Synthesis and biological evaluation of calcioic acid

Abstract

Herein, we describe the synthesis of calcioic acid following a recently developed synthetic strategy for calcitroic acid. Several improvements to reaction conditions were made, which resulted in higher yields. The improved workup and isolation procedures are described. Furthermore, we investigated the interaction between the vitamin D receptor (VDR) and calcioic acid. Calcioic acid was able to bind VDR with a binding constant of 71 μM. In cells, calcioic acid reduced the transcription of VDR target gene CYP24A1 in the presence 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) but did not induce the transcription of CYP24A1. Therefore, calcioic acid is a very weak VDR antagonist. With the generation of gram quantities, further studies are expected to reveal if calcioic acid is solely a water-soluble metabolite of vitamin D or if it mediates other biological functions through biomolecules other than VDR.

Graphical Abstract

1. Introduction

Calcioic acid is a metabolite of vitamin D first identified by Reddy et al. in 2006 [1]. It is formed from 25OHD₃ in conjunction with cholacalcioic acid in the kidney. The major metabolizing enzyme of 25OHD₃ and 1,25(OH)₂D₃ is the P450 enzyme, CYP24A1 [2, 3]. The expression of CYP24A1 is high in the kidney and urinary bladder [4]. Additionally, CYP24A1 can be transcriptionally induced by 1,25(OH)₂D₃ in many other cells and tissues including the immune system, endocrine tissue, brain, lung, gastrointestinal tract, and male- and female-specific tissues [5]. 1,25(OH)₂D₃ is the most active vitamin D metabolite and binds the vitamin D receptor (VDR) with very high affinity, which in turn mediates numerous physiological processes such as calcium homeostasis, cell proliferation and cell differentiation through gene transcription [6]. This includes the regulation of CYP24A1 by 1,25(OH)₂D₃ to enable a negative feedback control for 1,25(OH)₂D₃, which induces hypercalcemia at high concentrations [7]. The CYP24A1-mediated metabolism of 1,25(OH)₂D₃ generates calcitroic acid, which was isolated and characterized by DeLuca in 1979 [8]. Several synthetic methods to synthesize significant amounts of calcitroic acid have been introduced [9], including a high-yielding eleven-step synthesis by Meyer et al. [10]; however, a similar scalable method for calcioic acid has not been reported. Recently, we provided a small amount of calcioic acid to the Jones group to support the identification of calcioic acid in CYP24A^−/− and CYP24A^+/− mouse serum after 24,25(OH)₂D₃ administration [11]. Thus, the availability of synthetic standards is paramount to further explore the metabolism of vitamin D. Synthetically generated calcioic acid is expected to enable future in vitro and in vivo studies to elucidate possible physiological functions of this water-soluble vitamin D metabolite. Therefore, we report a scalable synthetic method to generate significant amounts of calcioic acid and evaluate the interaction between calcioic acid and VDR.

2. Experimental

2.1. Chemistry

Ergocalciferol was purchased from variable commercial sources (Alfa Aesar J62163, AstaTech 44109, Research Products International C20300, Sigma-Aldrich 95220, VWR 101172-472). ((Z)-2-((3S,5R)-3,5-bis((tert-butyldimethylsilyl)oxy)-2-methylenecyclohexylidene) ethyl) diphenylphosphine oxide was purchased from ChemScene (CS-M0003). All moisture or oxygen-sensitive reactions were carried out under a dry nitrogen atmosphere. Reaction temperatures refer to the containing bath temperatures. Reactions were monitored by thin-layer chromatography (TLC) using Merck 60 UV254 silica gel plates (Sigma-Aldrich). Visualization was performed with UV light, cerium molybdate general stain followed by heating, and other methods as noted. Synthesized compounds were purified by normal phase flash chromatography (SPI Biotage, silica gel 230-400 mesh) except where noted. Compound characterization was performed via Shimadzu 2020 LC-MS (single quadrupole) instrument. NMR spectra were recorded on a Bruker Avance 500MHz instrument with compounds dissolved in the specified deuterated solvent. Optical rotations were recorded using Jasco DIP-370 Digital Polarimeter instrument in LCMS grade chloroform or methanol.

2.2. (1R,3aR,4S,7aR)-1-((S)-1-hydroxypropan-2-yl)-7a-methyloctahydro-1H-inden-4-ol (2)

Ergocalciferol 1 (3 g, 7.5 mmol) was dissolved in dry pyridine (7 mL) and diluted in dry methanol (250 mL) in a 500 mL round bottom flask with a diffuse bubbler as the inlet for ozone and a slim outlet for the dissipating gas. The reaction mixture was cooled to −78°C and ozone was bubbled through the solution for 4-6 hours using an A2Z Ozone A2ZZ-5G 110 V with 38 g/m³ O₃ output. The reaction was monitored by TLC (UV and cerium molybdate). Many spots occurred initially but reduced to three main spots after 1 to 4 hours. One of the two lowest spots, partially overlapping with the UV-active pyridine spot, was identified as the product. When no more change was apparent after an additional hour of ozonolysis, the reaction mixture warmed up to 0°C and stirred vigorously. Sodium borohydride (2 g, 53 mmol) was added carefully. Once fully dissolved, the reaction was quenched with 70 mL of water and stirred for 5 minutes before concentrating by rotary evaporation. The crude reaction mixture was diluted with 100 mL of water and extracted with DCM (3 x 100 mL). The organic layers were washed with a 5% CuSO₄ solution (100 mL), 1 M HCl (2 x 100 mL) and saturated NaHCO₃ (100 mL), dried over MgSO₄, concentrated, and purified by column chromatography (EtOAc:hexanes 1:3) to yield the product 2 as a white solid (450 mg, 57%). Note that this reaction could not be scaled up, as larger percentages of pyridine were not tolerated, and larger reaction vessels could not be adequately cooled enough to allow for ozone saturation. R_f 0.09 (EtOAc:hexanes 1:3); ¹H-NMR (500 MHz, CDCl₃) δ 0.97 (s, 3H), 1.04 (d, J = 5.0, 3H), 1.14-1.23 (m, 2H), 1.31-1.40 (m, 2H), 1.41-1.53 (m, 5H), 1.54-1.62 (m, 2H), 1.78-1.91 (m, 3H), 1.98-2.04 (m, 1H), 3.38 (dd, J = 5.0, 3.5 Hz, 1H), 3.65 (dd, J = 10, 3.0 Hz, 1H), 4.10 (d, J = 2.0, 1H); ¹³C-NMR: (125 MHz, CDCl₃) δ 13.6, 16.6, 17.4, 22.6, 26.7, 33.5, 38.2, 40.2, 41.8, 52.4, 53.0, 67.6, 69.2; m/z calculated for C₁₃H₂₃O₂ 211 [M-H]^- found 211, ${\left[\alpha \right]}_D^{25}=+24.0$ (c = 1.0 mM, CHCl₃).

2.3. (S)-2-((1R,3aR,4S,7aR)-4-hydroxy-7a-methyloctahydro-1H-inden-1-yl)propyl 4-methyl benzenesulfonate (3)

To a stirred solution of 2 (0.38 g, 1.78 mmol) in dry DCM (20 mL), was added 4-dimethylaminopyridine (DMAP) (0.44 g, 3.58 mmol) and p-toluenesulfonyl chloride (0.36 g, 1.88 mmol). The reaction was stirred overnight at room temperature and then quenched with 1 M HCl (20 mL). The reaction mixture was extracted with DCM (3 x 50 mL), and combined organic layers were washed with water (50 mL), dried over MgSO₄, and purified by column chromatography (EtOAc:hexanes 3:7) to yield 3 as a white crystalline solid (410 mg, 84%). R_f 0.23 (EtOAc:hexanes 1:3); ¹H-NMR (500 MHz, CDCl₃) δ 0.92 (s, 3H), 1.00 (d, J = 5.0 Hz, 3H), 1.13-1.26 (m, 3H), 1.31-1.38 (m, 2H), 1.42-1.51 (m, 3H), 1.52-1.62 (m, 1H), 1.66-1.75 (m, 2H), 1.77-1.88 (m, 2H), 1.92-1.98 (m, 1H), 2.48 (s, 3H), 3.84 (dd, J = 6.5, 3.0 Hz, 1H), 3.98 (dd, J = 6.2, 3.0 Hz, 1H), 4.10 (s, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.5 Hz, 2H); ¹³C-NMR: (125 MHz, CDCl₃) δ 13.5, 16.7, 17.3, 21.6, 22.5, 26.4, 33.6, 40.1, 41.9, 52.2, 52.3, 69.1, 75.6, 127.9 (2C), 129.8 (2C), 133.1, 144.6; m/z calculated for C₂₀H₃₀O₄S 430 [M + Na⁺ + ACN], found 430; ${\left[\alpha \right]}_D^{25}=+19.0$ (c 1.0 mM, CHCl₃)

2.4. (S)-2-((1R,3aR,4S,7aR)-4-((tert-butyldimethylsilyl)oxy)-7a-methyloctahydro-1H-inden-1-yl)propyl 4-methylbenzenesulfonate (4)

To a solution of 3 (0.40 g, 1.09 mmol) in dry DCM (2 mL) at 0°C was added 2,6-lutidine (0.15 mL, 1.31 mmol) and tert-butyl dimethylsilyl trifluoromethanesulfonate (0.35 mL, 1.53 mmol). The reaction was monitored by TLC until completion (2 hours) and quenched with water (2 mL). The crude mixture was extracted with DCM (3 x 15 mL), washed with water (15 mL), dried over MgSO₄, concentrated, and purified by column chromatography (EtOAc: hexanes 1:19) to yield 4 as a clear or slightly pink oil, which solidified during storage (0.32 g, 62%). R_f 0.43 (EtOAc:hexanes 1:19); ¹H-NMR (500 MHz, CDCl₃) δ 0.00 (s, 3H), 0.02 (s, 3H), 0.88 (m, 12H), 0.97 (d, J = 6.5, 3H), 1.08-1.19 (m, 3H), 1.03-1.26 (m, 4H), 1.26-1.44 (m, 4H), 1.47-1.97 (m, 7H), 2.47 (s, 3H), 3.82 (dd, J = 6.5, 2.8 Hz, 1H), 3.95-4.02 (m, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 10.2 Hz, 2H); ¹³C-NMR: (125 MHz, CDCl₃) δ −5.2, −4.8, 13.7, 16.8, 17.6, 18.0, 21.6, 23.0, 25.8 (4C), 26.5, 34.3, 35.8, 40.4, 42.2, 52.4, 52.7, 69.2, 75.8, 127.9 (2C), 129.8 (2C), 133.2, 144.6; m/z calculated for C₂₆H₄₈O₄NSSi 499 [M + NH₄],⁺ found 499; m/z calculated for C₂₈H₄₇O₄NSSiNa 544 [M + Na + ACN],⁺ found 544; ${\left[\alpha \right]}_D^{25}=+34.0$ (c 1.0 mM, CHCl₃)

2.5. (R)-3-((1R,3aR,4S,7aR)-4-((tert-butyldimethylsilyl)oxy)-7a-methyloctahydro-1H-inden-1-yl)butanenitrile (5)

To a solution of 4 (2.74 g, 5.70 mmol) in dry acetonitrile (75 mL) was added NaOH (0.46 g, 11.4 mmol), potassium cyanide (0.74 g, 11.4 mmol), and 1,4,7,10,13,16-hexaoxacyclooctadecane (3.77 g, 14.25 mmol). The solution was stirred at 75°C for 2.5 hours or until the starting material disappeared by TLC. Acetonitrile was removed under reduced pressure and the residue was dissolved in water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine, dried over MgSO₄, concentrated, and purified by column chromatography (EtOAc :hexanes 3:97) to yield 5 (1.80 g, 94%) as a clear oil. R_f 0.20 (EtOAc: hexanes 3:97); ¹H-NMR (500MHz, CDCl₃) δ 0.02 (s, 3H), 0.04 (s, 3H), 0.91 (s, 9H), 0.95 (s, 3H), 1.16 (d, J = 6.6Hz, 3H), 1.18-1.66 (m, 8H), 1.67-1.97 (m, 6H), 2.25 (dd, J = 16.6, 7.0 Hz, 1H), 2.36 (dd, J = 16.6, 3.8 Hz, 1H), 4.01-4.04 (m, 1H); ¹³C-NMR: (125MHz, CDCl₃) δ −5.2, −4.8, 13.9, 17.5, 18.0, 19.3, 22.9, 24.7, 25.8 (3C), 27.2, 33.1, 34.2, 40.3, 42.2, 52.9, 55.4, 69.2, 119.1; m/z calculated for C₂₀H₄₁N₂OSi 354 [M + NH₄],⁺ found 354; ${\left[\alpha \right]}_D^{25}=+48.0$ (c 1.0 mM, CHCl₃).

2.6. (R)-3-((1R,3aR,4S,7aR)-4-((tert-butyldimethylsilyl)oxy)-7a-methyloctahydro-1H-inden-1-yl)butanal (6)

To a stirred solution of 5 (0.69 g, 2.06 mmol) in dry DCM (20 mL) at 0°C was added a solution of diisobutylaluminum hydride (1.5 M in toluene, 4.80 mL, 7.19 mmol) dropwise. The reaction was monitored at 0°C for 45 minutes until complete by TLC. The reaction was then carefully quenched with 10 mL of a saturated solution of NH₄Cl (aq) and stirred for an hour at 0°C. The viscous solution was broken up by stirring in TBME (60 mL), kept at 0°C for another two hours, and then allowed to warm up overnight. The aqueous layer formed a fluffy white ball, which was dried off directly with MgSO₄ and filtered off. The organic filtrate was concentrated under reduced pressure and purified via column chromatography (EtOAc:hexanes 5:95) to yield 6 (0.66 g, 94%) as a clear oil. R_f 0.41 (EtOAc:hexanes 7:93); ¹H-NMR (500 MHz, CDCl₃) δ 0.02 (s, 3H), 0.04 (s, 3H), 0.91 (s, 9H), 0.98 (s, 3H), 1.02 (d, J = 6.5 Hz, 3H), 1.12-1.19 (m, 2H), 1.23-1.30 (m, 2H), 1.36-1.43 (m, 3H), 1.56-1.65 (m, 1H), 1.67-1.73 (m, 1H), 1.76-1.86 (m, 2H), 1.94-2.00 (m, 1H), 2.02-2.09 (m, 1H), 2.13-2.19 (m, 1H), 2.47 (dd, J = 15.7, 2.5 Hz, 1H), 4.01-4.04 (m, 1H), 9.70 (dd, J = 3.6, 1.4 Hz, 1H); ¹³C-NMR: (125 MHz, CDCl₃) δ −5.2, −4.8, 13.7, 17.6, 18.0, 20.0, 23.0, 25.8 (3C), 27.6, 31.3, 34.3, 40.6, 42.3, 50.8, 53.0, 56.6, 69.3, 203.7; m/z calculated for C₂₀H₄₁N₂OSi 354 [M + NH₄],⁺ found 354; ${\left[\alpha \right]}_D^{25}=+27.0$ (c 1.0 mM, CHCl₃).

2.7. (R)-3-((1R,3aR,4S,7aR)-4-((tert-butyldimethylsilyl)oxy)-7a-methyloctahydro-1H-inden-1-yl)butanoic acid (7)

Sodium phosphate monobasic monohydride (3.39 g, 24.55 mmol) was dissolved in water (32 mL). To this was added sodium chlorite (3.41 g, 37.65 mmol). The aqueous solution was then added to a stirred solution of 6 (1.39 g, 4.09 mmol) in tert-butanol (77 mL) and 2-methyl-2-butene (19 mL). The reaction was monitored for 1 hour until completion and then concentrated under reduced pressure. The residue was dissolved in aqueous 0.1 M citric acid (20 mL) and extracted with DCM (3 x 50 mL). The organic layers were washed with water (20 mL), dried over MgSO₄, and concentrated to yield pure 7 (3.2g, 99%) as a white powder. R_f 0.19 (EtOAc:hexanes 1:4); ¹H-NMR (500MHz, CDCl₃) δ 0.02 (s, 3H), 0.03 (s, 3H), 0.91 (s, 9H), 0.97 (s, 3H), 1.03 (d, J = 6.4 Hz, 3H), 1.08-1.19 (m, 2H), 1.25-1.42 (m, 6H), 1.55-1.65 (m, 1H), 1.66-1.72 (m, 1H), 1.77-1.88 (m, 2H), 1.91-2.08 (m, 3H), 2.49 (d, J = 14.8, 3.2 Hz, 1H), 4.01-4.04 (m, 1H), 11.05 (bs, 1H); ¹³C-NMR: (125MHz, CDCl₃) δ −5.2, −4.8, 13.7, 17.6, 18.0, 19.5, 23.0, 25.8 (3C), 27.3, 33.2, 34.4, 40.6, 41.2, 42.3, 53.0, 56.5, 69.4, 179.7; m/z calculated for C₂₀H₃₈O₃Si 356 [M + H]⁺ , found 356; m/z calculated for C₂₀H₃₈O₃Si 353 [M-H]^-, found 353; ${\left[\alpha \right]}_D^{25}=+41.0$ (c 1.0 mM, CHCl₃).

2.8. (R)-methyl 3-((1R,3aR,4S,7aR)-4-((tert-butyldimethylsilyl)oxy)-7a-methyloctahydro-1H-inden-1-yl)butanoate (8)

To a stirred solution of 7 (1.01 g, 2.86 mmol) in toluene (16.5 mL) and methanol (11 mL) was added trimethylsilyl diazomethane (2.12 mL, 2 M in diethyl ether, 4.23 mmol). The reaction was stirred for 1.5 hours at room temperature and then quenched with acetic acid. The mixture was concentrated under reduced pressure and purified by column chromatography (EtOAc:hexanes 3:97) to yield 8 (1.05 g, quantitative) as a clear oil. R_f 0.28 (EtOAc:hexanes 3:97); ¹H-NMR (500 MHz, CDCl₃) δ 0.01 (s, 3H), 0.02 (s, 3H), 0.90 (s, 9H), 0.95-0.98 (m, 6H), 1.06-1.17 (m, 2H), 1.24-1.30 (m, 2H), 1.33-1.49 (m, 3H), 1.54-1.64 (m, 1H), 1.65-1.71 (m, 1H), 1.74-1.87 (m, 2H), 1.89-2.03 (m, 3H), 2.43 (dd, J = 14.2, 3.1 Hz, 1H), 3.67 (s, 3H), 3.99-4.03 (m, 1H); ¹³C-NMR: (125MHz, CDCl₃) δ −5.2, −4.8, 13.8, 17.6, 18.0, 19.5, 23.0, 25.8 (3C), 27.3, 33.4, 34.4, 40.6, 41.3, 42.2, 51.3, 53.0, 56.6, 69.4, 174.0; m/z calculated for C₂₁H₃₉O₃Si 367 [M-H]^-, found 367; ${\left[\alpha \right]}_D^{25}=+41.0$ (c 1.0 mM, CHCl₃).

2.9. (R)-methyl 3-((1R,3aR,4S,7aR)-4-hydroxy-7a-methyloctahydro-1H-inden-1-yl)butanoate (9)

To a stirred solution of 8 (1.48 g, 4.01 mmol) in dry DCM (45 mL) at 0°C was added trifluoroacetic acid (4.5 mL, 58.4 mmol). The reaction was stirred at 0°C for 1.5 hours, forming the product 9 (R_f 0.32) and a byproduct, the TFA ester of alcohol 9a (R_f 0.48). The reaction was concentrated under reduced pressure, and 9 was separated from the TFA ester by column chromatography (EtOAc: hexanes 1:4). The TFA ester was re-dissolved in THF (20 mL) at room temperature and tetrabutylammonium fluoride (1 M in THF, 4 mL, 4 mmol) was added. The reaction was stirred at room temperature for 1.5 hours at which point 9a converted into 9. After concentration and column chromatography (EtOAc:hexanes 1:4) the combined yield of 9 (white solid) was 0.93 g, 91%. ¹H-NMR (500 MHz, CDCl₃) δ 0.96-1.00 (m, 6H), 1.09-1.21 (m, 2H), 1.29-1.40 (m, 3H), 1.40-1.53 (m, 3H), 1.55-1.65 (m, 1H), 1.78-2.05 (m, 6H), 2.44 (dd, J = 14.2, 3.2 Hz, 1H), 3.68 (s, 3H), 4.08-4.11 (m, 1H), 4.30 (s, 1H) ; ¹³C-NMR: (125 MHz, CDCl₃) δ 13.6, 17.4, 19.4, 22.5, 27.2, 33.4, 33.6, 40.3, 41.3, 42.0, 51.4, 52.6, 56.4, 69.2, 174.0; m/z calculated for C₁₆H₂₇O₅ 299 [M + formic acid -H]^-, found 299; C₁₇H₂₆O₅F₃ 367 [M + TFA -H]^-, found 367; ${\left[\alpha \right]}_D^{25}=+30.0$ (c 1.0, CHCl₃).

2.10. (R)-methyl 3-((1R,3aR,7aR)-7a-methyl-4-oxooctahydro-1H-inden-1-yl)butanoate (10)

To a stirred solution of 9 (0.653 g, 2.57 mmol) in dry DCM (50 mL) was added freshly ground pyridinium dichromate (PDC) (1.93 g, 5.13 mmol). The reaction was monitored for two hours at room temperature, then diluted with TBME (50 mL) and filtered through celite. The filtered solid was resuspended twice more in fresh DCM (2 x 25 mL) and stirred vigorously for a half hour each before being filtered again. The combined filtrates were concentrated under reduced pressure and purified via column chromatography (EtOAc:hexanes 1:4) to yield 10 (0.565 g, 87%) as a white solid. R_f 0.35 (EtOAc:hexanes 1:4); ¹H-NMR (500 MHz, CDCl₃) δ 0.63 (s, 3H), 0.98 (d, J = 6.5, 3H), 1.26-1.60 (m, H), 1.66-1.76 (m, 1H), 1.78-1.93 (m, 3H), 1.94-2.04 (m, 2H), 2.04-2.09 (m, 1H), 2.14-2.27 (m, 2H), 2.36-2.46 (m, 2H), 3.62 (s, 3H); ¹³C-NMR: (125 MHz, CDCl₃) δ 12.5, 19.0, 19.5, 23.9, 27.4, 33.3, 38.8, 40.8, 41.0, 49.8, 51.4, 56.2, 61.8, 173.5, 211.5; m/z calculated for C₁₅H₂₄O₃ 253.1798 [M + H]⁺, found 253.1766; ${\left[\alpha \right]}_D^{25}=-5.0$ (c 1.0 mM, CHCl₃).

2.11. (R)-methyl 3-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-((tert-butyldimethylsilyl)oxy)-2-methylenecyclo hexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)butanoate (11)

A solution of n-butyllithium (1.6 M in hexanes, 0.55 mL, 0.565 mmol) was added dropwise to a solution of ((Z)-2-((3S,5R)-3,5-bis((tert-butyldimethylsilyl)oxy)-2-methylenecyclohexylidene)ethyl) diphenylphosphine oxide (500 mg, 0.715 mmol, dried over 3Å molecular sieves in 3 mL THF overnight) at −78°C. The solution turned deep red and was stirred for an hour at −78°C. Raising the solution out of the bath briefly deepened the red color and ensured that the deprotonation was complete. The solution was returned to the cold bath at −78°C and 10 (147 mg, 0.376 mmol, dried over 3Å molecular sieves in 2 mL THF overnight) was added dropwise. The solution was stirred for 5 hours at −78°C, and was then allowed to reach room temperature. The reaction mixture was then quenched with water (10 mL), diluted with TBME, and extracted (TBME, 2 x 50 mL). Combined organic layers were washed with brine, dried over MgSO₄, and concentrated. The crude product was purified by column chromatography (EtOAc:hexanes 5:95) to yield 11 as a white crystalline solid (117 mg, 41%). R_f 0.62 (EtOAc:hexanes 1:9); ¹H-NMR (500 MHz, CDCl₃) δ 0.06-0.10 (m, 6H), 0.60 (s, 2H), 0.90 (s, 9H), 1.00 (d, J = 4.0 Hz, 2H), 1.26-1.37 (m, 3H), 1.48-1.62 (m, 4H), 1.63-1.74 (m, 2H), 1.83-1.79 (m, 3H), 1.98-2.07 (m, 3H), 2.07-2.16 (m, 1H), 2.21-2.29 (m, 1H), 2.35-2.41 (m, 1H), 2.41-2.50 (m, 2H), 2.81-2.88 (m, 1H), 3.68 (s, 3H), 3.79-3.87 (m, 1H), 4.79 (s, 1H), 5.02 (s, 1H), 6.03 (d, J = 10.0 Hz, 1H), 6.17 (d, J = 10.0, 1H); ¹³C-NMR: (125 MHz, CDCl₃) δ −4.6, −4.5, 12.1, 18.2, 19.7, 22.2, 23.4, 25.9 (4C), 27.7, 28.8, 32.8, 34.1, 36.4, 40.4, 41.4, 45.8, 46.9, 51.4, 56.3 (2C), 70.6, 112.2, 118.0, 121.3, 136.6, 141.0, 145.4, 174.0; m/z calculated for C₃₀H₅₀O₃Si 487.3602 [M + H]⁺, found 487.3542; ${\left[\alpha \right]}_D^{25}=+61.0$ (c 1.0, CHCl₃).

2.12. (R)-methyl 3-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-hydroxy-2-methylenecyclohexylidene) ethylidene)-7a-methyloctahydro-1H-inden-1-yl)butanoate or calcioic acid methyl ester (12)

To a stirred solution of 11 (79 mg, 0.16 mmol) in dry THF (7 mL), tetrabutylammonium fluoride (1 M in THF, 1.6 mL, 1.6 mmol) was added. The reaction was stirred at room temperature overnight, then quenched with saturated NH₄Cl solution (10 mL). The mixture was extracted with EtOAc (3 x 20 mL) and the combined organic layers were washed with brine (30 mL), dried over MgSO₄, concentrated, and purified by column chromatography (EtOAc:hexanes 3:7) to yield the calcioic acid methyl ester 12 as a clear oil (55 mg, 91%). R_f 0.16 (EtOAc:hexanes 1:3); ¹H-NMR (500 MHz, CDCl₃) δ 0.59 (s, 3H), 1.00 (d, J = 6.5 Hz, 3H), 1.28-1.36 (m, 4H), 1.49-1.58 (m, 3H), 1.64-1.71 (m, 3H), 1.84-1.98 (m, 4H), 1.98-2.04 (m, 3H), 2.15-2.22 (m, 1H), 2.26-2.32 (m, 1H), 2.26-2.32 (m, 1H), 2.38-2.48 (m, 2H), 2.56-2.61 (m, 1H), 2.81-2.87 (m, 1H), 3.68 (s, 3H), 3.92-3.98 (m, 1H), 4.82-4.83 (m, 1H), 5.05-5.07 (m, 1H), 6.04 (d, J = 11.2, 1H), 6.24 (d, J = 11.2, 1H); ¹³C-NMR: (125 MHz, CDCl₃) δ 12.0, 19.7, 22.2, 23.5, 27.6, 28.9, 32.0, 34.1, 35.2, 40.4, 41.4, 45.9, 46.0, 51.4, 56.2, 56.3, 69.2, 112.4, 117.8, 122.3, 135.4, 141.7, 145.1, 174.0; m/z calculated for C₂₄H₃₆O₃ 373.2737 [M + H]⁺, found 373.2664; ${\left[\alpha \right]}_D^{25}=+18.0$ (c 1.0 mM, MeOH)

2.13. (R)-3-((1R,3aS,7aR,E)-4-((Z)-2-((3S,5R)-3,5-dihydroxy-2-methylenecyclohexylidene) ethylidene)-7a-methyloctahydro-1H-inden-1-yl)butanoic acid or calcioic acid (13)

To a stirred solution of 12 (44 mg, 0.12 mmol) in ethanol (1.5 mL) was added 10% aqueous NaOH (1 mL). The reaction was stirred at room temperature for 1 hour (conversion indicated by TLC) and subsequently neutralized with concentrated HCl to pH 7. Excess MeOH was evaporated off under reduced pressure. The reaction was then diluted with water (25 mL), acidified with concentrated HCl to pH 1, and extracted with EtOAc (3 × 5 mL). The organic layers were washed with brine (5 mL), dried over MgSO₄, concentrated, and purified by column chromatography (EtOAc:hexanes 4:1) to yield calcioic acid 13 (25 mg, 59%) as a white powder. R_f 0.53 (EtOAc); ¹H-NMR (500 MHz, CDCl₃) δ 0.61 (s, 3H), 1.06 (d, J = 6.5 Hz, 3H), 1.30-1.40 (m, 4H), 1.50-1.61 (m, 3H), 1.65-1.74 (m, 3H), 1.86-1.99 (m, 4H), 1.99-2.10 (m, 3H), 2.17-2.23 (m, 1H), 2.30 (dd, J = 12.8, 5.5 Hz, 1H), 2.39-2.46 (m, 1H), 2.51 (dd, J = 14.9, 3.0 Hz, 1H), 2.57-2.63 (m, 1H), 2.82-2.88 (m, 1H), 3.94-4.00 (m, 1H), 4.84 (s, 1H), 5.07 (s, 1H), 6.06 (d, J = 10 Hz, 1H), 6.25 (d, J = 10 Hz, 1H); ¹³C-NMR: (125 MHz, CDCl₃) δ 12.0, 19.7, 22.2, 23.4, 27.6, 28.9, 31.9, 33.9, 35.1, 40.3, 41.2, 45.8, 45.9, 56.2, 56.2, 69.3, 112.5, 117.7, 122.3, 135.3, 141.7, 145.1, 178.9; m/z calculated for C₂₃H₃₄O₃ 359.2581 [M + H]⁺, found 359.2524; ${\left[\alpha \right]}_D^{25}=-17.0$ (c 1.0 mM, CHCl₃).

2.14. Binding studies

Fluorescence Polarization Binding Assay:

The assay buffer contained 25 mM PIPES (piperazine-N,N’-bis(2-ethanesulfonic acid) (P6757, Sigma-Aldrich) at pH 6.75, 50 mM NaCl (BP358-1, Fisher Scientific), and 0.01% NP-40 (nonyl phenoxypolyethoxylethanol) (T1279500ML, Spectrum). Coactivator peptide SRC2-3 (CKKKENALLRYLLDKDDTKD), which had been labeled with cysteine-reactive AlexaFluor647 was added to yield 7.5 nM. To this solution was added 250 nM of recombinant VDR-LBD, which had been expressed and purified as reported previously [12]. The VDR agonist LG190178, which had been synthesized previously according to a published protocol, [13] was added at a concentration of 200 nM. Calcioic acid in DMSO was transferred using a 100 nL pin tool (V&P Scientific) with a Tecan Freedom EVO liquid handler system. After incubation of 1 hour, fluorescence polarization was detected at excitation (650 nm) and emission (665 nm) with a Tecan Infinite M1000 plate reader. The assay was carried out with an n = 4. Non-linear regression was carried out with GraphPad Prism using non-linear regression (log(inhibitor) vs. response – Variable slope (four parameters).

Transcription Assay Protocol:

Human embryonic kidney (HEK293, ATCC CRL-3249) T cells were cultured in DMEM/High Glucose (Hyclone, #SH3024301) with non-essential amino acids (Hyclone, #SH30238.01), 10 mM HEPES (Hyclone, #SH302237.01), penicillin and streptomycin (Hyclone, #SV30010), and 10% of fetal bovine serum (Gibco, #10082147). For transfection in 6-well plates, 0.99 mL of untreated media was combined with 0.78 μg of VDR-CMV plasmid, 7.9 μg of a CYP24A1-luciferase reporter gene, LipofectamineTM LTX (37.5 μL, Life Technologies #15338020), and PLUS™ reagent (12.5 μL) and incubated for 30 minutes followed by the addition to three of the six wells of a six-well plate. After 16 hours of incubation at 37°C with 5% CO₂, the cells were harvested with 0.3 mL of 0.05% Trypsin (Hyclone, #SH3023601), added to 5 mL of the assay buffer (DMEM/High Modified buffer without phenol red, Hyclone #SH30284.01). After centrifugation and resuspension of cells at 400,000 cell/mL, 40 μL of this suspension was added to each well of a sterile optical bottom 384-well plate, which was treated with a 0.25% Matrigel solution. The plate was centrifuged for two minutes at 1000 rpm. After four hours, plated cells were treated with 200 nL of calcioic acid or controls in DMSO using an EVO liquid handling system with a 100 nL pin tool (V&P Scientific). Controls used were 1,25(OH)₂D₃ (100 nM in DMSO, Endotherm) and DMSO. After 18 hours of incubation at 37°C with 5% CO₂, 20 μL of Bright-Glo™ (Promega, Madison, WI) were added and luminescence was detected with a Tecan Infinite M1000 reader. The assay was carried out with an n = 8 and non-linear regression was used to calculate IC₅₀.

Viability assay:

HEK293 cells were treated following the protocol for the transfection assay. After 18 hours of incubation at 37°C with 5% CO₂, 20 μl of CellTiter-Glo™ (Promega, Madison, WI) were added and luminescence was detected with a Tecan Infinite M1000 reader. The assay was carried out with an n = 8.

3. Results

In recent decades, vitamin D₂ isolated from yeast has become a cheap starting material for the synthesis of vitamin D analogs. In the presence of ozone at low temperature, the carbon-carbon double bonds of vitamin D₂ can be cleaved and the resulting carbonyl groups can be reduced with sodium borohydride to generate the Inhoffen Lythgoe diol 2 as reported previously [14]. Using this starting material, we performed further conversions to generate calcioic acid as outlined in Scheme 1.

Scheme 1.

Synthesis of calcioic acid. a) 1. Ozone, methanol, pyridine, −78°C, 6h; 2. NaBH₄, 0°C, 2h; b) DMAP, TsCl, DCM, rt, 12h; c) TBSOTf, lutidine, DCM, 0°C, 2h; d) KCN, NaOH, acetonitrile, 18-crown-6, 75°C, 2.5h; e) DIBAL-H (1.5 M in toluene), DCM, 0°C, 45 min; f) NaH₂PO₄, water, NaClO₂, t-BuOH, 2-methyl-2-butene, rt, 1h; g) trimethylsilyl diazomethane, toluene, methanol, rt, 1.5h; h) TFA, DCM, 0°C, 1.5h; i) pyridinium dichromate, DCM, rt, 2h; j) 1. n-BuLi (1.6 M in hexanes), ((Z)-2-((3S,5R)-3,5-bis((tert-butyldimethylsilyl) oxy)-2-methylenecyclohexylidene) ethyl) diphenylphosphine oxide, THF, −78°C, 1h; 2. 10, THF, −78°C to rt, 12h; k) TBAF (1 M in THF), THF, rt, 12h; l) NaOH (10% in water), methanol, rt, 1h.

Working from the reactions that were earlier introduced by Meyer et al.[10] for the synthesis of calcitroic acid, we changed several reaction conditions to improve yields and workup procedures. For the synthesis of the Inhoffen Lythgoe diol we introduced a 5% copper sulfate wash that completely removed pyridine from the crude product and simplified purification by column chromatography as well as crystallization. The primary and secondary alcohol functions of the diol were sequentially protected to form 4. The reported nucleophilic substitution of the tosyl group with cyanide in the presence of dimethyl sulfoxide and sodium hydroxide generated 5 in low yield (45% in our lab) due to degradation at elevated temperature and difficulties with a cumbersome workup necessary to remove traces of dimethyl sulfoxide. Instead, we applied acetonitrile as the low boiling solvent and 18-crown-6 to increase the nucleophilicity of the cyanide, which increased the yield to 94%. With these new reaction conditions, we observed no degradation due to a lower reaction temperature and shorter reaction time. The crude product was not contaminated by high boiling solvent dimethyl sulfoxide. The nitrile 5 was reduced to aldehyde 6 using DIBAL-H and as noted by Meyer et al.[10], we also found it very important to allow the aluminum/imine intermediate adequate time to fully react with a saturated NH₄Cl solution (1h) or risk precipitous drops in yield. Compound 6 was converted quantitatively to the corresponding acid using the Pinnick oxidation. This very clean reaction did not require purification of the acid beyond an aqueous workup, and methylation of compound 8 using trimethylsilyl diazomethane was thus achieved with a yield of 99% over two steps. We observed yields much lower than the reported 74% for the TFA-meditated conversion of the protecting TBS group to the alcohol 9 due to the formation of a less polar byproduct which was identified as the TFA ester of the intended product. Fortunately, we were able to convert this byproduct in the presence of TBAF to produce 9, which increased the combined yield of this conversion to 91%. Subsequently, oxidation with PCD yielded ketone 10 in excellent yield. The challenging Horner-Wadsworth-Emmons reaction to form 11 returned only 41%. For the synthesis of calcitroic acid using [(2Z)-2-[(3S,5R)-2-methylene-3,5-bis[(trimethylsilyl)oxy]cyclohexylidene] ethyl]diphenyl-phosphine oxide, higher yields were achieved for this reaction by Meyer (89%) and our lab (78%). Our findings agreed with the Meyer et al.[10] observation that decreasing the equivalents of the expensive phosphine oxide resulted in a decreased yield. However, we were able to recover the unreacted phosphine oxide nearly quantitatively during the column chromatography. Compound 11 was deprotected in the presence of TBAF in excellent yield. To avoid the degradation during the ester hydrolysis, which was observed using potassium hydroxide, methanol and water at 60°C, we used sodium hydroxide, methanol, and water at room temperature instead. The overall yield for our synthesis of calcioic acid 12 is 6.6%, or 11.6% starting from the Inhoffen-Lythgoe diol instead of ergocalciferol.

Next, we determined the ability of calcioic acid to interact with VDR. We employed a fluorescence polarization binding assay that consists of recombinantly expressed VDR-LBD protein, VDR ligand LG190178 [13], and fluorescently labeled SRC2-3 coactivator peptide. The ability of coactivator 2 to bind VDR has been reported by Xu et al. [15]. Furthermore, we have shown that the third LxxLL (leucine = L, any amino acid = x) motif has the strongest interaction with ligand-bound VDR [12]. The change of binding was determined by the change of fluorescence anisotropy as depicted in Figure 1.

Figure 1.

Interaction between VDR and calcioic acid. VDR, LG190178 and fluorescently labeled peptide SRC2-3 were incubated in the presence of different concentrations of calcioic acid and analyzed by fluorescence polarization at excitation wavelength of 650 nm and emission wavelength of 665 nm. Data is represented as means with standard deviations (n = 4). Nonlinear regression was used to determine IC₅₀.

For calcioic acid concentrations higher than 10 μM, we observed a change of fluorescence polarization due to the disruption of liganded VDR and coactivator peptide. Thus, calcioic acid is acting as a VDR antagonist with an IC₅₀ of 71.2 μM.

To investigate regulatory effects of calcioic acid in cells mediated by VDR, we carried out a transcription assay. VDR was overexpressed in HEK293 cells and transcription of CYP24A1, a known VDR target gene, was monitored using a luciferase gene fused to the CYP24A1 promotor. Transcription of luciferase was detected with Bright-Glo (Promega) and cell viability was determined by CellTiter-Glo (Promega). The results for different calcioic acid concentrations are depicted in Figure 2.

Figure 2.

Cellular effects of calcioic acid. A) Transcription assay with HEK293 cells using transient transfection of a CMV-VDR and a CYP24A1-luciferase plasmid followed by luminescence detected with addition of Bright-Glo (Promega). The cells were incubated with different concentration of calcioic acid in the absence and presence of 1,25(OH)₂D₃ (7.5 nM) for 18 hours. B) Viability assay of HEK293 cell incubated for 18 hours with different concentrations of calcioic acid and detection of ATP by CellTiter-Glo (Promega). Data is presented as means with standard average of the mean. (n = 8) Nonlinear regression was used to determine IC₅₀.

The results for the transcription assay showed a strong induction of the CYP24A1 in the presence of 7.5 nM 1,25(OH)₂D₃. This gene induction was reduced in the presence of increasing concentrations of calcioic acid with an IC₅₀ value of 114 μM. The luminescence values for the transcription assay with calcioic acid in the absence of 1,25(OH)₂D₃ did not change, thus calcioic acid is not promoting the transcription of CYP24A1. The viability assay showed no toxic effects of calcioic acid at 150 μM under these conditions.

4. Discussion:

Calcioic acid is one of four acidic vitamin D metabolites that are formed naturally from vitamin D.[1] Others include calcitroic acid [8], cholacalcioic acid (25,26,27-trisnor-vitamin D₃ 24-oic acid)[16], and 1α-hydroxycholacalcioic acid [16]. Calcitroic acid and cholacalcioic acid have been investigated in vivo. Calcitroic acid has been shown to bind VDR and increased calcification of epiphyseal plate in rats [17]. Cholacalcioic acid increased the intestinal calcium transport, and like calcitroic acid, did not increase serum calcium levels like 1,25(OH)₂D₃ [16]. In contrast, calcioic acid has not been investigated in vivo. The developed 11 step synthesis is expected to enable the generation of gram quantities of calcioic acid to conduct these studies. An overall yield of 6.6% was achieved. Although this implies an average yield of 78% for each step, we acknowledge that some yields can be improved by changing the reactions conditions. For instance, reaction a (Scheme 1) has been reported with a significantly higher yield of 90% [18], and for reactions b and c (Scheme 1) a combined 82% yield was achieved [10]. Furthermore, the phosphonate used for the Horner-Wadsworth-Emmons reaction was coupled to similar ketones with yields up to 94% [19]. Finally, we believe that a larger scale for the last step and application of a shorter column for chromatography will significantly improve the yield for calcioic acid. For the production of calcitroic acid, we observed a yield of 80% for the corresponding hydrolysis reaction (data not shown). However, the quantity of generated calcioic acid was enough to function as a reference compound to investigate 25-hydroxyvitamin D₃ metabolism in vivo and enabled initial binding studies with VDR [11]. We have shown that calcioic acid competes with 1,25(OH)₂D₃ for VDR binding at super physiological concentrations. Our cell-based assay confirmed this result. In the absence of 1,25(OH)₂D_3, calcioic acid did not induce CYP24A1, thus this vitamin D analog behaves like a very weak VDR antagonist. We can conclude that calcioic acid is predominately a water-soluble vitamin D metabolite that enables vitamin D secretion, or may mediate its effects through biomolecules other than VDR. Further studies will investigate these possibilities. Another important use of acidic vitamin D analogs is that of haptens for the generation of antibodies specific for 25-hydroxyvitamin D₃ and 1,25(OH)₂D₃. The acidic compounds are linked to serum albumin via peptide coupling reactions, which functions as an antigen. The application of calcioic acid has been described in a recently filed patent from Anciaux et al. [20]. In conclusion, we have developed a synthesis route for calcioic acid to enable future in vitro and in vivo studies for this molecule. Furthermore, this compound is expected to be used as analytical standard to further investigate the metabolism of vitamin D.

Abbreviation:

25OHD₃

25-hydroxyvitamin D₃

CYP24A1

1α,25(OH)₂D₃-24-hydroxylase

1,25(OH)₂D₃

1α,25-dihydroxyvitamin D₃

VDR

vitamin D receptor

TLC

thin-layer chromatography

DCM

dichloromethane

EtOAc

ethyl acetate

NMR

nuclear magnetic resonance

CHCl₃

chloroform

TBSOTf

tert-butyl dimethylsilyl trifluoromethanesulfonate

18-crown-6

1,4,7,10,13,16-hexaoxacyclooctadecane

DIBAL-H

diisobutylaluminum hydride

TBME

tert-butylmethylether

MeOH

methanol

t-BuOH

tert-butanol

TFA

trifluoroacetic acid

PDC

pyridinium dichromate

NH₄Cl

ammonium chloride

n-BuLi

n-butyllithium

THF

tetrahydrofuran

TBAF

tetrabutylammonium fluoride

PIPES

piperazine-N,N’-bis(2-ethanesulfonic acid)

NP-40

nonyl phenoxypolyethoxylethanol

LBD

ligand binding domain

DMSO

dimethyl sulfoxide

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

CMV

cytomegalovirus