Research paper
Discovery of 3-pyrazolyl-substituted pyrazolo[1,5-a]pyrimidine derivatives as potent TRK inhibitors to overcome clinically acquired resistance

https://doi.org/10.1016/j.ejmech.2022.114654Get rights and content

Highlights

  • TRKA G667C mutation still limit sensitivity to the second-generation inhibitors.

  • A novel inhibitor 5n was rationally designed to overcome the TRKA G667C mutation.

  • Compound 5n displayed favorable pharmacokinetic profile and antitumor efficacy.

Abstract

Several secondary tropomyosin receptor kinase (TRK) mutations located in the solvent front, xDFG, and gatekeeper regions, are a common cause of clinical resistance. Mutations in the xDFG motif in particular limit sensitivity to second-generation TRK inhibitors, which represent an unmet clinical need. We designed a series of 3-pyrazolyl-substituted pyrazolo[1,5-a]pyrimidine derivatives toward these secondary mutations using ring-opening and scaffold-hopping strategies. Compound 5n was the most potent, with IC50 values of 2.3 nM, 0.4 nM, and 0.5 nM against TRKAG667C, TRKAF589L, and TRKAG595R, compared to selitrectinib with IC50 values of 12.6 nM, 5.8 nM, and 7.6 nM, respectively (approximately 5.4, 14.5, and 15.2-fold increases). Furthermore, 5n displayed favorable pharmacokinetic properties and satisfactory antitumor efficacy (tumor growth inhibition of 97% at 30 mg/kg and 73% at 100 mg/kg) in TRKAWT and TRKAG667C xenograft mouse models. Collectively, 5n is a promising TRK inhibitor lead compound for overcoming clinically acquired resistance to second-generation inhibitors, particularly for resistant tumors harboring the TRKAG667C mutation in the xDFG motif.

Introduction

The highly homologous members of the tropomyosin receptor kinase (TRK) family, TRKA, TRKB, and TRKC, which are transmembrane receptor tyrosine kinases, are encoded by the genes NTRK1, NTRK2, and NTRK3, respectively [[1], [2], [3], [4], [5], [6], [7]]. Upon binding with different ligands, TRKs dimerize and autophosphorylate to trigger a cascade of signaling pathways, including the MAPK, PI3K, and PLCγ pathways, which are crucial for modulating cell proliferation, differentiation, and survival in the nervous system [[8], [9], [10], [11], [12], [13], [14], [15]]. The most common pathogenic mechanism of TRKs in malignant tumors is NTRK gene fusion, which is involved in carcinogenesis through rearrangements such as the ETS translocation variant 6-NTRK3 in secretory mesodermal congenital fibrosarcoma [16] and tropomyosin 3-NTRK1 in colorectal cancer [17]. Since the TRKs transmembrane fusions regularly result in the structural or functional loss of the extracellular domain, antibodies against TRK and its ligands (such as monoclonal antibody therapy) are not effective as anticancer agents [[18], [19], [20], [21], [22]]. Therefore, small molecule inhibitors have become a promising therapeutic strategy to target NTRK fusion genes.

To date, several small molecule TRK inhibitors have been reported to treat the cancer with NTRK gene fusions [[23], [24], [25]]. Representative TRK inhibitors are exhibited in Fig. 1, including Larotrectinib, Entrectinib, Selitrectinib, Repotrectinib, Milciclib, Cabozantinib, Altiratinib, and Belizatinib. Among them, Larotrectinib and Entrectinib (First-generation TRK inhibitors) were recently approved by U.S. Food and Drug Administration (FDA) for the patients with these cancers [26,27]. With prolonged treatment durations using first-generation TRK inhibitors, drug resistance generally develops, similar to other targeted antitumor drugs. Several secondary TRK mutations have emerged in patients [22]. Mutations of the solvent front region of TRKA (TRKAG595R), xDFG motif (TRKAG667C), and gatekeeper area (TRKAF589L) are confirmed as common mechanisms of clinical resistance [[28], [29], [30], [31], [32]]. The mutated amino acid residues create steric hindrance to the first-generation TRK inhibitors, which weaken the effect of the inhibitor [28,33]. Several second-generation inhibitors are being developed to address these mutations, such as selitrectinib [29,34] and repotrectinib [34,35], which have compact macrocyclic structures to mitigate the effects of mutated amino acid residues, and most other inhibitors have been limited their access to further development due to poor kinase selectivity or low potency [36]. These second-generation inhibitors can effectively circumvent clinical drug resistance resulting from G595R and F589L mutations. However, clinical case outcomes indicate that some patients treated with these compounds eventually became unresponsive due to mutations in the xDFG motif, suggesting that these mutations limit sensitivity to second-generation inhibitors [[37], [38], [39], [40], [41]]. No therapeutic drug is approved to date for the treatment of patients who develop resistance to first-generation TRK inhibitors due to the secondary mutations. Consequently, it is of great significance to explore novel TRK inhibitors with potent inhibitory activities for patients who harbor solvent front, xDFG, and gatekeeper region mutations.

Herein, we report our efforts to discover a series of pyrazolo[1,5-a]pyrimidine-5-amine derivatives, which were designed and synthesized through a ring-opening and scaffold-hopping strategy based on the structure of larotrectinib.

Section snippets

Molecular design

Larotrectinib (1) is a selective pan-TRK inhibitor approved by the US Food and Drug Administration (FDA) in 2018 for NTRK fusion-positive cancers. At present, the co-crystal structure of 1 and TRK protein remains undisclosed. To investigate the binding mode of 1 with mutant proteins TRKA G667C, F589L, and G595R, we first docked 1 with an existing wild TRK crystal structure (PDB ID: 4AOJ, a reported activated TRKA kinase targeted by type I inhibitors [42]) by using GOLD 3.0 [43]. Then, the Auto

Chemistry

The synthetic routes of compounds 5a-n are illustrated in Scheme 1 [46]. Commercially available (R)-1-(2,5-difluorophenyl)ethanaMine hydrochloride (2) was reacted with 5-chloropyrazolo[1,5-a]pyrimidine to generate (R)-N-(1-(2,5-difluorophenyl)ethyl)pyrazolo[1,5-a]pyrimidine-5-amine (3), which was then reacted with N-iodosuccinimide to generate (R)-N-(1-(2,5-difluorophenyl)ethyl)-3-iodopyrazolo [1,5-a]pyrimidine-5-amine (4). The Suzuki coupling reaction of intermediate 4 with corresponding

Results and discussion

To verify the rationality of our design strategy, compound 5a was first evaluated for inhibitory activity against TRKAG667C, TRKAF589L, and TRKAG595R, using larotrectinib and selitrectinib as double positive controls. Encouragingly, 5a displayed much stronger kinase inhibitory potencies than larotrectinib, with IC50 values of 15.3, 0.7, and 1.8 nM for TRKAG667C, TRKAF589L, and TRKAG595R, respectively (Table 1), providing a promising lead compound for further structural optimization. Compound 5a

Conclusions

In this study, a 3-pyrazolyl-substituted pyrazolo[1,5-a]pyrimidine derivative 5a was discovered as a hit compound through the a ring-opening and scaffold-hopping strategy. Compound 5a displayed strong kinase inhibition against TRKAF589L and TRKAG595R at a low nanomolar IC50 value, but it failed to improve the enzymatic inhibitory activity against TRKAG667C, located in the xDFG motif, over selitrectinib. Through detailed structural optimization of 5a, we identified compound 5n, which was more

General methods

Unless otherwise noted, reagents and solvents used in experiments were purchased from commercial sources and used without further purification. Flash chromatography was performed using 200–400 Mesh silica gel from Qingdao Makall Group Co., Ltd.; China. Silica gel plates-based thin-layer chromatography (TLC) was used to monitor all reactions with fluorescence F254 or F365 light. the reactions involving air- or moisture-sensitive reagents were performed under a nitrogen or argon atmosphere. 1H

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

We thank the National Key Research and Development Program of China (No. 2017YFA0505200) and the Key Research and Development Program of Hubei Province, China (2020BCB042) for financial support.

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    Y.G., F.W and. M.W. contributed equally to this work.

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