Chrong-Shiong Hwang

Synthesis and structure-activity relationship of 6-arylureido-3-pyrrol-2-ylmethylideneindolin-2-one derivatives as potent receptor tyrosine kinase inhibitors.

A series of new ureidoindolin-2-one derivatives were synthesized and evaluated as inhibitors of receptor tyrosine kinases. Investigation of structure-activity relationships at positions 5, 6, and 7 of the oxindole skeleton led to the identification of 6-ureido-substituted 3-pyrrolemethylidene-2-oxindole derivatives that potently inhibited both the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) families of receptor tyrosine kinases. Several derivatives showed potency against the PDGFR inhibiting both its enzymatic and cellular functions in the single-digit nanomolar range. Among them, compound 35 was a potent inhibitor against tyrosine kinases, including VEGFR and PDGFR families, as well as Aurora kinases. Inhibitor 36 (non-substituted on the pyrrole or phenyl ring) had a moderate pharmacokinetic profile and completely inhibited tumor growth initiated with the myeloid leukemia cell line, MV4-11, in a subcutaneous xenograft model in BALB/c nude mice.

Pharmacophore modeling and virtual screening to identify potential RET kinase inhibitors

Chemical features based 3D pharmacophore model for REarranged during Transfection (RET) tyrosine kinase were developed by using a training set of 26 structurally diverse known RET inhibitors. The best pharmacophore hypothesis, which identified inhibitors with an associated correlation coefficient of 0.90 between their experimental and estimated anti-RET values, contained one hydrogen-bond acceptor, one hydrogen-bond donor, one hydrophobic, and one ring aromatic features. The model was further validated by a testing set, Fischers randomization test, and goodness of hit (GH) test. We applied this pharmacophore model to screen NCI database for potential RET inhibitors. The hits were docked to RET with GOLD and CDOCKER after filtering by Lipinskis rules. Ultimately, 24 molecules were selected as potential RET inhibitors for further investigation.

Novel azulene-based derivatives as potent multi-receptor tyrosine kinase inhibitors.

A series of azulene-based derivatives were synthesized as potent inhibitors for receptor tyrosine kinases such as FMS-like tyrosine kinase 3 (FLT-3). Systematic side chain modification of prototype 1a was carried out through SAR studies. Analogue 22 was identified from this series and found to be one of the most potent FLT-3 inhibitors, with good pharmaceutical properties, superior efficacy, and tolerability in a tumor xenograft model.

Synthesis of 1-substituted 3-pyridinylmethylidenylindolin-2-ones and 1-substituted 3-quinolinylmethylidenylindolin-2-ones as the enhancers of ATRA-induced differentiation in HL-60 cells.

As part of our continuing search for potential differentiation agents, 1-benzyl-3-(4-pyridinylmethylidenyl)indolin-2-one (14) was selected as lead compound, and its new pyridinyl and quinolinyl analogs were synthesized and evaluated for differentiation-inducing activity toward HL-60 cells. Most of the tested compounds enhanced the ATRA-induced differentiation; among them, 1-(1-phenylethyl)-3-(3-quinolinylmethylidenyl)indolin-2-one (25) was the most promising one. The two isomers, 25Z and 25E; consisting 25 were found to have similar differentiation activity. The combination of 25 with all trans retinoic acid (ATRA) was found to induce complete differentiation of HL-60 cells and arrest the cells in the G(0)/G(1) phase of the cell cycle. Beside its excellent differentiation activity, 25 also exhibited relatively low cytotoxicity toward normal cells. Therefore, compound 25 is recommended as a candidate for further development of novel enhancer of ATRA-induced differentiation in HL-60 cells.

Design of novel FLT-3 inhibitors based on dual-layer 3D-QSAR model and fragment-based compounds in silico.

FMS-like tyrosine kinase 3 (FLT-3) is strongly correlated with acute myeloid leukemia, but no FLT-3-inhibitor cocomplex structure is available to assist the design of therapeutic inhibitors. Hence, we propose a dual-layer 3D-QSAR model for FLT-3 that integrates the pharmacophore, CoMFA, and CoMSIA. We then coupled the model with the fragment-based design strategy to identify novel FLT-3 inhibitors. In the first layer, the previously established model, Hypo02, was evaluated in terms of its correlation coefficient (r), RMS, cost difference, and configuration cost, with values of 0.930, 1.24, 106.45, and 16.44, respectively. Moreover, Fischers cross-validation test of data generated by Hypo02 yielded a 98% confidence level, and the validation of the testing set yielded a best r value of 0.87. The features of Hypo02 were separated into two parts and then used to screen the MiniMaybridge fragment compound database. Nine novel FLT-3 inhibitors were generated in this layer. In the second layer, Hypo02 was subjected to an alignment rule to generate CoMFA- and CoMSIA-based models, for which the partial least-squares validation method was utilized. The values of q(2), r(2), and predictive r(2) were 0.58, 0.98, and 0.76, respectively, derived from the CoMFA model with steric and electrostatic fields. The CoMSIA model with five different fields yielded values of 0.54, 0.97, and 0.76 for q(2), r(2), and predictive r(2), respectively. The CoMFA and CoMSIA models were used to constrain 3D structures of the nine novel FLT-3 inhibitors. This dual-layer 3D-QSAR model constitutes a valuable tool to easily and quickly screen and optimize novel potential FLT-3 inhibitors for the treatment of acute myeloid leukemia.

Abstract B186: ITRI-2531 - A novel kinase inhibitor for treating hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers and is also a leading cause of cancer-related deaths worldwide. Until now, treatments for HCC remain limited, especially for the shortage of promising systemic therapies to replace systemic chemotherapy. Sorafenib, the first drug applied in targeted therapy for HCC, was approved and drew considerable attention. However, the efficacy and side effects of sorafenib are still unsatisfied, which make sorafenib far from ideal for treatment of HCC. Thus, developing novel therapeutics for HCC is necessary. A novel compound, ITRI-2531, is developed to improve the efficacy from sorafenib. ITRI-2531 shows cytotoxicity in HCC cell lines (Huh-7 and PLC/PRF/5) and patient-derived HCC tissue cell lines. In studies of kinase activities, ITRI-2531 suppresses the phosphorylation of MEK and ERK in PLC/PRF/5 cells and shows inhibition of multiple kinases (FLT3, FLT4, PDGFRA, PGDFRB, and VEGFR2) in KINOMEscanTM study. In in vivo studies, oral treatment of ITRI-2531 repressed subcutaneous Huh-7 and PLC/PRF/5 tumor growth in severe combined immunodeficient (SCID) mice and shows better efficacy than sorafinib. Moreover, ITRI-2531 prolongs the survival of SCID mice with orthotopic patient-derived xenograft tumor and suppresses alpha-fetoprotein in serum. On the other hand, ITRI-2531 also has good pharmacokinetic profiles. According to these results, we believe that ITRI-2531 warrants further evaluation as a new anti-HCC drug. Citation Format: Tsung-Keng Kuo, On Lee, Mai-Wei Lin, Li-Zong Lin, Chun-Min Liu, Tai-ju Hsieh, Chun-Chung Wang, Shyh-Horng Lin, Chia-Ni Chang, Hui-Chun Hsu, Nien-Tzu Chou, Chin-Pen Lai, Chih-Hung Chen, Chia-Mu Tu, Shih-Ta Chen, Yuan-Jang Tsai, Chih-Peng Liu, Jenn-Tsang Hwang, Jui-Wen Huang, Yen-Chun Chen, Chrong-Shiong Hwang, Hsiang-Wen Tseng. ITRI-2531 - A novel kinase inhibitor for treating hepatocellular carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B186.

Abstract 5781: ITRI-260, a new azaazulenone class kinase inhibitor, induces complete tumor regression and prolongs survival in AML mice

The FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase with important roles in hematopoietic stem/progenitor cell survival and proliferation. FLT3 is over-expressed in the majority of acute myeloid leukemia (AML) and the presence of FLT3 active mutations is associated with poor prognosis, implicating FLT3 as a potential target for AML treatment. ITRI-260 is a new azaazulenone class kinase inhibitor that inhibited FLT3-ITD-dependent MV4-11 cell growth with IC50 of 21 nM and cellular phosphorylation with IC50 of 17 nM. In in vivo PK study, ITRI-260 showed favorable oral PK profiles. The maximum concentration of ITRI-260 was 700 nM after a single 100 mg/kg oral dosing in mice, with dose proportional exposure from 10 to 150 mg/kg. ITRI-260 showed therapeutic efficacy in a human AML orthotopic SCID mouse model, following a 100 mg/kg/qd oral dosing regimen. The treatment group showed 100% survival of animals at day 90 after tumor cell inoculation, while the vehicle control group showed 100% mortality. In the other study, ITRI-260 also increased survival in primary tumor cell engraftment model inoculated with cells isolated from sunitinib- and sorafenib-resistant AML patient. Through hERG assay with patch clamp technology, we found higher IC50 of ITIR-260 compared with sunitinib and MLN-518, which indicates that ITRI-260 might arise less cardiotoxic concern. In a 14-day subchronic toxicology study in mice, ITRI-260 was generally tolerated at 300 mg/kg/qd. The most prominent adverse effect appeared at 600 mg/kg/qd group is starry sky appearance in spleen, which is moderate and reversible. No specific clinical signs or body weight loss were attributed to the test article. In conclusion, the in vitro/in vivo PK/PD/toxicology studies have provided the basis for the clinical developmental potential of ITRI-260 in AML. The bridge studies from discovery to development such as CMC, GLP toxicology/safety pharmacology and pre-formulation are now in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5781.

Abstract 728: Development of pharmacophore-based RET inhibitors for thyroid cancer therapy

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The new therapeutic option is pivotal due to the restricted efficacy of current treatments (surgery, radioactive iodine treatment, and chemotherapy) in progressive thyroid carcinomas. Gain-of-function mutations of the receptor tyrosine kinase RET have been identified as driving oncogenic events in subsets of papillary and medullary thyroid cancers (PTC and MTC), but targeted therapy for RET-driven cancers is currently clinically unavailable. Some mulit-kinase inhibitors, such as sunitinib and vandetanib, which have shown efficacy against other neoplastic diseases, are being evaluated in clinical trials for thyroid cancer therapy. However, the anti-VEGFR activities of these mulit-kinase inhibitors have been considered to lead cardiotoxicity in some patients. Moreover, Ret mutation-associated resistance have been reported by some RET inhibitors in preclinical data. The “in silico model” was applied to design the specific RET target compounds through mapping the pharmacophore of RET ATP-binding pocket. Some novel scaffold compounds which fit the pharmacophore model were selected form ITRI library, and their anti-RET activities were evaluated by kinase assay. The in vitro anti-cell proliferation activities of the compounds to the TT cells (MTC specific cells) were demonstrated by MTT assay. From the screening of ITRI library, ITRI-305 showed the impressive anti-RET (IC50 ∼ 90 nM) and anti-cell proliferation (IC50 at low micro molar) activities and the activities are compatible to those of sunitinib and vandetanib. ITRI-305 also shows good solubility and favorable oral PK profiles. The xenograft study of TT cells with ITRI-305 is proceeding. In conclusion, a novel scaffold RET inhibitor, ITRI-305, with less potent VEGFR activity (anti-VEFGR IC50 ∼ 1 μM) is developed to overcome the cardiotoxicity and resistance problems which is caused by conventional RET inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 728.

Abstract LB-160: ITRI-260, a promising candidate for treatment of acute myeloid leukemia

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Acute myeloid leukemia (AML) is a highly malignant hematopoietic tumor characterized by an abnormal proliferation of myeloid progenitor cells, decreased rate of self-destruction and an arrest in cell differentiation. The internal tandem duplication mutations of FMS-like tyrosine kinase-3 (FLT3/ITD mutations) are common in AML and linked to poor prognosis. ITRI-260 generated from designed by computer-assisted rationale drug design and in silico prioritized approaches, was proved to be a potent cell growth inhibitor against FLT3/ITD mutated AML cells through FLT3 and c-Kit inhibition. The in vivo efficacy of ITRI-260 via oral route was demonstrated in MV4-11 xenograft mice model. Besides, two more orthotopic murine models were used to assess the in vivo therapeutic efficacy of ITRI-260. The first one is MV4-11 cell orthotopically engraft into NOD/SCID mice bone marrow and the second one is primary AML cells from AML patient engraft into NOD/SCID mice bone marrow. The results show that ITII-260 can decrease FLT3/ITD mutant leukemia blasts in peripheral blood and bone marrow in both animal models. Finally, ITRI-260 prolongs the survival in both bone marrow engraft mice models. These findings strongly suggest clinical benefits of ITRI-260 in patients with FLT3/ITD AML. Currently we are planning the bridged projects to push ITRI-260 toward IND status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-160.