Enzymatic Desymmetrization of 19-nor-Vitamin D3 A-Ring Synthon Precursor: Synthesis, Structure Elucidation, and Biological Activity of 1α,25-Dihydroxy-3-epi-19-nor-vitamin D3 and 1β,25-Dihydroxy-19-nor-vitamin D3

Article abstract of DOI:10.1002/adsc.201800481

In a search for novel vitamin D derivatives of potential therapeutic value, structurally simple but synthetically challenging A-ring epimers of the 19-nor-Calcitriol [19-nor-1α,25-(OH)₂-D₃] at C1 and C3 were efficiently synthesized. Both analogues (1-epi- and 3-epi-19-nor-Calcitriol) were obtained through a convergent synthesis starting from cis,cis-1,3,5-cyclohexanetriol and the protected 25-hydroxy Grundmann′s ketone. After Julia-Kocienski coupling of the corresponding C,D-ring/side chain sulfone fragment with the A-ring ketone moiety, both vitamin D analogues were isolated. The critical point was how to determine the structural configuration of both diastereoisomers since similar ¹H NMR spectra were observed. For that, a biocatalytic approach was crucial in the synthesis of orthogonally protected derivatives. NMR spectroscopy allows the unambiguous identification of these compounds and as a result the structural elucidation of the desired vitamin D diastereomeric analogues. Affinity studies demonstrated that these 1,25-19-nor analogues have a very low affinity for the vitamin D receptor compared with 1α,25-dihydroxyvitamin D₃ or 1α,25-dihydroxy-19-nor-vitamin D₃. In addition, these analogues have a lower binding affinity for the human vitamin D binding protein than the natural hormone. In vitro cell culture studies revealed that synthesized analogues were less active than 1α,25-dihydroxyvitamin D₃ in inhibiting cell proliferation. (Figure presented.).

Full text of DOI:10.1002/adsc.201800481

Title: Enzymatic Desymmetrization of 19-nor-Vitamin D3 A-Ring
Synthon Precursor: Synthesis, Structure Elucidation, and
Biological Activity of 1α,25-Dihydroxy-3-epi-19-nor-vitamin D3
and 1β,25-Dihydroxy-19-nor-vitamin D3
Authors: Tania González-García, Annemieke Verstuyf, Lieve
Verlinden, Susana Fernández, and Miguel Ferrero
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
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published online in Early View as soon as possible and may be different
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.201800481
Link to VoR: http://dx.doi.org/10.1002/adsc.201800481

10.1002/adsc.201800481
Advanced Synthesis & Catalysis
UPDATE
DOI: 10.1002/adsc.2018((will be filled in by the editorial staff))
Enzymatic Desymmetrization of 19-nor-Vitamin D3 A-Ring
Synthon Precursor: Synthesis, Structure Elucidation, and
Biological Activity of 1,25-Dihydroxy-3-epi-19-nor-vitamin D3
and 1β,25-Dihydroxy-19-nor-vitamin D3
Tania González-García,a,b Annemieke Verstuyf,c Lieve Verlinden,c Susana
Fernández,a,* and Miguel Ferreroa,*
a
Departamento de Química Orgánica e Inorgánica, Universidad de Oviedo, 33006-Oviedo (Asturias), Spain
Fax: (+34)-985-103-446; phone: (+34)-985-102-984 or (+34)-985-105-013; e-mail: fernandezgsusana@uniovi.es or
mferrero@uniovi.es
Current address: FAES FARMA, 48940-Leioa (Vizcaya), Spain
b
c
Laboratorium voor Experimentele Geneeskunde en Endocrinologie, Katholieke Universiteit Leuven,Gasthuisberg, B-
3000 Leuven, Belgium
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201800481 ((staff))
hypercalcemia and hypercalciuria hobble its
Abstract. In a search for novel vitamin D derivatives of
potential therapeutic value, structurally simple but pharmaceutical interest.[1]
synthetically challenging A-ring epimers of the 19-nor-
Calcitriol [19-nor-1α,25-(OH)2-D3] at C1 and C3 were
efficiently synthesized. Both analogues (1-epi- and 3-epi-
25
19-nor-Calcitriol) were obtained through a convergent
synthesis starting from cis,cis-1,3,5-cyclohexanetriol and
25
R
14
OH
the protected 25-hydroxy Grundmann´s ketone. After Julia-
H
H
Kocienski coupling of the corresponding C,D-ring/side
chain sulfone fragment with the A-ring ketone moiety, both
vitamin D analogues were isolated. The critical point was
6
19
X
5
how to determine the structural configuration of both
HO
R
1
3
1
OH
HO
3
diastereoisomers since similar 1H NMR spectra were
observed. For that, a biocatalytic approach was crucial in
the synthesis of orthogonally protected derivatives. NMR
spectroscopy allows the unambiguous identification of
these compounds and as a result the structural elucidation
of the desired vitamin D diastereomeric analogues. Affinity
studies demonstrated that these 1,25-19-nor analogues have
a very low affinity for the vitamin D receptor compared
with 1α,25-dihydroxyvitamin D3 or 1α,25-dihydroxy-19-
nor-vitamin D3. In addition, these analogues have a lower
binding affinity for the human vitamin D binding protein
than the natural hormone. In vitro cell culture studies
revealed that synthesized analogues were less active than
1α,25-dihydroxyvitamin D3 in inhibiting cell proliferation.
4, TX522 (1a,3b)
1, X= CH2, R= H; Vitamin D3
5, 3-epi-TX522 (1a,3a)
6, 1-epi-TX522 (1b,3b)
2, X= CH2, R= OH; 1a,25-(OH)2-D3
3, X= H2, R= OH; 1a,25-(OH)2-19-nor-D3
Figure 1. Vitamin D and 19-nor-analogues.
For this reason, research related to vitamin D and
especially focused at Calcitriol, has provided
synthetic routes and biological activities for over
3,000 new analogues. Nowadays, the emphasis is on
obtaining molecules with enhanced molecular activity
as well as reduced calcemic effects.[2] Simple
modifications on the structure of the natural hormone
led to interesting analogues. There are several
examples where the chirality importance is reflected
because of stereoisomers of the same analogue
having remarkably different biological profiles. For
example, the stereochemistry of C1 and C3 has a
strong impact on binding to the vitamin D receptor
(VDR) and also to the vitamin D binding protein
(DBP). Inversion of the natural orientation of the 3-
hydroxyl and 1α-hydroxyl resulted in a 4- to 90-fold
reduction in VDR binding. However, inversion of the
Keywords: Enzymatic desymmetrization; Vitamin D; 19-
nor analogues; A-Ring modified analogues; Calcitriol
Introduction
Due to its huge biological importance, 1α,25-(OH)2-
D3 (2, Calcitriol, Figure 1), the hormonally active
form of vitamin D3 (1), was considered as a candidate
for therapeutic usage. However, side effects like
1
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10.1002/adsc.201800481
Advanced Synthesis & Catalysis
1α-hydroxyl to the 1β-orientation results in a 65.7-
fold increase in DBP binding with respect to 1α,25-
(OH)2-D3, illustrating that the optimal ligand for DBP
is 25-OH-D3. Intriguingly, 1β,25-(OH)2-D3 was found
to be a specific inhibitor of the nongenomic responses
of 1α,25-(OH)2-D3 such as transcaltachia and Ca2+
uptake, making it the first analogue that displays
antagonist properties at the receptor level.[3]
to the corresponding di-silyl protected derivative 10,
which was next treated with Dess-Martin periodinane
reagent to achieve the ketone 11.
OH
OH
O
a
b
HO
OH
SO
OS
SO
OS
It is noteworthy that the removal of the 19-
exomethylene function is beneficial in itself. Thus,
1α,25-dihydroxy-19-nor-vitamin D3 (3) shows minor
calcemic effects while retaining good cell-
differentiating properties.[4] In an attempt to carry out
the preparation of the corresponding C1 and C3
diastereoisomers of 1α,25-(OH)2-14-epi-19-nor-D3
analogues, Wu et al.[5] described the preparation of 1-
epi and 3-epi derivatives of TX522 (4−6, Figure 1)
through a convergent process whose key step
involves the coupling between an aldehyde of A-ring
derivative and an allyl bromide CD-ring fragment.
The main drawback of this route is the low selectivity
obtained in the solvolysis of the coupling reaction
product (cyclovitamin intermediate). It proceeds
through an intermediate with two rotamers in
equilibrium around the 5,6-bond. This disadvantage
along with the challenging identification of both
isomers has been a barrier to expand the methodology
to the synthesis of 1-epi or 3-epi derivatives of 1α,25-
(OH)2-19-nor-D3.
10
11
9
S= TBDMS
Scheme 1. Preparation of symmetric A-ring synthon 11.
Reaction conditions: (a) TBDMSCl, Et3N, THF, −20 ºC,
2 h and then 16 h at r.t. (43%); (b) Dess-Martin reagent,
CH2Cl2, r.t., 30 min (97%).
Convergent coupling reaction between 11 and the
lithium enolate of sulfone 12 (Scheme 2) took place
with 51% yield, giving an equimolecular
diastereomeric mixture, which was hardly separated
by HPLC. For this reason, the mixture was treated
with tetrabutylammonium fluoride (TBAF), which
resulted in silyl ether deprotection, affording the
isomers 13 and 14. These compounds were easily
separated by semipreparative HPLC in an equimolar
ratio (see Figure S1, Supporting Information).
R
With this goal in mind, here we report a practical
approach for the preparation of the synthetically
elusive epimers C3 and C1 of 1α,25-(OH)2-19-nor-D3
(7 and 8, respectively, Figure 2).
R
R
O
O
H
S
N
S
H
H
12
a,b
+
+
11
25
OH
25
OH
3
1
3
1
OH
HO
OH
HO
H
H
13
14
R=
c
c
O
OEt
19
19
7
8
3
1
3
1
OH
HO
OH
HO
Scheme 2. Synthesis of 1-epi and 3-epi 19-nor-vitamin
D3 analogues. Reaction conditions: (a) LHMDS, THF, −78
ºC to −50 ºC, 7 h (51%); (b) TBAF, THF, r.t., 2 h (85%);
(c) EtOH sat HCl, r.t., 30 min (88% for 7 and 91% for 8).
7, 1a,25-(OH)2-3-epi-19-nor-D3
8, 1b,25-(OH)2-19-nor-D3
Figure 2. C1 and C3 epimers of 1α,25-(OH)2-19-nor-D3.
Results and Discussion
Structure determination from conventional
techniques was challenging due to the structural
similarity of both isomers. Accordingly, an extensive
NMR study was performed using a high resolution
NMR spectrometer. Despite the fact of using a 600
MHz apparatus, we were not able to distinguish the
diastereomers 13 and 14 by NMR methods (see
Figure S2, Supporting Information).
Chemistry
The convergent construction of the vitamin D
skeleton is based on an approach previously reported
by Kittaka.[6] The synthesis of compounds 7 and 8
was envisaged using a Julia-Kociensky olefination to
construct the diene unit between the A-ring and the
CD-ring/side chain fragment, which was previously
reported starting from Grundmann´s ketone.[7] The
synthesis of A-ring synthon 11 was developed as
shows in Scheme 1. Treatment of cis,cis-1,3,5-
cyclohexanetriol (9) with Et3N, and tert-
butyldimethylsilyl chloride (TBDMSCl) at –20 ºC led
To
deduce
unequivocally
the
correct
stereochemistry of both analogues, our efforts were
focused on the design of a new route for the synthesis
of an A-ring synthon in which orthogonal protection
of hydroxyl groups would be the key to solve the
2
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10.1002/adsc.201800481
Advanced Synthesis & Catalysis
problem of the structural determination (Scheme 3).
For this purpose, we take into account the pioneering
work of Wirz and co-workers,[8] who carried out the
acetylation of corresponding mono-TBDMS-
protected derivative of 9 catalyzed by QLM lipase to
afford the asymmetrically monoacylated compound
in quantitative yield and >99% ee. In our case, to
facilitate the analysis of the enzymatic reaction by
HPLC, we used as silyl ether the TBDPS (UV
visible) instead the TBDMS group.
Enzymatic catalysis in non-aqueous media
significantly extends the conventional aqueous-based
biocatalysis.[11] The effect of three different organic
solvents [THF, tert-butylmethyl ether (TBME), and
toluene] on the process was evaluated. The reactions
were carried out at 30 ºC using 5 equiv. of vinyl
acetate. Candida antarctica lipase B (CAL-B),
showed very high enantioselectivity in all solvents
(entries 4−6, Table 1) but different regioselectivities:
total conversion was achieved with THF and TBME,
after 12 and 10 h, respectively, but the diacetylated
product 20 was also obtained. Using toluene as
solvent, the reaction was slower and after 12 h
starting material was recovered.
OH
OH
OAc
a
b
On the other hand, when the acylation process of
15 was examined with Pseudomonas cepacia lipase
as catalyst and THF as solvent, lower reaction rate
and enantioselectivity were observed (entry 7, Table
1). On the contrary, excellent results were obtained
using toluene and TBME (entries 8−9, Table 1). Thus,
99% conversion was achieved after 4 h of reaction
and enantiopure (−)-16 was obtained (see Figure S3,
Supporting Information).
S'O
OH
HO
OH
S'O
OH
9
15
16
OAc
S= TBDMS
S'= TBDPS
c
S'O
OAc
20
O
OH
OAc
e
d
S'O
OS
S'O
OS
S'O
OS
19
18
17
Scheme 3. Preparation of orthogonally protected A-ring
synthon 19. Reaction conditions: (a) TBDPSCl, Et3N, NaH,
THF, 45 ºC, 48 h (85%); (b) Lipase, vinyl acetate, solvent,
30 ºC; (c) TBDMSCl, Imidazole, CH2Cl2, r.t., 3 h (94%);
(d) MeONa, MeOH, r.t., 4 h (95%); (e) Dess-Martin
reagent, CH2Cl2, r.t., 1 h (97%).
Thus, triol 9 was mono-TBDPS-protected by
treatment with tert-butyldiphenylsilyl chloride
(TBDPSCl), Et3N, and NaH in THF at 40 ºC to afford
the diol 15 in 85% yield.[9] At this point, two different
easily available lipases were tested for the
asymmetric acetylation of meso-diol 15 in vinyl
acetate: Candida antarctica lipase type B (CAL-B,
Novozyme 435) and Pseudomonas cepacia lipase
(PSL-IM). The choice of these enzymes was based on
the excellent results that they showed in enzymatic
resolution of secondary alcohols via enantioselective
acylation or hydrolysis of their esters.[10]
Scheme 4. Synthesis of enantiomerically pure diacetate
22. Reaction conditions: (a) Ph3P, DIAD, AcOH, THF, r.t.,
4 h (72%); (b) TBAF, THF, r.t., 3 h (79%).
Both enzymes showed the same stereochemical
preference. For the assignment of the absolute
configurations of 16, a sequence of chemical
transformations were carried out to convert the
optically pure compound (−)-16 into the previously
described[8] (+)-1,3-diacetoxy-5-hydroxycyclohexane
22 (Scheme 4). Inversion of the free hydroxyl group
in 16 under Mitsunobu conditions furnished 21.
Subsequent removal of the silyl ether with TBAF
afforded the corresponding diacylated diol (+)-22.
The comparison of the specific rotation of this
compound to that described in the literature led us to
determine the (1R,3S,5S)-absolute configuration for
the product (−)-16 obtained from the enzymatic
acylation.[8]
The synthesis of the A-ring synthon was completed
with three additional steps (Scheme 3). The treatment
of 16 with TBDMSCl and imidazole led us to achieve
the compound 17, which bears orthogonal protection
for its three hydroxyl groups. Sequentially, the
removal of acetyl group in basic conditions afforded
the alcohol 18, which was treated with Dess-Martin
Table 1. Lipase catalyzed acylation of 15.a)
Enzymeb) Solvent
t
15
16
16 ee 20
(h) (%)b) (%)b) (%)b)
(%)b)
1
2
3
4
5
6
7
8
9
-
-
-
THF
12 >99
-
-
-
-
-
-
-
-
-
8
6
10
2
-
Toluene 12 >99
TBME
THF
Toluene 12
TBME
THF
12 >99
CAL-B
CAL-B
CAL-B
PSL-IM
PSL-IM
PSL-IM
12
-
92
85
90
24
>99
>99
>99
98
97
85
>99
>99
9
-
10
12 74
Toluene
TBME
4
4
-
-
-
a) Ratio 15:enzyme (1:0.5, p/p), with vinyl acetate at 30 ºC.
b) Determined by chiral HPLC.
3
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10.1002/adsc.201800481
Advanced Synthesis & Catalysis
periodinane reagent to give the final A-ring synthon
19. The absolute configuration was maintained in all
steps, as it was verified by quiral HPLC analysis of
compound 18 (see Figure S4, Supporting
Information).
possible due to their low polarity, which was later
modulated through hydroxyl group deprotection.
On the other hand, the hydroxyl groups must be
differentiated from each other for stereochemical
determination. Therefore, different conditions were
tested to carry out the selective deprotection of
TBDMS vs TBDPS group. The best result was
achieved using a Lewis acid, Bi(OTf)3, in an organic
solvent. The removal of the ethoxymethyl protecting
group in the side chain took place simultaneously,
R1
R1
H
H
a
12 + 19
+
providing
a 1.37:1 diastereomeric mixture of
analogues 25 and 26 that were successfully separated
by HPLC. At this stage, we called diastereomer
compounds A and B according to the rate of elution
in the HPLC chromatogram (see Figure S5,
Supporting Information). The identification of the
synthesized analogues was performed by NMR. The
S= TBDMS
S'= TBDPS
3
1
3
1
OS
SO
OS'
S'O
24
23
b
R2
17
R2
1
protons at C6 and C7 in the H NMR spectrum, and
14
H
also the signals corresponding to the carbons C14 and
C17 could be identified by monodimensional DEPT-
135, DEPT-90 and bidimensional 13C-1H HMBC
analysis (see pp S55-S60, Supporting Information).
At this point, the 1D NMR selective NOE
experiments were the key to undoubtedly complete
the structure elucidation for both isomers. In the case
of compound A, the selective irradiation of H7
allowed us to identify H10 (Figure 3). This signal
was also showed in the 1D NMR selective NOE
experiment on the multiplet at 4.08 ppm. In this
second experiment we could also observe NOE
signals with the ortho aromatic protons of the TBDPS
group. This proved that the signal at 4.08 ppm
belonged to the proton adjacent to OTBDPS and that
this silyl ether was in the position C1. Moreover, this
aromatic signal and H7 were close enough to exhibit
a NOE enhancement. These data led us to identify the
isomer A with the structure of 25. This assignment
could be verified analyzing the selective NOE
experiment on H3 and H6, where we were able to
observe the signals of H4.
7 (from 25)
or
H
c
7
+
6
8 (from 26)
3
1
3
1
OH
HO
OS'
S'O
25
26
R1=
R2=
OEt
O
OH
Scheme 5. Synthesis of 19-nor-vitamin D analogues.
Reaction conditions: (a) LHMDS, THF, −78 ºC to −50 ºC,
7 h (63%); (b) Bi(OTf)3, CH2Cl2, 0 ºC, 2 h (75%); (c)
EtOH sat HCl, r.t., 30 min (89%).
A-Ring modified 19-nor-vitamin D3 analogues
were synthesized through the Julia-modified coupling
reaction as illustrated in Scheme 5. Reaction of
ketone 19 with the lithium enolate of sulfone 12 gave
a mixture of diastereomers 23 and 24. The separation
of 23 and 24 by semipreparative HPLC was not
Figure 3. Selective 1D NOE experiments on compound A.
4
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10.1002/adsc.201800481
Advanced Synthesis & Catalysis
Figure 4. Selective 1D NOE experiments on compound B.
The same argument could be applied for the
structure elucidation of compound B (Figure 4). The
ortho aromatic protons exhibited NOE enhancement
with H6, which in turn transferred nuclear spin
polarization through transverse relaxation with H4.
This effect was also observed between H4 and the
proton in alpha position to OTBDPS group. These
data ratified the assignment of compound B with the
structure of 26. In this case, the anisotropy effect of
the aromatic ring made possible to observe the ortho
protons when H6 was irradiated.
leukemia cell proliferation by 1α,25-(OH)2-D3, 1α,25-
(OH)2-19-nor-D3, and analogues 7 and 8 was
measured.
After successfully complete structure elucidation
of the isomers 25 and 26, the TBDPS group was
removed with a saturated solution of HCl in EtOH,
giving the final compounds 7 and 8, respectively.
Biological Evaluation
The biological activity of the newly synthesized
analogues was tested in vitro, and the results are
summarized in Table 2 and Figures 5-8.
Table 2. Biological activities of 19-nor analogues of
1α,25-(OH)2-D3.a)
Figure 5. Affinity of 1α,25-(OH)2-D3 and 19-nor
analogues for pig vitamin D receptor (VDR). Notes: 1α,25-
(OH)2-D3 (●);1α,25-(OH)2-19-nor-D3 (); 7 (); 8 (☐).
Compound
VDR hDBP
MFC-7
(%)
HL 60
(%)
(%)
(%)
1α,25-(OH)2-D3
19-nor-1α,25-(OH)2-D3
7
8
100
100
1.2
100
10
14
3
100
116
18
100
170
10
The 1α,25-(OH)2-19-nor-D3 analogue, possesing
the same configuration of the natural hormone at C-1
and C-3 positions, has the same potency in terms of
its ability to bind to the pig VDR, but analogues 7
and 8 have markedly reduced affinity for the VDR
(Table 2 and Figure 5).
When comparing hDBP binding among the three
1,25-19-nor analogues, compound 7 exhibited the
highest affinity, which was still seven times lower
than that of 1α,25-(OH)2-D3 (Table 2 and Figure 6).
0.4
12
6
a)
Values are expressed as percentages of activity EC50
concentration relative to 1α,25-(OH)2-D3 (100%).
The affinity of compounds 7 and 8 for pig mucosa
cytosol VDR and for human vitamin D binding
protein (hDBP) was evaluated in comparison to the
natural hormone and its 19-nor derivative. In addition,
inhibition of MCF-7 breast cancer and HL60
5
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10.1002/adsc.201800481
Advanced Synthesis & Catalysis
Figure 8. In vitro antiproliferative effects of 1α,25-(OH)2-
D3 and 19-nor analogues on promyelocytic HL 60
leukemia cells. Notes: Vehicle (); 1α,25-(OH)2-D3
(●);1α,25-(OH)2-19-nor-D3 (); 7 (); 8 (☐).
Figure 6. Affinity of 1α,25-(OH)2-D3 and 19-nor
analogues for human vitamin D binding protein (hDBP).
Notes: 1α,25-(OH)2-D3 (●);1α,25-(OH)2-19-nor-D3 (); 7
(); 8 (☐).
These findings prove the key role of the
stereochemistry in the biological activity and this
would be reflected in different pharmacokinetic
profiles of theses analogues.
As a measure of the antiproliferative activity of
1,25-19-nor analogues, [3H]thymidine incorporation
of MCF-7 breast cancer cells and HL 60 leukemia
cells was measured after a 72 h incubation period
with various concentrations of 1α,25-(OH)2-D3,
analogues or ethanol. The most active compound is
the 1α,25-(OH)2-19-nor-D3, which is slightly more
active than the natural hormone. Analogues 7 and 8
showed 6-10 and 8-17 fold lower activities than
1α,25, respectively, on MCF-7 and HL 60 cells (Table
1 and Figures 7 and 8).
Conclusion
The synthesis of A-ring diastereomers of 1α,25-
(OH)2-19-nor-D3 has been successfully accomplished.
For the construction of the diene unit, a strategy
based on a Julia-Kocienski olefination between the
sulfone 12 and the cyclohexanone derivative 11 was
employed.
cis,cis-1,3,5-Cyclohexanetriol,
a
commercial available compound with the appropriate
stereochemistry, was used as starting material for the
A-ring precursor. To elucidate the structure of the 19-
nor analogues, an alternative synthesis was carried
out through orthogonally protected derivatives. It is
noteworthy the importance of the biocatalytic
desymmetrization process of 15 to give different
chemical nature to the A-ring hydroxyl groups. This
step was successfully performed with Pseudomonas
cepacia lipase as catalyst, vinyl acetate as acylating
reagent, and toluene or TBME as solvents. The
preparation of these 19-nor-analogues has not been
described previously, probably due to the complexity
in structure elucidation of the diastereoisomers. The
structure of both compounds was established by
selective NOE NMR spectroscopy, homonuclear
(COSY, TOCSY) and heteronuclear (HSQC, HMBC)
correlations. Biological assays on 1,25-19-nor
analogues 7 and 8 have shown poor binding to VDR,
whereas 1α,25-(OH)2-19-nor-D3 has the same affinity
as the natural hormone. All three 1,25-19-nor
analogues have a lower affinity for hDBP than 1α,25-
(OH)2-D3. The most active compound in the
inhibition of MCF-7 cell proliferation and HL 60 cell
Figure 7. In vitro antiproliferative effects of 1α,25-(OH)2-
D3 and 19-nor analogues on breast cancer MCF-7 cells.
Notes: Vehicle (); 1α,25-(OH)2-D3 (●);1α,25-(OH)2-19-
nor-D3 (); 7 (); 8 (☐).
6
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10.1002/adsc.201800481
Advanced Synthesis & Catalysis
124.2 (CH, C6), 130.1 and 142.9 (2CH, C5 + C8) ppm; MS
(ESI+, m/z): 405 [(M+H)+, 100%].
differentiation was 1α,25-(OH)2-19-nor-D3, even
slightly higher than 1α,25.
(1r,3R,5S)-3,5-bis(tert-Butyldimethylsilyloxy)cyclo-
hexanol (10). To a solution of 9 (1.0 g, 7.57 mmol) in
anhydrous THF (30 mL) at −20 ºC, were successively
added anhydrous Et3N (2.1 mL, 15.14 mmol) and tert-
butyldimethylsilyl chloride (2.3 g, 15.14 mmol). The
reaction was stirred at this temperature for 2 h and then
16 h at r.t. The solvent was evaporated and the crude was
purified by column chromatography (30% Et2O/hexane) to
give the compound 10 as a white solid in 43% yield; Rf:
0.4 (30% Et2O/hexane); mp 78-80 °C; IR (KBr): ν 3269,
2943, 2928, 1471, 1463, 1378 cm-1; 1H NMR (300.13 MHz,
CDCl3): δ 0.05 (s, 12H, SiMe), 0.87 (s, 18H, SiCMe3), 1.32
(m, 3H, H2ax + H4ax + H6ax), 1.72 (s, 2H, OH), 1.99 (m, 1H,
H6eq), 2.11 (m, 2H, H2eq+ H4eq), 3.57 (m, 3H, H1 + H3 + H5)
ppm; 13C NMR (75.5 MHz, CDCl3): δ −4.6 (SiMe), −4.5
(SiMe), 18.3 (SiC), 26.0 (CH3, SiCMe3), 44.8 (C2 + C6),
45.1 (C4), 66.2 (C1), 66.7 (C3 + C5) ppm; MS (APCI+, m/z):
361 [(M+H)+, 25%], 383 [(M+Na)+, 100%].
Experimental Section
General Considerations. All reagents were at highest
commercial quality and used without further purification.
All non-aqueous reactions were carried out under argon
atmosphere in anhydrous, freshly destilled, solvents.
Candida antarctica lipase type B (CAL-B, Novozyme 435,
immobilized by adsorption in Lewatit, 9120 PLU/g) was
kindly donated by Novozyme. Pseudomonas cepacia
lipase (Lipase PS “Amano IM”, immobilized on
diatomaceous earth, 943 U/g) was purchased from Amano
Enzyme. Reactions were monitored by TLC carried out
using UV light as visualizing agent and/or spraying a 5%
aqueous sulphuric acid solution containing cerium(IV)
sulphate (1%) and molybdophosphoric acid (2.5%). Flash
chromatography was performed using silica gel 60 (230–
1
400 mesh). H, 13C NMR, and DEPT were obtained using
1
300.13, 400.13 or 600.13 MHz apparatus for H, and 75.5,
(3R,5S)-3,5-bis(tert-Butyldimethyllsilyloxy)cyclo-
90.61 or 150.90 MHz for 13C. The same spectrometers
were used for the acquisition of 1H-1H homonuclear
hexanone (11). To a solution of 10 (295 mg, 0.82 mmol)
in anhydrous CH2Cl2 (8.2 mL), Dess-Martin reagent was
added (381 mg, 0.898 mmol). The mixture was stirred at r.t.
for 30 min. Then, it was diluted with Et2O (9.5 mL) and a
mixture of saturated aqueous Na2S2O3 and NaHCO3
solution (1:1, v/v, 9.5 mL), obtaining a white precipitate
that disappeared after 10 min. The mixture was extracted
with Et2O. The combined organic fractions were dried
(Na2SO4), filtered and concentrated. Purification of the
crude by column chromatography (5% Et2O/hexane) gave
11 as a white solid in 97% yield; Rf: 0.4 (10%
Et2O/hexane); mp: 62-64 °C; IR (KBr): ν 2953, 2928, 2856,
(COSY, TOCSY, and NOESY) and H-13C heteronuclear
1
(HSQC and HMBC) correlations. Optical rotations were
recorded on a polarimeter, and values are reported as
T
follows: [α]λ (c: g/100 mL, solvent). IR spectra were
recorded as thin films on NaCl plates or KBr pellets on an
Infrared FT spectrophotometer. Mass spectra (MS) and
high resolution mass spectra (HRMS) were recorded on a
mass spectrometer under electron spray ionization (ESI) or
atmospheric pressure chemical ionization (APCI)
conditions.
1
1712, 1471, 1463, 1389 cm-1; H NMR (300.13 MHz,
1,25-Dihydroxy-3-epi-19-nor-vitamin D3 (7). To a
solution of 13 (13.86 mg, 0.03 mmol) or 25 (19.29 mg,
0.03 mmol) in EtOH (622 L), a saturated solution of HCl
in EtOH (7.6 mL) was added. The reaction was stirred for
30 min. Then, the solvent was removed, the mixture was
extracted with a saturated aqueous solution of NaHCO3
and Et2O, and the organic extract was washed with brine
and dried (Na2SO4). The residue was purified by column
chromatography using 80% EtOAc/hexane as eluent. Rf:
0.2 (90% EtOAc/hexane); 1H NMR (600.13 MHz, CDCl3):
δ 0.54 (s, 3H, Me18), 0.84 (m, 1H), 0.93 (d, 3H, Me21, J 6.4
Hz), 1.21 (s, 6H, Me26 + Me27), 1.25–2.10 (several m),
2.25 (dd, 1H, H4ax, J 13.2, 7.1 Hz), 2.40 (d, 1H, H10ax, J
13.1, 7.1 Hz), 2.48 (dd, 1H, H4eq, J 13.7, 3.6 Hz), 2.59 (dd,
1H, H10eq, J 13.5, 3.5 Hz), 2.81 (dd, 1H, H9eq, J 12.4, 4.0
Hz), 3.90 (m, 1H, H1 + H3), 5.85 (d, 1H, H7, J 11.2 Hz),
6.31 (d, 1H, H6, J 11.3 Hz) ppm; 13C NMR (150.5 MHz,
CDCl3): δ 12.2 (Me18), 19.0 (Me21), 21.0 (CH2), 22.4 (CH2),
CDCl3): δ −0.04 (s, 6H, SiMe), 0.04 (s, 6H, SiMe), 0.86 (s,
18H, SiCMe3), 1.76 (m, 1H, H4ax), 2.25 (m, 1H, H4eq), 2.23
(dd, 2H, H2ax + H6ax, J 13.7, 11.2 Hz), 2.53 (dd, 2H, J 13.7,
3.9 Hz, H2eq + H6eq), 3.78 (m, 2H, H3 + H5) ppm; 13C NMR
(75.5 MHz, CDCl3): δ −4.6 (SiMe), 18.1 (SiC), 25.9 (CH3,
SiCMe3), 45.2 (C4), 51.0 (C2 + C6), 66.0 (C3 + C5), 207
(C=O) ppm; MS (ESI+, m/z): 359 [(M+H)+, 5%], 383
[(M+Na)+, 100%].
Procedure for the synthesis of 13 and 14. To a solution
of 12 (167 mg, 0.30 mmol) in anhydrous THF (1.1 mL) at
−78 ºC was added dropwise LHMDS (305 μL, 1.0 M in
THF, 0.30 mmol), resulting in a deep red colour solution.
The mixture was stirred at the same temperature for 2 h,
and then, a solution of the corresponding ketone 11 (63 mg,
0.19 mmol) in anhydrous THF (1.2 mL) was added
dropwise to the mixture via cannula transfer. After being
stirred at −50 ºC during 5 h, the reaction mixture was
poured into a saturated aqueous solution of NH4Cl and
extracted with Et2O. The combined organic fractions were
dried (Na2SO4), filtered, and concentrated to give a crude
that was purified by column chromatography (4%
Et2O/hexane) to give an equimolecular diastereomeric
mixture in 51% yield. Then, TBAF (725 L, 1 M in THF,
0.70 mmol) was added dropwise to the epimeric mixture
(100 mg, 0.10 mmol) in anhydrous THF (1.5 mL) at 0 ºC
in darkness. After 5 min, the mixture was stirred at r.t. for
2 h. Then, solvent was evaporated and the mixture was
extracted with EtOAc. The combined organic fractions
were dried (Na2SO4), concentrated, and the residue
purified by column chromatography (60% Et2O/hexane).
The diastereoisomers were isolated by preparative HPLC
(SunFire® C18 Column, 100 Å, 5 m, 10 mm x 250 mm).
Conditions: 4.0 mL/min, 10% iPrOH/hexane.
23.6 (CH2), 27.8 (CH2), 29.0 (CH2), 29.3 and 29.5 (Me26
+
Me27), 36.2 (CH, C20), 36.5 (CH2), 37.0 (CH2), 40.6 (CH,
C2), 41.4 (CH2), 44.6 (CH2), 45.3 (CH2), 45.9 (C, C13),
56.5 and 56.7 (2CH, C14 and C17), 68.7 and 69.0 (2CH, C1
+ C3), 71.3 (C, C25), 115.6 (CH, C7), 124.2 (CH, C6), 130.1,
and 142.9 (2CH, C5 + C8) ppm; MS (ESI+, m/z): 405
[(M+H)+, 100%].
1β,25-Dihydroxy-19-nor-vitamin D3 (8). For the
synthesis of this derivative, we employed the same
procedure as 7 but starting from 14 or 26. Rf: 0.2 (90%
EtOAc/hexane); 1H NMR (600.13 MHz, CDCl3): δ 0.55 (s,
3H, Me18), 0.88 (m, 1H), 0.93 (d, 3H, Me21, J 6.5 Hz), 1.21
(s, 6H, Me26 + Me27), 1.25–2.07 (several m), 2.30 (dd, 1H,
H4ax, J 13.4, 6.0 Hz), 2.47 (apparent d, 2H, H4eq+ H10eq, J
13.6 Hz), 2.56 (dd, 1H, H10ax, J 13.7, 6.1 Hz), 2.81(dd, 1H,
H9eq, J 11.7, 4.1Hz), 4.00 (m, 2H, H1 + H3), 5.86 (d, 1H, H7,
J 11.2 Hz), 6.34 (d, 1H, H6, J 11.2 Hz) ppm; 13C NMR
(150.5 MHz, CDCl3): δ 12.2 (Me18), 19.0 (Me21), 21.0
(CH2), 22.4 (CH2), 23.6 (CH2), 27.8 (CH2), 29.1 (CH2),
29.4 and 29.5 (Me26 + Me27), 36.3 (CH, C20), 36.5 (CH2),
36.8 (CH2), 40.4 (CH, C2), 41.0 (CH2), 44.6 (CH2), 44.8
(CH2), 45.9 (C, C13), 56.4 and 56.6 (2CH, C14 + C17), 68.8
and 69.0 (2CH, C1 + C3), 71.3 (C, C25), 115.5 (CH, C7),
25-Ethoxymethyloxy-1-hydroxy-3-epi-19-nor-vitamin
D3 (13). Rf: 0.2 (60% EtOAc/hexane). IR (NaCl): ν 3367,
1
2943, 2872, 1463, 1380 cm-1; H NMR (600.13 MHz,
CDCl3): δ 0.54 (s, 3H, Me18), 0.92 (d, 3H, Me21, J 6.5 Hz),
1.02 (m, 1H, H22), 1.20 (t, 3H, H3´, J 4.0 Hz), 1.21 (s, 6H,
Me26 + Me27), 1.28 (m, 4H, H17 + H12 + H16), 1.39 (m, 5H,
H20 + 1H22 + 1H24 + H23), 1.51 (m, 6H, 2H15 + 1H24 + 3H),
1.66 (m, 3H, 1H9 + 2H11), 1.78 (m, 1H, H2ax), 1.88 (m, 1H),
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.201800481
Advanced Synthesis & Catalysis
1.99 (dd, 3H, H12, J 11.4, 8.1 Hz), 2.06 (dt, 1H, H2eq, J 12.9, EtOAc/hexane as gradient eluent) to give 16 in 99% yield.
3.3 Hz), 2.24 (dd, 2H, H4ax + OH, 13.2, 7.1 Hz), 2.39 (dd, Racemic derivative (for the HPLC analyses): To a solution
1H, H10ax, 13.5, 7.2 Hz), 2.48 (dd, 1H, H4ax, J 14.0, 3.2 Hz), of 15 (100 mg, 0.27 mmol) in anhydrous CH2Cl2 (2.7 mL)
2.48 (dd, 1H, H4eq, J 13.3, 3.6 Hz), 2.59 (dd, 1H, H10eq, J
13.5, 3.5 Hz), 2.80 (m, 1H, H9), 3.61 (q, 2H, J 7.1 Hz, H2´),
3.90 (m, 2H, H3 + H1), 4.75 (s, 2H, H1´), 5.85 (d, 1H, H7, J
11.3 Hz), 6.31 (d, 1H, H6, J 11.2 Hz) ppm; 13C NMR
(150.5 MHz, CDCl3): δ 12.2 (Me18), 15.3 (Me3´), 18.9
(Me21), 20.7 (CH2, C23), 22.4 (CH2, C15), 23.6 (CH2, C11),
26.5 y 26.6 (Me 26 + Me27) 27.8 (CH2, C16), 29.0 (CH2, C9),
36.3 (CH, C20), 36.6 (CH2, C22), 37.0 (CH2, C10), 40.6
at 0 ºC, Et3N (63 μL, 0.46 mmol), DMAP (4 mg, 0.033
mmol) and acetic anhydride (38 μL, 0.40 mmol, dropwise)
were successively added. The reaction was stirred at r.t. for
2 h and then the solvent was evaporated and the residue
purified
by
column
chromatography
(20-40%
EtOAc/hexane as gradient eluent) to give the racemic
derivative 16 (65% yield). Rf: 0.5 (40% EtOAc/hexane);
IR (NaCl): ν 3425, 3070, 3048, 2953, 2858, 1961, 1902,
(CH2, C12), 41.4 (CH2, C2), 42.4 (CH2, C24), 45.3 (CH2, C4), 1825, 1737, 1472, 1427 cm-1; [α]D20= −12 (c 1.0, CHCl3);
45.9 (C, C13), 56.4 (CH, C14), 56.7 (CH, C17), 63.1 (CH2,
C2´), 68.7 (CH, C3), 68.9 (CH, C1), 76.4 (C, C25), 89.6
(CH2, C1´), 115.5 (CH, C7), 123.8 (CH, C6), 130.3 (C, C5),
142.9 (C, C8) ppm; MS (ESI+, m/z): 485 [(M+Na)+, 50%].
1H NMR (300.13 MHz, CDCl3): δ 1.05 (s, 9H,SiCMe3),
1.24-1.67 (m, 4H, H2ax + H4ax +H6ax+ OH), 2.01 (s, 3H, Me,
OAc), 2.10 (m, 3H, H2eq + H4eq + H6eq), 3.50 (m, 1H, H5),
3.69 (m, 1H, H3), 4.56 (m, 1H, H1), 7.41 (m, 6H, Hmeta
+
Hpara), 7.65 (dd, 4H, Hortho, J 7.7, 1.6 Hz) ppm; 13C NMR
(75.5 MHz, CDCl3): δ 19.2 (SiC), 21.4 (Me, OAc), 27.0
(CH3, SiCMe3), 39.9 (CH2), 40.0 (CH2), 43.7 (CH2), 65.6
(CH, C5), 67.1 (CH, C3), 67.9 (CH, C1), 127.8 (4CH, Cmeta),
129.9 (2CH, Cpara), 133.9 (C, Cipso), 134.0 (C, Cipso), 135.9
(4CH, Cortho), 170.5 (C=O) ppm; MS (ESI+, m/z): 413
[(M+H)+, 100%]; HRMS (ESI+, m/z): calcd for
C24H32NaO4Si [(M+Na)+]: 435.1962, found: 435.1983.
25-Ethoxymethyloxy-1β-hydroxy-19-nor-vitamin
D3
(14). Rf: 0.2 (60% EtOAc/hexane). IR (NaCl): ν 3368,
1
2943, 2872, 1458, 1348 cm-1; H NMR (600.13 MHz,
CDCl3): δ 0.54 (s, 3H, Me18), 0.93 (d, 3H, Me21, J 6.5 Hz),
1.03 (dd, 1H, H22, J 20.0, 9.7 Hz), 1.20 (t, 3H, H3´, J 4.0
Hz,), 1.21 (s, 6H, Me26 + Me27), 1.29 (m, 4H, H17 + 2H12
+
H16), 1.40 (m, 4H, H20 + 1H22 + 1H24 + 1H23), 1.50 (m, 5H,
H15 + 1H24+ 2H), 1.66 (m, 3H, 1H9 + 2H11), 1.86 (m, 2H,
1H2 + 1H16), 1.99 (m, 3H, H2 + H14), 2.29 (dd, 1H, H4, J
13.4, 6.2 Hz), 2.34 (s, 1H, OH), 2.47 (dd, 1H, H10, J 14.0,
3.2 Hz), 2.48 (dd, 1H, H10, J 12.6, 2.7 Hz), 2.56 (dd, 1H,
H10, J 13.8, 6.1 Hz), 2.81 (dd, 1H, H9, J 13.6, 5.3 Hz), 3.61
(q, 2H, J 7.1 Hz, H2´), 4.00 (m, 2H, H3 + H1), 4.75 (s, 2H,
H1´), 5.86 (d, 1H, H7, J 11.3 Hz), 6.33 (d, 1H, H6, J 11.3
Hz) ppm; 13C NMR (150.5 MHz, CDCl3): δ 12.2 (Me18),
15.3 (Me3´), 18.9 (Me21), 20.6 (CH2, C23), 22.4 (CH2, C15),
23.7 (CH2, C11), 26.5 and 26.6 (Me26 + Me 27) 27.8 (CH2,
C16), 29.1 (CH2, C9), 36.3 (CH, C20), 36.6 (CH2, C22), 36.8
(CH2, C10), 40.4 (CH2, C2), 40.6 (CH2, C12), 42.4 (CH2,
C24), 45.2 (CH2, C4), 45.9 (C, C13), 56.4 (CH, C14), 56.7
(CH, C17), 63.1 (CH2, C2´). 68.8 (CH, C3), 69.0 (CH, C1),
76.4 (C, C25), 89.6 (CH2, C1´), 115.5 (CH, C7), 124.2 (CH,
C6), 130.1 (C, C5), 142.9 (C, C8) ppm; MS (ESI+, m/z): 387
[(M-OCH2OEt)+, 100%], 399 [(M-2OH-Et)+, 100%], 463
[(M+H)+, 75%], 485 [(M+Na)+, 50%].
(1R,3R,5S)-3-(tert-Butyldiphenylsilyloxy)-5-(tert-
butyldimethylsilyloxy)cyclohexyl acetate (17). To a
solution of 16 (500 mg, 1.21 mmol) in anhydrous CH2Cl2
(4.8 mL) at 0 ºC, were added imidazole (215 mg,
3.15 mmol) and tert-butyldimethylsilyl chloride (439 mg,
2.91 mmol). Afterwards, the reaction was stirred at r.t. for
3 h. Then, solvent was evaporated and the residue purified
by column chromatography (10% Et2O/hexane as eluent)
to give 17 in 94% yield. Rf: 0.5 (10% EtOAc/hexane); IR
(NaCl): ν 3425, 3070, 3048, 2953, 2858, 1961, 1902, 1825,
20
1
1737, 1472, 1427 cm-1; [α]D
=
3 (c 1.0, CHCl3); H
NMR (300.13 MHz, CDCl3): δ −0.04 (s, 3H, SiMe), −0.03
(s, 3H, SiMe), 0.85 (s, 9H, SiCMe3, TBDMS) 1.11 (s, 9H,
SiCMe3, TBDPS), 1.36 (m, 2H, H2ax + H6ax), 1.51 (dd, 1H,
H4ax, J 21.8, 10.3 Hz), 1.98 (m, 1H, H4eq), 2.00 (s, 6H, Me,
OAc), 2.09 (m, 1H, H2eq), 2.22 (m, 1H, H6eq) 3.40 (m, 1H,
H3), 3.65 (m, 1H, H5), 4.57 (m, 2H, H1), 7.41 (m, 6H, Hmeta
+ Hpara), 7.65 (dd, 4H, Hortho, J 7.8, 1.6 Hz) ppm; 13C NMR
(75.5 MHz, CDCl3): δ −4.7 (SiMe), −4.6 (SiMe), 18.1
(SiC), 19.2 (SiC), 21.3 (Me, OAc), 25.9 (SiCMe3,
TBDMS), 27.0 (SiCMe3, TBDPS), 40.6 (CH2, C6), 40.9
(CH2, C2), 45.0 (CH2, C4), 66.1 (CH, C3), 67.2 (CH, C5),
(1R,3S,5s)-5-(tert-Butyldiphenylsilyloxy)cyclohexane-
1,3-diol (15). To a solution of 9 (1 g, 7.18 mmol) in
anhydrous THF (20 mL) at r.t. were successively added
TBDPSCl (2.3 mL, 8.62 mmol) and anhydrous Et3N (1.2
mL, 8.62 mmol). After being stirred at this temperature for
30 min, NaH was added (60% w/w in mineral oil, 372 mg,
9.35 mmol). When the solution stopped bubbling, the
mixture was heated to 45 ºC for 48 h. After this time, it
was cooled to 0 ºC and filtered over Celite®. Solvents were
evaporated and the residue purified by column
chromatography (EtOAc as eluent) to give the diol 15 in
85% yield. Rf: 0.4 (80% EtOAc/hexane); mp: 94-96 °C; IR
(KBr): ν 3324, 3052, 2939, 2860, 1961, 1904, 1826, 1475
cm-1; 1H NMR (300.13 MHz, CDCl3): δ 1.06 (s, 9H,
SiCMe3), 1.30 (m, 1H, H2ax), 1.44 (dt, 2H, H4ax + H6ax, J
17.6, 8.8 Hz), 2.09 (m, 3H, H2eq + H4eq +H6eq), 2.24 (s, 2H,
68.0 (CH, C1), 127.6 (2CH, Cmeta), 127.7 (2CH, Cmeta
)
129.8 (2CH, Cpara), 134.2 (2C, Cipso), 134.4 (2C, Cipso),
135.8 (2CH, Cortho), 135.9 (2CH, Cortho), 170.2 (C=O) ppm;
MS (ESI+, m/z): 527 [(M+H)+, 100%].
(1R,3R,5S)-3-(tert-Butyldiphenylsilyloxy)-5-(tert-
butyldimethylsilyloxy)cyclohexan-1-ol (18). To
solution of 17 (520 mg, 0.99 mmol) in anhydrous MeOH
(4.8 mL), MeONa (140 mg, 2.47 mmol) was added,
showing a progressive cloudiness. After being stirred at r.t.
for 4 h, solid ammonium chloride was added until the
medium was neutralized. Then, the solvent was evaporated,
a
OH), 3.46 (ddd, 2H, H1 + H3, J 14.3, 10.2, 3.9 Hz), 3.67 (m, the residue was dissolved in EtOAc and it was filtered to
1H, H5), 7.40 (m, 6H, Hmeta + Hpara), 7.68 (dd, 4H, Hortho, J
7.7, 1.6 Hz) ppm; 13C NMR (75.5 MHz, CDCl3): δ 19.2
(SiC), 27.0 (CH3, SiCMe3), 43.4 (CH2, C2), 43.6 (2CH2, C4
+ C6), 65.9 (2CH, C1 + C3), 67.5 (CH, C5), 127.8 (4CH,
Cmeta), 129.8 (2CH, Cpara), 134.0 (2C, Cipso), 135.8 (4CH,
Cortho) ppm; MS (ESI+, m/z): 371 [(M+H)+, 100%]; HRMS
(ESI+, m/z): calcd for C22H30NaO3Si [(M+Na)+]: 393.1856,
found: 393.1860.
remove the salts. The crude was purified by column
chromatography (20% Et2O/hexane as eluent) to give 18 in
95% yield; Rf: 0.4 (20% Et2O /hexane); IR (NaCl): ν 3349,
3071, 3049, 2951, 2857, 1958, 1887, 1822, 1471, 1427
1
cm-1; [α]D20= +9 (c 1.0, CHCl3); H NMR (300.13 MHz,
CDCl3): δ −0.05 (s, 6H, SiMe), 0.84 (s, 9H, SiCMe3,
TBDMS) 1.07 (s, 9H, SiCMe3, TBDPS), 1.27 (m, 1H),
1.38 (m, 2H), 1.59 (s, 1H, OH), 1.92 (m, 1H), 2.05 (m, 1H),
2.15 (m, 1H), 3.38 (m, 2H, H1 + H5), 3.58 (m, 1H, H3),
7.41 (m, 6H, Hmeta + Hpara), 7.69 (ddd, 4H, Hortho, J 7.9, 4.5,
1.7 Hz) ppm; 13C NMR (75.5 MHz, CDCl3): δ −4.7 (SiMe),
−4.6 (SiMe), 18.3 (SiC), 19.2 (SiC), 26.0 (SiCMe3,
TBDMS), 27.1 (SiCMe3, TBDPS), 44.5 (CH2), 44.7 (CH2),
44.8 (CH2), 66.1 and 66.4 (2CH, C1 + C5), 67.3 (CH, C3),
127.7 (4CH, Cmeta), 129.76 (CH, Cpara), 129.81 (CH, Cpara),
134.31 (C, Cipso), 134.35 (C, Cipso), 135.86 (2CH, Cortho),
135.88 (2CH, Cortho) ppm; MS (ESI+, m/z): 485 [(M+H)+,
100%].
(1R,3S,5S)-3-(tert-Butyldiphenylsilyloxy)-5-
hydroxycyclohexyl acetate (16). Enzymatic reactions: A
mixture of compound 15 (540 mg, 1.46 mmol), lipase (270
mg) and vinyl acetate (672 μL, 7.29 mmol) in the
anhydrous solvent (0.2 M, 7.3 mL) was shaken at 30 ºC
and 250 rpm. The progress of the reaction was analyzed by
TLC (50% EtOAc/hexane) until the achievement of the
complete conversion (4-12 h). Then, the enzyme was
removed by filtration and washed with CH2Cl2. The crude
residue was purified by column chromatography (20-40%
8
This article is protected by copyright. All rights reserved.

10.1002/adsc.201800481
Advanced Synthesis & Catalysis
(3S,5R)-3-(tert-Butyldiphenylsilyloxy)-5-(tert-
crude was purified by column chromatography (30%
EtOAc/hexane) to give 22 in 79% yield. Rf: 0.3 (30%
AcOEt/hexane); IR (NaCl): ν 3444, 2958, 2865, 1738,
1734, 1716, 1435, 1372, 1256, 1238 cm-1; [α]D20= +13.8 (c
butyldimethylsilyloxy)cyclohexanone (19). To a solution
of 18 (452 mg, 0.93 mmol) in anhydrous CH2Cl2 (9.0 mL),
Dess-Martin reagent was added (448 mg, 1.02 mmol). The
mixture was stirred at r.t. for 1 h and then, it was diluted
with Et2O (9.5 mL) and a mixture of saturated aqueous
Na2S2O3 and NaHCO3 solution (1:1, v/v, 9.5 mL),
obtaining a white precipitate that disappeared after 10 min.
After that, the mixture was extracted with Et2O. The
combined organic fractions were dried (Na2SO4), filtered
and concentrated. Purification of the crude by column
chromatography (10% Et2O/hexane) gave 19 in 97% yield;
1
1.0, CHCl3); H NMR (300.13 MHz, CDCl3): δ 1.53 (m,
3H, H2ax + H4ax + H6ax), 1.96 (s, 1H, OH), 2.05 (s, 3H, Me,
OAc), 2.06 (s, 3H, Me, OAc), 2.08 (m, 2H), 2.28 (m, 1H),
4.03 (m, 1H, H5), 5.05 and 5.28 (2m, 2H, H1 + H3) ppm;
13C NMR (75.5 MHz, CDCl3): δ 21.3 (Me, OAc), 21.4 (Me,
OAc), 34.9 (CH2), 38.3 (CH2), 40.0 (CH2), 65.2 (CH, C5),
68.2 and 68.5 (2CH, C3 + C1), 170.3 (C=O), 170.4 (C=O)
ppm; MS (ESI+, m/z): 217 [(M+H)+, 100%].
Rf: 0.7 (20% Et2O/hexane); IR (KBr): ν 3064, 3049, 2951,
2890, 1958, 1894, 1822, 1717, 1467, 1427 cm-1; [α]D
=
20
Procedure for the synthesis of 25 and 26. To a solution
of 12 (300 mg, 0.55 mmol) in anhydrous THF (1.8 mL) at
−78 ºC was added dropwise LHMDS (520 μL, 1.0 M in
THF, 0.52 mmol), resulting in a deep red color solution.
The mixture was stirred at the same temperature for 2 h
and then, a solution of the corresponding ketone 19 (264
mg, 0.55 mmol) in anhydrous THF (1.2 mL) was added
dropwise via cannula transfer. After being stirred at −50 ºC
during 5 h, the reaction was poured into a saturated
aqueous solution of NH4Cl and extracted with Et2O. The
combined organic fractions were dried (Na2SO4), filtered,
and concentrated to give a crude that was purified by
column chromatography (4% Et2O/hexane). Then, the
resulting mixture (269 mg, 0.33 mmol) was dissolved in
anhydrous CH2Cl2 (3.3 mL), Bi(OTf)3 (238 mg, 0.36
mmol) was added and the mixture was stirred at 0 ºC for
2 h. The reaction was quenched by adding a saturated
aqueous solution of NaHCO3. Next, it was diluted with
CH2Cl2 and filtered over Celite®. Solvents were evaporated
under vacuum and the residue purified by column
chromatography (50% Et2O/hexane). The diastereoisomers
were isolated by preparative HPLC (SunFire® C18 Column,
100 Å, 5 m, 10 mm x 250 mm). Conditions: 5.0 mL/min,
5% iPrOH/hexane.
−1 (c 1.0, CHCl3); 1H NMR (300.13 MHz, CDCl3): δ
−0.03 (s, 6H, SiMe), 0.86 (s, 9H, SiCMe3, TBDMS) 1.11 (s,
9H, SiCMe3, TBDPS), 1.84 (m, 1H, H4ax), 2.17 (m, 1H,
H4eq), 2.35 (dd, 1H, H6ax, J 13.8, 11.1 Hz), 2.50 (dt, 2H,
H2ax + H6eq, J 14.0, 7.0 Hz), 2.63 (dd, 1H, H2eq, J 14.1, 5.1
Hz), 3.59 (m, 1H, H5), 3.84 (m, 1H, H3), 7.42 (m, 6H, Hmeta
+ Hpara) and 7.70 (ddd, 4H, Hortho, J 16.8, 7.6, 1.5 Hz) ppm;
13C NMR (75.5 MHz, CDCl3): δ −4.9 (SiMe), −4.8 (SiMe),
18.1 (CSi), 19.1 (SiC), 25.8 (SiCMe3, TBDMS), 27.0
(SiCMe3, TBDPS), 44.7 (CH2, C4), 50.6 (CH2, C2), 50.9
(CH2, C6), 65.6 (CH, C5), 66,6 (CH, C3), 127.8 (4CH,
Cmeta), 130.0 (2CH, Cpara),133.5 (C, Cipso), 133.8 (C, Cipso),
135.7 (2CH, Cortho), 135.8 (2CH, Cortho), 207.1 (C=O) ppm;
MS (ESI+, m/z): 483 [(M+H)+, 100%].
(1R,3S,5s)-5-(tert-Butyldiphenylsilyloxy)cyclohexan-1,3-
diyl diacetate (20). By-product of the enzymatic reaction.
Rf: 0.8 (40% EtOAc/hexane); IR (NaCl): ν 3071, 3049,
1
2954, 2930, 2857, 1959, 1887, 1738, 1471, 1427 cm-1; H
NMR (300.13 MHz, CDCl3): δ 1.05 (s, 9H, SiCMe3), 1.34
(q, 1H, H2ax, J 11.6 Hz), 1.48 (c, 2H, H4ax + H6ax, J 11.7
Hz), 2.00 (s, 6H, Me, OAc), 2.19 (m, 3H, H2eq + H4eq
+H6eq), 3.67 (m, 1H, H5), 4.53 (m, 2H, H1 + H3), 7.41 (m,
6H, Hmeta + Hpara), 7.65 (dd, 4H, Hortho, J 7.8, 1.6 Hz) ppm;
13C NMR (75.5 MHz, CDCl3): δ 19.2 (SiC), 21.2 (Me,
OAc), 27.0 (CH3, Me2CSi), 36.5 (CH2, C2), 40.5 (CH2, C4
+ C6), 66.7 (CH, C5), 67.3 (CH, C1 + C3), 127.8 (4CH,
Cmeta), 129.9 (2CH, Cpara), 133.8 (2C, Cipso), 135.8 (4CH,
Cortho), 170.1 (C=O) ppm; MS (ESI+, m/z): 455 [(M+H)+,
100%].
1-(tert-Butyldiphenylsilyloxy)-25-hydroxy-3-epi-19-
nor-vitamin D3 (25). Rf: 0.4 (70% Et O/hexane); 1H NMR
(600.13 MHz, CDCl3): δ 0.51 (s, 3H,2Me18), 0.88 (m, 1H),
0.94 (d, 3H, Me21, J 6.4 Hz), 1.06 (s, 9H, SiCMe3), 1.23 (s,
6H, Me26 + Me27), 1.25–2.07 (several m, 19H), 2.19 (d, 1H,
H10eq, J 13.8 Hz), 2.37 (dd, 2H, H4, J 10.2, 4.4 Hz), 2.47
(dd, 1H, H10ax, J 13.7, 5.7 Hz), 2.82 (m, 1H, H9), 3.16 (d,
1H, J 8.6 Hz), 3.85 (br s, 1H, H3), 4.08 (br s, 1H, H1), 5.73
(d, 1H, H7, J 11.2 Hz), 6.33 (d, 1H, H6, J 11.3 Hz), 7.38 (q,
4H, Hmeta, J 7.1 Hz), 7.43 (m, 2H, Hpara), 7.67 (d, 2H, J 6.6
(1R,3R)-5-(tert-Butyldiphenylsilyloxy)cyclohexan-1,3-
diyl diacetate (21). To a solution of (−)-16 (60 mg,
0.15 mmol) in anhydrous THF (250 μL), were successively
added PPh3 (57 mg, 0.22 mmol), DIAD (42 μL, 0.22 mmol, Hz, ), 7.71 (d, 2H, J 7.71 Hz) ppm; 13C NMR (150.5 MHz,
dropwise) and acetic acid (13 μL, 0.22 mmol, dropwise).
After being stirred at r.t. for 4 h, the solvent was
evaporated, and the residue purified by column
chromatography (20% Et2O/hexane) to give the diacetate
21 (72%). Rf: 0.2 (20% Et2O/hexane); IR (NaCl): ν 3071,
3049, 2958, 2857, 1961, 1892, 1741, 1734, 1471, 1427
CDCl3): δ 12.3 (Me18), 18.9 (Me21), 19.4 (CSi), 21.0 (CH2),
22.5 (CH2), 23.6 (CH2), 27.2 (SiCMe3), 27.8 (CH2), 29.0
(CH2) 29.3 y 29.5 (Me26 + Me27), 36.3 (CH, C20), 36.5
(CH2), 36.6 (CH2), 40.7 (CH2, C2), 40.5 (CH2), 44.6 (CH2),
45.5 (CH2), 45.9 (C, C13), 56.4 and 56.7 (2CH, C14 + C17),
68.9 (CH, C3), 70.6 (CH, C1), 71.5 (C, C25), 115.8 (CH, C7),
124.0 (CH, C6), 127.75 and 127.83 (2CH, Cmeta), 129.90
and 130.02 (2CH, Cpara), 130.6 (C5), 133.4 and 133.7 (2C,
Cipso) 135.97 and 136.06 (2CH, Cortho), 142.3 (C8) ppm.
1
cm-1; [α]D20= +1 (c 1.0, CHCl3); H NMR (300.13 MHz,
CDCl3): δ 1.05 (s, 9H, SiCMe3), 1.50 (m, 3H, H2ax + H4ax +
H6ax), 1.80 (s, 3H, Me, OAc). 1.86 (m, 1H, H6eq), 1.96 (m,
1H, H2eq), 2.02 (s, 3H, MeC=O), 2.26 (m, 1H, H4eq), 3.90
(m, 1H, H5), 4.88 (m, 1H, H3), 5.14 (m, 1H, H1) 7.39 (m,
6H, Hmeta + Hpara), 7.70 (dd, 4H, Hortho, J 7.7, 1.6 Hz) ppm;
13C NMR (75.5 MHz, CDCl3): δ 19.2 (SiC), 21.2 (Me,
OAc), 21.4 (Me, OAc), 27.0 (SiCMe3), 34.9 (CH2, C2),
38.6 (CH2, C6), 40.5 (CH2, C4), 66.4 (CH, C5), 68.0 (CH,
C3), 68.7 (CH, C1), 127.7 (2CH, Cmeta), 127.8 (2CH, Cmeta),
129.82 (CH, Cpara), 129.83 (CH, Cpara), 134.0 (C, Cipso),
134.1 (C, Cipso), 135.8 (2CH, Cortho),135.9 (2CH, Cortho),
170.0 (C=O), 170.4 (C=O) ppm; MS (ESI+, m/z): 455
[(M+H)+, 100%].
3-(tert-Butyldiphenylsilyl)-1β,25-dihydroxy-19-nor-
1
vitamin D3 (26). Rf: 0.4 (70% Et2O/hexane); H NMR
(600.13 MHz, CDCl3): δ 0.53 (s, 3H, Me18), 0.88 (m, 1H),
0.94 (d, 3H, Me21, J 6.4 Hz), 1.05 (s, 9H, SiCMe3), 1.22 (s,
6H, Me26 + Me27), 1.25–2.07 (several m, 20 H), 2.18 (m,
1H, H4eq), 2.40 (dd, 1H, H10eq, J 13.4, 3.3 Hz), 2.54 (dd, 1H,
H10ax, J 13.5, 6.3 Hz), 2.78 (dd, 1H, H9eq, J 14.2, 4.4 Hz),
2.94 (d, 1H, H2eq, J 9.1 Hz), 3.84 (br s, 1H, H1), 3.99 (m,
1H, H3), 5.14 (br s, 1H, OH), 5.86 (d, 1H, H7, J 11.2 Hz),
6.16 (d, 1H, H6, J 11.2 Hz), 7.38 (m, 4H, Hmeta), 7.43 (m,
2H, Hpara), 7.67 (d, 2H, J 7.9 Hz, ), 7.70 (d, 2H, J 6.7 Hz)
ppm; 13C NMR (150.5 MHz, CDCl3): δ 12.2 (Me18), 18.9
(Me21), 19.2 (CSi), 21.0 (CH2), 22.4 (CH2), 23.6 (CH2),
27.2 (SiCMe3), 27.9 (CH2), 29.0 (CH2), 29.4 and 29.5
(Me26 + Me27), 36.3 (CH, C20), 36.6 (CH2), 37.1 (CH2), 40.4
(CH2), 40.6 (CH2), 44.5 (CH2), 45.2 (CH2), 45.9 (C, C13),
56.4 and 56.6 (2CH, C14 + C17), 68.7 (CH, C3), 71.0 (CH,
C1), 71.3 (C, C25), 115.9 (CH, C7), 124.2 (CH, C6), 127.78
and 127.81 (2CH, Cmeta), 129.90 and 129.95 (2CH, Cpara),
(1S,3S)-5-Hydroxycyclohexan-1,3-diyl diacetate (22).
TBAF (219 L, 0.22 mmol, 1.0 M in THF) was added
dropwise to a solution of silyl ether 21 (40 mg, 0.09 mmol)
in anhydrous THF (437 L) at 0 ºC in darkness. The
reaction was stirred at r.t. until TLC (60% Et2O/hexane)
shows complete consumption of starting material (3 h).
Then, solvent was evaporated and the mixture was
extracted with EtOAc. The combined organic fractions
were dried (Na2SO4) and concentrated under vacuum. The
9
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10.1002/adsc.201800481
Advanced Synthesis & Catalysis
130.5 (C5), 133.7 and 134.0 (2C, Cipso) 136.00 and 136.09
(2CH, Cortho), 142.3 (C8) ppm.
[3] a) A. W. Norman, R. Bouillon, M. C. Farach-Carson, J.
E. Bishop, L.-X. Zhou, I. Nemere, J. Zhao, K. R.
Muralidharan, W. H. Okamura, J. Biol. Chem. 1993,
268, 20022-20030; b) K. R. Muralidharan, A. R. De
Lera, S. D. Isaeff, A. W. Norman, W. H. Okamura, J.
Org. Chem. 1993, 58, 1895–1899.
[4] a) S. Yang, C. Smith, H. F. DeLuca, Biochim. Biophys.
Acta 1993, 1158, 279-286; b) K. L. Perlman, R. E.
Swenson, H. E. Paaren, H. K. Schnoes, H. F. DeLuca,
Tetrahedron Lett. 1991, 32, 7663-7666; c) K. L.
Perlman, R. R. Sicinski, H. K. Schnoes, H. F. DeLuca,
Tetrahedron Lett. 1990, 31, 1823-1824.
In vitro Biological Evaluation
Affinity for VDR. The affinity of 1α,25-(OH)2-D3 and its
analogues to the vitamin D receptor was evaluated by their
ability to compete with [3H]1α,25-(OH)2-D3 for binding to
high speed supernatant from intestinal mucosa
homogenates obtained from normal pigs as described
previously.[12] The relative affinity of the analogues was
calculated from their concentration needed to displace 50%
of [3H]1α,25-(OH)2-D3 from its receptor compared with
the activity of 1α,25-(OH)2-D3 (assigned a 100% value).
Affinity for DBP. Binding of vitamin D metabolites and
analogues to hDBP was performed at 4 °C as described
previously.[13] [3H]1α,25-(OH)2-D3 and 1α,25-(OH)2-D3 or
its analogues were incubated with hDBP (0.18 μM) in a
final volume of 1 ml (0.01 M Tris-HCl buffer and 0.154 M
NaCl, pH 7.4) for 3 h at 4 °C. Phase separation was then
obtained by the addition of 0.5 ml of cold dextran-coated
charcoal.
[5] Y. Wu, Y. Zhao, H. Tian, P. DeClercq, M. Vandewalle,
M. Berthier, G. Pellegrino, P. Maillos, J.-C. Pascal, Eur.
J. Org. Chem. 2001, 3779–3788.
[6] K. Ono, A. Yoshida, N. Saito, T. Fujishima, S.
Honzawa, Y. Suhara, S. Kishimoto, T. Sugiura, K.
Waku, H. Takayama, A. Kittaka, J. Org. Chem. 2003,
68, 7407–7415.
[7] L. Sánchez-Abella, S. Fernández, A. Verstuyf, L.
Verlinden, V. Gotor, M. Ferrero, J. Med. Chem. 2009,
52, 6158–6162.
[8] B. Wirz, H. Iding, H. Hilpert, Tetrahedron: Asymmetry
2000, 11, 4171–4178.
Cell proliferation assays. As
a measure of cell
proliferation, [3H]-thymidine incorporation of breast
cancer MCF-7 cells (ATCC) was determined after a 72 h
incubation period with various concentrations of 1α,25-
(OH)2-D3, analogues or vehicle as described previously.[12]
[9] T. Seitz, K. Harms, U. Koert, Synthesis 2014, 46, 381-
386.
Cell
differentiation
assays.
Differentiation
of
promyeolocytic HL-60 leukemia cells (ATCC) was
measured by the nitro blue tetrazolium (NBT) reduction
assay after a 72 h incubation period in the presence of
1α,25-(OH)2-D3, analogues or vehicle.[12]
[10] a) D. Oves, V. Gotor-Fernández, S. Fernández, M.
Ferrero, V. Gotor, Tetrahedron: Asymmetry 2004, 15,
2881-2887; b) S. Fernández, V. Gotor-Fernández, M.
Ferrero, V. Gotor, Curr. Org. Chem. 2002, 6, 453-469;
c) M. Díaz, M. Ferrero, S. Fernández, V. Gotor,
Tetrahedron: Asymmetry 2002, 13, 539-546; d) V.
Gotor-Fernández, M. Ferrero, S. Fernández, V. Gotor, J.
Org. Chem. 2002, 67, 1266-1270; e) M. Díaz, V.
Gotor-Fernández, M. Ferrero, S. Fernández, V. Gotor, J.
Org. Chem. 2001, 66, 4227-4232; f) V. Gotor-
Fernández, M. Ferrero, S. Fernández, V. Gotor, J. Org.
Chem. 1999, 64, 7504-7510; g) M. Ferrero, S.
Fernández, V. Gotor, J. Org. Chem. 1997, 62, 4358-
4363; h) S. Fernández, M. Ferrero, V. Gotor, W. H.
Okamura, J. Org. Chem. 1995, 60, 6057-6061.
[11] a) A. Koskinen, A. Klibanov in Organic Synthesis
with Enzymes in Non-Aqueous Media, Springer
Netherlands, 2012; b) N. G. Munishwar, R. Ipsita, Eur.
J. Biochem. 2004, 271, 2575-2583.
Acknowledgements
Financial support by the Spanish Ministerio de Ciencia e
Innovación (MICINN) (Projects CTQ2011-24237 and CTQ2014-
55015-P) and Principado de Asturias (Project FC-15-
GRUPIN14-002) are gratefully acknowledged. T. G.-G. thanks
FICYT (Asturias) for a pre-doctoral fellowship. We also thank Dr.
I. Merino (NMR Facility, Universidad de Oviedo) for her
assistance in the NMR study.
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10
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10.1002/adsc.201800481
Advanced Synthesis & Catalysis
UPDATE
Enzymatic Desymmetrization of 19-nor-Vitamin
D3 A-Ring Synthon Precursor: Synthesis, Structure
Elucidation, and Biological Activity of 1,25-
Dihydroxy-3-epi-19-nor-vitamin D3 and 1β,25-
Dihydroxy-19-nor-vitamin D3
Adv. Synth. Catal. 2018, 360, 000 – 000
Tania González-García, Annemieke Verstuyf,
Lieve Verlinden, Susana Fernández,* and Miguel
Ferrero*
11
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