Alsaid, Mansour S.

Dinkova-Kostova, Albena T.

Ghorab, Mostafa M.

Higgins, Maureen

Naser, Almoqbl A.

Shahat, Abdelaaty A.

Acetamide derivatives and one pyridone derivative were prepared. The structure of the synthesized compounds was verified through elemental analyses,1H -NMR and13C- NMR, IR spectra. The NQO1 inducer activity of the synthesized compounds was evaluated using a quantitative bioassay in Hepa1c1c7 murine hepatoma cells. The acetamide derivatives showed weak activity, with the 3-ethylphenyl being more potent than the 2-ethylphenyl derivative. The pyridone derivative was inactive.</p

University of Dundee Online Publications

                                                              University of DundeeSynthesis and NAD(P)HGhorab, Mostafa M.; Shahat, Abdelaaty A.; Alsaid, Mansour S.; Higgins, Maureen; Dinkova-Kostova, Albena; Naser, Almoqbl A.Published in:Biomedical ResearchPublication date:2016Document VersionPublisher's PDF, also known as Version of recordLink to publication in Discovery Research PortalCitation for published version (APA):Ghorab, M. M., Shahat, A. A., Alsaid, M. S., Higgins, M., Dinkova-Kostova, A. T., & Naser, A. A. (2016).Synthesis and NAD(P)H: Quinone oxidoreductase 1 inducer activity of acetamide and pyridine-3-carbonitrilederivatives. Biomedical Research, 27(4), 1045-1048.General rightsCopyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or othercopyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated withthese rights. • Users may download and print one copy of any publication from Discovery Research Portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • You may freely distribute the URL identifying the publication in the public portal.Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Download date: 07. Nov. 2017Synthesis and NAD(P)H: quinone oxidoreductase 1 inducer activity ofacetamide and pyridine-3-carbonitrile derivatives.Mostafa M. Ghorab1,2*, Abdelaaty A. Shahat1,3, Mansour S. Alsaid1, Maureen Higgins4, Albena T.Dinkova-Kostova4,5, Almoqbl A. Naser61Department of Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, SaudiArabia2Department of Drug Radiation Research, National Center for Radiation Research and Technology, Atomic EnergyAuthority, P.O. Box 29 Nasr City, Cairo, Egypt3Phytochemistry Department, National Research Centre, 12311 Dokki, Cairo, Egypt4Jacqui Wood Cancer Centre, Division of Cancer Research, Medical Research Institute, University of Dundee, DundeeDD1 9SY, United Kingdom5Departments of Medicine and Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine,Baltimore, MD 21205, USA6Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud University,P.O. Box 2457, Riyadh 11451, Saudi ArabiaAbstractAcetamide derivatives and one pyridone derivative were prepared. The structure of the synthesizedcompounds was verified through elemental analyses, 1H -NMR and 13C- NMR, IR spectra. The NQO1inducer activity of the synthesized compounds was evaluated using a quantitative bioassay in Hepa1c1c7murine hepatoma cells. The acetamide derivatives showed weak activity, with the 3-ethylphenyl beingmore potent than the 2-ethylphenyl derivative. The pyridone derivative was inactive.Keywords Acetamides, Pyridone, NQO1, Electrophilicity.Accepted on March 23, 2016IntroductionThe antioxidant activity is related with compounds competentof protecting a biological system against the potential harmfuleffects of oxidative processes. The antioxidant components inthe last years have received enlarged care from medicalresearchers and nutritionists for their potential activities instopping cancer, cardiovascular disorders, as well as aging [1].Acetamide derivatives have been reported to possess a widevariety of important biological properties such as anti-malarial[2], antimicrobial [3-5], anti-proliferative [6], inhibitors ofPim-1 kinase [7], anti-inflammatory [8], anticonvulsant [9],antidyslipidemic, antioxidant [10], anti-HIV [11]. A largenumber of acetamide derivatives have been reported to showpotential anticancer activity in vitro and in vivo [12-14]. Inaddition compounds containing heteroaromatic rings regularlyare playing very important role as supports of bioactivesubstances. It is known that the pyridone and its derivatives areamong the most popular N-heteroaromatic compoundsintegrated into the structures of many pharmaceuticalcompounds and their structural units occur in severalmolecules showing diverse biological activities [15-20].Recently, a series of pyridine derivatives were reported topossess antioxidant, anticancer and angiotensin-I-convertingenzyme (ACE-I) inhibitory activity [21-23]. As part of ourcontinuing interest in this area, in the present work wesynthesised acetamide derivatives (3, 5), and, dihydropyridinederivative (6) to evaluated their cytoprotective activity. Weused induction of the cytoprotective enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a measure ofcytoprotective activity.ExperimentalThe melting points (°C, uncorrected) are determined in opencapillaries on a Gallenkemp melting point apparatus (SanyoGallenkemp, Southborough, UK). Pre-coated with Silica gelplates (silica gel, 60 G F 254, 0.25 mm, Merck, Germany) areused for TLC, methanol/dichloromethane (0.5: 9.5 mL)mixture was used as a developing solvent system. IR spectrawere recorded in KBr discs using IR-Shimadzu spectrometer(Shimadzu, Tokyo, Japan). NMR spectra in (DMSO-d6) wereISSN 0970-938Xwww.biomedres.infoBiomedical Research 2016; 27 (4): 1045-10481045Biomed Res- India 2016 Volume 27 Issue 4recorded on Bruker Ac-500 ultra-shield NMR spectrometer(Bruker, Flawil, Switzerland, δ ppm) at 500 MHz, using TMSas internal standard. Elemental analyses were performed onCarlo Erba 1108 Elemental Analyzer (Heraeus, Hanau,Germany). All compounds were within ± 0.4 % of thetheoretical values.2-Cyano-N-(2-ethylphenyl) acetamide (3)A mixture of 2-ethyl aniline 1 (1.21 g, 0.01 mol) and ethylcyanoacetate 2 (1.13 g, 0.01 mol) was refluxed for 2 h,concentrated and cooled. The obtained solid was filteredandcrystallized from benzene/ether (3:2) give compound 3[24].Yield, 80 %, m.p. 130-132 °C; IR (KBr, max, cm-1): 3180(NH), 2255 (CN), 1703 (CO). 1H- NMR spectrum of 3 in(DMSO-d6) δ : 1.09 [t, 3H, CH3], 2.23 [q, 2H, CH2], 3.85 [s,2H, CH2CN], 7.3-7.5 [m, 4H, Ar-H], 9.45 [s, 1H, NH,exchangeable with D2O], “Anal. Calculated; for C11H12N2OC, 70.21; H, 6.38, 14.89; found: Carbon, 70.50; Hydrogen,6.10; Nitrogen, 14.60.”2- Cyano-N-(3-ethylphenyl) acetamide (5)A mixture of 3-ethylaniline 4 (1.21 g, 0.01 mol) and ethylcyanoacetate 2 (1.13 g, 0.01 mol) was fused at 220 °C (for 3h). The concentrated reaction mixture was cooled. The gottenproduct was crystallized from the etahanol to afford compound5 [25] (Scheme 1). The yield as 88 %, m. p. 86-88 °C; IR (KBr,nmax, cm-1): 3317 (NH), 3100 (CH. arom.), 2960, 2870 (CHaliph.), 2260 (CºN), 1670 (C=O). 1H NMR spectrum of 3 in(DMSO-d6)δ : 1.2 [t, 3H, CH3], 2.6 [q, 2H, CH2], 3.6 [s, 2H,CH2], 4.2 [s, 1H, NH, exchangeable with D2O], 7.03-7.7 [m,4H, Ar-H]. “Anal. Calcd; for C11H12N2O: C, 70.21; H, 6.38,14.89; found: C, 70.50; H, 6.10; N, 14.60.1- (3-Ethylphenyl)-4, 6-dimethyl-2-oxo-1, 2-dihydropyridine-3 - carbonitrile (6)Interaction of component 3 (1.88 g, 0.01 mol) withacetylacetone (1.00 g, 0.01 mol) in ethyl alcohol (50 mL),piperidine (0.5 mL) was refluxed for 5 h. The reaction mixturewas triturated with ethyl alcohol and the obtained solid was re-crystallized from dioxane to yield compound 6 [26] (Scheme1). Yield: 86%. M.p.: 198200 °C. IR (KBr, ν, cm–1): 3100 (CHarom.), 2931, 2872 (CH aliph.), 2210 (C≡N), 1660 (C=O). 1HNMR (300 MHz, DMSOd6,δ, ppm): 1.2 (t, 3H, CH3, J=0.66Hz), 1.5 (2s, 6H, 2CH3), 2.6 (q, 2H, CH2, J=0.66 Hz), 5.6 (s,1H, CH pyridine), 7.27.7 (m, 4H, ArH). Anal. calcd. forC16H16N2O: C, 76.16; H, 6.39; N, 11.10. Found: C, 76.50; H,6.70; N, 11.40%”.Biological assayThe NQO1 inducer activity was determined using aquantitative microtiter plate bioassay [27,28]. Compoundswere prepared as stock solutions in DMSO, and then werediluted in the cell culture medium 1:1000. The finalconcentration of the solvent in the medium of the growing cellswas 0.1% (v/v). Each compound was tested at eight replicatesof 8 serial dilutions. The murine Hepa1c1c7 cell line wasmaintained at 37°C, 5% CO2, in α-MEM supplemented with10% (v/v) fetal bovine serum that had been heat-and charcoal-inactivated. Cells (104 per well) were plated in 96-well plates,and 24 h later, they were exposed to the compounds in freshmedium for a further 48 h. The cell culture medium was thenremoved; cells were washed 3 times with PBS, and lysed for30 min at 25°C in digitonin (0.1 g/L, pH 7.8). To measure theenzyme activity of NQO1 the quinone menadione was used asa substrate. Protein concentrations were determined by theBCA assay (Thermo Scientific), and the values were used tocalculate the specific NQO1 activity (Table 1 and Figure 1).Table 1. NQO1 inducer activity of acetamide and pyridine-3-carbonitrile derivatives.Compounds Induction Magnitude (Fold)“2-Cyano-N-(2-ethylphenyl) acetamide (3)” 1.2“2-Cyano-N-(3-ethylphenyl) acetamide (5)” 1.65“1-(3-Ethylphenyl)-4,6-dimethyl-2-oxo-1,2-dihydropyridine- 3-carbonitrile (6)”InactiveFigure 1. Dose-response of NQO1 inducer activity of acetamide andpyridine-3-carbonitrile derivatives.Results and DiscussionThe aim of this work was to design and synthesis of a series ofacetamides (3, 5) and pyridine (6) (Scheme 1) and evaluationof their potential NQO1 inducer activity. Interaction of 2-ethyl1 or 3-ethylaniline 4 with ethyl cyanoacetate 2 furnished thecorresponding acetamide derivatives 3 and 5, respectively. Thestructure of compounds 3 and 5 was proved in the basis ofelemental analysis and spectral data. The correspondingpyridine derivative 6 was obtained via reaction of compound 5with acetylacetone in the presence of a catalytic amount ofpiperedine. The structure of compound 6 was established byelemental analysis, spectral data.Ghorab/Shahat/Alsaid/Higgins/Dinkova-Kostova/Naser1046 Biomed Res- India 2016 Volume 27 Issue 4NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) is acytoprotective enzyme which is activated by electrophiliccompounds via the Keap1/Nrf2 pathway [29]. Numerouscompounds which have been shown to induce NQO1 havebeen subsequently found to the broadly cytoprotective and toeffectively inhibit tumor formation in animal models [30,31].We found that the acetamide derivatives have weak NQO1inducer activity. The 3-ethylphenyl (5) is more potent than the2-ethylphenyl (3) derivative. The pyridone derivative (6) isinactive.Scheme 1. Formation of acetamide and pyridone derivatives 3,5,6.ConclusionThe present study objective to synthesize and evaluate thepotential anticancer activities of some acetamide and pyridinederivatives. The acetamide derivative (5) was the most active.AcknowledgementsMostafa M. Ghorab1, Abdelaaty A. Shahat and Mansour S.Alsaid extend their truthful appreciation to the Deanship ofScientific Research at King Saud University for funding of thisresearch through the Research Group Project no. RGP-VPP-262. Maureen Higgins and Albena T. Dinkova-Kostovaare also grateful to Cancer Research UK (C20953/A10270) forfinancial support.References1. Asif M, Singh A, Lakshmayya. Development ofstructurally diverse antitubercular molecules withpyridazine ring. Chronicles of Young Scientists 2013; 4:1-8.2. Agarwal A, Srivastava K, Puri S, Sinha S, Chauhan P. Asmall library of trisubstituted pyrimidines as antimalarialand antitubercular agents. Bioorganic & medicinalchemistry letters 2015; 15: 5218-5221.3. Asif M, Singh A, Ratnakar. Antimicrobial Agents: BriefStudy of Pyridazine Derivatives against Some PhathogenicMicrorganisms. J Pharm Res 2011; 4: 664-667.4. Pedeboscq S, Gravier D, Casadebaig F, Hou G, Gissot A,De Giorgi F, Ichas F, Cambar J, Pometan JP. Synthesis andstudy of antiproliferative activity of novelthienopyrimidines on glioblastoma cells. European journalof medicinal chemistry 2010; 45; 2473-2479.5. Kassis P, Brzeszcz J, Bénéteau V, Lozach O, Meijer L, leGuével R, Guillouzo, C, Lewiński K, Bourg S, ColliandreL. Synthesis and biological evaluation of new 3-(6-hydroxyindol-2-yl)-5-(Phenyl) pyridine or pyrazine V-Shaped molecules as kinase inhibitors and cytotoxic agents.Eur. J. Med. Chem. 2011; 46: 5416-5434.6. Amir M, Javed S, Kumar H. Pyrimidine asantiinflammatory agent: A review. Indian journal ofpharmaceutical sciences 2007; 69: 337-343.7. Asif M, Singh A. In-vivo anticonvulsant and in-vitroantimycobacterial activities of 6-aryl pyridazine-3(2h)-onederivatives. American Journal of Pharmacological Sciences2014; 2, 1-6.8. Koneni V, Sashidhara, Gopala RP , Ranga P D, Ravi S,Khanna A K, Gitika B. Discovery of amide based fibratesas possible antidyslipidemic and antioxidant agentsEuropean Journal of Medicinal Chemistry 2012; 57:302-310.9. Ölgen, S. Recent development of new substituted indoleand azaindole derivatives as anti-HIVagents. Mini Rev.Med. Chem 2013; 13: 1700-1708.10. Xiang, Zhou PT, Wang L, Sun CY, Hu J, Zhao YL, Yang L.Novel Benzothiazole, Benzimidazole and BenzoxazoleDerivatives as Potential Antitumor Agents: Synthesis andPreliminary in Vitro Biological Evaluation. Molecules2012; 17: 873-883.11. Ahmed RA, El-Bendary E R, Ghaly MA, Shehata IA.Novel acetamid othiazole derivatives: Synthesis and invitro anticancer evaluation. European Journal of MedicinalChemistry. 2013; 69: 908-919.12. Situ Xue, Linlin Ma, Rongmei Gao, Yuhuan Lin , ZhuorongLin. Synthesis and antiviral activity of some novelindole-2-carboxylate derivatives. Acta PharmaceuticaSinica B. 2014; 4: 13–321.13. Ghorab MM, Ragab FA, Heiba HI, M. Agha H. Synthesisof Some Novel Sulfonamides Containing BiologicallyActive Alkanoic Acid, Acetamide, Thiazole, and PyrroleMoieties of Expected Antitumor and RadiosensitizingActivities. J. Basic. Appl .Chem.,2011; 1: 8-14.14. Ghorab MM, Al-Saidand MS, El-Hossary EM. In vitrocytotoxic evaluation of some new heterocyclic sulfonamidederivatives. Journal of Heterocyclic Chemistry 2011; 48:563–571.15. Taha NMH, Boray A AA, Saleh NM, Fouad SA. Synthesisand antitumor screening of some novel pyrrolo,pyrrolo[2,3-d] pyrimidinone and pyrrolo [2,3-b] pyridinonederivatives of sulfaquinoxaline. Journal of AppliedSciences Research. 2013; 9: 3108-3117.16. Akhtar T, Hameed S, Al-Masoudi NA, Lacolla RL. In vitroantitumor and antiviral activities of new benzothiazole and1,3,4-oxadiazole-2-thione derivatives. Acta Pharm. 2008;58: 135–149.17. El-Henawy AA, Mohamed S, Ma T, Ibrahim GAM, El-HagA. Synthesis of New Sulfonamide Scaffolds Acting AsSynthesis and NAD(P)H: quinone oxidoreductase 1 inducer activity of acetamide and pyridine-3-carbonitrilederivatives1047Biomed Res- India 2016 Volume 27 Issue 4Anticancer Targeting CAII Protein Based Docking Studies.New York Science Journal.2011; 4: 20-29.18. Mohammadi-Farania A, Heidarianb N, Aliabadi A. N-(5-Mercapto-1,3,4-Thiadiazol-2-yl)-2-PhenylacetamideDerivatives: Synthesis and In-vitro Cytotoxicity Evaluationas Potential Anticancer Agents. Iranian Journal ofPharmaceutical Research. 2014; 13: 487-492.19. El-Henawya AA, Mohamedb SI, Hassana AA, HalawaaAH, Elnassaga MA, Elhagea GA. Discovery Potent ofNovel Peptide Derivatives Containing Sulfonamide MoietyAs Inhibitors of CA Using Structure Based VirtualScreening and Binding Assays. New York Science Journal.2011; 4: 19-25.20. Chegwidden WR, Spencer IM. Sulphonamide inhlbitors ofcarbonic anhydrase inhibit the growth of human lymphomacells in culture. InflammoPharmacology.  1995; 3: 231-239.21. Miriana M, Zarghib A, Sadeghia S, Tabarakic P, TavallaeecM, Dadrassc O, Sadeghi-aliabadi H. Synthesis andCytotoxic Evaluation of Some Novel SulfonamideDerivatives Against a Few Human Cancer Cells. IranianJournal of Pharmaceutical Research 2011; 10: 741-748.22. Scozzafava A, Owa T, Mastrolorenzo A, Supuran CT.Anticancer and antiviral sulfonamides. Curr Med Chem,2003; 10 : 925-53.23. Ren Y, Zhang L, Zhou C-He, Geng R-X. RecentDevelopment of Benzotriazole-based Medicinal Drugs.Med chem 2014; 4: 640-662.24. Zareef M, Iqbal R, Mirza B, Khan KM, Manan A, Asim F,Khan SW. Synthesis and antimicrobial activity of somederivatives of acylhydrazine including novel benzenediazasulfonamides ARKIVOC, 2008; 141-152.25. Magda MF, Noaman E. Novel pirfenidone analogs asantifibrotic agents: Synthesis and antifibrotic evaluation of2-pyridones and fused pyridines. Med Chem Res 2005; 14:382-403.26. AlSaida MS, ElGazzarb MG, Ghorab MM. Invitrocytotoxic and radiosensitizing evaluation of novel2pyridone, isoquinoline, chromene and chromenopyridonederivatives. Eeu J Chem 2012; 3: 228234.27. Prochaska HJ, Santamaria AB. Direct measurement ofNAD(P)H:quinone reductase from cells cultured inmicrotiter wells: a screening assay for anticarcinogenicenzyme inducers. Anal Biochem 1988; 169: 328-336.28. Fahey JW, Dinkova-Kostova AT, Stephenson KK, TalalayP. The "Prochaska" microtiter plate bioassay for inducers ofNQO1. Methods Enzymol 2004; 382: 243-258.29. Dinkova-Kostova AT, Talalay P. NAD(P)H:quinoneacceptor oxidoreductase 1 (NQO1), a multifunctionalantioxidant enzyme and exceptionally versatilecytoprotector. Arch Biochem Biophys 2010; 501: 116-123.30. Liby KT, Sporn MB. Synthetic oleananetriterpenoids:multifunctional drugs with a broad range of applications forprevention and treatment of chronic disease Pharmacol Rev2012; 64: 972-1003.31. Liby KT, Yore MM, Sporn MB. Triterpenoids andrexinoids as multifunctional agents for the prevention andtreatment of cancer. Nat Rev Cancer 2007; 7: 357-369.Correspondence to:Mostafa M. M. GhorabDepartment of PharmacognosyCollege of PharmacyKing Saud UniversityRiyadh 11451Saudi Arabia Ghorab/Shahat/Alsaid/Higgins/Dinkova-Kostova/Naser1048 Biomed Res- India 2016 Volume 27 Issue 4

Synthesis and NAD(P)H:Quinone oxidoreductase 1 inducer activity of acetamide and pyridine-3-carbonitrile derivatives

                                                                    University of DundeeSynthesis and NAD(P)HGhorab, Mostafa M.; Shahat, Abdelaaty A.; Alsaid, Mansour S.; Higgins, Maureen; Dinkova-Kostova, Albena T.; Naser, Almoqbl A.Published in:Biomedical ResearchPublication date:2016Licence:CC BYDocument VersionPublisher's PDF, also known as Version of recordLink to publication in Discovery Research PortalCitation for published version (APA):Ghorab, M. M., Shahat, A. A., Alsaid, M. S., Higgins, M., Dinkova-Kostova, A. T., & Naser, A. A. (2016).Synthesis and NAD(P)H: Quinone oxidoreductase 1 inducer activity of acetamide and pyridine-3-carbonitrilederivatives. Biomedical Research, 27(4), 1045-1048. http://www.alliedacademies.org/articles/synthesis-and-nadph-quinone-oxidoreductase-1-inducer-activity-of-acetamide-and-pyridine3carbonitrile-derivatives.htmlGeneral rightsCopyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or othercopyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated withthese rights. • Users may download and print one copy of any publication from Discovery Research Portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • You may freely distribute the URL identifying the publication in the public portal.Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Download date: 18. Apr. 2023Synthesis and NAD(P)H: quinone oxidoreductase 1 inducer activity ofacetamide and pyridine-3-carbonitrile derivatives.Mostafa M. Ghorab1,2*, Abdelaaty A. Shahat1,3, Mansour S. Alsaid1, Maureen Higgins4, Albena T.Dinkova-Kostova4,5, Almoqbl A. Naser61Department of Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, SaudiArabia2Department of Drug Radiation Research, National Center for Radiation Research and Technology, Atomic EnergyAuthority, P.O. Box 29 Nasr City, Cairo, Egypt3Phytochemistry Department, National Research Centre, 12311 Dokki, Cairo, Egypt4Jacqui Wood Cancer Centre, Division of Cancer Research, Medical Research Institute, University of Dundee, DundeeDD1 9SY, United Kingdom5Departments of Medicine and Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine,Baltimore, MD 21205, USA6Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud University,P.O. Box 2457, Riyadh 11451, Saudi ArabiaAbstractAcetamide derivatives and one pyridone derivative were prepared. The structure of the synthesizedcompounds was verified through elemental analyses, 1H -NMR and 13C- NMR, IR spectra. The NQO1inducer activity of the synthesized compounds was evaluated using a quantitative bioassay in Hepa1c1c7murine hepatoma cells. The acetamide derivatives showed weak activity, with the 3-ethylphenyl beingmore potent than the 2-ethylphenyl derivative. The pyridone derivative was inactive.Keywords Acetamides, Pyridone, NQO1, Electrophilicity.Accepted on March 23, 2016IntroductionThe antioxidant activity is related with compounds competentof protecting a biological system against the potential harmfuleffects of oxidative processes. The antioxidant components inthe last years have received enlarged care from medicalresearchers and nutritionists for their potential activities instopping cancer, cardiovascular disorders, as well as aging [1].Acetamide derivatives have been reported to possess a widevariety of important biological properties such as anti-malarial[2], antimicrobial [3-5], anti-proliferative [6], inhibitors ofPim-1 kinase [7], anti-inflammatory [8], anticonvulsant [9],antidyslipidemic, antioxidant [10], anti-HIV [11]. A largenumber of acetamide derivatives have been reported to showpotential anticancer activity in vitro and in vivo [12-14]. Inaddition compounds containing heteroaromatic rings regularlyare playing very important role as supports of bioactivesubstances. It is known that the pyridone and its derivatives areamong the most popular N-heteroaromatic compoundsintegrated into the structures of many pharmaceuticalcompounds and their structural units occur in severalmolecules showing diverse biological activities [15-20].Recently, a series of pyridine derivatives were reported topossess antioxidant, anticancer and angiotensin-I-convertingenzyme (ACE-I) inhibitory activity [21-23]. As part of ourcontinuing interest in this area, in the present work wesynthesised acetamide derivatives (3, 5), and, dihydropyridinederivative (6) to evaluated their cytoprotective activity. Weused induction of the cytoprotective enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a measure ofcytoprotective activity.ExperimentalThe melting points (°C, uncorrected) are determined in opencapillaries on a Gallenkemp melting point apparatus (SanyoGallenkemp, Southborough, UK). Pre-coated with Silica gelplates (silica gel, 60 G F 254, 0.25 mm, Merck, Germany) areused for TLC, methanol/dichloromethane (0.5: 9.5 mL)mixture was used as a developing solvent system. IR spectrawere recorded in KBr discs using IR-Shimadzu spectrometer(Shimadzu, Tokyo, Japan). NMR spectra in (DMSO-d6) wereISSN 0970-938Xwww.biomedres.infoBiomedical Research 2016; 27 (4): 1045-10481045Biomed Res- India 2016 Volume 27 Issue 4recorded on Bruker Ac-500 ultra-shield NMR spectrometer(Bruker, Flawil, Switzerland, δ ppm) at 500 MHz, using TMSas internal standard. Elemental analyses were performed onCarlo Erba 1108 Elemental Analyzer (Heraeus, Hanau,Germany). All compounds were within ± 0.4 % of thetheoretical values.2-Cyano-N-(2-ethylphenyl) acetamide (3)A mixture of 2-ethyl aniline 1 (1.21 g, 0.01 mol) and ethylcyanoacetate 2 (1.13 g, 0.01 mol) was refluxed for 2 h,concentrated and cooled. The obtained solid was filteredandcrystallized from benzene/ether (3:2) give compound 3[24].Yield, 80 %, m.p. 130-132 °C; IR (KBr, max, cm-1): 3180(NH), 2255 (CN), 1703 (CO). 1H- NMR spectrum of 3 in(DMSO-d6) δ : 1.09 [t, 3H, CH3], 2.23 [q, 2H, CH2], 3.85 [s,2H, CH2CN], 7.3-7.5 [m, 4H, Ar-H], 9.45 [s, 1H, NH,exchangeable with D2O], “Anal. Calculated; for C11H12N2OC, 70.21; H, 6.38, 14.89; found: Carbon, 70.50; Hydrogen,6.10; Nitrogen, 14.60.”2- Cyano-N-(3-ethylphenyl) acetamide (5)A mixture of 3-ethylaniline 4 (1.21 g, 0.01 mol) and ethylcyanoacetate 2 (1.13 g, 0.01 mol) was fused at 220 °C (for 3h). The concentrated reaction mixture was cooled. The gottenproduct was crystallized from the etahanol to afford compound5 [25] (Scheme 1). The yield as 88 %, m. p. 86-88 °C; IR (KBr,nmax, cm-1): 3317 (NH), 3100 (CH. arom.), 2960, 2870 (CHaliph.), 2260 (CºN), 1670 (C=O). 1H NMR spectrum of 3 in(DMSO-d6)δ : 1.2 [t, 3H, CH3], 2.6 [q, 2H, CH2], 3.6 [s, 2H,CH2], 4.2 [s, 1H, NH, exchangeable with D2O], 7.03-7.7 [m,4H, Ar-H]. “Anal. Calcd; for C11H12N2O: C, 70.21; H, 6.38,14.89; found: C, 70.50; H, 6.10; N, 14.60.1- (3-Ethylphenyl)-4, 6-dimethyl-2-oxo-1, 2-dihydropyridine-3 - carbonitrile (6)Interaction of component 3 (1.88 g, 0.01 mol) withacetylacetone (1.00 g, 0.01 mol) in ethyl alcohol (50 mL),piperidine (0.5 mL) was refluxed for 5 h. The reaction mixturewas triturated with ethyl alcohol and the obtained solid was re-crystallized from dioxane to yield compound 6 [26] (Scheme1). Yield: 86%. M.p.: 198200 °C. IR (KBr, ν, cm–1): 3100 (CHarom.), 2931, 2872 (CH aliph.), 2210 (C≡N), 1660 (C=O). 1HNMR (300 MHz, DMSOd6,δ, ppm): 1.2 (t, 3H, CH3, J=0.66Hz), 1.5 (2s, 6H, 2CH3), 2.6 (q, 2H, CH2, J=0.66 Hz), 5.6 (s,1H, CH pyridine), 7.27.7 (m, 4H, ArH). Anal. calcd. forC16H16N2O: C, 76.16; H, 6.39; N, 11.10. Found: C, 76.50; H,6.70; N, 11.40%”.Biological assayThe NQO1 inducer activity was determined using aquantitative microtiter plate bioassay [27,28]. Compoundswere prepared as stock solutions in DMSO, and then werediluted in the cell culture medium 1:1000. The finalconcentration of the solvent in the medium of the growing cellswas 0.1% (v/v). Each compound was tested at eight replicatesof 8 serial dilutions. The murine Hepa1c1c7 cell line wasmaintained at 37°C, 5% CO2, in α-MEM supplemented with10% (v/v) fetal bovine serum that had been heat-and charcoal-inactivated. Cells (104 per well) were plated in 96-well plates,and 24 h later, they were exposed to the compounds in freshmedium for a further 48 h. The cell culture medium was thenremoved; cells were washed 3 times with PBS, and lysed for30 min at 25°C in digitonin (0.1 g/L, pH 7.8). To measure theenzyme activity of NQO1 the quinone menadione was used asa substrate. Protein concentrations were determined by theBCA assay (Thermo Scientific), and the values were used tocalculate the specific NQO1 activity (Table 1 and Figure 1).Table 1. NQO1 inducer activity of acetamide and pyridine-3-carbonitrile derivatives.Compounds Induction Magnitude (Fold)“2-Cyano-N-(2-ethylphenyl) acetamide (3)” 1.2“2-Cyano-N-(3-ethylphenyl) acetamide (5)” 1.65“1-(3-Ethylphenyl)-4,6-dimethyl-2-oxo-1,2-dihydropyridine- 3-carbonitrile (6)”InactiveFigure 1. Dose-response of NQO1 inducer activity of acetamide andpyridine-3-carbonitrile derivatives.Results and DiscussionThe aim of this work was to design and synthesis of a series ofacetamides (3, 5) and pyridine (6) (Scheme 1) and evaluationof their potential NQO1 inducer activity. Interaction of 2-ethyl1 or 3-ethylaniline 4 with ethyl cyanoacetate 2 furnished thecorresponding acetamide derivatives 3 and 5, respectively. Thestructure of compounds 3 and 5 was proved in the basis ofelemental analysis and spectral data. The correspondingpyridine derivative 6 was obtained via reaction of compound 5with acetylacetone in the presence of a catalytic amount ofpiperedine. The structure of compound 6 was established byelemental analysis, spectral data.Ghorab/Shahat/Alsaid/Higgins/Dinkova-Kostova/Naser1046 Biomed Res- India 2016 Volume 27 Issue 4NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) is acytoprotective enzyme which is activated by electrophiliccompounds via the Keap1/Nrf2 pathway [29]. Numerouscompounds which have been shown to induce NQO1 havebeen subsequently found to the broadly cytoprotective and toeffectively inhibit tumor formation in animal models [30,31].We found that the acetamide derivatives have weak NQO1inducer activity. The 3-ethylphenyl (5) is more potent than the2-ethylphenyl (3) derivative. The pyridone derivative (6) isinactive.Scheme 1. Formation of acetamide and pyridone derivatives 3,5,6.ConclusionThe present study objective to synthesize and evaluate thepotential anticancer activities of some acetamide and pyridinederivatives. The acetamide derivative (5) was the most active.AcknowledgementsMostafa M. Ghorab1, Abdelaaty A. Shahat and Mansour S.Alsaid extend their truthful appreciation to the Deanship ofScientific Research at King Saud University for funding of thisresearch through the Research Group Project no. RGP-VPP-262. Maureen Higgins and Albena T. Dinkova-Kostovaare also grateful to Cancer Research UK (C20953/A10270) forfinancial support.References1. Asif M, Singh A, Lakshmayya. Development ofstructurally diverse antitubercular molecules withpyridazine ring. Chronicles of Young Scientists 2013; 4:1-8.2. Agarwal A, Srivastava K, Puri S, Sinha S, Chauhan P. Asmall library of trisubstituted pyrimidines as antimalarialand antitubercular agents. Bioorganic & medicinalchemistry letters 2015; 15: 5218-5221.3. Asif M, Singh A, Ratnakar. Antimicrobial Agents: BriefStudy of Pyridazine Derivatives against Some PhathogenicMicrorganisms. J Pharm Res 2011; 4: 664-667.4. Pedeboscq S, Gravier D, Casadebaig F, Hou G, Gissot A,De Giorgi F, Ichas F, Cambar J, Pometan JP. Synthesis andstudy of antiproliferative activity of novelthienopyrimidines on glioblastoma cells. European journalof medicinal chemistry 2010; 45; 2473-2479.5. Kassis P, Brzeszcz J, Bénéteau V, Lozach O, Meijer L, leGuével R, Guillouzo, C, Lewiński K, Bourg S, ColliandreL. 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Anal Biochem 1988; 169: 328-336.28. Fahey JW, Dinkova-Kostova AT, Stephenson KK, TalalayP. The "Prochaska" microtiter plate bioassay for inducers ofNQO1. Methods Enzymol 2004; 382: 243-258.29. Dinkova-Kostova AT, Talalay P. NAD(P)H:quinoneacceptor oxidoreductase 1 (NQO1), a multifunctionalantioxidant enzyme and exceptionally versatilecytoprotector. Arch Biochem Biophys 2010; 501: 116-123.30. Liby KT, Sporn MB. Synthetic oleananetriterpenoids:multifunctional drugs with a broad range of applications forprevention and treatment of chronic disease Pharmacol Rev2012; 64: 972-1003.31. Liby KT, Yore MM, Sporn MB. Triterpenoids andrexinoids as multifunctional agents for the prevention andtreatment of cancer. Nat Rev Cancer 2007; 7: 357-369.Correspondence to:Mostafa M. M. GhorabDepartment of PharmacognosyCollege of PharmacyKing Saud UniversityRiyadh 11451Saudi Arabia Ghorab/Shahat/Alsaid/Higgins/Dinkova-Kostova/Naser1048 Biomed Res- India 2016 Volume 27 Issue 4

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Synthesis and NAD(P)H:Quinone oxidoreductase 1 inducer activity of acetamide and pyridine-3-carbonitrile derivatives

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