Here we report the synthesis of new PNA monomers for pseudocomplementary PNA (pcPNA) that are fully compatible with standard Fmoc chemistry. The thiocarbonyl group of the 2-thiouracil (sU) monomer was protected with the 4-methoxy-2-methybenzyl group (MMPM), while the exocyclic amino groups of diaminopurine (D) were protected with Boc groups. The newly synthesized monomers were incorporated into a 10-mer PNA oligomer using standard Fmoc chemistry for solid-phase synthesis. Oligomerization proceeded smoothly and the HPLC and MALDI-TOF MS analyses indicated that there was no remaining MMPM on the sU nucleobase. The new PNA monomers reported here would facilitate a wide range of applications, such as antigene PNAs and DNA nanotechnologies.Download high-res image (70KB)Download full-size image

•Two novel 7,8-cis-14-epi-1α,25-dihydroxy-19-norvitamin D3 analogs were synthesized.•HVDR binding affinity and osteocalcin promoter transactivation activity were tested.•2β-Substituted analog showed higher binding affinity for hVDR than the 2α-isomer.According to the binding mode of 14-epi-1α,25-dihydroxy-19-nortachysterol in the ligand binding domain of human vitamin D receptor (hVDR), i.e., 5,6- and 7,8-s-trans configuration that was shown by X-ray co-crystallographic analysis, 7,8-cis-locked 1α,25(OH)2D3 analogs were synthesized. In this paper, the synthesis and biological activity of 2α- and 2β-(3-hydroxypropyl)-7,8-cis-14-epi-1α,25-dihydroxy-19-norvitamin D3 are reported. The A-ring and CD-ring precursors for the Julia-Kociensky coupling reaction to create a diene system of the target molecules were prepared using our original methods. hVDR binding affinity and osteocalcin promoter transactivation activity of the new 7,8-cis-14-epi-vitamin D3 analogs were evaluated. Interestingly, the 2β-substituted 7,8-cis-analog was a better binder for hVDR than the 2α-isomeric counterpart.Download high-res image (136KB)Download full-size image

•Active vitamin D analog AH-1 was converted to the major metabolite M1 by CYP24A1.•M1 was chemically synthesized to determine the (24R)-OH stereochemistry.•The M1 diastereomer with (24S)-OH was also synthesized to compare their characters.Previously, we found that 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-dihydroxyvitamin D3 (AH-1) showed higher osteocalcin promoter transactivation activity in human osteosarcoma (HOS) cells and a greater therapeutic effect on ovariectomized (OVX) rats for enhancing bone mineral density than those of 1α,25(OH)2D3 without hypercalcemic side effects in vivo. Although CYP24A1 catalyzes multi-step oxidations toward the CD-ring side chain of the active vitamin D3 [1α,25(OH)2D3], the CYP24A1-dependent metabolism of AH-1 tended to stop at the first step hydroxylation at the C24-position of AH-1. Interestingly, the metabolite 24-hydroxy-AH-1 [24(OH)AH-1] showed potent VDR binding affinity, and the new chiral center of the 24-position might be the 24R configuration compared with the process of the natural 1α,25(OH)2D3 catabolism. This time, (24R)-2α-[2-(tetrazol-2-yl)ethyl]-1α,24,25-trihydroxyvitamin D3 [(24R-OH)AH-1] was synthesized as a candidate for the major metabolite of AH-1 using the Trost Pd-mediated coupling reaction between A-ring and CD-ring precursors to identify the metabolite and evaluate its biological activity. We confirmed that the CYP24A1-dependent major metabolite of AH-1 was (24R-OH)AH-1 by HPLC analyses.Download high-res image (94KB)Download full-size image

Four 7,8-cis-1α,25-dihydroxyvitamin D3 derivatives, 7,8-cis- and 7,8-cis-14-epi-1α,25-dihydroxy-19-norvitamin D3 as well as 7,8-cis- and 7,8-cis-14-epi-1α,25-dihydroxyvitamin D3 were synthesized, and their chemical stability was characterized. In our previous work, we disclosed that 14-epi-19-nortachysterol showed the unprecedented binding configuration in human vitamin D receptor (hVDR), that is, 5,6- and 7,8-s-trans configuration. However, this configuration is variable because of the rotation at the single bond between C7 and C8. For the precise discussion of the 7,8-s-trans configuration, we designed and synthesized the 7,8-cis-locked skeleton of vitamin D3 derivatives. Among four analogs, the 19-nor derivatives were stable at ambient temperature, and their hVDR binding affinity and co-crystallographic analysis of their hVDR complexes were studied. The other derivatives with the triene system were isomerized to corresponding previtamin D3 and vitamin D3.Vitamin D twistable?: 7,8-cis-19-norvitamin D3 with a 14-epi-19-nortachysterol structure is stable with keeping tiny receptor binding affinity, and 7,8-cis-vitamin D3 prefers to take the natural 1α,25-dihydroxyvitamin D3 skeleton.

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.Keywords: bone mineral density; in vivo antiosteoporotic effect; osteocalcin; Vitamin D; vitamin D receptor; X-ray cocrystallographic analysis;

In the study of the synthesis of 14-epi-19-norprevitamin D3, we found 14-epi-19-nortachysterol derivatives through C6,7-cis/trans isomerization. We also succeeded in their chemical synthesis and revealed their marked stability and potent VDR binding affinity. To the best of our knowledge, this is the first isolation of stable tachysterol analogues. Surprisingly, 14-epi-19-nortachysterol derivatives exhibited an unprecedented binding configurations for the ligand binding pocket in hVDR, C5,6-s-trans and C7,8-s-trans triene configurations, which were opposite the natural C7,8-ene-configuration of 1α,25(OH)2D3.

C15-Substituted 1α,25-dihydroxyvitamin D3 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.

A new vitamin D3 analogue, 2α-propoxy-1α,25-dihydroxyvitamin D3 (C3O1), was 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 studied the metabolism of the new analogue by human CYP24A1 and rat CYP24A1 to learn of species-based differences and found that the former has multiple metabolic pathways, but the latter has only a single pathway.Human CYP24A1 has multiple metabolic pathways and rat CYP24A1 has a single metabolic pathway for a highly active 2α-(3-hydroxyprpoxyl)-1α,25-dihydroxyvitamin D3 precursor C3O1.

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

We designed and synthesized 1α- and 1β-hydroxymethyl-2α-(3-hydroxypropyl)-25-hydroxyvitamin D3 (2a,b) and related analogues 2α-(3-hydroxypropyl)-25-hydroxyvitamin D3 (3), Posner’s analogues of 1α- and 1β-hydroxymethyl-25-hydroxyvitamin D3 (4a,b), as well as 2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D3 (5), to confirm the effect of the 1α-hydroxy group and/or 2α-(3-hydroxypropyl) group of vitamin D3 analogues with the modified A-ring moiety on the mutant vitamin D receptor, VDR(Arg274Leu). The 2α-(3-hydroxypropyl) group showed better effect on enhancement of the transcriptional activity through the mutant VDR than the 1α- and 1β-hydroxymethyl groups.

Novel 2α-methyl-, 2α-(3-hydroxypropyl)- and 2α-(3-hydroxypropoxy)-substituted 25-dehydro-1α-hydroxyvitamin D3-26,23-lactone derivatives were efficiently synthesized via Reformatsky type allylation and palladium-catalyzed alkenylative cyclization processes, and their biological activities were evaluated. Introducing functional groups into the 2α-position of the vitamin D3-26,23-lactones resulted in remarkable enhancement of their antagonistic activity on vitamin D receptor (VDR).

Novel A-ring analogues of the vitamin D receptor (VDR) antagonist (3a), ZK-159222, and its 24-epimer (3b) were convergently synthesized. Preparation of the CD-ring portions with the side chains of 3a,b, followed by palladium-catalyzed cross-coupling with the A-ring enyne precursors (15a,b), (3S,4S,5R)- and (3S,4S,5S)-bis[(tert-butyldimethylsilyl)oxy]-4-methyloct-1-en-7-yne, afforded the 2α-methyl-introduced analogues (4a,b) and their 3-epimers (5a,b). The biological profiles of the hybrid analogues were assessed in terms of affinity for VDR, and antagonistic activity to inhibit HL-60 cell differentiation induced by the natural hormone, 1α,25-dihydroxyvitamin D3. The analogue 4a showed an approximately fivefold higher antagonistic activity compared with 3a. The 2α-methyl introduction into 3a increased the receptor affinity, thereby enhancing VDR antagonism. This approach to design potent antagonists based on hybridization of structural motifs in the A-ring and in the side chain may prove to be valuable.Graphic

We found a concise route to the Trost A-ring precursor enyne for synthesizing 2α-alkylated 1α,25-dihydroxyvitamin D3 (1) from d-glucose. The enynes were coupled with the 20-epi-CD ring part to study the effect of the double modification of 2α-substitution and 20-epimerization upon biological activities of 1. The novel three analogues of 2α-alkyl- and four analogues of 2α-(ω-hydroxyalkyl)-20-epi-1α,25-dihydroxyvitamin D3 (5b–d and 6a–d) showed higher binding affinity for vitamin D receptor (VDR) and more potent activity in induction of HL-60 cell differentiation than those of the natural hormone 1.The novel three analogues of 2α-alkyl- and four analogues of 2α-(ω-hydroxyalkyl)-20-epi-1α,25-dihydroxyvitamin D3 showed higher binding affinity for vitamin D receptor (VDR) and more potent activity in induction of HL-60 cell differentiation than those of the natural hormone.

We here show that single-stranded oligonucleotides containing 5-formyl-2′-deoxyuridine (fdU) can crosslink the peptides derived from the DNA binding site of RecA protein through a Schiff base formation. The ability of crosslinking of fdU-containing oligonucleotides was investigated using a series of peptides whose amino acid residues spanning the center of the RecA-derived peptide were sequentially replaced with lysine. Circular dichroism (CD) spectroscopy, gel mobility shift assay and sedimentation experiment demonstrated that crosslinking reaction proceeded efficiently only when the peptides bound to the oligonucleotides.Schiff base-triggered aggregation of RecA-derived peptides onto fdU-containing ssDNA is reported.

•We synthesized four novel 2-[3-(tetrazolyl)propyl]-1α,25-dihydroxy-19-norvitamin D3.•Only 2α-[3-(tetrazol-1-yl)propyl] analog showed weak binding affinity for hVDR.•Metabolic stability of 2α-substituted analogs was higher than that of 2β-ones.Recently, we found that 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 in vivo than those of active vitamin D3, 1α,25(OH)2D3. We were interested in introducing a heterocyclic ring to the C2 position of the seco-steroidal structure via an alkyl linker, and four novel C2-(3-tetrazolylpropyl) substituted 1α,25-dihydroxy-19-norvitamin D3 analogs, 2α-[3-(tetrazol-1-yl)propyl]-, 2β-[3-(tetrazol-1-yl)propyl]-, 2α-[3-(tetrazol-2-yl)propyl]-, and 2β-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 were synthesized. Among them, 2α-[3-(tetrazol-1-yl)propyl]-19-nor-1α,25(OH)2D3 showed weak binding affinity for human vitamin D receptor (hVDR) (2.6% of 1α,25(OH)2D3 and ca. 15% of 19-nor-1α,25(OH)2D3) and weak VDR transactivation activity in HOS cells (EC50 7.3 nM, when 1α,25(OH)2D3 0.23 nM). Although the other three compounds could not act as VDR binders by evaluation of the competition assays, 2α-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 showed weak transactivation activity (EC50 12.5 nM). Metabolic stability of the 2α-substituted compounds 2α-[3-(tetrazol-1-yl)propyl]- and 2α-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 was higher than that of the 2β-substituted counterparts 2β-[3-(tetrazol-1-yl)propyl]- and 2β-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 against human CYP24A1. Introduction of a tetrazole ring to the C2-position of the 19-norvitamin D3 skeleton with the propyl linker led to weak VDR agonistic activity with stability against CYP24A1 metabolism.Download high-res image (172KB)Download full-size image

We report new analogs of 1α,25-dihydroxyvitamin D3 (1) in three categories. First, design and synthesis of ligands for a mutant vitamin D receptor (VDR)(Arg274Leu), which possess proper functional groups at both C1α and C2α positions of 1 to study the biological activity of the mutant VDR. Among our synthetic analogs, 1α-methyl-2α-(3-hydroxypropyl)-25-hydroxyvitamin D3 (8) showed 7.3-fold greater transcriptional activity for the VDR(Arg274Leu) than that of 1. Next, we examined the antiproliferative activity of 2-substituted 19-norvitamin D3 analogs on an immortalized normal prostate cell line, PZ-HPV-7, and we found MART 10 (14) showed the activity even at very low concentration of 10−10 to 10−11 M. We also synthesized 25-hydroxy-19-norvitamin D3 (13) using Julia-type olefination to connect between the C5 and C6 positions, effectively, to test it as a prohormone type agent for antiprostate diseases. Synthesized compound 13 showed potent antiproliferative activity in PZ-HPV-7, which has high 1α-hydroxylase activity. Finally, we describe design and synthesis of a new TEI-9647 analog, 2α-(3-hydroxypropoxy)-24-propyl-25-dehydro-1α-hydroxyvitamin D3-26,23-lactone (17), which showed the strongest VDR antagonism. Its IC50 value is 7.4 pM to inhibit differentiation of HL-60 cells induced by 10 nM of 1.

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’.Highlights▸ We synthesized a new stable 14-epi-19-nortachysterol derivative. ▸ We tested hVDR binding affinity of the 2α-substituted analog. ▸ The 2α-(3-hydroxypropoxy) group showed negative effects on the binding affinity for the hVDR.

2β-Substituted analogs of 14-epi-previtamin D3 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.

2β-Substituted analogs of 14-epi-previtamin D3 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.