research article nucleobase-based barbiturates:...

Research ArticleNucleobase-Based Barbiturates Their Protective Effectagainst DNA Damage Induced by Bleomycin-Iron Antioxidantand Lymphocyte Transformation Assay

Bhaveshkumar D Dhorajiya1 Bharatkumar Z Dholakiya1

Ahmed S Ibrahim2 and Farid A Badria3

1 Department of Applied Chemistry SV National Institute of Technology Ichchhanath Surat Gujarat 395007 India2Department of Biochemistry Faculty of Pharmacy Mansoura University Mansoura 35516 Egypt3 Department of Pharmacognosy Faculty of Pharmacy Mansoura University Mansoura 35516 Egypt

Correspondence should be addressed to Bharatkumar Z Dholakiya bharat281173gmailcom

Received 20 February 2014 Accepted 9 April 2014 Published 8 May 2014

Academic Editor Paul W Doetsch

Copyright copy 2014 Bhaveshkumar D Dhorajiya et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

A number of nucleobase-based barbiturates have been synthesized by combination of nucleic acid bases and heterocyclicamines and barbituric acid derivatives through green and efficient multicomponent route and one pot reaction This approachwas accomplished efficiently using aqueous medium to give the corresponding products in high yield The newly synthesizedcompounds were characterized by spectral analysis (FT-IR 1H NMR 13C NMR HMBC and UV spectroscopy) and elementalanalysis Representative of all synthesized compounds was tested and evaluated for antioxidant bleomycin-dependent DNAdamage and Lymphocyte Transformation studies Compounds TBC gt TBA gt TBG showed highest lymphocyte transformationassayTBC gtTBA gtBG showed inhibitory antioxidant activity using ABTSmethods andTBC gtBPA gtBAMT gtTBA gt 1 3-TBAmanifested the best protective effect against DNA damage induced by bleomycin

1 Introduction

The area of free radical biology and medicine is developingfast since the discovery of the involvement of free radicalsin oxidative tissue injury and diseases Free radicals andother reactive oxygen species such as superoxide radicalanion hydroxyl radical and hydrogen peroxide are con-stantly generated throughmany biological processes andmaybe considered as a measure of biological inefficiency [1]The human body uses an antioxidant system to neutralizethe excessive levels of reactive oxygen species that consistsof enzymes such as superoxide dismutases catalases andglutathione peroxidases in addition to numerous nonen-zymatic small molecules that are widely distributed in thebiological system such as glutathione 120572-tocopherol ascorbicacid 120573-carotene and selenium [2] In general the cell isable to maintain an appropriate balance between oxidants

and antioxidants under normal conditions The imbalancebetween reactive oxygen species production and the availableantioxidant defence leads to a widely accepted phenomenoncalled oxidative stress [3] Barbiturates have been in the centerof attention of researchers over many years because of thehigh practical value of these compounds In the first placebarbiturates are very important class of compounds for theirhigh reactivity in synthesis as key starting materials to formvarious classes of biologically and pharmacologically activecandidates The diverse biologic activity and coverage of abroad chemical spacemake barbituric acid and thiobarbituricacid derivatives excellent target compounds for organic andmedicinal chemists Owing to their ready availability andvarious functionalization possibilities the parent barbituricacid and thiobarbituric acid are convenient starting mate-rials for the preparation of different fused heterocycles andliterature survey also describe that 5-substituted derivatives

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 898670 7 pageshttpdxdoiorg1011552014898670

2 BioMed Research International

O O

O

O

O

O

EtO OEtCC

H2

X

X

X

NHR

NHR

N

N

N

N

R

R

R

R

+

NH-R1

(a)

(b)

R = ndashH or ndashCH3

X = O and S

Scheme 1 Synthesis of DNA base and heterocyclic primary amines-based barbiturates Reagents and conditions (a) NaOMeMeOH-refluxed(b) HCO

2H water reflux where R1 = adenine guanine cytosine and primary heterocyclic amine

are pharmacologically active compounds [4] The medicinalimportance of pyrimidine derivatives such as barbituric acidand thiobarbituric acid plays vital role among various hetero-cyclic compounds due to their antineoplastic [5 6] antiviral[7] antibiotic [8] and anti-inflammatory [9] activity Thepyrimidine ring system is present in various natural com-pounds such as nucleic acids vitamins coenzymes uric acidpurines and some marine microorganisms (eg sponge)[10] Cyclic imides are an important class of moleculesknown for their diverse array of bioactivitiesThese biologicalactivities and various pharmacological uses of cyclic imideshave been attributed to their unique structural features [11]In view of these facts and in continuation of our interest in thesynthesis of a variety of heterocycles of biological importancewe report here an efficient and convenient method forthe synthesis of novel nucleobased barbiturates derivativesattached to pyrimidine moiety The synthesized compoundswere evaluated for their antioxidant protection activitiesagainst DNA damage induced by bleomycin-iron assay andthis may help to reduce the side effects of chemotherapy forcancer patients

2 Materials and Methods

Chemicals and solvents were obtained from commercialsources and used as received throughout the investigationThe barbituric acid was synthesized by using diethyl mal-onate and urea using standard procedure Melting pointswere determined in open capillaries on a Veego electronicapparatus VMP-D (Veego Instrument CorporationMumbaiIndia) and are uncorrected IR spectra (4000ndash400 cmminus1) ofsynthesized compounds were recorded on a Perkin Elmer-Spectrum RX-IFTIR spectrophotometer using KBr pelletsThin layer chromatography was performed on object glass

slides (2 times 75 cm) coated with silica gel-G and spots werevisualized under UV irradiation 1H NMR and 13C NMRspectra were recorded on an Avance-II (Bruker) model usingDMSO as a solvent and TMS as internal standard with 1Hresonant frequency of 400MHz and 13C resonant frequencyof 100MHzThe 1HNMR and 13CNMR chemical shifts werereported as parts per million (ppm) downfield from TMS(Me4Si) The splitting patterns are designated as follows s

singlet br s broad singlet d doublet t triplet q quartetm multiplet UV spectra were recorded on Maya pro 2000(Ocean Optics USA) using DMSO as a solvent with 10minus5Msolution

21 Synthesis of Barbituric Acid As shown in Scheme 1 Toa solution of diethylmalonate (20 g 1189mmol) and ureathiourea and its methyl substituted analogues were (75 g985mmol) in methanol anhydrous sodium methoxide wasadded and refluxed at 65∘C for 8 h A white solid separatesThen in the above reaction mixture 125mL of hot (50∘C)water was added and hydrochloric acid was used to makethe solution acidic After the completion of the reactionthe resulting clear solution was filtered and cooled in anice bath overnight The white product formed was filteredwashed with 50mL of cold water dried and recrystallizedfrom acetone to afford compound as a white powder [12]

211 Synthesis of DNA-Based Barbiturates As shown inScheme 1 a solution of the corresponding DNA-bases (egadenine 1mmol) and formic acid (4mmol) in distilled wateras a green solvent was allowed to reflux at 60∘C over a periodof 2-3 h Then it was cooled down until the reaction mixturebecomes a clear solution The completion of reaction wasconfirmed by TLC In situ barbiturates were added againand refluxed further for 3-4 h As the reaction proceeds the

BioMed Research International 3

solid products were separated out in the form of suspensionand the precipitated DNA-based barbiturates were separatedby filtration washed with water three times followed by n-hexane and then dried in a desiccators [12]

22 Characterization Data of Synthesized Compounds

221 5-[(9H-Purin-6-ylamino)-methylene]-pyrimidine-246-trione (BA) Yellow powder yield 77 mp gt250∘C 1HNMR (400MHz DMSO) 402 120575 ppm (1H dd exocyclicNH of purine ring 119895 = 1680Hz) 688 120575 ppm (2H sendocyclic-NH of purine ring) 808 120575 ppm (1H dd exocyclicCH of pyrimidine ring 119895 = 2536Hz) 814 120575 ppm (1H ddNH of purine ring) 1121 120575 ppm (1H s NH of pyrimidinering) 1122 120575 ppm (1H s NH of pyrimidine ring) 13CNMR (400MHzDMSO) 77 (C-5) 11432 (C-9) 11467 (C-8)12295 (C-13) 12712 (C-12 C-14) 12927 (C-11 C-15) 132 (C-10) 155 (C-7) 16315 (C-4 C-6) 16805 (C-2) 120575 ppm FTIR(KBr) 120592max cmminus1 1212 (m -O-C stretching) 1631 (s =C-NH aliphatic amine) 1693 (s C=O) 2826 (exocyclic CH)3073 (m CH-NH stretching) 120582 max 30322 nm (C 110 times105 Lmolminus1 cmminus1) MW27321 ESIMS 119898119911 27425 (M + 1)Anal Calcd For C

10H7N7O3() C 4396 H 258 N 3589

Found () C 4394 H 257 N 3591

222 5-[(9H-Purin-6-ylamino)-methylene]-2-thioxo-dihydro-pyrimidine-46-dione (TBA) Yellow powder yield 58 mpgt 250∘C 1H NMR (400MHz DMSO) 404 120575 ppm (1H ddexocyclic NH of purine ring 119895 = 1585Hz) 692 120575 ppm(2H s -CH of purine ring) 812 120575 ppm (1H dd exocyclicCH of pyrimidine ring 119895 = 2460Hz) 820 120575 ppm (1H ddNH of purine ring) 1118 120575 ppm (1H s NH of pyrimidinering) 1121 120575 ppm (1H s NH of pyrimidine ring) 13C NMR(400MHzDMSO) 76 (C-5) 11510 (C-9) 11540 (C-8) 12282(C-13 C-14) 12672 (C-12 C-15) 12967 (C-11) 13189 (C-10)15515 (C-7) 16515 (C-4 C-6) 16825 (C-2) 120575 ppm FTIR(KBr) 120592max cmminus1 1216 (m -O-C stretching) 1628 (s =C-NH aliphatic amine) 1698 (s C=O) 2808 (exocyclic CH)3075 (m CH-NH stretching) 120582max 299 nm (C 103 times 105 Lmolminus1 cmminus1) MW28927 ESIMS 119898119911 28905 (M) AnalCalcd For C

10H7N7O2S () C 4152 H 244 N 3389 Found

() C 4248 H 248 N 3390

223 13-Dimethyl-5-[(9H-purin-6-ylamino)-methylene]-pyr-imidine-246-trione (13 BA) Yellow powder yield 82mpgt250∘C 1H NMR (400MHz DMSO) 272 (6H s two CH

3

group of pyrimidine ring) 567 120575 ppm (1H dd exocyclic NHof purine ring 119895 = 1408Hz) 685 120575 ppm (2H s -CH ofpurine ring) 809 120575 ppm (1H dd exocyclic CH of pyrimidinering 119895 = 2494Hz) 816 120575 ppm (1H dd NH of purine ring)13C NMR (400MHz DMSO) 78 (C-5) 11310 (C-9) 11338(C-8) 12139 (C-13) 12656 (C-12 C-14) 12960 (C-11 C-15)13329 (C-10) 15755 (C-7) 16315 (C-4 C-6) 16805 (C-2)120575 ppm FTIR (KBr) 120592max cmminus1 1208 (m -O-C stretching)1628 (s =C-NH aliphatic amine) 1695 (s C=O) 2805 (m-N-CH

3stretching) 2816 (exocyclic CH) 3070 (m CH-NH

stretching) 120582 max 30322 nm (C 100 times 105 Lmolminus1 cmminus1)

MW 30126 ESIMS 119898119911 30225 (M + 2) Anal Calcd ForC12H11N7O3() C 4784 H 368 N 3255 Found () C

4781 H 370 N 3256

224 13-Dimethyl-5-[(9H-purin-6-ylamino)-methylene]-2-thioxo-dihydro-pyrimidine-46-dione (13-TBA) Dark orangepowder yield 62 mp gt250∘C 1H NMR (400MHzDMSO) 278 (6H s two CH

3group of pyrimidine ring) 403

120575 ppm (1H dd exocyclic NH of purine ring 119895 = 1528Hz)690 120575 ppm (2H s -CH of purine ring) 814 120575 ppm (1H ddexocyclic CH of pyrimidine ring 119895 = 2334Hz) 818 120575 ppm(1H dd NH of purine ring) 13C NMR (400MHz DMSO)5808 (C-18) 7885 (C-5) 11330 (C-9) 11347 (C-8) 12295(C-11 C-15) 12927 (C-12 C-14) 13117 (C-10 C-13) 157 (C-7)16266 (C-4 C-6) 17205 (C-2) 120575 ppm 120582max 28821 nm (C090 times 105 Lmolminus1 cmminus1) MW 31733 FTIR (KBr) 120592max cm

minus11220 (m -O-C stretching) 1635 (s =C-NH aliphatic amine)1702 (s C=O) 2796 (m -N-CH

3stretching) 2812 (exocyclic

CH) 3081 (m CH-NH stretching) MW 31707 ESIMS 11989811991130906 (M + 2) Anal Calcd C

12H11N7O2S () C 4542 H

349 N 3090 Found () C 4240 H 352 N 3092

225 5-[(2-Hydroxy-9H-purin-6-ylamino)-methylene]-pyr-imidine-246-trione (BG) Yellow powder yield 74 mp250∘C 1H NMR (400MHz DMSO) 404 120575 ppm (1H ddexocyclic NH of purine ring 119895 = 1568Hz) 697 120575 ppm (1Hs -CH of purine ring) 806 120575 ppm (1H dd exocyclic CH ofpyrimidine ring 119895 = 2476Hz) 815 120575 ppm (1H dd NH ofpurine ring) 1115 120575 ppm (1H s NH of pyrimidine ring) 1127120575 ppm (1H s NH of pyrimidine ring) 1230 120575 ppm (1H s OHof purine ring) 13C NMR (400MHz DMSO) 5808 (C-18C-17) 7535 (C-5) 11456 (C-9) 11487 (C-8) 12295 (C-11 C-15) 12927 (C-12 C-14) 13117 (C-10 C-13) 158 (C-7) 16266(C-4 C-5) 16705 (C-2) 120575 ppm FTIR (KBr) 120592max cm

minus1 1221(m -O-C stretching) 1629 (s =C-NH aliphatic amine) 1691(s C=O) 3070 (m CH-NH stretching) 3345 (C-OH) 120582max276 nm (C 095 times 105 Lmolminus1 cmminus1) MW 28921 ESIMS119898119911 28920 (M) Anal Calcd For C

10H7N7O4() C 4153

H 244 N 3390 Found () C 4249 H 246 N 3389

226 5-[(2-Hydroxy-9H-purin-6-ylamino)-methylene]-2-thi-oxo-dihydro-pyrimidine-46-dione (TBG) Off-white yield84 mp gt250∘C 1HNMR (400MHz DMSO) 409 120575 ppm(1H dd exocyclic NH of purine ring 119895 = 1498Hz) 693120575 ppm (2H s -CH of purine ring) 803 120575 ppm (1H ddexocyclic CH of pyrimidine ring 119895 = 2326Hz) 1097 120575ppm (1H dd NH of purine ring) 1117 120575 ppm (1H s NH ofpyrimidine ring) 1126 120575 ppm (1H s NH of pyrimidine ring)1236 120575 ppm (1H s OH of purine ring) 13C NMR (400MHzDMSO) 7453 (C-5) 11326 (C-9) 11359 (C-8) 12292 (C-13C-14) 12651 (C-12 C-15) 12939 (C-11) 13178 (C-10) 15723(C-7) 16266 (C-4 C-6) 16862 (C-2) 120575 ppm FTIR (KBr)120592max cm

minus1 1215 (m -O-C stretching) 1362 (s C-NH aromaticamine) 1632 (s =C-NH aliphatic amine) 1698 (s C=O) 2821(exocyclic CH) 3076 (m CH-NH stretching) 3342 (b C-OH) 120582 max 29103 nm (C 095 times 105 Lmolminus1 cmminus1) MW30527 ESIMS119898119911 30629 (M+ 1) Anal Calcd C

10H7N7O3S


() C 3934 H 231 N 3212 Found () C 3931 H 235 N3209

227 5-(((2-Hydroxypyrimidin-4-yl)amino)methylene)pyr-imidine-246-(1H3H5H)-trione (BC) Yellow powder yield82 mp gt250∘C 1H NMR (400MHz DMSO) 406 120575ppm (1H dd exocyclic NH of purine ring 119895 = 1568Hz)698 120575 ppm (2H s -CH of cytosine ring) 811 120575 ppm (1Hdd exocyclic CH of pyrimidine ring 119895 = 2360Hz) 816 120575ppm (1H dd NH of cytosine ring) 1118 120575 ppm (1H s NHof pyrimidine ring) 1126 120575 ppm (1H s NH of pyrimidinering) 13C NMR (400MHz DMSO) 7853 (C-7) 11326(C-9) 11368 (C-8) 12129 (C-13) 12643 (C-12 C-14) 12958(C-11 C-15) 13318 (C-10) 15753 (C-7) 16135 (C-4 C-6)16934 (C-2) 120575 ppm FTIR (KBr) 120592max cm

minus1 1209 (m -O-Cstretching) 1354 (s C-NH aromatic amine) 1632 (s =C-NHaliphatic amine) 1694 (s C=O aromatic and 120572120573-unsaturatedketone) 3072 (m CH-NH stretching) 120582 max 28634 nm(C 114 times 105 Lmolminus1 cmminus1) MW 24918 ESIMS119898119911 25114(M + 2) Anal Calcd for C

9H7N5O4() C 4338 H 283 N

2811 Found () C 4340 H 279 N 2812

228 5-((2-Hydroxypyrimidine-4-ylamino)methylene)-dihy-dro-2-thioxopyrimidine-46(1H5H)-dione (TBC) Darkorange powder yield 75 mp gt250∘C 1HNMR (400MHzDMSO) 409 120575 ppm (1H dd exocyclic NH of purine ring119895 = 138Hz) 695 120575 ppm (2H s -CH of cytosine ring)807 120575 ppm (1H dd exocyclic CH of pyrimidine ring119895 = 2366Hz) 818 120575 ppm (1H dd NH of cytosine ring) 1112120575 ppm (1H s NH of pyrimidine ring) 1119 120575 ppm (1H s NHof pyrimidine ring) 13C NMR (400MHz DMSO) 77 (C-5)11345 (C-9) 11376 (C-8) 12178 (C-13) 12634 (C-12 C-14)12987 (C-11 C-15) 13222 (C-10) 15825 (C-7) 16615 (C-4C-6) 16926 (C-2) 120575 ppm FTIR (KBr) 120592max cmminus1 1213 (m-O-C stretching) 1626 (s =C-NH aliphatic amine) 1699 (sC=O) 2809 (exocyclic CH) 3078 (m CH-NH stretching)120582 max 28446 nm ESIMS 119898119911 28445 (M) (C 107 times105 Lmolminus1 cmminus1) MW 26525 Anal Calcd C

9H7N5O3S

() C 4075 H 266 N 2640 Found () C 4071 H 269 N2642

229 5-((16-Dihydro-2-hydroxypyrimidine-4-ylamino)-methylene)-13-dimethylpyrimidine-246(1H3H5H)-trione(13-BC) Yellow powder yield 65 mp gt250∘C 1HNMR (400MHz DMSO) 273 (6H s two CH

3group of

pyrimidine ring) 407 120575 ppm (1H dd exocyclic NH of purinering 119895 = 1452Hz) 692 120575 ppm (2H s -CH of cytosinering) 813 120575 ppm (1H dd exocyclic CH of pyrimidine ring119895 = 2362Hz) 815 120575 ppm (1H dd NH of cytosine ring) 13CNMR (400MHz DMSO) 266 (N-CH

3) 359 (C-11) 795

(C-5) 965 (C-12) 1409 (C-8) 1461 (C-7) 1624 (C-4 C-6)16954 (C-10) 1726 (C-2) 120575 ppm FTIR (KBr) 120592max cm

minus1 1217(m -O-C stretching) 1634 (s =C-NH aliphatic amine) 1708(s C=O) 2809 (m -N-CH

3stretching) 2824 (exocyclic CH)

3082 (m CH-NH stretching) 120582 max 29244 nm (C 105 times105 Lmolminus1 cmminus1) MW 27724 ESIMS 119898119911 27822 (M + 1)

Anal Calcd C11H13N5O4() C 4731 H 469 N 2508

Found () C 4729 H 474 N 2509

2210 5-((16-Dihydro-2-hydroxypyrimidin-4-ylamino)meth-ylene)-dihydro-13-dimethyl-2-thioxopyrimidine-46(1H5H)-dione (13-TBC) Orange powder yield 65 mp gt250∘C1H NMR (400MHz DMSO) 278 (6H s two CH

3group of


3) 397 (C-11) 788




3068 (m CH-NH stretching) 120582 max 28117 nm (C 095 times105 Lmolminus1 cmminus1) MW 29330 ESIMS119898119911 29527 (M + 2)Anal Calcd C

11H13N5O3S () C 4474 H 444 N 2371

Found () C 4471 H 446 N 2370

2211 5-((Pyridin-2-ylamino)methylene)pyrimidine-246(1H3H5H)-trione (2-BAP) Yellow powder yield 65 mpgt250∘C 1H NMR (400MHz DMSO) 405 120575 ppm (1H ddexocyclic NH of pyridine ring 119895 = 1638Hz) 659ndash809120575 ppm (4H m -CH of Pyridine ring) 823 120575 ppm (1Hdd exocyclic CH of pyrimidine ring 119895 = 2368Hz) 1089120575 ppm (1H s NH of pyrimidine ring) 1121 120575 ppm (1Hs NH of pyrimidine ring) 13C NMR (400MHz DMSO)802 (C-5) 1085 (C-13) 1137 (C-11) 1382 (C-12) 1478 (C-10) 1513 (C-2) 1544 (C-7) 1647 (C-4 C-6) 120575 ppm FT-IR (KBr) 120592max cmminus1 1218 (m -O-C stretching) 1622 (=C-NH aliphatic amine) 1697 (C=O) 2815 (exocyclic CH)3078 (CH-NH stretching) 120582 max 28817 nm (C 095 times105 Lmolminus1 cmminus1) MW 23220 ESIMS 119898119911 23218 (M)Anal Calcd C

10H8N4O3() C 5173 H 347 N 2413 Found

() C 5174 H 345 N 2415

2212 5-((5-Methylthiazol-2-ylamino)methylene)pyrimidine-246(1H3H5H)-trione (BAMT) Orange powder yield 65mp gt250∘C 1HNMR (400MHz DMSO) 239 (3H s -CH

3

group of thiazol ring) 406 120575 ppm (1H dd exocyclic NHof thiazol ring 119895 = 1538Hz) 717 120575 ppm (1H s -CH ofthiazol ring) 817 120575 ppm (1H dd exocyclic CH of pyrimidinering 119895 = 2436Hz) 1096 120575 ppm (1H s NH of pyrimidinering) 1126 120575 ppm (1H s NH of pyrimidine ring) 13C NMR(400MHzDMSO) 176 (-CH

3) 788 (C-5) 1207 (C-11) 1349

(C-12) 1504 (C-2) 1617 (C-9) 1653 (C-4 C-6) 1675 (C-7)120575 ppm FT-IR (KBr) 120592max cmminus1 1215 (m -O-C stretching)1256 (C-S) 1625 (=C-NH aliphatic amine) 1693 (C=O) 2236(C-N of thiazol ring) 2794 (-CH


CH) 3073 (CH-NH stretching) 120582max 28117 nm (C 095 times105 Lmolminus1 cmminus1) MW 25225 ESIMS 119898119911 25305 (M + 1)Anal Calcd C

9H8N4O3S () C 4285H 320N 2221 Found

() C 4282 H 321 N 2223


23 Pharmacology231 Biological Evaluation of the Isolated Compounds DNA(Calf Thymus type 1) bleomycin sulfate butylated hydrox-yanisole thiobarbituric acid (TBA) ethylenediaminete-traacetic acid (EDTA) and ascorbic acid were obtainedfrom Sigma 221015840-azo-bis-(2-amidinopropane) dihydrochlo-ride and ABTS were purchased fromWako Co USA

232 Antioxidant Screening Assay (ABTS Method) 221015840-azino-bis-3-ethylbenzthiazol-ine-6-sulfonic acid (ABTS)waspurchased from Wako Co USA L-ascorbic acid wasobtained from Sigma and all other chemicals were of thehighest quality available For each of the investigated com-pounds (2mL) ABTS solution (60 120583M) was added to 3mLMnO2solution (25mgmL) all prepared in (5mL) aqueous

phosphate buffer solution (pH 7 01M) The mixture wasshaken centrifuged and filtered and the absorbance of theresulting green-blue solution (ABTS radical solution) at734 nm was adjusted to approx 05 Then 50 120583L of (2mM)solution of the tested compound in spectroscopic gradeMeOHphosphate buffer (1 1) was added The absorbancewas measured and the reduction in color intensity wasexpressed as inhibition percentage L-ascorbic acid was usedas standard antioxidant (positive control) Blank samplewas run without ABTS using MeOHphosphate buffer (1 1)instead of tested compounds Negative control was runwith ABTS and MeOHphosphate buffer (1 1) only Theabsorbance 119860(test( was measured and the reduction in colorintensity was expressed as inhibition The inhibition foreach compound was calculated from the following equation

inhibition = [119860 (control) minus 119860 (test)119860 (control)

] times 100 (1)

Ascorbic acid (vitamin C) was used as standard antioxi-dant (positive control) Blank sample was run without ABTSusing methanolphosphate buffer (1 1) instead of sampleNegative control sample was run with methanolphosphatebuffer (1 1) instead of tested compound [13 14]

233 Antioxidant Activity Screening Assay 221015840-Azino-bis-3ethylbenzthiazoline-6-sulfonic Acid Method For each of theinvestigated compounds 2mL of ABTS solution (60120583M)was added to 3mL MnO

2solution (25mgmL) all prepared

in 5mL aqueous phosphate buffer solution (pH 7 01M)The mixture was shaken centrifuged and filtered and theabsorbance of the resulting green-blue solution (ABTS rad-ical solution) at 120582 734 nm was adjusted to approximately ca05 Then 50120583L of (2mM) solution of the tested compoundin spectroscopic grade MeOHphosphate buffer (1 1) wasadded The absorbance was measured and the reductionin color intensity was expressed as inhibition percentageL-ascorbic acid was used as standard antioxidant (positivecontrol) Blank sample was run without ABTS and usingMeOHphosphate buffer (1 1) instead of tested compounds[15 16]

234 Bleomycin-Dependent DNA Damage Assay The reac-tion mixture contained DNA (05mgmL) bleomycin sulfate

(005mgmL) MgCl2(5mM) FeCl

3(50mM) and samples

to be tested in a conc of 01mgmL L-ascorbic acid was usedas positive control The mixture was incubated at 37∘C for1 hour The reaction was terminated by addition of 005mLEDTA (01M) The color was developed by adding 05mLTBA (1wv) and 05mLHCl (25 vv) followed by heatingat 80∘C for 10 minutes After centrifugation the extent ofDNA damage was measured by increase in absorbance at532 nm [16]

235 Lymphocyte Transformation Assay The viable lympho-cytes were adjusted to a concentration of 2 times 106 cellsmLin RPMI-1640 medium supplemented with 600120583L peni-cillin 01mL streptomycin 1 glutamine 25 HEPES (N-2 hydroxyethylpiperazine-N-D2-ethanesulfonic acid)-bufferand 20 fetal calf serum (FCS)The lymphocytes were platedinto 96-well tissue culture plates (or Eppendorf tubes) 100120583Lof the volatile oil solution in DMF (100 120583LmL) and 20 120583g ofthe mitogen (PHA) were added to each well Cell cultureswere incubated at 37∘C in 5 CO

2atmosphere for 72 h

during which the mitogen produces its maximal effect onDNA synthesis After culture 237 cell films were stained byGiemsa stain and average count of percentage of transformed(proliferated) blasts was determined Aqueous Echinacea pur-purea extract (Immulon) and levamisole (Ketrax) were usedas positive control (standard immunostimulant) 100 120583gmLof each drug in DMSO [17]

3 Result and Discussion

31 Chemistry The synthetic strategy adopted to obtain thetarget compounds is depicted in Scheme 1 Various nucleicacid bases such as adenine guanine cytosine and hetero-cyclic amines were refluxed with formic acid in aqueousmedium at 60∘C to yield formamide derivatives of corre-sponding DNA bases Thereafter in situ barbituric acid andits analogs were refluxed giving the corresponding DNAbased barbiturates [18]

The important infrared spectral bands and their tentativeassignments for DNA-based barbiturates were recorded asKBr disks and are presented The IR spectra of synthesizedcompounds revealed characteristic bands between 2808 cmminus1and 2826 cmminus1 confirming the presence of exocyclic CHgroups IR spectrum of the compounds showed characteristicbands between 1691 cmminus1 and 1708 cmminus1 confirming thepresence of C=O groups1H NMR spectra revealed signals at 350 120575 ppm for

DMSO solvent between 42 and 409 120575 ppm for NH ofthe DNA base between 803 and 817 120575 ppm for exocyclicCH group and between 809 and 1127 120575 ppm for NH ofpyrimidine ring From 13CNMR spectra exocyclic CH signalwas observed between 1445 and 15825 120575 ppm The UVabsorption spectra were made using DMSO as a solventin concentrations (10minus5M) All synthesized new DNA-basedbarbiturates derivatives showed the strong absorption bands(120582max) in the range 275ndash305 nm owing to the 120587-120587lowast and 119899-120587lowasttransitions as well as presence of chromophoric exocyclic CHof pyrimidine ring in their UV spectra [19]


Table 1 Biological activities of compounds towards lymphocyte transformation ABTS (free radical scavenging) and bleomycin-dependentDNA damage activities

Compound codeLymphocyte

transformation assayat 50 120583mabg

ABTS (percentageof scavenging inhibition)cdefg

Bleomycin-dependentDNA damage assaygh

absorbance(120582max-532)

Negative control mdash mdash mdashButylated hydroxyanisole mdash mdash 00026Vitamin C (ascorbic acid) mdash mdash 00038 plusmn 001BA 19 343 plusmn 010 0210 plusmn 112TBA 53 605 plusmn 000 0014 plusmn 01513-BA 35 417 plusmn 115 0015 plusmn 02513-TBA 35 258 plusmn 130 0014 plusmn 131BG 37 495 plusmn 004 0017 plusmn 003TBG 45 332 plusmn 070 0016 plusmn 027BC 25 311 plusmn 238 0070 plusmn 020TBC 68 708 plusmn 010 0011 plusmn 00413-BC 20 354 plusmn 024 0017 plusmn 00513-TBC 10 133 plusmn 014 0210 plusmn 1182-BAP 35 307 plusmn 112 0011 plusmn 027BAMT 32 408 plusmn 010 0013 plusmn 005aConcentration showing 50 lymphocyte transformation bThe positive control was Echinacea purpurea (Ech) extract at concentration 50 giving 74lymphocyte transformation cABTS + scavenging activity () = [(Ac minusAs)Ac] times 100 where Ac is the absorbance value of the control and As is the absorbancevalue of the added samples test solution dThe concentration of the pure compounds was 2mM eThe concentration showing 50 inhibition is expressed inmM fThe positive control was vitamin C and showed 800 plusmn 104 gValues are means of 3 replicates plusmn SD and showed significant difference at 119875 lt 005 byStudentrsquos t-test hThe positive control was vitamin C (024mM) and showed absorbance 00038 plusmn 001 at the same concentration of the tested compounds

4 Pharmacology

A variety of DNA base and heterocyclic primary amines-based barbiturates were tested for antioxidant activity asreflected in the ability to control of ABTS assays The pro-oxidant activities of the aforementioned derivatives wereassessed by their effects on bleomycin-inducedDNAdamageTheDNAbase and heterocyclic primary amines-based barbi-turates manifested potent antioxidative activity in the ABTSassay

All compounds have been tested to bleomycin-dependentDNA damage The results indicated that they may have someprotective activity to DNA by certain mechanism A series ofcompounds (TBC TBA BG 1 3-BA and BAMT) exhibiteda high antioxidant activity On the other hand all compoundsshow potent protective effect on the DNA from the induceddamage by bleomycin with reference to ascorbic acid andbutylated hydroxyanisole (Table 1)

Lymphocyte transformation assay involves study of aspecific immune response at 50120583MThe assay investigates themitogenic effect of synthesized DNA base and heterocyclicprimary amines-based barbiturates on T-lymphocytes Thepositive control was Echinacea purpurea (Ech) extract atconcentration 50120583M giving 74 of lymphocyte transforma-tion Data obtained from Table 1 indicated compound TBCshows that 68 lymphocyte transformed at concentrationsimilar to standard while 35ndash55 lymphocyte transformed

by synthesized barbituratesTBATBG 13-BA 13-TBABGand BPA

Barbiturate is a versatile moiety that exhibits a widevariety of biological activities Barbiturate moiety acts asldquohydrogen binding domainrdquo and ldquoelectron donor systemrdquoIt also acts as a constrained pharmacophore Many drugscontaining barbituric acid nucleus are available on themarketsuch as allobarbital alphenal thiopental and cyclobarbital

Thioxodihydropyrimidine can act as the bioisostericreplacement of the pyrimidine moiety Members of this ringsystem have found their way into such diverse applica-tion as pharmaceuticals oxidation inhibitors cyanine dyesand metal complexing agents Barbiturate derivatives haveantimicrobial anti-inflammatory anticancer anticonvulsantantidepressant antioxidant radio protective and antileish-manial activities From the obtained results (Table 1) it canbe concluded that pyrimidine moiety is essential for the pro-tection activity against DNA damage induced by bleomycin-iron complex and pyrimidine ring is also required for theactivity and an unsubstituted phenyl ring exhibited betteractivity than those substituted derivatives This good activityof thioxodihydropyrimidine derivatives shows the hypothesisof a direct link between thiol function and an aromaticring to be a good one The thiol catches the radical andafterwards the aromatic ring permits the trapping of thisradical Moreover thiobarbituric acid derivative shows abetter activity than barbituric acid


5 Conclusion

In conclusion we have designed hybrid molecules on thebasis of the biological significance of nucleobase and barbi-turic acid and evaluated their anticancer activities All of thenew synthesized compounds were tested for their protectionactivity against DNA damage induced by bleomycin-ironcomplex Compounds TBC and 2-BAP showed the highestprotection activity against DNA damage On the other handcompounds BA and 13-TBC showed a very low activitywhereas the rest of the tested compounds exhibited well tomoderate activity It can be concluded that barbituric acidmoiety is essential for the protection activity against DNAdamage induced by bleomycin-iron complex and thiobarbi-turic acid ring is also required for the activity On the otherhand unsubstituted barbituric acid or thiobarbituric acidexhibited better activity than those substituted derivativesthat is 13-dimethyl substituted derivatives Out of a set of 12molecules four compoundsTBCTBABG andTBG exhibitsignificant antioxidant bleomycin-dependent DNA damageand lymphocyte transformation assay activities and could beused as leads for further investigations

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

All authors have equally contributed to this work

Acknowledgments

The authors are very much thankful to the Depart-ment of Applied Chemistry SVNIT Surat for provid-ing laboratory facilities and for the financial support byGujarat Council on Science and Technology (Project noGujcostMRP20204212-1304) For characterization theyare thankful to Mr D M Thumar from Amoli Organic PvtLtd Baroda Gujarat Avtarsingh and Manishkumar fromSAIF Panjab University Chandigarh India are gratefullyacknowledged

References

[1] M A Al-Omar K M Youssef M A El-Sherbeny S AAwadalla and H I El-Subbagh ldquoSynthesis and in vitro antiox-idant activity of some new fused pyridine analogsrdquo Archiv derPharmazie vol 338 no 4 pp 175ndash180 2005

[2] V P Memon and A R Sudheer ldquoAntioxidant and anti-inflammatory properties of curcuminrdquo inTheMolecular Targetsand Therapeutic Uses of Curcumin in Health and Disease B BAggarwal Y Surh and S Shishodia Eds pp 106ndash107 SpringerNew York NY USA 2007

[3] V B Djordjevic ldquoFree radicals in cell biologyrdquo InternationalReview of Cytology vol 237 pp 57ndash89 2004

[4] Z Nace and K Danijel ldquoPreparation and reactivity of 5-benzylidenebarbituric and 5-benzylidene-2-thiobarbituricacidsrdquo Acta Chimica Slovenica vol 58 no 1 pp 151ndash157 2011

[5] H S Basavaraja K V Jayadevaiah M M Hussain M MJ V Kumar and P Basavaraj ldquoSynthesis of novel piperazineand morpholine linked substituted pyrimidine derivatives asantimicrobial agentsrdquo Journal of Pharmaceutical Sciences andResearch vol 2 no 1 pp 5ndash12 2010

[6] A Holy I Votruba M Masojıdkova et al ldquo6-[2-(Phospho-nomethoxy)alkoxy]pyrimidines with antiviral activityrdquo Journalof Medicinal Chemistry vol 45 no 9 pp 1918ndash1929 2002

[7] P Andres and A Marhold ldquoA new synthesis of 5-trifluoro-methyluracilrdquo Journal of Fluorine Chemistry vol 77 no 1 pp93ndash95 1996

[8] J J Reddick S Saha J-M Lee J S Melnick J Perkinsand T P Begley ldquoThe mechanism of action of bacimethrina naturally occurring thiamin antimetaboliterdquo Bioorganic ampMedicinal Chemistry Letters vol 11 no 17 pp 2245ndash2248 2001

[9] Y Kashman S Carmely D Blasberger S Hirsch and DGreen ldquoMarine natural products new results from Red Seainvertebratesrdquo Pure and Applied Chemistry vol 61 no 3 pp517ndash520 1989

[10] B D Dhorajiya B S Bhakhar and B Z Dholakiya ldquoSynthesischaracterization solvatochromic properties and antimicro-bial evaluation of 5-acetyl-2-thioxo-dihydro-pyrimidine-46-dione-based chalconesrdquoMedicinal Chemistry Research vol 22no 9 pp 4075ndash4086 2013

[11] J R Patel B D Dhorajiya F A Badria A S Ibrahim andB Z Dholakiya ldquoIn-vitro cytotoxicity antioxidant bleomycin-dependent DNA damage and immunomodulatory evaluationof 1-(4-acetylphenyl)-3-aryloxypyrrolidine-2 5-dione basedderivativesrdquoMedicinal Chemistry Research 2014

[12] B D Dhorajiya and B Z Dholakiya ldquoGreen chemistry multi-component protocol for formylation and Knoevenagel conden-sation for synthesis of (Z)-5-arylaminomethylene pyrimidine 24 6-trione derivatives in waterrdquo Green Chemistry Letters andReviews vol 7 no 1 pp 1ndash10 2014

[13] B F Abdel-Wahab A-A S El-Ahl and F A Badria ldquoSynthesisof new 2-naphthyl ethers and their protective activities againstDNA damage induced by bleomycin-ironrdquo Chemical and Phar-maceutical Bulletin vol 57 no 12 pp 1348ndash1351 2009

[14] Y Morimoto K Tanaka Y Iwakiri S Tokuhiro S Fukushimaand Y Takeuchi ldquoProtective effects of some neutral aminoacids against hypotonic hemolysisrdquoBiologicalampPharmaceuticalBulletin vol 18 no 10 pp 1417ndash1422 1995

[15] E A Lissi B Modak R Torres J Escobar and A Urzua ldquoTotalantioxidant potential of resinous exudates from Heliotrapiumspecies and a comparison of the ABTS and DPPH methodsrdquoFree Radical Research vol 30 no 6 pp 471ndash477 1999

[16] A B A El-Gazzar M M Youssef A M S Youssef A A Abu-Hashem and F A Badria ldquoDesign and synthesis of azolopy-rimidoquinolines pyrimidoquinazolines as anti-oxidant anti-inflammatory and analgesic activitiesrdquo European Journal ofMedicinal Chemistry vol 44 no 2 pp 609ndash624 2009

[17] B R Mikhaeil G T Maatooq F A Badria andMM A AmerldquoChemistry and immunomodulatory activity of frankincenseoilrdquo Zeitschrift fur Naturforschung C vol 58 no 3-4 pp 230ndash238 2003

[18] B D Dhorajiya A S Ibrahim F A Badria and B Z DholakiyaldquoDesign and synthesis of novel nucleobase-based barbituratederivatives as potential anticancer agentsrdquoMedicinal ChemistryResearch vol 23 no 2 pp 839ndash847 2014

[19] R M Silverstein and F X Webster Spectrometric IdentificationofOrganic Compounds JohnWileyamp SonsNewYorkNYUSA6th edition 1997

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


O O

O

O

O

O

EtO OEtCC

H2

X

X

X

NHR

NHR

N

N

N

N

R

R

R

R

+

NH-R1

(a)

(b)

R = ndashH or ndashCH3

X = O and S

Scheme 1 Synthesis of DNA base and heterocyclic primary amines-based barbiturates Reagents and conditions (a) NaOMeMeOH-refluxed(b) HCO

2H water reflux where R1 = adenine guanine cytosine and primary heterocyclic amine

are pharmacologically active compounds [4] The medicinalimportance of pyrimidine derivatives such as barbituric acidand thiobarbituric acid plays vital role among various hetero-cyclic compounds due to their antineoplastic [5 6] antiviral[7] antibiotic [8] and anti-inflammatory [9] activity Thepyrimidine ring system is present in various natural com-pounds such as nucleic acids vitamins coenzymes uric acidpurines and some marine microorganisms (eg sponge)[10] Cyclic imides are an important class of moleculesknown for their diverse array of bioactivitiesThese biologicalactivities and various pharmacological uses of cyclic imideshave been attributed to their unique structural features [11]In view of these facts and in continuation of our interest in thesynthesis of a variety of heterocycles of biological importancewe report here an efficient and convenient method forthe synthesis of novel nucleobased barbiturates derivativesattached to pyrimidine moiety The synthesized compoundswere evaluated for their antioxidant protection activitiesagainst DNA damage induced by bleomycin-iron assay andthis may help to reduce the side effects of chemotherapy forcancer patients

2 Materials and Methods

Chemicals and solvents were obtained from commercialsources and used as received throughout the investigationThe barbituric acid was synthesized by using diethyl mal-onate and urea using standard procedure Melting pointswere determined in open capillaries on a Veego electronicapparatus VMP-D (Veego Instrument CorporationMumbaiIndia) and are uncorrected IR spectra (4000ndash400 cmminus1) ofsynthesized compounds were recorded on a Perkin Elmer-Spectrum RX-IFTIR spectrophotometer using KBr pelletsThin layer chromatography was performed on object glass

slides (2 times 75 cm) coated with silica gel-G and spots werevisualized under UV irradiation 1H NMR and 13C NMRspectra were recorded on an Avance-II (Bruker) model usingDMSO as a solvent and TMS as internal standard with 1Hresonant frequency of 400MHz and 13C resonant frequencyof 100MHzThe 1HNMR and 13CNMR chemical shifts werereported as parts per million (ppm) downfield from TMS(Me4Si) The splitting patterns are designated as follows s

singlet br s broad singlet d doublet t triplet q quartetm multiplet UV spectra were recorded on Maya pro 2000(Ocean Optics USA) using DMSO as a solvent with 10minus5Msolution

21 Synthesis of Barbituric Acid As shown in Scheme 1 Toa solution of diethylmalonate (20 g 1189mmol) and ureathiourea and its methyl substituted analogues were (75 g985mmol) in methanol anhydrous sodium methoxide wasadded and refluxed at 65∘C for 8 h A white solid separatesThen in the above reaction mixture 125mL of hot (50∘C)water was added and hydrochloric acid was used to makethe solution acidic After the completion of the reactionthe resulting clear solution was filtered and cooled in anice bath overnight The white product formed was filteredwashed with 50mL of cold water dried and recrystallizedfrom acetone to afford compound as a white powder [12]

211 Synthesis of DNA-Based Barbiturates As shown inScheme 1 a solution of the corresponding DNA-bases (egadenine 1mmol) and formic acid (4mmol) in distilled wateras a green solvent was allowed to reflux at 60∘C over a periodof 2-3 h Then it was cooled down until the reaction mixturebecomes a clear solution The completion of reaction wasconfirmed by TLC In situ barbiturates were added againand refluxed further for 3-4 h As the reaction proceeds the





10H7N7O3() C 4396 H 258 N 3589

Found () C 4394 H 257 N 3591


10H7N7O2S () C 4152 H 244 N 3389 Found

() C 4248 H 248 N 3390


3





4781 H 370 N 3256







12H11N7O2S () C 4542 H

349 N 3090 Found () C 4240 H 352 N 3092



10H7N7O4() C 4153

H 244 N 3390 Found () C 4249 H 246 N 3389



10H7N7O3S


() C 3934 H 231 N 3212 Found () C 3931 H 235 N3209



9H7N5O4() C 4338 H 283 N

2811 Found () C 4340 H 279 N 2812


9H7N5O3S

() C 4075 H 266 N 2640 Found () C 4071 H 269 N2642


3group of


3) 359 (C-11) 795






Found () C 4729 H 474 N 2509


3group of


3) 397 (C-11) 788





11H13N5O3S () C 4474 H 444 N 2371

Found () C 4471 H 446 N 2370


10H8N4O3() C 5173 H 347 N 2413 Found

() C 5174 H 345 N 2415


3


3) 788 (C-5) 1207 (C-11) 1349




9H8N4O3S () C 4285H 320N 2221 Found

() C 4282 H 321 N 2223






] times 100 (1)







3(50mM) and samples

















4 Pharmacology








5 Conclusion






Acknowledgments


References
























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





10H7N7O3() C 4396 H 258 N 3589

Found () C 4394 H 257 N 3591


10H7N7O2S () C 4152 H 244 N 3389 Found

() C 4248 H 248 N 3390


3





4781 H 370 N 3256







12H11N7O2S () C 4542 H

349 N 3090 Found () C 4240 H 352 N 3092



10H7N7O4() C 4153

H 244 N 3390 Found () C 4249 H 246 N 3389



10H7N7O3S


() C 3934 H 231 N 3212 Found () C 3931 H 235 N3209



9H7N5O4() C 4338 H 283 N

2811 Found () C 4340 H 279 N 2812


9H7N5O3S

() C 4075 H 266 N 2640 Found () C 4071 H 269 N2642


3group of


3) 359 (C-11) 795






Found () C 4729 H 474 N 2509


3group of


3) 397 (C-11) 788





11H13N5O3S () C 4474 H 444 N 2371

Found () C 4471 H 446 N 2370


10H8N4O3() C 5173 H 347 N 2413 Found

() C 5174 H 345 N 2415


3


3) 788 (C-5) 1207 (C-11) 1349




9H8N4O3S () C 4285H 320N 2221 Found

() C 4282 H 321 N 2223






] times 100 (1)







3(50mM) and samples

















4 Pharmacology








5 Conclusion






Acknowledgments


References
























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


() C 3934 H 231 N 3212 Found () C 3931 H 235 N3209



9H7N5O4() C 4338 H 283 N

2811 Found () C 4340 H 279 N 2812


9H7N5O3S

() C 4075 H 266 N 2640 Found () C 4071 H 269 N2642


3group of


3) 359 (C-11) 795






Found () C 4729 H 474 N 2509


3group of


3) 397 (C-11) 788





11H13N5O3S () C 4474 H 444 N 2371

Found () C 4471 H 446 N 2370


10H8N4O3() C 5173 H 347 N 2413 Found

() C 5174 H 345 N 2415


3


3) 788 (C-5) 1207 (C-11) 1349




9H8N4O3S () C 4285H 320N 2221 Found

() C 4282 H 321 N 2223






] times 100 (1)







3(50mM) and samples

















4 Pharmacology








5 Conclusion






Acknowledgments


References
























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






] times 100 (1)







3(50mM) and samples

















4 Pharmacology








5 Conclusion






Acknowledgments


References
























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of









4 Pharmacology








5 Conclusion






Acknowledgments


References
























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


5 Conclusion






Acknowledgments


References
























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

research article nucleobase-based barbiturates:...

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