Design, in silico studies, synthesis, and in vitro anticancer assessment of new naphthylidene isoxazolidinedione derivatives

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. In vitro biological evaluation revealed the most active compound 4d with an inhibitory concentration of 42.87 μM against K562 and 47.42 μM on A549 cells. The anticancer activity of this naphthylidene isoxazolidinedione derivative was evident by cell cycle and apoptosis assays, which showed arrest of the G2/M phase (65.6%) of the K562 cells and induction of apoptosis with 21.47% apoptotic cells as compared to 0.2% of untreated cells, respectively.

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Nội dung Text: Design, in silico studies, synthesis, and in vitro anticancer assessment of new naphthylidene isoxazolidinedione derivatives

Received: 21 April 2023 Revised: 27 September 2023 Accepted: 7 October 2023 DOI: 10.1002/vjch.202300148 RESEARCH ARTICLE Design, in silico studies, synthesis, and in vitro anticancer assessment of new naphthylidene isoxazolidinedione derivatives Neha Upadhyay1 Kalpana Tilekar1 Aditi Oak1 Vadim S. Pokrovsky2,3 Ramaa Subramanian Chelakara1 1 Department of Pharmaceutical Chemistry, Bharati Vidyapeeth’s College of Pharmacy, CBD Abstract Belapur, Navi Mumbai, India Cancer is the most destructive and fatal disease, representing an urgent med- 2 Laboratory of Biochemical Fundamentals of ical challenge to the world. The discovery of a new anticancer candidate may Pharmacology and Cancer Models, N. N. Blokhin help reduce or eliminate this alarming disease to a greater extent. On similar Cancer Research Center, Moscow, Russia lines, in silico research was carried out on novel naphthylidene isoxazolidinedione 3 Department of Biochemistry, People’s derivatives that were logically conceived, synthesized, purified, and structurally Friendship University, Moscow, Russia characterized. In vitro biological evaluation revealed the most active compound Correspondence 4d with an inhibitory concentration of 42.87 μM against K562 and 47.42 μM Ramaa Subramanian Chelakara, Department of on A549 cells. The anticancer activity of this naphthylidene isoxazolidinedione Pharmaceutical Chemistry, Bharati Vidyapeeth’s derivative was evident by cell cycle and apoptosis assays, which showed arrest of College of Pharmacy, CBD Belapur, Navi Mumbai 400614, India. the G2/M phase (65.6%) of the K562 cells and induction of apoptosis with 21.47% Email: sinharamaa@yahoo.in apoptotic cells as compared to 0.2% of untreated cells, respectively. Funding information KEYWORDS ASEAN-India S&T Development Fund (AISTDF), antiproliferative, apoptosis, bio-isostere, cell cycle, isoxazolidinedione Grant/Award Number: IMRC/AISTDF/CRD/2018/000001 1 INTRODUCTION incorporated a common pharmacophoric feature, which is a heterocyclic ring, “2,4-thiazolidinedione (TZD)” as a Cancer is a multigenic and multifactorial disease that is one central or peripheral moiety that significantly contributed of the most serious menaces to the human race. Though to antitumor activity by targeting single or multiple currently available therapies are effective for the treatment cancer hallmarks.5–15 However, we also published com- of early stages of cancer, patient survival remains limited pounds without the TZD nucleus showing good antipro- due to various factors such as toxicity, drug resistance, liferative activity (Figure 1).16–25 Interestingly, there are etc.1,2 Considering the cancer epidemiology and its notori- countless pieces of literature demonstrating compounds ously convoluted biology, it is an alarming condition as only with anticancer activity incorporating various heterocyclic scanty actives reach the clinical stage.3,4 Thus, continuous rings.17,19,25–30 On a similar path, we have recently reported efforts are ongoing to explore various new therapeutics to new small molecules incorporating heterocyclic rings, imi- successfully combat the disease. Among these discoveries, dazolidinedione, and pyrrolidinedione showing excellent the development of synthetic chemical entities has been antitumor activity.19,26 Therefore, with our previous expe- considered a source for the discovery of anticancer small rience and knowledge, by considering the structural mod- molecules. ification of our previously reported analogs, we developed We have previously reported numerous compounds compounds incorporating the TZD bio-isostere which is a with antiproliferative potential targeting various essen- heterocyclic ring isoxazolidinedione, to evaluate the effect tial cancer pathways such as PPARγ, HDAC4/8, VEGFR-2, of this bio-isosteric replacement over the anticancer activity GLUT1/4/5, etc. Most of our reported chemical moieties of these compounds (Figure 2). © 2024 Vietnam Academy of Science and Technology and Wiley-VCH GmbH. Vietnam J. Chem. 2024;62:217–226. wileyonlinelibrary.com/journal/vjch 217
25728288, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/vjch.202300148 by Readcube (Labtiva Inc.), Wiley Online Library on [01/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License UPADHYAY ET AL. F I G U R E 1 Previously reported anticancer molecules with and without TZD ring. HDAC - histone deacetylase; GLUT - glucose transporter; PPARg - peroxisome proliferator activated receptor gamma; VEGFR-2 - vascular endothelial growth factor receptor-2; TZD - thiazolidinedione. 218
25728288, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/vjch.202300148 by Readcube (Labtiva Inc.), Wiley Online Library on [01/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License UPADHYAY ET AL. 219 F I G U R E 2 Designing aspects of new small molecules with isoxazolidinedione ring. HDAC - histone deacetylase; PPARg - peroxisome proliferator activated receptor gamma. 1.1 Rationale of designing compounds. These sketches were then imported into the SwissADME environment to create molecular smiles, which Herein, we considered the major structural features of the were then used to run the program to provide the needed two different sets of molecules reported by Jadhav et al. and data. The data was retrieved from and analyzed from Tilekar et al. Interestingly, by playing around with the func- the readings of several physiological and pharmacokinetic tional moieties of these already published molecules, we parameters that were produced as CSV files. build a new set of compounds with antiproliferative poten- tial. The very first structural feature of the newly designed molecule was a terminal pharmacophore, “isoxazolidine- 2.2 Chemistry 3,5-dione”which was the result of bio-isosteric replacement of the “TZD” ring (similarly to Jadhav et al.) (Figure 2).31 Commercial grade solvents, chemicals, and reagents were Several chemical compounds incorporating heterocyclic obtained from various distributors in India (S D Fine, Sigma- rings have shown promising anticancer activity.32,33 Hence, Aldrich or Research Lab). Thin layer chromatography (TLC) we introduced heterocycle, isoxazolidine-3,5-dione, simi- was performed by using pre-coated Merck Silica Gel 60 lar to that of the compounds reported by Jadhav et al. F254. Melting points of all the synthesized compounds (antidiabetic activity), to determine the effect of this ring were obtained by using VEEGO, MODEL: VMP-DS Melting on the anticancer activity of compounds. The second Point apparatus. Fourier transform infrared (FTIR) spectra noticeable feature was the central aryl ring, which was were found using Schimadzu FT/IR-8400S with use of direct a naphthalene ring attached to the new pharmacophore sampling procedure. 1 H and 13 C NMR spectra were noted (isoxazolidine-3,5-dione) by a ─CH═C─ linker, giving a on a Bruker instrument at 500 MHz and 125 MHz instru- naphthylidene isoxazolidine-3,5-dione derivative (similarly ment respectively, abbreviations used in NMR interpreta- to Tilekar et al.).6 The third part of the molecule was tion include s-singlet, d-doublet, dd-doublet of doublet, substituted phenyl acetamide, which was retained in the t-triplet, m-multiplet, and q-quartet. Chemical shift values molecular framework similar to that of the two reported (δ) are given in ppm, whereas coupling constants (J values) molecules. With these designing considerations, we arrived are expressed in hertz (Hz). at naphthylidene isoxazolidinedione derivatives with possi- ble antiproliferative activity, which was further validated by Step 1-Synthesis of N-aryl-2-chloroacetamide (1a–1g). various in vitro experimental assays. Differently substituted aromatic amines and K2 CO3 (1:1.5) were stirred in a dry round bottom flask (RBF) for 30 min under icebath in the presence 2 MATERIALS AND METHODS of dichloromethane (DCM). Chloroacetyl chloride (2) was dropwise added to this reaction mixture 2.1 In silico SwissADME predictions using a dropping funnel. Stirring was then contin- ued at room temperature for another 4 h. After A trustworthy and cost-free online tool called SwissADME 4 h, the reaction mixture was quenched by adding is used to predict physicochemical properties of a small water (added several times to remove undissolved molecule. Bioavailability and pharmacokinetic character- K2 CO3 ). To extract crude solid, DCM was evapo- istics of any synthetic compound may be obtained by rated under vacuum before being filtered, dried, loading its structure on the SwissADME website (http:// and washed with ice cold water. This was recrystal- www.swissadme.ch/index.php#). Chemdraw Ultra version lized by ethanol to get respective titel intermediates 12.0 was used to create the 2D structures of the planned (1a-1g).
25728288, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/vjch.202300148 by Readcube (Labtiva Inc.), Wiley Online Library on [01/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 220 UPADHYAY ET AL. Step 2-Synthesis of N-aryl-2-(6-formylnaphthalen-2- (500 MHz, DMSO-d6 ) δ ppm 3.80 (s, 3H), 3.85 (s, 3H), 4.92 (s, yl)oxy)acetamide (2a–2g). DMF (10 mL) was stirred 2H), 7.07-7.11 (m, 1H), 7.33-7.39 (m, 3H), 7.46-7.48 (m, 2H), with 3 g K2 CO3 in a dry RBF for 10 min. To this mix- 7.72-7.77 (m, 1H), 7.80-7.87 (m, 2H), 7.94-7.96 (m, 1H), 8.16 ture, 2 g of 6-hydroxynaphthaldehyde was added (s, 1H), 10.01 (s, 1H). and the solution was stirred for 20 min. Phenyl Dimethyl-2-((6-(2-((3,4-dichlorophenyl)amino)−2-oxoet acetamides (2.5 g) was added to this reaction mix- hoxy)naphthalen-2-yl)methylene) malonate (3e). Light ture with continuous stirring. After 24 h, the reaction yellow color solid. Yield 0.5 g (58 %). MP 254–256 ◦ C. FTIR was immobilized by adding 20 mL of water to (cm−1 ) 1748, 1680, 1646, 1178, 1064. 1 H NMR (500 MHz, produce precipitate. This was then separated by vac- DMSO-d6 ) δ ppm3.33 (s, 6H), 4.92 (s, 2H), 7.44-7.46 (m, 1H), uum filtration, several times washed with water to 7.481-7.485 (m, 1H), 7.59-7.63 (m, 2H), 7.85-7.87 (m, 1H), remove excess solvent traces, and purified by col- 7.94-7.96 (m, 1H), 8.05-8.054 (m, 1H), 8.13-8.15 (m, 2H), 8.52 umn chromatography using ethyl acetate:hexane (s, 1H), 10.47 (s, 1H). (30:70) as the mobile phase to produce the respec- Dimethyl-2-((6-(2-((2,4-difluorophenyl)amino)−2-oxoet tive intermediates 2a–2g. Structural characteriza- hoxy)naphthalen-2-yl)methylene) malonate (3f). Pale yel- tion have been published elsewhere.6 low color solid. Yield 0.5 g (69 %). MP 258–260 ◦ C. FTIR Step 3-Knoevenagel condensation of N-aryl-2- (cm−1 ) 1731, 1669, 1635, 1558, 1268, 1180, 1075. 1 H NMR ((6-formylnaphtahen-2-yl) oxy) acetamide and (500 MHz, DMSO-d6 ) δ ppm 3.34 (s, 3H), 3.80 (s, 3H), 4.90 (s, dimethyl malonate (3a–3g). Step 2 intermedi- 2H), 7.07-7.11 (m, 1H), 7.33-7.39 (m, 3H), 7.46-7.48 (m, 1H), ate (1 g) and dimethyl malonate (2 mL) were 7.72-7.77 (m, 1H), 7.80-7.87 (m, 2H), 7.94-7.96 (m, 1H), 8.16 added in an RBF. To this mixture, toluene (5 mL) (s, 1H), 10.01 (s, 1H). was added followed by acetic acid and piperi- Dimethyl-2-((6-(2-((4-bromophenyl)amino)−2-oxoeth dine in catalytic amount and this mixture was oxy)naphthalen-2-yl)methylene) malonate (3g). Light yel- refluxed for 4–5 h. The reaction was stopped low color solid. Yield 0.6 g (73 %). MP 250–252 ◦ C. FTIR and the mixture was cooled to room tempera- (cm−1 ) 1757, 1684, 1646, 1180, 1061. 1 H NMR (500 MHz, ture. The respective intermediates (3a–3g) was DMSO-d6 ) δ ppm 3.80 (s, 3H), 3.84 (s, 3H), 4.87 (s, 2H), 7.35- produced by filtering the solid precipitate under 7.47 (m, 3H), 7.51-7.53 (m, 2H), 7.63-7.66 (m, 2H), 7.77-7.92 vacuum, washing it with distilled water, and recrys- (m, 2H), 7.94-7.96 (m, 1H), 8.16 (s, 1H), 10.30 (s, 1H). tallizing it with methanol. Dimethyl-2-((6-(2-((4- fluorophenyl) amino)−2-oxoethoxy)naphthalen- Step 4-Cyclization of Knoevenagel intermediates to 2-yl)methylene)malonate (3a). Buff white color afford isoxazolidin-3,5-dione derivatives (4a–4g). solid. Yield 1.2 g (75 %). MP 250–252 ◦ C. FTIR Knoevenagel intermediates (0.5 g) were added to (cm−1 )1730, 1658, 1558, 1179, 1394. 1 H NMR a clean and dry RBF into which hydroxylamine (500 MHz, DMSO-d6) δ ppm 3.80 (s, 3H), 3.85 (s, hydrochloride (5 mL) solution was added followed 3H), 4.90 (s, 2H), 7.08-7.12 (m, 1H), 7.33-7.39 (m, 3H), by Na2 CO3 (1 g) in the presence of 10 mL solvent 7.45-7.48 (m, 1H), 7.72-7.77 (m, 1H), 7.85-7.87 (m, THF: methanol (1:1). This reaction was refluxed for 5– 1H), 7.89 (s, 1H), 7.94-7.96 (m, 2H), 8.07 (s, 1H), 10.01 6 h and later cooled to RT followed by adding water (s, 1H). (5 mL) to precipitate solid. The crude obtained was filtered under vacuum and washed with water to Dimethyl-2-((6-(2-oxo-2-(p-tolylamino)ethoxy)naphthalen- remove the excess of solvent, recrystallized by chlo- 2-yl)methylene)malonate (3b). Yellow color solid. Yield roform:methanol (2:1) to afford respective target 0.98 g (70 %). MP 255–256 ◦ C. FTIR (cm−1 ) 1740, 1668, compounds. 1554, 1179, 1429. 1 H NMR (500 MHz, DMSO-d6 ) δ ppm 3.71 (s, 3H), 3.80 (s, 3H), 3.84 (s, 3H), 4.81 (s, 2H), 6.89-6.91 (m, 2-((6-((3,5-dioxoisoxazolidin-4-ylidene)methyl)naphtha 2H), 7.35-7.39 (m, 2H), 7.44-7.47 (m, 1H), 7.55-7.57 (m, 2H), len-2-yl)oxy)-N-(4-fluorophenyl)acetamide (4a). Buff white 7.77-7.88 (m, 2H), 7.90-7.96 (m, 1H), 8.06 (s, 1H), 10.01 (s, solid. Yield 0.5 g (60 %). MP 295–297 ◦ C. FTIR (cm−1 ) 1680, 1H). 1624, 1181, 1063, 984. 1 H NMR (500 MHz, DMSO-d6 ) δ ppm Dimethyl-2-((6-(2-((4-methoxyphenyl) amino)−2-oxoeth 4.83 (s, 2H), 7.16-7.19 (m, 2H), 7.33-7.36 (m, 2H), 7.67-7.70 oxy)naphthalen-2-yl)methylene) malonate (3c). Yellow (m, 2H), 7.77-7.81 (m, 2H), 7.89-7.91 (m, 1H), 7.95 (s, 1H), solid. Yield 0.5 g (52 %). MP 253–255 ◦ C. FTIR (cm−1 ) 1734, 8.24 (s, 1H), 10.21 (s, 1H), 11.22 (s, 1H). 13 C NMR (125 MHz, 1675, 1553, 1178, 1234. 1 H NMR (500 MHz, DMSO-d6 ) δ DMSO-d6 ): 67.10, 107.51, 115.11, 115.29, 119.05, 121.53, ppm 3.72 (s, 3H), 3.80 (s, 3H), 3.85 (s, 3H), 4.83-4.86 (m, 121.59, 123.06, 127.16, 127.27, 128.39, 128.75, 129.67, 2H), 6.90-6.91 (m, 2H), 7.35-7.40 (m, 2H), 7.45-7.57 (m, 3H), 134.39, 134.63, 148.13, 156.24, 157.23, 159.14, 166.16. 7.77-7.96 (m, 3H), 8.16 (s, 1H), 10.03 (s, 1H). 2-((6-((3,5-dioxoisoxazolidin-4-ylidene)methyl)naphtha Dimethyl-2-((6-(2-oxo-2-((4-(trifluoromethyl)phenyl) len-2-yl)oxy)-N-(p-tolyl)acetamide (4b). Buff white solid. amino)ethoxy)naphthalen-2-yl) methylene)malonate (3d). Yield 0.42 g (58 %). MP 296–298 ◦ C. FTIR (cm−1 ) 1675, 1653, Light yellow color solid. Yield 0.92 g (72 %). MP 262–264 1184, 1061, 1464. 1 H NMR (500 MHz, DMSO-d6 ) δ ppm 3.72 ◦ C. FTIR (cm−1 ) 1730, 1669, 1558, 1180, 1268. 1 H NMR (s, 3H), 4.80 (s, 2H), 6.90-6.91 (m, 2H), 7.33-7.36 (m, 2H),
25728288, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/vjch.202300148 by Readcube (Labtiva Inc.), Wiley Online Library on [01/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License UPADHYAY ET AL. 221 7.56-7.58 (m, 2H), 7.79-7.81 (m, 2H), 7.89-7.90 (m, 1H), 7.95 2.3 MTT cytotoxicity assay (s, 1H), 8.24 (s, 1H), 10.01 (s, 1H), 11.22 (s, 1H). 13 C NMR (125 MHz, DMSO-d6 ): 55.04, 67.14, 107.47, 113.72, 119.07, The MTT assay was used to examine how synthetic com- 121.32, 123.03, 127.14, 127.24, 128.35, 128.71, 129.64, pounds 4a−4g affected the viability of K562 and A549 cell 131.29, 134.38, 148.12, 155.46, 156.27, 165.69. lines.9 A 96-well flat-bottom microplate with about 5 × 103 2-((6-((3,5-dioxoisoxazolidin-4-ylidene)methyl)naphtha cells per well was seeded with the cells and kept at 37 ◦ C len-2-yl)oxy)-N-(4-methoxyphenyl)acetamide (4c). Off with 95 % humidity and 5 % CO2 overnight. Six different white color solid. Yield 0.3 g (45 %). MP 290–292 ◦ C. FTIR synthesized chemical concentrations (100, 75, 50, 25, 10, (cm−1 ) 1675, 1594, 1185, 1060, 1235. 1 H NMR (500 MHz, and 2.5 μM) were treated with the cells for 48 h. Before DMSO-d6 ) δ ppm 3.74 (s, 3H), 4.80 (s, 2H), 6.90-6.91 (m, adding 20 μL of the MTT staining solution (5 mg mL−1 in 2H), 7.33-7.36 (m, 2H), 7.56-7.57 (m, 2H), 7.77-7.81 (m, 2H), phosphate buffer solution) to each well and incubating the 7.89-7.90 (m, 1H), 7.95 (s, 1H), 8.24 (s, 1H), 10.01 (s, 1H), plates at 37 ◦ C, the cells in each well were twice rinsed with 11.22 (s, 1H). 13 C NMR (125 MHz, DMSO-d6 ): 55.06, 67.16, phosphate buffer solution. Dimethyl sulfoxide (DMSO) was 107.49, 113.74, 119.08, 121.33, 123.05, 127.16, 127.26, added to dissolve the formazan crystals in each well after 128.37, 128.72, 129.65, 131.31, 134.40, 148.13, 155.48, 4 h, and the absorbance at 570 nm was measured using a 156.29, 165.70. micro-plate reader. Graph Pad Prism Version 5.1 was used 2-((6-((3,5-dioxoisoxazolidin-4-ylidene)methyl)naphtha to calculate the IC50 . The DMSO concentration used for the len-2-yl)oxy)-N-(4-(trifluoromethyl)phenyl)acetamide (4d). experiments was less 1.5 %. All the concentrations were Buff white color solid. Yield 0.5 g (65 %). MP 297–299 ◦ C. used in duplicates. The formula used for determining the FTIR (cm−1 ) 1675, 1653, 1185, 1075, 1268. 1 H NMR (500 MHz, cell viability: Surviving cells (%) = [Mean OD of cells treated DMSO-d6 ) δ ppm 4.90 (s, 2H), 7.08-7.11 (m, 1H), 7.31-7.34 with the test compound/Mean OD of negative control] × (m, 1H), 7.35-7.38 (m, 2H), 7.72-7.81 (m, 3H), 7.89-7.91 (m, 100. 1H), 7.95 (s, 2H), 8.24 (s, 1H), 9.99 (s, 1H), 11.22 (s, 1H). 13 C- NMR (125 MHz, DMSO-d6 ): 66.80, 104.16, 104.38, 107.53, 111.03, 111.21, 118.98, 123.07, 126.66, 127.15, 127.24, 2.4 Cell cycle assay 128.38, 128.75, 129.67, 134.37, 148.11, 156.14, 166.71. N-(3,4-dichlorophenyl)−2-((6-((3,5-dioxoisoxazolidin-4- K562 cells were seeded on a 24-well flat-bottom microplate, ylidene)methyl)naphthalen-2-yl)oxy)acetamide (4e). Buff and they were then cultured in a CO2 incubator overnight white solid. Yield 0.28 g (45 %). MP 298–299 ◦ C. FTIR (cm−1 ) at 37 ◦ C for 24 h. The media was replaced with fresh media 1684, 1653, 1616, 1060, 808. 1 H NMR (500 MHz, DMSO-d6 ) δ and then for 24 h the cells were treated with IC50 concen- ppm4.88 (s, 2H), 7.32 (s, 4H), 7.58-8.24 (m, 6H), 10.81 (s, 1H), tration of 4d. Untreated cells were used as negative control. 11.35 (s, 1H). 13 C NMR (125 MHz, DMSO-d6 ): 67.08, 107.24, Post incubation, cells were washed with PBS, Centrifuge for 107.56, 118.57, 119.00, 119.73, 120.91, 123.07, 123.73, 5 min at 300 × g at 4 ◦ C and supernatant was discarded. Cells 124.07, 125.03, 126.49, 127.14, 127.31, 128.41, 128.63, were re-suspended in 0.5 mL PBS. The cells were fixed on 128.81, 129.16, 129.62, 130.57, 130.86, 133.67, 138.57, ice in 4.5 mL of ice cold 70% ethanol for 2 h, centrifuged for 155.54, 162.37, 167.01, 178.89. 5 min at 200 × g at 4 ◦ C and ethanol was decanted.34 Then N-(2,4-difluorophenyl)−2-((6-((3,5-dioxoisoxazolidin-4- the cell pellets were suspended in 5 mL PBS for 1 min, cen- ylidene)methyl)naphthalen-2-yl)oxy)acetamide (4f). Off trifuged for 5 min at 200 × g at 4 ◦ C and treated with 1 mL white solid. Yield 0.4 g (52 %). MP 298–300 ◦ C. FTIR (cm−1 ) propidium iodide staining solution for 15 min at 37 ◦ C. Cells 1717, 1684, 1646, 1586, 1179, 1064. 1 H NMR (500 MHz, were analyzed within 30 min (BD AccuriTM C5 flow cytome- DMSO-d6 ) δ ppm 4.89 (s, 2H), 7.08-7.11 (m, 1H), 7.31-7.34 ter, BD Biosciences, CA, USA). Cytometry data were analyzed (m, 1H), 7.35-7.38 (m, 2H), 7.72-7.81 (m, 3H), 7.89-7.91 (m, by FlowJo software (version 10.1, Ashland, OR, USA). 1H), 7.95 (s, 1H), 8.24 (s, 1H), 9.99 (s, 1H), 11.22 (s, 1H). 13 C NMR (125 MHz, DMSO-d6 ): 66.80, 104.38, 107.53, 111.21, 118.98, 123.07, 126.66, 127.24, 128.75, 129.67, 134.37, 2.5 Apoptosis 148.11, 156.14, 166.71. N-(4-bromophenyl)−2-((6-((3,5-dioxoisoxazolidin-4- By using flow cytometry, the beginning of apoptosis by ylidene)methyl)naphthalen-2-yl)oxy)acetamide (4g). Off compound 4d was studied. K562 cells were plated in a white solid. Yield 0.3 g (47 %). MP more than 300 ◦ C. FTIR 24-well flat-bottom micro-plate and left to grow for 24 h at (cm−1 ) 1684, 1646, 1179, 1064, 606. 1 H NMR (500 MHz, 37 ◦ C in CO2 incubator. Fresh medium was added, and cells DMSO-d6 ) δ ppm 4.84 (s, 2H), 7.33-7.36 (m, 2H), 7.51-7.53 were then cultured at an IC50 concentration of 4d for 24 h. (m, 2H), 7.64-7.77 (m, 2H), 7.76-7.81 (m, 2H), 7.89-7.90 (m, Cells that were not treated were used as a negative control. 1H), 7.95 (s, 1H), 8.24 (s, 1H), 10.29 (s, 1H), 11.22 (s, 1H). 13 C Post incubation, cells were washed with PBS, centrifuged NMR (125 MHz, DMSO-d6 ): 67.11, 107.50, 115.27, 119.01, for 5 min at 500 × g at 4 ◦ C, and the supernatant was 121.58, 123.06, 127.14, 127.25, 128.38, 128.75, 129.67, discarded. Cell pellets were re-suspended in ice-cold 1× 131.45, 134.37, 137.65, 148.11, 156.21, 166.41. binding buffer, to which 1 μL of annexin V-FITC solution
25728288, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/vjch.202300148 by Readcube (Labtiva Inc.), Wiley Online Library on [01/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 222 UPADHYAY ET AL. TA B L E 1 SwissADME prediction of physicochemical properties. Lipinski Bioavailability BBB P-gp Code TPSAa Log Pb Mol. wt. RBsc HBAd HBDe #violations score permeant substrate 4a 93.73 2.33 406.36 6 6 2 0 0.55 No No 4b 93.73 2.57 402.4 6 5 2 0 0.55 No No 4c 102.96 2.55 418.4 7 6 2 0 0.55 No No 4d 93.73 2.57 456.37 7 8 2 0 0.55 No No 4e 93.73 2.54 457.26 6 5 2 0 0.55 No No 4f 93.73 2.64 424.35 6 7 2 0 0.55 No No 4g 93.73 2.48 467.27 6 5 2 0 0.55 No No a Topological polar surface area (TPSA). b Log of the partition coefficient (P). c Rotatable bonds (RB). d Hydrogen bond acceptors (HBA). e Hydrogen bond donors (HBD). Blood brain barrier (BBB). and 5 μL PI (propidium iodide) was added.35 400 μL of blood-brain barrier (BBB) penetrants. These were also good ice-cold 1× binding buffer was poured to tubes that had candidates for passive oral absorption. According to the been placed on ice and allowed to sit in the dark for 15 min. predictions, the proposed compounds would not act as P- Using a BD AccuriTM C5 flow cytometer (BD Biosciences, gp substrates. An efflux transporter known as P-gp drives CA, USA), cell preparations were examined. The FlowJo xenobiotics out of the cells and reduces their beneficial software (version 10.1, Ashland, OR, USA) was used to effects.42 examine the cytometry data. SwissADME also offers an intrinsic model for predicting BBB access and passive GI absorption (HIA) called BOILED- Egg. It is a great technique that is based on the two descrip- 3 RESULTS AND DISCUSSION tors WLOGP (lipophilicity) and TPSA (apparent polarity). The white zone denotes a high likelihood of passive absorp- 3.1 SwissADME physicochemical property tion through the digestive system, but the yellow region prediction denotes a high likelihood of reaching the brain. The outside grey region stands for molecules with properties implying The capacity of a chemical candidate to reach the desired predicted low absorption as well as limited brain pene- target at a sufficient concentration, which is determined by tration. P-gp is a multidrug-resistance efflux pump that is their ADME qualities, determines how beneficial the chem- responsible for clearing the drug. In the BOILED-Egg plot, ical candidate will be therapeutically. A tiny molecule may whether the drug candidate is PGP+ or PGP- is indicated by be able to indicate drug likeliness, according to Lipinski’s blue or red color dots, respectively.43 The spread of the tar- rule of five. SwissADME is an online tool that provides quick, get compounds on BOILED-Egg showed that none of the trustworthy, and reliable prediction data on pharmacoki- molecules were present in the yellow region, suggesting netics, drug-likeness, and physicochemical parameters of small molecule (such as molecular weight, partition coeffi- cient, solubility, topological surface area, etc.)36 Hence, we used SwissADME model to predict these parameters for our newly designed molecules. All compounds were loaded (smiles) on this online tool, and the output suggested that all chemical candidates obey Lipinski’s rule of five, indicative of their drug-likeliness (Table 1). These substances likewise showed a range of tolerable clogP values between 2.00 and 5.00. The sur- face sum of all polar atoms in a molecule is represented by topological polar surface area (TPSA; the acceptable range is 20−130 Å2 ), and all compounds in the series fell within this range. The bioavailability score for these compounds was 0.55. Overall, all the compounds were pre- dicted to have desired drug-like properties (indicated by Lipinski’s rule of five,37 Muegge’s filter,38 Veber’s rule,39 Egan rule,40 and Ghose filter41 ). None of the molecules were FIGURE 3 Spread of the target compounds on the BOILED-Egg plot.
25728288, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/vjch.202300148 by Readcube (Labtiva Inc.), Wiley Online Library on [01/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License UPADHYAY ET AL. 223 S C H E M E 1 Synthesis of final compounds. (a) K2 CO3 , chloroform, 0 ◦ C, stir at RT, 24 h; (b) DMF, K2 CO3, stir at RT, 24 h; (c) Toluene, piperidine, acetic acid, reflux, 4–5 h; (d) Hydroxyl amine hydrochloride, Na2 CO3 , methanol: THF (1:1), refluxing time: 5–6 h. that they are non-BBB penetrants. All compounds were ently substituted phenyl acetamides in the presence of found in the white region, suggesting their suitability for anhydrous K2 CO3 and dimethylformamide (DMF) solvent passive absorption, and these were not subjected to the to obtain intermediate N-aryl-2-((6-formylnaphthalen- active efflux P-gp pump (PGP-), as indicated by the red dot 2-yl)oxy)acetamide (2a–2g). Step 2 intermediates were (Figure 3). reacted with dimethyl malonate under Knoevenagel conditions (in the presence of toluene, piperidine, and acetic acid) to afford step 3 intermediates, that 3.2 Chemistry is, dimethyl-2-((6-(2-arylamino)−2-oxoethyl)naphthalen- 2yl)methylene)malonate (3a–3g). Final step was cyclization The synthetic procedure for the target compounds (4a–4g) of the Knoevenagel intermediates which occurred in were outlined in Scheme 1. Substituted phenyl acetamide the presence of hydroxyl amine, methanol: tetrahydro- derivatives were prepared by commercially available aro- furan (THF) (1:1), Na2 CO3 to yield target compounds, matic amines (1a–1g) as previously reported by us.6,8–12,44 N-aryl-2((6-(3,5-dioxazolidin-4-ylidine)methyl)naphthalen- 6-Hydroxy-2-naphthaldehyde was reacted with differ- 2yl)oxy)acetamide (4a–4g).
25728288, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/vjch.202300148 by Readcube (Labtiva Inc.), Wiley Online Library on [01/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 224 UPADHYAY ET AL. TA B L E 2 Inhibitory concentration of test compounds on K562 3.4 Apoptosis assay (leukemia) and A549 (human lung cancer) cell lines. Sr. no. Compounds K562 IC50 (µm)a A549 IC50 (µm)a To determine whether the most active compound induced 1 4a 47.17 ± 1.38 52.78 ± 2.18 cytotoxicity due to the induction of apoptosis, we used 2 4b 54.97 ± 5.4 61.51 ± 3.60 annexin V-FITC/propidium iodide (PI) double staining of K562 cells at the IC50 concentration of compound 4d and 3 4c 51.02 ± 0.38 50.79 ± 4.65 used flow cytometry to estimate the rate of apoptosis. The 4 4d 42.87 ± 5.54 47.42 ± 0.9 results indicated that, 4d decreased the number of live cells 5 4e 63.54 ± 6.26 69.76 ± 3.29 to 56.1 % as compared to the control, which was 99.7 % 6 4f ND ND (Figure 4). Also, in the late apoptotic and dead conditions, 7 4g ND ND the cells were 21.3 % and 22.4 %, respectively, as com- 8 Paclitaxel 0.29 0.32 pared to the control. Thus, we could suggest that apoptotic cell death could be one of the mechanisms of cytotoxicity 9 Cisplatin 58.40 ± 1.40 16.68 ± 1.74 induced by these compounds. a Assays were performed in n = 6. ND—not determined. 3.5 Cell cycle assay 3.3 MTT cytotoxicity assay To investigate whether the antiproliferative activity of the most active compound was attributed to the arrest of cells The in vitro cytotoxicity of every compound was evaluated in a particular phase, flow cytometry analysis was used on against K562 (leukemia) and A549 (human lung cancer) K562 cells. The IC50 concentration of the test compound cell lines by MTT assay. Paclitaxel and Cisplatin were used was used and compared with that of untreated (control) as positive controls. All compounds displayed comparable K562 cells. The results suggested that there was as increase activity with Cisplatin on K562 cells; whereas, the cytotoxi- in the number of 4d-treated cells at the G2/M phase (65.6 %) city was many folds greater than Paclitaxel. All compounds as compared to untreated cells (19.4 %); whereas, in the G0- displayed poor activity on A549 cells as compared to both G1 phase, the number of 4d-treated cells decreased (12.8 the positive controls. Compound 4e with a dichlorophenyl %) as compared to untreated cells (64.1%). The percentage substituent showed inhibitory concentrations greater than of cells in S phase was found to have changed very slightly 60 μM on both K562 and A549 cell lines; whereas 4b as compared to the control (Figure 5). (with methylphenyl) and 4c (with methoxyphenyl) exhib- ited concentrations greater than 50 μM. Compound 4a (with fluorophenyl) displayed less than 50 μM on K562 and 4 CONCLUSIONS greater than 50 μM on A549 cells. Compound 4d was the most active of the series, with IC50 better than other com- In the present work, we have successfully developed pounds on both cell lines (Table 2). The best cytotoxic active (designed, synthesized, purified, and structurally charac- (4d) was further selected to determine whether cytotoxicity terized) seven new TZD bio-isosteres, naphthylidene isox- was induced by other mechanisms such as apoptosis, cell azolidinedione derivatives as potential anticancer agents. cycle, etc. Further in silico studies were performed on the SwissADME F I G U R E 4 Annexin V-FITC/PI flow cytometry analysis of K562 cells treated with test compound 4d at the IC50 concentration. (a) Cytogram of untreated K562 cells. (b) Cytogram of 4d-treated K562 cells.
25728288, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/vjch.202300148 by Readcube (Labtiva Inc.), Wiley Online Library on [01/05/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License UPADHYAY ET AL. 225 F I G U R E 5 Effect of compound 4d on different phases of cell cycle of K562 cells. (a) Histogram of untreated K562 cells. (b) Histogram of 4d-treated K562 cells. online tool, which revealed the drug-likeliness of these on molecules screened against NCI-60 cancer cell lines, BMC Cancer. compounds. The synthesized compounds were found to 2016, 16, 77. 5. V. Patil, K. Tilekar, S. Mehendale-Munj, R. Mohan, C. S. Ramaa. have considerable cytotoxicity towards K562 and A549 can- Synthesis and primary cytotoxicity evaluation of new 5-benzylidene- cer cells. Amongst these, 4d and 4a were found to have 2,4-thiazolidinedione derivatives, Eur. J. Med. Chem. 2010, 45, 4539. the greatest cytotoxicity towards both of these cell lines. 6. K. Tilekar, J. D. Hess, N. Upadhyay, A. L. Bianco, M. Schweipert, A. Apoptosis and cell cycle analysis were carried out for the Laghezza, F. Loiodice, F.-J. Meyer-Almes, R. J. Aguilera, A. Lavecchia, most active compound, 4d. The in vitro results suggested C. S. Ramaa. Thiazolidinedione “magic bullets” simultaneously target- ing PPARγ and HDACs: Design, synthesis, and investigations of their this representative compound was able to induce apoptosis in vitro and in vivo antitumor effects, J. Med. Chem. 2021, 64, 6949. in K562 cells. Moreover, this compound induced the arrest 7. K. Tilekar, J. D. Hess, N. Upadhyay, M. Schweipert, F. Flath, D. A. of K562 cells in the G2/M phase. Gutierrez, F. Loiodice, A. Lavecchia, F.-J. Meyer-Almes, R. J. Aguilera, C. In the present study, it was observed that these isoxazo- S. Ramaa. HDAC4 inhibitors with cyclic linker and non-hydroxamate lidinedione derivatives (TZD bio-isosteres) were endowed zinc binding group: Design, synthesis, HDAC screening and in vitro cytotoxicity evaluation, ChemistrySelect. 2021, 6, 6748. with anticancer potential, as evident by the in vitro assays. 8. N. Upadhyay, K. Tilekar, N. Jänsch, M. Schweipert, J. D. Hess, L. Henze However, the cytotoxicity potential was found to be less Macias, P. Mrowka, R. J. Aguilera, J. Choe, F.-J. Meyer-Almes, C. S. than that of the TZD derivatives. Thus, we could comment Rama. Discovery of novel N-substituted thiazolidinediones (TZDs) as that, considering the best active compound as the lead, a HDAC8 inhibitors: in-silico studies, synthesis, and biological evalua- further rigorous modification could be made to arrive at tion, Bioorg. Chem. 2020, 100, 103934. 9. K. Tilekar, N. Upadhyay, N. Jänsch, M. Schweipert, P. Mrowka, F. more optimized compounds as the extension of this series J. Meyer-Almes, C. S. Ramaa. Discovery of 5-Naphthylidene-2,4- could be developed and evaluated for their target-specific thiazolidinedione derivatives as selective HDAC8 inhibitors and eval- anticancer effects. uation of their cytotoxic effects in leukemic cell lines, Bioorg. Chem. 2019, 95, 103522. 10. K. Tilekar, N. Upadhyay, J. D. Hess, L. H. Macias, P. Mrowka, R. J. Aguilera, ACKNOWLEDGMENTS F.-J. Meyer-Almes, C. V. Iancu, J.-Y. Choe, C. S. Ramaa. Structure The Department of Science and Technology (DST), Govern- guided design and synthesis of furyl thiazolidinedione derivatives as ment of India, “Indo-ASEAN Collaborative Research Project inhibitors of GLUT 1 and GLUT 4, and evaluation of their anti-leukemic Grant” from ASEAN-India S&T Development Fund (AISTDF), potential, Eur. J. Med. Chem. 2020, 202, 112603. Project Reference Number IMRC/AISTDF/CRD/2018/000001 11. K. Tilekar, N. Upadhyay, M. Schweipert, J. D. Hess, L. H. Macias, P. Mrowka, F.-J. Meyer-Almes, R. J. Aguilera, C. V. Iancu, J.-Y. Choe, C. (to CSR), sponsored this study. S. Ramaa. Permuted 2,4-thiazolidinedione (TZD) analogs as GLUT inhibitors and their in-vitro evaluation in leukemic cells, Eur. J. Pharm. C O N F L I C T O F I N T E R E S T S TAT E M E N T Sci. 2020, 154, 105512. The authors declare no conflicts of interest. 12. H. Joshi, V. Patil, K. Tilekar, N. Upadhyay, V. Gota, C. S. Ramaa. Benzylidene thiazolidinediones: Synthesis, in vitro investigations of antiproliferative mechanisms and in vivo efficacy determination in REFERENCES combination with Imatinib, Bioorg. Med. Chem. Lett. 2020, 30, 127561. 1. N. Milman, L. Ginini, Z. Gil. Exosomes and their role in tumorigen- 13. U. Bhanushali, S. Rajendran, K. Sarma, P. Kulkarni, K. Chatti, S. esis and anticancer drug resistance, Drug Resistance Updates 2019, Chatterjee, C. S. Ramaa. 5-Benzylidene-2, 4-thiazolidenedione deriva- 45, 1. tives: Design, synthesis and evaluation as inhibitors of angiogenesis 2. A. Colombo, C. Cipolla, M. 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