Synthesis, anticancer activity and molecular docking studies of N-deacetylthiocolchicine and 4-iodo-N-deacetylthiocolchicine derivatives

Article abstract of DOI:10.1016/j.bmc.2021.116014

Colchicine is a plant alkaloid with a broad spectrum of biological and pharmacological properties. It has found application as an anti-inflammatory agent and also shows anticancer effects through its ability to destabilize microtubules by preventing tubulin dimers from polymerizing leading to mitotic death. However, adverse side effects have so far restricted its use in cancer therapy. This has led to renewed efforts to identify less toxic derivatives. In this article, we describe the synthesis of a set of novel double- and triple-modified colchicine derivatives. These derivatives were tested against primary acute lymphoblastic leukemia (ALL-5) cells and several established cancer cell lines including A549, MCF-7, LoVo and LoVo/DX. The novel derivatives were active in the low nanomolar range, with 7-deacetyl-10-thiocolchicine analogues more potent towards ALL-5 cells while 4-iodo-7-deacetyl-10-thiocolchicine analogues slightly more effective towards the LoVo cell line. Moreover, most of the synthesized compounds showed a favorable selectivity index (SI), particularly for ALL-5 and LoVo cell lines. Cell cycle analysis of the most potent molecules on ALL-5 and MCF-7 cell lines revealed contrasting effects, where M-phase arrest was observed in MCF-7 cells but not in ALL-5 cells. Molecular docking studies of all derivatives to the colchicine-binding site were performed and it was found that five of the derivatives showed strong β-tubulin binding energies, lower than ?8.70 kcal/mol, while the binding energy calculated for colchicine is ?8.09 kcal/mol. The present results indicate that 7-deacetyl-10-thiocolchicine and 4-iodo-7-deacetyl-10-thiocolchicine analogues constitute promising lead compounds as chemotherapy agents against several types of cancer.

Full text of DOI:10.1016/j.bmc.2021.116014

Bioorg. Med. Chem. 32 (2021) 116014
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, anticancer activity and molecular docking studies of
N-deacetylthiocolchicine and 4-iodo-N-deacetylthiocolchicine derivatives
Greta Klejborowskaa, Alicja Urbaniakb, Ewa Majc, Joanna Wietrzykc, Mahshad Mosharid,
,
f
b
a,*
Jordane Pretoe, Jack A. Tuszynskie , Timothy C. Chambers , Adam Huczynski
´
a
´
Department of Medical Chemistry, Faculty of Chemistry, Adam Mickiewicz University, Uniwersytetu Poznanskiego 8, 61-614 Poznan, Poland
b Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States
c Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wrocław, Poland
d Depertment of Chemistry, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada
e Depertment of Oncology, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada
f DIMEAS, Politecnico di Torino, Corso Duca degli Abruzzi 24, Turin, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
Colchicine is a plant alkaloid with a broad spectrum of biological and pharmacological properties. It has found
application as an anti-inflammatory agent and also shows anticancer effects through its ability to destabilize
microtubules by preventing tubulin dimers from polymerizing leading to mitotic death. However, adverse side
effects have so far restricted its use in cancer therapy. This has led to renewed efforts to identify less toxic de-
rivatives. In this article, we describe the synthesis of a set of novel double- and triple-modified colchicine de-
rivatives. These derivatives were tested against primary acute lymphoblastic leukemia (ALL-5) cells and several
established cancer cell lines including A549, MCF-7, LoVo and LoVo/DX. The novel derivatives were active in the
low nanomolar range, with 7-deacetyl-10-thiocolchicine analogues more potent towards ALL-5 cells while 4-
iodo-7-deacetyl-10-thiocolchicine analogues slightly more effective towards the LoVo cell line. Moreover,
most of the synthesized compounds showed a favorable selectivity index (SI), particularly for ALL-5 and LoVo
cell lines. Cell cycle analysis of the most potent molecules on ALL-5 and MCF-7 cell lines revealed contrasting
effects, where M-phase arrest was observed in MCF-7 cells but not in ALL-5 cells. Molecular docking studies of all
derivatives to the colchicine-binding site were performed and it was found that five of the derivatives showed
strong β-tubulin binding energies, lower than ꢀ 8.70 kcal/mol, while the binding energy calculated for colchicine
is ꢀ 8.09 kcal/mol. The present results indicate that 7-deacetyl-10-thiocolchicine and 4-iodo-7-deacetyl-10-thio-
colchicine analogues constitute promising lead compounds as chemotherapy agents against several types of
cancer.
Colchicine binding site inhibitor
Antimitotic agent
Antiproliferative activity
Thiocolchicine
1. Introduction
Despite numerous pre-clinical findings highlighting beneficial effects of
colchicine treatment for various types of cancers, its clinical application
1
Colchicine is an alkaloid isolated from Colchicum autumnale. It is
used for the treatment of acute gout, familial Mediterranean fever,
Behçet’s disease, pericarditis, and other medical conditions.2–12 It also
acts as an anticancer agent and its mechanism of action is well-described
in the scientific literature and is linked to its ability to inhibit mitosis.
Specifically, colchicine binds to β-tubulin and forms complexes with
tubulin dimers, which destabilizes microtubules and suppresses micro-
tubule dynamics preventing mitotic spindle formation. This conse-
quently leads to mitotic arrest and cell death typically via apoptosis.13–16
remains limited mainly to anti-inflammatory indications, due to its
associated side-effects. To overcome those limitations, efforts are
focused on developing more clinically-applicable colchicine
derivatives.17–40
.
Brossi et al. synthesized a series of N-acyl and N-aroyl derivatives
prepared from deacetylcolchicine. Several compounds showed high
potency in the lymphocytic leukemia P388 screens in vitro and in vivo.41
Later Kerekes et al. synthesized analogues of thiocolchicine, a very
potent inhibitor of tubulin polymerization and cell growth, including N-
* Corresponding author.
´
E-mail address: adhucz@amu.edu.pl (A. Huczynski).
https://doi.org/10.1016/j.bmc.2021.116014
Received 1 July 2020; Received in revised form 30 December 2020; Accepted 4 January 2021
Available online 11 January 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

G. Klejborowska et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116014
acyldeacetylthiocolchicines,
N-(alkoxycarbonyl)deacetylthiocolchi
(14–20) were synthesized analogously starting with the compound 13
(see Scheme 1). The structures and purity of all products 2–20 were
determined using the ESI-MS, FT-IR, 1H NMR and 13C NMR methods (see
exemplary NMR spectra Fig. S1-S8).
cines, thiodemecolchicine and its methyl carbamate, as well as O-ethyl
ethers of demethylthiocolchicines.42 Both novel and previously
described analogues were evaluated in vitro in a tubulin binding assay, in
vivo in mice for acute toxicity, and in the P388 lymphocytic leukemia
model.42 Sun et al. subsequently reported three series of novel thio-
colchicine analogs, N-acyl-, N-aroyl-, and N-(substituted benzyl)-
deacetylthiocolchicinoids.43 Those derivatives were evaluated for their
cytotoxicity against various tumor cell lines, with particular emphasis
on solid tumor cell lines, and for their inhibitory effects on tubulin
polymerization in vitro. In 2011, Takayama’s research group published
results of their studies on C-4 halogen substituted colchicine derivatives,
including 4-iodocolchicine.29 4-iodocolchicine showed in vitro similar
potency against A549, HT29 and HCT116 cancerous cell lines to other
halogenated colchicine derivatives in the C-4 position. Despite satis-
factory results, to the best of our knowledge, it has not been imple-
mented in in vivo studies or has not been further modified. In our
previous research, we developed the concept of double-33 and triple-
modified colchicine analogs with diversified carbamate34,35,44 or
amide45,46 substituents in the C-7 position. 4-iodothiocolchicine,
double-modified colchicine derivative in the C-4 and C-10 position,
showed very high potency against A549, MCF-7 and LoVo cancerous cell
lines in the nanomolar range, higher than the activity of unmodified
colchicine or 4-iodocolchicine. Interestingly, the high antiproliferative
activity of 4-iodothiocolchicine was combined with beneficial selectivity
index values.33 Also the majority of novel triple-modified derivatives
showed antiproliferative activity in the nanomolar range together with
beneficial selectivity index values when tested against normal cells.
Encouraged by the previously reported results, we sought to synthesize
two series of novel double-modified derivatives of N-deacetylth-
iocolchicine and triple-modified derivatives of 4-iodo-N-deacetylth-
iocolchicine. In this study, we describe their synthesis, molecular
docking, and anti-proliferative activities against several cancer cell
lines.
2.2. In vitro cytotoxic activity evaluation
The seven double-modified derivatives (4–10), triple-modified
colchicine derivatives (14–20), other colchicine derivatives (2–3,
11–13), and the starting material (1) were evaluated for their in vitro
antiproliferative effect on acute lymphoblastic leukemia cells (ALL-5)
and four human cancer cell lines: human lung adenocarcinoma (A549),
human breast adenocarcinoma (MCF-7), human colon adenocarcinoma
(LoVo) and its doxorubicin-resistant subline (LoVo/DX) as well as on
normal murine embryonic fibroblasts (BALB/3T3). The data, expressed
as IC50 ± SD of the tested compounds, are presented in Table 147,48 and
the viability curves for ALL cells are shown in Supplemental Figure S9.
Some general conclusions based on these data can be drawn. First, the
majority of novel double-modified analogs of 7-deacetyl-10-thiocolchi-
cine showed activity greater than or comparable to the unmodified
colchicine towards primary ALL-5, A549, MCF-7 and LoVo cells
(exception are compounds 9 and 10). Although the compounds 4–8 have
different substituents in the C-7 position, that does not seem to have a
significant impact on these derivatives’ activity, since the IC50 values
against all the tested cancer cell lines are quite similar. The situation is
different in case of triple-modified 4-iodo-7-deacetyl-10-thiocolchicine
analogs. Compounds 14 and 17 showed the highest activity toward
primary ALL-5, A549, MCF-7 and LoVo cells, and 16 showed moderate
activity. The structural differences in the C-7 position have bigger
impact on the IC50 values of triple-modified derivatives comparing to
double-modified ones. Second, the greatest improvement in the activity
of the new derivatives, in comparison to the colchicine, was observed for
the A549, LoVo and LoVo/DX cell lines. The most active compounds
were the following: against A549, 4–9, 14, 16–17 (approx. 9–12 fold
more active than 1); against LoVo, 4– 9, 14, 16–18 (approx. 12–15 fold
more active than 1, compound 6 had even 19 times lower IC50); against
LoVo/DX, 4–9, 14, 16–18 (approx. 10–20 fold more active than 1,
compound 6 had even 30 times lower IC50). For the primary acute
lymphoblastic leukemia cells (ALL-5) and MCF-7 cells only the moderate
improvement in the activity of the new derivatives was observed and the
most active compounds were the following: against ALL-5, 5–8 (approx.
1,5 fold more active than 1); and against MCF-7, 4–8, 14, 17 (approx.
1,5–2 fold more active than 1). All of the above-listed compounds had
very low, single- or double-nanomolar IC50 values, which are lower,
than those presented for doxorubicin and cisplatin, currently widely
2. Results and discussion
2.1. Chemistry
Compounds 2–3, 11–13 were obtained according to previously
described procedures.29,33,42 Double-modified (4–10) derivatives were
synthesized in one pot reaction of compound 3 with respective acyl
(4–9) or carbamoyl (10) chloride in the presence of triethylamine and 4-
dimethylaminopyridine (DMAP) (see Scheme 1). Compound 6 was
previously synthesized by Kerekes et al.42 Triple-modified derivatives
Scheme 1. Synthesis of colchicine derivatives (2–20). Reagents and conditions: (a) NIS, AcOH, 70 ◦C, 20 h (b) MeOH/H2O, CH3SNa, RT; (c) 2 M HCl, 90 ◦C, 72 h; (d)
Et3N, DMAP, respective acyl/carbamoyl chloride, THF, 0 ◦C → RT.
2

G. Klejborowska et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116014
used as antitumor agents in cancer chemotherapy. Third, all of the tested
derivatives were more active against LoVo cell line in comparison to its
drug-resistant cell line LoVo/DX. Compounds 13 and 19 showed the
weakest activity against all cancer cell lines tested (with the exception of
13 on LoVo/DX). When comparing double- (4–10) and triple-modified
(14–20) derivatives, in many cases the derivatives with the same sub-
stituents in C-7 position showed similar IC50 values as for compounds 4
and 14 (except BALB/3T3 against which compound 14 turned out to be
less potent), compounds 7 and 17, and compounds 10 and 20 (except
ALL-5 against which compound 20 turned out to be less potent). How-
ever, some differences between corresponding pairs should be high-
lighted. Compound 15 showed, depending on the cell line, 4–8 times
higher IC50 in comparison to compound 5. Compounds 6 and 16 showed
similar IC50 against A549, LoVo and LoVo/DX cell lines, but compound
16 was 4–5 times less potent against ALL-5, MCF-5 and BALB/3T3 cell
lines than 6. Compounds 8 and 18 showed similar IC50 against LoVo and
BALB/3T3, but compound 18 was about 4 times less potent against ALL-
5, A549 and MCF-7 cell lines. Surprisingly, compound 18 was more
active against LoVo/DX than 8. The biggest difference can be observed
derivative 16), propionamide (like 7, 17) and isobutyramide (like 8,
18).
In order to evaluate the activity of the new analogs against cells with
an MDR (multidrug resistance) phenotype, one drug resistant cancer cell
line, LoVo/DX, was tested, and the resistance index (RI) values were
calculated, as described in Materials and Methods and presented in
Table 1. However, none of the derivatives was able to overcome the drug
resistance of the LoVo/DX cell line, indicated by RI values ranging from
6.7 to 278.
Comparison of IC50 values between cancer cell lines and normal
murine fibroblasts (BALB/3T3) was made to calculate the Selectivity
Index (SI) as an initial indication of the compound’s therapeutic po-
tential (Fig. 1). Standard cancer chemotherapeutics utilized in this study
(doxorubicin and cisplatin) are characterized by very low SI values < 1
(with the exception of doxorubicin on ALL-5, SI = 4.2 and on LoVo, SI =
1.8). The majority of double- and triple-modified analogues as well as
their precursors showed favorable SI > 1.0 towards ALL-5 cells and
A549, MCF-7 and LoVo cell lines. The exceptions are compounds: 4 on
A549 and MCF-7; 10 on A549 and MCF-7; 13 on MCF-7; 19 on all cells
studied. A beneficial SI on LoVo/DX cell line was observed only for
compounds 3 and 13. In general, the parent (unmodified) colchicine is
characterized by higher SI values than doxorubicin and cisplatin (except
doxorubicin on the LoVo cell line). Despite that, the following analogues
showed higher SI values than parent colchicine: 2, 3, 8, 12 on ALL-5; 2,
3, 5–9, 11–18, 20 on A549; 2, 3, 12, on MCF-7; 2–18, 20 on LoVo; 2, 3,
5, 6, 8, 9, 12–16, 18 on LoVo/DX. This is important since high SI values
result from large differences between the cytotoxicity against cancer
versus normal cells, which might indicate that cancer cells will be
affected to a greater extent than normal cells.
between derivatives
9 and 19; the derivative of N-deacetylth-
iocolchicine bearing long alkyl chain (9) proved to be much more active
than the corresponding derivative of 4-iodo-N-deacetylthiocolchicine
(19). These differences are further discussed in the molecular docking
section in terms of binding affinities to the colchicine-binding pocket
and structural differences between colchicine derivatives.
In our previous papers, analogues of 4-chloro-N-deacetylth-
iocolchicine45 and 4-bromo- N-deacetylthiocolchicine46 were described.
The less potent derivatives in all 4-halo series were amides with long
hydrophilic alkyl chains (like 19 from 4-iodo derivatives). Interestingly,
the same substituent in the C-7 position did not decrease significantly
the activity of double-modified derivative (9). The moderate potency, in
all series, was also showed by ureas (like 10, 20), derivatives with 4-
chlorobutanamide moiety in the C-7 position (like 5, 15), and for 4-
iodo series also benzamide derivative (16). The highest activity, in all
series, showed compounds bearing given moieties in the C-7 position:
methoxyacetamide (like 4, 14), benzamide (like 6, except 4-iodo
2.3. The effect of colchicine and its double- and triple-modified analogues
on cell cycle progression in ALL-5 and MCF-7 cells
In order to further investigate the mechanism behind the favorable
activity of colchicine (1) and its analogues towards primary ALL-5 cells,
we assessed DNA content and DNA fragmentation via flow cytometry.
Table 1
Antiproliferative activity (IC50) and resistance index (RI) values of colchicine (1) and its derivatives (2–20) compared with antiproliferative activity of standard
anticancer drugs doxorubicin and cisplatin.32–35
Compound
ALL-5
A549
MCF-7
LoVo
LoVo/DX
IC50 (nM)
BALB/3T3
IC50 (nM)
IC50 (nM)
IC50 (nM)
IC50 (nM)
IC50 (nM)
RI
1
8.6 ± 0.2
3.1 ± 1.3
16.3 ± 4.9
8.5 ± 0.2
5.9 ± 0.1
5.7 ± 1.9
4.6 ± 0.6
5.5 ± 0.1
27.3 ± 0.5
17.4 ± 5.1
15.5 ± 1.6
8.7 ± 0.2
135 ± 35
15.0 ± 5.9
47.5 ± 9.0
25.4 ± 0.8
9.0 ± 1.1
19.9 ± 5.2
615 ± 181
81.9 ± 16.5
125 ± 13
20.7 ± 2.4
9.6 ± 2.4
108 ± 25
21.0 ± 5.5
16.9 ± 4.0
8.2 ± 1.3
8.4 ± 0.1
5.6 ± 3.2
8.5 ± 1.3
9.0 ± 0.1
7.6 ± 0.1
69.1 ± 11.7
10.2 ± 2.2
7.4 ± 1.8
126 ± 43
7.0 ± 0.1
39.2 ± 5.8
7.2 ± 1.0
7.2 ± 0.1
7.0 ± 0.1
568 ± 43
64.6 ± 1.0
1,694 ± 275
398 ± 75
15.7
19.0
8.6
106 ± 23
2
11.3 ± 1.4
24.1 ± 2.7
13.5 ± 0.1
13.3 ± 1.2
11.2 ± 1.2
13.2 ± 1.3
15.8 ± 3.9
13.3 ± 0.2
133 ± 6
137 ± 59
3
14.2 ± 1.6
12.7 ± 1.3
11.9 ± 1.2
11.2 ± 1.2
12.4 ± 1.3
13.5 ± 0.02
66.3 ± 25.5
113 ± 7
145 ± 21
223 ± 32
4
132 ± 44
16.0
12.2
10.4
19.1
19.3
11.2
16.0
278.0
91.7
6.7
12.0 ± 1.3
20.2 ± 10.5
21.6 ± 17.6
17.9 ± 1.3
69.9 ± 24.4
87.2 ± 25.1
88.2 ± 10.0
135 ± 56
5
102 ± 15
6
57.9 ± 13.5
163 ± 51
7
8
174 ± 46
9
84.6 ± 1.1
1,105 ± 191
2,776 ± 449
642 ± 84
10
11
12
13
14
15
16
17
18
19
20
93.9 ± 5.8
11.1 ± 1.8
866 ± 320
10.5 ± 1.8
82.8 ± 4.4
13.3 ± 4.4
11.4 ± 1.0
62.6 ± 5.6
833 ± 60
97.7 ± 29.3
16.6 ± 6.4
1,705 ± 361
10.5 ± 1.8
89.4 ± 6.6
47.5 ± 14.9
10.8 ± 1.8
44.5 ± 23.6
846 ± 117
94.7 ± 7.5
115 ± 44
844 ± 52
1,424 ± 304
51.2 ± 20.0
87.8 ± 23.2
76.2 ± 16.3
39.6 ± 13.9
69.7 ± 18.4
305 ± 138
107 ± 32
92.2 ± 19.3
529 ± 77
13.2
13.5
10.2
23.3
13.0
6.8
72.9 ± 5.0
168 ± 66
91.3 ± 16.8
3,866 ± 1,328
794 ± 134
81.3 ± 17.6
12.3
Doxorubicin
Cisplatin
39.1 ± 7.0
–*
258 ± 44
386 ± 118
92.0 ± 18.0
4,370 ± 73
4,7500 ± 990
5,700 ± 630
51.6
1.3
166 ± 74
6,367 ± 1,413
10,700 ± 753
3,900 ± 1,500
The IC50 value is defined as the concentration of a compound at which 50% growth inhibition is observed.
*Inhibition of proliferation did not exceed 50% at the highest concentration tested of 10 µM.
The RI indicates how many times a resistant subline is chemoresistant relative to its parental cell line. The RI was calculated for each compound using the formula: RI =
IC50 for LoVoDX/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.
3

G. Klejborowska et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116014
Fig. 1. Comparison of selectivity index (SI) values for the tested compounds. SI was calculated for each compound using the formula: SI = IC50 for normal cell line
BALB/3T3/IC50 for respective cancerous cell line. A beneficial SI > 1.0 indicates a drug with efficacy against tumor cells greater than the toxicity against normal cells.
Specifically, propidium iodide staining was employed to determine DNA
content and cells with sub-G1 (<2N) DNA were assessed as dead. Pri-
mary ALL-5 cells were treated for 24, 48 or 72 h with unmodified 1, the
most active synthesis precursors 2 and 12 (characterized by the lowest
IC50 values, see Table 1) as well as double- and triple-modified ana-
logues (4–8 and 17, respectively), each at concentrations equal to 5 ×
IC50 values. Treatment with 0.1% DMSO (vehicle) or 0.2 µM DX at
equivalent time intervals served as negative and positive controls,
respectively. For the full set of representative cytograms, see Supple-
mental Figure S10A. A graphical representation of cells in different
phases of the cell cycle has been summarized from the mean of 3 ex-
periments and presented in Fig. 2A. Statistically significant increases in
sub-G1 DNA content were observed after 48 h of treatment with all of
the compounds and further increased after 72 h (Fig. 2 A, orange bars).
However, double-modified analogues 5, 8 and synthesis precursor 12
induced DNA fragmentation more rapidly, as indicated by significant
sub-G1 DNA after 24 h. The increase in sub-G1 DNA (Fig. 2A, orange
bars) coincided with a decrease of the pool of cells in the G1 phase of the
cell cycle (Fig. 2A, green bars), suggesting that in response to treatments
ALL cells in G1 were susceptible to death. Since 1 is typically considered
to induce cell death in the M phase,49 it was of interest to investigate
whether it, and the novel analogues, caused mitotic arrest in these cells.
As shown in Fig. 2 A (red bars) the total amount of cells in the G2/M
phases (4N DNA) was maintained at a relatively low level throughout,
with a maximum of 20%, and no evidence of overt mitotic arrest was
observed. Thus it appears that 1 and the analogues developed here
induce death of primary ALL cells directly from the G1 not M phase, a
finding consistent with previous results where we reported that two
other microtubule destabilizing agents, vincristine and eribulin,
exhibited this same property.50 In contrast, when tested in MCF-7 cells,
treatment with 1 and its analogues caused mitotic arrest, as indicated by
accumulation of cells with 4N DNA (Fig. 2B, red bars; Supplemental
Figure S10 B). Noteworthy also was the lack of significant sub-G1 DNA
in treated MCF-7 cells, suggesting delayed death kinetics after arrest, or
that they may die through a mechanism independent of DNA fragmen-
tation, perhaps due to caspase-3 deficiency.51
2.4. The effect of colchicine, double-modified analogue 7 and triple-
modified analogue 17 on PARP cleavage in primary ALL-5 cells
In order to further assess apoptotic cell death we investigated poly
(ADP-ribose) polymerase (PARP) cleavage by immunoblotting. Primary
ALL-5 cells were treated with parent colchicine (1) or the most active
double- or triple-modified analogues (based on the lowest IC50 values,
Table 1), namely compounds 7 and 17, respectively, for 24 and 48 h.
Treatment with 0.1% DMSO (vehicle) or 0.2 µM DX represented nega-
tive and positive controls, respectively. Representative immunoblots are
shown in Fig. 3A and quantitation of PARP band intensities in Fig. 3B.
All of the studied compounds induced loss of 116 kDa PARP over 48 h
(Fig. 3 A, top panel and Fig. 3 B). The characteristic 85 kDa product of
PARP degradation can be clearly observed for each treatment condition
with 1 and its analogues. Treatment of ALL-5 cells with DX also induced
PARP cleavage as we previously reported.52,53 Glyceraldehyde 3-phos-
phate dehydrogenase (GAPDH) was used as a loading control (Fig. 3
A, lower panel).
2.5. Molecular docking
The primary mechanism of microtubule inhibitors involves binding
to tubulin. Tubulin heterodimers composed of tightly bound
α and ß
tubulin monomers are the building blocks of microtubules, which are
major components of the cytoskeleton of eukaryotic cells. Bundles of
microtubules form mitotic spindles and hence are indispensable for cell
division. Colchicine binds to β-tubulin, which results in microtubule
destabilization since colchicine-bound dimers are assembly incompe-
tent. Although most eukaryotic cells can express multiple isotypes of β
tubulin, βI is typically the most highly expressed and most popular target
for drug binding.54 Molecular docking was applied to predict the
4

G. Klejborowska et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116014
Fig. 2. ALL-5 (A) or MCF-7 (B) cells were treated with DMSO (vehicle), 1, or it’s selected double- and triple-modified analogues for 24, 48 or 72 h and subjected to
propidium iodide staining and flow cytometry. Percent of cells observed in different phases of cell cycle, determined by PI staining, is shown. Data are presented as a
mean ± SD (n = 3 for all ALL-5 time points and MCF-7 at 72 h; for MCF-7 at 24 and 48 h, n = 1) ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 control versus
dose. See Supplementa1 Fig. S9 for a full set of representative cytograms.
binding affinity of the new colchicine derivatives described in this paper
with the colchicine-binding pocket of βI tubulin (Fig. 4, Table 2).55
Based on binding energies of the docking results, 13 out of 19
modification on colchicine, resulted in the lower binding energies
compared to that of the unmodified colchicine, namely 2–9, 12, 14 and
16–18. All the double-modified compounds except 10 and all the triple-
modified ones except 15, 19 and 20 show stronger interactions than
colchicine alone. Based on the IC50 values obtained from cytotoxicity
experiments, described in section 2.2, the most active synthetic pre-
cursors are 2, 12, 4–8, 14 and 17, respectively.
colchicine, ꢀ 8.09 kcal/mol.
Based on the in silico results, Compounds 17 and 14 with a triplet
modification have the shared first and second position of the lowest
binding energies of modified derivatives, ꢀ 9.30 and ꢀ 9.20 kcal/mol,
respectively. As previously described, the above-mentioned compounds
also show two of the highest potency with regards to the LoVo, LoVo/
DX, A549 and MCF-7 cell lines.
Compounds 15, 16 and 18 also show strong activity toward LoVo,
LoVo/DX, A549 cell lines, although just compounds 16 and 18 were
reported with the binding energy values lower than that for colchicine, i.
e. ꢀ 8.30 kcal/mol.
While in agreement with compounds 4 to 8 having the highest po-
tency toward the investigated cell lines, the binding energies of com-
pounds 5 to 7 show the lowest values but not in the same order.
Compound 6 with ꢀ 9.30 kcal/mol has the lowest binding energy and
compounds 5 and 7 with values of ꢀ 8.78 kcal/mol and ꢀ 8.70 kcal/mol
come after. Compounds 4 and 8 also have stronger binding energies,
ꢀ 8.30 and ꢀ 8.25 kcal/mol, respectively compared with the unmodified
Consistently with the in vitro cytotoxic activity experiment results,
compounds 13 and 19 have been found to have the highest binding
energies, ꢀ 6.90 and ꢀ 7.05 kcal/mol.
None among the 2, 3, 11 and 12 compounds that show high potency
toward different cell lines are amongst compounds with the lowest
binding energies group. It is worth noting that except compound 11 that
5

G. Klejborowska et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116014
Fig. 3. (A) Cleavage of PARP. ALL-5 cells were
treated with 43 nM compound 1, 23 nM
compound 7, 45 nM compound 17, 0.2 µM
doxorubicin (DX), or 0.1% DMSO (vehicle) for
the times indicated, and extracts were pre-
pared and subjected to immunoblotting for
PARP. The intact (116 kDa) and cleaved (85
kDa) forms of PARP are shown. GAPDH was
used as a loading control. Images were quan-
tified by measuring the band intensity using
ImageJ software. (B) Bar diagram showing the
fold changes of PARP normalized to GAPDH.
Data represented as mean ± S.D. of three in-
A.
PARP 116 kDa
85 kDa
GAPDH 37 kDa
dependent determinations (n
=
3); 24
h
treatment was compared with 48
h
for
respective compound. **P < 0.005, *P < 0.05.
Compound
Harvest time (h)
vehicle
24
1
24
1
48
7
24
7
48
17
24
17
48
DX
24
B.
Fig. 4. A. The colchicine derivatives numbered 6, 17, 14, 5 and 7 show the strongest binding energies of ꢀ 9.30, ꢀ 9.30, ꢀ 9.20, ꢀ 8.78 and ꢀ 8.70 kcal/mol,
sequentially, unmodified colchicine added to the picture in white for comparison. B. compounds 13 and 19 have the highest binding energies, ꢀ 6.90 and ꢀ 7.05 kcal/
mol, colchicine is added in blue.
has a higher binding energy than ꢀ 8.09 the rest of the compounds still
have stronger binding energies, ꢀ 8.13, ꢀ 8.25 and ꢀ 8.13 kcal/mol
respectively, than the binding energies of unmodified colchicine
compounds.
of βI tubulin are most strongly involved in the ligand-tubulin in-
teractions. Met 259 and Lys 352 residues mostly interact with the
hydrogen of C-20 (side chain H-acceptor) and oxygen of the carbonyl
group (side chain H-donor) on ring C of the new colchicine derivatives,
respectively. Previous research showed that substituting the N-acetyl
group with an aliphatic, straight-chain acyl moiety group or an aromatic
group on the acetamido group of the B ring might show some strong
hydrophobic interactions with β tubulins. However, for example, adding
hydrophilic ether function to the chain of the acetamido group can
To sum up, as predicted by our in silico calculations, we conclude that
the colchicine derivatives numbered 6, 17, 14, 5 and 7 show the stron-
gest binding energies of ꢀ 9.30, ꢀ 9.30,
ꢀ 9.20, ꢀ 8.78 and ꢀ 8.70 kcal/mol, respectively.
The two Met 259 and Lys 352 residues present in the binding pocket
6

G. Klejborowska et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116014
compound 6 also shows the lowest binding energy among the novel
colchicine derivatives. It should be emphasized that while compound 16
has a binding energy which is higher than that for colchicine, the novel
derivative is not in the group of top 5 compounds with the lowest
binding energies. A side-by-side analysis of the 2D ligand-protein in-
teractions schemes of compounds 6 and 16 shows that the aromatic
group of compound 6 unlike that for compound 16, is close to the
proximity contour and deep in the receptor cavity, which might increase
its hydrophobic interactions with hydrophobic side chains of the resi-
dues such as Ala 317, Leu248 and Leu252. For compound 16, however,
the blue circle around the aliphatic carbons represents the exposure of
water to the functional group.
Table 2
Summary of the calculated binding energies for the interactions between βI
tubulin and N-deacetylthiocolchicine and 4-iodo-N-deacetylthiocolchicine ana-
logues, the values of compounds’ Moriguchi octanol-water partition coefficient
(MlogP), which have been investigated in this paper. The active residues (resi-
dues interacting with each ligand via hydrogen bonding or
binding pocket of βI tubulin are listed in the last column.
Compound
π-interactions) in the
Binding energy
(kcal/mol)
MlogP
Active residues
1
2
3
4
ꢀ 8.09
ꢀ 8.13
ꢀ 8.25
ꢀ 8.30
1.368
2.127
2.129
1.583
Asn258, Met259
Met259, Asn258, Lys352
Asn258, Met259
Ala 316, Lys352, Asn 258, Met
259
To investigate in more detail the non-bonded interactions of com-
pound 6, a contact preference map, electrostatic feature maps and the
protein-ligand interaction fingerprints (PLIF) based on surface contact
interactions were calculated (see figure S11). The data illustrate that
Leu248 and Leu252 present hydrophobic interactions with an aromatic
functional group in the C-7 position that might result in stronger binding
energy with βI tubulin and a lower value of IC50 again for ALL-5 and
LoVo cell lines.
5
ꢀ 8.78
ꢀ 9.30
ꢀ 8.70
ꢀ 8.25
ꢀ 8.36
ꢀ 7.40
ꢀ 7.53
ꢀ 8.13
ꢀ 6.90
ꢀ 9.20
ꢀ 7.94
2.761
2.944
2.342
2.553
3.753
2.813
1.794
2.553
2.570
2.001
3.166
Val 315, Met259, Lys254
Val 315, Met259, Ala250
Val 238, Met259, Lys352
Cys 241, Ala316
6
7
8
9
Val315
10
11
12
13
14
15
Lys352, Met259, Asn258, Val238
Ala317, Cys241
Met259, Lys352
Asn258, Cys241, Lys352
Lys 352, Met259 Ala316, Ala317
Lys 352, Met 259, Ala 250,
Lys254, Cys241
Triple-modified compound 17 also has the highest binding energy
and shares the first position in terms of the lowest binding energies for
the modified derivatives. The binding energy of double-modified com-
pound 7 also has the last position in the top 5 compounds with the
lowest binding energies. Replacing the methyl group of acetamido group
on carbon 7 on the B ring with ethyl group in compounds 7 and 17
improves the strength of their binding energies significantly. Based on
the 3D ligand-protein interactions scheme, Table 3, the rings C and B of
both compounds are in the same poses. Met259 has a sidechain H-
acceptor with the hydrogen of carbon 20 on ring C and Lys352 has a
sidechain H-donor with the oxygen of carbonyl on the same ring. The
residues that interact with ring A of compound 17 and 7, however, are
different and this might be the reason for the 0.6 kcal/mol difference
between their binding energies.
16
ꢀ 8.31
3.338
Asn258, Ala250, Lys254, Val238,
Met259
17
18
19
20
ꢀ 9.30
ꢀ 8.30
ꢀ 7.05
ꢀ 7.63
2.761
2.965
4.131
3.219
Lys 352, Met259, Ala316
Cys241
Lys254, Leu248
Lys352, Asn258, Met259
decrease the compound’s hydrophobicity.20,56 Table 3 and Table S2
depict these interactions (see Table S1. for 2D-ligand-protein in-
teractions representation).
In the 2D ligand-protein interactions scheme, see Table S1, greasy
residues, which do not have a polar or charged sidechain, are shown in
green. These residues are more likely to show hydrophobic interactions
either with other protein residues or ligands. A proximity contour,
shown as a dashed line, shows how deep a ligand is buried in the re-
ceptor cavity and if the ligand is surrounded by greasy residues, the most
probable interactions between the ligand and greasy residues are hy-
drophobic reactions. The other parameter that plays a key role in the
hydrophobic reactions is the ligand and receptor exposure, which shows
the exposed part of a ligand or a residue to the water.
Compound 14 ranks in the second position among the compounds
with the strongest interaction with βI tubulin. Adding hydrophilic ether
groups to the chain of a functional group can decrease the hydropho-
bicity, so theoretically the binding energies of compound 4 and 14
should be lower than those for compound 7 and 17. The binding energy
of compound 4 is still lower than that for colchicine, but is not among
the best modified compounds. To our surprise the binding energy for
compound 14 is almost as good as those for compounds 6 and 17. A
possible explanation of this finding could be that the interaction be-
tween iodine atom on at carbon atom 4 of ring A and Ala 317 put the
modified functional group of ring B in a position that can have a side-
chain H-acceptors interacting with Lysine 352, a residue with an
electrostatically-charged side chain, and Methionine 259. The above-
mentioned interactions cause the added ether group to be less exposed
to water and buried deeper into the cavity. It should be recalled that the
In the case of compound 6 and 16, an aromatic functional group was
substituted in the C-7 position that might induce the hydrophobic in-
teractions between aromatic rings and greasy residues and result in a
stronger interaction with βI tubulin. According to the in vitro cytotoxic
activity experiments reported here, compound 6 has 5 and 4 times
smaller IC50 values toward ALL-5 and LoVo cell lines than the IC50 values
for compound 16, respectively. Here, in the computational part,
Table 3
Exemplary 3D representations of the interactions between βI tubulin, colchicine and its derivatives (7 and 17).
Compound
1
7
17
3D representation
7

G. Klejborowska et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116014
IC50 values for compounds 17 and 14 are almost the same for the MCF-7,
A549 and LoVo cell lines.
experimental results, linear regression coefficients were calculated.
However, in numerous cases, the binding affinity alone is insufficient to
arrive at a close correlation between the results of biological assays (and
the values of IC50 given by them) and the binding free energies between
the ligands and their protein targets obtained from docking computa-
tions. In order to account for the novel compounds’ properties other
than the affinity for the target, the Moriguchi octanol-water partition
coefficients (MLogP) were calculated. MlogP is a useful factor to esti-
mate and compare the distribution of drugs within biological systems
such as the human body.
Compound 5 is placed in the third position among the top 5 tubulin-
binding compounds. Despite the fact that in both compounds 5 and 15,
methyl of acetamido group is replaced with 3-chloropropyl, the binding
energies of double-modified, ligand 5, and triple-modified, ligand 15,
are different as are their values of IC50 against all of the cell lines
investigated here. Either in experimental or theoretical results, com-
pound 5 shows to be a more desirable novel derivative than compound
15. In a side-by-side comparison of the 2D interaction scheme for
compound 5 and 15, it can be found that sulfur of the Cysteine residue
exhibits an interaction with sulfur of the substituted functional group on
ring B for compound 5. It is worth noting, that the interaction between
sulfurs is not a disulfide bridge due to the fact that the two sulfurs are
3.68 Å apart which is longer than 2.05 Å of the disulfide bond length and
hence can be categorized as a weak sulfur-sulfur interaction. Compound
15 has weaker binding energies than compound 5 even though it has a
few hydrogen donors and acceptors interacting with residues, Lys352,
Met259, Ala250, Lys 254 and Cys 241. The sidechain hydrogen acceptor
interaction between Cys241 and hydrogen of carbon 7 in the 4-iodosub-
stitued compound is caused by inducing electronegativity on the ortho
position of the subtitled iodine. This effect, puts the functional group of
ring B in a position that is more exposed to water and hence engages in
stronger hydrophilic interactions. The 3D molecular electrostatic po-
tential map included in Table 4 shows a larger negative charge cloud
over the oxygen of the ether group in the ortho position of iodine on the
A ring. In compound 5, the modified functional group is positioned in
the receptor cavity and has less interface with water, which leads to a
stronger interaction with the binding pocket of βI tubulin.
Thus, the two independent variables chosen in the compounds’
characterization have been: MlogP values and the binding free energies
with the tubulin βI isotype. Linear regression between IC50 values and
these two variables was then performed and analyzed. A value of 0.5 is a
good regression coefficient that has been found using this method for log
IC50 of BALB/3T3 and LoVo cell lines. An acceptable value of 0.4 was
obtained for LoVo/DX cell lines and the binding affinities for our com-
pounds and tubulin βI isotype. The regression coefficient values ob-
tained for the MCF-7, A549 and ALL-5 cell lines are very low. This may
be due to off-target interactions, P-glycoprotein-based efflux of these
compounds or additional complexities involved in the response of these
cells to the compounds tested.
To take into account the fact that, regardless of their redundancy, all
tubulin isotypes are expressed in a cell-based assay, the interaction en-
ergies involving the novel derivatives and the remaining isotypes of
tubulin, namely: βIIa, βIIb, βIII, βIVa, βIVb, and βVI were calculated
using the same docking method and comparisons of experimental data
with computational results were made. It is worth noting that the ALL-5
cell line shows an acceptable linear regression coefficient 0.4 with βIIa
and βVI isotypes (see Table 5). Low values of regression coefficients have
Consistently with experimental data, compound 13 and compound
19 are the weakest modified derivatives of colchicine that interact with
βI tubulin. To investigate in detail the poor performance of compound
13, 3D molecular electrostatic potential maps for compounds 3 and 13
were created for comparison. As is illustrated in Table 4, there is an
enlarged negative charge cloud around the subtitled iodine on carbon 4
of ring A and the ether group in its ortho position. The intensified
negative charge on ring A enhanced the hydrophilic interaction of ring A
with water and resulted in weaker binding energies with βI tubulin.
A comparison of 2D and 3D interaction schemes between compound
9 and 19 in Table S1 and Table S2 shows that the only differences in
their poses are the positions of their long hydrophobic aliphatic chains.
The long functional group on ring B of compound 9 is surrounded by
greasy residues and might result in an increased hydrophobic interac-
tion, which might also explain higher cytotoxicity of compound 9 in
comparison to compound 19. It should be noted that long hydrophobic
aliphatic chains can cause a steric effect which could jeopardize the
cytotoxic activity of the novel derivatives, which is particularly
confirmed by experimental data of compound 19.
Table 5
The docking binding free energy values for the ligand-tubulin complexes and the
MlogP predicted values for the ligands are two independent variables in the
linear regression calculations with log IC50 [nM] for different cancer cell lines.
The bolded value indicates the highest linear regression values.
Linear
ALL-
5
MCF-
7
LoVo
LoVo/
DX
A549
BALB/
3T3
regression of
colchicine
derivatives
(R2)
ßI
0.090
0.400
0.300
0.300
0.20
0.200
0.100
0.200
0.200
0.300
0.040
0.100
0.100
0.500
0.040
0.030
0.090
0.200
0.002
0.110
0.060
0.400
0.100
0.090
0.090
0.020
0.040
0.020
0.002
0.004
0.040
0.040
0.040
0.200
0.010
0.040
0.090
0.500
0.001
0.040
0.040
0.200
0.001
0.002
0.010
ßIIa
ßIIb
ßIII
ßIVa
ßIVb
ßV
0.200
0.200
0.400
ßVI
To provide
a comparison between the computational and
Table 4
3D molecular electrostatic potential maps of compound 3 and 13 and 15. The blue color represents negative charges and the red color represents positive charges.
Compound
3
13
15
3D-MEP map
8

G. Klejborowska et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116014
been found for the remaining tubulin isotypes. While these biological
assays include millions of cells in a culture, computational work only
focuses on a single protein at a time and its interaction with the phar-
macological agent binding to it. Other biopharmaceutical properties
should also be taken into account when theoretical and experimental
results are compared. The upregulation of MDR proteins that act as
efflux pumps for the tested compounds may be the main reason for some
discrepancies between computation and experiment. Another possibility
could involve off-target interactions whereby not only tubulin but also
other proteins present in the tested cell lines bind these compounds
lowering their measured potency.
used.
The ESI (Electrospray Ionisation) mass spectra were recorded also on
a Waters/Micromass (Waters Corporation, Manchester, UK) ZQ mass
spectrometer equipped with a Harvard Apparatus syringe pump. The
samples were prepared in dry acetonitrile (5 × 10–5 mol dmꢀ 3). The
sample was infused into the ESI source using a Harvard pump at a flow
rate of 20 ml minꢀ 1. The ESI source potentials were: capillary 3 kV, lens
0.5 kV, extractor 4 V. The standard ESI mass spectra were recorded at
the cone voltages: 10 and 30 V. The source temperature was 120 ◦C and
the desolvation temperature was 300 ◦C. Nitrogen was used as the
nebulizing and desolvation gas at flow-rates of 100 dm3 hꢀ 1. Mass
spectra were acquired in the positive ion detection mode with unit mass
resolution at a step of 1 m/z unit. The mass range for ESI experiments
was from m/z = 100 to m/z = 1000, as well as from m/z = 200 to m/z =
1500.
3. Conclusions
We synthesized and characterized a set of 7-deacetyl-10-thiocolchi-
cine and 4-iodo-7-deacetyl-10-thiocolchicine analogues. In total 19
colchicine derivatives, including 13 novel amide derivatives, were
developed with moderate to good yields. Most of the derivatives showed
activity against primary leukemia ALL-5 and established cancer cell
lines (MCF-7, LoVo, LoVo/DX) in the low nanomolar range. In general,
we conclude that 7-deacetyl-10-thiocolchicine analogues were more
active towards ALL-5 cells while 4-iodo-7-deacetyl-10-thiocolchicine
analogues were slightly more active towards the LoVo cell line. Most
of the synthesized compound showed favorable selectivity index values,
especially for ALL-5 and LoVo cell lines. Cell cycle progression studies
revealed that colchicine and its derivatives induce death of primary ALL
cells directly from G1 phase, as do other microtubule destabilizing
agents such as vincristine and eribulin. In contrast, a lack of sub-G1 DNA
after treatment of MCF-7 cells suggests delayed death kinetics after
mitotic arrest. In silico calculations demonstrated that colchicine de-
rivatives 6, 17, 14, 5 and 7 show the strongest binding energies of
ꢀ 9.30, ꢀ 9.30, ꢀ 9.20, 8.78 and ꢀ 8.70 kcal/mol, respectively. These also
exhibited very low nanomolar IC50 values in experimental assays.
Favourable linear regression coefficients (R2 = 0.5) were obtained for βI
tubulin and LoVo as well as BALB/3T3 cell lines emphasizing the utility
of molecular docking methodology for anticancer drug development.
4.3. Synthesis
4.3.1. Synthesis of thiocolchicine (2)
To a mixture of 1 (500 mg, 1.25 mmol) in MeOH/water (1/1, v/v, 5
ml), the sodium methanethiolate (solution 21% in H2O, 0.83 ml, 2.5
mmol) was added. The mixture was stirred in at RT for 72 h. Reaction
time was determined by TLC. After that time, the reaction mixture was
quenched by the addition of water (150 ml). The whole mixture was
extracted four times with CH2Cl2, and the combined organic layers were
dried over MgSO4, filtered, and evaporated under reduced pressure. The
residue was purified by CombiFlash® (hexane/EtOAc (1/1), then
EtOAc/MeOH, increasing concentration gradient) to give
2
57
(C22H25NO5S, MW = 415.5 g/mol) with yield 78%
.
1H NMR (403
MHz, CDCl3) δ 7.92 (s, 1H), 7.46 (s, 1H), 7.33 (d, J = 10.4 Hz, 1H), 7.10
(d, J = 10.5 Hz, 1H), 6.55 (s, 1H), 4.72–4.64 (m, 1H), 3.95 (s, 3H), 3.91
(s, 3H), 3.67 (s, 3H), 2.54 (dd, J = 13.0, 5.8 Hz, 1H), 2.45 (s, J = 5.7 Hz,
3H), 2.43–2.26 (m, 2H), 1.99 (s, 3H), 1.94 (dd, J = 11.8, 5.5 Hz, 1H)
ppm. 13C-NMR (101 MHz, CDCl3) δ 182.4, 170.0, 158.1, 153.6, 151.8,
151.1, 141.6, 138.6, 134.8, 134.4, 128.3,126.7, 125.6, 107.3, 61.6,
61.4, 56.1, 52.3, 36.4, 29.9, 22.8, 15.1 ppm. FT-IR (KBr pellet): 3283,
2935, 1660, 1605, 1541, 1485, 1461, 1425, 1404, 1349, 1321, 1286,
1236, 1195, 1155, 1138, 1095, 1023 cmꢀ 1. ESI-MS (m/z): [M+H]+
calcd. 416, found 416, [M+Na]+ calcd. 438, found 438, [M+K]+ calcd.
454 found 454, [2M+Na]+ calcd. 853, found 853, [3M+Na]+ calcd.
1268, found 1268.
4. Materials and methods
4.1. General
All precursors and solvents for the synthesis were obtained from
Sigma Aldrich (Merck KGaA, Saint Louis, MO, USA) and were used
without further purification. CDCl3 spectral grade solvent was stored
over 3 Å molecular sieves for several days. TLC was performed on pre-
coated plates (TLC silica gel 60 F254, Aluminium Plates Merck, Merck
KGaA, Saint Louis, MO, USA) visualized by illumination with an UV
lamp. HPLC grade solvents (without further purification) were used for
flash chromatography (CHROMASOLV from Sigma Aldrich, Merck
KGaA, Saint Louis, MO, USA). The elemental analysis of compounds was
performed on Vario ELIII (Elementar, Langenselbold, Germany).
4.3.2. Synthesis of N-deacetylthiocolchicine (3)
Compound 3 was prepared from 2 by hydrolisys with 2 N HCl. To a
solution of compound 2 (500 mg, 1.20 mmol) in MeOH (3 ml), the 2 N
HCl solution (5 ml) was added. The mixture was stirred at 90 ◦C for 72 h.
Reaction time was determined by TLC. After that time the reaction
mixture was quenched by the addition of water (100 ml). The whole
mixture was extracted four times with CH2Cl2, and the combined
organic layers were dried over MgSO4, filtered, and evaporated under
reduced pressure. The residue was purified by CombiFlash® (EtOAc/
MeOH, increasing concentration gradient) to give 3 (C20H23NO4S, MW
= 373.5 g/mol) with yield 86% 42. 1H NMR (403 MHz, CDCl3) δ 7.58 (s,
1H), 7.19 (d, J = 10.3 Hz, 1H), 7.03 (d, J = 10.7 Hz, 1H), 6.54 (s, 1H),
3.91 (s, 3H), 3.91 (s, 3H), 3.75–3.69 (m, 1H), 3.66 (s, 3H), 2.52–2.26 (m,
6H), 1.65–1.57 (m, 3H) ppm. 13C NMR (101 MHz, CDCl3) δ 182.5,
157.8, 153.7, 153.4, 150.6, 141.1, 138.1, 135.2, 134.1, 129.3, 125.9,
125.4, 106.9, 61.1, 61.0, 56.0, 53.6, 40.2, 30.5, 15.1 ppm. FT-IR (KBr
pellet): 3365, 3293, 2931, 2852, 2838, 1603, 1546, 1485, 1458, 1422,
1402, 1347, 1318, 1138, 1094, 1017 cmꢀ 1. ESI-MS (m/z): [M+H]+
calcd 374, found 374.
4.2. Spectroscopic measurements
The 1H, 13C spectra were recorded on a Varian VNMR-S 400 MHz
spectrometer (Varian, Inc., Palo Alto, CA, USA). 1H NMR measurements
of 2–20 (0.07 mol dmꢀ 3) in CDCl3 were carried out at the operating
frequency 402.64 MHz. The error of the chemical shift value was 0.01
ppm. The 13C NMR spectra were recorded at the operating frequency
101.25 MHz. The error of chemical shift value was 0.1 ppm. All spectra
were locked to deuterium resonance of CDCl3. The 1H and 13C NMR
spectra are shown in the Supplementary Materials.
The FT-IR spectra of 2–20 in the mid infrared region were recorded
in KBr. The spectra were taken with an IFS 113v FT-IR spectropho-
tometer (Bruker, Karlsruhe, Germany) equipped with a DTGS detector;
resolution 2 cmꢀ 1, NSS = 64. The Happ-Genzel apodization function was
4.3.3. General procedure for the synthesis of colchicine derivatives (4–10)
Compounds 4–10 were obtained directly from compound 3. To a
solution of compound 4 (100 mg, 0.27 mmol) in tetrahydrofuran (THF,
5 ml) cooled to the 0 ◦C temperature, the following compounds were
9

G. Klejborowska et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116014
added: Et3N (2 ml, 14 mmol) and DMAP (catalytic amount). The mixture
was first stirred at 0 ◦C temperature for a few minutes and then the
solution of respective acyl chloride (4–9) or dietyhylcarbamoyl chloride
(10) in THF (0.81 mmol in 2,5 ml) was added dropwise. The mixture was
stirred at RT for the next 24 h. The solution was filtered to remove
triethylamine hydrochloride. The THF was evaporated and the residue
was purified by CombiFlash® (hexane/ethyl acetate, increasing con-
centration gradient) to give respective compounds as amorphous yellow
solids with yield from 38% to 82% (4–10).
61.3, 56.1, 51.5, 36.7, 35.2, 30.0, 19.5, 19.5, 15.1 ppm. FT-IR: 3312,
2968, 2935, 1669, 1607, 1544, 1486, 1461, 1425, 1404, 1349, 1322,
1283, 1235, 1196, 1154, 1137, 1096, 1021 cmꢀ 1. ESI-MS (m/z):
[M+Na]+ calcd 466, found 466. Anal. Calcd. for C, 64.99; H, 6.59; N,
3.16; O, 18.04; S, 7.23; found C, 64.87; H, 6.56; N, 3.15; S, 7.31.
Compound 9: 1H NMR (403 MHz, CDCl3) δ 7.56 (d, J = 7.4 Hz, 1H),
7.45 (s, 1H), 7.31 (d, J = 10.4 Hz, 1H), 7.08 (d, J = 10.8 Hz, 1H), 6.54 (s,
1H), 4.71 (dt, J = 11.8, 6.9 Hz, 1H), 3.95 (s, 3H), 3.91 (s, 3H), 3.67 (s,
3H), 2.52 (dd, J = 13.3, 6.1 Hz, 1H), 2.46–2.34 (m, 4H), 2.30–2.21 (m,
3H), 1.89 (td, J = 11.9, 6.2 Hz, 1H), 1.58 (dd, J = 14.6, 7.2 Hz, 2H),
1.30–1.19 (m, 12H), 0.86 (t, J = 6.9 Hz, 3H) ppm. 13C NMR (101 MHz,
CDCl3) δ 182.3, 173.0, 158.0, 153.5, 151.7, 151.1, 141.5, 138.4, 134.6,
134.4, 128.6, 126.5, 125.7, 107.3, 61.6, 61.3, 56.0, 51.8, 36.6, 36.3,
31.8, 30.0, 29.3, 29.3, 29.2, 29.2, 25.5, 22.6, 15.1, 14.0 ppm. FT-IR:
3298, 2927, 2853, 1655, 1607, 1543, 1485, 1461, 1425, 1404, 1348,
1321, 1282, 1235, 1195, 1154, 1137, 1097, 1022 cmꢀ 1. ESI-MS (m/z):
[M+Na]+ calcd 550, found 550. Anal. Calcd. for C, 68.28; H, 7.83; N,
2.65; O, 15.16; S, 6.08; found C, 68.21; H, 7.98; N, 2.68; S, 5.98.
Compound 10: 1H NMR (403 MHz, CDCl3) δ 7.48 (s, 1H), 7.26 (d, J
= 10.4 Hz, 1H), 7.03–6.99 (d, J = 10.8 Hz, 1H), 6.50 (s, 1H), 5.90 (d, J
= 7.1 Hz, 1H), 4.66 (dt, J = 11.7, 6.7 Hz, 1H), 3.91 (s, 3H), 3.87 (s, 3H),
3.67 (s, 3H), 3.32–3.25 (m, 4H), 2.48 (dd, J = 13.3, 5.7 Hz, 1H),
Compound 4: 1H NMR (403 MHz, CDCl3) δ 7.26 (d, J = 10.3 Hz, 1H),
7.18 (s, 1H), 7.09 (d, J = 7.5 Hz, 1H), 7.02 (d, J = 10.6 Hz, 1H), 6.52 (s,
1H), 4.65 (dt, J = 11.8, 6.9 Hz, 1H), 3.92 (s, 3H), 3.88 (s, 3H), 3.83 (d, J
= 4.3 Hz, 2H), 3.63 (s, 3H), 3.41 (s, 3H), 2.59–2.51 (m, 1H), 2.48–2.37
(m, 4H), 2.22 (tt, J = 13.0, 6.6 Hz, 1H), 1.88 (ddd, J = 11.9, 8.9, 5.8 Hz,
1H) ppm. 13C NMR (101 MHz, CDCl3) δ 182.3, 167.0, 158.2, 153.5,
151.2, 150.0, 141.6, 137.7, 134.4, 134.1, 128.5, 126.1, 125.6, 107.3,
71.6, 61.4, 61.3, 59.1, 56.0, 51.2, 36.8, 29.8, 15.1 ppm. FT-IR: 3287,
2937, 1672, 1607, 1552, 1486, 1462, 1426, 1403, 1350, 1323, 1287,
1264, 1236, 1195, 1154, 1138, 1096, 1022 cmꢀ 1. ESI-MS (m/z):
[M+Na]+ calcd 468, found 468. Anal. Calcd. for C, 62.00; H, 6.11; N,
3.14; O, 21.55; S, 7.20; found C, 61.89; H, 6.05; N, 3.19; S, 7.35.
Compound 5: 1H NMR (403 MHz, CDCl3) δ 7.55 (d, J = 7.4 Hz, 1H),
7.45 (s, 1H), 7.34–7.29 (m, 1H), 7.08 (d, J = 10.8 Hz, 1H), 6.53 (s, 1H),
4.70 (dt, J = 11.8, 6.9 Hz, 1H), 3.94 (s, 3H), 3.90 (s, 3H), 3.66 (s, 3H),
3.51 (td, J = 6.6, 1.1 Hz, 2H), 2.55–2.47 (m, 1H), 2.45–2.33 (m, 6H),
2.26 (dt, J = 18.6, 6.3 Hz, 1H), 2.08–1.99 (m, 2H), 1.94–1.86 (m, 1H)
ppm. 13C NMR (101 MHz, CDCl3) δ 182.4, 171.5, 158.2, 153.6, 151.5,
151.1, 141.6, 138.4, 134.7, 134.3, 128.6, 126.6, 125.6, 107.3, 61.6,
61.4, 56.1, 51.9, 44.4, 36.8, 33.0, 30.0, 28.1, 15.12 ppm. FT-IR: 3273,
2937, 1669, 1604, 1531, 1486, 1461, 1428, 1403, 1368, 1346, 1320,
1282, 1234, 1194, 1156, 1138, 1092, 1020 cmꢀ 1. ESI-MS (m/z):
[M+Na]+ calcd 500, found 500. Anal. Calcd. for C, 60.30; H, 5.90; Cl,
7.42; N, 2.93; O, 16.74; S, 6.71; found C, 60.46; H, 6.01; Cl, 7.44; N,
2.98; S, 6.69.
2.41–2.20 (m, 5H), 1.97–1.86 (m, 1H), 1.11 (t, J = 7.1 Hz, 6H) ppm. 13
C
NMR (101 MHz, CDCl3) δ 182.2, 157.8, 156.0, 153.3, 152.6, 151.1,
141.5, 138.4, 134.6, 134.3, 129.2, 126.2, 125.8, 107.2, 61.7, 61.3, 56.0,
53.1, 40.9, 37.2, 30.2, 15.0, 13.9 ppm. FT-IR: 3372, 2970, 2935, 1641,
1607, 1550, 1523, 1486, 1460, 1425, 1404, 1349, 1322, 1282, 1269,
1236, 1195, 1152, 1138, 1096, 1021 cmꢀ 1. ESI-MS (m/z): [M+H]+
calcd 473, found 473, [M+Na]+ calcd 495, found 495, [M+K]+ calcd
511, found 511. Anal. Calcd. for C, 63.54; H, 6.82; N, 5.93; O, 16.93; S,
6.78; found C, 63.41; H, 6.77; N, 6.01; S, 6.61.
4.3.4. Synthesis of 4-iodocolchicine (11)
A mixture of N-iodosuccinimide (560 mg, 2.49 mmol) and 1 (500
mg, 1.25 mmol) in AcOH was stirred at 70 ◦C under nitrogen atmosphere
for the 20 h. Reaction time was determined by TLC. The reaction was
quenched with saturated aqueous Na2S2O3. The whole mixture was
extracted four times with CH2Cl2, and the combined organic layers were
dried over MgSO4, filtered, and evaporated under reduced pressure. The
residue was purified by CombiFlash® (EtOAc/MeOH, increasing con-
Compound 6: 1H NMR (403 MHz, CDCl3) δ 7.83–7.79 (m, 2H), 7.68
(d, J = 7.3 Hz, 1H), 7.49 (s, 1H), 7.40–7.28 (m, 4H), 7.08 (d, J = 10.8 Hz,
1H), 6.56 (s, 1H), 4.91 (dt, J = 11.7, 6.8 Hz, 1H), 3.96 (s, 3H), 3.91 (s,
3H), 3.74 (s, 3H), 2.57 (dd, J = 13.3, 5.9 Hz, 1H), 2.50–2.40 (m, 4H),
2.35 (td, J = 12.4, 6.2 Hz, 1H), 2.07 (td, J = 11.7, 5.4 Hz, 1H) ppm. 13
C
NMR (101 MHz, CDCl3) δ 182.2, 166.8, 158.2, 153.6, 151.2, 141.6,
138.3, 134.6, 134.4, 133.5, 131.5, 128.7, 128.4, 127.1, 126.4, 125.7,
107.3, 61.7, 61.4, 56.1, 52.5, 36.6, 30.0, 15.1 ppm. FT-IR: 3334, 2937,
1658, 1605, 1545, 1528, 1485, 1461, 1424, 1404, 1350, 1322, 1287,
1235, 1195, 1154, 1137, 1095, 1020 cmꢀ 1. ESI-MS (m/z): [M+H]+
calcd 478, found 478, [M+Na]+ calcd 500, found 500. Anal. Calcd. for
C, 67.90; H, 5.70; N, 2.93; O, 16.75; S, 6.71; found C, 67.81; H, 5.78; N,
2.89; S, 6.79.
centration gradient) to give 11 with yield 95% 29
.
1H NMR (403 MHz,
CDCl3) δ 8.22 (d, J = 5.6 Hz, 1H), 7.61 (s, 1H), 7.30 (d, J = 10.7 Hz, 1H),
6.89 (d, J = 11.2 Hz, 1H), 4.55–4.47 (m, 1H), 4.04 (s, 3H), 3.97 (s, 3H),
3.95 (s, 3H), 3.63 (s, 3H), 3.21–3.15 (m, 1H), 2.40 (dd, J = 12.7, 5.0 Hz,
1H), 1.99 (s, 3H), 1.87–1.81 (m, 1H) ppm. 13C NMR (101 MHz, CDCl3) δ
179.5, 170.2, 164.4, 153.4, 152.0, 151.4, 145.6, 136.7, 136.2, 135.6,
130.1, 129.5, 112.5, 92.1, 61.5, 61.3, 60.7, 56.5, 52.6, 34.4, 34.4, 22.7
ppm. FT-IR (KBr pellet): 3274, 2934, 1662, 1617, 1588, 1563, 1461,
Compound 7: 1H NMR (403 MHz, CDCl3) δ 7.39 (s, 1H), 7.30 (d, J =
10.3 Hz, 1H), 7.07 (d, J = 10.6 Hz, 1H), 6.52 (s, J = 4.6 Hz, 1H), 4.67 (dt,
J = 11.8, 6.7 Hz, 1H), 3.93 (s, 3H), 3.89 (s, 3H), 3.66 (s, 3H), 2.51 (dd, J
= 13.3, 6.2 Hz, 1H), 2.45–2.33 (m, 4H), 2.30–2.22 (m, 3H), 1.95–1.84
(m, 1H), 1.09 (t, J = 7.6 Hz, 3H) ppm. 13C NMR (101 MHz, CDCl3) δ
182.4, 173.6, 158.1, 153.5, 151.6, 151.1, 141.6, 138.4, 134.6, 134.4,
128.4, 126.6, 125.7, 107.3, 61.7, 61.3, 56.1, 51.9, 36.6, 29.9, 29.2, 15.1,
9.5 ppm. FT-IR: 3303, 2937, 1660, 1607, 1543, 1486, 1462, 1425, 1404,
1349, 1321, 1283, 1235, 1196, 1154, 1138, 1096, 1022 cmꢀ 1. ESI-MS
(m/z): [M+H]+ calcd 430, found 430, [M+Na]+ calcd 552, found 552
[M+K]+ calcd 468, found 468. Anal. Calcd. for C, 64.31; H, 6.34; N,
3.26; O, 18.62; S, 7.42; found C, 64.41; H, 6.46; N, 3.22; S, 7.48.
Compound 8: 1H NMR (403 MHz, CDCl3) δ 7.45 (s, 1H), 7.41 (d, J =
7.6 Hz, 1H), 7.32–7.28 (m, 1H), 7.06 (d, J = 10.8 Hz, 1H), 6.52 (s, 1H),
4.69 (dt, J = 11.8, 7.0 Hz, 1H), 3.93 (s, 3H), 3.89 (s, 3H), 3.67 (s, 3H),
2.51 (dt, J = 13.5, 6.8 Hz, 2H), 2.44–2.32 (m, 4H), 2.25 (dt, J = 18.6,
6.3 Hz, 1H), 1.89 (td, J = 11.8, 6.1 Hz, 1H), 1.12 (dd, J = 6.9, 4.0 Hz,
6H). 13C NMR (101 MHz, CDCl3) δ 182.3, 176.8, 158.0, 153.5, 151.7,
151.2, 141.6, 138.4, 134.5, 134.4, 128.6, 126.5, 125.7, 107.3, 61.7,
1406, 1393, 1346, 1318, 1266, 1249, 1171, 1136, 1078, 1015 cmꢀ 1
.
ESI-MS (m/z): [M+H]+ calcd 526, found 526 [M+Na]+ calcd 548, found
548.
4.3.5. Synthesis of 4-iodothiocolchicine (12)
To a mixture of 11 (500 mg, 0.95 mmol) in MeOH/water (1/1, v/v, 5
ml), the sodium methanethiolate (solution 21% in H2O, 0.72 ml, 1.9
mmol) was added. The mixture was stirred in at RT for 72 h. Reaction
time was determined by TLC. After that time the reaction mixture was
quenched by the addition of water (150 ml). The whole mixture was
extracted four times with CH2Cl2, and the combined organic layers were
dried over MgSO4, filtered, and evaporated under reduced pressure. The
residue was purified by CombiFlash® (hexane/EtOAc (1/1), then
EtOAc/MeOH, increasing concentration gradient) to give 12
(C22H24INO5S, MW = 541.4 g/mol) as amorphous yellow solid with
yield 71%.33,57 1H NMR (403 MHz, CDCl3) δ 7.75 (d, J = 6.9 Hz, 1H),
7.42 (s, 1H), 7.25 (d, J = 10.3 Hz, 1H), 7.09 (d, J = 10.8 Hz, 1H),
4.58–4.50 (m, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.63 (s, 3H), 3.18 (dd, J =
10

G. Klejborowska et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116014
13.7, 5.0 Hz, 1H), 2.46 (s, 3H), 2.40 (dd, J = 13.6, 6.2 Hz, 1H),
2.32–2.23 (m, 1H), 2.01 (s, 3H), 1.85–1.79 (m, 1H) ppm. 13C NMR (101
MHz, CDCl3) δ 182.4, 170.1, 159.1, 153.5, 151.4, 151.1, 145.6, 137.8,
136.8, 134.7, 129.7, 128.1, 126.3, 92.2, 61.6, 61.4, 60.8, 52.1, 34.5,
34.4, 22.9, 15.2 ppm. FT-IR (KBr pellet): 3288, 2936, 1660, 1607, 1547,
1154, 1137, 1081, 1054, 1019 cmꢀ 1. ESI-MS (m/z): [M+Na]+ calcd 626,
found 626. Anal. Calcd. for C, 47.73; H, 4.51; Cl, 5.87; I, 21.01; N, 2.32;
O, 13.25; S, 5.31; found C, 47.74; H, 4.53; I, 20.89; N, 2.26; S, 5.27.
Compound 16: 1H NMR (403 MHz, CDCl3) δ 7.93 (d, J = 7.1 Hz, 1H),
7.81 (dt, J = 8.5, 1.7 Hz, 2H), 7.52 (s, 1H), 7.37–7.32 (m, 1H), 7.29–7.23
(m, 3H), 7.10–7.05 (m, 1H), 4.79 (dt, J = 11.8, 6.9 Hz, 1H), 3.98 (s, 3H),
3.95 (s, 3H), 3.71 (s, 3H), 3.20 (dd, J = 13.7, 4.8 Hz, 1H), 2.52–2.42 (m,
4H), 2.31 (dt, J = 17.1, 4.9 Hz, 1H), 2.05 – 1.96 (m, 1H) ppm. 13C NMR
(101 MHz, CDCl3) δ 182.2, 167.1, 159.2, 153.5, 151.5, 150.9, 145.6,
137.7, 136.9, 134.6, 133.3, 131.6, 129.8, 128.5, 128.4, 127.1, 126.1,
92.2, 61.7, 61.4, 60.8, 52.3, 34.7, 34.5, 15.2 ppm. FT-IR: 3323, 3058,
2935, 1659, 1606, 1549, 1487, 1461, 1406, 1346, 1319, 1289, 1262,
1197, 1152, 1081, 1019 cmꢀ 1. ESI-MS (m/z): [M+H]+ calcd 604, found
604, [M+Na]+ calcd 626, found 626. Anal. Calcd. for C, 53.74; H, 4.34;
I, 21.03; N, 2.32; O, 13.26; S, 5.31; found C, 53.79; H, 4.46; I, 20.97; N,
2.28; S, 5.33.
1461, 1406, 1346, 1318, 1288, 1262, 1197, 1138, 1081, 1019 cmꢀ 1
.
ESI-MS (m/z): [M+H]+ calcd 542, found 542, [M+Na]+ calcd 564,
found 564, [M+K]+ calcd 580, found 580.
4.3.6. Synthesis of 4 -iododeacetylothiocolcicine (13)
Compound 13 was prepared from 12 by hydrolysis with 2 N HCl. To
a solution of compound 12 (500 mg, 0.92 mmol) in MeOH (3 ml), the 2
N HCl solution (5 ml) was added. The mixture was stirred at 90 ◦C for 72
h. Reaction time was determined by TLC. After that time the reaction
mixture was quenched by the addition of water (100 ml). The whole
mixture was extracted four times with CH2Cl2, and the combined
organic layers were dried over MgSO4, filtered, and evaporated under
reduced pressure. The residue was purified by CombiFlash® (EtOAc/
MeOH, increasing concentration gradient) to give 13 (C20H22INO4S,
MW = 499.4 g/mol) with yield 83% 42. 1H NMR (403 MHz, cdcl3) δ 7.59
(s, 1H), 7.11 (d, J = 10.3 Hz, 1H), 7.01 (d, J = 10.7 Hz, 1H), 3.93 (s, 6H),
3.62 (s, 3H), 3.57 (dd, J = 10.8, 6.2 Hz, 1H), 3.15–3.08 (m, 1H),
2.48–2.39 (m, 4H), 2.33–2.24 (m, 1H), 1.53–1.46 (m, 3H) ppm. 13C
NMR (101 MHz, cdcl3) δ 182.5, 158.7, 153.4, 153.1, 150.9, 145.1,
137.7, 137.5, 133.8, 129.5, 129.2, 125.5, 91.7, 61.2, 61.0, 60.8, 53.4,
38.2, 35.1, 15.1 ppm. FT-IR (KBr pellet): 3375, 3309, 2932, 1605, 1553,
1460, 1405, 1343, 1313, 1246, 1195, 1136, 1081, 1014 cmꢀ 1. ESI-MS
(m/z): [M+H]+ calcd 500, found 500. Anal. Calcd. for C, 48.10; H,
4.44; I, 25.41; N, 2.80; O, 12.82; S, 6.42; found C, 48.18; H, 4.54; I,
25.43; N, 2.75; S, 6.49.
Compound 17: 1H NMR (403 MHz, CDCl3) δ 7.53–7.48 (m, 1H), 7.40
(s, 1H), 7.24 (dd, J = 9.1, 4.0 Hz, 1H), 7.10––7.05 (m, 1H), 4.56 (dt, J =
11.9, 6.8 Hz, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.64 (s, 3H), 3.17 (dd, J =
14.0, 4.6 Hz, 1H), 2.49–2.37 (m, 4H), 2.35–2.20 (m, 3H), 1.79 (td, J =
12.0, 5.2 Hz, 1H), 1.11 (dd, J = 9.8, 5.3 Hz, 3H) ppm. 13C NMR (101
MHz, CDCl3) δ 182.3, 173.8, 159.1, 153.4, 151.4, 151.0, 145.6, 137.7,
136.8, 134.6, 129.7, 128.2, 126.2, 92.2, 61.6, 61.4, 60.8, 51.7, 34.6,
34.5, 29.2, 15.2, 9.6 ppm. FT-IR: 3301, 2938, 1660, 1608, 1567, 1462,
1406, 1346, 1319, 1283, 1262, 1231, 1198, 1138, 1081, 1019 cmꢀ 1
.
ESI-MS (m/z): [M+H]+ calcd 556, found 556, [M+Na]+ calcd 578,
found 578. Anal. Calcd. for C, 50.62; H, 4.96; I, 22.29; N, 2.46; O, 14.05;
S, 5.63; found C, 50.69; H, 4.91; I, 22.36; N, 2.41; S, 5.67.
Compound 18: 1H NMR (403 MHz, CDCl3) δ 7.47 (d, J = 8.3 Hz, 2H),
7.23 (d, J = 10.3 Hz, 1H), 7.06 (d, J = 10.6 Hz, 1H), 4.58 (dt, J = 11.8,
7.0 Hz, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.65 (s, 3H), 3.16 (dd, J = 13.8,
5.1 Hz, 1H), 2.55 (dt, J = 13.8, 6.9 Hz, 1H), 2.48–2.36 (m, 4H),
2.28–2.16 (m, 1H), 1.80 (td, J = 11.9, 5.3 Hz, 1H), 1.16 (dd, J = 6.9, 3.6
Hz, 6H) ppm. 13C NMR (101 MHz, CDCl3) δ 182.2, 177.0, 159.0, 153.4,
151.5, 151.1, 145.6, 137.7, 136.8, 134.5, 129.7, 128.5, 126.1, 92.2,
61.7, 61.3, 60.8, 51.3, 35.2, 34.7, 34.6, 19.5, 19.4, 15.1 ppm. FT-IR:
3331, 2970, 2935, 1669, 1608, 1552, 1461, 1406, 1345, 1319, 1284,
1262, 1239, 1198, 1153, 1137, 1081, 1019 cmꢀ 1. ESI-MS (m/z):
[M+H]+ [M+Na]+ calcd 592, found 592. Anal. Calcd. for C, 50.62; H,
4.96; I, 22.29; N, 2.46; O, 14.05; S, 5.63; found C, 50.71; H, 4.99; I,
22.31; N, 2.40; S, 5.59.
4.3.7. General procedure for the synthesis of colchicine derivatives (14–20)
Compounds 14–20 were obtained directly from compound 13. To a
solution of compound 13 (100 mg, 0.20 mmol) in tetrahydrofuran (THF,
5 ml) cooled to the 0 ◦C temperature, the following compounds were
added: Et3N (2 ml, 14 mmol) and DMAP (catalytic amount). The mixture
was first stirred at 0 ◦C temperature for a few minutes and then the
solution of respective acyl chloride (4–9) or dietyhylcarbamoyl chloride
(10) in THF (0.75 mmol in 2,5 ml) was added dropwise. The mixture was
stirred at RT for the next 24 h. The solution was filtered to remove
triethylamine hydrochloride. The THF was evaporated and the residue
was purified by CombiFlash® (hexane/ethyl acetate, increasing con-
centration gradient) to give respective compounds as amorphous yellow
solids with yield from 25% to 67% (14–20).
Compound 19: 1H NMR (403 MHz, CDCl3) δ 7.40 (s, 1H), 7.23 (d, J
= 10.3 Hz, 1H), 7.15 (d, J = 7.1 Hz, 1H), 7.06 (d, J = 10.8 Hz, 1H), 4.57
(dt, J = 11.9, 7.0 Hz, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.63 (s, 3H), 3.17
(dd, J = 13.9, 5.0 Hz, 1H), 2.47–2.18 (m, 7H), 1.75 (td, J = 12.0, 5.2 Hz,
1H), 1.60 (td, J = 14.8, 7.3 Hz, 2H), 1.35–1.20 (m, 12H), 0.86 (t, J = 7.2
Hz, 3H) ppm. 13C NMR (101 MHz, CDCl3) δ 182.3, 173.2, 159.1, 153.4,
151.5, 150.9, 145.6, 137.6, 136.7, 134.5, 129.7, 128.3, 126.2, 92.2,
61.6, 61.3, 60.8, 51.6, 36.4, 34.8, 34.5, 31.8, 29.4, 29.3, 29.3, 29.2,
25.5, 22.6, 15.1, 14.1 ppm. FT-IR: 3298, 2927, 2856, 1656, 1607, 1547,
1461, 1406, 1346, 1319, 1283, 1262, 1246, 1198, 1154, 1138, 1081,
1019 cmꢀ 1. ESI-MS (m/z): [M+H]+ calcd 654, found 654. Anal. Calcd.
for C, 55.13; H, 6.17; I, 19.42; N, 2.14; O, 12.24; S, 4.91; found C, 55.02;
H, 6.19; I, 19.44; N, 2.19; S, 4.86.
Compound 14: 1H NMR (403 MHz, CDCl3) δ 7.20 (d, J = 7.6 Hz, 1H),
7.19 (s, 1H), 7.16 (d, J = 6.6 Hz, 1H), 7.03 (d, J = 10.6 Hz, 1H), 4.53 (dt,
J = 11.8, 6.9 Hz, 1H), 3.97 (s, 3H), 3.94 (s, 3H), 3.87 (q, J = 15.0 Hz,
2H), 3.62 (s, 3H), 3.43 (s, 3H), 3.20 (dd, J = 13.9, 5.1 Hz, 1H), 2.52–2.42
(m, 4H), 2.28–2.17 (m, 1H), 1.80 (td, J = 12.0, 6.5 Hz, 1H) ppm. 13
C
NMR (101 MHz, CDCl3) δ 182.2, 169.2, 159.2, 153.4, 151.5, 149.4,
145.6, 137.0, 136.6, 134.3, 129.7, 128.3, 125.7, 92.1, 71.6, 61.4, 61.3,
60.7, 59.1, 51.1, 34.8, 34.4, 15.1 ppm. FT-IR: 3339, 2998, 2929, 1674,
1605, 1547, 1516, 1465, 1449, 1425, 1408, 1373, 1347, 1316, 1292,
1262, 1190, 1156, 1134, 1107, 1081, 1056, 1018 cmꢀ 1. ESI-MS (m/z):
[M+Na]+ calcd 594, found 594. Anal. Calcd. for C, 48.34; H, 4.59; I,
22.21; N, 2.45; O, 16.80; S, 5.61; found C, 48.22; H, 4.51; I, 22.36; N,
2.46; S, 5.66.
Compound 20: 1H NMR (403 MHz, CDCl3) δ 7.48 (s, 1H), 7.21–7.17
(m, 1H), 7.03–6.99 (m, 1H), 5.89–5.85 (m, 1H), 4.53 (dt, J = 11.7, 6.7
Hz, 1H), 3.94 (s, 3H), 3.92 (s, 3H), 3.64 (s, 3H), 3.30 (q, J = 7.1 Hz, 4H),
3.12 (dd, J = 13.8, 4.7 Hz, 1H), 2.44–2.33 (m, 4H), 2.28–2.18 (m, 1H),
1.81 (td, J = 12.0, 5.3 Hz, 1H), 1.12 (t, J = 7.1 Hz, 6H) ppm. 13C NMR
(101 MHz, CDCl3) δ 182.2, 158.8, 156.0, 153.2, 152.1, 151.4, 145.5,
137.6, 137.0, 134.3, 129.9, 128.9, 125.9, 92.1, 61.7, 61.3, 60.7, 52.9,
41.0, 35.2, 34.8, 15.1, 13.9 ppm. FT-IR: 3383, 2973, 2935, 1639, 1608,
1553, 1525, 1492, 1460, 1425, 1406, 1344, 1318, 1284, 1264, 1216,
1183, 1152, 1137, 1080, 1018 cmꢀ 1. ESI-MS (m/z): [M+H]+ calcd 599,
found 599, [M+Na]+ calcd 621, found 621. Anal. Calcd. for C, 50.17; H,
5.22; I, 21.20; N, 4.68; O, 13.37; S, 5.36; found C, 50.08; H, 5.19; I,
Compound 15: 1H NMR (403 MHz, CDCl3) δ 7.47 (d, J = 7.3 Hz, 1H),
7.42 (s, 1H), 7.23 (d, J = 10.4 Hz, 1H), 7.07 (d, J = 10.7 Hz, 1H), 4.57
(dt, J = 11.8, 6.9 Hz, 1H), 3.96 (s, 3H), 3.95 (s, 3H), 3.63 (s, 3H), 3.53 (t,
J = 6.4 Hz, 2H), 3.16 (dd, J = 13.7, 5.0 Hz, 1H), 2.52–2.35 (m, 7H),
2.29–2.18 (m, 1H), 2.11–2.02 (m, 2H), 1.78 (td, J = 12.0, 5.6 Hz, 1H)
ppm. 13C NMR (101 MHz, CDCl3) δ 182.3, 171.7, 159.2, 153.5, 151.4,
150.8, 145.6, 137.6, 136.7, 134.7, 129.6, 128.4, 126.2, 92.2, 61.6, 61.4,
60.8, 51.7, 44.4, 34.8, 34.5, 33.0, 28.1, 15.2 ppm. FT-IR: 3301, 2937,
1674, 1607, 1544, 1461, 1406, 1346, 1318, 1283, 1262, 1244, 1196,
11

G. Klejborowska et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116014
21.21; N, 4.70; S, 5.41.
cytotoxic activity of studied compounds towards adherent cell lines.
Cells (104 per well) were seeded in 96-well plates (Sarstedt, Nümbrecht,
Germany) in appropriate complete cell culture media and after 24 h
prior addition of tested compounds. Cells were subjected to the treat-
ment with tested agents or cisplatin (Teva Pharmaceuticals Polska,
Warsaw, Poland) or doxorubicin (Accord Healthcare Limited, Mid-
4.4. Cell lines and culturing conditions
Primary ALL-5 cells were derived from the bone marrow of a 37-year
old patient as previously described.58,59 Although these cells can be
cultured up to 6 months with no obvious change in their properties,58 in
the present study they were exclusively used at low passage for all ex-
periments, and are thus referred to as primary cells. Primary ALL-5 cells
were routinely maintained at 37 ◦C in a humidified 5% CO2 incubator in
dlesex, UK) in the concentration range 100–0.01
μg/ml for 72 h.
Treatment with DMSO (POCh, Gliwice, Poland) at concentrations cor-
responding to these present in tested agents’ dilutions was applied as a
control (100% cell viability). After 72 h of incubation with the tested
compounds, cells were fixed in situ by gently adding of 50 μL per well of
IMDM Modified (SH30228, HyClone) media supplemented with 10 μg
mLꢀ 1 cholesterol (C3045, Sigma-Aldrich), 6 mg mLꢀ 1 human serum
cold 50% trichloroacetic acid TCA (POCh, Gliwice, Poland) following
incubation at 4 ◦C for one hour.47 Next, wells were washed four times
albumin (HA1000, Golden West Biologicals),
2 mM L-glutamine
with water and air dried. 50 μL of 0.1% solution of sulforhodamine B
(25–005, Corning), 2% v/v amphotericin-B/penicillin/streptomycin
(Sigma–Aldrich, Merck KGaA, Saint Louis, MO, USA) in 1% acetic acid
(POCh, Gliwice, Poland) were added to each well and plates were
incubated at room temperature for 0.5 h. Unbounded dye was removed
by washing plates four times with 1% acetic acid. Stained cells were
solubilized with 10 mM Tris base (Sigma–Aldrich, Merck KGaA, Saint
Louis, MO, USA). Absorbance of each solution was read at Synergy H4
Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc.,
Winooski VT, USA) at the 540 nm wavelength.
(A2942, Sigma-Aldrich, 30–002, Corning),
1
μ
g
mLꢀ 1 insulin
(128–100, Cell Applications), 200
Sigma-Aldrich), and 50
μ
g mLꢀ 1 apo-transferrin (T1147,
μ
M β-mercaptoethanol, and were subcultured to
maintain a density of 1–3 × 106 cells mLꢀ 1. Human MCF-7 mammary
gland adenocarcinoma cells originally isolated from a 69 year old
Caucasian woman with several characteristics of differentiated mam-
mary epithelium were cultured in Eagle’s Minimum Essential Medium
(EMEM) (30-2003, ATCC, USA) supplemented with 10% (v/v) heat-
inactivated fetal bovine serum (FBS) (FP-0500-A, Atlas Biologicals,
USA), and 1% Penicillin/Streptomycin Solution 100x (30-002-Cl,
Corning, USA). MCF-7 cell line was tested via short tandem repeat
profiling in July 2018 by Genetica DNA Laboratories (Burlington, NC)
and verified as authentic, giving a 100% match when compared to the
known reference profile.60 Both primary ALL-5 cells and MCF-7 cell line
for cell cycle analysis were maintained in the Department of Biochem-
istry & Molecular Biology at University of Arkansas for Medical Sci-
ences, USA.
Results are presented as mean IC50 (concentration of the tested
compound, that inhibits cell proliferation by 50%) ± standard deviation.
IC50 values were calculated in Cheburator 0.4, Dmitry Nevozhay soft-
ware (version 1.2.0 software by Dmitry Nevozhay, 2004–2014, http:
//www.cheburator.nevozhay.com, freely available) for each experi-
ment.48 Compounds at each concentration were tested in triplicate in
individual experiment and each experiment was repeated at least three
times independently.
4.5.2. MTT assay
The BALB/3T3 cell line was purchased from the American Type
Culture Collection (ATCC Manassas, VA, USA), A549 and MCF-7 cell
lines – from European Collection of Authenticated Cell Cultures (Salis-
bury, UK), LoVo cell line was purchased from the American Type Culture
Collection (ATCC, Manassas, VA, USA), and LoVo/DX received courtesy
A 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT)-based assay,61 was used to evaluate the
effect of drugs on the viability of primary ALL-5 cells. Cells (105/well) in
100 µL of complete IMDM Modified medium were seeded in 96-well
plates (TPP, Switzerland) and treated with drugs at concentrations up
to 10 µM for 120 h with control cells receiving vehicle (0.1% DMSO)
´
´
of Prof. E. Borowski (Technical University of Gdansk, Gdansk, Poland).
All of the above-listed cell lines were maintained in the Institute of
Immunology and Experimental Therapy (IIET), Wroclaw, Poland.
Human lung adenocarcinoma cell line (A549) was cultured in the
mixture of OptiMEM and RPMI 1640 (1:1) medium (IIET, Wroclaw,
Poland), supplemented with 5% fetal bovine serum (GE Healthcare,
Logan UT, USA) and 2 mM L-glutamine (Sigma-Aldrich, Merck KGaA,
Saint Louis, MO, USA). Human colon adenocarcinoma cell lines (LoVo)
were cultured in mixture of OptiMEM and RPMI 1640 (1:1) medium
(IIET, Wroclaw, Poland), supplemented with 5% fetal bovine serum (GE
Healthcare, Logan, UT, USA), 2 mM L-glutamine, 1 mM sodium pyruvate
alone. After treatment, 10
μ
L of MTT solution (5 mg/mL) was added to
each well, and the plate was incubated at 37 ◦C for 24 h in a humidified
5% CO2 incubator. Then 100
μL of 10% SDS in 0.01 M HCl was added to
each well and the plate was incubated at 37 ◦C for a further 24 h. The
experiment was performed in quadruplate (n = 4). Absorbance was
recorded at 540 nm using a BioTek Plate Reader. Inhibition of formation
of colored MTT formazan was taken as an index of cytotoxicity activity.
IC50 values were determined by non-linear regression analysis using
GraphPad Prism 6 for Windows (GraphPad Software).
Selectivity index (SI) was calculated by dividing the IC50 value for
BALB/3T3 cells by the IC50 value for individual cancer cell lines, and
resistance index (RI) was calculated by dividing the IC50 for LoVo/DX
cells by the IC50 for LoVo cells. The Resistance Index (RI) was defined as
the ratio of IC50 for a given compound calculated for resistant cell line to
that measured for its parental drug sensitive cell line (Table 1).
(Sigma-Aldrich, Merck KGaA, Saint Louis, MO, USA) and 10 μg/100 ml
doxorubicin for LoVo/DX (Sigma-Aldrich, Merck KGaA, Saint Louis,
MO, USA). Murine embryonic fibroblast cells (BALB/3T3) were cultured
in Dulbecco medium (Life Technologies Limited, Paisley, UK), supple-
mented with 10% fetal bovine serum (GE Healthcare, Logan, UT, USA)
and 2 mM glutamine (Sigma-Aldrich, Merck KGaA, Saint Louis, MO,
USA). All cell culture media contained antibiotics: 100 U/ml penicillin
and 100 μg/ml streptomycin (Polfa-Tarchomin, Warsaw, Poland). All
4.6. DNA content analysis
cell lines were cultured during entire experiment in humid atmosphere
at 37 ◦C and 5% CO2. Cells were tested for mycoplasma contamination
by mycoplasma detection kit for conventional PCR: Venor GeM Classic
(Minerva Biolabs GmbH, Berlin, Germany) and negative results were
obtained. The procedure is repeated every year or in the case of less
frequently used lines after thawing.
ALL-5 (1.5 × 106) and MCF-7 (0.2 × cells 106) were seeded in 100
mm Petri dishes (Corning, NY) and incubated in the presence of vehicle
(0.1% DMSO) or compounds, at concentrations specified in the text, for
24, 48 or 72 h at 37 ◦C in a humidified 5% CO2 incubator. Cells were
then washed with 1 ml phosphate-buffer saline (PBS), fixed with 1–3 ml
of 70% ice-cold ethanol and stored at 4 ◦C prior to flow cytometric
4.5. Cell viability assays
analysis. Cells were centrifuged, treated with 500 μL propidium iodide/
RNase Staining buffer (BD Biosciences, San Jose, CA, USA) and stored in
the dark for 1 h at RT. The stained cells were subjected to a FacsAria IIIu
Flow Cytometer (BD Biosciences, San Jose, CA, USA) performed by
4.5.1. SRB assay
Sulforhodamine B (SRB) assay was performed to asses about
12

G. Klejborowska et al.
Bioorganic & Medicinal Chemistry 32 (2021) 116014
UAMS Flow Cytomery Core Facility and data were analyzed using
FlowJo software.
coefficient (MLogP) of every compound was calculated using the
ADMET Predictor 8.0 package (ADMET Predictor, Simulations Plus,
Lancaster, CA, USA). Both Vina scores and MlogP values were used as
inputs to build a two-variable linear regression model for every tubulin
isotype.
4.7. Western blot analysis
ALL-5 cells (15 × 106 cells/dish) were treated for 24 and 48 h with 1
and 5 at 5 × IC50 values concentration or with vehicle (0.1% DMSO) or
DX (0.2 µM) for 24 h. Cells were washed in PBS and lysed in lysis buffer
(25 mM HEPES, pH 7.5, 300 mM NaCl, 0.1% Triton X-100, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM DTT, EDTA-free complete protease in-
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
hibitor tablets (Roche), 20
μ
g mlꢀ 1 aprotinin, 50
μ
g mlꢀ 1 leupeptin, 10
μ
M pepstatin, 1 mM phenylmethylsulfonyl fluoride, 20 mM β-glycer-
ophosphate, 1 mM Na3VO4, and 1
μ
M okadaic acid. Protein content was
g) were separated
Acknowledgments
measured by Bradford assay and equal amounts (20
μ
by electrophoresis using Mini-PROTEAN® precast gels (Bio-Rad). Pro-
teins were electrophoretically transferred onto a PVDF membrane
(Immobilon-FL, Merck Millipore) and next stained with Ponceau S to
assess transfer efficiency and verify equal loading. The membrane was
blocked with 5% nonfat milk in Tris-buffered saline containing 0.1%
TWEEN-20 (TBS-T) for 1 h at RT and incubated overnight at 4 ◦C with
primary antibodies (Santa Cruz Biotechnology) against PARP (9532)
(1:2500 dilution) and GAPDH (2118) (1:10000 dilution). After washing
with TBS-T for 5 × 5 min the membrane was incubated with secondary
HRP-conjugated goat anti-rabbit IgG (H+L) antibody (1:5000 dilution)
(Bio-Rad) for 1 h at RT. After washing in TBS-T the membrane was
exposed to ClarityTM Western ECL Substrate luminol enhancer solution
and peroxide solution (Bio-Rad) for 5 min and visualized and quantified
using Image J software.
Financial support by grant of the Polish National Science Centre
(NCN) – No. 2016/21/B/ST5/00111 is gratefully acknowledged. GK
gratefully acknowledges the doctoral scholarship “ETIUDA” – No. 2018/
28/T/ST5/00041 financed by the Polish National Science Centre (NCN).
T.C.C. acknowledges support from Arkansas Breast Cancer Research
Program. The authors thank Ms. Andrea Harris from UAMS Flow
Cytometry Core. JAT acknowledges funding support from NSERC
(Canada), the Alberta Cancer Foundation and the Allard Foundation.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116014.
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