Report of the use of patient-derived xenograft models in the development of anticancer drugs in Japan.

Cell line-derived xenograft (CDX) models created by implanting cancer cell lines into immunodeficient mice have contributed largely to the development of cancer drug therapies. However, cell lines often lose their original biological characteristics through many passages and cancer tissues in CDX models have many cancer cells and few cancer stromal cells, therefore CDX models are currently considered not suitable for predicting the results of clinical studies. Conversely, patient-derived xenograft (PDX) models are gaining importance, as human cancer biological characteristics and microenvironments are recreated by implanting tumor tissue into immunodeficient mice. These highly expected, evidently beneficial PDX models have been used in some basic research and are becoming more generalized. However, quality control and quality assurance criteria have not been established for them, and challenges and problems in the utilization of valuable PDX models in drug development have yet to be clarified. In this report, we conducted a questionnaire survey among researchers in Japanese academic institutions and pharmaceutical companies to understand the current status of PDX models in Japan. Based on the questionnaire results, we summarized the situations surrounding respondent's utilization and quality control in the development of anticancer drugs and proposed several measures to facilitate the utilization of PDX models in the development of anticancer drugs.

Synthesis and biological evaluation of novel selective androgen receptor modulators (SARMs) Part III: Discovery of 4-(5-oxopyrrolidine-1-yl)benzonitrile derivative 2f as a clinical candidate.

We previously reported that 4-(pyrrolidin-1-yl)benzonitrile derivative 1b was a selective androgen receptor modulator (SARM) that exhibited anabolic effects on organs such as muscles and the central nervous system (CNS), but neutral effects on the prostate. From further modification, we identified that 4-(5-oxopyrrolidine-1-yl)benzonitrile derivative 2a showed strong AR binding affinity with improved metabolic stabilities. Based on these results, we tried to enhance the AR agonistic activities by modifying the substituents of the 5-oxopyrrolidine ring. As a consequence, we found that 4-[(2S,3S)-2-ethyl-3-hydroxy-5-oxopyrrolidin-1-yl]-2-(trifluoromethyl)benzonitrile (2f) had ideal SARM profiles in Hershberger assay and sexual behavior induction assay. Furthermore, 2f showed good pharmacokinetic profiles in rats, dogs, monkeys, excellent nuclear selectivity and acceptable toxicological profiles. We also determined its binding mode by obtaining the co-crystal structures with AR.

Synthesis and biological evaluation of novel selective androgen receptor modulators (SARMs). Part I.

To develop effective drugs for hypogonadism, sarcopenia, and cachexia, we designed, synthesized, and evaluated selective androgen receptor modulators (SARMs) that exhibit not only anabolic effects on organs such as muscles and the central nervous system (CNS) but also neutral or antagonistic effects on the prostate. Based on the information obtained from a docking model with androgen receptor (AR), we modified a hit compound A identified through high-throughput screening. Among the prepared compounds, 1-(4-cyano-1-naphthyl)-2,3-disubstituted pyrrolidine derivatives 17h, 17m, and 17j had highly potent AR agonistic activities in vitro and good tissue selectivity in vivo. These derivatives increased the weight of the levator ani muscle without influencing the prostate and seminal vesicle. In addition, these compounds induced sexual behavior in castrated rats, indicating that the compounds could also act as agonists on the CNS.

A simple and widely applicable hit validation strategy for protein-protein interaction inhibitors based on a quantitative ligand displacement assay.

Identification of inhibitors for protein-protein interactions (PPIs) from high-throughput screening (HTS) is challenging due to the weak affinity of primary hits. We present a hit validation strategy of PPI inhibitors using quantitative ligand displacement assay. From an HTS for Bcl-xL/Mcl-1 inhibitors, we obtained a hit candidate, I1, which potentially forms a reactive Michael acceptor, I2, inhibiting Bcl-xL/Mcl-1 through covalent modification. We confirmed rapid reversible and competitive binding of I1 with a probe peptide, suggesting non-covalent binding. The advantages of our approach over biophysical assays include; simplicity, higher throughput, low protein consumption and universal application to PPIs including insoluble membrane proteins.

Design, synthesis, and biological evaluation of 3-aryl-3-hydroxy-1-phenylpyrrolidine derivatives as novel androgen receptor antagonists.

We designed and synthesized a series of 3-aryl-3-hydroxy-1-phenylpyrrolidine derivatives D and evaluated their potential as novel androgen receptor (AR) antagonists therapeutically effective against castration-resistant prostate cancer (CRPC). Introduction of a methyl group at the 2-position (R(2)) of the pyrrolidine ring increased the AR binding affinity. The (2S,3R) configuration of the pyrrolidine ring was favorable for the AR antagonistic activity. It was found that introduction of an amide substituent (R(1)) and a pyridin-3-yl group (Q) was effective for reducing the AR agonistic activity which appeared during the optimization of lead compound 6. Compound 54 showed potent antitumor effects against a CRPC model of LNCaP-hr cell line in a mouse xenograft, in which bicalutamide exhibited only partial suppression of tumor growth. Thus, the pyrrolidine derivatives such as 54 are novel AR antagonists, and their properties having efficacy against CRPC are distinct from those of a representative first-generation antagonist, bicalutamide.

Prevention of body weight loss and sarcopenia by a novel selective androgen receptor modulator in cancer cachexia models.

Cancer cachexia is a syndrome that impairs the quality of life and overall survival of patients, and thus the effectiveness of anticancer agents. There are no effective therapies for cancer cachexia due to the complexity of the syndrome, and insufficient knowledge of its pathogenesis results in difficulty establishing appropriate animal models. Previously, promising results have been obtained in clinical trials using novel agents including the ghrelin receptor agonist anamorelin, and the selective androgen receptor modulator (SARM) enobosarm to treat cachexia in patients with cancer. The present study examined the pharmacological effects of SARM-2f, a novel non-steroidal small molecule SARM, in animal models. SARM-2f increased body and skeletal muscle weight without significantly increasing the weight of the seminal vesicles or prostates of the castrated male rats. In the mice with tumor necrosis factor α-induced cachexia, SARM-2f and TP restored body weight, carcass weight, and food consumption rate. In the C26 and G361 cancer cachexia animal models, body and carcass weight, lean body mass, and the weight of the levator ani muscle were increased by SARM-2f and TP treatments. Tissue selectivity of SARM-2f was also observed in these animal models. The results demonstrate the anabolic effects of SARM-2f in a cytokine-induced cachexia model and other cancer cachexia models, and suggest that SARM-2f may be a novel therapeutic option for cachexia in patients with cancer.

Highly potent and orally active CCR5 antagonists as anti-HIV-1 agents: synthesis and biological activities of 1-benzazocine derivatives containing a sulfoxide moiety.

Chemical modification has been performed on an orally bioavailable and potent CCR5 antagonist, sulfoxide compound 4, mainly focusing on replacement of the [6,7]-fused 1-benzazepine nucleus. We designed, synthesized, and evaluated the biological activities of ring-expanded [6,8]-, [6,9]-, and [6,10]-fused compounds containing S-sulfoxide moieties, which led to the discovery of 1-benzazocine and 1-benzazonine compounds that exhibited potent inhibitory activities (equivalent to compound 4) in a binding assay. In addition, 1-benzazocine compounds possessing the S-sulfoxide moiety ((S)-(-)-5a,b,d,e) showed greater potency than compound 4 in a fusion assay. From further investigation in a multi-round infection assay, it was found that 1-isobutyl-1-benzazocine compound (S)-(-)-5b, containing the S-{[(1-propyl-1H-imidazol)-5-yl]methyl}sulfinyl group, showed the most potent anti-HIV-1 activity (IC90=0.81 nM, in MOLT4/CCR5 cells). Compound (S)-(-)-5b (TAK-652) also inhibited the replication of six macrophage-tropic (CCR5-using or R5) HIV-1 clinical isolates in peripheral blood mononuclear cells (PBMCs) (mean IC90=0.25 nM). It was also absorbed after oral administration in rats, dogs, and monkeys and was thus selected as a clinical candidate. The synthesis and biological activity of the 1-benzazocine compound (S)-(-)-5b and its related derivatives are described.

Discovery of potent Mcl-1/Bcl-xL dual inhibitors by using a hybridization strategy based on structural analysis of target proteins.

Mcl-1 and Bcl-xL are crucial regulators of apoptosis, therefore dual inhibitors of both proteins could serve as promising new anticancer drugs. To design Mcl-1/Bcl-xL dual inhibitors, we performed structure-guided analyses of the corresponding selective Mcl-1 and Bcl-xL inhibitors. A cocrystal structure of a pyrazolo[1,5-a]pyridine derivative with Mcl-1 protein was successfully determined and revealed the protein-ligand binding mode. The key structure for Bcl-xL inhibition was further confirmed through the substructural analysis of ABT-263, a representative Bcl-xL/Bcl-2/Bcl-w inhibitor developed by Abbott Laboratories. On the basis of the structural data from this analysis, we designed hybrid compounds by tethering the Mcl-1 and Bcl-xL inhibitors together. The results of X-ray crystallographic analysis of hybrid compound 10 in complexes with both Mcl-1 and Bcl-xL demonstrated its binding mode with each protein. Following further optimization, compound 11 showed potent Mcl-1/Bcl-xL dual inhibitory activity (Mcl-1, IC50 = 0.088 µM; and Bcl-xL, IC50 = 0.0037 µM).

Orally active CCR5 antagonists as anti-HIV-1 agents. Part 3: Synthesis and biological activities of 1-benzazepine derivatives containing a sulfoxide moiety.

In order to develop orally active CCR5 antagonists, 1-propyl- or 1-isobutyl-1-benzazepine derivatives containing a sulfoxide moiety have been designed, synthesized, and evaluated for their biological activities. Sulfoxide compounds containing a 2-pyridyl group were first investigated, which led to discovering that the presence of a methylene group between the sulfoxide moiety and 2-pyridyl group was necessary for increased inhibitory activity in a binding assay. After further chemical modification, it was found that replacement of the pyridyl group with an imidazolyl or 1,2,4-triazolyl group enhanced activity in the binding assay and that S-sulfoxide compounds were more active than R-isomers. Particularly, compounds (S)-4r, (S)-4s, and (S)-4w exhibited highly potent CCR5 antagonistic activities (IC50=1.9, 1.7, 1.6 nM, respectively) and inhibitory effects (IC50=1.0, 2.8, 7.7 nM, respectively) in the HIV-1 envelope mediated membrane fusion assay, together with good pharmacokinetic properties in rats. In addition, we established the synthesis of (S)-4r and (S)-4w by asymmetric oxidation with titanium-(S)-(-)-1,1'-bi-2-naphthol complex.

A platinum-ruthenium dinuclear complex bridged by bis(terpyridyl)xanthene.

4,5-Bis(terpyridyl)-2,7-di-tert-butyl-9,9-dimethylxanthene (btpyxa) was prepared to serve as a new bridging ligand via Suzuki coupling of terpyridin-4'-yl triflate and 2,7-di-tert-butyl-9,9-dimethylxanthene-4,5-diboronic acid. The reaction of btpyxa with either 1 equiv or an excess of PtCl(2)(cod) (cod = 1,5-cyclooctadiene) followed by anion exchange afforded mono- and dinuclear platinum complexes [(PtCl)(btpyxa)](PF(6)) ([1](PF(6))) and [(PtCl)(2)(btpyxa)](PF(6))(2) ([2](PF(6))(2)), respectively. The X-ray crystallography of [1](PF(6)).CHCl(3) revealed that the two terpyridine units in the ligand are nearly parallel to each other. The heterodinuclear complex [(PtCl)[Ru((t)Bu(2)SQ)(dmso)](btpyxa)](PF(6))(2) ([4](PF(6))(2)) (dmso = dimethyl sulfoxide; (t)Bu(2)SQ = 3,5-di-tert-butyl-1,2-benzosemiquinone) and the monoruthenium complex [Ru((t)Bu(2)SQ)(dmso)(trpy)](PF(6)) ([5](PF(6))) (trpy = 2,2':6',2' '-terpyridine) were also synthesized. The CV of [2](2+) suggests possible electronic interaction between the two Pt(trpy) groups, whereas such an electronic interaction was not suggested by the CV of [4](2+) between Pt(trpy) and Ru((t)Bu(2)SQ) frameworks.

Orally active CCR5 antagonists as anti-HIV-1 agents 2: synthesis and biological activities of anilide derivatives containing a pyridine N-oxide moiety.

In order to develop orally active CCR5 antagonists, we investigated 1-benzoxepine derivatives containing new polar substituents, such as phosphonate, phosphine oxide or pyridine N-oxide moieties, as replacements for the previously reported quaternary ammonium moiety. Among these compounds, the 2-(alpha-hydroxybenzyl)pyridine N-oxide 5e exhibited moderate CCR5 antagonistic activity and had an acceptable pharmacokinetic profile in rats. Subsequent chemical modification was performed and compound (S)-5f possessing the (S)-configuration hydroxy group was found to be more active than the (R)-isomer. Replacement of the 1-benzoxepine ring with a 4-methylphenyl group by a 1-benzazepine ring with a 4-[2-(butoxy)ethoxy]phenyl group enhanced the activity in the binding assay. In addition, introduction of a 3-trifluoromethyl group on the phenyl group of the anilide moiety led to greatly increased activity in the HIV-1 envelope-mediated membrane fusion assay. In particular, compound (S)-5s showed the most potent CCR5 antagonistic activity (IC(50)=7.2 nM) and inhibitory effect (IC(50)=5.4 nM) in the fusion assay, together with good pharmacokinetic properties in rats.

Orally active CCR5 antagonists as anti-HIV-1 agents: synthesis and biological activity of 1-benzothiepine 1,1-dioxide and 1-benzazepine derivatives containing a tertiary amine moiety.

The search for orally active CCR5 antagonists was performed by chemical modification of the 1-benzothiepine 1,1-dioxide 3 and 1-benzazepine 4 lead compounds containing a tertiary amine moiety. Replacement of methyl group with a 2-(C(2-4) alkoxy)ethoxy group at the 4-position on the 7-phenyl group of the 1-benzothiepine ring resulted in both enhanced activity and significant improvement in the pharmacokinetic properties upon oral administration in rats. Introduction of C(2-4) alkyl, phenyl or (hetero)arylmethyl groups as the 1-substituent on the 1-benzazepine ring together with the 2-(butoxy)ethoxy group led to further increase of activity. Among the 1-benzazepine derivatives, the isobutyl (6i), benzyl (6o) or 1-methylpyrazol-4-ylmethyl (6s) compounds were found to exhibit highly potent inhibitory effects, equivalent to the injectable CCR5 antagonist 1, in the HIV-1 envelope-mediated membrane fusion assay. In particular, compound 6s showed the most potent CCR5 antagonistic activity (IC(50)=2.7 nM) and inhibitory effect (IC(50)=1.2 nM) on membrane fusion, together with good pharmacokinetic properties in rats. The synthesis of 1-benzothiepine 1,1-dioxide and 1-benzazepine derivatives and their biological activity are described.