Exploration of pyridine containing heteroaryl analogs of biaryl ureas as DGAT1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.07.109

The diacylglycerol acyltransferase enzyme, DGAT1, presents itself as a potential target for obesity as this enzyme is dedicated to the final committed step in triglyceride biosynthesis. Biphenyl ureas, exemplified by compound 4, have been reported to be potent hDGAT1 inhibitors. We have synthesized and evaluated 2-pyridyl and 3-pyridyl containing biaryl ureas as hDGAT1 inhibitors. Our aim was to incorporate a heteroaryl scaffold within these molecules thereby improving the c Log P profile and making these compounds more drug-like. Compounds within this series exhibited potent hDGAT1 inhibition when evaluated using an in vitro enzymatic assay. Selected compounds were also subjected to an oral fat tolerance test in mice where the percent triglyceride reduction versus a vehicle control was evaluated. Of the studied heteroaryl analogs compound 44 exhibited an in vitro IC₅₀ of 17 nM and a plasma triglyceride reduction of 79% along with a 12-fold improvement in solubility over the biphenyl urea compound 4.

Full text of DOI:10.1016/j.bmcl.2011.07.109

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5812–5817
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploration of pyridine containing heteroaryl analogs of biaryl ureas
as DGAT1 inhibitors
Hashim Motiwala a, Shivaji Kandre a, Vishal Birar a, Kishorkumar S. Kadam a, Atish Rodge a,
Ravindra D. Jadhav a, M. Mahesh Kumar Reddy b, Manoja K. Brahma b, Nitin J. Deshmukh b, Amol Dixit b,
a
a
b
Lalit Doshi b, Amol Gupte a, , Ashok K. Gangopadhyay , Ram A. Vishwakarma , Shaila Srinivasan ,
⇑
Mamta Sharma b, Kumar V.S. Nemmani b, Rajiv Sharma a
a Department of Medicinal Chemistry, Piramal Life Sciences Limited, Goregaon (E), Mumbai 400 063, Maharashtra, India
b Department of Pharmacology, Piramal Life Sciences Limited, Goregaon (E), Mumbai 400 063, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The diacylglycerol acyltransferase enzyme, DGAT1, presents itself as a potential target for obesity as this
enzyme is dedicated to the final committed step in triglyceride biosynthesis. Biphenyl ureas, exemplified
by compound 4, have been reported to be potent hDGAT1 inhibitors. We have synthesized and evaluated
2-pyridyl and 3-pyridyl containing biaryl ureas as hDGAT1 inhibitors. Our aim was to incorporate a het-
eroaryl scaffold within these molecules thereby improving the c Log P profile and making these com-
pounds more drug-like. Compounds within this series exhibited potent hDGAT1 inhibition when
evaluated using an in vitro enzymatic assay. Selected compounds were also subjected to an oral fat tol-
erance test in mice where the percent triglyceride reduction versus a vehicle control was evaluated. Of
the studied heteroaryl analogs compound 44 exhibited an in vitro IC50 of 17 nM and a plasma triglyceride
reduction of 79% along with a 12-fold improvement in solubility over the biphenyl urea compound 4.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 22 March 2011
Revised 11 July 2011
Accepted 28 July 2011
Available online 4 August 2011
Keywords:
Diacylglycerol acyltransferase (DGAT)
Obesity
Triglycerides
Fat tolerance test (FTT)
The World Health Organization (WHO) defines obesity as a con-
dition characterized by abnormal or excessive fat accumulation
that presents health risks to an individual. Obesity is associated
with an array of additional health problems, including increased
risk of insulin resistance, type 2 diabetes, fatty liver disease, ath-
erosclerosis, hypertension, and cardiovascular disease.1 The WHO
estimates that more than a billion adults worldwide are over-
weight, of whom 300 million are clinically obese.2 Clinical obesity
is one where an individual possesses a body mass index (BMI)
One of the strategies for development of anti-obesity drugs in-
cludes a reduction in energy intake through either central or
peripheral mechanisms. Orlistat, the only marketed anti-obesity
drug prevents absorption of fat from human diet thereby reducing
the intake of calories.4 However, this drug suffers from severe ad-
verse effects such as steatorrhea, fecal incontinence, and frequent
or urgent bowel movements.5 Hence there is a pressing need for
developing novel anti-obesity drugs possessing fewer side-effects
and targeting newer mechanisms of action. The Acyl CoA:diacyl-
glycerol acyltransferase 1 (DGAT1) is one of two known DGAT en-
zymes (DGAT1 and DGAT2) that catalyze the final committed step
of triglyceride biosynthesis.6 Of late, several potent inhibitors of
the hDGAT1 enzyme have been reported and some of these com-
pounds have also been studied clinically (Fig. 1).7–9 This particular
interest in hDGAT1 inhibition arises from a published phenotype of
DGAT1 deficient mice.10 These animals are viable, resistant to
weight gain when fed a high-fat diet, and exhibit increased insulin
and leptin sensitivity. Contrary to DGAT1, DGAT2 deficient mice
die soon after birth apparently from profound reductions in sub-
strates required for energy metabolism coupled with an impaired
permeability barrier function in the skin. Hence inhibition of
DGAT1 is an attractive target for developing anti-obesity agents.
A potent series of biphenyl keto acid derivatives, exemplified by
structure 3, for developing anti-obesity agents have been reported
by Bayer Pharmaceuticals.9 The reported IC50 value of compound 3
equal to or greater than 30 kg mÀ2 3
. It results from an energy
imbalance arising when the energy input exceeds energy output.
In humans, the excess calories consumed are converted into tri-
glycerides that are principal components of fat and in turn act as
a major repository for stored metabolic energy. Obese individuals
have significantly greater triglyceride levels. Because this ability
to make triglycerides is essential for the accumulation of adipose
tissue, inhibition of triglyceride synthesis may act as a strategy
to either prevent or reverse obesity and its related medical
consequences.
⇑
Corresponding author. Address:
Highway, Goregaon (E), Mumbai 400 063, India. Tel.: +91 22 30818312; fax: +91
22 30818036.
1 Nirlon Complex, Off Western Express
E-mail address: amol.gupte@piramal.com (A. Gupte).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.109

H. Motiwala et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5812–5817
5813
O
OH
O
OH
OH
O
O
NH2
N
O
NH2
N
F
N
N
O
N
N
N
S
N
H
F
O
3, BAY-744113
hDGAT1 IC50: 73 nM
2
, JTT-553
Figure 1. Few hDGAT1 inhibitors studied clinically.
1
, PF-4620110
is 73 nM when studied using an hDGAT1 enzymatic assay.11 Abbott
Laboratories discovered compound 4 by replacing the benzothia-
zole moiety of compound 3 with a phenyl urea resulting in
improved potency (IC50 = 7 nM).11 A series of similar compounds
have also been patented by Abbott Laboratories.12,13 Compound 4
was synthesized in-house using a reported procedure11 and in
our hands it exhibited an IC50 value of 20 nM.
modification was retained while synthesizing analogs displaying
the 2-pyridyl and 3-pyridyl orientations required in the study.
The synthesis of 3-pyridyl heteroaryl analogs retaining L-valine
as the acid head group were conducted similar to that of com-
pound 25 using either appropriately substituted phenyl isocya-
nates to obtain compounds 28 and 29 or a benzyl isocyanate to
yield compound 30.
We have investigated the effects of replacing phenyl ring ‘A’
within the biphenyl scaffold of compound 4 with a pyridine ring
(Fig. 2). This modification would result in reduced Log P of these
molecules. The pyridine ring can exist in either the 3-pyridyl orien-
tation, exemplified by structure 5, or the 2-pyridyl orientation,
exemplified by structure 6, as shown in Figure 2 and hence both
these orientations have been studied. Inspired by an earlier patent
filed by Bayer Pharmaceuticals14 we have also explored several
amino acid head groups for this heteroaryl scaffold. Uncharged po-
lar amino acid head groups such as serine and nonpolar amino acid
head groups such as glycine, alanine, valine, and leucine have been
studied. The selections of these amino acids were based on their
hydrophobicities, contributions towards Log P, and molecular
weights of the resulting analogs. Our goal was to develop a hetero-
aryl scaffold that allows for developing drug-like candidates while
retaining hDGAT1 inhibition.
To synthesize phenyl urea analogs of the 3-pyridyl heteroaryl
scaffold possessing different amino acid head groups (Scheme 1),
4-bromo benzoic acid (7) was subjected to esterification using sul-
furic acid and methanol to yield compound 8. This was further con-
verted to its corresponding boronic acid pinacol ester, compound 9,
using a palladium catalyzed reaction incorporating bispinacol
diboron. Compound 9 was then subjected to Suzuki coupling with
2-amino-5-bromopyridine yielding compound 10. Compound 10
was subsequently coupled with a substituted isocyanate to yield
the corresponding substituted urea derivative 11, followed by an
ester hydrolysis yielding 12. Compound 12 was coupled with a
variety of amino acid esters to yield compounds 13–21. Finally
compounds 13–21 were hydrolyzed to their corresponding acids
22–30 using lithium hydroxide. A total of six different amino acids
To synthesize the 2-pyridyl analogs (Scheme 2), compound 9
was subjected to Suzuki coupling with 2-bromo-5-nitropyridine
yielding compound 31. This was subsequently hydrolyzed using
lithium hydroxide to yield compound 32. Compound 32 was cou-
pled with the methyl ester of L-valine to yield compound 33.
Metallic reduction of the nitro group in compound 33 using Fe/
NH4Cl gave its corresponding amine 34. Compound 34 was then
coupled with various substituted phenyl isocyanates to yield com-
pounds 35–41 that on subsequent hydrolysis with lithium hydrox-
ide yielded the corresponding acids 42–48. These synthesized
compounds were studied for their hDGAT1 inhibitory activity
using an in vitro enzymatic assay that measures the output of tri-
olein from diolein and radiolabeled oleoyl-CoA.15 Results obtained
from these studies are discussed ahead.
The in vitro activities for different acid head groups (Table 1)
varied from 28% for
(compound 25) at a 10
groups with the exception of
less than 75% and were hence not subjected to IC50 evaluations.
The -valine head group, yielding the S enantiomer (compound
L
-serine (compound 24) to 88% for
M concentration. All of the acid head
-valine exhibited hDGAT1 inhibition
L-valine
l
L
L
25), displayed an IC50 of 120 nM for hDGAT1 enzyme. To confirm
the optimal stereochemistry of this valine head group its corre-
sponding R enantiomer (compound 26) was also synthesized using
D-valine and studied for its hDGAT1 inhibition. The R enantiomer,
exhibiting an hDGAT1 inhibition of 51% was however found to be
significantly less potent than the corresponding S enantiomer
thereby indicating a preference for
L-valine over
D-valine. As a
result of this preference we have retained
in our further studies.
L-valine as the acid head
Analogs incorporating the 2-pyridyl and 3-pyridyl heteroaryl
scaffolds have also been synthesized and evaluated in an effort to
understand the effect of pyridine on hDGAT1 inhibition. This mod-
ification leads to a decrease in c Log P of the molecules. Four ana-
logs possessing the 3-pyridyl heteroaryl scaffold and seven
were used for synthesis. These included glycine (22),
(23), -serine (24), -valine (25), -valine (26), and -leucine (27).
Since the biological results indicated -valine, incorporated as the
L-alanine
L
L
D
L
L
acid head group, to be optimum for hDGAT1 inhibition this
O
O
OH
O
R
O
OH
O
OH
N
H
B
B
B
O
O
O
F
N
A
A
X1
A
F
X2
N
H
N
H
N
H
N
H
S
N
H
1
2
4
5
6
3
,
Abbott Compound
In house hDGAT1 IC50: 20 nM
Percent TG reduction 10 mpk,p.o): 80.2%
, X =CH, X =N
,
BAY-744113
1
2
hDGAT1 IC50: 73 nM
, X =N, X =CH
(
Figure 2. Development of pyridine containing heteroaryl scaffold.

5814
H. Motiwala et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5812–5817
O
O
O
O
O
OH
O
O
b
a
c
O
Br
B
O
Br
7
8
10
9
H N
2
N
O
O
O
OH
O
d
O
e
f
R
R
N
H
N
H
N
N
N
N
H
H
11
12
R = Phenyl/ Substituted phenyl/ Benzyl
R = Phenyl/ Substituted phenyl/ Benzyl
1
1
O
R
O
R
O
OH
N
H
N
H
O
O
O
g
O
R
R
N
N
H
N
N
H
N
H
N
H
13-21
22-30
18: R = Ph, R1
=
27: R = Ph, R1
=
13: R = Ph, R1 = H
22: R = Ph, R1 = H
14: R = Ph, R1
15: R = Ph, R1
=
=
CH
23: R = Ph, R1
24: R = Ph, R1
=
=
CH
3
3
19: R = 4-Fluoro Phenyl,R1
=
28: R = 4-Fluoro Phenyl,R1
=
OH
OH
20: R = 2,4-Difluoro phenyl,R1 =
21: R = Benzyl, R1
29: R = 2,4-Difluoro phenyl,R1
30: R = Benzyl, R1
=
16: R = Ph, R1
17: R = Ph, R1
=
25: R = Ph, R1
26: R = Ph, R1
=
=
=
=
=
Scheme 1. Reagents and conditions: (a) H2SO4/MeOH reflux, 16 h (yield 77%); (b) Bis pinacol diboron/Potassium acetate/Pd(dppf)2: DCM in DMSO 80 °C, 3 h (yield 75%); (c)
2-amino 5-bromopyridine/2 M K2CO3/tetrakis in toluene/EtOH/H2O 80 °C, 5 h (yield 60%); (d) R-NCO in THF rt, 2–5 h (yield 80%); (e) LiOHÁH2O in THF/MeOH rt, 16 h (yield
80%); (f) Amino acid/EDCIÁHCI/HOBt/DIPEA in DMF rt, 16 h (yield 60–85%); (g) LiOHÁH2O in THF/MeOH rt, 16 h (yield 70–85%).
O
O
O
OH
O
O
a
b
c
O
B
O
N
32
N
31
O2N
O2N
9
O
O
O
O
O
N
H
N
H
e
d
O
N
N
O2N
33
H2N
34
O
O
O
OH
N
H
N
H
O
O
f
O
O
N
N
R
N
H
N
R
N
N
H
35-41
H
H
42-48
35: R = 2-Fluoro
36: R = 3-Fluoro
37: R = 4-Fluoro
42
:
R = 2-Fluoro
43: R = 3-Fluoro
44: R = 4-Fluoro
38: R = 2-Trifluoromethoxy
45: R = 2-Trifluoromethoxy
46: R = 4-Methoxy
39
:
R = 4-Methoxy
40: R = 2.4-Difluoro
41: R = 2-Methyl
47
:
R = 2.4-Difluoro
48: R = 2-Methyl
Scheme 2. Reagents and conditions: (a) 2-bromo 5-nitropyridine/2 M K2CO3/tetrakis in toluene/EtOH/H2O at 80 °C, 5 h (yield 69%); (b) LiOHÁH2O in THF/MeOH at rt, 5 h
(yield 87%); (c)
L
-Valine/EDCIÁHCI/HOBt/DIPEA in DMF at rt, 16 h (yield 47%); (d) Fe/NH4CI in EtOH/H2O at 75 °C, 4 h (yield 81%); (e) R-NCO in THF at rt, 2–5 h (yield 50–85%);
(f) LiOHÁH2O in THF/MeOH at rt, 16 h (yield 80–90%).
analogs possessing the 2-pyridyl heteroaryl scaffold have been
synthesized of which three compounds (25, 28, and 29) belonging
to the 3-pyridyl series and all seven compounds (42–48) belonging
to the 2-pyridyl series exhibited hDGAT1 inhibition greater than
75% in the primary screen conducted at 10 M. Compound 30
(hDGAT1 inhibition = 56%) belonging to the 3-pyridyl heteroaryl
l

H. Motiwala et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5812–5817
5815
Table 1
Table 3
In vitro evaluation of different amino acid head groups
In vitro evaluation of 2-pyridyl heteroaryl analogs
O
R
O
OH
OH
N
H
N
H
O
1
O
O
O
R
N
2
N
H
N
H
N
N
H
N
H
3
Compound
number
Parent amino
acid
R
hDGATI inhibition
IC50 (nM)
c Log Pa
Compound
number
R
hDGATI inhibition (% hDGATI
c Log Pa
inhibition 10
l
M)
Inhibition IC50
(nM)
22
23
Glycine
–H
CH
30
72
1.98
2.47
3
L
-Alanine
-Serine
42
92
14
3.22
OH
F
24
25
26
28
88
51
1.61
3.35
3.35
L
43
44
81
94
18
17
3.22
3.22
F
F
L
-Valine
D-Valine
45
46
47
79
93
82
64
25
14
4.59
2.93
3.38
27
32
3.70
L
-Leucine
O
F
F C
3
O
a
c Log P values were calculated using the Chemdraw version 12.0 ND.
F
Table 2
In vitro evaluation of 3-pyridyl heteroaryl analogs
O
48
78
ND
3.55
CH
3
OH
N
H
ND—Not Determined.
1
O
O
a
c Log P values were calculated using the Chemdraw version 12.0.
R
2
N
H
N
H
N
3
Compound
number
R
hDGATI
inhibition
(% inhibition
hDGATI Inhibition
IC50 (nM)
c Log Pa
Table 4
Plasma triglyceride reduction following an in vivo fat tolerance test and solubility
data
10
lM)
Compound
number
hDGATI inhibition IC50 FTT %TG
Solubilitya (mg/
mL)
(nM)
reduction
25
28
88
120
99
3.35
3.51
4
28
44
20
99
17
80
53
79
0.045
0.12
0.56
F
F
87
a
Solubility determined at pH 7.4.
29
30
90
56
57
3.67
3.42
F
ND
(IC50 = 25 nM) also exhibited greater enzymatic inhibition as
compared to the studied 3-pyridyl analogs. These results thereby
highlight the preference of hDGAT1 towards the 2-pyridyl
heteroaryl scaffold over the 3-pyridyl heteroaryl scaffold.
Compounds incorporating the pyridine scaffold also exhibit an
improvement in c Log P as compared to compound 4. Compound
44 (c Log P: 3.22), a potent 2-pyridyl analog exhibited a 3.5 log or-
der improvement over the Bayer compound 3 (c Log P: 6.72) and a
1 log order improvement over the Abbott compound 4 (c Log P:
4.22). However, the hDGAT1 enzymatic potency of compound 44
is retained when compared to the potent Abbott compound (4).
Compounds 42, 43, 46, and 47 exemplify other molecules within
the 2-pyridyl series displaying a trend similar to that of compound
44.
A confirmation of the in vitro results using an acute in vivo fat
tolerance test (FTT) was also undertaken. In an FTT fasted swiss
mice, belonging to the age range of 4–5 weeks and body weight
range of 25–30 g, were administered with either a vehicle (0.5%
CMC) or the test compound (10 mg/kg) by oral gavage. The test
compounds were formulated as a suspension in 0.5% CMC contain-
ND, Not Determined.
a
c Log P values were calculated using the Chemdraw version 12.0.
scaffold exhibited inhibition less than 75% in the primary screen
possibly because it possessed a benzyl urea as opposed to a phenyl
urea. Following the primary screening, IC50 values of these 2-
pyridyl heteroaryl analogs (Table 3) and 3-pyridyl heteroaryl
analogs (Table 2) were determined. In both the scaffolds, smaller
hydrophobic substituents appear to be well tolerated at all
positions of the terminal phenyl ring, as various methoxy, triflu-
oromethoxy, and halogen substituents exhibited good enzymatic
potency. Several compounds within the 2-pyridyl heteroaryl
scaffold were almost equipotent when compared with compound
4 (In house hDGAT1 IC50: 20 nM). Specifically compounds 44
(IC50 = 17 nM) and 47 (IC50 = 14 nM) belonging to the 2-pyridyl
series exhibited better enzymatic activity as compared to their
corresponding analogs 28 (IC50 = 99 nM) and 29 (IC50 = 57 nM)
within the 3-pyridyl series. Additionally three other 2-pyridyl
analogs, compounds 42 (IC50 = 14 nM), 43 (IC50 = 18 nM), and 46
ing Tween 80 (25 lL). An hour later, a bolus dose of olive oil

5816
H. Motiwala et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5812–5817
Compound- 4 (10 mg/kg)
Vehicle (0.5% CMC)
Compound- 4 (10 mg/kg)
Vehicle (0.5% CMC)
400
300
200
100
0
400
300
200
100
0
80.19%
0
1
2
3
4
Time (h)
Compound- 44 (10 mg/kg)
Vehicle (0.5% CMC)
Compound- 44 (10 mg/kg)
Vehicle (0.5% CMC)
500
400
300
200
100
0
800
600
400
200
0
78.92%
0
1
2
3
4
Time (h)
Figure 3. In vivo fat tolerance test (FTT) to measure the percent reduction in triglyceride levels following the administration of a test compound (10 mg/kg) by oral gavage.
Data analyses were performed using AUC0–4 h for the test compound and comparing it with AUC0–4 h of the vehicle (0.5% CMC) that is considered to be 100%.
(10 ml/kg) was given to the animals. Blood samples were subse-
quently collected at 1, 2, 3, and 4 h, the plasma was separated
and triglyceride levels were monitored using a commercially avail-
able kit (Diasys, Germany). Percent reduction in triglyceride levels
were calculated using the area under curve (AUC0–4 h) of the test
compound and comparing it along with the AUC0–4 h of the vehicle
group that is considered to be 100%.
DGAT1 has been reported to be expressed highly in the small
intestine of experimental animals and human subjects.16 DGAT1
shows significant expression within the enterocytes of the small
intestine. Enterocytes absorb fatty acids and monoacylglycerol
and thereby re-synthesize them into triglycerides. These triglycer-
ides are critical for assembly and secretion of chylomicrons that
transport triacylglycerol into the blood stream. So any inhibition
of the enzyme within the enterocytes should lead to a reduction
in triglyceride synthesis in these cells following an oral lipid load.
This in turn results in a decrease in the triglyceride levels of the
blood.
The 3-pyridyl analog 28 exhibited a plasma triglyceride reduc-
tion of 53% while its corresponding 2-pyridyl analog 44 displayed
a plasma triglyceride reduction of 79% (Table 4 and Fig. 3) when
dosed at 10 mg/kg. At a similar dose compound 4, discovered by
Abbott, displayed a plasma triglyceride reduction of 80% (Table 4
and Fig. 3). Thus, compound 44 is equi-efficacious at a 10 mg/kg
dose as compared to compound 4.
We also studied solubilities of the three compounds evaluated
in vivo in an attempt to understand if introducing a pyridine led
to an improvement in the drug-likeness of these molecules (Ta-
ble 4). Although modest, compound 44 (solubility: 0.56 mg/mL)
and compound 28 (solubility: 0.12 mg/mL) possessing the pyridine
containing heteroaryl scaffold exhibited a 12-fold and 2.4-fold
improvement in their respective solubilities over the biphenyl
compound 4 (solubility 0.045 mg/mL).
In this study, we have developed a pyridine containing hetero-
aryl scaffold possessing improved drug-like properties. We have
studied a few amino acid head groups using the 3-pyridyl hetero-
aryl scaffold and identified L-valine as the preferred acid head
group amongst those studied. A comparison between the 2-pyridyl
and 3-pyridyl scaffolds highlighted a preference of the 2-pyridyl
analogs as hDGAT1 inhibitors. Compounds possessing the 2-
pyridyl scaffold were found to be equi-efficacious and compound
44 also exhibited a 12-fold improvement in solubility as compared
to the Abbott compound (4). This indicated an improvement in the
drug-like characteristics of the molecule along with retention of
activity. The substantial in vitro hDGAT1 enzymatic inhibition cou-
pled along with in vivo plasma triglyceride reduction of some of
these 2-pyridyl heteroaryl analogs, exemplified by compound 44
(IC50 = 17 nM and 79% triglyceride reduction), marks them as
potential new leads for developing better hDGAT1 inhibitors for
therapeutic application.
Acknowledgments
The authors would like to thank Arno Enose and Nisha Potdar
for conducting solubility studies.
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5817
lg
of the protein from post nuclear
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supernatant (PNS) was preincubated with 100 ll of assay buffer [100 mM
Tris- HCl (pH 7.5), 250 mM sucrose, and 1.25 mg/ml fatty acid free BSA]
containing a given concentration of the inhibitor and supplemented using
9. Smith, R.; Campbell, A. M.; Coish, P.; Dai, M.; Jenkins, S.; Lower, D.; O’Connor, S.;
Wang, G.; Zhang; M.; Zhu, L. Z. U.S. Patent 7091228, 2006.
2047.5
16.8nci of [14C]-Oleoyl CoA. After incubation for 10 min at 37 °C, the reaction
was terminated following the addition of 300 l of alkaline ethanol stop
solution mix (AESSM) [12.5% of 100% non-denatured ethanol, 10% deionized
water, 2.5% NaOH, 75% stop solution (78.4% isopropanol, 19.6% n-heptane, 2%
deionized water)]. The reaction mixture was properly mixed and the 14C
lM 1, 2 dioleoylglycerol. The DGAT reaction was initiated by adding
10. Chen, H. C.; Farese, R. V. Arterioscler. Thromb. Vasc. Biol. 2005, 25(3), 482.
11. Zhao, G.; Souers, A. J.; Voorbach, M.; Falls, H. D.; Droz, B.; Brodjian, S.; Lau, Y. Y.;
Iyengar, R. R.; Gao, J.; Judd, A. S.; Wagaw, S. H.; Ravn, M. M.; Engstrom, K. M.;
Lynch, J. K.; Mulhern, M. M.; Freeman, J.; Dayton, B. D.; Wang, X.; Grihalde, N.;
Fry, D.; Beno, D. W. A.; Marsh, K. C.; Su, Z.; Diaz, G. J.; Collins, C. A.; Sham, H.;
Reilly, R. M.; Brune, M. E.; Kym, P. R. J. Med. Chem. 2008, 51, 380.
12. Iyengar, R.; Zhao, G.; Freeman, J. C.; Gao, J.; Judd, A. S.; Kym, P. R.; Lynch, J. K.;
Mulhern, M. M.; Souers, A. J. Patent WO2007137107, 2007.
l
triglyceride formed was extracted following the addition of 600
250 of heptane was added to the scintillation fluid and subjected to
radioactivity counting. The primary screening of hDGAT1 inhibitors was
undertaken at 10 M concentration whereas the IC50 evaluations were carried
ll of heptane.
l
l
l
13. Kym, P. R.; Zhao, G. Patent US20080015227, 2008.
out using nine different concentrations.
14. Smith, R.; O’Connor, S. J.; Coish, P.; Lowe, D.; Clark, R. B.; Stebbins, J.; Campbell,
A.; Akuche, C.; Shelkin, T. Patent WO2006044775, 2006.
16. Lee, B.; Fast, A. M.; Zhu, J.; Cheng, J. X.; Buhman, K. K. J. Lipid Res. 2010, 51(7),
1770.