Combretastatin A-4: The Antitubulin Agent that Inspired the Design and Synthesis of Styrene and Spiroisatin Hybrids as Promising Cytotoxic, Antifungal and Antiviral Compounds

Article abstract of DOI:10.21577/0103-5053.20190265

The design of a series of styrene and spiroisatin hybrids was based on the structure of combretastatin A-4 1. This library of 20 compounds were synthesized with the pharmacophoric units: 3,4,5-trimethoxy or/and 4-hydroxy-3-methoxy phenyl moities in their structure. Thereby, the libraries of β-nitrostyrenes 10a-10c, spiroisatin-dihydroquinolines 14a-14c, spiroisatinthiazolidinones 17a-17c and spiroisatin-nitropyrrolizidines 20a-20k were evaluated for their in vitro cytotoxic, anti-proliferative, antifungal and antiviral activities. Biological results revealed that among these compounds, β-nitrostyrenes 10a-10c exhibited significant cytotoxicity (HeLa and Jurkat tumor cells) and antifungal (T. mentagrophytes) activities. Moreover, the spiroisatin-dihydroquinoline 14a and 14c showed promising cytotoxicity (U937 cells). 14a-14c molecules were active against human herpesviruses serotypes 1 and 2 (HHV-1 and HHV-2), but only 14a and 14b were effective against dengue virus serotype 2 (DENV-2). The spiroisatin-nitropyrrolizidine 20c exhibited moderate anti-herpetic activity, while 17c spiroisatin-thiazolidinone derivative also reduced the infection of HHV-1 and DENV-2. Finally, the molecular docking showed that these kind of molecules interact with the subunit α/β-tubulin.

Full text of DOI:10.21577/0103-5053.20190265

http://dx.doi.org/10.21577/0103-5053.20190265
J. Braz. Chem. Soc., Vol. 00, No. 00, 1-12, 2019
Article
Printed in Brazil - ©2019 Sociedade Brasileira de Química
Combretastatin A-4: The Antitubulin Agent that Inspired the Design and
Synthesis of Styrene and Spiroisatin Hybrids as Promising Cytotoxic,
Antifungal and Antiviral Compounds
,
a
b
b,c
Yaneth M. Brand, * Vladimir V. Kouznetsov, Carlos E. Puerto,
a
a
a
Vicky C. R. Linares, Verónica T. Castaño and Liliana Betancur-Galvis
a
Group of Investigative Dermatology, Institute of Medicinal Research, Medicine Faculty,
University of Antioquia, 1226, 050022 Medellín, Colombia
b
Laboratorio de Química Orgánica y Biomolecular, Centro de Materiales y Nanociencias (CMN),
Parque Tecnológico Guatiguará, Universidad Industrial de Santander, km 2 vía Refugio,
A.A. 681011 Piedecuesta, Colombia
c
Laboratorio de Química Orgánica Aplicada, Universidad Manuela Beltrán,
Cl. 33 No. 26-34, A.A. 680002 Bucaramanga, Colombia
The design of a series of styrene and spiroisatin hybrids was based on the structure of
combretastatin A-4 1. This library of 20 compounds were synthesized with the pharmacophoric
units: 3,4,5-trimethoxy or/and 4-hydroxy-3-methoxy phenyl moities in their structure. Thereby,
the libraries of β-nitrostyrenes 10a-10c, spiroisatin-dihydroquinolines 14a-14c, spiroisatin-
thiazolidinones 17a-17c and spiroisatin-nitropyrrolizidines 20a-20k were evaluated for their
in vitro cytotoxic, anti-proliferative, antifungal and antiviral activities. Biological results revealed
that among these compounds, β-nitrostyrenes 10a-10c exhibited significant cytotoxicity (HeLa
and Jurkat tumor cells) and antifungal (T. mentagrophytes) activities. Moreover, the spiroisatin-
dihydroquinoline 14a and 14c showed promising cytotoxicity (U937 cells). 14a-14c molecules
were active against human herpesviruses serotypes 1 and 2 (HHV-1 and HHV-2), but only 14a and
1
2
4b were effective against dengue virus serotype 2 (DENV-2). The spiroisatin-nitropyrrolizidine
0c exhibited moderate anti-herpetic activity, while 17c spiroisatin-thiazolidinone derivative also
reduced the infection of HHV-1 and DENV-2. Finally, the molecular docking showed that these
kind of molecules interact with the subunit α/β-tubulin.
Keywords: combretastatin A-4, β-nitrostyrenes, spiroisatin hybrids, anti-proliferative,
antifungal and antiviral activities
Introduction
cell shape organization, chromosomal separation and
transportation of vesicles and mitochondria. With a
4
Combretastatin A-4 1 (CA-4), the most potent and
rather simple structure, CA-4 has shown to inhibit the
polymerization of tubulin by binding to the colchicine site
and thus, disrupting the tubulin-microtubule dynamics
leading to mitotic arrest and eventually the cell death by
studied compound isolated from the bark of the South
1
African willow tree Combretum caffrum, stands out
among other members of the combretastatin family due
to its cytotoxic properties as inhibitor of angiogenesis and
5
apoptosis. Nevertheless, the major drawbacks of CA-4 are
2
tubulin polymerization.
its poor aqueous solubility, low bioavailability, cumulative
toxicities and short biological half-life that have promoted
The constant assembly (polymerization) and
disassembly (depolymerization) of microtubules, formed
6
its derivatization during the last decades.
3
by a heterodimer constituted by α- and β-tubulin subunits,
Several structure-activity relationship studies of CA-4
has revealed that the cis configuration of the double
bond, the 3,4,5-trimethoxy groups on the A-ring, and the
meta-hydroxy and para-methoxy groups on the B-ring
plays a crucial role in mitosis and cell division where they
are involved during the spindle formation, migration,
7
*
e-mail: yaneth.miranda@udea.edu.co
are fundamental for its main biological activity. Thereby,

2
Combretastatin A-4
J. Braz. Chem. Soc.
CA-4 has inspired the synthesis and biological evaluation
of different cytotoxic analogues containing these privileged
pharmacophores: the disodium phosphate prodrug
early stages of infection of human immunodeficiency virus
(HIV).Additionally, the flaviviruses use these structures to
promote a reconfiguration of the cytoplasmic compartments
that are important for the genome replication and translation
8
9
10
11
CA-4P 2, colchicine 3, podophyllotoxin 4, eugenol 5
12
22
and some oxindole derivatives 6-7 (Figure 1).
of viral protein.
2
3
Besides the cytotoxic activity against a range of human
cancer cell lines, in which CA-4 1induces the destabilization
of proteins associated with the cytoskeleton, this compound
is responsible for altering the coordination of many cellular
processes, including the cell cycle, movement, adhesion,
Considering both the previously described data
regarding viral modulation of microtubules and the current
need to research on synthesis and biological evaluation
of small molecules inspired on the structure of CA-4 1,
2
3
our screening program for new bioactive heterocycles
1
3
morphogenesis and contraction. Several reports have
determined that these cellular processes are involved in
different infection processes of some pathogens such as
has focused this research on: (i) design and synthesis
of a 20-member library of styrene and spiroisatin
hybrids containing the 3,4,5-trimethoxyphenyl and/or
the 4-hydroxy-3-methoxyphenyl as pharmacophoric
subunits; (ii) evaluation of the cytotoxic activity in tumor cell
lines (HeLa (cervix epitheloid adenocarcinoma cell), Jurkat
(acute T cell leukemia), U937 (promonocytic cell) andVero
(African green monkey kidney, Cercopithecus aethiops));
(iii) study of the antifungal activities in pathogen
dermatophytes (T. mentagrophytes, T. rubrum and
F. oxysporum); (iv) determination of the antiviral activity
in human herpesvirus 1 and 2 (HHV-1 and HHV-2), and
dengue virus serotype 2 (DENV-2) of these molecules and
some related tubulin inhibitors, and (v) the study of the
possible interactions of these molecules with the active site
of the tubulin target through molecular docking studies.
1
4
15
16
17
bacteria, parasites, fungi and viruses.
In particular, CA-4 and some analogues have exhibited
antifungal and antiviral activities whose mechanism of
action involves the inhibition of tubulin polymerization
or arrest of the cell cycle at different stages. For
example, synthetic combretastatin analogues have
exhibited relevant activity against Trichophyton rubrum,
Trichophyton mentagrophytes, Aspergillus fumigatus
and Saccharomyces cerevisiae through the regulation of
1
8
signaling pathways.
On the other hand, the use of microtubule inhibitors
before the viral infection and during various phases of
the virus life cycle, delays the intracellular transport of
virions leading to the accumulation of viral particles at
1
9,20
intracellular level.
In fact, ribonucleic acid (RNA) and
Results and Discussion
deoxyribonucleic acid (DNA) viruses regulate microtubule
during the different stages of the replicative cycle. For
example, microtubule-associated proteins such as dynein,
kinesin as well as dynactin 1 direct the transport of human
herpesviruses serotype 1 (HHV-1), a DNA virus, inside the
The preparation of the first library, the β-nitrostyrenes
10a-10c, is shown in Scheme 1. The corresponding
benzaldehydes 8a-8c reacted with nitromethane 9 through
a Knoevenagel condensation catalyzed by a ethanolamine
21
cell (dynactin subunit 1 (DCTN1)). In the case of RNA
viruses, it has been described the role of microtubules in
supported on silica gel (ETAM/SiO ) at 90 °C to furnish
2
24
the styrene derivatives.
Figure 1. CA-4 (1) and some synthetic and natural derivatives (2-7) containing the pharmacophoric moieties related with its cytotoxic activity.

Vol. 00, No. 00, 2019
Brand et al.
3
Scheme 1. Synthesis of β-nitrostyrenes 10a-10c through a Knoevenagel condensation catalyzed by ethanolamine supported on silica gel (ETAM/SiO ).
2
a
Reaction conditions: 8a-8c (5 mmol), 9 (20 mmol), catalyst (100 mg). Products were obtained in quantitative yield and were purified by recrystallization.
The next group of molecules, the spiroisatin-
dihydroquinoline hybrids 14a-14c containing the
-hydroxy-3-methoxyphenyl moiety, were synthesized
anilines 12d-12f and thioglycolic acid 15 afforded
the respective 2,3-disubstituted-4-thiazolidinones as
intermediates (Scheme 3). Then, the latter compounds
underwent a Knoevenagel condensation catalyzed by
t-BuOK at 90 °C with 3,4,5-trimethoxybenzaldehyde 16,
leading to the desired compounds 17a-17c in good yields
(Scheme 3).
4
through a two-step protocol in which the respective
anilines 12a-12c were condensated with isatin 11 to
give the corresponding ketimine-isatin products. These
intermediates were reacted in a second step with trans-
isoeugenol 13 via Povarov reaction catalyzed by BF ∙OEt ,
Finally, the spiroisatin-nitropyrrolizidine hybrids
20a-20k were synthesized through a multicomponent and
regioselective 1,3-dipolar cycloaddition between isatin
derivatives 18a-18c, L-proline 19 and the β-nitrostyrenes
10a-10c under aqueous media to furnish the title
compounds 20a-20k with the 3,4,5-trimethoxyphenyl
3
2
given the desired adducts 14a-14c in moderate yields
2
5
(
Scheme 2).
Next, the spiroisatin-thiazolidinone hybrids 17a-17c
containing the 3,4,5-trimethoxyphenyl moiety were
prepared in another two-step synthetic methodology in
which the first multicomponent reaction between isatin 11,
26
moiety in excellent yields (Scheme 4).
Scheme 2. Two-step synthesis of spiroisatin-dihydroquinoline hybrids 14a-14c through a Povarov reaction with trans-isoeugenol 13. Reaction conditions:
first step: 11 (4 mmol), 12 (4.8 mmol), AcOH (5 equiv.) in MeOH (40 mL) at 70 °C for 8 h; the precipitated product (ketimine-isatin) was filtered and
washed with petroleum ether; second step: ketimine-isatin (2 mmol), BF3∙OEt (2 mmol), trans-isoeugenol 13 (3 mmol) in CH Cl (20 mL) at room
2
2
2
a
temperature for 5 h under inert atmosphere. Products were purified by recrystallization.

4
Combretastatin A-4
J. Braz. Chem. Soc.
Scheme 3. Two-step synthesis of spiroisatin-thiazolidinone hybrids 17a-17c through a Knoevenagel type condensation with 3,4,5-trimethoxybenzaldehyde
6. Reaction conditions: first step: 11 (4 mmol), 12d-f (4.2 mmol), 15 (6 mmol), AcOH (3 equiv.) in toluene (40 mL) at 110 °C for 8 h; the precipitated
product (spirothiazolidinone) was filtered and washed with petroleum ether; second step: spirothiazolidinone (2 mmol), 3,4,5-trimethoxybenzaldehyde
1
a
1
6 (2.5 mmol) and t-BuOK (3 mmol) in isopropanol (IPA) (15 mL) at 90 °C for 5 h under inert atmosphere. Products were purified by recrystallization.
In total, 20 molecules, having the main pharmacophoric
moieties of CA-4 1, were prepared following our described
IC values of 16.4 and 10 µM, respectively. Nonetheless,
50
the selectivity index (SI) was not significant with respect
to the controls (1, 3 and doxorubicine (DOX)) (Table 1).
Moreover, the β-nitrostyrene 10a resulted to be active only
for the Jurkat tumor cell line with an IC value of 14 µM
2
5,26
synthetic procedures.
All of these small molecules
were fully characterized, confirming their structure and
1
13
stereochemistry by 1D ( H and C)/2D (correlation
spectroscopy (COSY), heteronuclear multiple bond
correlation (HMBC), and heteronuclear single quantum
correlation (HSQC)) nuclear magnetic resonance (NMR),
infrared (IR) and mass spectroscopies; ensuring their purity
before they were tested on the biological assays described
below.
50
and with an SI of 2.4, being the higher magnitude among
the series 10a-10c, which were active and demonstrated
a dose-dependent inhibition of the growth of cell lines.
In addition, the spiroisatin-dihydroquinoline hybrids 14a
and 14c were selective and active against one tumor cell
line. Compound 14c, which contains a cyanide moiety
in the tetrahydroquinoline core and the 4-hydroxy-
3-methoxyphenyl pharmacophoric unit, exhibited a high
cytotoxic activity on U937 cell line with an IC value of
The target compounds 10a-10c, 14a-14c, 17a-17c and
20a-20k and the reference compounds CA-4 1, colchicine 3
and doxorubicine (adriamycin), were evaluated first for
their in vitro anti-proliferative activities (cytotoxicity)
against three different human tumor cell lines: cervix
epitheloid adenocarcinoma cell (HeLa), acute T cell
leukemia (Jurkat) and promonocytic cell (U937), as well as,
the non-tumor cell lineVero (African green monkey kidney,
Cercopithecus aethiops). For the determination of half
maximal inhibitory concentration (IC ) values, we used
50
13.5 µM.
Furthermore, the SI value of 14c (SI = 6) resulted to
be higher than the ones shown by the positive controls
CA-4 1 (SI = 4.9 for HeLa), colchicine 3 (SI = 0.1
for HeLa, SI = 2.1 for Jurkat and SI = 0.3 for U937)
and doxorubicine (SI = 2.4 for HeLa); suggesting that
compound 14c could be considered as a possible antitumor
agent (Table 1).
50
the in vitro tetrazolium dye assay (3-(4,5-dimethylthiazol-
2
-yl)-2,5-diphenyl tetrazolium bromide, MTT) according
The in vitro antifungal activity was evaluated
following the Clinical and Laboratory Standards Institute
27
to our modified protocol.
2
9
As shown in Table 1, compounds 17a-17c and 20a-20k
did not exhibit significant anti-proliferative activities against
different cancer cell lines, with an IC value superior to
M38-A, protocol for filamentous fungi, with some
3
0
modifications. The target compounds 10a-10c, 14a-14c,
17a-17c and 20a-20k, with the reference controls
amphotericin B and terbinafine, were evaluated against
different dermatophytes: Trichophyton mentagrophytes,
Trichophyton rubrum and the environmental fungi
Fusarium oxysporum (Table 2).
50
28
25 µM. However, the substituted β-nitrostyrenes 10a-10c
showed activity against HeLa and Jurkat tumor cells,
being compounds 10b and 10c the most active derivatives
among the tested compounds on the HeLa tumor line with

Vol. 00, No. 00, 2019
Brand et al.
5
Scheme 4. Synthesis of spiroisatin-nitropyrrolizidine hybrids 20a-20k through a regioselective 1,3-dipolar cycloaddition under aqueous media. Reaction
a
conditions: 18a-c (1 mmol), 19 (1.3 mmol), 10a-c (1.1 mmol) in H O/PC (1:0.2) at 90 °C for 1 h. Products were purified by SiO column chromatography;
combined yield of the isolated mixture of regioisomers.
2
2
b
We found that among the all tested compounds in all the
furan moiety have showed interesting antifungal activity
strains evaluated, only the group of β-nitrostyrenes 10a-10c
resulted to be active (minimal inhibitory concentration
against T. mentagrophytes and T. rubrum, giving MIC
values of 15.7 ± 6.8 and 15.8 ± 6.5 µg mL , respectively.
-1
32
(
MIC) ≤ 100 µM). These molecules were classified
according to their MIC values as follows: 10c > 10b > 10a
Table 2).
β-Nitrostyrene 10c showed the most important
Furthermore, the antifungal activity of 10c was previously
determined against C. albicansATCC 90028 giving an MIC
value of 32.0 µg mL .
-1 33
(
The results of this study confirmthat the 3,4,5-trimethoxy
phenyl moiety should be the fragment, which improves the
antifungal activity, especially against T. mentagrophytes,
allowing to conclude that this pharmacophoric subunit of
CA-4 1 is also relevant for the development of novel anti-
dermatophytes agents.
Finally, the in vitro antiviral activity of all the
synthesized compounds 10a-10c, 14a-14c, 17a-17c and
20a-20k, with the reference control acyclovir and CA-4,
were evaluated against human herpesvirus 1 (HHV-1
antifungal activity against T. mentagrophytes, T. rubrum
and F. oxysporum at concentrations 25.7, 40.6 and 32.4 µM,
respectively. The activity of β-nitrostyrenes 10a-10c against
F. oxysporum outstands over the other strains because this
fungus has been associated with systemic diseases that
show high mortality rates and strong resistance to several
antifungal agents.
We have found in previous studies that some CA-4 1
hybrids containing the (Z)-2-(3,4,5-trimethoxystyryl)
31

6
Combretastatin A-4
J. Braz. Chem. Soc.
Table 1. In vitro anti-proliferative activity (cytotoxicity) of all the synthesized compounds measured by the MTT test in four human tumor cell lines
a
Cell line
Compound
HeLa
Jurkat
U937
Vero
b
c
b
c
b
c
b
IC50 ± SD / µM
19.91 ± 3.3
0.01 ± 0.001
> 25
SI
IC50 ± SD / µM
SI
IC50 ± SD / µM
SI
IC50 ± SD / µM
97.0 ± 0.7
0.001 ± 0.0001
33.1 ± 2.6
15.7 ± 0.4
13.0 ± 0.4
70.9 ± 1.1
> 25
f
f
f
f
1
3
4.9
0.1
NT
NT
NT
NT
0.0006 ± 0.00001
14.0 ± 1.9
12.5 ± 2.2
> 25
2.1
2.4
1.3
0.005 ± 0.001
26.4 ± 0.2
29.1 ± 0.7
28.2 ± 0.6
14.7 ± 5.3
> 25
0.3
1.3
0.5
0.5
4.8
d
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
0a
0b
0c
4a
4b
4c
7a
7b
7c
0a
0b
0c
0d
0e
0f
NA
1.6
0.8
10.0 ± 1.06
16.4 ± 0.46
> 25
d
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
> 25
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
> 25
> 25
NA
6.0
> 25
> 25
13.5 ± 0.2
> 25
80.8 ± 2.98
> 25
d
> 25
> 25
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
d
d
d
d
d
d
d
d
d
d
d
d
d
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
0g
0h
0i
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
0j
> 25
> 25
> 25
> 25
0k
> 25
> 25
> 25
> 25
e
DOX
0.8 ± 0.1
2.4
0.1 ± 0.007
19.0
0.1 ± 0.01
19.0
1.9 ± 0.08
a
HeLa: human cervix epithelial adenocarcinoma cellsATCC CRL-1958; Jurkat: human acute T cell leukemiaATCC TIB-152; U937: human promonocytic
b
cell line ATCC CRL-1593.2; Vero: Cercopithecus aethiops African green monkey kidney cells ATCC CCL-81; IC : concentration of the compound
causing 50% cell growth inhibition after 48 h of compounds exposure, as determined by the MTT assay; SI: selectivity index defined as VERO IC over
either HeLa, Jurkat or U937 IC ; NA: not active; positive control; NT: not tested. SD: standard deviation.
5
0
c
5
0
d
e
f
5
0
CDC Atlanta acyclovir-sensitive strain) and human
herpesvirus 2 (HHV-2 VR-734-G acyclovir-sensitive
only the spiroisatin-dihydroquinoline hybrids 14b-14c
exhibited moderate anti-herpetic activity, classifying them
according to those parameters as follows: 14c > 14b > 14a.
strain). The determination of the reduction factor (R ),
f
which corresponds to the value obtained by dividing the
viral titer in the absence of compound over the viral titer
obtained in the presence of compound was employed as an
Interestingly, the potency of compounds 14a, 14b and 14c,
2
with an R of 1 × 10 , against HHV-2 at concentrations
f
25, 15.2 and 15.1 µM, respectively, can be correlated with
the in vitro anti-proliferative activity of these compounds
discussed above against U937.
34
end point of this antiviral bioassay (Table 3).
In a previous study carried out in our laboratory, we
reported the antiviral activity of combretastatinA-4 hybrid
32
It should be noted that the spiroisantin hybrids have
a broad-spectrum biological activities, which involves
-1
of HHV-2 at concentrations below 25.0 and 3.1 µg mL .
This background is important and establishes a guide for
35
antimicrobial. In the particular case of the spiro[indoline-
3,2’-quinolin]-2-one hybrid 14c, it is clear that the presence
of the nitrile (CN) moiety is important to improve the
cytotoxic and the anti-herpetic activity, while the ethyl
group and chlorine atoms at the same core decreases both
activities.
32
continue exploring antiviral activity.
In order to determine the relevant or moderate
antiviral activity of tested compounds, the reduction of
R was established as a criterion to classify their potency
f
3
0
according to the standard parameters. Nevertheless,

Vol. 00, No. 00, 2019
Brand et al.
7
Table 2. In vitro antifungal activity of all the synthesized compounds
Table 3. In vitro antiviral activity of all the synthesized compounds
Vero cell line
a,b
a
Antifungal activity (MIC) / µM
Compound
T. mentagrophytes
82.9
T. rubrum
> 100
59.8
F. oxysporum
> 100
59.8
HHV-1
HHV-2
Compound
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
0a
0b
0c
4a
4b
4c
7a
7b
7c
0a
0b
0c
0d
0e
0f
Antiviral
activity / µM
Antiviral
activity / µM
b
b
Rf
Rf
59.8
c
c
c
c
c
c
c
c
c
c
1
1
1
1
1
1
1
1
0a
0b
0c
4a
4b
4c
7a
7b
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
25.7
40.6
32.4
c
c
NA
NA
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
0.0268
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
0.0268
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
c
c
NA
NA
c
2
2
2
NA
10
10
10
25
c
NA
15.1
15.2
2
10
60.8
c
c
c
c
NA
NA
NA
NA
NA
NA
NA
NA
NA
c
c
c
c
c
c
c
NA
NA
2
c
17c
20a
20b
20c
20d
20e
20f
20g
20h
20i
20j
20k
1
10
99.1
NA
c
c
c
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
c
c
c
c
c
c
c
c
c
c
c
c
NA
NA
c
2
NA
10
31.8
c
c
c
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
c
c
c
c
c
c
c
c
c
0g
0h
0i
NA
NA
c
c
NA
NA
c
c
NA
NA
c
c
0j
NA
NA
c
c
0k
NA
NA
c
c
c
Terbinafine
NT
NA
NA
a
c
c
Geometric means of minimal inhibitory concentration (MIC) of tested
NA
NA
b
compounds; the other positive control, amphotericin B, showed an
antifungal activity of 1.1 µM against A. fumigatus and A. flavus; NT:
0
.5
2
4
10
149.5
6.7
10
10
74.8
6.7
c
4
not tested.
Acyclovir
10
a
Vero Cercopithecus aethiopsAfrican green monkey kidney cell lineATCC
b
CCL-81; reduction factor: ratio of the virus titer in the absence over virus
titer in the presence of the tested compound; NA: not active. HHV-1:
Other compound that exhibited good antiviral activity
belongs to the family of spiroisatin-nitropyrrolizidine
hybrids 20a-20k. With the 3,4-methylenedioxy phenyl
moiety, the derivative 20c resulted active against HHV-2,
reducing to one hundred times the challenged viral load
but at concentration of 31.8 µM. While the group of
β-nitrostyrenes 10a-10c showed no activity against human
herpesviruses, the spiroisatin-thiazolidinone hybrids, 17c,
revealed effectiveness against HHV-1 at concentration
c
human herpesvirus 1 CDC Atlanta strain; HHV-2: human herpesvirus 2
VR-734-G strain.
morphological alterations under the microscope (data
not shown), which are usually related to cytoskeleton
36,37
remodeling. Several studies have shown roles for tubulin
during DNA and RNA viral infections, cancers and other
pathological processes.
9
9.1 µM.
For anti-dengue activity, we highlight the effectiveness
This would explain the broad spectrum of biological
activity of the combretastatin hybrids included in this
study, regarding CA-4 inhibitory activity in microtubule
polymerization.
A key criterion for designing and developing new drugs
is considering broad activity against medically important
pathogens.
of the molecules 14a, 14b and 17c. In this case, 14a and 14b
molecules revealed an activity efficiency at a concentration
of 30.2 and 29.7 µM, respectively (Figure 2). In addition,
the 17c molecule showed activity at various concentrations
(198.2, 99.1 and 49.5 µM). Finally, for the antiviral assay
with dengue virus, we employed ribavirin as control for
each experiment.
The treatment with the 14a, 14b and 17c molecules
in dengue virus infected cells also caused visible
These results are important because the design and
development of new drugs must take their activity spectrum
into consideration, and a strong preference must be given to
drug candidates that are active against the largest number

8
Combretastatin A-4
J. Braz. Chem. Soc.
Figure 2. Molecules 14a, 14b and 17c were effective in DENV-2 infection. BHK-21 cells were incubated in the presence or absence of 14a (a); 14b (b)
and 17c (c) molecules for six days and plaque forming units (PFU) were count, using as control ribavirin (R). Mean and SD: standard deviation compared
to dimethyl sulfoxide (DMSO) and ribavirin control from three experiments are shown. p-Values were determined by two-tailed unpaired t-test (***:
p < 0.001; **: 0.001 < p < 0.01; *: 0.01 < p < 0.05).
of co-circulating viruses and other pathogens that use the
cell to perpetuate themselves and to cause pathology.
We carried out in silico assays using molecular docking
to establish the mechanism of action of the tested molecules,
that could explain their broad biological activities. The
results showed molecules group 10a-c binding to α-tubulin
through hidrofobic interactions in the residues: Ala12, Tyr
Arg48 and Lys 96 (Figure 3d) and the 14b showed Hb on
Arg42 (Figure 3e). The Hb interactions have high stability
which is relevant to explain the molecule activity. On the
contrary, the compounds exhibited hydrophobic bindings
that are less stable than Hb. Otherwise, the 14c molecule
interacts on the colchicine binding-site, nonetheless, the
interactions are hydrophobic (Figure 3f).
2
24, Gln 11, Gly 144, Gly 10, Thr 145, Gly 146, Glu 183,
The compound 17c binds to subunit α/β-tubulin with
-
1
Gly 142, Gly 143 and Ser 140 (Figures 3a-3c). The binding
affinities (in kcal mol ) obtained were: 10a (−7.2), 10b
an affinity −8.8 kcal mol (Figure 3g). Interestingly,
we observed an Hb on Arg 2 and Asn 50 residues.
According with the crystallographic structure reported by
-1
(
−6.7) and 10c (−7.1) (Figures 3a-3c).
36
38
In addition, the molecules 14a-14b were docked, and
Ravelli et al. and Nogales et al. the site of interaction
between microtubules and compounds is different to the
colchicine site. The results showed interaction in a region
near to the nucleotide exchange area of tubulin, this is
unlike the 10a-10c compounds, these structures formed
hydrogen bonds (Hb) with the α-tubulin. In the case of 14a,
the following Hb on the amino acids were shown: Leu 242,
Figure 3. Molecular docking of molecules 10a-10c (a-c); 14a-14c (d-f); 17c (g) and combretastatin A-4 (h). Each docking shows in parentheses the
-
1
binding affinities expressed in kcal mol . Hydrogen bounds are represented in a dotted line (green) and hydrophobic interactions are shown in red color.

Vol. 00, No. 00, 2019
Brand et al.
9
relevant because the exchange of guanine nucleotide is
required for the microtubule assembly.
temperature and the catalyst was filtered and the filtrate was
extracted three times with CH Cl (3 × 20 mL). The organic
2
2
Further, the control CA-4 was docked and as expected,
it showed interaction in the colchicine binding site
layer was separated, dried with Na SO , and concentrated
2 4
under reduced pressure to yield the pure product 10a-10c.
Characterization data for 10a-10c is included in the
Supplementary Information section associated to this article.
(
Figure 3h). Finally, as perspective, we suggest biological
validation of in silico results, i.e., to evaluate alterations
in the tubulin pattern in a treatment context as well as
determining if the possible changes are related to the
biological activities reported here.
General method for the synthesis of spiroisatin-thiazolidinone
hybrids 17a-17c
A mixture of isatin 11 (4 mmol), the corresponding
aniline 12d-12f (4.2 mmol) and acetic acid (3 equiv.)
in anhydrous toluene (40 mL) was refluxed for 3 h and
thioglycolic acid 15 (6.04 mmol) was then added after
15 min. The reaction mixture was refluxed with a Dean-Stark
apparatus for 8 h. After cooling, the reaction mixture was
Conclusions
In this study, a series of styrene 10a-10c and spiroisatin
hybrids, containing the dihydroquinoline 14a-14c,
thiazolidinone 17a-17c and nitropyrrolizidine 20a-20k
pharmacophoric subunits, were synthesized based on
the structure of combretastatin A-4 1. The cytotoxicity,
antifungal and antiviral activities of these libraries were
evaluated for the first time.
Biological results revealed that among the diverse
molecules tested with methoxy and/or hydroxy groups
substituting the benzene rings, some compounds resulted
active. β-Nitrostyrenes 10a-10c exhibited notable data in
two biological activities (cytotoxicity and antifungal); in
particular, 3,4-dimethoxy-β-nitrostyrene 10b displayed the
highest cytotoxic activity among the tested series having
the 3,4-methylenedioxy fragment.
concentrated and added to a saturated NaHCO solution. The
3
obtained product, the spirothiazolidinone, was filtered and
recrystallized. Without further purification, the respective
intermediate (2 mmol), 3,4,5-trimethoxybenzaldehyde
16 (2.5 mmol) and potassium tert-butoxide (3 mmol) in
isopropanol (IPA, 15 mL) was heated at 90 °C for 5 h
under nitrogen atmosphere. TLC monitoring confirmed the
end of the reaction, then the reaction mixture was cooled
at room temperature and acetic acid (1 mL) was added.
The precipitate was filtered and recrystallized to yield
the desired products. Characterization data for 17a-17c
is included in the Supplementary Information section
associated to this article.
Although none of the spiroisatin-thiazolidinone hybrids
1
7a-17c exhibited representative activities (cytotoxic,
antifungal or antiviral), our findings are relevant to
encourage search of novel antitumoral, antifungal and
antiherpetic agents. Additionally, the in silico studies
demonstrated that these molecules could interact with
tubulin in different areas compared to the colchicine
binding site, suggesting a different mechanism of action.
General procedure for the synthesis of the spirooxindoles
1’-nitro pyrrolizidines 20a-20k
Into a 15 mL round bottom flask equipped with a
condenser, a gas bubbler and a stir bar, it was added the
corresponding isatin 18a-18c (2.50 mmol, 1 equiv.), the
respective β-nitrostyrene 10a-10c (1.1 equiv.), 1 mL of
distilled water and 0.2 mL of propylene carbonate. The
reaction mixture was stirred at 90 °C for 15 min prior to
addition of the appropriate L-proline 19 (1.3 equiv.) and
then the mixture was stirred at this temperature for one
Experimental
Chemistry
hour controlling the bubbling of CO . TLC monitoring
2
General procedure for the synthesis of β-nitrostyrenes
confirmed the end of the reaction, then the reaction
mixture was cooled at room temperature and quenched
with saturated sodium bicarbonate solution (10 mL, 1 M)
and was extracted three times with CH Cl (3 × 20 mL).
10a-10c
Into a 10 mL round bottom flask equipped with a
condenser, a gas bubbler and a stir bar, it was added the
corresponding benzaldehyde 8a-8c (5 mmol, 1 equiv.)
and nitromethane 9 (20 mmol, 4 equiv.). The reaction
mixture was stirred at 90 °C and 0.1 g of catalyst
2
2
The organic layer was separated, dried with Na SO ,
2
4
and concentrated to afford the crude product, which
was purified by silica gel flash chromatography to yield
the corresponding spirooxindole 1’-nitro pyrrolizidines
20a-20k. Characterization data for 20a-20k is included
in the Supplementary Information section associated to
this article.
(
ETAM/SiO ) was added in one portion. The system was
2
stirred at this temperature for six hours until the thin layer
chromatography (TLC) monitoring confirmed the end of
the reaction, then the reaction mixture was cooled at room

1
0
Combretastatin A-4
J. Braz. Chem. Soc.
27
Biology
end-point titration technique (EPTT). The activity was
evaluated by determining the reduction factor (R ) that
f
In vitro anti-proliferative activity assay
corresponds to the ratio of viral titer treated with respect
to titer viral untreated. For this assay, two independent
experiments were carried out by quadruplicate and
acyclovir was included as positive control.According to the
The in vitro anti-proliferative activities of CA-4 1
and all of the target compounds were determined by
the MTT (Sigma, New Jersey, USA) assay as described
2
7
27
previously. Briefly, Vero (african green monkey kidney,
ATCC CCL-81) and HeLa (human cervix epithelial
established parameters, the relevant or moderate antiviral
activity of a purified natural product is one whose R of viral
f
3
2
28
carcinoma, ATCC CRL-1958) cell lines were seeded into
titer is ≥ 1 × 10 or 1 × 10 , respectively.
3
9
6-well plates at 2.0 × 10 cells per well and incubated
To determine the anti-DENV activity, baby hamster
for 24 h at 37 °C. After the incubation time, each diluted
compound was added to the appropriate wells and the plates
were incubated for further 48 h at 37 °C in a humidified
kidney (BHK-21) cell monolayers grown in 24-well plates
5.5 × 10 cells per well were infected with multiplicity
4
of infection (MOI) = 1. Treatments were performed by
adding compounds either simultaneously with virus (pre-
infection treatment) or after viral infection (post-infection
treatment). For pre-infection treatment, a virus/compound
mixture was added to cell monolayers and was incubated
incubator with 5% CO . Jurkat (acute T cell leukemia
2
human, ATCC TIB-152) and U937 (promonocytic,
ATCC CRL-1593.2) cell lines were centrifuged to remove
medium and were again suspended in culture medium
3
and plated at 3 × 10 cells per well in a 96-well round-
for 2 h at 37 °C (5% CO ). After incubation, a washing
2
bottomed plate and diluted compounds were added to
plates and incubated for 48 h at 37 °C. Subsequently, the
culture medium was removed and the MTT solution (24 µL,
step was performed with phosphate buffered saline (PBS,
pH 7.0) then it was added carboxymethyl cellulose (CMC)
1.5%. In post-infection treatment, virus was added on cell
-1
2
mg mL ) was added to each well. The minimal dilution
monolayer and incubated for 2 h at 37 °C (5% CO ). After,
2
of compound that induced 50% growth inhibition of the
cells was expressed as inhibitory concentration 50% (IC ).
washing was performed with PBS (pH 7.0), compounds
previously prepared in CMC 1.5% were added and plates
were incubated for six days. Cells were then fixed and
stained with a solution of 3.5% formaldehyde with 0.2%
crystal violet and viral plaques were counted. Dextran
sulfate was employed as positive control in pre-infection
assays. Ribavirin (RIBA) was used as positive control
in post-infection stages of DENV-2. The fifty effective
concentration (EC ) was defined as the concentration that
50
The IC values for each compound were obtained by linear
50
regression analysis of the dose-response curves generated
from the absorbance data with the statistical package
3
9
R. IC50 values were expressed as the mean ± standard
deviation (mean ± SD) of two independent experiments
done in quadruplicate. Also, the selectivity index (SI) was
calculated.
5
0
reduces the 50% of plaque forming units. EC for each
5
0
In vitro antifungal activity assay
compound were obtained by linear regression analysis of
the dose-response curves generated using the statistical
program GraphPad Prism 5.0.
The dilutions of the compounds were dispensed into
4
0
9
6-well flat-bottom microdilution plates in duplicate at
final concentrations between 100-20 µM.Amphotericine B
was used as control with Aspergillus fumigatus and
Aspergillus flavus at a range of 17.3-0.034 µM, and
terbinafine with dermatophytes were used as positive
controls at a range of 0.031-13.72 µM. The inoculum size
In silico (docking) studies
Molecular docking studies were perfomed using
4
1
AutoDock Vina to determine whether the synthesized
compounds have comparable binding mode to that of CA-4
on the α,β-tubulin subunit that corresponds to the colchicine
binding site. This area is formed by the following amino
acids: α-tubulin: Thr179, Val 181, Ser 178; β-tubulin:
Cys 241, Ala 250, Lys 254, Leu 255, Asn 258, Met 259,
Ala 316, Ala 317, Lys 352, Thr 353, Ile 378.
5
for microdilution plates were 0.2-2.5 × 10 colony forming
-
1
29
units (CFU) mL . For the CLSI M38-A method, the
MICs were determined after 48 h of incubation at 35 °C
to Aspergillus spp. and at 28 °C to Fusarium oxysporum.
The activity against the dermatophytes was determined
after 6 days of incubation at 28 °C.
In this study, the most active compounds (10a-10c,
1
4a-14c and 17c) were docked into colchicine binding site
In vitro antiviral activity assay
of the crystal structure of tubulin (PDB code: 3E22). For
+
The anti-herpetic activity of molecules was evaluated
on Vero cells against ten cell culture infectious dose fifty
percent (10TCID ) of HHV-1 and HHV-2, using the
the visualization of interactions, it was employed LigPlot
42
software.
5
0

Vol. 00, No. 00, 2019
Brand et al.
11
Supplementary Information
H.; Nat. Chem. 2010, 2, 735; Wang, S.; Zhao, Y.; Zhang, G.;
Lv,Y.; Zhang, N.; Gong, P.; Eur. J. Med. Chem. 2011, 46, 3509.
13. Kanthou, C.; Tozer, G. M.; Blood 2002, 99, 2060; Hsu, L.
C.; Durrant, D. E.; Huang, C. C.; Chi, N. W.; Baruchello, R.;
Rondanin, R.; Rullo, C.; Marchetti, P.; Grisolia, G.; Simoni,
D.; Lee, R. M.; Invest. New Drugs 2012, 30, 1379.
Supplementary information (experimental details,
1
13
characterization data, including copies of H NMR,
C
13
NMR, DEPT-135, COSY, C HMBC, HSQC and HSQC
charts, of all synthesized compounds) is available free of
charge at http://jbcs.sbq.org.br as PDF file.
14. Gahlmann, A.; Moerner, W. E.; Nat. Rev. Microbiol. 2014, 12,
9
.
Acknowledgments
15. Ferreira, D.; Cortez, M.; Atayde, V. D.; Yoshida, N.; Infect.
Immun. 2006, 74, 5522; Jewett, T. J.; Sibley, L. D.; Mol. Cell
2003, 11, 885.
We acknowledge the financial support from Colciencias
(
PatrimonioAutónomodelFondoNacionaldeFinanciamiento
16. Takeshita, N.; Manck, R.; Grün, N.; de Vega, S. H.; Fischer,
R.; Curr. Opin. Microbiol. 2014, 20, 34; Berepiki, A.; Lichius,
A.; Read, N. D.; Nat. Rev. Microbiol. 2011, 9, 876.
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para la Ciencia, la Tecnología y la Innovación, Francisco
José de Caldas-Colombia), grant 744 2016 (contract
6
Desarrollo de la Investigación-Universidad de Antioquia),
grant 2014-1041; and Estrategia de Sostenibilidad (CODI)
2
la República (agreement 201510). We thank Dr Miguel
A. González Cardenete, associate researcher at Consejo
Superior de Investigaciones Científicas (CSIC), Spain, for
his generous donation of CA-4 1.
48-2017 project 111574455595); CODI (Comité para el
016-2017 de la Universidad de Antioquia and Banco de
20. Mainou, B. A.; Zamora, P. F.; Ashbrook, A. W.; Dorset, D. C.;
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W.; Scholz, T.; Karger,A.; Sodeik, B.; PLoS Pathog. 2010, DOI
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Submitted: July 5, 2019
Published online: November 26, 2019
This is an open-access article distributed under the terms of the Creative Commons Attribution License.