Jinfeng Luo

Design, Synthesis, and Biological Evaluation of Novel Conformationally Constrained Inhibitors Targeting Epidermal Growth Factor Receptor Threonine790 → Methionine790 Mutant

The EGFR(T790M) mutant contributes approximately 50% to clinically acquired resistance against gefitinib or erlotinib. However, almost all the single agent clinical trials of the second generation irreversible EGFR inhibitors appear inadequate to overcome the EGFR(T790M)-related resistance. We have designed and synthesized a series of 2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidinyl derivatives as novel EGFR inhibitors. The most potent compounds, 2q and 2s, inhibited the enzymatic activities of wild-type and mutated EGFRs, with IC(50) values in subnanomolar ranges, including the T790M mutants. The kinase inhibitory efficiencies of the compounds were further validated by Western blot analysis of the activation of EGFR and downstream signaling in cancer cells harboring different mutants of EGFR. The compounds also strongly inhibited the proliferation of H1975 non small cell lung cancer cells bearing EGFR(L858R/T790M), while being significantly less toxic to normal cells. Moreover, 2s displayed promising anticancer efficacy in a human NSCLC (H1975) xenograft nude mouse model.

Discovery and Optimization of 3-(2-(Pyrazolo[1,5-a]pyrimidin-6-yl)ethynyl)benzamides as Novel Selective and Orally Bioavailable Discoidin Domain Receptor 1 (DDR1) Inhibitors

Discoidin domain receptor 1 (DDR1) is an emerging potential molecular target for new anticancer drug discovery. We have discovered a series of 3-(2-(pyrazolo[1,5-a]pyrimidin-6-yl) ethynyl)benzamides that are selective and orally bioavailable DDR1 inhibitors. The two most promising compounds (7rh and 7rj) inhibited the enzymatic activity of DDR1, with IC50 values of 6.8 and 7.0 nM, respectively, but were significantly less potent in suppressing the kinase activities of DDR2, Bcr-Abl, and c-Kit. Further study revealed that 7rh bound with DDR1 with a Kd value of 0.6 nM, while it was significantly less potent to the other 455 kinases tested. The S(35) and S(10) selectivity scores of 7rh were 0.035 and 0.008, respectively. The compounds also potently inhibited the proliferation of cancer cells expressing high levels of DDR1 and strongly suppressed cancer cell invasion, adhesion, and tumorigenicity. Preliminary pharmacokinetic studies suggested that they possessed good PK profiles, with oral bioavailabilities of 67.4% and 56.2%, respectively.

Identification of GZD824 as an orally bioavailable inhibitor that targets phosphorylated and nonphosphorylated breakpoint cluster region-Abelson (Bcr-Abl) kinase and overcomes clinically acquired mutation-induced resistance against imatinib.

Bcr-Abl(T315I) mutation-induced imatinib resistance remains a major challenge for clinical management of chronic myelogenous leukemia (CML). Herein, we report GZD824 (10a) as a novel orally bioavailable inhibitor against a broad spectrum of Bcr-Abl mutants including T315I. It tightly bound to Bcr-Abl(WT) and Bcr-Abl(T315I) with K(d) values of 0.32 and 0.71 nM, respectively, and strongly inhibited the kinase functions with nanomolar IC(50) values. The compound potently suppressed proliferation of Bcr-Abl-positive K562 and Ku812 human CML cells with IC(50) values of 0.2 and 0.13 nM, respectively. It also displayed good oral bioavailability (48.7%), a reasonable half-life (10.6 h), and promising in vivo antitumor efficacy. It induced tumor regression in mouse xenograft tumor models driven by Bcr-Abl(WT) or the mutants and significantly improved the survival of mice bearing an allograft leukemia model with Ba/F3 cells harboring Bcr-Abl(T315I). GZD824 represents a promising lead candidate for development of Bcr-Abl inhibitors to overcome acquired imatinib resistance.

Design, Synthesis, and Biological Evaluation of 3-(1H-1,2,3-Triazol-1-yl)benzamide Derivatives as Potent Pan Bcr-Abl Inhibitors Including the Threonine315→Isoleucine315 Mutant

A series of 3-(1H-1,2,3-triazol-1-yl)benzamide derivatives were designed and synthesized as new Bcr-Abl inhibitors by using combinational strategies of bioisosteric replacement, scaffold hopping, and conformational constraint. The compounds displayed significant inhibition against a broad spectrum of Bcr-Abl mutants including the gatekeeper T315I and p-loop mutations, which are associated with disease progression in CML. The most potent compounds 6q and 6qo strongly inhibited the kinase activities of Bcr-Abl(WT) and Bcr-Abl(T315I) with IC(50) values of 0.60, 0.36 and 1.12, 0.98 nM, respectively. They also potently suppressed the proliferation of K562, KU812 human CML cells, and a panel of murine Ba/F3 cells ectopically expressing either Bcr-Abl(WT) or any of a panel of other Bcr-Abl mutants that have been shown to contribute to clinical acquired resistance, including Bcr-Abl(T315I), with IC(50) values in low nanomolar ranges. These compounds may serve as lead compounds for further development of new Bcr-Abl inhibitors capable of overcoming clinical acquired resistance against imatinib.

Pyrimido[4,5‐d]pyrimidin‐4(1H)‐one Derivatives as Selective Inhibitors of EGFR Threonine790 to Methionine790 (T790M) Mutants

The epidermal growth factor receptor (EGFR, erbB1, HER1) has been well-validated as a molecular target in anticancer drug discovery. In non-small-cell lung cancer patients (NSCLCs) harboring active mutations in the EGFR tyrosine kinase domain (L858R and del E746-A750), the first generation inhibitors, gefinitib and erlotinib, have achieved significant clinical benefits but emerging acquired resistance to them has become a major clinical challenge. The “gatekeeper” T790M mutation (threonine!methionine) of EGFR, by which the binding of ATP with the kinase is favored, is one of the primary mechanisms for resistance and plays a role in the circa 50 % of NSCLC patients who acquired clinical resistance. 7] Although the Cys797-chelating irreversible EGFR inhibitors displayed promising potential to overcome EGFR related resistance in animal models, their non-selective inhibition against wild-type EGFR (EGFR) and/or other kinases results in a relatively low maximal-tolerated-dose (MTD) and poor clinical outcomes in human patients. Inhibitors selectively targeting EGFR mutants are an attractive strategy for the clinical management of NSCLC patients with acquired resistance. However, because EGFR and the EGFR mutants share highly similar three-dimensional structures and have almost identical binding affinities with ATP, nearly all of the reported irreversible EGFR inhibitors displayed equal potencies against the T790M mutants and the wild-type enzyme, highlighting the challenge in the search for EGFR mutant-selective inhibitors. Only recently, WZ4002 was reported as a new irreversible EGFR inhibitor displaying moderate selectivity on EGFR mutants over the wild-type kinase. A phase I clinical trial was recently initiated on another moderately mutant-selective EGFR inhibitor CO-1686 (Kd(EGFR )/Kd(EGFR ) = 25, NCT01526928) whose chemical structure was not disclosed, and PKC412 was reported as a novel reversible T790M mutant-selective EGFR inhibitor with promising in vivo efficacy. Herein, we wish to report the successful discovery of novel pyrimido[4,5-d]pyrimidin-4(1H)-onebased EGFR inhibitors with more than 100-fold selectivity over the wild-type kinase. We have successfully designed compounds 1 and 2 (Scheme 1) as novel EGFR inhibitors with low nanomolar IC50 and Kd values. However, these compounds only displayed four-fold selectivity on EGFR mutants over EGFR. The use of conformational constraint is a general strategy with which to improve ligand selectivity for a molecular target, and accordingly a series of pyrimido[4,5d]pyrimidin-4(1H)-one derivatives 3 a–3h with more rigid conformations based on the structure-activity relationship (SAR) studies of compounds 1 and 2 (Scheme 1) were designed. The compounds were readily synthesized by using the similar procedures to that of 3a (Scheme 2; Supporting Information, Scheme S1). Briefly, a direct nucleophilic coupling of commercially available ethyl 2,4-dichloropyrimidine5-carboxylate (4) with tert-butyl-3-aminophenylcarbamate (5) produced ethyl 4-[{3-[(tert-butoxycarbonyl)amino]phenyl}amino]-2-chloropyrimidine-5-carboxylate (6). Hydrolysis of compound 6 with 1m NaOH in a H2O/THF mixed solution yielded the carboxylic acid (7). The condensation of 7 and 8a in the presence of HATU and DIPEA in dry CH2Cl2 gave the intermediate 9a. Compound 9a was coupled with aniline by nucleophilic substitution followed by deprotection with 50% trifluoroacetic acid in CH2Cl2 to yield the key precursor 10a. The new inhibitor 3a was finally obtained by acryloylation of 10a with acryloyl chloride. The binding affinities of the compounds with EGFR and its T790M mutants were determined with an active-sitedependent competition binding assay conducted by Ambit Bioscience, San Diego, USA. The kinase inhibitory activities of the compounds were also evaluated by the well-established [*] T. Xu, L. Zhang, S. Xu, J. Luo, F. Ding, X. Lu, Y. Liu, Z. Tu, Prof. D. Pei, Q. Cai, X. Ren, Prof. K. Ding Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences 190 Kaiyuan Avenue, Guangzhou 510530 (China) E-mail: ding_ke@gibh.ac.cn T. Xu, S. Xu University of Chinese Academy of Sciences 19 Yuquan Road, Beijing 100049 (China)

New thiazole carboxamides as potent inhibitors of Akt kinases

A new series of 2-substituted thiazole carboxamides were identified as potent pan inhibitors against all three isoforms of Akt (Akt1, Akt2 and Akt3) by systematic optimization of weak screening hit N-(1-amino-3-phenylpropan-2-yl)-2-phenylthiazole-5-carboxamide (1). One of the most potent compounds, 5m, inhibited the kinase activities of Akt1, Akt2 and Akt3 with IC(50) values of 25, 196 and 24nM, respectively. The compound also potently inhibited the phosphorylation of downstream MDM2 and GSK3β proteins, and displayed strongly antiproliferative activity in prostate cancer cells. The inhibitors might serve as lead compounds for further development of novel effective anticancer agents.

Design, Synthesis, and Biological Evaluation of 2-Oxo-3,4-dihydropyrimido[4,5-d]pyrimidinyl Derivatives as New Irreversible Epidermal Growth Factor Receptor Inhibitors with Improved Pharmacokinetic Properties

Structural optimization of a series of 2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidinyl compounds, potential new irreversible EGFR inhibitors, was performed to improve pharmacokinetic properties of the compounds. This led to compound 2v with improved aqueous solubility and good pharmacokinetic properties which at the nanomolar level potently inhibits gefitinib-resistant EGFR(L858R/T790M) kinase and displays strong antiproliferative activity against H1975 nonsmall cell lung cancer cells. The new inhibitor also shows promising antitumor efficacy in a murine EGFR(L858R/T790M)-driven H1975 xenograft model without effect on body weight. These studies provide new lead compounds for further development of drugs for treatment of gefitinib-resistant nonsmall cell lung cancer patients.

Synthesis and Evaluation of Novel Erlotinib–NSAID Conjugates as More Comprehensive Anticancer Agents

A series of novel anticancer agents were designed and synthesized based on coupling of different nonsteroidal anti-inflammatory drugs (NSAIDs) with the epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitor, erlotinib. Both the antiproliferative and pharmacokinetic activity of the target compounds were evaluated using HCC827 and A431 tumor cell lines. Among the derivatives made, compounds 10a, 10c, and 21g showed superb potency, comparable to that of erlotinib. Furthermore, preliminary SAR analysis showed that when the NSAIDs were conjugated via linkage to C-6 OH versus linkage to C-7 OH of the quinazoline nucleus, superior anticancer activity was achieved. Finally, the in vitro pharmacokinetic profile of several conjugates demonstrated the desired dissociation kinetics as the coupled molecules were effectively hydrolyzed, releasing both erlotinib and the specific NSAID in a time-dependent manner. The conjugation strategy represents a unique and simplified approach toward combination therapy, particularly for the treatment of cancers where both EGFR overexpression and inflammation play a direct role in disease progression.

Spiro Meroterpenoids from Ganoderma applanatum

Spiroapplanatumines A-Q (1-12, 14-16, 18, and 20), new spiro meroterpenoids respectively bearing a 6/5/7 or 6/5/5 ring system, along with three known compounds, spirolingzhines A, B, and D, were isolated from the fruiting bodies of the fungus Ganoderma applanatum. Their structures including absolute configurations were assigned by using spectroscopic methods, ECD and 13C NMR calculations, and single-crystal X-ray diffraction analysis. Biological evaluation of all the compounds disclosed that compounds 7 and 8 inhibited JAK3 kinase with IC50 values of 7.0 ± 3.2 and 34.8 ± 21.1 μM, respectively.

Identification and Optimization of New Dual Inhibitors of B-Raf and Epidermal Growth Factor Receptor Kinases for Overcoming Resistance against Vemurafenib

Epidermal growth factor receptor (EGFR) amplification has been demonstrated to be critical for the inherent and/or acquired resistance against current B-Raf(V600E) inhibitor therapy for melanoma and colorectal cancer patients. We describe the discovery and structure-activity relationship study of a series of 1H-pyrazolo[3,4-b]pyridine-5-carboxamide analogues as novel dual inhibitors of EGFR and B-Raf(V600E) mutant. One of the most promising compounds, 6a, potently inhibited both of the kinases with IC50 values of 8.0 and 51 nM, respectively. The compound also strongly suppressed the proliferation of a panel of intrinsic and acquired resistant melanoma and/or colorectal cancer cells harboring overexpressed EGFR with submicromolar IC50 values. Further mechanism investigation revealed that 6a could sustainably inhibit the activation of the MAPK path way in the resistant SK-MEL-28 PR30 melanoma cancer cells and WiDr colorectal cancer cells with EGFR amplification. Our results support the hypothesis that the EGFR/B-Raf(V600E) dual inhibition might be a tractable strategy to overcome the intrinsic and acquired resistance of melanoma and/or colorectal cancers against the current B-Raf(V600E) inhibitor therapy.