Synthesis and biological evaluation of new antitubulin agents containing 2-(30,40,50-trimethoxyanilino)-3,6-disubstituted-4,5,6,7-tetrahydrothieno[2,3-c]pyridine Scaffold

Article abstract of DOI:10.3390/molecules25071690

Two novel series of compounds based on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine and 4,5,6,7-tetrahydrobenzo[b]thiophene molecular skeleton, characterized by the presence of a 3⁰,4⁰,5⁰-trimethoxyanilino moiety and a cyano or an alkoxycarbonyl group at its 2- or 3-position, respectively, were designed, synthesized, and evaluated for antiproliferative activity on a panel of cancer cell lines and for selected highly active compounds, inhibition of tubulin polymerization, and cell cycle effects. We have identified the 2-(3⁰,4⁰,5⁰-trimethoxyanilino)-3-cyano-6-methoxycarbonyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine derivative 3a and its 6-ethoxycarbonyl homologue 3b as new antiproliferative agents that inhibit cancer cell growth with IC₅₀ values ranging from 1.1 to 4.7 μM against a panel of three cancer cell lines. Their interaction with tubulin at micromolar levels leads to the accumulation of cells in the G2/M phase of the cell cycle and to an apoptotic cell death. The cell apoptosis study found that compounds 3a and 3b were very effective in the induction of apoptosis in a dose-dependent manner. These two derivatives did not induce cell death in normal human peripheral blood mononuclear cells, suggesting that they may be selective against cancer cells. Molecular docking studies confirmed that the inhibitory activity of these molecules on tubulin polymerization derived from binding to the colchicine site.

Full text of DOI:10.3390/molecules25071690

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molecules
Article
Synthesis and Biological Evaluation of New
Antitubulin Agents Containing 2-(30,40,50-
trimethoxyanilino)-3,6-disubstituted-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine Scaffold
Romeo Romagnoli 1,
*
, Filippo Prencipe 1, Paola Oliva 1, Barbara Cacciari 1, Jan Balzarini 2,
Sandra Liekens 2, Ernest Hamel 3, Andrea Brancale 4, Salvatore Ferla 4, Stefano Manfredini 5
,
Matteo Zurlo 5 , Alessia Finotti 5 and Roberto Gambari 5,6
1
Dipartimento di Scienze Chimiche e Farmaceutiche, University of Ferrara, Via L. Borsari 46, 44121 Ferrara,
Italy; prnfpp@unife.it (F.P.); lvopla@unife.it (P.O.); ccb@unife.it (B.C.)
2
3
4
5
Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, B-3000 Leuven,
Belgium; jan.balzarini@rega.kuleuven.be (J.B.); sandra.liekens@kuleuven.be (S.L.)
Screening Technologies Branch, Developmental for Cancer Research, National Cancer Institute, National
Institute of Health, Frederick, MD 21702, USA; hamele@dc37a.nci.nih.gov
School of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff CF10
3NB, UK; brancalea@cardiff.ac.uk (A.B.); ferlas1@cardiff.ac.uk (S.F.)
Dipartimento di Scienze della Vita e Biotecnologie, Università di Ferrara, 44121 Ferrara, Italy;
stefano.manfredini@unife.it (S.M.); matteo.zurlo@unife.it (M.Z.); alessia.finotti@unife.it (A.F.);
roberto.gambari@unife.it (R.G.)
Interuniversity Consortium for Biotechnology, Area di Ricerca, Padriciano, 34149 Trieste, Italy
Correspondence: rmr@unife.it; Tel.: +39-0532-455303
6
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 15 March 2020; Accepted: 4 April 2020; Published: 7 April 2020
Abstract: Two novel series of compounds based on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine
and 4,5,6,7-tetrahydrobenzo[b]thiophene molecular skeleton, characterized by the presence of a
30,40,50-trimethoxyanilino moiety and a cyano or an alkoxycarbonyl group at its 2- or 3-position,
respectively, were designed, synthesized, and evaluated for antiproliferative activity on a panel of
cancer cell lines and for selected highly active compounds, inhibition of tubulin polymerization, and
cell cycle effects. We have identified the 2-(30,40,50-trimethoxyanilino)-3-cyano-6-methoxycarbonyl-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine derivative 3a and its 6-ethoxycarbonyl homologue 3b as new
antiproliferative agents that inhibit cancer cell growth with IC50 values ranging from 1.1 to 4.7 µM
against a panel of three cancer cell lines. Their interaction with tubulin at micromolar levels leads to
the accumulation of cells in the G2/M phase of the cell cycle and to an apoptotic cell death. The cell
apoptosis study found that compounds 3a and 3b were very effective in the induction of apoptosis in a
dose-dependent manner. These two derivatives did not induce cell death in normal human peripheral
blood mononuclear cells, suggesting that they may be selective against cancer cells. Molecular
docking studies confirmed that the inhibitory activity of these molecules on tubulin polymerization
derived from binding to the colchicine site.
Keywords: microtubules; structure–activity relationship; antiproliferative activity; tubulin;
tetrahydrothieno[2,3-c]pyridine
1. Introduction
Cancer, which is the result of deviation of cell growth control mechanisms, has become a major
worldwide disease and the second leading cause of mortality in developed countries, with almost 20
Molecules 2020, 25, 1690; doi:10.3390/molecules25071690
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Molecules 2020, 25, 1690
2 of 23
million deaths annually [
surgery, radiation, and chemotherapy, have problems with toxicity and drug resistance [3,4
1
,
2
]. Many of the current treatments, involving an integrated treatment of
]. Therefore,
there is a strong demand for the discovery and development of effective chemotherapeutic agents with
high selectivity and low toxicity [5–7].
The central role of microtubules in cell division and mitosis makes them an important target for the
development of potential new anticancer agents [
wide structural diversity and derived from natural sources or obtained by chemical synthesis have been
identified within the last several decades [11 13] and have been shown to interfere with microtubules,
8–10]. A large number of small molecules displaying
–
polymeric structures composed of α- and β-tubulin heterodimers [14–16].
Prominent examples of tubulin-binding compounds used clinically for cancer treatment include
microtubule-stabilizers that inhibit microtubule function by promoting abnormally high levels of
tubulin polymerization. Examples of such drugs include paclitaxel (Taxol) and its semisynthetic
analogue docetaxel (Taxotere) [17
vinblastine destabilize microtubules and act as microtubule depolymerizers by inhibiting tubulin
polymerization [19]. Both taxoids and vinca alkaloids interact with -tubulin, but at different sites,
,18]. In contrast, the vinca alkaloids vincristine, vinorelbine, and
β
named the taxol and vinca sites, respectively [20]. The colchicine-site binders that inhibit tubulin
assembly [21] are another class of compounds in discovery and development, including combretastatin
A-4 phosphate (CA-4P) [22], that bind to the colchicine site located between the
subunits in the heterodimer, distinct from the vinca site located between adjacent
heterodimers [23].
α
- and
- and
β
-tubulin
-tubulin
α
β
Although many chemically diverse synthetic tubulin inhibitors have been synthesized, there
is still a need to identify novel small molecules that target microtubules [24 28]. Among
such compounds, thiophene [29 34] and fused thiophenes such as benzo[b]thiophenes [35 40],
thieno[3,2-d]pyrimidines [41 43], and thieno[2,3-b]pyridines [44] were present as molecular skeletons
–
–
–
–
in a significant number of synthetic inhibitors of microtubule polymerization, but there are limited
examples of small molecules showing interesting activities in inhibiting both microtubule assembly
and cell proliferation based on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine molecular skeleton as the
core structure.
In an earlier published study, a series of molecules with general structure 1 (Figure 1), based on
the 2-amino-3-(30,40,50-trimethoxybenzoyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine skeleton, yielded
the promising derivatives 1a and 1b. These compounds inhibit cancer cell growth with IC50 values
ranging from 25 to 440 nM against a panel of four cancer cell lines and exert strong inhibition of tubulin
polymerization by binding to the colchicine site of tubulin [45]. For this series of molecules, the presence
of a N-methoxy/ethoxycarbonyl moiety at the 6-position of the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine
system was needed for potent antiproliferative activity.
A second series of compounds with general structure
incorporated the 2-carboxamido-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridine system, and these
agents were active at micromolar concentrations (IC50 = 0.2–5 M) as antiproliferative agents against
human colon adenocarcinoma (RKOp27) cells. Bausch et al. [48] reported a molecule named 4SC-207
2a), characterized by the presence of the 2-[(pyridin-30-yl)acrylamido]-3-cyano-4,5,6,7-tetrahydrothieno
2, reported by Beckers et al. [46,47],
µ
(
[2,3-c]pyridine skeleton, able to inhibit microtubule polymerization at high concentrations and showing
strong antiproliferative activity at low nanomolar concentrations in a large panel of cancer cell
lines. These studies [46–48] showed that the presence of a cyano group at the 3-position of the
4,5,6,7-tetrahydrothieno[2,3-c]pyridine nucleus was required for antiproliferaive activity. These
findings prompted us to analyze this class of compounds in more detail.

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Figure 1. Structures of tetrahydrothieno[2,3-c]pyridines 1–3. Compounds with a 3,4,5-trimethoxyphenyl
moiety, such as colchicine, CA-4, podophyllotoxin, and poly-methoxychalcone.
Building upon these observations, since the 6-N-alkoxycarbonyl-4,5,6,7-tetrahydrothieno[2,3-c]
pyridine structure was demonstrated to be essential for bioactivity, we retained this pharmacophoric

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Molecules 2020, 25, 1690
4 of 23
unit throughout the present investigation and kept the 3-cyano group in compounds we synthesized.
We were intrigued by the idea of studying the biological effects of replacing the carboxamido group at
the 2-position of the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine ring of compounds with general structure
2
with a 30,40,50-trimethoxyanilino moiety to yield the 2-(30,40,50-trimethoxyanilino)-3-cyano-6-methoxy/
ethoxycarbonyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridines 3a and 3b. Previous studies have shown
that the 3,4,5-trimethoxyphenyl moiety is a pharmacophoric group that is an essential structural
requirement for the anticancer effect of numerous inhibitors of tubulin polymerization, such as
colchicine, combretastatin A-4 (CA-4), podophyllotoxin, and poly-methoxychalcone [49,50]. Starting
from compounds 3a and 3b, additional analogues were also generated by modification of the 3-cyano
function of the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine skeleton. The cyano group was replaced
with a 3-alkoxycarbonyl moiety, such as 3-methoxycarbonyl (3c
–3e), 3-ethoxycarbonyl (3f–3h), or
3-tert-butoxycarbonyl (3i 3j). Moreover, the influence on antiproliferative activity of the substituent
–
(R2) at the 6-position was evaluated with R2 as acetyl or methoxy/ethoxycarbonyl.
Additional structural modifications involved the nitrogen at the 6-position of 4,5,6,7-tetrahydrothi
eno[2,3-c]pyridine scaffold, by the classical bioisosteric replacement with a CH group to furnish a novel
series of 2-(30,40,50-trimethoxyanilino)-3,6-substituted-4,5,6,7-tetrahydrobenzo[b]thiophene derivatives
3k–3p. For this latter series of compounds, besides the C-3 cyan and methoxy/ethoxycarbonyl moieties,
the substituents examined at the C-6 position were methyl (3k, 3m, and 3o) and phenyl (3l, 3n, and
3p) groups.
2. Chemistry
The 2-(30,40,50-trimethoxyanilino)-3-cyano/alkoxycarbonyl-6-substituted-4,5,6,7-tetrahydrothieno
[2,3-c]pyridines 3a 3j and the corresponding 4,5,6,7-tetrahydrobenzo[b]thiophenes 3k 3p were
prepared through the three-step procedure shown in Scheme 1.
–
–
Scheme 1. Synthesis of compounds 3a-p. Reagents. a: appropriate ketone (4-methyl/phenylcyclohexan
one, N-acetyl-4-piperidone, or methoxy/ethoxycarbonyl-4-piperidone), malonitrile or me◦thyl/ethyl/t-
butylcyanoacetate, S8, TEA, MeOH or EtOH, reflux; b: t-BuONO, CuBr2, CH3CN, 65 C; c: 3,4,5-
trimethoxyaniline, Pd(OAc)2, BINAP, Cs2CO3, PhMe, 100 ◦C, 18 h.