20(
)-Hydroxyvitamin D3 (20(OH)D3) is an endogenous metabolite produced by the action of CYP11A1 on the side chain of vitamin D3 (D3). 20(OH)D3 can be further hydroxylated by CYP11A1, CYP27A1, ...CYP24A1 and/or CYP27B1 to several hydroxyderivatives. CYP11A1 also hydroxylates D3 to 22-monohydroxyvitamin D3 (22(OH)D3), which is detectable in the epidermis. 20-Hydroxy-7-dehydrocholesterol (20(OH)-7DHC) has been detected in the human epidermis and can be phototransformed into 20(OH)D3 following the absorption of ultraviolet B (UVB) energy by the B-ring. 20(OH)D3 and its hydroxyderivatives have anti-inflammatory, pro-differentiation and anti-proliferative effects, comparable to 1,25-dihydroxyvitamin D3 (1,25(OH)
D3). Since cytochromes P450 with 20- or 25-hydroxylase activity are found in insects participating in ecdysone synthesis from 7-dehydrocholesterol (7DHC), we tested whether D3-hydroxyderivatives are present in honey, implying their production in bees. Honey was collected during summer in the Birmingham area of Alabama or purchased commercially and extracted and analyzed using LC-MS. We detected a clear peak of
/
= 423.324 M + Na
for 20(OH)D3 corresponding to a concentration in honey of 256 ng/g. We also detected peaks of
/
= 383.331 M + H - H
O
for 20(OH)-7DHC and 25(OH)D3 with retention times corresponding to the standards. We further detected species with
= 407.329 M + Na
corresponding to the RT of 7DHC, D3 and lumisterol3 (L3). Similarly, peaks with
= 399.326 M + H - H
O
were detected at the RT of 1,25(OH)
D3 and 1,20-dihydroxyvitamin D3 (1,20(OH)
D3). Species corresponding to 20-monohydroxylumisterol3 (20(OH)L3), 22-monohydroxyvitamin D3 (22(OH)D3), 20,23-dihydroxyvitamin D3 (20,23(OH)
D3), 20,24/25/26-dihydroxyvitamin D3 (20,24/25/26(OH)
D3) and 1,20,23/24/25/26-trihydroxyvitamin D3 (1,20,23/24/25/26(OH)
D3) were not detectable above the background. In conclusion, the presence of 7DHC and D3 and of species corresponding to 20(OH)-7DHC, 20(OH)D3, 1,20(OH)
D3, 25(OH)D3 and 1,25(OH)
D3 in honey implies their production in bees, although the precise biochemistry and photochemistry of these processes remain to be defined.
Continuing our structure-activity studies on the vitamin D analogs with the altered intercyclic seco-B-ring fragment, we designed compounds possessing dienyne system conjugated with the benzene D ...ring. Analysis of the literature data and the docking experiments seemed to indicate that the target compounds could mimic the ligands with a good affinity to the vitamin D receptor (VDR). Multi-step synthesis of the C/D-ring building block of the tetralone structure was achieved and its enol triflate was coupled with the known A-ring fragments, possessing conjugated enyne moiety, using Sonogashira protocol. The structures of the final products were confirmed by NMR, UV and mass spectroscopy. Their binding affinities for the full-length human VDR were determined and it was established that compound substituted at C-2 with exomethylene group showed significant binding to the receptor. This analog was also able to induce monocytic differentiation of HL-60 cells.
CYP11A1 and CYP27A1 hydroxylate tachysterol3, a photoproduct of previtamin D3, producing 20S‐hydroxytachysterol3 20S(OH)T3 and 25(OH)T3, respectively. Both metabolites were detected in the human ...epidermis and serum. Tachysterol3 was also detected in human serum at a concentration of 7.3 ± 2.5 ng/ml. 20S(OH)T3 and 25(OH)T3 inhibited the proliferation of epidermal keratinocytes and dermal fibroblasts and stimulated the expression of differentiation and anti‐oxidative genes in keratinocytes in a similar manner to 1,25‐dihydroxyvitamin D3 1,25(OH)2D3. They acted on the vitamin D receptor (VDR) as demonstrated by image flow cytometry and the translocation of VDR coupled GFP from the cytoplasm to the nucleus of melanoma cells, as well as by the stimulation of CYP24A1 expression. Functional studies using a human aryl hydrocarbon receptor (AhR) reporter assay system revealed marked activation of AhR by 20S(OH)T3, a smaller effect by 25(OH)T3, and a minimal effect for their precursor, tachysterol3. Tachysterol3 hydroxyderivatives showed high‐affinity binding to the ligan‐binding domain (LBD) of the liver X receptor (LXR) α and β, and the peroxisome proliferator‐activated receptor γ (PPARγ) in LanthaScreen TR‐FRET coactivator assays. Molecular docking using crystal structures of the LBDs of VDR, AhR, LXRs, and PPARγ revealed high docking scores for 20S(OH)T3 and 25(OH)T3, comparable to their natural ligands. The scores for the non‐genomic‐binding site of the VDR were very low indicating a lack of interaction with tachysterol3 ligands. Our identification of endogenous production of 20S(OH)T3 and 25(OH)T3 that are biologically active and interact with VDR, AhR, LXRs, and PPARγ, provides a new understanding of the biological function of tachysterol3.
The biologically active metabolite of vitamin D3 - calcitriol – is a hormone involved in the regulation of calcium-phosphate homeostasis, immunological processes and cell differentiation, being ...therefore essential for the proper functioning of the human body. This suggests many applications of this steroid in the treatment of diseases such as rickets, psoriasis and some cancers. Unfortunately, using therapeutic doses of calcitriol is associated with high concentrations of this compound which causes hypercalcemia. For this reason, new calcitriol analogs are constantly sought, devoid of calcemic effects but maintaining its beneficial properties. In this study, we present the synthesis of vitamin D derivatives characterized by an enlarged (seven-membered) ring D. Preparation of the designed vitamin D compounds required separate syntheses of crucial building blocks (C/D-rings fragments with side chain and rings A) which were combined by different methods, including Wittig-Horner reaction and Suzuki coupling. Biological activities of the target vitamin D analogs were assessed both in vitro and in vivo, demonstrating their significant potency compared to the natural hormone. Furthermore, the successful crystallization of these compounds with the vitamin D receptor (VDR) enabled us to investigate additional molecular interactions with this protein.
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•First total synthesis of vitamin D3 analogs with 7-membered ring D.•X-ray crystal structures of zVDR LBD complexes of all synthesized analogs.•In vitro activity of all analogs in lung, breast and leukemia cell lines.•Synthesized analogs are active in stimulating calcium homeostasis in vivo.
The biologically active metabolite of vitamin D
- calcitriol - is a hormone involved in the regulation of calcium-phosphate homeostasis, immunological processes and cell differentiation, being ...therefore essential for the proper functioning of the human body. This suggests many applications of this steroid in the treatment of diseases such as rickets, psoriasis and some cancers. Unfortunately, using therapeutic doses of calcitriol is associated with high concentrations of this compound which causes hypercalcemia. For this reason, new calcitriol analogs are constantly sought, devoid of calcemic effects but maintaining its beneficial properties. In this study, we present the synthesis of vitamin D derivatives characterized by an enlarged (seven-membered) ring D. Preparation of the designed vitamin D compounds required separate syntheses of crucial building blocks (C/D-rings fragments with side chain and rings A) which were combined by different methods, including Wittig-Horner reaction and Suzuki coupling. Biological activities of the target vitamin D analogs were assessed both in vitro and in vivo, demonstrating their significant potency compared to the natural hormone. Furthermore, the successful crystallization of these compounds with the vitamin D receptor (VDR) enabled us to investigate additional molecular interactions with this protein.
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•A-Ring fragment’s synthesis started from cyclohexanone derived from quinic acid.•19-Norcalcitriol analogs with pegylated 2-alkylidene groups were synthesized.•2-Alkylidene chain is ...terminated by hydroxyl or 2-(pyridin-2′-yl)ethylamino group.•The affinities of the synthesized 19-norcalcitriol analogs to VDR were assessed.
The results presented in this paper constitute an extension of our synthetic efforts focused on 19-norvitamin D compounds possessing elongated 2-alkylidene substituents. Based on our previous results, molecular modeling studies, and docking experiments, we selected a novel 19-norcalcitriol analog with long chain at C-2 containing several ether moieties and terminated by 2-(pyridin-2′-yl)ethylamino fragment. It was expected that such structural modification might allow binding of transition metal by the ligand, increase solubility of the formed complexes as well as improve their affinity to the VDR. For comparison, a 19-norcalcitriol analog was also obtained with the terminal hydroxyl group at its pegylated 2-alkylidene substituent. The synthesis of the target vitamin D compounds described in this work was performed using the Wittig-Horner approach. The respective A-ring phosphine oxide was obtained starting from the D-(-)-quinic acid and then coupled with the known Grundmann ketone.
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•Starting from the natural compounds synthesis of the building blocks was achieved.•19-Norcalcitriol analog with elongated side chain obtained by Wittig-Horner coupling.•The side ...chain of the synthesized analog can protrude from the VDR binding pocket.
Pronounced biological potency of 19-norvitamin D compounds as well as interesting biological action of the vitamin D analogs possessing elongated side chains encouraged us to expand the scope of our structure-activity studies to encompass such modifications of the 1α,25-(OH)2D3 (calcitriol) molecule. The aim of our studies was the synthesis of calcitriol analog, designed on the basis of results of molecular modeling and docking experiments, and characterized by a presence of a long, nitrogen-containing substituent attached to carbon 26, and an exomethylene moiety transferred from C-10 to C-2. The convergent synthesis of such 19-norcalcitriol compound, described in this communication, consisted of the preparation and combining four building blocks. The crucial point of the synthesis, coupling of the known A-ring phosphine oxide and the synthesized Grundmann ketone analog, was achieved using Wittig-Horner protocol. It provided the protected analog of 1α,25-dihydroxy-2-methylene-19-norvitamin D3 which was further transformed into the target compound.
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•The building blocks required for the synthesis were prepared from natural compounds.•Epimeric at C-25 Gemini compounds with elongated side chains were synthesized.•Conjugate-type ...Pt(II) complexes of vitamin D analogs were obtained for the first time.•Cytotoxic activities of the synthesized 19-norcalcitrol analogs were evaluated.
Hormonally active vitamin D3 metabolite, calcitriol, plays an important role in calcium-phosphate homeostasis, immune system actions and cell differentiation. Although anticancer activity of calcitriol is well documented and thousands of its analogs have been synthesized, none has been approved as a potential drug against cancer. Therefore, we attempted to introduce the cytotoxic effect to the calcitriol molecule by its linking to cisplatin. Herein, we present the synthesis of vitamin D compounds, designed on the basis of molecular modeling and docking experiments to the vitamin D receptor, and characterized by the presence of significantly different two side chains attached to C-20. In this study, a new synthetic approach to Gemini analogs was developed. Preparation of the target 19-norcalcitriol compounds involved separate syntheses of several building blocks (the A-ring, C/D-rings and side-chain fragments). The convergent synthetic strategy was used to combine these components by the different coupling processes, the crucial one being Wittig-Horner reaction of the Grundmann ketone analog with the known 2-methylene A-ring phosphine oxide. Due to the nature of the constructed steroidal side chains (bidentate ligands), which allowed coordination of metal ions, the first conjugate-type platinum(II) complexes of the vitamin D analogs were also successfully prepared and characterized. The target vitamin D compounds, displaying significant affinity for a vitamin D receptor, were assessed in vitro for their anti-proliferative activities towards several cell lines.
CYP11A1 and CYP27A1 hydroxylate tachysterol
, a photoproduct of previtamin D
, producing 20S-hydroxytachysterol
20S(OH)T
and 25(OH)T
, respectively. Both metabolites were detected in the human ...epidermis and serum. Tachysterol
was also detected in human serum at a concentration of 7.3 ± 2.5 ng/ml. 20S(OH)T
and 25(OH)T
inhibited the proliferation of epidermal keratinocytes and dermal fibroblasts and stimulated the expression of differentiation and anti-oxidative genes in keratinocytes in a similar manner to 1,25-dihydroxyvitamin D
1,25(OH)
D
. They acted on the vitamin D receptor (VDR) as demonstrated by image flow cytometry and the translocation of VDR coupled GFP from the cytoplasm to the nucleus of melanoma cells, as well as by the stimulation of CYP24A1 expression. Functional studies using a human aryl hydrocarbon receptor (AhR) reporter assay system revealed marked activation of AhR by 20S(OH)T
, a smaller effect by 25(OH)T
, and a minimal effect for their precursor, tachysterol
. Tachysterol
hydroxyderivatives showed high-affinity binding to the ligan-binding domain (LBD) of the liver X receptor (LXR) α and β, and the peroxisome proliferator-activated receptor γ (PPARγ) in LanthaScreen TR-FRET coactivator assays. Molecular docking using crystal structures of the LBDs of VDR, AhR, LXRs, and PPARγ revealed high docking scores for 20S(OH)T
and 25(OH)T
, comparable to their natural ligands. The scores for the non-genomic-binding site of the VDR were very low indicating a lack of interaction with tachysterol
ligands. Our identification of endogenous production of 20S(OH)T
and 25(OH)T
that are biologically active and interact with VDR, AhR, LXRs, and PPARγ, provides a new understanding of the biological function of tachysterol
.
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•Series of 19-norcalcitriol analogs with pegylated 2-alkylidene groups were synthesized.•Pt(II) complexes of two vitamin D analogs were obtained and biologically tested.•Cytotoxic ...activity of the synthesized 19-norcalcitriol analogs was assessed.
Continuing our studies aimed at A-ring modified vitamin D compounds, we designed novel 19-norcalcitriol derivatives bearing at C-2 pegylated chains of different lengths. The terminal fragments of these substituents contain hydroxyls or moieties possessing nitrogen and/or sulfur atoms capable of transition metal ions complexation. Also, two conjugate–type platinum(II) complexes of 19-norcalcitriol were obtained in which l-methionine served as chelating moiety. The convergent synthesis of the target 19-norcalcitriol analogs involved several steps with the crucial one being condensation of A-ring phosphine oxide and the known Grundmann ketone by Wittig-Horner reaction. Further elaboration of the 2-alkylidene substituent provided all final compounds which were then tested to determine their affinity for the vitamin D receptor and cytotoxic activity.
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