Integrated Synthesis of Calcitriol and 26,27-Hexadeutero Calcitriol

Calcitriol (1 α ,25-dihydroxyvitamin D 3 , 1), a classical vitamin D drug, is indicated primarily in the treatment of patients with postmenopausal osteoporosis and renal osteodystrophy. In this study, a practical synthesis of calcitriol ( 1 ), from readily available commercial vitamin D 2 ( 5 ) via hub intermediate 18


Introduction
DeLuca's group reported the isolation and identification of calcitriol (1α,25-dihydroxyvitamin D 3 , 1) in 1971. 1,2In the same year, Kodicek's group suggested that this substance should properly be regarded as a hormone-controlling calcium metabolism. 3Since then, great progress has been made in the knowledge of vitamin D 3 (cholecalciferol, 2) metabolism.It has been established that vitamin D 3 is metabolized in the liver to the major circulating metabolite, 25-hydroxyvitamin D 3 (3), which is transported to the kidney where it is transformed into calcitriol (1), the active hormonal form of vitamin D 3 (►Scheme 1). 1 Calcitriol (1) was launched by F. Hoffmann-La Roche Ltd. (Roche) in 1978 under the brand name Rocaltrol.Approved by U.S. Food and Drug Administration, Rocaltrol is indicated in the management of secondary hyperparathyroidism and the resultant metabolic bone disease in patients with moderate to severe chronic renal failure not yet on dialysis, in the management of hypocalcemia and the resultant metabolic bone disease in patients undergoing chronic renal dialysis, and also in the management of hypocalcemia and its clinical manifestations in patients with postsurgical hypoparathyroidism, idiopathic hypoparathyroidism, and pseudohypoparathyroidism. 4 Nowadays, calcitriol is used primarily in the treatment of patients with postmenopausal osteoporosis and renal osteodystrophy.
As the vitamin D hormone, calcitriol (1) has been an active subject in experimental and clinical research in a wide variety of therapeutic fields, such as the treatment of advanced osteoarthritis, 5 its effect on bone turnover markers in people with type 2 diabetes (T2DM) and stage 3 chronic kidney disease, 6 combinations of calcitriol with anticancer treatments for breast cancer, 7 its modulatory effects on postischemic immunity response, 8 the potential treatment of age-related hypertension, 9 the immunoregulatory effect of calcitriol on experimental autoimmune encephalomyelitis mice, 10 the proliferation inhibition and potential apoptosis induction in B16-F10 melanoma cells, 11 the protection against the serotonin-depleting effects of neurotoxic doses of methamphetamine, 12 and the application potential of calcitriol (1) as an adjunctive treatment for T2DM-associated periodontitis. 136,27-Hexadeutero calcitriol (4), a deuterium-labeled calcitriol derivative, had been used as the internal standard in an immunoaffinity extraction-enabled sensitive LC/MS/MS (liquid chromatography with tandem mass spectrometry) method for quantification of calcitriol in serum. 14Therefore, 4 is useful in analyzing pharmacokinetic parameters and determining bioequivalence of calcitriol dosage forms.
the adduct in ethanolic sodium bicarbonate to retrieve the triene; (2) direct, regio-and stereoselective 1α-hydroxylation of 3β-TBS-protected (5E)-calciferol intermediate 19 using selenium dioxide in the presence of N-methylmorpholine N-oxide as a re-oxidant in a hot mixture of methylene chloride and methanol; (3) nickel(0)-mediated conjugate addition of the 22-iodide 23 to electron-deficient ethyl acrylate followed by Grignard reaction with methylmagnesium bromide to construct the calcitriol side chain; and (4) triplet-sensitized photoisomerization of 26 to access the bioactive (5Z,7E)-triene in calcitriol (1).The high-performance liquid chromatography purities of batches of the synthesized calcitriol (1) were consistently more than 99.9%, with related substances listed in the USP 2023 and EP 11.0 well controlled.This robust process proved amenable to pilot scale-up and industrial production.26,27-Hexadeutero calcitriol (4), a deuterium-labeled calcitriol derivative, is useful as the internal standard in the bioanalysis for the quantification of calcitriol in serum.4 was efficiently synthesized in an integrated manner from hub intermediate 18 in 48% yield.
Calcitriol (1) is a complex chiral molecule that contains six chiral carbons and the characteristic 5(Z),7(E),l0(l9)-triene motif.The practical synthesis of calcitriol ( 1) is challenging and requires expertise in vitamin D chemistry.This article covers the successful development of an integrated approach for the synthesis of both calcitriol (1) and its deuterated derivative 4 during our research and development campaign of generic calcitriol API (active pharmaceutical ingredient) and soft capsule.Furthermore, this article provides a tutorial review of the evolving strategy of calcitriol synthesis and valuable insights into vitamin D chemistry.

Results and Discussion
6][17] The semi-syntheses are the dominant strategy for the large-scale preparation of calcitriol.They can be further stratified into two sub-categories that use steroid precursors and vitamin D 2 (ergocalciferol, 5) as the starting material, respectively.
Historically, Roche relied on the semi-synthesis from 3βdehydroepiandrosterone (6) for the production of calcitriol (1) (►Scheme 2). 18,19The introduction of the 1α-hydroxyl group was accomplished by fermentation of 6 to reach diol 7, which was converted into tosylate 8 in multiple steps.The coupling of 8 with the tert-butyldimethylsilyl (TBS)-protected acetylenic alcohol 9 followed by Pt/C catalytic hydrogenation and subsequent tetrabutylammonium fluoride (TBAF) cleavage of the silyl protective groups in refluxing tetrahydrofuran (THF) or 1,2-dimethoxyethane gave rise to the key intermediate, 1α,25-dihydroxycholesterol (Ro 21-3245) (12) .Calcitriol (1) could be obtained after photochemical cleavage of ring B and thermal isomerization.The weaknesses of this Roche process lie in limited supply of the starting material 6 produced by fermentation, the use of pyrophoric reagent n-butyllithium, and the difficulty encountered in deprotecting the 25-TBS-protected alcohol group in the trisilyl ether 11.
2][23] This upgraded process has proven amenable to industrial production, thanks to four distinguished synthetic methods developed over time by genius scientists in the vitamin D field: (1) protection of the triene sensitive to ozonolysis by electrocyclic addition of sulfur dioxide (SO 2 ) and subsequent thermolysis of the SO 2 adduct in refluxing alcohol containing sodium hydrogen carbonate to restore the (5E,7E)-triene; 20 (2) direct, regio-and stereoselective lα-hydroxylation of the 3β-silyl-protected (5E)-calciferol derivative with selenium dioxide in the presence of N-methylmorpholine N-oxide (NMO) as a reoxidant in solvent mixtures containing methanol; 24 (3) installation of the calcitriol side chain by a nickelmediated conjugate addition of 22-iodide 23 to electrondeficient ethyl acrylate followed by Grignard reaction with methylmagnesium bromide; 23 and (4) photoisomerization in the presence of a triplet sensitizer to build the required (5Z,7E)-triene in calcitriol (1). 22,23nlike the semi-syntheses, the total syntheses of calcitriol (1) have so far found minimal if any usefulness, in the commercial production of calcitriol (1), primarily due to cost, length, and scalability.Mourino and colleagues, for example, reported a convergent synthesis of calcitriol (1) from vitamin D 3 (2) and vinyl triflate 33, which was prepared from (S)-carvone in 45% yield (►Scheme 4).However, this total synthesis was compromised by lengthy sequences, cryogenic reaction conditions, and expensive and/or hazardous reagents. 25In addition, bioconversion of vitamin D 3 into calcitriol (1) has been actively investigated for potential bioindustrial manufacture of calcitriol (1). 26,27n sharp contrast to the high volume of references dealing with the synthesis of calcitriol (1), there are only a handful of references pertinent to the preparation of 4. Mourino and colleagues documented a total synthesis of 4 from the lythgoeinhoffen diol (35) and ring-A enyne 40 (►Scheme 5) in 1993. 28he drawbacks of this synthesis were lengthy route, ultrasonic conditions, cryogenic reactions, hazardous reagents, and need of careful monitoring to avoid over-reduction of the triple bond.In addition, De Luca et al disclosed semi-syntheses of 4 from homocholenic acid methyl ester derivatives. 29The shortcomings of these syntheses were limited availability of starting materials, tedious operations, need for preparative thin layer chromatography (TLC) or normal-phase high-performance liquid chromatography (HPLC) for purifications, and low yields (<20%) for several steps.
2][23] This strategy also allowed for isotope labeling in the late stage of the synthesis by reacting deuterated Grignard reagent with hub intermediate 18, thus achieving an integrated synthesis of the title compounds, viz. 1 and 4 (►Scheme 3).
[23] Reported overall yields of 18 based on vitamin D 2 (5) of the green and violet routes are 18 and 16%, respectively.In both routes, 1α-hydroxylation is the step with the lowest yield.Considering the heavy loss of yield in the late-stage 1αhydroxylation of ester 17 in the green route, the violet route with an early-stage 1α-hydroxylation was selected for our inhouse optimization and development.Thus, sulfone 13 derived from vitamin D 2 (5) was heated in refluxing 95% ethanol in the presence of sodium bicarbonate to extrude SO 2 to afford 3β-TBS-protected (5E,7E)-triene 19, a direct 1αhydroxylation of which was accomplished with selenium dioxide/NMO combination in a hot mixture of methylene chloride and methanol (1:1) to give a ca.6:1 mixture of 1αand 1β-epimers, which was silylated with tert-butyldimethylsilyl chloride (TBSCl) and separated by silica gel chromatography to provide pure 1α-epimer 20 in 36% telescoping yield based on 5.After protection of the sensitive (5E,7E)triene motif with liquid SO 2 adduction, 20 were ozonized at -10°C in a mixture of methylene chloride and methanol (3:1), and the formed ozonides were subjected to one-pot reduction with powdered sodium borohydride to furnish the sulfone alcohol 22 in 87% yield.Iodination of 22 with I 2 / Ph 3 P/imidazole in methylene chloride delivered the sulfone iodides 23 in 86% yield, which was subjected to stoichiometric nickel(0)-mediated conjugate addition to ethyl acrylate to furnish 24 in 84% yield.This conjugate addition is the key reaction for the elaboration of calcitriol side chain.Interestingly, a related catalytic nickel(0)-mediated conjugate addition had been reported by using 8.6% equiv. of nickel chloride hexahydrate and 39% equiv. of added water in a mixture of THF and pyridine (1:1) to affect the side chain carbon-carbon bond-forming reaction. 18SO 2 was extruded from 24 in boiling 95% ethanol in the presence of sodium bicarbonate to deliver 18 in 86% yield.Taken together, our streamlined    1) and its deuterated derivative 4 was accomplished in a straightforward manner (►Scheme 3). 23,28A Grignard reaction of 18 with commercial methyl magnesium bromide solution in THF gave 25 in 86% yield, whose silylprotective groups were cleaved by the treatment of a TBAF solution in THF to afford the crystalline triol 26 in 81% yield.26 was subjected to photoisomerization by radiation with a tunable high-pressure mercury lamp in the presence of triplet-sensitizer 9-acetylanthracene (9-AA) to deliver calcitriol (1) in 60% yield after careful recrystallization from methyl formate.26,27-Hexadeutero calcitriol (4) was prepared in an analogous way in 48% yield from 18.It is worth noting that in the Grignard reaction of 18, methylmagnesium-d 3 iodide (CD 3 MgI) was conveniently prepared in situ from magnesium powders and trideuteriomethyl iodide (methyl-d 3 iodide), and used in excess to drive the reaction to complete. 30

Related Substances of Calcitriol
As per U.S. Pharmacopeia 46-NF 41 (USP 2023), triazoline adduct of pre-calcitriol (44), trans-calcitriol (26), 1β-calcitriol (45), and methylene calcitriol (46) are listed as specified organic impurities.As per European Pharmacopoeia (Ph.Eur.) 11th Edition (EP 11.0), 44, 26, and 45, but not 46, are listed as specified impurities.Since 44 and 46 are synthetic route-dependent impurities, they are not expected to appear in calcitriol (1) prepared in our process.Thus, we focused on related substances 26 and 45 in our quality control of synthesized calcitriol (1).As shown in ►Table 1, the related substances in three laboratory batches of calcitriol (1) conformed to the acceptance criteria defined in USP 2023 and EP 11.0.

Conclusion
A practical synthesis of calcitriol (1), from readily available commercial vitamin D 2 (5) via hub intermediate 18 in 9% overall yield, has been accomplished in our development campaign for calcitriol API.This semi-synthetic process featured four prominent components of vitamin D chemistry, which included (1) SO 2 adduction with 5,10(19)-diene 5 for the isomerization of (5Z,7E)-to (5E,7E)-triene, or with 5,10(19)-diene 20 for the protection of the sensitive triene before ozonolysis, (2) selective 1α-hydroxylation of 3β-TBSprotected (5E)-calciferol derivative 19 with the ratio of 1αand 1β-epimers being ca.6:1, (3) nickel(0)-mediated conjugate addition of the 22-iodide 23 to electron-deficient ethyl acrylate to construct the ester side chain in 24, and (4) triplet-sensitized photoisomerization to access the required (5Z,7E) configuration of the triene system in 1.The HPLC purities of three laboratory batches of calcitriol (1) were all more than 99.9%, with related substances listed in the USP 2023 and EP 11.0 well controlled.This rugged process proved amenable to pilot scale-up and commercial production.Furthermore, 26,27-hexadeutero calcitriol (4) was efficient-ly synthesized from hub intermediate 18 in 48% yield.In summary, we were able to achieve an integrated synthesis of both calcitriol (1) and its deuterium-labeled derivative 4, capitalizing on our relentless research and development in the vitamin D field.

Experimental Section General
All solvents and reagents were obtained from commercial suppliers and used without further purification unless otherwise noted.Reaction progress was monitored by TLC using precoated glass-backed silica gel plates and visualized with UV detection at 254 nm or 5% phosphomolybdic acid in ethanol.Silica gel 300-400 mesh was employed for column chromatography purification.Solvent ratios are volume ratios.Evaporation of solvents was carried out on a rotary evaporator under reduced pressure. 1H NMR and 13 C NMR were recorded on either a Bruker Avance III 400 or a Bruker Avance III 600 spectrometer, and were obtained in either deuterated chloroform (CDCl 3 ) or deuterated acetone (acetone-d 6 ). 1 H NMR and 13 C NMR spectra were referenced to protons (CHCl 3 , d 7.26 ppm; acetone, d 2.05 ppm) and carbons (CHCl 3 , d 77.0 ppm; acetone, d 29.84 ppm) in the deuterated solvents, respectively.Chemical shifts are given in d values and coupling constants are reported in hertz (Hz).
Elemental analysis was performed on a ThermoScientific FLASH 2000 Organic Elemental Analyzer.Triplet-sensitized photoisomerization was carried out in a BiLon BL-GHX-V photochemical reactor.

lα,25-Dihydroxyvitamin D 3 (calcitriol, 1)
A solution of 26 (1.04 g, 2.5 mmol) and 9-acetylanthracene (9-AA) (0.055 g, 0.25 mmol) in methanol (400 mL), contained in a 1-L vessel in the BiLon photochemical reactor, was cooled to -5°C while purging with argon, and irradiated with a tunable high-pressure 1000 W mercury lamp until the reaction was complete by HPLC monitoring.Caution!Light from the mercury lamp is damaging to the eyes and skin.The solution was transferred to a 1-L, round-bottomed flask and concentrated in vacuo to give a foam, which was chromatographed on silica gel with gradient eluting of 60 to 90% ethyl acetate in petroleum ether (60-90°C).The appropriate fractions were collected, evaporated in vacuo, and crystallized from methyl formate under argon and protected from light to give 1 (0.63 g, 60% yield) as a white solid. 1  To a stirred suspension of magnesium powders (1.33 g, 54.7 mmol) in dry diethyl ether (6 mL) under argon was added a solution of trideuteriomethyl iodide (methyl-d 3 iodide) (6.33 g, 43.66 mmol) in dry diethyl ether (26 mL) dropwise at 0 to 5°C.Upon completion of the addition, the mixture was allowed to stir under reflux for 30 minutes.To the resulting methylmagnesium-d 3 iodide (CD 3 MgI) solution was added a solution of 18 (3.2g, 4.85 mmol) in dry THF (16 mL) dropwise at 0 to 5°C. 23The reaction mixture was allowed to stir at 0°C for 30 minutes, room temperature for 1 hour, and then quenched under cooling with careful addition of saturated aqueous ammonium chloride (15 mL).The quenched reaction was diluted with ethyl acetate (300 mL), washed with brine (100 mL Â 3), dried over anhydrous sodium sulfate, and evaporated in vacuo to yield 27 in quantitative yield.The obtained 27 was free of 18, and used for the next reaction without further purification. 1 (1α,3β,5E,7E)-26,27-Hexadeuterio-9,10-secocholesta-5,7,10(19)-trien-1,3,25-triol (28) A 100-mL, three-necked, round-bottomed flask was charged with a solution of 27 obtained above (quantitative yield, 4.85 mmol) in anhydrous THF (36 mL) and 1 mol/L TBAF in THF (64 mL, 64 mmol).The resulting solution was stirred at room temperature under argon for 8 hours and concentrated in vacuo.The residue was partitioned between ethyl acetate (100 mL) and half-saturated brine (100 mL).The organic phase was separated, and the aqueous phase was backextracted with ethyl acetate (50 mL Â 2).The combined organic phase was washed with brine (50 mL Â 3), dried over anhydrous sodium sulfate, and evaporated in vacuo to give a residue, which was chromatographed on silica gel with 50% ethyl acetate in petroleum ether (60-90°C) as eluent to afford 28 (1.64 g, 80% yield) as a white solid. 1 26,27-Hexadeuterio-lα,25-dihydroxyvitamin D 3 (4)   A solution of 28 (0.35 g, 0.8 mmol) and 9-AA (0.018 g, 0.08 mmol) in methanol (250 mL), contained in a 500-mL vessel in the BiLon photochemical reactor, was cooled at -5 to 0°C while purging with argon, and irradiated with a tunable high-pressure 1000 W mercury lamp until the reaction was complete by HPLC monitoring.Caution!Light from the mercury lamp is damaging to the eyes and skin.The solution was transferred to a round-bottomed flask and concentrated in vacuo to give a foam, which was chromatographed on silica gel with gradient eluting of 60 to 90% ethyl acetate in petroleum ether (60-90°C).The appropriate fractions were collected, evaporated in vacuo, crystallized from methyl formate under argon and protected from light to give 4 (0.21 g, 60% yield) as a white solid. 1