Discovery of 6-Phenylpyrimido [4, 5-b][1, 4] oxazines as Potent and

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.

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INTRODUCTION Elevated plasma and tissue triglyceride levels are implicated in the pathogenesis of a variety of diseases and risk factors including obesity, insulin resistance syndrome, type II diabetes, dyslipidemia, hepatic steatosis, metabolic syndrome, and coronary heart disease.1−8 The modulation of triglyceride synthesis therefore constitutes an attractive strategy for therapeutic intervention, and enzymes within this pathway have emerged as potential molecular targets.9 Acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes catalyze the final and only committed step in triglyceride synthesis, the joining of 1,2-diacylglycerol and fatty acyl CoA at the endoplasmic reticulum.10,11 Two DGAT enzymes, DGAT1 and DGAT2, have been identified.12−14 Although both enzymes catalyze the formation of triglycerides (TG) from diacylglycerol and fatty acyl CoA, they share limited homology. In fact, DGAT1 is part of the acyl CoA:cholesterol acyltransferase (ACAT) gene family with mouse DGAT1 sharing 20% identity with mouse ACAT1.12 Recent evidence indicates that DGAT2 is responsible for the synthesis of TG from endogenous fatty acids whereas DGAT1 catalyzes the formation of TG using exogenous fatty acids.15,16 This may explain why DGAT2 © 2014 American Chemical Society

knockout mice (Dgat2−/−) die soon after birth because of severe reductions in plasma triglycerides and total carcass triglycerides.17 In contrast, DGAT1 knockout mice (Dgat1−/ −) are healthy and capable of synthesizing triglycerides.18 Dgat1−/− mice have been reported to be lean and remarkably resistant to diet-induced obesity. When fed a high fat diet (21% fat), Dgat1−/− mice maintain weights comparable to those of mice fed a regular diet (4% fat). Total triglyceride levels are lower in Dgat1−/− mice compared to wild type mice, and Dgat1−/− mice have increased insulin and leptin sensitivity compared to wild-type littermates. The resistance to dietinduced 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 Received: January 27, 2014 Published: March 26, 2014 3464

dx.doi.org/10.1021/jm500135c | J. Med. Chem. 2014, 57, 3464−3483

Journal of Medicinal Chemistry

Article

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 literature.9,20,22−40 Several of these inhibitors have been used to elucidate the pharmacology of DGAT1 inhibition in vivo and have successfully demonstrated the ability to reproduce the Dgat1−/− mouse phenotype in rodents. The observed in vivo 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 dietinduced obese mice. Since our initial disclosure of pyrimidooxazine 1,41 containing the phenylcyclohexylacetic acid functionality, 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, investigators from AstraZeneca reported results from their phase 1 study demonstrating that the treatment of healthy male subjects with DGAT1 inhibitor AZD7687 resulted in markedly reduced postprandial TG excursion in a dose-dependent manner.37a The DGAT1 inhibitor discovered by Novartis, pradigastat (LCQ908), is the most advanced clinical candidate and is currently being evaluated in phase II and phase III studies.45 Novartis has recently disclosed that pradigastat lowered the fasting plasma TG level in patients with familial chylomicronemia syndrome.45 Here, we report our efforts that led to the discovery of the first DGAT1 inhibitor to contain the phenylcyclohexylacetic acid functionality, compound 1, as well as on our medicinal chemistry program directed at improving upon the overall profile of this benchmark compound.

Figure 2. DGAT1 inhibitory activity of screening hit 2.

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 2position 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

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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 3465



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Table 1. DGAT1 IC50 Values of Pyrimidine Analogues 2−9

a

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

Table 2. DGAT1 IC50 Values of Oxazine Analogues 10−14

a


less potent than the corresponding unsaturated analogues 10 and 11. 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

higher IC50 value. We were able to replace the bromo atom with alkyl groups (18−23) and observed that potencies generally improved with increased alkyl size and additional branching. For example, potency increased from 410 nM for the methyl analogue 18 to ethyl (19) to propyl (20) to isopropyl (22) and finally to tert-butyl (23) possessing an IC50 value of 60 nM. Cyclohexyl analogue 24 also inhibited DGAT1 with an IC50 value of 60 nM, while phenyl derivative 25 was essentially equipotent to the bromo compound 11. Replacing the bromine of 11 with a hydroxy group (26) resulted in approximately a 40-fold decrease in DGAT1 potency. We 3466



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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 The promising potency, PK properties, and in vivo efficacy of 1 in rodents encouraged us to initiate preclinical development studies. We determined the PK of carboxylate 1 in beagle dogs and rhesus monkeys (Table 5). The bioavailability of 1 in dog was high, while clearance values were moderate in both dog and monkey, resulting in relatively short t1/2 values. We investigated the inhibitory activity of carboxylate 1 against several related enzymes, including human ACAT1, ACAT2, DGAT2, MGAT, and GPAT and demonstrated that 1 did not inhibit these enzymes at concentrations up to 10 μM. We also demonstrated that analogue 1 did not inhibit CYP1A2, CYP2D6, CYP2C9, or CYP3A4 at concentrations up to 10 μM. The IC50 value in the hERG inhibition assay of analogue 1 was greater than 10 μM. Satisfied with the selectivity of 1, a 14-day multiple-dose toxicity study was conducted in Sprague−Dawley rats using po doses of 30 and 100 mg/kg q.d. No significant clinical or histopathological findings were detected at the 30 mg/kg dose (AUC = 913 μM·h), while sebaceous gland atrophy was the only significant finding at the 100 mg/kg dose (AUC = 3752 μM·h). Sebaceous gland atrophy is an expected result of DGAT1 inhibition, as atrophic sebaceous glands have been characterized in Dgat1(−/−) mice.48 In contrast, significant elevation of the liver enzymes ALT and AST (∼10-fold) were observed in a single 100 mg/kg po dose (AUC = 1420 μM·h) in cynomolgus monkeys. We suspected that a difference in metabolism between the two species was responsible for the divergent results from the two toxicity studies. In order to investigate this possibility, we studied the metabolites formed during incubation of 1 with Sprague−Dawley rat and cynomolgus liver S9 fractions. Our investigation revealed that a single metabolite was produced in significantly larger quantities in cynomolgus liver S9 fractions compared to rat liver S9 fractions (Figure 4). Mass spectral analysis indicated that the metabolite was the product of a single oxidation, and further analysis by MS/MS suggested that the oxidation

Table 3. DGAT1 IC50 Values of Substituted Phenyl Analogues 15−30

compd

R

DGAT1 SPA IC50 (μM)a

11 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

bromo hydrogen chloro fluoro methyl ethyl propyl butyl isopropyl tert-butyl cyclohexyl phenyl hydroxyl methoxy N-piperidinyl N-morpholinyl 2-carboxypropan-2-yl

0.21 ± 0.03 2.9 ± 0.7 0.39 ± 0.05 3.3 ± 0.6 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 0.57 ± 0.10 0.13 ± 0.05 0.61 ± 0.09 0.05 ± 0.01

a


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 4,4difluoro analogues 31−33 had similar biochemical and cellular DGAT1 IC50 values compared to 24 (Table 4). Because of the potency of carboxylic acid 30, we prepared cyclohexanecarboxylic acid 34. To our surprise, 34 demonstrated a 6-fold increase in biochemical potency and a 3-fold increase in cellular potency compared to cyclohexane 24. A thorough investigation of the SAR around carboxylate 34 led us to the discovery of the ethanoic and propanoic acid homologues 1 and 35. The DGAT1 IC50 values of these two analogues in the biochemical assay were identical, but ethanoic acid 1 (IC50 = 40 nM) showed a clear advantage over propanoic acid 35 (IC50 = 100 nM) in the cellular assay. Biphenyl analogue 36 was also prepared and possessed good biochemical potency but had an IC50 value of >3 μM in the cellular assay. The excellent cellular activity of 1 encouraged us to investigate this compound’s ability to inhibit DGAT1 activity in vivo. We envisioned an initial study that would measure the uptake of triglyceride into plasma after an oral bolus of olive oil. We first assessed the pharmacokinetic profile of 1 in CD-1 mice 3467



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Table 4. DGAT1 IC50 Values of Cyclohexyl Analogues 1 and 31−36

a

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

Table 5. Pharmacokinetic Properties of Carboxylate 1 iv

po

species

dose iv/po (mg/kg)

CL (L h−1 kg−1)

Vdss (L/kg)

t1/2 (h)

AUC (μg·h/L)

F (%)

mouse rat dog rhesus

0.5/2.0 1/2.0 0.5/5.0 0.2/−

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

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 3468



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Figure 5. DGAT1 IC50 values and rat clearance of 2-substituted pyrimidines 1 and 37−38. IC50 values are reported as the average of a minimum of two determinations.

in the patch clamp assay. A 28-day multiple-dose toxicity study in Sprague−Dawley rats showed no treatment related changes in clinical pathology other than the expected atrophic sebaceous glands at the doses tested (100 and 500 mg kg−1 day−1 po). Analogue 37 was negative in the Ames test but positive in a chromosomal aberration test using Chinese hamster lung cells. Although a 2-hydroxy metabolite could not form, a 2- to 5-fold increase in plasma ALT and AST levels was detected at all doses (1, 10, and 100 mg kg−1 day−1 po) in a 28day multiple-dose cynomolgus toxicity study. Finally, two out of eight cynomolgus monkeys had prolonged QTc intervals at the 100 mg/kg dose while no effect was observed at the 1 and 10 mg/kg doses. Consideration of the small increase in liver enzyme levels, the positive chromosomal aberration result, and the prolonged QTc intervals taken together resulted in the termination of the development of methylpyrimidine 37. While we investigated the cause of the liver toxicity noted in cynomolgus monkeys, we further investigated the SAR of

Figure 3. Increase in plasma triglyceride concentrations 3 h after an oral gavage of olive oil in (A) C57BL/6J mice pretreated with vehicle or DGAT1 inhibitor 1 at 0.3 mg/kg and (B) Sprague−Dawley rats pretreated with vehicle or DGAT1 inhibitor 1 at 0.1, 0.3, and 1.0 mg/ kg: (∗) p < 0.05, (∗∗) p < 0.01 compared to vehicle (n = 6/group).

further evaluated in selectivity assays and was found to have IC50 values greater than 30 μM against ACAT1, DGAT2, MGAT, and GPAT. Furthermore, 37 had IC50 values greater than 10 μM for all of the 135 targets tested in the Panlabs panel and showed no inhibition of CYP1A2, CYP2D6, CYP2C9, or CYP3A4 when tested up to 10 μM. However, methylpyrimidine 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. 3469



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in vivo oxidation at the C2 position. Investigation of the pharmacokinetic properties of 42 indicated that it possessed a reasonable clearance of 0.21 L h−1 kg−1 in rats. We also approached blocking the formation of the toxic metabolite observed upon in vivo incubation of 1 by replacing the oxazine ring with a dihydropyran ring. We initially prepared analogues 43 and 44 possessing a hydrogen atom at C2 of the pyrimidine ring. Both of these analogues maintained excellent DGAT1 inhibitory activity in both the biochemical and cellular assay (Figure 8). The DGAT1 cellular potency of spiroindane

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

Figure 6. DGAT1 IC50 values of acid replacements 39−41. IC50 values are reported as the average of a minimum of two determinations.

maintained similar DGAT1 biochemical potencies compared to 1. Although amide 39 and sulfonamide 41 also had comparable cellular DGAT1 inhibitory activity, tetrazole 40 possessed a DGAT1 cellular IC50 value of 2.5 μM. 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 2trifluoromethylpyrimidine 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

Figure 8. DGAT1 IC50 values and rat clearance of dihydropyran analogues 43−45. IC50 values are reported as the average of a minimum of two determinations.

44 (9 nM) was significantly improved compared to cyclohexyl analogue 43 (50 nM). This result is consistent with the increase in potency achieved with spiroindane 42 in the oxazine series. However, both dihydropyran analogues 43 and 44 had higher clearance values in rats compared to the oxazine analogues 1, 37, 38, and 42. On the basis of the result in the oxazine series that installment of a methyl at the C2 position of the pyrimidine resulted in lower rat clearance (37 vs 1 Figure 5), a methyl was installed at the C2 position of the pyrimidine of dihydropyran analogue 43, providing compound 45. Unfortunately, replacing the C2 hydrogen with a methyl group in this compound resulted in a 4-fold reduction in DGAT1 biochemical activity and a significant decrease in cellular potency, resulting in an IC50 value greater than 1 μM. Although analogues 43 and 44 demonstrated that the nitrogen of the oxazine ring could be replaced by a carbon atom without losing DGAT1 inhibitory activity, these compounds possessed higher in vivo rat clearance values and thus provided no benefit over the oxazine analogues. The discovery of trifluoromethyl analogue 42 provided a molecule that could not undergo oxidative metabolism at C2 of the pyrimidine while improving the DGAT1 potency by an order of magnitude compared to 1. Evaluation of this molecule in the mouse oral triglyceride uptake test at doses of 0.3, 0.1, and 0.03 mg/kg demonstrated that 42 significantly reduced TG uptake after oral administration of olive oil in a dose dependent manner (Figure 9). The increased DGAT1 cellular potency of spiroindane 42 compared to 1 translated well in the TG uptake assay with 42 demonstrating a 99.7% and 105% inhibition of TG uptake compared to vehicle at doses of 0.1 and 0.3 mg/kg,

Figure 7. DGAT1 IC50 values and rat clearance of spiroindane 42. IC50 values are reported as the average of a minimum of two determinations.

the carbon−carbon bond joining the phenyl and cyclohexane ring systems and forces the rings into an orthogonal orientation. This structural modification resulted in a 20-fold increase in DGAT1 cellular potency of spiroindane analogue 42 compared to the cyclohexyl analogue 38, providing a single digit nanomolar DGAT1 inhibitor while blocking any potential 3470



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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.

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CHEMISTRY The key step of the synthesis of analogues 2−6 and 48 involved the formation of the 6-phenylpyrimido[4,5-b][1,4]oxazine core through a condensation of 2-bromo-1-(4-bromophenyl)ethanone (46) with an appropriate 2-aminophenol 47a−f under basic conditions (Scheme 1).50 N-Methylation of 2 using iodomethane in the presence of NaH in DMF provided analogues 7 and 8. Compound 9 was synthesized by reduction of the nitro group of 48 using SnCl2 in EtOH. Condensation of 4,5-diamino-6-hydroxypyrimidine (47a) with (±)-2-bromo-1(4-bromophenyl)propan-1-one (49) under the conditions used previously for the synthesis of analogues 2−6 provided 7methyl-6-phenylpyrimido[4,5-b][1,4]oxazine-4-amine analogue 10 (Scheme 2). Condensation of 2-bromo-2-methyl-1-phenylpropan-1-ones 50 with 4,5-diamino-6-hydroxypyrimidine (47a) under acidic conditions provided the 7,7-dimethyl-6-phenyl-7H-pyrimido[4,5-b][1,4]oxazine-4-amine analogues 11, 15−24, and 27−33 (Scheme 2).51 Demethylation of anisole 27 using BBr3 in dichloromethane provided phenol 26. Suzuki coupling of aryl bromide 11 using phenylboronic acid provided biphenyl analogue 25. Reduction of oxazines 2, 10, and 11 using NaBH4 in methanol provided the corresponding dihydrooxazines 12−14 in good yield (Scheme 3). Friedel−Crafts acylation of aryl esters 51 and 52 with 2bromo-2-methylpropanoyl bromide in the presence of AlCl3 in dichloromethane provided bromo ketones 53 and 54, respectively (Scheme 4). Condensation of the bromo ketones with 47a in the presence of HCl in aqueous EtOH at reflux followed by saponification of the corresponding esters provided carboxylic acid analogues 30 and 36. The preparation of cyclohexanecarboxylic acid 34 is outlined in Scheme 5. Saponification of ethyl 4-oxocyclohexanecarboxylate (55) with NaOH in aqueous EtOH provided the carboxylic acid, which was treated with 2 equiv of phenylmagnesium bromide in THF to yield the corresponding tertiary alcohol. Methylation of the carboxylate with iodomethane in the presence of K2CO3 in DMF provided methyl ester 56. Removal of the hydroxy group was attempted using triethylsilane and trifluoroacetic acid in chloroform;52 however, only the dehydration product was obtained. Hydrogenation of the alkene using 10% palladium on carbon in methanol provided a 1:1 mixture of cis and trans isomers. Treating this mixture with sodium methoxide in methanol at 70 °C provided ester 57 as a 4:1 mixture of trans/ cis isomers. Following the general procedure, Friedel−Crafts acylation of ester 57 followed by condensation with 47a and

Figure 9. Increase in plasma triglyceride concentrations 3 h after an oral gavage of olive oil in C57BL/6J mice pretreated with vehicle or DGAT1 inhibitor 42 at 0.03, 0.1, and 0.3 mg/kg: (∗∗) p < 0.01, (∗∗∗) p < 0.001 as compared to vehicle (n = 3/group).

respectively. Spiroindane 42 was more efficacious than carboxylate 1 despite having an approximately 4-fold lower plasma exposure when dosed at 0.3 mg/kg (42, AUC = 0.65 μM·h; 1, AUC = 2.6 μM·h). Because of the promising in vivo efficacy data from the mouse oral triglyceride uptake study, spiroindane 42 was evaluated in preclinical toxicity studies. The compound was shown to be selective against human ACAT1, ACAT2, DGAT2, MGAT, and GPAT, possessing IC50 values of >10 μM, and did not inhibit CYP1A2, CYP2D6, CYP2C9, or CYP3A4 at concentrations up to 10 μM. There was no inhibitory activity against the hERG channel at concentrations up to 5 μM in the patch clamp assay. Investigation of the pharmacokinetic properties in mouse, rat, dog, and cyno indicated that 42 had low clearance across species and good exposure and bioavailabilities in mouse, rat, and cyno (Table 6). A 14-day multiple-dose toxicity study in Sprague−Dawley rats was conducted at 10, 30, and 100 mg/kg q.d. po. No significant clinical or histopathological findings were detected at the 10 mg/kg (AUC = 11 μM·h) dose, while sebaceous gland atrophy was the only significant finding at the 30 and 100 mg/kg doses (AUC of 78 and 677 μM·h, respectively). Finally, no increase in ALT or AST levels, no prolonged QTc intervals, and no significant clinical or histopathological findings were detected in a 21-day multiple-dose toxicity study in cynomolgus monkeys conducted at 10 and 30 mg/kg q.d. po (AUC of 607 and 2561 μM·h, respectively). Herein we described the discovery of a potent, selective, and orally efficacious DGAT1 inhibitor 1 consisting of a pyrimidooxazine core and a phenylcyclohexylacetic acid substituent. Inhibitor 1 was a promising preclinical candidate shown to form a 2-hydroxy metabolite in cynomolgus monkeys, resulting in elevated levels of the liver enzymes ALT and AST. Molecules were designed to block the C2 position, thereby Table 6. Pharmacokinetic Properties of Spiroindane 42

iv

po

species

dose iv/po (mg/kg)

CL (L h−1 kg−1)

Vdss (L/kg)

t1/2 (h)

AUC (μg·h/L)

F (%)

mouse rat dog cyno

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 1.1 0.21 0.44

4.4 3.7 1.1 15.4

341 22862 3185 251581

77 98 22 99

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Scheme 1a

a

Reagents and conditions: (a) SnCl2·H2O, EtOH, reflux, 50%; (b) MeI, NaH, DMF, 13−32%.

Scheme 2a

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, condensation and saponification provided propanoic acid analogue 35. The synthesis of 5,6-diamino-4-hydroxy-2-trifluoromethylpyrimidine (67), required to prepare analogues 38 and 42, was completed on a several hundred gram scale, required no chromatography, and provided pyrimidine 67 in 61% yield over three steps (Scheme 7). Condensation of malonamidine hydrochloride 63 and ethyl trifluoroacetate 64 in THF with DBU as the base provided trifluoromethylpyrimidine 65 in 66% yield. Nitration of pyrimidine 65 was accomplished by using 90% nitric acid in concentrated sulfuric acid at 55 °C to provide 66 in 95% yield. Barone53 reported isolating the 4-Nnitroamine 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 accomplished 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 3472



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Scheme 4a

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%.

Scheme 5a

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). 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 containing 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. 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 W i t t i g r e a c t i o n o f 5 - b r o m o i n d a n o n e ( 7 3 ) w it h (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,βunsaturated ester 77. Palladium catalyzed carbonylation54 of aryl bromide 77 provided the benzyl ester which was removed by hydrogenolysis using hydrogen and 10% palladium on carbon in ethanol while also hydrogenating the alkene. Crystallization from ethyl acetate and hexanes provided the

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−Emmons (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 2position of the pyrimidine ring. Treatment of acid 1 with EDC, 3473



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Scheme 6a

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 7a

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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Scheme 8a

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%.

Scheme 9a

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.

a

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. 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

dichloromethane. The triflate was further transformed to boronate 84 using bis(pinacolato)diboron and a palladium catalyst. In a similar way aryl bromide 77 was converted to the boronate followed by hydrogenation of the α,β-unsaturated double bond to provide ester 85 as a 20:1 mixture of trans/cis isomers after successive crystallization from methanol and dichloromethane using methanol diffusion. The pyrimidinopyran ring structure was synthesized by the acid catalyzed condensation of bromoaldehyde 8655 with hydroxypyrimidine 87 or 88 to provide dihydropyran 89 or 90 (Scheme 11). Functional group conversion of hydroxypyrimidines 89 and 90 to chloropyrimidines 91 and 92 was accomplished using phosphorus oxychloride. Displacement of the chloride with 4-methoxybenzylamine in the presence of Hünigs 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.

■

EXPERIMENTAL SECTION

All solvents and chemicals used were reagent grade. Anhydrous solvents were purchased from Aldrich and used as such. Analytical thin layer chromatography (TLC) and flash chromatography were 3475



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Scheme 10a

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%.

6-(4-Bromophenyl)-7H-pyrimido[4,5-b][1,4]oxazin-4-amine (2). Following the general procedure, 4,5-diamino-6-hydroxypyrimidine (47a) and 2-bromo-1-(4-bromophenyl)ethanone (46) provided the title compound as a white solid in 31% yield: mp >200 °C. 1H 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 °C. 1H 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-diamine (4). Following the general procedure, 2,4,5-triamino-6hydroxypyrimidine sulfate (47c), 46, and 2.1 equiv of NaHCO3 provided the title compound as a white solid in 21% yield: mp >200 °C. 1H NMR (DMSO-d6) δ 5.25 (s, 2H), 6.24 (br s, 2H), 6.55 (br s, 2H), 7.63 (d, J = 12.0 Hz, 2H), 7.92 (d, J = 12.0 Hz, 2H); ESIMS m/z (rel intensity) 320 ([M + H], 100), 322 ([M + H], 100). 6-(4-Bromophenyl)-N2,N2-dimethyl-7H-pyrimido[4,5-b][1,4]oxazine-2,4-diamine (5). Following the general procedure, 47d, 46, and 3.1 equiv of NaHCO3 provided the title compound as a white solid in 23% yield: mp >200 °C. 1H NMR (DMSO-d6) δ 3.04 (s, 6H), 5.27 (s, 2H), 6.73 (br s, 2H), 7.63 (d, J = 6.0 Hz, 2H), 7.94 (d, J = 6.0 Hz, 2H); ESIMS m/z (rel intensity) 348 ([M + H], 100), 350 ([M + H], 100).

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) 3320, 2929, 1702, 1601; 1H 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). General Procedure for the Preparation of Pyrimidooxazines 2−10. To a suspension of an appropriate 5-amino-6-hydroxypyrimidine (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. 3476



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Scheme 11a

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. a

4-Amino-6-(4-bromophenyl)-7H-pyrimido[4,5-b][1,4]oxazin2-ol (6). Following the general procedure, 4,5-diamino-2,6-dihydroxypyrimidine (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). 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). 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

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). 3-(4-Bromophenyl)-2H-benzo[b][1,4]oxazin-5-amine (9). To a solution of 2-amino-3-nitrophenol (47f) (100 mg, 0.649 mmol) in DMF (2.0 mL) were added 46 (180 mg, 0.648 mmol) and K2CO3 (90 mg, 0.651 mmol), and the mixture was stirred at ambient temperature overnight. The mixture was diluted with EtOAc, washed with water and brine, dried over MgSO4, and concentrated to dryness to provide 3-(4-bromophenyl)-5-nitro-2H-benzo[b][1,4]oxazine (48) (217 mg). A mixture of (48) (100 mg, 0.300 mmol) and SnCl2·H2O (339 mg, 1.50 mmol) in EtOH was heated at reflux for 2 h and then cooled to ambient temperature. Concentrated HCl and CHCl3 were added to the mixture, and the organic layer was separated, dried over MgSO4, and concentrated in vacuo. Purification of the resulting residue by preparative TLC gave the title compound as a white solid (45.0 mg, 50% from 48): mp 75−77 °C. 1H 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)-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 3477



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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). General Procedure for the Preparation of Pyrimidooxazines 11, 15−36, 41, and 42. To a solution of an appropriately substituted benzene (1.2 equiv) in CH2Cl2 (2 L/mol bromide) were added AlCl3 (2 equiv) and 2-bromo-2-methylpropanoyl bromide (1 equiv) at 0 °C. After the mixture was stirred at ambient temperature for 1 h, it was poured onto ice−water, and the mixture was extracted with CHCl3. The organic layer was separated, washed with saturated aqueous NaHCO3 and brine, dried over MgSO4, and evaporated. The resulting residue was purified by column chromatography on silica gel to give the corresponding phenyl substituted 2-bromo-2-methyl-1-phenylpropan-1-one. A mixture of 4,5-diamino-6-hydroxypyrimidine 47a, 47b, or 67 (1.0 equiv), an appropriate 2-bromo-2-methyl-1-phenylpropan-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 bromobenzene, 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). (±)-6-(4-Bromophenyl)-6,7-dihydro-5H-pyrimido[4,5-b][1,4]oxazin-4-amine (12). To a solution of 2 (35 mg, 0.115 mmol) in MeOH (1 mL) and THF (1 mL) at 0 °C was added NaBH4 (8.7 mg, 0.23 mmol). The reaction mixture was warmed to ambient temperature and stirred for 1 h. The reaction mixture was poured into water, and the mixture was extracted with EtOAc. The organic layer was separated and washed with saturated aqueous NaHCO3 and brine, dried over MgSO4, and concentrated in vacuo. The resulting residue was slurried in toluene and the solid collected by vacuum filtration. This solid was slurried in MeOH and collected by vacuum filtration to provide the title compound as a white solid (23 mg, 65%): mp >200 °C. 1H 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). (±)-cis-6-(4-Bromophenyl)-7-methyl-6,7-dihydro-5Hpyrimido[4,5-b][1,4]oxazin-4-amine (13). To a solution of 10 (146 mg, 0.457 mmol) in MeOH (1.4 mL) and THF (1.4 mL) at 0 °C was added NaBH4 (34.6 mg, 0.914 mmol). The reaction mixture was stirred at ambient temperature for 1 h and then poured into water, and the mixture was extracted with EtOAc. The organic layer was separated, washed with saturated aqueous NaHCO3 and brine, dried over MgSO4, and concentrated in vacuo. The resulting residue was slurried in toluene and the solid collected by vacuum filtration. This solid was slurried in MeOH and collected by vacuum filtration to provide the title compound as a white solid (74.1 mg, 50%). 1H NMR (DMSO-d6) δ 0.99 (d, J = 6.0 Hz, 3H), 4.54 (br s, 2H), 5.36 (br s, 1H), 6.36 (br s, 2H), 7.28 (d, J = 9.0 Hz, 2H), 7.58 (d, J = 9.0 Hz, 2H), 7.65 (s, 1H); ESIMS m/z (rel intensity) 321 ([M + H], 100), 323 ([M + H], 100). (±)-6-(4-Bromophenyl)-7,7-dimethyl-6,7-dihydro-5Hpyrimido[4,5-b][1,4]oxazin-4-amine (14). To a solution of 11 (52.3 mg, 0.157 mmol) in MeOH (1.0 mL) and THF (1.0 mL) at 0 °C was added NaBH4 (11.9 mg, 0.314 mmol). The reaction mixture was stirred at ambient temperature for 1 h and then poured into water, and the mixture was extracted with EtOAc. The organic layer was separated, washed with saturated aqueous NaHCO3 and brine, dried over MgSO4, and concentrated in vacuo. The resulting residue was slurried in toluene and the solid collected by vacuum filtration. This solid was slurried in MeOH and collected by vacuum filtration to

provide the title compound as a white solid (50.1 mg, 95%): mp >220 °C. 1H 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). 7,7-Dimethyl-6-phenyl-7H-pyrimido[4,5-b][1,4]oxazin-4amine (15). Following the general procedure, 2-bromo-2-methyl-1phenylpropan-1-one (50a) (Aldrich) provided the title compound as a white solid in 45% yield: mp 189−190 °C. 1H 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 chlorobenzene, provided the title compound as a white solid in 42% yield: mp 210−211 °C. 1H 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 fluorobenzene, provided the title compound as a white solid in 17% yield: mp 189−190 °C. 1H 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-4amine (18). Following the general procedure, 2-bromo-2-methyl-1(p-tolyl)propan-1-one (50e), prepared from toluene, provided the title compound as a white solid in 45% yield: mp 161−162 °C. 1H 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-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 ethylbenzene, provided the title compound as a white solid in 59% yield: mp 136−137 °C. 1H 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-2methyl-1-(4-propylphenyl)propan-1-one (50g), prepared from propylbenzene, provided the title compound as a white solid in 40% yield: mp 186−187 °C. 1H 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). 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 butylbenzene, provided the title compound as a white solid in 67% yield: mp 158−161 °C. 1H 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: mp 176−179 °C. 1H NMR (DMSO-d6) δ 1.23 (d, J = 8.0 Hz, 6H), 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). 6-(4-(tert-Butyl)phenyl)-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-4-amine (23). Following the general procedure, 2bromo-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). 3478



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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, 2bromo-1-(4-cyclohexylphenyl)-2-methylpropan-1-one (50k), prepared from phenylcyclohexane, provided the title compound as a white solid in 55% yield: mp 186−187 °C. 1H NMR (DMSO-d6) δ 1.21−1.47 (m, 5H), 1.60 (s, 6H), 1.70−1.82 (m, 5H), 2.54−2.58 (m, 1H), 6.89 (br s, 2H), 7.29 (d, J = 9.0 Hz, 2H), 7.64 (d, J = 9.0 Hz, 2H), 7.94 (s, 1H); ESIMS m/z (rel intensity) 337 ([M + H], 100). 6-([1,1′-Biphenyl]-4-yl)-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-4-amine (25). To a solution of 11 (50 mg, 0.150 mmol) and PhB(OH)2 (22 mg, 0.180 mmol) in DMA (1.0 mL) were added a solution of Na2CO3 (40 mg, 0.375 mmol) in water (0.3 mL) and a suspension of Pd(PPh3)4 (17 mg, 0.015 mmol) in DMA (1.0 mL) at ambient temperature. The reaction mixture was stirred at 100 °C for 6.5 h, cooled to ambient temperature, and diluted with EtOAc and water. The insoluble material was removed by filtration, and the organic phase of the filtrate was separated, washed with saturated aqueous NaHCO3 and brine, dried over MgSO4, and concentrated. The resulting residue was triturated in diisopropyl ether to give a brown solid which was then recrystallized from EtOH to give the title compound as a pale yellow solid (23.4 mg, 47%): mp 218−219 °C. 1H NMR (DMSO-d6) δ 1.66 (s, 6H), 7.02 (br s, 2H), 7.39−7.53 (m, 3H), 7.73−7.77 (m, 4H), 7.83−7.86 (m, 2H), 7.97 (s, 1H); ESIMS m/z (rel intensity) 331 ([M + H], 100). 4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6yl)phenol (26). To a suspension of 27 (100 mg, 0.352 mmol) in CH2Cl2 (4.0 mL) was added 1.0 M BBr3 solution in CH2Cl2 (1.10 mL, 1.10 mmol) at −78 °C. The reaction mixture was stirred at ambient temperature for 3 days and was then poured into ice−water. The mixture was made basic with 1 M NaOH and washed with CHCl3 three times. The aqueous phase was neutralized with 2 M HCl and extracted with EtOAc. The organic phase was separated, successively washed with water and brine, dried over Na2SO4, and concentrated in vacuo. The resulting residue was slurried in toluene and then recrystallized from EtOH to give the title compound as a white solid (49.1 mg, 52%): mp 245−256 °C. 1H NMR (DMSO-d6) δ 1.60 (s, 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). 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 anisole, 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). 7,7-Dimethyl-6-(4-(piperidin-1-yl)phenyl)-7H-pyrimido[4,5b][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, 83%): mp 191−193 °C. 1H 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). 7,7-Dimethyl-6-(4-morpholinophenyl)-7H-pyrimido[4,5-b][1,4]oxazin-4-amine (29). Using the procedure for 28 with 2methyl-1-(4-morpholinophenyl)propan-1-one provided the title compound as a white solid in 21% yield: mp 214−219 °C. 1H 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).

2-(4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin6-yl)phenyl)-2-methylpropanoic Acid (30). Following the general procedure, ethyl 2-(4-(2-bromo-2-methylpropanoyl)phenyl)-2-methylpropanoate (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 the title compound as a white solid in 5.7% yield: mp >200 °C. 1H 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]oxazin6-yl)phenyl)cyclohexanone (31). Following the general procedure, 4-(4-(2-bromo-2-methylpropanoyl)phenyl)cyclohexanone (50o), prepared from 4-phenylcyclohexanone, provided the title compound as a white solid in 31% yield: mp >200 °C (dec). 1H 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-hydroxypyrimidine 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 g, 85%): mp 246−248 °C. 1H 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-7Hpyrimido[4,5-b][1,4]oxazin-4-amine (33). Following the general procedure, 50q provided the title compound as a white solid in 70% yield: mp 226−227 °C. 1H NMR (DMSO-d6) δ 1.60 (s, 6H), 1.64− 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). (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). 3-(trans)-(4-(4-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-yl)phenyl)cyclohexyl)propanoic Acid (35). Employing 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 (cm−1) 3310, 2922, 1702, 1611; 1H 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). 2-(4′-(4-Amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin6-yl)-[1,1′-biphenyl]-4-yl)acetic Acid (36). Following the general procedure, methyl 2-(4′-(2-bromo-2-methylpropanoyl)[1,1′-biphenyl]-4-yl)acetate (54), prepared from methyl 2-([1,1′-biphenyl]-4yl)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 3479



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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). 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 4,5diamino-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). 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 compound. 1H NMR (DMSO-d6) δ 1.10−1.20 (m, 2H), 1.42−1.90 (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 solution of 1 (80 mg, 0.203 mmol) in DMF (2.4 mL) were sequentially added HOBt·H2O (34.2 mg, 0.223 mmol), 28% aqueous NH3 (0.041 mL, 6.08 mmol), and EDC (42.8 mg, 0.278 mmol) at ambient temperature. After the mixture was stirred for 1 h, 28% aqueous NH3 (0.041 mL, 6.08 mmol) was added to the mixture, which was further stirred for 70 h at ambient temperature. The mixture was diluted with EtOAc and washed with saturated aqueous NaHCO3 solution, water, and brine. The organic layer was dried over MgSO4 and concentrated to give a residue that was slurried in Et2O. The resulting solid was collected by vacuum filtration and then slurried in EtOH. The resulting precipitate was collected by vacuum filtration and dried under reduced pressure to afford the title compound as a white solid (48.2 mg, 60%): mp 224−226 °C. 1H 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 compound as a pale yellow solid (363 mg, 54%): mp >200 °C. 1H 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)-5′-(4-Amino-7,7-dimethyl-2-(trifluoromethyl)-7Hpyrimido[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

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 compound (70 mg, 72%) as a white solid. 1H 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) provided the methyl ester as a white solid (1.09 g, 61%). 1H 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%). 1 H 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]-4yl)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 as an off-white solid (73 mg). 1H 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). 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%). 1 H NMR (DMSO-d6) δ 1.04−1.19 (m, 2H), 1.39−1.55 (m, 2H), 1.49 3480



Article

(s, 6H), 1.65−1.89 (m, 5H), 2.14 (d, J = 7.2 Hz, 2H), 2.21 (s, 3H), 2.47 (m, 1H), 6.55 (s, 1H), 6.78 (br s, 2H), 7.23 (d, J = 8.3 Hz, 2H), 7.27 (d, J = 8.3 Hz, 2H), 12.01 (br s, 1H); ESIMS m/z (rel intensity) 408 ([M + H], 100). Methyl 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)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 (DMSOd6) δ 1.18−1.27 (m, 2H), 1.48−1.86 (m, 13H), 1.98 (t, J = 7.3 Hz, 2H), 2.28 (d, J = 7.2 Hz, 2H), 2.89 (t, J = 7.1 Hz, 2H), 3.61 (s, 3H), 7.28 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.58 (s, 1H).

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cardiovascular events in women. JAMA, J. Am. Med. Assoc. 2007, 298 (3), 309−316. (8) Miller, M.; Stone, N. J.; Ballantyne, C.; Bittner, V.; Criqui, M. H.; Ginsberg, H. N.; Goldberg, A. C.; Howard, W. J.; Jacobson, M. S.; Kris-Etherton, P. M.; Lennie, T. A.; Levi, M.; Mazzone, T.; Pennathur, S. Triglycerides and cardiovascular disease: a scientific statement from the American Heart Association. Circulation 2011, 123, 2292−2333. (9) Koltun, D. O.; Zablocki, J. Enzymatic targets in the triglyceride synthesis pathway. Annu. Rep. Med. Chem. 2010, 45, 109−122. (10) Bell, R. M.; Coleman, R. A. Enzymes of glycerolipid synthesis in eukaryotes. Annu. Rev. Biochem. 1980, 49, 459−487. (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. (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. (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 DGAT1deficient 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 acyltransferase. 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. (23) Birch, A. M.; Buckett, L. A.; Turnbull, A. V. DGAT1 inhibitors as anti-obesity and anti-diabetic agents. Curr. Opin. Drug Discovery Dev. 2010, 13 (4), 489−496. (24) Yeh, V.; Judd, A. S.; Souers, A. J. Lipid-metabolizing enzymes as targets for dyslipidemia and insulin resistance. Annu. Rep. Med. Chem. 2007, 42, 161−175. (25) (a) King, A. J.; Segreti, J. A.; Larson, K. J.; Souers, A. J.; Kym, P. R.; Reilly, R. M.; Collins, C. A.; Voorbach, M. J.; Zhao, G.; Mittelstadt, S. W.; Cox, B. F. In vivo efficacy of acyl CoA:diacylglycerol acyltransferase (DGAT)1 inhibition in rodent models of postprandial hyperlipidemia. Eur. J. Pharmacol. 2010, 637, 155−161. (b) King, A. J.; Segreti, J. A.; Larson, K. J.; Souers, A. J.; Kym, P. R.; Reilly, R. M.;

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.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 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.

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ACKNOWLEDGMENTS Amgen Inc. and Japan Tobacco Inc. contributed equally to the work detailed in this paper.

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ABBREVIATIONS USED DGAT, acyl CoA:diacylglycerol acyltransferase; TG, triglyceride; ACAT, acyl CoA:cholesterol acyltransferase; SPA, scintillation proximity assay; MGAT, acyl CoA:monoacylglycerol acyltransferase; GPAT, glycerol 3-phosphate acyltransferase; ALT, alanine aminotransferase; AST, aspartate aminotransferase

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REFERENCES

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Discovery of 6-Phenylpyrimido [4, 5-b][1, 4] oxazines as Potent and

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