Copper-Catalyzed Syntheses of Pyrene-Pyrazole Pharmacophores

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Article Cite This: ACS Omega 2018, 3, 6378−6387

Copper-Catalyzed Syntheses of Pyrene-Pyrazole Pharmacophores and Structure Activity Studies for Tubulin Polymerization Dinabandhu Sar,†,∥,⊥ Indrajit Srivastava,†,∥,⊥ Santosh K. Misra,†,∥ Fatemeh Ostadhossein,†,∥ Parinaz Fathi,†,∥ and Dipanjan Pan*,†,‡,§,∥ †

Department of Bioengineering, ‡Department of Materials Science and Engineering, and §Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States ∥ Mills Breast Cancer Institute and Carle Foundation Hospital, 502 North Busey, Urbana, Illinois 61801, United States S Supporting Information *

ABSTRACT: Tubulin polymerization is critical in mitosis process, which regulates uncontrolled cell divisions. Here, we report a new class of pyrenepyrazole 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-1yl)-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.

1. INTRODUCTION

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-pyrazole compounds as antimitotic agents. Taking into account the biological importance of pyrazole10,11 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 triflate-catalyzed synthesis of new pyrene-pyrazole pharmacophore (PPP1−7) from alkenyl hydrazones via cross-dehydrogenative coupling14 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.

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 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 mitotic arrest and cell death.6 Importantly, changes in microtubule stability and the expression of different tubulin isotypes, as well as altered posttranslational 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 © 2018 American Chemical Society

Received: February 22, 2018 Accepted: June 5, 2018 Published: June 13, 2018 6378

DOI: 10.1021/acsomega.8b00320 ACS Omega 2018, 3, 6378−6387

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ACS Omega Scheme 1. Synthesis of Pyrene-Pyrazole Pharmacophore 2a−fa,b

a

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

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

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 1). In a set of oxidant screening, K2S2O8 and tertbutyl hydroperoxide give 2d in 42 and 38% yield, respectively. The syntheses of PPPs (2) from substituted alkenyl hydrazones (1) were achieved in presence of optimized reaction condition (Scheme 1). In a typical procedure, the substrate comprising a phenyl group at N-1 position and methyl group at 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

2. RESULTS AND DISCUSSION 2.1. Synthesis of Pyrene-Pyrazole Pharmacophore. We have designed seven new molecules possessing pyrenepyrazole core structure. Analogues were designed with sidechain modification at 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 6379


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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.

°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. The content of polymerized microtubules was monitored by measuring fluorescence intensity at 430 nm (λex = 350 nm) every 7 s for 1.5 h. Paclitaxel (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 1). 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 1. 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).

position in alkenyl hydrazone (1b) afforded product PPP2 (2b) in 53% yield. It is noteworthy that the reaction of benzothiazolyl alkenyl hydrazone (1c) readily occurred to furnish corresponding PPP3 (2c). In addition, substrate-bearing phenyl group at N-1 and C3 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 2). 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). 2.2. 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 1). In this protocol, 85 μL of 10 mg/mL tubulin stock was incubated with tubulin glycerol buffer for 1.5 h at 37 6380


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ACS Omega 2.3. 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 polyacrylamide gel electrophoresis (SDS-PAGE) was performed to separate both tubulin as well as polymerized tubulin from the mixture. Figure 2a shows 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 (GTP) in α-tubulin and guanosine5′-diphosphate (GDP) and Taxotere (TAX) bound to βtubulin (Figure 3a,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-, βtubulin on the basis of their structures. PPP1 (Figure 3c,d) and PPP3 were shown to bind into GDP pocket having a minimum binding energy of −7.97 and −8.09 kcal/mol, respectively (Table 1). However, PPP2, PPP4 (Figure 3e,f), PPP5, PPP6 (Figure 3g,h), and PPP7 (Figure 3i,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. 2.5. 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., cytotoxicity) needed to be verified in vitro. An in vitro cancer cell two-dimensional culture model was used for this study using 3-(4,5-dimethylthiazole-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay.19 It was found that all of the PPPs were effective in killing a significant population of MCF-7 cells. PPP4 was found to be causing the maximum damage (Figure 4a) 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. Taxol, 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 PPP4 (Figure 4b). 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 cancer origin, while their selectivity toward 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 MDAMB231 (Figure 5A,B) and C32 (Figure 5C,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 5B,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 2).

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.

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 FIJI17 to calculate the band intensities, as shown in Figure 2b. Fold change of band intensities was further calculated with respect to PPP5−tubulin mixture (Figure 2c). From the results, it was evident that the band intensity for PPP5−tubulin was the least amongst all PPP−tubulin mixtures, possibly indicating structural interferences from 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. 2.4. 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 6381


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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-, βtubulin 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.

3. CONCLUSIONS In conclusion, a new class of pyrene-pyrazole analogues were synthesized through a remarkably simple 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 pyrene-pyrazole heterocyclic core structure. Success of the synthesis followed by biological and docking

Effectivity of anticancer drugs not only depends on cancer inhibition property but minimum effect to noncancerous normal cells. A representative study was performed on MCF10A 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 5E). It indicates the selectivity of PPPs toward cancer cells, presumably due to higher variation in tubulin polymerization processes. 6382


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ACS Omega 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 PPP2 PPP3 PPP4 PPP5 PPP6 PPP7

−7.97 −7.90 −8.09 −8.14 −7.64 −6.97 −8.16

GDP TAX GDP TAX TAX TAX TAX

opportunities for the development of novel microtubuletargeting pharmacophores and next-generation antibody−drug conjugates for cancer therapy.

4. EXPERIMENTAL SECTION 4.1. General Information. Unless otherwise mentioned, the reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used without any purification. Aryl hydrazines, 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 ketones20a,b and their corresponding hydrazones21 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 13 C) 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. 4.2. Solubility. All of the pyrenylpyrazoles PPP1−7 (2a−g) compounds are soluble in dimethyl sulfoxide (DMSO) and methanol (MeOH) solvents at room temperature. Cell studies have been performed in DMSO solvent. 4.3. General Procedure for the Synthesis of Pyrenylpyrazoles PPP1−6 (2a−f). Alkenyl hydrazones 1a−f (0.5 mmol) were stirred with 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. 4.4. General Procedure for the Synthesis of Methyl (Z)-2-Hydroxy-4-oxo-4-(pyren-1-yl)but-2-enoate (A). A solution of acetophenone (3 mmol) and dimethyl oxalate (6 mmol) in 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. 4.5. General Procedure for the Synthesis of Methyl 1Phenyl-5-(pyren-1-yl)-1H-pyrazole-3-carboxylate (B). Compound A (1 mmol) was stirred with 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

a

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

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.

studies further provided a structural basis for the rational design of potent microtubule polymerization-modulating agents. The new 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 indepth preclinical study is warranted and will be undertaken in the near future. Results reported here may open up 6383


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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 MCF-7 MDA-MB231 C32

PPP1 (μM)

PPP2 (μM)

PPP3 (μM)

PPP4 (μM)

PPP5 (μM)

PPP6 (μM)

PPP7 (μM)

60 ± 10 >100 >100

34 ± 4 15 ± 5 >100

>100 >100 >100

1 ± 0.5 0.5 ± 0.2 5.0 ± 2.0

>100 >100 >100

43 ± 5 >100 >100

8±2 10 ± 2.0 14 ± 2.0

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. 4.6. General Procedure for the Synthesis of 1-Phenyl5-(pyren-1-yl)-1H-pyrazole-3-carboxylic Acid PPP7 (2g). In an oven-dried round bottom flask, KOH (1.75 mmol) and drops of 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). 4.6.1. 3-Methyl-1-phenyl-5-(pyren-1-yl)-1H-pyrazole PPP1 (2a). Analytical TLC on silica gel aluminum foil, 1:4 ethyl acetate/hexane Rf = 0.52; yellow liquid. Yield 58% (103 mg); 1 H 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. 4.6.2. 1-([1,1′-Biphenyl]-4-yl)-3-methyl-5-(pyren-1-yl)-1Hpyrazole PPP2 (2b). Analytical TLC on silica gel aluminum 6384


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ACS Omega 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. 4.6.3. 1-(Benzo[b]thiophen-2-yl)-3-methyl-5-(pyren-1-yl)1H-pyrazole PPP3 (2c). Analytical TLC on silica gel 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. 4.6.4. 1,3-Diphenyl-5-(pyren-1-yl)-1H-pyrazole PPP4 (2d). Analytical TLC on silica gel 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); 13 C 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. 4.6.5. 1-([1,1′-Biphenyl]-4-yl)-3-phenyl-5-(pyren-1-yl)-1Hpyrazole PPP5 (2e). Analytical TLC on silica gel 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. 4.6.6. 3-Methyl-5-(pyren-1-yl)-1H-pyrazole PPP6 (2f). Analytical TLC on silica gel 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. 4.6.7. 1-Phenyl-5-(pyren-1-yl)-1H-pyrazole-3-carboxylic Acid PPP7 (2g). Analytical TLC on silica gel 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. 4.7. Docking Studies. Protein−ligand docking studies were performed for compounds against tubulin α- and β-dimer (PDB code: 1tub) using Chimera.18 4.8. 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 bovine serum in antibiotic free media. The yellow 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.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00320. NMR, MS, fluorescence data, IC50 values of all of the PPP analogues, and functional activity of PPPs in different cells (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Indrajit Srivastava: 0000-0002-6864-0202 Santosh K. Misra: 0000-0002-3313-4895 Dipanjan Pan: 0000-0003-0175-4704 Author Contributions ⊥

D.S. and I.S. contributed equally to this work.

Funding

The authors gratefully acknowledge funding support from University of Illinois. Notes

The authors declare the following competing financial interest(s): Prof. Pan is the founder or co-founder of three University-based start-ups. None of these entities however support this research.

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ACKNOWLEDGMENTS Funding support from the University of Illinois is gratefully acknowledged. NMR spectroscopy measurements, tubulin 6385


Article

ACS Omega polymerization assay, fluorescence spectra of individual PPPs were performed in Roger Adams Lab, UIUC. Mass spectroscopy of the PPPs was carried out in Noyes Lab, UIUC.

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Copper-Catalyzed Syntheses of Pyrene-Pyrazole Pharmacophores

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