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Research ArticleSynthesis and In Vitro AMPK Activation ofCycloalkylAlkarylbiguanides with Robust In VivoAntihyperglycemic Action

Erika Gutierrez-Lara12 Carlos Martiacutenez-Conde1 Edgar Rosales-Ortega1

Juan Joseacute Ramiacuterez-Espinosa1 Julio C Rivera-Leyva1 David Centurioacuten2 Karla Carvajal3

Daniel Ortega-Cuellar3 Samuel Estrada-Soto1 and Gabriel Navarrete-Vaacutezquez1

1Facultad de Farmacia Universidad Autonoma del Estado de Morelos 62209 Cuernavaca MOR Mexico2Departamento de Farmacobiologıa Cinvestav-Coapa 14330 Ciudad de Mexico Mexico3Laboratorio de Nutricion Experimental Instituto Nacional de Pediatrıa 04150 Ciudad de Mexico Mexico

Correspondence should be addressed to Samuel Estrada-Soto enochuaemmxand Gabriel Navarrete-Vazquez gabriel navarreteuaemmx

Received 16 August 2017 Accepted 22 October 2017 Published 15 November 2017

Academic Editor Teodorico C Ramalho

Copyright copy 2017 Erika Gutierrez-Lara et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

This work describes the design synthesis in one step and the in vitro in vivo and in silico antidiabetic evaluation of a series of tenalicyclic and aromatic (alkyl +aryl alkaryl)biguanides analogues of metformin and phenformin The design was conceived usingisosteric replacement chain-ring transformation and lower and higher homologation strategies All compounds were obtainedas crystals and their structure was confirmed on the basis of their spectral data (NMR and mass spectra) and their purity wasascertained by microanalysis Compounds were in vitro evaluated as activators of AMP-Activated Protein Kinase (AMPK) Theresults indicated that compounds 4 5 and 6 showed similar or even better effect compared to metformin Docking analysis wasperformed with regulatory subunit 120574 of AMPK showing several interactions with nucleotide binding pocketThe in vivo evaluationof compounds 4ndash6 at a single dose of 50mgkg was performed in a murine experimental model of diabetes The results showedan important and robust decrease of plasmatic glucose levels (minus40) Compound 6 was selected for an oral glucose tolerance testshowing an antihyperglycemic effect similar to metformin The in vivo results indicated that compounds 4ndash6 may be effective intreating experimental T2DM

1 Introduction

Type 2 diabetes mellitus (T2DM) is a long-lasting and pro-gressive metabolic disease characterized by insulin resistancein several peripheral tissues such as livermuscle and adiposeas well as impaired insulin secretion by the pancreas [1]Metformin (a biguanide Figure 1) is the most extensivelyprescribed oral antidiabetic drug for the treatment of T2DM[2] The main effect of metformin is to decrease hepaticglucose production being a perfect agent for controlling fast-ing hyperglycemia Several mechanisms of action have beenproposed but a previous study [3] reported that metforminincreases phosphorylation of the AMP-Activated Protein

Kinase (AMPK) with subsequent activation of AMPK activ-ity in hepatocytes AMPK is a heterotrimeric protein kinasecomprised of three subunits 120572 catalytic subunit and theregulatory 120573 and 120574 subunits [3] AMPK is activated by phos-phorylation of Thr172 residue on the 120572-subunit by kinasesLKB1 and CaMKKb Furthermore AMP binds to nucleotidebinding sites on 120574-subunits to allosterically activate as wellas facilitate Thr172 phosphorylation AMPK plays a role ofldquofuel gaugerdquo of the cell as it works to guarantee that ATPlevels aremaintained under situations of energetic stress suchas exercise hypoxia and starvation [4] Due to the centralrole played by AMPK in cellular energy homeostasis it hasemerged as an attractive drug target for the treatment of a

HindawiJournal of ChemistryVolume 2017 Article ID 1212609 8 pageshttpsdoiorg10115520171212609

2 Journal of Chemistry

Metformin Phenformin

NH

NH

NH

NH

NH

N

NH NH

（2（2

Figure 1 Biguanides that activate AMPK

number of metabolic diseases such as type 2 diabetes mellitus(T2DM) [2]

Another biguanide phenformin (Figure 1) also activatesAMPK but in 1978 it was retired from the market due to itstoxicity producing lactic acidosis as a major side effect thatprompted the withdrawal of phenformin as a treatment fordiabetes [5]

In our ongoing research on molecules with antidiabeticactivity [6 7] we report in this manuscript the preparation often alicyclic and aromatic biguanides (Table 1) as well as thein vitro activation of AMPK We also describe the moleculardocking of the most active compounds in the nucleotidebinding site on 120574-subunit of AMPK and their in vivo anti-hyperglycemic effect using a streptozotocinndashnicotinamide ratmodel of noninsulin dependent diabetes mellitus

2 Materials and Methods

21 Chemicals and Analytical Methods Starting materialsand solvents were purchased from Sigma-Aldrich and wereused without any further purification Melting points weredetermined using an EZ-Melt MPA120 automated meltingpoint apparatus from Stanford Research Systems and areuncorrected Reactions were monitored by thin layer chro-matography on 02mm precoated silica gel 60 F

254Merck

plates 1H NMR spectra were recorded on Varian Oxford(400MHz) and 13C NMR (100MHz) as well as 1H NMR(200MHz) and 13C NMR (50MHz) instruments Chemicalshifts are given in ppm relative to tetramethylsilane (Me4Si120575 = 0) in DMSO-d

6and CDCl

3 119869 values are given in Hz

The following abbreviations are used s singlet d doubletq quartet dd doublet of doublet t triplet m multipletbs broad signal MS were recorded on a JEOL JMS-700spectrometer by electronic impact Element analyses havebeen carried out on an Elementar Vario ELIII instrument

22 General Procedure for the Synthesis of Compounds 1ndash10To a solution of dicyandiamide 21 (05 g 00060mol) intoluene (5mL) was added 11mol equiv of correspondingalkylamines 11ndash15 or arylamines 16ndash20 After the reactionmixture was stirred at room temperature for 15min amixture 50 50 of HCl diluted in water (25mL) was addeddropwise This mixture was stirred at reflux for 3 to 16 hAfter that the obtained residuewas neutralizedwith a dilutedsolution of NH

4OH Solvent was removed under vacuum

and the residues were washed with water The crude solidproducts were then recrystallized from ethanol affording titlecompounds (Figure 2 Table 1)

221 NN-Diethylimidodicarbonimidic Diamide (1) Yield37 White crystals obtained from ethanol Mp 190∘C (Dec)1H NMR (400MHz DMSO-1198896) 120575 116 (t 6H CH3 times 2)289 (q 4H N-CH2 times 2) 660 (bs 5H -NH times 5) ppm 13CNMR (100MHz DMSO-d6) 120575 111 (-CH3) 414 (CH2) 1629(C=NH) ppm MSEI mz ( int rel) 157 (M+ 25) 85(100) Anal Calcd for C6H15N5 C 4584 H 962 N 4455Found C 4580 H 958 N 4457

222 N-[Amino(imino)methyl]pyrrolidine-1-carboximidam-ide (2) Yield 46White crystals obtained from ethanolMp1564ndash1587∘C 1HNMR (400MHz DMSO-1198896) 120575 182 (q 4HCH2-CH2) 307 (t 4H N-CH2 times 2) 660 (bs 5H -NH times 5)ppm 13C NMR (100MHz DMSO-d6) 120575 237 (CH2-CH2)447 (CH2-N) 1629 (C=NH) ppm MSEI mz ( int rel)155 (M+ 2) 85 (100) Anal Calcd for C

6H13N5 C 4643

H 844 N 4512 Found C 4650 H 848 N 4501

223 N-[Amino(imino)methyl]piperidine-1-carboximidamide(3) Yield 63 White crystals obtained from ethanol Mp1165ndash1180∘C 1HNMR (400MHzDMSO-1198896) 120575 103 (m 4HCH2-CH2) 105 (m 2H CH

2) 340 (t 4H N-CH

2times 2) 658

(bs 5H -NH times 5) ppm 13C NMR (100MHz DMSO-d6) 120575185 (CH

2-CH2) 306 (CH

2) 561 (CH

2-N) 1629 (C=NH)

ppmMSEImz ( int rel) 169 (M+ 40) 85 (100) AnalCalcd for C

7H15N5 C 4968 H 893 N 4138 Found C

4962 H 890 N 4140

224 N-[Amino(imino)methyl]morpholine-4-carboximidam-ide (4) Yield 22White crystals obtained from ethanolMp1784ndash1805∘C 1HNMR (400MHzDMSO-d6) 120575 306 (t 4HCH2-O-CH

2) 375 (t 4H N-CH

2times 2) 661 (bs 5H NH times

5) ppm 13C NMR (100MHz DMSO-d6) 120575 432 (CH2-N)

637 (CH2-O) 1623 (C=NH) ppmMSEImz ( int rel) 171

(M+ 15) 85 (100) Anal Calcd for C6H13N5O C 4209

H 768 N 4091 Found C 4210 H 765 N 4088

225 N-[Amino(imino)methyl]-4-methylpiperazine-1-carbox-imidamide (5) Yield 54 White crystals obtained fromethanol Mp 148∘C (Dec) 1H NMR (400MHz DMSO-119889

6)

120575 248 (s 3H CH3) 273 (m 4H CH

2-N-CH

2) 338 (m

4H CH2-NMe-CH

2) 671 (bs 5H NH times 5) ppm 13C NMR

(100MHz DMSO-d6) 120575 425 (CH2-N) 433 (CH

2-N) 496

(CH3) 1633 (C=NH) ppm MSEImz ( int rel) 184 (M+

2) 85 (100) Anal Calcd for C7H16N6 C 4563 H 875

N 4561 Found C 4570 H 875 N 4558

226 N-Benzylimidodicarbonimidic Diamide (6) Yield 56White crystals obtained from ethanol Mp 1842ndash1868∘C 1HNMR (400MHz DMSO-1198896) 120575 399 (d 2H CH

2) 730 (m

2H H-2 H-6) 739 (m 2H H-3 H-5) 750 (m 1H H-4) 860(bs 6H NH times 6) ppm 13C NMR (100MHz DMSO-d6) 120575426 (CH

2-N) 1294 (C-4) 1288 (C-2 C-6) 1290 (C-3 C-5)

1345 (C-1) 1549 (C=NH) 1561 (C=NH) ppm MSEI mz( int rel) 191 (M+ 2) 91 (100) 85 (40) Anal Calcdfor C9H13N5 C 5653 H 685 N 3662 Found C 5650 H

685 N 3661

Journal of Chemistry 3

0-1

Toluene Δ

1ndash5

6ndash10

11ndash15

16ndash20

21

21

R

NH

R

NHN

NH

N

N HNH

N+

+NH

（2

NH

（2

NHNH

（2

NH

（2

NH

（2

NH

Toluene Δ

0-1

HCl（2O 50 50

HCl（2O 50 50

Figure 2 Scheme reaction to obtain compounds 1ndash10

Table 1 Physicochemical properties of biguanides 1ndash10

Compd R Yield () Mp (∘C) Mol Weight(gmol) Reaction time (h)

1 Diethylamine 37 190 (dec) 157 5

R NH

NH NH

（2

2 Pyrrolidine 46 1165ndash119 155 53 Piperidine 63 1564ndash1587 169 54 Morpholine 22 1784ndash1805 171 75 4-Methylpiperazine 54 148 (dec) 184 76 Benzylamine 56 1842ndash1868 191 77 Aniline 75 1853ndash1874 177 78 4-Nitroaniline 68 1455ndash1487 222 169 4-Chloroaniline 80 1619ndash1633 211 1010 4-Fluoroaniline 85 2075ndash2092 195 4

227 N-Phenylimidodicarbonimidic Diamide (7) Yield 75White crystals obtained from ethanol Mp 1853ndash1874∘C 1HNMR (400MHz DMSO-1198896) 120575 719 (m 5H H-2 to H-6) 991(bs 6H NH times 6) ppm 13C NMR (100MHz DMSO-d6) 1205751214 (C-4) 1237 (C-2 C-6) 1291 (C-3 C-5) 1391 (C-1) 1557(C=NH) 1616 (C=NH) ppm MSEI mz ( int rel) 177(M+ 70) 119 (100) 85 (20) Anal Calcd for C

8H11N5

C 5422 H 626 N 3952 Found C 5420 H 621 N 3960

228 N-(4-Nitrophenyl)imidodicarbonimidic Diamide (8)Yield 68 White crystals obtained from ethanol Mp1455ndash1487∘C 1HNMR (400MHz DMSO-1198896) 120575 768 (d 2HH-2 H-6 Jo = 92Hz) 821 (d 2H H-3 H-5 119869119900 = 92Hz)1107 (bs 6H NH times 6) ppm 13C NMR (100MHz DMSO-d6) 120575 1194 (C-2 C-6) 1255 (C-3 C-5) 1429 (C-1) 1445 (C-4) 1516 (C=NH) 1554 (C=NH) ppm MSEI mz ( intrel) 222 (M+ 70) 165 (100) 85 (5) Anal Calcd forC8H10N6O2 C 4324 H 454 N 3782 Found C 4331 H

450 N 3779

229 N-(4-Chlorophenyl)imidodicarbonimidic Diamide (9)Yield 80 White crystals obtained from ethanol Mp1619ndash1633∘C 1HNMR (200MHz DMSO-119889

6) 120575 730 (d 2H

H-2 H-6 119869119900 = 84Hz) 739 (d 2H H-3 H-5 119869119900 = 88Hz)999 (bs 6H NH times 6) ppm 13C NMR (50MHz DMSO-d6)120575 1225 (C-2 C-6) 12727 (C-3 C-5) 1289 (C-4) 1363 (C-1)1553 (C=NH) 1618 (C=NH)ppmMSEImz ( int rel) 211(M+ 90) 154 (100) 85 (10) Anal Calcd for C

8H10N5Cl

C 4540 H 476 N 3309 Found C 4538 H 478 N 3312

2210 N-(4-Fluorophenyl)imidodicarbonimidic Diamide (10)Yield 85 White crystals obtained from ethanol Mp2075ndash2092∘C 1H NMR (200MHz DMSO-1198896) 120575 718 (m2H H-3 H-5 119869119900 = 92Hz) 742 (m 2H H-2 H-6 119869119900 =92Hz) 991 (bs 6H NH times 6) ppm 13C NMR (50MHzDMSO-d6) 120575 1178 (d C-3 C-5 2119869119862-119865 = 1125Hz) 1256 (dC-2 C-6 3119869119862-119865 = 39Hz) 1376 (d C-1 4119869119862-119865 = 155Hz) 1609(d C-4 1119869

119862-119865 = 2947Hz) 1598 (C=NH) 1621 (C=NH)ppmMSEI mz ( int rel) 195 (M+ 80) 111 (100) 85 (70)Anal Calcd for C

8H10N5F C 4922 H 516 N 3588 Found

C 4921 H 520 N 3590

23 Biological Activity

231 In Vitro AMPK Activation Primary rat hepatocyteswere obtained by collagenase digestion as described by Berry


and Friend [8] Fresh isolated hepatocytes were equallydistributed in collagen-coated dishes and incubated at 37∘Cfor 2 h in medium 120572 MEM (Gibco cat 11900-024) 10FBS 100UmL penicillin and 100 120583gmL streptomycin Thecultured dishes were then washed three times with PBSto remove unattached dead cells Dishes were randomlyassigned to receive fresh FBS-free medium without or withdifferent concentrations of both metformin (10mM) and tenof its analogues (1 and 10mM) for 1 h After this time theplated cells were washed three times with cold PBS The cellswere then lysed using a buffer containing 50mM HEPES(pH 75) 50mM KCl 1mM EDTA 5mM EGTA 1mM glyc-erolphosphate 01 (volvol) Triton X-100 50mM NaPPi1 nM orthovanadate and 1 nM DTT a standard completeprotease inhibitor mixture (Roche) The lysates were thencentrifuged (10000119892 10min 4∘C) and the supernatants werestored at minus80∘C The activation of AMPK and its targetprotein acetyl-CoA carboxylase (ACC) was determinedby immunoblot detection with antibodies against phospho-Thr172 AMPK and phospho-Ser79 ACC1 as well as totalAMPK ACC and actin as loading controls All antibodieswere purchased from Cell Signaling

232 In Vivo Antidiabetic Activity Male Wistar rats weigh-ing an average of 300 g were used They were maintained at25∘C in a 12 h a lightdark cycle and at 45ndash65 of humidityduring experimentation time All animal procedures weredeveloped in accordance with the Mexican Federal Regu-lations for Animal Experimentation and Care ratified bythe Institutional Animal Care and Use Committee (UNAM)based on US National Institute of Health Publication 85-23[9 10]

233 Induction of Diabetes Streptozotocin (STZ) was dis-solved in citrate buffer (pH 45) and nicotinamide was dis-solved in normal physiological saline solution Non-insulin-dependent diabetes ratmodelwas induced in overnight fastedrats by a single intraperitoneal injection of 100mgkg STZ15min before the ip administration of 40mgkg of nicoti-namide Hyperglycemia was confirmed by elevated glucoseconcentration in plasma determined after 2 weeks by strip-glucometer The animals with blood glucose concentrationhigher than 200mgdL were used for the antidiabetic study[11 12]

234 Non-Insulin-Dependent Diabetes Mellitus Rat ModelThe diabetic animals were divided into three groups of fiveanimals each (119899 = 5) Rats of experimental groups weregiven a solution of compounds 4ndash6 (50mgkg body weightprepared in tween 80 10) Control group animals were alsotreated with saline plus tween 80 10Metformin (50mgkg)was used as antihyperglycemic reference drug Blood sampleswere collected from the caudal vein at 0 1 3 5 and 7 h aftervehicle compound and drug administration Blood glucoseconcentration was estimated by enzymatic glucose oxidasemethod using a commercial glucometer [13] The percentage

variation of glycemia for each groupwas calculated in relationto initial (0 h) level according to

variation of glycemia = (Glu119909 minus Glu0Glu0 ) times 100 (1)

where Glu0 were initial glycemia values and Glu119909 were theglycemia values at 1 3 5 and 7 h respectively All valueswere expressed as mean plusmn SEM Statistical significance wasestimated by analysis of variance (ANOVA) 119901 lt 005 impliessignificance

235 Glucose Tolerance Test Normoglycemic rats weredivided into groups of five animals each (119899 = 5) Thirtymin after administration of test compounds a dose of 2 gkgof glucose solution was orally administered to each animalCompound 6 (50mgkg) metformin (50mgkg) and vehicle(tween 80 10)were administered to rats in the same volumeof solution Blood samples were collected from the tail tip at 0(before oral administration) 1 15 2 25 and 3 h after vehiclepositive control or test compound administration

24 Docking Studies Discovery Studio version 35 and Pymolversion 10were used for visualizationThe crystal structure ofAMPK was retrieved from the PDB with the accession code2UV4 Docking calculations were conducted with AutoDockVina The program performs several runs in each dockingexperiment Each run provides one predicted binding modeAll water molecules and also cocrystal ligand (51015840-adenylicacid) were removed from the crystallographic structure TheAutoDock Vina plugin through Pymol program was usedwhere we generated the grid maps Each grid was centeredat the crystallographic coordinates of the cocrystal ligandThe grid dimensions were 20 times 20 times 20 A3 with pointsseparated by 10 A Also the protein file was selected as therigid part and the ligand file as the flexible one allowing allits torsions to rotate during docking AutoDock Vina usesdefault algorithms of searching and automatically preparesthe files for use as it adds charges and polar hydrogens to theprotein necessary to perform scoring calculations it clustersshowing only the main results The number of dockingruns was 10 After finishing the poses were visualized onPymol and compared against the cocrystalized ligand overthe protein

241 Docking Validation The molecular docking protocolwas validated by redocking of cocrystal ligand (51015840-adenylicacid) into the active site of the structure of AMPK Theroot mean square deviation between the cocrystal ligandand the docked structure was less than 25 A This valueindicates that the parameters for docking simulations aregood in reproducing orientation and conformation in the X-ray crystal structure of enzyme and receptors

3 Results and Discussion

31 Chemistry Compounds 1ndash10 were designed on the basisof the structure of metformin and phenformin (Figure 1


Table 1) maintaining the biguanide group removing bothdimethyl and phenylethyl side chains and substituting theproximal amino group with diethyl or cycloalkyl groupsusing a straightforward approach called chain-ring trans-formation attaining conformational constraint connectingalkyl substituents to give the corresponding cyclic analoguesThe homologation criteria were employed to pass frompyrrolidine to piperidine A homologous series is a groupof compounds that differ by a constant unit generally amethylene group [14] Morpholine and 4-methylpiperazinederivatives were selected as isosteric replacements of piperi-dine Benzyl or 4-substituted phenyl groups were designedas lower homologues with one or two methylene groups lessthan those presented by phenformin Some physicochemicalproperties of compounds 1ndash10 are described in Table 1

Compounds 1ndash10 were prepared in a single step start-ing from cyanoguanidine (21) which was condensed withseveral alkylamines 11ndash15 or aryl amines 16ndash20 under refluxconditions (Figure 2) Title compounds were recovered with22ndash85 yields and purified by recrystallization with ethanolTheir chemical structures were confirmed by spectral data(NMR and mass spectra) and their purity was ascertainedby elemental analysis

32 In Vitro Biological Activity To test the ability of eachderivative to activate AMPK an in vitro assay was performedon a primary culture of hepatocytes Aliquots of stocksolutions of the analogues (dissolved in DMSO) were dilutedwith the assay buffer usingmetformin as positive controlThephosphorylation of AMPK and its target ACC were assessedby immunoblot analysis where it is observed that aliphatic oralicyclic compounds 1ndash3were not able to activate the enzyme(data not shown) Conversely we found that AMPK phos-phorylation (activation) was increased in a concentration-dependent manner with compounds 4 5 and 6 being morepronounced with compound 6 in fact the concentrationsof three of the compounds required for activation of AMPKwere significantly lower than those of metformin (Figure 3)On the other hand aromatic biguanides 7ndash10 were unable toactivate theAMPKThese results are in accordancewith thosereported in a parallel work performed with closely relatedbiguanides [15]

In the immunoblot not only is the phosphorylatedAMPKobserved but also the phosphorylated ACC (acetyl-CoAcarboxylase) in serine 79 can be detected This enzyme isone of the targets of AMPK The phosphorylation of thisenzyme causes its inactivation and this leads to an increasein the oxidation of free fatty acids [16] The phosphorylationof ACC indicates that analogues induce the phosphorylation(activation) of AMPK which leads to modification of thedifferent metabolic pathways

33 In Silico Studies

331 Docking Analysis Based on the in vitro biologicalassay of AMPK activation the most active compounds wereselected to explain the experimental activities On this basisa preliminary molecular docking study was conducted toevaluate the putative binding mode of compounds 4ndash6

Compound Metformin

p-AMPK

P-ACC

Actin

t-AMPK

1 10 1 10 1 10 1 10

654

mM mdashmdash

MetforminC-6C-4

C-5

MetforminC-6C-4

C-5

05

10

15

20

25

p-A

MPK

t-A

MPK

(fol

d in

duct

ion)

05

10

15

20

25

p-AC

Cac

tin (f

old

indu

ctio

n)

1 10(mM)

1 10(mM)

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

Figure 3 AMPK phosphorylation by compounds 4ndash6 The ana-logues 4 and 5 are able to activate the enzyme in a similar wayto metformin whereas the analogue 6 appears to induce AMPKphosphorylation in a larger extent lowast119901 lt 005 lowastlowast119901 lt 001 andlowastlowastlowast119901 lt 0001

into the regulatory 120574-subunit of AMPK This moleculardocking reveals that compounds 4 and 5 internalize intothe nucleotide binding pocket of AMPK and interact byelectrostatic and hydrogen bonds with Asp-317 and Thr-200 and both residues are essential for the activation ofthis enzyme (Δ119866 = 52 kcalmol for both compounds)However compound 6 (the most active in vitro) showed an


Table 2 Toxicity profiles predicted for compounds 4ndash6 metformin and phenformin

CompdLD50(mgkg) Probability of inhibition

(IC50or 119870119894 lt 10 120583M)

Mouse Rat CYP450 isoform hERGip po ip po 3A4 2D6 1A2

4 400 510 260 850 001 006 001 0025 190 640 130 750 001 004 001 0036 170 710 240 850 001 010 003 005Metformin 247 810 220 960 001 002 001 001Phenformin 160 720 240 890 002 012 004 007

456

Figure 4 3D binding model of compounds 4ndash6 into the nucleotidebinding site of AMPK (120574 subunit) Compounds 4 (cyan) 5 (blue)and 6 (green) are shown as stick models whereas aminoacidsare depicted as lines A yellow dashed lines represent polar orelectrostatic interactions

additional interaction with Ser-226 increasing the dockingenergy to minus62 kcalmol Figure 4 shows the binding mode ofcompounds 4ndash6 found by AutoDock showing an extensivehydrogen bonds network

These results contribute to explaining at the molecularlevel the relevant activities of compounds 4ndash6 in the in vitrotest

332 In Silico Toxicity With the aim of anticipating potentialtoxicity issues of compounds4ndash6 a computational predictionof safety profiles was performed The toxicity parameters of4ndash6 metformin and phenformin were calculated throughthe ACDToxSuite software v 295 (Table 2)

The in silico calculation of inhibition for the three mainisoforms of CYP450 for compounds 4ndash6 was comparableto that of metformin at relevant clinical concentrations(lt10 120583M) showing low probabilities of drug-drug inter-actions and undesirable adverse effects [17] Several basicnitrogen compounds are associated with cardiovascular risksdue to human ether-a-go-go related gene (hERG) channelblockade [18ndash20] Compounds 4ndash6 showed low prediction of

VehicleMetformin4

56

1 2 3 4 5 6 7 80Time (h)

minus40

minus20

0

20

Varia

tion

of g

lyce

mia

()

lowast

lowast

lowast

lowastlowastlowast

lowastlowast

lowast

Figure 5 Effect of a single dose of compounds 4ndash6 and metformin(50mgKg intragastric 119899 = 5) or vehicle in streptozotocin-nicotinamide-induced diabetes rat model lowast119901 lt 0001 versus vehiclegroup

hERG channel blockage at clinically relevant concentrations(119870119894lt 10 120583M) being considered as potentially noncardiotoxic

compounds In the calculation of acute toxicity compounds4ndash6 demonstrated similar predicted LD

50than metformin

and phenformin by two different administration routes

34 In Vivo Antidiabetic Effect of Compounds 4ndash6 Com-pounds 4ndash6 were the most potent AMPK activators of theseries and they were selected in order to evaluate their invivo antidiabetic activity using an STZ-nicotinamide non-insulin-dependent diabetes mellitus rat model Metformin(50mgkg) was used as a positive control The antidiabeticactivity of compounds 4ndash6 was determined using a 50mgkgsingle dose by intragastric route (Figure 5)

The antidiabetic assay shows that analogue 4 significantlyreduced glucose levels compared to the vehicle and is asgood as to the control group (metformin) having at 7 hoursafter administration a percentage of glucose decrease of335 Analogue 5 also decreased glucose levels compared to


VehicleMetformin6

0

20

40

60

80

100

120

Varia

tion

of g

lyce

mia

()

1 15 2 305Time (h)

lowast

lowastlowast

lowastlowast

lowast

lowast

Figure 6 Oral glucose tolerance test Effect of a single dose ofcompound 6 and metformin (50mgKg intragastric 119899 = 5) orvehicle in streptozotocin-nicotinamide-induced diabetes rat modellowast119901 lt 005 versus vehicle

vehicle In addition its effect was similar to that presentedby metformin at the same dose The activity was retainedduring the 7 hours of experimentation At 7 hours afteradministration a 40 decrease in blood glucose levels wasobserved Analogue 6 which was the compound that showedthe best activity on AMPK activation was also active in thein vivo assay and its antihyperglycemic effect was retainedthroughout the assay

In order to verify the plausible antihyperglycemic effect ofcompound 6 glucose tolerance test curves in normoglycemicrats were obtained Dose of 50mgkg for 6 and metforminwas employed As shown in Figure 6 compound 6 displayeda significant reduction of hyperglycemic peak which wasattained at 05 h after glucose administration In Figure 6it can be seen that the animals treated with analogue 6reached a lower hyperglycemic value than the animals treatedwith metformin and compared to the vehicle 05 h after thetreatment

During the experiment glucose levels did not decreasebeyond baseline indicating that the antidiabetic effect ofcompound 6 is due to an antihyperglycemic action ratherthan a hypoglycemic effect Also compounds 4ndash6 did notincrease the lactic acid concentrations in plasma of ratstested (less than 19mM) so any evidence of lactic acidosiswas found With these results it can be concluded that themechanism of action of the analogues 4ndash6 that confer theirantidiabetic activity is similar to metformin through theactivation of AMPK and of some of the pathways that areregulated by this enzyme Previously in vitro reports agreewith the AMPK results obtained with compound 6 [15]However in our current study we have demonstrated therobust in vivo effect produced by this compound after an oraladministration Further studies are being conducted by us in

order to demonstrate the cardiovascular action of compound6 in a murine model of fructose-induced insulin resistance[21]

4 Conclusion

In summary ten alkarylbiguanides have been developed aspromising compounds for the treatment of type 2 diabetesmellitus Compounds 4ndash6 (a) exhibited AMPK activationsimilar to or greater than metformin (b) demonstrated arobust reduction of glucose levels with marked in vivo anti-hyperglycemic efficacy and (c) showed predicted low toxicityprofiles and any experimental evidence of lactic acidosisThese compounds could be an alternative to metformin theonly biguanide currently available

Disclosure

The paper is taken in part from the M Pharm thesis of EGutierrez-Lara

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported in part by the Consejo Nacional deCiencia y Tecnologıa (CONACyT) under Grant no 253814(CB-2015) The authors are in debt with Abraham Gutierrez-Hernandez M Pharm for technical assistanceThis article isdedicated to all the people mainly Pharmacist who helpedin the medication management and classification after therecent Mexicorsquos earthquake

References

[1] American Diabetes Association ldquoDiagnosis and classificationof diabetes mellitusrdquo Diabetes Care vol 32 supplement 1 ppS62ndashS67 2009

[2] S Meng J Cao Q He et al ldquoMetformin activates AMP-activated protein kinase by promoting formation of the 120572120573120574heterotrimeric complexrdquo The Journal of Biological Chemistryvol 290 no 6 pp 3393ndash3802 2015

[3] E Moreno-Arriola M El Hafidi D Ortega-Cuellar and KCarvajal ldquoAMP-activated protein kinase regulates oxidativemetabolism in Caenorhabditis elegans through the NHR-49and MDT-15 transcriptional regulatorsrdquo PLoS ONE vol 11 no1 Article ID 0148089 2016

[4] K O Cameron and R G Kurumbail ldquoRecent progress in theidentification of adenosine monophosphate-activated proteinkinase (AMPK) activatorsrdquo Bioorganic amp Medicinal ChemistryLetters vol 26 no 21 pp 5139ndash5148 2016

[5] L YangH Sha R LDavisson andLQi ldquoPhenformin activatesthe unfolded protein response in an AMP-activated proteinkinase (AMPK)-dependent mannerrdquo The Journal of BiologicalChemistry vol 288 no 19 pp 13631ndash13638 2013


[6] S Hidalgo-Figueroa J J Ramırez-Espinosa S Estrada-Soto etal ldquoDiscovery of thiazolidine-24-dionebiphenylcarbonitrilehybrid as dual ppar 120572120574 modulator with antidiabetic effect invitro in silico and in vivo approachesrdquoChemical BiologyampDrugDesign vol 81 no 4 pp 474ndash483 2013

[7] S Hidalgo-Figueroa G Navarrete-Vazquez S Estrada-Sotoet al ldquoDiscovery of new dual PPAR120574-GPR40 agonists withrobust antidiabetic activity Design synthesis and in combodrug evaluationrdquo Biomedicine amp Pharmacotherapy vol 90 pp53ndash61 2017

[8] M N Berry and D S Friend ldquoHigh-yield preparation ofisolated rat liver parenchymal cells a biochemical and finestructural studyrdquo The Journal of Cell Biology vol 43 no 3 pp506ndash520 1969

[9] U Albus ldquoGuide for the Care and Use of Laboratory Animals(8th edn)by the National Research Council of the NationalA-cademiesWashington DC National Academies Press 2011rdquoLaboratory Animals vol 46 no 3 pp 267-268 2012

[10] J A Garcıa-Dıaz G Navarrete-Vazquez S Garcıa-Jimenezet al ldquoAntidiabetic antihyperlipidemic and anti-inflammatoryeffects of tilianin in streptozotocin-nicotinamide diabetic ratsrdquoBiomedicine amp Pharmacotherapy vol 83 pp 667ndash675 2016

[11] E J Verspohl ldquoRecommended testing in diabetes researchrdquoPlanta Medica vol 68 no 7 pp 581ndash590 2002

[12] S N Goyal N M Reddy K R Patil et al ldquoChallengesand issues with streptozotocin-induced diabetes-a clinicallyrelevant animal model to understand the diabetes pathogenesisand evaluate therapeuticsrdquo Chemico-Biological Interactions vol244 pp 49ndash63 2016

[13] R R Ortiz-Andrade J C Sanchez-Salgado G Navarrete-Vazquez et al ldquoAntidiabetic and toxicological evaluations ofnaringenin in normoglycaemic and NIDDM rat models and itsimplications on extra-pancreatic glucose regulationrdquo DiabetesObesity amp Metabolism vol 10 no 11 pp 1097ndash1104 2008

[14] R B Silverman and M W Holladay The organic chemistry ofdrug design and drug action Academic Press San Diego CalifUSA 3rd edition 2014

[15] H R Bridges V A Sirvio A-N A Agip and J HirstldquoMolecular features of biguanides required for targeting ofmitochondrial respiratory complex I and activation of AMP-kinaserdquo BMC Biology vol 14 no 1 article 65 2016

[16] O Scudiero E Nigro M L Monaco et al ldquoNew syntheticAICAR derivatives with enhanced AMPK and ACC activationrdquoJournal of Enzyme Inhibition and Medicinal Chemistry vol 31no 5 pp 748ndash753 2016

[17] L Xu Y Chen Y Pan G L Skiles and M Shou ldquoPredictionof human drug-drug interactions from time-dependent inacti-vation of CYP3A4 in primary hepatocytes using a population-based simulatorrdquo Drug Metabolism and Disposition vol 37 no12 pp 2330ndash2339 2009

[18] O Taboureau and F S Joslashrgensen ldquoIn silico predictions ofhERG channel blockers in drug discovery from ligand-basedand target-based approaches to systems chemical biologyrdquoCombinatorial Chemistry amp High Throughput Screening vol 14no 5 pp 375ndash387 2011

[19] G Navarrete-Vazquez H Torres-Gomez S Hidalgo-Figueroaet al ldquoSynthesis in vitro and in silico studies of a PPAR120574 andGLUT-4 modulator with hypoglycemic effectrdquo Bioorganic ampMedicinal Chemistry Letters vol 24 no 18 pp 4575ndash4579 2014

[20] G Navarrete-Vazquez A Austrich-Olivares B Godınez-Chaparro et al ldquoDiscovery of 2-(34-dichlorophenoxy)-N-(2-morpholin-4-ylethyl)acetamide A selective 1205901 receptor ligand

with antinociceptive effectrdquo Biomedicine amp Pharmacotherapyvol 79 pp 284ndash293 2016

[21] E J Gutierrez-Lara G Navarrete-Vazquez A Sanchez-Lopezand D Centurion ldquoPharmacological evaluation of metforminand N- benzylbiguanide a novel analogue of metformin onthe vasopressor responses to adrenergic system stimulation inpithed rats with fructose-induced insulin resistancerdquo EuropeanJournal of Pharmacology vol 814 pp 313ndash323 2017

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

International Journal ofInternational Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal ofInternational Journal of


Journal of

Chemistry


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


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


Metformin Phenformin

NH

NH

NH

NH

NH

N

NH NH

（2（2

Figure 1 Biguanides that activate AMPK

number of metabolic diseases such as type 2 diabetes mellitus(T2DM) [2]

Another biguanide phenformin (Figure 1) also activatesAMPK but in 1978 it was retired from the market due to itstoxicity producing lactic acidosis as a major side effect thatprompted the withdrawal of phenformin as a treatment fordiabetes [5]

In our ongoing research on molecules with antidiabeticactivity [6 7] we report in this manuscript the preparation often alicyclic and aromatic biguanides (Table 1) as well as thein vitro activation of AMPK We also describe the moleculardocking of the most active compounds in the nucleotidebinding site on 120574-subunit of AMPK and their in vivo anti-hyperglycemic effect using a streptozotocinndashnicotinamide ratmodel of noninsulin dependent diabetes mellitus

2 Materials and Methods

21 Chemicals and Analytical Methods Starting materialsand solvents were purchased from Sigma-Aldrich and wereused without any further purification Melting points weredetermined using an EZ-Melt MPA120 automated meltingpoint apparatus from Stanford Research Systems and areuncorrected Reactions were monitored by thin layer chro-matography on 02mm precoated silica gel 60 F

254Merck

plates 1H NMR spectra were recorded on Varian Oxford(400MHz) and 13C NMR (100MHz) as well as 1H NMR(200MHz) and 13C NMR (50MHz) instruments Chemicalshifts are given in ppm relative to tetramethylsilane (Me4Si120575 = 0) in DMSO-d

6and CDCl

3 119869 values are given in Hz

The following abbreviations are used s singlet d doubletq quartet dd doublet of doublet t triplet m multipletbs broad signal MS were recorded on a JEOL JMS-700spectrometer by electronic impact Element analyses havebeen carried out on an Elementar Vario ELIII instrument

22 General Procedure for the Synthesis of Compounds 1ndash10To a solution of dicyandiamide 21 (05 g 00060mol) intoluene (5mL) was added 11mol equiv of correspondingalkylamines 11ndash15 or arylamines 16ndash20 After the reactionmixture was stirred at room temperature for 15min amixture 50 50 of HCl diluted in water (25mL) was addeddropwise This mixture was stirred at reflux for 3 to 16 hAfter that the obtained residuewas neutralizedwith a dilutedsolution of NH

4OH Solvent was removed under vacuum

and the residues were washed with water The crude solidproducts were then recrystallized from ethanol affording titlecompounds (Figure 2 Table 1)

221 NN-Diethylimidodicarbonimidic Diamide (1) Yield37 White crystals obtained from ethanol Mp 190∘C (Dec)1H NMR (400MHz DMSO-1198896) 120575 116 (t 6H CH3 times 2)289 (q 4H N-CH2 times 2) 660 (bs 5H -NH times 5) ppm 13CNMR (100MHz DMSO-d6) 120575 111 (-CH3) 414 (CH2) 1629(C=NH) ppm MSEI mz ( int rel) 157 (M+ 25) 85(100) Anal Calcd for C6H15N5 C 4584 H 962 N 4455Found C 4580 H 958 N 4457

222 N-[Amino(imino)methyl]pyrrolidine-1-carboximidam-ide (2) Yield 46White crystals obtained from ethanolMp1564ndash1587∘C 1HNMR (400MHz DMSO-1198896) 120575 182 (q 4HCH2-CH2) 307 (t 4H N-CH2 times 2) 660 (bs 5H -NH times 5)ppm 13C NMR (100MHz DMSO-d6) 120575 237 (CH2-CH2)447 (CH2-N) 1629 (C=NH) ppm MSEI mz ( int rel)155 (M+ 2) 85 (100) Anal Calcd for C

6H13N5 C 4643

H 844 N 4512 Found C 4650 H 848 N 4501

223 N-[Amino(imino)methyl]piperidine-1-carboximidamide(3) Yield 63 White crystals obtained from ethanol Mp1165ndash1180∘C 1HNMR (400MHzDMSO-1198896) 120575 103 (m 4HCH2-CH2) 105 (m 2H CH

2) 340 (t 4H N-CH

2times 2) 658

(bs 5H -NH times 5) ppm 13C NMR (100MHz DMSO-d6) 120575185 (CH

2-CH2) 306 (CH

2) 561 (CH

2-N) 1629 (C=NH)

ppmMSEImz ( int rel) 169 (M+ 40) 85 (100) AnalCalcd for C

7H15N5 C 4968 H 893 N 4138 Found C

4962 H 890 N 4140

224 N-[Amino(imino)methyl]morpholine-4-carboximidam-ide (4) Yield 22White crystals obtained from ethanolMp1784ndash1805∘C 1HNMR (400MHzDMSO-d6) 120575 306 (t 4HCH2-O-CH

2) 375 (t 4H N-CH

2times 2) 661 (bs 5H NH times

5) ppm 13C NMR (100MHz DMSO-d6) 120575 432 (CH2-N)

637 (CH2-O) 1623 (C=NH) ppmMSEImz ( int rel) 171

(M+ 15) 85 (100) Anal Calcd for C6H13N5O C 4209

H 768 N 4091 Found C 4210 H 765 N 4088

225 N-[Amino(imino)methyl]-4-methylpiperazine-1-carbox-imidamide (5) Yield 54 White crystals obtained fromethanol Mp 148∘C (Dec) 1H NMR (400MHz DMSO-119889

6)

120575 248 (s 3H CH3) 273 (m 4H CH

2-N-CH

2) 338 (m

4H CH2-NMe-CH

2) 671 (bs 5H NH times 5) ppm 13C NMR

(100MHz DMSO-d6) 120575 425 (CH2-N) 433 (CH

2-N) 496

(CH3) 1633 (C=NH) ppm MSEImz ( int rel) 184 (M+

2) 85 (100) Anal Calcd for C7H16N6 C 4563 H 875

N 4561 Found C 4570 H 875 N 4558

226 N-Benzylimidodicarbonimidic Diamide (6) Yield 56White crystals obtained from ethanol Mp 1842ndash1868∘C 1HNMR (400MHz DMSO-1198896) 120575 399 (d 2H CH

2) 730 (m

2H H-2 H-6) 739 (m 2H H-3 H-5) 750 (m 1H H-4) 860(bs 6H NH times 6) ppm 13C NMR (100MHz DMSO-d6) 120575426 (CH

2-N) 1294 (C-4) 1288 (C-2 C-6) 1290 (C-3 C-5)

1345 (C-1) 1549 (C=NH) 1561 (C=NH) ppm MSEI mz( int rel) 191 (M+ 2) 91 (100) 85 (40) Anal Calcdfor C9H13N5 C 5653 H 685 N 3662 Found C 5650 H

685 N 3661


0-1

Toluene Δ

1ndash5

6ndash10

11ndash15

16ndash20

21

21

R

NH

R

NHN

NH

N

N HNH

N+

+NH

（2

NH

（2

NHNH

（2

NH

（2

NH

（2

NH

Toluene Δ

0-1

HCl（2O 50 50

HCl（2O 50 50





R NH

NH NH

（2



8H11N5

C 5422 H 626 N 3952 Found C 5420 H 621 N 3960


450 N 3779


6) 120575 730 (d 2H


8H10N5Cl

C 4540 H 476 N 3309 Found C 4538 H 478 N 3312



8H10N5F C 4922 H 516 N 3588 Found

C 4921 H 520 N 3590























Compound Metformin

p-AMPK

P-ACC

Actin

t-AMPK

1 10 1 10 1 10 1 10

654

mM mdashmdash

MetforminC-6C-4

C-5

MetforminC-6C-4

C-5

05

10

15

20

25

p-A

MPK

t-A

MPK

(fol

d in

duct

ion)

05

10

15

20

25

p-AC

Cac

tin (f

old

indu

ctio

n)

1 10(mM)

1 10(mM)

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowastlowast








(IC50or 119870119894 lt 10 120583M)



456






VehicleMetformin4

56

1 2 3 4 5 6 7 80Time (h)

minus40

minus20

0

20

Varia

tion

of g

lyce

mia

()

lowast

lowast

lowast

lowastlowastlowast

lowastlowast

lowast




50than metformin





VehicleMetformin6

0

20

40

60

80

100

120

Varia

tion

of g

lyce

mia

()

1 15 2 305Time (h)

lowast

lowastlowast

lowastlowast

lowast

lowast






4 Conclusion


Disclosure




Acknowledgments


References

































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0-1

Toluene Δ

1ndash5

6ndash10

11ndash15

16ndash20

21

21

R

NH

R

NHN

NH

N

N HNH

N+

+NH

（2

NH

（2

NHNH

（2

NH

（2

NH

（2

NH

Toluene Δ

0-1

HCl（2O 50 50

HCl（2O 50 50





R NH

NH NH

（2



8H11N5

C 5422 H 626 N 3952 Found C 5420 H 621 N 3960


450 N 3779


6) 120575 730 (d 2H


8H10N5Cl

C 4540 H 476 N 3309 Found C 4538 H 478 N 3312



8H10N5F C 4922 H 516 N 3588 Found

C 4921 H 520 N 3590























Compound Metformin

p-AMPK

P-ACC

Actin

t-AMPK

1 10 1 10 1 10 1 10

654

mM mdashmdash

MetforminC-6C-4

C-5

MetforminC-6C-4

C-5

05

10

15

20

25

p-A

MPK

t-A

MPK

(fol

d in

duct

ion)

05

10

15

20

25

p-AC

Cac

tin (f

old

indu

ctio

n)

1 10(mM)

1 10(mM)

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowastlowast








(IC50or 119870119894 lt 10 120583M)



456






VehicleMetformin4

56

1 2 3 4 5 6 7 80Time (h)

minus40

minus20

0

20

Varia

tion

of g

lyce

mia

()

lowast

lowast

lowast

lowastlowastlowast

lowastlowast

lowast




50than metformin





VehicleMetformin6

0

20

40

60

80

100

120

Varia

tion

of g

lyce

mia

()

1 15 2 305Time (h)

lowast

lowastlowast

lowastlowast

lowast

lowast






4 Conclusion


Disclosure




Acknowledgments


References

































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





















Compound Metformin

p-AMPK

P-ACC

Actin

t-AMPK

1 10 1 10 1 10 1 10

654

mM mdashmdash

MetforminC-6C-4

C-5

MetforminC-6C-4

C-5

05

10

15

20

25

p-A

MPK

t-A

MPK

(fol

d in

duct

ion)

05

10

15

20

25

p-AC

Cac

tin (f

old

indu

ctio

n)

1 10(mM)

1 10(mM)

lowastlowast

lowastlowast

lowastlowast

lowast

lowastlowastlowast








(IC50or 119870119894 lt 10 120583M)



456






VehicleMetformin4

56

1 2 3 4 5 6 7 80Time (h)

minus40

minus20

0

20

Varia

tion

of g

lyce

mia

()

lowast

lowast

lowast

lowastlowastlowast

lowastlowast

lowast




50than metformin





VehicleMetformin6

0

20

40

60

80

100

120

Varia

tion

of g

lyce

mia

()

1 15 2 305Time (h)

lowast

lowastlowast

lowastlowast

lowast

lowast






4 Conclusion


Disclosure




Acknowledgments


References

































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




(IC50or 119870119894 lt 10 120583M)



456






VehicleMetformin4

56

1 2 3 4 5 6 7 80Time (h)

minus40

minus20

0

20

Varia

tion

of g

lyce

mia

()

lowast

lowast

lowast

lowastlowastlowast

lowastlowast

lowast




50than metformin





VehicleMetformin6

0

20

40

60

80

100

120

Varia

tion

of g

lyce

mia

()

1 15 2 305Time (h)

lowast

lowastlowast

lowastlowast

lowast

lowast






4 Conclusion


Disclosure




Acknowledgments


References

































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


VehicleMetformin6

0

20

40

60

80

100

120

Varia

tion

of g

lyce

mia

()

1 15 2 305Time (h)

lowast

lowastlowast

lowastlowast

lowast

lowast






4 Conclusion


Disclosure




Acknowledgments


References

































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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