A Novel Class of Pyrazoline Analogue of Combretastatin-A4 (CA-4): Synthesis Characterization and in-vitro Biological Testing

: A series of pyrazoline bridged combretastatin analogues were designed and synthesised from their precursor chalcone analogues, and all these compounds were ascertained by IR, 1 H NMR, and mass spectral analysis. Subsequently, all these compounds were evaluated for anticancer activities against breast cancer (MCF-7) and normal Vero (Monkey Kidney) cell lines, and five selected compounds from the series were evaluated against Hela (Human Cervical), MDA-MB-231 (Breast), and A-549 (Lung cancer) cell lines using the Sulforhodamine B (SRB) assay method. Compounds 3a, 6a, 6e, 5b, 7a, 5a, and 7d were found to be the most potent in the series, with a GI 50 value of 10 to 30 M in the MCF-7 cell line. Moreover, the same compounds 6a and 7a showed remarkable cytotoxicity against the A-549 (Lung) cell line with a GI 50 value ranging from 10 to 30 M, while compound 3a displayed moderate cytotoxicity against the Hela (Human Cervical) cell line. All these compounds were found nontoxic to the Vero (Monkey Kidney) normal cell line .


INTRODUCTION
ANCER is considered the most serious health issue all over the world. Since most of the currently available chemotherapeutic agents come with multi-drug resistance and acute toxicity. [1] To encounter the future threats arising due to cancer, progressive studies are being conducted towards understanding the biological processes involved in the development of cancer, which may aid in developing more potent and effective anticancer agents for the complete extinction of the disease. [2] Thus, in the pursuit of a more effective anticancer drug, a large number of structurally diverse synthetic and natural products have been explored for their anticancer potential. [3] Microtubules have been identified as an important structural component of the cytoskeleton and are involved in a wide range of critical cellular functions during cell division. [4] Microtubules are composed of α-and β-tubulin heterodimers that form a dynamic equilibrium in which the tubulin dimers polymerize into long cylindrical microtubules that then depolymerize into individual tubulin units. Therefore, microtubule dynamics is one of the most attractive strategies for the development of cancer therapeutics. [5,6] Any disruption in the dynamic equilibrium blocks cell division and may lead to the induction of mitochondrial apoptosis. [7,8] Small molecules that can disturb microtubule dynamics and cause mitotic arrest are extremely important in current cancer chemotherapy. [9] Over the past few decades, combretastatin-A4 (CA-4, 1, Figure 1) has been renowned for being simpler, small molecules, easy to synthesize, and displaying potency by binding with the colchicine binding site and disrupting microtubule polymerization, which induces rapid vascular disruption that eventually leads to tumour cell death. [10] Subsequently, a number of CA-4 analogues have been synthesised in the laboratory that showed substantial antitubulin activities. But, unfortunately, most of them failed to C receive appreciable results clinically due to poor bioavailability. [11][12][13] Consequently, it is now a challenge to synthesize CA-4 analogues with enhanced clinical potency.
Most combretastatin analogues that have been synthesised thus far have been modified at ring B or at the olefinic bridge. [14] Analysis of these analogues shed light on the fact that ring A with 3,4,5-trimethoxyphenyl groups displays a significant role in disrupting microtubule dynamics. While the cis-stilbene configuration at the bridge in the case of CA-4 has been reported to be essential for potent anti-tubulin polymerization. [15] However, the cis configuration of CA-4 is susceptible to isomerization during storage and after administration, which consequently results in a decrease in the anti-tubulin activity. [16] On the contrary, the CA-4 analogues have been synthesised with replacement of the olefinic bond either with a fivemembered or six-membered heterocyclic ring, which not only preserved the appropriate geometric orientation required between the two phenyl rings of CA-4 but is also effective for efficient interaction with the colchicine site of tubulin. [17] For instance, cyclohexanone, pyrazoline, thiophene, [13,18] pyrimidine, [19,20] pyridine, [21,22] (2, 3, and 4 Figure 1). Several of these compounds were synthesised in the author's laboratory and exhibited potent cytotoxic activity.
In continuation of our earlier work to identify possible anticancer agents [19,[22][23] , we herein aim to report combretastatin CA-4 analogues having the same substituents on the A-ring while altering the B-ring and stilbene bridgehead linker with pyrazoline, a 3-carbon linker, as possible anticancer agents.

Materials and Methods
All Chemicals required for the experiment were of AR/GR quality and purchased from Aldrich (Sigma-Aldrich), Spectrochem, or Lancaster (Alfa Aesar, Johnson Matthey Company) and used without any purification. The reaction course was monitored by TLC using chloroform/methanol as the mobile phase on pre-coated F254 Merck TLC plates and visualized in a UV/fluorescent analysis cabinet and/or iodine chamber. Organic solutions obtained after the reaction were dried over anhydrous Na2SO4. The melting points of compounds were determined with a digital thermometer and are uncorrected. Infrared (IR) spectra of compounds were recorded on the Infrared FT-IR Spectrometer, Nicolet iS10; Thermo Electron Scientific, USA and values were denoted in cm -1 . The mass spectra of compounds were established on the Shimadzu LCMS-2010 EV (Maharashtra, India). 1 H NMR and 13 C NMR spectra were recorded at 400 MHz on the FT-Nuclear Magnetic Resonance Spectrometer (FT-NMR), Bruker AVIII, Switzerland using CDCl3 solvent and chemical shift valves were noted in parts per million on δ scale. The coupling constant (J) is denoted in hertz (Hz). All the compounds showed 1 H NMR spectra in agreement with the assigned structures.

Anticancer Activity Experimental Procedure for SRB Assay
The cytotoxic activity of the compounds was measured against five human cell lines: MCF-7 (breast carcinoma) Hela (human Cervical), MDA-MB-231 (Breast), A-549 (Lung) and Vero (Monkey kidney normal cell line) (breast) according to the previously published standard SRB assay drug treatment experimental procedure. [24,25] The cell lines were grown in RPMI 1640 medium containing 10 % fetal bovine serum and 2 mM L-glutamine. For the present screening experiment, cells were inoculated into 96 well microtiter plates in 90 µL at 5000 cells per well. After cell inoculation, the microtiter plates were incubated at 37 °C, 5 % CO2, 95 % air, and 100 % relative humidity for 24 h prior to the addition of experimental drugs. Experimental drugs were solubilized in an appropriate solvent to prepare a stock of 10 -2 concentrations. At the time of the experiment, four 10-fold serial dilutions were made using the complete medium. Aliquots of 10 µL of these different drug dilutions were added to the appropriate microtiter wells already containing 90 µL of the medium, resulting in the required final drug concentrations.
After compound addition, plates were incubated at standard conditions for 48 hours and the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 µL of cold 30 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4 °C. The supernatant was discarded; the plates were washed five times with tap water and air-dried. Sulforhodamine B (SRB) solution (50 µL) at 0.4 % (w/v) in 1 % acetic acid was added to each of the wells, and plates were incubated for 20 minutes at room temperature. After staining, the unbound dye was recovered and the residual dye was removed by washing five times with 1 % acetic acid. The plates were air-dried. The bound stain was subsequently eluted with a 10 mM trizma base, and the absorbance was read on an Elisa plate reader at a wavelength of 540 nm with a 690 nm reference wavelength.
Percent growth was calculated on a plate-by-plate basis for test wells relative to control wells. Percent Growth was expressed as the ratio of average absorbance of the test well to the average absorbance of the control wells × 100. Using the six absorbance measurements [time zero (Tz), control growth (C), and test growth in the presence of drug at the four concentration levels (Ti)], the percentage growth was calculated at each of the drug concentrations levels. The dose-response parameters were calculated for each test article. Growth inhibition of 50 % (GI50) was calculated from [(Ti -Tz) / (C -Tz)] × 100 = 50, which is the drug concentration resulting in a 50 % reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz. The LC50 (concentration of drug resulting in a 50 % reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicates a net loss of cells following treatment is calculated from [(Ti -Tz) / Tz] × 100 = -50.
Values were calculated for each of these three parameters if the level of activity was reached; however, if the effect was not reached or was exceeded, the values for that parameter were expressed as greater or less than the maximum or minimum concentration tested.

Chemistry
In the current study, we report three categories of novel analogues of combretastatin-A4 with pyrazoline bridgehead linker having the same substituent on the A ring while the B ring is replaced with a 4-nitro-phenyl group, such as, N 1acetylated pyrazoline analogue of CA-4 (5a-c), N 1phenylpyrazoline of CA-4 (6a-e) and 3, 5-diaryl-1-carbothioamide-pyrazoline of CA-4 (7a-e). The synthesis of target compounds (5a-c) was achieved by treating precursor chalcone 3a separately with HCOOH, CH3COOH, and CH3CH2COOH in presence of hydrazine hydrate under reflux for 6 h (Scheme 1). Further, compounds (6a-e) were obtained by reacting 3a with differently substituted phenylhydrazine hydrochlorides in ethanol under the catalyst-free condition in good yield (Scheme 1). On the other hand, the synthesis of compounds (7a-e) was achieved by treating pyrazoline analogue 4a with differently substituted phenyl isothiocyanates. However, the pyrazoline analogue of CA-4 (4a) was synthesized by refluxing precursor chalcone 3a with hydrazine hydrate in absolute ethanol (Scheme 1).
The structures of all the newly synthesised-N 1acetylated pyrazoline analogues of CA-4 (5a-c), N 1 -phenyl pyrazolines (6a-e) and 3, 5-diaryl-1-carbothioamidepyrazoline (7a-e) derivatives were confirmed by 1 H NMR, 13 C NMR, IR and mass spectrometry. All the synthesised pyrazoline analogues show three characteristics doublet of doublet (dd) peaks in 1 H NMR spectrum for -CH proton present adjacent to -CH2 in pyrazoline ring, which vividly depicts the formation of pyrazoline ring. Besides this, 13 C NMR and DEPT spectrum also show peaks for -CH2, and -CH group, consistent with the suggested structures.

Cytotoxicity Study
The in vitro anticancer activity of the newly synthesised compounds N1-acetylated pyrazolines (5a-c), N1-phenyl pyrazolines (6a-e), 3,5-diaryl-1-carbothioamide-pyrazolines (7a-e), chalcone analogue (3a), and pyrazoline analogue of CA-4 (4a) was assessed using the sulforhodamine B (SRB) assay. The anticancer drug adriamycin was used as a reference standard. All the compounds were screened against two cancer cell lines, specifically, MCF-7 (breast) and Vero (monkey kidney normal cell line). Moreover, five selected compounds from the series were also investigated for their in vitro anticancer activity against Hela (Human Cervical), MDA-MB-231 (Breast), and A-549 (Lung). The screening process recorded three response parameters (GI50, TGI, and LC50). The GI50 value (growth inhibitory activity) unveils the concentration of a compound triggering 50 % reduction in the net cell growth, the TGI value (cytostatic activity) reveals the concentration of a compound required for the total growth inhibition while the LC50 value (cytotoxic activity) determines the concentration of a compound that cause the net 50 % loss of initial cells. The response parameters for all the compounds against MCF-7 and Vero (Monkey kidney normal cell line) along with the responses against Hela (Human Cervical), MDA-MB-231 (Breast), and A-549 (Lung) for selected five compounds are presented in the Table 1. The results obtained is related to the GI50 values, accordingly, the compound's activity is classified as: inactive, >100 µM; moderate, between >30 and <100 µM; good, between >10 and <30 µM; and active, <10 µM.
The screening process recorded three response parameters (GI50, TGI, and LC50). The GI50 value (growth inhibitory activity) reveals the concentration of a compound triggering a 50 % reduction in the net cell growth; the TGI value (cytostatic activity) reveals the concentration   of a compound required for the total growth inhibition; and the LC50 value (cytotoxic activity) determines the concentration of a compound that causes the net 50 % loss of initial cells. The response parameters for all the compounds against MCF-7 and Vero (monkey kidney normal cell line), along with the responses against Hela (Human Cervical), MDA-MB-231 (Breast), and A-549 (Lung) for selected five compounds are presented in Table 1. The results obtained are related to the GI50 values. Consequently, the compound's activity is classified as: inactive, >100 µM; moderate, between >30 and <100 µM; good, between >10 and <30 µM; and active, <10 µM.
The data obtained reveals that among the three categories of novel combretastatin-A4 analogues, most of the compounds exhibited significant cytotoxicity against the MCF-7 cell line, with the concentration of a compound that triggers 50 % inhibition of cell growth (GI50). In particular, compounds 3a, 6a, 6e, 5b, 7a, 5a, and 7d showed profound inhibitory effect against the MCF-7 cell line that exhibited a wide spectrum of activity with the GI50 value ranging from 10 to 30 µM. Whereas, compounds 5c and 7e demonstrated moderate cytotoxicity (GI50 = 60 µM) and the remaining compounds displayed weak cytotoxicity (GI50 = 90 -100 µM).
Out of the selected compounds tested against Hela (Human Cervical), MDA-MB-231 (Breast), and A-549 (Lung) cell lines, compounds 6a and 7a showed remarkable cytotoxicity against the A-549 (Lung) cell line with the GI50 value ranging from 10 to 30 µM, and compound 3a displayed moderate cytotoxicity against Hela (Human Cervical). Other compounds did not show significant inhibitory potential. Moreover, all the compounds in the series were tested on the Vero (monkey kidney) cell line, which represents the normal cell type. The results clearly show that these compounds did not show cytotoxicity, except for compounds 5a-c, which showed moderate cytotoxicity against the normal cell line. Unlike the reference standard drug adriamycin, it exhibited remarkable cytotoxicity against the Vero (Monkey kidney) normal cell line. The results reveal that compounds 6a, 6e, and 7a, 7d selectively target cancerous cells (MCF-7, A-549 Lung) and thus can be considered as a lead for the development of an effective anticancer drug.
A similar relationship has been established for the TGI concentrations of the compounds in comparison with adriamycin, the reference standard drug. All the tested compounds appeared to be inactive against all the tested cell lines. Likewise, the LC 50 concentrations of the compounds were compared with adriamycin to get an insight into the cytotoxic effects of these compounds against all the cell lines All the compounds (LC50 > 100 µM) like adriamycin (LC50 > 100 µM) were found to be inactive against all the cell lines, except adriamycin did show potency against MDA-MB-231 (LC50 > 20 µM).
The structure activity relationship (SAR) study reveals that the compound 3a (chalcone analogue of CA-4) with the same substituent on A ring and B ring replaced with a 4-nitro-phenyl group showed good cytotoxicity against the MCF-7 cell line (GI50 = 10.0 µM) and moderate cytotoxicity against the Hela cell line. However, the compound 4a (pyrazoline analogue of CA-4) showed moderate cytotoxicity against the MCF cell line (GI50= 60.0 µM) and poorly inhibited other cell lines. When the phenyl or phenyl carbothioamide group was substituted at the N 1 position of pyrazoline, the activity increased. Compounds 6a and 7a with a -CH3 group at the ortho position of the ring-C of N 1 -phenyl pyrazoline and 3,5-diaryl-1-carbothioamidepyrazoline showed better cytotoxicity against MCF-7 and A-549 cell lines than that of 4a. Moreover, compounds 6e and 7d with fluoro and cyano groups at the para position of the C-ring showed enhanced cytotoxicity against the MCF cell line. From this evidence, a general specific trend in structure and activity can be established. Since then, chalcone and pyrazoline analogues of CA-4 adopt twisted geometry [18,[26][27] like that of CA-4, which is indispensable to fit into the binding site of tubulin to inhibit their polymerization. Among the pyrazoline analogues (6a-e) and (7a-e), most of the compounds appeared to be potent. Since then, a small change in the structure of the CA-4 analogue has shown a surprising effect on other biological targets.

CONCLUSION
In conclusion, fourteen pyrazoline analogue of combretastatin A-4 (5a-c), (6a-e) and, (7a-e) were synthesised from the precursor chalcone analogue of combretastatin A-4 (3a). All the compounds were tested against two cancer cell lines viz, MCF-7 (breast) and Vero (monkey kidney normal cell line). Moreover, five compounds selected from the series were also tested for their anticancer activity against Hela (human Cervical), MDA-MB-231 (breast), and A-549 (lung) cell lines. Compounds 3a, 6a and 6e demonstrated the highest cytotoxicity followed by 5b, 7a, 5a and 7d against the MCF-7 cell line. Compounds 6a and 7a also displayed remarkable cytotoxic effect against A-549 (lung) cell line. Overall, MCF-7 and A-549 (lung) cell lines are susceptible to the set of synthesized compounds. In general, the presence of 4-F/4-CN/2-CH 3 groups on C-ring of N 1 -phenyl pyrazoline and 3,5diaryl-1-carbothioamide-pyrazoline showed good cytotoxicity against MCF-7 and A-549 cell line. The outcomes of the study undoubtedly reveal that a set of compounds 6a, 6e and 7a, 7d selectively target only cancerous cells (MCF-7, A-549 Lung) and thus, can be considered a lead for the development of an effective anticancer drug.