PK/PD Disconnect Observed with a Reversible Endothelial Lipase Inhibitor

Article abstract of DOI:10.1021/acsmedchemlett.8b00138

Screening of a small set of nonselective lipase inhibitors against endothelial lipase (EL) identified a potent and reversible inhibitor, N-(3-(3,4-dichlorophenyl)propyl)-3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamide (5; EL IC₅₀ = 6

Full text of DOI:10.1021/acsmedchemlett.8b00138

Letter
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
PK/PD Disconnect Observed with a Reversible Endothelial Lipase
Inhibitor
Jon J. Hangeland,* Lynn M. Abell, Leonard P. Adam, Ji Jiang, Todd J. Friends, Lauren E. Haque,
James Neels, Joelle M. Onorato, Alice Ye A. Chen, David S. Taylor, Xiaohong Yin, Thomas W. Harrity,
Michael D. Basso, Richard Yang, Paul G. Sleph, David A. Gordon, Christine S. Huang, Ruth R. Wexler,
Heather J. Finlay, and R. Michael Lawrence
Research and Development, Bristol-Myers Squibb, Princeton, New Jersey 08543, United States
S
* Supporting Information
ABSTRACT: Screening of a small set of nonselective lipase inhibitors against endothelial
lipase (EL) identified a potent and reversible inhibitor, N-(3-(3,4-dichlorophenyl)propyl)-3-
hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamide (5; EL IC50 = 61 nM, ELHDL
IC50 = 454 nM). Deck mining identified a related hit, N-(3-(3,4-dichlorophenyl)propyl)-4-
hydroxy-1-methyl-5-oxo-2,5-dihydro-1H-pyrrole-3-carboxamide (6a; EL IC50 = 41 nM,
ELHDL IC50 = 1760 nM). Both compounds were selective against lipoprotein lipase (LPL)
but nonselective versus hepatic lipase (HL). Optimization of compound 6a for EL inhibition using HDL as substrate led to N-(4-
(3,4-dichlorophenyl)butan-2-yl)-1-ethyl-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrole-3-carboxamide (7c; EL IC50 = 148 nM, ELHDL
IC50 = 218 nM) having improved PK over compound 6a, providing a tool molecule to test for the ability to increase HDL-
cholesterol (HDL-C) levels in vivo using a reversible EL inhibitor. Compound 7c did not increase HDL-C in vivo despite
achieving plasma exposures targeted on the basis of enzyme activity and protein binding demonstrating the need to develop more
physiologically relevant in vitro assays to guide compound progression for in vivo evaluation.
KEYWORDS: Endothelial lipase (EL), high density lipoprotein (HDL), reverse cholesterol transport (RCT),
coronary artery disease (CAD)
ndothelial lipase (EL; gene nomenclature LIPG)1,2 exerts
Epleiotropic effects on cardiovascular biology through its role
in high density lipoprotein (HDL) catabolism,3−6 vessel wall
inflammation,7−15 and subsequent effects on reverse cholesterol
transport (RCT).4,16−19 Inhibition of EL using neutralizing
polyclonal antibodies in mice20 or pharmacologically using
irreversible small molecule inhibitors XEN445 (1)21 and
compound 222 (Figure 1) has been reported to raise HDL-C
levels in mice, whereas loss of function EL variants in humans
(e.g., Asn396Ser) have been associated with increased HDL-C
levels.23 A recently published Mendelian randomization study
showed no correlation between increased HDL-C levels
resulting from a loss of function EL variant and the risk of
myocardial infarction despite an expected 13% reduction of risk
estimated from the amount of HDL-C increase associated with
the loss of function allele.24 This study throws doubt into the
hypothesis that raising HDL-C levels by inhibiting EL enzymatic
activity will decrease coronary artery disease (CAD). To fully
investigate the effect of raising HDL-C levels on CAD through
pharmacological inhibition of EL will require high quality drug
molecules with potent in vivo efficacy.
EL is a member of the family of enzymes that includes
lipoprotein lipase (LPL), hepatic lipase (HL), and pancreatic
lipase (PL). In contrast to LPL and PL, which selectively
hydrolyze triglycerides (TGs) and HL that hydrolyzes both TGs
and phospholipids, EL shows a preference for the hydrolysis of
the sn1 ester of phosphatidylcholines found in HDL producing
lyso-phosphatidylcholines (LPCs) and free fatty acids (FFAs),
resulting in lipid-depleted HDL particles that are cleared more
rapidly from the circulation.25
Several small molecule inhibitors of EL have been reported in
the literature, e.g., anthranilic acids (XEN445, 1),21 thiocarba-
mates (2),22 sulfanylfuran ureas (3),26,27 and phenyl boronic
acids (4)28 (Figure 1). We report herein our initial efforts to
identify potent, reversible inhibitors of EL for the purpose of
elevating HDL-C blood levels in vivo.
Received: March 23, 2018
Accepted: May 14, 2018
Figure 1. Examples of literature EL inhibitors.
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00138
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ACS Medicinal Chemistry Letters
Letter
In conjunction with virtual and high throughput screens,
nonselective in-house lipase inhibitors were screened for EL
inhibition using a mixed vesicle substrate (EL assay; see
Supporting Information). The screen led to the identification
of N-(3-(3,4-dichlorophenyl)propyl)-3-hydroxy-1-methyl-2-
oxo-1,2-dihydropyridine-4-carboxamide (compound 5, Figure
2) as a potent inhibitor of EL activity (EL IC50 = 61 nM)
Scheme 1. Synthesis of Compounds 7a−d
a
Reagents and conditions: TMSCH2N2, DIPEA, Et2O, rt, 48 h;
NaOH, MeOH/H2O (1:1), 70 °C, 1 h; (i) oxalyl chloride, CH2Cl2,
b
c
d
DMF(cat), (ii) R3-NH2, DIPEA, CH2Cl2, rt, 3 h; BCl3, CH2Cl2, rt, 5 h.
See Supporting Information for individual compound yield.
amines (R3-NH2) using the corresponding acid chloride
generated with oxalyl chloride. The resulting amides were
demethylated with boron trichloride to give the final products 7a
and 7b. Compounds 7c and 7d were obtained by chiral HPLC
separation of 7b. Compound 7c was also obtained from the
homochiral amine synthesized using the method of Ellman.32
Excellent stereochemical induction was observed (dr = 99:1)
when methylmagnesium bromide was combined with the (R)-N-
tert-butanesulfinyl imine of 3-(3,4-dichlorophenyl)propanal at
60 °C leading to a high enantiomeric excess of the final product
(see Supporting Information).
The compounds were evaluated using a PED-A1/DMPG
mixed vesicle assay (EL assay) and an HDL assay using purified
HDL particles as enzyme substrate with detection of the product,
linoleoyl-lyso-phosphatidylcholine, by LCMS (ELHDL assay; see
Supporting Information) in an effort to provide a bridging assay
between in vitro and in vivo activity.
Figure 2. Initial EL hits from focused deck screening.
containing a weakly acidic to neutral heterocycle (measured pKa
= 6.5). Compound 5 was also a potent inhibitor of HL (HL IC50
= 16 nM) but was found to be highly selective against LPL (LPL
IC50 > 90,000 nM). When EL was inhibited with a high
concentration of compound 5 (2 μM), enzymatic function was
restored by dialyzing away unbound inhibitor from the assay
buffer (data not shown), demonstrating the reversibility of
inhibition. Further deck mining around compound 5 identified a
structurally related EL inhibitor, N-(3-(3,4-dichlorophenyl)-
propyl)-4-hydroxy-1-methyl-5-oxo-2,5-dihydro-1H-pyrole-3-
cabox-amide (compound 6a, Figure 2), with EL IC50 = 41 nM
that contained a slightly more acidic heterocycle (measured pKa
= 4.8). Compound 6a was also not selective against HL (HL IC50
We first explored the SAR of the R1 position of the 1-methyl-5-
oxo-2,5-dihydro-1H-pyrrole core found on compound 6a. It was
observed that extending the R1 side chain with lipophilic or polar
groups maintained or improved EL potency (see compounds 7a
and 8−10, Table 2), ranging between 3.6 nM (compound 9, R1 =
benzyl) and 30 nM (compound 7a, R1 = ethyl). Addition of a
basic side chain (R1 = N-ethylmorpholine) reduced EL potency
by ca. 5-fold, whereas the acidic acyl sulfonamide 12 had similar
potency to compound 6a. When these compounds were tested
using the ELHDL assay, a significant right-shift to reduced potency
was observed giving ELHDL IC50/EL IC50 ratios between 11- and
150-fold.
= 76 nM) but had excellent selectivity versus LPL (LPL IC50
37,000 nM). Pharmacokinetic (PK) profiling of compounds 5
and 6a in CD1 mice (Table 1) showed both compounds to have
>
Table 1. Pharmacokinetic Parameters for Compounds 5 and
6a in CD1 Mice
compound 5
compound 6a
parameter
i.v.
p.o
i.v.
p.o.
a
dose (mpk)
1.0
25
1.0
2.0
16
1.0
32
1.0
8.2
18
C
max (μM)
AUCtotal (μM·h)
CL (mL/min/kg)
t1/2 (h)
19
24
The SAR for substitution at the R2 position was examined
within the context of R1 = CH3 (Table 3). A limited set of analogs
was examined (compounds 6b−6d). All were of similar potency
to the original hit. These analogs did not improve ELHDL potency
and the ratio of ELHDL to EL potencies ranged from 60- to 100-
fold. We concluded that further exploration of this position was
not warranted.
2.5
4.9
2.1
3.4
2.9
82
3.2
76
F (%)
a
Bolus administration i.v. and p.o. using 60% PEG400, 30% water, and
10% EtOH as vehicle. mpk = milligram per kilogram.
excellent oral bioavailability (82% and 76%, respectively). Owing
to slightly better selectivity against HL and facile synthetic
methods available for compound 6a, we focused our initial SAR
efforts on this chemotype, seeking to improve potency, to
maintain the favorable PK properties, and to establish a
pharmacodynamic (PD) profile and PK/PD relationship so the
EL mechanism of action could be evaluated pharmacologically in
vivo.
The synthesis of compounds 6a−g, 8−12, and 13a−d is
reported in the Supporting Information. Synthesis of the in vivo
candidate 7c and related compounds 7a, 7b, and 7d is shown in
Scheme 1. Ethyl 1-ethyl-4-hydroxy-5-oxo-2,5-dihydro-1H-pyr-
role-3- carboxylate (16)29,30 was first O-methylated with
trimethylsilyldiazomethane in the presence of DIPEA,31 followed
by saponification of the ester. The acid (17) was coupled with
Replacing the C-4 hydroxyl substituent with amines (R4-NH2)
in the context of R1 = ethyl and R3 = N-(3-(3,4-dichlorophenyl)-
prop-1-yl provided compounds 13a−d (Table 4). Modification
at this position provided analogs that were slightly more potent
or equipotent to 6a in the EL assay (IC50 range from <10 to 59
nM); however, a significant right-shift in potency in the ELHDL
assay was observed, with ELHDL/EL ratios ranging from 50- to
>300-fold.
Exploration of the SAR of the amide side chain (R3) was
accomplished in two series where R1 = CH3 or ethyl (Table 5). It
was generally observed that the most potent compounds in the
EL assay contained extended lipophilic R3 groups and two
chlorine atoms located at C-3 and C-4 on the terminal phenyl.
Thus, compound 6e (R3 = phenyethyl) was 10-fold less potent
than the bis-chloro compound 6f, and compound 6a was ca. 140-
B
DOI: 10.1021/acsmedchemlett.8b00138
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ACS Medicinal Chemistry Letters
Letter
Table 2. R1 Group SAR of 1-Methyl-5-oxo-2,5-dihydro-1H-
pyrrole core (compounds 7a and 8−12)
Table 4. R4 Group SAR of 1-Methyl-5-oxo-2,5-dihydro-1H-
pyrrole Core (Compounds 13a−d)
a
IC50 values are the average of at least two independent
determinations.
Table 5. R3 Group SAR of 1-Methyl-5-oxo-2,5-dihydro-1H-
pyrrole Core (Compounds 6a, 6e−g, and 7b−d)
a
IC50 values are an average of at least two independent determinations.
See Supporting Information for standard deviations. ND = not
b
c
determined.
Table 3. R2 Group SAR of 1-Methyl-5-oxo-2,5-dihydro-1H-
pyrrole Core (Compounds 6b−d)
a
IC50 values are an average of at least two independent determinations.
fold more potent than the des-chloro analog compound 6g.
Further SAR development of the R3 side chain identified a key
modification that provided an analog with more balanced EL and
ELHDL potency, compound 7b (EL IC50 = 44 nM, ELHDL IC50 =
264 nM). When the two enantiomers were obtained in
a
IC50 values are an average of at least two independent determinations.
See Supporting Information for standard deviations. ND = not
b
c
homochiral form, one enantiomer (compound 7c; EL IC50
=
148 nM, ELHDL IC50 = 218 nM) was significantly more potent
than the other (compound 7d) and was equally potent for mouse
EL (mouse ELHDL IC50 = 100 nM). Importantly, the potency of
compound 7c using human or mouse EL was relatively
insensitive to increasing concentration of HDL (tested at 40,
100, 300, and 1000 μg/mL HDL; see Supporting Information)
suggesting compound 7c is not competitive with HDL.
determined.
monoacylglycerol lipase (MAGL), and pancreatic lipase (PL).
Using the HDL-based assay, compound 7c was 12-fold selective
for EL vs HL (see Supporting Information).
Pharmacokinetic evaluation of compound 7c using male CD1
mice at a dose of 1.0 mpk i.v. or p.o. (Table 6 and Chart 1)
showed it to have excellent oral exposure (oral AUCtotal = 292
When tested against a panel of related enzymes, compound 7c
was found to be >250-fold selective for EL versus LDL,
C
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ACS Medicinal Chemistry Letters
Letter
μM; 96% bioavailability) and Cmax (15 μM) with a longer half-life
(t1/2 = 13 h) than compound 6a (t1/2 = 3.2 h).
Chart 2. . Plasma HDL Levels in C57BL/6J Mice Dosed with
Compound 7c
a
When compound 7c was dosed at 10 and 50 mpk, systemic
exposure was dose related but less than dose proportional for
Table 6. Pharmacokinetic Data for Compound 7c in CD1
Mice
parameter
i.v.
p.o.
a
dose (mpk)
1.0
39
1.0
15
C
T
max (μM)
max (h)
0.05
304
13
0.5
292
13
AUCtotal (μM·h)
1/2 (h)
t
F (%)
96
C 24 h (nM)
4310
99
b
C
free 24 h (nM)
a
Compound 7c was dosed at 10 and 50 mpk once daily p.o. using 60%
a
Dosing vehicle for i.v. and p.o. administration was 60% PEG400, 30%
PEG400/30% water/10% ethanol as dosing solution.
b
water, 10% ethanol. mpk = milligram per kilogram Adjusted for
mouse protein binding using 2.3% free fraction.
enzyme, suggests factors other than protein binding may be
involved in the inability of compound 7c to inhibit plasma EL. In
addition, these results point to the need to develop other, more
physiologically relevant in vitro assays to guide compound
progression for in vivo evaluation. The results of these
investigations will be reported separately.
Chart 1. Mean Plasma Concentrations of Compound 7a in
a
CD1 Mice
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00138.
■
S
Reaction scheme, experimental procedures and character-
ization data for compounds 6a−g, 7a−d, 8−12, and 13a−
d, standard deviations of EL and ELHDL IC50 data for
compounds 6a and 7c, dependence of ELHDL potency on
HDL concentration, mouse and hamster PK data, hamster
PD data, biochemical assay protocols, and mouse in vivo
protocol (PDF)
a
Compound 7c dosed p.o. at 1 mpk in CD1 mice using 60% PEG400/
30% water/10% ethanol as dosing solution.
AUTHOR INFORMATION
Corresponding Author
*E-mail: jon.hangeland@bms.com. Tel: (1) 609-466-5043.
both (7.5-fold and 18-fold increase in AUCtotal, respectively, vs
1.0 mpk dose; see Supporting Information). Protein binding in
mice was 97.7%.
■
When compound 7c was administered to C57BL/6J mice
once a day p.o. for 5 days at 10 and 50 mpk, high plasma
exposures were observed in the PK arm of the experiment (see
Supporting Information). Thus, on day 5 at 50 mpk, Cmax = 238
μM (6 h postdose) and Ctrough = 57 μM (24 h postdose). After
adjusting for protein binding, Cmax free (5.5 μM) and Ctrough free
(1.3 μM) were 55- and 13-fold, respectively, above the mouse
ELHDL IC50. Despite the high exposures achieved, there was no
effect on plasma HDL-C levels at either dose (Chart 2). A follow-
up study in normal hamsters dosed once a day for 7 days at 50
mpk gave the same outcome. In this study, drug exposures were
comparable to mouse. Accounting for protein binding in hamster
(98.4%), Cmax free = 5.1 μM (day 5, 6 h postdose), ca. 51-fold
above the IC50 for mouse EL (see Supporting Information).
In summary, optimizing potency of screening hit 6a using the
ELHDL assay resulted in the identification of compound 7c
(ELHDL IC50 = 218 nM; mouse ELHDL IC50 = 100 nM). The lack
of a pharmacodynamic effect in two animal models of increasing
plasma HDL-C levels, despite achieving levels of compound
exposure that would predict good inhibition of the target
ORCID
Jon J. Hangeland: 0000-0002-8234-8857
Heather J. Finlay: 0000-0003-2309-0136
Author Contributions
The manuscript was written by J.J.H. and through contributions
by all authors. All authors have given approval to the final version
of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to acknowledge Dr. Michael A. Walker
for helpful chemistry discussions and for generously supplying
large quantities of compound 15 and Mr. Robert A. Langish for
acquiring HRMS data contained in the Supporting Information.
ABBREVIATIONS
DIPEA, diisopropylethylamine; DMPG, 1,2-dimyristoyl-sn-glyc-
ero-3-[phosphor-rac-(1-glycerol)] sodium salt;; dr, diastereo-
■
D
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ACS Medicinal Chemistry Letters
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