Synthesis and evaluation of vitamin D3 analogues with C-11 modifications as inhibitors of Hedgehog signaling

Previous structure-activity relationship studies have provided potent and selective analogues of vitamin D3 as inhibitors of the Hedgehog (Hh) signaling pathway. These analogues contain both modified A- and seco-B ring motifs, and have been evaluated for anticancer therapeutic potential. To continue our studies on this scaffold, a new series of compounds were synthesized to explore additional interactions and spatial constraints. These compounds incorporate functional groups of varying size and hydrophobicity at the C-11 position. While large hydrophobic moieties (9c–e) resulted in significant loss of Hh inhibition, smaller or more flexible moieties (9a, 11) maintain anti-Hh activity. These results call for additional and continued studies to identify the binding pocket to better understand these structure-activity relationships.     

Molecular basis of the selective activity of vitamin D analogues

More than 2,000 synthetic analogues of the biological active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), are presently known. Basically, all of them interfere with the molecular switch of nuclear 1alpha,25(OH)(2)D(3) signaling, which is the complex of the vitamin D receptor (VDR), the retinoid X receptor (RXR), and a 1alpha,25(OH)(2)D(3) response element (VDRE). Central element of this molecular switch is the ligand-binding domain (LBD) of the VDR, which can be stabilized by a 1alpha,25(OH)(2)D(3) analogue either in its agonistic, antagonistic, or non-agonistic conformation. The positioning of helix 12 of the LBD is of most critical importance for these conformations. In each of the three conformations, the VDR performs different protein-protein interactions, which then result in a characteristic functional profile. Most 1alpha,25(OH)(2)D(3) analogues have been identified as agonists, a few are antagonists (e.g., ZK159222 and TEI-9647), and only Gemini and some of its derivatives act under restricted conditions as non-agonists. The functional profile of some 1alpha,25(OH)(2)D(3) analogues, such as EB1089 and Gemini, can be modulated by protein and DNA interaction partners of the VDR. This provides them with some selectivity for DNA-dependent and -independent signaling pathways and VDRE structures.     

Polymorphism of the vitamin D binding protein (DBP) among primates: an evolutionary analysis

The distribution of the DBP (vitamin D binding protein) polymorphism is now well characterized among human populations but for primates only limited results are known. The aim of this paper is to describe the electrophoretic polymorphism of this protein among various species. Using three different electrophoretic methods, we are able to detect an unknown polymorphism and to classify the different alleles observed. These results may be used to set an international nomenclature for further comparisons. The different electrophoretic mobilities between Old and New World Monkeys show that: 1) the Cercopithecoïdea are presenting the largest genetic heterogeneity; 2) the DBP among the Galago corresponds to the lowest isoelectric points observed among Primates; 3) during the evolution from nonhuman Primates to Man, the DBP is able to keep its affinity for vitamin D derivatives despite the occurrence of significant molecular modifications; 4) among Anthropoïdea, the electrophoretic patterns of DBP are very close to the human Gc 1 proteins. These results show that evolution at the DBP level can be considered as a continous mechanism of structural modifications. A significant transition occurs during the differentiation between Cercopithecoïdea and Anthropoïdea. It is not too speculative to consider that some electrophoretic forms detected among Gorilla, Pongo, or Pan may be identical to rare variants observed among humans.     

The vitamin D system in iguanian lizards

The apparent plasma concentration of vitamin D binding protein (DBP) in an iguanian lizard, Pogona barbata, and the affinity of this protein for 25-hydroxyvitamin D₃ (25(OH)D₃), 25-hydroxyvitamin D₂ (25(OH)D₂), and 1,25-dihydroxyvitamin D₃ (1,25(OH)D₃) was found to resemble more closely that of the domestic hen than that of the human. The characteristics of Pogona DBP, the pattern of vitamin D metabolites derived from injected radioactive vitamin D₃ and the plasma concentrations of endogenous 25-hydroxyvitamin D (25(OH)D) in a range of iguanian lizards have been examined. The findings suggest that 25-hydroxyvitamin D (25(OH)D) is the major metabolite of vitamin D, and that it may represent the storage form of vitamin D in these species in the same way as in mammals. High concentrations of vitamin D within iguanian embryos and egg yolks suggest a role for this compound in embryogenesis in these species, and perhaps indicates that there is a mechanism for vitamin D delivery to eggs comparable to that found in the domestic chicken.     

Synthesis and biological evaluation of novel tamoxifen analogues

A collection of compounds, structurally related to the anticancer drug tamoxifen, used in breast cancer therapy, were designed and synthesized as potential anticancer agents. McMurry coupling reaction was used as the key synthetic step in the preparation of these analogues and the structural assignment of E, Z isomers was determined on the basis of 2D-NOESY experiments. The compounds were evaluated for their antiproliferative activity on breast cancer (MCF-7), cervix adenocarcinoma (HeLa) and biphasic mesothelioma (MSTO-211H) human tumor cell lines. The estrogen like properties of the novel compounds were compared with those of the untreated controls using an estrogen responsive element-based (ERE) luciferase reporter assay and compared to 17β-estradiol (E2). Finally, with the aim to correlate the antiproliferative activity with an intracellular target(s), the effect on relaxation activity of DNA topoisomerases I and II was assayed.     

Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status

Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D₃ and its metabolic product—25(OH)D₃ has not been studied. We investigated this relationship in two separate populations: the first, individuals from Hawaii who received significant sun exposure; the second, subjects from a lactation study who received up to 6400 IU vitamin D₃/day for 6 months.

Results (1) the relationship between circulating vitamin D₃ and 25(OH)D in both groups was not linear, but appeared saturable and controlled; (2) optimal nutritional vitamin D status appeared to occur when molar ratios of circulating vitamin D₃ and 25(OH)D exceeded 0.3; at this point, the V_max of the 25-hydroxylase appeared to be achieved. This was achieved when circulating 25(OH)D exceeded 100 nmol.

We hypothesize that as humans live today, the 25-hydroxylase operates well below its V_max because of chronic substrate deficiency, namely vitamin D₃. When humans are sun (or dietary) replete, the vitamin D endocrine system will function in a fashion as do these other steroid synthetic pathways, not limited by substrate. Thus, the relationship between circulating vitamin D and 25(OH)D may represent what “normal” vitamin D status should be.     

Distribution of vitamin D binding protein expressing neurons in the rat hypothalamus

We observed immunostaining for vitamin D binding protein (DBP) in rat hypothalamus. Part of the supraoptic and of the paraventricular neurons showed DBP immunoreactivity, in part colocalized with Arg-vasopressin. DBP was also observed in widespread axonal projections throughout the lateral hypothalamus, the median eminence and the posterior pituitary lobe. A portion of ependymal cells, the choroids plexus epithelium and some of the endocrine cells in the anterior pituitary lobe contained DBP immunoreactivity. In situ hybridization of semithin sections with a synthetic oligonucleotide probe to DBP mRNA resulted in staining of magnocellular hypothalamic neurons, but not of ependymal cells or anterior lobe cells. Our observations indicate an intrinsic expression of DBP in the rat hypothalamus. DBP may be synthesized and transported along with the classical neurohypophyseal hormones. The multiple locations of DBP-expressing neurons indicate multiple functional properties: DBP may be released from in the posterior lobe, it may act as a hypophyseotropic factor and as a central neuroactive substance.     

Synthesis and composition of vitamin D-3 metabolites in Solanum malacoxylon

The synthesis of vitamin D-3 hydroxylated metabolites in Solanum malacoxylon was investigated. When crude leaf homogenates and subcellular fractions were incubated with [³H]vitamin D-3 and [³H]25-hydroxy-vitamin D-3 under conditions described for animal vitamin D-3-25-hydroxylase and 25-hydroxy-vitamin D-3-1α-hydroxylase, respectively, labelled metabolites identified on the basis of their chromatographic properties as 25-hydroxy-vitamin D-3 and 1,25-dyhydroxy-vitamin D-3 were formed. Other unidentified product metabolites were also detected. Vitamin D-3-25-hydroxylase activity was localized in microsomes and 25-hydroxy-vitamin D-3-1α-hydroxylase in mitochondria and microsomes. Chromatography of sterols isolated from leaf extracts preincubated with β-glucosidase on Sephadex LH-20 columns permitted the isolation of three biologically active fractions with elution properties similar to vitamin D-3, 25-hydroxy-vitamin D-3 and 1,25-dihydroxy-vitamin D-3, respectively. Ultraviolet spectra characteristic of vitamin D-3 and its metabolites were obtained after purification of the fractions by TLC. Co-chromatography of individual fractions with authentic metabolites on TLC provided further evidence that the plant contains vitamin D-3, 25-hydroxy-vitamin D-3 and 1,25-dihydroxy-vitamin D-3 as glucoside derivatives. These results suggest that a similar pathway of vitamin D-3 hydroxylation as in animals may be operative in S. malacoxylon.     

Synthesis and biological evaluation of novobiocin analogues as potential heat shock protein 90 inhibitors

Recent studies have shown that novobiocin (NB), a member of the coumermycin (CA) family of antibiotics with demonstrated DNA gyrase inhibitory activity, inhibits Heat shock protein 90 (HSP90) by binding weakly to a putative ATP-binding site within its C-terminus. To develop more potent HSP90 inhibitors that target this site and to define structure–activity relationships (SARs) for this class of compounds, we have synthesized twenty seven 3-amido-7-noviosylcoumarin analogues starting from NB and CA. These were evaluated for evidence of HSP90 inhibition using several biological assays including inhibition of cell proliferation and cell cycle arrest, induction of the heat shock response, inhibition of luciferase-refolding in vitro, and depletion of the HSP90 client protein c-erbB-2/HER-2/neu (HER2). This SAR study revealed that a substantial increase in biological activity can be achieved by introduction of an indole-2-carboxamide group in place of 4-hydroxy-isopentylbenzamido group at C-3 of NB in addition to removal/derivatization of the 4-hydroxyl group from the coumarin ring. Methylation of the 4-hydroxyl group in the coumarin moiety moderately increased biological activity as shown by compounds 11 and 13. Our most potent new analogue 19 demonstrated biological activities consistent with known HSP90-binding agents, but with greater potency than NB.     

Synthesis of VS-105: A novel and potent vitamin D receptor agonist with reduced hypercalcemic effects

We have synthesized a novel vitamin D receptor agonist VS-105 ((1R,3R)-5-((E)-2-((3αS,7αS)-1-((R)-1-((S)-3-hydroxy-2,3-dimethylbutoxy)ethyl)-7α-methyldihydro-1H-inden-4(2H,5H,6H,7H,7αH)-ylidene)ethylidene)-2-methylenecyclohexane-1,3-diol). Preparation of a-ring phenylphosphine oxide 11, followed by Wittig–Horner coupling of 11 with the protected 25-hydroxy Grundmann’s ketone 22 generated the precursor 12. Deprotection of the TBDMS groups of 12 produced the target compound VS-105. The biological profiles of VS-105 were evaluated using in vitro assays (VDR receptor binding, VDR reporter gene and HL-60 differentiation) in comparison to calcitriol (the endogenous hormone) or paricalcitol. Furthermore, the PTH suppressing potency and hypercalcemic side effects of VS-105 were evaluated in the 5/6 nephrectomized uremic rats in comparison to paricalcitol. Combining various changes at 20-epi, 22-oxa, 24-methyl, and 2-methylene yielded VS-105 that not only is highly potent in inducing functional responses in vitro, but also effectively suppresses PTH in a dose range that does not affect serum calcium in the 5/6 nephrectomized uremic rats.     

Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status

Circulating 25-hydroxyvitamin D [25(OH)D] is generally considered the means by which we define nutritional vitamin D status. There is much debate, however, with respect to what a healthy minimum level of circulation 25(OH)D should be. Recent data using various biomarkers such as intact parathyroid hormone (PTH), intestinal calcium absorption, and skeletal density measurements suggest this minimum level to be 80 nmol (32 ng/mL). Surprisingly, the relationship between circulating vitamin D₃ and its metabolic product—25(OH)D₃ has not been studied. We investigated this relationship in two separate populations: the first, individuals from Hawaii who received significant sun exposure; the second, subjects from a lactation study who received up to 6400 IU vitamin D₃/day for 6 months.Results (1) the relationship between circulating vitamin D₃ and 25(OH)D in both groups was not linear, but appeared saturable and controlled; (2) optimal nutritional vitamin D status appeared to occur when molar ratios of circulating vitamin D₃ and 25(OH)D exceeded 0.3; at this point, the V_max of the 25-hydroxylase appeared to be achieved. This was achieved when circulating 25(OH)D exceeded 100 nmol.We hypothesize that as humans live today, the 25-hydroxylase operates well below its V_max because of chronic substrate deficiency, namely vitamin D₃. When humans are sun (or dietary) replete, the vitamin D endocrine system will function in a fashion as do these other steroid synthetic pathways, not limited by substrate. Thus, the relationship between circulating vitamin D and 25(OH)D may represent what “normal” vitamin D status should be.     

Synthesis and biological evaluation of thielocin B1 analogues as protein-protein interaction inhibitors of PAC3 homodimer

The synthesis and biological evaluation of thielocin B1 analogues have been demonstrated. Fourteen analogues modified in the central core and terminal carboxylic acid moiety were concisely synthesized by simple esterification or etherification reaction. The evaluation of synthetic analogues as inhibitors of proteasome assembling chaperone (PAC) complexes (the PAC3 homodimer and PAC1/PAC2) revealed that the natural product-like bending structure and terminal carboxylic acid groups were crucial for its biological activity. Moreover, SAR and in silico docking studies indicated that all methyl groups on the diphenyl ether moiety of thielocin B1 contribute to the potent and selective inhibition of the PAC3 homodimer via hydrophobic interactions.     

Design and synthesis of potent tyr(OMe)5-gonadotropin-releasing hormone (GnRH) analogues with modifications at positions 6, 9 and 10

We have recently reported the synthesis and the conformational properties of some Gonadotropin-releasing hormone (GnRH) analogues in which the tyrosine residue at position 5 is substituted with tyrosine-O-methyl (Keramida et al., Let. Pept. Sci., 3 (1996) 257/Matsoukas et al., Eur. J. Med. Chem., 32 (1997) 927). The analogue [Tyr-(OMe)⁵]-GnRH was found to exert a lower degree of desensitization than the native GnRH peptides in terms of pituitary gonadotropin (GTH) release in goldfish. Compared to GnRH, however, [Tyr-(OMe)⁵]-GnRH exerted a lower GTH-release potency in cultured goldfish pituitary fragments, and was bound with a lower binding affinity to the rat pituitary GnRH receptors. In order to increase the potency of [Tyr-(OMe)⁵]-GnRH, we have synthesized a group of GnRH peptides containing Tyr-(OMe)⁵ in combination with other substitutions at positions 6, 9 and 10 and we have estimated their binding affinity for the rat pituitary receptors and gonadotropin (GTH) release potency in the goldfish pituitary. A selected number of these analogues was also tested for their ability to induce ovulation in seabass. The important structural modifications that increased the binding and gonadotropic activity of [Tyr(OMe)⁵]-GnRH in vitro and in vivo were found to include the replacement of the proline at position 9 with azetidine, glycine amide terminus with an alkyl amide group and Gly⁶ residue with hydrophilic D-amino acids such as D-Arg⁶. Overall, the findings provide SAR information on a group of novel GnRH peptides that can be also used to induce ovulation in teleosts.     

Synthesis of structurally diverse benzosuberene analogues and their biological evaluation as anti-cancer agents

Diversely functionalized, fused aryl–alkyl ring systems hold a prominent position as well-established molecular frameworks for a variety of anti-cancer agents. The benzosuberene (6,7 fused, also referred to as dihydro-5H-benzo[7]annulene and benzocycloheptene) ring system has emerged as a valuable molecular core component for the development of inhibitors of tubulin assembly, which function as antiproliferative anti-cancer agents and, in certain cases, as vascular disrupting agents (VDAs). Both a phenolic-based analogue (known as KGP18, compound 39) and its corresponding amine-based congener (referred to as KGP156, compound 45), which demonstrate strong inhibition of tubulin assembly (low micromolar range) and potent cytotoxicity (picomolar range for KGP18 and nanomolar range for KGP156) are noteworthy examples of such benzosuberene-based compounds. In order to extend the structure–activity relationship (SAR) knowledge base related to benzosuberene anti-cancer agents, a series of eleven analogues (including KGP18) were prepared in which the methoxylation pattern on the pendant aryl ring as well as functional group incorporation on the fused aryl ring were varied. The synthetic approach to these compounds featured a sequential Wittig olefination, reduction, Eaton’s reagent-mediated cyclization strategy to achieve the core benzosuberone intermediate, and represented a higher-yielding synthesis of KGP18 (which we prepared previously through a ring-expansion strategy). Incorporation of a fluorine or chlorine atom at the 1-position of the fused aryl ring or replacement of one of the methoxy groups with hydrogen (on the pendant aryl ring of KGP18) led to benzosuberene analogues that were both strongly inhibitory against tubulin assembly (IC₅₀ approximately 1.0 μM) and strongly cytotoxic against selected human cancer cell lines (for example, GI₅₀ = 5.47 nM against NCI-H460 cells with fluoro-benzosuberene analogue 37). A water-soluble phosphate prodrug salt of KGP18 (referred to as KGP265, compound 44) and a water-soluble serinamide salt (compound 48) of KGP156 were also synthesized and evaluated in this study.     

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.