Discovery of 6-phenylpyrimido[4,5- B ][1,4]oxazines as potent and selective Acyl CoA: Diacylglycerol acyltransferase 1 (DGAT1) inhibitors with in vivo efficacy in rodents

Article abstract of DOI:10.1021/jm500135c

The discovery and optimization of a series of acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) inhibitors based on a pyrimido[4,5-b][1,4]oxazine scaffold is described. The SAR of a moderately potent HTS hit was investigated resulting in the discovery of phenylcyclohexylacetic acid 1, which displayed good DGAT1 inhibitory activity, selectivity, and PK properties. During preclinical toxicity studies a metabolite of 1 was observed that was responsible for elevating the levels of liver enzymes ALT and AST. Subsequently, analogues were synthesized to preclude the formation of the toxic metabolite. This effort resulted in the discovery of spiroindane 42, which displayed significantly improved DGAT1 inhibition compared to 1. Spiroindane 42 was well tolerated in rodents in vivo, demonstrated efficacy in an oral triglyceride uptake study in mice, and had an acceptable safety profile in preclinical toxicity studies.

Full text of DOI:10.1021/jm500135c

Article
pubs.acs.org/jmc
Discovery of 6‑Phenylpyrimido[4,5‑b][1,4]oxazines as Potent and
Selective Acyl CoA:Diacylglycerol Acyltransferase 1 (DGAT1)
Inhibitors with in Vivo Efficacy in Rodents
Brian M. Fox,*,† Kazuyuki Sugimoto,‡ Kiyosei Iio,‡ Atsuhito Yoshida,‡ Jian (Ken) Zhang,† Kexue Li,†
Xiaolin Hao,† Marc Labelle,† Marie-Louise Smith,† Steven M. Rubenstein,† Guosen Ye,† Dustin McMinn,†
Simon Jackson,† Rebekah Choi,† Bei Shan,† Ji Ma,† Shichang Miao,† Takuya Matsui,‡ Nobuya Ogawa,‡
Masahiro Suzuki,‡ Akio Kobayashi,‡ Hidekazu Ozeki,‡ Chihiro Okuma,‡ Yukihito Ishii,‡
Daisuke Tomimoto,‡ Noboru Furakawa,‡ Masahiro Tanaka,‡ Mutsuyoshi Matsushita,‡
Mitsuru Takahashi,‡ Takashi Inaba,‡ Shoichi Sagawa,‡,§ and Frank Kayser†,§
†Amgen Inc., 1120 Veterans Boulevard, South San Francisco, California 94080, United States
‡Central Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1, Murasaki-cho, Takatsuki, Osaka 569-1125, Japan
S
* Supporting Information
ABSTRACT: The discovery and optimization of a series of acyl
CoA:diacylglycerol acyltransferase 1 (DGAT1) inhibitors based on
a pyrimido[4,5-b][1,4]oxazine scaffold is described. The SAR of a
moderately potent HTS hit was investigated resulting in the
discovery of phenylcyclohexylacetic acid 1, which displayed good
DGAT1 inhibitory activity, selectivity, and PK properties. During
preclinical toxicity studies a metabolite of 1 was observed that was
responsible for elevating the levels of liver enzymes ALT and AST.
Subsequently, analogues were synthesized to preclude the
formation of the toxic metabolite. This effort resulted in the
discovery of spiroindane 42, which displayed significantly
improved DGAT1 inhibition compared to 1. Spiroindane 42
was well tolerated in rodents in vivo, demonstrated efficacy in an oral triglyceride uptake study in mice, and had an acceptable
safety profile in preclinical toxicity studies.
knockout mice (Dgat2−/−) die soon after birth because of
severe reductions in plasma triglycerides and total carcass
triglycerides.17 In contrast, DGAT1 knockout mice (Dgat1−/
−) are healthy and capable of synthesizing triglycerides.18
Dgat1−/− mice have been reported to be lean and remarkably
resistant to diet-induced obesity. When fed a high fat diet (21%
fat), Dgat1−/− mice maintain weights comparable to those of
mice fed a regular diet (4% fat). Total triglyceride levels are
lower in Dgat1−/− mice compared to wild type mice, and
Dgat1−/− mice have increased insulin and leptin sensitivity
compared to wild-type littermates. The resistance to diet-
induced obesity in Dgat1−/− mice is not due to decreased
caloric intake but rather is the result of increased energy
expenditure. The similar expression pattern of DGAT1 in
humans and mice and the predicted comparable role of the
enzyme have created considerable enthusiasm for the inhibition
of DGAT1 in vivo as a target for the treatment of obesity,
dyslipidemia, and diabetes in humans.19−23
INTRODUCTION
■
Elevated plasma and tissue triglyceride levels are implicated in
the pathogenesis of a variety of diseases and risk factors
including obesity, insulin resistance syndrome, type II diabetes,
dyslipidemia, hepatic steatosis, metabolic syndrome, and
coronary heart disease.1−8 The modulation of triglyceride
synthesis therefore constitutes an attractive strategy for
therapeutic intervention, and enzymes within this pathway
have emerged as potential molecular targets.9 Acyl CoA:d-
iacylglycerol acyltransferase (DGAT) enzymes catalyze the final
and only committed step in triglyceride synthesis, the joining of
1,2-diacylglycerol and fatty acyl CoA at the endoplasmic
reticulum.10,11 Two DGAT enzymes, DGAT1 and DGAT2,
have been identified.12−14 Although both enzymes catalyze the
formation of triglycerides (TG) from diacylglycerol and fatty
acyl CoA, they share limited homology. In fact, DGAT1 is part
of the acyl CoA:cholesterol acyltransferase (ACAT) gene family
with mouse DGAT1 sharing 20% identity with mouse
ACAT1.12 Recent evidence indicates that DGAT2 is respon-
sible for the synthesis of TG from endogenous fatty acids
whereas DGAT1 catalyzes the formation of TG using
exogenous fatty acids.15,16 This may explain why DGAT2
Received: January 27, 2014
Published: March 26, 2014
© 2014 American Chemical Society
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Figure 1. Selected examples of DGAT1 inhibitors containing the phenylcyclohexylacetic acid moiety.
Given the attractiveness of this target, it is not surprising that
an increasing number of small molecule DGAT1 inhibitors of
varying structural types have been reported in the liter-
ature.9,20,22−40 Several of these inhibitors have been used to
elucidate the pharmacology of DGAT1 inhibition in vivo and
have successfully demonstrated the ability to reproduce the
Dgat1−/− mouse phenotype in rodents. The observed in vivo
Figure 2. DGAT1 inhibitory activity of screening hit 2.
effects include decreased plasma TG levels after administration
of an oral bolus of oil, inhibition of the formation of TG in
adipose tissue, reduced hepatic lipid levels, enhanced GLP-1
secretion, delayed gastric emptying, reduced food intake,
improved insulin sensitivity, reduced TG, fatty acid, and
cholesterol levels in plasma, and reduced weight gain in diet-
induced obese mice. Since our initial disclosure of pyrimidoox-
azine 1,41 containing the phenylcyclohexylacetic acid function-
ality, several DGAT1 inhibitors that share this privileged
structure have been nominated for clinical development and
entered clinical trials (Figure 1).35,38,42−44 Recently, inves-
tigators from AstraZeneca reported results from their phase 1
study demonstrating that the treatment of healthy male subjects
with DGAT1 inhibitor AZD7687 resulted in markedly reduced
postprandial TG excursion in a dose-dependent manner.37a The
DGAT1 inhibitor discovered by Novartis, pradigastat
(LCQ908), is the most advanced clinical candidate and is
currently being evaluated in phase II and phase III studies.45
Novartis has recently disclosed that pradigastat lowered the
fasting plasma TG level in patients with familial chylomicro-
nemia syndrome.45 Here, we report our efforts that led to the
discovery of the first DGAT1 inhibitor to contain the
phenylcyclohexylacetic acid functionality, compound 1, as
well as on our medicinal chemistry program directed at
improving upon the overall profile of this benchmark
compound.
2 to be a good candidate for lead optimization based on the low
molecular weight (305 g/mol), reasonable PSA (73.4) and
clogP values (2.7), and good binding efficiency46 (20.4). A brief
investigation of the pyrimidine ring indicated that the 2-
position tolerated hydrogen (2) and a methyl group (3), while
placing more polar amino (4), dimethylamino (5), and
hydroxyl (6) groups in that position resulted in a loss of
DGAT1 inhibitory activity (Table 1). Monomethylating (7) or
dimethylating (8) the 4-amino functionality resulted in the
complete loss of DGAT1 potency as did replacing both
nitrogen atoms of the pyrimidine ring with CH to provide
aniline 9.
Next we turned toward an investigation of the oxazine ring of
2. With concern about the possible chemical instability of the
oxazine ring system, a number of five- and six-membered ring
systems were investigated, such as furan, pyrrole, imidazole,
oxazole, benzene, pyridine, and pyridazine derivatives (data not
shown). However, no suitable replacement for the 1,4
arrangement of the nitrogen and oxygen atoms was found. In
an effort to stabilize the imine bond, we sought to introduce
substitution at the carbon adjacent to the oxygen. We were
pleased to find that the introduction of a methyl group (10) did
not negatively impact the potency and were gratified to see an
increase in inhibitory activity with the dimethyl analogue 11
(Table 2). Reduction of the carbon nitrogen double bond of
the oxazine ring provided morpholine 12, which displayed
DGAT1 potency comparable to that of 2. However, both
methyl analogue 13 and dimethyl analogue 14 were >10-fold
RESULTS AND DISCUSSION
■
A high-throughput screen (HTS) of our small molecule library
resulted in the identification of compound 2 as a novel,
moderately potent DGAT1 inhibitor (Figure 2). We considered
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Table 1. DGAT1 IC50 Values of Pyrimidine Analogues 2−9
a
Data are reported as the average of a minimum of two determinations.
Table 2. DGAT1 IC50 Values of Oxazine Analogues 10−14
a
Data are reported as the average of a minimum of two determinations.
less potent than the corresponding unsaturated analogues 10
and 11.
higher IC50 value. We were able to replace the bromo atom
with alkyl groups (18−23) and observed that potencies
generally improved with increased alkyl size and additional
branching. For example, potency increased from 410 nM for
the methyl analogue 18 to ethyl (19) to propyl (20) to
isopropyl (22) and finally to tert-butyl (23) possessing an IC50
value of 60 nM. Cyclohexyl analogue 24 also inhibited DGAT1
with an IC50 value of 60 nM, while phenyl derivative 25 was
essentially equipotent to the bromo compound 11. Replacing
the bromine of 11 with a hydroxy group (26) resulted in
approximately a 40-fold decrease in DGAT1 potency. We
Having investigated the pyrimidine and oxazine ring systems
of lead 2, we began optimizing the bromophenyl ring using
dimethyl analogue 11 as our starting point because of the
improvement in DGAT1 potency compared to lead 2.
Replacement of the bromine with hydrogen provided analogue
15, resulting in a 10-fold loss in DGAT1 inhibitory activity
(Table 3). Thus, the chloro and fluoro analogues, 16 and 17,
were prepared. While 16 maintained good DGAT1 inhibitory
potency compared to 11, the fluoro analogue 17 had a 10-fold
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and Sprague−Dawley rats. In general, carboxylate 1 had
reasonable clearance values and good exposure at the doses
administered (Table 5). With the confidence that we could
reach plasma levels that would allow us to determine the
efficacy of 1 in an in vivo mouse model, we conducted an oral
triglyceride uptake study. Mice (C57BL/6J) were dosed with
vehicle or 1 by oral gavage. Thirty minutes after administration
of vehicle or compound 1, the mice were given olive oil (5 mL/
kg) by oral gavage. Blood samples were taken, and plasma
triglyceride levels were measured before dosing with vehicle or
compound 1 and 3 h after olive oil administration. The results
are plotted as the difference in TG levels 3 h after olive oil
administration compared to TG levels before dosing vehicle or
1 (Figure 3). Administration of 0.3 mg/kg 1 to C57Bl/6J mice
resulted in a 93% reduction in plasma triglyceride levels 3 h
after olive oil oral gavage compared to vehicle treated mice.
This study was repeated in Sprague−Dawley rats using 0.1, 0.3,
and 1.0 mg/kg doses of 1, and once again, a dramatic decrease
in plasma triglyceride levels was observed. The reduction of
plasma triglycerides in rats was shown to be dose dependent
with 0.1 mg/kg providing a 68% reduction in plasma
triglycerides, 0.3 mg/kg providing an 88% reduction in plasma
triglycerides, and 1.0 mg/kg providing a 110% reduction in
plasma triglycerides relative to vehicle treated animals. These
results demonstrated that compound 1 effectively decreased
plasma TG levels after an olive oil bolus in both mice and rats
in agreement with triglyceride uptake studies conducted
elsewhere using compound 1.47
Table 3. DGAT1 IC50 Values of Substituted Phenyl
Analogues 15−30
a
compd
R
DGAT1 SPA IC50 (μM)
11
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
bromo
hydrogen
chloro
fluoro
0.21 0.03
2.9 0.7
0.39 0.05
3.3 0.6
methyl
ethyl
0.41 0.06
0.16 0.03
0.09 0.03
0.15 0.01
0.09 0.02
0.06 0.02
0.06 0.01
0.22 0.06
7.9 1.1
propyl
butyl
isopropyl
tert-butyl
cyclohexyl
phenyl
hydroxyl
methoxy
0.57 0.10
0.13 0.05
0.61 0.09
0.05 0.01
N-piperidinyl
N-morpholinyl
2-carboxypropan-2-yl
a
Data are reported as the average of a minimum of two
determinations.
The promising potency, PK properties, and in vivo efficacy of
1 in rodents encouraged us to initiate preclinical development
studies. We determined the PK of carboxylate 1 in beagle dogs
and rhesus monkeys (Table 5). The bioavailability of 1 in dog
was high, while clearance values were moderate in both dog and
monkey, resulting in relatively short t1/2 values. We investigated
the inhibitory activity of carboxylate 1 against several related
enzymes, including human ACAT1, ACAT2, DGAT2, MGAT,
and GPAT and demonstrated that 1 did not inhibit these
enzymes at concentrations up to 10 μM. We also demonstrated
that analogue 1 did not inhibit CYP1A2, CYP2D6, CYP2C9, or
CYP3A4 at concentrations up to 10 μM. The IC50 value in the
hERG inhibition assay of analogue 1 was greater than 10 μM.
Satisfied with the selectivity of 1, a 14-day multiple-dose
toxicity study was conducted in Sprague−Dawley rats using po
doses of 30 and 100 mg/kg q.d. No significant clinical or
histopathological findings were detected at the 30 mg/kg dose
(AUC = 913 μM·h), while sebaceous gland atrophy was the
only significant finding at the 100 mg/kg dose (AUC = 3752
μM·h). Sebaceous gland atrophy is an expected result of
DGAT1 inhibition, as atrophic sebaceous glands have been
characterized in Dgat1(−/−) mice.48 In contrast, significant
elevation of the liver enzymes ALT and AST (∼10-fold) were
observed in a single 100 mg/kg po dose (AUC = 1420 μM·h)
in cynomolgus monkeys. We suspected that a difference in
metabolism between the two species was responsible for the
divergent results from the two toxicity studies. In order to
investigate this possibility, we studied the metabolites formed
during incubation of 1 with Sprague−Dawley rat and
cynomolgus liver S9 fractions. Our investigation revealed that
a single metabolite was produced in significantly larger
quantities in cynomolgus liver S9 fractions compared to rat
liver S9 fractions (Figure 4). Mass spectral analysis indicated
that the metabolite was the product of a single oxidation, and
further analysis by MS/MS suggested that the oxidation
suspected that the increased polarity of 26 was responsible for
the decrease in potency and were satisfied to learn that
methylation of the phenol to provide anisole 27 resulted in
regaining most of the potency lost by the phenol. In an attempt
to balance the polarity and hydrophobicity in this part of the
molecule the piperidine and morpholine analogues 28 and 29
were prepared and subsequently shown to have moderate
DGAT1 inhibitory activities, which strongly suggested that this
was a viable approach. Encouraged by our findings, we prepared
analogue 30 containing a 2-carboxypropan-2-yl functionality at
C4 of the phenyl ring that exhibited potency comparable to that
of the tert-butyl analogue 23.
Subsequently, we chose to focus on analogues of 24
possessing a cyclohexyl group at C4 of the phenyl ring and
prepared several compounds having various groups at the 4
position of the cyclohexyl ring. The 4-oxo, 4-hydroxy, and 4,4-
difluoro analogues 31−33 had similar biochemical and cellular
DGAT1 IC50 values compared to 24 (Table 4). Because of the
potency of carboxylic acid 30, we prepared cyclohexanecarbox-
ylic acid 34. To our surprise, 34 demonstrated a 6-fold increase
in biochemical potency and a 3-fold increase in cellular potency
compared to cyclohexane 24. A thorough investigation of the
SAR around carboxylate 34 led us to the discovery of the
ethanoic and propanoic acid homologues 1 and 35. The
DGAT1 IC50 values of these two analogues in the biochemical
assay were identical, but ethanoic acid 1 (IC50 = 40 nM)
showed a clear advantage over propanoic acid 35 (IC50 = 100
nM) in the cellular assay. Biphenyl analogue 36 was also
prepared and possessed good biochemical potency but had an
IC50 value of >3 μM in the cellular assay.
The excellent cellular activity of 1 encouraged us to
investigate this compound’s ability to inhibit DGAT1 activity
in vivo. We envisioned an initial study that would measure the
uptake of triglyceride into plasma after an oral bolus of olive oil.
We first assessed the pharmacokinetic profile of 1 in CD-1 mice
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Table 4. DGAT1 IC50 Values of Cyclohexyl Analogues 1 and 31−36
a
b
Data are reported as the average of a minimum of two determinations. Not tested.
Table 5. Pharmacokinetic Properties of Carboxylate 1
iv
po
species
dose iv/po (mg/kg)
CL (L h−1 kg−1
)
Vdss (L/kg)
t1/2 (h)
AUC (μg·h/L)
F (%)
mouse
rat
0.5/2.0
1/2.0
0.11
0.55
0.66
0.22
0.79
1.65
1.90
0.46
4.7
1.7
2.4
1.8
6690
1010
4720
40
30
64
dog
0.5/5.0
0.2/−
rhesus
occurred at C2 of the pyrimidine ring to form the 2-hydroxy
compound. Sufficient quantity of the metabolite was isolated
from the incubation of 1 with cynomolgus liver S9 fractions to
obtain an NMR spectrum. The spectrum clearly indicated the
disappearance of the C2 proton, while the remainder of the
signals of 1 were still present. Having proven the structure of
the oxidized product, we set out to determine if this metabolite
was responsible for the liver toxicity observed in cynomolgus
monkeys. The 2-hydroxy metabolite was synthesized and
administered to Sprague−Dawley rats at a single dose of 50
mg/kg iv. A significant increase in plasma ALT and AST levels
was observed confirming the compound as the source of liver
toxicity. The toxicity of the 2-hydroxy metabolite is likely
explained by its ability to isomerize to a quinoneimine, a class
of molecules with a well documented history of causing
toxicity.49
Having demonstrated that the 2-hydroxy metabolite of 1 was
responsible for the observed liver toxicity in cynomolgus
monkeys, we set out to prepare molecules that blocked
oxidation at this site. We had shown previously that
replacement of the hydrogen at C2 with a methyl group as in
3 did not adversely affect potency; therefore, we prepared the
C2-methyl and C2-trifluoromethyl analogues of 1. Both methyl
analogue 37 and trifluoromethyl analogue 38 had potencies
comparable to that of 1 in both the biochemical and cellular
assays (Figure 5). Furthermore, both analogues demonstrated
the advantage of decreased in vivo clearance in rats compared
to 1.
Methyl analogue 37 was evaluated in the oral triglyceride
uptake study and was shown to reduce plasma triglyceride
levels by 91% in rats treated with a dose of 0.3 mg/kg
compared to rats administered vehicle. This compound was
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Figure 5. DGAT1 IC50 values and rat clearance of 2-substituted
pyrimidines 1 and 37−38. IC50 values are reported as the average of a
minimum of two determinations.
in the patch clamp assay. A 28-day multiple-dose toxicity study
in Sprague−Dawley rats showed no treatment related changes
in clinical pathology other than the expected atrophic
sebaceous glands at the doses tested (100 and 500 mg kg−1
day−1 po). Analogue 37 was negative in the Ames test but
positive in a chromosomal aberration test using Chinese
hamster lung cells. Although a 2-hydroxy metabolite could not
form, a 2- to 5-fold increase in plasma ALT and AST levels was
detected at all doses (1, 10, and 100 mg kg−1 day−1 po) in a 28-
day multiple-dose cynomolgus toxicity study. Finally, two out
of eight cynomolgus monkeys had prolonged QTc intervals at
the 100 mg/kg dose while no effect was observed at the 1 and
10 mg/kg doses. Consideration of the small increase in liver
enzyme levels, the positive chromosomal aberration result, and
the prolonged QTc intervals taken together resulted in the
termination of the development of methylpyrimidine 37.
While we investigated the cause of the liver toxicity noted in
cynomolgus monkeys, we further investigated the SAR of
Figure 3. Increase in plasma triglyceride concentrations 3 h after an
oral gavage of olive oil in (A) C57BL/6J mice pretreated with vehicle
or DGAT1 inhibitor 1 at 0.3 mg/kg and (B) Sprague−Dawley rats
pretreated with vehicle or DGAT1 inhibitor 1 at 0.1, 0.3, and 1.0 mg/
kg: (∗) p < 0.05, (∗∗) p < 0.01 compared to vehicle (n = 6/group).
further evaluated in selectivity assays and was found to have
IC50 values greater than 30 μM against ACAT1, DGAT2,
MGAT, and GPAT. Furthermore, 37 had IC50 values greater
than 10 μM for all of the 135 targets tested in the Panlabs panel
and showed no inhibition of CYP1A2, CYP2D6, CYP2C9, or
CYP3A4 when tested up to 10 μM. However, methylpyr-
imidine 37 demonstrated 12% inhibition of the hERG channel
Figure 4. HPLC−UV traces of samples obtained from the incubation of DGAT1 inhibitor 1 with cynomolgus and rat liver S9 fractions.
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carboxylate 1 in an attempt to further improve the DGAT1
potency and pharmacokinetic properties. Several carboxylic acid
isosteres were prepared including amide 39, tetrazole 40, and
sulfonamide 41 (Figure 6). All three of these isosteres
in vivo oxidation at the C2 position. Investigation of the
pharmacokinetic properties of 42 indicated that it possessed a
reasonable clearance of 0.21 L h−1 kg−1 in rats.
We also approached blocking the formation of the toxic
metabolite observed upon in vivo incubation of 1 by replacing
the oxazine ring with a dihydropyran ring. We initially prepared
analogues 43 and 44 possessing a hydrogen atom at C2 of the
pyrimidine ring. Both of these analogues maintained excellent
DGAT1 inhibitory activity in both the biochemical and cellular
assay (Figure 8). The DGAT1 cellular potency of spiroindane
Figure 6. DGAT1 IC50 values of acid replacements 39−41. IC50 values
are reported as the average of a minimum of two determinations.
maintained similar DGAT1 biochemical potencies compared
to 1. Although amide 39 and sulfonamide 41 also had
comparable cellular DGAT1 inhibitory activity, tetrazole 40
possessed a DGAT1 cellular IC50 value of 2.5 μM.
Figure 8. DGAT1 IC50 values and rat clearance of dihydropyran
analogues 43−45. IC50 values are reported as the average of a
minimum of two determinations.
We hypothesized that restricting the conformational
flexibility of the cyclohexyl ring present in our inhibitors
could provide an increase in DGAT1 activity if the molecule
adopted a conformation similar to the binding conformation.
Our approach was to inorporate a five-membered spirocyclic
ring system that tethered the cyclohexane ring to C3 and C4 of
the aromatic ring. In order to block the formation of the
potentially toxic oxidative metabolite observed with 1, the 2-
trifluoromethylpyrimidine ring was selected to incorporate with
the five-membered spirocyclic ring system to afford spiroindane
42 (Figure 7). The spirocyclic ring does not allow rotation of
44 (9 nM) was significantly improved compared to cyclohexyl
analogue 43 (50 nM). This result is consistent with the increase
in potency achieved with spiroindane 42 in the oxazine series.
However, both dihydropyran analogues 43 and 44 had higher
clearance values in rats compared to the oxazine analogues 1,
37, 38, and 42. On the basis of the result in the oxazine series
that installment of a methyl at the C2 position of the
pyrimidine resulted in lower rat clearance (37 vs 1 Figure 5), a
methyl was installed at the C2 position of the pyrimidine of
dihydropyran analogue 43, providing compound 45.
Unfortunately, replacing the C2 hydrogen with a methyl
group in this compound resulted in a 4-fold reduction in
DGAT1 biochemical activity and a significant decrease in
cellular potency, resulting in an IC50 value greater than 1 μM.
Although analogues 43 and 44 demonstrated that the nitrogen
of the oxazine ring could be replaced by a carbon atom without
losing DGAT1 inhibitory activity, these compounds possessed
higher in vivo rat clearance values and thus provided no benefit
over the oxazine analogues.
The discovery of trifluoromethyl analogue 42 provided a
molecule that could not undergo oxidative metabolism at C2 of
the pyrimidine while improving the DGAT1 potency by an
order of magnitude compared to 1. Evaluation of this molecule
in the mouse oral triglyceride uptake test at doses of 0.3, 0.1,
and 0.03 mg/kg demonstrated that 42 significantly reduced TG
uptake after oral administration of olive oil in a dose dependent
manner (Figure 9). The increased DGAT1 cellular potency of
spiroindane 42 compared to 1 translated well in the TG uptake
assay with 42 demonstrating a 99.7% and 105% inhibition of
TG uptake compared to vehicle at doses of 0.1 and 0.3 mg/kg,
Figure 7. DGAT1 IC50 values and rat clearance of spiroindane 42. IC50
values are reported as the average of a minimum of two
determinations.
the carbon−carbon bond joining the phenyl and cyclohexane
ring systems and forces the rings into an orthogonal
orientation. This structural modification resulted in a 20-fold
increase in DGAT1 cellular potency of spiroindane analogue 42
compared to the cyclohexyl analogue 38, providing a single
digit nanomolar DGAT1 inhibitor while blocking any potential
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abrogating the formation of this toxic metabolite while also
optimizing the potency and PK properties. This effort
culminated in the discovery of spiroindane 42 as a potent,
selective, and orally efficacious DGAT1 inhibitor that
demonstrated an acceptable safety profile in rat and
cynomolgus monkey toxicity studies.
CHEMISTRY
■
The key step of the synthesis of analogues 2−6 and 48 involved
the formation of the 6-phenylpyrimido[4,5-b][1,4]oxazine core
through a condensation of 2-bromo-1-(4-bromophenyl)-
ethanone (46) with an appropriate 2-aminophenol 47a−f
under basic conditions (Scheme 1).50 N-Methylation of 2 using
iodomethane in the presence of NaH in DMF provided
analogues 7 and 8. Compound 9 was synthesized by reduction
of the nitro group of 48 using SnCl2 in EtOH. Condensation of
4,5-diamino-6-hydroxypyrimidine (47a) with ( )-2-bromo-1-
(4-bromophenyl)propan-1-one (49) under the conditions used
previously for the synthesis of analogues 2−6 provided 7-
methyl-6-phenylpyrimido[4,5-b][1,4]oxazine-4-amine analogue
10 (Scheme 2).
Condensation of 2-bromo-2-methyl-1-phenylpropan-1-ones
50 with 4,5-diamino-6-hydroxypyrimidine (47a) under acidic
conditions provided the 7,7-dimethyl-6-phenyl-7H-pyrimido-
[4,5-b][1,4]oxazine-4-amine analogues 11, 15−24, and 27−33
(Scheme 2).51 Demethylation of anisole 27 using BBr3 in
dichloromethane provided phenol 26. Suzuki coupling of aryl
bromide 11 using phenylboronic acid provided biphenyl
analogue 25. Reduction of oxazines 2, 10, and 11 using
NaBH4 in methanol provided the corresponding dihydroox-
azines 12−14 in good yield (Scheme 3).
Friedel−Crafts acylation of aryl esters 51 and 52 with 2-
bromo-2-methylpropanoyl bromide in the presence of AlCl3 in
dichloromethane provided bromo ketones 53 and 54,
respectively (Scheme 4). Condensation of the bromo ketones
with 47a in the presence of HCl in aqueous EtOH at reflux
followed by saponification of the corresponding esters provided
carboxylic acid analogues 30 and 36. The preparation of
cyclohexanecarboxylic acid 34 is outlined in Scheme 5.
Saponification of ethyl 4-oxocyclohexanecarboxylate (55) with
NaOH in aqueous EtOH provided the carboxylic acid, which
was treated with 2 equiv of phenylmagnesium bromide in THF
to yield the corresponding tertiary alcohol. Methylation of the
carboxylate with iodomethane in the presence of K2CO3 in
DMF provided methyl ester 56. Removal of the hydroxy group
was attempted using triethylsilane and trifluoroacetic acid in
chloroform;52 however, only the dehydration product was
obtained. Hydrogenation of the alkene using 10% palladium on
carbon in methanol provided a 1:1 mixture of cis and trans
isomers. Treating this mixture with sodium methoxide in
methanol at 70 °C provided ester 57 as a 4:1 mixture of trans/
cis isomers. Following the general procedure, Friedel−Crafts
acylation of ester 57 followed by condensation with 47a and
Figure 9. Increase in plasma triglyceride concentrations 3 h after an
oral gavage of olive oil in C57BL/6J mice pretreated with vehicle or
DGAT1 inhibitor 42 at 0.03, 0.1, and 0.3 mg/kg: (∗∗) p < 0.01, (∗∗∗)
p < 0.001 as compared to vehicle (n = 3/group).
respectively. Spiroindane 42 was more efficacious than
carboxylate 1 despite having an approximately 4-fold lower
plasma exposure when dosed at 0.3 mg/kg (42, AUC = 0.65
μM·h; 1, AUC = 2.6 μM·h).
Because of the promising in vivo efficacy data from the
mouse oral triglyceride uptake study, spiroindane 42 was
evaluated in preclinical toxicity studies. The compound was
shown to be selective against human ACAT1, ACAT2,
DGAT2, MGAT, and GPAT, possessing IC50 values of >10
μM, and did not inhibit CYP1A2, CYP2D6, CYP2C9, or
CYP3A4 at concentrations up to 10 μM. There was no
inhibitory activity against the hERG channel at concentrations
up to 5 μM in the patch clamp assay. Investigation of the
pharmacokinetic properties in mouse, rat, dog, and cyno
indicated that 42 had low clearance across species and good
exposure and bioavailabilities in mouse, rat, and cyno (Table 6).
A 14-day multiple-dose toxicity study in Sprague−Dawley rats
was conducted at 10, 30, and 100 mg/kg q.d. po. No significant
clinical or histopathological findings were detected at the 10
mg/kg (AUC = 11 μM·h) dose, while sebaceous gland atrophy
was the only significant finding at the 30 and 100 mg/kg doses
(AUC of 78 and 677 μM·h, respectively). Finally, no increase in
ALT or AST levels, no prolonged QTc intervals, and no
significant clinical or histopathological findings were detected in
a 21-day multiple-dose toxicity study in cynomolgus monkeys
conducted at 10 and 30 mg/kg q.d. po (AUC of 607 and 2561
μM·h, respectively).
Herein we described the discovery of a potent, selective, and
orally efficacious DGAT1 inhibitor 1 consisting of a
pyrimidooxazine core and a phenylcyclohexylacetic acid
substituent. Inhibitor 1 was a promising preclinical candidate
shown to form a 2-hydroxy metabolite in cynomolgus monkeys,
resulting in elevated levels of the liver enzymes ALT and AST.
Molecules were designed to block the C2 position, thereby
Table 6. Pharmacokinetic Properties of Spiroindane 42
iv
po
species
dose iv/po (mg/kg)
CL (L h−1 kg−1
)
Vdss (L/kg)
t1/2 (h)
AUC (μg·h/L)
F (%)
mouse
rat
0.5/0.3
0.7/5.0
0.2/2.0
0.5/5.0
0.45
0.21
0.14
0.02
2.8
4.4
3.7
341
22862
3185
77
98
22
99
1.1
dog
0.21
0.44
1.1
cyno
15.4
251581
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Journal of Medicinal Chemistry
Article
a
Scheme 1
a
Reagents and conditions: (a) SnCl2·H2O, EtOH, reflux, 50%; (b) MeI, NaH, DMF, 13−32%.
a
Scheme 2
a
Reagents and conditions: (a) BBr3, DCM, rt, 52%; (b) PhB(OH)2, Pd(PPh3)4, Na2CO3, DMA, 100 °C, 47%.
provide aldehyde 60. Further homologation of aldehyde 60
followed by hydrogenation of the resulting alkene using 10%
palladium on carbon in ethanol provided ester 61. Following
the general procedure of Friedel−Crafts acylation, condensa-
tion and saponification provided propanoic acid analogue 35.
The synthesis of 5,6-diamino-4-hydroxy-2-trifluoromethyl-
pyrimidine (67), required to prepare analogues 38 and 42, was
completed on a several hundred gram scale, required no
chromatography, and provided pyrimidine 67 in 61% yield over
three steps (Scheme 7). Condensation of malonamidine
hydrochloride 63 and ethyl trifluoroacetate 64 in THF with
DBU as the base provided trifluoromethylpyrimidine 65 in 66%
yield. Nitration of pyrimidine 65 was accomplished by using
90% nitric acid in concentrated sulfuric acid at 55 °C to provide
66 in 95% yield. Barone53 reported isolating the 4-N-
nitroamine using similar conditions but at a lower temperature.
When we maintained a temperature of 0−5 °C, we also
Scheme 3
saponification provided cyclohexanecarboxylic acid analogue
34.
Homologation of 4-phenylcyclohexanone (59) was accom-
plished by using (methoxymethyl)triphenylphosphonium
chloride in THF with potassium tert-butoxide as the base to
provide the enol ether (Scheme 6). The enol ether was
hydrolyzed by heating at 70 °C in 80% aqueous AcOH to
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Journal of Medicinal Chemistry
Article
a
Scheme 4
a
Reagents and conditions: (a) 2-bromo-2-methylpropanoyl bromide, AlCl3, DCM, rt; (b) (i) 47a, EtOH, H2O, HCl, reflux, (ii) NaOH, EtOH, H2O,
40 °C, 6−20%.
a
HOBt, and aqueous ammonia in dimethylformamide provided
amide 39 that was further elaborated to tetrazole 40 using
thionyl chloride, sodium azide, and ammonium chloride in
DMF (Scheme 8).
Scheme 5
The preparation of sulfonamide 41, designed as a carboxylic
acid isostere, is shown in Scheme 9. Isomerization of aldehyde
60 was effected with DBU in toluene at 80 °C to provide a
3.7:1 mixture of trans and cis isomers. Reduction of the
aldehyde with sodium borohydride in isopropanol followed by
treatment with carbon tetrabromide and triphenylphosphine in
dichloromethane provided the corresponding bromide contain-
ing 11% of the cis isomer after column chromatography.
Subsequent displacement of the bromide with potassium
thioacetate provided thioacetate 70 as a 95:5 mixture of trans
and cis isomers after column chromatography. Thioacetate 70
was converted to the corresponding sulfonic acid with
hydrogen peroxide in acetic acid followed by precipitation of
the product to provide pure trans-sulfonic acid. Conversion of
the sulfonic acid to sulfonamide 71 was completed by preparing
the sulfonyl chloride using thionyl chloride and subsequent
displacement with ammonia in dioxane.
a
Reagents and conditions: (a) (i) NaOH, EtOH, H2O, rt, (ii)
PhMgBr, THF, 0 °C, (iii) MeI, K2CO3, DMF, rt, 59%; (b) (i) Et3SiH,
TFA, CHCl3, 65 °C, (ii) 10% Pd/C, H2, MeOH, rt, (iii) NaOMe,
MeOH, 70 °C; (c) 2-bromo-2-methylpropanoyl bromide, AlCl3,
DCM, rt; (d) 47a, EtOH, H2O, HCl, reflux, (ii) NaOH, EtOH, H2O,
40 °C, 34% from 57.
obtained the 4-N-nitroamine described by Barone that could
then be rearranged by heating in concentrated sulfuric acid. We
found that heating the mixture at 55 °C could achieve the
rearrangement of the initially formed nitroamine to the desired
5-nitropyrimidine 66 in one pot without isolating the
nitroamine intermediate. Reduction of the nitro group via
transfer hydrogenation with 10% palladium on carbon and
cyclohexene provided pyrimidine 67 in 97% yield.
Preparation of the bromoketone required for the synthesis of
analogues 1, 37, and 38 is described in Scheme 7. Starting with
4-phenylcyclohexanone (59), the Horner−Wadsworth−Em-
mons (HWE) reaction followed by hydrogenation of the
resulting alkene provided ester 68. Friedel−Crafts acylation
with 2-bromo-2-methylpropanoyl bromide provided bromo
ketone 69. Condensation of 69 with aminohydroxypyrimidines
47a, 47b, and 67 provided pyrimidooxazines with a proton (1),
methyl group (37), and trifluoromethyl group (38) at the 2-
position of the pyrimidine ring. Treatment of acid 1 with EDC,
Sulfonamide 41 was prepared by following the general
procedure of Friedel−Crafts acylation and condensation with
pyrimidine 47a.
The synthesis of five-membered spirocyclic analogue 42 was
accomplished through the condensation of bromo ketone 80
and pyrimidine 67 (Scheme 10). Synthesis of 80 began with the
Wittig reaction of 5-bromoindanone (73) with
(methoxymethyl)triphenylphosphonium chloride to provide
enol ether 74. Robinson annulation of 74 using methyl vinyl
ketone provided α,β-unsaturated ketone 75, which was
hydrogenated followed by HWE reaction to provide α,β-
unsaturated ester 77. Palladium catalyzed carbonylation54 of
aryl bromide 77 provided the benzyl ester which was removed
by hydrogenolysis using hydrogen and 10% palladium on
carbon in ethanol while also hydrogenating the alkene.
Crystallization from ethyl acetate and hexanes provided the
3473
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Journal of Medicinal Chemistry
Article
a
Scheme 6
a
Reagents and conditions: (a) (i) KO-t-Bu, Ph3P(Cl)CH2OMe, THF, rt, (ii) 80% aq AcOH, 70 °C; (b) DBU, Ph3P(Br)CH2CO2Me, toluene, 80−
100 °C, (ii) 10% Pd/C, H2, EtOH, rt, 77% from 59; (c) 2-bromo-2-methylpropanoyl bromide, AlCl3, DCM, rt; (d) 47a, EtOH, H2O, HCl, reflux,
(ii) NaOH, EtOH, H2O, 40 °C, 29% from 61.
a
Scheme 7
a
Reagents and conditions: (a) DBU, THF, 65 °C, 66%; (b) conc H2SO4, 90% HNO3, 55 °C, 95%; (c) 10% Pd/C, cyclohexene, EtOH, 65 °C, 97%;
(d) (i) NaH, DMF, (EtO)2P(O)CH2CO2Et, rt, (ii) 10% Pd/C, H2, EtOH, rt, 97%; (e) 2-bromo-2-methylpropanoyl bromide, AlCl3, DCM, rt, 92%;
(f) (i) 69 and 47a, 47b, or 67, EtOH, H2O, HCl, reflux, (ii) NaOH, EtOH, H2O, 40 °C.
desired trans isomer 78. Carboxylic acid 78 was converted to
the acid chloride using oxalyl chloride in dichloromethane
followed by the conversion to isopropyl ketone 79 using
isopropylmagnesium chloride and cuprous cyanide. Treatment
with cupric bromide in ethyl acetate and chloroform at reflux
provided bromoketone 80, which was condensed with
aminohydroxypyrimidine 67, employing the general procedure
to provide pyrimidooxazine 81. Finally, cleavage of the methyl
ester using lithium iodide in dimethylformamide at 125 °C
provided carboxylic acid 42.
Preparation of dihydropyran analogues was achieved by using
a convergent route utilizing a key Suzuki coupling to join the
two fragments (Scheme 11). Preparation of the nonspirocyclic
analogues started with commercially available ketone 82. The
ketone was transformed to the α,β-unsaturated ester using
HWE conditions followed by the hydrogenation of the double
bond using 10% Pd/C to provide methyl ester 83 as a 20:1
mixture of trans/cis isomers after recrystallization from EtOAc.
Conversion of phenol 83 to the corresponding triflate was
accomplished with triflic anhydride and triethylamine in
3474
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Journal of Medicinal Chemistry
Article
a
Scheme 8
a
Reagents and conditions: (a) EDC, HOBt, 28% aq NH3, DMF, rt, 60%; (b) (i) SOCl2, pyridine, rt, 85%, (ii) NaN3, NH4Cl, DMF, 110 °C, 54%.
a
Scheme 9
a
Reagents and conditions: (a) (i) DBU, toluene, 80 °C, (ii) NaBH4, i-PrOH, rt, (iii) CBr4, PPh3, DCM, 0 °C to rt, (iv) KSAc, ACN, rt, 58%; (b) (i)
30% H2O2, AcOH, rt, 82%, (ii) SOCl2, DMF, 90 °C, (iii) NH3, dioxane, rt, 85%; (c) 2-bromo-2-methylpropanoyl bromide, AlCl3, DCM, rt; (d) 47a,
EtOH, H2O, HCl, reflux, 50% from 71.
performed on Merck silica gel 60 (230−400 mesh). Removal of
solvents was conducted by using a rotary evaporator, and residual
solvent was removed from nonvolatile compounds using a vacuum
manifold maintained at approximately 1 Torr. All yields reported are
isolated yields. Preparative reversed-phase high pressure liquid
chromatography (RP-HPLC) was performed using an Agilent 1100
series HPLC and Phenomenex Gemini C18 column (5 μm, 100 mm ×
30 mm i.d.), eluting with binary solvent systems A and B using a
gradient elution [A, H2O with 0.1% TFA; B, CH3CN with 0.1% TFA]
with UV detection at 220 nm. All final compounds were purified to
≥95% purity as determined by an Agilent 1100 series HPLC
instrument with UV detection at 220 nm using the following method:
Zorbax SB-C8 column (3.5 μm, 150 mm × 4.6 mm i.d.), eluting with
binary solvent systems A and B using a 5−95% B (0−15 min) gradient
elution [A, H2O with 0.1% TFA; B, CH3CN with 0.1% TFA]; flow
rate 1.5 mL/min. Mass spectral data were recorded on an Agilent 1100
series LCMS with UV detection at 254 nm. NMR spectra were
recorded on a Varian Gemini 400 MHz or Bruker Avance 500 MHz
NMR spectrometer. Chemical shifts (δ) are reported in parts per
million (ppm) relative to residual undeuterated solvent as internal
reference, and coupling constants (J) are reported in hertz (Hz).
Splitting patterns are indicated as follows: s = singlet; d = doublet; t =
triplet; q = quartet; qn = quintet; dd = doublet of doublet; dt =
doublet of triplets; m = multiplet; br = broad peak.
dichloromethane. The triflate was further transformed to
boronate 84 using bis(pinacolato)diboron and a palladium
catalyst. In a similar way aryl bromide 77 was converted to the
boronate followed by hydrogenation of the α,β-unsaturated
double bond to provide ester 85 as a 20:1 mixture of trans/cis
isomers after successive crystallization from methanol and
dichloromethane using methanol diffusion.
The pyrimidinopyran ring structure was synthesized by the
acid catalyzed condensation of bromoaldehyde 8655 with
hydroxypyrimidine 87 or 88 to provide dihydropyran 89 or
90 (Scheme 11). Functional group conversion of hydroxypyr-
imidines 89 and 90 to chloropyrimidines 91 and 92 was
accomplished using phosphorus oxychloride. Displacement of
the chloride with 4-methoxybenzylamine in the presence of
Hunigs base followed by deprotection of the amine in TFA
̈
provided vinyl bromides 93 and 94 that would serve as the
coupling partners for boronates 84 and 85. Palladium catalyzed
coupling of boronate 84 with bromides 93 and 94 and boronate
85 with bromide 93 provided the corresponding methyl esters.
Hydrolysis of the methyl esters using lithium hydroxide in
methanol and water provided carboxylic acids 43−45.
2-(trans)-(4-(4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b]-
[1,4]oxazin-6-yl)phenyl)cyclohexyl)acetic Acid (1). A mixture of
47a (63.1 mg, 0.50 mmol) and 69 (395 mg, 1.00 mmol) in 1 N
aqueous HCl (0.50 mL), water (2 mL) and EtOH (2 mL) was heated
at reflux for 12 h. The reaction mixture was concentrated to half the
EXPERIMENTAL SECTION
■
All solvents and chemicals used were reagent grade. Anhydrous
solvents were purchased from Aldrich and used as such. Analytical thin
layer chromatography (TLC) and flash chromatography were
3475
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Journal of Medicinal Chemistry
Article
a
Scheme 10
a
Reagents and conditions: (a) KO-t-Bu, CH3OCH2P(Cl)Ph3, dioxane, rt, 98%; (b) methyl vinyl ketone, p-TsOH·H2O, toluene, 100 °C, 59%; (c)
10% Pd/C, AcOH, rt, 98%; (d) (MeO)2P(O)CH2CO2Me, NaH, rt, 79%; (e) (i) NEt3, BnOH, CO, Pd(PPh3)4, toluene, 90 °C, (ii) 10% Pd/C, H2,
EtOH, rt, (iii) crystallization, 47%; (f) (i) (COCl)2, DMF, DCM, 0 °C to rt, (ii) CuCN, i-PrMgCl, THF, −15 °C, 98%; (g) CuBr2, EtOAc, CHCl3,
reflux, 93%; (h) 67, MeOH, H2O, HCl, reflux, 61%; (i) LiI, DMF, 125 °C, 72%.
volume, and the pH was adjusted to 10 with 2 N aqueous NaOH. The
mixture was stirred at 40 °C for 4 h. Water (2 mL) was added, and the
solution was washed with EtOAc (2 mL). The aqueous layer was
adjusted to pH 4 with 10% aqueous citric acid and extracted with
EtOAc (5 mL). The organic layer was washed with water, brine, dried
over MgSO4, filtered, and concentrated in vacuo to provide a white
solid. This solid was recrystallized from EtOH to provide the title
compound as a white solid (92 mg, 23%): mp >270 °C. IR (cm−1)
6-(4-Bromophenyl)-7H-pyrimido[4,5-b][1,4]oxazin-4-amine
(2). Following the general procedure, 4,5-diamino-6-hydroxypyrimi-
dine (47a) and 2-bromo-1-(4-bromophenyl)ethanone (46) provided
1
the title compound as a white solid in 31% yield: mp >200 °C. H
NMR (DMSO-d6) δ 5.44 (s, 2H), 7.10 (br s, 2H), 7.69 (d, J = 8.6 Hz,
2H), 7.93 (s, 1H), 8.03 (d, J = 8.6 Hz, 2H); ESIMS m/z (rel intensity)
305 ([M + H], 100), 307 ([M + H], 100).
6-(4-Bromophenyl)-2-methyl-7H-pyrimido[4,5-b][1,4]-
oxazin-4-amine (3). Following the general procedure, 47b and 46
provided the title compound as a white solid in 42% yield: mp >200
1
3320, 2929, 1702, 1601; H NMR (DMSO-d6) δ 1.10−1.16 (m, 2H),
1.45−1.84 (m, 13H), 2.15 (d, J = 6.0 Hz, 2H), 2.53−2.56 (m, 1H),
6.97 (br s, 2H), 7.31 (d, J = 9.0 Hz, 2H), 7.65 (d, J = 9.0 Hz, 2H), 7.95
(s, 1H), 11.96 (br s, 1H); ESIMS m/z (rel intensity) 395 ([M + H],
100).
1
°C. H NMR (DMSO-d6) δ 2.25 (s, 3H), 5.40 (s, 2H), 7.05 (br s,
2H), 7.70 (d, J = 9.0 Hz, 2H), 8.03 (d, J = 9.0 Hz, 2H); ESIMS m/z
(rel intensity) 319 ([M + H], 100), 321 ([M + H], 100).
6-(4-Bromophenyl)-7H-pyrimido[4,5-b][1,4]oxazine-2,4-dia-
mine (4). Following the general procedure, 2,4,5-triamino-6-
hydroxypyrimidine sulfate (47c), 46, and 2.1 equiv of NaHCO3
provided the title compound as a white solid in 21% yield: mp >200
General Procedure for the Preparation of Pyrimidooxazines
2−10. To a suspension of an appropriate 5-amino-6-hydroxypyr-
imidine (1 equiv) and an appropriate 2-bromo-1-arylethanone (1
equiv) in EtOH (5 L/mol pyrimidine) was added NaHCO3 (1.1
equiv), and the mixture was heated at reflux for 3 h. After cooling to
ambient temperature, the mixture was concentrated and the residue
was suspended in a combined solvent of EtOAc (15.0 L/mol
pyrimidine), THF (15.0 L/mol pyrimidine), and water (25 L/mol
pyrimidine) for 15 min. The insoluble materials were removed by
vacuum filtration, and the organic phase of the filtrate was separated,
dried over MgSO4, and evaporated. The residue obtained was purified
by flash column chromatography and subsequently triturated in
diisopropyl ether to provide the desired products.
1
°C. H NMR (DMSO-d6) δ 5.25 (s, 2H), 6.24 (br s, 2H), 6.55 (br s,
2H), 7.63 (d, J = 12.0 Hz, 2H), 7.92 (d, J = 12.0 Hz, 2H); ESIMS m/z
(rel intensity) 320 ([M + H], 100), 322 ([M + H], 100).
6-(4-Bromophenyl)-N2,N2-dimethyl-7H-pyrimido[4,5-b][1,4]-
oxazine-2,4-diamine (5). Following the general procedure, 47d, 46,
and 3.1 equiv of NaHCO3 provided the title compound as a white
solid in 23% yield: mp >200 °C. 1H NMR (DMSO-d6) δ 3.04 (s, 6H),
5.27 (s, 2H), 6.73 (br s, 2H), 7.63 (d, J = 6.0 Hz, 2H), 7.94 (d, J = 6.0
Hz, 2H); ESIMS m/z (rel intensity) 348 ([M + H], 100), 350 ([M +
H], 100).
3476
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Journal of Medicinal Chemistry
Article
a
Scheme 11
a
Reagents and conditions: (a) (i) NaH, (MeO)2P(O)CH2CO2Me, THF, rt, (ii) 10% Pd/C, H2, EtOAc, rt, 96%; (b) (i) TfO2, NEt3, CH2Cl2, 0 °C
to rt, (ii) bis(pinacolato)diboron, PdCl2(dppf), dppf, KOAc, dioxane, 80 °C, 72%; (c) (i) bis(pinacolato)diboron, PdCl2(dppf), dppf, KOAc,
dioxane, 80 °C, (ii) 10% Pd/C, H2, MeOH, rt, 30%; (d) AcOH, 100 °C, 53−56%; (e) POCl3, 100 °C, 99%; (f) (i) 4-methoxybenzylamine, DIPEA,
THF, reflux, (ii) TFA, 50 °C, 83−92%; (g) (i) 84, PdCl2(dppf), Na2CO3, DMF, H2O, 80 °C or 85, Pd(PPh3)4, Na2CO3, DMF, H2O, 80 °C, (ii)
LiOH, MeOH, H2O, reflux, 44−86% over two steps.
4-Amino-6-(4-bromophenyl)-7H-pyrimido[4,5-b][1,4]oxazin-
2-ol (6). Following the general procedure, 4,5-diamino-2,6-dihydrox-
ypyrimidine (47e) and 46 provided the title compound as a white
solid in 22% yield: mp >200 °C. 1H NMR (DMSO-d6) δ 5.35 (s, 2H),
7.18 (br s, 2H), 7.65 (d, J = 9.0 Hz, 2H), 7.92 (d, J = 9.0 Hz, 2H),
10.67 (br s, 1H); ESIMS m/z (rel intensity) 321 ([M + H], 100), 323
([M + H], 100).
pooled, washed with saturated aqueous NH4Cl, dried over Na2SO4,
filtered, and concentrated in vacuo. Purification of the resulting residue
by flash chromatography on silica using 50% EtOAc/hexanes as the
eluent provided the title compound as a yellow solid (67 mg, 13%). 1H
NMR (DMSO-d6) δ 3.31 (s, 6H), 5.30 (s, 2H), 7.71 (d, J = 8.6 Hz,
2H), 7.88 (d, J = 8.6 Hz, 2H), 8.03 (s, 1H); ESIMS m/z (rel intensity)
333 ([M + H], 100), 335 ([M + H], 100).
6-(4-Bromophenyl)-N-methyl-7H-pyrimido[4,5-b][1,4]-
oxazin-4-amine (7). To a solution of 2 (103 mg, 0.338 mmol) in
DMF (2 mL) was added iodomethane (0.21 mL), and the reaction
mixture was heated at 40 °C for 16 h. The reaction mixture was
concentrated in vacuo, and to the residue was added 28% aqueous
NH4OH (5 mL). The reaction mixture was heated at 90 °C for 30
min, cooled to room temperature, and diluted with EtOAc. The layers
were separated, and the organic layer was dried over Na2SO4, filtered,
and concentrated in vacuo. Purification of the resulting residue by flash
column chromatography on silica using 50−66% EtOAc/hexanes as
the eluent provided the title compound as a yellow solid (35 mg,
32%). 1H NMR (DMSO-d6) δ 2.93 (d, J = 4.9 Hz, 3H), 5.43 (s, 2H),
7.50 (br q, J = 4.4 Hz, 1H), 7.71 (d, J = 8.6 Hz, 2H), 8.03 (s, 1H), 8.04
(d, J = 8.3 Hz, 2H); ESIMS m/z (rel intensity) 319 ([M + H], 100),
321 ([M + H], 100).
3-(4-Bromophenyl)-2H-benzo[b][1,4]oxazin-5-amine (9). To
a solution of 2-amino-3-nitrophenol (47f) (100 mg, 0.649 mmol) in
DMF (2.0 mL) were added 46 (180 mg, 0.648 mmol) and K2CO3 (90
mg, 0.651 mmol), and the mixture was stirred at ambient temperature
overnight. The mixture was diluted with EtOAc, washed with water
and brine, dried over MgSO4, and concentrated to dryness to provide
3-(4-bromophenyl)-5-nitro-2H-benzo[b][1,4]oxazine (48) (217 mg).
A mixture of (48) (100 mg, 0.300 mmol) and SnCl2·H2O (339 mg,
1.50 mmol) in EtOH was heated at reflux for 2 h and then cooled to
ambient temperature. Concentrated HCl and CHCl3 were added to
the mixture, and the organic layer was separated, dried over MgSO4,
and concentrated in vacuo. Purification of the resulting residue by
preparative TLC gave the title compound as a white solid (45.0 mg,
1
50% from 48): mp 75−77 °C. H NMR (DMSO-d6) δ 5.05 (s, 2H),
5.58 (br s, 2H), 6.06 (d, J = 8.0 Hz, 1H), 6.32 (d, J = 8.0 Hz, 1H), 6.85
(t, J = 8.0 Hz, 1H), 7.79 (d, J = 8.7 Hz, 2H), 8.03 (d, J = 8.7 Hz, 2H);
ESIMS m/z (rel intensity) 303 ([M + H], 100), 305 ([M + H], 100).
6-(4-Bromophenyl)-N,N-dimethyl-7H-pyrimido[4,5-b][1,4]-
oxazin-4-amine (8). To a suspension of NaH (132 mg, 3.3 mmol) in
anhydrous THF (18 mL) and DMF (10 mL) at room temperature was
added a solution of 2 (472 mg, 1.55 mmol) in anhydrous THF (18
mL) followed by iodomethane (1 mL). After 1 h the reaction was
quenched with water and extracted with EtOAc. The organics were
(
)-6-(4-Bromophenyl)-7-methyl-7H-pyrimido[4,5-b][1,4]-
oxazin-4-amine (10). Following the general procedure, 47a and
( )-2-bromo-1-(4-bromophenyl)propan-1-one (49) provided the
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desired compound as a white solid in 29% yield: mp 222−224 °C. 1H
NMR (DMSO-d6) δ 1.33 (d, J = 6.0 Hz, 3H), 5.98 (q, J = 6.0 Hz, 1H),
7.14 (br s, 2H), 7.69 (d, J = 6.0 Hz, 2H), 7.95 (s, 1H), 8.09 (d, J = 6.0
Hz, 2H); ESIMS m/z (rel intensity) 319 ([M + H], 100), 321 ([M +
H], 100).
provide the title compound as a white solid (50.1 mg, 95%): mp >220
°C. H NMR (DMSO-d6) δ 1.05 (s, 3H), 1.23 (s, 3H), 4.20 (br s,
1H), 5.33 (br s, 1H), 6.26 (br s, 2H), 7.40 (d, J = 9.0 Hz, 2H), 7.60 (d,
J = 9.0 Hz, 2H), 7.63 (s, 1H); ESIMS m/z (rel intensity) 335 ([M +
H], 100), 337 ([M + H], 100).
1
7,7-Dimethyl-6-phenyl-7H-pyrimido[4,5-b][1,4]oxazin-4-
amine (15). Following the general procedure, 2-bromo-2-methyl-1-
phenylpropan-1-one (50a) (Aldrich) provided the title compound as a
General Procedure for the Preparation of Pyrimidooxazines
11, 15−36, 41, and 42. To a solution of an appropriately substituted
benzene (1.2 equiv) in CH2Cl2 (2 L/mol bromide) were added AlCl3
(2 equiv) and 2-bromo-2-methylpropanoyl bromide (1 equiv) at 0 °C.
After the mixture was stirred at ambient temperature for 1 h, it was
poured onto ice−water, and the mixture was extracted with CHCl3.
The organic layer was separated, washed with saturated aqueous
NaHCO3 and brine, dried over MgSO4, and evaporated. The resulting
residue was purified by column chromatography on silica gel to give
the corresponding phenyl substituted 2-bromo-2-methyl-1-phenyl-
propan-1-one. A mixture of 4,5-diamino-6-hydroxypyrimidine 47a,
47b, or 67 (1.0 equiv), an appropriate 2-bromo-2-methyl-1-phenyl-
propan-1-one (2.0 equiv), and 1 M HCl (1.0 equiv) in 50% aqueous
EtOH (5 L/mol pyrimidine) was heated at reflux for 10 h. After
cooling to ambient temperature, the mixture was concentrated to half
volume, diluted with EtOAc, and washed with 1 M NaOH (3×) and
brine, dried over Na2SO4, and concentrated in vacuo. The residue was
triturated in diisopropyl ether to provide the title compounds.
6-(4-Bromophenyl)-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]-
oxazin-4-amine (11). Following the general procedure, 2-bromo-1-
(4-bromophenyl)-2-methylpropan-1-one (50b), prepared from bro-
mobenzene, provided the title compound as a white solid in 73% yield:
mp 201−203 °C (dec). 1H NMR (DMSO-d6) δ 1.60 (s, 6H), 7.03 (br
s, 2H), 7.65 (d, J = 9.0 Hz, 2H), 7.70 (d, J = 9.0 Hz, 2H), 7.96 (s, 1H);
ESIMS m/z (rel intensity) 333 ([M + H], 100), 335 ([M + H], 100).
1
white solid in 45% yield: mp 189−190 °C. H NMR (DMSO-d6) δ
1.60 (s, 6H), 6.93 (br s, 2H), 7.44−7.50 (m, 3H), 7.69−7.71 (m, 2H),
7.96 (s, 1H); ESIMS m/z (rel intensity) 225 ([M + H], 100).
6-(4-Chlorophenyl)-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]-
oxazin-4-amine (16). Following the general procedure, 2-bromo-1-
(4-chlorophenyl)-2-methylpropan-1-one (50c), prepared from chlor-
obenzene, provided the title compound as a white solid in 42% yield:
1
mp 210−211 °C. H NMR (DMSO-d6) δ 1.60 (s, 6H), 7.05 (br s,
2H), 7.52 (d, J = 9.0 Hz, 2H), 7.77 (d, J = 9.0 Hz, 2H), 7.96 (s, 1H);
ESIMS m/z (rel intensity) 289 ([M + H], 100).
6-(4-Fluorophenyl)-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]-
oxazin-4-amine (17). Following the general procedure, 2-bromo-1-
(4-fluorophenyl)-2-methylpropan-1-one (50d), prepared from fluo-
robenzene, provided the title compound as a white solid in 17% yield:
1
mp 189−190 °C. H NMR (DMSO-d6) δ 1.60 (s, 6H), 7.01 (br s,
2H), 7.29 (dd, J = 9.0 Hz, 2H), 7.81 (dd, J = 9.0 Hz, 2H), 7.96 (s,
1H); ESIMS m/z (rel intensity) 273 ([M + H], 100).
7,7-Dimethyl-6-(p-tolyl)-7H-pyrimido[4,5-b][1,4]oxazin-4-
amine (18). Following the general procedure, 2-bromo-2-methyl-1-
(p-tolyl)propan-1-one (50e), prepared from toluene, provided the title
1
compound as a white solid in 45% yield: mp 161−162 °C. H NMR
(DMSO-d6) δ 1.60 (s, 6H), 2.36 (s, 3H), 6.97 (br s, 2H), 7.27 (d, J =
9.0 Hz, 2H), 7.63 (d, J = 9.0 Hz, 2H), 7.95 (s, 1H); ESIMS m/z (rel
intensity) 269 ([M + H], 100).
(
)-6-(4-Bromophenyl)-6,7-dihydro-5H-pyrimido[4,5-b]-
[1,4]oxazin-4-amine (12). To a solution of 2 (35 mg, 0.115 mmol)
in MeOH (1 mL) and THF (1 mL) at 0 °C was added NaBH4 (8.7
mg, 0.23 mmol). The reaction mixture was warmed to ambient
temperature and stirred for 1 h. The reaction mixture was poured into
water, and the mixture was extracted with EtOAc. The organic layer
was separated and washed with saturated aqueous NaHCO3 and brine,
dried over MgSO4, and concentrated in vacuo. The resulting residue
was slurried in toluene and the solid collected by vacuum filtration.
This solid was slurried in MeOH and collected by vacuum filtration to
provide the title compound as a white solid (23 mg, 65%): mp >200
6-(4-Ethylphenyl)-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]-
oxazin-4-amine (19). Following the general procedure, 2-bromo-1-
(4-ethylphenyl)-2-methylpropan-1-one (50f), prepared from ethyl-
benzene, provided the title compound as a white solid in 59% yield:
1
mp 136−137 °C. H NMR (DMSO-d6) δ 1.21 (t, J = 7.0 Hz, 3H),
1.60 (s, 6H), 2.66 (q, J = 7.0 Hz, 2H), 6.90 (br s, 2H), 7.29 (d, J = 8.0
Hz, 2H), 7.64 (d, J = 8.0 Hz, 2H), 7.95 (s, 1H); ESIMS m/z (rel
intensity) 283 ([M + H], 100).
7,7-Dimethyl-6-(4-propylphenyl)-7H-pyrimido[4,5-b][1,4]-
oxazin-4-amine (20). Following the general procedure, 2-bromo-2-
methyl-1-(4-propylphenyl)propan-1-one (50g), prepared from pro-
pylbenzene, provided the title compound as a white solid in 40% yield:
1
°C. H NMR (DMSO-d6) δ 4.07 (dd, J = 7.6 and 10.7 Hz, 1H), 4.33
(dd, J = 1.6 and 10.7 Hz, 1H), 4.48 (m, 1H), 5.19 (br s, 1H), 6.27 (br
s, 2H), 7.40 (d, J = 8.6 Hz, 2H), 7.58 (s, 1H), 7.60 (d, J = 8.6 Hz, 2H);
ESIMS m/z (rel intensity) 307 ([M + H], 100), 309 ([M + H], 100).
1
mp 186−187 °C. H NMR (DMSO-d6) δ 0.91 (t, J = 8.0 Hz, 3H),
1.57−1.66 (m, 2H), 1.60 (s, 6H), 2.61 (t, J = 8.0 Hz, 2H), 6.89 (br s,
2H), 7.27 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H), 7.95 (s, 1H);
ESIMS m/z (rel intensity) 297 ([M + H], 100).
(
)-cis-6-(4-Bromophenyl)-7-methyl-6,7-dihydro-5H-
pyrimido[4,5-b][1,4]oxazin-4-amine (13). To a solution of 10
(146 mg, 0.457 mmol) in MeOH (1.4 mL) and THF (1.4 mL) at 0 °C
was added NaBH4 (34.6 mg, 0.914 mmol). The reaction mixture was
stirred at ambient temperature for 1 h and then poured into water, and
the mixture was extracted with EtOAc. The organic layer was
separated, washed with saturated aqueous NaHCO3 and brine, dried
over MgSO4, and concentrated in vacuo. The resulting residue was
slurried in toluene and the solid collected by vacuum filtration. This
solid was slurried in MeOH and collected by vacuum filtration to
provide the title compound as a white solid (74.1 mg, 50%). 1H NMR
(DMSO-d6) δ 0.99 (d, J = 6.0 Hz, 3H), 4.54 (br s, 2H), 5.36 (br s,
1H), 6.36 (br s, 2H), 7.28 (d, J = 9.0 Hz, 2H), 7.58 (d, J = 9.0 Hz,
2H), 7.65 (s, 1H); ESIMS m/z (rel intensity) 321 ([M + H], 100),
323 ([M + H], 100).
6-(4-Butylphenyl)-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]-
oxazin-4-amine (21). Following the general procedure, 2-bromo-1-
(4-butylphenyl)-2-methylpropan-1-one (50h), prepared from butyl-
benzene, provided the title compound as a white solid in 67% yield:
1
mp 158−161 °C. H NMR (DMSO-d6) δ 0.91 (t, J = 8.0 Hz, 3H),
1.27−1.36 (m, 2H), 1.54−1.61 (m, 2H), 1.60 (s, 6H), 2.63 (t, J = 8.0
Hz, 2H), 6.90 (br s, 2H), 7.27 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.0 Hz,
2H), 7.95 (s, 1H); ESIMS m/z (rel intensity) 311 ([M + H], 100).
6-(4-Isopropylphenyl)-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]-
oxazin-4-amine (22). Following the general procedure, 2-bromo-1-
(4-isopropylphenyl)-2-methylpropan-1-one (50i), prepared from
cumene, provided the title compound as a white solid in 43% yield:
1
mp 176−179 °C. H NMR (DMSO-d6) δ 1.23 (d, J = 8.0 Hz, 6H),
(
)-6-(4-Bromophenyl)-7,7-dimethyl-6,7-dihydro-5H-
1.61 (s, 6H), 2.91−2.98 (m, 1H), 6.89 (br s, 2H), 7.32 (d, J = 8.0 Hz,
2H), 7.64 (d, J = 8.0 Hz, 2H), 7.95 (s, 1H); ESIMS m/z (rel intensity)
297 ([M + H], 100).
pyrimido[4,5-b][1,4]oxazin-4-amine (14). To a solution of 11
(52.3 mg, 0.157 mmol) in MeOH (1.0 mL) and THF (1.0 mL) at 0
°C was added NaBH4 (11.9 mg, 0.314 mmol). The reaction mixture
was stirred at ambient temperature for 1 h and then poured into water,
and the mixture was extracted with EtOAc. The organic layer was
separated, washed with saturated aqueous NaHCO3 and brine, dried
over MgSO4, and concentrated in vacuo. The resulting residue was
slurried in toluene and the solid collected by vacuum filtration. This
solid was slurried in MeOH and collected by vacuum filtration to
6-(4-(tert-Butyl)phenyl)-7,7-dimethyl-7H-pyrimido[4,5-b]-
[1,4]oxazin-4-amine (23). Following the general procedure, 2-
bromo-1-(4-(tert-butyl)phenyl)-2-methylpropan-1-one (50j), prepared
from tert-butylbenzene, provided the title compound as a white solid in
52% yield: mp >200 °C. 1H NMR (DMSO-d6) δ 1.33 (s, 9H), 1.67 (s,
6H), 7.49 (d, J = 8.0 Hz, 2H), 7.58 (br s, 2H), 7.70 (d, J = 8.0 Hz,
2H), 8.15 (s, 1H); ESIMS m/z (rel intensity) 311 ([M + H], 100).
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6-(4-Cyclohexylphenyl)-7,7-dimethyl-7H-pyrimido[4,5-b]-
[1,4]oxazin-4-amine (24). Following the general procedure, 2-
bromo-1-(4-cyclohexylphenyl)-2-methylpropan-1-one (50k), prepared
from phenylcyclohexane, provided the title compound as a white solid
in 55% yield: mp 186−187 °C. 1H NMR (DMSO-d6) δ 1.21−1.47 (m,
5H), 1.60 (s, 6H), 1.70−1.82 (m, 5H), 2.54−2.58 (m, 1H), 6.89 (br s,
2H), 7.29 (d, J = 9.0 Hz, 2H), 7.64 (d, J = 9.0 Hz, 2H), 7.94 (s, 1H);
ESIMS m/z (rel intensity) 337 ([M + H], 100).
6-([1,1′-Biphenyl]-4-yl)-7,7-dimethyl-7H-pyrimido[4,5-b]-
[1,4]oxazin-4-amine (25). To a solution of 11 (50 mg, 0.150 mmol)
and PhB(OH)2 (22 mg, 0.180 mmol) in DMA (1.0 mL) were added a
solution of Na2CO3 (40 mg, 0.375 mmol) in water (0.3 mL) and a
suspension of Pd(PPh3)4 (17 mg, 0.015 mmol) in DMA (1.0 mL) at
ambient temperature. The reaction mixture was stirred at 100 °C for
6.5 h, cooled to ambient temperature, and diluted with EtOAc and
water. The insoluble material was removed by filtration, and the
organic phase of the filtrate was separated, washed with saturated
aqueous NaHCO3 and brine, dried over MgSO4, and concentrated.
The resulting residue was triturated in diisopropyl ether to give a
brown solid which was then recrystallized from EtOH to give the title
compound as a pale yellow solid (23.4 mg, 47%): mp 218−219 °C. 1H
NMR (DMSO-d6) δ 1.66 (s, 6H), 7.02 (br s, 2H), 7.39−7.53 (m, 3H),
7.73−7.77 (m, 4H), 7.83−7.86 (m, 2H), 7.97 (s, 1H); ESIMS m/z (rel
intensity) 331 ([M + H], 100).
2-(4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-
6-yl)phenyl)-2-methylpropanoic Acid (30). Following the general
procedure, ethyl 2-(4-(2-bromo-2-methylpropanoyl)phenyl)-2-meth-
ylpropanoate (53), prepared from ethyl 2-methyl-2-phenylpropanoate
(51), provided the ethyl ester of the desired product. The ethyl ester
was saponified using 1 M NaOH in aqueous EtOH at 40 °C to provide
1
the title compound as a white solid in 5.7% yield: mp >200 °C. H
NMR (DMSO-d6) δ 1.49 (s, 6H), 1.60 (s, 6H), 6.98 (br s, 2H), 7.40
(d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 7.95 (s, 1H), 12.4 (br s,
1H); ESIMS m/z (rel intensity) 341 ([M + H], 100).
4-(4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-
6-yl)phenyl)cyclohexanone (31). Following the general procedure,
4-(4-(2-bromo-2-methylpropanoyl)phenyl)cyclohexanone (50o), pre-
pared from 4-phenylcyclohexanone, provided the title compound as a
1
white solid in 31% yield: mp >200 °C (dec). H NMR (DMSO-d6) δ
1.61 (s, 6H), 1.89−2.13 (m, 4H), 2.26−2.34 (m, 2H), 2.55−2.62 (m,
2H), 3.09−3.17 (m, 1H), 6.97 (br s, 2H), 7.39 (d, J = 9.0 Hz, 2H),
7.68 (d, J = 9.0 Hz, 2H), 7.95 (s, 1H); ESIMS m/z (rel intensity) 351
([M + H], 100).
trans-4-(4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]-
oxazin-6-yl)phenyl)cyclohexanol (32). 4,5-Diamino-6-hydroxypyr-
imidine hemisulfate (47a) (32 g, 0.091 mol) was added to a solution
of 50p (58.5 g, 0.18 mol) in EtOH (350 mL) followed by the addition
of 2 N HCl (180 mL). The reaction mixture was heated at reflux for
18 h and then concentrated in vacuo to half the reaction volume. To
this mixture at ambient temperature was added 2 N NaOH (330 mL).
The resulting precipitate was collected by vacuum filtration and
washed with water to provide the title compound as a white solid (53.9
4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-
yl)phenol (26). To a suspension of 27 (100 mg, 0.352 mmol) in
CH2Cl2 (4.0 mL) was added 1.0 M BBr3 solution in CH2Cl2 (1.10 mL,
1.10 mmol) at −78 °C. The reaction mixture was stirred at ambient
temperature for 3 days and was then poured into ice−water. The
mixture was made basic with 1 M NaOH and washed with CHCl3
three times. The aqueous phase was neutralized with 2 M HCl and
extracted with EtOAc. The organic phase was separated, successively
washed with water and brine, dried over Na2SO4, and concentrated in
vacuo. The resulting residue was slurried in toluene and then
recrystallized from EtOH to give the title compound as a white solid
1
g, 85%): mp 246−248 °C. H NMR (DMSO-d6) δ 1.23−1.35 (m,
2H), 1.43−1.56 (m, 2H), 1.60 (s, 6H), 1.75−1.81 (m, 2H), 1.90−1.96
(m, 2H), 2.46−2.54 (m, 1H), 4.37 (m, 1H), 4.60 (d, J = 6.0 Hz, 1H),
6.97 (br s, 2H), 7.30 (d, J = 9.0 Hz, 2H), 7.64 (d, J = 9.0 Hz, 2H), 7.95
(s, 1H); ESIMS m/z (rel intensity) 353 ([M + H], 100).
6-(4-(4,4-Difluorocyclohexyl)phenyl)-7,7-dimethyl-7H-
pyrimido[4,5-b][1,4]oxazin-4-amine (33). Following the general
procedure, 50q provided the title compound as a white solid in 70%
1
(49.1 mg, 52%): mp 245−256 °C. H NMR (DMSO-d6) δ 1.60 (s,
1
yield: mp 226−227 °C. H NMR (DMSO-d6) δ 1.60 (s, 6H), 1.64−
6H), 6.82 (d, J = 9.0 Hz, 2H), 6.91 (br s, 2H), 7.62 (d, J = 9.0 Hz,
2H), 7.94 (s, 1H), 9.94 (s, 1H); ESIMS m/z (rel intensity) 271 ([M +
H], 100).
1.74 (m, 2H), 1.89−2.16 (m, 6H), 2.75−2.81 (m, 1H), 6.90 (br s,
2H), 7.33 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H), 7.95 (s, 1H);
ESIMS m/z (rel intensity) 373 ([M + H], 100).
6-(4-Methoxyphenyl)-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]-
oxazin-4-amine (27). Following the general procedure, 2-bromo-1-
(4-methoxyphenyl)-2-methylpropan-1-one (50l), prepared from ani-
sole, provided the title compound as a white solid in 70% yield: mp
183−184 °C. 1H NMR (DMSO-d6) δ 1.61 (s, 6H), 3.82 (s, 3H), 6.87
(br s, 2H), 6.99 (d, J = 6.0 Hz, 2H), 7.71 (d, J = 6.0 Hz, 2H), 7.94 (s,
1H); ESIMS m/z (rel intensity) 285 ([M + H], 100).
(trans)-4-(4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]-
oxazin-6-yl)phenyl)cyclohexanecarboxylic Acid (34). Using 57
(4:1 trans/cis) (69 mg) in the general procedure provided the methyl
ester of the desired product. The methyl ester was saponified using 1
M NaOH in aqueous EtOH at 40 °C to provide the title compound as
a white solid (54 mg): mp >250 °C. IR (cm−1) 3328, 2931, 1704,
1615; 1H NMR (DMSO-d6) δ 1.40−1.58 (m, 4H), 1.61 (s, 6H),
1.82−1.92 (m, 2H), 1.97−2.08 (m, 2H), 2.24−2.36 (m, 1H), 2.52−
2.61 (m, 1H), 6.91 (br s, 2H), 7.31 (d, J = 8.6 Hz, 2H), 7.64 (d, J = 8.6
Hz, 2H), 7.95 (s, 1H), 11.92 (br s, 1H); ESIMS m/z (rel intensity)
381 ([M + H], 100).
7,7-Dimethyl-6-(4-(piperidin-1-yl)phenyl)-7H-pyrimido[4,5-
b][1,4]oxazin-4-amine (28). To a mixture of 2-methyl-1-(4-
(piperidin-1-yl)phenyl)propan-1-one (64 mg, 0.277 mmol) in 47%
HBr (0.5 mL) was added Br2 (15.7 μL, 0.305 mmol), and the reaction
mixture was stirred at 60 °C for 30 min. After the mixture was cooled
to ambient temperature, 47a (17.5 mg, 0.139 mmol) was added to the
mixture, which was then diluted with H2O (2.0 mL) and EtOH (1.0
mL) and heated at reflux overnight. The reaction mixture was cooled
to ambient temperature and concentrated in vacuo. The resulting
residue was diluted with EtOAc, washed with 1 M NaOH and brine,
and concentrated in vacuo. The resulting residue was purified by
preparative TLC to give the title compound as a white solid (40.0 mg,
3-(trans)-(4-(4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b]-
[1,4]oxazin-6-yl)phenyl)cyclohexyl)propanoic Acid (35). Em-
ploying 61 (436 mg, 1.94 mmol) in the general procedure provided
the methyl ester of the desired product. The methyl ester was
saponified using 1 M NaOH in aqueous EtOH at 40 °C to provide the
title compound (231 mg, 29%) as a white solid: mp >250 °C. IR
1
(cm−1) 3310, 2922, 1702, 1611; H NMR (DMSO-d6) δ 1.04−1.09
(m, 2H), 1.43 (m, 1H), 1.43−1.50 (m, 4H), 1.60 (s, 6H), 1.83 (m,
4H), 2.25 (t, J = 7.7 Hz, 2H), 2.50 (m, 1H), 6.88 (br s, 2H), 7.29 (d, J
= 8.3 Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H), 7.94 (s, 1H), 11.84 (br s, 1H);
ESIMS m/z (rel intensity) 409 ([M + H], 100).
1
83%): mp 191−193 °C. H NMR (DMSO-d6) δ 1.58−1.62 (m, 6H),
1.62 (s, 6H), 3.26 (br s, 4H), 6.79 (br s, 2H), 7.29 (d, J = 9.0 Hz, 2H),
7.63 (d, J = 9.0 Hz, 2H), 7.91 (s, 1H); ESIMS m/z (rel intensity) 338
([M + H], 100).
2-(4′-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-
6-yl)-[1,1′-biphenyl]-4-yl)acetic Acid (36). Following the general
procedure, methyl 2-(4′-(2-bromo-2-methylpropanoyl)[1,1′-biphen-
yl]-4-yl)acetate (54), prepared from methyl 2-([1,1′-biphenyl]-4-
yl)acetate (52), provided the methyl ester of the desired product. The
methyl ester was saponified using 1 M NaOH in aqueous EtOH at 40
°C to provide the title compound as a white solid in 20% yield: mp
>200 °C. 1H NMR (DMSO-d6) δ 1.65 (s, 6H), 3.64 (s, 2H), 7.04 (br
7,7-Dimethyl-6-(4-morpholinophenyl)-7H-pyrimido[4,5-b]-
[1,4]oxazin-4-amine (29). Using the procedure for 28 with 2-
methyl-1-(4-morpholinophenyl)propan-1-one provided the title com-
1
pound as a white solid in 21% yield: mp 214−219 °C. H NMR
(DMSO-d6) δ 1.62 (s, 6H), 3.22 (t, J = 4.8 Hz, 4H), 3.75 (t, J = 4.8
Hz, 4H), 6.83 (br s, 2H), 6.96 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.4 Hz,
2H), 7.92 (s, 1H); ESIMS m/z (rel intensity) 340 ([M + H], 100).
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s, 2H), 7.39 (d, J = 9.0 Hz, 2H), 7.69 (d, J = 9.0 Hz, 2H), 7.75 (d, J =
9.0 Hz, 2H), 7.84 (d, J = 9.0 Hz, 2H), 7.97 (s, 1H), 12.32 (br s, 1H);
ESIMS m/z (rel intensity) 389 ([M + H], 100).
anhydrous lithium iodide (670 mg, 5 mmol). The reaction mixture was
heated at 125 °C for 24 h and then poured into water (20 mL). The
resulting precipitate was collected by filtration and washed with water.
The precipitate was recrystallized from ethanol to afford the title
2-((trans)-4-(4-(4-Amino-2,7,7-trimethyl-7H-pyrimido[4,5-b]-
[1,4]oxazin-6-yl)phenyl)cyclohexyl)acetic Acid (37). Using 4,5-
diamino-6-hydroxy-2-methylpyrimidine (47b) in the procedure used
for the preparation of 1 provided the title compound: mp >250 °C. 1H
NMR (DMSO-d6) δ 1.07−1.18 (m, 2H), 1.41−1.53 (m, 2H), 1.59 (s,
6H), 1.71−1.86 (m, 5H), 2.15 (d, J = 4.0 Hz, 2H), 2.24 (s, 3H), 2.51−
2.57 (m, 1H), 6.84 (br s, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 8.0
Hz, 2H), 12.04 (br s, 1H); ESIMS m/z (rel intensity) 409.2 ([M + H],
100).
1
compound (70 mg, 72%) as a white solid. H NMR (DMSO-d6) δ
1.17−1.26 (m, 2H), 1.48−1.82 (m, 16H), 1.98 (t, J = 7.4 Hz, 2H),
2.18 (d, J = 6.8 Hz, 2H), 2.90 (t, J = 7.2 Hz, 2H), 7.28 (d, J = 8.1 Hz,
1H), 7.37 (br s, 1H), 7.53 (d, J = 8.1 Hz, 1H), 7.58 (s, 1H), 7.80 (br s,
1H), 12.05 (br s, 1H); ESIMS m/z (rel intensity) 488 ([M + H], 100).
2-((trans)-4-(4-(4-Amino-7,7-dimethyl-7H-pyrano[2,3-d]-
pyrimidin-6-yl)phenyl)cyclohexyl)acetic Acid (43). A mixture of
93 (1.12 g, 4.38 mmol), 84 (2.0 g, 6.57 mmol), PdCl2(dppf) (107 mg,
0.132 mmol), and 2 M aqueous Na2CO3 (11 mL) in DMF (30 mL)
was heated at 80 °C under a nitrogen atmosphere for 24 h. The
solution was diluted with water (75 mL), and the resulting precipitate
was collected by vacuum filtration to provide an off-white solid.
Purification by flash chromatography (silica gel, 5% MeOH/CH2Cl2)
2-((trans)-4-(4-(4-Amino-7,7-dimethyl-2-(trifluoromethyl)-
7H-pyrimido[4,5-b][1,4]oxazin-6-yl)phenyl)cyclohexyl)acetic
Acid (38). Using 5,6-diamino-4-hydroxy-2-trifluoromethylpyrimidine
(67) in the procedure used for the preparation of 1 provided the title
1
compound. H NMR (DMSO-d6) δ 1.10−1.20 (m, 2H), 1.42−1.90
1
(m, 13H), 2.17 (d, J = 6.8 Hz, 2H), 2.45−2.60 (m, 1H), 7.34 (d, J =
8.0 Hz, 2H), 7.36 (br s, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.74 (br s, 1H),
12.04 (br s, 1H); ESIMS m/z (rel intensity) 462.2 ([M + H], 100).
2-((trans)-4-(4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b]-
[1,4]oxazin-6-yl)phenyl)cyclohexyl)acetamide (39). To a sol-
ution of 1 (80 mg, 0.203 mmol) in DMF (2.4 mL) were sequentially
added HOBt·H2O (34.2 mg, 0.223 mmol), 28% aqueous NH3 (0.041
mL, 6.08 mmol), and EDC (42.8 mg, 0.278 mmol) at ambient
temperature. After the mixture was stirred for 1 h, 28% aqueous NH3
(0.041 mL, 6.08 mmol) was added to the mixture, which was further
stirred for 70 h at ambient temperature. The mixture was diluted with
EtOAc and washed with saturated aqueous NaHCO3 solution, water,
and brine. The organic layer was dried over MgSO4 and concentrated
to give a residue that was slurried in Et2O. The resulting solid was
collected by vacuum filtration and then slurried in EtOH. The
resulting precipitate was collected by vacuum filtration and dried under
reduced pressure to afford the title compound as a white solid (48.2
provided the methyl ester as a white solid (1.09 g, 61%). H NMR
(DMSO-d6) δ 1.16 (m, 2H), 1.44 (m, 2H), 1.51 (s, 6H), 1.79 (m,
5H), 2.25 (d, J = 6.7 Hz, 2H), 2.47 (m, 1H), 3.61 (s, 3H), 6.57 (s,
1H), 6.86 (s, 2H), 7.24 (d, J = 8.1 Hz, 2 H), 7.29 (d, J = 8.0 Hz, 2H),
7.95 (s, 1H); ESIMS m/z (rel intensity) 408.5 ([M + H], 100). A
solution of the methyl ester (130 mg, 0.319 mmol) in MeOH (12 mL)
and 10% aqueous LiOH (4 mL) was heated at reflux for 4 h. The
MeOH was removed in vacuo and the resulting aqueous solution was
acidified with 1 N HCl to pH 1. The resulting precipitate was collected
by vacuum filtration to provide 43 as an off-white solid (111 mg, 88%).
1H NMR (DMSO-d6) δ 1.13 (m, 2H), 1.51 (m, 2H), 1.55 (s, 6H),
1.74 (m, 1H), 1.82 (m, 4H), 2.15 (d, J = 6.9 Hz, 2H), 2.47 (m, 1H),
6.61 (s, 1H), 7.26 (d, J = 8.1 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H), 7.33
(br s, 2H), 8.09 (s, 1H); ESIMS m/z (rel intensity) 394 ([M + H],
100).
2-((trans)-5′-(4-Amino-7,7-dimethyl-7H-pyrano[2,3-d]-
pyrimidin-6-yl)-2′,3′-dihydrospiro[cyclohexane-1,1′-inden]-4-
yl)acetic Acid (44). A mixture of 93 (100 mg, 0.391 mmol), 85 (150
mg, 0.391 mmol), Pd(PPh3)4 (23 mg, 0.020 mmol), and 2 M aqueous
Na2CO3 (1 mL) in DMF (10 mL) was heated at 80 °C for 3.5 h. The
reaction mixture was diluted with EtOAc (75 mL), washed with water
(3 × 50 mL), brine, dried (MgSO4), filtered, and concentrated in
vacuo to provide a yellow solid. Purification by flash chromatography
(silica gel, 5% MeOH/CH2Cl2) provided the methyl ester as a white
solid (103 mg). 1H NMR (DMSO-d6) δ 1.23 (m, 2H), 1.49 (m, 1H),
1.51 (s, 6H), 1.65 (m, 5H), 1.79 (m, 1H), 1.95 (t, J = 7.3 Hz, 2H),
2.28 (d, J = 7.0 Hz, 2H), 2.85 (t, J = 7.3 Hz, 2H), 3.61 (s, 3H), 6.56 (s,
1H), 6.84 (s, 2H), 7.18 (m, 3H), 7.95 (s, 1H); ESIMS m/z (rel
intensity) 434 ([M + H], 100). A solution of the methyl ester (103
mg, 0.238 mmol) in MeOH (9 mL) and 10% aqueous LiOH (3 mL)
was heated at reflux for 2 h. The MeOH was removed in vacuo, and
the resulting aqueous layer was acidified to pH 1 with 1 N HCl. The
resulting precipitate was collected by vacuum filtration to provide 44
1
mg, 60%): mp 224−226 °C. H NMR (DMSO-d6) δ 1.02−1.17 (m,
2H), 1.40−1.53 (m, 2H), 1.61 (s, 6H), 1.66−1.87 (m, 5H), 1.98 (d, J
= 7.1 Hz, 2H), 2.53 (m, 1H), 6.67 (br s, 1H), 6.89 (br s, 2H), 7.21 (br
s, 1H), 7.30 (d, J = 8.1 Hz, 2H), 7.63 (d, J = 8.1 Hz, 2H), 7.94 (s, 1H);
ESIMS m/z (rel intensity) 394 ([M + H], 100).
6-(4-((trans)-4-((2H-Tetrazol-5-yl)methyl)cyclohexyl)phenyl)-
7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-4-amine (40). To a
solution of 39 (1.25 g, 3.18 mmol) in pyridine (12.5 mL) was added
SOCl2 (1.16 mL, 15.9 mmol) at 0 °C. The reaction mixture was stirred
at ambient temperature for 25 min and was then quenched by the
dropwise addition of water at 0 °C. The deposited precipitates were
collected by filtration and dried under reduced pressure to give the
nitrile as a white solid (1.02 g, 85%). A mixture of the nitrile (600 mg,
1.60 mmol), NaN3 (1.56 g, 24.0 mmol), and NH4Cl (1.28 g, 24.0
mmol) in DMF (9.0 mL) was stirred at 110 °C for 4 days. The
mixture was concentrated in vacuo to provide a residue, which was
then diluted with EtOAc and washed with water three times. The
organic layer was concentrated in vacuo and the residue was purified
by flash column chromatography on silica gel to afford the title
1
as an off-white solid (73 mg). H NMR (DMSO-d6) δ 1.22 (m, 2H),
1.50 (m, 2H), 1.56 (s, 6H), 1.63 (m, 3H), 1.67 (m, 2H), 1.71 (m, 1H),
1.96 (t, J = 7.3 Hz, 2H), 2.17 (d, J = 6.8 Hz, 2H), 2.86 (t, J = 7.2 Hz,
2H), 6.61 (s, 1H), 7.20 (m, 3H), 7.50 (br s, 2H), 8.14 (s, 1H); ESIMS
m/z (rel intensity) 420 ([M + H], 100).
1
compound as a pale yellow solid (363 mg, 54%): mp >200 °C. H
NMR (DMSO-d6) δ 1.11−1.27 (m, 2H), 1.36−1.54 (m, 2H), 1.60 (s,
6H), 1.68−1.87 (m, 5H), 2.50−2.61 (m, 1H), 2.83 (d, J = 7.0 Hz,
2H), 6.93 (br s, 2H), 7.30 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.4 Hz,
2H), 7.95 (s, 1H); ESIMS m/z (rel intensity) 419 ([M + H], 100).
((trans)-4-(4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]-
oxazin-6-yl)phenyl)cyclohexyl)methanesulfonamide (41).
Using 71 (40.0 mg, 0.158 mmol) in the general procedure provided
the title compound as a white solid (30.0 mg, 50%): mp >200 °C. 1H
NMR (DMSO-d6) δ 1.16−1.29 (m, 2H), 1.43−1.56 (m, 2H), 1.61 (s,
6H), 1.80−2.11 (m, 5H), 2.53−2.59 (m, 1H), 2.94 (d, J = 6.0 Hz,
2H), 6.79 (s, 2H), 6.93 (br s, 2H), 7.31 (d, J = 9.0 Hz, 2H), 7.66 (d, J
= 9.0 Hz, 2H), 7.95 (s, 1H); ESIMS m/z (rel intensity) 430 ([M + H],
100).
2-((trans)-4-(4-(4-Amino-2,7,7-trimethyl-7H-pyrano[2,3-d]-
pyrimidin-6-yl)phenyl)cyclohexyl)acetic Acid (45). A mixture of
94 (1.02 g, 3.78 mmol), 84 (2.03 g, 5.67 mmol), PdCl2(dppf) (92.4
mg, 0.114 mmol), and 2 M aqueous Na2CO3 (11 mL) in DMF (30
mL) was heated at 80 °C under a nitrogen atmosphere for 24 h. The
solution was diluted with water (75 mL), and the resulting precipitate
was collected by vacuum filtration to provide an off-white solid.
Purification by flash chromatography (silica gel, 5% MeOH/CH2Cl2)
provided the methyl ester as a white solid (1.01 g). A solution of the
methyl ester (150 mg, 0.356 mmol) in MeOH (12 mL) and 10%
aqueous LiOH (4 mL) was heated at reflux for 4 h. The MeOH was
removed in vacuo, and the resulting aqueous solution was acidified
with 1 N HCl to pH 1. The resulting precipitate was collected by
vacuum filtration to provide 45 as an off-white solid (125 mg, 86%).
1H NMR (DMSO-d6) δ 1.04−1.19 (m, 2H), 1.39−1.55 (m, 2H), 1.49
2-((trans)-5′-(4-Amino-7,7-dimethyl-2-(trifluoromethyl)-7H-
pyrimido[4,5-b][1,4]oxazin-6-yl)-2′,3′-dihydrospiro-
[cyclohexane-1,1′-inden]-4-yl)acetic Acid (42). To a stirred
solution of 81 (100 mg, 0.199 mmol) in DMF (1 mL) was added
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Journal of Medicinal Chemistry
Article
(s, 6H), 1.65−1.89 (m, 5H), 2.14 (d, J = 7.2 Hz, 2H), 2.21 (s, 3H),
2.47 (m, 1H), 6.55 (s, 1H), 6.78 (br s, 2H), 7.23 (d, J = 8.3 Hz, 2H),
7.27 (d, J = 8.3 Hz, 2H), 12.01 (br s, 1H); ESIMS m/z (rel intensity)
408 ([M + H], 100).
cardiovascular events in women. JAMA, J. Am. Med. Assoc. 2007, 298
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(8) Miller, M.; Stone, N. J.; Ballantyne, C.; Bittner, V.; Criqui, M. H.;
Ginsberg, H. N.; Goldberg, A. C.; Howard, W. J.; Jacobson, M. S.;
Kris-Etherton, P. M.; Lennie, T. A.; Levi, M.; Mazzone, T.; Pennathur,
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Methyl 2-((trans)-5′-(4-Amino-7,7-dimethyl-2-(trifluoro-
methyl)-7H-pyrimido[4,5-b][1,4]oxazin-6-yl)-2′,3′-
dihydrospiro[cyclohexane-1,1′-inden]-4-yl)acetate (81). To 80
(0.5 g, 1.23 mmol) and 67 (0.26 g, 1.34 mmol) were added methanol
(13 mL), 2 M HCl (2 mL), and water (5 mL). The mixture was
heated at reflux for 24 h before concentrating in vacuo. To the residue
was added water (10 mL) and the resulting precipitate was collected
by filtration, washed with pentane, and dried under vacuum to afford
the title compound (375 mg, 61%) as a white solid. 1H NMR (DMSO-
d6) δ 1.18−1.27 (m, 2H), 1.48−1.86 (m, 13H), 1.98 (t, J = 7.3 Hz,
2H), 2.28 (d, J = 7.2 Hz, 2H), 2.89 (t, J = 7.1 Hz, 2H), 3.61 (s, 3H),
7.28 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.58 (s, 1H).
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Res. 1996, 35, 169−201.
(12) Cases, S.; Smith, S. J.; Zheng, Y.-W.; Myers, H. M.; Lear, S. R.;
Sande, E.; Novak, S.; Collins, C.; Welch, C. B.; Lusis, A. J.; Erickson, S.
K.; Farese, R. V., Jr. Identification of a gene encoding an acyl
CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol
synthesis. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 13018−13023.
(13) Lardizabal, K. D.; Mai, J. T.; Wagner, N. W.; Wyrick, A.;
Voelker, T.; Hawkins, D. J. DGAT2 is a new diacylglycerol
acyltransferase gene family. J. Biol. Chem. 2001, 276 (42), 38862−
38869.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures describing the preparation of
intermediates and in vivo protocols. This material is available
free of charge via the Internet at http://pubs.acs.org.
(14) Cases, S.; Stone, S. J.; Zhou, P.; Yen, E.; Tow, B.; Lardizabal, K.
D.; Voelker, T.; Farese, R. V., Jr. Cloning of DGAT2, a second
mammalian diacylglycerol acyltransferase, and related family members.
J. Biol. Chem. 2001, 276 (42), 38870−38876.
(15) Wurie, H. R.; Buckett, L.; Zammit, V. A. Diacylglycerol
acyltransferase 2 acts upstream of diacylglycerol acyltransferase 1 and
utilizes nascent diglycerides and de novo synthesized fatty acids in
HepG2 cells. FEBS J. 2012, 279, 3033−3047.
(16) Qi, J.; Lang, W.; Geisler, J. G.; Wang, P.; Petrounia, I.; Mai, S.;
Smith, C.; Askari, H.; Struble, G. T.; Williams, R.; Bhanot, S.; Monia,
B. P.; Bayoumy, S.; Grant, E.; Caldwell, G. W.; Todd, M. J.; Liang, Y.;
Gaul, M. D.; Demarest, K. T.; Connelly, M. A. The use of stable
isotope-labeled glycerol and oleic acid to differentiate the hepatic
functions of DGAT1 and -2. J. Lipid Res. 2012, 53, 1106−1116.
(17) Stone, S. J.; Myers, H. M.; Watkins, S. M.; Brown, B. E.;
Feingold, K. R.; Elias, P. M.; Farese, R. V., Jr. Lipopenia and skin
barrier abnormalities in DGAT2-deficient mice. J. Biol. Chem. 2004,
279 (12), 11767−11776.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: bmfox@amgen.com. Phone (650) 244-2221. Fax:
(650) 837-9369.
Author Contributions
§S.S. and F.K. contributed equally to the work presented in this
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Amgen Inc. and Japan Tobacco Inc. contributed equally to the
work detailed in this paper.
(18) Smith, S. J.; Cases, S.; Jensen, D. R.; Chen, H. C.; Sande, E.;
Tow, B.; Sanan, D. A.; Raber, J.; Eckel, R. H.; Farese, R. V., Jr. Obesity
resistance and multiple mechanisms of triglyceride synthesis in mice
lacking DGAT. Nat. Genet. 2000, 25, 87−90.
(19) Chen, H. C.; Farese, R. V., Jr. Inhibition of triacylglycerol
synthesis as a treatment strategy for obesity: lessons from DGAT1-
deficient mice. Arterioscler., Thromb., Vasc. Biol. 2005, 25 (3), 482−
486.
(20) Hubbard, B. K.; Enyedy, I.; Gilmore, T. A.; Serrano-Wu, M. H.
Antisense and small-molecule modulation of diacylglycerol acyltrans-
ferase. Expert Opin. Ther. Pat. 2007, 17 (11), 1331−1339.
(21) Zammit, V. A.; Buckett, L. A.; Turnbull, A. V.; Wure, H.;
Proven, A. Diacylglycerol acyltransferases: potential roles as
pharmacological targets. Pharmacol. Ther. 2008, 118, 295−302.
(22) King, A. J.; Judd, A. S.; Souers, A. J. Inhibitors of diacylglycerol
acyltransferase: a review of 2008 patents. Expert Opin. Ther. Pat. 2010,
20 (1), 19−29.
ABBREVIATIONS USED
■
DGAT, acyl CoA:diacylglycerol acyltransferase; TG, triglycer-
ide; ACAT, acyl CoA:cholesterol acyltransferase; SPA,
scintillation proximity assay; MGAT, acyl CoA:monoacylgly-
cerol acyltransferase; GPAT, glycerol 3-phosphate acyltransfer-
ase; ALT, alanine aminotransferase; AST, aspartate amino-
transferase
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