Identification of Novel ROS Inducers: Quinone Derivatives Tethered to Long Hydrocarbon Chains

We performed the first synthesis of the 17-carbon chain-tethered quinone moiety <b>22</b> (SAN5201) of irisferin A, a natural product exhibiting anticancer activity, and its derivatives. We found that <b>22</b> is a potent ROS inducer and cytotoxic agent. Compound <b>25</b> (SAN7401), the hydroquinone form of <b>22</b>, induced a significant release of intracellular ROS and apoptosis (EC₅₀ = 1.3–2.6 μM) in cancer cell lines, including A549 and HCT-116. Compared with the activity of a well-known ROS inducer, piperlongumine, <b>22</b> and <b>25</b> showed stronger cytotoxicity and higher selectivity over noncancerous cells. Another hydroquinone tethering 12-carbon chain, <b>26</b> (SAN4601), generated reduced levels of ROS but showed more potent cytotoxicity (EC₅₀ = 0.8–1.6 μM) in cancer cells, although it lacked selectivity over noncancerous cells, implying that the naturally occurring 17-carbon chain is also crucial for ROS production and a selective anticancer effect. Both <b>25</b> and <b>26</b> displayed strong, equipotent activities against vemurafenib-resistant SK-Mel2 melanoma cells and p53-deficient H1299 lung cancer cells as well, demonstrating their broad therapeutic potential as anticancer agents

Image_3_Anti-Tumor Activity of AZD4547 Against NTRK1 Fusion Positive Cancer Cells Through Inhibition of NTRKs.jpeg

Inhibitors of tropomyosin-related kinases (TRKs) display remarkable outcomes in the regression of cancers harboring the Neurotrophin Receptors Tyrosine Kinase (NTRK) fusion gene. As a result, TRKs have become attractive targets in anti-cancer drug discovery programs. Here, we demonstrate that AZD4547, a highly potent and selective inhibitor of fibroblast growth factor receptor (FGFR), displays anti-tumor activity against KM12(Luc) harboring the TPM3-NTRK1 fusion gene associated with its direct inhibition of TRKs. The results of profiling, using a 64-member in-house cancer cell panel, show that AZD4547 displays anti-proliferation activity against KM12(Luc) with a GI50 of 100 nM. In vitro biochemical assays reveal that AZD4547 has IC50 values of 18.7, 22.6 and 2.9 nM against TRKA, B and C, respectively. In a cellular context, AZD4547 blocks auto-phosphorylation of TRKs and phosphorylation of its downstream molecules including PLC-gamma and AKT in a dose dependent manner. Also, AZD4547 at 0.1 μM concentration downregulates expression of MAPK target genes (DUSP6, CCND1 and ETV1) as well as the E2F pathway. Furthermore, AZD4547 induces G0/G1 arrest and apoptosis, and suppresses anchorage independent growth of KM12(Luc). Oral administration of 40 mpk AZD4547 dramatically delays tumor growth in a KM12(Luc) implemented xenograft model, without promoting body weight changes. The capability of AZD4547 to inhibit TRKA, TRKB and clinically relevant mutants (TRKA G595R, G667S, G667C and G667A) was also evaluated using Ba/F3 cells harboring the ETV6-NTRKs fusion gene. The combined observations demonstrate the potential application of AZD4547 for treatment of NTRK fusion driven cancers.</p

DataSheet_1_Anti-Tumor Activity of AZD4547 Against NTRK1 Fusion Positive Cancer Cells Through Inhibition of NTRKs.docx

Inhibitors of tropomyosin-related kinases (TRKs) display remarkable outcomes in the regression of cancers harboring the Neurotrophin Receptors Tyrosine Kinase (NTRK) fusion gene. As a result, TRKs have become attractive targets in anti-cancer drug discovery programs. Here, we demonstrate that AZD4547, a highly potent and selective inhibitor of fibroblast growth factor receptor (FGFR), displays anti-tumor activity against KM12(Luc) harboring the TPM3-NTRK1 fusion gene associated with its direct inhibition of TRKs. The results of profiling, using a 64-member in-house cancer cell panel, show that AZD4547 displays anti-proliferation activity against KM12(Luc) with a GI50 of 100 nM. In vitro biochemical assays reveal that AZD4547 has IC50 values of 18.7, 22.6 and 2.9 nM against TRKA, B and C, respectively. In a cellular context, AZD4547 blocks auto-phosphorylation of TRKs and phosphorylation of its downstream molecules including PLC-gamma and AKT in a dose dependent manner. Also, AZD4547 at 0.1 μM concentration downregulates expression of MAPK target genes (DUSP6, CCND1 and ETV1) as well as the E2F pathway. Furthermore, AZD4547 induces G0/G1 arrest and apoptosis, and suppresses anchorage independent growth of KM12(Luc). Oral administration of 40 mpk AZD4547 dramatically delays tumor growth in a KM12(Luc) implemented xenograft model, without promoting body weight changes. The capability of AZD4547 to inhibit TRKA, TRKB and clinically relevant mutants (TRKA G595R, G667S, G667C and G667A) was also evaluated using Ba/F3 cells harboring the ETV6-NTRKs fusion gene. The combined observations demonstrate the potential application of AZD4547 for treatment of NTRK fusion driven cancers.</p

Image_1_Anti-Tumor Activity of AZD4547 Against NTRK1 Fusion Positive Cancer Cells Through Inhibition of NTRKs.jpeg

Inhibitors of tropomyosin-related kinases (TRKs) display remarkable outcomes in the regression of cancers harboring the Neurotrophin Receptors Tyrosine Kinase (NTRK) fusion gene. As a result, TRKs have become attractive targets in anti-cancer drug discovery programs. Here, we demonstrate that AZD4547, a highly potent and selective inhibitor of fibroblast growth factor receptor (FGFR), displays anti-tumor activity against KM12(Luc) harboring the TPM3-NTRK1 fusion gene associated with its direct inhibition of TRKs. The results of profiling, using a 64-member in-house cancer cell panel, show that AZD4547 displays anti-proliferation activity against KM12(Luc) with a GI50 of 100 nM. In vitro biochemical assays reveal that AZD4547 has IC50 values of 18.7, 22.6 and 2.9 nM against TRKA, B and C, respectively. In a cellular context, AZD4547 blocks auto-phosphorylation of TRKs and phosphorylation of its downstream molecules including PLC-gamma and AKT in a dose dependent manner. Also, AZD4547 at 0.1 μM concentration downregulates expression of MAPK target genes (DUSP6, CCND1 and ETV1) as well as the E2F pathway. Furthermore, AZD4547 induces G0/G1 arrest and apoptosis, and suppresses anchorage independent growth of KM12(Luc). Oral administration of 40 mpk AZD4547 dramatically delays tumor growth in a KM12(Luc) implemented xenograft model, without promoting body weight changes. The capability of AZD4547 to inhibit TRKA, TRKB and clinically relevant mutants (TRKA G595R, G667S, G667C and G667A) was also evaluated using Ba/F3 cells harboring the ETV6-NTRKs fusion gene. The combined observations demonstrate the potential application of AZD4547 for treatment of NTRK fusion driven cancers.</p

Image_2_Anti-Tumor Activity of AZD4547 Against NTRK1 Fusion Positive Cancer Cells Through Inhibition of NTRKs.jpeg

Inhibitors of tropomyosin-related kinases (TRKs) display remarkable outcomes in the regression of cancers harboring the Neurotrophin Receptors Tyrosine Kinase (NTRK) fusion gene. As a result, TRKs have become attractive targets in anti-cancer drug discovery programs. Here, we demonstrate that AZD4547, a highly potent and selective inhibitor of fibroblast growth factor receptor (FGFR), displays anti-tumor activity against KM12(Luc) harboring the TPM3-NTRK1 fusion gene associated with its direct inhibition of TRKs. The results of profiling, using a 64-member in-house cancer cell panel, show that AZD4547 displays anti-proliferation activity against KM12(Luc) with a GI50 of 100 nM. In vitro biochemical assays reveal that AZD4547 has IC50 values of 18.7, 22.6 and 2.9 nM against TRKA, B and C, respectively. In a cellular context, AZD4547 blocks auto-phosphorylation of TRKs and phosphorylation of its downstream molecules including PLC-gamma and AKT in a dose dependent manner. Also, AZD4547 at 0.1 μM concentration downregulates expression of MAPK target genes (DUSP6, CCND1 and ETV1) as well as the E2F pathway. Furthermore, AZD4547 induces G0/G1 arrest and apoptosis, and suppresses anchorage independent growth of KM12(Luc). Oral administration of 40 mpk AZD4547 dramatically delays tumor growth in a KM12(Luc) implemented xenograft model, without promoting body weight changes. The capability of AZD4547 to inhibit TRKA, TRKB and clinically relevant mutants (TRKA G595R, G667S, G667C and G667A) was also evaluated using Ba/F3 cells harboring the ETV6-NTRKs fusion gene. The combined observations demonstrate the potential application of AZD4547 for treatment of NTRK fusion driven cancers.</p

Image_4_Anti-Tumor Activity of AZD4547 Against NTRK1 Fusion Positive Cancer Cells Through Inhibition of NTRKs.jpeg

Inhibitors of tropomyosin-related kinases (TRKs) display remarkable outcomes in the regression of cancers harboring the Neurotrophin Receptors Tyrosine Kinase (NTRK) fusion gene. As a result, TRKs have become attractive targets in anti-cancer drug discovery programs. Here, we demonstrate that AZD4547, a highly potent and selective inhibitor of fibroblast growth factor receptor (FGFR), displays anti-tumor activity against KM12(Luc) harboring the TPM3-NTRK1 fusion gene associated with its direct inhibition of TRKs. The results of profiling, using a 64-member in-house cancer cell panel, show that AZD4547 displays anti-proliferation activity against KM12(Luc) with a GI50 of 100 nM. In vitro biochemical assays reveal that AZD4547 has IC50 values of 18.7, 22.6 and 2.9 nM against TRKA, B and C, respectively. In a cellular context, AZD4547 blocks auto-phosphorylation of TRKs and phosphorylation of its downstream molecules including PLC-gamma and AKT in a dose dependent manner. Also, AZD4547 at 0.1 μM concentration downregulates expression of MAPK target genes (DUSP6, CCND1 and ETV1) as well as the E2F pathway. Furthermore, AZD4547 induces G0/G1 arrest and apoptosis, and suppresses anchorage independent growth of KM12(Luc). Oral administration of 40 mpk AZD4547 dramatically delays tumor growth in a KM12(Luc) implemented xenograft model, without promoting body weight changes. The capability of AZD4547 to inhibit TRKA, TRKB and clinically relevant mutants (TRKA G595R, G667S, G667C and G667A) was also evaluated using Ba/F3 cells harboring the ETV6-NTRKs fusion gene. The combined observations demonstrate the potential application of AZD4547 for treatment of NTRK fusion driven cancers.</p

A Pyrazolo[3,4‑<i>d</i>]pyrimidin-4-amine Derivative Containing an Isoxazole Moiety Is a Selective and Potent Inhibitor of RET Gatekeeper Mutants

Aberrant RET kinase signaling plays critical roles in several human cancers such as thyroid carcinoma. The gatekeeper mutants (V804L or V804M) of RET are resistant to currently approved RET inhibitors such as cabozantinib and vandetanib. We, for the first time, report a highly selective and extremely potent RET inhibitor, <b>6i</b> rationally designed. Compound <b>6i</b> inhibits strongly RET gatekeeper mutants and other clinically relevant RET mutants as well as wt-RET. This substance also significantly suppresses growth of thyroid cancer-derived TT cell lines and Ba/F3 cells transformed with various RET mutants. Docking studies reveal that the isoxazole moiety in <b>6i</b> is responsible for binding affinity improvement by providing additional site for H-bonding with Lys758. Also, <b>6i</b> not only substantially blocks cellular RET autophosphorylation and its downstream pathway, it markedly induces apoptosis and anchorage-independent growth inhibition in TT cell lines while having no effect on normal thyroid Nthy ori-3-1 cells

A Pyrazolo[3,4‑<i>d</i>]pyrimidin-4-amine Derivative Containing an Isoxazole Moiety Is a Selective and Potent Inhibitor of RET Gatekeeper Mutants

Aberrant RET kinase signaling plays critical roles in several human cancers such as thyroid carcinoma. The gatekeeper mutants (V804L or V804M) of RET are resistant to currently approved RET inhibitors such as cabozantinib and vandetanib. We, for the first time, report a highly selective and extremely potent RET inhibitor, <b>6i</b> rationally designed. Compound <b>6i</b> inhibits strongly RET gatekeeper mutants and other clinically relevant RET mutants as well as wt-RET. This substance also significantly suppresses growth of thyroid cancer-derived TT cell lines and Ba/F3 cells transformed with various RET mutants. Docking studies reveal that the isoxazole moiety in <b>6i</b> is responsible for binding affinity improvement by providing additional site for H-bonding with Lys758. Also, <b>6i</b> not only substantially blocks cellular RET autophosphorylation and its downstream pathway, it markedly induces apoptosis and anchorage-independent growth inhibition in TT cell lines while having no effect on normal thyroid Nthy ori-3-1 cells

Discovery of a broad spectrum antiproliferative agent with selectivity for DDR1 kinase: cell line-based assay, kinase panel, molecular docking, and toxicity studies

<div><p></p><p>Herein, we report compound <b>KST9046</b>, a new agent possessing quinazoline-urea scaffold. Preliminary biological evaluation done by the National Cancer Institute (NCI), USA, showed a great inhibitory effect of <b>KST9046</b> over the 60 cell-line tumor panel. Accordingly, it was selected for a dose-response assay; a broad spectrum antiproliferative activity with GI₅₀ ranging from 1.3 to 3.9 µM was exerted. To explore a potential kinase inhibitory effect, <b>KST9046</b> was applied at a single dose of 10 µM against a kinase panel of 347 different enzymes representing >50% of the predicted human protein kinome. Interestingly, selective inhibition of 76% was observed on DDR1 kinase. Further, <b>KST9046</b> showed an IC₅₀ value of 4.38 µM for DDR1. A molecular docking model presented <b>KST9046</b> as a potential type III inhibitor for DDR1 kinase with an allosteric mode of interaction, which may offer an explanation for its selectivity. As further investigation, CYP450 assay was carried out for <b>KST9046</b>, it showed a promising toxicity profile against four different isoforms. Based on these findings, <b>KST9046</b> can be further evaluated as a promising safe new hit for the development of broad spectrum anticancer agents with a selectivity for DDR1 kinase.</p></div

Development of Highly Potent and Selective Steroidal Inhibitors and Degraders of CDK8

Cortistatin A is a natural product isolated from the marine sponge Corticium simplex and was found to be a potent and selective inhibitor of CDK8. Many synthetic groups have reported total syntheses of Cortistatin A; however, these syntheses require between 16 and 30 steps and report between 0.012–2% overall yields, which is not amenable to large-scale production. Owing to similarities between the complex core of Cortistatin A and the simple steroid core, we initiated a campaign to design simple, more easily prepared CDK8 inhibitors based on a steroid scaffold that would be more convenient for large-scale synthesis. Herein, we report the discovery and optimization of JH-VIII-49, a potent and selective inhibitor of CDK8 with a simple steroid core that has an eight-step synthesis with a 33% overall yield, making it suitable for large-scale preparation. Using this scaffold, we then developed a bivalent small molecule degrader, JH-XI-10-02, that can recruit the E3 ligase CRL4^Cereblon to promote the ubiquitination and proteosomal degradation of CDK8