Vitamin D receptor agonist/histone deacetylase inhibitor molecular hybrids

Article abstract of DOI:10.1016/j.bmc.2010.03.078

Incorporation of zinc-binding groups into the side-chain of 1α,25-dihydroxyvitamin D₃ (1,25D) fully bifunctional hybrid molecules which act both as vitamin D receptor agonists and histone deacetylase inhibitors. These bifunctional hybrids displ

Full text of DOI:10.1016/j.bmc.2010.03.078

Bioorganic & Medicinal Chemistry 18 (2010) 4119–4137
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Vitamin D receptor agonist/histone deacetylase inhibitor molecular hybrids
Marc Lamblin a, Basel Dabbas b, Russell Spingarn a, Rodrigo Mendoza-Sanchez a, Tian-Tian Wang b,
a,
Beum-Soo An b, Dao Chao Huang c, Richard Kremer c, John H. White b,c, , James L. Gleason
*
*
a Department of Chemistry, McGill University, Montreal, QC, Canada
b Department of Physiology, McGill University, Montreal, QC, Canada
c Department of Medicine, McGill University, Montreal, QC, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Incorporation of zinc-binding groups into the side-chain of 1
a
,25-dihydroxyvitamin D3 (1,25D) fully
Received 16 January 2010
Revised 30 March 2010
Accepted 31 March 2010
Available online 7 April 2010
bifunctional hybrid molecules which act both as vitamin D receptor agonists and histone deacetylase
inhibitors. These bifunctional hybrids display in vitro antiproliferative activity against the AT84 squa-
mous carcinoma cell line while lacking the in vivo hypercalcemic effects of 1,25D.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Vitamin D
Vitamin D receptor (VDR)
VDR agonists
Histone deacetylases (HDACs)
HDAC inhibitors
Transcriptional regulation
Zinc-binding groups
Hybrid molecules
Dual pharmacophores
1. Introduction
trichostatin A (TSA, 2; Fig. 1) and suberoylanilide hydroxamic acid
(SAHA, 3; Fig. 1), have been investigated as anti-cancer agents. Like
The hormonal form of vitamin D3, 1
a
,25-dihydroxyvitamin D3
VDR agonists, HDACi induce cell cycle arrest, cellular differentia-
tion and/or apoptosis.6–8 The potential of HDACi’s as therapeutics
is underscored by the recent approval of SAHA, under the trade
name Zolinza, for treatment of cutaneous T-cell lymphoma.9
(1,25D; 1; Fig. 1) has long been known for its central role in con-
trolling calcium homeostasis. A broad range of accumulating data
has provided evidence that 1,25D also has cancer chemopreventive
properties, and that it functions as a key regulator of innate and
adaptive immune responses.1–3 1,25D signals by binding to the
vitamin D receptor (VDR), a member of the nuclear receptor family
of ligand-regulated transcription factors.1,4 As the therapeutic
activity of 1,25D is limited by its capacity to induce hypercalcemia,
numerous laboratories have developed analogs of 1,25D that retain
VDR agonism but minimize its calcemic actions.5
Histone deacetylases (HDACs), along with the complementary
histone acetyl transferases (HATs), regulate the acetylation states
of histones and several other nuclear and non-nuclear proteins
such as tubulin and HSP90. HDACs and HATs act as modulators
of gene transcription by controlling DNA–histone interactions in
the nucleosome and through regulation of components of the tran-
scription machinery. Inhibitors of HDACs (HDACi), including
O
H
20
Me
25
Me
OH
N
OH
N
H
Me
H
Me
O
C
1
D
suberoylanilide hydroxamic acid
H
(3, SAHA)
19
O
A
1α,25-vitamin D3
OH
(1, calcitriol)
Me
Me
OH
3
HO
N
H
H
Me
O
O
H
OH
N
triciferol (4)
H
Me
Me Me
N
Me
HO
OH
trichostatin A (2,TSA)
* Corresponding authors. Tel.: +1 514 398 8498; fax: +1 514 398 7452 (J.H.W.);
tel.: +1 514 398 5596; fax: +1 514 398 3797 (J.L.G.).
E-mail addresses: john.white@mcgill.ca (J.H. White), jim.gleason@mcgill.ca (J.L.
Gleason).
Figure 1. Structures of 1,25D, HDACi’s and triciferol.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.078

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M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
We have been interested in developing bifunctional 1,25D
Inhoffen-Lythgoe diol 6 in 62% yield.14 Disilylation with TBSCl fol-
lowed by selective deprotection of the primary TBS group with
TBAF afforded alcohol 7 in 94% yield. To set the stage for side-chain
elongation, primary alcohol 7 was converted to the corresponding
iodide 8. In our hands, conversion of iodide 8 to either a cuprate or
organozinc reagent followed by conjugate addition to acrylate
derivatives was problematic.15,16 However, metal–halogen ex-
change with t-BuLi followed by addition to acrolein diethylacetal
cleanly effected an SN20 displacement to form enol ether 9 in a reli-
able 75% yield.17 An oxidation/deprotection sequence furnished ke-
tone 10 which subsequently underwent clean Horner coupling
with A-ring diphenylphosphine oxide (11) to furnish common pre-
cursor 12 as a single geometrical isomer.18
Ester 12 could be converted to a series of immediate hybrid pre-
cursors (Scheme 2). Simple saponification afforded acid 13 in high
yield. Homologation by one carbon could be achieved by reduction
of 12 with DIBAL to aldehyde 15 followed by Wittig olefination
with methoxymethylene triphenylphosphorane. Hydrolysis of the
resulting enol ether with TFA afforded aldehyde 17 which could
be oxidized to acid 18 in high yield. Primary amines 14, 16 and
19 could be generated, respectively, from 13 via Curtius rearrange-
ment using DPPA or from 15 and 17 via reductive amination of the
corresponding oximes.
As shown in Scheme 3, the precursors prepared above were
used to generate a wide variety of hybrids. Acids 13 and 18 were
transformed to their corresponding acid chlorides and then cou-
pled with either TBSONH2, or substituted versions thereof, to gen-
erate, after global removal of silicon protecting groups with HF in
acetonitrile, a series of hydroxamic acid and alkylhydroxamate hy-
brids. An N-hydroxyformamide (21) was also be prepared by treat-
ment of aldehyde 15 with hydroxylamine, reduction of the
resulting oxime with sodium cyanoborohydride and acylation with
trifluoroethyl formate. Additionally, an N-hydroxyurea (22) was
easily prepared by treatment of amine 16 with carbonyl diimidaz-
ole followed by addition of TBSONH2. ortho-Aminoanilides 23a,b
were prepared by coupling acid chlorides derived from 13 and 18
with 1,2-diaminobenzene. Amines 14 and 16 could also be acyl-
ated with S-acetylthioglycolic acid, bromoacetic acid, Boc-glycine
analogs that combine VDR agonism with inhibition of histone
deacetylase (HDAC) activity,10 as both 1,25D and HDACs are mod-
ulators of gene transcription. Notably, combinatorial effects of TSA
and 1,25D have been demonstrated on the proliferation of 1,25D-
resistant cancer cells.11 We have previously described the design,
synthesis and biochemical characterization of triciferol (4, Fig 1),
a molecule that combines agonism for the VDR with HDACi activity
and displays enhanced cytostatic and cytotoxic activity relative to
1,25D.10 The design of triciferol takes into account the need for a
bifunctional molecule to fit entirely within the ligand binding
pocket of the VDR. Simply linking 1,25D to TSA would create a mol-
ecule which would be too large to act as a VDR agonist. Thus, the
dienylhydroxamic acid, which provides affinity for the catalytic
zinc of HDAC active sites, was incorporated in the side-chain,
replacing the normal 25-OH of 1,25D.
In extending the scope of 1,25D/HDACi hybrids, we sought to
employ saturated, SAHA-like side-chains and, more importantly,
to incorporate a variety of terminal zinc-binding groups (ZBGs).
While hydroxamic acids are the most common ZBG in HDACi’s,
there is considerable interest in development of non-hydroxamate
HDACi’s, and a wide variety of alternative ZBGs have been re-
ported, most notably the ortho-aminoanilides.12,13 In the context
of a 1,25D/HDACi hybrid, it was not clear whether other ZBGs, par-
ticularly the large ortho-aminoanilides, could be incorporated
within the binding site of VDR. In this paper, we report the synthe-
sis a series of 1,25D/HDACi hybrids incorporating a wide variety of
ZBGs and show that there is indeed significant structural latitude
in the types of molecules that function effectively in this context.
2. Results
2.1. Chemistry
The synthesis of the hybrids makes use of a common precursor,
12, which may be prepared in nine steps from commercially avail-
able vitamin D2 (5, Scheme 1). Exhaustive ozonolysis of 5 followed
by a reductive workup with sodium borohydride furnished the
Me
Me
Me
Me
Me
Me
Me
Me
H
Me
OH
OH
O , MeOH/CH Cl ;
1) TBSCl, NaI, DMF
3
2
2
Ph P, I , imid.
3
2
H
H
NaBH
4
H
2) TBAF, NEt
3
87%
62%
H
H
7
94%
TBSO
HO
6
5
HO
Me
Me
Me
Me
Me
Me
OEt
OEt
I
1) PCC
t-BuLi;
2) HF, CH CN/CH Cl
2
H
H
H
O
3
2
OEt
OEt
75%
3) PCC
78%
86:14 E/Z
H
H
H
10
TBSO
TBSO
9
O
8
Me
Me
OEt
O
POPh
2
H
11
H
TBSO
OTBS
NaHMDS
71%
12
TBSO
OTBS
Scheme 1. Synthesis of a common intermediate for hybrids.

M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
4121
DPPA, NEt3;
NaOH
LiOH•H2O
Me
Me
OEt
THF/MeOH/H2O
OH
R* =
R*
R*
NH2
68%
97%
H
O
O
13
14
H
1) BnONH2
2) LiAlH4
H
NH2
DIBAL
R*
quant.
R*
80%
12
O
15
16
19
TBSO
OTBS
1) MeOCH2PPh3Br,
NaHMDS
2) TFA
84%
NaClO2
1) BnONH2
O
O
NaH2PO4
2) LiAlH4
R*
OH
R*
H
R*
NH2
74%
2-methyl-
2-butene
90%
17
18
Scheme 2. Synthesis of hybrid intermediates.
1) ClCOCOCl, DMF
2) TBSONHR1, iPr2NEt
or 2) R2ONHR1, iPr2NEt
1) EDC•HCl,
AcSCH2CO2H
O
R1
SH
R*
R*
NH2
R
R
N
OH
N
R*
R
OR2
n
n
H
2) NaOMe
3) HF, MeCN
n
n
3. HF, MeCN
O
O
14,16
24a n=1
24b n=2
13,18
20a n=1, R1,R2 = H
20b n=2, R1,R2 = H
20c n=2, R1 = Me, R2 = H
20d n=2, R1 = H, R2 = Me
20e n=2, R1,R2 = Me
1) EDC•HCl or DCC
RCH2CO2H
H
NH2
N
R
O
2) HF, MeCN or TBAF
1) NH2OH
2) NaCNBH3, AcOH
16
OH
25a R = NH2
25b R = NMe2
25c R = Br
H
N
H
R*
R*
R*
R
3) HCO2CH2CF3
4) HF, MeCN
EtSC(S)SNa
O
O
O
25d R = SC(S)SEt
15
16
21
O
S
1)
O
N
1) Im2CO
2) NH2OTBS
H
N
H
N
Boc
N
NH2
OH
R
R
OH
O
O
N
3) HF, MeCN
26
S
22
R
N
R
R*
NH2
H
n
n
or 1) RSO2Cl
2) HF, MeCN or TBAF
NH2
1) ClCOCOCl, DMF
2) o-Ph(NH2)2
27g n=2, R=Ph
H
N
27a n=1, R=Me
27b n=2, R=Me
27c n=2, R=CF3
27d n=2, R=Et
27e n=2, R=(CH2)2OH
27f n=2, R=nBu
14,16,19
27h n=2, R=4-CNPh
27i, n=3, R=Me
28a n=1, R=NH2
28b n=2, R=NH2
28c n=3, R=NH2
n
n
O
O
3) TBAF
13,18
23a n=1
23b n=2
O
P
O
Me
Me
OTBS
(MeO)2
OMe
OTBS
NaHMDS
Me
Me
H
H
H
R*
R*
CH2O2Me
R* =
R =
O
15
29
H
H
1) CsF, AcOH
2) MeNH2•HCl, Et3N
3) HF, MeCN
HF, MeCN
O
O
TBSO
OTBS
HO
OH
R
CO2Me
R
CONHMe
30b
30a
Scheme 3. Synthesis of VDR agonist/HDACi hybrids.
and N,N-dimethylglycine to afford other amide based hybrids. The
intermediate bromoacetamide above, prior to silicon protecting
group removal, could be further transformed into a trithiocarbon-
ate (25d) by addition of sodium ethyl trithiocarbonate (from eth-
2.2. Testing of bifunctional activities of hybrids
Compound function was screened using a series of well-defined
biochemical and cell-based assays to test for VDR agonism and
HDAC inhibition. Results of these screens are summarized in
ane thiol, NaHMDS and CS2).
A series of sulfonamides and
sulfamides were prepared easily by reaction of amines 14, 16
and 19 with alkyl and arylsulfonyl chlorides and sulfamidating
Table 1. Compounds were initially tested at 1 lM for their capacity
to induce expression of the CYP24 gene (Table 1 and Fig. 2A and B).
CYP24 encodes the enzyme that initiates catabolism of 1,1 and its
expression is exquisitely sensitive to VDR agonism. Hydroxamic
acid hybrids 20a and 20b, which differ only by one methylene unit,
displayed essentially identical potency, similar to that of 4, in
induction of CYP24 expression (Fig. 2A). Moreover, peak CYP24
agent 26, respectively. Finally,
a-ketoester and amide 30a and
30b were formed through a Horner–Wadsworth–Emmons olefin-
ation of aldehyde 15 with a silyloxy substituted phosphonate ester
followed by either full deprotection or partial deprotection fol-
lowed by aminolysis and final desilylation.

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M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
Table 1
Biochemical assessment of 1,25D/HDACi hybrids
Compound
Structure
VDR agonism
CYP24 FP (nM)
HDAC activity
Hypercalcemia
+++
Tubulin
Histone
HDAC2 (lM)
HDAC3 (
lM)
HDAC6 (lM)
OH
O
1
3
+++
13
ꢀ
ꢀ
SAHA
ꢀ
ꢀ
+++
+++
0.21
10.4
0.15
13.3
0.35
0.58
OH
4
+++
87
++
+
+
ꢀ
ꢀ
N
H
H
N
OH
20a
+++
ꢀ
3.2
9.3
3
O
O
O
OH
20b
20c
20d
20e
++
248
++
ꢀ
+
+
16.5
14.5
N
H
OH
++
ꢀ
ꢀ
ꢀ
N
Me
O
O
OMe
+++
+++
N
H
OMe
ꢀ
N
Me
OH
N
H
21
+++
ꢀ/+
++
ꢀ
+
O
H
N
H
N
OH
22
ꢀ
ꢀ
++
O
NH2
H
N
23a
524
185
++
460
104
330
ꢀ
ꢀ
O
O
O
23b
++
++
++
189
81.1
N
H
NH2
SH
24a
24b
25a
25b
25c
+
ꢀ
++
ꢀ
ꢀ
ꢀ
+
+
ꢀ
+
+
N
H
H
N
SH
++
ꢀ/+
ꢀ/+
ꢀ
36
96.3
32.3
1.75
ꢀ
O
O
O
O
H
N
NH2
NMe2
Br
H
N
H
N
H
N
S
SEt
25d
27a
27b
+++
+++
+++
ꢀ
++
+
ꢀ
O
S
O
O
S
213
321
+/ꢀ
++
730
718
71.3
191
N
H
Me
H
N
Me
ꢀ
S
O
O

M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
4123
Table 1 (continued)
Compound
Structure
VDR agonism
CYP24 FP (nM)
HDAC activity
HDAC2 ( M)
Hypercalcemia
Tubulin
+
Histone
++
l
HDAC3 (lM)
HDAC6 (
lM)
H
N
CF3
27c
27d
27e
27f
27g
++
14
695
153
S
S
S
O
O
H
N
Et
++
+
ꢀ
O
O
H
N
OH
++
+
ꢀ/+
ꢀ
O
O
H
N
Bu
S
+++
+++
29
15
ꢀ
++
725
508
101
293
ꢀ
ꢀ
O
O
H
N
Ph
S
++
O
O
CN
H
N
27h
+++
ꢀ/+
+
S
O
O
O
O
S
27i
+++
+++
++
ꢀ/+
+
+
N
Me
H
O
O
S
28a
28b
28c
ꢀ
++
+
N
NH2
H
H
N
NH2
S
280
196
+
470
605
50.4
31.6
O
O
O
O
S
+
++
N
H
NH2
O
O
OMe
30a
30b
+++
+++
ꢀ
+/ꢀ
O
NHMe
+
+
90.4
36
64
O
VDR agonism was assessed by RT-PCR analysis of CYP24 induction in SCC4 cells and direct binding to VDR was assessed using a fluorescence polarization (FP) assay. HDAC
activity in SCC4 cells was assessed by measuring induction of tubulin and histone hyperacetylation using Western blot analysis. Direct inhibition of purified HDACs was
measured using a fluorometric assay. Hypercalcemia assessed in mice at 240 or 1200 pmol/day.
levels induced by 20a and 20b were within a factor of two of that
induced by 1. Assaying other hybrids with wide-ranging ZBG’s
including ortho-aminoanilides 23a,b, thioglycolate amides 24a,b,
fying the functionality of chain terminal groups in analogs of 1
without markedly sacrificing affinity for the VDR.
As in our previous study,10 HDACi activity was initially assessed
sulfonamides 27a–i, sulfamides 28a–c and
a-ketocarbonyls
by screening the capacity of compounds at 1 lM concentration to
30a,b, showed moderate to strong agonism for the VDR. Con-
versely, amides 25a,b,c and N-hydroxyurea 22 exhibited little or
no VDR agonism and were not tested further.
induce acetylation of histones and tubulin (Table 1, Fig. 2D). The
potential function of hydroxamic acids 20a and 20b as HDACi’s
was tested initially by assessing of levels of acetylated tubulin
and histones H3 and H4 in SCC4 cells by Western blotting of ex-
tracts of vehicle- or compound-treated cells, using 2 and 4 as posi-
tive controls. Treatment for 24 h with 20a and 20b induced modest
increases in histone acetylation, similar to results previously ob-
tained with 4.10 Remarkably, while 20b induced robust hyperacet-
ylation of tubulin, 20a, which is shorter by only one methylene
unit, consistently had no effect (Fig. 2D). In addition to simple
hydroxamic acids, N-methyl, O-methyl and N,O-dimethyl hydroxa-
mates 20c, 20d and 20e were also screened. While all three com-
pounds displayed moderate VDR agonism (Table 1), their HDACi
activity was weak, as 20c and 20d only very poorly induced tubulin
and/or histone hyperacetylation while 20e was completely inac-
tive. Among hybrids with other ZBG’s, ortho-aminoanilides 23a
and 23b, and benzenesulfonamides and 27g all induced substantial
Direct binding to and affinity for the VDR of selected bifunc-
tional compounds (see below for assessment of HDACi activity)
was tested using a fluorescence polarization (FP) assay.10 Not sur-
prisingly, FP assays revealed a range of affinities for the VDR,
although IC50 values in FP assays were generally within a factor
of 20–25 of that of 1 (Fig. 2C; Table 1). This includes ortho-amino-
anilide 23b, indicating a surprising tolerance for larger groups in
the vicinity of the normal 25-OH binding region. Notably, four
compounds, sulfonamides 27c, 27f, and 27g and the thioglycolate
amide 24b, displayed affinities for the VDR in FP assays that were
within a factor of 3 of the native ligand. The high affinities of 27f
and 27g for the VDR were supported by the observation that they
induced CYP24 with a potency that was at least as high as that of 1
(Fig. 2B). These data confirm the broad latitude available in modi-

4124
M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
A
B
C
290
270
250
230
210
1
1
20b
290
13 nM
248 nM
270
250
230
210
190
170
cyp24
1
20a
20b
1
cyp24
gapdh
cyp24
cyp24
0.1
0.1
1
10 100 1000 nM
27f
gapdh
10 100 1000 nM
cyp24
gapdh
1000 nM
0.1
10
0
1
1000 nM
0.1
10
1
10 100 1000 nM
27g
380
360
340
320
300
280
260
350
330
310
290
270
23a
524 nM
27c
14 nM
cyp24
gapdh
0.1
1
10 100 1000 nM
D
10
1000 nM
0.1
1000 nM
0.1
10
6h
24h
6h
Ac-Tub
Ac-H3
Ac-H4
actin
Ac-Tub
actin
E
Tub
Ac-H3
Ac-H4
actin
-
-
2
4
20a
20a
100
20a
20b
100
-
9.3 µM
3.0 µM
2
20a
20a
80
60
40
20
80
60
40
20
24h
-
20b
24h
Ac-Tub
actin
Ac-H3
Ac-H4
2
4
µM
µM
100
0.01
1
100
0.01
1
actin
-
2
Figure 2. Representative VDR agonist and HDACi activities of hybrids. (A and B) Dose–response analysis of induction of CYP24 mRNA expression by 1,25D (1) and hybrids 20a
and 20b (A) and hybrids 27f and 27g (B). (C) Hybrids bind directly to the VDR ligand binding domain. Fluorescence polarization competition assays comparing displacement
of a fluorescent tracer from the VDR ligand binding domain are shown. (mP, milli-Polarization units.) Estimated IC50s for 1,25D and hybrids are indicated in the figure. (D)
Western blotting analysis of induction of histone H3, histone H4 or tubulin acetylation after 6 or 24 h of treatment with TSA (2) or hybrids 4, 20a or 20b, as indicated. (E)
Inhibition of isolated HDAC6 by hybrids 20a and 20b as measured using a fluorometric substrate.
tubulin and histone hyperacetylation while others were more
effective at inducing either tubulin (24b, 27a, 28c) or histone
(27b, 27c) hyperacetylation.
1.75
respectively.
lM, 32.3 lM and 96.3 lM against HDAC6, -3 and -2,
HDACi activities of a series of bifunctional compounds that in-
2.3. Lack of hypercalcemia in mice treated with hybrid compounds
duced histone and/or tubulin hyperacetylation at 1 lM concentra-
tion as assessed by Western blotting were further tested in a
standard fluorometric assay using purified HDACs (Fig. 2E).19,20
Consistent with its striking effects on tubulin, which is deacety-
lated primarily by HDAC6,21 4 inhibited purified HDAC6 deacetyl-
ase activity with sub-micromolar potency. The IC50 for 4 was very
similar to that of positive control 3. However, whereas 3 inhibited
all three HDACs tested with very similar potencies, the IC50’s of 4
for HDACs 2 and 3 were 20- to almost 100-fold higher than those
for HDAC6, suggesting that the secosteroidal ‘cap’ group of 4 con-
fers some degree of HDAC specificity on the compound. In agree-
ment with these findings, we found that 20b, a saturated side-
chain analog of 4, inhibited purified HDAC6 about fivefold more
potently than HDACs 2 or 3 (Table 1). Several other compounds
were tested in fluorometric assays and those found to have modest
As hypercalcemia is the dose-limiting toxicity associated with
1,25D treatment, 4 and seven selected second-generation com-
pounds were compared with 1,25D for their capacity to induce
hypercalcemia in mice (summarized in Table 1). Compounds cho-
sen had a range of affinities for the VDR and a variety of terminal
zinc-binding groups. Mice fed a normal calcium diet were infused
using minipumps with 1 (12 or 24 pmol/day) or hybrids (240 or
1200 pmol/day) over 6–7 days. Elevated concentrations of hybrid
were chosen in part because, based on the literature,22,23 we ex-
pected they would not induce hypercalcemia as readily as 1, but
also to account for the lower affinity for the VDR of some hybrids.
As expected, animals infused with 12 pmol/day 1 were hypercalce-
mic after 6–7 days of treatment (2.6–3.1 mM Ca vs 2.0–2.1 mM Ca
for controls), and all animals treated with a 24 pmol/day dose were
hypercalcemic within three days (2.9–3.4 mM Ca). In contrast, all
animals infused with 240 or 1200 pmol/day of hybrid compounds
appeared healthy (not shown). Moreover, most showed no sign of
hypercalcemia after 6–7 days (1.9–2.3 mM Ca). This includes ani-
mals infused with compounds 24b and 27g, which have 1,25D-
or near-1,25D-like affinities for the VDR as judged by FP assays
and CYP24 induction. Only hydroxamic acid hybrid 20a showed
HDAC6i activity (IC50: 10–100
lM) were ortho-aminoanilide 23b,
sulfonamide 27a, sulfamides 28b and c and
a-ketoamide 30b. As
with the hydroxamic acids, the general trend among the alterna-
tive ZBG’s was that they were more potent inhibitors of HDAC6,
although hydroxamic acid 20a and a-ketoamide 30b were notable
exceptions to this trend (Table 1). The most potent non-hydroxa-
mate HDACi was thioglycolate 24b, which displayed an IC50’s of

M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
4125
A
D
B
C 50
50
40
30
20
10
0
50
40
30
20
10
0
1
4
20b
40
30
20
10
0
0
100
1000
5000 nM
0
100
1000 nM
0
100
1000
5000 nM
50
40
30
20
10
0
50
40
30
20
10
0
60
50
40
30
20
10
0
E
F
23b
24b
30b
0
100
1000
5000 nM
0
100
1000
5000 nM
5000 nM
0
100
1000
Figure 3. Analysis of the antiproliferative activities of selected hybrids. Murine AT84 head and neck squamous carcinoma cells were cultured under the recommended
conditions and treated for 48 h with increasing doses, as indicated, of 1 (A) or hybrids 4 (B), 20b (C), 23b (D), 24b (E) or 30b (F). Cell proliferation was monitored using an EdU
incorporation assay.
any significant level of elevated calcium levels (2.3–2.5 mM at
1200 pmol/day), but this is still significantly lower than observed
with 1 and at a 100-fold higher concentration. Thus, all compounds
tested lacked the dose-limiting toxicity associated with treatment
with 1.
moderate to strong VDR agonism at 1 lM. Direct binding studies
revealed that some of these compounds even displayed 1,25D-like
affinity for the VDR. The VDR agonism of ortho-aminoanilides 23a
and 23b is most striking, indicating a remarkable capacity of the
VDR binding site to accommodate this large group. While aromatic
rings have been incorporated in the side-chain of a few VDR ago-
nists,26 they are more commonly found in antagonists, particularly
when appended to lactam rings.27 Most of the successful hybrids
containing alternative ZBG’s also possessed HDACi activity in the
micromolar range. In general, the hybrids were more potent to-
ward HDAC6 than HDAC3, suggesting some induction of selectivity
for this isoform by the secosteroidal vitamin D core.
One notable compound was the thioglycolate amide hybrid 24b,
which was a potent VDR agonist, and functioned with low micro-
molar potency as an HDAC6 inhibitor; its HDAC6i activity was
within a factor of 5 of that of 3 (Table 1). Consistent with data
for other secosteroidal hybrids, 24b inhibited HDAC6 18.5- and
>50-fold more potently than HDAC3 and HDAC2, respectively. Pre-
vious studies have suggested that thioglycolates (or mercaptoa-
mides) are bidentate chelators of zinc ions and have shown that
thioglycolate analogs of 3 could be developed with low micromolar
HDACi activity when measured against HDACs present in HeLa cell
extracts.28
We found that side-chain length was a critical parameter in
controlling HDACi activity of some hybrid compounds. Hybrids
20a and 20b, which retain similar VDR agonist activity, possess
saturated side-chains with 20a being shorter by one methylene
unit. Both compounds modestly induced histone hyperacetylation.
However, whereas 20b induced strong tubulin hyperacetylation in
living cells, 20a was inactive under the same conditions. Not unex-
pectedly, N- and O-alkylhydroxamates 20c–e displayed signifi-
cantly reduced HDAC activity, and inverting the hydroxamic acid
to form an N-hydroxyformate resulted in minimal HDACi activity.
We noted apparent discrepancies between the data obtained
from fluorometric assays with purified enzymes and those from
analysis of protein acetylation in intact cells. For example, satu-
rated hydroxamic acid 20b, but not 20a, induced robust tubulin
2.4. Antiproliferative activity of hybrid compounds
Selected compounds were further tested by analyzing their
capacity to inhibit the proliferation of the AT84 squamous carci-
noma cell line. AT84 cells were chosen because they are sensitive
to 1 in vitro and in vivo in tumor allografts.24 This model is of inter-
est for longer-term testing of compounds as, although 1 partially
inhibits AT84 tumor growth in vivo, its efficacy is limited by hyper-
calcemia.24 We tested a selection of bifunctional compounds
(Fig. 3) that combined a range of potencies as VDR agonists with
relatively potent HDACi activity in fluorometric assays. These in-
cluded hydroxamates 4 and 20b, ortho-aminoanilide 23b, thiogly-
colate 24b, and
a-ketoamide 30b. Remarkably, although 4
functioned comparably or better than 1 in previous cell growth as-
says,10 its potency was markedly weaker than that of 1 in AT84
cells (Fig. 3A and B). In contrast, hydroxamate 20b was a potent
inhibitor of AT84 proliferation (Fig. 3C) even though it is a ꢁ20-fold
less potent VDR agonist than 1 (Table 1). Compounds 23b, 24b, and
30b also markedly inhibited AT84 cell division, although at micro-
molar concentrations (Fig. 3D–F).
3. Discussion
The results above show that there is significant structural lati-
tude in the creation of hybrid molecules possessing VDR agonism
and HDACi activity, but that variation in chain length and ZBG
has significant effects on activity and selectivity. We found a
remarkable latitude in the types of ZBG’s that may be incorporated
into the side-chain while retaining VDR agonism.25 Various ortho-
aminoanilides, thioglycolates,
a-ketoesters and amides, sulfona-
mides, sulfamides, and -trithiocarbonyl amide 25d all showed
a

4126
M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
hyperacetylation in cells. Tubulin deacetylation is known to be
mediated by HDAC6.29 While 20a was found to be a stronger inhib-
itor of HDAC3 relative to HDAC6, one of only two cases of reversed
selectivity in all hybrids tested, the HDAC6 inhibition by 20a was
only a factor of three weaker than 20b. Moreover, 20a was more
potent in the fluorescence assay than several other hybrids which
did induce significant tubulin hyperacetylation such as 23a, 23b,
and 27g (Table 1; Fig. 2E). In particular, the latter three com-
pounds, which were selected for the fluorometric assay due to their
ability to induce strong histone and tubulin hyperacetylation in
on pre-coated silica gel plates (Ultra Pure Silica Gel Plates pur-
chased from Silicycle), visualized with a Spectroline UV254 lamp,
and stained with a 20% phosphomolybdic acid in ethanol solution,
or a basic solution of KMnO4. Solvent systems associated with Rf
values and flash column chromatography are reported as percent
by volume values.
1H and 13C NMR, recorded at 300 MHz and 75 MHz, respec-
tively, were performed on a Varian Mercury 300 spectrometer.
1H and 13C NMR, recorded at 400 MHz and 100 MHz, respectively,
were performed on a Varian Mercury 400 spectrometer. Proton
chemical shifts were internally referenced to the residual proton
resonance in CDCl3 (d 7.26 ppm), CD3OD (d 3.31 ppm), CD3CN (d
1.94 ppm), or DMSO-d6 (d 2.50 ppm). Carbon chemical shifts were
internally referenced to the deuterated solvent signals in CDCl3 (d
77.2 ppm), CD3OD (d 49.0 ppm), CD3CN (d 118.3 ppm and 1.3 ppm)
or DMSO-d6 (d 39.5 ppm). FT-IR spectra were recorded on a Nicolet
Avatar 360 ESP spectrometer with samples loaded as neat films on
NaCl plates. References following compound names indicate liter-
ature articles where 1H and 13C NMR data have previously been
reported.
cells at 1
lM, inhibited activity of purified HDACs 2, 3 or 6 with rel-
atively low potencies (IC50s generally >100
lM). There are several
possible explanations for these observations, including the fact
that HDACs function as components of multiprotein complexes
subject to multiple posttranslational modifications,30 which may
alter their conformation and susceptibility to inhibition relative
to isolated enzyme. In addition, only two Class I HDACs were as-
sayed in fluorometric assays, and these may not correspond to iso-
forms (e.g., HDAC1, HDAC8) controlling histone acetylation in SCC4
cells.30 Finally, the HDAC6 inhibition assay is further complicated
by the fact that it possesses two separate catalytic domains29 mak-
ing it difficult to directly correlate the fluorometric assay with
activity in cells.
There is also some discrepancy between the binding potency of
some of the hybrids with their observed affects on CYP24 induc-
tion. Notably, hybrids 24b and 27c displace fluorescent ligand from
the VDR at concentrations 10-fold lower than, for example, sulfon-
amides 27a and 27b, yet the latter were more potent inducers of
CYP24 induction. Similar results have been noted for other vitamin
D analogs31 and often reflect the capacity of different ligand–VDR
complexes to release co-repressors and recruit co-activators for
gene transcription in a manner which is reflective of the conforma-
tion of the ligand–receptor complex and not linearly correlated to
binding affinity.
4.1.1.1. Lythgoe-Inhoffen diol (6). A flame-dried 100 mL three-
necked flask was charged sequentially with 28 mg (0.33 mmol,
0.06 equiv) of NaHCO3, 20 mL of anhydrous MeOH, 60 mL of anhy-
drous CH2Cl2, and 2.0 g (5.12 mmol, 1 equiv) of ergocalciferol 5.
The solution was cooled to ꢀ78 °C and treated with O3 until a blue
color appears. The solution was subsequently flushed with Ar for
10–15 min until the blue color faded. Solid sodium borohydride
(1.68 g, 44.55 mmol, 8.7 equiv) was added portionwise over a per-
iod of 10 min at ꢀ78 °C until complete disappearance of starting
material was observed by TLC. The reaction mixture was warmed
to 0 °C and stirred for 3 h. After being stirred for an additional
30 min at room temperature, the mixture was quenched with 1 N
HCl (10 mL), extracted with EtOAc (3 ꢂ 50 mL), dried (MgSO4), fil-
tered, and concentrated in vacuo. Purification by silica gel chroma-
tography (30% EtOAc in hexanes) afforded 670 mg (3.17 mmol) of
Lythgoe-Inhoffen diol 6 in 62% yield as a white solid. Rf = 0.5
(50% EtOAc in hexanes); Mp 108–110 °C (lit.14 mp 109–110 °C);
1H NMR (400 MHz, CDCl3) d 4.08 (1H, br s), 3.63 (1H, dd, J = 10.4,
2.8 Hz), 3.37 (1H, J = 10.0, 6.8 Hz), 1.98 (1H, d, J = 12.8 Hz), 1.90–
1.75 (3H, m), 1.60–1.40 (5H, m), 1.38–1.29 (4H, m), 1.22–1.13
(2H, m), 1.02 (3H, d, J = 6.8 Hz), 0.95 (3H, s); 13C NMR (75 MHz,
CDCl3) d 69.1, 67.7, 52.9, 52.3, 41.8, 40.2, 38.2, 33.5, 26.6, 22.5,
Selected hybrids tested showed antiproliferative activity
against AT84 cells, with hydroxamic acid 20b, ortho-aminoanilide
23b and
a-ketoamide 30b showing the highest potency. While
none of the compounds tested were more potent than 1, it is
important to note that none of them induced hypercalcemia even
at concentrations that were 100-fold higher than a limiting dose
of 1 (Table 1). In follow-up studies it will therefore be of interest
to determine whether they display a greater therapeutic range
than 1 in inhibiting AT84 tumor growth in vivo.
In conclusion, we have demonstrated that it is possible to create
a wide range of bifunctional molecules which possess VDR agon-
ism and HDACi activity. The structural latitude is significant with
a wide variety of ZBGs amenable to incorporation into the side-
chain of vitamin D-like secosteroids. Importantly several of these
molecules function as antiproliferative agents against AT84 cells
in vitro while possessing minimal hypercalcemic activity in vivo.
17.4, 16.6, 13.5; IR (KBr)
m .
3621, 3464, 3017, 2943 cmꢀ1
4.1.1.2. (S)-2-((1R,3aR,4S,7aR)-4-(tert-Butyldimethylsilyloxy)-
7a-methyloctahydro-1H-inden-1-yl)propan-1-ol (7). To a solu-
tion of Lythgoe-Inhoffen diol 6 (2.017 g, 9.5 mmol, 1 equiv) in
60 mL of dry DMF under argon atmosphere was added tert-butyl-
dimethylsilylchloride (5.71 g, 38 mmol, 4 equiv) followed by NEt3
(5.54 mL, 42.7 mmol, 4.5 equiv) and sodium iodide (5.69 g,
38 mmol, 4 equiv). The reaction mixture was refluxed for 30 min
and then cooled and quenched with H2O (10 mL) and concentrated
in vacuo. The residue was dissolved in EtOAc (100 mL) and washed
with H2O (2 ꢂ 30 mL), the aqueous portion was extracted with
EtOAc (30 mL) and the combined organic layers were washed with
brine (30 mL), dried (MgSO4), filtered, concentrated by rotary evap-
oration, and immediately purified by silica gel chromatography
(10% EtOAc in hexanes) to afford 4.013 g (9.12 mmol) of bis-silylat-
ed diol intermediate in 96% yield. In a flame-dried round bottom
flask under argon, 4.013 g (9.12 mmol) of the bis-silylated diol
was dissolved in 80 mL of anhydrous THF and 4 mL of NEt3. To this
stirring solution was added TBAF (10.9 mL of a 1 M solution in THF,
10.9 mmol, 1.15 equiv). The resulting reaction mixture was stirred
at room temperature for 3 h, concentrated in vacuo, and purified
by silica gel column chromatography (20% EtOAc in hexanes) to af-
4. Experimental section
4.1. Synthesis of hybrid molecules
4.1.1. General experimental
MeCN, toluene and CH2Cl2 were distilled from CaH2 under ar-
gon. THF and Et2O were distilled from sodium metal/benzophe-
none ketyl under argon. All other commercial solvents and
reagents were used as received from the Aldrich Chemical Com-
pany, Fischer Scientific Ltd, EMD Chemicals Inc., Strem or BDH.
All glassware was flame dried and allowed to cool under a stream
of dry argon. Silica gel (60 Å, 230–400 mesh) used in flash column
chromatography was obtained from Silicycle and was used as re-
ceived. Analytical thin-layer chromatography (TLC) was performed

M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
4127
ford 2.91 g (8.94 mmol) of the desired alcohol 7 in 94% yield.
Rf = 0.5 (20% EtOAc in hexanes); 1H NMR (400 MHz, CDCl3) d 4.08
(1H, br s), 3.57 (1H, dd, J = 9.8, 3.0 Hz), 3.26 (1H, dd, J = 9.0,
7.8 Hz), 1.99 (1H, d, J = 13.6 Hz), 1.87–1.75 (3H, m), 1.58–1.40
(5H, m), 1.35–1.28 (3H, m), 1.40–1.09 (2H, m), 0.97 (3H, d,
J = 6.8), 0.94 (3H, s), 0.89 (9H, s), 0.025 (6H, s); 13C NMR
(75 MHz, CDCl3) d 69.3, 67.7, 53.2, 52.3, 41.8, 40.2, 38.5, 33.6,
4.1.1.5. (R)-Ethyl-5-((1R,3aR,7aR)-7a-methyl-4-oxo-octahydro-
1H-inden-1-yl)hexanoate (10). PCC (546 mg, 2.52 mmol, 2 equiv)
was added to a stirred solution of 9 (500 mg, 1.26 mmol) and Celite
(600 mg) in CH2Cl2 (20 mL). The solution was stirred for 3 h, and
then the mixture was filtered on a silica pad. The precipitate was
washed with CH2Cl2 (3 ꢂ 20 mL) then the combined organic frac-
tions were concentrated in vacuo to provide the crude ester as a col-
orless oil. The oil was diluted in CH2Cl2 (10 mL) and CH3CN (10 mL)
and a 48% solution of HF (1 mL) was added. The solution was stirred
for 24 h at room temperature. The reaction mixture was cautionly
quenched by the addition of a satd solution of NaHCO3 until no
effervescence was observed. The solution was extracted with CH2Cl2
(3 ꢂ 20 mL), the combined organic layers were then washed with
H20 (20 mL) and brine (20 mL), then dried (MgSO4). The solution
was concentrated in vacuo, then diluted in CH2Cl2 (20 mL), Celite
was added (500 mg) followed by PCC (546 mg, 2.52 mmol, 2 equiv).
After stirring for 3 h at room temperature, the reaction mixture was
filtered on a silica pad, the precipitate was washed with CH2Cl2
(3 ꢂ 20 mL) and the solution was concentrated in vacuo. The resid-
ual oil was purified by silica gel column chromatography (15%
EtOAc in hexanes) to afford 363 mg (1.23 mmol) of 10 in 78% yield.
Rf = 0.2 (15% EtOAc in hexanes); 1H NMR (400 MHz, CDCl3) d 4.08
(1H, q, J = 7.2 Hz), 2.46 (1H, dd, J = 11.6, 7.6 Hz), 2.30–2.12 (4H, m),
2.07 (1 h, d, J = 13.6 Hz), 2.02–1.76 (4H, m), 1.75–1.60 (2H, m),
1.60–1.31 (3H, m), 1.21 (3H, t, J = 6.8 Hz), 0.93 (3H, d, J = 6.4 Hz),
0.84 (3H, t, J = 6.8 Hz), 0.59 (3H, s); 13C NMR (75 MHz, CDCl3) d
212.2, 173.9, 62.1, 60.4, 56.5, 50.1, 41.1, 39.1, 35.4, 31.8, 27.6,
26.6, 26.0, 22.6, 18.4, 17.4, 16.8, 13.6, ꢀ5.3, ꢀ5.4; IR (KBr)
m
3300, 1470, 1250, 1160 cmꢀ1
.
4.1.1.3. (S)-2-((1R,3aR,4S,7aR)-4-(tert-Butyldimethylsilyloxy)-7a-
methyloctahydro-1H-inden-1-yl)-1-iodopropane (8). I2 (9.34 g,
36.8 mmol, 4.6 equiv) was added portionwise to an ice cooled solu-
tion of PPh3 (4.82 g, 18.4 mmol, 2.3 equiv) and imidazole (3.26 g,
48.0 mmol, 6 equiv) in 300 mL of CH2Cl2. The cooled mixture,
which become heterogeneous after 5 min, was stirred for 35 min
and treated with a solution of the primary alcohol 7 (2.61 g,
8 mmol, 1 equiv) in 100 mL of CH2Cl2 during 30 min and then the
mixture was stirred at room temperature for 4 h. The mixture
was quenched with a 2.5% solution of Na2SO3 (30 mL). The organic
layer was washed with H2O (30 mL), brine (30 mL), dried (MgSO4),
concentrated in vacuo, and then purified by silica gel column chro-
matography (5% EtOAc in hexanes) to afford 3.14 g (6.96 mmol) of
iodide 8 in 87% yield as a white solid. Rf = 0.6 (5% EtOAc in hex-
anes); Mp 40–41 °C (lit.32 mp 41–42 °C); 1H NMR (400 MHz, CDCl3)
d 3.99 (1H, s), 3.32 (1H, d, J = 9.6 Hz), 3.17 (1H, dd, J = 9.6, 5.2 Hz),
1.90 (1H, d, J = 12.8 Hz), 1.84–1.76 (2H, m), 1.66 (1H, d, J = 13.6 Hz),
1.59–1.54 (1H, m), 1.40–1.08 (8H, m), 0.98 (3H, d, J = 5.2 Hz), 0.94
(3H, s), 0.88 (9H, s), ꢀ0.01 (6H, s); 13C NMR (75 MHz, CDCl3) d 69.3,
56.0, 52.7, 42.1, 40.3, 36.4, 34.3, 26.6, 25.8, 22.9, 21.7, 20.7, 18.0,
25.5, 24.2, 22.8, 21.6, 19.2, 14.4, 12.6; IR (KBr)
m 2957, 2874, 1710,
1704, 1463, 1377, 1239, 1179, 1099, 1037 cmꢀ1; HRMS (ESI): m/z
calcd for [(M+Na)+] = 319.2093, found = 319.2086.
17.6, 14.6, ꢀ4.8, ꢀ5.2; IR (KBr)
m 2932, 2857, 1462, 1375, 1253,
.
4.1.1.6. (R)-Ethyl-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-bis(tert-
butyldimethylsilyloxy)cyclohexylidene)ethylidene)-7a-methyl-
1160, 1084, 1032, 836, 774 cmꢀ1
octahydro-1H-inden-1-yl)hexanoate (12). In
round bottom flask under argon atmosphere at ꢀ78 °C, NaHMDS
(514 L of a 1 M solution in THF, 0.514 mmol, 1.05 equiv) was
a
flame-dried
4.1.1.4. (S)-5-((1R,3aR,4S,7aR)-4-(tert-Butyldimethylsilyloxy)-7a-
methyloctahydro-1H-inden-1-yl)-1-ethoyhex-1-ene (9). In
a
l
flame-dried round bottom flask under argon atmosphere,
8
added to a solution of phosphine oxide 11 (307 mg, 0.539 mmol,
1.1 equiv) in dry THF (6 mL). The reaction vessel was suspended
above the ice bath for 5 min, then recooled to ꢀ78 °C. To this solu-
tion was added via cannula over a period of 5 min a solution of 10
(144 mg, 0.490 mmol, 1 equiv) in dry THF (2 mL). The reaction was
left to stir at ꢀ78 °C for 2 h then warmed to room temperature for
1 h, and quenched with satd NH4Cl (10 mL). The layers were sepa-
rated and the aqueous layer extracted with EtOAc (2 ꢂ 25 mL). The
organic layers were combined and extracted with satd NH4Cl
(2 ꢂ 10 mL), H2O (10 mL) and brine (10 mL), then dried (MgSO4),
and concentrated in vacuo. Purified by silica gel column chroma-
tography (20% EtOAc in hexanes) afforded 247 mg (0.382 mmol)
of 12 in 71% yield. Rf = 0.6 (20% EtOAc in hexanes); 1H NMR
(678 mg, 1.5 mmol, 1 equiv) was dissolved in dry Et2O (3 mL), the
solution was cooled to ꢀ78 °C, then t-BuLi (1.42 mL of a 2.22 M
solution in hexanes, 3.15 mmol, 2.1 equiv) was slowly added (ca.
1 h). The solution was stirred at ꢀ78 °C for 1 h, then warmed to
0 °C for 5 min and then recooled to ꢀ78 °C. A solution of acrolein
acetal (251 lL, 1.65 mmol, 1.1 equiv) in dry Et2O (2 mL) was slowly
added via cannula to the carbanion solution and the mixture was
warmed to room temperature. Stirring was continued for 30 min
then the solution was quenched with satd NH4Cl (10 mL). Extrac-
tion with Et2O (3 ꢂ 20 mL) afforded an organic phase that was
washed with H2O (20 mL), brine (20 mL) and dried (MgSO4), con-
centrated in vacuo, and then purified by silica gel column chroma-
tography (hexanes then 1% Et2O in hexanes) to afford 444 mg
(1.12 mmol) of the enol ether as a mixture 86/14 of E and Z in
75% yield. Rf = 0.2 (hexanes); 1H NMR (400 MHz, CDCl3) d (9 E):
6.21 (1H, d, J = 12.4 Hz), 4.74 (1H, dt, J = 12.4, 6.2 Hz), 3.98 (1H,
s), 3.69 (2H, q, J = 6.8 Hz), 1.95 (2H, br d, J = 12.4 Hz), 1.81–1.73
(3H, m), 1.65 (1H, br d, J = 13.2 Hz), 1.60–1.51 (1H, m), 1.42–1.29
(10H, m), 1.27–1.00 (3H, m), 0.95–0.80 (15H, m), 0.00 (3H, s),
ꢀ0.01 (3H, m); (9 Z): 5.90 (1H, d, J = 9.2 Hz), 4.30 (1H, dd, J = 9.2,
7.5 Hz), 3.98 (1H, br s), 3.77 (2H, q, J = 7.5 Hz), 1.95 (2H, br d,
J = 12.4 Hz), 1.81–1.73 (3H, m), 1.65 (1H, br d, J = 13.2 Hz), 1.60–
1.51 (1H, m), 1.42–1.29 (10H, m), 1.27–1.00 (3H, m), 0.95–0.80
(15H, m), 0.00 (3H, s), ꢀ0.01 (3H, m); 13C NMR (75 MHz, CDCl3) d
145.6, 104.8, 69.5, 64.5, 56.7, 53.0, 42.1, 40.7, 37.0, 34.8, 34.4,
27.3, 25.8, 24.5, 23.0, 18.5, 18.0, 17.7, 14.8, 13.7, ꢀ4.8, ꢀ5.2; IR
(400 MHz, CDCl3)
d 6.16 (1H, d, J = 11.2 Hz), 5.80 (1H, d,
J = 11.2 Hz), 4.20–4.00 (4H, m), 2.80 (1H, d, J = 11.6 Hz), 2.45–2.18
(4H, m), 2.09 (1H, dd, J = 12.8, 8.0 Hz), 1.98 (2H, d, J = 10.0 Hz),
1.80–1.25 (15H, m), 1.25 (3H, t, J = 6.8 Hz), 0.93 (3H, d,
J = 6.4 Hz), 0.86 (9H, s), 0.85 (9H, s), 0.52 (3H, s), 0.05 (12H, s);
13C NMR (75 MHz, CDCl3) d 173.9, 140.7, 133.6, 121.7, 116.1,
68.1, 67.9, 68.1, 60.1, 56.2, 46.0, 45.6, 43.7, 40.6, 36.7, 35.9, 35.3,
34.7, 28.7, 27.7, 25.9, 25.8, 23.4, 22.2, 21.6, 18.7, 18.13, 18.09,
14.3, 12.0, ꢀ4.7, ꢀ4.76, ꢀ4.83, ꢀ4.9; HRMS (ESI): m/z calcd for
[(M+Na)+] = 669.4710, found = 669.4744.
4.1.1.7. (5S)-5-((1R,3R,7E,17b)-1,3-Bis[tert-butyl(dimethyl)silyl-
oxy]-9,10-secoestra-5,7-dien-17-yl)hexanoic acid (13). LiOH.
H2O was added to
a stirred solution of ester 12 (128 mg,
(KBr)
m 2931, 2856, 1652, 1469, 1374, 1251, 1165, 1083, 1023,
0.197 mmol, 1 equiv) inn THF (2.5 mL), MeOH (0.75 mL) and H2O
(0.75 mmol). The solution was refluxed for 3 h, then cooled to
room temperature and diluted with EtOAc (20 mL), then quenched
977, 924, 836, 774 cmꢀ1; HRMS (ESI): m/z calcd for [(M+Na)+] =
417.3165, found = 417.3155.

4128
M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
with a 1 M solution of HCl (10 mL). The aqueous layer was ex-
tracted with EtOAc (2 ꢂ 10 mL) and then the combined organic
fractions were washed with H2O (10 mL), brine (10 mL) then dried
(Na2SO4). The solution was concentrated in vacuo to provide the
acid 13 in 97% yield without further purification (118 mg,
0.191 mmol). Rf = 0.6 (40% EtOAc in hexanes); 1H NMR (400 MHz,
CDCl3) d 6.16 (1H, d, J = 10.8 Hz), 5.81 (1H, d, J = 10.8 Hz), 4.15–
4.01 (2H, m), 2.81 (1H, br d, J = 11.6 Hz), 2.42–2.21 (5H, m),
2.12–1.05 (14H, m), 0.94 (3H, d, J = 7.2 Hz), 0.87 (9H, s), 0.86 (9H,
s), 0.53 (3H, s), 0.04 (12H, s); 13C NMR (75 MHz, CDCl3) d 180.0,
140.7, 133.7, 121.7, 116.1, 68.1, 68.0, 56.2, 46.0, 45.6, 43.7, 40.5,
36.7, 35.9, 35.3, 34.5, 28.7, 27.7, 25.9, 25.8, 23.4, 22.2, 21.3, 18.7,
1253, 1087, 1025, 836, 775 cmꢀ1; HRMS (ESI): m/z calcd for
[(M+Na)+] = 625.4448, found = 625.4434.
4.1.1.10. (R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Bis(tert-butyl-
dimethylsilyloxy)cyclohexylidene)ethylidene)-7a-methyl-octa-
hydro-1H-inden-1-yl)hexan-1-amine (16). A solution of alde-
hyde 15 (23 mg, 0.038 mmol, 1 equiv), O-benzylhydroxylamine
hydrochloride (6.1 mg, 0.038 mmol, 1 equiv) and sodium acetate
(3.1 mg, 0.03 mmol, 1 equiv) in 3 mL of toluene was refluxed for
30 min. The mixture was then concentrated and loaded directly
onto silica gel. The oximes were isolated by silica gel column chro-
matography (CH2Cl2) as a mixture of E and Z compounds. In a
flame-dried round bottom flask under argon atmosphere, a solu-
tion of the oximes in Et2O (1 mL) was added to a suspension of Al-
LiH4 (4.3 mg, 0.114 mmol, 3 equiv) in Et2O (1 mL) at 0 °C. The
solution was stirred at room temperature for 4 h then cooled to
18.2, 18.1, 12.0, ꢀ4.6, ꢀ4.7, ꢀ4.8, ꢀ4.9; IR (KBr)
m 3435 (br),
2951, 1637, 1459, 1247, 1086, 835, 780, 668 cmꢀ1; HRMS (ESI):
m/z calcd for [(M+Na)+] = 641.4397, found = 641.4383.
4.1.1.8. (R)-4-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Bis(tert-butyldi-
0 °C and quenched by addition of H2O (5
lL), then 15% NaOH
methylsilyloxy)cyclohexylidene)ethylidene)-7a-methyl-octahy-
(5 L) and then H2O (15 L). The mixture was diluted with Et2O
l
l
dro-1H-inden-1-yl)pentan-1-amine (14). To
carboxylic acid 13 (102 mg, 0.165 mmol, 1 equiv) in 1 mL of tolu-
ene was added via syringe Et3N (25 L, 0.181 mmol, 1.1 equiv), fol-
lowed by DPPA (35 L, 0.165 mmol, 1 equiv). The solution was
a
solution of
(10 mL), washed with water (5 mL), satd NaHCO3 (5 mL), brine
(5 mL) and dried (Na2SO4). The solution was concentrated in vacuo
then purified by silica gel column chromatography (10/10/80,
Et3N/MeOH/EtOAc) to provide 18 mg (0.030 mmol) of the amine
16 in 80% yield. Rf = 0.35 (10/10/80, Et3N/MeOH/EtOAc); 1H NMR
l
l
stirred at room temperature for 30 min then heated to reflux over-
night and then concentrated under reduced pressure. The crude
isocyanate was treated with 2 N NaOH (1.3 mL) and THF (3.3 mL)
then stirred for 20 min at room temperature. The resulting solution
was extracted with CH2Cl2 (2 ꢂ 10 mL), and the organic extract
was washed with brine and then dried (MgSO4). The solution
was concentrated in vacuo then purified by silica gel column chro-
matography (10/10/80, Et3N/MeOH/EtOAc) to obtain the amine 14
in 68% yield (66 mg, 0.111 mmol). Rf = 0.35 (10/10/80, Et3N/MeOH/
EtOAc); 1H NMR (400 MHz, CDCl3) d 6.14 (1H, d, J = 11.2 Hz), 5.79
(1H, d, J = 11.2 Hz), 4.11–4.01 (2H, m), 2.79 (1H, d, J = 12.0 Hz),
2.71–2.52 (2H, m), 2.42–2.30 (52H, m), 2.24 (1H, d, J = 12.4 Hz),
2.01–1.72 (5H, m), 1.71–0.98 (13H, m), 0.92 (3H, d, J = 8.0 Hz),
0.85 (9H, s), 0.84 (9H, s), 0.51 (3H, s), 0.04 (12H, s); 13C NMR
(75 MHz, CDCl3) d 140.7, 133.6, 121.7, 116.1, 68.1, 67.9, 56.4,
56.2, 46.0, 45.6, 43.7, 42.4, 40.6, 36.7, 36.0, 33.0, 29.7, 28.7, 27.7,
25.85, 25.83, 23.4, 22.2, 18.8, 18.13, 18.09, 12.0, ꢀ4.67, ꢀ4.77,
(400 MHz, CDCl3)
d 6.16 (1H, d, J = 11.1 Hz), 5.81 (1H, d,
J = 11.1 Hz), 4.12–4.01 (2H, m), 2.80 (1H, d, J = 10.5 Hz), 2.75–
2.62 (2H, m), 2.44–2.31 (2H, m), 2.25 (1H, d, J = 11.1 Hz), 2.10
(1H, dd, J = 12.6, 7.8 Hz), 2.04–1.92 (2H, m), 1.91–1.73 (2H, m),
1.70–0.98 (17H, m), 0.91 (3H, d, J = 6.3 Hz), 0.87 (9H, s), 0.86 (9H,
s), 0.53 (3H, s), 0.05 (12H, s); 13C NMR (75 MHz, CDCl3) d 141.1,
133.9, 122.0, 116.3, 68.3, 68.2, 56.7, 56.5, 46.2, 45.9, 43.9, 40.8,
37.0, 36.3, 36.0, 28.9, 28.0, 27.9, 26.10, 26.08, 23.6, 22.5, 19.0,
18.4, 18.3, 12.3, ꢀ4.4, ꢀ4.5, ꢀ4.6, ꢀ4.7; IR (KBr)
m 2930, 2857,
1469, 1377, 1362, 1253, 1085, 836, 775 cmꢀ1; HRMS (ESI): m/z
calcd for [(M+H)+] = 604.4945, found = 604.4948.
4.1.1.11. (6S)-6-((1R,3R,7E,17b)-1,3-Bis[tert-butyl(dimethyl)silyl-
oxy]-9,10-secoestra-5,7-dien-17-yl)heptanal (17). In a flame-
dried round bottom flask under argon atmosphere, NaHMDS
(1.0 mL of a 1 M solution in THF, 1.0 mmol, 10 equiv) was added
to a solution of (methoxymethyl)-triphenylphosphonium chloride
(360 mg, 1.0 mmol, 10 equiv) in dry THF (10 mL) at ꢀ78 °C. The
reaction vessel was suspended above the ice bath for 30 min, then
recooled to ꢀ78 °C. To this solution was added via cannula over a
period of 5 min a solution of 15 (60 mg, 0.100 mmol, 1 equiv) in
dry THF (1 mL). The reaction was left to stir at ꢀ78 °C for 1 h then
warmed to 0 °C for 4 h, and quenched with satd NH4Cl (25 mL). The
layers were separated and the aqueous layer extracted with EtOAc
(2 ꢂ 25 mL). The organic layers were combined and extracted with
satd NH4Cl (2 ꢂ 25 mL), H2O (25 mL) and brine (25 mL), then dried
(MgSO4), and concentrated in vacuo. The oil was then purified by
silica gel column chromatography (5% EtOAc in hexanes, Rf = 0.6)
to provide 53 mg of a mixture 7/3 of E/Z enol ethers. The enol
ethers mixture was dissolved in a solution of CHCl3 (1 mL), distilled
H2O (0.5 mL) and TFA (0.15 mL), and cooled to 0 °C. The reaction
was stirred at 0 °C for approx. 30 min and monitored by TLC until
complete consumption of the starting material, the reaction was
quenched with satd NaHCO3 (5 mL). CH2Cl2 (10 mL) was added
to the mixture, the layers were separated and the aqueous layer
extracted with CH2Cl2 (2 ꢂ 10 mL). The organic layers were com-
bined and washed with satd NaHCO3 (2 ꢂ 25 mL), distilled H2O
(25 mL) and brine (25 mL), then dried (MgSO4), and concentrated
in vacuo to give the crude product. 17 was isolated via FCC (10%
EtOAc in hexanes) as a clear oil in 84% yield (51.8 mg, 0.084 mmol).
Rf = 0.2 (10% EtOAc in hexanes); 1H NMR (400 MHz, CDCl3) d 9.77
(1H, t, J = 1.6 Hz), 6.16 (1H, d, J = 10.0 Hz), 5.81 (1H, d,
J = 10.0 Hz), 4.12–4.02 (2H, m), 2.81 (1H, d, J = 11.6 Hz), 2.43 (2H,
ꢀ4.84, ꢀ4.9; IR (KBr)
m
;
3350 (br), 2950, 2856, 1468, 1377, 1253,
1088, 836, 775 cmꢀ1
HRMS (ESI): m/z calcd for [(M+H)+] =
590.4789, found = 590.4792.
4.1.1.9. (5S)-5-((1R,3R,7E,17b)-1,3-Bis[tert-butyl(dimethyl)silyl-
oxy]-9,10-secoestra-5,7-dien-17-yl)hexanal (15). DIBALH (172
lL of a 1 M solution in toluene, 0.172 mmol, 1.1 equiv) was added
dropwise to a stirred solution of ester 12 (101 mg, 0.157 mmol,
1 equiv) in dry CH2Cl2 (1.5 mL) under Ar at ꢀ78 °C. The solution
was stirred at ꢀ78 °C for 1 h and warmed up to room temperature.
CH2Cl2 (10 mL) was added to the reaction mixture, then H2O
(0.16 mL) followed by 15% NaOH (0.16 mL) then more H2O
(0.48 mL). The solution was stirred for 30 min then MgSO4 was
added to the mixture. The solution was filtered, the precipitate
washed with CH2Cl2 (5 mL) and the combined organic fractions
were concentrated in vacuo to provide the aldehyde 15 in quanti-
tative yield without further purification (95 mg, 0.157 mmol).
Rf = 0.6 (CH2Cl2); 1H NMR (400 MHz, CDCl3) d 9.76 (1H, s), 6.16
(1H, d, J = 12.0 Hz), 5.81 (1H, d, J = 12.0 Hz), 4.40–4.43 (2H, m),
2.80 (1H, dd, J = 11.0, 3.1 Hz), 2.43–2.34 (4H, m), 2.24 (1H, br d,
J = 13.6 Hz), 2.89 (1H, dd, J = 12.8, 8.4 Hz), 2.05–1.85 (2H, m),
1.85–1.20 (15H, m), 1.20–1.05 (1H, m), 0.94 (3H, d, J = 6.0 Hz),
0.87 (9H, s), 0.86 (9H, s), 0.53 (3H, s), 0.05 (12H, s); 13C NMR
(75 MHz, CDCl3) d 202.9, 140.6, 133.7, 121.7, 116.2, 68.1, 67.9,
56.22, 56.20, 46.0, 45.6, 44.3, 43.7, 40.5, 36.7, 36.0, 35.4, 28.7,
27.7, 25.87, 25.84, 23.4, 22.2, 18.7, 18.15, 18.10, 12.0, ꢀ4.6, ꢀ4.7,
ꢀ4.8, ꢀ4.9; IR (KBr)
m 2950, 2883, 2856, 1728, 1468, 1377, 1361,

M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
4129
t, J = 7.2 Hz), 2.37 (2H, dd, J = 12.4, 5.2 Hz), 2.25 (1H, d, J = 11.6 Hz),
2.10 (1H, dd, J = 12.6, 8.2 Hz), 2.04–1.93 (2H, m), 1.93–1.75 (2H, m),
1.70–1.45 (8H, m), 1.45–1.15 (7H, m), 1.15–1.00 (1H, m), 0.91 (3H,
d, J = 6.0 Hz), 0.87 (9H, s), 0.86 (9H, s), 0.53 (3H, s), 0.05 (12H, s);
13C NMR (75 MHz, CDCl3) d 203.3, 141.0, 133.9, 121.9, 116.4,
68.3, 68.2, 56.6, 56.5, 46.3, 45.9, 44.3, 43.9, 40.8, 37.0, 36.2, 35.8,
28.9, 27.9, 26.11, 26.08, 25.9, 23.6, 22.8, 22.5, 19.0, 18.4, 18.3,
ldimethylsilylhydroxylamine (30 mg, 0.204 mmol, 2 equiv) in
CH2Cl2 (1 mL) was added to the reaction mixture. The solution
was left to stir at 0 °C for 1 h then at room temperature for 1 h. A
solution of citric acid (10 mL, 1 M) was then added, and the mixture
was extracted with EtOAc (3 ꢂ 10 mL) and the combined organic
layers were washed with H2O (10 mL), brine (10 mL) and dried
(MgSO4). The solution was concentrated in vacuo, then the crude
material was dissolved in CH2Cl2 (1 mL) and CH3CN (1 mL). A 48%
12.3, ꢀ4.4, ꢀ4.5, ꢀ4.6, ꢀ4.7; IR (KBr)
m 2949, 2884, 2856, 1728,
1468, 1252, 1052, 1025, 960, 920, 837, 776 cmꢀ1; HRMS (ESI): m/
z calcd for [(M+Na)+] = 639.4605, found = 639.4599.
solution of HF (70 lL, 2.04 mmol, 20 equiv) was added and the solu-
tion was stirred at room temperature for 2 h. The mixture was
quenched cautionly by the addition of satd NaHCO3 until no effer-
vescence was observed then acidified with a 1 M aq solution of citric
acid (5 mL). The solution was extracted with CH2Cl2 (3 ꢂ 5 mL) then
the combined organic layers were washed with H2O (5 mL), brine
(5 mL), dried (MgSO4) and then concentrated in vacuo. The residual
oil was purified by octadecyl-functionalized silica gel column chro-
matography (100% H2O to 100% MeOH) to afford the hydroxamic
acid 20a in 68% yield. Rf = 0.30 (88/10/2 CH2Cl2/MeOH/CH3COOH);
1H NMR (400 MHz, CD3OD) d 6.22 (1H, d, J = 11.0 Hz), 5.89 (1H, d,
J = 11.0 Hz), 4.08–3.92 (2H, m), 2.83 (1H, br d, J = 12.4 Hz), 2.59
(1H, br d, J = 13.2 Hz), 2.52–1.04 (27H, m), 0.97 (3H, d, J = 3.2 Hz),
0.58 (3H, s); 13C NMR (75 MHz, CD3OD) d 176.6, 140.9, 132.7,
122.3, 116.0, 66.8, 66.5, 56.6, 56.3, 45.6, 44.2, 41.5, 40.7, 36.5,
4.1.1.12. (6S)-6-((1R,3R,7E,17b)-1,3-Bis[tert-butyl(dimethyl)si-
lyl-oxy]-9,10-secoestra-5,7-dien-17-yl)heptanoic acid (18).
A
freshly prepared solution of NaClO2 (30 mg, 0.33 mmol, 3 equiv)
and NaH2PO4 (76 mg, 0.55 mmol, 5 equiv) in H2O (2 mL) was
added to a stirred solution of aldehyde 17 (69 mg, 0.11 mmol,
1 equiv) and 2-methyl-2-butene (0.5 mL) in tBuOH (2 mL) and
the mixture was stirred vigorously at room temperature for 1 h.
H2O (15 mL) was then added, and the mixture was extracted with
Et2O (3 ꢂ 20 mL). The combined organic layers were washed with
brine (10 mL) and dried (Na2SO4). The solution was concentrated
in vacuo then the oil was purified by silica gel column chromatog-
raphy (19/1/80 EtOAc/AcOH/hexanes) to afford 65 mg (0.10 mmol)
of acid 18 in 90% yield. Rf = 0.6 (40% EtOAc in hexanes); 1H NMR
36.1, 35.3, 34.2, 28.7, 27.5, 23.4, 22.1, 21.6, 18.1, 11.3; IR (KBr)
m
(400 MHz, CDCl3)
d
6.16 (1H, d, J = 11.0 Hz), 5.81 (1H, d,
3390 (br), 2941, 2870, 1709, 1439, 1376, 1212, 1046 cmꢀ1; HRMS
J = 11.0 Hz), 4.12–4.01 (2H, m), 2.80 (1H, d, J = 11.6 Hz), 2.44–
2.31 (4H, m), 2.25 (1H, d, J = 13.6 Hz), 2.10 (1H, dd, J = 12.8,
8.4 Hz), 2.03–1.94 (2H, m), 1.93–1.83 (1H, m), 1.83–1.75 (1H, m),
1.69–1.46 (9H, m), 1.45–1.34 (3H, m), 1.33–1.18 (4H, m), 1.12–
1.00 (1H, m), 0.91 (3H, d, J = 6.0 Hz), 0.87 (9H, s), 0.86 (9H, m),
0.53 (3H, s), 0.05 (12H, s); 13C NMR (75 MHz, CDCl3) d 179.9,
141.0, 133.9, 121.9, 116.4, 68.4, 68.2, 56.7, 56.5, 46.2, 45.9, 43.9,
40.8, 37.0, 36.2, 35.7, 34.3, 28.9, 27.9, 26.11, 26.08, 25.9, 25.4,
23.7, 22.5, 19.0, 18.4, 18.3, 12.3, ꢀ4.4, ꢀ4.5, ꢀ4.6, ꢀ4.7; IR (KBr)
(ESI): m/z calcd for [(M+Na)+] = 428.2777, found = 428.2769.
4.1.1.15.
(R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxycy-
clohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-yl)-
N-hydroxyheptanamide (20b). This was prepared from acid 18
by following the same procedure described for 20a. The reagents
used were as follows: acid 18 (30 mg, 0.047 mmol, 1 equiv), oxalyl-
chloride (5.9 lL, 0.070 mmol, 1.5 equiv), DIPEA (24.5 lL, 0.141,
3 equiv) and O-tert-butyldimethylsilylhydroxylamine (14 mg,
0.094 mmol, 2 equiv). Compound 20b was purified by octadecyl-
functionalized silica gel column chromatography (100% H2O to
100% MeOH) to afford 13 mg (0.032 mmol) of the hydroxamic acid
20b in 67% yield. Rf = 0.30 (88/10/2 CH2Cl2/MeOH/CH3COOH); 1H
NMR (400 MHz, CD3OD) d 6.21 (1H, d, J = 11.0 Hz), 5.88 (1H, d,
J = 11.0 Hz), 4.08–3.93 (2H, m), 2.83 (1H, d, J = 11.6), 2.59 (1H, dd,
J = 13.2, 3.2 Hz), 2.41 (1H, dd, J = 13.2, 3.2 Hz), 2.18–2.12 (2H, m),
2.12–2.05 (2H, m), 2.05–1.98 (2H, m), 1.98–1.89 (1H, m), 1.89–
1.80 (1H, m), 1.80–1.71 (1H, m), 1.71–1.15 (17H, m), 1.15–1.03
(1H, m), 0.95 (3H, d, J = 6.0 Hz), 0.57 (3H, s); 13C NMR (75 MHz,
CD3OD) d 171.9, 140.9, 132.7, 122.2, 116.0, 66.8, 66.5, 56.7, 56.3,
45.6, 44.2, 41.5, 40.7, 36.4, 36.2, 35.6, 32.7, 28.6, 27.5, 26.1, 25.5,
m
2950, 2857, 1710, 1468, 1253, 1088, 920, 836, 775 cmꢀ1; HRMS
(ESI): m/z calcd for [(MꢀH)ꢀ] = 631.4578, found = 631.4578.
4.1.1.13. (R)-6-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Bis(tert-butyl-
dimethylsilyloxy)cyclohexylidene)ethylidene)-7a-methyl-octa-
hydro-1H-inden-1-yl)heptan-1-amine (19). This was prepared
from aldehyde 17 by following the same procedure described for
16. The reagents used were as follows: 17 (30 mg, 0.048 mmol,
1 equiv), O-benzylhydroxylamine hydrochloride (7.7 mg, 0.048
mmol, 1 equiv), sodium acetate (4.9 mg, 0.03 mmol, 1 equiv) and
AlLiH4 (5.4 mg, 0.144 mmol, 3 equiv). This afforded 22 mg
(0.035 mmol) of amine 19 in 74% yield. Rf = 0.35 (10/10/80, Et3N/
MeOH/EtOAc); 1H NMR (400 MHz, CDCl3)
d
6.16 (1H, d,
23.3, 22.1, 18.1, 11.2; IR (KBr)
1456, 1046 cmꢀ1 HRMS (ESI): m/z calcd for [(M+Na)+] =
442.2933, found = 442.2925.
m 3370 (br), 2937, 2868, 1650,
J = 10.8 Hz), 5.81 (1H, d, J = 10.8 Hz), 4.15–4.01 (2H, m), 3.34 (1H,
t, J = 7.2 Hz), 2.80 (1H, d, J = 12.0 Hz), 2.70 (1H, d, J = 7.2 Hz),
2.42–2.30 (2H, m), 2.25 (1H, d, J = 12.4 Hz), 2.10 (1H, dd, J = 12.4,
8.0 Hz), 2.02–1.71 (5H, m), 1.70–0.98 (17H, m), 0.91 (3H, d,
J = 6.0 Hz), 0.87 (9H, s), 0.86 (9H, s), 0.53 (3H, s), 0.05 (12H, s);
13C NMR (75 MHz, CDCl3) d 140.9, 133.6, 121.7, 116.1, 68.1, 67.9,
56.5, 56.3, 46.0, 45.6, 43.7, 40.6, 36.7, 36.1, 28.7, 27.7, 27.4, 25.9,
25.8, 23.4, 22.2, 18.8, 18.15, 18.10, 12.0, ꢀ4.7, ꢀ4.6, ꢀ4.8, ꢀ4.9;
;
4.1.1.16.
(R)-6-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxycy-
clohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-
yl)-N-hydroxy-N-methylheptanamide (20c). This was prepared
from acid 18 by following the same procedure described for 20a.
The reagents used were as follows: acid 18 (6.5 mg, 0.010 mmol,
IR (KBr)
m
2930, 2855, 1468, 1377, 1252, 1086, 836, 775 cmꢀ1
;
1 equiv), oxalylchloride (1.2
(1.6 L, 0.027, 3 equiv) and O-tert-butyldimethylsilyl-N-methyl
hydroxylamine (2.26 L, 0.018 mmol, 2 equiv). Compound 20c
lL, 0.014 mmol, 1.5 equiv), DIPEA
HRMS (ESI): m/z calcd for [(M+H)+] = 618.5102, found = 618.5089.
l
l
4.1.1.14.
(R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxycy-
was purified by octadecyl-functionalized silica gel column chroma-
tography (100% H2O to 100% MeOH) to afford 2.2 mg (0.005 mmol)
of the hydroxamic acid 20c in 51% yield. Rf = 0.40 (88/10/2 CH2Cl2/
MeOH/CH3COOH); 1H NMR (400 MHz, CD3OD) d 6.21 (1H, d,
J = 11.2 Hz), 5.88 (1H, d, J = 11.2 Hz), 4.08–3.92 (2H, m), 3.19 (3H,
s), 2.83 (1H, d, J = 12.4 Hz), 2.59 (1H, dd, J = 13.2, 4.0 Hz), 2.52–
2.35 (3H, m), 2.26-2.12 (2H, m), 2.08–1.18 (19H, m), 1.15–1.02
(1H, m), 0.95 (3H, d, J = 6.0 Hz), 0.57 (3H, s); 13C NMR (75 MHz,
clohexylidene)ethylidene)-7a-methyl-octahydro-1-inden-1-yl)-
N-hydroxyhexanamide (20a). In a flame-dried round bottom flask
under argon atmosphere, oxalylchloride (12.9
1.5 equiv) was added to a solution of acid 13 (63 mg, 0.102 mmol,
1 equiv) in dry CH2Cl2 (2 mL) and dry DMF (1 L) at 0 °C then the
solution was left to stir at room temperature for 1 h and recooled
to 0 °C. A solution of DIPEA (53.3 L, 0.306, 3 equiv) and O-tert-buty-
lL, 0.153 mmol,
l
l

4130
M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
CDCl3) d 174.8, 140.7, 132.4, 122.0, 115.7, 66.5, 66.3, 56.5, 56.1,
45.4, 43.9, 41.2, 40.5, 36.2, 35.9, 35.4, 34.8, 31.7, 28.4, 27.3, 25.6,
in quantitative yield without further purification. The hydroxyl-
amine was dissolved in THF (1 mL) then trifluoroethylformate
(7.5 mg of a 86% wt solution in formic acid, 0.50 mmol, 10 equiv)
was added and the mixture was refluxed for 4 h. The solution was
concentrated in vacuo, then the crude material was dissolved in
24.9, 23.1, 21.8, 17.9, 11.0; IR (KBr)
m 3343 (br), 2934, 2848,
1620, 1455, 1200, 1045, 668 cmꢀ1; HRMS (ESI): m/z calcd for
[(M+Na)+] = 456.3090, found = 456.3089.
CH2Cl2 (1 mL) and CH3CN (1 mL). A 48% solution of HF (34 lL,
4.1.1.17.
(R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxycy-
1 mmol, 20 equiv) was added and the solution was stirred at
room temperature for 2 h. The mixture was quenched cautionly
by the addition of a satd NaHCO3 until no effervescence was ob-
served. The solution was extracted with CH2Cl2 (3 ꢂ 5 mL) then
the combined organic layers were washed with H2O (5 mL), brine
(5 mL), dried (MgSO4) and then concentrated in vacuo. The oil
was purified by octadecyl-functionalized silica gel column chro-
matography (100% H2O to 100% MeOH) to afford 8.2 mg
(0.019 mmol) of 21 in 40% yield. 1H NMR (400 MHz, CDCl3) d
6.16 (1H, d, J = 11.0 Hz), 5.81 (1H, d, J = 11.0 Hz), 4.13–4.01 (2H,
m), 2.94 (2H, t, J = 6.8 Hz), 2.81 (1H, d, J = 11.6 Hz), 2.44–2.33
(2H, m), 2.25 (1H, d, J = 13.2 Hz), 2.10 (1H, dd, J = 12.4, 8.4 Hz),
2.04–1.94 (2H, m), 1.93–1.75 (2H, m), 1.70–1.15 (15H, m), 1.12–
0.98 (1H, m), 0.92 (3H, d, J = 6.4 Hz), 0.87 (9H, s), 0.86 (9H, s),
0.53 (3H, s), 0.05 (12H, s); 13C NMR (75 MHz, CDCl3) d 141.0,
133.9, 122.0, 116.4, 68.3, 68.2, 56.7, 56.5, 54.2, 46.2, 45.9, 43.9,
40.8, 37.0, 36.3, 36.0, 28.9, 27.6, 26.10, 26.08, 23.9, 23.6, 22.5,
clohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-yl)-
N-methoxyheptanamide (20d). This was prepared from acid 18
by following the same procedure described for 20a. The reagents
used were as follows: acid 18 (23 mg, 0.036 mmol, 1 equiv), oxalyl-
chloride (4.5 lL, 0.054 mmol, 1.5 equiv), DIPEA (225.1 lL, 0.144,
4 equiv) and O-methyl hydroxylamine hydrochloride (6 mg,
0.072 mmol, 2 equiv). Compound 20d was purified by octadecyl-
functionalized silica gel column chromatography (100% H2O to
100% MeOH) to afford 12.5 mg (0.029 mmol) of the hydroxamic
acid 20d in 80% yield. Rf = 0.4 (1/9/90 AcOH/MeOH/CH2Cl2); 1H
NMR (400 MHz, CDCl3) d 8.05 (1H, br s), 6.31 (1H, d, J = 11.2 Hz),
5.85 (1H, d, J = 11.2 Hz), 4.17–4.09 (1H, m), 4.09–4.01 (1H, m),
3.77 (3H, m), 2.79 (1H, d, J = 12.0), 2.74 (1H, dd, J = 13.6, 3.6 Hz),
2.41 (1H, dd, J = 13.2, 3.2 Hz), 2.48 (1H, d, J = 13.6 Hz), 2.28–2.17
(2H, m), 2.03–1.75 (5H, m), 1.73–1.15 (16H, m), 1.12–1.00 (1H,
m), 0.91 (3H, d, J = 6.0 Hz), 0.53 (3H, s); 13C NMR (75 MHz, CDCl3)
d 171.0, 143.1, 131.1, 123.8, 115.2, 67.4, 67.2, 60.4, 56.4, 56.3,
45.8, 44.6, 42.1, 40.4, 37.2, 35.9, 35.5, 28.9, 27.6, 25.7, 23.5, 22.3,
19.0, 18.4, 18.3, 12.3, ꢀ4.4, ꢀ4.5, ꢀ4.6, ꢀ4.7; IR (KBr)
2857, 1468, 1253, 1087, 1052, 1026, 906, 835, 805, 775, 735
cmꢀ1 HRMS (ESI): m/z calcd for [(M+H)+] = 420.3114,
m 2931,
21.1, 18.8, 14.2, 12.1; IR (KBr)
m
3342 (br), 2931, 2867, 1651,
;
1437, 1048, 975, 734 cmꢀ1; HRMS (ESI): m/z calcd for [(M+H)+] =
found = 420.3106.
434.3270, found = 434.3270.
4.1.1.20. 1-((R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxy-
cyclohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-
yl)hexyl)-3-hydroxyurea (22). A solution of amine 16 (13.1 mg,
4.1.1.18.
(R)-6-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxycy-
clohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)-
N-methoxy-N-methylheptanamide (20e). This was prepared
from acid 18 by following the same procedure described for
20a. The reagents used were as follows: acid 18 (6.5 mg,
0.022 mmol, 1 equiv) in CH2Cl2 (130
stirred solution of 1,1-carbonyldiimidazole (25.6 mg, 0.22 mmol,
10 equiv) in THF (50 L) under argon cooled to 0 °C. The reaction
lL) was added dropwise to a
l
0.010 mmol, 1 equiv), oxalylchloride (1.3
l
L, 0.015 mmol,
mixture was stirred 16 h at room temperature, then O-tert-buty-
ldimethylsilylhydroxylamine (95 mg, 0.66 mmol, 30 equiv) was
added to the mixture, the solution was then allowed to stir a fur-
ther 16 h. The reaction mixture was diluted with EtOAc (5 mL),
washed with 0.1 M HCl (2 ꢂ 5 mL), water (5 mL), brine (5 mL)
and then dried (Na2SO4). The solution was then concentrated in va-
cuo to provide an oil which was purified by silica gel column chro-
matography (50% EtOAc in hexanes, Rf = 0.2) to give the O-tert-
butyldimethylsilylhydroxyurea. The product was then directly
submitted to deprotection: the oil was dissolved in CH2Cl2
1.5 equiv), DIPEA (11 L, 0.060, 6 equiv) and N,O-dimethylhydr-
l
oxylamine hydrochloride (4 mg, 0.040 mmol, 4 equiv). Compound
20e was purified by silica gel column chromatography (50%
EtOAc in hexanes) to afford 3.3 mg (0.0074 mmol) of the hydroxa-
mic acid 20e in 74% yield. Rf = 0.3 (50% EtOAc in hexanes); 1H
NMR (400 MHz, CDCl3) d 6.31 (1H, d, J = 11.2 Hz), 5.84 (1H, d,
J = 11.2 Hz), 4.11 (1H, br s), 4.04 (1H, br s), 3.68 (3H, m), 3.17
(3H, m), 2.84–2.79 (2H, m), 2.47 (1H, d, J = 13.2 Hz), 2.41 (2H, t,
J = 7.2 Hz), 2.27–2.15 (2H, m), 2.04–1.84 (5H, m), 1.83–1.74 (1H,
m), 1.73–1.16 (14H, m), 1.12–1.02 (2H, m), 0.91 (3H, d,
(100 lL) and CH3CN (100 lL) then a 48% solution of HF was added
J = 6.0 Hz), 0.53 (3H, s); 13C NMR (75 MHz, CDCl3)
143.4, 131.3, 124.1, 115.5, 67.6, 67.5, 61.4, 56.7, 56.5, 46.0, 44.9,
42.4, 40.7, 37.4, 36.2, 35.8, 32.2, 29.2, 27.8, 26.2, 25.3, 23.8,
d
175.2,
(two drops) and the solution was stirred overnight. The mixture
was quenched cautionly by the addition of satd NaHCO3 until no
effervescence was observed then acidified with a 1 M aq solution
of citric acid (5 mL). The solution was extracted with CH2Cl2
(3 ꢂ 5 mL) then the combined organic layers were washed with
H2O (5 mL), brine (5 mL), dried (MgSO4) and then concentrated
in vacuo. The oil was purified by octadecyl-functionalized silica
gel column chromatography (100% H2O to 100% MeOH) to afford
the hydroxyurea 22 in 44% yield (4.2 mg, 0.009 mmol). Rf = 0.2
(88/10/2 CH2Cl2/MeOH/CH3COOH); 1H NMR (400 MHz, CD3OD) d
6.21 (1H, d, J = 11.4 Hz), 5.88 (1H, d, J = 11.4 Hz), 4.08–4.01 (1H,
m), 4.00–3.93 (1H, m), 3.18 (2H, t, J = 6.8 Hz), 2.83 (1H, d,
J = 9.6 Hz), 2.59 (1H, d, J = 6.0 Hz), 2.41 (1H, d, J = 10.4 Hz), 2.25–
2.12 (2H, m), 2.06–1.98 (2H, m), 1.97–1.18 (17H, m), 1.15–1.04
(1H, m), 0.95 (3H, d, J = 6.0 Hz), 0.57 (3H, s); 13C NMR (75 MHz,
CD3OD) d 170.9, 140.9, 132.7, 122.2, 115.9, 66.8, 66.5, 56.7, 56.3,
45.6, 44.2, 41.4, 40.7, 39.9, 36.4, 36.2, 35.6, 30.5, 28.6, 27.5, 23.3,
22.5, 19.0, 12.2; IR (KBr)
m 3402 (br), 2938, 2876, 1645, 1440,
1380, 1049, 994, 732 cmꢀ1
;
HRMS (ESI): m/z calcd for
[(M+Na)+] = 470.3241, found = 470.3241.
4.1.1.19. N-((R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxy-
cyclohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-
yl)hexyl)-N-hydroxyformamide (21). A solution of aldehyde 15
(30 mg, 0.050 mmol, 1 equiv), hydroxylamine (9.2 lL of a 50%
wt solution in water, 0.50 mmol, 10 equiv) in 4 mL of toluene
was refluxed for 30 min. The mixture was then concentrated
and the crude residue was dissolved in AcOH/THF (2:1, 2 mL)
then cooled to 0 °C. NaBH3CN (6 mg, 0.10 mmol, 2 equiv) was
added portionwise. The mixture was stirred at 0 °C for 1 h then
0.5 M NaOH was slowly added until alkaline pH. The solution
was extracted with CH2Cl2 (3 ꢂ 10 mL), the combined organic lay-
ers were washed with brine (10 mL) then dried (Na2SO4). The
solution was concentrated and the hydroxylamine was obtained
22.1, 18.1, 11.2; (KBr)
735 cmꢀ1 HRMS (ESI): m/z calcd for [(M+Na)+] = 457.3042,
found = 457.3042.
m 3312 (br), 2937, 2841, 1651, 1457, 1049,
;

M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
4131
4.1.1.21. (R)-N-(2-Aminophenyl)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-
3,5-dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-
1H-inden-1-yl)hexanamide (23a). HBTU (4.6 mg, 0.012 mmol,
1.1 equiv) was added to a solution of acid 13 (6.8 mg, 0.011 mmol,
1 equiv), 1,2-phenylenediamine (1.2 mg, 0.011 mmol, 1 equiv),
(23 mg, 0.040 mmol, 1 equiv) and 2-(acetylthio)acetic acid
(5.9 mg, 0.044 mmol, 1.1 equiv) in CH2Cl2 (1 mL) at 0 °C. The solu-
tion was stirred at room temperature for 4 h then diluted with
CH2Cl2 (15 mL), washed with 0.5 M HCl (2 ꢂ 5 mL), with satd NaH-
CO3 (2 ꢂ 5 mL), with brine (5 mL) and dried (Na2SO4). The solution
was concentrated in vacuo to give the protected product as an oil
(27 mg, 0.046 mmol) and the product was carried forward without
further purification. In a flame-dried round bottom flask under ar-
HOBt (7.4 mg, 0.055 mmol, 5 equiv) and DIPEA (5.7
lL, 0.033
mmol, 3 equiv) in DMF (110 L). The mixture was stirred at room
l
temperature for 1 h, then diluted with EtOAc (10 mL). The organic
solution was washed with satd NaHCO3 (5 mL), water (5 mL), brine
(5 mL) and dried (Na2SO4). The solution was filtered, concentrated,
and the oil was purified by silica gel column chromatography (50/
45/5 EtOAc/hexanes/Et3N, Rf = 0.5) to afford 7 mg (0.010 mmol) of
protected o-aminoanilide. The product was dissolved under argon
gon atmosphere,
a deoxygenated solution of MeONa (2 mg
0.036 mmol, 1 equiv) in MeOH was added by cannula to the acet-
ylated mercaptoacetamide (25 mg, 0.036 mmol, 1 equiv). The solu-
tion was stirred at room temperature for 4 h then quenched by the
addition of AcOH (1 mL). The solution was concentrated in vacuo
then diluted in EtOAc (15 mL). The solution was washed with
water (5 mL), brine (5 mL), dried (Na2SO4) and concentrated to give
the deacetylated compound witch was dissolved in CH2Cl2 (1 mL)
in THF (0.5 mL), then TBAF (40
0.040 mmol, 4 equiv) and Et3N (5.6
l
l
L of a 1 M solution in THF,
L, 0.040 mmol, 4 equiv) were
added and the mixture was stirred for 48 h. The solution was con-
centrated in vacuo and loaded directly onto silica gel. Compound
23a was purified by octadecyl-functionalized silica gel column
chromatography (100% H2O to 100% MeOH) to afford 2.9 mg
(0.006 mmol) of 23a in 55% yield. Rf = 0.33 (90/10 EtOAc/Et3N);
1H NMR (500 MHz, CD3OD) d 7.07 (1H, d, J = 8.0 Hz), 7.02 (1H, t,
J = 7.6 Hz), 6.84 (1H, d, J = 8.0 Hz), 6.71 (1H, t, J = 7.5 Hz), 6.22
(1H, d, J = 11.0 Hz), 5.89 (1H, d, J = 11.0 Hz), 4.09–4.01 (1H, m),
4.00–3.96 (1H, m), 2.84 (1H, d, J = 12.0 Hz), 2.60 (1H, d,
J = 13.5 Hz), 2.46–2.31 (3H, m), 2.29–2.11 (3H, m), 2.09–1.91 (4H,
m), 1.88–1.73 (4H, m), 1.72–1.12 (9H, m), 1.00 (3H, d, J = 7.0 Hz),
0.59 (3H, s); 13C NMR (75 MHz, CD3OD) d 174.0, 142.1, 140.9,
132.7, 127.1, 126.0, 124.0, 122.2, 118.3, 117.4, 116.0, 66.8, 66.5,
56.6, 56.3, 45.6, 44.2, 41.5, 40.7, 36.4, 36.1, 35.4, 28.6, 27.5, 23.3,
and CH3CN (1 mL). HF (50 lL) was added to the solution and the
mixture was stirred at room temperature overnight. The reaction
mixture was quenched with satd NaHCO3 (2 mL) and extracted
with CH2Cl2 (3 ꢂ 10 mL). The combined organic fractions were
washed with brine (5 mL) and dried (Na2SO4). The solution was
concentrated in vacuo then purified by silica gel column chroma-
tography (5% MeOH in CH2Cl2) to give 24a in 69% yield. Rf = 0.5
(10% MeOH in CH2Cl2); 1H NMR (400 MHz, CDCl3) d 6.70 (1H, br
s), 6.31 (1H, d, J = 11.6 Hz), 5.85 (1H, d, J = 10.8 Hz), 4.19–4.08
(1H, m), 4.08–4.01 (1H, m), 3.35–3.17 (3H, m), 3.80 (1H, d,
J = 16.0 Hz), 2.74 (1H, d, J = 17.2 Hz), 2.48 (1H, d, J = 12.8 Hz),
2.26–2.15 (2H, m), 2.04–1.74 (6H, m), 1.73–1.00 (13H, m), 0.93
(3H, d, J = 6.4 Hz), 0.54 (3H, s); 13C NMR (75 MHz, CDCl3) d 169.4,
143.3, 131.3, 124.1, 115.4, 67.6, 67.5, 56.60, 56.58, 46.3, 44.9,
42.4, 40.7, 40.3, 37.2, 36.0, 35.7, 29.9, 29.1, 28.6, 23.7, 2.5, 22.5,
22.5, 22.1, 18.1, 11.2; IR (KBr)
m 3351 (br), 2943, 2872, 1653,
1503, 1454, 1376, 1306, 1047, 908, 732 cmꢀ1; HRMS (ESI): m/z
calcd for [(M+H)+] = 481.3426, found = 481.3430.
19.0, 12.3; IR (KBr) m 3311 (br), 2925, 2869, 1651, 1558, 1540,
1457, 1261, 1045, 802, 733 cmꢀ1; HRMS (ESI): m/z calcd for
4.1.1.22. (R)-N-(2-Aminophenyl)-6-((1R,3aS,7aR,E)-4-(2-((3R,5R)-
3,5-dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-
1H-inden-1-yl)heptanamide (23b). This was prepared from acid
18 by following the same procedure described for 23a. The re-
agents used were as follows: acid 18 (19 mg, 0.030 mmol, 1 equiv),
1,2-phenylenediamine (3.2 mg, 0.030 mmol, 1 equiv), HBTU
(12.5 mg, 0.033 mmol, 1.1 equiv), HOBt (20.2 mg, 0.150 mmol,
[(M+Na)+] = 458.2705, found = 458.2698.
4.1.1.24. N-((R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxy-
cyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-
yl)hexyl)-2-mercaptoacetamide (24b). This was prepared from
amine 16 by following the same procedure described for 24a.
The reagents used were as follows: amine 16 (29 mg,
5 equiv) and DIPEA (15.7
(1.2 L, 0.014 mmol, 1.5 equiv), DIPEA (1.6
O-tert-butyldimethylsilyl-N-methyl hydroxylamine
0.018 mmol, 2 equiv); TBAF (108 L of a 1 M solution in THF,
0.108 mmol, 4 equiv relative to the protected intermediate) and
Et3N (15 L, 0.108 mmol, 4 equiv relative to the protected interme-
diate). Compound 23b was purified by silica gel column chroma-
tography (50/45/5 EtOAc/hexanes/Et3N) to afford 19.3 mg
(0.027 mmol) of 23b in 84% yield. Rf = 0.33 (90/10 EtOAc/Et3N);
1H NMR (400 MHz, CD3OD) d 7.06 (1H, d, J = 8.0 Hz), 7.02 (1H, t,
J = 7.6 Hz), 6.84 (1H, d, J = 8.0 Hz), 6.71 (1H, t, J = 7.5 Hz), 6.22
(1H, d, J = 11.0 Hz), 5.89 (1H, d, J = 11.0 Hz), 4.10–4.01 (1H, m),
4.00–3.93 (1H, m), 2.83 (1H, dd, J = 12.8, 4.0 Hz), 2.59 (1H, dd,
J = 12.8, 3.6 Hz), 2.51–2.35 (3H, m), 2.30–2.10 (3H, m), 2.09–1.23
(17H, m), 1.22–1.08 (1H, m), 1.02 (1H, t, J = 7.2 Hz), 0.97 (3H, d,
J = 6.4 Hz), 0.58 (3H, s); 13C NMR (75 MHz, CD3OD) d 174.0,
140.9, 132.7, 127.1, 125.9, 124.0, 122.3, 118.3, 117.4, 116.0, 66.8,
66.5, 56.7, 56.3, 45.6, 44.2, 41.4, 40.7, 36.5, 36.2, 36.1, 35.6, 28.6,
l
L, 0.090 mmol, 3 equiv). oxalylchloride
L, 0.027, 3 equiv) and
(2.26 L,
0.048 mmol,
1 equiv),
2-(acetylthio)acetic
acid
(7.1 mg,
l
l
0.053 mmol, 1.1 equiv equiv), EDCꢃHCl (11 mg, 0.058 mmol,
1.2 equiv). Deprotection: acetylated mercaptoacetamide (30 mg,
0.041 mmol, 1 equiv), MeONa (2.2 mg 0.041 mmol, 1 equiv).
Compound 32 was purified by silica gel column chromatography
(5% MeOH in CH2Cl2) to give 13 mg (0.031 mmol) of 32 in 67%
yield. Rf = 0.5 (10% MeOH in CH2Cl2); 1H NMR (400 MHz, CDCl3)
l
l
l
d
6.68 (1H, br s), 6.31 (1H, d, J = 11.4 Hz), 5.85 (1H, d,
J = 11.4 Hz), 4.12 (1H, br s), 4.05 (1H, br s), 3.28 (2H, q,
J = 6.4 Hz), 3.24 (2H, d, J = 8.8 Hz), 2.80 (1H, d, J = 12.4 Hz), 2.73
(1H, d, J = 13.2 Hz), 2.48 (1H, d, J = 11.2 Hz), 2.27–2.15 (2H, m),
2.05–1.74 (6H, m), 1.73–1.16 (14H, m), 1.13–1.00 (1H, m), 0.92
(3H, d, J = 6.4 Hz), 0.54 (3H, s); 13C NMR (75 MHz, CDCl3) d
169.2, 143.3, 131.4, 124.1, 115.5, 67.6, 67.4, 56.7, 56.5, 46.0,
44.9, 42.4, 40.7, 40.2, 37.4, 36.2, 35.7, 30.1, 29.2, 28.6, 27.9,
23.71, 23.66, 22.5, 19.0, 12.3; IR (KBr)
m 3306 (br), 2935, 2869,
1650, 1550, 1439, 1048, 909, 732 cmꢀ1; HRMS (ESI): m/z calcd
for [(M+H)+] = 450.3042, found = 450.3043.
27.6, 26.4, 25.7, 23.6, 23.3, 22.1, 18.2, 11.2; IR (KBr)
m 3350 (br),
2943, 2872, 1652, 1503, 1456, 1376, 1306, 1046, 909, 732 cmꢀ1
;
HRMS (ESI): m/z calcd for [(M+H)+] = 495.3587, found = 495.3577.
4.1.1.25. 2-Amino-N-((R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-di-
hydroxycyclohexylidene)ethylidene)-7a-methyloctahydro-1H-
inden-1-yl)hexyl)acetamide (25a). DCC (2.2 mg, 0.0107 mmol,
1.05 equiv) then DMAP (catalytic amount) were added to a stir-
red solution of amine 16 (6.2 mg, 0.0102 mmol, 1 equiv) and N-
4.1.1.23. N-((R)-4-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxy-
cyclohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-
yl)pentyl)-2-mercaptoacetamide (24a). EDCꢃHCl (9.2 mg, 0.048
mmol, 1.2 equiv) was added to a stirred solution of the amine 14
Bocglycine (1.9 mg, 0.0107 mmol, 1.05 equiv) in CH2Cl2 (150 lL)

4132
M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
at 0 °C. The solution was then stirred at room temperature for
2 h then diluted with CH2Cl2 (10 mL), washed with satd NaHCO3
(2 ꢂ 5 mL), with brine (5 mL) and dried (Na2SO4). The solution
was concentrated in vacuo then purified by silica gel column
chromatography (50% EtOAc in hexanes) to obtain the protected
intermediate as a clear oil (7.3 mg, 0.0096 mmol). The product
tected 25c (10.1 mg, 0.014 mmol); Rf = 0.8 (50% EtOAc in hex-
anes); 1H NMR (400 MHz, CDCl3) d 6.47 (1H, br s), 6.15 (1H, d,
J = 10.8 Hz), 5.80 (1H, d, J = 10.8 Hz), 4.15–4.00 (2H, m), 3.88
(2H, s), 3.32–3.27 (2H, m), 3.18 (2H, br s), 2.79 (1H, d,
J = 12.0 Hz), 2.42–2.30 (2H, m), 2.25 (1H, d, J = 13.6 Hz), 2.09
(1H, dd, J = 12.8, 7.6 Hz), 2.03–1.01 (17H, m), 1.45 (9H, s), 0.90
(3H, d, J = 6.0 Hz), 0.86 (9H, s), 0.85 (9H, s), 0.52 (3H, s), 0.04
(12H, s); 13C NMR (75 MHz, CDCl3) d 165.3, 141.0, 133.9, 121.9,
116.4, 68.3, 68.2, 56.7, 56.5, 46.2, 45.9, 43.9, 40.8, 40.5, 37.0,
36.3, 35.7, 35.1, 30.0, 29.7, 28.9, 28.5, 28.0, 26.09, 26.08, 25.8,
24.9, 23.6, 22.4, 19.0, 18.38, 18.34, 12.3, ꢀ4.4, ꢀ4.5, ꢀ4.6, ꢀ4.7.
was then dissolved in CH3CN (100 lL) and CH2Cl2 (100 lL), then
HF (one drop of a 48% solution) was added to the solution and
the mixture was stirred at room temperature overnight. The
reaction mixture was quenched with satd NaHCO3 (2 mL) and
extracted with CH2Cl2 (3 ꢂ 10 mL). The combined organic frac-
tions were washed with brine (5 mL) and dried (Na2SO4). The
solution was concentrated in vacuo then purified by silica gel
column chromatography (10/20/17 Et3N/MeOH/EtOAc) to obtain
25a in 66% yield (3.3 mg, 0.0070 mmol). Rf = 0.15 (10/20/17
Et3N/MeOH/EtOAc); 1H NMR (400 MHz, CD3OD) d 6.21 (1H, d,
J = 11.2 Hz), 5.80 (1H, d, J = 11.2 Hz), 4.09–4.02 (1H, m), 4.01–
3.95 (1H, m), 3.25–3.15 (2H, m), 2.83 (1H, d, J = 11.2 Hz), 2.59
(1H, d, J = 10.0 Hz), 2.41 (1H, d, J = 10.8 Hz), 2.26–2.11 (2H, m),
2.09–1.80 (5H, m), 1.79–1.71 (1H, m), 1.70–1.02 (14H, m), 0.95
(3H, d, J = 6.0 Hz), 0.92–0.84 (2H, m), 0.57 (3H, s); 13C NMR
(75 MHz, CD3OD) d 169.3, 140.8, 132.7, 122.2, 116.0, 66.8, 66.5,
56.7, 56.3, 45.6, 44.2, 43.0, 41.5, 40.7, 39.2, 36.4, 36.2, 35.6,
IR (KBr)
m
2930, 2854, 2119, 1655, 1642, 1448, 1252, 1086, 835,
775 cmꢀ1
;
HRMS (ESI): m/z calcd for [(M+Na)+] = 746.3970,
found = 746.3970.
4.8 mg (0.0066 mmol) of TBS-O-protected 25c were dissolved
in CH3CN (100 lL) and CH2Cl2 (100 lL), then HF (one drop of a
48% solution) was added to the mixture was stirred at room tem-
perature overnight. The reaction mixture was quenched with satd
NaHCO3 (2 mL) and extracted with CH2Cl2 (3 ꢂ 10 mL). The com-
bined organic fractions were washed with brine (5 mL) and dried
(Na2SO4). The solution was concentrated in vacuo then purified
by silica gel column chromatography (40% acetone in hexanes)
to afford 2.5 mg (0.0050 mmol) of 25c in 65% yield. Rf = 0.15
(40% acetone in hexanes); 1H NMR (400 MHz, CDCl3) d 6.48 (1H,
br s), 6.31 (1H, d, J = 11.4 Hz), 5.85 (1H, d, J = 11.4 Hz), 4.12 (1H,
br s), 4.04 (1H, br s), 3.89 (2H, s), 3.29 (2H, q, J = 6.7 Hz), 2.79
(1H, dd, J = 12.2, 3.8 Hz), 2.74 (1H, dd, J = 13.6, 3.6 Hz), 2.26–
2.14 (5H, m), 2.04–1.02 (19H, m), 0.92 (3H, d, J = 6.8 Hz), 0.54
(3H, s); 13C NMR (75 MHz, CDCl3) d 165.4, 143.3, 131.3, 124.1,
115.5, 67.6, 67.5, 56.6, 56.5, 46.0, 44.9, 42.4, 40.5, 37.4, 36.2,
35.6, 30.0, 29.7, 29.5, 29.1, 27.9, 23.7, 22.5, 19.0, 12.3; IR (KBr)
29.7, 29.6, 28.6, 27.6, 23.3, 22.1, 18.1, 11.2; IR (KBr)
m 3301
(br), 2927, 2872, 1659, 1642, 1443, 1050, 978, 668 cmꢀ1; HRMS
(ESI): m/z calcd for [(M+H)+] = 433.3425, found = 433.3423.
4.1.1.26. N-((R)–5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxy-
cyclohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-
yl)hexyl)-2-(dimethylamino)acetamide (25b). EDCꢃHCl (1.6 mg,
0.011 mmol, 1.1 equiv) was added to a stirred solution of amine
16 (6.3 mg, 0.010 mmol, 1 equiv) and dimethylglycine (1.2 mg,
0.011 mmol, 1.1 equiv) in CH2Cl2 (0.2 mL) at 0 °C. The solution
was stirred at room temperature for 4 h then diluted with CH2Cl2
(10 mL), washed with satd NaHCO3 (2 ꢂ 5 mL), with brine (5 mL)
and dried (Na2SO4). The solution was concentrated in vacuo to give
m
3305 (br), 2926, 2868, 1659, 1552, 1442, 1216, 1046, 977,
734 cmꢀ1 HRMS (ESI): m/z calcd for [(M+Na)+] = 518.2240,
;
found = 518.2242.
4.1.1.28. 2-((R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxy-
cyclohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-
yl)hexylamino)-2-oxoethyl ethyl carbonotrithioate (25d). NaH-
the crude acetamide as a yellow oil. TBAF (4
lL of a 1 M solution in
THF, 0.040 mmol, 4 equiv) and Et3N (4 L, 0.033 mmol, 3 equiv)
l
were added to a stirred solution of the crude product in THF
(0.5 mL) under argon and the mixture was stirred overnight. The
solution was concentrated in vacuo and loaded directly onto silica
gel column chromatography (80/10/10 EtOAc/MeOH/Et3N) to ob-
tain 25b as a clear oil in 58% yield (2.9 mg, 0.006 mmol). Rf = 0.5
(80/10/10 EtOAc/MeOH/Et3N); 1H NMR (400 MHz, CDCl3) d 6.31
(1H, d, J = 11.2 Hz), 5.85 (1H, d, J = 11.2 Hz), 3.32–3.20 (2H, m),
2.93 (1H, s), 2.79 (1H, dd, J = 13.2, 4.0 Hz), 2.74 (1H, dd, J = 14.0,
4.0 Hz), 2.48 (1H, d, J = 14.0 Hz), 2.28 (6H, s), 2.28–2.16 (2H, m),
2.06–1.02 (18H, m), 0.91 (3H, d, J = 6.0 Hz), 0.88–0.80 (2H, m),
0.53 (3H, s); 13C NMR (75 MHz, CDCl3) d 170.7, 143.3, 131.3,
124.1, 115.5, 67.7, 67.5, 63.4, 56.7, 56.5, 46.3, 46.0, 44.9, 42.4,
40.7, 39.2, 37.4, 36.2, 35.7, 30.4, 29.1, 27.9, 23.7, 22.5, 19.0, 12.3;
MDS (42
added to a stirred solution of ethanethiol (3.1
1 equiv) in dry THF (200 L) cooled to 0 °C under argon. The
solution was stirred at 0 °C for 10 min then CS2 (3.3 L,
0.056 mmol, 4 equiv) was added dropwise and the reaction mix-
ture was stirred at room temperature for 1 h before a solution of
TBS-O-protected 25c (7 mg, 0.014 mmol, 1 equiv) in dry THF
l
L of a 1 M solution in THF, 0.042 mmol, 3 equiv) was
lL, 0.042 mmol,
l
l
(150 lL) was added. After stirring for an additional 1 h at room
temperature, the reaction mixture was quenched by addition of
satd NH4Cl (3 mL) and extracted with EtOAc (3 ꢂ 5 mL). The
combined organic fraction were dried (Na2SO4) and concentrated
in vacuo, the oil was then purified by silica gel column chroma-
tography (50% acetone in hexanes) to afford the trithiocarbonate
25d in 82% yield (6.4 mg, 0.011 mmol). Rf = 0.2 (50% acetone in
hexanes); 1H NMR (400 MHz, CDCl3) d 6.31 (1H, d, J = 11.4 Hz),
6.29 (1H, br s), 5.85 (1H, d, J = 11.4 Hz), 4.15–4.09 (1H, m),
4.07–4.01 (1H, m), 4.06 (2H, s), 3.39 (2H, q, J = 7.6 Hz), 3.23
(2H, d, J = 7.6 Hz), 2.79 (1H, dd, J = 12.4, 3.6 Hz), 2.74 (1H, d,
J = 13.2, 3.6 Hz), 2.47 (1H, dd, J = 13.2, 2.8 Hz), 2.25–2.16 (2H,
m), 2.04–1.10 (19H, m), 1.37 (3H, t, J = 7.6 Hz), 1.09–0.99 (1H,
m), 0.90 (3H, d, J = 6.4 Hz), 0.53 (3H, s); 13C NMR (75 MHz,
CDCl3) d 223.5, 166.8, 143.0, 131.3, 124.1, 115.5, 67.6, 67.5,
56.6, 56.5, 46.0, 44.9, 42.4, 40.7, 40.2, 39.4, 37.4, 36.2, 35.7,
IR (KBr)
m ;
3332 (br), 2931, 2869, 1657, 1528, 1454, 1047, 732 cmꢀ1
HRMS (ESI): m/z calcd for [(M+H)+] = 461.3738, found = 431.3735.
4.1.1.27. 2-Bromo-N-((R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-di-
hydroxycyclohexylidene)ethylidene)-7a-methyloctahydro-1H-
inden-1-yl)hexyl)acetamide (25c). DCC (3.5 mg, 0.017 mmol,
1.05 equiv) then DMAP (catalytic amount) were added to a stirred
solution of amine 16 (9.7 mg, 0.016 mmol, 1 equiv) and bromo-
acetic acid (2.33 mg, 0.017 mmol, 1.05 equiv) in CH2Cl2 (150 lL)
at 0 °C. The solution was then stirred at room temperature for
2 h then diluted with CH2Cl2 (10 mL), washed with satd NaHCO3
(2 ꢂ 5 mL), with brine (5 mL) and dried (Na2SO4). The solution
was concentrated in vacuo then purified by silica gel column
chromatography (50% EtOAc in hexanes) to afford the TBS-O-pro-
32.3, 30.0, 29.1, 27.9, 23.7, 23.5, 23.5, 19.0, 13.5, 12.3; (KBr)
3294 (br), 2929, 2869, 1653, 1551, 1446, 1375, 1079, 1046,
811 cmꢀ1 HRMS (ESI): m/z calcd for [(M+H)+] = 554.2796,
found = 554.2782.
m
;

M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
4133
4.1.1.29. N-((R)-4-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxy-
cyclohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-
yl)pentyl)methanesulfonamide (27a). Methylsulfonyl chloride
J = 12.0, 4.0 Hz), 2.74 (1H, dd, J = 13.2, 4.0 Hz), 2.48 (1H, dd,
J = 13.6, 3.2 Hz), 2.26–2.16 (2H, m), 2.04–1.75 (5H, m), 1.74–
1.18 (14H, m), 1.13–1.02 (1H, m), 0.92 (3H, t, J = 6.4 Hz), 0.54
(3H, s); 13C NMR (75 MHz, CDCl3) d 143.2, 131.4, 124.1, 115.5,
67.7, 67.5, 56.6, 56.5, 46.0, 44.9, 44.8, 42.4, 40.7, 37.4, 36.2,
(2.3
lL, 0.031 mmol, 1.1 equiv) was added to a stirred solution
of the amine 14 (16 mg, 0.028 mmol, 1 equiv) and Et3N (11
lL,
0.084 mmol, 3 equiv) in CH2Cl2 (1.5 mL) at 0 °C. The solution
was stirred at 0 °C for 1 h then at room temperature for 1 h.
The reaction mixture was then diluted with CH2Cl2 (5 mL),
washed with satd NaHCO3 (2 ꢂ 5 mL), water (5 mL), brine
(5 mL) then dried (Na2SO4). The solution was filtered, concen-
trated and the residual oil was loaded on silica gel (35% EtOAc
in hexanes) to give the sulfonamide as an oil. The oil was then
35.5, 31.0, 29.1, 27.9, 23.7, 23.1, 22.5, 18.9, 12.3; (KBr)
(br), 2940, 2871, 1435, 1371, 1221, 1188, 1148, 1045, 606 cmꢀ1
HRMS (ESI): m/z calcd for
[(M+Na)+] = 530.2528,
found = 530.2518.
m 3325
;
4.1.1.32. N-((R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxy-
cyclohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-
yl)hexyl)ethanesulfonamide (27d). This was prepared from
amine 16 by following the same procedure described for 27a.
The reagents used were as follows: amine 16 (14 mg, 0.024 mmol,
dissolved into CH2Cl2 (100 lL) and CH3CN (100 lL) and then a
48% solution of HF was added (one drop). The solution was stirred
at room temperature overnight then quenched cautionly by the
addition of a satd NaHCO3 until no effervescence was observed.
The solution was extracted with CH2Cl2 (3 ꢂ 5 mL) then the com-
bined organic layers were washed with H2O (5 mL), brine (5 mL),
dried (MgSO4) and then concentrated in vacuo. The oil was then
purified by silica gel column chromatography (5% MeOH in
EtOAc) to give 3.7 mg (0.0008 mmol) of the sulfonamide 27a in
30% yield. Rf = 0.7 (5% MeOH in EtOAc); 1H NMR (400 MHz, CDCl3)
d 6.31 (1H, d, J = 10.8 Hz), 5.85 (1H, d, J = 10.8 Hz), 4.19–4.00 (2H,
m), 3.11 (2H, t, J = 6.8 Hz), 2.96 (3H, s), 2.80 (1H, d, J = 12.4 Hz),
2.74 (1H, d, J = 13.2 Hz), 2.48 (1H, d, J = 12.8 Hz), 2.30–2.15 (3H,
m), 2.08–1.02 (17H, m), 0.93 (3H, d, J = 6.4 Hz), 0.54 (3H, s) 13C
NMR (75 MHz, CDCl3) d 143.1, 131.5, 124.0, 115.6, 67.6, 67.5,
56.53, 56.48, 46.0, 44.9, 44.1, 42.4, 40.7, 40.6, 37.4, 36.0, 33.0,
1 equiv), ethanesulfonyl chloride (4.5
lL, 0.048 mmol, 2 equiv) and
Et3N (10 L, 0.072 mmol, 3 equiv). Compound 27d was purified by
l
silica gel column chromatography (EtOAc) to give 6.0 mg
(0.013 mmol) the sulfonamide 27d in 35% yield. Rf = 0.2 (EtOAc);
1H NMR (400 MHz, CDCl3) d 6.31 (1H, d, J = 11.4 Hz), 5.85 (1H, d,
J = 11.4 Hz), 4.16–4.09 (1H, m), 4.08–4.01 (1H, m), 3.15–3.08 (2H,
m), 3.04 (2H, q, J = 7.2 Hz), 2.79 (1H, dd, J = 11.6, 4.0 Hz), 2.74
(1H, d, J = 13.2, 4.0 Hz), 2.48 (1H, dd, J = 13.6, 3.2 Hz), 2.27–2.16
(2H, m), 2.03–1.75 (5H, m), 1.73–1.16 (14H, m), 1.37 (3H, t,
J = 7.2 Hz), 1.12–1.08 (1H, m), 0.92 (3H, d, J = 6.4 Hz), 0.53 (3H,
s); 13C NMR (75 MHz, CDCl3) d 143.0, 131.1, 123.8, 115.3, 67.4,
67.2, 56.4, 56.3, 46.8, 45.8, 44.6, 43.4, 42.1, 40.4, 37.2, 36.0, 35.4,
30.9, 28.9, 27.7, 23.5, 23.1, 22.2, 18.8, 12.1, 8.4; (KBr)
m 3291 (br),
29.1, 27.9, 27.1, 23.7, 22.5, 19.0, 12.3; IR (KBr)
2929, 2871, 1445, 1316, 1261, 1149, 1045, 974, 801, 735 cmꢀ1
HRMS (ESI): m/z calcd for
[(M+Na)+] = 462.2624,
found = 462.2650.
m 3303 (br),
2939, 2876, 1451, 1371, 1141, 1046, 910, 732 cmꢀ1; HRMS (ESI):
;
m/z calcd for [(M+Na)+] = 490.2967, found = 490.2966.
4.1.1.33. N-((R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxy-
cyclohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-
yl)hexyl)-2-hydroxyethanesulfonamide (27e). This was prepared
from amine 16 by following the same procedure described for 27a.
The reagents used were as follows: amine 16 (14 mg, 0.023 mmol,
1 equiv), 2-hydroxyethanesulfonyl chloride (3.4 mg, 0.046 mmol,
4.1.1.30. N-((R)-5-((1R,3aS,7aR,E)–4-(2-((3R,5R)-3,5-Dihydroxy-
cyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-
yl)hexyl)methanesulfonamide (27b). This was prepared from
amine 16 by following the same procedure described for 27a.
The reagents used were as follows: amine 16 (5.5 mg, 0.009 mmol,
2 equiv) and Et3N (10 lL, 0.069 mmol, 3 equiv). Compound 27e
1 equiv), methylsulfonyl chloride (1
l
L, 0.01 mmol, 1.1 equiv) and
was purified by silica gel column chromatography (5% MeOH in
EtOAc) to give 4 mg (0.008 mmol) the sulfonamide 27e in 35% yield.
Rf = 0.2 (5% MeOH in EtOAc); 1H NMR (400 MHz, CDCl3) d 6.31 (1H,
d, J = 11.8 Hz), 5.85 (1H, d, J = 11.8 Hz), 4.19–4.09 (1H, m), 4.09–4.00
(1H, m), 4.07 (2H, t, J = 6.8 Hz), 3.17 (2H, t, J = 6.8 Hz), 3.03 (2H, t,
J = 6.8 Hz), 2.79 (1H, d, J = 13.6 Hz), 2.75 (1H, d, J = 12.8 Hz), 2.48
(1H, d, J = 12.0 Hz), 2.17–2.25 (2H, m), 2.03–1.15 (19H, m), 1.14–
1.01 (1H, m), 0.92 (3H, d, J = 6.4 Hz), 0.54 (3H, s); 13C NMR
(75 MHz, CDCl3) d 143.3, 131.3, 124.1, 115.5, 67.7, 67.5, 57.4, 56.7,
56.5, 53.6, 47.3, 46.0, 44.9, 42.4, 40.7, 37.4, 36.2, 36.0, 29.1, 28.3,
Et3N (3 L, 0.003 mmol, 3 equiv). Compound 27b was purified by
l
silica gel column chromatography (5% MeOH in EtOAc) to give
2 mg (0.004 mmol) the sulfonamide 27b in 48% yield. Rf = 0.7 (5%
MeOH in EtOAc); 1H NMR (400 MHz, CDCl3) d 6.31 (1H, d,
J = 11.2 Hz), 5.85 (1H, d, J = 11.2 Hz), 4.20–4.10 (1H, m), 4.09–4.00
(1H, m), 3.34 (2H, q, J = 6.4 Hz), 2.96 (3H, s), 2.80 (1H, d,
J = 12.0 Hz), 2.74 (1H, d, J = 13.6 Hz), 2.60 (1H, d, J = 13.6 Hz),
2.25–2.15 (2H, m), 2.08–0.85 (20H, m), 0.92 (3H, d, J = 6.4 Hz),
0.54 (3H, s) 13C NMR (75 MHz, CDCl3) d 131.3, 124.1, 115.5,
100.0, 67.6, 67.5, 56.6, 56.5, 46.0, 44.9, 43.6, 42.4, 40.6, 37.4, 36.2,
27.9, 23.9, 23.7, 22.5, 19.0, 12.3; (KBr)
m 3354 (br), 2934, 2867,
35.6, 30.9, 29.9, 29.1, 27.9, 23.7, 23.4, 22.5, 19.0, 12.3; IR (KBr)
m
1724, 1316, 1201, 1170, 1143, 1046, 732 cmꢀ1; HRMS (ESI): m/z
3310 (br), 2939, 2857, 1750, 1347, 1169, 1030, 698 cmꢀ1; HRMS
calcd for [(M+Na+] = 506.2916, found = 506.2919.
(ESI): m/z calcd for [(M+Na)+] = 476.2816, found = 476.2804.
4.1.1.34. N-((R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxy-
cyclohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-
yl)hexyl)butane-1-sulfonamide (27f). This was prepared from
amine 16 by following the same procedure described for 27a.
The reagents used were as follows: amine 16 (22.1 mg,
4.1.1.31. N-((R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxy-
cyclohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-
yl)hexyl)trifluoromethanesulfonamide (27c). This was pre-
pared from amine 16 by following the same procedure described
for 27a. The reagents used were as follows: amine 16 (20.9 mg,
0.036 mmol, 1 equiv), butylsulfonyl chloride (5.2
lL, 0.040 mmol,
0.035 mmol, 1 equiv), trifluoromethanesulfonyl chloride (4
lL,
1.1 equiv) and Et3N (15 L, 0.108 mmol, 3 equiv). Compound 27f
l
0.038 mmol, 1.1 equiv) and Et3N (14 L, 0.105 mmol, 3 equiv).
l
was purified by silica gel column chromatography (EtOAc) to
Compound 27c was purified by silica gel column chromatography
(5% MeOH in EtOAc) to give 6.2 mg (0.012 mmol) the sulfonamide
27c in 35% yield. Rf = 0.4 (EtOAc); 1H NMR (400 MHz, CDCl3) d
6.31 (1H, d, J = 11.4 Hz), 5.85 (1H, d, J = 11.4 Hz), 4.17–4.08 (1H,
m), 4.08–4.00 (1H, m), 3.30 (2H, q, J = 7.2 Hz), 2.79 (1H, dd,
give 10.1 mg (0.020 mmol) the sulfonamide 27f in 57% yield.
Rf = 0.3 (EtOAc); 1H NMR (400 MHz, CDCl3)
d 6.30 (1H, d,
J = 11.4 Hz), 5.84 (1H, d, J = 11.4 Hz), 4.15–4.08 (1H, m), 4.07–
3.98 (1H, m), 3.10 (2H, q, J = 6.8 Hz), 3.05–2.97 (2H, m), 2.79
(1H, dd, J = 12.0, 3.6 Hz), 2.74 (1H, dd, J = 13.6, 3.6 Hz), 2.47

4134
M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
(1H, dd, J = 13.6, 3.2 Hz), 2.27–2.16 (2H, m), 2.04–1.16 (23H, m),
1.13–1.01 (1H, m), 0.95 (3H, t, J = 7.6 Hz), 0.91 (3H, d, J = 6.8 Hz),
0.53 (3H, s); 13C NMR (75 MHz, CDCl3) d 143.2, 131.4, 124.0,
115.5, 67.6, 67.4, 56.6, 56.5, 52.6, 46.0, 44.9, 43.6, 42.4, 40.7,
37.4, 36.2, 35.6, 31.1, 29.1, 27.9, 25.9, 23.7, 23.4, 22.5, 21.8,
5.85 (1H, d, J = 11.2 Hz), 4.18–4.08 (1H, m), 4.07–4.00 (1H, m),
3.13 (2H, q, J = 6.8 Hz), 2.96 (3H, s), 2.80 (1H, d, J = 11.6 Hz),
2.74 (1H, d, J = 12.8 Hz), 2.62 (2H, br s), 2.48 (1H, d, J = 12.8 Hz),
2.26–2.16 (3H, m), 2.10–1.00 (19H, m), 0.91 (3H, d, J = 6.0 Hz),
0.54 (3H, s); 13C NMR (75 MHz, CDCl3) d 143.4, 131.3, 124.1,
115.5, 67.6, 67.5, 56.7, 56.5, 46.0, 44.9, 43.6, 42.4, 40.6, 37.4,
36.2, 35.9, 30.5, 29.2, 27.9, 27.3, 25.9, 23.7, 22.5, 19.0, 12.3; IR
19.0, 13.8, 12.3; (KBr)
m
3290 (br), 2937, 2871, 1449, 1320,
1141, 1047, 734 cmꢀ1
;
HRMS (ESI): m/z calcd for
[(M+Na)+] = 518.3280, found = 518.3267.
(KBr) m 3294 (br), 2933, 2869, 1453, 1318, 1149, 1048, 975,
732, 523 cmꢀ1; HRMS (ESI): m/z calcd for [(M+Na)+] = 490.2967,
found = 490.2967.
4.1.1.35. N-((R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxy-
cyclohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-
yl)hexyl)benzenesulfonamide (27g). This was prepared from
amine 16 by following the same procedure described for 27a. The re-
agents used were as follows: amine 16 (16.2 mg, 0.027 mmol,
4.1.1.38. N-((R)-5-((1R,3aS,7aR,E1R,3aS,7aR,E)–4-(2-((3R,5R)-3,5-
Dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-
1H -inden-1-yl)hexyl)methanesulfamide (28a). N-(tert-Butoxy-
carbonyl)-N-[4-(dimethylazaniumylidene)-1,4-dihydropyridin-1-
ylsulfonyl]azanide33 26 (8.6 mg, 0.028 mmol, 1.1 equiv) was added
to a stirred solution of amine 14 (15 mg, 0.026 mmol, 1 equiv) in
1 equiv), benzenesulfonyl chloride (6.8
lL, 0.052 mmol, 2 equiv)
and Et3N (11 L, 0.078 mmol, 3 equiv). Compound 27g was purified
l
by silica gel column chromatography (EtOAc) to give 5.1 mg
(0.010 mmol) the sulfonamide 27g in 37% yield. Rf = 0.4 (EtOAc);
1H NMR (400 MHz, CDCl3) d 7.87 (2H, d, J = 7.2 Hz), 7.58 (1H, t,
J = 7.2 Hz), 7.52 (2H, d, J = 7.2 Hz), 6.31 (1H, d, J = 11.6 Hz), 5.84
(1H, d, J = 11.6 Hz), 4.35 (1H, t, J = 6.0 Hz), 4.15–4.08 (1H, m), 4.08–
3.99 (1H, m), 2.95 (2H, q, J = 6.4 Hz), 2.79 (1H, dd, J = 12.4, 4.0 Hz),
2.74 (1H, dd, J = 13.2, 3.6 Hz), 2.48 (1H, dd, J = 13.6, 2.8 Hz), 2.28–
2.16 (2H, m), 2.04–1.91 (3H, m), 1.90–1.76 (2H, m), 1.65–1.59 (2H,
m), 1.58–1.06 (11H, m), 1.05–0.92 (1H, m), 0.86 (3H, d, J = 6.0 Hz),
0.51 (3H, s); 13C NMR (75 MHz, CDCl3) d 143.2, 140.2, 132.8, 131.4,
127.3, 124.1, 115.5, 67.6, 67.5, 56.5, 46.0, 44.9, 43.5, 42.4, 40.6,
CH2Cl2 (100 lL). The mixture was stirred at room temperature
for 16 h, then concentrated and directly loaded on silica gel (20%
EtOAc in hexanes) to give the protected tert-butylsulfamoylcarba-
mate. A 48% solution of HF (one drop) was added to a stirred solu-
tion of the product in CH2Cl2 (100 lL) and CH3CN (100 lL) and the
solution was stirred at room temperature overnight. The mixture
was quenched cautionly by the addition of satd NaHCO3 until no
effervescence was observed then acidified with a 1 M aq solution
of citric acid (5 mL). The solution was extracted with CH2Cl2
(3 ꢂ 5 mL) then the combined organic layers were washed with
H2O (5 mL), brine (5 mL), dried (MgSO4) and then concentrated
in vacuo. The oil was purified by silica gel column chromatography
(EtOAc) to afford 3.2 mg (0.007 mmol) of the sulfamide 28a in 27%
yield. 1H NMR (400 MHz, CDCl3) d 6.31 (1H, d, J = 11.4 Hz), 5.86
(1H, d, J = 11.4 Hz), 4.46 (2H, s), 4.24–4.16 (1H, m), 4.10–4.02
(1H, m), 3.20–3.02 (2H, m), 2.80 (1H, dd, J = 12.0, 4.0 Hz), 2.74
(1H, dd, J = 13.6, 4.0 Hz), 2.49 (1H, dd, J = 13.2, 3.2 Hz), 2.28–2.17
(2H, m), 2.04–1.70 (5H, m), 1.74–1.24 (12H, m), 1.18–1.05 (1H,
m), 0.94 (3H, d, J = 6.4 Hz), 0.55 (3H, s); 13C NMR (75 MHz, CDCl3)
d 143.1, 131.5, 124.1, 115.6, 67.7, 67.5, 56.51, 56.48, 46.0, 44.9,
44.4, 42.4, 40.7, 37.4, 36.0, 33.0, 29.1, 27.9, 26.4, 23.7, 22.5, 19.0,
37.4, 36.1, 35.5, 30.3, 29.1, 27.9, 23.2, 22.5, 18.9, 12.3; (KBr)
m 3290
(br), 2940, 2870, 1446, 1323, 1159, 1046, 905, 732, 689, 586 cmꢀ1
;
HRMS (ESI): m/z calcd for [(M+Na)+] = 538.2967, found = 538.2969.
4.1.1.36. 4-Cyano-N-((R)-5-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-
dihydroxycyclohexylidene)ethylidene)-7a-methyl-octahydro-1H-
inden-1-yl)hexyl)benzenesulfonamide (27h). This was prepared
from amine 16 by following the same procedure described for 27a.
The reagents used were as follows: amine 16 (14 mg, 0.024 mmol,
1 equiv), 4-cyanobenzenesulfonyl chloride (9.6 mg, 0.048 mmol,
12.3; IR (KBr)
m 3290 (br), 2940, 2872, 1706, 1434, 1329, 1156,
2 equiv) and Et3N (10 lL, 0.072 mmol, 3 equiv). Compound 27h
1045, 735, 668 cmꢀ1; HRMS (ESI): m/z calcd for [(M+Na)+] =
was purified by silica gel column chromatography (EtOAc) to give
8.0 mg (0.014 mmol) the sulfonamide 27h in 61% yield. Rf = 0.5
(EtOAc); 1H NMR (400 MHz, CDCl3) d 7.98 (2H, d, J = 8.0 Hz), 7.83
(2H, d, J = 8.0 Hz), 6.30 (1H, d, J = 11.6 Hz), 5.85 (1H, d, J = 11.6 Hz),
4.56 (1H, t, J = 6.0 Hz), 4.17–4.10 (1H, m), 4.10–4.01 (1H, m), 2.99
(2H, q, J = 6.7 Hz), 2.79 (1H, dd, J = 12.4, 4.0 Hz), 2.73 (1H, dd,
J = 13.2, 4.0 Hz), 2.48 (1H, dd, J = 13.2, 3.2 Hz), 2.17–2.29 (2H, m),
2.03–1.90 (3H, m), 1.76–1.88 (2H, m), 1.72–1.61 (2H, m), 1.61–
1.08 (11H, m), 1.06–0.92 (1H, m), 0.87 (3H, d, J = 6.0 Hz), 0.51 (3H,
s); 13C NMR (75 MHz, CDCl3) d 144.7, 143.1, 133.2, 131.5, 127.9,
124.0, 117.5, 116.6, 115.6, 67.6, 67.5, 56.53, 56.48, 45.9, 44.9, 43.6,
42.4, 40.6, 37.4, 36.1, 35.4, 30.3, 29.1, 27.9, 23.7, 23.2, 22.5, 18.9,
463.2596, found = 463.2606.
4.1.1.39. N-((R)-5-((1R,3aS,7aR,E)–4-(2-((3R,5R)-3,5-Dihydroxy-
cyclohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-
yl)hexyl)methanesulfamide (28b). This was prepared from
amine 16 by following the same procedure described for 28a.
The reagents used were as follows: 16 (7 mg, 0.011 mmol, 1 equiv)
and 26 (3.8 mg, 0.012 mmol, 1.1 equiv). Compound 28b was ob-
tained in 79% yield (3.6 mg, 0.008 mmol). Rf = 0.1 (EtOAc); 1H
NMR (400 MHz, CD3OD) d 6.22 (1H, d, J = 11.2 Hz), 5.88 (1H, d,
J = 11.2 Hz), 4.10–3.94 (2H, m), 3.01 (2H, t, J = 6.6 Hz), 2.84 (1H,
d; J = 11.6 Hz), 2.59 (1H, d, J = 10.8 Hz), 2.41 (1H, d, J = 12.0 Hz),
2.28–2.12 (3H, m), 2.08–1.99 (2H, m), 1.98–1.90 (1H, m), 1.89–
1.80 (1H, m), 1.80–1.72 (1H, m), 1.71–1.20 (12H, m), 1.15–1.05
(1H, m), 0.96 (3H, d, J = 6.4 Hz), 0.94–0.85 (1H, m), 0.57 (53H, s);
13C NMR (75 MHz, CD3OD) d 140.9, 132.7, 122.3, 116.0, 66.8,
66.5, 56.7, 56.3, 53.6, 45.6, 44.2, 43.1, 41.5, 40.7, 36.4, 36.2, 35.6,
12.3; (KBr)
m 3293 (br), 2939, 2869, 1434, 1332, 1160, 1091, 1045,
837, 735, 634, 574, 517 cmꢀ1
; HRMS (ESI): m/z calcd for
[(M+Na)+] = 563.2919, found = 563.2918.
4.1.1.37. N-((R)-6-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxy-
cyclohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-
yl)heptyl)methanesulfonamide (27i). This was prepared from
amine 19 by following the same procedure described for 27a.
The reagents used were as follows: amine 19 (10 mg, 0.016 mmol,
29.9, 28.6, 27.6, 23.3, 22.1, 18.1, 11.2; IR (KBr)
m 3302 (br), 2935,
2860, 1592, 1328, 1156, 1045, 669, 547 cmꢀ1; HRMS (ESI): m/z
calcd for [(M+Na)+] = 477.2757, found = 477.2760. 2606.
1 equiv), methylsulfonyl chloride (1.4
lL, 0.018 mmol, 1.1 equiv)
4.1.1.40. N-((R)-5-((1R,3aS,7aR,E)–4-(2-((3R,5R)-3,5-Dihydroxy-
cycloheptylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-
yl)hexyl)methanesulfamide (28c). This was prepared from
amine 19 by following the same procedure described for 28a.
and Et3N (7 L, 0.054 mmol, 3 equiv). 27i was purified by silica
l
gel column chromatography (5% MeOH in EtOAc) to give 5.5 mg
(0.012 mmol) the sulfonamide 27i in 73% yield. Rf = 0.7 (5% MeOH
in EtOAc); 1H NMR (400 MHz, CDCl3) d 6.31 (1H, d, J = 11.2 Hz),

M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
4135
The reagents used were as follows: 19 (10.3 mg, 0.016 mmol,
3380 (br), 2941, 2869, 1730, 1437, 1262, 1048, 733, 668 cmꢀ1
HRMS (ESI): m/z calcd for [(M+Na)+] = 469.2930, found = 469.2920.
;
1 equiv) and 26 (5.5 mg, 0.018 mmol, 1.1 equiv). Compound 28c
was obtained in 69% yield (5.2 mg, 0.011 mmol). 1H NMR
(400 MHz, CD3OD)
d
6.22 (1H, d, J = 11.2 Hz), 5.88 (1H, d,
4.1.1.43.
(R)-7-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-Dihydroxycy-
J = 11.2 Hz), 4.07–4.01 (1H, m), 4.00–3.95 (1H, m), 3.00 (2H, t,
J = 7.2 Hz), 2.83 (2H, d, J = 15.6 Hz), 2.59 (1H, dd, J = 13.2, 4.0 Hz),
2.44–2.35 (2H, m), 2.30–2.11 (3H, m), 2.05–1.00 (19H, m), 0.95
(3H, d, J = 6.4 Hz), 0.57 (3H, s); 13C NMR (75 MHz, CD3OD) d
140.2, 134.4, 121.9, 116.5, 67.6, 66.4, 56.7, 56.3, 45.2, 43.4, 40.7,
37.4, 36.2, 36.0, 28.5, 27.7, 27.4, 25.8, 23.5, 22.3, 18.6, 11.7; IR
clohexylidene)ethylidene)-7a-methyl-octahydro-1H-inden-1-yl)-
N-methyl-2-oxooctanamide (30b). To a solution of enol ether 29
(29 mg, 0.037 mmol, 1 equiv) and AcOH (11 lL, 0.185 mmol,
1 equiv) in 0.5 mL of MeCN and 0.5 mL of CH2Cl2 at 0 °C was added
CsF (14 mg, 0.092 mmol, 2.5 equiv) in one portion. The mixture
was allowed to warm to room temperature and then stirred for
an additional 75 min. The reaction mixture then was diluted with
10 mL of EtOAc and 5 mL of satd NaHCO3. The layers were sepa-
rated and the organic layer was washed with satd NaHCO3
(5 mL) and brine (5 mL), then dried (Na2SO4) and concentrated.
Purification of the residue on silica gel (5% EtOAc in hexanes,
(KBr)
667 cmꢀ1
found = 491.2914.
m
3298 (br), 2945, 2872, 1699, 1440, 1333, 1145, 1044, 732,
;
HRMS (ESI): m/z calcd for [(M+Na)+] = 491.2919,
4.1.1.41.
(R,Z)-Methyl-7-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-bis-
Rf = 0.4) gave the protected-a-ketoester. MeNH3Cl (3.4 mg,
(tert-butyldimethylsilyloxy)cyclohexylidene)ethylidene)-7a-meth-
yl-octahydro-1H -inden-1-yl)-2-(tert-butyldimethylsilyloxy)oct-
2-enoate (29). In a flame-dried round bottom flask under argon
0.051 mmol, 1.5 equiv) was added to a stirred solution of the prod-
uct in NEt3 (1 mL) at 0 °C. The solution was warmed to room tem-
perature and stirred for 2 days. The solution was concentrated and
the residue dissolved in EtOAc (10 mL). The organic layer was
washed with satd NaHCO3 (2 ꢂ 5 mL), H2O (5 mL), brine (5 mL)
and dried (Na2SO4). Purification by silica gel column chromatogra-
phy (5–10% EtOAc in hexanes) afforded the ketoamide [Rf = 0.2
(10% EtOAc in hexanes)] which was directly submitted to the alco-
hols deprotection using the same procedure as for 30a. Purification
by silica gel column chromatography (50% EtOAc in hexanes) gave
30b in 49% yield (8.0 mg, 0.018 mmol). Rf = 0.4 (50% EtOAc in hex-
anes); 1H NMR (400 MHz, CDCl3) d 6.95 (1H, br s), 6.31 (1H, d,
J = 10.8 Hz), 5.85 (1H, d, J = 10.8 Hz), 4.19–4.00 (2H, m), 2.97–2.85
(5H, m), 2.77 (2H, t, J = 14.1 Hz), 2.48 (1H, d, J = 12.0 Hz), 2.28–
2.16 (2H, m), 2.07–1.15 (19H, m), 1.14–1.00 (1H, m), 0.91 (3H, d,
J = 6.0 Hz), 0.53 (3H, s); 13C NMR (75 MHz, CDCl3) d 199.5, 161.1,
143.4, 131.3, 124.1, 115.5, 67.7, 67.5, 56.7, 56.5, 46.0, 44.9, 42.4,
40.7, 37.4, 37.0, 36.2, 35.8, 29.2, 27.9, 26.1, 25.8, 23.9, 23.7, 22.5,
atmosphere at ꢀ78 °C, NaHMDS (94
lL of a 1 M solution in THF,
0.095 mmol, 1.3 equiv) was added to a solution of methyl-2-
(dimethoxyphosphoryl)-2-methoxyacetate (34 mg, 0.110 mmol,
1.5 equiv) in dry THF (1 mL). The solution was stirred for
15 min at ꢀ78 °C, then
a solution of aldehyde 15 (44 mg,
0.073 mmol, 1 equiv) in dry THF (0.5 mL) was slowly added by
cannula. The solution was then slowly warmed to room temper-
ature and stirred overnight. The solution was quenched by the
addition of aq satd NH4Cl (2 mL) and extracted with Et2O
(3 ꢂ 5 mL), the combined organic layers were washed with water
(5 mL) and brine (5 mL) then dried (5 mL). The solution was con-
centrated in vacuo then purified by silica gel column chromatog-
raphy (5% EtOAc in hexanes) to obtain 29 in 71% yield (41 mg,
0.052 mmol). Rf = 0.7 (10% EtOAc in hexanes); 1H NMR
(400 MHz, CDCl3)
d 6.17 (1H, d, J = 10.0 Hz), 5.81 (1H, d,
J = 10.0 Hz), 5.51 (1H, t, J = 8.0 Hz), 4.13–4.02 (2H, m), 3.75 (3H,
s), 2.81 (1H, d, J = 12.0), 2.49–2.33 (4H, m), 2.25 (1H, d,
J = 14.0 Hz), 2.10 (1H, dd, J = 12.2, 8.2 Hz), 2.04–1.94 (2H, m),
1.93–1.83 (1H, m), 1.83–1.73 (1H, m), 1.72–1.22 (14H, m),
1.14–1.01 (1H, m), 1.00–0.91 (12H, m), 0.87 (9H, m), 0.86 (9H,
m), 0.53 (3H, s), 0.12 (6H, s), 0.05 (12H, s); 13C NMR (75 MHz,
CDCl3) d 165.5, 141.0, 140.1, 133.9, 126.6, 122.0, 116.3, 68.3,
68.2, 56.7, 56.5, 51.6, 46.2, 45.8, 43.9, 40.8, 37.0, 36.2, 35.7,
29.9, 28.9, 27.9, 27.4, 26.7, 26.10, 26.07, 25.8, 23.6, 22.4, 19.0,
19.0, 12.3; IR (KBr)
m 3376 (br), 2928, 1717, 1673, 1538, 1453,
1047, 668 cmꢀ1; HRMS (ESI): m/z calcd for [(M+Na)+] = 468.3096,
found = 468.3082.
4.2. Biochemical analysis of hybrid molecules
4.2.1. Cell culture
Cells were purchased from American Type Culture Collection
(Manassas, VA) and cultured under recommended conditions. Cells
were split at 60–70% confluence, as follows: cells were washed
with filtered PBS, split with a 5–10 min incubation with 1 mL Tryp-
sin–EDTA (Invitrogen, Carlsbad, CA) in PBS and collected with med-
ium. Following centrifugation, the supernatant was removed. Cell
pellets were resuspended with DMEM-F12, and distributed on
fresh media-containing dishes. For cell treatments, AT84 cells were
split and after 24 h the medium was changed to DMEM + 10% char-
coal-stripped FBS. After an additional 24 h, the media was changed
and cells were incubated in DMEM-F12 + 10% charcoal-stripped
FBS and treated with 1,25D (Sigma) or hybrid compounds, as indi-
cated in the figures.
18.44, 18.38, 18.34, 12.3, ꢀ4.4, ꢀ4.5, ꢀ4.6, ꢀ4.7; IR (KBr)
m
;
2951, 2857, 1727, 1636, 1468, 1360, 1252, 1087, 837, 777 cmꢀ1
HRMS (ESI): m/z calcd for [(M+Na)+] = 811.5524, found =
811.5509.
4.1.1.42. (R)-Methyl-7-((1R,3aS,7aR,E)-4-(2-((3R,5R)-3,5-dihydr-
oxycyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-
1-yl)-2-oxooctanoate (30a). A 48% solution of HF (one drop) was
added to a solution of 29 (7 mg, 0.009 mmol) in CH2Cl2 (0.3 mL)
and CH3CN (0.3 mL) and the solution was stirred at room temper-
ature for 2 h. The mixture was concentrated in vacuo and the res-
idue was loaded directly onto silica gel. Compound 30a was
isolated via FCC (50% EtOAc in hexanes) as a white solid in 85%
yield (3.9 mg, 0.007 mmol). 1H NMR (400 MHz, CDCl3) d 6.31 (1H,
d, J = 11.4 Hz), 5.85 (1H, d, J = 11.4 Hz), 4.18–4.08 (1H, m), 4.08–
4.01 (1H, m), 3.87 (3H, s), 2.84 (2H, t, J = 7.2 Hz), 2.79 (1H, dd,
J = 12.0, 3.6 Hz), 2.74 (1H, dd, J = 13.2, 3.6 Hz), 2.48 (1H, d,
J = 13.6 Hz), 2.28–2.16 (2H, m), 2.06–1.74 (5H, m), 1.72–1.16
(14H, m), 1.14–1.02 (1H, m), 0.91 (3H, m, J = 6.0 Hz), 0.54 (3H, s);
13C NMR (75 MHz, CDCl3) d 194.6, 161.8, 143.3, 131.3, 124.1,
115.5, 67.6, 67.5, 56.7, 53.1, 46.0, 44.9, 42.4, 40.7, 39.6, 37.4,
4.2.2. RT-PCR analysis
PCR primers were designed using Primer3 software found at
http://frodo.wi.mit.edu/ and have already been described.10 After
treatment, cells were washed with PBS, homogenized with 1 mL
TRIzol Reagent (Invitrogen, Carlsbad, CA), kept at room tempera-
ture for 2–5 min, collected and then kept at ꢀ80 °C for at least
1 h. After thawing, 0.2 mL of chloroform was added. After vigorous
mixing and storing for 10 min, mixtures were centrifuged
(10,000 rpm, 15 min, 4 °C) and the upper transparent layer was
transferred to a new tube. Isopropanol (0.5 mL) was added and
36.1, 35.7, 29.1, 27.9, 25.7, 23.7, 23.6, 22.5, 19.0, 12.3; IR (KBr)
m

4136
M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
the new solution was mixed. After centrifuging at 4 °C, the super-
natant was discarded and 1 mL of 75% ethanol was added. After
centrifuging and discarding the supernatant, the pellet was air-
dried for 5–10 min, and then dissolved in ddH2O. RNA concentra-
and diluted with HDAC buffer (15 mM Tris–HCl [pH 8.1], 250 lM
EDTA, 250 mM NaCl, 10% glycerol) to give 1 mM solutions contain-
ing 1.7% DMSO. HDAC enzyme solutions were prepared by diluting
HDAC2, -3 and -6 (Cayman Chemical) to concentrations of 0.04 ng/
tions were measured using a spectrophotometer, and 1–3
RNA were loaded for reverse transcriptase reactions (total volume:
20 L). ddH2O (80 L) was added to the RT-product, and 1.5 L of
the resulting solution was used as template for the PCR reaction.
Reverse transcriptase (Super Script II) was purchased from Invitro-
gen (Carlsbad, CA). DNA polymerase and dNTP’s were ordered from
Fermentas (Glen Burnie, MD).
l
g of
lL, 0.02 ng/lL and 0.05 ng/lL in HDAC buffer (above). A trypsin
solution 10 mg/mL in HDAC buffer was used for development. Re-
lease of AMC was monitored by measuring the fluorescence at
460 nm (lex = 390 nm) with a microplate reader (SpectraMax Gem-
ini) at 37 °C. The AMC signals were recorded against a blank with
buffer, substrate and trypsin but without the enzyme. All experi-
ments were carried out at least in triplicate.
l
l
l
For HDAC assays, inhibitor (or none for control) diluted in 50
of HDAC buffer was mixed with 10 L of diluted HDAC2, -3 or -6
solution in HDAC buffer at room temperature. The assay was begun
by adding 40 L of substrate solution in HDAC buffer followed by
incubation with stirring at 37 °C. After 30 min, 100 L of trypsin
solution was added. After a further 10 min incubation period with
stirring at 37 °C, the release of AMC was monitored by measuring
the fluorescence.
lL
4.2.3. Fluorescence polarization (FP) competition assay
The assay was performed using a vitamin D receptor competitor
assay kit (Polarscreen, Invitrogen, Carlsbad, CA) set up using
0.5 nM fluorescent tracer. The assay measures the decrease in FP
accompanying loss of binding to the relatively high molecular
weight VDR ligand binding domain of the fluorescent tracer due
to the presence of a competitor. FP was measured using an Analyst
HT fluorimeter (Molecular Devices) configured with absorption
and emissions filters as recommended by the kit manufacturers.
Dose–response curves and IC50 determination were determined
using XLfit (IDBS) Sigmoidal Dose–Response Model [fit = (A +
((B ꢀ A)/(1 + ((C/x)^D)))); inv = (C/((((B ꢀ A)/(y ꢀ A)) ꢀ 1)^(1/D)));
res = (y-fit)]. Note that use of concentrations of some analogs at
1 M or greater was precluded in the assay likely due to their insol-
ubility under assay conditions.
l
l
l
4.2.6. EdU Cell growth assay
Click-iT EdU Alexa Fluor high-throughput imaging (HCS) assay
was used for assessing cell proliferation. HCS assays were
performed on AT84 mouse squamous carcinoma cell line (ATCC)
following manufacturer’s instruction (Molecular Probes, Invitro-
gen). Images were analyzed for Hoechst 33342 (350/460 nm)
and Alexa Fluro 647 (620/700 nm) using Image Xpress Micro
(Molecular Devices, CA, USA). All samples were in triplicate.
4.2.4. Western blotting
The following primary (1st) antibodies (Ab) were used:
a-
4.2.7. Hypercalcemia assay
tubulin (Santa Cruz, sc-8035, Santa Cruz, CA), acetylated -tubu-
a
Eight-week old virgin female FVB mice (Charles River, Quebec,
Canada) were housed in a limited access room. Animal husbandry
adhered to Canadian Council on Animal Care Standards, and all
protocols were approved by the McGill University Health Center
Animal Care Utilization Committee. Mice were implanted with Al-
zet osmotic minipumps (Alzet, Cupertino, CA) containing 1,25D or
hybrids. In previous studies we determined that constant infusion
of 1,25D above 18 pmol/24 h results in hypercalcemia in the
majority of animals.34 We used a similar protocol here and treated
mice with a constant infusion of 12, 24 and 48 pmol/24 h of 1,25D
(Leo Pharmaceuticals, Ballerup, Denmark), as a positive control,
and increasing concentrations of the analogs (240 and
1200 pmol/24 h).35 Animals were treated for a total of two weeks.
Blood calcium and albumin concentrations (to correct calcium)
were monitored pre-treatment and at timed intervals (using
lin (Santa Cruz, sc-23950, Santa Cruz, CA), Ac-histone H4 (Santa
Cruz, sc-8660-R), b-actin (Santa Cruz, sc-47778, Santa Cruz, CA).
After treatments, cells were washed with PBS, scraped with
1 mL PBS, and pelleted by centrifugation (10,000 rpm, 10 min,
4 °C), and frozen at ꢀ80 °C after removing the supernatant. At
least 1 h later, cells were suspended for 20 min in lysis buffer
(10 mM Tris–Cl pH 8, 60 mM KCl, 1 mM EDTA, 1 mM DTT, and
0.5% NP-40 all in H2O). Cells were then centrifuged at 4 °C, and
supernatant was transferred to a new tube. Protein concentration
was measured using Bio-Rad protein assay, and samples were
mixed with equal volumes of 2ꢂ loading dye [100 mM Tris–Cl
pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 4% 2-
mercaptoethanol, and 200 mM DTT (latter two added before
use) in water]. Samples were loaded in 8–15% acrylamide/bis-
acrylamide gels immersed in running buffer (3 g Tris–Cl, 14.4 g
glycine, 1 g SDS in 1 L H2O), and electrophoresed (100 V) until
the bottom of samples had run all the way. Protein was trans-
ferred to methanol activated PVDF membranes (Bio-Rad, Missis-
sauga, ON) using a power source (100 V, 1 h) and transfer buffer
(3.03 g Tris–Cl, 14.4 g glycine, 0.037 g SDS in 1 L H2O. Membranes
where then blocked for 1 h with 7% skim milk in TTBS (TBS:
6.05 g Tris–Cl, 8.76 g NaCl, in 1 L H2O, TTBS = TBS + 0.1% Tween),
and incubated overnight with the 1st Ab solution (1:6000–
1:1000 diluted Ab in TBS). Membranes were washed three times
with TTBS, once with TBS, and incubated for 1 h with secondary
Ab solution (1:3000 diluted Ab in TBS). Membranes were washed
again and developed using Western Blot Luminol Reagent (Santa
Cruz Biotechnology, Santa Cruz, CA) and Hyperfilm (Amersham
Biosciences, Piscataway, NJ).
25 lL of plasma) by microchemistry (Kodak Ektachrome, Mississa-
uga, ON).
Acknowledgments
J.H.W. and J.L.G. thank the Canadian Institute of Health Research
(CIHR) for funding of this research through Grants MOP 43949 and
a Proof of Principle grant. J.H.W. thanks Fonds de la Recherche en
Santé Quebec (FRSQ) for a Bourse Nationale. R.K. thanks the CIHR
for support through Grant MOP 10839 and FRSQ for a Chercheur
Nationale. B.S.A. thanks FRSQ for a postdoctoral fellowship. R.M.S.
thanks Consejo Nacional de Ciencia y Tecnologia (Mexico) and
CIHR for postgraduate fellowships.
4.2.5. Fluorogenic HDAC assay
Supplementary data
Boc-Lys(Ac)-7-amino-4-methylcoumarin
(Boc-Lys(Ac)-AMC)
was used as substrate for the HDAC assays. Substrate solution
was prepared as follows: Boc-Lys(Ac)-AMC was dissolved in DMSO
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.03.078.

M. Lamblin et al. / Bioorg. Med. Chem. 18 (2010) 4119–4137
4137
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