Efficient and scalable total synthesis of calcitroic acid and its 13C-labeled derivative

Article abstract of DOI:10.1039/c4ra04322g

Calcitroic acid, the deactivated form of the physiological active vitamin D₃ metabolite calcitrol, and its ¹³C labeled derivative has been synthesized starting from the commercially available Inhoffen-Lythgoe diol in 11 steps with an

Full text of DOI:10.1039/c4ra04322g

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Efficient and scalable total synthesis of calcitroic
13
acid and its C-labeled derivative†
Cite this: RSC Adv., 2014, 4, 32327
*
Daniel Meyer, Lara Rentsch and Roger Marti
Calcitroic acid, the deactivated form of the physiological active vitamin D3 metabolite calcitrol, and its 13
C
Received 9th May 2014
Accepted 10th July 2014
labeled derivative has been synthesized starting from the commercially available Inhoffen-Lythgoe diol in 11
steps with an overall yield of 21%. The key steps in the synthesis were the formation of the C1-homologated
nitrile with KCN and K13CN as well as the Horner–Wittig reaction with the ring A phosphine oxide reagent.
DOI: 10.1039/c4ra04322g
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As of today, there are three total syntheses of calcitroic acid
(1) reported – the rst one published by De Luca et al. in 1981.3
In this work, and in the one by Whalley et al. in 1985,4 the total
syntheses of calcitroic acid (1) start from steroid derivatives and
introduce in a linear synthesis the 1a-hydroxy function, do a C1-
homologation of the side chain, and forms the calcitriol skel-
eton by photochemical ring opening. In a similar approach,
Caverley5 synthesized calcitroic acid (1) via a seleno-acetal
containing vitamin D synthon.
Introduction
To form the physiologically active form, vitamin D3 undergoes
two hydroxylations – one in the liver at C25 and a subsequent
one in the kidney at C1 to produce 1a,25-dihydroxy vitamin D3,
also called calcitriol (Fig. 1). Calcitriol regulates many biological
functions like calcium homeostasis and bone mineralization,
proliferation and differentiation of various types of cells, and
immune modulation. Its interesting biological mode of action
led to intense research and application in the eld of bone
disorder, as well as autoimmune disorders, cancer or psoriasis.1
Deactivation of calcitriol starts by hydroxylation on C24 and
by further metabolic degradation, the inactive water soluble
calcitroic acid (1) is formed, which is excreted via the liver into
the bile.2 For supporting analytical studies on the fate of vitamin
D3 and calcitriol in the body, there was a need for calcitroic acid
(1) and a 13C-labeled derivative as reference materials.
As all these syntheses take at least 15 to 18 steps, have a low
yield and produce only very small amounts of the desired
calcitroic acid (1), there is
a need for a concise and
scalable synthesis of calcitroic acid (1) and a 13C-labeled
derivative.
Our retrosynthetic approach is based on a convergent
synthesis via Horner–Wittig olenation of an A-ring Wittig
reagent and a C,D-ring carbonyl compound (Scheme 1).6 The
advantage of this approach is that the A-ring Wittig reagent7 is
commercially available as diphenylphosphine oxide whereas
the C,D-ring ketone can be prepared from the Inhoffen-
Lythgoe diol 2, which itself is commercially available or is
easily prepared from vitamin D by ozonolysis.8
´
´
´
´
HES-SO Haute ecole specialisee de Suisse occidentale, Ecole d'ingenieurs et
´
d'architectes de Fribourg, Institute ChemTech, Bd Perolles 80, CH-1700 Fribourg,
Switzerland. E-mail: roger.marti@hefr.ch; Fax: +41 26 429 6600; Tel: +41 26 429 6703
† Electronic supplementary information (ESI) available: Images of 1H and 13C
NMR of all products. See DOI: 10.1039/c4ra04322g
Fig. 1 Vitamin D3, calcitriol and calcitroic acid (1).
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the mild enzymatic hydrolysis failed due to the low solubility of
the nitrile 4 in the aqueous buffer system.14 So we decided to
rst reduce the nitrile 4 to the aldehyde followed by oxidation to
the corresponding carboxylic acid.
Results and discussion
Starting from the commercially available Inhoffen-Lythgoe diol
2 we rst transformed the primary alcohol into a tosylate9,10
followed by protecting the secondary hydroxyl group in the 7-
position as a tert-butyldimethylsilyl ether8 3 (Scheme 2). Next, by
a simple nucleophilic substitution reaction with cyanide the C1-
homologated nitrile 4 was prepared,10,11 while by using 13C-
labeled NaCN the corresponding 13C-labeled derivative 5 in
position C23 (based on steroidal numbering) as obtained. The
overall yield for these three reactions was 74%, independent
whether 13CN was used or not.
Next we studied the transformation of the nitrile 4 into the
corresponding carboxylic acid or methyl ester. All attempts to
do a direct hydrolysis of nitrile 4 under acidic conditions12 (HCl
in MeOH) were not successful and resulted in the cleavage of
the silyl ether and elimination of water in the 7-position. Under
basic conditions13 (KOH in MeOH), a mixture of acid and
primary amide was obtained (by LC-MS). The use of nitrilase for
Therefore, the nitrile 4 was reduced by DIBAL to form the
aldehyde.10,11 It is important to do the hydrolysis of the
aluminum/imine intermediate long enough otherwise the yield
drops dramatically. Oxidation of the aldehyde was accom-
plished by the method of Pinnick and Lindgren15 in quantitative
yield and the carboxylic acid was directly transformed into the
methyl ester 6 by using TMS-diazomethane in methanol.16
Again, we observed no difference in the isolated yields for this 3-
step sequence for the 13C-labled ester 7. Deprotection of the TBS
group was accomplished by treatment with TFA17 and aer
oxidation with PDC,18 the C,D-ring ketone 8 was obtained with a
73% yield, respectively the 13C-labeled C,D-ring ketone 9 with a
68% yield, over two steps.
As shown in Scheme 3, the Horner–Wittig reaction of the
phosphine oxide reagent 10 and the C,D-ring ketone 8,
Scheme 1 Retrosynthetic analysis of calcitroic acid (1).
Scheme 2 Synthesis of C,D-ring ketone intermediate 8 and 9.
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Scheme 3 Synthesis of calcitroic acid (1) and 13C-labeled calcitroic acid 13.
and its 13C-labeled derivative 13 as disclosed in our work, will
stimulates medical & pharmaceutical research on the metabo-
lism and fate of vitamin D in the body.
Experimental section
General
Inhoffen Lythgoe diol 2 and A-ring phosphine oxide 10 were
provided by Syncom BV. All reactions involving oxygen- or
moisture-sensitive compound were carried out under a dry
argon atmosphere. Reaction temperatures refer to external bath
temperatures. Column chromatography was performed with
Scheme 4 Synthesis of alcohol metabolite 14.
ꢀ
Fluka silica gel (pore size 60 A, 230–400 mesh particle size)
packed in glass columns. Reactions were monitored by thin
layer chromatography (TLC) using aluminum Merck 60 UV254
silica gel plates (0.2 mm thickness). Visualization was per-
formed by ultraviolet light or by KMnO4 stain, followed by
gentle heating. NMR spectra were recorded at 300 MHz (for
1H NMR) or 75 MHz (for 13C NMR) in CDCl3. Chemical shis are
reported as d (ppm) downeld from tetramethylsilane (d ¼ 0.00)
using residual solvent signal as an internal standard: d singlet
7.26 (1H), triplet 77.0 (13C). IR spectra were obtained on neat
samples (ATR spectroscopy). High-resolution mass spectra were
recorded on an ESI-TOF MS spectrometer. Optical rotations
were obtained at a wavelength of 589 nm using a 1.0 dm cell.
respectively 13C-labeled ketone 9, yields the calcitroic acid
skeleton. Aer some optimization, the yield for this step was
89%. The products 11 and 12 are stable and can be easily
puried by chromatography.18,19 All attempts to reduce the
equivalents of the expensive Horner–Wittig reagent 10 were not
successful. Using less than 2 equivalents, only a complex
mixture of products was obtained.
Finally, deprotection of the TBS group with TBAF18 and
hydrolysis of the methyl ester under basic conditions furnished
the calcitroic acid 1 and its 13C-derivative 13 with a 73% yield as
an amorphous solid.3 Its analytical data corresponds with the
literature values,3,4 while for the 13C-derivative 13, the typical
13C-coupling could be observed in the 1H and 13C NMR spectra.
O-Silylated C,D-ring tosylate 3
Additionally, alcohol 14, a metabolic precursor to calcitroic DMAP (2.27 g, 18.6 mmol) and para-toluenesulfonyl chloride
acid (1) was prepared by reduction with LiAlH4 from the bis- (1.82 g, 9.56 mmol) was added to a solution of Inhoffen Lythgoe
silylated ester 11 (76% yield over two steps, Scheme 4). The diol 2 (1.97 g, 9.28 mmol) in DCM (30 mL). The mixture was
analytical data (NMR, MS) corresponds with literature5 but we stirred for 21 h at room temperature. The reaction mixture was
observed a fast decomposition within hours of the alcohol 14.
treated with HCl 1 M (25 mL) and extracted with DCM (3 ꢀ 80
mL). The organic layers were washed with H2O (25 mL), dried
over MgSO4, and concentrated. The crude product was puried
by chromatographic ltration (EtOAc : cyclohexane 1 : 9 /
Conclusion
In conclusion, we developed a new scalable and facile synthesis 2.5 : 7.5) to yield the C,D-ring tosylate (3.05 g, 90%) as a color-
of calcitroic acid (1) and its 13C-labeled derivative 13 in 11 steps less oil: Rf 0.40 (EtOAc : cyclohexane 2.5 : 7.5). To a solution of
with an overall yield of 21%, starting from commercially avail- C,D-ring tosylate (3.05 g, 8.32 mmol) in dry DCM (100 mL), 2,6-
able materials. In a highly efficient and concise protocol we lutidine (1.07 g, 10.0 mmol) was added followed by a solution of
homologated the Inhoffen-Lythgoe diol (C,D-ring) with KCN or tert-butyldimethylsilyl triuoromethanesulfonate (3.08 g, 11.7
K13CN followed by a Wittig reaction using the A-ring phosphine mmol) in dry DCM (15 mL) at 0 ꢁC. Aer stirring 1.5 h at 0 C,
ꢁ
oxide as the key steps. The easy availability of calcitroic acid (1) the mixture was quenched with H2O (80 mL) and the water
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phase was extracted with DCM (80 mL). The organic layers were for C1913CH37NOSiNa 359.2570 [M + Na]+, found 359.2572; [a]2D0
washed with H2O (80 mL), dried over MgSO4, and concentrated. + 52.6 (c 2.0, CHCl3).
The crude product was puried by column chromatography
(EtOAc : cyclohexane 5 : 95) to yield 3 (3.62 g, 91%) as a slightly
yellow oil: Rf 0.40 (EtOAc : cyclohexane 5 : 95); 1H-NMR (300
O-Silylated C,D-ring methyl ester 6
MHz, CDCl3) d ꢂ0.03 (s, 3H), 0.00 (s, 3H), 0.87 (s, 3H), 0.87 (s, To a stirred solution of 4 (1.51 g, 4.50 mmol) in dry DCM
9H), 0.95 (d, J ¼ 6.6 Hz, 3H), 1.03–1.25 (m, 4H), 1.26–1.42 (m, (50 mL), a solution of DIBAL (1.0 M in toluene, 15.8 mL, 15.8
3H), 1.43–1.57 (m, 1H), 1.58–1.71 (m, 3H), 1.72–1.81 (m, 1H), mmol) was added dropwise at 0 ꢁC and stirred 4 h at this
1.83–1.92 (m, 1H), 2.45 (s, 3H), 3.79 (dd, J ¼ 9.2, 6.3 Hz, 1H), temperature. The reaction was quenched with a solution of
3.91–4.02 (m, 2H) 7.31–7.38 (m, 2H), 7.75–7.82 (m, 2H); saturated aqueous NH4Cl (15 mL) and stirred for 30 min at 0 ꢁC.
13C-NMR (75 MHz, CDCl3) d ꢂ5.2, ꢂ4.8, 13.7, 16.8, 17.6, 18.0, TBME (75 mL) was added to the viscous suspension and stirred
ꢁ
21.6, 23.0, 25.8, 26.5, 34.3, 35.8, 40.3, 42.1, 52.4, 52.7, 69.2, 75.7, for 30 min at 0 C. The mixture was dried over MgSO4, ltered
127.9, 129.8, 133.2, 144.6.
through celite, and concentrated. The crude product was puri-
ed by column chromatography (EtOAc : cyclohexane 3 : 97) to
yield the O-silylated C,D-ring aldehyde (1.10 g, 72%) as a slightly
yellow oil: Rf 0.45 (EtOAc : cyclohexane 3 : 97); 1H-NMR (300
O-Silylated C,D-ring nitrile 4
Sodium hydroxide (0.58 g, 14.6 mmol) and potassium cyanide MHz, CDCl3) d 0.00 (s, 3H), 0.01 (s, 3H), 0.89 (s, 9H), 0.96 (s, 3H),
(0.95 g, 14.6 mmol) were added to a solution of 3 (3.51, 7.30 0.99 (d, J ¼ 6.4 Hz, 3H), 1.07–1.21 (m, 2H), 1.22–1.32 (m, 2H),
mmol) in DMSO (100 mL) and heated to 90 ꢁC for 5 h. A solution 1.33–1.44 (m, 3H), 1.51–1.62 (m, 1H), 1.63–1.90 (m, 3H), 1.91–
of NaCl (2% in H2O, 200 mL) was added to the reaction mixture 1.99 (m, 1H), 2.00–2.08 (m, 1H), 2.09–2.19 (m, 1H) 2.40–2.49 (m,
and extracted with cyclohexane (4 ꢀ 100 mL). The organic layers 1H), 3.97–4.03 (m, 1H), 9.74 (dd, J ¼ 3.4, 1.4 Hz, 1H); 13C-NMR
were washed with a solution of NaCl (2% in H2O, 2 ꢀ 100 mL), (75 MHz, CDCl3) d ꢂ5.2, ꢂ4.8, 13.7, 17.6, 18.0, 20.0, 23.0, 25.8,
dried over MgSO4, and concentrated. The crude product was 27.6, 31.3, 34.3, 40.6, 42.3, 50.8, 53.0, 56.6, 69.3, 203.6. To a
puried by column chromatography (EtOAc : cyclohexane stirred solution of the O-silylated C,D-ring aldehyde (1.08 g, 3.19
3 : 97) to yield 4 (2.31 g, 89%) as a colorless oil: Rf 0.35 mmol) in tert-butanol (60 mL) and 2-methyl-2-butene (15 mL), a
(EtOA : cyclohexane 3 : 97); 1H-NMR (300 MHz, CDCl3) d 0.00 (s, mixture of sodium chlorite (2.66 g, 29.4 mmol) and mono-
3H), 0.01 (s, 3H), 0.89 (s, 9H), 0.93 (s, 3H), 1.13 (d, J ¼ 6.6 Hz, sodium phosphate monohydrate (2.66, 19.3 mmol) in H2O (25
3H), 1.15–1.27 (m, 3H), 1.28–1.33 (m, 1H), 1.34–1.46 (m, 3H), mL) was added at room temperature. The reaction mixture was
1.52–1.68 (m, 2H), 1.69–1.86 (m, 3H), 1.87–1.96 (m, 1H), 2.23 stirred for 1.5 h and concentrated under reduced pressure. The
(dd, J ¼ 16.7, 6.9 Hz, 1H), 2.34 (dd, J ¼ 16.7, 3.9 Hz, 1H), 3.97– residue was treated with H2O (100 mL) and extracted with
4.03 (m, 1H); 13C-NMR (75 MHz, CDCl3) d ꢂ5.2, ꢂ4.8, 13.9, 17.5, cyclohexane (100/50/50 mL). The organic layers were washed
18.0, 19.3, 22.9, 24.7, 25.8, 27.2, 33.1, 34.2, 40.3, 42.2, 52.9, 55.4, with H2O (50 mL), dried over MgSO4, and concentrated. The
69.2, 119.0; IR (neat) 772, 834, 1020, 1083, 1164, 1251, 2245, crude product was puried by column chromatography
2856, 2929 cmꢂ1; HRMS (ESI-TOF) m/z calcd for C20H37NOSiNa (EtOAc : cyclohexane 2 : 8) to yield the O-silylated C,D-ring acid
358.2537 [M + Na]+, found 358.2539; [a]2D0 + 53.0 (c 2.0, CHCl3). (1.11 g, 98%) as a white solid: Rf 0.35 (EtOAc : cyclohexane 2 : 8);
1H-NMR (300 MHz, CDCl3) d ꢂ0.01 (s, 3H), 0.01 (s, 3H), 0.89 (s,
9H), 0.95 (s, 3H), 1.01 (d, J ¼ 6.3 Hz, 3H), 1.06–1.19 (m, 2H),
[23-13C] O-Silylated C,D-ring nitrile 5
1.21–1.32 (m, 2H), 1.33–1.44 (m, 3H), 1.50–1.62 (m, 1H), 1.63–
Sodium hydroxide (1.86 g, 46.6 mmol) and potassium cyani- 1.71 (m, 1H), 1.72–1.86 (m, 2H), 1.87–2.06 (m, 3H), 2.47 (dd, J ¼
de-13C (3.08 g, 46.6 mmol) were added to a solution of 3 (11.18, 14.2, 2.7 Hz, 1H), 3.97–4.03 (m, 1H); 13C-NMR (75 MHz, CDCl3) d
ꢁ
23.3 mmol) in DMSO (300 mL) and heated to 90 C for 5 h. A ꢂ5.2, ꢂ4.8, 13.7, 17.6, 18.0, 19.5, 23.0, 25.8, 27.3, 33.2, 34.4,
solution of NaCl (2% in H2O, 600 mL) was added to the reaction 40.6, 41.3, 42.3, 53.1, 56.5, 69.4, 180.3. Trimethylsilyldiazo-
mixture and extracted with cyclohexane (4 ꢀ 300 mL). The methane (2 M in diethyl ether, 2.3 mL, 4.6 mmol) was added
organic layers were washed with a solution of NaCl (2% in H2O, dropwise to a solution of the O-silylated C,D-ring acid (1.10 g,
2 ꢀ 300 mL), dried over MgSO4, and concentrated. The crude 3.10 mmol) in toluene (18 mL) and methanol (12 mL) at room
product
was
puried
by
column
chromatography temperature. The reaction mixture was stirred for 1 h. Excess of
(EtOAc : cyclohexane 3 : 97) to yield 5 (7.10 g, 91%) as a colorless diazomethane was destroyed with acetic acid. The mixture was
oil: Rf 0.35 (EtOAc : cyclohexane 3 : 97); 1H-NMR (300 MHz, concentrated and the residue puried by column chromatog-
CDCl3) d 0.00 (s, 3H), 0.01 (s, 3H), 0.89 (s, 9H), 0.93 (s, 3H), 1.13 raphy (EtOAc : cyclohexane 3 : 97) to yield 6 (1.06 g, 93%) as a
(d, J ¼ 6.6 Hz, 3H), 1.15–1.27 (m, 3H), 1.28–1.33 (m, 1H), 1.34– slightly yellow liquid: Rf 0.50 (EtOAc : cyclohexane 3 : 97); 1H-
1.46 (m, 3H), 1.51–1.68 (m, 2H), 1.69–1.86 (m, 3H), 1.87–1.96 NMR (300 MHz, CDCl3) d ꢂ0.01 (s, 3H), 0.01 (s, 3H), 0.89 (s,
(m, 1H), 2.23 (ddd, J ¼ 16.6, 9.4, 6.9 Hz, 1H), 2.34 (ddd, J ¼ 16.6, 9H), 0.95 (s, 3H), 0.96 (d, J ¼ 5.8 Hz, 3H), 1.01–1.18 (m, 2H),
9.6, 3.9 Hz, 1H), 3.97–4.03 (m, 1H); 13C-NMR (75 MHz, CDCl3) d 1.20–1.31 (m, 2H), 1.32–1.43 (m, 3H), 1.50–1.62 (m, 1H), 1.63–
ꢂ5.2, ꢂ4.8, 13.9, 17.5, 18.0, 19.3 (d, J ¼ 1.5 Hz), 22.9, 24.7 (d, J ¼ 1.71 (m, 1H), 1.72–1.86 (m, 2H), 1.87–2.04 (m, 3H), 2.42 (dd, J ¼
55.5 Hz), 25.8, 27.2, 33.1 (d, J ¼ 2.4 Hz), 34.2, 40.3, 42.2, 52.9, 13.8, 2.6 Hz, 1H), 3.65 (s, 3H), 3.97–4.03 (m, 1H); 13C-NMR (75
55.4 (d, J ¼ 3.1 Hz), 69.2, 119.1; IR (neat) 772, 834, 1020, 1083, MHz, CDCl3) d ꢂ5.2, ꢂ4.8, 13.8, 17.6, 18.0, 19.5, 23.0, 25.8, 27.3,
1164, 1251, 2190, 2856, 2929 cmꢂ1; HRMS (ESI-TOF) m/z calcd 33.4, 34.4, 40.6, 41.4, 42.3, 51.3, 53.1, 56.6, 69.4, 174.1; IR (neat)
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772, 834, 1021, 1089, 1162, 1251, 1739, 2856, 2930 cmꢂ1; HRMS 2.04 (m, 3H), 2.42 (ddd, J ¼ 13.9, 7.3, 2.7 Hz, 1H), 3.65 (d, J ¼ 3.8
(ESI-TOF) m/z calcd for C21H40O3SiNa 391.2639 [M + Na]+, found Hz, 3H), 3.97–4.03 (m, 1H); 13C-NMR (75 MHz, CDCl3) d ꢂ5.2,
391.2635; [a]2D0 + 41.9 (c 2.0, CHCl3).
ꢂ4.8, 13.8, 17.6, 18.0, 19.5 (d, J ¼ 1.0 Hz), 23.0, 25.8, 27.3, 33.4
(d, J ¼ 1.5 Hz), 34.4, 40.6, 41.4 (d, J ¼ 57.3), 42.3, 51.3 (d, J ¼
2.8 Hz), 53.1, 56.6 (d, J ¼ 4.2 Hz), 69.4, 174.1; IR (neat) 772, 834,
1021, 1089, 1164, 1251, 1698, 2855, 2929 cmꢂ1; HRMS (ESI-TOF)
[23-13C] O-Silylated C,D-ring methyl ester 7
To a stirred solution of 5 (1.99 g, 5.91 mmol) in dry DCM (50 m/z calcd for C2013CH40O3SiNa 392.2672 [M + Na]+, found
mL), a solution of DIBAL (1.0 M in toluene, 21.4 mL, 21.4 mmol) 392.2677; [a]2D0 + 41.6 (c 2.0, CHCl3).
was added dropwise at 0 ꢁC and stirred 4 h at this temperature.
The reaction was quenched with a solution of saturated
aqueous NH4Cl (15 mL) and stirred for 30 min at 0 ꢁC. TBME (75
mL) was added to the viscous suspension and stirred for 30 min
at 0 ꢁC. The mixture was dried over MgSO4, ltered through
celite, and concentrated. The crude product was puried by
column chromatography (EtOAc : cyclohexane 3 : 97) to yield
the [23-13C] O-silylated C,D-ring aldehyde (1.50 g, 75%) as a
slightly yellow oil: Rf 0.45 (EtOAc : cyclohexane 3 : 97); 1H-NMR
(300 MHz, CDCl3) d 0.00 (s, 3H), 0.01 (s, 3H), 0.89 (s, 9H), 0.96 (s,
3H), 0.99 (d, J ¼ 6.5 Hz, 3H), 1.07–1.21 (m, 2H), 1.22–1.32 (m,
2H), 1.33–1.43 (m, 3H), 1.51–1.62 (m, 1H), 1.63–1.90 (m, 3H),
1.91–1.99 (m, 1H), 2.00–2.08 (m, 1H), 2.09–2.19 (m, 1H) 2.39–
2.50 (m, 1H), 3.97–4.03 (m, 1H), 9.74 (ddd, J ¼ 169.1, 3.4, 1.4 Hz,
1H); 13C-NMR (75 MHz, CDCl3) d ꢂ5.2, ꢂ4.8, 13.7, 17.6, 18.0,
20.0 (d, J ¼ 1.4 Hz), 23.0, 25.8, 27.6, 31.3, 34.3, 40.6, 42.3, 50.8 (d,
J ¼ 39.2 Hz), 53.0, 56.6 (d, J ¼ 3.7 Hz), 69.3, 203.6. To a stirred
solution of the [23-13C] O-silylated C,D-ring aldehyde (178 mg,
0.524 mmol) in tert-butanol (10 mL) and 2-methyl-2-butene (2.5
mL), a mixture of sodium chlorite (0.43 g, 4.76 mmol) and
monosodium phosphate monohydrate (0.43, 3.12 mmol) in
H2O (4 mL) was added at room temperature. The reaction
mixture was stirred for 1.5 h and concentrated under reduced
pressure. The residue was treated with H2O (30 mL) and
extracted with cyclohexane (30/15/15 mL). The organic layers
were washed with H2O (15 mL), dried over MgSO4, and
concentrated. The crude product was puried by column
chromatography (EtOAc : cyclohexane 2 : 8) to yield the [23-13C]
O-silylated C,D-ring acid (182 mg, 98%) as a white solid: Rf 0.35
C,D-Ring ketone 8
A solution of 6 (1.02 g, 2.77 mmol) in dry DCM (30 mL) was
treated with TFA (3 mL) at 0 ꢁC. The reaction mixture was stirred
for 90 min and then allowed to warm up to room temperature.
The mixture was concentrated and the residue was puried by
column chromatography (EOAc : cyclohexane 2 : 8) to yield the
C,D-ring alcohol (0.52 g, 74%) as a slightly yellow oil: Rf 0.30
(EtOAc : cyclohexane 2 : 8); 1H-NMR (300 MHz, CDCl3) d 0.96 (d,
J ¼ 6.2 Hz, 3H), 0.97 (s, 3H), 1.05–1.22 (m, 2H), 1.24–1.39 (m,
2H), 1.40–1.51 (m, 3H), 1.52–1.66 (m, 2H), 1.73–1.88 (m, 3H),
1.89–2.05 (m, 3H), 2.43 (dd, J ¼ 14.0, 2.9 Hz, 1H), 3.66 (s, 3H),
4.04–4.11 (m, 1H); 13C-NMR (75 MHz, CDCl3) d 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. PDC (1.48 g, 3.93 mmol) was added to a stirred solution
of C,D-ring alcohol (0.50 g, 1.97 mmol) in dry DCM (100 mL)
and stirred overnight at room temperature. TBME (100 mL) was
added and the suspension ltered through celite. The ltrate
was concentrated and the residue was puried by column
chromatography (EtOAc : cyclohexane 2 : 8) to yield 8 (0.49 g,
98%) as a slightly yellow oil: Rf 0.35 (EtOAc : cyclohexane 2 : 8);
1H-NMR (300 MHz, CDCl3) d 0.68 (s, 3H), 1.03 (d, J ¼ 6.4 Hz, 3H),
1.21–1.43 (m, 1H), 1.44–1.67 (m, 3H), 1.68–1.80 (m, 1H), 1.81–
2.15 (m, 6H), 2.16–2.34 (m, 2H), 2.38–2.53 (m, 2H), 3.67 (s, 3H);
13C-NMR (75 MHz, CDCl3) d 12.5, 19.1, 19.6, 24.0, 27.5, 33.4,
38.8, 40.9, 41.1, 49.8, 51.4, 56.3, 61.9, 173.6, 211.6; IR (neat)
1150, 1190, 1710, 1733, 2874, 2953 cmꢂ1; HRMS (ESI-TOF) m/z
calcd for C15H24O3Na 275.1618 [M + Na]+, found 275.1623; [a]2D0
ꢂ 6.9 (c 2.0, CHCl3).
1
(EtOAc : cyclohexane 2 : 8); H-NMR (300 MHz, CDCl3) d ꢂ0.01
(s, 3H), 0.01 (s, 3H), 0.89 (s, 9H), 0.95 (s, 3H), 1.00 (d, J ¼ 6.3 Hz,
[23-13C] C,D-Ring ketone 9
3H), 1.06–1.19 (m, 2H), 1.21–1.32 (m, 2H), 1.33–1.44 (m, 3H),
1.50–1.62 (m, 1H), 1.63–1.71 (m, 1H), 1.72–1.85 (m, 2H), 1.86– A solution of 7 (554 mg, 1.50 mmol) in dry DCM (15 mL) was
2.06 (m, 3H), 2.47 (ddd, J ¼ 14.0, 7.1, 2.6 Hz, 1H), 3.97–4.03 (m, treated with TFA (2 mL) at 0 ꢁC. The reaction mixture was stirred
1H); 13C-NMR (75 MHz, CDCl3) d ꢂ5.2, ꢂ4.8, 13.7, 17.6, 18.0, for 90 min and then allowed to warm up to room temperature.
19.5, 23.0, 25.8, 27.3, 33.2, 34.4, 40.6, 41.3 (d, J ¼ 55.0 Hz), 42.3, The mixture was concentrated and the residue puried by
53.1, 56.5 (d, J ¼ 4.1 Hz), 69.4, 180.1. Trimethylsilyldiazo- column chromatography (EOAc : cyclohexane 2 : 8) to yield the
methane (2 M in diethyl ether, 0.38 mL, 0.76 mmol) was added [23-13C]-C,D-ring alcohol (281 mg, 73%) as a slightly yellow oil:
dropwise to a solution of the [23-13C] O-silylated C,D-ring acid Rf 0.30 (EtOAc : cyclohexane 2 : 8); 1H-NMR (300 MHz, CDCl3) d
(182 mg, 0.512 mmol) in toluene (3 mL) and methanol (2 mL) at 0.96 (d, J ¼ 6.3 Hz, 3H), 0.96 (s, 3H), 1.05–1.22 (m, 2H), 1.24–1.40
room temperature. The reaction mixture was stirred for 1 h. (m, 2H), 1.41–1.53 (m, 3H), 1.54–1.67 (m, 1H), 1.73–1.88 (m,
Excess of diazomethane was destroyed with acetic acid. The 3H), 1.89–2.06 (m, 3H), 2.43 (ddd, J ¼ 13.5, 7.0, 2.7 Hz, 1H), 3.26
mixture was concentrated and the residue was puried by (s, 1H), 3.66 (d, J ¼ 3.8 Hz, 3H), 4.05–4.14 (m, 1H); 13C-NMR (75
column chromatography (EtOAc : cyclohexane 3 : 97) to yield 7 MHz, CDCl3) d 13.5, 17.4, 19.4 (d, J ¼ 1.0 Hz), 22.5, 27.2, 33.36
(168 mg, 89%) as
a
slightly yellow liquid: Rf 0.50 (d, J ¼ 1.5 Hz), 33.39, 40.2, 41.3 (d, J ¼ 57.3 Hz), 42.0, 51.4 (d, J ¼
(EtOAc : cyclohexane 3 : 97); 1H-NMR (300 MHz, CDCl3) d ꢂ0.01 2.8 Hz), 52.5, 56.4 (d, J ¼ 4.2 Hz), 69.5, 174.1. PDC (0.81 g, 2.15
(s, 3H), 0.01 (s, 3H), 0.89 (s, 9H), 0.94 (s, 3H), 0.95 (d, J ¼ 5.9 Hz, mmol) was added to a stirred solution of [23-13C]-C,D-ring
3H), 1.01–1.17 (m, 2H), 1.20–1.31 (m, 2H), 1.32–1.44 (m, 3H), alcohol (274 mg, 1.07 mmol) in dry DCM (50 mL) and stirred
1.49–1.62 (m, 1H), 1.63–1.71 (m, 1H), 1.72–1.86 (m, 2H), 1.87– overnight at room temperature. TBME (50 mL) was added and
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the suspension ltered through celite. The ltrate was concen- 0.08 (m, 12H), 0.57 (s, 3H), 0.88 (s, 18H), 0.99 (d, J ¼ 6.2 Hz, 3H),
trated and the residue was puried by column chromatography 1.21–1.37 (m, 3H), 1.41–1.59 (m, 3H), 1.60–1.69 (m, 2H), 1.70–
(EtOAc : cyclohexane 2 : 8) to yield 9 (252 mg, 93%) as a slightly 1.81 (m, 1H), 1.82–1.94 (m, 3H), 1.95–2.07 (m, 3H), 2.21 (dd, J ¼
yellow oil: Rf 0.35 (EtOAC : cyclohexane 2 : 8); 1H-NMR (300 13.1, 7.5 Hz, 1H), 2.37–2.50 (m, 2H), 2.75–2.87 (m, 1H), 3.66 (d, J
MHz, CDCl3) d 0.68 (s, 3H), 1.03 (d, J ¼ 6.4 Hz, 3H), 1.21–1.43 ¼ 3.8 Hz, 3H), 4.13–4.25 (m, 1H), 4.37 (dd, J ¼ 6.5, 3.6 Hz, 1H),
(m, 1H), 1.45–1.67 (m, 3H), 1.68–1.80 (m, 1H), 1.81–2.15 (m, 4.86 (d, J ¼ 2.4 Hz, 1H), 5.17 (dd, J ¼ 2.4, 0.8 Hz, 1H), 6.02 (d, J ¼
6H), 2.16–2.35 (m, 2H), 2.38–2.54 (m, 2H), 3.67 (d, J ¼ 3.8 Hz, 11.2 Hz, 1H), 6.24 (d, J ¼ 11.2 Hz, 1H); 13C-NMR (75 MHz,
3H); 13C-NMR (75 MHz, CDCl3) d 12.5, 19.1, 19.6 (d, J ¼ 1.0 Hz), CDCl3) d ꢂ5.1, ꢂ4.8, ꢂ4.68, ꢂ4.67, 12.0, 18.15, 18.22, 19.7, 22.1,
24.0, 27.5, 33.4 (d, J ¼ 1.4 Hz), 38.8, 40.9, 41.1 (d, J ¼ 57.5 Hz), 23.4, 25.8, 25.9, 27.7, 28.8, 34.1, 40.5, 41.4 (d, J ¼ 57.3 Hz), 44.8,
49.8, 51.4 (d, J ¼ 2.8 Hz), 56.3 (d, J ¼ 4.2 Hz), 61.9, 173.6, 211.6; 45.8, 46.1, 51.3 (d, J ¼ 2.7 Hz), 56.29, 56.31 (d, J ¼ 4.2), 67.5, 72.1,
IR (neat) 1138, 1189, 1691, 1710, 2874, 2953 cmꢂ1; HRMS (ESI- 111.3, 118.1, 123.1, 135.2, 140.6, 148.3, 174.0; IR (neat) 773, 831,
TOF) m/z calcd for C1413CH24O3Na 276.1651 [M + Na]+, found 1071, 1251, 1698, 2855, 2928, 2949 cmꢂ1; HRMS (ESI-TOF) m/z
276.1650; [a]2D0 ꢂ 6.9 (c 2.0, CHCl3).
calcd for C3513CH64O4Si2Na 640.4269 [M + Na]+, found 640.4275;
[a]2D0 + 24.2 (c 2.0, CHCl3).
O-Bis-silylated calcitroic acid methyl ester 11
A solution of n-BuLi (1.6 M in hexane, 0.30 mL, 0.480 mmol) was
added dropwise to a solution of A-ring phosphine oxide 10 (306
Calcitroic acid 1
A solution of tetrabutylammonium uoride (1 M in THF, 2.6 mL,
2.60 mmol) was added to a solution of 11 (159 mg, 0.258 mmol)
in dry THF (25 mL) at room temperature and stirred overnight.
The reaction mixture was treated with a saturated aqueous
NH4Cl solution (25 mL) and extracted with EtOAc (50/25 mL).
The organic layer were washed with brine (25 mL), dried over
MgSO4, and concentrated. The crude product was puried by
column chromatography (EtOAc : cyclohexane 7 : 3) to yield the
calcitroic acid methyl ester (97 mg, 97%) as a yellow oil: Rf 0.35
(EtOAc : cyclohexane 7 : 3); 1H-NMR (300 MHz, CDCl3) d 0.58 (s,
3H), 0.99 (d, J ¼ 6.3 Hz, 3H), 1.22–1.38 (m, 3H), 1.42–1.61 (m,
3H), 1.62–1.77 (m, 2H), 1.80–2.07 (m, 7H), 2.15 (s, 2H), 2.31 (dd, J
¼ 13.4, 6.5 Hz, 1H), 2.44 (dd, J ¼ 14.2, 2.9 Hz, 1H), 2.58 (dd, J ¼
13.4, 3.3 Hz, 1H), 2.82 (dd, J ¼ 11.7, 3.3 Hz, 1H), 3.66 (s, 3H),
4.15–4.26 (m, 1H), 4.42 (dd, J ¼ 7.7, 4.4 Hz, 1H), 4.95–5.01 (m,
1H), 5.28–5.35 (m, 1H), 6.02 (d, J ¼ 11.2 Hz, 1H), 6.36 (d, J ¼ 11.2
Hz, 1H),; 13C-NMR (75 MHz, CDCl3) d 12.0, 19.7, 22.2, 23.5, 27.6,
29.0, 34.1, 40.3, 41.3, 42.8, 45.2, 45.9, 51.4, 56.27 (2C), 66.7, 70.7,
111.8, 117.3, 124.7, 133.4, 142.5, 147.6, 174.0. The calcitroic acid
methyl ester (84 mg, 0.216 mmol) was dissolved in a solution of
KOH (10% in MeOH : H2O 9 : 1, 20 mL) and stirred for 1.5 h at
60 ꢁC. The reaction mixture was cooled to room temperature and
acidied with concentrated HCl solution to pH 2. The mixture
was concentrated, diluted with H2O (20 mL) and extracted with
EtOAc (4 ꢀ 20 mL). The organic layers were washed with brine (2
ꢀ 20 mL), dried over MgSO4, and concentrated. The crude
product was puried by column chromatography (EtOAc) to
ꢁ
mg, 0.525 mmol) in dry THF (6 mL) at ꢂ78 C. The deep red
solution was stirred at ꢂ78 ꢁC for 1 h, followed by the dropwise
addition of a solution of 8 (69.7 mg, 0.276 mmol) in dry THF (3
mL). The solution was stirred for 5 h at ꢂ78 ꢁC and then allowed
to warm up to room temperature. The reaction mixture was
quenched with H2O (10 mL) and extracted with TBME (2 ꢀ 30
mL). The organic layers were washed with brine (30 mL), dried
over MgSO4, and concentrated. The crude product was puried
by column chromatography (EtOAc : cyclohexane 3 : 97) to yield
11 (153 mg, 89%) as
a slightly yellow oil: Rf 0.45
(EtOAc : cyclohexane 3 : 97); 1H-NMR (300 MHz, CDCl3) d 0.04–
0.09 (m, 12H), 0.57 (s, 3H), 0.88 (s, 18H), 0.99 (d, J ¼ 6.2 Hz, 3H),
1.23–1.38 (m, 3H), 1.40–1.59 (m, 3H), 1.60–1.69 (m, 2H), 1.70–
1.80 (m, 1H), 1.81–1.94 (m, 3H), 1.95–2.07 (m, 3H), 2.21 (dd, J ¼
13.1, 7.5 Hz, 1H), 2.38–2.51 (m, 2H), 2.75–2.87 (m, 1H), 3.66 (s,
3H), 4.13–4.25 (m, 1H), 4.37 (dd, J ¼ 6.5, 3.6 Hz, 1H), 4.86 (d, J ¼
2.4 Hz, 1H), 5.17 (dd, J ¼ 2.4, 0.8 Hz, 1H), 6.02 (d, J ¼ 11.2 Hz,
1H), 6.24 (d, J ¼ 11.2 Hz, 1H); 13C-NMR (75 MHz, CDCl3) d ꢂ5.1,
ꢂ4.8, ꢂ4.68, ꢂ4.67, 12.0, 18.1, 18.2, 19.7, 22.1, 23.4, 25.8, 25.9,
27.7, 28.8, 34.1, 40.5, 41.4, 44.8, 45.8, 46.1, 51.3, 56.30 (2C), 67.5,
72.1, 111.3, 118.1, 123.1, 135.2, 140.6, 148.3, 174.0; IR (neat)
773, 831, 1071, 1251, 1740, 2855, 2928, 2949 cmꢂ1; HRMS (ESI-
TOF) m/z calcd for C36H64O4Si2Na 639.4235 [M + Na]+, found
639.4235; [a]2D0 + 25.3 (c 2.0, CHCl3).
[23-13C] O-Bis-silylated calcitroic acid methyl ester 12
A solution of n-BuLi (1.6 M in hexane, 0.29 mL, 0.46 mmol) was yield 1 (60 mg, 74%) as a white amorphous solid: Rf 0.40 (EtOAc);
added dropwise to a solution of A-ring phosphine oxide 10 (296 1H-NMR (300 MHz, CDCl3) d 0.58 (s, 3H), 1.04 (d, J ¼ 6.3 Hz, 3H),
ꢁ
mg, 0.507 mmol) in dry THF (5 mL) at ꢂ78 C. The deep red 1.22–1.40 (m, 3H), 1.42–1.61 (m, 3H), 1.62–1.77 (m, 2H), 1.80–
solution was stirred at ꢂ78 ꢁC for 1 h followed by the dropwise 2.11 (m, 7H), 2.15 (s, 2H), 2.31 (dd, J ¼ 13.4, 6.5 Hz, 1H), 2.48 (dd,
addition of a solution of 9 (67.6 mg, 0.267 mmol) in dry THF (2.5 J ¼ 14.6, 3.0 Hz, 1H), 2.59 (dd, J ¼ 13.4, 3.3 Hz, 1H), 2.82 (d, J ¼
mL). The solution was stirred for 5 h at ꢂ78 ꢁC and then allowed 11.8, 3.1 Hz, 1H), 4.18–4.28 (m, 1H), 4.43 (dd, J ¼ 7.7, 4.4 Hz, 1H),
to warm up to room temperature. The reaction mixture was 4.97–5.03 (m, 1H), 5.29–5.36 (m, 1H), 6.02 (d, J ¼ 11.2 Hz, 1H),
quenched with H2O (10 mL) and extracted with TBME (2 ꢀ 30 6.37 (d, J ¼ 11.2 Hz, 1H); 13C-NMR (75 MHz, CDCl3) d 12.0, 19.7,
mL). The organic layers were washed with brine (30 mL), dried 22.2, 23.5, 27.6, 29.0, 33.9, 40.3, 41.2, 42.8, 45.2, 45.9, 56.2, 56.3,
over MgSO4, and concentrated. The crude product was puried 66.9, 70.8, 111.9, 117.3, 124.9, 133.2, 142.7, 147.5, 178.9; IR (neat)
by column chromatography (EtOAc : cyclohexane 3 : 97) to yield 1051, 1704, 2871, 2927, 3344 cmꢂ1; HRMS (ESI-TOF) m/z calcd
12 (143 mg, 87%) as
a
slightly yellow oil: Rf 0.45 for C23H34O4Na 397.2349 [M + Na]+, found 397.2349; [a]D20 ꢂ 5.5
(EtOAc : cyclohexane 3 : 97); 1H-NMR (300 MHz, CDCl3) d 0.03– (c 2.0, CHCl3).
32332 | RSC Adv., 2014, 4, 32327–32334
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[23-13C] Calcitroic acid 13
ꢁ
dry THF (5 mL) at 0 C. The mixture was stirred for 15 min at
0 ꢁC and then allowed to warm up to room temperature. Aer 2
h stirring at room temperature the reaction mixture was
quenched with a saturated aqueous NH4Cl solution (20 mL) and
extracted with EtOAc (3 ꢀ 20 mL). The organic layers were
washed with brine (20 mL), dried over MgSO4, and concen-
trated. The crude product was puried by column chromatog-
raphy (EtOAc) to yield 14 (31.8 mg, 78%) as a white amorphous
solid: Rf 0.35 (EtOAc); 1H-NMR (300 MHz, CDCl3) d 0.56 (s, 3H),
0.96 (d, J ¼ 6.5 Hz, 3H), 1.20–1.38 (m, 4H), 1.42–1.59 (m, 4H),
1.62–1.80 (m, 3H), 1.80–2.07 (m, 8H), 2.31 (dd, J ¼ 13.4, 6.6 Hz,
1H), 2.59 (dd, J ¼ 13.4, 3.5 Hz, 1H), 2.82 (dd, J ¼ 11.6, 3.5 Hz,
1H), 3.57–3.78 (m, 2H), 4.18–4.28 (m, 1H), 4.42 (dd, J ¼ 7.8, 4.4
Hz, 1H), 4.97–5.02 (m, 1H), 5.28–5.35 (m, 1H), 6.02 (d, J ¼ 11.3
Hz, 1H), 6.37 (d, J ¼ 11.3 Hz, 1H); HRMS (ESI-TOF) m/z calcd for
A solution of tetrabutylammonium uoride (1 M in THF, 1.1
mL, 1.1 mmol) was added to a solution of 12 (64.4 mg, 0.107
mmol) in dry THF (11 mL) at room temperature and stirred
overnight. The reaction mixture was treated with a saturated
aqueous NH4Cl solution (25 mL) and extracted with EtOAc (2 ꢀ
25 mL). The organic layer were washed with brine (25 mL), dried
over MgSO4, and concentrated. The crude product was puried
by column chromatography (EtOAc : cyclohexane 7 : 3) to yield
the [23-13C] calcitroic acid methyl ester (41.1 mg, 98%) as a
yellow oil: Rf 0.35 (EtOAc : cyclohexane 7 : 3); 1H-NMR (300
MHz, CDCl3) d 0.58 (s, 3H), 0.99 (d, J ¼ 6.3 Hz, 3H), 1.23–1.38
(m, 3H), 1.42–1.61 (m, 3H), 1.62–1.77 (m, 2H), 1.80–2.07 (m,
9H), 2.31 (dd, J ¼ 13.4, 6.5 Hz, 1H), 2.44 (ddd, J ¼ 14.0, 7.5, 2.9
Hz, 1H), 2.59 (dd, J ¼ 13.4, 2.3 Hz, 1H), 2.82 (dd, J ¼ 11.8, 3.3 Hz,
1H), 3.66 (d, J ¼ 3.8 Hz, 3H), 4.15–4.26 (m, 1H), 4.42 (dd, J ¼ 7.5,
4.4 Hz, 1H), 4.95–5.02 (m, 1H), 5.28–5.35 (m, 1H), 6.02 (d, J ¼
11.2 Hz, 1H), 6.36 (d, J ¼ 11.2 Hz, 1H); 13C-NMR (75 MHz,
CDCl3) d 12.0, 19.7, 22.2, 23.5, 27.6, 29.0, 34.1 (d, J ¼ 1.3 Hz),
40.3, 41.3 (d, J ¼ 57.3 Hz), 42.8, 45.2, 45.9, 51.4 (d, J ¼ 2.8 Hz),
56.28, 56.28 (d, J ¼ 4.0 Hz), 66.8, 70.8, 111.8, 117.3, 124.8, 133.3,
142.6, 147.6, 174.0. The [23-13C] calcitroic acid methyl ester
(41.1 mg, 0.106 mmol) was dissolved in a solution of KOH (10%
in MeOH : H2O 9 : 1, 10 mL) and stirred for 1.5 h at 60 ꢁC. The
reaction mixture was cooled to room temperature and acidied
with concentrated HCl to pH 2. The mixture was concentrated,
diluted with H2O (15 mL) and extracted with EtOAc (4 ꢀ 15 mL).
The organic layers were washed with brine (2 ꢀ 15 mL), dried
over MgSO4, and concentrated. The crude product was puried
by column chromatography (EtOAc) to yield 13 (29.5 mg, 74%)
as a white amorphous solid: Rf 0.40 (EtOAc); 1H-NMR (300 MHz,
CDCl3) d 0.58 (s, 3H), 1.04 (d, J ¼ 6.3 Hz, 3H), 1.22–1.40 (m, 3H),
1.42–1.61 (m, 3H), 1.62–1.77 (m, 2H), 1.80–2.11 (m, 7H), 2.15 (s,
2H), 2.31 (dd, J ¼ 13.4, 6.6 Hz, 1H), 2.47 (ddd, J ¼ 14.3, 7.2, 2.8
Hz, 1H), 2.59 (dd, J ¼ 13.4, 3.1 Hz, 1H), 2.82 (dd, J ¼ 11.8, 3.1 Hz,
1H), 4.18–4.28 (m, 1H), 4.43 (dd, J ¼ 7.6, 4.4 Hz, 1H), 4.97–5.03
(m, 1H), 5.29–5.36 (m, 1H), 6.02 (d, J ¼ 11.2 Hz, 1H), 6.37 (d, J ¼
11.2 Hz, 1H); 13C-NMR (75 MHz, CDCl3) d 12.0, 19.7, 22.2, 23.5,
27.6, 29.0, 33.9, 40.3, 41.2 (d, J ¼ 55.1 Hz), 42.7, 45.1, 45.9, 56.2
(d, J ¼ 4.2 Hz), 56.3, 66.9, 70.8, 111.9, 117.3, 124.8, 133.2, 142.6,
147.5, 178.9; IR (neat) 1050, 1663, 2870, 2925, 3343 cmꢂ1; HRMS
(ESI-TOF) m/z calcd for C2213CH34O4Na 398.2383 [M + Na]+,
found 398.2380; [a]2D0 ꢂ 4.7 (c 2.0, CHCl3).
C
23CH36O3Na 383.2557 [M + Na]+, found 383.2554.
Conflict of interest
The authors declare no competing nancial interest.
Acknowledgements
We thank C. Cluse and S. Rossier for valuable lab support for
the synthesis of ketone intermediates as well as Dr O. Zoller and
H. Reinhard for analytical support. This work was supported by
the Federal Food Safety and Veterinary Office, Bern,
Switzerland.
References
1 Vitamin D, ed. D. Feldman, J. W. Pike and J. S. Adams,
Elsevier Science Publishing Co Inc, London, U.K., 2011.
2 (a) G. S. Reddy and K.-Y. Tserng, Biochemistry, 1989, 28,
1763–1769; (b) D. R. Zimmerman, T. A. Reinhardt,
R. Kremer, D. C. Beitz, G. S. Reddy and R. L. Horst, Arch.
Biochem. Biophys., 2001, 392, 14–22.
3 (a) R. P. Esvelt, M. A. Fivizzani, H. E. Paaren, H. K. Schnoes
and H. F. DeLuca, J. Org. Chem., 1981, 46, 456–458; (b)
H. F. DeLuca, H. K. Schnoes and R. P. Estvelt, US Patent,
US4260804, 1981.
4 B. R. De Costa, N. Makk, J. M. Midgley, N. T. Modi, R. A. Watt
and W. B. Whalley, J. Chem. Soc., Perkin Trans. 1, 1985, 7,
1331–1336.
Alcohol metabolite 14
5 M. J. Calverley, Synlett, 1990, 3, 155–157.
A solution of tetrabutylammonium uoride (1 M in THF, 1.2
mL, 1.20 mmol) was added to a solution of 11 (72.0 mg, 0.117
mmol) in dry THF (10 mL) at room temperature and stirred
overnight. The reaction mixture was treated with a saturated
aqueous NH4Cl solution (25 mL) and extracted with EtOAc (50/
25 mL). The organic layer were washed with brine (25 mL), dried
over MgSO4, and concentrated. The crude product was puried
by column chromatography (EtOAc : cyclohexane 7 : 3) to yield
calcitroic acid methyl ester (44.4 mg, 97%) as a yellow oil: Rf 0.35
(EtOAc : cyclohexane 7 : 3). LiAlH4 (8.8 mg, 0.232 mmol) was
added to calcitroic acid methyl ester (44.4 mg, 0.114 mmol) in
6 G.-D. Zhu and W. H. Okamura, Chem. Rev., 1995, 95, 1877–
1952.
7 (a) E. G. Baggiolini, J. A. Iacobelli, B. M. Hennessy,
A. D. Batcho, J. F. Sereno and M. R. Uskokovi&cacute, J.
˜
Org. Chem., 1986, 51, 3098–3108; (b) A. Mourino,
´
´
M. Torneiro, C. Vitale, S. Fernandez, J. Perez-Sestelo,
´
S. Anne and C. Gregorio, Tetrahedron Lett., 1997, 38, 4713–
4716.
8 G. H. Posner, J. K. Lee, M. C. White, R. H. Hutchings, H. Dai,
J. L. Kachinski, P. Dolan and T. W. Kensler, J. Org. Chem.,
1997, 62, 3299–3314.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 32327–32334 | 32333

View Article Online
RSC Advances
Paper
˜
9 F. J. Sardina, A. Mourino and L. Castedo, J. Org. Chem., 1986, 14 (a) D. Phaff, G. Nemecek and J. Podlech, Helv. Chim. Acta,
51, 1264–1269.
2012, 95, 1851–1856; (b) M. Winkler, D. Meischler and
N. Klempier, Adv. Synth. Catal., 2007, 349, 1475–1480.
15 S. Hanessian, J. Pan, A. Carnell, H. Bouchard and L. Lesage,
J. Org. Chem., 1997, 62, 465–473.
10 Y. Kato, Y. Hashimoto and K. Nagasawa, Molecules, 2003, 4,
488–499.
11 L. A. Sarandeses, M. J. Valles, L. Castedo and A. Mourino,
Tetrahedron, 1993, 49, 731–738.
´
˜
¨
16 A. Presser and A. Hufner, Monatsh. Chem., 2004, 135, 1015–
12 (a) F. A. Davis, K. R. Prasad and P. J. Carroll, J. Org. Chem.,
1022.
2002, 67, 7802–7806; (b) S. Chow, M. T. Fletcher, 17 T. Fujishima, L. Zhaopeng, K. Konno, K. Nakagawa,
L. K. Lambert, O. P. Gallagher, C. J. Moore, B. W. Cribb,
P. G. Allsopp and W. Kitching, J. Org. Chem., 2005, 70,
1808–1827.
T. Okano, K. Yamaguchi and K. Takayama, Bioorg. Med.
Chem., 2001, 9, 525–535.
˜
18 I. Cornella, J. P. Sestelo, A. Mourino and L. A. Sarandeses, J.
13 S. M. Sparks, C. P. Chow, L. Zhu and K. J. Shea, J. Org. Chem.,
2004, 69, 3025–3035.
Org. Chem., 2002, 67, 4707–4714.
19 M. Torneiro, Y. Fall, L. Castedo and A. Mourino, J. Org.
˜
Chem., 1997, 62, 6344–6352.
32334 | RSC Adv., 2014, 4, 32327–32334
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