Copper-Catalyzed Syntheses of Pyrene-Pyrazole Pharmacophores and Structure Activity Studies for Tubulin Polymerization.

Tubulin polymerization is critical in mitosis process, which regulates uncontrolled cell divisions. Here, we report a new class of pyrene-pyrazole pharmacophore (PPP) for targeting microtubules. Syntheses of seven pyrenyl-substituted pyrazoles with side-chain modification at N-1 and C-3 positions of the pyrazole ring were accomplished from alkenyl hydrazones via C-N dehydrogenative cross-coupling using copper catalyst under aerobic condition. Tubulin polymerization with PPPs was investigated using docking and biological tools to reveal that these ligands are capable of influencing microtubule polymerization and their interaction with α-, β-tubulin active binding sites, which are substituent specific. Furthermore, cytotoxicity response of these PPPs was tested on cancer cells of different origin, such as MCF-7, MDA-MB231, and C32, and also noncancerous normal cells, such as MCF-10A. All newly synthesized PPPs showed excellent anticancer activities. The anticancer activities and half-maximal inhibitory concentration (IC50) values of all PPPs across different cancer cell lines (MCF-7, MDA-MB231, and C32) have been demonstrated. 1,3-Diphenyl-5-(pyren-1-yl)-1H-pyrazole was found to be best among all other PPPs in killing significant population of all of the cancerous cell with IC50 values 1 ± 0.5, 0.5 ± 0.2, and 5.0 ± 2.0 μM in MCF-7, MDA-MB231, and C32 cells, respectively.

Introduction

Microtubules are dynamic protein filaments assembled from tubulin subunits, α and β.[1] They are generally involved in cell division, movement, intracellular trafficking, and mitosis.[2] Microtubules are long, hollow cylindrical rods that are formed by polymerization of subunits.[3] Microtubules targeting ligands affect their dynamics to act as strong inhibitor or facilitator for cell proliferation.[4] Tubulin dimers are known to bind with proteins as well as small molecules that either stabilize or inhibit microtubule polymerization.[5] In the context of n class="Gene">pan class="Disease">cancer, the tubulin families of proteins are recognized as the target of the tubulin-binding chemotherapeutics, which suppress the dynamics of the mitotic spindle to cause ne">pann> class="Disease">mitotic arrest and cell death.[6] Importantly, changes in microtubule stability and the expression of different tubulin isotypes, as well as altered post-translational modifications, have been reported for a range of cancers.[7] These changes have been correlated with poor prognosis and chemotherapy resistance in solid and hematological cancers.[8] Although, microtubule polymerization-influencing agents are widely used in combination therapy for the treatment of a few malignancies, its broad application is largely hampered by their relative toxicity, complex synthesis method, and developing multidrug resistance.[9] With this background, there has been a renewed interest in the development of potent microtubule polymerization-influencing agents that circumvent one or more of these issues.

In this work, we disclose a rational design and syntheses of a series of pyrene-pyrazolen> compounds as antimitotic agents. Taking into account the biological importance of pan class="Chemical">pyrazole[10,11] and fluorophoric behavior of a pyrene moiety,[12] including their formation of excimer in presence of tubulin,[13] we hypothesized that pyrene-pyrazole-based pharmacophores (PPPs) may modulate chemical extremities of the agent, which can further vary the interaction pattern during tubulin polymerization.

Herein, we report copper triflaten>-catalyzed synthesis of new pan class="Chemical">pyrene-pyrazole pharmacophore (PPP1–7) from alkenyl hydrazones via cross-dehydrogenative coupling[14] under aerobic condition. The effect of these new agents for microtubule polymerization was studied by biophysical methods and their molecular mechanism also been studied using biological and physicochemical experiments. Results indicate that members of this pyrene-pyrazole family of pharmacophores exhibited potent tubulin polymerization inhibition and cellular cytotoxicity in breast cancer cells.

Results and Discussion

Synthesis of Pyrene-Pyrazole Pharmacophore

We have designed seven new molecules possessing n class="Gene">pan class="Chemical">pyrene-pyrazole core structure. Analogues were designed with side-chain modification at pann> class="Gene">N-1 and C-3 positions of the pyrazole ring, introducing hydrogen, phenyl, methyl, biphenyl, benzothiazolyl, and carboxylic acid moieties. As an effort to develop highly efficient and mild reactions for chemical synthesis, a concept of copper-catalyzed cross-dehydrogenative coupling reaction was developed. First, we optimized the reaction with 1d as test substrate using copper triflate (Cu(OTf)2) as catalyst in the presence of varied oxidants in toluene at 80 °C. The reaction readily furnished to give 2d in 46% yield in presence of air (Scheme ). In a set of oxidant screening, K2S2O8 and tert-butyl hydroperoxide give 2d in 42 and 38% yield, respectively.

Scheme 1

Synthesis of Pyrene-Pyrazole Pharmacophore 2a–f,

Reaction conditions: substrate (0.5 mmol), Cu(OTf)2 (10 mol %), toluene (3 mL), 80 °C, air, 2 h.

Isolated yield.

Reaction conditions: substrate (0.5 mmol), pan class="Gene">n class="Chemical">Cu(OTf)2 (10 mol %), ne">pann> class="Chemical">toluene (3 mL), 80 °C, air, 2 h.

The syntheses of PPPs (2) from substituted alkenyl pan class="Chemical">hydrazonesn> (1) were achieved in presence of optimized reaction condition (Scheme ). In a typical procedure, the substrate comprising a phenyl group at N-1 position and methyl group at pan class="Gene">C-3 position in alkenyl hydrazone (1a) underwent reaction to produce the corresponding PPP1 (2a) in 58% yield, whereas substrate having biphenyl group in N-1 position in pan class="Chemical">alkenyl hydrazone (1b) afforded product PPP2 (2b) in 53% yield.

It is noteworthy that the reaction of benzothiazolyl alkenyl hydrazonen> (1c) readily occurred to furnish corresponding PPP3 (2c). In addition, substrate-bearing phenyl group at N-1 and pan class="Gene">C-3 position in alkenyl hydrazone (1d) was also compatible in this methodology to provide corresponding 1,3-diphenyl-5-(pyren-1-yl)-1H-pyrazole (PPP4) (2d), while gratifyingly, biphenyl group at N-1 position in alkenyl hydrazone moiety (1e) afforded PPP5 (2e) in 52% yield. A similar result was observed with substrate 1f bearing hydrogen atom in the N-1 position of alkenyl hydrazone, producing PPP6 (2f) in 43% yield. Furthermore, the reaction of acetophenone with dimethyl oxalate produced A. Next, compound A underwent reaction with phenyl hydrazine readily in this methodology to produce intermediate B via C–N bond formation in one pot. The intermediate B was treated with KOH/MeOH to produce PPP7 (2g) in 62% yield (Scheme ). However, in the absence of Cu(OTf)2, compound A failed to produce corresponding product, compound B. Therefore, our results indicated that a catalytic amount of Cu(OTf)2 is a critical requirement for the successful conversion. The crude PPPs were purified by silica gel (70–200 mesh) column chromatography using ethyl acetate and hexane as eluents. The structure of the purified ligands was confirmed by 1H and 13C NMR studies and mass spectrometry (MS) analyses (Figures S1–S9). Fluorescence spectroscopy measurements taken over a pH ranging from ∼3 to 8 found that all dyes exhibited a stable fluorescence emission in the blue range across all tested pH values (Figures S22–S24).

Scheme 2

Synthesis of 1-Phenyl-5-(pyren-1-yl)-1H-pyrazole-3-carboxylic Acid 2g

Effect of PPPs on Microtubule Polymerization

To gather further insight into the molecular mechanism of action of PPPs, an in vitro cytoskeleton assay was carried out to investigate the effect of these agents on microtubule polymerization (Figure ). In this protocol, 85 μL of 10 mg/mL tubulin stock was inn class="Chemical">cubated with tubulin pan class="Chemical">glycerol buffer for 1.5 h at 37 °C in the presence of various PPPs at a fixed concentration (20 mM). Following this, the degree of tubulin polymerization was evaluated by observing their fluorescence profile with time.

Figure 1

In vitro microtubule polymerization assay. Assays were conducted using purified tubulins to form polymerized tubules in presence of various PPPs. An increase in relative fluorescence intensity (RFU) was obtained for paclitaxel (PTXL), along with PPP1 to PPP7, barring PPP5, suggesting that the later supports microtubule formation. RFU decreased for PPP5 with time, suggesting that it inhibits microtubule formation.

In vitro microtubule polymerization assay. Assays were conducted using purified tubulins to form polymerized tubules in presence of various PPPs. An increase in relative fluorescence intensity (RFU) was obtained for paclitaxeln> (pan class="Chemical">PTXL), along with PPP1 to PPP7, barring PPP5, suggesting that the later supports microtubule formation. RFU decreased for PPP5 with time, suggesting that it inhibits microtubule formation.

The content of polymerized microtubules was monitored by measuring fluorescence intensity at 430 nm (λex = 350 nm) every 7 s for 1.5 h. Paclitaxeln> (pan class="Chemical">PTXL), a well-known microtubule stabilizer, was added to the assay as a positive control, whereas the tubulin glycerol buffer acted as a negative control. An increase in the fluorescence intensity was observed, indicating that PTXL is stabilizing the tubulin polymerization reaction. An increased fluorescence intensity was observed for PPP1 to PPP7, except PPP5, showing a similar trend to PTXL. This implies that addition of PPP1–7 into the assay led to a stabilization of microtubule polymerization. Interestingly, for PPP5, the fluorescence intensity was found to be decreasing over time, suggesting an inhibition of microtubule polymerization, similar to known microtubule destabilizing agents, such as nocodazole.[15] Further inspection to correlate between the obtained trend in tubulin polymerization and modifications attempted at the N-1 and C-3 positions yielded interesting observations. Keeping methyl group at C-3 position and changing the N-1 position from phenyl group (PPP1) to biphenyl group (PPP2) lead to an increased degree of tubulin polymerization (Figure ). Furthermore, keeping a phenyl group at N-1 position fixed and varying at C-3 positions from methyl (PPP1) to phenyl group (PPP4) lead to a drastic increase in the tubulin polymerization, as seen from Figure . Finally, keeping the phenyl group at C-3 positions fixed and changing the N-1 position from phenyl (PPP2) to biphenyl group (PPP5) lead to a decrease in the efficiency of tubulin polymerization. Hence, an interplay with the modifications at C-3 and N-1 positions leads to increased tubulin polymerization. However, substantial increase in the bulkiness of the groups at N-1 positions leads to decreased rate for tubulin polymerization (PPP5).

Effect of PPPs on Electrophoretic Mobility of Tubulin

With this promising result, we further focused on confirming the polymerization of microtubules. PPP–tubulin mixtures were collected from the abovementioned assay, and a sodium dodecyl sulfate n class="Gene">pan class="Chemical">polyacrylamide gel electrophoresis (ne">pann> class="Chemical">SDS-PAGE) was performed to separate both tubulin as well as polymerized tubulin from the mixture. Figure a shows the bands from SDS-PAGE gel, which was subsequently stained with Coomassie brilliant blue. Two distinct bands were obtained, one in the region of 40–55 kDa, which is consistent with the molecular weight of α-, β-tubulin,[16] and other in the region of 130 kDa. This is presumably a result of tubulin polymerization. A semiquantitative analysis of the polymerized tubulin bands was performed using FIJI[17] to calculate the band intensities, as shown in Figure b. Fold change of band intensities was further calculated with respect to PPP5–tubulin mixture (Figure c).

Figure 2

(a) Coomassie-stained gel lanes for tubulin–dye mixtures, showing two migrating populations, polymerized tubulin (∼130 kDa MW), and α-, β-tubulin (∼50 kDa MW). A representative molecular weight standard lane is also shown across the sample lanes for comparison. (b) Comparison of optical intensity of polymerized tubulin for different mixtures was calculated using FIJI and (c) corresponding fold change was calculated with respect to PPP5–tubulin mixture, which showed the least optical intensity.

(a) pan class="Chemical">Coomassien>-stained gel lanes for tubulin–dye mixtures, showing two migrating populations, polymerized tubulin (∼130 kDa MW), and α-, β-tubulin (∼50 kDa MW). A representative molecular weight standard lane is also shown across the sample lanes for comparison. (b) Comparison of optical intensity of polymerized tubulin for different mixtures was calculated using FIJI and (c) corresponding fold change was calculated with respect to pan class="Gene">PPP5–tubulin mixture, which showed the least optical intensity.

From the results, it was evident that the band intensity for PPP5n>–tubulin was the least amongst all PPP–tubulin mixtures, possibly indicating structural interferences from pan class="Gene">PPP5 hindering the polymerization process of tubulin. As evident, PPP5 had contrasting spectroscopic features compared to the rest of the PPP analogues presumably due to the presence of N-1 biphenyl moiety and a bulkier structure.

In Silico Response of PPPs on Tubulin Interaction

Target selectivity of synthesized PPPs could be studied with in silico model of tubulin protein. It also provides information about three-dimensional (3D) orientation and interaction pattern of PPP–tubulin protein complex. To achieve it, mechanistic understanding of PPPs binding to tubulin structure was obtained from molecular docking studies. For the simulation work, X-ray crystallographic 3D structure of tubulin α- and β-dimer (PDB code: 1tub) was downloaded from the online protein data bank (http://www.pdb.org). The structures of both α- and β-tubulin are similar, wherein each monomer is composed of two β-sheets covered by α-helices. The structure shows three possible binding sites for ligands, including bound guanosine-5′-triphosphate (n class="Gene">pan class="Chemical">GTP) in α-tubulin and guanosine-5′-diphosphate (ne">pann> class="Chemical">GDP) and Taxotere (TAX) bound to β-tubulin (Figure a,b). To provide insights into the binding site of different PPP molecules into α-, β-tubulin, a preliminary molecular docking was performed using Chimera.[18] The minimum binding energy indicated that α-, β-tubulin was successfully docked with PPPs. Interestingly, on the basis of this criterion, PPPs were shown to bind in different sites on α-, β-tubulin on the basis of their structures.

Figure 3

(a) Structure of α- and β-tubulin, highlighting the different active sites present, namely, GDP, TAX, and GTP; (b) side view of the α-, β-tubulin protein. (c–j) Docking studies were performed to see the binding location of PPP1, PPP4, PPP6, and PPP7 in α-, β-tubulin; and corresponding surface representation was used to show the binding pocket of the docked molecule in α-, β-tubulin.

(a) Structure of α- and β-tubulin, highlighting the different active sites present, namely, GDPn>, TAX, and GTP; (b) side view of the α-, β-tubulin protein. (c–j) Docking studies were performed to see the binding location of PPP1, PPP4, PPP6, and PPP7 in α-, β-tubulin; and corresponding surface representation was used to show the binding pocket of the docked molecule in α-, β-tubulin.

PPP1 (Figure c,d) and PPP3 were shown to bind into pan class="Chemical">GDPn> pocket having a minipan class="Gene">mum binding energy of −7.97 and −8.09 kcal/mol, respectively (Table ).

Table 1

Different PPP Molecules along with Their ΔG Values and Binding Location after the Docking Studies Were Performed Using Chimeraa

sample	ΔG (kcal/mol)	binding site
PPP1	–7.97	GDP
PPP2	–7.90	TAX
PPP3	–8.09	GDP
PPP4	–8.14	TAX
PPP5	–7.64	TAX
PPP6	–6.97	TAX
PPP7	–8.16	TAX

Abbreviation: GDP: guanosine-5′-diphosphate, GTP: guanosine-5′-triphosphate; TAX: taxotere.

Abbreviation: GDPn>: guanosine-5′-diphosphate, pan class="Chemical">GTP: guanosine-5′-triphosphate; TAX: taxotere.

However, PPP2, PPP4n> (Figure e,f), pan class="Gene">PPP5, PPP6 (Figure g,h), and PPP7 (Figure i,j) were shown to bind into the TAX pocket, which is similar to where PTXL binds. It is interesting to observe that C-1 substitution with a methyl group usually tends to direct the molecules (PPP1, 3) to GDP pocket. However, a substituent at N-1 presumably influences the molecule and introduces to the TAX pocket, as seen for the case of PPP2.

Cytotoxicity Response of PPPs on Cancer Cells of Different Origin

As the process of tubulin polymerization was found to be modulated by PPPs in biological assay, their functional activity (i.e., pan class="Disease">cytotoxicityn>) needed to be verified in vitro. An in vitro pan class="Disease">cancer cell two-dimensional culture model was used for this study using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.[19]

It was found that all of the PPPs were effective in killing a significant population of ne">n class="CellLine">MCF-7 cells. ne">pann> class="Gene">PPP4 was found to be causing the maximum damage (Figure a) with a cell growth inhibition by 50% at a concentration of 1 ± 0.5 μM. When compared for % cell population inhibited by drug molecules at 10 μM, PPP4 with only 30 ± 3% viable cells was significantly less than PPP1, PPP2, PPP3, PPP5, PPP6, and PPP7 with 50 ± 5, 45 ± 3, 52 ± 5, 70 ± 10, 55 ± 5, and 50 ± 5% of viable cells, respectively.

Figure 4

Functional activity of PPPs in ER (+) breast cancer cells MCF-7. (a) MTT assay results from 48 h treatment of MCF-7 cells at different concentrations. (b) Comparison of cell growth inhibition for different formulations at 10 μM incubation.

Functional activity of PPPs in ER (+) pan class="Disease">breast cancern> cells pan class="CellLine">MCF-7. (a) MTT assay results from 48 h treatment of pan class="CellLine">MCF-7 cells at different concentrations. (b) Comparison of cell growth inhibition for different formulations at 10 μM incubation.

Taxoln>, a well-established tubulin polymerization drug, was used as a positive control and found to be inhibiting the cell growth level to only 42 ± 10%, very much comparable with pan class="Gene">PPP4 (Figure b). Similar results were obtained from a cell growth density study where maximum loss in MCF-7 cell growth density was obtained from PPP4 incubations at 1 μM, which was very comparable with Taxol treatment (Figure S25).

Broad spectrum effect of these molecules was studied by introducing more cells of n class="Gene">pan class="Disease">cancer origin, while their selectivity toward ne">pann> class="Disease">cancer was established by studying noneffective nature in normal cells of same origin. It was found that all of the PPPs were effective in killing a significant population of MDA-MB231 (Figure A,B) and C32 (Figure C,D) cells. Similar to MCF-7 cells, PPP4 was found to be causing the maximum damage with second best effect by PPP7. It was found that MDA-MB231 and C32 cell growth inhibition by 50% was achieved by PPP4 at a concentration of 0.5 ± 0.2 and 5.0 ± 2.0 μM, respectively, whereas by PPP7 at a concentration of 10 ± 2.0 and 14 ± 2.0 μM, respectively (Figure B,D). Comparative half-maximal inhibitory concentration (IC50) values for all of the molecules showed a trend of PPP4 < PPP7 < PPP1 < PPP2 < PPP3 < PPP5 < PPP6 across all of the cancer cell lines used (Table ).

Figure 5

Functional activity of PPPs in breast cancer cells MDA-MB231 and human melanoma C32 cells. (A) MTT assay results from 48 h treatment of MDA-MB231 cells at different concentrations and (B) comparison of cell growth inhibition for different formulations at 10 μM incubation. (C) MTT assay results from 48 h treatment of C32 cells at 10 and 100 μM and (D) comparison of cell growth inhibition for different formulations at 10 μM incubation. (E) Lower functional activity of PPPs in breast cells MCF-10A of noncancerous origin at 10 μM concentration of representative PPPs for 48 h.

Table 2

IC50 Values of All of the PPP Analogues across MCF-7, MDA-MB231, and C32 Cell Lines

	PPP1 (μM)	PPP2 (μM)	PPP3 (μM)	PPP4 (μM)	PPP5 (μM)	PPP6 (μM)	PPP7 (μM)
MCF-7	60 ± 10	34 ± 4	>100	1 ± 0.5	>100	43 ± 5	8 ± 2
MDA-MB231	>100	15 ± 5	>100	0.5 ± 0.2	>100	>100	10 ± 2.0
C32	>100	>100	>100	5.0 ± 2.0	>100	>100	14 ± 2.0

Functional activity of PPPs in breast cancern> cells pan class="CellLine">MDA-MB231 and human melanoma C32 cells. (A) MTT assay results from 48 h treatment of MDA-MB231 cells at different concentrations and (B) comparison of cell growth inhibition for different formulations at 10 μM incubation. (C) MTT assay results from 48 h treatment of C32 cells at 10 and 100 μM and (D) comparison of cell growth inhibition for different formulations at 10 μM incubation. (E) Lower functional activity of PPPs in breast cells MCF-10A of noncancerous origin at 10 μM concentration of representative PPPs for 48 h.

Effectivity of anticancern> drugs not only depends on pan class="Disease">cancer inhibition property but minimum effect to noncancerous normal cells. A representative study was performed on MCF-10A cells of noncancerous nature and human breast origin. It was found that representative molecules of PPP1, PPP4, and PPP7 did not result in any significant cell death at a treatment concentration of 10 μM where similar concentration of PPPs in MCF-7, MDA-MB231, and C32 showed cell growth inhibition of more than 50% (Figure E). It indicates the selectivity of PPPs toward cancer cells, presumably due to higher variation in tubulin polymerization processes.

Conclusions

In conclusion, a new class of pyrene-pyrazolen> analogues were synthesized through a remarkably simple pan class="Chemical">copper-catalyzed cross-dehydrogenative coupling. As an effort to develop highly efficient and mild reactions for chemical synthesis, copper triflate-catalyzed reaction afforded seven potent microtubule polymerization-modulating agents under aerobic condition.

Our results outline a distinct molecular mechanism of action for the inhibition of microtubule assembly by a new class of agents based on n class="Gene">pan class="Chemical">pyrene-pyrazole heterocyclic core structure. Success of the synthesis followed by biological and docking studies further provided a structural basis for the rational design of potent microtubule polymerization-modulating agents. The new ne">pann> class="Chemical">pyrene-pyrazole analogues showed excellent activity in the tubulin assembly assay and significantly decreased activity against MCF-7 cell proliferation. It turns out that the substitution at C-1 and sterically demanding substituents at N-1 are key factors to drive the location of their binding inside tubulin sites. The most active agent showed comparable cell growth inhibition to Taxol. The full medicinal potential of these lipophilic agents warrants future studies. This can be addressed by formulation and other drug delivery approaches, and it is beyond the scope of this preliminary report. Our in vitro results demonstrated significant anticancer activity with these newly identified and synthesized compounds. We envision nanoparticle-enabled delivery of these agents to address their solubility. To fully understand their biological activity, an in-depth preclinical study is warranted and will be undertaken in the near future. Results reported here may open up opportunities for the development of novel microtubule-targeting pharmacophores and next-generation antibody–drug conjugates for cancer therapy.

Experimental Section

General Information

Unless otherwise mentioned, the reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used without any purification. Aryl hydrazinesn>, pan class="Chemical">hydrazine hydrate, aryl aldehydes, dimethyl oxalate, and Cu(OTf)2 were purchased from Aldrich and used as received. Tubulin polymerization assay kit was purchased from Cytoskeleton Inc. and used in accordance to the instruction manual. α-,β-Unsaturated ketones[20a,20b] and their corresponding hydrazones[21] were synthesized according to a reported literature procedure. Analytical thin-layer chromatography (TLC) was performed on a Merck silica gel (60 F254) plastic sheet for progress of the reaction, and column chromatography was performed using Alfa Aesar 70–200 mesh silica gel. VARIAN UNITY 500 (Varian, Inc., Palo Alto, CA) spectrometer operating at 500 MHz equipped with 5 mm Nalorac QUAD probe was used for recording NMR (1H and 13C) spectra using CDCl3 as a solvent and Me4Si as an internal standard. Chemical shifts (δ) and spin–spin coupling constant (J) are reported in ppm and Hz, respectively. Multiplicities are reported as follows: s = singlet, d = doublet, and m = multiplet. High-resolution mass spectra (HRMS) were recorded on a quad time-of-flight electrospray ionization (ESI)-MS instrument.

Solubility

All of the pyrenylpyrazolesn> PPP1–7 (2a–g) compounds are soluble in pan class="Chemical">dimethyl sulfoxide (DMSO) and methanol (MeOH) solvents at room temperature. Cell studies have been performed in DMSO solvent.

General Procedure for the Synthesis of Pyrenylpyrazoles PPP1–6 (2a–f)

Alkenyl hydrazonesn> 1a–f (0.5 mmol) were stirred with pan class="Gene">Cu(OTf)2 (10 mol %) in toluene (3 mL) at 80 °C for 2 h under air. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled at room temperature and diluted with ethyl acetate. The resultant solution was passed through a short pad of celite. The evaporation of the solvent under vacuum through rotary evaporator gave a residue that was purified on silica gel column chromatography using hexane and ethyl acetate as eluent to afford the corresponding pyrenylpyrazoles product.

General Procedure for the Synthesis of Methyl (Z)-2-Hydroxy-4-oxo-4-(pyren-1-yl)but-2-enoate (A)

A solution of acetophenonen> (3 mmol) and dimethyl oxalate (6 mmol) in pan class="Chemical">methanol (5 mL) was added dropwise to a solution of sodium methoxide (6 mmol) in methanol (1 mL). The resultant reaction mixture was refluxed for 5 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled at room temperature and poured in water. The resultant reaction mixture was acidified with dilute HCL (1 M) to reach pH 3–4 and extracted with diethyl ether. The organic layer was dried over Na2SO4 and evaporated under vacuo to give a crude residue that was used directly in the next step without further purification.

General Procedure for the Synthesis of Methyl 1-Phenyl-5-(pyren-1-yl)-1H-pyrazole-3-carboxylate (B)

Compound A (1 mmol) was stirred with ne">n class="Chemical">Cu(OTf)2 (10 mol %) in ne">pann> class="Chemical">toluene (3 mL) at 80 °C for 2 h under air. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled at room temperature and diluted with ethyl acetate. The resultant solution was passed through a short pad of celite. The evaporation of the solvent under vacuum through a rotary evaporator gave a residue that was purified on silica gel column chromatography using hexane and ethyl acetate as eluent to afford the corresponding pyrenylpyrazoles product B.

General Procedure for the Synthesis of 1-Phenyl-5-(pyren-1-yl)-1H-pyrazole-3-carboxylic Acid PPP7 (2g)

In an oven-dried round bottom flask, KOHn> (1.75 mmol) and drops of pan class="Chemical">water (1 mL) were added in the solution of compound B (0.5 mmol) in methanol (10 mL). The resultant mixture was refluxed for 2 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled at room temperature and diluted with water. The resultant solution was acidified with dilute HCL (1 M) to reach pH 3–4. The organic layer was extracted with ethyl acetate, dried over Na2SO4, and evaporated under vacuo to give the compound PPP7 (2g).

3-Methyl-1-phenyl-5-(pyren-1-yl)-1H-pyrazole PPP1 (2a)

Analytical TLC on silica geln> pan class="Chemical">aluminum foil, 1:4 ethyl acetate/hexane Rf = 0.52; yellow liquid. Yield 58% (103 mg); 1H NMR (500 MHz, CDCl3) δ 8.22 (d, J = 8.0 Hz, 1H), 8.19 (d, J = 7.5 Hz, 1H), 8.15–8.02 (m, 6H), 7.79 (d, J = 8.0 Hz, 1H), 7.19 (d, J = 7.0 Hz, 2H), 7.08–7.04 (m, 3H), 6.52 (s, 1H), 2.53 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 149.6, 142.1, 140.2, 131.6, 131.4, 131.0, 129.8, 128.8, 128.4, 128.3, 127.4, 126.7, 126.4, 126.1, 125.7, 125.6, 124.9, 124.6, 124.1, 110.7, 13.9; HRMS (ESI) m/z [M + H]+ calcd for C26H18N2: 359.1548, found: 359.1536.

1-([1,1′-Biphenyl]-4-yl)-3-methyl-5-(pyren-1-yl)-1H-pyrazole PPP2 (2b)

Analytical TLC on silica geln> pan class="Chemical">aluminum foil, 1:9 ethyl acetate/hexane Rf = 0.54; yellow liquid. Yield 53% (115 mg); 1H NMR (500 MHz, CDCl3) δ 8.23 (d, J = 7.5 Hz, 1H), 8.20–8.17 (m, 1H), 8.16 (d, J = 10.0 Hz, 1H), 8.12–8.10 (m, 4H), 7.83 (d, J = 7.5 Hz, 1H), 7.41–7.29 (m, 10H), 6.53 (s, 1H), 2.53 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 149.3, 145.5, 141.2, 135.7, 131.5, 130.8, 128.8, 128.7, 128.5, 127.7, 127.4, 127.0, 126.8, 126.4, 126.2, 125.8, 125.7, 124.3, 122.2, 115.9, 113.5, 110.9, 16.1; HRMS (ESI) m/z [M + H]+ calcd for C32H22N2: 435.1861, found: 435.1857.

1-(Benzo[b]thiophen-2-yl)-3-methyl-5-(pyren-1-yl)-1H-pyrazole PPP3 (2c)

Analytical TLC on silica geln> pan class="Chemical">aluminum foil, 1:3 ethyl acetate/hexane Rf = 0.43; yellow liquid. Yield 39% (81 mg); 1H NMR (500 MHz, CDCl3) δ 8.26 (d, J = 9.5 Hz, 1H), 8.23 (d, J = 7.5 Hz, 1H), 8.20–8.18 (m, 3H), 8.10 (d, J = 8.0 Hz, 1H), 8.05–8.00 (m, 3H), 7.89 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.17–7.14 (m, 1H), 7.06–7.03 (m, 1H), 6.47 (s, 1H), 2.16 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 163.8, 155.1, 152.7, 134.4, 131.7, 131.5, 131.1, 130.8, 128.1, 127.6, 127.5, 126.2, 125.8, 125.6, 125.4, 125.0, 122.6, 121.8, 120.9, 120.1, 106.9, 16.2; HRMS (ESI) m/z [M + H]+ calcd for C27H17N3S: 416.1221, found: 416.1214.

1,3-Diphenyl-5-(pyren-1-yl)-1H-pyrazole PPP4 (2d)

Analytical TLC on silican> gel pan class="Chemical">aluminum foil, 1:9 ethyl acetate/hexane Rf = 0.48; yellow liquid. Yield 46% (96 mg); 1H NMR (500 MHz, CDCl3) δ 8.25 (d, J = 8.0 Hz, 1H), 8.14–8.12 (m, 1H), 8.06–8.04 (m, 1H), 7.99–7.91 (m, 5H), 7.84–7.82 (m, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.50–7.43 (m, 3H), 7.38–7.35 (m, 2H), 7.17–7.16 (m, 2H), 7.06–7.04 (m, 2H), 6.98 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 153.4, 141.7, 139.1, 138.6, 133.0, 131.5, 130.9, 130.6, 128.9, 128.8, 128.5, 127.9, 127.5, 126.5, 126.3, 126.1, 125.2, 124.4, 124.2, 123.7, 122.8, 107.0; HRMS (ESI) m/z [M + H]+ calcd for C31H20N2: 421.1699, found: 421.1695.

1-([1,1′-Biphenyl]-4-yl)-3-phenyl-5-(pyren-1-yl)-1H-pyrazole PPP5 (2e)

Analytical TLC on silica geln> pan class="Chemical">aluminum foil, 1:4 ethyl acetate/hexane Rf = 0.57; yellow liquid. Yield 52% (129 mg); 1H NMR (500 MHz, CDCl3) δ 8.25 (d, J = 7.5 Hz, 1H), 8.22–8.19 (m, 2H), 8.15 (d, J = 7.5 Hz, 2H), 8.10–8.07 (m, 1H), 8.06–8.04 (m, 3H), 7.90 (d, J = 8.0 Hz, 1H), 7.51–7.48 (m, 3H), 7.43–7.39 (m, 4H), 7.35–7.31 (m, 6H), 7.05 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 152.2, 142.8, 140.1, 139.7, 139.4, 133.2, 131.8, 131.4, 131.0, 129.9, 128.97, 128.90, 128.6, 128.5, 128.3, 127.5, 127.0, 126.5, 126.1, 125.8, 125.7, 124.8, 124.7, 124.4, 108.2; HRMS (ESI) m/z [M + H]+ calcd for C37H24N2: 497.2018, found: 497.2036.

3-Methyl-5-(pyren-1-yl)-1H-pyrazole PPP6 (2f)

Analytical TLC on silican> gel pan class="Chemical">aluminum foil, 1:4 ethyl acetate/hexane Rf = 0.39; yellow liquid. Yield 43% (60 mg); 1H NMR (500 MHz, CDCl3) δ 9.64 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.13–7.94 (m, 5H), 7.91 (d, J = 9.0 Hz, 1H), 7.58–7.54 (m, 1H), 6.59 (s, 1H), 2.59 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 143.3, 140.6, 136.5, 131.5, 131.4, 129.0, 128.1, 127.6, 126.5, 126.3, 126.2, 125.3, 124.5, 123.4, 118.0, 110.5; HRMS (ESI) m/z [M + H]+ calcd for C20H14N2: 283.1230, found: 283.1239.

1-Phenyl-5-(pyren-1-yl)-1H-pyrazole-3-carboxylic Acid PPP7 (2g)

Analytical TLC on silica geln> pan class="Chemical">aluminum foil, 1:4 ethyl acetate/hexane Rf = 0.25; yellow liquid. Yield 62% (256 mg); 1H NMR (500 MHz, CDCl3) δ 8.25 (d, J = 7.5 Hz, 1H), 8.22 (d, J = 7.5 Hz, 1H), 8.16 (d, J = 9.0 Hz, 1H), 8.11–8.05 (m, 4H), 7.78 (d, J = 8.0 Hz, 1H), 7.32 (s, 1H), 7.26–7.12 (m, 7H); 13C NMR (125 MHz, CDCl3) δ 165.6, 143.8, 143.5, 139.4, 132.1, 131.4,130.9, 129.9, 129.0, 128.8, 128.3, 127.4, 126.6, 126.1, 125.9, 124.8, 124.2, 124.0, 113.0; HRMS (ESI) m/z [M + Na]+ calcd for C26H16N2O2: 411.1104, found: 411.1124.

Docking Studies

Protein–ligand docking studies were performed for compounds against tubulin α- and β-dimer (PDB code: 1tub) using Chimera.[18]

MTT Assay

The cell viability of PPPs was investigated using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay in the presence of 10% fetal n class="Gene">pan class="Species">bovine serum in antibiotic free media. The yellow ne">pann> class="Chemical">tetrazolium salt (MTT) is modified by mitochondrial dehydrogenases to its purple formazan derivative (MTT-formazan) with maximum absorbance at 560–570 nm. The intensity of purple formazans indirectly reveals the mammalian cell survival and proliferation. Experiment was performed in 96-well plates (Cellstar, Germany) growing 10 000 cells (MCF-7, MDA-MB231, and MCF-10A) per well for 24 h before treatments. Experiments were performed for various concentrations of PPPs ranging from 105 to 100 nM and compared with Taxol as positive control ranging from 102 to 10–3 nM, as described in particular experiment. Cells were incubated for 48 h before performing the MTT assay. At the end of the incubation period, cells were treated with MTT (20 μL, 5 mg/mL) per well and further incubated for 4 h. At the end of the incubation, the entire medium was removed from the wells and 200 μL of dimethyl sulfoxide (DMSO) was added to dissolve blue-colored formazan crystals produced by mitochondrial respirations. The percentage cell viability was obtained by calculating absorbance values and calculated using the formula %viability = {[A630(treated cells) – (background)]/[A630(untreated cells) – background]} × 100.