Novel 9-Alkyl- and 9-Alkylidene-Substituted 1Î±,25-Dihydroxyvitamin

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Journal of Medicinal Chemistry

Novel 9-Alkyl- and 9-Alkylidene-substituted 1α α,25-Dihydroxyvitamin D3 Analogues: Synthesis and Biological Examinations

Urszula Kulesza,† Lori A. Plum,‡ Hector F. DeLuca,‡ Antonio Mouriño,*,¶ and Rafal R. Sicinski*,† †

Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland

‡

Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin

53706, United States ¶

Departamento de Química Orgánica, Laboratorio de Investigación Ignacio Ribas, Universidad

de Santiago de Compostela, 15782 Santiago de Compostela, Spain

AUTHOR INFORMATION Corresponding Authors [email protected] [email protected]

Keywords: Vitamin D analogues; Functionalization at C-9; 19-Norvitamin D analogues; Vitamin D receptor; Cellular differentiation; Transcriptional activity.

ACS Paragon Plus Environment


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ABSTRACT Continuing the structure-activity relationship studies in the vitamin D area, we designed and synthesized novel C-9 substituted calcitriol analogues, possessing different non-polar groups at this position. 9α-Methyl-1α,25-(OH)2D3, both epimers of 9-methylene-10,19-dihydro-1α,25(OH)2D3 as well as the parent vitamin with the “reversed” triene system, 9-methylene-19-nor1α,25-(OH)2D3, were obtained from the previtamin D precursors, constructed by either SuzukiMiyaura, Sonogashira or Stille couplings of the corresponding A- and C,D-ring fragments. An alternative synthetic path, leading to the latter vitamin and its homologue with 9-ethylidene group, involved formation of dienynes as precursors of the respective 19-norprevitamin D compounds. 9β-Methyl-19-nor-1α,25-(OH)2D3 was prepared by homogeneous hydrogenation with Wilkinson catalyst, and this analogue was found to be the most active in vitro. Moreover, 9α-methyl-1α,25-(OH)2D3 and 9-methylene-19-nor-1α,25-(OH)2D3 showed some in vitro activity, however, the in vivo assays indicated only weak calcemic potency of these compounds in the intestinal calcium transport.


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INTRODUCTION 1α,25-Dihydroxyvitamin D3 [calcitriol, 1α,25-(OH)2D3 (1), Figure 1], being the active form of vitamin D3 in vivo in the regulation of calcium and phosphorous,1 is also active in many other biological processes, for example, induction of cell differentiation and apoptosis as well as inhibition of cell proliferation and angiogenesis.2 The genomic actions of the natural hormone 1 are mediated through the vitamin D receptor (VDR),3 a transcription factor and member of the nuclear receptor superfamily,4 whose presence has been detected in numerous tissues.5 These findings make calcitriol potentially useful in various biomedical applications, including treatment of metabolic bone diseases, psoriasis, some kinds of cancers and immune disorders.6 However, the broader clinical application of 1α,25-(OH)2D3 is limited by its strong calcemic potency.7 In an effort to enhance selective biological actions and minimize undesired side effects, more than 3000 vitamin D analogues, modified in different parts of the molecule, have been developed in the recent years, but only few of them have found clinical applications.8 So far, only few vitamin D3 analogues functionalized at C-9 have been described in the literature. The first 9-substituted analogue of calcitriol with a different configuration of the intercyclic diene moiety, (7Z)-9α-hydroxyvitamin D3, was synthesized by Mouriño and coworkers9 and later by Dauben and Greenfield.10 Wittig-Horner coupling of 9α-benzoyloxy or 9αfluoro Grundmann ketone with the anion of the A-ring phospine oxide, due to the steric interaction between H-6 and 9α-substituent, led to the compound characterized by 7E-geometry of the newly formed double bond. In 2006, a series of 9α-alkylated derivatives of 19-norcalcitriol (2) was prepared by Shimizu11 using Wittig-Horner and Julia olefination approach but almost no information was disclosed about the biological properties of these compounds. Very recently,



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Japanese chemists synthesized few 9α-alkylated and 9α-hydroxyalkylated 2-substituted 19norcalcitriols and analyzed their crystal complexes with W282R mutant of rVDR.12 On the basis of the limited knowledge of the biological effect caused by modification at C-9 position in the vitamin D3 molecule, and as an extension of the structure-activity research conducted in our laboratories, we designed new homologues of calcitriol and 19-norcalcitriol (49) substituted with non-polar (alkyl and alkylidene) groups at C-9 (Figure 1). As the direct precursors of these vitamin D analogues, the corresponding 9-alkylated previtamin D compounds 10-12 (or their derivatives) were chosen. These, in turn, could be constructed from the synthons described in the literature - the protected 25-hydroxy Grundmann ketone 1313 and vinyl iodide 14,14 derived from vitamin D3,15 as well as two enynes 15 and 16, readily obtainable from quinic acid16 and S-carvone,17 respectively.18

RESULTS AND DISCUSSION Chemistry. As we reported previously, the conjugated triene systems of vitamins 5, 7 and 8 were obtained by thermal sigmatropic [1,7]-hydrogen shift from the same previtamin D compound 11, whereas the dienyne precursor of the required previtamin was efficiently constructed employing three alternative approaches: Sonogashira, Stille and stereoselective dehydration of a tertiary 8β-alcohol by Burgess reagent.14 Here we also describe a new mild and efficient convergent route to the previtamin D system that circumvents the troublesome semihydrogenation of dienynes. The new method generates the previtamin D skeleton by SuzukiMiyaura coupling19 between an alkenyl-boronic ester and a vinyl iodide. For the conversion of enyne 16 to the A-ring precursor, cis-alkenyl boronate 18 (Scheme 1), we applied the procedure based on hydroboration of alkynes developed by Miyaura, employing pinacolborane in the


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presence of Rh(I)-Pi-Pr3 complex and Et3N.20 Use of more than one equivalent of Et3N and excess of alkyne with respect to the borane reagent were critical for achieving the high cisselectivity. Under these conditions, terminal alkyne 16 was transformed to the desired (Z)-1alkenyl boronate 18 (74% yield) along with its geometric isomer 17 (18% yield). The double bond stereochemistries in the reaction products were determined by analysis of their 1H NMR data, namely, by comparison of the vicinal coupling constants of doublets derived from the olefinic protons resonating at 5.51 and 7.45 ppm (J = 18 Hz) for the trans-boronate 17, and at 5.40 and 6.89 ppm (J = 15 Hz) for the cis-isomer 18, respectively. The vinyl boronate 18 was then coupled with the known vinyl iodide 1414 in the presence of bis[triphenylphosphine]palladium(II) chloride and 2M aqueous solution of K3PO4,21 to give directly the desired previtamin D compound 19. Although the desired previtamin D analogue was obtained in high yield, the side product of the A-ring homocoupling reaction was also observed. Finally, the previtamin 19 was easily converted, by subsequent deprotection of hydroxyl groups and thermolysis, into the target vitamin D compounds 5, 7 and 8, which were purified by HPLC. To examine the synergetic biological effect of C-ring substitution of the vitamin D analogue with 9-alkylidene group and 19-nor modification of its ring A, compounds 6 and 9 were prepared using enyne 15 as the A-ring building block.18 Thus, the vinyl iodide 14 was coupled with either the enyne 15 or its tributyltin derivative 20 (Scheme 2) using Sonogashira or Stille reactions. The yields of the dienyne 21 prepared by both methods were high, although it was necessary to use a 5-fold molar excess of the starting enyne 15 in the case of Sonogashira coupling due to the concomitant homocoupling process. Partial hydrogenation of the triple bond of dienyne 21 using Lindlar catalyst poisoned with quinoline afforded 9-methyl-19-norprevitamin D compound 22 in 91% yield. In an alternative attempt, Pd-catalyzed Suzuki coupling of iodide 14 with (Z)-alkenyl boronate 24, prepared from enyne precursor 15, led also directly to the



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previtamin 22. The presence of methyl group attached to the terminal C-9 of the conjugated hexatriene moiety made such previtamin D analogue capable of equilibration with the corresponding vitamin D isomer 23. Final deprotection of hydroxyl groups in this product provided the desired 19-norcalcitriol analogue 6, possessing an ‘unnatural’ triene system. The synthesis of 9-ethylidene-19-norcalcitriol (9) began from the α-alkylation of protected 25-hydroxy Grundmann ketone performed analogously as described for the preparation of 26.14 Thus, α-ethylation of 13 was achieved by treatment with LDA in the presence of DMPU, followed by addition of EtI to afford the single product 27 in 66% yield (Scheme 3). Attempts to transform the resulting ketone 27 into the corresponding vinyl iodide were unsuccessful. For the construction of dienynes 30 and 31, the precursors of 19-norcalcitriol analogues 6 and 9, we applied the procedure based on the addition of an acetylide ion (generated from 15 by nbutyllithium) to the alkylated Grundmann ketones 26 and 27 in the presence of cerium trichloride. Stereoselective dehydration of the resulted propargylic alcohols 28 and 29 with Burgess reagent afforded the desired dienynes 30 (95%) and 31 (96%).14 Further conversion of dienynes 30 and 31 to the respective 9-alkylidene analogues of 19-norcalcitriol 6 and 9 with an ‘unnatural’ triene system was accomplished as described above. The thermal isomerization process of previtamin 33 led to the vitamin analogue 35 as a single isomer with E-configuration of the ethylidene group as assigned by NOE experiment. On irradiation of the vinylic proton signal from the 9-ethylidene substituent (CH3CH=) at δ 5.33 ppm, 2% enhancement was observed for the olefinic H-6 signal at 6.37 ppm. A similar preferential formation of more stable geometrical isomers of vitamin D analogues during the reversible thermal isomerization process was observed in the series of 19-functionalized compounds.22 The UV spectra of compounds 6


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and 9, possessing the ‘reversed’ triene chromophore in their structures, were similar to the spectrum of 1α,25-dihydroxyvitamin D3 (1). The successful preparation of the vitamin D analogues with 9α-methyl and 9-alkylidene groups encouraged us to extend the studies on the effect of non-polar substituents at C-9 on the biological activity of these compounds, and to synthesize also the 9β-alkylated vitamin D compound 4. We expected that introduction of such an equatorial substituent at C-9, could increase the steric strain between this group and C(6)-H and influence hydrophobic interactions between the ligand and the amino acids in the ligand binding pocket of the VDR. As we previously established,14 9β-methyl-calcitriol was not formed during the thermal rearrangement of 1α,25-dihydroxy-9-methylprevitamin D3 (11). Other methods of direct synthesis of the vitamin D triene system (for instance, Wittig-Horner approach) would require 9β-alkylated Grundmann ketone derivatives as precursors of the CD-ring building blocks. As previously observed, alkylation of the Grundmann ketone led exclusively to 9α-substituted products and, moreover, formation of products with the reversed configuration of the intercyclic diene moiety could be expected in the Wittig-Horner coupling.9,10 We decided to prepare such a vitamin D analogue by selective reduction of 9-exomethylene group. Such chemoselectivity was achieved by the homogeneous hydrogenation of 9-methylene-19-norcalcitriol (6), carried out in the presence of tris(triphenylphosphine)rhodium(I) chloride (Wilkinson catalyst). The desired 9βmethyl-19-norcalcitriol (4) was formed in 56% yield as single diene product (Scheme 4), which was successfully separated from the unreacted starting material (36%) by reversed-phase HPLC. The structure of compound 4 was assigned on the basis of its 1H NMR spectra. Spin decoupling experiment performed with the multiplet derived from the methine proton at C-9 (2.29 ppm), allowed measuring its vicinal coupling constants (9.1 and 2.8 Hz) with the neighboring protons at



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C-11. Their values, being in agreement with these resulting from molecular modeling (HyperChem and PCModel), indicated an axial disposition of H-9 and, consequently, equatorial β-orientation of the introduced methyl group.23 Also, a comparison of the 1H NMR spectrum of the vitamin analogue 4 with the respective spectral data of its 9-epimer 3, as reported in the Japanese patent,11 also supported our configurational assignment. Interestingly, the UV spectrum of the vitamin D compound 4 exhibits three absorption maxima (λmax 244, 252 and 261 nm), characteristic of the planar 5,7-diene chromophore present in 19-norvitamin D compounds. This finding seems to indicate that, despite severe interaction of equatorial 9β-methyl group with the vinyl hydrogen at C-6, there is no significant deviation of the intercyclic C(5)=C(6)-C(7)=C(8) moiety from planarity. Further confirmation of the ascribed structure of the synthesized compound 4, including its 9S-configuration and the geometry of molecule, was obtained by X-ray crystallographic analysis. Thus, X-ray diffraction data indicate that vitamin 4 crystallizes with its ring A fixed in the β-chair conformation,24 and the ring C exists in the chair conformation with an equatorially oriented 9β-methyl group (Figure 2). The torsion angle of the intercyclic 5,7-diene moiety was found to be 168o, that explained the UV absorption data of the examined compound. Biological Evaluation. Biological in vitro activities of the novel vitamin D analogues are compared to the natural hormone and summarized in Table 1.25 In the first assay, the synthesized derivatives of 1α,25-(OH)2D3 were tested for their ability to bind the full-length recombinant rat vitamin D receptor. The competition binding assays revealed that the 9α-methyl group in 5 significantly reduced VDR binding ability by 200-fold from that of the natural hormone. Among the compounds with a “reversed” triene system, the best binder was the 9-methylene-19norcalcitriol (6) characterized by similar binding activity as 5. Both A-ring methylated derivatives 7 and 8, as well as homologue 9 with 9-ethylidene substituent, were practically


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devoid of binding activity. Interestingly, 19-norvitamin D compound 4 with 9β-methyl substituent exhibited affinity for VDR similar to 1α,25-(OH)2D3 and this analogue was also found to be very active in inducing differentiation of HL-60 cells and in transcriptional activity. Analogue 6 had weak HL-60 cell differentiating potency whereas activities of other tested compounds were reduced by ca. four orders of magnitude from that of 1α,25-(OH)2D3. With a notable exception for 4, the analogues also exhibited low activities in the transcriptional assay, being 200-300 times (5, 6 and 8) less active than 1α,25-(OH)2D3 or decreased by ca. four orders of magnitude (7 and 9). Rather surprisingly, the results of the in vivo tests clearly demonstrated that 9β-methyl-19norcalcitriol (4), being the most active in vitro, was characterized by a weak calcemic potency. Its activity in intestinal calcium transport was lower than that of calcitriol itself, and the analogue showed only marginal activity in bone calcium mobilization when tested at very large doses (Figure 3). 9α-Methyl-calcitriol (5) showed some intestinal calcium transport activity, being inactive in mobilizing calcium from bone (Figure 4). When tested in vivo, compounds possessing 9-alkylidene substituents gave no response in bone calcium mobilization (Figure 4). Analogue 6 showed very low intestinal calcium transport, while a slightly increased intestinal activity was observed for compound 9 possessing 9ethylidene group. Studies on docking the analogues 4-6 into the VDR. Binding studies of the newly synthesized 9-substituted calcitriol analogues showed that only vitamins 4-6 exerted significant affinity for the VDR. To gain better understanding of this important biological function, we performed docking simulations of these compounds to the ligand binding pocket (LBP) of the VDR using Gold (5.1 version) software. When analyzing the calculated complexes, we



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considered the energy criterion, formation of hydrogen bonds and the parallel orientation of the ligand intercyclic 5,7-diene moiety in respect to the aromatic rings of Trp-286. This unique tryptophan residue plays an essential role in anchoring the ligand in the binding pocket and transcription of genes controlled by VDR, which was demonstrated by mutation26 and NMR27 experiments. The analysis of docking experiments shows that 9β-methyl-19-norcalcitriol (4), 9αmethyl-calcitriol (5) and 9-methylene-19-norcalcitriol (6) anchor the VDR cavity in an analogous fashion to 1α,25-(OH)2D3 in the crystalline VDR(LBD)-1 complex28 (1DB1) (Figure 5a-c) and they similarly interact with the receptor. All six hydrogen bonds present in the crystalline VDR(LBD)-1 complex28 were also found in these modeled complexes with the synthetic ligands. However, in the case of complexes with analogues 5 and 6, the interactions between their 25hydroxyl groups and His-305 and His-397 were much weaker than for the natural hormone. Moreover, inspection of the complex VDR(LBD)-6 indicated a poor contact between Ser-278 and the ligand’s 3β-hydroxyl (3.35 Å). In the analyzed complexes, the intercyclic diene moieties of all analogues were approximately parallel to the Trp-286 rings. However, in the VDR(LBD)-4 complex, the tryptophan indole ring was positioned little closer to the ligand’s 5,7-diene fragment (3.79 Å) than in the crystalline complex VDR(LBD)-1 (3.88 Å). The distance between Trp-286 and C(7)=C(8) bond of analogue 6 was larger, and in VDR(LBD)-5 exceeded 4 Å. Evidently, the presence of the 9α-methyl substituent in the compound 5 causes that the seco-B ring of this analogue is significantly moved away from the tryptophan moiety. Thus, interestingly, it seems that the observed differences between the compared complexes can be potentially explained by considering the distances of the modeled ligands from Trp-286.


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CONCLUSIONS C-Ring modification of calcitriol and 19-norcalcitriol derivatives represented an interesting target for our continued structure-activity studies on 1α,25-(OH)2D3 analogues. An attractive goal for the planned synthetic efforts in this area seemed to be vitamin D analogues possessing non-polar groups at the rather unexplored position C-9. Such 9-substitution could potentially enhance interactions of the novel compounds with hydrophobic residues in the ligand binding pocket of the vitamin D receptor. Also introduction of 9-alkylidene (methylene and ethylidene) substituents to the 19-norcalcitriol skeleton seemed to be an interesting endeavor, because it could lead to 1α,25-(OH)2D3 analogues with the ‘reversed’ triene system. In the present work, we described an efficient syntheses of such compounds and evaluated their biological activity. It was established that compounds with a non-natural triene system are characterized by significantly decreased biological in vitro and in vivo activity. In strong contrast to the 9α-alkylated calcitriol 5, analogue 4 representing the first 9β-substituted vitamin D compound described in the literature, was found as active as calcitriol in the in vitro tests. However, despite its strong binding affinity to the VDR and high transcriptional activity, this analogue was devoid of bone calcium mobilization ability and poorly supported intestinal calcium transport. Further studies on this interesting compound should be undertaken. Thus, it would be highly desirable to examine the crystal structure of its complex with VDR(LBD) and establish which conformational changes of the receptor are responsible for the decreased calcemic activity of the ligand.

EXPERIMENTAL SECTION Chemistry. Melting points (uncorrected) were determined on a SMP10 Stuart Scientific capillary melting point apparatus. Optical rotations were measured in chloroform using a Perkin-



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Elmer model 343 polarimeter at 24 °C. Ultraviolet (UV) absorption spectra were obtained on a Shimadzu UV-1800 UV spectrophotometer in absolute ethanol. Nuclear magnetic resonance spectra were recorded in CDCl3 solutions using Varian Unity plus (200 MHz), Bruker WM-250, Varian Inova (400 MHz), Varian Unity Inova 500 and Bruker DMX-500 or Varian 700 Active Shield (700 MHz) instruments. Chemical shifts (δ) are reported in parts per million relative to CH3Si (δ 0.00) or solvent signal as an internal standard. COSY, NOESY, HMBC and HMQC spectra, as well as spin decoupling, DEPT 90, DEPT 135 and differential NOE experiments were used to assign particular signals in the 1H and 13C NMR spectra. Signals in 1H NMR spectra are described using the following abbreviations: s - singlet, d - doublet, t - triplet, q – quartet, m – multiplet, br – broad, narr – narrow. High-resolution mass spectra were recorded on LCT (TOF) or Mass Quattro LC spectrometers using electrospray ionization (ESI) technique. Reactions were usually carried out with magnetic stirring. All reactions involving moisture- or oxygen-sensitive compounds were carried out under dry argon atmosphere. Reaction temperatures refer to external bath temperatures. Tetrahydrofuran was distilled from Na/benzophenone; dichloromethane and toluene were distilled from P2O5, whereas pyridine, diisopropylamine, diethylamine and triethylamine were distilled from CaH2. The organic extracts were dried over anhydrous MgSO4 or Na2SO4, filtered and concentrated using a rotary evaporator at a water aspirator pressure (20-30 mm Hg). Reactions were monitored by thin-layer chromatography (TLC) using aluminum-backed MERCK 60 silica gel plates (0.2 mm thickness). The chromatograms were visualized first with ultraviolet light (254 nm) and then by immersion in a cerium-molybdenum solution [10 g Ce(SO4)2 × 4 H2O, 25 g phosphomolybdic acid, 60 mL H2SO4 and 940 mL H2O] or p-anisaldehyde solution (5 mL H2SO4, 1.5 mL glacial HOAc, 3.7 mL p-anisaldehyde, 135 mL H2O) followed by heating. Flash column chromatography was


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performed with MERCK 60 (230-400 mesh) silica gel and C-18 reversed phase silica gel. Highperformance liquid chromatography (HPLC) purifications were performed on Waters Associates liquid chromatograph equipped with a Model 486 tunable absorbance detector and refractive index detector (R 401) and Shimadzu UFLS liquid chromatograph equipped with SPD-20A tunable absorbance detector. The purity of final compounds was determined by HPLC, and they were judged at least 99% pure. Two HPLC columns (9.4 mm × 25 cm Zorbax-Sil and Zorbax RX-C18) were used as indicated in Table 2 (Supporting Information). The purity and identity of the synthesized vitamins were additionally confirmed by inspection of their 1H NMR and high-resolution mass spectra. 2-{(E)-2’-[(3”S,5”R)-3”,5”-bis-[(tert-butyldimethylsilyl)oxy]-2”-methyl-cyclohex-1”-enyl]vinyl}-4,4,5,5-tetramethyl-[1,3,2]-dioxaborolane (17) and 2-{(Z)-2’-[(3”S,5”R)-3”,5”-Bis[(tert-butyldimethylsilyl)oxy]-2”-methyl-cyclohex-1”-enyl]-vinyl}-4,4,5,5-tetramethyl[1,3,2]-dioxaborolane (18). Et3N (564 µL, 4.0 mmol), i-Pr3P (1 drop), [RhCl2(cod)]2 (6 mg, 0.012 mmol) and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1 M in THF, 0.80 mL, 0.80 mmol) were dissolved in cyclohexane (4 mL). After 30 min a solution of the enyne 16 (400 mg, 1.052 mmol) in cyclohexane (4 mL) was added via cannula and the reaction mixture was stirred at room temperature for 1 h. The solvent was removed under reduced pressure to give a residue which was applied to a silica Sep-Pak (10 g). Elution with hexane/Et2O (99.5:0.5) provided unreacted substrate 16 (200 mg) and a mixture of isomeric boronates (252 mg). Final separation of the boronates 17 and 18 was achieved by HPLC (10 mm × 25 cm Phenomenex Luna Silica column, 4 mL/min) using a hexane/ethyl acetate (97:3) solvent system. The E-isomer 17 was



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collected at Rv 34 mL (48 mg, 9%; 18% considering the recovered substrate) and the Z-isomer 18 at Rv 28 mL (198 mg, 37%; 74% considering the recovered substrate). 1α-[(tert-Butyldimethylsilyl)oxy]-25-hydroxy-9-methyl-previtamin D3 tert-butyldimethylsilyl ether (19). An aqueous solution of K3PO4 (2M; 1.36 mL) and PdCl2(PPh3)2 (4 mg, 4.8 µmol) were added to a solution of the boronate 18 (198 mg, 0.39 mmol) and iodide 14 (40 mg, 0.1 mmol) in THF (10 mL). The reaction mixture was stirred vigorously for 2 h and then extracted with Et2O. The combined organic layers were dried (MgSO4) and concentrated. The residue was purified by flash chromatography on silica gel. Elution with hexane/Et2O (98:2) afforded the previtamin 19 (51 mg, 78%) as a colorless oil. 1α,25-Dihydroxy-9-methyl-previtamin D3 (11). Tetrabutylammonium fluoride (1.0 M in THF; 1.6 mL, 1.6 mmol) was added to a solution of protected previtamin D compound 19 (51 mg, 0.08 mmol) in dry THF (4 mL) at room temperature. The reaction mixture was stirred overnight and quenched by addition of brine, extracted with EtOAc, dried (MgSO4) and concentrated. The residue was purified by flash chromatography on silica gel. Elution with EtOAc gave the deprotected previtamin 11 (31 mg, 91%) as a colorless oil. 1α,25-Dihydroxy-9α-methyl-vitamin

D3

(5),

1α,25-dihydroxy-9-methylene-10(S),19-

dihydrovitamin D3 (7) and 1α,25-dihydroxy-9-methylene-10(R),19-dihydrovitamin D3 (8). A solution of the previtamin 11 (31 mg, 0.073 mmol) in isooctane (18 mL) was refluxed under argon in the darkness for 14 h and then concentrated. The isomeric compounds were separated by HPLC (9.4 mm × 25 cm Eclipse XDB-C18 column, 4 mL/min) using a methanol/water (86:14) solvent system. The 10α-methyl analogue 8 was collected at Rv 40 mL (4.6 mg, 15%), 10βmethyl vitamin 7 at Rv 43 mL (8.7 mg, 28%), 9α-methyl analogue 5 at Rv 46 mL (1.9 mg, 6%) and the unreacted previtamin 11 at Rv 55 mL (12.4 mg, 40%). Taking into account the recovered


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substrate, the yields of the isolated products 5, 7 and 8 were 10, 46 and 25%, respectively, based on recovered starting material. (3S,5R)-3,5-Bis-[(tert-butyldimethylsilyl)oxy]-1-tributylstannylethynyl-cyclohexene (20). nHexyllithium (2.3 M in hexane; 0.18 mL, 0.41 mmol) was slowly added to a stirred solution of the enyne 15 (138 mg, 0.38 mmol) in dry THF (3 mL) at -78 ºC. After 1 h, freshly distilled tri-nbutyltin chloride (0.13 mL, 0.45 mmol) was added, and the mixture was stirred at -78 ºC for 15 min and then at room temperature for 2 h. The reaction was quenched by addition of water. The mixture was diluted with Et2O and washed with saturated NaHCO3. The organic phase was dried (MgSO4) and concentrated. The residue was purified by flash chromatography on C-18 reversed phase silica gel. Elution with acetonitrile gave the compound 20 (190 mg, 77%). 1α,3β-Bis[(tert-butyldimethylsilyl)oxy]-9-methyl-25-hydroxy-19-nor-9,10-secocholesta5(10),8-dien-6-yne (21). (a) A catalytic amount of Pd(PPh3)4 (5 mg, 4 µmol) and LiCl (16 mg, 0.38 mmol; dried under vacuum) were successively added to a solution of compound 20 (36 mg, 0.054 mmol) and iodide 14 (20 mg, 0.050 mmol) in dry THF (3 mL). The resulting mixture was refluxed in the darkness for 3 h, poured into water and extracted with Et2O. The combined organic extracts were dried (MgSO4) and concentrated. The residue was purified by flash chromatography on silica gel. Elution with hexane/Et2O (98:5) gave the dienyne 21 (29 mg, 83%) as a colorless oil. (b) CuI (6 mg, 0.02 mmol) and Pd(PPh3)2Cl2 (6 mg, 0.006 mmol) were successively added to a solution of the iodide 14 (10 mg, 0.025 mmol) and the enyne 15 (37 mg, 0.1 mmol) in dry Et2NH (0.2 mL) at 0 ºC. The mixture (protected from light) was stirred at this temperature for 1 h and then at room temperature for 3 h. Saturated solution of NH4Cl was added and the mixture was extracted with Et2O. The combined organic layers were dried (MgSO4) and concentrated to give



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an oily residue which was purified by flash chromatography on silica gel. Elution with hexane/Et2O (95:5) afforded the dienyne 21 (15 mg, 92%) as a colorless oil. 1α-[(tert-Butyldimethylsilyl)oxy]-25-hydroxy-9-methyl-19-norprevitamin

D3

tert-

butyldimethylsilyl ether (22). (a) Lindlar catalyst (20 mg) and a 0.5% (v/v) solution of quinoline in hexane (38 µL) were successively added to a solution of the dienyne 21 (15 mg, 0.023 mmol) in hexane (7 mL). The mixture was stirred under a hydrogen atmosphere for 4 h until all starting material disappeared (TLC control). The reaction mixture was filtered through Celite and the filtrate was concentrated under vacuum. The residue was purified by flash chromatography on silica gel. Elution with hexane/Et2O (95:5) furnished the previtamin 22 (13.5 mg, 91%) as a colorless oil. (b) An aqueous solution of K3PO4 (2 M, 0.7 mL) and PdCl2(PPh3)2 (2 mg, 2.5 µmol) were successively added to a solution of the boronate 24 (33 mg, 0.067 mmol; prepared as described below) and iodide 14 (18 mg, 0.049 mmol) in dry THF (3 mL). The mixture was stirred vigorously at room temperature for 2 h and then extracted with Et2O. The combined organic layers were dried (MgSO4) and concentrated in vacuo to give a residue which was purified by flash chromatography on silica gel. Elution with hexane/Et2O (99:1) gave the previtamin 22 (25 mg, 79%). 1α-[(tert-Butyldimethylsilyl)oxy]-25-hydroxy-9-methylene-19-norvitamin

D3

tert-

butyldimethylsilyl ether (23). A solution of the previtamin 22 (13.5 mg, 0.021 mmol) in isooctane (7 mL) was refluxed in the darkness for 6.5 h and then concentrated under vacuum. The resulting product was purified by flash chromatography on silica gel. Elution with hexane/Et2O (95:5) gave the protected vitamin 23 (13.5 mg, 100%) as a colorless oil.


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1α,25-Dihydroxy-9-methylene-19-norvitamin D3 (6). Tetrabutylammonium fluoride (1.0 M in THF; 1.05 mL, 1.05 mmol) was added to a solution of the protected vitamin D analogue 23 (13.5 mg, 0.021 mmol) in dry THF (3 mL) at room temperature. The reaction mixture was stirred overnight and quenched by the addition of brine, extracted with EtOAc, dried (MgSO4) and concentrated in vacuum. The residue was purified by HPLC [(10 mm × 25 cm Phenomenex Luna Silica column, 4 mL/min, hexane/2-propanol (8:2)]. The vitamin D compound 6 was collected at Rv 67 mL. Final purification of the product was achieved by reversed-phase HPLC [(9.4 mm × 25 cm Eclipse XDB-C18 column, 4 mL/min, methanol/water (88:12)]. The 9-methylene-19norcalcitriol (6) was collected at Rv 28 mL (7.5 mg, 88%). 2-{(Z)-2’-[(3”S,5”R)-3”,5”-bis-[(tert-butyldimethylsilyl)oxy]-cyclohex-1”-enyl]-vinyl}4,4,5,5-tetramethyl-[1,3,2]-dioxaborolane (24) and

2-{(E)-2’-[(3”S,5”R)-3”,5”-bis-[(tert-

butyldimethylsilyl)oxy]-cyclohex-1”-enyl]-vinyl}-4,4,5,5-tetramethyl-[1,3,2]-dioxaborolane (25). Dry Et3N (290 µL, 2.1 mmol), i-Pr3P (1 drop), [RhCl2(cod)]2 (3 mg, 0.006 mmol) and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1 M in THF, 0.42 mL, 0.42 mmol) were successively dissolved in cyclohexane (1 mL). After 30 min, a solution of the enyne 15 (200 mg, 0.545 mmol) in cyclohexane (2 mL) was added via cannula and the resulting mixture was stirred for 1 h. The solvent was removed under vacuum to give a residue which was applied to silica Sep-Pak (5 g). Elution with hexane/Et2O (99.5:0.5) provided the unreacted substrate 15 (44%) and a mixture of isomeric boronates. Final separation of the boronates 24 and 25 was achieved by HPLC [(10 mm × 25 cm Phenomenex Luna Silica column, 4 mL/min, hexane/ethyl acetate (98.5:1.5)]. The Eisomer 25 was collected at Rv 55 mL (8%; 15% based on recovered substrate) and the Z-isomer 24 at Rv 60 mL (37%; 66% based on recovered substrate).



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(1R,3aR,5R,7aR)-1-[(R)-1’,5’-Dimethyl-5’-[(triethylsilyl)oxy]-hexyl]-5-ethyl-7a-methyloctahydro-inden-4-one (27). A solution of the ketone 13 (500 mg, 1.27 mmol) in dry THF (7 mL) was added dropwise to a solution of LDA (2.0 M in THF/heptane/ethylbenzene; 1.27 mL, 2.53 mmol) in dry THF (7 mL) under argon at -78 °C, followed by addition of DMPU (0.12 mL, 1.01 µmol). The solution was warmed to -30 °C and stirred at this temperature for 1 h. Then it was cooled to -78 °C and ethyl iodide (0.41 mL, 5.09 mmol) was added. The reaction mixture was allowed to reach 10 °C during 1 h, stirred at this temperature for 5 h and then quenched by addition of water. The product was extracted with Et2O, the combined organic extracts were washed with brine, dried (MgSO4) and concentrated in vacuo. The oily residue was purified by flash chromatography on silica gel. Elution with hexane/Et2O (98:2) gave the ethylated ketone 27 (354 mg, 66%) as a colorless oil. 1α,3β-Bis[(tert-butyldimethylsilyl)oxy]-9α-methyl-25-[(triethylsilyl)oxy]-19-nor-9,10secocholest-5(10)-en-6-yn-8β-ol (28). n-Butyllithium (1.6 M in hexane; 165 µL, 0.264 mmol) was added dropwise to a stirred solution of the enyne 15 (88 mg, 0.24 mmol) in anhydrous THF (1.5 mL) at -78 ºC. After 5 min, CeCl3 (65 mg, 0.264 mmol) was added and the resulting suspension was stirred at -78 ºC for 30 min. A solution of ketone 26 (88 mg, 0.216 mmol) in dry THF (1.5 mL) was added. The mixture was stirred for 15 min and warmed to room temperature. The reaction was quenched after 1 h by addition of saturated NH4Cl. The resulting mixture was extracted with Et2O and the combined organic layers were washed with brine, dried (MgSO4) and concentrated in vacuo. The residue was purified by flash chromatography on silica gel. Elution with hexane/Et2O (98:2) gave the enynol 28 (112 mg, 60%) as a colorless oil. 1α,3β-Bis[(tert-butyldimethylsilyl)oxy]-9α-ethyl-25-[(triethylsilyl)oxy]-19-nor-9,10secocholest-5(10)-en-6-yn-8β-ol (29). Following the procedure described above for preparation of 28, the alcohol 29 was obtained by coupling of the ketone 27 with the anion of the enyne 15,


Page 19 of 45

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performed analogously to the process described above for the preparation of 28. The product was purified by flash chromatography on silica gel. Elution with hexane/Et2O (98:2) gave the enynol 29 (68%) as a colorless oil. 1α,3β-Bis[(tert-butyldimethylsilyl)oxy]-9-methyl-25-[(triethylsilyl)oxy]-19-nor-9,10secocholesta-5(10),8-dien-6-yne (30). A solution of alcohol 28 (112 mg, 0.144 mmol) in dry toluene

(5

mL)

was

added

dropwise

to

a

solution

of

methyl

N-

(triethylammoniumsulfonyl)carbamate (69 mg, 0.289 mmol) in dry toluene (5 mL). The reaction mixture was heated at 50 ºC under stirring for 30 min. The mixture was poured into brine and extracted with Et2O. The combined organic layers were dried (MgSO4) and evaporated to give a residue, which was purified by flash chromatography on silica gel. Elution with hexane/Et2O (98:2) gave the dienyne 30 (134 mg, 95%) as a colorless oil. 1α,3β-Bis[(tert-butyldimethylsilyl)oxy]-9-ethyl-25-[(triethylsilyl)oxy]-19-nor-9,10secocholesta-5(10),8-dien-6-yne (31). Dehydration of the alcohol 29 with methyl N(triethylammoniumsulfonyl)carbamate was performed as described above for the preparation of 30. The product was purified by flash chromatography on silica gel. Elution with hexane/Et2O (98:2) gave the dienyne 31 (96%) as a colorless oil. 1α-[(tert-Butyldimethylsilyl)oxy]-9-methyl-25-[(triethylsilyl)oxy]-19-norprevitamin D3 tertbutyldimethylsilyl ether (32). The previtamin 32 was obtained by semihydrogenation of the dienyne 30, as described above for the conversion of 21 to 22. The product was purified by flash chromatography on silica gel. Elution with hexane furnished the previtamin 32 (89% yield) as a colorless oil. 1α-[(tert-Butyldimethylsilyl)oxy]-9-ethyl-25-[(triethylsilyl)oxy]-19-norprevitamin

D3

tert-

butyldimethylsilyl ether (33). The previtamin 33 was obtained by hydrogenation of the dienyne 31, performed analogously to the process described above for the conversion of 21 to 22.



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Purification of the product by flash chromatography (hexane) on silica gel gave the previtamin 33 (78% yield) as a colorless oil. 1α-[(tert-Butyldimethylsilyl)oxy]-25-[(triethylsilyl)oxy]-9-methylene-19-norvitamin D3 tertbutyldimethylsilyl ether (34). A solution of the previtamin 32 (119 mg, 0.157 mmol) in isooctane (50 mL), protected from light, was refluxed for 6 h and then concentrated. The resulting product was purified by flash chromatography on silica gel. Elution with hexane furnished the protected vitamin 34 (119 mg, 100%) as a colorless oil. (9E)-1α-[(tert-Butyldimethylsilyl)oxy]-25-[(triethylsilyl)oxy]-9-ethylidene-19-norvitamin D3 tert-butyldimethylsilyl ether (35). A solution of the previtamin 33 (99 mg, 0.128 mmol) in isooctane (40 mL), protected from light, was refluxed for 7 h and then concentrated. The resulting product was purified by flash chromatography (hexane) on silica gel to give the protected vitamin 35 (99 mg, 100%) as a colorless oil. 1α,25-Dihydroxy-9-methylene-19-norvitamin D3 (6). The silyl protecting groups in the vitamin D compound 34 were removed by treatment with tetrabutylammonium fluoride, following the same experimental procedure as described above for vitamin 23. HPLC purification afforded the vitamin D analogue 6 in 89% yield. (9E)-1α,25-Dihydroxy-9-ethylidene-19-norvitamin D3 (9). Removal of silyl protecting groups in 35 by treatment with TBAF, following the same procedure as for the diprotected vitamin D compound 23, afforded a product that was purified by HPLC [(10 mm × 25 cm Phenomenex Luna Silica column, 4 mL/min, hexane/2-propanol (8:2)]. The vitamin D compound 9 was collected at Rv 54 mL. Final purification of the product was achieved by reversed-phase HPLC [(9.4 mm × 25 cm Eclipse XDB-C18 column, 4 mL/min, methanol/water (85:15)]. 9-Ethylidene19-norcalcitriol (9) was collected at Rv 54 mL (90%).


Page 21 of 45

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1α,25-Dihydroxy-9β-methyl-19-norvitamin

D3

(4).

Tris(triphenylphosphine)-rhodium(I)

chloride (31 mg, 0.034 mmol) was added to dry benzene (freshly distilled from CaH2; 20 mL) presaturated with hydrogen. The mixture was stirred under H2 at room temperature until homogeneous solution was formed (ca. 30 min). A solution of the vitamin compound 6 (14 mg, 0.034 mmol) in benzene (5 mL) was then added and the reaction mixture was allowed to proceed under a continuous stream of H2 for 3 h. Benzene was removed under vacuum and the residue was purified by HPLC [(10 mm × 25 cm Phenomenex Luna Silica column, 4 mL/min, hexane/2propanol (8:2)]. The vitamin D compound 4 was collected at Rv 36 mL. Final purification of the product was achieved by reversed-phase HPLC [(9.4 mm × 25 cm Eclipse XDB-C18 column, 4 mL/min, methanol/water (85:15)]. 9β-Methyl-19-norcalcitriol (4) was collected at Rv 49 mL (8 mg, 56%).

Biological Studies 1. In vitro Studies. Measurement of VDR Binding Activity. Measurement of binding ability of the synthesized vitamin D analogues to the VDR was carried out using purified full-length rat recombinant receptor.29 The protein was diluted in TEDK50 (50 mM Tris, 1.5 mM EDTA, pH 7.4, 5 mM DTT, 150 mM KCl) with 0.1% Chaps detergent. The radiolabeled ligand [3H-1α,25(OH)2D3, ~ 159 Ci/mmol] was added in ethanol at a final concentration of 1 nM. The radiolabeled and unlabeled ligands were added to 100 µL of the diluted protein at a final ethanol concentration of ≤ 10%, then mixed and incubated overnight on ice to reach binding equilibrium. The following day, 100 µL of hydroxylapatite slurry (50%) was added to each tube and mixed at 10-min intervals for 30 min. The hydroxylapatite was collected by centrifugation and then washed three times with Tris–EDTA buffer (50 mM Tris, 1.5 mM EDTA, pH 7.4) containing



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0.5% Triton X-100. After the final wash, the pellets were transferred to scintillation vials containing 4 mL of Biosafe II scintillation cocktail, mixed and placed in a scintillation counter. Total binding was determined from the tubes containing only the radiolabeled ligand. The experiments were carried out in duplicate on two to three different occasions. The Ki values are derived from dose-response curves and represent the inhibition constant when radiolabeled 1α,25-(OH)2D3 is present at 1 nM and a Kd of 0.2 nM is used. Measurement of Cellular Differentiation. Human promyelocytic leukemia (HL-60) cells were grown in RPMI-1640 medium containing 10% fetal bovine serum at 37 °C in the presence of 5% CO2. HL-60 cells were plated at 1.2 × 105 cells/plate. Eighteen hours after plating, cells in duplicate were treated with the tested compound so that the final concentration of ethanol was less than 0.2%. Four days later, the cells were harvested and a nitro blue tetrazolium (NBT) reduction assay was performed. The percentage of differentiated cells was determined by counting a total of 200 cells and recording the number that contained intracellular black-blue formazan deposits.30 The ED50 values are derived from the dose-response curves and represent the tested compound concentration capable of inducing 50% maturation. The experiment was repeated 2 to 3 times, and the results are reported as the mean. Verification of differentiation to monocytic cells was determined by measuring phagocytic activity (data not shown). Measurement of Transcriptional Activity. The ability of the vitamin D compounds to induce transcription in rat osteosarcoma cells was established using 24-hydroxylase (CYP-24) luciferase reporter gene system. Transcriptional activity was measured in ROS 17/2.8 (bone) cells that were stably transfected with a 24-hydroxylase (24OHase) gene promoter upstream of a luciferase reporter gene.31 Cells were subjected to a range of doses. Sixteen hours after dosing, the cells were harvested and luciferase activities were measured using a luminometer. The ED50 values are derived from dose-response curves and represent the analogue concentration capable


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of increasing the luciferase activity by 50%. Each experiment was performed in duplicate two to three separate times. 2. In vivo Studies. Bone calcium mobilization and intestinal calcium transport. Male, weanling SpragueDawley rats were purchased from Harlan (Indianapolis, IN). The animals were group housed and placed on Diet 11 (0.47% Ca) + AEK oil for one week followed by Diet 11 (0.02% Ca) + AEK oil for 3 weeks. The rats were then switched to a diet containing 0.47% Ca3221 for one week, followed by two weeks on a diet containing 0.02% Ca. Dose administration began during the last week on 0.02% Ca diet. Four consecutive intraperitoneal doses were given approximately 24 hours apart. Twenty four hours after the last dose, blood was collected from the severed neck and the concentration of serum calcium determined as a measure of bone calcium mobilization. The first 10 cm of the intestine was also collected for the intestinal calcium transport analysis using the everted gut sac method.29 All animals were managed in accordance with University of Wisconsin standards and protocols for animal care and use. Our experiments were approved by the College of Agricultural and Life Sciences Institutional Animal Care and Use Committee. Molecular Modeling. The synthesized analogues 4, 5 and 6 were docked into vitamin D receptor using Gold (release 5.1) software package. The Builder module, available in the InsightII suite of programs,33 allowed us to construct new analogues by modifying the structure of 1α,25-(OH)2D3 extracted from crystalline VDR(LBD)-1 complex (PDB Code: 1DB1). The new structures were energy-minimized (cvff potentials) with the Discover module in InsightII. In the next step, the minimized structures of the new ligands 4, 5 and 6 (in all cases with their Aring assuming the β-conformation with equatorially oriented 1α-hydroxyl groups) were docked



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Page 24 of 45

into the VDR(LBP). For final consideration, only the lowest energy complexes were selected, possessing tryptophan oriented parallel to the ligand’s 5,7-diene moiety. Crystallographic studies Crystal data for 9β-methyl-19-norcalcitriol (4). C27H46O3, M = 417.63, T = 100 K, orthorhombic, space group P212121, Z = 4, a = 6.5288(8), b = 19.093(2), c = 19.969(2) Å, αβγ = 90°, V = 2489.2(5) Å3, Dx = 1.114 g·cm-3, 4381 unique data (2θmax = 25.000), 2429 with F02 > 2σ (F02), R = 0.0935, Rw = 0.2518, S = 1.049. Structure determination. The structure of the vitamin 4 was determined in a singlecrystal X-ray diffraction measurement on a Kuma KM4CCD κ-axis diffractometer with graphitemonochromated Mo Kα radiation (λ = 0.71073 A, 50.0 kV, 40.0 mA) at 100 K, using the Oxford Cryostream cooling device. The 1369 frames were measured with scan width of 1.00o and counting time of 60 s. Indexing, integration and scaling were performed with original KUMA Diffraction software.34 The 11866 reflections were measured and merged to 8607 unique reflections, of which 4226 were rejected during the refinement. The analytical absorption correction was applied in the scaling procedure. The structure solution and refinement were performed using merged reflections. The structure was solved by direct approach35 using the SHELXS-97 program and refined with the SHELXL-97.36 The refinement was based on F2 for all reflections except those with negative intensities. Weighted R factors wR and all goodness-of-fit S values were based on F2, whereas conventional R factors were based on the amplitudes, with F set to zero for negative F2. The F02> 2σ(F02) criterion was applied only for R factors calculation and was not relevant to the choice of reflections for the refinement. The R factor based on F2 is about twice as large as the one based on F. The hydrogen atoms were located in idealized geometrical positions. Scattering


Page 25 of 45

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factors were taken from Tables 4.2.6.8 and 6.1.1.4 from the International Tables for Crystallography.37 Crystallographic data for the structure reported in this paper have been deposited at the Cambridge Crystallographic Data Centre with the deposition number CCDC-1413876.

ASSOCIATED CONTENT Supporting Information Available: Purity criteria of the newly synthesized vitamin D analogues 4, 6 and 9, their 1H and

13

C NMR spectra, and spectral data of all the synthesized

compounds, figures with either competitive binding curves or dose-response curves derived from cellular differentiation and transcriptional assays of the vitamin D analogue 4. CCDC-1413876 contains the supplementary crystallographic data for this article. These data can be obtained free of

charge

from

the

Cambridge

Crystallographic

Data

Centre

via

www.ccdc.cam.ac.uk/data_request/cif. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *Prof. Antonio Mouriño. Telephone: +34 981 563100 ext. 14254. Fax: +34 983 595012. E-mail: [email protected]. *Prof. Rafal R. Sicinski. Telephone: +48 22 8220211 ext. 216. Fax: +48 22 8225996. E-mail: [email protected].

ACKNOWLEDGMENTS



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Page 26 of 45

U.K. thanks the Foundation for Polish Science; the MPD Program has been co-financed by the EU European Regional Development Fund. H.F.D and L.A.P. thank the Wisconsin Alumni Research Foundation for financial support. A.M. thanks the Spanish Ministry of Education and Innovation (SAF2010-15291) and Xunta de Galicia (project GPC2014/001) for financial support. The authors gratefully acknowledge William Blaser and Erin Gudmundson from University of Wisconsin-Madison (USA) for their excellent technical assistance.

ABBREVIATION USED 1α,25-(OH)2D3, 1α,25-dihydroxyvitamin D3; VDR, vitamin D receptor.


Page 27 of 45

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rearrangement of the first analogue of (7Z)-vitamin D. J. Org. Chem. 1991, 56, 3582-3587. 10

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14


Kulesza U.; Sigüeiro R.; Mouriño A.; Sicinski R. R. Synthesis of 9-alkylated calcitriol and two

1α,25-dihydroxy-9-methylene-10,19-dihydrovitamin D3 analogues with a non-natural triene system by thermal sigmatropic rearrangements. J. Org. Chem. 2013, 78, 1444-1450. 15

Sicinski, R. R.; DeLuca, H. F. Ruthenium tetroxide oxidation of Grundmann’s ketone derived

from vitamin D3. Bioorg. Med. Chem. Lett. 1995, 5, 159-162. 16

Sarandeses, L. A.; Mascareñas, J. L.; Castedo, L.; Mouriño, A. Synthesis of 1,25-dihydroxy-

19-norprevitamin D3. Tetrahedron Lett. 1992, 33, 5445-5448. 17

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dihydroxyvitamin D synthons. Tetrahedron Lett. 1987, 28, 2099-2102. (b) Aurrecoechea, J. M.; Okamura, W. H. A short, enantiospecific synthesis of the 1α-hydroxyvitamin D enyne A-ring synthon. Tetrahedron Lett. 1987, 28, 4947-4950. (c) Okamura, W. H.; Aurrecoechea, J. M.; Gibbs, R. A.; Norman, A. W. Synthesis and biological activity of 9,11-dehydrovitamin D3 analogues: stereoselective preparation of 6β-vitamin D vinylallenes and a concise enynol synthesis for preparing the A-ring. J. Org. Chem. 1989, 54, 4072-4083. 18

Part of this work has previously been communicated: Kulesza U.; Mouriño A.; Plum L. A.;

DeLuca H. F.; Sicinski R. R. Synthesis of novel 19-norvitamin D3 analogs with unnatural triene system. J. Steroid Biochem. Mol. Biol. 2013, 136, 23-26. 19

(a) Oh-e, T.; Miyaura, N.; Suzuki, A. Palladium-catalyzed cross-coupling reaction of aryl or

vinylic triflates with organoboron compounds. Synlett 1990, 4, 221-223. (b) Oh-e, T.; Miyaura, N.; Suzuki, A. Palladium-catalyzed cross-coupling reaction of organoboron compounds with organic triflates. J. Org. Chem. 1993, 58, 2201-2208.



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20

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Ohmura, T.; Yamamoto, Y.; Miyaura, N. Rhodium- or iridium-catalyzed trans-hydroboration

of terminal alkynes, giving (Z)-1-alkenylboron compounds. J. Am. Chem. Soc. 2000, 122, 49904991. 21

Gogoi, P.; Sigüeiro, R.; Eduardo, S.; Mouriño, A. An expeditious route to 1α,25-

dihydroxyvitamin D3 and its analogues by an aqueous tandem palladium-catalyzed A-ring closure and Suzuki coupling to the C/D unit. Chem. Eur. J. 2010, 16, 1432-1435. 22

Sicinski, R. R. Photochemical and thermal isomerizations of C-19 functionalized previtamin D

analogs in the androstane series. Acta Chim. Hung. 1992, 129, 191-200. 23

Huitric, A. C.; Carr, J. B.; Trager, W. F.; Nist, B. J. Configurational and conformational

analysis: axial-axial and axial-equatorial coupling constants in six-membered ring compounds. Tetrahedron 1963, 19, 2145-2151. 24

(a) Wing, R. M.; Okamura, W. H.; Sine, S. M.; Norman, A. W. Vitamin D in solution:

conformations of vitamin D3, 1α,25-dihydroxyvitamin D3, and dihydrotachysterol D3. Science 1974, 186, 939-941. (b) Bouillon, R.; Okamura, W. H.; Norman, A. W. Structure-function relationship in the vitamin D endocrine system. Endocr. Rev. 1995, 16, 200-257. 25

In vitro studies (VDR binding, HL-60 differentiation and transcriptional assay) as well as

measurement of intestinal calcium transport and bone calcium mobilization were performed as previously described: Glebocka, A.; Sicinski, R.R.; Plum, L.A.; Clagett-Dame, M.; DeLuca, H.F. J. Med. Chem. 2006, 49, 2909-2920. 26

Yamada, S.; Yamamoto, K. Ligand recognition by vitamin D receptor: Total alanine scanning

mutational analysis of the residues lining the ligand binding pocket of vitamin D receptor. Curr. Top. Med. Chem. 2006, 6, 1255-1265.


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27


Sicinska, W.; Westler, W. M.; DeLuca, H. F. NMR assignments of tryptophan residue in apo

and holo LBD-rVDR. Proteins 2005, 61, 461-467. 28

Rochel, N.; Wurtz, J. M.; Mitschler, A.; Kloholz, B.; Moras, D. The crystal structure of the

nuclear receptor for vitamin D bound to its natural ligand. Mol. Cell 2000, 5, 173-179. 29

Martin, D. L.; DeLuca, H. F. Influence of sodium on calcium transport by the rat small

intestine. Am. J. Physiol. 1969, 216, 1351-1359. 30

Ostrem, V. K.; Lau, W. F.; Lee, S. H.; Perlman, K.; Prahl, J.; Schnoes, H. K.; DeLuca, H. F.;

Ikekawa, N. Induction of monocytic differentiation of HL-60 cells by 1,25-dihydroxyvitamin D analogues. J. Biol. Chem. 1987, 262, 14164-14171. 31

Arbour, N. C.; Ross, T. K.; Zierold, C.; Prahl, J, M.; DeLuca, H. F. A highly sensitive method

for large-scale measurements of 1,25-dihydroxyvitamin D. Anal. Biochem. 1998, 255, 148-154. 32

Suda, T.; DeLuca, H. F.; Tanaka, Y. Biological activity of 25-hydroxyergocalciferol in rats. J.

Nutr. 1970, 100, 1049-1052. 33

InsightII, Discover, and Search and Compare Biopolymer are trademarked software of

Accelrys Inc., San Diego, CA. 34

Oxford Diffraction CrysAlis CCD, Oxford Diffraction Ltd., Version 1.171.33.66 and Oxford

Diffraction CrysAlis RED, Oxford Diffraction Ltd., Version 1.171.28cycle2beta; analytical numeric absorption correction using a multifaceted crystal model based on expressions derived by R. C. Clark & J. S. Reid. 35

Sheldrick, G. M. Phase annealing in SHELX-90: direct methods for larger structures. Acta

Crystallogr., Sect. A 1990, 46, 467-473. 36

G. M. Sheldrick. SHELXL93. Program for the Refinement of Crystal Structures; Univ. of

Göttingen, Germany.



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International Tables for Crystallography; A. J. C. Wilson, Ed., Kluwer: Dordrecht, 1992; Vol.

C.


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Figure captions Figure 1. Chemical structures of 1α,25-(OH)2D3 (calcitriol, 1), 19-norcalcitriol (2), their analogues and precursors used in the synthesis. Figure 2. ORTEP drawing derived from the single-crystal X-ray analysis of 9β-methyl-19norcalcitriol (4). Ellipsoids are drawn at the 50% probability level. Figure 3. Bone calcium mobilization and intestinal calcium transport of 1α,25-(OH)2D3 (1) and 9β-methyl-19-norcalcitriol (4). Figure 4. Bone calcium mobilization and intestinal calcium transport of 1α,25-(OH)2D3 (1), 9α-methyl-calcitriol (5), and 9-alkylidene-19-norcalcitriols 6 and 9. Figure 5. Calculated structures of complexes VDR(LBD)-4 (a), VDR(LBD)-5 (b) and VDR(LBD)-6 (c). Ligand interactions with Trp-286, Tyr-295 and hydrogen-bond forming residues (pink) are shown; hydrogen bonds are marked in yellow. Hydrogens are omitted for clarity.



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Table 1. Relative VDR Binding Activities,a HL-60 Differentiating Activities,b and Transcriptional Activitiesc of the Vitamin D Hormone (1) and the Vitamin D Analogues 4-9.

Compound structure

VDR bindinga

24-OHase transcriptionc

No.

Ki (M)

ratio

ED50 (M)

ratio

ED50 (M)

ratio

1

1 × 10-10

1

2 × 10-9

1

3 × 10-10

1

2 × 10-10

0.5

9 × 10-9

0.2

3 × 10-10

1

5

2 × 10-8

0.005

5 × 10-6

~ 10-4

5 × 10-8

0.006

6

2 × 10-8

0.005

1× 10-6

0.002

5 × 10-8

0.006

7

> 10-5

< 10-5

~ 10-5

~ 10-4

~ 10-6

~ 10-4

8

> 10-5

< 10-5

~ 10-5

~ 10-4

9 × 10-8

0.003

9

> 10-5

< 10-5

~ 10-5

~ 10-4

~ 10-6

~ 10-4

4

a

HL-60 differentationb

Competitive binding of 1α,25-(OH)2D3 (1) and the synthesized vitamin D analogues to the full-

length recombinant rat vitamin D receptor. The experiments were carried out in duplicate on two


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different occasions. The Ki values are derived from the dose-response curves and represent the inhibition constant when radiolabeled 1α,25-(OH)2D3 is present at 1 nM and a Kd of 0.2 nM is used. The numbers shown in the Table are expressed as the average ratio of the 1α,25-(OH)2D3 Ki to the Ki for the analogue. bInduction of differentiation of HL-60 promyelocytes to monocytes by 1α,25-(OH)2D3 (1) and the synthesized vitamin D analogues. Differentiation state was determined by measuring the percentage of cells reducing nitro blue tetrazolium (NBT). The experiment was repeated in duplicate two times. The ED50 values are derived from the doseresponse curves and represent the analogue concentration capable of inducing 50% maturation. The numbers shown in the Table are expressed as the average ratio of the 1α,25-(OH)2D3 ED50 to the ED50 for the analogue. cTranscriptional assay in rat osteosarcoma cells stably transfected with a 24-hydroxylase gene reporter plasmid. The ED50 values are derived from dose-response curves and represent the analogue concentration capable of increasing the luciferase activity by 50%. The numbers shown in the Table are expressed as the average ratio of the 1α,25-(OH)2D3 ED50 to the ED50 for the analogue.



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Figure 1


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Figure 2



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Figure 3


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Figure 4



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Figure 5


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Scheme 1



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R

SnBu3 14, Pd(PPh3)4, LiCl THF, reflux

77%

TBSO

R=

H

1. n-HexLi, THF 2. Bu3SnCl -78 oC rt OTBS

H

83%

20 TBSO 14, PdCl2(PPh3)2, Et2NH, CuI, 0 oC

OTBS 21

H2, Lindlar's catalyst quinoline, hexane, rt 91%

rt

92%

15

B O

PinB-H, [RhCl(cod)]2 i-Pr3P, Et3N, CyH, rt

OH

R

14, PdCl2(PPh3)2, K3PO4 (2M, aq), THF, rt

O

H

TBSO H

TBSO

79%

OTBS 24 (66%) + 25 (E-isomer, 15%)

TBSO

R

R

H

H

TBAF, THF, rt 89%

HO

OH 6

isooctane 100 oC

H

H

TBSO

OTBS 23

Scheme 2


22

100%

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Scheme 3



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H

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OH

H H2, RhCl(PPh3)3 benzene, rt

H

H

56% HO

OH

HO

6

OH 4

Scheme 4


OH

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Table of Contents Graphic Novel 9-Alkyl- and 9-Alkylidene-substituted 1α,25-Dihydroxyvitamin D3 Analogues: Synthesis and Biological Examinations Urszula Kulesza, Lori A. Plum, Hector F. DeLuca, Antonio Mouriño, and Rafal R. Sicinski


Novel 9-Alkyl- and 9-Alkylidene-Substituted 1Î±,25-Dihydroxyvitamin

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