{Synthesis of novel phthalimido oxime esters and evaluation of their cytotoxicity} J. Serb. Chem. Soc. 85 (1) 1–8 (2020) UDC 547.584+547.574.2–326+542.913:576+ JSCS–5278 615.9:57–188 Original scientific paper 1 Synthesis of novel phthalimido oxime pseudoesters and evaluation of their cytotoxicity ASMA MEHREZ1, IBTISSEM CHAKROUN2, DALILA MTAT1, HÉDI BEN MANSOUR3 and RIDHA TOUATI1* 1Laboratory of Asymmetric Organic Synthesis and Homogeneous Catalysis (UR11ES56), Faculty of Sciences of Monastir, Avenue of the Environment, 5019 Monastir, Tunisia, 2Labor- atory for Analysis, Treatment and Recovery of Environmental Pollutants and Products, Fac- ulty of Pharmacy, Avicenne Street, 5000 Monastir, Tunisia and 3Research Unit Analyzes and Processes Applied to the Environment (UR17ES32), Higher Institute of Applied Sciences and Technology of Mahdia, 5111 Mahdia, Tunisia (Received 2 March 2018, revised 16 July 2019, accepted 1 August 2019) Abstract: A series of novel optically pure oxime pseudoesters derivatives were synthesized by the reaction of substitute keto oximes with various N-substi- tuted α-amino acids chlorides in the presence of triethylamine and dichloro- methane at 0 °C, and their structures were characterized by IR and 1D-NMR methods. The synthesized compounds were tested for their ability to inhibit the proliferation of human colon cancer cells and human epithelial cells. Some of them were revealed to have a significant cytotoxic effect. Keywords: oxime esters; α-amino acids; stereoselective; cytotoxic; biological activity; enantiomer. INTRODUCTION Cancer remains a serious human health problem despite considerable pro- gress in the understanding of its biology and pharmacology. The main problem is that cancer is not one disease, but a group of diseases affecting different organs and systems of the body. Cancer develops due to abnormal and uncontrolled cell division, frequently at a rate greater than that of most normal body cells.1 For some types of disseminated cancers, chemotherapy is the only effective therapy because it distributes anticancer drugs through the circulatory system. Oxime pseudoesters are a small, but important, class of biologically useful com- pounds for the synthesis of fragrances,2 crop protection, and therapeutic studies.3 They are useful building blocks in peptide synthesis.4 Oxime pseudoesters are * Corresponding author. E-mail: ridhato@yahoo.fr https://doi.org/10.2298/JSC180302082M ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. 2 MEHREZ et al. selective covalent inhibitors of serine hydrolase retinoblastoma-binding protein 9 (RBBP9) and cleave DNA under photolytic conditions.5,6 They also possess fun- gicidal,7 herbicidal,8 insecticidal and antitumor activity.9 Oxime pseudoesters of dihydrocoumaric acid have been synthesized and they were reported to have anti- bacterial activity.10 Aromatic benzophenone oxime esters and dibenzosuberone oxime esters are pharmacologically important.11 Vanillin derived piperidin-4-one oxime esters have been tested for their antioxidant and antimicrobial potential.12 The oxime pseudoesters derived from nafimidone have been tested as potential anticonvulsant compounds.13 Several methods have been developed for the preparation of oxime pseudo- esters derivatives.3 The most common method is the condensation of acid chlo- rides with oximes under basic conditions or the use of acid anhydrides in pre- sence of strong acids.2,6,10,12,13 Oxime pseudoesters can be prepared using α,β-unsaturated aldehydes and oximes employing a N-heterocyclic carbene as a redox esterification catalyst,14 or by treatment of alkyl- or aryl-substituted oximes with aliphatic or aromatic acids in the presence of the N-[3-(dimethylamino)propyl]-N′-ethylcarbodiimide hydrochloride (EDCI) reagent.15 A large number of studies on their synthesis and biological activities have been reported during the last thirty years.16 However, no attention was paid to the stereoselective synthesis of chiral oxime pseudoesters derivatives. Hence, in con- tinuation of our research aimed at the preparation of natural and non-natural com- pounds of biomedical importance,17 and in connection with ongoing investig- ations on the reactivity of natural amino acids,18 herein, an efficient and easy methodology is reported for the synthesis of a series of new optically pure oxime pseudoesters 5a–k starting from the commercially available acetophenone deri- vatives 1a–f and natural amino acids 3a–d, which are of considerable interest as chiral pool agents since they are easily accessible and inexpensive enantiomeric- ally pure compounds. EXPERIMENTAL Reagent grade chemicals and solvents were purchased from commercial supplier and used without purification. TLC was performed on silica gel F254 plates (Merck). Silica gel (100–200 mesh) was used for column chromatographic purification. Melting points are uncor- rected and were measured in open capillary tubes, using a Rolex melting point apparatus. IR spectra were recorded as KBr pellets on Perkin Elmer RX spectrometer. 1H-NMR and 13C- -NMR spectral data were recorded on Advance Bruker 300 spectrometer (300 MHz) with CDCl3 as solvent and TMS as internal standard. J values are in Hz. Mass spectra were obtained by Agilent 5973- network mass selective detector (EI). General procedure for the preparation of oximes 2a–f Acidic hydroxylamine (NH2OH·HCl, 0.1 mol) was added dropwise to a stirred solution of substituted acetophenone (0.02 mol) in 95 % EtOH (150 mL) and pyridine (8 mL, 0.1 mol) ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. SYNTHESIS OF OXIME PSEUDOESTERS 3 at room temperature. The resulting mixture was refluxed for 0.5–2 h (until the starting mat- erial was completely consumed as indicated by TLC), and cooled in cold water for1 h. The precipitate was collected by suction, washed with warm water (3×50 mL) and dried in a vacuum oven. The crude compound was recrystallized in ethanol to give a white solid. Synthesis of N-phthaloyl-L-amino acids 4a–d A solution of the required amino acid (1 equiv.) in toluene was added to a solution of phthalic anhydride (1 equiv.) and triethylamine (1.2 equiv.). The reaction mixture was then refluxed on a water bath for about 4–5 h. After completion of the reaction, the resulting solut- ion was separated. The organic phase was washed with water until neutral, dried over MgSO4 and filtered. The filtrate was evaporated and the residue purified by column chromatography on silica gel to give the required compounds (4a–d). Synthesis of oxime pseudoesters 5a–k A mixture of an N-phthaloyl-L-amino acid 4a–d (1 equiv.) and thionyl chloride (2 mL, slow addition) were mixed together and the contents were heated at 55 °C for 4 h. The react- ion mixture was cooled to room temperature and kept in an ice bath. Then, a solution of oxime 2 (1 equiv.), triethylamine (1.2 equiv.) in dichloromethane (CH2Cl2, 30 mL) was added sub- sequently to the reaction mixture and the contents were stirred at room temperature for 2 h. On completion of the reaction, checked by thin-layer chromatography (TLC) analysis, the sol- vent was removed under reduced pressure and extracted with dichloromethane (3×30 mL). The organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The thus obtained residue was purified by silica gel chromatography (hexane/ethyl acetate) to afford the desired oxime pseudoesters derivatives (5a–k). Cell lines and culture medium The human colon carcinoma cells (Caco-2; ECACC 86010202) and the epidermoid carcinoma epithelial cells (Hep-2; ATCC CCL-23) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10 % of fetal bovine serum, 1 % non-essential amino acids and 1 % penicillin/streptomycin (Invitrogen). At 85–90 % confluence, the cells were harvested using a 0.25 % trypsin/EDTA solution and sub-cultured onto 96-well plates according to the experimental requirements. Cytotoxicity screening assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay described earlier19 was used to screen the cytotoxic activity of the isolated compound. Briefly, the Caco-2 and Hep-2 cell lines (1×105 cells/well) were grown overnight on 96-well flat bottom cell culture plates, incubated 24 h. When a partial monolayer had formed, the supernatant was flicked off, the monolayer washed once with medium and 100 μL of different concentrations (10, 5, 2.5 and 1.25 mg mL-1) of pure compound were added to the cells.in the microtitre plates. After 24 h, the cells were washed and treated with 0.01 mL MTT reagent (Invtrogen) prepared in 5.0 mg mL-1 phosphate buffered saline (PBS) per well. The plates were incubated at 37 °C in a 5 % CO2 atmosphere for 4 h, and 0.1 mL dimethyl sulfoxide (DMSO) was added. After overnight incubation at 37 °C, the absorbance was measured at 550 nm using an ELISA reader (Thermo Scientific Multiskan FC) and was compared with the con- trol cultures without compound. The results were generated from 3 independent experiments and each experiment was performed in triplicate. The percentage growth inhibition was cal- culated using following formula: Cell inhibition, % = 100–100{(At–Ab)/(Ac–Ab)} ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. 4 MEHREZ et al. where, At = absorbance value of the test compound, Ab = absorbance value of the blank and Ac = absorbance value of the control. Stock solutions (5 mg/mL) of pure compounds were prepared in dimethyl sulfoxide (DMSO) and the final concentration of this solvent was kept constant at 0.25 %. Serial dilu- tions with culture media were prepared just prior to the test. Statistical analysis The results are expressed as mean ± SEM. The data were statistically analyzed by one- way analysis of variance (ANOVA) to determine differences among the groups and the Tukey test as a post-hoc. All the statistical analysis was conducted using Statistical Package for Social Science (SPSS for Windows; v. 19.0, USA) and differences were considered statistic- ally significant at p < 0.05. Analytical and spectral data of the synthesized compounds are given in the Supplement- ary material to this paper. RESULTS AND DISCUSSION The substituted oximes 2a–f were synthesized according to the literature procedure6b,20 shown in Scheme 1. The condensation of acetophenone deri- vatives 1a–f with hydroxylamine hydrochloride and pyridine gave white shiny coloured oximes 2a–f in 90 % yield. O O O NO O R1 O OH H 2 a-f Toluene, Et3N, reflux 1) SOCl2, 55°C, 4h CH3 O R CH3 N OH R R= H, CH3, OCH3, F, Cl, Br 1 a-f NH2OH.HCl, 100°C 4 a-d 5 a-k N OO O O NR1 H CH3 R 2) oxime 2, Et3N, 0°C, CH2Cl2 95% EtOH, pyridine R1 O OH NH2 H 3 a-d 5a: R= CH3, R1= CH3 5e: R= Br, R1= CH3 5i: R= H, R1= CH2C6H3(OH)2 5b: R= OCH3, R1= CH3 5f: R= CH3 , R1= CH2CH(CH3)2 5j: R= OCH3, R1= CH2C6H3(OH)2 5c: R= F, R1= CH3 5g: R= Cl, R1= CH2CH(CH3)2 5k: R= Cl, R1= CH2C6H3(OH)2 5d: R= Cl, R1= CH3 5h: R= CH3, R1= CH(CH3)CH2CH3 Scheme 1. Synthesis of oxime pseudoesters 5a–k. The α-N-phthalimido amino acids (compounds 4a–d) were synthesized according to a method described in the literature18a by allowing phthalic anhyd- ride to react with a number of commercially available amino acids in refluxing apolar solvents, such as toluene, in the presence of triethylamine and separation of the formed water (Scheme 1). ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. SYNTHESIS OF OXIME PSEUDOESTERS 5 Treatment of 4a–d with thionyl chloride followed by treatment with keto oxime in anhydrous dichloromethane in the presence of Et3N at 0 °C to room temperature provided the corresponding N-substituted phthaloyl derivatives 5a–k with an average yield of 76 % in two steps and after purification. Their structures were established by IR, 1H-NMR, 13C-NMR and mass spectrometry. The antiproliferative potential of the synthesized compounds 5a–d, 5f and 5h–j was determined in vitro against two cancer cell lines, i.e., Hep-2 and Caco-2. The cytotoxicity values were obtained as inhibition of different concentrations and the results are summarized in Tables I and II. TABLE I. Cytotoxic activity of some derivatives against Caco-2 cells; the 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay described earlier19 was used to screen the cytotoxic activity of the isolated compound Compound Concentration, mg mL-1 10 5 2.5 1.25 5a < 20 % < 20 % < 20 % < 20 % 5b 59.67±1.91 40.98±1.22 22.56±2.03 25.17±1.15 5c 52.94±1.22 43.01±2.16 41.37±0.9 35.01±0.85 5d 56.19±0.78 44.41±1.08 37.96±1.2 29.06±0.63 5f < 20 % < 20 % < 20 % < 20 % 5h < 20 % < 20 % < 20 % < 20 % 5i 41.03±3.54 40.15±1.56 28.92±1.94 14.02±0.76 5j < 20 % < 20 % < 20 % < 20 % TABLE II. Cytotoxic activity of some derivatives against Hep-2 cells Compound Concentration, mg mL-1 10 5 2.5 1.25 5a < 20 % < 20 % < 20 % < 20 % 5b 48.22±1.12 20.15±0.86 11.02±1.61 7.89±0.74 5c 57.85±2.13 43.21±1.92 21.11±0.83 27.02±2.35 5d 61.02±1.69 56.12±2.01 31.12±0.77 10.25±0.92 5f < 20 % < 20 % < 20 % < 20 % 5h < 20 % < 20 % < 20 % < 20 % 5i 53.91±1.6 50.77±2.51 17.01±1.38 9.49±1.02 5j < 20 % < 20 % < 20 % < 20 % The results demonstrated that only four compounds, 5b–d and 5i, have moderate potency of around 40 % inhibition at 10 mg mL–1 against Hep-2 and Caco-2, while the other compounds, 5a, 5f, 5h and 5j were inactive against the two cancer cell lines (inhibition effect < 20 %). It was observed that when the methoxy group was attached at the para position of the phenyl ring (compound 5b), the activity reduced to 48.22 % against the Hep-2 cell line. Replacing the substituent at the para position by an electron withdrawing group caused a dec- rease in the anticancer activity as compared to compound 5b against the Caco-2 ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. 6 MEHREZ et al. cell line. This could be justified by the fact that compounds bearing electron withdrawing groups, such as fluoro (5c) and chloro (5d) substituents at the para position of the phenyl ring exhibited activity at inhibitory ratios values 52.94 and 56.19 %, respectively, against the Caco-2 cell line and 57.85 and 61.02 %, res- pectively, against the Hep-2 cell line. Compound 5i also showed activity against Hep-2 at 53.91 and 41.03 % against Caco-2 cell lines. It could be concluded that the cytotoxicities of the resulting oxime pseudoesters derivatives are significantly correlated with the nature of the substituent group. CONCLUSIONS In summary, a new series of optically pure phthalimido oxime pseudoesters derivatives were prepared and their cytotoxic activities against two human cancer cells lines, Caco-2 and Hep-2, evaluated. Some derivatives exhibited strong cyto- toxic activity and therefore, further structural modifications and in vivo anti- -tumor activity studies are to be undertaken. The present findings could provide new evidence showing the relationship between chemical structure and biological activity and may be useful for the design of novel chemotherapeutic drugs. SUPPLEMENTARY MATERIAL Analytical and spectral data of the synthesized compounds are available electronically at the pages of journal website: http://www.shd-pub.org.rs/index.php/JSCS, or from the corres- ponding author on request. И З В О Д СИНТЕЗА НОВИХ ПСЕУДОЕСТАРА ФТАЛИМИДО ОКСИМА И ПРОЦЕНА ЊИХОВЕ ЦИТОТОКСИЧНОСТИ ASMA MEHREZ1, IBTISSEM CHAKROUN2, DALILA MTAT1, HÉDI BEN MANSOUR3 и RIDHA TOUATI1 1 Laboratory of Asymmetric Organic Synthesis and Homogeneous Catalysis (UR11ES56), Faculty of Sciences of Monastir, Avenue of the Environment, 5019 Monastir, Tunisia, 2 Laboratory for analysis, treatment and recovery of environmental pollutants and products, Faculty of Pharmacy, Avicenne Street, 5000 Monastir, Tunisia и 3 Research Unit Analyses and Processes Applied to the Environment (UR17ES32), Higher Institute of Applied Sciences and Technology of Mahdia, 5111 Mahdia, Tunisia Синтетисана је серија нових, оптички чистих деривата псеудоестара оксима, реак- цијом супституисаних кето-оксима са различитим хлоридима N-супституисаних α-амино киселина, у присуству триетил-амина, у дихлорметану на 0 °C. Њихове структуре су утврђене IR и 1D-NMR спектроскопским методама. 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