Discovery of PF-04620110, a potent, selective, and orally bioavailable inhibitor of DGAT-1

Article abstract of DOI:10.1021/ml200051p

Acyl-CoA:diacylglycerol acyltransferase-1 (DGAT-1) catalyzes the final committed step in the biosynthesis of triglycerides. DGAT-1 knockout mice have been shown to be resistant to diet-induced obesity and have increased insulin sensitivity. Thus, inhibiti

Full text of DOI:10.1021/ml200051p

LETTER
pubs.acs.org/acsmedchemlett
Discovery of PF-04620110, a Potent, Selective, and Orally Bioavailable
Inhibitor of DGAT-1
Robert L. Dow,* Jian-Cheng Li, Michael P. Pence, E. Michael Gibbs, Jennifer L. LaPerle, John Litchfield,
David W. Piotrowski, Michael J. Munchhof, Tara B. Manion, William J. Zavadoski, Gregory S. Walker,
R. Kirk McPherson, Susan Tapley, Eliot Sugarman, Angel Guzman-Perez, and Paul DaSilva-Jardine
Pfizer Global Research and Development, Groton, Connecticut 06340, United States
S Supporting Information
b
ABSTRACT: Acyl-CoA:diacylglycerol acyltransferase-1 (DGAT-1) catalyzes the final
committed step in the biosynthesis of triglycerides. DGAT-1 knockout mice have been
shown to be resistant to diet-induced obesity and have increased insulin sensitivity. Thus,
inhibition of DGAT-1 may represent an attractive target for the treatment of obesity or type
II diabetes. Herein, we report the discovery and characterization of a potent and selective
DGAT-1 inhibitor PF-04620110 (3). Compound 3 inhibits DGAT-1 with an IC50 of 19 nM
and shows high selectivity versus a broad panel of off-target pharmacologic end points. In
vivo DGAT-1 inhibition has been demonstrated through reduction of plasma triglyceride
levels in rodents at doses of g0.1 mg/kg following a lipid challenge. On the basis of this
pharmacologic and pharmacokinetic profile, compound 3 has been advanced to human
clinical studies.
KEYWORDS: DGAT-1, diabetes, obesity, triglyceride, phototoxicity, acyl glucuronide
ysregulation of fatty acid metabolism is recognized as a
major determinant in a number of human diseases including
focus our efforts on a pyrimidinooxazine-based series reported by
scientists at Japan Tobacco and Tularik.14 A representative
example for this series is pyrimidinooxazine 1, which is a potent
(IC50 = 72 nM), selective (DGAT-2 IC50 > 10 μM) inhibitor of
the human DGAT-1 enzyme and has a logD7.4 of 1.4.15
D
obesity, insulin resistance, and hepatic steatosis.1 Excess disposi-
tion of triglycerides in various tissues has been implicated in
driving these disease end points. On the basis of this connection,
there has been considerable interest in the acyl-CoA:diacylgly-
cerol acyltransferase (DGAT) family of enzymes that catalyze the
final committed step in triglyceride synthesis.2 Two members of
this enzyme family have been reported, DGAT-13 and DGAT-2.4
Mice lacking DGAT-1 (DGAT-1À/À) are resistant to diet-
induced obesity and have increased insulin sensitivity and energy
expenditure.5,6 Furthermore, transplantation of white adipose
tissue from these mice into the wild-type strain confers the
enhanced metabolic profile observed in the DGAT-1 knockout
mice.7 These findings have spurred research efforts to determine
whether selective, small molecule inhibitors of DGAT-1 can
produce the same improved metabolic profile observed in
DGAT-1À/À mice.
While possessing an encouraging profile, two chemical fea-
tures of this series raised concerns. Compound 1 and related
analogues exhibit substantial absorption of light at wavelengths
greater than 290 nm, which can be indicative of phototoxicity
potential.16,17 The molar extinction coefficient for 1 is 7693 at
320 nm, with both solution and solid phase instabilities observed
for 1.18 On the basis of this data, design strategies based on
mimicking the spatial arrangement of the key pharmacophore
elements while eliminating the pyrimidinooxazine core were
prioritized. The second issue that we chose to evaluate with
analogues of this series is the potential to form reactive acyl
glucuronide metabolites.19 During the course of our studies,
workers at Astra Zeneca reported that the major route of in vivo
clearance of 1 is via the acyl glucuronide, although stability data
are not reported.20 However, it has been shown that aliphatic
acids containing no R-branching (e.g., 1) tend to form the most
reactive acyl glucuronides.21 To address this issue, the Astra
Zeneca group utilized increased steric hindrance in the vicinity of
the carboxylic acid (2) as a means of increasing the stability of the
acyl glucuronide metabolite.20 Part of our program strategy
included monitoring both the formation and the stability of the
Several research groups have disclosed potent and selective
DGAT-1 inhibitors from several distinct chemical series.8,9 Pre-
clinical studies with these compounds have confirmed that small
molecule DGAT-1 inhibitors can elicit metabolic outcomes
comparable to those observed in DGAT-1À/À mice.10,11 While
a number of distinct chemotypes have been reported as inhibitors
of DGAT-1, a substantial portion of the early chemical matter
reported at the time that we began our work in 2006 were high
molecular weight and lipophilic in nature.12 For example, >65% of
the compounds disclosed in patent applications up to this point
were in property space, which violated at least two of Lipinski's
rule of five cutoffs.13 On the basis of this analysis, we chose to
Received: February 17, 2011
Accepted: March 15, 2011
Published: March 18, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Pyrimidooxazepinone 3a
a Reagents and conditions: (a) Pd(OAc)2, CsCO3, XPhos, toluene, 120 °C. (b) Et3N, THF, 0 °C. (c) MeOH, aqueous HCl(c), 25 °C. (d) Et3N, DMF,
80 °C. (e) NH3, p-dioxane, 25 °C. (f) LiOH, H2O, p-dioxane.
potential acyl glucuronide metabolites of key analogues to
minimize the associated safety concerns.
and spatial features that need to be retained based on previously
reported structureÀactivity relationship studies.14 The 10-fold
loss in potency when the oxazine carbonÀnitrogen bond is
reduced to the corresponding dihydrooxazine suggests that an
in-plane orientation of the bicyclic core to the phenylcyclohexyl
side chain is critical. For the aminopyrimidine portion of the
bicyclic core, replacement of either one or both of the aminopyr-
imidine ring nitrogens with carbon produces a 10-/100-fold
reduction in potency, respectively. In evaluating alternative de-
signs that would retain these pharmacophore requirements,
pyrimidooxazepinone 3 provided a good overlay with the key
pharmacophore features with 1. We also speculated that the
lactam-based bicyclic core present in 3 would potentially have a
different UV/vis absorbance/stability profile relative to 1.
In part driven by synthetic limitations, there are no reported
analogues of 1 where the oxygen atom of the pyrimidooxazine
ring is replaced with carbon have been reported to date.14,20
Because of the resulting uncertainty surrounding the importance
of this group, the pyrimidodihydropyridone-based analogue 4
was also a goal in our efforts. This communication details the
syntheses and DGAT-1 inhibitory profiles of these novel ami-
nobicyclic targets. A summary of pharmacokinetic, efficacy, and
In evaluating alternative designs in which the pyrimidinoox-
azine core of 1 is modified, there are multiple pharmacophore
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Scheme 2. Synthesis of Pyrimidodihydropyridinone 4a
a Reagents and conditions: (a) β-Alanine ethyl ester, Cs2CO3, X-Phos, Pd(OAc)2 toluene, reflux. (b) Cyanoacetyl chloride, triethylamine, 0 °C. (c) DBU,
MeOH, reflux. (d) Oxalyl chloride, DMF, CH2Cl2, 25 °C; then MeOH, reflux. (e) Cyanamide, NaH, p-dioxane, 25 °C; then 4 M HCl in p-dioxane, 100 °C.
(f) 20% Pd(OH)2 on carbon, H2 (800 psi), 25 °C. (g) 2,3-Dichloro-5,6-dicyanobenzoquinone, CH3CN, 25 °C. (h) LiOH, H2O, p-dioxane, 45 °C.
Table 1. In Vitro Pharmacology and Microsomal Stability
Data for 1, 3, and 4
safety parameters is also discussed, leading to the advancement of
PF-04620110 (3) to clinical trials.
Scheme 1 details the synthetic route developed for the
preparation of the pyrimidooxazepinone 3. Amination of triflate
514 with the protected aminoalcohol 622 utilizing Buchwald
conditions23 affords 7 in 70% yield. Acylation with acid chloride
IC50 (nM)
HLM CLapp
compound
DGAT-1a
DGAT-2b
TG synthesisc
(mL/min/kg)
8
24 followed by deprotection of the silyl protecting group with
1
3
4
72
19
60
>10000
>30000
>30000
16
8
<8
<8
12
aqueous hydrochloric acid provides alcohol 10. Protection of the
alcohol was necessary since substantial amounts of O-acylation
are observed in its absence. Treatment of 10 with triethylamine
at 80 °C forms the pyrimidooxazepinone core, with 11 isolated
in 83% overall yield from 7. Treatment of this chloropyrimidine
with ammonia in p-dioxane at room temperature affords 12 in a
near quantitative yield. Lithium hydroxide-mediated hydrolysis
provided the target carboxylic acid 3 in 95% isolated yield as a
crystalline solid.
Preparation of pyrimidodihydropyridinone 4 (Scheme 2) is
initiated through coupling of ethyl 3-aminopropanoate utilizing
the conditions employed for the synthesis of intermediate 7.
Acylation of the resulting amine with cyanoacetyl chloride affords
amide 15 in 77% overall yield. Base-catalyzed cyclization gen-
erates the dihydropyridinone core, and conversion of the 4-hy-
droxy group of 16 to the corresponding methyl ether (17) is
achieved in 64% overall yield for this two-step process. Con-
struction of the aminopyrimidine ring follows the generalized
route developed by Schmidt and co-workers.25 Reaction of 17
with cyanamide, followed by cyclization with hydrogen chloride
in p-dioxane, creates the fused 2-chloro-4-aminopyrimidine ring.
Hydrogenolysis of the 2-chloro substituent requires somewhat
forcing conditions [Pd(OH)2/800 psi H2], leading to a 12:1
71
a Average of gfive determinations run in triplicate. b Average of two
determinations run in triplicate. c Inhibition of triglyceride synthesis deter-
mined in HT-29 cells. Average of gthree determinations run in triplicate.
mixture of 18:19 in 63% combined yield. Tetrahydropyrimido-
pyridinone 18 can be converted to 19 by oxidation with DDQ,
and subsequent hydrolysis provides pyrimidodihydropyridinone
4 in 62% overall yield.
Pyrimidooxazepinone 3 is a potent inhibitor of human
DGAT-1 possessing an IC50 of 19 nM (Table 1). This result
confirmed our design hypothesis that the oxazepinone ring
system could function as an effective replacement for the fused
oxazine ring of 1. This result answered two additional pharma-
cophore questions that were part of our design analysis leading
up to the pursuit of 3. The first of these was whether the lactam
carbonyl moiety would affect potency through steric encum-
brance in a spatial vector that is not occupied in 1. This concern
was enhanced by the previously mentioned significant reduction
in potency observed when the oxazine ring of 1 is reduced,
placing a hydrogen in space occupied by the carbonyl oxygen of 3.
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Scheme 3. Synthesis of Acyl Glucuronide 21a
a Reagents and conditions: (a) HATU, N-methylmorpholine, acetonitrile, 25 °C. (b) Pd(Ph3)4, pyrrolidine, THF, 25 °C.
The second question revolved around the criticality of the gem-
dimethyl substituents in determining the DGAT-1 inhibitory
potency of 1. On the basis of the potency of 3, it appears that
these methyl groups do not contribute significantly to the
potency of 1, which is consistent with the trends reported for
des-cyclohexylacetic acid analogues of 1.14
Compound 3 is a highly selective inhibitor of DGAT-1 with
>100-fold selectivity against a panel of lipid processing enzymes
(human DGAT-2, human acyl-CoA:cholesterol acyltransferase-
1, human acyl-CoA:wax alcohol acyltransferase-1/-2, and human
acyl-CoA:monacylglycerol acyltransferase-2/-3 and mouse MG-
AT-1) . The lack of significant activity (IC50 > 10 μM) against
200 enzyme, ion channel, and receptor pharmacology end points
confirmed high DGAT-1 specificity for 3. Analysis of inhibition
of triglyceride synthesis of 3 in the intestinal-derived HT-29 cell
line shows good translation of potency from the isolated enzyme
to a whole cell environment (IC50 = 8 nM, Table 1). With
3 possessing both a low logD (À0.15) and passive permeability
(1 Â 10À6cm/sÀ1),26 there was some concern that this profile
might limit cellular uptake and thus inhibition of triglyceride
synthesis in the HT-29 assay. This is in comparison to 1, which
also shows good translation of DGAT-1 enzymatic inhibitory
activity in this assay but has a logD of 1.4 and a high passive
Figure 1. Rat triglyceride tolerance test with 3.
preparation of 21 via ester coupling of 3 with ally R/β-D-
glucopyranuronate27 followed by removal of the allyl protecting
group. Exposure of 3 to both rat and human liver microsomal
fractions did not produce detectible quantities of 21. In rat, 3% of
an oral dose is excreted in the bile as a 1:1 mixture of 3 and 21.
While 19% of the dose is excreted in urine of these rats, with no
acyl glucuronide or rearrangement products were observed. To
further understand the potential risk of any acyl glucuronide
formed in vivo, the rates of acyl migration and/or hydrolytic
cleavage of 21 were measured by NMR.28 In pH 7.4 aqueous
sodium phosphate at 37 °C, the T1/2 for loss of 21 was greater
than 9.5 h with only direct hydrolysis to 3, and no rearrangement
products observed. Half lives of greater than 1.5 h under these
experimental conditions have been proposed to be associated
with a low risk of adverse side effects.29
In anticipation of advancing 3 to preclinical in vivo efficacy
studies, pharmacokinetic profiling was performed in the rat. Both
plasma clearance and volume in the rat are in the moderate range
at 6.7 mL/min/kg and 1.8 L/kg, respectively. High oral bioavail-
ability (100%) and a moderate half-life of 6.8 h were observed
with an oral dose of 5 mg/kg of crystalline 3 in 0.5% methylcel-
lulose vehicle.30 To confirm that this pharmacokinetic profile
translated into the pharmacodynamic effect via DGAT-1 inhibi-
tion in vivo, 3 was evaluated in an acute lipid challenge model
measuring plasma triglycerides after a corn oil bolus. Spra-
gueÀDawley rats (n = 7 per dose) were treated with vehicle
(5% methyl cellulose), 0.1, 1, or 10 mg/kg of 3 30 min prior to
corn oil dosing. Blood samples for triglyceride and pharmacoki-
netic analyses were taken at 1, 2, and 4 h postlipid challenge. All
three doses produced a statistically significant (p < 0.05) reduc-
tion in plasma triglyceride excursion at 2 h to near prelipid load
levels (Figure 1). Free Cavg plasma concentrations31 of 3 over the
permeability of 10 Â 10À6cm/sÀ1
.
Pyrimidodihydropyridinone 4 is also a relatively potent
DGAT-1 inhibition with enzymatic and whole-cell IC50 values
of 60 and 71 nM, respectively. However, when compound 4 was
advanced to an in vitro ADME screening panel, it was found to
have moderate turnover in human liver microsomes (12 mL/
min/kg). This is significantly higher oxidative metabolism than is
observed for either 1 or 3, which are <8 mL/min/kg in this high-
throughput assay. Based on the combination of DGAT-1 po-
tency and projected oxidative metabolism, compound 3 was
selected for further profiling.
While 1 has substantial absorbance in the near visible light
region, pyrimidinooxazepinone 3 has a greatly reduced absor-
bance profile with an MEC of 1315 L/mol cmÀ1, which returns
to baseline by 340 nM. Compound 3 was advanced to photo-
stability testing where no degradation was observed in solution
or solid state.
Because of the potential for idiosyncratic toxicology associated
with the acyl glucuronide of 1 and the structural equivalency of
the carboxylic acid side chain of 3, analysis of the metabolite
profile was initiated. An authentic sample of the acyl glucuronide
of 3 was prepared to serve as a standard for in vitro and in vivo
metabolite identification as well for stability measurements
of this potential reactive intermediate. Scheme 3 details the
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course of the 4 h study were 8, 37, and 398 nM for the 0.1, 1, and
10 mg/kg doses, respectively. While the plasma free Cavg for the
two lower doses were below the rat DGAT-1 IC50 (64 nM), it is
likely that in the enterocytes, a key DGAT-1 expressing tissue,
concentrations of 3 were considerably higher. On the basis of this
excellent potency and efficacy in an acute setting, compound 3
was advanced to a chronic preclinical model of obesity and
diabetes, which will be reported separately.
In conclusion, PF-04620110 (3) is a potent and selective
inhibitor of DGAT-1. Results for this compound demonstrate
the low risk of potential adverse effects mediated via the acyl
glucuronide metabolite or via photochemical processes. This
compound has excellent pharmacokinetic properties that are
consistent with once daily oral administration.32 Compound 3
has been advanced to human clinical trials for the treatment of
type II diabetes.
(8) King, A. J.; Judd, A. S.; Souers, A. J. Inhibitors of diacylglycerol
acyltransferase: A review of 2008 patents. Expert Opin. Ther. Patents
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’ ASSOCIATED CONTENT
(12) See the Supporting Information for a property space analysis of
DGAT-1 patent literature for 2004À2006.
S
Supporting Information. Experimental details for the
b
(13) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Experimental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv. Drug
Delivery Rev. 2001, 46, 3–26.
syntheses and pharmacological characterization of 3, 4, and 21.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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Jackson, S. M.; Kayser, F.; Labelle, M.; Kexue, M.; Matsui, T.; McMinn,
D. L.; Ogawa, N.; Rubenstein, S. M.; Sagawa, S.; Sugimoto, K.; Suzuki,
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’ AUTHOR INFORMATION
Corresponding Author
*Tel: 860-441-4423. Fax: 860-441-0548. E-mail: robert.l.dow@
pfizer.com.
(15) LogD measurements were determined by shake flask method
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’ ACKNOWLEDGMENT
We thank Chris Wood and Chris Foti for UVÀvis spectral/
photostability analyses and Kay Ahn for MGAT enzyme
inhibition data.
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