3rd annual international conference on pharmaceutical sciences

Design, Docking, Synthesis and in vitro Binding Studies of Few

Benzamide Derivatives with Glucokinase Enzyme

PHA2016/2662075

Dr. V. A. ChatpalliwarM Pharm. (Pharm. Chem.), Ph. D.

Prof., Pharm. Chem.Head, Dept. Pharm. Chem.

S. N. J. B. Coll. Pharm.Neminagar, Chandwad

Dist.: Nashik

Global Prevalence of Diabetes mellitus

3rd Annual International Conference on Pharmaceutical Sciences, ATINER

Facts about diabetes

In 2014 the global prevalence of diabetes * was estimated to be 9% among adults aged 18+ years (1).

In 2012, an estimated 1.5 million deaths were directly caused by diabetes .

More than 80% of diabetes deaths occur in low- and middle-income countries.

WHO projects that diabetes will be the 7th leading cause of death in 2030.


Secondary complications

Prolonged exposure of tissues to sustained high levels of glucose in blood result in developing

secondary complications

Available Medicines and some new Strategies


No medicine guarantee normal life to diabetic patients

Single medicine is unable to provide strict glucose levels

β-cell apoptosis cannot be avoided

No medicine comes without a side-effect

• Sulphonylureas

• Glinides

• Biguanides

• Incretin mimetic (GLP-1)

• Biguanides

• Thiazolidinediones

(Pioglitazone, Rosiglitazone)

• α-Glucosidase inhibitor

• (Acarbose, Miglitol)

Search for novel strategies with safer drugs are required to provide near-to-normal life

Tripathi B. K., et al., Medical Science Monitoring, 12(7), 2006, 130-147Tahrani A. A., et al., New Horizons, 378, 2011, 182-197

Matschinsky F.M., et al., Medicine Reports, 2010, 1-5


Dual role in glucose homeostasis

Matschinsky F.M. et al., Medicine Reports, 2010, 1-5

Sarabu R., et.al., J. Med. Chem., 55, 2012, 7021-7036

http://www.google.co.in/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjIgdbnkfXLAhXCQI4KHeSyBjcQjRwIBw&url=http://www.medscape.com/viewarticle/438144_3&psig=AFQjCNFYGLqojDl9U8mtMnsZwMt3DorTtQ&ust=1459864613127050

http://www.google.co.in/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjIgdbnkfXLAhXCQI4KHeSyBjcQjRwIBw&url=http://www.medscape.com/viewarticle/438144_3&psig=AFQjCNFYGLqojDl9U8mtMnsZwMt3DorTtQ&ust=1459864613127050

http://www.google.co.in/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjIyYjpkvXLAhXVkY4KHWDNAlIQjRwIBw&url=http://themedicalbiochemistrypage.org/insulin.php&psig=AFQjCNHeCg_6_4CElKS5iGezfDlYQpXLmg&ust=1459864771373950

http://www.google.co.in/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjIyYjpkvXLAhXVkY4KHWDNAlIQjRwIBw&url=http://themedicalbiochemistrypage.org/insulin.php&psig=AFQjCNHeCg_6_4CElKS5iGezfDlYQpXLmg&ust=1459864771373950

http://www.google.co.in/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjTt4eWlfXLAhXKbY4KHcu1BB8QjRwIBw&url=http://intranet.tdmu.edu.te.ua/data/kafedra/internal/clinlab/classes_stud/en/med/lik/ptn/4/03. DIABETES MELLITUS. LIPOPROTEINS. CLINICAL TOXICOLOGY.htm&psig=AFQjCNFHOTE_rh5jn43oGSed1MaGL4xD3g&ust=1459865459294234

http://www.google.co.in/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjTt4eWlfXLAhXKbY4KHcu1BB8QjRwIBw&url=http://intranet.tdmu.edu.te.ua/data/kafedra/internal/clinlab/classes_stud/en/med/lik/ptn/4/03. DIABETES MELLITUS. LIPOPROTEINS. CLINICAL TOXICOLOGY.htm&psig=AFQjCNFHOTE_rh5jn43oGSed1MaGL4xD3g&ust=1459865459294234

http://www.google.co.in/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiX_8mslvXLAhUBA44KHfNqCfcQjRwIBw&url=http://physrev.physiology.org/content/90/1/1&psig=AFQjCNFHOTE_rh5jn43oGSed1MaGL4xD3g&ust=1459865459294234

http://www.google.co.in/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiX_8mslvXLAhUBA44KHfNqCfcQjRwIBw&url=http://physrev.physiology.org/content/90/1/1&psig=AFQjCNFHOTE_rh5jn43oGSed1MaGL4xD3g&ust=1459865459294234

GK Protein Structure

Schematic Representation of for GK demonstrating active (closed) and inactive (super-open)

conformations

0.4 mM

≥ 15 mM

≥ 7.5 mM

T. O. Johnson, et al., Annual Reports in Med. Chem., 41, 2006, 141-154

Strategy


Crystal Structure of Human Glucokinase

Filipski, K. J., et al., Bioorg. Med. Chem. Lett.,23, 2013, 4571-78

Catalytic domain

Allosteric binding site


Details of Protein

Web Source: RCSB PDB

Resolution: 2.3 Å

Crystal Code: 1V4S

Chain Length: 455 AA

Originally Bound Molecules

2-amino-4-fluoro-5-[(1-methyl-1H-imidazol-

2-yl)sulfanyl]-N-(1, 3-thiazol-2-yl)benzamide

α-D-glucose

Kamata, K., et al., Structure, 12, 2004, 429-438

http://www.google.co.in/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwj1vOzyl_XLAhXPCI4KHSjiB_MQjRwIBw&url=http://www.rcsb.org/pdb/explore.do?structureId=1v4s&psig=AFQjCNFVrr08LxMOQafXSDamNp8F6FniCg&ust=1459866254477210

http://www.google.co.in/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwj1vOzyl_XLAhXPCI4KHSjiB_MQjRwIBw&url=http://www.rcsb.org/pdb/explore.do?structureId=1v4s&psig=AFQjCNFVrr08LxMOQafXSDamNp8F6FniCg&ust=1459866254477210

Serendipitous discovery of small molecule GK activator: RO-28-1675


RO-28-1675

NH

O

S

O

OCH3

S

N

First GKA patented in year 2000, was used to validate the clinical relevance of

targeting GK to treat T2D , had potential cardiovascular risk, caused reversible

hepatic lipidosis in repeat dose toxicity studies due to oxidative metabolism of

thiazole group. Thus further development was abandoned.

NH

O

NH2

S

N

CH3

SN

N

N

CH3

Tyr215

Arg63

NH

O

N

N

Cl

S

O

OCH3

O

RO-4389620

Banyu•A 2-Aminobenzamide derivative

•Good binding abilities

•Good glucose lowering activity in diabetic models of rodents

T. O. Johnson, et al., Annl. Rep. Med. Chem., 41, 2006, 141-154

Piraglatin GK2 or R1440 and patented in 2007, reached phase II trial stage

Improves the OGTT in patients with T2DM by increasing insulin secretion

and glucose uptake and lowering hepatic glucose output

9

T. Nishimura, et al., Bioorg. Med. Chem. Lett., 19, 2009, 1357–1360

overall structure of GK protein with a ligand, having 2-amino group, bound to it and glucose molecule trapped 20 Ǻ away

Ligand shown in orange mesh, nitrogen in thiazole ring and amidine -NH interacting with ARG63 backbone

Amino –NH forming hydrogen bond (purple colour) with TYR215

3FRO

N

N

F

NH

O S

N

NH2

Importance of amino group



Kumari, V., et al., Curr. Chem. Genomics, 2, 2008, 76-89

Pocket 1: Val62, Arg63, Ile159, Val255, Val452

Pocket 2: Pro66, Tyr 215

Pocket 3: Met210, Met235, Tyr 214

Amino Acid Residues in the Allosteric site

N

N

F

NH

O S

N

NH2

N

NNH

O S

N

NH2

N

NNH

O

NH2

FF

F

N

NNH

O

NH2

NH2


GKA showing good glycemic control in rodents

TASK

Mutagenic liabilitiesOGTT in Diabetic rodents

List of Crystal structures of Glucokinase enzymes

Sr. No. Protein PDB ID Resolution (Å) Residue Count

1 1V4S 2.30 455

2 1V4T 3.40 451

3 3FGU 2.15 470

4 3IDH 2.14 470

5 3QIC 2.20 470

6 3ID8 2.40 470

7 4IWV 2.10 456

8 4IXC 2.00 456

9 4DHY 2.38 469

10 3F9M 1.50 470

11 4ISE 1.78 458

12 3FRO 2.70 455

13 4ISF 2.09 458

14 3VEY 2.25 455

15 4ISG 2.64 458

16 3AOI 2.20 455

17 3H1V 2.11 451

18 3IMX 2.00 455

19 3GOI 2.52 455

20 4BB9 1.47 633

21 4BBA 1.92 633

22 3S41 2.18 469

23 3VEV 1.80 470

24 3VF6 1.86 470

25 4DCH 1.79 473


Computational Details

CADD software Schrodinger’s

Molecular Modeling Suit

After initial cleaning and correction, the

allosteric site was maintained in flexible

mode.

Structures were drawn using Build panel in

Maestro GUI & optimized using LigPrep

(v2.2) module

where low energy conformations of all

ligands were prepared

using OPLS force field.

Ligands were docked into the active site of

crystal structures using Glide. The best

suited conformations of few ligands, which

were successful in reversing the protein in

its original conformation and produced

maximum dock score were studied

precisely.

N

N

F

NH

O S

N

NH2

Crystal Structure: 3FGU

Banyu


Trials of docking in different crystal structures with Banyu

Dock score: -3.593

N

N

F

NH

O S

N

NH2

Crystal Structure: 1V4T

Banyu



Dock score: -8.048


N

N

F

NH

O S

N

NH2

Crystal Structure: 1V4S

Banyu


Dock score: -11.534

Hydrophobic group

NH

H

ANH

O N

S

CH3

Design on benzamide template


Modification Dock Score Replacement Dock Score

-9.3462 -2.2765

-8.3462 -1.363

-8.3462 -2.7439

-10.021 -3.1282

Cyclization

in an

heterocycle

-8.578 Cyclization

in a fused

aromatic

nucleus

-3.845

R

O

Ar-

O

Ar-

OR

S O

O

R

Ar-

O

Ar-

Protein: 1V4S

Cut-off: -7.000

Ligands: 1400

S O

O

R

Design and Docking



GK2f

Dock score: -9.121

NH

O N

S

CH3

N

H

NH

H


Design and Docking


GK3l

Dock score: - -8.578

NH

N

S

CH3

N

H

NH

H

Synthesis


*Spectral and Elemental data obtained from SAIF, Chandigarh, well supported the proposed structures for the compounds

*Problematic compounds, very few, were re-purified using chromatography, and re-submitted for the analysis before in vitro tests

Total 23 benzamide derivatives, either with secondary amino function, amide function, or sulphonamido function

were selected for synthesis, based on high dock score.

B. S. Furniss, B. S. ,et al., Vogel’s Textbook of Practical Organic Chemistry, V (ed.), 1989, 413-69

Khadse S. C. et al., Arabian Journal of Chemistry, 2012, http://dx.doi.org/10.1016/j.arabjc.2012.12.020

N+

O-

O

N+

O-

O

O

O 2-Amino-4-methylthiazole

Et3N, reflux, 90° C, 4hN

+

O-

O

N+

O-

O

O

NH

N

S

O

NH

NH2

NH2

N

S

(NH4)2S, NH3

6 h

Schotten-Bowman’s Reaction

GK2 series

NH

O N

S

CH3

N

H

NH

H

http://dx.doi.org/10.1016/j.arabjc.2012.12.020

N+

O-

O

NH

NH2

Ethyl acetoacetate

Glacial acetic acid

Synthesis


*Spectral and Elemental data obtained from SAIF, Chandigarh, well supported the proposed structures for the compounds

*Problematic compounds, very few, were re-purified using chromatography, and re-submitted for the analysis before in vitro tests

B. S. Furniss, B. S. ,et al., Vogel’s Textbook of Practical Organic Chemistry, V (ed.), 1989, 413-69

Khadse S. C. et al., Arabian Journal of Chemistry, 2012, http://dx.doi.org/10.1016/j.arabjc.2012.12.020

reflux, 4 hNH

O

N+

O-

O

2-Amino-4-methylthiazole

Et3N, reflux, 90° C, 4h

NH

ON

N+

O-

O

S

(NH4)2S, NH3 reflux, 4 h

NH

ON

SNH2

N-alkylation

NH

N

S

CH3

N

H

NH

H

CH3GK3

http://dx.doi.org/10.1016/j.arabjc.2012.12.020

in vitro glucokinase assay

GK activity

Spectrophotometrically 340 nm

Room temperature (22 – 25 °C)

Contents of assay mixture

KCl 25 mmol

Hepes pH 7.1 25 mmol

β-mercaptoethanol 5 mmol

NADP 1 mmol

ATP 2.5 mmol

MgCl2 2.5 mmol

Glucose 6-phosphate

dehydrogenase 2 units/mL

GK 60 nmol

Glucose 10 mM

(simulating fed condition)

Brocklehurst, K. J., et al., Diabetes, 53 (2004), 535-541

Ligand EC 50 (µM)* E max† Ligand EC 50 (µM)* E max†

RO-28-1675 0.387------ GK3f 4.53 0.36

PSN-GK1 0.04 0.93 GK3h 4.82 0.37

GK1d 13.367 0.16 GK3j 4.98 0.36

GK1g 13.20 0.04 GK3k 5.38 0.23

GK1h 13.45 0.07 GK3l 0.89 0.68

GK1h 18.75 0.03 GK3m 4.86 0.02

GK2e 4.56 0.14 GK3p 5.65 0.02

GK2f 1.02 0.76 GK3r 3.41 0.02

GK2g 2.69 0.13 GK3v 6.45 0.03

GK2h 2.87 0.45 GK4b > 10 -

GK2m 2.93 0.45 G4g > 10 -

GK2p 2.75 0.82 GK4s > 10 -

* Values are mean of three readings

† Ratio of maximal activating response of ligand to standard at each concentration point


Progress so far

Docking studies assured replacing toxic primary amino function from Benzamide scaffold

could yield glucokinase activators.

in vitro Glucokinase assay differentiated the potent ligands that can be developed further

newer binding modes in GK enzyme was indicated

Ligands GK2f and GK3l have been subjected to glucose lowering

assays in rats with induced diabetes.

A report has been submitted to the Sponsorers, B. C. U. D., S. P. P. U.

Pune

Status


O

NH

NH

N

N

S

R

H

R

GK2f

NH

N

S

CH3

N

H

NH

H

GK3l

Board of Colleges and University Development, Savitribai Phule Pune University

Athens Institute of Education and Research, Athens, Greece

3rd annual international conference on pharmaceutical sciences

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