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

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.¹⁷In 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.¹⁻⁸The modulation of triglyceride

synthesis therefore constitutes an attractive strategy for

therapeutic intervention, and enzymes within this pathway

have emerged as potential molecular targets.⁹Acyl 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.^10,11Two DGAT enzymes, DGAT1 and DGAT2,

have been identiﬁed.¹²⁻¹⁴Although 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

ACAT1.¹²Recent 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.^15,16This may explain why DGAT2

Received: January 27, 2014

Published: March 26, 2014

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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.^9,20,22−40Several 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 1,⁴¹containing 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.^37aThe

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.⁴⁵Here, 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 IC₅₀Values of Pyrimidine Analogues 2−9

a

Data are reported as the average of a minimum of two determinations.

Table 2. DGAT1 IC₅₀Values 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 IC₅₀value. 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 IC₅₀value 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 IC₅₀Values 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 t_1/2values. 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 IC₅₀value 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.⁴⁸In 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 IC₅₀values 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 IC₅₀values 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

IC₅₀value 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 IC₅₀Values 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⁻¹kg⁻¹

)

Vd_ss(L/kg)

t_1/2(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 IC₅₀values and rat clearance of 2-substituted

pyrimidines 1 and 37−38. IC₅₀values 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⁻¹po). 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⁻¹day⁻¹po) 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

IC₅₀values greater than 30 μM against ACAT1, DGAT2,

MGAT, and GPAT. Furthermore, 37 had IC₅₀values 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⁻¹kg⁻¹in 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 IC₅₀values of acid replacements 39−41. IC₅₀values

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 IC₅₀value of 2.5 μM.

Figure 8. DGAT1 IC₅₀values and rat clearance of dihydropyran

analogues 43−45. IC₅₀values 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 IC₅₀value 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 IC₅₀values 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).⁵⁰N-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 SnCl₂in 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).⁵¹Demethylation of anisole 27 using BBr₃in

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

NaBH₄in 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 AlCl₃in

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 K₂CO₃in

DMF provided methyl ester 56. Removal of the hydroxy group

was attempted using triethylsilane and triﬂuoroacetic acid in

chloroform;⁵²however, 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 IC₅₀values 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⁻¹kg⁻¹

)

Vd_ss(L/kg)

t_1/2(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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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. Barone⁵³reported 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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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 carbonylation⁵⁴of

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

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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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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 86⁵⁵with

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

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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 NaHCO₃provided 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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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 CHCl₃were 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

NaHCO₃and 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 NaHCO₃and 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 NaHCO₃and 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 NaHCO₃and 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 NaHCO₃and 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 BBr₃solution 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 NaHCO₃solution, water,

and brine. The organic layer was dried over MgSO₄and 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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(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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Ginsberg, H. N.; Goldberg, A. C.; Howard, W. J.; Jacobson, M. S.;

Kris-Etherton, P. M.; Lennie, T. A.; Levi, M.; Mazzone, T.; Pennathur,

S. Triglycerides and cardiovascular disease: a scientific statement from

the American Heart Association. Circulation 2011, 123, 2292−2333.

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eukaryotes. Annu. Rev. Biochem. 1980, 49, 459−487.

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).

(11) Lehner, R.; Kuksis, A. Biosynthesis of triacylglycerols. Prog. Lipid

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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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

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