Synthesis and initial biological evaluation of new mimics of the LXR-modulator 22(S)-hydroxycholesterol

Article abstract of DOI:10.1016/j.bmc.2013.10.024

The generic, synthetic oxysterol 22(S)-hydroxycholesterol (22SHC) has shown antagonistic effects towards liver X receptor (LXR) in vitro and promising effects on plasma triacylglycerol level and body weight-gain in animal studies.^1,2 On the contrary, the endogenic LXR agonist 22(R)-hydroxycholesterol (22RHC) and synthetic LXR agonists convincingly have shown agonistic effects on genes involved in lipogenesis, and inhibitory effects on cell proliferation in vitro and in vivo.³ We hypothesized that the carbon side chain containing the hydroxyl group at the 22-position was a pharmacophore affecting these opposite effects on LXR. This prompted us to initiate a rational drug design incorporating the 22-hydroxylated 20-27 cholesterol moiety into cholesterol-mimicking building blocks. The two enantiomers of the 22-hydroxylated 20-27 cholesterol moiety were synthesized with an excellent enantiomeric excess and the stereochemistry are supported by X-ray crystallography. Molecular modelling of the new compounds showed promising LXR selectivity (LXRβ over LXRα) and initial in vitro biological evaluation in human myotubes showed that compound 16b had agonistic effects on the gene expression of SCD1 and increased lipogenesis.

Full text of DOI:10.1016/j.bmc.2013.10.024

Bioorganic & Medicinal Chemistry 22 (2014) 643–650
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and initial biological evaluation of new mimics of the
LXR-modulator 22(S)-hydroxycholesterol
O. Alexander H. Åstrand a, Marcel Sandberg b, Ingebrigt Sylte c, Carl Henrik Görbitz d, G. Hege Thoresen d,e
,
Eili T. Kase d, Pål Rongved a,
⇑
a Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, PO Box 1068, Blindern, N-0315 Oslo, Norway
b Synthetica AS, Oslo Innovation Center, Gaustadalleen 21, 0349 Oslo, Norway
c Medicinal Pharmacology and Toxicology, Department of Medical Biology, Faculty of Health Sciences, The Arctic University of Norway, , Tromsø, Norway
d Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway
e Department of Pharmacology, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo and Oslo University Hospital, Oslo, Norway
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 July 2013
Revised 10 October 2013
Accepted 18 October 2013
Available online 5 November 2013
The generic, synthetic oxysterol 22(S)-hydroxycholesterol (22SHC) has shown antagonistic effects
towards liver X receptor (LXR) in vitro and promising effects on plasma triacylglycerol level and body
weight-gain in animal studies.1,2 On the contrary, the endogenic LXR agonist 22(R)-hydroxycholesterol
(22RHC) and synthetic LXR agonists convincingly have shown agonistic effects on genes involved in lipo-
genesis, and inhibitory effects on cell proliferation in vitro and in vivo.3 We hypothesized that the carbon
side chain containing the hydroxyl group at the 22-position was a pharmacophore affecting these oppo-
site effects on LXR. This prompted us to initiate a rational drug design incorporating the 22-hydroxylated
20–27 cholesterol moiety into cholesterol-mimicking building blocks. The two enantiomers of the
22-hydroxylated 20–27 cholesterol moiety were synthesized with an excellent enantiomeric excess
and the stereochemistry are supported by X-ray crystallography. Molecular modelling of the new
Keywords:
Liver X receptor agonists
Molecular modeling
New chemical entities
SAR
compounds showed promising LXR selectivity (LXRb over LXRa) and initial in vitro biological evaluation
Gene regulation
in human myotubes showed that compound 16b had agonistic effects on the gene expression of SCD1
and increased lipogenesis.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
very flexible ligand-binding pocket (LBP) of the LXRs. Their LBP al-
lows binding of compounds of highly different structures such as
An increasing number of reports document a repertoire of bio-
logical roles of the liver X receptor (LXR) within major disease
areas.1–6 The biological effects of steroidal-as well as synthetic
LXR modulators now establish LXR as a promising target for several
T0901317, GW3965 and oxygenated sterols (Fig. 1).
The endogenic LXR agonist 22(R)-hydroxycholesterol (22RHC)
and synthetic LXR agonists convincingly have shown inhibitory ef-
fects on cell proliferation in vitro and in vivo.15 LXR agonists have
earlier been shown to have anti proliferative effects on LNCaP hu-
man prostate cancer cells and activation of LXR also inhibited the
proliferation of different other prostate and breast cancer cell
lines.16 In the same study, oral administration of the LXR agonist
T0901317 inhibited the growth of LNCaP tumours in athymic nude
mice.16 Oxysterols have also been shown to induce apoptosis in a
wide range of models such as human leukaemia cell lines15 and pan-
creatic beta-cells.17 Likewise, pharmacological activation of LXRs
leads to increase apoptosis of LNCaP xenografted prostate cancer
new therapeutic opportunities. The two isoforms LXR
a and LXRb
are relatively new drug targets within the nuclear receptor super
family and they are important because they are key players in cho-
lesterol and lipid metabolism, and also influence glucose metabo-
lism.7,8 LXR agonists have been developed as potential drugs for
treatment of metabolic and cardiovascular diseases regulate
inflammatory responses and immunity, skin diseases and are effec-
tive for treatment of murine models of atherosclerosis, diabetes,
and Alzheimer’s disease.9–11 The agonists also display anti-inflam-
matory activities12–14 and inhibit cell proliferation in a number of
major cancer forms.9,10 However, lack of selectivity between the
cells.3 In another study, oxygenated steroids and nonsteroidal LXR
agonists at low micro molar concentrations inhibited proliferation
of breast carcinoma cell lines in culture.18 LXR
agonists are de-
a
two isoforms LXR
a
and LXRb hampers most of the agents so far
a
approaching clinical use. Another complexing factor concerns the
scribed to show promising results in hormone-dependent cancers
such as prostate,3,19 ovarian,20 certain types of breast cancer,18,21
benign prostate hyperplasia11 and LXR agonists have been reported
to reduce cell proliferation in colon cancer.22 These results have also
⇑
Corresponding author. Tel.: +47 95700897; fax: +47 22855947.
E-mail address: pal.rongved@farmasi.uio.no (P. Rongved).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.024

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O. Alexander H. Åstrand et al. / Bioorg. Med. Chem. 22 (2014) 643–650
Figure 1. Overview of known modulators of the liver X receptor.
been supported by others15,23,24 and recent reviews broadly docu-
ment the key role of LXR in cancer and other diseases.9–11
Based on the different effect the change in stereochemistry at
the 22-position of 22-hydroxycholesterol afforded, it was assumed
that the stereochemistry is an essential part of the pharmacophore
for LXR regulators. This has also been supported by molecular
modelling.1 In order to mimic the structure and binding of
22SHC, while keeping the pharmacophore intact, a series of poten-
tial modulators were generated (Fig. 2) and subjected to in vitro
evaluation.
It has recently been shown that the synthetic oxysterol 22SHC
behaved like an LXR antagonist in skeletal muscle cells (human
myotubes), SGBS (preadipocytes), HepG2 (human hepatoblastoma
cell line) and CaCo-2 (human epithelial colorectal adenocarcinoma
cells), in contrast to its diastereomer, the agonistic 22RHC
(Fig. 1).1,2 22SHC repressed genes involved in lipogenesis (i.e.
SREBP, FAS, ASCL1, SCD1) and lipid handling that resulted in re-
duced synthesis of complex lipids even when compared to un-
treated myotubes.25 It was shown that exposure to a synthetic
LXR agonist (T0901317, Fig. 1) increased lipogenesis more in myo-
tubes from type 2 diabetic subjects than in myotubes from lean
subjects,1,2 and that these effects could be counteracted by
22SHC. Further, 22SHC reduced de novo lipogenesis below basal,
reduced fatty acid uptake and oxidation at the same time as glu-
cose uptake and oxidation were increased.2 Thus, the two stereo-
isomers 22SHC (synthetic antagonist) and 22RHC (endogenous
agonist) seem to have opposite physiological effects.
2. Results and discussion
2.1. Synthesis
The synthetic pathway to the key building block 5 and all tested
compounds are given in Scheme 1. The moderate yield in the PCC
oxidation of 1 to 2 was related to loss of 2 because of solvent aze-
otropic effects during distillation. The diol 5 was then used in the
synthesis of the screening compounds (7, 9, 13 and 16a–d). Build-
ing blocks comprising substituted aromatic or alicyclic groups
Figure 2. Pharmacophore analysis based on 22-hydroxycholesterol.

O. Alexander H. Åstrand et al. / Bioorg. Med. Chem. 22 (2014) 643–650
645
Scheme 1. Experimental conditions: (a) PCC, CH2Cl2, rt 60%. (b) nBu2BOTf, DIPEA, CH2Cl2, 0 °C. (c) Compound 2, CH2Cl2, À78 °C, (d) MeOH, H2O2, rt, 62%. (e) LiBH4, Et2O, 0 °C,
67%. (f) NaH, THF, 5, 0 °C–rt, 28%. (g) NaH, THF, 5, 0 °C–rt, 8%. (h) PPh3, CBr4, CH2Cl2, 0 °C, 48%. (i) NaH, THF, 5, 0 °C–rt, 18%. (j) AcOH, H2O, 0 °C, 44%. (k) HNR1R2, TEA, CH2Cl2,
0 °C, 20–50%. (l) Compound 5, NaH, THF, DMF, 0 °C–rt, 10–50%.
were identified in molecular modelling as synthesis candidates
with respect to mimicking the cholesterol A and B rings in
22SHC. Direct alkylation of 5 with benzyl bromides NaH in THF/
DMF at 0 °C gave good regioselectivity for primary hydroxyl group
alkylation with minimal alkylation of the secondary hydroxyl
group, in low to moderate yields. This method was sufficient for
the purpose and not optimized further at the present stage.
No metabolism or degradation studies have been conducted for
the new modulators.
with excellent enantiomeric excess shown by chiral HPLC of the
tosylates 17 and R,R-17, see Scheme 2. Correspondingly, the tosyl-
ate R,R-17 was prepared in similar yields from (S)-4-benzyl-3-pro-
pionyloxazolidin-2-one (S-3). The absolute stereochemistry was
supported by single crystal X-ray structure analysis of 4 and 5
(Fig. 3).26
2.2. Molecular modelling
Using the auxiliary (R)-4-benzyl-3-propionyloxazolidin-2-one
(3), the protocol conveniently gave the enantiomer 5 in good yield
The binding affinities for the new LXR receptors were predicted
in silico using the Internal Coordinate Mechanics (ICM) program.
Scheme 2. Preparation of 17 and R,R-17 for HPCL evaluation of 5.
Figure 3. X-ray structures of compounds 4 (right) and 5 (left).

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O. Alexander H. Åstrand et al. / Bioorg. Med. Chem. 22 (2014) 643–650
Table 1
16a had any functional effects, we tested its effect on lipogenesis.
Both T0901317 and compound 16b increased lipogenesis signifi-
cantly, while T0901317 and compound 16a in combination in-
creased lipogenesis even more. 22SHC did not alter lipogenesis
itself, but seemed to abolish the T0901317-induced effect. These
results show that the new compound 16b acted as an LXR agonist
in these biological assays, similar to the known LXR agonist
T0901317. Extended biological evaluation should be performed
in other test systems, like cancer cell assays.
Structures and docking scores of the compounds presented
Compound
number
Chemical structure
Docking score
R =
LXR
a
LXRb
7
9
À11.68 À14.94
À13.83 À19.81
3. Conclusion
Seven new synthetic compounds with potential LXR modulator
properties were prepared in decent overall yield and the stereo-
chemical configuration of the key intermediate 5 was confirmed
by X-ray analysis. The molecular modelling data suggests that
the new modulators will bind weakly to the LXR binding pocket,
and based on in vitro testing on SCD1, only small changes are ob-
served in the gene expression. Of the new modulators tested, 16b
showed significant agonistic behaviour in SCD1 gene expression
experiments and this was also confirmed in lipogenesis assays in
myotubes. The lipogenesis was more than two-fold increased
when treated with 16b alone as compared to control. The effect
of 16b co-administered with T0901317 also seemed to give an
additional increase in lipogenesis as compared to T0901317 alone.
13
À20.18 À28.98
À16.76 À22.99
16a
16b
16c
16d
À18.31 À19.74
À10.35 À22.61
À14.68 À24.75
4. Experimental protocols
4.1. General
All reagents were purchased from Sigma-Aldrich and used
without further purification unless otherwise noted. For column
chromatography and filtration trough a pad of silica Merck silica
60 mesh (35–70 l
m) is used unless noted otherwise. 1H and 13C
22SHC
À17.5
À29.2
NMR were recorded on Bruker DPX 300 and Bruker AVII 400
instrument equipped with a BACS-60 and a BACS-120 automatic
sample changer, respectively. All experiments were performed at
25 °C in DMSO-d6 and CDCl3.
4.2. Synthesis
The results are found in Table 1 and the best docking pose of the
ligands docked into the ligand binding pocket of LXRb is seen in
Figure 4. All compounds fit well inside the defined ligand binding
pocket, however the docking scores show that the binding affinity
4.2.1. 4-Methylpentanal (2)
A solution of 4-methylpentanol (1, 4.0 g, 39.15 mmol) in CH2Cl2
(133 ml) under nitrogen atmosphere was added PCC (12.67 g,
58.78 mmol) in portions and the mixture was stirred for 24 h at
room temperature. The reaction mixture was added celite, filtered
through a pad of silica and concentrated in vacuo at room temper-
ature. Distillation yielded the title compound as a colorless oil. The
organic layer was dried (MgSO4) and distilled. Distillation gave
2.35 g (60%) of the title compound as a colorless oil. 1H NMR
(300 MHz, CDCl3): d 9.77 (t, J = 1.9 Hz, 1H), 2.52–2.23 (m, 2H),
1.71–1.46 (m, 3H), 0.91 (d, J = 6.2 Hz, 6H), which are in consensus
with published data.5
is generally low towards both LXR
a and LXRb, see Table 1.
(A strong binder needs a docking score of <À32). Only compound
13 and 22SHC show good docking score towards LXRb.
The X-ray crystal structures of the LXRa ligand binding domain
(PDB entry: 1UHL) and LXRb ligand binding domain (PDB entry:
1PQ6) were used as targets for docking. The docking scores were
then calculated using the ICM Virtual Ligand Screening add-on
and the docking results are the best of three docking runs. The pro-
cesses were identical for both LXRa and LXRb.
2.3. Gene expression studies and lipogenesis
4.2.2. 4-(R)-Benzyl-3-(3-(S)-hydroxy-2(S),6-dimethyl-
heptanoyl)-oxazolidin-2-one (4)
In an indicative first study, the compounds were compared to
the characteristic gene expression pattern of 22SHC and
T0901317 on stearyl-CoA desaturase 1 (SCD1) (Hessvik et al.,
2012). SCD1 is a direct LXR target and an important enzyme in lipid
metabolism and lipogenesis. The results confirmed previous find-
ings (Hessvik et al., 2012), showing that the synthetic LXR-agonist
T0901317 up-regulated SCD1, while 22SHC tended to down-regu-
late (p = 0.1) (Fig. 5). Only compound 16b seemed to behave as an
agonist of LXR and up-regulated gene expression of SCD1. To test if
To a cooled (0 °C) stirred solution of (R)-(À)-4-benzyl-3-propio-
nyl-2-oxazolidinone (3, 1.17 g, 5 mmol) in dry CH2Cl2 (10 ml) was
added slowly di-n-butylbortriflate (1 M in CH2Cl2, 5.5 ml,
5.5 mmol) followed by N,N-diisopropylethylamine (1.05 ml,
6 mmol). The reaction mixture was stirred at 0 °C for 30 min and
cooled to À78 °C. Freshly distilled 4-methylpentanal (2, 0.55 g,
5.5 mmol) was added in one portion. The reaction mixture kept
at À78 °C for 30 min, allowed to reach room temperature and stir-
red for 2 h. The reaction mixture was quenched by addition of 4 ml

O. Alexander H. Åstrand et al. / Bioorg. Med. Chem. 22 (2014) 643–650
647
Figure 4. Molecular docking into the ligand binding pocket of LXRb. All new modulators were docked and the best confirmations were superimposed in the binding pocket of
LXRb (PDB: 1PQ6).
Figure 5. Biological effects on gene expression and lipogenesis. (A) Myotubes were treated with DMSO (0.1%), 1
16b and 16c for 4 days. Total RNA was isolated from the cells and analyzed by qPCR as described in Materials and Methods. Gene expressions were normalized to 36B4.
Values represent fold change relative to control given as mean SEM (n = 3–6). SCD1, stearoyl-CoA desaturase 1. (B) Myotubes were treated with DMSO (0.1%), 10 M 22SHC
lM T0901317, 10 lM 22SHC, compounds 9, 14, 15, 21, 16a,
l
and compounds 22
1 l lCi/ml, 100 lM) for 24 h before lipids were isolated by filtration through
M T0901317 for 4 days. The cells were incubated with [1-14C]acetate (1
hydrophobic MultiScreenÒ HTS plates. The levels of lipids were determined by scintillation counting. Values represent fold change relative to control for total lipids
synthesized from acetate given as means SEM (n = 3–5). ⁄P < 0.05 versus control (DMSO) and #P < 0.05 for all other treatments.
phosphate buffer (pH 7.2) and methanol (12 ml). This solution was
treated with 12 ml methanol: 30% H2O2 (2:1) and stirred for 1 h at
room temperature. Water was added and the aqueous phase was
extracted with CH2Cl2 (Â3). The organic phase was dried (Na2SO4),
filtered and evaporated under reduced pressure. Flash chromatog-
raphy on silica gel eluting with heptane/EtOAc (70:30) yielded
1.04 g (62%) of the title compound as a colorless solid. 1H NMR
(300 MHz, CDCl3) d 0.90 (s, 3H), 0.92 (s, 3H), 1.15–1.34 (m, 4H),
1.34–1.50 (m, 2H), 1.50–1.62 (m, 2H), 2.81 (dd, J = 13.4 Hz,
9.4 Hz, 1H), 2.92 (s, 1H), 3.27 (dd, J = 13.5 Hz, 3.2 Hz, 1H),
3.63–3.87 (m, 1H), 3.89–4.03 (m, 1H), 4.09–4.32 (m, 2H), 4.68–
4.75 (m, 1H), 7.21–7.23 (m, 2H), 7.26–7.37 (m, 3H). 13C NMR
(75 MHz, CDCl3) d 10.4, 22.6, 22.7, 28.1, 31.8, 35.2, 37.9, 42.1,
55.2, 66.3, 71.9, 127.5, 129.1, 129.5, 135.1, 153.1, 177.7 HRMS
(EI) calcd for C19H27NO4 333.1940, found 333.1942.
EtOAc (90:10)–(80:20)–(70:30)–(50:50) yielded 0.32 g (67%) of the
title compound as a colorless oil. 1H NMR (300 MHz, CDCl3) d
0.82–0.86 (m, 9H), 1.02–1.17 (m, 1H), 1.25–1.57 (m, 4H),
1.66–1.75 (m, 1H), 3.31 (br s, 1H), 3.60 (d, J = 6 Hz, 2H), 3.74 (bs,
2H). 13C NMR (75 MHz, CDCl3) d 10.1, 22.7 (2Â CH3), 28.2, 31.8,
35.5, 39.0, 66.7, 74.3. HRMS (EI) calcd for C9H18O [MÀH2O]+
142.1358, found 142.1347.
4.2.4. (2S,3S)-2,6-Dimethyl-1-(pyridin-3-ylmethoxy)heptan-3-ol
(7)
A
suspension of NaH dispersion in mineral oil (0.053 g,
1.33 mmol) in dry THF (14 ml) was cooled to 0 °C under N2-atmo-
sphere before a solution of 5 (0.202 g, 1.26 mmol) in dry THF (3 ml)
was added. The reaction mixture was stirred at 0 °C for 30 min. A
suspension of NaH dispersion in mineral oil (0.048 g, 1.20 mmol)
in dry THF (14 ml) was cooled to 0 °C under N2-atmosphere before
commercially available 3-(bromomethyl)-pyridine hydrobromide
(6, 0.285 g, 1.13 mmol) was added. The mixture was stirred for
30 min and then added to the solution of 5. The cooling bath was
removed and the mixture was stirred for 71 h. Water (10 ml)
was added, the mixture was extracted with EtOAc (50 ml  2),
dried (Na2SO4), filtered and evaporated under reduced pressure.
Flash chromatography on silica gel eluting with heptane—hep-
tane/EtOAc (90:10)–(50:50) yielded 0.080 g (28%) of the title com-
pound as a colorless oil. 1H NMR (300 MHz, CD3OD) d 8.52 (s, 1H),
4.2.3. (2S,3S)-2,6-Dimethylheptane-1,3-diol (5)
To a cooled (0 °C) solution of 4-(R)-benzyl-3-(3-(S)-hydroxy-2
(S),6-dimethyl-heptanoyl)-oxazolidin-2-one (4, 0.99 g, 2.97 mmol)
in dry diethyl ether (60 ml) was added water (1 ml) followed by
LiBH4 (0.17 g, 7.75 mmol). The reaction mixture was stirred at
0 °C and then 1 h at room temperature. Water was added and the
water phase was extracted with diethyl ether. The organic phase
was dried (Na2SO4), filtered and evaporated under reduced pres-
sure. Flash chromatography on silica gel eluting with heptane/

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O. Alexander H. Åstrand et al. / Bioorg. Med. Chem. 22 (2014) 643–650
8.46 (d, J = 4.2 Hz, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.43 (dd, J = 7.7,
4.9 Hz, 1H), 4.56 (s, 2H), 3.64 (td, J = 6.4, 3.6 Hz, 1H), 3.55 (dd,
J = 9.1, 6.9 Hz, 1H), 3.40 (dd, J = 9.1, 6.2 Hz, 1H), 1.83 (ddd,
J = 13.3, 6.8, 3.6 Hz, 1H), 1.05–1.65 (m, 6H), 0.79–0.96 (m, 9H).
13C NMR (75 MHz, CD3OD) d 149.28, 149.13, 137.61, 136.54,
125.16, 74.85, 72.98, 71.25, 39.83, 36.65, 33.43, 29.30, 23.12,
22.98, 11.26. MS (electrospray) (pos): 252 (M+H)+/274 (M+Na)+.
HRMS (EI) calcd for C15H25NO2, 251.1885, found 251.1891.
yielded 0.306 g (18%) of the title compound as a colorless oil.
13C NMR (300 MHz, CD3OD) d 7.86 (d, J = 8.4 Hz, 2H), 7.52 (d,
J = 8.4 Hz, 2H), 4.79 (s, 4H), 4.53–4.63 (m, 2H), 3.63–3.69 (m,
1H), 3.56 (dd, J = 9.1, 6.9 Hz, 1H), 3.35–3.50 (m, 5H), 1.80–1.88
(m, 1H), 1.14–1.65 (m, 5H), 0.87–0.97 (m, 10H), 0.72–0.86 (m,
4H), À0.04–0.02 (m, 18H). 13C NMR (75 MHz, CD3OD) d 145.76,
141.61, 128.58, 128.45, 77.93, 74.97, 73.02, 72.99, 66.79, 39.88,
36.68, 33.48, 29.33, 23.15, 23.02, 18.69, 11.34, À1.31. MS (elec-
trospray) (pos): 612/613/614 (M+Na)+. HRMS (EI) calcd for
4.2.5. (2S,3S)-1-(Benzo[d][1,3]dioxol-5-ylmethoxy)-2,6-dimethyl
heptan-3-ol (9)
C17H26O4Na, 612.3187, found 612.3202.
A suspension of NaH 60% dispersion in mineral oil (0.146 g,
3.66 mmol) in dry THF (20 ml) and dry DMF (20 ml) was cooled to
0 °C under N2-atmosphere before a solution 5 (0.551 g, 3.44 mmol)
in dry THF (3 ml) was added drop wise. After 30 min at 0 °C, 5-(bro-
momethyl)benzo[d][1,3]dioxole27 (8, 0.665 g, 3.09 mmol) in dry
THF (3 ml) was added. The cooling bath was removed and the reac-
tion mixture was stirred for 130 min. NH4Cl (aq) satd (25 ml) was
added, the mixture was extracted with EtOAc (50 ml), dried
(Na2SO4), filtered and evaporated under reduced pressure. Flash
chromatography on silica gel eluting with heptane—heptane/EtOAc
(95:5) yielded 0.534 g (53%) of the title compound as a colorless oil
as well as 0.079 g (8%) of the regioisomer 3(S)-(1,3-dihydro-
isobenzofuran-5-ylmethoxy)-2(S),6-dimethyl-heptan-1-ol (9a) as
colorless oils. Compound 9a was not used in the further work. 9:
1H NMR (300 MHz, CD3OD) d 6.73–6.86 (m, 3H), 5.93 (s, 2H), 4.29–
4.48 (m, 2H), 3.60 (td, J = 6.4, 3.7 Hz, 1H), 3.47 (dd, J = 9.2, 6.7 Hz,
1H), 3.32–3.38 (m, 1H), 1.78 (ddd, J = 13.3, 6.7, 3.7 Hz, 1H), 1.27–
1.62 (m, 4H), 0.99–1.25 (m, 1H), 0.81–0.97 (m, 9H). 13C NMR
(75 MHz, CD3OD) d 149.18, 148.57, 133.73, 122.45, 109.37, 108.85,
102.31, 74.25, 73.96, 73.30, 39.76, 36.62, 33.39, 29.31, 23.13,
22.98, 11.42. MS (electrospray) (pos): 317 (M+Na)+. HRMS (EI) calcd
for C17H26O4, 294.1831, found 294.1826.
4.2.6.3. (c) 4-((((2S,3S)-3-Hydroxy-2,6-dimethylheptyl)oxy)methyl)
benzenesulfonamide (13). A mixture of 12 (0.221 g, 0.38 mmol)
in CH3CO2H (6.8 ml) and water (3.4 ml) was stirred at 70 °C under
N2-atmosphere for 3 h. The reaction mixture was cooled to room
temperature and evaporated under reduced pressure. MeOH
(15 ml) was added and the mixture was evaporated under reduced
pressure, this was repeated once. Flash chromatography on silica
gel eluting with heptane–heptane/EtOAc (50:50) yielded 0.055 g
(44%) of the title compound as a colorless oil. 1H NMR (300 MHz,
CD3OD) d 7.88 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 4.52–
4.66 (m, 2H), 3.63–3.68 (m, 1H), 3.49–3.60 (m, 1H), 3.35–3.43
(m, 1H), 1.80–1.88 (m, 1H), 1.04–1.67 (m, 6H), 0.82–0.97 (m,
9H). 13C NMR (75 MHz, CD3OD) d 144.69, 143.93, 128.70, 127.15,
74.81, 73.06 (2C), 39.76, 36.61, 33.38, 29.25, 23.13, 22.99, 11.28.
MS (electrospray) (pos): 352/353/354 (M+Na)+. HRMS (EI) calcd
for C16H27NO4SNa, 352.1559, found 352.1568.
4.2.7. General procedure for condensation products between
(2S,3S)-2,6-dimethylheptane-1,3-diol (5) and the building
blocks with benzylbromide functionality1-((4-(bromomethyl)-
phenyl)sulfonamides (15a–d)
4.2.7.1. General procedure part I: 1-((4-(Bromomethyl)phenyl)
sulfonamides (15a–d). To a solution of commercially available
4-(bromomethyl)benzenesulfonyl chloride (14, 5 mmol) in dry
CH2Cl2 (10 ml) under argon at 0 °C was added triethylamine
(10 mmol) followed by the amine (5 mmol) with stirring. After
75 min at 0 °C, 2 M HCl was added and the aqueous phase ex-
tracted with CH2Cl2 (Â2). The combined organic phase was washed
with 2 M HCl, brine, dried (Na2SO4), filtered and concentrated un-
der reduced pressure. This allowed the isolation of the sulfona-
mides in low to moderate yields (20–50%) as pale yellow to
white solids, used without further purification or reaction
optimization.
4.2.6. 4-(((2S,3S)-3-Hydroxy-2,6-dimethylheptyl)oxy)methyl)ben
zenesulfonamide (13)
4.2.6.1. (a) 4-(Bromomethyl)-N,N-bis((2-(trimethylsilyl)-eth-
oxy)-methyl)-benzenesulfonamide
(11). 4-(Hydroxymethyl)-
N,N-bis((2-(trimethylsilyl)-ethoxy)-methyl)-benzenesulfonamide
(10, prepared according to literature,28) was dissolved in dry CH2-
Cl2 (60 ml) was cooled to 0 °C under N2-atmosphere before PPh3
(2.173 g, 8.29 mmol) and CBr4 (2.023 g, 6.10 mmol) was added.
The mixture was stirred at 0 °C for 2 h and then diluted with
100 mL CH2Cl2, washed with 50 mL water, dried (Na2SO4), filtered
and evaporated under reduced pressure. Flash chromatography on
silica gel eluting with heptane/EtOAc (90:10) yielded 1.35 g (48%)
4.2.7.2. General procedure part II:. Condensation products (16a–
d) between (2S,3S)-2,6-dimethylheptane-1,3-diol (5) and the
building blocks with benzylbromide functionality1-((4-(bromo-
methyl)-phenyl)sulfonamides (15a–d)
of the title compound as
a
sticky, colorless oil. 1H NMR
(300 MHz, CDCl3) d 7.86 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H),
4.77 (s, 4H), 4.48 (s, 2H), 3.38–3.55 (m, 4H), 0.77–0.89 (m, 4H),
À0.07–0.00 (m, 18H). 13C NMR (75 MHz, CDCl3) d 142.50, 141.28,
129.54, 127.91, 76.41, 65.90, 31.68, 17.94, À1.25. MS (electrospray)
(pos): 532/533/534/535/536/537 (M+Na)+.
To a solution of 5 (1 mmol) in THF/DMF (4 mL, 50:50) under ar-
gon at 0 °C was added sodium hydride (1.15 mmol). After stirring
at 0 °C for 20 min, the respective sulfonamide 15a–d (1 mmol) in
THF (2 mL) was added. The reaction mixture was allowed to reach
room temperature and stirred overnight. NH4Cl (satd) was added
and the aqueous phase extracted with ether (Â3). The combined
organic extract was washed with brine, dried (Na2SO4), filtered
and concentrated. Purification by flash chromatography (hep-
tane/EtOAc 70:30) afforded the title compounds in low yields, that
were subjected to analysis and biological evaluation without fur-
ther method optimization.
4.2.6.2. (b) 4-((((2S,3S)-3-Hydroxy-2,6-dimethylheptyl)oxy)
methyl)-N,N-bis((2-(trimethylsilyl)-ethoxy)methyl)-benzene-
sulfonamide (12). A suspension of NaH dispersion in mineral
oil (0.124 g, 3.10 mmol) in dry THF (20 ml) and dry DMF
(20 ml) was cooled to 0 °C under N2-atmosphere before a solu-
tion of 5 (0.472 g, 2.94 mmol) in dry THF (3 ml) was added drop
wise. The reaction mixture was stirred at 0 °C for 35 min before a
solution of 11 (1.35 g, 2.64 mmol) in dry THF (3 ml) was added.
The cooling bath was removed and the reaction mixture was
stirred for 3 h. NH4Cl (aq) satd (25 ml) was added, the mixture
was extracted with EtOAc (50 ml), dried (Na2SO4), filtered and
evaporated under reduced pressure. Flash chromatography on
silica gel eluting with heptane–heptane/EtOAc (98:2)–(95:5)
4.2.8. 4-(Bromomethyl)-N,N-diethylbenzenesulfonamide (16a)
1H NMR (300 MHz, chloroform-d) d 7.73 (d, J = 8.4 Hz, 2H), 7.40
(d, J = 8.4 Hz, 2H), 4.53 (s, 2H), 3.70 (ddd, J = 7.9, 4.6, 2.7 Hz, 1H),
3.64–3.42 (m, 2H), 3.19 (q, J = 7.1 Hz, 4H), 2.49 (s, 1H), 1.96–1.75
(m, 1H), 1.61–1.00 (m, 12H), 0.99–0.75 (m, 9H). 13C NMR

O. Alexander H. Åstrand et al. / Bioorg. Med. Chem. 22 (2014) 643–650
649
(75 MHz, chloroform-d) d 143.0, 139.5, 127.6, 127.2, 75.2, 73.9,
72.4, 42.1, 38.0, 35.5, 32.1, 28.1, 22.7, 22.6, 14.2, 10.6. HRMS (EI)
calcd for C20H35NO4S, 385.2287, found 385.2276.
mic mixture were also analysed using identical conditions. The re-
sults are uncorrected.
4.4. Culturing of human myotubes
4.2.9. 1-((4-(Bromomethyl)phenyl)sulfonyl)piperidine (16b)
1H NMR (300 MHz, chloroform-d) d 7.77 (d, J = 8.3 Hz, 2H), 7.45
(d, J = 8.4 Hz, 2H), 4.55 (s, 2H), 3.72 (ddd, J = 7.9, 4.7, 2.7 Hz, 1H),
3.65–3.43 (m, 2H), 3.20 (t, J = 6.8 Hz, 4H), 2.71 (s, 1H), 1.96–1.81
(m, 1H), 1.80–1.64 (m, 4H), 1.62–0.99 (m, 6H), 0.92 (d, J = 7.1 Hz,
3H), 0.86 (dd, J = 6.6, 1.3 Hz, 6H). 13C NMR (75 MHz, chloroform-
d) d 143.7, 136.4, 128.0, 128.0, 75.7, 74.4, 72.8, 48.3, 38.3, 35.8,
32.4, 28.5, 25.6, 23.0, 23.0, 11.0. HRMS (EI) calcd for C20H33NO4S,
383.2134, found 383.2126.
Satellite cells were isolated as previously described (Gaster M,
Kristensen SR, Beck-Nielsen H, Schroder HD (2001) A cellular mod-
el system of differentiated human myotubes. APMIS 109:735–744)
from the M. obliquus internus abdominis from healthy volunteers.
The biopsies were obtained with informed written consent and ap-
proval by the National Committee for Research Ethics (Oslo, Nor-
way). The research performed in this study was approved, as a
part of a larger project, by the National Committee for Research
Ethics (Oslo, Norway). The cells were cultured in DMEM-Gluta-
max-I with 5.5 mM glucose supplemented 2% foetal bovine serum,
4.2.10. (2S,3S)-2,6-Dimethyl-1-((4-(morpholinosulfonyl)benzyl)
oxy)heptan-3-ol (16c)
2% Ultroser G, penicillin (100 units/ml) and streptomycin (100
lg/
1H NMR (300 MHz, chloroform-d) d 7.69 (d, J = 8.3 Hz, 2H), 7.45
(d, J = 8.4 Hz, 2H), 4.55 (s, 2H), 3.72 (ddd, J = 7.9, 4.6, 2.7 Hz, 1H),
3.64–3.46 (m, 2H), 3.04–2.84 (m, 4H), 2.43 (s, 1H), 1.89 (dd,
J = 4.8, 1.6 Hz, 1H), 1.74–0.99 (m, 12H), 0.99–0.79 (m, 9H). 13C
NMR (75 MHz, chloroform-d) d 143.4, 135.4, 127.9, 127.6, 75.4,
74.0, 72.5, 47.0, 38.0, 35.5, 32.1, 28.2, 25.2, 23.5, 22.7, 22.7,
10.64. HRMS (EI) calcd for C21H35NO4S, 397.2287, found 397.2291.
ml), amphotericin B (1.25 g/ml) and 5.5 mM sodium pyruvate for
l
proliferation. At 70–80% confluence the growth medium was re-
placed by DMEM-Glutamax-I with 5.5 mM glucose supplemented
with 2% foetal bovine serum, penicillin (100 units/ml), streptomy-
cin (100 lg/ml), amphotericin B (1.25 lg/ml), 5.5 mM sodium
pyruvate and insulin (25 pM) to induce differentiation. The cells
were cultured in humidified 5% CO2 atmosphere at 37 °C, and the
medium was changed every 2–3 days. Experiments were per-
formed after 7 days of differentiation.
4.2.11. 4-((4-(Bromomethyl)phenyl)sulfonyl)morpholine (16d)
1H NMR (300 MHz, chloroform-d) d 7.70 (d, J = 8.4 Hz, 2H), 7.48
(d, J = 8.4 Hz, 2H), 4.57 (s, 2H), 3.71 (q, J = 4.8 Hz, 5H), 3.54 (qd,
J = 9.0, 5.7 Hz, 2H), 3.06–2.87 (m, 4H), 2.42 (s, 1H), 1.89 (dd,
J = 4.9, 1.9 Hz, 1H), 1.68–1.01 (m, 5H), 1.01–0.77 (m, 9H). 13C
NMR (75 MHz, chloroform-d) d 144.1, 134.2, 128.1, 127.7, 75.3,
73.9, 72.4, 66.1, 46.0, 38.0, 35.5, 32.1, 28.2, 22.7, 22.7, 10.6. HRMS
(EI) calcd for C20H33NO5SNa, 422.1978, found 422.1986.
4.5. RNA isolation and analysis of gene expression by qPCR
After proliferation and differentiation in DMEM-media, cells
were harvested and total RNA was isolated by RNeasy Mini kit
(Qiagen Sciences, Oslo, Norway) according to the supplier’s total
RNA isolation protocol. Equal amount of RNA obtained from myo-
tubes from different donors were reversely transcribed with a High
4.2.12. (2S,3S)-3-Hydroxy-2,6-dimethylheptyl 4-methylbenzene-
sulfonate (17)
Capacity cDNA Archive Kit. Total RNA (1 lg/ll) was reversely tran-
scribed with hexamere primers using a PerkinElmer Thermal Cy-
cler 9600 (25 °C for 10 min, 37 °C for 1 h, 99 °C for 5 min) and a
TaqMan reverse-transcription reagents kit (Applied Biosystems).
Primers (36B4 and SCD1) were designed using Primer ExpressÒ
(Applied Biosystems). Primer sequences are available upon re-
quest. Each target gene were quantified in triplicates and carried
To a cooled (0 °C) solution of 2-(S)-6-dimethyl-heptane-1,3-(S)-
diol (5, 1.76 g, 11 mmol) in CH2Cl2 (80 ml) was added pyridine
(3.55 ml, 44 mmol) followed by tosyl chloride (2.30 g, 12.1 mmol).
The reaction mixture was stirred at room temperature for 2 days.
The reaction mixture was quenched with water and the aqueous
phase was extracted with diethyl ether (Â2). The combined organic
phase was washed with 2 M HCl and brine, dried (Na2SO4), filtered
and evaporated under reduced pressure. Flash chromatography on
silica gel eluting with heptane/EtOAc (80:20)–(70:30) yielded
2.59 g (75%) of the title compound as a colorless oil. 1H NMR
out in a 25 ll reaction volume according to the supplier’s protocol.
All assays were run for 40 cycles (95 °C for 12 s followed by 60 °C
for 60 s). The transcription levels were normalized to the reference
control gene 36B4.
(300 MHz, CDCl3)
d
0.82–0.88 (m, 9H), 1.07–1.19 (m, 1H),
4.6. Lipogenesis
1.23–1.45 (m, 3H), 1.48–1.54 (m, 1H), 1.84–1.92 (m, 1H), 2.43 (s,
3H), 3.62–3.67 (m, 1H), 3.88 (dd, J = 9.6 Hz, 6.0 Hz, 1H), 4.07 (dd,
J = 9.6 Hz, 7.8 Hz, 1H), 7.33 (dd, J = 8.6 Hz, 0.6 Hz, 2H), 7.73–7.80
(m, 2H). 13C NMR (75 MHz, CDCl3) d 9.5, 21.7, 22.62, 22.64, 28.1,
32.3, 35.3, 37.8, 70.9, 73.0, 127.9, 130.0, 133.1, 144.9.
(2R,3R)-3-Hydroxy-2,6-dimethylheptyl 4-methylbenzenesulfo-
nate (R,R-17) was prepared from 4-(S)-benzyl-3-(3-(S)-hydroxy-
2(S),6-dimethyl-heptanoyl)-oxazolidin-2-one using the same
protocol as for 17, and was used for analytical purposes only in
the present work.
For lipogenesis, cells were incubated in DMEM-media with
[U-14C]glucose (1
lCi/ml, 200 lM) for 24 h and harvested in
dH2O, assayed for protein (Bradford MM (1976) A rapid and sensi-
tive method for the quantitation of microgram quantities of pro-
tein utilizing the principle of protein–dye binding. Anal. Biochem.
72: 248–254), and total lipids were isolated by filtration of the cell
lysate through hydrophobic MultiScreenÒ HTS plates (Millipore,
Billerica, MA, USA). The amount of lipids was determined by liquid
scintillation counting, and lipogenesis was related to cell protein
content and given as nmol/mg protein.
4.3. Determination of enantiomeric excess by chiral HPLC
4.7. Presentation of data and statistical analysis
The enantiomeric excess of compound 5 was determined based
on analysis of the corresponding tosylate 17 using a CHIRAPAK
AS-H column with 0.6 mL/min 9:1 iso-hexane/2-propanol at room
temperature equipped with a UV detector. The UV absorption was
measured at 254 nm and the enantiomeric excess was determined
by comparing the integral of the peaks with retention time 16.5
and 20.1 min respectively. The opposite diastereomer and the race-
Data in text and figures are given as mean ( SEM) from
n = number of separate experiments, all performed on muscle cells
established from separate cell donors. At least 3 parallels were
included in each experiment. For lipogenesis, comparisons of dif-
ferent treatments were evaluated by two-tailed, paired, Student’s
t-test, and P < 0.05 was considered significant. For gene expression

650
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a
Acknowledgments
FORNY: This work was supported by Grants from the Norwe-
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