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Design, synthesis and biological evaluation of novel benzodioxole derivatives as COX inhibitors and cytotoxic agents

Design, synthesis and biological evaluation of novel benzodioxole derivatives as COX inhibitors... Non-steroidal anti-inflammatory drugs are among the most used drugs. They are competitive inhibitors of cyclooxy- genase (COX). Twelve novel compounds (aryl acetate and aryl acetic acid groups) were synthesized in this work in order to identify which one was the most potent and which group was most selective towards COX1 and COX2 by using an in vitro COX inhibition assay kit. The cytotoxicity was evaluated for these compounds utilizing MTS assay against cervical carcinoma cells line (HeLa). The synthesized compounds were identified using FTIR, HRMS, H-NMR, and C-NMR techniques. The results showed that the most potent compound against the COX1 enzyme was 4f with IC = 0.725 µM. The compound 3b showed potent activity against both COX1 and COX2 with IC = 1.12 and 1.3 µM, 50 50 respectively, and its selectivity ratio (0.862) was found to be better than Ketoprofen (0.196). In contrast, compound 4d was the most selective with a COX1/COX2 ratio value of 1.809 in comparison with the Ketoprofen ratio. All com- pounds showed cytotoxic activity against the HeLa Cervical cancer cell line at a higher concentration ranges (0.219– 1.94 mM), and the most cytotoxic compound was 3e with a CC value of 219 µM. This was tenfold more than its IC 50 50 values of 2.36 and 2.73 µM against COX1 and COX2, respectively. In general, the synthesized library has moderate activity against both enzymes (i.e., COX1 and COX2) and ortho halogenated compounds were more potent than the meta ones. Keywords: Benzodioxole, COX, Ketoprofen Introduction a 100  years [1, 2]. The biosynthesis of prostaglandin H2 Some of the most used analgesics are non-steroidal anti- from arachidonic acid is catalysed by COX enzymes [3]. inflammatory drugs (NSAIDs) that target the cyclooxy - Prostaglandin H2 is the main component in the forma- genase (COX) enzymes. NSAIDs are used for various tion of other prostaglandins, such as thromboxane and therapeutic purposes globally. Due to their wide pharma- prostacyclin, which play important roles in different bio - cological effects, including analgesic, anti-inflammatory logical responses [4, 5]. In fact, COX1 and COX2 are the and antipyretic effects, they are investigated as being two major isoforms of COX membrane-bound enzymes some of the best choices for treating different diseases [6]. COX1 is involved in the biosynthesis of important like arthritis and rheumatism, and they are widely used prostaglandins which maintain the constant functions in as analgesics. Actually, acetyl salicylic acid (ASA), one of the body, essentially in the cardiovascular and gastroin- the members of this family, has been used for more than testinal systems [7]. Moreover, COX2 is an enzyme cata- lyst that is overexpressed in several pathophysiological events such as hyperalgesia, inflammation, and cancer *Correspondence: mohawash@najah.edu 1 [8, 9]. The structures of COX1 and COX2 enzymes are Department of Pharmacy, Faculty of Medicine and Health Sciences, An- Najah National University, P.O. Box 7, Nablus 00970, Palestine 67% identical in amino acid chains. The main difference Full list of author information is available at the end of the article between the two enzymes is the presence of isoleucine © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hawash et al. BMC Chemistry (2020) 14:54 Page 2 of 9 (Ilu523) in COX1 instead of valine (Val523) in COX2. associated with lung cancer, fruits and vegetables con- This allows 25% greater available space in the binding taminated with pesticides and phyto-growth hormones, region of COX2 in comparison to COX1 [10]. All of these and the unhealthy lifestyles of modern people as well data encourage the researchers to focus their efforts to as some physical carcinogens such as radiation, some the find COX2 selective inhibitors in order to improve chronic diseases such as diabetes, and some infectious ill- treatment efficacy and to reduce the side effects that are nesses such Hepatitis B and C viral infections [19]. associated with the use of non-selective inhibitors of The heterocycle-containing agents have several phar - these enzymes [11–13]. macological effects including anticancer [20, 21], anti- COX2 enzyme is associated with carcinogenesis and inflammatory [22], antioxidant [23] and analgesic effects inflammatory diseases. It is suspected to induce tis - [24]. Therefore, the Benzodioxole containing compounds sue invasion of tumours, angiogenesis, and resistance to (Fig.  2) have different biological activities such as anti - apoptosis. Moreover, COX2 plays an important role in cancer, anti-tuberculosis, anti-microbial, anti-epileptic, the innate and adaptive immune response, and it contrib- and analgesic activity [25–30]. Various tricyclic com- utes to immune evasion and resistance to cancer immu- pounds and Ketoprofen like structures were synthesized notherapy. However, COX inhibitors can facilitate a and evaluated as COX enzyme inhibitors [31, 32]. The benefit to patients from addition of COX inhibitors when current work aims to synthesize new compounds with a compared to standard chemotherapy [14]. Benzodioxole core structure in two final product groups A large number of agents with different structural with different halogen atoms and aryl acetate and aryl features were produced in the discovery efforts of new acetic acid (Fig.  3), to evaluate their COX1 and COX2 COX2 selective inhibitors. A lot of classical non-selective NSAIDs were synthesized, approved, and used broadly, such as Ibuprofen, Naproxen, and Ketoprofen (Fig.  1), but their selectivity is too low against COX2/COX1 [15], and the previous studies were implemented to synthesize more selective agents as COX2 inhibitors by using differ - ent methods and structures [16]. According to the World Health Organization (WHO) surveys, cancer is one of the leading causes of death around the globe, and it was responsible for about 10 million deaths in 2018 [17, 18]. Around 1 in 6 peo- ple died from cancer, which is considered the largest cause of death. This is a considerably alarming estimate. WHO has recognized that 1.16 trillion US dollars were spent on the prevention and treatment of cancer in 2010 alone, and that number has increased dramatically over the years [17]. These important statistics are the result of erratic human behaviours such as smoking, which is Fig. 2 Structures of benzodioxol derivatives having various biological Fig. 1 Classical NSAIDs with COOH functional group activities Ha wash et al. BMC Chemistry (2020) 14:54 Page 3 of 9 compounds were synthesized by dissolving the ester (2) in dichloromethane with benzoic acid derivatives in the presence of an excess of phosphorus pentoxide and stir- ring at room temperature for approximately 18  h. The H-NMR spectrum data of these compounds showed 5–7 protons (depend on the Halogen atoms for each com- pound) in the aromatic area, 2 protons around 6.13 ppm singlet peaks for O–CH –O of benzodioxole and 5 pro- tons were observed in area 3.40 and 3.80 ppm for –CH – CO–CH . According to the C-NMR spectrum, C signal of carbonyl groups was found around 195 and 171 ppm, Fig. 3 Halogenated Ketoprofen analogues as aryl acetic acid and aryl and at 37–51  ppm two signals of aliphatic carbon were acetate observed. The Benzodioxole acetic acid derivatives (4a– 4f) were synthesized by hydrolysis reaction of the ester compounds 3a–3f using NaOH [35] (see Scheme 1). The H-NMR spectrum data showed one proton with sin- glet peak around 12  ppm (–COOH), 2 protons around 6.13  ppm singlet peaks for O-CH -O of benzodioxole and 2 protons were observed in area 3.40–3.78 ppm for – CH –COOH. However, C-NMR spectrum data showed C signal of carbonyl groups around 197 and 172 ppm. Cyclooxygenase inhibition activity The synthesized compounds have a structure that is simi - lar to Ketoprofen, and because of that Ketoprofen was used as a positive control in the COX inhibition analy- sis of the synthesized library. All Benzodioxole acetate structures with halogens (Br, Cl, I; 3b–3f) on the phenyl ring showed better activity against COX1 (IC 1.12– 27.06  µM) than acetic acid Benzodioxole with halogens (IC 4.25–33.7 µM; 4b–4e), except 4f which showed the most potent inhibitory activity (IC = 0.725  µM) against the COX1 enzyme. However, the acetic acid Benzodiox- Scheme 1 The reaction steps a methanol, oxalyl chloride b DCM, ole compound without a halogen (4a) showed stronger P O , aryl-carboxylic acid, c MeOH/THF/H O, NaOH reflux 2 5 2 inhibition activity toward cyclooxygenase enzymes COX1 and COX2 (1.45 and 3.34  µM, respectively) than acetate Benzodioxole without a halogen compound (3a) inhibitory activity and to evaluate the synthesized com- toward COX1 and COX2 (12.32 and 14.34  µM, respec- pounds’ cytotoxic effects. tively). However, all Benzodioxole acetate structures with halogens (3b–3f) showed better activity against COX2 Results and discussion (IC 1.30–37.45 µM) than acetic acid Benzodioxole with Chemistry halogens (IC 2.35–39.14  µM; 4b–4f) as presented in The Benzodioxole aryl acetate derivatives (3a-3f) and Table 1. acetic acid derivatives (4a–4f) were synthesized as out- lined in Scheme 1. The methyl 3,4-(methylenedioxy) phe - Cytotoxic evaluation nylacetate (2) was generated by an esterification reaction An MTS assay was used to determine the cytotoxic effect of 3,4-(methylenedioxy) phenylacetic acid (1). To pro- of Benzodioxole derivatives on HeLa (cervical carcinoma duce the ester (2), oxalyl chloride was added dropwise cells). As shown in Table  1, four different concentra - to methanol solvent and stirred for half an hour in an tions were used (2, 1, 0.5, and 0.1 mM) to investigate the ice bath [33, 34]. The IR spectra of the ester (2) showed cytotoxicity of the compounds. Actually, all compounds the disappearance of the broad band that belonged showed inhibition of cell growth at relatively high con- to the acetic acid group of (1). The aryl acetate 3a–3f centrations in comparison to the IC of COX enzyme. 50 Hawash et al. BMC Chemistry (2020) 14:54 Page 4 of 9 Table 1 IC inhibition of  COX1 and  COX2, Selectivity ratio for  COX1/COX2, and  the  CC on  HeLa cancer cell line 50 50 of the synthesized compounds The IC in µM of COX 1 COX1/COX2 Ratio HeLa Cell CC 50 50 and COX2 in milli-molar Codes X R1 R2 R3 COX1 COX2 Selectivity CC 3a O-CH3 H H H 12.320 14.340 0.859 1.49 3b O-CH3 I H H 1.120 1.300 0.862 0.228 3c O-CH3 H I H 27.060 37.450 0.723 1.79 3d O-CH3 Br H H 1.3 1.45 0.897 1.61 3e O-CH3 H Br H 2.360 2.730 0.864 0.219 3f O-CH3 Cl H Cl 5.180 4.100 1.263 0.949 4a OH H H H 1.450 3.340 0.434 1.94 4b OH I H H 7.670 30.700 0.250 0.697 4c OH H I H 33.700 39.140 0.861 1.049 4d OH Br H H 4.250 2.350 1.809 0.547 4e OH H Br H 7.110 49.300 0.144 0.437 4f OH Cl H Cl 0.725 4.290 0.169 1.019 Ketoprofen 0.031 0.158 0.196 P-values for the experiments p < 0.05 The CC were in the range between 0.219 and 1.79 mM. There is no clear relationship between the ortho ver - The most cytotoxic compound was 3e with a CC value sus meta halogen and the cytotoxicity results. Generally, of 219 µM. the halogenated compounds are more cytotoxic than non-halogenated (3a & 4a). The most cytotoxic com - SAR study pound was compound 3e (ester with Br on meta position; All ortho halogenated compounds 3b, 3d, 4b, and 4d CC = 0.219 mM). It was more toxic than compound 3d showed better activity with lower IC values than their (ester with Br on ortho), and the same relation was found meta halogenated compounds 3c, 3e, 4c and 4d. For between 4e and 4d, respectively. In contrast, the ortho example the IC values of compound 3b (ortho-halo- iodo halogenated compounds (3b and 4b) were more genated) against both COX1 and COX2 were 1.120 and toxic than meta iodo halogenated compounds (3c & 4c). 1.300  µM in comparison with 3d (meta-halogenated) In this study we can observe that our synthesized com- which were 27.060 and 37.450  µM, respectively. This pounds have inhibition activity against both COX1 and depended on the theory that the ortho-halogenated com- COX2 enzymes better than some tricyclic compounds pounds can make the second aromatic ring non-coplanar synthesized by other research teams. As published by with the first aromatic ring, which is ideal for the COX Caliskan et  al., one of pyrazol-3-propanoic acids deriva- inhibitory activity. All ester-mono halogenated com- tives was the most active compound in this series, and pounds (ortho or meta; 3b, 3c, 3d & 3e) have better COX it showed a selectivity ratio of 0.93 and activity against inhibitory activity than acetic acid mono-halogenated COX1 and COX2 with an IC value relatively close to compounds (ortho or meta; 4b, 4c, 4d & 4e). Except for our results (1.5 and 1.6  µM, respectively). However, the compound 4b, all other ortho-halogenated compounds inhibitory activity against COX1 for most of our synthe- (3b, 3d, and 4d) showed better selectivity ratios (COX1/ sized compounds were very close to or better than their COX2) than meta-halogenated compounds. The most tested compound [36]. In another study by Assali et  al., potent compound against COX1 enzyme was the acetic a series of pyrazole and triazole derivatives were synthe- acid di-halogenated (2,4-dichloro) compound 4f. The sized, and one of their triazole derivatives was considered ortho-iodo ester compound 3b was potent against COX2 to be a highly selective COX2 inhibitor with a high selec- enzyme with a good selectivity ratio (0.862). tivity ratio (162.5) [16]. Comparing our results with other studies, the results of this study clearly demonstrate Ha wash et al. BMC Chemistry (2020) 14:54 Page 5 of 9 that the synthesized agents have good inhibition activ- column had a pore size of 60  Å and 230–400  mesh par- ity against both COX1 and COX2 enzymes with rela- ticle size, 40–63  μm particle size. The inhibitory activity tively low IC values, and the COX selectivity ratio of the of ovine COX1 and human recombinant COX2 enzymes compounds synthesized in this study were better than was determined using a COX inhibitor screening assay approved drugs like ketoprofen or aspirin. kit No. 560131 (Cayman Chemical, USA). The yellow product of this enzymatic reaction was determined using Conclusion a UV spectrophotometer with a Microplate Reader (Bio- The synthesized compounds showed moderate activ - Rad, Japan) at a wavelength of 415  nm. HeLa Cervical ity against COX1 and COX2 enzymes. However, most Carcinoma cell line was purchased from ATCC (ATCC ® ™ compounds have better COX2 inhibition selectivity com- CCL-2 ), and the cyototoxicty test of the cell viability pared to Ketoprofen. The results showed a promising was assessed by the CellTilter 96 Aqueous One Solution group of compounds having a Benzodioxole moiety. They Cell Proliferation (MTS) assay according to the manufac- had better COX2 selectivity compared with Ketoprofen, turer’s instructions (Promega Corporation, Madison, WI) and this may be due to the bigger moiety (Benzodioxole) (Additional file 1). in the synthesized compound in comparison with phenyl moiety in Ketoprofen. Future plans should include dock- Chemistry method ing studies and synthesizing more analogues of this core Synthesis of methyl 2‑(2H‑1,3‑benzodioxol‑5‑yl) acetate structure to study the structure–activity relationship. synthesis 2 This is required in order to improve their COX inhibitory The 3,4-(methylenedioxy)phenylacetic acid (1) (8  g, activity and to achieve a better COX2 selectivity ratio. All 44.40  mmol) was dissolved in methanol, then it was compounds 3a–4f showed cytotoxic activity on the HeLa cooled in an ice bath to 0 °C. Then oxalyl chloride (4 mL, cancer cell line at higher doses. However the effective 46.80 mmol) was added dropwise, and the reaction mix- doses towards COX enzyme were at least lesser 10 times ture was stirred for 30–45 min. The reaction mixture was greater than the cytotoxic concentrations. then evaporated under vacuum and the resulting residue was diluted with ethyl acetate solvent and washed with Experimental section saturated sodium bicarbonate (NaHCO ) and distilled Chemicals and instruments water, sequentially. The organic layer was dried with All chemicals were purchased from Sigma-Aldrich and sodium sulphate, then filtered and evaporated again to Alfa Aesar. Melting points were determined with an concentrate it. In the last step, it was purified by silica gel SMP-II Digital Melting Point Apparatus and are uncor- column chromatography by using a hexane:ethyl acetate rected. IR spectra were obtained using a Perkin Elmer solvent system (50%:50%). The resulting compound (2) Spectrum 400 FTIR/FTNIR spectrometer. H-NMR and was a yellow oil with 94% yield. C-NMR spectra were recorded in DMSO-d6 and were performed on two NMR instruments. The first was a General synthesis procedure for ketoester (3a–3f ) derivatives Bruker 500  MHz-Avance III High-Performance Digital The benzoic acid derivatives (1.46  g, 6.68  mmol) and FT-NMR spectrometer at the Faculty of Science, Depart- phosphorus pentoxide (5 g) were added to a stirred solu- ment of Chemistry, The University of Jordan, Jordan (it tion of dichloromethane (60  mL) and compound (2) was used for the H-NMR of just one compound, 3e). (1  g, 5.14  mmol). Then, the mixture was stirred at room The second was a Bruker 300  MHz-Avance III High- temperature for 18  h before distilled water (60  mL) was Performance Digital FT-NMR spectrometer at the NMR cautiously added, and the mixture was extracted with facility at the Doping and Narcotics Analysis Laboratory ethyl acetate twice (60  mL). Then, the organic layer was of the faculty of pharmacy, Anadolu University, Turkey (it separated and treated with 1  M NaOH (60  mL), brine 1 13 was used for both H-NMR and C-NMR for the other (60  mL), and twice with 60  mL of distilled water. The compounds). Tetramethylsilane was used as the internal organic layer was dried with sodium sulphate, filtered, standard. All chemical shifts were recorded as d (ppm). evaporated under vacuum, and then purified by silica gel High resolution mass spectral data (HRMS) were col- column chromatography with different solvent systems. lected using a Waters LCT Premier XE Mass Spectrome- ter (high sensitivity orthogonal acceleration time-of-flight Methyl 2‑(6‑benzoylbenzo[d][1,3]dioxol‑5‑yl)acetate instrument) using ESI (+) method (The instrument was (3a) Purified by silica gel column chromatography using coupled to an AQUITY Ultra Performance Liquid Chro- n-hexane: ethyl acetate solvent system (3:2). Crude yellow matography system (Waters Corporation, Milford, MA, semi solid, Yield 75%; ESI–MS: 299.0919 (100), 300 (20), −1 USA) at the Pharmacy Faculty Gazi University Ankara- 301 (2), For C H O . IR (FTIR/FTNIR-ATR): 1737 cm 17 15 5 −1 Turkey. The silica gel used for the flash chromatography ester carbonyl (C=O), 1661 cm keton carbonyl (C=O). Hawash et al. BMC Chemistry (2020) 14:54 Page 6 of 9 H NMR (DMSO-d , 300 MHz) δ ppm: 7.62–7.67 (3H, m, chromatography using n-hexane: ethyl acetate solvent Ar–H), 7.52 (2H, t, J = 7.8 Hz, Ar–H), 7.05 (1H, s, Ar–H), system (3:2). Powder product, mp: 72.5–74.5  °C, Yield 6.89 (1H, s, Ar–H), 6.12 (2H, s, O–CH –O), 3.74 (2H, s, – 79%; ESI–MS: 377.00 (100), 379 (98), 380 (20), for 13 −1 CH –C=O), 3.47 (3H, s, O–CH ). C-NMR (DMSO-d , C H BrO . IR (FTIR/FTNIR-ATR): 1742  cm ester 2 3 6 17 14 5 −1 1 75  MHz) δ ppm: 196.53, 171.61, 149.66, 146.04, 138.08, carbonyl (C=O), 1655  cm keton carbonyl (C=O). H 133.39, 131.59, 130.21, 130.00, 129.73, 129.44, 128.95, NMR (DMSO-d , 500 MHz) δ ppm: 7.86 (1H, d, J = 8 Hz, 112.66, 110.34, 102.45, 51.89, and 38.36. Ar–H), 7.77 (1H, s, Ar–H), 7.64 (1H, d, J = 8  Hz, Ar–H), 7.50 (1H, t, J = 8 Hz, Ar–H), 7.06 (1H, s, Ar–H), 6.95 (1H, Methyl 2‑(6‑(2‑iodobenzoyl)benzo[d][1,3]dioxol‑5‑yl)ace ‑ s, Ar–H), 6.15 (2H, s, O–CH –O), 3.77 (2H, s, –CH – 2 2 tate (3b) Purified by silica gel column chromatography C=O), 3.49 (3H, s, O–CH ). using n-hexane: ethyl acetate solvent system (1:1). Semi solid product, Yield 90%. ESI–MS: 424.9875 (100), 425.99 Methyl 2‑(6‑(2,4‑dichlorobenzoyl)benzo[d][1, 3] −1 (20), for C H IO . IR (FTIR/FTNIR-ATR): 1740  cm dioxol‑5‑yl)acetate (3f) Purified by silica gel column 17 14 5 −1 ester carbonyl (C = O), 1659 cm keton carbonyl (C=O). chromatography using n-hexane: ethyl acetate solvent H NMR (DMSO-d , 300  MHz) δ ppm: 7.95 (1H, d, system (1:1). Powder product, mp: 95–97  °C, Yield 83%; J = 7 Hz, Ar–H), 7.51 (1H, t, J = 7.5 Hz, Ar–H), 7.23–7.30 ESI–MS: 367.01 (100), 369 (67), for C H Cl O . IR 17 13 2 5 −1 (2H, m, Ar–H), 7.11 (1H, s, Ar–H), 6.64 (1H, s, Ar–H), (FTIR/FTNIR-ATR): 1760  cm ester carbonyl (C=O), −1 1 6.13 (2H, s, O-CH -O), 3.92 (2H, s, –CH –C=O), 3.59 1633  cm keton carbonyl (C=O). H NMR (DMSO-d , 2 2 6 (3H, s, O–CH ). C-NMR (DMSO-d , 75  MHz) δ ppm: 300 MHz) δ ppm: 7.79–7.82 (2H, m, Ar–H), 7.59 (1H, d, 3 6 197.19, 171.49, 151.23, 146.51, 145.12, 139.78, 134.05, J = 8.4 Hz, Ar–H), 7.07 (1H, s, Ar–H), 7.00 (1H, s, Ar–H), 133.30, 131.97, 128.95, 128.60, 113.60, 112.03, 102.93, 6.14 (2H, s, O–CH –O), 3.78 (2H, s, –CH –C=O), 3.48 2 2 93.46, 51.98. (3H, s, O-CH ). C-NMR (DMSO-d , 75  MHz) δ ppm: 3 6 194.29, 171.68, 150.13, 146.17, 138.56, 136.07, 131.70, 131.34, 130.62, 130.52, 130.33, 116.68, 112.82, 110.64, Methyl 2‑(6‑(4‑iodobenzoyl)benzo[d][1,3]dioxol‑5‑yl)ace ‑ 102.06, 52.05, 38.33. tate (3c) Purified by silica gel column chromatography using n-hexane: ethyl acetate solvent system (1:1). Pow- der product mp: 119–121 °C, Yield 87%. ESI–MS: 424.96 General synthesis procedure of 2‑(6‑benzoyl‑2H‑1,3‑ben ‑ (100), 425.99 (20), for C H IO . IR (FTIR/FTNIR-ATR): zodioxol‑5‑yl)acetic acid (4a–4f) The ketoesters 3a–3f 17 14 5 −1 −1 1735  cm ester carbonyl (C=O), 1660  cm keton car- (450  mg, 1.35  mmol) were dissolved in methanol/H O/ bonyl (C=O). H NMR (DMSO-d , 300  MHz) δ ppm: THF (12/12/12  mL), then NaOH (540.9  mg, 13.5  mmol) 7.92 (2H, d, J = 8.4  Hz, Ar–H), 7.41 (2H, d, J = 8.8  Hz, was added. The solution was heated in an oil bath and Ar–H), 7.06 (1H, s, Ar–H), 6.92 (1H, s, Ar–H), 6.13 (2H, refluxed for 4  h before being cooled to room tempera - s, O–CH –O), 3.75 (2H, s, –CH –C=O), 3.47 (3H, s, O– ture. The solution was then evaporated, and the residue 2 2 CH ). C-NMR (DMSO-d , 75  MHz) δ ppm: 195.94, was made acidic by adding HCl 2 N (pH = 2). The pre - 3 6 171.61, 149.79, 138.52, 137.89, 137.40, 131.94, 131.20, cipitate was filtered and concentrated under vacuum to 130.14, 112.70, 110.38, 102.49, 102.05, 52.02, 38.29. give the crude products 4a–4f. Methyl 2‑(6‑(2‑bromobenzoyl)benzo[d][1,3]dioxol‑5‑yl) 2‑(6‑benzoylbenzo[d][1,3]dioxol‑5‑yl)acetic acid acetate (3d) Purified by silica gel column chromatog - (4a) Purified by silica gel column chromatography raphy using n-hexane: ethyl acetate solvent system (4:1). using n-hexane: ethyl acetate solvent system (3:2). Pow- Powder product, mp: 85–87  °C, Yield 85%; ESI–MS: der product, mp: 184.5–186.5  °C, Yield 97%; ESI–MS: 377.00 (100), 379 (98), 380 (20), For C H BrO . IR 285.07 (100), 286 (20), for C H O . IR (FTIR/FTNIR- 17 14 5 16 13 5 −1 −1 −1 (FTIR/FTNIR-ATR): 1740  cm ester carbonyl (C=O), ATR): 1770 cm acetic acid carbonyl (C=O), 1655 cm −1 1 1 1658  cm keton carbonyl (C=O). H NMR (DMSO-d , keton carbonyl (C=O). H NMR (DMSO-d , 300  MHz) 6 6 300 MHz) δ ppm: 7.34–7.77 (4H, m, Ar–H), 7.11 (1H, s, δ ppm: 12.18 (1H, s, OH), 7.49–7.70 (5H, m, Ar–H), Ar–H), 6.69 (1H, s, Ar–H), 6.14 (2H, s, O–CH –O), 3.93 7.03 (1H, s, Ar–H), 6.86 (1H, s, Ar–H), 6.11 (2H, s, O– 13 13 (2H, s, –CH –C=O), 3.59 (3H, s, O–CH ). C-NMR CH –O), 3.67 (2H, s, –CH –C=O). C-NMR (DMSO- 2 3 2 2 (DMSO-d , 75  MHz) δ ppm: 196.53, 171.61, 149.66, d , 75 MHz) δ ppm: 96.64, 172.65, 149.48, 138.12, 133.68, 6 6 146.04, 138.08, 133.39, 131.59, 130.21, 130.00, 129.73, 133.33, 131.73, 130.68, 130.26, 130.01, 129.20, 128.90, 129.44, 128.95, 112.66, 110.34, 102.45, 51.89, 38.36. 112.62, 110.16, 102.32, 38.59. Methyl 2‑(6‑(3‑bromobenzoyl)benzo[d][1, 3] 2‑(6‑(2‑iodobenzoyl)benzo[d][1,3]dioxol‑5‑yl)acetic acid dioxol‑5‑yl)acetate (3e) Purified by silica gel column (4b) Purified by silica gel column chromatography Ha wash et al. BMC Chemistry (2020) 14:54 Page 7 of 9 using n-hexane: ethyl acetate solvent system (3:2). Pow- 6.12 (2H, s, O–CH –O), 3.70 (2H, s, –CH –C=O). 2 2 der product, mp: 147–149 °C, Yield 92%; ESI–MS: 410.97 C-NMR (DMSO-d , 75  MHz) δ ppm: 195.25, 172.72, (100), 411 (20), for C H IO . IR (FTIR/FTNIR-ATR): 149.80, 145.88, 140.39, 135.85, 132.47, 131.17, 131.09, 16 12 5 −1 −1 1754 cm acetic acid carbonyl (C=O), 1653 cm keton 131.04, 129.34, 122.12, 112.70, 110.31, 102.44, 38.58. carbonyl (C=O). H NMR (DMSO-d , 300 MHz) δ ppm: 7.95 (1H, d, J = 7.8  Hz, Ar–H), 7.50 (1H, t, J = 7.8  Hz, 2‑(6‑(2,4‑dichlorobenzoyl)benzo[d][1,3]dioxol‑5‑yl)acetic Ar–H), 7.23–7.31 (2H, m, Ar–H), 7.06 (1H, s, Ar–H), acid (4f ) Purified by silica gel column chromatography 6.61 (1H, s, Ar–H), 6.11 (2H, s, O–CH –O), 3.83 (2H, using n-hexane: ethyl acetate solvent system (1:1). Solid s, –CH –C=O). C-NMR (DMSO-d , 75  MHz) δ ppm: product, mp: 168.5–170  °C, Yield 91%; ESI–MS: 352.99 2 6 197.14, 172.60, 150.97, 146.17, 145.23, 139.81, 134.23, (100), 354 (67) for C H Cl O . IR (FTIR/FTNIR-ATR): 16 12 2 5 −1 −1 131.97, 129.31, 129.14, 128.54, 113.48, 111.77, 102.75, 1768 cm acetic acid carbonyl (C=O), 1657 cm keton 93.51. carbonyl (C=O). H NMR (DMSO-d , 300 MHz) δ ppm: 12.25 (1H, s, OH), 7.77–7.81 (2H, m, Ar–H), 7.60 (1H, 2‑(6‑(4‑iodobenzoyl)benzo[d][1,3]dioxol‑5‑yl)acetic acid dd, J = 8.3, 1.8  Hz, Ar–H), 7.05 (1H, s, Ar–H), 6.98 (1H, (4c) Purified by silica gel column chromatography using s, Ar–H), 6.13 (2H, s, O–CH –O), 3.71 (2H, s, –CH – 2 2 n-hexane: ethyl acetate solvent system (3:2). Powder prod- C=O). C-NMR (DMSO-d , 75  MHz) δ ppm: 194.43, uct, mp: 239.5–241.5 °C, Yield 89%; ESI–MS: 410.97 (100), 172.73, 149.41, 145.94, 138.60, 135.99, 131.75, 131.29, −1 411 (20), for C H IO . IR (FTIR/FTNIR-ATR): 1760 cm 131.20, 130.74, 130.36, 129.84, 112.74, 110.40, 102.47, 16 12 5 −1 acetic acid carbonyl (C=O), 1660  cm keton carbonyl 38.56 (Additional file 1). (C=O). H NMR (DMSO-d , 300  MHz) δ ppm: 12.20 (1H, s, OH), 7.91 (2H, d, J = 8.7  Hz, Ar–H), 7.42 (2H, d, J = 8.4 Hz, Ar–H), 7.03 (1H, s, Ar–H), 6.90 (1H, s, Ar–H), Biological COX assay method 6.11 (2H, s, O–CH –O), 3.67 (2H, s, –CH –C=O). C- The ability of the synthesized a series to prevent the con - 2 2 NMR (DMSO-d , 75 MHz) δ ppm: 196.04, 172.63, 149.60, version of arachidonic acid (AA) to PGH2 by human 145.85, 138.14, 137.83, 137.44, 131.99, 131.30, 130.77, recombinant COX2 and bovine COX1 was assessed using 112.63, 110.17, 102.37, 101.95, 38.56. a COX inhibitor screening assay kit (Item No: 560131) according to the Cayman chemical manufacturer’s guide- 2‑(6‑(2‑bromobenzoyl)benzo[d][1,3]dioxol‑5‑yl)acetic lines (USA). The 50% inhibitory concentration (IC ) of acid (4d) Purified by silica gel column chromatography COX1/COX2 activity of the compounds was carried out. using n-hexane: ethyl acetate solvent system (3:2). Pow- The assay was run in duplicate with three concentrations der product, mp: 145–147 °C, Yield 87%; ESI–MS: 362.99 (50, 20, and 5 µM). A standard curve of eight concentra- (100), 364 (98), 365 (20) for C H BrO . IR (FTIR/ tions of prostaglandin, a non-specific binding sample, 16 12 5 −1 FTNIR-ATR): 1766  cm acetic acid carbonyl (C=O), and a maximum binding sample was used, as instructed −1 1 1664  cm keton carbonyl (C=O). H NMR (DMSO-d , in the kit manual, to determine the inhibition of sample 300 MHz) δ ppm: 7.41–7.73 (4H, m, Ar–H), 6.96 (1H, s, compound by applying the multiple regression generated Ar–H), 6.61 (1H, s, Ar–H), 6.07 (2H, s, O-CH -O), 3.68 best-fit line. The percentage inhibition of the three con - (2H, s, –CH –C=O), 3.42 (1H, bs, O–H). C-NMR centrations was used to calculate the IC [16]. (DMSO-d , 75  MHz) δ ppm: 195.59, 172.70, 150.48, 133.34, 132.50, 132.01, 130.31, 127.99, 119.14, 112.82, Cell culture and cytotoxicity assay 112.45, 111.03, 105.10, 102.38, 101.57. HeLa Cervical Carcinoma was cultured in RPMI-1640 media and supplemented with 10% fetal bovine serum, 2‑(6‑(3‑bromobenzoyl)benzo[d][1, 3]dioxol‑5‑yl)acetic 1% penicillin/streptomycin antibiotics and 1% l-glu - acid (4e) Purified by silica gel column chromatography tamine. Cells were grown in a humidified atmosphere using n-hexane: ethyl acetate solvent system (3:2). Pow- with 5% C O at 37 °C, and they were seeded in 2.6 × 104 der product, mp: 154–156 °C, Yield 96%; ESI–MS: 362.98 cells/well in a 96-well plate. After 48  h, the cells were (100), 364 (98), 365 (20) for C H BrO . IR (FTIR/ confluent, the media was changed, and cells were incu - 16 12 5 −1 FTNIR-ATR): 1759  cm acetic acid carbonyl (C=O), bated with four concentrations (2, 1, 0.5, and 0.2  mM) −1 1 1658  cm keton carbonyl (C=O). H NMR (DMSO- of the tested compounds for 24  h. Cell viability was d , 300 MHz) δ ppm: 12.24 (1H, s, O–H) 7.75–7.78 (2H, assessed by the CellTilter 96 Aqueous One Solution m, Ar–H), 7.64 (1H, d, J = 8.1  Hz, Ar–H), 7.48 (1H, t, Cell Proliferation (MTS) Assay according to the manu- J = 8.1 Hz, Ar–H), 7.04 (1H, s, Ar–H), 6.92 (1H, s, Ar–H), facturer’s instructions (Promega Corporation, Madison, WI). Briefly, at the end of the treatment, 20  μL of MTS Hawash et al. BMC Chemistry (2020) 14:54 Page 8 of 9 3. Smith WL, Urade Y, Jakobsson PJ (2011) Enzymes of the cyclooxygenase solution per 100 μL of media was added to each well and pathways of prostanoid biosynthesis. Chem Rev 111(10):5821–5865 incubated at 37 °C for 2 h. Absorbance was measured at 4. Engblom D, Ek M, Saha S, Ericsson-Dahlstrand A, Jakobsson PJ, Blomqvist 490 nm [37]. A (2002) Prostaglandins as inflammatory messengers across the blood– brain barrier. J Mol Med (Berl). 80(1):5–15 5. Fitzpatrick FA (2004) Cyclooxygenase enzymes: regulation and function. Supplementary information Curr Pharm Des 10(6):577–588 Supplementary information accompanies this paper at https ://doi. 6. Garavito RM, DeWitt DL (1999) The cyclooxygenase isoforms: structural org/10.1186/s1306 5-020-00706 -1. insights into the conversion of arachidonic acid to prostaglandins. Bio- chim Biophys Acta 1441(2–3):278–287 7. Kirkby NS, Lundberg MH, Harrington LS, Leadbeater PDM, Milne GL, Pot- Additional file 1: The data in the addtional file include NMR spectrum ter CMF et al (2012) Cyclooxygenase-1, not cyclooxygenase-2, is responsi- files and HRMS file of all newly synthesized compounds described in this ble for physiological production of prostacyclin in the cardiovascular article. system. Proc Natl Acad Sci 109(43):17597–17602 8. Grosser T, Yu Y, FitzGerald GA (2010) Emotion recollected in tranquility: lessons learned from the COX-2 saga. Annu Rev Med 61(1):17–33 Abbreviations 9. Méric J-B, Rottey S, Olaussen K, Soria J-C, Khayat D, Rixe O et al (2006) COX: Cyclooxygenase; FTIR: Fourier-transform infrared spectroscopy; HRMS: Cyclooxygenase-2 as a target for anticancer drug development. Crit Rev High resolution mass spectroscopy; H NMR: Proton nuclear magnetic reso- Oncol Hematol 59(1):51–64 nance; C NMR: Carbon nuclear magnetic resonance; µM: Micro molar; mM: 10. Blobaum AL, Marnett LJ (2007) Structural and functional basis of Mili molar; NSAIDs: Non-steroidal anti-inflammatory drugs; ASA: acetyl salicylic cyclooxygenase inhibition. J Med Chem 50(7):1425–1441 acid; WHO: World Health Organization; HeLa: Cervical carcinoma cells; IC : 11. Hsiao G, Shen M-Y, Chou D-S, Chang Y, Lee L-W, Lin C-H et al (2004) 50% Inhibition concentration; CC : 50% Cytotoxic concentration; NaHCO : 50 3 Mechanisms of antiplatelet and antithrombotic activity of midazolam in Sodium bicarbonate; NaOH: Sodium hydroxide; DMSO: Dimethyl sulfoxide; AA: in vitro and in vivo studies. Eur J Pharmacol 487(1–3):159–166 Arachidonic acid. 12. Qneibi M, Hamed O, Fares O, Jaradat N, Natsheh A-R, AbuHasan Q et al (2019) The inhibitory role of curcumin derivatives on AMPA receptor Acknowledgements subunits and their effect on the gating biophysical properties. Eur J The authors would like to thank An-Najah National University for funding this Pharm Sci 136:104951 study (grant number ANNU-1920-Sc013), the Dean of Scientific Research, Gazi 13. Grossi G, Di Braccio M, Roma G, Ballabeni V, Tognolini M, Calcina F et al University and Anadolu University for their support in chemical analysis. (2002) 1,5-Benzodiazepines: part XIII. Substituted 4H-[1,2,4] triazolo [4,3-a] [1,5] benzodiazepin-5-amines and 4H-imidazo [1,2-a][1,5] benzodiazepin- Authors’ contributions 5-amines as analgesic, anti-inflammatory and/or antipyretic agents with M.H., N.J., S.H., and A.M. conceived and designed the current study and low acute toxicity. Eur J Med Chem 37(12):933–944 analyzed the data obtained. This paper was written by M.H., N.J., and S.H., and 14. Liu B, Qu L, Yan S (2015) Cyclooxygenase-2 promotes tumor growth and drafted by all authors. All authors read and approved the final manuscript. suppresses tumor immunity. Cancer Cell Int 15(1):106 15. Botting R (2006) Inhibitors of cyclooxygenases: mechanisms, selectivity Funding and uses. J Physiol Pharmacol 57:113 This research was supported by the Dean of Scientific Research, An-Najah 16. Assali M, Abualhasan M, Sawaftah H, Hawash M, Mousa A (2020) Synthe- National University (grant number ANNU-1920-Sc013). sis, biological activity, and molecular modeling studies of pyrazole and triazole derivatives as selective COX-2 inhibitors. J Chem 2020:6393428 Availability of data and materials 17. Ferlay J, Colombet M, Soerjomataram I, Mathers C, Parkin D, Piñeros M The datasets used and/or analysed during the current study available from the et al (2019) Estimating the global cancer incidence and mortality in 2018: corresponding author on reasonable request. GLOBOCAN sources and methods. Int J Cancer 144(8):1941–1953 18. Hawash M (2019) Highlights on specific biological targets; cyclin- Ethics approval and consent to participate dependent kinases, epidermal growth factor receptors, Ras protein, and Not applicable. cancer stem cells in anticancer drug development. Drug Res. 69:471–478 19. World Health Organization. Cancer Geneva: WHO; 2018 https ://www. Consent for publication who.int/news-room/fact-sheet s/detai l/cance r. Accessed 29 Aug 2019 Not applicable. 20. Hawash M, Baytas S (2017) Antiproliferative activities of some biologically important scaffold. FABAD J Pharm Sci 43(1):59–77 Competing interests 21. Hawash MM, Kahraman DC, Eren F, Cetin Atalay R, Baytas SN (2017) Syn- The authors declare that they have no competing interests. thesis and biological evaluation of novel pyrazolic chalcone derivatives as novel hepatocellular carcinoma therapeutics. Eur J Med Chem 129:12–26 Author details 22. Bandgar BP, Totre JV, Gawande SS, Khobragade CN, Warangkar SC, Kadam Department of Pharmacy, Faculty of Medicine and Health Sciences, An-Najah PD (2010) Synthesis of novel 3,5-diaryl pyrazole derivatives using combi- National University, P.O. Box 7, Nablus 00970, Palestine. Department of Bio- natorial chemistry as inhibitors of tyrosinase as well as potent anticancer, medical Sciences, Faculty of Medicine and Health Sciences, An-Najah National anti-inflammatory agents. Bioorg Med Chem 18(16):6149–6155 University, Nablus 00970, Palestine. 23. Abu-Hashem AA, El-Shehry MF, Badria FA (2010) Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine Received: 8 June 2020 Accepted: 2 September 2020 derivatives as antioxidant and antitumor agent. Acta Pharm 60:311–323 24. Abdellatif KR, Lamie PF, Omar HA (2016) 3-methyl-2-phenyl-1-substi- tuted-indole derivatives as indomethacin analogs: design, synthesis and biological evaluation as potential anti-inflammatory and analgesic agents. J Enzyme Inhib Med Chem 31(2):318–324 References 25. Deshpande SR, Nagrale SN, Patil MV, Chavan SS (2015) Novel 3,4-meth- 1. Zarghi A, Arfaei S (2011) Selective COX-2 inhibitors: a review of their ylenedioxybenzene scaffold incorporated 1,3,5-trisubstituted-2-pyrazo - structure-activity relationships. Iran J Pharm Res 10(4):655 lines: synthesis, characterization and evaluation for chemotherapeutic 2. Fiorucci S, Meli R, Bucci M, Cirino G (2001) Dual inhibitors of cyclooxyge- activity. Indian J Pharm Sci 77(1):24–28 nase and 5-lipoxygenase. A new avenue in anti-inflammatory therapy? 26. Rollas S, Küçükgüzel SG (2007) Biological activities of hydrazone deriva- Biochem Pharmacol 62(11):1433–1438 tives. Molecules 12:1910–1939 Ha wash et al. BMC Chemistry (2020) 14:54 Page 9 of 9 27. Miller EC, Swanson AB, Phillips DH, Fletcher TL, Liem A, Miller JA (1983) 34. Qneibi M, Jaradat N, Hawash M, Olgac A, Emwas N (2020) Ortho versus Structure-activity studies of the carcinogenicities in the mouse and rat meta chlorophenyl-2,3-benzodiazepine analogues: synthesis, molecular of some naturally occurring and synthetic alkenylbenzene derivatives modeling, and biological activity as AMPAR antagonists. ACS Omega related to safrole and estragol. Cancer Res 43:1124–1134 5:3588–3595 28. Lima PC, Lima LM, Silva KCM, Léda PHO, Miranda ALP, Fraga CAM et al 35. Jin G, Lee S, Choi M, Son S, Kim GW, Oh JW et al (2014) Chemical (2000) Synthesis and analgesic activity of novel N-acylarylhydrazones and genetics-based discovery of indole derivatives as HCV NS5B polymerase isosters, derived from natural safrole. Eur J Med Chem 35:187–203 inhibitors. Eur J Med Chem 75:413–425 29. Khayyat SA (2013) Photosynthesis of dimeric cinnamaldehyde, eugenol, 36. çaliŞkan Ergün B, Nuñe MT, Labeaga L, Ledo F, Darlington J, Bain G et al and safrole as antimicrobial agents. J Saudi Chem Soc 17(1):61–65 (2010) Synthesis of 1,5-diarylpyrazol-3-propanoic acids towards inhibition 30. Espahbodinia M, Ettari R, Wen W, Wu A, Shen YC, Niu L et al (2017) of cyclooxygenase-1/2 activity and 5-lipoxygenase-mediated LTB4 forma- Development of novel N-3-bromoisoxazolin-5-yl substituted 2,3-ben- tion. Arzneimittelforschung 60(08):497–505 zodiazepines as noncompetitive AMPAR antagonists. Bioorg Med Chem 37. Jaradat NA, Al-lahham S, Zaid AN, Hussein F, Issa L, Abualhasan MN et al 25(14):3631–3637 (2019) Carlina curetum plant phytoconstituents, enzymes inhibitory and 31. Irannejad H, Kebriaieezadeh A, Zarghi A, Montazer-Sadegh F, Shafiee A, cytotoxic activity on cervical epithelial carcinoma and colon cancer cell Assadieskandar A et al (2014) Synthesis, docking simulation, biologi- lines. Eur J Integrat Med 30:100933 cal evaluations and 3D-QSAR study of 5-Aryl-6-(4-methylsulfonyl)-3- (metylthio)-1,2,4-triazine as selective cyclooxygenase-2 inhibitors. Bioorg Publisher’s Note Med Chem 22(2):865–873 Springer Nature remains neutral with regard to jurisdictional claims in pub- 32. Zarghi A, Ghodsi R (2010) Design, synthesis, and biological evaluation of lished maps and institutional affiliations. ketoprofen analogs as potent cyclooxygenase-2 inhibitors. Bioorg Med Chem 18(16):5855–5860 33. Bandyopadhyay S, Pal BC, Parasuraman J, Roy S, Chakrabotry JB, Mukher- jee C, et al (2016) Inventors inhibitors of phosphatidylinositol-3-kinase (PI3) and inducers of nitric oxide (NO). United States 2016 Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Chemistry Central Journal Springer Journals

Design, synthesis and biological evaluation of novel benzodioxole derivatives as COX inhibitors and cytotoxic agents

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Abstract

Non-steroidal anti-inflammatory drugs are among the most used drugs. They are competitive inhibitors of cyclooxy- genase (COX). Twelve novel compounds (aryl acetate and aryl acetic acid groups) were synthesized in this work in order to identify which one was the most potent and which group was most selective towards COX1 and COX2 by using an in vitro COX inhibition assay kit. The cytotoxicity was evaluated for these compounds utilizing MTS assay against cervical carcinoma cells line (HeLa). The synthesized compounds were identified using FTIR, HRMS, H-NMR, and C-NMR techniques. The results showed that the most potent compound against the COX1 enzyme was 4f with IC = 0.725 µM. The compound 3b showed potent activity against both COX1 and COX2 with IC = 1.12 and 1.3 µM, 50 50 respectively, and its selectivity ratio (0.862) was found to be better than Ketoprofen (0.196). In contrast, compound 4d was the most selective with a COX1/COX2 ratio value of 1.809 in comparison with the Ketoprofen ratio. All com- pounds showed cytotoxic activity against the HeLa Cervical cancer cell line at a higher concentration ranges (0.219– 1.94 mM), and the most cytotoxic compound was 3e with a CC value of 219 µM. This was tenfold more than its IC 50 50 values of 2.36 and 2.73 µM against COX1 and COX2, respectively. In general, the synthesized library has moderate activity against both enzymes (i.e., COX1 and COX2) and ortho halogenated compounds were more potent than the meta ones. Keywords: Benzodioxole, COX, Ketoprofen Introduction a 100  years [1, 2]. The biosynthesis of prostaglandin H2 Some of the most used analgesics are non-steroidal anti- from arachidonic acid is catalysed by COX enzymes [3]. inflammatory drugs (NSAIDs) that target the cyclooxy - Prostaglandin H2 is the main component in the forma- genase (COX) enzymes. NSAIDs are used for various tion of other prostaglandins, such as thromboxane and therapeutic purposes globally. Due to their wide pharma- prostacyclin, which play important roles in different bio - cological effects, including analgesic, anti-inflammatory logical responses [4, 5]. In fact, COX1 and COX2 are the and antipyretic effects, they are investigated as being two major isoforms of COX membrane-bound enzymes some of the best choices for treating different diseases [6]. COX1 is involved in the biosynthesis of important like arthritis and rheumatism, and they are widely used prostaglandins which maintain the constant functions in as analgesics. Actually, acetyl salicylic acid (ASA), one of the body, essentially in the cardiovascular and gastroin- the members of this family, has been used for more than testinal systems [7]. Moreover, COX2 is an enzyme cata- lyst that is overexpressed in several pathophysiological events such as hyperalgesia, inflammation, and cancer *Correspondence: mohawash@najah.edu 1 [8, 9]. The structures of COX1 and COX2 enzymes are Department of Pharmacy, Faculty of Medicine and Health Sciences, An- Najah National University, P.O. Box 7, Nablus 00970, Palestine 67% identical in amino acid chains. The main difference Full list of author information is available at the end of the article between the two enzymes is the presence of isoleucine © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hawash et al. BMC Chemistry (2020) 14:54 Page 2 of 9 (Ilu523) in COX1 instead of valine (Val523) in COX2. associated with lung cancer, fruits and vegetables con- This allows 25% greater available space in the binding taminated with pesticides and phyto-growth hormones, region of COX2 in comparison to COX1 [10]. All of these and the unhealthy lifestyles of modern people as well data encourage the researchers to focus their efforts to as some physical carcinogens such as radiation, some the find COX2 selective inhibitors in order to improve chronic diseases such as diabetes, and some infectious ill- treatment efficacy and to reduce the side effects that are nesses such Hepatitis B and C viral infections [19]. associated with the use of non-selective inhibitors of The heterocycle-containing agents have several phar - these enzymes [11–13]. macological effects including anticancer [20, 21], anti- COX2 enzyme is associated with carcinogenesis and inflammatory [22], antioxidant [23] and analgesic effects inflammatory diseases. It is suspected to induce tis - [24]. Therefore, the Benzodioxole containing compounds sue invasion of tumours, angiogenesis, and resistance to (Fig.  2) have different biological activities such as anti - apoptosis. Moreover, COX2 plays an important role in cancer, anti-tuberculosis, anti-microbial, anti-epileptic, the innate and adaptive immune response, and it contrib- and analgesic activity [25–30]. Various tricyclic com- utes to immune evasion and resistance to cancer immu- pounds and Ketoprofen like structures were synthesized notherapy. However, COX inhibitors can facilitate a and evaluated as COX enzyme inhibitors [31, 32]. The benefit to patients from addition of COX inhibitors when current work aims to synthesize new compounds with a compared to standard chemotherapy [14]. Benzodioxole core structure in two final product groups A large number of agents with different structural with different halogen atoms and aryl acetate and aryl features were produced in the discovery efforts of new acetic acid (Fig.  3), to evaluate their COX1 and COX2 COX2 selective inhibitors. A lot of classical non-selective NSAIDs were synthesized, approved, and used broadly, such as Ibuprofen, Naproxen, and Ketoprofen (Fig.  1), but their selectivity is too low against COX2/COX1 [15], and the previous studies were implemented to synthesize more selective agents as COX2 inhibitors by using differ - ent methods and structures [16]. According to the World Health Organization (WHO) surveys, cancer is one of the leading causes of death around the globe, and it was responsible for about 10 million deaths in 2018 [17, 18]. Around 1 in 6 peo- ple died from cancer, which is considered the largest cause of death. This is a considerably alarming estimate. WHO has recognized that 1.16 trillion US dollars were spent on the prevention and treatment of cancer in 2010 alone, and that number has increased dramatically over the years [17]. These important statistics are the result of erratic human behaviours such as smoking, which is Fig. 2 Structures of benzodioxol derivatives having various biological Fig. 1 Classical NSAIDs with COOH functional group activities Ha wash et al. BMC Chemistry (2020) 14:54 Page 3 of 9 compounds were synthesized by dissolving the ester (2) in dichloromethane with benzoic acid derivatives in the presence of an excess of phosphorus pentoxide and stir- ring at room temperature for approximately 18  h. The H-NMR spectrum data of these compounds showed 5–7 protons (depend on the Halogen atoms for each com- pound) in the aromatic area, 2 protons around 6.13 ppm singlet peaks for O–CH –O of benzodioxole and 5 pro- tons were observed in area 3.40 and 3.80 ppm for –CH – CO–CH . According to the C-NMR spectrum, C signal of carbonyl groups was found around 195 and 171 ppm, Fig. 3 Halogenated Ketoprofen analogues as aryl acetic acid and aryl and at 37–51  ppm two signals of aliphatic carbon were acetate observed. The Benzodioxole acetic acid derivatives (4a– 4f) were synthesized by hydrolysis reaction of the ester compounds 3a–3f using NaOH [35] (see Scheme 1). The H-NMR spectrum data showed one proton with sin- glet peak around 12  ppm (–COOH), 2 protons around 6.13  ppm singlet peaks for O-CH -O of benzodioxole and 2 protons were observed in area 3.40–3.78 ppm for – CH –COOH. However, C-NMR spectrum data showed C signal of carbonyl groups around 197 and 172 ppm. Cyclooxygenase inhibition activity The synthesized compounds have a structure that is simi - lar to Ketoprofen, and because of that Ketoprofen was used as a positive control in the COX inhibition analy- sis of the synthesized library. All Benzodioxole acetate structures with halogens (Br, Cl, I; 3b–3f) on the phenyl ring showed better activity against COX1 (IC 1.12– 27.06  µM) than acetic acid Benzodioxole with halogens (IC 4.25–33.7 µM; 4b–4e), except 4f which showed the most potent inhibitory activity (IC = 0.725  µM) against the COX1 enzyme. However, the acetic acid Benzodiox- Scheme 1 The reaction steps a methanol, oxalyl chloride b DCM, ole compound without a halogen (4a) showed stronger P O , aryl-carboxylic acid, c MeOH/THF/H O, NaOH reflux 2 5 2 inhibition activity toward cyclooxygenase enzymes COX1 and COX2 (1.45 and 3.34  µM, respectively) than acetate Benzodioxole without a halogen compound (3a) inhibitory activity and to evaluate the synthesized com- toward COX1 and COX2 (12.32 and 14.34  µM, respec- pounds’ cytotoxic effects. tively). However, all Benzodioxole acetate structures with halogens (3b–3f) showed better activity against COX2 Results and discussion (IC 1.30–37.45 µM) than acetic acid Benzodioxole with Chemistry halogens (IC 2.35–39.14  µM; 4b–4f) as presented in The Benzodioxole aryl acetate derivatives (3a-3f) and Table 1. acetic acid derivatives (4a–4f) were synthesized as out- lined in Scheme 1. The methyl 3,4-(methylenedioxy) phe - Cytotoxic evaluation nylacetate (2) was generated by an esterification reaction An MTS assay was used to determine the cytotoxic effect of 3,4-(methylenedioxy) phenylacetic acid (1). To pro- of Benzodioxole derivatives on HeLa (cervical carcinoma duce the ester (2), oxalyl chloride was added dropwise cells). As shown in Table  1, four different concentra - to methanol solvent and stirred for half an hour in an tions were used (2, 1, 0.5, and 0.1 mM) to investigate the ice bath [33, 34]. The IR spectra of the ester (2) showed cytotoxicity of the compounds. Actually, all compounds the disappearance of the broad band that belonged showed inhibition of cell growth at relatively high con- to the acetic acid group of (1). The aryl acetate 3a–3f centrations in comparison to the IC of COX enzyme. 50 Hawash et al. BMC Chemistry (2020) 14:54 Page 4 of 9 Table 1 IC inhibition of  COX1 and  COX2, Selectivity ratio for  COX1/COX2, and  the  CC on  HeLa cancer cell line 50 50 of the synthesized compounds The IC in µM of COX 1 COX1/COX2 Ratio HeLa Cell CC 50 50 and COX2 in milli-molar Codes X R1 R2 R3 COX1 COX2 Selectivity CC 3a O-CH3 H H H 12.320 14.340 0.859 1.49 3b O-CH3 I H H 1.120 1.300 0.862 0.228 3c O-CH3 H I H 27.060 37.450 0.723 1.79 3d O-CH3 Br H H 1.3 1.45 0.897 1.61 3e O-CH3 H Br H 2.360 2.730 0.864 0.219 3f O-CH3 Cl H Cl 5.180 4.100 1.263 0.949 4a OH H H H 1.450 3.340 0.434 1.94 4b OH I H H 7.670 30.700 0.250 0.697 4c OH H I H 33.700 39.140 0.861 1.049 4d OH Br H H 4.250 2.350 1.809 0.547 4e OH H Br H 7.110 49.300 0.144 0.437 4f OH Cl H Cl 0.725 4.290 0.169 1.019 Ketoprofen 0.031 0.158 0.196 P-values for the experiments p < 0.05 The CC were in the range between 0.219 and 1.79 mM. There is no clear relationship between the ortho ver - The most cytotoxic compound was 3e with a CC value sus meta halogen and the cytotoxicity results. Generally, of 219 µM. the halogenated compounds are more cytotoxic than non-halogenated (3a & 4a). The most cytotoxic com - SAR study pound was compound 3e (ester with Br on meta position; All ortho halogenated compounds 3b, 3d, 4b, and 4d CC = 0.219 mM). It was more toxic than compound 3d showed better activity with lower IC values than their (ester with Br on ortho), and the same relation was found meta halogenated compounds 3c, 3e, 4c and 4d. For between 4e and 4d, respectively. In contrast, the ortho example the IC values of compound 3b (ortho-halo- iodo halogenated compounds (3b and 4b) were more genated) against both COX1 and COX2 were 1.120 and toxic than meta iodo halogenated compounds (3c & 4c). 1.300  µM in comparison with 3d (meta-halogenated) In this study we can observe that our synthesized com- which were 27.060 and 37.450  µM, respectively. This pounds have inhibition activity against both COX1 and depended on the theory that the ortho-halogenated com- COX2 enzymes better than some tricyclic compounds pounds can make the second aromatic ring non-coplanar synthesized by other research teams. As published by with the first aromatic ring, which is ideal for the COX Caliskan et  al., one of pyrazol-3-propanoic acids deriva- inhibitory activity. All ester-mono halogenated com- tives was the most active compound in this series, and pounds (ortho or meta; 3b, 3c, 3d & 3e) have better COX it showed a selectivity ratio of 0.93 and activity against inhibitory activity than acetic acid mono-halogenated COX1 and COX2 with an IC value relatively close to compounds (ortho or meta; 4b, 4c, 4d & 4e). Except for our results (1.5 and 1.6  µM, respectively). However, the compound 4b, all other ortho-halogenated compounds inhibitory activity against COX1 for most of our synthe- (3b, 3d, and 4d) showed better selectivity ratios (COX1/ sized compounds were very close to or better than their COX2) than meta-halogenated compounds. The most tested compound [36]. In another study by Assali et  al., potent compound against COX1 enzyme was the acetic a series of pyrazole and triazole derivatives were synthe- acid di-halogenated (2,4-dichloro) compound 4f. The sized, and one of their triazole derivatives was considered ortho-iodo ester compound 3b was potent against COX2 to be a highly selective COX2 inhibitor with a high selec- enzyme with a good selectivity ratio (0.862). tivity ratio (162.5) [16]. Comparing our results with other studies, the results of this study clearly demonstrate Ha wash et al. BMC Chemistry (2020) 14:54 Page 5 of 9 that the synthesized agents have good inhibition activ- column had a pore size of 60  Å and 230–400  mesh par- ity against both COX1 and COX2 enzymes with rela- ticle size, 40–63  μm particle size. The inhibitory activity tively low IC values, and the COX selectivity ratio of the of ovine COX1 and human recombinant COX2 enzymes compounds synthesized in this study were better than was determined using a COX inhibitor screening assay approved drugs like ketoprofen or aspirin. kit No. 560131 (Cayman Chemical, USA). The yellow product of this enzymatic reaction was determined using Conclusion a UV spectrophotometer with a Microplate Reader (Bio- The synthesized compounds showed moderate activ - Rad, Japan) at a wavelength of 415  nm. HeLa Cervical ity against COX1 and COX2 enzymes. However, most Carcinoma cell line was purchased from ATCC (ATCC ® ™ compounds have better COX2 inhibition selectivity com- CCL-2 ), and the cyototoxicty test of the cell viability pared to Ketoprofen. The results showed a promising was assessed by the CellTilter 96 Aqueous One Solution group of compounds having a Benzodioxole moiety. They Cell Proliferation (MTS) assay according to the manufac- had better COX2 selectivity compared with Ketoprofen, turer’s instructions (Promega Corporation, Madison, WI) and this may be due to the bigger moiety (Benzodioxole) (Additional file 1). in the synthesized compound in comparison with phenyl moiety in Ketoprofen. Future plans should include dock- Chemistry method ing studies and synthesizing more analogues of this core Synthesis of methyl 2‑(2H‑1,3‑benzodioxol‑5‑yl) acetate structure to study the structure–activity relationship. synthesis 2 This is required in order to improve their COX inhibitory The 3,4-(methylenedioxy)phenylacetic acid (1) (8  g, activity and to achieve a better COX2 selectivity ratio. All 44.40  mmol) was dissolved in methanol, then it was compounds 3a–4f showed cytotoxic activity on the HeLa cooled in an ice bath to 0 °C. Then oxalyl chloride (4 mL, cancer cell line at higher doses. However the effective 46.80 mmol) was added dropwise, and the reaction mix- doses towards COX enzyme were at least lesser 10 times ture was stirred for 30–45 min. The reaction mixture was greater than the cytotoxic concentrations. then evaporated under vacuum and the resulting residue was diluted with ethyl acetate solvent and washed with Experimental section saturated sodium bicarbonate (NaHCO ) and distilled Chemicals and instruments water, sequentially. The organic layer was dried with All chemicals were purchased from Sigma-Aldrich and sodium sulphate, then filtered and evaporated again to Alfa Aesar. Melting points were determined with an concentrate it. In the last step, it was purified by silica gel SMP-II Digital Melting Point Apparatus and are uncor- column chromatography by using a hexane:ethyl acetate rected. IR spectra were obtained using a Perkin Elmer solvent system (50%:50%). The resulting compound (2) Spectrum 400 FTIR/FTNIR spectrometer. H-NMR and was a yellow oil with 94% yield. C-NMR spectra were recorded in DMSO-d6 and were performed on two NMR instruments. The first was a General synthesis procedure for ketoester (3a–3f ) derivatives Bruker 500  MHz-Avance III High-Performance Digital The benzoic acid derivatives (1.46  g, 6.68  mmol) and FT-NMR spectrometer at the Faculty of Science, Depart- phosphorus pentoxide (5 g) were added to a stirred solu- ment of Chemistry, The University of Jordan, Jordan (it tion of dichloromethane (60  mL) and compound (2) was used for the H-NMR of just one compound, 3e). (1  g, 5.14  mmol). Then, the mixture was stirred at room The second was a Bruker 300  MHz-Avance III High- temperature for 18  h before distilled water (60  mL) was Performance Digital FT-NMR spectrometer at the NMR cautiously added, and the mixture was extracted with facility at the Doping and Narcotics Analysis Laboratory ethyl acetate twice (60  mL). Then, the organic layer was of the faculty of pharmacy, Anadolu University, Turkey (it separated and treated with 1  M NaOH (60  mL), brine 1 13 was used for both H-NMR and C-NMR for the other (60  mL), and twice with 60  mL of distilled water. The compounds). Tetramethylsilane was used as the internal organic layer was dried with sodium sulphate, filtered, standard. All chemical shifts were recorded as d (ppm). evaporated under vacuum, and then purified by silica gel High resolution mass spectral data (HRMS) were col- column chromatography with different solvent systems. lected using a Waters LCT Premier XE Mass Spectrome- ter (high sensitivity orthogonal acceleration time-of-flight Methyl 2‑(6‑benzoylbenzo[d][1,3]dioxol‑5‑yl)acetate instrument) using ESI (+) method (The instrument was (3a) Purified by silica gel column chromatography using coupled to an AQUITY Ultra Performance Liquid Chro- n-hexane: ethyl acetate solvent system (3:2). Crude yellow matography system (Waters Corporation, Milford, MA, semi solid, Yield 75%; ESI–MS: 299.0919 (100), 300 (20), −1 USA) at the Pharmacy Faculty Gazi University Ankara- 301 (2), For C H O . IR (FTIR/FTNIR-ATR): 1737 cm 17 15 5 −1 Turkey. The silica gel used for the flash chromatography ester carbonyl (C=O), 1661 cm keton carbonyl (C=O). Hawash et al. BMC Chemistry (2020) 14:54 Page 6 of 9 H NMR (DMSO-d , 300 MHz) δ ppm: 7.62–7.67 (3H, m, chromatography using n-hexane: ethyl acetate solvent Ar–H), 7.52 (2H, t, J = 7.8 Hz, Ar–H), 7.05 (1H, s, Ar–H), system (3:2). Powder product, mp: 72.5–74.5  °C, Yield 6.89 (1H, s, Ar–H), 6.12 (2H, s, O–CH –O), 3.74 (2H, s, – 79%; ESI–MS: 377.00 (100), 379 (98), 380 (20), for 13 −1 CH –C=O), 3.47 (3H, s, O–CH ). C-NMR (DMSO-d , C H BrO . IR (FTIR/FTNIR-ATR): 1742  cm ester 2 3 6 17 14 5 −1 1 75  MHz) δ ppm: 196.53, 171.61, 149.66, 146.04, 138.08, carbonyl (C=O), 1655  cm keton carbonyl (C=O). H 133.39, 131.59, 130.21, 130.00, 129.73, 129.44, 128.95, NMR (DMSO-d , 500 MHz) δ ppm: 7.86 (1H, d, J = 8 Hz, 112.66, 110.34, 102.45, 51.89, and 38.36. Ar–H), 7.77 (1H, s, Ar–H), 7.64 (1H, d, J = 8  Hz, Ar–H), 7.50 (1H, t, J = 8 Hz, Ar–H), 7.06 (1H, s, Ar–H), 6.95 (1H, Methyl 2‑(6‑(2‑iodobenzoyl)benzo[d][1,3]dioxol‑5‑yl)ace ‑ s, Ar–H), 6.15 (2H, s, O–CH –O), 3.77 (2H, s, –CH – 2 2 tate (3b) Purified by silica gel column chromatography C=O), 3.49 (3H, s, O–CH ). using n-hexane: ethyl acetate solvent system (1:1). Semi solid product, Yield 90%. ESI–MS: 424.9875 (100), 425.99 Methyl 2‑(6‑(2,4‑dichlorobenzoyl)benzo[d][1, 3] −1 (20), for C H IO . IR (FTIR/FTNIR-ATR): 1740  cm dioxol‑5‑yl)acetate (3f) Purified by silica gel column 17 14 5 −1 ester carbonyl (C = O), 1659 cm keton carbonyl (C=O). chromatography using n-hexane: ethyl acetate solvent H NMR (DMSO-d , 300  MHz) δ ppm: 7.95 (1H, d, system (1:1). Powder product, mp: 95–97  °C, Yield 83%; J = 7 Hz, Ar–H), 7.51 (1H, t, J = 7.5 Hz, Ar–H), 7.23–7.30 ESI–MS: 367.01 (100), 369 (67), for C H Cl O . IR 17 13 2 5 −1 (2H, m, Ar–H), 7.11 (1H, s, Ar–H), 6.64 (1H, s, Ar–H), (FTIR/FTNIR-ATR): 1760  cm ester carbonyl (C=O), −1 1 6.13 (2H, s, O-CH -O), 3.92 (2H, s, –CH –C=O), 3.59 1633  cm keton carbonyl (C=O). H NMR (DMSO-d , 2 2 6 (3H, s, O–CH ). C-NMR (DMSO-d , 75  MHz) δ ppm: 300 MHz) δ ppm: 7.79–7.82 (2H, m, Ar–H), 7.59 (1H, d, 3 6 197.19, 171.49, 151.23, 146.51, 145.12, 139.78, 134.05, J = 8.4 Hz, Ar–H), 7.07 (1H, s, Ar–H), 7.00 (1H, s, Ar–H), 133.30, 131.97, 128.95, 128.60, 113.60, 112.03, 102.93, 6.14 (2H, s, O–CH –O), 3.78 (2H, s, –CH –C=O), 3.48 2 2 93.46, 51.98. (3H, s, O-CH ). C-NMR (DMSO-d , 75  MHz) δ ppm: 3 6 194.29, 171.68, 150.13, 146.17, 138.56, 136.07, 131.70, 131.34, 130.62, 130.52, 130.33, 116.68, 112.82, 110.64, Methyl 2‑(6‑(4‑iodobenzoyl)benzo[d][1,3]dioxol‑5‑yl)ace ‑ 102.06, 52.05, 38.33. tate (3c) Purified by silica gel column chromatography using n-hexane: ethyl acetate solvent system (1:1). Pow- der product mp: 119–121 °C, Yield 87%. ESI–MS: 424.96 General synthesis procedure of 2‑(6‑benzoyl‑2H‑1,3‑ben ‑ (100), 425.99 (20), for C H IO . IR (FTIR/FTNIR-ATR): zodioxol‑5‑yl)acetic acid (4a–4f) The ketoesters 3a–3f 17 14 5 −1 −1 1735  cm ester carbonyl (C=O), 1660  cm keton car- (450  mg, 1.35  mmol) were dissolved in methanol/H O/ bonyl (C=O). H NMR (DMSO-d , 300  MHz) δ ppm: THF (12/12/12  mL), then NaOH (540.9  mg, 13.5  mmol) 7.92 (2H, d, J = 8.4  Hz, Ar–H), 7.41 (2H, d, J = 8.8  Hz, was added. The solution was heated in an oil bath and Ar–H), 7.06 (1H, s, Ar–H), 6.92 (1H, s, Ar–H), 6.13 (2H, refluxed for 4  h before being cooled to room tempera - s, O–CH –O), 3.75 (2H, s, –CH –C=O), 3.47 (3H, s, O– ture. The solution was then evaporated, and the residue 2 2 CH ). C-NMR (DMSO-d , 75  MHz) δ ppm: 195.94, was made acidic by adding HCl 2 N (pH = 2). The pre - 3 6 171.61, 149.79, 138.52, 137.89, 137.40, 131.94, 131.20, cipitate was filtered and concentrated under vacuum to 130.14, 112.70, 110.38, 102.49, 102.05, 52.02, 38.29. give the crude products 4a–4f. Methyl 2‑(6‑(2‑bromobenzoyl)benzo[d][1,3]dioxol‑5‑yl) 2‑(6‑benzoylbenzo[d][1,3]dioxol‑5‑yl)acetic acid acetate (3d) Purified by silica gel column chromatog - (4a) Purified by silica gel column chromatography raphy using n-hexane: ethyl acetate solvent system (4:1). using n-hexane: ethyl acetate solvent system (3:2). Pow- Powder product, mp: 85–87  °C, Yield 85%; ESI–MS: der product, mp: 184.5–186.5  °C, Yield 97%; ESI–MS: 377.00 (100), 379 (98), 380 (20), For C H BrO . IR 285.07 (100), 286 (20), for C H O . IR (FTIR/FTNIR- 17 14 5 16 13 5 −1 −1 −1 (FTIR/FTNIR-ATR): 1740  cm ester carbonyl (C=O), ATR): 1770 cm acetic acid carbonyl (C=O), 1655 cm −1 1 1 1658  cm keton carbonyl (C=O). H NMR (DMSO-d , keton carbonyl (C=O). H NMR (DMSO-d , 300  MHz) 6 6 300 MHz) δ ppm: 7.34–7.77 (4H, m, Ar–H), 7.11 (1H, s, δ ppm: 12.18 (1H, s, OH), 7.49–7.70 (5H, m, Ar–H), Ar–H), 6.69 (1H, s, Ar–H), 6.14 (2H, s, O–CH –O), 3.93 7.03 (1H, s, Ar–H), 6.86 (1H, s, Ar–H), 6.11 (2H, s, O– 13 13 (2H, s, –CH –C=O), 3.59 (3H, s, O–CH ). C-NMR CH –O), 3.67 (2H, s, –CH –C=O). C-NMR (DMSO- 2 3 2 2 (DMSO-d , 75  MHz) δ ppm: 196.53, 171.61, 149.66, d , 75 MHz) δ ppm: 96.64, 172.65, 149.48, 138.12, 133.68, 6 6 146.04, 138.08, 133.39, 131.59, 130.21, 130.00, 129.73, 133.33, 131.73, 130.68, 130.26, 130.01, 129.20, 128.90, 129.44, 128.95, 112.66, 110.34, 102.45, 51.89, 38.36. 112.62, 110.16, 102.32, 38.59. Methyl 2‑(6‑(3‑bromobenzoyl)benzo[d][1, 3] 2‑(6‑(2‑iodobenzoyl)benzo[d][1,3]dioxol‑5‑yl)acetic acid dioxol‑5‑yl)acetate (3e) Purified by silica gel column (4b) Purified by silica gel column chromatography Ha wash et al. BMC Chemistry (2020) 14:54 Page 7 of 9 using n-hexane: ethyl acetate solvent system (3:2). Pow- 6.12 (2H, s, O–CH –O), 3.70 (2H, s, –CH –C=O). 2 2 der product, mp: 147–149 °C, Yield 92%; ESI–MS: 410.97 C-NMR (DMSO-d , 75  MHz) δ ppm: 195.25, 172.72, (100), 411 (20), for C H IO . IR (FTIR/FTNIR-ATR): 149.80, 145.88, 140.39, 135.85, 132.47, 131.17, 131.09, 16 12 5 −1 −1 1754 cm acetic acid carbonyl (C=O), 1653 cm keton 131.04, 129.34, 122.12, 112.70, 110.31, 102.44, 38.58. carbonyl (C=O). H NMR (DMSO-d , 300 MHz) δ ppm: 7.95 (1H, d, J = 7.8  Hz, Ar–H), 7.50 (1H, t, J = 7.8  Hz, 2‑(6‑(2,4‑dichlorobenzoyl)benzo[d][1,3]dioxol‑5‑yl)acetic Ar–H), 7.23–7.31 (2H, m, Ar–H), 7.06 (1H, s, Ar–H), acid (4f ) Purified by silica gel column chromatography 6.61 (1H, s, Ar–H), 6.11 (2H, s, O–CH –O), 3.83 (2H, using n-hexane: ethyl acetate solvent system (1:1). Solid s, –CH –C=O). C-NMR (DMSO-d , 75  MHz) δ ppm: product, mp: 168.5–170  °C, Yield 91%; ESI–MS: 352.99 2 6 197.14, 172.60, 150.97, 146.17, 145.23, 139.81, 134.23, (100), 354 (67) for C H Cl O . IR (FTIR/FTNIR-ATR): 16 12 2 5 −1 −1 131.97, 129.31, 129.14, 128.54, 113.48, 111.77, 102.75, 1768 cm acetic acid carbonyl (C=O), 1657 cm keton 93.51. carbonyl (C=O). H NMR (DMSO-d , 300 MHz) δ ppm: 12.25 (1H, s, OH), 7.77–7.81 (2H, m, Ar–H), 7.60 (1H, 2‑(6‑(4‑iodobenzoyl)benzo[d][1,3]dioxol‑5‑yl)acetic acid dd, J = 8.3, 1.8  Hz, Ar–H), 7.05 (1H, s, Ar–H), 6.98 (1H, (4c) Purified by silica gel column chromatography using s, Ar–H), 6.13 (2H, s, O–CH –O), 3.71 (2H, s, –CH – 2 2 n-hexane: ethyl acetate solvent system (3:2). Powder prod- C=O). C-NMR (DMSO-d , 75  MHz) δ ppm: 194.43, uct, mp: 239.5–241.5 °C, Yield 89%; ESI–MS: 410.97 (100), 172.73, 149.41, 145.94, 138.60, 135.99, 131.75, 131.29, −1 411 (20), for C H IO . IR (FTIR/FTNIR-ATR): 1760 cm 131.20, 130.74, 130.36, 129.84, 112.74, 110.40, 102.47, 16 12 5 −1 acetic acid carbonyl (C=O), 1660  cm keton carbonyl 38.56 (Additional file 1). (C=O). H NMR (DMSO-d , 300  MHz) δ ppm: 12.20 (1H, s, OH), 7.91 (2H, d, J = 8.7  Hz, Ar–H), 7.42 (2H, d, J = 8.4 Hz, Ar–H), 7.03 (1H, s, Ar–H), 6.90 (1H, s, Ar–H), Biological COX assay method 6.11 (2H, s, O–CH –O), 3.67 (2H, s, –CH –C=O). C- The ability of the synthesized a series to prevent the con - 2 2 NMR (DMSO-d , 75 MHz) δ ppm: 196.04, 172.63, 149.60, version of arachidonic acid (AA) to PGH2 by human 145.85, 138.14, 137.83, 137.44, 131.99, 131.30, 130.77, recombinant COX2 and bovine COX1 was assessed using 112.63, 110.17, 102.37, 101.95, 38.56. a COX inhibitor screening assay kit (Item No: 560131) according to the Cayman chemical manufacturer’s guide- 2‑(6‑(2‑bromobenzoyl)benzo[d][1,3]dioxol‑5‑yl)acetic lines (USA). The 50% inhibitory concentration (IC ) of acid (4d) Purified by silica gel column chromatography COX1/COX2 activity of the compounds was carried out. using n-hexane: ethyl acetate solvent system (3:2). Pow- The assay was run in duplicate with three concentrations der product, mp: 145–147 °C, Yield 87%; ESI–MS: 362.99 (50, 20, and 5 µM). A standard curve of eight concentra- (100), 364 (98), 365 (20) for C H BrO . IR (FTIR/ tions of prostaglandin, a non-specific binding sample, 16 12 5 −1 FTNIR-ATR): 1766  cm acetic acid carbonyl (C=O), and a maximum binding sample was used, as instructed −1 1 1664  cm keton carbonyl (C=O). H NMR (DMSO-d , in the kit manual, to determine the inhibition of sample 300 MHz) δ ppm: 7.41–7.73 (4H, m, Ar–H), 6.96 (1H, s, compound by applying the multiple regression generated Ar–H), 6.61 (1H, s, Ar–H), 6.07 (2H, s, O-CH -O), 3.68 best-fit line. The percentage inhibition of the three con - (2H, s, –CH –C=O), 3.42 (1H, bs, O–H). C-NMR centrations was used to calculate the IC [16]. (DMSO-d , 75  MHz) δ ppm: 195.59, 172.70, 150.48, 133.34, 132.50, 132.01, 130.31, 127.99, 119.14, 112.82, Cell culture and cytotoxicity assay 112.45, 111.03, 105.10, 102.38, 101.57. HeLa Cervical Carcinoma was cultured in RPMI-1640 media and supplemented with 10% fetal bovine serum, 2‑(6‑(3‑bromobenzoyl)benzo[d][1, 3]dioxol‑5‑yl)acetic 1% penicillin/streptomycin antibiotics and 1% l-glu - acid (4e) Purified by silica gel column chromatography tamine. Cells were grown in a humidified atmosphere using n-hexane: ethyl acetate solvent system (3:2). Pow- with 5% C O at 37 °C, and they were seeded in 2.6 × 104 der product, mp: 154–156 °C, Yield 96%; ESI–MS: 362.98 cells/well in a 96-well plate. After 48  h, the cells were (100), 364 (98), 365 (20) for C H BrO . IR (FTIR/ confluent, the media was changed, and cells were incu - 16 12 5 −1 FTNIR-ATR): 1759  cm acetic acid carbonyl (C=O), bated with four concentrations (2, 1, 0.5, and 0.2  mM) −1 1 1658  cm keton carbonyl (C=O). H NMR (DMSO- of the tested compounds for 24  h. Cell viability was d , 300 MHz) δ ppm: 12.24 (1H, s, O–H) 7.75–7.78 (2H, assessed by the CellTilter 96 Aqueous One Solution m, Ar–H), 7.64 (1H, d, J = 8.1  Hz, Ar–H), 7.48 (1H, t, Cell Proliferation (MTS) Assay according to the manu- J = 8.1 Hz, Ar–H), 7.04 (1H, s, Ar–H), 6.92 (1H, s, Ar–H), facturer’s instructions (Promega Corporation, Madison, WI). Briefly, at the end of the treatment, 20  μL of MTS Hawash et al. BMC Chemistry (2020) 14:54 Page 8 of 9 3. Smith WL, Urade Y, Jakobsson PJ (2011) Enzymes of the cyclooxygenase solution per 100 μL of media was added to each well and pathways of prostanoid biosynthesis. Chem Rev 111(10):5821–5865 incubated at 37 °C for 2 h. Absorbance was measured at 4. Engblom D, Ek M, Saha S, Ericsson-Dahlstrand A, Jakobsson PJ, Blomqvist 490 nm [37]. A (2002) Prostaglandins as inflammatory messengers across the blood– brain barrier. J Mol Med (Berl). 80(1):5–15 5. Fitzpatrick FA (2004) Cyclooxygenase enzymes: regulation and function. Supplementary information Curr Pharm Des 10(6):577–588 Supplementary information accompanies this paper at https ://doi. 6. Garavito RM, DeWitt DL (1999) The cyclooxygenase isoforms: structural org/10.1186/s1306 5-020-00706 -1. insights into the conversion of arachidonic acid to prostaglandins. Bio- chim Biophys Acta 1441(2–3):278–287 7. Kirkby NS, Lundberg MH, Harrington LS, Leadbeater PDM, Milne GL, Pot- Additional file 1: The data in the addtional file include NMR spectrum ter CMF et al (2012) Cyclooxygenase-1, not cyclooxygenase-2, is responsi- files and HRMS file of all newly synthesized compounds described in this ble for physiological production of prostacyclin in the cardiovascular article. system. Proc Natl Acad Sci 109(43):17597–17602 8. Grosser T, Yu Y, FitzGerald GA (2010) Emotion recollected in tranquility: lessons learned from the COX-2 saga. Annu Rev Med 61(1):17–33 Abbreviations 9. Méric J-B, Rottey S, Olaussen K, Soria J-C, Khayat D, Rixe O et al (2006) COX: Cyclooxygenase; FTIR: Fourier-transform infrared spectroscopy; HRMS: Cyclooxygenase-2 as a target for anticancer drug development. Crit Rev High resolution mass spectroscopy; H NMR: Proton nuclear magnetic reso- Oncol Hematol 59(1):51–64 nance; C NMR: Carbon nuclear magnetic resonance; µM: Micro molar; mM: 10. Blobaum AL, Marnett LJ (2007) Structural and functional basis of Mili molar; NSAIDs: Non-steroidal anti-inflammatory drugs; ASA: acetyl salicylic cyclooxygenase inhibition. J Med Chem 50(7):1425–1441 acid; WHO: World Health Organization; HeLa: Cervical carcinoma cells; IC : 11. Hsiao G, Shen M-Y, Chou D-S, Chang Y, Lee L-W, Lin C-H et al (2004) 50% Inhibition concentration; CC : 50% Cytotoxic concentration; NaHCO : 50 3 Mechanisms of antiplatelet and antithrombotic activity of midazolam in Sodium bicarbonate; NaOH: Sodium hydroxide; DMSO: Dimethyl sulfoxide; AA: in vitro and in vivo studies. 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Bandyopadhyay S, Pal BC, Parasuraman J, Roy S, Chakrabotry JB, Mukher- jee C, et al (2016) Inventors inhibitors of phosphatidylinositol-3-kinase (PI3) and inducers of nitric oxide (NO). United States 2016 Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

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