2-Hydroxy-N-arylbenzenesulfonamides as ATP-citrate lyase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.03.017

A novel series of 2-hydroxy-N-arylbenzenesulfonamides were identified to be ATP-citrate lyase (ACL) inhibitors with compound 9 displaying potent in vitro activity (IC₅₀ = 0.13 μM). Chronic oral dosing of compound 9 in high-fat fed mice lowered

Full text of DOI:10.1016/j.bmcl.2007.03.017

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3208–3211
2-Hydroxy-N-arylbenzenesulfonamides as
ATP-citrate lyase inhibitors
James J. Li,a,* Haixia Wang,a Joseph A. Tino,a Jeffrey A. Robl,a Timothy F. Herpin,a
R. Michael Lawrence,a Scott Biller,a Haris Jamil,a Randy Ponticiello,a Luping Chen,a
Ching-hsuen Chu,a Neil Flynn,a Dong Cheng,a Rulin Zhao,a Bangchi Chen,a
Dora Schnur,a Mary T. Obermeier,a Vito Sasseville,a Ramesh Padmanabha,b
Kristen Pikeb and Thomas Harritya
aBristol-Myers Squibb Pharmaceutical Research Institute, PO Box 5400, Princeton, NJ 08543-5400, USA
bBristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT 06492, USA
Received 1 February 2007; revised 6 March 2007; accepted 6 March 2007
Available online 12 March 2007
Abstract—A novel series of 2-hydroxy-N-arylbenzenesulfonamides were identified to be ATP-citrate lyase (ACL) inhibitors with
compound 9 displaying potent in vitro activity (IC50 = 0.13 lM). Chronic oral dosing of compound 9 in high-fat fed mice lowered
plasma cholesterol, triglyceride, and glucose, as well as inhibited weight gain.
Ó 2007 Elsevier Ltd. All rights reserved.
Cardiovascular disease remains one of the leading
causes of morbidity and mortality in developed coun-
tries with hypercholesterolemia contributing as a major
risk factor.1 Multiple clinical trials in dyslipidemic pa-
tients have shown that aggressive LDL cholesterol low-
ering can achieve significant reduction of coronary
artery disease (CAD) events.2 However, there is growing
evidence that correlates CAD with many other indepen-
dent risk factors such as diabetes, obesity, low HDL,
and high triglyceride levels.3,4 Thus, new therapeutic
agents that can treat multiple risk factors continue to
be an area of intensive medical research. ATP-citrate
lyase (ACL) is an extramitochondrial enzyme that is
expressed in lipogenic tissues such as liver and adipose.5
Since ACL is the primary enzyme responsible for the
production of cytosolic acetyl-CoA, a precursor re-
quired for de novo biosyntheses of cholesterol and fatty
acids, inhibition of ACL has the potential to reduce cho-
lesterol and triglyceride levels and possibly exert an im-
pact on obesity via reduction of lipogenic factors.6–8
There are several literature reports of ACL inhibitors
(Fig. 1) including (À)-hydroxycitrate (1) (Ki = 0.15 lM)9
and the succinic acid derivative 2 (Ki = 1.0 lM).10–14
However, in a HepG2 cell-based assay both compounds
showed no inhibitory activity of lipid synthesis at con-
centrations up to 100 lM, probably due to poor cell-per-
meability of these polar compounds. When tested as its
lactone, prodrug 3, compound 2 exhibited 82–91% inhi-
bition of cholesterol and fatty acid syntheses at 30 lM.10
Oral treatment with 3 in chow fed rats also showed a de-
crease in plasma cholesterol and triglyceride levels.14
In an attempt to identify a cell-permeable ACL inhibi-
tor, a high throughput screen of our internal compound
collection was initiated. The primary goal was to iden-
Cl
Cl
OH
OH
CO2H
CO2H
HO2C
OH CO2H
2
3
HO CO2H
Cl
Cl
1
OH
CO2H
Keywords: ATP-citrate lyase (ACL) inhibitors; Lipid; Glycemic pro-
files and weight loss.
O
O
*
Corresponding author. Tel.: +1 609 818 7124; e-mail: James.Li@
bms.com
Figure 1. Examples of ACL inhibitors in the literature.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.017

J. J. Li et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3208–3211
3209
Table 1. In vitro SAR summary
tify an orally active tool compound that could be used to
evaluate the efficacy potential of ACL inhibition in an
animal model. Through these efforts, the 2-hydroxy-N-
arylbenzenesulfonamide 4 was identified as a modest
inhibitor of ACL (IC50 = 1.1 lM).15 Subsequent
similarity deck mining of the compound collection based
on the 2-hydroxy-N-phenylbenzenesulfonamide phar-
macophore identified an additional 50 analogs for test-
ing, of which 11 showed greater than 50% inhibition at
10 lM. IC50 values are depicted in Table 1.15,16 The
SAR trend was unremarkable based on this limited set
of compounds, with the exception of the 2-substituted
anilines (i.e., compounds 8, 9, 13 or 14) which appeared
to be more potent than other analogs in the set. Among
them, compound 9 was the most potent with an IC50 of
0.13 lM.17
Cl
Cl
OH
O
O
S
NHR
a
Compound
R
IC50 (lM)
1
2
0.15
2.1
4
5
6
7
8
9
1.1
t-Bu
t-Bu
2.3
Cl
F
Cl
In HepG2 cells, compound 9 showed inhibition of total
lipid syntheses with an IC50 of 8 lM.18 A cell based Ala-
mar Blue cytotoxicity assay was used in parallel to dif-
ferentiate the effect on the inhibition of lipid synthesis
versus potential cytotoxicity.19 Under identical incuba-
tion conditions, compound 9 showed no cytotoxicity
up to 50 lM, indicating the observed inhibition of lipid
synthesis was not a result of compound-induced
cytotoxicity.
7.6
F
t-Bu
5.6
t-Bu
Ph
Ph
To assess if compound 9 was suitable for oral dosing,
a
0.19
0.13
standard pharmacokinetic assessment was per-
formed. In mice, 9 showed an oral bioavailability of
55% but a relatively short half-life of 2.1 h.20 We
therefore decided to dose 9 admixed in the food to as-
sure greater duration of exposure in subsequent
chronic efficacy studies.
Ph
OMe
Ph
In contrast to the reported hypolipidemic effect of com-
pound 3 in normal fed rats,14 no clear trend for lipid
lowering or hepatic lipid synthesis inhibition was ob-
served when compound 9 was dosed chronically in mice
that were fed a chow diet (data not shown). We subse-
quently examined the effect of 9 in high-fat fed mice, a
model which more closely mimics typical western dietary
intake. There were a total of four groups in the study;
mice on normal diet and high-fat diet controls, and
two treated groups that were supplemented with 9 in
their high-fat diet to an equivalent daily dose of 10 or
100 mg/kg. The study was continued for a total of
34 days. Food consumption and body weight gain were
tracked along with weekly assessment of lipid and glu-
cose plasma chemistries21.
10
11
6.9
n-Hex
10.9
N
Me
COMe
12
13
14
8.1
OPh
Me
O
O
0.37
0.34
As shown in Figure 2, there was a modest lowering of
both plasma cholesterol and triglycerides after 20 days
of treatment. A reduction in fasting plasma glucose
was observed from day 7 to completion of the study.
At day 29, cholesterol was lowered by 19% with the
100 mg/kg dose and triglycerides were lowered by 26%
in both the 10 and 100 mg/kg treatment groups. Fasting
plasma glucose was lowered by 48% and 32% for 10 and
100 mg/kg doses, respectively, as summarized in Table 2.
i-Pr
O
O
Ph
a In vitro data are at least two separate measurements using recom-
binant hACL, see Ref. 11 for detail assay condition.
same length of time gained 7.8% (Fig. 3 and Table 3).
The 10 and 100 mg/kg treatment groups gained only
7.2% and 3.0%, respectively, from their starting weights.
The high-fat diet produced a 13.8% weight gain over the
course of the study while mice on normal diet for the

3210
J. J. Li et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3208–3211
Effect of Compound 9 on Plasma Cholesterol
15.00
13.00
11.00
9.00
normal diet
control
0
-5
10 mpk
10 mpk
100 mpk
-10
-15
-20
100 mpk
*
*
7.00
0
5
10 15 20 25 30 35
Days of Oral Treatment
5.00
3.00
Effect of Compopund 9 on Plasma Triglyceride
0
-5
1.00
-1.00
-10
-15
-20
-25
-30
0
3
6
9
12
15
18
21
24
27
30
10 mpk
Days of Oral Treatment
100 mpk
Figure 3. Effects of compound 9 on body weight.
*
0
5
10 15 20 25 30 35
Days of Oral Treatment
Table 3. Effect of compound 9 on body weight and fat
Dose
Body weight
gaina (%)
Fat % as whole
bodyb
Effect of Compound 9 on Plasma Glucose
Normal diet
High-fat diet
7.8 1.2
13.8 1.5
7.2 2.5c
3.0 0.7d
13.7 1.3
25.4 3.4
12.9 1.6c
7.3 1.1d
0
-10
-20
-30
-40
-50
-60
*
High-fat diet + 10 mpk/day
High-fat diet + 100 mpk/day
10 mpk
*
*
*
*
*
*
100 mpk
a Percent (%) of body weight gain SEM for day 27 versus day 0.
b Percent (%) SEM, DEXA analysis was performed at day 34.
c Not statistically significant (p > 0.05) versus high-fat diet group.
d Statistically significant (p < 0.05) versus high-fat diet group.
0
5
10
15
20
25
30
Days of Oral Treatment
35
30
25
20
15
10
5
Figure 2. The reduction (%) of plasma lipids and glucose are expressed
as compound 9 treated group versus high-fat fed controls. Data points
with an asterisk were statistically significant (p < 0.05) versus non-
treated high-fat fed mice.
Table 2. Effect of compound 9 on plasma lipids and glucose
Dose
(mpk/day)
Cholesterola
(%)
Triglyceridesa
(%)
Glucosea
(%)
10
100
À9b
À19
À26
À26
À48
À32
0
normal diet
high-fat diet
10 mg/kg
100 mg/kg
a Percent reduction versus high-fat diet control group as measured at
day 29.
b Not statistically significant (p > 0.05); all other values were statisti-
cally significant versus high-fat fed control group (p < 0.05).
Figure 4. Fat percentage relative to whole body weight at day 34 as
determined by DEXA analysis.
Relative to high-fat fed controls, treatment with 10 mg/kg
of 9 showed a trend toward decreased weight gain,
whereas it became significant at the 100 mg/kg dose
(Table 3). Importantly, no apparent changes in food
consumption among high-fat fed groups were
observed,21 indicating that the reduction in body weight
gain and lipid/glucose parameters was not attributed
simply to a difference in food intake between the treated
groups. Additionally, no overt toxicities, including
changes in locomotor activity, plasma transaminases
or liver composition, were observed in any group (data
not shown).
increase in adiposity as compared to the normal fed
group (13.7% body weight as fat for normal diet vs
25.4% body weight as fat for high-fat diet group, an
85% increase; Table 3). In contrast, the 10 mg/kg treat-
ment resulted in a comparable fat composition to the
lean, normal-chow fed control, while the 100 mg/kg
group experienced a 71% reduction in adiposity relative
to high-fat control. The epididymal fat pads from these
mice were also weighed, and the reduction in adiposity
was qualitatively similar to the results from the DEXA
analysis. Furthermore, a reduction of fat tissue was seen
across several anatomical areas including the neck, kid-
ney, and in the subcutaneous depot (data not shown).
Body composition was determined by DEXA analysis at
the completion of the study (Fig. 4 and Table 3). As ex-
pected, mice on the high-fat diet exhibited a significant
In summary, we have identified a 2-hydroxy-N-arylben-
zenesulfonamide 9 as a cell-permeable ACL inhibitor

J. J. Li et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3208–3211
3211
with modest potency.22 When administered to mice fed
on a high-fat diet at 10 and 100 mg/kg/day, it produced
an approximate 20–30% lowering in plasma cholesterol
and triglycerides, as well as a 30–50% decrease in fasting
plasma glucose. More intriguingly, chronic treatment
with 9 showed a gradual inhibition of weight gain along
with a reduction in adiposity without apparent changes
in food intake. Using this high-fat diet mouse model,
our preliminary results suggest that inhibition of ACL
results in improved lipid and glycemic profiles as well
as decreased adipogenesis, ultimately leading to a reduc-
tion in body weight gain. However, future studies will be
required to clarify the mechanism and probe the role of
ACL in the regulation of metabolic pathway and its
therapeutic potential.
13. Dolle, R. E.; McNair, D.; Hughes, J.; Kruse, L. I.;
Eggleston, D.; Saxty, B. A.; Wells, T. N. C.; Groot, P. H.
E. J. Med. Chem. 1992, 35, 4875.
14. Pearce, N. J.; Yates, J.; Berkhout, T. A.; Jackson, B.; Tew,
D.; Boyd, H.; Camilleri, P.; Sweeney, P.; Gribble, A. D.;
Shaw, A.; Groot, P. H. Biochem. J. 1998, 334, 113.
15. The in vitro enzyme assay was performed using recombi-
nant human ACL, as described in Ref. 11.
16. General synthetic scheme for the preparation of 2-
hydroxy-N-phenylbenzenesulfonamides 4–14 is described
herein.
Cl
Cl
Cl
Cl
NH2
OH
poly(4-vinylpyridine)
O
O
S
+
OH
NH
O
O
S
Cl
R
R
Acknowledgments
17. Compound 9. 1H NMR (CDCl3) d 8.66 (br s, 1H), 7.68 (d,
J = 2.2 Hz, 1 H), 7.30–7.55 (m, 8H), 7.20 (br s, 1H), 6.84
(d, J = 8.5 Hz, 1H), 3.71 (s, 3H); LC/MS m/z = 422
(MÀH); Anal. C19H15Cl2NO4S: Calcd C, 53.78; H, 3.56;
N, 3.30; S, 7.55; Cl, 16.71. Found: C, 53.63; H, 3.57; N,
3.17; S, 7.86; Cl, 16.62.
The authors thank Drs. Robert Zahler and Mary Ann
Pelleymounter for helpful discussions during the manu-
script preparation.
18. Inhibition of total lipid synthesis assay. HepG2 cells were
incubated for 6 h after addition of the testing compound
and [14C]-alanine was added for the last 4 h of incubation.
Cell lipids were extracted, separated by TLC. The incor-
poration of [14C]-labeled in the total lipids was measured
using Packard Imager.
References and notes
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1987, 317, 1237.
4. Cullen, P. Am. J. Cardiol. 2000, 86, 943.
19. Alamar Blue assay kits were purchased from BioSource
Back, S. A.; Khan, R.; Gan, X.; Rosenberg, P. A.; Volpe,
J. J. J. Neurosci. Methods 1999, 91, 47.
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Wells, T. N.; Groot, P. H. J. Biol. Chem. 1990, 265, 1430.
6. Srere, P. A. Adv. Enzymol. 1975, 43, 57.
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10. Gribble, A. D.; Ife, R. J.; Shaw, A.; McNair, D. J.;
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11. Gribble, A. D.; Dolle, R. E.; Shaw, A.; McNair, D.;
Novelli, R.; Novelli, C. E.; Slingsby, B. P.; Shah, V. P.;
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20. Pharmacokinetics study of 9. Mice were dosed at 10 mg/kg
(iv and po) in a vehicle containing 10% ethanol, 45%
water,
45%
PEG-400.
F% = 55%,
T1/2 = 2.1 h,
Cmax = 13 lM (po), Tmax = 0.5 h (po), total body clear-
ance 17.3 mL/min/kg.
21. Eight-week-old C57BL/6 mice with an average 30 g of
body weight were used with five mice per group. The high-
fat diet contained 30% coconut oil and 45% sucrose,
whereas the normal-diet group was fed with Teklad
Global 18% protein rodent diet. The average food
consumption for the high-fat control and 9 treated group
was 3.2 0.2 g/day/mouse. The average food consumption
for normal diet mice was 4.0 0.1 g/day/mouse.
22. In a broad screen against other metabolic disease related
targets such as PTP-1B, PPAR, and ACC, it was deter-
mined that compound 9 had an IC50 of 6 lM and 12 lM
against human ACC1 and ACC2, respectively. While it is
unlikely that the in vivo efficacy of 9 in the high-fat feeding
model was the result of ACC inhibition, contribution of
this mechanism or other potential off-target mechanisms
in the pharmacological response cannot be completely
ruled out.