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Melatonin and serotonin, products of tryptophan metabolism, are endogenous neurotransmitters and hormones. We have identified and quantified these metabolites in natural honey from Australia, USA, and Poland using a Xevo G2 XS qTof LC–MS. To help ensure correct product identification, some samples were prepurified by RP-HPLC based on the retention times of standards, prior to LC–MS. The concentrations of the metabolites of interest depended on the source of the honey. For Australian honey, levels for melatonin and 2-hydroxymelatonin were 0.91 and 0.68 ng/g, respectively. Melatonin was detected in one brand of US commercial honey at 0.48 ng/g, while a second brand contained serotonin at 88.2 ng/g. In Polish natural honey, 20.6 ng/g of serotonin and 40.8 ng/g of N-acetylserotonin (NAS) were detected, while in Polish commercial honey 25.9 ng/g of serotonin and 7.30 ng/g of NAS were present. We suggest that addictive and health-related properties of honey may be in part dependent on the presence of serotonin, melatonin, and their metabolites, and that these compounds may play a role in the colony activities of bees.

Absorption of ultraviolet B (UVB) radiation by the B ring of 7-dehydrocholesterol (7-DHC) leads to the transformation of 7-DHC to previtamin D3, which after absorption of additional UVB isomerizes to tachysterol3 (T3) and lumisterol3 (L3). Previously we demonstrated that CYP11A1 can hydroxylate the side chain of vitamin D3 (D3), 7-DHC and L3. Similarly CYP27A1 can hydroxylate the side chain of L3 to biologically active hydroxyderivatives. In a continuation of these studies, we report that CYP11A1 and CYP27A1 hydroxylate T3 to 20S(OH)T3 and 25(OH)T3, respectively, plus minor unidentified hydroxyderivatives. Both 20S(OH)T3 and 25(OH)T3were detected in the human epidermis and serum. T3 was also detected in human serum and was present 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 . They acted on the vitamin D receptor (VDR) as demonstrated by image flow cytometry and the translocation of VDR-coupled GFP (VDR-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 revealed marked activation of AhR by 20S(OH)T3, a smaller effect by 25(OH)T3 and only minimal activation by T3. The T3 hydroxyderivatives showed high affinity binding to the ligand binding domain (LBD) of the liver X receptor (LXR) α and β, and to the peroxisome proliferator-activated receptor γ (PPARg) 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 previously characterized ligands. The scores for binding to the non-genomic site of the VDR were very low indicating a lack of interaction with hydroxy-T3 ligands. In conclusion, we have identified 20S(OH)T3 and 25(OH)T3 in the human epidermis and serum, identified CYP enzymes responsible for their production, demonstrated phenotypic effects on skin cells, and identified VDR, AhR, LXRs and PPARγ, and possibly RORs, as their genomic receptor targets. Thus CYP11A1, aside from its role in steroidogenesis, not only metabolizes vitamin D3, 7-DHC and L3 to biologically active metabolites, but also activates T3 to biologically active 20S(OH)T3. Similarly, CYP27A1, previously reported to act on L3, D3 and 7DHC, also activates T3 via hydroxylation at C25. We believe that these novel findings open new areas for future studies on the role of active forms of T3, not only in the skin, but also in systemic physiology and pathology. Presentation: Sunday, June 12, 2022 12:54 p.m. - 12:59 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

A new series of highly biologically active (20S,22R)-1α,25-dihydroxy-22-methyl- 2-methylene-vitamin D3 analogs, possessing different side chains, have been efficiently prepared as potential agents for medical therapy. Design of these synthetic targets was based on the analysis of the literature data and molecular docking experiments. The synthetic strategy involved Sonogashira coupling of the known A-ring dienyne with the C,D-ring enol triflates, obtained from the corresponding Grundmann ketones. All synthesized vitamin D compounds were characterized by high in vitro potency and, moreover, they proved to be very calcemic in vivo exerting high activity on bone with particularly elevated intestinal calcium transport.

New and more efficient routes of chemical synthesis of vitamin D(3) (D(3)) hydroxy (OH) metabolites, including 20S(OH)D(3), 20S,23S(OH)(2)D(3) and 20S,25(OH)(2)D(3), that are endogenously produced in the human body by CYP11A1, and of 20S,23R(OH)(2)D(3) were established. The biological evaluation showed that these compounds exhibited similar properties to each other regarding inhibition of cell proliferation and induction of cell differentiation but with subtle and quantitative differences. They showed both overlapping and differential effects on T-cell immune activity. They also showed similar interactions with nuclear receptors with all secosteroids activating vitamin D and aryl hydrocarbon receptors in functional assays and also as indicated by molecular modeling. They functioned as substrates for CYP27B1 with enzymatic activity being the highest towards 20S,25(OH)(2)D(3) and the lowest towards 20S(OH)D(3). In conclusion, defining new routes for large scale synthesis of endogenously produced D(3)-hydroxy derivatives by pathways initiated by CYP11A1 opens an exciting era to analyze their common and differential activities in vivo, particularly on the immune system and inflammatory diseases.

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.

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.

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.

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.

On the basis of the literature data, our previous research work and docking experiments, we designed novel 19-norvitamin D compounds having elongated 2-alkylidene substituents. These 19-norcalcitriol derivatives have attached 2-(3'-aminopropylidene) substituent in which the nitrogen atom bears acyl residue derived from succinic acid and l-methionine. Both compounds were obtained by the same synthetic strategy involving Julia coupling of the A-ring ketone with the known C/D-ring sulfone. In the obtained 1α,25-dihydroxy-19-norvitamin D3 derivative, the alkylidene substituent at C-2 was further elaborated to the desired structures.