Microtubule destabilizing sulfonamides as an alternative to taxane-based chemotherapy

Article abstract of DOI:10.3390/ijms22041907

Pan-Gyn cancers entail 1 in 5 cancer cases worldwide, breast cancer being the most com-monly diagnosed and responsible for most cancer deaths in women. The high incidence and mortality of these malignancies, together with the handicaps of taxanes —first-l

Full text of DOI:10.3390/ijms22041907

International Journal of
Molecular Sciences
Article
Microtubule Destabilizing Sulfonamides as an Alternative to
Taxane-Based Chemotherapy
Myriam González 1,2,3,†, María Ovejero-Sánchez 2,4,5,† , Alba Vicente-Blázquez 1,2,3, Raquel Álvarez 1,2,3
,
Ana B. Herrero 2,4,5 , Manuel Medarde 1,2,3, Rogelio González-Sarmiento 2,4,5,
*
and Rafael Peláez 1,2,3,
*
1
Laboratorio de Química Orgánica y Farmacéutica, Departamento de Ciencias Farmacéuticas,
Facultad de Farmacia, Universidad de Salamanca, 37007 Salamanca, Spain; mygondi@usal.es (M.G.);
avicenteblazquez@usal.es (A.V.-B.); raquelalvarez@usal.es (R.Á.); medarde@usal.es (M.M.)
Instituto de Investigación Biomédica de Salamanca (IBSAL), Hospital Universitario de Salamanca,
37007 Salamanca, Spain; maria.os@usal.es (M.O.-S.); anah@usal.es (A.B.H.)
Centro de Investigación de Enfermedades Tropicales de la Universidad de Salamanca (CIETUS),
Facultad de Farmacia, Universidad de Salamanca, 37007 Salamanca, Spain
Unidad de Medicina Molecular, Departamento de Medicina, Facultad de Medicina,
Universidad de Salamanca, 37007 Salamanca, Spain
2
3
4
5
Laboratorio de Diagnóstico en Cáncer Hereditario, Laboratorio 14, Centro de Investigación del Cáncer,
Universidad de Salamanca-CSIC, 37007 Salamanca, Spain
*
Correspondence: gonzalez@usal.es (R.G.-S.); pelaez@usal.es (R.P.); Tel.: +34-923-294500 (R.G.-S.);
+34-677-554-890 (ext. 1837) (R.P.)
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Pan-Gyn cancers entail 1 in 5 cancer cases worldwide, breast cancer being the most
commonly diagnosed and responsible for most cancer deaths in women. The high incidence and
mortality of these malignancies, together with the handicaps of taxanes—first-line treatments—
turn the development of alternative therapeutics into an urgency. Taxanes exhibit low water sol-
ubility that require formulations that involve side effects. These drugs are often associated with
dose-limiting toxicities and with the appearance of multi-drug resistance (MDR). Here, we pro-
pose targeting tubulin with compounds directed to the colchicine site, as their smaller size of-
fer pharmacokinetic advantages and make them less prone to MDR efflux. We have prepared
52 new Microtubule Destabilizing Sulfonamides (MDS) that mostly avoid MDR-mediated resis-
tance and with improved aqueous solubility. The most potent compounds, N-methyl-N-(3,4,5-
trimethoxyphenyl-4-methylaminobenzenesulfonamide 38, N-methyl-N-(3,4,5-trimethoxyphenyl-4-
methoxy-3-aminobenzenesulfonamide 42, and N-benzyl-N-(3,4,5-trimethoxyphenyl-4-methoxy-3-
aminobenzenesulfonamide 45 show nanomolar antiproliferative potencies against ovarian, breast,
and cervix carcinoma cells, similar or even better than paclitaxel. Compounds behave as tubulin-
binding agents, causing an evident disruption of the microtubule network, in vitro Tubulin Poly-
merization Inhibition (TPI), and mitotic catastrophe followed by apoptosis. Our results suggest that
these novel MDS may be promising alternatives to taxane-based chemotherapy in chemoresistant
Pan-Gyn cancers.
Citation: González, M.;
Ovejero-Sánchez, M.;
Vicente-Blázquez, A.; Álvarez, R.;
Herrero, A.B.; Medarde, M.;
González-Sarmiento, R.; Peláez, R.
Microtubule Destabilizing
Sulfonamides as an Alternative to
Taxane-Based Chemotherapy. Int. J.
Mol. Sci. 2021, 22, 1907. https://
doi.org/10.3390/ijms22041907
Academic Editor: Peter Van Dam
Received: 12 January 2021
Accepted: 11 February 2021
Published: 14 February 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Keywords: tubulin; sulfonamide; antitumor; taxane; combretastatin A-4; breast cancer; gyneco-
logic cancer
Copyright:
© 2021 by the authors.
1. Introduction
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/).
Gynecologic (i.e., ovarian, uterine, vulva, and vagina) and women breast cancer
share relevant similarities at the molecular level that cluster them together (Pan-Gyn)
and distinguish them from other tumor types [
Pan-Gyn cancers accounted for 19% of new diagnoses worldwide in 2018 and entailed 40%
of new cancer cases among females [ ]. 39% of women with Pan-Gyn cancers died from
them, representing 30% of all female cancer deaths and 13% of total cancer deaths globally.
1,2]. According to GLOBOCAN estimates,
3
Int. J. Mol. Sci. 2021, 22, 1907. https://doi.org/10.3390/ijms22041907
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The high incidence and mortality of female breast [4,5], ovarian [6,7], and uterine [8,9]
cancers and the acquired resistance to first-line treatments highlight the need for new
therapeutic alternatives.
Since the FDA approved the use of Taxol® for advanced ovarian carcinoma in 1992
and metastatic breast cancer in 1994 [10], taxane-based chemotherapy has become the basis
for treating breast and ovarian cancers [11]. First-line chemotherapy involves a platinum-
taxane combination for ovarian cancer [12
taxanes sequenced or combined with anthracyclines for advanced and metastatic breast
cancer [16 19]. Furthermore, the combination of cisplatin and paclitaxel is considered
–14], preferably carboplatin/paclitaxel [15], and
–
a standard regimen in the palliative setting for patients with metastatic, recurrent, or
persistent uterine cancer [9,20–22].
However, the clinical efficacy of taxanes is limited by an intrinsic low aqueous sol-
ubility (<2
tance [26]. The low solubility (remaining challenging) requires complex solvent-system
formulations that can alter the pharmacokinetic profiles [27 28] and have been associated
with toxicity [29 30]. The development of resistance to these drugs is usually ascribed to
Pgp-mediated efflux and alterations in tubulin (mutations in the tubulin genes, altered
tubulin isotype expression, or aberrant levels of tubulin) [31 33]. Specific resistance to
taxane-based chemotherapy has been described for female breast cancer, with an approx-
imately 30% of recurrence [34 35], ovarian cancer, in which 50% of patients relapse with
µg/mL [23–25]), dose-limiting toxicities, and the development of drug resis-
,
,
–
,
the chemoresistant disease within the first two years post-diagnosis [36
cancer [38].
,37], and uterine
Taxanes bind to microtubules, stabilizing lateral contacts between protofilaments, thus
promoting assembly and inhibiting depolymerization at high concentrations. At lower con-
centrations, they alter microtubule dynamics and preclude cells from properly completing
mitosis [39
,40], which leads to cell cycle arrest at G2/M and, subsequently, to cell death
mainly triggered by apoptosis [41
,
42]. There are other drug binding sites in tubulin that
affect microtubule dynamics similarly to taxanes, but whose engagement results in micro-
tubule depolymerization at high concentrations, such as the colchicine, the vinca alkaloids,
the maytansine, the pironetin, and the hemiasterlin sites [43]. The only approved drugs
belong to the vinca alkaloids site binding ligands, which are used against hematological
malignancies and solid tumors, including breast cancer. However, they share with taxanes
the problems of toxic side effects, difficult pharmacokinetics, and complex hydrophobic
structures that make them substrates of drug efflux pumps [44].
The colchicine site offers advantages for drug design since its ligands exhibit simple
chemical structures and are synthetically accessible. Colchicine-site ligands display an-
tiproliferative effects (characterized by cell cycle arrest at G2/M phase followed by the
induction of apoptosis [45]) and act as vascular disrupting agents, affording additional
odds against solid tumors [46]. Representatives such as the stilbene combretastatin A-4
(CA-4) and the sulfonamide ABT-751 have reached clinical trials [47]. However, the Z
olefin of combretastatins is chemically unstable and readily isomerizes to the more stable
but inactive E isomer. The low solubility of CA-4 requires the use of phosphate prodrugs
on the phenolic hydroxyl group, which is, in turn, the target for metabolic inactivation by
glucuronidation in resistant cells, such as the colon adenocarcinoma HT-29 cell line [48].
ABT-751 is an orally administered sulfonamide with modest potency against human cancer
cell lines and xenograft models. Despite its favorable pharmacokinetics, ABT-751 has not
found clinical application due to insufficient potency [49].
In this work, we have designed and synthesized a new family of Microtubule Desta-
bilizing Sulfonamides (MDS) hybrids of CA-4 and ABT-751. The effects of replacing the
chemically unstable CA-4 olefin with a sulfonamide bridge, the removal or substitution
of the phenolic hydroxyl group, and the introduction of several modifications on the
aromatic rings and the sulfonamide bridge have been explored while maintaining the 3,4,5-
trimethoxyphenyl ring that has been long considered essential for high potency [50
,51]
(Figure 1). The resulting compounds have been evaluated for tubulin inhibition in vitro

Int. J. Mol. Sci. 2021, 22, 1907
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and antiproliferative activity against several human tumor cell lines. We have also stud-
ied whether MDR1 pumps could compromise their effectiveness by the pharmacological
inhibition of Pgp using verapamil. After a comprehensive preliminary evaluation, three
promising MDS have been further screened against several representative cancer cell
lines representative of the tumor types that are associated with the highest mortalities:
breast, ovarian, and uterine, accounting for 51%, 15%, and 32% of cancer deaths in women,
respectively. The effect of the compounds on cancer cell proliferation has been studied
and compared with paclitaxel, CA-4, and ABT-751. The mechanism of action of these
novel MDS has been studied by ascertaining their effect on the microtubule network
in vitro. These MDS induce mitotic arrest, followed by apoptotic cell death with differ-
ences arising from different genetic backgrounds of the studied cell lines. The favorable
pharmacodynamic and pharmacokinetic profiles compared to reference drugs, including
solubility, absence of Pgp-mediated resistance, and improved potency indicate that MDS
are promising candidates for the treatment of this kind of malignancies.
Figure 1. Representative ligands binding at the colchicine site used as a starting point for the rational design of new
Microtubule Destabilizing Sulfonamides (MDS). General structure and structure variations of new MDS.
2. Results
2.1. Synthesis of MDS
52 new MDS (Figure 1) were prepared following the synthetic approach shown in
Figure 2 (detailed synthetic procedures and NMR spectra can be found in Supplemen-
tal Figure S1 and Methods SP1,2). The synthesized compounds were divided into three
series according to the substituents on the aromatic B ring (ArB): series 1 (compounds
1a-24), series 2 (25–38), and series 3 (39–48b) (Table 1). Sulfonamides were built up by the
reaction between 4-methoxy- (series 1), 4-nitro- (series 2), or 4-methoxy-3-nitro- (series 3)

Int. J. Mol. Sci. 2021, 22, 1907
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benzenesulfonyl chlorides and 3,4,5-trimethoxyaniline, providing crystalline products in
excellent yields (90–96%). Nitro groups were reduced to amines by palladium-catalyzed
hydrogenation (82–98% yields). The subsequent amino derivatization by alkylation, acy-
lation, and/or formylation-reduction sequences allowed the introduction of varied Z, R,
and Y substituents (Figure 1). Substitutions at the sulfonamide nitrogen were conducted
by alkylation reactions with alkyl halides in KOH/CH3CN (methylations with methyl
iodide in 63–98% yields) or K2CO3/DMF (ethyl, acetyl, acetonitrile, benzyl, or ethyl acetate
substituents in 40–99% yields).
Figure 2. General synthetic approach. Reagents, conditions, and yields: (a) Pyridine, CH2Cl2, rt,
4–8 h, 90–96% ( ) RN = CH3, CH3I, KOH, CH3CN, rt, 24 h, 63–98%; RN = Ac, acetic anhydride,
b
pyridine, CH2Cl2, reflux, 8–12 h, 61–83%; RN
=
CH3 and Ac, RN-halogen, K2CO3, dry DMF, rt, 24–48
) H2, Pd/C, EtOAc, rt, 48–72 h, 82–98% (
) Trichloroacetic acid, NaBH4, dry THF, 0◦C, 24 h, 97%
) Paraformaldehyde, NaBH3CN, AcOH, MeOH, rt, 72–96 h, 95% ( ) Ethyl 2-bromoacetate, NaI,
acetone/THF 1:1, reflux, 48 h, 19% (i) NBS, CH2Cl2, rt, 6 h, 43%.
◦
h, 40–99% (c) tert-Butyl nitrite, CH3CN, 45 C, 24 h, 60% (
d
e)
Formic acid, CH2Cl2, rt, 24–48 h, 62–82% (
f
(g
h
Table 1. Chemical structure, antiproliferative activity against human tumor cell lines, and Tubulin Polymerization Inhibition
(TPI) of novel Microtubule Destabilizing Sulfonamides (MDS). Compounds have been divided into three different series
according to the substituents on the aromatic B ring (ArB). TM: 3,4,5-trimethoxyphenyl.
Antiproliferative Activity
TPI
IC50 (nM)
TPI %
Series 1:
Z
RN
Compound
HeLa
MCF7
10 µM
IC50 (µM)
H
H
H
H
H
H
H
H
H
H
1a
1b
2a
2b
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
240
>1000
71
>1000
99
94
287
143
217
>1000
750
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
375
>1000
127
>1000
87
340
270
275
335
>1000
830
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
0
0
35
0
25
30
0
0
21
0
21
11
10
0
0
0
0
0
4
0
0
0
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
SO2–4-OMePh
Me
CH2-Dim
Et
EtBr
Ac
CH2CN
CH2COOEt
CH2COOH
Benzyl
COOBenzyl
H
H
H
NO2
NH2
NH2
NH2
NHAc
NHAc
NHCHO
N(CH3)2
N(CH3)2
Br
H
H
Me
CH2-Dim
H
Me
H
H
Me
H
Gly-tBOC
H
0

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Table 1. Cont.
Succinic
-N=N-
H
22
23
24
>1000
>1000
>1000
>1000
>1000
>1000
0
0
0
>20
>20
>20
-N=CH-
Antiproliferative Activity
IC50 (nM)
TPI
TPI %
Series 2:
R
RN
Compound
HeLa
MCF7
10 µM
IC50 (µM)
NO2
NH2
NH2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
NHCHO
NHCH3
NHCH3
H
H
Me
H
25
26
27
>1000
>1000
>1000
230
63
>1000
66
183
55
99
>1000
330
>1000
>1000
>1000
173
30
>1000
81
135
120
91
>1000
100
0
0
0
0
40
0
45
38
17
14
6
72
0
>20
>20
>20
>20
>20
>20
>20
14
>20
>20
>20
5.3
28
Me
29a
29b
30
31
32
33
34
35a
35b
36
CH2-Dim
Et
Ac
CH2CN
CH2COOEt
CH2COOH
Benzyl
COOBenzyl
H
>1000
>1000
607
>1000
>1000
>1000
61
>20
>20
>20
10
0
7
47
H
Me
37
38
44
Antiproliferative Activity
IC50 (nM)
TPI
TPI %
Series 3:
Y
RN
Compound
HeLa
MCF7
10 µM
IC50 (µM)
NO2
NH2
NH2
NH2
NH2
H
H
Me
40
41
42
43
44
45
46
47
48a
48b
>1000
260
23
38
60
>1000
96
26
14
8
48
48
>1000
1190
>1000
0
15
41
37
5
75
13
0
>20
>20
12
>20
>20
3.7
>20
>20
>20
>20
Et
CH2CN
Benzyl
Me
CH2COOEt
Me
NH2
25
NHCH2COOEt
NHCH2COOEt
NHCH3
N(CH3)2
210
>1000
440
>1000
2
0
Me
1
Paclitaxel [52]
Combretastatin A-4
ABT-751
2.6
2
388
2.5
1
180
n.d.
3
4.4
n.d.
100
69
1
n.d.: not determined.
2.2. Replacement of CA-4 Olefin by a Sulfonamide Highly Increased Aqueous Solubility
Aqueous solubility of representative compounds was spectrophotometrically mea-
sured in pH 7.0 phosphate buffer (Table 2). UV absorbance at three maximum absorbance
wavelengths of selected compounds was measured after 48 h shaking and subsequent
microfiltration. Replacement of the CA-4 olefinic bridge by a sulfonamide group highly
increased aqueous solubility when compared to paclitaxel and to CA-4 (4- up to 1700-fold
increase). Similar or even better results were obtained when compared with the orally
administered sulfonamide ABT-751 (up to 42-fold increase). The best results were achieved
by the introduction of amino acid derivatives at the Z position (compounds 21 and 22).

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Table 2. Aqueous solubility in µg/mL of representative compounds.
Compound Solub (µg/mL) Compound Solub (µg/mL) Compound Solub (µg/mL)
1a
1b
2a
3
5
6
11
12
15
83
108
27
30
38
33
88
158
230
22
23
26
27
28
29a
29b
30
1690
41
28
25
15
7
4
6
18
37
38
41
42
43
44
45
43
16
89
108
58
46
14
1
CA-4
ABT-751
Paclitaxel
[23–25]
–
31
40
20
21
87
32
36
8
<2
–
357
27
2.3. MDS Inhibit Cell Proliferation in Breast, Cervix, and Ovarian Tumor Cells
The in vitro antiproliferative activity of MDS at 1 M after 72 h treatments was
µ
evaluated by a colorimetric method against the human tumor cell lines HeLa (cervix
epithelioid carcinoma) and MCF7 (breast adenocarcinoma). Compounds showing more
than 40% inhibition at 1
µM in at least three independent assays were evaluated in a
range of concentrations between 0.1 nM and 10
µM for the determination of half-maximal
inhibitory concentrations (IC50) (Table 1). Paclitaxel [52], CA-4, and ABT-751 were used
as references.
HeLa and MCF7 cells showed similar sensitivities to MDS, both cell lines displayed
less than two-fold changes in IC50 values between them in most cases. The most potent
compounds (e.g., 29a, 30, 38, 42, 43, 44, and 45) showed IC50 values in the double-digit
nanomolar range, less potent than paclitaxel and CA-4 but more than ABT-751. Substitu-
tions on the trimethoxyphenyl ring resulted in inactive compounds (11–24). Alkylations of
the sulfonamide improved the potency, with methyl groups yielding the lowest IC50 values
in most cases (e.g., 29a, 30, and 42). Larger substituents gave worse results except for some
benzyl (e.g., 45) and cyanomethyl (e.g., 44) derivatives. Replacement of the 4-methoxy
group on the B ring in series 1 by an amino substituent (series 2) and introduction of an
amino group on position 3 as a primary amine (series 3) gave favorable results. Substituents
with a hydrogen bond donor, such as methylamines (38) in series 2, or primary amines in
series 3 (42–45), resulted in the most potent MDS. Based on antiproliferative results, we
selected 3 out of the 7 most potent compounds (38
,
42, and 45) as representative leaders for
further investigation.
The in vitro antiproliferative activity of compounds 38
,
42, and 45 against a panel of
five human ovarian carcinoma cell lines (SKOV3, IGROV-1, A2780, OVCAR-8, and OVCAR-
3) was evaluated by a colorimetric method (Table 3) and compared with paclitaxel [14].
Paclitaxel and MDS showed a similar comparative behavior against the highly paclitaxel-
sensitive A2780 and OVCAR-8 and the less sensitive IGROV-1 cell line, with paclitaxel
being one order of magnitude more potent than MDS. Concerning OVCAR-3, paclitaxel is
less potent, but MDS maintain their potency range, thus resulting in similar effects of both
types of drugs. For SKOV3, the potency reduction observed for paclitaxel together with
a sustained or improved (42 with a 7 nM IC50 value) potency for MDS results in a more
favorable profile of the MDS compared to paclitaxel.
2.4. Lead Compounds Overcome MDR-Mediated Resistance
The antiproliferative activity of MDS against the CA-4-resistant cell line HT-29 (colon
adenocarcinoma) in the absence or the presence of 10
Pgp/MDR1 inhibitor, was measured 72 h post-treatment by a colorimetric method
Supplementary Table S1). Verapamil itself did not affect cell proliferation at that con-
µM verapamil, a non-selective
(
centration. Detecting changes in drug potency when inhibiting MDR-mediated efflux
reflect that these transporters reduce the intracellular drug concentration. HT-29 has been

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reported to express moderate levels of MDR1, accounting for a resistant phenotype [53].
A comparison of the antiproliferative IC50 values of each MDS in the presence or absence
of verapamil provides an estimate of the susceptibility of each compound to efflux pumps.
Most of the compounds, including leading derivatives 38
mentary Table S1), showed no significant differences (up to two-fold change) between pairs
of IC50 values, thus suggesting that they are not substrates of MDR pumps. Compounds
, 42, and 45 (Table 4 and Supple-
3,
29a, and 30 showed higher potencies against HT-29 cells when combined with verapamil
(comparables to that of obtained against sensitive HeLa an MCF7 cells), thus validating the
assay as positive controls.
Table 3. Antiproliferative activity of the lead compounds and paclitaxel against several human
ovarian tumor cell lines.
Antiproliferative Activity IC50 (nM)
Compound
SKOV3
IGROV-1
A2780
OVCAR-8
OVCAR-3
38
42
45
46
7
48
248
400
492
39
68
42
104
3
74
37
48
6
31
72
58
17
Paclitaxel 1
81
1
IC50 values against A2780, OVCAR-8, and OVCAR-3 cell lines from Bicaku, E. et al. evaluation [14].
Table 4. Antiproliferative activity against the CA-4 resistant cancer cell line HT-29 and sensitivity of
the lead MDS to MDR pumps.
Antiproliferative Activity IC50 (nM)
Compound
HT-29
HT-29 Verapamil 10 µM
38
42
45
59
81
300
305
50
79
276
327
CA-4
2.5. Lead Compounds Induce G2/M Arrest in Breast, Ovarian and Cervix Tumor Cells
The effect of the lead MDS on the cell cycle of MCF7, SKOV3, and HeLa cells was
analyzed in time-course experiments at 24, 48, and 72 h post-treatment by DNA staining
and flow cytometry quantification. The working concentration for each compound (600 nM
for 38, 50 nM for 42, and 400 nM for 45) was established at the complete inhibition of cell
proliferation in view of individually assays run in parallel for each cell line.
For breast adenocarcinoma MCF7 cells (Figure 3a) a pronounced and sustained G2/M
arrest was observed for all the treatments from 24 h (70–80%) to 72 h (60–76%), compared
to untreated MCF7 cells, which maintained a 32–40% population at G2/M throughout
the time course. A moderate increase of the percentage of cells at the SubG0/G1 region
was observed, which slightly increased over time (from 4–6% at 24 h to 6–12% at 72 h)
accompanied by the decrease in the G2/M population.
Compounds 38, 42, and 45 (Figure 3b) arrested ovarian carcinoma SKOV3 cells at the
G2/M phase 24 h post-treatment (44%) compared to untreated cells (30%). The percentage
of SubG0/G1 treated SKOV3 cells was already elevated at 24 h (20% to 22%) compared to
untreated controls (always <14%). Treatments with compounds 38 and 42 increased the
percentage of SubG0/G1 modestly at 48 h (35% and 34%, respectively), and notably at 72 h
(43% and 53%, respectively). This increase presumably occurred at the expense of cells
arrested at the G2/M phase. The effect of compound 45 was notably higher, with 46% of
cells at SubG0/G1 region 48 h post-treatment and 63% at 72 h.
Treatment of HeLa cells with 38 and 45 (Figure 3c) caused a significant arrest at the
G2/M phase after 24 h (80%) and a more modest one for compound 42 (44%) accompanied
by a limited increase in the percentage of cells of the SubG0/G1 population (9–16% vs. <4%
in untreated controls). At subsequent time points, around 50% of the cells for all three

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treatments were allocated to the SubG0/G1 region of the cell cycle histogram at the expense
of the G2/M phase.
Figure 3. Cell cycle analysis. (
distribution. Cells arrested in G2/M phase experienced time-dependent cell death (SubG0/G1 region). (
a
) MCF7 cell cycle distribution. Cells were arrested in G2/M phase. (
b
) SKOV3 cell cycle
) HeLa cell cycle
c
distribution. Cells were arrested in the G2/M phase followed by cell death (SubG0/G1 region). Cells were incubated with
the lead compounds at 600 nM (38), 50 nM (42), or 400 nM (45) for 24, 48, and 72 h, stained with propidium iodide (PI) and
their DNA content was analyzed by flow cytometry. Untreated samples were analyzed in parallel. The different cell cycle
populations were quantified, expressed in percentages, and represented in bar charts. Data shown are representative of
three independent experiments.

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2.6. Lead Compounds Trigger Apoptotic Cell Death
To determine whether the cytotoxic/antiproliferative effect of the MDS was due to
apoptosis induction, cells were treated with variable concentrations of the drug for 72 h or
6 days, stained with Annexin V-FITC (AnV) and propidium iodide (PI), and analyzed by
flow cytometry. Double negative cells (AnV−/PI−) were classified as live cells (L), cells
−
positive for AnV and negative for PI (AnV+/PI ) were considered in Early Apoptosis (EA),
cells negative for AnV and positive for PI (AnV−/PI+) were considered necrotic (N), and
double-positive cells (AnV+/PI+) as Late Apoptosis (LA). In all cases, the compounds were
used at the same concentrations previously used in cell cycle studies (Table 5).
Table 5. Cell death quantification in Annexin V-FITC/PI double staining experiments. MCF7, SKOV3, and HeLa cells were
incubated with the lead compounds for 72 h and/or 6 days, stained with Annexin V-FITC (AnV) and PI, and analyzed by
flow cytometry. The results are expressed in percentage as the average of three independent experiments. Untreated samples
were analyzed in parallel. Cells were classified into double-negatives (live cells), AnV-positive cells (early apoptosis),
double-positives (late apoptosis), and PI-positive cells (necrosis).
Annexin V-FITC/PI 72 h (%)
Annexin V-FITC/PI 6 Days (%)
Compound
Live
EA 1
LA 2
Necrosis
Live
EA
LA
Necrosis
38 (600 nM)
42 (50 nM)
45 (400 nM)
CA-4 (50 nM)
Control
60.5
87.7
70.5
58.3
93.6
29.0
8.5
20.1
31.7
1.8
5.4
2.3
7.5
3.3
2.9
5.1
1.4
1.9
6.7
1.7
52.0
79.1
56.7
44.5
97.4
45.0
18.9
39.3
52.4
0.2
2.7
1.7
3.8
2.7
0.8
0.3
0.3
0.3
0.4
1.6
MCF7
38 (600 nM)
42 (50 nM)
45 (400 nM)
Paclitaxel (10 nM)
Control
62.3
85.1
72.7
90.7
99.2
30.5
12.2
23.4
3.8
5.4
2.0
2.6
3.1
0.3
1.8
0.7
1.3
2.4
0.1
SKOV3
HeLa
0.5
38 (600 nM)
42 (50 nM)
45 (400 nM)
Control
0.6
7.3
0.8
1.1
1.4
1.5
1.7
87.6
82.0
84.9
5.5
10.6
9.4
12.7
2.4
90.5
1
Early Apoptosis (EA). 2 Late Apoptosis (LA).
Breast cancer MCF7 cells treated with compound 42 for 72 h were mostly classified
as live cells (88%), whereas lower percentages of live cells were found when treated with
compounds 38 (60%), 45 (71%) and the reference compound CA-4 (58%). This lower
percentage of live cells was accompanied by a higher proportion of EA cells for 38 (29%),
45 (20%), and CA-4 (32%) compared to the treatment with 42 (9%). Longer treatment
exposures of 6 days confirmed this trend, with 38, 45, and CA-4 showing significantly
lower percentages of live cells (45–57%) and higher EA populations (39–52%) than 42
,
which showed live-cell percentages of 79% and EA of 19%.
Similar results were found 72 h post-treatment for SKOV3 ovarian tumor cells for
MCF7 cells. SKOV3 cells treated with compounds 38 and 45 showed modest percentages
of EA cells (31% and 23%, respectively) whereas a lower apoptotic population was found
for compound 55 (12%). These percentages of apoptotic cells for compounds 38 and 55
were accompanied by low percentages of live cells (62% and 73%, respectively), with 10 nM
paclitaxel showing high percentages of live cells (91%).

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Treatment of the cervical cancer HeLa cells with compounds 38
,
42, and 45 resulted in
a severe reduction of live cells down to less than 7% and an accumulation of LA (82–88%)
and necrotic (9–13%) cells after 72 h.
2.7. Lead Compounds Disrupt the Microtubule Network and Inhibit Tubulin Polymerization
The effect of MDS 38 and 45 on the tubulin cytoskeleton was analyzed in MCF7,
SKOV3, and HeLa cells after 72-h treatments. The microtubule network was visualized
by confocal microscopy staining α-tubulin in green, and cell nuclei were stained in blue
with DAPI (4’,6-diamidino-2-phenylindole) (Figure 4). Compounds 38 and 45 disrupted
the microtubule system of MCF7, SKOV3, and HeLa cells (Figure 4a), which loses the
hairy appearance of untreated cells and shows up as a diffuse gauze. Treated cells showed
multilobulated nuclei resembling bunches of grapes (Figure 4b).
The effect of all the synthesized compounds on the in vitro tubulin polymerization
was evaluated at 10
were also assayed at 20
tubulin polymerization by at least 50% at 20
Most antiproliferative N-alkylated sulfonamides interfere to a greater or lesser extent with
tubulin polymerization, with the highest effect shown by compound 45 (IC50 = 3.7 M),
with a benzyl group on the sulfonamide nitrogen, close to CA-4 (IC50 = 3 M) and better
than ABT-751 (IC50 = 4.4 M). Compounds 38 and 42 showed moderate to good IC50 values
of 10 and 12 µM, respectively.
µ
M. All the compounds that inhibited polymerization more than 30%
M. IC50 values were calculated for those compounds inhibiting
M (Table 1 and Supplemental Figure S2).
µ
µ
µ
µ
µ
The binding mode of the active compounds at the colchicine site of tubulin was studied
by flexible docking studies. The protein conformational variability was represented by
using 55 protein structures with different binding sites, as previously described [54,55].
Two frequently used docking programs with different scoring functions were used for the
flexible docking of the ligands in the binding sites. Several thousand poses were generated
for each ligand which were automatically classified according to their occupation of the
subsites of the colchicine domain, and the poses with the best consensus scores were
selected (Figure 5). The selected ligands bind in similar dispositions to combretastatin A-4,
with a good overlap of the trimethoxyphenyl ring, which is inserted edgewise towards
the surface of sheets S8 and S9 between the sidechains of Ala316
and covered by helices H7 and H8 and the H7-H8 loop and interacting with Cys241
Leu242 , Leu248 , Ala250 , and Leu255
to the olefin binding zone of CA-4, with the other aromatic ring behind helix H8 and
above the sidechains of Ala316 and the methylenes of Lys352 , with the substituents
β, Val318
β
, Ala354
β,
β,
β
β
β
β. The sulfonamide bridges are placed close
β
β
on the sulfonamide nitrogen projecting towards a hydrophobic region of the interdimer
interface, thus explaining the preference for small hydrophobic substituents such as methyl
or cyanomethyl groups.

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(a)
MCF7
SKOV3
HeLa
Control
38
(600 nM)
45
(400 nM)
(b)
MCF7
Control
38 (600 nM)
38 (600 nM)
45 (400 nM)
45 (400 nM)
Figure 4. Confocal microscopy experiments in MCF7, SKOV3, and HeLa cells. Cells were incubated in the absence (Control)
or the presence of 38 (600 nM) and 45 (400 nM) for 72 h. ( ) Visualization of the microtubule network. -Tubulin was stained
in green, and nuclei were stained with DAPI (blue fluorescence). ( ) Multilobulated nuclei (blue) resembling bunches of
grapes in MCF7 cells. Photomicrographs are representative of three independent experiments. Scale bar: 25 µm.
a
α
b

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Figure 5. Docking poses for compounds 38, 42, and 45 at the colchicine site of tubulin. Combretastatin A-4 (CA-4) is shown
in diffuse green as a reference.
3. Discussion
Taxanes—usually paclitaxel alone or in combination regimens—entail the first-line
chemotherapy for breast and gynecological cancers [ 13 18 20]. However, limitations
9
, , ,
related to toxicity, pharmacokinetic problems, and the appearance of resistance [31] prompt
the search for new treatments. This work has yielded a new family of tubulin inhibitors with
a sulfonamide scaffold as hybrids of combretastatin A-4 and ABT-751, two colchicine site
ligands in clinical trials that present shortcomings of opposite nature whose hybridization
might alleviate (Figure 1).
Therefore, a focused library of sulfonamides was designed, and 52 representatives
were synthesized following a synthetic methodology that involves first the assembly of
the sulfonamide followed by functional group manipulations at different points of the
main scaffold. A breadth of substituents was thus introduced to explore the chemical
space for optimal pharmacokinetic and pharmacodynamic properties (Figure 1). One of the
main drawbacks of taxanes and CA-4 is their low water solubility, making necessary drug
administration as formulations with several associated adverse effects [27,28,47]. New
sulfonamide derivatives show moderate to good solubilities (Table 2). Improvements up to
1700 times in solubility were found when compared with reference drugs paclitaxel and
CA-4 and even surpass the solubility of the orally administered ABT-751 up to 42 times.
The antiproliferative activity of the synthesized compounds was evaluated against
two Pan-Gyn representative human tumor cell lines (HeLa and MCF7) (Table 1). Many
compounds showed growth inhibitory activities at submicromolar concentrations, thus
validating the adequacy of the selected diarylsulfonamide scaffold. Most potent derivatives
inhibited cell proliferation at very low drug concentrations, with IC50 values in the low to
medium nanomolar range. Compounds 2a, 29a, 30, 32, 38, and 42–45 display antiprolif-
erative IC50 values comparable to those of paclitaxel and CA-4 and better than ABT-751,
a tubulin colchicine-site inhibiting sulfonamide in clinical trials used as a reference [49].
Alkylation of the sulfonamide nitrogen significantly increases potency compared to the
unsubstituted sulfonamides, probably due to a combination of a favoring of the cisoid
disposition of both aromatic rings, an essential requirement for colchicine-site-binding
drugs, with a more favorable interaction with the target [56]. Small alkyl groups, such as
methyl or ethyl (e.g., 2a, 29a, 38, or 42), are usually preferred over longer chains such as
carboxylic acid derivatives. All the modifications attempted on the trimethoxyphenyl ring
abolished the activity, whereas the introduction of hydrogen bond donor amines on the B
ring translated into more potent analogs, such as the secondary amines in the para position
of the sulfonamide group (38) and the primary amines ortho to the methoxy group of B
ring (42–45). Both modifications improve the polarity and aqueous solubility. The potency
improvement suggests favorable hydrogen bonding with the target. Thus, B-ring amines
improve both the pharmacokinetics and the pharmacodynamics.
The antiproliferative IC50 values of each compound against HeLa and MFC7 cell lines
were quite similar, whereas the CA-4-resistant HT-29 cell line was less or at best equally
sensitive to MDS (Supplementary Table S1). When differences in antiproliferative potencies

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occur, they are not due to multidrug-resistance by the described moderate expression of
MDR1 or MDR3 proteins in HT-29 colon carcinoma cell line [48
compounds experienced a potency increase in the presence of the Ppg/MDR1 inhibitor,
verapamil [58]. Therefore, none of our lead compounds (38 42, and 45) seemed to be MDR
,53,57], as only a handful of
,
substrates and even compounds 38 and 42 overcame the resistance shown by CA-4 in the
HT-29 cell line (IC50 = 59, 81, and 305 nM, respectively) (Table 4).
The microtubule disruption caused by the lead compounds in MCF7 cells winded
up in the accumulation of cells in the G2/M region (Figure 3a), as previously reported for
CA-4 and ABT-751, used as references [59,60]. This sustained mitotic arrest lasted at least
72 h with percentages of cells showing DNA fragmentation below 12%. The mild response
regarding this apoptotic marker is in line with the results observed in AnV/PI staining
experiments (Table 5). Compounds 38 and 45 triggered phosphatidylserine exposure in
28–34% of MCF7 cells; 11% in the case of 42. The lack of PI staining within the apoptotic
population reveals the integrity of the plasma membrane in most Annexin V-positive cells.
This slight apoptotic response might be related to the expression levels of caspase 3, the
hub effector of the apoptotic machinery. MCF7 cells have been reported to be deficient in
caspase 3 [61,62]. Despite MCF7 cells suffer a weak apoptotic response upon treatment with
the lead compounds for 72 h, the percentage of early apoptotic cells increases after 6-day
treatments, still preserving the integrity of the plasma membrane. This slow build-up of
the apoptotic response in MCF7 cells may putatively rely on the other two effector caspases
6 and 7, as described for DNA damaging agents [11,63,64].
Lead compounds 38, 42, and 45 were then assayed against a panel of human ovar-
ian tumor cell lines. Ovarian serous adenocarcinoma cell lines (SKOV3, OVCAR-8, and
OVCAR-3), the most common type of ovarian cancer, were more sensitive than endometri-
oid cell lines (IGROV-1 and A2780). The metastatic cell line SKOV3 was found quite
resistant to paclitaxel and CA-4 (IC50 of 81 and 380 nM [65], respectively) but was sensitive
to MDS (Table 3). Time-course analysis of the cell cycle distribution (Figure 3b) showed a
strong mitotic arrest for 42, 38, and 45 24 h after treatment. The mitotic arrest readily trig-
gered apoptosis 48 h after treatment. Compound 45 elicits a higher SubG0/G1 population
than 38 and 42 at 72 h. Dual staining experiments confirm the apoptotic response with
Annexin V-positive cells at 72 h ranging from 14–36%, notably higher when compared with
paclitaxel (7%).
Studies on the mechanism of action of MDS on the cervical cancer cell line HeLa were
also performed (Figure 3c). After the common mitotic arrest at G2/M phase 24 h after
treatment (more pronounced for 38 and 45 than for 42) HeLa cells exhibited strikingly
high percentages of SubG0/G1 cells (around 50%) from 48 h after drug exposure. AnV/PI
staining experiments revealed a strong and advanced apoptotic response with rates of
double-positives cells above 82%, indicating the permeabilization of the plasma membrane.
At this point, we wished to verify if the observed effect was due to the disruption of mi-
crotubule assembly. Tubulin immunofluorescence analysis of cells treated with compounds
38 and 45 revealed a clear disruption in the microtubule network (Figure 4a). Furthermore,
as interference with microtubule dynamics is expected to affect chromatin organization,
cell nuclei were stained with DAPI. Accumulation of multiple micronuclei characteristic of
mitotic catastrophe was observed, widely linked to tubulin-binding drugs [66,67]. In vitro
tubulin polymerization inhibition experiments confirmed the effect of the compounds on
tubulin, and molecular docking experiments suggest a similar binding mode to that of
CA-4 to the colchicine site of tubulin (Figure 5). However, no correlation was observed be-
tween TPI and antiproliferative IC50 values, as has previously been found for many tubulin
polymerization inhibitors [68]. This discrepancy can be explained by an antiproliferative
effect dependent on the alteration of microtubule dynamics and not on polymer mass, as
measured in TPI assays.
In conclusion, a new family of MDS has been designed and synthesized. MDS an-
tiproliferative effects against several representative cell lines of Pan-Gyn cancers have
been studied. The effects of the compounds vary depending on the structural modifi-

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cations, the most potent derivatives being those with a small alkyl substituent on the
sulfonamide nitrogen. The molecular mechanism of action studies revealed a pronounced
G2/M arrest mainly followed by a SubG0/G1 increase over time. The different behavior
between MCF7, SKOV3 and HeLa cells seems to be related to the rate at which each cell
line builds up its apoptotic response to the initial unsolved mitotic arrest. Inhibition of
tubulin polymerization in vitro was observed for most potent MDS, as well as disruption
of microtubule network by immunofluorescence, probably by binding similarly to CA-4
to the colchicine site of tubulin. The improved aqueous solubility, lack of Pgp-mediated
resistance, and antiproliferative potencies comparable to paclitaxel—first-line treatment of
Pan-Gyn cancers—make MDS interesting for future application as antitumor agents.
4. Materials and Methods
4.1. Chemistry
4.1.1. Chemical Synthesis
Detailed synthetic procedures and characterization of compounds can be found in
Supplemental methods SP1 and SP2.
4.1.2. Chemical Characterization of Lead MDS 38, 42, and 45
MDS 38. M.p.: 147–150 ◦C (CH2Cl2/Hexane). IR (KBr): 3390, 1599, 1502, 835 cm−1
1H NMR (400 MHz, CDCl3):
2.83 (3H, s), 3.07 (3H, s), 3.71 (6H, s), 3.80 (3H, s), 6.28 (2H,
s), 6.51 (2H, d, J = 8.8), 7.35 (2H, d, J = 8.8). 13C NMR (100 MHz, CDCl3):
30.0 (CH3),
.
δ
δ
38.4 (CH3), 56.1 (2CH3), 60.8 (CH3), 104.6 (2CH), 110.8 (2CH), 122.6 (C), 130.0 (2CH), 137.2
(C), 137.8 (C), 152.7 (C), 152.8 (2C). HRMS (C17H22N2O5S + H+): calcd 367.1322 (M + H+),
found 367.1325.
MDS 42. M.p.: 124–126 ◦C (MeOH). 1H NMR (400 MHz, CDCl3):
δ
3.11 (3H, s), 3.73
(6H, s), 3.83 (3H, s), 3.90 (3H, s), 3.94 (2H, s), 6.30 (2H, s), 6.78 (1H, d, J = 8.8), 6.90 (1H, d, J
= 2.4), 6.99 (1H, dd, J = 8.8 and 2.4). 13C NMR (50 MHz, CDCl3):
38.5 (CH3), 55.7 (CH3),
δ
56.0 (2CH3), 60.8 (CH3), 104.6 (2CH), 109.1 (CH), 113.2 (CH), 118.8 (CH), 128.0 (C), 136.5
(C), 137.3 (C), 137.7 (C), 150.2 (C), 152.8 (2C). HRMS (C17H22N2O6S + H+): calcd 383.1271
(M + H+), found 383.1263.
MDS 45. 1H NMR (200 MHz, CDCl3):
δ
3.63 (6H, s), 3.78 (3H, s), 3.93 (3H, s), 4.63 (2H,
s), 6.14 (2H, s), 6.83 (1H, d, J = 8.4), 7.02 (1H, d, J = 2), 7.11 (1H, dd, J = 8.4 and 2), 7.23 (5H,
bs). 13C NMR (100 MHz, CDCl3):
55.3 (CH2), 55.8 (CH3), 55.9 (2CH3), 60.8 (CH3), 106.7
δ
(2CH), 109.3 (CH), 113.1 (CH), 118.8 (CH), 127.6 (CH), 128.3 (2CH), 128.7 (2CH), 130.5 (C),
134.9 (C), 136.2 (C), 136.6 (C), 137.6 (C), 150.3 (C), 152.7 (2C). HRMS (C23H26N2O6S + H+):
calcd 459.1584 (M + H+), found 459.1589.
4.1.3. Aqueous Solubility
The aqueous solubility of the sulfonamides was determined using an approach based
on the saturation shake-flask method. Tested compounds (1–2 mg) were stirred in pH
7.0 phosphate buffer (300
was filtered over a 45 m filter to discard insoluble residues, and the concentration in the
supernatant was measured by UV absorbance. To determine the concentration, a scan
between 270 and 400 nm was performed in a Helios- UV-320 Spectrophotometer (Thermo-
µL) for 48 h at room temperature. The resulting suspension
µ
α
Spectronic, Thermo Fischer Scientific, Waltham, MA, USA) for each tested compound.
Then, three maximum wavelengths of absorbance per compound were selected from the
previous scan and calibration curves were performed at these three wavelengths. Solubility
results are given as the average of the three measurements.
4.2. Biological Evaluation
4.2.1. Cell Lines and Cell Culture Conditions
MCF7 (human breast carcinoma), SKOV-3 (human ovarian carcinoma), OVCAR-3
(human ovarian carcinoma), OVCAR-8 (human ovarian carcinoma), and HeLa (human cer-
vical carcinoma) cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)

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(Gibco, Thermo Fischer Scientific) containing 10% (v/v) heat-inactivated fetal bovine serum
(HIFBS) (Lonza-Cambrex, Karlskoga, Sweden), 2 mM L-glutamine (Lonza-Cambrex, Karl-
skoga, Sweden), 100 µg/mL streptomycin, and 100 IU/mL penicillin (Lonza-Cambrex)
at 37 ◦C in humidified 95% air and 5% CO2. HT-29 (human colon carcinoma), IGROV-1
(human ovarian carcinoma), and A2780 (human ovarian carcinoma) cell lines were cultured
in RPMI 1640 medium (Gibco, Thermo Fischer Scientific)◦supplemented with 10% HIFBS,
100 µg/mL streptomycin, and 100 IU/mL penicillin at 37 C in humidified 95% air and 5%
CO2 atmosphere. The presence of mycoplasma was routinely checked with MycoAlert kit
(Lonza-Cambrex) and only mycoplasma-free cells were used in the experiments. Ovarian
tumor cell lines were originally acquired from Dr Atanasio Pandiella from Centro de Inves-
tigación del Cáncer (CIC), Salamanca, Spain. HeLa, HT-29 and MCF7 tumor cell lines were
provided by Dr Faustino Mollinedo from Centro de Investigaciones Biológicas Margarita
Salas, Madrid, Spain.
4.2.2. Cell Proliferation Assay
MCF7, HeLa, and HT-29 cell proliferation, when treated with the corresponding
compounds, were determined using the XTT (sodium 3’-[1(phenylaminocarbonyl)-3,4-
tetrazolium]-bis(4-methoxy-6-nitro)-benzenesulfonic acid hydrate) cell proliferation kit
(Roche Molecular Biochemicals, Mannheim, Germany). A freshly prepared mixture of
XTT labeling reagent with 0.02% (v/v) PMS (N-methyl-dibenzopyrazine methyl sulfate)
electron coupling reagent was added to cells (50
µL/well in 96-well plates, total volume of
160 L/well). Cells were incubated under standard culture conditions for 4 h (MCF7 and
µ
HeLa) or 6 h (HT-29). The absorbance of the formazan product generated was measured
at 450 nm using a multi-well plate reader. SKOV-3, OVCAR-3, OVCAR-8, IGROV-1, A-
2780 cell proliferation, when treated with the corresponding compound, were determined
using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) (Sigma-
Aldrich, St. Louis, MO, USA). MTT in Phosphate Buffer Saline (PBS) (5 mg/ml) was added
to cells (110
incubation, the medium was aspirated, and formazan violet crystals were dissolved in
dimethyl sulfoxide (DMSO) (500 L/well). Absorbance was measured at 570 nm in a plate
reader (Ultra Evolution, Tecan, Männedorf, Suiza). To determine cell viability, cells in
exponential growth phase were seeded (100 L/well in 96-well plates and 1000 L/well in
24-well plates) with appropriate cell line concentration (1.5
104 cells/mL for MCF7 and
µL/well in 24-well plates, total volume of 1110 µL/well). After 1 hour of
µ
µ
µ
·
HeLa, 3
·
104 cells/mL for HT-29, and 1 104 cells/mL for SKOV-3, OVCAR-3, OVCAR-8,
·
IGROV-1, and A-2780) in complete RPMI 1640 or DMEM medium at 37◦C and 5% CO2
atmosphere. After 24 h incubation, to allow cells to attach to the plates, all compounds
were added at 1 µM concentration and the effect on the proliferation was evaluated 72 h
post-treatment. Compounds showing antiproliferative effects at tested concentration were
selected for IC50 calculation (50% inhibitory concentration with respect to the untreated
controls) from 10−5 to 10−10 M concentration. Non-linear curves fitting the experimental
data were carried out for each compound. Compounds were dissolved in DMSO and the
final solvent concentrations never exceeded 0.5% (v/v). The control wells included treated
cells with 0.5% (v/v) DMSO and the positive control. 10 µM verapamil was included as
a control for the HT-29 cell line. Measurements were performed in triplicate, and each
experiment was repeated three times.
4.2.3. Cell Cycle Analysis
Cell cycle analysis was performed in MCF7, SKOV-3, and HeLa cells by quantifying the
DNA content by flow cytometry. Cells in the exponential growth phase at 2
104 cells/mL
·
were seeded in 24-well plates (1 mL/well). After 24 h incubation, cells were treated with
different concentrations of the selected compounds and the effect of treated and untreated
cells was measured 24, 48, and 72 h post-treatment. Live and dead cells were collected and
fixed in ice-cold ethanol/PBS (7:3) and stored at 4 ◦C for later use. Cells were rehydrated
with PBS, treated with 100 µg/mL RNase A (Sigma-Aldrich Co., St. Louis, MO, USA) and

Int. J. Mol. Sci. 2021, 22, 1907
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stained overnight in darkness at room temperature with 50
µg/mL PI (Sigma-Aldrich Co.).
Cell cycle profiles were then analyzed by flow cytometry using BD Accuri
™ C6 Plus Flow
Cytometer (BD Biosciences, San José, CA, USA). Data were analyzed with BD Accuri
™
C6 Software (version 1.0.264.21, BD Biosciences, San José, CA, USA) and compared to
control cells. Compounds were dissolved in DMSO and the final solvent concentration
never exceeded 0.5% (v/v). Control wells included cells with 0.5% (v/v) DMSO.
4.2.4. Apoptotic Cell Death Quantification
MCF7, SKOV-3, and HeLa apoptotic cells were quantified using an Annexin V-FITC/PI
apoptosis detection kit (Immunostep, Salamanca, Spain) according to the manufacturer’s
guidelines. 1.5 mL/well of cells in the exponential growth phase at 2
104 cells/mL were
·
seeded onto 12-well plates and left to attach overnight. After 24 h incubation, cells were
treated with selected compounds. After 72 h (MCF7, SKOV-3, and HeLa) and 6 days
(MCF7) post-treatment, attached and floating cells of treated and untreated wells were
collected, centrifuged, resuspended in Annexin V binding buffer, and stained with Annexin
V-FITC/PI. Cells were then incubated in darkness at room temperature for 15 min and a
total of 30,000 cells were acquired and analyzed using the BD Accuri
™ C6 Plus Flow Cy-
tometer and Software (version 1.0.264.21, BD Biosciences, San José, CA, USA), respectively.
Compounds were dissolved in DMSO and the final solvent concentration never exceeded
0.5% (v/v). Control wells included cells with 0.5% (v/v) DMSO.
4.2.5. Immunofluorescence
MCF7, SKOV-3, and HeLa cells were grown on glass coverslips coated with poly-L-
lysine (12 mm diameter). To reach appropriate cell confluence, coverslips were manipulated
in 6-well plates (3 coverslips/well) seeding 3 mL/well of cells in the exponential growth
phase at 2
×
104 cells/mL. After 24 h incubation, cells were treated with selected com-
pounds and incubated for 72 h. Cells were washed with PBS, fixed with 4% formaldehyde
in PBS for 10 min, permeabilized with 0.5% Triton X-100 (Boehringer Mannheim, Ingelheim
am Rhein, Germany) in PBS for 10 min and blocked with 10% BSA in PBS for 30 min. Mi-
crotubule detection was performed by incubation with anti-α-tubulin mouse monoclonal
antibody (1:200 in 3% BSA/PBS) (Sigma-Aldrich) for 1.5 h. After PBS washing, coverslips
were incubated with fluorescent secondary antibody Alexa Fluor 488 goat anti-mouse
IgG (1:400 in 1% BSA/PBS) (Molecular Probes, Invitrogen, Thermo Fischer Scientific) for
1 h in darkness. After being washed with PBS, cell nuclei were stained with DAPI (di-
hydrochloride of 40,6-diamidino-2-phenylindole) (diluted 1:10,000 in mq H2O) (Roche,
Basel, Switzerland) for 5 min in darkness. DAPI excess was removed by washing with PBS.
Mowiol reagent (Calbiochem, Sigma-Aldrich) was used to mount preparations on slides.
Cells were analyzed by confocal microscopy using a LEICA SP5 microscope DMI-6000V
model coupled to a LEICA LAS AF software computer.
4.2.6. Tubulin Isolation
Microtubular protein was isolated from calf brain according to the modified Shelan-
ski method [69
,70] by two cycles of temperature-dependent assembly/disassembly and
stored at
−
80 ◦C. Before each use, protein concentration was determined by the Bradford
method [71] taking BSA as standard.
4.2.7. Tubulin Polymerization Inhibition (TPI) Assay
Tubulin polymerization was monitored using a Helios
suring the increase in turbidity at 450 nm, caused by a shift from 4 C to 37 C, which
allows the in vitro microtubular protein to depolymerize and polymerize, respectively. The
assay was carried out in quartz cuvettes containing 1.5 mg/mL microtubular protein and
the ligand (except for control cuvette with only DMSO at the same concentration) in a
α
spectrophotomet◦er by mea-
◦
mixture of 0.1 M MES buffer, 1 mM EGTA, 1 mM MgCl2, 1 mM
β-ME, and 1.5 mM GTP
at pH 6.7 (final volume 500
µ
L). Cuvettes were preincubated at 20 ◦C for 30 min, to allow

Int. J. Mol. Sci. 2021, 22, 1907
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ligand binding to tubulin, and subsequently cooled on ice for 10 min. Then, the experiment
◦
starts at 4 C to establish the initial baseline. The assembly process was initiated by a
temperature shift to 37 ◦C and the turbidity produced by tubulin polymerization can be
measured by an absorbance increase. After reaching a stable plateau, the temperature was
switched back to 4 ◦C to return to the initial absorption values (to confirm the reversible
nature of the monitored process and to determine whether or not the microtubular tubular
protein has stabilized). The difference in amplitude between the stable plateau and the
initial baseline of the curves was taken as the degree of tubulin polymerization for each ex-
periment. Comparison with control curves under identical conditions but without ligands
yielded TPI as a percentage value. All compounds were tested at 10
Compounds that inhibited tubulin polymerization by at least 30% at 10
at 20 M. IC50 values were calculated for compounds that inhibit tubulin polymerization
by more than 50% at 20 M. Compounds were dissolved in DMSO, and the final solvent
µM concentration.
µM were tested
µ
µ
concentration never exceeded 4% (v/v), which has been reported not to interfere with the
assembly process. All the measurements were carried out in at least two independent
experiments using microtubular protein from different preparations.
4.3. Computational Studies
Docking studies were carried out as previously described [45]. Briefly, dockings
were performed in parallel with PLANTS [72] with default settings and 10 runs per lig-
and and with AutoDock 4.2 [73] runs applying the Lamarckian genetic algorithm (LGA)
100−300 times for a maximum of 2.5
×
106 energy evaluations, 150 individuals, and
27,000 generations maximum. The retrieved poses were automatically assigned to the
colchicine subzones using in-house KNIME pipelines [74]. The programs’ docking scores
were converted into Z-scores and the poses with best consensus scores were selected
as the docking results. Docked poses were analyzed with Chimera [75], Marvin [76],
OpenEye [77], and JADOPPT [78].
Supplementary Materials: The following are available online at https://www.mdpi.com/1422-006
7/22/4/1907/s1, Figure S1: Synthetic procedures of new Microtubule Destabilizing Sulfonamides,
Table S1: Antiproliferative activity against HT-29 and sensitivity to MDR pumps, Figure S2: Tubulin
Polymerization Assay data. IC50 calculation, Methods SP1: Chemistry. General chemical techniques
1
and chemical synthesis, Methods SP2: H NMR and 13C NMR spectra
Author Contributions: Conceptualization, M.G. and R.P.; Data curation, M.G. and R.P.; Formal
analysis, M.G., M.O.-S., A.V.-B. and A.B.H.; Funding acquisition, R.G.-S. and R.P. Investigation, R.Á.,
M.M., R.G.-S. and R.P.; Methodology, M.G. and M.O.-S.; Project administration, R.G.-S. and R.P.;
Resources, R.G.-S. and R.P.; Software, R.P.; Supervision, R.G.-S. and R.P.; Validation, M.M., R.G.-S.
and R.P.; Visualization, M.G. and M.O.-S.; Writing—original draft, M.G.; Writing—review and editing,
A.V.-B., A.B.H., M.M., R.G.-S. and R.P.; All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Consejería de Educación de la Junta de Castilla y León
(SA030U16, SA262P18 and SA116P20), co-funded by the EU’s European Regional Development
Fund-FEDER, the Spanish Ministry of Science, Innovation and Universities (RTI2018-099474-BI00)
and the health research program of the Instituto de Salud Carlos III (Spanish Ministry of Economy
and Competitiveness, PI16/01920 and PI20/01569) co-funded with FEDER founds.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data that support the findings of this study are available from the
corresponding author upon reasonable request.
Acknowledgments: We thank the people at Frigoríficos Salamanca S.A slaughterhouse for providing
us with the calf brains, “Servicio General de NMR” and “Servicio General de Espectrofotometría de
Masas” of the University of Salamanca for equipment. M.G. acknowledges a predoctoral fellowship
from the Junta de Castilla y León (ORDEN EDU/529/2017 de 26 de junio). M.O.-S. acknowledges

Int. J. Mol. Sci. 2021, 22, 1907
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a predoctoral fellowship from the IBSAL (IBpredoc17/00010). A.V.-B. acknowledges a predoctoral
fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (FPU15/02457).
Conflicts of Interest: The authors declare no conflict of interest.
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