Heat shock protein Hsp is a
Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a central role in regulating the maturation, activation and stability of numerous “client proteins” that drive the development and progression of many cancers. Therefore, inhibition of Hsp90 would result in degradation of the client proteins and suppress the growth of cancers [14,15]. As EML4-ALK is one of the most sensitive “client proteins” of Hsp90, inhibition of Hsp90 could decrease the level of EML4-ALK. Recently, Hsp90 inhibitors have been proposed as an alternative strategy for overcoming ALK inhibitors-induced resistance [, , ]. Data from clinical development of several Hsp90 inhibitors have demonstrated that Hsp90 inhibitors are efficient in NSCLC patients with EML4–ALK gene fusion, whether sensitive or resistant to crizotinib [, , ]. More clinical trials of Hsp90 inhibitors are being conducted to evaluate the anticancer efficacy in combination with ALK inhibitors [2,22,23]. Apart from combined use of ALK inhibitors and Hsp90 inhibitors, we envision that ALK/Hsp90 dual inhibitors by simultaneously targeting both targets are an attractive strategy with more therapeutic benefit .
Analysis of the X-ray crystal structure of Hsp90 inhibitor AUY922 (Fig. 2A) reveals that the resorcinol moiety binds deep into the pocket . Phenolic hydroxyl groups and the N-, O-atoms of the isoxazole ring form a tight network of hydrogen bonds with the key residue Asp93 and surrounding water molecules. The isoxazole 3-amide group and morpholine moiety extend to the solvent region where larger space exists for further modification. In addition, compound 6 is a high potency second-generation ALK inhibitor reported previously by our group . Molecular modeling of 6 was conducted based upon the reported crystal structure of ceritinib (3) with the kinase domain of ALK  (Fig. 2B). Compound 6 forms two key hydrogen bonds at the hinge area via the pyrimidine nitrogen ACP-196 and NH with the backbone NH and oxygen of Met1199 respectively. The isopropyl group fits into a small hydrophobic pocket. The glycine moiety extends to the solvent region where larger space exists allowing for further structural modification. The larger space in the solvent-interaction region of either ALK or Hsp90 might be used to incorporate the other target interaction motif. Based on these analysis, we decided to develop ALK/Hsp90 dual inhibitors by tethering the terminal amino group of ALK inhibitor 6 with the solvent-interaction region of the Hsp90 inhibitor AUY922.
Chemistry The synthesis of compounds 10a-k is described in Scheme 1, Scheme 2, Scheme 3. The key ALK intermediates 7a-f  and Hsp90 inhibitor intermediates 8a-d [24,27] were prepared according to previously reported procedures. The synthesis of compounds 10a-e was conducted as shown in Scheme 1. Condensation of acid 8a with anilines 7c-e in the presence of HATU, HOAt and DIPEA afforded products 9a-c in 81–90% yields, which were then converted to compounds 10a-c in 25–30% yields through O-debenzylation using BCl3. Compound 7e was condensed with acid 8b followed by O-debenzylation using BCl3 to afford compound 10d in 27% overall yield. Similarly, compound 10e was obtained by following similar procedures in 33% overall yield. As shown in Scheme 2, the intermediate 11 was formed through reductive amination using glycine hydrochloride and NaCNBH3, followed by N-Boc protection, and hydrolysis with NaOH in 60% overall yield (three steps). Condensation of 11 with 7a-b using HATU, HOAt and DIPEA, followed by removal of N-Boc under acidic condition provided compounds 9f-g in 83% overall yields, which were then converted to final compounds 10f-g through O-debenzylation using BCl3 in 41% and 33% yields respectively. Meanwhile, compound 12 was prepared in 62% overall yield by reductive amination of 8c with N-Boc-piperazine and NaCNBH3, followed by N-Boc deprotection with TFA, nucleophilic substitution with tert-butyl bromoacetate and hydrolysis with TFA. Subsequent condensation of 12 with 7b, followed by O-debenzylation using BCl3 gave 10h in 40% overall yield. Compound 10i was obtained in 33% overall yield by condensation of 12 with 7e followed by O-debenzylation.