Synthesis of Deuterium-Labeled Zaleplon-d5

Posted by Tylor Keller from the Technology category at 18 Jun 2025 09:18:03 am.

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Introduction

Zaleplon is a pyrazolopyrimidine sedative-hypnotic agent licensed for the short-term treatment of insomnia. Due to its potential for abuse and addiction, zaleplon is classified as a controlled substance in some countries. Accurate analysis and identification of zaleplon in drug abuse cases requires the use of isotopically labeled internal standards, such as zaleplon-d₅.

Here, we present the synthesis of deuterium-labeled zaleplon-d₅, which can serve as an internal standard for the GC-MS analysis of zaleplon.

Synthesis of Zaleplon-d₅ by Scheme 1

Our first route to zaleplon-d₅ shown in Scheme 1.

Initially, 3nitroacetophenone 1 was treated with N,N-dimethylformamide dimethylacetal under reflux to yield the intermediate, enamide 2.
Another key intermediate, 5-amino-1H-pyrazole-4-carbonitrile (4) was obtained by refluxing ethoxymethylenemalononitrile (3) and hydrazine hydrate in ethanol.
In the following step, compounds 4 and 2 underwent cyclization under mild acidic condition at reflux to yield 7-(3-nitro-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carbonitrile (5).
An efficient reduction of 5 using 10% Pd/C catalyst at an H pressure of 60 psi gave 7-(3-amino-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carbonitrile (6).
The 3-amino phenyl pyrazolopyrimidine 6 was treated with an acetic anhydride and pyridine to produce acetamide 7.
Introduction of the isotopic label was attempted by treating acetamide 7 with ethyl iodide-din the presence of sodium hydride, under an inert atmosphere at 50℃.

Unfortunately, the yield of zaleplon-d₅ obtained was negligible. Various alternative conditions were tried, but the maximum yield obtained was only 20%. The purification of zaleplon-d₅ was also problematic, as zaleplon-d₅ could not be successfully crystallized and was difficult to elute from a silica gel column. Therefore, this route was abandoned.

Synthesis of Zaleplon-d₅ by Scheme 2

The synthesis started from compound 1, which is readily available and inexpensive. 3'-Nitroacetophenone 1 was reduced to 3'-aminoacetophenone 9.
Subsequently, Acylation to generate acetamide 10, which in turn was treated with N,N-dimethylformamide dimethylacetal to produce enamide 11.
Enamide 11 was alkylated using ethyl iodide-d₅ and sodium hydride as base at room temperature to yield the N-ethylated enamide 12.
In the final step, N-ethylenamide 12 was coupled with 5-aminopyrazole 4 in aqueous acid at 50℃ to produce zaleplon-d₅ 8 in 85% yield.
Further purification by recrystallization of the crude product from 30% aqueous acetic acid yielded colorless crystals of zaleplon-d₅ 8.

In conclusion, this work provides an elegant route to zaleplon-d₅, an internal standard for zaleplon analysis. This synthesis paves the way for the quantitative detection of zaleplon in drug abusers.

Reference

Shaikh, A. C., & Chen, C. Synthesis of deuterium-labeled zaleplon-d₅ as an internal standard. Journal of Labelled Compounds and Radiopharmaceuticals: The Official Journal of the International Isotope Society. 2008, 51(1), 72-76.

Tags: Zaleplon, Deuterium Labeling, Synthesis, Isotopic Standards, GC-MS Analysis, Controlled Substances

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