Biological and Pharmaceutical Bulletin
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Synthesis of New N1-Substituted-5-aryl-3-(3,4,5-trimethoxyphenyl)-2-pyrazoline Derivatives as Antitumor Agents Targeting the Colchicine Site on Tubulin
Salwa Elmeligie Nadia Abdalla KhalilEman Mohamed AhmedSoha Hussein EmamSawsan Abo-Bakr Zaitone
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2016 Volume 39 Issue 10 Pages 1611-1622

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Abstract

A series of pyrazoline derivatives 2ae, 3ae and 4ae structurally related to combretastatin A4 (CA-4) were synthesized and characterized by spectroscopic means and elemental analyses. In these compounds, the cis double bond of CA-4 was replaced with the pyrazoline ring aiming to enhance the cytotoxic effects displayed by CA-4 and to prevent the cis/trans isomerization that is associated with inactivation of CA-4. The cytotoxic activity of all new compounds was investigated in vitro against MCF-7 and HCT-116 cell lines. The inhibition of tubulin polymerization by the most active compounds 3d, 4a and e was evaluated. The cytotoxicity of 4e was correlated with induction of apoptosis and caspase-3 activation in vitro thus indicating the apoptotic pathway of anticancer effect of these compounds. Furthermore, in vivo evaluation of the synthesized compounds was carried out against Ehrlich’s ascites carcinoma (EAC) solid tumor grown in mice. Compounds 2c, 3a and e showed significant reduction in tumor weight, and about 2–4 fold increase in caspase-3 expression.

Microtubules are attractive targets for cancer chemotherapy. They are composed of α and β tubulin heterodimers that are involved in numerous cellular functions including motility, division and intracellular transport.16) A number of small molecules bind tubulin interfering with microtubule polymerization and depolymerization, thereby inducing cell cycle arrest leading to apoptosis. It is known that the antitumor efficacy of many chemotherapeutic agents is correlated to their ability to promote apoptosis in cancer cells.7) One of the important pathways for apoptosis induction is the activation of caspase-3, which belongs to a family of cysteine protease proteins that are crucial mediators of apoptosis. Therefore, compounds that induce apoptosis in cancer cells is an attractive approach in cancer treatment.8,9)

The majority of microtubule targeting agents are derived from natural sources. Colchicine is a natural tubulin depolymerizing agent however, its therapeutic value against cancer is restricted due to poor bioavailability, high toxicity and low therapeutic index.10) Its structure is formed of three rings; a trimethoxy phenyl ring (A), a seven membered ring with an acetamido group at C7 (B) and a methoxy tropone ring (C). Rings A and C comprise the main pharmacophoric scaffold important for tubulin binding, whereas the central ring B was found to be not essential for tubulin interaction11) (Fig. 1).

Fig. 1. Correlation between Colchicine, CA-4, A-105972 and the Newly Synthesized Compounds 2ae, 3ae, and 4ae

Lately, new tubulin targeting agents have been intensively investigated and developed. Combretastatin A4 acts as both cytotoxic and vascular disrupting agent acting through inhibition of endothelial cell proliferation and vascular tube formation important for angiogenesis.1215) It is structurally related to colchicine where the ring B in colchicine was replaced by a cis-double bond, which allows placement of two benzene rings at right distance and dihedral angle to get maximum interaction with the colchicine binding site.16) Although CA-4 exhibits high cytotoxic and vascular disrupting activities in vitro, it does not show efficacy in vivo because of its poor aqueous solubility as well as isomerization of its cis-double bond into the inactive trans-isomer.17) CA-4 phosphate (Zybrestat) is a water soluble prodrug that is currently in phase III clinical trials.1820) One of the strategies to prevent in vivo cis/trans isomerization associated with the inactivation of CA-4 was the replacement of the olefinic bridge in CA-4 with an appropriate heterocyclic ring aiming to rigidify the structure and restrict its rotation.21,22)

In the present study, the design of the new compounds was based upon the potent cytotoxic activity displayed by the oxadiazoline derivative A-105972 that interacts with the microtubules and induces apoptosis.23) Herein, we performed the synthesis of certain CA-4 analogues containing pyrazoline core (2ae, 3ae and 4ae) (Fig. 1). These compounds possess a two ring scaffold; one of them contains a trimethoxyphenyl group for anchorage, which was reported to efficiently inhibit tubulin polymerization.16,21) Moreover, due to limited water solubility of CA-4, the cis-double bond was replaced with the more polar pyrazoline ring. Furthermore, incorporation of formyl, carboxamide or carbothioamide residues at N1 of pyrazoline moiety is supposed to provide additional hydrogen binding interactions with the colchicine binding site.

RESULTS AND DISCUSSION

Chemistry

As shown in Chart 1, the key intermediate chalcones 1ae have been synthesized by Claisen–Schmidt condensation of 3,4,5-trimethoxyacetophenone and different substituted benzaldehydes in ethanol containing potassium hydroxide as a catalyst.24) Cyclization of 1ae with hydrazine hydrate in formic acid afforded the corresponding N-formyl pyrazolines 2ae. On the other hand, cyclization of 1ae with semicarbazide hydrogen chloride (HCl) or thiosemicarbazide in alkaline medium afforded 3ae and 4ae, respectively.

Chart 1. General Synthetic Pathways of Derivatives 2ae, 3ae and 4ae

Reagents and conditions: a) KOH, stirring; b) NH2NH2·H2O, HCOOH, reflux 8–12 h; c) NH2CONHNH2·HCl, NaOH, reflux 8–12 h; d) NH2CSNHNH2, NaOH, reflux 8–12 h.

The structures of the new compounds 2ae, 3ae and 4ae were confirmed by elemental analyses and spectral data. IR spectra of the carboxaldehyde derivatives 2ae and the carboxamide derivatives 3ae showed a prominent band in the range of 1683–1670 cm−1 corresponding to the carbonyl function. Further, the IR spectra of 3ae and 4ae revealed a forked band in the region of 3473–3253 cm−1 indicating NH2 group. In addition, compounds 4ae showed a characteristic band at 1367–1357 cm−1 corresponding to C=S. All compounds displayed bands corresponding to C=N due to ring closure.

Moreover, the formation of 2-pyrazoline ring in 2ae, 3ae and 4ae was confirmed by the appearance of ABX system in their 1H-NMR spectra as a result of geminal-vicinal coupling. The pyrazoline ring system displayed three signals; a doublet of doublets in the range of 3.07–3.28 ppm (Ha), a multiplet in the range of 3.71–3.94 ppm (Hb) and a doublet of doublets downfield in the range of 5.28–5.89 ppm (Hx).

The protons belonging to the aromatic system and phenyl substituents were observed at the expected chemical shifts and integral values. The aldehydic proton in compounds 2ae was displayed at 8.86–8.93 ppm. D2O exchangeable signals corresponding to NH2 protons in 3ae and 4ae were observed in the range of 6.48–6.56 and 7.96–8.10 ppm, respectively.

Mass spectra of the compounds 2a, 3a and 4a showed the characteristic M and M+2 peaks confirming the presence of Br. In addition, compounds 3ae showed a characteristic fragmentation peak corresponding to M−CONH2, however the mass spectra of the compounds 4ae displayed a peak at m/z 60 corresponding to CSNH2. The synthetic route for the preparation of the new compounds 2ae, 3ae and 4ae is outlined in Chart 1.

Biological Study

In Vitro Screening

Cytotoxic Activity All the synthesized compounds 2ae, 3ae and 4ae were evaluated for cytotoxic activity against breast cancer cell line MCF-7 and colon cancer cell line HCT-116 using sulphorhodamine B stain (SRB) colorimetric assay. The results were summarized and represented graphically (Table 1, Fig. 2).

Table 1. IC50 Values (µM) for the Compounds 2ae, 3ae and 4ae against MCF-7 and HCT-116 Cancer Cell Lines Using SRB Assay
2a–e, 3a–e and 4a–e
Compound No.RR1R2R3XIC50 (µM)
MCF-7HCT-116
2aHBrHHO42.9318.60
2bHOCH3HHO56.699.17
2cOCH3OCH3HHO16.739.24
2dOCH3OCH3OCH3HO86.6531.82
2eHN(CH3)2HHO39.1112.51
3aHBrHNH2O8.5112.43
3bHOCH3HNH2O32.958.56
3cOCH3OCH3HNH2O36.3420.21
3dOCH3OCH3OCH3NH2O8.088.08
3eHN(CH3)2HNH2O77.2942.66
4aHBrHNH2S8.216.88
4bHOCH3HNH2S30.1310.71
4cOCH3OCH3HNH2S53.9946.81
4dOCH3OCH3OCH3NH2S19.5020.80
4eHN(CH3)2HNH2S6.859.64
Colchicine12.313.50
Fig. 2. Cytotoxic Activity of the New Compounds 2ae, 3ae, 4ae and Colchicine against MCF-7 and HCT-116 Cell Lines

Compounds 3a, d, 4a and e displayed the highest inhibitory activity (IC50=6.88–12.43 µM) against both breast and colon cancer cell lines. These compounds showed reasonable cytotoxic activity against MCF-7 cell line and a moderate activity against HCT-116 cell line in comparison to colchicine as a reference compound. Moreover, compounds 2c and 4d revealed moderate cytotoxic activity against MCF-7 with IC50 of 16.73 and 19.50 µM respectively.

Among the pyrazoline derivatives 2ae, compound 2c containing 3,4-dimethoxy substitution on the ring B showed the highest cytotoxic activity against both tested cell lines, whereas compounds 3a and d with the 4-bromo and 3,4,5-trimethoxy substitution respectively were the most active in 3ae series against MCF-7 cell line. Furthermore, compounds 4a and e containing the 4-bromo and 4-dimethylamino substitution respectively showed comparable cytotoxic activity against both tested cancer cell lines.

Data in Table 1 revealed that the carboxamide derivatives 3ae and the carbothioamide derivatives 4ae were more active than the carboxaldehyde derivatives 2ae against both tested cancer cell lines. This may be attributed to presence of NH2 group that could provide additional hydrogen bonding interaction with the colchicine binding site.

Immunohistochemistry Three of the most active compounds in the SRB assay (3d, 4a and e) were subjected for immunohistochemistry evaluation to establish the effect of these compounds on cellular microtubules. MCF-7 cells were treated with 30% of the IC50 of the tested compounds for 48 h, fixed and immunostained for microtubules, then compared to the untreated cells (negative control). The resulted microscope images are presented in Fig. 3.

Fig. 3. Apotome Fluorescence Microscope Images of MCF-7 Cells Treated with DMSO (A), 3d (B), 4a (C) and e (D) for 48 h

Cells were stained with an α-tubulin antibody.

Tubulin analysis in the untreated MCF-7 cells (A) showed living cells with no signs of toxicity as well as distinct acridine stained nucleus with homogenous and normal tubulin expression pattern and normal cytoplasm/nucleous proportion. The cell–cell microtubules mesh was intact and healthy active with a homogenous intracellular distribution. On the other hand, cells treated with the tested compounds 3d, 4a and e showed moderate to high malformed cellular structure with shrinked nuclei, abnormal reduced cytoplasm/nucleous proportion, abnormal tubulin expression pattern and reduction in the cell–cell microtubules mesh. The photo micrographs illustrated that the cytotoxicity of these compounds may be attributed to interference with the microtubule assembly.

Tubulin Polymerization Inhibition Assay Compounds 3d, 4a and e were further investigated for inhibition of tubulin polymerization following enzyme linked immunosorbent assay (ELISA) using human β-tubulin assay kit SEB870Hu (Cloud-Clone Corp., U.S.A.). Colchicine was used as a positive control and the results are summarized in Table 2. The results revealed that the tested compounds produced significant tubulin suppression compared to the reference antimitotic agent colchicine. Therefore, cytotoxic activity of the tested compounds may be referred to their tubulin inhibitory activity.

Table 2. Percentage Inhibition of Tubulin Polymerization at IC50 Concentration for the Compounds 3d, 4a and e
Compound No.IC50 (µg/mL)IC50 (µM)% Inhibition of tubulin polymerization (MCF-7)
3d3.608.0875.70
4a3.708.2184.40
4e2.846.8581.60
Colchicine4.9212.3174.50

Caspase-3 Activation Assay Activation of caspases plays a crucial role in induction of apoptosis. Caspase-3 is one of the key effector caspases that is essential for certain biochemical events and cell morphological changes associated with the execution of programmed cell death. Compound 4e was selected to correlate its cytotoxic activity with the induction of apoptosis through activation of caspase-3. The amount of activated caspase-3 was investigated following ELISA using human active caspase-3 Invitrogen EIA kit, Catalogue No. KHO1091 (Camarillo, CA, U.S.A.). Colchicine was used as a reference and the results are summarized in Table 3 and Fig. 4. The results showed that 4e produced about 5 fold increases in cleaved caspase-3.

Table 3. Caspase-3 Concentration of 4e, Colchicine and Control MCF-7 Cells
Compound No.Concentration of active caspase-3 in ng/mL
4e0.645
Colchicine0.483
Control0.134
Fig. 4. Effect of 4e and Colchicine on Caspase-3 Activity

MCF-7 cells were treated with the tested compounds at IC50 concentration values for 24 h.

In Vivo Evaluation of Antitumor Activity

All newly synthesized compounds 2a–e, 3a–e and 4ae were tested for in vivo antitumor activity against solid Ehrlich’s ascites tumors (EAT) grown in mice and compared to colchicine as the reference compound. The results showed that colchicine produced marginally non-significant decrease in tumor mass compared to EAT control group, which may be attributed to its poor bioavailability.25) On the other hand, all tested compounds except 4c, produced significant reduction in tumor masses in comparison to the control (Fig. 5). Some selected compounds that showed significant reduction in tumor masses; 2c, 3a and e were further investigated for their proapoptotic potential via induction of the expression of caspase-3.

Fig. 5. Effect of Colchicine and the Tested Compounds 2ae, 3ae and 4ae on Tumor Mass in Ehrlich’s Tumor Bearing Mice

Mice were inoculated with Ehrlich’s tumor at day 1 while treatment with different drugs was initiated at day 8. Data were presented as the mean±S.E.M. and analyzed using one-way ANOVA followed by post-hoc analysis at p<0.05 employing SPSS program. * Compared to EAT control. $ Compared to colchicine.

Microscopic examination of solid tumor sections stained with hematoxylin and eosin (H&E) revealed that colchicine did not significantly reduce the total histological score compared to EAT control group. However, the three selected highly-acting compounds 2c, 3a and e showed reduction in the total histological score compared to both EAT control group and colchicine-treated group as well (p<0.05, Table 4, Fig. 6).

Table 4. Scoring for Sections from Solid EAT Grown in Mice Treated with Colchicine or the Tested Compounds
GroupsNecrosis areaGiant cellMitotic pictureTotal score
EAT control3±03±03±09±0
Colchicine2.7±0.22.2±0.22.3±0.27.2±0.5
2c1.8±0.2*$0±0*$1.83±0.2*3.7±0.2*$
3a1.8±0.2*$0±0*$1.83±0.2*3.7±0.2*$
3e1.8±0.2*$0±0*$1.83±0.2*3.7±0.2*$

EAT: Ehrlich’s ascites tumor. Scoring for H&E stained tumors was done as (0) absent, (1) low or weak, (2) mild to moderate and (3) high or frequent and the total score was calculated. Results were presented as the mean±S.E.M. and analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test at p<0.05. * Significantly different from EAT control. $ Significantly different from colchicine group.

Fig. 6. Photographs Showing Sections from Ehrlich’s Solid Tumors Stained with H&E

Photos from upper panel illustrate mitotic picture (dashed circles). Photo from the left side of the lower panel demonstrates tumor giant cell (solid arrow) and the right side demonstrates necrosis area (outlined by the square).

Immunostaining for caspase-3 activity demonstrated low degree of staining in both EAT control group and the colchicine group (Fig. 7a). On the other hand, treatment with the selected new compounds 2c, 3a and e resulted in significant increase in tumoral expression of caspase-3, leading to stronger antitumor activity compared to EAT control group and colchicine-treated group (Fig. 7b).

Fig. 7. Effect of Colchicine and the Tested Compounds 2c, 3a and e on Tumoral Expression of Caspase-3

Mice were inoculated with Ehrlich’s tumor at day 1 while treatment with different drugs was initiated at day 8 for 2 weeks. Data were presented as the mean±S.E.M. and analyzed using one-way ANOVA followed by post-hoc analysis at p<0.05 employing SPSS program. * Compared to EAT control. $ Compared to colchicine.

In vivo results highlighted that colchicine was not efficient in reducing the growth of solid EAT in mice. However, all new derivatives, except 4c, produced significant antitumor activity. Treatment with the three potent compounds 2c, 3a or e resulted in 2–4 fold raise in tumoral caspase-3 expression. Therefore, the antitumor activity produced by the new compounds may be, at least in part, mediated through induction of apoptotic machinery.

Molecular Modelling

Docking simulation was performed to predict the probable binding mode of the most active compounds 3d, 4a and e with the crystal structure of tubulin at the colchicine binding site (PDB ID: 3E22) using discovery studio software version 2.55. The co-crystallized ligand (colchicine) revealed H-bond interaction of the 4-methoxy group in ring A with βCyst241 (distance 2.15 Å). To gain better understanding on the important interactions with the colchicine binding site, we proceeded to examine CA-4 and A-105972 binding modes. CA-4 was docked into the active site of the enzyme and showed three H-bond interactions with two amino acid residues βCyst241 and αAsn101. SH of βCyst241 interacted with oxygen atom of 4-methoxy group in ring A through one H-bond (distance 2.18 Å). In addition, αAsn101 NH2 and C=O groups interacted with oxygen atom of 3-methoxy group and hydrogen atom of OH group in ring B respectively (distance 2.80, 2.05 Å, respectively) through two H-bonds with an estimated binding energy score=−15.50 kcal/mol (Fig. 8).

Fig. 8. 3D Interaction of CA-4 with the Colchicine Binding Site (3E22)

H-Bonds are represented as dashed lines and amino acids involved in interaction with CA-4 (ASN101 and CYS241) are labelled bold.

Both lead compounds, CA-4 and A-105972 showed comparable H-bond interactions and binding energy scores. Docking simulation of A-105972 revealed two H-bond interactions between oxygen atom of 3- and 4-methoxy groups in ring A and SH of βCyst241 (distance 2.28, 2.79 Å, respectively), a H-bond between oxygen of 3-methoxy group in ring B and NH2 of αAsn101 (distance 2.63 Å). In addition, βLys254 was also involved in π–cation interaction with the ring B with an estimated binding energy score=−16.07 kcal/mol (Fig. 9).

Fig. 9. 2D Interaction of A-105972 with the Colchicine Binding Site (3E22)

Similarly, all docked compounds were capable of occupying the colchicine binding site while maintaining the essential key interaction with the same amino acid residue (βCyst241). The trimethoxy phenyl (ring A) in compounds 3d, 4a and e was superimposed with that of colchicine. Compound 3d was bound to tubulin at the colchicine binding site through five H-bonds with three amino acid residues; βCyst241, βAsn258 and αAsn101 with an estimated binding energy score=−11.68 kcal/mol. βCyst241 forms two SH…O H-bonds (distance 2.97, 2.24 Å) with oxygens of 3- and 4-methoxy groups of ring A respectively, while βAsn258 forms two H-bonds (distance 2.47, 2.86 Å) with the NH2 hydrogens. Furthermore, αAsn101 was bound to 3-methoxy group of ring B through one H-bond (distance 2.13 Å). In addition, βLys352 was involved in π–cation interaction with the ring B (trimethoxyphenyl) (Fig. 10). Although 3d was involved in several H-bonding interactions with the colchicine binding site, it showed lower binding energy score compared to 4a and e. This may be referred to steric hindrance associated with the presence of trimethoxy group on ring B leading to twisting of the compound 3d to an unfavourable orientation.

Fig. 10. 2D Interaction of 3d with the Colchicine Binding Site (3E22)

Compounds 4a and e showed comparable both cytotoxic activity and free binding energy scores. Compound 4a was bound appropriately through four H-bonds with βCyst241, αThr179 and βLys254 with an estimated interaction energy=−15.27 kcal/mol. βCyst241 forms a H-bond with the oxygen of 4-methoxy group in ring A (distance 2.34 Å). It is worth to mention that 4a takes a different orientation so that carbothioamide moiety is in proximity to αThr179 forming two H-bonds with hydrogens of the NH2 group (distance 2.13, 2.63 Å) (Fig. 9). H-Bonding with αThr179 amino acid residue at the colchicine binding site was reported to increase the cytotoxic activity.26) βLys254 was bound to the bromine atom in ring B through a H-bond (distance 2.71 Å). It was also involved in π–cation interaction with 4-bromophenyl ring (Fig. 11).

Fig. 11. 2D Interaction of 4a with the Colchicine Binding Site (3E22)

Binding model of 4e revealed three H-bond interactions with βCyst241 and βAsn258 with an estimated binding free energy score=−16.28 kcal/mol. SH of βCyst241 forms one H-bond (distance 2.35 Å) with the oxygen of 4-methoxy group in ring A, while βAsn258 forms two H-Bonds; one with the hydrogen atom of NH2 group (distance 2.18 Å) and the other with the sulphur atom of C=S (distance 2.87 Å) (Fig. 12). H-Bonding with the polar amide amino acid residue βAsn258 increases stability of the ligand–colchicine binding site complex.27,28) The binding modes of 3d, 4a and e were similar to that of CA-4 and A-105972, they interacted with similar amino acid residues including βCyst241, αAsn101 and βLys254 and showed comparable binding energy scores=−11.68 to −16.28 kcal/mol. The length of all H-bond interactions was less than 3 Å. It is clear that these compounds fit well in the colchicine binding site showing good interactions and binding free energy scores compared to colchicine.

Fig. 12. 3D Interaction of 4e with the Colchicine Binding Site (3E22)

H-Bonds are represented as dashed lines and amino acids (CYS241 and ASN258) involved in interaction with 4e are labelled bold.

CONCLUSION

In this study, modifications of prior lead compound A-105972 resulted in the synthesis of three series of 2-pyrazoline derivatives as a novel class of tubulin polymerization inhibitors that bind to the colchicine site on tubulin. Four compounds; 3a, d, 4a and e showed reasonable cytotoxic activity compared to colchicine against MCF-7 cell line with IC50 (6.85–8.51 µM) range, and a moderate effect against HCT-116 cell line with IC50 (6.88–12.43 µM) range. Three most active compounds 3d, 4a and e showed percentage suppression of tubulin comparable to that of colchicine in breast cancer cells (MCF-7) (75.7, 84.4, 81.6%, respectively). Immunohistochemistry assay showed that the cytotoxic effect exerted by these compounds may be referred to the interference with the microtubule assembly.

In vivo studies against Ehrlich’s ascites (EAT) solid tumor grown in mice revealed that significant reduction in tumor weight has been shown by the compounds 2c, 3a and e. These compounds demonstrated 2–4 fold increases in tumoral expression of caspase-3 compared to control.

Aiming to rationalize the biological results, molecular docking was further performed. After analysis of the binding modes of the compounds 3d, 4a and e with the colchicine site of tubulin and comparing the results with that of CA-4 and A-105972, it was obvious that these compounds could appropriately bind to the colchicine binding site of α- and β-tubulin through H-bond interactions with βCyst241, which may play a crucial role in its antitubulin polymerization and antiproliferative activities. In addition, the NH2 group in the pyrazoline derivatives 3ae and 4ae also interacted with βAsn258 or αThr179. Therefore, tubulin could be considered a good target for pyrazoline derivatives containing a trimethoxyphenyl scaffold.

MATERIALS AND METHODS

Chemistry

The starting materials 1ae were prepared as reported. Other chemicals and reagents were obtained from Aldrich, Fluca or Merck and were used without further purification. Progress of the reactions was monitored using TLC sheets precoated with UV fluorescent silica gel Merck 60F 254. The solvent system was benzene–chloroform–methanol (5 : 9 : 1) and spots were visualized using UV lamp. IR spectra were determined on Shimadzu FT-IR 8400 s spectrophotometer (KBr, cm−1). 1H-NMR spectra were carried out using a Mercury 300-BB 300 MHz and Bruker 400 MHz spectrophotometers using tetramethylsilane (TMS) as internal standard. 13C-NMR spectra were carried out using a Mercury 300-BB 75 MHz and Bruker 100 MHz using TMS as internal standard. Chemical shifts (δ) are recorded in ppm on δ scale, Microanalytical Unit, Faculty of Pharmacy, Cairo University, Egypt. Mass spectra were performed on Shimadzu QP-2010 plus mass spectrophotometer at 70 eV. Microanalytical Unit, Faculty of Science, Cairo University, Egypt. Elemental analyses were carried out at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Egypt. Melting points were determined on Stuart apparatus and the values given are uncorrected.

General Procedure for the Preparation of 2a–e

To a mixture of the appropriate chalcone 1ae (0.001 mol) and formic acid (5 mL), hydrazine hydrate 99% (0.001 mol, 0.05 g) was added. The reaction mixture was heated under reflux for 8–12 h, then cooled and poured onto ice-cold water. The solid product formed was filtered and crystallized from ethanol.

5-(4-Bromophenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbaldehyde (2a)

Yield 90%; mp 147–148°C; 1H-NMR (DMSO-d6, 300 MHz) δ (ppm): 3.26 (1H, dd, Jax=5 Hz, Jab=18 Hz), 3.71 (3H, s), 3.82 (6H, s), 3.88–3.94 (1H, m), 5.51 (1H, dd, Jax=5 Hz, Jbx=12 Hz), 7.06 (2H, s), 7.19 (2H, d, J=7.5 Hz), 7.54 (2H, d, J=7.5 Hz), 8.90 (1H, s); IR (KBr) cm−1: 3074, 2968, 2918, 2798, 1670; electron ionization (EI)-MS m/z (% rel. int.): 419.80 (M+2), 418.85 (M+H), 417.80 (M+), 234.95, 208.95, 193.95. Anal. Calcd for C19H19BrN2O4: C, 54,43; H, 4.57; N, 6.68. Found: C, 54.58; H, 4.62; N, 6.81.

5-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbaldehyde (2b)

Yield 69%; mp 129–130°C; 1H-NMR (DMSO-d6, 300 MHz) δ (ppm): 3.23 (1H, dd, Jax=4 Hz, Jab=18 Hz), 3.69 (3H, s), 3.72 (3H, s), 3.82 (6H, s), 3.86–3.90 (1H, m), 5.46 (1H, dd, Jax=5 Hz, Jbx=12 Hz), 6.89 (2H, d, J=7 Hz), 7.08 (2H, s), 7.16 (2H, d, J=7 Hz), 8.88 (1H, s); IR (KBr) cm−1: 3062, 2931, 2868, 2829, 1674; EI-MS m/z (% rel. int.): 370.85 (M+H), 369.95 (M+, 100), 340.90, 193.95; Anal. Calcd for C20H22N2O5: C, 64.85; H, 5.99; N, 7.56. Found: C, 65.07; H, 6.07; N, 7.63.

5-(3,4-Dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbaldehyde (2c)

Yield 81%; mp 156–157°C; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 3.25 (1H, dd, Jax=4.8 Hz, Jab=18 Hz), 3.64 (3H, s), 3.73 (3H, s), 3.74 (3H, s), 3.83 (6H, s), 3.84–3.90 (1H, m), 5.46 (1H, dd, Jax=5 Hz, Jbx=12 Hz), 6.70 (1H, d, J=8 Hz), 6.84 (1H, s), 6.90 (1H, d, J=8 Hz), 7.09 (2H, s), 8.90 (1H, s); IR (KBr) cm−1: 3001, 2941, 2887, 2829, 1674; EI-MS m/z (% rel. int.): 401.00 (M+H), 400.00 (M+), 371, 234.95, 208.95, 91.00; Anal. Calcd for C21H24N2O6: C, 62.99; H, 6.04; N, 7.00. Found: C, 63.17; H, 6.08; N, 7.08.

5-(3,4,5-Trimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbaldehyde (2d)

Yield 90%; mp 149–150°C; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 3.28 (1H, dd, Jax=4 Hz, Jab=18 Hz), 3.64 (3H, s), 3.70 (3H, s), 3.75 (6H, s), 3.83 (6H, s), 3.83–3.87 (1H, m), 5.46 (1H, dd, Jax=5 Hz, Jbx=12 Hz), 6.51 (2H, s), 7.06 (2H, s), 8.93 (1H, s); 13C-NMR (DMSO-d6) ppm: 43.09, 56.47, 59.23, 60.60, 103.08, 104.65, 126.65, 137.13, 137.64, 140.00, 153.61, 156.57, 159.45, 160.20; IR (KBr) cm−1: 3074, 2937, 2887, 2825, 1670; EI-MS m/z (% rel. int.): 430.95 (M+H), 430.00 (M+), 429.05 (M−H), 401, 234.95, 194.00; Anal. Calcd for C22H26N2O7: C, 61.39; H, 6.09; N, 6.51. Found: C, 61.54; H, 6.13; N, 6.62.

5-(4-Dimethylaminophenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbaldehyde (2e)

Yield 71%; mp 144–145°C; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 2.85 (6H, s), 3.22 (1H, dd, Jax=4 Hz, Jab=18 Hz), 3.71 (3H, s), 3.79 (6H, s), 3.83–3.86 (1H, m), 5.40 (1H, dd, Jax=4 Hz, Jbx=11.5 Hz), 6.67 (2H, d, J=8.5 Hz), 7.02 (2H, d, J=8.5 Hz), 7.06 (2H, s), 8.86 (1H, s); IR (KBr) cm−1: 3009, 2939, 2862, 2794, 1680; EI-MS m/z (% rel. int.): 384.00 (M+H), 383.05 (M+), 382.05 (M−H), 208.95, 147.05, 146.05, 134.05, 121.05; Anal. Calcd for C21H25N3O4: C, 65.78; H, 6.57; N, 10.96. Found: C, 65.89; H, 6.59; N, 11.13.

General Procedure for the Preparation of 3a–e and 4a–e

To a solution of the appropriate chalcone 1ae (0.001 mol) and semicarbazide HCl or thiosemicarbazide (0.001 mol) in ethanol (5 mL), was added a solution of sodium hydroxide (0.08 g, 0.002 mol) in water (1 mL), then the reaction mixture was heated under reflux for 8–12 h, cooled and poured into crushed ice. The separated solid was filtered, dried and crystallized from ethanol.

5-(4-Bromophenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (3a)

Yield 78%; mp 209–210°C; 1H-NMR (DMSO-d6, 300 MHz) δ (ppm): 3.08 (1H, dd, Jax=5 Hz, Jab=18 Hz), 3.67 (3H, s), 3.72–3.78 (1H, m), 3.81(6H, s), 5.36 (1H, dd, Jax=5 Hz, Jbx=12 Hz), 6.53 (2H, s, D2O exchangeable), 7.08 (2H, s), 7.12 (2H, d, J=8 Hz), 7.55 (2H, d, J=8 Hz); IR (KBr) cm−1: 3462, 3277, 3064, 2993, 2929, 1670; EI-MS m/z (% rel. int.): 434.80 (M+2), 433.80 (M+H), 432.75 (M+), 389.80, 234.90, 103, 90.95; Anal. Calcd for C19H20BrN3O4: C, 52.55; H, 4.64; N, 9.68. Found: C, 52.70; H, 4.71; N, 9.79.

5-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (3b)

Yield 68%; mp 161–162°C; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 3.08 (1H, dd, Jax=5 Hz, Jab=18 Hz), 3.69 (3H, s), 3.72 (3H, s), 3.74–3.78 (1H, m), 3.82 (6H, s), 5.34 (1H, dd, Jax=5 Hz, Jbx=12 Hz), 6.55 (2H, s, D2O exchangeable), 6.87 (2H, d, J=8 Hz), 7.09 (2H, s), 7.64 (2H, d, J=8 Hz); IR (KBr) cm−1: 3454, 3275, 3066, 2954, 2908, 1670; EI-MS m/z (% rel. int.): 385.90 (M+H), 384.90 (M+), 340.90, 234.90, 208.95, 193.95, 91.00; Anal. Calcd for C20H23N3O5: C, 62.33; H, 6.01; N, 10.90. Found: C, 62.67; H, 6.12; N, 11.08.

5-(3,4-Dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (3c)

Yield 69%; mp 175–176°C; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 3.09 (1H, dd, Jax=5 Hz, Jab=18 Hz), 3.68 (3H, s), 3.72 (3H, s), 3.73 (3H, s), 3.74–3.77 (1H, m), 3.82 (6H, s), 5.33 (1H, dd, Jax=5 Hz, Jbx=12 Hz), 6.56 (2H, s, D2O exchangeable), 6.65 (1H, d, J=8 Hz), 6.80 (1H, s), 6.88 (1H, d, J=8 Hz), 7.09 (2H, s); 13C-NMR (DMSO-d6) ppm: 42.42, 55.44, 55.56, 55.96, 59.60, 60.06, 105.62, 109.58, 111.99, 117.07, 127.07, 136.23, 138.90, 147.82, 148.77, 150.48, 152.91, 155.07; IR (KBr) cm−1: 3431, 3292, 3010, 2960, 2831, 1683; EI-MS m/z (% rel. int.): 415.05 (M+), 373.05, 372.05, 371.05, 235.00, 194.00; Anal. Calcd for C21H25N3O6: C, 60.71; H, 6.07; N, 10.11. Found: C, 60.87; H, 6.18; N, 10.27.

5-(3,4,5-Trimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (3d)

Yield 84%; mp 171–172°C; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 3.12 (1H, dd, Jax=5.8 Hz, Jab=18 Hz), 3.64 (3H, s), 3.68 (s, 3H), 3.71 (6H, s), 3.74–3.80 (1H, m), 3.83 (6H, s), 5.34 (1H, dd, Jax=5.8 Hz, Jbx=12 Hz), 6.48 (2H, s), 6.62 (2H, s, D2O exchangeable), 7.10 (2H, s); 13C-NMR (DMSO-d6) ppm: 43.09, 56.30, 56.46, 60.37, 60.57, 102.86, 104.50, 127.54, 136.82, 140.13, 151.03, 153.40, 153.48, 155.70, 157.28; IR (KBr) cm−1: 3473, 3358, 3074, 2937, 2837, 1681; EI-MS m/z (% rel. int.): 446.00 (M+H, 26.92), 445.00 (M+), 444.05 (M−H), 402.05, 401.05, 235.00, 209.00, 194.00, 91.00; Anal. Calcd for C22H27N3O7: C, 59.32; H, 6.11; N, 9.43. Found: C, 59.47; H, 6.17; N, 9.48.

5-(4-Dimethylaminophenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (3e)

Yield 77%; mp 181–182°C; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 2.85 (6H, s), 3.07 (1H, dd, Jax=5 Hz, Jab=18 Hz), 3.68 (3H, s), 3.71–3.75 (1H, m), 3.82 (6H, s), 5.28 (1H, dd, Jax=5 Hz, Jbx=12 Hz), 6.49 (2H, s, D2O exchangeable), 6.98 (2H, d, J=8 Hz), 7.09 (2H, s), 7.49 (2H, d, J=8 Hz); 13C-NMR (DMSO-d6) ppm: 40.22, 42.29, 55.98, 59.35, 60.07, 103.93, 112.45, 127.67, 131.32, 138.84, 140.37, 149.63, 152.92, 154.93, 156.92; IR (KBr) cm−1: 3471, 3263, 3095, 2972, 2846, 1683; EI-MS m/z (% rel. int.): 399.05 (M+H), 398.10 (M+), 354.10, 205.00, 134.10, 121.10; Anal. Calcd for C21H26N4O4: C, 63.30; H, 6.58; N, 14.06. Found: C, 63.43; H, 6.62; N, 14.35.

5-(4-Bromophenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (4a)

Yield 65%; mp 199–200°C; 1H-NMR (DMSO-d6, 300 MHz) δ (ppm): 3.20 (1H, dd, Jax=3 Hz, Jab=18 Hz), 3.69 (3H, s), 3.83 (6H, s), 3.85–3.89 (1H, m), 5.89 (1H, dd, Jax=3 Hz, Jbx=11 Hz), 7.13–7.19 (6H, m), 8.04 (2H, s, D2O exchangeable); IR (KBr) cm−1: 3462, 3280, 3061, 2987, 2931, 1357; EI-MS m/z (% rel. int.): 450.75 (M+2), 449.75 (M+H), 448.75 (M+), 59.90; Anal. Calcd for C19H20BrN3O3S: C, 50.67; H, 4.48; N, 9.33. Found: C, 50.81; H, 4.53; N, 9.41.

5-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (4b)

Yield 59%; mp 210–211°C; 1H-NMR (DMSO-d6, 300 MHz) δ (ppm): 3.17 (1H, dd, Jax=3 Hz, Jab=18 Hz), 3.68 (3H, s), 3.71 (3H, s), 3.76–3.79 (1H, m), 3.82 (6H, s), 5.83 (1H, dd, Jax=3 Hz, Jbx=11 Hz), 6.85 (2H, d, J=8.5 Hz), 7.03 (2H, d, J=8.5 Hz), 7.18 (2H, s), 8.01 (2H, s, D2O exchangeable); IR (KBr) cm−1: 3439, 3273, 3074, 2995, 2933, 1367; EI-MS m/z (% rel. int.): 400.80 (M+), 206.90, 192.90, 59.90; Anal. Calcd for C20H23N3O4S: C, 59.83; H, 5.77; N, 10.47. Found: C, 59.99; H, 5.83; N, 10.65.

5-(3,4-Dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (4c)

Yield 75%; mp 177–178°C; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 3.21 (1H, dd, Jax=3 Hz, Jab=18 Hz), 3.69 (3H, s), 3.71 (3H, s), 3.72 (3H, s), 3.73–3.82 (1H, m), 3.83 (6H, s), 5.85 (1H, dd, Jax=3 Hz, Jbx=11 Hz), 6.57 (1H, d, J=8 Hz), 6.79 (1H, s), 6.87 (1H, d, J=8 Hz), 7.18 (2H, s), 8.04 (2H, s, D2O exchangeable); 13C-NMR (DMSO-d6) ppm: 42.95, 55.98, 56.03, 56.54, 60.59, 63.14, 105.15, 110.19, 112.38, 117.37, 126.84, 136.02, 140.01, 148.24, 149.17, 153.45, 155.37, 176.46; IR (KBr) cm−1: 3446, 3321, 3061, 2933, 2833, 1367; EI-MS m/z (% rel. int.): 432.00 (M+H), 431.00 (M+), 430.05 (M−H), 371.00, 222.95, 163, 91.00, 59.95; Anal. Calcd for C21H25N3O5S: C, 58.45; H, 5.84; N, 9.74. Found: C, 58.62; H, 5.88; N, 9.89.

5-(3,4,5-Trimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (4d)

Yield 79%; mp 189–190°C; 1H-NMR (DMSO-d6, 400 MHz) δ (ppm): 3.25 (1H, dd, Jax=3.5 Hz, Jab=18 Hz), 3.64 (3H, s), 3.70 (3H, s), 3.73 (6H, s), 3.74–3.83 (1H, m), 3.84 (6H, s), 5.86 (1H, dd, Jax=3.5 Hz, Jbx=11 Hz), 6.43 (2H, s), 7.19 (2H, s), 8.10 (2H, s, D2O exchangeable); 13C-NMR (DMSO-d6) ppm: 43.01, 56.34, 56.54, 60.38, 60.58, 63.53, 102.94, 105.20, 126.76, 136.82, 139.35, 140.04, 153.44, 155.46, 176.70 (C=S); IR (KBr) cm−1: 3446, 3331, 3074, 2937, 2840, 1367; EI-MS m/z (% rel. int.): 461.95 (M+H), 461.00 (M+), 460.00 (M−H), 401.00, 267.95, 266.95, 234.95, 91.00, 59.95; Anal. Calcd for C22H27N3O6S: C, 57.25; H, 5.90; N, 9.10. Found: C, 57.49; H, 5.98; N, 9.23.

5-(4-Dimethylaminophenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (4e)

Yield 69%; mp 169–170°C; 1H-NMR (DMSO-d6, 300 MHz) δ (ppm): 2.83 (6H, s), 3.20 (1H, dd, Jax=3 Hz, Jab=18 Hz), 3.68 (3H, s), 3.82 (6H, s), 3.84–3.92 (1H, m), 5.89 (1H, dd, Jax=3 Hz, Jbx=11 Hz), 6.66–7.58 (6H, m), 7.96 (2H, s, D2O exchangeable); IR (KBr) cm−1: 3410, 3253, 3061, 2993, 2931, 1361; EI-MS m/z (% rel. int.): 413.90 (M+), 219.90, 146.00, 59.95; Anal. Calcd for C21H26N4O3S: C, 60.85; H, 6.32; N, 13.52. Found: C, 61.04; H, 6.41; N, 13.74.

Biological Study

In Vitro Screening

Growth Inhibition Assay The response of two different carcinoma cell lines; colon cancer cell line (HCT-116) and breast cancer cell line (MCF-7) to the newly synthesized compounds was evaluated by the determination of cell survival using SRB assay following the method of Skehan et al.29) Colchicine was used as a positive control. Cells from different cell lines were cultured in 96-well plate (104 cells/well) for 24 h before treatment with the compounds, to allow cell attachment. Different concentrations of each compound (0, 1, 2.5, 5, 10 µg/mL) were added to the cell monolayer triplicate wells, which were prepared for each individual dose. Cells were incubated with the compounds for 48 h at 37°C and in atmosphere of 5% CO2, then they were fixed, washed, and stained with sulphorhodamine B stain. Excess stain was washed with acetic acid and the attached stain was recovered with tris ethylenediaminetetraacetic acid (EDTA) buffer. Color intensity was measured using an ELISA reader. The relation between surviving fraction and drug concentration was plotted to get the survival curve of each tumor cell line. The IC50 value was calculated using sigmoidal dose response curve-fitting models (GraphPad, Prizm Software Incorporated).

Immunohistochemistry Human breast carcinoma cells were cultured onto multi-well confocal adhesive slides until confluence. The cells were treated with 30% of the IC50 of the tested compounds for 48 h then fixed on slides by glacial acetic acid–methanol mixture. Antigen retrieval was performed, then the slides were incubated overnight at 4°C with polyclonal rabbit α-tubulin antibody (1 : 1000) [Abcam, U.S.A.]. After washing, slides were soaked in conjugated fluorescein isothiocyanate (FITC) goat immunoglobulin G (IgG) antibodies (1 : 2000) [Abcam] then in acridine orange solution (100 µg/mL in phosphate buffered saline (PBS)) for 10 min, and washed by PBS. Fluorescent images were visualized using Apotome fluorescence microscope (Axiostar Plus, Zeiss, Gottingen, Germany) equipped with image analyzer and digital camera (PowerShot A20, Canon, U.S.A.).30)

In Vitro Tubulin Polymerization Assay MCF-7 cells were obtained from American Type Culture Collection, and cultured using Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen/Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 10 µg/mL of insulin (Sigma-Aldrich, MO, U.S.A.), and 1% penicillin–streptomycin. All other chemicals and reagents were purchased from Sigma, or Invitrogen. MCF-7 cells were cultured in 96 well plate (10000 cells/well, cells density 1.2–1.8). The cells were incubated with the tested compounds at IC50 concentration of each compound (Table 2) 24 h before enzyme assay. Tubulin polymerization inhibitory activity was determined using human β-tubulin SEB870HU assay kit (Cloud-Clone Corp., U.S.A.). The procedure of the used kit was performed according to the manufacturer’s instructions.

In Vitro Caspase-3 Activation Assay MCF-7 cells were purchased from American Type Culture Collection. Cells were grown in Roswell Park Memorial Institute medium (RPMI) 1640 containing 10% foetal bovine serum at 37°C, stimulated with the compounds to be tested for caspase-3 at their IC50 concentration and lysed with cell extraction buffer. This lysate was diluted in standard diluent buffer and measured for human active caspase-3 content. The assay was performed using human active caspase-3 Invitrogen EIA kit, Catalogue No. KHO1091 (Camarillo, CA, U.S.A.). The procedure of the used kit was done according to the manufacturer’s instructions.

In Vivo Evaluation of Antitumor Activity

Induction of Solid EAT in Mice In this study, adult female Swiss albino mice were used under fixed housing conditions under normal dark/light cycle. Mice were housed in clean polyethylene cages with food and water provided ad libitum. The study protocol was approved by the Research Ethics Committee at the Faculty of Pharmacy, Suez Canal University (license number 201412A2). A mouse carrying EAT cell line was purchased from the Department of Tumor Biology at the National Cancer Institute (Cairo, Egypt). First, Trypan blue exclusion test was employed to detect the viability of EAT cells according to the method reported previously.31) Second, a suspension of EAT cells was prepared in sterile saline to obtain a concentration equals 2.5 million of EAT cells/0.1 mL. At the beginning of the experiment, mice were shaved at their back and inoculated subcutaneously with 0.1 mL of the EAT suspension in their back.

Experimental Design EAT is commonly employed as a solid form to test antitumor activity of drugs and natural compounds.32,33) Seven days after inoculation with the tumor cells, mice were randomly divided into 17 groups. The therapeutic regimens (or vehicle) were initiated at day 8 for 14 d as follows, Group 1: mice treated with DMSO (5 mL/kg, intraperitoneally (i.p.)) daily, Group 2: mice treated with the reference compound, colchicine (1 mg/kg, i.p.) daily,25) Groups 3–7: mice were treated with compounds 2a, b, c, d and e (1 mg/kg, i.p.) daily, Groups 8–12: mice were treated with compounds 3a, b, c, d and e (1 mg/kg, i.p.) daily, Groups 13–17: mice were treated with compounds 4a, b, c, d and e (1 mg/kg, i.p.) daily. All therapies were continued for 14 d.

Measurement of Tumor Mass and Histopathological Examination At day 22, mice were anesthetized by ether, sacrificed by cervical dislocation and the skin on the back was cut to dissect the implanted solid tumors. The tumors were weighed and immersed in 10% formalin solution then embedded in paraffin and sectioned at 4 µm for routine staining with H&E. Histopathological examination for the tumor sections determined the extension of necrotic area, the presence of neoplastic giant cells and demonstrated the typical mitotic picture under the light microscope. It was done blindly and each of these findings was scored according to their frequency in the microscopic fields as: (0) absent, (1) weak or low, (2) mild to moderate and (3) high or frequent. The total of these scores was calculated and processed for statistical comparison.32)

Immunohistochemical Staining for Caspase-3 and Image Analysis Immunohistochemical staining for caspase-3 was done on another 4 µm tissue section. Primary antibodies against caspase-3 [ab4051], supplied by Abcam Company (Cambridge Science Park, Cambridge CB4 0FL, U.K.), were added to each section and incubated at 4°C. The biotinylated secondary antibodies were added followed by the enzyme conjugate and 3,3-diaminobenzidine, which was used as a chromogen. Images from different areas covering the surface of the cut section were captured under a light microscope and processed for analysis using the Image J 1.45 system (National Institute of Health, U.S.A.). The percent of the area of caspase-3 immunostaining was determined and compared.

Statistical Analysis Data of the experiment were collected and presented as the mean±standard error of the mean (S.E.M.). One-way ANOVA was employed for statistical analysis and was followed by Bonferroni’s post-hoc analysis at p value <0.05. Statistical tests were performed using the statistical package for social science (Chicago, U.S.A.), version 16.

Molecular Docking

The X-ray crystal structure of DAMA–colchicine–tubulin complex (PDB code 3E22) was downloaded from http://www.rscb.org/pdb. All molecular modelling calculations and docking studies were carried out using Discovery Studio software (version 2.55). Automatic protein preparation was done using CHARMm forcefield. 3D structures of target compounds were built using ChemBioDraw Ultra 11.0. Our ligands were prepared using Accelry’s discovery studio prepare ligands protocol. The validation results showed the same binding interactions of the co-crystallized and the re-docked ligand with rmsd of 0.50 Å and docking score of −6.66 kcal/mol.

Acknowledgments

The authors would like to thank Dr. Amira A. Mohammed, Histology Department, Faculty of Veterinary Medicine, Suez Canal University for her help in histopathology and photomicrography and Dr. Mohammed Saad, Pathology Department, Faculty of Medicine, Suez Canal University, Egypt for his help in caspase-3 immunostaining. Authors are also thankful to Prof. Amira M. Gamal-Eldeen, Head of Cancer Biology laboratory, Center of Excellence for Advanced Sciences, National Research Center, Egypt for carrying out immunohistochemical analysis of tubulin and Dr. Essam Rashwan, Head of Confirmatory Diagnostic Unit, Vacsera, Egypt for performing ELISA assay for tubulin and caspase-3.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES
 
© 2016 The Pharmaceutical Society of Japan
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