Synthesis and in Vitro AMPK Activation of Cycloalkyl/Alkarylbiguanides with Robust in Vivo Antihyperglycemic Action

Article abstract of DOI:10.1155/2017/1212609

This work describes the design, synthesis in one step, and the in vitro, in vivo, and in silico antidiabetic evaluation of a series of ten alicyclic and aromatic (alkyl +aryl: alkaryl)biguanides, analogues of metformin and phenformin. The design was conceived using isosteric replacement, chain-ring transformation, and lower and higher homologation strategies. All compounds were obtained as crystals and their structure was confirmed on the basis of their spectral data (NMR and mass spectra), and their purity was ascertained by microanalysis. Compounds were in vitro evaluated as activators of AMP-Activated Protein Kinase (AMPK). The results indicated that compounds 4, 5, and 6 showed similar or even better effect compared to metformin. Docking analysis was performed with regulatory subunit γ of AMPK, showing several interactions with nucleotide binding pocket. The in vivo evaluation of compounds 4-6 at a single dose of 50 mg/kg was performed in a murine experimental model of diabetes. The results showed an important and robust decrease of plasmatic glucose levels (-40%). Compound 6 was selected for an oral glucose tolerance test, showing an antihyperglycemic effect similar to metformin. The in vivo results indicated that compounds 4-6 may be effective in treating experimental T2DM.

Full text of DOI:10.1155/2017/1212609

Hindawi
Journal of Chemistry
Volume 2017, Article ID 1212609, 8 pages
https://doi.org/10.1155/2017/1212609
Research Article
Synthesis and In Vitro AMPK Activation of
Cycloalkyl/Alkarylbiguanides with Robust In Vivo
Antihyperglycemic Action
Erika Gutierrez-Lara,1,2 Carlos Martínez-Conde,1 Edgar Rosales-Ortega,1
Juan José Ramírez-Espinosa,1 Julio C. Rivera-Leyva,1 David Centurión,2 Karla Carvajal,3
Daniel Ortega-Cuellar,3 Samuel Estrada-Soto,1 and Gabriel Navarrete-Vázquez1
1
´
Facultad de Farmacia, Universidad Autonoma del Estado de Morelos, 62209 Cuernavaca, MOR, Mexico
2
3
´
´
Departamento de Farmacobiologıa, Cinvestav-Coapa, 14330 Ciudad de Mexico, Mexico
´
´
´
Laboratorio de Nutricion Experimental, Instituto Nacional de Pediatrıa, 04150 Ciudad de Mexico, Mexico
Correspondence should be addressed to Samuel Estrada-Soto; enoch@uaem.mx
and Gabriel Navarrete-Va´zquez; gabriel navarrete@uaem.mx
Received 16 August 2017; Accepted 22 October 2017; Published 15 November 2017
Academic Editor: Teodorico C. Ramalho
Copyright © 2017 Erika Gutierrez-Lara et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
is work describes the design, synthesis in one step, and the in vitro, in vivo, and in silico antidiabetic evaluation of a series of ten
alicyclic and aromatic (alkyl +aryl: alkaryl)biguanides, analogues of metformin and phenformin. e design was conceived using
isosteric replacement, chain-ring transformation, and lower and higher homologation strategies. All compounds were obtained
as crystals and their structure was confirmed on the basis of their spectral data (NMR and mass spectra), and their purity was
ascertained by microanalysis. Compounds were in vitro evaluated as activators of AMP-Activated Protein Kinase (AMPK). e
results indicated that compounds 4, 5, and 6 showed similar or even better effect compared to metformin. Docking analysis was
performed with regulatory subunit ꢀ of AMPK, showing several interactions with nucleotide binding pocket. e in vivo evaluation
of compounds 4–6 at a single dose of 50 mg/kg was performed in a murine experimental model of diabetes. e results showed
an important and robust decrease of plasmatic glucose levels (−40%). Compound 6 was selected for an oral glucose tolerance test,
showing an antihyperglycemic effect similar to metformin. e in vivo results indicated that compounds 4–6 may be effective in
treating experimental T2DM.
1. Introduction
Kinase (AMPK), with subsequent activation of AMPK activ-
ity in hepatocytes. AMPK is a heterotrimeric protein kinase
comprised of three subunits: ꢁ catalytic subunit and the
regulatory ꢂ and ꢀ subunits [3]. AMPK is activated by phos-
phorylation of r172 residue on the ꢁ-subunit by kinases
LKB1 and CaMKKb. Furthermore, AMP binds to nucleotide
binding sites on ꢀ-subunits to allosterically activate as well
as facilitate r172 phosphorylation. AMPK plays a role of
“fuel gauge” of the cell as it works to guarantee that ATP
levels are maintained under situations of energetic stress such
as exercise, hypoxia, and starvation [4]. Due to the central
role played by AMPK in cellular energy homeostasis, it has
emerged as an attractive drug target for the treatment of a
Type 2 diabetes mellitus (T2DM) is a long-lasting and pro-
gressive metabolic disease characterized by insulin resistance
in several peripheral tissues such as liver, muscle, and adipose,
as well as impaired insulin secretion by the pancreas [1].
Metformin (a biguanide, Figure 1) is the most extensively
prescribed oral antidiabetic drug for the treatment of T2DM
[2]. e main effect of metformin is to decrease hepatic
glucose production, being a perfect agent for controlling fast-
ing hyperglycemia. Several mechanisms of action have been
proposed, but a previous study [3] reported that metformin
increases phosphorylation of the AMP-Activated Protein

2
Journal of Chemistry
2.2.1. N,N-Diethylimidodicarbonimidic Diamide (1). Yield
NH
NH
NH
NH
37%. White crystals obtained from ethanol. Mp 190∘C (Dec).
．（2
1H NMR (400 MHz, DMSO-ꢆ6) ꢃ: 1.16 (t, 6H, CH × 2),
N
H
N
H
．（2
N
H
N
3
2.89 (q, 4H, N-CH × 2), 6.60 (bs, 5H, -NH × 5) ppm. 13C
2
Phenformin
Metformin
NMR (100 MHz, DMSO-d6) ꢃ: 11.1 (-CH ), 41.4 (CH ), 162.9
3
2
(C=NH) ppm. MS/EI: m/z (% int. rel). 157 (M+, 25%), 85
Figure 1: Biguanides that activate AMPK.
(100%). Anal. Calcd for C H N : C, 45.84; H, 9.62; N, 44.55.
6
15
5
Found: C, 45.80; H, 9.58; N, 44.57.
2.2.2. N-[Amino(imino)methyl]pyrrolidine-1-carboximidam-
ide (2). Yield 46%. White crystals obtained from ethanol. Mp
156.4–158.7∘C. 1H NMR (400 MHz, DMSO-ꢆ6) ꢃ: 1.82 (q, 4H,
CH -CH ), 3.07 (t, 4H, N-CH × 2), 6.60 (bs, 5H, -NH × 5)
number of metabolic diseases such as type 2 diabetes mellitus
(T2DM) [2].
Another biguanide, phenformin (Figure 1), also activates
AMPK, but in 1978 it was retired from the market due to its
toxicity, producing lactic acidosis as a major side effect that
prompted the withdrawal of phenformin as a treatment for
diabetes [5].
In our ongoing research on molecules with antidiabetic
activity [6, 7], we report in this manuscript the preparation of
ten alicyclic and aromatic biguanides (Table 1), as well as the
in vitro activation of AMPK. We also describe the molecular
docking of the most active compounds in the nucleotide
binding site on ꢀ-subunit of AMPK, and their in vivo anti-
hyperglycemic effect using a streptozotocin–nicotinamide rat
model of noninsulin dependent diabetes mellitus.
2
2
2
ppm. 13C NMR (100 MHz, DMSO-d6) ꢃ: 23.7 (CH -CH ),
2
2
44.7 (CH -N), 162.9 (C=NH) ppm. MS/EI: m/z (% int. rel).
2
155 (M+, 2%), 85 (100%). Anal. Calcd for C H N : C, 46.43;
6
13
5
H, 8.44; N, 45.12. Found: C, 46.50; H, 8.48; N, 45.01.
2.2.3. N-[Amino(imino)methyl]piperidine-1-carboximidamide
(3). Yield 63%. White crystals obtained from ethanol. Mp
116.5–118.0∘C. 1H NMR (400 MHz, DMSO-ꢆ6) ꢃ: 1.03 (m, 4H,
CH -CH ), 1.05 (m, 2H, CH ) 3.40 (t, 4H, N-CH × 2), 6.58
2
2
2
2
(bs, 5H, -NH × 5) ppm. 13C NMR (100 MHz, DMSO-d6) ꢃ:
18.5 (CH -CH ), 30.6 (CH ), 56.1 (CH -N), 162.9 (C=NH)
2
2
2
2
ppm. MS/EI: m/z (% int. rel). 169 (M+, 40%), 85 (100%). Anal.
Calcd for C H N : C, 49.68; H, 8.93; N, 41.38. Found: C,
7
15
5
2. Materials and Methods
49.62; H, 8.90; N, 41.40.
2.1. Chemicals and Analytical Methods. Starting materials
and solvents were purchased from Sigma-Aldrich and were
used without any further purification. Melting points were
determined using an EZ-Melt MPA120 automated melting
point apparatus from Stanford Research Systems and are
uncorrected. Reactions were monitored by thin layer chro-
2.2.4. N-[Amino(imino)methyl]morpholine-4-carboximidam-
ide (4). Yield 22%. White crystals obtained from ethanol. Mp
178.4–180.5∘C. 1H NMR (400 MHz, DMSO-d6) ꢃ: 3.06 (t, 4H,
CH -O-CH ), 3.75 (t, 4H, N-CH × 2), 6.61 (bs, 5H, NH ×
2
2
2
5) ppm. 13C NMR (100 MHz, DMSO-d6) ꢃ: 43.2 (CH -N),
2
63.7 (CH -O), 162.3 (C=NH) ppm. MS/EI: m/z (% int. rel). 171
2
matography on 0.2 mm precoated silica gel 60 F Merck
254
(M+, 15%), 85 (100%). Anal. Calcd for C H N O: C, 42.09;
1
6
13
5
plates. H NMR spectra were recorded on Varian Oxford
H, 7.68; N, 40.91. Found: C, 42.10; H, 7.65; N, 40.88.
(400 MHz) and 13C NMR (100 MHz), as well as H NMR
1
(200 MHz) and 13C NMR (50 MHz) instruments. Chemical
2.2.5. N-[Amino(imino)methyl]-4-methylpiperazine-1-carbox-
shifs are given in ppm relative to tetramethylsilane (Me Si,
4
imidamide (5). Yield 54%. White crystals obtained from
ꢃ ꢄ =) in DMSO-d and CDCl ; ꢅ values are given in Hz.
6
3
ethanol. Mp 148∘C (Dec). H NMR (400 MHz, DMSO-ꢆ6)
1
e following abbreviations are used: s, singlet; d, doublet;
q, quartet; dd, doublet of doublet; t, triplet; m, multiplet;
bs, broad signal. MS were recorded on a JEOL JMS-700
spectrometer by electronic impact. Element analyses have
been carried out on an Elementar Vario ELIII instrument.
ꢃ: 2.48 (s, 3H, CH ), 2.73 (m, 4H, CH -N-CH ), 3.38 (m,
3
2
6
2
4H, CH -NMe-CH ), 6.71 (bs, 5H, NH × 5) ppm. 13C NMR
2
2
(100 MHz, DMSO-d6) ꢃ: 42.5 (CH -N), 43.3 (CH -N), 49.6
2
2
(CH ), 163.3 (C=NH) ppm. MS/EI: m/z (% int. rel). 184 (M+,
3
2%), 85 (100%). Anal. Calcd for C H N : C, 45.63; H, 8.75;
7
16
N, 45.61. Found: C, 45.70; H, 8.75; N, 45.58.
2.2. General Procedure for the Synthesis of Compounds 1–10.
To a solution of dicyandiamide 21 (0.5 g, 0.0060 mol) in
toluene (5 mL) was added 1.1 mol equiv of corresponding
alkylamines 11–15 or arylamines 16–20. Afer the reaction
mixture was stirred at room temperature for 15 min, a
mixture 50 : 50 of HCl diluted in water (2.5 mL) was added
dropwise. is mixture was stirred at reflux for 3 to 16 h.
Afer that, the obtained residue was neutralized with a diluted
2.2.6. N-Benzylimidodicarbonimidic Diamide (6). Yield 56%.
White crystals obtained from ethanol. Mp 184.2–186.8∘C. 1H
NMR (400 MHz, DMSO-ꢆ6) ꢃ: 3.99 (d, 2H, CH ), 7.30 (m,
2
2H, H-2, H-6), 7.39 (m, 2H, H-3, H-5), 7.50 (m, 1H, H-4), 8.60
(bs, 6H, NH × 6) ppm. 13C NMR (100 MHz, DMSO-d6) ꢃ:
42.6 (CH -N), 129.4 (C-4), 128.8 (C-2, C-6), 129.0 (C-3, C-5),
2
solution of NH OH. Solvent was removed under vacuum,
134.5 (C-1), 154.9 (C=NH), 156.1 (C=NH) ppm. MS/EI: m/z
(% int. rel). 191 (M+, 2%), 91 (100%), 85 (40%). Anal. Calcd
for C H N : C, 56.53; H, 6.85; N, 36.62. Found: C, 56.50; H,
4
and the residues were washed with water. e crude solid
products were then recrystallized from ethanol affording title
compounds (Figure 2, Table 1).
9
13
5
6.85; N, 36.61.

Journal of Chemistry
3
NH
NH
NH
N
Toluene, Δ
+
N
H
．（2
N
H
N
．（2
N
H
21
HCl/（2O 50 : 50
1–5
11–15
．（2
NH
NH
Toluene, Δ
NH
0-1
．（2
N
H
NH
N
+
HCl/（2O 50 : 50
．（2
N
H
0-1
R
6–10
16–20
21
R
Figure 2: Scheme reaction to obtain compounds 1–10.
Table 1: Physicochemical properties of biguanides 1–10.
Mol. Weight
Compd.
R
Yield (%)
Mp (∘C)
Reaction time (h)
(g/mol)
157
1
2
3
4
5
6
7
8
9
10
Diethylamine
Pyrrolidine
Piperidine
37
46
63
22
54
56
75
68
80
85
190 (dec)
116.5–119
156.4–158.7
178.4–180.5
148 (dec)
184.2–186.8
185.3–187.4
145.5–148.7
161.9–163.3
207.5–209.2
5
5
5
7
7
7
7
16
10
4
155
169
171
184
191
177
222
211
195
NH
NH
Morpholine
4-Methylpiperazine
Benzylamine
Aniline
4-Nitroaniline
4-Chloroaniline
4-Fluoroaniline
N
H
．（2
R
2.2.7. N-Phenylimidodicarbonimidic Diamide (7). Yield 75%.
White crystals obtained from ethanol. Mp 185.3–187.4∘C. 1H
NMR (400 MHz, DMSO-ꢆ6) ꢃ: 7.19 (m, 5H, H-2 to H-6), 9.91
(bs, 6H, NH × 6) ppm. 13C NMR (100 MHz, DMSO-d6) ꢃ:
121.4 (C-4), 123.7 (C-2, C-6), 129.1 (C-3, C-5), 139.1 (C-1), 155.7
(C=NH), 161.6 (C=NH) ppm. MS/EI: m/z (% int. rel). 177
(M+, 70%), 119 (100%), 85 (20%). Anal. Calcd for C H N :
H-2, H-6, ꢅꢇ ꢄ 8.4 Hz), 7.39 (d, 2H, H-3, H-5, ꢅꢇ ꢄ 8.8 Hz),
9.99 (bs, 6H, NH × 6) ppm. 13C NMR (50 MHz, DMSO-d6)
ꢃ: 122.5 (C-2, C-6), 127.27 (C-3, C-5), 128.9 (C-4), 136.3 (C-1),
155.3 (C=NH), 161.8 (C=NH) ppm. MS/EI: m/z (% int. rel). 211
(M+, 90%), 154 (100%), 85 (10%). Anal. Calcd for C H N Cl:
8
10
5
C, 45.40; H, 4.76; N, 33.09. Found: C, 45.38; H, 4.78; N, 33.12.
8
11
5
C, 54.22; H, 6.26; N, 39.52. Found: C, 54.20; H, 6.21; N, 39.60.
2.2.10. N-(4-Fluorophenyl)imidodicarbonimidic Diamide (10).
Yield 85%. White crystals obtained from ethanol. Mp
1
207.5–209.2∘C. H NMR (200 MHz, DMSO-ꢆ6) ꢃ: 7.18 (m,
2.2.8. N-(4-Nitrophenyl)imidodicarbonimidic Diamide (8).
Yield 68%. White crystals obtained from ethanol. Mp
145.5–148.7∘C. 1H NMR (400 MHz, DMSO-ꢆ6) ꢃ: 7.68 (d, 2H,
H-2, H-6, Jo = 9.2 Hz), 8.21 (d, 2H, H-3, H-5, ꢅꢇ ꢄ 9.2 Hz),
11.07 (bs, 6H, NH × 6) ppm. 13C NMR (100 MHz, DMSO-
d6) ꢃ: 119.4 (C-2, C-6), 125.5 (C-3, C-5), 142.9 (C-1), 144.5 (C-
4), 151.6 (C=NH), 155.4 (C=NH) ppm. MS/EI: m/z (% int.
rel). 222 (M+, 70%), 165 (100%), 85 (5%). Anal. Calcd for
C H N O : C, 43.24; H, 4.54; N, 37.82. Found: C, 43.31; H,
2H, H-3, H-5, ꢅꢇ ꢄ 9.2 Hz), 7.42 (m, 2H, H-2, H-6, ꢅꢇ ꢄ
9.2 Hz), 9.91 (bs, 6H, NH × 6) ppm. 13C NMR (50 MHz,
DMSO-d6) ꢃ: 117.8 (d, C-3, C-5, 2ꢅꢀ-ꢁ 4ꢄ 11.25 Hz), 125.6 (d,
C-2, C-6, 3ꢅꢀ-ꢁ ꢄ 3.9 Hz), 137.6 (d, C-1, ꢅꢀ-ꢁ ꢄ 1.55 Hz), 160.9
(d, C-4, 1ꢅꢀ-ꢁ ꢄ 294.7 Hz), 159.8 (C=NH), 162.1 (C=NH) ppm.
MS/EI: m/z (% int. rel). 195 (M+, 80%), 111 (100%), 85 (70%).
Anal. Calcd for C H N F: C, 49.22; H, 5.16; N, 35.88. Found:
8
10
5
C, 49.21; H, 5.20; N, 35.90.
8
10
6
2
4.50; N, 37.79.
2.3. Biological Activity
2.2.9. N-(4-Chlorophenyl)imidodicarbonimidic Diamide (9).
Yield 80%. White crystals obtained from ethanol. Mp
161.9–163.3∘C. 1H NMR (200 MHz, DMSO-ꢆ6) ꢃ: 7.30 (d, 2H,
2.3.1. In Vitro AMPK Activation. Primary rat hepatocytes
were obtained by collagenase digestion as described by Berry

4
Journal of Chemistry
and Friend [8]. Fresh isolated hepatocytes were equally
distributed in collagen-coated dishes and incubated at 37∘C
for 2 h in medium ꢁ MEM (Gibco; cat 11900-024), 10%
FBS, 100 U/mL penicillin, and 100 ꢈg/mL streptomycin. e
cultured dishes were then washed three times with PBS
to remove unattached dead cells. Dishes were randomly
assigned to receive fresh FBS-free medium without or with
different concentrations of both metformin (10 mM) and ten
of its analogues (1 and 10 mM) for 1 h. Afer this time, the
plated cells were washed three times with cold PBS. e cells
were then lysed using a buffer containing 50 mM HEPES
(pH 7.5), 50 mM KCl, 1 mM EDTA, 5 mM EGTA, 1 mM glyc-
erolphosphate, 0.1% (vol/vol) Triton X-100, 50 mM NaPPi,
1 nM orthovanadate, and 1 nM DTT, a standard complete
protease inhibitor mixture (Roche). e lysates were then
centrifuged (10,000ꢉ, 10 min, 4∘C), and the supernatants were
stored at −80∘C. e activation of AMPK and its target
protein, acetyl-CoA carboxylase (ACC), was determined
by immunoblot detection with antibodies against phospho-
variation of glycemia for each group was calculated in relation
to initial (0 h) level, according to
Gluꢋ − Glu=
% variation of glycemia ꢄ (
) × 1==, (1)
Glu=
where Glu0 were initial glycemia values and Gluꢋ were the
glycemia values at 1, 3, 5, and 7 h, respectively. All values
were expressed as mean SEM. Statistical significance was
estimated by analysis of variance (ANOVA); ꢌ < =.=5 implies
significance.
2.3.5. Glucose Tolerance Test. Normoglycemic rats were
divided into groups of five animals each (ꢊ ꢄ 5). irty
min afer administration of test compounds, a dose of 2 g/kg
of glucose solution was orally administered to each animal.
Compound 6 (50 mg/kg), metformin (50 mg/kg), and vehicle
(tween 80, 10%) were administered to rats in the same volume
of solution. Blood samples were collected from the tail tip at 0
(before oral administration), 1, 1.5, 2, 2.5, and 3 h afer vehicle,
positive control, or test compound administration.
r172 AMPK and phospho-Ser79 ACC1, as well as total
AMPK, ACC, and actin as loading controls. All antibodies
were purchased from Cell Signaling.
2.4. Docking Studies. Discovery Studio version 3.5 and Pymol
version 1.0 were used for visualization. e crystal structure of
AMPK was retrieved from the PDB with the accession code:
2UV4. Docking calculations were conducted with AutoDock
Vina. e program performs several runs in each docking
experiment. Each run provides one predicted binding mode.
All water molecules and also cocrystal ligand (5ꢂ-adenylic
acid) were removed from the crystallographic structure. e
AutoDock Vina plugin through Pymol program was used,
where we generated the grid maps. Each grid was centered
2.3.2. In Vivo Antidiabetic Activity. Male Wistar rats weigh-
ing an average of 300 g were used. ey were maintained at
25∘C in a 12 h a light/dark cycle and at 45–65% of humidity
during experimentation time. All animal procedures were
developed in accordance with the Mexican Federal Regu-
lations for Animal Experimentation and Care, ratified by
the Institutional Animal Care and Use Committee (UNAM)
based on US National Institute of Health Publication #85-23
[9, 10].
at the crystallographic coordinates of the cocrystal ligand.
3
˚
e grid dimensions were 2= × 2= × 2= A with points
˚
separated by 1.0 A. Also, the protein file was selected as the
2.3.3. Induction of Diabetes. Streptozotocin (STZ) was dis-
solved in citrate buffer (pH 4.5) and nicotinamide was dis-
solved in normal physiological saline solution. Non-insulin-
dependent diabetes rat model was induced in overnight fasted
rats by a single intraperitoneal injection of 100 mg/kg STZ,
15 min before the i.p. administration of 40 mg/kg of nicoti-
namide. Hyperglycemia was confirmed by elevated glucose
concentration in plasma, determined afer 2 weeks by strip-
glucometer. e animals with blood glucose concentration
higher than 200 mg/dL were used for the antidiabetic study
[11, 12].
rigid part, and the ligand file as the flexible one, allowing all
its torsions to rotate during docking. AutoDock Vina uses
default algorithms of searching and automatically prepares
the files for use as it adds charges and polar hydrogens to the
protein necessary to perform scoring calculations; it clusters
showing only the main results. e number of docking
runs was 10. Afer finishing, the poses were visualized on
Pymol and compared against the cocrystalized ligand over
the protein.
2.4.1. Docking Validation. e molecular docking protocol
was validated by redocking of cocrystal ligand (5ꢂ-adenylic
acid) into the active site of the structure of AMPK. e
root mean square deviation between the cocrystal ligand
2.3.4. Non-Insulin-Dependent Diabetes Mellitus Rat Model.
e diabetic animals were divided into three groups of five
animals each (ꢊ ꢄ 5). Rats of experimental groups were
given a solution of compounds 4–6 (50 mg/kg body weight,
prepared in tween 80, 10%). Control group animals were also
treated with saline plus tween 80, 10%. Metformin (50 mg/kg)
was used as antihyperglycemic reference drug. Blood samples
were collected from the caudal vein at 0, 1, 3, 5, and 7 h afer
vehicle, compound, and drug administration. Blood glucose
concentration was estimated by enzymatic glucose oxidase
method using a commercial glucometer [13]. e percentage
˚
and the docked structure was less than 2.5 A. is value
indicates that the parameters for docking simulations are
good in reproducing orientation and conformation in the X-
ray crystal structure of enzyme and receptors.
3. Results and Discussion
3.1. Chemistry. Compounds 1–10 were designed on the basis
of the structure of metformin and phenformin (Figure 1,

Journal of Chemistry
5
Compound
p-AMPK
4
5
6
Metformin
Table 1), maintaining the biguanide group, removing both
dimethyl and phenylethyl side chains, and substituting the
proximal amino group with diethyl or cycloalkyl groups
using a straightforward approach called chain-ring trans-
formation, attaining conformational constraint connecting
alkyl substituents to give the corresponding cyclic analogues.
e homologation criteria were employed to pass from
pyrrolidine to piperidine. A homologous series is a group
of compounds that differ by a constant unit, generally a
methylene group [14]. Morpholine and 4-methylpiperazine
derivatives were selected as isosteric replacements of piperi-
dine. Benzyl or 4-substituted phenyl groups were designed
as lower homologues, with one or two methylene groups less
than those presented by phenformin. Some physicochemical
properties of compounds 1–10 are described in Table 1.
Compounds 1–10 were prepared in a single step start-
ing from cyanoguanidine (21), which was condensed with
several alkylamines 11–15 or aryl amines 16–20 under reflux
conditions (Figure 2). Title compounds were recovered with
22–85% yields and purified by recrystallization with ethanol.
eir chemical structures were confirmed by spectral data
(NMR and mass spectra), and their purity was ascertained
by elemental analysis.
t-AMPK
P-ACC
Actin
mM
2.5
—
1
10
1
10
1
10
—
1
10
∗∗∗
2.0
1.5
1.0
0.5
∗∗∗
∗∗∗
∗∗
∗∗
∗
1
10
(mM)
3.2. In Vitro Biological Activity. To test the ability of each
derivative to activate AMPK, an in vitro assay was performed
on a primary culture of hepatocytes. Aliquots of stock
solutions of the analogues (dissolved in DMSO) were diluted
with the assay buffer, using metformin as positive control. e
phosphorylation of AMPK and its target ACC were assessed
by immunoblot analysis, where it is observed that aliphatic or
alicyclic compounds 1–3 were 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 more
pronounced with compound 6; in fact, the concentrations
of three of the compounds required for activation of AMPK
were significantly lower than those of metformin (Figure 3).
On the other hand, aromatic biguanides 7–10 were unable to
activate the AMPK. ese results are in accordance with those
reported in a parallel work performed with closely related
biguanides [15].
In the immunoblot not only is the phosphorylated AMPK
observed, but also the phosphorylated ACC (acetyl-CoA
carboxylase) in serine 79 can be detected. is enzyme is
one of the targets of AMPK. e phosphorylation of this
enzyme causes its inactivation, and this leads to an increase
in the oxidation of free fatty acids [16]. e phosphorylation
of ACC indicates that analogues induce the phosphorylation
(activation) of AMPK, which leads to modification of the
different metabolic pathways.
C-4
C-5
C-6
Metformin
2.5
2.0
1.5
1.0
0.5
∗∗
∗∗
∗∗
1
10
(mM)
C-4
C-5
C-6
Metformin
Figure 3: AMPK phosphorylation by compounds 4–6. e ana-
logues 4 and 5 are able to activate the enzyme in a similar way
to metformin, whereas the analogue 6 appears to induce AMPK
∗
∗∗
ꢌ
ꢌ
phosphorylation in a larger extent.
< =.=5,
< =.=1, and
∗∗∗
ꢌ
< =.==1.
into the regulatory ꢀ-subunit of AMPK. is molecular
docking reveals that compounds 4 and 5 internalize into
the nucleotide binding pocket of AMPK and interact by
electrostatic and hydrogen bonds with Asp-317 and r-
200, and both residues are essential for the activation of
3.3. In Silico Studies
3.3.1. Docking Analysis. Based on the in vitro biological
assay of AMPK activation, the most active compounds were
selected to explain the experimental activities. On this basis,
a preliminary molecular docking study was conducted to
evaluate the putative binding mode of compounds 4–6
this enzyme (Δꢍ
ꢄ
5.2 kcal/mol for both compounds).
However, compound 6 (the most active in vitro) showed an

6
Journal of Chemistry
Table 2: Toxicity profiles predicted for compounds 4–6, metformin, and phenformin.
Probability of inhibition
LD (mg/kg)
50
(IC or ꢎꢏ < 1= ꢈM)
50
Compd.
Mouse
Rat
CYP450 isoform
hERG
i.p.
400
190
170
247
160
p.o.
510
640
710
810
720
i.p.
260
130
240
220
240
p.o.
850
750
850
960
890
3A4
0.01
0.01
0.01
0.01
0.02
2D6
0.06
0.04
0.10
0.02
0.12
1A2
0.01
0.01
0.03
0.01
0.04
4
5
6
0.02
0.03
0.05
0.01
0.07
Metformin
Phenformin
4
5
6
20
0
−20
−40
∗
∗
∗
∗
∗
∗
∗
∗
∗
0
1
2
3
4
5
6
7
8
Time (h)
Figure 4: 3D binding model of compounds 4–6 into the nucleotide
binding site of AMPK (ꢀ subunit). Compounds 4 (cyan), 5 (blue),
and 6 (green) are shown as stick models, whereas aminoacids
are depicted as lines. A yellow dashed lines represent polar or
electrostatic interactions.
Vehicle
5
6
Metformin
4
Figure 5: Effect of a single dose of compounds 4–6 and metformin
(50 mg/Kg, intragastric, ꢊ
ꢄ
5), or vehicle in streptozotocin-
nicotinamide-induced diabetes rat model. ∗ < =.==1 versus vehicle
ꢌ
group.
additional interaction with Ser-226, increasing the docking
energy to −6.2 kcal/mol. Figure 4 shows the binding mode of
compounds 4–6 found by AutoDock showing an extensive
hydrogen bonds network.
hERG channel blockage at clinically relevant concentrations
(ꢎꢃ < 1= ꢈM), being considered as potentially noncardiotoxic
compounds. In the calculation of acute toxicity, compounds
ese results contribute to explaining at the molecular
level the relevant activities of compounds 4–6 in the in vitro
test.
4–6 demonstrated similar predicted LD than metformin
50
and phenformin by two different administration routes.
3.3.2. In Silico Toxicity. With the aim of anticipating potential
toxicity issues of compounds4–6, a computational prediction
of safety profiles was performed. e toxicity parameters of
4–6, metformin, and phenformin were calculated through
the ACD/ToxSuite sofware, v. 2.95 (Table 2).
e in silico calculation of inhibition for the three main
isoforms of CYP450 for compounds 4–6 was comparable
to that of metformin at relevant clinical concentrations
(<10 ꢈM), showing low probabilities of drug-drug inter-
actions and undesirable adverse effects [17]. Several basic
nitrogen compounds are associated with cardiovascular risks
due to human ether-a-go-go related gene (hERG) channel
blockade [18–20]. Compounds 4–6 showed low prediction of
3.4. In Vivo Antidiabetic Effect of Compounds 4–6. Com-
pounds 4–6 were the most potent AMPK activators of the
series, and they were selected in order to evaluate their in
vivo antidiabetic activity using an STZ-nicotinamide non-
insulin-dependent diabetes mellitus rat model. Metformin
(50 mg/kg) was used as a positive control. e antidiabetic
activity of compounds 4–6 was determined using a 50 mg/kg
single dose, by intragastric route (Figure 5).
e antidiabetic assay shows that analogue 4 significantly
reduced glucose levels compared to the vehicle and is as
good as to the control group (metformin), having at 7 hours
afer administration a percentage of glucose decrease of
33.5%. Analogue 5 also decreased glucose levels compared to

Journal of Chemistry
7
120
order to demonstrate the cardiovascular action of compound
6 in a murine model of fructose-induced insulin resistance
[21].
100
∗
∗
80
∗
∗
4. Conclusion
∗
In summary, ten alkarylbiguanides have been developed as
promising compounds for the treatment of type 2 diabetes
mellitus. Compounds 4–6 (a) exhibited AMPK activation
similar to or greater than metformin, (b) demonstrated a
robust reduction of glucose levels with marked in vivo anti-
hyperglycemic efficacy, and (c) showed predicted low toxicity
profiles and any experimental evidence of lactic acidosis.
ese compounds could be an alternative to metformin, the
only biguanide currently available.
60
∗
40
20
∗
0
0.5
1
1.5
Time (h)
2
3
Vehicle
Metformin
6
Disclosure
e paper is taken in part from the M. Pharm. thesis of E.
Gutierrez-Lara.
Figure 6: Oral glucose tolerance test. Effect of a single dose of
compound 6 and metformin (50 mg/Kg, intragastric, ꢊ ꢄ 5), or
vehicle in streptozotocin-nicotinamide-induced diabetes rat model.
∗
ꢌ
< =.=5 versus vehicle.
Conflicts of Interest
e authors declare that there are no conflicts of interest
regarding the publication of this paper.
vehicle. In addition, its effect was similar to that presented
by metformin at the same dose. e activity was retained
during the 7 hours of experimentation. At 7 hours afer
administration, a 40% decrease in blood glucose levels was
observed. Analogue 6, which was the compound that showed
the best activity on AMPK activation, was also active in the
in vivo assay, and its antihyperglycemic effect was retained
throughout the assay.
Acknowledgments
is work was supported in part by the Consejo Nacional de
Ciencia y Tecnolog´ıa (CONACyT) under Grant no. 253814
(CB-2015). e authors are in debt with Abraham Gutierrez-
Hernandez, M. Pharm. for technical assistance. is article is
dedicated to all the people, mainly Pharmacist, who helped
in the medication management and classification afer the
recent Mexico’s earthquake.
In order to verify the plausible antihyperglycemic effect of
compound 6, glucose tolerance test curves in normoglycemic
rats were obtained. Dose of 50 mg/kg for 6 and metformin
was employed. As shown in Figure 6, compound 6 displayed
a significant reduction of hyperglycemic peak which was
attained at 0.5 h afer glucose administration. In Figure 6,
it can be seen that the animals treated with analogue 6
reached a lower hyperglycemic value than the animals treated
with metformin and compared to the vehicle, 0.5 h afer the
treatment.
During the experiment, glucose levels did not decrease
beyond baseline, indicating that the antidiabetic effect of
compound 6 is due to an antihyperglycemic action rather
than a hypoglycemic effect. Also, compounds 4–6 did not
increase the lactic acid concentrations in plasma of rats
tested (less than 1.9 mM), so any evidence of lactic acidosis
was found. With these results, it can be concluded that the
mechanism of action of the analogues 4–6 that confer their
antidiabetic activity is similar to metformin, through the
activation of AMPK and of some of the pathways that are
regulated by this enzyme. Previously in vitro reports agree
with the AMPK results obtained with compound 6 [15].
However, in our current study, we have demonstrated the
robust in vivo effect produced by this compound afer an oral
administration. Further studies are being conducted by us in
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