2-(3'-Hydroxypropylidene)-1alpha-hydroxy-19-norvitamin D compounds with truncated side chains

Our recent studies with 2-(3'-hydroxypropylidene) analogs of 1alpha,25-dihydroxy-19-norvitamin D(3) showed that this 2-substituent creates compounds with very potent biological activity. In the continuing search for vitamin D compounds with selective activity profiles, we prepared a series of 1alpha-hydroxy-19-norvitamin D analogs characterized by the presence of a 3'-hydroxypropylidene substituent at C-2 and a truncated side chain. These vitamin D compounds were efficiently prepared using convergent syntheses. The C,D-fragments, namely the Grundmann ketones 19, 20, 27, 36 and 37 were synthesized from the known 8beta-benzoyloxy-22-aldehydes 12 and 29. These hydrindanones were subjected to Lythgoe type Wittig-Horner coupling with phosphine oxide 21, prepared by us previously, and after hydroxyl deprotection the set of 19-norvitamins 7-11 was successfully obtained. According to our expectations, all analogs (with an exception of the 20R-compound 7) have pronounced in vitro activity. When compared to the natural hormone 1alpha,25-(OH)(2)D(3) (1), they show the same or only slightly reduced affinity for the vitamin D receptor while being similarly effective as 1 in differentiation of HL-60 cells into monocytes.     

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

In pursuit of novel biologically active Vitamin D compounds of potential therapeutic value, 1alpha,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 1alpha,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.     

Structure-activity relationships of 19-norvitamin D analogs having a fluoroethylidene group at the C-2 position

We have synthesized four new geometric isomers of 1alpha,25-dihydroxy-2-(2'-fluoroethylidene)-19-norvitamin D analogs 1 and 2 having a 20R- and 20S-configuration, whose structures are correlated with 2MD possessing high potencies in stimulating bone formation in vitro and in vivo. The E-isomers of (20R)- and (20S)-2-fluoroethylidene analogs 1a and 1b were comparable with the natural hormone 1alpha,25-(OH)(2)D(3) in binding to the vitamin D receptor (VDR), while two Z-isomers 2a and 2b were about 15-20 times less active than the hormone. In inducing expression of the vitamin D responsive element-based luciferase reporter gene, the E-isomers 1a and 1b were 1.2- and 8.6-fold more potent than the hormone, respectively, while the Z-isomers 2a and 2b had 27-55% of the potency. On the basis of the biological activities and a docking simulation based on X-ray crystallographic analysis of the VDR ligand-binding pocket, the structure-activity relationships of the fluorinated 19-norvitamin D analogs are discussed.     

2-Methylene analogs of 1alpha-hydroxy-19-norvitamin D3: synthesis, biological activities and docking to the ligand binding domain of the rat vitamin D receptor

In continuing efforts towards the synthesis of biologically active vitamin D compounds of potential therapeutic value, new 2-methylene-1alpha-hydroxy-19-norvitamin D(3) analogs 3 and 4 with modified alkyl side chains have been synthesized. The key synthetic step involved Lythgoe-type Wittig-Horner coupling of Windaus-Grundmann type ketones 9, possessing different 17beta-alkyl substituents, with the phosphine oxide 10 prepared from (-)-quinic acid. The prepared vitamins 3 and 4 were ca. eight times less potent than 1alpha,25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) (1) in binding to the rat intestinal vitamin D receptor (VDR). In comparison with the hormone 1 they exhibited slightly lower cellular HL-60 differentiation activity. When tested in vivo; the analog 3 was characterized by very high bone calcium mobilizing potency and intestinal calcium transport activity. Unexpectedly, the 25-methyl compound 4 showed marked calcemic activity in both assays. Computational docking of the vitamin 3 into the binding pocket of the rat vitamin D receptor is also reported.     

Selective analogs of 1alpha,25-dihydroxyvitamin D3 for the study of specific functions of Vitamin D

New analogs of 1alpha,25-dihydroxyvitamin D(3) synthesized in our research group that show selective activity in vivo are presented along with supporting biological results. Compounds that act preferentially on intestine are 2-(3'-propylidene-19-nor-(20S or 20R))-1alpha,25-dihydroxyvitamin D(3) and 2-methylene-19-21-dinor-1alpha,25-dihydroxyvitamin D(3). Compounds that act anabolically to induce bone formation are 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D(3) (2MD), the 2alpha-methyl derivative, the 26,27-dimethyl derivative, and the 26-dimethylene derivative. Compounds that act preferentially on parathyroid glands are 2-methylene-19-nor-1alpha-hydroxy-homopregnacalciferol, the 20S-bishomo derivative and the 2-methylene-19,26,27-trinor-1alpha,25-dihydroxyvitamin D(3). These latter compounds do not elevate serum calcium until doses of the order of >300 microg/kg body weight are used, while parathyroid hormone levels are suppressed at much lower doses. Some of these novel analogs may ultimately be useful as new and safer therapeutic agents. Regardless of their clinical utility, they represent valuable research tools that can be used to study the specific functions of the Vitamin D hormone in vivo.     

Methyl substitution of the 25-hydroxy group on 2-methylene-19-nor-1alpha,25-dihydroxyvitamin D3 (2MD) reduces potency but allows bone selectivity

The discovery of 2-methylene-19-nor-1alpha,25-dihydroxyvitamin D3 (2MD) as a bone selective and bone anabolic form of vitamin D has stimulated an investigation of structure/function of bone selectivity. Four new 2-substituted-19-norvitamin D analogs 3-6 have been developed to study the structure-activity relationship at C-25. As predicted, removing the 25-hydroxy group (compound 3) from the very potent analog 2MD and its 2-methyl derivatives (5 and 6) dramatically reduces in vitro activities, but biological potency is nearly fully restored in vivo likely due to in vivo 25-hydroxylation. The introduction of a methyl group at C-25 (compound 4) that blocks in vivo 25-hydroxylation reduces biological activity both in vitro and in vivo. However, analog 4 retains bone selectivity making it interesting as a possible therapeutic for bone loss diseases.     

New highly calcemic 1 alpha,25-dihydroxy-19-norvitamin D(3) 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 Grundmann's 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).     

2-(3′-Hydroxypropylidene)-1α-hydroxy-19-norvitamin D compounds with truncated side chains

Our recent studies with 2-(3′-hydroxypropylidene) analogs of 1α,25-dihydroxy-19-norvitamin D₃ showed that this 2-substituent creates compounds with very potent biological activity. In the continuing search for vitamin D compounds with selective activity profiles, we prepared a series of 1α-hydroxy-19-norvitamin D analogs characterized by the presence of a 3′-hydroxypropylidene substituent at C-2 and a truncated side chain. These vitamin D compounds were efficiently prepared using convergent syntheses. The C,D-fragments, namely the Grundmann ketones 19, 20, 27, 36 and 37 were synthesized from the known 8β-benzoyloxy-22-aldehydes 12 and 29. These hydrindanones were subjected to Lythgoe type Wittig–Horner coupling with phosphine oxide 21, prepared by us previously, and after hydroxyl deprotection the set of 19-norvitamins 7–11 was successfully obtained. According to our expectations, all analogs (with an exception of the 20R-compound 7) have pronounced in vitro activity. When compared to the natural hormone 1α,25-(OH)₂D₃ (1), they show the same or only slightly reduced affinity for the vitamin D receptor while being similarly effective as 1 in differentiation of HL-60 cells into monocytes.     

Enhanced biological activity of 1alpha,25-dihydroxy-20-epi-vitamin D3, the C-20 epimer of 1alpha,25-dihydroxyvitamin D3, is in part due to its metabolism into stable intermediary metabolites with significant biological activity

1alpha,25-dihydroxy-20-epi-vitamin D3 (1alpha,25(OH)2-20-epi-D3), the C-20 epimer of the natural hormone 1alpha,25(OH)2D3, is several fold more potent than the natural hormone in inhibiting cell growth and inducing cell differentiation. At present, the various mechanisms responsible for the enhanced biological activities of this unique vitamin D3 analog are not fully understood. In our present study we compared the target tissue metabolism of 1alpha,25(OH)2D3 with that of 1alpha,25(OH)2-20-epi-D3 using the technique of isolated perfused rat kidney. The results indicated that the C-24 oxidation pathway plays a major role in the metabolism of both compounds in the rat kidney. However, it was noted that the concentrations of two of the intermediary metabolites of 1alpha,25(OH)2-20-epi-D3, namely, 1alpha,24(R),25(OH)3-20-epi-D3 and 1alpha,25(OH)2-24-oxo-20-epi-D3 in the kidney perfusate, exceeded the concentrations of the corresponding intermediary metabolites of 1alpha,25(OH)2D3. Furthermore, 1alpha,25(OH)2-24-oxo-20-epi-D3 induces the conformation of the vitamin D receptor similar to that induced by its parent analog and is nearly as potent as its parent in inducing transactivation of a gene construct containing the human osteocalcin vitamin D-responsive element. We conclude that 1alpha,25(OH)2-20-epi-D3 by itself is not metabolically stable when compared to 1alpha,25(OH)2D3, but it acquires its metabolic stability because of the reduced rate of catabolism of its intermediary metabolites. Furthermore, 1alpha,25(OH)2-24-oxo-20-epi-D3, the stable bioactive intermediary metabolite plays a significant role in generating the enhanced biological activities ascribed to 1alpha,25(OH)2-20-epi-D3.     

Synthesis and biological properties of 2-methylene-19-nor-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactones--weak agonists

In a continuing effort to explore the 2-methylene-1alpha-hydroxy-19-norvitamin D(3) class of pharmacologically important vitamin D compounds, two novel 2-methylene-19-nor-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactones, GC-3 and HLV, were synthesized and biologically tested. Based on reports of similarly structured molecules, it was hypothesized that these compounds might act as antagonists, at least in vitro. The pathway designed to synthesize these compounds was based on two key steps: first, the Lythgoe-type Wittig-Horner coupling of Windaus-Grundmann-type ketone 18, with phosphine oxide 15, followed, later in the synthesis, by the Zn-mediated Reformatsky-type allylation of aldehyde 20 with methylbromomethylacrylate 8. Our biological data show that neither compound has antagonistic activity but acts as weak agonists in vitro and in vivo.     

New analogs of 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 with conformationally restricted side chains: evaluation of biological activity and structural determination of VDR-bound conformations

We have successfully prepared E- and Z- isomers of 17-20 dehydro analogs of 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D3 (2MD). Both isomers bind to the recombinant rat vitamin D receptor (VDR) with high affinity. The Z-isomer (Vit-III 17-20Z) displays activity in vivo and in vitro that is similar to 2MD. The in vitro activity of the E-isomer (Vit-III 17-20E) is comparable to the natural hormone, though in vivo this analog is significantly less calcemic. Crystal structures of the rat VDR ligand binding domain complexed with the analogs demonstrate that the Vit-III 17-20Z analog is oriented almost identically to 2MD, with only minor differences induced by the planar configuration around the C17-C20 double bond. The Vit-III 17-20E analog is oriented in a conformation distinct from both 2MD and the natural hormone. The structural comparisons suggest that the position of C21 in the ligand binding site may be an important determinant of biological activity.