Steroids are important components of cell membranes and are involved in several physiological functions. A diphenylmethane (DPM) skeleton has recently been suggested to act as a mimetic of the steroid skeleton. However, difficulties are associated with efficiently introducing different substituents between two phenyl rings of the DPM skeleton, and, thus, further structural development based on the DPM skeleton has been limited. We herein developed an efficient synthetic method for introducing different substituents into two phenyl rings of the DPM skeleton. We also synthesized DPM-based estrogen receptor (ER) modulators using our synthetic method and evaluated their ER transcriptional activities.Download high-res image (78KB)Download full-size image

Estrogen receptors (ERs) are a family of nuclear receptors (NRs) that regulate physiological effects such as reproduction and bone homeostasis. It has been reported that approximately 70% of human breast cancers are hormone-dependent and ERα-positive. Recently, novel anti-breast cancer drugs based on different mechanisms of action have received significant attention. In this article, we have designed and synthesized a selective ER degradation inducer based on the diphenylheptane skeleton. Western blotting analysis revealed that PBP-NC10 degraded ERα through the ubiquitin–proteasome system. We also performed computational docking analysis to predict the binding mode of PBP-NC10 to ERα.

Estrogen receptors (ERs) play a major role in the growth of human breast cancer cells. A selective estrogen receptor down-regulator (SERD) that acts as not only an inhibitor of ligand binding, but also induces the down-regulation of ER, would be useful for the treatment for ER-positive breast cancer. We previously reported that tamoxifen derivatives, which have a long alkyl chain, had the ability to down-regulate ERα. With the aim of expanding range of the currently available SERDs, we designed and synthesized raloxifene derivatives, which had various lengths of the long alkyl chains, and evaluated their SERD activities. All compounds were able to bind ERα, and RC10, which has a decyl group on the amine moiety of raloxifene, was shown to be the most potent compound. Our findings suggest that the ligand core was replaceable, and that the alkyl length was important for controlling SERD activity. Moreover, RC10 showed antagonistic activity and its potency was superior to that of 4,4′-(heptane-4,4-diyl)bis(2-methylphenol) (18), a competitive antagonist of ER without SERD activity. These results provide information that will be useful for the development of promising SERDs candidates.

The manipulation of protein stability with small molecules has great potential as a technique for aiding the development of clinical therapies, including treatments for cancer. In this study, BCR-ABL protein degradation inducers called SNIPER(ABL) (Specific and Non-genetic inhibitors of apoptosis protein [IAP]-dependent Protein Erasers) were developed. The designed molecules contained two biologically active scaffolds: one was an imatinib derivative that binds to BCL-ABL and the other was a methyl bestatin that binds to cellular IAP 1 (cIAP1). The hybrid molecules, SNIPER(ABL), were expected to recruit BCR-ABL to cIAP1 for removal by proteasomes. In fact, SNIPER(ABL) induced the degradation of BCR-ABL protein and a subsequent reduction in cell growth. Thus, the degradation of BCR-ABL by SNIPER(ABL) is one potential strategy for treating BCR-ABL driven chronic myelogenous leukemia.

A peptide-based protein knockdown system for inducing nuclear receptors degradation via the ubiquitin–proteasome system was developed. Specifically, the designed molecules were composed of two biologically active scaffolds: a peptide that binds to the estrogen receptor α (ERα) surface and an MV1 molecule that binds to cellular inhibitors of apoptosis proteins (IAP: cIAP1/cIAP2/XIAP) to induce ubiquitylation of the ERα. The hybrid peptides induced IAP-mediated ubiquitylation followed by proteasomal degradation of the ERα. Those peptides were also applicable for inducing androgen receptor (AR) degradation.

We designed and synthesized two dodecapeptides, Boc-(l-Leu-l-Leu-Aib-d-Leu-d-Leu-Aib)2-OMe (5) and Boc-l-Leu-l-Leu-Aib-(d-Leu-d-Leu-Aib)2-l-Leu-l-Leu-Aib-OMe (6), that contain equal amounts of l-Leu, d-Leu, and achiral Aib residues. The conformations of peptides 5 and 6 in the crystalline state were studied using X-ray crystallographic analysis. Peptide 5 formed a left-handed (M) α-helical structure, whereas peptide 6 was composed of a combination of fused (M) α-helical and right-handed (P) 310-helical structures. In solution, roughly equivalent amounts of (P) and (M) helices were present in 5, whereas the (M) α-helix was present in 6 as its dominant conformation.

A new bis-cationic cyclic amino acid, 9-amino-3,7-diazabicyclo[3.3.1]nonane-9-carboxylic acid (9-amino-bispidine-9-carboxylic acid; Abp), which is available for both solution phase and solid phase peptide synthesis, was designed and synthesized. Furthermore, a heterotripeptide Cbz-Leu-Abp-Ala-OMe (9) containing Abp was prepared, and its dominant conformation was analyzed by examining its nuclear magnetic resonance and infrared spectra and performing molecular modeling. The tripeptide 9 formed a β-turn structure as its preferred conformation in solution.

The heptaarginine (R7)-conjugated peptide 5 was designed and synthesized as an inhibitor of ER-coactivator interactions and ER-mediated transcription at the cellular level. The R7-conjugated peptide 5 was able to enter ER-positive T47D cells efficiently, and treatment with 3 μM of 5 downregulated the mRNA expression of pS2 (an ER-mediated gene) by 87%.

We synthesized four types of arginine-based amphipathic nonapeptides, including two homochiral peptides, R-(l-Arg-l-Arg-Aib)3-NH2 (R = 6-FAM-β-Ala: FAM-1; R = Ac: Ac-1) and R-(d-Arg-d-Arg-Aib)3-NH2 (R = 6-FAM-β-Ala: ent-FAM-1; R = Ac: ent-Ac-1); a heterochiral peptide, R-(l-Arg-d-Arg-Aib)3-NH2 (R = 6-FAM-β-Ala: FAM-2; R = Ac: Ac-2); and a racemic mixture of diastereomeric peptides, R-(rac-Arg-rac-Arg-Aib)3-NH2 (R = 6-FAM-β-Ala: FAM-3; R = Ac: Ac-3), and then investigated the relationship between their secondary structures and their ability to pass through cell membranes. Peptides 1 and ent-1 formed stable one-handed α-helical structures and were more effective at penetrating HeLa cells than the non-helical peptides 2 and 3.

We designed and synthesized an estrogen receptor (ER) down-regulator (5), which is a derivative of tamoxifen with a long alkyl side chain. Compound 5 effectively reduced ER protein levels in MCF-7 cells and had an antagonistic effect.

We synthesized stapled helical leucine-based peptides (DPI-01-07) containing 2-aminoisobutyric acid and a covalent cross-linked unit as inhibitors of vitamin D receptor (VDR)–coactivator interactions. The effects of these peptides on the human VDR were examined in an inhibition assay based on the receptor cofactor assay system, and one of them, DPI-07, exhibited potent inhibitory activity (IC50: 3.2 μM).

We investigated the preferred conformations of two nonapeptides, Boc-(l-Leu-d-Leu-Aib)3-OMe (2) and its enantiomer Boc-(d-Leu-l-Leu-Aib)3-OMe (ent-2), four dodecapeptides, Boc-(l-Leu-d-Leu-Aib)4-OMe (3), Boc-(l-Leu-Aib-d-Leu)4-OMe (4), Boc-(Aib-l-Leu-d-Leu)4-OMe (5), and Boc-(l-Leu-Aib-d-Leu-Aib)3-OMe (6), and a decapeptide, Boc-l-Leu-(d-Leu-l-Leu-Aib)3-OMe (7), in solution and in the crystalline state. The nonapeptide 2 formed a right-handed (P) α-helix, and its enantiomer ent-2 formed a left-handed (M) α-helix. The dodecapeptides 3 and 5 were folded into (P) helices, and 4 formed an (M) helical structure. As for 6, roughly equivalent amounts of (P) and (M) helices were observed in solution, and two (M) α-helices were detected in the crystalline state. Furthermore, the decapeptide 7, which possesses four l-Leu residues and three d-Leu residues, was folded into an (M) α-helix.

We have developed new helical oligomers using a combination of (1S,2S)-cyclopentane-1,2-diamine [(S,S)-CPDA] and 2,2-dimethylmalonic acid (DMM) residues as building blocks. In solution, the preferred secondary structure of the (S,S) tetramer 6 was a right-handed (P) helix, and that of the (R,R) tetramer ent-6 was a left-handed (M) helix. In the crystalline state, both 6 and the (S,S) pentamer 7 folded into (P) 11-helices, and ent-6 folded into an (M) 11-helix with hydrogen bonds that were oriented in alternating directions.

We designed and synthesized estrogen receptor (ER) degradation inducers 5, 6, and 7, which crosslink the ER and the cellular inhibitor of apoptosis protein 1 (cIAP1). Compounds 5, 6, and 7 induced cIAP1-mediated ubiquitylation of ERα resulting in its proteasomal degradation.

We designed and synthesized a C2-symmetric cyclic hexapeptide, cyclo(l-Leu-d-Leu-Aib)2 (2), which contains l- and d-amino acids and achiral Aib residues. The conformation of 2 was analyzed in the crystalline state and in solution, which was a unique figure-eight-shaped conformation.

We designed and synthesized nonsecosteroidal vitamin D receptor (VDR) ligands that formed H-bonds with six amino acid residues (Tyr143, Ser233, Arg270, Ser274, His301 and His393) of the VDR ligand-binding domain. The ligand YR335 exhibited potent transcriptional activity, which was comparable to those of 1α,25-dihydroxyvitamin D3 and YR301. The crystal structure of the complex formed between YR335 and the VDR ligand-binding domain was solved, which revealed that YR335 formed H-bonds with the six amino acid residues mentioned above.

Stabilized short helical heptapeptides containing a combination of an α-aminoisobutyric acid as a helical promoter and l/d-serine derivatives to produce cross-linked units were synthesized. The cyclic peptide R3,7R-2, which had d-serine derivatives at its 3rd and 7th positions, formed a stable right-handed (P) α-helix in solution and the crystalline state. Furthermore, its N-terminal free helical peptide catalyzed the enantioselective epoxidation of (E)-chalcone to afford the epoxide in a high yield and moderate enantioselectivity.

Three diastereomeric -Leu-Leu-Aib-Leu-Leu-Aib- peptides composed of the same numbers of l-Leu, d-Leu, and Aib residues were synthesized: Boc-l-Leu-l-Leu-Aib-d-Leu-d-Leu-Aib-OMe (1), Boc-l-Leu-d-Leu-Aib-l-Leu-d-Leu-Aib-OMe (2), and Boc-l-Leu-d-Leu-Aib-d-Leu-l-Leu-Aib-OMe (3). The crystals of the three peptides were characterized by X-ray crystallographic analysis as follows: (1) orthorhombic, P212121, a = 21.383 Å, b = 11.070 Å, c = 19.560 Å, Z = 4, R1 = 0.0527, and Rw = 0.1562; (2) monoclinic, P21, a = 9.391 Å, b = 21.278 Å, c = 11.662 Å, β = 99.125, Z = 2, R1 = 0.0507, and Rw = 0.1447; and (3) triclinic, P1, a = 12.545 Å, b = 14.913 Å, c = 15.330 Å, α = 77.622, β = 66.601, γ = 78.839, Z = 2, R1 = 0.0775, and Rw = 0.1971. The three diastereomeric peptides, 1, 2, and 3, showed unique conformations. That is to say, 1 was folded into a left-handed (M) 310-helical structure, 2 was folded into a distorted β-hairpin nucleated by a type II′ β-turn-like structure, and 3 was folded into an S-shape turn structure based on two type II/III β-turns.

Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of the skin, prostate, colon, and breast as well as leukemia. LG190178 is a novel non-secosteroidal ligand for VDR. We synthesized and evaluated stereoisomers of LG190178 and found that only an (2S,2′R)-analogue of LG190178 (YR301) had strong activity.YR301 was the (2S,2′R)-analogue of LG190178 and had strong activity.

Retinoids are natural and synthetic analogues of all-trans retinoic acid (ATRA). Cancer and other serious hyperproliferative diseases are attractive therapeutic targets for retinoids. We report here the design and synthesis of novel cyclic urea compounds with retinoidal activity. YR105 exhibited potent differentiation-inducing ability toward human promyelocytic leukemia HL-60 cells at the concentration of 10−9 M: its potency was almost equal to that of the native ligand, all-trans retinoic acid.YR105 exhibited potent differentiation-inducing ability toward human promyelocytic leukemia HL-60 cells at the concentration of 10−9 M: its potency was almost equal to that of the native ligand, all-trans retinoic acid.