Discovery of a potent, selective, and orally efficacious pyrimidinooxazinyl bicyclooctaneacetic acid diacylglycerol acyltransferase-1 inhibitor

Article abstract of DOI:10.1021/jm801507v

Inhibition of DGAT-1 is increasingly seen as an attractive mechanism with the potential for treatment of obesity and other elements of the metabolic syndrome. We report here a bicyclooctaneacetic acid derivative in the pyrimidinooxazine structural class o

Full text of DOI:10.1021/jm801507v

1558
J. Med. Chem. 2009, 52, 1558–1568
Discovery of a Potent, Selective, and Orally Efficacious Pyrimidinooxazinyl Bicyclooctaneacetic
Acid Diacylglycerol Acyltransferase-1 Inhibitor
Alan M. Birch,* Susan Birtles, Linda K. Buckett, Paul D. Kemmitt, Graham J. Smith, Tim J. D. Smith, Andrew V. Turnbull,
and Steven J. Y. Wang
AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom
ReceiVed December 1, 2008
Inhibition of DGAT-1 is increasingly seen as an attractive mechanism with the potential for treatment of
obesity and other elements of the metabolic syndrome. We report here a bicyclooctaneacetic acid derivative
in the pyrimidinooxazine structural class of DGAT-1 inhibitors that has good potency, selectivity, and
pharmacokinetic characteristics across a variety of species. This compound is an effective inhibitor of DGAT-1
in both intestinal and adipose tissue, which results in a reduction in body weight or body weight gain following
oral administration in both mouse and rat models of dietary-induced obesity.
Introduction
selective DGAT-1 inhibitor (Table 1). It also proved to have
an acceptable PK profile, in terms of bioavailability and half-
life, in rodents (Table 2). We sought to improve on the overall
profile of this compound, with a specific aim to reduce the
clearance of the compound in rat and dog PK studies. We had
evidence that the major route of metabolism of 2 in these species
was via conjugation of the acid group to form the acylglucu-
ronide. In the light of recent concerns that the reactivity of such
metabolites can lead to covalent protein adducts, which may
give rise to idiosyncratic toxicity,7 we sought to introduce a
greater degree of steric crowding around the acid group by
replacing the cyclohexane ring with various bi- and tricyclic
systems. Certain sterically hindered carboxylic acid acyl glu-
curonides have been shown8 to be inherently more stable both
to hydrolysis and to rearrangement to more reactive isomers,
making them less likely to react with proteins in vivo. We
therefore prepared a short series of adamantane, bicyclo[2.2.2]-
octane, and bicyclo[3.2.2]nonane-pyrimido[4,5-b][1,4]oxazine
derivatives, as described below.
Current therapeutic options for the treatment of obesity target
a reduction of energy intake either through inhibition of appetite
or through reduction in lipid absorption from the small intestine.1
The continuing need for improved therapies, which are either
more efficacious or give rise to fewer side effects or both, has
led to a variety of newer mechanistic targets, including those
which seek to inhibit triacylglycerol synthesis and storage in
adipose tissue. The diacylglycerol acyltransferase enzymes
DGAT-1a and DGAT-2 are potential targets in such an ap-
proach,2 as they are dedicated to the final committed step in
trigylceride synthesis. Inhibitors of varying structural types of
DGAT-1 are claimed and reported with increasing frequency
as potential treatments for obesity and other disorders.3 This
particular interest in DGAT-1 inhibition stems from the
published phenotype of DGAT-1 deficient (Dgat1-/-) mice.4
These animals are viable, resistant to weight gain when fed a
high-fat diet, and show increased insulin and leptin sensitivity.
Resistance to weight gain results from increased energy
expenditure rather than decreased food intake (the animals are
in fact hyperphagic) and is associated with loss of adipose rather
than lean tissue mass. A recent paper5 has demonstrated that
most aspects of this phenotype can be reproduced in rodents
by treatment with a potent and selective small molecule inhibitor
of DGAT-1 such as compound 1. We now report a further
structural class of potent and selective DGAT-1 inhibitors, see
Figure 1, one example of which is highly effective in rodent
pharmacodynamic and disease models.
Chemistry
The phenylbicyclo[2.2.2]octane core was constructed accord-
ing to either method A or B as illustrated in Scheme 1. Method
A starts with a base-catalyzed condensation of acetophenone
and diethyl (ethoxymethylene)malonate, giving the intermediate
diethyl (3-oxo-3-phenylpropylidene)malonate, which was con-
verted into the pyrone 9 on treatment with acetyl chloride and
DMF. The Diels-Alder reaction of 9 with ethene was effected
at 75 bar rather than the reported 3000 bar9-11 to give
bicyclooctene 10, which was hydrogenated to afford 11.
The procedure described by Chapman et al.12 (Method B)
was also used to make the phenylbicyclo[2.2.2]octane as in 15,
and this method could be extended to the bicyclo[3.2.2]nonane
system as in 19. Selective hydrolysis of the diesters 1213 and
1614 with barium hydroxide gave the acids 13 and 17, which
were converted into the corresponding bromides 14 and 18 under
Hunsdiecker conditions. The bromobicycles 14 and 18 were
treated with aluminum chloride in benzene to provide 15 and
19.
The first published patent application, which described small
molecule inhibitors of DGAT-1, resulted from a collaboration
between the Japan Tobacco and Tularik companies.6 It claimed
a series of pyrimido[4,5-b][1,4]oxazine derivatives, and one
interesting example, compound 2, proved to be a potent and
* To whom correspondence should be addressed. Phone: +44 (0)1625
514650. Fax: +44 (0)1625 516667. E-mail: alan.birch@astrazeneca.com.
a Abbreviations: ACAT-1, acyl-cholesterol acyltransferase-1; AG, acyl-
glucuronide; Clp, plasma clearance; DAG, diacylglycerol; DGAT-1, dia-
cylglycerol acyltransferase-1; DGAT-2, diacylglycerol acyltransferase-2;
DIO, diet-induced obesity; ED50, effective dose, dose effects a 50% reduction
in biological end point; GI, gastrointestinal; HuTu 80, human gastrointestinal
tumor cell line; iv, intravenous; MDCK, Madin-Darby canine kidney cell
line; OLTT, oral lipid tolerance test; PK, pharmacokinetic; SEM, standard
error of the mean; TAG, triacylglycerol; Vdss, volume of distribution at steady
state.
The synthesis of compounds 3-5 are outlined in Scheme 2.
The esters 20 and 22 were obtained by using the modified
Arndt-Eistert protocol,15 and 20 was alkylated with LDA and
methyl iodide to give 21. The pyrimido ring system of the test
compounds was built up via a Friedel-Crafts acylation with
10.1021/jm801507v CCC: $40.75
2009 American Chemical Society
Published on Web 03/03/2009

Pyrimidinooxazinyl Bicyclooctaneacetic Acid DGAT-1 Inhibitor
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1559
Figure 1. Literature and novel DGAT-1 inhibitors.
2-bromoisobutyryl bromide, followed by cyclization with 5,6-
diaminopyrimidin-4-ol and final basic hydrolysis gave com-
pounds 3-6.
Compounds 7 and 8 were made using analogous chemistry
to that described for compounds 3-6 (Scheme 3).
marmoset, and human, respectively, during 1 h incubation. These
data are consistent with the relative in vivo clearance data in
the rat, dog, and marmoset (Table 2). Furthermore, in dog, 82%
of parent-related material was excreted as the acyl glucuronide
in a 2 h urine sample.
It is believed that in some circumstances the initially formed
1ꢀ-isomer of an acyl glucuronide metabolite can directly lead
to drug-protein covalent conjugates through acylation, while
the 2- and 4-O acyl rearrangement products can give rise to
adducts through Schiff base formation with protein amino
groups.7 To assess the potential of the acylglucuronide metabo-
lite of compound 4 to enter into such reactions its hydrolytic
stability (as an indication of acylation potential) and propensity
to form the O-acyl rearrangement products was investigated.
Compound 4 acyl glucuronide was found to undergo hydrolytic
cleavage only very slowly at 37 °C in pH 7.4 buffer (Figure 2)
compared to various standard drugs reported in the literature.8
In addition, only ∼15% rearrangement to acyl migrated isoforms
was noted under these conditions. Taken together, these data
suggest that compound 4 would be unlikely to give rise to
idiosyncratic toxicity as a result of covalent protein binding
involving its acyl glucuronide metabolite.
Compound 4 was inactive (<25% inhibition @30 µM) versus
the cytochrome P450 enzymes CYP1A2, CYP2C9, CYP2C19,
CYP2D6, and CYP3A4. Similarly, the compound showed no
significant activity against the hERG encoded potassium chan-
nel. The key physical properties of 4 are given in Table 3 and
are typical for a moderately lipophilic carboxylic acid. Although
aqueous solubility is lower than ideal, this is counterbalanced
by good cellular permeability, resulting in no issues with
absorption in animal studies.
Results and Discussion
Compounds in this series showed good to very good potency
(15-250 nM) as inhibitors of recombinant human DGAT-1
(Table 1). The bicyclooctane and bicyclononane compounds,
which have a 1,4- relationship between the aryl and acid groups,
were more potent than the adamantane derivatives that have a
corresponding 1,3-relationship. All compounds showed much
lower potency against human ACAT-1, the nearest enzyme in
terms of structural homology and where tested were essentially
inactive versus human DGAT-2, which, although serving the
same function, is only distantly related to DGAT-1 structurally.16
Where tested, compounds showed little difference between their
IC50 values against rat microsomal and recombinant human
DGAT-1. The bicyclo[2.2.2]octaneacetic acid 4 was the most
potent of the new compounds, giving an equivalent IC50 to
compound 2. These two compounds also showed excellent
potency in inhibiting triacylglycerol synthesis in human HuTu
80 cells, which are relevant to the gut as a target organ.
Comparison of the compounds in PK studies after oral and
iv dosing to rats showed reduced clearance for 4 compared to
compound 2 (Table 2). This ranking was repeated in dogs with
compound 4 again showing a superior profile, with reduced
clearance compared to 2. Compound 4 also showed a very good
PK profile in marmosets. Acyl glucuronidation is the major route
of metabolism of 4 in hepatocytes (18%, 38%, and 17%
conversion during 2 h incubation in rat, dog, and human,
respectively). In alamethicin-activated microsomes, the conver-
sion to glucuronide was 9%, 76%, 15%, and 25% in rat, dog,
Although ubiquitously expressed, adipocytes and the entero-
cytes of the small intestine are two major tissue types in which
DGAT-1 has significant expression and is therefore thought to

1560 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Birch et al.
Table 1. Enzyme and Cellular Inhibitory Potencies for Target Compoundsa
a Mean values from a minimum n ) 3 experiments. b Inhibition of triacylglycerol synthesis in HuTu 80 cells. NT: not tested.
Table 2. Pharmacokinetic Parameters for Selected DGAT-1 Inhibitorsa
Inhibition of the enzyme in enterocytes should lead to reduced
TAG synthesis in these cells also, which should in turn result
in a measurable effect on TAG levels in the systemic circulation
following an oral lipid load. Encouraged by the potency,
selectivity, and PK profile of compound 4, we tested it further
in a variety of animal models designed to demonstrate the effects
described above.
Clp
Vdss iv half-life
oral
bioavailability
(%)
compd species (mL/min/kg) (L/kg)
(h)
half-life (h)
2
4
rat
dog
5.2
12
0.69
2.0
3.0
3.9
5.6
2.8
>100b
>100b
rat
dog
1.5
3.3
0.78
0.70
1.0
7.0
8.6
5.4
9.9
9.5
72
>100b
61
marmoset
0.88
13
In an oral lipid tolerance test (OLTT), fasted rats were
administered compound by oral gavage at a range of doses 2 h
before a bolus dose of corn oil. Plasma TAG levels were
measured 1.5 h later. Figure 3A shows that compound 4 was
highly efficacious in reducing plasma TAG excursion in this
model (statistically significant effect at 0.3 mg/kg), indicating
a reduction in enterocyte lipid transport. This compound also
showed good, but less potent, effects on adipose TAG synthesis
(Figure 3B), giving a statistically significant decrease in the TAG
to DAG ratio in adipose cells at 1 mg/kg, thereby demonstrating
inhibition of DGAT-1 in these cells. The greater potency of 4
in oral lipid tolerance test is to be anticipated from its presumed
relatively higher local unbound concentration in the gut from a
given dose compared to that appearing in the blood, giving
greater DGAT-1 inhibition in the enterocytes compared to that
a Compounds were dosed at between 1 and 3 mg/kg in either solution
(DMSO/hydroxy-ꢀ-cyclodextrin)orsuspension(HPMC/Tween).b Bioavailabilities
>100% are believed to reflect enterohepatic recirculation of acyl glucuronide
metabolite.
play a major role in lipid metabolism. In adipocytes, DGAT-1
catalyzes the condensation of diacylglycerol (DAG) and a range
of fatty acids (FA) to form triacylglycerol (TAG) for high-
density energy storage. In the GI tract, dietary sources of FA
and monoacylglycerol absorbed by the enterocyte are resyn-
thesized to TAG, which is critical for the assembly and secretion
of chylomicrons that transport triacylglycerol into the blood
stream for utilization by high energy requiring tissues such as
muscle. Inhibition of DGAT-1 in adipocytes would be expected
to result in measurable effects on TAG synthesis in these cells.

Pyrimidinooxazinyl Bicyclooctaneacetic Acid DGAT-1 Inhibitor
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1561
Scheme 1. Synthesis of Phenylbicycloalkanecarboxylatesa
a Reagents and conditions: (a) (i) NaOEt, EtOH, diethyl (ethoxymethylene)malonate, DME; (ii) AcCl, DMF, 56%. (b) Ethene, 200 °C, 15 h, 75 bar, 51%.
(c) H2, 10% Pt on carbon, EtOH, 84%. (d) Ba(OH)2, MeOH/H2O. (e) (i) NaOH, acetone; (ii) AgNO3; (iii) Br2, pet ether (bp 40-60 °C). (f) AlCl3, PhH.
Scheme 2. Synthesis of Compounds 3-6a
a Reagents and conditions: (a) NaOH, MeOH. (b) (i) Oxalyl chloride, DCM; (ii) TMSCHN2/MeCN/THF/Et3N; (iii) PhCO2Ag/Et3N/MeOH. (c) LDA,
MeI, THF, -70 °C. (d) 2-Bromoisobutyryl bromide, AlCl3, DCM. (e) 5,6-diaminopyrimidin-4-ol, 1 M HCl, EtOH. (f) 2 M NaOH, MeOH. (g) KOTMS,
THF.
in adipocytes. These results demonstrate that DGAT-1 inhibitors
can elicit a dual pharmacodynamic effect through inhibition of
the enzyme in both the enterocyte and adipose tissue, with
potential beneficial clinical effects on both postprandial plasma
TAG and adiposity.
To assess its activity in a model of diet-induced obesity,
compound 4 was tested for effects on body weight loss in DIO
mice (Figure 4). Treatment for 3 days resulted in a modest but
statistically significant reduction in body weight compared to
start weight (2%) and in comparison with age-matched vehicle-
treated control mice on the same diet (3%).
superior PK profile in rat and dog compared to compound 2.
Compound 4 shows potent effects in rodent pharmacodynamic
models, demonstrating efficacy in both gastrointestinal and
adipose tissues, and is effective in reducing body weight in a
rodent model. These results provide further evidence that small
molecule DGAT-1 inhibitors can reproduce key aspects of the
DGAT-1 ko phenotype and have potential in the treatment of
obesity and other elements of metabolic syndrome.
Experimental Section
All solvents and chemicals used were reagent grade. Anhydrous
solvents tetrahydrofuran (THF), benzene, and dimethoxyethane
(DME) were purchased from Aldrich. Flash column chromatogra-
In summary, the novel DGAT-1 inhibitor 4 has been shown
to be a potent and selective inhibitor of the enzyme, with a

1562 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Scheme 3. Synthesis of Compounds 7 and 8a
Birch et al.
a Reagents and conditions: (a) (i) Oxalyl chloride, DCM; (ii) TMSCHN2/MeCN/THF/Et3N, 87%; (iii) PhCO2Ag/Et3N/MeOH, 51%. (b) 2-Bromoisobutyryl
bromide, AlCl3, DCM. (c) 5,6-diaminopyrimidin-4-ol, 1 M HCl, EtOH. (d) 2 M NaOH, MeOH. (e) TMSCHN2, MeOH, PhMe, 94%.
precoated thin layer chromatography (TLC) plates (silica gel 60
F
254, 0.25 mm, art. 5715) were used for TLC analysis. Solutions
were dried over anhydrous magnesium sulfate, and the solvent was
removed by rotary evaporation under reduced pressure.
Method A: Ethyl 2-Oxo-6-phenyl-2H-pyran-3-carboxylate
(9). To a solution of sodium ethoxide (21% solution in ethanol;
116 mL, 358.33 mmol) in anhydrous DME (300 mL) was added
diethyl (ethoxymethylene)malonate (66 mL, 325.75 mmol). The
reaction mixture was heated to reflux, and 1-phenylethanone (38
mL, 325.75 mmol) was added dropwise and heating maintained
for 1 h. The reaction mixture was allowed to cool to ambient
temperature and added to a stirred solution of 5 M HCl (500 mL),
and the resulting yellow solution was extracted into ether (3 × 500
mL). The organic extracts were combined, washed with brine (300
mL), dried over MgSO4, and concentrated to leave a red oil. Acetyl
chloride (60 mL) was added followed by DMF (2 mL), and the
mixture was heated to 70 °C for 2 h, allowed to cool, and then
concentrated to leave a dark residue that formed a crystalline solid
on standing. This was recrystallized from absolute EtOH (50 mL)
to give 9 (49.16 g, 201.2 mmol, 62%) as a yellow solid. 1H NMR
(CDCl3) δ: 1.39 (t, J ) 7.1 Hz, 3H), 4.39 (q, J ) 7.1, 2H), 6.77 (d,
J ) 7.3, 1H), 7.47-7.56 (m, 3H), 7.89-7.91 (m, 2H), 8.29 (d, J
) 7.4,1H). GC-MS EI m/z: (M+•) 244.
Ethyl 4-Phenylbicyclo[2.2.2]oct-2-ene-1-carboxylate (10). Eth-
yl 2-oxo-6-phenyl-2H-pyran-3-carboxylate 9 (49.1 g, 201.2 mmol)
was dissolved in toluene saturated with ethylene and heated at 200
°C for 15 h at 75 bar pressure. The solvent was concentrated to
leave a pale-yellow gum (49 g), which was purified by silica
chromatography (ether/isohexane ) 5:95) to provide 10 (26.4 g,
103 mmol, 51%) as a colorless oil. 1H NMR (CDCl3) δ: 1.31 (t, J
) 7.1, 3H), 1.56-1.66 (m, 4H), 1.90-2.06 (m, 4H), 4.23 (q, J )
7.1, 2H), 6.40-6.43 (m, 1H), 6.59 (d, J ) 8.6, 1H), 7.18-7.26
(m, 1H), 7.31-7.43 (m, 5H). GC-MS CI m/z: (M+ + H) 257.
Ethyl 4-Phenylbicyclo[2.2.2]octane-1-carboxylate (11). Ethyl
4-phenylbicyclo[2.2.2]oct-2-ene-1-carboxylate 10 (26.3 g, 102.5
mmol) in absolute ethanol (600 mL) and 10% Pt on carbon (2.4 g)
were stirred under a balloon of hydrogen at ambient temperature
overnight. The reaction mixture was filtered and the solvent
removed under reduced pressure to leave a 11 as a colorless oil,
which rapidly formed a white solid on standing (22.179 g, 85.96
mmol, 84%). 1H NMR (CDCl3) δ: 1.25 (t, J ) 7.1, 3H), 1.83-1.96
(m, 12H,), 4.12 (q, J ) 7.1, 2H), 7.15-7.20 (m, 1H), 7.28-7.31
(m, 4H). GC-MS EI m/z: (M+•) 258.
Figure 2. Hydrolysis half-life of compound 4 acyl glucuronide
compared with those of standard drugs. Compound 4 acyl glucuronide
was biosynthesised utilizing dog activated microsomes, followed by
isolation and purification via solid phase extraction. It was then
incubated at 37 °C in pH 7.4 phosphate buffer and samples taken at
regular intervals to determine (via HPLC-MS) the half-life of hydrolysis.
phy was carried out using prepacked silica cartridges (from 4 g up
to 330 g) from Redisep, Biotage, or Crawford and eluted using an
Isco Companion system. Purity and characterization of compounds
were established by a combination of liquid chromatography-mass
spectroscopy (LC-MS), gas chromatography-mass spectroscopy
(GC-MS), and NMR analytical techniques and was >95% for all
test compounds. Combustion analysis was also employed for
1
compound 4. H NMR were recorded on a Varian Gemini 2000
(300 MHz) or a Bruker Avance DPX400 (400 MHz) and were
determined in CDCl3 or DMSO-d6. Chemical shifts are reported in
ppm relative to tetramethylsilane (TMS) (0.00 ppm) or solvent peaks
as the internal reference and coupling constant (J) values are
reported in Hertz (Hz). Splitting patterns are indicated as follows:
s, singlet; d, doublet; t, triplet; m, multiplet; br, broad peak. Merck

Pyrimidinooxazinyl Bicyclooctaneacetic Acid DGAT-1 Inhibitor
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1563
Table 3. Physical Properties of Compound 4
aqueous solubility
pH 7.4 (µM)
rat plasma protein
bindinga (% free)
dog plasma protein
bindinga (% free)
human plasma protein
bindinga (% free)
MDCKb permeability
logD
1.9
Papp (× 10-6 cm·s-1
)
12 (n ) 2)
0.91 (n ) 1)
0.60 (n ) 2)
1.0 (n ) 2)
18 (A-B); 26 (B-A) (n ) 1)
a PPB was assessed by equilibrium dialysis in the appropriate species plasma at 37 °C. Free and bound concentrations were quantified by LC-UVMS.
b Compounds were incubated at 10 µM in cultured MDCK cells. Permeability was measured in both the A to B and B to A direction
Figure 3. Pharmacodynamic effects of compound 4. (A) Dose-related reduction in plasma triacylglycerol excursion at 1.5 h post bolus corn oil
administration; n ) 6. (B) Dose-related reduction in adipose triacylglycerol synthesis as determined by changes in the ratio of radiolabel incorporation
into TAG and DAG; n ) 10. Mean values ( SEM (* P < 0.05, **P < 0.01).
°C) (230 mL) under nitrogen, and bromine (3.2 mL, 61.88 mmol)
was added dropwise. The resulting orange suspension was allowed
to stir at ambient temperature for 30 min and then at 60 °C for 40
min. The reaction mixture was allowed to cool to ambient
temperature, the suspension was filtered, and the solid was washed
with ether (3 × 100 mL) and then 1 M Na2CO3 (3 × 150 mL).
The organic phase was separated, washed with brine (100 mL),
dried over MgSO4, and concentrated to give 14 (15.3 g, 61.8 mmol,
1
89%) as a pale-yellow oil, which formed a solid on standing. H
NMR (CDCl3) δ: 1.94-1.98 (m, 6H), 2.23-2.27 (m, 6H), 3.64 (s,
3H). GC-MS m/z: (M+ + H) 247.
Methyl 4-Phenylbicyclo[2.2.2]octane-1-carboxylate (15). A
solution of methyl 4-bromobicyclo[2.2.2]octane-1-carboxylate 14
(15.3 g, 61.91 mmol) in anhydrous benzene (50 mL) was added
dropwise to an ice-water cooled suspension of aluminum chloride
(30.5 g, 229.1 mmol) in benzene (100 mL) under nitrogen. The
resulting reaction mixture was allowed to stir in the ice bath for 30
min and then at ambient temperature overnight. The mixture was
heated to 60 °C for 4 h and then allowed to cool to ambient
temperature and cautiously poured onto ice (300 g) and concentrated
HCl (50 mL) and the mixture was extracted into ether (4 × 300
mL). The ether extracts were combined, washed with brine (200
mL), separated, and dried over MgSO4 to leave an orange-brown
solid. Purification by silica chromatography (ether/isohexane ) 1:9)
Figure 4. Percent reduction in body weight compared to start weight
in DIO mice receiving high energy cafeteria diet. Day 0 is first treatment
day; mice dosed daily at 16:00 and weighed at this time. In mice
receiving compound 4, statistical differences from DIO controls were
seen at all time points; n ) 12 (P < 0.001).
Method B: 4-(Methoxycarbonyl)bicyclo[2.2.2]octane-1-car-
boxylic Acid (13). A solution of dimethyl bicyclo[2.2.2]octane-
1,4-dicarboxylate13 12 (18.36 g, 81.14 mmol), barium hydroxide
octahydrate (12.8 g, 40.57 mmol) in methanol (200 mL), and water
(40 mL) was stirred vigorously at ambient temperature overnight.
The reaction mixture was diluted with water (100 mL) and washed
with isohexane (4 × 250 mL), and the aqueous phase was acidified
with 2 M HCl to pH 2 and extracted into AcOEt (5 × 300 mL).
The organic extracts were combined, dried over MgSO4, and
concentrated to leave a white solid. This was re-dissolved in toluene
(500 mL), filtered, and the filtrate evaporated to dryness to give 13
as a white solid (9.64 g, 45.41 mmol, 56%). 1H NMR (DMSO) δ:
1.71 (s, 12H), 3.58 (s, 3H), 12.03 (s, 1H). GC-MS m/z: (M+ + H)
213.
Methyl 4-Bromobicyclo[2.2.2]octane-1-carboxylate (14). To
a stirred suspension of 4-(methoxycarbonyl)bicyclo[2.2.2]octane-
1-carboxylic acid 13 (14.64 g, 68.98 mmol) in acetone (120 mL)
was added 1 M NaOH (70 mL), and the resulting clear solution
was allowed to stir at ambient temperature for 10 min. A 4 M
solution of silver nitrate (12.4 g, 73.11 mmol) in water (18 mL)
was added dropwise and an immediate white suspension formed,
which was allowed to stir at ambient temperature for a further hour.
The suspension was filtered, washed with water (100 mL), acetone
(100 mL), ether (100 mL), and dried under vacuum overnight at
60 °C to leave 19.74 g (61.88 mmol, 89%) of the silver salt as a
pale-brown solid. This was suspended in petroleum ether (bp 40-60
1
gave 15 (8.45 g, 34.63 mmol, 56%) as a yellow solid. H NMR
(CDCl3) δ: 1.86-1.93 (m, 12H), 3.67 (s, 3H), 7.28-7.31 (m, 5H).
GC-MS EI m/z: (M+•) 244.
Methoxycarbonylbicyclo[3.2.2]nonane-5-carboxylic Acid (17).
To a stirred solution of dimethyl bicyclo[3.2.2]nonane-1,5-dicar-
boxylate14 16 (2.02 g, 8.41 mmol) and barium hydroxide octahy-
drate (1.33 g, 4.20 mmol) was added methanol (20 mL) and water
(4 mL). The reaction mixture was stirred vigorously at ambient
temperature overnight. The reaction mixture was diluted with water
(20 mL) and washed with isohexane (2 × 100 mL), and the aqueous
phase was acidified with 2 M HCl to pH 2 and extracted into AcOEt
(2 × 100 mL). The organic extracts were combined, dried over
MgSO4, and concentrated to give 17 (931 mg, 4.11 mmol, 49%)
1
as a colorless gum, which slowly formed a white solid. H NMR
(CDCl3) δ: 1.72-1.75 (m, 2H), 1.87-1.92 (m, 12H), 3.65 (s, 3H).
MS m/z: (M+ + H) 226.
Methyl 5-Bromobicyclo[3.2.2]nonane-1-carboxylate (18). To
a stirred suspension methoxycarbonylbicyclo[3.2.2]nonane-5-car-
boxylic acid 17 (3.43 g, 15.16 mmol) in acetone (25 mL) was added
1 M NaOH (16 mL), and the resulting clear pale-yellow solution
was allowed to stir at ambient temperature for 10 min. A 4 M

1564 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Birch et al.
solution of silver nitrate (2.73 g, 16.07 mmol) in water (4 mL)
was added dropwise, and an immediate thick brown suspension
formed, which was allowed to stir at ambient temperature for a
further 60 min. The suspension was filtered, washed with water
(100 mL), acetone (100 mL), ether (100 mL), and dried under
vacuum overnight at 45 °C to leave 4.42 g of a brown solid.
To a suspension of the silver salt (4.24 g, 12.73 mmol) in
petroleum ether (40-60) (50 mL) under nitrogen was added
bromine (0.65 mL, 12.73 mmol) dropwise. The resulting orange
suspension was allowed to stir at ambient temperature for 30 min
and then at 60 °C for 40 min. The reaction mixture was allowed to
cool to ambient temperature, the suspension was filtered, and the
solid was washed with ether (3 × 100 mL) and then 1 M Na2CO3
(3 × 150 mL). The organic phase was separated, washed with brine
(100 mL), dried over MgSO4, and concentrated to give 18 (1.22 g,
4.67 mmol, 37%) as a pale-yellow oil. 1H NMR (CDCl3) δ:
1.69-1.75 (m, 2H), 1.83-2.00 (m, 6H), 2.43-2.50 (m, 6H), 3.65
(s, 3H). MS m/z: (M+ + H) 261.
6H), 1.84 (s, 12H), 3.61 (s, 3H), 6.98 (s, 2H), 7.41 (d, J ) 8.5, 2H),
7.66 (d, J ) 8.4, 2H), 7.95 (s, 1H). MS m/z: (M+ + H) 421.
4-[4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-
yl)phenyl]bicyclo[2.2.2]octane-1-carboxylic Acid (3). To a solution
of methyl 4-[4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-
6-yl)phenyl]bicyclo[2.2.2]octane-1-carboxylate 24 (140 mg, 0.33
mmol) in MeOH (10 mL) was added 2 M NaOH (0.82 mL, 1.66
mmol). The reaction mixture was allowed to stir at ambient
temperature overnight, and then after the addition of EtOH (5 mL)
and 1 M NaOH (2 mL) the reaction mixture was heated to 50 °C
for 5.5 h. The organic solvent was removed by evaporation under
reduced pressure, and the residue was acidified to pH 2 with 2 M
HCl. AcOEt was added, and the suspension was filtered and dried
1
to provide 3 (97 mg, 2.39 mmol, 72%) as a white solid. H NMR
(DMSO) δ: 1.60 (s, 6H), 1.82 (s, 12H), 6.97 (s, 2H), 7.40 (d, J )
8.7, 2H), 7.65 (d, J ) 8.7, 2H), 7.95 (s, 1H). MS m/z: (M+ + H)
407. HRMS (ES+) for C23H27N4O3 (M+ + H): calcd, 407.20777;
found, 407.20773.
5-Phenylbicyclo[3.2.2]nonane-1-carboxylic Acid. A solution of
methyl 5-bromobicyclo[3.2.2]nonane-1-carboxylate 18 (1.22 g, 4.67
mmol) in anhydrous benzene (5 mL) was added dropwise to an
ice-water cooled suspension of aluminum chloride (2.3 g, 17.28
mmol) in benzene (5 mL) under nitrogen. The resulting reaction
mixture was allowed to stir in the ice bath for 30 min and then
removed from the cooling bath and allowed to warm to and stir at
ambient temperature overnight. Then the reaction mixture was
heated to 60 °C for 4 h and allowed to cool to ambient temperature.
It was cautiously poured onto ice (30 g) and concentrated HCl (5
mL), and the mixture was extracted into ether (4 × 100 mL). The
ether extracts were combined, washed with brine (50 mL),
separated, and dried over MgSO4 to leave methyl 5-phenylbicyclo-
[3.2.2]nonane-1-carboxylate 19 as an orange-brown gum (821 mg),
which was submitted to the following step without purification.
To methyl 5-phenylbicyclo[3.2.2]nonane-1-carboxylate (821 mg)
19 in MeOH (20 mL) was added 2 M NaOH (8 mL) and allowed
to stir at ambient temperature overnight. The solvent was removed,
and the residue was washed with ether, the aqueous phase was
acidified with 2 M HCl, and extracted into AcOEt. The organic
extracts were dried over MgSO4 and concentrated to give the title
4-Phenylbicyclo[2.2.2]octane-1-carboxylic Acid. To a solution of
methyl 4-phenylbicyclo[2.2.2]octane-1-carboxylate 15 (8.45 g,
34.58 mmol) in methanol (500 mL) was added 2 M NaOH (86
mL, 173.2 mmol). The reaction mixture was allowed to stir at
ambient temperature overnight, the methanol was removed by
evaporation, and the residue acidified to pH 2 with 2 M HCl and
then extracted with AcOEt (250 mL). The organic phase was
washed with brine (50 mL), separated, dried over MgSO4, and
concentrated to give 37 as a pale-yellow solid, (5.76 g, 25.0 mmol,
1
72%). H NMR (DMSO) δ: 1.86 (s, 12H), 7.19-7.23 (m, 1H),
7.31-7.39 (m, 4H). MS EI m/z: (M- - H) 229. HRMS (ES-) for
C15H17O2 (M - H): calcd, 229.1229; found, 229.1218.
Methyl (4-Phenylbicyclo[2.2.2]oct-1-yl)acetate (20). To an
ice-water cooled solution of 4-phenylbicyclo[2.2.2]octane-1-car-
boxylic acid (5.76 g, 25.00 mmol) in DCM (200 mL) was added
DMF (∼5 drops) followed by oxalyl chloride (3.3 mL, 37.51 mmol).
The reaction mixture was allowed to stir at 0 °C for 30 min and
then at ambient temperature overnight, and the solvent was
evaporated under reduced pressure to leave crude acid chloride that
was used directly. This was redissolved in a 1:1 solution of MeCN
and THF (100 mL) and added dropwise to an ice-water cooled
solution of 2 M trimethylsilyldiazomethane (20 mL, 40 mmol),
triethylamine (4.4 mL, 31.26 mmol) in a 1:1 solution of MeCN,
and THF (200 mL). The reaction mixture was allowed to stir at 0
°C for 1 h and then overnight at ambient temperature. The solvent
was removed under reduced pressure and the residue redissolved
in AcOEt (250 mL) and washed with water (100 mL), saturated
NaHCO3 (150 mL), and brine (150 mL). The organic phase was
dried over MgSO4 and concentrated to leave a brown gum.
Chromatography (isohexane/AcOEt ) 8:2) gave 2-diazo-1-(4-
phenylbicyclo[2.2.2]oct-1-yl)ethanone (5.74 g, 22.59 mmol, 90%)
1
compound (534 mg, 2.18 mmol, 47%) as a brown gum. H NMR
(DMSO) δ: 1.34-1.38 (m, 6H), 1.61-1.65 (m, 6H), 2.40 (s, 2H),
7.04-7.07 (m, 2H), 7.16-7.20 (m, 1H), 7.24-7.28 (m, 2H), 11.87
(s, 1H). MS (EI) m/z: MH+• 244. HRMS (EI) for C16H20O2 (MH+):
calcd, 244.1463; found, 244.1478.
Methyl-4-[4-(2-bromo-2-methylpropanoyl)phenyl]bicyclo[2.2.2]-
octane-1-carboxylate (23). To an ice/IPA cooled solution of methyl
4-phenylbicyclo[2.2.2]octane-1-carboxylate 15 (412 mg, 1.69 mmol)
in DCM (20 mL) was added aluminum chloride (680 mg, 5.06
mmol) followed by the dropwise addition of 2-bromoisobutyryl
bromide (0.21 mL, 1.69 mmol). The reaction mixture was allowed
to stir at 0 °C for 30 min and then poured onto ice-water (∼50
mL). The aqueous mixture was extracted into DCM (3 × 100 mL),
the organic extracts were combined, washed with brine (100 mL),
dried over MgSO4, and concentrated to leave crude product.
Chromatography (isohexane/ether ) 9:1) gave 23 (507 mg, 1.29
1
as an orange-yellow gum, which formed a solid on standing. H
NMR (CDCl3) δ: 1.82-1.91 (m, 12H), 5.38 (s, 1H), 7.15-7.21
(m, 2H), 7.29-7.31 (m, 4H). GC-MS ES+ m/z: M+• 255. HRMS
(EI) for C16H21N2O (M+): calcd, 255.1497; found, 255.1490.
2-Diazo-1-(4-phenylbicyclo[2.2.2]oct-1-yl)ethanone (5.74 g, 22.59
mmol) in methanol (250 mL) and placed in an ultrasound bath, a
solution of silver benzoate (1.03 g, 4.52 mmol, 0.2 equiv) in
triethylamine (12.6 mL, 90.39 mmol, 4 equiv) was added dropwise,
and the mixture was sonicated for 1 h. The methanol was removed
by evaporation and the residue dissolved in AcOEt (200 mL) and
washed with NaHCO3 (100 mL), citric acid (2 M, 100 mL), brine
(100 mL), dried over MgSO4, and concentrated to leave 20 (5.59
g, 21.66mmol, 96%) as a yellow oil, which formed a solid on
standing. 1H NMR (CDCl3) δ: 1.63-1.71 (m, 6H), 1.83-1.93 (m,
6H), 2.17 (s, 2H), 3.67 (s, 3H), 7.14-7.19 (m, 1H), 7.29-7.32
(m, 4H). GC-MS EI m/z: M+• 258. HRMS (EI) for C17H22O2 (M+);
calcd, 258.1620; found, 258.1640.
1
mmol, 76%) as an orange solid. H NMR (CDCl3) δ: 1.87-1.95
(m, 12H), 2.04 (s, 6H), 3.68 (s, 3H), 7.38 (d, J ) 8.4, 2H), 8.11 (d,
J ) 8.4, 2H). MS m/z: MH+ 407.
Methyl 4-[4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-
6-yl)phenyl]bicyclo[2.2.2]octane-1-carboxylate (24). To a solution
of methyl 4-[4-(2-bromo-2-methylpropanoyl)phenyl]bicyclo[2.2.2]octane-
1-carboxylate 23 (507 mg, 1.29 mmol) in absolute EtOH (10 mL)
was added 5,6-diaminopyrimidin-4-ol (179 mg, 1.42 mmol),followed
by 1 M HCl (1.4 mL). The suspension was heated under reflux
overnight, and the reaction mixture was allowed to cool to ambient
temperature and then evaporated to dryness. The residue treated with
2 M K2CO3 to adjust the pH to 10, and then the mixture was extracted
into AcOEt (4 × 50 mL). The organic extracts were combined, dried
over MgSO4, and concentrated, and the residue was purified by
chromatography (DCM/MeOH ) 0-5%) to provide 24 (150 mg,
Methyl 2-(4-Phenylbicyclo[2.2.2]oct-1-yl)propanoate (21). To a
solution of diisopropylamine (0.9 mL, 6.39 mmol) in anhydrous
THF (10 mL) cooled to -78 °C was added butyllithium (3.2 mL,
6.4 mmol, 2 M solution in cyclohexane), and the reaction mixture
wasallowedtostirat-78°Cfor5min.Methyl(4-phenylbicyclo[2.2.2]oct-
1
0.357 mmol, 27%) as a white solid. H NMR (DMSO) δ: 1.61 (s,

Pyrimidinooxazinyl Bicyclooctaneacetic Acid DGAT-1 Inhibitor
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1565
1-yl)acetate 20 (1.5 g, 5.81 mmol) in anhydrous THF (2 mL) was
added, and the mixture was allowed to stir for 30 min at -78 °C.
Methyl iodide (0.4 mL, 6.39 mmol) was added, and the reaction
mixture was allowed to warm to ambient temperature over 4 h.
Saturated NH4Cl (50 mL) was added and the mixture extracted into
AcOEt (2 × 150 mL), and the organic extracts were combined,
dried over MgSO4, and concentrated to leave crude product. This
was an inseparable mixture of the 20 and 21 and was submitted to
the following step without purification.
was purified by chromatography (isohexane/AcOEt ) 8:2) to give
25 (5.18 g, 12.72 mmol, 85%) as a white solid. 1H NMR (CDCl3)
δ: 1.64-1.69 (m, 6H), 1.83-1.88 (m, 6H), 2.03 (s, 6H), 2.18 (s,
2H), 3.66 (s, 3H), 7.35-7.38 (d, J ) 8.6, 2H), 8.07-8.12 (m, 2H).
Solid probe MS m/z: M+ 407. HRMS (EI) (M-OMe) (M+): calcd,
375.0998; found, 375.0960.
Methyl {4-[4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]-
oxazin-6-yl)phenyl]bicyclo[2.2.2]oct-1-yl}acetate (28). To a solution
of 5,6-diaminopyrimidin-4-ol (280 mg, 2.2 mmol) in water (8 mL),
absolute ethanol (25 mL), and 1 M HCl (2.2 mL, 2.2 mmol) was
added a solution of methyl {4-[4-(2-bromo-2-methylpropanoyl)-
phenyl]bicyclo[2.2.2]oct-1-yl}acetate 25 (877 mg, 2.15 mmol) in
absolute EtOH (80 mL). The solution was heated to 100 °C
overnight and then allowed to cool to ambient temperature,
evaporated to dryness, and the residue treated with 2 M NaOH to
adjust the pH to 10. The resulting suspension was filtered to provide
a yellow solid, which was purified by chromatography (DCM/
MeOH ) 0-5%) to provide 28 (601 mg, 1.38 mmol, 64%) as a
white solid. 1H NMR δ: 1.59-1.63 (m, 12H), 1.79-1.83 (m, 6H),
2.16 (s, 2H), 3.59 (s, 3H), 6.91 (s, 2H), 7.39 (d, J ) 8.6, 2H), 7.64
(d, J ) 8.6, 2H), 7.95 (s, 1H). MS m/z: (M+ + H) 435.
{4-[4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-
yl)phenyl]bicyclo[2.2.2]oct-1-yl}acetic Acid (4). To a solution of
methyl {4-[4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-
6-yl)phenyl]bicyclo[2.2.2]oct-1-yl}acetate 28 (601 mg, 1.38 mmol)
in MeOH (40 mL) and THF (40 mL) was added 2 M NaOH solution
(3.5 mL, 6.9 mmol). The reaction mixture was stirred at 60 °C
overnight, the organic solvent was concentrated, and the residue
acidified with 2 M HCl to pH 2. The suspension was filtered and
the solid purified by reverse phase chromatography 5-95% water
(+0.5% NH3)-acetonitrile to give 4 (302 mg, 0.717 mmol, 52%)
as a white solid. 1H NMR (DMSO) δ: 1.61 (6H, s), 1.59-1.64 (m,
6H), 1.79-1.83 (m, 6H), 2.06 (s, 2H), 6.90 (s, 2H), 7.40 (d, J )
8.4, 2H), 7.64 (d,, J ) 8.4, 2H), 7.95 (s, 1H). MS m/z: (M+ + H)
421. HRMS (ES+) for C24H29N4O3 (M+ + H): calcd, 421.22342;
found, 421.22357.
Methyl2-[1-[4-(2-Bromo-2-methylpropanoyl)phenyl]-4-bicyclo[2.2.2]-
octanyl]propanoate (26). To an ice-water cooled solution of a
mixture of 20 and 21 (961 mg) in DCM (10 mL) was added
aluminum chloride (1.4 g, 10.58 mmol), followed by the dropwise
addition of 2-bromoisobutyryl bromide (0.45 mL, 3.53 mmol). The
reaction mixture was allowed to stir at 0 °C for 40 min, and then
it was then poured onto ice-water (100 mL). The organic phase
was separated and the aqueous phase washed with DCM (3 × 50
mL), the organic washings were combined, dried over MgSO4, and
concentrated to leave a yellow gum. This was purified by chro-
matography (isohexane/AcOEt ) 8:2) to provide the 26 (705 mg,
1
1.67mmol, 47%) as a white solid. H NMR (CDCl3) δ: 1.09 (d, J
) 7.1, 3H), 1.50-1.57 (m, 3H), 1.67-1.74 (m, 3H), 1.82-1.86
(m, 6H), 2.03 (s, 6H), 2.27 (q, J ) 7.1, 1H), 3.67 (s, 3H), 7.35-7.38
(m, 2H), 8.09-8.12 (m, 2H). MS m/z: MH+• 421.
Methyl 2-{4-[4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-
6-yl)phenyl]bicyclo[2.2.2]oct-1-yl}propanoate (29). To a solution of
methyl {4-[4-(2-bromo-2-methylpropanoyl)phenyl]bicyclo[2.2.2]-
oct-1-yl}acetate 26 (705 mg, 1.67 mmol) in absolute EtOH (20
mL) was added 5,6-diaminopyrimidin-4-ol (233 mg, 1.84 mmol),
followed by 1 M HCl (2.0 mL). The suspension was heated under
gentle reflux overnight. The reaction mixture was allowed to cool
to ambient temperature, evaporated to dryness, and treated with 2
M NaOH to adjust the pH to 10. The suspension was filtered to
provide a yellow solid, the filtrate was extracted into AcOEt (2 ×
100 mL), the organic extracts were separated, dried over MgSO4,
and concentrated to leave a pale-yellow gum. The solid was purified
by chromatography (DCM/MeOH ) 0-5%) to provide 29 (220
1
mg, 0.491 mmol, 29%) as a cream solid. H NMR (DMSO) δ:
Methyl (5-Phenylbicyclo[3.2.2]non-1-yl)acetate (22). To a solu-
tion of 5-phenylbicyclo[3.2.2]nonane-1-carboxylic acid (534 mg,
2.19 mmol) in DCM (10 mL) was added DMF (2 drops), followed
by oxalyl chloride (0.3 mL, 3.28 mmol). The reaction mixture was
allowed to stir at ambient temperature overnight, and the solvent
was evaporated under reduced pressure to leave crude acid chloride,
which was used directly.
1.02 (d, J ) 7.1, 3H), 1.44-1.52 (m, 3H), 1.61 (s, 6H), 1.58-1.67
(m, 3H), 1.77-1.79 (m, 6H), 2.24 (q, J ) 7.1, 1H), 3.60 (s, 3H),
6.89 (s, 2H), 7.39 (d, J ) 8.5, 2H), 7.64 (d, J ) 8.5, 2H), 7.95 (s,
1H). MS m/z: (M+ + H) 449.
2-{4-[4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-
6-yl)phenyl]bicyclo[2.2.2]oct-1-yl}propanoic Acid (5). A mixture of
methyl 2-{4-[4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-
6-yl)phenyl]bicyclo[2.2.2]oct-1-yl}propanoate 29 (220 mg, 0.49
mmol) and potassium trimethylsilanolate (315 mg, 2.45 mmol) in
THF (5 mL) was heated in a microwave for 35 min at 100 °C and
then allowed to stir at ambient temperature for 2 days. The solvent
was removed under reduced pressure and the residue partitioned
between 2 M HCl (20 mL) and AcOEt (50 mL) and the aqueous
phase was re-extracted into AcOEt (50 mL). The organic extracts
were combined, dried over MgSO4, and concentrated to leave a
gum. This was purified by reverse phase chromatography eluting
5-95% water-acetontrile (+0.2% TFA) to provide 5 (45 mg, 0.104
This was redissolved in a 1:1 solution of MeCN and THF (10
mL) and added dropwise to an ice-water cooled solution of 2 M
trimethylsilyldiazomethane (1.5 mL, 3.0 mmol) and triethylamine
(0.4 mL, 2.73 mmol) in a 1:1 solution of MeCN and THF (20 mL).
The reaction mixture was allowed to stir at 0 °C for 1 h and then
for 5 h and ambient temperature and allowed to stand for 48 h.
The solvent was removed under reduced pressure and the residue
redissolved in AcOEt (250 mL) and washed with water (100 mL),
saturated NaHCO3 (150 mL), and brine (150 mL). The organic
phase was dried over MgSO4 and concentrated to leave a brown
gum. The residue purified by chromatography (isohexane/AcOEt
) 8:2) to provide 2-diazo-1-(4-phenylbicyclo[3.2.2]non-1-yl)etha-
1
mmol, 21%) as a white solid. H NMR (DMSO) δ: 1.01 (d, J )
7.1, 3H), 1.49-1.55 (m, 3H), 1.62 (s, 6H), 1.64-1.69 (m, 3H),
1.78-1.81 (m, 6H), 2.12 (q, J ) 7.0, 1H), 7.05 (s, 1H), 7.40 (d, J
) 8.5, 2H), 7.65 (d, J ) 8.5, 2H), 7.99 (s, 1H). MS m/z: (M+ + H)
435. HRMS (ES+) for C25H31N4O3 (M+ + H): calcd, 435.23907;
found, 435.23889.
1
none (252 mg, 0.940 mmol, 43%) as an orange gum. H NMR
(CDCl3) δ 1.24-1.29 (m, 2H), 1.43-1.47 (m, 6H), 1.61-1.67 (m,
6H), 5.28 (s,1H), 7.05-7.24 (m, 5H). MS m/z MH+• 268.
A solution of 2-diazo-1-(4-phenylbicyclo[3.2.2]non-1-yl)ethanone
(252 mg, 0.94 mmol) in methanol (10 mL) was placed in an
ultrasound bath, a solution of silver benzoate (44 mg, 0.19 mmol,
0.2 equiv) in triethylamine (0.52 mL, 3.76 mmol, 4 equiv) was
added dropwise, and the mixture was sonicated for 1 h. The
methanol was removed by evaporation and the residue dissolved
in AcOEt (20 mL), filtered through a pad of celite, and washed
with NaHCO3 (10 mL), citric acid (2M, 10 mL), brine (10 mL),
dried over MgSO4, and concentrated to leave 22 as a brown gum,
229 mg which was which was submitted to the following step
without purification.
Methyl {4-[4-(2-Bromo-2-methylpropanoyl)phenyl]bicyclo[2.2.2]oct-
1-yl}acetate (25). To an ice-water cooled solution of methyl (4-
phenylbicyclo[2.2.2]oct-1-yl)acetate 20 (3.88 g, 15.02 mmol) in
DCM (150 mL) was added aluminum chloride (6.01 g, 45.05
mmol), followed by the dropwise addition of 2-bromoisobutyryl
bromide (1.86 mL, 15.02 mmol). The reaction mixture was allowed
to stir at 0 °C for 30 min and then poured onto ice-water (20
mL). The organic phase was separated and the aqueous phase
washed with DCM (2 × 150 mL), the organic washings combined,
dried over MgSO4, and concentrated to leave a yellow gum. This

1566 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Birch et al.
Methyl {5-[4-(2-Bromo-2-methylpropanoyl)phenyl]bicyclo[3.2.2]non-
1-yl}acetate (27). To an ice-water cooled solution of methyl (5-
phenylbicyclo[3.2.2]non-1-yl)acetate 22 (229 mg, 0.79 mmol) in
DCM (10 mL) was added aluminum chloride (318 mg, 2.38 mmol),
followed by the dropwise addition of 2-bromoisobutyryl bromide
(0.10 mL, 0.79 mmol). The reaction mixture was allowed to stir at
0 °C for 40 min, and it was then poured onto ice-water (100 mL).
The organic phase was separated and the aqueous phase washed
with DCM (3 × 50 mL), the organic washings were combined,
dried over MgSO4, and concentrated to leave a yellow gum. The
residue purified by chromatography (isohexane/AcOEt ) 8:2) to
m/z: MH+ 281. HRMS (ES+) for C18H21N2O (M+ + H): calcd,
281.1654; found, 281.1626. IR ν cm-1 2101 (strong, broad).
The diazoketone (699 mg, 2.49 mmol) was dissolved in methanol
(25 mL) and placed in an ultrasound bath, and a solution of silver
benzoate (114 mg, 0.498 mmol, 0.2 equiv) in triethylamine (1.4
mL, 9.96 mmol, 4 equiv) was added dropwise and the mixture was
sonicated for 1 h. The methanol was removed by evaporation and
the residue dissolved in AcOEt (50 mL) and washed with NaHCO3
(40 mL), citric acid (2M, 40 mL), brine (40 mL), dried over MgSO4,
and concentrated to leave a yellow oil. This was purified by
chromatography (isohexane/AcOEt ) 8:2) to provide 31 (421 mg
1.48 mmol, 59%, from the diazoketone, 51% from the starting acid)
1
provide 27 (85 mg, 0.202 mmol, 26%) as a pale-yellow gum. H
1
NMR (CDCl3) δ: 1.41-1.51 (m, 14H), 2.03 (s, 6H), 2.45 (s, 2H),
3.67 (s, 3H), 7.37 (d, J ) 8.3, 2H), 8.11 (d, J ) 8.3, 2H). MS m/z:
MH+• 421. HRMS (EI) for C22H29BrO3 (M+•): calcd, 420.1300;
found, 420.1334.
as a pale-yellow gum. H NMR (CDCl3) δ: 1.64-1.70 (m, 5H),
1.76 (s, 2H), 1.86-1.96 (m, 5H), 2.17 (s, 2H), 2.18-2.19 (m, 2H),
3.65 (s, 3H), 7.15-7.20 (m, 1H), 7.29-7.37 (m, 4H). MS GC-
MS-EI m/z: MH+• 284.
Methyl{5-[4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-yl)-
phenyl]bicyclo[3.2.2]non-1-yl}acetate (30). To a solution of methyl
{5-[4-(2-bromo-2-methylpropanoyl)phenyl]bicyclo[3.2.2]non-1-
yl}acetate 27 (80 mg, 0.19 mmol) in absolute EtOH (20 mL) was
added 5,6-diaminopyrimidin-4-ol (30 mg, 0.21 mmol), followed
by 1 M HCl (0.21 mL). The suspension was heated under gentle
reflux overnight, the mixture was allowed to cool, the solvent was
removed, and the residue was treated with 2 M NaOH (2 mL).
The aqueous phase was extracted into AcOEt (3 × 50 mL), the
organic extracts were combined, dried over MgSO4, and concen-
trated to leave a pale-yellow gum. The residue purified by
chromatography (DCM/MeOH ) 0-5-10% MeOH) to give 30
(55 mg, 0.123mmol, 65%) as a pale-yellow gum. 1H NMR (CDCl3)
δ: 1.42-1.64 (m, 14H), 1.70 (s, 6H), 2.07 (s, 2H), 3.61 (s, 3H),
7.11 (d, J ) 8.3, 2H), 7.51 (d, J ) 8.3, 2H), 8.14 (1H, s). MS m/z:
(M+ + H) 449.
Methyl{3-[4-(2-Bromo-2-methylpropanoyl)phenyl]-1-adamantyl}-
acetate (32). To an ice-water cooled solution of methyl (3-phenyl-
1-adamantyl)acetate 31 (247 mg, 0.868 mmol) in DCM (15 mL)
was added aluminum chloride (350 mg, 2.61 mmol), followed by
the dropwise addition of 2-bromoisobutyryl bromide (0.11 mL, 0.87
mmol). The reaction mixture was allowed to stir at 0 °C for 15
min, and it was then poured onto ice-water (20 mL). The organic
phase was separated and the aqueous phase washed with DCM (2
× 50 mL), and the organic washings were combined, washed with
brine (100 mL), dried over MgSO4, and concentrated to leave a
yellow gum. This was purified by chromatography (isohexane/
AcOEt ) 8:2) to provide 32 (133 mg, 0.307 mmol, 35%) as a
colorless oil. 1H NMR (CDCl3) δ: 1.65-1.67 (m, 4H), 1.71 (s, 2H),
1.77 (s, 2H), 1.87 (s, 4H), 2.04 (s, 6H), 2.18 (s, 2H), 2.20-2.22
(m, 2H), 3.65 (s, 3H), 7.40-7.42 (m, 2H), 8.10-8.14 (m, 2H).
MS m/z: (M+ + H) 433.
{5-[4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-
yl)phenyl]bicyclo[3.2.2]non-1-yl}acetic Acid (6). To a solution of
methyl {5-[4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-
6-yl)phenyl]bicyclo[3.2.2]non-1-yl}acetate 30 (55 mg, 0.12 mmol)
in MeOH (5 mL) was added 2 M NaOH (0.3 mL) and the reaction
mixture was allowed to stir at ambient temperature overnight and
then at 50 °C for 8 h and ambient temperature overnight. The
reaction mixture was allowed to cool, the solvent was removed
under reduced pressure, the residue was acidified to pH 2 with 2
M HCl, and the filtrate was extracted into AcOEt (2 × 50 mL).
The organic extracts were combined, dried over MgSO4, and
concentrated to leave a yellow gum. This was purified by reverse
phase chromatography eluting 5-95% water-acetontrile 0.2% TFA
to provide 6 (31 mg, 0.0714 mmol, 59%) as a pale-yellow solid.
1H NMR (DMSO) δ: 1.34-1.56 (m, 14H), 1.63 (s, 6H), 1.95 (s,
2H), 7.15 (d, J ) 8.2, 2H), 7.64 (d, J ) 8.2, 2H), 8.02 (1H, s). MS
m/z (M+ + H) 435. HRMS (ES+) for C25H31N4O3 (M+ + H): calcd,
435.23907; found, 435.23892.
Methyl (3-Phenyl-1-adamantyl)acetate (31). To an ice-water
cooled solution of 3-phenyladamantane-1-carboxylic acid (735 mg,
2.87 mmol) in DCM (50 mL) was added DMF (2-3 drops),
followed by oxalyl chloride (0.25 mL). The reaction mixture was
allowed to stir at 0 °C for 6 h, and the solvent was evaporated
under reduced pressure to leave crude acid chloride that was used
directly. This was redissolved in a 1:1 solution of MeCN and THF
(10 mL) and added dropwise to an ice-water cooled solution of 2
M trimethylsilyldiazomethane (2.0 mL) and triethylamine (0.50 mL,
3.58 mmol) in a 1:1 solution of MeCN and THF (20 mL). The
resulting yellow reaction mixture was allowed to warm to ambient
temperature overnight. The solvent was removed under reduced
pressure and the residue redissolved in AcOEt (50 mL) and washed
with water (50 mL), NaHCO3 (50 mL), and brine (50 mL). The
organic phase was dried over MgSO4 and concentrated to leave a
red-brown gum (344 mg), which was purified by chromatography
(isohexane/AcOEt ) 8:2) to give 2-diazo-1-(3-phenyl-1-adaman-
tyl)ethanone (699 mg, 2.49 mmol, 87%) as a yellow gum. 1H NMR
(CDCl3) δ: 1.74 (s, 2H), 1.83 (s, 4H), 1.91-1.94 (m, 6H), 2.27 (s,
2H), 5.43 (s, 1H), 7.18-7.22 (m, 2H), 7.31-7.38 (m, 4H). MS
Methyl {3-[4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-
6-yl)phenyl]-1-adamantyl}acetate (33). Preparation according to the
procedure described for methyl {4-[4-(4-amino-7,7-dimethyl-7H-
pyrimido[4,5-b][1,4]oxazin-6-yl)phenyl]bicyclo[2.2.2]oct-1-
yl}acetate 28 using methyl {3-[4-(2-bromo-2-methylpropanoyl)phe-
nyl]-1-adamantyl}acetate 32 isolated the title compound in 44%
yield. 1H NMR (DMSO) δ: 1.62 (s, 6H), 1.65-1.69 (m, 6H), 1.71
(s, 2H), 1.83 (s, 4H), 2.15 (s, 2H), 2.17 (s, 2H), 3.58 (s, 3H), 6.92
(s, 2H), 7.42 (d, J ) 8.5, 2H), 7.68 (d, J ) 8.5, 2H), 7.96 (s, 1H).
MS m/z: (M+ + H) 461.
{3-[4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-
yl)phenyl]-1-adamantyl}acetic Acid (7). To a solution of methyl
{3-[4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-yl)phe-
nyl]-1-adamantyl}acetate 33 (292 mg, 0.633 mmol) in EtOH (20
mL) and THF (20 mL) was added 2 M NaOH (1.6 mL, 3.17 mmol).
The reaction mixture was allowed to stir at ambient temperature
overnight, and a further 1 mL of 1 M NaOH (1 mmol) was added
and the reaction mixture was heated to 50 °C, for 6 h. The reaction
mixture was allowed to cool, the solvent was removed under
reduced pressure, and the residue was acidified to pH 2 with 2 M
HCl and the suspension was filtered to provide a yellow solid, which
was dried under vacuum over the weekend to leave 229 mg of
crude product. The residue purified by reverse phase chromatog-
raphy 17-40% water (+1% NH3)-acetonitrile to give 7 (163 mg,
0.365 mmol, 58%) as a white solid. 1H NMR (DMSO) δ: 1.67 (s,
6H), 1.61 (s, 6H), 1.78 (s, 2H), 1.88 (s, 4H), 2.12 (s, 2H), 2.20 (s,
2H), 6.96 (s, 2H), 7.42 (d, J ) 8.5, 2H), 7.68 (d, J ) 8.5, 2H),
7.95 (s, 1H). MS m/z: (M+ + H) 447. HRMS (ES+) for C26H31N4O3
(M+ + H): calcd, 447.23907; found, 447.23907.
Methyl 3-[4-(2-Bromo-2-methylpropanoyl)phenyl]adamantane-
1-carboxylate (35). Compound 35 was prepared in 89% yield
according to the procedure described for methyl {4-[4-(2-bromo-
2-methylpropanoyl)phenyl]bicyclo[2.2.2]oct-1-yl}acetate 25 using
methyl 3-phenyladamantane-1-carboxylate 34.17 Isolated 35: H
1
NMR (CDCl3) δ: 1.91-1.93 (m, 8H), 2.04 (s, 6H), 2.23-2.27 (m,
6H), 4.52 (s, 3H), 7.42 (d, J ) 8.7, 2H), 8.13 (d, J ) 8.7, 2H). MS
m/z: (M+ + H) 421.

Pyrimidinooxazinyl Bicyclooctaneacetic Acid DGAT-1 Inhibitor
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1567
Methyl 3-[4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-
6-yl)phenyl]adamantane-1-carboxylate (36). Prepared according to
the procedure described for methyl {4-[4-(4-amino-7,7-dimethyl-
7H-pyrimido[4,5-b][1,4]oxazin-6-yl)phenyl]bicyclo[2.2.2]oct-1-
yl}acetate 28 using methyl 3-[4-(2-bromo-2-methylpropanoyl)phe-
nyl]adamantane-1-carboxylate 35 to provide 36 in 44% yield, which
was submitted to the following step without purification. MS m/z
MH+: (M+ + H) 447.
2 h. The cells were washed in phosphate buffered saline and
solubilized in 1% sodium dodecyl sulfate. An aliquot was removed
for protein determination using a protein estimation kit (Perbio)
based on the method of Lowry.20 The lipids were extracted into
the organic phase using a heptane:propan-2-ol:water (80:20:2)
mixture followed by aliquots of water and heptane according to
the method of Coleman.18 The organic phase was collected and
the solvent evaporated under a stream of nitrogen. The extracts
solubilized in iso-hexane:acetic acid (99:1) and lipids separated via
normal phase high performance liquid chromatography (HPLC)
using a Lichrospher diol-5, 4 mm × 250 mm column, and a gradient
solvent system of iso-hexane:acetic acid (99:1) and iso-hexane:
propan-2-ol:acetic acid (85:15:1), flow rate of 1 mL/min according
to the method of Silversand and Haux.22 Incorporation of radiolabel
into the triacylglycerol fraction was analyzed using a Radiomatic
Flo-one Detector (Packard) connected to the HPLC machine.
Adipose Triacylglycerol Synthesis in the Rat. Male Han-Wistar
rats (∼300 g) maintained on a chow diet were dosed by oral gavage
with compound suspended in HPMC/Tween. After 1.5 h, animals
were anaesthetised using inactin, a canula was introduced into the
right jugular vein, and [3H]-palmitic acid was infused as a bolus.
Twenty minutes later, rats were euthanized and epididymal fat pads
removed. Then 100 mg segments of fat (in duplicate) were
homogenized with microbeads (Fast PrepTM) in the presence of 1
mL of methanol and addition of [14C]-oleic acid internal standard.
Following centrifugation, an aliquot of the organic phase was
combined with 4 mL of chloroform mixed and incubated for 1 h at
40 °C, after which 1.2 mL of 0.73% sodium chloride was added.
Following separation of the phases, the lower organic layer was
removed and dried under nitrogen and redissolved in hexane:glacial
acetic acid (99:1) to a final volume of 500 µL. Lipid classes were
separated by HPLC according to the method described in HuTu
80 cells above and radiolabel incorporation into triacylglycerol
measured by in-line scintillography (Radiomatic Flo-one Detector;
Packard). Following correction for recovery of internal standard
and fat weight, TAG synthesis was calculated as the dpm/mg tissue.
DGAT-1 inhibitory activity was calculated as the ratio of
incorporation of radiolabeled FA into newly synthesized TAG and
DAG. Efficacy was calculated as the percentage reduction of this
ratio in animals treated with DGAT-1 inhibitor compared to vehicle
control animals.
Oral Lipid Tolerance Test (OLTT) in the Rat. Male Han-Wistar
rats (∼230 g), previously maintained on a chow diet, were fasted
for 14 h. Animals were dosed by oral gavage with compound
suspended in HPMC/Tween and 2 h later dosed with a bolus of
corn oil (5 mL/kg). After a further 1.5 h, animals were euthanized
and a blood sample rapidly collected from the abdominal vena cava.
Plasma triacylglycerol levels were determined using a commercially
available colorimetric assay (GPO-PAP, Roche). Efficacy was
calculated as the percentage reduction in plasma triacylglycerol
compared to vehicle-treated control animals.
3-[4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-
yl)phenyl]adamantane-1-carboxylic Acid (8). Prepared according
to the procedure described for {4-[4-(4-amino-7,7-dimethyl-7H-
pyrimido[4,5-b][1,4]oxazin-6-yl)phenyl]bicyclo[2.2.2]oct-1-
yl}acetic acid 2 using methyl 3-[4-(4-amino-7,7-dimethyl-7H-
pyrimido[4,5-b][1,4]oxazin-6-yl)phenyl]adamantane-1-
1
carboxylate 36 to provide 8 in 25% yield. H NMR (DMSO) δ:
1.62 (s, 6H), 1.70-1.72 (m, 2H), 1.83-1.88 (m, 7H), 2.16-2.19
(m, 2H), 6.92 (s, 2H), 7.45 (d, J ) 8.4, 2H), 7.68 (d, J ) 8.4, 2H),
7.96 (s, 1H). HRMS (ES+) for C25H29O3N4 (M+ + H): calcd
433.22342; found, 433.22333.
Enzyme Assays. The in vitro assay to identify DGAT-1 inhibitors
uses human DGAT-1 expressed in insect cell membranes as the
enzyme source.21 Briefly, sf9 cells were infected with recombinant
baculovirus containing human DGAT-1 coding sequences and
harvested after 48 h. Cells were lysed by sonication and membranes
isolated by centrifuging at 28000 rpm for 1 h at 4 °C on a 41%
sucrose gradient. The membrane fraction at the interphase was
collected, washed, and stored at -80 °C.
DGAT-1 activity was assayed by a modification of the method
described by Coleman.18 Compound at 0.00003-10 µM (final conc)
was incubated with 25 µg/mL (final conc) membrane protein, 5
mM MgCl2, and 100 µM 1,2 dioleoyl-sn-glycerol in a total assay
volume of 200 µL in a 96-deep well plate. The reaction was started
by adding 14C oleoyl coenzyme A (30 µM final concentration) and
incubated at room temperature for 30 min. The reaction was stopped
by adding 300 µL 2-propanol:heptane 7:1. Radioactive triolein
product was separated into the organic phase by adding 200 µL of
heptane and 200 µL of 0.1 M carbonate buffer pH 9.5. DGAT-1
activity was quantified by counting aliquots of the upper heptane
layer by liquid scintillography.
DGAT-2 activity was assayed by an adaptation of the above
method. Compound at 0.01-10 µM was incubated with 0.6 µg of
membrane protein, 5 mM MgCl2, 100 µM 1,2 dioleoyl-sn-glycerol,
and 10 mM dithiothreitol in a total assay volume of 200 µL in
96-deep well plates. The reaction was started by adding 14C oleoyl
coenzyme A (30 µM final concentration) and incubated at room
temperature for 30 min. The reaction was stopped by adding 0.3
mL of 2-propanol:heptane (12:1). Radioactive triolein product was
separated into the organic phase by adding 0.2 mL of heptane and
0.2 mL of 1 M carbonate buffer pH 9.5. DGAT-1 activity was
quantified by counting aliquots of the upper heptane layer by liquid
scintillography.
ACAT-1 activity was assayed by a modification of the method
described by Billheimer.19 Compound at 0.005-30 µM was
incubated with 10 µg of membrane protein, 0.25 mM cholesterol
solubilized in Triton WR-1339, and 100 mM glutathione in a total
assay volume of 200 µL in 96-well plates. The reaction was started
by adding 14C oleoyl coenzyme A (50 µM final concentration) and
incubated at 37 °C for 30 min. The reaction was stopped by adding
0.3 mL of 2-propanol:heptane(12:1). Radioactive cholesteryl oleate
product was separated into the organic phase by adding 0.2 mL of
heptane and 0.2 mL of 1 M carbonate buffer pH 9.5. ACAT-1
activity was quantified by counting aliquots of the upper heptane
layer by liquid scintillography.
Diet-Induced Obesity in the Mouse. Female C57BlJ/6 mice (∼20
g) were housed in groups of three animals and allowed free access
to water and high calorie cafeteria diet consisting of chocolate,
cheese, and chocolate coconut balls (Delecato, Sweden) for 15
weeks to accelerate weight gain. When mice had achieved a mean
weight of ∼29 g, they were randomized by mean body weight into
groups of 12 and dosed once per day by oral gavage (10 mL/kg)
for 3 d, in a subacute protocol, with compound 2 h prior to start of
the dark phase of the light cycle. Body weights were measured
daily just prior to dose administration. Results are presented as the
percentage daily increase in body weight compared to day 0 start
weight.
Measurement of Triacylglycerol Synthesis in HuTu 80 Cells.
HuTu 80 cells were cultured to confluency in 6-well plates in
minimum essential media containing 10% fetal calf serum. For the
experiment, the medium was changed to serum-free medium and
the cells preincubated with compound solubilized in DMSO (final
concentration 0.1%) for 30 min. De novo lipogenesis was measured
by the addition of 0.12 mM sodium oleate plus 1 µCi/mL 14C-
oleic acid complexed to 0.03 mM BSA to each well for a further
Acknowledgment. Mark Denn’s expert technical assistance
in generating DMPK data is acknowledged. Graeme Moody is
thanked for helpful discussions regarding all aspects of DMPK
interpretation. Usha Chauhan’s expert technical assistance in
generating enzyme inhibition data is also acknowledged.

1568 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Birch et al.
(11) Whitney, J. G.; Gregory, W. A., Kauer, J. C.; Roland, J. R., Snyder,
J.A.;Benson,R.E.;Hermann,E.C.Antiviralagents.I.Bicyclo[2.2.2]octan-
and-oct-2-enamines J. Med. Chem., 1970, 13, 254-260.
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Supporting Information Available: Combustion analysis data
for compound 4, HPLC data for compounds 2-8, HPLC traces
for compounds 2-7. This material is available free of charge via
the Internet at http://pubs.acs.org.
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