Chemoenzymatic synthesis and biological evaluation of C-3 carbamate analogues of 1α,25-dihydroxyvitamin D3

Article abstract of DOI:10.1016/j.bmc.2004.07.042

The synthesis of new analogues of 1α,25-dihydroxyvitamin D ₃ containing a carbamate function at the A-ring fragment has been described using the cross-coupling approach. The carbamate group was selectively introduced at the C-3 position by regi

Full text of DOI:10.1016/j.bmc.2004.07.042

Bioorganic & Medicinal Chemistry 12 (2004) 5443–5451
Chemoenzymatic synthesis and biological evaluation of C-3
carbamate analogues of 1a,25-dihydroxyvitamin D3
a
Vicente Gotor-Fernandez, Susana Fernandez, Miguel Ferrero, Vicente Gotor,
a
a
a,
*
´
´
Roger Bouillonb and Annemieke Verstuyf b
a
´
´
´
´
Departamento de Quımica Organica e Inorganica, Facultad de Quımica, Universidad de Oviedo, 33071 Oviedo, Spain
bLaboratorium voor Experimentele Geneeskunde en Endocrinologie, Katholieke Universiteit Leuven,
Gasthuisberg, B-3000 Leuven, Belgium
Received 14 May 2004; accepted 20 July 2004
Available online 20 August 2004
Abstract—The synthesis of new analogues of 1a,25-dihydroxyvitamin D3 containing a carbamate function at the A-ring fragment
has been described using the cross-coupling approach. The carbamate group was selectively introduced at the C-3 position by reg-
ioselective enzymatic alkoxycarbonylation of A-ring enyne 3 and subsequent treatment with ammonia, amines, amino alcohols, and
amino acids. Biological studies to evaluate the potency of all five of these carbamate analogues were performed and demonstrated
very low binding affinity for the vitamin D receptor compared with 1a,25-dihydroxyvitamin D3. Moreover, all the carbamate ana-
logues were less active than 1a,25-dihydroxyvitamin D3 in inhibiting cell proliferation or stimulating cell differentiation. Of all the
five analogues, the 3-O-carbamoyl-1a,25-(OH)2-D3 analogue 10a was the most potent one in vitro. However, all investigated carb-
amate analogues demonstrated lower calcemic effects in vivo than the parent compound.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
ited by inherent toxicity due to adverse calcium absorp-
tion and mobilization effects.1c,d For this reason, there is
a continued effort stimulated by medical needs to design
analogues of the natural hormone with dissociation of
effects on cell differentiation compared with their calce-
mic effects.2
The seco-steroid 1a,25-dihydroxyvitamin D3 [calcitriol,
1a,25-(OH)2-D3 (1), Fig. 1], the biologically active form
of vitamin D3 (2), functions as a regulator of calcium
and phosphorous homeostasis, and also shows a broad
spectrum of biological activities such as modulation of
cell proliferation and differentiation.1 Unfortunately,
the therapeutic value of 1a,25-(OH)2-D3 has been lim-
Carbamate derivatives are compounds of considerable
interest in some areas of medicinal chemistry.3 This
functional group is present in antineoplastic agents such
as mitomycins4 and bleomycins.5 The alkaloid
physostigmine, which has a carbamate moiety in its
structure, is under investigation as potential drug in
the therapy of AlzheimerÕs disease.6 Also, potent inhibi-
tors of HIV-1 protease posses this group in their struc-
tures.7 An important application of the carbamate
group is to increase the permeability across the cellular
membranes. Thus, certain b-adrenergic blockers are
introduced into cells as carbamate derivatives.8
Recently, Liu and co-workers have described the synthe-
sis of amino and polyamino carbamate analogues of
vitamin D2 and vitamin D3 as useful cationic lipids for
gene delivery in vitro.9
25
Side Chain
2
R
CD-Ring
H
Triene
A-Ring
1
HO
R
1
3
1
2
1, R = R = OH; 1α,25-(OH) -D
2
3
1
2
2, R = R = H; Vitamin D
3
Figure 1.
*
Most analogues of 1a,25-(OH)2-D3 involved modifica-
tions of the side chain, while modifications of the A-ring
Corresponding author. Tel./fax: +34-98-510-3448; e-mail: vgs@
sauron.quimica.uniovi.es
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.042

´
V. Gotor-Fernandez et al. / Bioorg. Med. Chem. 12 (2004) 5443–5451
5444
are less extensive due to its more complicated synthetic
routes required to obtain the adequate precursors. The
A-ring fragment possesses two hydroxyl groups of sim-
ilar reactivity, and as a result it is very difficult to discern
between these two groups from a chemical point of view.
However, we have applied the synthetic potential of en-
zymes in this field to selectively modify polyfunctional-
ized compounds.10
a
b
O
HO
OH
3
O
O
c
OH
5
3
4
O
O
2
1
R
R
d (f or5d)
Taking into account the interesting features showed by
the carbamate moiety, here we report the chemoenzy-
matic synthesis of C-3 carbamates analogues of the
steroid hormone 1a,25-(OH)2-D3. Furthermore, differ-
ent hydrophilic groups such as alcohol, amino, and
acid (from an amino acid) were introduced at the end
of the alkyl chain of the carbamate function. These
interesting products can provide information about
the mode of action of 1a,25-(OH)2-D3 and the influence
of the several functional groups of the molecule in
the biological responses associated to the hormone.
Amino acids linked to vitamin D3 would be promising
products.
N
N
H
O
OTBDMS
O
1
OH
H
6a-e
5a-e
2
a, R = H
b, R = Bu
a, R = H
b, R = Bu
c, R = (CH ) OH
d, R = (CH ) NH
2
1
2
1
c, R = (CH ) OTBDMS
d, R = (CH ) NHBoc
e, R = CH CO Na
2 2
2 2
2
1
2 3
2 3
2
2
1
2
2
e, R = CH CO Na
2
2
Scheme 1. Reagents and conditions: (a) CAL-B, acetone O-[(vinyl-
oxy)carbonyl]oxime, toluene, 30ꢁC, 4h (quantitative); (b) R1NH2,
THF (DMF for 5e), 60ꢁC [18h (quantitative) for 5a, 22h (91%) for 5b,
48h (80%) for 5c, 48h (76%) for 5d, 48h (87%) for 5e]; (c) TBDMSCl,
imidazol, DMAP, CH2Cl2, rt, 12h (91% for 6a, 97% for 6b, 86% for 6c,
88% for 6e); (d) Boc2O, CHCl3, NaHCO3 (aq), rt, 3h (67% for 6d,
steps c and d).
2. Results and discussion
To synthesize the C-3 carbamate derivatives we used a
convergent route11 based on the palladium-catalyzed
coupling of an A-ring enyne with the CD-ring vinyl tri-
flate fragment, which are separately synthesized. In this
approach, a dienyne is semihydrogenated to a previta-
min structure that undergoes rearrangement to the cor-
responding vitamin D analogue.
generated previtamin 9a. Reaction was carefully
A
monitored by TLC to avoid over-reduction.
critical factor is the state of the catalyst; so previ-
ously it was pre-dried at 60ꢁC in vacuum. Thermolysis
of compound 9a at 80ꢁC for 4h afforded 3-O-
carbamoyl-1a,25-(OH)2-D3 (10a) in 62% yield from
8a.
Previously, we have reported12 the synthesis of several
carbamate derivatives of the 1a,25-(OH)2-D3 A-ring
precursor through a two-step chemoenzymatic process
(Scheme 1). First, enzymatic alkoxycarbonylation
reaction of synthon 313 with acetone O-[(vinyloxy)carb-
onyl] oxime catalyzed by Candida antarctica lipase B
(CAL-B) gave rise selectively to carbonate 4 in quantita-
tive yield. This versatile vinylcarbonate reacts with
ammonia, amines, amino alcohols, diamines, and amino
acids to obtain the corresponding 1a,25-(OH)2-D3
A-ring C-514 carbamate synthons 5a–e. In order to
improve the yield of the coupling reaction, enynes 5 were
conveniently protected as silyl ethers with tert-butyl-
dimethylsilyl chloride (TBDMSCl) given place to
derivatives 6.
Similarly,
3-O-[N-(butyl)carbamoyl]-1a,25-(OH)2-D3
(10b) and 3-O-[N-(2-hydroxyethyl)carbamoyl]-1a,25-
(OH)2-D3 (10c) were synthesized from A-ring precursors
6b and 6c, respectively.
Firstly, the preparation of N-(3-aminopropyl)carbamoyl
derivative 11 was carried out with the A-ring synthon in
an unprotected state, but only low yields were obtained.
The presence of a free amino group led to difficulties in
the isolation and purification of the products; for exam-
ple, it was necessary to use aqueous ammonia for puri-
fication by flash chromatography. Accordingly, the
terminal amino group in ring A was protected as the
tert-butyloxycarbonyl (Boc) derivative to afford deriva-
tive 6d. Excellent yields were obtained in the coupling
reaction between this A-ring fragment and the enol tri-
flate 7. Subsequent catalytic hydrogenation, isomeriza-
tion, and deprotection of the amino group with HCl in
ethanol afforded 3-O-[N-(3-aminopropyl)carbamoyl]-
1a,25-(OH)2-D3 (11), which was isolated as its hydro-
chloride salt.
The synthesis of 3-O-carbamoyl-1a,25-(OH)2-D3 (10a)
(Scheme 2) starts with the coupling of A-ring precursor
6a and vinyl triflate 7, prepared according to the pub-
lished procedure,15 in the presence of catalytic amount
of bis(triphenylphosphine)palladium(II) acetate–cop-
per(I) iodide. The protected dienyne was unstable, and
for this reason desilylation with tetrabutylammonium
fluoride (TBAF) was performed immediately to afford
dienyne 8a in 90% yield (for coupling and desilylation
steps).
For the synthesis of 3-O-[N-(carboxymethyl)carb-
amoyl]-1a,25-(OH)2-D3 sodium salt (10e) a larger
amount of Pd(PPh3)2(OAc)2 and CuI was needed to im-
prove the yield of the cross-coupling reaction of 7 and
6e. Moreover, the thermolysis process was performed
at lower temperature (65ꢁC) and in less reaction time
(3h).
Catalytic hydrogenation of diol 8a in methanol, in
the presence of Lindlar catalyst and quinoline poison,

´
V. Gotor-Fernandez et al. / Bioorg. Med. Chem. 12 (2004) 5443–5451
5445
OH
OTMS
a, b
OH
OH
OH
H
H
H
TfO
O
7
d
c
H
3
R
+
N
H
O
O
O
3
9a-e
3
R
R
6a-e
N
H
N
H
O
OH
O
OH
8a-e
10a-e
3
a, R = H
e (f or10d)
3
b, R = Bu
3
11, R = (CH ) NH /HCl
2 3
2
3
c, R = (CH ) OH
2 2
3
d, R = (CH ) NHBoc
2 3
3
e, R = CH CO Na
2
2
Scheme 2. Reagents and conditions: (a) Pd(PPh3)(OAc)2, CuI, Et2NH, DMF, rt, 1h; (b) Bu4NF, THF, rt, 12h (90% for 8a, 71% for 8b, 90% for 8c,
96% for 8d, 61% for 8e, two steps); (c) H2, Lindlar cat., quinoline, MeOH, rt, 1h; (d) acetone, 80ꢁC, 4h (65ꢁC, 3h for 9e) (62% for 10a, 54% for 10b,
71% for 10c, 54% for 10d, 62% for 10e, two steps); (e) HCl(g), EtOH, rt, 30 min (69%).
3. Biological evaluation
could inhibit the cell proliferation for 80% at this con-
centration (Table 1, Fig. 2A). The other analogues
10b, 10e, and 11 demonstrated no effects on the MCF-
7 cell proliferation. All the evaluated analogues inhib-
ited the proliferation of keratinocytes but all were less
potent (1.3–11-fold) than the parent compound (Table
1, Fig. 2B).
Analogues 10a–c, 10e, and 11 were evaluated in vitro in
terms of their ability to bind to the calf thymus vitamin
D receptor in comparison to the natural hormone and to
inhibit the cell proliferation (MCF-7, keratinocytes) or
to induce cell differentiation (HL 60) (Table 1). The rel-
ative affinity of the analogues was calculated from their
concentration required for 50% displacement of
[3H]1a,25-(OH)2-D3 from the receptor protein com-
pared with the activity of 1a,25-(OH)2-D3 (assigned a
value of 100 by definition). In this assay, all the
analogues were less effective than 1a,25-(OH)2-D3 for
binding to the VDR. As shown in Table 1, the N-(2-hy-
droxyethyl)carbamoyl derivative 10c was at least three
times more potent than the other analogues.
Differentiation of HL 60 cells (ATCC) was measured by
the nitro blue tetrazolium reduction assay after a 72h
incubation period in presence of 1a,25-(OH)2-D3, ana-
logues or vehicle.16 Again all the analogues were less ac-
tive than 1a,25-(OH)2-D3 (Table 1). The analogues 10a
and 10e induced only differentiation at 10À6 M of,
respectively, 50% and 30% whereas nearly 100% of the
HL 60 cells were differentiated after incubation with
10À6 M 1a,25-(OH)2-D3 (Fig. 2C).
As a measure of cell proliferation, [3H]-thymidine incor-
poration of MCF-7 and keratinocytes was determined
after a 72h incubation period with various concentra-
tions of 1a,25-(OH)2-D3, analogues or vehicle (arachis
oil). The most active analogue to inhibit MCF-7 cell
proliferation was the carbamate analogue 10a, however
this compound was 10 times less potent than 1a,25-
(OH)2-D3. The analogue 10c inhibited the MCF-7 cell
proliferation only at the highest concentration of
10À6 M and was not able to inhibit the cell proliferation
for 50% but only for 30% whereas 1a,25-(OH)2-D3
Besides the in vitro screening, the in vivo calcemic effects
of the carbamate analogues were evaluated in mice. All
the analogues were much less calcemic than 1a,25-
(OH)2-D3 even at 10–100-fold higher doses (Fig. 3A
and B) than 1a,25-(OH)2-D3 (0.1lg/kg/d).
4. Conclusions
In summary, we synthesized several carbamate deriva-
tives of 1a,25-(OH)2-D3 (A-ring modified analogues)
through a chemoenzymatic process based in the selective
functionalization catalyzed by CAL-B of the C-5 hydr-
oxyl group in A-ring enyne 3. The versatility of the route
allows the introduction of several functional groups at
the end of the alkyl chain of the carbamate moiety such
as alcohol, amino, or acid. The results of VDR binding
affinity indicated that these analogues exhibited less po-
tency than 1a,25-(OH)2-D3. The most potent analogue
to inhibit the cell proliferation of MCF-7 cells or kera-
tinocytes or to stimulate the HL 60 cell differentiation
is the analogue 3-O-carbamoyl-1a,25-(OH)2-D3 (10a)
however this compound is still less potent than the par-
ent compound 1a,25-(OH)2-D3 but has lower calcemic
effects in vivo.
Table 1. Biological activity of carbamate analogues of 1a,25-(OH)2-D3
Compound
VDR
(%)
MCF-7
(%)
Keratinocytes
(%)
HL 60
(%)
1a,25-(OH)2-D3
100
<1
<1
3
100
10
0
100
75
55
65
20
9
100
5
10a
10b
10c
10e
11
0
4
0
<1
<1
0
2
0
0
Summary of the in vitro effects of carbamate analogues of 1a,25-
(OH)2-D3 on receptor binding (VDR), HL 60 differentiation, MCF-7,
and keratinocyte proliferation. The in vitro effect is expressed as per-
centage activity (at EC50 or EC30 if analogues do not reach EC50) in
comparison with 1a,25-(OH)2-D3 (= 100% activity).

´
V. Gotor-Fernandez et al. / Bioorg. Med. Chem. 12 (2004) 5443–5451
5446
120
100
80
60
40
20
0
120
100
80
60
40
20
0
10-10
10-9
10-8
10-7
10-6
10-10 10-9
10-8
10-7
10-6
10-5
CONCENTRATION (M)
(A)
CONCENTRATION (M)
(B)
120
100
80
60
40
20
0
10-9
10-8
10-7
10-6
(C)
CONCENTRATION (M)
Figure 2. Prodifferentiating and antiproliferating effects of carbamate analogues on: (A) MCF-7; (B) keratinocytes; and (C) HL 60 cells. 1a,25-
(OH)2-D3 (d); 10a (s); 10b (Ä); 10c (h); 10e (m); 11 (n).
5. Experimental section
5.2. Synthesis of carbamates 6a–e
5.1. General spectroscopic and experimental data
Imidazole (54mg, 0.793mmol), DMAP (3.9mg,
0.032mmol), and tert-butyldimethylsilyl chloride
(96mg, 0.634mmol) were added to a stirred solution of
carbamates 512 (0.317mmol) in CH2Cl2 (2.5mL) under
nitrogen atmosphere. In case of 5c, which possesses
an extra hydroxyl group at the end of the alkyl chain,
double amount of reagents were used. The reaction mix-
ture was stirred at room temperature for 12h. Then, a
saturated solution of NH4Cl was added, and the aque-
ous phase was extracted three times with CH2Cl2. Sol-
vents were evaporated under reduce pressure, and the
residue was purified by flash chromatographic column
(20% EtOAc/hexane for 6a, 5% EtOAc/hexane for 6b
and 6c, 1% NH3(aq)/MeOH for silyl ether precursor of
6d, 60% EtOAc/MeOH for 6e). To a solution of silyl
ether precursor of 6d (39mg, 0.106mmol) in CHCl3
(2mL), were added Boc2O (26mg, 0.117mmol) and
0.1mL of an aqueous saturated solution of NaHCO3.
The reaction mixture was stirred for 3h at room temper-
ature. After this time, H2O was added and the aqueous
layer was extracted three times with CHCl3. Solvents
were evaporated, and the residue was purified by flash
Melting points were taken on samples in open capil-
lary tubes and are uncorrected. IR spectra were recorded
on a Infrared Fourier Transform spectrophotometer
using NaCl plates or KBr pellets. Flash chromatog-
raphy was performed using silica gel 60 (230–400 mesh).
1H, 13C NMR, and DEPT were obtained using
AC-200 (1H, 200.13MHz and 13C, 50.3MHz), AC-300
(1H, 300.13MHz, and 13C, 75.5MHz) or DPX 300
(1H, 300.13MHz and 13C, 75.5MHz) spectrometers for
routine experiments. AMX-400 spectrometer operating
1
at 400.13 and 100.61MHz for H and 13C, respectively,
1
was used for the acquisition of H–13C heteronuclear
correlation experiments. The chemical shifts are given
in delta (d) values and the coupling constants (J) in
Hertz (Hz). EI (70eV), FAB+ (nitrobenzyl alcohol as
matrix), and ES+ were used to record mass spectra
(MS). EI was used to record HRMS Microanalyses were
performed on a Perkin–Elmer model 2400 instrument.
HPLC was performed using UV detector and a Spheri-
sorb W, 5lm silica gel column, 250 · 10mm.

´
V. Gotor-Fernandez et al. / Bioorg. Med. Chem. 12 (2004) 5443–5451
5447
3
H14, JHH 7.4Hz), 1.34 (m, 2H, H13), 1.46 (m, 2H,
20
16
12
8
1,25(OH)2D3
H12), 1.87 (m, 1H, H4), 1.94 (s, 3H, H9), 1.96 (m, 1H,
H4), 2.19 (d, 1H, H6, JHH 17.6Hz), 2.59 (d, 1H, H6,
10a
10b
10c
10e
11
2
2JHH 17.6Hz), 3.05 (s, 1H, H8), 3.15 (apparent q, 2H,
3
H11, JHH 6.5Hz), 4.24 (m, 1H, H3), and 5.06 (m, 1H,
H5); 13C NMR (CDCl3, 100.6MHz): d À4.9 (MeSi),
À4.4 (MeSi), 13.7 (C14), 18.0 (Me3CSi), 18.5 (C9), 19.8
(C13), 25.7 (Me3CSi), 32.0 (C12), 35.2 (C6), 36.8 (C4),
40.6 (C11), 67.3 (C5), 68.4 (C3), 79.8 (C8), 83.3 (C7),
112.9 (C1), 144.2 (C2), and 155.8 (C10); MS (FAB+, m/
z): 388 [(M+Na)+, 30%], 364 [(M À H)+, 3], 308 (12),
234 (38), and 73 (100); Anal Calcd (%) for C20H35NO3-
Si: C, 65.71; H, 9.66; N, 3.83. Found: C, 65.9; H, 9.7; N,
3.8.
4
0
vehicle 0.1 1.0 10.0 1.0 10.0 1.0 10.0 1.0 10.0 1.0 10.0
Dose (µg/kg/day)
(A)
0.6
0.4
0.2
0.0
5.2.3. (3S,5R)-3-[(tert-Butyldimethylsilyl)oxy]-5-[N-[[2-
(tert-butyldimethyl-silyl)oxyethyl]-carbamoyl]oxy]-1-eth-
ynyl-2-methylcyclohex-1-ene (6c). Rf (5% EtOAc/hex):
0.2; Colorless oil; IR (NaCl): m 3361, 3314, 2953, 2930,
2857, 2094, 1722, 1512, 1471, 1360, 1072, and
1,25(OH)2D3
10a
10b
10c
10e
11
1
835cmÀ1; H NMR (CDCl3, 200MHz): d 0.05 (s, 6H,
2MeSi), 0.09 (s, 6H, 2MeSi), 0.88 (s, 9H, Me3CSi), 0.89
(s, 9H, Me3CSi), 1.87 (m, 2H, H4), 1.93 (s, 3H, H9),
2
2.17 (d, 1H, H6, JHH 16.0Hz), 2.59 (d, 1H, H6, JHH
3
3
17.2Hz), 3.04 (s, 1H, H8), 3.27 (q, 2H, H11, JHH
3
10.4Hz), 3.66 (t, 2H, H12, JHH 5.1Hz), 4.23 (t, 1H,
3
H3, JHH 5.3Hz), and 5.00 (m, 1H, H5); 13C NMR
vehicle 0.1 1.0 10.0 1.0 10.0 1.0 10.0 1.0 10.0 1.0 10.0
Dose (µg/kg/day)
(B)
(CDCl3, 75.5MHz): d À5.4 (Me2Si), À4.9 (MeSi), À4.4
(MeSi), 17.9 (Me3CSi), 18.2 (Me3CSi), 18.6 (C9), 25.7
(Me3CSi), 25.8 (Me3CSi), 35.2 (C6), 36.8 (C4), 43.1
(C11), 62.0 (C12), 67.3 (C5), 68.5 (C3), 79.8 (C8), 83.2
(C7), 112.9 (C1), 144.0 (C2), and 155.8 (C10); MS (EI,
Figure 3. In vivo biological activity of carbamate analogues deter-
mined by measuring serum and urine calcium levels in mice after
intraperitoneally injections after 7 consecutive days. Mice were injected
with vehicle (arachis oil), 1a,25-(OH)2-D3 (0.1lg/kg/d) or analogues (1
or 10lg/kg/d).
t
m/z): 410 [(M À Bu)+, 4%], 249 (12), 248 (13), 162 (40),
117 (20), 115 (24), 100 (17), and 75 (100); HRMS (m/z)
t
Calcd for C20H36NO4Si2 (M À Bu): 410.2183. Found:
chromatography column (20% EtOAc/hexane) to afford
6d. Yields are given in Scheme 1.
410.2176; Anal Calcd (%) for C24H45NO4Si2: C, 61.62;
H, 9.47; N, 2.99. Found: C, 61.8; H, 9.4; N, 3.1.
5.2.1. (3S,5R)-3-[(tert-Butyldimethylsilyl)oxy]-5-(carba-
moyloxy)-1-ethynyl-2-methylcyclohex-1-ene (6a). Rf
(20% EtOAc/hex): 0.3; Mp: 114–116ꢁC; IR (KBr): m
3456, 3282, 2955, 2931, 2859, 2095, 1696, 1607, 1407,
5.2.4. (3S,5R)-5-[N1-[3-(N2-Boc-aminopropyl)carbamo-
yl]oxy]-3-[(tert-butyldimethylsilyl)oxy]-1-ethynyl-2-meth-
ylcyclohex-1-ene (6d). Rf (25% EtOAc/hex): 0.2;
Colorless oil; IR (NaCl): m 3347, 3311, 2953, 2931,
1363, 1075, and 837cmÀ1
;
1H NMR (MeOH-d4,
2858, 2094, 1696, and 1516cmÀ1 1H NMR (CDCl3,
;
300MHz): d 0.32 (s, 6H, 2MeSi), 1.11 (s, 9H, Me3CSi),
2.07 (m, 1H, H4), 2.10 (s, 3H, H9), 2.17 (m, 1H, H4), 2.33
200MHz): d 0.09 (s, 6H, 2MeSi), 0.88 (s, 9H, Me3CSi),
1.42 (s, 9H, Me3C), 1.90 (m, 2H, H4), 1.93 (s, 3H, H9),
2.16 (d, 1H, H6, JHH 5.9Hz), 2.58 (dd, 1H, H6, JHH
2
(d, 1H, H6, JHH 17.3Hz), 2.72 (d, 1H, H6, JHH
2
3
2
3
3
17.2Hz), 3.63 (s, 1H, H8), 4.51 (t, 1H, H3, JHH
16.7, JHH 2.8Hz), 3.04 (s, 1H, H8), 3.18 (m, 4H,
4.9Hz), and 5.15 (m, 1H, H5); 13C NMR (MeOH-d4,
75.5MHz): d À4.1 (MeSi), À3.7 (MeSi), 19.4 (C9), 19.6
(Me3CSi), 26.8 (Me3CSi), 37.0 (C6), 38.6 (C4), 68.8
(C5), 70.6 (C3), 82.3 (C8), 84.4 (C7), 115.1 (C1), 144.9
2H11+2H13), 4.22 (m, 1H, H3), 4.88 (s, 2H, 2NH), and
5.05 (m, 1H, H5); 13C NMR (CDCl3, 75.5MHz): d
À4.9 (MeSi), À4.5 (MeSi), 17.9 (Me3CSi), 18.5 (C9),
25.7 (Me3CSi), 28.3 (Me3C), 30.6 (C12), 35.2 (C6), 36.8
(C4), 37.1 (C13), 37.50 (C11), 67.4 (C5), 68.5 (C3), 79.2
(Me3C), 79.8 (C8), 83.2 (C7), 112.9 (C1), 144.0 (C2),
156.2 (C10), and 156.3 (C14); MS (EI, m/z): 409
t
(C2), and 159.8 (C10); MS (EI, m/z): 252 [(M À Bu)+,
100%], 249 (21), 248 (62), 234 (16), and 233 (56); HRMS
(m/z) Calcd for C16H27NO3Si: 309.1760. Found:
309.1753; Anal Calcd (%) for C16H27NO3Si: C, 61.75;
H, 8.52; N, 4.53. Found: C, 62.1; H, 8.8; N, 4.5.
t
[(M À Bu)+, 20%], 335 (2), 309 (5), 249 (16), 248 (62),
115 (35), and 75 (100); HRMS (m/z) Calcd for
t
C20H33N2O5Si (M À Bu): 409.2159. Found: 409.2163;
5.2.2. (3S,5R)-5-[N-(Butylcarbamoyl)oxy]-3-[(tert-but-
yldimethylsilyl)oxy]-1-ethynyl-2-methylcyclohex-1-ene
(6b). Rf (5% EtOAc/hex): 0.2; Mp: 43–45ꢁC; IR (NaCl):
m 3314, 2952, 2932, 2859, 2095, 1696, 1530, 1464, 1363,
Anal Calcd (%) for C24H42N2O5Si: C, 61.76; H, 9.08;
N, 6.01. Found: C, 61.8; H, 9.2; N, 6.1.
5.2.5. (3S,5R)-3-[(tert-Butyldimethylsilyl)oxy]-5-[N-
[(carboxymethyl)carbamoyl]oxy]-1-ethynyl-2-methylcyclo-
hex-1-ene sodium salt (6e). Rf (70% EtOAc/MeOH): 0.2;
1
1071, and 836cmÀ1; H NMR (CDCl3, 400.13MHz): d
0.10 (s, 6H, 2MeSi), 0.90 (s, 9H, Me3CSi), 0.92 (t, 3H,

´
V. Gotor-Fernandez et al. / Bioorg. Med. Chem. 12 (2004) 5443–5451
5448
Mp: 148–150ꢁC; IR (KBr): m 3420, 3313, 2957, 2931,
2858, 2096, 1701, 1595, 1406, 1362, 1257, 1073,
5.3.2. 3-O-(N-Butylcarbamoyl)-6,7-dehydro-1a,25-dihyd-
roxyvitamin D3 (8b). Rf (40% EtOAc/hex): 0.1; Mp:
74–76ꢁC; IR (KBr): m 3348, 2964, 2872, 2187, 1703,
1
and 837cmÀ1; H NMR (MeOH-d4, 300MHz): d 0.32
(s, 6H, 2MeSi), 1.11 (s, 9H, Me3CSi), 2.10 (m, 2H,
1535, 1466, 1379, and 1253cmÀ1 1H NMR (CDCl3,
;
2
H4), 2.11 (s, 3H, H9), 2.36 (dd, 1H, H6, JHH 16.4,
200MHz): d 0.70 (s, 3H, H18), 0.92 (m, 6H, 3H21+
3H32), 1.21 (s, 6H, 3H26+3H27), 1.11–2.70 (m, 25H,
2
3
3JHH 6.1Hz), 2.74 (dd, 1H, H6, JHH 17.1, JHH
6.0Hz), 3.64 (s, 1H, H8), 3.88 (s, 2H, H11), 4.51
(m, 1H, H3), and 5.17 (m, 1H, H5); 13C NMR
(MeOH-d4, 75.5MHz): d À4.2 (MeSi), À3.7 (MeSi),
19.4 (Me3CSi), 19.5 (C9), 26.8 (Me3CSi), 37.1 (C6),
38.7 (C4), 45.8 (C11), 69.1 (C5), 70.6 (C3), 82.3 (C8),
84.5 (C7), 115.1 (C1), 144.8 (C2), 158.8 (C10), and 178.0
(C12); MS (ES+, m/z): 413 [(MH+Na)+, 100%] and 390
[(MH)+, 12]; Anal Calcd (%) for C18H28NNaO5Si:
C, 55.50; H, 7.25; N, 3.6. Found: C, 55.8; H, 7.1;
N, 3.3.
2H2+2H4+2H11+2H12+H14+2H15+2H16+H17+H20
+
2H22+2H23+2H24+2H30+2H31), 1.99 (s, 3H, H19), 3.18
(m, 2H, H29), 4.24 (m, 1H, H1), 4.63 (m, 1H, NH),
5.03 (m, 1H, H3), and 5.98 (m, 1H, H9); 13C NMR
(CDCl3, 75.5MHz): d 10.9, 13.6, 18.4, 18.6, 19.8, 20.6,
20.7, 24.0, 25.1, 27.9, 29.0, 29.2, 31.9, 35.8, 36.0, 36.2,
36.7, 40.5, 41.7, 44.2, 49.9, 54.6, 67.0, 68.4, 71.0, 87.3,
93.1, 115.0, 122.3, 133.5, 140.1, and 155.9; MS (FAB+,
m/z): 536 [(M+Na)+, 97%], 496 [(M À OH)+, 10], 413
(12), 396 (90), 379 (30), and 361 (42); Anal Calcd (%)
for C32H51NO4: C, 74.80; H, 10.01; N, 2.73. Found:
C, 74.6; H, 10.1; N, 2.9.
5.3. Synthesis of dienynes 8a–e
5.3.3. 6,7-Dehydro-3-O-[N-(2-hydroxyethyl)carbamoyl]-
1a,25-dihydroxyvitamin D3 (8c). Rf (20% PrOH/hex):
CuI (1.7mg, 0.009mmol), Pd(PPh3)2(OAc)2 (2.0mg,
0.003mmol), and Et2NH (0.55mL) were added to a stir-
i
0.1; Mp: 129–131ꢁC; IR (KBr): m 3435, 3202, 2957,
red solution of
6
(0.097mmol) and
7
(43mg,
2872, 2186, 1702, 1677, and 1355cmÀ1 1H NMR
;
0.088mmol) in DMF (0.55mL) under nitrogen atmos-
phere. In order to improve the yield in the coupling reac-
tion of 8e, 0.2equivof CuI and 0.15equivof
Pd(PPh3)2(OAc)2 were used. After 1h at room tempera-
ture, the resulting mixture was diluted with H2O, and
the aqueous phase was extracted three times with
Et2O. The organic layer was dried (Na2SO4) and evapo-
rated to dryness to give a crude, which was sufficiently
pure for the next step. TBAF (0.44mL, 0.441mmol,
1.0M in THF) was added dropwise to a solution of this
crude in THF (3mL) at 0ꢁC and the reaction was stirred
for 12h at room temperature. THF was evaporated and
the residue was poured into water/EtOAc. The aqueous
layer was extracted with EtOAc, solvents were evapo-
rated, and the residue was purified by flash chromatog-
(CDCl3, 200MHz): d 0.67 (s, 3H, H18), 0.93 (d, 3H,
H21, JHH 6.2Hz), 1.24 (s, 6H, 3H26+3H27), 1.00–2.30
3
(m, 21H, 2H2+2H4+2H11+2H12+H14+2H15+2H16+
H17+H20+2H22+2H23+2H24), 1.96 (s, 3H, H19), 2.57
(d, 1H, H4, JHH 13.6Hz), 3.28 (m, 2H, H29), 3.65 (m,
2
2H, H30), 4.19 (m, 1H, H1), 4.99 (m, 1H, H3), 5.46 (m,
3
1H, NH), and 5.94 (d, 1H, H9, JHH 2.6Hz); 13C
NMR (CDCl3, 75.5MHz): d 11.0, 18.5, 18.6, 20.8,
24.1, 25.1, 25.2, 27.9, 29.0, 29.2, 35.8, 36.0, 36.3, 36.7,
41.7, 43.2, 44.2, 49.9, 54.6, 61.7, 67.4, 68.4, 71.1, 87.2,
96.3, 115.1, 122.3, 133.7, 140.0, and 156.7; MS (ES+,
m/z): 524 [(M+Na)+, 100%] and 540 [(M+K)+, 43]; Anal
Calcd (%) for C30H47NO5: C, 71.82; H, 9.44; N, 2.79.
Found: 71.6; H, 9.5; N, 2.8.
i
raphy column (20% PrOH/hexane for 8a, 40% EtOAc/
hexane for 8b, 20% PrOH/hexane for 8c, 65% EtOAc/
5.3.4. 3-O-[N1-(3-N2-Boc-aminopropyl)carbamoyl]-6,7-
dehydro-1a,25-dihydroxyvitamin D3 (8d). Rf (65% EtO-
Ac/hex): 0.3; Mp: 68–70ꢁC; IR (KBr): m 3405, 2964,
i
hexane for 8d, 50% EtOAc/MeOH for 8e). Yields are
given in Scheme 2.
2880, 2191, 1690, 1530, 1451, and 1366cmÀ1
;
1H
NMR (CDCl3, 200MHz): d 0.68 (s, 3H, H18), 0.93 (d,
3H, H21, JHH 6.4Hz), 1.20 (s, 6H, 3H26+3H27), 1.42
(s, 9H, Me3C), 1.98 (s, 3H, H19), 2.58 (dd, 1H, H4,
3
5.3.1. 3-O-Carbamoyl-6,7-dehydro-1a,25-dihydroxyvita-
i
min D3 (8a). Rf (20% PrOH/hex): 0.3; Mp: 138–140ꢁC;
2JHH 16.7, JHH 3.3Hz), 1.00–2.20 (m, 22H, 2H2+H4+
3
IR (KBr): m 3387, 2951, 2872, 2187, 1700, 1541, 1458,
1
1377, and 1269cmÀ1; H NMR (CDCl3, 200MHz): d
0.68 (s, 3H, H18), 0.94 (d, 3H, H21, JHH 6.4Hz), 1.20
2H11+2H12+H14+2H15+2H16+H17+H20+2H22+2H23+
2H24+2H30), 3.16 (m, 4H, H29+2H31), 4.23 (m, 1H, H1),
4.94 (m, 2H, 2NH), 5.22 (m, 1H, H3), and 5.95 (d, 1H,
3
(s, 6H, 3H26+3H27), 1.00–2.65 (m, 21H, 2H2+2H4+
2H11+2H12+H14+2H15+2H16+H17+H20+2H22+2H23+
2H24), 1.99 (s, 3H, H19), 4.21 (t, 1H, H1, JHH 4.6Hz),
3
H9, JHH 3.1Hz); 13C NMR (CDCl3, 75.5MHz): d
3
11.0, 18.4, 18.6, 20.8, 24.1, 25.1, 27.9, 28.3 (3C), 29.1,
29.3, 30.5, 35.8, 36.1, 36.3, 36.7, 37.1, 37.5, 41.8, 44.3,
49.9, 54.6, 67.1, 68.5, 71.0, 79.2, 87.2, 93.3, 115.3,
122.3, 133.7, 139.9, and 156.3 (2C); MS (ES+, m/z):
637 [(M+Na)+, 100%] and 638 [(MH+Na)+, 47]; Anal
Calcd (%) for C36H58N2O6: C, 70.31; H, 9.51; N, 4.56.
Found: C, 70.3; H, 9.5; N, 4.6.
4.79 (s, 4H, 2NH+2OH), 5.00 (m, 1H, H3), and 5.96
(m, 1H, H9); 13C NMR (CDCl3, 75.5MHz): d 11.0,
18.5, 18.6, 20.7, 24.1, 25.1, 27.9, 29.1, 29.3, 35.6, 25.8,
36.0, 36.2, 36.5, 41.7, 44.2, 49.9, 54.6, 67.5, 68.5, 71.1,
87.1, 93.4, 115.2, 122.2, 133.8, 139.8, and 156.4; 13C
NMR (CDCl3, 75.5MHz): d 11.0, 18.5, 18.6, 20.7,
24.1, 25.1, 27.9, 29.1, 29.3, 35.6, 25.8, 36.0, 36.2, 36.5,
41.7, 44.2, 49.9, 54.6, 67.5, 68.5, 71.1, 87.1, 93.4, 115.2,
122.2, 133.8, 139.8, and 156.4; MS (ES+, m/z): 480
[(M+Na)+, 100%] and 413 (40); Anal Calcd (%) for
C28H43NO4: 73.47; H, 9.48; N, 3.06. Found: C, 73.3;
H, 9.5; N, 3.1.
5.3.5. 3-O-[N-(Carboxymethyl)carbamoyl]-6,7-dehydro-
1a,25-dihydroxyvitamin D3 sodium salt (8e). Rf (60% Et-
OAc/MeOH): 0.3; Mp: 130–132ꢁC; IR (KBr): m 3420,
2963, 2940, 2878, 2185, 1701, 1686, 1596, 1406, and
1
1279cmÀ1; H NMR (MeOH-d4, 300.13MHz): d 0.92

´
V. Gotor-Fernandez et al. / Bioorg. Med. Chem. 12 (2004) 5443–5451
5449
(s, 3H, H18), 1.18 (d, 3H, H21, 3JHH 6.6Hz), 1.37 (s, 6H,
3H26+3H27), 1.00–2.60 (m, 20H, 2H2+H4+2H11+2H12+
H14+2H15+2H16+H17+H20+2H22+2H23+2H24), 2.14 (s,
3H26+3H27), 1.20–2.90 (m, 29H, 2H2+H4+2H9+2H11+
2H12+H14+2H15+2H16+H17+H20+2H22+2H23+2H24),
3.16 (m, 2H, H29), 4.36 (m, 1H, H1), 4.65 (s, 1H, NH),
5.00 (s, 1H, H19), 5.10 (m, 1H, H3), 5.35 (s, 1H, H19),
2
3H, H19), 2.74 (d, 1H, H4, JHH 13.6Hz), 3.86 (s, 2H,
H29), 4.41 (m, 1H, H1), 5.20 (m, 1H, H3), and 6.10 (d,
3
6.03 (d, 1H, H7, JHH 11.3Hz), and 6.32 (d, 1H, H6,
3
1H, H9, JHH 3.1Hz); 13C NMR (MeOH-d4,
3JHH 11.3Hz); 13C NMR (CDCl3, 75.5MHz): d 11.9,
13.7, 18.8, 19.8, 20.7, 22.2, 23.6, 27.6, 29.1 (2C), 29.3,
31.9, 36.0, 36.3, 40.2, 40.4, 40.6, 41.8, 44.3, 45.9, 56.2,
56.4, 69.8, 70.2, 71.1, 111.0, 117.0, 124.3, 133.0, 143.0,
147.7, and 155.9; MS (ES+, m/z): 538 [(M+Na)+,
100%]; HRMS (m/z) Calcd for C32H52NO3 (M À OH):
498.3947. Found: 498.3923; Anal Calcd (%) for
C32H53NO4: C, 74.51; H, 10.36; N, 2.72. Found: C,
74.4; H, 10.5; N, 2.6.
100.6MHz): d 11.9, 19.3, 19.7, 22.4, 25.9, 26.6, 29.6
(2C), 29.8, 37.5, 37.7, 38.0, 38.2, 38.4, 43.5, 45.8, 46.1,
51.9, 56.6, 69.1, 69.5, 72.0, 89.0, 94.5, 116.7, 124.4,
134.6, 141.8, 158.8, and 177.7; MS (ES+, m/z): 560
[(M+Na)+, 6%], 538 [(MH)+, 5], 413 (16), 242 (100);
Anal Calcd (%) for C30H44NNaO6: C, 67.00; H, 8.25;
N, 2.61. Found: C, 66.8; H, 8.4; N, 2.7.
5.4. Synthesis of C-3 carbamate analogues of 1a,25-
dihydroxyvitamin D3 10a–d
5.4.3. 3-O-[N-(2-Hydroxyethyl)carbamoyl]-1a,25-dihydr-
oxyvitamin D3 (10c). Rf (20% PrOH/hex): 0.2; Mp: 95–
i
A flask containing Lindlar catalyst (50mg, 0.024mmol,
pre-dried at 60ꢁC during 4h in vacuo) was exposed to
a positive pressure of hydrogen gas (balloon). A mixture
of dienyne 8 (0.039mmol) and quinoline (14lL,
0.024mmol, 0.17M in hexane) in deoxygenated MeOH
(2.3mL) was added and the reaction was stirred for 1h
at room temperature. The mixture was filtered on Celite
and concentrated to afford crude 9. 1H NMR analysis of
the latter material showed signals of the compound 9. A
solution of the crude previtamin 9 in acetone (3mL) was
placed in a screw-capped vial and heated for 4h in a
constant temperature bath set at 80ꢁC. For 9e, thermo-
lysis was performed at 65ꢁC during 3h. The residue was
concentrated under vacuum and purified by flash chro-
97ꢁC; IR (KBr): m 3419, 2946, 2872, 1700, 1538, 1376,
1
and 1262cmÀ1; H NMR (CDCl3, 300MHz): d 0.53 (s,
3H, H18), 0.92 (d, 3H, H21, JHH 6.3Hz), 1.20 (s, 6H,
3
3H26+3H27), 1.00–2.90 (m, 21H, 2H2+2H4+2H11+
2H12+H14+2H15+2H16+H17+H20+2H22+2H23+2H24),
3.31 (m, 2H, H30), 3.68 (m, 1H, H29), 4.35 (q, 1H, H1,
3JHH 8.5Hz), 5.00 (s, 1H, H19), 5.11 (m, 1H, H3), 5.24
3
(m, 1H, NH), 5.34 (s, 1H, H19), 6.02 (d, 1H, H7, JHH
3
10.9Hz), and 6.32 (d, 1H, H6, JHH 11.4Hz); 13C
NMR (CDCl3, 50.3MHz): d 11.9, 18.7, 20.7, 22.2,
23.5, 27.6, 29.1 (2C), 29.3, 36.0, 36.3, 40.1, 40.4, 41.7,
43.3, 44.3, 45.9, 56.3, 56.5, 62.3, 70.2, 70.4, 71.1, 111.3,
117.0, 124.5, 132.7, 143.1, 147.4, and 156.7; MS (ES+,
m/z): 526 [(M+Na)+, 100%], and 242 (42); Anal Calcd
(%) for C30H49NO5: C, 71.52; H, 9.81; N, 2.78. Found:
C, 71.7; H, 9.9; N, 2.5.
i
matography column (15% PrOH/hexane for 10a and
10c, 40% EtOAc/hexane for 10b, 60% EtOAc/hexane
i
for 10d, 50% PrOH/hexane for 10e). Further purifica-
tion by HPLC (Spherisorb W, 5lm silica gel column,
250 · 10mm, 4mL/min, 12% iPrOH/hexane for 10a
and 10c, 30% EtOAc/hexane for 10b and 10d, 50%
iPrOH/hexane for 10e).
5.4.4.
3-O-[N1-(3-N2-Boc-aminopropyl)carbamoyl]-1a,
25-dihydroxyvitamin D3 (10d). Rf (60% EtOAc/hex):
1
0.4; Colorless oil; H NMR (CDCl3, 200MHz): d 0.54
(s, 3H, H18), 0.93 (d, 3H, H21, JHH 6.3Hz), 1.21 (s,
3
6H, 3H26+3H27), 1.42 (s, 9H, Me3C), 1.00–2.90 (m,
25H,
5.4.1. 3-O-Carbamoyl-1a,25-dihydroxyvitamin D3 (10a).
2H2+2H4+2H9+2H11+2H12+H14+2H15+2H16+
i
Rf (15% PrOH/hex): 0.2; Mp: 98–100ꢁC; IR (KBr): m
3396, 2947, 2871, 1718, 1604, 1397, 1327, and
H17+H20+2H22+2H23+2H24+2H30), 3.17 (m, 4H,
2H29+2H31), 4.38 (m, 1H, H1), 4.87 (s, 2H, 2NH), 5.01
(s, 1H, H19), 5.07 (m, 1H, H3), 5.34 (s, 1H, H19), 6.03
1
1262cmÀ1; H NMR (CDCl3, 300MHz): d 0.54 (s, 3H,
H18), 0.93 (d, 3H, H21, JHH 6.2Hz), 1.21 (s, 6H,
3
3
3
(d, 1H, H7, JHH 12.0Hz), and 6.32 (d, 1H, H6, JHH
11.1Hz); 13C NMR (CDCl3, 75.5MHz): d 11.9, 18.7,
20.7, 22.2, 23.5, 27.6, 28.3 (3C), 29.0, 29.1, 29.3, 30.5,
36.0, 36.3, 37.1, 37.5, 40.1, 40.4, 41.8, 44.3, 45.9, 56.3,
56.4, 70.1, 70.2, 71.0, 79.2, 111.3, 117.0, 124.4, 132.8,
143.0, 147.4, and 156.3 (2C).
3H26+3H27), 1.00–2.70 (m, 22H, 2H2+H4+2H9+2H11+
2H12+H14+2H15+2H16+H17+H20+2H22+2H23+2H24),
2.81 (dd, 1H, H4, JHH 11.5, JHH 4.0Hz), 4.39 (c, 1H,
2
3
3
H1, JHH 8.8Hz), 4.63 (s, 4H, 2NH+2OH), 5.01 (s,
1H, H19), 5.10 (m, 1H, H3), 5.35 (s, 1H, H19), 6.02 (d,
3
3
1H, H7, JHH 11.6Hz), and 6.34 (d, 1H, H6, JHH
11.5Hz); 13C NMR (CDCl3, 75.5MHz): d 11.9, 18.7,
20.7, 22.2, 23.5, 27.6, 29.0, 29.1, 29.3, 36.0, 36.3, 39.9,
40.4, 41.6, 44.3, 45.9, 56.3, 56.4, 70.2, 70.4, 71.1, 111.4,
116.9, 124.5, 132.5, 143.2, 147.4, and 156.2; MS (ES+,
m/z): 482 [(M+Na)+, 100%]; Anal Calcd (%) for
C28H45NO4: C, 73.15; H, 9.87; N, 3.05. Found: C,
73.4; H, 9.7; N, 3.1.
5.4.5. 3-O-[N-(Carboxymethyl)carbamoyl]-1a,25-dihydr-
oxyvitamin D3 sodium salt (10e). Rf (80% EtOAc/
MeOH): 0.1; Mp: 140–142ꢁC; IR (KBr): m 3407, 2945,
2868, 1701, 1597, 1403, 1379, and 1279cmÀ1
;
1H
NMR (MeOH-d4, 200MHz): d 0.77 (s, 3H, H18), 1.16
(d, 3H, H21, JHH 6.0Hz), 1.36 (s, 6H, 3H26+3H27),
3
1.00–2.80 (m, 22H, 2H2+H4+2H9+2H11+2H12+H14+
2H152+2H16+H17+H20+2H22+2H23+2H24), 3.04 (d, 1H,
H4, JHH 11.4Hz), 3.89 (s, 2H, H29), 4.52 (m, 1H, H1),
5.12 (s, 1H, H19), 5.26 (m, 1H, H3), 5.55 (s, 1H, H19),
5.4.2. 3-O-(N-Butylcarbamoyl)-1a,25-dihydroxyvitamin
D3 (10b). Rf (40% EtOAc/hex): 0.2; Mp: 126–128ꢁC;
IR (KBr): m 3360, 2943, 2872, 1695, 1531, 1444, 1377,
3
6.26 (d, 1H, H7, JHH 10.8Hz), and 6.49 (d, 1H, H6,
1
and 1259cmÀ1; H NMR (CDCl3, 200MHz): d 0.54 (s,
3JHH 11.4Hz); 13C NMR (MeOH-d4, 75.5MHz): d
12.9, 19.9, 22.4, 23.9, 25.2, 29.2, 29.6, 29.8, 30.5, 38.0,
3
3H, H18), 0.92 (d, 3H, H21, JHH 6.1Hz), 1.20 (s, 6H,

´
V. Gotor-Fernandez et al. / Bioorg. Med. Chem. 12 (2004) 5443–5451
5450
41.7, 42.4, 43.3, 45.5, 45.8, 47.5, 52.6, 58.1, 58.5, 71.0,
72.0, 72.2, 111.8, 119.3, 125.4, 135.7, 143.4, 150.3,
158.8, and 177.1; MS (ES+, m/z): 540 [(MH)+, 43%],
and 413 (100); Anal Calcd (%) for C30H46NNaO6: C,
66.75; H, 8.60; N, 2.60. Found: C, 66.9; H, 8.8; N, 2.5.
oil) for 7 consecutive days to groups of six mice. Serum
and urinary calcium were measured as calcemic param-
eters using a commercially available kit (Sigma Diagnos-
tics).
5.5. Synthesis of 3-O-[N-(aminopropyl)carbamoyl]-1a,25-
dihydroxyvitamin D3 hydrochloride (11)
Acknowledgements
This work has been supported by grants from MCYT
(Spain; Project PPQ-2001-2683) and the Principado de
Asturias (Spain; Project GE-EXP01-05). S.F. thanks
A solution of 10d (16.6mg, 0.027mmol) in EtOH (1mL)
was added to a solution of HCl/EtOH [12mL, generated
by bubbled HCl(g) in EtOH]. The reaction was stirred
for 0.5h, then the EtOH was evaporated and the residue
was washed with Et2O, affording 11 as a white solid
(69%). Mp: 150–152ꢁC; IR (KBr): m 3422, 2942, 2868,
´
MCYT for a personal grant (Ramon y Cajal Program).
We thank Solvay-Duphar (Wesps, The Netherlands) for
the generous gift of vitamin D3, which was used for
preparation of GrundmannÕs ketone.
1701, 1655, 1542, 1377, and 1263cmÀ1 1H NMR
;
(MeOH-d4, 200MHz): d 0.77 (s, 3H, H18), 1.16 (d, 3H,
H21, JHH 6.0Hz), 1.36 (s, 6H, 3H26+3H27), 1.00–3.00
3
References and notes
(m, 25H, 2H2+2H4+2H9+2H11+2H12+H14+2H15+
2H16+H17+H20+2H22+2H23+2H24+2H30),
3.10–3.50
(m, 4H, 2H29+2H31), 4.51 (m, 1H, H1), 5.12 (s, 1H,
H19), 5.26 (m, 1H, H3), 5.55 (s, 1H, H19), 6.24 (d, 1H,
1. (a) 1st International Conference on Chemistry and Biol-
ogy of Vitamin D Analogues, Steroids-Special Issue 2001,
66, 127–472; (b) Vitamin D Endocrine System: Structural
Biological Genetic and Clinical Aspects; Norman, A. W.,
Bouillon, R., Thomasset, M., Eds.; University of Califor-
nia Riverside: Riverside CA, 2000; (c) Vitamin D; Feld-
man, D., Glorieux, F. H., Pike, J. W., Eds.; Academic:
New York, 1997; (d) Ettinger, R. A.; DeLuca, H. F. Adv.
Drug Res. 1996, 28, 269–312; (e) Christakos, S.; Raval-
Pandya, M.; Wernyj, R. P.; Yang, W. Biochem. J. 1996,
316, 361–371; (f) Bouillon, R.; Okamura, W. H.; Norman,
A. W. Endocrine Rev. 1995, 16, 200–257.
3
2
H7, JHH 10.8Hz), and 6.48 (d, 1H, H6, JHH 11.4Hz);
13C NMR (MeOH-d4, 75.5MHz): d 12.8, 19.9, 22.4,
23.8, 25.2, 27.1, 29.2, 29.6, 29.8, 30.5, 37.9, 38.2, 38.4,
41.6, 42.4, 43.3, 45.8, 47.5, 58.1, 58.5, 71.0, 72.0, 72.4,
112.0, 119.2, 125.4, 131.8, 135.6, 150.2, and 159.8; MS
(ES+, m/z): 567 [(M À Cl)+, 100%] and 499 (28); Anal
Calcd (%) for C31H53ClN2O4: C, 67.35; H, 9.67; N,
5.07. Found: C, 67.5; H, 9.8; N, 4.7.
2. (a) Zhu, G.-D.; Okamura, W. H. Chem. Rev. 1995, 95,
1877–1952; (b) Dai, H.; Posner, G. H. Synthesis 1994,
1383–1398.
5.6. In vitro and in vivo biological evaluation
3. (a) Matassa, V. G.; Maduskuie, T. P.; Shapiro, H. S.;
Hesp, B.; Snyder, D. W.; Aharony, D.; Krell, R. D.;
Keith, R. A. J. Med. Chem. 1990, 33, 1781–1790; (b)
Firestone, R. A.; Pisano, J. M.; Falck, J. R.; McPhaul, M.
M.; Krieger, M. J. Med. Chem. 1984, 27, 1037–1043.
4. Webb, J. S.; Cosulich, D. B.; Mowat, J. H.; Patrick, J. B.;
Broschard, R. W.; Meyer, W. E.; Williams, R. P.; Wolf, C.
F.; Fulmor, W.; Pidacks, C.; Lancaster, J. E. J. Am. Chem.
Soc. 1962, 84, 3185–3187.
5.6.1. VDR binding assays. Vitamin D receptor binding
assays were performed with calf thymus cells. These
VDRs were saturated with [3H]-calcitriol. Then solu-
tions of vitamin D analogues were added whereby triti-
ated calcitriol was removed from the VDR. Affinity
toward the VDR was measured by counting the amount
of radioactive calcitriol that remains specifically bound
to the VDR.
5. Takita, Y.; Maeda, K.; Umezawa, H.; Omoto, S.; Ume-
zawa, S. J. Antibiot. 1969, 22, 237–239.
5.6.2. Cell proliferation assays. As a measure of cell pro-
liferation, [3H]-thymidine incorporation of breast cancer
MCF-7 (ATCC, Rockville, MD) and human keratino-
cytes were determined after a 72h incubation period
with various concentrations of 1a,25-(OH)2-D3, ana-
logues or vehicle as described previously.16
6. Moeller, H. J.; Hampel, H.; Hegerl, U.; Schmitt, W.;
Walter, K. Pharmacopsychiatry 1999, 32, 99–106.
7. Ghosh, A. K.; McKee, S. P.; Thompson, W. J.; Darke, P.
L.; Zugay, J. C. J. Org. Chem. 1993, 58, 1025–1029.
8. Alexander, J.; Cargill, R.; Michelson, S. R.; Schwam, H.
J. Med. Chem. 1988, 31, 318–322.
9. Ren, T.; Zhang, G.; Liu, F.; Liu, D. Bioorg. Med. Chem.
Lett. 2000, 10, 891–894.
5.6.3. Cell differentiation assays. Differentiation of
promyeolocytic HL 60 leukemia cells (ATCC) was
measured by the nitro blue tetrazolium reduction assay
after a 72h incubation period in presence of 1,25-
(OH)2-D3, analogues or vehicle.16
´
´
10. (a) Fernandez, S.; Gotor-Fernandez, V.; Ferrero, M.;
Gotor, V. Curr. Org. Chem. 2002, 6, 453–469; (b) Ferrero,
M.; Gotor, V. Chem. Rev. 2000, 100, 4319–4347; (c)
Ferrero, M.; Gotor, V. In Stereoselective Biocatalysis;
Patel, R. N., Ed.; Marcel Dekker: New York, 2000,
Chapter 20, pp 579–631.
5.6.4. In vivo calcemic activity. Eight weeks old, male,
NMRI mice were obtained from the Proefdierencentrum
of Leuven (Belgium) and fed with a vitamin D-replete
diet (1% calcium, 1% phosphate, and 2500units vitamin
D/kg; Hope Farms, Woerden, The Netherlands). The
calcemic effects of the analogues were tested by daily
injections intraperitoneally of 1a,25-(OH)2-D3 (0.1lg/
kg/d), analogues (1 or 10lg/kg/d), or vehicle (arachis
11. (a) Mascaren˜as, J. L.; Sarandeses, L. A.; Castedo, L.;
Mourin˜o, A. Tetrahedron 1991, 47, 3485–3498; (b)
Barrack, S. A.; Gibbs, R. A.; Okamura, W. H. J. Org.
Chem. 1988, 53, 1790–1796.
´
12. (a) Ferrero, M.; Fernandez, S.; Gotor, V. J. Org. Chem.
1997, 62, 4358–4363; Related papers: (b) Dıaz, M.; Gotor-
´
´
Fernandez, V.; Ferrero, M.; Fernandez, S.; Gotor, V.
J. Org. Chem. 2001, 66, 4227–4232; (c) Gotor-Fernandez,
´
´

´
V. Gotor-Fernandez et al. / Bioorg. Med. Chem. 12 (2004) 5443–5451
5451
´
V.; Ferrero, M.; Fernandez, S.; Gotor, V. J. Org. Chem.
1999, 64, 7504–7510.
different numbering, compare Figure 1 with Scheme 1.
Thus, carbons 1 and 3 in vitamin D structures correspond
with carbons 3 and 5 in the A-ring, respectively.
13. Compound 3 has been synthesized by Okamura from (S)-
(+)-carvone. (a) Okamura, W. H.; Aurrecoechea, J. M.;
Gibbs, R. A.; Norman, A. W. J. Org. Chem. 1989, 54,
4072–4083; (b) Aurrecoechea, J. M.; Okamura, W. H.
Tetrahedron Lett. 1987, 28, 4947–4950.
15. (a) Maynard, D. F.; Trankle, W. G.; Norman, A. W.;
Okamura, W. H. J. Med. Chem. 1994, 37, 2387–2393; (b)
´
Dıaz, M.; Ferrero, M.; Fernandez, S.; Gotor, V. J. Org.
Chem. 2000, 65, 5647–5652; (c) Also see Ref. 11.
´
14. Note that a (down) and b (up) have their usual definitions
for the structures of vitamin D3 and its analogues.
However the A-ring synthon 3 and its derivatives has
16. Verstuyf, A.; Verlinden, L.; Van Baelen, H.; Sabbe, K.;
DÕHalleweyn, C.; De Clercq, P.; Vandewalle, M.; Bouil-
lon, R. J. Bone Miner. Res. 1998, 13, 549–558.