Synthesis and biological activity of previtamin D3 analogues with A-ring modifications

Article abstract of DOI:10.1016/j.bmc.2008.10.053

Synthesis of two novel 6-s-cis analogues of 1α,25-dihydroxyvitamin D₃ are described using shikimic acid and its 4-epi isomer as versatile chiral starting materials. These derivatives contain a 2β-(3′-hydroxypropoxy) moiety or a 2β,3β-epoxy grou

Full text of DOI:10.1016/j.bmc.2008.10.053

Bioorganic & Medicinal Chemistry 16 (2008) 10244–10250
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activity of previtamin D3 analogues
with A-ring modifications
Laura Sánchez-Abella a, Susana Fernández a, Annemieke Verstuyf b, Lieve Verlinden b, Vicente Gotor a,
,
*
Miguel Ferrero a,
*
a Departamento de Química Orgánica e Inorgánica and Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006 Oviedo (Asturias), Spain
b Laboratorium voor Experimentele Geneeskunde en Endocrinologie, Katholieke Universiteit Leuven, Gasthuisberg, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of two novel 6-s-cis analogues of 1a,25-dihydroxyvitamin D3 are described using shikimic acid
and its 4-epi isomer as versatile chiral starting materials. These derivatives contain a 2b-(30-hydroxyprop-
Received 15 July 2008
Revised 17 October 2008
Accepted 22 October 2008
Available online 26 October 2008
oxy) moiety or a 2b,3b-epoxy group into 1a,25-(OH)2-19-nor-pre-D3. The synthesized analogues were
found to be not suitable for binding to the vitamin D receptor and showed weak binding affinity toward
the vitamin D-binding protein. The new derivatives failed to inhibit cell proliferation.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Previtamin D3
Novel A-ring analogues
Calcitriol
1a,25-dihydroxyprevitamin D3 analogues
1. Introduction
The conformational flexibility of vitamin D3 and its metabolites
is unique among the steroid hormones.11 This seco steroids can
In the last decade, conformational, biological, physiological and
chemical studies have enhanced our understanding of vitamin D
mechanism of action.1 The discovered biological activities of
undergo a rotation around the 6,7 carbon-carbon single bond
25
1a,25-dihydroxyvitamin D3 [1a,25-(OH)2-D3 (1), Fig. 1], the hor-
R2
monally active form, allow for application of vitamin D3 (2) for
the treatment of diseases such as cancer, psoriasis, osteoporosis,
rheumatoid arthritis, and multiple sclerosis.2 The biological
OH
H
H
7
6
responses of 1a,25-(OH)2-D3 are mediated via binding to the nu-
clear vitamin D receptor (VDR),3 which belongs to the nuclear
receptor superfamily and acts as a ligand-dependent transcription
factor. In addition to these relatively slow (hours to days) genomic
HO
R1
HO
OH
3
1
effects,4 1
a,25-(OH)2-D3 generates a variety of non-genomic, rapid
O
OH
1, R1= R2= OH
1α,25-Dihydroxyvitamin D3
2, R1= R2= H
responses (seconds to minutes); some examples include the stim-
ulation of intestinal Ca2+ transport (transcaltachia),5 secretion of
insulin by pancreatic b-cells,6 opening of voltage–gated Ca2+ and
Clꢀ channels,7 and the rapid migration of endothelial cells.8 There
is controversy as to the nature of the receptor that initiates non-
genomic actions. In 1994 it was postulated a plasma membrane-
associated receptor (VDRmem).9 However, new evidence indicates
that these rapid actions are mediated by the classical VDR located
near or associated with the plasma membrane or its caveolae
components.10
3, ED-71
Vitamin D3
R=
OH
OH
R
R
HO
HO
O
H
O
H
HO
4
5
* Corresponding authors. Tel.: +34 98 510 3448/5013; fax: +34 98 510 3448.
E-mail addresses: vgs@uniovi.es (V. Gotor), MFerrero@uniovi.es (M. Ferrero).
Figure 1. Structure of 1a,25-dihydroxyvitamin D3 and its derivatives.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.053

L. Sánchez-Abella et al. / Bioorg. Med. Chem. 16 (2008) 10244–10250
10245
which generates a wide array of molecular shapes extending from
the 6-s-cis (steroid-like conformation) to the more stable extended
6-s-trans conformation. It is well documented that a 6-s-trans con-
formation is required for efficient gene transcription, while 6-s-cis
locked metabolites activate a variety of non-genomic responses.12
Scarce examples of 6-s-cis locked derivatives of vitamin D are de-
scribed in the literature. This include the synthesis of several pro-
vitamin D diastereoisomers,12c stable previtamin D derivatives
which are unable to undergo rearrangement to the respective vita-
min D form by virtue of the absence of the C-19 methyl group,13 or
the A-ring (5). Results of preliminary evaluation of their biological
properties are included.
2. Results and discussion
For the synthesis of both analogues 4 and 5, standard Sonogash-
ira coupling26 of A-ring precursors 6 and 7 with an enol triflate of
the CD-ring/side chain fragment (8)14,27 was employed (Fig. 2).
The A-ring precursor 6 was synthesized from methyl 4-epi-shi-
kimate (9), a versatile chiral building block with the correct hydro-
xy-stereochemistry. This compound was previously reported by us
from shikimic acid through an efficient approach.28 The prepara-
tion of 6 started with the selective protection of hydroxyl groups
at C-3 and C-5 position of 9 (Scheme 1). Treatment of the latter
with tert-butyldimethylsilyl chloride afforded compound 10.
Transformation of the ester into the aldehyde was best carried
out via a two-step sequence. Thus, reduction of 10 with DIBALH
gave the alcohol 11, which upon oxidation of the allylic alcohol
with MnO2 yielded the aldehyde 12.
a 9,19-methano-bridged analogue of 1a
,25-(OH)2-D3.14 Surpris-
ingly, the first previtamin D analogue, characterized by the pres-
ence of a trans-fused decalin CD-ring system, with genomic
activities equivalent to 1a
,25-(OH)2-D3 has been described.15 This
analogue interacted as efficiently as the natural hormone with the
VDR and uses the same contact points within the receptor as did
1a,25-(OH)2-D3.
There are multiple vitamin D analogues currently used as treat-
ment for a variety of diseases as well as several others in clinical
trials. Of note is the ED-71 (3, Fig. 1), an analogue developed by
Chugai Pharmaceuticals Co., which is currently under phase III clin-
ical studies for the treatment of osteoporosis and bone fracture
prevention.16 This derivative has a 2b-(30-hydroxypropoxy) group
The reaction of the aldehyde with lithium trimethylsilyldia-
zomethane generates the alkyne 13. Alkylation of the hydroxyl
group at C-2 position was carried out by treatment with 1-bro-
mo-3-[(tert-butyldimethylsilyl)oxy]propane to afford the key pre-
cursor 6 in high yield.
attached to the C-2 position of 1a,25-(OH)2-D3. As a consequence
The 2b-(30-hydroxypropoxy)-1
a,25-(OH)2-19-nor-pre-D3 ana-
logue (4) was successfully synthesized according to the reaction
sequence shown in Scheme 2. The vinyl triflate 8 was treated with
of their interesting biological profile, synthesis of ED-71 analogues
has received considerable attention. Thus, synthesis of 19-nor,17 3-
epi,18 2-hydroxyalkyl,19 2-fluoroalkyl,20 and 2-hydroxyalkoxy21
derivatives, or analogues of ED-71 having side chain modifica-
tions22 have been described.
To elucidate further the structure–activity relationships of the
natural hormone and its analogues we focused our attention on
examining the introduction of a 2b-(30-hydroxypropoxy) group at
the A-ring synthon
6 in the presence of bis(triphenylphos-
phine)palladium (II) acetate-copper (I) iodide catalyst and Et2NH
in DMF affording the dienyne 14, which after silyl ether deprotec-
tion with camphor sulfonic acid (CSA) in MeOH gave tetraol 15 in
70% yield. Careful catalytic hydrogenation of 15 in the presence of
Lindlar catalyst and quinoline provided the desired previtamin D
analogue 4.
Synthesis of analogue 5 begins with A-ring precursor 7, which
was prepared starting from shikimic acid (Scheme 3). Transforma-
tion of 16 into 20 was performed in a similar manner as above de-
scribed for 13. Commercial shikimic acid was esterified and
selectively protected to afford 17. Reduction of the ester to alde-
hyde 19 was followed by formation of the enyne 20 by reaction
C-2 in 1
analogue (4). This derivative possess the same configuration in
the A-ring that the previously synthesized analogue 1 ,2b,25-
a,25-(OH)2-19-nor-pre-D3, a 6-s-cis locked previtamin D3
a
(OH)3-19-nor-pre-D3, the most potent diastereomer in inhibiting
proliferation on MCF-7 cells of a series of 2-hydroxy substituted
1a
,25-(OH)2-pre-D3 derivatives.13a On the other hand, limited
examples are available for vitamin D analogues possessing an
epoxide in its skeleton. Introduction of an epoxy group in the side
chain created some interesting vitamin D analogues whose cell dif-
ferentiating activity exceeded their calcemic effects more than
100-fold.23 Furthermore, some derivatives with the epoxy group
at the triene system have been reported.24 In addition, 3b-(1,2-
epoxypropyl)ether-25-hidroxyvitamin D3 has been described as
an affinity labeling reagent of human DBP.25 Herein, we also syn-
thesized a previtamin D analogue containing an epoxy group in
CO2Me
CO2Me
CH2OH
a
b
HO
OH
ΣO
OΣ
ΣO
OΣ
OH
OH
OH
9
10
11
Methyl 4-epi-shikimate
Σ= TBDMS
c
TBDMSO
OTBDMS
OTBDMS
TBDMSO
OTBDMS
CHO
O
OMs
e
d
7
6
ΣO
OΣ
ΣO
OΣ
ΣO
OΣ
OH
OH
O
OΣ
OTMS
6
13
12
H
Scheme 1. Reagents and conditions: (a) TBDMSCl, Et3N, DMAP, DMF, 0 °C ? rt, 2 h
(65%); (b) DIBALH, Et2O, ꢀ78 °C, 2 h (81%); (c) MnO2, CH2Cl2, 18 h (88%); (d)
TMSCHN2, nBuLi, THF, ꢀ78 °C ? rt, 8 h (56%); (e) Br(CH2)3OTBDMS, NaH, DMF,
ꢀ10 °C, 24 h (80%).
TfO
8
Figure 2. A-ring and CD-ring/side chain precursors.

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L. Sánchez-Abella et al. / Bioorg. Med. Chem. 16 (2008) 10244–10250
+
7
8
OR2
c
a
H
a
+
6
8
4
OR2
c
OH
R1O
OR1
H
H
O
OR1
14, R1= TBDMS; R2= TMS
15, R1= R2= H
b
R1O
OR1
OH
O
OMs
Scheme 2. Reagents and conditions: (a) (PPh3)2Pd(OAc)2, CuI, Et2NH, DMF, 4 h; (b)
(ꢀ)-CSA, MeOH, overnight (70%, two steps); (c) H2, Lindlar catalyst, quinoline,
MeOH, 20 min (82% yield).
23
21, R1= TBDMS; R2= TMS
22, R1= R2= H
b
d
CO2H
CO2Me
CH2OH
a,b
c
5
HO
OH
ΣO
OΣ
ΣO
OΣ
Scheme 4. Reagents and conditions: (a) (PPh3)2Pd(OAc)2, CuI, Et2NH, DMF, 4 h; (b)
(ꢀ)-CSA, MeOH, overnight; (c) DBU, THF, 24 h (58%, three steps); (d) H2, Lindlar
catalyst, quinoline, MeOH, 20 min (85% yield).
OH
OH
OH
16
17
18
Shikimic acid
Σ= TBDMS
Table 1
d
Biological activity of 19-nor-pre-D3 analogues
Compound
VDR (%)
hDBP (%)
MCF-7 (%)
CHO
1
1
4
5
a
,25-(OH)2-D3
100
2
0
100
8
0.5
0.9
100
8
0
f
e
13a
a,2b,25-(JY)3-19-nor-pre-D3
0
0
ΣO
OΣ
ΣO
OΣ
ΣO
OΣ
OH
OH
OMs
Summary of the in vitro effects of A-ring modified 19-nor-pre-D3 analogues on
receptor binding (VDR), interaction with human vitamin D-binding protein (hDBP),
and inhibition of breast cancer MCF-7 cell proliferation. The in vitro effect is
7
20
19
expressed as percentage activity at EC50 in comparison with 1a,25-(OH)2-D3 (=100%
activity).
Scheme 3. Reagents and conditions: (a) HCl, MeOH, 60 °C, 6 h (quantitative); (b)
TBDMSCl, Et3N, DMAP, DMF, 0 °C ? rt, 4 h (75%); (c) DIBALH, Et2O, ꢀ78 °C, 3 h
(75%); (d) MnO2, CH2Cl2, 16 h (91%); (e) TMSCHN2, nBuLi, THF, ꢀ78 °C ? rt, 7 h
(56%); (f) MsCl, pyridine, 2 h (89%).
120
100
80
60
40
20
0
with TMSCHN2. The resulting product 20 was treated with mesyl
chloride to give mesylate 7.
Sonogashira coupling of 7 with the CD-ring/side chain fragment
8 followed by direct desilylation afforded the corresponding dien-
yne 22 (Scheme 4). The latter was converted to epoxide 23 by
treatment with DBU in THF at room temperature in 58% overall
yield for the three steps. Compound 23 was next subjected to
hydrogenation under standard conditions (Lindlar catalyst) to give
the 2b,3b-epoxy-1a,25-(OH)2-19-nor-pre-D3 analogue 5.
3. Biological evaluation
10-10
10-9
10-8
10-7
The synthesized 6-s-cis locked analogues 4 and 5 with the struc-
tural modification at the A-ring were examined for the binding
affinity to the vitamin D receptor (VDR) and vitamin D-binding
protein (DBP). In addition, the capacity to inhibit breast cancer
MCF-7 cell proliferation was evaluated. The results are summa-
rized in Table 1, the activities being shown as percentages of that
Concentration (M)
Figure 3. Affinity of 1
a
,25-(OH)2-D3 and 19-nor-pre-D3 analogues for pig vitamin D
receptor. 1 ,25-(OH)2-D3 (d); 1
a
a
,2b,25-(JY)3-19-nor-pre-D3 (}); 4 ( ); 5 (N).
r
13a
30-hydroxypropyl group into the 1
decreased drastically its binding affinity. Similarly, 2b,3b-epoxy-
,25-(OH)2-19-nor-pre-D3 analogue 5 did not bind to the VDR.
a,2b,25-(OH)3-19-nor-pre-D3
of 1
The 2b-(30-hydroxypropoxy)-1
logue 4 possessed no affinity for the VDR (Fig. 3). Introduction of a
a,25-(OH)2-D3.
a,25-(OH)2-19-nor-pre-D3 ana-
1a

L. Sánchez-Abella et al. / Bioorg. Med. Chem. 16 (2008) 10244–10250
10247
Both analogues displayed also markedly decreased affinity to
(400.13 MHz, CDCl3): d 0.69 (s, 3H, Me18), 0.95 (s, 3H, Me21,
3JHH
the human DBP compared with the binding affinity of the analogue
carrying a 2b-OH substituent. The epoxy derivative 5 probed to be
6.4 Hz), 1.04 (m, 1H), 1.21 (s, 6H, Me26 + Me27), 1.2–1.5 (m, 12H),
3
1.7–2.0 (5H, m), 2.1–2.3 (m, 4H), 2.66 (d, 1H, H4e, |2JHH| 17.6, JHH
3
8-fold and 100-fold less potent than 1
a
,2b,25-(OH)3-19-nor-pre-
,25-(OH)2-D3,15 respectively, to bind to DBP.
Since the 19-nor-pre-D3 analogues 4 and 5 have no affinity at all
5.9 Hz), 3.28 (dd, 1H, H2, JHH 9.8, 4.4 Hz), 3.73 (m, 1H), 3.8–3.9
13a
3
D3
and 1a
(m, 3H), 4.04 (ddd, 1H, H3, JHH 9.8, 9.8, 4.4 Hz), 4.46 (dd, 1H, H1,
3JHH 4.4, 4.4 Hz), 5.99 (s, 1H, H10) and 6.01 (s, 1H, H9) ppm; (ESI+,
m/z): 476 [(M)+, 45%], 499 [(M+Na)+, 20%].
for the VDR, these analogues were unable to inhibit MCF-7 breast
cancer cell proliferation (Fig. 4).
5.3. 2b,3b-Epoxy-1a,25-dihydroxy-3-deoxy-19-nor-previtamin
D3 (5)
4. Conclusions
A similar procedure as that described for 4 afforded 5 in 85%
yield. Flash chromatography (EtOAc) was performed using silica
We have described the synthesis and biological evaluation of 6-
s-cis locked vitamin D analogues with structural modifications at
the A-ring. These novel target compounds have been prepared in
order to investigate important structure–activity features. We have
demonstrated the versatility of shikimic acid and its 4-epi isomer
for the synthesis of vitamin D analogues. Data from biological as-
60 Å (32–63
PrepHT, 10 mL/min, hexane/IPA, 90:10). 1H NMR (600.13 MHz,
CDCl3): d 0.74 (s, 3H, Me18), 0.99 (s, 3H, Me21
3JHH 6.1 Hz), 1.10
lm) pH 7. Further purification by HPLC (Zorbax sil
,
(m, 1H), 1.24 (s, 6H, Me26 + Me27), 1.3–1.6 (m, 11H), 1.9–2.0 (m,
2H), 2.0–2.1 (m, 2H), 2.3–2.4 (m, 3H). 2.72 (d, 1H, H4, |2JHH| 19.7),
2.81 (d, 1H, H4, |2JHH| 19.6), 3.30 (s, 1H, H2), 3.38 (s, 1H, H3), 4.59
(s, 1H, H1), 5.44 (s, 1H, H9), 5.69 (s, 1H, H10) and 5.81 (s, 2H,
H6 + H7) ppm; 13C NMR (150.9 MHz, CDCl3): d 11.3 (C18), 18.7
(C21), 20.8 (CH2), 23.2 (CH2), 24.7 (CH2), 27.5 (C4), 28.3 (CH2),
29.2 (CH), 29.3 (CH), 36.1 (CH2), 36.2 (C26 and C27), 36.4 (CH2),
42.1 (C), 44.4 (CH2), 50.8 (C3), 51.1 (CH) 54.3 (C2), 63.9 (C1), 71.1
(C),, 119.6 (C3), 124.2 (C10), 125.6 (C9), 129.6 (C6), 131.9 (C7),
134.0 (C5) and 136.4 (C8) ppm; (ESI+, m/z): 423 [(M+Na)+, 100%].
says indicate that 2b-(30-hydroxypropoxy)-1
pre-D3 and 2b,3b-epoxy-1 ,25-(OH)2-19-nor-pre-D3 possessed no
a,25-(OH)2-19-nor-
a
affinity for the vitamin D receptor and bound very poorly to the
vitamin D-binding protein. Unfortunately these analogues showed
no antiproliferative activity.
5. Experimental
5.1. General
5.4. (3R,4R,5R)-3,5-Di[(tert-butyldimethylsilyl)oxy]-4-[((30-tert-
butyldimethylsilyl)oxy)propoxy]-1-ethynylcyclohex-1-ene (6)
Synthesis of 814,27 and 928 was previously reported. HPLC semi-
preparative was performed using a Zorbax Sil PrepHT column,
7
l
m, 250 ꢁ 21.2 mm. Column chromatography was performed
NaH (246 mg, 60% in mineral oil, 5.89 mmol) was added to a
solution of 13 (75 mg, 0.20 mmol) in anhydrous DMF at ꢀ10 °C.
After 10 min, it was added dropwise 1-bromo-3-[(tert-butyldi-
over silica 60 Å (230–400 mesh) or silica 60 Å (32–63 m) pH 7.
l
5.2. 1
previtamin D3 (4)
a
,25-Dihydroxy-2b-(30-hydroxypropoxy)-19-nor-
methylsilyl)oxy]propane (220 lg, 1 mmol). The mixture was stir-
red at this temperature during 24 h. The reaction was quenched
with water and allowed to reach room temperature. The aqueous
layer was extracted with Et2O. The combined organic fractions
were dried (Na2SO4) and concentrated, and the residue purified
A flask containing Lindlar catalyst (45 mg) was exposed to a po-
sitive pressure of hydrogen gas (balloon). A solution of 15 (17 mg,
0.036 mmol) in MeOH (1.8 mL) and quinoline (130 lL of 0.17 M in
hexane, 0.022 mmol) were added. The reaction was stirred vigor-
ously during 20 min. The mixture was filtered on Celite, concen-
trated, and the crude subjected to flash chromatography using
by flash chromatography using silica 60 Å (32–63
ent elution with hexane–2% Et2O/hexane) affording 6 as a colorless
oil in 80% yield. IR (NaCl): 3455, 3316, 2955, 2885 and
2858 cmꢀ1 1H NMR (400.13 MHz, CDCl3): d 0.06 (s, 6H, 2SiMe),
lm) pH 7 (gradi-
t
;
silica 60 Å (32–63 lm) pH 7 (gradient elution with 10–30% ace-
0.08 (s, 3H, SiMe), 0.09 (s, 6H, 2SiMe), 0.10 (s, 3H, SiMe), 0.88 (s,
tone/CH2Cl2). Further purification by HPLC (Zorbax sil PrepHT,
3
0
10 mL/min, hexane/IPA, 70:30) afford 4 in 82% yield. 1H NMR
9H, SiCMe3), 0.91 (s, 9H, SiCMe3), 1.78 (q, 2H, 2H2 , JHH 6.4 Hz),
3
2.21 (dd, 1H, H6e, |2JHH| 16.4, JHH 4.4 Hz), 2.39 (dd, 1H, H6a, |2JHH
|
3
3
0
16.4, JHH 8 Hz), 2.86 (s, 1H, H8), 3.63 (t, 2H, H3 , JHH 6.4 Hz),
0
3.69 (t, 2H, 2H1 ), 3.74 (br s, 1H, H4), 3.79 (br s, 1H, H3), 3.94
120
100
80
60
40
20
0
3
(ddd, 1H, H5, JHH 5.2, 5.2, 1.6 Hz) and 6.08 (br s, 1H, H2) ppm;
13C NMR (100.13 MHz, CDCl3): d ꢀ5.3 (SiMe), ꢀ4.8 (SiMe), ꢀ4.7
(SiMe), ꢀ4.5 (SiMe), ꢀ4.4 (SiMe), 18.1 (SiC), 18.2 (SiC), 18.3 (SiC),
0
0
25.8 (SiCMe3), 26.0 (SiCMe3), 31.0 (C2 ), 33.4 (C6), 59.8 (C3 ), 66.7
0
(C1 ), 68.5 (C5), 72.3 (C3), 76.1 (C8), 78.3 (C4), 84.2 (C7), 123.7 (C1)
and 132.0 (C2) ppm; (ESI+, m/z): 577 [(M+Na)+, 100%].
5.5. (3R,4S,5R)-3,5-Di[(tert-butyldimethylsilyl)oxy]-1-ethynyl-
4-(methanesulfonyloxy)cyclohex-1-ene (7)
To a stirred solution of compound 20 (65 mg, 0.17 mmol) in
anhydrous pyridine (1 mL), was added methanesulfonyl chloride
dropwise (21 lL, 0.26 mmol). Then, the solution was stirred for
2 h at room temperature. The reaction was quenched by adding
an aqueous saturated solution of NaHCO3 and extracting with
Et2O. The combined organic fractions were dried (Na2SO4) and con-
centrated, and the residue purified by flash chromatography using
silica 60 Å (32–63
yielding 7 as a colorless oil in 89%. IR (NaCl):
10-10 10-9
10-8
10-7
10-6
Concentration (M)
Figure 4. In vitro antiproliferative effects of 1
on breast cancer MCF-7 cells. 1 ,25-(OH)2-D3 (d); 1
(}); 4 ( ); 5 (N).
a,25-(OH)2-19-nor-pre-D3 analogues
lm) pH 7 (gradient eluent, 5–20% Et2O/hexane)
a
a,2b,25-(OH)3-19-nor-pre-D3
r
t
3277, 2950, 2929,

10248
L. Sánchez-Abella et al. / Bioorg. Med. Chem. 16 (2008) 10244–10250
2881 and 2851 cmꢀ1
SiMe), 0.13 (s, 3H, SiMe), 0.15 (s, 3H, SiMe), 0.15 (s, 3H, SiMe),
0.91 (s, 9H, SiCMe3), 0.92 (s, 9H, SiCMe3), 2.40 (m, 2H, 2H6), 2.92
;
1H NMR (300.13 MHz, CDCl3): d 0.12 (s, 3H,
was filtered through a short column of Celite and washed with
CH2Cl2. The filtrate was concentrated to afford 12 as a white solid
(88% yield), which was sufficiently pure for direct use in the next
step. This aldehyde is instable and should be kept in the refrigera-
3
(s, 1H, H8), 3.10 (s, 3H, MeS), 4.30 (ddd, 1H, H5, JHH 5.4, 5.4,
3
3
1.3 Hz), 4.47 (dd, 1H, H3, JHH 4.2, 4.2 Hz), 4.54 (dd, 1H, H3, JHH
1.5, 4.8 Hz) and 5.98 (s, 1H, H2) ppm; 13C NMR (100.13 MHz,
CDCl3): d ꢀ4.9 (SiMe), ꢀ4.8 (SiMe), ꢀ4.7 (SiMe), 18.0 (SiC), 18.1
(SiC), 25.7 (SiCMe3), 25.8 (SiCMe3), 35.4 (C6), 38.3 (MeS) 66.1 (C5),
68.2 (C3), 83.2 (C7), 83.5 (C4), 120.2 (C1) and 133.4 (C2) ppm;
(ESI+, m/z): 483 [(M+Na)+, 100%].
tor. Mp: 52–54 °C; IR(KBr):
t 3506, 2957, 2928, 2894, 2853 and
1665 cmꢀ1 1H NMR (400.13 MHz, CDCl3): d 0.09 (s, 3H, SiMe),
;
0.11 (s, 3H, SiMe), 0.16 (s, 6H, SiMe), 0.89 (s, 9H, SiCMe3), 0.93 (s,
3
9H, SiCMe3), 2.30 (dd, 1H, H6a, |2JHH| 18, JHH 6.4 Hz), 2.48 (dd,
3
1H, H6e, |2JHH| 18, JHH 4.8 Hz), 3.70 (br s, 1H, H4), 4.14 (ddd, 1H,
3
3
H5, JHH 4.8, 4.8, 2 Hz), 4.49 (dd, 1H, H3, JHH 3.6, 3.6 Hz), 6.51 (s,
1H, H2) and 9.52 (s, 1H, H7) ppm; 13C NMR (100.13 MHz, CDCl3):
d ꢀ4.9 (SiMe), ꢀ4.8 (SiMe), ꢀ4.7 (SiMe), ꢀ4.6 (SiMe), 18.1 (SiC),
18.0 (SiC), 25.7 (SiCMe3), 25.8 (SiCMe3), 27.6 (C6), 67.8 (C5), 70.0
(C3), 74.9 (C4), 138.7 (C1), 147.0 (C2) and 193.6 (C7) ppm; (ESI+,
m/z): 409 [(M+Na)+, 30%] and 795 [(2M+Na)+, 20%].
5.6. Methyl (3R,4R,5R)-3,5-di[(tert-butyldimethylsilyl)oxy]-4-
hydroxycyclohex-1-enecarboxylate (10)
Anhydrous Et3N (1.2 mL, 8.92 mmol), DMAP (155 mg,
1.27 mmol) and TBDMSCl (1.1 g, 7.65 mmol) were added to a solu-
tion of 9 (480 mg, 2.55 mmol) in anhydrous DMF (5 mL) at 0 °C.
The mixture was stirred at room temperature during 2 h. Next,
the reaction was quenched with water and the aqueous layer
was extracted with Et2O. The combined organic fractions were
dried (Na2SO4) and concentrated, and the residue purified by flash
chromatography using silica 60 Å (230–400 mesh) (gradient elu-
tion with 3–5% EtOAc/hexane) affording 10 as a white solid in
5.9. (3R,4R,5R)-3,5-Di[(tert-butyldimethylsilyl)oxy]-1-ethynyl-
4-hydroxycyclohex-1-ene (13)
nBuLi (0.38 mL of 1.6 M in hexane, 0.60 mmol) was added to a
solution of TMSCHN2 (0.29 mL of 2.0 M in hexane, 0.57 mmol) at
ꢀ78 °C. To this solution was added 12 (255 mg, 0.51 mmol) in
anhydrous THF (2 mL). The mixture was stirred and allowed to
reach room temperature during 8 h. The reaction was poured into
H2O/Et2O and the aqueous layer extracted with Et2O. The com-
bined organic fractions were dried (Na2SO4) and concentrated,
and the residue purified by flash chromatography using silica
65% yield. Mp: 70–72 °C; IR(NaCl):
2854 and 1711 cmꢀ1 1H NMR (400.13 MHz, CDCl3): d 0.09 (s, 3H,
3SiMe), 0.10 (s, 3H, SiMe), 0.13 (s, 3H, 3SiMe), 0.13 (s, 3H, SiMe),
t 3508, 2958, 2927, 2895,
;
0.90 (s, 9H, SiCMe3), 0.91 (s, 9H, SiCMe3), 2.36 (dd, 1H, H6a, |2JHH
|
3
3
17.8, JHH 6.7 Hz), 2.54 (dd, 1H, H6e, |2JHH| 17.8, JHH 4.9 Hz), 3.65
60 Å (32–63
hexane) to afford 13 as a colorless oil in 56% yield. IR(NaCl):
3466, 3316, 2955, 2930, 2895 and 2857 cmꢀ1 1H NMR
lm) pH 7 (gradient elution with hexane–20% Et2O/
3
(dd, 1H, H4, JHH 4.4, 2.2 Hz), 3.75 (s, 3H, H8), 4.11 (ddd, 1H, H5,
t
3
3JHH 7.0, 4.8, 2.2 Hz), 4.35 (dd, 1H, H3, JHH 3.7, 3.7 Hz) and 6.71
;
(br s, 1H, H2) ppm; 13C NMR (100.13 MHz, CDCl3): d ꢀ4.9 (SiMe),
ꢀ4.8 (SiMe), ꢀ4.7 (SiMe), ꢀ4.6 (SiMe), 18.1 (SiC), 25.8 (SiCMe3),
30.3 (C6), 51.9 (C8), 67.9 (C5), 69.7 (C3), 74.3 (C4), 128.3 (C1),
137.1 (C2) and 167.1 (C7) ppm; (ESI+, m/z): 417 [(M+H)+, 100%].
(400.13 MHz, CDCl3): d 0.10 (s, 3H, 2SiMe), 0.10 (s, 3H, SiMe),
0.11 (s, 3H, SiMe), 0.12 (s, 3H, SiMe), 0.90 (s, 9H, SiCMe3), 0.91 (s,
9H, SiCMe3), 2.32 (m, 2H, 2H6), 2.86 (s, 1H, H8), 3.64 (br s, 1H,
3
3
H4), 4.10 (ddd, 1H, H5, JHH 5.6, 5.6, 2 Hz), 4.28 (dd, 1H, H3, JHH
4, 4 Hz) and 5.99 (br s, 1H, H2) ppm; 13C NMR (100.13 MHz, CDCl3):
d ꢀ4.8 (SiMe), ꢀ4.7 (SiMe), ꢀ4.6 (SiMe), 18.1 (SiC), 25.8 (SiCMe3),
34.8 (C6), 67.5 (C5), 69.7 (C3), 74.2 (C4), 76.3 (C8), 83.8 (C7), 119.3
(C1) and 134.5 (C2) ppm; (ESI+, m/z): 405 [(M+Na)+, 100%].
5.7. (3R,4R,5R)-3,5-Di[(tert-butyldimethylsilyl)oxy]-4-hydroxy-
1-hydroxymethylcyclohex-1-ene (11)
DIBALH (2 mL of 1.0 M in toluene, 2 mmol) was added dropwise
to a solution of 10 (210 mg, 0.50 mmol) in anhydrous Et2O (3 mL)
at ꢀ78 °C, and the reaction was stirred for 2 h at the same temper-
ature. An aqueous solution of potassium and sodium tartrate
(1.0 M) was added and the mixture was warmed to room temper-
ature, diluted with Et2O, and filtered through a short column of sil-
ica gel, using additional Et2O to elute the column. The filtrate was
concentrated and the crude was purified by flash chromatography
with silica 60 Å (230–400 mesh) (gradient elution with 20–30%
EtOAc/hexane) to afford 11 as a white solid in 81% yield. Mp:
5.10. 1a
,25-dihydroxy-2b-(30-hydroxypropoxy)-6,7-dehydro-
19-nor-previtamin D3 (15)
CuI (1 mg, 0.005 mmol), (PPh3)2Pd(OAc)2 (1 mg, 0.001 mmol),
and anhydrous Et2NH (350
(25 mg, 0.045 mmol) and 8 (24 mg, 0.049 mmol) in anhydrous
DMF (350 L). The reaction was monitored by TLC (hexane). After
lL) were added to a solution of 6
l
4 h, the mixture was poured into H2O/Et2O and the aqueous layer
extracted with Et2O. The combined organic fractions were dried
(Na2SO4) and concentrated. (ꢀ)-CSA (63 mg, 0.27 mmol) was
added to a solution of this crude in MeOH (1.4 mL) at 0 °C and
the reaction was stirred overnight a room temperature. The mix-
ture was poured into NaHCO3 (aqueous)/EtOAc and the aqueous
layer extracted with EtOAc. The combined organic fractions were
dried (Na2SO4) and concentrated, and the residue was subjected
97–99 °C; IR(KBr):
t 3505, 2947, 2928, 2895, 2856 and
1666 cmꢀ1 1H NMR (400.13 MHz, CDCl3): d 0.10 (s, 3H, SiMe),
;
0.11 (s, 3H, SiMe), 0.11 (s, 3H, SiMe), 0.12 (s, 3H, SiMe), 0.91 (s,
18H, 2SiCMe3), 2.2 (m, 3H, 2H6+OH), 3.68 (br s, 1H, H4), 3.64 (br
3
s, 1H, H4), 4.04 (m, 2H, H7), 4.14 (ddd, 1H, H5, JHH 7.2, 7.2,
2.4 Hz), 4.26 (br s, 1H, H3) and 5.59 (s, 1H, H2) ppm; 13C NMR
(100.13 MHz, CDCl3): d ꢀ4.8 (SiMe), ꢀ4.7 (SiMe), ꢀ4.6 (SiMe),
ꢀ4.5 (SiMe), 18.1 (SiC), 18.2 (SiC), 25.8 (SiCMe3), 25.8(SiCMe3),
31.1 (C6), 66.2 (C7), 67.9 (C5), 69.8 (C3), 74.9 (C4), 121.6 (C2) and
137.8 (C1) ppm; (ESI+, m/z): 411 [(M+Na)+, 50%] and 799
[(2M+Na)+, 50%].
to flash chromatography using silica 60 Å (32–63 lm) pH 7 (gradi-
ent elution with 90% EtOAc/hexane–EtOAc) to afford 15 as a color-
less oil in 70% yield. 1H NMR (400.13 MHz, CDCl3): d 0.70 (s, 3H,
Me18), 0.96 (s, 3H, Me21,
3JHH 6.5 Hz, 1.04 (m, 1H), 1.23 (s, 6H,
Me26 + Me27), 1.2–1.5 (m, 9H), 1.7–2.1 (5H, m), 2.1–2.3 (m, 4H),
2.40 (d, 1H, |2JHH| 17.4 Hz), 2.52 (d, 1H, |2JHH| 17.4 Hz), 3.73 (m,
2H), 3.8–3.9 (m, 3H), 4.08 (br s, 1H), 4.16 (br s, 1H) and 5.99 (br
s, 2H, H9 + H10) ppm; 13C NMR (100.13 MHz, CDCl3): d 11.0 (C18),
18.7 (C21), 20.8 (CH2), 23.9 (CH2), 25.2 (CH2), 28.0 (CH2), 29.2 y
29.4 (C26+C27), 30.9 (CH), 32.0 (CH2), 35.9 (CH), 36.1 (CH2), 36.4
(CH), 41.8 (C), 44.4 (CH2), 49.9 (CH), 54.7 (CH), 61.6 (CH2), 68.0
(CH), 68.4 (CH2), 71.1 (C), 72.4 (CH), 87.6 (C), 89.6 (C), 120.6 (C),
5.8. (3R,4R,5R)-3,5-Di[(tert-butyldimethylsilyl)oxy]-4-
hydroxycyclohex-1-enecarbaldehyde (12)
MnO2 (224 mg, 2.60 mmol) was added to a solution of 11
(100 mg, 0.26 mmol) in anhydrous CH2Cl2 (2.5 mL). The reaction
mixture was stirred at room temperature for 18 h. The mixture

L. Sánchez-Abella et al. / Bioorg. Med. Chem. 16 (2008) 10244–10250
10249
122.3 (C), 129.2 (CH) y 134.3 (CH) ppm; (ESI+, m/z): 497 [(M+Na)+,
centrated. (ꢀ)-CSA (182 mg, 0.78 mmol) was added to a solution of
this crude in MeOH (1.4 mL) at 0 °C and the reaction was stirred
overnight a room temperature. The mixture was poured into
NaHCO3 (aqueous)/EtOAc and the aqueous layer extracted with
EtOAc. The combined organic fractions were dried (Na2SO4) and
concentrated. To a solution of this crude in anhydrous THF
100%], 971 [(2M+Na)+, 50%].
5.11. Methyl (3R,4S,5R)-3,5-di[(tert-butyldimethylsilyl)oxy]-4-
hydroxycyclohex-1-enecarboxylate (17)
A similar procedure as that described for 10 afforded 17. This
(1.3 mL) was added dropwise DBU (40 lL, 0.26 mmol). After stir-
ring the mixture at room temperature for 24 h, the solvent was
compound was previously described.28
concentrated in vacuo, and the residue purified by flash chroma-
5.12. (3R,4S,5R)-3,5-Di[(tert-butyldimethylsilyl)oxy]-4-
tography using silica 60 Å (32–63 lm) pH 7 (gradient elution with
hydroxy-1-hydroxymethylcyclohex-1-ene (18)
50% EtOAc/hexane–EtOAc) to afford 23 as a colorless oil in 58%
yield. 1H NMR (600.15 MHz, CDCl3): d 0.70 (s, 3H, Me18), 0.96 (s,
A similar procedure as that described for 11 afforded 18 as a
3H, Me21,
3JHH 6.4 Hz), 1.09 (m, 1H), 1.23 (s, 6H, Me26+Me27), 1.3–
white solid in 75% yield. Mp: 116–117 °C; IR(KBr):
t
3323, 2952,
1.8 (m, 13H), 1.9–2.1 (2H, m), 2.1–2.2 (m, 3H), 2.64 (d, 1H, H4,
|2JHH| 19.2 Hz), 2.72 (d, 1H, H4, |2JHH| 20.4 Hz), 3.27 (m, 1H, H2),
3.35 (s, 1H, H3), 4.57 (s, 1H, H1,), 5.92 (br s, 1H, H10) and 6.0 (br
s, 1H, H9) ppm; 13C NMR (150.9 MHz, CDCl3): d 11.0 (C18), 18.7
(C21), 20.8 (CH2), 23.9 (CH2), 25.2 (CH2), 27.9 (CH2), 29.2
(C26 + C27), 29.3 (C4), 29.4 (CH), 35.8 (CH2), 36.2 (CH), 36.3 (CH2),
41.8 (C), 44.4 (CH2), 49.9 (CH), 50.2 (C3), 52.6 (C2), 63.5 (C1), 71.1
(C), 87.8 (C6), 90.2 (C7), 119.6 (C5), 122.0 (C8), 128.4 (C10) and
134.8 (C9) ppm; (ESI+, m/z): 421 [(M+Na)+, 100%].
2927, 2892 and 2858 cmꢀ1 1H NMR (300.13 MHz, CDCl3): d 0.09
;
(s, 3H, SiMe), 0.10 (s, 3H, SiMe), 0.14 (s, 6H, 2SiMe), 0.89 (s, 9H,
SiCMe3), 0.93 (s, 9H, SiCMe3), 1.94 (dd, 1H, H6a, |2JHH| 17.7, JHH
3
3
4.5 Hz), 2.42 (ddd, 1H, H6e, |2JHH| 17.7, JHH 3.8, |4JHH| 2.1 Hz),
3
3.64 (br s, 1H, H4), 4.04 (br s, 2H, H7), 4.13 (ddd, 1H, H5, JHH
11.1, 4.8, 4.8 Hz), 4.43 (s, 1H, H3) and 5.53 (s, 1H, H2) ppm; 13C
NMR (75.5 MHz, CDCl3): d ꢀ4.8 (SiMe), ꢀ4.4 (SiMe), 18.2 (SiC),
18.0 (SiC), 25.9 (SiCMe3), 27.8(SiCMe3), 31.91 (C6), 66.2 (C7), 67.7
(C3), 68.6 (C5), 71.9 (C4), 121.6 (C2) and 137.4 (C1) ppm; (ESI+, m/
z): 411 [(M+Na)+, 30%] and 799 [(2M+Na)+, 40%].
6. In vitro biological evaluation
6.1. Cell proliferation assay
5.13. (3R,4S,5R)-3,5-Di[(tert-butyldimethylsilyl)oxy]-4-
hydroxycyclohex-1-enecarbaldehyde (19)
As a measure of cell proliferation, [3H]thymidine incorporation
of breast cancer MCF-7 (ATCC, Rockville, MD) was determined after
A similar procedure as that described for 12 afforded 19 as a
white solid in 91% yield. Mp: 72–73 °C; IR(KBr):
t 3947, 2952,
a 72 h incubation period with various concentrations of 1
a,25-
2925, 2883, 2849 and 1668 cmꢀ1 1H NMR (300.13 MHz, CDCl3):
;
(OH)2-D3, analogues or vehicle as described previously.29
d 0.08 (s, 3H, SiMe), 0.09 (s, 3H, SiMe), 0.18 (s, 3H, SiMe), 0.19 (s,
3H, SiMe), 0.86 (s, 9H, SiCMe3), 0.96 (s, 9H, SiCMe3), 2.24 (d, 1H,
3
H6a, |2JHH| 13.5 Hz), 2.51 (ddd, 1H, H6e, |2JHH| 13.5, JHH 0.9, |4JHH
|
6.2. Binding studies
3
0.9 Hz), 2.58 (s, 1H, OH), 3.80 (dd, 1H, H4, JHH 3, 3 Hz), 4.25
3
The affinity of 1
D receptor was evaluated by their ability to compete with
[3H]1
,25-(OH)2-D3 for binding to high speed supernatant from
a,25-(OH)2-D3 and its analogues to the vitamin
(ddd, 1H, H5, JHH 5.7, 3, 3 Hz), 4.68 (br s, 1H, H3), 6.43 (s, 1H, H2)
and 9.50 (s, 1H, H7) ppm; 13C NMR (75.5 MHz, CDCl3): d ꢀ4.8
(SiMe), ꢀ4.7 (SiMe), ꢀ5.0 (SiMe), 17.9 (SiC), 18.2 (SiC), 25.6
(SiCMe3), 25.8 (SiCMe3), 26.5 (C6), 67.8 (C3), 68.0 (C5), 71.2 (C4),
138.8 (C1), 147.4 (C2) and 194.0 (C7) ppm; (ESI+, m/z): 409
[(M+Na)+, 35%] and 795.0 [(2M+Na)+, 15%].
a
intestinal mucosa homogenates obtained from normal pigs as de-
scribed previously.29 The relative affinity of the analogues was cal-
culated from their concentration needed to displace 50% of
[3H]1
of 1
a
,25-(OH)2-D3 from its receptor compared with the activity
a
,25-(OH)2-D3 (assigned a 100% value).
5.14. (3R,4S,5R)-3,5-Di[(tert-butyldimethylsilyl)oxy]-1-ethynyl-
4-hydroxycyclohex-1-ene (20)
Binding of vitamin D analogues to the human vitamin D-bind-
ing protein (hDBP) was performed at 4 °C as described
previously.30
A similar procedure as that described for 13 afforded 20 as a col-
orless oil in 56% yield. IR(NaCl):
t 3452, 3316, 2954, 2937, 2888
and 2858 cmꢀ1 1H NMR (400.13 MHz, CDCl3): d 0.09 (s, 6H,
;
Acknowledgments
2SiMe), 0.12 (s, 3H, SiMe), 0.13 (s, 3H, SiMe), 0.89 (s, 9H, SiCMe3),
0.93 (s, 9H, SiCMe3), 2.06 (d, 1H, H6a, |2JHH| 18 Hz), 2.58 (d, 2H,
H6e + OH), 2.86 (s, 1H, H8), 3.64 (br s, 1H, H4), 4.11 (ddd, 1H, H5,
3JHH 5.6, 5.6, 5.6 Hz), 4.44 (s, 1H, H3) and 5.93 (s, 1H, H2) ppm;
13C NMR (100.61 MHz, CDCl3): d ꢀ4.9 (SiMe), ꢀ4.8 (SiMe), ꢀ4.6
(SiMe), 18.1 (SiC), 18.0 (SiC), 25.8 (SiCMe3), 25.7 (SiCMe3), 34.7
(C6), 67.36 (C3), 68.1 (C5), 70.9 (C4), 76.2 (C8), 84.07 (C7), 119.20
(C1) and 134.74 (C2) ppm; (ESI+, m/z): 405 [(M+Na)+, 100%].
Financial support of this work by the Spanish Ministerio de
Educación y Ciencia (MEC-07-CTQ-61126) is gratefully acknowl-
edged. L.S.-A. thanks MEC for a pre-doctoral fellowship. The Fonds
voor Wetenschappelijk Onderzoek (FWO Grant G.0508.05 and
G.0553.06) is gratefully acknowledged.
Supplementary data
5.15. 2b,3b-Epoxy-1a,25-dihydroxy-6,7-dehydro-3-deoxy-19-
nor-previtamin D3 (23)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.10.053.
CuI (2.5 mg, 0.013 mmol), (PPh3)2Pd(OAc)2 (3 mg, 0.004 mmol),
and anhydrous Et2NH (1 mL) were added to a solution of 7 (70 mg,
0.130 mmol) and 8 (70 mg, 0.143 mmol) in anhydrous DMF (1 mL).
The reaction was monitored by TLC (hexane). After 4 h, the mixture
was poured into H2O/Et2O and the aqueous layer extracted with
Et2O. The combined organic fractions were dried (Na2SO4) and con-
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