Design and synthesis of 2-amino-6-(1H,3H-benzo[de]isochromen-6-yl)-1,3,5-triazines as novel Hsp90 inhibitors.

A novel series of 2-amino-1,3,5-triazines bearing a tricyclic moiety as heat shock protein 90 (Hsp90) inhibitors is described. Molecular design was performed using X-ray cocrystal structures of the lead compound CH5015765 and natural Hsp90 inhibitor geldanamycin with Hsp90. We optimized affinity to Hsp90, in vitro cell growth inhibitory activity, water solubility, and liver microsomal stability of inhibitors and identified CH5138303. This compound showed high binding affinity for N-terminal Hsp90α (Kd=0.52nM) and strong in vitro cell growth inhibition against human cancer cell lines (HCT116 IC50=0.098µM, NCI-N87 IC50=0.066µM) and also displayed high oral bioavailability in mice (F=44.0%) and potent antitumor efficacy in a human NCI-N87 gastric cancer xenograft model (tumor growth inhibition=136%).

Enhanced antitumor activity of erlotinib in combination with the Hsp90 inhibitor CH5164840 against non-small-cell lung cancer.

Inhibition of heat shock protein 90 (Hsp90) can lead to degradation of multiple client proteins, which are involved in tumor progression. Epidermal growth factor receptor (EGFR) is one of the most potent oncogenic client proteins of Hsp90. Targeted inhibition of EGFR has shown clinical efficacy in the treatment of patients with non-small-cell lung cancer (NSCLC). However, primary and acquired resistance to the existing EGFR inhibitors is a major clinical problem. In the present study, we investigated the effect of the novel Hsp90 inhibitor CH5164840 on the antitumor activity of erlotinib. The NSCLC cell lines and xenograft models were treated with CH5164840 and erlotinib to examine their mechanisms of action and cell growth inhibition. We found that CH5164840 showed remarkable antitumor activity against NSCLC cell lines and xenograft models. The addition of CH5164840 enhanced the antitumor activity of erlotinib against NCI-H292 EGFR-overexpressing xenograft models. Phosphorylation of Stat3 increased with erlotinib treatment in NCI-H292 cells, which was abrogated by Hsp90 inhibition. Furthermore, in a NCI-H1975 T790M mutation erlotinib-resistant model, CH5164840 enhanced the antitumor activity of erlotinib despite the low efficacy of erlotinib treatment alone. In addition, ERK signaling was effectively suppressed by combination treatment with erlotinib and CH5164840 in a NCI-H1975 erlotinib-resistant model. Taken together, these data indicate that CH5164840 has potent antitumor activity and is highly effective in combination with erlotinib against NSCLC tumors with EGFR overexpression and mutations. Our results support the therapeutic potential of CH5164840 as a Hsp90 inhibitor for combination therapy with EGFR-targeting agents against EGFR-addicted NSCLC.

Preclinical antitumor activity of the novel heat shock protein 90 inhibitor CH5164840 against human epidermal growth factor receptor 2 (HER2)-overexpressing cancers.

Heat shock protein 90 (Hsp90), a molecular chaperone that plays a significant role in the stability and maturation of client proteins, including oncogenic targets for cell transformation, proliferation, and survival, is an attractive target for cancer therapy. We identified the novel Hsp90 inhibitor, CH5164840, and investigated its induction of oncogenic client protein degradation, antiproliferative activity, and apoptosis against an NCI-N87 gastric cancer cell line and a BT-474 breast cancer cell line. Interestingly, CH5164840 demonstrated tumor selectivity both in vitro and in vivo, binding to tumor Hsp90 (which forms active multiple chaperone complexes) in vitro, and being distributed effectively to tumors in a mouse model, which, taken together, supports the decreased levels of phosphorylated Akt by CH5164840 that we observed in tumor tissues, but not in normal tissues. As well as being well tolerated, the oral administration of CH5164840 exhibited potent antitumor efficacy with regression in NCI-N87 and BT-474 tumor xenograft models. In addition, CH5164840 significantly enhanced antitumor efficacy against gastric and breast cancer models when combined with the human epidermal growth factor receptor 2 (HER2)-targeted agents, trastuzumab and lapatinib. These data demonstrate the potent antitumor efficacy of CH5164840 when administered alone, and its significant combination efficacy when combined with trastuzumab or lapatinib, supporting the clinical development of CH5164840 as an Hsp90 inhibitor for combination therapy with HER2-targeted agents against HER2-overexpressing tumors.

Design and synthesis of novel macrocyclic 2-amino-6-arylpyrimidine Hsp90 inhibitors.

Macrocyclic compounds bearing a 2-amino-6-arylpyrimidine moiety were identified as potent heat shock protein 90 (Hsp90) inhibitors by modification of 2-amino-6-aryltriazine derivative (CH5015765). We employed a macrocyclic structure as a skeleton of new inhibitors to mimic the geldanamycin-Hsp90 interactions. Among the identified inhibitors, CH5164840 showed high binding affinity for N-terminal Hsp90α (K(d)=0.52nM) and strong anti-proliferative activity against human cancer cell lines (HCT116 IC(50)=0.15µM, NCI-N87 IC(50)=0.066µM). CH5164840 displayed high oral bioavailability in mice (F=70.8%) and potent antitumor efficacy in a HCT116 human colorectal cancer xenograft model (tumor growth inhibition=83%).

[Investigation of susceptibility testing methods for micafungin: comparison of two microdilution methods].

As there is not yet a standardized in vitro susceptibility test of micafungin (MCFG), we evaluated the methods of such testing, focusing on the judgment method of MIC, based on the National Committee for Clinical Laboratory Standards (NCCLS) M27-A2, M38-A, the proposed standards of The Japanese Society for Medical Mycology (JSMM) for yeast (JSMM-Y) and for filamentous fungi (JSMM-F) against Candida spp. and Aspergillus spp. The judgment of MIC value was performed spectrophotometrically and visually in both (NCCLS and JSMM) assays. Only the spectrophotometric MIC judgment against Aspergillus spp. in the NCCLS assay used two end points: 80% inhibitory concentration (IC80) of the growth control and 50% inhibitory concentration (IC50). The end point for the visual judgment against Aspergillus spp. in the NCCLS assay was determined to be no growth from the small clumps of altered hyphae in the microtiter plate. The other MIC judgments used an IC80 end point. The MICs of MCFG for Candida spp. were 4mug/ml and 0.0078-0.0313mug/ml). However, the MICs using the IC50 end point and those by JSMM assay agreed with the result of the visual assessment. Therefore, we recommend the JSMM assay, the NCCLS assay using the IC50 end point or the novel visual judgment for the susceptibility testing of MCFG against Aspergillus spp.

[Epidemiology of visceral mycoses in patients with leukemia and MDS - Analysis of the data in annual of pathological autopsy cases in Japan in 1989, 1993, 1997 and 2001].

To study the changes of visceral mycoses in autopsy cases, data on visceral mycosis cases with leukemia and myelodysplastic syndrome (MDS) reported in the Annual of the Pathological Autopsy Cases in Japan in 1989, 1993, 1997 and 2001 were analyzed. The frequency rate of visceral mycoses with leukemia and MDS was 27.9% (435/1,557) in 1989, 23.0% (319/1,388) in 1993, 22.3% (246/1,105) in 1997 and 25.1% (260/ 1,037) in 2001, which was clearly higher than the rate of cases without leukemia and MDS: 3.4%, 2.7%, 3.5% and 3.7%, respectively. Furthermore, in comparing the rate of mycoses in recipients of stem cell transplantation with that of non-recipients, that of recipients was about 10% higher. The predominant causative agents were Candida and Aspergillus, at approximately the same rate (Candida 33.6%, Aspergillus 33.3%) as in 1989. However, Aspergillus increased conspicuously in 1993 (Candida 22.3% Aspergillus 44.5%), and continued to increase (Candida 22.8%, Aspergillus 50.8% in 1997; Candida 16.9%, Aspergillus 54.2% in 2001). In aspergillosis and zygomycosis, the lung and bronchi comprised the most commonly infected organs: 74.7% and 75.6% of the total cases, respectively. Among a total of 1,260 cases with mycotic infections in the four years studied, acute lymphatic leukemia and acute myeloid leukemia were the major diseases (35.5% and 33.5%, respectively) followed by MDS (29.0%). Given these facts, we emphasize that a greater interest in mycoses should be taken by clinicians, and immunocompromised patients should be protected from opportunistic invasive fungal infections, especially aspergillosis.

The dual RAF/MEK inhibitor CH5126766/RO5126766 may be a potential therapy for RAS-mutated tumor cells.

Although melanoma is the most aggressive skin cancer, recent advances in BRAF and/or MEK inhibitors against BRAF-mutated melanoma have improved survival rates. Despite these advances, a treatment strategy targeting NRAS-mutated melanoma has not yet been elucidated. We discovered CH5126766/RO5126766 as a potent and selective dual RAF/MEK inhibitor currently under early clinical trials. We examined the activity of CH5126766/RO5126766 in a panel of malignant tumor cell lines including melanoma with a BRAF or NRAS mutation. Eight cell lines including melanoma were assessed for their sensitivity to the BRAF, MEK, or RAF/MEK inhibitor using in vitro growth assays. CH5126766/RO5126766 induced G1 cell cycle arrest in two melanoma cell lines with the BRAF V600E or NRAS mutation. In these cells, the G1 cell cycle arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27 and down-regulation of cyclinD1. CH5126766/RO5126766 was more effective at reducing colony formation than a MEK inhibitor in NRAS- or KRAS-mutated cells. In the RAS-mutated cells, CH5126766/RO5126766 suppressed the MEK reactivation caused by a MEK inhibitor. In addition, CH5126766/RO5126766 suppressed the tumor growth in SK-MEL-2 xenograft model. The present study indicates that CH5126766/RO5126766 is an attractive RAF/MEK inhibitor in RAS-mutated malignant tumor cells including melanoma.

Epidemiology of visceral mycoses in autopsy cases in Japan: comparison of the data from 1989, 1993, 1997, 2001, 2005 and 2007 in Annual of Pathological Autopsy Cases in Japan.

The data on visceral mycoses reported in the " Annual of Pathological Autopsy Cases in Japan " were analyzed epidemiologically every four years from 1989 to 2005, and in 2007. The frequency rates of visceral mycoses dropped sharply between 1989 (4.5%) and 1994 (3.2%), but by 2001 had risen again and have remained (4.4-4.6%) generally stable since then. The predominant causative agents were Candida and Aspergillus. Although the rate of candidosis showed a gradual decrease, the rate of aspergillosis showed an increase by degrees. Furthermore, the rate of aspergillosis exceeded that of candidosis in 1994, and the difference in the rates between the two conditions apparently further increased until 2001. After 2005, however no changes in this difference were observed. For complicated infections, the incidence of coinfection with Aspergillus and Candida showed a decreasing, and that with Aspergillus and Zygomycetes showed an increasing tendency. Severe infections with Zygomycetes showed a clear increase from 57.4% in 1989 to 88.9% in 2007. Comparing underlying diseases with mycoses in 1989 and 2007, leukemia (including myelodysplastic syndrome) decreased from 26.1% to 18.8% and bacterial infections (including interstitial pneumonia) increased from 11.1% to 22.1%. By age, the highest frequency rate of mycoses was observed in the range of 60-79 years, and the frequency rate of exogenous fungal infections such as aspergillosis, cryptococcosis, zygomycosis and trichosporonosis showed an increasing trend in the less than one-year old group.

The selective class I PI3K inhibitor CH5132799 targets human cancers harboring oncogenic PIK3CA mutations.

PURPOSE: The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in cell proliferation and survival in human cancer. PIK3CA mutations, which are found in many cancer patients, activate the PI3K pathway, resulting in cancer development and progression. We previously identified CH5132799 as a novel PI3K inhibitor. Thus, this study aimed to clarify the biochemical and antitumor activity of CH5132799 and elucidate the correlation between CH5132799 response and genetic alterations in the PI3K pathway. EXPERIMENTAL DESIGN: Kinase inhibitory activity was profiled in cell-free assays. A large panel of human breast, ovarian, prostate, and endometrial cancer cell lines, as well as xenograft models, were used to evaluate the antitumor activity of CH5132799, followed by analysis for genetic alterations. Effects on Akt phosphorylation induced by mTORC1 inhibition were tested with CH5132799 and compared with mTORC1 and PI3K/mTOR inhibitors. RESULTS: CH5132799 selectively inhibited class I PI3Ks and PI3Kα mutants in in vitro kinase assays. Tumors harboring PIK3CA mutations were significantly sensitive to CH5132799 in vitro and were remarkably regressed by CH5132799 in in vivo mouse xenograft models. In combination with trastuzumab, tumors disappeared in the trastuzumab-insensitive breast cancer model with the PIK3CA mutation. Moreover, CH5132799 did not reverse a negative feedback loop of PI3K/Akt/mTOR signaling and induced regression against tumors regrown after long-term mTORC1 inhibitor treatment. CONCLUSIONS: CH5132799 is a selective class I PI3K inhibitor with potent antitumor activity against tumors harboring the PIK3CA mutations. Prediction of CH5132799 response on the basis of PIK3CA mutations could enable patient stratification in clinical settings.

In vitro activity of isavuconazole against 140 reference fungal strains and 165 clinically isolated yeasts from Japan.

The in vitro susceptibilities of 140 laboratory reference strains of fungi, including type strains, and 165 clinical yeast isolates from Japan towards isavuconazole compared with fluconazole (FLC), itraconazole (ITC), voriconazole and amphotericin B were measured. Broth microdilution methods based on Clinical and Laboratory Standards Institute (CLSI) methods were used for yeasts, and RPMI-MOPS medium semi-solidified with 0.2% low-melting-point agarose based on CLSI guidelines was used for moulds. The range of isavuconazole minimum inhibitory concentrations (MICs) was 0.0004-0.21 mg/L for Candida albicans, 0.0036-0.4 mg/L for Candida glabrata, 0.023-0.058 mg/L for Candida krusei, 0.0026-0.032 mg/L for Cryptococcus neoformans, 0.1-0.39mg/L for Aspergillus fumigatus and 0.2-0.39 mg/L for Aspergillus terreus. Isavuconazole was as active as ITC against the dimorphic true pathogenic fungi, with a range of MICs from <0.0004 mg/L to 0.0063 mg/L for Blastomyces dermatitidis and Histoplasma capsulatum. It was also active against uncommon dematiaceous fungi such as Exophiala spp. and Phialophora spp. as well as against dermatophytic species. Isavuconazole showed very good in vitro antifungal activity with a broad spectrum, including against FLC-resistant Candida spp., Aspergillus spp. and uncommon opportunistic fungal species. This is the first report of the in vitro susceptibility of Japanese clinical yeast isolates to isavuconazole. No cross-resistance was found to isavuconazole amongst FLC-resistant strains.

Increase in aspergillosis and severe mycotic infection in patients with leukemia and MDS: comparison of the data from the Annual of the Pathological Autopsy Cases in Japan in 1989, 1993 and 1997.

To study the relationship between the changes in visceral mycoses rates and recently advanced medical care in hematological settings, data on visceral mycosis cases with leukemia and myelodysplastic syndrome (MDS) that had been reported in the Annual of the Pathological Autopsy Cases in Japan in 1989, 1993 and 1997 were analyzed. The frequency rate of visceral mycoses with leukemia and MDS was 27.9% (435/1557) in 1989, 23.0% (319/1388) in 1993 and 22.3% (246/1105) in 1997. In comparing the rate of mycoses in recipients of organ or bone marrow transplantation with that of non-recipients, that of recipients was approximately 10% higher. The predominant causative agents were Candida and Aspergillus, at approximately the same rate as in 1989. The rate of candidosis decreased to one-half that of aspergillosis by 1993. Furthermore, severe mycotic infections clearly increased from 58.9% in 1989 to 75.6% in 1997. Among a total of 1000 cases with mycotic infection in those 3 years, acute lymphatic leukemia and acute myeloid leukemia were the major diseases (40.6% and 34.8%, respectively), followed by MDS (26.1%). The reasons for increased rates of aspergillosis and of severe mycotic infection can be surmised to be: (i) candidosis had become controllable by prophylaxis and by empiric therapy for mycoses with effective antifungal drugs; (ii) the marketed antifungal drugs were not sufficiently effective against severe infections or Aspergillus infections; and (iii) the number of patients surviving in an immunocompromised state had increased due to developments in chemotherapy and progress in medical care.

Synthesis of novel water soluble benzylazolium prodrugs of lipophilic azole antifungals.

Water soluble N-benzyltriazolium or N-benzylimidazolium salt type prodrugs of several highly lipophilic triazole or imidazole antifungals have been synthesized. They were designed to undergo an enzymatic activation followed by a self-cleavage to release a parent drug. The prodrugs such as 16 had enough chemical stability and water solubility for parenteral use and were rapidly and quantitatively converted to the active substance in human plasma.

A controllable gene-expression system for the pathogenic fungus Candida glabrata.

A system for controlling gene expression was established in the pathogenic fungus Candida glabrata to elucidate the physiological functions of genes. To control the expression of the gene of interest, the C. glabrata cells were first transformed with the plasmid carrying the tetracycline repressor-transactivator fusion tetR::GAL4, then with the DNA fragment containing the controllable cassette, the tetracycline operator chimeric promoter (tetO::ScHOP1). The peptide elongation factor 3 (CgTEF3) and DNA topoisomerase II (CgTOP2) genes from C. glabrata were cloned and their expression assessed using this system. When the promoter of CgTEF3 or CgTOP2 was replaced with tetO::ScHOP1, doxycycline almost completely repressed the expression of both mRNAs, and impaired growth. Repression of the TOP2 or TEF3 gene by doxycycline also hampered the survival of C. glabrata cells in mice; in mouse kidneys the number of C. glabrata cells, in which the TOP2 or TEF3 promoter was replaced with the tetO::ScHOP1 controllable cassette, did not increase when the mice were given doxycycline. Thus, it appears that the gene repression mediated by doxycycline occurred not only in culture media but also in animals; therefore, this system can be used to elucidate the function of the gene in fungal infections and pathogenesis.

Design, synthesis and antifungal activity of a novel water soluble prodrug of antifungal triazole.

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.