JSH-150

Phthalimide Conjugations for the Degradation of Oncogenic PI3K

Wenlu Lia,b, Chunmei Gaob,d, Lei Zhaoa,b, Zigao Yuanb,f*, Yuzong Chend,e,f, Yuyang Jiangb,c,d*

Abstract

PI3K/Akt/mTOR pathway is crucial for carcinogenesis and its inhibitors have made a great progress in cancer treatment. However, there is still a great developing space for PI3K inhibitors as the acquired drug resistance hindered their application in clinical. Proteolysis-targeting chimeras (PROTACs) with the potential to handle the challenges faced in drug development could be an alternative therapeutic strategy. Moreover, the past two years have witnessed remarkable advances in the development of phthalimide conjugation as a strategy for the degradation instead of inhibition of the targets, including BET family proteins, Sirtuin 2, CDK 9, Smad 3, and BCR-ABL proteins. Here, we designed and synthesized a series of potential small molecular PROTACs for the degradation of PI3K. Four compounds induced remarkable PI3K degradation and down-regulated the phosphorylation of Akt, S6K and GSK-3β in liver cancer cells HepG2. Furthermore, the representative compound D proved to inhibit tumor cells proliferation by the induction of autophagy instead of apoptosis or cell cycle arrest.

1. Introduction

Phosphatidylinositol 3-kinases (PI3Ks) are a family of intracellular lipid kinases, which have crucial functions in the survival, proliferation, growth, differentiation and migration of cells.[1-6] According to the primary structural characteristics and substrate specificity, PI3Ks are usually divided into three classes, among which Class I PI3Ks were the most commonly studied enzyme.[2, 7] Class I PI3Ks can be activated by G protein-coupled receptor (GPCR) or receptor tyrosine kinase (RTK), thus transducing signals from growth factors and cytokines into intracellular receptors.[8] The activated Class I PI3Ks can phosphorylate inositol lipids to form the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3), which further activate the serine-threonine protein kinase Akt and other downstream targets to regulate cellular processes. In mammals, based on the modes of regulation, Class I PI3Ks are divided into IA and IB subclasses.[9] Class IA PI3Ks are heterodimers that consist of a p110 catalytic subunit and a p85 regulatory subunit. There are three highly homologous isoforms of p110, including α, β, and δ, constituting the corresponding enzymes PI3Kα, PI3Kβ, and PI3Kδ, respectively. Class IB PI3Ks, expressed in white blood cells, are heterodimers that consist of a catalytic subunit p110γ and a regulatory subunit p101 or p84.[10]
As PI3K plays an important role in carcinogenesis, it has become a famous anticancer target and its inhibitors have achieved great success in chemotherapy.[9, 11-15] ZSTK474 (Fig. 1) is a pan Class I PI3K inhibitor, which indicates high selectivity over other classes of PI3Ks and protein kinases.[16] It inhibited the growth of many human cancer cell lines and block cell cycle progression at G1 phase.[17] Moreover, it inhibited migration and invasion of prostate cancer cells PC-3, and also induced autophagy in breast cancer cells MCF-7. ZSTK474 is now in phase I/II clinical trails for treatment of solid tumors. However, acquired resistance has been observed in tumor cells after long-term treatment with ZSTK474.
Proteolysis-targeting chimeras (PROTACs) are heterobifunctional compounds, which consist of two recruiting ligands connected by a linker.[18-24] One ligand binds specific to the protein of interest (POI), while the other one recruits an E3 ligase. By hijacking the E3 ligase around target protein, this strategy can selectively knock down the level of POI. Unlike small-molecule inhibitors, which only control a specific activity, PROTACs can lead to the loss function of many activities.[25] This approach has been successfully applied to the degradation of PI3K in a reported work.[26, 27] The peptidic PROTACs showed dose-dependent toxicity in MCF-7 and induced reduction of tumor size in a mouse xenograft model. However, as peptide lacked traditional drug-like properties, the development of small-molecule candidates with increased stability, better biodistribution and improved potency are urgently needed. Pomalidomide was identified as the ligand of cereblon (CRBD), a component of the cullin-4-containig E3 ubiquitin ligase complex (CRL4), which is ubiquitously expressed in physiologic and pathophysiologic tissues.[28-30] Phthalimide conjugation as a strategy for target degradation instead of inhibition has been successful in targets, including bromodomain and extraterminal (BET) family proteins[31-33], Sirtuin 2[34], CDK 9[35], Smad 3[36], and BCR-ABL proteins[37].
Our group has been doing some researches on protein degradation[38] and small molecular anticancer agents including PI3K inhibitors[39], which were proved to be efficient anticancer therapies. Based on pomalidomide and ZSTK474, we have that introducing other biological active groups to the second morpholine group might be tolerable for PI3K activity. Moreover, Ross’s[40, 41] works on PI3K and MEK dual inhibitors, which introduced the MEK inhibitor to one of the morpholine groups via different linkers, have already proved the above assumption. Based on these results, we introduced pomalidomide to the piperazine derivative of ZSTK474 via different linkers to afford new small-molecule PROTACs (Fig. 2) for the degradation gave compound 6. The condensation product of compound 5 and 6-(boc-amino)carproic acid gave compound 7, which could afford compound 8 after a step of deprotection. After the treatment of compound 8 with succinic anhydride, intermediate 9 was achieved. Compound 5 could give alkyne 10 after a step of nucleophilic substitution reaction with propargyl bromide. The nucleophilic substitution reaction of compound 5 with intermediate 2b gave target compound A. Other target compounds were given by the condensation of different carboxylic acids 3 anhydride, dichloromethane (DCM), Et3N, rt, 24 h; (e) 6-((tert-butoxycarbonyl)amino)hexanoic acid, DMF, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), N,N-Diisopropylethylamine (DIEA), rt, overnight; (f) CF3COOH, DCM, rt, 2 h; (g) succinic anhydride, DCM, Et3N, rt, 24 h; (h) propargyl bromide, K2CO3, DMF, reflux, 6 h; (i) MeCN, Et3N, reflux, overnight; (j-l) DMF, HATU, DIEA, rt, 4 h; (m-n) sodium ascorbate, CuSO4 5H2SO4, t-BuOH, H2O, 60 oC, 4 h.

2.3 Enzyme inhibition

To determine the optimal linker of the PI3K degraders we have synthesized, the kinase assays were performed to evaluate their binding affinities with targeted protein. ZSTK474 inhibited the growth of tumor cells by binding with the catalytic subunit of PI3K[42]. In this work, we chose one of the most ubiquitously expressed PI3Kα[1] as our study model. The inhibition efficiencies of our compounds on PI3Kα were shown in Table 1. Most of the compounds indicated great potency on PI3Kα with IC50 values in nanomolar scale except compound A, which confirmed our consumption that introducing other biological active groups to the second morpholine group was tolerable for PI3K activity. The results also indicated the linkers longer than hexanamide were appropriate. The most potent compound against PI3Kα, B showed similar IC50 (18 nM) with the positive control drug PI103 (17 nM) and ZSTK474 (16 nM as reported).[43] Further slightly increase the length of the linker, we achieved compound D with the IC50 of 24 nM, the inhibitory activity showed only a little decrease. While inserted a six-carbon length group into compound B to yield C, the IC50 doubled (the IC50 of C was 38 nM), which indicated that the nine-atom length linker was the optimal one. As for the compounds E and F with triazoles, which were expected to act as bioisosteres, the IC50 values were 90 nM and 86 nM, respectively.
Since our target compounds proved to be effective PI3Kα degraders, we examined other related proteins in PI3K pathway to find out its anticancer mechanism. We first tested the expression level of two representative proteins in PI3K pathway, phosphorylated Akt (p-Akt) and phosphorylated S6K (p-S6K).[44] Both p-Akt and p-S6K were decreased as the increase of drug concentration. In particular, the expression level of p-Akt showed a great decrease for B, E, or F treated cells. Then we investigated other related proteins downstream of the pathway, including

4.2 Bioassay

4.2.1 PI3Kα kinase assay

The PI3Kα kinase assay was carried out by Shanghai Chempartner Co., Ltd in Shanghai, China. The six newly synthesized compounds were tested for their activities against PI3Kα using Kinase-Glo® Luminescent Kinase Assay. The kinase assay was performed in a 384-well black plate with adding different reagents in order, that were 2.5 mL of different concentration of compounds solutions in DMSO (4% DMSO as control), 2.5 mL PI3Kα solution (final concentration of 1.65 nM) and 5 mL
PIP2 substrate solution (final concentration of 50 mM) and ATP solution in kinase buffer (final concentration of 25 mM). The assay plate was incubated at room temperature for 1 h before adding 10 mL Kinase-Glo reagent to each well to stop the reaction. The assay plate was read on a plate reader Envision program for before boiled at 100 oC with 5×loading buffer for 10 min. 10% SDS-PAGE jelly was used to analysis the lysate before transferred the protein samples to membrane completely. After blocking the membrane with 5% dry milk in TBST for 1 h, incubating them in primary antibody dilution (rabbit anti-p110α, Abcam; rabbit anti-p-Akt (Ser 473), rabbit anti-pp70 S6K (Thr 389), mouse anti-cyclin D1, mouse anti-β-actin, Cell Signaling Technology; rabbit anti-LC3B, Beyotime) overnight at 4

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