Synthesis of Combretastatin A-4 and 30-Aminocombretastatin A-4 derivatives with Aminoacid Containing Pendants and Study of their Interaction with Tubulin and as Downregulators of the VEGF, hTERT and c-Myc Gene Expression

Article abstract of DOI:10.3390/molecules25030660

Natural product combretastatin A-4 (CA-4) and its nitrogenated analogue 3⁰-aminocombretastatin A-4 (AmCA-4) have shown promising antitumor activities. In this study, a range of CA-4 and AmCA-4 derivatives containing amino acid pendants have been synthesized in order to compare their biological actions with those of their parent compounds. Thus, inhibition of cell proliferation on tumor cell lines HT-29, MCF-7 and A-549, as well as on the nontumor cell line HEK-273; in vitro tubulin polymerization; mitotic cell arrest; action on the microtubule cell network and inhibition of VEGF, hTERT, and c-Myc genes have been evaluated. Some AmCA-4 derivatives bearing L-amino acids exhibited inhibition of cell proliferation at low nanomolar levels exceeding the values shown by AmCA-4. Furthermore, while CA-4 and AmCA-4 derivatives do not show significant effects on the in vitro tubulin polymerization and cell cycle arrest, some selected CA-4 and AmCA-4 derivatives are able to cause total depolymerization of the microtubule network on A-549 cells. The best results were obtained in the inhibition of gene expression, particularly on the VEGF gene, in which some AmCA-4 derivatives greatly exceeded the inhibition values achieved by the parent compound.

Full text of DOI:10.3390/molecules25030660

Article
Synthesis of Combretastatin A-4 and 3′-
Aminocombretastatin A-4 derivatives with
Aminoacid Containing Pendants and Study of Their
Interaction with Tubulin and as Downregulators of
the VEGF, hTERT and c-Myc Gene Expression
1
1,
1
1
1
1
Raül Agut , Eva Falomir *, Juan Murga , Celia Martín-Beltrán , Raquel Gil-Edo , Alberto Pla ,
1,
2
Miguel Carda * and J. Alberto Marco
1
Departamento de Química Inorgánica y Orgánica, Universidad Jaume I, E-12071 Castellón, Spain;
ragut@uji.es (R.A.); jmurga@uji.es (J.M.); beltranc@uji.es (C.M.-B.); ragil@uji.es (R.G.-E.); apla@uji.es (A.P.)
Departamento de Química Orgánica, Universidad de Valencia, E-46100 Valencia, Spain;
alberto.marco@uv.es
2
* Correspondence: efalomir@uji.es (E.F.); mcarda@uji.es (M.C.),
Tel.: +34 964 728 240 (E.F.); +34 964 728 242 (M.C.).
Academic editor: Wolfgang Kreis and Jennifer Munkert
Received: 18 December 2019; Accepted: 1 February 2020; Published: 4 February 2020
Abstract: Natural product combretastatin A-4 (CA-4) and its nitrogenated analogue 3′-
aminocombretastatin A-4 (AmCA-4) have shown promising antitumor activities. In this study, a
range of CA-4 and AmCA-4 derivatives containing amino acid pendants have been synthesized in
order to compare their biological actions with those of their parent compounds. Thus, inhibition of
cell proliferation on tumor cell lines HT-29, MCF-7 and A-549, as well as on the nontumor cell line
HEK-273; in vitro tubulin polymerization; mitotic cell arrest; action on the microtubule cell network
and inhibition of VEGF, hTERT, and c-Myc genes have been evaluated. Some AmCA-4 derivatives
bearing L-amino acids exhibited inhibition of cell proliferation at low nanomolar levels exceeding
the values shown by AmCA-4. Furthermore, while CA-4 and AmCA-4 derivatives do not show
significant effects on the in vitro tubulin polymerization and cell cycle arrest, some selected CA-4
and AmCA-4 derivatives are able to cause total depolymerization of the microtubule network on A-
549 cells. The best results were obtained in the inhibition of gene expression, particularly on the
VEGF gene, in which some AmCA-4 derivatives greatly exceeded the inhibition values achieved by
the parent compound.
Keywords: Combretastatin A-4; 3′-aminocombretastatin A-4; cytotoxicity; tubulin; microtubules;
cell cycle; VEGF; hTERT; c-Myc
1. Introduction
Cancer is a disease characterized by the growth and spread by the body of aberrant cells. Cancer,
along with cardiovascular diseases, are the two leading causes of mortality in developed countries.
The ageing of the populations in these countries cause an increase in cancer mortality so that there is
considerable interest in the discovery of new anticancer agents. A range of natural products or direct
derivatives are used in the treatment of cancer, and over the last 35 years, more than half of the new
compounds indicated for the treatment of cancer are natural products or derivatives thereof [1].
Molecules 2020, 25, 660; doi:10.3390/molecules25030660
www.mdpi.com/journal/molecules

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Combretastatins are a class of natural phenolic stilbene compounds that have been isolated from the
African willow tree Combretum caffrum. The most potent member of these natural stilbenes is
combretastatin A-4 (CA-4, see Figure 1), whose antimitotic action is based on the inhibition of tubulin
polymerization. CA-4 shows strong cytotoxicity against a variety of cancer cells, including multidrug
resistant cancer cell lines [2–4]. In addition, CA-4 has also been demonstrated to exert effects in the
proliferation of endothelial cells [5] and to act as a vascular-disrupting agent [6].
Figure 1. Structures of combretastatin A-4, combretastatin A-4 disodium phosphate and 3′-
aminocombretastatin A-4.
Recently, Steinmetz and colleagues have confirmed, by resolution of the crystal structure of a
tubulin-combretastatin A-4 complex, that the binding site of CA-4 to β-tubulin is the colchicine site
[7]. Combretastatin A-4 disodium phosphate (CA4DP, see Figure 1) is a water-soluble prodrug of
CA-4 that shows potent antivascular and antitumor effects in a wide variety of tumor cell lines [8].
3′-Aminocombretastatin (AmCA-4, see Figure 1) is a non-natural combretastatin analogue that also
shows strong cytotoxicity, inhibition of tubulin polymerization and antivascular activity [9–11].
The formation of new blood vessels from pre-existing vasculature is known as angiogenesis.
Tumoral angiogenesis causes tumor growth and dissemination of tumor cells, which ends up as
metastasis [12]. There are several “on” and “off” switches that regulate the angiogenesis process.
Vascular endothelial growth factor (VEGF) is an inducer of angiogenesis and promotes endothelial
cell survival, proliferation and migration. Overexpression in the production of VEGF has been
reported to occur in various types of tumors [13,14]; therefore, it is not surprising that VEGF and
angiogenesis process have become targets in cancer therapy [15].
Telomerase is a reverse transcriptase enzyme that carries a short RNA molecule used as a
template to elongate telomeres. Telomerase is inactive in most somatic cells but is active in normal
stem cells and most cancer cells [16]. Telomerase does not incite the oncogenic process, but its action
is needed for the growth and development of many cancers. Since telomerase is not expressed in most
normal human cells, this has led to the development of targeted telomerase anticancer agents [17].
The reverse transcriptase subunit of human telomerase (hTERT) catalyzes the nucleotide
polymerization process. Human telomerase is regulated during development and differentiation,
mainly through transcriptional control of the hTERT gene, the expression of which is restricted to
cells that exhibit telomerase activity. For the expression of the hTERT gene, several transcriptional
factors such as c-Myc, a proto-oncoprotein [18], have been found to play an important role through
upregulation of the mRNA encoding the hTERT protein subunit [19,20]. Moreover, deregulation of
c-Myc has been described as an upregulator of VEGF and hTERT gene expression and as one of the
major factors responsible for increased tumor malignancy [21].
During the past few years, we have been investigating a range of analogues of natural products
for their potential values in anticancer therapy as multitarget agents [22,23]. We have published
several reports on the biological properties of combretastatin and aminocombretastatin A-4
derivatives [24–29]. In addition to their cytotoxicity, we have also investigated their ability to inhibit
the expression of certain genes related to the angiogenesis process and the telomerase activation, such
as c-Myc, VEGF and hTERT genes, as well as their actions as vascular disrupting agents. Therefore,
based on the aspects commented above and in continuation of our research on novel natural product
analogues with multitarget actions in cancer therapy, we wanted to ascertain whether the
introduction of amino acid subunities in combretastatin A-4 and 3′-aminocombretastatin A-4 might
give rise to noticeable changes in the biological activity. For these reasons, we have conceived the

Molecules 2020, 25, 660
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synthesis and biological evaluation of a range of combretastatin and 3′-aminocombretastatin A-4
derivatives having amino acid pendants.
2. Results
2.1. Chemistry
In order to obtain CA-4 derivatives carrying amino acids, we have used an acetic acid linkage to
connect the CA-4 and the amino acid fragments, as indicated in Scheme 1. Thus, CA-4 was converted
into compound 1 by reaction with ethyl bromoacetate in the presence of K2CO3. Saponification of 1
led to acid 2, which was coupled with a range of methyl L- and D-amino esters in the presence of
EDCI·HCl and DMAP [30] to afford compounds of general structures 3 and 5, respectively.
Saponification of these compounds afforded combretastatin A-4/amino acid conjugates 4 and 6.
Scheme 1. Synthesis of combretastatin A-4 (CA-4) derivatives. Reagents and conditions: (a) K2CO3
and DMF, RT 1 h., then BrCH2COEt, RT 24 h, 94%. (b) NaOH and EtOH, RT 1 h, 91%. (c) L-amino
ester, EDCI·HCl, DMAP and DMF, 0 °C, 2 h, then RT 2 h. (d) LiOH and MeOH/H2O, RT 1 h. (e) D-
amino ester, EDCI·HCl, DMAP and DMF, 0 °C, 2 h, then RT 2 h.
The structures of the CA-4 derivatives, as well as the esters from which they are derived, are
indicated in Figure 2 (for yields and spectral data, see Supplementary Materials).

Molecules 2020, 25, 660
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Figure 2. Structures of CA-4 derivatives.
As regards the AmCA-4 derivatives carrying amino acids, they were obtained by means of direct
coupling with N-fluorenylmethoxycarbonyl (N-Fmoc)-protected amino acids [31], followed by N-
Fmoc deprotection [32] (see Scheme 2; for yields and spectral data, see Supplementary Materials).
Scheme 2. Synthesis of 3′-aminocombretastatin A-4 (AmCA-4) derivatives. Reagents and conditions:
(a) N-Fmoc-L-amino acid or N-Fmoc-D-amino acid, DCC, HOBt and DMF, RT 16 h. (b) KF, 18-crown-
6 and DMF, RT 4 h.
2.2. Inhibition of Cell Proliferation
The ability of the synthetic compounds to inhibit cell proliferation was established by means of
their IC50 values towards the tumor cell lines HT-29 (human colon adenocarcinoma), MCF-7 (breast
adenocarcinoma) and A-549 (human pulmonary adenocarcinoma), as well as towards the nontumor
cell line HEK-293 (human embryonic kidney cells). Results for compounds derived from CA-4 are
presented in Table 1, along with the calculated selectivity indexes SIA (for HT-29 cell line), SIB (for
MCF-7 cell line) and SIc (for A-549 cell line), obtained by dividing the IC50 values of the nontumor cell
line (HEK-293) by those of the corresponding tumor cell line. The higher the SI index, the higher the
therapeutic safety margin. By comparison, the IC50 values of CA-4 are also indicated in Table 1.
All derivatives indicated in Table 1 show an antiproliferative activity in the micromolar range,
with values similar to those of the natural product CA-4. For the HT-29 cell line, the most active
compounds are 1 (7 μM); 3.6 (L-Ser-OMe, 9 μM) and 4.1 (Gly, 5 μM), although their selectivity indexes
are lower than 1. On the contrary, compounds 4.5 (L-Pro), 4.9 (L-Met), 6.2 (D-Leu), 6.3 (D-Phe), 6.24
(D-Pro) and 6.25 (D-Ser) show SI values greater than 1, although they do not reach the high SI values
displayed by CA-4. In the MCF-7 cell line, compounds 3.6 (L-Ser-OMe, 7 μM); 4.1 (Gly, 6 μM); 4.2 (L-
Val, 7 μM) and 5.5 (D-Ser-OMe, 7 μM) are the most active, showing 3.6 and 5.5 selectivity indexes
above 1. In the A-549 cell line, IC50 values are, in general, comparable to those of the other cell lines,
with compounds 1, 3.3 (L-Leu-OMe), 3.4 (L-Phe-OMe), 3.9 (L-Met-OMe) and 5.1 (D-Val-OMe)
exhibiting IC50 values less than 10 μM. In this cell line, compounds 3.3 (L-Leu-OMe) and 4.8 (L-Tyr)
exhibit SI values greater than 1.
Table 1. IC50 values (μM) and SI indexes for CA-4 derivatives a.

Molecules 2020, 25, 660
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b
c
d
Comp. HT-29
CA-4 4.2 ± 0.5 1.0 ± 0.2 0.428 ± 0.004
MCF-7
A-549
HEK-293 SIA
SIB
25
0.4
1.1
1.1
0.4
0.5
SIc
58
25 ± 3
3.6 ± 0.5
12 ± 5
9 ± 1
5 ± 2
9 ± 2
5.9
0.5
0.4
0.7
0.2
0.3
1
2
7 ± 4
33 ± 6
12 ± 2
24 ± 4
29 ± 6
28 ± 7
48 ± 5
9 ± 3
18 ± 7
16 ± 5
16 ± 7
5 ± 1
12 ± 5
15 ± 5
54 ± 9
26 ± 13
39 ± 16
34 ± 11
55 ± 5
32 ± 6
27 ± 7
26 ± 1
12 ± 4
50
11 ± 2
45 ± 5
47 ± 9
27 ± 5
50 ± 11
13 ± 1
8 ± 4
11 ± 5
8 ± 2
12 ± 6
19 ± 6
8 ± 2
24 ± 2
7 ± 3
12 ± 4
12 ± 4
10 ± 3
6 ± 1
9 ± 1
38 ± 5
26 ± 2
24 ± 1
8 ± 1
0.4
0.3
0.3
0.2
1.1
0.4
0.4
0.4
0.7
0.6
0.9
0.2
0.3
0.6
0.5
0.4
0.5
0.6
1.1
<0.1
0.8
0.6
0.4
0.4
0.3
0.7
0.7
0.8
0.6
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
5.1
5.2
5.3
5.4
5.5
6.1
6.2
6.3
6.4
6.5
9 ± 2
4 ± 1
0.14 0.5
50 ± 10
18 ± 4
19 ± 1
18 ± 1
10 ± 2
24 ± 4
17 ± 2
14 ± 2
100 ± 20
110 ± 10
36 ± 4
41 ± 3
30 ± 7
>300
20 ± 1
7 ± 2
14 ± 2
10 ± 2
8 ± 1
5 ± 1
4 ± 1
9 ± 2
47 ± 3
45 ± 16
17 ± 1
23 ± 9
33 ± 1
40 ± 4
3.9 ± 0.6
13 ± 3
3.7 ± 1.6
5.7 ± 0.8
9 ± 3
39 ± 6
49 ± 12
46 ± 6
53 ± 9
14 ± 4
0.4
0.8
0.8
0.6
0.5
0.9
0.4
0.6
0.9
1.7
0.4
0.7
0.6
1.3
0.1
0.5
0.3
0.1
0.8
0.9
1.0
1.7
1.1
1.1
0.8
1.0
1.2
0.8
0.8
0.8
0.6
0.6
1.0
0.5
0.6
0.6
0.8
3.1
0.4
0.8
0.7
0.6
1.3
0.8
2.0
1.1
1.0
7 ± 1
16 ± 6
45 ± 4
83 ± 16
27 ± 9
40 ± 9
43 ± 7
13 ± 2
9 ± 3
16 ± 3
6 ± 1
10 ± 4
7 ± 2
46 ± 3
25 ± 2
43 ± 3
55 ± 13
12 ± 1
5 ± 1
22 ± 3
10 ± 3
15 ± 1
28 ± 1
54 ± 7
68 ± 1
60 ± 4
95 ± 14
>150
1.2 <0.09
a IC50 values are expressed as the compound concentration (μM) that inhibits the cell growth by 50%
after 48 h of treatment. Data are the average (±SD) of three experiments: b SIA = IC50 (HEK-293)/IC50
(HT-29). c SIB = IC50 (HEK-293)/IC50 (MCF-7). d SIB = IC50 (HEK-293)/IC50 (A-549).
IC50 values and SI indexes for AmCA-4 derivatives are presented in Table 2. AmCA-4 derivatives
show important differences in antiproliferative activity, which depends on the configuration of the
amino acid.
Table 2. IC50 values (nM) and SI indexes for AmCA-4 derivatives a.
b
c
d
Comp.
CA-4
AmCA-4
7.1
HT-29
4200 ± 500
22 ± 1
MCF-7
1000 ± 200
8 ± 1
4.8 ± 0.5
1.9 ± 0.8
100 ± 50
9 ± 2
A-549
428 ± 4
117 ± 7
186 ± 11
242 ± 13
870 ± 60
490 ± 13
270 ± 50
1330 ± 90
422 ± 8
HEK-293
25000 ± 3000 5.9
SIA
SIB
25
SIc
60
7 ± 1
3.5 ± 1.9
1.5 ± 0.6
60 ± 20
14 ± 3
2.3 ± 2
130 ± 30
13 ± 2
0.3
0.5
1.4
0.5
0.6
0.3
0.4
1.4
0.9
0.9 0.06
0.7 0.02
0.8 0.01
0.6 0.07
1.6 0.03
0.2 0.01
7 ± 4
7.2
7.3
7.4
7.5
7.6
7.7
7.8
1.1 ± 0.4
130 ± 40
23 ± 10
8 ± 3
320 ± 140
9 ± 3
14 ± 4
58 ± 12
25 ± 12
12 ± 2
2.2
0.1
0.5 0.03
1.3 0.01
18 ± 5
8900 ± 1200
16 ± 2

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7.9
8.1
8.2
8.3
8.4
8.5
8.6
8.7
8.8
33 ± 20
10 ± 2
5.1 ± 0.3
9 ± 4
4.8 ± 0.5
13 ± 2
1.8 ± 1.2
2.3 ± 1.5
3100 ± 600
12 ± 1
4.9 ± 0.2
3.4 ± 1.2
4800 ± 500
407 ± 23
400 ± 70
611 ± 16
490 ± 100
167 ± 23
141 ± 3
1200 ± 300
410 ± 40
206 ± 20
90 ± 30
8 ± 4
10 ± 2
0.2
1.0
1.3
0.6
0.9
0.8
3.4
1.8
1.2
0.7
0.9
1.6 0.02
1.1 0.02
0.8 0.01
0.6 0.02
0.9 0.01
1.5 0.02
3.0 ± 0.3
13.3 ± 0.5
1.8 ± 0.4
4.4 ± 2.5
700 ± 200
8 ± 2
2.2 ± 1.2
4.9 ± 0.1
5500 ± 700
4.0 ± 0.1
7.6 ± 0.3
1.7 ± 0.8
3.5 ± 1.9
2400 ± 1400
14 ± 3
2.7 ± 1.4
3.7 ± 0.3
5100 ± 700
2400 ± 400
0.8
2.0
1.1 0.03
0.5 0.01
1.1 0.04
8.9
9.1
9.2
7340 ± 230
1.1
0.7
44000 ± 6000 3100 ± 500 48000 ± 3000
0.05 0.8 0.05
9.3
9.4
10.1
10.2
10.3
10.4
13000 ± 5000 4500 ± 1700 18500 ± 1700 11200 ± 1500 0.9
2.5
0.6
0.5
0.2
1.8
5.0
0.6
0.2
0.2
2.7
1.2
0.4
5300 ± 300
4800 ± 600
1050 ± 140
3900 ± 800
4400 ± 1900
5300 ± 1100 12800 ± 800
3100 ± 1100
2400 ± 300
430 ± 40
3500 ± 900
3000 ± 600
0.6
0.5
0.4
0.9
0.7
4700 ± 700
2000 ± 900
2000 ± 800
600 ± 140
9900 ± 1300
157 ± 7
2900 ± 500
7030 ± 220
a IC50 values are expressed as the compound concentration (nM) that inhibits the cell growth by 50%
after 48 h of treatment. Data are the average (±SD) of three experiments: b SIA = IC50 (HEK-293)/IC50
(HT-29). c SIB = IC50 (HEK-293)/IC50 (MCF-7). d SIB = IC50 (HEK-293)/IC50 (A-549).
Those derivatives that incorporate amino acids of the L series exhibit IC50 values in the
nanomolar range of the same order of magnitude, in general, as AmCA-4 and are much more active
than the natural product CA-4. Compounds 7.2 (Fmoc- L-Ala), 8.2 (L-Ala), 8.4 (L-Leu), 8.5 (L-Phe), 8.8
(L-Tyr) and 8.9 (L-Met) show IC50 values lower than 5 nM in HT-29, MCF-7 and HEK-293 cell lines.
The least active of the L series are those that contain the amino acid proline (7.6 and 8.6). As for the
selectivity indexes, the highest values in the HT-29 cell line are observed for derivatives 7.2 (Fmoc-L-
Ala), 7.7 (Fmoc-L-Thr), 8.2 (L-Ala), 8.6 (L-Pro), 8.7 (L-Thr) and 8.8 (L-Tyr), with values higher than 1;
thus, higher than those of AmCA-4 but lower than those of CA-4. For the A-549 cell line, the IC50
values are some orders of magnitude higher than those obtained in the other two tumor cell lines,
with SI values similar to those of AmCA-4. On the other hand, the derivatives of D series 9.1–10.4
exhibit IC50 values in the micromolar range; therefore, with activities similar to that of CA-4, or even
lower. Only 10.2 (D-Leu) exhibits antiproliferative activity in the high nanomolar range in the A-549
cell line, together with a high SI value (2.7).
2.3. Effect on Microtubule Assembly
The effect on the self-assembly of tubulin was determined by measuring the critical
concentration (CrC). All derivatives were tested at a concentration of 27.5 μM in a glycerol (GAB)
and GTP assembly buffer, to which tubulin was added at a concentration of 25 μM. Table 3 shows
the results achieved with CA-4 derivatives and compares them with those obtained in the absence of
ligand (control) and in the presence of CA-4 as a reference compound.
Table 3. Critical concentration (CrC) values for the assembly of purified tubulin in GAB in the
presence of CA-4 derivatives.
Comp.
Control
CA-4
1
CrC (µM)
7.9 ± 1.2
21.5 ± 0.8
13.6 ± 0.3
12.0 ± 0.7
9.52 ± 0.05
Comp. CrC (µM)
Comp.
5.1
5.2
5.3
5.4
CrC (µM)
13.31 ± 0.01
13.2 ± 0.5
13.1 ± 0.8
9 ± 3
3.8
3.9
4.1
4.2
4.3
13.26 ± 0.19
8.06 ± 0.04
9.3 ± 0.4
9.3 ± 0.3
8 ± 3
2
3.1
5.5
9.2 ± 0.9

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3.2
3.3
3.4
3.5
3.6
3.7
11.7 ± 2.3
9.4 ± 1.4
13.20 ± 0.06
10.4 ± 2.0
13.00 ± 0.10
10 ± 3
4.4
4.5
4.6
4.7
4.8
4.9
11.4 ± 0.7
9.2 ± 0.5
7.9 ± 1.6
10.1 ± 1.8
8.5 ± 2.0
8.6 ± 0.4
6.1
6.2
6.3
6.4
6.5
9.4 ± 1.3
11 ± 3
9.2 ± 0.4
10 ± 3
9.25 ± 0.16
Unexpectedly, Table 3 shows that, while CA-4 is a potent microtubule destabilizer, most of the
synthetic CA-4 derivatives do not exhibit significant differences in effects, as compared with the
control. The highest CrC values were obtained by compounds 1, 3.4 (L-Phe-OMe), 3.6 (L-Ser-OMe),
3.8 (L-Tyr-OMe), 5.1 (D-Val-OMe), 5.2 (D-Leu-OMe) and 5.3 (D-Phe-OMe) (values around 13 μM) very
far from the CA-4 value, which is 21.5 μM. In addition, the time needed to start tubulin
polymerization is similar to that of the control. Thus, it can be concluded that these CA-4 derivatives
do not inhibit in vitro tubulin polymerization.
Table 4 shows the critical concentration values achieved by AmCA-4 derivatives and compares
them with those obtained in the absence of ligand (control) and in the presence of CA-4 and AmCA-
4 as reference compounds. Most of the AmCA-4 derivatives do not show CrC values different from
the control value. It should be noted that compounds 7.1 (Fmoc-Gly), 7.7 (Fmoc-L-Thr), 8.1 (Gly), 8.2
(L-Ala), 8.7 (L-Thr), 9.4 (Fmoc-D-Pro) and 10.1 (D-Val) present CrC values around 10–14 μM, higher
than those shown by the control, although much lower than those exhibited by the reference
compounds CA-4 and AmCA-4.
Table 4. Critical Concentration (CrC) values for the assembly of purified tubulin in GAB in the
presence of AmCA-4 derivatives.
Comp.
Control
CA-4
AmCA-4
7.1
CrC (µM)
7.9 ± 1.2
21.5 ± 0.8
23 ± 3
Comp.
7.8
7.9
8.1
8.2
8.3
8.4
8.5
8.6
CrC (µM)
10.1 ± 1.1
7.1 ± 0.4
10.2 ± 1.8
13.9 ± 1.5
9.3 ± 0.5
8.3 ± 0.3
9.1 ± 0.9
8.4 ± 0.1
12.3 ± 0.6
7.7 ± 0.8
Comp.
8.9
9.1
9.2
9.3
CrC (µM)
8.5 ± 1.2
8.9 ± 1.5
6.5 ± 0.7
6.6 ± 0.2
11.4 ± 0.2
12.8 ± 0.3
7.0 ± 0.5
7.7 ± 2.1
8.7 ± 1.2
11 ± 4
7.2
7.3
7.4
7.5
7.6
7.7
9.4 ± 0.3
6.9 ± 0.3
9.9 ± 0.8
8.1 ± 1.6
6.0 ± 0.5
12.0 ± 0.3
9.4
10.1
10.2
10.3
10.4
8.7
8.8
o
The tubulin polymerization process was studied by means of turbidimetry at 350 nm and 37 C
and is shown in Figure 3 for compounds 7.1 (Fmoc-Gly), 7.7 (Fmoc-L-Thr), 8.1 (Gly), 8.2 (L-Ala), 8.7
(L-Thr), 9.4 (Fmoc-D-Pro) and 10.1 (D-Val), which are those having the highest CrC values. Figure 3
also includes polymerization curves in the presence of CA-4, AmCA-4 and paclitaxel. This test was
also performed using paclitaxel, a microtubule stabilizing agent, as a control for the complete
formation of microtubules (see Figure 3).

Molecules 2020, 25, 660
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0.10
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
Paclitaxel
Control
CA-4
AmCA-4
3.38
3.44
3.39
3.29, 3.50
3.51
3.35
0
10 20 30 40 50 60 70 80 90
t (min)
Figure 3. Effects of paclitaxel, CA-4, AmCA-4 and derivatives on the in vitro tubulin
polymerization.
It can be seen in Figure 3 that almost no absorbance is detected in the presence of CA-4 and
AmCA-4. In fact, the polymerization curve for AmCA-4 overlaps on the x axis. This is indicative of
the inhibition of the polymerization process that these two compounds exert. On the other hand, it
can be seen the high absorbance displayed when the polymerization process is carried out in the
presence of paclitaxel, a compound that promotes tubulin polymerization. In this case, the plateau
phase is reached 30 min after the beginning of the process. CA-4 and AmCA-4 derivatives do not
exert significant effects. Only derivatives 8.2 (L-Ala) and 8.7 (L-Thr) are able to delay the
polymerization process.
Values for It50, which indicate the time required to reach 50% of the polymerization, are given in
Table 5.
Table 5. It50 values.
Compound
Control
Paclitaxel
CA-4
AmCA-4
7.1
It50 (min)
16.6 ± 1.0
3.2 ± 0.4
21.5 ± 0.8
23 ± 3
16 ± 3
7.7
8.1
8.2
19.2 ± 0.3
18.5 ± 2.1
48 ± 5
8.7
9.4
10.1
65 ± 7
16.5 ± 0.3
17.1 ± 1.3
With control (absence of ligand) and with the majority of derivatives, 50% of the polymerization
is reached between 16–20 min. However, with derivative 8.2 (L-Ala), the process suffers a delay of
almost 50 min, while for derivative 8.7 (L-Thr), a 65-min delay is measured. These two compounds
therefore could be classified as partial inhibitors of the polymerization process.
2.4. Effect on the Cell Cycle
The effects of some CA-4 derivatives on the cell cycle were evaluated in A-549 cells. Cell cycle
results for CA-4 derivatives are shown in Table 6, as well as the ligand concentrations at which the
test was conducted (see Supplementary Information for cell cycle histograms). The derivatives were
selected after observing the cell morphology, discarding the compounds that did not show a high
concentration of cells stopped in mitosis, which are those that are characterized by having a rounded
shape and being slightly detached from the lower base of the plate. It can be seen in Table 6 that the

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control sample shows very little accumulation of cells in the G2/M phase, while for CA-4, a majority
of cells were observed in the G2/M phase (60%). This study was performed using several
concentrations for the selected compounds and Table 6 collects the lowest effective concentration for
each compound without causing cell death. Compounds 1, 3.4 (L-Phe-OMe), 3.6 (L-Ser-OMe), 3.7 (L-
Thr-OMe), 3.8 (L-Tyr-OMe), 4.2 (L-Val), 4.3 (L-Leu) and 5.5 (D-Pro-OMe) accumulate between 50–70%
of cells in the G2/M phase, in some cases improving the results obtained for CA-4 (60%). It should be
noted, however, that these compounds exert their actions at concentrations between 1000–1500 times
higher than those of CA-4. Effects of 3.2 (L-Val-OMe), 3.3 (L-Leu-OMe), 3.4 (L-Phe-OMe), 3.9 (L-Met-
OMe), 5.3 (D-Phe-OMe) and 5.4 (D-Pro-OMe) are similar to CA-4, but only when they were tested at
concentrations between 1000-1500 times higher than those of the natural product. In turn, compounds
3.5 (L-Pro-OMe), 4.1 (Gly), 4.5 (L-Pro), 4.9 (L-Met), 6.1 (D-Val), 6.2 (D-Leu), 6.3 (D-Phe), 6.4 (D-Pro) and
6.5 (D-Ser) do not exert any significant effect on the cells.
Table 6. Cell cycle distribution for CA-4 and selected compounds after 24 h of treatment.
Comp. Conc. (µM) G0/G1
S
G2/M
Control
CA-4
1
-
0.05
50
75
75
50
100
75
75
75
75
75
50
75
75
75
25
100
50
75
100
75
75
75
75
75 ± 4 11 ± 2 14 ± 2
25 ± 1 15 ± 1 60 ± 2
23 ± 5 22 ± 4 55 ± 8
42 ± 3 14 ± 4 44 ± 1
45 ± 6 19 ± 2 36 ± 4
33 ± 4 16 ± 5 51 ± 2
77 ± 2 16 ± 2 7 ± 1
30 ± 9 17 ± 6 53 ± 3
25 ± 5 18 ± 1 57 ± 6
17 ± 2 18 ± 1 65 ± 1
47 ± 4 17 ± 3 36 ± 1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
4.1
4.2
4.3
4.5
4.9
5.1
5.2
5.3
5.4
6.1
6.2
6.3
6.4
6.5
68
12
20
17 ± 2 16 ± 3 67 ± 4
22 ± 6 15 ± 2 63 ± 5
72
76
12
11
16
13
53 ± 8 16 ± 2 31 ± 7
43 ± 4 23 ± 4 34 ± 1
38 ± 2 19 ± 2 43 ± 1
36 ± 4 14 ± 3 50 ± 1
67 ± 2 20 ± 2 13 ± 1
85 ± 1
3 ± 2 12 ± 1
74 ± 3 11 ± 6 14 ± 2
71
64
14
18
15
18
The effects of some AmCA-4 derivatives on the cell cycle were also evaluated in A-549 cells. The
derivatives were selected after observing the cell morphology, and the assay was carried out only in
those compounds in which cells stopped in mitosis were detected; that is, cells rounded off and
slightly detached from the lower base of the plate. Compound 3.51 (D-Val), which exerted a lower
antimitotic effect, was also studied in order to compare its effect with those of the other derivatives.
Cell cycle results for selected AmCA-4 derivatives are presented in Table 7, as well as the ligand
concentrations at which the test was conducted. It can be seen in Table 7 that for AmCA-4 a majority
of cells remain accumulated in the G2/M phase, a situation similar to that previously observed for
CA-4. Derivatives 7.1 (Fmoc-Gly), 7.2 (Fmoc-L-Ala), 8.1 (Gly), 8.2 (L-Ala), 8.8 (L-Tyr), 8.9 (L-Met) and
10.1 (D-Val) were capable of accumulating more than 50% of the cells in the G2/M phase, values much
higher than those of the control (14%) and similar to those of CA-4 and AmCA-4, although at
concentrations higher than those of these compounds. Derivatives 7.9 (Fmoc-L-Met), 8.3 (L-Val) and

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10.2 (D-Leu) do not accumulate a high percentage of cells in phase G2/M, and the rest of the
derivatives are in an intermediate situation between those commented above.
Table 7. Cell cycle distribution for CA-4, AmCA-4 and selected compounds after 24 h of treatment.
Compound Conc. (µM) G0/G1
S
G2/M
14 ± 2
60 ± 2
59 ± 2
54 ± 1
53 ± 2
46 ± 3
10 ± 3
46 ± 4
58 ± 5
15 ± 2
48 ± 1
Control
CA-4
AmCA-4
7.1
-
0.05
0.125
0.5
0.5
2
2
2
3
2
75 ± 4
25 ± 1
26 ± 1
28 ± 1
28 ± 1
34 ± 2
72 ± 2
32 ± 5
28 ± 1
65 ± 2
35 ± 1
11 ± 2
15 ± 1
15 ± 2
18 ± 2
19 ± 2
20 ± 1
18 ± 1
22 ± 2
14 ± 4
20 ± 1
17 ± 2
7.2
7.5
7.9
8.1
8.2
8.3
8.4
8.5
0.5
0.5
2
46 ± 2 28 ± 11 26 ± 10
8.7
31 ± 1
30 ± 2
30 ± 1
26 ± 2
63 ± 4
18 ± 1
18 ± 1
19 ± 1
14 ± 1
16 ± 3
51 ± 2
52 ± 3
51 ± 2
60 ± 1
22 ± 2
8.8
8.9
10.1
10.2
0.5
0.2
40
0.5
2.5. Effect on the Cellular Microtubule Network
The effect on the cellular microtubule network of CA-4 derivatives was studied for compounds
1, 3.7 ( -Thr-OMe), 3.8 ( -Tyr-OMe), 4.2 ( -Val) and 4.3 ( -Leu), which were those accumulating more
L
L
L
L
cells in the G2/M phase. Thus, A-549 cells were incubated for 24 h in the presence of the above
compounds and CA-4 at the concentrations indicated in Figure 4. CA-4 (see panel B) greatly
depolymerizes microtubules at a concentration of 50 nM. In addition, derivatives 1 (50 μM, panel C);
3.7 (L
-Thr-OMe, 75 μM, panel D); 3.8 (
L-Tyr-OMe, 75 μM, panel E); 4.4 (L-Val, 50 μM, panel F) and
4.3 (L-Leu, 50 μM, panel G) produce disorganization in the mitotic spindle, although this effect is
exerted at much higher concentrations than in the case of the natural product CA-4.
Figure 4. Effects on the microtubule network. (A) Cellular microtubules with DMSO, (B) 50 nM CA-
4, (C) 50 μM 1, (D) 75 μM 3.7 (
75 μM 4.3 ( -Leu).
L-Thr-OMe), (E) 75 μM 3.8 (L-Tyr-OMe), (F) 50 μM 4.2 (L-Val) and (G)
L

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The effect of some derivatives on the cellular microtubule network were also studied on A-549
cells. The selected compounds were 7.1 (Fmoc-Gly) (0.5 μM), 8.1 (Gly) (2 μM), 8.2 ( -Ala) (3 μM) and
10.1 ( -Val) (40 μM), which were those exerting a higher effect on the cell cycle. In addition, other
derivatives selected were compound 8.5 ( -Phe) (0.5 μM), as it exhibited a low IC50, and compound
10.2 ( -Leu) (0.5 μM), as it did not stand out either for its antiproliferative activity or for its action on
L
D
L
D
the assembly of microtubules or in the cell cycle.
It can be seen in Figure 5 (panel B) that AmCA-4 at a concentration of 50 nM caused a total
depolymerization of microtubules. On the other hand, derivatives 7.1 (Fmoc-Gly, panel C); 8.1 (Gly,
panel D); 8.2 (
of the microtubule network, although at concentrations higher than AmCA-4. Derivatives 8.5 (L-Phe,
panel F) and 10.1 ( -Leu, panel H) are much less active, although some alterations in the microtubule
L-Ala, panel E) and 10.1 (D-Val, panel G) also caused the almost total depolymerization
D
network can be observed.
Figure 5. Effects on the microtubule network. (A) Cellular microtubules with DMSO, (B) 50 nM
AmCA-4, (C) 0.5 μM 7.1 (Fmoc-Gly), (D) 2 μM 8.1 (Gly), (E) 3 μM 8.2 (
(G) 40 μM de 10.1 ( -Val) and (H) 0.5 μM 10.2 ( -Leu).
L-Ala), (F) 0.5 μM 8.5 (L-Phe),
D
D
2.6. Effects on VEGF, hTERT and c-Myc Genes
The effect on the expression of VEGF, hTERT and c-Myc genes has been studied for CA-4 and its
derivatives that show selectivity indexes higher than 0.9 on the HT-29 cell line. The studied
derivatives, together with the concentrations to which they have been tested, are CA-4 (15 μM); 4.1
(Gly, 5 μM); 4.5 (
25 μM) and 6.5 (
L
-Pro, 25 μM); 4.9 (
L-Met, 25 μM); 6.2 (D-Leu, 25 μM); 6.3 (D-Phe, 25 μM); 6.4 (D-Pro,
D
-Ser, 10 μM). The expression of the genes was determined by RT-qPCR
methodology, as described in the Materials and Methods section. The results are presented in Figure
6, which shows the gene expression percentage after 48 h of incubation in the presence of DMSO
(control), CA-4 and each of the selected derivatives.

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Figure 6. Percentages of gene expression (normalized to β-actin). Concentrations: CA-4 (15 μM); 4.1
(Gly, 5 μM); 4.5 (L-Pro, 25 μM); 4.9 (L-Met, 25 μM); 6.2 (D-Leu, 25 μM); 6.3 (D-Phe, 25 μM); 6.4 (D-Pro,
25 μM) and 6.5 (D-Ser, 10 μM).
Some derivatives are quite active in the inhibition of VEGF gene expression, such as compounds
6.2 (D-Leu), which shows 40% of gene expression and 6.4 (D-Pro), which improves (25% gene
expression) the inhibition caused by CA-4 (40%). In the case of the hTERT gene, almost all the
derivatives provoke better percentages of inhibition than CA-4, although it is worth mentioning that
this compound only manages to reduce the expression of the gene up to 85%. Derivative 4.9 (L-Met)
is the most active, expressing only 38% with respect to control. In the case of the c-Myc gene, none of
the derivatives improves the inhibitory capacity of CA-4, which expresses only 35% of this gene.
However, 4.9 (L-Met) downregulates the expression of the c-Myc gene to 37%. Thus, 4.9 (L-Met) can
be considered the most active compound of these CA-4 derivatives, as it reduces the expression of
the three genes studied to 37–55%.
As regards AmCA-4 derivatives, the effect on the expression of the VEGF, hTERT and c-Myc
genes has been studied on those that show IC50 in the low nanomolar range on the HT-29 cell line
and/or on compounds whose selectivity indexes with respect to this cell line are higher than 1. The
studied compounds, together with the concentrations to which they have been tested, are: CA-4 (15
μM), AmCA-4 (15 nM), 7.1 (7 nM), 7.2 (1.5 nM), 7.3 (4 nM), 7.4 (15 nM), 7.5 (2 nM), 7.7 (7 nM), 7.8 (15
nM), 7.9 (30 nM), 8.1 (7 nM), 8.2 (3 nM), 8.3 (10 nM), 8.4 (1.5 nM), 8.5 (4 nM), 8.7 (7 nM), 8.8 (3 nM)
and 8.9 (4 nM). In all cases, the concentration chosen for a given compound is slightly lower than its
IC50 value. The results are presented in Figure 7, which shows the gene expression percentage after
48 h of incubation in the presence of DMSO (control), CA-4, Am-CA-4 and each of the selected
derivatives.

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CA-4 AmCA-4 7.1
7.2 7.3 7.4 7.5 7.7
7.8 7.9
8.1 8.2 8.3 8.4 8.5
8.7 8.8 8.9
CA-4 AmCA-4 7.1 7.2 7.3 7.4 7.5 7.7 7.8 7.9
8.1 8.2 8.3 8.4 8.5 8.7 8.8 8.9
CA-4 AmCA-4
7.1
7.2 7.3 7.4 7.5 7.7 7.8 7.9 8.1 8.2 8.3 8.4 8.5
8.7 8.8 8.9
Figure 7. Percentages of gene expression. Concentrations: CA-4 (15 μM), AmCA-4 (15 nM), 7.1 (7 nM),
7.2 (1.5 nM), 7.3 (4 nM), 7.4 (15 nM), 7.5 (2 nM), 7.7 (7 nM), 7.8 (15 nM), 7.9 (30 nM), 8.1 (7 nM), 8.2 (3
nM), 8.3 (10 nM), 8.4 (1.5 nM), 8.5 (4 nM), 8.7 (7 nM), 8.8 (3 nM) and 8.9 (4 nM).
As regards the expression of the VEGF gene, half of the tested AmCA-4 derivatives show
expression percentages lower than 40%, values lower than those of CA-4 and AmCA-4. The most
outstanding percentages are those of 7.2 (Fmoc-L-Ala, 25%), 7.3 (Fmoc-L-Val, 23%), 7.5 (Fmoc-L-Phe,
16%), 8.1 (Gly, 24%) and 8.7 (L-Thr) (15%). In the expression of the hTERT gene, AmCA-4 derivatives
are not as active as in the case of the VEGF gene. The most prominent compound is 8.8 (L-Tyr, 46%),
which improves the percentages of both CA-4 and AmCA-4, while derivatives 7.1 (Fmoc-Gly), 7.5
(Fmoc-L-Phe), 7.7 (Fmoc-L-Thr) and 8.3 (L-Val), with expression percentages lower than 70%, are
more active than CA-4 and at the same level as AmCA-4. For the c-Myc gene, most derivatives exert
a prominent effect, particularly 7.2 (Fmoc-L-Ala), which reduces the expression up to 22% and is more
active than the reference compounds CA-4 and AmCA-4. Compounds 7.5 (Fmoc-L-Phe, 40%), 8.4 (L-
Leu, 48%), 8.7 (L-Thr, 37%) and 8.8 (L-Tyr, 42%) also deserve mention, as they are able to reduce the
expression of the c-Myc gene by half. The most notable compound in the inhibition of gene expression
is 7.5 (Fmoc-L-Phe), which shows very active in the inhibition of the three genes studied.
3. Discussion
As regards antiproliferative activities, CA-4 derivatives show IC50 values in the low micromolar
range for all the cell lines studied. Derivatives that incorporate D-amino acids, together with the
derivative 4.9 (L-Met), exhibit, in general, the best therapeutic safety margins, with values higher than
1 for HT-29 and MCF-7 and close to this value on the A-549 line. AmCA-4 derivatives show
antiproliferative activities ranging from low nanomolar values for L-derivatives (except for L-Pro
derivatives, which show IC50 values in the high nanomolar range) to low micromolar values for D-
derivatives. Figure 8 logarithmically compares IC50 values on the HT-29 line for L- and D-AmCA-4.

Molecules 2020, 25, 660
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Thus, differences of 2-4 orders of magnitude can be seen in the antiproliferative activities of L-and D-
AmCA-4 derivatives, even though these differences are much more attenuated in the case of proline
derivatives.
Figure 8. IC50 values (μM) in the logarithmic scale for the HT-29 cell line.
In relation to antimitotic activities, CA-4 derivatives bearing D-amino acids do not exert an
appreciable effect on the cell cycle, while those with D-amino esters do exert a certain antimitotic
effect, accumulating between 30–50% of cells in the G2/M phase. However, derivatives with L-amino
acids exert a greater effect on the cell cycle, even improving the effect of CA-4. The most active
derivatives are those bearing hydroxylated chain amino esters, such as 3.7 (L-Thr-OMe) and 3.8 (L-
Tyr-OMe), as well as those bearing amino acids with short hydrophobic side chains, such as 4.2 (L-
Val) and 4.3 (L-Leu). AmCA-4 derivatives tested have generally been quite active at concentrations
lower than those used with CA-4 derivatives. Thus, F-moc-protected derivatives with short chains,
7.1 (Fmoc-Gly), 7.2 (Fmoc-L-Ala) and 8.2 (L-Ala), and those containing hydroxylated amino acids (8.7
L-Thr and 8.8 L-Tyr) or sulfur-containing amino acids (8.9 L-Met) accumulate more than 50% of cells
in the G2/M phase. In addition, compounds 8.2 (L-Ala) and 8.7 (L-Thr) are partial inhibitors of the in
vitro polymerization of tubulin.
In gene inhibition expression, CA-4 derivatives are generally more active against the VEGF gene,
with 6.2 (D-Leu) being particularly active. The most active compound of that group, however, is 4.9
(L-Met), which downregulates the expression of the three genes studied more than 55%. The most
active AmCA-4 derivatives on VEGF and c-Myc genes are 7.2 (Fmoc-L-Ala), 7.5 (Fmoc-L-Phe) and 8.7
(L-Thr), since they downregulate the expression of these two genes more than 60%. For the set of the
three genes, the most notable derivative is 7.5 (Fmoc-L-Pro), since it is able to reduce VEGF expression
to 16%, and c-Myc and hTERT expression to 56%.
4. Materials and Methods
4.1. Chemistry
All chemicals were supplied by Sigma-Aldrich Inc. (Darmstadt, Germany). NMR spectra were
measured at 25 °C in an Avance III HD NMR spectrometer (Bruker Biospin AG, Bruker Española,
Madrid, Spain). The signals of the deuterated solvent (CDCl3) were taken as the reference.
13
Multiplicity assignments of C signals were made by means of the DEPT pulse sequence. Complete
1
13
signal assignments in H and C NMR spectra were made with the aid of 2D homo- and
heteronuclear pulse sequences (COSY, HSQC and HMBC). High-resolution mass spectra were run
by the electrospray mode (ESMS). IR data were measured with oily films on NaCl plates (oils) and
given only for relevant functional groups (C=O and NH). Experiments which required an inert
atmosphere were carried out under dry N2 in flame-dried glassware. Commercially available
reagents were used as received.

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4.1.1. Synthesis of Ethyl (Z)-2-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)acetate 1.
K2CO3 (2 mmol) was added to a solution of combretastatin A-4 (316 mg, 1 mmol) in anhydrous
DMF (20 mL), and the reaction mixture was stirred, under N2 atmosphere, for one hour at room
temperature. Then, ethyl bromoacetate (1.5 mmol) was added, and the resulting mixture was stirred
at room temperature for 24 h. The mixture was then poured into a saturated aqueous NH4Cl solution
and extracted with Et2O (3 × 30 mL), and the combined organic extracts were dried over anhydrous
MgSO4. After filtration and evaporation of the solvent under reduced pressure, the resulting residue
was chromatographed on silica gel with Hexanes-AcOEt (7:3) to afford 378 mg (94%) of compound
1.
4.1.2. Synthesis of (Z)-2-(2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)acetic acid 2.
NaOH (10 mmol) in H2O (2.6 mL) was added to a solution of ester 1 (201 mg, 0.5 mmol) in EtOH
(25 mL). The mixture was stirred for one hour at room temperature, and then EtOH was removed
under reduced pressure. The residue was acidified with 2 M HCl and extracted with CH2Cl2 (3 × 30
mL), and the combined organic extracts were dried over anhydrous MgSO4. After filtration and
evaporation of the solvent under reduced pressure, the resulting residue was recrystallized from the
Hexanes-AcOEt mixture (8:2) to afford 170 mg (91%) of compound 2.
4.1.3. General Procedure for the Synthesis of N-acyl-amino Ester Derivatives 3 and 5.
DMAP (1.14 mmol, 2.28 eq.) and the corresponding amino ester hydrochloride (0.57 mmol, 1.14
eq.) were added to a solution of acid 2 (0.5 mmol, 1 eq) in anhydrous DMF (10 mL). The reaction
mixture was cooled at 0 °C, and then EDCI·HCl (0.55 mmol, 1.1 eq.) was added. The resulting mixture
was stirred for 2 h at 0 °C and then brought to room temperature and left to react for 2 h. The mixture
was then poured into a saturated aqueous NH4Cl solution and extracted with AcOEt (3 × 20 mL). The
combined organic extracts were washed with H2O and brine and dried over anhydrous MgSO4. After
filtration and evaporation of the solvent under reduced pressure, the resulting residue was
chromatographed on silica gel with Hexanes-AcOEt (6:4), affording the corresponding N-acyl-
aminoester derivatives 3 and 5.
4.1.4. General Procedure for the Synthesis of N-acyl-amino Acid Derivatives 4 and 6.
LiOH·H2O (0.45 mmol, 3 eq.) in H2O (2.2 mL) was added to a solution of amino ester 3 or 5 (0.15
mmol, 1 eq.) in MeOH (2.2 mL). The mixture was stirred for one hour at room temperature and then
poured into H2O and washed with Et2O. The resulting aqueous phase was acidified with 2M HCl and
extracted with AcOEt (3 × 30 mL). The combined organic extracts were dried over anhydrous MgSO4.
Filtration and evaporation of the solvent under reduced pressure afforded the corresponding amino
acid derivatives 4 and 6.
4.1.5. General Procedure for the Synthesis of N-Fmoc-Protected Derivatives 7 and 9.
DCC (0.47 mmol, 1.2 eq.); the N-Fmoc-protected amino acid (0.472 mmol, 1.2 eq) and HOBt·H2O
(0.47 mmol, 1.2 eq) were added to a solution of the 3′-aminocombretastatin (0.39 mmol, 1 eq) in
anhydrous DMF (1.3 mL). The reaction mixture was stirred under N2 atmosphere for 16 h. Then, it
was filtered, and the filtrate was poured into a saturated aqueous NH4Cl solution and then extracted
with AcOEt (3 × 15 mL). The combined organic extracts were washed 2 times with water and brine
and dried over anhydrous MgSO4. After filtration and evaporation of the solvent under reduced
pressure, the resulting residue was chromatographed on silica gel with Hexanes-AcOEt (6:4) to afford
the corresponding N-Fmoc-protected derivatives 7 and 9.
4.1.6. General Procedure for the Synthesis of 8 and 10 Derivatives by N-Fmoc Deprotection
Anhydrous KF (1.5 mmol, 10 eq) and 18-crown-6 (0.015 mmol, 0.1 eq.) were added to a solution
of the N-Fmoc-protected derivative (0.15 mmol, 1 eq.) in anhydrous DMF (5.5 mL). The resulting

Molecules 2020, 25, 660
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mixture was stirred at room temperature under N2 atmosphere for 4 h. Then, the reaction mixture
was poured into a saturated aqueous NH4Cl solution and extracted with AcOEt (3 × 25 mL). The
combined organic extracts were washed sequentially with water (×2) and with brine and dried over
anhydrous MgSO4. After filtration and evaporation of the solvent under reduced pressure, the
resulting residue was chromatographed on silica gel with AcOEt to afford derivatives 8 and 10.
4.2. Biological Assays
4.2.1. Cell culture
Cell culture media were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum
(FBS) was a product of Harlan-Seralab (Belton, U.K.). Supplements and other chemicals not listed in
this section were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Plastics for cell culture
were supplied by Thermo Fischer Scientific Inc., Madrid, Spain. All tested compounds were
dissolved in DMSO at a concentration of 10 μg/mL and stored at −20 °C until use. HT-29, MCF-7, A-
549 and HEK-293 cell lines were maintained in a Dulbecco’s modified Eagle’s medium (DMEM)
containing glucose (1 g/L), glutamine (2 mM), penicillin (50 IU/mL), streptomycin (50 μg/mL) and
amphotericin B (1.25 μg/mL), supplemented with 10% FBS. Cells from human non-small lung
carcinoma (clone A549; ATCC n°: CCL2, MD, USA) were routinely grown at 37 °C in a humidified
atmosphere of 5% CO2. Cells were maintained in a standard medium composed of RPMI 1640 (Lonza,
Group Ltd., Basel, Switzerland), supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine
and 1% penicillin and streptomycin (Invitrogen). Cells were free from mycoplasma as determined by
MycoAlert tests (Lonza, Group Ltd., Basel, Switzerland).
4.2.2. Cell Proliferation
For cell viability assay on the cell line A549, cells harvested from subconfluent monolayers were
seeded at 25,000/mL in a 96-well microtiter plate (Cambridge Technology, France) and cultured 24 h
under standard conditions. Standard medium was then replaced by fresh medium containing no
drugs (control or “0”) or compounds at different concentrations. The surviving cells were quantified
after 72 h by the MTT assay, according to the manufacturer’s instructions. Briefly, 20 μL of 3-(4,5-
−1
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution at 5 mg·mL were added to
cells for 2 h at 37 °C. The supernatant was discarded and replaced by 200 μL of DMSO to dissolve
formazan crystals. The absorbance was then read at 540 nm by spectrophotometry. For all
concentrations of the compound, cell viability was expressed as the percentage of the ratio between
the mean absorbance of treated cells and the mean absorbance of untreated cells. Three independent
experiments were performed, and the IC50 values (i.e., concentration half inhibiting cell proliferation)
were graphically determined.
3
A total of 5 × 10 HT-29, MCF-7 or HEK-293 cells in a total volume of 100 μL of their respective
growth media were incubated with serial dilutions of the tested compounds. The 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co.) dye reduction
assay in a 96-well microplate was used, as previously described [33]. After 3 days of incubation (37
°C, 5% CO2 in a humid atmosphere), 10 μL of MTT (5 mg/mL in phosphate-buffered saline; PBS) was
added to each well, and the plate was incubated for a further 4 h (37 °C). The resulting formazan was
dissolved in 150 μL of 0.04 N HCl/2-propanol and read at 550 nm. All determinations were carried
out in triplicate.
4.2.3. Fluorescence Microscopy
6
For immunofluorescence microscopy of the microtubule network, 10 cells were plated on cover
glass and incubated with the different drugs. Cells were then fixed in 3.7% formaldehyde (in PBS pH
7.4) for 20 min at room temperature and permeabilized with PBS-Triton X-100 0.5% for 10 min at
room temperature. Direct immunostaining was carried out for 2 h at room temperature with a
primary FITC-conjugated anti-α-tubulin antibody (dilution 1:400 in PBS-BSA 1% from a 1 mg/mL
solution; monoclonal antibody, clone DM1A; Sigma-Aldrich, Darmstadt, Germany.). Next, cells were

Molecules 2020, 25, 660
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incubated for 45 min at room temperature in darkness with secondary Alexa Fluor 488-conjugated
antibodies (dilution 1:200 each from 1.5 mg/mL; Molecular Probe, France). Then, cells were washed
®
in PBS and cover glasses were mounted with a drop of ProLong antifade solution (Invitrogen). The
cytoskeleton was imaged by a confocal laser scanning microscope (CLSM) Leica SP5 with a Leica
inverted microscope, equipped with a Plan-Apochromat 63× oil immersion objective (NA = 1.4). Each
image was recorded with the CLSM’s spectral mode selecting specific domains of the emission
spectrum. The FITC fluorophore was excited at 488 nm with an argon laser, and its fluorescence
emission was collected between 496 nm and 535 nm.
4.2.4. Cell Cycle
5
For flow cytometric analysis of DNA content, 4 × 10 cells in exponential growth were treated
with the IC50 value and ten times this value of each compound; thus, 14 nM and 140 nM for CLC, 18
nM and 180 nM compound 2, 133 nM and 1.33 μM compound 3, 25 nM and 250 nM compound 4, 62
nM and 620 nM compound 5 and 72 nM and 720 nM compound 6 for 24 h. The supernatant and
trypsinated cells were harvested and then centrifuged (1200 rpm, 5 min, 4 °C). The cell pellet was
resuspended in 1 mL of cold 70% ethanol for 30 min at −20 °C. Cells were centrifuged (2000 rpm, 5
min, 4 °C) to remove ethanol, and then the cell pellet was resuspended in the staining mix containing
50 μg/mL propidium iodide (Molecular Probes, France) and 100 μg/mL RNAse A (Sigma, France) in
PBS for 20 min at room temperature in darkness. Samples were analyzed on a Becton Dickinson
FACScan flow cytometer using the CellQuest software, which was also used to determine the
percentage of cells in the G2/M phases of the cell cycle. PI was excited at 488 nm, and fluorescence
analyzed at 620 nm on channel Fl-2.
4.2.5. Gene Expression Evaluation by RT-qPCR-assay
HT-29 cells at 70–80% confluence were collected, and 1.5 × 105 cells were placed in a six-well
plate in 1.5 mL of the medium. After 24 h, cells were incubated with the corresponding compounds
for 48 h. Cells were collected, and the total cellular RNA from the HT-29 cells was isolated using an
Ambion RNA Extraction Kit, according to the manufacturer’s instructions. The cDNA was
synthesized by MMLV-RT with 1–21 μg of extracted RNA and oligo(dT)15, according to the
®
manufacturer’s instructions. Genes were amplified by a thermal cycler and StepOnePlus™ TaqMan
®
probes. TaqMan Gene Expression Master Mix Fast, containing the appropriate buffer for the
amplification conditions, dNTPs, thermostable DNA polymerase enzyme and a passive reference
probe, was used. To amplify each of the genes, the predesigned primers sold by Life Technologies
®
TaqMan Gene Expression Assays Hs99999903-m1 (β-actin), Hs00900055-m1 (VEGF), Hs00972646-
m1 (hTERT) and Hs00153408-m1 (c-Myc) were used.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1: cell cycle histograms;
spectroscopic data and 1H-NMR, 13C-NMR and HR-MS spectra of compounds 1, 2, 3.1–3.9, 4.1–4.9, 5.1–5.5, 6.1–
6.5, 7.1–7.9, 8.1–8.9, 9.1–9.4 and 10.1–10.4.
Author Contributions: Ideas and experiment design: E.F., J.M. and M.C.; chemistry: J.M., R.A., R.G.-E. and A.P.;
biology: E.F., R.A and C.M.-B.; analysis and interpretation of data: R.A., E.F. and M.C.; writing and review of the
manuscript: E.F., M.C. and J.A.M. and study supervision: E.F., M.C. and J.A.M. All authors have read and agreed
to the published version of the manuscript.
Funding: This research was funded by the Spanish Ministerio de Economia y Competitividad (project CTQ2014-
52949-P), by the Spanish Ministerio de Ciencia, Innovación y Universidades (project RTI2018-097345-B-I00) and
by Jaume I University (project UJI-B2018-38).
Acknowledgments: R. A. thanks the Universitat Jaume I for a predoctoral fellowship. R. G.-E. thanks the
Asociación Española Contra el Cancer (AECC) for a predoctoral fellowship. The authors are also grateful to the
SCIC of the Universitat Jaume I for providing NMR and mass spectrometry facilities.
Conflicts of Interest: The authors declare no conflicts of interest.

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Sample Availability: Samples of all compounds are available from the authors.
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).