Synthesis and anti-inflammatory properties of 1 α,25-dihydroxy-16- ene-20-cyclopropyl-24-oxo-vitamin d3, a hypocalcemic, stable metabolite of 1α,25-dihydroxy-16-ene-20-cyclopropylvitamin D3

Article abstract of DOI:10.1021/jm801365a

1α,25(OH)₂-16-ene-20-cyclopropyl-vitamin D₃ (13) is several fold more potent than the natural hormone 1α,25- dihydroxyvitamin D₃ (1) as an anti-inflammatory agent. Here, we have further analyzed the antiinflammatory proper

Full text of DOI:10.1021/jm801365a

2204
J. Med. Chem. 2009, 52, 2204–2213
Synthesis and Anti-inflammatory Properties of 1r,25-Dihydroxy-16-ene-20-cyclopropyl-24-
oxo-vitamin D3, a Hypocalcemic, Stable Metabolite of 1r,25-Dihydroxy-16-ene-20-cyclopropyl-
vitamin D3
Gilles Laverny,† Giuseppe Penna,† Milan Uskokovic,‡ Stanislaw Marczak,‡ Hubert Maehr,‡ Pawel Jankowski,‡
Caroline Ceailles,§ Paul Vouros,§ Brenden Smith,| Matthew Robinson,| G. Satyanarayana Reddy,|, and Luciano Adorini*,†
BioXell, 20132 Milan, Italy, BioXell Inc., Nutley, New Jersey 07110, The Barnett Institute, Department of Chemistry, Northeastern UniVersity,
Boston Massachusetts 02115, Epimer LLC, ProVidence, Rhode Island 02906, Department of Chemistry, Brown UniVersity, Box H,
ProVidence, Rhode Island 02912
ReceiVed October 28, 2008
1R,25(OH)2-16-ene-20-cyclopropyl-vitamin D3 (13) is several fold more potent than the natural hormone
1R,25-dihydroxyvitamin D3 (1) as an anti-inflammatory agent. Here, we have further analyzed the anti-
inflammatory properties of 13, confirming it as the most potent analogue tested within this family. We then
determined the structures of all the natural metabolites of 13, including the 24-oxo metabolite 14, and carried
out its synthesis. A comparison of 13 with 14 showed a similar induction of the primary VDR target genes
CYP24A1 and CAMP and comparable anti-inflammatory properties as revealed by a similar inhibition of
TNF-R, IL-12/23p40, IL-6, and IFN-γ production. Interestingly, 14 displays a 3-fold lower calcemic activity
in vivo compared to 13. Collectively, these findings indicate that the strong potency of 13 can be explained
by the accumulation of its stable 24-oxo metabolite, which shows immunoregulatory and anti-inflammatory
properties superimposable to those exerted by 13 itself.
Introduction
biological properties. Both classes of VDRa agonists have been
shown to decrease cell proliferation and promote cell dif-
ferentiation with a potency several orders of magnitude greater
than that of 1.14,16 The potency of 20-cyclopropyl-vitamin D3
analogues can be increased both in terms of their calcemic and
immunomodulatory activities by the addition of a 16-ene
moiety.14 In the present study, we have further characterized
the potency of this 16-ene-20-cyclopropyl family with respect
to their anti-inflammatory properties by studying the inhibition
of interferon-γ (IFN-γ) and tumor necrosis factor-R (TNF-R)
in two different in vitro models. On the basis of these
experiments, we have identified 1R,25(OH)2-16-ene-20-cyclo-
propyl-vitamin D3 (13) as the most potent anti-inflammatory
compound among all the members of the 16-ene-20-cyclopropyl
family tested.
Over a decade ago, we proposed that one of the mechanisms
responsible for the enhanced biological activities of 1R,25(OH)2-
16-ene-vitamin D3, when compared to 1, is the difference in
their target tissue metabolism.15,16 On the basis of our earlier
observationsrelatedtothetargettissuemetabolismof1R,25(OH)2-
16-ene-vitamin D3, we anticipated similarities between this
compound and 13 in their target tissue metabolism. Therefore,
we investigated the metabolism of 13 in rat osteosarcoma cells
(UMR-106) and found 13 to be metabolized in the same way
as 1R,25(OH)2-16-ene-vitamin D3.14 We noted that 13 is
metabolized in UMR-106 cells into a metabolite tentatively
identified as the 24-oxo derivative.14 Here, we unequivocally
identify the stable metabolite of 13 as 1R,25(OH)2-16-ene-20-
cyclopropyl-24-oxo-vitamin D3 (14). Chemical synthesis of 14
allowed a comparison of VDR-mediated activities and anti-
1R,25-dihydroxyvitamin D3 (1), the biologically active form
of vitamin D3, is a secosteroid hormone with a central role in
calcium and bone metabolism. 1 also regulates growth and
differentiation of many cell types and possesses exquisite
immunoregulatory and anti-inflammatory properties.1 The bio-
logical effects of 1 are mediated by the vitamin D receptor
(VDR), a member of the superfamily of nuclear receptors.2 VDR
functions as a ligand-activated transcription factor that binds
to specific vitamin D response elements in the promoter region
of its primary target genes, modulating their rate of expression.3
VDR agonists, which show benefit in several models of
autoimmune and chronic inflammatory diseases, are the most
used class of topical agents for the treatment of psoriasis, an
autoimmune disease of the skin,4 and are also recognized to
have potential therapeutic value in other clinical conditions.5-7
Because supraphysiologic concentrations of 1 and its analogues
are usually required to exert immunosuppressive and antipro-
liferative effects, more than 3000 vitamin D3 analogues with
several different structural modifications have been synthesized
during the past two decades.8-10 Some of these analogues, such
as elocalcitol (BXL-628), were found to have enhanced biologi-
cal activity with less calcemic liability compared to 1.11
Elocalcitol was recently proposed for the treatment of benign
prostatic hyperplasia12 and clinically tested with good efficacy
and excellent safety.13
Two classes of analogues with 16-ene or 20-cyclopropyl
modification have become prominent because of their unique
* To whom correspondence should be addressed. Phone: +39-075-
7921934. Fax: +39-075-7921934. Mobile: +39-348-0172983. E-mail:
LAdorini@interceptpharma.com. Address: Intercept Pharma, Via P. Togliatti
22 bis, 06073 Corciano (Perugia), Italy.
a Abbreviations: IFN-γ, interferon-γ; TNF-R, tumor necrosis factor-R;
CYP24A1, 25-hydroxyvitamin D-24-hydroxylase; CAMP, cathelicidin
antimicrobial peptide; UMR-106, rat osteosarcoma cells; IL, interleukin;
VDR, vitamin D receptor; CAOV, carcinoma ovarian; PBMC, peripheral
blood mononuclear cells; THP-1, human acute monocytic leukemia cell
line; LPS, lipopolysaccharide; MLR, mixed lymphocyte reaction; MTD,
maximal tolerated dose; TLR4, Toll like receptor 4; Th, helper T cells.
†
BioXell.
BioXell Inc.
‡
§
The Barnett Institute, Department of Chemistry, Northeastern University.
Epimer LLC.
|
Department of Chemistry, Brown University.
10.1021/jm801365a CCC: $40.75
2009 American Chemical Society
Published on Web 03/23/2009

1R,25-Dihydroxy-16-ene-20-cyclopropyl-24-oxo-Vitamin D3
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2205
Figure 1. Chemical structures of 1R,25(OH)2-vitamin D3 (1) and various 16-ene-20-cyclopropyl-vitamin D3 analogues used in this study.
Table 1. Inhibition of Pro-inflammatory Cytokines by 16-ene-20-cyclopropyl-vitamin D3 Analoguesa
MLR IFN-γ
IC50(pM)
LPS TNF-R
IC50(nM)
chemical name
compd
1,25-dihydroxy-vitamin D3
1
29
8
1,25-dihydroxy-16-ene-23-yne-20-cyclopropyl-vitamin D3
2
3
4
5
6
7
8
9
10
11
12
13
34
25
16
562
14
45
585
12
40
65
0.001
0.001
>100
ND
>100
1
1,25-dihydroxy-16-ene-23-yne-20-cyclopropyl-19-nor-vitamin D3
1R-fluoro-25-hydroxy-16-ene-23-yne-20-cyclopropyl-vitamin D3
3-desoxy-1,25-dihydroxy-16-ene-23-yne-20-cyclopropyl-vitamin D3
1,25-dihydroxy-16-ene-20-cyclopropyl-23-yne-26,27-hexafluoro-19-nor-vitamin D3
1,25-dihydroxy-16-ene-20-cyclopropyl-23-yne-26,27-hexafluoro-vitamin D3
1R-fluoro-25-hydroxy-16-ene-20-cyclopropyl-23-yne-26,27-hexafluoro-vitamin D3
1,25-dihydroxy-16,23E-diene-20-cyclopropyl-26,27-hexafluoro-19-nor-vitamin D3
1,25-dihydroxy-16,23E-diene-20-cyclopropyl-26,27-hexafluoro-vitamin D3
1R-fluoro-25-hydroxy-16,23E-diene-20-cyclopropyl-26,27-hexafluoro-vitamin D3
1,25-dihydroxy-16-ene-20-cyclopropyl-19-nor-vitamin D3
ND
>100
>100
>100
6
31
<0.00001
1,25-dihydroxy-16-ene-20-cyclopropyl-vitamin D3
0.0002
a IFN-γ and TNF-R secretion were used as read-out for the MLR assay and LPS-stimulated PBMC assay, respectively. The results demonstrate that 13
is the most potent inhibitor of IFN-γ and TNF-R secretion among the 16-ene-20-cyclopropyl-vitamin D3 analogues tested. Assays were performed as described
in the Experimental Section. ND, not determined.
inflammatory properties of 14 with its parent compound 13. The
available data indicate that the accumulation of the active 24-
oxo metabolite 14 in target tissues could explain the high
potency of 13. Further, we also found that the potency of 14 to
increase serum calcium levels is three times lower than that of
13, indicating that 14 represents an anti-inflammatory agent with
a wider therapeutic window than its parent 13.
assessing their ability to inhibit two key pro-inflammatory
cytokines, namely TNF-R and IFN-γ; the results are summarized
in Table 1. Most of the 16-ene-20-cyclopropyl-vitamin D3
analogues inhibited IFN-γ with an IC50 similar to that of 1 (IC50
) 29 pM), but 13 showed a much stronger potency in the
inhibition of IFN-γ production (IC50 < 10-5 pM). While 16-
ene-20-cyclopropyl-vitamin D3 analogues inhibited IFN-γ simi-
larly to 1, the inhibition of TNF-R showed wide differences.
Some compounds failed to induce TNF-R inhibition like 1R-
fluoro-25-hydroxy-16-ene-23-yne-20-cyclopropyl-vitamin D3
(4), 1R,25(OH)2-16-ene-20-cyclopropyl-23-yne-26,27-hexafluoro-
19-nor-vitamin D3 (6), 1R,25(OH)2-16,23E-diene-20-cyclopro-
pyl-26,27-hexafluoro-19-nor-vitamin D3 (9), 1R,25(OH)2-16,23E-
diene-20-cyclopropyl-26,27-hexafluoro-vitamin D3 (10), and 1R-
fluoro-25-hydroxy-16,23E-diene-20-cyclopropyl-26,27-hexafluoro-
vitamin D3 (11) (IC50 > 10-7 M), whereas others inhibited
TNF-R more efficiently than 1, irrespective of a similar
inhibitory capacity of IFN-γ production. Conversely, 13 inhib-
Results
Potent Anti-inflammatory Properties of 1r,25(OH)2-16-ene-
20-cyclopropyl-vitamin D3 (13) Compared to Other 16-ene-
20-cyclopropyl Vitamin D3 Analogues. In our recent study, we
assessed the anti-inflammatory property of several 16-ene-20-
cyclopropyl-vitamin D3 analogues in an assay measuring the
inhibition of IFN-γ production and found that 13 is several fold
more potent than 1.14 In the present study, we reassessed 13,
along with several other 16-ene-20-cyclopropyl-vitamin D3
analogues (Figure 1), for their anti-inflammatory properties by

2206 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
LaVerny et al.
Figure 2. Identification of the various metabolites of 1R,25-dihydroxyvitamin D3 (1) and 1R,25(OH)2-16-ene-20-cyclopropyl-vitamin D3 (13). (A)
HPLC profiles of the lipid extracts of various vitamin D metabolites produced in CAOV cells incubated with 1 (upper panel) and 13 (lower panel).
HPLC was performed using a Zorbax-SIL column (9-250 nm) eluted with hexane-isopropanol (90:10) at a flow rate of 2 mL/min. The various
metabolites of 1 and 13 were monitored by their UV absorbance at 265 nm, and each metabolite is represented by a number. The peak represented
by the asterisk is pre-1 in the upper panel and pre-13 in the lower panel. (B) Chemical structures of various metabolites of 1 and 13 are as follows:
1R,25(OH)2-24-oxo-vitamin D3 (15), 1R,23,25(OH)3-vitamin D3 (16), 1R,23,25(OH)3-24-oxo-vitamin D3 (17), 1R,24,25(OH)3-vitamin D3
(18),1R,23(OH)2-24,25,26,27-tetranor-vitamin D3 (19), 1R,25(OH)2-3-epi-vitamin D3 (20), 1R,24,25(OH)3-16-ene-20-cyclopropyl-vitamin D3 (21),
1R,25(OH)2-24-oxo-16-ene-20-cyclopropyl-vitamin D3 (14), 1R,25(OH)2-16-ene-20-cyclopropyl-3-epi-vitamin D3 (22).
ited IFN-γ and TNF-R production with an IC50 < 10-17 M and
2 × 10-13 M, respectively.
C-23 and C-24 oxidation pathways as well as through the C-3
epimerization pathway. The pattern of metabolism of 1 into its
various metabolites is similar to those reported previously.33
However, the pattern of the metabolism of 13 is different. Upon
careful examination, it becomes obvious that most of the starting
substrate of 13 is converted into 24-hydroxy- (21) and 24-oxo-
(14) metabolites, which accumulate significantly. Along with
metabolites 21 and 14, we also noted the production of a less
polar metabolite 22, which is identified as 1R,25(OH)2-16-ene-
20-cyclopropyl-3-epi-vitamin D3.
Thus, the present data provide convincing evidence to indicate
13 as the most potent anti-inflammatory agent of all the 16-
ene-20-cyclopropyl-vitamin D3 analogues tested.
Production of Various Metabolites of 13 in CAOV
Cells. In our previous study, we compared the metabolism of
two 20-cyclopropyl analogues, namely 1R,25(OH)2-20-cyclo-
propyl-vitamin D3 and 13 in UMR 106 cells.14 The metabolism
data generated in UMR 106 cells indicated that 1R,25(OH)2-
20-cyclopropyl-vitamin D3 was rapidly metabolized through
three different pathways (24-oxidation, C-3 epimerization, and
C-1 esterification pathways) and the pattern of metabolism was
similar to that of 1R,25(OH)2-20-epi-vitamin D3 previously
studied in our laboratory (17). Conversely, 13 was mainly
metabolized through only two pathways (C-24 oxidation and
C-3 epimerization pathways). On the basis of the preliminary
data, we concluded that 13 attains higher biological potency
over 1R,25(OH)2-20-cyclopropyl-vitamin D3 because of its
metabolic stability. However, in our previous study, we could
not unequivocally identify all the metabolites of 13, including
its 24-oxo metabolite 14. Therefore, we undertook the present
study to produce the various natural metabolites of 13 in human
ovarian cancer cells (CAOV cells), which were previously
shown to express high 24-hydroxylase activity (S. Reddy et al.,
unpublished observations).
In conclusion, it appears that a significant proportion of the
biological activity of 13 may be due to its conversion into stable
intermediary metabolites.
Structure Identification of All the Natural Metabolites
of 13. Finally, we succeeded in producing all three metabolites
of 13 in quantities sufficient for their structure identification.
Data in Figure 3 show the mass spectra obtained for the
trimethylsilylated 13 substrate and its three metabolites.
The mass spectrum of trimethylsilylated 13 substrate shown
in Figure 3A exhibited a molecular ion at m/z 642. The
fragments at m/z 552 and 462 were formed by sequential
elimination of one and two trimethylsilanol moieties from m/z
642. Ion fragments at m/z 511 (loss of 131 Da from the
molecular ion) and m/z 217 arised from cleavage of the A ring,
as depicted in Figure 3A. Cleavage of the side chain (C24-C25)
yielded a characteristic ion fragment at m/z 131.
The HPLC profiles of all of the metabolites of 1 (upper panel)
and 13 (lower panel) produced by CAOV cells during a 24 h
incubation period are shown in Figure 2A. The chemical
structures of all the metabolites of 1 and 13 are shown in Figure
2B. 1 was metabolized into several polar metabolites via the
The trimethylsilylated metabolite 22 (Figure 3B) exhibited
virtually identical mass spectral characteristics as the trimeth-
ylsilylated substrate (Figure 3A), but the GC retention time
(15.16 min) did not match that of the trimethylsilylated substrate
(14.51 min). These observations indicated that both compounds

1R,25-Dihydroxy-16-ene-20-cyclopropyl-24-oxo-Vitamin D3
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2207
Figure 3. Structure identification of all the natural metabolites of 13. GC-MS analysis was performed using an Agilent GC System 6890 equipped
with a mass selective detector MSD5973. All analyses were performed as described in the Experimental Section.
must be stereoisomers, in agreement with the proposed identi-
fication of metabolite 22 as the 3-epimer of the substrate, namely
1R,25(OH)2-16-ene-20-cyclopropyl-3-epi-vitamin D3 (22), prod-
uct of the C-3 epimerization pathway.
was secured by a series of standard reactions, as shown in
Scheme 1. Condensation of the CD-ring moiety 23 with acetone
in the presence of butyl lithium as base produced the tertiary
alcohol 24. The desired 24-oxo group in 25 was then formed
by alkyne hydration with sulfuric acid and mercury oxide.
Oxidation of the secondary alcohol and protection of the tertiary
alcohol generated 26 needed for the coupling with ring A
precursor 27 to produce 14 after deprotection.
Similar Induction of Primary VDR Target Genes by 13
and its 24-oxo Metabolite 14. To address the possibility that
the metabolism of 13 into a stable bioactive metabolite could
explain its higher potency, we first compared the ability of 13
and its 24-oxo metabolite 14 to induce primary VDR target
genes in two different human cell culture systems, peripheral
blood mononuclear cells (PBMCs) and human acute monocytic
leukemia cells (THP-1 cells). As shown in Figure 4A, a
dose-response titration at low concentrations of VDR agonists
in PBMCs showed at 10 pM and 100 pM, but not at 1 pM, a
significantly higher increase of transcripts encoding 25-hydrox-
yvitamin D-24-hydroxylase (CYP24A1) and cathelicidin anti-
microbial peptide (CAMP) by both 13 and 14 compared to 1.
Then, to study if the higher potency of these two compounds
The mass spectrum of trimethylsilylated metabolite 21 shown
in Figure 3C revealed a molecular ion at m/z 730, which, when
compared to the substrate, indicated the incorporation of a
hydroxyl group (-OTMS after derivatization). The peak at m/z
599 (M-131) and the presence of a fragment at m/z 217
confirmed that the A ring remained unmodified. These observa-
tions were in agreement with the assigned structure, 21.
Identical mass spectral characteristics were observed for the
trimethylsilylated 14 synthetic standard (Figure 3E) and for the
trimethylsilylated metabolite 14 (Figure 3D). Both trimethyl-
silylated derivatives exhibited molecular ions at m/z 656 as well
as the characteristic ion fragments at m/z 525 (M-131) and m/z
217 (A ring cleavage) and m/z 131 (side chain cleavage). In
addition to the identical mass spectra, the GC retention time
(rt) on GC-MS confirmed the identity of 14 (rt ) 15.19 min),
which was identical to that of the synthetic standard.
Synthesis of 14. The synthesis of 13 was recently disclosed
by our group.14 The preparation of its 24-oxo metabolite 14

2208 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Scheme 1. Summary of the Synthesis of 14
LaVerny et al.
results in a modification of the kinetics of VDR signaling, we
measured the induction of CYP24A1 mRNA at a saturating dose
(10 nM) of 13, 14, and 1 over a time course of 8 h. As shown
in Figure 4B, all the three VDR agonists induced CYP24A1
mRNA similarly at all time points analyzed, indicating that the
higher potency of both 13 and 14 when compared to 1 is mainly
due to the capacity of 13 and 14 to activate the VDR at lower
concentrations.
Similar Immunomodulatory Properties of 13 and its 24-
oxo Metabolite 14. To compare the immunomodulatory proper-
ties of 13 with 14, we evaluated the potency of the three VDR
agonists 13, 14, and 1 to inhibit the production of two key pro-
inflammatory cytokines, IFN-γ and TNF-R. The inhibitory
activities of 13 and 14 on IFN-γ production in the MLR assay
are perfectly superimposable, and both compounds are signifi-
cantly more potent than 1 (IC50 < 10-12 M and 2.5 × 10-11 M,
respectively) (Figure 6A). Production of TNF-R was also
similarly inhibited by 13 and 14 in LPS-stimulated PBMCs,
and both compounds were again significantly more potent than
1 (Figure 6B). We then studied the expression of TNF-R mRNA
using the same conditions previously used to study the expres-
sion of CYP24A1 mRNA (Figure 4B). We noted that the
expression of TNF-R mRNA in LPS-activated PBMCs was
inhibited to the same extent and with similar kinetics by all the
three VDR agonists (13, 14, and 1) at 10 nM concentration
(Figure 6C).
Potential differences in VDR signaling could be masked by
the variability between donors and by the saturation of the
system. For these reasons, we performed a time-course analysis
of the induction of primary VDR target genes in THP-1 cells
using a common nonsaturating concentration of each VDR
agonist and determined EC50 value for each compound. Results
obtained using this model system (Figure 5A) confirmed that
13 and 14 possess a similar capacity to induce the primary VDR
target genes CYP24A1 and CAMP (EC50 values of 6.8 × 10-10
M and 6.9 × 10-10 M, respectively), with a potency significantly
higher than that of 1 (EC50 ) 1 × 10-8 M). As previously shown
in PBMCs (Figure 4B), induction of CYP24A1 and CAMP
mRNA in THP-1 cells proceeded with the same kinetics for
the three VDR agonists tested at their EC50 concentrations,
confirming that 13 and 14 are indeed more potent than 1 (Figure
5B).
To further compare the anti-inflammatory properties of 13
and 14, we also carried out extended dose-response curves
analyzing the production of two other key pro-inflammatory
cytokines, IL-12/23p40 (Figure 7A) and IL-6 (Figure 7B). 13
and 14 are again indistinguishable in their inhibitory activity in
LPS-activated PBMCs and showed a significantly lower IC50
Figure 4. Induction of primary VDR response genes by 1, 13, and its 24-oxo metabolite 14. (A) Induction of CYP24A1 and CAMP transcripts
after overnight incubation of PBMCs with the indicated concentrations of VDR agonists. (B) Time course of CYP24A1 mRNA induction in
24 h LPS-stimulated PBMCs by 10 nM of the indicated VDR agonists. Data have been obtained as described in Experimental Section.
Values are expressed in arbitrary units where 1 represents gene expression in the absence of VDR agonists. 13 vs 1, ***P < 0.001; 14 vs 1,
∆∆P < 0.01, ∆∆∆P < 0.001.

1R,25-Dihydroxy-16-ene-20-cyclopropyl-24-oxo-Vitamin D3
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2209
without increased hypercalcemic properties.17 Crystal structure
analysis of the VDR-1R,25(OH)2-20-epi-vitamin D3 complex
indicated that additional contacts of the ligand within the ligand-
binding pocket of the VDR contributed to enhanced stability
and longer half-life.18 This led us to the synthesis of 20-
cyclopropyl analogues, which mimic the methyl groups of 1
and its 20-epi analogue. During the course of these studies, we
noted that introduction of the 16-ene modification into our VDR
agonist library of 20-cyclopropyl vitamin D3 analogues increases
their potency both in terms of calcemic and immunomodulatory
activities.14
In the present study, we reassessed the immunomodulatory
actions of several 16-ene-20-cyclopropyl-vitamin D3 analogues
using two different in vitro assays, the MLR and the activation
of PBMCs induced by LPS, and identified 13 as the analogue
with the highest potency to inhibit the production of two key
pro-inflammatory cytokines, IFN-γ and TNF-R.
The MLR assay is used as an in vitro model of adaptive
immunity and is widely applied to monitor diseases, such as
AIDS,19 to predict transplant rejection, especially in renal
transplantation,20 and to screen for novel immunosuppressive
drugs.21 An important cytokine produced in a MLR assay is
IFN-γ, a cytokine directly regulated by VDR agonists.22 IFN-γ
is the signature cytokine of Th1 cells, a cell subset pathogenic
in different conditions, including autoimmune diseases.23 Th1
cells are strongly inhibited by VDR agonists, both directly and
indirectly via inhibition of IL-12 production by antigen-
presenting cells.24,25 The very potent inhibitory activity exerted
on IFN-γ production by 16-ene-20-cyclopropyl compounds, and
in particular by 13, demonstrates their marked immunoregulatory
properties.
Figure 5. Equal potency of 13 and its 24-oxo metabolite 14 in the
induction of primary VDR response genes in THP-1 cells. (A)
Expression of CYP24A1 and CAMP mRNA in THP-1 cells after a 4 h
incubation with the indicated concentrations of VDR agonists. (B) Time
course of CYP24A1 and CAMP mRNA induction by 10 nM 1, 1 nM
13, or 1 nM 14. Data have been obtained as described in the
Experimental Section. Values are expressed in arbitrary units, where 1
represents gene expression in the absence of VDR agonists. 13 vs 1,
**P < 0.01, ***P < 0.001; 14 vs 1, ∆∆P < 0.01, ∆∆∆P < 0.001.
One of the mechanisms regulating innate immunity involves
the TLRs, pattern recognition receptors playing a crucial role
in early host defense against invading pathogens.26 In our
experiments, we focused on toll-like receptor 4 (TLR4) because
its ligand, the bacterial endotoxin LPS, is a potent inducer of
immune responses.27 We have analyzed the pro-inflammatory
cytokine TNF-R, which is produced after the activation of TLR4.
TNF-R plays a key role in many disorders, such as rheumatoid
arthritis, acute lung injury, and inflammatory bowel disease,28
and anti-TNF-R biologicals are clinically used for the treatment
of several autoimmune diseases.29 Our data show that 13 is a
potent inhibitor of TNF-R. These findings indicate that 13 is an
extremely potent and versatile immunomodulatory compound
targeting key pathogenic effector cells. Moreover, its MTD,
which is three times higher than 1, would provide a wider
therapeutic window for 13 over 1.
It is now well established that 1 is metabolized by CYP24A1,
a multicatalytic enzyme, in various target tissues.33,34 Over a
decade ago, we identified differences in the metabolism between
1R,25(OH)2-16-ene-vitamin D3 and 1 and recognized that minor
alterations in the structure of 1 can produce major changes in
its target tissue metabolism. We noted that the introduction of
16-ene modification into 1 possibly produces a conformational
change in the side chain, which allows efficient C-24 hydroxy-
lation and C-24 oxidation but not C-23-hydroxylation.15,16 As
a result, the 24-oxo metabolite of 1R,25(OH)2-16-ene-vitamin
D3 accumulates in increasing amounts in target tissues when
compared to the corresponding 24-oxo metabolite of 1. We then
investigated the biological activity of the stable 24-oxo me-
tabolite of 1R,25(OH)2-16-ene-vitamin D3 to clarify if some of
the actions of 1R,25(OH)2-16-ene-vitamin D3 may indeed be
accounted for by its stable 24-oxo metabolite. Indeed, 1R,25(OH)2-
16-ene-vitamin D3 and its 24-oxo metabolite were found to be
value compared to 1 for production of IL-12/23p40 (13: <10-12
M; 14: < 10-12 M; 1: 10-9 M) and IL-6 (13: 3.2 × 10-9 M; 14:
3.5 × 10-9 M; 1: 1.2 × 10-8 M). These data, together with the
similar up-regulation of primary VDR response genes, indicate
that 13 and 14 possess a similar activity in all the assays
performed. These findings suggest that the VDR-dependent
effects induced by 13 may actually be mediated by its 24-oxo
metabolite.
Lower Calcemic Activity of 14 Compared to 13 and 1.
The in vivo calcemic activities of 14, 13, and 1 were assessed
by measuring the capacity of each compound at different doses
to induce hypercalcemia in the mouse after a single oral
administration of each dose for four consecutive days. Results
in Figure 8 show that the maximum tolerated dose (MTD) for
1 is 0.3 µg/kg, and for 13 is 1 µg/kg, whereas the MTD for 14
is 3 µg/kg. These results indicate that 14 is the least hypercal-
cemic among the three VDR agonists tested.
Discussion
Structural and stereochemical modifications at C-20 signifi-
cantly influence the biological effects of 1 and its analogues.14
1R,25(OH)2-20-epi-vitamin D3 exhibits up to 5000 times greater
antiproliferative activity than 1 in various cancer cell lines

2210 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
LaVerny et al.
Figure 6. Equal potency of 13 and its 24-oxo metabolite 14 in the inhibition of IFN-γ and TNF-R production. (A) Allogeneic PBMCs were
cultured in a bidirectional MLR assay for 5 days with the indicated concentrations of VDR agonists. At the end of the culture period, IFN-γ
production was measured in culture supernatants by two-site ELISA. Assays were performed as described in the Experimental Section. Results are
expressed as percent of cytokine production in the absence of VDR agonists. (B) Inhibition of TNF-R secretion in LPS stimulated PBMCs. (C)
Inhibition of TNF-R mRNA by 10 nM of the indicated VDR agonists. Each point represents the mean ( SEM of at least three independent
experiments. 13 vs 1, ***P < 0.001; 14 vs 1, ∆∆P < 0.01, ∆∆∆P < 0.001.
Figure 7. Equal potency of 13 and its 24-oxo metabolite 14 in the
inhibition of IL-12/23p40 and IL-6 in LPS stimulated PBMCs. (A)
Inhibition of IL-12/23p40 secretion. (B) Inhibition of IL-6 secretion.
Data have been obtained as described in the Experimental Section.
Results are expressed as percent of cytokine production in the absence
of VDR agonists. Each point represents the mean ( SEM of at least
three independent experiments. 13 vs 1 *P < 0.05, **P < 0.01, ***P
< 0.001; 14 vs 1, ∆P < 0.05, ∆∆P < 0.01, ∆∆∆P < 0.001.
Figure 8. Calcemic activity of 14 compared to 13. Eight week-old
female C57BL/6 mice (3 mice/group) were dosed orally (0.1 mL/mouse)
with various concentrations of VDR agonists daily for four days. Serum
calcium levels were determined on day five using a colorimetric assay.
Dotted plot represent the maximum normo-calcemic concentration ()
10.7 mg/mL) 13 vs 1, ***P < 0.001; 14 vs 1, ∆∆∆P < 0.001; 14 vs
13, OP < 0.05, OOOP < 0.001.
equipotent in inducing growth inhibition and promoting dif-
ferentiation of RWLeu-4 human myeloid leukemic cells.16 These
data thus support the concept that one of the important
mechanisms responsible for the increase in the potency of some
of the vitamin D analogues can be due to their metabolism
through alternative pathways leading to the production of stable
and bioactive metabolites. On the basis of these earlier observa-
tions, we recently compared the metabolism of 1R,25(OH)2-
20-cyclopropyl-vitamin D3 with that of 13 and showed that
1R,25(OH)2-20-cyclopropyl-vitamin D3 is rapidly metabolized
through three different pathways (24-oxidation, C-3 epimeriza-
tion and C-1 esterification pathways),14 with a pattern of
metabolism similar to that of 1R,25(OH)2-20-epi-vitamin D3.17
Conversely, 13 is mainly metabolized through only two
pathways (C-24 oxidation and C-3 epimerization pathways). On
the basis of these preliminary data, we concluded that 13 can
attain higher biological potency over 1R,25(OH)2-20-cyclopro-
pyl-vitamin D3 because of its metabolic stability. In the present
study, we investigated the metabolism of 1R,25(OH)2-20-
cyclopropyl-16-ene-vitamin D3 in CAOV cells and confirmed
that the 24-oxo metabolite 14 is indeed the final, stable
metabolite. The chemical synthesis of 14 allowed us to assess
its various biological properties.
To clarify if some of the actions of 13 may indeed be
accounted for by its 24-oxo metabolite 14, we first determined
the potency of both 13 and 14 to modulate the induction of
primary VDR target genes. Expression of CYP24A1, the most
responsive primary VDR target gene, is similarly induced by
14 and 13 at the mRNA level both in terms of dose-response
curves and timing of gene induction. Expression of CAMP,
another primary VDR target gene is also modulated by 13 and
14 in the same way as CYP24A1.35 As cathelicidin plays an
important role in innate immunity,36-38 these data indicate
comparable properties for 13 and 14 in the regulation of innate
immune responses. We then compared the anti-inflammatory
properties of 13 and 14 by evaluating their ability to inhibit

1R,25-Dihydroxy-16-ene-20-cyclopropyl-24-oxo-Vitamin D3
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2211
mL) at -78 °C was added n-BuLi (2.72 mL, 4.35 mmol, 1.6 M in
hexane).37 After stirring at -78 °C for 1 h, acetone (2.5 mL, 34.6
mmol) was added and stirring was continued for 2.5 h. A saturated
aqueous solution of ammonium chloride was added (15 mL), and
the mixture was stirred for 15 min at room temperature and then
extracted with ethyl acetate (2-50 mL). The combined extracts
were washed with brine (50 mL) and dried (Na2SO4). The residue,
obtained after evaporation of the solvent (2.4 g), was flash-
chromatographed using 10% ethyl acetate in hexane as mobile phase
to give the tertiary alcohol (1.05 g, 2.61 mmol), which was treated
with tetrabutylammonium fluoride (6 mL, 6 mmol, 1.0 M in THF)
and stirred at 65-75 °C for 48 h. The mixture was diluted with
ethyl acetate (25 mL) and washed with water (5 × 25 mL) and
brine (25 mL). The combined aqueous washes were extracted with
ethyl acetate (25 mL), and the combined organic extracts were dried
(Na2SO4). The residue, after evaporation (1.1 g), was flash-
chromatographed (20% ethyl acetate in hexane) to give 24 (0.75
g, 2.59 mmol, 90%) [R]30D +2.7 (c 0.75, CHCl3). 1H NMR (CDCl3):
5.50 (1H, m), 4.18 (1H, m), 2.40 (2H, s), 2.35-1.16 (11H, m),
1.48 (6H, s), 1.20 (3H, s), 0.76-0.50 (4H, m). 13C NMR (CDCl3):
156.39, 125.26, 86.39, 80.19, 69.21, 65.16, 55.14, 46.94, 35.79,
33.60, 31.67, 29.91, 27.22, 19.32, 19.19, 17.73, 10.94, 10.37. HRMS
(EI) calcd for C22H28O2 (M+) 288.2089, obsd 288.2091.
4-Hydroxy-1-[1-((3aR,4S,7aS)-4-hydroxy-7a-methyl-3a,4,5,6,7,7a-
hexahydro-3H-inden-1yl)-cyclopropyl]-4-methyl-pentan-3-one (25).
The mixture of 24 (250 mg, 0.87 mmol), THF (4 mL), and 6 drops
of mercuric salts mixture (made by stirring at 65-70 °C of: 150
mg of HgO, 0.1 mL of concentrated sulfuric acid, 1.3 mL water,
and 3.8 mL of methanol) was stirred at room temperature for 7 d
with addition of 6 drops of mercuric salts mixture each day. Sodium
hydrogen carbonate (0.5 g) was added and the reaction mixture
filtered after 3 h; the solids were washed with Et2O (50 mL). The
filtrates were evaporated and the residue (280 mg) was flash-
chromatographed (25 g, 25% AcOEt in hexane) to give 25 (60 mg).
(3aR,7aS)-7a-Methyl-1-[1-(4-methyl-4-trimethylsilanyloxy-3-oxo-
pentyl)-cyclopropyl]-3a,4,5,6,7,7a-hexahydro-3H-inden-4-one (26).
To a stirred suspension of (100 mg, 0.33 mmol) and celite (0.4 g)
in dichloromethane (5 mL) at room temperature wad added
pyridinium dichromate (0.25 g, 0.66 mmol). The resulting mixture
was stirred for 3 h filtered through silica gel (5 g), and then silica
gel pad was washed with 20% ethyl acetate in hexane. The
combined filtrate and washes were evaporated to give a crude 26
(96 mg). This material was taken up in dichloromethane (4 mL),
and trimethylsilyl-imidazole (0.1 mL, 0.75 mmol) was added. The
resulting mixture was stirred for 2.5 h, filtered through silica gel
(10 g), and the silica gel pad was washed with 10% ethyl acetate
in hexane. The combined filtrate and washes were evaporated to
give 26 (100 mg, 0.27 mmol, 82%).
IFN-γ and TNF-R along with two additional pro-inflammatory
cytokines, IL-12/23p40 and IL-6. Both IL-12/23p40 and IL-6
also play an important role in immunomodulation. IL-12/23p40
is a component of the IL-12 heterodimer, which drives Th1 cell
development,30 but the IL-12p40 subunit can also bind to p19
and form IL-23, a cytokine sustaining the amplification of Th17
cells, a pathogenic T cell subset recognized to play a major
role in the pathogenesis of autoimmune diseases.31 IL-6 is a
pleiotropic inflammatory cytokine that is also involved in the
induction of Th17 cells, which in addition to IL-17 also produce
IL-6 and TNF-R.32 Interestingly, we noted that both 14 and 13
were equipotent in their ability to inhibit the production of TNF-
R, IL-12/23p40, and IL-6, whereas 13 inhibited the production
of IFN-γ more efficiently than 14 at their lowest concentrations.
This difference could be due to the target cytokine or possibly
to the assay used because, while effects on mRNA up-regulation
and protein inhibition are similar in a 24 h assay, a slight
difference was observed in the 5-d MLR model.
In summary, we have shown that the strong induction of
primary VDR response genes and immuno-regulatory activities
of 14 are comparable to those exerted by its parent compound
13. Collectively, these findings indicate that the strong potency
of 13 can be explained by the accumulation of its stable 24-
oxo metabolite that displays immunoregulatory and anti-
inflammatory properties superimposable to those exerted by 13
itself. However, we found that the MTD of 14 is three times
higher than its parent compound 13, indicating that the potency
of the metabolite compared to the parent compound to induce
hypercalcemia was reduced. On the basis of these findings, we
now propose that 14 represents a better anti-inflammatory agent
than its parent 13 due to its wider therapeutic window.
Conclusion
We have identified 13 as a VDR agonist with the highest
potency to inhibit pro-inflammatory cytokine production among
members of the 16-ene-20-cyclopropyl family. This compound
is metabolized to 14, which resists further metabolism. As a
result, this 24-oxo metabolite of 13 accumulates in tissues. By
combining our observations of equipotency between 13 and 14
in transcript regulation, and similar inhibition of cytokine
production, we conclude that accumulation of 14 can explain
the strong potency of 13, supporting the concept that specific
differences in the target tissue metabolism of VDR agonists can
play a critical role to increase their potency.
1r,25-Dihydroxy-16-ene-20-cyclopropyl-24-oxo-cholecalciferol
(14). To a stirred solution of 27 (310 mg, 0.53 mmol) in
tetrahydrofuran (2 mL) at -78 °C was added n-BuLi (0.34 mL,
0.54 mmol). The resulting mixture was stirred for 10 min and
solution of 26 (95 mg, 0.25 mmol, in tetrahydrofuran (2 mL) was
added dropwise. The reaction mixture was stirred at -72 °C for
3.5 h, diluted with hexane (25 mL), washed brine (20 mL), dried
over Na2SO4, and evaporated. The residue (850 mg) was flash-
chromatographed (1:9 EtOAc - hexane) to give 1R,3ꢀ-di(tert-butyl-
dimethyl-silanyloxy)-25-trimethylsilanyloxy-16-ene-20-cyclopropyl-
24-oxo-cholecalciferol (152 mg, 0.21 mmol). This material was
dissolved in a tetrabutylammonium fluoride solution (3 mL, 1 M
solution in THF), and the solution was stirred for 40 h, diluted
with ethyl acetate (25 mL), washed with water (5-20 mL) and
brine (20 mL), dried over Na2SO4, and evaporated. The residue
(130 mg) was flash-chromatographed (2:1 EtOAc-hexane and ethyl
acetate) to give the 14 (74 mg, 0.17 mmol, 85%); [R]30D) -3.5
CHCl3, c 0.4; UV λmax (EtOH): 241 nm (ε 11080), 273 nm (ε
13283). 1H NMR, 400 MHz (DMSO): 6.19 (1H, d, J ) 11.3 Hz),
6.07 (1H, d, J ) 11.3 Hz), 5.36 (1H, s), 5.24 (1H, s), 4.77 (1H, s),
4.19 (1H, m), 3.99 (1H, m), 2.76 (1H, m), 2.64 (2H, m), 2.39-1.24
(17H, m), 1.14 (3H, s), 1.13 (3H, s), 0.73 (3H, s),0.64-0.26 (4H,
m). 13C NMR (DMSO-d6): 215.68, 156.33, 149.42, 139.29, 135.98,
Experimental Section
Chemistry. Synthesis and Characterization (NMR, NP-HPLC,
MS, Elemental Analysis) of Target Compounds. NMR spectra were
obtained with a Varian 400 MHZ spectrometer and the chemical
shifts are reported in parts per million (ppm). The abbreviations
used are as follows: s, singlet; bs, broad singlet; d, doublet; dd,
double doublet; m, multiplet. Flash column chromatography was
performed using Merck silica gel 60 (0.040-0.063 mm). TLCs were
carried out on precoated TLC plates with silica gel 60 F-254
(Merck). All reactions were carried out under a nitrogen atmosphere.
The purity of the tested compounds was determined by HPLC
analysis and results were g95% purity unless otherwise stated. The
analytical HPLC measurements were made according to: method
(A) NP-HPLC, Zorbax-SIL, 9.4 mm × 25 cm, 9:1 hexane:
isopropanol, flow rate 2 mL/min, UV 265 nM, charge 10 µg;
method (B) RP-HPLC, Zorbax-ODS, 4.6 mm × 25 cm, 85:15
acetonitrile:methylene chloride, flow rate 2 mL/min, UV 265 nM,
charge 10 µg.
(3aR,4S,7aS)-1-[1-(4-Hydroxy-4-methyl-pent-2-ynyl)-cyclopro-
pyl]-7a-methyl-3a,4,5,6,7,7a-hexahydro-3H-inden-4-ol (24). To a
stirred solution of 23 (1.0 g, 2.90 mmol) in tetrahydrofuran (15

2212 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
LaVerny et al.
124.45, 122.37, 117.73, 109.77, 75.89, 68.35, 65.15, 58.90, 49.59,
44.83, 43.11, 34.94, 33.85, 30.93, 28.80, 27.99, 26.53, 26.50, 23.12,
20.87, 17.50, 12.75, 10.00. HRMS (ES) calcd for C28H40O4 M +
Na: 463.2819; obsd M + Na: 463.2818.
Biological Studies. Compound 1 and Vitamin D Analogues.
Purity of all compounds was determined by NMR spectra and was
found to be g95%.
Relative quantification of target cDNA was determined by
arbitrarily setting the control value to 1 and changes in cDNA
content of a sample were expressed as a multiple thereof. Differ-
ences in cDNA input were corrected by normalizing to glyceral-
dehyde 3-phosphate dehydrogenase (GAPDH) signal. To exclude
amplification of genomic DNA, RNA samples were treated with
DNase (Sigma-Aldrich, USA).
Metabolism Studies and HPLC. Confluent CAOV cells were
incubated with 1 µM concentrations of 1 and 13 in 50 mL media
containing 10% FCS. The incubations were carried out at 37 °C in
a humidified atmosphere under 5% CO2 and were stopped at 24 h
with 10 mL of methanol. The lipids from both cells and media
were extracted for HPLC analysis using the extraction procedure
described earlier.11 The lipid extract of media and cells was
subjected directly to high performance liquid chromatography
(HPLC) using the chromatographic conditions described in the
legend of Figure 2. The various vitamin D3 metabolites were
monitored by a photodiode array detector, and the metabolites were
identified based on the elution positions of known standards.
GC/MS Analysis. GC-MS analysis was performed using an
Agilent GC System 6890 equipped with a mass selective detector
MSD5973. Prior to analysis, the vitamin D3 compounds were
trimethylsilylated in a 1:1 acetonitrile:Tri-Sil TBT (Pierce, Rock-
ford, IL) mixture and incubated at 70 °C for 15 min to ensure
complete derivatization. The final concentration of each trimeth-
ylsilylated compound was 50 µg/mL. All analyses were performed
on a HP-Ultra1 GC column (25 mm × 0.2 mm × 0.11 µm film
thickness, 100% dimethylpolysiloxane), using UHP helium as a
carrier gas at a flow rate of 0.8 mL/min. Oven temperature program
was as follows: initial temperature 150 °C, then 10 °C/min until
reaching a final temperature of 300 °C, which was held for 5 min.
Full scan electron spectra across the mass range of m/z 50-800
were acquired in each run and the published spectra were averaged
and background subtracted.
Crystalline 1,25(OH)2D3 (1) and its analogues were reconstituted
in absolute ethanol, at the concentration of 1 mg/mL and stored in
concentrated solutions at -80 °C under nitrogen atmosphere. 1 and
its analogues were freshly diluted before each experiment, and the
ethanol concentration in the test conditions did not exceed
0.00025%.
Mice. Eight-week-old C57BL/6 mice (Charles River, Italy) were
housed in plastic cages with water absorbent bedding. Litter was
changed at least twice a week. The animal room was temperature
controlled and had a 12 h light/dark cycle. Food and water were
supplied ad libitum. All procedures were reviewed and approved
by local ethical committee.
Assessment of the MTD. Eight-week-old female C57BL/6 mice
(3 mice/group) were dosed orally (0.1 mL/mouse) with various
concentrations of VDR agonists daily for four days. Analogues were
formulated in miglyol for a final concentration of 0.01, 0.03, 0.1
0.3, 1, 3, 10 30, 100, and 300 µg/kg when given at 0.1 mL/mouse
po. Blood for serum calcium assay was drawn by tail bleed on day
five, the final day of the study. Serum calcium levels were
determined using a colorimetric assay (Sigma Diagnostics, proce-
dure no. 597). The highest dose of analogue tolerated without
inducing hypercalcemia (serum calcium >10.7 mg/dL) was taken
as the MTD and expressed in µg/kg.
Cell Cultures. PBMC were separated from buffy coats by Ficoll
gradient. For the bidirectional MLR, the same number (3 × 105/
200 µL/well) of allogeneic PBMC from two different donors were
cocultured in 96-well flat-bottom plates in RPMI 1640 with
Glutamax and 5% (v/v) heat inactivated Fetal Clone-1 (FC-1,
Charles River) with 1% non essential amino acids, 0.5 mg/mL
gentamicin, and 1 mM of sodium pyruvate. After 5 days, prolifera-
tion and cytokine production were measured. For LPS-activated
cells, PBMCs (2 × 105/200 µL/well) were cultured in RPMI 1640
with Glutamax and 10% (v/v) heat inactivated fetal bovine serum
(FBS, Charles River) with 1% non essential amino acids, 0.5 mg/
mL gentamicin, and 1 mM of sodium pyruvate in 96-well flat-
bottom plates in presence of 100 ng/mL of LPS from Escherichia
coli 0111:B4 (Sigma-Aldrich, USA). After 24 h, cytokine produc-
tion was measured. THP-1 cells were maintain in culture in phenol
red-free RPMI 1640 with Glutamax and 10% (v/v) heat inactivated
FBS with 0.1 mg/mL streptomycin and 100 U/mL of penicillin.
Prior to the treatment, THP-1 (106/mL) were grown overnight in
phenol red-free RPMI 1640 with Glutamax and 5% (v/v) charcoal-
treated heat inactivated FBS with 0.1 mg/mL streptomycin and 100
U/mL of penicillin. Then after 4 h treatment with the corresponding
concentration of VDR agonist, mRNAs were extracted. CAOV cells
were maintained in McCoy’s culture media supplemented with 10%
FCS. For the metabolism studies 3-106 cells were seeded in T150
culture bottles and were grown to confluence.
Statistics
Data were analyzed with graph prism version 5.0 and curves
were generated with the appropriate nonlinear fit regression.
Statistical significance was determined by a two-way analysis
of variance (ANOVA), followed by Bonferroni t-test. Differ-
ences were considered significant at P < 0.05.
Acknowledgment. This paper is dedicated to Professor
Anthony W. Norman in recognition of his many fundamental
contributions to vitamin D research, including the discovery of
1R,25-dihydroxy D3 and its first clinical use in treatment of renal
osteodystrophy. This work was supported in part by the
European Community grant NucSys to L.A. (MRTN-CT-
019496).
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