Identification of 4H,6H-[2]benzoxepino[4,5-c][1,2]oxazoles as novel squalene synthase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.04.092

Novel squalene synthase inhibitors are disclosed. The design, synthesis, SAR and pharmacological profile of the compounds are discussed.

Full text of DOI:10.1016/j.bmcl.2011.04.092

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3648–3653
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of 4H,6H-[2]benzoxepino[4,5-c][1,2]oxazoles as novel
squalene synthase inhibitors
Nils Griebenow a, , Anja Buchmueller b, Peter Kolkhof b, Jens Schamberger a, Hilmar Bischoff b
⇑
a Bayer Pharma AG, Global Drug Discovery, Medicinal Chemistry Wuppertal, Building 460, D-42096 Wuppertal, Germany
b Bayer Pharma AG, Global Drug Discovery, Cardiology Research, Building 500, D-42096 Wuppertal, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel squalene synthase inhibitors are disclosed. The design, synthesis, SAR and pharmacological profile
of the compounds are discussed.
Received 19 March 2011
Revised 18 April 2011
Accepted 20 April 2011
Available online 29 April 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Squalene synthase inhibitor
SAR
Atherosclerosis
4H,6H-[2]Benzoxepino[4,5-c][1,2]oxazole
A large number of epidemiological studies have shown a causal
connection between dyslipidemias and cardiovascular disorders.
Elevated plasma cholesterol, mainly LDL-cholesterol, is one of the
highest risk factors for cardiovascular disorders, such as athero-
sclerosis.1 This relates both to an isolated hypercholesterolemia
and to hypercholesterolemia combined with elevated plasma tri-
glycerides or low plasma HDL-cholesterol. The standard of care
in hypercholesterolemia is the use of statins which can reduce
mortality and morbidity among patients with cardiovascular disor-
ders. Statins block one of the key enzymes, HMG-CoA reductase, in
the cholesterol biosynthesis pathway which leads to a compensa-
tory up-regulation of hepatic LDL-receptors.
non-response, additional lipid lowering therapies are necessary.
Squalene synthase (EC 2.5.1.21) catalyzes the conversion of far-
nesyl pyrophosphate into squalene by reductive condensation. This
is another crucial step in cholesterol biosynthesis. Whereas HMG-
CoA reductase inhibitors also block the synthesis of essential non-
sterol isoprenoids like farnesyl pyrophosphate which are also of
importance for other cellular metabolic pathways and reactions,
squalene serves as the exclusive precursor for cholesterol. Inhibi-
tion of squalene synthase leads directly to a reduction in choles-
terol biosynthesis and thus to a fall in plasma cholesterol levels. It
has already been shown in animal models and in humans that
plasma LDL-cholesterol and triglycerides are lowered by squalene
synthase inhibitors. Substances which have a cholesterol or com-
bined cholesterol and triglyceride lowering effect ought therefore
However, since about half of patients treated with statins do not
reach target LDL levels due to efficacy limitations or even
O
O
O
O
O
O
OH
OH
O
O
Cl
Cl
O
O
N
N
N
N
TAK-475
T-91485
O
O
O
O
HO
Figure 1. Chemical structure of lapaquistat acetate (TAK-475) and its active metabolite (T-91485).
⇑
Corresponding author. Tel.: +49 202 36 5866.
E-mail addresses: nils.griebenow@bayer.com, nils.griebenow@bayerhealthcare.com (N. Griebenow).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.092

N. Griebenow et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3648–3653
3649
to be suitable for the treatment and prevention of cardiovascular
disorders.1c
Herein, we describe a structure based design approach to a
novel class of squalene synthase inhibitors. Starting point for
our investigations was the most advanced squalene synthase
inhibitor lapaquistat acetate (TAK-475), which is so far the only
squalene synthase inhibitor progressed into phase III clinical tri-
als (Fig. 1).2
Contemplating the backbone of lapaquistat acetate,3 our design
plan was to replace the central amide group by an amide bioisoste-
re, such as a five membered heterocyclic moiety.4 In detail, we
envisioned to replace the amide by an isoxazole,4 whereby the
nitrogen of the heterocycle should be able to act as a hydrogen
bond acceptor like the carbonyl group of the amide (Fig. 2).5
A good match of electronic and geometrical properties was sup-
ported by molecular modeling (Fig. 3).6 It was proposed that the
introduction of this bioisosteric group would improve pharmaco-
logical and physicochemical properties, because such a scaffold
has a reduced number of rotational bonds and the presumably
metabolic labile amide bond is replaced.
Figure 3. Superimposition of T-91485 (gray) and 13d (yellow).
O
H
Further insight into the role of the amide carbonyl was gained
by analysis of the published X-ray crystal structure of squalene
synthase by Pandit et al.7 The co-crystallized ligand CP-320473
exhibits the same core structure as TAK-475 (Fig. 4). The crystal
structure revealed a water mediated hydrogen bond between the
carbonyl group of the amide and Asn 215. Virtual docking of 13d
confirmed that this water bridge could well be established by the
benzoxepino-oxazole scaffold (Fig. 5).8
Cl
O
N
COOH
COOH
N
O
CP-320473
Figure 4. Structure of CP-320473, which was co-crystallized with squalene
synthase (PDB entry code 1EZF).
Inspired by a report of Moore et al.9 describing the synthesis of
trisubstituted isoxazole 4-boronates with high levels of regiocon-
trol (Scheme 1), we considered isoxazole 1 as a suitable key build-
ing block for the synthesis of the projected benzoxepino-oxazole
scaffold.
In detail, such a building block should allow for facile C–C bond
formation between an aromatic moiety and the isoxazole via Suzu-
ki cross-coupling, as well as for the further elaboration of the di-
methyl acetal group towards the required acrylate for ring
closure reaction (see Scheme 2).
Thus isoxazole 1 was synthesized as outline in Scheme 3.10 Ini-
tially, glyoxal dimethyl acetal (6) was reacted with hydoxylamine
to provide the aldoxime, which was converted into the N-hydroxy-
imidoyl chloride 2 upon treatment with N-chloro-succinimide. In
parallel, alkyne 7 was transformed into the alkynylboronate 3
via deprotonation with n-butyl lithium followed by reaction
with 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane. Final-
ly [2+3] cycloaddition reaction of 2 and 3 yielded the target
isoxazole 1 as a single regioisomer.11 Instead of the typical
Huisgen-protocol,12 the nitrile oxide from 2 was generated with
potassium bicarbonate in dimethoxyethane according to the
method of Moore et al.9
Figure 5. (a) X-ray crystal structure of squalene synthase in complex with inhibitor
CP-320473; (b) Best virtual docking pose of 13d.
oxide to minimize unwanted nitrile oxide dimerization. However
the yield of the cycloaddition remains low to moderate.13 The sec-
ond key intermediate 5 was built-up starting from commercial 2-
amino-5-chloro-benzoic acid (8) as shown in Scheme 4.
Because of its low basicity and solubility in dimethoxyethane,
potassium bicarbonate permits the slow generation of the nitrile
Anthranilic acid 8 was converted to benzoxazinone 9 upon
treatment with acetic anhydride under refluxing conditions. Addi-
tion of lithiated 1,2-dimethoxybenzene to 9 and refluxing in
hydrochloric acid yielded 10. Conversion of the amino functional-
ity of 10 via anhydrous diazotiziation followed by treatment with
sodium iodide in acetone afforded the iodo-compound 5.9
Having both key intermediates in hand we investigated the fur-
ther elaboration to the benzoxepino-oxazole scaffold as outlined in
Scheme 2. Initially building blocks 1 and 5 were reacted in a Suzuki
cross-coupling reaction14 to the biaryl compound 11 (Scheme 5).
Elaboration of the acetal 11 to the acrylate 4 was performed via
treatment with aq HCl followed by Wittig-olefination15 of the
formed aldehyde with ethoxy-carbonylmethyltriphenylphospho-
rane. Selective reduction of the ketone moiety of 4 and subsequent
O
O
O
O
O
O
O
O
O
R2
Cl
R
Cl
N
N
O
R1O
cyclization of the alcohol via 1,4-addition upon treatment
Figure 2. Design approach yielding the benzoxepino-oxazole scaffold derived from
TAK-475 and T-91485.
16
with Schwesinger phosphazene base tert-Bu-P2
yielded the

3650
N. Griebenow et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3648–3653
O
B
OH
Cl
O
a
Cl
O
B
N
+
O
Cl
N
O
OH
N
O
B
O
B
O
N
b
O
O
O
+
Cl
O
O
1
2
3
O
Scheme 1. Reagents and conditions: (a) KHCO3, DME, 16 h, 50 °C (44%). For details see Moore et al. Ref. 9 (b) Proposed synthetic access to target intermediate 1.
O
B
O
N
O
O
O
O
O
O
O
N
O
1
O
O
b
a
Cl
Cl
R
O
O
+
O
O
N
4
O
O
Cl
O
5
I
Scheme 2. (a) Retrosynthetic approach: Suzuki cross-coupling of building blocks 1 and 5 and further elaboration of the dimethyl acetal to the acrylate 4; (b) Selective
reduction at the ketone of intermediate 4 and cyclization of the formed alcohol by 1,4-addition to the acrylate.
O
O
O
N
H
a
b,c
Cl
Cl
O
6
OH
NH2
O
7
O
N
8
9
a,b
O
c,d
O
O
HO
O
d,e
O
Cl
Cl
O
O
O
B
Cl
O
O
NH2
10
2
3
I
5
e
Scheme 4. Reagents and conditions: (a) Ac2O, reflux, 2 h (83%); (b) 1,2-dimethoxy-
benzene, THF, n-BuLi, 0 °C, 1 h; (c) concn aq HCl, EtOH, H2O, reflux, 3 h (47% over
two steps); (c) THF, boron trifluoride–diethyl ether complex, isoamyl nitrite,
À10 °C, 30 min; (d) NaI, acetone, rt, 20 min (41% over two steps).
O
O
O
B
O
benzoxepino-oxazole 12 as
a
mixture of diastereomers
N
1
O
(ds = 58:42). Saponification followed by separation of the four ster-
eoisomers by chiral HPLC provided the isolated isomers 13a–d.10
The IC50 values of the isomers 13a–d were determined by a bio-
chemical assay measuring the conversion of farnesyl pyrophos-
phate to squalene by squalene synthase.17 The absolute
stereochemistry of the benzoxepino-oxazole 13d was assigned by
Scheme 3. (a) Glyoxal dimethylacetal (45% in tert-butyl methyl ether), hydroxyl-
amine (prepared from hydroxylamine hydrochloride and NaOMe), methanol, rt,
16 h; (b) DMF, N-chloro-succinimide, 3 h, rt (28% over two steps); (c) n-BuLi, THF,
À78 °C, 30 min; (d) 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, 2 h,
À78 °C (88%); (e) KHCO3, DME, 50 °C, 56 h (17%).

N. Griebenow et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3648–3653
3651
O
O
O
O
B
O
N
a
Cl
O
O
O
O
O
+
Cl
O
O
11
O
N
I
O
5
1
O
O
O
N
O
O
b,c
d,e
Cl
Cl
O
O
O
O
O
N
4
12
O
O
O
O
O
f,g
Cl
O
OH
stereoisomers:
13a, 13b, 13c
+
N
13d
O
Scheme 5. Reagents and conditions: (a) K3PO4, dioxane [1,1’-bis(diphenylphosphino)-ferrocene)di-chloropalladium(II) (1:1 complex with DCM), 85 °C, 72 h (42%); (b) THF,
10% aq HCl, reflux, 42 h; (c) ethoxy-carbonylmethyltriphenylphosphorane, DCM, rt, 16 h (72% over two steps); (d) lithium tri(tert-butyloxy)aluminum hydride THF, 0 °C, 2 h;
(e) 1-tert-butyl-2,2,4,4,4-pentakis(dimethylamino)-25,45-catenodi(phosphazene) (phosphazene base tert-Bu-P2), THF, 0 °C, 1 h, (36%, mixture of two couples of diastereo-
mers, dr = 58:42); (f) dioxane, water, concn HCl, 80 °C, 19 h (51%); (g) separation of the four stereoisomers by preparative HPLC on a chiral phase (13d: 14%).
Table 1
SAR of the benzoxepino-oxazoles
O
O
O
O
O
O
O
O
O
O
N
O
O
O
Cl
Cl
Cl
R
R
R
N
N
scaffold C
1st metabolite
O
O
scaffold A
scaffold B
TAK-475
O
O
HO
HLMc (%)
b
Compounda
Scaffold
Isomer
R
IC50 (nM)
Sterol biosynthesisd (%)
3 mg/kg po
1 mg/kg po
13a
13b
13c
13d
A
A
A
A
1e
2e
3e
4e
OH
OH
OH
OH
1420
5500
169
56
nd
nd
64
100
nd
nd
31
58
nd
nd
nd
nd
OH
14d
A
4
162
nd
91
nd
nd
N
*
O
OH
15d
A
4
131
80
nd
N
N
*
*
O
16d
A
B
4
112
213
98
7
79
73
71
OH
O
OH
TAK-475
—
59
N
*
(continued on next page)

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N. Griebenow et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3648–3653
Table 1 (continued)
b
Compounda
Scaffold
Isomer
R
IC50 (nM)
HLMc (%)
Sterol biosynthesisd (%)
3 mg/kg po 1 mg/kg po
16
C
C
—
—
OH
223
260
nd
37
nd
nd
O
OH
T-91485
nd
nd
N
*
a
For experimental data of compounds 13a–d. 14d, 15d and 16d see Ref. 10.
Values are means of three experiments. For a detailed description of the biochemical assay see Ref. 17.
Human liver microsomal stability, % compound remaining after 60 min.
b
c
d
e
Inhibition of sterol biosynthesis in % (in vivo). For details see Ref. 19.
1H NMR spectra indicated that 13a and 13d as well as 13b and 13c are pairs of enantiomers.
O
O
O
O
References and notes
1. (a) Carpender, K. I.; Taylor, S. E.; Ballantine, J. A.; Fussell, B.; Halliwell, B.;
Mitchinson, M. J. Biochim. Biophys. Acta 1993, 1167, 121; (b) Brown, A. J.;
Mander, E. L.; Gelissen, I. C.; Kritharides, L.; Dean, R. T.; Jessup, W. J. Lipid Res.
2000, 41, 226; (c) Kourounakis, A. P.; Charitos, C.; Rekka, E. A.; Kourounakis, P.
N. J. Med. Chem. 2008, 51, 5861.
2. (a) Burnett, J. Curr. Opin. Investig. Drugs. 2006, 7, 850; (b) Seiki, S.; Frishman, W.
H. Cardiol. Rev. 2009, 17, 70; (c) Elsayed, R. K.; Evans, J. D. Expert Opin. Emerging
Drugs 2008, 13, 309.
3. (a) Miki, T.; Kori, M.; Fujishima, A.; Mabuchi, H.; Tozawa, R.; Nakamura, M.;
Sugiyama, Y.; Yukimasa, H. Bioorg. Med. Chem. 2002, 10, 385; (b) Miki, T.; Kori,
M.; Mabuchi, H.; Banno, H.; Tozawa, R.; Nakamura, M.; Itokawa, S.; Sugiyama,
Y.; Yukimasa, H. Bioorg. Med. Chem. 2002, 10, 401.
4. (a) Pevarello, P.; Amici, R.; Brasca, M. G.; Villa, M.; Varasi, M. Targets in
Heterocycl. Syst. 1999, 3, 301; (b) Mogensen, J. P.; Roberts, S. M.; Bowler, A. N.;
Thomsen, C.; Knutsen, L. J. S. Bioorg. Med. Chem. Lett. 1998, 8, 1767.
5. (a) Nobeli, I.; Price, S. L.; Lommerse, J. P. M.; Taylor, R. J. Comput. Chem. 1997, 18,
2060; (b) Böhm, H.-J.; Klebe, G.; Brode, S.; Hesse, U. Chem. Eur. J. 1996, 2, 1509;
(c) Liu, G.; Xin, Z.; Pei, Z.; Hajduk, P. J.; Abad-Zapatero, C.; Hutchins, C. W.; Zhao,
H.; Lubben, T. H.; Ballaron, S. J.; Haasch, D. L.; Kaszubska, W.; Rondinone, C. M.;
Trevillyan, J. M.; Jirousek, M. R. J. Med. Chem. 2003, 46, 4232; (d) Sharp, S. Y.;
Prodromou, C.; Boxall, K.; Powers, M. V.; Holmes, J. L.; Box, G.; Matthews, T. P.;
Cheung, K.-M. J.; Kalusa, A.; James, K.; Hayes, A.; Hardcastle, A.; Dymock, B.;
Brough, P. A.; Barril, X.; Cansfield, J. E.; Wright, L.; Surgenor, A.; Foloppe, N.;
Hubbard, R. E.; Aherne, W.; Pearl, L.; Jones, K.; McDonald, E.; Raynaud, F.;
Eccles, S.; Drysdale, M.; Workman, P. Mol. Cancer Ther. 2007, 6, 1198.
6. (a) Flexible structural alignment was carried out using the program Surflex Sim
provided with SYBYL-X 1.2 (Tripos Int., St. Louis, MO, USA).; (b) Jain, A. N. J.
Comput.-Aided Mol. Des. 2000, 14, 199; Structures were optimized prior to
alignment using the MMFF94s force field, supplied with SYBYL-X 1.2, see (c)
Halgren, T. J. Comp. Chem. 1999, 20, 720.
O
N
O
N
O
O
a,b
Cl
Cl
OH
R
13d
14d-16d
O
O
Scheme 6. Reagents and conditions: (a) Ethyl piperidine-4-carboxylate or ethyl
piperidin-4-ylacetate or (3R)-pyrrolidin-3-ol, PyBOP, DIEA, rt, 16 h. (b) In case of
amines bearing an ester group: dioxane, water, concn aq HCl, 60 °C, 16 h.
analogy to the stereoselective inhibitory potencies of 4,1-ben-
zoxazepine derivatives (Table 1).3b
The stereoisomer 13d was elaborated further at the carboxylic
acid to the amides 14d–16d as outlined in Scheme 6. Because of
the previously established structure activity relationship,18 we
considered only small variations at the carboxylic acid. Amide
formation was accomplished under standard coupling conditions
(PyBOP, DIEA).
Surprisingly, all the amides 14d–16d showed a higher in vitro
potency compared to lapaquistat acetate (Table 1). Compounds
13d, 15d and 16d were progressed further to in vivo animal stud-
ies. Inhibition of hepatic cholesterol biosynthesis was investigated
in NMRI-mice.
7. Protein Data Bank, Research Collaboratory for Structural Bioinformatics,
Rutgers University, New Brunswick, NJ (http://www.rcsb.org) with reference
code 1EZF. See: Pandit, J.; Danley, D. E.; Schulte, G. K.; Mazzalupo, S. J. Biol.
Chem. 2000, 275, 30610.
8. Docking studies were carried out employing the Schrödinger suite of programs.
The X-ray crystal structure 1EZF was used as a glide receptor grid after protein
preparation. The water forming a bridge to ASN 215 was included into the
receptor grid. Ligands were optimized before docking via the ligprep routine.
Docking was carried out in SP and XP mode with similar results. Figures were
prepared with PyMol.; (b) Glide, version 5.6, Schrödinger, LLC, New York, NY,
2010.; (c) LigPrep, version 2.4, Schrödinger, LLC, New York, NY, 2010.; (d) The
PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC.
9. (a) Moore, J. E.; Davies, M. W.; Goodenough, K. M.; Wybrow, R. A. J.; York, M.;
Johnson, C. N.; Harrity, J. P. A. Tetrahedron 2005, 61, 6707; (b) Moore, J. E.;
Goodenough, K. M.; Spinks, D.; Harrity, J. P. A. Synlett 2002, 2071.
10. For experimental details and characterization of the compounds see: (a)
Griebenow, N.; Buchmueller, A.; Kolkhof, P.; Bischoff, H. PCT Int. Appl., WO
2008003424, 2008. (b) Griebenow, N.; Buchmueller, A.; Kolkhof, P.; Bischoff, H.
US 2010016299, 2010.
After po administration of 3 mg/kg, the compounds 13d, 15d
and 16d showed a reduction in sterol biosynthesis of 58%, 80%
and 79% respectively (Table 1).19
The best match of in vitro potency and microsomal stability
combined with a pronounced reduction in sterol biosynthesis
was displayed by compound 16d.20 Thus 16d, was investigated at
lower dosage in NMRI-mice (1 mg/ kg po) showing a reduction in
sterol biosynthesis of 71%.
In conclusion, we have identified a novel series of substituted
benzoxepino-oxazoles which have a superior profile compared to
lapaquistat acetate (TAK-475) and its active metabolite T-91485,
regarding in vitro potency and microsomal stability as well as
reduction of sterol biosynthesis. Further pharmacological data of
16d will be reported in due course.
11. The geometry of the isoxazole 1 was reasoned from the subsequent synthesis
steps providing the 4,1-benzoxazepine scaffold. The regioisomeric 3-
(dimethoxymethyl)-4-isopropyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-1,2-oxazole will not yield such a cyclization pruduct.
12. Generation of nitrile oxides from N-hydroxyimidoyl chlorides by slow addition
of triethylamine, where the stationary concentration of nitrile oxide is kept low
to avoid dimerization: Huisgen, R. Angew. Chem., Int. Ed. 1963, 2, 565.
13. Yields were low due to dimerization of the formed nitrile oxide and proto-
deborylation of the product.
14. For comprehensive reviews, see: (a) Suzuki, A. Pure Appl. Chem. 1994, 66, 213;
(b) Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457; (c) Bellina, F.; Carpita, A.;
Rossi, R. Synthesis 2004, 15, 2419.
Acknowledgements
We thank our Analytical Chemistry colleagues for structure and
purity determination. In addition, we are grateful to Dr. Klemens
Lustig for providing microsomal stability data, and Dr. Stuart Ince
and Dr. Hartmut Schirok for critical reading of the manuscript
and helpful discussions.
15. Wittig, G.; Schöllkopf, U. Chem. Ber. 1954, 87, 1318.
16. Schwesinger, R.; Schlemper, H. Angew. Chem., Int. Ed. 1987, 26, 1167.

N. Griebenow et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3648–3653
3653
17. Squalene synthase catalyses the reductive condensation of farnesyl
pyrophosphate to squalene during cholesterol biosynthesis. Turnover of
trans,trans-[1-3H]-farnesyl pyrophosphate to [3H]-squalene by squalene
mevalonolactone. One hour after mevalonolactone application, livers were
removed and the amount of hepatic
14C]-cholesterol synthesis was
[
determined after sterol extraction by liquid scintillation counting. See also:
Duncan, I. W.; Culbreth, P. H.; Burtis, C. A. J. Chromatogr. 1979, 162, 281.
20. Experimental data for 16d: 1H NMR (400 MHz, CDCl3): d = 1.36 (d, J = 6.9,
3H), 1.48–1.98 (m, 4H), 1.51 (d, J = 6.9, 3H), 2.49–2.58 (m, 1H), 2.79–3.02
(m, 2H), 3.07–3.20 (m, 2H), 3.44 (s, 3H), 3.46 (sept, J = 6.9, 1H), 3.84–3.89
(m, 4H), 4.32–4.46 (m, 1H), 5.23–5.37 (m, 1H), 5.87 (s, 1H), 6.71–6.73 (m,
1H), 6.91–6.95 (m, 1H), 7.14–7.18 (m, 1H), 7.24–7.35 (m, 3H). LC/MS (for
instrumentation and conditions see Ref. 21): 2.59 min, 100% (214 nm), m/
z = 569 [M+H]+.
synthase was determined under the following conditions: 0.03 lg
recombinant SQS, 1 mM NADPH, 5 mM DTT, 10% PBS, 10 mM sodium
fluoride, 0.5 mM MgCl2, pH 7.5. Test compounds were dissolved in DMSO
and added in defined concentrations. The assay was started by adding farnesyl
pyrophosphate (final conc.
pyrophosphate and incubated for 10 min at 37 °C. Subsequently, 100
solution was extracted with 200 l chloroform, 200 l methanol and 60
5 l
M) and 0.2 nM trans,trans-[1-3H]-farnesyl
ll
l
l
l
l
5 N sodium hydroxide and adjusted to 2 mM squalene. An aliquot of the
organic phase was mixed with scintillation fluid and measured for b-emission.
18. Griebenow, N.; Flessner, T.; Buchmueller, A.; Raabe, M.; Bischoff, H.; Kolkhof, P.
Bioorg. Med. Chem. Lett. 2011, 21, 2554.
19. The compounds were administered orally to NMRI-mice (n = 8) 1 h before
monitoring of the cholesterol synthesis started. Biosynthesis of [14C]-labeled
cholesterol and its precursors was monitored after ip application of [14C]-
21. Intrument: Micromass Quattro LCZ with HPLC Agilent Series 1100; column:
Phenomenex Synergi
2 l Hydro-RP Mercury, 20 Â 4 mm; eluent A: 1 l
water + 0.5 ml 50% formic acid; eluent B: 1 l acetonirile + 0.5 ml 50% formic
acid; gradient: 0.0 min 90% A?2.5 min 30% A?3.0 min 5% A?4.5 min 5% A;
flow rate: 0.0 min 1 ml/min?2.5 min/3.0 min/4.5 min 2 ml/min; oven: 50 °C;
UV detection: 208–400 nm.