In Silico Molecular Docking and In Vitro Antidiabetic ... · 2 BioMedResearchInternational OH N NN HO OH R NN N N R N NNO O O OH N N R NN HO OH NN R R N N N OO Cl Cl Cl I II III IV

Research ArticleIn Silico Molecular Docking and In Vitro Antidiabetic Studies ofDihydropyrimido[45-a]acridin-2-amines

A Bharathi1 Selvaraj Mohana Roopan1 C S Vasavi2 Punnagai Munusami2

G A Gayathri3 and M Gayathri3

1 Chemistry Research Laboratory Organic Chemistry Division School of Advanced Sciences VIT University VelloreTamil Nadu 632 014 India

2 Bioinformatics Division School of Bio Sciences and Technology VIT University Vellore Tamil Nadu 632 014 India3 Division of Industrial Biotechnology School of Bio Sciences and Technology School of Advanced Sciences VIT UniversityVellore Tamil Nadu 632 014 India

Correspondence should be addressed to Selvaraj Mohana Roopan mohanaroopansgmailcom

Received 19 February 2014 Revised 18 March 2014 Accepted 1 April 2014 Published 2 June 2014

Academic Editor Kota V Ramana

Copyright copy 2014 A Bharathi et al This 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

An in vitro antidiabetic activity on 120572-amylase and 120572ndashglucosidase activity of novel 10-chloro-4-(2-chlorophenyl)-12-phenyl-56-dihydropyrimido[45-a]acridin-2-amines (3andash3f) were evaluated Structures of the synthesized molecules were studied by FT-IR1H NMR 13C NMR EI-MS and single crystal X-ray structural analysis data An in silico molecular docking was performed onsynthesized molecules (3andash3f) Overall studies indicate that compound 3e is a promising compound leading to the developmentof selective inhibition of 120572-amylase and 120572-glucosidase

1 Introduction

Pyrimidine is a well-known biologically active nitrogen con-taining heterocyclic compound In recent years researchersare much interested in the synthesis of pyrimidine analoguesPyrimidine derivatives posse fungicidal [1] herbicidal [2]antidepressant [3] and antitumor properties [4 5] Syn-thetically prepared amino pyrimidine derivatives display awide range of biological activities such as antibacterial [6]antitumor [7] and antiviral [8 9] Therefore the substitutedamino pyrimidine structure can be found in diverse clinicallyapproved drugs Interestingly a substituted amino pyrimi-dinemoiety was also suggested to account for the antioxidantactivity [10] Among those amino pyrimidine heterocycle 2-aminopyrimidines have beenwidely used as pharmacophoresfor drug discovery 2-Aminopyrimidines constitute a partof the DNA base pair molecules [11] Compounds havingpotent anticancer activity CDK inhibitory activity I [12ndash14]antiproliferative activity [15] kinase inhibitor II [16] antibac-terial agents [17] antitumor III antidiabetic activity [18]

antimalarial IV antiplasmodial agents [19] antimicrobialac-tivity V [20] and anti-inflammatory activity VI [21](Figure 1) contain amino pyrimidine moiety in the struc-ture The tricyclic planar acridine moiety is responsible forintercalation between base pairs of double-stranded DNAthrough 120587-120587 interactions and therefore causes alteration inthe cellular machinery [22] Intercalative interaction of struc-turally related well-known intercalators 9-aminoacridine(9AA) and proflavine (PF) was determined bymeans of fluo-rescence quenching study [23]

Due to numerous biological applications of 2-aminopy-rimidine and acridine amine and its analogues we havefocused our research on synthesis of 2-aminopyrimidine byusing pharmacologically important structural scaffold acri-dine pharmacophore

Protein-ligand interaction is comparable to the lock-and-key principle in which the lock encodes the protein and thekey is grouped with the ligand The major driving force forbinding appears to be hydrophobic interaction [24] In silicotechniques helps identifying drug target via bioinformatics

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 971569 10 pageshttpdxdoiorg1011552014971569

2 BioMed Research International

OH

N

NN

HO OH

R

N N

N

N

R

N

N N

N

O

O

O

OH

N

N

R

N N

HO OH

N NR R

N

N

NO O

Cl

Cl

Cl

I

II

III

IV

V

VI

NH2

H2N

H3CO

NH2

NH2

NH2NH2

NH2

H2N

R = H Me Et etc

R = H Cl CH3 NO2

R = H 2-Cl 4-Cl Br OMe NO2

R = Cl Br Me H

CH3

CH3

H3C

Figure 1 I CDK inhibitor II kinase inhibitor III antitumor IV antimalarial V antimicrobial and VI anti-inflammatory

tools They can also be used to explore the target structuresfor possible active sites generate candidate molecules dockthese molecules with the target rank them according totheir binding affinities and further optimize themolecules toimprove binding characteristics [25] Diabetes mellitus (DM)is a leading noncommunicable disease which affects morethan 100 million people worldwide and is considered as oneof the fine leading diseases which causes death in the world[26] Type-2 diabetes mellitus is a chronic metabolic disorderthat results from defects in both insulin secretion and insulinaction Management of type-2 diabetes by conventionaltherapy involves the inhibition of degradation of dietarystarch by glucosidases such as 120572-amylase and 120572-glucosidase[27]The pathogenesis of type-2 diabetes involves progressivedevelopment in insulin resistance associated with a defect ininsulin secretion leading to overt hyperglycemia Howevercompounds which improve insulin sensitivity and glucoseintolerance are somewhat limited warranting the discoveryand characterization of novel molecules targeting variouspathways involved in the pathogenesis of type-2 diabetes [28]Currently there are few drugs that are able to counteractthe development of the associated pathologiesTherefore theneed to search for new drug candidates in this field appearsto be critical Aromatic amines and thiazolidinones nucleiwould produce new compounds with significant antidiabeticproperties [29]

In our research group we have already reportedlarvicidal activity of 7-chloro-34-dihydro-9-phenylacridin-1(2H)-one and (E)-7-chloro-34-dihyro-phenyl-2-[(pyridin-2-yl)methylene]acridin-1(2H)-one [30] In continuation ofour research work on acridine moiety presently we focus onthe synthesis of 10-chloro-4-(2-chlorophenyl)-12-phenyl-56-dihydropyrimido[45-a]acridin-2-amine 3andash3f derivativesAll the synthesized dihydropyrimido[45-a]acridin-2-aminesanalogues 3andash3f were evaluated for docking and in vitroantidiabetic activity

2 Materials and Methods

21 Chemistry Melting points were determined by opencapillary method and are corrected with standard benzoicacid All solvents were distilled and dried prior to use TLCwas performed on silica gel G and the spots were exposedto iodine vapour for visualization A mixture of petroleumether and ethyl acetate was used as an eluent at differentratio Column chromatography was performed by usingsilica gel (60ndash120mesh) 1HNMR and 13CNMR spectra wererecorded in CDCl

3on a Bruker advance 400MHz instru-

ment Chemical shifts are reported in ppm using TMS asthe internal standard IR spectra were obtained on a Perkin-Elmer spectrum RXI FT-IR spectrometer (400ndash4000 cmminus1

BioMed Research International 3

N

Cl

O

R

N

Cl

N

R

N

Cl

Cl

1a-f

NaOHEtOH

3a-f2

(a) (b) (c) (d) (e) (f)

+ guanidine carbonate reflux 5h

H3CO

NH2

OCH3

OCH3 OCH3 OCH3

R =

Scheme 1 Synthesis of 10-chloro-412-diphenyl-56-dihydropyrimido[45-a]acridin-2-amines 3andash3f

resolution 1 cmminus1) using KBr pellets Molecular mass wasdetermined using ESI-MS THERMO FLEET spectometer

22 Biological Assays The dialysis membrane 14-120572-Dglucan-glucanohydrolase 120572-amylase 120572-glucosidase P-nitro-phenyl-120572-D-glucopyranoside and acarbose were purchasedfrom Himedia Laboratories Mumbai India All otherchemicals and reagents were AR grade purchased locally

23 General Synthesis of 10-Chloro-412-diphenyl-56-dihydro-pyrimido[45-a]acridin-2-amine 3andash3f 10-Chloro-412-di-phenyl-56-dihydropyrimido[45-a]acridin-2-amines 3andash3fwere synthesized via (E)-2-benzylidene-7-choloro-34-dihydro-9-phenylacridin-1(2H)-ones 1andash1f (39511 g001mol) were mixed with guanidine carbonate 2 (09008 g001mol) and 10mL of 10 alc NaOH (1 g in 10mLethanol) and then heated under reflux condition for 5 hAfter the completion of the reaction reaction mixturewas cooled and poured into crushed ice The crudeproduct was separated by column chromatographyusing 10ndash15 of ethyl acetate and pet ether solvent toget the target compounds 3andash3f The synthetic schemewas presented in Scheme 1 and physical data of allsynthesized derivatives were summarized in Table 1

Spectral data of the synthesized compounds are describedbelow

10-Chloro-412-diphenyl-56-dihydropyrimido[45-a]acridin-2-amine (3a) Yellow solid Yield 70 mp 194ndash196∘C FT-IR(KBr) ]max (cmminus1) 345030 (ndashNH

2) 1HNMR (400MHz

CDCl3) 120575 (ppm) 299 (s 2H ndashCH

2) 318 (s 2H ndashCH

2)

443 (s 2H ndashNH2) 725 (m 6H) 746 (d 2H) 753 (m 2H)

760ndash765 (m 2H) 799ndash802 (d 119869 = 88Hz 1H) 13CNMR(400MHz CDCl

3) 120575 (ppm) 238 338 1185 1250 1262

1273 1279 1283 1287 1288 1292 1293 1302 1311 13211378 1379 1461 1469 1604 1607 1609 1655 EI-MS mz43530 [M+1]

Table 1 Summary of synthesized 2-aminopyrimidine derivatives(3andashf) via Scheme 1

S no Compounds R MP (∘C) Yield1 3a -H 194ndash196 702 3b -34-OCH3 218ndash220 813 3c -25-OCH3 212ndash214 754 3d -2-OCH3 168ndash170 695 3e -4-Cl 210ndash212 716 3f -2-Cl 238ndash240 69

10-Chloro-4-(34-dimethoxyphenyl)-12-phenyl-56-dihydropy-rimido[45-a]acridin-2-amine (3b) Yellow solid Yield 81mp 218ndash220∘C FT-IR (KBr) ]max (cm

minus1) 344679 (ndashNH2)

293180ndash295880 (ndashOCH3) 1HNMR (400MHz CDCl

3) 120575

(ppm) 303ndash306 (m 2H ndashCH2) 318ndash321 (m 2H ndashCH

2)

393-394 (d 119869 = 32Hz 6H 2-OCH3) 434 (s 2H ndashNH

2)

693ndash695 (d 119869 = 8Hz 1H) 711 (d 119869 = 2Hz 1H) 713 (d119869 = 2Hz 1H) 715 (d 119869 = 2Hz 1H) 723ndash726 (m 2H)744ndash750 (m 2H) 761 (d 119869 = 24Hz 1H) 764 (d 119869 = 24Hz1H) 766 (d 119869 = 24Hz 1H) 13CNMR (400MHz CDCl

3)

120575 (ppm) 239 328 559 1109 1172 1179 1193 1202 12061253 1275 1279 1281 1284 1289 1290 1301 1303 13231350 1385 1403 1407 1449 1486 1493 1578 1584 EI-MSmz 49552 [M+1]

10-Chloro-4-(25-dimethoxyphenyl)-12-phenyl-56-dihydropy-rimido[45-a]acridin-2-amine (3c) White solid Yield 75mp 212ndash214∘C FT-IR (KBr) ]max (cmminus1) 348537 (ndashNH

2)


3) 120575

(ppm) 264 (d 119869 = 68Hz 1H ndashCH2) 287 (d 119869 = 32Hz

1H ndashCH2) 314ndash322 (dd 119869 = 68Hz 119869 = 172Hz 2H

ndashCH2) 375ndash379 (d 119869 = 184Hz 6H 2-OCH

3) 430 (s 2H

ndashNH2) 689ndash697 (m 3H) 731-732 (d 119869 = 48Mz 1H) 746

(m 3H) 757-758 (d 119869 = 24Mz 1H) 762-763 (d 119869 = 24Mz1H) 765 (d 119869 = 24Hz 1H) 799ndash801 (d 119869 = 88Hz 1H)13C NMR (400MHz CDCl

3) 120575 (ppm) 230 336 558 560

1121 1154 1158 1205 1249 1262 1271 1278 1288 1301


C18

C19

C20C21

C16

C12

C15C12

C11

C10C9

N1C4

C3

C2

C1

C11C6C5

C7

C22

C23

C24C25

C26N2

C27C13

C8

C14

N4

N3

Figure 2 ORTEP diagram of compound 3f

1310 1319 1380 1460 1469 1506 1537 1594 1604 16121637 EI-MSmz 49535 [M+1]

10-Chloro-4-(3-methoxyphenyl)-12-phenyl-56-dihydropyrim-ido[45-a]acridin-2-amine (3d) Yellow solid Yield 69mp 168-170∘C FT-IR (KBr) ]max (cmminus1) 347573 (ndashNH

2)


3) 120575

(ppm) 239-240 (m 2H ndashCH2) 271ndash283 (m 2H ndashCH

2)

382 (s 3H ndashOCH3) 483 (s 2H ndashNH

2) 691 (d 119869 = 12Hz

1H) 692-693 (s 1H) 697ndash699 (d 119869 = 72Hz 1H) 702ndash704(d 119869 = 84Hz 1H) 712ndash714 (m 1H) 715 (m 1H) 720ndash722(m 2H) 724 (m 2H) 731ndash733 (d 119869 = 84Hz 1H) 734ndash736(d 119869 = 72Hz 1H) 13CNMR (400MHz CDCl

3) 120575 (ppm)

237 338 553 1141 1150 1185 1211 1249 1262 1272 12791287 1293 1294 1302 1311 1320 1378 1393 1461 14681595 1604 1606 1609 1654 EI-MSmz 46731 [M+3]

10-Chloro-4-(4-chlorophenyl)-12-phenyl-56-dihydropyrimi-do[45-a]acridin-2-amine (3e) White solid Yield 71mp 210-212∘C FT-IR (KBr) ]max (cmminus1) 349501 (ndashNH

2)

1HNMR (400MHz CDCl3) 120575 (ppm) 297ndash300 (m 2H

ndashCH2) 318ndash321 (m 2H ndashCH

2) 435 (s 2H ndashNH

2) 723ndash726

(m 2H) 743ndash751 (m 7H) 760 (d 119869 = 2Hz 1H) 764-765(d 119869 = 24Hz 1H) 766-767 (d 119869 = 24Hz 1H) 13C NMR(400MHz CDCl

3) 120575 (ppm) 238 337 1184 1248 1262

1273 1279 1286 1287 1293 1302 1312 1321 1354 13631377 1461 1470 1604 1607 1609 1643 EI-MS mz 47003[M+1]

10-Chloro-4-(2-chlorophenyl)-12-phenyl-56-dihydropyrimi-do[45-a]acridin-2-amine (3f ) Light yellow Yield 69mp 238-240∘C FT-IR (KBr) ]max (cmminus1) 348151 (ndashNH

2)


2-CH2) 572 (s 2H ndashNH

2) 726-727 (d 119869 = 72Hz 2H)

737 (s 1H) 741ndash744 (m 1H) 746ndash751 (m 5H) 756ndash758(d 119869 = 76Hz 1H) 779ndash781 (dd 119869 = 2Hz 119869 = 2Hz 1H)804ndash806 (d 119869 = 88Mz 1H) 13CNMR (400MHz CDCl

3)

120575 (ppm) 238 337 1184 1248 1262 1273 1279 1286 12871293 1302 1312 1321 1354 1363 1377 1461 1470 16041607 1609 1643 EI-MSmz 46923 [M+]

24 Single Crystal X-RayDiffraction (XRD)Analysis Crystalssuitable for X-ray analysis were obtained by slow evapora-tion of a solution of the 10-chloro-4-(2-chlorophenyl)-12-phenyl-56-dihydropyrimido[45-a]acridin-2-amine 3f inethyl acetateThemeasurements were made on Enraf NoniusCAD4-MV31

single crystal X-ray diffractometer The dia-grams and calculations have been performed using SAINT(APEX II) for frame integration SHELXTL for structuresolution and refinement software programs The structurewas refined using the full-matrix least squares procedures onF2 with anisotropic thermal parameters for all nonhydrogenatomsThe crystal data and details concerning data collectionand structure refinement of compound 3f single crystalare summarized in Table 2 As we can see compound 3fcrystallizes in triclinic space group P-1 The single crystalstructure and atomic numbering chosen for compound 3f aredemonstrated in Figure 2 Selected bond lengths and bondangles are compiled in Table 3 It can be observed that thebond lengths of all kind atoms on the whole molecule areabsorbed The typical CndashC single (155 A) and C=C double(138 A) bonds CndashN typical single (135 A) and C=N double(133 A) bonds This means that the carbon-carbon bond andthe carbon-nitrogen bond have double bond character andcontribute to form conjugated system Furthermore carbonattached with strong electronegative atom chlorine bondlength is CndashCl (168A) in case all CndashH (097A) and C=H(093A) are absorbed The primary amine hydrogen bondlength is NndashH (086A) respectively (Table 3) Concerninginspection of the torsion angles the 3f molecule has nearlyplanar core Figure 3 illustrates a representative view ofcompound 3f crystal packing structure The two moleculesof compound 3f are packed in face-to-face arrangement dueto the intermolecular hydrogen bonds The crystal packingstructure demonstrates the existence of two intermolecularhydrogen bonds Interesting result were absorbed from crys-tal packing structure is the intermolecular hydrogen bondingoccurs at C(23)ndashH(23) and C(24)ndashH(24) of one molecule toanother neighbouring molecule of C(23)ndashH(23) and C(24)ndashH(24) not in the case of primary amine containing twohydrogen N(4)ndashH(A) and N(4)ndashH(B) Figure 3


Table 2 The crystallographic data and structure refinement parameters of compound 3f

Empirical formula C27 H18 Cl2 N4

Formula weight 46935Temperature 293(2) KWavelength 071073 ACrystal system space group Triclinic P-1

Unit cell dimensions119886 = 99421(5) A 120572 = 67248(2)∘119887 = 109553(5) A 120573 = 78530(2)∘119888 = 115126(5) A 120574 = 89875(2)∘

Volume 112939(9) A3

119885 2Calculated density 1380Mgm3

Absorption coefficient 0311mmminus1

119865(000) 484Crystal size 035 times 030 times 025mmTheta range for data collection 196 to 2500∘Limiting indices minus11 le ℎ le 11 minus12 le 119896 le 12 minus13 le 119897 le 13Reflections collectedunique 195033929 [119877(int) = 00276]Completeness to theta = 2500 988Absorption correction Semiempirical from equivalentsMax and min transmission 09635 and 08623Refinement method Full-matrix least squares on 1198652

Datarestraintsparameters 39296308Goodness-of-fit on 1198652 1200Final 119877 indices [119868 gt 2 sigma(119868)] 119877

1= 00569 119908119877

2= 01944

119877 indices (all data) 1198771= 00712 119908119877

2= 02074

Largest diff peak and hole 0383 and minus0385 119890sdotAminus3

b

a

c

Figure 3The packing of themolecules in the unit cell viewed downthe a-axis with the hydrogen bond geometry of compound 3f

25 Glucose Diffusion Inhibitory Test

Sample Preparation Four different concentrations (100 200300 and 400 120583gmL) of samples were prepared 1mL ofthe sample was placed in a dialysis membrane (12000MWHimedia laboratoriesMumbai) alongwith a glucose solution(022mM in 015M NaCl) Then it was tied at both endsand immersed in a beaker containing 40mL of 015M NaCland 10mL of distilled water The control contained 1mL of015M NaCl containing 022mM glucose solution and 1mLof distilled water The beaker was then placed in an orbitalshaker The external solution was monitored every half anhourThree replications of this testwere done for 3 hrs [31 32]

26 Inhibition Assay for 120572-Amylase Activity Four differentconcentrations (100 200 300 and 400 120583gmL) of samplesand standard drug acarbose were prepared and made up to1mL with DMSO A total of 500120583L of sample and 500 120583L of002M sodium phosphate buffer (pH 69 with 0006MNaCl)containing 120572-amylase solution (05mgmL) were incubatedfor 10 minutes at 25∘C After preincubation 500 120583L of 1starch solution in 002M sodium phosphate buffer (pH 69with 0006M NaCl) was added to each tube This reactionmixture was then incubated for 10 minutes at 25∘C 1mLof DNSA colour reagent was added to stop the reactionThese test tubes were then incubated in a boiling waterbath for 5 minutes and cooled to room temperature Finallythis reaction mixture was again diluted by adding 10mL ofdistilled water of inhibition by 120572-amylase can be calculatedby using the following formula Absorbance was measured at540 nm [32 33]

inhibition =119860540

control minus 119860540

sample119860540

controltimes 100 (1)

Triplicates were done for each sample at different concentra-tions

27 Inhibition Assay for 120572-Glucosidase Activity Various con-centrations of samples and standard drug acarbose wereprepared 120572-Glucosidase (0075 units) was premixed with


Table 3 Some important bond lengths (A) and bond angles (∘) of compound 3f

Bond Bond length Bond Bond angleC(1)ndashC(6) 1371(6) N(1)ndashC(4)ndashC(5) 1232(3)C(2)ndashH(2) 09300 N(1)ndashC(4)ndashC(3) 1171(4)C(11)ndashH(11A) 09700 N(1)ndashC(9)ndashC(8) 1241(4)C(11)ndashH(11A) 09700 N(1)ndashC(9)ndashC(10) 1164(3)C(11)ndashH(11B) 09700 C(8)ndashC(9)ndashC(10) 1194(3)C(11)ndashH(11B) 09700 C(9)ndashC(10)ndashH(10A) 1095C(13)ndashN(2) 1330(4) C(11)ndashC(10)ndashH(10A) 1095C(14)ndashN(2) 1332(4) C(12)ndashC(11)ndashC(10) 1090(3)C(14)ndashN(3) 1344(4) H(11A)ndashC(11)ndashH(11B) 1083C(14)ndashN(4) 1354(4) C(13)ndashC(12)ndashC(15) 1158(3)C(15)ndashN(3) 1339(4) C(15)ndashC(12)ndashC(11) 1243(3)N(4)ndashH(4A) 08600 N(2)ndashC(13)ndashC(12) 1230(3)N(4)ndashH(4B) 08600 N(2)ndashC(13)ndashC(8) 1178(3)C(4)ndashN(1) 1361(6) C(12)ndashC(13)ndashC(8) 1191(3)C(9)ndashN(1) 1317(5) N(2)ndashC(14)ndashN(3) 1261(3)C(9)ndashC(10) 1496(6) N(2)ndashC(14)ndashN(4) 1170(3)C(10)ndashC(11) 1525(6) N(3)ndashC(14)ndashN(4) 1169(3)C(11)ndashC(12) 1508(5) N(3)ndashC(15)ndashC(12) 1224(3)C(26)ndashH(26) 09300 N(3)ndashC(15)ndashC(16) 1167(3)C(27)ndashH(27) 09300 C(13)ndashN(2)ndashC(14) 1164(3)C(9)ndashN(1) 1317(5) C(15)ndashN(3)ndashC(14) 1162(3)C(1)ndashCl(11015840) 1681(11) C(14)ndashN(4)ndashH(4B) 1200C(1)ndashCl(1) 199(4) H(4A)ndashN(4)ndashH(4B) 1200C(17)ndashCl(2) 1732(4)

Table 4 Release of glucose through dialysis membrane to external solution (mgdL)

Time (min) Control 3a 3b 3c 3d 3e 3f30 1833 plusmn 000 1515 plusmn 001 1661 plusmn 000 1830 plusmn 000 15 plusmn 000 1830 plusmn 000 2 plusmn 000

60 2670 plusmn 003 1831 plusmn 000 1831 plusmn 000 2 plusmn 001 183 plusmn 001 2 plusmn 001 216 plusmn 000





Values are mean plusmn SEM for groups of 3 observations

sample 3mM p-nitrophenyl glucopyranoside used as asubstrate was added to the reaction mixture to start thereaction [34] The reaction was incubated at 37∘C for 30minand stopped by adding 2mL of Na

2CO3 The 120572-glucosidase

activity was measured by p-nitrophenol release from PNPGat 400 nm of inhibition can be calculated by using (1)

Triplicates are done for each sample at different concen-trations [31ndash33]

28 Statistical Analysis Statistical analysis was performedusing one-way analysis of variance (ANOVA) Results areexpressed as mean plusmn SD and 119899 = 3

3 Results and Discussion

31 Glucose Diffusion Inhibitory Test The results are summa-rized in Tables 4 and 5 The diffused glucose concentrationis given in Table 4 and of relative movement is given in

Table 5 The movement of glucose from inside of membraneto external solution monitored and compared in Figures 4and 5 Among the six samples only the 3e retains the glucoseand shows minimum of relative movement 4106 over180 minutes All other samples show the highest of relativemovement in glucose diffusion from 30 to 180 minutes

311 120572-Amylase Inhibitory Assay The results are given inTable 6 All samples show gradual increase in inhibitionwhere the sample concentration increased from 100 to400 120583gmL Sample 3e shows maximum inhibition of 57and sample 3d shows 2355 of inhibition All other samplesshow varying less significant inhibition

312 120572-Glucosidase Inhibition Assay The results are shownin Table 7 Among the six samples 3e shows maximuminhibitory activity of 6025 at higher concentration


Table 5 of relative movement in glucose diffusion inhibitory assay

Time (min) 3a 3b 3c 3d 3e 3f30 8183 plusmn 001 9056 plusmn 003 9983 plusmn 000 8133 plusmn 000 7490 plusmn 000 10911 plusmn 001

60 6866 plusmn 007 6853 plusmn 000 7491 plusmn 007 6866 plusmn 001 7320 plusmn 002 8090 plusmn 002






Table 6 inhibition of 120572-amylase assay

Concentration (120583gmL) Acarbose 3a 3b 3c 3d 3e 3f100 3201 plusmn 009 797 plusmn 001 543 plusmn 006 326 plusmn 000 1883 plusmn 002 1920 plusmn 001 1223 plusmn 000


300 7003 plusmn 025 1122 plusmn 007 652 plusmn 004 5070 plusmn 008 2173 plusmn 001 5362 plusmn 003lowastlowast1485 plusmn 002


Values are mean plusmn SEM for groups of 3 observationslowastlowast119875 lt 005

40 60 80 100 120 140 160 180 20012

14

16

18

20

22

24

26

28

30

32

Con

cent

ratio

n of

glu

cose

(mg

dL)

Time (min)

Cont3a3b3c

3d3e3f

Figure 4 Concentration of glucose (mgdL)

(400 120583gmL) Sample 3d shows 2301 of inhibition All theother samples are less significant

32 Molecular Docking Studies The synthesized molecules3andash3f were constructed usingVEGA ZZmolecular modelingpackage [35] The obtained structures were then geometri-cally optimized using AM1 Hamiltonian in MOPAC soft-ware package [36] The X-ray structure of pig 120572-pancreatic120572-amylase (PDB 3L2M) and N-terminal human maltase-glucoamylase with Casuarina (PDB 3CTT) [37] obtained

20 40 60 80 100 120 140 160 180 200

404550556065707580859095

100105110

Relat

ive m

ovem

ent o

f glu

cose

()

Time (min)

Cont3a3b

3c3d3e

Figure 5 of relative movement in glucose diffusion inhibitoryassay

from Brookhaven Protein Data Bank were used for dock-ing calculations Docking calculations were performed withAutoDock 40 [38]

The crystal structures were refined by removing watermolecules and repeating coordinates Hydrogen atoms wereadded and charges were assigned to the protein atoms usingKollman united atoms force field by using AutoDockTools-156 For docking calculations Gasteiger partial atomiccharges were added to the synthesized structures and allpossible flexible torsion angles of the ligand were defined byusing AUTOTORS The structures were saved in a PDBQTformat for AutoDock calculations


Table 7 inhibition of 120572-glucosidase assay

Concentration (120583gmL) Acarbose 3a 3b 3c 3d 3e 3f100 3008 plusmn 005 602 plusmn 001 629 plusmn 000 429 plusmn 001 2091 plusmn 000 2145 plusmn 001

lowast1402 plusmn 002




Values are mean plusmn SEM for groups of 3 observationslowast119875 lt 001lowastlowast119875 lt 005

Table 8 The binding energy (Δ119866BE) and intermolecular energy (Δ119866intermol) of the structures 3d and 3e are given The docking energy andbinding energy are reported in Kcalmol

Structures Δ119866BEΔ119866intermol

Δ119866internal Δ119866torΔ119866vdw hb desol Δ119866elec

120572-Amylase3d minus452 minus531 minus040 minus157 1193e minus483 minus535 minus037 minus154 089

120572-Glucosidase3d minus651 minus634 minus137 minus144 1193e minus661 minus695 minus056 minus141 089

AutoDock requires precalculated grid maps one for eachatom type present in the structure being docked The auxil-iary program AutoGrid generated the grid maps Lennard-Jones parameters 12-10 and 12-6 implemented with theprogramwere used formodelingH-bonds and van derWaalsinteractions respectively The Lamarckian genetic algorithmmethod was applied for docking calculations using defaultparameters AutoDock uses a semiempirical free energy forcefield to evaluate conformations during docking simulationsThe optimized orientations represent possible bindingmodesof the ligand within the site

Δ119866 = Δ119866vdw + Δ119866hbond + Δ119866elec + Δ119866tor + Δ119866desolv (2)

The first three terms are van der Waals hydrogen bondingand electrostatics respectively The term Δ119866tor is for rotationand translation and Δ119866desolv is for desolvation upon bindingand the hydrophobic effect

After docking the 50 solutions were clustered into groupswith RMS deviations lower than 10 A The clusters wereranked by the lowest energy representative of each cluster

321 Molecular Docking Study on 120572-Amylase The bindingsite of the structures was not identified because of theabsence of the crystal structure of the ligand and a blinddocking was performed for all the structures 3andash3f withthe protein structure 3L2M [39] Two interacting bindingsites were identified [37] one near the entrance of the cen-tral beta-barrel of the enzyme and the other near the N-terminal of the protein [39] The docked structures showedbinding energy in the range of minus42 to minus48 Kcalmol It wasobserved that the structure 3e exhibits high binding energy ofminus48 Kcalmol and the structure 3d exhibits binding energyof minus45 Kcalmol respectively The binding energies (Δ119866BE)and intermolecular energies (Δ119866intermol) of the structures 3d

and 3e obtained for 120572-amylase are given in Table 8 Whencomparing 3d and 3e structures 3e exhibits high bindingenergyThe structure 3e shows interaction with the residuesTrp58 Trp59 Tyr62 Asp197 and Asp300 present in thebinding site of 120572-amylase The amino acids Trp58 and Trp59were the key residues interacting with all the structures Thedocking studies revealed that the van derWaals electrostaticand desolvation energies play a key role in binding It wasobserved that the Pyrimido ring was fitted well with bindingsite via hydrogen bonds through the hydrogen atom ofthe amino group of 3e with carboxylate group of Asp300The hydrophobic interactions were formed by Trp58 Trp59Gln63 Val163 Asp300 His305 and Asp356 in 3e

322 Molecular Docking Study on 120572-Glucosidase All thebinding modes of the structures 3andash3f were explored usingthe docking calculation in AutoDock The binding site of thestructures was identified using the crystal structure ofCasua-rina The docked structures showed binding energy in therange of minus63 to minus66 Kcalmol The binding energies (Δ119866BE)and intermolecular energies (Δ119866intermol) of the structures 3dand 3e obtained for 120572-glucosidase are given in Table 8 Thestructure 3e showed high binding energy when comparedto 3d Based on docking calculations the synthesized com-pounds 3d and 3e show better inhibition of 120572-glucosidasecompared to 120572-amylase which is in a good agreement within vitro studies The following key residues Asp203 Tyr299Trp406 Asp443 Met444 Phe450 Lys480 Asp542 Phe575and Gln603 present in the binding site of 120572-glucosidaseshow noncovalent interactions with 3e It was observed thatthe Pyrimido ring of 3e formed hydrogen bonds throughthe hydrogen atom of the amino group with carboxylategroup of Asp203 at a distance of 19 A The residues Tyr299


Trp406 Asp443 Met444 Phe450 Lys480 Asp542 Phe575and Gln603 formed hydrophobic interactions with 3e

In diabetes mellitus control of postprandial plasma glu-cose level is critical in the early treatment [34] Inhibitionof enzymes involved in the metabolism of carbohydrates areone of the therapeutic approaches for reducing postprandialhyperglycemia The inhibition by natural products is moresafe than synthetic drugs120572-Amylase and 120572-glucosidase are significant enzymes

which cleave carbohydrates responsible for absorption ofglucose in the blood stream For this reason syntheticdrug such as acarbose miglitol and voglibose are someinhibitors which inhibit 120572-amylase and 120572-glucosidase [40]However these agents have their limitations because they arenonspecific and produce serious side effects such as GI tractinflammation and to elevate diabetic complications [41]

4 Conclusion

Thepresent study revealed that we have successfully achievedour target title compound 10-chloro-412-diphenyl-56-di-hydropyrimido[45-a]acridin-2-amine derivatives All com-pounds were confirmed by suiTable experimental andspectroscopic techniques Synthesized derivatives (3andash3f)were evaluated in vitro 120572-amylase and 120572-glucosidaseinhibitory activity and glucose diffusion test of samplesAmong all other derivatives compounds 10-chloro-4-(4-chlorophenyl)-12-phenyl-56-dihydropyrimido[45-a]acri-din-2-amine 3e and 10-chloro-4-(3-methoxyphenyl)-12-phenyl-56-dihydropyrimido[45-a]acridin-2-amine 3dshow good inhibitory activity for 120572-amylase and 120572-glucosi-dase with the values of 5796 6027 2355 and 2301 forcompounds 3e and 3d respectively The docking studies arein good agreement with the in vitro studies The dockingcalculations showed that van der Waals electrostatic anddesolvation energies play a key role in binding These factorsare considered for designing new inhibitors for 120572-amylaseand 120572-glucosidase

Conflict of Interests

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

Acknowledgments

This study was supported by DST-SERB Fast Track (Sanctionno SRFTCS-2642012) Government of India New DelhiThe authors gratefully acknowledge DST-FIST for providingNMR facilities to VIT The authors acknowledge the supportextended by VIT-SIF for GC-MS and NMR analysis

References

[1] RJMilling andCJ Richardson ldquoMode of action of the anilino-pyrimidine fungicide pyrimethanil 2 Effects on enzyme secre-tion in Botrytis cinereardquo Pesticidal Science vol 45 no 1 pp 33ndash48 2006

[2] M Y Liu and D Q Shi ldquoSynthesis and herbicidal activityof 2-aroxy-propanamides containing pyrimidine and 1 3 4-thiadiazole ringsrdquo Journal of Heterocyclic Chemistry vol 51 no2 pp 432ndash435 2014

[3] J L Bernier J P Henichart V Warin and F Baert ldquoSyn-thesis and structure-activity relationship of a pyrimido[45-d]pyrimidine derivative with antidepressant activityrdquo Journal ofPharmaceutical Sciences vol 69 no 11 pp 1343ndash1345 1980

[4] B Gong F Hong C Kohm et al ldquoSynthesis SAR and anti-tumor properties of diamino-CN-diarylpyrimidine positionalisomers Inhibitors of lysophosphatidic acid acyltransferase-120573rdquoBioorganic and Medicinal Chemistry Letters vol 14 no 9 pp2303ndash2308 2004

[5] L S Jeong L X Zhao W J Choi et al ldquoSynthesis and antitu-mor activity of fluorocyclopentenyl-pyrimidinesrdquo NucleosidesNucleotides andNucleic Acids vol 26 no 6-7 pp 713ndash716 2007

[6] S Hawser S Lociuro and K Islam ldquoDihydrofolate reductaseinhibitors as antibacterial agentsrdquo Biochemical Pharmacologyvol 71 no 7 pp 941ndash948 2006

[7] J Lee K-H Kim and S Jeong ldquoDiscovery of a novel class of 2-aminopyrimidines as CDK1 and CDK2 inhibitorsrdquo Bioorganicand Medicinal Chemistry Letters vol 21 no 14 pp 4203ndash42052011

[8] V R Gadhachanda B Wu Z Wang et al ldquo4-Aminopyrim-idines as novel HIV-1 inhibitorsrdquo Bioorganic and MedicinalChemistry Letters vol 17 no 1 pp 260ndash265 2007

[9] D Hockova A Holy M Masojıdkova et al ldquo5-Substituted-24-diamino-6-[2-(phosphonomethoxy) ethoxy]pyrimidines-acyclic nucleoside phosphonate analogues with antiviralactivityrdquo Journal of Medicinal Chemistry vol 46 no 23 pp5064ndash5073 2003

[10] B M Rezk G R M M Haenen W J F Van Der Vijgh and ABast ldquoTetrahydrofolate and 5-methyltetrahydrofolate are folateswith high antioxidant activity Identification of the antioxidantpharmacophorerdquo FEBSLetters vol 555 no 3 pp 601ndash605 2003

[11] T Kitamura A Hikita H Ishikawa and A FujimotoldquoPhotoinduced amino-imino tautomerization reaction in 2-aminopyrimidine and its methyl derivatives with acetic acidrdquoSpectrochimica Acta A Molecular and Biomolecular Spec-troscopy vol 62 no 4-5 pp 1157ndash1164 2005

[12] F Xie H Zhao L Zhao L Lou and Y Hu ldquoSynthesisand biological evaluation of novel 245-substituted pyrimidinederivatives for anticancer activityrdquo Bioorganic and MedicinalChemistry Letters vol 19 no 1 pp 275ndash278 2009

[13] C D Jones D M Andrews A J Barker et al ldquoImida-zole pyrimidine amides as potent orally bioavailable cyclin-dependent kinase inhibitorsrdquo Bioorganic and Medicinal Chem-istry Letters vol 18 no 24 pp 6486ndash6489 2008

[14] Y-S Seong C Min L Li et al ldquoCharacterization of a nov-el cyclin-dependent kinase 1 inhibitor BMI-1026rdquo CancerResearch vol 63 no 21 pp 7384ndash7391 2003

[15] M A Vilchis-Reyes A Zentella M A Martınez-Urbina etal ldquoSynthesis and cytotoxic activity of 2-methylimidazo[12-a]pyridine- and quinoline-substituted 2-aminopyrimidinederivativesrdquo European Journal of Medicinal Chemistry vol 45no 1 pp 379ndash386 2010

[16] R Capdeville E Buchdunger J Zimmermann and A MatterldquoGlivec (ST1571 imatinib) a rationally developed targetedanticancer drugrdquo Nature Reviews Drug Discovery vol 1 no 7pp 493ndash502 2002

[17] P-P Kung M D Casper K L Cook et al ldquoStructure-activityrelationships of novel 2-substituted quinazoline antibacterial


agentsrdquo Journal of Medicinal Chemistry vol 42 no 22 pp4705ndash4713 1999

[18] N Singh S K Pandey N Anand et al ldquoSynthesis molec-ular modeling and bio-evaluation of cycloalkyl fused 2-aminopyrimidines as antitubercular and antidiabetic agentsrdquoBioorganic and Medicinal Chemistry Letters vol 21 no 15 pp4404ndash4408 2011

[19] K Singh H Kaur K Chibale J Balzarini S Little and PV Bharatam ldquo2-Aminopyrimidine based 4-aminoquinolineanti-plasmodial agents Synthesis biological activity structure-activity relationship and mode of action studiesrdquo EuropeanJournal of Medicinal Chemistry vol 52 pp 82ndash97 2012

[20] A Nagaraj and C Sanjeeva Reddy ldquoSynthesis and bio-logical study of novel bis-chalcones bis-thiazines and bis-pyrimidinesrdquo Journal of the Iranian Chemical Society vol 5 no2 pp 262ndash267 2008

[21] N Kumar A Chauhan and S Drabu ldquoSynthesis of cyanopyri-dine and pyrimidine analogues as new anti-inflammatory andantimicrobial agentsrdquo Biomedicine and Pharmacotherapy vol65 no 5 pp 375ndash380 2011

[22] J H Kwon H J Park N Chitrapriya et al ldquoEffect of numberand location of amine groups on the thermodynamic param-eters on the acridine derivatives to DNArdquo The Bulletin of theKorean Chemical Society vol 34 no 3 pp 810ndash814 2013

[23] L S LERMAN ldquoThe structure of the DNA-acridine complexrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 49 pp 94ndash102 1963

[24] H Kubinyi ldquoCombinatorial and computational approachesin structure-based drug designrdquo Current Opinion in DrugDiscovery and Development vol 1 no 1 pp 16ndash27 1998

[25] S Amuthalakshmi and A Anton Smith ldquoInsilico design of aligand for DPP IV in type II diabetesrdquo Advances in BiologicalResearch vol 7 no 6 pp 248ndash252 2013

[26] P Z Zimmet ldquoDiabetes epidemiology as a tool to triggerdiabetes research and carerdquoDiabetologia vol 42 no 5 pp 499ndash518 1999

[27] H P Rang M M Dale J M Ritter and P K MoorePharmacology Churchill Living Stone 5th edition 2003

[28] M R Kandadi P K Rajanna M K Unnikrishnan et al ldquo2-(34-Dihydro-2H-pyrrolium-1-yl)-3oxoindan-1-olate (DHPO)a novel synthetic small molecule that alleviates insulin resis-tance and lipid abnormalitiesrdquo Biochemical Pharmacology vol79 no 4 pp 623ndash631 2010

[29] R Shashikant P Kekareb P Ashwini and A Nikaljec ldquoStudieson the synthesis of novel 24-thiazolidinedione derivativeswith antidiabetic activityrdquo Iranian Journal of PharmaceuticalSciences vol 5 no 4 pp 225ndash230 2009

[30] R Subashini A Bharathi S Mohana Roopan and G Rajaku-mar ldquoSynthesis spectral characterization and larvicidal activityof acridin-1(2H)-one analoguesrdquo Spectrochimica Acta A Molec-ular and Biomolecular Spectroscopy vol 95 pp 442ndash445 2012

[31] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959

[32] A M Gray and R R Flatt ldquoNaturersquos own pharmacy thediabetes perspectiverdquo Proceedings of the Nutrition Society vol56 no 1B pp 507ndash517 1997

[33] G Ramırez M Zavala J Perez and A Zamilpa ldquoIn vitroscreening of medicinal plants used in Mexico as antidiabeticswith glucosidase and lipase inhibitory activitiesrdquo Evidence-Based Complementary and Alternative Medicine vol 2012Article ID 701261 6 pages 2012

[34] P Senthil Kumar and S Sudha ldquoEvaluation of 120572-amylase or 120572-glucosidase inhibitory prosperities of selected seaweeds fromgulf ofmannarrdquo International Research Journal of Pharmacy vol3 no 8 pp 128ndash130 2012

[35] A Pedretti L Villa and G Vistoli ldquoVEGA an open platformto develop chemo-bio-informatics applications using plug-inarchitecture and script programmingrdquo Journal of Computer-Aided Molecular Design vol 18 no 3 pp 167ndash173 2004

[36] M J S Dewar E G Zoebisch E F Healy and J J P StewartldquoAM1 a new general purpose quantum mechanical molecularmodelrdquo Journal of the American Chemical Society vol 107 no13 pp 3902ndash3909 1985

[37] E Kashani-Amin B Larijani and A E Habibi ldquoNeohesperidindihydrochalcone presentation of a small molecule activatorof mammalian alpha-amylase as an allosteric effectorrdquo FEBSLetter vol 587 pp 652ndash658 2013

[38] G M Morris D S Goodsell R S Halliday et al ldquoAutomateddocking using a Lamarckian genetic algorithm and an empiricalbinding free energy functionrdquo Journal of Computational Chem-istry vol 19 no 14 pp 1639ndash1662 1998

[39] T Liu Y M Yip L Song and S Feng ldquoInhibiting enzymaticstarch digestion by the phenolic compound diboside A amechanistic and in silico studyrdquo Food Research Internationalvol 54 pp 595ndash600 2013

[40] L J Scott and C M Spencer ldquoMiglitol a review of its thera-peutic potential in type 2 diabetes mellitusrdquo Drugs vol 59 no3 pp 521ndash549 2000

[41] A Y Y Cheng and I G Fantus ldquoOral antihyperglycemictherapy for type 2 diabetes mellitusrdquo Canadian MedicinalAssociation Journal vol 172 no 2 pp 213ndash226 2005


OH

N

NN

HO OH

R

N N

N

N

R

N

N N

N

O

O

O

OH

N

N

R

N N

HO OH

N NR R

N

N

NO O

Cl

Cl

Cl

I

II

III

IV

V

VI

NH2

H2N

H3CO

NH2

NH2

NH2NH2

NH2

H2N

R = H Me Et etc

R = H Cl CH3 NO2

R = H 2-Cl 4-Cl Br OMe NO2

R = Cl Br Me H

CH3

CH3

H3C

Figure 1 I CDK inhibitor II kinase inhibitor III antitumor IV antimalarial V antimicrobial and VI anti-inflammatory

tools They can also be used to explore the target structuresfor possible active sites generate candidate molecules dockthese molecules with the target rank them according totheir binding affinities and further optimize themolecules toimprove binding characteristics [25] Diabetes mellitus (DM)is a leading noncommunicable disease which affects morethan 100 million people worldwide and is considered as oneof the fine leading diseases which causes death in the world[26] Type-2 diabetes mellitus is a chronic metabolic disorderthat results from defects in both insulin secretion and insulinaction Management of type-2 diabetes by conventionaltherapy involves the inhibition of degradation of dietarystarch by glucosidases such as 120572-amylase and 120572-glucosidase[27]The pathogenesis of type-2 diabetes involves progressivedevelopment in insulin resistance associated with a defect ininsulin secretion leading to overt hyperglycemia Howevercompounds which improve insulin sensitivity and glucoseintolerance are somewhat limited warranting the discoveryand characterization of novel molecules targeting variouspathways involved in the pathogenesis of type-2 diabetes [28]Currently there are few drugs that are able to counteractthe development of the associated pathologiesTherefore theneed to search for new drug candidates in this field appearsto be critical Aromatic amines and thiazolidinones nucleiwould produce new compounds with significant antidiabeticproperties [29]

In our research group we have already reportedlarvicidal activity of 7-chloro-34-dihydro-9-phenylacridin-1(2H)-one and (E)-7-chloro-34-dihyro-phenyl-2-[(pyridin-2-yl)methylene]acridin-1(2H)-one [30] In continuation ofour research work on acridine moiety presently we focus onthe synthesis of 10-chloro-4-(2-chlorophenyl)-12-phenyl-56-dihydropyrimido[45-a]acridin-2-amine 3andash3f derivativesAll the synthesized dihydropyrimido[45-a]acridin-2-aminesanalogues 3andash3f were evaluated for docking and in vitroantidiabetic activity

2 Materials and Methods

21 Chemistry Melting points were determined by opencapillary method and are corrected with standard benzoicacid All solvents were distilled and dried prior to use TLCwas performed on silica gel G and the spots were exposedto iodine vapour for visualization A mixture of petroleumether and ethyl acetate was used as an eluent at differentratio Column chromatography was performed by usingsilica gel (60ndash120mesh) 1HNMR and 13CNMR spectra wererecorded in CDCl

3on a Bruker advance 400MHz instru-

ment Chemical shifts are reported in ppm using TMS asthe internal standard IR spectra were obtained on a Perkin-Elmer spectrum RXI FT-IR spectrometer (400ndash4000 cmminus1


N

Cl

O

R

N

Cl

N

R

N

Cl

Cl

1a-f

NaOHEtOH

3a-f2

(a) (b) (c) (d) (e) (f)


H3CO

NH2

OCH3

OCH3 OCH3 OCH3

R =







2) 1HNMR (400MHz



2)



3) 120575 (ppm) 238 338 1185 1250 1262

1273 1279 1283 1287 1288 1292 1293 1302 1311 13211378 1379 1461 1469 1604 1607 1609 1655 EI-MS mz43530 [M+1]






3) 120575


2)


2)


3)

120575 (ppm) 239 328 559 1109 1172 1179 1193 1202 12061253 1275 1279 1281 1284 1289 1290 1301 1303 13231350 1385 1403 1407 1449 1486 1493 1578 1584 EI-MSmz 49552 [M+1]


2)


3) 120575




3) 430 (s 2H



3) 120575 (ppm) 230 336 558 560

1121 1154 1158 1205 1249 1262 1271 1278 1288 1301


C18

C19

C20C21

C16

C12

C15C12

C11

C10C9

N1C4

C3

C2

C1

C11C6C5

C7

C22

C23

C24C25

C26N2

C27C13

C8

C14

N4

N3


1310 1319 1380 1460 1469 1506 1537 1594 1604 16121637 EI-MSmz 49535 [M+1]


2)


3) 120575


2)


2) 691 (d 119869 = 12Hz


3) 120575 (ppm)

237 338 553 1141 1150 1185 1211 1249 1262 1272 12791287 1293 1294 1302 1311 1320 1378 1393 1461 14681595 1604 1606 1609 1654 EI-MSmz 46731 [M+3]


2)




2) 723ndash726


3) 120575 (ppm) 238 337 1184 1248 1262

1273 1279 1286 1287 1293 1302 1312 1321 1354 13631377 1461 1470 1604 1607 1609 1643 EI-MS mz 47003[M+1]


2)



2) 726-727 (d 119869 = 72Hz 2H)


3)

120575 (ppm) 238 337 1184 1248 1262 1273 1279 1286 12871293 1302 1312 1321 1354 1363 1377 1461 1470 16041607 1609 1643 EI-MSmz 46923 [M+]








Volume 112939(9) A3





1= 00569 119908119877

2= 01944

119877 indices (all data) 1198771= 00712 119908119877

2= 02074


b

a

c







sample119860540








































40 60 80 100 120 140 160 180 20012

14

16

18

20

22

24

26

28

30

32

Con

cent

ratio

n of

glu

cose

(mg

dL)

Time (min)

Cont3a3b3c

3d3e3f




20 40 60 80 100 120 140 160 180 200

404550556065707580859095

100105110

Relat

ive m

ovem

ent o

f glu

cose

()

Time (min)

Cont3a3b

3c3d3e




























4 Conclusion




Acknowledgments


References













































N

Cl

O

R

N

Cl

N

R

N

Cl

Cl

1a-f

NaOHEtOH

3a-f2

(a) (b) (c) (d) (e) (f)


H3CO

NH2

OCH3

OCH3 OCH3 OCH3

R =







2) 1HNMR (400MHz



2)



3) 120575 (ppm) 238 338 1185 1250 1262

1273 1279 1283 1287 1288 1292 1293 1302 1311 13211378 1379 1461 1469 1604 1607 1609 1655 EI-MS mz43530 [M+1]






3) 120575


2)


2)


3)

120575 (ppm) 239 328 559 1109 1172 1179 1193 1202 12061253 1275 1279 1281 1284 1289 1290 1301 1303 13231350 1385 1403 1407 1449 1486 1493 1578 1584 EI-MSmz 49552 [M+1]


2)


3) 120575




3) 430 (s 2H



3) 120575 (ppm) 230 336 558 560

1121 1154 1158 1205 1249 1262 1271 1278 1288 1301


C18

C19

C20C21

C16

C12

C15C12

C11

C10C9

N1C4

C3

C2

C1

C11C6C5

C7

C22

C23

C24C25

C26N2

C27C13

C8

C14

N4

N3


1310 1319 1380 1460 1469 1506 1537 1594 1604 16121637 EI-MSmz 49535 [M+1]


2)


3) 120575


2)


2) 691 (d 119869 = 12Hz


3) 120575 (ppm)

237 338 553 1141 1150 1185 1211 1249 1262 1272 12791287 1293 1294 1302 1311 1320 1378 1393 1461 14681595 1604 1606 1609 1654 EI-MSmz 46731 [M+3]


2)




2) 723ndash726


3) 120575 (ppm) 238 337 1184 1248 1262

1273 1279 1286 1287 1293 1302 1312 1321 1354 13631377 1461 1470 1604 1607 1609 1643 EI-MS mz 47003[M+1]


2)



2) 726-727 (d 119869 = 72Hz 2H)


3)

120575 (ppm) 238 337 1184 1248 1262 1273 1279 1286 12871293 1302 1312 1321 1354 1363 1377 1461 1470 16041607 1609 1643 EI-MSmz 46923 [M+]








Volume 112939(9) A3





1= 00569 119908119877

2= 01944

119877 indices (all data) 1198771= 00712 119908119877

2= 02074


b

a

c







sample119860540








































40 60 80 100 120 140 160 180 20012

14

16

18

20

22

24

26

28

30

32

Con

cent

ratio

n of

glu

cose

(mg

dL)

Time (min)

Cont3a3b3c

3d3e3f




20 40 60 80 100 120 140 160 180 200

404550556065707580859095

100105110

Relat

ive m

ovem

ent o

f glu

cose

()

Time (min)

Cont3a3b

3c3d3e




























4 Conclusion




Acknowledgments


References













































C18

C19

C20C21

C16

C12

C15C12

C11

C10C9

N1C4

C3

C2

C1

C11C6C5

C7

C22

C23

C24C25

C26N2

C27C13

C8

C14

N4

N3


1310 1319 1380 1460 1469 1506 1537 1594 1604 16121637 EI-MSmz 49535 [M+1]


2)


3) 120575


2)


2) 691 (d 119869 = 12Hz


3) 120575 (ppm)

237 338 553 1141 1150 1185 1211 1249 1262 1272 12791287 1293 1294 1302 1311 1320 1378 1393 1461 14681595 1604 1606 1609 1654 EI-MSmz 46731 [M+3]


2)




2) 723ndash726


3) 120575 (ppm) 238 337 1184 1248 1262

1273 1279 1286 1287 1293 1302 1312 1321 1354 13631377 1461 1470 1604 1607 1609 1643 EI-MS mz 47003[M+1]


2)



2) 726-727 (d 119869 = 72Hz 2H)


3)

120575 (ppm) 238 337 1184 1248 1262 1273 1279 1286 12871293 1302 1312 1321 1354 1363 1377 1461 1470 16041607 1609 1643 EI-MSmz 46923 [M+]








Volume 112939(9) A3





1= 00569 119908119877

2= 01944

119877 indices (all data) 1198771= 00712 119908119877

2= 02074


b

a

c







sample119860540








































40 60 80 100 120 140 160 180 20012

14

16

18

20

22

24

26

28

30

32

Con

cent

ratio

n of

glu

cose

(mg

dL)

Time (min)

Cont3a3b3c

3d3e3f




20 40 60 80 100 120 140 160 180 200

404550556065707580859095

100105110

Relat

ive m

ovem

ent o

f glu

cose

()

Time (min)

Cont3a3b

3c3d3e




























4 Conclusion




Acknowledgments


References

















































Volume 112939(9) A3





1= 00569 119908119877

2= 01944

119877 indices (all data) 1198771= 00712 119908119877

2= 02074


b

a

c







sample119860540








































40 60 80 100 120 140 160 180 20012

14

16

18

20

22

24

26

28

30

32

Con

cent

ratio

n of

glu

cose

(mg

dL)

Time (min)

Cont3a3b3c

3d3e3f




20 40 60 80 100 120 140 160 180 200

404550556065707580859095

100105110

Relat

ive m

ovem

ent o

f glu

cose

()

Time (min)

Cont3a3b

3c3d3e




























4 Conclusion




Acknowledgments


References
















































































40 60 80 100 120 140 160 180 20012

14

16

18

20

22

24

26

28

30

32

Con

cent

ratio

n of

glu

cose

(mg

dL)

Time (min)

Cont3a3b3c

3d3e3f




20 40 60 80 100 120 140 160 180 200

404550556065707580859095

100105110

Relat

ive m

ovem

ent o

f glu

cose

()

Time (min)

Cont3a3b

3c3d3e




























4 Conclusion




Acknowledgments


References



























































40 60 80 100 120 140 160 180 20012

14

16

18

20

22

24

26

28

30

32

Con

cent

ratio

n of

glu

cose

(mg

dL)

Time (min)

Cont3a3b3c

3d3e3f




20 40 60 80 100 120 140 160 180 200

404550556065707580859095

100105110

Relat

ive m

ovem

ent o

f glu

cose

()

Time (min)

Cont3a3b

3c3d3e




























4 Conclusion




Acknowledgments


References




































































4 Conclusion




Acknowledgments


References
















































4 Conclusion




Acknowledgments


References






































































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