Synthesis, antiproliferative activity and molecular docking studies of novel doubly modified colchicine amides and sulfonamides as anticancer agents

molecules

Article

Synthesis, Antiproliferative Activity and Molecular

Docking Studies of Novel Doubly Modified Colchicine

Amides and Sulfonamides as Anticancer Agents

Julia Krzywik ¹

,2

, Witold Mozga , Maral Aminpour , Jan Janczak , Ewa Maj ,

2

3

4

5

Joanna Wietrzyk ⁵ , Jack A. Tuszy n´ ski

3,6

and Adam Huczy n´ ski *

1,

1

Department of Medical Chemistry, Faculty of Chemistry, Adam Mickiewicz University,

Uniwersytetu Pozna n´ skiego 8, 61–614 Pozna n´ , Poland; julia.krzywik@amu.edu.pl

TriMen Chemicals, Piłsudskiego 141, 92–318 Łód z´ , Poland; mozga@trimen.pl

Department of Oncology, University of Alberta, Edmonton, AB T6G 1Z2, Canada;

aminpour@ualberta.ca (M.A.); jack.tuszynski@gmail.com (J.A.T.)

2

3

4

5

Institute of Low Temperature and Structure Research, Polish Academy of Sciences, PO Box 1410,

5

0–950 Wrocław, Poland; j.janczak@intibs.pl

Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12

3–114 Wrocław, Poland; ewa.maj@hirszfeld.pl (E.M.); joanna.wietrzyk@hirszfeld.pl (J.W.)

,

5

6

DIMEAS, Politecnico di Torino, Corso Duca degli Abruzzi, 24, 10129 Torino, Italy

*

Correspondence: adhucz@amu.edu.pl; Tel.: +48618291673

ꢀ

ꢁꢂꢀꢃꢄꢅꢆꢇ

Academic Editor: Qiao-Hong Chen

Received: 16 March 2020; Accepted: 11 April 2020; Published: 14 April 2020

ꢀ

ꢁꢂꢃꢄꢅꢆ

Abstract: Colchicine is a well-known compound with strong antiproliferative activity that has had

limited use in chemotherapy because of its toxicity. In order to create more potent anticancer agents,

a series of novel colchicine derivatives have been obtained by simultaneous modiﬁcation at C7 (amides

and sulfonamides) and at C10 (methylamino group) positions and characterized by spectroscopic

methods. All the synthesized compounds have been tested in vitro to evaluate their cytotoxicity

toward A549, MCF-7, LoVo, LoVo/DX and BALB/3T3 cell lines. Additionally, the activity of the

studied compounds was investigated using computational methods involving molecular docking of

the colchicine derivatives to β-tubulin. The majority of the obtained derivatives exhibited higher

cytotoxicity than colchicine, doxorubicin or cisplatin against tested cancer cell lines. Furthermore,

molecular modeling studies of the obtained compounds revealed their possible binding modes into

the colchicine binding site of tubulin.

Keywords: anticancer agents; colchicine amide; colchicine sulfonamide; tubulin inhibitors; docking

studies; crystal structure

1

. Introduction

Microtubules, which are composed of α- and β-tubulin heterodimers, are involved in a large

number of processes, such as intracellular transport, cell shape development, cell division and cell

motility. During cell division microtubules form the mitotic spindle that in normal cells correctly

separates the chromosomes into two daughter cells. In cancer cells the rate of mitosis is typically

increased but chromosome segregation is imperfect leading to aneuploidy. Microtubules formed

during mitosis are considered as an ideal target for anticancer drugs since no cell can divide without

the force generated by microtubules. Therefore, many inhibitors of microtubule dynamics have been

investigated for their potential use as cancer chemotherapy drugs [

colchicine (see Scheme 1), a well–known tropolone alkaloid isolated from Colchicum autumnale, which

has been shown to exhibit very high cytotoxic eﬀects. It binds to tubulin at the colchicine binding

1–7]. One of these compounds is

1

Molecules 2020, 25, 1789; doi:10.3390 /molecules25081789

www.mdpi.com /journal /molecules

Molecules 2020, 25, 1789

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site and induces conformational change in the tubulin dimer making it incompetent for microtubule

assembly. As a result, the cell cycle is blocked and apoptosis is induced [ 14]. Unfortunately, colchicine

is too toxic to be useful as an antitumor agent [ 15 21]. Nevertheless, it has found use in therapy, e.g.,

for the treatment of familial Mediterranean fever, Behcet’s disease or acute gout [22–27].

8

–

8

,

–

Scheme 1. Synthesis of doubly modiﬁed colchicine derivatives (

2

–

21), changes at C7 and C10

) NHCH /EtOH, reflux; ( ) 2M HCl,

) RCOOH,

positions are highlighted in red. Reagents and conditions: (

a

b

d

3

◦

reflux; (

EDCI, DCM, RT for

,4-dihydropyridin-1-ylsulfonyl]azanide, DCM, RT, (2) 4M HCl/EtOAc, RT for 17; (

c

) RC(O)Cl or RSO Cl, Et N, DCM, 0 C to RT for

4,

8,

13,

15

–16, 18 and 21; (

2

3

5–7, 9–12 and 14; (e) (1) N-(tert-butoxycarbonyl)-N-[4-(dimethylazaniumylidene)-

1

f

) ClCH CH SO Cl,

2

◦

Et N, DCM, 0 C to RT; (g) morpholine, DCM, RT.

3

Therefore, over the past few decades much interest has been focused on structural modiﬁcations

in the hope of improving its therapeutic index [28 31]. Numerous double-modiﬁed colchicine

of

1

–

Molecules 2020, 25, 1789

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derivatives have been synthesized with the group at position C7 substituted by various amide

and sulfonamide moieties as well as with replacement of the methoxy group at position C10 by

a group containing either a nitrogen or a sulfur atom and their biological activities have been

determined [28,29,32–36]. These results have shown that new derivatives may have lower toxicity

with respect to normal cells while maintaining high antitumor activity. In addition, from the chemical

point of view, such a change at position C10 allows obtaining compounds more resistant to acid

hydrolysis [29,30,33]. We have decided to check various amide and sulfonamide moieties because they

have long been valued for their rich biological and chemical proﬁles and have emerged as a promising

class of compounds in drug discovery [37–41].

Herein, we report the synthesis, crystallographic and spectroscopic analysis of a series of

structurally diﬀerent derivatives of colchicine obtained by its modiﬁcation at position C7 (various

amide, sulfonamide or sulfamide moieties) and at position C10 (methylamino group). We also describe

the results of in vitro antiproliferative activity evaluation of colchicine (

derivatives ( 21) against four human cancer cell lines and one normal murine embryonic ﬁbroblast

cell line. To acquire more knowledge about the molecular mechanism of action of the investigated

compounds ( 21), we also present results of in silico molecular docking study of the colchicine binding

site (CBS) of β-tubulin.

1) and the obtained colchicine

2–

1–

2

. Results and Discussion

2

.1. Chemistry

To investigate the eﬀect of methylamino group at position C10 and, at the same time, various

amide, sulfonamide and sulfamide moieties at position C7 of colchicine

eighteen new derivatives ( 21) were synthesized. To facilitate the structure-activity relationship

analysis (SAR) we designed compounds with diﬀerent side chains at position C7: alkyl chains of

various length, straight and branched ( ), unsaturated alkyl chain (19), alkyl chains of various

lengths containing halogen atoms ( 11), an aromatic group without or with substituents (12 16 21),

1 on its antiproliferative activity,

4–

4–8

9–

– ,

and compounds containing an amino group 17–18 and 20.

The general route for the synthesis of colchicine derivatives 2–21 is depicted in Scheme 1. Colchicine

(

1

2

) was treated with methylamine solution in ethanol to give 10-methylamino-10-demethoxycolchicine

) with 80% yield, according to the method described earlier [42]. The replacement of water

solution of methylamine by ethanol solution eliminated the work up after the reaction and

permitted obtaining comparable ﬁnal yields. Next, hydrolysis of

N-deacetyl-10-methylamino-10-demethoxycolchicine ( ). Compounds

readily available from

in the presence of triethylamine or with the corresponding carboxylic acid and carbodiimide

as a condensing agent. Compound 17 was prepared using a N-(tert-butoxycarbonyl)-N-

4-(dimethylazaniumylidene)-1,4-dihydropyridin-1-ylsulfonyl]azanide and further modiﬁed by

removal of the tert-butoxycarbonyl group from amine with HCl [43]. Vinylsulfonamide 19 was

synthesized from 2-chloroethanesulfonyl chloride and through sulfonylation and in situ -elimination

2

with 2M HCl yielded

3

4–

16 18 19 and 21 were

,

–

3

by treatment with respective acid/ sulfonamide/ sulfamide chloride

[

3

β

of HCl. Compound 19 was used as the Michael acceptor with an electron-deﬁcient double bond for the

reaction with morpholine to produce compound 20 [44,45]. All synthesized compounds were isolated

in pure form after column chromatography.

The purity and structures of the obtained compounds

2–21 were determined using the LC-MS,

¹H and ¹³C NMR methods and are shown in the Supplementary Materials and discussed below.

1

13

The characteristic signals of -OCH group at position C10 of

1

in the H NMR and C NMR spectra

were observed as a singlet at 4.0 ppm and at 56.5 ppm, respectively. These signals vanish after the

reaction of colchicine with methylamine proving the substitution of the -OCH group in the tropolone

3

ring of

1. After the introduction of -NHCH at position C10 the signals of this group in the obtained

3

derivatives (2–21) were visible approx. at 3.1 ppm as a doublet and approx. at 7.4 ppm as a quartet in

Molecules 2020, 25, 1789

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1

H NMR and approx. at 29.5 ppm in ¹³C NMR. The chemical shifts of the amide moiety can be found

1

13

in the range 7.9–9.7 ppm in H NMR and in the range 164.2–175.2 ppm in C NMR, depending on the

substituent used. The ESI mass spectrometry conﬁrmed the structure of the obtained compounds by the

presence of an m/z signals assigned to the corresponding pseudomolecular ions of these compounds.

2

.2. X-ray Crystal Analysis

Structural characterization of the colchicine derivatives is very important in order to understand

their anticancer properties stemming from their interaction with tubulin as well as to enable

structure–activity relationship analysis (SAR) and related investigation. Therefore, structural analyses

of all crystals that were suitable for X-ray analysis of single crystals were performed. Crystals of

6,

1

,

12 14 18 and 19 suitable for the X-ray single crystal analysis were obtained by recrystallization of

,

the respective colchicine derivatives from acetonitrile, whereas crystals 15 and 16 from ethyl acetate

solutions. All crystals were measured at room (295 K) and low (100 K) temperature. Details of the

data collection parameters, crystallographic data and the ﬁnal agreement parameters are listed in

Supplementary Table S1. In the temperature range from 295 K to 100 K, no structural phase transitions

were observed in the crystals studied, although for colchicine derivative 11 at low temperature some

disorder of the -CF group in the -CH -CH -CF group at atom C21 could be observed. Colchicine

3

2

3

derivatives

6, 11, 12, 14, 15, and 16 crystallize in the P3 21 space group of the trigonal system while

2

derivative 18 crystallizes in the P2 2 2 space group of the orthorhombic system and derivative 19

1

1 1

crystallizes in the P2 space group of the monoclinic system. These space groups are chiral since the

1

compounds contain an asymmetric carbon (C7) atom. The absolute conﬁguration at the C7 atom is

S in all structures. The molecular structures of all colchicine derivatives (

) are illustrated in Supplementary Figure S60. The planar phenyl A and tropolone C rings in all

colchicine derivatives ( 11 12 14 15 16 18 and 19) are twisted around the C13–C16 bond with the

6, 11, 12, 14, 15, 16, 18 and

1

9

6

,

◦

torsion angle describing the twisting conformation C1–C16–C13–C12 between ~53 and ~56 at 100

K and they do not diﬀer signiﬁcantly from the values at room temperature (Table 1). Ring B in all

colchicine derivatives exhibits a similar puckering pattern and the extent of its non-planarity is such

that it adopts a conformation, which is close to the twist-boat with a ﬂattening caused by the fusion of

rings A and C (see Supplementary Figure S60). So the conformation of the fused A, B and C rings of

colchicine skeleton in the investigated derivatives is quite similar to that in colchicine itself [46].

◦

Table 1. Selected torsion angles ( ) of colchicine derivatives

6,

11,

12,

14

,

15,

16,

18 and 19 obtained by

X-ray analysis and DFT computation for a comparison.

6

11

12

14

15

16

18

19

1

00 K

53.1(4)

55.2(4)

53.1

55.5(6)

52.3

53.7(3)

55.0(3)

53.4

54.0(5)

54.4(4)

53.5

53.6(4)

54.7(4)

52.9

54.1(3)

55.0(3)

53.3

56.1(4)

54.5

55.3(3)

55.9(4)

56.0

C1

–C16

–

C13–C12

295 K

DFT value

1

00 K

−79.0(4) −86.4(6) −89.3(3) −90.6(5) −87.3(4) −86.8(4)

−

108.6(3)

66.2(3)

65.6(4)

59.2

−102.7(3)

−100.6(4)

−77.2

7.7(4)

6.8(5)

3.1

C17–O1–C1–C2

C18–O2–C2–C3

C19–O3–C3–C4

295 K

DFT value

1

295 K

DFT value

−86.5(4) −86.4(6) −86.4(4) −87.4(5) −87.8(4) −86.4(4)

−106.8(3)

−73.0

108.0(3)

106.9(4)

70.1

−70.7

105.2(6)

68.8

−80.3

96.1(2)

98.7(3)

82.1

8.6(4)

6.9(6)

−2.0

−80.0

95.5(4)

98.0(4)

82.3

14.4(6)

11.4(7)

−1.9

−72.9

99.7(4)

101.4(4)

71.1

−79.4

94.5(3)

98.7(3)

81.1

7.4(6)

6.9(6)

−1.9

−74.8

−77.5(4)

−74.0(4)

−59.6

1.2(5)

0.2(5)

0.7

−0.2(5)

−1.7(5)

0.2

00 K

100 K

−14.1(5) −10.5(9)

−

6.0(6)

295 K

DFT value

1

295 K

DFT value

−8.1(7)

−4.0

−

10.5(10)

−3.7

−

2.3(6) 3.8

−

00 K

7.7(5)

6.8(6)

−0.9

8.8(8)

8.7(9)

−0.9

4.6(3)

7.5(4)

−1.4

4.4(6)

5.0(6)

−1.0

5.0(5)

5.2(5)

−1.2

6.6(4)

7.5(4)

−1.3

4.7(4)

4.0(7)

−1.6

C20–N1

–C10

–

C11

The methoxy group -OCH linked to the phenyl ring at C3 is almost coplanar with the ring

3

in all structures of colchicine derivatives, whereas the other two methoxy groups linked at C1 and

C2 atoms of the phenyl ring have diﬀerent orientations in some colchicine derivatives (Table 1).

The N-methylamino group (-NHCH ) linked to tropolone C ring at C10 atom is almost coplanar

3

Molecules 2020, 25, 1789

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with the ring. The diﬀerences between the conformations of the investigated colchicine derivatives

are illustrated in Figure 1 and Table 1. For clarity, the colchicine derivatives have been divided

into two groups, according to the substituent in ring B at position C7; one group comprises the

derivatives with a chain substituent (Figure 1a) and the other group comprises the derivatives with

a substituent containing an aromatic ring (Figure 1b). Analysis of these results reveals a signiﬁcant

◦

diﬀerence in the torsion angle C17-O1-C1-C2 in compounds 18 (~

−

109 ) and 19 (~66 ) or the diﬀerence

77 ) torsion angle C18-O2 -C2-C3 in compound

9. These diﬀerences result from diﬀerent approach to the description of the molecule conformations,

◦

between the calculated (~

−

103 ) and measured (~

−

1

the X-ray analysis values refer to the conformation of molecules in crystals, in which the intermolecular

interactions play a signiﬁcant role and leads crystallization and speciﬁc crystal packing, while the DFT

values refer to a single isolated molecule in the gas state with the intermolecular interactions not taken

into account.

Figure 1. Comparison of the X-ray structure of (a) derivatives with a chain substituent at position C7:

6

(blue), 11 (red), 18 (pink) and 19 (green) and (b) derivatives with a substituent containing an aromatic

ring at position C7: 12 (pink), 14 (blue), 15 (green) and 16 (red) showing the conformation of the colchicine

skeleton. The molecules are overlapped one over another so that the ring C1-C2-C3-C4-C15-C16 is at

the same position.

2

.3. Molecular Electrostatic Potential Map Analysis

The role of the trimethoxyphenyl ring A of colchicine as well as that of the tropolone ring C in

tubulin binding have been studied in great details. The tropolonoic ring C of the colchicine skeleton

Molecules 2020, 25, 1789

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is found to be crucial for the interaction with tubulin [47

,48]. The molecular electrostatic potential

map (MESP) correlated with the electronic density in a molecule and is a powerful tool for analyzing

interactions [49 51]. It was, therefore, calculated for all structurally characterized colchicine derivatives.

–

Additionally, the gas-phase structures of all molecules were determined using the DFT optimization

with the Gaussian09 program package [52]. All calculations were carried out by the DFT method using

the Becke3-Lee–Yang–Parr correlation functional (B3LYP) [53–56] with the 6–31 + G basis set, starting

from the X-ray geometry of molecules. The gas-phase optimized conformations of all colchicine

derivatives are, in general, in good agreement with those obtained from the X-ray single crystal

investigation, however the optimized torsion angle C18–O2–C2–C3 describing the orientation of the

methoxy group is signiﬁcantly smaller than that provided by the X-ray analysis (Table 1 and for more

details see Supplementary Table S2).

The region of tubulin that interacts with colchicine is near the αβ-tubulin/dimer interface [57].

In order to better understand the interaction of the colchicine derivatives with tubulin, the molecular

electrostatic potential was calculated for all structurally characterized colchicine derivatives as well

as, for comparison, for colchicine itself. The three-dimensional MESP maps for colchicine derivatives

and for colchicine itself were calculated on the basis of the DFT (B3LYP) optimized geometries of

−3

molecules and mapped onto the total electron density isosurface (0.008 eÅ ) for both molecules using

the GaussView 5.0 program (Figure 2). The color coding of MESP is in the range of 0.05 (red) to

.05 eÅ (blue). For all colchicine derivatives, the regions of negative MESP are usually associated with

−

-1

0

the lone pair of electronegative atoms (O and N), whereas the regions of positive MESP are associated

with the electropositive atoms (Figure 2).

Figure 2. Cont.

Molecules 2020, 25, 1789

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Figure 2. Cont.

Molecules 2020, 25, 1789

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Figure 2. Optimized conformation (left) and three-dimensional molecular electrostatic potential map

−1

(

right) for colchicine derivatives (

red) to +0.05 eÅ (blue).

a–

h

) and colchicine itself (

i

), for comparison. Color code:

−0.05 eÅ

−1

The nucleophilic regions in colchicine derivatives are observed near oxygen atoms of all methoxy

and carbonyl groups. In addition, signiﬁcantly less negative value of MESP than that near the

oxygen atoms spreads across the aromatic phenyl rings. The planar conformation of tropolone ring

C, showing the alternating single and double C-C bonds, is manifested as partial delocalization of

the

π electrons resulting in a slightly negative value of MESP on both sides of the planar fragment of

colchicine derivatives. The molecular electrostatic potential for colchicine itself was also calculated for

comparison (Figure 2i). In all colchicine derivatives the methoxy (-OCH ) substituent in the tropolone

3

C ring is replaced by N-methylamino substituent (-NHCH ), therefore, MESP maps show a less

3

negative area near this group (Figure 2a–h) compared to the MESP map of colchicine itself (Figure 2i).

Molecules 2020, 25, 1789

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Additionally, the replacement in colchicine molecule of the -NHC(O)CH group in ring B at C7 atom

3

with various substituents modiﬁed the size of the molecule and the maps of electrostatic potential.

This is particularly visible for derivatives 11

, 14 and 18, 19 in which the -NHC(O)CH group is replaced

3

by -NHC(O)C H CF , -NHC(O)C H F in 11 and 14, respectively, and in 18 and 19 by the substituents

2

4

3

6

4

containing sulfonyl group (-SO R).

2

The DFT results, especially the three-dimensional molecular electrostatic potential map (MESP)

providing information on the distribution of the electron density of molecules are useful for predictions

of interactions between the tested compounds and homology modeled tubulin βI. The formation of

guest-host complexes (colchicine derivatives as guest and tubulin as host) depends on the guest’s ﬁt into

the host cavity and their interactions that result from the mutual matching of electrostatic interactions.

2

.4. In Vitro Determination of Drug-Induced Inhibition of Human Cancer Cell Line Growth

The synthesized colchicine derivatives 21 and starting material were evaluated for their

2

–

1

in vitro antiproliferative eﬀect on four human cancer cell lines, including one cell line displaying

various levels of drug resistance and additionally one normal murine embryonic ﬁbroblast cells.

The majority of new derivatives of

and were characterized by lower IC₅₀values than unmodiﬁed colchicine

and cisplatin, commonly used as antitumor agents in cancer chemotherapy (Table 2). From the set

of tested compounds, the ones most active against A549 tumor cell line were 14 and 18 19

IC₅₀15 nM), against MCF-7 tumor cell line-were 17 and 19 (IC₅₀13 nM), against LoVo

tumor cell line-were 16 18 19 (IC₅₀11 nM) of which the lowest IC values were shown

by compounds 10 and 13 (IC₅₀= 0.7–1.8 nM). Moreover, compound 13 was observed to be most

active towards the LoVo/DX line (IC₅₀= 9.6 nM), approx. 170 times more potent than unmodiﬁed

colchicine (IC₅₀= 1646.6 nM). Compound from amides and compound 20 from sulfonamides

showed the weakest activity (the highest IC₅₀values) against all cancer cell lines tested. The decrease

in cytotoxicity of compound could be related to an increase in hydrophobicity (high calculated

1

showed antiproliferative activity in the nanomolar range

1, as well as doxorubicin

2,

4–

6,

8–

–

(

≤

2

,

9

–

≤

2

,

4–

6,

8

–

,

–

≤

50

9–

1

7

octanol/water partition coeﬃcient clogP = 8.7, see Table 3). It is well known that high clogP value

and therefore low hydrophilicity are responsible for poor absorption and permeation to the colchicine

binding pocket in

β-tubulin. The high IC₅₀value for compound 20 may be due to the presence of a

morpholine ring which is a large volume substituent and can adopt diﬀerent conformations. Although

these compounds were the least potent out of the whole series of tested derivatives (

1–21), their IC₅₀

values were in the micromolar range (see Table 2).

Table 2. Antiproliferative activity (IC ) of colchicine (1) and its derivatives (2–21) compared with

50

antiproliferative activity of standard anticancer drugs doxorubicin and cisplatin and the calculated

values of the resistance index (RI) of tested compounds.

A549

MCF-7

LoVo

LoVo/DX

IC50 [nM]

BALB/3T3

Compound

IC50 [nM]

RI

IC50 [nM]

1

2

3

4

5

6

7

8

9

115.3 ± 23.6

10.8 ± 1.3

16.9 ± 2.8

14.6 ± 2.4

14.1 ± 2.4

13.6 ± 1.4

613.8 ± 194.4

11.7 ± 1.4

11.6 ± 2.8

8.6 ± 1.3

22.6 ± 1.3

8.6 ± 1.3

17.5 ± 2.5

4.3 ± 1.3

14.0 ± 1.7

9.7 ± 1.5

9.6 ± 0.5

6.8 ± 3.9

62.4 ± 16.9

7.0 ± 1.4

1.8 ± 0.4

1.5 ± 0.5

10.4 ± 1.3

8.5 ± 0.4

0.7 ± 0.1

8.6 ± 0.8

10.8 ± 1.3

1646.6 ± 314.0

271.3 ± 99.9

129.2 ± 11.8

271.8 ± 104.4

194.8 ± 51.9

102.3 ± 20.7

2435.7 ± 923.4

171.2 ± 41.3

62.4 ± 6.7

93.9

63.0

9.2

115.3 ± 36.8

10.8 ± 1.3

19.7 ± 7.0

19.4 ± 4.1

16.4 ± 3.5

13.6 ± 2.3

545.9 ± 104.4

28.1 ± 10.8

11.6 ± 2.3

10.7 ± 1.3

81.3 ± 20.4

13.0 ± 3.3

6.2 ± 1.6

19.7 ± 1.7

14.6 ± 1.5

14.1 ± 1.4

15.9 ± 6.6

464.8 ± 186.7

18.8 ± 9.9

9.2 ± 1.4

28.0

20.2

15.0

39.0

24.3

35.2

25.8

27.8

11.8

14.0

12.0

76.8

87.4

10

11

12

13

14

15

16

8.6 ± 1.2

38.5 ± 21.6

14.6 ± 2.1

13.0 ± 1.3

6.3 ± 3.2

12.7 ± 0.4

13.0 ± 1.3

9.2 ± 0.8

289.6 ± 165.2

99.9 ± 10.0

9.6 ± 3.3

10.7 ± 0.6

36.8 ± 12.1

17.3 ± 3.7

12.6 ± 1.3

13.0 ± 2.2

12.8 ± 0.9

102.5 ± 24.9

832.1 ± 292.7

946.9 ± 260.5

12.6 ± 2.1

43.3 ± 29.7

52.0 ± 29.3

Molecules 2020, 25, 1789

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Table 2. Cont.

1

2

7

8

9

0

1

56.6 ± 14.7

10.6 ± 0.6

10.3 ± 3.9

15.5 ± 1.7

21.1 ± 17.5

9.3 ± 1.1

6466.0 ± 264.2

540.2 ± 107.2

306.0

57.8

36.7

165.4

83.3

100.1 ± 24.1

14.2 ± 13.2

8.3 ± 3.8

11.4 ± 1.7

13.0 ± 4.3

8.4 ± 0.7

306.7 ± 144.9

800.3 ± 130.0

150.0 ± 25.3

268.4 ± 94.0

44385.7 ± 23852.0

991.1 ± 280.5

87.6 ± 13.5

85.5 ± 5.4

134.3 ± 41.5

73.5 ± 15.7

6122.0 ± 825.1

Doxorubicin

Cisplatin

141.7 ± 46.0

204.2 ± 47.8

99.4 ± 41.0

8732.0 ± 2540.7

8336.5 ± 1119.2

87.9

1.2

149.0 ±126.8

5741.0 ± 968.0

7139.8 ± 1218.7

7076.3 ± 1596.2

5665.1 ± 31.8

The IC50 value is deﬁned as the concentration of a compound at which 50% growth inhibition is observed. The IC50

values shown are mean SD. Human lung adenocarcinoma (A549), human breast adenocarcinoma (MCF-7), human

±

colon adenocarcinoma cell line (LoVo) and doxorubicin-resistant subline (LoVo/DX), normal murine embryonic

ﬁbroblast cell line (BALB/3T3). The RI (Resistance Index) indicates how many times more chemoresistant is a

resistant subline relative to its parental cell line. The RI was calculated for each compound using the formula: RI =

(

IC50 for LoVo/DX cell line)/(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 cells shows moderate sensitivity to the drug tested, RI above 10 indicates strong

drug resistance.

Table 3. Computational predictions of interactions between tested compounds (1–21) and homology

modeled tubulin I. 3D representation and 2D layout of colchicine derivatives–tubulin protein

β

complex, binding energy (BE), calculated octanol/water partition coeﬃcient (clogP) and active residues

are tabulated.

Binding

Energy

kcal/mol]

3

D Representation of the

2D Representation of the

Interactions

Active

Residues

Compound

clogP

Interactions

[

Ala179

Val180

Cys674

Leu688

Asn691

Ala749

Lys785

1

−41.0

−39.3

−4.0

1.1

Cys674

Ala683

Leu688

Lys785

2

3

4

1.6

0.9

1.9

Cys674

Lys687

Leu688

Asn691

Cys674

Ala683

Leu688

Asn691

Met692

Ala749

Lys785

−43.4

Molecules 2020, 25, 1789

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Table 3. Cont.

Binding

Energy

kcal/mol]

3

D Representation of the

2D Representation of the

Interactions

Active

Residues

Compound

clogP

Interactions

[

Ala179

Val180

Cys674

Leu688

Asn691

Ala749

Lys785

5

−43.0

2.5

Asn100

Ser177

Ala179

Leu681

Lys687

Leu688

Asn691

Lys785

6

−37.2

3.0

Gln10

Asn100

Ser177

Thr178

Cys674

Leu681

Asn682

Ala683

Lys687

Leu688

Asn691

Ala749

Lys785

7

−53.7

8.7

Ser177

Leu681

Ala683

Leu688

Lys785

8

−32.3

−44.0

−41.8

2.7

2.2

2.7

Ala179

Cys674

Ala683

Lys687

Leu688

Asn691

Met693

Ala749

Lys785

9

Ala179

Cys674

Ala683

Leu688

Asn691

Asn782

Lys785

10

Molecules 2020, 25, 1789

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Table 3. Cont.

Binding

Energy

kcal/mol]

3

D Representation of the

2D Representation of the

Interactions

Active

Residues

Compound

clogP

Interactions

[

Ala179

Cys674

Leu681

Ala683

Lys687

Leu688

Asn691

Lys785

1

−34.7

3.0

Ser177

Ala179

Leu681

Lys687

Leu688

Asn691

Lys785

12

−35.2

3.3

Cys674

Leu681

Ala683

Lys687

Leu688

Asn691

Lys785

13

14

15

16

−40.1

−34.3

−43.9

−38.3

3.9

Val176

Ser177

Thr178

Ala179

Gln680

Leu688

Lys785

3.4

2.0

Asn100

Ser177

Cys674

Leu681

Asn682

Ala683

Lys687

Leu688

Asn691

Lys785

Ser177

Thr178

Cys674

Leu681

Ala683

Leu688

Asn691

Lys785

Molecules 2020, 25, 1789

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Table 3. Cont.

Binding

Energy

kcal/mol]

3

D Representation of the

2D Representation of the

Interactions

Active

Residues

Compound

clogP

Interactions

[

Gln10

Ser177

Thr178

Ala179

Asn682

Ala683

Asp684

Lys687

Asn691

Lys785

1

7

−34.8

1.9

Gln10

Ala99

Asn100

Gly142

Gly143

Thr144

Ser177

Thr178

Ala179

Leu681

1

8

−54.6

2.5

Ser177

Thr178

Leu681

Ala683

Asp684

Lys687

Asn691

Lys785

1

9

−23.4

2..8

Gly9

Gln10

Ala11

Asp68

Ala99

Asn100

Gly142

Gly143

Thr144

Gly145

Ser177

Thr178

Lys687

20

−59.6

2.4

Ala179

Val180

Cys674

Leu688

Asn691

Ala749

Lys785

21

−29.8

4.9

Although the synthesized compounds are eﬀective toward cancer cells, their potential is limited

against cells with developed drug resistance. The data presented in Table 2 show that unmodiﬁed

colchicine 1 and all of the colchicine derivatives 2–21 less eﬀectively inhibited the proliferation of the

doxorubicin-resistant subline LoVo/DX than the sensitive LoVo cell line. The calculated values of RI

clearly conﬁrmed that none of the tested amides and sulfonamides was able to overcome the drug

resistance of LoVo/DX cell line (RI ranges from 11.8 to 306.0). It can be explained by the upregulated

expression of eﬄux transporters in these cells, playing an important role in drug transport in many

Molecules 2020, 25, 1789

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organs and determining the drug resistance of cancer cells. Because this type of resistance is one of the

mechanisms of cancer resistance [58], compounds and 21 are probably good substrates for such

1–

2

4–

pumps. Increased eﬄux of compounds makes it impossible to reach their adequate concentrations in

the cell and consequently to exert eﬃcient cytotoxic eﬀect. The only compound which showed RI < 10

was derivative

0 nM for three cancer cells, see Table 2) it can be still considered as a good starting point for the chase

after antitumor agents active against drug resistant lines.

3, having at position C7 a free amino group. Keeping in mind its good activity (IC₅₀<

2

The selectivity index (SI) was calculated to evaluate the toxicity of the compounds studied against

normal cells and to predict their therapeutic potential (see Figure 3). High SI values result from large

diﬀerences between the cytotoxicity against cancer and normal cells and this means that cancer cells

will be killed at a higher rate than normal ones. From the set of tested compounds with methylamino

group at position C10 and alkyl chains at position C7 (

MCF-7 and LoVo cells (SI = 1.5–4.0) showed with short and branched substituent (isobutyric acid

derivative). The 4,4,4-triﬂuorobutyric acid derivative 11 (from compounds with alkyl chains of various

lengths containing halogen atoms 11) showed outstanding selectivity for three out of four cancer

cells (SI = 5.6–7.8). It should be emphasized that compound 11 was the only compound with SI values

greater than that of unmodiﬁed colchicine for all tumor cell lines. Despite the fact that compounds

0, and 13 stood out from the synthesized compounds in terms of IC₅₀values, their selectivity indices

were high only for LoVo lines (SI = 6.5–9.1), they were very cytotoxic also to non-cancerous BALB/3T3

cells (IC₅₀= 6.2–11.6 nM). Among aryl amides and sulfonamides 12 16 and 21, distinctive SI values were

2, 4–8, 19) the best selectivity index for A549,

8

9–

1

9,

1

–

derived for colchicine derivatives containingnicotinic and isonicotinic amide residue (SI ranges 3.0 to 4.8).

These results are noteworthy and suggest that extended and more detailed research of similar derivatives

is necessary to determine the importance of ring aromaticity, its size or heteroatom type. Especially high

SI values for MCF-7 were obtained for sulfamide 17 (SI = 9.8) and sulfonamide containing a morpholine

ring 20 (SI = 6.6). These results deserve special attention and require further studies to determine whether

the conversion of an amide bond to a sulfonamide bond with an appropriate substituent would allow

obtaining compounds highly selective towards MCF7 cells, compared to colchicine

1. As many as thirteen

of the obtained derivatives exhibited SI 2 for LoVo cell line (see Figure 3). The results indicated that

≥

properly designed doubly modified (at C7 and C10 positions) colchicine derivatives can have greater

selectivity towards cancer cells than the parental compound.

Figure 3. Comparison of selectivity index (SI) values of the tested compounds. The SI was calculated

for each compound using the formula: SI = (IC₅₀for normal cell line BALB/3T3)/(IC₅₀for respective

cancerous cell line). A favorable SI > 1.0 indicates a drug with eﬃcacy against tumor cells greater than

the toxicity against normal cells.

Molecules 2020, 25, 1789

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2

.5. In Silico Determination of the Molecular Mode of Action

In the present study, computational investigation including molecular docking, molecular

dynamics (MD) simulations, molecular mechanics generalized Born/surface area (MM/GBSA) binding

free energy calculations and decomposition of pair-wise free energy on a per-residue basis were

conducted to deeply explore the molecular basis for the binding of twenty novel double modiﬁed

colchicine amides and sulfonamides to β-tubulin. The latter is one of the subunits of microtubules

in the cytoskeleton structure of every eukaryotic cell, which is the target of many anticancer drugs.

The twenty structures of colchicine derivatives described above were docked into the βI-tubulin (the

most abundant isotype in most cancer tumors) colchicine binding site.

On the basis of our computational predictions, according to increasing binding energy,

the compounds are ordered as follows: 20

(

−

59.6), 18

(

−

54.6),

7

(

−

53.7),

37.2), 12

9

(

−

44.0), 15

(

−

43.9),

34.9), 11

4

(

−

43.4),

5

(

−

43.1), 10

34.3),

(

−

41.8),

32.3), 21

1

(

−

41.1), 13

(

−

40.1),

2

(

3

39.3), 16

(

38.3),

6

(−

35.2), 17

(

−

34.7),

1

4

(

−

8

(−

(

−

29.8), 19

(

−

23.4),

(−4.0) kcal/mol with the binding energies in the parenthesis

given in units of kcal/mol. Binding energies of these compounds are shown in Figure 4 and Table 3.

Figure 4. Comparison of binding energies of the tested compounds complexed with tubulin

energies have been estimated using the MM/GBSA method.

βI. Binding

In view of the calculated binding energies, we can conclude that there is no strong correlation

(

the lower the binding energy, the more biologically active the chemical compound) between in silico

computer calculations (BE values, Figure 4) and in vitro activity results (IC₅₀values, Table 2).

The lowest binding energies, 59.6 and 53.7 kcal/mol, were shown by sulfonamide 20 (derivative

with the morpholine ring) and amide (derivative of palmitic acid), respectively. However, these

−

7

compounds showed the weakest antiproliferative activity (highest IC₅₀values) among all compounds

tested (1–21).

Compound 18 has the third lowest energy (

.3 to 15.5 nM for LoVo, A549 and MCF-7 cancer cells. The molecular-level computations indicate

that 18 ﬁts well to I-tubulin and probably uses this binding site as a target. The majority of the

other derivatives 17 exhibited binding energy less than 30.0 kcal/mol, so close to the energy

of unmodiﬁed colchicine (BE = 41.1 kcal/mol), which is a compound known to inhibit tubulin

polymerization. These compounds bind to the active sites of I-tubulin isotype and may therefore

−54.6 kcal/mol) and good cytotoxicity with IC₅₀from

9

β

2

,

4–6

,

8

–

1

−

β

Molecules 2020, 25, 1789

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have a mechanism of action similar to

1

and may be colchicine binding site inhibitors, although their

in vitro activities towards various cells characterized by an IC₅₀value (Table 2) were lower than that of

colchicine 1.

Derivative

antiproliferative activity is not exclusively the result of interaction with tubulin. This is an important

ﬁnding because compound was the only one which showed any possibility of breaking the drug

3

showed the highest binding energy value (BE =

−

4.0 kcal/mol), therefore its

3

resistance of the LoVo/DX line (RI = 9.2, see Table 2).

Therefore, we can see that the results only partly correlate with in vitro determined biological

activity of these compounds as indicated though the corresponding IC₅₀values. This may be explained

by several additional eﬀects taking place in living cells compared to the computational simulations

that focus only on the binding mode of the compounds to the target. Primarily oﬀ-target interactions

involving eﬄux transporters with diﬀerent aﬃnities for the individual compounds and diﬀerences

in solubility of these colchicine derivatives or their membrane permeability. Additionally, the lack

of signiﬁcant correlation may be due to the fact that in various cancer cells the expression of speciﬁc

β-tubulin isotypes signiﬁcantly varies [59]. We propose the inclusion of binding aﬃnity calculations

for these compounds with regard to not only other tubulin isotypes but also with respect to most

important eﬄux transporters in order to minimize them and hence increase the activity of the drug

in vitro and in vivo.

Schematic interactions of the compounds with CBS of βI-tubulin residues are shown in Table 3.

In 3D representation, the interacting residues predicted from pairwise per-residue binding free

energy decomposition calculations (E < 2 kcal/mol) are shown in stick presentation and their carbons

−

and the ribbon are colored as green. Tubulin is shown in cartoon representation. Hydrogen bonds and

their directionality are represented as black dashed arrows. The structures are color-coded as follows:

tubulin

as O (red), C (gray), N (blue), S (yellow), Cl (green) and F (pink). Binding energy deﬁnes the aﬃnity

of binding for colchicine derivatives complexed with tubulin I. Binding energies are predicted by

MM/GBSA method. The last column contains information about the active residues with the binding

free energy decomposition (E_d_e_c_o_m_p) less than 2 kcal/mol (the residues with E_d_e_c_o_m_p3 kcal/mol

αI, brown; tubulin βI, beige. Compounds are displayed with sticks and the atoms are colored

β

−

<

−

are highlighted in boldface). The last line contains the graphical key to help interpret the 2D part of the

ligand interactions panel.

Derivative compounds are designed with diﬀerent side chains at C7 position of colchicine

Compounds have alkyl chains of various lengths, straight and branched and compound 19 has

an unsaturated alkyl chain. The second lowest binding energy compound within all the compounds

appears to be engaged in some interactions with

2 kcal/mol). Most of the compounds in this group are bound through

1.

2, 4–8

7

is in this group (BE =

−

53.6 kcal/mol). Compound

7

α

-tubulin (

−

3 < E

<

−

decomp

Lys785, Lys687 and Leu688 residues.

Compounds 11 were designed with alkyl chains of various length containing halogen atoms

side chains at position C7 of colchicine

E_d_e_c_o_m_p2 kcal/mol) are Ala179, Cys674, Asn691, while the strongest binding residues (E

3 kcal/mol) are Leu688 and Lys785.

Compounds 12 16, 21 were designed with an aromatic group side chain without or with

substituents at C7 position of colchicine . The same trend of strong binding of Leu688 and Lys785

9

–

1

. In this group, the common binding residues with (

−

3 <

<

−

decomp

−

–

1

residues with the compounds is seen in this group. The binding energies in this group are on the

higher side compared to the other compounds.

As the last group, compounds 17

colchicine . The strongest binding residues in this group belong to both

in compound 17, (Gln10, Gly142, Gly143, Thr144 and Ser177) in compound 18 and (Asp68 and Asn100)

in compound 20 with (E_d_e_c_o_m_p3 kcal/mol) and -tubulin (Asp684, Asn691, Lys785) in compound

and Lys687 in compound 20. The lowest binding energy between all the compounds characterizes

compounds 20 and 18, which mainly bind to α-tubulin.

–18 and 20 contain the sulfonamide moieties at position C7 of

1

α

-tubulin (Gln10 and Ser177)

<

−

β

1

7

Molecules 2020, 25, 1789

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In the experimental part of the study, the highest IC₅₀values (the weakest activity) were found for

compound from the amides and compound 20 from the sulfonamides studied against all cancer cell

lines tested. According to our computational analysis, although these compounds show the lowest

7

binding energies, they do not bind to the CBS, which is located in β-tubulin and has the tendency to go

beyond and also bind to α-tubulin.

3

. Materials and Methods

3

.1. General

All solvents, substrates and reagents were obtained from TriMen Chemicals (Poland) or Sigma

Aldrich and were used without further puriﬁcation. CDCl and CD Cl spectral grade solvents were

3

2

stored over 3 Å molecular sieves for several days. TLC analysis was performed using pre-coated glass

plates (0.2 mm thickness, GF-254, pore size 60 Å) from Agela Technologies and spots were visualized

by UV-light. Products were puriﬁed by ﬂash chromatography using high-purity grade silica gel (pore

size 60 Å, 230–400 mesh particle size, 40–63

was performed on LC-20AP Shimadzu with ELSD-LTII detector equipped with Phenomenex Luna C18

50 21 mm, 5 m column eluted with 20 mL/min ﬂow over 20 min of acetonitrile in water. Solvents

were removed using a rotary evaporator.

µm particle size) from SiliCycle Inc. Preparative HPLC

2

×

µ

3

.2. Spectroscopic Measurements

NMR spectra were recorded on Bruker Avance DRX 500 ( H NMR at 500 MHz and ¹³C NMR

at 126 MHz) magnetic resonance spectrometers. ¹H NMR spectra are reported in chemical shifts

1

downfield from TMS using the respective residual solvent peak as internal standard (CDCl₃

δ 7.26 ppm,

1

CD Cl₂

δ

5.32 ppm, (CD ) SO

δ

2.50 ppm). H NMR spectra are described as follows: chemical shift (

δ,

2

3 2

ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, dt = doublet

of triplets, dq = doublet of quartets, m = multiplet), coupling constant (J) in Hz, and integration. ¹³C NMR

spectra are described in chemical shifts downfield from TMS using the respective residual solvent peak as

internal standard (CDCl δ 77.16 ppm, CD Cl δ 53.84 ppm and (CD ) SO δ 39.52 ppm).

3

2

3 2

Electrospray ionization (ESI) mass spectra were obtained on a Waters Alliance 2695 separation

module with a PDA 2996 UV detector and Waters Micromass ZQ 2000 mass detector equipped with

Restek Ultra Biphenyl 50

×

3 mm, 3

µm column eluted with 0.3 mL/min ﬂow of 3–100% gradient (over

6

3

min) of acetonitrile in water.

.3. Synthesis

.3.1. Synthesis of 2

To a solution of 1 (1.0 equiv.) in EtOH, a methylamine (solution 33% in EtOH, 10.0 equiv.) was

added. The mixture was stirred at reﬂux for 24 h and then concentrated under reduced pressure to

dryness. The residue was puriﬁed using column ﬂash chromatography (silica gel; DCM/MeOH) and

next lyophilized from dioxane to give the pure product 2 as a yellow solid with a yield of 80%.

+

ESI-MS for C H N O (m/z): [M + H] 399, [M + Na] 421, [2M + H] 797, [2M + Na] 819, [M –

22

26

− −

2

5

−

H] 397, [M + HCOO ] 443.

1

H NMR (500 MHz, CDCl₃)

δ 8.70 (d, J = 6.4 Hz, 1H), 7.58 (s, 1H), 7.46 (d, J = 11.1 Hz, 1H),

7

.28–7.25 (m, 1H), 6.58 (d, J = 11.3 Hz, 1H), 6.52 (s, 1H), 4.73–4.64 (m, 1H), 3.93 (s, 3H), 3.88 (s, 3H), 3.61

(s, 3H), 3.08 (d, J = 5.4 Hz, 3H), 2.47–2.43 (m, 1H), 2.37–2.31 (m, 1H), 2.29–2.22 (m, 1H), 2.02–1.96 (m,

1

H), 1.94 (s, 3H).

¹³C NMR (126 MHz, CDCl₃)

δ 175.11, 170.19, 155.23, 152.91, 151.61, 151.10, 141.51, 139.37, 134.66,

1

30.42, 126.93, 122.81, 108.28, 107.20, 61.46, 61.40, 56.16, 52.72, 37.06, 30.15, 29.53, 22.74.

Molecules 2020, 25, 1789

18 of 31

3

.3.2. Synthesis of 3

To a solution of compound

2

(1.0 equiv.) in dioxane, 2M HCl (10.0 equiv.) was added and

the mixture was stirred at reﬂux. Reaction progress was monitored by LC-MS. Then the reaction

mixture was neutralized with 4M NaOH to pH~10 and extracted four times with EtOAc. The organic

layers were combined, washed with brine, dried over Na SO , ﬁltered and evaporated under reduced

2

4

pressure. The residue was puriﬁed using column ﬂash chromatography (silica gel; DCM/MeOH) and

next lyophilized from dioxane to give the pure product 3 as a yellow solid with a yield of 73%.

+

ESI-MS for C H N O (m/z): [M + H] 357, [M + Na] 379, [2M + Na] 735.

H NMR (500 MHz, CDCl₃)

20

24

2

4

1

δ 7.61 (s, 1H), 7.33 (d, J = 11.1 Hz, 1H), 7.23–7.21 (m, 1H), 6.50 (s, 1H),

6

.50 (d, J = 11.4 Hz, 2H), 3.87 (s, 3H), 3.87 (s, 3H), 3.75–3.72 (m, 1H), 3.59 (s, 3H), 3.05 (d, J = 5.5 Hz, 3H),

2

.41–2.37 (m, 1H), 2.33–2.31 (m, 2H), 2.25 (s, 2H), 1.71–1.61 (m, 1H).

13

C NMR (126 MHz, CDCl₃)

δ 175.57, 154.99, 153.25, 152.76, 150.59, 141.06, 138.73, 135.41, 129.74,

1

26.63, 123.68, 107.35, 106.94, 61.19, 60.84, 56.06, 54.01, 40.90, 30.69, 29.48.

3

.3.3. General Procedure for the Synthesis of Colchicine Derivatives 4, 8, 13, 15–16, 18 and 21

Compounds 13 15 16 18 and 21 were obtained directly from compound . To a solution

of compound (1.0 equiv.) and Et N (3.0 equiv.) in DCM in an ice bath, the corresponding

4,

8,

,

–

,

3

acid/sulfonyl/sulfamide chloride (1.1 equiv.) diluted with DCM was added slowly. Next the ice bath

was removed and the reaction mixture was stirred at RT. Reaction progress was monitored by LC-MS.

Then the reaction mixture was diluted with EtOAc, washed with H O, 1M K CO , brine and dried

2

3

over Na SO . The residue was puriﬁed using column ﬂash chromatography (silica gel; DCM/MeOH)

2

4

and next lyophilized from dioxane to give respective compound.

Compound 4

Yellow solid, yield 86%.

+

ESI-MS for C H N O (m/z): [M + H] 413, [M + Na] 435, [2M + H] 825, [2M + Na] 847,

2

3

28

2

5

−

− −

[

M−H] 411, [M + HCOO ] 457.

1

H NMR (500 MHz, CDCl ) δ 8.46 (s, 1H), 7.53 (s, 1H), 7.44 (d, J = 11.1 Hz, 1H), 7.25–7.21 (m, 1H),

3

6.55 (d, J = 11.4 Hz, 1H), 6.50 (s, 1H), 4.72–4.65 (m, 1H), 3.91 (s, 3H), 3.86 (s, 3H), 3.61 (s, 3H), 3.06 (d, J = 5.4

Hz, 3H), 2.44–2.40 (m, 1H), 2.33–2.30 (m, 1H), 2.26–2.17 (m, 3H), 1.94–1.88 (m, 1H), 1.05 (t, J = 7.6 Hz, 3H).

13

C NMR (126 MHz, CDCl₃)

δ 175.09, 173.89, 155.20, 152.86, 151.64, 151.09, 141.49, 139.23, 134.67,

1

30.33, 126.95, 122.94, 108.14, 107.20, 61.44, 56.14, 52.37, 37.20, 30.18, 29.53, 29.11, 9.72.

Compound 8

Yellow solid, yield 96%.

ESI-MS for C H N O (m/z): [M + H] 427, [M + Na] 449, [2M + H] 853, [2M + Na] 875,

+

2

4

30

2

5

−

− −

[

M−H] 425, [M + HCOO ] 471.

1

H NMR (500 MHz, CDCl )

δ 7.48 (d, J = 11.2 Hz, 1H), 7.45 (s, 1H), 7.31–7.28 (m, 1H), 6.67 (d, J =

3

7

3

.3 Hz, 1H), 6.59 (d, J = 11.3 Hz, 1H), 6.53 (s, 1H), 4.75–4.63 (m, 1H), 3.94 (s, 3H), 3.89 (s, 3H), 3.63 (s,

H), 3.10 (d, J = 5.4 Hz, 3H), 2.53–2.43 (m, 2H), 2.38–2.32 (m, 1H), 2.28–2.20 (m, 1H), 1.93–1.82 (m, 1H),

1

.14 (t, J = 6.7 Hz, 6H).

¹³C NMR (126 MHz, CDCl₃)

δ 176.86, 175.21, 155.19, 152.85, 151.15, 141.56, 139.08, 134.55, 130.09,

26.97, 123.07, 107.91, 107.22, 61.51, 61.45, 56.13, 51.96, 37.56, 35.21, 30.19, 29.54, 19.70, 19.60.

Compound 13

Yellow solid, yield 86%.

−

ESI-MS for C H ClN O (m/z): [M + H] 495/497, [M + Na] 517, [2M + H] 989/991, [M-H]

+

2

7

27

2

5

− −

4

93/495, [M + HCOO ] 540.

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1

H NMR (500 MHz, CD Cl )

δ

8.12 (d, J = 7.5 Hz, 1H), 7.60 (s, 1H), 7.42–7.39 (m, 2H), 7.29–7.22

2

(

2

m, 2H), 7.19 (t, J = 7.5 Hz, 1H), 7.06 (t, J = 7.4 Hz, 1H), 6.60 (s, 1H), 6.53 (d, J = 11.3 Hz, 1H), 4.88–4.83

m, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 3.71 (s, 3H), 3.03 (d, J = 5.2 Hz, 3H), 2.52–2,48 (m, 1H), 2.42–2.31 (m,

H), 2.14–2.04 (m, 1H).

13

C NMR (126 MHz, CD Cl )

δ

174.51, 165.99, 155.24, 153.11, 150.94, 150.51, 141.52, 139.19, 135.71,

34.71, 130.64, 130.43, 130.01, 129.60, 129.58, 126.83, 126.79, 123.26, 107.90, 107.46, 61.08, 61.07, 56.02,

2.86, 37.83, 30.11, 29.35.

2

1

5

Compound 15

Yellow solid, yield 23%.

+

ESI-MS for C H N O (m/z): [M + H] 462, [M + Na] 484, [2M + H] 923, [2M + Na] 945,

2

6

27

3

5

−

− −

[

M-H] 460, [M + HCOO ] 506.

1

H NMR (500 MHz, CDCl₃)

δ 9.35 (d, J = 7.0 Hz, 1H), 9.07 (d, J = 1.8 Hz, 1H), 8.48 (dd, J = 4.8,

1

7

3

.5 Hz, 1H), 8.13–8.06 (m, 1H), 7.65 (s, 1H), 7.51 (d, J = 11.2 Hz, 1H), 7.31 (q, J = 5.1 Hz, 1H), 7.05 (dd, J =

.9, 4.9 Hz, 1H), 6.61 (d, J = 11.4 Hz, 1H), 6.54 (s, 1H), 4.99–4.86 (m, 1H), 3.95 (s, 3H), 3.89 (s, 3H), 3.70 (s,

H), 3.08 (d, J = 5.4 Hz, 3H), 2.51–2.45 (m, 1H), 2.40–2.34 (m, 1H), 2.33–2.27 (m, 1H), 2.17–2.09 (m, 1H).

13

C NMR (126 MHz, CDCl₃)

39.62, 134.89, 134.61, 130.41, 129.34, 126.86, 123.08, 122.79, 108.49, 107.27, 61.53, 61.48, 56.15, 53.22,

6.83, 30.28, 29.58.

δ 174.69, 165.09, 155.37, 153.01, 151.82, 151.42, 151.15, 148.87, 141.59,

1

3

Compound 16

Yellow solid, yield 39%.

+

ESI-MS for C H N O (m/z): [M + H] 462, [M + Na] 484, [2M + H] 923, [2M + Na] 945,

2

6

27

3

5

−

− −

[

M-H] 460, [M + HCOO ] 506.

1

H NMR (500 MHz, CDCl₃) δ 9.71 (d, J = 6.6 Hz, 1H), 8.37 (d, J = 5.8 Hz, 2H), 7.69 (s, 1H), 7.64

(d, J = 5.8 Hz, 2H), 7.54 (d, J = 11.2 Hz, 1H), 7.32–7.28 (m, 1H), 6.65 (d, J = 11.4 Hz, 1H), 6.53 (s, 1H),

4

2

.98–4.81 (m, 1H), 3.95 (s, 3H), 3.87 (s, 3H), 3.71 (s, 3H), 3.10 (d, J = 5.4 Hz, 3H), 2.50–2.41 (m, 1H),

.41–2.25 (m, 2H), 2.16–2.11 (m, 1H).

13

C NMR (126 MHz, CDCl₃)

δ 174.51, 164.91, 155.37, 153.05, 151.64, 151.17, 150.18, 141.59, 140.46,

1

39.81, 134.59, 130.64, 126.78, 122.68, 120.99, 108.68, 107.27, 61.55, 61.47, 56.14, 53.46, 36.50, 30.29, 29.60.

Compound 18

Yellow solid, yield 38%.

ESI-MS for C H N O S (m/z): [M + H] 464, [M + Na] 486, [2M + H] 927, [2M + Na] 949,

+

22

29

3

6

−

[

M-H] 462.

¹H NMR (500 MHz, CDCl₃)

δ

7.85 (s, 1H), 7.45–7.41 (m, 2H), 6.58 (d, J = 11.3 Hz, 1H), 6.54 (s, 1H),

6

.49–6.46 (m, 1H), 4.33–4.19 (m, 1H), 3.93–3.89 (m, 6H), 3.57 (s, 3H), 3.10 (d, J = 5.1 Hz, 3H), 2.58 (s, 6H),

2

.49–2.39 (m, 2H), 2.35–2.26 (m, 1H), 2.01–1.90 (m, 1H).

13

C NMR (126 MHz, CDCl₃)

δ 174.73, 155.46, 153.08, 150.77, 150.08, 141.39, 139.41, 134.59, 129.64,

1

26.34, 124.53, 108.22, 107.43, 61.34, 60.80, 56.48, 56.05, 39.91, 37.95, 30.50, 29.57.

Compound 21

Yellow solid, yield 83%.

ESI-MS for C H F N O S (m/z): [M + H] 565, [M + Na] 587, [2M + H] 1129, [2M + Na]

+

27

3

2

6

−

1

151, [M-H] 563.

¹H NMR (500 MHz, CDCl₃)

δ

8.72 (d, J = 7.5 Hz, 1H), 8.07 (s, 1H), 7.92 (s, 1H), 7.84 (d, J = 8.2 Hz,

2

8

H), 7.53 (d, J = 8.3 Hz, 2H), 7.48 (d, J = 11.5 Hz, 1H), 6.66 (d, J = 11.8 Hz, 1H), 6.46 (s, 1H), 4.16 (q, J =

.8 Hz, 1H), 3.91 (s, 3H), 3.86 (s, 3H), 3.58 (s, 3H), 3.08 (d, J = 5.2 Hz, 3H), 2.45–2.31 (m, 1H), 2.31–2.18

(m, 2H), 2.16–2.10 (m, 1H).

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¹³C NMR (126 MHz, CDCl₃)

δ

170.13, 155.43, 153.53, 150.65, 150.15, 144.53, 141.35, 134.75, 133.34,

131.89, 127.57, 125.82, 125.79, 125.49, 124.38, 123.51, 122.21, 111.26, 107.42, 61.29, 60.95, 56.63, 56.04,

3

8.98, 30.26, 29.78.

3

.3.4. General Procedure for the Synthesis of Colchicine Derivatives 5–7, 9–12 and 14

Compounds 12 and 14 were obtained directly from compound . To a solution of compound

5–

7,

9–

3

(1.0 equiv.) in DCM corresponding carboxylic acid (1.1 equiv.) and 1-(3-dimethylaminopropyl)-

3-ethylcarbodiimide hydrochloride (EDCI, 1.1 equiv.) were added. Reaction progress was monitored

by LC-MS. Then the reaction mixture was diluted with EtOAc, washed with H O, 1M K CO ,

2

3

brine and dried over Na SO . The residue was puriﬁed using column ﬂash chromatography (silica gel;

2

4

DCM/MeOH) and next lyophilized from dioxane to give respective compound.

Compound 5

Yellow solid, yield 93%.

+

ESI-MS for C H N O (m/z): [M + H] 427, [M + Na] 449, [2M + H] 853, [2M + Na] 875,

2

4

30

2

5

−

− −

[

M-H] 425, [M + HCOO ] 471.

1

H NMR (500 MHz, CDCl₃)

δ 7.88 (s, 1H), 7.51 (s, 1H), 7.45 (d, J = 11.1 Hz, 1H), 7.27–7.25 (m, 1H),

6

5

2

.56 (d, J = 11.3 Hz, 1H), 6.51 (s, 1H), 4.76–4.68 (m, 1H), 3.92 (s, 3H), 3.87 (s, 3H), 3.61 (s, 3H), 3.08 (d, J =

.4 Hz, 3H), 2.45–2.41 (m, 1H), 2.35–2.27 (m, 1H), 2.25–2.17 (m, 3H), 1.91 –1.85 (m, 1H), 1.65–1.55 (m,

H), 0.87 (t, J = 7.4 Hz, 3H).

13

C NMR (126 MHz, CDCl₃)

δ 175.10, 172.96, 155.22, 152.87, 151.43, 151.13, 141.52, 139.20, 134.61,

1

30.31, 126.94, 123.10, 108.13, 107.22, 61.45, 56.13, 52.21, 38.22, 37.45, 30.18, 29.54, 19.09, 13.93.

Compound 6

Yellow solid, yield 73%.

+

ESI-MS for C H N O (m/z): [M + H] 441, [M + Na] 463, [2M + H] 881, [2M + Na] 903,

2

5

32

2

5

−

− −

[

M-H] 439, [M + HCOO ] 485.

1

H NMR (500 MHz, CDCl₃)

δ 7.86 (s, 1H), 7.50 (s, 1H), 7.44 (d, J = 11.1 Hz, 1H), 7.26–7.21 (m, 1H),

6

5

2

.55 (d, J = 11.3 Hz, 1H), 6.51 (s, 1H), 4.76–4.68 (m, 1H), 3.93 (s, 3H), 3.88 (s, 3H), 3.61 (s, 3H), 3.08 (d, J =

.4 Hz, 3H), 2.45–2.41 (m, 1H), 2.36–2.30 (m, 1H), 2.25–2.18 (m, 3H), 1.93–1.84 (m, 1H), 1.58–1.51 (m,

H), 1.30–1.22 (m, 2H), 0.81 (t, J = 7.3 Hz, 3H).

13

C NMR (126 MHz, CDCl₃)

δ 175.19, 173.02, 155.19, 152.85, 151.32, 151.15, 141.54, 139.11, 134.59,

1

30.19, 126.98, 123.13, 107.97, 107.20, 61.44, 56.13, 52.20, 37.46, 36.05, 30.19, 29.53, 27.70, 22.46, 13.76.

Compound 7

Yellow solid, yield 24%.

ESI-MS for C H N O (m/z): [M + H] 595, [2M + H] 1189, [M-H] 593, [M + HCOO ] 639.

+

−

− −

36

54

2

5

1

H NMR (500 MHz, CDCl₃)

δ 8.18–7.90 (m, 2H), 7.73–7.53 (m, 2H), 6.88 (d, J = 10.1 Hz, 1H), 6.55

(

s, 1H), 4.74 (s, 1H), 3.93 (s, 3H), 3.89 (s, 3H), 3.62 (s, 3H), 3.19 (s, 3H), 2.47 (d, J = 7.4 Hz, 1H), 2.39–2.08

m, 5H), 1.54–1.49 (m, 2H), 1.28–1.16 (m, 26H), 0.86 (t, J = 7.0 Hz, 3H).

13

C NMR (126 MHz, CDCl₃)

30.35, 126.91, 123.12, 108.24, 107.23, 61.45, 56.12, 52.16, 37.56, 36.39, 31.95, 30.18, 29.73, 29.69, 29.54,

9.53, 29.43, 29.39, 25.64, 22.72, 14.16.

δ 175.09, 173.07, 155.23, 152.89, 151.32, 151.14, 141.55, 139.25, 134.58,

1

2

Compound 9

Yellow solid, yield 83%.

ESI-MS for C H ClN O (m/z): [M + H] 433/435, [M + Na] 455/457, [2M + H] 865, [2M +

+

22

25

2

5

+

−

Na] 887, [M-H] 431.

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¹H NMR (500 MHz, CDCl₃)

δ

8.60–8.38 (m, 1H), 7.48–7.39 (m, 2H), 7.29 (q, J = 5.2 Hz, 1H), 6.56 (d,

J = 11.4 Hz, 1H), 6.52 (s, 1H), 4.75–4.63 (m, 1H), 4.08–3.95 (m, 2H), 3.92 (s, 3H), 3.87 (s, 3H), 3.60 (s, 3H),

.07 (d, J = 5.3 Hz, 3H), 2.50–2.43 (m, 1H), 2.40–2.31 (m, 1H), 2.28–2.21 (m, 1H), 2.04–1.93 (m, 1H).

3

13

C NMR (126 MHz, CDCl₃)

δ 175.04, 166.12, 155.33, 152.97, 151.10, 150.18,141.57, 139.37, 134.41,

1

29.97, 126.80, 122.73, 108.18, 107.27, 61.45, 61.33, 56.14, 52.91, 42.63, 37.17, 30.02, 29.55.

Compound 10

Yellow solid, yield 46%.

+

ESI-MS for C H Cl N O (m/z): [M + H] 467/469, [M + Na] 489/491, [2M + H] 933/935, [2M

2

24

2

5

+

−

+

Na] 957/959, [M-H] 465/467.

1

H NMR (500 MHz, CDCl₃)

δ 9.53 (d, J = 7.2 Hz, 1H), 7.56 (s, 1H), 7.53 (d, J = 11.2 Hz, 1H), 7.34 (q,

J = 5.2 Hz, 1H), 6.64 (d, J = 11.4 Hz, 1H), 6.52 (s, 1H), 6.20 (s, 1H), 4.77–4.69 (m, 1H), 3.94 (s, 3H), 3.89 (s,

H), 3.63 (s, 3H), 3.12 (d, J = 5.4 Hz, 3H), 2.46–2.44 (m, 1H), 2.38–2.23 (m, 2H), 1.97–1.91 (m, 1H).

3

13

C NMR (126 MHz, CDCl₃)

δ 174.73, 164.19, 155.51, 153.12, 151.17, 150.51, 141.69, 139.81, 134.31,

1

30.45, 126.66, 122.71, 108.80, 107.36, 66.56, 61.52, 61.48, 56.17, 53.28, 37.17, 30.06, 29.66.

Compound 11

Yellow solid, yield 89%.

ESI-MS for C H F N O (m/z): [M + H] 481, [M + Na] 503, [2M + H] 961, [2M + Na] 983,

+

2

4

27

3

2

5

−

− −

[

M-H] 479, [M + HCOO ] 525.

1

H NMR (500 MHz, CDCl₃) δ 8.77–8.65 (m, 1H), 7.56 (s, 1H), 7.49 (d, J = 11.2 Hz, 1H), 7.35–7.28

(

3

m, 1H), 6.61 (d, J = 11.4 Hz, 1H), 6.51 (s, 1H), 4.75–4.70 (m, 1H), 3.94 (s, 3H), 3.88 (s, 3H), 3.62 (s, 3H),

.10 (d, J = 5.4 Hz, 3H), 2.56–2.46 (m, 2H), 2.45–2.27 (m, 4H), 2.25–2.20 (m, 1H), 1.90–1.84 (m, 1H).

13

C NMR (126 MHz, CDCl₃)

δ 174.99, 169.79, 155.36, 152.97, 151.33, 151.12, 141.57, 139.53, 134.54,

1

30.36, 127.98, 126.81, 122.89, 108.41, 107.25, 61.46, 61.34, 56.13, 52.52, 37.37, 30.16, 29.56, 29.33, 28.17.

Compound 12

Yellow solid, yield 89%.

+

ESI-MS for C H N O (m/z): [M + H] 461, [M + Na] 483, [2M + H] 921, [2M + Na] 943,

2

7

28

2

5

−

− −

[

M-H] 459, [M + HCOO ] 505.

1

H NMR (500 MHz, CDCl₃) δ 8.36 (d, J = 7.3 Hz, 1H), 7.81 (d, J = 7.2 Hz, 2H), 7.68 (s, 1H), 7.49

(d, J = 11.2 Hz, 1H), 7.29–7.23 (m, 2H), 7.16 (t, J = 7.7 Hz, 2H), 6.57 (d, J = 11.4 Hz, 1H), 6.53 (s, 1H),

4

2

.99–4.93 (m, 1H), 3.96 (s, 3H), 3.89 (s, 3H), 3.70 (s, 3H), 3.06 (d, J = 5.4 Hz, 3H), 2.50–2.45 (m, 1H),

.41–2.37 (m, 1H), 2.34–2.26 (m, 1H), 2.11–2.06 (m, 1H).

13

C NMR (126 MHz, CDCl₃)

δ 175.02, 167.10, 155.26, 152.91, 151.30, 151.20, 141.57, 139.18, 134.65,

133.84, 131.31, 130.17, 128.28, 127.17, 127.01, 123.23, 108.01, 107.28, 61.53, 61.49, 56.14, 52.85, 37.19, 30.30, 29.53.

Compound 14

Yellow solid, yield 57%.

+

ESI-MS for C H FN O (m/z): [M + H] 479, [M + Na] 501, [2M + H] 957, [2M + Na] 979,

2

7

27

2

5

−

− −

[

M-H] 477, [M + HCOO ] 523.

1

H NMR (500 MHz, CDCl₃)

δ 8.75 (s, 1H), 7.68 (s, 1H), 7.59 (d, J = 7.5 Hz, 1H), 7.53–7.47 (m, 2H),

7

.31–7.26 (m, 1H), 7.10–7.05 (m, 1H), 6.97–6.92 (m, 1H), 6.61 (d, J = 11.4 Hz, 1H), 6.55 (s, 1H), 4.99–4.90

(

m, 1H), 3.96 (s, 3H), 3.90 (s, 3H), 3.70 (s, 3H), 3.09 (d, J = 5.4 Hz, 3H), 2.53–2.47 (m, 1H), 2.43–2.37 (m,

1

H), 2.35–2.27 (m, 1H), 2.18–2.10 (m, 1H).

13

C NMR (126 MHz, CDCl₃)

30.36, 129.84, 126.92, 123.06, 122.53, 118.29, 118.12, 114.70, 114.52, 108.29, 107.27, 61.52, 61.50, 56.15,

3.09, 37.00, 30.31, 29.56.

δ 174.84, 165.56, 155.31, 152.98, 151.38, 151.20, 141.59, 139.42, 134.63,

1

5

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3

.3.5. Synthesis of 17

Toasolutionofcompound

3

(1.0equiv.) inDCM,N-(tert-Butoxycarbonyl)-N-[4-(dimethylazaniumylidene)-

1,4-dihydropyridin-1-ylsulfonyl]azanide (4.0 equiv.) was added and the mixture was stirred at RT. Reaction

progress was monitored by LC-MS. Then the reaction mixture was diluted with EtOAc, washed with H O,

2

1

0% citric acid, 1M K CO , brine and dried over Na SO . The residue was purified using column flash

2 3 2 4

chromatography (silica gel; EtOAc/hexanes). To the obtained compound, 4M HCl/EtOAc was added

and the reaction mixture was stirred at RT overnight. Next the solvent was removed under reduced

pressure and the residue was puriﬁed using preparative HPLC (MeCN/H O) and lyophilized from

2

dioxane to give the pure hydrochloride product 17 as a yellow solid with a yield of 21%.

+

ESI-MS for C H N O S (m/z): [M + H] 436, [M + Na] 458, [2M + H] 871, [2M + Na] 893,

2

0

25

3

6

−

[

M-H] 434.

¹H NMR (500 MHz, CDCl₃)

H), 3.94 (m, 6H), 3.56 (s, 3H), 3.11 (s, 3H), 2.59–2.46 (m, 2H), 2.56–2.49 (s, 1H), 2.21 (s, 1H).

δ

8.19 (s, 2H), 7.50–7.39 (m, 2H), 6.66 (s, 1H), 6.57 (s, 1H), 4.41–4.32 (m,

1

3

.3.6. Synthesis of 19

To a solution of compound

3 (1.0 equiv.) and Et N (3.0 equiv.) in DCM in an ice bath,

3

2

-chloroethanesulfonyl chloride (1.2 equiv.) diluted with DCM was slowly added. Next, the ice bath

was removed and the reaction mixture was stirred at RT. Reaction progress was monitored by LC-MS.

Then the reaction mixture was diluted with EtOAc, washed with 1M K CO , brine and dried over

2

3

Na SO . The residue was puriﬁed using column ﬂash chromatography (silica gel; EtOAc/MeOH) and

2

4

lyophilized from dioxane to give the pure product 19 as a yellow solid with a yield of 64%.

+

−

ESI-MS for C H N O S (m/z): [M + H] 447, [2M + H] 893, [2M + Na] 915, [M-H] 445.

22

26

2

6

1

H NMR (500 MHz, (CD ) SO)

δ

8.26 (d, J = 7.5 Hz, 1H), 8.04 (s, 1H), 7.41 (s, 1H), 7.23 (d, J = 11.2

Hz, 1H), 6.71 (s, 1H), 6.64 (d, J = 11.5 Hz, 1H), 6.48 (dd, J = 16.4, 9.9 Hz, 1H), 5.71 (dd, J = 21.1, 13.1 Hz, 2H),

.84–3.76 (m, 4H), 3.73 (s, 3H), 3.37 (s, 3H), 2.95 (s, 3H), 2.49 (s, 1H), 2.11–2.01 (m, 2H), 1.88–1.82 (m, 1H).

3

2

3

13

C NMR (126 MHz, (CD ) SO)

δ

173.10, 155.58, 153.04, 150.65, 149.28, 141.12, 139.36, 137.66,

3

2

1

34.78, 128.80, 126.48, 125.64, 123.98, 108.88, 108.07, 61.19, 60.73, 56.26, 55.99, 38.70, 29.82.

3

.3.7. Synthesis of 20

Compound 20 was obtained directly from compound 19. To 19 in DCM, morpholine was added in

excess. Reaction progress was monitored by LC-MS. Then the mixture was diluted with EtOAc, washed

with H O, brine and dried over Na SO . The residue was puriﬁed using column ﬂash chromatography

2

4

(

silica gel; DCM/MeOH) and subsequently lyophilized from dioxane to give the pure product 20 as a

yellow solid with a yield of 63%.

ESI-MS for C H N O S (m/z): [M + H] 534, [2M + H] 1067, [M-H] 532.

+

−

26

35

3

7

1

H NMR (500 MHz, CDCl₃)

δ 7.72 (s, 1H), 7.43 (d, J = 11.2 Hz, 1H), 7.40–7.38 (m, 1H), 6.84–6.50

(m, 1H), 6.56 (d, J = 11.3 Hz, 1H), 6.53 (s, 1H), 4.48–4.35 (m, 1H), 3.93 (s, 3H), 3.90 (s, 3H), 3.63 (t, J =

4

.5 Hz, 4H), 3.58 (s, 3H), 3.17–3.11 (m, 1H), 3.10 (d, J = 5.4 Hz, 3H), 3.07–2.99 (m, 1H), 2.83–2.76 (m, 1H),

2

.75–2.69 (m, 1H), 2.51–2.33 (m, 7H), 1.99–1.91 (m, 1H).

13

C NMR (126 MHz, CDCl₃)

δ 175.05, 155.45, 153.09, 150.89, 149.61, 141.54, 139.38, 134.35, 129.35,

129.06, 128.25, 126.38, 124.07, 108.00, 107.38, 61.37, 61.02, 56.09, 55.65, 53.37, 52.68, 51.08, 39.95, 30.38, 29.56.

3

.4. Single Crystal X-ray Measurement

The single crystals of colchicine derivatives (6, 11, 12, 14, 15, 16, 18 and 19) were used for data

collection on a four-circle KUMA KM4 diﬀractometer equipped with two-dimensional CCD area

detector at room (295(1) K) and low temperature (100(1) K. The graphite monochromatized Mo-K

α

◦

radiation (

λ

= 0.71073 Å) and the

ω-scan technique (∆ω = 1 ) were used for data collection. Lattice

parameters were reﬁned by the least-squares methods at all reﬂection positions. One image was

monitored as a standard after every 40 images for a control of the crystal stability. Data collection and

Molecules 2020, 25, 1789

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reduction along with absorption correction were performed using the CrysAlis software package [60].

The structures were solved by direct methods using SHELXT [61] giving positions of almost all

non-hydrogen atoms. The structures were reﬁned using SHELXL–2018 [62] with the anisotropic thermal

displacement parameters. Hydrogen atoms were reﬁned as rigid. Visualizations of the structures were

made with the Diamond 3.0 program [63]. Details of the data collection parameters, crystallographic

data and ﬁnal agreement parameters are collected in Supplementary Table S1. The structures have

been deposited with the Cambridge Crystallographic Data Center in the CIF format, no. CCDC

1980349–1980354 for 6, 11, 12, CCDC 1980341–1980344 for 14 and 15, CCDC 1980339–1980340 for 16

and CCDC 1980345–1980348 for 18 and 19 at 100 K and RT, respectively. Copies of this information can

be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax:

+

44 1223 336 033); email: deposit@ccdc.cam.ac.uk or www:http://www.ccdc.cam.ac.uk ).

3

.5. DFT Molecular Modeling

Molecular orbital calculations with full geometry optimization of colchicine derivatives (

6

,

11,

12,

1

4

,

15, 16, 18 and 19) were performed with the Gaussian09 program package [52]. All calculations were

carried out with the DFT level using the Becke3-Lee–Yang–Parr correlation functional (B3LYP) [53

–

56]

with the 6–31+G basis set assuming the geometry resulting from the X-ray diﬀraction study as the

starting structure. As convergence criteria, the threshold limits of 0.00025 and 0.0012 a.u. were

applied for the maximum force and the displacement, respectively. The three-dimensional molecular

electrostatic potential (3D MESP) maps are obtained on the basis of the DFT (B3LYP/6–31G) optimized.

-3

The calculated 3D MESP is mapped onto the total electron density isosurface (0.008 eÅ ) for each

molecule. The color code of MESP maps is in the range of −0.05 eÅ⁻(red) to +0.05 eÅ (blue).

1

−1

3

.6. In Vitro Antiproliferative Activity

3

.6.1. Cell Lines and Culturing Conditions

Four human cancer cell lines and one murine normal cell line were used to evaluate antiproliferative

activity of colchicine and its derivatives

adenocarcinoma (MCF-7), human colon adenocarcinoma cell lines sensitive and resistant to doxorubicin

LoVo) and (LoVo/DX) respectively, and normal murine embryonic ﬁbroblast cell line (BALB/3T3).

1–21: human lung adenocarcinoma (A549), human breast

(

The A549 cell line was purchased from the European Collection of Authenticated Cell Cultures (ECACC,

Salisbury, UK). The MCF-7, LoVo and LoVo/DX cell lines was purchased from American Type Culture

Collection (ATCC, Manassas, VA, USA). All the cell lines are maintained in the Institute of Immunology

and Experimental Therapy (IIET), Wroclaw, Poland.

Human lung adenocarcinoma cell line was cultured in a mixture of OptiMEM and RPMI 1640

(1:1) medium (IIET, Wroclaw, Poland), supplemented with 5% fetal bovine serum HyClone (GE

Healthcare, USA) and 2 mM L-glutamine (Sigma-Aldrich, Germany). Human breast adenocarcinoma

cell line was cultured in a mixture of Eagle medium (IIET, Wroclaw, Poland), supplemented with

10% fetal bovine serum, 2 mM l-glutamine, 8 µg/mL insulin and 1% amino acids (Sigma-Aldrich,

Germany). Human colon adenocarcinoma cell lines were cultured in a mixture of OptiMEM and

RPMI 1640 (1:1) medium (IIET, Wroclaw, Poland), supplemented with 5% fetal bovine serum HyClone

(

GE Healthcare, USA), 2 mM l-glutamine, 1 mM sodium pyruvate (Sigma-Aldrich, Germany) and

g/100 mL doxorubicin (Accord) for LoVo/DX. Murine embryonic ﬁbroblast cells were cultured in

Dulbecco medium (Gibco), supplemented with 10% fetal bovine serum (GE Healthcare, USA) and

mM L-glutamine (Sigma-Aldrich, Germany). All culture media contained antibiotics: 100 U/mL

10 µ

2

penicillin (Polfa-Tarchomin, Poland) and 0.1 mg/mL streptomycin (Sigma Aldrich, Germany). All cell

◦

lines were cultured during entire experiment in humid atmosphere at 37 C and 5% CO .

2

Molecules 2020, 25, 1789

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3

.6.2. Cell Viability Assays

Twenty-four hours before adding the tested compounds, all cell lines were seeded in 96-well

4

plates (Sarstedt, Germany) in appropriate media with 0.5

×

10 cells per well for A549 cell line, 0.75

×

4

1

0 cells per well for MCF-7 cell line and 1.0

lines. All cell lines were exposed to each tested agent at diﬀerent concentrations in the range 100–0.001

g/mL for 72 h. The cells were also exposed to the reference drug cisplatin (Teva Pharmaceuticals,

×

10 cells per well for LoVo, LoVo/DX and BALB/3T3 cell

µ

Poland) and doxorubicin (Accord Healthcare Limited, UK). Additionally, all cell lines were exposed to

DMSO (solvent used for tested compounds) (POCh, Poland) at concentrations corresponding to those

present in dilutions of tested agents. After 72 h sulforhodamine B assay (SRB) was performed [64].

SRB

After 72 h of incubation with the tested compounds, the cells were ﬁxed in situ by gently

adding of 50 µL per well of cold 50% trichloroacetic acid TCA (POCh, Poland) and were incubated

at room temperature for one hour. Then the wells were washed four times with water and air dried.

Next, 50 µL of 0.1% solution of sulforhodamine B (Sigma-Aldrich, Germany) in 1% acetic acid (POCh,

Poland) were added to each well and were incubated at room temperature for 0.5 h. After incubation

time, unbound dye was removed by washing plates four times with 1% acetic acid, whereas the

stain bound to cells was solubilized with 150

µL of 10 mM Tris base (Sigma-Aldrich, Germany).

Absorbance of each solution was read from a Synergy H4 Hybrid Multi-Mode Microplate Reader

(BioTek Instruments, USA) at the 540 nm wavelength.

Results are presented as mean IC₅₀(concentration of the tested compound that inhibits cell

proliferation by 50%)

±

standard deviation. IC₅₀values were calculated in Cheburator 0.4, Dmitry

Nevozhay software (version 1.2.0 software by Dmitry Nevozhay, 2004–2014, http://www.cheburator.

nevozhay.com , freely available for each experiment [65]. Compounds at each concentration were

tested in triplicates in a single experiment and each experiment was repeated at least three times

independently. Results are summarized in Table 2.

3

.7. Molecular Docking Studies

3

.7.1. Ligand Preparation

The ligand structures were prepared using Ligprep from the Schrödinger suite [66]. Conformations

and tautomeric states were assigned to the ligands by following the ligand preparation protocol

implemented in Schrödinger suite with default settings. LigPrep generates variants of the same ligand

with diﬀerent tautomeric, stereochemical, and ionization properties.

3

.7.2. Tubulin Model

The tubulin crystal structures available in the PDB are those for bovine protein. The bovine

tubulin structure of tubulin (PDB ID: 1SA0) [67] was used as a template to construct the homology

model of human αβ-tubulin isotypes ( I (UniProtKb: P07437), which is the most abundant isotype in

β

most tumors using the Molecular Operating Environment (MOE) software package [68]. The sequence

corresponding to the gene TUBA1A (UniProt ID: Q71U36) was chosen as a reference sequence for

human tubulin, whereas gene TUBB associated to I isoform (UniProt ID: P07437) was chosen for

human tubulin. Homology modeling was performed using MOE by setting the number of generated

models to 10 and by selecting the ﬁnal model based on MOE’s generalized Born/volume integral

(

GB/VI) scoring function. The models used in the simulations performed for the present study were

developed earlier and described in our previous publications [69]. These earlier investigations can also

be considered to be validations of the computational model employed here since they were directly

compared to the corresponding experimental data.

Molecules 2020, 25, 1789

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3

.7.3. Molecular Dynamics Simulations

The missing hydrogens for heavy atoms were added using the tLEAP module of AMBER 14 with

the AMBER14SB force ﬁeld [70]. The protonation states of all ionizable residues were determined at

pH = 7 using the MOE program [68]. Each protein model was solvated in a 12 Å box of TIP3P water.

+

−

In order to bring the salt concentration to the physiological value of 0.15 M, 93 Na ions and 57 Cl ions

were added. Minimization of the structure was carried out in two steps, using the steepest descent and

conjugate gradient methods successively. At ﬁrst, minimization was made in 2 ps on solvent atoms

only, by restraining the protein-ligand complex. Next, minimization was run without the restraint in

1

0 ps. After minimization, the molecular dynamics (MD) simulations were carried out in three steps:

heating, density equilibration, and production. At ﬁrst, each solvated system was heated to 298 K for

5

0 ps, with weak restraints on all backbone atoms. Next, density equilibration was carried out for

0 ps of constant pressure equilibration at 298 K, with weak restraints. Finally, MD production runs

were performed for all systems for 70 ns. The root-mean-square deviation (RMSD) of both the entire

tubulin structure and the colchicine binding site were found to reach a plateau after 40 ns. Clustering

analysis of the last 30 ns of the generated MD trajectory was carried out using the Amber’s CPPTRAJ

program [71] to identify representative conformations of the tubulin dimer. Clustering was made via

the hierarchical agglomerative approach using the RMSD of atoms in the colchicine binding site as a

metric. An RMSD cutoﬀ of 1.0 Å was set to diﬀerentiate the clusters. On the basis of the clustering

analysis, three representative structures of the tubulin dimer were found. The docking was performed

on all the three representative structures and the one with the highest docking score was selected,

which was the largest cluster (about 70% of the simulation) conformation of the tubulin structure.

During the modeling, the cofactors including GTP, GDP, colchicine, and the magnesium ion located at

the interface between

reﬁnement step.

α–and β–monomers were kept as part of the environment and included in the

3

.7.4. Docking Simulations

We used the AutoDock Vina [72] program to predict the binding pose of the ligands under ﬂexible

ligand and rigid receptor conditions. Dockbox package was used to facilitate preparing docking inputs

and post-processing of the docking results [73]. Docking simulations performed with a cubic box (size

30.0 Å) were centered at the center of binding pockets and the docking was run separately on tubulin

structure. Every generated pose was energy-minimized using Amber14 by keeping the protein ﬁxed

and was re-scored using the MOE’s GBVI/WSA dG scoring function [68]. No constraints were applied

in the docking studies. For each compound/protein-structure pair, the pose with the best score was

identiﬁed and used as an initial conﬁguration for molecular mechanics Gibbs–Boltzmann surface area

MM/GBSA computations.

3

.7.5. Binding Energy and Pairwise Per-Residue Free Energy Decomposition Calculations Using

MM/GBSA Method

The MM/GBSA technique is used to calculate the free energy associated with the binding of double

modiﬁed colchicine amides and sulfonamides [74]. This method combines molecular mechanics with

continuum solvation models. We performed MM-GBSA integrated in Amber. The binding free energy

is estimated as:

h

i

∆

G_b_i_n_d= G_c_o_m_p_l_e_x− G

+ G_l_i_g_a_n_d

(1)

protein

where G is the average free energy of the complex, protein, and ligand, are calculated according to the equation:

G = E_M₊G_s_o_l_v_a_t_i_o_n− TS

(2)

Molecules 2020, 25, 1789

26 of 31

where EMM are determined with the SANDER program and represent the internal energy (bond,

angle, and dihedral), van der Waals and electrostatic interactions (see Equation (3)). TS is the entropy

contribution estimated using normal mode (nmode) analysis.

E_M= E_i_n_t+ E_e_l_e_c+ E_v_d_W

(3)

The solvation free energy can be calculated as the sum of polar and nonpolar contributions.

The polar parts are obtained by using the generalized-born (GB) model—resulting in the MM/GBSA

method, whereas the nonpolar terms are estimated from a linear relation (Equation (4)) to the solvent

accessible surface area (SASA).

G

= γ SASA + b

(4)

non−polar

In the present study, a 2 ns-duration MD trajectory was run in TIP3P water using Amber14,

for every top pose generated at the end of the docking step. It is worth noting that to assess the

performance of MM/GBSA methodology [75], we evaluated the prediction accuracy of this method

by various simulation protocols including 1 ns MD production calculations using PDBbind data set.

Too long an MD simulation could be prejudicious for the overall success of the MM/GBSA method.

According to this study and the common practice to calculate binding energies using MM/GBSA,

we have decided to run MD production simulation for 2 ns. The MM/GBSA calculations were

performed on a subset of 200 frames collected at regular time intervals from the trajectory. For PB

calculations, an ionic strength of 0.0 nM (istrng = 0.0) and a solvent probe radius of 1.6 Å (prbrad =

1

.6) were used. For GB calculations, the igb parameter was set to 5 that corresponds to a modiﬁed GB

model equivalent to model II in reference. Pairwise free energy decomposition analysis was performed

using the MM/GBSA decomposition process by the MM/GBSA program in AMBER 14 to compute

the interaction between the inhibitors and each residue. For each of the tested compounds, the active

residues estimated from MM/GBSA decomposition process and the best GB score out of the trajectories

associated with the representative structures of the tubulin dimer were collected and are reported in

Table 3 and Figure 4.

3

.8. Calculation clogP

We used the Molinspiration online database (http://www.molinspiration.com , free of charge) to

predict the clogP values for all synthesized compounds and collected them in Table 3 [76].

4

. Conclusions

In an attempt to discover novel potent inhibitors of microtubule dynamics, eighteen novel double

modiﬁed colchicine analogs comprising methylamine substituent at position C10 of ring C and amide

or sulfonamide substituents at position C7 of ring B were successfully synthesized, puriﬁed and their

structures were conﬁrmed by spectroscopic analyses as well as X-ray measurements. Four human

cancer cell lines (A549, MCF-7, LoVo, LoVo/DX) were used to evaluate the anticancer potency of all

synthesized compounds.

Compared with unmodiﬁed colchicine, the majority of its studied derivatives exhibited excellent

potency against A549, MCF-7, and LoVo cell lines. The antiproliferative activity of colchicine derivatives

was in the nanomolar range and they also were characterized by lower IC₅₀values also than doxorubicin

and cisplatin, commonly used as antitumor agents in cancer chemotherapy. The preliminary SAR

revealed that the type of substituent at position C7 in ring B and the presence of –NHCH group at

3

position C10 in ring C of colchicine 1 were of cardinal importance to the compounds’ cytotoxicity.

The calculated values of the selectivity index (SI) clearly show that it is possible to design more

selective compounds than colchicine 1. The most active double-modiﬁed colchicine derivatives were

the compounds that included chloroacetamide and dichloroacetamide (

13) moiety (Table 2). However, in vitro tests showed that they did not have high SI values (except

towards the LoVo cells) and in vivo testing should be carried out to determine their therapeutic potential.

9, 10) and 2–chlorobenzamide

(

Molecules 2020, 25, 1789

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But of all tested compounds, 4,4,4-triﬂuorobutyric amide of 10-N-methylaminocolchicine 11 was the

most prominent. Compound 11 showed high activity towards A549, MCF7 and LoVo cell lines,

with antiproliferative IC₅₀values ranging between 10.4 and 14.6 nM as well as SI values higher than 5

for these three cell lines. The most selective towards MCF-7 cell line was compound 17 (SI was almost

10) and towards LoVo cell line the most selective were compounds 7, 9, 10, and 13 (SI were over 6).

So we conclude that the appropriate modiﬁcation of colchicine molecule and synthesis of its analogs

might overcome the toxicity, which is a major challenge in designing a potential colchicine-based

drug candidate.

Compound 11 was subsequently identiﬁed as the that with potentially the best therapeutic index

and therefore the most promising candidate for further development, as it showed strong in vitro

anticancer activities with high SI against three human cancer cell lines. However, further in vivo

studies should be conducted for the successful development of this compound.

Molecular docking study showed the ability of the obtained colchicine derivatives to bind into the

active sites of

βI-tubulin isotype with the well-deﬁned binding modes. It is worthy to mention that

10-N-methylaminocolchicine based compounds serve as useful templates for the further development

of the anticancer and antimitotic agents. Further evaluation should help to ﬁnd more detailed

structure-activity relationships of microtubule-targeting drugs and CBS inhibitors, which can help in

rational drug design in future.

Supplementary Materials: The following are available online, Table S1: Details of the data collection parameters,

crystallographic data and the ﬁnal agreement parameters. (for compounds 6, 11, 12, 14, 15, 16, 18 and 19);

Table S2: Optimized parameters for colchicine derivatives (

6

,

11

,

12

,

14

,

15

,

16

,

18 and 19), Figure S1: The LC-MS

1

13

chromatogram and mass spectra of

2; Figure S2: The H NMR spectrum of

2

in CDCl ; Figure S3: The C NMR

3

1

spectrum of

and mass spectra of

2

3

in CDCl ; Figure S4: The LC-MS chromatogram and mass spectra of

; Figure S5: The H NMR

3

13

in CDCl ; Figure S6: The C NMR spectrum of

3

in CDCl ; Figure S7: The LC-MS chromatogram

3

1

13

4

; Figure S8: The H NMR spectrum of

4

in CDCl , Figure S9: The C NMR spectrum of

4

in

5

3

1

CDCl , Figure S10: The LC-MS chromatogram and mass spectra of

5; Figure S11: The H NMR spectrum of

3

1

3

in CDCl , Figure S12: The C NMR spectrum of

spectra of

5

in CDCl ; Figure S13: The LC-MS chromatogram and mass

3

1

13

6

; Figure S14: The H NMR spectrum of

6

in CDCl ; Figure S15: The C NMR spectrum of

6

7

in CDCl₃;

3

7

1

Figure S16: The LC-MS chromatogram and mass spectra of

; Figure S17: The H NMR spectrum of

1

3

Figure S18: The C NMR spectrum of

7

in CDCl ; Figure S19: The LC-MS chromatogram and mass spectra of

8;

3

1

13

Figure S20: The H NMR spectrum of

8

in CDCl ; Figure S21:. The C NMR spectrum of

8

in CDCl ; Figure S22:

3

1

The LC-MS chromatogram and mass spectra of

9

; Figure S23: The H NMR spectrum of

9

in CDCl ; Figure S24:

3

1

3

The C NMR spectrum of

9

in CDCl ; Figure S25: The LC-MS chromatogram and mass spectra of 10; Figure S26:

3

1

13

The H NMR spectrum of 10 in CDCl ; Figure S27: The C NMR spectrum of 10 in CDCl ; Figure S28. The LC-MS

3

1

13

chromatogram and mass spectra of 11; Figure S29. The H NMR spectrum of 11 in CDCl ; Figure S30: The

C

3

NMR spectrum of 11 in CDCl ; Figure S31. The LC-MS chromatogram and mass spectra of 12; Figure S32: The

3

1

13

H NMR spectrum of 12 in CDCl ; Figure S33: The C NMR spectrum of 12 in CDCl ; Figure S34: The LC-MS

3

1

13

chromatogram and mass spectra of 13; Figure S35: The H NMR spectrum of 13 in CD Cl ; Figure S36: The

C

2

NMR spectrum of 13 in CD Cl . Figure S37: The LC-MS chromatogram and mass spectra of 14; Figure S38: The

2

1

13

H NMR spectrum of 14 in CDCl₃_;Figure S39: The C NMR spectrum of 14 in CDCl ; Figure S40: The LC-MS

3

1

13

chromatogram and mass spectra of 15; Figure S41: The H NMR spectrum of 15 in CDCl ; Figure S42: The

C

3

NMR spectrum of 15 in CDCl₃_;Figure S43: The LC-MS chromatogram and mass spectra of 16; Figure S44: The

1

13

H NMR spectrum of 16 in CDCl . Figure S45: The C NMR spectrum of 16 in CDCl ; Figure S46: The LC-MS

3

1

chromatogram and mass spectra of 17; Figure S47: The H NMR spectrum of 17 in CDCl ; Figure S48: The LC-MS

chromatogram and mass spectra of 18;Figure S49: The H NMR spectrum of 18 in CDCl ; Figure S50: The

3

1

13

1

C

3

NMR spectrum of 18 in CDCl ; Figure S51: The LC-MS chromatogram and mass spectra of 19; Figure S52: The H

3

1

3

NMR spectrum of 19 in (CD ) SO; Figure S53: The C NMR spectrum of 19 in (CD ) SO; Figure S54: The LC-MS

3

2

3 2

1

13

chromatogram and mass spectra of 20; Figure S55

NMR spectrum of 20 in CDCl ; Figure S57: The LC-MS chromatogram and mass spectra of 21; Figure S58: The

H NMR spectrum of 21 in CDCl . Figure S59: The C NMR spectrum of 21 in CDCl ; Figure S60: Molecular

structure of colchicine derivatives (6, 11, 12, 14, 15, 16, 18 and 19) at 295 K and 100 K.

:

The H NMR spectrum of 20 in CDCl . Figure S56: The

C

3

1

13

3

Author Contributions: Conceptualization, J.K., and A.H.; methodology, J.K., A.H., J.J., J.W. and J.A.T.; software,

J.J.; M.A.; validation, A.H., J.A.T. and J.J.; formal analysis, A.H. and J.K.; investigation, J.K., E.M., J.J. and M.A;

resources, A.H.; J.K. and W.M.; data curation, J.K., M.A. and E.M.; writing—original draft preparation, J.K., J.J.

and M.A.; writing—review and editing, W.M., A.H. J.W. and J.A.T.; visualization, J.K., J.J. and M.A.; supervision,

Molecules 2020, 25, 1789

28 of 31

A.H.; project administration, A.H.; funding acquisition, A.H. and W.M. All authors have read and agreed to the

published version of the manuscript.

Acknowledgments: Financial support by a grant No. 2016/21/B/ST5/00111 of the Polish National Science Centre

(

NCN) –is gratefully acknowledged. JAT gratefully acknowledges research support from NSERC and the Allard

Foundation for his research.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Synthesis, antiproliferative activity and molecular docking studies of novel doubly modified colchicine amides and sulfonamides as anticancer agents

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