J. Krzywik et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128197
1
–
–
simplest urea (4) and thiourea (15) analogues. A variety of side chains
have been selected to facilitate the preliminary structure–activity rela-
tionship (SAR) analysis, which should help in designing colchicines
showing improved biological properties.
H NMR and approx. at 157.0 ppm (C O) in 13C NMR spectra. In the
NMR spectra of derivatives 15–26, the signals corresponding to the
–
thiourea moiety were observed in the range 7.4–9.4 ppm (–NH(C S)
–
N–) in H NMR and approx. at 182.0 ppm (C S) in 13C NMR ones. The
1
–
–
Our synthetic strategy to obtain colchicine derivatives 2–28 is
illustrated in Scheme 1 and a detailed description of the synthetic pro-
cedures is provided in the Supplementary Materials. The key interme-
diate 3 was available from colchicine 1 by treatment with methylamine
followed by hydrolysis with 2 M HCl.38,43 New analogs were synthesized
from 3 by treatment with respective isocyanate (6, 11–14), isothiocya-
nate (17, 23–24) or carbamoyl chloride in the presence of triethylamine
(7–10). The thioureas 15–16, 18–22 and 25 were prepared by com-
pound 3, thiophosgene and corresponding primary or secondary amine,
based on the method described earlier.42 Compounds 5 and 27 were
obtained in the reaction with N-succinimidyl N-methylcarbamate and N,
ESI mass spectrometry confirmed the structure of the synthesized
compounds by the presence of m/z signals assigned to the corresponding
pseudomolecular ions of these analogs.
A library of newly synthesized derivatives (4–28), starting com-
pounds (1–3) and commonly used anticancer agents doxorubicin and
cisplatin were screened for their antiproliferative activity against four
human cancer cell lines (A549, MCF-7, LoVo, LoVo/DX) and normal
cells (BALB/3T3) following the previously published procedures.38
Detailed information concerning the biological assay can be found in the
Supplementary Materials. The results are collected in Table 1.
Resistance indexes (RI) were calculated (ratio IC50 value for LoVo/
DX cell line to IC50 value for LoVo cell line) for evaluation of the activity
of the studied compounds against the cells with MDR (multidrug resis-
tance) phenotype.44 RI values are shown in Table 1. According to the RI
value, the cells can be classified to one of the three categories: drug-
sensitive for RI ranging from 0 to 2, moderate drug-sensitive for RI
2–10 and strong drug resistant for RI above 10.44,45
N’-di-Boc-1H-pyrazole-1-carboxamidine,
respectively.
Protecting
groups from compounds 25 and 27 were removed with HCl in MeOH or
EtOAc, giving derivatives 26 and 28, respectively. All compounds were
isolated in pure form after column flash chromatography on silica gel.
The purity and structures of the obtained compounds 2–28 were
determined using LC-MS, 1H and 13C NMR methods and are shown in the
Supplementary Materials. The characteristic signals of the –OCH3 group
of the tropolone C ring of colchicine 1 in the 1H NMR and 13C NMR
spectra were observed at 4.0 ppm and at 56.5 ppm, respectively. After
the reaction with methylamine, these signals were no longer visible, but
new ones appeared: approx. at 3.1 ppm (–CH3-) and approx. at 7.3 ppm
(–NH-) in 1H NMR and approx. at 29.5 ppm in 13C NMR, corresponding
to the –NHCH3 group. The chemical shifts of the amide moiety of the
The effects of the obtained compounds were studied also toward the
non-cancerous murine embryonic fibroblasts (BALB/3T3) in order to
estimate the therapeutic potential. Selectivity indexes were calculated as
the ratio of IC50 value for normal cell line (BALB/3T3) to IC50 value for a
respective cancer cell line.46 High SI values (at least greater than 2)
mean that cancer cells will be killed at a higher rate than normal
(healthy) ones (see Figure 1).
starting compounds 1 and 2 can be found at 1.9 ppm (–CH3) and 8.6 ppm
The majority of new derivatives 4–28 showed cytotoxicity against
cancerous cells in the nanomolar range and were characterized by lower
IC50 values than the conventional chemotherapeutics: cisplatin (except
10 and 28 against LoVo/DX cells) and doxorubicin (except 10 and
26–28 against A549, 26–28 against MCF-7 and LoVo cells and only 28
1
–
(–NH–) in H NMR and at 23.0 ppm (–CH ) and at 170.0 ppm (C O) in
–
3
13C NMR and also were not visible in the spectra of new analogs 4–28. In
the NMR spectra of derivatives 4–14, the signals corresponding to the
–
–
urea moiety were observed in the range 7.0–8.5 ppm (–NH(C O)N–) in
Scheme 1. Synthesis of doubly modified colchicine derivatives (2–28), changes at C7 and C10 positions are highlighted in red. Reagents and conditions: (a)
NH2CH3/EtOH, reflux; (b) 2 M HCl, reflux; (c) 1) C(O)Cl2/PhCH3 or C(S)Cl2, Et3N, THF, 0 ◦C, 2) NH3(gas), THF, 0 ◦C to RT for 4 or 15; (d) N-succinimidyl N-
methylcarbamate, Et3N, DCM, RT for 5; (e) RNCO, THF, RT for 6, 11–14; (f) R1R1C(O)Cl, Et3N, DCM, reflux for 7–10; (g) 1) C(S)Cl2, Et3N, DCM, 0 ◦C to RT, 2)
R1R2NH, DCM, RT for 16, 18–22 and 25; (h) RNCS, THF, RT for 17, 23–24; (i) N,N’-di-Boc-1H-pyrazole-1-carboxamidine, Et3N, DCM/MeOH, RT for 27; (j) 4 M HCl/
EtOAc for 28.
2