Synthesis of deuterium-labeled crizotinib , a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK)

Wangwei Ao^, Yuan Li^, Yinsheng Zhang*

Abstract:

To more accurately and rapidly achieve quantitative detection of clinical crizotinib samples, stable isotope labeled crizotinib was required as an internal standard. We have developed a method to prepare racemic [D9]crizotinib using a base-catalyzed H/D exchange of both nitro compound 2 and the acetophenone compound 6 with D2O and NaBD4 reduction of 7 as the key steps to introduce the nine deuterium atoms. Starting with 4-hydroxypiperidine, 14 step-synthesis furnished the desired racemic [D9]crizotinib 18. The deuterium-labeled compound 18 with the chemical purity of 99.62% was applicable for use as internal standards in the drug clinical study.

Key words: Deuteration; H-D exchange; Internal Standard; Crizotinib; c-MET; ALK

Introduction

In 2011, crizotinib (brand name Xalkori, Fig.1) became the first ALK inhibitor approved by the USA Food and Drug Administration (FDA) as a first-line treatment for ALK-positive lung cancer patients.[1-4] Stable isotope-labeled compounds have been proven to be ideal internal standards for use in a human absorption, distribution, metabolism, and excretion (ADME) studies.[5] Among all of the internal standard compounds developed, deuterium-labeled compounds are well known to be the first choice for such applications due to their similarity of physical and chemical properties with original compounds and easy availability in terms of deuterium source and synthetic approach.[6]
To our knowledge, there were few procedures described in literature for the preparation of deuterium-labeled crizotinib.[7-8] The known approaches either need expensive deuterium sources, such as CDCl3 and DCOD, or only can offer one or two deuterium labeled crizotinib. For crizotinib, the abundance of the M+5 (454 amu) isotopologues comes to 1.5%, considering the natural isotope distribution of 35Cl/ 37Cl and 12C/13C. Therefore, a minimum of six deuterated sites for the internal standard appeared to be sufficient to prevent unfavorable interferences between signals of non-deuterated and deuterated crizotinib in a quantitative bioanalytical liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay. Considering the structure and possible synthetic approach of crizotinib (Fig. 1), a 14-step labeling process (Scheme 1 , 2 & 3) was developed to prepare the desired deuterium-labeled R/S-crizotinib 18.

RESULTS AND DISCUSSION

Methods for the synthesis of crizotinib have been reported in the literature,[3, 9-10] but the key approach is basically the same. According to the existing synthetic route of the original drug, we designed the synthetic routes shown in Scheme 1, 2 & 3 to successfully prepare the deuterated crizotinib 18. To activate the ortho-hydrogen of the nitrogen atom, nitroso group was first introduced into the nitrogen atom of 4-hydroxypiperidine 1 to form nitroso compound 2 in 78% yield, and then four deuterium atoms can be easily introduced to give compound 3 after the hydrogen-deuterium (H-D) exchange with D2O under basic (NaOD) conditions.[11] Removal of nitroso group by using aluminum-nickel (Al-Ni) alloy under alkaline condition afforded 4-[D4]hydroxypiperidine 4. Without isolation of 4, direct N-acylation of 4 with di-tert-butyl dicarbonate furnished 5 with a protection group in overall 25% yield (3 steps). From 2, 5 was prepared in one pot without the isolation of intermediates 3 & 4 since H-D exchange, (Al-Ni) alloy reduction and Boc protection reactions used the same basic solution as a solvent.
Although Hesk12 et al. has described the three-step approach toward the synthesis of 5, it needed expensive dueterated formaldehyde and lacked analytical data. Our new approch to 5 uses much cheaper and more readily availabe D2O as only deuterium source to offer >95% deuterium enrichment. Furthermore, four deuterium atoms were introduced into 7 after the H-D exchange in a mixed solution of D2O and 1,4-dioxane under a basic (NaOD) condition in a 92% yield.[13-14] Unlike the literature,7 our NaOD promoted H-D exchange reaction allowed not only deutrium atoms to be incorpoarted into α methyl group of the ketone 7, but also into 3 position of phenyl ring. To avoid the back-exchange of 7 with the non-deuterated agents and solvents, deuterated sodium boron hydride (NaBD4) in a mixed solution of MeOD and THF was applied to reduce the labeled ketone 7 to racemic 8 in a 82% yield. Based on HNMR of 8, it was observed that the base-catalyzed D-H exchange of 6 with D2O gave >95% D at 2 position and >93% D at 3’ position, respectively, and NaBD4 reduction of the ketone 7 offered 98% D enrichment (Scheme 2). Mitsunobu reaction of 8 with 9 provided 10 in 42.8% yield, and then 10 was further reduced with zinc powder in acetic acid under reflux to 11. Bromination of 11 with NBS in acetonitrile produced 12 in 78% yield.
With the key deuterium-labeled intermediates 5 & 12 in hand, deuterated crizotinib was successfully prepared via a 5-step synthetic sequence starting from 5 as depicted in Scheme 3. The N-mesylation of 5 with methane sulfonyl chloride under a basic (Et3N) condition provided 13 in 86% yield. N-Alkylation of 13 with 3-iodopyrazole 14 generated iodo compound 15 in 70.7% yield. Palladium-catalyzed coupling reaction of 15 with bis(pinacilato)diboron gave the boronic ester derivative 16 in 62.9% yield. Suzuki cross-coupling reaction of two labeled intermediates 12 & 16 offered boc-protected crizotinib 17 that was de-protected with 5% HCl methanol solution to generate R/S-[D9]crizotinib 18 in a fair yield. It was not necessary to separated R/S isomers since the mixture of two enantiomers as an internal standard (Figure 2) does not affect the accuracy in a quantitative bioanalytical LC/MS/MS assay.

Conclusion

The key points of our deuterium-labeling synthetic approach to R/S-[D9]crizotinib 18 are summarized as follows: 1) activating the adjacent hydrogen atom of the piperidine moiety by introducing the nitroso group into the secondary amine of 4-hydroxypiperidine, which make the nitroso compound easily exchanged with D2O to incorporate four deuterium atoms into the key intermediate 5; and 2) carrying out H-D exchange of acetophenone 6 with D2O followed by reduction of carbonyl with NaBD4, which allows five deuterium atoms introduced into another intermediate 8. Although the entire synthetic route is long (14 steps), the reactions involved are relatively conventional and several steps do not require post-treatment. So far, we have developed a practical method for synthesizing deuterium-labeled crizotinib. The total yield of the 14-step synthesis was 6.2%, which provided a much-needed stable isotope internal standard for clinical studies of crizotinib.

Experimental General

All reagents were purchased from commercial sources and were used as received. Routine monitoring of reactions was performed by thin layer chromatography (TLC) using pre-coated Haiyang GF254 silica gel TLC plates. NMR spectra were recorded on a Bruker AVANCE 500 spectrometer at 500 MHz with tetramethylsilane used as an internal reference. High resolution mass spectra (HRMS) were performed on Agilent Acrrurate-Mass Q-ToF LC/MS 6520 mass spectrometer with electron spray ionization (ESI) mode. Intermediates 1, 7 & 9 were purchased from Meryer (Shanghai) Co., Ltd. Intermediates 14 were purchased from Energy chemical Ltd. Deuterium agents, D2O (99.9% D) and NaBD4 (98.5% D) were obtained from Sigma-Aldrich.

References

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