Identification and preliminary characterization of a potent, safe, and orally efficacious inhibitor of acyl-CoA:Diacylglycerol acyltransferase 1

Article abstract of DOI:10.1021/jm201524g

A high-throughput screen against human DGAT-1 led to the identification of a core structure that was subsequently optimized to afford the potent, selective, and orally bioavailable compound 14. Oral administration at doses ≥0.03 mg/kg significantly reduced postprandial triglycerides in mice following an oral lipid challenge. Further assessment in both acute and chronic safety pharmacology and toxicology studies demonstrated a clean profile up to high plasma levels, thus culminating in the nomination of 14 as clinical candidate ABT-046.

Full text of DOI:10.1021/jm201524g

Article
pubs.acs.org/jmc
Identification and Preliminary Characterization of a Potent, Safe, and
Orally Efficacious Inhibitor of Acyl-CoA:Diacylglycerol
Acyltransferase 1
Vince S. C. Yeh, David W. A. Beno, Sevan Brodjian, Michael E. Brune, Steven C. Cullen, Brian D. Dayton,
Madhup K. Dhaon, Hugh D. Falls, Ju Gao, Nelson Grihalde, Philip Hajduk, T. Matthew Hansen,
Andrew S. Judd, Andrew J. King, Russel C. Klix, Kelly J. Larson, Yau Y. Lau, Kennan C. Marsh,
Scott W. Mittelstadt, Dan Plata, Michael J. Rozema, Jason A. Segreti, Eric J. Stoner, Martin J. Voorbach,
Xiaojun Wang, Xili Xin, Gang Zhao, Christine A. Collins, Bryan F. Cox, Regina M. Reilly, Philip R. Kym,
and Andrew J. Souers*
Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064-6100,
United States
S
* Supporting Information
ABSTRACT: A high-throughput screen against human DGAT-1 led to the identification of a
core structure that was subsequently optimized to afford the potent, selective, and orally
bioavailable compound 14. Oral administration at doses ≥0.03 mg/kg significantly reduced
postprandial triglycerides in mice following an oral lipid challenge. Further assessment in both
acute and chronic safety pharmacology and toxicology studies demonstrated a clean profile up
to high plasma levels, thus culminating in the nomination of 14 as clinical candidate ABT-046.
knockout mice.12 Additionally, Pfizer13 and Novartis14 have
advanced DGAT-1 inhibitors into the clinic for diabetes and
INTRODUCTION
Excess accumulation of triglycerides in the blood and in tissues
■
dyslipidemia, respectively; thus, results from larger powered
is a hallmark of diverse metabolic disturbances including
trials may provide human proof of concept for this novel
atherogenic dyslipidemia, insulin resistance, and hepatic
steatosis. In addition, deposition of triglycerides in adipose
tissue causes obesity, and evidence is mounting that the
accumulation of triglycerides in nonadipose tissue is a principal
mechanism in the near future.
We previously reported the identification and character-
ization of compound A-922500 (1), which was used as a tool to
provide early preclinical validation for the hypothesis that
cause of the constellation of cardiovascular risk factors
associated with the Metabolic Syndrome.1 The final and only
DGAT-1 inhibitors could induce weight loss in DIO mice and
lower triglyceride excursions in the postprandial state in
committed step in the synthesis of triglycerides is common to
multiple rodent models (Figure 1).15−17 To identify additional
both major triglyceride biosynthesis pathways and is catalyzed
by acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes.2
pharmacophores that could serve as inhibitors of DGAT-1, we
conducted a high-throughput screen (HTS) against a
This family is comprised of DGAT-1 and DGAT-2, which share
limited homology.3,4
DGAT-1, in particular, has received significant attention as a
recombinant form of the human enzyme. Compounds 2 and
3 were identified as hits with >50% enzyme inhibition at 10
μM, and subsequent validation experiments determined IC50
values of 1.58 and 0.044 μM, respectively. While the more
active analogue 3 was rapidly metabolized in human liver
therapeutic target for cardiometabolic diseases following the
disclosure of the DGAT-1 knockout mice phenotype.5,6 Mice
lacking the DGAT-1 gene were shown to be viable and resistant
to the effects of diet-induced obesity (DIO) and hepatic
steatosis when fed a high-fat diet. Additionally, these animals
were reported to have an increased sensitivity to both insulin
and leptin, decreased levels of tissue triglycerides, and increased
energy expenditure. Further studies demonstrated that DGAT-
microsomes (HLM), we were intrigued by the high binding
efficiency (BE) of this compact inhibitor.18
The 40-fold increase in enzyme potency of analogue 3
relative to 2 highlighted the critical nature of the fused
pyrazolo[1,5-a]pyrimidine ring system in the former com-
1−/− mice had dramatically reduced levels of intestinal
pound. Additionally, a rapid scan of des-amino, methylamino,
and dimethylamino analogues indicated that the exocyclic 7-
triglyceride synthesis and chylomicron secretion following an
oral lipid challenge.7−9
amino moiety was essential, as all three compounds showed
Since the initial disclosure of DGAT-1 inhibitors by Bayer10
significant diminution of enzyme inhibition (data not shown).
and the Tularik/Japan Tobacco group,11 several companies
have reported orally active inhibitors of this enzyme that
Received: December 2, 2011
Published: January 20, 2012
recapitulate several metabolic aspects of the DGAT-1−/−
© 2012 American Chemical Society
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Figure 1. DGAT-1 inhibitor A-922500 (1) and two structurally related hits from an HTS against human DGAT-1.
Figure 2. Synopsis of the design of putative DGAT-1 inhibitor 14.
a
Table 1. DGAT-1 Inhibition and Microsomal Stability of Selected Compounds
b
c
compd
R
hDGAT-1 IC50 (μM)
HLM (% parent)
3
4
4-tert-butyl
H
0.0440
>10.0
1.86
16
ND
<5
5
4-bromo
6
3-bromo
>10.0
>10.0
0.250
0.110
>10.0
<5
7
2-bromo
69
8
4-isopropyl
4-cyclohexyl
4-carboxylic acid
4-methylsulfone
<5
9
ND
100
91
10
11
>10.0
a
b
1
All compounds were >95% pure by HPLC and characterized by H NMR and MS. Values are the means of a series of separate assays, each
c
performed in triplicate. 60 min. ND, not determined.
This prompted us to retain the 6-phenylpyrazolo[1,5-a]-
pyrimidin-7-amine pharmacophore as a starting point for
optimization while rapidly probing the importance of the
hydrophobic tert-butyl moiety. To this end, we prepared the
nonsubstituted phenyl analogue 4, as well as analogues with a
simple hydrophobe (bromine) placed systematically around the
terminal phenyl ring. As shown in Table 1, phenyl analogue 4
was inactive against DGAT-1, as were the meta-bromo and
ortho-bromo substituted analogues 6 and 7. The para-bromo
analogue 5 showed an IC50 of 1.86 μM, thus confirming the
preference for substitution at this position. In HLM, the
metabolic stabilities of these compounds were similar or worse
than the hit structure 3, with the interesting exception of ortho-
bromo-substituted analogue 7.
in DGAT-1 inhibition (8), whereas the cyclohexyl derivative
(9) was within 2-fold of analogue 3. Similar to the parent
structure, both analogues possessed very poor metabolic
stability in HLM. While analogues with ionizable (10) or
polarizeable (11) groups at the para position were devoid of
DGAT-1 inhibition, both carboxylic- and sulfone-containing
moieties conferred excellent stability in HLM, thus indicating
the beneficial effect of increased polarity on this metabolic
parameter.19
Given the clear requirements of hydrophobic para-
substitution for obtaining good enzyme inhibition and polar
terminal substitution for obtaining acceptable microsomal
stability, we were intrigued by the possibility of merging
these two structural features via an extended linker region to
afford putative inhibitor 14 (Figure 2).20 This was additionally
inspired by the literature compound 13, which was first
disclosed in 2004 by the Tularik/Japan Tobacco group and has
We next performed a rapid scan of analogues with
hydrophobic and hydrophilic substitutions at the para position
(Table 1). Substitution of a terminal isopropyl group for the
tert-butyl moiety in 3 lead to an approximately 6-fold decrease
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subsequently served as the structural foundation of multiple
series from other companies.13,21
of hit structure 3. Additionally, this compound showed no
inhibition against human DGAT-2 and inhibited triglyceride
formation in HeLa cells expressing human DGAT-1 with an
IC50 of 78 nM. Compound 14 demonstrated negligible
turnover in microsome preparations from mouse and human
livers, indicating that the hybrid design conferred both potent
enzyme inhibition and microsomal stability. Finally, 14
exhibited high in vitro permeability values in Caco-2 cells
with no evidence of active efflux (efflux ratio = 1.4 and 1.1 at
0.5 and 5 μM, respectively).
CHEMISTRY
■
Analogues 3−11 were prepared according to Scheme 1. Briefly,
known benzyl nitriles (12, Scheme 1) were condensed with
a
Scheme 1.
We next assessed the pharmacokinetic properties of 14 in
mouse and rat in advance of acute efficacy studies (Table 3).
The properties following a single intravenous dose were
characterized by low volumes of distribution and plasma
clearance values and moderate terminal plasma elimination half-
lives of 4.6 and 3.8 h in mouse and rat, respectively. The oral
absorption was high for both species, affording excellent levels
of bioavailability.
To investigate the in vivo effects of specific inhibition of
intestinal DGAT-1, compound 14 was evaluated in a model of
postprandial hyperlipidemia measuring chylomicron-derived
triglycerides. CD-1 mice were fasted overnight and then
administered a single dose of 0.03, 0.3, or 3 mg/kg via oral
gavage. One hour later, the mice were given an oral corn oil
bolus (6 mL/kg), and serum triglycerides and drug
concentrations were measured after 2 h. As demonstrated in
Figure 3A, 14 showed a dose-dependent reduction in serum
triglycerides starting at 0.03 mg/kg and increasing through the
higher doses (40, 60, and 90% reduction from vehicle at 0.03,
0.3, and 3.0 mg/kg, respectively). The ascending pharmacody-
namics correlated well with a linear increase in plasma exposure
going from 0.03 to 3 mg/kg (C2h = 0.033, 0.36, and 3.10 μg/
mL at 0.03, 0.3, and 3.0 mg/kg, respectively).
a
(i) 2-tert-Butoxy-N,N,N′,N′-tetramethylethane-1,1-diamine, toluene,
reflux. (ii) 3-Aminopyrazole, HOAc, toluene, reflux.
Bredereck's reagent in toluene and subsequently condensed
with 3-aminopyrazole.
The synthesis of compound 14 (Scheme 2) was initiated via
Horner−Wadsworth−Emmons olefination of 4-phenylcyclo-
hexanone to afford the α,β-unsaturated ester 15. Subsequent
hydrogenation afforded intermediate 16a as a mixture of
isomers (2.4/1, trans/cis). Following saponification, the trans
isomer was then preferentially crystallized as a lithium salt and
re-esterified to provide pure ester 16b in 60% yield. Friedel−
Crafts acylation afforded the corresponding acid chloride,
which was hydrogenated over palladium on carbon to furnish
the benzylic alcohol 17. Subsequent bromination and displace-
ment via sodium cyanide provided nitrile 18 in 80% yield.
Condensation with Bredereck's reagent in refluxing toluene
followed by a second condensation with 2-amino pyrazole
afforded the penultimate ester 19. Finally, base-mediated ester
hydrolysis and acidification furnished the desired analogue 14
in 76% yield.
We subsequently measured the ability of DGAT-1 inhibitor
14 to reduce serum triglycerides throughout the time span
covering the postprandial excursion in DIO mice. A dose of 0.3
mg/kg, which showed significant efficacy in nonobese CD-1
mice, was chosen. One hour after oral dosing of the DGAT-1
inhibitor, a corn oil bolus (6 mL/kg) was administered, and
serum triglycerides subsequently were measured each hour for
RESULTS AND DISCUSSION
■
As shown in Table 2, hybrid structure 14 showed potent
inhibition against both human and mouse isoforms of DGAT-1
(IC50 = 8 nM) and retained much of the high BE (BE = 23.1)
a
Scheme 2.
a
(i) Triethyl phosphonoacetate, NaOtBu, THF. (ii) H2, Pd(C), EtOH. (iii) LiOH, EtOH/H2O. (iv) EtOH, H2SO4. (v) AlCl3, oxalyl chloride, DCM.
(vi) H2, Pd (C), TEA, THF. (vii) HBr, HOAc. (viii) NaCN, ACN/water, 40 °C. (ix) 2-tert-Butoxy-N,N,N′,N′-tetramethylethane-1,1-diamine,
toluene, reflux and then 3-aminopyrazole, HOAc, toluene, reflux. (x) NaOH, MeOH, THF, 50 °C.
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Table 2. Selected Enzyme Inhibition and in Vitro ADME Results for Compound 14
microsomal stability (%
a
ab
c
d
DGAT IC50 (nM)
triglyceride synthesis IC50 (nM)
HeLa cells
parent)
permeability
hDGAT-1
8
mDGAT-1
8
hDGAT-2
>10000
HLM
97
MLM
99
Caco-2 (cm/s)
78
84 × 10−6
a
b
Values are the means of a series of separate assays, each performed in triplicate. Inhibition of triglyceride synthesis determined in HeLa cells. See
c
d
the Supporting Information for experimental details and references. 60 min. 0.5 μM, pH 7.4.
a
Table 3. Selected Pharmacokinetic Properties of 14
mouse (10 mg/kg)
rat (5 mg/kg)
b
iv
T1/2 (h)
4.6
0.3
0.1
3.8
Vss (L/kg)
Clp (L/h/kg)
0.3
0.05
b
po
T1/2 (h)
5.1
17.4
151
78
5.6
9.3
Cmax (μg/mL)
AUC (μg h/mL)
F (%)
130
91
Figure 4. Serum triglyceride concentrations measured in overnight
fasted DIO mice pretreated with vehicle or DGAT-1 inhibitor 14 (0.3
mg/kg) prior to and for 5 h after an oral gavage of corn oil (n = 4/
group). *p < 0.05 as compared to vehicle.
a
All values are mean values SEMs (n = 3 unless specified otherwise).
1% Tween-80 in water.
b
5 h following the lipid challenge. As demonstrated in Figure 4,
oral administration of 14 afforded a sustained reduction in
serum triglyceride concentrations throughout the experiment.
The favorable in vitro and pharmacokinetic properties of
compound 14 in addition to the potent triglyceride reductions
observed in models of postprandial hyperlipidemia prompted
further assessment of this compound in acute and chronic
secondary pharmacology assays. In a battery of 80 G-protein-
coupled receptors, enzymes, and ion channels, 14 showed no
inhibition greater than 80% at 10 μM (CEREP). Negligible
blockade of the ion channel derived from the human ether-a-
go-go-related gene (hERG) gene was observed in a manual
patch clamp assay (IC50 > 300 μM), and no prolongation of the
APD90 was observed up to the highest concentration tested in a
dog Purkinje fiber repolarization assay. As an assessment of
cardiovascular safety, 14 was infused in three ascending doses
into comprehensively instrumented anesthetized dogs (n = 6).
No physiologically relevant hemodynamic effects were
observed through the highest achieved plasma concentration
of 20.47 μg/mL. Additionally, no emesis was observed in a
ferret model, nor were any changes in GI transit noted in rats
through the highest plasma concentrations achieved (30.19 μg/
mL). Finally, 2 weeks of oral dosing in rat and dog afforded no
significant toxicological findings up to terminal concentrations
of 3455 μg h/mL (female) and 789 μg h/mL, respectively.
CONCLUSION
■
In summary, optimization of the HTS hit 3 lead to the design
and preparation of hybrid structure 14. This compound is a
potent and selective inhibitor of DGAT-1 and possesses good
oral pharmacokinetics in rodents. Upon oral administration,
significant reduction of postprandial triglycerides was observed
at doses as low as 0.03 mg/kg in CD-1 mice, and a single dose
at 0.3 mg/kg abolished the postprandial triglyceride excursion
in DIO mice. Compound 14 shows little cross-reactivity at the
hERG channel and other off-targets as assessed by CEREP and
no remarkable effects in acute in vivo assays for cardiovascular
function, GI transit, and emesis. Similarly, no general toxicology
findings were noted up to high plasma levels following
prolonged dosing in rat and dog species. As a result of the
favorable efficacy and safety profile, compound 14 was
advanced to clinical candidacy. A further description of the in
vivo profiling in multiple models, cross-species metabolism, and
Figure 3. (A) Serum triglyceride concentrations measured in overnight fasted, untreated CD-1 mice and 2 h after an oral gavage of corn oil in mice
pretreated with vehicle or DGAT-1 inhibitor 14 at 0.03, 0.3, and 3 mg/kg (n = 10/group). #p < 0.05 as compared to fasted, **p < 0.05 as compared
to vehicle. (B) Plasma concentrations of 14 measured 3 h after oral dosing and 2 h after the oral lipid challenge.
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solution of 16b [54.4 g, 46.9 wt %, 25.5 g of product, 94% yield against
a standard prepared by bulb to bulb distillation (0.2 Torr, 150 °C oven
temperature), ratio 99:1 trans- to cis-]. 1H NMR (400 MHz, CDCl3): δ
7.31−7.10 (m, 5H), 4.13 (q, J = 7.1 Hz, 2H), 2.46 (tt, J = 12.3, 3.3 Hz,
1H), 2.23 (d, J = 6.7 Hz, 2H), 1.98−1.80 (m, 5H), 1.58−1.42 (m,
2H), 1.26 (t, J = 7.1 Hz, 4H), 1.23−1.07 (m, 2H). 13C NMR (101
MHz, CDCl3): δ 172.43, 146.85, 127.96, 126.45, 125.58, 60.20, 44.22,
42.22, 34.77, 34.09, 33.40, 14.61. MS (DCI − NH3) (M + 18) 262.2
m/e. FTIR 2923, 1694, 1293, 695 cm−1.
projected human pharmacokinetics will be disclosed in due
course.
EXPERIMENTAL SECTION
■
General. Proton NMR spectra were obtained on a Varian Mercury
plus 300 or Varian UNITY plus 300 MHz instrument with chemical
shifts (δ) reported relative to tetramethylsilane as an internal standard.
Target compounds possess a purity of at least 95% based on
combustion analysis (< 0.4), performed by QTI (Quantitative
Technologies, Inc.). Column chromatography was carried out either
with an Analogix IntelliFlash 280 chromatography apparatus using
SuperFlash Compound Purification Columns or on silica gel 60 (230−
400 mesh). Thin-layer chromatography was performed using 250 mm
silica gel 60 glass-backed plates with F254 as indicator.
trans-Ethyl 2-4-(4-(Hydroxymethyl)phenyl)cyclohexyl)-
acetate (17). To a slurry of AlCl3 (31.2 g, 234 mmol) in
dichloromethane (250 mL) was added a solution of ester 16b in
heptanes (54.2 g, 51.1% w/w, 112 mmol) while maintaining the
temperature below 5 °C. The flask was again cooled to −5 °C, and
oxalyl chloride (17.8 g, 140 mmol) was added while the temperature
was maintained below 10 °C. The solution was stirred for 45 min at
which time the reaction was complete by HPLC. The reaction mixture
was poured slowly into an aqueous solution of CaCl2 (13 g of CaCl2 in
170 mL of water) precooled to −5 °C. The internal temperature of the
quench was maintained at or below 10 °C, and the quench solution
was stirred at room temperature for 30 min. The organic layer was
separated, and the aqueous layer was extracted with DCM (50 mL).
The combined organic layers were washed with CaCl2 (13 g CaCl2 in
170 mL of water). The organic layer was then dried over MgSO4 and
filtered sequentially through Celite and a carbon-impregnated filter.
The solution was concentrated under reduced pressure to 75 g and
chased with approximately 250 g of THF to give the product as a
Ethyl 2-(4-Phenylcyclohexylidene)acetate (15). To a solution
of NaOtBu (17.0 g, 176.9 mmol, 1.1 equiv) in THF (100 mL) was
added triethyl phosphonoacetate (39.6 g, 176.6 mmol, 1.1 equiv)
dropwise over 1 h at 0 °C, and the resulting mixture was stirred at 0 °C
for 1 h. A THF (100 mL) solution of 4-phenylcyclohexanone (28.0 g,
160.7 mmol, 1.0 equiv) was then added dropwise over 1 h, and the
reaction mixture was warmed to 25 °C and stirred for 90 min. Upon
completion of the reaction as indicated by HPLC, heptane (100 mL)
and water (100 mL) were added, the contents were mixed for 10 min,
and the layers were separated. The organic layer was washed with
aqueous NaHCO3 (6.5 wt %, 100 mL) and water (100 mL) and
concentrated to a volume of ∼80 mL by rotary evaporation. EtOH
(100 mL) was added to the residue, and the resultant solution was
reconcentrated to a volume of ∼80 mL by rotary evaporation. This
process was repeated to afford an ethanolic solution of ethyl 2-(4-
phenylcyclohexylidene)acetate (74.6 g, 52.0 wt %, 38.8 g product, 98%
yield against a standard prepared by bulb to bulb distillation (0.2 Torr,
150 °C oven temperature). 1H NMR (400 MHz, CDCl3): δ 7.33−7.10
(m, 5H), 5.67 (s, 1H), 4.15 (q, J = 7.1 Hz, 2H), 4.03−3.92 (m, 1H),
2.77 (tt, J = 12.2, 3.4 Hz, 1H), 2.43−2.27 (m, 2H), 2.10−1.96 (m,
3H), 1.72−1.54 (m, 2H), 1.28 (t, J = 7.1 Hz, 3H). 13C NMR (101
MHz, CDCl3): δ 166.13, 161.22, 145.52, 128.06, 126.41, 125.84,
113.47, 59.60, 44.17, 37.83, 35.72, 34.94, 29.63, 14.61. MS (DCI −
NH3) (M + 18) 262.2 m/e FTIR 2930, 1709, 1647, 1141, 698 cm−1.
Anal. calcd for C16H22O2: C, 78.65%; H, 8.25%. Found: C, 78.72%; H,
8.46%.
1
33.7% w/w solution in THF (26.0 g, 79%). H NMR (400 MHz,
CDCl3): δ 8.05 (d, J = 8.5, 2H), 7.32 (d, J = 8.5, 2H), 4.41 (dd, J =
14.2, 7.11, 2H), 2.61−2.54 (m, 1H), 2.25 (d, J = 6.8, 2H), 1.97−1.85
(m, 4H), 1.57−1.47 (m, 2H), 1.27 (t, J = 7, 3H), 1.24−1.11 (m, 2H).
A 250 mL stainless steel pressure canister was charged with THF (19
g, 13 volumes), acid chloride (8.77 g, 16.6% w/w, 4.7 mmol, 1 equiv),
triethylamine (2.0 g, 2.75 mL, 19.8 mmol, 2 equiv), and 5% Pd/C
(0.62 g) and was agitated for 18 h at room temp under an atmosphere
of H2 (40 psi). The reaction was monitored by HPLC. Upon
completion, the catalyst was filtered away using a 0.45 μM filter rinsing
with THF (5 g, 3.5 volumes) to give the desired alcohol as a solution
in THF (38 g, 3.67% w/w) in 50.1% assay yield. 1H NMR (400 MHz,
CDCl3) δ 7.28 (d, J = 8.2, 2H), 7.19 (d, J = 8.2, 2H), 4.65 (s, 1H),
4.13 (dd, J = 7.1, 7.1, 2H), 2.51−2.41 (m, 1H), 2.23 (d, J = 6.7, 2H),
1.93−1.85 (m, 4H), 1.70−1.45 (m, 4H), 1.27 (t, J = 5.0, 3H), 1.22−
1.11 (m, 2H).
trans-Ethyl 2-4-(4-(Cyanomethyl)phenyl)cyclohexyl)acetate
(18). A solution of alcohol 17 in THF (483 g, 1.79% w/w, 33
mmol) was diluted with MTBE (130 g, 15 volumes) and washed
sequentially with 1% Na2CO3 aqueous solution (2 × 60 mL), 2 N
aqueous solution of HCl (2 × 130 mL), water (2 × 130 mL), and a 5%
aqueous NaCl solution (130 mL). The organic layer was dried over
MgSO4. The solution was concentrated under reduced pressure to
afford an oil that was dissolved in AcOH (26 mL), and a 33% aqueous
solution of HBr (13 mL, 3 equiv) was added with stirring. The
reaction mixture was stirred for 1.5 h, and the progress was monitored
by HPLC. Upon completion of the reaction, water (160 mL) was
added slowly to the stirring reaction mixture during which time a
brown precipitate formed. The solids were filtered, washed with water
(50 mL), 2.5% NaHCO3 (50 mL), and water (50 mL), and dried in an
oven overnight at 50 °C to give the desired product as a white solid
(8.6 g, 80.9% w/w, 88% yield). 1H NMR (400 MHz, CDCl3): δ 7.3 (d,
J = 8.2, 2H), 7.16 (d, J = 8.2, 2H), 4.48 (s, 2H), 4.14 (dd, J = 14.2, 7.1,
2H), 2.52−2.40 (m, 1H), 2.23 (d, J = 6.7, 2H), 1.95−1.83 (m, 5H),
1.57−1.42 (m, 2H), 1.27 (t, J = 7.1, 3H), 1.22−1.09 (m, 2H). A
solution of the resultant bromide (10.1 g, 29.9 mmol) in acetonitrile
(90 mL) was warmed to 40 °C, and an aqueous solution of NaCN (4.9
g, 100 mmol) in water (10 mL) was added over 15 min while keeping
the internal temperature below 50 °C. The reaction mixture was
stirred at 40 °C for 6 h. The reaction was monitored by HPLC. Upon
completion, the mixture was cooled to room temperature and
partitioned between water (225 mL) and MTBE (100 mL). The
aqueous layer was separated and extracted with MTBE (60 mL). The
trans-Ethyl 2-4-(4-(Hydroxymethyl)phenyl)cyclohexyl)-
acetate (16b). A solution of ethyl 2-(4-phenylcyclohexylidene)-
acetate (38.8 g) in ethanol (200 mL) was hydrogenated over 5%
Pd(C) at 50 °C and 40 psi for 2.5 h, filtered, and concentrated by
rotary evaporation to a volume of approximately 120 mL to afford a
2.4/1 mixture of trans and cis isomers. An aqueous solution of LiOH
(20.0 g in 100 g water) was added, and the resulting reaction mixture
was warmed to 50
5 °C. After 1 h, the trans Li-carboxylate
crystallized from the reaction mixture as a thick slurry, and additional
water (287 g) was added to facilitate mixing, which continued at 50
5 °C for an additional 6 h. Analysis by HPLC revealed that the
hydrolysis was complete, and the reaction mixture was cooled to room
temperature. The trans Li salt was collected by filtration and washed
with aqueous 5% LiOH (2 × 50 g). The wet cake was partitioned
between EtOAc (250 g) and 6 N HCl (100 g), and the suspension was
mixed until all solids were dissolved. After separation of the layers, the
EtOAc layer was concentrated by rotary evaporation, and the residue
was treated with EtOH (100 g), which was then removed in the same
manner. The residue was dissolved in EtOH (300 mL), and H2SO4
(0.3 mL) was added. The reaction mixture was heated to reflux and
mixed overnight. After this time, HPLC analysis revealed that the re-
esterification was complete, and the solution was cooled to room
temperature. After it was concentrated to a volume of approximately
60 mL by rotary evaporation, heptane (195 g) was added, the layers
were separated, and the organic layer was washed with saturated
aqueous NaHCO3 (50 mL) and brine (50 mL). The organic layer was
reduced to a volume of approximately 60 mL by rotary evaporation.
Heptane (100 g) was added, and the resulting solution was
concentrated to ∼60 mL by rotary evaporation affording a heptane
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combined organic extracts were washed with brine (2 × 100 mL),
treated with active charcoal, and filtered through MgSO4 and Celite.
The MTBE solution was concentrated to by rotary evaporation, and
the residue was crystallized from MTBE/heptane (1/3), filtered,
washed with heptane, and dried in a vacuum oven to afford the
terminal His6-epitope tag was produced in the baculovirus expression
system. Insect cells (e.g., Sf 9 or High Five) were infected for 24−72 h
and collected by centrifugation. Cell pellets were resuspended in
homogenization buffer [250 mM sucrose, 10 mM Tris-HCl (pH 7.4),
and 1 mM EDTA] and lysed using a homogenization apparatus, such
as a Microfluidizer (single pass, 4 °C). Cell debris was removed by
centrifugation at 10000g for 30 min, and microsomal membranes were
collected by ultracentrifugation at 100000g for 30 min. Mouse DGAT-
1 was also produced in insect cells and purified by the same
procedures as described above.
1
product as a white crystalline solid (5.7 g, 67%); mp 70−77 °C. H
NMR (400 MHz, CDCl3): δ 7.25−7.15 (m, 4H), 4.13 (q, J = 7.1 Hz,
2H), 3.69 (s, 2H), 2.47 (tt, J = 12.2, 3.0 Hz, 1H), 2.23 (d, J = 6.8 Hz,
2H), 1.95−1.79 (m, 5H), 1.58−1.42 (m, 2H), 1.26 (t, J = 7.1 Hz, 3H),
1.22−1.09 (m, 2H). 13C NMR (101 MHz, CDCL3): δ 172.24, 146.77,
127.49, 127.13, 126.99, 117.69, 60.12, 43.72, 42.04, 34.58, 33.94,
33.18, 23.35, 14.53. MS (DCI − NH3) (M + 18) 303.2 m/e. FTIR
2918, 2248, 1725, 1160, 826 cm−1. Anal. calcd for C18H22NO2: C,
75.76%; H, 8.12%. Found: C, 75.78%; H, 8.33%.
The DGAT-1 activity was determined as follows: Assay buffer [20
mM HEPES (pH 7.5), 2 mM MgCl2, and 0.04% BSA] containing 50
μM enzyme substrate (didecanoyl glycerol) and 7.5 μM radiolabeled
acyl-CoA substrate.[1-14C]decanoyl-CoA) was added to each well of a
phospholipid FlashPlate (PerkinElmer Life Sciences). A small aliquot
of membrane (1 μg/well) was added to start the reaction, which was
allowed to proceed for 60 min. The reaction was terminated upon the
addition of an equal volume (100 μL) of isopropanol. The plates were
sealed, incubated overnight, and counted the next morning on a
TopCount Scintillation Plate Reader (PerkinElmer Life Science).
DGAT-1 catalyzes the transfer of the radiolabeled decanoyl group
onto the sn-3 position of didecanoyl glycerol. The resultant
radiolabeled tridecanoyl glycerol (tricaprin) preferentially binds to
the hydrophobic coating on the phospholipid FlashPlate. The
proximity of the radiolabeled product to the solid scintillant
incorporated into the bottom of the FlashPlate induced fluor release
from the scintillant, which was measured in the TopCount Plate
Reader. Various concentrations (e.g., 0.0001, 0.001, 0.01, 0.1, 1.0, and
10.0 μM) of the representative compounds were added to individual
wells prior to the addition of membranes. The potencies of DGAT-1
inhibition for the compounds were determined by calculating the IC50
values defined as the inhibitor concentration from the sigmoidal dose−
response curve at which the enzyme activity was inhibited 50%.
In Vivo Characterization. All protocols were approved by the
Abbott Laboratories Institutional Animal Care and Use Committee
and performed in accordance with the National Institutes of Health
Guide for the Care and Use of Laboratory Animals. All animals were
housed under standard laboratory conditions with a 12 h light/dark
cycle, in a temperature- and humidity-controlled room. Male CD-1
mice were obtained from Charles River Laboratories (Portage, MI).
DIO was induced in male C57BL/6J mice (Jackson Laboratories, Bar
Harbor, ME) by feeding a high-fat diet [D-12492i (60 kcal% fat)
Research Diets] for 18 weeks beginning at 5 weeks of age. After
shipment to our facilities, they were acclimated to a pathogen-free
barrier facility with a 12 h light/12 h dark cycle, single housed in small
mouse shoebox cages with bedding, enrichment, and standard mouse
water limits, and were fed the same high-fat diet until reaching greater
than 40 g of body weight, usually at the age of 22 weeks. All animals
were treated in accordance with IACUC guidelines and the Guide for
the Care and Use of Laboratory Animals. Serum triglycerides were
measured in the clinical pathology laboratory at Abbott Laboratories
on an Aeroset c8000 clinical chemistry analyzer (Abbott Laboratories,
Abbott Park, IL) using photometric methods.
trans-Ethyl 2-(4-(4-((Z)-1-Cyano-2-(dimethylamino)vinyl)-
phenyl)cyclohexyl)acetate (19). To a 250-mL, three-necked,
round bottomed flask was charged nitrile 18 (13.4 g, 35.5 mmol),
toluene (100 mL), and Bredereck's reagent (20.42 g, 3.3 equiv), and
the reaction mixture was heated to 70 °C. The mixture was monitored
by HPLC until the starting material was consumed. The reaction
mixture was then cooled to 40 °C, and acetic acid (51.04 g, 24 equiv)
and 3-aminopyrazole (5.88 g, 2.0 equiv) in 4 mL of CH3CN were
added. The reaction mixture was heated to 75 5 °C for 21 h. The
mixture was monitored by HPLC until the starting material was
consumed. The acetic acid was then removed by azeotropic distillation
with toluene, and the solvent was switched to acetonitrile by
concentration and chasing with acetonitrile on the rotary evaporator.
The residue was suspended in acetonitrile (200 mL), heated to reflux,
and then slowly cooled to 10 °C. The product was collected by
filtration and washed with cold acetonitrile (20 mL), cold acetonitrile/
water (1/1, 20 mL), and cold water (20 mL). After it was dried in a
vacuum oven at 50 °C for 18 h, the product was obtained as a white
crystalline solid (10.9 g, 85%); mp 145−147 °C. 1H NMR (400 MHz,
DMSO): δ 8.14 − 8.06 (m, 2H), 7.42 (dd, J = 6.3, 1.9 Hz, 4H), 7.33
(d, J = 8.2 Hz, 2H), 6.44 (d, J = 2.3 Hz, 1H), 4.07 (q, J = 7.1 Hz, 2H),
2.58−2.43 (m, 1H), 2.23 (d, J = 6.7 Hz, 2H), 1.91−1.71 (m, 5H), 1.50
(tt, J = 13.5, 6.9 Hz, 2H), 1.27−1.07 (m, 2H), 1.20 (t, J = 7.1 Hz, 3H).
13C NMR (101 MHz, DMSO): δ 171.32, 149.46, 147.55, 145.38,
144.37, 143.29, 131.12, 128.54, 126.86, 101.10, 94.09, 59.49, 43.03,
41.20, 34.04, 33.42, 32.52, 14.30. FTIR 3282, 2920, 1725, 1532, 1091,
772 cm−1. Anal. calcd for C22H26N4O2: C, 69.82%; H, 6.92%; N,
14.80%. Found: C, 69.88%; H, 7.03%; N, 14.61%.
trans-{4-[4-(7-Aminopyrazolo[1,5-a]pyrimidin-6-yl)phenyl]-
cyclohexyl}acetic Acid (14). A solution of ester 19 (2.9 g, 7.7
mmol) in THF (35 mL) and MeOH (35 mL) was treated with a
solution of NaOH (0.92 g, 23 mmol) at 50 °C for 75 min. After this
time, the solution was cooled to room temperature, and the volume
was reduced by rotary evaporation to ∼35 mL. The suspension of
solids was heated to dissolve the solids and then cooled slowly to 2 °C
and held at this temperature for 1.5 h. The solid product was collected
by vacuum filtration and washed with water (10 mL). The wet cake
was dissolved in water (150 mL) and added slowly to a dilute solution
of HCl (0.25 M, 100 mL) at 50 °C. After it was cooled to room
temperature, the product was collected, washed with water (10 mL),
and dried in a vacuum oven at 50 °C for 18 h. The product (2.1 g,
1
78%) was obtained as a white crystalline solid; mp 264−266 °C. H
NMR (400 MHz, DMSO): δ 12.05 (s, 1H), 8.12 (d, J = 2.3 Hz, 1H),
8.10 (s, 1H), 7.44 (s, 2H), 7.38 (dd, J = 36.6, 8.2 Hz, 4H), 6.45 (d, J =
2.3 Hz, 1H), 2.57−2.43 (m, 1H), 2.16 (d, J = 6.9 Hz, 2H), 1.85 (d, J =
11.4 Hz, 4H), 1.76 (ddd, J = 15.6, 11.3, 7.6 Hz, 1H), 1.58−1.41 (m,
2H), 1.21−1.03 (m, 2H). 13C NMR (101 MHz, DMSO): δ 172.99,
149.48, 147.56, 145.47, 144.42, 143.33, 131.11, 128.56, 126.88, 101.15,
94.12, 43.09, 41.47, 33.91, 33.51, 32.65. MS (ESI) (M + 1) 351.3 m/e
FTIR 3315, 2919, 1702, 1289, 770 cm−1. Anal. calcd for C20H22N4O2:
C, 68.55%; H, 6.33%; N, 15.99%. Found: C, 68.74%; H, 6.59%; N,
15.90%.
Effect of DGAT-1 Inhibition on Postprandial Hyperlipidemia.
Either CD-1 or DIO mice were fasted overnight. Animals were
randomly assigned to receive either vehicle or DGAT-1 inhibitor 14 at
0.03, 0.3, or 3.0 mg/kg by oral gavage (6 mL/kg). One hour after
administration of vehicle or DGAT-1 inhibitor, all animals were given
an oral bolus of corn oil (6 mL/kg). Serum triglyceride levels were
then measured 2 h later in CD-1 mice and every hour for 5 h in DIO
mice.
Statistical Analysis. The effect of DGAT-1 inhibition on
postprandial serum triglyceride concentrations was assessed using a
one-way ANOVA, with posthoc multiple comparisons made using
Dunnett's procedure to compare each dose group (0.03, 0.3, and 3
mg/kg) to the vehicle group.
DGAT-1 Phospholipid Flasphplate Assay. The inhibition of
DGAT-1 was readily determined via a FlashPlate assay. The same
procedure was also used to assess activity of compounds against
DGAT-2. In this assay, recombinant human DGAT-1 containing an N-
1756
dx.doi.org/10.1021/jm201524g | J. Med. Chem. 2012, 55, 1751−1757

Journal of Medicinal Chemistry
Article
and use of aryl alkyl acid derivatives for the treatment of obesity.
US2005/07091228.
ASSOCIATED CONTENT
■
S
* Supporting Information
(11) Fox, B. M.; Furukawa, N. H.; Hao, X.; Lio, K.; Inaba, T.;
Jackson, S. M.; Kayser, F.; Labelle, M.; Kexue, M.; Matsui, T.;
McMinn, D. L.; Ogawa, N.; Rubenstein, S. M.; Sagawa, S.; Sugimoto,
K.; Suzuki, M.; Tanaka, M.; Ye, G. Yoshida, A.; Zhang, J. A.
Preparation of fused bicyclic nitrogen containing heterocycles, useful
in the treatment or prevention of metabolic and cell proliferative
diseases. WO 2004/047755A2, CAN 141:38623.
(12) Birch, A. M.; Buckett, L. K.; Turnbull, A. V. DGAT1 inhibitors
as anti-obesity and anti-diabetic agents. Curr. Opin. Drug Discovery Dev.
2010, 13 (4), 489−496.
Experimental details for the syntheses of compounds 3−11, in
vitro ADME, and pharmacological details/characterization of
14. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 847-937-5312. Fax: 847-935-5165. E-mail: Andrew.
souers@abbott.com.
(13) Dow, R. L.; Li, J.-C.; Pence, M. P.; Gibbs, E. M.; LaPerle, J. L.;
Litchfield, J.; Piotrowski, D. W.; Munchhof, M. J.; Manion, T. B.;
Zavadoski, W. J.; Walker, G. S.; McPherson, R. K.; Tapley, S.;
Sugarman, E.; Guzman-Perez, A.; Dasilva-Jardine, P. Discovery of PF-
04620110, a Potent, Selective, and Orally Bioavailable Inhibitor of
DGAT-1. ACS Med. Chem. Lett. 2011, 2, 407−412.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(14) http://clinicaltrials.gov/ct2/show/NCT01146522?term=LCQ-
908&rank=1.
1
We thank Jan Waters for H NMR analysis and support and
Michael Wendt for valuable discussion.
(15) 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.; Raven, 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. Validation of diacyl glycerolacyl-
transferase 1 as a novel target for the treatment of obesity and
dyslipidemia using a potent and selective small molecule inhibitor. J.
Med. Chem. 2008, 51, 380−383.
ABBREVIATIONS USED
■
DGAT, acyl-CoA:diacylglycerol acyltransferase; HTS, high-
throughput screen; hDGAT, human DGAT; mDGAT, mouse
DGAT-1; BE, binding efficiency; HLM, human liver micro-
somes; MLM, mouse liver microsomes; hERG, human ether-a-
go-go-related gene; DIO, diet-induced obesity
(16) King, A. J.; Segreti, J. A.; Larson, K. J.; Souers, A. J.; Kym, P. R.;
Reilly, R. M.; Zhao, G.; Mittelstadt, S. W.; Cox, B. F. Diacylglycerol
acyltransferase 1 (DGAT-1) inhibition lowers serum triglycerides in
the Zucker fatty rat and the hyperlipidemic hamster. J. Pharmacol. Exp.
Ther. 2009, 330 (2), 526−531.
(17) King, A. J.; Segreti, J. A.; Larson, K. J.; Souers, A. J.; Kym, P. R.;
Reilly, R. M.; Collins, C. A.; Voorbach, M. J.; Zhao, G.; Mittelstadt, S.
W.; Cox, B. F. In vivo efficacy of acyl CoA:diacylglycerol
acyltransferase (DGAT) 1 inhibition in rodent models of postprandial
hyperlipidemia. Eur. J. Pharmacol. 2010, 637 (1−3), 155−161.
(18) Abad-Zapatero, C.; Metz, J. M. Ligand efficiency indices as
guideposts for drug discovery. Drug Discovery Today 2005, 10, 464−
469.
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1757
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