5-Benzylidene-2,4-thiazolidenedione derivatives: Design, synthesis and evaluation as inhibitors of angiogenesis targeting VEGR-2

Bioorganic Chemistry 67 (2016) 139–147

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5-Benzylidene-2,4-thiazolidenedione derivatives: Design, synthesis and evaluation as inhibitors of angiogenesis targeting VEGR-2 Umesh Bhanushali a, Saranya Rajendran b, Keerthana Sarma c, Pushkar Kulkarni c, Kiranam Chatti c, Suvro Chatterjee b, C.S. Ramaa a,⇑ a b c

Department of Pharmaceutical Chemistry, Bharati Vidyapeeth’s College of Pharmacy, C.B.D Belapur, Navi Mumbai 400614, Maharashtra, India Vascular Biology Lab, AU KBC Research Center, MIT Campus, Chromepet, Chennai 600044, Tamil Nadu, India Dr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500046, Andhra Pradesh, India

a r t i c l e

i n f o

Article history: Received 14 May 2016 Revised 27 June 2016 Accepted 28 June 2016 Available online 30 June 2016 Keywords: 5-Benzylidene-2,4-thiazolidinediones VEGFR2 Docking CAM assay Zebrafish embryo

a b s t r a c t A series of novel 5-benzylidene-2,4-thiazolidinediones were designed as inhibitors of angiogenesis targeting VEGFR-2. In docking study, molecules showed similar way of binding with VEGFR-2 as that of the co-crystallized ligand. Compounds were then synthesized, purified and characterized by spectroscopic techniques. Compounds 3f and 3i were found to be most active in the series showing good inhibition of angiogenesis in both CAM and in zebrafish embryo assays. Compound 3i also exhibited IC50 of 0.5 lM against VEGFR-2. Ó 2016 Published by Elsevier Inc.

1. Introduction 2,4-Thiazolidenedione derivatives viz., troglitazone, rosiglitazone, pioglitazone are well known for their anti-diabetic activity. Their insulin sensitizing activity was attributed to their potency to activate nuclear receptor, peroxisome proliferating activating receptor-gamma (PPAR-c) [1]. Efforts have been made to unearth the various applications of glitazones. More recently a TZD derivative efatutazone is in phase II clinical trials as maintenance therapy in patients with advanced colorectal cancer [2]. TZDs have exhibited anti-tumor activity in a wide variety of experimental cancer models by affecting cell cycle, induction of cell differentiation and apoptosis as well as by inhibiting tumor angiogenesis [3]. A study reported by Divya et al., showed that TZD derivative ciglitazone significantly decrease the VEGF production in human granulosa cells in an in vitro model [4]. The vascular endothelial growth factor (VEGF) is a positive regulator with its distinct specificity for vascular endothelial cells. The biological action of VEGF is mediated by two structurally related receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). The biological responses via these two distinct receptors to VEGF appear to be different.

⇑ Corresponding author. E-mail addresses: [email protected], [email protected] (C.S. Ramaa). http://dx.doi.org/10.1016/j.bioorg.2016.06.006 0045-2068/Ó 2016 Published by Elsevier Inc.

Prominently, the activation of VEGFR-2 leads to proliferation and migration of endothelial cells, whereas VEGFR-1 fails to transduce such signals when stimulated by VEGF [5–7]. Hence, inhibition of angiogenesis via blocking VEGFR-2 pathway has attracted widespread interest as an approach to anti-cancer therapy. In addition, inhibition of VEGF signaling can also change or destroy tumor vessels. Several small molecule inhibitors of VEFR-2 like vandetinib, vatalanib, sorafenib, sunitinib, pazopanib have been approved for treatment of various cancers [8]. However, recent reports on their efficiencies cited that KDR inhibitors like sorafenib developed resistance in advanced hepatocellular carcinoma (HCC). The exact mechanism was not known but thought primarily due to genetic heterogeneity of HCC and acquired resistance. Sorafenib was developed as multikinase inhibitor, hence it acts on several cellular signaling pathways and simultaneously activates the addiction switches and compensatory pathways. Several mechanisms were found to involve in the acquired resistance to sorafenib, such as crosstalks involving PI3K/Akt and JAK-STAT pathways, hypoxia-inducible pathways, epithelial-mesenchymal transition, etc. Based on the investigated mechanisms, some other molecular targeted drugs have been applied as second-line treatment for treat HCC after the failure of sorafenib therapy and more are under evaluation in clinical trials [9]. This fact encouraged us to develop newer inhibitor of KDR that could further be develop an as anti-cancer agent.

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Recently Kyungik et al. reported structural requirements for adenine region of ATP binding site of VEGFR-2; the core structure of all VEGFR-2 inhibitors consists of a flat aromatic ring system with the hydrogen bond acceptor group which will accept hydrogen from the backbone NH of Cys-917 [10].

2.2.2. 2-Chloro-N-[3-(trifluoromethyl) phenyl] acetamide (2b) Yield 56.0%, white crystalline, mp 132 °C. (Lit.mp [13]-129– 130 °C) IR [KBr cm
1]: 3304 (NAH), 1691 (C@O). 1H NMR [600 MHz, d, ppm, DMSO-d6]: 10.66 (s, 1H, NH), 8.65 (s, 1H, Arproton), 7.79 (d, J = 8.4 Hz, 1H Ar-proton), 7.59 (t, J = 7.8 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H Ar-proton), 4.30 (s 2H, ACH2A).

2. Materials and methods Melting points were determined using a Veego melting point apparatus in open capillaries and were uncorrected. IR spectra were recorded as KBr discs, using Shimadzu 8400S FTIR spectrophotometer. 1H NMR (300, 400 and 500 MHz) and 13C NMR (75 and 100 MHz) spectra were recorded on Varian Mercury Plus spectrometer, with DMSO-d6 or CDCl3 as solvents and TMS as an internal standard. Elemental analysis was recorded on Thermo finnigan-FLASH EA 1112 series. All reactions were monitored by TLC using Merk pre-coated silica gel 60 F254 plates and spots were visualized by observing in UV cabinet under short UV. All reagents were used as received unless otherwise stated. For the synthesis of the TZD class of compounds, we followed Knoevenagel condensation. It involved condensation of phydroxybenzaldehyde with TZD in the presence of piperidinium benzoate to yield 5-(4-hydroxybenzylidene)-2,4-thiazolidinedione (1) as a common intermediate. For compounds 3a-3j, chloroacetamides were condensed with 5-(4-hydroxybenzylidene)-2,4-thia zolidinedione in presence of K2CO3 in DMF. The intermediate chloracetamides (2a-2j) were obtained by a base-mediated chloroacetylation of substituted aryl or heteroaryl amines with chloroacetyl chloride in chloroform or dichloromethane. The compounds were purified by recrystallization from ethanol (Fig. 2) [11]. 2.1. Procedure for synthesis of 5-(4-hydroxybenzylidene)-2,4thiazolidinedione (1) [11] The mixture of 4-hydroxybenzaldehyde (1 mmol) and 2,4-TZD (1 mmol) in toluene was refluxed with catalytic quantity of piperidinium benzoate using Dean-Stark apparatus for 4 h. The reaction mixture was cooled and solid obtained was filtered. It was dissolved in 10% aqueous NaOH and solution was filtered. To this filtered solution, conc. HCl was added until it was neutralized to pH 7. The solid thus obtained as filtered, washed cold water, dried and was recrystallized from ethanol. Yield 93%, shiny dark yellow crystalline, mp 302–304 °C (Lit.mp [11] 301–303 °C). IR [KBr v cm
1] 3404 (NAH), 1718 (C@O, TZD ring), 1683 (C@O, amide). 1H NMR [500 MHz, d, ppm, DMSO-d6]: 9.84 (bs, 1H, NH), 7.36 (d, J = 8.5 Hz, 2H, Ar-protons), 7.26 (s, 1H, benzylidene proton), 6.83 (d, J = 8.5 Hz, 2H, Ar-protons).

2.2.3. 2-Chloro-N-(3-chlorophenyl)acetamide (2c) Yield 68.0%, brown crystalline, mp 125 °C (Lit.mp [13] 122– 124 °C). IR [KBr cm
1]: 3315 (NAH), 1687 (C@O). 1H NMR [400 MHz, d, ppm, CDCl3]: 8.28 (bs 1H, NH), 7.66 (t, J = 2 Hz, 1H, Ar-proton), 7.39 (d, J = 8.1 Hz, 1H Ar-proton), 7.26 (t, J = 8.1 Hz, 1H), 7.14 (d, J = 8.1 Hz, 1H, Ar-proton), 4.17 (s, 2H, ACH2A). 2.2.4. 2-Chloro-N-(4-chlorophenyl)acetamide (2d) Yield 68.0%, brown crystalline, mp 122 °C (Lit.mp [13] 122– 124 °C). IR [KBr cm
1]: 3308 (NAH), 1683 (C@O). 1H NMR [400 MHz, d, ppm, CDCl3]: 8.23 (bs 1H, NH), 7.50 (d, J = 9.4 Hz, 2H Ar-proton), 7.32 (d, J = 9.4 Hz, 2H Ar-proton), 4.19 (s 2H, ACH2A). 2.2.5. 2-Chloro-N-(5-chloropyridin-2-yl) acetamide (2e) Yield 68.0%, white crystalline, mp 139 °C (Lit.mp [13] 136– 137 °C). IR [KBr cm
1]: 3273 (NAH), 1674 (C@O). 1H NMR [400 MHz, d, ppm, CDCl3]: 8.86 (bs, 1H, NH), 8.27 (d, J = 2.4 Hz 1H, pyridine proton), 8.19 (d, J = 8.8 Hz, 1H, pyridine proton), 7.70 (dd, J = 8.4 Hz & 2.5 Hz, 1H, pyridine proton), 4.20 (s, 2H, ACH2A). 2.2.6. 2-Chloro-N-[pyridin-2-y] acetamide (2f) Yield 39.0%, pinkish white crystalline, mp 114–115 °C (Lit.mp [13] 178–180 °C). IR [KBr cm
1]: 3294 (NAH), 1666 (C@O). 1H NMR [400 MHz, d, ppm, CDCl3]: 8.92 (bs, 1H, NH), 8.33 (d, J = 6.2 Hz, 1H, pyridine proton), 8.19 (d, J = 8.3 Hz, 1H, pyridine proton), 7.74 (dt, 1H, J = 7.84 Hz, & 1.88 Hz, pyridine proton), 7.10 (dt, J = 6.08 Hz & 0.92 Hz, 1H, pyridine proton), 4.20 (s, 2H, ACH2A). 2.2.7. 2-Chloro-N-(5-methylisoxazol-3-yl) acetamide (2g) Yield 63%, white crystalline needles, mp 189 °C (Lit.mp [13] 190–191 °C). IR [KBr cm
1]: 3294 (NAH), 1670 (C@O). 1H NMR [300 MHz, d, ppm, DMSO-d6]: 10.45 (s, 1H, NH), 6.61 (s, 1H, isoxazole proton), 4.03 (s, 2H, ACH2), 2.36 (s, 3H, CH3).

2.2. General procedure for synthesis of 2a-2j [12]

2.2.8. 2-Chloro-N-(5-methylthiazole-2-yl) acetamide (2h) Yield 65%, white crystalline, mp 189–191 °C (Lit.mp [13] 191– 192 °C). IR [KBr cm
1]: 3186 (NAH), 1704 (C@O). 1H NMR [300 MHz, d, ppm, DMSO-d6]: 11.0 (bs 1H), 7.15 (s 1H, thiazole proton), 4.24 (s, 2H, ACH2A), 2.43 (s, CH3, 3H).

A solution of Aryl-or heteroary-amine (1.0 mol) in chloroform or dichloromethane was stirred with potassium carbonate (1.5 mol). To this solution was then added 1.5 mol of chloroacetyl chloride under cold. Reaction was stirred overnight at room temperature. After completion of reaction (monitored by TLC), solvent was removed under reduced pressure. To the solid mass, ice cold water was added. The solid thus obtained was then filtered dried and recrystallized from ethanol.

2.2.9. N-(Benzo[d]thiazol-2-yl)-2-chloroacetamide (2i) Yield 65%, white crystals, mp 166–168 °C (Lit.mp [13] 143– 144 °C). IR [KBr cm
1]: 3296 (NAH), 1683 (C@O). 1H NMR [300 MHz, d, ppm, DMSO-d6]: 12.79 (s 1H, NH), 8.00 (d, J = 8.1 Hz, 1H, benzothiazole proton), 7.77 (d, J = 8.1 Hz 1H, benzothiazole proton), 7.45 (dt, J = 7.2 Hz & 1.5 Hz, 1H, benzothiazole proton), 7.33 (dt, J = 7.95 Hz & 1.5 Hz, 1H, benzothiazole proton), 4.22 (s, 2H, ACH2).

2.2.1. 2-Chloro-N-phenylacetamide (2a) Yield 56.0%, white crystalline, mp 163–165 °C. (Lit.mp [13] 164 °C) IR [KBr cm
1]: 3306 (NAH), 1683 (C@O). 1H NMR [300 MHz, d, ppm, DMSO-d6]: 8.25 (bs 1H, NH), 7.54, (d, J = 6.9 Hz, 2H, Ar-proton) 7.36 (t J = 6.9 Hz, 2H, Ar-proton), 7.17 (t J = 6.9 Hz, 1H, Ar-proton), 4.18 (s 2H, ACH2A).

2.2.10. 2-Chloro-N-(4-methylbenzothiazol-2-yl)acetamide (2j) Yield 76%, Yellow crystalline, mp 144 °C (Lit.mp [13] 138– 140 °C). IR [KBr cm
1]: 3304 (NAH), 1685 (C@O). 1H NMR [300 MHz, d, ppm, DMSO-d6]: 9.66 (bs, 1H, NH), 7.67 (dd, J = 7.16 Hz & 1.6 Hz, 1H, benzothiazole proton), 7.22–7.28 (m, 2H, benzothiazole proton), 4.32 (s 2H, ACH2). 2.65 (s, 3H, CH3).

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2.3. Synthesis of 2-[4-(2,4-dioxothiazolidin-5-ylidenemethyl) phenoxy]-N-arylacetamide (3a-3j) The mixture of 5-(4-hydroxybenzylidene)-2,4-thiazolidene dione (1) (1.0 mol) and 1.5 mol of substituted aryl/heteroaryl chloracetamides (2a-2j) was dissolved in dimethyl formamide (DMF). The solution was stirred overnight with 1.5 mol of potassium carbonate at room temperature. The reaction was monitored by TLC. After completion of reaction, ice cold water was added to precipitate crude product (3a-3j). The compounds were the purified by recrystallization from absolute ethanol. 2.3.1. 2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]-Nphenylacetamide (3a) Yield 58%, Yellow colored solid, mp 285 °C. IR [KBr cm
1]: 3306 (NAH), 1788 (C@O, of 2,4-TZD), 1666 (C@O of amide). 1H NMR [300 MHz, d, ppm, DMSO-d6]: 10.39 (s, 2H, TZD-NH & amide NH), 7.88 (s, 1H, benzylidene proton), 7.56–7.51 (m, 4H, Ar-protons), 7.32 (t, J = 7.2 Hz, 2H, Ar-protons), 7.07 (t, 1H, J = 7.5 Hz, Arproton), 6.94 (d, 2H, J = 8.7 Hz, Ar-protons), 4.49 (s, 2H, ACH2A). 13 C NMR [100 MHz, d, ppm, DMSO-d6]: 167.14 (C@O), 165.40 (C@O), 163.64 (C@O), 138.35 (benzylidene carbon), 160.24, 133.95, 132.44, 128.60, 123.69, 123.48, 119.07, 116.30, 116.27, 43.77 (methylene carbon). Anal. Calcd. for C18H14N2O4S: C, 61.01; H, 3.95; N, 7.90. Found: C, 59.96; H, 3.89; N, 7.85. 2.3.2. 2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-phenoxy]-N-(3trifluoromethyl-phenyl) acetamide (3b) Yield 88%, Pale Yellow Solid, mp 234–237 °C. IR [KBr cm
1]: 3398 (NAH), 1788 (C@O, of 2,4-TZD), 1699 (C@O of amide). 1H NMR [300 MHz, d, ppm, DMSO-d6]: 10.75 (s, 1H, NH), 10.41 (s, 1H, NH), 8.05 (s, 1H, Ar-proton), 7.89 (s, 1H, benzylidene proton), 7.73 (d, J = 8.7 Hz, 1H, Ar-proton), 7.51–7.60 (m, 3H, Ar-protons), 7.44 (d, J = 7.5 Hz, 1H, Ar-proton), 6.94 (d, J = 9 Hz, 2H, Ar-protons), 4.54 (s, 2H, ACH2A). 13C NMR [75 MHz, d, ppm, DMSO-d6]: 167.34 (C@O), 165.48 (C@O), 164.67 (C@O), 139.14 (benzylidene carbon), 160.35, 134.18, 132.77, 130.22, 129.80, 125.83, 123.78, 122.76, 116.48, 115.22, 120.09, 44.39 (methylene carbon). Anal. Calcd. for C19H13F3N2O4S: C, 54.02; H, 3.08; N, 6.63. Found: C, 53.60; H, 3.02; N, 6.59. 2.3.3. N-(3-chlorophenyl)-2-[4-(2,4-dioxothiazolidin-5ylidenemethyl) phenoxy] acetamide (3c) Yield 64%, pale yellow solid, mp 250–251 °C. IR [KBr cm
1]: 3377 (NAH), 1788 (C@O, of 2,4-TZD), 1695 (C@O of amide). 1H NMR [400 MHz, d, ppm, DMSO-d6]: 10.63 (s, 1H, NH), 7.89 (s, 1H, benzylidene proton), 7.74 (t, J = 2 Hz, 1H, Ar-proton), 7.53 (d, J = 8.8 Hz, 2H, Ar-protons), 7.41 (d, J = 2 Hz, 1H, Ar-proton), 7.38– 7.34 (m, 1H, Ar-proton), 7.15 (d, J = 8.8 Hz, 1H, Ar-proton), 6.94 (d, J = 8.8 Hz, 2H, Ar-protons), 4.51 (s, 2H, ACH2A). 13C NMR [100 MHz, d, ppm, DMSO-d6]: 167.81 (C@O), 165.93 (C@O), 163.85 (C@O), 140.21 (benzylidene carbon), 160.78, 134.60, 133.64, 133.21, 131.12, 124.20, 119.13, 188.04, 116.93, 116.71, 44.39 (methylene carbon). Anal. Calcd. for C18H13ClN2O4S: C, 55.59; H, 3.34; N, 7.20. Found: C, 55.63; H, 3.26; N, 7.10. 2.3.4. N-(4-chlorophenyl)-2-[4-(2,4-dioxothiazolidin-5ylidenemethyl)phenoxy] acetamide (3d) Yield 42%, Pale Yellow Solid, mp 242–243 °C. IR [KBr cm
1]: 3416 (NAH), 1790 (C@O, of 2,4-TZD), 1701 (C@O of amide). 1H NMR [400 MHz, d, ppm, DMSO-d6]: 10.54 (s, 2H, TZD-NH & amide NH), 7.89 (s, 1H, benzylidene proton), 7.59 (d, J = 8 Hz, 2H, Ar-protons), 7.53 (d, J = 8.4 Hz, 2H, Ar-protons) 7.38 (d, J = 8 Hz, 2H, Ar-proton), 6.95 (d, J = 8.4 Hz, 2H, Ar-proton), 4.50 (s, 2H, ACH2A). 13C NMR [100 MHz, d, ppm, DMSO-d6]: 167.78 (C@O), 165.94 (C@O), 164.58 (C@O), 137.81 (benzylidene car-

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bon), 160.78, 134.57, 133.21, 129.28, 127.75, 124.23, 121.20, 116.92, 116.75, 44.40 (methylene carbon). Anal. Calcd. for C18H13ClN2O4S: C, 55.59; H, 3.34; N, 7.20. Found: C, 55.55; H, 3.28; N, 7.12. 2.3.5. N-(5-chloropyridin-2-yl)-2-[4-(2,4-dioxothiazolidin-5ylidenemethyl)phenoxy]acetamide (3e) Yield 58%, Pale Yellow Solid, mp 238–240 °C. IR [KBr cm
1]: 3365 (NAH), 1788 (C@O, of 2,4-TZD), 1683 (C@O of amide). 1H NMR [300 MHz, d, ppm, DMSO-d6]: 11.17 (bs, 1H, NH), 8.40 (d, J = 2.7 Hz, 1H, pyridine proton), 8.00 (d, J = 9.3 Hz, 1H, pyridine proton), 7.91 (dd, J = 8.8 & 2.7 Hz, 1H, pyridine proton), 7.86 (s, 1H, benzylidene proton), 7.50 (d, J = 8.4 Hz, 2H, Ar-protons), 6.90 (d, J = 8.7 Hz, 2H, Ar-protons), 4.50 (s, 2H, ACH2A). 13C NMR [100 MHz, d, ppm, DMSO-d6]: 167.73 (C@O), 165.33 (C@O), 164.57 (C@O), 134.09 (benzylidene carbon), 160.48, 132.49, 123.56, 116.36, 116.06, 150.00, 146.21, 137.62, 125.54, 114.52 (pyridine carbon). Anal. Calcd. for C17H12ClN3O4S: C, 52.37; H, 3.08; N, 10.78. Found: C, 52.35; H, 3.00; N, 10.69. 2.3.6. 2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-phenoxy]-Npyridin-2-yl-acetamide (3f) Yield 88%, Pale Yellow Solid, mp 181–182 °C. IR [KBr cm
1]: 3392 (NAH), 1797 (C@O, of 2,4-TZD), 1689 (C@O of amide). 1H NMR [300 MHz, d, ppm, DMSO-d6]: 10.97 (s, 1H, NH), 10.41 (s, 1H, NH) 8.34 (d, J = 4.2 Hz, 1H, pyridine proton), 7.97 (d, J = 6.9 Hz, 1H, pyridine proton), 7.88 (s, 1H, benzylidene proton), 7.78 (t, J = 7.2 Hz, 1H, pyridine proton), 7.52 (d, J = 8.7 Hz, 2H, Arprotons), 7.13 (t, J = 6.6 Hz, 1H, pyridine proton), 7.52 (d, J = 8.7 Hz, 2H, Ar-protons), 4.57 (s, 2H, ACH2A). 13C NMR [75 MHz, d, ppm, DMSO-d6]: 167.34 (C@O), 165.50 (C@O), 164.83 (C@O), 134.09 (benzylidene carbon), 160.32, 132.74, 123.80, 116.46, 116.35, 151.41, 148.12, 138.43, 132.74, 113.51, 43.92 (methylene carbon). Anal. Calcd. for C17H13N3O4S: C, 57.46; H, 3.66; N, 11.83. Found: C, 57.84; H, 3.89; N, 11.70. 2.3.7. 2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]-N-(5methylisoxazol-3-yl) acetamide (3g) Yield 75%, Pale Yellow Solid, mp 244–246 °C. IR [KBr cm
1]: 3400 (NAH), 1788 (C@O, of 2,4-TZD), 1697 (C@O of amide). 1H NMR [300 MHz, d, ppm, DMSO-d6]: 11.40 (s, 1H, NH), 10.40 (s, 1H, NH), 7.88 (s, 1H, benzylidene proton), 7.52 (d, J = 8.7 Hz, 2H, Ar-protons), 6.93 (d, J = 8.4 Hz, 2H, Ar-protons), 6.56 (s, 1H, isoxazole proton), 4.51 (s, 2H, ACH2A), 2.37 (s, 3H, CH3). 13C NMR [75 MHz, d, ppm, DMSO-d6]: 167.30 (C@O), 165.41 (C@O), 164.52 (C@O), 134.14 (benzylidene carbon), 160.33, 132.76, 123.78, 116.46, 116.29, 169.94, 157.63, 96.16, 43.66 (methylene carbon), 12.14 (methyl carbon). Anal. Calcd. for C16H13N3O5S: C, 53.48; H, 3.62; N, 11.69. Found: C, 53.40; H, 3.57; N, 3.51. 2.3.8. 2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl) phenoxy]-N-(5methylthiazol-2-yl) acetamide (3h) Yield 88%, Pale Yellow Solid, mp 230–235 °C. IR [KBr cm
1]: 3315 (NAH), 1790 (C@O, of 2,4-TZD), 1660 (C@O of amide). 1H NMR [300 MHz, d, ppm, DMSO-d6]: 12.40 (bs, 1H, NH), 10.40 (s, 1H, NH), 7.90 (s, 1H, benzylidene proton), 7.52 (d, J = 9.0 Hz, 2H, Ar-protons), 7.16 (s, 1H, thiazole proton), 6.94 (d, J = 8.4 Hz, 2H, Ar-proton), 4.88 (s, 2H, ACH2A), 2.40 (s, 3H, CH3). 13C NMR [100 MHz, d, ppm, DMSO-d6]: 167.17 (C@O), 165.31 (C@O), 164.11 (C@O), 134.09 (benzylidene carbon), 160.54, 132.62, 123.55, 116.46, 116.44, 116.23, 155.82, 134.18, 126.58, 43.17 (methylene carbon), 12.07 (methyl carbon). Anal. Calcd. for C16H13N3O4S2: C, 55.97; H, 3.79; N, 12.24. Found: C, 55.92; H, 3.72; N, 12.19.

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2.3.9. 2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl) phenoxy]-N(benzothiazol-2-yl) acetamide (3i) Yield 51%, Pale Yellow Solid, mp above 300 °C. IR [KBr cm
1]: 3400 (NAH), 1788 (C@O, of 2,4-TZD), 1697 (C@O of amide). 1H NMR [300 MHz, d, ppm, DMSO-d6]: 12.85 (s, 1H, NH), 10.43 (s, 1H, NH), 7.99 (d, J = 7.8 Hz, 1H, benzothiazole proton), 7.90 (s, 1H, benzylidene proton), 7.77 (d, J = 7.5 Hz, 1H, benzothiazole proton), 7.54 (d, J = 8.7 Hz, 2H, Ar-protons), 7.45 (d, J = 7.5 Hz, 1H, benzothiazole proton), 7.33 (d, J = 7.5 Hz, 1H, benzothiazole proton), 6.96–6.93 (d, J = 8.7 Hz, 2H, Ar-proton), 4.51 (s, 2H, ACH2A). 13 C NMR [75 MHz, d, ppm, DMSO-d6]: 167.32 (C@O), 165.69 (C@O), 164.39 (C@O), 134.32 (benzylidene carbon), 160.39, 132.79, 123.85, 120.56, 116.50, 116.22 (Ar-Carbon), 157.51, 148.51, 126.29, 126.14, 123.58, 121.85 (benzothiazole carbon), 43.54 (methylene carbon). Anal. Calcd. for C19H13N3O4S: C, 55.47; H, 3.16; N, 10.21. Found: C, 55.39; H, 3.10; N, 10.12. 2.3.10. 2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl) phenoxy]-N-(4methylbenzothiazol-2-yl) acetamide (3j) Yield 59%, Pale Yellow Solid, mp above 300 °C. IR [KBr cm
1]: 3275 (NAH), 1780 (C@O, of 2,4-TZD), 1681 (C@O of amide). 1H NMR [300 MHz, d, ppm, DMSO-d6]: 12.93 (s, 1H, NH), 10.46 (s, 1H, NH), 7.90 (s, 1H, benzylidene proton), 7.79 (d, J = 6.6 Hz, 1H, benzothiazole proton), 7.53 (d, J = 8.4 Hz, 2H, Ar-proton), 7.277.18 (m, 2H, benzothiazole proton), 6.94 (d, J = 8.4 Hz, 2H, Arproton), 4.66 (s, 2H, ACH2A), 2.45 (s, 3H, ACH3). 13C NMR [75 MHz, d, ppm, DMSO-d6]: 167.08 (C@O), 165.23 (C@O), 164.09 (C@O), 134.25 (benzylidene carbon), 160.31, 132.50, 123.64, 118.79, 116.33, 116.11, 156.33, 147.55, 126.49, 123.55, 43.24 (methylene carbon), 17.90 (methyl carbon). Anal. Calcd. for C20H15N3O4S2: C, 56.47; H, 3.52; N, 9.88. Found: C, 56.38; H, 3.47; N, 9.85. 2.4. Anti-angiogenic activity evaluation 2.4.1. Chick chorioallontoic membrane (CAM) assay Fertilized eggs of White Leghorn breed were acquired from Poultry Research Station, Nandanam, Chennai and incubated at 37 °C with 80% humidity. On day 4, these eggs were inoculated with the concentration of the test compounds (6 eggs per concentration, 1 lM, 10 lm, 100 lM) through a window made in the eggshell in upward position near the air sac and sealed with parafilm. DMSO control was maintained parallel and sorafanib (2 lg/ml) was used as positive control. The eggs were incubated till 8 h. After 8 h, eggs were opened and the CAM was isolated. The CAM was then transferred to petridish containing saline. Images were taken using Nikon Cool Pix camera adapted to a stereomicroscope. Images were analysed using AngioQuant software. AngioQuant uses networks of connected tubules as a basic unit in the quantification. These networks are called tubule complexes. AngioQuant provides quantitative and repeatable measurements of the lengths and sizes of tubule complexes as well as the numbers of junctions (branching points) in the tubule complexes [14]. 2.4.2. Zebrafish assay Zebrafish (wildtype) were obtained from local suppliers and maintained at 28 °C on a 14 h light/10 h dark cycle in 40 L glass tanks with four females and eight males in separate tanks. Embryos were collected by natural spawning with 2:1 male to female ratio and staged. Embryo stage is denoted as hours postfertilization (hpf). Compounds (3a-3j) were dissolved in DMSO at stock concentrations of 10 mM and then diluted with dose concentration of 0.3, 1, 3, 10, 30 lM directly to the embryo media in a 6 well culture plate in which the synchronized embryos 24 hpf (hours post-fertilization), were arranged by pipette, 20 embryos per well containing 2 mL of embryo medium in each well.

After drug treatment, the embryos were maintained in individual wells of culture plates at 28 °C until 72 hpf. After 48 and 72 hpf of drug addition to the wells, embryos were visually inspected for viability, gross morphological defects, heart rate and circulation. Circulation was assayed by visually comparing the movement of blood cells in treated and control embryos to assess the relative flow rate. Embryos of 48 hpf and 72 hpf were dechorionated with protease, washed with phosphate buffer saline (PBS) for three to four times and fixed in 4% paraformaldehyde (pH–7.5) for 2 h at room temperature. Embryos were then again washed three to four times in PBS and then it was dehydrated by immersing in 25%, 50%, 75% and 100% methanol each with 5 min in phosphate buffer saline with tween (PBST). It was then rehydrated in 25%, 50%, 75% and 100% PBST each with 5 min in methanol. For staining, embryos were equilibrated in alkaline phosphatase (NTMT) buffer thrice each with 15 min duration (0.1 M Tris-HCl; pH 9.5; 50 mM MgCl2; 0.1 M NaCl; 0.1% Tween-20) at room temperature. Once the embryos were equilibrated in NTMT, 4.5 lL of 75 mg/mL NBT and 3.5 lL of 50 mg/mL BCIP were added. After staining for 20 min, the reaction was stopped by adding PBST. Embryos were then immersed in a solution of 5% formamide and 10% hydrogen peroxide in PBS for 20 min, which removed endogenous melanin in the pigment cells and allowed full visualization of stained vessels. It was then examined by compound microscope and photographed [15]. 2.5. In vitro VEGFR Kinase inhibition The inhibition was carried out in 96-well polystyrene round bottomed plates. These plates were precoated with 100 ll per well of 50 lg per ml poly(Glu:Tyr, 4:1) peptide (Sigma) in PBS. The kinase reaction was performed in the plates by addition of 50 ll of kinase buffer (50 mM HEPES, 125 mM NaCl, 10 mM MgCl2, pH 7.4) containing 100 lM of ATP, 10 ng of KDR (Invitrogen, catalytic domain of VEGFR2), and the compound to be tested (0.1 lM, 0.5 lM, 1 lM, 5 lM and 10 lM). The compounds were dissolved in DMSO and were further diluted with PBS. After 30 min of exposure, the plates were washed 2–3 times with PBS and inoculated with 50 ll per well of 0.2 lg per ml HRP conjugated antiphosphotyrosine antibody (Santa Cruz). After two washes, the plates were developed by addition of 50 ll per well tetramethylbenzidine (Sigma) and the reaction was terminated by addition of 50 ll per well of 2 N H2SO4. The absorbance at 450 nm was measured by a 96-well plate reader (Tecan) [16]. 2.6. Statistical analysis Single comparisons between the different conditions studied were done using Student’s t-test, and differences between groups were tested using two-way analysis of variance. Statistical analysis was done using GraphPad Prism version 5. The level of significance in all the statistical analyses was set at P < 0.05. 2.7. Docking studies Molecular docking studies were performed using SYBYL-X 2.0 software on a Windows 7 platform with Intel Core i5 processor computer. To investigate the possible binding of compounds in the ATP binding site of KDR, we used the co-crystallized protein structure of KDR with 1-{4-[4-amino-6-(4-methoxyphenyl)-furo[2,3-d]pyrimidin-5-yl]-phenyl}-3-(2-fluoro-5-trifluoromethylphe>nyl) urea (PDB entry 1YWN). Surflex-Dock module was used for docking of our ligands at the KDR active site. All the parameters for protomol generation and docking run were set to default. Hydrogens were added to the protein, side-chain amides were

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2. One hydrogen bond donor (D): Donates its hydrogen to Glu883. 3. Two hydrogen bond acceptors (A): Accept hydrogen from backbone amine of Cys-917 of adenine binding region and Asp-1044 of DFG motif.

fixed and a staged minimization was performed on the protein using AMBER7 FF99 force field. 3. Result and discussion Our laboratory is actively involved in the development of novel thiazolidinediones. We have reported anti-diabetic [11], histone deacetylase (HDAC) inhibitory potential [17] of our synthesized TZD molecules. Recently we reported VEGFR-2 inhibitory activity of 5-pyridin-4-yl-2-thioxo-1,3,4-oxadiazole derivatives [18]. With the view to develop and repurpose novel 2,4-thiazolidinedione derivative, we performed thorough literature survey and obtained pharmacophoric requirements for KDR inhibitors.

In accordance with the structural requirements for the adenine region of ATP, the core structure of most VEGFR-2 kinase inhibitors consists of a flat aromatic ring system with a hydrogen bond acceptor group which will accept hydrogen from the backbone NH of Cys-917. The VEGFR-2 inhibitors approved for treatment in various cancers possess common structural features and these features are shown in Fig. 1A. All the molecules contain a hydrogen bond acceptor head (A) attached to a central aryl ring (HB). This central aryl ring then connected to hydrophobic tail (HB) via hydrogen bond acceptor donor system (A–D). Fig. 1B depicts how Sorafenib an approved small molecular inhibitor of VEGFR-2, satisfies these pharmacophoric requirements. A review of our in-house chemical library revealed that 5-benzylidene-2,4-thiazolidinediones derivatives also satisfy the above mentioned pharmacophore (Fig. 1C). We proposed that, the 2,4-thiazolidinedione ring arrests into the

3.1. Design of novel molecules The pharmacophoric requirements for VEGFR-2 inhibitors have been reported by Lee et al. [10] (Fig. 1), which are as follows: 1. Presence of two hydrophobic groups (HB): A central aryl ring and hydrophobic end.

B

A

Hydrogen Bond Acceptor

C

O

O

Hydrogen Bond Doner

O

S

HN

N H

Ar

Aryl or Heteroaryl Tail

O

Central Aryl Ring Fig. 1. Pharmacophore model for VEGFR 2 inhibitors (A) and Sorafenib (B). Generalized structure of novel 5-benzylidene-2,4-thiazolidinediones (C). A = hydrogen bond acceptor, HB = hydrophobic group, D = hydrogen bond donor.

CHO

Compd.

O

Ar

Compd.

3a

2,4-TZD, Reflux Piperidinium Benzoate OH 4- Hydroxy benzaldehyde

Ar

3f

NH

S

OH O 5-(4-Hydroxybenzylidene) thiazolidine-2,4-dione (1)

K2CO3 Substituted Aryl or Heteroaryl Chloracetamide DMF 2a-2j

N

CF3

CH3

3g 3b

NO Cl

N

3h H3 C

3c

N

3i O

O O Ar

NH

3a-3j

S

Cl

S

3d CH3

NH O

S

Cl

3j

N

3e N

Fig. 2. Experimental scheme for synthesis.

S

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3.2. Docking analysis

Table 1 The docking scores for compounds 3a-3j. Compd.

Docking score

3a 3b 3c 3d 3e 3f 3g 3h 3i 3j

4.83 3.38 3.34 4.31 4.61 5.64 4.88 4.29 5.96 5.51

Sorafenib

5.96

adenine binding site and accepts hydrogen of Cys-917. The lipophilic side chain securely pierces the hydrophobic pocket of DFG loop forming the hydrogen bonds with the gatekeepers Glu-883 and Asp-1044 residue. To investigate the possible binding of compounds in the ATP binding site of KDR, we used the co-crystallized protein structure of VEGFR-2 with 1-{4-[4-amino-6-(4-methoxy-phenyl)-furo-[2,3d]pyrimidin-5-yl]phenyl}-3-(2-fluoro-5-trifluoromethylphenyl)ur ea (PDB entry 1YWN). Ten molecules were synthesized and further screened for anti-angiogenic potential.

All the parameters for protomol generation and docking run were set to default. According to the crystal structure of VEGFR-2 with 1-{4-[4-Amino-6-(4-methoxyphenyl)-furo[2,3-d]pyrimidin-5 -yl]-phenyl}-3-(2-fluoro-5-trifluoromethylphenyl) urea, as shown in Fig. 2, the NH and CO of the urea form interactions with the backbone of Asp-1044 and the carboxylic acid residue of Glu883, respectively. The NH2 and nitrogen of the amino-pyrimidine occupies adenine binding region of hinge region and form interactions with Glu-915 and Cys-917 respectively. Surface Dock module of Sybyl X 2.0 was used for docking of ligand at the active side of VEGFR2. Docking score was used as the scoring function for evaluation of receptor ligand interactions and is tabulated in Table 1. Docking with program Surflex-Dock module successfully reproduces the X-ray pose of co-crystallized ligand with a root-meansquare deviation (RMSD) of 0.30 Å. As per reported by Söderholm et al. [19], this RMSD of the pose was regarded as a good reproduction of the crystal structure. The binding mode of these molecules (3a-3j) into active pocket of VEGFR2 shows similar types of interaction. Two representative molecules (3i and 3f) showing their binding with receptor are given Fig. 3. The TZD nucleus occupies the adenine binding region and interacts with side chain NH and amide carbonyl of Cys-917 with the carbonyl oxygen and NH of TZD as shown in Fig. 3. The benzothiazole moiety of compound 3i penetrates the hydrophobic cavity

A

B

Fig. 3. (A) 4-Amino-furo[2,3-d]pyrimidine derivative and its binding mode with active site of VEGR2. (B) Binding mode of compound 3i and 3f in active side of VEGFR2. Yellow dotted line represents the hydrogen bonds.

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3.3. Chemistry

formed by the DFG loop of the VEGFR-2 and the amide NH of molecule forms hydrogen bond with side chain carbonyl of the carboxylic group of Glu-883. The benzothiazole nitrogen accepts a hydrogen bond from the backbone NH of Asp-1044. In case of 3f, the pyridine ring does not rest securely into the hydrophobic pocket albeit showing the desired interactions which explain its lesser activity as compared to 3i.

Fold difference in Length

A

The synthetic route of the compounds is outlined in experimental scheme of synthesis showed in Fig. 2. A series of 5-benzylidene2,4-thiazolidinediones (3a-3j) were synthesized in three steps. 5(4-Hydroxybenzylidene)-2,4-thiazolidinedione (1) was prepared by Knoevenagel condensation reaction between 4-hydroxy ben-

Length of Tubule Complexes

2.5 2.0 1.5 1.0 0.5

3f

3g

3h

3i

Std

100µM

Control

1µ M

10µ M

100µM

1µ M

10µM

10µM

100µM

1µ M

100µ M

1µM

10µM

100µ M

1µ M

3e

10µM

100µ M

1µ M

3d

10µM

100µ M

1µ M

3c

10µM

100µM

1µ M

3b

10µ M

100µ M

1µ M

3a

10µM

10µ M

100µ M

1µ M

0.0

3j

Compound Concentration

Fold difference in Length

B

Length of Tubule Complexes

2.5 2.0 1.5 1.0 0.5

3f

3g

3h

3i

3j

Std

10 µM

100 µM

1µM

10 µM

100 µM

1µM

100 µM

10 µM

1µ M

10µM

100µM

1µ M

10 µM

3e

100 µM

1 µM

10µM

100 µ M

1µM

10µ M

3d

100 µM

1µM

100 µM

1µM

10µM

3c

Control

3b

100 µM

1µ M

10 µ M

10 µM

3a

100 µ M

1 µM

0.0

Compound Concentration

C

DMSO Control

3f

3i

Sorafanib Fig. 4. Angiogenesis inhibitory effect of compound 3a-3j on CAM after 4 h (A) and 8 h (B) of compound exposure and (C) snapshot of CAM after 8 h of exposure with compound 3f and 3i at a concentration of 10 lM.

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zaldehyde and 2,4-TZD. This intermediate was common to all molecules being synthesized. Further, this moiety was condensed with various chloroacetylated hetero or aryl-amides. The chloroacetylated moieties (2a-2j) were prepared by acetylation of respective amines with chloroacetyl chloride under basic conditions. The structures of all synthesized compounds were determined by spectral data (FTIR, 1H NMR and 13C NMR). In the 1H NMR spectra, the presence of characteristic singlet at d ppm 7.88–7.90 for benzylidene proton provided evidence for formation of (1). Formation of the chloroacetylated moieties (2a-2j) was characterized by a peculiar singlet for ACH2A protons resonating

Table 2 Average number of blood vessels of CAM treated with compounds 3a-3j after 8 h of exposure. Compd. # Conc. ! 3a 3b 3c 3d 3e 3f 3g 3h 3i 3j

Average no. of blood vessels 1 lM 41.10 ± 5.09 43.2 ± 3.75 41.44 ± 6.15 43.16 ± 5.18 41.22 ± 4.07 42.28 ± 5.15 41.23 ± 2.10 41.17 ± 7.11 20.68 ± 5.19 44.21 ± 6.59

Sorafanib (2 lg/ml) Control

10 lM

100 lM

21.58 ± 6.05 21.60 ± 4.17 22.55 ± 6.22 19.20 ± 7.93 26.44 ± 1.17 13.80 ± 2.07 27.29 ± 4.05 20.41 ± 2.12 10.43 ± 5.60 19.30 ± 3.72

11.69 ± 3.12 15.75 ± 6.10 17.55 ± 1.21 19.6 ± 2.18 18.83 ± 8.12 9.64 ± 1.07 16.44 ± 4.17 19.41 ± 6.12 5.23 ± 2.02 16.77 ± 4.59

16.35 ± 5.31 54.50 ± 0.069

Fig. 5. Development of the vasculature in zebrafish. At prim stage 5 (approximately 24 h pf), the dorsal aorta (DA) and axial vein (AV) have formed. Blood cells circulate over the yolk sac via the Ducts of Cuvier (DC). By 72 h pf, angiogenic vessels have formed, including the subintestinal vessels (SIVs). Heart (H).

at 4.0–4.8 ppm. In the IR spectra of 3a-3j the COANHACO and C@O bands were observed in the region 3460–3446 cm
1 and 1735–1670 cm
1 respectively. In the 1H NMR spectra, presence of a singlet at d ppm 4.50–4.60 of resonance assigned to the ACH2A protons provides evidence for formation of ACH2AOA linkage in final thiazolidinedione analogs (3a-3j). The benzylidene proton i.e. the methine proton appears in the 1H NMR at d 7.90, which indicates that the molecules are in Z configuration, as per the observations reported by Momose et al. [20]. 3.4. Anti-angiogenic activity evaluation 3.4.1. Chick chorioallontoic membrane (CAM) assay The strategy of this study was to initially evaluate compounds 3a-3j for their angiogenesis inhibitory effect on growing chick chorioallontoic membrane (CAM). We treated CAM for 4 h and 8 h with either DMSO (control) or with compounds in DMSO solution. As shown in Fig. 4, compounds 3f and 3i exhibited significant reduction of angiogenic responses as compared to control after 4 h and 8 h of compound exposure. When we observed CAM treated with DMSO, it was surrounded by allantoic vessels as newlyformed capillaries. Among all CAMs treated with TZD derivatives, compounds 3f and 3i excellently inhibited angiogenesis at 10 lM. It was observed that compound 3i showed angiogenesis inhibition potential at a dose of 1 lM. However other compounds showed no inhibitory effects on growing CAM (see Table 2). 3.4.2. Anti-angiogenic effects on developing zebrafish embryos With this observation, we extended our study to observe the angiogenesis inhibition effect of these compounds on zebrafish. Blood vessel patterning is highly characteristic in the developing zebrafish embryo and the subintestinal vessels (SIVs) can be stained and visualized microscopically as a primary screen for compounds that affect angiogenesis (Fig. 5). Small molecules added directly to the fish culture media diffuse into the embryo and induce observable, dose-dependent effects. The subintestinal vessels form on the dorsolateral surface of the yolk on both sides of the embryo in the shape of a basket that extends 50 ± 100 lm from the ventral edge of the somite over the yolk. For this screen, anti-angiogenic effects were defined as either the complete

Vehicle Control

Compound 3f At 3.0 µM

Compound 3i At 1.0 µM

Fig. 6. Compound 3f and 3i blocks both angiogenic and vasculogenic vessel formation in zebrafish embryos. Lateral view of AP stained embryos at72 h pf.

U. Bhanushali et al. / Bioorganic Chemistry 67 (2016) 139–147

absence of these vessels or the loss of either the lateral or ventral vessels of the basket [21]. To determine the in vivo inhibition of angiogenesis, we treated the embryos with two compounds which have been shown to inhibit angiogenesis in CAM. The compounds were added to the media at 24 hpf and the embryos were assessed after 72 hpf. The compounds 3f and 3i were added in concentrations of 0.3, 1, 3, 10, 30 lM on the growing embryos of zebrafish and the embryos were observed for the growth of SIVs. During treatment, embryos were both mobile and responsive to stimuli. As shown in Fig. 6 compound 3f was found to inhibit the growth of SIVs at a concentration of 3 lM. However, compound 3i showed inhibition of growth of SIVs at 1 lM. Treatment with 0.1% DMSO (vehicle control) had no effect on vessel formation. 3.4.3. In vitro VEGFR kinase inhibition assay Compound 3f and 3i exhibited notable inhibition potential in CAM and zebrafish assay; hence these compounds were selected for the VEGFR kinase inhibition. Compound 3i was found to inhibit the kinase at IC50 of 0.5 lM, however compound 3f did not show any significant inhibitory activity at tested concentrations. The reason could be empty hydrophobic pocket also known as allosteric side form by the DFG motif. As seen in Fig. 2, the benzothiazole tail of 3i rests into this pocket, but the pyridine ring was not able to penetrate this side. This signifies the importance of the allosteric binding side in the kinase activity. Sorafenib, however found to inhibit the KDR an IC50 of 0.1 lM. 4. Conclusion In conclusion, we have successfully synthesized a series of 5-b enzylidene-2,4-thiazolidinediones derivatives as new candidates for KDR inhibitors using structure based drug design systems (SBDD). Among the compounds synthesized, we found that compound 3f and 3i showed significant inhibition of angiogenesis in both CAM and in zebrafish embryo at a given concentration. Amongst these two compounds, compound 3i is the most potent compound which showed the inhibition of angiogenesis in CAM as well as zebrafish assay at a concentration of 1 lM. With these observations, compound 3i and 3f was taken further for the VEGFR kinase inhibition potential. Compound 3i was found to show IC50 of 0.5 lM. The current findings suggest that compound 3i would be a candidate for future molecular modification to serve as a potent KDR inhibitor. Acknowledgment Help extended by Mr. Vijay Patil, Ph.D. student of Bharati Vidyapeeth’s College of Pharmacy towards the synthetic part of this work is highly acknowledged. We extend our thanks to Director SAIF IIT Mumbai for carrying out the NMR study.

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