Rafal R. Sicinski

1α,25-dihydroxy-19-nor-vitamin D3, a novel vitamin D-related compound with potential therapeutic activity

1α,25-Dihydroxy-19-nor-vitamin D3 has been synthesized via oxidative degradation of the 1α-hydroxycylcovitamin intermediate. Preliminary studies indicate that the new analog induces the differentiation of human leukemia HL-60 cells, with little or no calcemic activity.

A ligand for the aryl hydrocarbon receptor isolated from lung.

The aryl hydrocarbon receptor (AHR) is a ligand-inducible transcription factor that is best known because it mediates the actions of polycyclic and halogenated aromatic hydrocarbon environmental toxicants such as 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. We report here the successful identification of an endogenous ligand for this receptor; ≈20 μg was isolated in pure form from 35 kg of porcine lung. Its structure was deduced as 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester from extensive physical measurements and quantum mechanical calculations. In a reporter gene assay, this ligand activates the AHR with a potency five times greater than that of β-naphthoflavone, a prototypical synthetic AHR ligand. 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester competes with 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin for binding to human, murine, and fish AHRs, thus showing that AHR activation is caused by direct receptor binding, and that recognition of this endogenous ligand is conserved from early vertebrates (fish) to humans.

New highly calcemic 1α,25-dihydroxy-19-norvitamin D3 compounds with modified side chain: 26,27-dihomo- and 26,27-dimethylene analogs in 20S-series

New highly potent 2-substituted (20S)-1 alpha,25-dihydroxy-19-norvitamin D(3) analogs with elongated side chain were prepared by Wittig-Horner coupling of A-ring phosphine oxide with the corresponding protected (20S)-25-hydroxy Grundmanns ketones. Biologic evaluation in vitro and in vivo of the synthesized compounds was accomplished. All the synthesized vitamins possessing a 25-hydroxylated saturated side chain were slightly less active (3-5X) than 1 alpha,25-dihydroxyvitamin D(3) in binding to the porcine intestinal vitamin D receptor and significantly more potent (12-150X) in causing differentiation of HL-60 cells. In vivo, 2-methylene-26,27-dihomo and 2 alpha-methyl-26,27-dimethylene analogs were at least 10 times more active, and 2 alpha-methyl-26,27-dihomo compound at least 5 times more active than the vitamin D hormone both in stimulating intestinal calcium transport and bone calcium mobilization (serum calcium increase). It was also established that a 260 pmol dose of the corresponding 2 beta-methyl analogs had a similar effect on intestinal calcium transport and a much more pronounced effect on bone calcium mobilization as the same dose of 1 alpha,25-dihydroxyvitamin D(3).

Removal of the 20-methyl group from 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD) selectively eliminates bone calcium mobilization activity

The 18-nor (7), 21-nor (8) and 18,21-dinor (9) analogs of (20S)-1alpha,25-dihydroxy-2-methylene-19-norvitamin D(3) (6, 2MD) were prepared by convergent syntheses. The known phosphine oxide 10 was coupled by the Wittig-Horner process with the corresponding C,D-fragments (13-15), obtained by a multi-step procedure from commercial vitamin D(2). The goal of our studies was to examine the influence of removal of the methyl groups located at carbons 13 and 20 on the biological potency of 2MD in the hope of finding analogs with improved therapeutic profiles. Replacement of the 20-methyl with hydrogen in 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D(3) (2MD) did not affect binding to the rat vitamin D receptor and had little effect on transcription activity and on HL-60 differentiation. However, the mobilization of calcium from bone was largely eliminated while intestinal calcium transport remained strong. Curiously, removal of both the C-13-methyl and 20-methyl restored slightly the bone calcium mobilizing activity. Thus, the 21-nor analog of 2MD may provide a potent analog with a greater margin of safety than 2MD.

Synthesis of 1α,25-dihydroxyvitamin D2, its 24 epimer and related isomers, and their binding affinity for the 1,25-dihydroxyvitamin D3 receptor

Abstract The synthesis of 1α-25-dihydroxyvitamin D2 and of several stereoisomers (5,6-trans and 1β-hydroxy isomers and the 24R-epimers of these compounds) was reported. Synthesis was accomplished from two different starting materials, 25-hydroxyvitamin D2 and 25,25-ethylenedioxy-26-norvitamin D2, and involved C-1-hydroxylation via 3,5-cyclovitamin D intermediates. Synthetic 1α,25-dihydroxyvitamin D2 was found to be identical with the biologically generated natural product. An analysis of the binding affinity of the synthetic products for the 1α,25-dihydroxyvitamin D3 receptor protein showed that isomerization of the 5,6 double bond from cis to trans, or epimerization of the 24-methyl group from S to R, reduced ligand binding to the receptor only slightly, while both changes together led to a 100-fold reduction of binding affinity. The epimerization of the 1-hydroxy function from 1α to 1β attenuated binding dramatically (ca. 1000-fold).

New 1α,25-Dihydroxy-19-norvitamin D3 Compounds Constrained in a Single A-Ring Conformation: Synthesis of the Analogues by Ring-Closing Metathesis Route and Their Biological Evaluation

Vitamin D compounds possessing A rings prohibited from flipping to the alternative chair form (i.e., analogues 2 and 26) were synthesized. The bicyclic fragment 22 consisting of the fused cyclohexane and dihydropyran rings was constructed via the ring-closing metathesis route. Also, a homologous synthon 23 with an attached dihydropyran ring was successfully synthesized using this strategy. The carbonyl deprotection in 22 yielded cyclohexanone 5 that was subjected to Julia coupling with the anion of the phenylthiazoline sulfone 25. In the resulting isomeric 19-norvitamins 2 and 26, their A rings can exist only in the alpha- and beta-conformation. The analogue 26 was 300 times more active in binding to the vitamin D receptor protein, 30 times more effective in causing HL-60 differentiation, and 10 times more active in transcription. These results confirm that the beta-chair form of the vitamin D ring A is necessary for the binding to the receptor.

Design, Synthesis, and Biological Evaluation of a 1α,25-Dihydroxy-19-norvitamin D3 Analogue with a Frozen A-Ring Conformation

To establish the conformation of vitamin D compounds responsible for biological activity, a 1alpha,25-dihydroxy-19-norvitamin D analogue 4 possessing a 1alpha-hydroxy group fixed in the axial orientation (beta-chair form) was synthesized. The starting compounds were bicyclic lactones 6, 7a, and 7b, derived from the quinic acid lactone, which were converted to the bicyclic ketone 13. Julia coupling of this compound with sulfone 15 produced the 19-norvitamin D analogue 4, possessing an additional ring connecting the 3beta-oxygen and C-2, and the isomeric 3beta-hydroxy compound 5. In vitro, both analogues 4 and 5 exhibit reduced activity compared to the natural hormone 1, but the binding, differentiation, and transcriptional activities of analogue 5 are markedly higher than that of 4 constrained in the alpha-chair conformation. Surprisingly, in vivo tests in mice showed that the analogue 4 significantly increases serum calcium at dose levels similar to 1alpha,25-(OH)2D3. These seemingly discordant results are discussed.

1-Desoxy analog of 2MD: Synthesis and biological activity of (20S)-25-hydroxy-2-methylene-19-norvitamin D3

During our ongoing structure-activity studies in the vitamin D area, we obtained (20S)-1alpha,25-dihydroxy-2-methylene-19-norvitamin D3 (5). This analog, designated 2MD, is characterized by a significantly enhanced calcemic activity and is currently evaluated as a potential drug for osteoporosis. Therefore, it was of interest to synthesize also its 1-desoxy analog and to evaluate its biological action. These studies were aimed at solving an intriguing problem: can such a vitamin also be hydroxylated in vivo at the allylic C-1 position despite lack of the exomethylene moiety at C-10? The Wittig-Horner coupling of the known protected (20S)-25-hydroxy Grundmann ketone 17 and the phosphine oxides 16 and 33, differing in their hydroxyls protection, provided the target 1-desoxy-2MD (6) after removal of the silyl protecting groups. Two synthetic paths have been elaborated leading to the desired A-ring synthons and starting from commercially available compounds: 1,4-cyclohexanedione monoethylene acetal (7) and (-)-quinic acid (19). The biological activity in vitro of the synthesized 1-desoxy-2MD (6) was evaluated and this analog was found to have an affinity for the vitamin D receptor (VDR) similar as its parent compound 2MD (5) while being much less active in the transcriptional assay. The results of the biological tests in vivo are also discussed.

New derivative of 1α,25-dihydroxy-19-norvitamin D3 with 3′-alkoxypropylidene moiety at C-2: synthesis, biological activity and conformational analysis

Abstract In pursuit of novel biologically active Vitamin D compounds of potential therapeutic value, 1α,25-dihydroxy-2-[3′-(methoxymethoxy)propylidene]-19-norvitamin D 3 ( 7 ) was efficiently prepared in a convergent synthesis, starting with (−)-quinic acid and the protected 25-hydroxy Grundmann ketone 16 . The key synthetic step involved Lythgoe type Wittig–Horner coupling of 16 , with the phosphine oxide 15 . Molecular modeling was employed to establish the A-ring conformation of the synthesized Vitamin 7 . Also, preliminary modeling of its complex with the rVDR was performed and interactions between ligand and the binding domain analyzed. Analog 7 was found to be only six times less potent than 1α,25-(OH) 2 D 3 ( 1 ) in binding to the rat recombinant Vitamin D receptor (VDR). In comparison with hormone 1 , it also showed slightly lower cellular HL-60-differentiation activity. Preliminary in vivo tests indicated unusually high calcemic activity of 7 .

Synthesis and biological activity of 22-iodo- and (E)-20(22)-dehydro analogues of 1α, 25-dihydroxyvitamin D3

Construction of 25-hydroxy-steroidal side chain substituted with iodine at C-22 was elaborated on a model PTAD-protected steroidal 5,7-diene and applied to a synthesis of (22R)- and (22S)-22-iodo-1α,25-dihydroxyvitamin D3. Configuration at C-22 in the iodinated vitamins, obtained by nucleophilic substitution of the corresponding 22S-tosylates with sodium iodide, was determined by comparison of their iodine-displacement processes and cyclizations leading to isomeric five-membered (22,25)-epoxy-1α-hydroxyvitamin D3 compounds. Also, 20(22)-dehydrosteroids have been obtained and their structures established by 1H NMR spectroscopy. When compared to the natural hormone, (E)-20(22)-dehydro-1α,25-dihydroxyvitamin D3 was found 4 times less potent in binding to the porcine intestinal vitamin D receptor (VDR) and 2 times less effective in differentiation of HL-60 cells. 22-Iodinated vitamin D analogues showed somewhat lower in vitro activity, whereas (22,25)-epoxy analogues were inactive. Interestingly, it was established that (22S)-22-iodo-1α,25-dihydroxyvitamin D3 was 3 times more potent than its (22R)-isomer in binding to VDR and four times more effective in HL-60 cell differentiation assay. The restricted mobility of the side chain of both 22-iodinated vitamin D compounds was analyzed by a systematic conformational search indicating different spatial regions occupied by their 25-oxygen atoms. Preliminary data on the in vivo calcemic activity of the synthesized vitamin D analogues indicate that (E)-20(22)-dehydro-1α,25-dihydroxyvitamin D3 and 22-iodo-1α,25-dihydroxyvitamin D3 isomers were ca. ten times less potent than the natural hormone 1α,25-(OH)2D3 both in intestinal calcium transport and bone calcium mobilization.