Synthesis of 1α,25-Dihydroxyvitamin D Analogues Featuring a S2-symmetric CD-ring Core

Three analogues of 1α,25-dihydroxyvitamin D3 (calcitriol), featuring a trans-fused decalin C,D-core with local S2-symmetry, and possessing identical side-chain and seco-B,A-ring structures, have been synthesized starting from readily available (4aR,8aS)-octahydronaphthalene-1,5-dione (7). The very short sequences involve the simultaneous introduction of the side-chain and seco-B,A-ring fragments via Suzuki and Sonogashira coupling reactions. The analogues are devoid of relevant biological activity.


Introduction
Since the discovery that the biological action of vitamin D 3 originates from the dihydroxylated metabolite 1α,25-dihydroxyvitamin D 3 (1, calcitriol) and that, next to its classical role in the regulation of calcium homeostasis, these actions also involve immunomodulation, cell differentiation and antiproliferation, there has been an intense search for structural analogues of calcitriol that might show a separation in calcemic and antiproliferative-prodifferentiating activities (Scheme 1) [1]. In this context various successful structural modifications have been introduced in each one of the three parts that one may distinguish in its structure: the rigid central C,D-ring system and the flexible parts of the molecule, comprising the side chain and the seco-B,A-ring [2].

OPEN ACCESS
At the molecular level calcitriol generates biological responses via signal transduction pathways in which interaction with the nuclear receptor (n-VDR) leads to gene transcription regulation (genomic pathway) and in which interaction with a putative membrane receptor (m-VDR) leads to rapid actions such as transcaltachia (non-genomic pathway) [3]. A turning point in the rational development of analogues has been the disclosure of the detailed structure of the ligand binding domain of the n-VDR, obtained via X-ray diffraction analysis of the complex between calcitriol and a truncated mutant of the n-VDR [4]. Interestingly, this study revealed that the position of the ligand within the binding site was opposite to the location that was previously suggested on the basis of extensive molecular modeling studies [5]. This could indicate that the two polar parts of the molecule are interchangeable so that each bonding type could be associated with a different biological action. In this context we became interested in the development of analogues featuring structural symmetry so that the two flexible parts would become indistinguishable. Herein we wish to describe analogues 2a, 2b and 2c which are characterized by the presence of a trans-fused decalin C,D-core with local S 2 -symmetry (Scheme 1) [6].

Results and Discussion
The choice of a S 2 -symmetric trans-decalin core functionalized as in 2 was dictated both by structural and synthetic considerations. Indeed symmetrical non-steroidal derivatives have been described that were found to possess vitamin D-like activity ( Figure 1). In particular 3 is able to induce VDR-mediated transactivation, albeit much less potently than calcitriol [7]. Also in the context of structural modifications the following are relevant to the present work ( Figure 2): (i) cyclic motifs in the side chain (e.g. 4a) [8,9]; (ii) unsaturation at C16,C17 (e.g. 4a, 4b) [10]; (iii) unsaturation at C8,C9 (e.g. 5a, 5b) [11]; and (iv) analogues featuring an enlarged six-membered D-ring [12]. In principle the structural features of analogues 2 should allow for very direct and convergent syntheses in which the two identical fragments representing the side chain and the seco-B,A-ring are introduced simultaneously on a suitable functionalized trans-decalin core in a process were stereoisomers would not be formed. This is in contrast with the often lengthy sequences and difficult separations that are otherwise required [13][14][15]. Finally, whereas most efforts in the development of analogues have been directed towards modifications in the flexible parts, our laboratories have always focused on the central less accessible part of the molecule, and almost as a rule, such modifications have led to a reduction in calcemic activity [16].
The procedure developed by Castedo and Mouriño for coupling of a C-ring vinyl triflate with an Aring terminal alkyne led to the desired 11b in quantitative yield [26]. Semi-hydrogenation of the ynemoieties with Lindlar catalyst in the presence of quinoline gave 11c in 78% yield. Eventual deprotection with TBAF yielded the crystalline analogues 2b (51%) and 2c (81%).
The biological evaluation of the analogues includes the determination of the binding affinity for the porcine intestinal VDR, and the in vitro antiproliferative activity on breast cancer MCF-7 cells [27]. The analogues 2a, 2b and 2c did not show any relevant biological activity. This result shows that the side-chain fragment of the analogues in this series is probably too large.

Conclusions
In the present work advantage is taken of the S 2 -symmetry of the readily available bisenol triflate 8 to introduce simultaneously fragments that may be considered as structural variations of the classical side-chain and of the seco-B,A-ring of calcitriol. The attachment of these fragments proceed in an efficient way via palladium catalyzed cross-couling processes involving either a Sonogashira reaction or a Suzuki coupling. Unfortunately the prepared calcitriol analogues 2a, 2b and 2c were devoid of biological activity.

Experimental
General TLC were run on glass plates precoated with silica gel (Merck, 60F-254). Column chromatography was performed on silica gel (Merck, 230-400 mesh) or Florisil (100-200 mesh). IR spectra were recorded on a Perkin-Elmer series 1600 FT-IR spectrometer. 1 H-NMR and 13 C-NMR spectra were recorded on a Bruker AM-500 spectrometer. Mass spectra (EI) were recorded on a Hewlett-Packard 5898A spectrometer at 70 eV.
(4aR,8aS)-octahydronaphthalene-1,5-dione (7). To a solution of decalin-1,5-diol 6 (8.86 g, 52 mmol) in benzene (70 mL) is slowly added dropwise the Cr(VI) oxidant over a period of 3 hours, during which period the temperature is kept at 6 °C. The oxidant is prepared separately by the successive addition to water (68 mL) of glacial acetic acid (11.5 mL), concentrated sulfuric acid (21 mL) and sodium dichromate dihydrate (15.   and Lindlar catalyst (120 mg, purchased from Aldrich and dried for 3 hours in vacuo). The mixture is stirred under an atmosphere of hydrogen at room temperature and the reaction followed by TLC. After disappearance of starting material diethyl ether is added and the mixture filtered over Celite ® . After concentration in vacuo the residue is purified by column chromatography on silica gel with isooctane/ethyl acetate (98:2) as the eluent and by HPLC with 0.2% methyl tert-butyl ether in isooctane as the eluent to yield 157 mg of 11c as a colorless oil (78%) which is further subjected to deprotection.