Discovery of potent, selective sulfonylfuran urea endothelial lipase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.11.033

Endothelial lipase (EL) activity has been implicated in HDL catabolism, vascular inflammation, and atherogenesis, and inhibitors are therefore expected to be useful for the treatment of cardiovascular disease. Sulfonylfuran urea 1 was identified in a high-throughput screening campaign as a potent and non-selective EL inhibitor. A lead optimization effort was undertaken to improve potency and selectivity, and modifications leading to improved LPL selectivity were identified. Radiolabeling studies were undertaken to establish the mechanism of action for these inhibitors, which were ultimately demonstrated to be irreversible inhibitors.

Full text of DOI:10.1016/j.bmcl.2008.11.033

Bioorganic & Medicinal Chemistry Letters 19 (2009) 27–30
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of potent, selective sulfonylfuran urea endothelial lipase inhibitors
a
a
d
a
Krista B. Goodman a, , Michael J. Bury , Mui Cheung , Maria A. Cichy-Knight , Sarah E. Dowdell ,
Allison K. Dunn d, Dennis Lee a, Jeffrey A. Lieby d, Michael L. Moore d, Daryl A. Scherzer d, Deyou Sha a,
Dominic P. Suarez d, Dennis J. Murphy c, Mark R. Harpel c, Eric S. Manas e, Dean E. McNulty e,
Roland S. Annan e, Rosalie E. Matico f, Benjamin K. Schwartz f, John J. Trill f, Thomas D. Sweitzer f,
Da-yuan Wang f, Paul M. Keller g, John A. Krawiec b, Michael C. Jaye b
*
a Department of Chemistry, Cardiovascular and Urogenital Center of Excellence in Drug Discovery, GlaxoSmithKline, 709 Swedeland Road, UW2430, King of Prussia, PA 19406, USA
b Department of Biology, Cardiovascular and Urogenital Center of Excellence in Drug Discovery, GlaxoSmithKline, 709 Swedeland Road, UW2430, King of Prussia, PA 19406, USA
c Department of Biochemistry, Cardiovascular and Urogenital Center of Excellence in Drug Discovery, GlaxoSmithKline, 709 Swedeland Road, UW2430, King of Prussia, PA 19406, USA
d Department of Discovery Medicinal Chemistry, Molecular Discovery Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, 19426 PA, USA
e Department of Computational and Structural Chemistry, Molecular Discovery Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, 19426 PA, USA
f Department of Biological Reagents and Assay Development, Molecular Discovery Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, 19426 PA, USA
g Department of Screening and Compound Profiling, Molecular Discovery Research, King of Prussia, PA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Endothelial lipase (EL) activity has been implicated in HDL catabolism, vascular inflammation, and ath-
erogenesis, and inhibitors are therefore expected to be useful for the treatment of cardiovascular disease.
Sulfonylfuran urea 1 was identified in a high-throughput screening campaign as a potent and non-selec-
tive EL inhibitor. A lead optimization effort was undertaken to improve potency and selectivity, and mod-
ifications leading to improved LPL selectivity were identified. Radiolabeling studies were undertaken to
establish the mechanism of action for these inhibitors, which were ultimately demonstrated to be irre-
versible inhibitors.
Received 7 October 2008
Revised 6 November 2008
Accepted 10 November 2008
Available online 14 November 2008
Keywords:
Endothelial lipase
Phospholipases
Irreversible inhibitors
Selectivity
Ó 2008 Elsevier Ltd. All rights reserved.
Furan
Endothelial lipase (EL), a serine-phospholipase, is a member of
the triglyceride lipase family first cloned in 1999.1 Unlike other tri-
glyceride lipases, EL has a dramatic difference in substrate prefer-
ence, possessing predominantly phospholipase activity rather than
triglyceride lipase activity.2 Importantly, a role for EL in the regu-
lation of HDL cholesterol in mice has been well-documented. EL
knockout mice have a pronounced elevation in HDL cholesterol rel-
ative to wild type mice.3 Moreover, recent studies suggest that EL
may have a proinflammatory effect4 and may be involved in ath-
erogenesis.5 Taken together, this evidence suggests that an EL
inhibitor could have benefit in the treatment of cardiovascular
disease.
A high-throughput screening campaign identified compound 1
as a potent inhibitor of EL (Fig. 1). Although the EL potency of com-
pound 1 was promising, poor lipase selectivity was a concern, since
other lipases such as lipoprotein lipase (LPL) and hepatic lipase
(HL) also affect lipoprotein metabolism and their inhibition could
confound interpretation of in vivo data.6 Consequently, improving
lipase selectivity became a critical goal for the program. Com-
pounds were tested in parallel against both EL and LPL to evaluate
lipase selectivity. Since an EL inhibitor would be used for chronic
treatment, reversibility was also an important criterion for pro-
gression to minimize potential selectivity issues.
Sulfonylfuran ureas were synthesized in a straightforward man-
ner from methyl 2-methyl-3-furancarboxylate (Scheme 1). Sulfo-
nation with chlorosulfonic acid was followed by treatment with
PCl5 to afford the sulfonyl chloride 3. Reaction of sulfonyl chloride
H
N
H
N
O
N
O
S
O
O
1
EL IC50 = 0.13 uM
LPL IC50 = 0.10 uM
* Corresponding author. Tel.: +1 610 270 6635.
E-mail address: krista.b.goodman@gsk.com (K.B. Goodman).
Figure 1. Initial sulfonylfuran urea lead.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.033

28
K. B. Goodman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 27–30
Table 1
CO2Me
b
CO2Me
Lipase activity for urea analogs and replacementsa,b
CO2Me
a
Cl
R2N
S
S
O
O
O
R
O
O
O O
3
4
2
N
c
S
O
O
O
H
N
Ph
H
COOH
N
d
Compound
R
EL IC50
0.13
(lM)
LPL IC50
0.10
(
lM)
R2N
O
S
R2N
O
S
O
O
O
H
N
H
N
O
O
1
5
Ph
6
O
O
Scheme 1. Synthesis of sulfonylfuran ureas. Reagents: (a) ClSO2OH, DCM, then
pyridine, PCl5; (b) R2NH, Et3N, DCM; (c) NaOH, MeOH; (d) DPPA, Et3N, toluene, then
PhNH2.
H
N
H
N
7
0.13
0.20
0.13
>50
0.06
0.16
>50
>50
25
0.13
0.13
0.32
>50
0.40
2.5
OMe
Cl
H
N
H
N
8
Table 2
O
Lipase activity for sulfonamide analogsa
Cl
H
N
H
N
9
H
H
N
N Ph
O
O
H
N
H
N
O
R
O
10
11
12
13
14
15
S
O
O
Ph
H
N
H
N
Compound
R
EL IC50
0.13
(lM)
LPL IC50
0.10
(lM)
Ph
O
O
1
H
N
H
N
N
Ph
Ph
O
17
18
19
20
0.13
1.3
0.04
0.50
0.05
0.40
0.10
Me
N
H
N
N
>50
>50
16
Ph
O
O
N
Me
N
H
N
N
Cl
MeO
Ph
0.25
1.0
N
Me
N
H
N
Ph
O
N
O
H
16
>50
>50
Ph
N
H
N
21
0.3
a
Data represent the average value for two or more measurements. Standard
error is typically within two-fold of the reported mean.
b
a
Inhibition by the most potent compounds may be driven in part by a covalent
Data represent the average value for two or more measurements. Standard
error is typically within two-fold of the reported mean.
interaction between enzyme and inhibitor (vide infra). Although the kinetics for
this process are still under investigation, the IC50 values were determined after a
15-min preincubation of enzyme with the inhibitor in order to minimize the effects
of time-dependent inhibition that may arise from this process.
LPL. Additional substitution at the urea nitrogens was not toler-
ated, and modification of the urea to an amide similarly led to loss
of activity (entries 13–16).
Exploration of the sulfonamide functional group revealed a
broad tolerance for substitution. A range of sulfonamides (Table
2, entries 17–21) maintained activity against EL with little effect
on LPL selectivity.
Substitution and replacement of the furan core was also ex-
plored. Addition of a methyl group at C3 (22, Table 3) resulted in
a marked decrease in activity. Similarly, removal of the C5 methyl
3 with amines under standard conditions afforded sulfonamide 4,
which was hydrolyzed to provide acid 5. Curtius rearrangement
and trapping of the intermediate isocyanate with an amine affor-
ded the desired urea products 6.
Initially, substitution around the phenyl urea was investigated.
EL activity and LPL selectivity were relatively insensitive to aryl
substitution (Table 1, entries 7–9), although ortho-substitution re-
sulted in a modest improvement in LPL selectivity. An unsubstitut-
ed alkyl group (entry 10) was not tolerated; however, branched
alkyl groups (entries 11–12) markedly improved selectivity against

K. B. Goodman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 27–30
29
group significantly decreased EL activity with little effect on selec-
H
N
N
Cl
H
H
tivity. A range of aryl and heteroaryl furan replacements were ex-
plored (24–28), and in all cases, replacement of the furan resulted
in a dramatic loss of activity. Based on these data, we conclude that
the furan is required for activity.
Radiolabeling experiments were conducted to definitively
establish the mechanism of action for compound 1.7 Three radi-
olabeled compounds were employed, including 1, a negative
control (14), and a positive control ([3H]-DFP, an irreversible ser-
ine hydrolase inhibitor) (Fig. 2). Tritium-labeled compounds
were incubated with EL for 20 min, then incubates were precip-
itated and separated by gel electrophoresis. Fluorography of the
N
N
O
3H
O
3H
N
O
N
O
S
S
O
O
O
O
[3H]-1
[3H]-14
O
P
F
O
O
[3H]-DFP
Figure 2. Compounds employed in radiolabeling studies.
resulting gel revealed significant radiolabeling of
a band at
68 kDa, corresponding to intact EL, in the presence of [3H]-1.
(Fig. 3, lane 3). Similar radiolabeling was observed for the posi-
tive control, [3H]-DFP, while significantly less radiolabeling was
observed for the negative control 14. Based on these observa-
1
2
3
4
250
140
tions, we conclude that
1 covalently modifies EL, and that
Table 3
Lipase activity for alternative core analogsa
Compound
R
EL IC50
0.13
(l
M)
LPL IC50
0.10
(lM)
EL
H
60
42
N Ph
H
N
1
O
N
O
S
S
S
S
O
O
O
O
O
O
O
H
N Ph
H
N
Figure 3. Fluorograph resulting from the incubation of endothelial lipase (EL) with
either radiolabeled and/or unlabeled inhibitors of EL. Lane 1: EL + buffer only, lane
2: EL + [1,3-3H] DFP, lane 3: EL + [3H]-1, lane 4: EL + [3H]-14. Incubations contained
22
23
24
25
>50
2.5
>50
4.0
O
N
O
3.5 lg EL and either 5.5 l l
M [1,3-3H] DFP or 10 M [3H]-1 1 or [3H]-14. Reactions
H
were incubated for 20 min at ambient temperature after which they were acetone
precipitated, resuspended in 1Â Laemmli sample buffer and electrophoresed on an
SDS Tris glycine 4–20% polyacrylamide gradient gel. Following SDS–PAGE, gels were
fixed in 10% acetic acid, 30 min, and impregnated with Amplify Fluorographic
Reagent (Amersham) for another 30 min. The gel was then dried and fluorographed
at À80 °C for 4 days.
N Ph
H
N
O
N
O
H
N Ph
H
N
>50
>50
>50
>50
O
N
O
specific and irreversible inhibition contributes to the observed
activity for these compounds.
N
H
In order to investigate a possible explanation for the SAR
described in Table 3 based on covalent modification of the active
site serine, quantum chemical calculations were employed to
determine attachment energies for methanol (a surrogate for the
active site serine) at various furan ring positions. Although these
calculations neglect vibrational effects as well as the energetic bar-
riers between reactants and products, they have been shown to
provide a reasonable estimate of electrophilicity in some cases.8
Results indicate that attachment energies are much more favorable
for the 2- and 3-positions of compound 1 relative to compounds
22, 25, as well as for an inactive thiophene analog (data not
shown). This suggests that electrophilicity trends alone are suffi-
cient to explain the SAR amongst these isosteric analogs, presum-
ing a roughly equivalent fit for the attached covalent intermediate
in the active site.
H
N Ph
H
N
O
N
O
S
S
N
N
O
H
H
N
N Ph
26
27
>50
>50
>50
>50
O
N
O
N
O
S
H
N
H
N
Ph
O
O
N
O
O
In conclusion, a novel series of EL inhibitors featuring a sulfonyl
furan urea core have been disclosed. Studies have demonstrated
that both the furan and urea cores are required for EL activity.
Selectivity against key lipases can be modulated by appropriate
functionalization of the urea group. Radiolabeling studies have
established that these inhibitors covalently modify EL and that this
likely contributes to the observed activity.
H
H
N
N
Ph
28
>50
>50
O
N
S
O
N
O
a
Data represent the average value for two or more measurements. Standard
error is typically within two-fold of the reported mean.

30
K. B. Goodman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 27–30
3. Ishida, T.; Choi, S.; Kunda, R. K.; Hirata, K.; Rubin, E. M.; Cooper, A. D.;
Supplementary data
Quertermous, T. J. Clin. Invest. 2003, 347.
4. Wang, X.; Jin, W.; Rader, D. J. Circ. Res. 2007, 1008.
Synthetic schemes for all analogs. EL and LPL assay conditions.
Experimental procedures for radiolabeling experiments. Supple-
mentary data associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmcl.2008.11.033.
5. (a) Azumi, H.; Hirata, K.; Ishida, T.; Kojima, Y.; Rikitake, Y.; Takeuchi, S.; Inoue,
N.; Kawashima, S.; Hayashi, Y.; Itoh, H. Cardiovasc. Res. 2003, 647; (b) Ishida, T.;
Choi, S. Y.; Kundu, R. K.; Spin, J.; Yamashita, T.; Hirata, K.; Kojima, Y.; Yokoyama,
M.; Cooper, A. D.; Quertermous, T. J. Biol. Chem. 2004, 45085; (c) Ko, K. W.; Paul,
A.; Ma, K.; Li, L.; Chan, L. J. Lipid Res. 2005, 2586.
6. For a review on lipoprotein lipase, see: (a) Goldberg, I. J. J. Lipid Res. 1996, 693;
For a review on hepatic lipase, see: (b) Santamarina-Fojo, S.; Haudenschild, C.;
Amar, M. Curr. Opin. Lipid. 1998, 211.
References and notes
7. For examples of radiolabelling studies with LPL employing [3H]-DFP, see: (a)
Parkin, S. M.; Speake, B. K.; Robinson, D. S. Biochem. J. 1982, 485; (b) Reddy, M.
N.; Maraganore, J. M.; Meredith, S. C.; Heinrikson, R. L.; Kezdy, F. J. J. Biol. Chem.
1986, 9678.
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K.; South, V.; Amin, D.; Perrone, M.; Rader, D. Nat. Genet. 1999, 424; (b)
Hirata, K.; Dichek, H. L.; Cioffi, J. A.; Choi, S. Y.; Leeper, N. J.; Quintana,
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b B3LYP/6-31G(d,p)
2. Maugeais, C.; Tietge, U. J. F.; Broedl, U. C.; Marchadier, D.; Cain, W.; McCoy, M.
G.; Lund-Katz, S.; Glick, J. M.; Rader, D. J. Circulation 2003, 108, 2121.
calculations were performed utilizing a PBF solvation model, as implemented
in Jaguar 7.0 (Schrodinger, L.L.C.).