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PI3Ks are frequently hyper-activated in breast cancer and targeting PI3Kα has exhibited promising but variable response in preclinical and clinical settings. CYH33 is a novel PI3Kα-selective inhibitor in phase I clinical trial. We investigated the efficacy of CYH33 against breast cancer and explored potential predictive biomarkers. CYH33 potently restrained tumor growth in mice bearing human breast cancer cell xenografts and in R26-Pik3caH1047R;MMTV-Cre transgenic mice. CYH33 significantly inhibited proliferation of a panel of human breast cancer cells, while diversity in sensitivity has been observed. Cells harboring activating PIK3CA mutation, amplified HER2 were more responsive to CYH33 than their counterparts. Besides, cells in HER2-enriched or luminal subtype were more sensitive to CYH33 than basal-like breast cancer. Sensitivity to CYH33 has been further revealed to be associated with induction of G1 phase arrest and simultaneous inhibition of Akt and ERK. Sensitivity of patient-derived xenograft to CYH33 was also positively correlated with decrease in phosphorylated ERK. Taken together, CYH33 is a promising PI3Kα inhibitor for breast cancer treatment and decrease in ERK phosphorylation may indicate its efficacy, which provides useful clues for rational design of the ongoing clinical trials.

Cynotophyllosides M–O, three new polyhydroxypregnane glycosides, were obtained from the roots of Cynanchum otophyllum, together with four known compounds, deacetylmetaplexignin, sarcostion, cynotophylloside J and kidjoranin 3-O-α-diginopyranosyl-(1→4)-β-cymaropyranoside. Their structures were elucidated using extensive spectroscopic methods (in particular, 2D-NMR techniques) and acid-catalysed hydrolysis.

// Jin-Xue He 1, * , Meng Wang 2, * , Xia-Juan Huan 1 , Chuan-Huizi Chen 1 , Shan-Shan Song 1 , Ying-Qing Wang 1 , Xue-Mei Liao 1 , Cun Tan 2 , Qian He 2 , Lin-Jiang Tong 1 , Yu-Ting Wang 1 , Xiao-Hua Li 1 , Yi Su 1 , Yan-Yan Shen 1 , Yi-Ming Sun 1 , Xin-Ying Yang 1 , Yi Chen 1 , Zhi-Wei Gao 3 , Xiao-Yan Chen 3 , Bing Xiong 2 , Xiu-Lian Lu 4 , Jian Ding 1 , Chun-Hao Yang 2 , Ze-Hong Miao 1 1 Division of Anti-Tumor Pharmacology and State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China, University of Chinese Academy of Sciences, Beijing 100049, China 2 Department of Medicinal Chemistry, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China, University of Chinese Academy of Sciences, Beijing 100049, China 3 Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China 4 Cisen Pharmaceutical Co., LTD, Jining 272073, Shandong, China * These authors contributed equally to this work Correspondence to: Ze-Hong Miao, email: zhmiao@simm.ac.cn Chun-Hao Yang, email: chyang@simm.ac.cn Keywords: MPH, PARP inhibitor, homologous recombination, antitumor activity, synthetic lethality Received: April 01, 2016 Accepted: November 23, 2016 Published: December 01, 2016 ABSTRACT The approval of poly(ADP-ribose) polymerase (PARP) inhibitor AZD2281 in 2014 marked the successful establishment of the therapeutic strategy targeting homologous recombination repair defects of cancers in the clinic. However, AZD2281 has poor water solubility, low tissue distribution and relatively weak in vivo anticancer activity, which appears to become limiting factors for its clinical use. In this study, we found that mefuparib hydrochloride (MPH) was a potent PARP inhibitor, possessing prominent in vitro and in vivo anticancer activity. Notably, MPH displayed high water solubility (> 35 mg/ml) and potent PARP1/2 inhibition in a substrate-competitive manner. It reduced poly(ADP-ribose) (PAR) formation, enhanced γH2AX levels, induced G2/M arrest and subsequent apoptosis in homologous recombination repair (HR)-deficient cells. Proof-of-concept studies confirmed the MPH-caused synthetic lethality. MPH showed potent in vitro and in vivo proliferation and growth inhibition against HR-deficient cancer cells and synergistic sensitization of HR-proficient xenografts to the anticancer drug temozolomide. A good relationship between the anticancer activity and the PARP inhibition of MPH suggested that PAR formation and γH2AX accumulation could serve as its pharmacodynamic biomarkers. Its high bioavailability (40%~100%) and high tissue distribution in both monkeys and rats were its most important pharmacokinetic features. Its average concentrations were 33-fold higher in the tissues than in the plasma in rats. Our work supports the further clinical development of MPH as a novel PARP1/2 inhibitor for cancer therapy.

In various human cancers, PI3Ks pathway is ubiquitously dysregulated and thus become a promising anti-cancer target. To discover new potent and selective PI3K inhibitors as potential anticancer drugs, new pyrrolo[2,1-f][1,2,4]triazines were designed, leading to the discovery of compound 37 (CYH33), a selective PI3Kα inhibitor (IC50 = 5.9 nM, β/α, δ/α,γ/α = 101-, 13-, 38-fold). Western blot analysis confirmed that compound 37 could inhibit phosphorylation of AKT in human cancer cells to modulate the cellular PI3K/AKT/mTOR pathway. And further evaluation in vivo against SKOV-3 xenograft models demonstrated that a dose-dependent antitumor efficacy was achieved.

A one-pot copper(II)-catalyzed tandem synthesis of 2-substituted pyrrolo[1,2-b]pyridazin-4(1H)-ones from N-aminopyrroles was developed. This tandem reaction involves a Conrad–Limpach-type reaction, including the thermal condensation of N-aminopyrroles with the carbonyl group of β-oxo esters followed by the cyclization of Schiff base intermediates. Compared to the traditional Conrad–Limpach quinoline synthesis, we herein successfully applied copper(II) as a catalyst in this transformation to furnish 2-substituted pyrrolo[1,2-b]pyridazin-4(1H)-ones for the first time. Most of the substrates bearing electron-donating (EDG) and electron-withdrawing (EWG) groups worked well with this procedure. The corresponding products could be converted directly into diverse pyrrolo[1,2-b]pyridazine for drug discovery and materials science.

The nuclear protein poly(ADP-ribose) polymerases-1/2 (PARP-1/2) are involved in DNA repair damaged by endogenous or exogenous process. And PARP-1/2 inhibitors have been proved to be clinically efficacious for DNA repair deficient tumors in the past decade. We have developed a series of 4,5,6,7-tetrahydrothienopyridin-2-yl benzimidazole carboxamides as novel and potent PARP-1/2 inhibitors. The best compound resulted from this series is compound 27 which displays excellent PARP-1 and PARP-2 inhibitory activity with IC50 of 18 nM and 42 nM, respectively. Furthermore, it can selectively kill BRCA2 deficient V-C8 cells with a CC50 of 920 nM. In the MDA-MB-436 (BRCA-1 mutant) xenograft model, this compound was well tolerated and showed single-agent activity. Based on the results above, compound 27 has been selected as a lead candidate targeting PARP-1/2 and its preclinical characterization is also underway.

Two series of novel 2-substituted 5,7-dihydroxyanthra[2,1-d]thiazole-6,11-dione derivatives from natural rhein were designed, synthesized and evaluated for their antitumour activities against human cancer cell lines A549 and HeLa in vitro.

Esophageal cancer is the seventh most common cancer and the sixth leading cause of cancer-related mortality in the world. More than 80% of esophageal cancers are esophageal squamous cell carcinomas (ESCCs), and no molecularly targeted therapy is currently available. Aberrant activation of PI3Kα occurs frequently in ESCC, making it a promising target for the treatment of ESCC. CYH33 is a novel PI3Kα-selective inhibitor and displays promising efficacy against ESCC in preclinical settings, which is currently in phase I clinical trial for the treatment of ESCC. Acquired resistance is likely to develop after continuous administration of PI3Kα-selective inhibitor in breast cancer patients. It is of great importance to study the mechanism conferring resistance to CYH33 in ESCC. We established a CYH33-resistant cell line, namely KYSE180C, by incubating ESCC KYSE180 cells with increasing concentrations of CYH33. Though CYH33 was able to inhibit Akt phosphorylation, it was much less potent to inhibit the cell cycle progression and proliferation in KYSE180C cells compared to its activity in parental cells. To dissect the mechanism rendering resistance to CYH33, we conducted whole-genome sequencing in KYSE180 and KYSE180C cells. 29 new somatic mutations were detected in KYSE180C cells, including HRASG12S mutant. Accordingly, heterozygous mutation of HRASG12S and hyper-activated ERK signaling were detected in monoclonal cells derived from KYSE180C cells. Similar to KYSE180C cells, these monoclonal cells were resistant to CYH33. Overexpression of HRASG12S conferred KYSE180 cells tolerant to CYH33, which was accompanied with enhanced phosphorylated ERK and S6. Moreover, overexpression of HRASG12S also rendered other ESCC cells, including KYSE70, KYSE410, and KYSE510 cells resistant to CYH33. By contrast, down-regulation of HRAS with SiRNA restored the sensitivity of KYSE180C monoclonal cells to CYH33, which was associated with the induction of apoptosis. Combination of CYH33 with MEK inhibitor MEK162 or ERK inhibitor GDC09994 but not RAF inhibitor GSK2118436 inhibited phosphorylation of ERK and S6 in KYSE180C cells, which was consistent with the enhanced inhibition on cell proliferation. In summary, continuous treatment of CYH33 might induce genomic instability and HRASG12S mutant mediated resistance to CYH33 in ESCC cells. Combination of MAPK inhibitors with CYH33 are able to overcome resistance mediated by HRAS hyper-activation. Citation Format: Yuxiang Wang, Xu Zhang, Qingyang Ma, Xi Zhang, Chunhao Yang, Cun Tan, Landing Hu, Jian Ding, Linghua Meng. HRASG12S mutant mediates resistance to a PI3Kα inhibitor CYH33 in esophageal squamous cell carcinomas [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A116. doi:10.1158/1535-7163.TARG-19-A116

An efficient tandem method for the annulation of 1-amino-1H-pyrrole-2-amides with various substituted benzaldehydes, heteroaryl aldehydes, alkyl aldehydes or even acetals, promoted by cupric chloride, to synthesize 2-substituted pyrrolo[2,1-f][1,2,4]triazin-4(3H)-ones is reported. This approach provides a useful method for constructing the privileged structure in medicinal chemistry. Electron-donating groups on both partners could accelerate the reaction.

Phosphatidylinositol 3-kinaseα isoform (PI3Kα) is frequently deregulated in a wide variety of human solid tumors. A series of new pyrrolo[2,1-f][1,2,4]triazines has been prepared in an effort to discover new PI3Kα-selective inhibitors. Intensive structure-activity relationship (SAR) and structure-pharmacokinetic relationship (SPR) studies on this new series led to the discovery of compound 40 (CYH33), a potent and highly selective PI3Kα inhibitor. CYH33 displays the most potent activity against PI3Kα among the class I PI3K isoforms, with IC50s of 5.9 nM/598 nM/ 78.7 nM/225 nM against α/β/δ/γ isoform respectively. Kinome profiling revealed that CYH33 showed little activity among more than 300 kinases. The selectivity of CYH33 against PI3Kα was further confirmed with a panel of Rh30-Myr-p110s isogenic cells, which express constitutively active PI3K isoform respectively. Accordingly, CYH33 inhibited PI3K/AKT/mTOR signaling in glioblastoma U87MG and rhabdomyosarcoma Rh30 cells, which is associated with its potent anti-proliferative activity in against cell proliferation in a panel cancer cell lines originated from breast, lung, ovary and colon, prostate etc. It is noteworthy that most of sensitive cells were originated from breast cancer, which was consistent with the fact that PI3K is frequently hyper-activated in breast cancer. Oral administration of CYH33 to mice bearing ovarian cancer SKOV-3 xenografts resulted in decreased phosphorylation of AKT in tumor tissue, which is consistent with the significant efficacy of CYH33 to inhibit the growth of SKOV-3 xenograft. Moreover, CYH33 did not cause significant weight loss and toxic symptoms during the treatment period. We also detected the glucose tolerance after mice bearing SKOV-3 xenografts were administered with CYH33 for 18 days. Though CYH33 treatment delayed the restoration of blood glucose to normal level, blood glucose of the CYH33-treated groups were similar to that of control group 2 h after the injection of glucose. CYH33 displays a favorable pharmacokinetic profile with an oral bioavailability of 36.9% and little activity against the major cytochrome P450 isozymes. In summary, CYH33 emerged as a novel selective PI3Kα inhibitor with potent activity against human solid tumors and favorable pharmaceutical profile, which makes it a promising drug candidate and enter clinical trials in China. Citation Format: Haoyue Xiang, Xiang Wang, Yanhong Chen, Xi Zhang, Yi Chen, Cun Tan, Yi Wang, Jian Ding, Ling-Hua Meng, Chunhao Yang. Discovery of clinical candidate methyl (5-(6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-4-morpholinopyrrolo[2,1-f][1,2,4]triazin-2-yl)-4-(trifluoromethyl)pyridin-2-yl)carbamate (CYH33) : A highly potent and selective PI3K alpha inhibitor for the treatment of advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-268.

A one-pot copper(II)-catalyzed tandem synthesis of 2-substituted pyrrolo[1,2-b]pyridazin-4(1H)-ones from N-aminopyrroles was developed. This tandem reaction involves a Conrad–Limpach-type reaction, including the thermal condensation of N-aminopyrroles with the carbonyl group of β-oxo esters followed by the cyclization of Schiff base intermediates. Compared to the traditional Conrad–Limpach quinoline synthesis, we herein successfully applied copper(II) as a catalyst in this transformation to furnish 2-substituted pyrrolo[1,2-b]pyridazin-4(1H)-ones for the first time. Most of the substrates bearing electron-donating (EDG) and electron-withdrawing (EWG) groups worked well with this procedure. The corresponding products could be converted directly into diverse pyrrolo[1,2-b]pyridazine for drug discovery and materials science.

An efficient tandem method for the annulation of 1-amino-1H-pyrrole-2-amides with various substituted benzaldehydes, heteroaryl aldehydes, alkyl aldehydes or even acetals, promoted by cupric chloride, to synthesize 2-substituted pyrrolo[2,1-f][1,2,4]triazin-4(3H)-ones is reported. This approach provides a useful method for constructing the privileged structure in medicinal chemistry. Electron-donating groups on both partners could accelerate the reaction.

A novel Pd/Cu catalyzed synthesis of pyrrolopyridazines under mild conditions is developed.

An efficient palladium/copper-catalyzed domino reaction was developed to yield functionalized naphthalenes, quinolines, and isoquinolines. The reactions of various substituted 1-phenylprop-2-yn-1-ols containing electron-donating (EDG) and electron-withdrawing (EWG) groups and 1-heteroarylprop-2-yn-1-ols worked well with this procedure. Both electron-rich and -deficient aryl halides were satisfactory substrates for this reaction. This domino benzannulation process involves a Sonogashira cross-coupling reaction, followed by isomerization to a chalcone, and finally an intramolecular condensation reaction.

An efficient palladium/copper-catalyzed domino reaction was developed to yield functionalized naphthalenes, quinolines, and isoquinolines. The reactions of various substituted 1-phenylprop-2-yn-1-ols containing electron-donating (EDG) and electron-withdrawing (EWG) groups and 1-heteroarylprop-2-yn-1-ols worked well with this procedure. Both electron-rich and -deficient aryl halides were satisfactory substrates for this reaction. This domino benzannulation process involves a Sonogashira cross-coupling reaction, followed by isomerization to a chalcone, and finally an intramolecular condensation reaction.

A novel Pd/Cu catalyzed domino reaction for the synthesis of functionalized pyrrolo[1,2-b]pyridazines from readily accessible (hetero)aryl propargyl alcohols and 1-amino-2-bromopyrroles was developed. This cascade process involves a Sonogashira cross-coupling reaction, an isomerization and an intramolecular condensation.