Design, synthesis, and evaluation of hybrid vitamin D3 side chain analogues as hedgehog pathway inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.12.005

Vitamin D3 (VD3) is a moderately potent and non-selective inhibitor of the Hedgehog (Hh) signaling cascade. Previous studies have established that the CD-ring region of VD3 serves as the Hh inhibitory pharmacophore. Subsequently, compound 3, an ester link

Full text of DOI:10.1016/j.bmc.2014.12.005

Bioorganic & Medicinal Chemistry 23 (2015) 548–555
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and evaluation of hybrid vitamin D3 side chain
analogues as hedgehog pathway inhibitors
⇑
Upasana Banerjee, Albert M. DeBerardinis, M. Kyle Hadden
Department of Pharmaceutical Sciences, University of Connecticut, 69 N Eagleville Rd, Storrs, CT 06269, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Vitamin D3 (VD3) is a moderately potent and non-selective inhibitor of the Hedgehog (Hh) signaling cas-
cade. Previous studies have established that the CD-ring region of VD3 serves as the Hh inhibitory phar-
macophore. Subsequently, compound 3, an ester linked aromatic A-ring and CD-ring derivative was
identified as an improved and selective Hh inhibitor. Herein, we report modifications of the CD-ring side
chain that afford enhancement of selectivity for Hh modulation thereby diminishing the detrimental
effects of concomitant vitamin D receptor activation. In general, linear or moderately branched alkyl
chains of five or six carbons were optimal for potent and selective inhibition of Hh signaling. Moreover,
hybrid VD3 side chain derivative 20 demonstrated 4-fold improvement in Hh antagonistic activity over
Received 29 September 2014
Revised 24 November 2014
Accepted 3 December 2014
Available online 10 December 2014
Keywords:
Vitamin D3
Hedgehog signaling
Gli1
Basal cell carcinoma
Medulloblastoma
VD3(IC50 = 1.1–1.6
lM) while gaining greater than a 1000-fold selectivity for Hh signaling over canonical
activation of the vitamin D receptor pathway.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Significant efforts to develop cancer chemotherapeutics that act
by targeting Hh signaling have been undertaken in the last
decade.1,2 Consequently, GDC-0449 (Vismodegib/Erivedge™), the
first small molecule Hh inhibitor was approved for clinical use
against advanced/metastatic BCC in early 2012.5 In recent years,
several members of the vitamin D class of seco-steroids have been
identified as modulators of Hh pathway signaling.6,7 Previous stud-
ies undertaken in our lab revealed that vitamin D3 (cholecalciferol,
VD3, Fig. 1) mediated inhibition of Hh signaling in vitro is modest
Targeting embryonic signaling pathways is an emerging
approach to selectively treat several forms of human malignancy.
One such signaling cascade that has been amenable to therapeutic
intervention is the Hedgehog (Hh) signaling pathway.1,2 During the
embryonic stages of life, this critical signaling mechanism dictates
tissue patterning, proliferation and differentiation; however, it is
much less active and primarily responsible for maintaining stem
cell populations in skin and the central nervous system among
healthy adults. The key upstream mediators of the pathway
include a twelve transmembrane protein called Patched (Ptch)
receptor which responds to the presence or absence of Hh ligands
by correspondingly activating or repressing the action of a seven
transmembrane receptor called Smoothened (Smo). Under normal
conditions, Smo along with other proteins and protein complexes
tightly regulate the production of activator and repressor forms
of Hh target genes such as glioma-associated transcription factors
(Gli1, Gli2 and Gli3).1,2 Dysregulation and constitutive activation of
the pathway through mutations in either Ptch or Smo has been
linked to cancer initiation and progression; most notably, in basal
cell carcinoma (BCC) and medulloblastoma (MB).3,4 In addition,
ligand dependent over activation of Hh signaling has been associ-
ated with various other forms of cancers such as pancreatic ductal
adenocarcinoma, colon, ovarian, prostate, breast, and lung cancer.
(IC50 = 4.1 lM) and non-selective due to its ability to concurrently
activate canonical vitamin D receptor (VDR) signaling presumably
through metabolic conversion to an active VDR ligand (i.e.,
calcitriol).8,9 Thereafter, a medicinal chemistry approach was
undertaken to design analogues of VD3 that selectively target Hh
signaling.
Initial attempts to improve the potency and selectivity with
analogues prepared by facile modification of the 30-hydroxyl func-
tionality of the A-ring proved inadequate.8 Next, as a part of our
ongoing structure–activity relationship (SAR) studies to elucidate
the pharmacophore for selective inhibition of Hh signaling by
VD3, we probed the structural requirements of the alkyl side chain
appended to the CD-ring.10 Interestingly, truncation of the side
chain as in analogue 2 completely abolished inhibition of Hh sig-
naling, suggesting the aliphatic moiety was essential for Hh inhibi-
tion. A parallel series of SAR studies identified analogue 3 as a
potent (IC50 = 0.74 l
M) and selective inhibitor of Hh signaling.9,11
The structurally related steroidal natural product cyclopamine
(Cyc, 4) is also a well-known inhibitor of pathway signaling.
⇑
Corresponding author. Tel.: +1 (860) 486 8446; fax: +1 (860) 486 1553.
E-mail address: kyle.hadden@uconn.edu (M.K. Hadden).
http://dx.doi.org/10.1016/j.bmc.2014.12.005
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

U. Banerjee et al. / Bioorg. Med. Chem. 23 (2015) 548–555
549
O
OH
H
N
OH
( )5
O
O
H
H
H
O
O
H
6
a
H
5
8
10
, R = TBS
, R = H
b
3
1
VD3,
2
HO
HO
HO
OTBS
OR
6
RN
H
O
H
N
OH
H
O
( )5
O
O
H
O
H
N
4, Cyc; R=H
Ph
H
5, KAAD-Cyc; R=
H
H
( )5
a
N
H
H
HO
O
9
, R = TBS
11
b
Figure 1. Vitamin D3 and cyclopamine analogues.
, R = H
TBSO
7
RO
Attempts to optimize the potency and drug-like properties of Cyc
led to the identification of KAAD-Cyc, which incorporates an
extended ‘phenyl amino’ side chain moiety onto the piperidine ring
as an improved inhibitor of pathway signaling (approximate IC50
values = 300 and 30 nM, respectively).12,13 Using each of these
compounds as initial leads, we report herein the design, synthesis
and evaluation of a series of VD3-based analogues that incorporate
modified side chains as potent Hh pathway inhibitors.
Scheme 1. Synthesis of KAAD-VD3 hybrid analogues. Reagents and conditions: (a)
KAAD carboxylic acid,14 DCC, DMAP, anhydrous DCM, 80–89%; (b) TBAF, THF, 45–
60%.
analogues described below maintained the ester linkage and aro-
matic A-ring substituent of 3.
Complete ozonolysis of vitamin D2 (VD2) and subsequent
reduction of the resultant ozonide via a one-pot procedure pro-
vided the previously characterized Inhoffen–Lythgoe diol
(Scheme 2).15 Selective tosylation of the primary hydroxyl moiety
proceeded in excellent yield to afford the key CD-ring intermediate
12.16 For the synthesis of the A-ring aromatic mimic, the phenol of
methyl 3-hydroxybenzoate was protected as the methoxymethyl
ether, followed by hydrolysis of the methyl ester to provide 14 in
excellent yield.17
With the necessary precursors in hand, standard esterification
protocols afforded the intact ester 15 that we envisioned as a key
intermediate that could be directly derivatized through nucleo-
philic displacement of the tosyl moiety with alkyl and aryl Grig-
nard reagents (Scheme 3).18 Initial attempts to generate side
chain analogues through this method proved difficult. Overall
yields of the reaction were poor (<10%) and not reproducible even
following extensive modifications to the solvent system, reaction
time, temperature, and copper catalyst. Even with these difficul-
ties, a small series of analogues were generated through this
method (19–21, Scheme 3). In addition, the tosyl group of 15
was removed to provide ‘des-side chain’ analogue 22.19
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of KAAD-vitamin D3 side chain analogues
Initial analogues prepared to probe the side chain region for
optimal Hh pathway inhibition focused on incorporation of the
KAAD side chain into the VD3 scaffold. The steroidal nature of
Cyc, along with our structural overlays of VD3 and Cyc (Fig. 2, Spar-
tan) and the previous reports that VD3 and Cyc shared a binding
site on Smo,6 suggested that the KAAD and alkyl side chains could
potentially bind in a similar location and orientation within Smo.
To access the KAAD derivatives of VD3, we coupled the KAAD side
chain14 directly to the primary alcohol of either 6 or 710 via
standard esterification conditions to afford both the trans- and
cis-KAAD-VD3 hybrids 10 and 11 (5,6-olefin) (Scheme 1).
2.1.2. Synthesis of hybrid side chain analogues: Route 1
As noted, a concurrent SAR study identified analogue 3 as an
improved Hh pathway inhibitor.9 In addition to its increased
potency and selectivity, the synthetic route to access 3 and related
analogues is significantly reduced compared to compounds gener-
ated from intermediate 7 that incorporate the intact VD3 triene
and cyclohexyl A-ring. For these reasons subsequent side chain
2.1.3. Synthesis of hybrid side chain analogues: Route 2
Based on the difficulties associated with displacement of the
tosyl moiety of 15, we sought to explore whether improvements
in both yield and the number of Grignard reagents amenable to
Figure 2. Structural overlay of Cyc (blue) and VD3 (green).

550
U. Banerjee et al. / Bioorg. Med. Chem. 23 (2015) 548–555
2.1.4. Additional hybrid analogues
The preparation of additional hybrid side chain VD3 analogues
proceeded via the selective oxidative cleavage of vitamin D2
(VD2) to afford the unsaturated CD-ring/side chain secondary alco-
hol 44 (Scheme 5).21 Coupling of the alcohol with carboxylic acid
14 was followed by removal of the methoxymethyl ether to pro-
vide unsaturated side chain analogue 46. Subsequent reduction
of the olefin generated the saturated side chain analogue 47.
Finally, coupling of the TMS-protected Inhoffen–Lythgoe diol 4822
to the aromatic A-ring mimic provided analogue 50, which incor-
porated a primary hydroxyl in the side chain region (Scheme 6).
OTs
OH
a,b
c
H
H
H
HO
HO
Inhoffen-Lythgoe (IL) Diol
12
HO
VD2
O
O
OMe
OMe
O
OH
d
e
2.2. Biological evaluation
MOMO
HO
MOMO
14
13
The initial evaluation of all VD3 side chain analogues as Hh
pathway inhibitors was performed at a single dose (5 lM) by mon-
Scheme 2. Synthesis of CD- and A-ring precursors. Reagents and conditions: (a) O3,
anhydrous DCM/MeOH (3:1), (b) NaBH4, 40%; (c) TsCl, DMAP, Et3N, anhydrous DCM,
85%; (d) MOMCl, DIPEA, anhydrous DCM, 98%; (e) 10% NaOH, MeOH, 76%.
itoring endogenous Gli1 mRNA levels in C3H10T1/2 cells following
standard protocols.8–10 At this concentration, neither of the KAAD–
VD3 analogues (10 and 11) was active against Hh signaling. This
data, coupled with our recent findings that VD3 does not displace
BODIPY-Cyc from Smo, suggests that these two structures do not
share a similar binding orientation and SAR cannot be applied
R2
across the two scaffolds.11 At 5
lM, the majority of hybrid ana-
logues down-regulated Gli1 mRNA by approximately 90%, a level
comparable to that of lead 3. The subsequent evaluation of each
analogue at 2.5 lM provided more definitive SAR for this series
b
H
O
16
O
; R1 = MOM, R2 = Et
17; R1 = MOM, R2 = i-Bu
18
; R1 = MOM, R2 = Bn
OTs
c
19; R1 = H, R2 = Et
of analogues (Table 1).
20; R1 = H, R2 = i-Bu
R1O
21
; R1 = H, R2 = Bn
Analogues with linear side chains that were reduced (19, 22, 37,
38) or extended (41) in length compared to the natural VD3 chain
demonstrated reduced activity compared to lead analogue 3. Not
surprisingly, 39, which retains the 6-carbon chain length, demon-
strated activity comparable to 3. Several interesting effects on
the Hh inhibition were observed following introduction of
12
a
H
+
O
O
14
d,c
MOMO
15
H
O
O
22
HO
Scheme 3. Initial route to hybrid side chain analogues. Reagents and conditions: (a)
DCC, DMAP, anhydrous DCM, 40%; (b) CuI, RMgX, anhydrous THF, 5–10%; (c) TFA,
DCM, 80–89%; (d) NaBH4, DMSO, 80 °C, 56%.
a,b
c
VD2
H
O
O
H
HO
44
45
coupling could be achieved by carrying out this transformation
prior to formation of the ester bond.20 Our hypothesis was that
the steric bulk associated with the ester/aromatic A-ring moiety
hinders formation of the ligand–Cu complex, leading to poor reac-
tion progress and low yield. By contrast, the Cu–alkyl complex
should more readily displace the tosyl group in the presence of
the unsubstituted secondary alcohol of 12. While using this route
increased the number of synthetic steps required to generate each
analogue, it resulted in reaction reproducibility and improved
yields. Following displacement of the tosyl moiety of 12 with a ser-
ies of alkyl Grignards, coupling to the aromatic A-ring mimic pro-
vided intermediates 30–36. Removal of the methoxymethyl ether
groups from 30–36 provided corresponding hybrid VD3 analogues
37–43 in modest yields (Scheme 4).
MOMO
d
e
H
H
O
O
O
O
46
47
HO
HO
Scheme 5. Synthesis of VD2 hybrid side chain analogues. Reagents and conditions:
(a) KMnO4, THF, H2O, 65%; (b) Pb(OAc)4, DCM, Vitride™, DCM, 89% (c) DCC, DMAP,
anhydrous DCM, 14, 70%; (d) TFA, DCM, 65%; (e) H2/Pd(OH)2, MeOH; THF, 75%.
R
OH
37
; R = Me
OTMS
38; R = n-Pr
R
a
b,c
39
; R = n-Bu
H
H
IL Diol
a
b,c
O
O
O
O
40; R = i-Pr
12
H
41; R = n-Hex
OH
48
42
; R = t-Bu
H
OH
43; R = CH(Et)2
50
23 29
-
HO
HO
Scheme 4. Second route to hybrid side chain analogues. Reagents and conditions:
(a) CuI, RMgX, anhydrous THF 40–60%; (b) DCC, DMAP, 14, anhydrous DCM 60–70%;
(c) TFA, DCM, 80–90%.
Scheme 6. Synthesis of hydroxyl hybrid side chain. Reagents and conditions: (a)
TMSCl, TEA, DMAP, anhydrous DCM, 48%; (b) DCC, DMAP, anhydrous DCM, 14, 70%;
(c) TFA, DCM, 65%.

U. Banerjee et al. / Bioorg. Med. Chem. 23 (2015) 548–555
551
Table 1
It has been demonstrated previously that while the IC50 for VD3
inhibition of Hh signaling correlated well between the C3H10T1/2
and ASZ001 cell lines, lead analogue 3 was less active in the lat-
ter.9,11 Consistent with that observation, correlation of anti-Hh
activity for the hybrid side chain analogues between the two cell
lines was minimal (Table 1, Fig. 3). Among the potent, selective
analogues identified in the C3H10T1/2 cell line, only analogue 20
retained its anti-Hh activity in the BCC cell line. Several analogues
(19, 21, 38, 41, 50) that demonstrated modest activity in the
C3H10T1/2 cells were active in the BCC cell line, while the inhibi-
tory activity of others (37, 40, 46, 47) was completely abolished. It
is noteworthy, among the compounds showing improved activity
in this cell line, 38 and 41 demonstrated detrimental up-regulation
of Cyp24A1. In addition, compounds 21 and 50 demonstrated sig-
Selective in vitro inhibition of Hh signaling
Compd
Gli1 mRNAa (%)
2.5 lM
Cyp24A1b
VDRc
(lM)
5
l
M
OHC
VD3
2
100
1.0
ND
31.7 0.7
80.0 10.4
1.9 0.5
126 6.8
78.6 1.4
7.7 5.5
2.9 0.3
7.2 1.8
1.3 0.1
0.7 0.3
38.4 4.2
1.1 0.3
74.4 0.7
29.9 16.1
32.4 28.9
0.5 0.2
2.2 0.5
9.2 6.1
10.2 6.1
71.4 2.1
ND
15.0 2.0
ND
8336 38
1354 61
10.5 2.1
0.7 0.4
22.7 7.7
4.8 0.9
2.8 0.03
3.8 0.01
0.7 0.2
1.1 0.3
4.9 0.5
4.4 0.03
2.5 1.0
3.8 0.7
7.9 1.4
0.6 0.02
1.1 0.02
6.8 0.02
0.4 0.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3
10
11
19
20
21
22
37
38
39
40
41
42
43
46
47
50
ND
69.8 27.2
23.0 4.3
88.4 55.4
49.8 1.8
33.9 13.1
52.3 13.2
17.3 3.6
90.1 12.5
54.4 9.7
49.1 19.6
17.4 4.5
15.6 0.9
40.4 9.5
56.9 10.2
nificant anti-proliferative effects at concentrations of 5 lM. These
results indicate that additional non-specific mechanisms of activ-
ity/toxicity exist for these scaffolds in the ASZ001 cells and are
being investigated further.
Based on our preliminary results at a single concentration, three
compounds (20, 39, 43) that demonstrated selective and enhanced
anti-Hh activity consistently were evaluated for their ability to
inhibit Hh signaling in a concentration-dependent fashion. Each
of these demonstrated low micromolar IC50 values for inducing
Gli1 down-regulation in both C3H10T1/2 and ASZ cells (Table 2),
suggesting modest concentration-dependent inhibition of Hh sig-
naling in these model systems.
a
b
c
Relative Gli1mRNA levels compared to oxysterol (OHC) control.
Fold up-regulation relative to OHC treated controls.
Calcitriol (IC50 = 290 nM) is utilized as a positive control for VDR binding.
branched alkyl chains in this region. Analogues with short,
branched chains (40 and 42) were less potent; however, medium
length branched chains (20 and 43) salvaged the loss in potency
seen for the corresponding unbranched analogues with similar side
chain length. Analogue 47 combined optimal length and non-ter-
minal branching in the same structure; however, instead of a syn-
ergistic increase in activity for this compound, overall activity was
slightly reduced. Interestingly, slight modification through the
addition of an unsaturated olefin (46) restored the diminished
activity seen for the saturated analogue, resulting in a 2-fold
increase in activity for the more rigid side chain. Finally, incorpo-
rating an aromatic benzyl side chain (21) or a primary hydroxyl
(50) resulted in modest decreases in activity when compared to
the most active analogues.
After establishing the Hh inhibitory effects of modified side
chains, the selectivity profile of all the analogues was assessed by
measuring their ability to (1) bind recombinant VDR and (2) up-
regulate Cyp24A1 mRNA in C3H10T1/2 cells.9 Unlike calcitriol
(IC50 290 nM), none of these analogues or VD3, directly bind to
VDR even at high concentrations (P100 lM). As demonstrated
previously, C3H10T1/2 cells respond to VDR activation with robust
up-regulation of Cyp24A1, a well-characterized target gene of
canonical vitamin D signaling.8 Overall, each of the hybrid side
chain analogues maintained the high level of selectivity for Hh
inhibition previously demonstrated by analogue 3. Based on the
results, it appears that the changes to the A-ring region of the scaf-
fold are sufficient to mitigate any VDR-mediated effects associated
with modification to the side chain region.
Previous studies evaluating Hh pathway inhibition in cultured
cancer cells have suggested that the majority of in vitro cancer cell
lines fail to appropriately model in vivo Hh signaling.8 Therefore,
we pursued the evaluation of VD3 analogues in a cell line,
ASZ001, which has shown initial promise as in vitro model of onco-
genic Hh signaling. The ASZ001 cell line was developed from a vis-
ible BCC tumor isolated from a Ptch1+/ꢀ mouse.23 These cells
demonstrate loss of the wildtype Ptch1 allele, high baseline
expression of Gli1, and cellular morphology similar to Hh-depen-
dent BCC tumors. In addition, treatment of these cells with either
Cyc or VD3 resulting in Gli1 down-regulation has been previously
reported.9,24
Figure 3. Selective inhibition of Hh signaling in ASZ001 cells. ASZ001 cells were
incubated with analogue (2.5 lM, 48 h) or control and down-regulation of
endogenous Gli1mRNA (A) or up-regulation of Cyp24A1 (B) was determined
relative to DMSO control.

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U. Banerjee et al. / Bioorg. Med. Chem. 23 (2015) 548–555
Table 2
1.89 (m, 3H), 1.67 (m, 12H), 1.35 (m, 10H), 1.04 (d, J = 6.4 Hz,
3H), 0.59 (s, 3H). 13C NMR (125 MHz, CDCl3) d 173.8, 156.7,
149.1, 144.1, 140.9, 135.1, 126.2, 120.8, 116.2, 108.2, 69.3, 68.9,
56.2, 53.2, 49.1, 45.9, 40.3, 39.2, 38.6, 37.1, 36.1, 34.7, 34.1, 33.9,
31.8, 31.1, 29.7, 29.1, 28.9, 27.1, 26.3, 25.6, 24.9, 24.4, 23.4, 22.3,
17.3, 12.1. IR(film) vmax 3325, 2905, 2850, 2352, 2330, 2250,
1729, 1650, 1629, 1555, 1538, 1455, 1446, 1428, 1417, 1088,
1045, 1028, 755, 702, 641. microTOF-HRMS: (a) m/z calculated
for C37H54NO4: 576.4053 [MH]+. Found: 576.4035.
IC50 values for inhibition of Hh signaling
Compd
IC50 valuea
(lM)
C3H10T1/2
ASZ001
VD3
3
20
39
43
4.1 0.3
0.74 0.1
1.1 0.6
1.1 0.4
1.1 0.5
2.1 0.1
5.2 0.2
1.6 0.2
6.6 2.8
4.9 2.9
a
IC50 values represent down-regulation of Gli1 mRNA. All values are mean SEM
for at least two separate experiments performed in triplicate.
4.2.1.2. (S)-2-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-Hydroxy-2-methyl-
enecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-
1-yl)propyl 2-(3-phenylpropanamido)acetate (11). Analogue
11 was prepared in the same fashion as described above for ana-
logue 10 (45% yield, off-white solid). 1H NMR (500 MHz, CDCl3/
MeOD 19:1) d 7.18 (m, 2H), 7.10 (m, 3H), 6.60 (br s, 1H), 6.13
(m, 1H), 5.94 (m, 1H), 4.95 (m, 1H), 4.72 (m, 1H), 4.00 (m, 2H),
3.72 (m, 2H), 3.37 (m, 1H), 3.06 (m, 2H), 2.83 (m, 2H), 2.74 (m,
1H), 2.55 (m, 1H), 2.51 (m, 1H), 2.48 (m, 1H), 2.37 (m, 3H), 2.29
(m, 1H), 2.21 (m, 3H), 2.04 (m, 1H), 1.80 (m, 8H), 1.45 (m, 15H),
0.93 (d, J = 6.4 Hz, 3H), 0.47 (s, 3H). 13C NMR (125 MHz, CDCl3/
MeOD 19:1) d 174.1, 172.9, 157.6, 144.8, 141.0, 140.5, 138.4,
135.6, 128.1, 126.2, 125.8, 125.1, 121.5, 117.6, 112.1, 69.2, 68.9,
55.6, 55.1, 52.8, 45.5, 44.8, 40.1, 38.9, 37.9, 35.9, 33.8, 33.4, 28.6,
26.8, 25.9, 25.3, 24.6, 24.2, 23.1, 18.1, 16.9, 11.7. IR(film) vmax
3319, 3028, 2935, 2925, 2853, 2359, 2336, 2243, 1730, 1643,
1642, 1559, 1534, 1471, 1461, 1436, 1375, 1254, 1163, 1089,
909, 836, 774, 733, 698. DART-HRMS: (a) m/z calculated for
3. Conclusion
With the goal of identifying improved Hh pathway inhibitors
based on the VD3 scaffold, we have synthesized and evaluated a
series of analogues that incorporate modifications to the natural
alkyl side chain. During the initial phase of this study, a VD3-based
analogue incorporating an aromatic A-ring analogue (3) was iden-
tified in a parallel SAR study. Subsequently, side chain region ana-
logues focused on the preparation and evaluation of compounds
that incorporated this modified and improved VD3 scaffold. Over-
all, linear or moderately branched alkyl chains of five or six carbons
were optimal for potent and selective inhibition of Hh signaling.
The most potent analogue, 20, demonstrated inhibition of Hh sig-
naling in MEFs and BCC cells, comparable to lead compound 3.
The continued exploration of VD3 analogues as improved Hh path-
way inhibitors is ongoing.
C
37H54NO4: 576.4053 [MH]+. Found: 576.3989; (b) m/z calculated
for C37H51O3N: 558.3947 [MꢀH2O]+. Found: 558.3906.
4. Experimental section
4.1. General
4.2.2. Hybrid analogues: Route 1
4.2.2.1.
(1R,4S,7aR)-7a-Methyl-1-((S)-1-(tosyloxy)propan-2-
yl) octahydro-1H-inden-4-yl 3-(methoxymethoxy)benzoate
(15). Dicyclohexylcarbodiimide (0.34 g, 1.65 mmol), dimethyl-
aminopyridine (0.2 g, 1.64 mmol) and 12 (0.2 g, 0.55 mmol) were
dissolved in anhydrous CH2Cl2 and cooled to 0 °C. Carboxylic acid
14 (0.3 g, 1.65 mmol) in CH2Cl2 was added and the reaction mix-
ture warmed to rt and stirred for 12 h. The reaction mixture was
diluted with CH2Cl2 (30 mL), washed with 0.1 N HCl (30 mL) and
saturated NaCl (30 mL), dried over sodium sulfate and concen-
trated. Column chromatography (SiO2, 5–25% EtOAc in Hex) affor-
ded 15 as a clear oil in modest yield (40%). 1H NMR (500 MHz,
CDCl3) d 7.82 (s, 1H), 7.80 (s, 1H), 7.73 (dd, J = 2.6, 1.5 Hz, 1H),
7.70 (dt, J = 7.7, 1.3 Hz, 1H), 7.40–7.35 (m, 3H), 7.25 (m, 1H), 5.22
(d, J = 0.7 Hz, 2H), 3.99 (dd, J = 9.3, 3.0 Hz, 1H), 3.84 (dd, J = 9.3,
6.2 Hz, 2H), 3.50 (s, 3H), 2.48 (s, 3H), 2.02 (dt, J = 12.5, 3.2 Hz,
2H), 1.85–1.63 (m, 4H), 1.60–1.41 (m, 3H), 1.31–1.11 (m, 4H),
1.04 (s, 3H), 0.99 (d, 3H). 13C NMR (125 MHz, CDCl3) d 166.1,
157.2, 144.6, 132.2, 129.8, 129.4, 127.9, 123.0, 120.9, 117.2, 94.5,
75.4, 72.0, 56.1, 52.1, 51.3, 42.0, 39.6, 35.9, 30.4, 26.3, 22.6, 21.7,
17.9, 16.8, 13.5. DART-HRMS: m/z calculated for C29H38O7SNH4:
548.2682 [M+NH4]. Found: 548.2686.
VD2 and VD3 used for chemical synthesis were purchased from
HBC Chem, Inc. Methyl ester phenol and other chemicals were pur-
chased from either Sigma–Aldrich or Fisher Scientific. ACS or HPLC
grade methanol, acetone, and tetrahydrofuran were purchased
from Fisher Scientific. Anhydrous DCM (low water, <50 ppm water)
was purchased from BrandNu Laboratories, Inc. (J.T. Baker solvent).
Modeling and structural overlays were performed in Spartan ’10
Software. NMR data was performed on
a Bruker AVANCE
500 MHz spectrometer and analysis was done on MestReNova ver-
sion 8.0.0. HRMS data was collected on AccuTOF mass spectrome-
ter (JEOL USA, Peabody, MA) with a DART ionization source
(IonSense, Saugus, MA) and analyzed at the Mass Spectrometry
Facility at the University of Connecticut. Infrared (IR) analysis
was performed on a Shimadzu FTIR-8400S spectrophotometer
with IR Solution software.
4.2. Synthesis
4.2.1. KAAD–VD3 analogues
4.2.1.1. (S)-2-((1R,3aS,7aR,E)-4-((E)-2-((S)-5-Hydroxy-2-methyl-
enecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-
1-yl)propyl 2-(3-phenylpropanamido)acetate (10). To a solution
of 8 (12.6 mg, 0.018 mmol) in anhydrous THF (1 mL) was added
4.2.2.2.1. General procedure for tosyl group displacement. Com-
mercially purchased Grignard reagent (1.5 mmol) was added to a
solution of CuI (0.5 mmol) in anhydrous THF (2 mL) under argon
and cooled to 0 °C. To this mixture was added ester 15 (0.3 mmol)
in anhydrous THF and the solution stirred for 1 h at 0 °C and
quenched by the addition of H2O (10 mL). The solution was washed
with EtOAc (3 ꢁ 10 mL), 0.1 N HCl (3 ꢁ 10 mL), dried over sodium
sulfate, and concentrated. Column chromatography (SiO2, 0–20%
EtOAc in Hex) afforded coupled products as oils in poor yields
(5–10%).
tetrabutylammonium fluoride (40 lL, 0.037 mmol). The mixture
was stirred at rt for 12 h and subsequently stirred at 60 °C for
4 h. The mixture was diluted with EtOAc (10 mL), washed with
H2O (10 mL), and concentrated. Column chromatography (SiO2,
5–65% EtOAc in Hex) afforded off-white solid product 10 in modest
yield (60%). 1H NMR (500 MHz, CDCl3) d 7.31 (m, 2H), 7.22 (m, 3H),
6.56 (m, 1H), 5.90 (m, 1H), 5.39 (br s, 1H), 5.00 (m, 1H), 4.71 (m,
1H), 3.91 (m, 1H), 3.83 (m, 1H), 3.51 (m, 2H), 3.23 (m, 2H), 2.99
(m, 2H), 2.89 (m, 1H), 2.48 (m, 3H), 2.31 (m, 3H), 2.15 (m, 1H),
4.2.2.2.2. General procedure for methoxymethyl removal. To a
solution of coupled ester (0.1 mmol) in CH2Cl2, was added trifluro-
acetic acid (0.5 mL). The reaction mixture was stirred at rt and pro-

U. Banerjee et al. / Bioorg. Med. Chem. 23 (2015) 548–555
553
gress monitored by thin layer chromatography. Upon completion,
the reaction was neutralized with sodium carbonate (5 mL) and
washed with CH2Cl2 (2 ꢁ 10 mL). The combined organic layers
were dried over sodium sulfate and concentrated. Column chroma-
tography (SiO2, 0–15% EtOAc in Hex) provided final analogues as
clear oils.
4.2.3. Hybrid analogues: Route 2
Copper-mediated displacement of the tosyl moiety in 12, ester-
ification with the aromatic A-ring carboxylic acid, and removal of
the methoxymethyl protecting group was as described above for
Route 1.
4.2.3.1. (1R,4S,7aR)-1-((R)-sec-Butyl)-7a-methyloctahydro-1H-
inden-4-yl 3-hydroxybenzoate (37). Clear oil, yield 87%, 1H
NMR (500 MHz, CDCl3) d 7.65 (m, 1H), 7.61 (dd, J = 2.6, 1.5 Hz,
1H), 7.38–7.31 (m, 1H), 7.08 (m, 1H), 5.57 (s, 1H), 5.43 (q,
J = 2.8 Hz, 1H), 2.12–2.06 (m, 1H), 2.05–1.98 (m, 1H), 1.91–1.77
(m, 2H), 1.64–1.43 (m, 6H), 1.41–1.30 (m, 2H), 1.25 (m, 1H),
1.21–1.07 (m, 2H), 1.06 (s, 3H), 0.95 (d, J = 6.4 Hz, 3H), 0.89–0.83
(m, 3H). 13C NMR (125 MHz, CDCl3) d 166.7, 156.0, 132.5, 129.9,
122.1, 120.2, 116.6, 72.9, 56.1, 51.8, 42.1, 40.1, 36.8, 30.7, 29.9,
28.3, 27.1, 22.2, 18.2, 13.8, 10.5. IR (film) mmax 3329 (br s), 3146,
3017, 2950, 2931, 2867, 1671, 1613, 1584, 1485, 1466, 1250,
1208, 1137, 1088, 1061, 1032, 937, 917, 755, 702, 666. DART-
4.2.2.2. (1R,4S,7aR)-7a-Methyl-1-((R)-pentan-2-yl)octahydro-
1H-inden-4-yl 3-hydroxybenzoate (19). Clear oil, yield 85%,
1H NMR (500 MHz, CDCl3) d 7.65 (m, 1H), 7.59 (d, J = 6.9 Hz, 1H),
7.34 (m, 1H), 7.11–7.05 (m, 1H), 5.51 (s, 1H), 5.45–5.40 (m, 1H),
2.13–2.06 (m, 1H), 1.92–1.77 (m, 2H), 2.01 (m, 1H), 1.69–1.50
(m, 5H), 1.50–1.34 (m, 3H), 1.29 (t, J = 2.3 Hz, 3H), 1.21–1.11 (m,
1H), 1.06 (d, J = 2.7 Hz, 4H), 0.95 (m, 3H), 0.93–0.87 (m, 3H). 13C
NMR (125 MHz, CDCl3) d 166.6, 155.9, 132.5, 129.8, 122.1, 120.1,
116.5, 72.8, 56.7, 51.8, 42.1, 40.1, 38.3, 35.4, 30.7, 29.9, 27.3,
22.9, 19.4, 18.8, 18.2, 13.8. IR (film) mmax 3400 (br s), 2976, 2947,
2867, 2866, 1684, 1600, 1579, 1296, 1215, 1145, 1110, 946, 756,
680, 659. DART-HRMS: m/z calculated for
C22H32O3NH4:
HRMS: m/z calculated for C
21H30O3NH4: 348.2539 [M+NH4]+.
362.2695 [M+NH4]+. Found: 362.2714.
Found: 348.2564.
4.2.2.3. (1R,4S,7aR)-7a-Methyl-1-((R)-5-methylhexan-2-yl)octa-
hydro-1H-inden-4-yl 3-hydroxybenzoate (20). Clear oil, yield
89%, 1H NMR (500 MHz, CDCl3) d 7.65 (d, J = 7.5 Hz, 1H), 7.59 (d,
J = 6.8 Hz, 1H), 7.35 (t, J = 8.0 Hz, 1H), 7.13–7.03 (m, 1H), 5.42 (m,
1H), 2.08 (m, 1H), 2.05–1.97 (m, 1H), 1.93–1.71 (m, 2H), 1.56 (m,
4H), 1.49–1.30 (m, 6H), 1.28–1.09 (m, 4H), 1.05 (s, 3H), 0.96 (d,
J = 13.2, 6.2 Hz, 3H), 0.91 (dd, J = 6.6, 1.9 Hz, 6H). 13C NMR
(125 MHz, CDCl3) d 166.3, 155.7, 132.4, 129.7, 122.0, 119.9, 116.3,
72.6, 58.5, 51.6, 41.9, 39.8, 35.5, 31.9, 30.9, 30.7, 30.5, 29.7, 27.3,
23.0, 22.7, 22.4, 18.0, 13.7. IR (film) mmax 3385 (br s), 3019, 2951,
2935, 2868, 1689, 1599, 1589, 1466, 1453, 1366, 1344, 1295,
1215, 1157, 1106, 1075, 1062, 982, 946, 886, 755, 680, 668. DART-
HRMS: m/z calculated for C24H36O3NH4: 391.2848 [M+NH4]+.
Found: 391.2869.
4.2.3.2. (1R,4S,7aR)-1-((R)-Hexan-2-yl)-7a-methyloctahydro-1H-
inden-4-yl 3-hydroxybenzoate (38). Clear oil, yield 85%, 1H
NMR (500 MHz, CDCl3)
d 7.65 (d, J = 7.6 Hz, 1H), 7.58 (d,
J = 2.6 Hz, 1H), 7.39–7.31 (m, 1H), 7.12–7.04 (m, 1H), 5.43 (s, 1H),
5.41–5.31 (m, 1H), 2.14–2.05 (m, 1H), 2.05–1.96 (m, 1H), 1.92–
1.75 (m, 2H), 1.74–1.51 (m, 5H), 1.51–1.30 (m, 4H), 1.29–1.08
(m, 9H), 1.06 (s, 3H), 1.02–0.96 (m, 3H), 0.89 (t, J = 3.9 Hz, 4H).
13C NMR (125 MHz, CDCl3) d 166.4, 155.9, 132.5, 129.9, 122.2,
120.1, 116.5, 72.8, 58.7, 51.8, 42.1, 40.0, 30.9, 30.7, 29.9, 27.5,
23.2, 22.9, 22.6, 22.2, 18.2, 14.2, 13.9. IR (film) mmax 3305 (br s),
3234, 2951, 2931, 2867, 2775, 1610, 1584, 1485, 1466, 1364,
1346, 1250, 1208, 1137, 1086, 1062, 1032, 937, 917, 755, 715,
656. DART-HRMS: m/z calculated for C23H34O3NH4: 376. 2887
[M+NH4]+. Found: 376.2891.
4.2.2.4. (1R,4S,7aR)-7a-Methyl-1-((R)-4-phenylbutan-2-yl)octa-
hydro-1H-inden-4-yl 3-hydroxybenzoate (21). Clear oil, yield
80%, 1H NMR (500 MHz, CDCl3) d 7.64 (d, J = 7.6 Hz, 1H), 7.60–
7.57 (m, 1H), 7.33 (dd, J = 21.9, 7.9 Hz, 3H), 7.22–7.17 (m, 3H),
7.08 (dd, J = 8.1, 2.6 Hz, 1H), 5.57 (s, 1H), 5.42 (q, J = 2.7 Hz, 1H),
2.73 (m, 1H), 2.50 (m, 1H), 2.11 (m, 1H), 2.02 (m, 1H), 1.96–1.71
(m, 4H), 1.67 (q, J = 4.1 Hz, 1H), 1.64–1.45 (m, 4H), 1.44–1.33 (m,
1H), 1.29 (s, 2H), 1.24 (d, J = 7.3 Hz, 1H), 1.07 (d, J = 6.6 Hz, 3H),
1.05 (s, 3H). 13C NMR (125 MHz, CDCl3) d 166.4, 155.8, 143.3,
132.3, 129.7, 128.4, 128.3, 125.5, 121.9, 120.0, 116.3, 72.6, 56.2,
51.6, 42.0, 40.0, 37.9, 35.3, 32.5, 30.5, 27.0, 22.7, 18.6, 18.0, 13.6.
IR (film) mmax 3170, 2949, 2917, 2849, 1692, 1496, 1484, 1453,
4.2.3.3. (1R,4S,7aR)-1-((R)-Heptan-2-yl)-7a-methyloctahydro-
1H-inden-4-yl 3-hydroxybenzoate (39). Clear oil, yield 82%,
1H NMR (500 MHz, CDCl3) d 7.65 (dd, J = 7.5, 1.6 Hz, 2H), 7.35 (t,
J = 8.0 Hz, 1H), 7.13–7.03 (m, 1H), 5.79 (s, 1H), 5.42 (dd, J = 4.1,
2.2 Hz, 1H), 2.08 (m, 1H), 2.05–1.97 (m, 1H), 1.93–1.71 (m, 2H),
1.56 (m, 4H), 1.49–1.30 (m, 6H), 1.28–1.09 (m, 7H), 1.05 (s, 3H),
0.96 (dd, J = 13.2, 6.2 Hz, 3H), 0.91 (t, J = 7.1 Hz, 3H). 13C NMR
(125 MHz, CDCl3) d 166.7, 156.0, 132.4, 129.9, 122.1, 120.2,
116.6, 72.9, 56.6, 51.8, 42.1, 40.1, 35.8, 35.6, 32.5, 30.7, 27.2,
25.9, 22.9, 22.9, 18.8, 18.2, 14.3, 13.8. IR (film) mmax 3375 (br s),
3138, 3011, 2951, 2931, 2867, 1613, 1584, 1485, 1466, 1364,
1346, 1250, 1208, 1137, 1086, 1062, 1032, 937, 917, 755, 704,
657. DART-HRMS: m/z calculated for C24H36O3NH4: 390.3008
[M+NH4]+. Found: 390.3045.
1250, 1224, 756. DART-HRMS: m/z calculated for
C27H33O3:
405.2430 [MꢀH]+. Found: 405.2437; calculated for C27H34O3NH4:
424.2852, [M+NH4]+. Found: 424.2869.
4.2.3.4.
(1R,4S,7aR)-7a-Methyl-1-((R)-4-methylpentan-2-yl)
4.2.2.5. (1R,4S,7aR)-1-Isopropyl-7a-methyloctahydro-1H-inden-
4-yl 3-hydroxybenzoate (22). Clear oil, yield 84%, 1H NMR
(500 MHz, CDCl3) d 7.65 (m, 1H), 7.58 (m, 1H), 7.35 (m, 1H), 7.07
(m, 1H), 5.42 (m, 1H), 5.36 (s, 1H), 2.13–2.04 (m, 2H), 2.01 (dd,
J = 14.5, 3.8 Hz, 1H), 1.84 (m, 3H), 1.56 (m, 7H), 1.50–1.38 (m,
1H), 1.36–1.30 (m, 1H), 1.26–1.18 (m, 1H), 1.06 (d, J = 2.2 Hz,
4H), 0.98 (dd, J = 6.5, 2.2 Hz, 3H), 0.88 (dd, J = 6.6, 2.2 Hz, 3H). 13C
NMR (125 MHz, CDCl3) d 166.5, 155.8, 132.4, 129.7, 122.0, 119.9,
116.3, 72.6, 58.5, 51.6, 41.9, 39.8, 30.7, 30.5, 29.7, 27.3, 23.0,
22.7, 22.4, 18.0, 13.7. IR (film) mmax 3430 (br s), 2956, 2939, 2868,
2858, 1700, 1684, 1601, 1589, 1294, 1217, 1160, 1110, 946, 756,
octahydro-1H-inden-4-yl 3-hydroxybenzoate (40). Clear oil,
yield 88%, 1H NMR (500 MHz, CDCl3) d 7.65 (d, J = 7.5 Hz, 1H),
7.59 (d, J = 6.8 Hz, 1H), 7.35 (q, J = 7.9, 7.3 Hz, 1H), 7.07 (d,
J = 7.8 Hz, 1H), 5.42 (s, 2H), 2.17–2.08 (m, 1H), 2.02 (d,
J = 14.7 Hz, 1H), 1.94–1.77 (m, 2H), 1.55 (m, 9H), 1.22–1.10 (m,
1H), 1.08 (s, 3H), 1.06–0.97 (m, 1H), 0.93 (d, J = 6.7 Hz, 3H), 0.90
(d, J = 7.3 Hz, 4H), 0.87–0.82 (m, 3H). 13C NMR (125 MHz, CDCl3)
d 166.5, 155.9, 132.5, 129.8, 122.1, 120.1, 116.5, 72.8, 57.6, 51.8,
45.7, 42.2, 40.2, 33.7, 30.7, 29.9, 27.5, 24.9, 22.9, 21.5, 18.8, 18.2,
13.8. IR (film) mmax 3400 (br s), 3215, 2953, 2941, 2867, 2775,
1610, 1584, 1485, 1466, 1364, 1301, 1250, 1208, 1137, 1086,
1062, 1032, 937, 917, 715. DART-HRMS: m/z calculated for C23H34-
O3NH4: 376.2853 [M+NH4]+. Found: 376.2868.
680, 669. DART-HRMS: m/z calculated for
C20H28O3NH4:
334.2382 [M+NH4]+. Found: 334.2373.

554
U. Banerjee et al. / Bioorg. Med. Chem. 23 (2015) 548–555
4.2.3.5. (1R,4S,7aR)-7a-Methyl-1-((R)-nonan-2-yl)octahydro-1H-
inden-4-yl 3-hydroxybenzoate (41). Clear oil, yield 85%, 1H
NMR (500 MHz, CDCl3) d 7.65 (dd, J = 7.8, 1.3 Hz, 1H), 7.59 (dd,
J = 2.6, 1.5 Hz, 1H), 7.34 (t, J = 7.9 Hz, 1H), 7.10–7.04 (m, 1H), 5.45
(s, 2H), 5.43 (s, 1H), 2.12–2.05 (m, 2H), 2.05–1.98 (m, 1H), 1.93–
1.76 (m, 3H), 1.65 (s, 2H), 1.62–1.41 (m, 9H), 1.40–1.19 (m, 7H),
1.04 (d, J = 10.2 Hz, 3H), 0.99 (t, J = 6.9 Hz, 3H), 0.89 (dd, J = 6.8,
2.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) d 166.7, 156.1, 132.5,
129.8, 122.1, 120.2, 116.5, 72.9, 58.7, 51.8, 42.1, 40.0, 33.9, 30.9,
30.7, 30.3, 29.9, 29.8, 29.6, 27.5, 22.9, 22.6, 18.8, 18.2, 13.9, 13.8.
IR (film) mmax 3456 (br s), 2992, 2950, 2926, 2907, 1712, 1699,
1485, 1466, 1364, 1346, 1250, 1208, 1137, 1086, 1062, 1032,
937, 917, 715, 656. DART-HRMS: m/z calculated for C26H40O3NH4:
418.3982 [M+NH4]+. Found: 418.3301.
J = 6.6 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H), 0.78 (dd, J = 6.8, 3.4 Hz, 6H).
13C NMR (125 MHz, CDCl3) d 166.4, 155.7, 132.2, 129.6, 121.9,
119.9, 116.3, 72.6, 56.2, 51.5, 41.9, 39.8, 39.0, 35.8, 33.4, 31.4,
30.5, 30.4, 29.7, 27.0, 22.6, 20.5, 18.7, 18.0, 17.5, 15.4, 13.5. IR (film)
mmax 3400, 2956, 2939, 2868, 2858, 1700, 1684, 1601, 1589, 1294,
1217, 1160, 1110, 946, 756, 680, 669. DART-HRMS: m/z calculated
for C26H40O3NH4: 418.3321 [M+NH4]+. Found: 418.3306.
4.2.4.3. (1R,4S,7aR)-1-((S)-1-Hydroxypropan-2-yl)-7a-methyloc-
tahydro-1H-inden-4-yl 3-hydroxybenzoate (50)
Clear oil, yield 65%, alcohol 48 was esterified with carboxylic
acid 15 and the methoxymethyl protecting group removed using
the previously described protocols to afford final analogue 50 in
excellent yield as a clear oil (65%). 1H NMR (500 MHz, CDCl3) d
7.63 (d, J = 7.6 Hz, 1H), 7.56 (d, J = 2.1 Hz, 1H), 7.34 (t, J = 7.8 Hz,
1H), 7.09 (dd, J = 8.2, 2.8 Hz, 1H), 5.66 (s, 1H), 5.39 (d, J = 3.3 Hz,
1H), 4.76 (s, 1H), 4.32 (dd, J = 10.8, 3.6 Hz, 1H), 4.12 (m, 1H), 2.10
(m, 1H), 2.04–1.85 (m, 2H), 1.83–1.74 (m, 3H), 1.61 (m, 4H), 1.44
(m, 2H), 1.14 (d, J = 6.6 Hz, 3H), 0.97 (d, J = 7.8 Hz, 3H). 13C NMR
(125 MHz, CDCl3) d 166.7, 156.1, 132.1, 129.8, 122.0, 120.3,
116.4, 74.8, 70.0, 53.1, 51.0, 42.2, 39.6, 35.8, 30.4, 26.2, 22.6,
17.6, 17.4, 13.0. IR (film) mmax 3621, 3464, 3385 (br s), 3019,
2951, 2868, 1599, 1589, 1466, 1453, 1366, 1295, 1215, 1157,
1106, 1075, 1062, 982, 946, 886, 755, 680, 668. DART-HRMS: m/z
calculated for C20H27O3: 315.1960 [MꢀOH]+, Found: 315.1983.
4.2.3.6. (1R,4S,7aR)-1-((R)-4,4-Dimethylpentan-2-yl)-7a-methyl-
octahydro-1H-inden-4-yl 3-hydroxybenzoate (42). Clear oil,
yield 90%, 1H NMR (500 MHz, CDCl3) d 7.79–7.54 (m, 2H), 7.44–
7.31 (m, 1H), 7.18–6.99 (m, 1H), 5.80 (d, J = 12.1 Hz, 1H), 5.42 (s,
1H), 2.17–2.07 (m, 1H), 2.07–1.98 (m, 1H), 1.97–1.67 (m, 3H),
1.66–1.43 (m, 6H), 1.37 (dd, J = 14.4, 7.0 Hz, 2H), 1.25 (d,
J = 10.1 Hz, 1H), 1.20–1.11 (m, 1H), 1.09 (d, J = 1.7 Hz, 1H), 1.06–
0.99 (m, 1H), 0.99–0.85 (m, 11H). 13C NMR (125 MHz, CDCl3) d
166.5, 155.8, 132.3, 129.7, 121.9, 120.0, 116.4, 72.8, 57.8, 51.7,
49.7, 42.0, 40.0, 32.4, 31.1, 30.5, 30.4, 30.4, 29.7, 27.7, 22.7, 22.6,
18.0, 13.4. IR (film) mmax 3430 (br s), 3146, 3017, 2950, 2931,
2867, 1671, 1613, 1584, 1485, 1466, 1374, 1346, 1250, 1208,
1137, 1088, 1061, 1032, 937, 917, 755, 702, 666. DART-HRMS: m/z
calculated for C24H36O3NH4: 390.3008 [M+NH4]+. Found: 390.3030.
4.3. Biological evaluation
4.3.1. Cell line maintenance
C3H10T1/2 cells were purchased from American Type Culture
Collection (ATCC). ASZ001 cells were a generous gift of Dr. Ervin
Epstein (Children’s Hospital of Oakland Research Institute). Gibco
by Life Technologies culture media was purchased from ABI.
C3H10T1/2 cells were cultured in BME (Gibco) supplemented with
4.2.3.7. (1R,4S,7aR)-1-((R)-4-Ethylhexan-2-yl)-7a-methyloctahy-
dro-1H-inden-4-yl 3-hydroxybenzoate (43). Clear oil, yield
86%, 1H NMR (500 MHz, CDCl3) d 7.65 (m, 1H), 7.58 (dd, J = 2.7,
1.5 Hz, 1H), 7.35 (m, 1H), 7.07 (m, 1H), 5.42 (q, J = 2.9 Hz, 1H),
5.29 (d, J = 4.4 Hz, 1H), 2.16–1.98 (m, 2H), 1.98–1.76 (m, 2H),
1.64–1.29 (m, 8H), 1.28–1.04 (m, 8H), 1.01–0.79 (m, 10H). 13C
NMR (125 MHz, CDCl3) d 166.5, 155.8, 132.6, 129.84, 122.2,
120.0, 116.5, 72.8, 57.8, 51.8, 42.2, 39.8, 37.4, 33.6, 30.7, 29.9,
27.6, 26.8, 24.6, 22.9, 18.9, 18.2, 13.9, 11.5, 10.2. IR (film) mmax
3324 (br s), 3215, 2953, 2941, 2867, 2775, 1610, 1584, 1485,
1466, 1364, 1341, 1250, 1208, 1137, 1086, 1062, 1032, 937, 917,
10% FBS (Atlanta Biologicals, Premium Select), 1%
gro; 200 nM solution), and 0.5% penicillin/streptomycin (Cellgro;
10,000 I.U./mL penicillin, 10,000 g/mL). ASZ001 cells were cul-
L-glutamine (Cell-
l
tured in 154CF media, supplemented with 2% FBS (chelexed, heat-
inactivated), 1.0% penicillin/streptomycin, and a final concentration
of 0.05 mM CaCl2. Cells were maintained using the media described
above (denoted ‘growth’ media’). Media denoted as ‘low FBS’ con-
tained 0.5% FBS and the same percentage of other supplements as
specified for growth media (‘low FBS’ media was used for
C3H10T1/2 cell assays). Following plating and 24 h growth period,
no FBS supplemented media was used for ASZ001 cell assays. All
cells were grown in Corning Cell Culture, canted neck T75 or T150
flasks (Fisher Scientific) in an Autoflow IR water-jacketed CO2 incu-
bator (37 °C, 5% CO2). Experiments with C3H10T1/2 cells were per-
formed in BD Falcon sterile 60 mm dishes. Experiments with
ASZ001 cells were performed in BD Falcon 35 mm dishes or in 6-
well, 35 mm plates. DMSO was used as solvent to prepare all drug
solutions and the final DMSO concentration did not exceed 0.3%.
715, 656. DART-HRMS: m/z calculated for
C25H38O3NH4:
404.3208 [M+NH4]+. Found: 404.3230.
4.2.4. Additional side chain analogues
4.2.4.1. (1R,3aR,4S,7aR)-1-((2R,5R,E)-5,6-Dimethylhept-3-en-2-
yl)-7a-methyloctahydro-1H-inden-4-yl 3-hydroxybenzoate
(46). Clear oil, yield 65%, 1H NMR (500 MHz, CDCl3) d 7.61 (m,
2H), 7.31 (m, 1H), 7.06 (m, 1H), 5.98 (br s, 1H), 5.39 (m, 1H),
5.18 (m, 2H), 2.01 (m, 3H), 1.81 (m, 2H), 1.68 (m, 1H), 1.49 (m,
6H), 1.24 (m, 4H), 1.15 (m, 1H), 1.04 (m, 3H), 1.02 (d, J = 6.5 Hz,
3H), 0.91 (d, J = 6.8 Hz, 3H), 0.73 (m, 6H). 13C NMR (125 MHz,
CDCl3) d 166.3, 155.6, 135.4, 132.3, 132.0, 129.6, 122.0, 119.8,
116.2, 72.6, 56.3, 51.7, 42.8, 41.8, 39.9, 39.8, 33.0, 30.5, 27.5,
22.6, 20.8, 19.9, 19.6, 18.0, 17.6, 13.8.IR (film) mmax 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 calculated for
20a-Hydroxycholesterol, 22(S)-hydroxycholesterol (OHC) and
VD3, for biological studies, were purchased from Sigma–Aldrich.
4.3.2. Hh and VDR target gene expression in C3H10T1/2 cells
Cells (500 K, 5 mL) were plated in growth media at approxi-
mately 80% confluence. Once cells reached confluence (approxi-
mately 24 h post-plating), growth media was removed and
replaced with low FBS media (5 mL). This was followed by addition
of OHC, OHC and analogue, or DMSO (vehicle control). Cells were
incubated (37 °C, 5% CO2) for 24 h and RNA was isolated and eval-
uated by qRT-PCR analysis as described previously.8 Data was ana-
lyzed using GraphPad Prism 5 and IC50 values computed as
mean SEM from at least three separate experiments performed
in triplicate.
C
26H38O3NH4: 416.3165 [M+NH4]+. Found: 416.3171.
4.2.4.2. (1R,3aR,4S,7aR)-1-((2R,5S)-5,6-Dimethylheptan-2-yl)-
7a-methyloctahydro-1H-inden-4-yl 3-hydroxybenzoate (47)
Clear oil, yield 75%, 1H NMR (500 MHz, CDCl3) d 7.62 (m, 1H),
7.58 (m, 1H), 7.32 (m, 1H), 7.05 (m, 1H), 5.58 (m, 1H), 5.39 (m,
1H), 2.05 (m, 1H), 1.98 (m, 1H), 1.81 (m, 2H), 1.65 (m, 1H), 1.53
(m, 5H), 1.40 (m, 4H), 1.29 (m, 8H), 1.02 (s, 3H), 0.93 (d,

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Acknowledgements
We gratefully acknowledge support of this work by the Charles
H. Hood Foundation, the University of Connecticut Research
Foundation, and the American Cancer Society (RSG-13-131-01).
ASZ001 cells were provided by Dr. Ervin Epstein (Children’s
Hospital Oakland Research Institute).
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Supplementary data
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Supplementary data (intermediate synthesis and characteriza-
tion and NMR spectra for final analogues) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2014.12.005.
References and notes
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