Accepted Manuscript Synthesis, antiproliferative activity and molecular docking of Colchicine derivatives Adam Huczyński, Urszula Majcher, Ewa Maj, Joanna Wietrzyk, Jan Janczak, Mahshad Moshari, Jack A. Tuszynski, Franz Bartl PII: DOI: Reference:
S0045-2068(16)30002-5 http://dx.doi.org/10.1016/j.bioorg.2016.01.002 YBIOO 1870
To appear in:
Bioorganic Chemistry
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 e
Department of Oncology, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada 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: [email protected], 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
O 18
4
3
15
O
O 2
7 NH 14 8
16 1 O 13 12
19
23
6 O 21
5
22
O
O
O
24
O
25
NH 2
O
O
(c)
H 3 CO
O
H 3 CO
(2)
NH
O
(d)
11 10 9O 20
21
NH
O
H 3 CO
(3)
(4)
(a) (b) 17
O 18
O 19
O
A O
B
7
O O
NH (e)
1
O
21 22
O 20
(f)
20
21
NH
O
NH O
O
21
O
C 10
H 3 CO
O
20
colchicine (1)
H2N
O
O
HN 22
(5)
(6)
HN
O 23
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 vincristineresistant 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 4dimethylaminopyridine (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 structures of all products 2-6 were determined using the EI MS, ESI-MS, FT-IR, 1H and
13
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 P2 1212 1 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 C13C16 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-YangParr 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 CC 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 -1
-1
Fig. 2. Three-dimensional molecular electrostatic potential (right) (-0.05eÅ , red and +0.05 eÅ , blue) mapped -3 on the surface of total electron density (0.008eÅ ) for the N-Boc-deacetylColchicine (a),10-N-phenylurea-10demethoxyColchicine (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
S0045-2068(16)30002-5 http://dx.doi.org/10.1016/j.bioorg.2016.01.002 YBIOO 1870
To appear in:
Bioorganic Chemistry
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 e
Department of Oncology, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada 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: [email protected], 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
O 18
4
3
15
O
O 2
7 NH 14 8
16 1 O 13 12
19
23
6 O 21
5
22
O
O
O
24
O
25
NH 2
O
O
(c)
H 3 CO
O
H 3 CO
(2)
NH
O
(d)
11 10 9O 20
21
NH
O
H 3 CO
(3)
(4)
(a) (b) 17
O 18
O 19
O
A O
B
7
O O
NH (e)
1
O
21 22
O 20
(f)
20
21
NH
O
NH O
O
21
O
C 10
H 3 CO
O
20
colchicine (1)
H2N
O
O
HN 22
(5)
(6)
HN
O 23
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 vincristineresistant 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 4dimethylaminopyridine (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 structures of all products 2-6 were determined using the EI MS, ESI-MS, FT-IR, 1H and
13
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 P2 1212 1 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 C13C16 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-YangParr 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 CC 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 -1
-1
Fig. 2. Three-dimensional molecular electrostatic potential (right) (-0.05eÅ , red and +0.05 eÅ , blue) mapped -3 on the surface of total electron density (0.008eÅ ) for the N-Boc-deacetylColchicine (a),10-N-phenylurea-10demethoxyColchicine (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
