Discovery of pyrrolopyridazines as novel DGAT1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.08.066

A new structural class of DGAT1 inhibitors was discovered and the structure-activity relationship was explored. The pyrrolotriazine core of the original lead molecule was changed to a pyrrolopyridazine core providing an increase in potency. Further exploration resulted in optimization of the propyl group at C7 and the discovery that the ester at C6 could be replaced by five-membered heterocyclic rings. The analogs prepared have DGAT1 IC ₅₀ values ranging from >10 μM to 48 nM.

Full text of DOI:10.1016/j.bmcl.2010.08.066

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6030–6033
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of pyrrolopyridazines as novel DGAT1 inhibitors
Brian M. Fox a, Kiyosei Iio b, Kexue Li a, Rebeka Choi a, Takashi Inaba b, Simon Jackson a, Shoichi Sagawa b,
Bei Shan a, Masahiro Tanaka b, Atsuhito Yoshida b, Frank Kayser a,
⇑
a Amgen Inc., Amgen South San Francisco, 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
b Japan Tobacco Inc., Central Pharmaceutical Research Institute, 1-1, Murasaki-cho Takatsuki, Osaka 569 1125, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A new structural class of DGAT1 inhibitors was discovered and the structure–activity relationship was
explored. The pyrrolotriazine core of the original lead molecule was changed to a pyrrolopyridazine core
providing an increase in potency. Further exploration resulted in optimization of the propyl group at C7
and the discovery that the ester at C6 could be replaced by five-membered heterocyclic rings. The analogs
Received 13 July 2010
Revised 12 August 2010
Accepted 12 August 2010
Available online 18 August 2010
prepared have DGAT1 IC50 values ranging from >10
lM to 48 nM.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
DGAT1 inhibitors
Pyrrolopyridazines
DGAT1
Acyl CoA:diacylglycerol acyltransferase
Acyl CoA:diacylglycerol acyltransferase (DGAT) is a microsomal
enzyme widely expressed in mammalian tissues that catalyzes the
esterification of 1,2-diacylglycerol with fatty acyl CoA to form
triglycerides at the endoplasmic reticulum.1,2 Two DGAT enzymes,
DGAT1 and DGAT2, have been identified.3–5 Both DGAT1 and
DGAT2 catalyze the formation of triglyceride from diacylglycerol
and fatty acyl CoA but they share no homology. In fact, DGAT1 is
part of the acyl CoA:cholesterol acyltransferase (ACAT) gene family
consisting of enzymes that synthesize cholesterol esters from cho-
lesterol and fatty acyl CoA substrates. Mouse DGAT1 shares 20%
identity with mouse ACAT1.3 Dgat1 null mice (Dgat1ꢀ/ꢀ) are lean
and resistant to diet-induced obesity.6 Total triglyceride and leptin
levels are lower in Dgat1ꢀ/ꢀ mice compared to their wild type lit-
termates and they have lower glucose and insulin levels after a glu-
cose tolerance test, indicating that Dgat1ꢀ/ꢀ mice have increased
insulin sensitivity. The totality of data indicates that loss of DGAT1
activity can counteract obesity and obesity-induced diabetes.1,6,7
Disorders or imbalances in triglyceride metabolism are impli-
cated in the pathogenesis of a variety of diseases and risk factors
including obesity, insulin resistance syndrome, type II diabetes,
dyslipidemia, metabolic syndrome and coronary heart disease.7–
erol to triglyceride by specifically inhibiting the activity of the hu-
man homolog of DGAT1 may find use in lowering corporeal
concentrations and absorption of triglycerides, thereby therapeuti-
cally counteracting the pathogenic effects caused by abnormal
metabolism of triglycerides in these disorders.
A high-throughput screen (HTS) of our small molecule library
resulted in the identification of pyrrolotriazine 1 as a novel, mod-
erately potent DGAT1 inhibitor (Fig. 1). Early work exploring cores
similar to pyrrolotriazine 1 resulted in the discovery of pyrrolopy-
ridazine 2a possessing increased DGAT1 potency, as well as im-
proved selectivity against ACAT1. We wished to improve the
selectivity over ACAT1 in order to study the effect of DGAT1 inhi-
bition without the need to deconvolute the contribution from each
enzyme in any potential in vivo results. This report will focus on
the synthesis of pyrrolopyridazine derivatives 2a and the evalua-
tion of their potency in DGAT1 biochemical15 and cellular assays16
and their selectivity in an ACAT1 biochemical assay.17
O
O
1
1
7
7
N
N
N
N
O
O
N
N
2
2
4
6
4
6
N
14
OEt
OEt
The finding that multiple enzymes catalyze the synthesis of tri-
OEt
OEt
glyceride from diacylglycerol provides the opportunity to modu-
late one catalytic mechanism of this biochemical reaction to
achieve therapeutic results in an individual with minimal adverse
side effects. Compounds that inhibit the conversion of diacylglyc-
O
O
1
2a
DGAT1 IC50 = 0.25 µM
ACAT1 IC50 = 0.5 µM
DGAT1 IC50 = 0.12 µM
ACAT1 IC50 = 1.3 µM
⇑
Corresponding author.
E-mail address: fkayser@amgen.com (F. Kayser).
Figure 1. Structures of original screening hit pyrrolotriazine 1 and pyrrolopyrid-
azine 2a.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.066

B. M. Fox et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6030–6033
6031
O
O
Preparation of analogs 2a–w (Table 1) and 9a–e (Table 2) pos-
CO2K
CN
sessing a phenyl group at C4 began by treatment of phenylmaleic
anhydride (5) with hydrazine followed by reaction with POCl3 to
provide dichloropyridazine 6 (Scheme 1). Displacement of the
chloride in the 6-position with secondary amines followed by
hydrogenation provided 5-phenylpyridazines 8. Selective N-alkyl-
ation with various halides followed by 1,3-dipolar addition of the
resulting ylide to diethyl acetylenedicarboxylate18 provided pyrro-
lopyridazine analogs 2a–w and 9a–e. The synthesis of analogs 9f–
h containing substituted phenyls at C4 of the pyrrolopyridazine
core required the preparation of arylmaleic anhydrides 5 that
was accomplished by the condensation of benzonitriles 3 with gly-
oxylic acid followed by the hydrolysis of the resulting nitriles 4
(Scheme 1). Conversion of anhydrides 5 into pyrrolopyridazines
9f–h was accomplished as described for analogs 2a–w and 9a–e.
a
c,d
b
CN
Cl
R'
R'
R'
R'
O
3
6
4
5
8
R1
R1
N
N
N
N
N
N
e
f
R'
N
R'
Cl
Cl
7
O
R
R1
N
N
O
O
N
N
g,h
OEt
OEt
OEt
OEt
R2
O
O
Table 1
DGAT1 and ACAT1 IC50 values of 2a–w
9a-h
2a-w
Compd
R
DGAT1 IC50
M)
ACAT1 IC50
M)
(
l
(
l
Scheme 1. Reagents and conditions: (a) glyoxylic acid, K2CO3, MeOH; (b) HCO2H,
H2SO4, reflux; (c) NH2NH2ꢁHCl, EtOH, H2O, reflux; (d) POCl3, reflux; (e) R1H, i-
Pr2NEt, dioxane, reflux; (f) HCO2NH4, Pd/C, MeOH, 48 °C; (g) RCH2X, CH3CN, reflux;
(h) diethyl acetylenedicarboxylate, TBAF, THF, EtOH, reflux.
2a
2b
2c
2d
2e
2f
2g
2h
2i
Propyl
Hydrogen
Methyl
Ethyl
0.12 0.06a
>3
1.3 0.04b
0.52
0.24
0.075
0.11
0.16
0.22
0.62
2.5
>1.0
0.24
0.27
>1
2.59
2.17
1.4
0.92
<0.3
0.3
Allyl
Preparation of analogs 9i–m containing alkyl groups at C4 of
the pyrrolopyridazine core began by alkylating 3,6-dichloropyrid-
azine (10) to provide 4-alkyl-3,6-dichloropyridazines 11 (Scheme
2).19 Conversion of pyridazines 11 to the corresponding 4-
alkylpyrrolopyridazines 9i–m was accomplished using the chemis-
try shown in Scheme 1.
Selective hydrolysis of 2a using NaOH in EtOH and H2O pro-
vided the carboxylate. Subsequent conversion to the acid chloride,
displacement with hydroxyamidines and cyclization in the pres-
ence of TBAF provided 1,2,4-oxadiazoles 12a–n (Scheme 3).
Systematic exploration of the pyrrolopyridazine SAR included
replacing the 7-propyl group of 2a with various alkyl and aryl
groups to provide analogs 2b–w (Table 1). Propyl was found to
be the optimal chain length for DGAT1 potency although larger
and more lipophilic groups such as trifluoropropyl 2f, butyl 2g
and pentyl 2h had similar IC50 values. Sterically more demanding
3,3,3-Trifluoropropyl
Butyl
Pentyl
Cyclopropyl
Cyclobutyl
3-Hydroxypropyl
Methoxymethyl
3-Methoxypropyl
3(N,N-
3.25
2j
2kc
2l
8.75
2.56
2m
2nc
Dimethylamino)propyl
Phenyl
2o
2p
2q
2r
0.081
0.47
0.29
0.068
0.57
0.052
0.63
0.55
0.47
0.31
0.22
1.29
0.6
1.05
<0.3
<0.1
3-OMe-phenyl
3-Cl-phenyl
4-Cl-phenyl
4-OH-phenyl
4-OMe-phenyl
4-NH2-phenyl
4-Ph-phenyl
4-OBn-phenyl
2sd
2t
2ue
2v
0.25
0.060
2w
a
b
c
n = 17.
n = 2.
Hydroboration oxidation of 2e provided 2k followed by mesylation and dis-
Cl
Cl
O
placement with dimethylamine providing 2n.
N
N
d
O
Hydrogenolysis of 2w provided 2s.
N
N
N
N
N
a
b,c,d,e
e
Hydrogenation of the corresponding nitroaryl provided 2u.
R2
OEt
R2
Cl
Cl
OEt
O
10
11
9i-m
Table 2
DGAT1 and ACAT1 IC50 values of 9a–m
Scheme 2. Reagents and conditions: (a) R2CO2H, H2SO4, AgNO3, H2O, (NH4)2S2O8,
72 °C; (b) morpholine, i-Pr2NEt, dioxane, reflux; (c) HCO2NH4, Pd/C, MeOH, 48 °C;
(d) butyl iodide, CH3CN, reflux; (e) diethyl acetylenedicarboxylate, TBAF, THF, EtOH,
reflux.
Compd
R1
R2
DGAT1
IC50 M)
ACAT1
IC50 (lM)
(l
2a
9a
Morpholin-4-yl
cis-2,6-Dimethyl-
morpholin-4-yl
Azetidin-1-yl
Phenyl
Phenyl
0.12 0.06
>3
1.3 0.04
9b
9c
9d
9e
9f
9g
9h
9i
Phenyl
Phenyl
Phenyl
Phenyl
2-F-phenyl
3-F-phenyl
4-F-phenyl
Propyl
1.36
0.12
6.1
0.83
0.57
0.27
0.29
>3
>3
>3
>3
>3
1.49
1.3
O
O
Pyrrolidin-1-yl
Piperidin-1-yl
Dimethylamino
Morpholin-4-yl
Morpholin-4-yl
Morpholin-4-yl
Morpholin-4-yl
Morpholin-4-yl
Morpholin-4-yl
Morpholin-4-yl
Morpholin-4-yl
R
N
N
N
N
O
N
O
N
N
0.66
1.49
0.38
0.99
a,b,c,d
N
OEt
OEt
OEt
O
O
9j
9k
9l
t-Butyl
2a
12a-n
Cyclobutyl
Cyclopentyl
Cyclohexyl
Scheme 3. Reagents and conditions: (a) NaOH, EtOH, H2O, reflux; (b) (COCl)2, DMF
(cat), CH2Cl2; (c) RC(@NOH)NH2, NEt3, THF; (d) TBAF, THF.
9m

6032
B. M. Fox et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6030–6033
Table 3
alkyl groups such as cyclopropyl 2i and cyclobutyl 2j were less pre-
ferred in this position. The 7-ethyl derivative 2d also retained
activity but shortening the 7-alkyl group to methyl 2c and deleting
the group altogether 2b caused a significant loss in DGAT1 inhibi-
tory activity. Allyl analog 2e was the most potent in the alkyl ser-
ies, possibly due to its increased ability to form van der Waals
DGAT1, CaCO2 and ACAT1 IC50 values of 12a–n
Compd
R
DGAT1
CaCO2
IC50
ACAT1
IC50 (lM)
IC50
(lM)
(
l
M)
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
Methyl
Ethyl
Propyl
i-Propyl
Butyl
t-Butyl
Pentyl
Phenyl
0.38
0.11
<1
0.065
0.057
0.073
0.048
0.11
0.18
0.12
0.048
0.056
0.10
0.26
<1
<1
0.083
<1
interactions through its diffuse p-electron cloud.
0.051
0.069
0.019
Heteroatoms were tolerated in the alkyl chain as demonstrated
by the DGAT1 IC50 values of ethers 2l and 2m; however, introduc-
tion of significant polarity as with an alkyl alcohol 2k or alkyl
amine 2n completed abrogated DGAT1 inhibitory activity.
Replacement of the propyl group in 2a by aryl groups was well
tolerated and in fact the para-methoxyphenyl derivative 2t is the
most potent analog in the series. para-Substitution was preferred
over meta-substitution in all cases tested as exemplified by meta-
methoxyphenyl derivative 2p being about 10-fold less potent than
2t. The para-position of the phenyl ring tolerated a wide variety of
substituents excluding polar groups as shown by the reduced
inhibitory activity of the phenol 2s and the aniline 2u.
0.2
1.59
0.077
0.23
0.69
0.77
0.19
1.05
Benzyl
Cyclopentyl
Cyclohexyl
Pyrrolidin-1-yl
Dimethylamino
2-OH-ethyl
<0.03
0.031
0.10
0.14
0.18
0.26
Early selectivity studies indicated that pyrrolotriazine 1 and
pyrrolopyridazine 2a did not inhibit DGAT2; whereas, they po-
tently inhibited ACAT1. Pyrrolopyridazine 2a is about 11-fold
selective for DGAT1 over ACAT1. Replacement of the 7-propyl
group of 2a with the methoxymethyl group in 2l provided 36-fold
DGAT1 selectivity compared to ACAT1 and was the most selective
compound prepared. The para-methoxyphenyl analog 2t and the
allyl analog 2e also had improved selectivities of 12- and 19-fold,
respectively. Alterations of the morpholine at C2 failed to provide
an increase in DGAT1 selectivity. Pyrrolidine 9c had similar DGAT1
selectivity compared to 2a while the dimethylamino derivative 9e
showed increased activity for ACAT1 over DGAT1. The oxadiazole
analogs also had a range of DGAT1 selectivities including nine-
and 12-fold selectivity for phenyl analog 12h and cyclohexyl ana-
log 12k, respectively; whereas, benzyl analog 12i possessed a low-
er IC50 for ACAT1 than for DGAT1.
The bulk of the data indicates that the optimal groups at C7 are
hydrophobic and capable of forming van der Waals interactions.
Any significant polarity dramatically reduces DGAT1 inhibitory
activity. Furthermore, this area of the binding pocket is able to
accommodate very large groups as shown by analog 2w, which
possesses a para-benzyloxyphenyl group. There may however be
a fairly narrow channel as analogs that force groups orthogonal
to the phenyl ring such as 2v or have any branching close to the
pyrrolopyridazine core such as cycloalkyl derivatives 2i and 2j
have significantly reduced DGAT1 IC50 values.
Changes at C2 of pyrrolopyridazine 2a proved to be much less tol-
erated than at C7. This area of the molecule appeared to have very
strict steric parameters in order to retain DGAT1 inhibitory activity.
Simply replacing the morpholino ring of 2a with a piperidinyl group
such as in 9d resulted in a 50-fold reduction in potency. Similarly,
2,6-dimethylmorpholine derivative 9a proved to be at least 24-fold
less potent than 2a. In contrast, sterically less demanding groups
such as pyrrolidine analog 9c retained potency while azetidine 9b
and dimethylamine derivative 9e were moderately potent.
All alkyl replacements of the phenyl group at C4 of 2a provided
analogs that were unable to inhibit DGAT1 activity at concentra-
tions up to 3 lM (analogs 9i–m). Furthermore, replacement of
any of the phenyl hydrogens of 2a resulted in decreased DGAT1
inhibitory activity (analogs 9f–h).
There are many heterocyclic bioisosteric replacements for es-
ters reported in the literature.20 We evaluated the ability of 5-
methyloxazole, 5-methyl-1,3,4-oxadiazole and 3-methyl-1,2,4-
oxadiazole to act as such replacements for the ethyl ester at C6
of pyrrolopyridazine 2a. All three of these heterocycles possessed
some DGAT1 inhibitory activity with 3-methyl-1,2,4-oxadiazole
12a being the most potent inhibitor (data not shown). Further
exploration of the SAR of the 1,2,4-oxadiazoles resulted in com-
pounds 12b–n (Table 3).
In summary, novel DGAT1 inhibitor pyrrolotriazine 1 was dis-
covered. Design of pyrrolopyridazine 2a and systematic investiga-
tion of the SAR resulted in the finding that groups having
p-
electron density such as allyl, phenyl, and 4-substituted phenyls
increased DGAT1 potency relative to the original propyl group at
C7 of 2a. The morpholino substituent at C2 and the phenyl ring
at C4 of pyrrolopyridazine 2a proved to be optimal within the ser-
ies described in this report. It was discovered that the ethyl ester at
C6 could be replaced by oxadiazoles 12 resulting in an increase in
DGAT1 inhibitory activity. In fact, the t-butyl 12f and cyclopentyl
12j analogs are the most potent DGAT1 inhibitors described in this
report with DGAT1 IC50 values of 48 nM. Selectivity for DGAT1 over
ACAT1 was modestly increased for methoxymethyl analog 2l and
allyl analog 2e but for the majority of compounds selectivity did
not improve.21
References and notes
Propyl analog 12c was the most potent inhibitor of the un-
branched alkyl derivatives with the butyl derivative 12e being
essentially equipotent. Longer chains such as pentyl 12g or shorter
chains such as ethyl 12b caused DGAT1 inhibitory activity to de-
crease slightly while the methyl derivative 12a was about a sixfold
weaker DGAT1 inhibitor. Branched and cyclic alkyl groups proved
to be quite potent with the t-butyl 12f and cyclopentyl 12j analogs
possessing DGAT1 IC50 values of 48 nM. Compounds possessing
hydrophilic groups such as amines 12l and 12m, as well as hydro-
xyl groups 12n showed decreased DGAT1 inhibitory activity. The
oxadiazoles were evaluated in a DGAT1 cellular assay16 to deter-
mine the amount of triglyceride uptake into CaCO2 cells. All of
the oxadiazole analogs tested in the cell-based assay possessed
similar IC50 values to that determined in the biochemical assay.
1. Chen, H. C.; Farese, R. V., Jr. Trends Cardiovasc. Med. 2000, 10, 188.
2. Farese, R. V., Jr.; Cases, S.; Smith, S. J. Curr. Opin. Lipidol. 2000, 11, 229.
3. Cases, S.; Smith, S. J.; Zheng, Y.-W.; Myers, H. M.; Lear, S. R.; Sande, E.; Novak, S.;
Collins, C.; Welch, C. B.; Lusis, A. J.; Erickson, S. K.; Farese, R. V., Jr. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 13018.
4. Lardizabal, K. D.; Mai, J. T.; Wagner, N. W.; Wyrick, A.; Voelker, T.; Hawkins, D. J.
J. Biol. Chem. 2001, 276, 38862.
5. Cases, S.; Stone, S. J.; Zhou, P.; Yen, E.; Tow, B.; Lardizabal, K. D.; Voelker, T.;
Farese, R. V., Jr. J. Biol. Chem. 2001, 276, 38870.
6. Smith, S. J.; Cases, S.; Jensen, D. R.; Chen, H. C.; Sande, E.; Tow, B.; Sanan, D. A.;
Raber, J.; Eckel, R. H.; Farese, R. V., Jr. Nat. Genet. 2000, 25, 87.
7. Chen, H. C.; Smith, S. J.; Ladha, Z.; Jensen, D. R.; Ferreira, L. D.; Pulawa, L. K.;
McGuire, J. G.; Pitas, R. E.; Eckel, R. H.; Farese, R. V., Jr. J. Clin. Invest. 2002, 109,
1049.
8. Kahn, R. C. Nat. Genet. 2000, 25, 6.
9. Yanovski, S. C.; Yanovski, J. A. N. Eng. J. Med. 2002, 346, 591.
10. Lewis, G. F.; Carpentier, A.; Adeli, K.; Giacca, A. Endocr. Rev. 2002, 23, 201.
11. Brazil, M. Nat. Rev. Drug Disc. 2002, 1, 408.

B. M. Fox et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6030–6033
6033
12. Malloy, M. J.; Kane, J. P. Adv. Inter. Med. 2001, 47, 111.
13. Subauste, A.; Burant, C. F. Curr. Drug Targets Immune Endocr. Metabol. Disord.
2003, 3, 263.
0.5 ml hexane/isopropyl alcohol (3:2 v/v) for 5 min twice. Lipid extracts were
evaporated to dryness, dissolved in chloroform and the lipid classes were
resolved by TLC using hexane/ether/glacial acetic acid (80:20:1 v/v/v). The
14. Yu, Y.-H.; Ginsberg, H. Ann. Med. 2004, 36, 252.
plates were exposed to a phosphorimager screen and the amount of
15. DGAT1 was cloned from a human liver cDNA library. PCR was used to add a
restriction site and flag epitope at the most 50end and a restriction site at the 30
end of the sequence. Thereafter, human flagtag (FT) DGAT1 baculovirus was
generated using a Bac-to-Bac Baculovirus Expression SystemÒ (Invitrogen).
Insect cells (e.g., sf9, sf2 1, or High Five) were infected for 24–72 h and collected
by centrifugation. Cell pellets were re-suspended in homogenization buffer and
lysed using a homogenization device, such as a microfluidizer. Total cell
membranes were collected by ultracentrifugation at 45,000 rpm for 1 h. A small
radioactivity incorporated in to the triglyceride fraction was determined.
17. Full length human ACAT1 was cloned and modified by PCR to add a restriction
site and flag epitope at the most 50 end and a restriction site at the 30 end of the
sequence. Thereafter, human flagtag (FT) ACAT1 baculovirus was generated
using a Bac-to-Bac Baculovirus Expression SystemÒ (Invitrogen). Insect cells,
sf21, were infected for 24–72 h and collected by centrifugation. Cell pellets
were re-suspended in homogenization buffer and lysed using
homogenization device, such as a microfluidizer. Total cell membranes were
collected by ultracentrifugation at 45,000 rpm for 1 h. Membrane (0.5–2 g)
a
aliquot (0.2
of compound or mercuric chloride (as positive control for inhibition) in the
presence of enzyme substrate, dioleoyl glycerol (200 M) in 384-well plates,
final volume 50 L per well. The reaction was started by the addition of
radioactive substrate, 14C acyl coenzyme A (25
M, such as decanoyl CoA,
l
g/well) of membrane was incubated with varying concentrations
l
was incubated with varying concentrations of compound or SA-58035 (5–
50 nM, as positive control for inhibition) in the presence of substrate,
l
l
cholesterol/cyclodextrin (500
volume of 100 L. The reaction was started by the addition of radioactive
substrate, 14C palmitoyl CoA (25
M), and incubated at ambient temperature
lM cholesterol), in microfuge tubes at a final
l
l
palmitoyl CoA, oleoyl CoA), and incubated at room temperature for 2 h. The
reaction was stopped by adding Wheat Germ Agglutinin (WGA) SPA beads
(0.2 mg) in mercuric chloride. Cell membranes were allowed to couple to the
beads overnight. The signal was measured using a TopCount device.
l
for 10 min. The reaction was stopped by adding chloroform (1 mL) and
vortexing. After brief centrifugation, the upper aqueous phase was removed
and the organic phase applied to an aminopropyl Sep-Pak cartridge.
Radioactive product, cholesterol palmitate, was eluted with chloroform
(1 mL) into a scintillation vial and counted.
16. Human colon tumor CaCO2 cells were cultured until confluent in 24-well
plates. The medium was replaced with serum-free medium and the cells
incubated for a further 24 h. Medium was replaced with serum-free medium
18. Kanemasa, S.; Kobira, S.; Kajigaeshi, S. Heterocycles 1980, 14, 1107.
19. Samaritoni, J. G. Org. Prep. Proced. Int. 1988, 20, 117.
20. Patani, G. A.; LaVoie, E. J. Chem. Rev. 1996, 96, 3147.
21. ACAT1 activity would presumably not pose a problem in a clinical setting, see
example, Chang, C.; Dong, R.; Miyazaki, A.; Sakashita, N.; Zhang, Y.; Liu, J.; Guo,
M.; Li, B.-L.; Chang, T.-Y. Acta Biochim. Biophys. Sin. 2006, 38, 151.
containing 400
compound at varying doses in a final volume of 200
incubated for 30 min before adding 0.1
Ci of 14C oleic acid directly to the cells
lM
oleic acid (complexed with BSA, 2:1 mol/mol) and
ll per well. Cells were
l
and the incubation continued for 30 min. Cells were washed two times with
1 ml PBS and air-dried at 37 °C for 10 min. Cell lipids were extracted with