Chemical synthesis, pharmacological evaluation and in silico analysis of new 2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole derivatives as potential anti-mitotic agents

Article abstract of DOI:10.1016/j.bmcl.2016.07.025

We have synthesized new, biologically active mono- and di-substituted 2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole derivatives bearing electron withdrawing groups and electron donating groups. These derivative structures were characterized by their spectra

Full text of DOI:10.1016/j.bmcl.2016.07.025

Accepted Manuscript
Chemical synthesis, pharmacological evaluation and in silico analysis of new
2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole derivatives as potential anti-mi-
totic agents
Maninder Minu, Deepti Singh, Tejashree Mahaddalkar, Manu Lopus, Philip
Winter, Ahmed Taha Ayoub, Kristal Missiaen, Tatiana Martins Tilli, Manijeh
Pasdar, Jack Tuszynski
PII:
S0960-894X(16)30738-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.07.025
BMCL 24069
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Accepted Date:
23 May 2016
8 July 2016
Please cite this article as: Minu, M., Singh, D., Mahaddalkar, T., Lopus, M., Winter, P., Ayoub, A.T., Missiaen, K.,
Tilli, T.M., Pasdar, M., Tuszynski, J., Chemical synthesis, pharmacological evaluation and in silico analysis of new
2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole derivatives as potential anti-mitotic agents, Bioorganic & Medicinal
Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.07.025
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Chemical synthesis, pharmacological evaluation and in silico analysis of new
2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole derivatives as potential anti-mitotic
agents
Maninder Minua,I,*, Deepti Singha, Tejashree Mahaddalkarb, Manu Lopusb, Philip
Winterc, Ahmed Taha Ayoubc, Kristal Missiaenc, Tatiana Martins Tillid , Manijeh Pasdarc
and Jack Tuszynskic
a Department of Chemistry, Dayalbagh Educational Institute, Dayalbagh, Agra, India-282110
b Experimental Cancer Therapeutics and Chemical Biology, UM-DAE Centre for Excellence in
Basic Sciences, University of Mumbai, Kalina, Mumbai, India-400098
c Division of Experimental Oncology, Cross Cancer Institute, University of Alberta, Edmonton,
AB, Canada
d Laboratório de Modelagem de Sistemas Biológicos, National Institute of Science and
Technology for Innovation in Neglected Diseases (INCT/IDN, CNPq), Centro de
Desenvolvimento Tecnológico em Saúde, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
I Lecturer, Chemistry and Biochemistry Department, Old Dominion University, Norfolk, VA,
USA
* Author for correspondence; E-mail: minu.maninder@gmail.com
1

Abstract
We have synthesized new, biologically active mono- and di-substituted 2,3,3a,4,5,6-
hexahydrocyclopenta[c]pyrazole derivatives bearing electron withdrawing groups and electron
donating groups. These derivative structures were characterized by their spectral and analytical
data. The newly synthesized hexahydropyrazole analogues were evaluated for their in vitro
anticancer activity against breast and lung cancer cell lines using a cytotoxicity bioassay. To
understand their mechanism of action, tubulin binding assays were performed which pointed to
their binding to microtubules in a mode similar to but not identical to colchicine, as evidenced by
their KD value evaluation. Computational docking studies also suggested binding near the
colchicine binding site on tubulin. These results were further confirmed by colchicine-binding
assays on the most active compounds, which indicated that they bound to tubulin near but not at
the colchicine site. The moderate cytotoxic effects of these compounds may be due to the
presence of electron donating groups on the para-position of the phenyl ring, along with the
hexahydropyrazole core nucleus. The observed anti-cancer activity based on inhibition of
microtubule formation may be helpful in designing more potent compounds with a
hexahydropyrazole moiety.
Abbreviations
EDG: electron donating group; EWG: electron withdrawing groups; MT: microtubule; SAR:
structure activity relationship
2

Cancer, a spectrum of diseases characterized by the uncontrolled and abnormal division
of cells in the body by escaping the normal cell cycle control and programmed cell death is the
second leading cause of death worldwide1. It is estimated that 12 million people will die from
cancer by 20302. In the past few years, many efforts have been made to develop new strategies
for finding safe and effective ways of treating these diseases, which must be based on an
improved understanding of the biological processes involved in cancer cell survival and the
search for novel chemotherapeutic agents1. The conventional methods to treat metastatic cancer,
i.e. chemotherapy and radiation therapy, have a major drawback of producing severe side effects.
These methods inflict “collateral damage” as they are unable to precisely differentiate between
the cancerous and normal cells.
Normal cells affected adversely by systemically administered chemotherapy are mostly
the cells in the mouth, blood cells, hair follicles, stomach and bowel, which usually results in
nausea, diarrhea, mouth sores, low blood counts, and/or hair loss1. Therefore, there is an urgent
need to focus on novel targeted cancer therapies that attack preferentially the cancer cells,
thereby avoiding any serious damage to normal cells.
One such molecular target are microtubules (MTs), which are ubiquitous protein polymer
structures representing key components of the cytoskeletal architecture. MTs are involved in
many important cellular processes including mitosis, morphogenesis, intracellular transport and
secretion3. MTs are hollow cylinders consisting of α- and β-tubulin heterodimers. The functions
of MTs are regulated by their continuously alternating polymerization and depolymerization
processes, a unique property of microtubules referred to as dynamic instability4. Tubulin-binding
molecules interfere with the dynamic instability of MTs leading to the formation of abnormal
spindles, cell cycle arrest in the M-phase, and ultimately apoptotic cell death5,6. A variety of
3

compounds which were obtained from natural sources, such as paclitaxel, vinblastine,
combretastatin A-4 and colchicine, are known to interfere with the dynamic instability of MTs
and result in mitotic arrest7. These drugs, however, are often associated with severe side effects
due to their adverse effects on normal cells (which also express tubulin and form microtubules)
and the development of drug resistance in cancer cells8. Therefore, there is still an urgent need to
find tubulin-binding pharmacological agents which would have an improved profile in terms of
specificity and selectivity for cancer versus normal cells.
Heterocyclic compounds comprise the largest and most diverse group of organic
medicinal compounds. More than 75% of the top 200 branded drugs from the pharmaceutical
industry have heterocyclic fragments in their structures. Among all the heterocyclic compounds
discovered so far, those containing nitrogen in their core structure are the most significant and
appear in most of the drugs marketed worldwide. For these reasons, heterocycles have an
enormous potential to be used as scaffolds for the design of new drugs.
Pyrazoles are an important class of nitrogen containing five-membered heterocyclic
compounds. The pyrazole scaffold represents a common motif in many pharmaceutically active
compounds that demonstrate a wide range of pharmacological activities including anti-cancer,
anti-microbial9,10, anti-inflammatory11,12, anti-convulsant, anti-histaminic13, anti-viral14 and anti-
HIV 15. Because of their clinical significance, the synthesis of new lead compounds retaining the
‘core’ pyrazole chromophore has attracted considerable attention of the medicinal chemistry
research community16–18
.
As a part of our ongoing drug discovery program, we have synthesized various pyrazole
scaffold-bearing heterocyclic compounds as potential novel anti-cancer agents with a focus on
the features important for anti-mitotic activity.
4

This novel series of 2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole derivatives bearing
both electron donating and electron withdrawing substituents in the para-position of the
sulfonylaryl moiety has been synthesized keeping in mind their structural simplicity. These
compounds, obtained by convenient synthetic methods, have been shown to strongly bind to
MTs as evidenced by our biological assays and in silico docking studies.
The synthetic approach to obtaining mono-substituted 2,3,3a,4,5,6-hexahydrocyclopenta
[c]pyrazole derivatives (5-8) and 2,3-disubstituted-2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole
derivatives (9-28) followed the reactions shown in Scheme 1. We accomplished the synthesis of
substituted (E)-benzylidene cyclopentanones by cyclopentanone reaction with p-substituted
benzaldehydes in the presence of ethanolic sodium hydroxide19. Heating of benzylidene
cyclopentanones with hydrazine hydrate resulted in the formation of 3-substituted-3,3a,4,5,6,7-
hexahydro-2H-indazoles (1-3)20. Further, the sulfonation of 3-substituted-hexahydrocyclo-
pentapyrazoles with appropriate sulfonyl chlorides in pyridine yielded 2,3-disubstituted-
2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazoles (4-21)21.
Scheme I. Synthesis of mono- and di-substituted 2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazoles.
O
O
N
NH
NH2NH2, C2H5OH
MeOH
aq.NaOH
R1
R1
1-4
5-9
R2SO2Cl,
Pyridine
O
N
N
S
R2
O
R1
10-28
5

In general, the benzylidene cyclopentanones exhibited UV maxima between 300-362 nm.
Their infrared spectra (FT-IR) showed bands at 2900-2955, 1629-1691 and 1529-1599 cm-1
corresponding to the presence of C-H str., C=O str. and C=C str. bonds, respectively. In the
proton NMR spectra (1H-NMR), the signals of the respective protons of the title compounds
were verified on the basis of their chemical shifts, multiplicities and coupling constants. The
spectra showed multiplets and triplets in the range of δ 1.05-3.12 ppm corresponding to aliphatic
ring protons, doublets at δ 7.10-7.61 ppm corresponding to aromatic protons, and singlets in the
range of δ 7.26-7.58 ppm corresponding to vinylic protons. The latter is the characteristic peak in
the benzylidene compounds confirming the geometry of the vinylic bond as E22,23
.
The 3-substituted hexahydropyrazoles were prepared by reacting (E)-2-benzylidene-
cyclopentanones (1-4) and hydrazine hydrate by a Michael type of addition reaction (Step-II). In
general, their UV maxima appeared within the range of 228-319 nm. In the infrared spectra (FT-
IR) 3-substituted hexahydropyrazoles showed the absence of C=O str. band at 1629-1691 cm-1,
the appearance of secondary amine group stretching at 3300-3490 cm-1, and the presence of C=N
str. and C-N str. band at 1550-1688 and 1095-1195 cm-1 respectively, which confirmed the
formation of 3-substituted hexahydropyrazoles. In the 1H-NMR, the appearance of singlets in the
range of δ 7.12-8.64 ppm in the proton spectrum, corresponding to NH of hexahydropyrazoles,
which disappeared upon D2O exchange, further supported the formation of the title compounds.
Similarly, the appearance of doublets for the 3-CH proton at δ 4.27-4.69 ppm and the absence of
singlets at δ 7.26-7.58 ppm corresponding to vinylic proton were also in favour of the formation
of 3-substituted hexahydropyrazoles.
6

The reaction between the 3-substituted hexahydropyrazoles (5-8) with p-substituted
sulfonylchlorides resulted in the formation of 2,3-disubstituted-2,3,3a,4,5,6,- hexahydro-2H-
pyrazoles (Step-III). In general, the product showed UV maxima from 305-384 nm. In the
infrared spectra (FT-IR) of 2,3,3a,4,5,6,- hexahydro-2H- pyrazoles showed the absence of
secondary amine group at 3300-3490 cm-1 and the presence of asymmetric and symmetric SO2
stretching at 1292-1384 and 1100-1192 cm-1 respectively, confirming the reaction between
substituted sulfonylchlorides and NH of hexahydropyrazole ring. In the 1H-NMR. the absence of
singlets at δ 7.12-8.64 ppm corresponding to NH of hexahydropyrazoles and the appearance of
peaks obtained from δ 6.80-8.66 ppm corresponding to aromatic protons confirmed the
substitution of NH group of hexahydropyrazole ring with substituted sulfonylchlorides.
Moreover, the molecular ion peaks along with fragmentation pattern from mass spectral
studies also indicated the formation of synthesized compounds. The elemental (C, H, N)
analysis confirmed that the calculated and observed values were within the acceptable limits (±
0.4%).
The physicochemical parameters and molecular structures of the hexahydropyrazole
derivatives used in the present study are given in Table 1. Additional information on the
chemical synthesis and analysis is provided in the Supplementary Material.
Table 1. Characterization data of hexahydropyrazoles 1-28.
Compound
R1
R2
Molecular formula M.Wt. MP (oC) Rf-value* Yield(%)
F
-
C12H11OF
C13H14O2
C15H18O
190.21 200-202 0.58
202.24 198-200 0.75
214.30 120-122 0.46
206.66 205-207 0.64
204.24 100-102 0.62
91.03
63.54
94.48
94.48
39.20
27.50
48.49
1
2
3
4
5
6
7
OCH3
CH(CH3)2
Cl
-
-
-
C12H11OCl
C12H13FN2
C13H16N2O
C15H20N2
F
H
H
H
OCH3
CH(CH3)2
216.27 98-100
0.87
228.33 113-115 0.86
7

Cl
H
C12H13ClN2
220.69 90-92
0.47
48.49
45.81
63.20
79.70
53.00
80.00
37.83
22.35
73.50
54.50
40.85
37.04
53.20
80.60
44.30
61.10
38.04
53.00
80.00
71.50
60.00
8
F
4-NO2C6H4 C18H16FN3O4S
4-CH3C6H4 C19H19FN2O2S
389.40 180-182 0.78
358.42 200-202 0.67
282.33 168-170 0.55
378.84 228-230 0.51
344.40 162-164 0.60
401.43 212-215 0.60
370.46 163-165 0.79
294.36 180-182 0.76
390.88 170-172 0.81
356.43 143-145 0.67
413.49 205-207 0.62
382.51 195-197 0.67
306.42 185-187 0.66
402.93 190-192 0.57
368.49 163-165 0.74
405.85 110-112 0.46
374.88 195-197 0.47
298.78 188-190 0.52
395.30 200-202 0.61
360.85 189-191 0.74
9
F
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
F
4-CH3
C13H15FN2O2S
C18H16ClFN2O2S
C18H17FN2O2S
F
4-ClC6H4
C6H5
F
OCH3
OCH3
OCH3
OCH3
OCH3
4-NO2C6H4 C19H19N3O5S
4-CH3C6H4 C20H22N2O3S
4-CH3
C14H18N2O3S
C19H19ClN2O3S
C19H20N2O3S
4-ClC6H4
C6H5
CH(CH3)2 4-NO2C6H4 C21H23N3O4S
CH(CH3)2 4-CH3C6H4 C22H26N2O2S
CH(CH3)2 4-CH3
CH(CH3)2 4-ClC6H4
CH(CH3)2 C6H5
C16H22N2O2S
C21H23ClN2O2S
C21H24N2O2S
Cl
Cl
Cl
Cl
Cl
4-NO2C6H4 C18H16ClN3O4S
4-CH3C6H4 C19H19ClN2O2S
4-CH3
C13H15ClN2O2S
C18H16Cl2N2O2S
C18H17ClN2O2S
4-ClC6H4
C6H5
*TLC mobile phase – Benzene: Diethyl ether (10 mL : 1 drop)
The synthesized hexahydropyrazoles were tested for their in vitro pharmacological
properties regarding their anti-cancer activity using the MTS assay protocol. The human lung
adenocarcinoma cell line (A549) and breast cancer carcinoma cell lines MDA-MB-231, MDA-
MB-468, T-47D as well as MCF-7 were treated with the compounds and assessed for cell
proliferation/viability and cytotoxicity using the MTS assay24. The IC50 values (concentration
with 50% growth inhibition) were determined by fitting the data to a dose-response model. A
detailed description of the assay and the analysis performed are given in the Supplementary
Material.
The preliminary screening of the synthesized hexahydropyrazoles on human breast
cancer carcinoma cells (MDA-MB-468, MDA-MB-231, T-47D and MCF-7) and human lung
8

carcinoma cells (A549) showed a great difference in cytotoxic activity strictly related to the
pyrazole substituents. The presence of hexahydropyrazole moiety yielded active compounds
showing anti-proliferative activity in all the cell lines tested. The analyses of concentration-
response curves of each cell line treated with our compounds together with the resulting
calculation of mean IC50 values displayed a broad range of sensitivity (from 0.8 ± 0.5 µM to
more than 100 µM, Table 2). Thiocolchicine and paclitaxel were included as controls.
Table 2. The IC50 values of synthesized hexahydropyrazole derivatives
IC50 (µM)a
Compound
MDA-MB-231 MDA-MB-468
T-47D
4.1 ± 17.7
0.2 ± 1.1
37.2 ± 24.8
N/D
MCF-7
0.1 ± 0.2
A549
0.6 ± 1.9
N/A
0.1 ± 0.2
0.1 ± 0.5
30.9 ± 21.3
N/D
0.2 ± 0.6
0.2 ± 0.4
14.8 ± 39.2
20.0 ± 14.3
186.2 ± 184.4
10.2 ± 7.3
3.8 ± 2.8
70.8 ± 48.9
N/D
Thiocolchicine
0.1 ± 0.2
Paclitaxel
38.0 ± 26.2
24.0 ± 13.8
30.9 ± 20.6
44.7 ± 27.8
N/D
5
6
144.5 ± 495.8
25.1 ± 20.2
1.1 ± 0.8
51.3 ± 39.0
9.3 ± 10.1
N/D
128.8 ± 127.5 N/D
7
29.5 ± 21.7
3.3 ± 2.5
N/D
51.3 ± 47.2
47.9 ± 32.0
5.8 ± 4.1
81.3 ± 54.3
N/D
8
50.1 ± 175.3
75.9 ± 52.4
N/D
10
11
12
14
15
16
19
20
21
22
24
89.1 ± 209.3
N/D
N/D
151.4 ± 125.5 N/D
177.8 ± 429.9 N/D
N/D
N/D
N/D
8.7 ± 7.4
N/D
1.0 ± 0.7
28.2 ± 17.5
7.9 ± 5.3
120.2 ± 99.6
15.5 ± 21.8
43.7 ± 139.9
0.8 ± 0.6
N/D
15.8 ± 76.4
N/D
19.1 ± 11.0
N/D
N/D
N/D
N/D
N/D
15.5 ± 26.1
N/D
40.7 ± 40.3
N/D
N/D
N/D
N/D
N/D
N/D
42.7 ± 73.7
N/D
N/D
a
Each IC50 value represents the effective concentration of the drug at which the growth of the cells is 50% of the
growth of untreated cells. ± values are the estimated standard errors of the IC50 parameter determined by a Monte
Carlo method; N/D: no activity detected. N/A: data not available.
9

The mono-substituted hexahydropyrazoles containing both electron withdrawing groups
(EWG) and electron donating groups (EDG) in the p-position of the phenyl ring (R1) yielded
active cytotoxic compounds 5-8. Among these, the derivatives 5 and 8 with EWGs, showed the
most cytotoxic activity against MDA-MB-468 at the lowest IC50 values against the MCF-7 cell
line. On the other hand, the derivative 7, containing iso-propyl group, was the least active on all
three breast cancer cell lines and showed no anti-proliferative effect on the A459 cell line.
In the 2,3-disubstituted hexahydropyrazoles, the substituents at R2 also drastically
affected the activity. Observing which substituents result in the most active compounds can make
structure-activity relationship (SAR) considerations possible. We note that phenyl and sulphonyl
substituents together played a fundamental role in determining the potency of the activity.
Compound 16 with a p-methoxyphenyl at R1 and a methyl at R2 exhibiting the highest level of
activity among the disubstituted pyrazoles on all the cell lines. Similarly, the second most active
compound 10 also had both EDG and EWG groups at R1 and R2 positions, respectively.
Interestingly, the presence of a chloro group at R1 resulted in inactive compounds, except
for derivative 24, which was active on MDA-MB-231 and MDA-MB-468 cell lines. On the
other hand, a fluoro group in the same position in compounds 10-12 yielded cytotoxic
compounds. Derivative 11, with a methyl group in the sulphonyl moiety showed cytotoxicity
against four cell lines. However, isopropyl substation on R1 did not generate very active
compounds (19-22), except 20 with a IC50 value of 8 µM against the MDA-MB-468 cell line.
Compound 16, having a methyl substitution on R2, showed the best activity against most
cell lines. Interestingly, replacing this group with p-nitrophenyl (14) or p-tolyl (15) drastically
reduced the activity, and its replacement with 4-chlorophenyl (17) and phenyl (18) resulted in a
total loss of cytotoxic activity.
10

In order to determine if the anti-proliferative action of the synthesized hexahydropyrazole
derivatives was due to their effect on MTs through their interactions with tubulin25–27, we
selected the compounds endowed with a higher activity for the next assay, namely protein-
binding determination. Notably, the cytotoxic activity of these compounds was in the micromolar
range compared to the nanomolar range observed with clinically approved MT-binding agents
such as paclitaxel or vincristine. While the potency of these new drugs is vastly lower than the
reference compounds, it is possible to substantially improve their performance by subsequent
derivatization based on the mode of action, which is a standard approach in medicinal chemistry.
To determine the tubulin binding parameters of the drugs, we performed
spectrofluorimetric titrations and a tryptophan fluorescence-quenching assay. The relative
intrinsic fluorescence intensity of the tryptophan residues of tubulin was monitored. The samples
were excited at 295 nm and the emission peaks recorded at 335 nm. KD was calculated as
described in the experimental protocols section28,29
.
Compounds 10, 11 and 16 were selected to test if they bind to tubulin. Both 11 and 16
showed concentration-dependent binding to tubulin (Fig. 1). By plotting the inverse of fractional
occupancy of the drug to tubulin versus inverse of free drug concentration, this assay has
revealed higher binding affinities to tubulin, as suggested by the equilibrium dissociation
constants (KD) of the compounds, which were determined to be 127 ± 8 µM for 11 (Fig. 1A) and
85 ± 2 µM for 16 (Fig. 1B), respectively. The compound 10 also showed potential binding to
tubulin, as shown by its ability to quench the intrinsic tryptophan fluorescence of tubulin.
However, due to a non-linear quenching pattern and higher absorbance at the excitation and
emission wavelengths, more accurate measurement of the KD values was not possible. Of these
11

tested compounds, the overall most active compound, 16, binds to tubulin with the highest
affinity, with a KD of 85 ± 2 µM. This result is consistent with the anti-proliferative assay studies.
Fig. 1. (A) Double reciprocal plot showing a dissociation constant (KD) of 127 ± 8 µM for 11 binding to tubulin. (B)
Double reciprocal plot showing a dissociation constant (KD) of 85 ± 2 µM for 16 binding to tubulin.
A
B
20
10
0
0
0.05 0.1 0.15 0.2 0.25
1/[Free Compound 16]
1/[Free Compound 11]
To learn more about the possible binding of the compounds, computational docking
studies were employed. First, blind docking simulations of the compounds to tubulin were
performed, i.e. docking without assuming a specific binding site. This was followed by flexible
docking on six putative binding sites obtained from the blind docking of the ligand to  tubulin,
including the colchicine binding site. Models of the tubulin structure in both straight and curved
conformations were used.
The blind docking runs performed on both the straight and curved tubulin models are
summarized in Tables 3 and 4. The straight-dimer model is based on the epothilone-stabilized
structure of tubulin, and is consistent with the microtubule-bound dimers, while the curved-dimer
model is based on colchicine-bound free tubulin, which is assembly-incompetent30. As
12

mentioned before, we analyzed the top ten poses and clustered them on the binding sites selected
for docking. The curved tubulin dimer model produced six different putative binding sites, only
one of which is known (site Cc; colchicine binding site). The straight tubulin dimer model
produced only three binding sites, with most of the poses being clustered in the top one (site As).
Comparison of the energies in the two tables showed, at the first glance, that straight model sites
bind more strongly to the ligands than the curved ones do. To verify the actual binding sites, a
second docking protocol was used where the ligands were directed to the putative binding sites
and receptor flexibility was taken into account. The details of the computational methods used
are provided in the Supplementary Material.
Table 3. Results of the blind docking of 11 and 16 to the curved (PDB entry: 4O2B)
tubulin model. The table shows the rank and energy in kcal/mol (in parentheses) for each pose in
each binding site. Cc is the colchicine binding site, the other sites are previously unknown
putative binding sites.
11
16
Site Ac
1 (-5.1)
1 (-4.9)
3 (-4.9)
5 (-4.7)
6 (-4.7)
9 (-4.6)
-
2 (-4.9)
8 (-4.4)
9 (-4.4)
-
5 (-4.5)
6 (-4.5)
7 (-4.5)
3 (-4.7)
-
Site Bc
Site Cc
-
-
2 (-4.9)
4 (-4.7)
8 (-4.6)
7 (-4.6)
-
-
Site Dc
Site Ec
4 (-4.7)
10 (-4.4)
Site Fc
10 (- 4.5)
-
13

Table 4. Results of the blind docking of 11 and 16 to the straight (PDB entry: 1TVK)
tubulin model. The table shows the rank and energy in kcal/mol (in parentheses) for each pose in
each binding site. Cc is the colchicine binding site, the other sites are previously unknown
putative binding sites.
11
16
Site As
1 (-5.1)
1 (-5.3)
2 (-5.0)
3 (-5.0)
4 (-4.9)
5 (-4.9)
6 (-4.8)
7 (-4.7)
8 (-4.7)
9 (-4.7)
10 (-4.6)
-
2 (-5.2)
3 (-5.1)
4 (-5.1)
5 (-5.1)
7 (-5.1)
9 (-5.0)
10 (-5.0)
-
-
Site Bs
Site Cs
6 (-5.1)
8 (-5.0)
-
The results of the second docking protocol are given in Tables 5 and 6. The compounds
are ranked according to the binding energy of the top-scoring pose for each compound.
Comparing the nine putative binding sites, it is clear that, after considering receptor flexibility,
site Cc (the colchicine binding site) is the site with the highest affinity for the compounds. In fact,
the lowest affinity ligand for this site, compound 11, still scored higher than almost all poses in
the other sites.
Since the colchicine binding site had the highest affinity towards our ligands, it was
predicted to be the primary binding site for these ligands, although we cannot rule out the
possibility of a ligand having more than one binding site.
14

Table 5. Results of the target docking of all the ligands to the curved tubulin model.
Energies are given in kcal/mol.
Site Ac
Site Bc
Site Cc
Site Dc
Site Ec
Site Fc
-6.6
-6.8
-8.9
-6.1
-5.43
-5.26
17
17
17
17
10
10
-6.5
-5.6
-5.6
-6.8
-6.3
-5.9
-8.0
-7.3
-7.1
-5.9
-5.6
-5.1
-5.43
-5.14
-4.55
-5.10
-4.88
-5.10
10
11
16
10
16
11
10
16
11
10
16
11
17
16
11
17
16
17
Table 6. Results of the target docking of all the ligands to the straight tubulin model.
Energies are given in kcal/mol.
Site As
Site Bs
Site Cs
-5.6
-7.2
-5.6
10
17
10
-5.4
-5.3
-5.2
-6.8
-6.6
-6.1
-5.5
-5.0
-4.9
16
17
11
10
16
11
17
16
11
However, the ranking of the binding energies of the compounds at the colchicine binding site
obtained from our computational analysis did not seem to agree with the microtubule stability
experiments. Since binding at the colchicine binding site usually imparts instability30,31, we
expected the compounds that scored best in docking to colchicine binding site to score worst in
MT stability assays and vice versa.
Figure 2 shows the top scoring pose of compound 16 in the colchicine binding site. The
figure shows that the ligand made an arene-H interaction with Asn101 as well as a hydrogen
bond with Asn258.
Fig. 2. Ligand 16 in the colchicine binding site
15

In order to test in vitro the in silico prediction that the active compounds bind to tubulin
at the colchicine binding, a competitive inhibition binding assay to tubulin with colchicine was
performed. Compounds 11 and 16 were selected for this assay. In this assay, tubulin was
incubated with different concentrations (0-70 µM) of the test compound (11 or 16) for 30 min,
then colchicine (10 μM) was added and the samples were incubated for an additional 60 min at
35˚C32. The samples were excited at 360 nm and the fluorescence was measured in the range
390-500 nm (Fig. 3). Each experiment was performed at least three times. Both drugs increased
the tubulin-colchicine fluorescence in a concentration-dependent manner, indicating that the
compounds, although they did not appear to bind to the colchicine site, stabilized colchicine
binding to tubulin, indicating that they bind to tubulin near to but not at the colchicine site.
Fig. 3. Stabilization of colchicine binding to tubulin by 11 and 16. (A) Effects of 11 on colchicine binding to
tubulin. Tubulin was incubated in the absence (solid squares), or in the presence of 30 µM (open circles), 50 µM
(solid triangles), or 70 µM (open triangles) of 11 and the fluorescence measurements were obtained as described in
the Materials and Methods. (B) Effects of 16 on colchicine binding to tubulin. Tubulin was incubated in the absence
(open circles), or in the presence of 10 µM (open squares), 30 µM (asterisks), 50 µM (solid triangles), or 70 µM
16

(open triangles). Both drugs increased the tubulin-colchicine fluorescence in a concentration-dependent manner,
indicating that the compounds stabilized colchicine binding to tubulin.
A
B
400000
300000
200000
100000
0
300000
250000
200000
150000
100000
50000
400
420
440
460
400
420
440
460
wavelength (nm)
Wavelength (nm)
Consequently, it appears that the mode of action of these novel compounds is similar to but not
identical to colchicine with binding in a similar location to  tubulin, which very likely differs in
the effects on tubulin conformation. Colchicine causes a conformation of the tubulin dimer that
is assembly incompetent by the virtue of the induced curvature in its shape. The strength of
binding correlates with anti-proliferatic activity. The new compounds assessed here appear to
have multiple binding sites on tubulin, the highest affinity one is close to the colchicine binding
site but identical to it. The effect of such a displacement on the activity may be a less pronounced
conformational change of the dimer and hence a weaker anti-proliferative effect on cancer cells.
A precedent already exists for a similar behavior since noscapinoids share a binding site close to
the colchicine site on  tubulin, but not exactly at this site. A similar situation exists with
peloruside and laulimalide compounds 33
Acknowledgements
The authors MM and DS are grateful to the Dayalbagh Educational Institute, Dayalbagh, Agra,
India and UGC-BSR for providing the financial support and for providing necessary research
17

facilities to carry out this work. JAT acknowledges support from the Natural Sciences and
Engineering Research Council of Canada and the Allard Foundation. The research in MP
laboratory is supported by the Canadian Breast Cancer Foundation, Prairie-Northwest Chapter.
ML thanks UM-DAE CBS for financial support. T. M. Tilli is supported by a fellowship from
CAPES-Fiocruz (cooperation term 001/2012 CAPES-Fiocruz), and the National Institute for
Science and Technology on Innovation on Neglected Diseases (INCT/IDN, CNPq, 573642/2008-
7).
Author contributions
Conceived and designed the study: MM and DS. Performed the experiments: MM, DS, TM,
TMT, KM. Conducted in silico work: ATA, PW. Contributed to the experimental design and
analysis of the results: JAT, MM, ML, MP, TMT. All authors contributed to writing the
manuscript.
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