Synthesis of novel vitamin D3 analog with an additional ring annulated to A and seco-B rings

Article abstract of DOI:10.1016/j.steroids.2014.05.016

A simple method for the synthesis of yet unknown 5E-vitamin D₃ analogs with an additional six-membered ring connecting C-6 and C-19 was developed. Ring-closing metathesis (RCM) was used for efficient formation thereof from the corresponding 5E-isomers of 6-alkenyl vitamin D₃ compounds which in turn were prepared from the 6-oxo-3,5-cyclovitamin D ₃. Reinvestigation of the Grignard reactions of this latter compound as well as the following acid-catalyzed cycloreversions showed discrepancies with the literature data describing the course of such processes.

Full text of DOI:10.1016/j.steroids.2014.05.016

Accepted Manuscript
Synthesis of novel vitamin D3 analog with an additional ring annulated to A and
seco-B rings
Katarzyna Sokolowska, Rafal R. Sicinski
PII:
S0039-128X(14)00129-9
http://dx.doi.org/10.1016/j.steroids.2014.05.016
STE 7578
DOI:
Reference:
Steroids
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Revised Date:
Accepted Date:
12 February 2014
23 April 2014
25 May 2014
Please cite this article as: Sokolowska, K., Sicinski, R.R., Synthesis of novel vitamin D3 analog with an additional
ring annulated to A and seco-B rings, Steroids (2014), doi: http://dx.doi.org/10.1016/j.steroids.2014.05.016
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Synthesis of novel vitamin D3 analog with an additional ring annulated to A and seco-B
rings
Katarzyna Sokolowska and Rafal R. Sicinski*
Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
*
To whom correspondence should be addressed:
Prof. Rafal R. Sicinski
Department of Chemistry
University of Warsaw
Pasteura 1
02-093 Warsaw, Poland
Telephone: (+48) 22 822-0211 ext. 216
Fax: (+48) 22 822-5996
Email: rasici@chem.uw.edu.pl
1

Abstract
A simple method for the synthesis of yet unknown 5E-vitamin D3 analogs with an additional six-
membered ring connecting C-6 and C-19 was developed. Ring-closing metathesis (RCM) was
used for efficient formation thereof from the corresponding 5E-isomers of 6-alkenyl vitamin D3
compounds which in turn were prepared from the 6-oxo-3,5-cyclovitamin D3. Reinvestigation of
the Grignard reactions of this latter compound as well as the following acid-catalyzed
cycloreversions showed discrepancies with the literature data describing the course of such
processes.
Keywords: Vitamin D analogs; 5E-Vitamin D3, B-seco-Steroids; 3,5-Cyclovitamin D3; Ring-
closing metathesis
2

1. Introduction
Since a long time, there is an established view that vitamin D3 (1, Figure 1) undergoes two-
step activation in the living organisms [1] by two sequential enzymatic hydroxylations occurring in
the liver [1], and then in the kidney [2,3], resulting in the introduction of 25- and 1α-hydroxyl
groups, respectively. The final product of these transformations, 1α,25-dihydroxyvitamin D3 [2,
1α,25-(OH)2D3, calcitriol] is commonly considered as biologically active, hormonal form
responsible for all actions associated with calcium and phosphorus homeostasis [4]. In addition to
its well-known, classical role in mineral metabolism and bone growth, calcitriol exerts pleiotropic
actions in multiple organs and tissues [5-7]. Of particular interest are its antiproliferative functions,
which have stimulated numerous synthetic efforts to obtain the analogs characterized by selective
biological activities [8]. Taking into account the potential risk of hypercalcemia, the main goal of
these studies was to obtain low-calcemic compounds possessing elevated antiproliferative and
differentiation activity against various types of malignant cells [9]. Among thousands of calcitriol
analogs synthesized to the date and characterized by a wide array of structural changes [10],
modifications of the intercyclic 5,7-diene fragment were introduced only into several compounds
[11-15]. We have recently described the synthesis and biological evaluation of the series of 6-
substituted analogs of 1α,25-dihydroxyvitamin D3 and 1α,25-dihydroxy-19-norvitamin D3 [16,17].
Particularly noteworthy is one of them, 1α,25-dihydroxy-6-methylvitamin D3 (3), that binds the
vitamin D receptor (VDR) very effectively. Taking into consideration the tendency of this analog
to rearrange to its previtamin D form [16], we decided to direct our continued structure-activity
studies to the respective 5E-isomers of 6-substituted vitamin D3 compounds in which such a
thermal sigmatropic reaction could not take place.
3

The 5E-configuration “transforms” the natural 3β-hydroxyl group into a pseudo-1α-OH
group, that plays a crucial role in binding VDR. It has been established that an analog of the
natural hormone, (5E)-1α,25-dihydroxyvitamin D3 (4), in which the exocyclic methylene group is
transposed, compared to 2, from the right side to the left side of ring A, is still characterized by a
good affinity to VDR [18]. Thus, the presence of the C(10)=C(19) methylene group, playing a role
of a substituent at C-4, decreased the binding affinity of such modified analog ca. 8-fold; even a
smaller difference in VDR affinities between the 5Z- and “unnatural” 5E-isomers (only 2-fold) was
found in the case of 1α,25-dihydroxyvitamin D2 [19]. Interestingly, analog 4 was less calcemic
than 2 [18] but exerted similar inhibition of clonal proliferation of several tumor cells [20]. It was
also established that configurational 5Z→5E modification combined with some other structural
changes in the vitamin D molecule could result in analogs having unique activity profiles. (5E)-16-
Dehydro-calcitriol (5) may serve as an example, being at least 40-fold less calcemic than 2,
nevertheless exerting antiproliferative activities against cancer cell lines (HL-60, MCF-7 and
LNCaP) greater by two orders of magnitude [20]. Studies on the A-ring stereoisomers of 2-
methyl-1α,25-dihydroxyvitamin D3 proved that decreased VDR binding affinity of some 5E-
isomers, especially of those characterized by side chain alterations, does not exclude their
substantial cell differentiation-inducing potency [21].
Here, we would like to present the results of our work concerning the synthesis of the 6-
substituted 5E- and 5Z-vitamin D3 acetates 6-11, as well as of the analog 12, possessing an
additional ring connecting C-6 and C-19.
4

2. Experimental
2.1. General
All reactions involving oxygen- or moisture-sensitive compounds were carried out under
dry argon atmosphere. Reaction temperatures refer to external bath temperatures. Tetrahydrofuran
(THF) was distilled from Na/benzophenone, whereas diethyl ether (Et2O), dichloromethane
(CH2Cl2) and toluene were distilled from P2O5. Liquid reagents or solutions of reagents were
added via a syringe or a cannula. Reactions were monitored by thin-layer chromatography (TLC)
using aluminum-backed MERCK 60 silica gel plates (0.2 mm thickness); the chromatograms were
visualized first with ultraviolet light (254 nm) and then by immersion in a cerium-molybdenum
solution [10 g Ce(SO4)2 × 4 H2O, 25 g phosphomolybdic acid, 60 mL H2SO4 and 940 mL H2O),
followed by heating. Flash column chromatography was performed using MERCK Silica Gel 60
(230-400 mesh). High-performance liquid chromatography (HPLC) was performed on a Waters
Associates liquid chromatograph equipped with a Model 486 tunable absorbance detector or
Shimadzu UFLS liquid chromatograph equipped with SPD-20A tunable absorbance detector.
Ultraviolet spectra were obtained on a Perkin-Elmer Lambda 3B UV-VIS spectrophotometer in
100% ethanol (EtOH).
All nuclear magnetic resonance spectra were recorded using Varian Unity plus 200 MHz,
and Bruker DMX 500, using solutions in CDCl3 and Me4Si (δ 0.00) as an internal standard. To
1
describe the signals in H NMR spectra, the following abbreviations were used: s - singlet, d -
doublet, t - triplet, q - quartet, b - broad, narr. – narrow, w/2 = half-width. High resolution mass
spectra were recorded on LCT (TOF) or Mass Quattro LC spectrometers.
5

The starting 6-oxo-3,5-cyclovitamin D3 (13) was prepared according to the published
procedure [22].
2.1.1. (6S)- and (6R)-6-Allyl-3,5-cyclovitamin D3 (14 and 15)
Allylmagnesium bromide (1 M in THF; 0.28 mL, 0.28 mmol) was added to a solution of
the keto cyclovitamin 13 (44 mg, 0.115 mmol) in anhydrous THF (1 mL) and the mixture was
stirred at room temperature for 1 h under argon. The reaction was quenched by addition of water,
extracted with ethyl acetate, dried (MgSO4), and evaporated. The residue was purified by flash
chromatography on silica gel. Elution with hexane/Et2O (9:1) gave a mixture of epimeric 6-
hydroxy cyclovitamins 14 and 15 (ratio of 2:1; 47 mg, 97%) as a colorless oil.
1
14 and 15: H NMR (200 MHz, CDCl3) δ 0.537 and 0.585 (2H and 1H, each s, 18-H3), 0.647
(1H, t, J = 4.4 Hz, one of 4-H2), 0.868 (6H, d, J = 6.7 Hz, 26- and 27-H3), 0.913 (3H, d, J = 6.2
Hz, 21-H3), 3.19 and 3.28 (0.67H and 0.33H, each br d, J ~ 10 Hz), 4.9-5.2 (4H, m, 3’-H2 and 19-
H2), 5.16 and 5.42 (0.67H and 0.33H, each br s, 7-H), 5.84 (1H, ddt, J = 17.5, 12.0, 7.4 Hz, 2’-
H); HRMS (ESI) exact mass calculated for C30H48ONa (M + Na)+ 447.3603, measured 447.3609.
2.1.2. (6S)- and (6R)-6-(But-3’-enyl)-3,5-cyclovitamin D3 (16 and 17)
4-Bromo-1-butene (0.38 mL, 3.60 mmol) was dropwise added to a vigorously stirred
mixture of magnesium turnings (2.72 g, 3.60 mmol) in anhydrous THF (25 mL) under argon.
When magnesium was dissolved, the resulted solution of the Grignard reagent was transferred to a
solution of the keto cyclovitamin 13 (300 mg, 0.79 mmol) in anhydrous THF (10 mL) and the
mixture was stirred at room temperature for 2.5 h. The reaction was quenched by addition of
water and extracted with ethyl acetate, dried (MgSO4), and evaporated. The residue was purified
6

by flash chromatography on silica gel. Elution with hexane/Et2O (9:1) gave a mixture of isomers
16 and 17 (ratio of 5.5:1; 96%) as a colorless oil.
1
16 and 17: H NMR (200 MHz, CDCl3) δ 0.553 and 0.590 (2.54H and 0.46H, each s, 18-H3),
0.641 (1H, br t, J = 4.4 Hz, one of 4-H2), 0.851 and 0.883 (3H and 3H, each d, J = 6.6 Hz, 26-
and 27-H3), 0.918 (3H, d, J = 6.2 Hz, 21-H3), 3.14 and 3.31 (0.85H and 0.15H, each br d, J ~ 10
Hz), 4.86-5.06 (4H, m, 4’-H2 and 19-H2), 5.18 and 5.36 (0.85H and 0.15H, each br s, 7-H), 5.84
(1H, ddt, J = 16.8, 10.1, 6.6 Hz, 3’-H); HRMS (ESI) exact mass calculated for C31H50ONa (M +
Na)+ 461.3759, measured 461.3763.
2.1.3. (6S)- and (6R)-6-(Pent-4’-enyl)-3,5-cyclovitamin D3 (18 and 19)
Reaction of the keto cyclovitamin 13 with 4-pentenylmagnesium bromide was carried out
as described above for the preparation of 16 and 17. The crude products were purified by flash
chromatography on silica gel. Elution with hexane/Et2O (9:1) gave epimeric alcohols 18 and 19
(ratio of 6.1:1; 97%) as a colorless oil.
18 and 19: 1H NMR (200 MHz, CDCl3) δ 0.545 and 0.584 (2.58H and 0.42, each s, 18-H3), 0.625
(1H, br t, J = 4.4 Hz, one of 4-H2), 0.863 (6H, d, J = 6.6 Hz, 26- and 27-H3), 0.913 (3H, d, J =
6.0 Hz, 21-H3), 3.13 and 3.25 (0.86H and 0.14H, each br d, J ~ 10 Hz), 4.86-5.06 (4H, m, 5’-H2
and 19-H2), 5.16 and 5.35 (0.86H and 0.14H, each br s, 7-H), 5.80 (1H, ddt, J = 17.0, 10.2, 6.5
Hz, 4’-H); HRMS (ESI) exact mass calculated for C32H52ONa (M + Na)+ 475.3916, measured
475.3932.
2.1.4. (5E)- and (5Z)-6-Allyl-vitamin D3 acetate (6 and 7)
A solution of the cyclovitamins 14 and 15 (45 mg, 0.102 mmol) in glacial acetic acid (1.7
mL) was heated at 60 °C for 25 min, cooled to room temperature, quenched by slow addition of
7

ice-cold aqueous solution of NaHCO3 and extracted with Et2O. The organic layers were washed
with saturated NaHCO3 and brine, dried (MgSO4), and evaporated. The residue was purified by
flash chromatography on silica gel. Elution with hexane/Et2O (98:2) gave an oily mixture of
isomeric vitamins 6 and 7 (ratio of 1.5:1; 48 mg, 98%) which were separated by HPLC (9.4 mm ×
25 cm Zorbax-Sil column, 4 mL/min) using hexane/ethyl acetate (99:1). The vitamins 6 and 7 were
collected at Rv 35 mL and Rv 47 mL, respectively.
1
6: [α]24 +42o (c 0.38, CHCl3); H NMR (200 MHz, CDCl3) δ 0.581 (3H, s, 18-H3), 0.868 and
D
0.872 (3H and 3H, each d, J = 6.6 Hz, 26- and 27-H3), 0.920 (3H, d, J = 6.0 Hz, 21-H3), 2.01
(3H, s, OCOCH3), 2.59 (1H, dd, J = 13.4, 4.2 Hz, 4α-H), 2.98 (2H, m, 1’-H2), 4.81 (1H, d, J =
2.6 Hz, one of 19-H2), 4.88 (1H, m, 3α-H), 4.90-5.02 (3H, m, 3’-H2 and one of 19-H2), 5.28 (1H,
13
br s, 7-H), 5.74 (1H, ddt, J = 16.8, 10.2, 6.5 Hz, 2’-H); C NMR (50 MHz) δ 12.32 (C-18),
19.18 (C-21), 21.53 (OCOCH3), 22.83, 23.16 (C-26 and C-27), 24.24, 28.01, 28.35, 30.24,
32.97, 33.42, 33.62, 33.80, 36.50, 38.14, 39.82, 40.63, 44.95, 55.88, 56.93, 72.21, 111.02,
114.40, 120.22, 132.03, 133.96, 139.12, 142.47, 147.09, 170.97 (OCOCH3); HRMS (ESI) exact
mass calculated for C32H50O2Na (M + Na)+ 489.3709, measured 489.3703.
7: [α]24D +26o (c 0.29, CHCl3); 1H NMR (200 MHz, CDCl3) δ 0.534 (3H, s, 18-H3), 0.866 (6H, d,
J = 6.6 Hz, 26- and 27-H3), 0.907 (3H, d, J = 6.2 Hz, 21-H3), 2.03 (3H, s, OCOCH3), 2.59 (1H,
dd, J = 14.0, 4.0 Hz, 4α-H), 2.86 (2H, m, 1’-H2), 4.70 and 4.87 (1H and 1H, each br d, J = 2.4
Hz, 19-H2), 4.90-5.05 (3H, m, 3α- and 3’-H2), 5.42 (1H, br s, 7-H), 5.74 (1H, ddt, J = 16.8, 10.2,
6.5 Hz, 2’-H); 13C NMR (50 MHz) δ 12.34 (C-18), 19.04 (C-21), 21.60 (OCOCH3), 22.73, 22.98
and 23.05 (C-26 and C-27), 24.08, 27.89, 28.19, 30.28, 32.05, 32.43, 32.80, 33.10, 36.07, 36.31,
39.69, 40.70, 46.23, 55.75, 56.73, 72.28, 112.96, 114.57, 121.95, 131.71, 133.59, 139.45,
8

139.58, 146.72, 170.76 (OCOCH3); HRMS (ESI) exact mass calculated for C32H50O2Na (M +
Na)+ 489.3709, measured 489.3705.
2.1.5. (5E)- and (5Z)-6-(But-3’-enyl)-vitamin D3 acetate (8 and 9)
Solvolysis of the isomeric compounds 16 and 17 with glacial acetic acid was performed as
described above for the cyclovitamins 14 and 15. The crude products were purified by flash
chromatography on silica. Elution with hexane/Et2O (98:2) gave a mixture of vitamins 8 and 9 as a
colorless oil (ratio of 2.5:1; 99% yield). Isomeric products were separated by HPLC (9.4 mm × 25
cm Zorbax-Sil column, 4 mL/min) using hexane/ethyl acetate (99:1). The vitamins 8 and 9 were
collected at Rv 31 mL and Rv 38 mL, respectively.
1
8: [α]24 +39o (c 1.30, CHCl3); H NMR (200 MHz, CDCl3) δ 0.598 (3H, s, 18-H3), 0.867 and
D
0.870 (3H and 3H, each d, J = 6.6 Hz, 26- and 27-H3), 0.925 (3H, d, J = 6.0 Hz, 21-H3), 2.00
(3H, s, OCOCH3), 2.60 (1H, dd, J = 13.0, 3.6 Hz, 4α-H), 4.74 (1H, d, J = 2.4 Hz, one of 19-H2),
4.86 (1H, tt, J ~ 8 and 4 Hz, 3α-H), 4.91 (1H, ddd, J = 10.1, 1.9, 1.2 Hz, 4’-H(E)), 4.95 (1H, d, J
= 2.4 Hz, one of 19-H2), 4.97 (1H, ddt, J = 16.9, 1.9, 1.6 Hz, 4’-H(Z)), 5.28 (1H, br s, 7-H), 5.79
(1H, ddt, J = 16.9, 10.1, 6.6 Hz, 3’-H); 13C NMR (50 MHz) δ 12.29 (C-18), 19.06 (C-21), 21.57
(OCOCH3), 22.64, 22.78, 23.05 (C-26 and C-27), 24.12, 27.89, 28.22, 30.12, 32.85, 33.30,
33.49, 33.67, 36.38, 38.01, 39.71, 40.58, 45.52, 55.75, 56.79, 72.07, 111.04, 114.25, 120.13,
131.73, 133.80, 139.14, 142.39, 147.15, 170.64 (OCOCH3); HRMS (ESI) exact mass calculated
for C33H52O2Na (M + Na)+ 503.3865, measured 503.3866.
9: [α]24D +27o (c 0.58, CHCl3); 1H NMR (200 MHz, CDCl3) δ 0.540 (3H, s, 18-H3), 0.868 (6H, d,
J = 6.6 Hz, 26- and 27-H3), 0.911 (3H, d, J = 6.2 Hz, 21-H3), 2.05 (3H, s, OCOCH3), 2.66 (1H,
dd, J = 13.5, 3.5 Hz, 4α-H), 4.67 (1H, d, J = 2.4 Hz, one of 19-H2), 4.86 (1H, br d, J ~ 10 Hz, 4’-
9

H(E)), ca. 4.9 (1H, m, 3α-H; overlapped), 4.97 (1H, d, J = 2.4 Hz, one of 19-H2), 5.00 (1H, br dd,
J = 16.9, 1.8 Hz, 4’-H(Z)), 5.38 (1H, br s, 7-H), 5.82 (1H, ddt, J = 16.9, 10.3, 6.5 Hz, 3’-H); 13C
NMR (50 MHz) δ 12.40 (C-18), 19.07 (C-21), 21.62 (OCOCH3), 22.69, 22.79, 23.04 and 23.09
(C-26 and C-27), 24.12, 27.93, 28.23, 30.32, 32.10, 32.48, 32.85, 33.14, 36.11, 36.36, 39.73,
40.75, 46.28, 55.79, 56.77, 72.47, 112.95, 114.63, 121.89,131.75, 133.33, 138.91, 139.71,
146.70, 170.72 (OCOCH3); HRMS (ESI) exact mass calculated for C33H52O2Na (M + Na)+
503.3865, measured 503.3872.
2.1.6. (5E)- and (5Z)-6-(Pent-3’-enyl)-vitamin D3 acetate D3 (10 and 11)
Glacial acetic acid solvolysis of the isomeric compounds 18 and 19 was performed as
described above for the cyclovitamins 14 and 15. The crude products were purified by flash
chromatography on silica gel. Elution with hexane/Et2O (98:2) gave a mixture of vitamins 10 and
11 (ratio of 3:1; 99% yield) as a colorless oil. Isomeric products were separated by HPLC (9.4 mm
× 25 cm Zorbax-Sil column, 4 mL/min) using the hexane/ethyl acetate (99:1). The vitamins 10 and
11 were collected at Rv 31 mL and Rv 37 mL, respectively.
1
10: [α]24 +69o (c 1.06, CHCl3); H NMR (200 MHz, CDCl3) δ 0.590 (3H, s, 18-H3), 0.869 and
D
0.873 (3H and 3H, each d, J = 6.6 Hz, 26- and 27-H3), 0.922 (3H, d, J = 6.2 Hz, 21-H3), 2.00
(3H, s, OCOCH3), 2.59 (1H, dd, J = 12.8, 4.0 Hz, 4α-H), 4.72 (1H, d, J = 2.4 Hz, one of 19-H2),
4.86 (1H, tt, J ~ 8 and 4 Hz, 3α-H), 4.92 (1H, ddd, J = 10.1, 2.2. 1.2 Hz, 5’-H(E)), 4.94 (1H, one
of 19-H2, overlapped), 4.97 (1H, ddt, J = 17.2, 2.2, 1.4 Hz, 5’-H(Z)), 5.28 (1H, br s, 7-H), 5.79
(1H, ddt, J = 17.2, 10.1, 6.6 Hz, 4’-H); 13C NMR (50 MHz) δ 12.38 (C-18), 19.19 (C-21), 21.71
(OCOCH3), 22.90 and 23.17 (C-26 and C-27), 24.17, 24.84, 25.41, 28.02 (C-25), 30.04, 33.67,
36.52, 39.83, 40.59, 45.53, 55.00, 55.74, 72.11, 110.95, 114.43, 120.31, 131.35, 134.37, 139.23,
10

142.13, 147.27, 171.53 (OCOCH3); HRMS (ESI) exact mass calculated for C34H54O2Na (M +
Na)+ 517.4022, measured 517.4009.
1
11: [α]24 +51o (c 0.36, CHCl3); H NMR (200 MHz, CDCl3) δ 0.537 (3H, s, 18-H3), 0.866 and
D
0.868 (6H, each d, J = 6.6 Hz, 26- and 27-H3), 0.911 (3H, d, J = 6.2 Hz, 21-H3), 2.04 (3H, s,
OCOCH3), 2.62 (1H, dd, J = 13.0, 3.4 Hz, 4α-H), 4.67 and 4.85 (1H and 1H, each br d, J = 2.4
Hz, 19-H2), ca. 4.9 (1H, m, 3α-H, overlapped), 4.94 (1H, ddd, J = 10.1, 2.1, 1.2 Hz, 5’-H(E)),
4.99 (1H, ddt, J = 17.1, 2.1, 1.6 Hz, 5’-H(Z)), 5.37 (1H, s, 7-H), 5.80 (1H, ddt, J = 17.1, 10.1, 6.5
13
Hz, 4’-H); C NMR (50 MHz) δ 12.39 (C-18), 19.07 (C-21), 21.63 (OCOCH3), 22.71, 22.79,
23.05 and 23.10 (C-26 and C-27), 24.12, 27.85, 27.93, 28.23, 30.28, 31.99, 32.44, 32.98, 33.92,
36.00, 36.37, 39.73, 40.75, 46.18, 55.78, 56.77, 72.37, 112.85, 114.59, 122.08, 131.48, 133.93,
139.14, 139.48, 146.84, 170.77 (OCOCH3); HRMS (ESI) exact mass calculated for C34H54O2Na
(M + Na)+ 517.4022, measured 517.4012.
2.1.7. Isomerization of vitamin 11 to its geometrical E-isomer 10
A solution of vitamin 11 (10 mg, 0.019 mmol) in Et2O (10 mL) containing a catalytic
amount of iodine (0.2 mg, 0.002 mmol) was kept under diffuse daylight. The ratio (1:5.3) of the
equilibrium concentration of 11 and its E-isomer 10 was achieved after 1 h.
2.1.8. Thermal isomerization of vitamin 11 to its previtamin form 20
A solution of vitamin 11 (20 mg, 0.039 mmol) in hexane (5 mL) was heated at 40 °C for 6
h. The ratio of vitamin 11 to its previtamin form 20 was determined by HPLC as 1.5:1 after 30 min
and 1:2.2 after 3 h. The equilibrium was reached after 6 h and the ratio of 11:20 was established as
1:5.2, respectively. Isomeric products were separated by HPLC (9.4 mm × 25 cm Zorbax-Sil
11

column, 4 mL/min) using the hexane/ethyl acetate (99:1). The compounds 20 and 11 were
collected at Rv 34 mL and Rv 37 mL, respectively.
1
20: H NMR (500 MHz, CDCl3, 23 °C) δ 0.669 (3H, br s, 18-H3), 0.865 and 0.870 (3H and 3H,
each d, J = 6.5 Hz, 26- and 27-H3), 0.934 (3H, d, J = 6.5 Hz, 21-H3), 1.55 (3H, br s, 19-H3), 2.03
(3H, s, OCOCH3), 2.59 (1H, dd, J = 12.8, 4.0 Hz, 4α-H), 4.90 (0.5H, narr m, 3α-H), 4.92 (1H,
ddd, J = 10.5 Hz, 5’-H(E)), 5.00 (1H, dm, J = 17.3 Hz, 5’-H(Z)), 5.05 (0.5H, narr m, 3α-H), 5.44
and 5.53 (0.5H and 0.5H, each narr m, 9-H), 5.53 (1H, s, 7-H), 5.80 (1H, ddt, J = 17.3, 10.5, 6.5
Hz, 4’-H); 13C NMR (125 MHz) δ 171.51 and 171.48 (OCOCH3), 139.12, 135.68, 135.54,
127.38, 125.29, 125.05, 122.77, 122.17, 114.65, 102.55, 99.83, 91.24, 83.68, 77.48, 77.23,
76.98, 71.06, 70.09, 63.17, 58.25, 54.82, 53.08, 51.49, 51.07, 42.39, 39.74, 37.87, 37.74, 36.49,
36.42, 35.15, 34.27, 33.76, 29.94, 29.77, 28.59, 28.25, 27.50, 27.27, 25.25, 24.12, 23.90, 23.05
and 22.79(C-26 and C-27), 21.73 (OCOCH3), 20.45, 19.01 (C-21), 14.35 (C-19), 11.39 (C-18).
1
20: H NMR (500 MHz, CDCl3, - 23 °C) δ 0.649 and 0.659 (1.5H and 1.5H, each s, 18-H3),
0.857 and 0.861 (3H and 3H, each d, J = 6.5 Hz, 26- and 27-H3), 0.926 (3H, d, J = 6.5 Hz, 21-
H3), 1.54 and 1.56 (1.5H and 1.5H, each s, 19-H3), 2.07 (3H, s, OCOCH3), 2.28 (1H, br d, J =
17.5 Hz, 4α-H), 4.87 (0.5H, narr m, 3α-H), 4.97 (1H, dd, J = 10.0, 2.5 Hz, 5’-H(E)), 5.02 (1H,
dm, J = 17.0 Hz, 5’-H(Z)), 5.07 (0.5H, narr m, 3α-H), 5.44 (0.5H, narr m, 9-H), 5.52 (1H, s, 7-H),
5.54 (0.5H, narr m, 9-H), 5.82 (1H, ddt, J = 17.0, 10.0, 6.5 Hz, 4’-H);
1
20: H NMR (500 MHz, CDCl3, 55 °C) δ 0.678 (3H, s, 18-H3), 0.868 and 0.873 (3H and 3H,
each d, J = 6.5 Hz, 26- and 27-H3), 0.939 (3H, d, J = 6.5 Hz, 21-H3), 1.55 (1H, br s, 19-H3), 2.01
(3H, s, OCOCH3), 2.30 (1H, m, 4α-H), 4.94 (1H, dm, J = 10.0 Hz, 5’-H(E)), 4.99 (1H, dm, J =
17.0 Hz, 5’-H(Z)), 5.01–4.92 (1H, m, 3α-H overlapped with 5’-H(Z) and 5’-H(E)), 5.48 (1H, br s, 9-
H), 5.54 (1H, s, 7-H), 5.80 (1H, ddt, J = 17.0, 10.0, 6.5 Hz, 4’-H); HRMS (ESI) exact mass
calculated for C34H54O2Na (M + Na)+ 517.4022, measured 517.4016.
12

2.1.9. (5E)-1α-Hydroxy- and (5E)-1β-hydroxy-(6-pent-4’-enyl)-vitamin D3 acetate (21 and 22)
tert-Butyl hydroperoxide (5.5 M in decane; 0.123 mmol, 0.022 mL) was added to a
mixture of SeO2 (8.6 mg, 0.078 mmol) in anhydrous CH2Cl2 (1 mL) and stirred vigorously at
room temperature for 30 min. The mixture was cooled to 15 °C and a solution of 10 (10.2 mg,
0.021 mmol) in anhydrous CH2Cl2 (1 mL) was dropwise added. After 4 h of stirring at room
temperature, the reaction was quenched by slow addition of aqueous 10% solution of NaOH (10
mL) and extracted with Et2O. The organic layers were washed with 10% aqueous solution of
NaOH, then with brine, dried (MgSO4), and evaporated. The residue was purified by flash
chromatography on silica gel. Elution with hexane/ethyl acetate (8:2) gave the mixture of the
vitamins 21 and 22 (ratio of 2.1:1; 7.2 mg, 68%). The isomeric vitamins were purified and
separated by HPLC (9.4 mm × 25 cm Zorbax-Sil column, 4 mL/min) using hexane/ethyl acetate
(85:15). The 1β-hydroxy compound 22 and its 1α-hydroxy isomer 21 were collected at Rv 51 mL
and Rv 56 mL, respectively.
21: 1H NMR (200 MHz, CDCl3) δ 0.596 (3H, s, 18-H3), 0.870 and 0.874 (3H and 3H, each d, J =
6.6 Hz, 26- and 27-H3), 0.924 (3H, d, J = 6.0 Hz, 21-H3), 1.99 (3H, s, OCOCH3), 4.39 (1H, m,
1β-H), 4.90 (1H, narr m, one of 19-H2), 4.92 (1H, br dd, J = 10.3, 2.1 Hz, 5’-H(E)), 4.98 (1H, ddt,
J = 17.1, 2.1, 1.6 Hz, 5’-H(Z)), 5.13 (1H, m, 3α-H), 5.21 (1H, t, J = 1.6 Hz, one of 19-H2), 5.27
(1H, br s, 7-H), 5.79 (1H, ddt, J = 17.1, 10.3, 6.6 Hz, 4’-H); HRMS (ESI) exact mass calculated
for C34H54O3Na (M + Na)+ 533.3971, measured 533.3983.
22: 1H NMR (200 MHz, CDCl3) δ 0.593 (3H, s, 18-H3), 0.870 and 0.874 (3H and 3H, each d, J =
6.6 Hz, 26- and 27-H3), 0.925 (3H, d, J = 6.0 Hz, 21-H3), 2.02 (3H, s, OCOCH3), 2.62 (1H, dd, J
= 13.2, 4.0 Hz, 4α-H), 4.17 (1H, m, 1α-H), 4.90 (2H, narr m, 3α-H and one of 19-H2), 4.92 (1H,
13

br dd, J = 10.5, 2.0 Hz, 5’-H(E)), 4.97 (1H, ddt, J = 16.9, 2.0, 1.6 Hz, 5’-H(Z)), 5.23 (1H, t, J = 1.6
Hz, one of 19-H2), 5.29 (1H, br s, 7-H), 5.78 (1H, ddt, J = 16.9, 10.5, 6.6 Hz, 4’-H); HRMS
(ESI) exact mass calculated for C34H54O3Na (M + Na)+ 533.3971, measured 533.3984.
2.1.10. 6,19-Dimethylene-vitamin D3 acetate (23)
(a) The 2nd generation Grubbs catalyst (7.2 mg, 0.008 mmol) was added to a solution of
the vitamin 8 (40 mg, 0.008 mmol) in anhydrous toluene (5 mL) at room temperature. Then the
mixture was stirred at 70 °C for 2 h, cooled to room temperature and applied on a silica Sep-Pak
(2 g). The product was eluted with hexane/ethyl acetate (95:5) and it was further purified by
HPLC (9.4 mm × 25 cm Zorbax-Sil column, 4 mL/min) using hexane/ethyl acetate (99.5:0.5). The
tetracyclic vitamin 23 (31 mg, 83%) was collected at Rv 55 mL.
(b) Metathesis reaction of the vitamin 10 was performed in the presence of the 2nd
generation Grubbs catalyst and carried out as described above for the vitamin 8. The crude
product was purified on a silica Sep-Pak and then by HPLC (9.4 mm × 25 cm Zorbax-Sil column,
4 mL/min) using hexane/ethyl acetate (99.5:0.5) to give the vitamin 23 in 46% yield.
1
23: UV (in EtOH) λmax 282 nm; H NMR (500 MHz, CDCl3) δ 0.586 (3H, s, 18-H3), 0.870 and
0.874 (3H and 3H, each d, J = 6.7 Hz, 26- and 27-H3), 0.927 (3H, d, J = 6.5 Hz, 21-H3), 2.03
(3H, s, OCOCH3), 2.64 (1H, dd, J = 15.0, 4.5 Hz, 4α-H), 4.93 (1H, tt, J = 8.5, 4.4 Hz, 3α-H),
13
5.51 (1H, narr m, 19-H), 5.53 (1H, s, 7-H); C NMR (50 MHz) δ 12.19 (C-18), 19.10 (C-21),
21.67 (OCOCH3), 22.71, 22.79, 22.90, 23.05, 23.51, 24.15, 27.92, 28.25, 28.46, 29.63, 29.85,
30.96, 33.14, 36.40, 39.74, 40.68, 45.79, 56.12, 56.84, 71.52, 119.56, 120.47, 126.54, 131.74,
134.50, 141.73, 170.84 (OCOCH3); HRMS (ESI) exact mass calculated for C31H48O2Na (M +
Na)+ 475.3552, measured 475.3570.
14

2.1.11. 6,19-Dimethylene-vitamin D3 (12)
Lithium aluminum hydride (2.6 mg, 0.069 mmol) was added to a stirred solution of 23 (4.2
mg, 0.009 mmol) in anhydrous THF (1 mL) at -40°C under argon. The mixture was warmed up to
0°C during 1 h and stirred at this temperature for next 3 h. The excess of the reagent was
decomposed with saturated aqueous Na2SO4, and the mixture was extracted with ethyl acetate.
The organic layers were washed with brine, dried (MgSO4), and evaporated. The oily residue was
applied on a silica Sep-Pak (2 g). Elution with hexane/ethyl acetate (9:1) gave the compound 12
that was further purified by HPLC (9.4 mm × 25 cm Zorbax-Sil column, 4 mL/min) using
hexane/ethyl acetate (85:15). The deprotected vitamin 12 (3.6 mg, 98%) was collected at Rv 26
mL. The purity of the final compound was confirmed by reversed-phase HPLC (9.4 mm × 25 cm
Zorbax-C18 column, 4 mL/min) using methanol as a solvent: the tetracyclic vitamin D analog 12
was eluted as a single sharp peak at Rv 64 mL.
1
12: UV (in EtOH) λmax 285 nm (ε 19 900); H NMR (500 MHz, CDCl3) δ 0.601 (3H, s, 18-H3),
0.871 and 0.876 (3H and 3H, each d, J = 6.5 Hz, 26- and 27-H3), 0.930 (3H, d, J = 6.5 Hz, 21-
H3), 2.69 (1H, br dd, J ~ 15, 4 Hz, 4α-H), 3.87 (1H, tt, J = 8.5, 4.2 Hz, 3α-H), 5.52 (1H, narr m,
19-H), 5.59 (1H, s, 7-H); 13C NMR (50 MHz) δ 12.21 (C-18), 19.10 (C-21), 22.71, 22.82, 22.89,
23.07, 23.53, 24.18, 27.92, 28.22, 28.54, 29.63, 29.89, 31.00, 33.16, 36.42, 39.83, 40.59, 45.80,
56.11, 56.82, 71.41, 119.54, 120.41, 126.42, 131.43, 135.64, 145.71; HRMS (ESI) exact mass
calculated for C29H46ONa (M + Na)+ 433.3446, measured 433.3452.
3. Results and discussion
We chose 6-oxo-3,5-cyclovitamin D3 (13, Scheme 1) as a starting material, which was
obtained from vitamin D3 (1) using the procedure described by Mazur [22]. Treatment of this
15

compound with the respective Grignard reagent, derived from allyl, 3-butenyl or 4-pentenyl
bromide, resulted in the formation of 2:1, 5.5:1 and 6.1:1 mixtures of 6S- and 6R-hydroxy-3,5-
cyclovitamins: 14 and 15, 16 and 17, 18 and 19, respectively. The structures of the obtained
cyclovitamin D products were established on the basis of their spectral data and mechanistic
rationale.
Since an attack of nucleophiles at a carbonyl center should occur from the less hindered
side of a molecule, we decided to use molecular modeling studies to determine the stereochemistry
of the major product. The calculations of optimized geometry and steric energies of the ketone 13,
carried out using PCModel (v9.0, Serena Software) program, led to two low-energy conformers
(A and B) differing in steric energy by only 0.13 kcal/mol (Figure 2). Careful inspection of the
conformer A structure indicates that hydrogens at C-4 and C-9 hinder the access of nucleophiles
to the 6-carbonyl group from the si face of the molecule. In the conformation B, the presence of a
bulky exomethylene group at C-10 also shields the si face, while attack from re face, as in the
previous case, seems to be easier. Thus, results of molecular modeling of the ketone 13 suggest
that approach of RMgX from re face should be preferred, especially for the bulky Grignard
reagents, resulting in formation of 6S-hydroxy cyclovitamins.
In the next acetolysis step, the mixtures of epimeric alcohols 14 and 15, 16 and 17, 18 and
19 were used, and the corresponding pairs of 5E- and 5Z-vitamin D3 acetates 6 and 7, 8 and 9, 10
and 11 (Scheme 2) were obtained in the ratio of 1.5:1, 2.5:1 and 3:1, respectively. In order to
1
establish the double bond stereochemistry of the obtained compounds, the H NOE difference
experiments were performed for isomers 10 and 11. The irradiation of 7-H (5.37 ppm) in the 5Z-
analog 11 resulted in the expected enhancement (2.6%) of the signal (4.67 ppm) derived from the
vinylic 19-H(Z). In agreement with expectations, the analogous experiment performed for 5E-
16

isomeric compound 10 (irradiated 7-H signal at 5.28 ppm) did not change the intensity of the
signals of protons from 10-exomethylene group.
Earlier, Ray and collaborators reported that acidic solvolysis of the analogous epimeric
mixture of 6-hydroxy-3,5-cyclovitamin D3 compounds gave predominantly 5Z-vitamin D3
derivatives [13]. However, the results of our studies suggest that configurations at C-6 assigned by
these Authors for 3,5-cyclovitamins and, consequently, configurations of the triene products,
resulting from their cycloreversion processes, are incorrect.1
Literature data indicate that cycloreversion step may occur according to mixed SN1’/SN2’
mechanism [23]. In the latter case, the protonated hydroxyl group of 6-alkylated 6R-cyclovitamin
leaves simultaneously to nucleophilic attack at 3β-position (Scheme 3). A necessary condition for
SN2’ process, resulting in the stereoselective formation of 5Z-vitamin, is anti-relationship of C(3)-
C(5) with respect to C(6)-OH (energetically preferred in the studied compounds). On the contrary,
such a nucleophilic substitution of the 6S-cyclovitamin should provide exclusively the 5E-product.
However, due to the fact that in the lowest energy conformation, C(3)-C(5) and C(6)-OH bonds
of the 6S-epimer are in the syn-relationship (anti-orientation is less stable by ca. 2 kcal/mol), SN2’
mechanism can be less favored. Alternatively, if 6-hydroxyl group dissociates from the
cyclovitamin compound (SN1’ mechanism) during the solvolysis, the formed carbocation may react
with a solvent molecule in a nonstereospecific fashion, yielding 6R- or 6S-cyclovitamin or it may
produce the corresponding vitamin product. Taking into account the ratios of the 5,7,10(19)-
trienes, obtained by us from acetolysis of the respective mixtures of 6S- and 6R-cyclovitamins, and
assuming that 6R-cyclovitamins formed almost exclusively 5Z-trienes, it can be calculated that the
majority (ca. 7/8) of 6S-cyclovitamins were converted to the isomeric 5E-products. These
1
Although the paper [13] indicates (6S)-hydroxy-3,5-cyclovitamins D3 as the major products of reaction of 6-
ketone 13 with Grignard reagents, the structures of these compounds shown in the included figures have the
opposite 6R-configuration.
17

findings, being in agreement with the mechanism of cycloreversion of 6-substituted alcohols
proposed by DeLuca [23] and Mazur [24], prove the highly stereospecific nature of the
investigated process and support our configurational assignment of cyclovitamins. If 6R-
cyclovitamins were the prevailing products of Grignard reaction of the ketone 13, their acetolysis
would more likely result in a higher content of the 5Z-vitamins. However, in order to
unequivocally establish the configurations of the isomeric trienes formed by the cycloreversion
reaction, two of them (10 and 11) were chosen as the representative products and subjected to
additional experiments.
At first, isomerization of vitamin 11 to its geometrical isomer 10 (ratio of the equilibrium
concentrations of 1:5.3, respectively) was achieved (Scheme 4), induced by visible light and
catalytic amount of iodine. It is well known, that reversible iodine-catalyzed isomerization of
vitamin D compounds strongly favors E-isomers [25].
Also, thermal isomerization of vitamin 11 to its previtamin form 20 (ratio of the
equilibrium concentrations of 1:5.2, respectively) was accomplished by its heating in 40 °C
solution. It is evident that such reversible thermal sigmatropic [1,7]-hydrogen shift may occur only
between 5Z-vitamins and their previtamin D counterparts. Interestingly, the anomaly in the proton
NMR spectra of the triene 20 was detected indicating its conformational isomerism and existence
of two rotamers in approximately equal populations (Scheme 4). Thus, under normal temperature
conditions the signal of 3α-H (and also the olefinic 9-H) appeared as two broad peaks; signals of
o
methyl groups at C-10 and C-13 were significantly broadened. At higher temperature (55 C) the
o
separated peaks coalesced to the single signals whereas at low temperature (-23 C) the signal
splitting of the above mentioned methine signals was more intense, and each of the methyl signals
appeared as two singlets.
Finally, the correctness of our structural assignment was confirmed by comparison of the
18

reaction of 5E- (10) and 5Z-vitamin (11) with oxidizing system of selenium dioxide and tert-butyl
hydroperoxide [26]. In the case of the isomer 10, such oxidation led to the epimeric mixture of 1-
hydroxylated vitamins 21 and 22 (ratio of 2.1:1; Scheme 5), that was in a full agreement with the
reported reactivity of 5E-vitamin D compounds, efficiently undergoing allylic oxidation [27]. On
the contrary, the same reaction conditions applied to the analog 11 resulted in formation of a
complex mixture of products, that was consistent with the literature data indicating that, in the
case of the 5Z-vitamin D compounds, concomitant overoxidation and isomerization processes
drastically reduce the yields of the desired 1α-hydroxylated derivatives [23].
The strategy of forming a bridge connecting C-6 and C-19 was based on the ring closing
metathesis (RCM) of 5E-vitamin D3 analogs [28]. The reaction was carried out in toluene in the
presence of the 2nd generation Grubbs catalyst. As a result of cyclization of compound 6, we
expected to obtain the analog possessing an additional 5-membered ring. Since, despite many
introduced modifications of the reaction conditions, we were unable to obtain the pure product in
reasonable yield, we decided to synthesize the homologous compound having the less-strained 6-
membered ring. For this purpose, we used 5E-vitamin D3 acetate with 3’-butenyl substituent at C-
6 (8), that after the RCM reaction, followed by deprotection of hydroxyl group in the formed
tetracyclic product 23 (Scheme 6), provided the desired target vitamin D3 analog 12. In agreement
with the assigned structure, the 1H NMR spectrum of the latter compound showed the presence of
two olefinic protons at C-7 and C-19 (singlet at 5.59 ppm and a narrow multiplet at 5.52 ppm) and
a triplet of triplets at 3.87 ppm, originating from the 3α-H. Since the observed vicinal coupling
constant J3α,4β was 8.5 Hz, the prevailing A-ring conformer should have an equatorially oriented
hydroxyl; molecular modeling fully supported this conclusion. In the calculated lowest-energy
conformation of compound 12, its ring A, condensed with the cyclohexadiene unit, preferentially
19

assumes the half-chair conformation with an equatorial hydroxyl group, four neighboring atoms
(C-1, C-10, C-5 and C-4) lying in one plane, and two other (C-2 and C-3) located above and
below that plane, respectively.
We decided to extend our studies by using also the homologous compound 10 as a
substrate for the ring closure process. However, as a result of cyclization, instead of the expected
product possessing an additional 7-membered ring, we obtained the analog 23 again, albeit in
lower yield. Its formation is obviously the result of a double bond isomerization and subsequent
ring closing metathesis with the liberation of propene. Such alternative reaction course is known
[29,30] and it has been attributed to the presence of ruthenium hydride complexes in the reaction
matrix [31,32].
4. Conclusions
We have established a convenient route for the preparation of vitamin D3 analogs possessing an
additional 6-membered ring, by applying the ring-closing metathesis (RCM) as a method of its
construction. Additionally, in the search for the suitable precursors of 6-substituted 5E-vitamin D3
structure, we reexamined the method described in literature and involving the reaction of 6-oxo-
3,5-cyclovitamin D3 with Grignard reagents, followed by acid-catalyzed cycloreversion of the
formed tertiary 6-alcohols. Contrary to the literature data, the desired 5E-isomers were prevailing
products of this two-step reaction sequence. Our findings may be useful for the design of novel B-
seco steroids possessing the additional rings sharing the carbon atoms with A and B rings.
Acknowledgements
20

This research was supported by Statutory Research Grant BST from the Faculty of
Chemistry, University of Warsaw.
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24

Figure legends
Figure 1. Chemical structures of 25-dihydroxyvitamin D3 (1), 1α,25-dihydroxyvitamin D3
(calcitriol, 2) and their analogs.
Figure 2. Stereomodels of the ketone 13 in its low-energy conformations A and B (viewed
from the re face of carbonyl moiety).
25

OH
OH
OH
H
H
H
H
H
H
H
H
OH
OH
OH
HO
HO
HO
HO
1
2
3
4
OH
H
R
R
AcO
OH
OH
OAc
HO
5
6: R= -CH2CH=CH2
7: R= -CH2CH=CH2
12
8: R= -CH2CH2CH=CH2
9: R= -CH2CH2CH=CH2
10: R= -CH2CH2CH2CH=CH2
11: R= -CH2CH2CH2CH=CH2
Figure 1
26

Figure 2
27

O
HO
H
1
13
RMgBr, THF
96-97%
HO
HO
6S
R
R
6R
14: R = -CH2CH=CH2
15: R = -CH2CH=CH2
H
H
16: R = -CH2CH2CH=CH2
18: R = -CH2CH2CH2CH=CH2
17: R = -CH2CH2CH=CH2
19: R = -CH2CH2CH2CH=CH2
Scheme 1
HO
CH3COOH
98-99%
R
R
R
+
H
AcO
OAc
a) 14 and 15: R = -CH2CH=CH2
6: R = -CH2CH=CH2
7: R = -CH2CH=CH2
b) 16 and 17: R = -CH2CH2CH=CH2
c) 18 and 19: R = -CH2CH2CH2CH=CH2
8: R = -CH2CH2CH=CH2
9: R = -CH2CH2CH=CH2
11: R = -CH2CH2CH2CH=CH2
10: R = -CH2CH2CH2CH=CH2
Scheme 2
28

XOH (SN2)
R
XO
R
+
6R
SN1
R
5Z
XO
XO
R
XO
R
R
+
6S
SN1
∆
R
5E
OX
X = H, HCO, CH3CO, etc.
XOH (SN2)
Scheme 3
AcO
I2, Et2O, hν
rotation
AcO
AcO
OAc
10
11
20
Scheme 4
29