Structure-activity relationships for vitamin D3-based aromatic a-ring analogues as hedgehog pathway inhibitors

Article abstract of DOI:10.1021/jm401812d

A structure-activity relationship study for a series of vitamin D3-based (VD3) analogues that incorporate aromatic A-ring mimics with varying functionality has provided key insight into scaffold features that result in potent, selective Hedgehog (Hh) pathway inhibition. Three analogue subclasses containing (1) a single substitution at the ortho or para position of the aromatic A-ring, (2) a heteroaryl or biaryl moiety, or (3) multiple substituents on the aromatic A-ring were prepared and evaluated. Aromatic A-ring mimics incorporating either single or multiple hydrophilic moieties on a six-membered ring inhibited the Hh pathway in both Hh-dependent mouse embryonic fibroblasts and cultured cancer cells (IC₅₀ values 0.74-10 μM). Preliminary studies were conducted to probe the cellular mechanisms through which VD3 and 5, the most active analogue, inhibit Hh signaling. These studies suggested that the anti-Hh activity of VD3 is primarily attributed to the vitamin D receptor, whereas 5 affects Hh inhibition through a separate mechanism.

Full text of DOI:10.1021/jm401812d

Article
pubs.acs.org/jmc
Structure−Activity Relationships for Vitamin D3-Based Aromatic
A‑Ring Analogues as Hedgehog Pathway Inhibitors
Albert M. DeBerardinis, Daniel J. Madden, Upasana Banerjee, Vibhavari Sail, Daniel S. Raccuia,
Daniel De Carlo, Steven M. Lemieux, Adam Meares, and M. Kyle Hadden*
Department of Pharmaceutical Sciences, University of Connecticut, 69 North Eagleville Road, Unit 3092, Storrs, Connecticut
06269-3092, United States
S
* Supporting Information
ABSTRACT: A structure−activity relationship study for a
series of vitamin D3-based (VD3) analogues that incorporate
aromatic A-ring mimics with varying functionality has provided
key insight into scaffold features that result in potent, selective
Hedgehog (Hh) pathway inhibition. Three analogue sub-
classes containing (1) a single substitution at the ortho or para
position of the aromatic A-ring, (2) a heteroaryl or biaryl
moiety, or (3) multiple substituents on the aromatic A-ring
were prepared and evaluated. Aromatic A-ring mimics
incorporating either single or multiple hydrophilic moieties on a six-membered ring inhibited the Hh pathway in both Hh-
dependent mouse embryonic fibroblasts and cultured cancer cells (IC50 values 0.74−10 μM). Preliminary studies were conducted
to probe the cellular mechanisms through which VD3 and 5, the most active analogue, inhibit Hh signaling. These studies
suggested that the anti-Hh activity of VD3 is primarily attributed to the vitamin D receptor, whereas 5 affects Hh inhibition
through a separate mechanism.
Chart 1. Hh Pathway Inhibitors
INTRODUCTION
■
The Hedgehog (Hh) signaling pathway is a developmental
pathway essential for directing normal growth and tissue
patterning during embryonic development. Dysregulation of
Hh signaling contributes to the onset and progression of several
forms of human cancer, most notably basal cell carcinoma
(BCC) and medulloblastoma (MB).1−3 Both BCCs and MBs
are widely recognized as Hh-dependent, and a significant
number of BCCs (70%) and MBs (25%) exhibit detectable
genetic mutations in Hh pathway components.4−6 These
mutations result in constitutive activation of the pathway and
increased expression of Hh target genes, including several forms
of the glioma-associated oncogene (Gli) family of signaling
proteins.
Over the past decade, much attention across academia and
industry has been targeted toward the identification of selective
Hh signaling inhibitors as potential chemotherapeutic agents.7,8
These studies have primarily focused on the development of
synthetic small molecules as direct inhibitors of Smoothened
(Smo), a GPCR-like 7-transmembrane protein that serves as a
key regulator of Hh signaling and is the most druggable target
in the pathway. The most advanced of these small molecules,
GDC-0449 (Vismodegib, 1, Chart 1), was approved by the
FDA in early 2012 as the first-in-class Hh-specific antagonist for
the treatment of locally advanced BCCs. While treatment of
two MB patients with 1 also demonstrated initial positive
results, relapse occurred when a point mutation in Smo
(D473H) rendered the patient insensitive to further treatment
with 1.9,10 In addition, a recent study reported that 21% of
patients receiving continuous treatment of 1 for BCC
developed regrowth of at least one BCC, suggesting resistance
in BCC patients will also be an issue moving forward.11 Finally,
over half (54%) of basal cell nevus syndrome (BCNS) patients
receiving 1 to treat BCCs voluntarily withdrew from the study
due to adverse side effects.12
Received: November 22, 2013
Published: April 14, 2014
© 2014 American Chemical Society
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a
Table 1. Hh Inhibitory Activity for VD3 and Initial Analogues
C3H10T1/2
ASZ
DAOY
b
c
b
c
b
c
analogue
Gli1
Cyp24A1
Gli1
Cyp24A1
Gli1
Cyp24A1
VD3
4.1 0.3
3.1 0.2
0.74 0.1
2.8 0.6
8336 38
3.6 0.5
22.2 4.7
2.1 0.1
>10
764 24
2.4 0.9
1.9 0.1
2.5 1.4
>10
>10
149 90
0.8 0.1
0.4 0.1
0.5 0.1
2
5
6
5.2 0.2
3.7 0.04
9.2 1.7
24.3
3
7.1
1
a
b
Data reproduced from refs 15 and 16. IC50 values (μM) represent the av SEM of at least two separate experiments performed in triplicate.
c
Values represent Cyp24A1 mRNA expression relative to DMSO control (24 h, 5 μM).
a
Vitamin D3 (cholecalciferol, VD3, 3) is a metabolic
precursor to 1α,25-dihydroxyvitamin D3 (calcitriol, 4),
generally acknowledged as the hormonally active form of
vitamin D. Both VD3 and calcitriol down-regulate Hh signaling
in Hh-dependent cell culture and murine models of BCC;
however, neither compound is a selective inhibitor of pathway
signaling.13,14 As calcitriol is known to exert a range of
physiological effects via direct binding and activation of the
nuclear vitamin D receptor (VDR), its nonselective nature is
not surprising. By contrast, VD3 does not bind VDR and is
commonly perceived to be physiologically inactive. This
intriguing new activity for VD3 prompted a series of
preliminary structure−activity relationship studies designed to
identify the pharmacophore for VD3 inhibition of Hh signaling
and serve as a starting point for developing selective inhibitors
of Hh signaling based on the VD3 scaffold.15 These initial
studies identified Grundmann’s alcohol (2) as comparable to
VD3 in its ability to inhibit Hh signaling without activating
canonical VDR signaling (Table 1). On the basis of these
results, we designed, synthesized, and evaluated a small series of
VD3 analogues in which the natural A-ring was replaced with
an aromatic mimic.16 Two compounds containing either a 3-
phenol (5) or 3-aniline (6) linked to Grundmann’s alcohol
through an ester bond were identified as potent and selective
inhibitors of pathway signaling in several Hh-dependent cell
lines (Table 1). Further evaluation of analogues 2 and 5
demonstrated that the intact ester-linked scaffold was required
for potent pathway inhibition, verifying this scaffold as an
intriguing lead for further development. Herein, we report on
an in-depth SAR study for this series of compounds and
provide preliminary information as to the mechanistic aspects
that govern its inhibition of Hh signaling.
Scheme 1. Protection Schemes for Aromatic A-Ring Mimics
a
Reagents and conditions: (a) BnBr, K2CO3 (80−95%); (b) KOH or
LiOH (50−80%); (c) BnBr, Cs2CO3, (50−65%); (d) 2 N NaOH
(80%); (e) Boc2O, dioxane, 5% NaHCO3 (70%).
(Scheme 1A,B). Similar protection of hydroxy-pyridine
carboxylic acids 25−26 yielded both mono- and dibenzylated
intermediates. The bis-protected compounds were readily
separated via chromatography and sodium hydroxide-mediated
hydrolysis afforded the carboxylic acids 29−30 (Scheme 1C).
Finally, the aniline substituent of 31 was protected as the t-
butoxycarbonyl using standard conditions (Scheme 1D).
Standard esterification conditions (DCC and DMAP) were
utilized to couple Grundmann’s alcohol 2 to the requisite
benzoic acid (Schemes 2 and 3A). Intermediates that required
deprotection were submitted to the appropriate conditions and
final analogues were isolated in good to excellent yields.
Installing indole-2-carboxylic as the A-ring mimic was
accomplished by preparing the acyl chloride in situ (oxalyl
chloride and catalytic DMF), followed by coupling with 2 in the
presence of DMAP (Scheme 3B).
RESULTS AND DISCUSSION
■
Chemistry. We envisioned expanding the scope of the ester-
linked series through the design and synthesis of a number of
analogue subclasses based on modifications made to the
aromatic motif. The ester linkage between Grundmann’s
alcohol and the aromatic A-ring mimics was maintained to
explore this class of pathway inhibitors in an expeditious
manner. Commercially available aromatic acids and esters were
purchased and either coupled directly to 2 (acids) or coupled
following standard protection/hydrolysis strategies shown in
Scheme 1. For these studies, we prepared three series of
analogues containing either (1) a single substitution at the ortho
or para position of the aromatic A-ring, (2) a heteroaryl or
biaryl moiety, or (3) multiple substituents on the aromatic A-
ring.
Benzyl bromide was utilized to protect the free phenols of
methyl ester benzoates 7−10 and 19−20 as the benzyl ethers.
Potassium or lithium hydroxide-mediated hydrolysis afforded
carboxylic acids 15−18 and 23−24 in moderate to good yields
Biological Evaluation. The initial evaluation of VD3
analogues as Hh pathway inhibitors was performed at a single
dose (5 μM) by monitoring endogenous Gli1 mRNA levels in
C3H10T1/2 cells, an Hh-dependent mouse embryonic
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Scheme 2. Monosubstituted Aromatic A-Ring Analogues:
Subclass I
Table 2. Hh Inhibition for Monosubstituted VD3 Analogues
a
a
b
c
analogue
Gli1 mRNA (%)
Cyp24A1 mRNA
1.0
DMSO
OHCs
VD3
1.0
100
35.7 0.3
4.8 0.2
46.4 3.5
1.9 0.5
2.4 0.5
8336 38
1.0 0.02
3.6 0.5
22.2 4.7
24.3 3.0
0.9 0.1
0.8 0.01
0.9 0.3
0.9 0.2
1.4 0.2
1.2 0.1
18.8 10
140 10
d
1
2
5
6
33
34
35
36
40
41
42
43
109
3
77.7 5.7
98.0 14
113 17
107 11
115 6.0
2.4 0.5
15.4 1.1
a
Reagents and conditions: (a) DCC, DMAP, DCM, (X)-ArCO2H
(60−95%); (b) 37 or 38, Pd(OH)2 (10%), H2, MeOH:THF (2:1)
(75%); (c) 39, TFA:DCM (1:1) (65%).
a
b
Scheme 3. Hetero- and Biaryl A-Ring Analogues: Subclass
II
All analogues evaluated at 5 μM and 24 h. Values represent % Gli1
a
c
mRNA expression relative to OHC control (set as 100%). Values
represent up-regulation of Cyp24A1 mRNA normalized to DMSO
(set to 1.0). Compound 1 was evaluated at 0.5 μM.
d
(Table 1).16 To more fully explore SAR for the aromatic A-ring
substituent, we first evaluated a series of analogues that
incorporated a single functional group at either the 2- or 4-
position. Interestingly, while incorporation of the phenol in the
4-position resulted in an analogue (42) that maintained potent
inhibition of Hh signaling, shifting the phenol to the 2-position
(41) resulted in complete loss of activity. There are two
possible explanations for this observed result. First, placing the
hydroxyl in the ortho-position may simply prevent it from
accessing key interaction(s) available to the 3- or 4-phenol due
to increased distance or orientation. Second, it is possible that
being ortho to the ester linker orients the phenol such that it
can form an intramolecular hydrogen bond with the carbonyl of
the ester bond, preventing it from interacting with the binding
site. Incorporating an amine in the 4-position (43) resulted in
an analogue with slight decrease in activity when compared to
the 3-position amine (6) (Gli1 expression = 15.4% and 2.4%,
respectively). Similar to the 3-substituted analogues, incorpo-
rating a chlorine (33−34) or masking the phenol as the
methoxy (35−36) resulted in analogues that did not inhibit
pathway signaling. In addition, masking the 4-amine as the
dimethylated amine (40) completely abolished the inhibitory
activity. Similar to the results previously identified for 5 and 6,
analogues that decreased Gli1 expression also moderately up-
regulated Cyp24A1 (42−43).
The hetero- and biaryl analogue subclass was designed to (1)
study whether incorporating functionality within the aromatic
ring results in enhanced Hh inhibitory activity and (2) to
explore steric constraints associated within the aromatic A-ring
binding pocket (Table 3). An interesting trend was observed
for the analogues incorporating a pyridine ring as the aromatic
substituent (44−46). Whereas the 2-phenol did not inhibit Hh
signaling, the 2-pyridyl substitution (44) provided an analogue
that retained modest inhibition (Gli1 expression = 14.1%).
Shifting the nitrogen to the 3- or 4-position resulted in a
concomitant decrease in activity against the Hh pathway. This
result is the opposite of that seen above when the substituent
was appended to the ring. Finally, each of the pyridyl-
containing analogues modestly up-regulated Cyp24A1 (14−57-
fold) when compared to the other analogues in this subclass.
a
Reagents and conditions: (a) DCC, DMAP, DCM, Aryl-CO2H, (60−
95%); (b) 53 or 54, Pd(OH)2 (10%), H2, MeOH:THF (2:1), (75%);
(c) (i) oxalyl chloride, DMF, DCM, (ii) 2, DMAP, DCM (45%).
fibroblast (MEF). In addition, we have previously established
that C3H10T1/2 cells respond to VDR activation with robust
up-regulation of Cyp24A1, a well-characterized target gene of
canonical vitamin D signaling; therefore, this assay provides a
dual function for the determination of potency (Gli1 mRNA
expression) and selectivity (Cyp24a1 mRNA expression) for
VD3 analogues.13 The protocol utilized for this assay was
described previously,15,16 and oxysterol activators of Hh
signaling (OHCs; 20α-hydroxycholesterol and 22(S)-hydroxy-
cholesterol, 5 μM each) were utilized to up-regulate Gli1
expression. Gli1 and Cyp24a1 mRNA expression levels were
monitored following concomitant analogue and OHC addition.
Results are provided in Tables 2−4 according to the VD3
analogue subclass.
As described previously, the addition of an aromatic A-ring
mimic into the VD3 scaffold significantly enhances the Hh
inhibitory properties of this class of VD3 analogues when
compared to VD3 and 2.16 Our initial SAR for this class of
compounds focused on the incorporation of a variety of
functional groups in the 3-position and identified both 5 and 6
as potent inhibitors of Hh signaling in MEFs that show
promising anti-Hh activity in two cultured cancer cell lines
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Table 3. Hh Inhibition for Hetero- and Biaryl VD3
Analogues
Table 4. Hh Inhibition for Analogues with Multiple
Substitutions
a
b
c
a
b
c
analogue
Gli1 mRNA (%)
Cyp24A1 mRNA
analogue
Gli1 mRNA (%)
Cyp24A1 mRNA
44
45
46
47
48
49
50
51
52
55
56
58
14.1 10
20.7 3.7
51.1 11
95 30
57.1 10.3
17.1 1.4
14.7 6.7
1.7 0.5
2.2 1.3
1.6 0.4
1.3 0.6
2.7 1.0
3.0 0.2
63
64
65
66
8.6 2.2
5.1 1.3
23.0 1.0
36.5 3.6
19
11
1
2
2
1
7.5
3.1
a
b
91.5 23
All analogues evaluated at 5 μM and 24 h. Values represent % Gli1
c
109
3
mRNA expression relative to OHC control (set as 100%). Values
represent up-regulation of Cyp24A1 mRNA normalized to DMSO
(set to 1.0).
109 0.6
82.3 0.8
77.4
103
91.9
4
9
3
Chart 2. Potential Isomerization of Phenolic-Substituted
Pyridine Analogue 66
4.7
4
0.8 0.1
2.7 0.3
84.5 1.0
a
b
All analogues evaluated at 5 μM and 24 h. Values represent % Gli1
c
mRNA expression relative to OHC control (set as 100%). Values
represent up-regulation of Cyp24A1 mRNA normalized to DMSO
(set to 1.0).
The five-membered heterocycles (47−50) did not inhibit Hh
signaling nor did they activate VDR. Finally, the bicyclic aryl
analogues (51−52, 55−56, 58) had no effects on Hh or VDR
signaling. Taken together, these results indicate that a six-
membered ring is the optimal size for the aromatic A-ring
mimic.
The results for the first two subclasses of aromatic A-ring
analogues suggest that potent Hh inhibition can be achieved by
either incorporating a hydrogen bond donating substituent at
the 3- or 4-position or utilizing a 2- or 3-pyridine moiety. On
the basis of this SAR, a third class of analogues was designed to
incorporate multiple substituents within the aromatic A-ring
mimic to determine whether an additive effect could be
achieved. These analogues contained either multiple hydroxyl
groups (63−64) or a hydroxyl-substituted pyridine moiety
(65−66) (Scheme 4). Interestingly, while the 3,5-dihydroxy
substituted analogues that retained Hh inhibition at this
concentration (63−64) also demonstrated modest VDR
activation (Cyp24A1 up-regulation = 11−19-fold).
Following the single concentration studies, we chose
analogues from each subclass that demonstrated potent anti-
Hh activity for concentration-dependent evaluation in
C3H10T1/2, ASZ001 (murine BCC), and DAOY (human
MB) cells. We also performed a more extensive analysis of
VD3, 5, and 6 in these cellular models.16 Both ASZ and DAOY
cells have been utilized by our group and others as early stage
models of Hh signaling in cultured cancer cells. For these
studies, we chose to evaluate both Gli1 and Ptch expression to
provide further support that down-regulation of Gli1 is related
to Hh inhibition and not a result of other cellular mechanisms.
Each analogue down-regulated Gli1 in a concentration-
dependent fashion in C3H10T1/2 cells (Table 5). IC50 values
for these analogues (IC50 range = 1.0−4.7 μM) were
comparable to those previously identified for 5 and 6. In
addition, IC50 values for down-regulation of Ptch in C3H10T1/
2 cells closely followed those obtained for Gli1, highlighting the
correlation between the two target genes in MEFs and
providing strong support that this class of VD3 analogues is
reducing Gli1 via inhibition of Hh signaling in this model.
The results of the concentration-dependent studies in
cultured ASZ and DAOY cells provided an interesting contrast
to the data from the MEFs. First, while the IC50s for down-
regulation of Gli1 in ASZ for VD3 and the analogues (IC50
range = 2.1−8.1 μM) more closely mirrored those obtained in
C3H10T1/2 cells, VD3 and several of the analogues had no
effect on Gli1 expression in DAOY cells. In addition, there was
decreased correlation between down-regulation of Gli1 and
Ptch in both ASZ and DAOY cells. This may be the result of
alterations within the gene encoding Ptch in these particular
cancer cell lines. For example, loss of the wild-type PTCH1
allele is characteristic of the ASZ line and drives Gli1
overexpression.14 It is reasonable that Ptch1 mRNA modulation
will not reflect an appropriate picture of Hh signaling and thus
cannot be used to monitor Hh activity in these cells. Our
previous analysis with known Hh signaling pathway inhibitors
Scheme 4. Aromatic A-Ring Analogues with Multiple
a
Substitutions: Subclass III
a
Reagents and conditions: (a) DCC, DMAP, DCM, 17−18 or 29−30
(60−95%); (b) Pd(OH)2 (10%), H2, MeOH:THF (2:1) (10−75%).
(63) and 3,4,5-trihydroxy (64) benzene derivatives demon-
strated inhibition comparable to the singly substituted
analogues, the hydroxyl-substituted pyridines (65−66) ex-
hibited a significant decrease in activity (Table 4). It is possible
that the decrease in potency demonstrated for these analogues
is a result of isomerization in the aqueous environment between
the pyridinol and a pyridinone moiety (Chart 2). This was
observed in the benzylation of 26, where N-benzylation
competes with phenolic-benzylation, as reported in the
literature. Similar to the data for subclasses I and II, multiply
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Table 5. Concentration-Dependent Hh Inhibition for Representative VD3 Analogues
C3H10T1/2
ASZ
DAOY
Ptch
a
a
a
b
a
b
c
analogue
Gli1
Ptch
Gli1
Cyp24A1
Gli1
Cyp24A1
149 90
VDR binding
VD3
5
4.1 0.3
0.74 0.1
1.3 0.3
2.8 0.6
1.0 0.04
4.7 1.3
1.8 0.6
1.1 0.1
2.9 0.3
1.1 0.3
2.5 0.1
3.3 0.3
2.9 1.4
12.6 1.7
4.5 3.2
1.3 0.03
2.1 0.1
5.2 0.2
764 24
1.9 0.1
2.5 1.4
6.3 0.1
2.1 0.9
4.8 1.6
3.6 0.3
1.3 0.5
>10
3.7 0.04
9.2 1.7
>10
>10
3.6 0.02
>10
>100
>100
>100
>100
>100
>100
>100
>100
1.9 0.9
0.5 0.1
1.5 0.4
2.2 1.4
1.1 0.2
0.3 0.01
1.0 0.3
6
7.1
1
42
43
44
63
64
6.6 0.7
5.3 0.4
4.8 1.2
4.1 0.4
8.1 0.2
>10
>10
>10
>10
>10
5.7 0.9
9.2 1.7
>10
>10
a
b
IC50 values (μM) represent the Mean SEM of at least two separate experiments performed in triplicate. Values represent Cyp24A1 mRNA
c
expression relative to DMSO control (24 h, 5 μM). Values represent binding affinity for VD3 and VD3 analogues from at least two separate
experiments. Calcitriol (4) is used as a positive control for VDR binding.
Figure 1. Displacement of BODIPY-Cyc by VD3 and 5 in HEK293T cells overexpressing Smo in intact cells (A) and isolated membranes (B). Data
are from a representative experiment performed in triplicate.
(cyclopamine and 1) indicated that Gli1 down-regulation in
ASZ cells correlates well with their reported activities in the
standard Hh-dependent assay in MEFs;16 however, measure-
ment of Ptch1 expression failed to demonstrate significant
down-regulation even at high concentrations of 1, VD3, or
analogue (10 μM, data not shown). Similar observations were
made in the DAOY cells, with only analogue 5 demonstrating
Ptch1 down-regulation which correlated to the Gli1 data. As
noted above with the ASZ cells, mutations within PTCH1 have
been implicated in MB formation and this may explain these
inconsistencies across cell lines. Not surprisingly, none of the
analogues significantly induced up-regulation of Cyp24A1in
either cancer cell line. Finally, the ability of these analogues to
bind VDR was also analyzed in a fluorescence polarization-
based competition assay.13,15,16 We have demonstrated that
neither VD3 nor any of the analogues tested displace the tight-
binding fluorescent VDR ligand from full length human VDR in
this assay at concentrations up to 100 μM, suggesting a lack of
affinity for VDR (4 was used as a positive control in these
experiments).
The only reported studies designed to characterize the
mechanism through which VD3 inhibits Hh signaling have
focused on the ability of cyclopamine (Cyc) to displace tritiated
VD3 from an inducible form of human Smo in cultured yeast.17
In an attempt to adapt a similar assay to a cultured mammalian
cell system and to more fully explore the ability of VD3 and its
analogues to bind Smo, we evaluated the ability of VD3 and 5
to displace BODIPY-Cyc from HEK293T cells overexpressing
full length human Smo. Initially, we utilized flow cytometry to
evaluate BODIPY-Cyc bound to intact cells. While nonlabeled
Cyc displaced BODIPY-Cyc in a concentration-dependent
fashion, neither VD3 nor 5 demonstrated the ability to affect
Cyc-Smo binding interactions up to 10 μM (Figure 1A). As
these results were in direct contrast to those previously
reported and to verify that these results were not assay specific,
we also performed a fluorescence polarization assay with
membranes isolated from the same Smo overexpressing
HEK293T cells (Figure 1B). The results from this assay were
similar to those observed with the intact cells, i.e., neither VD3
nor 5 displaced BODIPY-Cyc at concentrations up to 10 μM.
In addition, we performed combination studies to determine
whether VD3 or 5 could potentially synergize with GDC-0449
to enhance anti-Hh activity (Supporting Information Figure 1).
Treatment with a single concentration of GDC-0449 (100 or
25 nM) did not result in a significant change in the IC50 value
of either VD3 or 5; however, GDC-0449 did enhance the
overall efficacy of both VD3 and 5, suggesting the potential for
additive effects. Similar results were demonstrated for the ability
of VD3 (5 μM) or 5 (1 μM) to affect the Hh inhibitory activity
of GDC-0449. While these results do not preclude the binding
of VD3 or 5 to Smo as the means through which they regulate
Hh signaling, they do suggest that neither compound binds in
the Cyc/GDC-0449 binding site.
As noted above, several of the VD3 analogues identified as
inhibitors of Hh signaling (5, 6, 43, and 44) also demonstrated
modest activation of VDR, as measured by Cyp24A1 up-
regulation (22−140-fold induction). Taken with our data
demonstrating that VD3 and its analogues do not bind to the
Cyc binding site on Smo, we sought to explore whether the
anti-Hh activity for VD3 and this class of analogues is a
downstream effect of VDR activation. For this study, we
knocked down VDR expression in C3H10T1/2 cells with
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VDR-specific siRNA to probe whether decreased expression of
VDR affected the overall Hh inhibitory activity for VD3,
calcitriol, and 5. Overall, treatment of C3H10T1/2 cells with
VDR-specific siRNA significantly reduced VDR expression and
did not affect Gli1expression (Figure 2). Throughout our
Figure 2. Expression of VDR, Gli1, and Cyp24A1 (mRNA) following
nontargeting (filled) or VDR-specific (unfilled) RNAi transfection.
Multiple experiments (>5) were performed, and data shown are from a
representative experiment performed in triplicate.
experiments, treatment with VDRi resulted in consistent
knockdown of VDR mRNA expression to 30−45% of
nontargeting controls. Not surprisingly, the decrease in VDR
expression resulted in a concomitant decrease in Cyp24A1
expression.
Once a suitable protocol was in place to reproducibly
knockdown VDR in C3H10T1/2 cells, we evaluated the effects
of Hh activation (OHCs) or inhibition (OHC + VD3, OHC +
calcitriol, or OHC + 5) on VDR, Gli1, and Cyp24A1 expression
(Figure 3). Neither activation of the Hh pathway nor pathway
inhibition with 5 had an effect on VDR expression (Figure 3A);
however, treatment with VD3 and calcitriol resulted in modest
up-regulation of VDR expression in cells treated with both
nontargeting and VDR-specific siRNA. VDR knockdown had
no effect on the ability of OHCs to activate Hh signaling;
however, concomitant treatment with either VD3, calcitriol, or
5 demonstrated interesting results. Knockdown of VDR
completely abolished the ability of VD3 to decrease Gli1
expression and inhibit Hh signaling (Figure 3B). VD3
maintained its ability to significantly decrease Gli1 expression
in cells treated with nontargeting siRNA, indicating the loss of
Hh inhibition for VD3 is not a general result of the transfection
protocol. By contrast, reduced VDR expression had no effect on
the ability of either calcitriol or analogue 5 to inhibit Hh
signaling. Finally, the reduced expression of VDR did not affect
the ability of VD3 or calcitriol to significantly up-regulate
Cyp24A1; however, the modest up-regulation of Cyp24A1 by 5
in nontargeting treated cells was completely abrogated in the
presence of VDRi.
Figure 3. Effect of VDR expression level on Hh inhibition and VDR
activation for VD3, 5, and calcitriol in C3H10T1/2 cells. Relative
expression of VDR (A), Gli1 (B), and Cyp24A1 (C) in response to
nontargeting (filled) and VDR-specific (open) RNAi transfection.
demonstrated that incorporation of a hydrophilic moiety in
either the 3- or 4-position of the aromatic ring resulted in
enhanced Hh inhibition and increased selectivity. Incorporating
five-membered or biaryl aromatic moieties significantly reduced
activity, suggesting that the six-membered phenyl ring is the
optimal size for the aromatic A-ring mimic. Finally, the addition
of multiple hydrophilic residues resulted in analogues that
retained potent pathway inhibition; however, no additive effects
were seen for analogues with multiple substituents. Concen-
tration-dependent analysis of several analogues in Hh-depend-
ent cancer cell lines further demonstrated the ability of this
DISCUSSION AND CONCLUSIONS
■
The initial identification of 5 as a potent, selective inhibitor of
Hh signaling (IC50 = 0.74 0.1) prompted the synthesis and
evaluation of a series of related analogues to identify structure−
activity relationships for this class of Hh pathway inhibitor. The
preparation of this class of VD3 analogues was performed in an
expeditious manner by linking aromatic functionalities designed
to mimic the A-ring of VD3 to Grundmann’s alcohol via an
ester bond. Overall, SAR for this class of compounds
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class of compounds to selectively inhibit Hh signaling in vitro.
Future generations of VD3-based analogues will explore the
role of the ester linkage, modify the side chain region and
incorporate functionality in the C-ring of the trans-hydrindane
core of Grundmann’s alcohol; all modifications designed to
develop a more complete understanding of SAR for Hh
inhibition of the VD3 scaffold.
these anti-Hh effects of calcitriol were not abrogated in Vdr−/−
fibroblasts.41 More specifically, the ability of calcitriol to inhibit
Hh signaling in vitro has been localized to a step downstream of
Ptch, suggesting its effects may also be mediated via Smo. As
our assays (C3H10T1/2) and those previously reported (ASZ
and fibroblasts) were performed in different cell lines, it is clear
that cellular context is essential for determining the
mechanisms through which VD3, calcitriol, and their analogues
inhibit Hh signaling. In addition, the development of chemical
probes that can definitively identify binding interactions
between these seco-steroids and the Smo CRD will be necessary
to further our understanding of whether the anti-Hh effects of
these sterols are mediated via direct binding to Smo, or
another, as yet unidentified, mechanism.
In conclusion, the synthesis and evaluation of a full series of
aromatic A-ring VD3 analogues has provided insight into the
structural requirements necessary for their potent, selective
inhibition of Hh signaling. Ongoing SAR studies for this class of
pathway inhibitors include modification to the ester linker as
well as the aliphatic side chain. The results from these studies
will provide a more comprehensive image of structural
modalities that enhance pathway inhibition for the VD3
scaffold.
An ester linkage between the northern CD-ring region and
the aromatic A-ring isostere was chosen for these VD3
analogues due to its ease of formation and because analogue
generation could be performed rapidly based on the parallel
coupling of 2 with a library of commercially available aromatic
acids. Esters are commonly used as prodrugs to provide
enhanced bioavailability for carboxylic acids; however, they are
not generally utilized as tethers in small molecule drugs due to
the abundance of esterases in the human body that can
contribute to their rapid metabolism. More recently, studies
have demonstrated that esterase activity can be both cell-type
and organelle-specific, resulting in enhanced stability of the
parent compound.34−37 Data from our studies provides strong
evidence that our ester-linked VD3 analogues are stable in cell
culture and that their anti-Hh activity is dependent on the
intact scaffold. The parent alcohol 2 is a modest inhibitor of Hh
signaling in C3H10T1/2 cells and is comparable to VD3 (IC50
values = 3.1 and 4.1 μM, respectively). Analogue 5 is
significantly more potent in this cell line with an IC50 value
of 0.74 μM. In addition, alcohol 2 is inactive against Hh
signaling at concentrations up to 10 μM in ASZ and DAOY cell
lines; however, analogue 5 demonstrates modest activity in
these cells (IC50 values = 5.2 and 3.7 μM, respectively). Finally,
if 2 were responsible for the activity of the ester-linked
analogues (through hydrolysis of the ester bond in the parent
scaffold), it would be reasonable to expect there would be no
discernible SAR and that our compounds would all
demonstrate anti-Hh activity comparable to 2. In fact, we see
clear SAR for each different substitution pattern. Taken
together, these data indicate that the ester-linked analogues
are significantly more potent than the parent alcohol in
multiple cellular contexts, providing strong evidence that the
intact scaffold and not Grundmann’s alcohol is primarily
responsible for the anti-Hh activity of the compounds.
Studies designed to explore the cellular mechanisms through
which VD3 and 5 inhibit Hh signaling focused on the two
cellular receptors previously associated with VD3, VDR and
Smo. As noted above, a tritiated analogue of VD3 was shown to
selectively bind yeast cultures in which expression of human
Smo had been chemically induced.17 In addition, this binding
was antagonized by the addition of Cyc. Results from our
binding experiments suggest that neither VD3 nor 5 bind Smo
in the Cyc binding pocket; however, this data does not preclude
binding to another region of Smo. The recent identification of a
second sterol binding site within the cysteine-rich domain
(CRD) of Smo provides another region to explore for VD3/
Smo binding interactions.38−40 Preliminary analysis of VDR-
mediated Hh inhibition for VD3 and 5 suggested that VDR is
required for the anti-Hh effects of VD3 but not for calcitriol or
analogue 5. The inability of VD3 to maintain Hh inhibition in
the presence of VDR knockdown was surprising for several
reasons. First, similar studies performed in ASZ cells utilizing
VDR-specific shRNA demonstrated that VDR knockdown did
not affect the ability of VD3 to down-regulate Gli1 mRNA
expression.11 In addition, as demonstrated by our lab13 and
others,41 calcitriol inhibits Hh signaling in vitro and in vivo and
EXPERIMENTAL SECTION
■
Chemical Synthesis. General Information. VD3 used for
chemical synthesis was purchased from HBCChem, Inc. Methyl
ester phenols were purchased from Sigma-Aldrich or Fisher Scientific.
ACS or HPLC grade methanol, acetone, tetrahydrofuran, and
anhydrous DMF were purchased from Fisher Scientific. Anhydrous
DCM (low water, <50 ppm water) was purchased from BrandNu
Laboratories, Inc. (J.T. Baker solvent). All reactions were run under an
inert atmosphere. NMR data was collected on a Bruker AVANCE 500
or 300 MHz spectrometer and analysis performed using MestReNova.
HRMS data was analyzed at the Mass Spectrometry Facility at the
University of Connecticut. Infrared (IR) analysis was performed on a
Shimadzu FTIR-8400S spectrophotometer and analyzed with IR
Solution software. The purity of all final compounds was determined
to be ≥95% by HPLC on an LC-6AD Shimadzu LC instrument,
equipped with Prominence auto sampler (SIL-20AC), UV/vis detector
(SPD-20A), and communications bus module (CBM-20A). Com-
pound purity was measured on 10 μL of sample (dissolved in MeOH
at 5−10 mM) on a Phenomenex Luna C18(2) 100A column (250 mm
× 4.60 mm, 5 μm) with precolumn (guard). One of four isocratic
methods were used depending on the analogue (method A, 100%
MeOH, 1.0 mL/min; method B, 98% MeOH:2% H2O, 1.0 mL/min;
method C, 90% MeOH:10% H2O, 1 mL/min; or method D, 90%
MeOH:10% H2O, 1.5 mL/min). Eluent was continuously monitored
(254 nm), and data analysis was performed with Shimadzu LC
Solution software.
General Procedure for Benzylation of Phenolic Esters. To a
solution of methyl ester phenol (6 mmol) in acetone (30 mL) was
added benzyl bromide (15 mmol) followed by potassium carbonate as
an anhydrous powder (36 mmol). The reaction was stirred at room
temperature until the starting phenol was consumed (by TLC, 12−18
h). When the reaction was complete, the mixture was filtered over a
Celite pad, washed with EtOAc, and concentrated. The crude residue
was purified by column chromatography (SiO2, 5% EtOAc in hexanes)
to afford the benzyl-protected phenols as a pale-yellow or clear oil
(11−12) or off-white solid (13−14, 23−24) in good yields (70−
95%). Characterization of intermediates 11−14 and 22 was consistent
with that previously reported.18−22
1
Methyl 6-(benzyloxy)-1-naphthoate (21). H NMR (500 MHz,
CDCl3) δ 8.97 (d, J = 9.4 Hz, 1H), 7.99 (dd, J = 7.3, 1.3 Hz, 1H), 7.80
(dd, J = 8.3 Hz, 1H), 7.38 (m, 2H), 7.35 (m, 1H), 7.30 (m, 2H), 7.23
(m, 2H), 7.17 (m, 1H), 5.10 (m, 2H). 13C NMR (126 MHz CDCl3) δ
169.9, 156.4, 136.4, 135.1, 132.0, 128.4, 128.1, 127.8, 127.4, 127.3,
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127.1, 126.8, 124.9, 120.2, 107.6, 69.8. DART-HRMS: m/z calcd for
C25H38ClO2NH3 [MNH4]+, 422.2826; found, 422.2803.
4-Chloro-VD3 Ester (34). HPLC: 98%, Rt = 14.8 min (method B).
1H NMR (500 MHz, CDCl3) δ 7.98 (d, J = 8.6 Hz, 2H), 7.41 (d, J =
8.5 Hz, 2H), 5.39 (m, 1H), 2.07 (m, 1H), 1.98 (m, 1H), 1.82 (m, 2H),
1.54 (m, 6H), 1.38 (m, 4H), 1.24 (m, 3H), 1.13 (m, 4H), 1.01 (s, 3H),
0.93 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 2.0 Hz, 3H), 0.86 (d, J = 2.0 Hz,
3H). 13C NMR (126 MHz CDCl3) δ 166.6, 139.0, 130.9, 129.3, 128.6,
72.6, 56.4, 51.5, 41.9, 39.8, 39.4, 35.9, 35.4, 30.5, 28.0, 27.0, 23.7, 22.8,
22.6, 22.5, 18.6, 18.0, 13.5. IR (film) vmax 2949, 2930, 2866, 1720,
1716, 1286, 1266, 1256, 1113, 1102, 1092, 1015, 752. DART-HRMS:
m/z calcd for C25H38ClO2NH3 [MNH4]+, 422.2826; found, 422.2803.
2-Methoxy-VD3 Ester (35). HPLC: 95%, Rt = 8.7 min (method B).
1H NMR (500 MHz, CDCl3) δ 7.81 (m, 1H), 7.46 (m, 1H), 6.97 (m,
2H), 5.39 (m, 1H), 3.89 (s, 3H), 2.02 (m, 2H), 1.81 (m, 2H), 1.51 (m,
7H), 1.34 (m, 3H), 1.23 (m, 2H), 1.11 (m, 4H), 0.97 (s, 3H), 0.92 (d,
J = 6.4 Hz, 3H), 0.87 (d, J = 2.2 Hz, 3H), 0.86 (d, J = 2.2 Hz, 3H). 13C
NMR (126 MHz CDCl3) δ 166.0, 159.1, 133.1, 131.5, 120.8, 119.9,
111.8, 71.9, 56.5, 55.7, 51.5, 41.8, 40.1, 39.4, 35.9, 35.4, 30.6, 28.0,
27.1, 23.7, 22.8, 22.6, 22.5, 18.6, 18.1, 13.2. IR (film) vmax 2948, 2932,
2866, 2852, 1723, 1699, 1599, 1490, 1465, 1435, 1301, 1250, 1162,
1129, 1071, 753. DART-HRMS: m/z calcd for C26H41O3 [MH]+,
401.3055; found, 401.3100.
General Procedure for Benzylation of (Hydroxy)pyridine Carbox-
ylic Acids. To a solution of carboxylic acid (10 mmol) in anhydrous
DMF (30 mL) was added benzyl bromide (15 mmol) and cesium
carbonate (60 mmol). The solution was stirred for 24 h at RT and
diluted with water (100 mL) and EtOAC (100 mL). The organic layer
was washed sequentially with saturated sodium bicarbonate (100 mL),
water, 10% citric acid (100 mL), and saturated sodium chloride (100
mL). The EtOAc was dried (Na2SO4), filtered, concentrated, and
purified by column chromatography (SiO2, 1−5% MeOH in DCM) to
yield the benzylated methyl esters as either a yellow or white solid in
moderate yield (65−70%). Characterization of intermediates 27−28
was consistent with that previously reported.23,24
4-((tert-Butoxycarbonyl)amino)benzoic Acid (32). 4-Aminoben-
zoic acid (1.5 g, 11 mmol) was dissolved in degassed (Ar-purged) 1,4-
dioxane (20 mL) and 5% sodium bicarbonate (25 mL). The solution
was cooled to 0 °C. While stirring vigorously, a solution of di-tert-butyl
dicarbonate (3.6 g, 16.5 mmol) in degassed (Ar-purged) 1,4-dioxane
(10 mL) was added dropwise. Following complete addition, the
reaction was allowed to warm to RT and stirred for an additional 24 h.
The solution was concentrated, redissolved in water (50 mL), and
acidified to pH ∼ 2 with dropwise addition of 3 N HCl. The aqueous
layer was washed with EtOAc (2 × 100 mL), the combined organics
were dried (Na2SO4), filtered, and concentrated. The crude residue
was purified by crystallization (EtOAc) and rinsed with cold EtOAc
and copious amounts of hexanes to provide 32 as an off-white solid in
good yield (70%).25
4-Methoxy-VD3 Ester (36). HPLC: >98%, Rt = 10.7 min (method
B). 1H NMR (500 MHz, CDCl3) δ 8.01 (d, J = 8.8 Hz, 2H), 6.93 (d, J
= 8.8 Hz, 2H), 5.37 (m, 1H), 3.86 (s, 3H), 2.06 (m, 1H), 1.98 (m,
1H), 1.82 (m, 2H), 1.52 (m, 6H), 1.39 (m, 4H), 1.24 (m, 2H), 1.14
(m, 4H), 1.03 (s, 3H), 0.93 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 2.0 Hz,
3H), 0.86 (d, J = 1.9 Hz, 3H). 13C NMR (126 MHz CDCl3) δ 166.2,
163.1, 131.5, 123.3, 113.5, 71.8, 56.4, 55.4, 51.6, 41.9, 39.9, 39.4, 35.9,
35.4, 30.6, 28.0, 27.1, 23.7, 22.8, 22.6, 22.5, 18.6, 18.0, 13.5. IR (film)
vmax 2948, 2931, 2866, 1709, 1606, 1511, 1271, 1252, 1166, 1160,
1109, 1100, 1032, 846, 769. DART-HRMS: m/z calcd for C26H41O3
[MH]+, 401.3055; found, 401.3101.
General Procedure for Saponification of Methyl or Benzyl Esters.
To a stirred solution of ester (5 mmol) in THF (20 mL) was added
potassium or lithium hydroxide (20%, aq; 20 mL). The solution was
stirred at room temperature or refluxed for 12−24 h and adjusted to
pH 7 with the dropwise addition of 3 N HCl upon completion of
reaction. The pH of the solution was then brought to ∼1−3 using 1 N
HCl (note: in the case of substrates 65 and 66, pH was maintained at
7 for the extraction). The aqueous layer was washed with EtOAc (2 ×
100 mL), and the combined organic layers were dried (Na2SO4),
filtered, and concentrated. The crude residue was purified by column
chromatography (SiO2, 30−50% EtOAc in hexanes) or crystallized
from EtOAc to afford pure acids as white to off-white solids in good to
excellent yield (45−90%). Characterization of intermediates 15−18
and 24 was consistent with that previously reported.22,26−29
1
2-Benzyloxy-VD3 Ester (37). H NMR (500 MHz, CDCl3) δ 7.80
(m, 1H), 7.47 (m, 2H), 7.38 (m, 3H), 7.30 (m, 1H), 6.98 (m, 2H),
5.42 (m, 1H), 5.19 (s, 2H), 1.98 (m, 2H), 1.81 (m, 1H), 1.69 (m, 1H),
1.51 (m, 5H), 1.42 (m, 2H), 1.34 (m, 3H), 1.26 (s, 1H), 1.14 (m, 6H),
1.00 (m, 1H), 0.93 (s, 3H), 0.91 (d, J = 6.5 Hz, 3H), 0.87 (dd, J = 6.6,
2.2 Hz, 6H). 13C NMR (126 MHz CDCl3) δ 166.0, 157.9, 136.7,
132.8, 131.3, 128.5, 127.7, 127.0, 121.8, 120.3, 113.6, 71.9, 70.5, 56.4,
51.5, 41.8, 40.0, 39.4, 35.9, 35.3, 30.6, 27.9, 27.1, 23.7, 22.8, 22.7, 22.5,
18.6, 18.0, 13.3. DART-HRMS: m/z calcd for C32H44O3 [MH]+,
477.3369; found, 477.3421.
1
4-Benzyloxy-VD3 Ester (38). H NMR (500 MHz, CDCl3) δ 8.06
1
6-(Benzyloxy)-1-naphthoic Acid (23). Yield 45%. H NMR (500
(d, J = 8.6 Hz, 2H), 7.46 (m, 4H), 7.37 (m, 1H), 7.04 (d, J = 8.6 Hz,
2H), 5.43 (m, 1H), 5.16 (s, 2H), 2.11 (m, 1H), 2.03 (m, 1H), 1.87 (m,
2H), 1.56 (m, 5H), 1.44 (m, 3H), 1.31 (m, 3H), 1.18 (m, 4H), 1.08 (s,
3H), 0.99 (d, J = 6.5 Hz, 3H), 0.92 (dd, J = 6.5, 1.9 Hz, 6H). 13C
NMR (126 MHz CDCl3) δ 166.2, 162.3, 136.3, 131.5, 128.6, 128.1,
127.4, 123.6, 114.4, 71.9, 70.0, 56.5, 51.6, 41.9, 39.5, 35.9, 35.4, 30.6,
28.0, 27.1, 23.7, 22.8, 22.6, 22.5, 18.6, 18.0, 13.5. DART-HRMS: m/z
calcd for C32H45O3 [MH]+, 477.3369; found, 477.3385.
MHz, CDCl3:MeOD) δ 8.97 (d, J = 9.4 Hz, 1H), 7.99 (dd, J = 7.3, 1.3
Hz, 1H), 7.80 (dd, J = 8.3 Hz, 1H), 7.38 (m, 2H), 7.35 (m, 1H), 7.30
(m, 2H), 7.23 (m, 2H), 7.17 (m, 1H), 5.10 (m, 2H). 13C NMR (126
MHz, CDCl3:MeOD) δ 169.9, 156.4, 136.4, 135.1, 132.0, 128.4, 128.1,
127.8, 127.4, 127.3, 127.1, 126.8, 124.9, 120.2, 107.6, 69.8. DART-
HRMS: m/z calcd for C25H38ClO2NH3 [MNH4]+, 422.2826; found,
422.2803.
General Ester Coupling Protocol. Grundmann’s alcohol 2 (50 mg,
0.19 mmol), DCC (117 mg, 0.57 mmol), and DMAP (70 mg, 0.57
mmol) were dissolved in anhydrous DCM (4 mL) in an oven-dried
round-bottom flask. Aromatic acid (0.57 mmol) was added and the
solution stirred at RT for 12 h. The solvent was reduced to
approximately 1.5 mL and the crude mixture purified with column
chromatography (SiO2, 4−60% EtOAc in hexanes) to yield coupled
products as clear oils in moderate to good yields (30−95%).
2-Chloro-VD3 Ester (33). HPLC: >98%, Rt = 16.0 min (method D).
1H NMR (500 MHz, CDCl3) δ 7.79 (m, 1H), 7.45 (m, 1H), 7.40 (m,
1H), 7.31 (m, 1H), 5.44 (m, 1H), 2.04 (m, 2H), 1.81 (m, 2H), 1.52
(m, 5H), 1.34 (m, 3H), 1.24 (m, 2H), 1.12 (m, 4H), 1.00 (m, 1H),
0.92 (s, 3H), 0.90 (s, 3H), 0.87 (d, J = 2.2 Hz, 3H), 0.85 (d, J = 2.2
Hz, 3H). 13C NMR (126 MHz CDCl3) δ 165.5, 133.4, 132.0, 131.0,
130.9, 126.4, 73.0, 56.4, 51.4, 41.8, 39.9, 39.4, 35.9, 35.3, 30.5, 27.9,
27.0, 23.7, 22.7, 22.7, 22.5, 18.5, 18.0, 13.2. IR (film) vmax 2949, 2938,
2866, 1730, 1470, 1434, 1294, 1266, 1250, 1135, 1116, 1048, 937, 918,
746. DART-HRMS: m/z calcd for C25H38ClO2NH3 [MNH4]+,
422.2826; found, 422.2805.
4-((tert-Butoxycarbonyl)amino)-VD3 Ester (39). 1H NMR (500
MHz, CDCl3) δ 7.98 (m, 2H), 7.42 (m, 2H), 6.62 (br s, 1H), 5.37 (m,
1H), 2.06 (m, 1H), 1.97 (m, 1H), 1.81 (m, 2H), 1.54 (m, 6H), 1.53 (s,
9H), 1.42 (m, 2H), 1.33 (m, 3H), 1.23 (m, 3H), 1.13 (m, 4H), 1.01 (s,
3H), 0.93 (d, J = 6.5 Hz, 3H), 0.86 (m, 6H). 13C NMR (126 MHz
CDCl3) δ 166.1, 152.1, 142.4, 130.8, 125.2, 117.3, 72.0, 69.4, 56.4,
51.6, 41.9, 39.9, 39.5, 35.9, 35.4, 30.6, 29.7, 28.2, 28.0, 27.0, 23.7, 22.8,
22.6, 22.5, 18.6, 18.0, 13.5. DART-HRMS: m/z calcd for C30H47NO4
[M]+, 485.3505; found, 485.3509.
4-Dimethylamino-VD3 Ester (40). Analogue 40 was afforded in
80% yield using the following alternate method: 4-Dimethylamino-
carboxylic acid (1 mmoL) was reacted with oxalyl chloride (2.6
mmoL) in DCM (10 mL) at 0 °C with 2 drops of DMF for 2 h. The
reaction was then stirred at room temperature for 16 h. The solvent
was removed and the mixture redissolved in DCM (6 mL), and 2 (0.5
mmoL in DCM, 3 mL) was added dropwise at room temperature and
stirred for 24 h. The reaction was quenched by addition of sodium
bicarbonate (satd aq; 30 mL), washed with DCM (1 × 50 mL), and
purified with column chromatography (SiO2, 5% EtOAc in hexanes).
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HPLC: 99%, Rt = 11.3 min (method B). 1H NMR (500 MHz, CDCl3)
δ 7.93 (m, 2H), 6.66 (m, 2H), 5.37 (m, 1H), 3.03 (s, 6H), 2.06 (m,
1H), 1.98 (m, 1H), 1.81 (m, 2H), 1.51 (m, 5H), 1.34 (m, 3H), 1.24
(m, 3H), 1.12 (m, 4H), 1.04 (s, 3H), 1.00 (m, 1H), 0.93 (d, J = 6.5
Hz, 3H), 0.88 (d, J = 2.0 Hz, 3H), 0.87 (d, J = 2.0 Hz, 3H). 13C NMR
(126 MHz, CDCl3) δ 166.8, 153.1, 131.1, 117.8, 110.7, 71.0, 56.5,
51.7, 41.9, 40.0, 39.4, 35.9, 35.3, 30.7, 29.6, 27.9, 27.1, 23.7, 22.7, 22.6,
22.5, 18.6, 18.0, 13.5. IR (film) vmax 3011, 3002, 2940, 2933, 2924,
2911, 2903, 2890, 2866, 2854, 1694, 1606, 1526, 1467, 1445, 1366,
1343, 1317, 1285, 1273, 1233, 1216, 1183, 1159, 1109, 1063, 946, 829,
757, 667. DART-HRMS: m/z calcd for C27H44NO2 [MH]+, 414.3372;
found, 414.3388.
2-Pyridyl-VD3 Ester (44). HPLC: >98%, Rt = 19.0 min (method D).
1H NMR (500 MHz, CDCl3) δ 8.76 (m, 1H), 8.04 (m, 1H), 7.80 (m,
1H), 7.43 (m, 1H), 5.47 (m, 1H), 2.04 (m, 2H), 1.82 (m, 2H), 1.52
(m, 5H), 1.40 (m, 2H), 1.31 (m, 2H), 1.22 (m, 2H), 1.11 (m, 4H),
1.02 (s, 3H), 0.91 (d, J = 6.6 Hz, 3H), 0.85 (d, J = 1.8 Hz, 3H), 0.84
(d, J = 1.9 Hz, 3H). 13C NMR (126 MHz CDCl3) δ 164.5, 150.1,
148.6, 136.8, 126.4, 124.6, 73.1, 56.3, 51.5, 41.8, 39.8, 39.4, 35.8, 35.3,
30.4, 27.9, 27.0, 23.7, 22.7, 22.6, 22.5, 18.5, 17.9, 13.4. IR (film) vmax
3060, 2946, 2942, 2920, 2905, 2863, 1737, 1716, 1583, 1571, 1466,
1436, 1383, 1366, 1305, 1276, 1244, 1130, 1135, 1086, 1060, 1044,
993, 939, 918, 745, 704, 618. DART-HRMS: m/z calcd for
C32H44NO2 [MH]+, 372.2903; found, 372.2889.
3-Pyridyl-VD3 Ester (45). HPLC: >98%, Rt = 25.0 min (method D).
1H NMR (500 MHz, CDCl3) δ 9.25 (br s, 1H), 8.78 (br s, 1H), 8.29
(m, 1H), 7.39 (m, 1H), 5.43(m, 1H), 2.07 (m, 1H), 1.99 (m, 1H),
1.81 (m, 2H), 1.54 (m, 5H), 1.40 (m, 2H), 1.33 (m, 2H), 1.24 (m,
3H), 1.12 (m, 4H), 1.01 (s, 3H), 0.92 (d, J = 6.5 Hz, 3H), 0.86 (d, J =
2.0 Hz, 3H), 0.85 (d, J = 1.9 Hz, 3H). 13C NMR (126 MHz CDCl3) δ
165.1, 153.1, 150.8, 137.0, 126.7, 123.3, 73.0, 56.4, 51.4, 41.8, 39.7,
39.4, 35.8, 35.3, 30.5, 27.9, 27.0, 23.7, 22.7, 22.6, 22.5, 18.5, 17.9, 13.5.
IR (film) vmax 2929, 2921, 2907, 2896, 2867, 1723, 1717, 1590, 1467,
1418, 1362, 1325, 1287, 1274, 1266, 1235, 1122, 1117, 741, 702.
DART-HRMS: m/z calcd for C32H44NO2 [MH]+, 372.2903; found,
372.2897.
4-Pyridyl-VD3 Ester (46). HPLC: >98%, Rt = 27.9 min (method D).
1H NMR (500 MHz, CDCl3) δ 8.77 (m, 2H), 7.84 (m, 2H), 5.42 (m,
1H), 2.07 (m, 1H), 1.98 (m, 1H), 1.81 (m, 2H), 1.53 (m, 5H), 1.36
(m, 4H), 1.23 (m, 3H), 1.12 (m, 4H), 1.01 (s, 3H), 0.92 (d, J = 6.5
Hz, 3H), 0.86 (d, J = 2.0 Hz, 3H), 0.85 (d, J = 1.9 Hz, 3H). 13C NMR
(126 MHz CDCl3) δ 164.9, 150.5, 138.0, 122.8, 73.4, 56.3, 51.4, 41.8,
39.7, 39.4, 35.8, 35.3, 30.4, 27.9, 26.9, 23.7, 22.7, 22.6, 22.5, 18.5, 17.9,
13.5. IR (film) vmax 2955, 2948, 2935, 2870, 2868, 1723, 1653, 1635,
1623, 1467, 1407, 1323, 1287, 1274, 1213, 1121, 1063, 938, 917, 849,
749, 707, 670. DART-HRMS: m/z calcd for C32H44NO2 [MH]+,
372.2903; found, 372.2892.
1381, 1309, 1216, 1149, 1111, 1064, 1032, 985, 873, 760, 667. DART-
HRMS: m/z calcd for C23H36O3 [M]+, 360.2664; found, 360.2668.
2-Thiophenyl-VD3 Ester (49). HPLC: >98%, Rt = 25.9 min
1
(method C). H NMR (500 MHz, CDCl3) δ 7.79 (m, 1H), 7.53 (m,
1H), 7.10 (m, 1H), 5.35 (m, 1H), 2.06 (m, 1H), 1.98 (m, 1H), 1.81
(m, 2H), 1.52 (m, 5H), 1.41 (m, 2H), 1.33 (m, 2H), 1.12 (m, 4H),
1.02 (s, 3H), 0.93 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 2.0 Hz, 3H), 0.86
(d, J = 2.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 162.0, 134.6,
133.0, 131.9, 127.6, 72.5, 56.4, 51.5, 41.8, 39.9, 39.4, 35.9, 35.3, 34.9,
30.5, 27.9, 27.0, 23.7, 22.7, 22.6, 22.5, 18.5, 17.9, 13.3. IR (film) vmax
2939, 2925, 2908, 2895, 2866, 2855, 2118, 1703, 1700, 1525, 1467,
1444, 1418, 1382, 1366, 1357, 1286, 1257, 1231, 1224, 1160, 1090,
1075, 1034, 938, 917, 861, 750, 715. DART-HRMS: m/z calcd for
C23H36O2SNH4 [MNH4]+, 394.2780; found, 394.2775.
3-Thiophenyl-VD3 Ester (50). HPLC: >99%, Rt = 39.6 min
1
(method C). H NMR (500 MHz, CDCl3) δ 8.08 (m, 1H), 7.53 (m,
1H), 7.30 (m, 1H), 5.35 (m, 1H), 2.06 (m, 1H), 1.97 (m, 1H), 1.81
(m, 2H), 1.51 (m, 5H), 1.38 (m, 4H), 1.24 (m, 3H), 1.13 (m, 4H),
1.02 (s, 3H), 0.93 (d, J = 6.5 Hz, 3H), 0.88 (d, J = 1.9 Hz, 3H), 0.86
(d, J = 1.9 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 162.6, 134.5,
132.2, 127.9, 125.8, 71.9, 56.5, 51.5, 41.9, 39.9, 39.4, 35.9, 35.4, 30.5,
27.9, 27.0, 23.7, 22.7, 22.6, 22.5, 18.6, 18.0, 13.4. IR (film) vmax 3146,
2977, 2947, 2929, 2924, 2896, 2863, 2856, 2845, 1722, 1704, 1523,
1519, 1463, 1444, 1408, 1382, 1374, 1261, 1253, 1203, 1187, 1159,
1100, 1060, 982, 943, 917, 873, 858, 841, 821, 747, 695. DART-
HRMS: m/z calcd for C23H36O2SNH4 [MNH4]+, 394.2780; found,
394.2779.
1-Naphthyl-VD3 Ester (51). HPLC: 97%, Rt = 15.9 min (method
B). 1H NMR (500 MHz, CDCl3) δ 8.99 (m, 1H), 8.19 (m, 1H), 8.01
(m, 1H), 7.88 (m, 1H), 7.62 (m, 1H), 7.52 (m, 2H), 5.54 (m, 1H),
2.09 (m, 2H), 1.86 (m, 2H), 1.57 (m, 6H), 1.36 (m, 3H), 1.27 (m,
2H), 1.15 (m, 4H), 1.03 (m, 1H), 1.01 (s, 3H), 0.94 (d, J = 6.5 Hz,
3H), 0.89 (d, J = 2.1 Hz, 3H), 0.88 (d, J = 2.3 Hz, 3H). 13C NMR (126
MHz, CDCl3) δ 167.2, 133.8, 132.9, 131.4, 129.6, 128.4, 127.9, 127.5,
126.1, 125.9, 124.4, 72.1, 56.5, 51.6, 41.9, 40.0, 39.4, 35.9, 35.4, 30.7,
28.0, 27.1, 23.7, 22.8, 22.8, 22.5, 18.6, 18.1, 13.4. IR (film) vmax 3051,
2947, 2933, 2923, 2909, 2866, 2850, 1713, 1594, 1510, 1466, 1382,
1374, 1365, 1274, 1244, 1235, 1215, 1160, 1132, 1076, 1063, 1003,
937, 812, 782, 757, 508. DART-HRMS: m/z calcd for C29H40O2 [M]+,
420.3028; found, 420.3022.
2-Naphthyl-VD3 Ester (52). HPLC: 96%, Rt = 17.0 min (method
B). 1H NMR (500 MHz, CDCl3) δ 8.62 (m, 1H), 8.09 (m, 1H), 7.96
(m, 1H), 7.88 (m, 2H), 7.56 (m, 2H), 5.49 (m, 1H), 2.09 (m, 2H),
1.89 (m, 2H), 1.57 (m, 6H), 1.44 (m, 1H), 1.36 (m, 2H), 1.27 (m,
3H), 1.16 (m, 3H), 1.13 (s, 3H), 0.96 (d, J = 6.5 Hz, 3H), 0.89 (d, J =
1.9 Hz, 3H), 0.88 (d, J = 1.9 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
166.6, 135.3, 132.4, 130.9, 129.3, 128.1, 128.0, 127.6, 126.5, 125.2,
72.3, 56.4, 51.6, 41.9, 39.9, 39.4, 35.9, 35.4, 30.6, 27.9, 27.0, 23.7, 22.7,
22.7, 22.5, 18.6, 18.0, 13.5. IR (film) vmax 3058, 2986, 2965, 2898,
2855, 2849, 2843, 2116, 1722, 1705, 1629, 1601, 1463, 1456, 1383,
1373, 1365, 1352, 1289, 1254, 1206, 1185, 1159, 1130, 1091, 954, 918,
860, 778, 761, 473. DART-HRMS: m/z calcd for C29H40O2 [M]+,
420.3028; found, 420.3024.
2-Furyl-VD3 Ester (47). HPLC: 99%, Rt = 7.5 min (method B). 1H
NMR (500 MHz, CDCl3) δ 7.58 (m, 1H), 7.11 (m, 1H), 6.50 (m,
1H), 5.38 (m, 1H), 2.05 (m, 1H), 1.96 (m, 1H), 1.81 (m, 2H), 1.50
(m, 5H), 1.38 (m, 4H), 1.23 (m, 2H), 1.12 (m, 4H), 1.00 (m, 1H),
0.99 (s, 3H), 0.92 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 2.0 Hz, 3H), 0.86
(d, J = 2.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 158.7, 146.2,
144.9, 117.2, 111.6, 72.2, 56.2, 51.3, 41.8, 39.7, 39.3, 35.8, 35.3, 30.4,
27.9, 27.0, 23.6, 22.8, 22.5, 18.5, 17.8, 13.1. IR (film) vmax 3141, 3121,
2962, 2945, 2934, 2927, 2920, 2912, 2887, 2851, 2253, 1794, 1712,
1580, 1568, 1461, 1397, 1374, 1301, 1277, 1228, 1174, 1158, 1115,
1013, 981, 941, 915, 763, 733, 615, 596. DART-HRMS: m/z calcd for
C23H36O3NH4 [MNH4]+, 378.3008; found, 378.2983.
3-Furyl-VD3 Ester (48). HPLC: >98%, Rt = 16.3 min (method C).
1H NMR (500 MHz, CDCl3) δ 7.98 (m, 1H), 7.41 (m, 1H), 6.74 (m,
1H), 5.33 (m, 1H), 2.04 (m, 1H), 1.95 (m, 1H), 1.79 (m, 2H), 1.50
(m, 5H), 1.37 (m, 3H), 1.22 (m, 2H), 1.12 (m, 4H), 1.00 (m, 1H),
0.97 (s, 3H), 0.92 (d, J = 6.4 Hz, 3H), 0.87 (d, J = 2.0 Hz, 3H), 0.86
(d, J = 2.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 162.8, 147.4,
143.5, 120.0, 109.8, 71.6, 56.5, 51.4, 41.8, 39.9, 39.4, 35.9, 35.3, 30.5,
27.9, 27.0, 23.7, 22.7, 22.6, 22.5, 18.5, 17.9, 13.2. IR (film) vmax 2932,
2925, 2901, 2895, 2865, 2858, 2851, 1726, 1704, 1699, 1637, 1457,
6-Benzyloxy-1-naphthyl-VD3 Ester (53). 1H NMR (500 MHz,
CDCl3) δ 8.92 (m, 1H), 8.03 (m, 1H), 7.88 (m, 1H), 7.49 (m, 2H),
7.42 (m, 3H), 7.35 (2H), 7.25 (m, 2H), 5.51 (m, 1H), 5.21 (s, 2H),
2.07 (m, 1H), 1.86 (m, 2H), 1.57 (m, 7H), 1.31 (m, 8H), 1.15 (m,
4H), 1.02 (m, 1H), 0.99 (s, 3H), 0.93 (d, J = 6.4 Hz, 3H), 0.88 (d, J =
2.2 Hz, 3H), 0.86 (d, J = 2.2 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
167.4, 156.6, 136.7, 135.2, 131.7, 128.6, 128.0, 127.8, 127.7, 127.5,
127.4, 127.1, 125.1, 120.4, 107.7, 72.1, 69.9, 56.5, 51.6, 41.9, 40.0, 39.4,
35.9, 35.4, 30.7, 29.7, 28.0, 27.1, 23.7, 22.8, 22.8, 22.5, 18.6, 18.1, 13.5.
DART-HRMS: m/z calcd for C36H46O3NH4 [MNH4]+, 544.3791;
found, 544.3792.
6-Benzyloxy-2-naphthyl-VD3 Ester (54). 1H NMR (500 MHz,
CDCl3) δ 8.53 (m, 1H), 8.04 (m, 1H), 7.86 (m, 1H), 7.75 (m, 1H),
7.49 (m, 2H), 7.42 (m, 2H), 7.35 (m, 1H), 7.28 (m, 1H), 7.25 (m,
1H), 5.46 (m, 1H), 5.21 (s, 2H), 2.10 (m, 1H), 2.04 (m, 1H), 1.86 (m,
2H), 1.54 (m, 6H), 1.42 (m, 1H), 1.35 (m, 2H), 1.25 (m, 3H), 1.15
(m, 3H), 1.11 (s, 3H), 1.03 (m, 1H), 0.95 (d, J = 6.5 Hz, 3H), 0.88 (d,
3732
dx.doi.org/10.1021/jm401812d | J. Med. Chem. 2014, 57, 3724−3736

Journal of Medicinal Chemistry
Article
J = 1.9 Hz, 3H), 0.87 (d, J = 1.9 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ 166.7, 158.5, 136.9, 136.4, 131.0, 130.7, 128.6, 128.1, 128.0,
127.5, 126.8, 126.1, 126.0, 119.8, 106.9, 72.2, 70.1, 56.5, 51.7, 41.9,
40.0, 39.4, 35.9, 35.4, 30.6, 28.0, 27.1, 23.7, 22.8, 22.7, 22.5, 18.6, 18.1,
13.6. DART-HRMS: m/z calcd for C36H46O3 [M]+, 526.3447; found,
526.3453.
4-Hydroxy-VD3 Ester (42). HPLC: 95%, Rt = 7.7 min (method A).
1H NMR (500 MHz, CDCl3) δ 7.96 (m, 2H), 6.87 (m, 2H), 5.80 (br
s, 1H), 5.37 (m, 1H), 2.07 (m, 1H), 1.98 (m, 1H), 1.81 (m, 2H), 1.51
(m, 5H), 1.34 (m, 3H), 1.23 (m, 3H), 1.12 (m, 4H), 1.03 (s, 3H), 0.93
(d, J = 6.4 Hz, 3H), 0.87 (dd, J = 6.6, 2.0 Hz, 6H). 13C NMR (126
MHz CDCl3) δ 166.5, 159.7, 131.8, 123.4, 115.2, 72.1, 56.4, 51.6, 41.9,
39.9, 39.4, 35.9, 35.4, 30.6, 28.0, 27.1, 23.7, 22.8, 22.6, 22.5, 18.6, 18.0,
13.5. IR (film) vmax 3329 (br s), 3018, 2949, 2937, 2931, 2868, 1679,
1608, 1592, 1514, 1466, 1443, 1383, 1367, 1311, 1276, 1216, 1164,
1160, 1122, 1100, 1062, 938, 851, 754, 700, 668. DART-HRMS: m/z
calcd for C25H37O3NH4 [MNH4]+, 404.3165; found, 404.3170.
6-Hydroxy-1-naphthyl-VD3 Ester (55). HPLC: 99%, Rt = 8.5 min
3,5-Bis(benzyloxy)-VD3 Ester (59). 1H NMR (500 MHz, CDCl3) δ
7.45 (m, 4H), 7.40 (m, 4H), 7.35 (m, 2H), 7.33 (m, 2H), 6.83 (m,
1H), 5.39 (m, 1H), 5.09 (s, 4H), 2.08 (m, 1H), 1.99 (m, 1H), 1.82 (m,
2H), 1.54 (m, 5H), 1.40 (m, 5H), 1.26 (m, 3H), 1.15 (m, 4H), 1.03 (s,
3H), 0.95 (d, J = 6.4 Hz, 3H), 0.90 (d, J = 2.3 Hz, 3H), 0.89 (d, J = 2.2
Hz, 3H). 13C NMR (126 MHz CDCl3) δ 166.0, 159.7, 136.4, 132.8,
128.5, 128.0, 127.4, 108.2, 107.1, 70.2, 56.4, 51.5, 41.9, 39.8, 39.4, 35.9,
35.4, 30.4, 27.9, 27.0, 23.7, 22.7, 22.6, 22.5, 18.5, 18.0, 13.5. DART-
HRMS: m/z calcd for C39H51O4 [MH]+, 583.3787; found, 583.3760.
3,4,5-Tris(benzyloxy)-VD3 Ester (60). 1H NMR (500 MHz, CDCl3)
δ 7.43 (m, 5H), 7.39 (m, 6H), 7.34 (m, 2H), 7.28 (m, 2H), 5.34 (m,
1H), 5.15 (m, 6H), 2.07 (m, 1H), 1.97 (m, 1H), 1.85 (m, 1H), 1.73
(m, 1H), 1.52 (m, 5H), 1.38 (m, 4H), 1.26 (m, 3H), 1.16 (m, 4H),
1.04 (m, 1H), 0.98 (s, 3H), 0.95 (d, J = 6.4 Hz, 3H), 0.90 (d, J = 2.1
Hz, 3H), 0.89 (d, J = 2.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
165.9, 152.4, 142.0, 137.4, 136.6, 128.5, 128.5, 128.1, 127.9, 127.9,
127.3, 125.9, 108.9, 77.2, 75.1, 72.3, 71.0, 56.4, 51.5, 41.9, 39.8, 39.4,
35.9, 35.4, 30.4, 28.0, 27.0, 23.7, 22.8, 22.5, 22.5, 18.5, 17.9, 13.4.
DART-HRMS: m/z calcd for C46H57O5 [MH]+, 689.4206; found,
689.4202.
1
(method B). H NMR (500 MHz, CDCl3) δ 8.90 (m, 1H), 8.04 (m,
1H), 7.82 (m, 1H), 7.45 (m, 1H), 7.23 (m, 2H), 6.04 (br s, 1H), 5.55
(m, 1H), 2.10 (m, 2H), 1.87 (m, 2H), 1.58 (m, 6H), 1.38 (m, 3H),
1.28 (m, 3H), 1.16 (m, 4H), 1.04 (m, 1H), 1.02 (s, 3H), 0.96 (d, J =
6.5 Hz, 3H), 0.91 (d, J = 2.0 Hz, 3H), 0.89 (d, J = 2.1 Hz, 3H. 13C
NMR (126 MHz, CDCl3) δ 167.8, 153.6, 135.3, 131.5, 127.8, 127.8,
127.4, 126.6, 125.1, 119.4, 110.0, 72.4, 56.4, 51.6, 41.9, 39.9, 39.4, 35.9,
35.3, 30.6, 27.9, 27.1, 23.7, 22.8, 22.7, 22.5, 18.6, 18.1, 13.4. IR (film)
vmax 3400, 2953, 2929, 2917, 2896, 2865, 1706, 1684, 1624, 1597,
1516, 1465, 1374, 1263 1233, 1217, 1196, 1133, 1063, 1007, 937, 834,
755, 720, 659. DART-HRMS: m/z calcd for C29H40O3NH4 [MNH4]+,
454.3321; found, 454.3347.
6-Hydroxy-2-naphthyl-VD3 Ester (56). HPLC: 96%, Rt = 8.8 min
1
(method B). H NMR (500 MHz, CDCl3) δ 8.54 (m, 1H), 8.03 (m,
2-Benzyloxy-3-pyridyl-VD3 Ester (61). 1H NMR (500 MHz,
CDCl3) δ 8.13 (m, 1H), 7.82 (m, 1H), 7.35 (m, 5H), 6.57 (m,
1H), 5.28 (m, 1H), 5.15 (m, 2H), 2.02 (m, 1H), 1.90 (m, 1H), 1.83
(m, 1H), 1.65 (m, 1H), 1.49 (m, 6H), 1.22 (m, 10H), 1.01 (m, 1H),
0.92 (d, J = 6.5 Hz, 3H), 0.88 (d, J = 2.1 Hz, 3H), 0.86 (d, J = 2.0 Hz,
3H), 0.84 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 164.0, 162.4,
142.2, 138.4, 135.3, 129.0, 128.6, 128.4, 119.8, 110.5, 72.5, 56.3, 52.5,
51.3, 41.8, 39.6, 39.4, 35.8, 35.3, 30.4, 27.9, 26.9, 23.7, 22.7, 22.5, 18.5,
17.8, 13.4. DART-HRMS: m/z calcd for C31H44NO3 [MH]+,
478.3321; found, 478.3329.
1H), 7.86 (m, 1H), 7.70 (m, 1H), 7.19 (m, 2H), 5.81 (br s, 1H), 5.48
(m, 1H), 2.10 (m, 1H), 2.05 (m, 1H), 1.86 (m, 2H), 1.55 (m, 6H),
1.42 (m, 1H), 1.36 (m, 2H), 1.27 (m, 3H), 1.15 (m, 3H), 1.11 (s, 3H),
1.02 (m, 1H), 0.95 (d, J = 6.5 Hz, 3H), 0.88 (d, J = 1.9 Hz, 3H), 0.87
(d, J = 1.9 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 167.0, 155.6,
137.0, 131.4, 131.0, 127.8, 126.4, 126.0, 125.8, 118.6, 109.4, 77.2, 72.4,
56.4, 51.6, 41.9, 39.9, 39.4, 35.9, 35.4, 30.6, 28.0, 27.1, 23.7, 22.8, 22.7,
22.5, 18.6, 18.1, 13.6. IR (film) vmax 3379, 2946, 2920, 2914, 2902,
2869, 1685, 1680, 1624, 1480, 1436, 1394, 1365, 1288, 1276, 1200,
1158, 1096, 943, 865, 758, 753, 668, 473. DART-HRMS: m/z calcd for
C29H40O3 [M]+, 436.2977; found, 436.2972.
2-Indyl-VD3 Ester (58). Analogue 58 was prepared according to the
alternate method listed for analogue 40. HPLC: 98%, Rt = 46.9 min
(method D). 1H NMR (500 MHz, CDCl3) δ 8.95 (br s, 1H), 7.69 (m,
1H), 7.42 (m, 1H), 7.32 (m, 1H), 7.19 (m, 1H), 7.15 (m, 1H), 5.43
(m, 1H), 2.09 (m, 1H), 2.02 (m, 1H), 1.86 (m, 2H), 1.55 (m, 6H),
1.43 (m, 2H), 1.35 (m, 2H), 1.15 (m, 4H), 1.09 (s, 3H), 1.02 (m, 1H),
0.95 (d, J = 6.5 Hz, 3H), 0.88 (d, J = 1.9 Hz, 3H), 0.87 (d, J = 1.8 Hz,
3H). 13C NMR (126 MHz, CDCl3) δ 161.9, 136.7, 127.9, 127.5, 125.2,
122.5, 120.7, 111.8, 108.2, 72.5, 56.5, 51.6, 42.0, 39.9, 39.4, 35.9, 35.4,
30.6, 29.7, 28.0, 27.0, 23.7, 22.8, 22.6, 22.5, 18.6, 18.0, 13.4. IR (film)
vmax 3480, 3436, 3323, 2953, 2932, 2919, 2867, 2854, 2848, 1683,
1528, 1466, 1457, 1395, 1382, 1341, 1309,1251, 1208, 1158, 1147,
1062, 985, 937, 817, 774, 751, 737. DART-HRMS: m/z calcd for
C27H42NO2 [MH]+, 410.3059; found, 410.3088.
2-Pyridyl-3-benzyloxy-VD3 Ester (62). 1H NMR (500 MHz,
CDCl3) δ 7.74 (m, 1H), 7.69 (m, 1H), 7.51 (m, 2H), 7.39 (m,
2H), 7.33 (m, 1H), 6.97 (m, 1H), 5.48 (m, 3H), 2.06 (m, 2H), 1.85
(m, 2H), 1.54 (m, 6H), 1.38 (m, 4H), 1.26 (m, 2H), 1.15 (m, 3H),
1.12 (s, 3H), 1.02 (m, 1H), 0.95 (d, J = 6.5 Hz, 3H), 0.89 (d, J = 2.1
Hz, 3H), 0.88 (d, J = 2.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
164.9, 163.1, 145.9, 138.9, 137.0, 128.3, 128.2, 127.8, 118.4, 115.1,
72.7, 67.7, 56.4, 51.5, 41.9, 40.0, 39.4, 35.8, 35.3, 30.5, 27.9, 27.0, 23.7,
22.7, 22.6, 22.5, 18.5, 18.0, 13.3. DART-HRMS: m/z calcd for
C31H44NO3NH4 [MNH4]+, 478.3321; found, 478.3350.
General Procedure for Benzyl Deprotection. To a solution of
benzyl-protected ester (0.15 mmoL) in MeOH:THF (2:1, 6 mL) was
added palladium hydroxide (10% on carbon; 10 mg). The mixture was
sealed with a rubber septum, sequentially purged with argon and
hydrogen, and stirred under positive hydrogen pressure (1 atm) at RT
for 12−18 h. The mixture was filtered through a Celite pad, rinsed
with EtOAC (10 mL), and concentrated. The crude residue was
purified using column chromatography (SiO2, 10−40% EtOAc in
hexanes) to yield final VD3 analogues in good yields (60−85%).
3-Hydroxy-VD3 Ester (5). Previously characterized.16 HPLC: >98%,
Rt = 7.4 min (method A).
2-Hydroxy-VD3 Ester (41). HPLC: 95%, Rt = 14.5 min (method B).
1H NMR (500 MHz, CDCl3) δ 11.0 (s, 1H), 7.88 (m, 1H), 7.48 (m,
1H), 7.01 (m, 1H), 6.91 (m, 1H), 5.46 (m, 1H), 2.12 (m, 1H), 2.04
(m, 1H), 1.86 (m, 2H), 1.57 (m, 5H), 1.45 (m, 3H), 1.36 (m, 2H),
1.28 (m, 3H), 1.17 (m, 4H), 1.06 (s, 3H), 0.96 (d, J = 6.8 Hz, 3H),
0.89 (d, J = 6.2 Hz, 6H). 13C NMR (126 MHz CDCl3) δ 170.2, 161.8,
135.4, 129.7, 119.1, 117.6, 113.0, 73.1, 56.4, 51.5, 41.9, 39.8, 39.4, 35.9,
35.4, 30.4, 28.0, 27.0, 23.7, 22.8, 22.6, 22.5, 18.6, 17.9, 13.5. IR (film)
vmax 3184, 3146, 3017, 2950, 2931, 2867, 1671, 1613, 1584, 1485,
1466, 1374, 1346, 1325, 1301, 1250, 1208, 1137, 1088, 1061, 1032,
937, 917, 883, 800, 755, 702, 666. DART-HRMS: m/z calcd for
C25H37O3 [M − H]+, 385.2743; found, 385.2736.
3,5-Bis(hydroxy)-VD3 Ester (63). HPLC: >98%, Rt = 16.6 min
1
(method C). H NMR (500 MHz, CDCl3) δ 7.14 (m, 2H), 6.58 (s,
1H), 5.57 (br s, 2H), 5.37 (m, 1H), 2.04 (m, 1H), 1.96 (m, 1H), 1.79
(m, 2H), 1.69 (m, 1H), 1.50 (m, 5H), 1.40 (m, 2H), 1.33 (m, 3H),
1.22 (m, 4H), 1.10 (m, 4H), 1.00 (s, 3H), 0.92 (d, J = 6.4 Hz, 3H),
0.86 (d, J = 6.8 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 166.0, 156.8,
133.1, 109.1, 107.2, 72.8, 56.4, 51.5, 41.9, 39.8, 39.4, 35.9, 35.4, 30.4,
28.0, 27.0, 23.7, 22.8, 22.6, 22.5, 18.6, 17.9, 13.6. IR (film) vmax 3430,
3019, 2947, 2934, 2866, 2399,1699, 1605, 1347, 1302, 1215, 780, 755,
669, 653, 627. DART-HRMS: m/z calcd for C25H38O4 [M]+,
402.2770; found, 402.2770.
3,4,5-Tris(Hydroxy)-VD3 Ester (64). Analogue 64 was ultimately
purified by using the following crystallization method: following two
column chromatographic separations that afforded an impure oily
mixture (30%), crude 64 was dissolved in diethyl ether inside a 1 dram
vial. This was subsequently placed inside a 30 mL vial that was filled
with 10 mL of pentanes. The cap was placed on the outer vial,
parafilmed, and covered with aluminum foil. After 3 days, pure white
crystalline compound had formed within the inner vial, which was
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filtered and repeatedly washed with pentanes. HPLC: >98%, Rt = 9.5
min (method D). 1H NMR (500 MHz, MeOD:CDCl3) δ 7.35 (s, 2H),
5.53 (m, 1H), 3.61 (s, 1H), 2.30 (m, 1H), 2.16 (m, 1H), 2.06 (m, 2H),
1.75 (m, 5H), 1.65 (m, 2H), 1.57 (m, 2H), 1.48 (m, 2H), 1.36 (m,
4H), 1.28 (s, 3H), 1.23 (m, 1H), 1.17 (d, J = 6.4 Hz, 3H), 1.10 (d, J =
2.0 Hz, 3H), 1.09 (d, J = 2.0 Hz, 3H). 13C NMR (126 MHz,
MeOD:CDCl3) δ 166.9, 144.3, 137.5, 120.6, 108.6, 71.6, 55.9, 51.1,
41.3, 39.4, 38.9, 35.3, 34.8, 29.9, 27.3, 26.4, 23.1, 22.0, 21.8, 21.6, 17.8,
17.3, 12.7. IR (film) vmax 3456, 3020, 2969, 2952, 2869, 1653, 1617,
1458, 1443, 1214, 1158, 1034, 768, 754, 749, 669. DART-HRMS: m/z
calcd for C25H37O4 [M − H]+, 417.2641; found, 417.2648.
BODIPY-Cyc (5 nM), and various concentrations of Cyc, VD3, or 5 at
37 °C for 4 h. Cells were trypsinized, centrifuged, and resuspended in
500 μL of phosphate buffered saline (PBS) for flow cytometry analysis.
To determine transfection efficiency, cells were fixed with 2% PFA (15
min), permeabilized with 90% ethanol (30 min, 4 °C), and blocked for
nonspecific binding with 10% FBS in PBS (15 min, RT). Cells were
incubated with anti-Myc antibody (1:800, Cell Signaling Technology)
for 1 h at RT, followed by Alexa Flour-647 donkey anti-rabbit antibody
(1:1000, Invitrogen) for 30 min at RT. Finally, cells were analyzed for
green fluorescence using flow cytometry.
For isolated membrane experiments, HEK293T cells were seeded
and transfected as described above. Cells were collected 24 h post-
transfection and washed with PBS (2×) and water (1×). Cells were
resuspended in hypotonic buffer (50 mM Tris-HCl, 1 mM EDTA, pH
7.5, protease inhibitor cocktail) and incubated at 4 °C for 10 min.
Cells were permeabilized by repeatedly passing them through a syringe
(25 times, 26 gauge). Cells were centrifuged (500g, 10 min) to pellet
the larger cellular components. The supernatant was removed and
recentrifuged (20000g, 30 min) to isolate the membranes incorporat-
ing the full-length Smo. The pellet was resuspended in hypotonic
buffer and stored (−80 °C). For the fluorescence polarization assay,
processed Smo-transfected membranes (10 μg/well) were incubated
with BODIPY-Cyc (5 nM) and various concentrations of Cyc, VD3, or
5 in hypotonic buffer (total volume of 100 μL) in a 96-well black-
sided, flat bottom plate (Costar). Membranes were incubated at 30 °C
for 5 h before FP was analyzed on a Synergy H1 hybrid multi-mode
microplate reader (excitation 485 nM, emission 528 nM).
VDRi Assay. C3H10T1/2 cells were seeded into 24-well tissue
culture treated plates 24 h prior to transfection. Lipofectamine 2000
(2.1 μL, Invitrogen) was mixed with mouse VDR-specific siRNA (2.5
μL of a 20 μM stock solution, Dharmacon) in OptimMEM reduced
serum media (95.4 μL, Life Technologies). Transfection solution (100
μL/well) was added dropwise to cells supplemented with BME (400
μL/well) containing 0.5% fetal bovine serum (Atlanta Biologicals) and
1% penicillin/streptomycin/L-glutamine (Cellgro) and plates returned
to the incubator (37 °C, 5% CO2). Following a 6 h incubation period,
transfection media was replaced with complete growth media and cells
incubated overnight. Cells were treated with VD3 (5 μM), calcitriol
(0.5 μM), 5 (5 μM), or control 24 h post-transfection. RNA isolation
and cDNA synthesis was performed 24 h post-treatment (48 h post-
transfection) using the Cells-to-Ct kit (Ambion) following the
manufacturer’s protocol and qPCR analysis of target genes followed
standard procedures.15,16
2-Hydroxy-3-pyridyl-VD3 Ester (65). HPLC: 97%, Rt = 15.8 min
1
(method D). H NMR (500 MHz, CDCl3) δ 12.92 (br s, 1H), 8.16
(m, 1H), 8.02 (m, 1H), 6.59 (m, 1H), 5.35 (m, 1H), 2.05 (m, 1H),
1.94 (m, 1H), 1.84 (m, 1H), 1.69 (m, 2H), 1.52 (m, 5H), 1.35 (m,
5H), 1.23 (m, 5H), 1.14 (m, 4H), 1.00 (m, 1H), 0.95 (s, 3H), 0.92 (d,
J = 6.5 Hz, 3H), 0.87 (d, J = 2.0 Hz, 3H), 0.86 (d, J = 1.9 Hz, 3H). 13C
NMR (126 MHz, CDCl3) δ 165.4, 163.9, 141.1, 139.3, 119.5, 111.7,
72.7, 56.3, 51.4, 41.8, 39.7, 39.4, 35.8, 35.3, 30.4, 29.6, 27.9, 27.0, 23.7,
22.8, 22.6, 22.5, 18.5, 17.9, 13.6. IR (film) vmax 3503 (br, weak), 3205
(br, weak), 2962, 2940, 2908, 2872, 2862, 2854, 1719, 1708, 1648,
1637, 1560, 1459, 1430, 1387, 1336, 1295, 1266, 1246, 1213, 1157,
1063, 1011, 979, 876, 757, 751, 725. DART-HRMS: m/z calcd for
C24H38NO3 [MH]+, 388.2852; found, 388.2856.
2-Pyridyl-3-hydroxy-VD3 Ester (66). HPLC: >98%, Rt = 16.2 min
(method D). 1H NMR (500 MHz, CDCl3) δ 10.3 (br s, 1H), 7.44 (m,
1H), 6.91 (m, 1H), 6.80 (m, 1H), 5.39 (m, 1H), 2.05 (m, 1H), 1.96
(m, 1H), 1.84 (m, 1H), 1.72 (m, 1H), 1.52 (m, 5H), 1.34 (m, 4H),
1.22 (m, 3H), 1.11 (m, 4H), 0.99 (m, 1H), 0.96 (s, 3H), 0.91 (d, J =
6.5 Hz, 3H), 0.86 (d, J = 2.0 Hz, 3H), 0.84 (d, J = 2.0 Hz, 3H). 13C
NMR (126 MHz, CDCl3) δ 162.9, 160.9, 139.7, 134.1, 126.8, 108.9,
74.7, 56.1, 51.1, 41.7, 39.4, 39.3, 35.7, 35.2, 30.1, 27.9, 26.8, 23.6, 22.7,
22.5, 22.4, 18.4, 17.7, 13.3. IR (film) vmax 3360 (br, w), 3017, 2952,
2933, 2891, 2869, 2855, 1723, 1657, 1611, 1467, 1296, 1277, 1260,
1215, 1158, 1139, 1060, 1001, 915, 819, 767, 753, 668. DART-HRMS:
m/z calcd for C24H38NO3NH4 [MNH4]+, 388.2852; found, 388.2854.
4-Amino-VD3 Ester (43). To 39 (58 mg, 0.15 mmol) in DCM (2
mL) was added trifluoroacetic acid (1 mL), and the solution stirred for
16 h. The solution was diluted with saturated sodium bicarbonate (20
mL) and the aqueous layer washed with DCM (2 × 30 mL). The
combined organic fractions were dried (Na2SO4), filtered, and
concentrated. The crude residue was purified using column
chromatography (SiO2, 30−60% EtOAc in hexanes) to afford 43 as
a clear oil in modest yield (65%). HPLC: 97%, Rt = 18.5 min (method
D). 1H NMR (500 MHz, CDCl3) δ 7.86 (d, J = 8.4 Hz, 2H), 6.64 (d, J
= 8.3 Hz, 2H), 5.35 (m, 1H), 4.03 (bs, 2H), 2.05 (m, 1H), 1.95 (m,
1H), 1.81 (m, 2H), 1.51 (m, 6H), 1.36 (m, 4H), 1.23 (m, 3H), 1.12
(m, 4H), 1.03 (s, 3H), 0.93 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 2.0 Hz,
3H), 0.86 (d, J = 2.0 Hz, 3H). 13C NMR (126 MHz CDCl3) δ 166.5,
131.5, 120.6, 113.7, 71.4, 60.3, 56.5, 51.7, 41.9, 40.0, 39.4, 35.9, 35.4,
31.5, 30.6, 28.0, 27.1, 23.7, 22.8, 22.6, 22.5, 18.6, 18.1, 14.1, 13.5. IR
(film) vmax 3451 (br, s), 3018, 2952, 2868, 1683, 1622, 1272, 1215,
1172, 1113, 851, 769, 749, 668. DART-HRMS: calcd for C25H39NO2
[M]+, 385.2981; found, 385.2968.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra for all new intermediates and final
VD3 analogues. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: 1-806-486-8446. Fax: 1-860-486-6857. E-mail: kyle.
hadden@uconn.edu.
Biological Assay Protocols. General Information. Protocols for
general cell culture, qPCR (Hh pathway and VDR), and VDR binding
assays are as previously described.15,16 20α-Hydroxycholesterol and
22(S)-hydroxycholesterol (OHCs) were purchased from Sigma-
Aldrich. VD3 for biological studies was purchased from Sigma-Aldrich.
Data was analyzed using GraphPad Prism 5, and reported values
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
represent mean
SEM for at least two separate experiments
Notes
performed in triplicate.
Fluorescence Binding Assays..30−33 For flow cytometry experi-
ments, HEK293T cells were seeded in 10 cm cell culture dishes (2.5 ×
106 cells) and incubated overnight (37 °C, 5% CO2). Cells were
transfected with Smo-Myc3 expression plasmid30 (24 μg) using
Lipofectamine 2000 reagent (60 μL) was added and the cells returned
to the incubator overnight. The next day, cells were split into a single
6-well plate and incubated overnight. The next day (2 days post-
transfection) cells were incubated in DMEM containing 10% FBS,
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge support of this work by the V
Foundation for Cancer Research (V Scholar, M.K.H.), the
Charles H. Hood Foundation, the University of Connecticut
Research Foundation, and the American Cancer Society (RSG-
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inhibition of hedgehog signaling. Bioorg. Med. Chem. Lett. 2012, 22,
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13-131-01). Daniel Madden, Daniel DeCarlo, and Steven M.
Lemieux recognize the University of Connecticut for Under-
graduate Research support. ASZ001 cells were provided by Dr.
Ervin Epstein (Children’s Hospital Oakland Research In-
stitute). The Smo-Myc3 expression plasmid was a kind gift from
James Chen (Stanford University).
(16) DeBerardinis, A. M.; Banerjee, U.; Hadden, M. K. Identification
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ABBREVIATIONS USED
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