1. INTRODUCTION AND OBJECTIVE New thiourea derivatives were synthesized by reaction between 2-(4-ethylphenoxymethyl)benzoyl isothiocyanate obtained in situ and aromatic amines. The new synthesized compounds were characterized using IR, NMR and elemental analysis. The results of viability and cell proliferation suggest that 1a, 1b and 1c did not display cytotoxic effects on the 3T3-E1 preosteoblastes embedded in layer shaped alginate hydrogels and therefore they could be used for developing biomedical applications. 2-((4-Ethylphenoxy)methyl)-N-(3,5-dimethoxyphenylcarbamo- thioyl)benzamide (1a); yield 74%; mp 135- 136.7°C; 1 H-RMN (dmso-d6, δ ppm): 12.43 (s, 1H, NH, deuterable); 11.80 (br s, 1H, NH, deuterable); 7.61 (br d, J= 7.4 Hz, 1H, H-7); 7.58 (dd, J= 1.6 Hz, J= 7.4 Hz, 1H, H-4); 7.55 (td, J= 1.4 Hz, J= 7.4 Hz, 1H, H-5); 7.46 (td, J= 1.4 Hz, J= 7.8 Hz, 1H, H-6); 7.05 (d, J= 8.6 Hz, 2H, H-11- 13); 6.89 (d, J= 8.6 Hz, 2H, H-10-14); 6.88 (d, J= 2.2 Hz, 2H, H-18, H-22); 6.42 (t, J= 2.2 Hz, 1H, H-20); 5.26 (s, 2H, H-8); 3.75 (s, 6H, H-19’ and H-21’); 2.51 (q, J= 7.6 Hz, 2H, H-15); 1.12 (t, J= 7.6 Hz, 3H, H-15’). 13 C-RMN (dmso-d6, δ ppm): 179.28 (C-16); 170.84 (C-1); 160.97 (C-19, C-21); 156.97 (C-9); 140.09 (Cq); 136.92 (Cq); 136.48 (Cq); 133.96 (Cq); 131.72 (C-5); 129.28 (C-11, C-13); 129.15 (C-4); 129.06 (C-7); 128.43 (C-6); 115.30 (C-10, C-14); 102.85 (C-18, C-22); 98.93 (C-20); 68.23 (C-8); 56.03 (C-19’, C-21’); 27.92 (C-15); 16.37 (C-15’). FT-IR (solid in ATR, ν cm-1): 3279w; 3025w; 2958w; 2930w; 2836w; 1673w; 1574s; 1511vs; 1456m; 1360w; 1328s; 1235s; 1196m; 1153vs; 1120m; 1041m; 892w; 816m; 741w; 684m; 596w. Anal. Calcd for C 25 H 26 N 2 O 4 S (450.55): C, 66.65; H, 5.82; N, 6.22; S, 7.12%; Found: C, 66.51; H, 5.89; N, 6.31; S 7.18%. 2-((4-Ethylphenoxy)methyl)-N-(3-nitro-4-methylphenylcarbamo- thioyl)benzamide (1b); yield 81%; mp 126.3- 127.6°C 2-((4-Ethylphenoxy)methyl)-N-(3,4,5-trichlorophenylcarbamo- thioyl)benzamide (1c); yield 74 %; mp 152- 153.2°C ; The synthetic route to the target compounds, 2-((4- ethylphenoxy)methyl)-N-(arylcarbamothioyl)benzamides (1a-c), is shown in Scheme 1. Structural elucidation of these compounds was performed by IR, NMR spectroscopy and elemental analysis.. Viability and cell proliferation In order to examine the cell survival, the viability of the 3T3-E1 preosteoblastes was assessed after 48 h of exposure to the tested compounds by fluorescence microscopy (fig. 1), based on the simultaneous detection of the live (green labeled) and dead (red labeled) cells inside the 3D hydrogels. The resulting 3T3-E1/ alginate hydrogels (AlgH) were placed in DMEM culture medium supplemented with 1 mg/mL from 1a-c. The viability of 3T3-E1 cells within 3T3-E1/AlgH-1a, 3T3-E1/AlgH-1b, 3T3-E1/AlgH-1c and 3T3-E1/AlgH bioconstructs was evaluated by fluorescence microscopy using Live/Dead Kit. The viability of the 3T3-E1 cells within the alginate hydrogels was quantitatively assessed by MTT spectrophotometric assay at 48 h post exposure to 1a, 1b and 1c compounds. 3T3-E1/AlgH was subjected to the same procedure at 72 h post seeding. The result was a sensitive assay with a colorimetric signal proportional to the viable cell number. SYNTHESIS, CHARACTERIZATION AND CYTOTOXIC EVALUATION OF 2-(4-ETHYLPHENOXY)METHYL)-N-(ARYLCARBAMOTHIOYL)BENZAMIDES Carmen Limban 1, Bianca Gălăţeanu 2, Marieta Costache 2, Alexandru Vasile Missir 1, Laurenţiu Moruşciag 1, Cornel Chiriţă 1, Miron Teodor Căproiu 3, Diana Camelia Nuţă 1 1 Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania 2 Biochemistry and Molecular Biology Department, University of Bucharest,Bucharest, Romania 3 The Organic Chemistry Center of Romanian Academy "Costin C.D. Nenitescu” Bucharest,Romania MATERIAL AND METHOD RESULTS CONCLUSIONS The thiourea skeleton can be effectively used to prepare a large number of new compounds with biological activities such as antiviral, anticancer, anti- inflammatory, antimicrobial, anticonvulsant, and anthelmintic activities. In order to evaluate for their viability and cell proliferation, new thiourea derivatives were designed and prepared. Scheme 1. Synthetic pathway for the new N- phenylcarbamothioylbenzamides (1a-c) Fig. 1. Fluorescence microscopy micrographs of 3T3-E1 preosteoblastes embedded in layer shaped alginate hydrogels, in plain culture medium (ctrl) and exposed to 1a, 1b and 1c, stained with calcein AM (green fluorescence) and ethidium bromide (red fluorescence). Fig. 2. The MTT spectrophotometric quantification of 3T3-E1 preosteoblastes viability after 48 h exposure to 1a, 1b and 1c, as compared to the unexposed reference (ctrl).[***p<0.001 3T3-E1/AlgH-1a bioconstruct vs ctrl, 3T3- E1/AlgH-1b bioconstruct vs ctrl, 3T3-E1/AlgH-1c bioconstruct vs ctrl].