Synthesis, antiproliferative activity and molecular docking of Colchicine derivatives

Article abstract of DOI:10.1016/j.bioorg.2016.01.002

In order to create more potent anticancer agents, a series of five structurally different derivatives of Colchicine have been synthesised. These compounds were characterised spectroscopically and structurally and their antiproliferative activity against f

Full text of DOI:10.1016/j.bioorg.2016.01.002

Accepted Manuscript
Synthesis, antiproliferative activity and molecular docking of Colchicine deriv-
atives
Adam Huczyński, Urszula Majcher, Ewa Maj, Joanna Wietrzyk, Jan Janczak,
Mahshad Moshari, Jack A. Tuszynski, Franz Bartl
PII:
S0045-2068(16)30002-5
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.01.002
YBIOO 1870
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 November 2015
7 January 2016
8 January 2016
Please cite this article as: A. Huczyński, U. Majcher, E. Maj, J. Wietrzyk, J. Janczak, M. Moshari, J.A. Tuszynski,
F. Bartl, Synthesis, antiproliferative activity and molecular docking of Colchicine derivatives, Bioorganic
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.01.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Bioorganic Chemistry- Regular Articles
Synthesis, antiproliferative activity and molecular docking of Colchicine
derivatives
Adam Huczyński a,* Urszula Majcher a, Ewa Maj b, Joanna Wietrzyk b, Jan Janczak c,
Mahshad Moshari d, Jack A. Tuszynski d, Franz Bartl e
a
Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznań,
Poland
b Ludwik Hierszfeld Institute of Immunology and Experimental Therapy, Polish Academy of
Sciences, Rudolfa Weigla 12, 53-114 Wrocław, Poland
c Institute of Low Temperature and Structure Research, Polish Academy of Sciences, PO Box
1410, 50-950 Wrocław, Poland
d Department of Oncology, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada
e
Institut für Medizinische Physik und Biophysik Charité – Universitätsmedizin Berlin,
Campus Charité Mitte, Charitéplatz 1, 10117 Berlin, Germany
Abstract
In order to create more potent anticancer agents, a series of five structurally different
derivatives of Colchicine have been synthesised. These compounds were characterised
spectroscopically and structurally and their antiproliferative activity against four human
tumour cell lines (HL-60, HL-60/vinc, LoVo, LoVo/DX) was evaluated. Additionally the
activity of the studied compounds was calculated using computational methods involving
molecular docking of the Colchicine derivatives to β-tubulin. The experimental and
computational results are in very good agreement indicating that the antimitotic activity of
Colchicine derivatives can be readily predicted using computational modeling methods.
Corresponding author: adhucz@amu.edu.pl, tel. +48 618291297, fax. +48 618291555 (A.
Huczyński)
Key words: Antimitotic agent; Tubulin; Colchicine derivatives; Crystal structure;
Cytotoxicity; Docking
1

1. Introduction
Colchicine (1), the major alkaloid isolated from Colchicum autumnale and Gloriosa
superba is a well-known antimitotic agent, which has been shown to exhibit very high
cytotoxic effects in vivo.1-2 Microtubules, formed by polymerization of tubulin hetero-dimers,
are essential components of the cytoskeleton in eukaryotic cells and are involved in many
important cellular processes, including mitosis.3-4 Colchicine binds at a unique binding site
close to the interface between α- and β-tubulin but makes contact solely with β-tubulin.
Colchicine binding results in the formation of a tubulin–Colchicine complex that prevents the
formation of microtubules due to induced conformational change in the tubulin dimer making
it incompetent for microtubule assembly. As a result the cells exposed to Colchicine exhibit
apparent mitotic arrest during the cell cycle.5-7 Colchicine has been shown to have very high
anti-cancer (specifically anti-mitotic) activity in vitro but its use as an anticancer drug have
not led to clinical applications because of its relatively high toxicity, although Colchicine is
currently used in therapy8,9, i.e. for gout3,10,11, acute pericarditis12,13 and Familial
Mediterranean fever (FMF).14,15 Colchicine remains a molecule of great research interest
since Colchicine site inhibitors (CSI) as a class have been most widely explored compared to
the other binding sites of tubulin.16,17 Colchicine is a compound whose structure is still being
optimized, by designing new analogues Some derivatives of 1, such as thiocolchicoside
(Neoflax™, Muscoril™) show improved therapeutic properties and clinical significance as
anti-inflammatory, analgesic and anti-cancer drugs.18,19
Therefore, currently much interest has focused on structural modifications of 1 in the
hope of improving its anti-cancer activity. Numerous studies have shown previously that
changing of the -OCH3 group at the C(10) position of C-ring into an amine, as well as
substitution of the acyl group at C(7) position of B-ring into several new derivatives may
2

reduce the toxicity of 1 toward normal cell; thus, this kind of modifications may lead to
improved anti-cancer agents.20-27
The main purpose of our study was to design and synthesize (Scheme 1) a few
derivatives of Colchicine showing considerable activity against human cancer cell lines.
17
23
22
4
O
O
O
5
O
O
21
6
21
O
15
18
3
24
NH
O
NH
2
O
NH2
O
NH
16
13
7
14
25
1
O
19
O
O
(d)
(c)
8
12
11
10
9
20
H3CO
O
O
O
H3CO
(3)
H3CO
(4)
(2)
(b)
(a)
17
O
O
O
20
O
O
O
20
21
21
21
22
18
NH
O
A
O
NH
7
O
NH
B
(f)
(e)
1
O
O
O
19
C
10
O
O
HN
O
H2N
22
H3CO
20
HN
O
(5)
(6)
23
colchicine (1)
Scheme 1. Synthesis of Colchicine derivatives (2-6). Reagents and conditions: (a) Boc2O, DMAP, triethylamine,
CH3CN, reflux; (b) MeONa, MeOH, RT; (c). trifluoroacetic acid, DCM, reflux; (d) phenyl isocyanate,
tetrahydrofuran, reflux; (e) NH3(aq), C2H5OH, reflux; (f) phenyl isocyanate, acetone, reflux.
Below, we report the synthesis, crystallographic and spectroscopic analysis of a series
of structurally different derivatives of 1 obtained by the modification at C(10) and C(7)
positions of 1 as well as evaluation of these novel compounds as cytotoxic, tubulin-targeting
agents. The antiproliferative effect of five Colchicine derivatives (2-6) was tested in vitro
using four cancer cell lines, i.e.: human promyelocytic leukemia (HL-60) and its vincristine-
resistant subline (HL-60/vinc), human colon adenocarcinoma cell line (LoVo) and
doxorubicin resistant subline (LoVo/DX), and one normal murine embryonic fibroblast cell
line (BALB/3T3). To better understand the interactions between the Colchicine derivatives
and tubulin, we investigated potential binding modes of all studied compounds (1-6) docked
into the Colchicine binding site (CBS) of βІ tubulin using Autodock4 program under flexible
3

ligand and rigid receptor condition. A detailed discussion regarding the differences between
the structures of the synthesized compounds and their ability to form complexes with
Colchicine binding site (CBS) is provided herein.
2. Results and Discussion
2.1. Chemistry
The synthesis routes to Colchicine derivatives 2-6 are outlined in Scheme 1.
Compounds 2, 3 and 5 were prepared according to the methods developed earlier.26,27
Compounds 4 and 6, containing N-phenyl urea substituent, were synthesized and
characterized for the first time.
According to Bagnato’s method26 N-deacetylColchicine (3) was synthesized in three
steps as follows: Colchicine (1) reacted with Boc-anhydride (Boc2O) using 4-
dimethylaminopyridine (DMAP) forming N-Boc-Colchicine (not isolated), followed by
selective methanolysis of the acetate group giving N-Boc-deacetylColchicine (2) and the
subsequent reaction with trifluoroacetic acid (TFA) yielded the desired N-deacetylColchicine
(3). Isolated compound 2 was crystallized from acetonitrile producing crystals, which are
analyzed by X-ray diffraction. A simple reaction between 3 and phenyl isocyanate in
tetrahydrofuran at room temperature gives compound 4 with a 68 % yield. Compound 5 was
readily available from 1 by treatment with excess of aqueous ammonia solution in ethanol and
heat under reflux for 12 hours27. The reaction proceeds quantitatively. Compound 6 was
obtained from 5 by a reaction with phenyl isocyanate in acetone at room temperature and then
after chromatographic purification and crystallization from acetonitrile (final yield 55%). The
crystals are suitable for X-ray analysis whose results are presented and discussed below. The
1
13
structures of all products 2-6 were determined using the EI MS, ESI-MS, FT-IR, H and C
4

NMR methods and are shown in Supplementary data (Fig. S1-Fig. S18) and discussed below.
All the spectroscopic and mass spectrometry data presented in Supplementary data
confirm the structure of the studied compounds. Additionally, the structures of compounds 2
and 6 are also determined strictly from X-ray analysis presented also in Supplementary data
(Tables S1-S3, Fig. S19 and S20).
The 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.28-32
Therefore, we have characterized two Colchicine derivatives 2 and 6 that produce appropriate
crystals suitable for the single crystal X-ray analysis. The single crystals of 2 and 6 were
grown by crystallization in acetonitrile solution (Table S1, Fig. S19).
Compound 2 crystallizes in the C2 space group of the monoclinic system while
compound 6 in the P212121 space group of the orthorhombic system. Both space groups are
chiral since the compounds contain an asymmetric carbon (C7) atom. The planar phenyl A
and tropolone C rings in both Colchicine derivatives (2 and 6) are twisted around the
C13C16 bond with the torsion angle describing the twisting conformation C1—C16—
C13—C12 of 57.17(23) and 52.06(26)o in 2 and 6, respectively. The ring B in both
compounds exhibits a similar puckering pattern and the extent of its non-planarity and adopts
a conformation, which is close to the twist-boat with a flattening caused by fusion of rings A
and C (Fig. S19).
5

Fig. 1. Comparison of the X-ray structure of 2 (capped sticks) and 6 (wireframe) showing the conformation of
the Colchicine skeleton.
The extension of the whole molecule 6 is about 50% larger than molecule 2 due to the
greater N-phenylurea substituent (NHC(O)NH(C6H5)) in relation to the methoxy group
(OCH3) in 2 linked to the tropolone ring C at the carbon (C10) atom (Fig. 1). The role of the
trimethoxyphenyl ring A of Colchicine as well as the role of the tropolonic ring C towards
tubulin binding has been studied in great details. The tropolonoic ring C of Colchicine
skeleton is found to be crucial for interaction with tubulin.44-45 The molecular electrostatic
potential map is a powerful tool for analyzing the interactions, and was therefore calculated
for both, Colchicine 2 and 6 derivatives. Additionally the gas-phase structures of molecules 2
and 6 using the DFT optimization were were performed with the Gaussian09 program
package.46 All calculations were carried out by the DFT method using the Becke3-Lee-Yang-
Parr correlation functional (B3LYP)47-49 with the 6-31+G basis set, starting from the X-ray
geometry of molecules. The gas-phase optimized conformations of 2 and 6 molecules are, in
general, in good agreement with those obtained from the X-ray single crystal investigation

(Table S2 and Fig. S21). The region of tubulin that interacts with Colchicine is near the α-β
interface tubulin/dimer. Therefore, to better understand the interaction of the Colchicine
derivatives 2 and 6, molecular electrostatic potential for both Colchicine derivatives and, for
comparison, for Colchicine itself was calculated.51-61 Therefore, the three-dimensional MESP
maps for both Colchicine derivatives 2 and 6 were calculated on the basis of the DFT
(B3LYP) optimized geometries of molecules and mapped onto the total electron density
isosurface (0.008 eÅ-3) for both molecules using the GaussView 5.0 program. (Fig. 2).The
color coding of MESP is in the range of –0.05 (red) to 0.05 eÅ-1 (blue). For both molecules,
regions of negative MESP are usually associated with the lone pair of electronegative atoms
(O and N), whereas the regions of positive MESP are associated with the electropositive
atoms (Fig. 2a and 2b). The nucleophilic regions in molecules 2 and 6 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,
which is clearly evidenced in 6 (Fig. 2b). The planar conformation of the 10-N-phenylurea
substituent that is coplanar with the 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 derivative
6 (Fig. 2b). The molecular electrostatic potential for Colchicine itself was also calculated for
comparison, (Fig. 2c).
The MESP of Colchicine derivative 2 exhibits similar nucleophilic and electrophilic regions
as that of Colchicine itself (see Fig. 2a and c), since in 2 ring B is modified (the NHC(O)CH3
substituent was replaced by NHCOOC(CH)3). Changing the substituent at the C(10) position
of the tropolone C ring from methoxy to N-phenylurea, as in Colchicine derivative 6, leads to
modification of the MESP when compared to that of Colchicine itself (Fig. 2b and Fig 2c).
7

(a)
(b)
(c)
-0.05 eÅ-1
+0.05 eÅ-1
-0. 05eÅ-1
+0.05eÅ-1
Fig. 2. Three-dimensional molecular electrostatic potential (right) (-0.05eÅ-1, red and +0.05 eÅ-1, blue) mapped
on the surface of total electron density (0.008eÅ-3) for the N-Boc-deacetylColchicine (a),10-N-phenylurea-10-
demethoxyColchicine (b) and for Colchicine (c).
8

3. Biological activity
3.1. In vitro determination of drug-induced inhibition of human cancer cell line growth
Colchicine (1) and its derivatives 2-6 synthesized were evaluated for their
antiproliferative activity against four cancer cell lines. Each compound was tested on two
human cancer cell lines displaying various levels of drug resistance, namely human
promyelocytic leukemia (HL-60) and its vincristine-resistant subline (HL-60/vinc), and
human colon adenocarcinoma cell line (LoVo), and doxorubicin resistant subline (LoVo/DX).
To illustrate the agents’ activity against the cells with MDR (multidrug resistance) phenotype
the Resistance Index (RI) was calculated (Table 1). The RI value indicates the enhancement
of the subline resistance in comparison to its parental cell line.
The antiproliferative effect was also studied on normal murine embryonic fibroblast cell line
(BALB/3T3) for better description of cytotoxic activity of the compounds studied. The mean
IC50 SD of the tested compounds are collected in Table 1.
The results shown in Table 1 indicate that almost all studied compounds (1-6) had
higher cytotoxicity against the HL-60 cancer cell line (0.007 <IC50<24.1 µM) than against
normal cells BALB/3T3 (0.18 <IC50 <156 µM).
These high differences between the cytotoxicity against cancer and normal cells means
that cancer cells will be killed preferentially before normal ones. The values of the calculated
Selectivity Index (SI) especially for compounds 1, 4 and 5 (22.5 <SI < 44.2) are higher than
that observed for typical anticancer drugs like cisplatin and doxorubicin (SI = 4.2 and 5.0,
respectively). However, the introduction of N-phenylurea moiety at C(10) position in
compound 6 generally attenuated the antiproliferative activity against cancer and normal cells
9

as well. Although the synthesized compounds are effective towards HL-60 cells, their
potential is limited against cells with developed drug resistance.
10

Table 1. Antiproliferative activity (IC50) of Colchicine (1) and its derivatives (2-6) compared with antiproliferative activity of standard
anticancer drugs doxorubicin and cisplatin and the calculated values of the resistance index (RI) and selectivity (SI) of tested compounds.
HL-60
IC50 [µM]
0.0073 0.0002 24.14
5
HL-60vinc
LoVo
IC50 [µM]
0.078 0.025
LoVoDX
BALB 3T3
Compound
SI
IC50 [µM]
SI
0.15
RI1
SI
2.26
IC50 [µM]
SI
0.27
RI2
IC50 [µM]
0.18 0.025
1.18 0.23
162
0.65 0.05
8.4
1
0.033 0.013
0.050 0.011
0.040 0.017
0.010 0.0039
24.07 6.81
5.33
3.33
44.21
22.50
6.48
5.00
4.18
0.59 0.15
1.06 0.28
6.85 1.49
3.75 2.13
uv
0.30
0.16
0.26
0.063
—
18
21
0.068 0.015
0.076 0.022
0.59 0.26
2.58
2.22
3.00
0.625
2.48
1.8
0.11 0.022
0.53 0.084
4.28 0.63
3.49 0.91
uv
1.6
0.32
0.41
0.067
—
1.6
7.0
7.3
9.3
—
0.175 0.022
0.168 0.028
1.76 0.44
2
3
4
5
6
171
360
—
0.37 0.20
0.23 0.026
155.85 23.06
0.166 0.074
3.90 1.50
62.91 12.13
0.092 0.018
4.37 0.73
0.033 0.0092
0.93 0.27
0.68 0.18
9.27 1.47
0.24
0.42
21
4.75 0.99
5.70 0.63
0.035
0.68
51.6
1.3
doxorubicin
cisplatin
10
0.89
The IC50 value is defined as the concentration of a compound that corresponds to a 50% growth inhibition. Human promyelocytic leukemia (HL-60) and its vincristine-
resistant subline (HL-60/vinc);human colon adenocarcinoma cell line (LoVo) and doxorubicin resistant subline (LoVo/DX); normal murine embryonic fibroblast cell line
(Balb 3T3). Data are expressed as the mean SD;
uv - unavailable in range of concentrations used, IC50 could not be calculated due to weak antiproliferative activity of compound across the tested concentrations.
The RI (Resistance Index) indicates how many times a resistant subline is chemoresistant relative to its parental cell line. RI were calculated for each compounds using
formula: RI1 = IC50 for HL-60 / IC50 for HL-60vinc cell line and RI2 = IC50 for LoVoDX / IC50 for LoVo cell line.
When RI is 0-2 the cells are sensitive to tested compound; RI of the range 2-10 means that the cell shows moderate sensitivity to a drug; RI above 10 indicates strong drug-
resistance.
The SI (Selectivity Index) was calculated for each compounds using formula: SI = IC50 for normal cell line (BALB/3T3) / IC50 for respective cancerous cell line. A beneficial
SI > 1.0 indicates a drug with efficacy against tumour cells greater than toxicity against normal cells.
11

The data presented in Table 1 show that most of the studied compounds including
unmodified Colchicine and two reference anticancer drugs less effectively inhibit the
proliferation of the resistant HL-60/vinc cell line. The calculated values of RI clearly
confirmed that these compounds (RI ranges from 10 to over 360) are not able to overcome the
drug resistance of HL-60/vinc cell line.
On the other hand, compounds 2 and 3 exhibit very high antiproliferative activity (IC50
= 0.11 µM and 0.51 µM, respectively) and compounds 4-5 show good antiproliferative
activity (IC50 = 4.3 µM and 3.5 µM, respectively) against the human colon adenocarcinoma
cell line (LoVo). It is worth noting that compounds 2 and 3 are more active than the
commonly used anticancer drug cisplatin and revealed similar activity to doxorubicin and
unmodified Colchicine (1). In case of the doxorubicin resistant subline (LoVo/DX), all
compounds showed good to moderate activity except for compound 6 which was inactive.
The resistance indexes calculated for (LoVo/DX) line are lower than 10 for all Colchicine
derivatives analyzed here 2-6.
3.2. Molecular docking
Microtubules are the primary target for many anti-cancer drugs including Colchicine
and its derivatives, the majority of which bind specifically to β-tubulin. On the basis of the
previous findings and as an attempt to discover a molecular mechanism of action of the
cytotoxic activity for the synthesized compounds, Colchicine (1) and all the newly
synthesized derivatives were docked to the Colchicine-binding site in βІ tubulin. In order to
use only the best structures of the derivatives, single point energies were calculated at the
RHF/cc-pVDZ level of theory and then, the structures were fully optimized on the RHF/RM1
level of theory in GAMESS-US version 2010-10-01.
12

2D structures of compounds 1-6 shown in Scheme 1 were docked into the Colchicine
binding site of βІ tubulin using Autodock4 program under flexible ligand and rigid receptor
conditions. βІ Tubulin sequence data (TBB5_HUMAN) were obtained from UniProt ID.
Moreover, a homology model was constructed for βІ tubulin on the basis of the tubulin
structure in RCSB Protein Data Bank (1SA0.pdb) by MOE2013.0802. This structure includes
Colchicine bound to tubulin and reflects the conformation of the dimer in the presence of this
ligand.
The binding energies were determined for the binding location of Colchicine and its
derivatives with βІ tubulin. Moreover, the probability of existence of ligands in a specific
binding location was assumed to correspond to the number of conformations at this location.
To estimate the expectation value of the binding energy for the ensemble of most probable
conformations, a summation of the respective binding energies multiplied by the frequency
(probability) of each conformation for all ligands was evaluated and is shown in Table 2 and
denoted as M2. The M2 parameter is defined as M2 = Σ BEiρ where index i enumerates the
conformations found. Fig.3 presents the interactions between the six compounds and tubulin
in the vicinity of the binding site. Only the most dominant conformations are shown. It is
important to note that while an overall composition of the interacting residues is similar
between the six compounds, subtle but important differences occur.
Table 2. Binding energies for highest cluster size, the highest cluster size probability and M2
for Colchicine (1) and its five derivatives (2-6).
Compound
Bonding
energy(Kcal/mol)
-6.45
Highest Cluster size
M2
93%
74%
-6.4641
-6.2261
1
2
-6.52
-6.23
-7.5
85%
37%
77%
34%
-6.2255
-6.8552
-6.312
-8.43
3
4
5
6
-6.17
-8.5
13

1
Cluster size =93%
2
Cluster size =74%
3
Cluster size =85%

4
Cluster size =37%
5
Cluster size =77%
6
cluster size =34%
Fig. 3. Interaction diagrams for the Colchicine derivatives and the parent compound with tubulin
residues in the vicinity of the binding site. Only the most dominant poses of the compounds are
shown.

Colchicine (1) makes the most important interactions with Leu 245 (backbone donor)
and Glu 245 (backbine donor), compound 2 with Cys 239 (sidechain donor) and Leu 246
(backbone donor), compound 3 with Cys 239 (sidechain donor) compound 4 with Gln 245
(backbone donor) and Leu 246 (backbone donor), compound 5 with Leu 246 (backbone
donor) and compound 6 with Ala 314 (arene H) and Asn 256 (sidechain acceptor). Fig. 4
illustrates the 3D orientation of the compounds in the Colchicine binding site on the surface
of β-tubulin. It is well known that the proliferation rate of cancer cells is in most cases
higher than that of non-cancer ones. The in vitro determined antiproliferative activity of 1 and
compounds 2-5 demonstrates that these compounds more effectively inhibit proliferation of
HL-60 and LoVo cancer cells than its vincristine-resistant subline (HL-60/vinc) and
doxorubicin resistant subline (LoVo/DX), respectively. Therefore, we observed better
correlation between experimental antiproliferative activity, defined as logIC50, and the
calculated M2 parameters for the drug sensitive cancer lines (HL-60 and LoVo), and also the
normal cell line (BALB/3T3) than the drug resistant cell lines (HL-60/vinc and LoVo/DX).
Fig. 4. Colchicine and its derivatives docked to the binding site in tubulin.
16

Analysis of a comparison between logIC50 and the calculated M2 values presented in Fig. S22
(Supplementary data) indicates that the data for HL-60, LoVo and BALB/3T3 fit a
statistical model of linear regression with a high correlation coefficient (R2 ranging from 0.85
to 0.97). For the drug resistant cell lines (HL-60/vinc and LoVo/DX) the R2 coefficient was
found as 0.52 and 0.36, respectively, indicating a weak correlation between the theoretically
calculated M2 and logIC50. This can be readily explained by the upregulated expression in
these cell lines of efflux transporters like P-glycoprotein, playing an important role in drug
transport in many organs and determining the drug resistance in cancer. Because this type of
resistance is one of the mechanisms of cancer resistance also to anti-mitotic drugs63 the
compounds tested are probably good substrates for such pumps. Increased efflux of
compounds makes it impossible to reach their adequate concentrations in the cell and
consequently to bind tubulin and exert efficient cytotoxic effect. Since M2 correlates directly
to the energy of binding to tubulin, this latter aspect becomes less important than P-
glycoprotein affinity of these compounds, when assessing their cytotoxicity.
In view of the binding mode and the interaction energy, we can conclude that
compounds 1-6 can bind well to the Colchicine site of β-tubulin of HL-60, LoVo and
BALB/3T3 cells, and this effect is more pronounced than that observed for HL-60/vinc and
LoVo/DX cells. Additionally, these differences can be easily explained by the fact that in
various cancer cells the expression of specific β-tubulin isotopes significantly varies and each
isotype has different specific structure, also at the Colchicine binding site, leading to different
effects of these compounds for different cancer cell lines.64
Nonetheless, our computational work for Colchicine and the novel derivatives
indicates that the binding energy estimates weighted by the probability of occurrence of a
particular arrangement correlate reasonably well with their biological activity as measured by
the IC50 values. The correlation is stronger for wild type cells than for drug-resistant ones. We
17

explain this by the overexpression of multidrug resistance proteins e.g. ABC transporter or
P–glycoprotein in drug resistant cells and by a possible change in the expression of tubulin
isotypes in different cancer and normal cell lines.
4. Conclusions
We synthesized five structurally different derivatives 2-6 of Colchicine (1) and
evaluated their biological activity according to the in vitro antiproliferative tests as well as the
preliminary structure-activity relationship (SAR). From the molecular-level calculations
several conclusions can be drawn. Firstly, the derivatives of Colchicine with a free amine
group at C(7) or C(10) positions in compound 3 and 5, respectively, exhibit rather strong
cytotoxic activity, which is relatively lower than the antiproliferative activity of unmodified 1,
but still stronger than that of the reference drugs cisplatin and doxorubicin. The calculated
binding energy indicates that both compounds 3 and 5 fit well sterically into the Colchicine
site in βI tubulin. Secondly, the derivatives with large size substituents at the C(7) position
such as tert-butyl carbamate (2) or N-phenylurea (4) exhibit interesting antiproliferative
activity. Compound 4 has a very high selectivity index value (approximately 44) towards HL-
60 cancer cells. The most cytotoxic derivative of 1 is 2 which is in the majority of cases more
active than unmodified 1 and the tested reference drugs. The molecular-level computations
indicate that compound 2 fits better to βI tubulin than compound 4. Our studies clearly show
that various modifications of Colchicine at the C(7) position may lead to more potent anti-
cancer derivatives. Moreover, large substituents at the C(10) positions can decrease the
activity of the starting compound as observed for compound 6 which exhibits the lowest
antiproliferative activity of all tested compounds. On the other hand, our calculations indicate
that this compound does not fit well to the Colchicine binding site in βI tubulin because of a
very low value of the M2 parameter. Thirdly, both biological assays and theoretical
18

calculations demonstrate that the same type modifications of 1, but at different positions, such
as in the pairs of compounds 2 and 5 or 4 and 6 could produce to some extent different
bioactivity.
Microtubules are the primary target for many anti-cancer drugs or drug candidates,
many of which bind specifically to the Colchicine site in β-tubulin. Using this binding site as
a target, we have computationally probed Colchicine and five of its novel derivatives for
affinity to βI-tubulin, which is the most common isotype in most cancer cells. We observed a
correlation between computational-binding predictions and in vitro determined IC50 values,
especially against the drug sensitive cancer lines HL-60 and LoVo as well as the normal cell
line BALB/3T3. The correlation between the in vitro determined IC50 and the calculated
binding energy is low for the drug resistant cell lines HL-60/vinc and LoVo/DX. The
consistency of the experimental and theoretical results for the derivatives of 1 demonstrates
the utility of computational screening in the elucidation of the activity of derivatives of 1.
Also, these finding can assist in the rational design of its more active derivatives. The
similarity of the binding energies obtained in the computational method with the experimental
results for Colchicine and its derivatives indicates that computational methods can be used to
design new Colchicine binding site inhibitors (CBSI). Further work is still required to
experimentally confirm the binding location of the most active compound on βI-tubulin and
its isotypes. Finally, our studies show that attempts at different modifications of the
Colchicine structure should be continued in order to create compounds with an enhanced
antiproliferative activity against diverse human cancer cell lines, especially drug resistant
ones. We propose inclusion of binding affinity calculations for these compounds with regard
to not only tubulin isotypes but also with respect to P-glycoprotein and/or other most
important efflux transporters in order to minimize them and hence increase the activity of the
19

drug in vitro and in vivo. These results will help us to develop drug candidates on the basis of
this novel class of tubulin inhibitors targeting the Colchicine site in further studies.
5. Experimental
5.1. General
All precursors for the synthesis of Colchicine derivatives and solvents were obtained
from Aldrich or Fluka and were used as received without further purification. CDCl3 spectral–
grade solvents were stored over 3Å molecular sieves for several days. All the solvent used in
flash chromatography were of HPLC grade (CHROMASOLV from Sigma-Aldrich) and were
used as received. The elemental analyses of 2-6 were carried out on Vario ELIII (Elementar,
Germany).
5.2. Synthesis
5.2.1. General procedure for the synthesis of Colchicine derivatives 2, 3 and 5
Compounds 2, 3 and 5 were prepared from Colchicine according to literature.26-27 The
spectroscopic data of these compounds data were in agreement with previously published
assignments and additionally are shown in the Supplementary material.
5.2.2. General procedure for the synthesis of Colchicine derivatives 4 and 6
Compounds 4 and 6 were obtained directly from compounds 3 and 5, respectively.
Phenyl isocyanate (1.5 mmol) and compound 3 (0.25 mmol) were dissolved in
tetrahydrofuran (100ml) while phenyl isocyanate and compound 5 (0.25 mmol) were
dissolved in dry acetone (100ml). Both mixtures were stirred at room temperature. The course
of the reaction was followed by silica gel TLC. After a two days, the reaction appeared
complete and the reaction mixture was evaporated to dryness under reduced pressure and
purified using dry-column flash chromatography (Silica gel 60 from Fluka) to give respective
urea derivatives of Colchicine 4 and 6 with 68 % and 55% yield. The structures of both
20

compounds were confirmed by spectroscopic methods. Examples of the obtained spectra are
included in the Supplementary Material. Additionally, the crystals of compound 6 were
studied by X-ray methods.
5.3. Spectroscopic measurements
The 1H, 13C spectra were recorded on a Bruker Avance DRX 400 spectrometer.
1H NMR measurements of 1-6 (0.07 mol dm-3) in CDCl3 were carried out at the operating
frequency 600.055 MHz; flip angle, pw = 450; spectral width, sw = 4500 Hz; acquisition time,
at = 2.0 s; relaxation delay, d1=1.0 s; T = 293.0 K and using TMS as the internal standard. No
window function or zero filling was used. Digital resolution was 0.2 Hz per point. The error
of the chemical shift value was 0.01 ppm. The 13C NMR spectra were recorded at the
operating frequency 150.899 MHz; pw = 600; sw = 19000 Hz; at = 1.8 s; d1=1.0 s; T = 293.0
K and TMS as the internal standard. Line broadening parameters were 0.5 or 1 Hz. The error
in the chemical shift value was 0.1 ppm. All spectra were locked to deuterium resonance of
CDCl3.
The FT-IR spectra of 1-6 in the mid infrared region were recorded in KBr. The spectra
were taken on an IFS 113v FT-IR spectrophotometer (Bruker, Karlsruhe) equipped with a
DTGS detector; resolution 2 cm–1, NSS = 64. The Happ-Genzel apodization function was
used.
The ESI (Electrospray Ionization) mass spectra were recorded on a Waters/Micromass
(Manchester, UK) ZQ mass spectrometer equipped with a Harvard Apparatus syringe pump.
The samples were prepared in dry acetonitrile (5×10-5 mol dm-3). A sample was infused into
the ESI source using a Harvard pump at a flow rate of 20 µl min-1. The ESI source potentials
were: capillary 3 kV, lens 0.5 kV, extractor 4 V. The standard ESI mass spectra were recorded
at the cone voltages: 10 and 30 V. The source temperature was 120 °C and the desolvation
temperature was 300 °C. Nitrogen was used as the nebulizing and desolvation gas at flow-
21

rates of 100 dm3 h-1. Mass spectra were acquired in the positive ion detection mode with unit
mass resolution at a step of 1 m/z unit. The mass range for ESI experiments was from m/z =
300 to m/z = 1200.
Low-resolution EI-mass spectra were recorded using AMD-Intectra GmbH (Harpstedt) D-
27243 model 402 double-focusing sector mass spectrometer (ionizing voltage 70 eV;
accelerating voltage 8 kV; resolution 10,000; 10% valley definition). Samples of 1-6 were
introduced using direct insertion probe at a source temperature of 150 °C.
5.4. Single crystal X-ray measurement
X-ray intensity data for the 2 and 6 crystals were collected using graphite
monochromatized MoKα radiation (λ=0.71073 Å) on a four-circle κ geometry KUMA KM-4
diffractometer with a two dimensional CCD detector. The ω-scan technique with ∆ω=1.0o for
each image was used for data collection. One image was used as a standard after every 50
images for monitoring of the crystal stability and the data collection. No correction to the
relative intensity variations was necessary. Data collections were made using the CrysAlis
CCD program.65 Integration, scaling of the reflections, correction for Lorenz and polarization
effects and absorption corrections were performed using the CrysAlis Red program.65 The
structures were solved by the direct methods using SHELXS-97 and refined using SHELXL-
97 program.66 The hydrogen atoms were introduced in their geometrical positions. The final
difference Fourier maps showed no peaks of chemical significance. Details of the data
collection parameters, crystallographic data and the final agreement parameters are listed in
Table S1. Visualizations of the structures were made with the Diamond 3.1 program.67 The
structures have been deposited with the Cambridge Crystallographic Data Center in the CIF
format, no. CCDC 1428748 and 1428749 for 2 and 6 respectively. Copies of this information
can be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge, CB2
22

1EZ, UK (fax: +44-1223-336-033); email: deposit@ccdc.cam.ac.uk or www:
http://www.ccdc.cam.ac.uk).
5.5. Antiproliferative activity of Colchicine and its derivatives
Four human cancer cell lines and one murine normal cell line were used to evaluate
antiproliferative activity of Colchicine and its derivatives: human acute promyelocytic
leukemia (HL-60) and its vincristine resistant subline – (HL-60/vinc), human colon
adenocarcinoma cell lines sensitive and resistant to doxorubicin (LoVo) and (LoVo/DX)
respectively, and also normal murine embryonic fibroblast cell line (BALB/3T3). The
BALB/3T3 cell line was purchased from the American Type Culture Collection (ATCC
Rockville, Maryland, USA), HL-60 cell line - from European Type Culture Collection by
courtesy of Professor Spik and Dr Mazurier (Laboratory of Biological Chemistry USTL,
Lille, France) and HL-60/vinc, LoVo and LoVo/DX by courtesy of Prof. E. Borowski
(Technical University of Gdańsk, Poland). All the cell lines are maintained at the Institute of
Immunology and Experimental Therapy (IIET), Wrocław, Poland.
Human leukaemia cells were cultured in RPMI 1640 medium (IIET, Wroclaw)
containing 10% fetal bovine serum, glucose (5.5 g/l), 2mM L-glutamine, 1mM sodium
puryvate (Sigma-Aldrich, Germany) and 1µg/100ml doxorubicin for HL-60/vinc (Sigma-
Aldrich, Germany). Human colon adenocarcinoma cell lines were cultured in mixture of
OptiMEM and RPMI 1640 (1:1) medium (Optimem from Gibco, RPMI 1640 from PAA,
Austria), supplemented with 5% fetal bovine serum (PAA, Austria), 2mM L-glutamine, 1mM
sodium puryvate (Sigma-Aldrich, Germany) and 10µg/100ml doxorubicin for LoVo/DX
(Sigma-Aldrich, Germany). Murine embryonic fibroblast cells were cultured in Dulbecco
medium (IIET, Wroclaw), supplemented with 10% fetal bovine serum (PAA, Austria), 4mM
glutamine and glucose 4.5 g/l (Sigma-Aldrich, Germany). All culture media contained
23

antibiotics: 100U/ml penicillin and 100µg/ml streptomycin (Polfa-Tarchomin, Poland). All
cell lines were cultured during entire experiment in humid atmosphere at 37ºC and 5% CO2.
The antiproliferative assays in vitro: Twenty four hours before adding the tested
compounds, all cell lines were seeded in 96-well plates (Sarstedt, Germany) in appropriate
media with 104 cells per well. All cell lines were exposed to each tested agent at four different
concentrations in the range 100 to 0.1 µg/ml for 72 hours. Cells were also exposed to the
reference drug cisplatin (Ebewe, Austria) and doxorubicin (IBA, Poland). Additionally, all
cell lines were exposed to DMSO (solvent used for tested compounds) (Sigma-Aldrich,
Germany) at concentrations corresponding to these present in tested agents’ dilutions. For
adherent cells, a sulforodamine B assay was performed and an MTT assay for leukemia cells.
SRB: After 72 hours of incubation with the tested compounds, cells were fixed in situ
by gently adding of 50µl per well of cold 50% trichloroacetic acid TCA (POCh, Poland) and
were incubated at 4ºC for one hour. Following, wells were washed four times with water and
air dried. Next, 50 µl of 0.4% solution of sulforodamine B (Sigma-Aldrich, Germany) in 1%
acetic acid (POCh, Poland) were added to each well and plates were incubated at room
temperature for 0,5 hour. After incubation time, unbound dye was removed by washing plates
four times with 1% acetic acid whereas stain bound to cells was solubilized with 10mM Tris
base (Sigma-Aldrich, Germany). Absorbance of each solution was read at Multiskan RC
photometer (Labsystems, Finland) at the 540 nm wavelength.
MTT: Proliferation inhibition of leukemia cells by the tested compounds was measured
by means of an MTT assay. Thus, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide solution (Sigma-Aldrich, Germany) was added to each well and plates
were left in cell incubator for four hours to allow the cells to metabolize yellow MTT to blue
formazan. Then, the lysing mixture consisting of 225 ml dimethylformamide, 67.5 g sodium
dodecyl sulfate (both from Sigma-Aldrich, Germany) and 275 ml of distilled water, was
24

added in 80 µl volume per well. Plates were incubated for 24 hours for the formazan crystals
to be released from cells and dissolved and then absorbance of each well was read at
Multiskan RC photometer (Labsystems, Finland) at the 570 nm wavelength.
Results are presented as mean IC50 (concentration of the tested compound, that inhibits
cell proliferation by 50%) standard deviation. IC50 values were calculated in Cheburator 0.4,
Dmitry Nevozhay software for each experiment.68 Compounds at each concentration were
tested in triplicates in single experiment and each experiment was repeated at least three times
independently. Results are summarized in Table 1. The RI was defined as the ratio of IC50 for
a given compound calculated for resistant cell line to that measured for its parental drug
sensitive cell line (Table 1).
5.7. Molecular modeling studies
Molecular orbital calculations with full geometry optimization of N-Boc-
deacetylColchicine (2) and 10-N-phenylurea-10-demethoxyColchicine (6) were performed
with the Gaussian09 program package.46 All calculations were carried out with the DFT level
using the Becke3-Lee-Yang-Parr correlation functional (B3LYP)1 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.
5.8. Molecular Docking Simulations
3D structures of Colchicine and its five derivatives (shown in Fig. 5) were docked into
the Colchicine binding site of βІ tubulin using the Autodock4 program under flexible ligand
and rigid receptor conditions. AutoDock4 is designed to predict how drug candidates bind to a
receptor of a known 3D structure and consists of two main programs: (a) autodock which
performs the docking of the ligand to a set of grids describing the target protein; (b) autogrid
25

which pre-calculates these grids. The initial structures of ligands were first minimized using
the Amber12: EHT force field (in MOE2013.0802). Single point energies were calculated in
RHF/cc-pVDZ level of theory for those structures and then, they were fully optimized on
RHF/RM1 level of theory in GAMESS-US version 2010-10-01. βІ Tubulin sequence data
(TBB5_HUMAN) were obtained from UniProt ID and a homology model was constructed
for βІ tubulin on the basis of the bovine tubulin structure in RCSB Protein Data Bank
(1SA0.pdb) by MOE2013.0802.
Supplementary data
Supplementary data (examples of NMR and ESI spectra) associated with this article
can be found in the online version.
References
[1] A. Slobodnick, B. Shah, M.H. Pillinger, S. Krasnokutsky, Am. J. Med. 128 (2015) 461-
470.
[2] N. Nerlekar, A. Beale, R.W.Harper, Med. J. Aust. 201 (2014) 687-688.
[3] I. Grattagliano, L. Bonfrate, V. Ruggiero, G. Scaccianoce, G. Palasciano P. Portincasa
Clin. Pharmacol. Ther. 95 (2014) 89-97.
[4] N.G. Vindya, N.Sharma, M. Yadav, K.R. Ethiraj. Curr. Top. Med. Chem. 15 (2015) 73-
82.
[5] Y. Lu, J. Chen, M. Xiao W. Li, D.D. Miller, Pharm. Res. 29 (2012) 2943-2971.
[6] Y.T. Ji, Y.N. Liu, Z.PLiu, Curr. Med. Chem. 22 (2015) 1348-1360.
[7] R.A. Stanton, K.M. Gernert, J.H. Nettles, R. Aneja, Med. Res. Rev. 31 (2011) 443-481.
[8] G. Cocco, D.C. Chu, S.Pandolfi, Eur. J. Intern. Med. 21 (2010) 503-508.
[9] L. P. Yang, Drugs 70 (2010) 1603-1613.
[10] N. Schlesinger, Expert. Opin. Drug. Saf. 6 (2007) 625-629.
26

[11] N. Schlesinger, R. Schumacher, M. Catton, L. Maxwell , Cochrane Database Syst. Rev.
18 (2006) CD006190.
[12] U. Lange, C. Schumann, K. L.Schmidt , Eur. J. Med. Res. 6 (2001) 150-160.
[13] Y. Karaaslan, I. Simsek, N. Engl. J. Med. 370 (2014) 780-781.
[14] G. Markel, M. Imazio, A, Brucato Y., Adler , Clin. Cardiol. 36 (2013) 125-128.
[15] C. Korkmaz, Clin. Exp. Rheumatol. (2012) S104-S107.
[16] K. Nepali, R. Ojha, S. Sharma, P.M. Bedi, K. L. Dhar, Rec. Pat. Anticancer Drug
Discov. 9 (2014) 176-220.
[17] Y. Lu, J. Chen, M. Xiao, W. Li, D. D.Miller, Pharm. Res. 29 (2012) 2943-2971.
[18] T. Kallinich, D. Haffner , T. Niehues, K. Huss, E. Lainka, U. Neudorf, C. Schaefer, S.
Stojanov, C. Timmann, R. Keitzer , H. Ozdogan, S. Ozen, Pediatrics. 119 (2007) e474-e483.
[19] G. Lahti, J. Indian Med. Assoc. 110 (2012) 56-58.
[20] X. Zhang, Y. Kong, J. Zhang, M. Su, Y. Zhou, Y. Zang, J. Li, Y. Chen, Y. Fang, X.
Zhang, W. Lu, Eur. J. Med. Chem. 95 (2015) 127-135.
[21] A. Huczyński, J. Rutkowski, K. Popiel, E. Maj, J. Wietrzyk, J. Stefańska, U. Majcher, F.
Bartl, Eur. J. Med. Chem. 90 (2015) 296-301.
[22] K. C. Nicolaou, R. A. Valiulin, J. K. Pokorski, V.Chang, J.S. Chen, Bioorg. Med. Chem.
Lett. 22 (2012) 3776-3780.
[23] D. J Chang, E.Y. Yoon, G. B. Lee, S.O. Kim, W.J. Kim, Y. M. Kim, J. W. Jung, H. An,
Y.G. Suh, Bioorg. Med. Chem. Lett. 19 (2009) 4416-4420.
[24] B. Singh, A. Kumar, P. Joshi, S.K. Guru, S. Kumar, Z.A Wani, G. Mahajan, A.
Hussain, A. K. Qazi, A. Kumar, S. S. Bharate, B.D. Gupta P. R. Sharma, A. Hamid, A.K.
Saxena, D.M. Mondhe, S. Bhushan, S.B. Bharate R.A. Vishwakarma, Org. Biomol. Chem.
13 (2015) 5674-89.
27

[25] K. Nakagawa-Goto, C.X. Chen, E. Hamel, C. C. Wu, K. F. Bastow, A. Brossi, K. H.
Lee, Bioorg. Med. Chem. Lett. 15 (2005) 235-238.
[26] J. D. Bagnato, A. L. Eilers, R. A. Horton, C. B..Grissom, J. Org.Chem. 69 (2004) 8987 –
8996.
[27] R. M. Horowitz, G. E. Ullyot, J. Am. Chem. Soc. 74 (1952) 587-592.
[28] S.B Hastie, Pharmacol. Ther. 51 (1991) 377-401.
[29] R.B.G. Ravelli, B. Gigant, P.A. Curmi, I. Jourdain, S. Lachkar, A. Sobel, M. Knossow,
Nature 428 (2004) 198-202.
[30] M. Banerjee, A. Poddar, G. Mitra, A. Surolia, T. Owa, B. Bhattacharyya, J. Med. Chem.
48 (2005) 547-555.
[31] Y. Lu, J. Chen, M. Xiao, W. Li, D. D.Miller, Pharm. Res. 29 (2012) 2943-2971.
[32] D.E. McNamara, S. Senese, T.O. Yeates, J.Z. Torres, Protein Science 24 (2015) 1164-
1172.
[33] L. Lessinger, T.N. Margulis, Acta Cryst. B34 (1978) 578-584.
[34] M.V. King, J.L. De Vries, J. Peponsky, Acta Cryst. 5 (1952) 437-440.
[35] B. Busetta, F. Leroy, M. Hospital, J. Elguero, A. Blade-Font, Acta Cryst. B39 (1979)
1525-1527.
[36] C. Miravitlles, J. Ruis, A. Blade-Font, G. Germain, Acta Cryst. C41 (1983) 1709-1711.
[37] P.N. Sharma, A. Brossi, J.V. Silverton, Acta Cryst. C41 (1985) 1069-1072.
[38] P. Kerekes, A. Brossi, J.L. Flippen-Anderson, C.F. Chignell, Helv. Chim. Acta 68 (1985)
571-580.
[39] R. Dumont, A. Brossi, J.V. Silverton, J. Org. Chem. 51 (1986) 2515-2521.
[40] J.V. Silverton, R. Dumont, A. Brossi, Acta Cryst. C43 (1987) 1802-1805.
[41] M.G. Banwell, S.C. Peters, R.J. Greenwood, M.F. Mackay. E. Hamel, C.M. lin, Aust. J.
Chem. 45 (1992) 1577-1588.
28

[42] U. Berg, H. Bladh, M. Hoff, C. Svensson, J. Chem. Soc., Perkin Trans 2 (1997) 1697-
1704.
[43] L. Yu. Izotova, K.M. Beketov, B.T. Ibragimov, M.K. Yusupov, J. Inclusion Phenom.
Mol. Recog. Chem. 28 (1997) 33-37.
[44] W.O. McClure, J.C. Paulson, Mol. Pharmacol. 13 (1977) 560-575.
[45] S.B. Hastie, R.C.Jr. Williams, D. Puett, T.L. MacDonald, J. Biol.Chem. 264 (1989)
6682-6688.
[46] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G.
Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P.
Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K.
Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T.
Vreven, J.A. Montgomery, Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers,
K.N. Kudin, V.N. Staroverov, T. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A.
Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene,
J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O.
Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma,
V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O.
Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, and D.J. Fox, Gaussian, Inc., Wallingford
CT, 2013. Gaussian09, Revision D.01, Programme, Gaussian, Inc., Wallingford, CT, 2013.
[47] A.D. Becke, J. Chem. Phys. 104 (1996) 1040-1046.
[48] C. Lee, W. Yang, R.G. Parr, Phys. Rev. B37 (1988) 785-789.
[49] B.G. Parr, W. Yang, Density-functional Theory of Atoms and Molecules, Oxford
University Press, New York, 1989.
[50] R. Bai, X. Pei, O. Boye, Z. Getanunt, S. Grover, J. Nekisz, N. Nguyen, A. Brossi, E.
Hamel, J. Mol. Biol. 272 (1996) 12639-12645.
29