Synthesis of N-substituted N-nitrosohydroxylamines as inhibitors of Mushroom Tyrosinase

doi:10.1016/S0968-0896(01)00003-7

Bioorganic & Medicinal Chemistry

Volume 9, Issue 5, May 2001, Pages 1233-1240

https://doi.org/10.1016/S0968-0896(01)00003-7 Get rights and content

Abstract

A series of N-substituted N-nitrosohydroxylamines including six new compounds were synthesized and examined for inhibition of mushroom tyrosinase. Corresponding hydroxylamines were reacted with n-butyl nitrite to give substituted nitrosohydroxylamines as their ammonium salt. The N-substituted hydroxylamines were prepared from the primary amines via the oxaziridine, or from the carbonyl compounds via the oxime. Most of the nitrosohydroxylamines tested inhibited mushroom tyrosinase. Among them, N-cyclopentyl-N-nitrosohydroxylamine exhibited the most potent activity (IC₅₀=0.6 μM), as powerful as that of tropolone, one of the most powerful inhibitors. As removal of nitroso or hydroxyl moiety, the enzyme inhibitory activity was completely diminished. Both N-nitroso group and N-hydroxy group were suggested to be essential for the activity, probably by interacting with the copper ion at the active site of the enzyme. Lineweaver–Burk plotting showed that cupferron was a competitive inhibitor but that N-cyclopentyl-N-nitrosohydroxylamine was not.

A series of N-substituted N-nitrosohydroxylamines were synthesized and their inhibitory effects on mushroom tyrosinase were examined. Among them, N-cyclopentyl-N-nitrosohydroxylamine (9) was found to be the most potent inhibitor(IC₅₀=0.6 μM).

Introduction

Tyrosinase (E. C.1.14.8.1) is a copper-containing enzyme widely distributed in plants and animals. It catalyses the o-hydroxylation of monophenols and also the oxidation of o-diphenols to o-quinones. Tyrosinase is known to be a key enzyme for melanin biosynthesis in plants and animals. Therefore, tyrosinase inhibitors should be clinically useful for the treatment of some dermatological disorders associated with melanin hyperpigmentation¹ and also important in cosmetics for whitening and depigmentation after sunburn.2, 3 In addition, tyrosinase is known to be involved in the molting process of insects⁴ and adhesion of marine organisms.⁵

Several N-substituted N-nitrosohydroxylamines are known to inhibit various types of enzymes; e.g., dopastin (1) is an inhibitor of dopamine-β-monohydroxylase (E. C.1.14.2.1),⁶ nitrosoxacins inhibit 5-lipoxygenase (E. C.1.13.11.12),⁷ and cupferron (2) and neocupferron (3), which are metal-chelating agents, inhibit superoxide dismutase (E. C.1.15.1.1)⁸ (Fig. 1). In the present paper, we found that dopastin (1) also inhibited mushroom tyrosinase effectively. The inhibitory activity of dopastin (1) is likely to be derived from the N-nitroso-N-hydroxylamino group, which can interact with the copper ion at the active site of the enzyme. Therefore, we synthesized several N-nitroso-N-hydroxylamines and examined their inhibitory activity on mushroom tyrosinase to study the structure–activity relationship.

Section snippets

Synthesis of N-substituted N-nitrosohydroxylamines

Eleven N-substituted N-nitrosohydroxylamines (4–14) including six new compounds (5–8, 12 and 13) were synthesized. The procedure for the synthesis of the N-substituted N-nitrosohydroxylamines is outlined in Scheme 1. The N-nitrosohydroxylamines were prepared by nitrosation of the corresponding hydroxylamine, which were obtained by procedures A or B in Scheme 1. Procedure A included condensation of the primary amine with benzaldehyde and oxidation of the resulting imine with m-chloroperbenzoic

Conclusion

We found that N-substituted N-nitrosohydroxylamines inhibited mushroom tyrosinase. Their potency was affected by the structure of N-substitutent. N-Substitution might cause steric hindrance for the approach of inhibitors to the active site of the enzyme. As a mechanism of inhibition, we suggested that both N-nitroso and N-hydroxyl groups were essential to inhibit the enzyme, interacting with the copper ion at the active site of the enzyme. Furthermore, kinetic analysis of the enzyme indicated

Materials

Cupferron (2) and neocupferron (3) were purchased from Tokyo Kashei. Dopastine (1) was kindly supplied by Dr. H. Iinuma, Institute of Microbial Chemistry, Tokyo. Tyrosinase was obtained from Sigma.

Syntheses

The structures were determined by 60 MHz ¹H NMR (Hitachi R-24), IR (Jasco A-100, Bio-Rad FTS-60A(FTIR)), HRMS (JEOL JMS-700 MSstaition) and the elemental analysis. The typical procedure for the preparation of N-nitrosohydroxylamines 5 and 8 (procedure A) is described for 5.

Acknowledgements

The authors wish to thank Dr. H. Iinuma, Institute of Microbial Chemistry, for the gift of dopasitin and valuable suggestions.

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Synthesis of N-substituted N-nitrosohydroxylamines as inhibitors of Mushroom Tyrosinase

Abstract

Introduction

Section snippets

Synthesis of N-substituted N-nitrosohydroxylamines

Conclusion

Materials

Syntheses

Acknowledgements

Biochim. Biophys. Acta

Biochim. Biophysica Acta

Tetrahedron

Phytochemistry

Phytochemistry

Polyhedron

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Soc. Cosmet. Chem.

Fragrance J.

FEBS Lett.ers

Agric. Biol. Chem.

J. Antibiot.

Ind. J. Biochem. Biophys.