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Kinase fusions involving tropomyosin receptor kinases (TRKs) have been proven to act as strong oncogenic drivers and are therefore recognized as attractive therapeutic targets. We screened an in-house kinase-focused library and identified a promising hit compound with a unique tetracyclic scaffold. Compound

Members of the tropomyosin receptor kinase (TRK) family are expressed in their constitutively activated forms as a result of a gene fusion that occurs across a wide variety of cancer types. We have identified CH7057288 as a potent and selective TRK inhibitor that belongs to a novel chemical class. CH7057288 showed selective inhibitory activity against TRKA, TRKB, and TRKC in cell-free kinase assays and suppressed proliferation of TRK fusion–positive cell lines, but not that of TRK-negative cell lines. Strong in vivo tumor growth inhibition was observed in subcutaneously implanted xenograft tumor models of TRK fusion–positive cells. Furthermore, in an intracranial implantation model mimicking brain metastasis, CH7057288 significantly induced tumor regression and improved event-free survival. Recently, resistant mutations in the kinase domain of TRK have been reported in patients who show disease progression after treatment with the TRK inhibitors now under clinical development. Our compound maintained similar levels of in vitro and in vivo activity against one of these resistant mutants as it did to wild-type TRK. An X-ray crystal structure of the TRKA and CH7057288 complex supported the activity against the mutant. In addition, gene expression analysis revealed that CH7057288 suppressed MAPK and E2F pathways as downstream signaling of TRK fusion. Therefore, CH7057288 could be a promising therapeutic agent for TRK fusion–positive cancer.

Figure S1. Chemical structure, Figure S2. Kinase selectivity, Figure S3. PK, Figure S4. Inhibition of proliferation and induction of apoptosis in vivo, Figure S5. TP53 sequencing, Table S2. Crystallographic data collection, Table S3. Genes changed by 1 uM CH7057288 treatment, Table S4. Upstream regulators, Table S5. The Caco-2 permeability assay

Cell line information and sensitivity to CH7057288 in high-throughput assay systems.

Members of the tropomyosin receptor kinase (TRK) family are expressed in their constitutively activated forms as a result of a gene fusion that occurs across a wide variety of cancer types. We have identified CH7057288 as a potent and selective TRK inhibitor that belongs to a novel chemical class. CH7057288 showed selective inhibitory activity against TRKA, TRKB, and TRKC in cell-free kinase assays and suppressed proliferation of TRK fusion–positive cell lines, but not that of TRK-negative cell lines. Strong in vivo tumor growth inhibition was observed in subcutaneously implanted xenograft tumor models of TRK fusion–positive cells. Furthermore, in an intracranial implantation model mimicking brain metastasis, CH7057288 significantly induced tumor regression and improved event-free survival. Recently, resistant mutations in the kinase domain of TRK have been reported in patients who show disease progression after treatment with the TRK inhibitors now under clinical development. Our compound maintained similar levels of in vitro and in vivo activity against one of these resistant mutants as it did to wild-type TRK. An X-ray crystal structure of the TRKA and CH7057288 complex supported the activity against the mutant. In addition, gene expression analysis revealed that CH7057288 suppressed MAPK and E2F pathways as downstream signaling of TRK fusion. Therefore, CH7057288 could be a promising therapeutic agent for TRK fusion–positive cancer.

Microphthalmia-associated transcription factor (MITF) is expressed in melanomas and has a critical role in melanocyte development and transformation. Because inhibition of MITF inhibits cell growth in melanoma, MITF is a potential therapeutic target molecule. Here, we report the identification of CH6868398, which has a novel chemical structure and suppresses MITF expression at the protein level in melanoma cells. CH6868398 showed cell growth inhibition activity against MITF-dependent melanoma cells both with and without BRAF mutation and also exhibited anti-tumor efficacy in a melanoma xenograft model. Because selective BRAF inhibitors are standard therapeutics for BRAF-mutated melanoma, we investigated the effect of CH6868398 with a BRAF inhibitor, PLX4720, on cell growth inhibition. The addition of CH6868398 enhanced the cell growth inhibition activity of PLX4720 in melanoma cell lines. Furthermore, combination of CH6868398 and PLX4720 efficiently suppressed MITF protein and enhanced cleavage of Caspase3 and poly (ADP-ribose) polymerase (PARP) in melanoma cell lines. These data support the therapeutic potential of CH6868398 as an anti-melanoma agent that reduces MITF protein levels in combination with BRAF inhibitors.

9-Substituted 6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazoles were discovered as highly selective and potent anaplastic lymphoma kinase (ALK) inhibitors by structure-based drug design. The high target selectivity was achieved by introducing a substituent close to the E(0) region of the ATP binding site, which has a unique amino acid sequence. Among the identified inhibitors, compound 13d showed highly selective and potent inhibitory activity against ALK with an IC(50) value of 2.9 nM and strong antiproliferative activity against KARPAS-299 with an IC(50) value of 12.8 nM. The compound also displayed significant antitumor efficacy in an established ALK fusion gene-positive anaplastic large-cell lymphoma (ALCL) xenograft model in mice without body weight loss.

SummaryAnaplastic lymphoma kinase (ALK) is a tyrosine kinase that is constitutively activated in certain cancers, following gene alterations such as chromosomal translocation, amplification, or point mutation. Here, we identified CH5424802, a potent, selective, and orally available ALK inhibitor with a unique chemical scaffold, showing preferential antitumor activity against cancers with gene alterations of ALK, such as nonsmall cell lung cancer (NSCLC) cells expressing EML4-ALK fusion and anaplastic large-cell lymphoma (ALCL) cells expressing NPM-ALK fusion in vitro and in vivo. CH5424802 inhibited ALK L1196M, which corresponds to the gatekeeper mutation conferring common resistance to kinase inhibitors, and blocked EML4-ALK L1196M-driven cell growth. Our results support the potential for clinical evaluation of CH5424802 for the treatment of patients with ALK-driven tumors.

A series of tumor-activated prodrugs of the inhibitors of dihydropyrimidine dehydrogenase (DPD), an enzyme catabolizing 5-fluorouracil (5-FU: 4g), has been designed and synthesized. RO0094889 (11c) is a prodrug of 5-vinyluracil (4c), a known DPD inhibitor, and was designed to generate 4c selectively in tumor tissues by sequential conversion of 11c by three enzymes: esterase, cytidine deaminase and thymidine phosphorylase, the latter two of which are known to be highly expressed in various tumor tissues. When capecitabine (1), a tumor-activated prodrug of 5-FU, was co-administered orally with 11c, 5-FU in tumor tissues was significantly increased with only a slight increase of 5-FU in plasma as compared with oral capecitabine alone.

TRK receptor tyrosine kinases are expressed as fusion proteins encoded by various fusion genes across a wide variety of cancer types, including lung and colorectal cancer. These fusion proteins have potent oncogenic activity and are thought to be an attractive therapeutic target. In a kinase inhibitor screening we identified CH7057288, a potent and selective TRK inhibitor belonging to a novel chemical class. Our inhibitor showed selective inhibitory activity against TRKA, TRKB, and TRKC in cell-free kinase assays and suppressed proliferation of TRK fusion-positive cell lines, but not that of TRK-negative cell lines. In subcutaneously implanted xenograft models of TRK fusion-positive cells, strong tumor growth inhibition was observed. Furthermore, CH7057288 induced regression of intracranial tumors and greatly improved event-free survival in an intracranial implantation model mimicking brain metastasis. Recently, resistant mutations in TRK have been reported in patients showing disease progression after treatment with a TRK inhibitor under clinical development. Our compound maintained similar levels of in vitro and in vivo activity against some of the resistant mutants as it did to wild-type TRK. In summary, CH7057288 could be a promising therapeutic agent for TRK fusion-positive cancer. Citation Format: Hiroshi Tanaka, Hitoshi Sase, Toshiyuki Tsukaguchi, Hiromi Tanimura, Masami Hasegawa, Kiyoshi Hasegawa, Yoshiyuki Ono, Nobuhiro Oikawa, Hiroshi Sakamoto, Toshiyuki Mio. Potent and selective TRK inhibitor CH7057288 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4179. doi:10.1158/1538-7445.AM2017-4179

An effective and convenient protocol has been developed for the conversion of d -glucose and 6-O-α- d -glucopyranosyl- d -fructose (palatinose®, isomaltulose) into 5-[(1′S)-1′,2′-dihydroxyethyl]-1-phenylpyrazole-3-carboxaldehyde (4) and 5-[(1S)-2-(α- d -glucopyranosyloxy)-1-hydroxethyl])-1-phenylpyrazole-3-carboxaldehyde (5), key steps being the acetic anhydride-promoted dehydrative cyclization of the respective phenylosazones, and subsequent liberation of the N-acetylphenylhydrazone-blocked aldehyde function. Exploitation of the ensuing chemistry of 4 and 5 led to a variety of pyrazole building blocks with a diverse level of hydrophilic substituents (hydroxymethyl, dihydroxyethyl or glucosyl residues) and useful functional groups, such as chloro, cyano, aminomethyl, vinyl and acryloyl moieties.

The ability to stimulate N-acetylglucosamine (GluNAc) incorporation in-vitro of a number of N-tert-butyl-N,N′-dibenzoylhydrazines having various substituents on both phenyl rings was measured in cultured integument excised from the rice stem borer (Chilo suppressalis Walker). The relationship between in-vitro and larvicidal potency was approximately linear. The substituent effects on variations in the potency were similar between in-vitro and larvicidal activities. An inhibitor of oxidative detoxication, piperonyl butoxide, had no synergistic effects on the in-vitro potency. The ability of some dibenzoylhydrazines to inhibit GluNAc incorporation at exposure periods longer than the optimum for stimulation was also measured in a similar cultured integument system. The relationship between the inhibitory and stimulatory potency indices was linear, indicating that the larvicidal activity of dibenzoylhydrazines is closely related to its ability to stimulate as well as to inhibit GluNAc incorporation into the larval cuticle.

The anaplastic lymphoma kinase (ALK) inhibitor is one of the most successful oncology drug discoveries of recent years. The kinase has only recently emerged as an attractive drug target for cancer therapy after a novel oncogenic fusion gene of ALK with echinoderm microtubule-associated protein-like 4 (EML4) was identified in nonsmall cell lung cancer (NSCLC) in 2007. The fusion gene was found to be a key oncogenic driver in EML4-ALK-positive NSCLC, like breakpoint cluster region-Abelson (BCR-ABL) in chronic myeloid leukemias (CML). A number of research groups have identified promising drugs, where Pfizer succeeded in launching a potent ALK inhibitor, crizotinib, for treatment of EML4-ALK-positive NSCLC. However, acquired drug resistance caused by mutations of ALK has been identified in patients who were treated with crizotinib; therefore, many clinical and preclinical second-generation ALK inhibitors are now required to have efficacy against ALK mutants.

The larvicidal activity of a number of 1-(substituted benzoyl)-2-benzoyl–1 -ten-butylhydrazines against the rice stem borer (Chilo suppressalis Walk.) was measured. Variations in the activity were examined quantitatively using physico-chemical substituent and molecular parameters and regression analysis. The results indicated that the molecular hydrophobicity and the electron-withdrawing inductive/ field effect of ontho substituents are favourable to larvicidal activity. The bulkiness of substituents at the meta and para positions was unfavourable to activity, substitution at the para position being more unfavourable than that at the meta position in terms of van der Waals' volume. The 2,3–, 2,5- and 2,6-disubstitution patterns were also unfavourable to activity. Reductions in larvicidal activity caused by the 2,6-,- 2,3,5- and 2,3,4,5-substitutions were greater than those induced by the 2,3- and 2,5-disubstitutions. When the sum of contributions from favourable effects is greater than that from unfavourable effects, the larvicidal activity is expected to be superior to that of the unsubstituted compound.

The larvicidal activity of 1- tert -butyl-1-(2-chlorobenzoyl)-2-(substituted benzoyl)hydrazines was measured against the rice stem borer ( Chilo suppressalis) in the presence of piperonyl butoxide, an inhibitor of oxidative metabolism. Variations in the activity were quantitatively examined by use of physicochemical substituent parameters and regression analysis. The results indicated that the hydrophobicity of substituents is favorable in general but the bulkiness of substituents is unfavorable position-specifically. The most favorable substitution pattern is the para substitution with lower alkyls and halogens. Compounds in which substitution patterns are simultaneously optimized in two benzene rings in the molecule were designed and prepared according to the present and previous quantitative analyses. Some of them, including 1- tert -butyl-1-(3,5-dichloro- and 3,5-dimethylbenzoyl)-2-(4-ethylbenzoyl)hydrazines, the second being RH-5992, were about 20-30 times as larvicidal as the unsubstituted compound, RH-5849, against the rice stem borer.

Anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is considered an attractive therapeutic target for human cancers, especially non-small cell lung cancer (NSCLC). Our previous study revealed that 8,9-side-chains of 6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazole scaffold crucially affected kinase selectivity, cellular activity, and metabolic stability. In this work, we optimized the side-chains and identified highly selective, orally active and potent ALK inhibitor CH5424802 (18a) as the clinical candidate.

The effects of 20-hydroxyecdysone and dibenzoylhydrazine insecticides related to RH-5849 (1,2-dibenzoyl-1- tert -butylhydrazine) were tested on the integument fragments of the rice stem borer, Chilo suppressalis . The fragments were first exposed 10 the compounds. Then cultured in fresh medium containing 14 C-labeled N -acetylglucosamine ([ 14 C]GluNAc). These compounds enhanced the incorporation of [ 14 C]GluNAc into the integument fragments in a dose-dependent manner in the range of 10 −6 ∼ 10 −7 M . When the concentration of the compounds was above this range or if the fragments were exposed for over 48 hr, the degree of the enhancement was smaller than that observed under the optimum conditions. By extracting the cultured integument in hot alkali, the incorporated radioactivity was partially released. The degree of this was not affected by the concentration of the compounds. The ability of compounds to enhance the incorporation of [ 14 C]GluNAc into the cultured integument was evaluated in terms of the concentration required to incorporate half maximum of the control level.

The larvicidal activity of benzoylphenylureas with various multiple substitution patterns at the anilide moiety including chlorfluazuron and teflubenzuron was estimated against the rice stem borer, Chilo suppressalis , under conditions in which the oxidative metabolism was eliminated as far as possible by using piperonyl butoxide. The activity was quantitatively analyzed using physicochemical parameters of substituents at each of the anilide aromatic positions. The results indicated that the total hydrophobicity (with an optimum) and electron-withdrawing ability of substituents are favorable to the activity. Small size of substituents was advantageous but the size effect was specific to their positions. The molecular mode of interaction with the possible receptor was suggested so that the compounds may recognize the possible receptor wall from the side of the vicinal pair of ortho and meta positions occupied by smaller substituents, and the receptor site corresponding to the ortho substituent is hydrophilic and constrained.

To select a novel orally administered VEGFR-2 (KDR/flk-1) specific tyrosine kinase inhibitor in a murine model of choroidal neovascularization (CNV).From a compound library, potent VEGFR2 inhibitors were selected by VEGF-induced phosphorylation of VEGFR-2 and RAF kinases and the proliferation analysis by HUVEC cultures and in vitro tube formation assay. CNV was induced in C57/BL6 mice using diode laser photocoagulation. The antiangiogenic effect of selected compounds was assessed by angiographic examination, in which extent of fluorescein leakage was scored and histological analysis, allowing for measurement of CNV membrane under light microscope. In addition, C57/BL6 mice were treated with daily oral administration of selected compounds for 14 days and body weights were measured.Six compounds that potently inhibited VEGFR-2 were selected for further investigation. Selected compounds-treated conditions showed a dose-dependent inhibition of phosphorylation of VEGFR-2 tyrosine kinase with an IC50 of 0.0022 to 0.098 microm. Selected compounds did not inhibit the HCT116 proliferation but did demonstrate a strong inhibition effect for VEGFR-2 dependant HUVEC (IC50=0.0018 to 0.058 microm). Selected compounds treatment also resulted in a dose-dependent attenuation of in vitro tube formation. In the murine CNV model, #0451 is the most effective compound. The intensity of fluorescein leakage was significantly lower in doses of 12.5, 25, 50, and 100 mg/kg #0451-treated eyes compared to controls. Histologically, CNV membrane volumes were significantly reduced in #0451-treated eyes in a dose-dependent manner. At therapeutic doses of 100 mg/kg or less, there was no significant weight loss between the treated and untreated groups.Oral administration of #0451, a novel VEGFR-2 (KDR/flk-1)-specific tyrosine kinase inhibitor, demonstrates anti-angiogenic effects in our murine model of CNV. #0451 may be useful to treat the choroidal neovascularization associated with AMD.

DNA microarray analysis comparing human tumor tissues with normal tissues including hematopoietic progenitor cells resulted in identification of membrane dipeptidase as a prodrug activation enzyme. Novel prodrugs of 2'-deoxy-2'-methylidenecytidine (DMDC) including compound 23 that are activated by membrane dipeptidase (MDP) preferentially in tumor tissue were designed and synthesized to generate the active drug, DMDC, after hydrolysis of the dipeptide bond followed by spontaneous cyclization of the promoiety.

In this study, 172 diacylhydrazine analogs were examined for their ability to activate an ecdysone (molting hormone)-dependent reporter gene in a silkworm (Bombyx mori) cell-based high-throughput screening assay. The measured EC(50) values (concentration required to cause an effect in 50% of the cells) were used to construct a 3-D QSAR model that describes the ecdysone agonist activities of the diacylhydrazine analogs. Of these compounds, 14 exhibited no activity and were excluded from the 3-D QSAR analysis. The resulting equation described approximately 74% of the activity for 158 compounds. The final equation consisted of 42% electrostatic and 58% steric effects (r(2) = 0.74 and q(2) = 0.45). Comparative molecular field analysis (CoMFA) was used to visualize the steric and electrostatic potential fields that were favorable and unfavorable for biological activity. Of particular interest was the observation that the hydrophobic parameter (logP) was not necessary for describing the observed activities, although previous studies have cited the importance of hydrophobic parameters in both classical and 3-D QSAR analyses of these compounds. Modeling studies of the B. mori ecdysone receptor supported the observed physicochemical parameters required for activity reported by the CoMFA models. Comparison of the present analysis with those performed using other lepidopteran assay systems evidenced a high degree of correlation (r(2) = 0.81 for a Sf-9 cell-based assay and r(2) = 0.89 for a Chilo suppressalis integument-based assay), indicating that it is valid to compare the results generated with the B. mori cell-based system to those generated with previous lepidopteran assays. This novel assay system is amendable to a high-throughput screening format and should greatly increase our ability to discover novel agonists of molting hormone (ecdysone) activity.

A highly potent water soluble triazole antifungal prodrug, RO0098557 (1), has been identified from its parent, the novel antifungal agent RO0094815 (2). The prodrug includes a triazolium salt linked to an aminocarboxyl moiety, which undergoes enzymatic activation followed by spontaneous chemical degradation to release 2. Prodrug 1 showed high chemical stability and water solubility and exhibited strong antifungal activity against systemic candidiasis and aspergillosis as well as pulmonary aspergillosis in rats.

Recent development of targeted protein kinase inhibitors provides new opportunity in cancer treatment. On the other hand, there are some cases limiting the efficacy of cancer therapies, owing to narrow therapeutic index by inhibiting multiple kinase, and the emergence of resistant mutants. Thus, the development of kinase inhibitors with more potent and selective properties and effectiveness to resistant mutants is needed. Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that is constitutively activated in some cancers, due to gene alterations such as chromosomal translocation, amplification, or point mutation. Here we have identified CH5424802, a potent, selective and orally available ALK inhibitor with a new chemical scaffold. CH5424802 showed preferential antitumor activity against cancers with gene alterations of ALK, such as non-small cell lung cancer (NSCLC) cells expressing EML4-ALK fusion, anaplastic large-cell lymphoma (ALCL) cells expressing NPM-ALK fusion, and neuroblastoma with gene amplification of ALK in vitro and in vivo. Also, CH5424802 could inhibit ALK L1196M, which corresponds to the gatekeeper mutation conferring common resistance to kinase inhibitors, and block the growth of EML4-ALK L1196M-driven cells. CH5424802 is currently being investigated in phase I/II clinical trials for patients with ALK-positive NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr DDT01-02. doi:10.1158/1538-7445.AM2011-DDT01-02

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that is constitutively activated in some cancers, due to gene alterations such as chromosomal translocation, amplification, or point mutation. It has been recognized as an attractive solid tumor target since the discovery in 2007 of the fusion gene, comprised of portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the ALK gene, which is detected in ca. 6.7% of non-small-cell lung cancer (NSCLC) patients. We have identified a lead compound as an ALK inhibitor through kinase panel screening of in-house kinase-oriented library. The lead compound had a unique scaffold but showed a rather broad kinase inhibition profile. Therefore, we examined structure-activity relationship from the viewpoint of the kinase selectivity as well as ALK inhibition potency. Finally, we identified CH5424802 as a clinical candidate having high selectivity over other kinases including c-Met, c-Kit and KDR. CH5424802 has a preferable PK profile and good oral bioavailability in rats and monkeys. CH5424802 showed preferential antitumor activity against cancers with gene alterations of ALK, such as non-small cell lung cancer (NSCLC) cells expressing EML4-ALK fusion both in vitro and in vivo. CH5424802 is currently being investigated in Phase I/II clinical trials for patients with ALK-positive NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3593. doi:10.1158/1538-7445.AM2011-3593