224
U. Galli et al. / Bioorg. Med. Chem. Lett. 17 (2007) 220–224
Chem. Soc. 1972, 94, 7154; (c) Stephenson, L. M.; Speth,
D. R. J. Org. Chem. 1979, 44, 4683.
7. Fairlamb, I. J. S.; Dickinson, J. M.; Pegg, M. Tetrahedron
Lett. 2001, 42, 2205.
8. (a) Needles, H. L.; Whitfield, R. E. J. Org. Chem. 1966, 31,
HPLC analysis using a SYNERGI MAX-RP 80A
(150 · 4.6 mm, 4 lm particle size) column on a Shimadzu
HPLC system. Solvents for the separation were: solvent A:
1
1
water (0.01% w/v H
w/v H PO ) at a flow rate of 0.7 ml/min and a sample
3 4
PO ) ; solvent B: acetonitrile (0.01%
3
4
9
89; (b) Woodbury, R. P.; Rathke, M. W. J. Org. Chem.
injection volume of 20 ll, k = 220 nm. For all compounds,
eluants A and B were delivered isocratically at a 20:80
ratio. All four compounds displayed a purity of at least
95%.
1977, 42, 1688; (c) Woodbury, R. P.; Rathke, M. W.
J. Org. Chem. 1978, 43, 881.
+
9. Compound 6: yellow oil. MS (ESI) m/z: 226 (M+H) ; H
1
1
NMR (300 MHz, CDCl ): d 5.35 (br t, J = 6.87 Hz, 1H),
21. The S. cerevisiae strains: SMY8[pBJ1.21], expressing the
OSC of T. cruzi; SMY8[pSM61.21], expressing the wild-
type yeast OSC; SMY8[pBJ4.21], expressing the P. carinii
OSC were kindly provided by Professor S. P. T. Matsuda
[Department of Chemistry and Biochemistry and Cell
Biology, Rice University, Houston, TX, USA. For refer-
ences and cultural conditions see Refs. 11, 13, and 24. The
enzymatic activity was determined as described in Refs. 13
and 24.
3
5.07 (br t, J = 6.60 Hz, 1H), 4.10 (d, J = 6.90 Hz, 2H), 2.96
(
br s, OH), 2.22 (br s, 8H), 2.08–1.96 (m, 4H), 1.91 (t,
1
3
J = 7.68 Hz, 2H), 1.64 (s, 3H), 1.55 (br s, 5H); C NMR
75 MHz, CDCl ): d 138.5, 134.9, 124.4, 124.3, 59.3, 59.1,
(
3
4
5.3, 39.5, 37.2, 26.2, 25.5, 16.3, 16.2.
0. Compound 7: yellow oil. MS (ESI) m/z: 344 (M+H) ; H
NMR (300 MHz, CDCl ): d 7.30–7.10 (m, 5-H), 5.33 (t,
+
1
2
3
J = 6.86 Hz, 1H), 5.11 (t, J = 5.76, 1H), 3.97 (d,
J = 6.60 Hz, 2H), 3.42 (t, J = 6.30 Hz, 2H), 2.69 (t,
J = 7.40 Hz, 2H), 2.30 (br s, 8H), 2.11–1.89 (m, 6H),
22. Ruf, A.; Muller, F.; D’Arcy, B.; Stihle, M.; Kusznir, E.;
Handschin, C.; Morand, O. H.; Thoma, R. Biochem.
Biophys. Res. Commun. 2004, 315, 247.
1
3
1.87 (q, J = 6.33 Hz, 2H), 1.66 (br s, 5H), 1.59 (s, 3H);
C
NMR (75 MHz, CDCl ): d 142.1, 140.0, 135.1, 128.6,
23. Pichia pastoris cells expressing human OSC were kindly
provided by Ralf Thoma from Hoffmann-La Roche Ltd,
Pharma Research Discovery (CH-4070 Basel, Switzer-
land). Cultural conditions were as in Ref. 22 and cell-free
homogenates were prepared as described for S. cerevisiae
cells in Ref. 13. Enzymatic assays were determined in
phosphate buffer, pH 8, at 45 ꢁC as described in Ref. 13.
24. Oliaro-Bosso, S.; Viola, F.; Matsuda, S. T. P.; Cravotto,
G.; Tagliapietra, S.; Balliano, G. Lipids 2004, 39, 1007.
25. Thoma, R.; Schulz-Gasch, T.; D’Arcy, B.; Benz, J.; Aebi,
J.; Dehmlow, H.; Hennig, M.; Stihle, M.; Ruf, A. Nature
2004, 432, 118.
3
1
3
28.4, 125.8, 124.1, 121.1, 69.4, 67.4, 59.6, 45.6, 39.7, 37.5,
2.5, 31.5, 26.3, 26.1, 16.6, 16.0. Compound 8: yellow oil.
+
1
MS (ESI) m/z: 394 (M+H) ; H NMR (300 MHz, CDCl ):
3
d 8.05 (d, J = 7.44 Hz, 1H), 7.84 (dd, J = 7.14/2.2 Hz, 1H),
7.70 (d, J = 7.68, 1H), 7.52–7.32 (m, 4H), 5.37 (br t,
J = 5.49 Hz, 1H), 5.11 (br t, J = 5.76 Hz, 1H), 3.99 (d,
J = 6.90 Hz, 2H), 3.48 (t, J = 6.30 Hz, 2H), 3.45 (t,
J = 7.60 Hz, 2H), 2.41 (br s, 8H), 2.10–1.90 (m, 8H),
1
3
1
.66 (br s, 5H), 1.60 (s, 3H); C NMR (75 MHz, CDCl
3
):
d 139.8, 138.2, 134.1, 133.9, 132.0, 128.8, 126.7, 126.2,
1
4
25.8, 125.6, 125.5, 125.0, 124.0, 121.2, 69.6, 67.5, 58.8,
4.5, 39.6, 37.0, 30.8, 29.6, 26.3, 24.4, 16.6, 15.9. Com-
1
4
26. Incorporation of [2- C]acetate into cholesterol and
biosynthetic intermediates was determined in 3T3 fibro-
blasts (ATCC CCL 92 3T3 Swiss albino) as previously
+
1
pound 9: yellow oil. MS (ESI) m/z: 420 (M+H) ; H NMR
300 MHz, CDCl ): d 7.58 (d, J = 8.25 Hz, 2H), 7.51 (d,
J = 8.28 Hz, 2H), 7.42 (t, J = 7.35, 2H), 7.34–7.32 (m, 1H),
(
3
4
described in Ref. 4d. Briefly, 7 · 10 cells were suspended
7.27 (d, J = 7.95, 2H), 5.38 (t, J = 6.75 Hz, 1H), 5.12 (t,
and grown for 24 h in 950 ll DMEM containing 10%
(v/v) lipid-depleted serum. After a 3 h preincubation in
the presence of inhibitor, labeled acetate (1 lCi/well) was
added. After further 3 h incubation, the medium was
removed and the cells were lysed with 500 ll of 0.1 N
NaOH for 30 min. After saponification with 1.5 ml of
10% (w/v) methanolic KOH for 1 h at 80 ꢁC, nonsapon-
ifiable lipids were extracted with petroleum ether and
separated on TLC plates (Alufolien Kieselgel 60F254,
Merck, Darmstadt, Germany) using n-hexane/ethyl ace-
tate (85:15 v/v) as a developing solvent and authentic
standards of cholesterol, lanosterol, dioxidosqualene,
oxidosqualene, and squalene as reference compounds.
Radioactivity in separated bands was measured using a
System 200 Imaging Scanner (Hewlett-Packard, Palo
Alto, CA, USA).
J = 5.82 Hz, 1 H), 3.99 (d, J = 6.72 Hz, 2H), 3.46 (t,
J = 6.42 Hz, 2H), 2.74 (t, J = 7.35 Hz, 2H), 2.25 (br s,
1
3
8H), 2.16–1.90 (m, 8H), 1.68 (s, 3H), 1.60 (br s, 5H);
C
3
NMR (75 MHz, CDCl ): d 141.3, 141.2, 140.0, 138.8,
134.9, 129.0, 128.8, 127.2, 127.1, 124.3, 121.1, 69.4, 67.4,
5
9.4, 45.4, 39.7, 37.4, 32.2, 31.5, 26.3, 25.8, 16.6, 16.0.
+
1
Compound 10: yellow oil. MS (ESI) m/z: 310 (M+H) ; H
NMR (300 MHz, CDCl ): d 5.32 (t, J = 6.60 Hz, 1H), 5.08
t, J = 6.33, 1H), 3.94 (d, J = 6.87 Hz, 2H), 3.38 (t,
J = 6.87 Hz, 2 H), 2.22 (br s, 8H), 2.10–2.01 (m, 4H),
3
(
1
1
.95 (t, J = 7.68 Hz, 2H), 1.64 (s, 3H), 1.60–1.50 (m, 7H),
.36–1.23 (m, 6H), 0.86 (br t, J = 6.87 Hz 3H); C NMR
1
3
(
75 MHz, CDCl ): d 139.8, 134.9, 124.2, 121.2, 70.5, 67.3,
3
5
1
9.5, 45.4, 39.6, 37.4, 31.8, 29.9, 26.3, 26.0, 25.8, 22.7, 16.6,
6.0, 14.2. Purity of target compounds was checked by