Synthesis of 5-chloro-N-aryl-1H-indole-2-carboxamide derivatives as inhibitors of human liver glycogen phosphorylase a

doi:10.1016/j.bmc.2008.04.010

Bioorganic & Medicinal Chemistry

Volume 16, Issue 10, 15 May 2008, Pages 5452-5464

https://doi.org/10.1016/j.bmc.2008.04.010 Get rights and content

Abstract

A series of 5-chloro-N-aryl-1H-indole-2-carboxamide derivatives were prepared and evaluated as inhibitors of human liver glycogen phosphorylase a (hLGPa). One compound, 5-chloro-N-[4-(1,2-dihydroxyethyl)phenyl]-1H-indole-2-carboxamide (2f), inhibited hLGPa with an IC₅₀ of 0.90 μM. The pyridine analogue of 2f showed inhibitory activity of glucagon-induced glucose output in cultured primary hepatocytes with an IC₅₀ of 0.62 μM and oral hypoglycemic activity in diabetic db/db mice. Crystallographic determination of the complex of 2f with hLGPa showed binding of the inhibitor in a solvent cavity at the dimer interface, with the two hydroxyl groups making favorable electrostatic interactions with hLGPa.

Graphical abstract

5-Chloro-N-[4-(1,2-dihydroxyethyl)phenyl]-1H-indole-2-carboxamide and its pyridine analog are potent inhibitors of human liver glycogen phosphorylase a (hLGPa) that binds in a solvent cavity at the hLGPa dimer interface.

Introduction

Non-insulin-dependent diabetes mellitus (type 2 diabetes) is a severe and prevalent metabolic disorder. Tight control of blood glucose levels reduces the extent and progression of diabetic complications such as nerve and kidney damage, blindness, and heart disease,1, 2 but it is difficult to achieve exact glycemic control with oral hypoglycemic agents because of limited efficacy, tolerability, and hypoglycemic risk.³ In type 2 diabetic patients, high blood glucose levels are in part caused by elevated hepatic glucose output (HGO) due to gluconeogenesis (de novo synthesis of glucose from 2- and 3-carbon precursors) and glycogenolysis (phosphorolysis of α-(1,4)-linkages within glycogen molecules),4, 5, 6, 7 and inhibition of glycogenolysis has recently emerged as an attractive therapeutic target for treatment of type 2 diabetes.8, 9, 10, 11 Glycogen phosphorylase (GP) catalyzes the first step of the breakdown of glycogen to yield glucose-1-phosphate. In the liver, phosphoglucomutase and glucose-6-phosphatase convert glucose-1-phosphate to glucose, which is then released into the blood to be supplied to the tissues (Fig. 1).12, 13, 14, 15 Therefore, inhibition of liver GP (LGP) is an alternative target for development of new drugs for type 2 diabetes.

GP is a homodimeric enzyme that exists in two conformational states (the GPa and GPb forms) that are interconverted by phosphorylation–dephosphorylation reactions at Ser14.¹⁶ Only GPa has significant potency.¹⁶ The activity of GP is regulated by ligand binding at several sites, including an allosteric site, a catalytic site and a caffeine-binding site.8, 17, 18, 19, 20, 21, 22 Several studies have revealed a further site (a new allosteric site or dimer interface site) at which 5-chloroindole-2-carboxamide derivatives are able to interact as GP inhibitors.23, 24 Among this new class of inhibitors, CP-320626, which has 5-chloroindole and phenylalanine moieties, shows potent activity against human liver GPa (hLGPa) and may act synergistically with glucose.¹⁰ As the blood glucose concentration decreases, the potency of GP inhibitors decreases, and so the risk of hypoglycemia seems to be minimized. This hypothesis prompted us to modify the structure of CP-320626 to identify novel and potent GP inhibitors.

A crystal structure of the CP-320626 inhibitor–GP complex shows that the planar 5-chloroindole moiety docks into a hydrophobic pocket, and suggests that there is little room for modification of this part of the structure.²³ However, the relatively flexible phenylalanine moiety is located close to a solvent-filled gap between the two subunits of the GP dimer and could be modified to maintain or improve the potency.23, 24, 25 The hydroxyl group and the nitrogen atom of the 4-hydroxypiperidyl moiety form a hydrogen bond to GP through a water molecule.²⁶ Therefore, to discover more potent inhibitors, we focused on replacement of the phenylalanine of CP-320626 with substituents having hydroxyl group(s) that might form hydrophilic interactions with the enzyme (Fig. 2).

In this paper, we report the discovery of a novel class of GP inhibitors and discuss their binding site based on a crystal structure of one of the inhibitors with GP.

Section snippets

Chemistry

The 5-chloro-N-phenyl-1H-indole-2-carboxamide analogues 2a–i were prepared as shown in Scheme 1.

Coupling reactions of anilines 1a–n with 5-chloro-1H-indole-2-carboxylic acid gave the corresponding products in moderate to high yield. In the case of branched diol derivative 2i, the starting material 1i was synthesized from 4-nitrobenzaldehyde through three steps, as shown in Scheme 2.²⁷

Compound 1h as starting material for 2h was obtained by hydrogenation of 2-methoxy-1-(4-nitrophenyl)ethanol²⁸

Results and discussion

The structure of the synthesized compounds and their hLGPa inhibitory activities in vitro are summarized in Table 1, Table 2, Table 3, Table 4. The 4-hydroxymethyl (2a) and 4-(2-hydroxyethyl) (2b) derivatives inhibited hLGPa with IC₅₀ values of 1.6 and 1.2 μM, respectively, whereas the 4-(3-hydroxypropyl) derivative (2c) did not show strong inhibitory activity. For 2-hydroxyethyl substitution, the three positions showed different activities and the 4-position was the most effective (2d, 2e vs 2b

Conclusion

In conclusion, we synthesized a series of N-aryl-1H-indole-2-carboxamide derivatives with a 1,2-dihydroxyethyl moiety as a new class of hLGPa inhibitors. The inhibitory activity of these compounds was explained using the structure of the hLGPa–2f complex. Compound 13a, which was the most potent in vitro hLGPa inhibitor in the study, inhibited glucose output from hepatocytes and reduced blood glucose in a db/db mouse model of diabetes.

Chemistry

¹H NMR spectra were recorded on a JEOL JNM-LA300 or a JEOL JNM-EX400 spectrometer and the chemical shifts are expressed in δ (ppm) values with tetramethylsilane as an internal standard (in NMR description, s, singlet; d, doublet; dd, double doublet; t, triplet; q, quartet; m, multiplet; and br, broad peak). Mass spectra were recorded on a JEOL JMS-700T or micromass Q-Tof Ultima API spectrometer. The elemental analyses were performed with a Yanaco MT-5 microanalyzer (C, H, N) and were within

Acknowledgments

The authors wish to thank Ms. Akiko Matsuyama-Yokono for helpful support in the preparation of this manuscript and the staff of the Division of Analytical Science Laboratories for the elemental analysis and spectral measurements. The authors also wish to thank Dr. Hitoshi Sakashita, Dr. Tetsuo Matsui, Mr. Tatsuya Maruyama and Dr. Minoru Okada for their support of this work.

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Synthesis of 5-chloro-N-aryl-1H-indole-2-carboxamide derivatives as inhibitors of human liver glycogen phosphorylase a

Abstract

Graphical abstract

Introduction

Section snippets

Chemistry

Results and discussion

Conclusion

Chemistry

Acknowledgments

Structure

Mol. Cell

Chem. Biol.

Structure

Bioorg. Med. Chem.

Ann. Intern. Med.

Nature

Diabetes

Curr. Pharm. Des.

Diabetes Care

Horm. Metab. Res.

Exp. Opin. Invest. Drugs

Curr. Protein Pept. Sci.

Proc. Natl. Acad. Sci. U.S.A.

Arch. Biochem. Biophys.

Crit. Rev. Biochem. Mol. Biol.