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Research ArticleSynthesis X-Ray Crystal Structures Biological Evaluation andMolecular Docking Studies of a Series of Barbiturate Derivatives
Assem Barakat12 Hazem A Ghabbour34 Abdullah Mohammed Al-Majid1
Qurat-ul-ain5 Rehan Imad5 Kulsoom Javaid5 Nimra Naveed Shaikh5 Sammer Yousuf5
M Iqbal Choudhary15 and Abdul Wadood6
1Department of Chemistry College of Science King Saud University PO Box 2455 Riyadh 11451 Saudi Arabia2Department of Chemistry Faculty of Science Alexandria University PO Box 426 Ibrahimia Alexandria 21321 Egypt3Department of Pharmaceutical Chemistry College of Pharmacy King Saud University PO Box 2457 Riyadh 11451 Saudi Arabia4Department of Medicinal Chemistry Faculty of Pharmacy Mansoura University Mansoura 35516 Egypt5HEJ Research Institute of Chemistry International Center for Chemical and Biological Sciences University of KarachiKarachi 75270 Pakistan6Department of Biochemistry Abdul Wali Khan University Mardan 23200 Pakistan
Correspondence should be addressed to Assem Barakat ambarakatksuedusa
Received 4 February 2016 Revised 6 April 2016 Accepted 10 April 2016
Academic Editor Gustavo Portalone
Copyright copy 2016 Assem Barakat et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
A series of barbiturates derivatives synthesized and screened for different set of bioassays are describedThemolecular structures ofcompounds 5a 5d and 5f were solved by single-crystal X-ray diffraction techniquesThe results of bioassay show that compounds4a 4b 4c 4d 4e 4f and 4g are potent antioxidants in comparison to the tested standards butylated hydroxytoluene (BHT) and119873-acetylcysteine Compounds 4andash4e (IC
50= 1018plusmn08ndash1244plusmn44 120583M) and4g (IC
50= 1041plusmn19 120583M)weremore potent antioxidants
than the standard (BHT IC50= 1288 plusmn 21 120583M)The enzyme inhibition potential of these compounds was also evaluated in vitro
against thymidine phosphorylase 120572-glucosidase and 120573-glucuronidase enzymes Compounds 4c 4h 4o 4p 4q 5f and 5m werefound to be potent 120572-glucosidase inhibitors and showed more activity than the standard drug acarbose whereas compounds 4vand 5h were found to be potent thymidine phosphorylase inhibitors more active than the standard drug 7-deazaxanthine Allbarbiturates derivatives (4andash4x 4z and 5andash5m) were found to be noncytotoxic against human prostate (PC-3) Henrietta Lackscervical (HeLa) andMichigan Cancer Foundation-7 breast (MCF-7) cancer cell lines and 3T3 normal fibroblast cell line except 4ywhich was cytotoxic against all the cell lines
1 Introduction
Derivatives of 13-dimethylbarbituric acid including pyrim-idine-246-trione (PYT) heterocycles play an important rolein the pharmaceutical chemistry [1 2] Barbituric acid and itsderivatives are well known as hypotensive antibacterial andantisclerotic hypnotic sedative anticonvulsant antispas-modic anticancer matrix metalloproteinase inhibitors anti-convulsants and anti-inflammatory and anxiolytic agents aswell as being used in local anesthetic drugs [2ndash9] Barbituricacid derivatives are also known for their use as clinicaldrugs such as Seconal Veronal phenobarbital bucolome
and sodium pentothal (Figure 1 1ndash5) [10ndash13] The interestingmechanism of action of barbital and phenobarbital againstepilepsy made them attractive targets for medicinal chemistsThey have also been investigated as anticancer and anti-AIDS agents [14] Reactive oxygen species (ROS) are oxygen-centered free radicals such as reactive oxygen species (ROS)which are peroxyls (ROO∙) superoxides (O
2
∙minus) hydroxyls(HO∙) alkoxyls (RO∙) and nitric oxides (NO∙) [15] whilehypochlorous acid (HOCl) hydrogen peroxide (H
2O2) and
singlet oxygen (O2) are classified as nonradical ROSTheROS
may react in various environments [15] However overpro-duction of ROS species has been linked to cell membrane
Hindawi Publishing CorporationJournal of ChemistryVolume 2016 Article ID 8517243 11 pageshttpdxdoiorg10115520168517243
2 Journal of Chemistry
O OS Na
O O O O ON OH
HN HN NH
O
O O
HN NH
O
O O
O O O
O
OO
OO
O
O
OO N N
H
NN
O
O
O O O
O
O O O
OO
N
NH
HN NH
NH
Cl
HN N
1 (Bucolome) 2 (Sodium pentothal) 3 (Seconal) 4 (Phenobarbital)
HO N NNHN
5 (Veronal) 6 (Hsp90 inhibitor)
HNNH
NH
HN
NH
NH
7 (Ro 28-2653 MMP inhibitor) 8 (EM20-25 BCL-2 inhibitor) 9 (TACE inhibitor)
Potent nitric oxide scavenger
NH2Et2
NO2
NO2
oplus
oplus
⊖
⊖
10 IC50 = 69 plusmn 166120583MStd ascorbic acid IC50 = 618 plusmn 20 120583M
Figure 1 Structure of biologically active barbituric acid derivatives
damage protein and enzyme modifications and DNA dam-age within the body [16] These types of changes accelerateaging and play a causative role in a variety of degenerativediseases such as cancers atherosclerosis brain disfunctioninflammation shock and heart diseases Recently Barakatet al [17] have synthesized some novel salts ofMichael adductderived from PYT and evaluated them for NO (nitric oxide)scavenging activity Compound 10 was found to possessseveral hundredfold more activity against nitric oxide radical(IC50values of 69plusmn166 120583M) than the standard drug ascorbic
acid (IC50= 618 plusmn 20 120583M)
Because of the important biological activities PYT iswidely used as a synthon in the design of antitumor agentsIt has a high efficacy to form hydrogen bonding with drugtargets (6ndash9 Figure 1) Singh et al have reported the activityof a series of new N-benzyl indole-pyrimidine-246-trionehybrids against a panel of 60 human tumor cell lines Severalof these analogs were also found to be inhibitors of DNArepair and replication stress response polymerases [18]
The discovery and development of effective antioxidantscapable of supplementing bodyrsquos antioxidant system is animportant area of health care research Similarly researchefforts are focused on the identification of effective and safeinhibitors of 120572-glucosidase as potential antidiabetic agents
The focus in recent years is on the development ofresource-efficient environmentally benign and sustainablechemistry practices In continuation of our work in thisfield we describe here the synthesis of a series of previouslyreported [17 19 20] and new PYT adductsThese compoundswere evaluated for their antioxidant activity by using DDPH
scavenging assay as well as for in vitro enzyme inhibitionactivities against alpha-glucosidase thymidine phosphory-lase and 120573-glucuronidase enzymes
2 Materials and Methods
The chemical reagents used for the preparation of the targetcompounds were commercially available and used withoutfurther purification 1H-NMR and 13C-NMR were measuredusing Bruker Avance AV-600 NMR spectrometer MS werecarried out with a Jeol JMS-600H instrument and single-crystal X-ray diffractionwas collected using a Bruker SMARTAPEX II CCD diffractometer
21 General Procedure for Aldol Condensation Michael Addi-tion for the Synthesis of 4 and 5 (GP1) Amixture of aldehyde3 (15mmol) and 1 and2 (3mmol) aswell as Et
2NH(15mmol
155 120583L) in 3mL of degassed H2O (bubbling nitrogen through
the water) was stirred at room temperature for 1ndash5 h untilTLC showed complete disappearance of the reactants Theprecipitate was removed by filtration and washed with ether(3 times 20mL) The solid product was dried to afford pureproducts 4 and 5
The full characterization of the synthesized compounds 4and 5 and the X-ray crystal data of 5a 5d and 5f can be seenin the SupplementaryMaterials (see supplementarymaterialsavailable online at httpdxdoiorg10115520168517243)
22 Biological Activity The samples of the synthesized com-pound were screened against DPPH radical 120573-glucuronidase
Journal of Chemistry 3
5a 5d
C27C26
C25
01C1
C6
C7C8
C13C12
C15C14
C19
C17C18
C16
C506N1
050204
C2C3
C4
C21
C20C24C23
C11 C9C10C22
01W
03
N2
C21
C4 02
04
C3 C2
C5C6
C7C8
C9
C25
C13C12
C23C11
C22C10
0103
C1
C14C15
C26N1
C16
C17 C27C28
C24
05
C19
C18
C20
5f
C2203
0401
06
05
N1
N2
02
C10
C23 C11
C12 C13
C9
C5
C4
C3
C2C1
C27
C26C16C19
C18 C17
C25 C24
C6C7
C14 C15
C21
C20
C8
Figure 2 ORTEP diagram of 5a 5d and 5f Displacement ellipsoids are plotted at the 40 probability level for non-H atoms
inhibition assay thymidine phosphorylase inhibition assayand 120572-glucosidase inhibition assay and full methods descrip-tions are provided in Supplementary Materials
3 Results and Discussion
31 Chemistry We have been focusing on the developmentof highly expedient methods for the synthesis of diverseheterocyclic compounds of biological importance via one-pot multicomponent reactions (MCRs) and avoiding organicsolvents in organic synthesis [17 19 20] This had led tothe development of several efficient environmentally benignclean and economical process technology (Green ChemistryConcepts) In current reaction strategy equimolar amountsof barbituric acid (1) and dimedone (2) with aldehyde(3) in the presence of aqueous diethylamine medium atRT yielded salts of PYT adducts 4andash4y and 5andash5m wereobtained in quantitative yields by simple filtration [17ndash20]
Products 4s 4t and 5g were identified as new products(Scheme 1)
32 X-Ray Diffraction The molecular structures of com-pounds 5a 5d and 5f were unambiguously deduced bysingle-crystal X-ray diffraction technique (Figure 2) Thecrystals of the synthesized molecules 5a 5d and 5f weregrown by slow diffusion of diethyl ether solution of puresamples 5a 5d and 5f in DCMEtOH at room temperaturefollowed by allowing standing for 24 h The structures wereresolved by direct methods by using the SHELXL97 program[21 22] in the SHELXTL-plus package and refined by afull-matrix least-squares procedure on 1198652 using SHELXS97Diffraction data were collected on a Bruker APEX-II CCDdiffractometer (Bruker AXS GmbH) The crystal structureand refinement data of compounds 5a 5d and 5f are listedin Table 1 The final atomic coordinates for all atoms andcomplete listing of bond distance and angles are presented
4 Journal of Chemistry
O
O O O O
RT
RT RT
ON N
2 1a b
1a b
O
O
O
O O
O
R R
O O O O O
O O
O
O
HOHO
NH
O
HO
O
N NN
NHN
O O HO
HO HO
HO
O O
O
O O
OO
O O O O O
O O
O
OO
O
OO
O O
ONH
O
4l
4z
NH
NH
NH N
NN
NN
H
NN
N NN
H
NH
HN
H NN
NH
N
O O O O
O
O O HO O
OO
N N
NN N
NO2 2
2
3
O
HO
HO
O
ON
N
CHO5l
4h
HOHO
R
NH2Et2
R2
R2
R2
R2
oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus
NH2Et2oplus
⊖
⊖ NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
⊖
NH2Et2oplus
NH2Et2oplus
⊖R1 R1R1
R1
R1
R1
R2R3
R4
R1
R1
R1
R1 R1
H2ONHEt2
H2ONHEt2 H2ONHEt2
CH3
CH3CH3
CH3 CH3
CH3
CH3CH3
H3C
H3CH3C
H3C
H3CH3CH3C
H3C
H3CH3C
R3
R2
R4
4a R=CHOR1=H4b R=HR1=CH34c R=NO2R1=H4d R=OCH3R1=H4e R=HR1=Br4f R=OHR1=H4g R=CH3R1=H
4i R=CH34j R=Cl4k R=OCH3
4m R1=R2=R3=R4=H4n R1=R2=R4=HR3=CH34o R1=R2=R4=HR3=OCH34p R1=R2=R4=HR3=Cl4q R1=R2=R4=HR3=Br4r R1=R3=R4=HR2=Br4s R1=NO2R2=R3=R4=H4t R1=R2=R4=HR3=NMe24v R1=R2=R4=HR3=OH4w R1=R3=ClR2=R4=H4x R1=R2=R4=HR3=CHO4y R1=R4=ClR2=R3=H
5a R1=NO2R2=R3=R4=H5b R1=R3=R4=HR2=CH35c R1=R3=R4=CH3R2=H5d R1=R2=R4=HR3=OCH35e R1=R4=ClR2=R3=H5f R1=R2=R4=HR3=NO25g R1=R2=R4=HR3=CHO5h R1=R2=R4=HR3=OH
5i R=Cl5j R=H5k R=Br5l R=CH35m R=CHO
Scheme 1 Synthesis of the target compounds
Journal of Chemistry 5
Table 1 The crystal and experimental data of compounds 5a 5d and 5f
5a 5d 5fEmpirical formula C
23H26NO6sdotC4H12NsdotH2O C
24H29O5sdotC4H12N C
23H26NO6sdotC4H12N
Formula weight 50461 47162 48659Temperature 100K 100K 100KWavelength Mo K120572 radiation 120582 = 071073 A Mo K120572 radiation 120582 = 071073 A Mo K120572 radiation 120582 = 071073 ACrystal system Orthorhombic Monoclinic MonoclinicSpace group P2
12121
P21n P2
1n
119886 116155 (5) A 102288 (5) A 101137 (4) A119887 120514 (5) A 180048 (7) A 181225 (7) A119888 193665 (7) A 150893 (7) A 154853 (6) A120573 9000∘ 106425 (2)∘ 106730 (1)∘
Volume 271098 (19) A3 26656 (2) A3 271809 (18) A3
119885 4 4 4Calculated density 1236Mgmminus3 1175Mgmminus3 1189Mgmminus3
Absorption coefficient 009mmminus1 008mmminus1 008mmminus1
119865 (000) 1088 1024 1048Crystal size 057 times 041 times 007mm 066 times 033 times 025mm 033 times 029 times 022mm120579 range 24ndash242∘ 22ndash297∘ 22ndash321∘
Reflections collected 28531 67389 25488Reflections unique 4501 6194 4829Independent reflections 6224 11203 6233Parameters 350 326 334119877int 0090 0122 0053R [F2 gt 2120590(F2)] 0054 0069 0055119908R (F2) 0109 0156 0132Goodness of fit 105 101 109Maxmin 120588 e Aminus3 042 and minus027 043 and minus030 029 and minus026CCDC number 1444058 1443783 1444050
in supplementary information ORTEP drawings of final X-ray model of compounds 5a 5d and 5f with the atomicnumbering scheme are presented in Figure 2 while crystalpacking presentation of compounds 5a 5d and 5f is shownin Figure 3
Compound 5a crystallizes in orthorhombic crystal sys-tem in a space group of P2
12121 on the other hand com-
pounds 5d and 5f crystalize in monoclinic crystal system ina space group of P2
1n It was observed that the compound
5a has hydrogen bonding between the C5-O2sdot sdot sdotHsdot sdot sdotO
4-C13
while compounds 5d and 5f exist only in the enol formThereare two strong intramolecular hydrogen bonds on compound5a between O
4mdashH1O4sdot sdot sdotO2and N
2mdashH2N2sdot sdot sdotO1 while
compounds 5d and 5f have no intramolecular hydrogenbonding The crystal structures of the tested compounds 5a5d and 5f were formed by different hydrogen bonds betweenthemolecules and diethyl amine and water solvent moleculesin case of 5a to form network structures Crystallographicdata for the solved structures 5a 5d and 5f were deposited tothe Cambridge Crystallographic Data Center as supplemen-tary publication numbers CCDC-1444058 CCDC-1443783and CCDC-1444050 respectively which can be obtained freeof charge from the Cambridge Crystallographic Data Centrevia httpwwwccdccamacukdata requestcif
33 Biological Activity Evaluation All synthesized diethylammonium salts of barbiturates having bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (4andash4h)bis(6-hydroxypyrimidine-24(1H3H)-dione (4indash4l) and(2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dim-ethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate (4mndash4z) as well as bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) (5andash5m) as basic nuclei were screened for theirantioxidant potential in vitro by using DPPH radicalscavenging assay and cytotoxicity against PC-3 HeLa andMCF-7 cancer cell lines and 3T3 normal fibroblast cell linesThese derivatives 4andash4z and 5andash5m were also evaluated forin vitro enzyme inhibition activities against 120572-glucosidasethymidine phosphorylase and 120573-glucuronidase enzymesThe results are summarized in Table 2
331 Antioxidant Activity (DPPH Radical Scavenging Assay)Among series of compounds (4andash4h) having bis(6-hydroxy-1 3-dimethyl-2 4-dioxo-1 2 3 4-tetrahydropyrimidin-5-yl)ring as basic nucleus compounds 4a (IC
50= 1188plusmn21 120583M)
4b (IC50
= 117 plusmn 24 120583M) 4c (IC50
= 1119 plusmn 19 120583M) 4d(IC50
= 1244 plusmn 44 120583M) 4e (IC50
= 1011 plusmn 08 120583M) 4f(IC50
= 1486 plusmn 26 120583M) and 4g (IC50
= 1041 plusmn 19 120583M)were found to be potent antioxidants in comparison to the
6 Journal of Chemistry
5f
5d5a
Figure 3Molecular packing of 5a 5d and 5f viewed approximately three-dimensional network along 119887 axis and three-dimensional networkrespectively Intermolecular interaction is drawn as dashed lines
standards BHT (IC50= 1288 plusmn 21 120583M) andN-acetylcysteine
(IC50
= 1076 plusmn 28 120583M) Compounds 4andash4e (IC50
=1018 plusmn 08ndash1244 plusmn 44 120583M) were found to be more activecompared to the standard BHT (IC
50= 1288 plusmn 21 120583M) The
meta-bromo-substituted phenyl ring containing compound4e (IC
50= 1011 plusmn 08 120583M) was the most potent member of
the series (4andash4e) followed by the para-methyl-substitutedphenyl ring containing compound 4g (IC
50= 1041plusmn19 120583M)
A gradual decrease in activity was observed for compounds4c (IC
50= 1199 plusmn 19 120583M) and 4a (IC
50= 1188 plusmn 31 120583M)
having para-nitro- and para-aldehyde-substituted phenylring attached to central bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) ring A decrease inDPPH radical scavenging activity was due to positional effectof methyl substituent on phenyl ring which was observed forcompound 4b (IC
50= 117plusmn31 120583M) havingmeta-substituted
phenyl ring instead of para-methyl-substituted phenyl ring(4g IC
50= 1041 plusmn 19 120583M) The electron donating para-
hydroxyl-substituted phenyl ring containing compound 4f(IC50= 1486 plusmn 26 120583M) was found to be a last active member
of the series However a considerable increase in activity was
observed for compound 4d (IC50
= 1244 plusmn 44 120583M) havinga para-methoxy group instead of hydroxyl functionalityon phenyl ring whereas naphthalene ring containingbarbiturate 4h (IC
50= 1336 plusmn 57 120583M) was also found to be
a weak antioxidant agent against DPPH radicalsAmong second series of the tested compounds 4indash4l
having bis(6-hydroxypyrimidine-24(1H3H)-dione as back-bone compounds having para-methyl- (4i IC
50= 3849 plusmn
66 120583M) para-chloro- (4j IC50= 3843 plusmn 88 120583M) and para-
methoxy- (4l IC50= 3979 plusmn 09 120583M) substituted phenyl ring
attached to the central bis(6-hydroxypyrimidine-24(1H3H)-dione) showed better antioxidant activity as compared toBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) whereas naphthalene containing compound4l was found to be inactive
(2-Hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate ringcontaining compounds 4mndash4z were also evaluated for theirantioxidant potential in vitro in DPPH radical scavengingassayThese compounds were found to be significant to weakinhibitors of free radicals (IC
50= 1332plusmn47ndash4396plusmn101 120583M)
Journal of Chemistry 7
Table2Re
sultof
invitro
biologicalevaluatio
nof
thes
ynthesized
compo
unds
Num
berD
PPHradicalscaveng
ingassay
IC50plusmnSE
Ma(120583M)
Anticancercytotoxicityassay
IC50plusmnSE
M(120583M)
120572-G
lucosid
aseinh
ibition
IC50plusmnSE
M(120583M)
Thym
idinep
hospho
rylase
inhibitio
nIC50plusmnSE
M(120583M)
120573-G
lucuronidase
inhibitio
nIC50plusmnSE
M(120583M)
PC3b
Helac
MCF
-7d
3T3
4a118
8plusmn31
gt30
gt30
NA
gt30
NA
NA
20481plusmn60
4b117plusmn24
gt30
gt30
NA
gt30
NA
NA
NA
4c1119plusmn19
gt30
gt30
NA
gt30
355plusmn10
NA
1721plusmn
21
4d1244plusmn44
gt30
gt30
NA
gt30
NA
2309plusmn08
NA
4e1011plusmn08
gt30
gt30
NA
gt30
NA
NA
3389plusmn64
4f1486plusmn26
gt30
gt30
NA
gt30
NA
2179plusmn20
NA
4g1041plusmn
19gt30
gt30
NA
gt30
NA
NA
NA
4h1336plusmn57
gt30
gt30
NA
gt30
230plusmn39
NA
NA
4i3849plusmn66
gt30
gt30
NA
gt30
NA
NA
NA
4j3843plusmn88
gt30
gt30
NA
gt30
NA
NA
NA
4kNA
gt30
gt30
NA
gt30
NA
1826plusmn10
NA
4l3979plusmn01
gt30
gt30
NA
gt30
NA
NA
NA
4mNA
gt30
gt30
NA
gt30
NA
1602plusmn45
NA
4nNA
gt30
gt30
NA
gt30
NA
2269plusmn15
NA
4oNA
gt30
gt30
NA
gt30
3009plusmn65
NA
2621plusmn
43
4pNA
gt30
gt30
NA
gt30
1868plusmn81
2420plusmn15
NA
4qNA
gt30
gt30
NA
gt30
2148plusmn72
1413plusmn15
29217plusmn10
4rNA
gt30
gt30
NA
gt30
201plusmn
55
168plusmn12
2621plusmn
44
4s4396plusmn101
gt30
gt30
NA
gt30
NA
NA
NA
4t2782plusmn100
gt30
gt30
NA
gt30
NA
NA
NA
4v1332plusmn47
gt30
gt30
NA
gt30
NA
576plusmn12
NA
4w4116plusmn06
gt30
gt30
NA
gt30
NA
NA
1435plusmn12
4x3888plusmn79
gt30
gt30
NA
gt30
NA
239plusmn20
NA
4yNA
1071plusmn
03
347plusmn025
49plusmn001
320plusmn02
NA
NA
NA
4z2230plusmn39
gt30
gt30
NA
gt30
NA
558plusmn15
NA
5a4616plusmn88
gt30
gt30
NA
gt30
NA
NA
NA
5bNA
gt30
gt30
NA
gt30
NA
2169plusmn05
NA
5cNA
gt30
gt30
NA
gt30
NA
1614plusmn15
NA
5dNA
gt30
gt30
NA
gt30
NA
605plusmn18
7NA
5e289plusmn09
gt30
gt30
NA
gt30
NA
NA
NA
5fNA
gt30
gt30
NA
gt30
238plusmn93
NA
1850plusmn25
5g2704plusmn23
gt30
gt30
NA
gt30
NA
1866plusmn12
NA
5hNA
gt30
gt30
NA
gt30
NA
569plusmn16
NA
5iNA
gt30
gt30
NA
gt30
NA
NA
3236plusmn64
5jNA
gt30
gt30
NA
gt30
NA
318plusmn25
NA
5k1276plusmn16
gt30
gt30
NA
gt30
NA
1664plusmn12
NA
5l1697plusmn93
gt30
gt30
NA
gt30
NA
1781plusmn
21
3875plusmn70
5mNA
gt30
gt30
NA
gt30
4534plusmn42
1471plusmn12
NA
STD
e BHT(288plusmn21)
Doxorub
icin
Doxorub
icin
Doxorub
icin
Cyclo
heximide
Acarbo
se7-Deazaxanthine
D-Saccharicacid
1-4lacton
eN-acetylcysteine(
1076plusmn28)
0912plusmn012
0506plusmn015
156plusmn005
026plusmn01
840plusmn17
341plusmn16
44575plusmn216
a SEM
stand
arderrorof
mean
b PC3
hum
anprostate
cancer
celllin
ec H
eLaHenrie
ttaLackscervical
cancer
celllin
ed M
CF-7M
ichiganCa
ncer
Foun
datio
n-7breastcancer
celllin
esand
e BHT
butylated
hydroxytoluene
8 Journal of Chemistry
except compounds 4mndash4r and 4y which were found to beinactive
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) (5andash5h) and 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) (5indash5k) rings con-taining compounds were also evaluated for their antioxidantpotential by using DPPH radical scavenging assay Para-bromo-substituted phenyl ring containing compound5k (IC
50= 1276 plusmn 16 120583M) was found as a potent anti-
oxidant agent against the tested standards BHT (IC50
=1288 plusmn 21 120583M) The compound showed similar antioxidantpotential as BHT and showed a significant antioxidant agentagainst N-acetyl cysteine (IC
50= 1076 plusmn 28 120583M) The
gradual and drastic decrease in activity was for compounds5l (IC
50= 1697 plusmn 53 120583M) 5g (IC
50= 2704 plusmn 23 120583M)
5e (IC50
= 289 plusmn 09 120583M) and 5a (IC50
= 4616 plusmn 88 120583M)having para-methyl- para-aldehyde- ortho-chloro- andortho-nitro-substituted phenyl rings attached to the centralbarbiturate moieties Compounds 5bndash5d 5f and 5hndash5j werefound to be inactive
On the basis of the observed antioxidant abilities ofthe above five different series of pyridinium adducts itwas concluded that bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) ring containing com-pounds 4andash4h are the most active one The activity may bedue to the methyl substituted nitrogen atoms of the tetrahy-dropyrimidine rings that facilitate the electronic conjugationwithin the ring However the variation of antioxidantproperties within the most important series is may be due tothe presence of electron donating (-OH (4f) -OCH
3(4d)
and -CH3(4b 4g)) electron withdrawing (-NO
2(4a) and
-CHO (4c)) and halogen (-Br (4e)) substituents on thephenyl ring attached to the bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) moiety Howeverdetailed studies are required to study the mechanism ofaction of these compounds
332 Cytotoxic Activity All synthesized diethyl ammoniumsalts of barbiturates (4andash4z and 5andash5m) were evaluated fortheir in vitro cytotoxicity against PC-3 HeLa MCF-7 cancerand 3T3 normal cell lines All compounds were found to benoncytotoxic against PC-3 HeLaMCF-7 cancer and 3T3 celllines except dichloro-substituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tet-rahydropyrimidin-4-olate ring containing compounds 4ywhich was found to be significantly toxic against all the celllines (PC3 IC
50= 1071plusmn026 120583MHeLa IC
50= 347plusmn03 120583M
MCF-7 IC50= 49 plusmn 001 120583M and 3T3 IC
50= 320 plusmn 02 120583M)
Doxorubicin was used as a standard drug to compare theactivities against PC3 (IC
50= 170 plusmn 029 120583M) HeLa (IC
50=
051 plusmn 015 120583M) and MCF-3 (IC50= 092 plusmn 001 120583M) cancer
cell lines while cycloheximide (IC50
= 026 plusmn 012 120583M) wasused as a standard-in 3T3 cell line (Table 2)
333 120572-Glucosidase Inhibition Activity Synthesized diethylammonium salts of barbiturates 4andash4z and 5andash5m were alsoevaluated for their in vitro 120572-glucosidase inhibition activityin comparison to the standard drug acarbose (IC
50= 841 plusmn
173 120583M) Among dimethyl ammonium salts (4andash4h) nitro-substituted phenyl ring containing 4c (IC
50= 355 plusmn 10 120583M)
and naphthalene ring containing 4h (IC50= 230 plusmn 392 120583M)
were found to be potent inhibitors of 120572-glucosidase enzymeand showed more activity than acarbose (IC
50= 841 plusmn
173 120583M) All other compounds were found to be inactiveAmong diethyl ammonium salts having (2-hydroxy-
44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate ring (4mndash4z)para-OCH
3- (40 IC
50= 3009 plusmn 65 120583M) para-chloro-
(4p IC50
= 1868 plusmn 081 120583M) para-bromo- (4q IC50
=2148plusmn072 120583M) andmeta-bromo- (4r IC
50= 201plusmn55 120583M)
substituted phenyl ring containing pyridinium adducts werefound to be potent 120572-glucosidase inhibitors and showedmore activity than standard acarbose (IC
50= 841plusmn 173 120583M)
Other members of the series (4m-4n and 4sndash4z) were foundto be inactive The results showed the substitution effects ofvarious functionalities of phenyl ring on the potential activityof tested series The para-bromo-substituted phenyl ringcontaining compound 4q (IC
50= 2148 plusmn 072 120583M) showed
more activity than tested standard However the replacementof bromo group with that of methylNN-dimethyl hydroxyland aldehyde functionalities resulted in complete loss ofactivity as observed in compounds 4n 4t 4v and 4xrespectively The presence of ortho-nitro- ortho-choloro-and para-choloro-substituted phenyl ring also contributedtowards inactivity as observed for compounds 4t 4w and4y
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring containing diethyl ammonium pyridinium salts(5andash5h) were also evaluated for their in vitro 120572-glucosidaseenzyme inhibition activity The series nitro-substitutedphenyl ring containing 5f was found to be the only potent120572-glucosidase inhibitor All other compounds (5andash5e and5g-5h) were found to be inactive
Among series of diethylaminium salts of pyridiniumadducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-di-oxo-1234-tetrahydropyrimidin-5-yl)methyl) central moietyattached with substituted phenyl ring the benzaldehydecontaining 5m (IC
50= 4534 plusmn 42 120583M) was found to be the
only potent inhibitor compound in comparison to against thestandard drug acarbose (IC
50= 841plusmn173 120583M)All other com-
poundswere found to be inactiveThe results are summarizedin Table 2
334 Thymidine Phosphorylase Inhibition Activity Five dif-ferent series of diethyamonium salts of barbiturates 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evalu-ated for their in vitro thymidine phosphorylase inhibition7-Deazaxanthine was used as a standard inhibitor (IC
50
= 41 plusmn 146 120583M) Phenol and naphthalene rings sub-stituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olatemoiety containing compounds 4v (IC
50= 576 plusmn 120120583M)
and 4z (IC50
= 558 plusmn 150 120583M) were found to be the potentthymidine phosphorylase inhibitors However a drasticdecrease in activity was observed when para-hydroxygroup on phenyl ring was replaced with methyl (4n IC
50
Journal of Chemistry 9
= 2269 plusmn 150 120583M) chloro (4n IC50
= 242 plusmn 15 120583M)bromo (4q IC
50= 1413 plusmn 15 120583M) and aldehyde (4x
IC50
= 239 plusmn 20 120583M) groups whereas the compounds4o 4s 4t 4w and 4y have para-methoxy- ortho-nitro-ortho-NN-dimethyl amine- ortho- para-dichloro- andortho-ortho-dichloro-substituted phenyl ring respectively
Among series of compounds (5andash5h) having bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring as centralmoiety electron donating hydroxy- andmethoxy-substitutedphenyl ring containing compounds 5h (IC
50= 569plusmn16 120583M)
and 5d (IC50
= 605 plusmn 187 120583M) respectively were foundas potent inhibitors of thymidine phosphorylase enzymeagainst the tested standard 7-deazaxanthine which was(IC50
= 41 plusmn 146 120583M) used as standard Again a drasticdecrease in activity was observed for compounds 5b (IC
50
= 2169 plusmn 05 120583M) 5c (IC50
= 1614 plusmn 15 120583M) and 5g(IC50= 1866 plusmn 12 120583M) havingmeta-methyl- ortho-methyl-
and para-aldehyde-substituted benzene ring respectivelyinstead of para-hydroxyl phenyl ring The replacement ofortho-methyl with chloro-group makes the compound 5ecompletely inactive Similarly the replacement of electrondonating para-hydroxy-group of phenyl ring with electronwithdrawing nitro-functionality also contributed towardsinactivity of compound as observed for 5f Phenol substi-tuted bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ringcontaining 5h (IC
50= 569 plusmn 16 120583M) was found to be the
potent thymidine phosphorylase inhibitorsAll members of the series of diethylaminium salts of pyri-
dinium adducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) central moi-ety attached with substituted phenyl ring were found to beweak inhibitors of thymidine phosphorylase enzyme againstthe tested standard compound 7-deazaxanthine (IC
50= 41 plusmn
146 120583M) with IC50
values 318 plusmn 25 120583M 1664 plusmn 12 120583M1781 plusmn 21 120583M and 1471 plusmn 123 120583M for 5j 5k 5l and 5mrespectively whereas para-chloro-substituted phenyl ringcontaining compound 5i was found to be inactiveThe resultsare summarized in Table 1
335 120573-Glucuronidase Inhibition Activity Compounds 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evaluatedfor their in vitro 120573-glucuronidase inhibitory activity by usingD-saccharic acid 14-lactone as standard inhibitor (IC
50=
4575 plusmn 216 120583M) Compounds 4a (IC50
= 2048 plusmn 601 120583M)4c (IC
50= 1721 plusmn 21 120583M) 4e (IC
50= 3389 plusmn 642 120583M) 4o
(IC50
= 2621 plusmn 435 120583M) 4q (IC50
= 2922 plusmn 103 120583M) 4r(IC50= 2621plusmn44 120583M) 4w (IC
50= 1435plusmn124 120583M) 5f (IC
50
= 185 plusmn 25 120583M) 5i (IC50
= 2236 plusmn 64 120583M) and 5l (IC50
=3875plusmn70 120583M) showed veryweak120573-glucuronidase inhibitionpotential whereas pyridinium adducts 4andash4c 4e 4gndash4j 4l4o 4s-4t 4w 4y 5a 5e-5f and 5i were found to be inactive(Table 2)
34 Molecular Docking Studies
341 Binding Interactions of 120572-Glucosidase Enzyme To de-fine inhibitors interactions and their specificity the dockingstudy of the most active compound 4p against 120572-glucosidase
Figure 4 Three-dimensional representation of compound 4p withtarget macromolecule 120572-glucosidase
Figure 5 Three-dimensional representation of compound 4v withtarget macromolecule thymidine phosphorylase
was carried out [23ndash25] Ten different conformations ofprotein-ligand interactions were generated and the topranked pose was selected to explore the binding interactionsIn our docking study we have observed two interactions withthe active site residues of 120572-glucosidase The polar imidazolering of His 239 showed hydrogen bond through its H atomwith the lone pair of oxygen of the pyrimidinedionemoiety ofthe compound whereas Arg 312 formed hydrogen bond withthe OH group of the same moiety of compound (Figure 4)The presence of polar and electron rich center in the formof pyrimidinedione and chlorobenzene groups of the com-pound was observed here as key factors for the interactionbetween compound andprotein Anumber of hydrogen bonddonor and acceptor pairs with their suitable orientationswere observed in the interaction pose Furthermore severalhydrophobic interactions between the nonpolar active siteresidues and the compound 4p were also observed
342 Binding Interactions of Thymidine PhosphorylaseEnzyme To explore the binding modes of these newly syn-thesized compounds in the active site of thymidine phos-phorylase the most active compound 4v was docked againstthis enzyme The docking results showed that compound4v well accommodated in the active site of thymidinephosphorylase (Figure 5) The active site residue Ala 206established a hydrogen bond through its lone pair of
10 Journal of Chemistry
Figure 6 Three-dimensional representation of compound 4w withtarget enzyme 120573-glucuronidase
oxygen with the active hydrogen of hydroxyl moiety ofthe pyrimidinedione group of the compound Howeverthe substituted phenolic group and the other electron richcenters of the pyrimidinedione showed poor behaviorregarding interaction which may depend on the nature andorientation of the surrounding active site residues Moreoverseveral weak hydrophobic interactions between the activesite residues and the compound were also observed
343 Binding Interaction of 120573-Glucuronidase Enzyme Thepredicted bindingmode ofmost active compound 4w against120573-glucuronidase showed that a hydrogen bond is formedbetween the lone pair of oxygen of cyclohexanedione compo-nent of the compound and imidazole hydrogen of the impor-tant active site residue His 509 An arene-arene and arene-cation interactionwas also observed between the pi electroniccloud of dichlorobenzene ring of compound and imidazolering of His 509 (Figure 6) Besides hydrogen bonding andarene-arene interactions several hydrophobic interactionsbetween compound 4w and the active site residues werealso observed The pyrimidinedione moiety in this case wasobserved to be passive regarding interactions
4 Conclusions
In conclusion barbiturate salts were synthesized by one-potmulticomponent reactions via tandem AldolMichael addi-tion reactions between barbituric acid and dimedone withaldehydes mediated by aqueous diethylamine The molec-ular structures of 5a 5d and 5f were deduced by single-crystal X-ray diffraction techniques All of the synthesizedcompounds were evaluated for their antioxidant potentialin vitro by using DPPH radical scavenging assay as well asenzyme inhibition activities against120572-glucosidase thymidinephosphorylase and 120573-glucuronidase enzymes Compounds4andash4g (IC
50= 1018 plusmn 08ndash1244 plusmn 44 120583M) were found to
be the potent antioxidants as compared to the standardsBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) Compound 4p (IC50= 1868 plusmn 81 120583M) was
found to be a potent 120572-glucosidase inhibitor Compound 4v(IC50
= 87 plusmn 12 120583M) was found to be a potent thymidinephosphorylase inhibitor Promising results indicate that thesecompounds need to be investigated as antioxidant agentsto treat various associated oxidative disorders Similarlytheir enzyme inhibitory potential can be reported on drugdiscovery program
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Assem Barakat Hazem A Ghabbour Abdullah MohammedAl-Majid Qurat-ul-ain Rehan Imad Kulsoom Javaid NimraNaveed Shaikh Sammer Yousuf M Iqbal Choudhary andAbdul Wadood contributed equally to this work
Acknowledgments
The authors would like to extend their sincere appreciation tothe Deanship of Scientific Research at King Saud Universityfor funding this Research Group no (RGP-257-1435-1436)
References
[1] K Undheim T Bennecke A R Katritzky C W Rees andE F V Scriven Comprehensive Heterocyclic Chemistry vol 6supplement 93 Elsevier Pergamon Oxford UK 1996
[2] B TaylorModern Medical Chemistry Prentice Hall New YorkNY USA 1994
[3] R Y Levina and F K Velichko ldquoAdvances in the chemistry ofbarbituric acidsrdquo Russian Chemical Reviews vol 29 no 8 pp437ndash459 1960
[4] P Sing and K Paul ldquoA simple synthesis of 5-spirobarbituricacids and transformations of spirocyclopropane barbiturates to5-substitutated barbituratesrdquo Indian Journal of Chemistry B vol45 pp 247ndash251 2006
[5] X Chen K Tanaka and F Yoneda ldquoSimple newmethod for thesynthesis of 5-deaza-10-oxaflavin a potential organic oxidantrdquoChemical and Pharmaceutical Bulletin vol 38 no 2 pp 307ndash311 1990
[6] L R Morgan B S Jursic C L Hooper D M Neumann KThangaraj and B LeBlanc ldquoAnticancer activity for 4 41015840-di-hydroxybenzophenone-2 4-dinitrophenylhydrazone (A-007)analogues and their abilities to interact with lymphoendothelialcell surfacemarkersrdquo Bioorganic ampMedicinal Chemistry Lettersvol 12 no 23 pp 3407ndash3411 2002
[7] G Orzalesi P Gratteri and S Selleri ldquoSynthesis of new 13-dicyclohexyl barbituric acid derivatives with anti-inflammatorypotential activityrdquoEuropean Journal ofMedicinal Chemistry vol25 no 2 pp 197ndash201 1990
[8] D T Puerta and S M Cohen ldquoA bioinorganic perspectiveson matrix metalloproteinase inhibitionrdquo Current Topics inMedicinal Chemistry vol 4 no 15 pp 1551ndash1573 2004
[9] M E Wolff Burgerrsquos Medicinal Chemistry and Drug DiscoveryJohn Wiley amp Sons New York NY USA 1997
Journal of Chemistry 11
[10] E Fischer and J von Mering ldquoUeber eine neue klasse vonschlafmittelnrdquo in Untersuchungen aus Verschiedenen GebietenM Bergmann Ed pp 671ndash679 Springer Berlin Germany1924
[11] W J Doran ldquoBarbituric acid hypnoticsrdquo Medicinal Chemistryvol 4 pp 164ndash167 1959
[12] B Bobranski ldquoProgress in chemistry of barbituric acidrdquoWiad-omosci Chemiczne vol 31 pp 231ndash278 1977
[13] S Senda H Izumi and H Fujimura ldquoUracil derivaives andrelated compounds 6 Derivatives of 5-alkyle-246-trioxoper-hydropyrimidine as antiphlogisticsrdquo Arzneimittel Forschungvol 17 no 12 p 151 1967
[14] N Moussier L Bruche F Viani and M Zanda ldquoFluorinatedbarbituric acid derivatives synthesis and bio-activityrdquo CurrentOrganic Chemistry vol 7 no 11 pp 1071ndash1080 2003
[15] P-G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000
[16] N Ramarathnam T Osawa H Ochi and S Kawakishi ldquoThecontribution of plant food antioxidants to human healthrdquoTrends in Food Science amp Technology vol 6 no 3 pp 75ndash821995
[17] A Barakat A M Al-Majid H J Al-Najjar et al ldquoZwitterionicpyrimidinium adducts as antioxidants with therapeutic poten-tial as nitric oxide scavengerrdquo European Journal of MedicinalChemistry vol 84 pp 146ndash154 2014
[18] P Singh M Kaur and P Verma ldquoDesign synthesis andanticancer activities of hybrids of indole and barbituric acids-Identification of highly promising leadsrdquo Bioorganic andMedic-inal Chemistry Letters vol 19 no 11 pp 3054ndash3058 2009
[19] A M Al-Majid A Barakat H J Al-Najjar Y N Mabkhot HA Ghabbour and H-K Fun ldquoTandem aldol-michael reactionsin aqueous diethylamine medium a greener and efficientapproach to bis-pyrimidine derivativesrdquo International Journalof Molecular Sciences vol 14 no 12 pp 23762ndash23773 2013
[20] A Barakat A M Al-Majid G Lotfy et al ldquoSynthesis and dy-namics studies of barbituric acid derivatives as urease inhibi-torsrdquo Chemistry Central Journal vol 9 no 1 article 63 2015
[21] G M Sheldrick ldquoA short history of SHELXrdquo Acta Crystallo-graphica A vol 64 pp 112ndash122 2008
[22] A L Spek ldquoStructure validation in chemical crystallographyrdquoActaCrystallographica SectionD Biological Crystallography vol65 no 2 pp 148ndash155 2009
[23] F Rahim K Ullah H Ullah et al ldquoTriazinoindole analogs aspotent inhibitors of 120572-glucosidase synthesis biological evalua-tion and molecular docking studiesrdquo Bioorganic Chemistry vol58 pp 81ndash87 2015
[24] F Rahim F Malik H Ullah et al ldquoIsatin based Schiff basesas inhibitors of 120572-glucosidase synthesis characterization invitro evaluation and molecular docking studiesrdquo BioorganicChemistry vol 60 article 1803 pp 42ndash48 2015
[25] A Barakat A M Al-Majid M S Islam et al ldquoMolecular struc-ture investigation and biological evaluation of Michael adductsderived from dimedonerdquo Research on Chemical Intermediatesvol 42 pp 4041ndash4053 2016
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
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Carbohydrate Chemistry
International Journal of
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Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
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Analytical Methods in Chemistry
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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
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CatalystsJournal of
2 Journal of Chemistry
O OS Na
O O O O ON OH
HN HN NH
O
O O
HN NH
O
O O
O O O
O
OO
OO
O
O
OO N N
H
NN
O
O
O O O
O
O O O
OO
N
NH
HN NH
NH
Cl
HN N
1 (Bucolome) 2 (Sodium pentothal) 3 (Seconal) 4 (Phenobarbital)
HO N NNHN
5 (Veronal) 6 (Hsp90 inhibitor)
HNNH
NH
HN
NH
NH
7 (Ro 28-2653 MMP inhibitor) 8 (EM20-25 BCL-2 inhibitor) 9 (TACE inhibitor)
Potent nitric oxide scavenger
NH2Et2
NO2
NO2
oplus
oplus
⊖
⊖
10 IC50 = 69 plusmn 166120583MStd ascorbic acid IC50 = 618 plusmn 20 120583M
Figure 1 Structure of biologically active barbituric acid derivatives
damage protein and enzyme modifications and DNA dam-age within the body [16] These types of changes accelerateaging and play a causative role in a variety of degenerativediseases such as cancers atherosclerosis brain disfunctioninflammation shock and heart diseases Recently Barakatet al [17] have synthesized some novel salts ofMichael adductderived from PYT and evaluated them for NO (nitric oxide)scavenging activity Compound 10 was found to possessseveral hundredfold more activity against nitric oxide radical(IC50values of 69plusmn166 120583M) than the standard drug ascorbic
acid (IC50= 618 plusmn 20 120583M)
Because of the important biological activities PYT iswidely used as a synthon in the design of antitumor agentsIt has a high efficacy to form hydrogen bonding with drugtargets (6ndash9 Figure 1) Singh et al have reported the activityof a series of new N-benzyl indole-pyrimidine-246-trionehybrids against a panel of 60 human tumor cell lines Severalof these analogs were also found to be inhibitors of DNArepair and replication stress response polymerases [18]
The discovery and development of effective antioxidantscapable of supplementing bodyrsquos antioxidant system is animportant area of health care research Similarly researchefforts are focused on the identification of effective and safeinhibitors of 120572-glucosidase as potential antidiabetic agents
The focus in recent years is on the development ofresource-efficient environmentally benign and sustainablechemistry practices In continuation of our work in thisfield we describe here the synthesis of a series of previouslyreported [17 19 20] and new PYT adductsThese compoundswere evaluated for their antioxidant activity by using DDPH
scavenging assay as well as for in vitro enzyme inhibitionactivities against alpha-glucosidase thymidine phosphory-lase and 120573-glucuronidase enzymes
2 Materials and Methods
The chemical reagents used for the preparation of the targetcompounds were commercially available and used withoutfurther purification 1H-NMR and 13C-NMR were measuredusing Bruker Avance AV-600 NMR spectrometer MS werecarried out with a Jeol JMS-600H instrument and single-crystal X-ray diffractionwas collected using a Bruker SMARTAPEX II CCD diffractometer
21 General Procedure for Aldol Condensation Michael Addi-tion for the Synthesis of 4 and 5 (GP1) Amixture of aldehyde3 (15mmol) and 1 and2 (3mmol) aswell as Et
2NH(15mmol
155 120583L) in 3mL of degassed H2O (bubbling nitrogen through
the water) was stirred at room temperature for 1ndash5 h untilTLC showed complete disappearance of the reactants Theprecipitate was removed by filtration and washed with ether(3 times 20mL) The solid product was dried to afford pureproducts 4 and 5
The full characterization of the synthesized compounds 4and 5 and the X-ray crystal data of 5a 5d and 5f can be seenin the SupplementaryMaterials (see supplementarymaterialsavailable online at httpdxdoiorg10115520168517243)
22 Biological Activity The samples of the synthesized com-pound were screened against DPPH radical 120573-glucuronidase
Journal of Chemistry 3
5a 5d
C27C26
C25
01C1
C6
C7C8
C13C12
C15C14
C19
C17C18
C16
C506N1
050204
C2C3
C4
C21
C20C24C23
C11 C9C10C22
01W
03
N2
C21
C4 02
04
C3 C2
C5C6
C7C8
C9
C25
C13C12
C23C11
C22C10
0103
C1
C14C15
C26N1
C16
C17 C27C28
C24
05
C19
C18
C20
5f
C2203
0401
06
05
N1
N2
02
C10
C23 C11
C12 C13
C9
C5
C4
C3
C2C1
C27
C26C16C19
C18 C17
C25 C24
C6C7
C14 C15
C21
C20
C8
Figure 2 ORTEP diagram of 5a 5d and 5f Displacement ellipsoids are plotted at the 40 probability level for non-H atoms
inhibition assay thymidine phosphorylase inhibition assayand 120572-glucosidase inhibition assay and full methods descrip-tions are provided in Supplementary Materials
3 Results and Discussion
31 Chemistry We have been focusing on the developmentof highly expedient methods for the synthesis of diverseheterocyclic compounds of biological importance via one-pot multicomponent reactions (MCRs) and avoiding organicsolvents in organic synthesis [17 19 20] This had led tothe development of several efficient environmentally benignclean and economical process technology (Green ChemistryConcepts) In current reaction strategy equimolar amountsof barbituric acid (1) and dimedone (2) with aldehyde(3) in the presence of aqueous diethylamine medium atRT yielded salts of PYT adducts 4andash4y and 5andash5m wereobtained in quantitative yields by simple filtration [17ndash20]
Products 4s 4t and 5g were identified as new products(Scheme 1)
32 X-Ray Diffraction The molecular structures of com-pounds 5a 5d and 5f were unambiguously deduced bysingle-crystal X-ray diffraction technique (Figure 2) Thecrystals of the synthesized molecules 5a 5d and 5f weregrown by slow diffusion of diethyl ether solution of puresamples 5a 5d and 5f in DCMEtOH at room temperaturefollowed by allowing standing for 24 h The structures wereresolved by direct methods by using the SHELXL97 program[21 22] in the SHELXTL-plus package and refined by afull-matrix least-squares procedure on 1198652 using SHELXS97Diffraction data were collected on a Bruker APEX-II CCDdiffractometer (Bruker AXS GmbH) The crystal structureand refinement data of compounds 5a 5d and 5f are listedin Table 1 The final atomic coordinates for all atoms andcomplete listing of bond distance and angles are presented
4 Journal of Chemistry
O
O O O O
RT
RT RT
ON N
2 1a b
1a b
O
O
O
O O
O
R R
O O O O O
O O
O
O
HOHO
NH
O
HO
O
N NN
NHN
O O HO
HO HO
HO
O O
O
O O
OO
O O O O O
O O
O
OO
O
OO
O O
ONH
O
4l
4z
NH
NH
NH N
NN
NN
H
NN
N NN
H
NH
HN
H NN
NH
N
O O O O
O
O O HO O
OO
N N
NN N
NO2 2
2
3
O
HO
HO
O
ON
N
CHO5l
4h
HOHO
R
NH2Et2
R2
R2
R2
R2
oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus
NH2Et2oplus
⊖
⊖ NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
⊖
NH2Et2oplus
NH2Et2oplus
⊖R1 R1R1
R1
R1
R1
R2R3
R4
R1
R1
R1
R1 R1
H2ONHEt2
H2ONHEt2 H2ONHEt2
CH3
CH3CH3
CH3 CH3
CH3
CH3CH3
H3C
H3CH3C
H3C
H3CH3CH3C
H3C
H3CH3C
R3
R2
R4
4a R=CHOR1=H4b R=HR1=CH34c R=NO2R1=H4d R=OCH3R1=H4e R=HR1=Br4f R=OHR1=H4g R=CH3R1=H
4i R=CH34j R=Cl4k R=OCH3
4m R1=R2=R3=R4=H4n R1=R2=R4=HR3=CH34o R1=R2=R4=HR3=OCH34p R1=R2=R4=HR3=Cl4q R1=R2=R4=HR3=Br4r R1=R3=R4=HR2=Br4s R1=NO2R2=R3=R4=H4t R1=R2=R4=HR3=NMe24v R1=R2=R4=HR3=OH4w R1=R3=ClR2=R4=H4x R1=R2=R4=HR3=CHO4y R1=R4=ClR2=R3=H
5a R1=NO2R2=R3=R4=H5b R1=R3=R4=HR2=CH35c R1=R3=R4=CH3R2=H5d R1=R2=R4=HR3=OCH35e R1=R4=ClR2=R3=H5f R1=R2=R4=HR3=NO25g R1=R2=R4=HR3=CHO5h R1=R2=R4=HR3=OH
5i R=Cl5j R=H5k R=Br5l R=CH35m R=CHO
Scheme 1 Synthesis of the target compounds
Journal of Chemistry 5
Table 1 The crystal and experimental data of compounds 5a 5d and 5f
5a 5d 5fEmpirical formula C
23H26NO6sdotC4H12NsdotH2O C
24H29O5sdotC4H12N C
23H26NO6sdotC4H12N
Formula weight 50461 47162 48659Temperature 100K 100K 100KWavelength Mo K120572 radiation 120582 = 071073 A Mo K120572 radiation 120582 = 071073 A Mo K120572 radiation 120582 = 071073 ACrystal system Orthorhombic Monoclinic MonoclinicSpace group P2
12121
P21n P2
1n
119886 116155 (5) A 102288 (5) A 101137 (4) A119887 120514 (5) A 180048 (7) A 181225 (7) A119888 193665 (7) A 150893 (7) A 154853 (6) A120573 9000∘ 106425 (2)∘ 106730 (1)∘
Volume 271098 (19) A3 26656 (2) A3 271809 (18) A3
119885 4 4 4Calculated density 1236Mgmminus3 1175Mgmminus3 1189Mgmminus3
Absorption coefficient 009mmminus1 008mmminus1 008mmminus1
119865 (000) 1088 1024 1048Crystal size 057 times 041 times 007mm 066 times 033 times 025mm 033 times 029 times 022mm120579 range 24ndash242∘ 22ndash297∘ 22ndash321∘
Reflections collected 28531 67389 25488Reflections unique 4501 6194 4829Independent reflections 6224 11203 6233Parameters 350 326 334119877int 0090 0122 0053R [F2 gt 2120590(F2)] 0054 0069 0055119908R (F2) 0109 0156 0132Goodness of fit 105 101 109Maxmin 120588 e Aminus3 042 and minus027 043 and minus030 029 and minus026CCDC number 1444058 1443783 1444050
in supplementary information ORTEP drawings of final X-ray model of compounds 5a 5d and 5f with the atomicnumbering scheme are presented in Figure 2 while crystalpacking presentation of compounds 5a 5d and 5f is shownin Figure 3
Compound 5a crystallizes in orthorhombic crystal sys-tem in a space group of P2
12121 on the other hand com-
pounds 5d and 5f crystalize in monoclinic crystal system ina space group of P2
1n It was observed that the compound
5a has hydrogen bonding between the C5-O2sdot sdot sdotHsdot sdot sdotO
4-C13
while compounds 5d and 5f exist only in the enol formThereare two strong intramolecular hydrogen bonds on compound5a between O
4mdashH1O4sdot sdot sdotO2and N
2mdashH2N2sdot sdot sdotO1 while
compounds 5d and 5f have no intramolecular hydrogenbonding The crystal structures of the tested compounds 5a5d and 5f were formed by different hydrogen bonds betweenthemolecules and diethyl amine and water solvent moleculesin case of 5a to form network structures Crystallographicdata for the solved structures 5a 5d and 5f were deposited tothe Cambridge Crystallographic Data Center as supplemen-tary publication numbers CCDC-1444058 CCDC-1443783and CCDC-1444050 respectively which can be obtained freeof charge from the Cambridge Crystallographic Data Centrevia httpwwwccdccamacukdata requestcif
33 Biological Activity Evaluation All synthesized diethylammonium salts of barbiturates having bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (4andash4h)bis(6-hydroxypyrimidine-24(1H3H)-dione (4indash4l) and(2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dim-ethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate (4mndash4z) as well as bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) (5andash5m) as basic nuclei were screened for theirantioxidant potential in vitro by using DPPH radicalscavenging assay and cytotoxicity against PC-3 HeLa andMCF-7 cancer cell lines and 3T3 normal fibroblast cell linesThese derivatives 4andash4z and 5andash5m were also evaluated forin vitro enzyme inhibition activities against 120572-glucosidasethymidine phosphorylase and 120573-glucuronidase enzymesThe results are summarized in Table 2
331 Antioxidant Activity (DPPH Radical Scavenging Assay)Among series of compounds (4andash4h) having bis(6-hydroxy-1 3-dimethyl-2 4-dioxo-1 2 3 4-tetrahydropyrimidin-5-yl)ring as basic nucleus compounds 4a (IC
50= 1188plusmn21 120583M)
4b (IC50
= 117 plusmn 24 120583M) 4c (IC50
= 1119 plusmn 19 120583M) 4d(IC50
= 1244 plusmn 44 120583M) 4e (IC50
= 1011 plusmn 08 120583M) 4f(IC50
= 1486 plusmn 26 120583M) and 4g (IC50
= 1041 plusmn 19 120583M)were found to be potent antioxidants in comparison to the
6 Journal of Chemistry
5f
5d5a
Figure 3Molecular packing of 5a 5d and 5f viewed approximately three-dimensional network along 119887 axis and three-dimensional networkrespectively Intermolecular interaction is drawn as dashed lines
standards BHT (IC50= 1288 plusmn 21 120583M) andN-acetylcysteine
(IC50
= 1076 plusmn 28 120583M) Compounds 4andash4e (IC50
=1018 plusmn 08ndash1244 plusmn 44 120583M) were found to be more activecompared to the standard BHT (IC
50= 1288 plusmn 21 120583M) The
meta-bromo-substituted phenyl ring containing compound4e (IC
50= 1011 plusmn 08 120583M) was the most potent member of
the series (4andash4e) followed by the para-methyl-substitutedphenyl ring containing compound 4g (IC
50= 1041plusmn19 120583M)
A gradual decrease in activity was observed for compounds4c (IC
50= 1199 plusmn 19 120583M) and 4a (IC
50= 1188 plusmn 31 120583M)
having para-nitro- and para-aldehyde-substituted phenylring attached to central bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) ring A decrease inDPPH radical scavenging activity was due to positional effectof methyl substituent on phenyl ring which was observed forcompound 4b (IC
50= 117plusmn31 120583M) havingmeta-substituted
phenyl ring instead of para-methyl-substituted phenyl ring(4g IC
50= 1041 plusmn 19 120583M) The electron donating para-
hydroxyl-substituted phenyl ring containing compound 4f(IC50= 1486 plusmn 26 120583M) was found to be a last active member
of the series However a considerable increase in activity was
observed for compound 4d (IC50
= 1244 plusmn 44 120583M) havinga para-methoxy group instead of hydroxyl functionalityon phenyl ring whereas naphthalene ring containingbarbiturate 4h (IC
50= 1336 plusmn 57 120583M) was also found to be
a weak antioxidant agent against DPPH radicalsAmong second series of the tested compounds 4indash4l
having bis(6-hydroxypyrimidine-24(1H3H)-dione as back-bone compounds having para-methyl- (4i IC
50= 3849 plusmn
66 120583M) para-chloro- (4j IC50= 3843 plusmn 88 120583M) and para-
methoxy- (4l IC50= 3979 plusmn 09 120583M) substituted phenyl ring
attached to the central bis(6-hydroxypyrimidine-24(1H3H)-dione) showed better antioxidant activity as compared toBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) whereas naphthalene containing compound4l was found to be inactive
(2-Hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate ringcontaining compounds 4mndash4z were also evaluated for theirantioxidant potential in vitro in DPPH radical scavengingassayThese compounds were found to be significant to weakinhibitors of free radicals (IC
50= 1332plusmn47ndash4396plusmn101 120583M)
Journal of Chemistry 7
Table2Re
sultof
invitro
biologicalevaluatio
nof
thes
ynthesized
compo
unds
Num
berD
PPHradicalscaveng
ingassay
IC50plusmnSE
Ma(120583M)
Anticancercytotoxicityassay
IC50plusmnSE
M(120583M)
120572-G
lucosid
aseinh
ibition
IC50plusmnSE
M(120583M)
Thym
idinep
hospho
rylase
inhibitio
nIC50plusmnSE
M(120583M)
120573-G
lucuronidase
inhibitio
nIC50plusmnSE
M(120583M)
PC3b
Helac
MCF
-7d
3T3
4a118
8plusmn31
gt30
gt30
NA
gt30
NA
NA
20481plusmn60
4b117plusmn24
gt30
gt30
NA
gt30
NA
NA
NA
4c1119plusmn19
gt30
gt30
NA
gt30
355plusmn10
NA
1721plusmn
21
4d1244plusmn44
gt30
gt30
NA
gt30
NA
2309plusmn08
NA
4e1011plusmn08
gt30
gt30
NA
gt30
NA
NA
3389plusmn64
4f1486plusmn26
gt30
gt30
NA
gt30
NA
2179plusmn20
NA
4g1041plusmn
19gt30
gt30
NA
gt30
NA
NA
NA
4h1336plusmn57
gt30
gt30
NA
gt30
230plusmn39
NA
NA
4i3849plusmn66
gt30
gt30
NA
gt30
NA
NA
NA
4j3843plusmn88
gt30
gt30
NA
gt30
NA
NA
NA
4kNA
gt30
gt30
NA
gt30
NA
1826plusmn10
NA
4l3979plusmn01
gt30
gt30
NA
gt30
NA
NA
NA
4mNA
gt30
gt30
NA
gt30
NA
1602plusmn45
NA
4nNA
gt30
gt30
NA
gt30
NA
2269plusmn15
NA
4oNA
gt30
gt30
NA
gt30
3009plusmn65
NA
2621plusmn
43
4pNA
gt30
gt30
NA
gt30
1868plusmn81
2420plusmn15
NA
4qNA
gt30
gt30
NA
gt30
2148plusmn72
1413plusmn15
29217plusmn10
4rNA
gt30
gt30
NA
gt30
201plusmn
55
168plusmn12
2621plusmn
44
4s4396plusmn101
gt30
gt30
NA
gt30
NA
NA
NA
4t2782plusmn100
gt30
gt30
NA
gt30
NA
NA
NA
4v1332plusmn47
gt30
gt30
NA
gt30
NA
576plusmn12
NA
4w4116plusmn06
gt30
gt30
NA
gt30
NA
NA
1435plusmn12
4x3888plusmn79
gt30
gt30
NA
gt30
NA
239plusmn20
NA
4yNA
1071plusmn
03
347plusmn025
49plusmn001
320plusmn02
NA
NA
NA
4z2230plusmn39
gt30
gt30
NA
gt30
NA
558plusmn15
NA
5a4616plusmn88
gt30
gt30
NA
gt30
NA
NA
NA
5bNA
gt30
gt30
NA
gt30
NA
2169plusmn05
NA
5cNA
gt30
gt30
NA
gt30
NA
1614plusmn15
NA
5dNA
gt30
gt30
NA
gt30
NA
605plusmn18
7NA
5e289plusmn09
gt30
gt30
NA
gt30
NA
NA
NA
5fNA
gt30
gt30
NA
gt30
238plusmn93
NA
1850plusmn25
5g2704plusmn23
gt30
gt30
NA
gt30
NA
1866plusmn12
NA
5hNA
gt30
gt30
NA
gt30
NA
569plusmn16
NA
5iNA
gt30
gt30
NA
gt30
NA
NA
3236plusmn64
5jNA
gt30
gt30
NA
gt30
NA
318plusmn25
NA
5k1276plusmn16
gt30
gt30
NA
gt30
NA
1664plusmn12
NA
5l1697plusmn93
gt30
gt30
NA
gt30
NA
1781plusmn
21
3875plusmn70
5mNA
gt30
gt30
NA
gt30
4534plusmn42
1471plusmn12
NA
STD
e BHT(288plusmn21)
Doxorub
icin
Doxorub
icin
Doxorub
icin
Cyclo
heximide
Acarbo
se7-Deazaxanthine
D-Saccharicacid
1-4lacton
eN-acetylcysteine(
1076plusmn28)
0912plusmn012
0506plusmn015
156plusmn005
026plusmn01
840plusmn17
341plusmn16
44575plusmn216
a SEM
stand
arderrorof
mean
b PC3
hum
anprostate
cancer
celllin
ec H
eLaHenrie
ttaLackscervical
cancer
celllin
ed M
CF-7M
ichiganCa
ncer
Foun
datio
n-7breastcancer
celllin
esand
e BHT
butylated
hydroxytoluene
8 Journal of Chemistry
except compounds 4mndash4r and 4y which were found to beinactive
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) (5andash5h) and 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) (5indash5k) rings con-taining compounds were also evaluated for their antioxidantpotential by using DPPH radical scavenging assay Para-bromo-substituted phenyl ring containing compound5k (IC
50= 1276 plusmn 16 120583M) was found as a potent anti-
oxidant agent against the tested standards BHT (IC50
=1288 plusmn 21 120583M) The compound showed similar antioxidantpotential as BHT and showed a significant antioxidant agentagainst N-acetyl cysteine (IC
50= 1076 plusmn 28 120583M) The
gradual and drastic decrease in activity was for compounds5l (IC
50= 1697 plusmn 53 120583M) 5g (IC
50= 2704 plusmn 23 120583M)
5e (IC50
= 289 plusmn 09 120583M) and 5a (IC50
= 4616 plusmn 88 120583M)having para-methyl- para-aldehyde- ortho-chloro- andortho-nitro-substituted phenyl rings attached to the centralbarbiturate moieties Compounds 5bndash5d 5f and 5hndash5j werefound to be inactive
On the basis of the observed antioxidant abilities ofthe above five different series of pyridinium adducts itwas concluded that bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) ring containing com-pounds 4andash4h are the most active one The activity may bedue to the methyl substituted nitrogen atoms of the tetrahy-dropyrimidine rings that facilitate the electronic conjugationwithin the ring However the variation of antioxidantproperties within the most important series is may be due tothe presence of electron donating (-OH (4f) -OCH
3(4d)
and -CH3(4b 4g)) electron withdrawing (-NO
2(4a) and
-CHO (4c)) and halogen (-Br (4e)) substituents on thephenyl ring attached to the bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) moiety Howeverdetailed studies are required to study the mechanism ofaction of these compounds
332 Cytotoxic Activity All synthesized diethyl ammoniumsalts of barbiturates (4andash4z and 5andash5m) were evaluated fortheir in vitro cytotoxicity against PC-3 HeLa MCF-7 cancerand 3T3 normal cell lines All compounds were found to benoncytotoxic against PC-3 HeLaMCF-7 cancer and 3T3 celllines except dichloro-substituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tet-rahydropyrimidin-4-olate ring containing compounds 4ywhich was found to be significantly toxic against all the celllines (PC3 IC
50= 1071plusmn026 120583MHeLa IC
50= 347plusmn03 120583M
MCF-7 IC50= 49 plusmn 001 120583M and 3T3 IC
50= 320 plusmn 02 120583M)
Doxorubicin was used as a standard drug to compare theactivities against PC3 (IC
50= 170 plusmn 029 120583M) HeLa (IC
50=
051 plusmn 015 120583M) and MCF-3 (IC50= 092 plusmn 001 120583M) cancer
cell lines while cycloheximide (IC50
= 026 plusmn 012 120583M) wasused as a standard-in 3T3 cell line (Table 2)
333 120572-Glucosidase Inhibition Activity Synthesized diethylammonium salts of barbiturates 4andash4z and 5andash5m were alsoevaluated for their in vitro 120572-glucosidase inhibition activityin comparison to the standard drug acarbose (IC
50= 841 plusmn
173 120583M) Among dimethyl ammonium salts (4andash4h) nitro-substituted phenyl ring containing 4c (IC
50= 355 plusmn 10 120583M)
and naphthalene ring containing 4h (IC50= 230 plusmn 392 120583M)
were found to be potent inhibitors of 120572-glucosidase enzymeand showed more activity than acarbose (IC
50= 841 plusmn
173 120583M) All other compounds were found to be inactiveAmong diethyl ammonium salts having (2-hydroxy-
44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate ring (4mndash4z)para-OCH
3- (40 IC
50= 3009 plusmn 65 120583M) para-chloro-
(4p IC50
= 1868 plusmn 081 120583M) para-bromo- (4q IC50
=2148plusmn072 120583M) andmeta-bromo- (4r IC
50= 201plusmn55 120583M)
substituted phenyl ring containing pyridinium adducts werefound to be potent 120572-glucosidase inhibitors and showedmore activity than standard acarbose (IC
50= 841plusmn 173 120583M)
Other members of the series (4m-4n and 4sndash4z) were foundto be inactive The results showed the substitution effects ofvarious functionalities of phenyl ring on the potential activityof tested series The para-bromo-substituted phenyl ringcontaining compound 4q (IC
50= 2148 plusmn 072 120583M) showed
more activity than tested standard However the replacementof bromo group with that of methylNN-dimethyl hydroxyland aldehyde functionalities resulted in complete loss ofactivity as observed in compounds 4n 4t 4v and 4xrespectively The presence of ortho-nitro- ortho-choloro-and para-choloro-substituted phenyl ring also contributedtowards inactivity as observed for compounds 4t 4w and4y
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring containing diethyl ammonium pyridinium salts(5andash5h) were also evaluated for their in vitro 120572-glucosidaseenzyme inhibition activity The series nitro-substitutedphenyl ring containing 5f was found to be the only potent120572-glucosidase inhibitor All other compounds (5andash5e and5g-5h) were found to be inactive
Among series of diethylaminium salts of pyridiniumadducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-di-oxo-1234-tetrahydropyrimidin-5-yl)methyl) central moietyattached with substituted phenyl ring the benzaldehydecontaining 5m (IC
50= 4534 plusmn 42 120583M) was found to be the
only potent inhibitor compound in comparison to against thestandard drug acarbose (IC
50= 841plusmn173 120583M)All other com-
poundswere found to be inactiveThe results are summarizedin Table 2
334 Thymidine Phosphorylase Inhibition Activity Five dif-ferent series of diethyamonium salts of barbiturates 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evalu-ated for their in vitro thymidine phosphorylase inhibition7-Deazaxanthine was used as a standard inhibitor (IC
50
= 41 plusmn 146 120583M) Phenol and naphthalene rings sub-stituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olatemoiety containing compounds 4v (IC
50= 576 plusmn 120120583M)
and 4z (IC50
= 558 plusmn 150 120583M) were found to be the potentthymidine phosphorylase inhibitors However a drasticdecrease in activity was observed when para-hydroxygroup on phenyl ring was replaced with methyl (4n IC
50
Journal of Chemistry 9
= 2269 plusmn 150 120583M) chloro (4n IC50
= 242 plusmn 15 120583M)bromo (4q IC
50= 1413 plusmn 15 120583M) and aldehyde (4x
IC50
= 239 plusmn 20 120583M) groups whereas the compounds4o 4s 4t 4w and 4y have para-methoxy- ortho-nitro-ortho-NN-dimethyl amine- ortho- para-dichloro- andortho-ortho-dichloro-substituted phenyl ring respectively
Among series of compounds (5andash5h) having bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring as centralmoiety electron donating hydroxy- andmethoxy-substitutedphenyl ring containing compounds 5h (IC
50= 569plusmn16 120583M)
and 5d (IC50
= 605 plusmn 187 120583M) respectively were foundas potent inhibitors of thymidine phosphorylase enzymeagainst the tested standard 7-deazaxanthine which was(IC50
= 41 plusmn 146 120583M) used as standard Again a drasticdecrease in activity was observed for compounds 5b (IC
50
= 2169 plusmn 05 120583M) 5c (IC50
= 1614 plusmn 15 120583M) and 5g(IC50= 1866 plusmn 12 120583M) havingmeta-methyl- ortho-methyl-
and para-aldehyde-substituted benzene ring respectivelyinstead of para-hydroxyl phenyl ring The replacement ofortho-methyl with chloro-group makes the compound 5ecompletely inactive Similarly the replacement of electrondonating para-hydroxy-group of phenyl ring with electronwithdrawing nitro-functionality also contributed towardsinactivity of compound as observed for 5f Phenol substi-tuted bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ringcontaining 5h (IC
50= 569 plusmn 16 120583M) was found to be the
potent thymidine phosphorylase inhibitorsAll members of the series of diethylaminium salts of pyri-
dinium adducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) central moi-ety attached with substituted phenyl ring were found to beweak inhibitors of thymidine phosphorylase enzyme againstthe tested standard compound 7-deazaxanthine (IC
50= 41 plusmn
146 120583M) with IC50
values 318 plusmn 25 120583M 1664 plusmn 12 120583M1781 plusmn 21 120583M and 1471 plusmn 123 120583M for 5j 5k 5l and 5mrespectively whereas para-chloro-substituted phenyl ringcontaining compound 5i was found to be inactiveThe resultsare summarized in Table 1
335 120573-Glucuronidase Inhibition Activity Compounds 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evaluatedfor their in vitro 120573-glucuronidase inhibitory activity by usingD-saccharic acid 14-lactone as standard inhibitor (IC
50=
4575 plusmn 216 120583M) Compounds 4a (IC50
= 2048 plusmn 601 120583M)4c (IC
50= 1721 plusmn 21 120583M) 4e (IC
50= 3389 plusmn 642 120583M) 4o
(IC50
= 2621 plusmn 435 120583M) 4q (IC50
= 2922 plusmn 103 120583M) 4r(IC50= 2621plusmn44 120583M) 4w (IC
50= 1435plusmn124 120583M) 5f (IC
50
= 185 plusmn 25 120583M) 5i (IC50
= 2236 plusmn 64 120583M) and 5l (IC50
=3875plusmn70 120583M) showed veryweak120573-glucuronidase inhibitionpotential whereas pyridinium adducts 4andash4c 4e 4gndash4j 4l4o 4s-4t 4w 4y 5a 5e-5f and 5i were found to be inactive(Table 2)
34 Molecular Docking Studies
341 Binding Interactions of 120572-Glucosidase Enzyme To de-fine inhibitors interactions and their specificity the dockingstudy of the most active compound 4p against 120572-glucosidase
Figure 4 Three-dimensional representation of compound 4p withtarget macromolecule 120572-glucosidase
Figure 5 Three-dimensional representation of compound 4v withtarget macromolecule thymidine phosphorylase
was carried out [23ndash25] Ten different conformations ofprotein-ligand interactions were generated and the topranked pose was selected to explore the binding interactionsIn our docking study we have observed two interactions withthe active site residues of 120572-glucosidase The polar imidazolering of His 239 showed hydrogen bond through its H atomwith the lone pair of oxygen of the pyrimidinedionemoiety ofthe compound whereas Arg 312 formed hydrogen bond withthe OH group of the same moiety of compound (Figure 4)The presence of polar and electron rich center in the formof pyrimidinedione and chlorobenzene groups of the com-pound was observed here as key factors for the interactionbetween compound andprotein Anumber of hydrogen bonddonor and acceptor pairs with their suitable orientationswere observed in the interaction pose Furthermore severalhydrophobic interactions between the nonpolar active siteresidues and the compound 4p were also observed
342 Binding Interactions of Thymidine PhosphorylaseEnzyme To explore the binding modes of these newly syn-thesized compounds in the active site of thymidine phos-phorylase the most active compound 4v was docked againstthis enzyme The docking results showed that compound4v well accommodated in the active site of thymidinephosphorylase (Figure 5) The active site residue Ala 206established a hydrogen bond through its lone pair of
10 Journal of Chemistry
Figure 6 Three-dimensional representation of compound 4w withtarget enzyme 120573-glucuronidase
oxygen with the active hydrogen of hydroxyl moiety ofthe pyrimidinedione group of the compound Howeverthe substituted phenolic group and the other electron richcenters of the pyrimidinedione showed poor behaviorregarding interaction which may depend on the nature andorientation of the surrounding active site residues Moreoverseveral weak hydrophobic interactions between the activesite residues and the compound were also observed
343 Binding Interaction of 120573-Glucuronidase Enzyme Thepredicted bindingmode ofmost active compound 4w against120573-glucuronidase showed that a hydrogen bond is formedbetween the lone pair of oxygen of cyclohexanedione compo-nent of the compound and imidazole hydrogen of the impor-tant active site residue His 509 An arene-arene and arene-cation interactionwas also observed between the pi electroniccloud of dichlorobenzene ring of compound and imidazolering of His 509 (Figure 6) Besides hydrogen bonding andarene-arene interactions several hydrophobic interactionsbetween compound 4w and the active site residues werealso observed The pyrimidinedione moiety in this case wasobserved to be passive regarding interactions
4 Conclusions
In conclusion barbiturate salts were synthesized by one-potmulticomponent reactions via tandem AldolMichael addi-tion reactions between barbituric acid and dimedone withaldehydes mediated by aqueous diethylamine The molec-ular structures of 5a 5d and 5f were deduced by single-crystal X-ray diffraction techniques All of the synthesizedcompounds were evaluated for their antioxidant potentialin vitro by using DPPH radical scavenging assay as well asenzyme inhibition activities against120572-glucosidase thymidinephosphorylase and 120573-glucuronidase enzymes Compounds4andash4g (IC
50= 1018 plusmn 08ndash1244 plusmn 44 120583M) were found to
be the potent antioxidants as compared to the standardsBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) Compound 4p (IC50= 1868 plusmn 81 120583M) was
found to be a potent 120572-glucosidase inhibitor Compound 4v(IC50
= 87 plusmn 12 120583M) was found to be a potent thymidinephosphorylase inhibitor Promising results indicate that thesecompounds need to be investigated as antioxidant agentsto treat various associated oxidative disorders Similarlytheir enzyme inhibitory potential can be reported on drugdiscovery program
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Assem Barakat Hazem A Ghabbour Abdullah MohammedAl-Majid Qurat-ul-ain Rehan Imad Kulsoom Javaid NimraNaveed Shaikh Sammer Yousuf M Iqbal Choudhary andAbdul Wadood contributed equally to this work
Acknowledgments
The authors would like to extend their sincere appreciation tothe Deanship of Scientific Research at King Saud Universityfor funding this Research Group no (RGP-257-1435-1436)
References
[1] K Undheim T Bennecke A R Katritzky C W Rees andE F V Scriven Comprehensive Heterocyclic Chemistry vol 6supplement 93 Elsevier Pergamon Oxford UK 1996
[2] B TaylorModern Medical Chemistry Prentice Hall New YorkNY USA 1994
[3] R Y Levina and F K Velichko ldquoAdvances in the chemistry ofbarbituric acidsrdquo Russian Chemical Reviews vol 29 no 8 pp437ndash459 1960
[4] P Sing and K Paul ldquoA simple synthesis of 5-spirobarbituricacids and transformations of spirocyclopropane barbiturates to5-substitutated barbituratesrdquo Indian Journal of Chemistry B vol45 pp 247ndash251 2006
[5] X Chen K Tanaka and F Yoneda ldquoSimple newmethod for thesynthesis of 5-deaza-10-oxaflavin a potential organic oxidantrdquoChemical and Pharmaceutical Bulletin vol 38 no 2 pp 307ndash311 1990
[6] L R Morgan B S Jursic C L Hooper D M Neumann KThangaraj and B LeBlanc ldquoAnticancer activity for 4 41015840-di-hydroxybenzophenone-2 4-dinitrophenylhydrazone (A-007)analogues and their abilities to interact with lymphoendothelialcell surfacemarkersrdquo Bioorganic ampMedicinal Chemistry Lettersvol 12 no 23 pp 3407ndash3411 2002
[7] G Orzalesi P Gratteri and S Selleri ldquoSynthesis of new 13-dicyclohexyl barbituric acid derivatives with anti-inflammatorypotential activityrdquoEuropean Journal ofMedicinal Chemistry vol25 no 2 pp 197ndash201 1990
[8] D T Puerta and S M Cohen ldquoA bioinorganic perspectiveson matrix metalloproteinase inhibitionrdquo Current Topics inMedicinal Chemistry vol 4 no 15 pp 1551ndash1573 2004
[9] M E Wolff Burgerrsquos Medicinal Chemistry and Drug DiscoveryJohn Wiley amp Sons New York NY USA 1997
Journal of Chemistry 11
[10] E Fischer and J von Mering ldquoUeber eine neue klasse vonschlafmittelnrdquo in Untersuchungen aus Verschiedenen GebietenM Bergmann Ed pp 671ndash679 Springer Berlin Germany1924
[11] W J Doran ldquoBarbituric acid hypnoticsrdquo Medicinal Chemistryvol 4 pp 164ndash167 1959
[12] B Bobranski ldquoProgress in chemistry of barbituric acidrdquoWiad-omosci Chemiczne vol 31 pp 231ndash278 1977
[13] S Senda H Izumi and H Fujimura ldquoUracil derivaives andrelated compounds 6 Derivatives of 5-alkyle-246-trioxoper-hydropyrimidine as antiphlogisticsrdquo Arzneimittel Forschungvol 17 no 12 p 151 1967
[14] N Moussier L Bruche F Viani and M Zanda ldquoFluorinatedbarbituric acid derivatives synthesis and bio-activityrdquo CurrentOrganic Chemistry vol 7 no 11 pp 1071ndash1080 2003
[15] P-G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000
[16] N Ramarathnam T Osawa H Ochi and S Kawakishi ldquoThecontribution of plant food antioxidants to human healthrdquoTrends in Food Science amp Technology vol 6 no 3 pp 75ndash821995
[17] A Barakat A M Al-Majid H J Al-Najjar et al ldquoZwitterionicpyrimidinium adducts as antioxidants with therapeutic poten-tial as nitric oxide scavengerrdquo European Journal of MedicinalChemistry vol 84 pp 146ndash154 2014
[18] P Singh M Kaur and P Verma ldquoDesign synthesis andanticancer activities of hybrids of indole and barbituric acids-Identification of highly promising leadsrdquo Bioorganic andMedic-inal Chemistry Letters vol 19 no 11 pp 3054ndash3058 2009
[19] A M Al-Majid A Barakat H J Al-Najjar Y N Mabkhot HA Ghabbour and H-K Fun ldquoTandem aldol-michael reactionsin aqueous diethylamine medium a greener and efficientapproach to bis-pyrimidine derivativesrdquo International Journalof Molecular Sciences vol 14 no 12 pp 23762ndash23773 2013
[20] A Barakat A M Al-Majid G Lotfy et al ldquoSynthesis and dy-namics studies of barbituric acid derivatives as urease inhibi-torsrdquo Chemistry Central Journal vol 9 no 1 article 63 2015
[21] G M Sheldrick ldquoA short history of SHELXrdquo Acta Crystallo-graphica A vol 64 pp 112ndash122 2008
[22] A L Spek ldquoStructure validation in chemical crystallographyrdquoActaCrystallographica SectionD Biological Crystallography vol65 no 2 pp 148ndash155 2009
[23] F Rahim K Ullah H Ullah et al ldquoTriazinoindole analogs aspotent inhibitors of 120572-glucosidase synthesis biological evalua-tion and molecular docking studiesrdquo Bioorganic Chemistry vol58 pp 81ndash87 2015
[24] F Rahim F Malik H Ullah et al ldquoIsatin based Schiff basesas inhibitors of 120572-glucosidase synthesis characterization invitro evaluation and molecular docking studiesrdquo BioorganicChemistry vol 60 article 1803 pp 42ndash48 2015
[25] A Barakat A M Al-Majid M S Islam et al ldquoMolecular struc-ture investigation and biological evaluation of Michael adductsderived from dimedonerdquo Research on Chemical Intermediatesvol 42 pp 4041ndash4053 2016
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Chemistry 3
5a 5d
C27C26
C25
01C1
C6
C7C8
C13C12
C15C14
C19
C17C18
C16
C506N1
050204
C2C3
C4
C21
C20C24C23
C11 C9C10C22
01W
03
N2
C21
C4 02
04
C3 C2
C5C6
C7C8
C9
C25
C13C12
C23C11
C22C10
0103
C1
C14C15
C26N1
C16
C17 C27C28
C24
05
C19
C18
C20
5f
C2203
0401
06
05
N1
N2
02
C10
C23 C11
C12 C13
C9
C5
C4
C3
C2C1
C27
C26C16C19
C18 C17
C25 C24
C6C7
C14 C15
C21
C20
C8
Figure 2 ORTEP diagram of 5a 5d and 5f Displacement ellipsoids are plotted at the 40 probability level for non-H atoms
inhibition assay thymidine phosphorylase inhibition assayand 120572-glucosidase inhibition assay and full methods descrip-tions are provided in Supplementary Materials
3 Results and Discussion
31 Chemistry We have been focusing on the developmentof highly expedient methods for the synthesis of diverseheterocyclic compounds of biological importance via one-pot multicomponent reactions (MCRs) and avoiding organicsolvents in organic synthesis [17 19 20] This had led tothe development of several efficient environmentally benignclean and economical process technology (Green ChemistryConcepts) In current reaction strategy equimolar amountsof barbituric acid (1) and dimedone (2) with aldehyde(3) in the presence of aqueous diethylamine medium atRT yielded salts of PYT adducts 4andash4y and 5andash5m wereobtained in quantitative yields by simple filtration [17ndash20]
Products 4s 4t and 5g were identified as new products(Scheme 1)
32 X-Ray Diffraction The molecular structures of com-pounds 5a 5d and 5f were unambiguously deduced bysingle-crystal X-ray diffraction technique (Figure 2) Thecrystals of the synthesized molecules 5a 5d and 5f weregrown by slow diffusion of diethyl ether solution of puresamples 5a 5d and 5f in DCMEtOH at room temperaturefollowed by allowing standing for 24 h The structures wereresolved by direct methods by using the SHELXL97 program[21 22] in the SHELXTL-plus package and refined by afull-matrix least-squares procedure on 1198652 using SHELXS97Diffraction data were collected on a Bruker APEX-II CCDdiffractometer (Bruker AXS GmbH) The crystal structureand refinement data of compounds 5a 5d and 5f are listedin Table 1 The final atomic coordinates for all atoms andcomplete listing of bond distance and angles are presented
4 Journal of Chemistry
O
O O O O
RT
RT RT
ON N
2 1a b
1a b
O
O
O
O O
O
R R
O O O O O
O O
O
O
HOHO
NH
O
HO
O
N NN
NHN
O O HO
HO HO
HO
O O
O
O O
OO
O O O O O
O O
O
OO
O
OO
O O
ONH
O
4l
4z
NH
NH
NH N
NN
NN
H
NN
N NN
H
NH
HN
H NN
NH
N
O O O O
O
O O HO O
OO
N N
NN N
NO2 2
2
3
O
HO
HO
O
ON
N
CHO5l
4h
HOHO
R
NH2Et2
R2
R2
R2
R2
oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus
NH2Et2oplus
⊖
⊖ NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
⊖
NH2Et2oplus
NH2Et2oplus
⊖R1 R1R1
R1
R1
R1
R2R3
R4
R1
R1
R1
R1 R1
H2ONHEt2
H2ONHEt2 H2ONHEt2
CH3
CH3CH3
CH3 CH3
CH3
CH3CH3
H3C
H3CH3C
H3C
H3CH3CH3C
H3C
H3CH3C
R3
R2
R4
4a R=CHOR1=H4b R=HR1=CH34c R=NO2R1=H4d R=OCH3R1=H4e R=HR1=Br4f R=OHR1=H4g R=CH3R1=H
4i R=CH34j R=Cl4k R=OCH3
4m R1=R2=R3=R4=H4n R1=R2=R4=HR3=CH34o R1=R2=R4=HR3=OCH34p R1=R2=R4=HR3=Cl4q R1=R2=R4=HR3=Br4r R1=R3=R4=HR2=Br4s R1=NO2R2=R3=R4=H4t R1=R2=R4=HR3=NMe24v R1=R2=R4=HR3=OH4w R1=R3=ClR2=R4=H4x R1=R2=R4=HR3=CHO4y R1=R4=ClR2=R3=H
5a R1=NO2R2=R3=R4=H5b R1=R3=R4=HR2=CH35c R1=R3=R4=CH3R2=H5d R1=R2=R4=HR3=OCH35e R1=R4=ClR2=R3=H5f R1=R2=R4=HR3=NO25g R1=R2=R4=HR3=CHO5h R1=R2=R4=HR3=OH
5i R=Cl5j R=H5k R=Br5l R=CH35m R=CHO
Scheme 1 Synthesis of the target compounds
Journal of Chemistry 5
Table 1 The crystal and experimental data of compounds 5a 5d and 5f
5a 5d 5fEmpirical formula C
23H26NO6sdotC4H12NsdotH2O C
24H29O5sdotC4H12N C
23H26NO6sdotC4H12N
Formula weight 50461 47162 48659Temperature 100K 100K 100KWavelength Mo K120572 radiation 120582 = 071073 A Mo K120572 radiation 120582 = 071073 A Mo K120572 radiation 120582 = 071073 ACrystal system Orthorhombic Monoclinic MonoclinicSpace group P2
12121
P21n P2
1n
119886 116155 (5) A 102288 (5) A 101137 (4) A119887 120514 (5) A 180048 (7) A 181225 (7) A119888 193665 (7) A 150893 (7) A 154853 (6) A120573 9000∘ 106425 (2)∘ 106730 (1)∘
Volume 271098 (19) A3 26656 (2) A3 271809 (18) A3
119885 4 4 4Calculated density 1236Mgmminus3 1175Mgmminus3 1189Mgmminus3
Absorption coefficient 009mmminus1 008mmminus1 008mmminus1
119865 (000) 1088 1024 1048Crystal size 057 times 041 times 007mm 066 times 033 times 025mm 033 times 029 times 022mm120579 range 24ndash242∘ 22ndash297∘ 22ndash321∘
Reflections collected 28531 67389 25488Reflections unique 4501 6194 4829Independent reflections 6224 11203 6233Parameters 350 326 334119877int 0090 0122 0053R [F2 gt 2120590(F2)] 0054 0069 0055119908R (F2) 0109 0156 0132Goodness of fit 105 101 109Maxmin 120588 e Aminus3 042 and minus027 043 and minus030 029 and minus026CCDC number 1444058 1443783 1444050
in supplementary information ORTEP drawings of final X-ray model of compounds 5a 5d and 5f with the atomicnumbering scheme are presented in Figure 2 while crystalpacking presentation of compounds 5a 5d and 5f is shownin Figure 3
Compound 5a crystallizes in orthorhombic crystal sys-tem in a space group of P2
12121 on the other hand com-
pounds 5d and 5f crystalize in monoclinic crystal system ina space group of P2
1n It was observed that the compound
5a has hydrogen bonding between the C5-O2sdot sdot sdotHsdot sdot sdotO
4-C13
while compounds 5d and 5f exist only in the enol formThereare two strong intramolecular hydrogen bonds on compound5a between O
4mdashH1O4sdot sdot sdotO2and N
2mdashH2N2sdot sdot sdotO1 while
compounds 5d and 5f have no intramolecular hydrogenbonding The crystal structures of the tested compounds 5a5d and 5f were formed by different hydrogen bonds betweenthemolecules and diethyl amine and water solvent moleculesin case of 5a to form network structures Crystallographicdata for the solved structures 5a 5d and 5f were deposited tothe Cambridge Crystallographic Data Center as supplemen-tary publication numbers CCDC-1444058 CCDC-1443783and CCDC-1444050 respectively which can be obtained freeof charge from the Cambridge Crystallographic Data Centrevia httpwwwccdccamacukdata requestcif
33 Biological Activity Evaluation All synthesized diethylammonium salts of barbiturates having bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (4andash4h)bis(6-hydroxypyrimidine-24(1H3H)-dione (4indash4l) and(2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dim-ethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate (4mndash4z) as well as bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) (5andash5m) as basic nuclei were screened for theirantioxidant potential in vitro by using DPPH radicalscavenging assay and cytotoxicity against PC-3 HeLa andMCF-7 cancer cell lines and 3T3 normal fibroblast cell linesThese derivatives 4andash4z and 5andash5m were also evaluated forin vitro enzyme inhibition activities against 120572-glucosidasethymidine phosphorylase and 120573-glucuronidase enzymesThe results are summarized in Table 2
331 Antioxidant Activity (DPPH Radical Scavenging Assay)Among series of compounds (4andash4h) having bis(6-hydroxy-1 3-dimethyl-2 4-dioxo-1 2 3 4-tetrahydropyrimidin-5-yl)ring as basic nucleus compounds 4a (IC
50= 1188plusmn21 120583M)
4b (IC50
= 117 plusmn 24 120583M) 4c (IC50
= 1119 plusmn 19 120583M) 4d(IC50
= 1244 plusmn 44 120583M) 4e (IC50
= 1011 plusmn 08 120583M) 4f(IC50
= 1486 plusmn 26 120583M) and 4g (IC50
= 1041 plusmn 19 120583M)were found to be potent antioxidants in comparison to the
6 Journal of Chemistry
5f
5d5a
Figure 3Molecular packing of 5a 5d and 5f viewed approximately three-dimensional network along 119887 axis and three-dimensional networkrespectively Intermolecular interaction is drawn as dashed lines
standards BHT (IC50= 1288 plusmn 21 120583M) andN-acetylcysteine
(IC50
= 1076 plusmn 28 120583M) Compounds 4andash4e (IC50
=1018 plusmn 08ndash1244 plusmn 44 120583M) were found to be more activecompared to the standard BHT (IC
50= 1288 plusmn 21 120583M) The
meta-bromo-substituted phenyl ring containing compound4e (IC
50= 1011 plusmn 08 120583M) was the most potent member of
the series (4andash4e) followed by the para-methyl-substitutedphenyl ring containing compound 4g (IC
50= 1041plusmn19 120583M)
A gradual decrease in activity was observed for compounds4c (IC
50= 1199 plusmn 19 120583M) and 4a (IC
50= 1188 plusmn 31 120583M)
having para-nitro- and para-aldehyde-substituted phenylring attached to central bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) ring A decrease inDPPH radical scavenging activity was due to positional effectof methyl substituent on phenyl ring which was observed forcompound 4b (IC
50= 117plusmn31 120583M) havingmeta-substituted
phenyl ring instead of para-methyl-substituted phenyl ring(4g IC
50= 1041 plusmn 19 120583M) The electron donating para-
hydroxyl-substituted phenyl ring containing compound 4f(IC50= 1486 plusmn 26 120583M) was found to be a last active member
of the series However a considerable increase in activity was
observed for compound 4d (IC50
= 1244 plusmn 44 120583M) havinga para-methoxy group instead of hydroxyl functionalityon phenyl ring whereas naphthalene ring containingbarbiturate 4h (IC
50= 1336 plusmn 57 120583M) was also found to be
a weak antioxidant agent against DPPH radicalsAmong second series of the tested compounds 4indash4l
having bis(6-hydroxypyrimidine-24(1H3H)-dione as back-bone compounds having para-methyl- (4i IC
50= 3849 plusmn
66 120583M) para-chloro- (4j IC50= 3843 plusmn 88 120583M) and para-
methoxy- (4l IC50= 3979 plusmn 09 120583M) substituted phenyl ring
attached to the central bis(6-hydroxypyrimidine-24(1H3H)-dione) showed better antioxidant activity as compared toBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) whereas naphthalene containing compound4l was found to be inactive
(2-Hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate ringcontaining compounds 4mndash4z were also evaluated for theirantioxidant potential in vitro in DPPH radical scavengingassayThese compounds were found to be significant to weakinhibitors of free radicals (IC
50= 1332plusmn47ndash4396plusmn101 120583M)
Journal of Chemistry 7
Table2Re
sultof
invitro
biologicalevaluatio
nof
thes
ynthesized
compo
unds
Num
berD
PPHradicalscaveng
ingassay
IC50plusmnSE
Ma(120583M)
Anticancercytotoxicityassay
IC50plusmnSE
M(120583M)
120572-G
lucosid
aseinh
ibition
IC50plusmnSE
M(120583M)
Thym
idinep
hospho
rylase
inhibitio
nIC50plusmnSE
M(120583M)
120573-G
lucuronidase
inhibitio
nIC50plusmnSE
M(120583M)
PC3b
Helac
MCF
-7d
3T3
4a118
8plusmn31
gt30
gt30
NA
gt30
NA
NA
20481plusmn60
4b117plusmn24
gt30
gt30
NA
gt30
NA
NA
NA
4c1119plusmn19
gt30
gt30
NA
gt30
355plusmn10
NA
1721plusmn
21
4d1244plusmn44
gt30
gt30
NA
gt30
NA
2309plusmn08
NA
4e1011plusmn08
gt30
gt30
NA
gt30
NA
NA
3389plusmn64
4f1486plusmn26
gt30
gt30
NA
gt30
NA
2179plusmn20
NA
4g1041plusmn
19gt30
gt30
NA
gt30
NA
NA
NA
4h1336plusmn57
gt30
gt30
NA
gt30
230plusmn39
NA
NA
4i3849plusmn66
gt30
gt30
NA
gt30
NA
NA
NA
4j3843plusmn88
gt30
gt30
NA
gt30
NA
NA
NA
4kNA
gt30
gt30
NA
gt30
NA
1826plusmn10
NA
4l3979plusmn01
gt30
gt30
NA
gt30
NA
NA
NA
4mNA
gt30
gt30
NA
gt30
NA
1602plusmn45
NA
4nNA
gt30
gt30
NA
gt30
NA
2269plusmn15
NA
4oNA
gt30
gt30
NA
gt30
3009plusmn65
NA
2621plusmn
43
4pNA
gt30
gt30
NA
gt30
1868plusmn81
2420plusmn15
NA
4qNA
gt30
gt30
NA
gt30
2148plusmn72
1413plusmn15
29217plusmn10
4rNA
gt30
gt30
NA
gt30
201plusmn
55
168plusmn12
2621plusmn
44
4s4396plusmn101
gt30
gt30
NA
gt30
NA
NA
NA
4t2782plusmn100
gt30
gt30
NA
gt30
NA
NA
NA
4v1332plusmn47
gt30
gt30
NA
gt30
NA
576plusmn12
NA
4w4116plusmn06
gt30
gt30
NA
gt30
NA
NA
1435plusmn12
4x3888plusmn79
gt30
gt30
NA
gt30
NA
239plusmn20
NA
4yNA
1071plusmn
03
347plusmn025
49plusmn001
320plusmn02
NA
NA
NA
4z2230plusmn39
gt30
gt30
NA
gt30
NA
558plusmn15
NA
5a4616plusmn88
gt30
gt30
NA
gt30
NA
NA
NA
5bNA
gt30
gt30
NA
gt30
NA
2169plusmn05
NA
5cNA
gt30
gt30
NA
gt30
NA
1614plusmn15
NA
5dNA
gt30
gt30
NA
gt30
NA
605plusmn18
7NA
5e289plusmn09
gt30
gt30
NA
gt30
NA
NA
NA
5fNA
gt30
gt30
NA
gt30
238plusmn93
NA
1850plusmn25
5g2704plusmn23
gt30
gt30
NA
gt30
NA
1866plusmn12
NA
5hNA
gt30
gt30
NA
gt30
NA
569plusmn16
NA
5iNA
gt30
gt30
NA
gt30
NA
NA
3236plusmn64
5jNA
gt30
gt30
NA
gt30
NA
318plusmn25
NA
5k1276plusmn16
gt30
gt30
NA
gt30
NA
1664plusmn12
NA
5l1697plusmn93
gt30
gt30
NA
gt30
NA
1781plusmn
21
3875plusmn70
5mNA
gt30
gt30
NA
gt30
4534plusmn42
1471plusmn12
NA
STD
e BHT(288plusmn21)
Doxorub
icin
Doxorub
icin
Doxorub
icin
Cyclo
heximide
Acarbo
se7-Deazaxanthine
D-Saccharicacid
1-4lacton
eN-acetylcysteine(
1076plusmn28)
0912plusmn012
0506plusmn015
156plusmn005
026plusmn01
840plusmn17
341plusmn16
44575plusmn216
a SEM
stand
arderrorof
mean
b PC3
hum
anprostate
cancer
celllin
ec H
eLaHenrie
ttaLackscervical
cancer
celllin
ed M
CF-7M
ichiganCa
ncer
Foun
datio
n-7breastcancer
celllin
esand
e BHT
butylated
hydroxytoluene
8 Journal of Chemistry
except compounds 4mndash4r and 4y which were found to beinactive
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) (5andash5h) and 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) (5indash5k) rings con-taining compounds were also evaluated for their antioxidantpotential by using DPPH radical scavenging assay Para-bromo-substituted phenyl ring containing compound5k (IC
50= 1276 plusmn 16 120583M) was found as a potent anti-
oxidant agent against the tested standards BHT (IC50
=1288 plusmn 21 120583M) The compound showed similar antioxidantpotential as BHT and showed a significant antioxidant agentagainst N-acetyl cysteine (IC
50= 1076 plusmn 28 120583M) The
gradual and drastic decrease in activity was for compounds5l (IC
50= 1697 plusmn 53 120583M) 5g (IC
50= 2704 plusmn 23 120583M)
5e (IC50
= 289 plusmn 09 120583M) and 5a (IC50
= 4616 plusmn 88 120583M)having para-methyl- para-aldehyde- ortho-chloro- andortho-nitro-substituted phenyl rings attached to the centralbarbiturate moieties Compounds 5bndash5d 5f and 5hndash5j werefound to be inactive
On the basis of the observed antioxidant abilities ofthe above five different series of pyridinium adducts itwas concluded that bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) ring containing com-pounds 4andash4h are the most active one The activity may bedue to the methyl substituted nitrogen atoms of the tetrahy-dropyrimidine rings that facilitate the electronic conjugationwithin the ring However the variation of antioxidantproperties within the most important series is may be due tothe presence of electron donating (-OH (4f) -OCH
3(4d)
and -CH3(4b 4g)) electron withdrawing (-NO
2(4a) and
-CHO (4c)) and halogen (-Br (4e)) substituents on thephenyl ring attached to the bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) moiety Howeverdetailed studies are required to study the mechanism ofaction of these compounds
332 Cytotoxic Activity All synthesized diethyl ammoniumsalts of barbiturates (4andash4z and 5andash5m) were evaluated fortheir in vitro cytotoxicity against PC-3 HeLa MCF-7 cancerand 3T3 normal cell lines All compounds were found to benoncytotoxic against PC-3 HeLaMCF-7 cancer and 3T3 celllines except dichloro-substituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tet-rahydropyrimidin-4-olate ring containing compounds 4ywhich was found to be significantly toxic against all the celllines (PC3 IC
50= 1071plusmn026 120583MHeLa IC
50= 347plusmn03 120583M
MCF-7 IC50= 49 plusmn 001 120583M and 3T3 IC
50= 320 plusmn 02 120583M)
Doxorubicin was used as a standard drug to compare theactivities against PC3 (IC
50= 170 plusmn 029 120583M) HeLa (IC
50=
051 plusmn 015 120583M) and MCF-3 (IC50= 092 plusmn 001 120583M) cancer
cell lines while cycloheximide (IC50
= 026 plusmn 012 120583M) wasused as a standard-in 3T3 cell line (Table 2)
333 120572-Glucosidase Inhibition Activity Synthesized diethylammonium salts of barbiturates 4andash4z and 5andash5m were alsoevaluated for their in vitro 120572-glucosidase inhibition activityin comparison to the standard drug acarbose (IC
50= 841 plusmn
173 120583M) Among dimethyl ammonium salts (4andash4h) nitro-substituted phenyl ring containing 4c (IC
50= 355 plusmn 10 120583M)
and naphthalene ring containing 4h (IC50= 230 plusmn 392 120583M)
were found to be potent inhibitors of 120572-glucosidase enzymeand showed more activity than acarbose (IC
50= 841 plusmn
173 120583M) All other compounds were found to be inactiveAmong diethyl ammonium salts having (2-hydroxy-
44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate ring (4mndash4z)para-OCH
3- (40 IC
50= 3009 plusmn 65 120583M) para-chloro-
(4p IC50
= 1868 plusmn 081 120583M) para-bromo- (4q IC50
=2148plusmn072 120583M) andmeta-bromo- (4r IC
50= 201plusmn55 120583M)
substituted phenyl ring containing pyridinium adducts werefound to be potent 120572-glucosidase inhibitors and showedmore activity than standard acarbose (IC
50= 841plusmn 173 120583M)
Other members of the series (4m-4n and 4sndash4z) were foundto be inactive The results showed the substitution effects ofvarious functionalities of phenyl ring on the potential activityof tested series The para-bromo-substituted phenyl ringcontaining compound 4q (IC
50= 2148 plusmn 072 120583M) showed
more activity than tested standard However the replacementof bromo group with that of methylNN-dimethyl hydroxyland aldehyde functionalities resulted in complete loss ofactivity as observed in compounds 4n 4t 4v and 4xrespectively The presence of ortho-nitro- ortho-choloro-and para-choloro-substituted phenyl ring also contributedtowards inactivity as observed for compounds 4t 4w and4y
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring containing diethyl ammonium pyridinium salts(5andash5h) were also evaluated for their in vitro 120572-glucosidaseenzyme inhibition activity The series nitro-substitutedphenyl ring containing 5f was found to be the only potent120572-glucosidase inhibitor All other compounds (5andash5e and5g-5h) were found to be inactive
Among series of diethylaminium salts of pyridiniumadducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-di-oxo-1234-tetrahydropyrimidin-5-yl)methyl) central moietyattached with substituted phenyl ring the benzaldehydecontaining 5m (IC
50= 4534 plusmn 42 120583M) was found to be the
only potent inhibitor compound in comparison to against thestandard drug acarbose (IC
50= 841plusmn173 120583M)All other com-
poundswere found to be inactiveThe results are summarizedin Table 2
334 Thymidine Phosphorylase Inhibition Activity Five dif-ferent series of diethyamonium salts of barbiturates 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evalu-ated for their in vitro thymidine phosphorylase inhibition7-Deazaxanthine was used as a standard inhibitor (IC
50
= 41 plusmn 146 120583M) Phenol and naphthalene rings sub-stituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olatemoiety containing compounds 4v (IC
50= 576 plusmn 120120583M)
and 4z (IC50
= 558 plusmn 150 120583M) were found to be the potentthymidine phosphorylase inhibitors However a drasticdecrease in activity was observed when para-hydroxygroup on phenyl ring was replaced with methyl (4n IC
50
Journal of Chemistry 9
= 2269 plusmn 150 120583M) chloro (4n IC50
= 242 plusmn 15 120583M)bromo (4q IC
50= 1413 plusmn 15 120583M) and aldehyde (4x
IC50
= 239 plusmn 20 120583M) groups whereas the compounds4o 4s 4t 4w and 4y have para-methoxy- ortho-nitro-ortho-NN-dimethyl amine- ortho- para-dichloro- andortho-ortho-dichloro-substituted phenyl ring respectively
Among series of compounds (5andash5h) having bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring as centralmoiety electron donating hydroxy- andmethoxy-substitutedphenyl ring containing compounds 5h (IC
50= 569plusmn16 120583M)
and 5d (IC50
= 605 plusmn 187 120583M) respectively were foundas potent inhibitors of thymidine phosphorylase enzymeagainst the tested standard 7-deazaxanthine which was(IC50
= 41 plusmn 146 120583M) used as standard Again a drasticdecrease in activity was observed for compounds 5b (IC
50
= 2169 plusmn 05 120583M) 5c (IC50
= 1614 plusmn 15 120583M) and 5g(IC50= 1866 plusmn 12 120583M) havingmeta-methyl- ortho-methyl-
and para-aldehyde-substituted benzene ring respectivelyinstead of para-hydroxyl phenyl ring The replacement ofortho-methyl with chloro-group makes the compound 5ecompletely inactive Similarly the replacement of electrondonating para-hydroxy-group of phenyl ring with electronwithdrawing nitro-functionality also contributed towardsinactivity of compound as observed for 5f Phenol substi-tuted bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ringcontaining 5h (IC
50= 569 plusmn 16 120583M) was found to be the
potent thymidine phosphorylase inhibitorsAll members of the series of diethylaminium salts of pyri-
dinium adducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) central moi-ety attached with substituted phenyl ring were found to beweak inhibitors of thymidine phosphorylase enzyme againstthe tested standard compound 7-deazaxanthine (IC
50= 41 plusmn
146 120583M) with IC50
values 318 plusmn 25 120583M 1664 plusmn 12 120583M1781 plusmn 21 120583M and 1471 plusmn 123 120583M for 5j 5k 5l and 5mrespectively whereas para-chloro-substituted phenyl ringcontaining compound 5i was found to be inactiveThe resultsare summarized in Table 1
335 120573-Glucuronidase Inhibition Activity Compounds 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evaluatedfor their in vitro 120573-glucuronidase inhibitory activity by usingD-saccharic acid 14-lactone as standard inhibitor (IC
50=
4575 plusmn 216 120583M) Compounds 4a (IC50
= 2048 plusmn 601 120583M)4c (IC
50= 1721 plusmn 21 120583M) 4e (IC
50= 3389 plusmn 642 120583M) 4o
(IC50
= 2621 plusmn 435 120583M) 4q (IC50
= 2922 plusmn 103 120583M) 4r(IC50= 2621plusmn44 120583M) 4w (IC
50= 1435plusmn124 120583M) 5f (IC
50
= 185 plusmn 25 120583M) 5i (IC50
= 2236 plusmn 64 120583M) and 5l (IC50
=3875plusmn70 120583M) showed veryweak120573-glucuronidase inhibitionpotential whereas pyridinium adducts 4andash4c 4e 4gndash4j 4l4o 4s-4t 4w 4y 5a 5e-5f and 5i were found to be inactive(Table 2)
34 Molecular Docking Studies
341 Binding Interactions of 120572-Glucosidase Enzyme To de-fine inhibitors interactions and their specificity the dockingstudy of the most active compound 4p against 120572-glucosidase
Figure 4 Three-dimensional representation of compound 4p withtarget macromolecule 120572-glucosidase
Figure 5 Three-dimensional representation of compound 4v withtarget macromolecule thymidine phosphorylase
was carried out [23ndash25] Ten different conformations ofprotein-ligand interactions were generated and the topranked pose was selected to explore the binding interactionsIn our docking study we have observed two interactions withthe active site residues of 120572-glucosidase The polar imidazolering of His 239 showed hydrogen bond through its H atomwith the lone pair of oxygen of the pyrimidinedionemoiety ofthe compound whereas Arg 312 formed hydrogen bond withthe OH group of the same moiety of compound (Figure 4)The presence of polar and electron rich center in the formof pyrimidinedione and chlorobenzene groups of the com-pound was observed here as key factors for the interactionbetween compound andprotein Anumber of hydrogen bonddonor and acceptor pairs with their suitable orientationswere observed in the interaction pose Furthermore severalhydrophobic interactions between the nonpolar active siteresidues and the compound 4p were also observed
342 Binding Interactions of Thymidine PhosphorylaseEnzyme To explore the binding modes of these newly syn-thesized compounds in the active site of thymidine phos-phorylase the most active compound 4v was docked againstthis enzyme The docking results showed that compound4v well accommodated in the active site of thymidinephosphorylase (Figure 5) The active site residue Ala 206established a hydrogen bond through its lone pair of
10 Journal of Chemistry
Figure 6 Three-dimensional representation of compound 4w withtarget enzyme 120573-glucuronidase
oxygen with the active hydrogen of hydroxyl moiety ofthe pyrimidinedione group of the compound Howeverthe substituted phenolic group and the other electron richcenters of the pyrimidinedione showed poor behaviorregarding interaction which may depend on the nature andorientation of the surrounding active site residues Moreoverseveral weak hydrophobic interactions between the activesite residues and the compound were also observed
343 Binding Interaction of 120573-Glucuronidase Enzyme Thepredicted bindingmode ofmost active compound 4w against120573-glucuronidase showed that a hydrogen bond is formedbetween the lone pair of oxygen of cyclohexanedione compo-nent of the compound and imidazole hydrogen of the impor-tant active site residue His 509 An arene-arene and arene-cation interactionwas also observed between the pi electroniccloud of dichlorobenzene ring of compound and imidazolering of His 509 (Figure 6) Besides hydrogen bonding andarene-arene interactions several hydrophobic interactionsbetween compound 4w and the active site residues werealso observed The pyrimidinedione moiety in this case wasobserved to be passive regarding interactions
4 Conclusions
In conclusion barbiturate salts were synthesized by one-potmulticomponent reactions via tandem AldolMichael addi-tion reactions between barbituric acid and dimedone withaldehydes mediated by aqueous diethylamine The molec-ular structures of 5a 5d and 5f were deduced by single-crystal X-ray diffraction techniques All of the synthesizedcompounds were evaluated for their antioxidant potentialin vitro by using DPPH radical scavenging assay as well asenzyme inhibition activities against120572-glucosidase thymidinephosphorylase and 120573-glucuronidase enzymes Compounds4andash4g (IC
50= 1018 plusmn 08ndash1244 plusmn 44 120583M) were found to
be the potent antioxidants as compared to the standardsBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) Compound 4p (IC50= 1868 plusmn 81 120583M) was
found to be a potent 120572-glucosidase inhibitor Compound 4v(IC50
= 87 plusmn 12 120583M) was found to be a potent thymidinephosphorylase inhibitor Promising results indicate that thesecompounds need to be investigated as antioxidant agentsto treat various associated oxidative disorders Similarlytheir enzyme inhibitory potential can be reported on drugdiscovery program
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Assem Barakat Hazem A Ghabbour Abdullah MohammedAl-Majid Qurat-ul-ain Rehan Imad Kulsoom Javaid NimraNaveed Shaikh Sammer Yousuf M Iqbal Choudhary andAbdul Wadood contributed equally to this work
Acknowledgments
The authors would like to extend their sincere appreciation tothe Deanship of Scientific Research at King Saud Universityfor funding this Research Group no (RGP-257-1435-1436)
References
[1] K Undheim T Bennecke A R Katritzky C W Rees andE F V Scriven Comprehensive Heterocyclic Chemistry vol 6supplement 93 Elsevier Pergamon Oxford UK 1996
[2] B TaylorModern Medical Chemistry Prentice Hall New YorkNY USA 1994
[3] R Y Levina and F K Velichko ldquoAdvances in the chemistry ofbarbituric acidsrdquo Russian Chemical Reviews vol 29 no 8 pp437ndash459 1960
[4] P Sing and K Paul ldquoA simple synthesis of 5-spirobarbituricacids and transformations of spirocyclopropane barbiturates to5-substitutated barbituratesrdquo Indian Journal of Chemistry B vol45 pp 247ndash251 2006
[5] X Chen K Tanaka and F Yoneda ldquoSimple newmethod for thesynthesis of 5-deaza-10-oxaflavin a potential organic oxidantrdquoChemical and Pharmaceutical Bulletin vol 38 no 2 pp 307ndash311 1990
[6] L R Morgan B S Jursic C L Hooper D M Neumann KThangaraj and B LeBlanc ldquoAnticancer activity for 4 41015840-di-hydroxybenzophenone-2 4-dinitrophenylhydrazone (A-007)analogues and their abilities to interact with lymphoendothelialcell surfacemarkersrdquo Bioorganic ampMedicinal Chemistry Lettersvol 12 no 23 pp 3407ndash3411 2002
[7] G Orzalesi P Gratteri and S Selleri ldquoSynthesis of new 13-dicyclohexyl barbituric acid derivatives with anti-inflammatorypotential activityrdquoEuropean Journal ofMedicinal Chemistry vol25 no 2 pp 197ndash201 1990
[8] D T Puerta and S M Cohen ldquoA bioinorganic perspectiveson matrix metalloproteinase inhibitionrdquo Current Topics inMedicinal Chemistry vol 4 no 15 pp 1551ndash1573 2004
[9] M E Wolff Burgerrsquos Medicinal Chemistry and Drug DiscoveryJohn Wiley amp Sons New York NY USA 1997
Journal of Chemistry 11
[10] E Fischer and J von Mering ldquoUeber eine neue klasse vonschlafmittelnrdquo in Untersuchungen aus Verschiedenen GebietenM Bergmann Ed pp 671ndash679 Springer Berlin Germany1924
[11] W J Doran ldquoBarbituric acid hypnoticsrdquo Medicinal Chemistryvol 4 pp 164ndash167 1959
[12] B Bobranski ldquoProgress in chemistry of barbituric acidrdquoWiad-omosci Chemiczne vol 31 pp 231ndash278 1977
[13] S Senda H Izumi and H Fujimura ldquoUracil derivaives andrelated compounds 6 Derivatives of 5-alkyle-246-trioxoper-hydropyrimidine as antiphlogisticsrdquo Arzneimittel Forschungvol 17 no 12 p 151 1967
[14] N Moussier L Bruche F Viani and M Zanda ldquoFluorinatedbarbituric acid derivatives synthesis and bio-activityrdquo CurrentOrganic Chemistry vol 7 no 11 pp 1071ndash1080 2003
[15] P-G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000
[16] N Ramarathnam T Osawa H Ochi and S Kawakishi ldquoThecontribution of plant food antioxidants to human healthrdquoTrends in Food Science amp Technology vol 6 no 3 pp 75ndash821995
[17] A Barakat A M Al-Majid H J Al-Najjar et al ldquoZwitterionicpyrimidinium adducts as antioxidants with therapeutic poten-tial as nitric oxide scavengerrdquo European Journal of MedicinalChemistry vol 84 pp 146ndash154 2014
[18] P Singh M Kaur and P Verma ldquoDesign synthesis andanticancer activities of hybrids of indole and barbituric acids-Identification of highly promising leadsrdquo Bioorganic andMedic-inal Chemistry Letters vol 19 no 11 pp 3054ndash3058 2009
[19] A M Al-Majid A Barakat H J Al-Najjar Y N Mabkhot HA Ghabbour and H-K Fun ldquoTandem aldol-michael reactionsin aqueous diethylamine medium a greener and efficientapproach to bis-pyrimidine derivativesrdquo International Journalof Molecular Sciences vol 14 no 12 pp 23762ndash23773 2013
[20] A Barakat A M Al-Majid G Lotfy et al ldquoSynthesis and dy-namics studies of barbituric acid derivatives as urease inhibi-torsrdquo Chemistry Central Journal vol 9 no 1 article 63 2015
[21] G M Sheldrick ldquoA short history of SHELXrdquo Acta Crystallo-graphica A vol 64 pp 112ndash122 2008
[22] A L Spek ldquoStructure validation in chemical crystallographyrdquoActaCrystallographica SectionD Biological Crystallography vol65 no 2 pp 148ndash155 2009
[23] F Rahim K Ullah H Ullah et al ldquoTriazinoindole analogs aspotent inhibitors of 120572-glucosidase synthesis biological evalua-tion and molecular docking studiesrdquo Bioorganic Chemistry vol58 pp 81ndash87 2015
[24] F Rahim F Malik H Ullah et al ldquoIsatin based Schiff basesas inhibitors of 120572-glucosidase synthesis characterization invitro evaluation and molecular docking studiesrdquo BioorganicChemistry vol 60 article 1803 pp 42ndash48 2015
[25] A Barakat A M Al-Majid M S Islam et al ldquoMolecular struc-ture investigation and biological evaluation of Michael adductsderived from dimedonerdquo Research on Chemical Intermediatesvol 42 pp 4041ndash4053 2016
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
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International Journal ofPhotoenergy
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Carbohydrate Chemistry
International Journal of
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Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
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Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
4 Journal of Chemistry
O
O O O O
RT
RT RT
ON N
2 1a b
1a b
O
O
O
O O
O
R R
O O O O O
O O
O
O
HOHO
NH
O
HO
O
N NN
NHN
O O HO
HO HO
HO
O O
O
O O
OO
O O O O O
O O
O
OO
O
OO
O O
ONH
O
4l
4z
NH
NH
NH N
NN
NN
H
NN
N NN
H
NH
HN
H NN
NH
N
O O O O
O
O O HO O
OO
N N
NN N
NO2 2
2
3
O
HO
HO
O
ON
N
CHO5l
4h
HOHO
R
NH2Et2
R2
R2
R2
R2
oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus
NH2Et2oplus
⊖
⊖ NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
NH2Et2oplus⊖
⊖
NH2Et2oplus
NH2Et2oplus
⊖R1 R1R1
R1
R1
R1
R2R3
R4
R1
R1
R1
R1 R1
H2ONHEt2
H2ONHEt2 H2ONHEt2
CH3
CH3CH3
CH3 CH3
CH3
CH3CH3
H3C
H3CH3C
H3C
H3CH3CH3C
H3C
H3CH3C
R3
R2
R4
4a R=CHOR1=H4b R=HR1=CH34c R=NO2R1=H4d R=OCH3R1=H4e R=HR1=Br4f R=OHR1=H4g R=CH3R1=H
4i R=CH34j R=Cl4k R=OCH3
4m R1=R2=R3=R4=H4n R1=R2=R4=HR3=CH34o R1=R2=R4=HR3=OCH34p R1=R2=R4=HR3=Cl4q R1=R2=R4=HR3=Br4r R1=R3=R4=HR2=Br4s R1=NO2R2=R3=R4=H4t R1=R2=R4=HR3=NMe24v R1=R2=R4=HR3=OH4w R1=R3=ClR2=R4=H4x R1=R2=R4=HR3=CHO4y R1=R4=ClR2=R3=H
5a R1=NO2R2=R3=R4=H5b R1=R3=R4=HR2=CH35c R1=R3=R4=CH3R2=H5d R1=R2=R4=HR3=OCH35e R1=R4=ClR2=R3=H5f R1=R2=R4=HR3=NO25g R1=R2=R4=HR3=CHO5h R1=R2=R4=HR3=OH
5i R=Cl5j R=H5k R=Br5l R=CH35m R=CHO
Scheme 1 Synthesis of the target compounds
Journal of Chemistry 5
Table 1 The crystal and experimental data of compounds 5a 5d and 5f
5a 5d 5fEmpirical formula C
23H26NO6sdotC4H12NsdotH2O C
24H29O5sdotC4H12N C
23H26NO6sdotC4H12N
Formula weight 50461 47162 48659Temperature 100K 100K 100KWavelength Mo K120572 radiation 120582 = 071073 A Mo K120572 radiation 120582 = 071073 A Mo K120572 radiation 120582 = 071073 ACrystal system Orthorhombic Monoclinic MonoclinicSpace group P2
12121
P21n P2
1n
119886 116155 (5) A 102288 (5) A 101137 (4) A119887 120514 (5) A 180048 (7) A 181225 (7) A119888 193665 (7) A 150893 (7) A 154853 (6) A120573 9000∘ 106425 (2)∘ 106730 (1)∘
Volume 271098 (19) A3 26656 (2) A3 271809 (18) A3
119885 4 4 4Calculated density 1236Mgmminus3 1175Mgmminus3 1189Mgmminus3
Absorption coefficient 009mmminus1 008mmminus1 008mmminus1
119865 (000) 1088 1024 1048Crystal size 057 times 041 times 007mm 066 times 033 times 025mm 033 times 029 times 022mm120579 range 24ndash242∘ 22ndash297∘ 22ndash321∘
Reflections collected 28531 67389 25488Reflections unique 4501 6194 4829Independent reflections 6224 11203 6233Parameters 350 326 334119877int 0090 0122 0053R [F2 gt 2120590(F2)] 0054 0069 0055119908R (F2) 0109 0156 0132Goodness of fit 105 101 109Maxmin 120588 e Aminus3 042 and minus027 043 and minus030 029 and minus026CCDC number 1444058 1443783 1444050
in supplementary information ORTEP drawings of final X-ray model of compounds 5a 5d and 5f with the atomicnumbering scheme are presented in Figure 2 while crystalpacking presentation of compounds 5a 5d and 5f is shownin Figure 3
Compound 5a crystallizes in orthorhombic crystal sys-tem in a space group of P2
12121 on the other hand com-
pounds 5d and 5f crystalize in monoclinic crystal system ina space group of P2
1n It was observed that the compound
5a has hydrogen bonding between the C5-O2sdot sdot sdotHsdot sdot sdotO
4-C13
while compounds 5d and 5f exist only in the enol formThereare two strong intramolecular hydrogen bonds on compound5a between O
4mdashH1O4sdot sdot sdotO2and N
2mdashH2N2sdot sdot sdotO1 while
compounds 5d and 5f have no intramolecular hydrogenbonding The crystal structures of the tested compounds 5a5d and 5f were formed by different hydrogen bonds betweenthemolecules and diethyl amine and water solvent moleculesin case of 5a to form network structures Crystallographicdata for the solved structures 5a 5d and 5f were deposited tothe Cambridge Crystallographic Data Center as supplemen-tary publication numbers CCDC-1444058 CCDC-1443783and CCDC-1444050 respectively which can be obtained freeof charge from the Cambridge Crystallographic Data Centrevia httpwwwccdccamacukdata requestcif
33 Biological Activity Evaluation All synthesized diethylammonium salts of barbiturates having bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (4andash4h)bis(6-hydroxypyrimidine-24(1H3H)-dione (4indash4l) and(2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dim-ethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate (4mndash4z) as well as bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) (5andash5m) as basic nuclei were screened for theirantioxidant potential in vitro by using DPPH radicalscavenging assay and cytotoxicity against PC-3 HeLa andMCF-7 cancer cell lines and 3T3 normal fibroblast cell linesThese derivatives 4andash4z and 5andash5m were also evaluated forin vitro enzyme inhibition activities against 120572-glucosidasethymidine phosphorylase and 120573-glucuronidase enzymesThe results are summarized in Table 2
331 Antioxidant Activity (DPPH Radical Scavenging Assay)Among series of compounds (4andash4h) having bis(6-hydroxy-1 3-dimethyl-2 4-dioxo-1 2 3 4-tetrahydropyrimidin-5-yl)ring as basic nucleus compounds 4a (IC
50= 1188plusmn21 120583M)
4b (IC50
= 117 plusmn 24 120583M) 4c (IC50
= 1119 plusmn 19 120583M) 4d(IC50
= 1244 plusmn 44 120583M) 4e (IC50
= 1011 plusmn 08 120583M) 4f(IC50
= 1486 plusmn 26 120583M) and 4g (IC50
= 1041 plusmn 19 120583M)were found to be potent antioxidants in comparison to the
6 Journal of Chemistry
5f
5d5a
Figure 3Molecular packing of 5a 5d and 5f viewed approximately three-dimensional network along 119887 axis and three-dimensional networkrespectively Intermolecular interaction is drawn as dashed lines
standards BHT (IC50= 1288 plusmn 21 120583M) andN-acetylcysteine
(IC50
= 1076 plusmn 28 120583M) Compounds 4andash4e (IC50
=1018 plusmn 08ndash1244 plusmn 44 120583M) were found to be more activecompared to the standard BHT (IC
50= 1288 plusmn 21 120583M) The
meta-bromo-substituted phenyl ring containing compound4e (IC
50= 1011 plusmn 08 120583M) was the most potent member of
the series (4andash4e) followed by the para-methyl-substitutedphenyl ring containing compound 4g (IC
50= 1041plusmn19 120583M)
A gradual decrease in activity was observed for compounds4c (IC
50= 1199 plusmn 19 120583M) and 4a (IC
50= 1188 plusmn 31 120583M)
having para-nitro- and para-aldehyde-substituted phenylring attached to central bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) ring A decrease inDPPH radical scavenging activity was due to positional effectof methyl substituent on phenyl ring which was observed forcompound 4b (IC
50= 117plusmn31 120583M) havingmeta-substituted
phenyl ring instead of para-methyl-substituted phenyl ring(4g IC
50= 1041 plusmn 19 120583M) The electron donating para-
hydroxyl-substituted phenyl ring containing compound 4f(IC50= 1486 plusmn 26 120583M) was found to be a last active member
of the series However a considerable increase in activity was
observed for compound 4d (IC50
= 1244 plusmn 44 120583M) havinga para-methoxy group instead of hydroxyl functionalityon phenyl ring whereas naphthalene ring containingbarbiturate 4h (IC
50= 1336 plusmn 57 120583M) was also found to be
a weak antioxidant agent against DPPH radicalsAmong second series of the tested compounds 4indash4l
having bis(6-hydroxypyrimidine-24(1H3H)-dione as back-bone compounds having para-methyl- (4i IC
50= 3849 plusmn
66 120583M) para-chloro- (4j IC50= 3843 plusmn 88 120583M) and para-
methoxy- (4l IC50= 3979 plusmn 09 120583M) substituted phenyl ring
attached to the central bis(6-hydroxypyrimidine-24(1H3H)-dione) showed better antioxidant activity as compared toBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) whereas naphthalene containing compound4l was found to be inactive
(2-Hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate ringcontaining compounds 4mndash4z were also evaluated for theirantioxidant potential in vitro in DPPH radical scavengingassayThese compounds were found to be significant to weakinhibitors of free radicals (IC
50= 1332plusmn47ndash4396plusmn101 120583M)
Journal of Chemistry 7
Table2Re
sultof
invitro
biologicalevaluatio
nof
thes
ynthesized
compo
unds
Num
berD
PPHradicalscaveng
ingassay
IC50plusmnSE
Ma(120583M)
Anticancercytotoxicityassay
IC50plusmnSE
M(120583M)
120572-G
lucosid
aseinh
ibition
IC50plusmnSE
M(120583M)
Thym
idinep
hospho
rylase
inhibitio
nIC50plusmnSE
M(120583M)
120573-G
lucuronidase
inhibitio
nIC50plusmnSE
M(120583M)
PC3b
Helac
MCF
-7d
3T3
4a118
8plusmn31
gt30
gt30
NA
gt30
NA
NA
20481plusmn60
4b117plusmn24
gt30
gt30
NA
gt30
NA
NA
NA
4c1119plusmn19
gt30
gt30
NA
gt30
355plusmn10
NA
1721plusmn
21
4d1244plusmn44
gt30
gt30
NA
gt30
NA
2309plusmn08
NA
4e1011plusmn08
gt30
gt30
NA
gt30
NA
NA
3389plusmn64
4f1486plusmn26
gt30
gt30
NA
gt30
NA
2179plusmn20
NA
4g1041plusmn
19gt30
gt30
NA
gt30
NA
NA
NA
4h1336plusmn57
gt30
gt30
NA
gt30
230plusmn39
NA
NA
4i3849plusmn66
gt30
gt30
NA
gt30
NA
NA
NA
4j3843plusmn88
gt30
gt30
NA
gt30
NA
NA
NA
4kNA
gt30
gt30
NA
gt30
NA
1826plusmn10
NA
4l3979plusmn01
gt30
gt30
NA
gt30
NA
NA
NA
4mNA
gt30
gt30
NA
gt30
NA
1602plusmn45
NA
4nNA
gt30
gt30
NA
gt30
NA
2269plusmn15
NA
4oNA
gt30
gt30
NA
gt30
3009plusmn65
NA
2621plusmn
43
4pNA
gt30
gt30
NA
gt30
1868plusmn81
2420plusmn15
NA
4qNA
gt30
gt30
NA
gt30
2148plusmn72
1413plusmn15
29217plusmn10
4rNA
gt30
gt30
NA
gt30
201plusmn
55
168plusmn12
2621plusmn
44
4s4396plusmn101
gt30
gt30
NA
gt30
NA
NA
NA
4t2782plusmn100
gt30
gt30
NA
gt30
NA
NA
NA
4v1332plusmn47
gt30
gt30
NA
gt30
NA
576plusmn12
NA
4w4116plusmn06
gt30
gt30
NA
gt30
NA
NA
1435plusmn12
4x3888plusmn79
gt30
gt30
NA
gt30
NA
239plusmn20
NA
4yNA
1071plusmn
03
347plusmn025
49plusmn001
320plusmn02
NA
NA
NA
4z2230plusmn39
gt30
gt30
NA
gt30
NA
558plusmn15
NA
5a4616plusmn88
gt30
gt30
NA
gt30
NA
NA
NA
5bNA
gt30
gt30
NA
gt30
NA
2169plusmn05
NA
5cNA
gt30
gt30
NA
gt30
NA
1614plusmn15
NA
5dNA
gt30
gt30
NA
gt30
NA
605plusmn18
7NA
5e289plusmn09
gt30
gt30
NA
gt30
NA
NA
NA
5fNA
gt30
gt30
NA
gt30
238plusmn93
NA
1850plusmn25
5g2704plusmn23
gt30
gt30
NA
gt30
NA
1866plusmn12
NA
5hNA
gt30
gt30
NA
gt30
NA
569plusmn16
NA
5iNA
gt30
gt30
NA
gt30
NA
NA
3236plusmn64
5jNA
gt30
gt30
NA
gt30
NA
318plusmn25
NA
5k1276plusmn16
gt30
gt30
NA
gt30
NA
1664plusmn12
NA
5l1697plusmn93
gt30
gt30
NA
gt30
NA
1781plusmn
21
3875plusmn70
5mNA
gt30
gt30
NA
gt30
4534plusmn42
1471plusmn12
NA
STD
e BHT(288plusmn21)
Doxorub
icin
Doxorub
icin
Doxorub
icin
Cyclo
heximide
Acarbo
se7-Deazaxanthine
D-Saccharicacid
1-4lacton
eN-acetylcysteine(
1076plusmn28)
0912plusmn012
0506plusmn015
156plusmn005
026plusmn01
840plusmn17
341plusmn16
44575plusmn216
a SEM
stand
arderrorof
mean
b PC3
hum
anprostate
cancer
celllin
ec H
eLaHenrie
ttaLackscervical
cancer
celllin
ed M
CF-7M
ichiganCa
ncer
Foun
datio
n-7breastcancer
celllin
esand
e BHT
butylated
hydroxytoluene
8 Journal of Chemistry
except compounds 4mndash4r and 4y which were found to beinactive
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) (5andash5h) and 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) (5indash5k) rings con-taining compounds were also evaluated for their antioxidantpotential by using DPPH radical scavenging assay Para-bromo-substituted phenyl ring containing compound5k (IC
50= 1276 plusmn 16 120583M) was found as a potent anti-
oxidant agent against the tested standards BHT (IC50
=1288 plusmn 21 120583M) The compound showed similar antioxidantpotential as BHT and showed a significant antioxidant agentagainst N-acetyl cysteine (IC
50= 1076 plusmn 28 120583M) The
gradual and drastic decrease in activity was for compounds5l (IC
50= 1697 plusmn 53 120583M) 5g (IC
50= 2704 plusmn 23 120583M)
5e (IC50
= 289 plusmn 09 120583M) and 5a (IC50
= 4616 plusmn 88 120583M)having para-methyl- para-aldehyde- ortho-chloro- andortho-nitro-substituted phenyl rings attached to the centralbarbiturate moieties Compounds 5bndash5d 5f and 5hndash5j werefound to be inactive
On the basis of the observed antioxidant abilities ofthe above five different series of pyridinium adducts itwas concluded that bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) ring containing com-pounds 4andash4h are the most active one The activity may bedue to the methyl substituted nitrogen atoms of the tetrahy-dropyrimidine rings that facilitate the electronic conjugationwithin the ring However the variation of antioxidantproperties within the most important series is may be due tothe presence of electron donating (-OH (4f) -OCH
3(4d)
and -CH3(4b 4g)) electron withdrawing (-NO
2(4a) and
-CHO (4c)) and halogen (-Br (4e)) substituents on thephenyl ring attached to the bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) moiety Howeverdetailed studies are required to study the mechanism ofaction of these compounds
332 Cytotoxic Activity All synthesized diethyl ammoniumsalts of barbiturates (4andash4z and 5andash5m) were evaluated fortheir in vitro cytotoxicity against PC-3 HeLa MCF-7 cancerand 3T3 normal cell lines All compounds were found to benoncytotoxic against PC-3 HeLaMCF-7 cancer and 3T3 celllines except dichloro-substituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tet-rahydropyrimidin-4-olate ring containing compounds 4ywhich was found to be significantly toxic against all the celllines (PC3 IC
50= 1071plusmn026 120583MHeLa IC
50= 347plusmn03 120583M
MCF-7 IC50= 49 plusmn 001 120583M and 3T3 IC
50= 320 plusmn 02 120583M)
Doxorubicin was used as a standard drug to compare theactivities against PC3 (IC
50= 170 plusmn 029 120583M) HeLa (IC
50=
051 plusmn 015 120583M) and MCF-3 (IC50= 092 plusmn 001 120583M) cancer
cell lines while cycloheximide (IC50
= 026 plusmn 012 120583M) wasused as a standard-in 3T3 cell line (Table 2)
333 120572-Glucosidase Inhibition Activity Synthesized diethylammonium salts of barbiturates 4andash4z and 5andash5m were alsoevaluated for their in vitro 120572-glucosidase inhibition activityin comparison to the standard drug acarbose (IC
50= 841 plusmn
173 120583M) Among dimethyl ammonium salts (4andash4h) nitro-substituted phenyl ring containing 4c (IC
50= 355 plusmn 10 120583M)
and naphthalene ring containing 4h (IC50= 230 plusmn 392 120583M)
were found to be potent inhibitors of 120572-glucosidase enzymeand showed more activity than acarbose (IC
50= 841 plusmn
173 120583M) All other compounds were found to be inactiveAmong diethyl ammonium salts having (2-hydroxy-
44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate ring (4mndash4z)para-OCH
3- (40 IC
50= 3009 plusmn 65 120583M) para-chloro-
(4p IC50
= 1868 plusmn 081 120583M) para-bromo- (4q IC50
=2148plusmn072 120583M) andmeta-bromo- (4r IC
50= 201plusmn55 120583M)
substituted phenyl ring containing pyridinium adducts werefound to be potent 120572-glucosidase inhibitors and showedmore activity than standard acarbose (IC
50= 841plusmn 173 120583M)
Other members of the series (4m-4n and 4sndash4z) were foundto be inactive The results showed the substitution effects ofvarious functionalities of phenyl ring on the potential activityof tested series The para-bromo-substituted phenyl ringcontaining compound 4q (IC
50= 2148 plusmn 072 120583M) showed
more activity than tested standard However the replacementof bromo group with that of methylNN-dimethyl hydroxyland aldehyde functionalities resulted in complete loss ofactivity as observed in compounds 4n 4t 4v and 4xrespectively The presence of ortho-nitro- ortho-choloro-and para-choloro-substituted phenyl ring also contributedtowards inactivity as observed for compounds 4t 4w and4y
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring containing diethyl ammonium pyridinium salts(5andash5h) were also evaluated for their in vitro 120572-glucosidaseenzyme inhibition activity The series nitro-substitutedphenyl ring containing 5f was found to be the only potent120572-glucosidase inhibitor All other compounds (5andash5e and5g-5h) were found to be inactive
Among series of diethylaminium salts of pyridiniumadducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-di-oxo-1234-tetrahydropyrimidin-5-yl)methyl) central moietyattached with substituted phenyl ring the benzaldehydecontaining 5m (IC
50= 4534 plusmn 42 120583M) was found to be the
only potent inhibitor compound in comparison to against thestandard drug acarbose (IC
50= 841plusmn173 120583M)All other com-
poundswere found to be inactiveThe results are summarizedin Table 2
334 Thymidine Phosphorylase Inhibition Activity Five dif-ferent series of diethyamonium salts of barbiturates 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evalu-ated for their in vitro thymidine phosphorylase inhibition7-Deazaxanthine was used as a standard inhibitor (IC
50
= 41 plusmn 146 120583M) Phenol and naphthalene rings sub-stituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olatemoiety containing compounds 4v (IC
50= 576 plusmn 120120583M)
and 4z (IC50
= 558 plusmn 150 120583M) were found to be the potentthymidine phosphorylase inhibitors However a drasticdecrease in activity was observed when para-hydroxygroup on phenyl ring was replaced with methyl (4n IC
50
Journal of Chemistry 9
= 2269 plusmn 150 120583M) chloro (4n IC50
= 242 plusmn 15 120583M)bromo (4q IC
50= 1413 plusmn 15 120583M) and aldehyde (4x
IC50
= 239 plusmn 20 120583M) groups whereas the compounds4o 4s 4t 4w and 4y have para-methoxy- ortho-nitro-ortho-NN-dimethyl amine- ortho- para-dichloro- andortho-ortho-dichloro-substituted phenyl ring respectively
Among series of compounds (5andash5h) having bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring as centralmoiety electron donating hydroxy- andmethoxy-substitutedphenyl ring containing compounds 5h (IC
50= 569plusmn16 120583M)
and 5d (IC50
= 605 plusmn 187 120583M) respectively were foundas potent inhibitors of thymidine phosphorylase enzymeagainst the tested standard 7-deazaxanthine which was(IC50
= 41 plusmn 146 120583M) used as standard Again a drasticdecrease in activity was observed for compounds 5b (IC
50
= 2169 plusmn 05 120583M) 5c (IC50
= 1614 plusmn 15 120583M) and 5g(IC50= 1866 plusmn 12 120583M) havingmeta-methyl- ortho-methyl-
and para-aldehyde-substituted benzene ring respectivelyinstead of para-hydroxyl phenyl ring The replacement ofortho-methyl with chloro-group makes the compound 5ecompletely inactive Similarly the replacement of electrondonating para-hydroxy-group of phenyl ring with electronwithdrawing nitro-functionality also contributed towardsinactivity of compound as observed for 5f Phenol substi-tuted bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ringcontaining 5h (IC
50= 569 plusmn 16 120583M) was found to be the
potent thymidine phosphorylase inhibitorsAll members of the series of diethylaminium salts of pyri-
dinium adducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) central moi-ety attached with substituted phenyl ring were found to beweak inhibitors of thymidine phosphorylase enzyme againstthe tested standard compound 7-deazaxanthine (IC
50= 41 plusmn
146 120583M) with IC50
values 318 plusmn 25 120583M 1664 plusmn 12 120583M1781 plusmn 21 120583M and 1471 plusmn 123 120583M for 5j 5k 5l and 5mrespectively whereas para-chloro-substituted phenyl ringcontaining compound 5i was found to be inactiveThe resultsare summarized in Table 1
335 120573-Glucuronidase Inhibition Activity Compounds 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evaluatedfor their in vitro 120573-glucuronidase inhibitory activity by usingD-saccharic acid 14-lactone as standard inhibitor (IC
50=
4575 plusmn 216 120583M) Compounds 4a (IC50
= 2048 plusmn 601 120583M)4c (IC
50= 1721 plusmn 21 120583M) 4e (IC
50= 3389 plusmn 642 120583M) 4o
(IC50
= 2621 plusmn 435 120583M) 4q (IC50
= 2922 plusmn 103 120583M) 4r(IC50= 2621plusmn44 120583M) 4w (IC
50= 1435plusmn124 120583M) 5f (IC
50
= 185 plusmn 25 120583M) 5i (IC50
= 2236 plusmn 64 120583M) and 5l (IC50
=3875plusmn70 120583M) showed veryweak120573-glucuronidase inhibitionpotential whereas pyridinium adducts 4andash4c 4e 4gndash4j 4l4o 4s-4t 4w 4y 5a 5e-5f and 5i were found to be inactive(Table 2)
34 Molecular Docking Studies
341 Binding Interactions of 120572-Glucosidase Enzyme To de-fine inhibitors interactions and their specificity the dockingstudy of the most active compound 4p against 120572-glucosidase
Figure 4 Three-dimensional representation of compound 4p withtarget macromolecule 120572-glucosidase
Figure 5 Three-dimensional representation of compound 4v withtarget macromolecule thymidine phosphorylase
was carried out [23ndash25] Ten different conformations ofprotein-ligand interactions were generated and the topranked pose was selected to explore the binding interactionsIn our docking study we have observed two interactions withthe active site residues of 120572-glucosidase The polar imidazolering of His 239 showed hydrogen bond through its H atomwith the lone pair of oxygen of the pyrimidinedionemoiety ofthe compound whereas Arg 312 formed hydrogen bond withthe OH group of the same moiety of compound (Figure 4)The presence of polar and electron rich center in the formof pyrimidinedione and chlorobenzene groups of the com-pound was observed here as key factors for the interactionbetween compound andprotein Anumber of hydrogen bonddonor and acceptor pairs with their suitable orientationswere observed in the interaction pose Furthermore severalhydrophobic interactions between the nonpolar active siteresidues and the compound 4p were also observed
342 Binding Interactions of Thymidine PhosphorylaseEnzyme To explore the binding modes of these newly syn-thesized compounds in the active site of thymidine phos-phorylase the most active compound 4v was docked againstthis enzyme The docking results showed that compound4v well accommodated in the active site of thymidinephosphorylase (Figure 5) The active site residue Ala 206established a hydrogen bond through its lone pair of
10 Journal of Chemistry
Figure 6 Three-dimensional representation of compound 4w withtarget enzyme 120573-glucuronidase
oxygen with the active hydrogen of hydroxyl moiety ofthe pyrimidinedione group of the compound Howeverthe substituted phenolic group and the other electron richcenters of the pyrimidinedione showed poor behaviorregarding interaction which may depend on the nature andorientation of the surrounding active site residues Moreoverseveral weak hydrophobic interactions between the activesite residues and the compound were also observed
343 Binding Interaction of 120573-Glucuronidase Enzyme Thepredicted bindingmode ofmost active compound 4w against120573-glucuronidase showed that a hydrogen bond is formedbetween the lone pair of oxygen of cyclohexanedione compo-nent of the compound and imidazole hydrogen of the impor-tant active site residue His 509 An arene-arene and arene-cation interactionwas also observed between the pi electroniccloud of dichlorobenzene ring of compound and imidazolering of His 509 (Figure 6) Besides hydrogen bonding andarene-arene interactions several hydrophobic interactionsbetween compound 4w and the active site residues werealso observed The pyrimidinedione moiety in this case wasobserved to be passive regarding interactions
4 Conclusions
In conclusion barbiturate salts were synthesized by one-potmulticomponent reactions via tandem AldolMichael addi-tion reactions between barbituric acid and dimedone withaldehydes mediated by aqueous diethylamine The molec-ular structures of 5a 5d and 5f were deduced by single-crystal X-ray diffraction techniques All of the synthesizedcompounds were evaluated for their antioxidant potentialin vitro by using DPPH radical scavenging assay as well asenzyme inhibition activities against120572-glucosidase thymidinephosphorylase and 120573-glucuronidase enzymes Compounds4andash4g (IC
50= 1018 plusmn 08ndash1244 plusmn 44 120583M) were found to
be the potent antioxidants as compared to the standardsBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) Compound 4p (IC50= 1868 plusmn 81 120583M) was
found to be a potent 120572-glucosidase inhibitor Compound 4v(IC50
= 87 plusmn 12 120583M) was found to be a potent thymidinephosphorylase inhibitor Promising results indicate that thesecompounds need to be investigated as antioxidant agentsto treat various associated oxidative disorders Similarlytheir enzyme inhibitory potential can be reported on drugdiscovery program
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Assem Barakat Hazem A Ghabbour Abdullah MohammedAl-Majid Qurat-ul-ain Rehan Imad Kulsoom Javaid NimraNaveed Shaikh Sammer Yousuf M Iqbal Choudhary andAbdul Wadood contributed equally to this work
Acknowledgments
The authors would like to extend their sincere appreciation tothe Deanship of Scientific Research at King Saud Universityfor funding this Research Group no (RGP-257-1435-1436)
References
[1] K Undheim T Bennecke A R Katritzky C W Rees andE F V Scriven Comprehensive Heterocyclic Chemistry vol 6supplement 93 Elsevier Pergamon Oxford UK 1996
[2] B TaylorModern Medical Chemistry Prentice Hall New YorkNY USA 1994
[3] R Y Levina and F K Velichko ldquoAdvances in the chemistry ofbarbituric acidsrdquo Russian Chemical Reviews vol 29 no 8 pp437ndash459 1960
[4] P Sing and K Paul ldquoA simple synthesis of 5-spirobarbituricacids and transformations of spirocyclopropane barbiturates to5-substitutated barbituratesrdquo Indian Journal of Chemistry B vol45 pp 247ndash251 2006
[5] X Chen K Tanaka and F Yoneda ldquoSimple newmethod for thesynthesis of 5-deaza-10-oxaflavin a potential organic oxidantrdquoChemical and Pharmaceutical Bulletin vol 38 no 2 pp 307ndash311 1990
[6] L R Morgan B S Jursic C L Hooper D M Neumann KThangaraj and B LeBlanc ldquoAnticancer activity for 4 41015840-di-hydroxybenzophenone-2 4-dinitrophenylhydrazone (A-007)analogues and their abilities to interact with lymphoendothelialcell surfacemarkersrdquo Bioorganic ampMedicinal Chemistry Lettersvol 12 no 23 pp 3407ndash3411 2002
[7] G Orzalesi P Gratteri and S Selleri ldquoSynthesis of new 13-dicyclohexyl barbituric acid derivatives with anti-inflammatorypotential activityrdquoEuropean Journal ofMedicinal Chemistry vol25 no 2 pp 197ndash201 1990
[8] D T Puerta and S M Cohen ldquoA bioinorganic perspectiveson matrix metalloproteinase inhibitionrdquo Current Topics inMedicinal Chemistry vol 4 no 15 pp 1551ndash1573 2004
[9] M E Wolff Burgerrsquos Medicinal Chemistry and Drug DiscoveryJohn Wiley amp Sons New York NY USA 1997
Journal of Chemistry 11
[10] E Fischer and J von Mering ldquoUeber eine neue klasse vonschlafmittelnrdquo in Untersuchungen aus Verschiedenen GebietenM Bergmann Ed pp 671ndash679 Springer Berlin Germany1924
[11] W J Doran ldquoBarbituric acid hypnoticsrdquo Medicinal Chemistryvol 4 pp 164ndash167 1959
[12] B Bobranski ldquoProgress in chemistry of barbituric acidrdquoWiad-omosci Chemiczne vol 31 pp 231ndash278 1977
[13] S Senda H Izumi and H Fujimura ldquoUracil derivaives andrelated compounds 6 Derivatives of 5-alkyle-246-trioxoper-hydropyrimidine as antiphlogisticsrdquo Arzneimittel Forschungvol 17 no 12 p 151 1967
[14] N Moussier L Bruche F Viani and M Zanda ldquoFluorinatedbarbituric acid derivatives synthesis and bio-activityrdquo CurrentOrganic Chemistry vol 7 no 11 pp 1071ndash1080 2003
[15] P-G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000
[16] N Ramarathnam T Osawa H Ochi and S Kawakishi ldquoThecontribution of plant food antioxidants to human healthrdquoTrends in Food Science amp Technology vol 6 no 3 pp 75ndash821995
[17] A Barakat A M Al-Majid H J Al-Najjar et al ldquoZwitterionicpyrimidinium adducts as antioxidants with therapeutic poten-tial as nitric oxide scavengerrdquo European Journal of MedicinalChemistry vol 84 pp 146ndash154 2014
[18] P Singh M Kaur and P Verma ldquoDesign synthesis andanticancer activities of hybrids of indole and barbituric acids-Identification of highly promising leadsrdquo Bioorganic andMedic-inal Chemistry Letters vol 19 no 11 pp 3054ndash3058 2009
[19] A M Al-Majid A Barakat H J Al-Najjar Y N Mabkhot HA Ghabbour and H-K Fun ldquoTandem aldol-michael reactionsin aqueous diethylamine medium a greener and efficientapproach to bis-pyrimidine derivativesrdquo International Journalof Molecular Sciences vol 14 no 12 pp 23762ndash23773 2013
[20] A Barakat A M Al-Majid G Lotfy et al ldquoSynthesis and dy-namics studies of barbituric acid derivatives as urease inhibi-torsrdquo Chemistry Central Journal vol 9 no 1 article 63 2015
[21] G M Sheldrick ldquoA short history of SHELXrdquo Acta Crystallo-graphica A vol 64 pp 112ndash122 2008
[22] A L Spek ldquoStructure validation in chemical crystallographyrdquoActaCrystallographica SectionD Biological Crystallography vol65 no 2 pp 148ndash155 2009
[23] F Rahim K Ullah H Ullah et al ldquoTriazinoindole analogs aspotent inhibitors of 120572-glucosidase synthesis biological evalua-tion and molecular docking studiesrdquo Bioorganic Chemistry vol58 pp 81ndash87 2015
[24] F Rahim F Malik H Ullah et al ldquoIsatin based Schiff basesas inhibitors of 120572-glucosidase synthesis characterization invitro evaluation and molecular docking studiesrdquo BioorganicChemistry vol 60 article 1803 pp 42ndash48 2015
[25] A Barakat A M Al-Majid M S Islam et al ldquoMolecular struc-ture investigation and biological evaluation of Michael adductsderived from dimedonerdquo Research on Chemical Intermediatesvol 42 pp 4041ndash4053 2016
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Carbohydrate Chemistry
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Analytical Methods in Chemistry
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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Analytical ChemistryInternational Journal of
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Quantum Chemistry
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CatalystsJournal of
Journal of Chemistry 5
Table 1 The crystal and experimental data of compounds 5a 5d and 5f
5a 5d 5fEmpirical formula C
23H26NO6sdotC4H12NsdotH2O C
24H29O5sdotC4H12N C
23H26NO6sdotC4H12N
Formula weight 50461 47162 48659Temperature 100K 100K 100KWavelength Mo K120572 radiation 120582 = 071073 A Mo K120572 radiation 120582 = 071073 A Mo K120572 radiation 120582 = 071073 ACrystal system Orthorhombic Monoclinic MonoclinicSpace group P2
12121
P21n P2
1n
119886 116155 (5) A 102288 (5) A 101137 (4) A119887 120514 (5) A 180048 (7) A 181225 (7) A119888 193665 (7) A 150893 (7) A 154853 (6) A120573 9000∘ 106425 (2)∘ 106730 (1)∘
Volume 271098 (19) A3 26656 (2) A3 271809 (18) A3
119885 4 4 4Calculated density 1236Mgmminus3 1175Mgmminus3 1189Mgmminus3
Absorption coefficient 009mmminus1 008mmminus1 008mmminus1
119865 (000) 1088 1024 1048Crystal size 057 times 041 times 007mm 066 times 033 times 025mm 033 times 029 times 022mm120579 range 24ndash242∘ 22ndash297∘ 22ndash321∘
Reflections collected 28531 67389 25488Reflections unique 4501 6194 4829Independent reflections 6224 11203 6233Parameters 350 326 334119877int 0090 0122 0053R [F2 gt 2120590(F2)] 0054 0069 0055119908R (F2) 0109 0156 0132Goodness of fit 105 101 109Maxmin 120588 e Aminus3 042 and minus027 043 and minus030 029 and minus026CCDC number 1444058 1443783 1444050
in supplementary information ORTEP drawings of final X-ray model of compounds 5a 5d and 5f with the atomicnumbering scheme are presented in Figure 2 while crystalpacking presentation of compounds 5a 5d and 5f is shownin Figure 3
Compound 5a crystallizes in orthorhombic crystal sys-tem in a space group of P2
12121 on the other hand com-
pounds 5d and 5f crystalize in monoclinic crystal system ina space group of P2
1n It was observed that the compound
5a has hydrogen bonding between the C5-O2sdot sdot sdotHsdot sdot sdotO
4-C13
while compounds 5d and 5f exist only in the enol formThereare two strong intramolecular hydrogen bonds on compound5a between O
4mdashH1O4sdot sdot sdotO2and N
2mdashH2N2sdot sdot sdotO1 while
compounds 5d and 5f have no intramolecular hydrogenbonding The crystal structures of the tested compounds 5a5d and 5f were formed by different hydrogen bonds betweenthemolecules and diethyl amine and water solvent moleculesin case of 5a to form network structures Crystallographicdata for the solved structures 5a 5d and 5f were deposited tothe Cambridge Crystallographic Data Center as supplemen-tary publication numbers CCDC-1444058 CCDC-1443783and CCDC-1444050 respectively which can be obtained freeof charge from the Cambridge Crystallographic Data Centrevia httpwwwccdccamacukdata requestcif
33 Biological Activity Evaluation All synthesized diethylammonium salts of barbiturates having bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (4andash4h)bis(6-hydroxypyrimidine-24(1H3H)-dione (4indash4l) and(2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dim-ethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate (4mndash4z) as well as bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) (5andash5m) as basic nuclei were screened for theirantioxidant potential in vitro by using DPPH radicalscavenging assay and cytotoxicity against PC-3 HeLa andMCF-7 cancer cell lines and 3T3 normal fibroblast cell linesThese derivatives 4andash4z and 5andash5m were also evaluated forin vitro enzyme inhibition activities against 120572-glucosidasethymidine phosphorylase and 120573-glucuronidase enzymesThe results are summarized in Table 2
331 Antioxidant Activity (DPPH Radical Scavenging Assay)Among series of compounds (4andash4h) having bis(6-hydroxy-1 3-dimethyl-2 4-dioxo-1 2 3 4-tetrahydropyrimidin-5-yl)ring as basic nucleus compounds 4a (IC
50= 1188plusmn21 120583M)
4b (IC50
= 117 plusmn 24 120583M) 4c (IC50
= 1119 plusmn 19 120583M) 4d(IC50
= 1244 plusmn 44 120583M) 4e (IC50
= 1011 plusmn 08 120583M) 4f(IC50
= 1486 plusmn 26 120583M) and 4g (IC50
= 1041 plusmn 19 120583M)were found to be potent antioxidants in comparison to the
6 Journal of Chemistry
5f
5d5a
Figure 3Molecular packing of 5a 5d and 5f viewed approximately three-dimensional network along 119887 axis and three-dimensional networkrespectively Intermolecular interaction is drawn as dashed lines
standards BHT (IC50= 1288 plusmn 21 120583M) andN-acetylcysteine
(IC50
= 1076 plusmn 28 120583M) Compounds 4andash4e (IC50
=1018 plusmn 08ndash1244 plusmn 44 120583M) were found to be more activecompared to the standard BHT (IC
50= 1288 plusmn 21 120583M) The
meta-bromo-substituted phenyl ring containing compound4e (IC
50= 1011 plusmn 08 120583M) was the most potent member of
the series (4andash4e) followed by the para-methyl-substitutedphenyl ring containing compound 4g (IC
50= 1041plusmn19 120583M)
A gradual decrease in activity was observed for compounds4c (IC
50= 1199 plusmn 19 120583M) and 4a (IC
50= 1188 plusmn 31 120583M)
having para-nitro- and para-aldehyde-substituted phenylring attached to central bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) ring A decrease inDPPH radical scavenging activity was due to positional effectof methyl substituent on phenyl ring which was observed forcompound 4b (IC
50= 117plusmn31 120583M) havingmeta-substituted
phenyl ring instead of para-methyl-substituted phenyl ring(4g IC
50= 1041 plusmn 19 120583M) The electron donating para-
hydroxyl-substituted phenyl ring containing compound 4f(IC50= 1486 plusmn 26 120583M) was found to be a last active member
of the series However a considerable increase in activity was
observed for compound 4d (IC50
= 1244 plusmn 44 120583M) havinga para-methoxy group instead of hydroxyl functionalityon phenyl ring whereas naphthalene ring containingbarbiturate 4h (IC
50= 1336 plusmn 57 120583M) was also found to be
a weak antioxidant agent against DPPH radicalsAmong second series of the tested compounds 4indash4l
having bis(6-hydroxypyrimidine-24(1H3H)-dione as back-bone compounds having para-methyl- (4i IC
50= 3849 plusmn
66 120583M) para-chloro- (4j IC50= 3843 plusmn 88 120583M) and para-
methoxy- (4l IC50= 3979 plusmn 09 120583M) substituted phenyl ring
attached to the central bis(6-hydroxypyrimidine-24(1H3H)-dione) showed better antioxidant activity as compared toBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) whereas naphthalene containing compound4l was found to be inactive
(2-Hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate ringcontaining compounds 4mndash4z were also evaluated for theirantioxidant potential in vitro in DPPH radical scavengingassayThese compounds were found to be significant to weakinhibitors of free radicals (IC
50= 1332plusmn47ndash4396plusmn101 120583M)
Journal of Chemistry 7
Table2Re
sultof
invitro
biologicalevaluatio
nof
thes
ynthesized
compo
unds
Num
berD
PPHradicalscaveng
ingassay
IC50plusmnSE
Ma(120583M)
Anticancercytotoxicityassay
IC50plusmnSE
M(120583M)
120572-G
lucosid
aseinh
ibition
IC50plusmnSE
M(120583M)
Thym
idinep
hospho
rylase
inhibitio
nIC50plusmnSE
M(120583M)
120573-G
lucuronidase
inhibitio
nIC50plusmnSE
M(120583M)
PC3b
Helac
MCF
-7d
3T3
4a118
8plusmn31
gt30
gt30
NA
gt30
NA
NA
20481plusmn60
4b117plusmn24
gt30
gt30
NA
gt30
NA
NA
NA
4c1119plusmn19
gt30
gt30
NA
gt30
355plusmn10
NA
1721plusmn
21
4d1244plusmn44
gt30
gt30
NA
gt30
NA
2309plusmn08
NA
4e1011plusmn08
gt30
gt30
NA
gt30
NA
NA
3389plusmn64
4f1486plusmn26
gt30
gt30
NA
gt30
NA
2179plusmn20
NA
4g1041plusmn
19gt30
gt30
NA
gt30
NA
NA
NA
4h1336plusmn57
gt30
gt30
NA
gt30
230plusmn39
NA
NA
4i3849plusmn66
gt30
gt30
NA
gt30
NA
NA
NA
4j3843plusmn88
gt30
gt30
NA
gt30
NA
NA
NA
4kNA
gt30
gt30
NA
gt30
NA
1826plusmn10
NA
4l3979plusmn01
gt30
gt30
NA
gt30
NA
NA
NA
4mNA
gt30
gt30
NA
gt30
NA
1602plusmn45
NA
4nNA
gt30
gt30
NA
gt30
NA
2269plusmn15
NA
4oNA
gt30
gt30
NA
gt30
3009plusmn65
NA
2621plusmn
43
4pNA
gt30
gt30
NA
gt30
1868plusmn81
2420plusmn15
NA
4qNA
gt30
gt30
NA
gt30
2148plusmn72
1413plusmn15
29217plusmn10
4rNA
gt30
gt30
NA
gt30
201plusmn
55
168plusmn12
2621plusmn
44
4s4396plusmn101
gt30
gt30
NA
gt30
NA
NA
NA
4t2782plusmn100
gt30
gt30
NA
gt30
NA
NA
NA
4v1332plusmn47
gt30
gt30
NA
gt30
NA
576plusmn12
NA
4w4116plusmn06
gt30
gt30
NA
gt30
NA
NA
1435plusmn12
4x3888plusmn79
gt30
gt30
NA
gt30
NA
239plusmn20
NA
4yNA
1071plusmn
03
347plusmn025
49plusmn001
320plusmn02
NA
NA
NA
4z2230plusmn39
gt30
gt30
NA
gt30
NA
558plusmn15
NA
5a4616plusmn88
gt30
gt30
NA
gt30
NA
NA
NA
5bNA
gt30
gt30
NA
gt30
NA
2169plusmn05
NA
5cNA
gt30
gt30
NA
gt30
NA
1614plusmn15
NA
5dNA
gt30
gt30
NA
gt30
NA
605plusmn18
7NA
5e289plusmn09
gt30
gt30
NA
gt30
NA
NA
NA
5fNA
gt30
gt30
NA
gt30
238plusmn93
NA
1850plusmn25
5g2704plusmn23
gt30
gt30
NA
gt30
NA
1866plusmn12
NA
5hNA
gt30
gt30
NA
gt30
NA
569plusmn16
NA
5iNA
gt30
gt30
NA
gt30
NA
NA
3236plusmn64
5jNA
gt30
gt30
NA
gt30
NA
318plusmn25
NA
5k1276plusmn16
gt30
gt30
NA
gt30
NA
1664plusmn12
NA
5l1697plusmn93
gt30
gt30
NA
gt30
NA
1781plusmn
21
3875plusmn70
5mNA
gt30
gt30
NA
gt30
4534plusmn42
1471plusmn12
NA
STD
e BHT(288plusmn21)
Doxorub
icin
Doxorub
icin
Doxorub
icin
Cyclo
heximide
Acarbo
se7-Deazaxanthine
D-Saccharicacid
1-4lacton
eN-acetylcysteine(
1076plusmn28)
0912plusmn012
0506plusmn015
156plusmn005
026plusmn01
840plusmn17
341plusmn16
44575plusmn216
a SEM
stand
arderrorof
mean
b PC3
hum
anprostate
cancer
celllin
ec H
eLaHenrie
ttaLackscervical
cancer
celllin
ed M
CF-7M
ichiganCa
ncer
Foun
datio
n-7breastcancer
celllin
esand
e BHT
butylated
hydroxytoluene
8 Journal of Chemistry
except compounds 4mndash4r and 4y which were found to beinactive
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) (5andash5h) and 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) (5indash5k) rings con-taining compounds were also evaluated for their antioxidantpotential by using DPPH radical scavenging assay Para-bromo-substituted phenyl ring containing compound5k (IC
50= 1276 plusmn 16 120583M) was found as a potent anti-
oxidant agent against the tested standards BHT (IC50
=1288 plusmn 21 120583M) The compound showed similar antioxidantpotential as BHT and showed a significant antioxidant agentagainst N-acetyl cysteine (IC
50= 1076 plusmn 28 120583M) The
gradual and drastic decrease in activity was for compounds5l (IC
50= 1697 plusmn 53 120583M) 5g (IC
50= 2704 plusmn 23 120583M)
5e (IC50
= 289 plusmn 09 120583M) and 5a (IC50
= 4616 plusmn 88 120583M)having para-methyl- para-aldehyde- ortho-chloro- andortho-nitro-substituted phenyl rings attached to the centralbarbiturate moieties Compounds 5bndash5d 5f and 5hndash5j werefound to be inactive
On the basis of the observed antioxidant abilities ofthe above five different series of pyridinium adducts itwas concluded that bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) ring containing com-pounds 4andash4h are the most active one The activity may bedue to the methyl substituted nitrogen atoms of the tetrahy-dropyrimidine rings that facilitate the electronic conjugationwithin the ring However the variation of antioxidantproperties within the most important series is may be due tothe presence of electron donating (-OH (4f) -OCH
3(4d)
and -CH3(4b 4g)) electron withdrawing (-NO
2(4a) and
-CHO (4c)) and halogen (-Br (4e)) substituents on thephenyl ring attached to the bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) moiety Howeverdetailed studies are required to study the mechanism ofaction of these compounds
332 Cytotoxic Activity All synthesized diethyl ammoniumsalts of barbiturates (4andash4z and 5andash5m) were evaluated fortheir in vitro cytotoxicity against PC-3 HeLa MCF-7 cancerand 3T3 normal cell lines All compounds were found to benoncytotoxic against PC-3 HeLaMCF-7 cancer and 3T3 celllines except dichloro-substituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tet-rahydropyrimidin-4-olate ring containing compounds 4ywhich was found to be significantly toxic against all the celllines (PC3 IC
50= 1071plusmn026 120583MHeLa IC
50= 347plusmn03 120583M
MCF-7 IC50= 49 plusmn 001 120583M and 3T3 IC
50= 320 plusmn 02 120583M)
Doxorubicin was used as a standard drug to compare theactivities against PC3 (IC
50= 170 plusmn 029 120583M) HeLa (IC
50=
051 plusmn 015 120583M) and MCF-3 (IC50= 092 plusmn 001 120583M) cancer
cell lines while cycloheximide (IC50
= 026 plusmn 012 120583M) wasused as a standard-in 3T3 cell line (Table 2)
333 120572-Glucosidase Inhibition Activity Synthesized diethylammonium salts of barbiturates 4andash4z and 5andash5m were alsoevaluated for their in vitro 120572-glucosidase inhibition activityin comparison to the standard drug acarbose (IC
50= 841 plusmn
173 120583M) Among dimethyl ammonium salts (4andash4h) nitro-substituted phenyl ring containing 4c (IC
50= 355 plusmn 10 120583M)
and naphthalene ring containing 4h (IC50= 230 plusmn 392 120583M)
were found to be potent inhibitors of 120572-glucosidase enzymeand showed more activity than acarbose (IC
50= 841 plusmn
173 120583M) All other compounds were found to be inactiveAmong diethyl ammonium salts having (2-hydroxy-
44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate ring (4mndash4z)para-OCH
3- (40 IC
50= 3009 plusmn 65 120583M) para-chloro-
(4p IC50
= 1868 plusmn 081 120583M) para-bromo- (4q IC50
=2148plusmn072 120583M) andmeta-bromo- (4r IC
50= 201plusmn55 120583M)
substituted phenyl ring containing pyridinium adducts werefound to be potent 120572-glucosidase inhibitors and showedmore activity than standard acarbose (IC
50= 841plusmn 173 120583M)
Other members of the series (4m-4n and 4sndash4z) were foundto be inactive The results showed the substitution effects ofvarious functionalities of phenyl ring on the potential activityof tested series The para-bromo-substituted phenyl ringcontaining compound 4q (IC
50= 2148 plusmn 072 120583M) showed
more activity than tested standard However the replacementof bromo group with that of methylNN-dimethyl hydroxyland aldehyde functionalities resulted in complete loss ofactivity as observed in compounds 4n 4t 4v and 4xrespectively The presence of ortho-nitro- ortho-choloro-and para-choloro-substituted phenyl ring also contributedtowards inactivity as observed for compounds 4t 4w and4y
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring containing diethyl ammonium pyridinium salts(5andash5h) were also evaluated for their in vitro 120572-glucosidaseenzyme inhibition activity The series nitro-substitutedphenyl ring containing 5f was found to be the only potent120572-glucosidase inhibitor All other compounds (5andash5e and5g-5h) were found to be inactive
Among series of diethylaminium salts of pyridiniumadducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-di-oxo-1234-tetrahydropyrimidin-5-yl)methyl) central moietyattached with substituted phenyl ring the benzaldehydecontaining 5m (IC
50= 4534 plusmn 42 120583M) was found to be the
only potent inhibitor compound in comparison to against thestandard drug acarbose (IC
50= 841plusmn173 120583M)All other com-
poundswere found to be inactiveThe results are summarizedin Table 2
334 Thymidine Phosphorylase Inhibition Activity Five dif-ferent series of diethyamonium salts of barbiturates 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evalu-ated for their in vitro thymidine phosphorylase inhibition7-Deazaxanthine was used as a standard inhibitor (IC
50
= 41 plusmn 146 120583M) Phenol and naphthalene rings sub-stituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olatemoiety containing compounds 4v (IC
50= 576 plusmn 120120583M)
and 4z (IC50
= 558 plusmn 150 120583M) were found to be the potentthymidine phosphorylase inhibitors However a drasticdecrease in activity was observed when para-hydroxygroup on phenyl ring was replaced with methyl (4n IC
50
Journal of Chemistry 9
= 2269 plusmn 150 120583M) chloro (4n IC50
= 242 plusmn 15 120583M)bromo (4q IC
50= 1413 plusmn 15 120583M) and aldehyde (4x
IC50
= 239 plusmn 20 120583M) groups whereas the compounds4o 4s 4t 4w and 4y have para-methoxy- ortho-nitro-ortho-NN-dimethyl amine- ortho- para-dichloro- andortho-ortho-dichloro-substituted phenyl ring respectively
Among series of compounds (5andash5h) having bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring as centralmoiety electron donating hydroxy- andmethoxy-substitutedphenyl ring containing compounds 5h (IC
50= 569plusmn16 120583M)
and 5d (IC50
= 605 plusmn 187 120583M) respectively were foundas potent inhibitors of thymidine phosphorylase enzymeagainst the tested standard 7-deazaxanthine which was(IC50
= 41 plusmn 146 120583M) used as standard Again a drasticdecrease in activity was observed for compounds 5b (IC
50
= 2169 plusmn 05 120583M) 5c (IC50
= 1614 plusmn 15 120583M) and 5g(IC50= 1866 plusmn 12 120583M) havingmeta-methyl- ortho-methyl-
and para-aldehyde-substituted benzene ring respectivelyinstead of para-hydroxyl phenyl ring The replacement ofortho-methyl with chloro-group makes the compound 5ecompletely inactive Similarly the replacement of electrondonating para-hydroxy-group of phenyl ring with electronwithdrawing nitro-functionality also contributed towardsinactivity of compound as observed for 5f Phenol substi-tuted bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ringcontaining 5h (IC
50= 569 plusmn 16 120583M) was found to be the
potent thymidine phosphorylase inhibitorsAll members of the series of diethylaminium salts of pyri-
dinium adducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) central moi-ety attached with substituted phenyl ring were found to beweak inhibitors of thymidine phosphorylase enzyme againstthe tested standard compound 7-deazaxanthine (IC
50= 41 plusmn
146 120583M) with IC50
values 318 plusmn 25 120583M 1664 plusmn 12 120583M1781 plusmn 21 120583M and 1471 plusmn 123 120583M for 5j 5k 5l and 5mrespectively whereas para-chloro-substituted phenyl ringcontaining compound 5i was found to be inactiveThe resultsare summarized in Table 1
335 120573-Glucuronidase Inhibition Activity Compounds 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evaluatedfor their in vitro 120573-glucuronidase inhibitory activity by usingD-saccharic acid 14-lactone as standard inhibitor (IC
50=
4575 plusmn 216 120583M) Compounds 4a (IC50
= 2048 plusmn 601 120583M)4c (IC
50= 1721 plusmn 21 120583M) 4e (IC
50= 3389 plusmn 642 120583M) 4o
(IC50
= 2621 plusmn 435 120583M) 4q (IC50
= 2922 plusmn 103 120583M) 4r(IC50= 2621plusmn44 120583M) 4w (IC
50= 1435plusmn124 120583M) 5f (IC
50
= 185 plusmn 25 120583M) 5i (IC50
= 2236 plusmn 64 120583M) and 5l (IC50
=3875plusmn70 120583M) showed veryweak120573-glucuronidase inhibitionpotential whereas pyridinium adducts 4andash4c 4e 4gndash4j 4l4o 4s-4t 4w 4y 5a 5e-5f and 5i were found to be inactive(Table 2)
34 Molecular Docking Studies
341 Binding Interactions of 120572-Glucosidase Enzyme To de-fine inhibitors interactions and their specificity the dockingstudy of the most active compound 4p against 120572-glucosidase
Figure 4 Three-dimensional representation of compound 4p withtarget macromolecule 120572-glucosidase
Figure 5 Three-dimensional representation of compound 4v withtarget macromolecule thymidine phosphorylase
was carried out [23ndash25] Ten different conformations ofprotein-ligand interactions were generated and the topranked pose was selected to explore the binding interactionsIn our docking study we have observed two interactions withthe active site residues of 120572-glucosidase The polar imidazolering of His 239 showed hydrogen bond through its H atomwith the lone pair of oxygen of the pyrimidinedionemoiety ofthe compound whereas Arg 312 formed hydrogen bond withthe OH group of the same moiety of compound (Figure 4)The presence of polar and electron rich center in the formof pyrimidinedione and chlorobenzene groups of the com-pound was observed here as key factors for the interactionbetween compound andprotein Anumber of hydrogen bonddonor and acceptor pairs with their suitable orientationswere observed in the interaction pose Furthermore severalhydrophobic interactions between the nonpolar active siteresidues and the compound 4p were also observed
342 Binding Interactions of Thymidine PhosphorylaseEnzyme To explore the binding modes of these newly syn-thesized compounds in the active site of thymidine phos-phorylase the most active compound 4v was docked againstthis enzyme The docking results showed that compound4v well accommodated in the active site of thymidinephosphorylase (Figure 5) The active site residue Ala 206established a hydrogen bond through its lone pair of
10 Journal of Chemistry
Figure 6 Three-dimensional representation of compound 4w withtarget enzyme 120573-glucuronidase
oxygen with the active hydrogen of hydroxyl moiety ofthe pyrimidinedione group of the compound Howeverthe substituted phenolic group and the other electron richcenters of the pyrimidinedione showed poor behaviorregarding interaction which may depend on the nature andorientation of the surrounding active site residues Moreoverseveral weak hydrophobic interactions between the activesite residues and the compound were also observed
343 Binding Interaction of 120573-Glucuronidase Enzyme Thepredicted bindingmode ofmost active compound 4w against120573-glucuronidase showed that a hydrogen bond is formedbetween the lone pair of oxygen of cyclohexanedione compo-nent of the compound and imidazole hydrogen of the impor-tant active site residue His 509 An arene-arene and arene-cation interactionwas also observed between the pi electroniccloud of dichlorobenzene ring of compound and imidazolering of His 509 (Figure 6) Besides hydrogen bonding andarene-arene interactions several hydrophobic interactionsbetween compound 4w and the active site residues werealso observed The pyrimidinedione moiety in this case wasobserved to be passive regarding interactions
4 Conclusions
In conclusion barbiturate salts were synthesized by one-potmulticomponent reactions via tandem AldolMichael addi-tion reactions between barbituric acid and dimedone withaldehydes mediated by aqueous diethylamine The molec-ular structures of 5a 5d and 5f were deduced by single-crystal X-ray diffraction techniques All of the synthesizedcompounds were evaluated for their antioxidant potentialin vitro by using DPPH radical scavenging assay as well asenzyme inhibition activities against120572-glucosidase thymidinephosphorylase and 120573-glucuronidase enzymes Compounds4andash4g (IC
50= 1018 plusmn 08ndash1244 plusmn 44 120583M) were found to
be the potent antioxidants as compared to the standardsBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) Compound 4p (IC50= 1868 plusmn 81 120583M) was
found to be a potent 120572-glucosidase inhibitor Compound 4v(IC50
= 87 plusmn 12 120583M) was found to be a potent thymidinephosphorylase inhibitor Promising results indicate that thesecompounds need to be investigated as antioxidant agentsto treat various associated oxidative disorders Similarlytheir enzyme inhibitory potential can be reported on drugdiscovery program
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Assem Barakat Hazem A Ghabbour Abdullah MohammedAl-Majid Qurat-ul-ain Rehan Imad Kulsoom Javaid NimraNaveed Shaikh Sammer Yousuf M Iqbal Choudhary andAbdul Wadood contributed equally to this work
Acknowledgments
The authors would like to extend their sincere appreciation tothe Deanship of Scientific Research at King Saud Universityfor funding this Research Group no (RGP-257-1435-1436)
References
[1] K Undheim T Bennecke A R Katritzky C W Rees andE F V Scriven Comprehensive Heterocyclic Chemistry vol 6supplement 93 Elsevier Pergamon Oxford UK 1996
[2] B TaylorModern Medical Chemistry Prentice Hall New YorkNY USA 1994
[3] R Y Levina and F K Velichko ldquoAdvances in the chemistry ofbarbituric acidsrdquo Russian Chemical Reviews vol 29 no 8 pp437ndash459 1960
[4] P Sing and K Paul ldquoA simple synthesis of 5-spirobarbituricacids and transformations of spirocyclopropane barbiturates to5-substitutated barbituratesrdquo Indian Journal of Chemistry B vol45 pp 247ndash251 2006
[5] X Chen K Tanaka and F Yoneda ldquoSimple newmethod for thesynthesis of 5-deaza-10-oxaflavin a potential organic oxidantrdquoChemical and Pharmaceutical Bulletin vol 38 no 2 pp 307ndash311 1990
[6] L R Morgan B S Jursic C L Hooper D M Neumann KThangaraj and B LeBlanc ldquoAnticancer activity for 4 41015840-di-hydroxybenzophenone-2 4-dinitrophenylhydrazone (A-007)analogues and their abilities to interact with lymphoendothelialcell surfacemarkersrdquo Bioorganic ampMedicinal Chemistry Lettersvol 12 no 23 pp 3407ndash3411 2002
[7] G Orzalesi P Gratteri and S Selleri ldquoSynthesis of new 13-dicyclohexyl barbituric acid derivatives with anti-inflammatorypotential activityrdquoEuropean Journal ofMedicinal Chemistry vol25 no 2 pp 197ndash201 1990
[8] D T Puerta and S M Cohen ldquoA bioinorganic perspectiveson matrix metalloproteinase inhibitionrdquo Current Topics inMedicinal Chemistry vol 4 no 15 pp 1551ndash1573 2004
[9] M E Wolff Burgerrsquos Medicinal Chemistry and Drug DiscoveryJohn Wiley amp Sons New York NY USA 1997
Journal of Chemistry 11
[10] E Fischer and J von Mering ldquoUeber eine neue klasse vonschlafmittelnrdquo in Untersuchungen aus Verschiedenen GebietenM Bergmann Ed pp 671ndash679 Springer Berlin Germany1924
[11] W J Doran ldquoBarbituric acid hypnoticsrdquo Medicinal Chemistryvol 4 pp 164ndash167 1959
[12] B Bobranski ldquoProgress in chemistry of barbituric acidrdquoWiad-omosci Chemiczne vol 31 pp 231ndash278 1977
[13] S Senda H Izumi and H Fujimura ldquoUracil derivaives andrelated compounds 6 Derivatives of 5-alkyle-246-trioxoper-hydropyrimidine as antiphlogisticsrdquo Arzneimittel Forschungvol 17 no 12 p 151 1967
[14] N Moussier L Bruche F Viani and M Zanda ldquoFluorinatedbarbituric acid derivatives synthesis and bio-activityrdquo CurrentOrganic Chemistry vol 7 no 11 pp 1071ndash1080 2003
[15] P-G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000
[16] N Ramarathnam T Osawa H Ochi and S Kawakishi ldquoThecontribution of plant food antioxidants to human healthrdquoTrends in Food Science amp Technology vol 6 no 3 pp 75ndash821995
[17] A Barakat A M Al-Majid H J Al-Najjar et al ldquoZwitterionicpyrimidinium adducts as antioxidants with therapeutic poten-tial as nitric oxide scavengerrdquo European Journal of MedicinalChemistry vol 84 pp 146ndash154 2014
[18] P Singh M Kaur and P Verma ldquoDesign synthesis andanticancer activities of hybrids of indole and barbituric acids-Identification of highly promising leadsrdquo Bioorganic andMedic-inal Chemistry Letters vol 19 no 11 pp 3054ndash3058 2009
[19] A M Al-Majid A Barakat H J Al-Najjar Y N Mabkhot HA Ghabbour and H-K Fun ldquoTandem aldol-michael reactionsin aqueous diethylamine medium a greener and efficientapproach to bis-pyrimidine derivativesrdquo International Journalof Molecular Sciences vol 14 no 12 pp 23762ndash23773 2013
[20] A Barakat A M Al-Majid G Lotfy et al ldquoSynthesis and dy-namics studies of barbituric acid derivatives as urease inhibi-torsrdquo Chemistry Central Journal vol 9 no 1 article 63 2015
[21] G M Sheldrick ldquoA short history of SHELXrdquo Acta Crystallo-graphica A vol 64 pp 112ndash122 2008
[22] A L Spek ldquoStructure validation in chemical crystallographyrdquoActaCrystallographica SectionD Biological Crystallography vol65 no 2 pp 148ndash155 2009
[23] F Rahim K Ullah H Ullah et al ldquoTriazinoindole analogs aspotent inhibitors of 120572-glucosidase synthesis biological evalua-tion and molecular docking studiesrdquo Bioorganic Chemistry vol58 pp 81ndash87 2015
[24] F Rahim F Malik H Ullah et al ldquoIsatin based Schiff basesas inhibitors of 120572-glucosidase synthesis characterization invitro evaluation and molecular docking studiesrdquo BioorganicChemistry vol 60 article 1803 pp 42ndash48 2015
[25] A Barakat A M Al-Majid M S Islam et al ldquoMolecular struc-ture investigation and biological evaluation of Michael adductsderived from dimedonerdquo Research on Chemical Intermediatesvol 42 pp 4041ndash4053 2016
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
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Carbohydrate Chemistry
International Journal of
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Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
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Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
6 Journal of Chemistry
5f
5d5a
Figure 3Molecular packing of 5a 5d and 5f viewed approximately three-dimensional network along 119887 axis and three-dimensional networkrespectively Intermolecular interaction is drawn as dashed lines
standards BHT (IC50= 1288 plusmn 21 120583M) andN-acetylcysteine
(IC50
= 1076 plusmn 28 120583M) Compounds 4andash4e (IC50
=1018 plusmn 08ndash1244 plusmn 44 120583M) were found to be more activecompared to the standard BHT (IC
50= 1288 plusmn 21 120583M) The
meta-bromo-substituted phenyl ring containing compound4e (IC
50= 1011 plusmn 08 120583M) was the most potent member of
the series (4andash4e) followed by the para-methyl-substitutedphenyl ring containing compound 4g (IC
50= 1041plusmn19 120583M)
A gradual decrease in activity was observed for compounds4c (IC
50= 1199 plusmn 19 120583M) and 4a (IC
50= 1188 plusmn 31 120583M)
having para-nitro- and para-aldehyde-substituted phenylring attached to central bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) ring A decrease inDPPH radical scavenging activity was due to positional effectof methyl substituent on phenyl ring which was observed forcompound 4b (IC
50= 117plusmn31 120583M) havingmeta-substituted
phenyl ring instead of para-methyl-substituted phenyl ring(4g IC
50= 1041 plusmn 19 120583M) The electron donating para-
hydroxyl-substituted phenyl ring containing compound 4f(IC50= 1486 plusmn 26 120583M) was found to be a last active member
of the series However a considerable increase in activity was
observed for compound 4d (IC50
= 1244 plusmn 44 120583M) havinga para-methoxy group instead of hydroxyl functionalityon phenyl ring whereas naphthalene ring containingbarbiturate 4h (IC
50= 1336 plusmn 57 120583M) was also found to be
a weak antioxidant agent against DPPH radicalsAmong second series of the tested compounds 4indash4l
having bis(6-hydroxypyrimidine-24(1H3H)-dione as back-bone compounds having para-methyl- (4i IC
50= 3849 plusmn
66 120583M) para-chloro- (4j IC50= 3843 plusmn 88 120583M) and para-
methoxy- (4l IC50= 3979 plusmn 09 120583M) substituted phenyl ring
attached to the central bis(6-hydroxypyrimidine-24(1H3H)-dione) showed better antioxidant activity as compared toBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) whereas naphthalene containing compound4l was found to be inactive
(2-Hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate ringcontaining compounds 4mndash4z were also evaluated for theirantioxidant potential in vitro in DPPH radical scavengingassayThese compounds were found to be significant to weakinhibitors of free radicals (IC
50= 1332plusmn47ndash4396plusmn101 120583M)
Journal of Chemistry 7
Table2Re
sultof
invitro
biologicalevaluatio
nof
thes
ynthesized
compo
unds
Num
berD
PPHradicalscaveng
ingassay
IC50plusmnSE
Ma(120583M)
Anticancercytotoxicityassay
IC50plusmnSE
M(120583M)
120572-G
lucosid
aseinh
ibition
IC50plusmnSE
M(120583M)
Thym
idinep
hospho
rylase
inhibitio
nIC50plusmnSE
M(120583M)
120573-G
lucuronidase
inhibitio
nIC50plusmnSE
M(120583M)
PC3b
Helac
MCF
-7d
3T3
4a118
8plusmn31
gt30
gt30
NA
gt30
NA
NA
20481plusmn60
4b117plusmn24
gt30
gt30
NA
gt30
NA
NA
NA
4c1119plusmn19
gt30
gt30
NA
gt30
355plusmn10
NA
1721plusmn
21
4d1244plusmn44
gt30
gt30
NA
gt30
NA
2309plusmn08
NA
4e1011plusmn08
gt30
gt30
NA
gt30
NA
NA
3389plusmn64
4f1486plusmn26
gt30
gt30
NA
gt30
NA
2179plusmn20
NA
4g1041plusmn
19gt30
gt30
NA
gt30
NA
NA
NA
4h1336plusmn57
gt30
gt30
NA
gt30
230plusmn39
NA
NA
4i3849plusmn66
gt30
gt30
NA
gt30
NA
NA
NA
4j3843plusmn88
gt30
gt30
NA
gt30
NA
NA
NA
4kNA
gt30
gt30
NA
gt30
NA
1826plusmn10
NA
4l3979plusmn01
gt30
gt30
NA
gt30
NA
NA
NA
4mNA
gt30
gt30
NA
gt30
NA
1602plusmn45
NA
4nNA
gt30
gt30
NA
gt30
NA
2269plusmn15
NA
4oNA
gt30
gt30
NA
gt30
3009plusmn65
NA
2621plusmn
43
4pNA
gt30
gt30
NA
gt30
1868plusmn81
2420plusmn15
NA
4qNA
gt30
gt30
NA
gt30
2148plusmn72
1413plusmn15
29217plusmn10
4rNA
gt30
gt30
NA
gt30
201plusmn
55
168plusmn12
2621plusmn
44
4s4396plusmn101
gt30
gt30
NA
gt30
NA
NA
NA
4t2782plusmn100
gt30
gt30
NA
gt30
NA
NA
NA
4v1332plusmn47
gt30
gt30
NA
gt30
NA
576plusmn12
NA
4w4116plusmn06
gt30
gt30
NA
gt30
NA
NA
1435plusmn12
4x3888plusmn79
gt30
gt30
NA
gt30
NA
239plusmn20
NA
4yNA
1071plusmn
03
347plusmn025
49plusmn001
320plusmn02
NA
NA
NA
4z2230plusmn39
gt30
gt30
NA
gt30
NA
558plusmn15
NA
5a4616plusmn88
gt30
gt30
NA
gt30
NA
NA
NA
5bNA
gt30
gt30
NA
gt30
NA
2169plusmn05
NA
5cNA
gt30
gt30
NA
gt30
NA
1614plusmn15
NA
5dNA
gt30
gt30
NA
gt30
NA
605plusmn18
7NA
5e289plusmn09
gt30
gt30
NA
gt30
NA
NA
NA
5fNA
gt30
gt30
NA
gt30
238plusmn93
NA
1850plusmn25
5g2704plusmn23
gt30
gt30
NA
gt30
NA
1866plusmn12
NA
5hNA
gt30
gt30
NA
gt30
NA
569plusmn16
NA
5iNA
gt30
gt30
NA
gt30
NA
NA
3236plusmn64
5jNA
gt30
gt30
NA
gt30
NA
318plusmn25
NA
5k1276plusmn16
gt30
gt30
NA
gt30
NA
1664plusmn12
NA
5l1697plusmn93
gt30
gt30
NA
gt30
NA
1781plusmn
21
3875plusmn70
5mNA
gt30
gt30
NA
gt30
4534plusmn42
1471plusmn12
NA
STD
e BHT(288plusmn21)
Doxorub
icin
Doxorub
icin
Doxorub
icin
Cyclo
heximide
Acarbo
se7-Deazaxanthine
D-Saccharicacid
1-4lacton
eN-acetylcysteine(
1076plusmn28)
0912plusmn012
0506plusmn015
156plusmn005
026plusmn01
840plusmn17
341plusmn16
44575plusmn216
a SEM
stand
arderrorof
mean
b PC3
hum
anprostate
cancer
celllin
ec H
eLaHenrie
ttaLackscervical
cancer
celllin
ed M
CF-7M
ichiganCa
ncer
Foun
datio
n-7breastcancer
celllin
esand
e BHT
butylated
hydroxytoluene
8 Journal of Chemistry
except compounds 4mndash4r and 4y which were found to beinactive
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) (5andash5h) and 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) (5indash5k) rings con-taining compounds were also evaluated for their antioxidantpotential by using DPPH radical scavenging assay Para-bromo-substituted phenyl ring containing compound5k (IC
50= 1276 plusmn 16 120583M) was found as a potent anti-
oxidant agent against the tested standards BHT (IC50
=1288 plusmn 21 120583M) The compound showed similar antioxidantpotential as BHT and showed a significant antioxidant agentagainst N-acetyl cysteine (IC
50= 1076 plusmn 28 120583M) The
gradual and drastic decrease in activity was for compounds5l (IC
50= 1697 plusmn 53 120583M) 5g (IC
50= 2704 plusmn 23 120583M)
5e (IC50
= 289 plusmn 09 120583M) and 5a (IC50
= 4616 plusmn 88 120583M)having para-methyl- para-aldehyde- ortho-chloro- andortho-nitro-substituted phenyl rings attached to the centralbarbiturate moieties Compounds 5bndash5d 5f and 5hndash5j werefound to be inactive
On the basis of the observed antioxidant abilities ofthe above five different series of pyridinium adducts itwas concluded that bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) ring containing com-pounds 4andash4h are the most active one The activity may bedue to the methyl substituted nitrogen atoms of the tetrahy-dropyrimidine rings that facilitate the electronic conjugationwithin the ring However the variation of antioxidantproperties within the most important series is may be due tothe presence of electron donating (-OH (4f) -OCH
3(4d)
and -CH3(4b 4g)) electron withdrawing (-NO
2(4a) and
-CHO (4c)) and halogen (-Br (4e)) substituents on thephenyl ring attached to the bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) moiety Howeverdetailed studies are required to study the mechanism ofaction of these compounds
332 Cytotoxic Activity All synthesized diethyl ammoniumsalts of barbiturates (4andash4z and 5andash5m) were evaluated fortheir in vitro cytotoxicity against PC-3 HeLa MCF-7 cancerand 3T3 normal cell lines All compounds were found to benoncytotoxic against PC-3 HeLaMCF-7 cancer and 3T3 celllines except dichloro-substituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tet-rahydropyrimidin-4-olate ring containing compounds 4ywhich was found to be significantly toxic against all the celllines (PC3 IC
50= 1071plusmn026 120583MHeLa IC
50= 347plusmn03 120583M
MCF-7 IC50= 49 plusmn 001 120583M and 3T3 IC
50= 320 plusmn 02 120583M)
Doxorubicin was used as a standard drug to compare theactivities against PC3 (IC
50= 170 plusmn 029 120583M) HeLa (IC
50=
051 plusmn 015 120583M) and MCF-3 (IC50= 092 plusmn 001 120583M) cancer
cell lines while cycloheximide (IC50
= 026 plusmn 012 120583M) wasused as a standard-in 3T3 cell line (Table 2)
333 120572-Glucosidase Inhibition Activity Synthesized diethylammonium salts of barbiturates 4andash4z and 5andash5m were alsoevaluated for their in vitro 120572-glucosidase inhibition activityin comparison to the standard drug acarbose (IC
50= 841 plusmn
173 120583M) Among dimethyl ammonium salts (4andash4h) nitro-substituted phenyl ring containing 4c (IC
50= 355 plusmn 10 120583M)
and naphthalene ring containing 4h (IC50= 230 plusmn 392 120583M)
were found to be potent inhibitors of 120572-glucosidase enzymeand showed more activity than acarbose (IC
50= 841 plusmn
173 120583M) All other compounds were found to be inactiveAmong diethyl ammonium salts having (2-hydroxy-
44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate ring (4mndash4z)para-OCH
3- (40 IC
50= 3009 plusmn 65 120583M) para-chloro-
(4p IC50
= 1868 plusmn 081 120583M) para-bromo- (4q IC50
=2148plusmn072 120583M) andmeta-bromo- (4r IC
50= 201plusmn55 120583M)
substituted phenyl ring containing pyridinium adducts werefound to be potent 120572-glucosidase inhibitors and showedmore activity than standard acarbose (IC
50= 841plusmn 173 120583M)
Other members of the series (4m-4n and 4sndash4z) were foundto be inactive The results showed the substitution effects ofvarious functionalities of phenyl ring on the potential activityof tested series The para-bromo-substituted phenyl ringcontaining compound 4q (IC
50= 2148 plusmn 072 120583M) showed
more activity than tested standard However the replacementof bromo group with that of methylNN-dimethyl hydroxyland aldehyde functionalities resulted in complete loss ofactivity as observed in compounds 4n 4t 4v and 4xrespectively The presence of ortho-nitro- ortho-choloro-and para-choloro-substituted phenyl ring also contributedtowards inactivity as observed for compounds 4t 4w and4y
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring containing diethyl ammonium pyridinium salts(5andash5h) were also evaluated for their in vitro 120572-glucosidaseenzyme inhibition activity The series nitro-substitutedphenyl ring containing 5f was found to be the only potent120572-glucosidase inhibitor All other compounds (5andash5e and5g-5h) were found to be inactive
Among series of diethylaminium salts of pyridiniumadducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-di-oxo-1234-tetrahydropyrimidin-5-yl)methyl) central moietyattached with substituted phenyl ring the benzaldehydecontaining 5m (IC
50= 4534 plusmn 42 120583M) was found to be the
only potent inhibitor compound in comparison to against thestandard drug acarbose (IC
50= 841plusmn173 120583M)All other com-
poundswere found to be inactiveThe results are summarizedin Table 2
334 Thymidine Phosphorylase Inhibition Activity Five dif-ferent series of diethyamonium salts of barbiturates 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evalu-ated for their in vitro thymidine phosphorylase inhibition7-Deazaxanthine was used as a standard inhibitor (IC
50
= 41 plusmn 146 120583M) Phenol and naphthalene rings sub-stituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olatemoiety containing compounds 4v (IC
50= 576 plusmn 120120583M)
and 4z (IC50
= 558 plusmn 150 120583M) were found to be the potentthymidine phosphorylase inhibitors However a drasticdecrease in activity was observed when para-hydroxygroup on phenyl ring was replaced with methyl (4n IC
50
Journal of Chemistry 9
= 2269 plusmn 150 120583M) chloro (4n IC50
= 242 plusmn 15 120583M)bromo (4q IC
50= 1413 plusmn 15 120583M) and aldehyde (4x
IC50
= 239 plusmn 20 120583M) groups whereas the compounds4o 4s 4t 4w and 4y have para-methoxy- ortho-nitro-ortho-NN-dimethyl amine- ortho- para-dichloro- andortho-ortho-dichloro-substituted phenyl ring respectively
Among series of compounds (5andash5h) having bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring as centralmoiety electron donating hydroxy- andmethoxy-substitutedphenyl ring containing compounds 5h (IC
50= 569plusmn16 120583M)
and 5d (IC50
= 605 plusmn 187 120583M) respectively were foundas potent inhibitors of thymidine phosphorylase enzymeagainst the tested standard 7-deazaxanthine which was(IC50
= 41 plusmn 146 120583M) used as standard Again a drasticdecrease in activity was observed for compounds 5b (IC
50
= 2169 plusmn 05 120583M) 5c (IC50
= 1614 plusmn 15 120583M) and 5g(IC50= 1866 plusmn 12 120583M) havingmeta-methyl- ortho-methyl-
and para-aldehyde-substituted benzene ring respectivelyinstead of para-hydroxyl phenyl ring The replacement ofortho-methyl with chloro-group makes the compound 5ecompletely inactive Similarly the replacement of electrondonating para-hydroxy-group of phenyl ring with electronwithdrawing nitro-functionality also contributed towardsinactivity of compound as observed for 5f Phenol substi-tuted bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ringcontaining 5h (IC
50= 569 plusmn 16 120583M) was found to be the
potent thymidine phosphorylase inhibitorsAll members of the series of diethylaminium salts of pyri-
dinium adducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) central moi-ety attached with substituted phenyl ring were found to beweak inhibitors of thymidine phosphorylase enzyme againstthe tested standard compound 7-deazaxanthine (IC
50= 41 plusmn
146 120583M) with IC50
values 318 plusmn 25 120583M 1664 plusmn 12 120583M1781 plusmn 21 120583M and 1471 plusmn 123 120583M for 5j 5k 5l and 5mrespectively whereas para-chloro-substituted phenyl ringcontaining compound 5i was found to be inactiveThe resultsare summarized in Table 1
335 120573-Glucuronidase Inhibition Activity Compounds 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evaluatedfor their in vitro 120573-glucuronidase inhibitory activity by usingD-saccharic acid 14-lactone as standard inhibitor (IC
50=
4575 plusmn 216 120583M) Compounds 4a (IC50
= 2048 plusmn 601 120583M)4c (IC
50= 1721 plusmn 21 120583M) 4e (IC
50= 3389 plusmn 642 120583M) 4o
(IC50
= 2621 plusmn 435 120583M) 4q (IC50
= 2922 plusmn 103 120583M) 4r(IC50= 2621plusmn44 120583M) 4w (IC
50= 1435plusmn124 120583M) 5f (IC
50
= 185 plusmn 25 120583M) 5i (IC50
= 2236 plusmn 64 120583M) and 5l (IC50
=3875plusmn70 120583M) showed veryweak120573-glucuronidase inhibitionpotential whereas pyridinium adducts 4andash4c 4e 4gndash4j 4l4o 4s-4t 4w 4y 5a 5e-5f and 5i were found to be inactive(Table 2)
34 Molecular Docking Studies
341 Binding Interactions of 120572-Glucosidase Enzyme To de-fine inhibitors interactions and their specificity the dockingstudy of the most active compound 4p against 120572-glucosidase
Figure 4 Three-dimensional representation of compound 4p withtarget macromolecule 120572-glucosidase
Figure 5 Three-dimensional representation of compound 4v withtarget macromolecule thymidine phosphorylase
was carried out [23ndash25] Ten different conformations ofprotein-ligand interactions were generated and the topranked pose was selected to explore the binding interactionsIn our docking study we have observed two interactions withthe active site residues of 120572-glucosidase The polar imidazolering of His 239 showed hydrogen bond through its H atomwith the lone pair of oxygen of the pyrimidinedionemoiety ofthe compound whereas Arg 312 formed hydrogen bond withthe OH group of the same moiety of compound (Figure 4)The presence of polar and electron rich center in the formof pyrimidinedione and chlorobenzene groups of the com-pound was observed here as key factors for the interactionbetween compound andprotein Anumber of hydrogen bonddonor and acceptor pairs with their suitable orientationswere observed in the interaction pose Furthermore severalhydrophobic interactions between the nonpolar active siteresidues and the compound 4p were also observed
342 Binding Interactions of Thymidine PhosphorylaseEnzyme To explore the binding modes of these newly syn-thesized compounds in the active site of thymidine phos-phorylase the most active compound 4v was docked againstthis enzyme The docking results showed that compound4v well accommodated in the active site of thymidinephosphorylase (Figure 5) The active site residue Ala 206established a hydrogen bond through its lone pair of
10 Journal of Chemistry
Figure 6 Three-dimensional representation of compound 4w withtarget enzyme 120573-glucuronidase
oxygen with the active hydrogen of hydroxyl moiety ofthe pyrimidinedione group of the compound Howeverthe substituted phenolic group and the other electron richcenters of the pyrimidinedione showed poor behaviorregarding interaction which may depend on the nature andorientation of the surrounding active site residues Moreoverseveral weak hydrophobic interactions between the activesite residues and the compound were also observed
343 Binding Interaction of 120573-Glucuronidase Enzyme Thepredicted bindingmode ofmost active compound 4w against120573-glucuronidase showed that a hydrogen bond is formedbetween the lone pair of oxygen of cyclohexanedione compo-nent of the compound and imidazole hydrogen of the impor-tant active site residue His 509 An arene-arene and arene-cation interactionwas also observed between the pi electroniccloud of dichlorobenzene ring of compound and imidazolering of His 509 (Figure 6) Besides hydrogen bonding andarene-arene interactions several hydrophobic interactionsbetween compound 4w and the active site residues werealso observed The pyrimidinedione moiety in this case wasobserved to be passive regarding interactions
4 Conclusions
In conclusion barbiturate salts were synthesized by one-potmulticomponent reactions via tandem AldolMichael addi-tion reactions between barbituric acid and dimedone withaldehydes mediated by aqueous diethylamine The molec-ular structures of 5a 5d and 5f were deduced by single-crystal X-ray diffraction techniques All of the synthesizedcompounds were evaluated for their antioxidant potentialin vitro by using DPPH radical scavenging assay as well asenzyme inhibition activities against120572-glucosidase thymidinephosphorylase and 120573-glucuronidase enzymes Compounds4andash4g (IC
50= 1018 plusmn 08ndash1244 plusmn 44 120583M) were found to
be the potent antioxidants as compared to the standardsBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) Compound 4p (IC50= 1868 plusmn 81 120583M) was
found to be a potent 120572-glucosidase inhibitor Compound 4v(IC50
= 87 plusmn 12 120583M) was found to be a potent thymidinephosphorylase inhibitor Promising results indicate that thesecompounds need to be investigated as antioxidant agentsto treat various associated oxidative disorders Similarlytheir enzyme inhibitory potential can be reported on drugdiscovery program
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Assem Barakat Hazem A Ghabbour Abdullah MohammedAl-Majid Qurat-ul-ain Rehan Imad Kulsoom Javaid NimraNaveed Shaikh Sammer Yousuf M Iqbal Choudhary andAbdul Wadood contributed equally to this work
Acknowledgments
The authors would like to extend their sincere appreciation tothe Deanship of Scientific Research at King Saud Universityfor funding this Research Group no (RGP-257-1435-1436)
References
[1] K Undheim T Bennecke A R Katritzky C W Rees andE F V Scriven Comprehensive Heterocyclic Chemistry vol 6supplement 93 Elsevier Pergamon Oxford UK 1996
[2] B TaylorModern Medical Chemistry Prentice Hall New YorkNY USA 1994
[3] R Y Levina and F K Velichko ldquoAdvances in the chemistry ofbarbituric acidsrdquo Russian Chemical Reviews vol 29 no 8 pp437ndash459 1960
[4] P Sing and K Paul ldquoA simple synthesis of 5-spirobarbituricacids and transformations of spirocyclopropane barbiturates to5-substitutated barbituratesrdquo Indian Journal of Chemistry B vol45 pp 247ndash251 2006
[5] X Chen K Tanaka and F Yoneda ldquoSimple newmethod for thesynthesis of 5-deaza-10-oxaflavin a potential organic oxidantrdquoChemical and Pharmaceutical Bulletin vol 38 no 2 pp 307ndash311 1990
[6] L R Morgan B S Jursic C L Hooper D M Neumann KThangaraj and B LeBlanc ldquoAnticancer activity for 4 41015840-di-hydroxybenzophenone-2 4-dinitrophenylhydrazone (A-007)analogues and their abilities to interact with lymphoendothelialcell surfacemarkersrdquo Bioorganic ampMedicinal Chemistry Lettersvol 12 no 23 pp 3407ndash3411 2002
[7] G Orzalesi P Gratteri and S Selleri ldquoSynthesis of new 13-dicyclohexyl barbituric acid derivatives with anti-inflammatorypotential activityrdquoEuropean Journal ofMedicinal Chemistry vol25 no 2 pp 197ndash201 1990
[8] D T Puerta and S M Cohen ldquoA bioinorganic perspectiveson matrix metalloproteinase inhibitionrdquo Current Topics inMedicinal Chemistry vol 4 no 15 pp 1551ndash1573 2004
[9] M E Wolff Burgerrsquos Medicinal Chemistry and Drug DiscoveryJohn Wiley amp Sons New York NY USA 1997
Journal of Chemistry 11
[10] E Fischer and J von Mering ldquoUeber eine neue klasse vonschlafmittelnrdquo in Untersuchungen aus Verschiedenen GebietenM Bergmann Ed pp 671ndash679 Springer Berlin Germany1924
[11] W J Doran ldquoBarbituric acid hypnoticsrdquo Medicinal Chemistryvol 4 pp 164ndash167 1959
[12] B Bobranski ldquoProgress in chemistry of barbituric acidrdquoWiad-omosci Chemiczne vol 31 pp 231ndash278 1977
[13] S Senda H Izumi and H Fujimura ldquoUracil derivaives andrelated compounds 6 Derivatives of 5-alkyle-246-trioxoper-hydropyrimidine as antiphlogisticsrdquo Arzneimittel Forschungvol 17 no 12 p 151 1967
[14] N Moussier L Bruche F Viani and M Zanda ldquoFluorinatedbarbituric acid derivatives synthesis and bio-activityrdquo CurrentOrganic Chemistry vol 7 no 11 pp 1071ndash1080 2003
[15] P-G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000
[16] N Ramarathnam T Osawa H Ochi and S Kawakishi ldquoThecontribution of plant food antioxidants to human healthrdquoTrends in Food Science amp Technology vol 6 no 3 pp 75ndash821995
[17] A Barakat A M Al-Majid H J Al-Najjar et al ldquoZwitterionicpyrimidinium adducts as antioxidants with therapeutic poten-tial as nitric oxide scavengerrdquo European Journal of MedicinalChemistry vol 84 pp 146ndash154 2014
[18] P Singh M Kaur and P Verma ldquoDesign synthesis andanticancer activities of hybrids of indole and barbituric acids-Identification of highly promising leadsrdquo Bioorganic andMedic-inal Chemistry Letters vol 19 no 11 pp 3054ndash3058 2009
[19] A M Al-Majid A Barakat H J Al-Najjar Y N Mabkhot HA Ghabbour and H-K Fun ldquoTandem aldol-michael reactionsin aqueous diethylamine medium a greener and efficientapproach to bis-pyrimidine derivativesrdquo International Journalof Molecular Sciences vol 14 no 12 pp 23762ndash23773 2013
[20] A Barakat A M Al-Majid G Lotfy et al ldquoSynthesis and dy-namics studies of barbituric acid derivatives as urease inhibi-torsrdquo Chemistry Central Journal vol 9 no 1 article 63 2015
[21] G M Sheldrick ldquoA short history of SHELXrdquo Acta Crystallo-graphica A vol 64 pp 112ndash122 2008
[22] A L Spek ldquoStructure validation in chemical crystallographyrdquoActaCrystallographica SectionD Biological Crystallography vol65 no 2 pp 148ndash155 2009
[23] F Rahim K Ullah H Ullah et al ldquoTriazinoindole analogs aspotent inhibitors of 120572-glucosidase synthesis biological evalua-tion and molecular docking studiesrdquo Bioorganic Chemistry vol58 pp 81ndash87 2015
[24] F Rahim F Malik H Ullah et al ldquoIsatin based Schiff basesas inhibitors of 120572-glucosidase synthesis characterization invitro evaluation and molecular docking studiesrdquo BioorganicChemistry vol 60 article 1803 pp 42ndash48 2015
[25] A Barakat A M Al-Majid M S Islam et al ldquoMolecular struc-ture investigation and biological evaluation of Michael adductsderived from dimedonerdquo Research on Chemical Intermediatesvol 42 pp 4041ndash4053 2016
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Chemistry 7
Table2Re
sultof
invitro
biologicalevaluatio
nof
thes
ynthesized
compo
unds
Num
berD
PPHradicalscaveng
ingassay
IC50plusmnSE
Ma(120583M)
Anticancercytotoxicityassay
IC50plusmnSE
M(120583M)
120572-G
lucosid
aseinh
ibition
IC50plusmnSE
M(120583M)
Thym
idinep
hospho
rylase
inhibitio
nIC50plusmnSE
M(120583M)
120573-G
lucuronidase
inhibitio
nIC50plusmnSE
M(120583M)
PC3b
Helac
MCF
-7d
3T3
4a118
8plusmn31
gt30
gt30
NA
gt30
NA
NA
20481plusmn60
4b117plusmn24
gt30
gt30
NA
gt30
NA
NA
NA
4c1119plusmn19
gt30
gt30
NA
gt30
355plusmn10
NA
1721plusmn
21
4d1244plusmn44
gt30
gt30
NA
gt30
NA
2309plusmn08
NA
4e1011plusmn08
gt30
gt30
NA
gt30
NA
NA
3389plusmn64
4f1486plusmn26
gt30
gt30
NA
gt30
NA
2179plusmn20
NA
4g1041plusmn
19gt30
gt30
NA
gt30
NA
NA
NA
4h1336plusmn57
gt30
gt30
NA
gt30
230plusmn39
NA
NA
4i3849plusmn66
gt30
gt30
NA
gt30
NA
NA
NA
4j3843plusmn88
gt30
gt30
NA
gt30
NA
NA
NA
4kNA
gt30
gt30
NA
gt30
NA
1826plusmn10
NA
4l3979plusmn01
gt30
gt30
NA
gt30
NA
NA
NA
4mNA
gt30
gt30
NA
gt30
NA
1602plusmn45
NA
4nNA
gt30
gt30
NA
gt30
NA
2269plusmn15
NA
4oNA
gt30
gt30
NA
gt30
3009plusmn65
NA
2621plusmn
43
4pNA
gt30
gt30
NA
gt30
1868plusmn81
2420plusmn15
NA
4qNA
gt30
gt30
NA
gt30
2148plusmn72
1413plusmn15
29217plusmn10
4rNA
gt30
gt30
NA
gt30
201plusmn
55
168plusmn12
2621plusmn
44
4s4396plusmn101
gt30
gt30
NA
gt30
NA
NA
NA
4t2782plusmn100
gt30
gt30
NA
gt30
NA
NA
NA
4v1332plusmn47
gt30
gt30
NA
gt30
NA
576plusmn12
NA
4w4116plusmn06
gt30
gt30
NA
gt30
NA
NA
1435plusmn12
4x3888plusmn79
gt30
gt30
NA
gt30
NA
239plusmn20
NA
4yNA
1071plusmn
03
347plusmn025
49plusmn001
320plusmn02
NA
NA
NA
4z2230plusmn39
gt30
gt30
NA
gt30
NA
558plusmn15
NA
5a4616plusmn88
gt30
gt30
NA
gt30
NA
NA
NA
5bNA
gt30
gt30
NA
gt30
NA
2169plusmn05
NA
5cNA
gt30
gt30
NA
gt30
NA
1614plusmn15
NA
5dNA
gt30
gt30
NA
gt30
NA
605plusmn18
7NA
5e289plusmn09
gt30
gt30
NA
gt30
NA
NA
NA
5fNA
gt30
gt30
NA
gt30
238plusmn93
NA
1850plusmn25
5g2704plusmn23
gt30
gt30
NA
gt30
NA
1866plusmn12
NA
5hNA
gt30
gt30
NA
gt30
NA
569plusmn16
NA
5iNA
gt30
gt30
NA
gt30
NA
NA
3236plusmn64
5jNA
gt30
gt30
NA
gt30
NA
318plusmn25
NA
5k1276plusmn16
gt30
gt30
NA
gt30
NA
1664plusmn12
NA
5l1697plusmn93
gt30
gt30
NA
gt30
NA
1781plusmn
21
3875plusmn70
5mNA
gt30
gt30
NA
gt30
4534plusmn42
1471plusmn12
NA
STD
e BHT(288plusmn21)
Doxorub
icin
Doxorub
icin
Doxorub
icin
Cyclo
heximide
Acarbo
se7-Deazaxanthine
D-Saccharicacid
1-4lacton
eN-acetylcysteine(
1076plusmn28)
0912plusmn012
0506plusmn015
156plusmn005
026plusmn01
840plusmn17
341plusmn16
44575plusmn216
a SEM
stand
arderrorof
mean
b PC3
hum
anprostate
cancer
celllin
ec H
eLaHenrie
ttaLackscervical
cancer
celllin
ed M
CF-7M
ichiganCa
ncer
Foun
datio
n-7breastcancer
celllin
esand
e BHT
butylated
hydroxytoluene
8 Journal of Chemistry
except compounds 4mndash4r and 4y which were found to beinactive
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) (5andash5h) and 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) (5indash5k) rings con-taining compounds were also evaluated for their antioxidantpotential by using DPPH radical scavenging assay Para-bromo-substituted phenyl ring containing compound5k (IC
50= 1276 plusmn 16 120583M) was found as a potent anti-
oxidant agent against the tested standards BHT (IC50
=1288 plusmn 21 120583M) The compound showed similar antioxidantpotential as BHT and showed a significant antioxidant agentagainst N-acetyl cysteine (IC
50= 1076 plusmn 28 120583M) The
gradual and drastic decrease in activity was for compounds5l (IC
50= 1697 plusmn 53 120583M) 5g (IC
50= 2704 plusmn 23 120583M)
5e (IC50
= 289 plusmn 09 120583M) and 5a (IC50
= 4616 plusmn 88 120583M)having para-methyl- para-aldehyde- ortho-chloro- andortho-nitro-substituted phenyl rings attached to the centralbarbiturate moieties Compounds 5bndash5d 5f and 5hndash5j werefound to be inactive
On the basis of the observed antioxidant abilities ofthe above five different series of pyridinium adducts itwas concluded that bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) ring containing com-pounds 4andash4h are the most active one The activity may bedue to the methyl substituted nitrogen atoms of the tetrahy-dropyrimidine rings that facilitate the electronic conjugationwithin the ring However the variation of antioxidantproperties within the most important series is may be due tothe presence of electron donating (-OH (4f) -OCH
3(4d)
and -CH3(4b 4g)) electron withdrawing (-NO
2(4a) and
-CHO (4c)) and halogen (-Br (4e)) substituents on thephenyl ring attached to the bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) moiety Howeverdetailed studies are required to study the mechanism ofaction of these compounds
332 Cytotoxic Activity All synthesized diethyl ammoniumsalts of barbiturates (4andash4z and 5andash5m) were evaluated fortheir in vitro cytotoxicity against PC-3 HeLa MCF-7 cancerand 3T3 normal cell lines All compounds were found to benoncytotoxic against PC-3 HeLaMCF-7 cancer and 3T3 celllines except dichloro-substituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tet-rahydropyrimidin-4-olate ring containing compounds 4ywhich was found to be significantly toxic against all the celllines (PC3 IC
50= 1071plusmn026 120583MHeLa IC
50= 347plusmn03 120583M
MCF-7 IC50= 49 plusmn 001 120583M and 3T3 IC
50= 320 plusmn 02 120583M)
Doxorubicin was used as a standard drug to compare theactivities against PC3 (IC
50= 170 plusmn 029 120583M) HeLa (IC
50=
051 plusmn 015 120583M) and MCF-3 (IC50= 092 plusmn 001 120583M) cancer
cell lines while cycloheximide (IC50
= 026 plusmn 012 120583M) wasused as a standard-in 3T3 cell line (Table 2)
333 120572-Glucosidase Inhibition Activity Synthesized diethylammonium salts of barbiturates 4andash4z and 5andash5m were alsoevaluated for their in vitro 120572-glucosidase inhibition activityin comparison to the standard drug acarbose (IC
50= 841 plusmn
173 120583M) Among dimethyl ammonium salts (4andash4h) nitro-substituted phenyl ring containing 4c (IC
50= 355 plusmn 10 120583M)
and naphthalene ring containing 4h (IC50= 230 plusmn 392 120583M)
were found to be potent inhibitors of 120572-glucosidase enzymeand showed more activity than acarbose (IC
50= 841 plusmn
173 120583M) All other compounds were found to be inactiveAmong diethyl ammonium salts having (2-hydroxy-
44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate ring (4mndash4z)para-OCH
3- (40 IC
50= 3009 plusmn 65 120583M) para-chloro-
(4p IC50
= 1868 plusmn 081 120583M) para-bromo- (4q IC50
=2148plusmn072 120583M) andmeta-bromo- (4r IC
50= 201plusmn55 120583M)
substituted phenyl ring containing pyridinium adducts werefound to be potent 120572-glucosidase inhibitors and showedmore activity than standard acarbose (IC
50= 841plusmn 173 120583M)
Other members of the series (4m-4n and 4sndash4z) were foundto be inactive The results showed the substitution effects ofvarious functionalities of phenyl ring on the potential activityof tested series The para-bromo-substituted phenyl ringcontaining compound 4q (IC
50= 2148 plusmn 072 120583M) showed
more activity than tested standard However the replacementof bromo group with that of methylNN-dimethyl hydroxyland aldehyde functionalities resulted in complete loss ofactivity as observed in compounds 4n 4t 4v and 4xrespectively The presence of ortho-nitro- ortho-choloro-and para-choloro-substituted phenyl ring also contributedtowards inactivity as observed for compounds 4t 4w and4y
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring containing diethyl ammonium pyridinium salts(5andash5h) were also evaluated for their in vitro 120572-glucosidaseenzyme inhibition activity The series nitro-substitutedphenyl ring containing 5f was found to be the only potent120572-glucosidase inhibitor All other compounds (5andash5e and5g-5h) were found to be inactive
Among series of diethylaminium salts of pyridiniumadducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-di-oxo-1234-tetrahydropyrimidin-5-yl)methyl) central moietyattached with substituted phenyl ring the benzaldehydecontaining 5m (IC
50= 4534 plusmn 42 120583M) was found to be the
only potent inhibitor compound in comparison to against thestandard drug acarbose (IC
50= 841plusmn173 120583M)All other com-
poundswere found to be inactiveThe results are summarizedin Table 2
334 Thymidine Phosphorylase Inhibition Activity Five dif-ferent series of diethyamonium salts of barbiturates 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evalu-ated for their in vitro thymidine phosphorylase inhibition7-Deazaxanthine was used as a standard inhibitor (IC
50
= 41 plusmn 146 120583M) Phenol and naphthalene rings sub-stituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olatemoiety containing compounds 4v (IC
50= 576 plusmn 120120583M)
and 4z (IC50
= 558 plusmn 150 120583M) were found to be the potentthymidine phosphorylase inhibitors However a drasticdecrease in activity was observed when para-hydroxygroup on phenyl ring was replaced with methyl (4n IC
50
Journal of Chemistry 9
= 2269 plusmn 150 120583M) chloro (4n IC50
= 242 plusmn 15 120583M)bromo (4q IC
50= 1413 plusmn 15 120583M) and aldehyde (4x
IC50
= 239 plusmn 20 120583M) groups whereas the compounds4o 4s 4t 4w and 4y have para-methoxy- ortho-nitro-ortho-NN-dimethyl amine- ortho- para-dichloro- andortho-ortho-dichloro-substituted phenyl ring respectively
Among series of compounds (5andash5h) having bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring as centralmoiety electron donating hydroxy- andmethoxy-substitutedphenyl ring containing compounds 5h (IC
50= 569plusmn16 120583M)
and 5d (IC50
= 605 plusmn 187 120583M) respectively were foundas potent inhibitors of thymidine phosphorylase enzymeagainst the tested standard 7-deazaxanthine which was(IC50
= 41 plusmn 146 120583M) used as standard Again a drasticdecrease in activity was observed for compounds 5b (IC
50
= 2169 plusmn 05 120583M) 5c (IC50
= 1614 plusmn 15 120583M) and 5g(IC50= 1866 plusmn 12 120583M) havingmeta-methyl- ortho-methyl-
and para-aldehyde-substituted benzene ring respectivelyinstead of para-hydroxyl phenyl ring The replacement ofortho-methyl with chloro-group makes the compound 5ecompletely inactive Similarly the replacement of electrondonating para-hydroxy-group of phenyl ring with electronwithdrawing nitro-functionality also contributed towardsinactivity of compound as observed for 5f Phenol substi-tuted bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ringcontaining 5h (IC
50= 569 plusmn 16 120583M) was found to be the
potent thymidine phosphorylase inhibitorsAll members of the series of diethylaminium salts of pyri-
dinium adducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) central moi-ety attached with substituted phenyl ring were found to beweak inhibitors of thymidine phosphorylase enzyme againstthe tested standard compound 7-deazaxanthine (IC
50= 41 plusmn
146 120583M) with IC50
values 318 plusmn 25 120583M 1664 plusmn 12 120583M1781 plusmn 21 120583M and 1471 plusmn 123 120583M for 5j 5k 5l and 5mrespectively whereas para-chloro-substituted phenyl ringcontaining compound 5i was found to be inactiveThe resultsare summarized in Table 1
335 120573-Glucuronidase Inhibition Activity Compounds 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evaluatedfor their in vitro 120573-glucuronidase inhibitory activity by usingD-saccharic acid 14-lactone as standard inhibitor (IC
50=
4575 plusmn 216 120583M) Compounds 4a (IC50
= 2048 plusmn 601 120583M)4c (IC
50= 1721 plusmn 21 120583M) 4e (IC
50= 3389 plusmn 642 120583M) 4o
(IC50
= 2621 plusmn 435 120583M) 4q (IC50
= 2922 plusmn 103 120583M) 4r(IC50= 2621plusmn44 120583M) 4w (IC
50= 1435plusmn124 120583M) 5f (IC
50
= 185 plusmn 25 120583M) 5i (IC50
= 2236 plusmn 64 120583M) and 5l (IC50
=3875plusmn70 120583M) showed veryweak120573-glucuronidase inhibitionpotential whereas pyridinium adducts 4andash4c 4e 4gndash4j 4l4o 4s-4t 4w 4y 5a 5e-5f and 5i were found to be inactive(Table 2)
34 Molecular Docking Studies
341 Binding Interactions of 120572-Glucosidase Enzyme To de-fine inhibitors interactions and their specificity the dockingstudy of the most active compound 4p against 120572-glucosidase
Figure 4 Three-dimensional representation of compound 4p withtarget macromolecule 120572-glucosidase
Figure 5 Three-dimensional representation of compound 4v withtarget macromolecule thymidine phosphorylase
was carried out [23ndash25] Ten different conformations ofprotein-ligand interactions were generated and the topranked pose was selected to explore the binding interactionsIn our docking study we have observed two interactions withthe active site residues of 120572-glucosidase The polar imidazolering of His 239 showed hydrogen bond through its H atomwith the lone pair of oxygen of the pyrimidinedionemoiety ofthe compound whereas Arg 312 formed hydrogen bond withthe OH group of the same moiety of compound (Figure 4)The presence of polar and electron rich center in the formof pyrimidinedione and chlorobenzene groups of the com-pound was observed here as key factors for the interactionbetween compound andprotein Anumber of hydrogen bonddonor and acceptor pairs with their suitable orientationswere observed in the interaction pose Furthermore severalhydrophobic interactions between the nonpolar active siteresidues and the compound 4p were also observed
342 Binding Interactions of Thymidine PhosphorylaseEnzyme To explore the binding modes of these newly syn-thesized compounds in the active site of thymidine phos-phorylase the most active compound 4v was docked againstthis enzyme The docking results showed that compound4v well accommodated in the active site of thymidinephosphorylase (Figure 5) The active site residue Ala 206established a hydrogen bond through its lone pair of
10 Journal of Chemistry
Figure 6 Three-dimensional representation of compound 4w withtarget enzyme 120573-glucuronidase
oxygen with the active hydrogen of hydroxyl moiety ofthe pyrimidinedione group of the compound Howeverthe substituted phenolic group and the other electron richcenters of the pyrimidinedione showed poor behaviorregarding interaction which may depend on the nature andorientation of the surrounding active site residues Moreoverseveral weak hydrophobic interactions between the activesite residues and the compound were also observed
343 Binding Interaction of 120573-Glucuronidase Enzyme Thepredicted bindingmode ofmost active compound 4w against120573-glucuronidase showed that a hydrogen bond is formedbetween the lone pair of oxygen of cyclohexanedione compo-nent of the compound and imidazole hydrogen of the impor-tant active site residue His 509 An arene-arene and arene-cation interactionwas also observed between the pi electroniccloud of dichlorobenzene ring of compound and imidazolering of His 509 (Figure 6) Besides hydrogen bonding andarene-arene interactions several hydrophobic interactionsbetween compound 4w and the active site residues werealso observed The pyrimidinedione moiety in this case wasobserved to be passive regarding interactions
4 Conclusions
In conclusion barbiturate salts were synthesized by one-potmulticomponent reactions via tandem AldolMichael addi-tion reactions between barbituric acid and dimedone withaldehydes mediated by aqueous diethylamine The molec-ular structures of 5a 5d and 5f were deduced by single-crystal X-ray diffraction techniques All of the synthesizedcompounds were evaluated for their antioxidant potentialin vitro by using DPPH radical scavenging assay as well asenzyme inhibition activities against120572-glucosidase thymidinephosphorylase and 120573-glucuronidase enzymes Compounds4andash4g (IC
50= 1018 plusmn 08ndash1244 plusmn 44 120583M) were found to
be the potent antioxidants as compared to the standardsBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) Compound 4p (IC50= 1868 plusmn 81 120583M) was
found to be a potent 120572-glucosidase inhibitor Compound 4v(IC50
= 87 plusmn 12 120583M) was found to be a potent thymidinephosphorylase inhibitor Promising results indicate that thesecompounds need to be investigated as antioxidant agentsto treat various associated oxidative disorders Similarlytheir enzyme inhibitory potential can be reported on drugdiscovery program
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Assem Barakat Hazem A Ghabbour Abdullah MohammedAl-Majid Qurat-ul-ain Rehan Imad Kulsoom Javaid NimraNaveed Shaikh Sammer Yousuf M Iqbal Choudhary andAbdul Wadood contributed equally to this work
Acknowledgments
The authors would like to extend their sincere appreciation tothe Deanship of Scientific Research at King Saud Universityfor funding this Research Group no (RGP-257-1435-1436)
References
[1] K Undheim T Bennecke A R Katritzky C W Rees andE F V Scriven Comprehensive Heterocyclic Chemistry vol 6supplement 93 Elsevier Pergamon Oxford UK 1996
[2] B TaylorModern Medical Chemistry Prentice Hall New YorkNY USA 1994
[3] R Y Levina and F K Velichko ldquoAdvances in the chemistry ofbarbituric acidsrdquo Russian Chemical Reviews vol 29 no 8 pp437ndash459 1960
[4] P Sing and K Paul ldquoA simple synthesis of 5-spirobarbituricacids and transformations of spirocyclopropane barbiturates to5-substitutated barbituratesrdquo Indian Journal of Chemistry B vol45 pp 247ndash251 2006
[5] X Chen K Tanaka and F Yoneda ldquoSimple newmethod for thesynthesis of 5-deaza-10-oxaflavin a potential organic oxidantrdquoChemical and Pharmaceutical Bulletin vol 38 no 2 pp 307ndash311 1990
[6] L R Morgan B S Jursic C L Hooper D M Neumann KThangaraj and B LeBlanc ldquoAnticancer activity for 4 41015840-di-hydroxybenzophenone-2 4-dinitrophenylhydrazone (A-007)analogues and their abilities to interact with lymphoendothelialcell surfacemarkersrdquo Bioorganic ampMedicinal Chemistry Lettersvol 12 no 23 pp 3407ndash3411 2002
[7] G Orzalesi P Gratteri and S Selleri ldquoSynthesis of new 13-dicyclohexyl barbituric acid derivatives with anti-inflammatorypotential activityrdquoEuropean Journal ofMedicinal Chemistry vol25 no 2 pp 197ndash201 1990
[8] D T Puerta and S M Cohen ldquoA bioinorganic perspectiveson matrix metalloproteinase inhibitionrdquo Current Topics inMedicinal Chemistry vol 4 no 15 pp 1551ndash1573 2004
[9] M E Wolff Burgerrsquos Medicinal Chemistry and Drug DiscoveryJohn Wiley amp Sons New York NY USA 1997
Journal of Chemistry 11
[10] E Fischer and J von Mering ldquoUeber eine neue klasse vonschlafmittelnrdquo in Untersuchungen aus Verschiedenen GebietenM Bergmann Ed pp 671ndash679 Springer Berlin Germany1924
[11] W J Doran ldquoBarbituric acid hypnoticsrdquo Medicinal Chemistryvol 4 pp 164ndash167 1959
[12] B Bobranski ldquoProgress in chemistry of barbituric acidrdquoWiad-omosci Chemiczne vol 31 pp 231ndash278 1977
[13] S Senda H Izumi and H Fujimura ldquoUracil derivaives andrelated compounds 6 Derivatives of 5-alkyle-246-trioxoper-hydropyrimidine as antiphlogisticsrdquo Arzneimittel Forschungvol 17 no 12 p 151 1967
[14] N Moussier L Bruche F Viani and M Zanda ldquoFluorinatedbarbituric acid derivatives synthesis and bio-activityrdquo CurrentOrganic Chemistry vol 7 no 11 pp 1071ndash1080 2003
[15] P-G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000
[16] N Ramarathnam T Osawa H Ochi and S Kawakishi ldquoThecontribution of plant food antioxidants to human healthrdquoTrends in Food Science amp Technology vol 6 no 3 pp 75ndash821995
[17] A Barakat A M Al-Majid H J Al-Najjar et al ldquoZwitterionicpyrimidinium adducts as antioxidants with therapeutic poten-tial as nitric oxide scavengerrdquo European Journal of MedicinalChemistry vol 84 pp 146ndash154 2014
[18] P Singh M Kaur and P Verma ldquoDesign synthesis andanticancer activities of hybrids of indole and barbituric acids-Identification of highly promising leadsrdquo Bioorganic andMedic-inal Chemistry Letters vol 19 no 11 pp 3054ndash3058 2009
[19] A M Al-Majid A Barakat H J Al-Najjar Y N Mabkhot HA Ghabbour and H-K Fun ldquoTandem aldol-michael reactionsin aqueous diethylamine medium a greener and efficientapproach to bis-pyrimidine derivativesrdquo International Journalof Molecular Sciences vol 14 no 12 pp 23762ndash23773 2013
[20] A Barakat A M Al-Majid G Lotfy et al ldquoSynthesis and dy-namics studies of barbituric acid derivatives as urease inhibi-torsrdquo Chemistry Central Journal vol 9 no 1 article 63 2015
[21] G M Sheldrick ldquoA short history of SHELXrdquo Acta Crystallo-graphica A vol 64 pp 112ndash122 2008
[22] A L Spek ldquoStructure validation in chemical crystallographyrdquoActaCrystallographica SectionD Biological Crystallography vol65 no 2 pp 148ndash155 2009
[23] F Rahim K Ullah H Ullah et al ldquoTriazinoindole analogs aspotent inhibitors of 120572-glucosidase synthesis biological evalua-tion and molecular docking studiesrdquo Bioorganic Chemistry vol58 pp 81ndash87 2015
[24] F Rahim F Malik H Ullah et al ldquoIsatin based Schiff basesas inhibitors of 120572-glucosidase synthesis characterization invitro evaluation and molecular docking studiesrdquo BioorganicChemistry vol 60 article 1803 pp 42ndash48 2015
[25] A Barakat A M Al-Majid M S Islam et al ldquoMolecular struc-ture investigation and biological evaluation of Michael adductsderived from dimedonerdquo Research on Chemical Intermediatesvol 42 pp 4041ndash4053 2016
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
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Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
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Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
8 Journal of Chemistry
except compounds 4mndash4r and 4y which were found to beinactive
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) (5andash5h) and 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) (5indash5k) rings con-taining compounds were also evaluated for their antioxidantpotential by using DPPH radical scavenging assay Para-bromo-substituted phenyl ring containing compound5k (IC
50= 1276 plusmn 16 120583M) was found as a potent anti-
oxidant agent against the tested standards BHT (IC50
=1288 plusmn 21 120583M) The compound showed similar antioxidantpotential as BHT and showed a significant antioxidant agentagainst N-acetyl cysteine (IC
50= 1076 plusmn 28 120583M) The
gradual and drastic decrease in activity was for compounds5l (IC
50= 1697 plusmn 53 120583M) 5g (IC
50= 2704 plusmn 23 120583M)
5e (IC50
= 289 plusmn 09 120583M) and 5a (IC50
= 4616 plusmn 88 120583M)having para-methyl- para-aldehyde- ortho-chloro- andortho-nitro-substituted phenyl rings attached to the centralbarbiturate moieties Compounds 5bndash5d 5f and 5hndash5j werefound to be inactive
On the basis of the observed antioxidant abilities ofthe above five different series of pyridinium adducts itwas concluded that bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) ring containing com-pounds 4andash4h are the most active one The activity may bedue to the methyl substituted nitrogen atoms of the tetrahy-dropyrimidine rings that facilitate the electronic conjugationwithin the ring However the variation of antioxidantproperties within the most important series is may be due tothe presence of electron donating (-OH (4f) -OCH
3(4d)
and -CH3(4b 4g)) electron withdrawing (-NO
2(4a) and
-CHO (4c)) and halogen (-Br (4e)) substituents on thephenyl ring attached to the bis(6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) moiety Howeverdetailed studies are required to study the mechanism ofaction of these compounds
332 Cytotoxic Activity All synthesized diethyl ammoniumsalts of barbiturates (4andash4z and 5andash5m) were evaluated fortheir in vitro cytotoxicity against PC-3 HeLa MCF-7 cancerand 3T3 normal cell lines All compounds were found to benoncytotoxic against PC-3 HeLaMCF-7 cancer and 3T3 celllines except dichloro-substituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tet-rahydropyrimidin-4-olate ring containing compounds 4ywhich was found to be significantly toxic against all the celllines (PC3 IC
50= 1071plusmn026 120583MHeLa IC
50= 347plusmn03 120583M
MCF-7 IC50= 49 plusmn 001 120583M and 3T3 IC
50= 320 plusmn 02 120583M)
Doxorubicin was used as a standard drug to compare theactivities against PC3 (IC
50= 170 plusmn 029 120583M) HeLa (IC
50=
051 plusmn 015 120583M) and MCF-3 (IC50= 092 plusmn 001 120583M) cancer
cell lines while cycloheximide (IC50
= 026 plusmn 012 120583M) wasused as a standard-in 3T3 cell line (Table 2)
333 120572-Glucosidase Inhibition Activity Synthesized diethylammonium salts of barbiturates 4andash4z and 5andash5m were alsoevaluated for their in vitro 120572-glucosidase inhibition activityin comparison to the standard drug acarbose (IC
50= 841 plusmn
173 120583M) Among dimethyl ammonium salts (4andash4h) nitro-substituted phenyl ring containing 4c (IC
50= 355 plusmn 10 120583M)
and naphthalene ring containing 4h (IC50= 230 plusmn 392 120583M)
were found to be potent inhibitors of 120572-glucosidase enzymeand showed more activity than acarbose (IC
50= 841 plusmn
173 120583M) All other compounds were found to be inactiveAmong diethyl ammonium salts having (2-hydroxy-
44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olate ring (4mndash4z)para-OCH
3- (40 IC
50= 3009 plusmn 65 120583M) para-chloro-
(4p IC50
= 1868 plusmn 081 120583M) para-bromo- (4q IC50
=2148plusmn072 120583M) andmeta-bromo- (4r IC
50= 201plusmn55 120583M)
substituted phenyl ring containing pyridinium adducts werefound to be potent 120572-glucosidase inhibitors and showedmore activity than standard acarbose (IC
50= 841plusmn 173 120583M)
Other members of the series (4m-4n and 4sndash4z) were foundto be inactive The results showed the substitution effects ofvarious functionalities of phenyl ring on the potential activityof tested series The para-bromo-substituted phenyl ringcontaining compound 4q (IC
50= 2148 plusmn 072 120583M) showed
more activity than tested standard However the replacementof bromo group with that of methylNN-dimethyl hydroxyland aldehyde functionalities resulted in complete loss ofactivity as observed in compounds 4n 4t 4v and 4xrespectively The presence of ortho-nitro- ortho-choloro-and para-choloro-substituted phenyl ring also contributedtowards inactivity as observed for compounds 4t 4w and4y
Similarly bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring containing diethyl ammonium pyridinium salts(5andash5h) were also evaluated for their in vitro 120572-glucosidaseenzyme inhibition activity The series nitro-substitutedphenyl ring containing 5f was found to be the only potent120572-glucosidase inhibitor All other compounds (5andash5e and5g-5h) were found to be inactive
Among series of diethylaminium salts of pyridiniumadducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-di-oxo-1234-tetrahydropyrimidin-5-yl)methyl) central moietyattached with substituted phenyl ring the benzaldehydecontaining 5m (IC
50= 4534 plusmn 42 120583M) was found to be the
only potent inhibitor compound in comparison to against thestandard drug acarbose (IC
50= 841plusmn173 120583M)All other com-
poundswere found to be inactiveThe results are summarizedin Table 2
334 Thymidine Phosphorylase Inhibition Activity Five dif-ferent series of diethyamonium salts of barbiturates 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evalu-ated for their in vitro thymidine phosphorylase inhibition7-Deazaxanthine was used as a standard inhibitor (IC
50
= 41 plusmn 146 120583M) Phenol and naphthalene rings sub-stituted (2-hydroxy-44-dimethyl-6-oxocyclohex-1-en-1-yl)-13-dimethyl-26-dioxo-1236-tetrahydropyrimidin-4-olatemoiety containing compounds 4v (IC
50= 576 plusmn 120120583M)
and 4z (IC50
= 558 plusmn 150 120583M) were found to be the potentthymidine phosphorylase inhibitors However a drasticdecrease in activity was observed when para-hydroxygroup on phenyl ring was replaced with methyl (4n IC
50
Journal of Chemistry 9
= 2269 plusmn 150 120583M) chloro (4n IC50
= 242 plusmn 15 120583M)bromo (4q IC
50= 1413 plusmn 15 120583M) and aldehyde (4x
IC50
= 239 plusmn 20 120583M) groups whereas the compounds4o 4s 4t 4w and 4y have para-methoxy- ortho-nitro-ortho-NN-dimethyl amine- ortho- para-dichloro- andortho-ortho-dichloro-substituted phenyl ring respectively
Among series of compounds (5andash5h) having bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring as centralmoiety electron donating hydroxy- andmethoxy-substitutedphenyl ring containing compounds 5h (IC
50= 569plusmn16 120583M)
and 5d (IC50
= 605 plusmn 187 120583M) respectively were foundas potent inhibitors of thymidine phosphorylase enzymeagainst the tested standard 7-deazaxanthine which was(IC50
= 41 plusmn 146 120583M) used as standard Again a drasticdecrease in activity was observed for compounds 5b (IC
50
= 2169 plusmn 05 120583M) 5c (IC50
= 1614 plusmn 15 120583M) and 5g(IC50= 1866 plusmn 12 120583M) havingmeta-methyl- ortho-methyl-
and para-aldehyde-substituted benzene ring respectivelyinstead of para-hydroxyl phenyl ring The replacement ofortho-methyl with chloro-group makes the compound 5ecompletely inactive Similarly the replacement of electrondonating para-hydroxy-group of phenyl ring with electronwithdrawing nitro-functionality also contributed towardsinactivity of compound as observed for 5f Phenol substi-tuted bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ringcontaining 5h (IC
50= 569 plusmn 16 120583M) was found to be the
potent thymidine phosphorylase inhibitorsAll members of the series of diethylaminium salts of pyri-
dinium adducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) central moi-ety attached with substituted phenyl ring were found to beweak inhibitors of thymidine phosphorylase enzyme againstthe tested standard compound 7-deazaxanthine (IC
50= 41 plusmn
146 120583M) with IC50
values 318 plusmn 25 120583M 1664 plusmn 12 120583M1781 plusmn 21 120583M and 1471 plusmn 123 120583M for 5j 5k 5l and 5mrespectively whereas para-chloro-substituted phenyl ringcontaining compound 5i was found to be inactiveThe resultsare summarized in Table 1
335 120573-Glucuronidase Inhibition Activity Compounds 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evaluatedfor their in vitro 120573-glucuronidase inhibitory activity by usingD-saccharic acid 14-lactone as standard inhibitor (IC
50=
4575 plusmn 216 120583M) Compounds 4a (IC50
= 2048 plusmn 601 120583M)4c (IC
50= 1721 plusmn 21 120583M) 4e (IC
50= 3389 plusmn 642 120583M) 4o
(IC50
= 2621 plusmn 435 120583M) 4q (IC50
= 2922 plusmn 103 120583M) 4r(IC50= 2621plusmn44 120583M) 4w (IC
50= 1435plusmn124 120583M) 5f (IC
50
= 185 plusmn 25 120583M) 5i (IC50
= 2236 plusmn 64 120583M) and 5l (IC50
=3875plusmn70 120583M) showed veryweak120573-glucuronidase inhibitionpotential whereas pyridinium adducts 4andash4c 4e 4gndash4j 4l4o 4s-4t 4w 4y 5a 5e-5f and 5i were found to be inactive(Table 2)
34 Molecular Docking Studies
341 Binding Interactions of 120572-Glucosidase Enzyme To de-fine inhibitors interactions and their specificity the dockingstudy of the most active compound 4p against 120572-glucosidase
Figure 4 Three-dimensional representation of compound 4p withtarget macromolecule 120572-glucosidase
Figure 5 Three-dimensional representation of compound 4v withtarget macromolecule thymidine phosphorylase
was carried out [23ndash25] Ten different conformations ofprotein-ligand interactions were generated and the topranked pose was selected to explore the binding interactionsIn our docking study we have observed two interactions withthe active site residues of 120572-glucosidase The polar imidazolering of His 239 showed hydrogen bond through its H atomwith the lone pair of oxygen of the pyrimidinedionemoiety ofthe compound whereas Arg 312 formed hydrogen bond withthe OH group of the same moiety of compound (Figure 4)The presence of polar and electron rich center in the formof pyrimidinedione and chlorobenzene groups of the com-pound was observed here as key factors for the interactionbetween compound andprotein Anumber of hydrogen bonddonor and acceptor pairs with their suitable orientationswere observed in the interaction pose Furthermore severalhydrophobic interactions between the nonpolar active siteresidues and the compound 4p were also observed
342 Binding Interactions of Thymidine PhosphorylaseEnzyme To explore the binding modes of these newly syn-thesized compounds in the active site of thymidine phos-phorylase the most active compound 4v was docked againstthis enzyme The docking results showed that compound4v well accommodated in the active site of thymidinephosphorylase (Figure 5) The active site residue Ala 206established a hydrogen bond through its lone pair of
10 Journal of Chemistry
Figure 6 Three-dimensional representation of compound 4w withtarget enzyme 120573-glucuronidase
oxygen with the active hydrogen of hydroxyl moiety ofthe pyrimidinedione group of the compound Howeverthe substituted phenolic group and the other electron richcenters of the pyrimidinedione showed poor behaviorregarding interaction which may depend on the nature andorientation of the surrounding active site residues Moreoverseveral weak hydrophobic interactions between the activesite residues and the compound were also observed
343 Binding Interaction of 120573-Glucuronidase Enzyme Thepredicted bindingmode ofmost active compound 4w against120573-glucuronidase showed that a hydrogen bond is formedbetween the lone pair of oxygen of cyclohexanedione compo-nent of the compound and imidazole hydrogen of the impor-tant active site residue His 509 An arene-arene and arene-cation interactionwas also observed between the pi electroniccloud of dichlorobenzene ring of compound and imidazolering of His 509 (Figure 6) Besides hydrogen bonding andarene-arene interactions several hydrophobic interactionsbetween compound 4w and the active site residues werealso observed The pyrimidinedione moiety in this case wasobserved to be passive regarding interactions
4 Conclusions
In conclusion barbiturate salts were synthesized by one-potmulticomponent reactions via tandem AldolMichael addi-tion reactions between barbituric acid and dimedone withaldehydes mediated by aqueous diethylamine The molec-ular structures of 5a 5d and 5f were deduced by single-crystal X-ray diffraction techniques All of the synthesizedcompounds were evaluated for their antioxidant potentialin vitro by using DPPH radical scavenging assay as well asenzyme inhibition activities against120572-glucosidase thymidinephosphorylase and 120573-glucuronidase enzymes Compounds4andash4g (IC
50= 1018 plusmn 08ndash1244 plusmn 44 120583M) were found to
be the potent antioxidants as compared to the standardsBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) Compound 4p (IC50= 1868 plusmn 81 120583M) was
found to be a potent 120572-glucosidase inhibitor Compound 4v(IC50
= 87 plusmn 12 120583M) was found to be a potent thymidinephosphorylase inhibitor Promising results indicate that thesecompounds need to be investigated as antioxidant agentsto treat various associated oxidative disorders Similarlytheir enzyme inhibitory potential can be reported on drugdiscovery program
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Assem Barakat Hazem A Ghabbour Abdullah MohammedAl-Majid Qurat-ul-ain Rehan Imad Kulsoom Javaid NimraNaveed Shaikh Sammer Yousuf M Iqbal Choudhary andAbdul Wadood contributed equally to this work
Acknowledgments
The authors would like to extend their sincere appreciation tothe Deanship of Scientific Research at King Saud Universityfor funding this Research Group no (RGP-257-1435-1436)
References
[1] K Undheim T Bennecke A R Katritzky C W Rees andE F V Scriven Comprehensive Heterocyclic Chemistry vol 6supplement 93 Elsevier Pergamon Oxford UK 1996
[2] B TaylorModern Medical Chemistry Prentice Hall New YorkNY USA 1994
[3] R Y Levina and F K Velichko ldquoAdvances in the chemistry ofbarbituric acidsrdquo Russian Chemical Reviews vol 29 no 8 pp437ndash459 1960
[4] P Sing and K Paul ldquoA simple synthesis of 5-spirobarbituricacids and transformations of spirocyclopropane barbiturates to5-substitutated barbituratesrdquo Indian Journal of Chemistry B vol45 pp 247ndash251 2006
[5] X Chen K Tanaka and F Yoneda ldquoSimple newmethod for thesynthesis of 5-deaza-10-oxaflavin a potential organic oxidantrdquoChemical and Pharmaceutical Bulletin vol 38 no 2 pp 307ndash311 1990
[6] L R Morgan B S Jursic C L Hooper D M Neumann KThangaraj and B LeBlanc ldquoAnticancer activity for 4 41015840-di-hydroxybenzophenone-2 4-dinitrophenylhydrazone (A-007)analogues and their abilities to interact with lymphoendothelialcell surfacemarkersrdquo Bioorganic ampMedicinal Chemistry Lettersvol 12 no 23 pp 3407ndash3411 2002
[7] G Orzalesi P Gratteri and S Selleri ldquoSynthesis of new 13-dicyclohexyl barbituric acid derivatives with anti-inflammatorypotential activityrdquoEuropean Journal ofMedicinal Chemistry vol25 no 2 pp 197ndash201 1990
[8] D T Puerta and S M Cohen ldquoA bioinorganic perspectiveson matrix metalloproteinase inhibitionrdquo Current Topics inMedicinal Chemistry vol 4 no 15 pp 1551ndash1573 2004
[9] M E Wolff Burgerrsquos Medicinal Chemistry and Drug DiscoveryJohn Wiley amp Sons New York NY USA 1997
Journal of Chemistry 11
[10] E Fischer and J von Mering ldquoUeber eine neue klasse vonschlafmittelnrdquo in Untersuchungen aus Verschiedenen GebietenM Bergmann Ed pp 671ndash679 Springer Berlin Germany1924
[11] W J Doran ldquoBarbituric acid hypnoticsrdquo Medicinal Chemistryvol 4 pp 164ndash167 1959
[12] B Bobranski ldquoProgress in chemistry of barbituric acidrdquoWiad-omosci Chemiczne vol 31 pp 231ndash278 1977
[13] S Senda H Izumi and H Fujimura ldquoUracil derivaives andrelated compounds 6 Derivatives of 5-alkyle-246-trioxoper-hydropyrimidine as antiphlogisticsrdquo Arzneimittel Forschungvol 17 no 12 p 151 1967
[14] N Moussier L Bruche F Viani and M Zanda ldquoFluorinatedbarbituric acid derivatives synthesis and bio-activityrdquo CurrentOrganic Chemistry vol 7 no 11 pp 1071ndash1080 2003
[15] P-G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000
[16] N Ramarathnam T Osawa H Ochi and S Kawakishi ldquoThecontribution of plant food antioxidants to human healthrdquoTrends in Food Science amp Technology vol 6 no 3 pp 75ndash821995
[17] A Barakat A M Al-Majid H J Al-Najjar et al ldquoZwitterionicpyrimidinium adducts as antioxidants with therapeutic poten-tial as nitric oxide scavengerrdquo European Journal of MedicinalChemistry vol 84 pp 146ndash154 2014
[18] P Singh M Kaur and P Verma ldquoDesign synthesis andanticancer activities of hybrids of indole and barbituric acids-Identification of highly promising leadsrdquo Bioorganic andMedic-inal Chemistry Letters vol 19 no 11 pp 3054ndash3058 2009
[19] A M Al-Majid A Barakat H J Al-Najjar Y N Mabkhot HA Ghabbour and H-K Fun ldquoTandem aldol-michael reactionsin aqueous diethylamine medium a greener and efficientapproach to bis-pyrimidine derivativesrdquo International Journalof Molecular Sciences vol 14 no 12 pp 23762ndash23773 2013
[20] A Barakat A M Al-Majid G Lotfy et al ldquoSynthesis and dy-namics studies of barbituric acid derivatives as urease inhibi-torsrdquo Chemistry Central Journal vol 9 no 1 article 63 2015
[21] G M Sheldrick ldquoA short history of SHELXrdquo Acta Crystallo-graphica A vol 64 pp 112ndash122 2008
[22] A L Spek ldquoStructure validation in chemical crystallographyrdquoActaCrystallographica SectionD Biological Crystallography vol65 no 2 pp 148ndash155 2009
[23] F Rahim K Ullah H Ullah et al ldquoTriazinoindole analogs aspotent inhibitors of 120572-glucosidase synthesis biological evalua-tion and molecular docking studiesrdquo Bioorganic Chemistry vol58 pp 81ndash87 2015
[24] F Rahim F Malik H Ullah et al ldquoIsatin based Schiff basesas inhibitors of 120572-glucosidase synthesis characterization invitro evaluation and molecular docking studiesrdquo BioorganicChemistry vol 60 article 1803 pp 42ndash48 2015
[25] A Barakat A M Al-Majid M S Islam et al ldquoMolecular struc-ture investigation and biological evaluation of Michael adductsderived from dimedonerdquo Research on Chemical Intermediatesvol 42 pp 4041ndash4053 2016
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Chemistry 9
= 2269 plusmn 150 120583M) chloro (4n IC50
= 242 plusmn 15 120583M)bromo (4q IC
50= 1413 plusmn 15 120583M) and aldehyde (4x
IC50
= 239 plusmn 20 120583M) groups whereas the compounds4o 4s 4t 4w and 4y have para-methoxy- ortho-nitro-ortho-NN-dimethyl amine- ortho- para-dichloro- andortho-ortho-dichloro-substituted phenyl ring respectively
Among series of compounds (5andash5h) having bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ring as centralmoiety electron donating hydroxy- andmethoxy-substitutedphenyl ring containing compounds 5h (IC
50= 569plusmn16 120583M)
and 5d (IC50
= 605 plusmn 187 120583M) respectively were foundas potent inhibitors of thymidine phosphorylase enzymeagainst the tested standard 7-deazaxanthine which was(IC50
= 41 plusmn 146 120583M) used as standard Again a drasticdecrease in activity was observed for compounds 5b (IC
50
= 2169 plusmn 05 120583M) 5c (IC50
= 1614 plusmn 15 120583M) and 5g(IC50= 1866 plusmn 12 120583M) havingmeta-methyl- ortho-methyl-
and para-aldehyde-substituted benzene ring respectivelyinstead of para-hydroxyl phenyl ring The replacement ofortho-methyl with chloro-group makes the compound 5ecompletely inactive Similarly the replacement of electrondonating para-hydroxy-group of phenyl ring with electronwithdrawing nitro-functionality also contributed towardsinactivity of compound as observed for 5f Phenol substi-tuted bis(2-hydroxy-44-dimethyl-6-oxocyclohex-1-ene) ringcontaining 5h (IC
50= 569 plusmn 16 120583M) was found to be the
potent thymidine phosphorylase inhibitorsAll members of the series of diethylaminium salts of pyri-
dinium adducts (5indash5m) having 4-((6-hydroxy-13-dimethyl-24-dioxo-1234-tetrahydropyrimidin-5-yl) (6-hydroxy-24-dioxo-1234-tetrahydropyrimidin-5-yl)methyl) central moi-ety attached with substituted phenyl ring were found to beweak inhibitors of thymidine phosphorylase enzyme againstthe tested standard compound 7-deazaxanthine (IC
50= 41 plusmn
146 120583M) with IC50
values 318 plusmn 25 120583M 1664 plusmn 12 120583M1781 plusmn 21 120583M and 1471 plusmn 123 120583M for 5j 5k 5l and 5mrespectively whereas para-chloro-substituted phenyl ringcontaining compound 5i was found to be inactiveThe resultsare summarized in Table 1
335 120573-Glucuronidase Inhibition Activity Compounds 4andash4g 4indash4k 4mndash4z 5andash5h and 5indash5m were also evaluatedfor their in vitro 120573-glucuronidase inhibitory activity by usingD-saccharic acid 14-lactone as standard inhibitor (IC
50=
4575 plusmn 216 120583M) Compounds 4a (IC50
= 2048 plusmn 601 120583M)4c (IC
50= 1721 plusmn 21 120583M) 4e (IC
50= 3389 plusmn 642 120583M) 4o
(IC50
= 2621 plusmn 435 120583M) 4q (IC50
= 2922 plusmn 103 120583M) 4r(IC50= 2621plusmn44 120583M) 4w (IC
50= 1435plusmn124 120583M) 5f (IC
50
= 185 plusmn 25 120583M) 5i (IC50
= 2236 plusmn 64 120583M) and 5l (IC50
=3875plusmn70 120583M) showed veryweak120573-glucuronidase inhibitionpotential whereas pyridinium adducts 4andash4c 4e 4gndash4j 4l4o 4s-4t 4w 4y 5a 5e-5f and 5i were found to be inactive(Table 2)
34 Molecular Docking Studies
341 Binding Interactions of 120572-Glucosidase Enzyme To de-fine inhibitors interactions and their specificity the dockingstudy of the most active compound 4p against 120572-glucosidase
Figure 4 Three-dimensional representation of compound 4p withtarget macromolecule 120572-glucosidase
Figure 5 Three-dimensional representation of compound 4v withtarget macromolecule thymidine phosphorylase
was carried out [23ndash25] Ten different conformations ofprotein-ligand interactions were generated and the topranked pose was selected to explore the binding interactionsIn our docking study we have observed two interactions withthe active site residues of 120572-glucosidase The polar imidazolering of His 239 showed hydrogen bond through its H atomwith the lone pair of oxygen of the pyrimidinedionemoiety ofthe compound whereas Arg 312 formed hydrogen bond withthe OH group of the same moiety of compound (Figure 4)The presence of polar and electron rich center in the formof pyrimidinedione and chlorobenzene groups of the com-pound was observed here as key factors for the interactionbetween compound andprotein Anumber of hydrogen bonddonor and acceptor pairs with their suitable orientationswere observed in the interaction pose Furthermore severalhydrophobic interactions between the nonpolar active siteresidues and the compound 4p were also observed
342 Binding Interactions of Thymidine PhosphorylaseEnzyme To explore the binding modes of these newly syn-thesized compounds in the active site of thymidine phos-phorylase the most active compound 4v was docked againstthis enzyme The docking results showed that compound4v well accommodated in the active site of thymidinephosphorylase (Figure 5) The active site residue Ala 206established a hydrogen bond through its lone pair of
10 Journal of Chemistry
Figure 6 Three-dimensional representation of compound 4w withtarget enzyme 120573-glucuronidase
oxygen with the active hydrogen of hydroxyl moiety ofthe pyrimidinedione group of the compound Howeverthe substituted phenolic group and the other electron richcenters of the pyrimidinedione showed poor behaviorregarding interaction which may depend on the nature andorientation of the surrounding active site residues Moreoverseveral weak hydrophobic interactions between the activesite residues and the compound were also observed
343 Binding Interaction of 120573-Glucuronidase Enzyme Thepredicted bindingmode ofmost active compound 4w against120573-glucuronidase showed that a hydrogen bond is formedbetween the lone pair of oxygen of cyclohexanedione compo-nent of the compound and imidazole hydrogen of the impor-tant active site residue His 509 An arene-arene and arene-cation interactionwas also observed between the pi electroniccloud of dichlorobenzene ring of compound and imidazolering of His 509 (Figure 6) Besides hydrogen bonding andarene-arene interactions several hydrophobic interactionsbetween compound 4w and the active site residues werealso observed The pyrimidinedione moiety in this case wasobserved to be passive regarding interactions
4 Conclusions
In conclusion barbiturate salts were synthesized by one-potmulticomponent reactions via tandem AldolMichael addi-tion reactions between barbituric acid and dimedone withaldehydes mediated by aqueous diethylamine The molec-ular structures of 5a 5d and 5f were deduced by single-crystal X-ray diffraction techniques All of the synthesizedcompounds were evaluated for their antioxidant potentialin vitro by using DPPH radical scavenging assay as well asenzyme inhibition activities against120572-glucosidase thymidinephosphorylase and 120573-glucuronidase enzymes Compounds4andash4g (IC
50= 1018 plusmn 08ndash1244 plusmn 44 120583M) were found to
be the potent antioxidants as compared to the standardsBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) Compound 4p (IC50= 1868 plusmn 81 120583M) was
found to be a potent 120572-glucosidase inhibitor Compound 4v(IC50
= 87 plusmn 12 120583M) was found to be a potent thymidinephosphorylase inhibitor Promising results indicate that thesecompounds need to be investigated as antioxidant agentsto treat various associated oxidative disorders Similarlytheir enzyme inhibitory potential can be reported on drugdiscovery program
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Assem Barakat Hazem A Ghabbour Abdullah MohammedAl-Majid Qurat-ul-ain Rehan Imad Kulsoom Javaid NimraNaveed Shaikh Sammer Yousuf M Iqbal Choudhary andAbdul Wadood contributed equally to this work
Acknowledgments
The authors would like to extend their sincere appreciation tothe Deanship of Scientific Research at King Saud Universityfor funding this Research Group no (RGP-257-1435-1436)
References
[1] K Undheim T Bennecke A R Katritzky C W Rees andE F V Scriven Comprehensive Heterocyclic Chemistry vol 6supplement 93 Elsevier Pergamon Oxford UK 1996
[2] B TaylorModern Medical Chemistry Prentice Hall New YorkNY USA 1994
[3] R Y Levina and F K Velichko ldquoAdvances in the chemistry ofbarbituric acidsrdquo Russian Chemical Reviews vol 29 no 8 pp437ndash459 1960
[4] P Sing and K Paul ldquoA simple synthesis of 5-spirobarbituricacids and transformations of spirocyclopropane barbiturates to5-substitutated barbituratesrdquo Indian Journal of Chemistry B vol45 pp 247ndash251 2006
[5] X Chen K Tanaka and F Yoneda ldquoSimple newmethod for thesynthesis of 5-deaza-10-oxaflavin a potential organic oxidantrdquoChemical and Pharmaceutical Bulletin vol 38 no 2 pp 307ndash311 1990
[6] L R Morgan B S Jursic C L Hooper D M Neumann KThangaraj and B LeBlanc ldquoAnticancer activity for 4 41015840-di-hydroxybenzophenone-2 4-dinitrophenylhydrazone (A-007)analogues and their abilities to interact with lymphoendothelialcell surfacemarkersrdquo Bioorganic ampMedicinal Chemistry Lettersvol 12 no 23 pp 3407ndash3411 2002
[7] G Orzalesi P Gratteri and S Selleri ldquoSynthesis of new 13-dicyclohexyl barbituric acid derivatives with anti-inflammatorypotential activityrdquoEuropean Journal ofMedicinal Chemistry vol25 no 2 pp 197ndash201 1990
[8] D T Puerta and S M Cohen ldquoA bioinorganic perspectiveson matrix metalloproteinase inhibitionrdquo Current Topics inMedicinal Chemistry vol 4 no 15 pp 1551ndash1573 2004
[9] M E Wolff Burgerrsquos Medicinal Chemistry and Drug DiscoveryJohn Wiley amp Sons New York NY USA 1997
Journal of Chemistry 11
[10] E Fischer and J von Mering ldquoUeber eine neue klasse vonschlafmittelnrdquo in Untersuchungen aus Verschiedenen GebietenM Bergmann Ed pp 671ndash679 Springer Berlin Germany1924
[11] W J Doran ldquoBarbituric acid hypnoticsrdquo Medicinal Chemistryvol 4 pp 164ndash167 1959
[12] B Bobranski ldquoProgress in chemistry of barbituric acidrdquoWiad-omosci Chemiczne vol 31 pp 231ndash278 1977
[13] S Senda H Izumi and H Fujimura ldquoUracil derivaives andrelated compounds 6 Derivatives of 5-alkyle-246-trioxoper-hydropyrimidine as antiphlogisticsrdquo Arzneimittel Forschungvol 17 no 12 p 151 1967
[14] N Moussier L Bruche F Viani and M Zanda ldquoFluorinatedbarbituric acid derivatives synthesis and bio-activityrdquo CurrentOrganic Chemistry vol 7 no 11 pp 1071ndash1080 2003
[15] P-G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000
[16] N Ramarathnam T Osawa H Ochi and S Kawakishi ldquoThecontribution of plant food antioxidants to human healthrdquoTrends in Food Science amp Technology vol 6 no 3 pp 75ndash821995
[17] A Barakat A M Al-Majid H J Al-Najjar et al ldquoZwitterionicpyrimidinium adducts as antioxidants with therapeutic poten-tial as nitric oxide scavengerrdquo European Journal of MedicinalChemistry vol 84 pp 146ndash154 2014
[18] P Singh M Kaur and P Verma ldquoDesign synthesis andanticancer activities of hybrids of indole and barbituric acids-Identification of highly promising leadsrdquo Bioorganic andMedic-inal Chemistry Letters vol 19 no 11 pp 3054ndash3058 2009
[19] A M Al-Majid A Barakat H J Al-Najjar Y N Mabkhot HA Ghabbour and H-K Fun ldquoTandem aldol-michael reactionsin aqueous diethylamine medium a greener and efficientapproach to bis-pyrimidine derivativesrdquo International Journalof Molecular Sciences vol 14 no 12 pp 23762ndash23773 2013
[20] A Barakat A M Al-Majid G Lotfy et al ldquoSynthesis and dy-namics studies of barbituric acid derivatives as urease inhibi-torsrdquo Chemistry Central Journal vol 9 no 1 article 63 2015
[21] G M Sheldrick ldquoA short history of SHELXrdquo Acta Crystallo-graphica A vol 64 pp 112ndash122 2008
[22] A L Spek ldquoStructure validation in chemical crystallographyrdquoActaCrystallographica SectionD Biological Crystallography vol65 no 2 pp 148ndash155 2009
[23] F Rahim K Ullah H Ullah et al ldquoTriazinoindole analogs aspotent inhibitors of 120572-glucosidase synthesis biological evalua-tion and molecular docking studiesrdquo Bioorganic Chemistry vol58 pp 81ndash87 2015
[24] F Rahim F Malik H Ullah et al ldquoIsatin based Schiff basesas inhibitors of 120572-glucosidase synthesis characterization invitro evaluation and molecular docking studiesrdquo BioorganicChemistry vol 60 article 1803 pp 42ndash48 2015
[25] A Barakat A M Al-Majid M S Islam et al ldquoMolecular struc-ture investigation and biological evaluation of Michael adductsderived from dimedonerdquo Research on Chemical Intermediatesvol 42 pp 4041ndash4053 2016
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
10 Journal of Chemistry
Figure 6 Three-dimensional representation of compound 4w withtarget enzyme 120573-glucuronidase
oxygen with the active hydrogen of hydroxyl moiety ofthe pyrimidinedione group of the compound Howeverthe substituted phenolic group and the other electron richcenters of the pyrimidinedione showed poor behaviorregarding interaction which may depend on the nature andorientation of the surrounding active site residues Moreoverseveral weak hydrophobic interactions between the activesite residues and the compound were also observed
343 Binding Interaction of 120573-Glucuronidase Enzyme Thepredicted bindingmode ofmost active compound 4w against120573-glucuronidase showed that a hydrogen bond is formedbetween the lone pair of oxygen of cyclohexanedione compo-nent of the compound and imidazole hydrogen of the impor-tant active site residue His 509 An arene-arene and arene-cation interactionwas also observed between the pi electroniccloud of dichlorobenzene ring of compound and imidazolering of His 509 (Figure 6) Besides hydrogen bonding andarene-arene interactions several hydrophobic interactionsbetween compound 4w and the active site residues werealso observed The pyrimidinedione moiety in this case wasobserved to be passive regarding interactions
4 Conclusions
In conclusion barbiturate salts were synthesized by one-potmulticomponent reactions via tandem AldolMichael addi-tion reactions between barbituric acid and dimedone withaldehydes mediated by aqueous diethylamine The molec-ular structures of 5a 5d and 5f were deduced by single-crystal X-ray diffraction techniques All of the synthesizedcompounds were evaluated for their antioxidant potentialin vitro by using DPPH radical scavenging assay as well asenzyme inhibition activities against120572-glucosidase thymidinephosphorylase and 120573-glucuronidase enzymes Compounds4andash4g (IC
50= 1018 plusmn 08ndash1244 plusmn 44 120583M) were found to
be the potent antioxidants as compared to the standardsBHT (IC
50= 1288 plusmn 21 120583M) and N-acetylcysteine (IC
50=
1076 plusmn 28 120583M) Compound 4p (IC50= 1868 plusmn 81 120583M) was
found to be a potent 120572-glucosidase inhibitor Compound 4v(IC50
= 87 plusmn 12 120583M) was found to be a potent thymidinephosphorylase inhibitor Promising results indicate that thesecompounds need to be investigated as antioxidant agentsto treat various associated oxidative disorders Similarlytheir enzyme inhibitory potential can be reported on drugdiscovery program
Competing Interests
The authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Assem Barakat Hazem A Ghabbour Abdullah MohammedAl-Majid Qurat-ul-ain Rehan Imad Kulsoom Javaid NimraNaveed Shaikh Sammer Yousuf M Iqbal Choudhary andAbdul Wadood contributed equally to this work
Acknowledgments
The authors would like to extend their sincere appreciation tothe Deanship of Scientific Research at King Saud Universityfor funding this Research Group no (RGP-257-1435-1436)
References
[1] K Undheim T Bennecke A R Katritzky C W Rees andE F V Scriven Comprehensive Heterocyclic Chemistry vol 6supplement 93 Elsevier Pergamon Oxford UK 1996
[2] B TaylorModern Medical Chemistry Prentice Hall New YorkNY USA 1994
[3] R Y Levina and F K Velichko ldquoAdvances in the chemistry ofbarbituric acidsrdquo Russian Chemical Reviews vol 29 no 8 pp437ndash459 1960
[4] P Sing and K Paul ldquoA simple synthesis of 5-spirobarbituricacids and transformations of spirocyclopropane barbiturates to5-substitutated barbituratesrdquo Indian Journal of Chemistry B vol45 pp 247ndash251 2006
[5] X Chen K Tanaka and F Yoneda ldquoSimple newmethod for thesynthesis of 5-deaza-10-oxaflavin a potential organic oxidantrdquoChemical and Pharmaceutical Bulletin vol 38 no 2 pp 307ndash311 1990
[6] L R Morgan B S Jursic C L Hooper D M Neumann KThangaraj and B LeBlanc ldquoAnticancer activity for 4 41015840-di-hydroxybenzophenone-2 4-dinitrophenylhydrazone (A-007)analogues and their abilities to interact with lymphoendothelialcell surfacemarkersrdquo Bioorganic ampMedicinal Chemistry Lettersvol 12 no 23 pp 3407ndash3411 2002
[7] G Orzalesi P Gratteri and S Selleri ldquoSynthesis of new 13-dicyclohexyl barbituric acid derivatives with anti-inflammatorypotential activityrdquoEuropean Journal ofMedicinal Chemistry vol25 no 2 pp 197ndash201 1990
[8] D T Puerta and S M Cohen ldquoA bioinorganic perspectiveson matrix metalloproteinase inhibitionrdquo Current Topics inMedicinal Chemistry vol 4 no 15 pp 1551ndash1573 2004
[9] M E Wolff Burgerrsquos Medicinal Chemistry and Drug DiscoveryJohn Wiley amp Sons New York NY USA 1997
Journal of Chemistry 11
[10] E Fischer and J von Mering ldquoUeber eine neue klasse vonschlafmittelnrdquo in Untersuchungen aus Verschiedenen GebietenM Bergmann Ed pp 671ndash679 Springer Berlin Germany1924
[11] W J Doran ldquoBarbituric acid hypnoticsrdquo Medicinal Chemistryvol 4 pp 164ndash167 1959
[12] B Bobranski ldquoProgress in chemistry of barbituric acidrdquoWiad-omosci Chemiczne vol 31 pp 231ndash278 1977
[13] S Senda H Izumi and H Fujimura ldquoUracil derivaives andrelated compounds 6 Derivatives of 5-alkyle-246-trioxoper-hydropyrimidine as antiphlogisticsrdquo Arzneimittel Forschungvol 17 no 12 p 151 1967
[14] N Moussier L Bruche F Viani and M Zanda ldquoFluorinatedbarbituric acid derivatives synthesis and bio-activityrdquo CurrentOrganic Chemistry vol 7 no 11 pp 1071ndash1080 2003
[15] P-G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000
[16] N Ramarathnam T Osawa H Ochi and S Kawakishi ldquoThecontribution of plant food antioxidants to human healthrdquoTrends in Food Science amp Technology vol 6 no 3 pp 75ndash821995
[17] A Barakat A M Al-Majid H J Al-Najjar et al ldquoZwitterionicpyrimidinium adducts as antioxidants with therapeutic poten-tial as nitric oxide scavengerrdquo European Journal of MedicinalChemistry vol 84 pp 146ndash154 2014
[18] P Singh M Kaur and P Verma ldquoDesign synthesis andanticancer activities of hybrids of indole and barbituric acids-Identification of highly promising leadsrdquo Bioorganic andMedic-inal Chemistry Letters vol 19 no 11 pp 3054ndash3058 2009
[19] A M Al-Majid A Barakat H J Al-Najjar Y N Mabkhot HA Ghabbour and H-K Fun ldquoTandem aldol-michael reactionsin aqueous diethylamine medium a greener and efficientapproach to bis-pyrimidine derivativesrdquo International Journalof Molecular Sciences vol 14 no 12 pp 23762ndash23773 2013
[20] A Barakat A M Al-Majid G Lotfy et al ldquoSynthesis and dy-namics studies of barbituric acid derivatives as urease inhibi-torsrdquo Chemistry Central Journal vol 9 no 1 article 63 2015
[21] G M Sheldrick ldquoA short history of SHELXrdquo Acta Crystallo-graphica A vol 64 pp 112ndash122 2008
[22] A L Spek ldquoStructure validation in chemical crystallographyrdquoActaCrystallographica SectionD Biological Crystallography vol65 no 2 pp 148ndash155 2009
[23] F Rahim K Ullah H Ullah et al ldquoTriazinoindole analogs aspotent inhibitors of 120572-glucosidase synthesis biological evalua-tion and molecular docking studiesrdquo Bioorganic Chemistry vol58 pp 81ndash87 2015
[24] F Rahim F Malik H Ullah et al ldquoIsatin based Schiff basesas inhibitors of 120572-glucosidase synthesis characterization invitro evaluation and molecular docking studiesrdquo BioorganicChemistry vol 60 article 1803 pp 42ndash48 2015
[25] A Barakat A M Al-Majid M S Islam et al ldquoMolecular struc-ture investigation and biological evaluation of Michael adductsderived from dimedonerdquo Research on Chemical Intermediatesvol 42 pp 4041ndash4053 2016
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Chemistry 11
[10] E Fischer and J von Mering ldquoUeber eine neue klasse vonschlafmittelnrdquo in Untersuchungen aus Verschiedenen GebietenM Bergmann Ed pp 671ndash679 Springer Berlin Germany1924
[11] W J Doran ldquoBarbituric acid hypnoticsrdquo Medicinal Chemistryvol 4 pp 164ndash167 1959
[12] B Bobranski ldquoProgress in chemistry of barbituric acidrdquoWiad-omosci Chemiczne vol 31 pp 231ndash278 1977
[13] S Senda H Izumi and H Fujimura ldquoUracil derivaives andrelated compounds 6 Derivatives of 5-alkyle-246-trioxoper-hydropyrimidine as antiphlogisticsrdquo Arzneimittel Forschungvol 17 no 12 p 151 1967
[14] N Moussier L Bruche F Viani and M Zanda ldquoFluorinatedbarbituric acid derivatives synthesis and bio-activityrdquo CurrentOrganic Chemistry vol 7 no 11 pp 1071ndash1080 2003
[15] P-G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000
[16] N Ramarathnam T Osawa H Ochi and S Kawakishi ldquoThecontribution of plant food antioxidants to human healthrdquoTrends in Food Science amp Technology vol 6 no 3 pp 75ndash821995
[17] A Barakat A M Al-Majid H J Al-Najjar et al ldquoZwitterionicpyrimidinium adducts as antioxidants with therapeutic poten-tial as nitric oxide scavengerrdquo European Journal of MedicinalChemistry vol 84 pp 146ndash154 2014
[18] P Singh M Kaur and P Verma ldquoDesign synthesis andanticancer activities of hybrids of indole and barbituric acids-Identification of highly promising leadsrdquo Bioorganic andMedic-inal Chemistry Letters vol 19 no 11 pp 3054ndash3058 2009
[19] A M Al-Majid A Barakat H J Al-Najjar Y N Mabkhot HA Ghabbour and H-K Fun ldquoTandem aldol-michael reactionsin aqueous diethylamine medium a greener and efficientapproach to bis-pyrimidine derivativesrdquo International Journalof Molecular Sciences vol 14 no 12 pp 23762ndash23773 2013
[20] A Barakat A M Al-Majid G Lotfy et al ldquoSynthesis and dy-namics studies of barbituric acid derivatives as urease inhibi-torsrdquo Chemistry Central Journal vol 9 no 1 article 63 2015
[21] G M Sheldrick ldquoA short history of SHELXrdquo Acta Crystallo-graphica A vol 64 pp 112ndash122 2008
[22] A L Spek ldquoStructure validation in chemical crystallographyrdquoActaCrystallographica SectionD Biological Crystallography vol65 no 2 pp 148ndash155 2009
[23] F Rahim K Ullah H Ullah et al ldquoTriazinoindole analogs aspotent inhibitors of 120572-glucosidase synthesis biological evalua-tion and molecular docking studiesrdquo Bioorganic Chemistry vol58 pp 81ndash87 2015
[24] F Rahim F Malik H Ullah et al ldquoIsatin based Schiff basesas inhibitors of 120572-glucosidase synthesis characterization invitro evaluation and molecular docking studiesrdquo BioorganicChemistry vol 60 article 1803 pp 42ndash48 2015
[25] A Barakat A M Al-Majid M S Islam et al ldquoMolecular struc-ture investigation and biological evaluation of Michael adductsderived from dimedonerdquo Research on Chemical Intermediatesvol 42 pp 4041ndash4053 2016
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
