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Non-secosteroidal VDR ligands without any assymmetric carbon were designed and synthesized based on the structure of the previously reported non-secosteroidal VDR agonist LG190178. The VDR-agonistic activity of all synthesized compounds was evaluated, and 7b emerged as a potent agonist activity with an EC50 value of 9.26 nM. Moreover, a docking simulation analysis was also performed to determine the binding mode of 7b with VDR-LBD.

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An analytical expression for impedance spectra in the case of double injection (both electrons and holes are injected into an organic semiconductor thin film) has been derived from the basic transport equations (the current density equation, the continuity equation and the Possion's equation). Capacitance-frequency characteristics calculated from the analytical expression have been examined at different recombination constants and different values of mobility balance defined by a ratio of electron mobility to hole mobility. Negative capacitance appears when the recombination constant is lower than the Langevin recombination constant and when the value of the mobility balance approaches unity. These results are consistent with the numerical results obtained by a device simulator (Atlas, Silvaco).

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The p-type organic semiconductor (OSC) material tetrathieno[2,3-a:3',2'-c:2″,3″-f:3‴,2‴-h]naphthalene (2TTN) and its alkyl-substituted derivatives C(n)-2TTNs (n = 6, 8, and 10) have been developed based on the results of theoretical calculation-inspired investigation. A hole mobility for amorphous C(n)-2TTNs (10(-2)-10(-3) cm(2) V(-1) s(-1)) was accurately predicted by using a novel statistical method in which the geometric mean of the mobilities for many individual small molecular flocks in an amorphous solid was obtained by using molecular mechanical molecular dynamics simulations and quantum chemical calculations. The simulation also suggests that upon increasing the length of alkyl chains in C(n)-2TTNs the mobilities become smaller as a consequence of a decrease in transfer integral values. C(n)-2TTNs are synthesized in a microflow reactor through photoreactions of the corresponding precursors. C(n)-2TTNs are then utilized in the fabrication of organic field-effect transistors (OFETs). Although spin-coated thin films of C(n)-2TTNs are crystalline, the hole mobilities (10(-2)-10(-3) cm(2) V(-1) s(-1)) of trial OFETs decrease upon elongation of the alkyl chains. This finding parallels the results of theoretical simulation. The simulation method for amorphous solids developed in this effort should become a useful tool in studies aimed at designing new OSC materials.

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We synthesized and evaluated novel vitamin D3 derivatives with cyanoalkyl side chain at C-2 position on the basis of our previous research for 2α side chain which bears nitrogen atom-containing functional group. Through a study of X-ray co-crystal structures of human VDR and compound 3, we demonstrated that the 2α alkyl side chain in compound 3 shows a novel interaction in the complex of hVDR-LBD and ligand. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

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Three different A-ring perhydroxylated trihydroxyvitamin D3 metabolites were synthesized from their appropriate A-ring precursors and CD-ring for their potential therapeutic applications. We first chemically synthesized 1α,2α,25-trihydroxyvitamin D3 [1α,2α,25(OH)3D3] to study its VDR binding affinity because this metabolite is a product of recombinant human CYP3A4 catalysis when 2α-(3-hydroxypropoxy)-1α,25-dihydroxyvitamin D3 (O2C3), a more potent vitamin D receptor (VDR) binder than 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], is used as the substrate. We found that this metabolite retained 27.3% of the VDR binding affinity compared to 1α,25(OH)2D3. The kcat/Km value of CYP24A1 for 1α,2α,25(OH)3D3 is 60% of that for 1α,25(OH)2D3. Since the biological activity and the metabolic fate of a naturally occurring C4-hydroxylated vitamin D2 metabolite found in the serum of rats treated with pharmacological doses of vitamin D2 have never been described, we next synthesized 1α,4α,25-trihydroxyvitamin D3 and its diastereoisomer, 1α,4ß,25-trihydroxyvitamin D3, to study their metabolism and biological activities. Both 4-hydroxylated isomers showed weaker VDR binding affinity than 1α,25(OH)2D3. Although either 4-hydroxylated isomer can be metabolized by CYP24A1 almost at the same level as 1α,25(OH)2D3, their metabolic patterns catalyzed by uridine 5'-diphosphoglucuronosyltransferase (UGT) are different; only the 4α-hydroxylated analog can be metabolized by UGT to produce a glucuronate conjugate. The results provide important information for the synthesis of new novel chemotherapeutic vitamin D analogs which would be less subjective to degradation and therefore more bioavailable than 1α,25(OH)2D3. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

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X-ray cocrystallographic studies of the human vitamin D receptor (hVDR)-[2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3 (O1C3)] complex showed that the terminal hydroxy group of the 2α-functional group of O1C3 formed a hydrogen bond with Arg274 in the ligand binding domain (LBD) of hVDR to stabilize the complex; therefore, O1C3 showed 3-times greater binding affinity for VDR than the natural hormone. Here, the effects of a heteroaromatic ring on binding to hVDR instead of the terminal OH group of O1C3 and also on preliminary biological activities were studied. We synthesized 2α-[2-(tetrazol-2-yl)ethyl]-1α,25(OH)2D3 (1a) and its regioisomer 2α-[2-(tetrazol-1-yl)ethyl]-1α,25(OH)2D3 (1b), in which 1a showed much higher hVDR binding affinity and greater osteocalcin promoter transactivation activity in human osteosarcoma (HOS) cells than those of 1b. X-ray cocrystallographic analysis of the hVDR-1a complex showed new hydrogen bond formation between one of the nitrogen atoms of the tetrazole ring and Arg274. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

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Up to the present, numerous vitamin D derivatives have been synthesized, but most of them have straight side chains, and there are few publications described about in vitro and in vivo evaluations on bone by vitamin D derivatives. In our previous paper, we reported the synthesis of various C-2 substituted vitamin D derivatives (2b-2i) with a 2,2-dimethylcyclopentanone unit in the CD-ring side chains, and that the derivatives have strong activity for enhancing bone growth. On the basis of results, this time, we report the synthesis of 2α-substituted vitamin D3 derivatives with chiral cyclopentanone (3-6 and 12-16). These derivatives were obtained by Pd-coupling reaction with A-ring precursor and CD-rings precursor. We evaluated novel derivatives in vitro assays, for affinities for VDR and transactivation assays by human osteosarcoma (HOS) cells. In this research, we demonstrated that some novel vitamin D derivatives (12-MP, 13-MP, 15-MP and 16-LP) have strong transactivation activities in spite of lower affinity for VDR than 1. In addition, we also demonstrated that these derivatives have strong activities for enhancing bone growth using OVX therapeutic rats. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

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2α-Heteroarylethyl-1α,25-dihydroxyvitamin D3 analogues, which were designed to form a hydrogen bond between Arg274 of human vitamin D receptor (hVDR) and a nitrogen atom of the heteroaromatic ring at the 2α-position, were synthesized. Among them, 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-dihydroxyvitamin D3 showed higher osteocalcin promoter transactivation activity in human osteosarcoma (HOS) cells and a greater therapeutic effect in ovariectomized (OVX) rats, osteoporosis model animals, on enhancing bone mineral density than those of active vitamin D3. X-ray cocrystallographic analysis of the hVDR-ligand complex confirms that the new hydrogen bond formation stabilized the complex.

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Recently, we evaluated a novel skeleton in the vitamin D family, 14-epi-1α,25(OH)2-19-nortachysterol, and discovered its unique binding configuration in the human vitamin D receptor (VDR) with the C5,6- and C7,8-s-trans triene configuration. Because of its unprecedented form, this skeleton has a promising characteristic profile for clinical use, and also the synthesis of its derivatives should be versatile. Therefore, we synthesized the novel analog, 2α-hydroxypropoxy substituted 14-epi-1α,25(OH)2-19-nortachysterol, and evaluated its human VDR binding affinity. Although this substitution is one of the promising modification of vitamin D3 such as eldecalcitol (ED-71), it had negative effects on the binding affinity, and the compound showed lower affinity than 1α,25(OH)2D3 and its parent compound, 14-epi-1α,25(OH)2-19-nortachysterol. It was thought that the unprecedented binding configuration of this skeleton should not allow the terminal hydroxyl group of the 2α-substituent to construct effective hydrogen bond networks around the amino acid residues in the binding pocket. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

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A previous report has demonstrated the existence of a C4-hydroxylated vitamin D(2) metabolite in serum of rats treated with pharmacological doses of vitamin D(2). However, the biological significance and metabolic fate of this metabolite have not been described. To explore its potential biological activities, we therefore synthesized 1α,4α,25-trihydroxyvitamin D(3) and its diastereoisomer, 1α,4ß,25-trihydroxyvitamin D(3), using Trost Pd-mediated coupling reaction, and studied their vitamin D receptor (VDR) binding affinity, osteocalcin promoter transactivation activity, and their further metabolism by human CYP24A1 as well as by human liver microsomal fraction based on CYP- and UDP-glucuronosyltransferases (UGTs)-reactions.

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The Δ(16) structure as a vitamin D analog enhanced vitamin D receptor (VDR) binding affinity and induced significant cell differentiation, whereas its relative calcemic activity was reduced compared to 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)). Methodologies available to introduce a double bond at C16-C17 of the D-ring on the seco-steroidal skeleton were limited; therefore, a new synthetic strategy was developed to obtain not only the Δ(16) structure, but also a new C15-functional group. Since C15-functionalization was unprecedented in vitamin D analog studies, the hybrid structure of Δ(16) and the C15-OH group at the D-ring may provide important information on the structure-activity relationship with vitamin D analogs. The synthesized 16-ene-2α-methyl-1α,15α,25-trihydroxyvitamin D(3) showed almost 3-times higher VDR binding affinity and an equipotent level of osteocalcin promoter transactivation activity in human osteosarcoma cells as compared to 1α,25(OH)(2)D(3).

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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 D(3) 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.

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C15-Substituted 1α,25-dihydroxyvitamin D(3) analogs were synthesized for the first time to investigate the effects of the modified CD-ring on biological activity concerning the agonistic positioning of helix-3 and helix-12 of the vitamin D receptor (VDR). X-ray cocrystallographic analysis proved that 0.6 Å shifts of the CD-ring and shrinking of the side chain were necessary to maintain the position of the 25-hydroxy group for proper interaction with helix-12. The 15-hydroxy-16-ene derivative showed higher binding affinity for hVDR than the natural hormone.

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In order to obtain vitamin D derivatives, which have strong activity for enhancing bone growth, we designed vitamin D derivatives with various substitutions at the C-2 position. Novel 2 α-substituted vitamin D derivatives were synthesized starting from d-glucose as a chiral template of the A-ring with a CD-ring bromoolefin unit using the Trost coupling method. We evaluated these compounds by two in vitro assays, affinity to VDR and transactivation assays, using human osteosarcoma (Hos) cells, and demonstrated the SAR of the C-2 position of VD(3). Furthermore, by using the OVX model, we found that compound 5c, which has a hydroxypropoxy side chain at C-2 and 2,2-dimethyl cyclopentanone in the CD-ring side chain, has a strong activity for enhancing bone growth, same as the reported compound, 2α-(3-hydroxypropoxy)-1α,25-dihydroxyvitamin D(3)1d, and this derivative shows a possibility that calcemic activity is less than 1d in vivo.

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2beta-substituted analogs of 14-epi-previtamin D(3) were synthesized for the first time by the thermal isomerization of the corresponding 14-epi-vitamin D3 that were available using coupling reaction between the A-ring phosphine oxide derived from a chiral epoxide and CD-ring cis-hydrindanone. The VDR binding affinity and transactivation activity of osteocalcin promoter in HOS cells were evaluated, and the new analogs were found to be less active, 0.01-0.18% of VDR binding affinity compared with the natural hormone and EC50 1.0-9.1 nM for transactivation activity, than 14-epi-previtamin D3 with 0.5% (VDR) and EC50 0.46 nM, respectively.

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Vitamin D plays an important role in regulating bone and calcium metabolism. The actions of vitamin D are mediated through the nuclear vitamin D receptor (VDR), and gene disruption of the VDR in mice causes skeletal disorders. However, the precise role of the VDR in each stage of osteoblastogenesis is not well understood. To address this issue, we used a biochemical approach to identify an osteoblast-specific coregulator of the VDR. Using a GST-fused VDR ligand-binding domain as bait, proteins associated with liganded VDR were purified from nuclear extracts of HOS osteoblastic cells and compared with those of HeLa cells. Among the interactants identified by mass fingerprinting, CCAAT displacement protein (CDP) was found as a novel ligand-dependent VDR interactant in HOS cells, together with other previously reported DRIP/TRAP complex components. Further biochemical analysis showed that complex formation between the VDR and CDP was distinct from the previously known DRIP/TRAP complex and the p160 family coactivator complexes. Transient expression of CDP potentiated VDR-mediated transcriptional activation in HOS cells. Furthermore, modulation of CDP expression levels in osteoblastic SaM-1 cells affected vitamin D-dependent osteoblast differentiation before the maturation (mineralization) stage. These findings suggest that CDP is a novel differentiation stage-specific coactivator of the VDR in osteoblasts.

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We synthesized the 4-hydroxy and 4-methoxy analogs of active vitamin D(3) (1alpha,25(OH)(2)D(3), 1) and its C14-epimer with the previtamin D(3) form of 14-epi-1alpha,25(OH)(2)preD(3) (14-epi-pre1). Their vitamin D receptor (VDR) binding affinity and osteocalcin promoter transactivation activity in HOS cells were evaluated, and had lower activity than the natural hormone (1) and 14-epi-pre1, respectively.

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The 14-epimer of MART-10, namely 14-epi-MART-10 (14-epi-2alpha-(3-hydroxypropyl)-1alpha,25-dihydroxy-19-norvitamin D3) and its 2-epimeric analog (14-epi-MART-11) were efficiently synthesized using the Julia coupling reaction to connect between the C5 and C6 positions (steroid numbering). An A-ring precursor was prepared from (-)-quinic acid as shown in the previous MART-10 synthesis. The novel 14-epi-CD-ring coupling partner with an elongated two carbon unit as a sulfone was synthesized from 14-epi-25-hydroxy Grundmann's ketone in good yield. The subsequent coupling reaction followed by a deprotection step afforded a mixture of 14-epi-MART-10 and 14-epi-MART-11 in 40% yield. To separate 14-epi-MART-10 and 14-epi-MART-11, each primary hydroxyl group was esterified with a pivaloyl group and the resulting pivalates 2alpha and 2beta were separated by high performance liquid chromatography. After the separation, the C2-stereochemistry of each (2alpha or 2beta) was determined by 1H NMR (nuclear magnetic resonance) studies including NOE (nuclear Overhauser effect) experiments. The pivaloyl group was removed under basic conditions to obtain the target molecules of 14-epi-MART-10 and 14-epi-MART-11, respectively. The VDR (vitamin D receptor)-binding affinity, HL-60 (human promyelocytic leukemia) cell differentiation activity, antiproliferative activity in PZ-HPV-7 (immortalized normal prostate) cells and transactivation activity of the osteocalcin promoter in HOS (human osteoblast cell line) cells (serum-free conditions) were investigated. In addition, the effects on bone mineral density (BMD) and the blood and urine calcium concentrations of ovariectomized (OVX) rats were examined. 14-epi-MART-10 has much greater antiproliferative and cell differentiation activities compared to 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3).

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We synthesized and isolated 2 alpha-substituted analogs of 14-epi-previtamin D3 after thermal isomerization at 80 degrees C for the first time. The VDR binding affinity and transactivation activity of osteocalcin promoter in HOS cells were evaluated, and the 2 alpha-methyl-substituted analog was found to have greater genomic activity than 14-epi-previtamin D3.

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