Synthesis and bioacivity screening of some novel N-(Adamantan-1-yl)-1-Aryl-Methanimines

Synthesis and bioactivity testing of some novel N-(adamantan-1-yl)-1-arylmethanimines derivatives. Methods: The novel of N-(adamantan-1-yl)-1-aryl-methanimines were synthesized by the condensation of 1-aminoadamantane with aromatic aldehydes in absolute ethanol under the catalyzation of acetic acid. Synthesized compounds were bioactivity tested as inhibition of acetylcholinesterase, antimicrobial, anticancer in vitro. Results: A new series of N- (adamantan-1-yl)-1- aryl-methanimines was designed, synthesized and evaluated for their inhibition of acetylcholinesterase, antimicrobial, anticancer. Representative two compounds did not show the inhibition of acetylcholinesterase and antimicrobials (S. aureus, B. subtilis, L. fermentum, S. enterica, E. coli, P. aureus). However, synthesize compounds were shown to be promising in at least three tumor cell lines with IC50 from 3.6 - 82.2 µg/mL. Otherwise, five per ten compounds possessed the activity against C. albican. Conclusions: At the present work, it had synthesized ten novel N-(adamantan-1-yl)-1-aryl-methanimine derivatives, between them, some compounds were identified against C. albican and with significant cytotoxic activity in testing tumor cell lines.

Journal of military pharmaco-medicine n02-2019 SYNTHESIS AND BIOACIVITY SCREENING OF SOME NOVEL N-(ADAMANTAN-1-YL)-1-ARYL-METHANIMINES Pham Van Hien1; Vu Binh Duong1; Cao Dang Nam1; Nguyen Van Tu1 Phan Thi Phuong Dung2; Nguyen Quynh Hoa3; Phan Dinh Chau4 SUMMARY Objectives: Synthesis and bioactivity testing of some novel N-(adamantan-1-yl)-1-arylmethanimines derivatives Methods: The novel of N-(adamantan-1-yl)-1-aryl-methanimines were synthesized by the condensation of 1-aminoadamantane with aromatic aldehydes in absolute ethanol under the catalyzation of acetic acid Synthesized compounds were bioactivity tested as inhibition of acetylcholinesterase, antimicrobial, anticancer in vitro Results: A new series of N(adamantan-1-yl)-1- aryl-methanimines was designed, synthesized and evaluated for their inhibition of acetylcholinesterase, antimicrobial, anticancer Representative two compounds did not show the inhibition of acetylcholinesterase and antimicrobials (S aureus, B subtilis, L fermentum, S enterica, E coli, P aureus) However, synthesize compounds were shown to be promising in at least three tumor cell lines with IC50 from 3.6 - 82.2 µg/mL Otherwise, five per ten compounds possessed the activity against C albican Conclusions: At the present work, it had synthesized ten novel N-(adamantan-1-yl)-1-aryl-methanimine derivatives, between them, some compounds were identified against C albican and with significant cytotoxic activity in testing tumor cell lines * Keywords: N-(adamantan-1-yl)-1-aryl-methanimines; Bioactivity INTRODUCTION Finding for new biological agents such as antimicrobial, inhibition of acetylcholinesterase, anticancer is becoming the major interest in the huge of academic and industry research laboratories all over the world [1] This is due to discover new biological agents with more specificity and less toxicity [2], in addition to the various types of new disease-caused agents that are recently discovered which is becoming a great challenge for the scientists [3] The adamantane nucleus was found to be important constituent in many ingredients [4] The incorporation of an adamantyl moiety into a pharmacologically-activemolecule resulted in improving the therapeutic profile of the parent drugs [5, 6, 7] Since the discovery of amantadine (I) [8] as a first antiviral therapy for systemic use, several hundreds of 1-adamantyl derivatives were synthesized and tested for various biological activities including anticancer, Vietnam Military Medical University Hanoi University of Pharmacy National Centralized Drug Procurement Center Hanoi University of Science and Technology Corresponding author: Pham Van Hien (phamvanhien181288@gmail.com) Date received: 20/12/2018 Date accepted: 16/01/2019 88 Journal of military pharmaco-medicine n02-2019 antiviral (HIV, influenza, Hespes simple virus), antimicrobial and so on [9] However, most of them, the synthesized routes focused to get oxadiazoles [10], isoxazoles [11], thiadiazoles [10], urea/thiourea derivatives and other components To date, there are no findings of 1adamantylimine via condensation of 1-aminoadamantane with aromatic aldehydes as well as their bioactivity The present study aimed to synthesize and biological test the novel N-(adamantan-1yl)-1-aryl-methanimines derivatives MATERIALS AND METHODS General All reagents were commercially available and used without further purification Reactions were magnetically stirred with reflux heating and monitored by thin-layer chromatography (TLC) on Merck silica gel 60F-254 by fluorescence and Dragendorf reagent 1H-NMR and 13C-NMR spectra were obtained Brucker AV-500 system at 500 MHz Chemical shilfs were given in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard Multiplicities were abbreviated as follows: single (s), doublet (d), multiplet (m), and broad signal (br s) High resolution mass spectral (HR-MS) data were acquired on a SCIEX X500 QTOF system Melting points were measured on Stuart, SMP-10 micromelting and were really corrected * Antimicrobial screening: The in vitro antimicrobial testing was performed at Laboratory of Applied Biochemistry, Institute of Chemistry, Vietnamese Academy of Science and Technology The following standard bacteria were obtained: Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 13709), Lactobacillus fermentum (N4): Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 15442), Salmonella enterica, Candida albicans (ATCC 10231) The media used were MHB (Mueller-Hinton Broth), MHA (MuellerHinton Agar); TSB (Tryptic Soy Broth); TSA (Tryptic Soy Agar) for antibacterial testing; SDB (Sabourand - 2% dextrose broth) and SA (Sabourand - 4% dextrose agar) for antifungal evaluation IC50 (the half maximal inhibitory concentration) of compounds was determined by turdibity measurement and calculated on RawData software * Acetylcholinesterase activity inhibition: The activity of AChE, a marker for cholinergic neurotransmission, was determined as triplicate, in standard spectrophotometric bioassay in 96 well microplate Doneperil was used as standard inhibitor, and control test was performed without the presence of acetylcholinesterase inhibitors In brief, ten microliters of the test solution were added in the test wells and twenty-five microliters of AChE soulution (0.22 U/ml in PBS) were added in the test and control wells Filled up to 50 µL by adding of 0,1M PBS buffer pH Then, acetylthiocholine chloride 15 mM (125 µL) and mM Ellman’s reagent (5,5'-dithiobis 2-nitrobenzoic acid, DTNB) were added in each of well Absorbance was measured immediately and then after 30 of reaction at 410 nm on Microplate reader BIOTEK (USA) IC50 of the testing 89 Journal of military pharmaco-medicine n02-2019 compounds was determined by the linearity method * Anti-cancer assay: KB (CCL-17TM), Hep G2 (HB-8065TM), LU-1 (HTB-57TM), MCF-7 (HTB-22TM) cells were seeded in 96-well plates at a density of x 104 cells/well The cell lines were exposed to testing solution in a concentration range of - 128 μg/mL for 72h Then the cells were washed with 1X PBS and incubated in 10 μL of mg/mL MTT at 37°C for hours Then, the media in every well was removed, the dark blue crystals of formazan (MTT metabolites) were dissolved with 100 μL DMSO and incubate at 37°C for 30 mins Absorbance was measured at 540 nm using a microplate reader spectrophotometer At last, the IC50 values (concentrations required to inhibit 50% of cell growth) were calculated using Rawdata Software RESULTS AND DISCUSSIONS N-(adamantan-1-yl)-1-(4-nitrophenyl) methanimine (1): Yield 77.5%, m.p 116.3 - 117.1 1H-NMR (500 MHz, DMSO-d6, δ ppm): 8.46 (1H, s, N=CH-); 8.29 (2H, t, 90 Chemistry A mixture of the appropriate 1-aminoadamantane (0.004 moL) and aromatic aldehydes (0.002 moL) in absolute ethanol or benzene (10 mL) was heated under reflux for hours The reactions were monitored by TLC on Merck silica gel 60 F-254 by fluorescence and Dragendorf reagent (the mobile phase as n-hexane/acetone = 4:1, v/v) At the reaction finishing, the solvent was evaporated under reduced pressure The resulted residue was washed by acetone (15 mL x 2) and filtered off and removed the solid The filtrate was evaporated under vacuum and the residue was recystallized from ethanol and washed by water Finally, the crystals were dried at 500C for hours to get the pure compounds N-(adamantane-1-yl)-1aryl-methanimine (1-12) in 62.5 - 82.0% yields J1 = Hz, J2 = Hz, H3 Phenyl & H5 Phenyl); 8.02 (2H, t, J1 = Hz, J2 = Hz, H2 Phenyl & H6 Phenyl); 2.13 (3H, m, CH-ad); 1.77 (6H, m, CH2-ad); 1.74 - 1.66 (6H, m, CH2-ad) 13C-NMR (500 MHz, DMSO-d6, δ ppm): 153.7 (1C, CH=N); 148.3 - 123.8 Journal of military pharmaco-medicine n02-2019 (6C, Cphenyl); 58,0 (1C, C-N); 42.5 - 28.9 (15C, CAd) HR-MS [m/z]: 285.1578 [M+H+] N-(adamantan-1-yl)-1-(3-nitrophenyl) methanimine (2): Yield 76% M.p 117.7 118.3°C 1H-NMR (500 MHz, DMSO-d6, δ ppm): 8.57 (1H, s, N=CH-), 8.47 (H, s, H2’), 8.27 (H, dd, J1 = Hz, J2 = Hz, H6 Phenyl); 8.18 (H, d, J = 7.5 Hz, H4 Phenyl); 7.3 (H, t, J1 = Hz, J2 = 16 Hz, H5 Phenyl); 2.13 (3H, m, CH-ad); 1.77 (6H, m, CH2-ad); 1.73 - 1.66 (6H, m, CH2-ad) HR-MS [m/z]: 285.1574 [M+H+] N-(adamantan-1-yl)-1-(2-hydroxy-5methylphenyl) methanimine (3): Yield 73% M.p 123.3 - 124.2°C 1H-NMR (500MHz, DMSO-d6, δ ppm): 14.0 (1H, s, OH); 8.47 (1H, s, N=CH-); 7.25 (1H, d, J = Hz, H6 Phenyl); 8.02 (1H, dd, J1 = Hz, J2 = 7.5 Hz, H4 Phenyl); 6.73 (1H, d, J1 = Hz, H3 Phenyl); 2.23 (3H, s, CH3); 2.13 (3H, m, CH-ad); 1.78 (6H, m, CH2-ad); 1.72 1.65 (6H, m, CH2-ad) 13C-NMR (500 MHz, DMSO-d6, δ ppm): 160.5 (1C, C-OH); 158.9 (1C, CH=N); 132.6, 131.7, 126.5, 118.4 (Cphenyl); 56.6 (1C, C-N); 42.4 (3C, CH2-Ad); 35.7 (3C, CH2-Ad); 28.8 (3C, CHAd), 19.9 (C, CH3) HR-MS [m/z]: 270.2079 [M+H+] N-(adamantan-1-yl)-1-(4-ethoxyphenyl) methanimine (4): Yield 78.5% m.p 125.3 - 126.6°C 1H-NMR (500 MHz, DMSO-d6, δ ppm): 8.23 (1H, s, N=CH-); 7.66 (2H, d, J = 8.5 Hz, H2 Phenyl, H6 Phenyl); 6.95 (2H, d, J = 8.5 Hz, H3 Phenyl, H5 Phenyl); 4.08 4.04 (2H, m, CH2-O); 2.11 (3H, m, CH-ad); 1.73 - 1.72 (6H, m, CH2-ad); 1.69 - 1.64 (6H, m, CH2-ad); 1.35 - 1.32 (3H, t, J1 = 7,0 Hz, J2 = 13,5 Hz) 13C-NMR (500 MHz, DMSO-d6, δ ppm): 160.1 (1C, C-O-); 153.8 (1C, CH=N); 129.6, 129.1, 114.3 (Cphenyl); 63.1 (1C, C-N); 56.6 (1C, CH2-O); 42.9 (3C, CH2-Ad); 36.0 (3C, CH2-Ad); 28.9 (3C, CH-Ad), 14.5 (1C, CH3) HR-MS [m/z]: 284.1988 [M+H+] N-(adamantan-1-yl)-1-(2,5-dimethylphenyl) methanimine (5): Yield 87% M.p 100.6 101.6°C 1H-NMR (500 MHz, DMSO-d6, δ ppm):8.51 (1H, s, N=CH-); 7.66 (2H, d, J = 8.5 Hz, H5 Phenyl); 7.12 - 7.08 (2H, m, H3 Phenyl , H4 Phenyl); 2.41 (3H, s, O-CH3); 2.28 (3H, s, m-CH3); 2.12 (3H, m, CH-ad); 1.75 - 1.73 (6H, m, CH2-ad); 1.70 - 1.65 (6H, m, CH2-ad) 13C-NMR (500 MHz, DMSO-d6, δ ppm): 153.5 (1C, CH=N); 135.2, 134.9, 134.7, 131.1, 130.7, 127.4 (CPhenyl); 57,9 (1C, C-N); 43.3 (3C, CH2Ad); 36.6 (3C, CH2-Ad); 29.5 (3C, CH-Ad), 21.0 (1C, m-CH3); 18.9 (1C, o-CH3) MS [m/z]: 268.2038 [M+H+] N-(adamantan-1-yl)-1-(3 nitro-4-chloro phenyl) methanimine (6): Yield 78.8% m.p 123.7 - 124.4°C 1H-NMR (500 MHz, DMSO-d6, δ ppm): 8.40 (1H, s, HC=N); 8.38 (1H, d, J = 1,5 Hz, H5 Phenyl); 8.06 8.04 (1H, dd, J1 = 2.0 Hz, J2 = 8.5 Hz, H2 Phenyl); 7.85 - 7.83 (1H, d, J = 8.5 Hz, H3 Phenyl); 2.12 (3H, m, CH-ad); 1.75 (6H, m, CH2-ad); 1.73 - 1.64 (6H, m, CH2-ad) 13CNMR (500 MHz, DMSO-d6, δ ppm): 152.5 (1C, CH=N); 147.7 (1C, C-NO2); 137.2, 132.3, 132.0, 126.3, 123.9 (Cphenyl); 57.8 (1C, C-N); 42.5 (3C, CH2-Ad); 35.9 (3C, CH2-Ad); 28.8 (3C, CH-Ad) MS [m/z]: 319.1198 [M+H+] N-(adamantan-1-yl)-1-(2-hydroxyphenyl) methanimine (7): Yield 82% M.p 90.2 91.8°C 1H-NMR (500 MHz, DMSO-d6, δ ppm):14.39 (1H, s, HO-); 8.54 (1H, s, HC=N); 7.47 - 7.45 (1H, dd, J1 = 2.0 Hz, J2 = 8.0 Hz, H5 Phenyl); 7.31 - 7.28 (1H, m, 91 Journal of military pharmaco-medicine n02-2019 H4 Phenyl); 6.87 - 6.81 (2H, m, H3 Phenyl, H5Phenyl); 2.14 (3H, m, CH-ad); 1.80 (6H, m, CH2-ad); 1.73 - 1.66 (6H, m, CH2-ad) 13 C-NMR (500 MHz, DMSO-d6, δ ppm): 161.5 (1C, C-OH); 160.7 (1C, CH=N); 132.0, 131.8, 118.6, 117.9, 116.6 (CPhenyl); 56.6 (1C, C-N); 42.4 (3C, CH2-Ad); 35.7 (3C, CH2-Ad); 28.8 (3C, CH-Ad) MS [m/z]: 256.1908 [M+H+] N-(adam ant an - 1- yl)- 1-(3-nitro-4bromophenyl) methanimine (8): Yield 67.5% m.p 126.7 - 128.0°C 1H-NMR (500 MHz, DMSO-d6, δ ppm):8.39 (1H, s, HC=N); 8.33 (1H, d, J = 2,0 Hz, H2 Phenyl); 8.00 - 7.98 (1H, d, J = 8.0 Hz, H6 Phenyl); 7.96 - 7.94 (1H, dd, J1 = 1.5 Hz, J2 = 8.0 Hz, H5 Phenyl); 2.12 (3H, m, CH-ad); 1.75 (6H, m, CH2-ad); 1.73 - 1.64 (6H, m, CH2-ad) 13 C-NMR (500 MHz, DMSO-d6, δ ppm): 152.6 (1C, CH=N); 149.7, 137.7, 135.1, 132.2, 123.7, 114.6 (CPhenyl); 57.8 (1C, C-N); 42.5 (3C, CH2-Ad); 35.9 (3C, CH2-Ad); 28.8 (3C, CH-Ad) HR-MS [m/z]: 363.0689 [M+H+] N-(adamantan-1-yl)-1-(4-chlorophenyl) methanimine (9): Yield 62.7% m.p 128.0 - 129.1°C 1H-NMR (500 MHz, DMSO-d6, δ ppm): 8.31 (1H, s, HC=N); 7.78 - 7.76 (2H, d, J = 7.5 Hz, H2 Phenyl, H6 Phenyl); 7.49 - 7.48 (2H, d, J = 8.5 Hz, H3 Phenyl, H5 Phenyl); 2.11 (3H, m, CH-ad); 1.74 - 1.72 (6H, m, CH2-ad); 1.70 - 1.64 (6H, m, CH2-ad) 13 C-NMR (500 MHz, DMSO-d6, δ ppm): 154.2 (1C, CH=N); 136.3, 135.2, 129.8, 129.1 (Cphenyl); 57.74 (1C, C-N); 43.20 (3C, CH2-Ad); 36.50 (3C, CH2-Ad); 29.43 (3C, CH-Ad) HR-MS [m/z]: 274.1345 [M+H+] N-(adamantan-1-yl)-1-(4-methoxyphenyl) methanimine (10): Yield 82.5% m.p 92 106.3 - 107.7°C 1H-NMR (500 MHz, DMSO-d6, δ ppm): 8.24 (1H, s, HC=N); 7.69 - 7.67 (2H, d, J = 8,5 Hz, H2 Phenyl, H6 Phenyl); 6.98 - 6.96 (2H, d, J = 8.0 Hz, H3 Phenyl, H5 Phenyl); 3.78 (3H, s, CH3); 2.11 (3H, m, CH-ad); 1.73 (6H, m, CH2-ad); 1.70 - 1.64 (6H, m, CH2-ad) 13C-NMR (500 MHz, DMSO-d6, δ ppm):, 153.8 (1C, CH=N); 160.8, 129.8, 129.1, 113.8 (Cphenyl); 56.6 (1C, CH3); 55.2 (1C, C-N); 42.8 (3C, CH2-Ad); 36.0 (3C, CH2-Ad); 28.9 (3C, CH-Ad) HR-MS [m/z]: 271.1871 [M+H+] Biologycal testing results * Acetylcholinesterase activity inhibition: Synthesized compounds were assayed by Ellman’s method so as to investigate their inhibitory activity against AChE In all of twelve synthesized compounds, two derivatives were probed AChE Tested compounds were found to be inactive on AChE with IC50 more than 128 µg/mL * Antimicrobial screening: In the present work, two compounds were probed for their antimicrobial activity against the gram positive bacteria S aureus, B substilis, L fermentum; the Gram negative bacteria S enterica, E coli, P aeruginosa; and the pathogenic fungi C albicans The results showed that all synthesized derivatives were devoid of any nhibitory effect against the tested gram negative and gram positive bacteria So, synthesized others were not tested further for antibacterials However, compounds 1, 2, 3, and showed variable activity against C albicans with IC50 as 78.3, 78.6, 14.59, 74.36, 16.97 µg/mL, respectively Journal of military pharmaco-medicine n02-2019 * Anti-cancer activity: Six synthesized adamantane derivatives were assessed in tumor cell lines KB (CCL -17TM), Hep G2 (HB - 8065TM), LU-1 (HTB - 57TM), MCF-7 (HTB - 22TM) and SK-Mel (HTB - 68TM) cells Target compounds 3, 4, 6, 7, 9, and 10 were evaluated for in vitro cytotoxicity against four human tumour cell lines Ellipticine was used as a reference The results showed that five per six compounds possessed considerable anticancer activity (table 1) At the cytotoxicity against KB cells, the present of -OH group at C-2 position seem requirement for potential effect such as compound (IC50 = 3.6 µg/mL) and compoud (IC50 = 16.4 µg/mL) The only 4-chloro derivatives showed the unactive with all tested cell lines The present of 4-methoxy showed moderate activities On the in vitro test on MCF cell lines, compound - the derivative contained ethoxy group at C-4 position showed the higher activation than others (IC50 = 32 µg/mL) The simultaneous present of chloro group at C-4 position and NO2 group at C-3 position showed the higher toxycity to Lu and HepG2 than others (IC50 = 12.42 and 15.14 µg/mL, respectively) Table 1: Results of cytotoxity against tested cancer cell lines of N-(adamantan-1-yl)1-aryl-methanimine derivatives IC50 (g/mL) Comp R1 R2 KB MCF7 Lu HepG2 OH 6-CH3 16.4 77.8 23.3 26.7 H 4-OC2H5 20.7 32 14.8 15.71 3-NO2 4-Cl 24.0 71.5 12.42 15.14 H 2-OH 3.6 37.5 59.4 31.4 H 4-Cl > 128 > 128 > 128 > 128 10 H 4-OCH3 81.54 > 128 83.2 79.06 0.32 0.35 0.42 0.43 Ellipticine 93 Journal of military pharmaco-medicine n02-2019 CONCLUSIONS Ten N-(adamantan-1-yl)-1-arylmethanimines were designed, synthesized and evaluated antimicrobial, inhibition of AChE and cytotoxy of cancer cell lines in vitro Their structures were determined via the data of 1H-NMR, 13C-NMR, and MS spectra showed these are the novel derivatives Among them, five per ten testing compounds showed are the potential agent for antifungus of C albicans with IC50 as 16.97 - 78.6 µg/mL; five per six testing compounds showed toxicity on cancer cell lines including KB (CCL - 17TM), HepG2 (HB - 8065TM), LU-1 (HTB - 57TM), MCF-7 (HTB - 22TM) with IC50 as 3.6 83.2 µg/mL and clinical trials Revista Brasileira de Psiquiatria (AHEAD) 2018 Al-Abdullah E.S, Al-Tuwaijri H.M, Hassan H.M et al Synthesis, antimicrobial and hypoglycemic activities of novel N-(1adamantyl)carbothioamide derivatives Molecules 2015, 20 (5), pp.8125-8143 Chuchkov K.N, Georgiev R.K, Ivanova G et al New adamantane analoguessynthesis and antiviral activit Mathematics and Natural Science, Proceedings of the Fifth International Scientific Conference SouthWest University, Bulgaria 2013, pp.3-9 Kazimierczuk Z, Gorska A, Świtaj T et al Adamantylaminopyrimidines and pyridines are potent inducers of tumor necrosis factor-α Bioorganic & Medicinal Chemistry Letters 2001, 11 (9), pp.1197-1200 REFERENCES Stetter H Advances in the chemistry of organic ring systems with adamantane type structures Angewandte Chemie International Edition in English 1962, (6), pp.286-298 Safavi M, Ashtari A, Khalili F et al Novel quinazolin‐4 (3H)‐one linked to 1, 2, 3‐triazoles: Synthesis and anticancer activity Chemical Biology & Drug Design 2018, 91 (1), pp.13731381 Al Abdullah E.S Synthesis and Biological Testing of New 1-adamantyl derivatives, Ph.D thesis Pharmaceutical Chemistry, College of Pharmacy King Saud University 2007 Cragg G.M, Grothaus P.G, Newman D.J Impact of natural products on developing new anti-cancer agents Chemical Reviews 2009, 109 (7), pp.3012-3043 10 Kadi A.A, El-Brollosy N.R, Al-Deeb O.A et al Synthesis, antimicrobial, and antiinflammatory activities of novel 2-(1adamantyl)-5-substituted-1,3,4-oxadiazoles and 2-(1-adamantylamino)-5-substituted-1,3,4thiadiazoles Eur J Med Chem 2007, 42 (2), pp.235-242 Kim W, Kaelin W.G The von HippelLindau tumor suppressor protein: New insights into oxygen sensing and cancer Current Opinion in Genetics & Development 2003, 13 (1), pp.55-60 Raupp-Barcaro I.F, Vital M.A, Galduróz J.C et al Potential antidepressant effect of amantadine: A review of preclinical studies 94 11 Burmistrov V, Morisseau C, Danilov D et al 1, 3-disubstituted and 1, 3, 3-trisubstituted adamantyl-ureas with isoxazole as soluble epoxide hydrolase inhibitors Bioorganic & Medicinal Chemistry Letters 2015, 25 (23), pp.5514-5519  ... micromelting and were really corrected * Antimicrobial screening: The in vitro antimicrobial testing was performed at Laboratory of Applied Biochemistry, Institute of Chemistry, Vietnamese Academy of Science... as standard inhibitor, and control test was performed without the presence of acetylcholinesterase inhibitors In brief, ten microliters of the test solution were added in the test wells and twenty-five... DTNB) were added in each of well Absorbance was measured immediately and then after 30 of reaction at 410 nm on Microplate reader BIOTEK (USA) IC50 of the testing 89 Journal of military pharmaco-medicine