed
D’Agostino–Pearson normality (GraphPad Prism 5 utility software, San Diego, CA, USA).
All experiments were repeated at least twice with similar results.
3. Results
3.1. Synthesis of Title Compounds
The synthesis of triazolo–thiadiazoles was initiated by the conversion of substituted
ethyl benzoates (Figure 1A (1a–e)) to their corresponding hydrazides (2a–e). The esters
(1a–e) were refluxed with hydrazine hydrate in ethanol, which resulted in the formation
of hydrazides (2a–e) as the intermediates. Compounds 2a–e were treated with carbon
disulphide under basic conditions using potassium hydroxide resulted in the formation of
potassium salts (3a–e). The potassium salts were further treated with hydrazine to generate

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4-amino-5-substituted phenyl-4H-1,2,4-triazole-3-thiols (4a–e). Compounds 4a–e were
reacted with substituted salicylic acid in the presence of phosphorous oxychloride under
refluxing conditions to obtain triazolo–thiadiazoles, as shown in Figure 1B. The structures
of all the target compounds presented in Table 1 were characterized by LCMS, 1H NMR,
and 13C NMR spectrometry.
Table 1. List of synthesized substituted [1,2,4]triazolo[3,4-b] [1,3,4]thiadiazoles.
Triazole Amino Thiol
Acid
Products
Derivatives
5a
4a
4-NDI
3-BDI
3-CDI
4-CDI
4-MDI
4b
5a
4c
5a
5a
4d
4e
5a
4e

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Table 1. Cont.
Triazole Amino Thiol
Derivatives
Acid
Products
5b
4a
4-NMI
3-BMI
3-CMI
4b
5b
4c
5b
5b
4d
4-CMI
4e
5b
4-MMI

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Figure 1. (A) Synthesis of 4-amino-5-substituted phenyl-4H-1,2,4-triazole-3-thiol. (B) Synthesis of substituted [1,2,4]triazolo
[1,3,4]thiadiazole (where R1 = 4-NO2, 3-Br, 3-Cl, 4-Cl, 4-CH3 and R2 = 3,5-diiodo (5a) and 5-iodo (5b)).
3.2. Newly Synthesized Triazolo–Thiadiazoles Inhibit Heparanase Enzymatic Activity
The newly synthesized compounds presented in Table 1 were examined for their
ability to inhibit the enzymatic activity of recombinant heparanase. For the initial screening
of compounds, we applied a colorimetric assay based on the cleavage of the synthetic
heparin pentasaccharide fondaparinux (Arixtra) [23]. The assay measures the appearance
of a disaccharide product of heparanase-catalyzed fondaparinux cleavage, using the tetra-
zolium salt WST-1 [23]. All compounds were first tested for inhibition of heparanase at
10 and 50 µg/mL (not shown). Compounds that yielded inhibition at these high concen-
trations were further examined at lower concentrations to determine their IC50 (Figure 2).
Among these compounds, 2-(3-(4-chlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-
yl)-4-iodophenol (4-CMI), 2,4-diiodo-6-(3-(p-tolyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-
6-yl)phenol (4-MDI), and 4-iodo-2-(3-(p-tolyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)
phenol (4-MMI) showed good heparanase inhibitory activity with IC50 values of 3, 12.5,
and 3.1 µg/mL, respectively. Compound #98, used as a positive control, displayed an IC50
value of 0.7 µg/mL (Figure 2, bottom table).
To validate the results and better resemble the in vivo situation, we applied metaboli-
cally sulfate [Na235SO4] labeled extracellular matrix (ECM) deposited by cultured endothe-
lial cells. This naturally produced substrate closely resembles the subendothelial basement
membrane in its composition, biological function, and barrier properties [25]. Years of
experience indicate that compounds that effectively inhibit the enzyme in this assay are also
effective in preclinical animal models [9]. This semi-quantitative assay measures release of
radioactive heparan sulfate (HS) degradation fragments from an insoluble extracellular
matrix (ECM) that is firmly bound to a culture dish [26]. As demonstrated in Figure 3A,
compounds 4-CMI, 4-MMI, and 4-MDI inhibited (50–70%) heparanase activity at 25 µg/mL,
but there was little or no inhibition at 5 µg/mL.

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Figure 2. Screening of compounds for inhibition of heparanase enzymatic activity applying the
fondaparinux (Arixtra) heparanase assay. Compound #98 (red) = N-(4-phenyl)-benzamide = positive
control. Newly synthesized compounds were first tested for inhibition of heparanase at 10 and
50
tested at lower concentrations to determine their IC50. Several compounds (i.e., 4-CMI; 4-MMI;
4-MDI; IC50 = 3, 3.1, 12.5 g/mL, respectively) inhibited the enzyme better than 2,4-Diiodo-6-(3-
µg/mL (not shown). Compounds that yielded inhibition at these concentrations were further
µ
phenyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6yl)phenol (DTP; IC50 = 30 µg/mL) (bottom table).
3.3. Compounds 4-CMI and 4-MMI Inhibit Glioma Cell Invasion
The causal involvement of heparanase in cancer metastasis has long been demon-
strated in various types of cancer [12
we investigated the effect of 4-CMI and 4-MMI on human U87 glioma cell invasion. As
demonstrated in Figure 3B, treatment with 4-CMI or 4-MMI (10 g/mL) significantly
,13,24]. Applying Matrigel-coated transwell filters,
µ
inhibited the invasion capacity of U87 cells. A similar result was obtained with compound
4-MDI (not shown). A dose–response effect of compound 4-MMI is presented in Figure 3C.

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Figure 3. Inhibition of ECM degradation and cell invasion. (
A
) ECM degradation. Active heparanase
(200 ng) was incubated for 5 h without (red) or with 5 and 25
µg/mL of 4-CMI, 4-MDI, or 4-MMI on
dishes coated with sulfate-labeled ECM. Labeled material released from the ECM was subjected to
gel filtration (Sepharose 6B). Heparan sulfate degradation fragments are typically eluted in fractions

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18–30. (
B
) Cell invasion. U87 human glioma cells (2
×
105) were plated onto Matrigel-coated 8
µm
◦
transwell filters and incubated (6 h, 37 C) in the absence (Con) or presence of 4-MMI or 4-CMI
(10 µg/mL). (C) Dose–response showing the effect of compound 4-MMI (2.5, 10, and 40 µg/mL) on
U87 cell invasion. Invading cells adhering to the lower side of the membrane were visualized (top
panels) and quantified (bottom panel) in 7 random fields as described in ‘Materials and Methods’.
The structures of compounds 4-CMI and 4-MMI are presented in Table 1.
3.4. Compounds 4-CMI and 4-MMI Mitigate Breast Carcinoma Experimental Metastasis
We evaluated the effect of 4-CMI and 4-MMI on 4T1 mouse breast carcinoma exper-
imental metastasis. Briefly, luciferase-labeled 4T1 breast carcinoma cells were injected
i.v. into BALB/c mice. Compound 4-CMI or 4-MMI was injected (i.p.) 20 min before
cell inoculation, and metastases formation was inspected and quantified by IVIS fluores-
cence imaging. As demonstrated in Figure 4 and Figure S1, compound 4-MMI and, to
a lesser extent, compound 4-CMI inhibited the extravasation of 4T1 cells and their sub-
sequent colonization in the mouse lungs. The effect of 4-MMI was comparable to that
exerted by Roneparstat (=SST), a well-characterized N-acetylated, glycol-split heparin [17
]
that recently completed phase I clinical trial in myeloma patients [33]. These results in-
dicate that triazolo–thiadiazoles attenuate the ability of blood-borne carcinoma cells to
extravasate through the subendothelial basement membrane, attributed to their ability to
inhibit heparanase enzymatic activity, ECM degradation, and cell invasion.
Figure 4. Inhibition of 4T1 breast carcinoma experimental metastasis. Luciferase-labeled 4T1 breast
carcinoma cells (1.5
Vehicle (DMSO alone), positive control (150
×
105/mouse) were injected i.v. into BALB/c mice (n = 6 BALB/c mice/group).
µg/mouse, SST), 4-CMI, or 4-MMI (500 µg/mouse) were
injected (i.p.; 0.1 mL/mouse) 20 min prior to cell inoculation. Metastasis was inspected by IVIS on
days 7 (not shown) and 13 after cell inoculation. Quantification of the luciferase intensities is shown
graphically in the lower panel, as described in ‘Section 2. Materials and Methods’. * p = 0.02 (SST vs.
DMSO alone); * p = 0.02 (4-MMI vs. DMSO alone).

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3.5. Compound 4-MMI Reduces Tumor Burden in a Myeloma Model
Next, we examined the capacity of compound 4-MMI to inhibit myeloma primary
tumor growth. For this purpose, we applied the MPC-11 syngeneic myeloma model [20 32].
Briefly, BALB/c mice were implanted subcutaneously with MPC-11 mouse myeloma
cells (0.5
106/mouse) and treated with either DMSO (0.1 mL/mouse) or 4-MMI (daily
200 g/mouse, i.p.), starting on day two after tumor cell inoculation. Xenograft Tumor
,
×
µ
size was measured on days 8 and 12 (Figure 5A), resected, and weighed (Figure 5B) (see
also Figure S2). Strikingly, compound 4-MMI effectively and markedly decreased the rate
of tumor growth and the tumor burden. Immunostaining of fixed paraffin-embedded
tissue sections with anti-VEGF antibodies revealed a marked decrease in the levels of
VEGF in tumors derived from mice treated with 4-MMI (Figure S3), likely reflecting an
anti-angiogenesis effect of the drug.
Figure 5. MPC-11 tumor growth. Cells (MPC-11 mouse myeloma) from exponential cultures
were washed with PBS and brought to a concentration of 5
(5
105/0.1 mL) was inoculated subcutaneously at the right flank of 6-week-old female BALB/c mice
(n = 6). Mice were treated with either DMSO (0.1 mL/mouse) or test compound (daily 200 g/mouse,
i.p.), starting on day 2 after the injection of tumor cells, for 12 days. Tumor size was determined on
×
106 cells/mL. Cell suspension
×
µ
days 8 and 12 by externally measuring the tumors in two dimensions using a caliper (
A
, p < 0.001).
At the end of the experiment, mice were sacrificed and the tumors resected, weighed (
and photographed.
B, p = 0.003),
4. Discussion
Heparanase is a multifaceted enzyme that together with heparan sulfate drives signal
transduction, immune cell activation, exosome formation, autophagy, and gene transcrip-
tion via enzymatic and non-enzymatic activities [8,34,35]. Heparanase releases a myriad
of HS-bound growth factors, cytokines, and chemokines that are sequestered by heparan
sulfate in the glycocalyx and ECM. Collectively, the heparan sulfate–heparanase axis plays
pivotal roles in creating a permissive environment for cell proliferation, differentiation,
and function, often resulting in the pathogenesis of diseases, such as cancer, chronic inflam-
mation, sepsis, endotheliitis, kidney dysfunction, diabetic nephropathy, diabetes, tissue

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fibrosis, bone osteolysis, thrombosis, atherosclerosis, and viral infection [36]. Heparanase
represents a druggable target because (i) there is only a single enzymatically active hep-
aranase expressed in humans; (ii) the enzyme is present in low levels in normal tissues but
is markedly elevated in cancer, inflammation, and other pathologies; and (iii) heparanase
deficient mice appear normal [37]. Thus, properly designed heparanase inhibitors will
likely have few, if any, negative side effects. The development of heparanase inhibitors
has focused predominantly on carbohydrate-based compounds with heparin-like proper-
ties [38–40]. These compounds bind to the heparin-binding domains that flank the active
site of heparanase thereby inhibiting cleavage of heparan sulfate. Four different heparin
mimics are currently in clinical trials in human cancer patients. However, all of these
mimics have the disadvantage that they are not specific for heparanase and likely inter-
act with different heparin-binding proteins with unknown consequences and off-target
effects [38,39]. Therefore, even if they prove efficacious in patients, it will be impossible to
attribute their effect solely to heparanase inhibition. Moreover, three of the four mimics are
heterogeneous in structure, adding to their complexity and uncertainty as viable drugs for
use in humans. Small-molecule inhibitors of heparanase have the potential to overcome
some of the limitations of polysaccharides, offering an opportunity for specificity, favorable
pharmacokinetics, and oral availability [36,41]. Numerous heparanase-inhibiting small
molecules were reported [39 42 43], but none entered clinical testing. Surprisingly, only
,
,
one compound was found to inhibit experimental metastasis (B16 melanoma model) [44],
and there are no reports of their preclinical testing in xenograft tumor models.
We have previously reported the results of an in vitro screening of a library of
small molecules characterized by a variety of scaffolds. More than 150 compounds were
tested for their ability to inhibit the enzymatic activity of human heparanase, identify-
ing [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives as active substances. The most potent
anti-heparanase derivative, DTP, was found to inhibit tumor cell proliferation, migration,
and invasion in vitro with IC50 values in the micromolar range [22]. The present study
focuses on the development of related triazolo–thiadiazole compounds that inhibit hep-
aranase enzymatic activity, ECM degradation, cell invasion, experimental metastasis, and
tumor growth in mouse models. We used DTP as a template structure for the synthesis of
substituted triazolo–thiadiazoles endowed with better heparanase inhibitory activity. The
structure of DTP was modified by removing iodine at the second position of the phenyl
ring and the addition of a methyl group on the terminal benzene ring, which resulted in the
formation of compound 4-MMI. MMI showed relatively potent heparanase inhibitory and
antitumor activity compared to its parent compound. Other structural analogs showed var-
ious degrees of heparanase inhibition. The newly designed compounds were examined for
their capacity to inhibit heparanase enzymatic activity, using as substrates heparin-derived
pentasaccharide [23] and HS naturally embedded in the ECM produced by endothelial
cells [25]. To translate the in vitro inhibition of heparanase enzymatic activity and cell
invasion to preclinical models, the most promising triazolo–thiadiazole compounds were
examined in the 4T1 mouse mammary carcinoma model of experimental metastasis and
the MPC-11 mouse model of myeloma tumor growth [20,32]. Breast cancer metastasis is the
leading cause of female mortality worldwide and accounts for 25% of the total number of
cancer cases and 15% of all cancer-associated female mortality [45]. Importantly, compound
4-MMI yielded a nearly fourfold inhibition of 4T1 breast carcinoma metastasis, comparable
to the effect exerted by Roneparstat (=SST), a well-characterized heparin-like heparanase
inhibitor (Figure 4) [15,17,33,36,39,41].
Multiple myeloma is a devastating cancer of plasma cells that is highly dependent
on the bone marrow microenvironment for growth and survival [46]. Heparanase has
been shown to critically accelerate myeloma tumor growth and bone colonization [47],
and the heparanase inhibitor Roneparstat has been examined in myeloma patients, yield-
ing promising results [33]. Strikingly, treatment with compound 4-MMI resulted in a
profound inhibition of tumor growth and burden in the aggressive MPC-11 myeloma
model (Figure 5). We also noted a profound reduction in VEGF levels in tumor sections

Cancers 2021, 13, 2959
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derived from 4-MMI treated vs. untreated mice, likely due to inhibition of heparanase-
mediated VEGF gene expression and/or release [6,7,10]. Due to limited amounts of the
4-MMI compound, we could not perform dose–response and pharmacokinetic studies. Yet,
there were no signs of distress and toxicity in response to the daily administration of 200
ug/mouse/day, a dose that is well below the amounts of anti-cancer small molecules given
to mice and the equivalent dose in humans. In subsequent studies we will examine the
effect of 4-MMI in additional tumor models, with emphasis on orthotopic models of breast
and pancreatic carcinomas. We will also apply medicinal chemistry and computational
principles to further optimize the compound for improved potency; specificity; selectiv-
ity; and pharmacokinetic properties, including stability, permeability, solubility, and oral
bioavailability.
5. Conclusions
To the best of our knowledge, this is the first report showing a marked decrease in
primary tumor growth in mice treated with a small molecule that inhibits heparanase
enzymatic activity. Given these encouraging results, studies are underway to better eluci-
date the mode of action and clinical significance of compound 4-MMI and related triazolo–
thiadiazoles. Among other aspects, the compound is being examined for an effect on human
tumor xenografts, primarily myeloma and breast carcinoma, pharmacokinetic characteris-
tics, efficacy, and mode of administration. Docking and SAR studies are being performed,
applying the recently resolved crystal structure of the heparanase protein [48–50]. Selected
molecules exerting little or no side effects will then be examined for oral availability and
anti-cancer effects in combination with currently available treatments. We will also study
the effect of our lead compounds on other pathological indications involving heparanase,
such as chronic inflammation (i.e., colitis, pancreatitis) [51
dysfunction (i.e., AKI, diabetic nephropathy) [54 57], and diabetes [58]. Of particular
relevance are recent studies on the presumed involvement of heparanase and heparan
sulfate in the pathogenesis of COVID-19 [59 62]. Studies on the effect of 4-MMI and related
,52], tissue fibrosis [53], kidney
–
–
compounds on SARS-CoV-2 infectivity are ongoing.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/cancers13122959/s1. Figure S1: Inhibition of 4T1 breast carcinoma experimental metastasis,
Figure S2: MPC-11 tumor growth, Figure S3: Immunostaining The spectra and other information are
freely available with this paper.
Author Contributions: Conceptualization and design, K.S.R., C.D.M., B.B. and I.V.; experimental
work, U.B., I.B., D.V. and S.R.; writing—original draft, C.D.M., S.R., D.V., B.B., I.B. and U.B.; writing—
review and editing, C.D.M., B.B., K.S.R. and I.V.; supervision, K.S.R., C.D.M., B.B. and I.V. All authors
have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Israel Science Foundation (ISF) and the University Grants
Commission (UGC), India, awarded to I.V. and K.S.R. within the ISF-UGC joint research program
framework (grant No. 2277/15). It was also supported by grants from the Israel Science Foundation
(1021/19) and the Israel Cancer Research Fund (ICRF). I.V. is a Research Professor of the Israel Cancer
Research Fund (ICRF). B. thanks the Department of Biotechnology (BT/PR24978/NER/95/938/2017)
and Vision Group on Science and Technology (CESEM), Government of Karnataka for funding. This
project was supported by DST PhD Fellowship from KSTePS to D.V.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the Institutional Review Board (018-08-18).
Informed Consent Statement: Not applicable.
Data Availability Statement: All the data associated with this study are freely available.
Acknowledgments: K.S.R. thanks the Institution of Excellence, DST-PURSE, University of Mysore,
for providing infrastructure and other research facilities. K.S.R. also thanks the Council of Scientific
and Industrial Research for providing an emeritus scientist fellowship.
Conflicts of Interest: The authors declare no conflict of interest.

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