Docking, Synthesis and Anti-Diabetic Activity of Novel Sulfonylhydrazone Derivatives Designed as...

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Current Topics in Medicinal Chemistry, 2012, 12, 2037-2048 2037

Docking, Synthesis and Anti-Diabetic Activity of Novel Sulfonylhydrazone Derivatives Designed as PPAR-Gamma Agonists

Gisele Zapata-Sudo1,*, Lídia M. Lima

1,2, Sharlene L. Pereira

1, Margarete M. Trachez

1,

Filipe P. da Costa1,2

, Beatriz J. Souza1, Carlos E. S. Monteiro

1, Nelilma C. Romeiro

2,

Éverton D. D’Andréa1,2

, Roberto T. Sudo1 and Eliezer J. Barreiro

1,2

1Programa de Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Ja-neiro, Rio de Janeiro, RJ, Brazil; 2Laboratório de Avaliação e Síntese de Substâncias Bioativas, Faculdade de Far-mácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil

Abstract: Diabetes is a metabolic disorder characterized by hyperglycemia. When not properly controlled, complications

include neuropathy, coronary artery disease, and renal failure. Several drugs are approved for diabetes treatment; however

their use is associated with side effects and lack of efficacy in attenuating the development of long-term complications.

This work describes the virtual screening and synthesis of a novel series of sulfonylhydrazone derivatives designed as

peroxisome proliferator-activated receptor gamma (PPAR ) agonists and investigation of the analogs for hypoglycemic

activity in a murine model of diabetes. Docking studies identified LASSBio-331 (5) as having theoretical affinity for

PPAR similar to the prototype (S)-rosiglitazone. Several structural modifications were proposed for the structure of

LASSBio-331, resulting in the synthesis of five novel compounds, which showed experimental affinity for PPAR .

Among these new compounds, LASSBio-1471 (15) had the best theoretical binding energy for PPAR and was selected

for testing in STZ-induced diabetes. Four weeks after single intravenous injection of STZ (60 mg/kg), Wistar rats were

treated with vehicle (DMSO) or LASSBio-1471 (20 mg/kg, i.p.) for 7 days. The blood glucose levels of rats treated with

LASSBio-1471 were reduced from 548.4 ± 26.0 mg/dL before treatment to 259.6 ± 73.1 mg/dL (P < 0.05). Paw with-

drawal threshold was significantly reduced in diabetic rats and was restored from 21.9 ± 1.7 g to 36.7 ± 1.2 g after 7 days

of treatment with LASSBio-1471 (P < 0.05). Thus, the novel sulfonylhydrazone derivative is a PPAR ligand that is ef-

fective for treatment of diabetic neuropathy in STZ-injected rats.

Keywords: Diabetic neuropathy, virtual screening, sulfonylhydrazone, hypoglycemic activity, PPAR .

INTRODUCTION

Diabetes mellitus (DM) is a metabolic disorder character-

ized by hyperglycemia resulting from defects in insulin se-

cretion, insulin action, or both. Along with hyperglycemia

and abnormalities in serum lipids, DM is associated with

cardiovascular diseases such as coronary heart disease and

congestive heart failure, nephropathy, neuropathy and reti-

nopathy. These complications are the main causes of mor-

bidity and mortality of diabetic patients [1].

Currently available antidiabetic agents include sulfony-

lureas, biguanides, thiazolidinediones, -glycosidase inhibi-

tors, and dipeptidyl peptidase-4 inhibitors. These drugs are

widely used to control hyperglycemia, but fail to alter the

course of diabetic complications and have limited use be-

cause of undesirable side effects and high rates of secondary

failure [2-4].

At least five major pathways—metabolic, vascular, im-

munologic, neurohormonal growth factor deficiency, and

extracellular matrix remodeling—are involved in the devel-

*Address correspondence to this author at the Universidade Federal do Rio

de Janeiro, Centro de Ciencias da Saude, Instituto de Ciencias Biomedicas,

Bloco J, Sala 14, Rio de Janeiro, RJ, Brazil, 21941-590; Tel/Fax: 55-21-

25626505; Email: [email protected]

opment of diabetic neuropathy [5]. Neuropathic pain is a

significant cause of impairment among DM patients and pe-

ripheral neuropathy has been associated with an increased

risk of mortality in this population. Only two medications are

formally approved by the US Food and Drug Administration

to treat painful diabetic neuropathy: duloxetine, a serotonin-

norepinephrine reuptake inhibitor, and pregabalin, a voltage-

sensitive calcium channel modulator [6]. Several other com-

pounds such as antiepileptic drugs and tricyclic antidepres-

sants (TCAs) have been shown to have efficacy in treating

diabetic neuropathy pain [7]. Nevertheless, adverse effects

such as weight gain, postural hypotension, sedation, dry

mouth and constipation greatly limit the use of TCAs for

diabetic patients. TCAs also have a cardiovascular risk pro-

file that is unfavorable in a population already at risk for

cardiovascular events [8]. Thus, more effective antidiabetic

agents with fewer side effects that are efficacious for diabetic

neuropathy pain are essential.

Peroxisome proliferator-activated receptor gamma

(PPAR ) is expressed primarily in adipose tissue with less

expression in cardiac, skeletal, and smooth muscle cells, islet

cells, macrophages, and vascular endothelial cells [9,10].

Along with adipocyte differentiation, PPAR activity pro-

motes uptake of circulating fatty acids into fat cells and the

shifting of lipid stores from extra-adipose to adipose tissue.

1873-5294/12 $58.00+.00 © 2012 Bentham Science Publishers

https://www.researchgate.net/publication/51567988_Exploring_an_optimal_approach_to_the_use_of_oral_hypoglycemic_agents_based_on_CGM_results_implications_for_combination_therapy_with_oral_hypoglycemic_agents?el=1_x_8&enrichId=rgreq-c6b29fec-9abf-4a81-8c63-fe9a0ad592a5&enrichSource=Y292ZXJQYWdlOzIzMzczNjg4MztBUzoxMzQwODU1MDI1NzQ1OTJAMTQwODk3OTg4MzIwMQ==

https://www.researchgate.net/publication/51533871_Macrovascular_Effects_and_Safety_Issues_of_Therapies_for_Type_2_Diabetes?el=1_x_8&enrichId=rgreq-c6b29fec-9abf-4a81-8c63-fe9a0ad592a5&enrichSource=Y292ZXJQYWdlOzIzMzczNjg4MztBUzoxMzQwODU1MDI1NzQ1OTJAMTQwODk3OTg4MzIwMQ==

2038 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 19 Zapata-Sudo et al.

One consequence of these cellular responses to PPAR acti-

vation is increased tissue sensitivity to insulin. This is the

basis for the pharmacological application of thiazolidinedi-

ones in DM patients [10].

Despite improvements in the treatment of DM in recent

years, the prevalence of this clinical condition continues to

increase. DM and its complications have a significant eco-

nomic impact on health systems and countries [11]. We con-

sidered the potential of PPAR agonists for DM treatment

and the minimum structural requirements for PPAR ligands,

which include an acidic head attached to an aromatic scaf-

fold and a hetero-aromatic hydrophobic tail [12] (Chart 1),

and identified compounds that fulfill these structural re-

quirements. Initially, 10 compounds were selected from our

database for docking studies with PPAR (Chart 2) accord-

ing to previously described pharmacophoric prerequisites

[13]. These molecules represented distinct chemical classes.

The reference compound used in the docking studies was the

S-isomer of rosiglitazone co-crystallized with PPAR [14].

This virtual screening study with flexible docking deter-

mined a structure-based design strategy of a new series of

PPAR ligands. We describe the synthesis and the investiga-

tion of hypoglycemic activity of novel sulfonylhydrazone

derivatives in a murine model of diabetes induced by strep-

tozotocin.

RESULTS

Molecular Docking Studies with PPAR

Theoretical Gbind (kJ/mol) values obtained in docking

studies of PPAR with the 10 molecules selected from our

database are in (Fig. 1).

Analysis of Gbind values from PPAR docking studies

indicated that selected database molecules had features that

led to more energetically favorable interactions with the

agonist binding site than the reference compound, (S)-

rosiglitazone ( Gbind= -36.8 kJ/mol, Fig. 1). The best theo-

retical binding energy obtained for the 10 molecules was for

compound 5 (LASSBio-331; E-isomer, -47.6 kJ/mol, Fig. 1),

which formed hydrogen bonds to Tyr473, His323, Tyr327,

Ser289, His449, and Arg288 of PPAR , specifically by

means of a carboxylate group that is an essential pharma-

cophore in the design of PPAR agonists. Additional interac-

tions of LASSBio-331 (5) with the hydrophobic residues

Leu330, Cys285, Phe282, and Phe363 of PPAR , seemed to

contribute to its excellent Gbind (Fig. 2B).

Another compound that was evaluated in our preliminary

virtual screening was LASSBio-289 ( Gbind= -45.6 kJ/mol

for the R isomer and -44.1 kJ/mol for the S isomer; Fig. 3).

The isomers of LASSBio-289 (4; Fig. 3A-B) showed bin-

ding modes with PPAR that were similar to LASSBio-331,

with hydrogen bonding to Tyr473, His449, Ser289, Tyr327,

and Arg288 of PPAR in the same region bound by rosigli-

tazone. A slightly better binding energy was observed for the

R isomer of this compound than the S isomer ( Gbind = -45.6

for R vs. -44.1 kJ/mol for S, Fig. 3A), due to a stronger inter-

action with Arg288 (Fig. 3A). This interaction seemed to be

less geometrically suitable with the S-isomer (Fig. 3B). Both

isomers bound in the vicinity of hydrophobic residues

Leu453, Phe282, Phe363, and Leu330 of PPAR (Fig. 3A-

B).

Prototype Selection for Synthesis

Two compounds emerged in the virtual screening study

with PPAR : the R-isomer of LASSBio-289 (4) and the E-

isomer of LASSBio-331 (5) ( Gbind = -47.6 for LASSBio-

331 and -45.6 kJ/mol for LASSBio-289, Fig. 1, 2B and 3A).

LASSBio-331 was selected as the prototype molecule for

synthetic development of a series of new PPAR ligands

because of its better theoretical binding energy; the presence

of a pharmacophoric carboxylic acid group that was suscep-

tible to bioisosteric changes, which are useful for structure-

activity studies; its low synthetic cost; and its easier struc-

tural determination compared to LASSBio-289 (R), which

has a chiral carbon atom.

Structural Modifications Proposed for Prototype LASSBio-331 (5)

After the selection of LASSBio-331(5) as the ligand with

the best theoretical affinity for the target PPAR , alterations

in its acidic subunity were proposed to build a new series of

compounds (Chart 3). This new series was planned using

nonclassical bioisosterism [15] between carboxylic acid (5),

tretrazole (16) and suphonic acid (17) functional groups; and

exploring an isomerism strategy to design the regioisomers

14 and 15 (Chart 3).

Following design, flexible molecular docking studies

were performed to investigate the putative interactions of the

compounds with PPAR to support the design strategy (Fig.

4). The Gbind values (kJ/mol) of the new series of analogs

were very similar to those observed previously for the proto-

type compound (E)-LASSBio-331 (5) (Fig. 4).

Interactions with the PPAR binding site that were ob-

served previously were maintained (Fig. 5A-B) and all com-

pounds also showed a better Gbind value than (S)-

rosiglitazone, the reference compound.

(E)-LASSBio-1470 (14) had a Gbind energy of -40.7

kJ/mol (Fig. 5) with hydrogen bonds to Tyr473, His323,

Tyr327, Ser289, His449, Arg288, and the amino acid resi-

dues on top of the agonist binding site of PPAR (Fig. 5A).

Furthermore, the carboxylate group of (E)-LASSBio-1470

interacted with His323, His449, and Tyr473 of PPAR ,

which was important for binding. Additional hydrophobic

interactions involved Leu330, Leu453, Leu468, Phe282, and

Ile326 of PPAR (Fig. 5A).

Interestingly, LASSBio-1471 (15; Gbind = -49.4 kJ/mol,

Fig. 5) formed hydrogen bonds to Tyr473, His323, Ser289,

His449 and Arg288 of PPAR in the site occupied by rosigli-

tazone. However, compared to the other compounds (Fig.

5A-B), LASSBio-1471 showed an alternative inverted bind-

ing site in which its carboxylate group preferably interacted

with Arg288 of PPAR , forming a stronger ionic bond. This

may be due to a better fit of the 3-carboxylate group which

does not seem to be so favored energetically when it is pre-

sent in position 2 as in LASSBio-1470, probably because of

classic ortho effects or bumps with hydrophobic amino acids

that line the ligand binding channel. Hydrophobic interacti-

ons of LASSBio-1471 involved Leu330, Leu469, Phe363,

Phe282 and Ile326 of PPAR (Fig. 5B). These results led to

the synthesis of the new series of compounds that were ana-

logs of LASSBio-331 (5).

Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 19 2039

Chart 1. Structural requirements for PPAR ligands.

Chart 2. Selected compounds for docking studies with PPAR .

Synthesis of LASSBio-331 and Analogs

Compound 5 (LASSBio-331) and its new analogs (14-

17) were synthesized using previously described methodol-

ogy [16,17], exploring 6-methyl-3,4-methylenedioxyphenyl-

sulphonylhydrazide (19) as an intermediate (Scheme 1). The

sulfonylhydrazone derivatives (5, 14-17) were obtained in

elevated yields by acid condensation of 19 with appropriate

functionalized benzaldehydes.

1H and

13C NMR spectra of compounds 14-17 showed a

single signal from the hydrogen and carbon of imine group

(HC=N), indicating the presence of only one isomer, attrib-

uted to the E-diastereoisomer.

Blood Glucose Levels in Streptozotocin-Induced Diabetes in Rats

Male Wistar rats received an intravenous injection of

streptozotocin (STZ) (60 mg/kg) to induce diabetes. A

N N

CH3

O

NHS

O

O

rosiglitazone, 1

O

NHS

O

ON

H3C

pioglitazone, 2

O

NHS

O

OO

HO

CH3

H3C

CH3

troglitazone, 3

a

b

c

c

c

b

a

a

b

a = hetero-aromatic hydrophobic tail b = linkerc = an acidic head attached to an aromatic scaffold

O

O SNH

CH3

O OO

O

OH

CH3

(R,S)-4 (E)-5O

O SNH

CH3

O ON

O

OH

N

O

O

O O

OH6

N

O

O

O O

OHH3C(R,S)-7

N

O

O O

OOH

CH3(R,S)-8

O

O O

O

OH

9 O

O NH

O

N

O

OH

(E)-10

Cl

O

NH

N

O

OH

(E)-11Cl

O

NH

O

OH

O

NH

O

OH

Cl12 13

(LASSBio-1312)(LASSBio-1313)(LASSBio-1314)

(LASSBio-1315)(LASSBio-561)

(LASSBio-482) (LASSBio-485) (LASSBio-541)

(LASSBio-331)(LASSBio-289)


Fig. (1). Theoretical G binding free energy values ( Gbind, kJ/mol) obtained for the database with AM1 charges (PC Spartan Pro, Wave-

function, Inc., License number 1-001259) in the flexible docking software FlexE and PPAR (Sybyl 8.0, Tripos, Inc., License number 7512).

Fig. (2). The structures of PPAR ligand binding domain in complex with 1 (S-rosiglitazone, A) and 5 (LASSBio-331, E-isomer, B) (stick

representations) obtained by flexible docking. Hydrogen bonds in dashed yellow lines. White legends= hydrogen bonding residues; Yellow

legends= putative hydrophobic interactions. Co-crystallized rosiglitazone in blue carbon atoms. Docked ligands in gray carbon atoms.


Fig. (3). The structures of PPAR ligand binding domain in complex with 4 (LASSBio-289, R-, A and S-isomer, B) (stick representations)

obtained by flexible docking. Hydrogen bonds in dashed yellow lines. White legends = hydrogen bonding residues; Yellow legends = putati-

ve hydrophobic interactions. Docked ligands in gray carbon atoms.

Chart 3. Structural modifications proposed for prototype LASSBio-331.

significant increase in blood glucose concentration was ob-

served 4 weeks after diabetes induction compared to the glu-

cose levels of nondiabetic animals. The blood glucose levels

of diabetic rats were increased from 122.0 ± 4.8 and 126.7 ±

6.6 mg/dL before STZ injection to 486.2 ± 42.8 and 548.4 ±

26.0 mg/dL (P < 0.05) at 4 weeks after the induction, respec-

tively (Table 1). In addition, the STZ-injected rats had no

significant weight gain over 4 weeks compared to the

nondiabetic group. The body weight of diabetic rats was

209.0 ± 23.3 and 219.7 ± 10.2 g (before STZ injection) and

249.0 ± 16.4 and 209.0 ± 14.3 g at 4 weeks after the induc-

tion, respectively (Table 1).

Mechanical allodynia was evaluated by measurement of

the paw withdrawal threshold, which was significantly re-

duced in diabetic rats compared to the nondiabetic group or

to values before diabetes induction. Paw withdrawal thresh-

old of diabetic rats was decreased from 35.5 ± 2.7 and 37.9 ±

2.0 g before STZ injection to 27.1 ± 3.1 and 21.9 ± 1.7 g (P

< 0.05) at 4 weeks after the induction, respectively (Table 2).

Thus, diabetic neuropathy was observed 4 weeks after STZ

administration by appearance of allodynia.

Single Intraperitoneal Injection of LASSBio-1471 Re-duced Pain of Diabetic Neuropathy

Four weeks after STZ injection, we evaluated the me-

chanical allodynia of diabetic rats after a single intraperito-

neal injection of LASSBio-1471 or vehicle (DMSO). Paw

withdrawal threshold was significantly reduced in

O

O SNH

CH3

O O

N

O

OH

5 (LASSBio-331)

acid subunit

regioisomers

O

O SNH

CH3

O O

N

O

OH

O

O SNH

CH3

O O

N

O OH

15 (LASSBio-1471)

14 (LASSBio-1470)

non-classicalbioisosterism

O

O SNH

CH3

O O

N

NH

NNN

16 (LASSBio-1503) 17 (LASSBio-1523)O

O SNH

CH3

O O

N

OCH3

SOH

O O


Fig. (4). Theoretical G binding values ( Gbind, kJ/mol) obtained for the database with AM1 charges (PC Spartan Pro, Wavefunction, Inc.,

License number 1-001259) in the flexible docking software FlexE and PPAR (Sybyl 8.0, Tripos, Inc., License number 7512).

Fig. (5). The structures of PPAR ligand binding domain in complex with 14 (LASSBio-1470, A) and 15 (LASSBio-1471, B), E-isomers

(stick representations), obtained by flexible docking. Hydrogen bonds in dashed yellow lines. White legends= hydrogen bonding residues;

Yellow legends= putative hydrophobic interactions. Docked ligands in gray carbon atoms.

diabetic rats compared to the nondiabetic group. This pa-

rameter was partially recovered in rats treated with LASS-

Bio-1471. At 30 minutes after LASSBio-1471 administra-

tion, the paw withdrawal threshold increased from 21.9 ± 1.7

(before injection) to 34.3 ± 1.5 g (P < 0.05). Reduced allo-

dynia was observed again after 180 minutes of LASSBio-

1471 administration, when the paw withdrawal threshold

decreased to 21.3 ± 0.9 g (Fig. 6).


Scheme 1. Synthesis of functionalized sulfonylhydrazone derivatives (5, 14-17).

Fig. (6). Mechanical allodynia of nondiabetic rats and diabetic rats

treated either with DMSO (diabetic + vehicle) or LASSBio-1471

(20 mg/kg, i.p.) (diabetic + LASSBio-1471) after a single dose

injection. * P < 0.05 before treatment; # P < 0.05 compared to

nondiabetic; † P < 0.05 compared to diabetic + vehicle. Values are

mean ± S.E.M. n = 4-8 per group.

Repeated Administration of LASSBio-1471 Reduced Blood Glucose and Diabetic Neuropathy Pain

Four weeks after induction of diabetes, rats were treated

with vehicle (DMSO) or LASSBio-1471 (20 mg/kg, i.p.) for

7 days. Rats treated with vehicle had glucose levels of 486.2

± 42.8 before and 557.0 ± 24.9 mg/dL after 7 days of treat-

ment. The blood glucose levels of rats treated with LASS-

Bio-1471 (20 mg/kg, i.p.) were reduced from 548.4 ± 26.0

mg/dL before treatment to 259.6 ± 73.1 mg/dL at 7 days

after the beginning of treatment (P < 0.05, Table 1). Daily

intraperitoneal administration of LASSBio-1471 for 7 days

produced a significant reduction in blood glucose levels

compared to vehicle-treated diabetic rats, indicating the hy-

poglycemic activity of this compound. The body weight of

diabetic rats treated with LASSBio-1471 (20 mg/kg, i.p.)

was 209.0 ± 14.3 before treatment and 206.5 ± 16.8 g after

treatment (Table 1).

Mechanical allodynia was observed in diabetic rats in-

jected with vehicle. The paw withdrawal threshold was 27.1

± 3.1 g before treatment and 16.8 ± 0.6 g at 7 days after the

beginning of treatment. The paw withdrawal threshold of

LASSBio-1471-treated diabetic rats was 21.9 ± 1.7 g before

treatment and 36.7 ± 1.2 g after 7 days of treatment (P <

0.05; Table 2).

Oral Glucose Tolerance Test

Long-term administration of LASSBio-1471 for 7 days in

diabetic rats resulted in a significant improvement in oral

glucose tolerance following oral glucose loading. Vehicle-

treated rats had glucose levels of 375.0 ± 20.5 mg/dL (Fig. 7)

after 120 min of oral glucose loading.

In contrast, rats treated with LASSBio-1471 had a sig-

nificant reduction in glucose levels to 237.1 ± 46.1 mg/dL

(Fig. 7) after 120 minutes of oral glucose administration.

These results indicated the anti-hyperglycemic activity of the

sulfonylhydrazone derivative.

Table 1. Evaluation of Blood Glucose Levels and Body Weight in Diabetic Rats Treated with Vehicle (DMSO) or LASSBio-1471

(20 mg/kg, i.p.) for 7 Days.

Diabetic rats

Before treatment + Vehicle Before treatment + LASSBio-1471

Blood Glucose (mg/dL) 486.2 ± 42.8 557.0 ± 24.9 548.4 ± 26.0 259.6 ± 73.1*#

Body weight (g) 249.0 ± 16.4 238.8 ± 6.5 209.0 ± 14.3 206.5 ± 16.8

STZ: streptozotocin * P < 0.05 compared to before treatment; # P < 0.05 compared to diabetic + vehicle. Values are mean ± SEM.

O

O

Lima et al. 1999 O

O CH3

S

HN

O ONH2

O

O SNH

CH3

O O

N

W

W1

W2

95%; W = CO2H; W1=W2=H; 589%; W=W1=H; W2 = CO2H; 1493%; W=W2 =H; W1 = CO2H; 1594%; W= tetrazole; W1=W2=H; 1674%; W=OCH3; W1=SO3H; W2=H; 17

W

W1

W2

OHC

EtOH/HCl cat/r.t./1h

5,14-1718 (safrole) 19

0 30 60 90 120 150 1800

10

20

30

40

50

Nondiabetic

Diabetic + vehicle

Diabetic + LASSBio-1471

* *#

#

#

#

#

#

# ## #

Time (min)

Paw

withdra

wal th

reshold

(g)


Fig. (7). Effects of long-term administration of DMSO (diabetic +

vehicle) or LASSBio-1471 (20 mg/kg, i.p.) (diabetic + LASSBio-

1471) on the oral glucose tolerance in diabetic rats. * P < 0.05

compared to nondiabetic; # P < 0.05 compared to diabetic + vehi-

cle. Values are mean ± S.E.M. n = 5-7 per group.

DISCUSSION

In this study, LASSBio-1471 exhibited hypoglycemic

activity in rats with STZ-induced diabetes, as indicated by

reduced blood glucose levels after prolonged treatment. Ad-

ditionally, LASSBio-1471 exhibited analgesic effects, as

demonstrated by improvement in mechanical allodynia in

STZ-induced neuropathy.

The activation of PPAR controls energy metabolism and

plays a key role in insulin sensitivity as well as glucose and

lipid metabolism. The stimulation of this receptor leads to

increased glucose uptake through increased tissue insulin

sensitivity, mainly in fat cells but also in liver and muscle

cells, without affecting insulin secretion [18]. Consequently

PPAR agonists such as LASSBio-1471 may have beneficial

effects on hyperglycemic rats. Thiazolidinediones are phar-

macological PPAR agonists that increase insulin sensitivity,

reduce blood glucose and circulating free fatty acid levels,

and inhibit inflammatory pathways [19-22]. Our results indi-

cated that LASSBio-1471 had a hypoglycemic effect in STZ-

injected rats by activating PPAR suggested by molecular

docking studies.

Whereas persistent hyperglycemia appears to be the pri-

mary factor in the pathogenesis of neuropathy, several func-

tional disturbances are found in the microvasculature of the

nerves of diabetic patients, before the onset of structural

damage [23]. These include decreased neural blood flow,

increased vascular resistance and altered vascular permeabil-

ity. Control of glucose levels has been the best method for

preventing and treating diabetic neuropathy. Nevertheless,

there is no clear evidence that hyperglycemic control results

in the reversal of established small fiber injury in diabetic

neuropathy, although the progression of the disease may be

slowed [24].

In addition to controlling energy metabolism, PPAR has

been implicated in inflammation. In mono-

cytes/macrophages, PPAR is proposed to reduce production

of cytokines (TNF , interleukin-1 , interleukin-6) by inhib-

iting the activity of pro-inflammatory transcription factors

such as AP-1, STAT and NF- [25]. This anti-

inflammatory effect of PPAR could be beneficial in the

treatment of diabetic neuropathy. This suggests that LASS-

Bio-1471 promoted improvement of mechanical allodynia in

diabetic rats by activating PPAR and consequently decreas-

ing cytokine production and promoting anti-inflammatory

action. Previously, the analgesic and anti-inflammatory ef-

fects of the lead compound LASSBio-331 were reported to

be mediated by the activation of PPAR [16]. Therefore, we

propose the evaluation of this class of compounds in animal

models of DM.

The novel PPAR agonist LASSBio-1471 is a promising

substance, with beneficial effects on reducing hyperglycemia

and diabetic neuropathy.

Therefore this compound could be of great therapeutic

benefit in the management of DM. However, the presence of

1,3-benzodioxole subunit in the structure of LASSBio-1471,

described to inhibit and/or induce CYP450, anticipates the

importance to evaluate its toxicity to characterize its poten-

tial as new anti-diabetes drug candidate and to establish the

need for further structural optimization step.

METHODS

Molecular Modeling

General Procedures

All calculations were performed on a PC using Linux

Red Hat Enterprise version 4.0 platform. Structural manipu-

Table 2. Evaluation of Mechanical Allodynia in Diabetic Rats Treated with Vehicle (DMSO) or LASSBio-1471 (20 mg/kg, i.p.) for

7 Days.

Paw withdrawal threshold (g)

Before STZ After STZ STZ + treatment

Vehicle group 35.5 ± 2.7 27.1 ± 3.1* 16.8 ± 0.6*

LASSBio-1471 group 37.9 ± 2.0 21.9 ± 1.7* 36.7 ± 1.2†

STZ: streptozotocin, * P < 0.05 vs before STZ injection; † P < 0.05 vs diabetic + vehicle. Values are mean ± SEM.

0 30 60 90 1200

100

200

300

400

500

600

*

Diabetic + vehicle

Nondiabetic

#

Diabetic + LASSBio-1471

* #

*

*

*

*

*

* *

*

Time (min)

Gly

cem

ia (

mg/d

L)


lations were performed using SYBYL 8.0 (Tripos Associa-

tes, St. Louis, MO, 2007). Docking procedures used FlexE

[26].

Preparation of Ligands

The preparation of the ligands for FlexE was performed

using Sybyl version 8.0. The molecular structure of rosiglita-

zone bound to PPAR (PDB code: 1FM6) was extracted

[14]. Other ligands’ coordinates were generated using the

program Spartan Pro. The correct atom types (including

hybridization states) and correct bond types were defined,

hydrogen atoms were added and charges were assigned to

each atom. Carboxylate groups were modeled in their ioni-

zed states at physiological pH and, when applicable, possible

stereoisomers were constructed. Finally, structures were e-

nergy-minimized using the semiempirical AM1 method [27]

available in the MOPAC module of Sybyl 8.0 software. Af-

ter this procedure, AM1 point charges were assigned to the

ligands.

Selection of Protein Crystal Structures

Ligand-bound crystallographic structures of PPAR are

available in the Protein Data Bank [28]. In this study, 2F4B,

2GTK, 1KNU, 1ZEO, 2ATH and 1FM6 were selected for

flexible docking with FlexE, available in Sybyl 8.0 [14,29-

31]. The active recognition site of the ensemble was defined

as the collection of residues within 10.0 Å of the bound inhi-

bitor and comprised the union of all ligands of the ensemble.

All atoms located less than 10.0 Å from any ligand atom

were considered. 2GTK was used as a reference structure for

the united protein preparation. All carboxylic acid and amino

groups were modeled in their ionized forms. Proteins were

prepared for the docking studies using the Biopolymer mo-

dule of Sybyl 8.0. Amber7. FF99 charges were attributed to

protein atoms. A biopolymer protein analysis tool was used

in a stepwise process of analysis and correction of geometry

parameters. The side chains of lysine and arginine, and the

carboxylate groups of aspartic and glutamic acid side chains

were modeled in their ionized states. Water molecules con-

tained in the PDB file were removed.

Chemistry

Reactions were routinely monitored by thin-layer chro-

matography in silica gel (F245 Merck plates) and products

visualized with iodine or an ultraviolet lamp (254 and 365

nm). 1H and

13C nuclear magnetic resonance (NMR) spectra

were determined in DMSO-d6 solutions using a Bruker AC-

200 spectrometer. Peak positions are in parts per million ( )

using tetramethylsilane as an internal standard, and coupling

constant values (J) are in Hz. Signal multiplicities are repre-

sented by: s (singlet), d (doublet), dd (double doublet), m

(multiplet) and br (broad signal). Infrared (IR) spectra were

obtained using an ABB FTLA2000-100 IR spectrometer.

Samples were examined as potassium bromide (KBr) disks.

Elemental microanalyses were obtained on an Elemental

Analyzer (Flash EA 1112 Series, Thermo Scientific) from

vacuum-dried samples. The analytical results for C, H, and

N, were within ± 0.4% of the theoretical values. Melting

points were determined using a Quimis instrument and are

uncorrected. All described products showed NMR spectra

according to the assigned structures. All organic solutions

were dried over anhydrous sodium sulphate and all organic

solvents were removed under reduced pressure in a rotatory

evaporator.

General Procedure for the Preparation of 6-Methyl-3,4-

Methylenedioxybenzene-Sulphonylhydrazone Derivatives

(5, 14-17)

To a solution of 1 mmol of 6-methyl-3,4-

methylenedioxybenzene-sulphonylhydrazide (19) in absolute

ethanol (25 mL) containing one drop of 37% HCl, 1 mmol of

the corresponding functionalized benzaldehyde was added.

The mixture was stirred at room temperature for 1 h. At the

end of the reaction, cold water and ice were added and the

resulting precipitate was collected by filtration and dried

under vacuum to give the desired sulfonylhydrazones (5, 14-

17) in elevated yields.

4’-[(E)-[2-[(6-Methyl-1,3-Benzodioxol-5-yl)Sulfonyl] hy-

drazinylidene]Methyl]-Benzoic Acid (5, LASSBio-331)

The title compound was obtained by condensation of 19

with 4-carboxybenzaldehyde as white amorphous powder, in

95% yield, mp 212-213oC. The melting point,

1H NMR,

13C

NMR and IR data for compound 5 are in agreement with

previews reports [16].

IR (KBr) (cm-1

): 3445 (O-H); 3191 (N-H); 2922 (CH);

1685 (C=O); 1612 (C=C); 1355 and 1121 (S-N); 1320

(S=O); 1255 (C-O-C); 1153 (C-S-O).

1H NMR (200 MHz, DMSO-d6, TMS) (ppm): 12.10

(br,CO2H); 11.83 (1H, s, NH); 7.99 (1H, s, HC=N); 7.94

(2H, d, H-3’ and H-5’, J = 8.2 Hz); 7.63 (2H, d, H-2’ and H-

6’, J = 8.2 Hz); 7.36 (1H, s, H-4); 6.96 (1H, s, H-7); 6.11

(2H, s, H-2); 2.55 (3H, s, ArCH3).

13C NMR (50 MHz, DMSO-d6, TMS) (ppm): 166.8

(CO2H); 150.9 (C-7a); 145.4 (C-3a); 144.7 (HC=N); 137.7

(C-1’); 133.2 (C-4’); 131.6 (C-5); 129.8 (C-3’ and C-5’);

129.7 (C-6); 126.6 (C-2’ and C-6’); 111.9 (C-4); 109.0 (C-

7); 102.3 (C-2); 20.0 (ArCH3).

Anal. Calcd. for C16H14N2O6S: C, 53.03; H, 3.89; N,

7.73. Found: C, 53.22; H, 3.90; N, 7.56.

2’-[(E)-[2-[(6-Methyl-1,3-Benzodioxol-5-yl)Sulfonyl] hy-drazinylidene]Methyl]-Benzoic Acid (14, LASSBio-1470)



89% yield, mp 180-181oC.

IR (KBr) (cm-1

): 3154 (N-H); 2918 (CH); 1687 (C=O);

1608 (C=C); 1353 and 1120 (S-N); 1321 (S=O); 1233 (C-O-

C); 1148 (C-S-O).

1H NMR (200 MHz, DMSO-d6, TMS) (ppm): 12.15

(br,CO2H); 11.77 (1H, s, NH); 8.72 (1H, s, HC=N); 7.86

(1H, d, H-3’, J = 7.5 Hz); 7.70 (1H, d, H-5’, J = 7.5 Hz);

7.51 (2H, m, H-4’ and H-5’); 7.36 (1H, s, H-4); 6.97 (1H, s,

H-7); 6.11 (2H, s, H-2); 2.55 (3H, s, ArCH3).


(CO2H); 150.8 (C-7a); 145.4 (C-3a); 144.9 (HC=N); 134.0

(C-1); 133.1 (C-5’); 132.0 (C-5); 130.3 (C-2’); 130.2 (C-4’);

130.0 (C-6); 130.0 (C-6’); 126.0 (C-3’); 111.8 (C-4); 109.0

(C-7); 102.3 (C-2); 20.0 (ArCH3).


7.73. Found: C, 52.78; H, 3.87; N, 7.66.


3’-[(E)-[2-[(6-Methyl-1,3-Benzodioxol-5-yl)Sulfonyl] hy-

drazinylidene]Methyl]-Benzoic Acid (15, LASSBio-1471)



93% yield, mp 227oC.

IR (KBr) (cm-1

): 3450 (O-H); 3226 (N-H); 2918 (C-H);

1696 (C=O); 1612 (C=C); 1351 and 1123 (S-N); 1322

(S=O); 1252 (C-O-C); 1158 (C-S-O).

1H NMR (200 MHz, DMSO-d6, TMS) (ppm): 12.15 (br,

CO2H); 11.73 (1H, s, NH); 8.09 (1H, s, HC=N); 8.02 (1H, s,

H-2’); 7.93 (1H, d, H-4’, J = 7.4 Hz); 7.75 (1H, d, H-6’, J =

7.5 Hz); 7.52 (1H, dd, H-5’, J = 7.5 and 7.4 Hz); 7.36 (1H, s,

H-4); 6.96 (1H, s, H-7); 6.11 (2H, s, H-2); 2.56 (3H, s,

ArCH3).


(CO2H); 150.9 (C-7a); 145.4 (C-3a); 145.0 (HC=N); 134.2

(C-6’); 133.2 (C-1’); 131.3 (C-4’); 130.8 (C-5); 130.4 (C-3’);

129.9 (C-6); 129.1 (C-2’); 127.2 (C-5’); 111.8 (C-4); 109.0

(C-7); 102.3 (C-2); 20.0 (ArCH3).


7.73. Found: C, 52.88; H, 4.00; N, 7.73.

6-Methyl-N’-[(E)-[4’-(1H-Tetrazol-5-yl)Phenyl]Methylene}

-1,3-Benzodioxol-5-yl)Sulfonyl]hydrazide (16, LASSBio-

1503)


with 4-(1H-tetrazol-5yl)benzaldehyde as white powder, in

94% yield, mp 112oC.

IR (KBr) (cm-1

): 3549 (N-H); 3092 (C-H); 2925 (C-H);

1614 (C=C); 1352 and 1121 (S-N); 1307 (S=O); 1251 (C-O-

C); 1155 (C-S-O).

1H NMR (200 MHz, DMSO-d6, TMS) (ppm): 11.84 (1H,

s, NH); 8.07 (1H, s, HC=N); 8.02 (2H, d, H-2’ and H-6’, J =

7.9 Hz); 7,75 (2H, d, H-3’ and H-5’, J = 7.9 Hz); 7.37 (1H, s,

H-7); 6.98 (1H, s, H-4); 6,11 (2H, s, H-2); 2.56 (3H, s,

ArCH3).

13C NMR (50 MHz, DMSO-d6, TMS) (ppm): 151.5 (C-

5’’); 146.0 (C-7a); 145.2 (C-3a); 136.8 (HC=N); 133.8 (C-1’

and C-4’); 130.4 (C-5); 128.0 (C-2’and C-6’); 128.3 (C-6);

127.9 (C-3’ and C-5’); 112.5 (C-4); 109.6 (C-7); 102.9 (C-

2); 20.6 (ArCH3).

Anal. Calcd. for C16H14N6O4S. 2H2O: C, 45.76; H, 4.18;

N, 19.78; Found: C, 45.82; H, 4.20; N, 19.82.

4’-Methoxy,3’-[(E)-[2-[(6-Methyl-1,3-Benzodioxol-yl) Sul-fonyl]hydrazinylidene]Methyl]-Sulfonic Acid (17, LASS-

Bio-1523)


with 5-formyl-2-methoxybenzenesulfonic acid as white pow-

der, in 74% yield, mp 80oC.

IR (KBr) (cm-1): 3464 (O-H); 2907 (C-H); 1606 (C=C);

1352 and 1122 (S-N); 1317 (S=O); 1258 (C-O-C); 1159 (C-

S-O).

1H NMR (200 MHz, DMSO-d6, TMS) (ppm): 11.43 (1H,

s, NH); 7.92 (1H, s, HC=N); 7.89 (1H, s, H-2’); 7.46 (1H, d,

H-6’, J = 8.6 Hz); 7,32 (1H, s, H-4); 7,01 (1H, d, H-5’, J =

8.6 Hz); 6.95 (1H, s, H-7); 6.11 (2H, s, H-2); 3.78 (3H, s,

OCH3); 2.56 (3H, s, ArCH3).

13C NMR (50 MHz, DMSO-d6, TMS) (ppm): 158.2 (C-

4’); 151.3 (C-7a); 146.7 (C-3a); 146.0 (HC=N); 136.3 (C-

6’); 133.8 (C-5); 130.7 (C-3’); 130.0 (C-6); 127.2 (C-1’);

125.5 (C-2’); 112.6 (C-5’); 112.4 (C-4); 109.4 (C-7); 102.8

(C-2); 56.3 (OCH3); 20.6 (ArCH3).

Anal. Calcd. for C16H16N2O8S2: C, 48.86; H, 3.76; N,

6.54; Found: C, 48.69; H, 3.95; N, 6.82.

Pharmacology

Animals

Male Wistar rats weighing 180–220 g were housed under

an artificial 12-h light/dark cycle in controlled temperature

(23 °C) and humidity (60%) conditions with food and water

available ad libitum. The Animal Care and Use Committee

of Universidade Federal do Rio de Janeiro approved the pro-

tocols used.

Induction of Experimental Diabetes and Protocols

Each rat received a single intravenous injection of STZ

(60 mg/kg) to induce diabetes. STZ was dissolved in citrate

buffer (pH 4.5) and injected immediately to avoid degrada-

tion. STZ-treated rats were randomly divided into two

groups (n = 6 each) treated with vehicle (dimethylsulfoxide,

DMSO) or LASSBio-1471 (20 mg/kg, i.p.). These groups

were compared with a control group of normal rats. Plasma

glucose levels were examined in blood samples collected by

tail-vein puncture, using the Accu-Check®

Performa moni-

toring system (Roche, Mannheim, Germany).

Body weight, blood glucose and mechanical allodynia

were evaluated before and weekly after the STZ injection

during 4 weeks. Four weeks after the induction of diabetes

with STZ, rats with glucose levels >200 mg/dL were treated

with either vehicle or LASSBio-1471 for 7 days.

After the first administration of vehicle or LASSBio-

1471, mechanical allodynia was measured 30, 60, 120 and

180 min after injection. At the end of the period of treatment,

mechanical allodynia and blood glucose concentration were

measured in diabetic rats. Also, the animals were submitted

to an oral glucose tolerance test in which after overnight fast-

ing (14 h), blood glucose was determined before and 15, 30,

60, 90, and 120 min after oral glucose administration (2

g/kg).

Mechanical Allodynia

The withdrawal threshold to pressure applied to the hind

paw, expressed in grams, was measured using a digital anal-

gesimeter (model EFF301, Insight, SP, Brazil) [32]. The

pressure meter consisted of a hand-held force transducer

fitted with a 0.7 mm2 polipropylene tip. Upon paw with-

drawal, the pressure was released immediately, and the noci-

ceptive threshold was read on a scale. The stimulation of the

paw was repeated five times, and readings were used if the

difference between the highest and the lowest measure was

less than 10 g. An upper limit of 150 g was used to avoid

potential tissue injury in the absence of response.


Chemicals

Streptozotocin was from Sigma Chemical (St. Louis,

MO, USA). LASSBio-1471 was synthesized and provided

by Laboratório de Avaliação e Síntese de Substâncias

Bioativas (UFRJ, Brazil). The compound was dissolved in

dimethylsulfoxide (DMSO) (Merck Darmstadt, Germany).

Statistical Analysis

Data are presented as mean ± standard error of the mean.

Comparisons between non-diabetic, diabetic + LASSBio-

1471 and diabetic + DMSO groups were determined using

two-way ANOVA followed by the Bonferroni post-test. Dif-

ferences among post-treatment and pre-treatment effects in

the same group were conducted using one-way ANOVA

followed by the Dunnett post-test. A P value of less than

0.05 was considered significant.

CONFLICT OF INTEREST

The authors declared no conflict of interest. All co-

authors have agreed with the submission of the final manu-

script and all authors participated in the research and article

preparation.

ACKNOWLEDGEMENTS

This work was supported by Coordenação de Aperfeiço-

amento de Pessoal de Nível Superior (CAPES), Fundação

Universitária Jose Bonifácio (FUJB), Instituto Nacional de

Ciência e Tecnologia (INCT), Fundação Carlos Chagas Filho

de Amparo à Pesquisa do Estado do Rio de Janeiro (FA-

PERJ), Conselho Nacional de Desenvolvimento Científico e

Tecnológico (CNPq), Programa de Apoio a Núcleos de Ex-

celência (PRONEX).

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Received: July 23, 2012 Accepted: October 11, 2012

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