Synthesis, characterization and biological evaluation

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European Journal of Medicinal Chemistry 155 (2018) 13e23

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

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Synthesis, characterization and biological evaluation of tertiary sulfonamide derivatives of pyridyl-indole based heteroaryl chalcone as potential carbonic anhydrase IX inhibitors and anticancer agents Mudasir Nabi Peerzada a, Parvez Khan b, Kamal Ahmad b, Md Imtaiyaz Hassan b, Amir Azam a, * a b

Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, 110 025, New Delhi, India Centre for Interdisciplinary Research in Basic Science, Jamia Nagar, 110 025, New Delhi, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 March 2018 Received in revised form 3 May 2018 Accepted 20 May 2018 Available online 22 May 2018

In the quest for novel effective carbonic anhydrase inhibitors, some sulfonamide derivatives of pyridylindole based chalcone were synthesized and screened in vitro for inhibitory activity against human carbonic anhydrase IX isoform. Among all the synthesized compounds (SC2 -SC11), only three compounds SC3, SC7 and SC10 were found to have better binding affinity as shown by molecular docking and fluorescence binding studies. Further, the enzyme inhibition assay and in vitro anti-tumor evaluation against MCF-7 and HepG-2 cell lines revealed that the compounds SC3, SC7 and SC10 inhibited the CA IX selectively, possessed predominant anti-proliferative potential and significantly induced apoptosis in cancerous cells. © 2018 Elsevier Masson SAS. All rights reserved.

Keywords: Tertiary sulfonamide Chalcone Carbonic anhydrase inhibition Apoptosis Molecular docking

1. Introduction Cancer is a major global health problem and is the second leading cause of mortality after cardiovascular diseases [1]. There is an immense need for the development of safe and efficacious chemotherapeutic agent for such a dreadful disease [2]. The inhibition of carbonic anhydrase activity is one of the important areas of anticancer drug discovery as out of sixteen isoforms found in humans, the transmembrane human carbonic anhydrase form (hCA) IX is exclusively over expressed in hypoxic tumors and is involved in the promotion of many adaptive changes in solid tumors [3e6]. Thus CA IX emerged as potential anticancer drug target. The sulfonamides have emerged as the significant class of compounds explored for drug discovery and have been found to be the most effective CA inhibitors [7e9]. A large number of sulfonamide derivatives mostly primary and some secondary have proven to possess effective carbonic anhydrase inhibitory activity and some of them are clinically approved like acetazolamide, dichlorphenamide and ethoxzolamide (Fig. 1) [8,10].

* Corresponding author. E-mail addresses: [email protected], [email protected] (A. Azam). https://doi.org/10.1016/j.ejmech.2018.05.034 0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

Rare focus has been depicted towards the carbonic anhydrase inhibitory activity of the tertiary sulfonamides in comparison to primary and secondary, however such a class has recently been observed to be the promising selective anti-tumor agents and act via unknown mechanism on CAs [11,12]. Probenecid (Fig. 1) and some other tertiary sulfonamide derivatives (Fig. 1AeC) have been found to inhibit the activity of CA IX selectively [12,13]. The indole and pyridine are the attractive drug oriented scaffolds and their derivatives possess the broad spectrum of biological activities including anticancer and hCA inhibition [14,15]. The pyridine based derivatives (Fig. 1D) have been reported earlier to inhibit the hypoxemic tumor associate CA IX isform selectively [16,17]. Chalcones are well known for their diverse biological activities and have been also reported to be the hCA inhibitors [18,19]. The various pharmacological activities of chalcones are attributed to the binding of a, b unsaturated carbonyl group to the receptor site in the cells [20,21]. It is well known that the combination of biologically active moieties in the single pharmacophore enhances the bioactivity of the compound [22,23]. Therefore, encouraged by these findings, it was speculated to synthesize tertiary-sulfonamide derivatives of pyridyl indole based chalcones and to screen them against human carbonic anhydrase IX isoform and the off target CA II isoform. In

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Fig. 1. Some representatives of carbonic anhydrase inhibitors.

order to assess the anticancer profile of the synthesised compounds, all the title compounds were subjected for the determination of apoptosis (in MCF-7) and antiproliferative potential in MCF-7 and HepG-2 cancerous cells. 2. Results and discussion 2.1. Chemistry The synthetic strategy employed for the synthesis of target compounds is depicted in Scheme 1. The chalcone (M1) was synthesized by piperidine catalyzed Knoevenagel condensation by reacting 4-acetylpyridine with indole-3-carbaldehyde [24]. Further the treatment of (M1) with different sulfonyl chlorides in basic medium furnished the targeted sulfonamides (SC 2- SC 11). The structures of all the compounds (M1-SC11) were elucidated on the basis of FT-IR, 1H NMR, 13C NMR, ESI-MS and the purity of was confirmed by CHNS elemental analysis. 2.2. Biological activity 2.2.1. Enzyme inhibition assay Initial screening of all the synthesized compounds have been carried out by enzyme inhibition assay. Enzyme inhibition potential of synthesized compounds against CA II and CA IX was analyzed by measuring the esterase activity of CA using p-nitrophenol acetate

(4-PNA) as a substrate. Acetazolamide was used as a positive control in enzyme inhibition studies. It was found that though all the synthesized compounds inhibited the activity of CA IX but compounds SC3, SC7 and SC10 exhibited the significant inhibition of CA IX activity having IC50 value of 0.15 mM, 0.13 mM and 0.15 mM respectively. It is interesting to note that if activity of these compounds is compared against CA II, less inhibition was noticed with following IC50 values, 12.10 mM, 9.10 mM and 14.10 mM, respectively (Table 1). The compound bearing 2,4 dichloro phenyl substituent was found to be most active with IC50 value of 0.13 mM. Compound SC3 possess the strongly deactivated p-nitro phenyl substituent and SC10 have the strongly activating p-tertiary butyl phenyl substituent attached. Both compounds SC3 and SC10 have equal IC50 value of 0.15 mM. Therefore in terms of structure activity relationship (SAR), it can be construed that the activity of these compounds is substituent dependent. 2.2.2. Cell viability assay Antiproliferative potential of synthesized compounds on MCF-7 and HepG-2 cells was studied with the help of MTT assay. Treatment of compounds inhibited the viability of MCF-7 cell line in a dose-dependent manner (Fig. 2). IC50 values of each compound after 48 h treatment are given in Table 1. Interestingly, it was found that all compounds hindered the viability of cells as the concentration of compounds increases, but SC3, SC7 and SC10 inhibits the viability of MCF-7 cells more prominently as suggested by their low

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Scheme 1. Synthesis of tertiary sulfonamide derivatives of pyridyl-indole based chalcone. Reagents and conditions (a) Piperidine, methanol, reflux 80  C, 16 h (b) Sulfonyl chlorides, Na2CO3, 50% THF:H2O, room temperature, 24e48 h.

IC50 values. The IC50 value of SC3, SC 7 and SC10 is found to be 24 mM, 12 mM and 14.5 mM, respectively. On the other hand in case of HEK-293 cells, it was found that studied compounds did not inhibit the proliferation of HEK-293 cells in the studied concentration range (0e150 mM). These results suggested that the synthesized compounds specifically show significant toxicity towards cancerous cells (MCF-7 and HepG-2) only. 2.2.3. Apoptosis assay To further extend our interpretations regarding the functional influence of selected CA IX inhibitors in terms of apoptotic potential, MCF-7 cells were treated with the respective IC50 dose of each compound for 24 h. Annexin-V and 7-AAD double staining method was used to study early apoptosis (annexin-V staining) and late apoptosis (PE-staining/7-AAD). It was found that treatment of cells with compounds SC3, SC7 and SC10 induces apoptosis in MCF7 cells (Fig. 3). Consistent with the cell viability studies as described in earlier section, it was found that treatment of compounds SC3, SC7 and SC10 induces apoptosis in MCF-7 cells as the treated cells are found to be positive for annexin-V and PE-staining (Fig. 3). As compared to untreated or control cells, the treatment of compounds SC3, SC7 and SC10 induces apoptosis in 41.75%, 89.33% and 21.57% of cells, respectively. These results clearly indicated that studied compounds induce the apoptosis in MCF-7 cells. Evasion of apoptosis is an emergent and important hallmark of cancer [25], so for the development of effective anticancer agents it would be highly significant if the synthesized molecule induces apoptosis. It was found in earlier studies that some of the 4/3-((4-oxo-5-(2oxoindolin-3-ylidene)thiazolidin-2-ylidene) amino) benzenesulfonamide derivatives inhibited the hCA IX and induced the apoptosis in MCF-7 cells [26]. Similarly, various research groups studied the different types of compounds and observed that some CA IX inhibitors induced the significant apoptosis in some cancerous cells [27e29]. Interestingly, our results are in consistence with these studies that among all the synthesized compounds, three compounds which inhibited the hCA IX, also induced the appreciable apoptosis in MCF-7 cells. 2.2.4. Fluorescence binding studies To evaluate the actual binding

affinity of

synthesized

compounds with CA IX, fluorescence measurements were performed. Binding affinity (in terms of binding constants) of selected compounds towards purified CA IX was assessed with the help of fluorescence-monitored compound-CA IX titration by using modified Stern-Volmer equation (equation (1)). When CA IX was titrated with synthesized compounds, the significant quenching was observed in case of compounds SC3, SC7 and SC10 (Fig. 4). Binding constant of these compounds to CA IX were found Ka ¼ 1.0  106 M1, Ka ¼ 5.1  107 M1 and Ka ¼ 2.8  105 M1 for compounds SC3, SC 7 and SC10, respectively. These observations suggested that studied compounds bind with CA IX significantly, which may be further exploited as potential inhibitors of CA IX. 2.2.5. Molecular docking Auto dock 4.2 was used to determine the orientation of inhibitors bound in the active site of the CA IX and the conformation with the highest binding energy value for each molecule was chosen for further analysis and results of these studies are given in Table 2. The binding mode of CA IX inhibitors are visualized by PyMOL. The binding site of CA IX has been used to elucidate the interactions as reported earlier [30]. Docking analysis of compounds M1-SC11 was carried with CA IX to elucidate the interaction pattern of synthesized inhibitors. Results showed that the compounds SC3, SC7 and SC10 bind effectively into the active site of CA IX with minimum binding energy of (DG) 10.7 kcal/mol, (DG) 11.5 kcal/mol and 10.1 kcal/mol respectively as with reference to acetazolamide (DG) 6.43 kcal/mol [30] (Table 2). The binding mode of compound SC3 in the active site of CA IX is stabilized by eight H-bond interaction with Trp5, Pro11, Arg60, Gln67, Gly98, Arg246 (Fig. 5AeB). Active site residues Gln28, Leu148, Glu150, Thr199 and His224 are involved in hydrophobic interaction and His224 is stabilized by phenyl moiety of compound SC3 via pep interaction (Fig. 5AeB). Compound SC7 formed four H-bonds with Arg60, Thr69, Thr200 and Pro201 residues of active site (Fig. 5CeD). The active site residues Trp5, His94, Gly98, His119, Val121, Trp209 and Thr198 are involved in hydrophobic interaction whereas His94 and Trp209 residues are stabilized by phenyl and indole motif of compound SC7 respectively due to pep interaction (Fig. 5CeD). At the active site of CA IX, compound SC10 forms six Hbond interactions with Gln67, His94, His96, Thr199, whereas

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Table 1 Biological activity of the compounds against various cell lines, hCA II and hCA IX. Compound No.

HEK-293

MCF-7

HepG-2

Esterase assay IC50 (mM),

IC50 (mM)±S.D.

IC50 (mM)±S.D.

IC50 (mM)±S.D.

hCA IX

hCA II

>150 >150

45.3 ± 1.21 65.8 ± 1.35

49.3 ± 1.31 26.0 ± 2.47

N.D. 26.15

N.D 29.35

SC3

>150

24.0 ± 1.16

27 ± 1.67

0.15

12.10

SC4

>150

36.9 ± 0.79

31.7 ± 1.10

57.31

36.21

SC5

>150

48.9 ± 1.19

44.8 ± 1.35

29.9

38.40

SC6

>150

37.5 ± 1.34

35.1 ± 1.47

14.41

13.50

SC7

>150

12.2 ± 1.02

14.8 ± 1.28

0.13

9.10

SC8

>150

58.0 ± 2.18

62.9 ± 1.34

44.08

65.22

SC9

>150

36.3 ± 1.16

42.6 ± 1.41

37.56

24.32

SC10

>150

14.5 ± 1.15

18.3 ± 1.44

0.15

14.10

11

>150

52.4 ± 1.22

46.2 ± 1.14

44.52

>100.00

142.8 ± 1.62 N.D

20.2 ± 1.70 N.D

18.7 ± 1.37 N.D

N.D 0.027

N.D 0.013

M1 SC2

12 13

R

e

Doxorubicin AZM

residues Leu70, Leu81, Tyr88, Arg89 and Gln92 are involved in hydrophobic interaction (Fig. 5E and F). The indole ring of compound SC10 is stabilized by pep interaction of Tyr88. Molecular docking result concluded that interaction and environment at the binding site of CA IX is more favourable for SC3, SC7 and SC10 as compared to other compounds. Additionally, docking studies showed that the synthesized compounds have emerged as a novel class of selective hCA IX inhibitors and exhibited excellent binding pattern. 3. Conclusion This study led to the finding of novel potential hCA IX inhibitors SC3, SC7 and SC10 which indicates that tertiary arylsulfonamides may act as the lead compounds in the discovery of non-zincbinding inhibitors, with an anticancer pharmacological profile.

Moreover, the biological evaluation revealed that synthesized tertiary arylsulfonamides inhibited the activity of transmembrane isoform hCA IX selectively and have mere effect on active cystosolic isoform hCA II off target, which is the significant breakthrough for anticancer drug discovery as such a class of compounds possess least side effects. Lamentably, the exact inhibition mechanism of such inhibitors has not been yet elucidated [12]. The results assessed for the pharmacological effects of cell apoptosis and cytotoxicity on MCF-7 cell line were adequate and divulged that such sulfonamides may be construed as lead compounds for the quest of anticancer chemotherapeutic molecules in future. 4. Experimental protocols All the required chemicals were purchased from Merck and Aldrich Chemical Company (USA). Precoated aluminium sheets

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doublet; m, multiplet. Chemical shift values are given in ppm. Mass spectra of all the compounds were recorded by ESI-MS (AB-Sciex, 2000, Applied Biosystem), 4.1. Procedure for the synthesis of pyridyl-indole chalcone (M1) Indole-3-carboxaldehyde (13.56 mmol) was reacted with 4Acetyl-pyridine (13.56 mmol) in presence of piperidine (6.77 mmol) using methanol as solvent. The reaction mixture was stirred under reflux for 16 h. After the completion of the reaction, yellow colored precipitate was filtered, washed, dried and recrystallized from ethanol-dichloromethane to furnish precursor (M1).

Fig. 2. Cell proliferation studies. Effect of SC3, SC7 and SC10 compounds on the viability of MCF-7 cells; Cells were treated with increasing concentration of compounds as indicated on x-axis for 48 h. Cell viabilities were presented as a percentage of the number of viable cells to that of the control. Each data point shown is the mean ± SD from n ¼ 3. (For anticancer activities doxorubicin has been taken as positive control).

(Silica gel 60 F254, Merck Germany) were employed for thin-layer chromatography (TLC). The melting points of all the compounds were observed on Veego instrument with model specifications REC-22038 A2 and are uncorrected. IR spectra were acquired from Bruker FT-IR spectrophotometer. 1H NMR and 13C NMR were recorded on a Bruker Spectrospin DPX 300 MHz and Bruker Spectrospin DPX 75 MHz spectrometer respectively, using CDCl3 or DMSO-d6 as a solvent and trimethylsilane (TMS) as the internal standard. Splitting patterns are designated as follows; s, singlet; d,

4.1.1. (2E)-3-(1H-indol-3-yl)-1-(pyridin-4-yl)prop-2-en-1-on (M1) Yield: 93%; yellow solid; m. p: 266e268  C; Anal. Calc. C16H12N2O: C 77.40, H 4.87, N 11.28, O 6.44%. found: C 77.28, H 4.73, N 11.17%; FT-IR nmax (cm1): 3039 (N-H), 1648 (C]O), 1607 (C]C); 1 H NMR (DMSO-d6) d (ppm): 12.02 (s, 1H, NH), 8.83 (d, 2H, J ¼ 6.0 Hz), 8.18e8.09 (m, 3H, Ar-H), 7.98 (d, 2H, J ¼ 6.0Hz, Ar-H), 7.60 (d, 1Hb, J ¼ 15.6 Hz), 7.53e7.50 (m, 1H, Ar-H), 7.29e7.22 (m, 2H, Ar-H); 13C NMR (DMSO-d6) d (ppm): 188.89, 151.07, 145.23, 141.33, 138.12, 134.89, 125.55, 123.39, 121.94, 121.88, 121.02, 115.21, 113.35, 113.04; ESI-MS: m/z ¼ 249 [Mþþ1]. 4.2. synthesis of sulphonamide derivatives of indole-pyridine chalcone (SC2-SC11) The synthesized chalcone M1 (0.80 mmol) was stirred in 50% THF: H2O with sodium carbonate (0.80 mmol) for 30 min. The different substituted sulfonyl chlorides (0.80 mmol) dissolved in THF was added drop wise. Progress of the reaction was monitored by TLC. The reaction was completed in 24e48 h at room temperature. The reaction mixture was poured onto ice cold water, the solid

Fig. 3. Inhibition of CA IX by studied compounds induces apoptosis in MCF-7 cells. MCF-7 cells were treated with IC50 concentrations of each compound for 24 h and processed for apoptosis analysis using Annexin-V-PI apoptosis kit and were quantified by flow cytometry. (A) Representative flow cytometry images showing FITC-Annexin-V labelled cells, which directly corresponds to the percentage of apoptotic cells. (B) Bar graphs represents the percentage of apoptotic MCF-7 cells stained with Annexin-V for duplicate measurements ± SD. Statistical analysis was done using two-tailed Student t-test for uPNAired samples.**p < 0.001, as compared to control (untreated cells).

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Fig. 4. Binding studies of compounds SC3, SC7 and SC10 with CA IX using fluorescence spectroscopy. Fluorescence emission spectra of CA IX (10 mM) with the increasing concentration of; (A) compound SC3 (B) compound SC7 (C) compound SC10. Modified Stern-Volmer plot showing fluorescence quenching of FASTK by (D) compound SC3 (E) compound SC7 (F) compound SC10, used to calculate binding affinity (Ka) and number of binding sites (n).

product was filtered, dried and recrystallized from ethanol and dichloromethane. 4.2.1. (2E)-3-[1-(4-Methoxybenzene-1-sulfonyl)-1H-indol-3-yl]-1(pyridin-4-yl)prop-2-en-1-one (SC2) Yield: 60%; light brown solid; m. p: 216  C; Anal. Calc. for C23H18N2O4S: C 66.01, H 4.34, N 6.69, O 15.29, S 7.66%. found: C

65.94, H 4.13, N 6.54, S 7.47%; FT-IR nmax (cm1): 1667 (C]O), 1601 (C]C), 1344 (-SO2-N); 1H NMR (DMSO-d6) d (ppm): 8.86 (d, 2H, J ¼ 5.7 Hz Ar-H), 8.70 (s, 1H, Ar-H), 8.14 (d, 1H, J ¼ 7.8 Hz, Ar-H), 8.04e7.97 (m, 6H, Ar-H), 7.89 (d, 1Ha, J ¼ 15.6 Hz), 7.48e7.37 (m, 2H, Ar-H), 7.12 (d, 2H, J ¼ 9.0 Hz, Ar-H), 3.78 (s, 3H, -OCH3); 13C NMR (DMSO-d6) d (ppm): 189.37, 164.59, 151.20, 144.13, 137.18, 135.25, 131.71, 129.88, 128.27, 128.17, 126.13, 124.81, 122.06, 121.65,

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Table 2 Binding energy and specific interaction of CA IX with compounds. Compounds

Binding energy (kcal/mol)

Protein ligands interaction No. of H bonds

Amino acid residues

Distance (Å)

Thr200 Gln67 Trp5 Ser3 Trp5 Pro11 Arg60 Gln67 Gly98 Arg246 Arg60 Thr200 Pro201 Trp5 Gly9 Pro11 Gln67 Arg246 Gln67 Cys23 Asp10 Thr199 Arg60 Thr69 Thr200 Pro201 His94 Cys23 Asp10 Gly9 Pro11 Gln67 His94 Gln67 His94 His96 Thr199 Thr200 Pro11 Cys23 Asp10 His64 Gln67 His94 His96 Thr199 Thr200

2.0 2.8 3.1 3.4 2.9 3.4 3.1 2.9, 2.8 2.6, 3.2 2.7 3.1 3.1 2.9 3.5 3.0, 2.9, 2.9 3.2 3.0 2.8, 3.2 2.7 2.4 3.4 2.1 2.2, 2.8, 2.2 3.7 2.1 3.4 2.1 2.8 2.3 2.8, 2.1 2.7 3.1 2.2, 1.6 1.9 1.9 3.1 2.9, 2.9,

M1 SC2

7.5 8.9

1 3

SC3

10.7

8

SC4

9.1

3

SC5

8.8

7

SC6

8.3

6

SC7

11.5

4

SC8

9.0

5

SC9

8.1

4

SC10

10.1

6

SC11

9.3

5

Acetazolamide

6.43

9

121.48,118.45, 115.69,113.84, 56.37; ESI-MS: m/z ¼ 403.1 [Mþþ1]. 4.2.2. (2E)-3-[1-(4-Tert-butylbenzene-1-sulfonyl)-1H-indol-3-yl]1-(pyridin-4-yl)prop-2-en-1-one (SC3) Yield: 90%; light yellow solid; m. p: 244  C; Anal. Calc. for C26H24N2O3S: C 70.25, H 5.44, N 6.30, O 10.80, S 7.21%. found: C 70.31, H 5.32, N 6.15, S 7.08%; FT-IR nmax (cm1): 1669 (C]O), 1603 (C]C), 1372 (-SO2-N); 1H NMR (DMSO-d6) d (ppm): 8.86 (d, 2H, J ¼ 5.1 Hz Ar-H), 8.74 (s, 1H, Ar-H), 8.13 (d, 2H, J ¼ 7.5 Hz, Ar-H), 8.044e7.96 (m, 4H, Ar-H), 7.91 (d, 1Ha, J ¼ 15.9 Hz), 7.62 (d, 2H, J ¼ 8.4 Hz, Ar-H), 7.49e7.37 (m, 2H, Ar-H), 1.18 (s, 9H, -CH3); 13C NMR (DMSO-d6) d (ppm): 189.35, 158.84, 151.21, 144.10, 137.04, 135.28, 134.32, 131.54, 128.19, 127.42, 127.32, 126.24, 124.90, 122.06, 121.68, 118.62, 113.54, 35.51, 30.91; ESI-MS: m/z ¼ 445.0 [Mþþ1]. 4.2.3. (2E)-3-[1-(2-Nitrobenzene-1-sulfonyl)-1H-indol-3-yl]-1(pyridin-4-yl)prop-2-en-1-one (SC4) Yield: 85%; brown solid; m. p: 198  C; Anal. Calc. for C22H15N3O5S: C 60.96, H 3.49, N 9.69, O 18.46, S 7.40%. found: C

3.0 3.4

3.4 3.4

2.9, 2.9

3.6 3.0

2.4, 3.2

3.0

2.9, 3.2 2.9

60.85, H 3.62, N 9.45, S 7.21%; FT-IR nmax (cm1): 1672 (C]O), 1603 (C]C), 1367 (-SO2-N); 1H NMR (CDCl3) d (ppm): 8.87 (d, 2H, J ¼ 5.7 Hz, Ar-H), 8.11e8.06 (m, 3H, Ar-H), 8.00 (s, 1H, Ar-H), 7.94e7.87 (m, 2H, Ar-H), 7.82e7.71 (m, 4H, Ar-H), 7.60 (d, 1Hb, J ¼ 15.9 Hz), 7.47e7.41 (m, 2H, Ar-H); 13C NMR (DMSO-d6) d (ppm): 189.50, 151.19, 147.70, 144.07, 137.42, 136.92, 135.16, 134.11, 131.96, 130.76, 129.36, 128.08, 126.51, 126.11, 125.36, 122.21, 122.11, 121.96, 118.53, 113.72; ESI-MS: m/z ¼ 434.0 [Mþþ1]. 4.2.4. (2E)-3-[1-(4-Methylbenzene-1-sulfonyl)-1H-indol-3-yl]-1(pyridin-4-yl)prop-2-en-1-one (SC5) Yield: 80%; light brown solid; m. p: 203  C; Anal. Calc. for C23H18N2O3S: C 68.64, H 4.51, N 6.96, O 11.93, S 7.97%. found: C 68.45, H 4.36, N 6.94, S 7.80%; FT-IR nmax (cm1): 1669 (C]O), 1603 (C]C), 1343 (-SO2-N); 1H NMR (DMSO-d6) d (ppm): 8.86 (d, 2H, J ¼ 5.7 Hz, Ar-H), 8.72 (s, 1H, Ar-H), 8.15 (d, 1H, J ¼ 7.5 Hz, Ar-H), 8.03e7.98 (m, 4H, Ar-H), 7.95 (d, 2H, J ¼ 8.4 Hz, Ar-H), 7.90 (d, 1Ha, J ¼ 15.9Hz), 7.48 (d, 1H, J ¼ 6.6 Hz, Ar-H), 7.42e7.37 (m, 3H, ArH), 2.31 (s, 3H, -CH3); 13C NMR (DMSO-d6) d (ppm): 189.35, 151.21,

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Fig. 5. Molecular Docking studies of CA IX with compounds SC3, SC7 and SC10: Cartoon view of (A) compound SC3 (C) compound SC7 (E) compound SC10 docked with CA IX. Active site residue interactions with (B) compound SC3 (D) compound SC7 (F) compound SC10. Residues are shown with ball and stick and compound SC3, SC7 and SC10 is shown with stick model. Hydrogen bonds are shown as broken lines (black).

146.53, 144.08, 137.09, 135.24, 134.08, 131.67, 130.91, 128.18, 127.39, 126.19, 124.90, 122.07, 121.68, 121.57, 118.65, 113.84, 21.49; ESI-MS: m/z ¼ 403.2 [Mþþ1]. 4.2.5. (2E)-3-[1-(4-Chlorobenzene-1-sulfonyl)-1H-indol-3-yl]-1(pyridin-4-yl)prop-2-en-1-one (SC6) Yield: 85%; brown solid; m. p: 216  C; Anal. Calc. for C22H15ClN2O3S: C 62.48, H 3.58, N 6.62, O 11.35, S 7.58, Cl 8.38%. found: C 62.65, H 3.47, N 6.55, S 7.49%; FT-IR nmax (cm1): 1665 (C] O), 1607 (C]C), 1371 (-SO2-N); 1H NMR (CDCl3) d (ppm): 8.86 (d, 2H, J ¼ 6.0 Hz, Ar-H), 8.02e7.93 (m, 2H, Ar-H), 7.89e7.83 (m, 4H, ArH), 7.79e7.67 (m, 2H, Ar-H), 7.55 (d, 1H, J ¼ 15.9 Hz), 7.46e7.37 (m,

4H, Ar-H); 13C NMR (DMSO-d6) d (ppm): 189.38, 151.18, 144.08, 140.71, 136.86, 135.74, 135.22, 131.44, 130.70, 129.27, 128.26, 126.37, 125.10, 122.06, 121.93, 121.77, 119.09, 113.81; ESI-MS: m/z ¼ 423.1 [Mþþ1]. 4.2.6. (2E)-3-[1-(2,4-Dichlorobenzene-1-sulfonyl)-1H-indol-3-yl]1-(pyridin-4-yl)prop-2-en-1-one (SC7) Yield: 80%; light green solid; m. p: 220  C; Anal. Calc. for C22H14Cl2N2O3S: C 57.78, H 3.09, N 6.13, O 10.50, S 7.01, Cl 15.50%. found: C 57.77, H 2.99, N 6.06, S 6.99%; FT-IR nmax (cm1): 1667 (C] O), 1603 (C]C), 1372 (-SO2-N); 1H NMR (DMSO-d6) d (ppm): 8.85 (d, 2H, J ¼ 5.4 Hz, Ar-H), 8.78 (s, 1H, Ar-H), 8.42 (s, 1H, Ar-H),

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8.19e8.16 (m, 1H, Ar-H), 8.07e7.99 (m, 3H, Ar-H), 7.92 (d, 1Ha, J ¼ 15.9 Hz), 7.86e7.82 (m, 1H, Ar-H), 7.78e7.75 (m, 1H, Ar-H), 7.70 (d, 1H, J ¼ 8.7 Hz Ar-H), 7.44e7.39 (m, 2H, Ar-H); 13C NMR (DMSOd6) d (ppm): 189.46, 151.16, 144.11, 137.05, 136.94, 135.95, 134.97, 134.79, 133.47, 132.93, 131.66, 130.78, 127.97, 126.34, 125.18, 122.10, 121.88, 117.79, 113.52; ESI-MS: m/z ¼ 457.1 [Mþ]. 4.2.7. (2E)-3-[1-(Naphthalene-1-sulfonyl)-1H-indol-3-yl]-1(pyridin-4-yl)prop-2-en-1-one (SC8) Yield: 80%; light green solid; m. p: 248  C; Anal. Calc. for C26H18N2O3S: C 71.22, H 4.14, N 6.39, O 10.95, S 7.31%. found: C 71.10, H 3.94, N 6.18, 7.20%; FT-IR nmax (cm1): 1667 (C]O), 1603 (C]C), 1372 (-SO2-N); 1H NMR (DMSO-d6) d (ppm): 8.92 (s, 1H, ArH), 8.85e8.83 (m, 2H, Ar-H), 8.79 (s, 1H, Ar-H), 8.23 (d, 1H, J ¼ 7.5 Hz, Ar-H), 8.12e8.04 (m, 3H, Ar-H), 8.00e7.96 (m, 4H, Ar-H), 7.92 (s, 1H, Ar-H), 7.90 (d, 1Ha, J ¼ 15.9 Hz) 7.71e7.67 (m, 2H, Ar-H), 7.44e7.36 (m, 2H, Ar-H); 13C NMR (DMSO-d6) d (ppm): 189.39, 151.19, 144.10, 137.09, 135.42, 135.25, 133.84, 131.89, 131.76, 130.84, 130.57, 130.13, 129.65, 128.70, 128.41, 128.16, 126.23, 124.94, 122.06, 121.70, 121.53, 118.67, 113.85; ESI-MS: m/z ¼ 438.9 [Mþþ1]. 4.2.8. (2E)-3-[1-(3-Nitrobenzene-1-sulfonyl)-1H-indol-3-yl]-1(pyridin-4-yl)prop-2-en-1-one (SC9) Yield: 90%; light yellow solid; m. p: 250  C; Anal. Calc. for C22H15N3O5S: C 60.96, H 3.49, N 9.69, O 18.46, S 7.40%. found: C 60.82, H 3.61, H 3.61, S 7.25%; FT-IR nmax (cm1): 1669 (C]O), 1600 (C]C), 1343 (-SO2-N); 1H NMR (DMSO-d6) d (ppm): 8.85 (d, 2H, J ¼ 5.7 Hz, Ar-H), 8.79 (s, 1H, Ar-H), 8.70 (s, 1H, Ar-H), 8.52e8.49 (m, 2H, Ar-H), 8.15 (d, 1H, J ¼ 7.8 Hz, Ar-H), 8.07 (d, 1H, J ¼ 8.4Hz, Ar-H), 8.00e7.95 (m, 3H, Ar-H), 7.92 (s, 1H, Ar-H), 7.89 (d, 1Ha J ¼ 15.6 Hz), 7.52e7.40 (m, 2H, Ar-H); 13C NMR (DMSO-d6) d (ppm): 189.40, 151.20, 148.70, 144.01, 138.30, 136.70, 135.20, 133.12, 132.64, 131.39, 130.08, 128.30, 126.61, 125.31, 122.16, 122.05, 121.87, 119.47, 113.82; ESI-MS: m/z ¼ 434.0 [Mþþ1]. 4.2.9. (2E)-3-[1-(4-Nitrobenzene-1-sulfonyl)-1H-indol-3-yl]-1(pyridin-4-yl)prop-2-en-1-one (SC10) Yield: 90%; brown solid; m. p: 260  C; Anal. Calc. for C22H15N3O5S: C 60.96, H 3.49, N 9.69, O 18.46, S 7.40%. found: C 60.75, H 3.38, N 9.44, S 7.23%; FT-IR nmax (cm1): 1669 (C]O), 1600 (C]C), 1344 (-SO2-N); 1H NMR (CDCl3) d (ppm): 8.87 (s, 2H, Ar-H), 8.33 (d, 2H, J ¼ 8.7 Hz, Ar-H), 8.13 (d, 2H, J ¼ 9.0 Hz, Ar-H), 8.04 (d, 1H, J ¼ 7.2 Hz, Ar-H), 7.97 (s, 1H, Ar-H), 7.92e9.87 (m, 2H, Ar-H), 7.80 (s, 2H, Ar-H), 7.56 (d, 1Ha, J ¼ 15.9 Hz), 7.47e7.40 (m, 2H, Ar-H); 13C NMR (DMSO-d6) d (ppm): 189.32, 150.90, 144.12, 142.79, 136.86, 135.55,128.90, 128.29, 128.22, 126.43, 125.08, 124.79, 121.95, 121.49, 121.02, 119.79, 113.79; ESI-MS: m/z ¼ 434.0 [Mþþ1]. 4.2.10. (2E)-3-[1-(Propane-1-sulfonyl)-1H-indol-3-yl]-1-(pyridin4-yl)prop-2-en-1-one (SC11) Yield: 60%; light yellow solid; m. p: 264  C; Anal. Calc. for C19H18N2O3S: C 64.39, H 5.12, N 7.90, O 13.54, S 9.05%. found: C 64.14, H 4.92, N 7.79, S 8.93%; FT-IR nmax (cm1): 1672 (C]O), 1594 (C]C), 1367 (-SO2-N); 1H NMR (CDCl3) d (ppm): 8.86 (d, 2H, J ¼ 5.7 Hz, Ar-H), 8.02 (d, 1Ha J ¼ 15.9 Hz), 7.96e7.94 (m, 2H, Ar-H), 7.87 (s, 1H, Ar-H), 7.81e7.79 (m, 2H, Ar-H), 7.58 (d, 1Hb J ¼ 15.9 Hz), 7.50e7.43 (m, 2H, Ar-H), 3.35e3.30 (m, 2H, -CH2), 1.80e1.67 (m, 2H, -CH2), 1.00 (t, 3H, J ¼ 7.5 Hz, -CH3); 13C NMR (CDCl3) d (ppm): 189.44, 150.88, 144.28, 141.33, 137.43, 135.91, 135.58, 126.08, 124.68, 121.46, 121.34, 120.85, 118.94, 113.86, 77.23, 58.44, 18.46; ESI-MS: m/ z ¼ 340.9 [Mþþ1]. 5. Pharmacological evaluation N-lauroyl sarcosine, p-nitrophenyl acetate (PNA) and 3-[4,5-

21

dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), were purchased from Sigma Aldrich (St. Louis, MO). Ni-NTA column and gel filtration column (Superdex-75) were purchased from GE healthcare (GE Healthcare Life Sciences, Uppsala, Sweden). Human breast cancer cells (MCF-7), human heptoma cells (HepG-2) and human embryonic kidney cells (HEK293) were procured from National Centre for Cell Sciences (NCCS), Pune, India. FITC-Annexin-V detection kit was purchased from BD-Pharmingen, BD Biosciences (USA). Dulbecco minimal essential medium (DMEM), RPMI-1640 and Ham's F-12 nutrients mix cell culture medium and fetal bovine serum (FBS) were purchased from Gibco life sciences. All reagents used were of molecular biology grade. 5.1. Expression and purification of proteins The CA IX and CA II were successfully expressed in E. coli (BL21 D3 strain) and subsequently purified [5,31]. In brief, the recombinant cells bearing the CA IX expression construct were grown and induced by IPTG at 37  C. The pellet obtained from this culture was dissolved in lysis buffer (50 mM Tris, 0.1 mM EDTA, 250 mM NaCl, 0.1 mM PMSF and 1% Triton-100) and inclusion bodies were prepared, subsequently these inclusion bodies were dissolved in sarcosine buffer (50 mM Tris, 1.5% N-laurosyl sarcosine, pH 8.0) and centrifuged for 30 min at 19680  g and the supernatant was collected. Finally, the filtered supernatant so obtained was purified using Ni-NTA affinity chromatography. In case of CA II, the culture has been grown as that of CA IX, only the difference is that here we do not prepared inclusion bodies as after cell lysis, the CA II comes in the solubilized form into the supernatant. After cell lysis, filtered supernatant was directly loaded on Ni-NTA affinity column, preequilibrated with Tris-HCl buffer (pH 7.5, 500 mM NaCl and 5% Glycerol). The eluted CA II was further purified by gel-filtration chromatography (Superdex 200 pg connected to the Akta purifier, GE Healthcare). The purity of eluted CA IX and CA II was then checked by running SDS-PAGE and confirmed by antiHis antibodies using luminol method [32]. 5.2. CA inhibitory assay Enzyme inhibition assays of CA IX and CA II were performed by using our earlier reported method [5,33]. This assay spectrophotometrically measured the p-nitrophenol, a yellow colored product which is formed by the hydrolysis of p-nitrophenyl acetate (4-PNA) catalyzed by CA. Absorbance was measured at 400 nm with the help of UV/visible spectrophotometer (Jasco V-660, Model B028661152) equipped with peltier-type temperature regulator. The IC50 values for each synthesized compounds were determined by analyzing the absorption data using Graph Pad Prism (Version 6.0) software. 5.3. Cell proliferation study Cell cytotoxicity and antiproliferative properties of synthesized compounds were determined through MTT assay as described previously [34,35]. Briefly, 9e10  103 freshly passaged MCF-7, HepG2 and HEK293 cells were seeded per well of a 96 well cell culture plates. The cells were exposed with increasing concentration of selected compounds (for MCF-7 and HepG2, 2.5e80 mM, for HEK293 2.5e150 mM). After 48 h of incubation, mixture of medium and compound were removed and the cells were washed twice with phosphate buffer saline (PBS). Followed the washing of cells, 20 ml MTT (from 5 mg/ml stock) and 100 ml DMEM was added into each well and plate was further incubated for 4e5 h at 37  C in a CO2 incubator. Finally, the residual MTT and medium was removed carefully and the resultant formazan crystals were dissolved by

22

M.N. Peerzada et al. / European Journal of Medicinal Chemistry 155 (2018) 13e23

adding 100 ml DMSO to each well. The plates were then agitated for 15e20 min on an orbital shaker. Absorbance was measured at 570 nm on a titerplate reader (BioRad). The absorbance values so obtained were transformed into percentage viability in comparison to the control cells. Doxorubicin has been taken as positive control for anticancer activities.

having lower occupancy were deleted, and the side chains that were incomplete were then replaced by using Auto Dock Tools (ADT) version 1.5.6 from the Scripps Research Institute. Further, to each atom having Gasteiger charges were added and the non-polar hydrogen atoms were merged to the protein structure. After that the structures constructed were saved in PDBQT file format, for further analysis in ADT [38].

5.4. Cell apoptotic assay 6.2. Molecular docking Cell apoptosis was determined with the help of Annexin-V staining by following the standard procedures [36]. Briefly, MCF7 cells were treated with IC50 dose (concentration at which cell viability decreases by 50%) for 48 h at 37  C, and the control cells were treated with the media only. After the treatment, approximately 2  106 cells collected by trypsinization and washed twice with 5 ml of PBS by centrifugation (1800 rpm for 4e5 min). FITCAnnexin-V staining was performed with the help of FITCAnnexin-V kit by following the manufacturer's instructions (BDBiosciences, USA). For each sample 10,000 events were collected by flow cytometry on BD FACS Canto and data analysis was performed with help of flowJo software.

Conflicts of interest Authors have declared that there is not any conflict of interest. Acknowledgement The author Mudasir Nabi Peerzada is highly thankful to University Grants Commission, Government of India for fellowship.

5.5. Fluorescence measurements The binding properties of selected synthesized compounds with CA IX were carried out by monitoring changes in intensity of fluorescence maxima of protein. Jasco spectroflourimeter (FP6200) was used to perform the fluorescence experiments by using a 5 mm quartz cuvette at 25 ± 0.1  C. The synthesized compounds were dissolved in DMSO, and diluted to 1 mM/ml working concentration in 50 mM Tris buffer, pH 8.0. The protein was excited at 280 nm and the fluorescence emission was recorded in 300e400 nm range. The characteristic emission peak was seen at 346 nm. As CA IX consists of significant number of tryptophan residues, which absorb at 280 nm and give their characteristic maxima of emission near to 346, that why fluorescence quenching (due to binding of compound with protein) has been selected as a criteria to determine the binding affinity. The final spectra were obtained by subtracting with the corresponding blank measurements. The experiments were performed in triplicates and the average data was used for analysis. The decreased fluorescence intensity as a function of ligand concentration forms the basic criteria to determine the binding constant (Ka) and number of binding sites present for a compound on protein (n) using the modified Stern-Volmer equation [37]:

log ðFo  FÞ=F ¼ log Ka þ nlog½L

Molecular docking stimulation using the ligand molecules with CA IX (PDB ID: 3IAI) was conducted using Autodock 4.2 docking suite by employing Lamarckian genetic algorithm as described previously [5,40].

(1)

where, Fo ¼ Fluorescence intensity of native protein, F ¼ Fluorescence intensity of protein in the presence of ligand, Ka ¼ Binding constant, n ¼ number of binding sites, L ¼ concentration of ligand. The values for binding constant (Ka) and number of binding sites (n) were derived from the intercept and slope, respectively. 6. Molecular docking studies 6.1. Preparation of ligands and Protein molecule The structures of ligands were drawn by using the software ChemBioDraw Office 12.0 (licensed @ Cambridge's soft). The PDB file of ligand was then generated and converted into PDBQT file by process like detect root, chose the torsion and set the number of torsion by using ADT [38]. Crystal structure of CA IX was taken from the Protein Data Bank (www.rcsb.org) [39]. Then, the hydrogen atoms having polar nature were added, the residue structures

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