Synthesis and antiproliferative screening of novel analogs of regioselectively demethylated colchicine and thiocolchicine

Article abstract of DOI:10.3390/molecules25051180

Colchicine, a pseudoalkaloid isolated from Colchicum autumnale, has been identified as a potent anticancer agent because of its strong antimitotic activity. It was shown that colchicine modifications by regioselective demethylation affected its biological properties. For demethylated colchicine analogs, 10-demethylcolchicine (colchiceine, 1) and 1-demethylthiocolchicine (3), a series of 12 colchicine derivatives including 5 novel esters (2b-c and 4b-d) and 4 carbonates (2e-f and 4e- f) were synthesized. The antiproliferative activity assay, together with in silico evaluation of physicochemical properties, confirmed attractive biological profiles for all obtained compounds. The substitutions of H-donor and H-acceptor sites at C1 in thiocolchicine position provide an efficient control of the hydration affinity and solubility, as demonstrated for anhydrate 3, hemihydrate 4e and monohydrate 4a.

Full text of DOI:10.3390/molecules25051180

Article
Synthesis and Antiproliferative Screening Of Novel
Analogs of Regioselectively Demethylated
Colchicine and Thiocolchicine
1
2
3
3
Dominika Czerwonka , Szymon Sobczak , Ewa Maj , Joanna Wietrzyk ,
2
1,
Andrzej Katrusiak and Adam Huczyński
*
1
Department of Medical Chemistry, Faculty of Chemistry, Adam Mickiewicz University, Uniwersytetu
Poznańskiego 8, 61-614 Poznan, Poland; dominika.czerwonka@amu.edu.pl
Department of Materials Chemistry, Faculty of Chemistry Adam Mickiewicz University, Uniwersytetu
Poznańskiego 8, 61-614 Poznań, Poland; szymon.sobczak@amu.edu.pl (S.S.); katran@amu.edu.pl (A.K.)
Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa
Weigla 12, 53-114 Wrocław, Poland; ewa.maj@hirszfeld.pl (E.M.); joanna.wietrzyk@hirszfeld.pl (J.W.)
2
3
* Correspondence: adhucz@amu.edu.pl; Tel.: +48618291673
Academic Editor: Qiao-Hong Chen
Received: 29 January 2020; Accepted: 03 March 2020; Published: 5 March 2020
Abstract: Colchicine, a pseudoalkaloid isolated from Colchicum autumnale, has been identified as a
potent anticancer agent because of its strong antimitotic activity. It was shown that colchicine
modifications by regioselective demethylation affected its biological properties. For demethylated
colchicine analogs, 10-demethylcolchicine (colchiceine, 1) and 1-demethylthiocolchicine (3), a series
of 12 colchicine derivatives including 5 novel esters (2b–c and 4b–d) and 4 carbonates (2e–f and 4e–
f) were synthesized. The antiproliferative activity assay, together with in silico evaluation of
physicochemical properties, confirmed attractive biological profiles for all obtained compounds.
The substitutions of H-donor and H-acceptor sites at C1 in thiocolchicine position provide an
efficient control of the hydration affinity and solubility, as demonstrated for anhydrate 3,
hemihydrate 4e and monohydrate 4a.
Keywords: colchicine analogs; thiocolchicine; colchiceine; antimitotic agents; antiproliferative
activity; hydrates
1. Introduction
Colchicine (Figure 1) is a pseudoalkaloid produced by Colchicum autumnale [1]. Its beneficial
applications have been known for centuries, as colchicine was used by ancient Greeks and Egyptians
to treat swelling and inflammation [2]. Presently, colchicine is considered as the first-line therapy for
gout, pericarditis and familial Mediterranean fever [1,3–5]. The most interesting property of
colchicine is its high affinity for binding to the tubulin in cancer cells, leading to the inhibition of
microtubule polymerization and mitosis arrest [6–8]. These unique properties have attracted great
interest to colchicine as an anticancer-drug candidate, however its use is limited by its toxicity
towards normal cells [9–11]. For this reason, new derivatives obtained through chemical
modifications guided by structure–activity relations are intensively investigated. This research is
aimed at reducing the colchicine toxicity and preserving the antimitotic and anticancer properties
[12–24]. As it turns out, tubulin interacts with trimethoxyphenyl ring A and tropolone ring C, making
the methoxy groups at C1, C2 and C10, as well as the C9-keto group, crucial for colchicine’s
Molecules 2020, 25, 1180; doi:10.3390/molecules25051180
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antimitotic activity [25–27]. Unfortunately, colchicine is also prone to isomerization, resulting in
isocolchicine (Figure 1) with 500 times lower affinity for binding to tubulin than colchicine itself [28].
Figure 1. Structures of colchicine, lumicolchicine, isocolchicine, colchiceine, isocolchiceine and
thiocolchicine.
Colchiceine (Figure 1) is a C10 demethylated colchicine analog characterized by much lower
toxicity [29,30]. It is, next to lumicolchicine (Figure 1), the main product of colchicine degradation
and can naturally occur in plant extracts together with colchicine [31]. Elguero et al. [32] have
established that colchiceine (as well as its C10-acylated analog) can occur in two tautomeric forms,
from which the isoform is slightly more dominant. This equilibrium between colchiceine and
isocolchiceine (Figure 1) exists also in the solid state, which has been confirmed by X-ray diffraction
studies [33]. The tendency of colchiceine towards tautomerization, in combination with the loss of
the C10-methoxy group, may be responsible for its poor antimitotic activity. In contrast, colchiceine
shows better bacteriostatic and antifungal activity than colchicine, because its free hydroxyl group
may increase its binding affinity towards the cell walls of pathogens [29,33].Interestingly, the
replacement of the C10-methoxy group with thiomethyl in a colchicine analog thiocolchicine (Figure
1) decreases its isomerization. Thiocolchicine is more stable, exhibits lower toxicity and has a higher
binding affinity to tubulin [34–36]. As could be expected, demethylation of thiocolchicine at C1/C2/C3
positions leads to a lower binding ability to tubulin and lower toxicity [37–39]. As the removal of the
methoxy groups from colchicine’s structure reduces its toxicity but also lowers its binding affinity to
tubulin, we decided to replace these groups with a bigger substituent, such as an ester or carbonate
moiety, to investigate an effect of this modification on biological activity [40].
For this reason, we carried out a series of regioselective demethylation reactions of colchicine
and thiocolchicine, in order to to obtain colchiceine (1) and 1-demethylthiocolchicine (3). On the
basis of 1 and 3, we synthesized a series of 12 mono- and double-modified colchicine analogs. Within
these analogs, we present 5 novel esters (2b–c and 4b–d) and 4 novel carbonates (2e–f and 4e–f). The
pathways used for these syntheses are depicted in Scheme 1.
We also evaluated the antiproliferative activity of these derivatives using three human cancer
cell lines, human lung adenocarcinoma (A549), human breast adenocarcinoma (MCF-7) and human
colon adenocarcinoma cell line (LoVo), and one normal murine embryonic fibroblast cell line
(BALB/3T3). Moreover, on the basis of the in silico calculations, we were able to predict the
physicochemical properties of the obtained compounds and check their compliance with Lipiński’s
rule of 5, permitting evaluation of its drug-likeness.
2. Results and Discussion
2.1. Chemistry
A series of colchicine esters and carbonates substituted at C1 and C10 positions in colchicine
were synthesized by performing regioselective demethylation reactions of C1 and C10 methoxy
groups, respectively. At first, we performed regioselective demethylation of the colchicine C10
methoxy group by treating colchicine with a mixture of glacial acetic acid and 0.1N hydrochloric acid
(i). Reaction resulted in precipitation of yellow solid[41], which was further recrystallized in acetone,
leading to growth of good quality, large single crystals. Performed X-ray diffraction analysis proved
them to be a colchiceine (1, Scheme 1) hemihydrate of the same structure as that reported by Mackay
et al. [33], in which the unit-cell accommodates two tautomeric forms: colchiceine and isocolchiceine.

Molecules 2020, 25, 1180
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Scheme 1. Synthesis of novel colchicine analogs. The obtained crystal structure of 1 was identical to
the one proposed by Mackey et al. [33]. Reagents and conditions: (i) glacial acetic acid, hydrochloric
acid, 100 °C, 6h; (iii) MeOH/H O, CH SNa, RT, 24h; (iv) DCM, acetyl chloride, SnCl , 0 °C RT, 48h;
O, LiOH, RT, 1h; (ii,vi) DCM, Et N, respective acyl chloride/chloroformate, 0 °C RT,
2
3
4
(v) MeOH/H
2
3
24h.
Colchicine was also treated with sodium methanethiolate (iii) to obtain thiocolchicine [42]. By
using a slightly modified method proposed by Bladé-Font [43] (iv,v), thiocolchicine was
regioselectively demethylated at the C1 methoxy group, leading to 1-demethylthiocolchicine (3) with
40% yield. This regioselective demethylation was confirmed by X-ray analysis (Figure 2). Finally,
compounds 1 and 3 were treated with respective acyl chlorides or chloroformates (ii, vi) to give single
modified derivatives 2a–f and double modified derivatives 4a–f with 38%–58% yields (Scheme 1).
Figure 2. (a) Molecule of 1-demethylthiocolchicine (3) and (b) its crystal structure projected along the
crystal direction [100].
The purity and structure of synthesized colchicine derivatives 1, 2a–f, 3 and 4a–f were
determined on the basis of ESI-MS, NMR and X-ray analysis. The specific data can be found in the
Materials and Methods section and in Supplementary Materials. The disappearance of the signal at
13
about 56.5 ppm in the C NMR spectrum of colchicine, assigned to the C10 methoxy group, is the
evidence of the formation of colchiceine (1). Moreover, in the spectra of derivatives 2a–2f, an
additional signal from the carbonyl group can be observed in the range 164.1–174.3 ppm for esters
2a–2d, and 151.9–152.5 ppm for carbonates 2e–f. A shift of the signal corresponding to the C10
methoxy group from 56.5 to 15.1 ppm is evidence for the introduction of the thiomethyl group at
position C10, and the absence of one of the signals around 61 ppm assigned to the C1 methoxy group
is evidence of 1-demethylthiocolchicine (3) formation. An additional signal corresponding to the

Molecules 2020, 25, 1180
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carbonyl carbon atom can be observed in the range 168.3–174.7 ppm for esters 4a–4d and in the range
152.8–152.9 ppm for carbonates 4e–f.
2.2. In Silico Calculations of the Physicochemical Properties
We used the Molinspiration online database (http://www.molinspiration.com, free of charge) to
predict the physicochemical properties of all synthesized compounds and collected them in Table 1
[44].
According to Lipinski’s rule of 5, most “drug-like” molecules should have logP ≤ 5, molecular
weight ≤ 500, number of hydrogen bond acceptors ≤ 10 and number of hydrogen bond donors ≤ 5.
Additionally, it was demonstrated that a total polar surface area (tPSA) < 140 and number of rotatable
bonds < 10, in combination with logP< 5, are crucial for both good bioavailability and permeability
through biological membranes [45].
Table 1. Physicochemical properties of the synthesized compounds based on the Molinspiration
database [44].
Compound
MW
clogP
0.83
0.60
1.27
1.51
2.92
0.95
1.33
1.89
1.66
2.33
2.57
3.98
2.02
2.39
1.10
tPSA
94.10
n(O,N)
N(OH,NH)
Rotb
MV
1
2a
2b
2c
2d
2e
2f
3
4a
4b
4c
4d
4e
4f
385.42
427.45
441.48
455.51
489.52
443.45
457.48
401.48
443.52
457.55
471.57
505.59
459.52
473.55
399.44
7
8
8
8
8
9
9
6
7
7
7
7
8
8
7
2
1
1
1
1
1
1
2
1
1
1
1
1
1
1
4
6
7
7
7
7
8
4
6
7
7
7
7
8
5
346.63
383.14
399.94
416.53
437.99
392.12
408.93
355.77
392.28
409.08
425.67
447.13
401.27
418.07
364.15
100.18
100.18
100.18
100.18
109.41
109.41
84.86
90.94
90.94
90.94
90.94
100.18
100.18
83.11
colchicine
MW: Molecular weight; clogP: calculated log octanol/water partition coefficient; tPSA: total polar
surface area; n(O,N): number of hydrogen acceptors; n(OH,NH): number of hydrogen donors; Rotb:
rotatable bonds; MV: molecular volume.
The demethylation of the C1 and C10 position of colchicine, both in 1 and 3, increases the number
of H-donor sites while the substitution of colchicine’s C10 methoxy group with thiomethyl results in
a decreased number of H-acceptor sites in 3. Additionally, the demethylation of the C1 and C10
position of colchicine reduces the number of rotatable bonds. Substitution of a hydroxyl group, both
in 1 and 3, with an ester or carbonate substituent, increases the number of H-acceptor sites and
reduces the number of H-donor sites to that of colchicine. According to the performed calculations,
all synthesized compounds 2a–e and 4a–e have a rather lipophilic character, with the clogP values
between 0.60 and 3.98. Conducted syntheses allowed us to obtain compounds with less lipophilic
character than that of colchicine for compounds 1, 2a and 2e, with clogP values 0.83, 060 and 0.95,
respectively. Interestingly, esters and carbonates in the C10 position (2a–e) are in general less
lipophilic than corresponding esters and carbonates in the C1 position (4a–e). Except for 4d, whose
molecular weight is above 500, the calculations for all colchicine analogs are in agreement with
Lipinski’s descriptors. Importantly, in order to be active, a drug should not have more than one
violation [46]. This, in combination with the number of rotatable bonds between 4 and 8, as well as
tPSA between 84.86 and 100.18, confirm good physicochemical profiles of all obtained compounds.

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2.3. X-Ray Analysis
The structures of compounds 3, 4a and 4e were additionally confirmed by X-ray diffraction.
Selected crystallographic data are collected in Table 2, and more detailed information can be found
in Supplementary Information. The single crystals of 3, 4a, 4e were obtained by slow evaporation
from an ethyl acetate mixture. The 1-Demethylthiocolchicine (3) crystallizes in the orthorhombic
space group P2
12
1
2
1
(Figure 2) while the crystals of 4a and 4e are monoclinic, space group P2 . The
1
substitution at the C1 atom with acetyl ester (4a) and methyl carbonate (4e), and the following change
in the ratio between H-bond acceptors and donors, increases the tendency of these colchicine
derivatives to co-crystallize with water; hence, monohydrate 4a‧H
2
O and hemihydrate 4e‧½H O were
2
obtained.
Molecules 4a and 4e significantly differ in conformation (Figure 3).The arrangement of the C2
methoxy group, which can be described by the C1-C2-O2-C18 torsion angle, changed from −122.36°
to 108.75° in 4a and 4e, respectively. Together with increasing mass of the C1 moiety, the twist
between the planar phenyl and tropolone rings around the C13-C16 bond increases, altering the C1-
C16-C13-C12 angle from 45.43° in 3, through 58.54° in 4a, to 60.11° in 4e.
Figure 3. Comparison of the molecular conformation from the X-ray analysis studies between (a)
molecule 4a and (b) 4e, superimposed with respect to the benzene ring on the 1-
demethylthiocolchicine (molecule 3), highlighted with a light-green color.
Table 2. Selected crystallographic data for compounds 3, 4a‧H
2
O and 4e‧½H
2
O.
Label
Formula
CCDC number
Crystal system
Space group
3
4a
‧
H
6
2
O
4e
25NO
‧
½H
S· ½H
1966195
monoclinic
P2
2
O
C21
H
22NO
5
S
C23
H
25NO
S · H
1966194
monoclinic
P2
2
O
C23
H
7
2
O
1966196
orthorhombic
P2
12
1
2
1
1
1
a (Å)
9.1005(17)
11.866(2)
17.881(4)
90
1930.8(7)
4/1
10.600(2)
6.9635(11)
16.712(3)
107.07(2)
1179.2(4)
2/1
10.8835(10)
9.2158(6)
12.0632(12)
104.613(9)
1170.80(18)
2/1
Unit cell
dimensions
b(Å)
c (Å)
β (°)
3
Volume (Å )
Z/Z’
(g/cm )
3
D
x
1.378
1.300
1.329
The presence of water significantly alters the pattern of H-bonds between 3 and 4a‧H
2
O and
4e‧½H O. Although in all structures amide nitrogen participates in the formation of intermolecular
2

Molecules 2020, 25, 1180
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contacts, only in 3 are the NH···S, of 2.793(2) Å, and NH···O4, of 2.370(3) Å,bonds formed. In a
monohydrate crystal 4a‧H2O, water interrupts the NH···O4interaction observed in the crystal
structure of 3, accepting NH amide proton, of 2.086(1) Å and further donating its H-atoms to the HO-
H···O4’, of 2.007(3) Å, and HO-H···O5′, of 1.994(3) Å bond. Much weaker H-bonds are present in the
crystal structure of 4e‧½H2O. Despite the presence of a water molecule in the crystal structure of
4e‧½H2O, the strong NH···O5′ bond, of 2.033(2) Å is present again. The disordered by location at half-
occupied sites water molecule is involved in formation of a weak HO-H···O5′’ interaction of 2.538(1)
Åand the H-bond at the disordered terminal carbonyl at C9. Due to the HO-H···O4′ of 2.241(1) Å
bond, the occupational disorder of H2O is passed onto the orientation of 4e molecules as illustrated
in Figure 3. Depending on the presence or absence of this water molecule, its adjacent molecule 4e is
slightly shifted in the crystal structure. This disorder is most apparent for the terminal carbonyl at C9
(dislocated by 0.358 Å) and thiomethyl at C10 (dislocated by 0.625 Å).
2.4. Antiproliferative Activity
The antiproliferative activity of all synthesized colchicine analogs 1, 2a–f, 3, 4a–f was tested in
vitro against three human cancer cell lines: human lung adenocarcinoma (A549), human breast
adenocarcinoma (MCF-7) and human colon adenocarcinoma cell line (LoVo). For a more accurate
evaluation of cytotoxic activity, the effect on normal murine embryonic fibroblast cell line (BALB/3T3)
was also tested according to the previously published procedure [12]. Detailed information
concerning antiproliferation assay can be found in Supplementary Materials. The mean values of IC50
± SD of the tested compounds are collected in Table 3.
Table 3. Antiproliferative activity (IC50) of 10-demethylcolchicine (colchiceine, 1), 1-
demethylthiocolchicine (3) and their derivatives(2a–f, 4a–f) compared to the antiproliferative activity
of colchicine and standard anticancer drugs doxorubicin and cisplatin and the calculated values of
selectivity index (SI).
A549
IC50 (μM)
MCF-7
IC50 (μM)
LoVo
BALB/3T3
IC50 (μM)
Compound
SI
SI
SI
IC50 (μM)
6.00 ± 1.88
6.18 ± 1.48
2.39 ± 1.20
6.47 ± 0.35
4.87 ± 2.17
0.48 ± 0.32
6.41 ± 1.57
0.11 ± 0.03
0.11 ± 0.02
0.50 ± 0.19
0.56 ± 0.06
0.10 ± 0.08
0.43 ± 0.19
0.79 ± 0.16
0.01 ± 0.01
3.80 ± 0.28
0.08 ± 0.03
1
2a
2b
2c
2d
2e
2f
3
4a
4b
4c
4d
4e
12.99 ± 1.79
12.47 ± 2.77
9.97 ± 0.70
20.39 ± 11.30
8.75 ± 0.33
0.98 ± 0.16
9.67 ± 0.84
0.82 ± 0.02
0.48 ± 0.15
0.97 ± 0.04
0.89 ± 0.07
0.60 ± 0.31
0.90 ± 0.09
0.96 ± 0.13
0.07 ± 0.01
3.60 ± 0.25
0.16 ± 0.03
0.79
0.68
0.75
0.41
1.02
0.84
0.86
0.74
1.36
0.77
0.80
0.75
0.87
1.08
0.63
0.81
0.25
11.23 ± 2.52
10.03 ± 2.18
7.56 ± 2.84
11.95 ± 1.54
8.21 ± 3.35
0.98 ± 0.33
9.07 ± 1.90
0.12 ± 0.05
0.10 ± 0.02
0.96 ± 0.13
0.86 ± 0.10
0.78 ± 0.22
1.02 ± 0.10
0.95 ± 0.20
0.01 ± 0.01
3.05 ± 0.67
0.15 ± 0.05
0.91
0.85
0.99
0.70
1.09
0.84
0.92
4.92
6.85
0.78
0.83
0.58
0.77
1.09
4.30
0.95
0.26
1.71
1.37
3.13
1.30
1.83
1.70
1.30
5.38
5.92
1.53
1.27
4.71
1.84
1.31
5.38
0.77
0.49
10.25 ± 0.96
8.50 ± 0.50
7.47 ± 0.50
8.42 ± 1.14
8.92 ± 3.93
0.82 ± 0.11
8.36 ± 0.17
0.61 ± 0.14
0.65 ± 0.12
0.75 ± 0.10
0.71 ± 0.03
0.45 ± 0.23
0.78 ± 0.06
1.03 ± 0.20
0.04 ± 0.01
2.91 ± 1.83
0.04 ± 0.03
4f
colchicine
cisplatin
doxorubicin
The IC50 value is defined as the concentration of a compound at which 50% growth inhibition is
observed. Human lung adenocarcinoma (A549), human breast adenocarcinoma (MCF-7), human
colon adenocarcinoma cell line (LoVo) and normal murine embryonic fibroblast cell line (BALB/3T3).
The SI (Selectivity Index) was calculated for each compound, using the formula SI = IC50 for normal
cell line BALB/3T3/IC50 for the respective cancerous cell line. A beneficial SI > 1.0 indicates a drug with
efficacy against tumor cells greater than the toxicity against normal cells.

Molecules 2020, 25, 1180
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All colchicine analogs showed stronger antiproliferative activity against all tested cancer cell
lines than the conventional chemotherapeutic, cisplatin. In general, the IC50 values are better for
colchicines doubly modified at C1 and C10 positions (3, 4a–f) than for the analogs singly modified at
position C10 only (1, 2a–f). Compound 4a, with a thiomethyl group at position C10 and an acetyl
ester substituent at C1, showed the highest activity against all tested cell lines (IC50 = 0.48 ± 0.15, 0.10
± 0.02, 0.11 ± 0.02 μM for A549, MCF-7, LoVo cancer cell lines, respectively).
All tested colchicine analogs exhibit higher SI values than doxorubicin, which indicates the
therapeutic potential of the synthesized compounds. Compound 3, 1-demethylthiocolchicine, as well
as its acylated analog 4a, showed high SI values against MCF-7 (SI = 4.92, SI = 6.88) as well as LoVo
cell lines (SI = 5.36, SI = 5.95). From among analogs modified exclusively at position C10, compound
2b with propionyl ester substituent revealed a higher SI value towards LoVo cell line (SI = 3.13).
3. Materials and Methods
3.1. General
Information concerning reagents and solvents, as well as equipment used for measurements, can
be found in the Supplementary Materials.
3.1.1. Synthesis and Characterization of 10-Demethylcolchicine (colchiceine, 1), Thiocolchicine and
1-Demethylthiocolchicine (3)
Information concerning the synthesis of 10-demethylcolchicine (colchiceine, 1), thiocolchicine
and 1-demethylthiocolchicine (3) can be found in Supplementary Materials.
3.1.2. General Route for Synthesis of Compounds 2a–f and 4a–f
To a solution of 1 or 3 (100 mg, 0.25 mmol) in dichloromethane (DCM, 10 mL) cooled to 0 °C,
Et3N (1 mL, 7 mmol) and respective acyl chloride or chloroformate were added (excess). The mixture
was first stirred at 0 °C for 30 min and then for the next 24h at RT. Reaction time was determined by
TLC. After that time the solution was filtered to remove triethylamine hydrochloride, the DCM was
®
evaporated under reduced pressure and the residue was purified by CombiFlash
(chloroform/acetone, increasing concentration gradient) to give respective compounds as amorphous
yellow solids.
3.1.3. Characterization of Acetyl Ester of Colchiceine 2a
1
Amorphous yellow solid, yield 49%, m.p 120–122 °C; H NMR (CDCl3, 400 MHz) δ 7.50 (1H, s),
7.31 (1H, d, J = 11.3 Hz), 7.22 (1H, d, J = 11.3 Hz), 6.53 (1H, s), 4.60 (1H, dt, J = 12.7, 6.5 Hz), 3.91 (3H,
s), 3.88 (3H, s), 3.66 (3H, s, 2.50 (1H, dd, J = 13.6, 6.3 Hz), 2.35 (3H, s), 2.26–2.17 (1H, m), 1.96 (3H, s),
13
1.87 (1H, td, J = 12.1, 6.9 Hz). C NMR (CDCl3, 101 MHz) δ 169.9, 168.2, 153.9, 151.0, 148.6, 141.8, 141.5,
+
137.0, 134.5, 131.2, 130.3, 125.2, 107.4, 61.6, 61.3, 56.0, 52.1, 36.8, 29.7, 22.7, 20.7. ESI-MS (m/z): [M + H]
428, [M + Na] 450, [M+K] 466, [2M + Na] 877.
+
+
+
3.1.4. Characterization of Propionyl Ester of Colchiceine 2b
1
Amorphous yellow solid, yield 52%, m.p 133-135 °C; H NMR (CDCl3, 400 MHz) δ 7.50 (1H, s),
7.30 (1H, d, J = 11.3 Hz), 7.22 (1H, d, J = 11.2 Hz), 6.52 (1H, s), 4.66–4.57 (1H, m), 3.91 (3H, s), 3.88 (3H,
s), 3.66 (3H, s), 2.66 (2H, q, J = 7.4 Hz), 2.50 (1H, dd, J = 13.5, 6.3 Hz), 2.35 (1H, ddd, J = 12.7, 10.3, 6.0
13
Hz), 2.24–2.16 (1H, m), 1.97 (3H, s), 1.87 (2H, td, J = 12.0, 6.7 Hz), 1.26 (3H, t, J = 7.6 Hz). C NMR
(CDCl3, 101 MHz) δ 171.8, 169.9, 153.9, 151.1, 148.9, 141.7, 141.6, 136.7, 134.5, 130.9, 130.5, 125.2, 107.4,
+
+
61.7, 61.4, 56.1, 52.1, 36.9, 29.8, 27.4, 22.8, 9.0. ESI-MS (m/z): [M + Na] 464, [2M + Na] 905.
3.1.5. Characterization of Isobutyryl Ester of Colchiceine 2c
1
Yellowish brown oil, yield 55%; H NMR (CDCl3, 400 MHz) δ 7.56 (1H, s), 7.28 (1H, d, J = 11.0
Hz), 7.23 (1H, d, J = 10.6 Hz), 6.52 (1H, s), 4.64 (1H, dt, J = 12.2, 6.8 Hz), 3.91 (3H, s), 3.88 (3H, s), 3.65

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(3H, s), 2.90–2.82 (1H, m), 2.54–2.47 (1H, m), 2.36 (1H, td, J = 13.2, 6.9 Hz), 2.24–2.12 (2H, m), 1.96 (3H,
13
s), 1.92–1.85 (1H, m), 1.35 (3H, d, J = 1.4 Hz), 1.33 (3H, d, J = 1.3 Hz). C NMR (CDCl3, 101 MHz) δ
174.3, 170.0, 153.9, 151.1, 150.3, 142.1, 141.5, 136.0, 134.5, 131.6, 130.1, 125.2, 107.4, 61.7, 61.3, 56.0, 52.1,
+
+
36.6, 34.0, 29.8, 26.4, 22.7, 19.0. ESI-MS (m/z): [M + Na] 478, [2M + Na] 933.
3.1.6. Characterization of Benzyl Ester of Colchiceine 2d
1
Amorphous yellow solid, yield 41%, m.p 123–125 °C; H NMR (CDCl3, 400 MHz) δ 8.18 (2H, d,
J = 7.4 Hz), 7.65–7.60 (2H, m), 7.49 (1H, ddd, J = 4.8, 3.1, 1.1 Hz), 7.34 (2H, s), 6.53 (1H, s), 4.66–4.58
(1H, m), 3.93 (3H, d, J = 0.9 Hz,), 3.90 (3H, d, J = 0.7 Hz), 3.68 (3H, d, J = 1.1 Hz), 2.49 (1H, dd, J = 13.4,
13
4.9 Hz), 2.42–2.32 (1H, m), 2.17–2.06 (1H, m), 1.86 (3H, d, J = 4.0 Hz), 1.84–1.78 (1H, m). C NMR
(CDCl3, 101 MHz) δ 169.9, 164.1, 153.9, 151.1, 142.0, 141.6, 134.5, 133.8, 130.4, 128.9, 128.5, 125.3, 107.4,
+
+
+
61.7, 61.4, 56.1, 52.0, 36.9, 29.8, 22.7. ESI-MS (m/z): [M + H] 490, [M + Na] 512, [M + K] 528.
3.1.7. Characterization of Methyl Carbonate of Colchiceine 2e
1
Amorphous yellowish brown solid, yield 58%, m.p 115–118 °C; H NMR (CDCl3, 400 MHz) δ
7.57 (1H, s), 7.32 (1H, d, J = 20.5 Hz), 6.54 (1H, s), 4.62 (1H, dt, J = 11.8, 6.0 Hz), 3.92 (3H, s)*, 3.92 (3H,
s)*, 3.89 (3H, s), 3.68 (3H, s), 2.52 (1H, dd, J = 13.4, 6.1 Hz), 2.37 (1H, td, J = 13.0, 6.7 Hz), 2.29–2.18 (1H,
13
m), 1.99 (3H, s), 1.91 (1H, dd, J = 19.0, 12.2 Hz). C NMR (CDCl3, 101 MHz) δ 169.9, 154.1, 152.5, 151.1,
+
141.6, 134.5, 125.1, 107.4, 61.7, 61.3, 56.1, 55.8, 52.2, 37.2, 29.8, 22.8. ESI-MS (m/z): [M + H] 444, [M +
+
+
+
Na] 466, [M + K] 482, [2M + Na] 909.
3.1.8. Characterization of Ethyl Carbonate of Colchiceine 2f
1
Yellowish brown oil, yield 55%; H NMR (CDCl3, 400 MHz) δ 7.46 (1H, s), 7.40–7.32 (1H, m, J =
6.2 Hz), 7.30–7.21 (1H, m), 6.55 (1H, s), 4.62 (1H, dt, J = 12.6, 6.4 Hz), 4.36 (2H, q, J = 7.1 Hz), 3.93 (3H,
s, J = 2.9 Hz), 3.90 (3H, s), 3.68 (3H, s), 2.54 (1H, dd, J = 13.6, 6.4 Hz), 2.41 (1H, td, J = 13.1, 7.1 Hz), 2.32
13
– 2.21 (1H, m), 2.01 (3H, s), 1.94–1.84 (1H, m), 1.41 (3H, t, J = 7.1 Hz). C NMR (CDCl3, 101 MHz) δ
169.9, 154.0, 151.9, 151.1, 141.6, 134.5, 125.1, 107.4, 65.4, 61.7, 61.3, 56.1, 52.1, 37.2, 29.8, 22.8, 14.1. ESI-
+
+
+
+
MS (m/z): [M + H] 458, [M + Na] 480, [M + K] 496, [2M + Na] 937.
3.1.9. Characterization of Acetyl Ester of 1-demethylthiocolchicine 4a
1
Amorphous yellow solid, yield 45%, m.p 168-172 °C; H NMR (DMSO-d6, 400 MHz) δ 8.51 (1H,
d, J = 7.7 Hz), 7.23 (1H, d, J = 10.8 Hz), 7.02 (1H, s), 6.97 (1H, s), 6.92 (1H, d, J = 10.3 Hz), 3.89 (3H, s),
3.74 (3H, s), 2.65 (1H, dd, J = 13.5, 6.0 Hz), 2.41 (3H, s), 2.32–2.24 (1H, m), 2.22 (3H, s), 2.12–2.01 (1H,
13
m), 1.85 (3H, s, J = 6.3 Hz), 1.84–1.77 (1H, m, J = 12.1, 6.8 Hz). C NMR (DMSO-d6, 101 MHz) δ 181.2,
168.9, 168.2, 157.7, 152.8, 150.4, 140.8, 139.3, 136.4, 134.4, 132.7, 128.2, 126.4, 124.6, 109.9, 60.1, 56.0, 50.7,
+
+
36.2, 29.1, 22.5, 20.3, 14. ESI-MS (m/z): [M + Na] 466, [2M + Na] 909.
3.1.10. Characterization of Propionyl Ester of 1-demethylthiocolchicine 4b
1
Amorphous yellow solid, yield 47%, m.p 136-140 °C; H NMR (DMSO-d6, 400 MHz) δ 8.52 (1H,
d, J = 7.7 Hz), 7.23 (1H, d, J = 10.7 Hz), 7.03 (1H, s), 6.98 (1H, s), 6.92 (1H, d, J = 10.2 Hz), 4.34 (1H, dt,
J = 11.8, 7.1 Hz), 3.90 (3H, s), 3.76–3.71 (3H, m), 2.66 (1H, dd, J = 13.2, 6.4 Hz), 2.41 (3H, s), 2.33–2.19
13
(2H, m), 2.08 (2H, ddd, J = 11.4, 9.8, 5.4 Hz), 1.84–1.78 (1H, m), 1.11 (3H, t, J = 7.5 Hz). C NMR (DMSO-
d6, 101 MHz) δ 181.2, 172.3, 168.3, 157.7, 152.8, 150.5, 140.9, 139.2, 136.4, 134.4, 132.9, 128.3, 126.4, 124.7,
+
+
109.9, 60.1, 56.0, 50.7, 36.2, 29.1, 26.7, 22.6, 14.5, 9.2. ESI-MS (m/z): [M + H] 458, [M + Na] 480, [M +
K] 496, [2M + Na] 937.
+
+
3.1.11. Characterization of Isobutyryl Ester of 1-demethylthiocolchicine 4c
1
Amorphous yellow solid, yield 49%, m.p 165-167 °C; H NMR (DMSO-d6, 101 MHz) δ 8.50 (1H,
d, J = 7.8 Hz), 7.22 (1H, d, J = 10.6 Hz), 7.03 (1H, s), 6.98 (1H, s), 6.91 (1H, d, J = 8.8 Hz), 4.35 (1H, dt, J
= 11.7, 7.3 Hz), 3.90 (3H, s), 3.72 (3H, s), 2.74 (1H, dt, J = 13.9, 6.9 Hz), 2.66 (1H, dd, J = 13.6, 6.1 Hz),

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2.39 (3H, s), 2.34–2.25 (1H, m), 2.09 (1H, td, J = 12.7, 5.9 Hz), 1.86 (3H, s), 1.81 (1H, dd, J = 12.0, 6.9 Hz,
13
1.16 (3H, d, J = 3.5 Hz), 1.14 (3H, d, J = 3.5 Hz). C NMR (DMSO-d6, 101 MHz) δ 181.2, 174.7, 168.3,
157.7, 152.7, 150.4, 140.8, 139.2, 136.4, 134.4, 132.9, 128.3, 126.3, 124.8, 109.9, 60.1, 56.1, 50.7, 36.2, 33.3,
+
+
29.1, 22.6, 18.8, 14.6. ESI-MS (m/z): [M + Na] 494, [2M + Na] 965.
3.1.12. Characterization of Benzyl Ester of 1-demethylthiocolchicine 4d
1
Amorphous yellow solid, yield 38%, m.p 166-170 °C; H NMR (DMSO-d6, 101 MHz) δ 8.52 (1H,
s), 8.07 (2H, d, J = 7.3 Hz), 7.77–7.71 (1H, m), 7.63–7.56 (2H, m), 7.22–7.12 (1H, m), 7.06 (1H, s), 7.02
(1H, s), 4.49–4.40 (1H, m), 3.94 (3H, s), 3.74 (3H, s), 2.72 (1H, dd, J = 13.0, 5.9 Hz), 2.36 (1H, dd, J = 7.4,
13
5.5 Hz,), 2.31 (3H, s), 2.13 (2H, ddd, J = 19.4, 12.8, 6.9 Hz,), 1.87 (3H, s). C NMR (DMSO-d6, 101 MHz)
δ 181.1, 168.3, 157.7, 152.9, 150.6, 141.0, 139.4, 136.3, 134.6, 134.2, 129.8, 129.1, 128.4, 128.3, 126.2, 124.9,
+
+
110.2, 60.3, 56.1, 50.8, 36.3, 29.2, 22.6, 14.5. ESI-MS (m/z): [M + Na] 528, [2M + Na] 1033.
3.1.13. Characterization of Methyl Carbonate of 1-demethylthiocolchicine 4e
1
Amorphous yellow solid, yield 53%, m.p 158-162 °C; H NMR (DMSO-d6, 101 MHz) δ 8.55 (1H
d, J = 7.6 Hz), 7.26 (1H, d, J = 10.6 Hz), 7.05–6.99 (3H, m), 4.34 (1H, dt, J = 11.8, 7.0 Hz), 3.91 (3H, s),
3.82 (3H, s), 3.79 (3H, s), 2.69 (1H, dd, J = 13.5, 6.2 Hz), 2.43 (3H, s), 2.29 (1H, ddd, J = 18.6, 12.3, 5.4
13
Hz), 2.09 (1H, ddd, J = 18.7, 12.5, 6.2 Hz), 1.88 (3H, s), 1.86–1.80 (1H, m). C NMR (DMSO-d6, 101
MHz) δ 181.2, 168.4, 157.9, 152.9, 152.8, 150.5, 140.8, 139.3, 136.0, 134.5, 133.2, 128.2, 126.4, 124.3, 110.2,
+
+
+
60.4, 56.14, 56.07, 50.8, 36.1, 29.1, 22.6, 14.4. ESI-MS (m/z): [M + H] 460, [M + Na] 482, [M + K] 498,
[2M + Na] 941.
+
3.1.14. Characterization of Ethyl Carbonate of 1-demethylthiocolchicine 4f
1
Amorphous yellow solid, yield 48%, m.p 165-167 °C; H NMR (DMSO-d6, 400 MHz) δ 8.52 (1H,
d, J = 7.6 Hz), 7.23 (1H, d, J = 10.7 Hz), 7.01 (3H, t, J = 10.3 Hz), 4.33 (1H, dt, J = 12.6, 7.5 Hz), 4.27–4.20
(2H, m), 3.90 (3H, s), 3.78 (3H, s), 2.67 (1H, dd, J = 13.2, 6.6 Hz), 2.41 (3H, s), 2.34–2.23 (1H, m), 2.08
13
(3H, s), 1.87 (3H, s), 1.85–1.78 (1H, m), 1.22 (3H, t, J = 7.1 Hz). C NMR (DMSO-d6, 101 MHz) δ 181.2,
168.4, 157.9, 152.8, 152.4, 150.5, 140.8, 139.3, 136.2, 134.5, 133.1, 128.2, 126.3, 124.4, 110.2, 65.1, 60.3, 56.1,
+
+
+
50.8, 36.1, 29.1, 22.6, 14.4, 14.0. ESI-MS (m/z): [M + Na] 496, [M + K] 512, [2M + Na] 969.
3.2. Antiproliferative Activity
Detailed information concerning antiproliferative activity assay is given in Supplementary
Materials.
3.3. X-Ray Measurements
X-Ray measurements and detailed crystallographic data are given in Supplementary Materials.
4. Conclusions
On the basis of regioselectively demethylated colchicine analogs, colchiceine (1) and 1-
demethylthiocolchicine (3), we have designed and synthesized a series of 12 colchicine derivatives
bearing ester and carbonate substituents, including 9 entirely novel derivatives (2b,c,e,f and
4b,c,d,e,f). Derivatives 4a·H2O and 4e·½H2O, with acetyl ester and methyl carbonate moieties,
display reduced hydrophilic properties and crystallize as hydrates, which affects their biological
activity and increases their solubility. The control over water co-crystallization from monohydrate to
hemihydrate has been achieved through the number of H-donor and acceptor sites. These features
can be invaluable for the regulation of the bioaccessibility and pharmaceutical processing of this
group of compounds [47]. The synthesized derivatives exhibit a considerable in vitro antiproliferative
activity against three human cancer cell lines. Compound 4a, carrying the thiomethyl group at
position C10 and acetyl ester substituent at C1, showed the highest activity and selectivity index

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values. The biological evaluation has been supported by prediction of physicochemical properties,
which are consistent with Lipinski’s rule of five for all synthesized analogs.
Supplementary Materials: The following are available online www.mdpi.com/1420-3049/25/5/1180/s1: General
procedures, experimental details, as well as an elaborate description of the X-ray diffraction sudies and in vitro
methods used.
Author Contributions: Conceptualization, D.C. and A.H.; methodology, D.C., A.H., S.S. and E.M. validation,
A.H., A.K. and J.W.; investigation, D.C, S.S. and E.M. resources, A.H., A.K. and J.W. data curation, D.C., S.S and
E.M writing—original draft preparation, D.C. and S.S writing—review and editing, A.H., A.K. and J.W.
visualization, D.C.; supervision, A.H.; project administration, A.H.; funding acquisition, A.H. All authors have
read and agreed to the published version of the manuscript.
Funding: Financial support with
a grant from the Polish National Science Centre (NCN)—No.
2016/21/B/ST5/00111 is gratefully acknowledged.
Acknowledgments: D.C. and S.S. acknowledge a scholarship (No. POWR. 03.02.00-00-I026/16 and POWR.
03.02.00-00-I023/17, respectively) co-financed by the European Union through the European Social Fund under
the Operational Program Knowledge Education Development.
Conflicts of Interest: The authors declare that they have no conflict of interest.
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Sample Availability: Samples of the compounds 1, 2a–f, 3 and 4a–f are available from the authors.
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