New series of double-modified colchicine derivatives: Synthesis, cytotoxic effect and molecular docking

Article abstract of DOI:10.3390/molecules25153540

Colchicine is a well-known anticancer compound showing antimitotic effect on cells. Its high cytotoxic activity against different cancer cell lines has been demonstrated many times. In this paper we report the syntheses and spectroscopic analyses of novel colchicine derivatives obtained by structural modifications at C7 (carbon-nitrogen single bond) and C10 (methylamino group) positions. All the obtained compounds have been tested in vitro to determine their cytotoxicity toward A549, MCF-7, LoVo, LoVo/DX, and BALB/3T3 cell lines. The majority of obtained derivatives exhibited higher cytotoxicity than colchicine, doxorubicin and cisplatin against the tested cancerous cell lines. Additionally, most of the presented derivatives were able to overcome the resistance of LoVo/DX cells. Additionally, their mode of binding to β-tubulin was evaluated in silico. Molecular docking studies showed that apart from the initial amides 1 and 2, compound 14, which had the best antiproliferative activity (IC₅₀ = 0.1–1.6 nM), stood out also in terms of its predicted binding energy and probably binds best into the active site of βI-tubulin isotype.

Full text of DOI:10.3390/molecules25153540

molecules
Article
New Series of Double-Modified Colchicine
Derivatives: Synthesis, Cytotoxic Effect and
Molecular Docking
Julia Krzywik 1
,2
, Maral Aminpour , Ewa Maj
3
4
, Witold Mozga , Joanna Wietrzyk 4
,
2
Jack A. Tuszy n´ ski 3
,5
and Adam Huczy n´ ski *
1,
1
Department of Medical Chemistry, Faculty of Chemistry, Adam Mickiewicz University, Uniwersytetu
Pozna n´ skiego 8, 61-614 Pozna n´ , Poland; julia.krzywik@amu.edu.pl
TriMen Chemicals, Piłsudskiego 141, 92-318 Łód z´ , Poland; mozga@trimen.pl
Department of Oncology, University of Alberta, Edmonton, AB T6G 1Z2, Canada;
aminpour@ualberta.ca (M.A.); jack.tuszynski@gmail.com (J.A.T.)
2
3
4
Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla
1
2, 53-114 Wrocław, Poland; ewa.maj@hirszfeld.pl (E.M.); joanna.wietrzyk@hirszfeld.pl (J.W.)
5
DIMEAS, Politecnico di Torino, Corso Duca degli Abruzzi, 24, 10129 Torino, Italy
*
Correspondence: adhucz@amu.edu.pl; Tel.: +48-61-8291673
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Bartosz Tylkowski, Anna Bajek and Krzysztof Roszkowski
Received: 14 July 2020; Accepted: 31 July 2020; Published: 2 August 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Colchicine is a well-known anticancer compound showing antimitotic effect on cells. Its
high cytotoxic activity against different cancer cell lines has been demonstrated many times. In this
paper we report the syntheses and spectroscopic analyses of novel colchicine derivatives obtained by
structural modifications at C7 (carbon-nitrogen single bond) and C10 (methylamino group) positions.
All the obtained compounds have been tested in vitro to determine their cytotoxicity toward A549,
MCF-7, LoVo, LoVo/DX, and BALB/3T3 cell lines. The majority of obtained derivatives exhibited
higher cytotoxicity than colchicine, doxorubicin and cisplatin against the tested cancerous cell lines.
Additionally, most of the presented derivatives were able to overcome the resistance of LoVo/DX cells.
Additionally, their mode of binding to
β-tubulin was evaluated in silico. Molecular docking studies
showed that apart from the initial amides
1
and , compound 14, which had the best antiproliferative
2
activity (IC50 = 0.1–1.6 nM), stood out also in terms of its predicted binding energy and probably
binds best into the active site of βI-tubulin isotype.
Keywords: anticancer agents; colchicine derivatives; reductive alkylation; tubulin inhibitors;
docking studies
1
. Introduction
Tubulin is the target of some of the most widely used anticancer anti-mitotic agents, such as
vinblastine or paclitaxel [1,2]. Unfortunately, clinical usefulness of many microtubule disrupting agents
has been reduced as a result of tumor cell multidrug-resistance (MDR). The main role in MDR is played
by P-glycoprotein, which is overexpressed in various types of cancers and is known to be associated
with poor response to chemotherapy [3–5]. Colchicine 1 (see Scheme 1) is a plant-based alkaloid
extracted from Colchicum autumnale and Gloriosa superba that shows antimitotic effects on a number of
cancer cell lines. It binds to tubulin, inhibiting its assembly and microtubule polymerization and finally
arresting cell division at metaphase [
6–12]. However, in addition to the problem with cancer cells
developing drug resistance, colchicine was found to have some toxic effects, including accumulation in
the gastrointestinal tract, as well as neurotoxicity [
limit the use of colchicine in chemotherapy.
6
,13
–
19]. These are the two main disadvantages that
Molecules 2020, 25, 3540; doi:10.3390/molecules25153540
www.mdpi.com/journal/molecules

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Scheme 1. Synthesis of double-modified colchicine derivatives (2–17), changes at C7 and C10 positions
are highlighted in red. Reagents and conditions: ( ) NHCH /EtOH, reflux; ( ) 2M HCl, reflux;
c) respective aldehyde, NaBH CN, MeOH, RT for 4–9 and 11–17; (d) acrylonitrile, MeOH, RT for 10.
a
b
3
(
3
Despite the above limitations, colchicine exhibits high antiproliferative activity and represents
an interesting research area for understanding of relationship between the structure and biological
activity (SAR) of compounds interacting with tubulin. Many colchicine analogues containing amide or
urethane at position C7 have been studied [20–27]. Such groups may be hydrolyzed in vivo causing a
change in pharmacological activity [28 29]. Conversion of such moieties into single carbon-nitrogen
,
bond prevents hydrolysis of the side chain in the cells and creates a new class of colchicine derivatives.
Numerous modified colchicine and thiocolchicine derivatives with substituted benzyl moieties at
carbon C7 have been synthesized and their biological activity has been evaluated. Some of the described
derivatives showed antiproliferative activity superior to that of colchicine [22,30]. In addition, as
shown in our earlier studies, the replacement of -OCH group at position C10 with -NHCH group
3
3
leads to generation of compounds more active than unmodified colchicine [20]. On the basis of these
findings, we decided to develop the concept, design, and synthesize a new series of double-modified
colchicine derivatives with different amine moieties at C7 atom (aromatic but also alkyl substituents
that have not been previously widely studied) and with a methylamino group at C10 atom in the
tropolone ring C.
Herein, we report the syntheses and spectroscopic analyses of a series of structurally different
derivatives of colchicine obtained by its modification at C7 (amide bond replacement by a
carbon-nitrogen single bond) and C10 (methylamino group) positions. We also describe the results of
in vitro antiproliferative activity evaluation of the starting compounds
derivatives ( 17) against four human cancer cell lines, namely A549, MCF-7, LoVo, and LoVo/DX
as well as normal murine cells BALB/3T3. To gain more knowledge about molecular mechanism of
action of the investigated compounds ( 17), we also present in silico results of a molecular docking
1–2 and the obtained colchicine
3–
1
–

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study into the colchicine-binding site (CBS) of
β-tubulin. The derivatives presented in this work are
double-modified colchicine derivatives with the amine groups at C7 and C10 positions. On the basis of
the obtained preliminary structure–activity relationship (SAR) studies, we will be able to design new
promising compounds for more extended research.
2
. Results and Discussion
2
.1. Chemistry
To determine the effect of replacement of the amide moiety at position C7 by a single
nitrogen-carbon bond and, at the same time, -OCH group replacement by -NHCH at position
3
3
C10 of colchicine
1
, on its bioactivity, fourteen new derivatives (
4–
17) were designed. The obtained
compounds contained different moieties (replacing acetamide of unmodified compound
nitrogen at position C7, such as: alkyl chains of various length, straight or branched (
containing various substituents ( 12), aromatic moiety with or without substituents (13
heteroatom in the ring (16 17). Diversity of groups at position C7 is intended to facilitate the analysis
1
) attached to
), alkyl chains
15) or with
4–8
9–
–
–
of the structure–activity relationship. The synthetic routes to colchicine derivatives 2–17 are outlined
in Scheme 1.
Compound
hydrolysis with 2M HCl [20
starting material for the synthesis of new derivatives (
previously [22], reductive alkylation of in a reaction with the corresponding aldehyde in the presence of
sodium cyanoborohydride allowed us to obtain double-modified compounds and 11 17. However,
3
was readily available from
31]. N-deacetyl-10-methylamino-10-demethoxycolchicine
17). On the basis of the method described
1
by treatment with methylamine and followed by
,
3
became the
4–
3
4–
9
–
aldehydes used in the present paper did not require activation with acid, so the reactions were carried
out without the addition of acetic acid, which eliminated the formation of unidentified by-products.
Most of the aldehydes necessary for the synthesis of the designed compounds were purchased, but
aldehydes for the synthesis of compounds
4 and 11 were obtained by oxidation of the corresponding
alcohols using pyridinium chlorochromate, PCC [32]. Compound 10 was synthesized via addition of
amine (
pure form after column flash chromatography on silica gel.
The structures and purities of all products
3
) to the Michael acceptors (acrylonitrile). All obtained compounds (
2
–
17) were isolated in
1
2
–
17 were determined using the LC-MS, H and
13
C NMR analyses, and are shown in Supplementary Materials and discussed below. The signals
1
13
corresponding to -OCH group at position C10 of
1
in the H NMR and C NMR spectra were observed
3
as singlets at 4.0 ppm and at 56.5 ppm, respectively. After replacement of this group in the tropolone
ring of colchicine by -NHCH group in compounds
doublet and 7.2–7.3 ppm as a quartet in H NMR spectra and at 29.5–29.9 ppm in C NMR spectra.
2
–
17, new signals appeared, at 3.0–3.1 ppm as a
3
1
13
The signals characteristic of the acetyl group in colchicine
1
appeared at 1.9 ppm (-CH ) and 8.6 ppm
3
1
13
(
-NH-) in H NMR and at 22.7 ppm (-CH ) and 170.3 ppm (-C=O) in C NMR, respectively. These
3
signals are no longer observed in the NMR spectra of
hydrogen atom at C7 has been shifted from 4.6 ppm for unmodified colchicine
compounds 17. The ESI mass spectrometry confirmed the structure of the obtained analogs by the
presence of m/z signals assigned to the corresponding pseudomolecular ions of these compounds.
3
–17 derivatives. The signal assigned to the
1
to 3.4–3.6 ppm for
3–
2
.2. In Vitro Determination of Drug-Induced Inhibition of Human Cancer Cell Line Growth
The tested compounds ( 17) were evaluated for their in vitro antiproliferative effect on four
1–
human cancer cell lines and one normal murine embryonic fibroblast cell line according to the previously
published procedure [20]. Detailed information concerning antiproliferation assay can be found in
Supplementary Materials.
All obtained derivatives showed better antiproliferative activity than those of the commonly used
antitumor agents doxorubicin and cisplatin. It is also worth noting that all compounds (except
7 with

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long alkyl chain) were characterized by very high cytotoxicity with IC50
four tumor lines tested (see Table 1).
≤
10 nM towards three of the
Table 1. Antiproliferative activity (IC50) [nM] of colchicine (
1
) and its derivatives (
2–
17) compared with
that of standard anticancer drugs doxorubicin and cisplatin.
A549
MCF-7
LoVo
LoVo/DX
BALB/3T3
Compound
IC50 [nM]
IC50 [nM]
IC50 [nM]
IC50 [nM]
IC50 [nM]
1
2
3
4
5
6
7
8
9
45.2 ± 18.6
7.2 ± 1.3
8.6 ± 3.9
9.4 ± 3.2
10.5 ± 0.7
8.1 ± 2.6
88.4 ± 3.2
9.6 ± 1.3
9.2 ± 2.2
9.0 ± 1.0
8.4 ± 2.2
9.5 ± 0.2
4.6 ± 1.8
1.1 ± 0.7
8.9 ± 1.4
8.9 ± 1.7
9.4 ± 0.5
10.9 ± 0.9
1.6 ± 0.5
9.8 ± 4.5
8.7 ± 3.3
10.4 ± 0.6
8.7 ± 3.1
75.2 ± 10.8
9.7 ± 1.5
10.1 ± 1.2
7.5 ± 0.7
7.6 ± 2.8
9.0 ± 1.7
7.3 ± 4.4
0.7 ± 0.4
8.5 ± 0.9
9.6 ± 1.8
9.3 ± 0.6
9.9 ± 1.0
1.2 ± 0.2
7.4 ± 2.7
7.3 ± 2.5
5.0 ± 0.1
7.8 ± 1.3
46.6 ± 15.5
7.8 ± 1.0
5.6 ± 1.5
2.0 ± 0.6
1.3 ± 0.3
2.3 ± 1.3
0.9 ± 0.4
0.1 ± 0.02
5.2 ± 0.5
5.2 ± 1.9
0.9 ± 0.3
702.2 ± 38.2
80.6 ± 18.7
39.5 ± 18.6
62.0 ± 7.5
17.2 ± 6.7
13.3 ± 5.8
99.5 ± 35.5
8.5 ± 1.1
33.1 ± 16.0
7.5 ± 1.0
7.1 ± 3.6
8.0 ± 1.6
8.6 ± 0.4
7.5 ± 1.6
70.4 ± 1.2
7.5 ± 1.5
8.2 ± 0.9
6.7 ± 1.3
5.9 ± 2.5
7.9 ± 0.4
6.2 ± 0.7
4.5 ± 2.7
7.3 ± 0.7
6.9 ± 1.3
4.9 ± 2.8
132.0 ± 37.5
51.5 ± 8.6
12.8 ± 3.3
8.2 ± 0.6
10
11
12
13
14
15
16
17
6.0 ± 0.8
1.6 ± 1.0
72.2 ± 12.5
48.1 ± 14.5
7.9 ± 1.0
Doxorubicin
Cisplatin
172.0 ± 58.0
131.5 ± 63.7
83.2 ± 61.1
4814.7 ± 1996.9
32.3 ± 28.6
4916.9 ± 1852.4
5812.2 ± 2610.6
5463.2 ± 1412.4
5013.9 ± 1562.2
3968.8 ± 995.1
The IC50 value is defined as the concentration of a compound at which 50% growth inhibition is observed. The IC50
values shown are mean
±
SD. Human lung adenocarcinoma (A549), human breast adenocarcinoma (MCF-7), human
colon adenocarcinoma cell line (LoVo), and doxorubicin-resistant subline (LoVo/DX), normal murine embryonic
fibroblast cell line (BALB/3T3).
Even though compound
7 has a weak potential compared to the other derivatives with an
amine moiety on C7 carbon and methylamino group on C10 carbon, its IC50 values were in the
nanomolar range. The activities of the remaining derivatives against A549 cells were better than that
of colchicine
0-methylaminocolchicine
by the lowest IC50 values towards the A549 line. In relation to the MCF-7 cell line, the newly designed
derivatives ( 17) showed activities similar to that of colchicine and only one compound—a derivative
with a 2-chlorobenzyl moiety 14—stood out from them having a lower IC50 = 0.7 nM, comparable with
that of amide (IC50 = 1.6 nM). In relation to the LoVo cells, most of the new derivatives exhibited higher
activities than unmodified colchicine , and five of them had IC50 comparable to amide . Derivative
turned out to be the most cytotoxic towards the LoVo cell line (IC50 = 0.1 nM). As many as five of
the obtained derivatives ( 12 14 and 17) exhibited IC50 10 nM towards LoVo/DX cells. Moreover,
compound 14 (IC50 = 1.6 nM) was observed to be the most active towards the doxorubicin-resistant
subline LoVo/DX, approx. 440 times more potent than unmodified colchicine (IC50 = 702.2 nM).
1
and for the majority of compounds comparable with the second starting compound
1
2. Compounds 13 (IC50 = 4.6 nM) and 14 (IC50 = 1.1 nM) were characterized
4–
1
2
1
2
1
4
8,
–
≤
1
Comparison of the results for C7-amine double-modified analogues and those of previously published
studies of C7-amide double-modified analogues [20] showed that in both cases some of the most
cytotoxic compounds were derivatives containing a 2-chlorobenzyl ring at position C7. These results
are noteworthy and suggest that extended and more detailed research is necessary to determine the
meaning and role of the above mentioned substituent. Additionally, it should be mentioned that the
antiproliferative activity depends on the type of the tested cell lines and is different for different cells.
In vitro tests allow the initial determination of biological activity, but in vivo studies are necessary to
determine the therapeutic potential.

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Selectivity index (SI) is a major challenge in drug discovery, because it defines the ability of a
particular compound to preferentially kill tumor cells in relation to normal cells. Therefore, the obtained
compounds were tested not only against cancerous cells but also against the normal murine embryonic
fibroblast cell line (BALB/3T3). The majority of the tested derivatives were characterized with low SI
values (see Figure 1), smaller than those found for the starting compounds
(with 2-chlorobenzyl moiety at C7 carbon), which stands out from the obtained compounds is
noteworthy. Its SI values were especially high for all tested cancer cell lines (SI = 4.1 for A549 cells, SI
6.1 for MCF-7 cells, SI = 93.7 for LoVo cells, SI = 2.8 for LoVo/DX cells) and were higher than SI of
parental amides and . N-(2-chlorobenzyl)-10-methylaminocolchicine 14 therefore emerges as a very
promising molecule (the lowest IC50 and the highest SI) for further more detail research (in vitro but
also in vivo) as a potential anticancer agent. Moreover, compounds 10 13 and 17 exhibited SI > 3 for
LoVo cells. The results indicated that conversion of acetyl at C7 atom of the starting compounds
and into a single carbon-nitrogen bond did not bring (except the derivatives mentioned above, in
1–2. However, derivative
1
4
=
1
2
–
1
2
particular 14) more selective compounds in relation to the cancer cells tested.
Figure 1. Comparison of selectivity index (SI) values of the tested compounds. The SI (Selectivity Index)
was calculated for each compound using the formula: SI = (IC50 for normal cell line BALB/3T3)/(IC50
for respective cancerous cell line). A favorable SI > 1.0 indicates a drug with efficacy against tumor
cells greater than the toxicity against normal cells.
The data presented in Table 1 show that the studied compounds inhibited the proliferation of
the doxorubicin-resistant subline LoVo/DX less effectively than that of the sensitive LoVo cell line.
However, all new derivatives
an acetyl at position C7. In addition, as many as eleven of the tested colchicine derivatives (
13 16 17) showed RI 10 (see Figure 2). The best values of the calculated resistance index, RI
1.1–2.1 were obtained for derivatives (N-butyl/N-decyl/N-isobutyl 10-methylaminocolchicine),
3–17 exhibited lower RI than the starting compounds 1 and 2 with
3–8,
1
=
1
–
,
–
≤
6–8
which means that the cells are very sensitive to these compounds. The results indicate that some of
the double-modified colchicine derivatives are able to overcome the drug resistance of the LoVo/DX
cell line, but this desirable property was offset by poor SI values (see Figure 1). It is possible that a
replacement of the amide moiety at position C7 by a single nitrogen-carbon bond will solve one of

Molecules 2020, 25, 3540
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the main problems of using colchicine in chemotherapy (multidrug resistance) and therefore, such
derivatives should be taken into account for further investigation aimed at structure optimization.
7
1.1
70
60
50
40
30
20
10
0
6
4.9
5
7.9
3
4.0
2
5.9
2
3.6
1
3.9
1
0.0
9
.3 8.9
8
.5
7.0
5
.4
3
.5
3.6
1
.7 2.1
1
.1
0.9
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 DX CP
Figure 2. Comparison of resistance index (RI) values of tested compounds. RI indicates how many
times more chemoresistant is a resistant subline relative to its parental cell line. The RI was calculated
for each compound using the formula: RI = (IC50 for LoVo/DX)/(IC50 for LoVo) cell line. When RI is 0–2,
the cells are sensitive to the compound tested, RI in the range 2–10 means that the cell shows moderate
sensitivity to the drug tested, RI above 10 indicates strong drug resistance.
2
.3. In Silico Determination of the Molecular Mode of Action
In our study, computational methodology (according to the previously published procedure [20])
has been employed to precisely explore the molecular basis for the binding of the fourteen novel
colchicines to -tubulin. The -tubulin monomer together with the highly homologous -tubulin
monomer form a stable dimer, which is the building block of microtubules in the cytoskeleton structure
of every eukaryotic cell. Tubulin, in particular -tubulin, is the target of many antiproliferative agents.
In our simulations, compounds described above were docked into the most abundant isotype of
-tubulin in most cancer cells ( I-tubulin) in the colchicine-binding site and ranked according to their
predicted binding affinity.
According to our computational predictions, the compounds from the lowest binding energy to
the highest are ordered as follows: 39.1), 37.5), 14 31.5), 16 27.4), 15 27.1), 11 19.9),
19.4), 17.2), 16.9), 4.9), 4.5), 17 3.6), 2.6), 1.0), 10 (5.0), and 12 (8.7) with the
β
β
β
β
β
β
1
3
(
(
−
2
(
(
−
(
−
−
(
−
(
−
(
−
6
(−
4
(−
7
(−
5
(
−
−
−
9
(
8
(−
binding energies in the parentheses given in kcal/mol units. Binding energies of these compounds are
shown in Figure 3 and in Supplementary Materials Table S1.

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8
.7
5
.0
5
-1.0
-2.6
-
5
-4.5
-3.6
-4.9
-
-
-
-
15
25
35
45
-17.2
-16.9
-19.4
-19.9
-21.4
-27.1 -27.4
-31.5
-37.5
-39.1
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17
Figure 3. Comparison of binding energies of the tested compounds complexed with tubulin
I. Binding energies are estimated using the molecular mechanics generalized Born/surface area
MM/GBSA) method.
β
(
Replacement of the methoxy group in colchicine 1 by the methylamino group in derivative
C10 carbon significantly improved the antiproliferative activity of unmodified (Table 1) but their BE
values did not differ significantly ( 39.1 kcal/mol for and 37.5 kcal/mol for ). Compound 14 was the
most toxic to tumor cells (IC50 = 0.1–1.6 nM) and has the third lowest energy ( 31.5 kcal/mol). These
observations may indicate that the new double-modified colchicine 14 fits well to I-tubulin and may
have a similar mechanism of action to , despite significantly lower IC50 values. However, if we look at
subsequent derivatives with a relatively low BE, 15 16 (approx. 27.0 kcal/mol) close to BE for 14 and
compare with their IC50 values, we notice here the absence of any correlation—compounds 15 and 16
were definitely less active in vitro than compound 14. In turn, compounds 10 12 and 17 had the
2 at
1
−
1
−
2
−
β
1
–
−
5, 8– ,
highest BE from among the new derivatives with a single carbon-nitrogen bond at position C7 (4–17)
even though their IC50 values were comparable or lower than those of compounds 15 and 16 with low
binding energies. Thus, the trend in the computed binding energy values is not the same as for IC50
values that were determined from the experimental assays. However, it should be emphasized that IC50
is strongly dependent on the ability of these compounds to enter cancer cells through their membranes
without being removed by efflux pumps and cause cell death. Since there is little correlation here,
we conclude that this ability depends mainly on: solubility and membrane permeability plus affinity
to P-glycoproteins (P-gp). It is worth mentioning that colchicine is subject to multidrug resistance
in tumor cells as a P-gp substrate. It induces P-gp activity causing a conformational change, which
subsequently leads to its rapid efflux from tumor cells [4,33,34]. Binding energy to its primary target,
tubulin, is a secondary factor affecting the value of IC50 and most of these compounds fall into a fairly
narrow range due to a similar mode of binding to their target. On the other hand, SI is the ratio of the
0
IC50 s for normal cells versus cancer cells. It largely depends on the three main factors listed above of
which solubility and membrane permeability do not change between cancer and normal cells, so it
mainly depends on the affinity to P-gp, which is not known to us at present.
Therefore, we can see that the in silico results do not correlate with the in vitro determined
biological activity of these compounds as indicated by the corresponding IC50 values. The inclusion

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of binding affinity calculations for these compounds with regard to the other tubulin isotypes is still
informative in a broader context of tubulin-binding agents and with respect to most important efflux
transporters, in order to minimize interactions with the latter and maximize with the former, and hence
increase the activity of the chemical compound.
Schematic interactions of the compounds with CBS residues of
and Supplementary Materials Table S1. Derivatives were designed with different alkyl chains at
position C7 of 10-methylaminocolchicine . In this group, the common binding residues were Asn100,
Leu688, Asn691, and Lys785. Compound showed additionally some interactions with -tubulin
Gln10, Asn100, Ser177, Ala179). Another series with alkyl chains having various substituents ( 12) at
C7 carbon were bound mostly through Asn100, Thr178, Leu681, Leu688, and Lys785. Compounds
17 have been designed with an aromatic group side chain without or with substituents at C7
position. The binding residues in this group belong to both -tubulin (Asn100, Thr178) and -tubulin
Leu681, Asn682, Asp684, Leu688, Lys785).
βI-tubulin are shown in Table 2
4–8
2
7
α
(
9–
13–
α
β
(
Table 2. Computational predictions of interactions between starting compounds
and homology modeled tubulin I. 3D representation and 2D layout of colchicine derivatives–tubulin
protein complex, binding energy and active residues are tabulated.
1–3, most cytotoxic 14
β
Binding
Energy
kcal/mol]
3
D Representation
2D Representation
of the Interactions
Active
Residues
Compound
of the Interactions
[
Cys674
Leu681
Ala683
Leu688
Met692
Ala749
Lys785
1
−39.1
Cys674
Ala683
Leu688
Asn691
Lys785
2
−37.5
Ser177
Cys674
Leu681
Ala683
Asp684
Leu688
Asn691
Lys785
3
−4.5

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Table 2. Cont.
Binding
Energy
kcal/mol]
3
D Representation
2D Representation
of the Interactions
Active
Residues
Compound
of the Interactions
[
Asn100
Ser177
Cys674
Leu681
Asn682
Asp684
Leu688
Lys785
−
31.5
1
4
In 3D illustrations the interacting residues predicted from pairwise per-residue binding free energy decomposition
calculations (E < 2 kcal/mol) are shown in stick representation and their carbons and the ribbon are colored as
green. Tubulin is shown in cartoon representation. Hydrogen bonds and their directionality are represented as
black dashed arrows. The structures are color coded as follows: Tubulin I, brown; tubulin I, beige. Ligands are
displayed with stick and the atoms are colored as O (red), C (gray), N (blue), S (yellow), Cl (green), and F (pink).
Binding energy defines the affinity of binding of colchicine derivatives complexed with tubulin I. Binding energies
are predicted by the MM/GBSA method. The last column contains information about active residues with binding
free energy decomposition (E < 2 kcal/mol) and the residues with (E < 3 kcal/mol) are highlighted in bold. The
−
α
β
β
−
−
last line contains the graphical key to help interpret the 2D part of the ligand interactions panel.
3
. Materials and Methods
3
.1. General
Information concerning reagents and solvents can be found in the Supplementary Materials.
3
.2. Spectroscopic Measurements
Information concerning equipment used for measurements can be found in the
Supplementary Materials.
3
.3. Synthesis
3
.3.1. Synthesis of 2 and 3
Synthesisof10-N-methylaminocolchicine2andN-deacetyl-10-methylamino-10-demethoxycolchicine
3
was performed according to the previously published procedure [20] and can be found in
Supplementary Materials.
3
.3.2. General Procedure for the Synthesis of Colchicine Derivatives 5–9 and 12–17
Compound and 12 17 were obtained directly from compound . To a solution of compound
(1.0 equiv.) in MeOH, the corresponding aldehyde (1.0 equiv.) was added followed in 15 min by
5–
9
–
3
3
NaBH CN (1.2 equiv.). Reaction progress was monitored by LC-MS. Then the mixture was evaporated
3
under reduced pressure, diluted with EtOAc, washed with 5% NaHCO brine and dried over Na SO .
3
2
4
The residue was purified using column flash chromatography (silica gel; EtOAc/MeOH, 20/1 v/v) and
next lyophilized from dioxane to give respective compounds.
Compound 5
Yellow solid, yield 47%.

Molecules 2020, 25, 3540
10 of 16
+
+
+
+
ESI-MS for C H N O (m/z): [M + H] 399, [M + Na] 421, [2M + H] 797, [2M + Na] 819.
23
30
2
4
1
H NMR (500 MHz, CDCl3)
m, 2H), 3.91 (s, 3H), 3.88 (s, 3H), 3.55 (s, 3H), 3.41–3.37 (m, 1H), 3.06 (d, J = 5.4 Hz, 3H), 2.51–2.47 (m,
H), 2.45–2.35 (m, 1H), 2.33–2.27 (m, 1H), 2.24–2.15 (m, 2H), 1.72–1.65 (m, 1H), 1.46–1.37 (m, 2H), 0.82
δ 7.64 (s, 1H), 7.36 (d, J = 11.0 Hz, 1H), 7.27–7.25 (m, 1H), 6.53–6.48
(
1
(
t, J = 7.4 Hz, 3H).
13C NMR (126 MHz, CDCl3)
δ 175.7, 155.1, 152.6, 150.9, 150.6, 141.1, 138.6, 135.5, 130.5, 127.0,
1
24.4, 107.2, 107.1, 61.3, 61.0, 60.6, 56.1, 49.9, 39.4, 30.5, 29.5, 23.4, 11.8.
Compound 6
Yellow solid, yield 44%.
ESI-MS for C H N O (m/z): [M + H] 413, [M + Na] 435, [2M + H] 825, [2M + Na] 847.
+
+
+
+
24
32
2
4
1
H NMR (500 MHz, CDCl3)
d, J = 12.3 Hz, 2H), 3.89 (s, 3H), 3.87 (s, 3H), 3.54 (s, 3H), 3.39–3.37 (m, 1H), 3.05 (d, J = 5.3 Hz, 3H),
.49–2.46 (m, 1H), 2.39–2.37 (m, 1H), 2.30–2.28 (m, 1H), 2.22–2.18 (m, 2H), 1.73–1.64 (m, 1H), 1.39–1.36
m, 2H), 1.28–1.25 (m, 2H), 0.80 (t, J = 7.3 Hz, 3H).
δ 7.63 (s, 1H), 7.35 (d, J = 11.0 Hz, 1H), 7.28–7.26 (s, 1H), 6.50–6.48
(
2
(
1
3
C NMR (126 MHz, CDCl3)
δ 175.7, 155.1, 152.6, 150.7, 150.5, 141.1, 138.6, 135.5, 130.5, 126.9,
1
24.3, 107.3, 107.1, 61.3, 61.0, 60.6, 56.0, 47.6, 39.3, 32.3, 30.5, 29.5, 20.4, 14.0.
Compound 7
Yellow solid, yield 40%.
ESI-MS for C H N O (m/z): [M + H] 497, [M + Na] 519, [2M + H] 993, [2M + Na] 1015.
+
+
+
+
30
44
2
4
1
H NMR (500 MHz, CDCl3) δ 7.64 (s, 1H), 7.36 (d, J = 11.1 Hz, 1H), 7.32–7.27 (m, 1H), 6.52–6.48 (m,
2
H), 3.90 (s, 3H), 3.87 (s, 3H), 3.55 (s, 3H), 3.44–3.37 (m, 1H), 3.06 (d, J = 5.4 Hz, 3H), 2.55–2.48 (m, 1H),
2
.43–2.37 (m, 1H), 2.34–2.26 (m, 1H), 2.25–2.17 (m, 2H), 1.75–1.67 (m, 1H), 1.44–1.36 (m, 2H), 1.26–1.15
(
m, 14H), 0.83 (t, J = 7.0 Hz, 3H).
13
C NMR (126 MHz, CDCl3)
δ 175.6, 155.1, 152.7, 150.5, 141.1, 138.7, 135.4, 130.5, 126.9, 124.3,
1
07.3, 107.1, 61.3, 61.1, 60.6, 56.0, 48.0, 39.3, 31.9, 30.4, 29.6, 29.5, 29.3, 27.3, 22.7, 14.1.
Compound 8
Yellow solid, yield 45%.
ESI-MS for C H N O (m/z): [M + H] 413, [M + Na] 435, [2M + H] 825, [2M + Na] 847.
+
+
+
+
24
32
2
4
1
H NMR (500 MHz, CDCl3)
m, 2H), 3.92 (s, 3H), 3.89 (s, 3H), 3.56 (s, 3H), 3.38–3.34 (m, 1H), 3.07 (d, J = 5.4 Hz, 3H), 2.42–2.38 (m,
H), 2.33–2.29 (m, 2H), 2.24–2.15 (m, 1H), 2.08–2.04 (m, 1H), 1.73–1.68 (m, 1H), 1.64–1.62 (m, 1H), 0.88
d, J = 6.6 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H).
δ 7.68 (s, 1H), 7.36 (d, J = 11.0 Hz, 1H), 7.25–7.21 (m, 1H), 6.55–6.47
(
1
(
1
3
C NMR (126 MHz, CDCl3)
δ 175.8, 155.1, 152.6, 151.1, 150.6, 141.1, 138.5, 135.5, 130.5, 127.0,
1
24.6, 107.2, 107.0, 61.3, 61.2, 60.6, 56.0, 56.0, 39.4, 30.5, 29.5, 28.8, 20.8, 20.7.
Compound 9
Yellow solid, yield 50%.
ESI-MS for C H N O (m/z): [M + H] 401, [M + Na] 423, [2M + H] 801, [2M + Na] 823.
+
+
+
+
22
28
2
5
1
H NMR (500 MHz, CDCl3)
m, 2H), 3.90 (s, 3H), 3.88 (s, 3H), 3.64–3.57 (m, 2H), 3.55 (s, 3H), 3.48–3.44 (m, 1H), 3.13 (s, 1H), 3.07 (d,
J = 5.4 Hz, 3H), 2.7–2.74 (m, 1H), 2.45–2.40 (m, 2H), 2.35–2.25 (m, 2H), 1.75–1.71 (m, 1H).
δ 7.62 (s, 1H), 7.39 (d, J = 11.1 Hz, 1H), 7.35–7.33 (m, 1H), 6.57–6.50
(
1
3
C NMR (126 MHz, CDCl3)
δ 175.4, 155.2, 152.8, 150.5, 150.4, 141.4, 139.1, 135.3, 130.7, 126.7,
1
24.0, 107.8, 107.2, 61.3, 61.2, 60.8, 60.7, 56.1, 49.6, 39.3, 30.4, 29.5.
Compound 12
Yellow solid, yield 55%.

Molecules 2020, 25, 3540
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+
+
+
+
ESI-MS for C H F N O (m/z): [M + H] 453, [M + Na] 475, [2M + H] 905, [2M + Na] 927.
H NMR (500 MHz, CDCl3)
23
27
3
2
4
1
δ 7.66 (s, 1H), 7.37 (d, J = 11.1 Hz, 1H), 7.29–7.26 (m, 1H), 6.53–6.50
(
m, 2H), 3.91 (s, 3H), 3.89 (s, 3H), 3.57 (s, 3H), 3.43–3.38 (m, 1H), 3.07 (d, J = 5.4 Hz, 3H), 2.80–2.68 (m,
1
H), 2.58–2.50 (m, 1H), 2.43–2.37 (m, 1H), 2.35–2.29 (m, 1H), 2.27–2.14 (m, 3H), 1.73–1.68 (m, 1H).
13
C NMR (126 MHz, CDCl3)
δ 175.7, 155.1, 152.8, 150.6, 150.0, 141.2, 138.9, 135.3, 130.3, 127.7,
1
26.7, 124.0, 107.3, 107.1, 61.3, 60.9, 60.6, 56.0, 40.8, 40.8, 39.3, 30.4, 29.5.
Compound 13
Yellow solid, yield 47%.
ESI-MS for C H N O (m/z): [M + H] 447, [M + Na] 469, [2M + H] 893, [2M + Na] 915.
+
+
+
+
27
30
2
4
1
H NMR (500 MHz, CDCl3)
m, 4H), 7.18–7.13 (m, 1H), 6.55–6.49 (m, 2H), 3.90 (s, 3H), 3.88 (s, 3H), 3.78–3.72 (m, 1H), 3.50 (s, 3H),
.49–3.46 (m, 1H), 3.42–3.40 (m, 1H), 3.07 (d, J = 5.4 Hz, 3H), 2.45–2.28 (m, 2H), 2.26–2.15 (m, 1H),
δ 7.83 (s, 1H), 7.37 (d, J = 11.0 Hz, 1H), 7.31–7.27 (m, 1H), 7.26–7.19
(
3
1
.77–1.71 (m, 1H).
13C NMR (126 MHz, CDCl3)
δ 175.8, 155.1, 152.6, 150.6, 150.5, 141.1, 140.0, 138.7, 135.4, 130.5,
1
28.3, 128.2, 127.0, 126.9, 124.5, 107.2, 107.0, 61.3, 60.6, 60.4, 56.0, 51.9, 39.4, 30.5, 29.5.
Compound 14
Yellow solid, yield 33%.
ESI-MS for C H ClN O (m/z): [M + H] 481/483, [M + Na] 503, [2M + H] 961, [2M + Na]
+
+
+
+
27
29
2
4
9
83/985.
1H NMR (500 MHz, CDCl3)
δ
7.87 (s, 1H), 7.37 (d, J = 11.0 Hz, 1H), 7.33 (dd, J = 7.2, 1.6 Hz, 1H),
7
3
.30–7.23 (m, 2H), 7.16–7.08 (m, 2H), 6.53–6.49 (m, 2H), 3.90 (s, 3H), 3.88 (s, 3H), 3.81–3.75 (m, 1H),
.58–3.55 (m, 4H), 3.49–3.44 (m, 1H), 3.07 (d, J = 5.4 Hz, 3H), 2.44–2.31 (m, 2H), 2.26–2.16 (m, 1H),
1
.81–1.73 (m, 1H).
13C NMR (126 MHz, CDCl3)
δ 175.8, 155.1, 152.6, 150.6, 150.4, 141.1, 138.7, 137.4, 135.4, 133.6,
1
30.4, 130.2, 129.3, 128.3, 126.8, 124.6, 107.2, 107.0, 61.3, 60.7, 60.3, 56.0, 49.3, 39.3, 30.5, 29.5.
Compound 15
Yellow solid, yield 51%.
+
+
+
+
ESI-MS for C H N O (m/z): [M + H] 463, [M + Na] 485, [2M + H] 925, [2M + Na] 947, [M
2
7
30
2
5
−
1
− −
−
H] 461, [M + HCOO ] 507.
H NMR (500 MHz, CDCl3)
δ 7.94 (s, 1H), 7.45 (d, J = 11.2 Hz, 1H), 7.40–7.36 (m, 1H), 7.03 (d, J =
8
3
2
.2 Hz, 2H), 6.74 (d, J = 8.1 Hz, 2H), 6.62 (d, J = 11.4 Hz, 1H), 6.52 (s, 1H), 3.92 (s, 3H), 3.88 (s, 3H),
.59–3.56 (m, 1H), 3.55 (s, 3H), 3.52–3.46 (m, 1H), 3.27 (d, J = 12.0 Hz, 1H), 3.09 (d, J = 5.4 Hz, 3H),
.43–2.36 (m, 1H), 2.35–2.18 (m, 2H), 1.77–1.73 (m, 1H).
13
C NMR (126 MHz, CDCl3)
δ 175.1, 156.4, 155.4, 152.8, 151.3, 150.6, 141.1, 139.6, 135.5, 131.4, 130.5
1
29.5, 126.7, 124.2, 115.6, 108.6, 107.2, 61.4, 61.0, 60.8, 56.1, 51.8, 39.3, 30.5, 29.6.
Compound 16
Yellow solid, yield 56%.
ESI-MS for C H N O (m/z): [M + H] 448, [M + Na] 470, [2M + Na] 917.
+
+
+
26
29
3
4
1
H NMR (500 MHz, CD CN)
δ
8.35 (d, J = 5.7 Hz, 2H), 7.66 (s, 1H), 7.36–7.31 (m, 1H), 7.25 (d, J =
1.0 Hz, 1H), 7.17 (d, J = 5.8 Hz, 2H), 6.61 (s, 1H), 6.55 (d, J = 11.2 Hz, 1H), 3.80 (s, 3H), 3.76 (s, 3H), 3.68
d, J = 14.9 Hz, 1H), 3.47 (d, J = 14.9 Hz, 1H), 3.39 (s, 3H), 3.35–3.29 (m, 1H), 3.00 (d, J = 5.4 Hz, 3H),
.44–2.36 (m, 1H), 2.26–2.13 (m, 2H), 1.74–1.63 (m, 1H).
3
1
(
2
1
3
C NMR (126 MHz, CD CN)
δ
176.2, 156.1, 153.6, 151.3, 150.5, 150.4, 150.2, 141.9, 139.3, 136.2,
3
1
30.7, 127.5, 124.6, 123.9, 108.2, 107.7, 61.3, 61.0, 60.9, 56.5, 50.7, 39.5, 30.8, 29.7.

Molecules 2020, 25, 3540
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Compound 17
Yellow solid, yield 40%.
ESI-MS for C H N O S (m/z): [M + H] 453, [M + Na] 475, [2M + H] 905, [2M + Na] 927.
+
+
+
+
25
28
2
4
1
H NMR (500 MHz, CDCl3)
δ 7.74 (s, 1H), 7.37 (d, J = 11.1 Hz, 1H), 7.29–7.27 (m, 1H), 7.10 (dd, J =
5
3
2
.0, 0.9 Hz, 1H), 6.83–6.80 (m, 1H), 6.76 (d, J = 2.9 Hz, 1H), 6.54–6.50 (m, 2H), 3.93 (d, J = 14.1 Hz, 1H),
.90 (s, 3H), 3.88 (s, 3H), 3.65 (d, J = 14.1 Hz, 1H), 3.57–3.53 (m, 1H), 3.51 (s, 3H), 3.08 (d, J = 5.4 Hz, 3H),
.45–2.29 (m, 2H), 2.25–2.16 (m, 1H), 1.78–1.71 (m, 1H).
13
C NMR (126 MHz, CDCl3)
δ 175.7, 155.1, 152.7, 150.6, 150.2, 143.8, 141.1, 138.8, 135.4, 130.5,
1
26.8, 126.5, 124.7, 124.4, 124.3, 107.3, 107.0, 61.3, 60.7, 59.9, 56.0, 46.3, 39.4, 30.5, 29.5.
3
.3.3. General Procedure for the Synthesis of Colchicine Derivatives 4 and 11
Compound and 11 were obtained in a two-step procedure. The first step involved the synthesis
4
of aldehyde and the second was the reaction of the obtained aldehyde with the starting compound 3.
To a solution of appropriate alcohol (10.0 eqiuv.) in DCM was added PCC (30.0 equiv.) and the
reaction mixture was stirred at RT for 4 h. After this time the mixture was filtered through thin pad of
silica gel and the filtrate containing aldehyde was used in the next step (without further purification).
To a solution of compound
3 (1.0 equiv.) in MeOH, one-second of the previously prepared portion
of aldehyde was added followed in 15 min by NaBH CN (1.2 equiv.). After two hours, a further
3
portion of the prepared aldehyde and NaBH CN (1.2 equiv.) were added. Reaction progress was
3
monitored by LC-MS. Then the mixture was evaporated under reduced pressure, diluted with EtOAc,
washed with 5% NaHCO , brine and dried over Na SO . The residue was purified using column
3
2
4
flash chromatography (silica gel; DCM/MeOH, 30/1 v/v) and next lyophilized from dioxane to give
respective compounds.
Compound 4
Yellow solid, yield 13%.
+
+
+
ESI-MS for C H N O (m/z): [M + H] 385, [M + Na] 407, [2M + Na] 791.
22
28
2
4
1
H NMR (500 MHz, CDCl3)
m, 2H), 3.93 (s, 3H), 3.91 (d, J = 4.6 Hz, 3H), 3.90–3.84 (m, 1H), 3.60 (s, 3H), 3.12 (d, J = 5.3 Hz, 3H),
.86–2.69 (m, 1H), 2.59–2.30 (m, 4H), 1.87 (s, 1H), 1.13 (s, 3H).
δ 7.77 (s, 1H), 7.43 (d, J = 11.1 Hz, 1H), 7.26–7.24 (m, 1H), 6.64–6.53
(
2
Compound 11
Yellow solid, yield 43%.
ESI-MS for C H N O (m/z): [M + H] 433/435, [M + Na] 455/457, [2M + Na] 887/889.
+
+
+
23
29
2
4
1
H NMR (500 MHz, CD Cl ) δ 7.59 (s, 1H), 7.33 (d, J = 11.0 Hz, 1H), 7.25–7.22 (m, 1H), 6.59–6.47
2
2
(m, 2H), 3.86 (d, J = 1.9 Hz, 6H), 3.62–3.55 (m, 2H), 3.54 (s, 3H), 3.38–3.36 (m, 1H), 3.06 (d, J = 5.5
Hz, 3H), 2.63–2.60 (m, 1H), 2.50–2.39 (m, 2H), 2.31–2.29 (m, 1H), 2.26–2.16 (m, 1H), 1.87–1.82 (m, 2H),
1
1
.74–1.65 (m, 1H).
13
C NMR (126 MHz, CD Cl )
24.6, 107.6, 107.4, 61.5, 61.5, 60.9, 56.5, 45.4, 43.8, 39.8, 33.6, 30.8, 29.9.
δ
176.0, 155.5, 153.3, 151.1, 151.0, 141.7, 138.9, 135.9, 130.7, 127.3,
2
2
3
.3.4. Synthesis of 10
Compound 10 was obtained directly from compound 3. To a solution of compound 3 (1.0 equiv.)
in MeOH, acrylonitrile (5.0 equiv.) was added. Reaction progress was monitored by LC-MS. Then the
reaction mixture was diluted with EtOAc, washed with 5% NaHCO , brine and dried over Na SO .
3
2
4
The residue was purified using column flash chromatography (silica gel; DCM/MeOH) and next
lyophilized from dioxane to give the pure product 10 as a yellow solid with a yield of 19%.
+
+
+
−
H] 408, [M +
ESI-MS for C H N O (m/z): [M + H] 410, [M + Na] 432, [2M + Na] 841, [M
HCOO ] 454.
−
23
27
3
4
−
−

Molecules 2020, 25, 3540
13 of 16
1H NMR (500 MHz, CDCl3)
δ
7.63 (s, 1H), 7.39 (d, J = 11.1 Hz, 1H), 7.3–7.27 (m, 1H), 6.60–6.49 (m,
2
H), 3.92 (s, 3H), 3.91 (s, 3H), 3.59 (s, 3H), 3.51–3.40 (m, 1H), 3.09 (d, J = 5.4 Hz, 3H), 2.84–2.80 (m, 1H),
2
.68–2.57 (m, 1H), 2.49–2.40 (m, 3H), 2.36–2.30 (m, 1H), 2.28–2.21 (m, 1H), 1.77–1.71 (m, 1H).
3
.4. In Vitro Antiproliferative Activity
Cytotoxic activity was tested according to the previously published procedure [20] and detailed
information can be found in Supplementary Materials.
3
.5. Molecular Docking Studies
Molecular docking studies was performed according to the previously published procedure [20
]
and detailed information can be found in Supplementary Materials.
4
. Conclusions
In summary, a series of novel double-modified colchicine analogues derivatized at their C7 (single
carbon-nitrogen bond) and C10 (methylamine substituent) positions, were devised. All compounds
were successfully synthesized, purified and their structures were confirmed by spectroscopic analyses.
All target compounds were screened for their in vitro cytotoxicity against A549, MCF-7, LoVo, and
LoVo/DX cells to evaluate their anticancer potency.
All presented derivatives were shown to be active at nanomolar concentrations and exhibited
better antiproliferative activity than the commonly used anticancer agents doxorubicin and cisplatin.
The introduction of methylamino group at position C10 significantly increased the cytotoxicity of a
relevant derivative in comparison to that of unmodified colchicine
1. In addition, the majority of the
investigated double-modified derivatives exhibited excellent potency (IC50
≤
10 nM) against A549,
MCF-7, LoVo cell lines. The most toxic to all tumor cells tested was derivative 14 with 2-chlorobenzyl
moiety at C7 carbon (IC50 = 0.1–1.6 nM). Thus, preliminary conclusions revealed that the choice of
the type of chemical bond or substituent at C7 and C10 positions was critical for the cytotoxicity of
the compounds.
The calculated values of resistance index (RI) indicated that colchicine-based compounds can be
effective against drug-resistant cancer cells. Eleven new derivatives (
3
–
8
,
11
–
13
,
16
–
17) showed RI
≤
10,
which means that LoVo/DX cells are sensitive to these compounds. Although these compounds have
low RI values, they have proved to be non-selective for cancer cells in relation to normal cells (low SI
values). Further evaluation should help to find more detailed structure–activity relationships for the
drugs overcoming the resistance of tumor cells, which can help in rational drug design efforts in future.
In terms of selectivity for cancerous versus normal cells, only one derivative 14 was superior to parent
amides 1 and 2. Replacement of acetyl at C7 carbon with a 2-chlorobenzyl moiety and simultaneous
substitution of methoxy group to methylamino group at position C10 allowed obtaining a compound
selective towards all four cancer cell lines tested (high SI values from 2.8 to 93.7).
Molecular docking studies showed that apart from the initial amides
which had the best antiproliferative activity, stood out also in terms of its predicted binding energy and
probably binds best into the active site of I-tubulin isotype. Otherwise, no correlation between in vitro
1 and 2, compound 14,
β
and in silico results was observed indicating that other factors than affinity to the target determine their
biological activity. These factors may include solubility, membrane permeability as well as off-target
interactions including the affinity to P-glycoprotein.
Derivative 14 with strong in vitro antiproliferative activity and high SI values, represents the most
promising lead for further development. Test results for this compound are noteworthy and suggest
that extended and more detail in vivo research of 14 and similar derivatives is necessary to determine
their therapeutic potential as anticancer drugs.
Supplementary Materials: The following are available online, Table S1: Computational predictions of interactions
between tested compounds (1–17) and homology modeled tubulin βI, Figure S1: The LC-MS chromatogram

Molecules 2020, 25, 3540
14 of 16
1
13
and mass spectra of
2
, Figure S2: The H NMR spectrum of
2
in CDCl , Figure S3: The C NMR spectrum of
3
1
2
3
in CDCl , Figure S4: The LC-MS chromatogram and mass spectra of
in CDCl , Figure S6: The C NMR spectrum of
spectra of 4, Figure S8: The LC-MS chromatogram and mass spectra of
in CDCl , Figure S10: The C NMR spectrum of
, Figure S12: The H NMR spectrum of
CDCl , Figure S14: The LC-MS chromatogram and mass spectra of
3,
Figure S5: The H NMR spectrum of
3
13
3
in CDCl , Figure S7: The LC-MS chromatogram and mass
3
3
1
5
, Figure S9: The H NMR spectrum
in CDCl , Figure S11: The LC-MS chromatogram and
in CDCl , Figure S13: The C NMR spectrum of
13
of
5
5
3
3
1
13
mass spectra of
6
6
6
in
7
3
1
7
, Figure S15: The H NMR spectrum of
in CDCl , Figure S17: The LC-MS chromatogram and mass
in CDCl , Figure S19: The C NMR spectrum of
3
1
3
in CDCl , Figure S16: The C NMR spectrum of
7
8
3
3
1
13
spectra of
8
, Figure S18:The H NMR spectrum of
8
9
in CDCl3,
in CDCl3,
3
1
Figure S20: The LC-MS chromatogram and mass spectra of
9
, Figure S21: The H NMR spectrum of
in CDCl , Figure S23: The LC-MS chromatogram and mass spectra of
Figure S22: The 13C NMR spectrum of
9
3
1
1
0
, Figure S24: The H NMR spectrum of 10 in CDCl , Figure S25: The LC-MS chromatogram and mass spectra
of 11, Figure S26: The H NMR spectrum of 11 in CD Cl , Figure S27: The C NMR spectrum of 11 in CD Cl ,
3
1
13
2
2
2
2
1
Figure S28: The LC-MS chromatogram and mass spectra of 12, Figure S29: The H NMR spectrum of 12 in CDCl3,
Figure S30: The 13C NMR spectrum of 12 in CDCl , Figure S31: The LC-MS chromatogram and mass spectra
3
1
13
of 13, Figure S32: The H NMR spectrum of 13 in CDCl , Figure S33: The C NMR spectrum of 13 in CDCl3,
3
1
Figure S34: The LC-MS chromatogram and mass spectra of 14, Figure S35: The H NMR spectrum of 14 in CDCl3,
Figure S36: The 13C NMR spectrum of 14 in CDCl , Figure S37: The LC-MS chromatogram and mass spectra
3
1
13
of 15, Figure S38: The H NMR spectrum of 15 in CDCl , Figure S39: The C NMR spectrum of 15 in CDCl3,
3
1
Figure S40: The LC-MS chromatogram and mass spectra of 16, Figure S41: The H NMR spectrum of 16 in CD CN,
3
Figure S42: The 13C NMR spectrum of 16 in CD CN, Figure S43: The LC-MS chromatogram and mass spectra of
3
1
13
1
7, Figure S44: The H NMR spectrum of 17 in CDCl , Figure S45: The C NMR spectrum of 17 in CDCl3.
3
Author Contributions: Conceptualization, J.K. and A.H.; methodology, J.K., A.H., J.W. and J.A.T.; software,
M.A.; validation, A.H. and J.A.T.; formal analysis, A.H. and J.K.; investigation, J.K., E.M. and M.A.; resources,
A.H.; J.K. and W.M.; data curation, J.K., M.A. and E.M.; writing—original draft preparation, J.K. and M.A.;
writing—review and editing, W.M., A.H., J.W. and J.A.T.; visualization, J.K. and M.A.; supervision, A.H.; project
administration, A.H.; funding acquisition, A.H. and W.M. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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