Rational design of 6-(2,4-diaminopyrimidinyl)-1,4-benzoxazin-3-ones as small molecule renin inhibitors

Bioorganic & Medicinal Chemistry 15 (2007) 5912–5949

Rational design of 6-(2,4-diaminopyrimidinyl)-1,4-benzoxazin-3-

ones as small molecule renin inhibitors

Noel A. Powell,^*,ꢀFred L. Ciske,^ꢁCuiman Cai, Daniel D. Holsworth, Ken Mennen,^§

Chad A. Van Huis,^–Mehran Jalaie, Jacqueline Day, Michelle Mastronardi,

Pat McConnell, Igor Mochalkin, Erli Zhang, Michael J. Ryan, John Bryant,

Wendy Collard, Suzie Ferreira,^§§Chungang Gu, Roxane Collins and Jeremy J. Edmunds^––

Pﬁzer Global Research & Development, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, USA

Received 2 May 2007; revised 25 May 2007; accepted 29 May 2007

Available online 2 June 2007

Abstract—We report the design and synthesis of a series of 6-(2,4-diaminopyrimidinyl)-1,4-benzoxazin-3-ones as orally bioavailable

small molecule inhibitors of renin. Compounds with a 2-methyl-2-aryl substitution pattern exhibit potent renin inhibition and good

permeability, solubility, and metabolic stability. Oral bioavailability was found to be dependent on metabolic clearance and cellular

permeability, and was optimized through modulation of the sidechain that binds in the S3^spsubsite.

1. Introduction

Angiotensinogen

Angiotensin I

Renin

Hypertension is a leading risk factor for cardiovascular

disease, such as congestive heart failure, stroke, and

myocardial infarction, and is a major cause of death in

the Western world.¹The renin angiotensin system

(RAS) is well established as an endocrine system in-

volved in blood pressure regulation and ﬂuid electrolyte

balance (Fig. 1).²Activation of the RAS is stimulated by

several signals, including a drop in blood pressure, a de-

crease in the circulating volume, or a reduction in plas-

ma sodium concentration. These signals stimulate the

release of renin, which cleaves angiotensinogen at the

peptide bond between Leu10 and Val11 to form angio-

Bradykinin

Inactives

Chymase

ACE

Angiotensin II

AT₁Receptor

Blood Volume

Peripheral Resistance

Vasoconstriction

Central pressor activation

Sympathetic tone

Aldosterone release

Sodium re-absorption

Thirst

Keywords: Hypertension; Renin; Structure-based drug design; Oral

bioavailability.

*

Corresponding author. Tel.: +1 617 679 2000; fax: +1 617 679

2617; e-mail: powellns@netzero.com

Figure 1. The renin angiotensin system (RAS).

ꢀ

Present address: Biogen Idec, 14 Cambridge Center, Cambridge, MA

02142, USA.

tensin I (AngI). Angiotensin converting enzyme (ACE)

then converts AngI into the vasopressor octapeptide

angiotensin II (AngII). The binding of AngII to the

AT₁receptor initiates a number of physiological eﬀects,

such as sodium and water retention and vasoconstric-

tion, ultimately leading to an increase in blood pressure.

Since renin is the rate-limiting step in the RAS cascade

and angiotensinogen is the only known renin substrate,

ꢁ

Present address: Cayman Chemical Company, Ann Arbor, MI

48108, USA.

§

Present address: Array Biopharma, Boulder, CO 80301, USA.

Present address: Lycera Corporation, Ann Arbor, MI, USA.

–

§§

Present address: Novartis Institute for Biomedical Research, Cam-

bridge, MA 02139, USA.

––

Present address: Abbott Labs, 381 Plantation Street, Worchester,

MA 01605, USA.

doi:10.1016/j.bmc.2007.05.069

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

5913

renin inhibition is considered to be an attractive antihy-

pertensive strategy. Furthermore, inhibition of renin

prevents the formation of AngI and AngII, unlike the

angiotensin receptor blockers (ARBs) and ACE inhibi-

tors, which increase AngI levels but do not block

ACE-independent AngII production through the hydro-

lysis of AngI by chymase in human heart tissue. In addi-

tion, ACE has been shown to degrade the

pharmacologically active peptide bradykinin which has

been implicated in a chronic cough side eﬀect that aﬀects

5–35% of patients treated with ACE inhibitors.³There-

fore, renin inhibitors have been predicted to provide bet-

ter kidney and heart protection than ACE inhibitors and

ARBs while oﬀering superior blood pressure lowering.⁴

O

OH

O O

S

H

N

H

N

H

O

OH

NH₂

S

CI-992

IC₅₀= 0.38 nM

MW = 708

Rotatable bonds = 17

clogP = 4.40

%F = 1% (monkey)

MeO

OH

H

H₂N

N

NH₂

O

Many pharmaceutical companies have advanced renin

inhibitors to the clinic during the past few decades. Most

programs were based on peptidic or peptidomimetic

scaﬀolds that were designed to mimic the N-terminal se-

quence of angiotensinogen (Fig. 2). Although potent

in vitro renin inhibitory activity was obtained, these

scaﬀolds, exempliﬁed by CI-992 and others, frequently

exhibited poor pharmacokinetic properties and low oral

bioavailability likely due to high molecular weights

(MW > 600), conformational ﬂexibility (>15 rotatable

bonds), dissolution limited absorption, and high meta-

bolic clearance.^5,6Research on these scaﬀolds reached

an apex with the discovery and development of aliskerin

by scientists at Novartis and Speedel.^4c,7Aliskerin was

recently approved by the FDA for the treatment of

hypertension under the trade name Tekturna^ꢁin

March, 2007. Like CI-992, aliskerin is a transition-state

mimic with high MW = 552 and large number of rotat-

able bonds (21), but it takes advantage of a subpocket

accessible from the S3 region (S3^sp) that is unique for re-

nin to provide additional potency and speciﬁcity against

other aspartic proteases. In spite of the low human oral

bioavailability (3%), aliskerin exhibits comparable

blood pressure reduction to the AT1-receptor antagonist

losartan (300 mg daily vs 100 mg daily) due to very po-

tent inhibition of human renin (IC₅₀= 0.6 nM), low

plasma protein binding, and an extended in vivo half-life

(24 h).⁸Aliskerin has also demonstrated superior protec-

tion from AngII-induced end organ damage in a double

transgenic rat model, illustrating the potential of renin

inhibitors to combat cardiovascular disease.⁹Since the

initial disclosure of aliskerin, work around similar tem-

plates has been reported.¹⁰In 1999, the ﬁrst non-pepti-

domimetic renin inhibitors, exempliﬁed by Ro-X1,

were disclosed by researchers at Roche.¹¹The trans,-

trans-3,5-dialkoxy-4-aryl-piperidine scaﬀold of Ro-X1

binds in the renin active site in an induced-ﬁt ﬂap-open

conformation with the 3-(2-methoxybenzyloxy)-propan-

oxy sidechain binding in a large lipophilic pocket that is

revealed upon the conformational change in the ﬂap re-

gion of the protein. Ro-X1 lowered BP in marmoset

monkeys, and prevented cardiac hypertrophy and albu-

minuria in double transgenic rats that express both hu-

man angiotensinogen and renin proteins.^11dAlthough

a non-peptidomimetic scaﬀold, Ro-X1 exhibited poor

oral bioavailability (6% in dog) due to rapid ﬁrst pass

metabolism of the highly lipophilic compound by CY-

P3A4.^11eThe trans,trans-3,5-dialkoxy-4-aryl-piperidine

MeO

Tekturna^®(Aliskiren)

IC₅₀= 0.6 nM

MW = 552

Rotatable bonds = 21

clogP = 3.51

%F = 2% (Rat), 33% (Dog), 3% (Human)

H

N

MeO

O

OH

MeO

O

OMe

Ro-X1

IC₅₀= 0.07 nM

MW = 646

Rotatable bonds = 18

clogP = 6.01

%F = 6% (dog)

H

N

H

N

H

N

O

Cl

O

Cl

1

IC₅₀= 0.3 nM

MW = 649

Rotatable bonds = 13

clogP = 8.35

Figure 2. Large molecule renin inhibitors.

scaﬀold of Ro-X1 has served as the inspiration for the

design of similar templates,¹²including compound 1

and previous reports from these laboratories on the de-

sign of a 6-alkoxymethyl-1-aryl-2-ketopiperazine scaf-

fold (2, Fig. 3).¹³Although compounds based on the

4-aryl-piperidine scaﬀold have recently entered human

clinical trials, these scaﬀolds frequently possess high

molecular weights and clogP values and large number

of rotatable bonds, reducing the likelihood of attaining

good drug-like properties. Due to the lipophilic nature

of the large renin active site, the design of small molecule

renin inhibitors with good drug-like properties has been

5914

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

the S3^spsubsite sidechain led to a >5-fold improvement

NH₂

O

in potency, the target potency range (<20 nM) could not

be achieved solely through manipulation of the S3^spsub-

site sidechain. In addition, the introduction of an ester

group was viewed as a potential metabolic liability, as

the corresponding acids were inactive against renin.¹⁴

Consequently, we sought to improve potency through

modiﬁcations at other sites of the template. Our SAR

around the early HTS lead 3 indicated that the renin

inhibition activity was driven largely by substituents

around the benzylamine ring.¹⁴Crystal structures of 3

bound in the ﬂap-closed conformation of renin indicated

the 3,5-diﬂuorobenzylamine ring occupies the S4 pocket

and partly extends into solvent. Overlap of the crystal

structures of 3 and 4 indicated that addition of substit-

uents at the tetrahydroquinoline C-3 position might in-

crease potency through additional van der Waals

contacts in the S4 pocket (Fig. 4). As 3-substituted tetra-

hydroquinolines are synthetically challenging targets

and less amenable to rapid analoging, we envisioned

that replacing the C-4 carbon atom with an oxygen

atom would allow for a modular synthesis using readily

available a-hydroxyacetate derivatives to introduce the

desired substituent (Fig. 5). Our previously reported

SAR in the ketopiperazine series (i.e., compound 2) indi-

cated that 1,4-benzoxazin-3-ones were readily accepted

in the S3 pocket.^13dAccordingly, we targeted a series

of 2-substituted-1,4-benzoxazines and 1,4-benzoxazin-

3-ones with the goal of optimizing renin inhibitory

activity.

N

H

N

H₂N

O

N

SBDD

O

N

+

HN

O

OMe

F

2

IC₅₀= 7 nM

3

IC₅₀= 4000 nM

4

O

IC₅₀= 650 nM

MW = 341

Rotatable bonds = 6

clogP = 3.35

H₂N

N

C_s= 59.3 μg/mL

Caco2 = 26×10^-6cm/s

NH₂

CYP3A4 (BFC) IC₅₀= 2.8 uM

Figure 3. Design of the novel small molecule renin inhibitor 4.

perceived as diﬃcult to attain, and consequently, there is

a clear need for the discovery of novel renin inhibitor

templates.

We have recently reported the structure-based design of

a novel 7-(2,4-diamino-6-ethyl-pyrimidinyl)-tetrahydro-

quinoline-based inhibitor of renin, 4 (Fig. 3).¹⁴This

compound was designed based on the overlap observed

in the crystal structures of the 6-alkoxymethyl-1-aryl-2-

ketopiperazine 2 and 5-aryl-2,4-diamino-6-ethyl-pyrimi-

dine 3 bound within the active site of renin. Compound

4 exhibited submicromolar renin inhibitory activity with

a molecular weight of 341 and 6 rotatable bonds, as well

as moderate lipophilicity, good solubility, and cellular

permeability. In addition, 4 exhibited only modest inhi-

bition of CYP3A4 compared to compounds in our pre-

viously reported ketopiperazine series,¹³and was

therefore viewed as a promising lead for further struc-

ture-activity studies. The 2-aminoheterocycle motif ap-

pears to be a new and potentially general template for

binding to the catalytic groups of aspartic proteases,

as 2-aminopyridines and 2-aminoquinolines have re-

cently been reported to bind in a similar manner to

the catalytic aspartate residues in b-secretase.¹⁵In this

paper, we report our structure-activity studies on the

7-(2,4-diamino-6-ethyl-pyrimidinyl)-tetrahydroquinoline

template to optimize the renin inhibitory activity and

pharmacokinetic properties.

3. Chemistry

The unsubstituted 1,4-benzoxazine and 1,4-benzoxazin-

3-one analogs 5 and 6 were synthesized as shown in

Scheme 1. Alkylation of 4-bromo-2-nitrophenol 48 with

ethyl 2-bromoacetate and reduction of the nitro group

with Fe/acetic acid followed by in situ cyclization

yielded 7-bromo-1,4-benzoxazin-3-one 49. The 3-meth-

oxypropyl sidechain was introduced by deprotonation

of the amide with NaH in DMF, followed by addition

2. Design of 6-(2,4-diaminopyrimidinyl)-1,4-benzoxazin-

3-ones

The initial goal of our studies was to improve the po-

tency of 4 to low nanomolar levels. The potency of 4

was driven largely by the extension of the 3-methoxy-

propyl group into the S3^spsubpocket, as analogs that

did not access the S3^spsubsite were >10-fold less ac-

tive.¹⁴While introduction of an ester functionality in

˚

Figure 4. Overlap of the crystal structures of 3 (yellow stick, 1.9 A

˚

resolution, PDB code: 2G24) and 4 (purple stick, 2.2 A resolution,

PDB code: 2G21) in the renin active site (ﬂap-closed conformation).

The protein Connolly surface is colored by hydrophobicity. Brown,

hydrophobic; blue, hydrophilic.

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

5915

55, 56, and 58. The racemic 2-methyl-1,4-benzoxazin-

3-one 71 was prepared by NaH deprotonation of 50

and alkylation of the resulting enolate with MeI. The

2-methoxypropyl sidechain was introduced by deproto-

nation of the amide with NaH and treatment with 2-bro-

mo-1-methoxypropane in the presence of 15-crown-5 or

alkylation with 2-bromo-1-methoxypropane in the pres-

ence of K₂CO₃. While the chirality of 63 and 64 was pre-

served under the NaH conditions to give the

corresponding tertiary amides 72 and 73 with no loss

of enantiomeric purity, the more acidic methine proton

in 66 was easily deprotonated under these conditions to

give 75 as a racemic mixture. Intermediates 81–89 were

converted to the desired analogs 10–18 via the

previously described two-step Miyaura borylation and

Suzuki coupling method. The racemic 2-methyl-1,4-ben-

zoxazine analog 7 and (2R)-8 were prepared by BH₃–

SMe₂reduction of the 1,4-benzoxazin-3-ones 10 and

11, respectively. The corresponding (2S)-9 analog was

prepared by BH₃–SMe₂reduction of intermediate 73,

followed by boronate formation and Suzuki coupling.

NH₂

N

NH₂

N

NH₂

N

H₂N

+

N

2

O

HN

N

X

F

O

3

O

4

F

IC₅₀= 0.66 uM

3

X = H,H or O

IC₅₀= 4.0 uM

Figure 5. Design of the 1,4-benzoxazine and 1,4-benzoxazin-3-one

scaﬀold.

of 1-bromo-3-methoxypropane to give 50. Bromide 50

was converted to the boronate 52 by a Pd-catalyzed

Miyaura borylation with bis(pinacolato)diboron. Suzu-

ki coupling with 5-bromo-2,4-diamino-6-ethyl-pyrimi-

dine 54¹⁷in a 1,4-dioxane/H₂O solvent mixture yielded

the desired 1,4-benzoxazin-3-one analog 6. Best yields

in the Suzuki coupling reaction were obtained using

CsOH as base in the presence of 300 mol% LiCl. Reduc-

tion of the amide bond in intermediate 50 with BH₃–

SMe₂provided the corresponding 1,4-benzoxazine 51,

which was converted to the 1,4-benzoxazine analog 5

in an analogous two-step fashion. Our synthetic

approach toward the desired 2-substituted-1,4-benzoxa-

zin-3-ones relied on a Mitsunobu coupling with 2-hy-

droxy carboxylic ester derivatives and a deprotonation

of intermediate 50 and alkylation of the resulting eno-

late with the appropriate electrophile to introduce the

desired substituent (Scheme 2). Mitsunobu coupling of

4-bromo-2-nitrophenol 48 and 2-hydroxy carboxylic es-

ters 55–62, followed by reduction of the nitro group and

in-situ cyclization, yielded the corresponding 2-substi-

tuted-1,4-benzoxazin-3-ones 63–70. No loss of enantio-

meric purity was observed in the Mitsunobu coupling

and reduction/cyclization of the chiral 2-hydroxy esters

The 2,2-dimethyl analogs 19 and 20 were prepared as

shown in Scheme 3. Alkylation of phenol 48 with ethyl

2-bromoisobutyrate under concentrated conditions and

nitro reduction/cyclization provided the 2,2-dimethyl-

1,4-benzoxazin-3-one 93 in excellent yield. Intermediate

93 was transformed into analog 19 by the standard pro-

tocol of alkylation with 1-bromo-3-methoxypropane,

boronate formation, and Suzuki coupling. As part of

our strategy to improve the metabolic stability, we were

also interested in replacing the 3-methoxypropyl side-

chain with an N-(2-ethyl)acetamide sidechain. This was

installed by alkylation of intermediate 93 with bromo-

acetonitrile to aﬀord nitrile 95, which was then hydroge-

nated over Raney nickel in the presence of acetic

anhydride to aﬀord the acetamide 96. Miyaura boryla-

tion and Suzuki coupling yielded analog 20. Other more

highly substituted 2,2-disubstituted-1,4-benzoxazin-3-

one ring intermediates 95–111 were prepared through

a convergent nucleophilic aromatic substitution/reduc-

tion method using the corresponding a-hydroxyacetate

O

H

Br

NO₂

OH

Br

N

O

Br

N

O

X

CO₂Et

i, ii

Br

B

N

O

X

iii

v

O

48

49

50 X = O

51 X = H, H

52 X = O

53 X = H, H

iv

O

X

H₂N

N

H₂N

N

NH₂

Br

54

N

O

NH₂

vi

6 X = O

5 X = H, H

Scheme 1. Reagents and conditions: (i) K₂CO₃, acetone, reﬂux; (ii) Fe, AcOH, 50 ꢂC; (iii) 1-bromo-3-methoxy propane, NaH, 15-crown-5, DMF, rt;

(iv) BH₃–SMe₂, THF, 50 ꢂC; (v) Bis(pinacolato)diboron, Pd(dppf)₂Cl₂, KOAc, 1,4-dioxane, 90 ꢂC; (vi) Pd(PPh₃)₄, CsOH, LiCl, 1,4-dioxane/H₂O,

reﬂux.

5916

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

O

CO₂Me

R

O

B

H

Br

NO₂

OH

Br

N

O

R

Br

N

O

R

i, ii

O

R

vii

N

O

HO

iii

O

or iv

O

48

R

(

S

R

)-CH₃

55

56

(

(R

rac)-CH₃

)-CH₃

(

R

S

)-CH₃

65 CH₂CH₂CH₃

66 ( )-phenyl

(

rac)-CH₃

)-CH₃

81

82

63

64 (

71

72

(

R

57 CH₂CH₂CH₃

58 )-phenyl

73 (

S

83 (

S

(

R

S

74 CH₂CH₂CH₃

75 phenyl

76 3,4-difluorophenyl

77 4-fluorophenyl

78 3,5-difluorophenyl

79 2,3-difluorophenyl

84 CH₂CH₂CH₃

85 phenyl

86 3,4-difluorophenyl

87 4-fluorophenyl

88 3,5-difluorophenyl

89 2,3-difluorophenyl

59 3,4-difluorophenyl

60 4-fluorophenyl

61 3,5-difluorophenyl

62 2,3-difluorophenyl

67 3,4-difluorophenyl

68 4-fluorophenyl

69 3,5-difluorophenyl

70 2,3-difluorophenyl

vi

O

Br

N

Br

N

O

v

71

54, viii

O

80

50

(1) vii

(2) 54, viii

O

H₂N

N

NH₂

H₂N

N

NH₂

vi

N

O

R

O

R

(

rac)

7

8

9

(R)

(S)

(

rac)-CH₃

)-CH₃

10

11

(R

12 (S)-CH₃

13 CH₂CH₂CH₃

14 phenyl

15 3,4-difluorophenyl

16 4-fluorophenyl

17 3,5-difluorophenyl

18 2,3-difluorophenyl

Scheme 2. Reagents and conditions: (i) PS–PPh₃, DIAD, CH₂Cl₂, rt; (ii) Fe, AcOH, 50 ꢂC; (iii) NaH, 15-crown-5, DMF, rt, then 1-bromo-3-

methoxy propane; (iv) 1-bromo-3-methoxy propane, K₂CO₃, CH₃CN, reﬂux; (v) NaH, DMF, 0 ꢂC; then MeI; (vi) BH₃-SMe₂, THF, 50 ꢂC; (vii)

bis(pinacolato)diboron, Pd(dppf)₂Cl₂, KOAc, 1,4-dioxane, 90 ꢂC; (viii) Pd(PPh₃)₄, CsOH, LiCl, 1,4-dioxane/H₂O, reﬂux.

O

H₂N

N

NH₂

H

N

Br

O

Br

N

O

N

O

Br

NO₂

OH

Br

CO₂Et

54 iv,

iii

v

O

vi

i, ii

19

94

93

48

O

HN

CN

H₂N

N

NH₂

vii

54 iv,

Br

N

O

Br

N

O

N

O

v

95

96

20

Scheme 3. Reagents and conditions: (i) K₂CO₃, CH₃CN, reﬂux; (ii) Fe, AcOH, 50 ꢂC; (iii) 1-bromo-3-methoxy propane, NaH, 15-crown-5, DMF, rt;

(iv) bis(pinacolato)diboron, Pd(dppf)₂Cl₂, KOAc, 1,4-dioxane, 90 ꢂC; (v) Pd(PPh₃)₄, CsOH, LiCl, 1,4-dioxane/H₂O, reﬂux; (vi) K₂CO₃,

bromoacetonitrile, CH₃CN, reﬂux; (vii) H₂, Raney Ni, Ac₂O, THF.

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

5917

R¹R²

NO₂

Br

CO₂Et

HO

Br

NO₂

F

ii

CO₂Et

R²

i

O

R¹

R¹R²

97

98 CH₃CF₃

99 CH₃Et

100 Et

Et

101

-CH₂CH₂-

102 CH₃3,5-difluorophenyl

S)-103 CH₃phenyl

(

R

)-104 CH₃phenyl

O

H₂N

N

NH₂

H

N

O

Br

N

O

Br

O

Br

OMe

54 iv,

v

R²

iii

O

R¹

R¹R²

112 CH₃Et

113

114

R¹R²

105 CH₃CF₃

106 CH₃Et

22 CH₃Et

23 Et Et

Et

107 Et

Et

CH₃3,5-difluorophenyl

25 CH₃3,5-difluorophenyl

108

-CH₂CH₂-

109 CH₃3,5-difluorophenyl

S)-110 CH₃phenyl

(

R

)-111 CH₃phenyl

O

iii

Br

CN

HN

CN

H₂N

N

NH₂

vi

54 iv,

O

Br

O

N

O

N

O

N

O

v

R²

R¹

R¹R²

21 CH₃CF₃

-CH₂CH₂-

26 CH₃3,5-difluorophenyl

115 CH₃CF₃

116

120 CH₃CF₃

121

24

-CH₂CH₂-

117 CH₃3,5-difluorophenyl

)-118 CH₃phenyl

122 CH₃3,5-difluorophenyl

(

S

(

S

)-123 CH₃phenyl

)-124 CH₃phenyl

(

S

)-33 CH₃phenyl

(R)-34 CH₃phenyl

(

R

)-119 CH₃phenyl

(

R

Scheme 4. Reagents and conditions: (i) NaH, 15-crown-5-THF, 0 ꢂC; (ii) Fe, AcOH, 50 ꢂC; (iii) K₂CO₃, CH₃CN, reﬂux; (iv) bis(pinacolato)diboron,

Pd(dppf)₂Cl₂, KOAc, 1,4-dioxane, 90 ꢂC; (v) Pd(PPh₃)₄, CsOH, LiCl, 1,4-dioxane/H₂O, reﬂux; (vi) H₂, Raney Ni, Ac₂O, THF.

esters (Scheme 4). Our standard alkylation, boronate

formation, and Suzuki coupling route aﬀorded the de-

sired analogs 21–26, 33, and 34. Analogs 27 and 28 were

prepared by NaH deprotonation of the mono-substi-

tuted intermediates 78 and 76, and alkylation with

iodomethane or iodoethane, respectively (Scheme 5).

Analogs 29–32 with the N-ethylacetamide sidechain

were synthesized from the corresponding intermediates

127–130, which were prepared as described in Scheme

2, via a similar deprotonation/alkylation sequence utiliz-

ing iodomethane.

substituent at the C2 position of the 1,4-benzoxazine

ring led to a 2-fold improvement in the renin inhibition

activity (7, IC₅₀= 0.235 lM) relative to the lead

compound 4. Both of the 2-methyl-1,4-benzoxazine

enantiomers (2R)-8 and (2S)-9 showed equivalent renin

inhibitory activity and were within the 2-fold experimen-

tal error range of the racemic analog 7, indicating that

the 2-methyl-1,4-benzoxazine template exhibited no

strong enantiomeric preference. The corresponding

racemic 2-methyl-1,4-benzoxazin-3-one analog 10

showed a modest decrease in renin inhibition activity

(IC₅₀= 0.520 lM). However, the 2-methyl-1,4-benzoxa-

zin-3-one enantiomers possessed a marked diﬀerence in

4. Results and discussion

activity. The (R)-enantiomer 11 exhibited an IC₅₀=

0.125 lM, while the (S)-enantiomer 12 was approxi-

mately 10-fold less potent (IC₅₀= 1.04 lM). We hypoth-

esized that the carbonyl group imposed additional

conformational restraints on the 2-methyl-1,4-benzoxa-

zin-3-one ring structure and regidiﬁed the 3D conforma-

tion of 11, whereas the corresponding 2-methyl-1,4-

benzoxazine analogs 8 and 9 are more conformationally

ﬂexible and can adjust to ﬁt the renin active site. The

1,4-benzoxazin-3-ones 11 and 12 also showed a substan-

Surprisingly, the unsubstituted 1,4-benzoxazine ring and

1,4-benzoxazin-3-one ring analogs 5 and 6, respectively,

were 4- to 6-fold less potent than the tetrahydroquino-

line ring analog 4 (Table 1). This contrasted with our

previously reported SAR work in the 6-alkoxymethyl-

1-aryl-2-ketopiperazine scaﬀold that indicated a 1,4-

benzoxazin-3-one ring was readily accepted in the S3

pocket.^13dHowever, introduction of

a

methyl

5918

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

O

H₂N

N

NH₂

N

O

Br

N

O

Br

N

O

i

54 ii,

iii

R²

R¹

R²

R¹

R²

R¹

R²

R¹

78

76

H

3,5-difluorophenyl

3,4-difluorophenyl

125 CH₂CH₃3,5-difluorophenyl

27 CH₂CH₃3,5-difluorophenyl

126 CH₃

3,4-difluorophenyl

28 CH₃

3,4-difluorophenyl

O

HN

H₂N

N

NH₂

N

O

Br

N

O

Br

N

O

54 ii,

i

iii

R³

O

R³

131 3-trifluoromethylphenyl

132 4-chlorophenyl

127 3-trifluoromethylphenyl

128 4-chlorophenyl

29 3-trifluoromethylphenyl

30 4-chlorophenyl

129 3-chlorophenyl

133 3-chlorophenyl

31 3-chlorophenyl

130 2,5-difluorophenyl

134 2,5-difluorophenyl

32 2,5-difluorophenyl

Scheme 5. Reagents and conditions: (i) iodomethane or ethyl iodide, NaH, 15-crown-5, DMF, rt; (ii) bis(pinacolato)diboron, Pd(dppf)₂Cl₂, KOAc,

1,4-dioxane, 90 ꢂC; (iii) Pd(PPh₃)₄, CsOH, LiCl, 1,4-dioxane/H₂O, reﬂux.

tial increase in metabolic stability, as assessed by the

half-life in human liver microsomes (HLM), over the

corresponding 1,4-benzoxazines 8 and 9, presumably

due to the ꢀ1 log unit decrease in clogP. In general,

we found that the half-lives of compounds in human li-

ver microsomes (HLM) and rat liver microsomes

(RLM) exhibited excellent correlation (Table 3). There-

fore, the HLM data were used to guide SAR decisions

prior to in vivo PK experiments. Due to the increase

in potency and metabolic stability, we decided to focus

upon the 1,4-benzoxazin-3-one scaﬀold in all further

analogs. A further increase in the length of the C2-alkyl

substituent to a 3-carbon n-propyl group resulted in a

modest increase in renin inhibitory activity (13,

IC₅₀= 0.43 lM) compared to the racemic 2-methyl ana-

log 10. On the other hand, a C2-phenyl substituent re-

sulted in a 2-fold increase in renin inhibition activity

(14, IC₅₀= 0.220 lM) and substantial decrease in

HLM stability. Recognizing that unsubstituted phenyl

rings are often metabolic liabilities, we examined the ef-

fect of introducing one or more electron-withdrawing

ﬂuorine substituents on the C2 phenyl ring on renin

inhibition activity and HLM stability. The 3,4-diﬂuoro

and 4-ﬂuorophenyl analogs 15 and 16 exhibited similar

renin inhibition potencies and HLM stability as the

unsubstituted phenyl analog 14. However, the 3,5-

diﬂuorophenyl and 2,3-diﬂuorophenyl analogs 17 and

18 were 2- to 3-fold more potent (IC₅₀= 0.095

and 0.072 lM, respectively), but still exhibited

poor HLM stability. Analogs 17 and 18 validated our

original hypothesis that an increase in renin inhibition

activity could be achieved through manipulation of

substituents at the C2 position of a 1,4-benzoxazin-3-

one scaﬀold.

methoxypropyl sidechain extending into the S3^spsub-

site, the 3,5-diﬂuorophenyl ring occupying the S4 pock-

et, and the ﬂuorine atoms extending toward solvent

(Fig. 6). The carbonyl of the 1,4-benzoxazin-3-one ring

makes no hydrogen bond contacts directly with the pro-

tein, but does make a hydrogen bond with a crystallo-

graphic water molecule. We believe that the carbonyl

group serves to rigidify the 1,4-benzoxazin-3-one ring

scaﬀold to properly orient the 3,5-diﬂuorophenyl substi-

tuent. Interestingly, the (S)-enantiomer of 17 was ob-

served to bind preferentially in the renin active site as

predicted by the initial design envisioned in Fig. 4. This

result contrasts with the observed 10-fold diﬀerence in

activity between the (2R)-methyl analog 11 and (2S)-

methyl analog 12.

Although a phenyl substituent at the 2-position of the

1,4-benzoxazin-3-one improved renin inhibition activity,

analogs 14–18 exhibited decreased in vitro metabolic

stability, presumably through an increase in lipophilicity

as measured by clogP (Table 1). The addition of elec-

tron-withdrawing ﬂuorine substituents around the

C2-phenyl ring resulted in no improvement in metabolic

stability, indicating that metabolism was occurring else-

where in the molecule. Accordingly, the site of metabolic

liability of 6, a compound which showed a medium-risk

of metabolism in the in vitro HLM screen, was deter-

mined using LC/MS/MS methods to identify the metab-

olites formed in the microsomal incubations. The two

main observed metabolites were identiﬁed as the oxida-

tive hydroxylation at C2 of the 1,4-benzoxazin-3-one

ring to give the C2-hydroxyl metabolite 91 and demeth-

ylation of the 3-methoxypropyl sidechain to aﬀord

metabolite 92 (Fig. 7). We believe that the 2-methyl sub-

stituent in analogs 10–12 resulted in improved metabolic

stability by steric hindrance of the C2 position. A similar

metabolite identiﬁcation analysis of 10 supported this

X-ray crystallography of the 17/renin co-crystal complex

revealed that 17 bound in the renin active site with the 3-

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

Table 1. SAR of 2-monosubstituted-1,4-benzoxazine and 1,4-benzoxazin-3-ones

5919

O

H₂N

N

O

X

R

NH₂

ID

R^a

H

X

RENIN IC₅₀(lM)^b

HLM T_1/2(min)

clogP

5

H,H

2.70

26

2.77

1.70

3.29

2.22

3.28

6

7

H

O

3.90

32

30

CH₃

H,H

O

0.235

0.325

0.310

0.520

0.125

1.04

8

(R)-CH₃

(S)-CH₃

CH₃

26

9

32

10

11

12

13

>40

60

(R)-CH₃

(S)-CH₃

CH₂CH₂CH₃

O

>40

31

O

0.430

14

15

16

O

0.220

0.206

0.325

12

10

16

3.5

F

3.72

3.62

F

17

18

O

0.095

0.072

9

3.79

3.72

F

21

^aUnless otherwise denoted, all analogs are achiral or racemic mixtures.

^bIC₅₀values obtained in duplicate using a ﬂuorescent tGFP assay.^13a

hypothesis as sidechain demethylation was the major

metabolite observed with only a minor amount of the

C2 hydroxylation metabolite. While the sterically larger

2-phenyl substituent in analogs 14–18 provides addi-

tional steric bulk around the C2 position, these analogs

show reduced metabolic stability presumably because

the phenyl ring results in a substantial increase in lipo-

philicity and increased acidity of the C2 methine hydro-

gen atom, making this position more susceptible to

oxidation through proton abstraction.

mer of the 2-methyl analogs 11 and 12 exhibited greater

renin inhibition activity (compare 11 and 12, Table 1).

To explain this dichotomy, we hypothesized that the

methyl substituent of (R)-11 and the 3,5-diﬂuorophenyl

substituent of (S)-17 occupied diﬀerent space within the

renin active site. Closer examination of the crystal struc-

ture of 17 indicated a small hydrophobic indentation in

the S3 pocket formed by the sidechains of the amino

acid residues Phe117, Ala115, and Leu114 that might

accept a sterically small hydrophobic substituent.

Introduction of a second substituent at C2 of the 1,4-

benzoxazin-3-one ring would block the oxidative metab-

olism, as well as potentially improving renin potency by

additional van der Waals contacts with the renin pro-

tein.¹⁶Accordingly, we targeted a series of 2,2-disubsti-

tuted-1,4-benzoxazin-3-ones, such as 25 (Fig. 8), for

synthesis.

Fortunately, the crystal structure of 17 bound in the ac-

tive site of renin (Fig. 6) provided an important clue to

removing the site of oxidative hydroxylation at C2 of the

1,4-benzoxazin-3-one ring. The S-enantiomer of 17 was

observed to bind preferentially in the renin active site.

However, earlier IC₅₀data indicated that the R-enantio-

5920

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

compared to (R)-2-methyl-1,4-benzoxazin-3-one 11

(IC₅₀= 0.125 lM, Table 1), conﬁrming our hypothesis

that the pro-(S) C2 methyl substituent would improve

potency through additional van der Waals contacts with

the protein surface. As previously observed, conversion

of the 3-methoxypropyl S3^spsidechain to a N-(2-ethyl)-

acetamide resulted in a further >4-fold improvement in

renin inhibition activity (20, IC₅₀= 0.020 lM).^11d,13

Analog 20 represented the ﬁrst compound to meet our

potency range criteria. Both 19 and 20 exhibited good

HLM stability and solubility, and no appreciable inhibi-

tion of cytochrome P450 isozymes, although the N-(2-

ethyl)-acetamide sidechain of 20 resulted in a substantial

reduction in cellular permeability. While the 2,2-di-

methyl-1,4-benzoxazin-3-one analogs 19 and 20 exhib-

ited good HLM stability, this may be due to their low

lipophilicity as measured by clogP, as 19 still possessed

a site of potential metabolism in the 3-methoxypropyl

sidechain. The 2,2-diethyl-1,4-benzoxazin-3-one analog

23 illustrated that HLM stability decreased in more

lipophilic analogs with a 3-methoxypropyl sidechain.

Adding a methyl at the 2-position of the 2-(3,5-diﬂuoro-

phenyl)-1,4-benzoxazin-3-one template resulted in ana-

log 25, which possessed single digit nanomolar renin

inhibition activity (IC₅₀= 0.007 lM) as a racemic mix-

ture, a >13-fold improvement in potency compared to

the corresponding des-methyl analog 17. Analog 25

exhibited a decreased HLM stability, presumably be-

cause the 3-methoxypropyl sidechain still presented a

potential site of metabolism within a lipophilic molecule

(clogP = 4.31). Conversion to the N-(2-ethyl)-acetamide

sidechain resulted in a further improvement in renin

inhibition activity (26, IC₅₀= 0.001 lM) and excellent

HLM stability through a reduction in clogP (3.41)

and by removing a potential site of metabolism. The

2-methyl substituent was optimal, as the 2-ethyl analog

27 exhibited a 25-fold reduction in renin inhibition

activity. X-ray crystallography of the 25/renin complex

(Fig. 9) shows the 2-(3,5-diﬂuorophenyl)-2-methyl-1,4-

benzoxazin-3-one ring snuggly ﬁlling the S3 and S4

pockets, with the methyl substituent making van der

Waals contacts with the Phe117, Ala115, and Leu114

residues as designed. In addition, the 3,5-diﬂuorophenyl

group is moved slightly to make additional van der

Waals contacts with the Pro111 residue. The additional

hydrophobic contacts with the protein surface formed

upon introduction of the 2-methyl substituent are re-

ﬂected in the greater enthalpic contributions to the over-

all energy of binding observed in the previously reported

isothermal calorimetry experiments.¹⁸

˚

Figure 6. Crystal structure of the 17/renin complex (2.5 A resolution,

PDB code: 2G1S). The protein Connolly surface is colored by

hydrophobicity. Brown, hydrophobic; blue, hydrophilic.

NH₂

N

H₂N

NH₂

N

C2 oxidative

hydroxylation

N

O

H₂N

OH

91

HLM

incubation

N

NH₂

N

O

N

6

Sidechain

demethylation

H₂N

N

O

OH

92

Figure 7. Metabolic analysis of 6.

NH₂

N

NH₂

N

NH₂

N

H₂N

+

N

O

F

O

Although analogs 25–27 demonstrated that addition of

a methyl substituent at the C2 position of the 1,4-benz-

oxazin-3-one resulted in improved renin inhibition activ-

ity and blocked a potential site of metabolism, more

worrisome was the observation that these analogs also

exhibited an increased inhibition of CYP3A4. We

hypothesized that variation of the substituents about

the C2 aryl ring might result in decreased CYP3A4 inhi-

bition. Accordingly, we prepared analogs 29–32 with the

N-(2-ethyl)-acetamide sidechain to maintain good HLM

stability. Analogs with a clogP < 4 exhibited good

HLM stability, while a clogP > 4 resulted in poor

11

F

25

(S)-17

Figure 8. Design of the 2,2-disubstituted 1,4-benzoxazin-3-one

scaﬀold.

We were quite pleased to ﬁnd that the 2,2-dimethyl-1,4-

benzoxazin-3-one 19 (IC₅₀= 0.090 lM, Table 2) re-

sulted in an improvement in renin inhibition activity

Table 2. SAR of 2,2-disubstituted-1,4-benzoxazin-3-ones

H₂N

N

R₃

N

O

R²

NH₂

O

R¹

In vitro ADME data

Cytochrome P450 inhibition data

ID

C-2 Conﬁg.

—

R¹

R²

R³

RENIN IC₅₀

(lM)^a

clogP

HLM T_1/2

(min)

Solubility^b

Caco-2^cAB Perm

19

CYP3A4 IC₅₀

(lM)

CYP2D6 IC₅₀

(lM)

CYP2C9 IC₅₀

(lM)

(lg/mL)

19

CH₃

0.090

2.74

>40

34

>30

O

20

21

22

23

24

25

—

rac

—

–

CH₃

0.020

1.86

2.62

3.27

3.80

1.13

4.31

>40

11

200

54

1.7

2.4

14

>30

>22

>30

3.0

>30

30

N

H

O

CH₃

CF₃

0.017

0.052

0.245

0.100

0.007

>120

>40

16

>30

8.2

12

N

H

CH₃

CH₂CH₃

–CH₂–

CH₃

O

16

32

15

–CH₂–

>40

12

24

5.4

27

>30

1.14

>30

6.3

>30

10.5

N

H

F

rac

87

O

F

26

27

rac

CH₃

0.001

0.175

3.41

4.84

>60

18

133

1.4

0.86

0.46

>30

3.3

>30

4.9

N

H

F

CH₂CH₃

O

2.6

17

F

(continued on next page)

Table 2 (continued)

In vitro ADME data

Cytochrome P450 inhibition data

ID

C-2 Conﬁg.

R¹

R²

R³

RENIN IC₅₀

(lM)^a

clogP

HLM T_1/2

(min)

Solubility^b

Caco-2^cAB Perm

CYP3A4 IC₅₀

(lM)

CYP2D6 IC₅₀

(lM)

CYP2C9 IC₅₀

(lM)

(lg/mL)

F

28

rac

CH₃

0.066

0.060

>0.040

0.007

0.0008

0.86

4.24

10

3

13

17

0.92

5.7

8.3

O

F₃C

29

30

31

32

33

34

rac

S

CH₃

4.00

3.84

3.41

3.12

12

8.8

0.12

>30

>22

N

H

O

>60

>40

>60

34

0.59

19

12

N

H

Cl

133

2.3

119

40

0.0

86% at 3 lM

7% at 3 lM

11% at 3 lM

N

H

F

—

1.90

>30

N

H

F

2.1

1.9

>30

N

H

R

0.53

>30

N

H

Fluorometric CYP substrates: BFC (CYP3A4), AMMC (CYP2D6), MFC (CYP2C9).

^aIC₅₀values determined in duplicate using a ﬂuoresence tGFP assay.^13a

^bAqueous solubility at pH 6.5.

^cPermeability expressed as ·10^ꢁ6cm/s.

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

5923

ﬁrst-pass metabolism and high in vivo clearance

(48 mL/min/kg). Analog 26 was metabolically stable

against HLM and RLM, but showed only slightly im-

proved oral bioavailability (F = 8%) due to poor absorp-

tion across the gut wall that was reﬂected in the low

Caco-2 cellular permeability caused by the polarity of

the N-(2-ethyl)-acetamide sidechain. Analog (S)-33,

which also contained the polar N-(2-ethyl)-acetamide

sidechain, also exhibited a similar proﬁle of low rat oral

bioavailability (F = 10%) likely due to poor cellular per-

meability. In contrast, the metabolite of the 3-methoxy-

propyl sidechain in 25, the 3-hydroxypropyl 35,

exhibited an excellent proﬁle of metabolic stability and

cellular permeability, and possessed excellent rat oral

bioavailability (F = 44%). Although 35 showed reduced

renin inhibition activity, we were encouraged that good

rat oral bioavailability could be obtained in the 6-(2,4-

diaminopyrimidinyl)-2-aryl-2-methyl-1,4-benzoxazin-3-

one template with a metabolically stable and permeable

S3^spsidechain. Consequently, we conducted further

SAR to identify an S3^spsidechain that possessed the best

proﬁle of renin inhibition activity, metabolic stability,

cellular permeability, and rat oral bioavailability.

˚

Figure 9. X-ray structure of the 25/renin complex (2.3 A resolution,

PDB core: 2I4Q), Protein surface of active site is colored by

hydrophobicity (Brown, hydrophobic; blue, hydrophilic).

metabolic stability (29, HLM T_1/2= 3 min). The 3-

chlorophenyl analog 31 and 2,5-diﬂuorophenyl analog

32 exhibited the best renin inhibition activity and good

metabolic stability, however these analogs still inhibited

CYP3A4. All of the analogs 25–32 were prepared as

racemic mixtures due to ease and speed of preparation.

We were able to prepare the discrete enantiomers of the

unsubstituted 2-phenyl ring, analogs 33 and 34. The (S)-

enantiomer 33 exhibited the most potent renin inhibition

activity in this series (IC₅₀= 0.0008 lM), while the (R)-

enantiomer 34 was approximately 1000-fold less potent.

The renin inhibition activity of (S)-33 is comparable to

the activity of aliskerin, with a MW = 461 compared

to MW = 551. Surprisingly, the chiral (S)-2-methyl-2-

phenyl analog (S)-33 exhibited a clean CYP inhibition

proﬁle in the ﬂuorometric assays, with no appreciable

inhibition of CYP3A4, 2D6, or 2C9. On the other hand,

the (R)-34 enantiomer exhibited signiﬁcant inhibition of

CYP3A4, indicating that the CYP3A4 inhibition liabili-

ties noted in the racemic compounds may reside solely in

the less active and undesired R-enantiomer.

We ﬁrst attempted to improve the potency of alcohol 35

by varying the length of the S3^spsidechain. Analog 36

with a 2-hydroxyethyl sidechain resulted in poor renin

inhibition activity, presumably because the 2-hydroxy-

ethyl chain does not penetrate far enough into the

S3^sp. A longer 4-hydroxybutyl sidechain extended fur-

ther in the S3^spand resulted in a >20-fold increase in re-

nin inhibition activity (37, IC₅₀= 0.043 lM), but

decreased HLM and RLM stability and exhibited poor

rat oral bioavailability. Introduction of an amide into

the alcohol sidechain resulted in single digit renin inhibi-

tion activity (38, IC₅₀= 0.003 lM), but reduced HLM

and RLM stability and increased clearance. We then

turned to improving the cellular permeability of the met-

abolically stable amide sidechain. The reverse amide

sidechain analog 39 showed single digit nanomolar renin

inhibition, as well as good HLM stability, moderate

RLM stability, and improved cellular permeability,

but showed no decrease in the in vivo clearance (not

dosed orally), and potent inhibition of CYP3A4. An

ethyl acetamide reverse amide sidechain resulted in re-

duced renin inhibition (40, IC₅₀= 0.20 lM), as did

replacing the N-acetamide with a N-sulfonamide (41,

IC₅₀= 0.63 lM). Within the amide sidechain group of

compounds, the methyl N-(2-ethyl)-carbamate 42 exhib-

Since 2,4-diaminopyrimidines are common templates for

kinase inhibitors, we screened representative compounds

for kinase inhibition activity against 19 kinases selected

from the Dundee kinase panel to give an initial overview

of kinase activity. Compounds 9, 14, and 20 showed no

signiﬁcant inhibition of 18/19 kinases, with only moder-

ate kinase activity at 10 lM concentration observed for

Met kinase (9, 50%; 14, 40%; and 20, 61% kinase activ-

ity).¹⁹Additional proﬁling in the CEREP panel of recep-

tors, enzymes, and kinases revealed no signiﬁcant oﬀ-

target activity (data not shown).

ited

the

best

proﬁle

of

renin

inhibition

(IC₅₀= 0.002 lM), HLM stability, and rat oral bioavail-

ability (F = 34%). Disappointingly, 42 was again a po-

tent inhibitor of CYP3A4 (IC₅₀= 0.67 lM).

We also investigated improving the metabolic stability

of the 3-methoxypropyl sidechain. Replacement of the

methoxy ether with a nitrile group resulted in a 10-fold

reduction in renin inhibition activity, but good HLM

and RLM stability and an improved CYP inhibition

proﬁle, in vivo clearance, and oral bioavailability in

the rat (43, IC₅₀= 0.062 lM, F = 12%). Attempting to

block the demethylation of the methoxy ether, we in-

stalled a 3-triﬂuoromethoxypropyl sidechain, but analog

While we had been successful in designing small mole-

cule renin inhibitors with MW < 500 that met our po-

tency range goal with good ADME and CYP

inhibition properties, our lead compounds 25 and 26

exhibited poor oral bioavailability in the rat (Table 3).

Analog 25 exhibited short HLM and RLM half-lives

and <1% oral bioavailability presumably due to rapid

Table 3. Optimization of PK parameters

H₂N

N

NH₂

R

N

O

F

O

F

In vitro ADME data

Renin IC₅₀HLM T_1/2RLM T_1/2Solubility^bCaco-2

CYP450 inhibition data^a

Rat in vivo PK parameters

ID

R

CYP3A4 IC50 CYP2D6 IC50 Cl (ml/min/kg) Vd_ss(L/kg) T_1/2(h) C_max(ng/mL) F (%)

(lM)

(nM)

(min)

(lg/mL)

AB Perm^c(lM)

25^d

0.007

16

13

87

32

1.14

6.3

48

37

44

32

22

69

44

42

ND

45

4.5

2.1

3.1

3.3

2.3

2.9

2.1

3.1

ND

3.5

1.3

2

23

0.4

8

O

H

26^d

35^d

36^e

37^e

38^d

39^d

40^f

41

0.001

0.89

>60

>40

>60

14

39

>40

50

7

95

1.4

10

0.86

30

220

180

ND

186

ND

60

N

O

28

30

1.6

1.8

1.6

1.2

1.1

1.4

ND

1.6

44

OH

1.08

89

6.3

10.6

1.5

5.1

21

8% at 3 lM

4.79

8% at 3 lM

6.96

>30

ND

7

OH

0.043

0.003

0.006

0.200

0.630

0.002

59

H

N

19

11

26

>60

25

—

59

1.67

ND

14

OH

O

37

133

89

0.16

>30

N

H

N

>60

34

>30

1.89

15.1

>30

O

H

N

O

200

—

1.8

2.1

2.18

ND

280

ND

34

S

O

H

N

42^e

66

0.67

O

43^e

0.062

>60

3.5

—

>30

27.9

28

1.9

1.6

237

12

CN

44^e

45^f

0.182

0.041

31

56

23

16

7.8

59

—

13

0.27

1.06

3.22

20.6

40

41

6.3

2.0

2.2

0.9

200

<5

28

0

CF₃

O

46^e

0.188

0.141

7

9

27

2.8

9

46% at 3 lM

54% at 3 lM

37% at 3 lM

34% at 3 lM

40

27

6

2.3

2.5

110

160

15

12

O

CF₃

rac-47^e

>60

34

5.2

5.1

CF₃

(S)-47^e

0.048

>60

—

1.7^g

0.43^g

26

5.7

3.0

380

74

ND, not dosed.

^aFluorometric CYP substrates: BFC (CYP3A4), AMMC (CYP2D6).

^bAqueous solubility at pH 6.5.

^cPermeability expressed as ·10^ꢁ6cm/s.

^dIV dose = 1 mg/kg, po dose = 5 mg/kg.

^eIV dose = 1 mg/kg, po dose = 10 mg/kg.

^fIV dose = 0.33 mg/kg, po dose = 1.67 mg/kg.

^gDeﬁnitive CYP substrates: midazolam (CYP3A4) and dextromethorphan (CYP2D6).

5926

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

(S)-47 exhibited low micromolar inhibition of CYP3A4

(IC₅₀= 2 lM), but potent inhibition of CYP2D6

(IC₅₀= 0.43 lM). In contrast, the ﬂuorometric assays

that were used to routinely screen for CYP inhibition

activity had predicted that rac-47 would show little inhi-

bition of CYP2D6 (Table 3).

5. Conclusion

We have designed a novel series of small molecule inhib-

itors of renin based on a 6-(2,4-diaminopyrimidinyl)-

1,4-benzoxazin-3-one template, which represents

a

signiﬁcant structural diﬀerence from the previously re-

ported peptidomimetic or large-molecule renin inhibitor

templates. Improved renin inhibition activity was ob-

tained through the addition of substituents at the 2-po-

sition of the 1,4-benzoxazin-3-one ring that provide

additional van der Waals contacts with the protein sur-

face in the S3 and S4 pockets. A 2-methyl-2-aryl substi-

tution pattern resulted in compounds with the most

potent renin inhibition activity and improved metabolic

stability through direct blockade of a site of metabolism.

The S-enantiomer binds preferentially in the renin active

site and exhibits improved CYP inhibition activity rela-

tive to the R-enantiomer, as measured by ﬂuorometric

assays. The 3-methoxypropyl sidechain that binds in

the S3^spsubsite utilized in early SAR studies resulted

in low oral bioavailability due to rapid ﬁrst pass metab-

olism. While the N-(2-ethyl)-acetamide S3^spsubsite side-

chain resulted in a ꢀ10-fold improvement in renin

inhibition and good microsomal metabolic stability, oral

bioavailability was limited by cellular permeability. Im-

proved metabolic stability, cellular permeability, CYP

isozyme inhibition, and rat oral bioavailability could

be obtained through modulation of the S3^spsubsite side-

chain. The best oral bioavailability was obtained with

sidechains that exhibited good metabolic stability, cellu-

lar permeability, and increased volume of distribution.

The in vivo clearance of this series does not directly cor-

relate to the metabolic stability and may be reﬂective of

the removal of the compound from the plasma by non-

metabolic processes such as tissue distribution or pro-

tein binding, as compounds with increased volume of

distribution exhibit improved oral bioavailability. While

this project was terminated prior to identiﬁcation of a

clinical candidate or assessment of in vivo eﬃcacy, we

believe that this work will signiﬁcantly aid in the design

of future renin inhibitors with improved oral

bioavailability.

Figure 10. Mean plasma concentrations ( SD) of (S)-47 in the rat.

Figure 11. Mean plasma concentrations ( SD) of (S)-47 in the dog.

44 showed only a slight improvement in HLM and

RLM stability and no signiﬁcant change in the in vivo

clearance, although the oral bioavailability improved

to F = 28% as a result of the increased volume of distri-

bution. A 3-ethoxypropyl sidechain was installed to ste-

rically block the metabolism of the ether functionality.

Although 45 showed improved HLM stability, no

improvement in the in vivo clearance or oral bioavail-

ability was observed, presumably as a result of the low

RLM stability. A 3-triﬂuoroethoxypropyl sidechain re-

sulted in a further loss of renin inhibition activity (46,

IC₅₀= 0.188 lM).

A

4,4,4-triﬂuorobutyl sidechain

exhibited a similar level of renin inhibition (47,

IC₅₀= 0.141 lM), however this analog exhibited lower

in vivo clearance with moderate oral bioavailability

(F = 12%). We were encouraged that 47 also exhibited

reasonable aqueous solubility, cellular permeability,

and only modest inhibition of CYP isozymes in the ﬂuo-

rometric assays as the racemate. On the basis of the de-

creased in vivo clearance, the two enantiomers were

separated to aﬀord (S)-47, which exhibited a renin

IC₅₀= 0.048 lM, and excellent oral bioavailability in

the rat (F = 74%) with low in vivo clearance and good

in vivo half-life (Fig. 10). Analog (S)-47 also exhibited

a long in vivo half-life in the dog (T_1/2= 10 h) with a

moderate oral bioavailability (F = 19%, Fig. 11). Sur-

prisingly, deﬁnitive CYP inhibition studies indicated

6. Experimental

6.1. Chemistry

6.1.1. General methods. All commercially available

chemicals were used without further puriﬁcation. Anhy-

drous solvents were purchased from Aldrich Chemical

Co. Polymer-supported reagents were swelled by rinsing

with anhydrous CH₃CN (3·), THF (3·), and CH₂Cl₂

(3·), and dried under vacuum at room temperature.

Melting points were determined on a Mel-temp melting

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

5927

1

point apparatus and are uncorrected. H NMR spectra

were recorded on a Bruker 400 MHz FT NMR spec-

trometer. Chemical shifts are reported as d values rela-

tive to residual solvent (CHCl₃= 7.26 ppm and

DMSO = 2.52 ppm). Purity of ﬁnal compounds was as-

sessed by analytical HPLC on a Waters photodiode ar-

ray detector system using the following conditions

Column, Symmetry^ꢁC-18 4.6 · 150 mm; Solvent A,

H₂O/0.1% TFA; Solvent B, CH₃CN/0.1% TFA; ﬂow

rate, 2.0 mL/min; run time 25 min; gradient, from 0%

to 90% solvent B. IR spectra were recorded an a

diamond single-bounce horizontal attenuated total

reﬂectance (ATR) accessory installed in a Avatar 370

FT-IR spectrometer. Optical rotations were recorded

on an Autopol IV polarimeter. Combustion analyses

(C, H, N, F) were performed by Quantitative Technolo-

gies, Inc.

(21.0 mmol) of 1-bromo-3-methoxypropane were added,

and the reaction mixture was allowed to warm to room

temperature and stirred for 18 h. Excess hydride was

quenched by the slow dropwise addition of H₂O, and

the mixture was poured into 800 mL of H₂O. The aque-

ous layer was extracted with EtOAc (3·). The combined

organic layers were washed with H₂O and brine, dried

over MgSO₄, ﬁltered, and concentrated under vacuum.

Puriﬁcation by ﬂash column chromatography (SiO₂,

5% EtOAc/hexanes then gradient to 20% EtOAc/hex-

anes) yields 4.33 g (82%) of 6-bromo-4-(3-methoxypro-

1

pyl)-4H-benzo[1,4]oxazin-3-one 50 as a white solid. H

NMR (400 MHz, CDCl₃)

d ppm 1.94 (quintet,

J = 6.1 Hz, 2H), 3.35 (s, 3H), 3.41 (t, J = 5.9 Hz, 2H),

3.99 (m, 2H), 4.57 (s, 2H), 6.85 (d, J = 8.6 Hz, 1H),

7.09 (dd, J = 8.6, 2.2 Hz, 1H), 7.26 (d, J = 3.0 Hz, 2H).

MS(ESI+): m/z 300.0 (M+1) and 302.0 (M+2).

6.1.2. 6-Ethyl-5-[4-(3-methoxypropyl)-3,4-dihydro-2H-

benzo[1,4]oxazin-6-yl]-pyrimidine-2,4-diamine (5). Step

1. A 500 mL round-bottomed ﬂask was charged with

25.5 g (117 mmol) of 4-bromo-2-nitrophenol 48,

300 mL of acetone, and 19.4 g (140 mmol) of K₂CO₃.

Ethyl bromoacetate (15.6 mL, 140 mmol) was added,

and the suspension was heated at reﬂux for 18 h. After

cooling to room temperature, the suspension was ﬁltered

through Celite and rinsed with acetone, and the ﬁltrate

was concentrated. The solid residue was slurried with

hexanes, and the solid was collected on a medium frit

funnel and dried under vacuum to provide 31.14 g

(88%) 4-bromo-2-nitrophenoxy-acetic acid ethyl ester

Step 4. A solution of 837 mg (2.79 mmol) of 50 in 30 mL

of anhydrous THF was treated with 5.58 mL

(11.2 mmol) of 2.0 M BH₃ÆMe₂S in THF and heated in

a 50 ꢂC oil bath for 22 h. After the reaction mixture

was cooled to room temperature, excess borane was

quenched by the slow dropwise addition of methanol.

The resulting solution was concentrated and azeotrop-

ped from methanol. The crude residue was puriﬁed by

ﬂash column chromatography (SiO₂, 5% EtOAc/hex-

anes then gradient to 15% EtOAc/hexanes) to aﬀord

773 mg (97%) of 6-bromo-4-(3-methoxypropyl)-3,4-

dihydro-2H-benzo[1,4]oxazine 51 as a viscous oil. ¹H

NMR (400 MHz, CDCl₃)d 1.85 (quintet, J = 5.9 Hz,

2H), 3.35 (s, 3H), 3.34 (m, 4H), 3.42 (t, J = 5.7 Hz,

2H), 4.19 (m, 2H), 6.62 (d, J = 8.6 Hz, 1H), 6.68 (dd,

J = 8.3, 2.2 Hz, 1H), 6.81 (d, J = 2.2 Hz, 1H). MS

(ESI+): m/z 286.0 and 288.0 (⁷⁹BrꢁM+1 & ⁸¹BrꢁM+2).

1

which was used without further puriﬁcation. H NMR

(400 MHz, CDCl₃) d 1.28 (t, J = 7.2 Hz, 3H), 1.53

(s, 1H), 4.26 (q, J = 7.1 Hz, 2 H) 4.75 (s, 2H), 6.88

(d, J = 8.8 Hz, 1H,) 7.61 (dd, J = 9.0, 2.4 Hz, 1H), 8.00

(d, J = 2.4 Hz, 1H).

Step 5. A suspension of 773 mg (2.70 mmol) of 51,

823 mg (3.24 mmol) of bis(pinacolato)diboron, and

819 mg (8.34 mmol) of KOAc in 27 mL of anhydrous

1,4-dioxane was degassed by Ar sparge for 10 min.

PdCl₂(dppf)–CH₂Cl₂complex (441 mg, 0.54 mmol)

was added, and the suspension was heated to reﬂux

for 4 h. After cooling to room temperature, the mixture

was diluted with EtOAc, ﬁltered through a Celite pad,

and concentrated. Puriﬁcation by ﬂash column chroma-

tography (SiO₂, 5% EtOAc/hexanes then gradient to

25% EtOAc/hexanes) yielded 530 mg (59%) of 4-(3-

methoxypropyl)-6-(4,4,5,5-tetramethyl-[1,3,2]dioxa-

borolan-2-yl)-3,4- dihydro-2H-benzo[1,4]oxazine 53 as a

viscous oil. ¹H NMR (400 MHz, CDCl₃) d 1.30 (s, 12H),

1.90 (quintet, J = 6.8 Hz, 2H), 3.33 (m, 2H), 3.34

(s, 3H), 3.43 (m, 2H), 3.45 (t, J = 5.9 Hz, 2H), 4.25

(m, 2H), 6.77 (d, J = 7.8 Hz, 1H), 7.12 (m, 1H), 7.17

(m, 1H). MS(ESI+): m/z 334.1 (M+1).

Step 2. A solution of (4-bromo-2-nitrophenoxy)-acetic

acid ethyl ester (25.01 g, 82.2 mmol) in 400 mL of glacial

acetic acid was treated by the slow addition of iron pow-

der (91.9 g, 1.6 mol). The resulting black suspension was

heated at an internal temperature of 50 ꢂC for 6 h. After

cooling to room temperature, the resulting gray suspen-

sion was ﬁltered through a Celite pad, rinsing with

EtOAc. The combined ﬁltrates were concentrated under

vacuum. The residue was diluted with EtOAc, and

washed with deionized H₂O and brine. The organic layer

was dried over MgSO₄, ﬁltered, and concentrated until a

white precipitate formed. The slurry was diluted with

hexanes. The precipitate was collected on a medium frit

and dried under reduced pressure to provide 17.24 g

(92%) of 6-bromo-4H-benzo[1,4]oxazin-3-one 49. ¹H

NMR (400 MHz, DMSO-d₆) d 4.53 (s, 2H), 6.86 (d,

J = 8.5 Hz, 1H), 6.97 (d, J = 2.2 Hz, 1H), 7.02 (dd,

J = 8.5, 2.4 Hz, 1H), 10.74 (s, 1H). MS(ESI+): m/z

227.9 (M+1) and 229.9 (M+2).

Step 6. A round bottomed ﬂask was charged with

529 mg (1.59 mmol) of 53, 414 mg (1.90 mmol) of 5-bro-

mo-6-ethyl-pyrimidine-2,4-diamine 54, and 1.55 g

(4.76 mmol) of Cs₂CO₃, and 8 mL of anhydrous 1,4-

dioxane. The suspension was degassed by Ar sparge

for 10 min. Pd(PPh₃)₄(183 mg, 0.16 mmol) was added,

and the yellow suspension was degassed again and

then heated to reﬂux for 18 h. After cooling to room

Step 3. A solution of 49 (4.00 g, 17.5 mmol) in 100 mL

of anhydrous DMF was cooled in an ice bath under

an argon atmosphere. A 60% dispersion of NaH in min-

eral oil (807 mg, 20.2 mmol) was added in a single por-

tion, and the resulting suspension was stirred in an ice

bath for 15 min. 15-Crown-5 (0.10 mL) and 3.22 g

5928

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

temperature, the black mixture was diluted with EtOAc,

ﬁltered through Celite, and concentrated. Puriﬁcation

by ﬂash column chromatography (SiO₂, 100:0 CH₂Cl₂/

CH₃OH then gradient to 85:15 CH₂Cl₂/CH₃OH) gave

114 mg (21%) of 5 as a white solid. ¹H NMR

(400 MHz, DMSO-d₆) d 0.93 (t, J = 7.5 Hz, 3H), 1.68

(quintet, J = 6.4 Hz, 2H), 2.09 (q, J = 7.3 Hz, 2H), 3.14

(s, 3H), 3.24 (m, 4H), 3.30 (t, J = 6.1 Hz, 2H), 4.13 (br

s, 2H), 5.29 (s, 2H), 5.71 (s, 2H), 6.23 (dd, J = 8.0,

1.6 Hz, 1H), 6.37 (d, J = 1.6 Hz, 1H), 6.65

(d, J = 7.8 Hz, 1H). MS(ESI+): m/z 344.1 (M+1). Elem.

Anal. Calcd for C₁₈H₂₅N₅O₂Æ0.05CH₂Cl₂: C, 62.36; H,

7.28; N, 20.14. Found: C, 62.49; H, 7.04; N, 19.94.

heated at 60 ꢂC for 18 h. Excess hydride was quenched

by the dropwise addition of MeOH, and the mixture

was concentrated. Puriﬁcation by ﬂash column chroma-

tography (SiO₂, 100% EtOAC then gradient to 20%

MeOH/EtOAc) provided 23 mg (52%) of 7. HRMS Calcd

for C₁₉H₂₅N₅O₃(M+1): 358.2243. Found: 358.2248.

6.1.5. (2R)-6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-4-(3-

methoxy-propyl)-2-methyl-4H-benzo[1,4]oxazine (8). A

solution of 82 mg (0.22 mmol) (2R)-6-(2,4-diamino-6-

ethyl-pyrimidin-5-yl)-4-(3-methoxypropyl)-2- methyl-2H-

benzo[b][1,4]oxazin-3(4H)-one (11) in 6 mL of anhydrous

THF was treated with 0.44 mL (0.88 mmol) of 2 M

BH₃SMe₂in THF and heated in a 50 ꢂC oil bath for

16 h. After cooling to room temperature, excess borane

was quenched by the dropwise addition of MeOH. The

mixture was concentrated and azeotropped from MeOH

(2·). Puriﬁcation by ﬂash column chromatography

(SiO₂, 100:0 CH₂Cl₂/MeOH then gradient to 90:10

CH₂Cl₂/MeOH) aﬀorded 40 mg of 8 as a white solid.

¹H NMR (400 MHz, DMSO-d₆) d 0.98 (t, J = 7.6 Hz,

6.1.3. 6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-4-(3-meth-

oxy-propyl)-4H-benzo[1,4]oxazin-3-one (6). Step 1. A

50 mL round bottomed ﬂask was ﬂushed with Ar and

charged with 640 mg (2.50 mmol) of bis(pinacola-

to)diboron, 560 mg (0.69 mmol) of PdCl₂(dppf)–CH₂Cl₂

complex, and 680 mg (6.9 mmol) of KOAc. A solution

of 690 mg (2.30 mmol) of 49 in 10 mL of anhydrous

1,4-dioxane was added, and the resulting dark red sus-

pension was heated in a 110 ꢂC oil bath for 16 h. The

mixture was ﬁltered through Celite and concentrated,

and the residue was partitioned between EtOAc and

H₂O. The organic layer was washed with brine, dried

over MgSO₄, ﬁltered, and concentrated. Puriﬁcation

by ﬂash column chromatography (SiO₂, 100% hexanes

then gradient to 50% EtOAc/hexanes) provided 720 mg

3

H) 1.29 (d, J = 6.2 Hz, 3H), 1.72 (quintet,

J = 7.4 Hz, 2H), 2.15 (q, J = 7.6 Hz, 2H), 2.97–3.07

(m, 2H), 3.19 (s, 3H), 4.14–4.23 (m, 2H), 5.37 (br s,

2H), 5.78 (br s, 2H), 6.29 (dd, J = 8.0, 2.0 Hz, 1H),

6.43 (d, J = 2.0 Hz, 1H), 6.71 (d, J = 7.8 Hz, 1 H). MS:

m/z 358.2 (M+1).

6.1.6. (2S)-6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-4-(3-

methoxy-propyl)-2-methyl-4H-benzo[1,4]oxazine (9).

Step 1. A solution of 750 mg (2.39 mmol) of 73 in

20 mL of THF was treated with 4.77 mL (9.55 mmol)

of 2 M BH₃ÆSMe₂in THF and heated in a 50 ꢂC oil bath

for 7 h. Excess hydride was carefully quenched by the

dropwise addition of MeOH. The mixture was concen-

trated and azeotropped from MeOH. Puriﬁcation by

ﬂash column chromatography (SiO₂, 5% EtOAc/hex-

1

(90%) of 52 as an oil. H NMR (400 MHz, CDCl₃) d

1.34 (s, 12H) 1.98 (quintet, J = 6.4 Hz, 2H), 3.35 (s,

3H), 3.44 (t, J = 6.1 Hz, 2H), 4.09 (t, J = 7.0 Hz, 2H),

4.60 (s, 2H), 6.97 (d, J = 8.0 Hz, 1H), 7.47 (dd,

J = 8.0 Hz, 2.0 Hz, 1H), 7.53 (d, J = 2.0 Hz, 1H). MS(E-

SI+): m/z 348.1 (M+1).

Step 2. A mixture of 670 mg (1.93 mmol) of 52, 420 mg

(1.94 mmol) of 54, and 67 mg (0.058 mmol) of

Pd(PPh₃)₄in 15 mL of anhydrous DMF and 7 mL of

anhydrous toluene was degassed by Ar sparge for

10 min. K₃PO₄(620 mg, 2.9 mmol) was added, and the

mixture was heated at reﬂux for 18 h. The mixture was

diluted with EtOAc, ﬁltered through Celite, washed with

H₂O, dried over MgSO₄, ﬁltered, and concentrated.

Puriﬁcation by ﬂash column chromatography (SiO₂,

100% EtOAC then gradient to 40% MeOH/EtOAc) pro-

anes then gradient to 20% EtOAc/hexanes) aﬀorded

24:9

D

691 mg (96%) of 80 as a clear viscous oil. ½aꢂ

+2.6ꢂ

1

(c 9.7, CH₂Cl₂); H NMR (400 MHz, CDCl₃) d 1.36

(d, J = 6.4 Hz, 3H), 1.80–1.91 (m, 2H), 3.09 (dd,

J = 12.0, 8.3 Hz, 1H), 3.24 (dd, J = 11.7, 2.4 Hz, 1H),

3.30 (dd, J = 14.2, 6.6 Hz, 1H), 3.36 (s, 3H), 3.40 (d,

J = 4.0 Hz, 1H), 3.43 (ddd, J = 5.7, 5.7, 1.7 Hz, 1H),

4.13–4.23 (m, 1H), 6.64 (d, J = 8.0 Hz, 1H), 6.70 (dd,

J = 8.0, 2.0 Hz, 1H), 6.83 (d, J = 2.0 Hz, 1H); MS(E-

SI+): m/z 300.0, 302.0 ([M+1], 1:1 ratio of Br isotopes).

1

vided 156 mg (23%) of 5. H NMR (400 MHz, DMSO-

d₆) d 0.98 (t, J = 7.6 Hz, 3H), 1.76 (quintet, J = 6.5 Hz,

2H), 2.13 (q, J = 7.4 Hz, 2H), 3.17 (s, 3H), 3.34 (t,

J = 3.1 Hz, 2H), 3.93 (m, 2H), 4.65 (q, J = 7.3 Hz,

2H), 5.52 (br s, 2H), 5.82 (s, 2H), 6.78 (dd, J = 8.1,

1.7 Hz, 1H), 6.95 (d, J = 1.7 Hz, 1H), 7.04 (d,

J = 8.1 Hz, 1H). MS: m/z 358.1 (M+1). Elem. Anal.

Calcd for C₁₈H₂₃N₅O₃Æ0.2EtOAc: C, 60.25; H, 6.59;

N, 18.81. Found: C, 60.27; H, 6.39; N, 18.85.

Step 2. A suspension of 680 mg (2.27 mmol) of 80,

690 mg (2.72 mmol) of bis(pinacolato)diboron, and

667 mg (6.8 mmol) of KOAc in 12 mL of anhydrous

1,4-dioxane was degassed by an Ar sparge for 10 min.

PdCl₂(dppf)–CH₂Cl₂complex (93 mg (0.11 mmol)) was

added, and the resulting dark red suspension was heated

in a 110 ꢂC oil bath for 4 h. The mixture was ﬁltered

through Celite and concentrated. Puriﬁcation by ﬂash

column chromatography (SiO₂, 5% EtOAc/hexanes then

gradient to 15% EtOAc/hexanes) provided 561 mg

(71%) of 90 as a yellow viscous oil. MS(ESI+): m/z

348.2 (M+1).

6.1.4. 6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-4-(3-meth-

oxy-propyl)-2-methyl-4H-benzo[1,4]oxazine (7). A solu-

tion of 45 mg (0.12 mmol) of 10 in 10 mL of anhydrous

THF was treated with 0.24 mL (0.48 mmol) of 2.0 M

BH₃ÆSMe₂in THF, and the reaction mixture was heated

at 50 ꢂC for 2 h. An additional 0.48 mL of 2.0 M

BH₃ÆSMe₂in THF was added, and the mixture was

Step 3. A mixture of 561 mg (1.62 mmol) of 90, 421 mg

(1.94 mmol) of 54, and 1.58 g (4.85 mmol) of Cs₂CO₃in

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

5929

8 mL of anhydrous 1,4-dioxane was degassed by freeze–

pump–thaw techniques (2·). Pd(PPh₃)₄(187 mg,

0.16 mmol) was added, and the yellow suspension was

heated at 100 ꢂC for 18 h. The mixture was diluted with

EtOAc, ﬁltered through Celite, and concentrated. Puri-

ﬁcation by ﬂash column chromatography (SiO₂, 100:0

with EtOAc and ﬁltered through Celite. The ﬁltrate was

washed with H₂O, dried over Na₂SO₄, ﬁltered, and con-

centrated. Puriﬁcation by ﬂash column chromatography

(SiO₂, 100% EtOAc then gradient to 40% MeOH/

EtOAc) gave 130 mg (22%) of 10. ¹H NMR

(400 MHz, DMSO-d₆) d 0.98 (t, J = 7.6 Hz, 3H), 1.44

(d, J = 6.8 Hz, 3H), 1.74 (dd, J = 13.1, 6.5 Hz, 2H),

2.13 (q, J = 7.5 Hz, 2H), 3.16 (s, 3H), 3.32 (t,

J = 6.0 Hz, 2H), 3.92 (m, 2H), 4.71 (m, 1H), 5.57 (br s,

2H), 5.83 (s, 2H), 6.78 (d, J = 7.6 Hz, 1H), 6.94 (s,

1H), 7.04 (d, J = 7.8 Hz, 1H). MS: m/z 372.1 (M+1).

Elem. Anal. Calcd for C₁₉H₂₅N₅O₃Æ0.5 H₂O: C, 61.29;

H, 6.79; N, 18.81. Found: C, 61.38; H, 6.59; N, 18.41.

CH₂Cl₂/MeOH then gradient to 85:15 CH₂Cl₂/MeOH)

24

gave 191 mg (33%) of 9 as a white solid. ½aꢂ ꢁ9.4 (c

D

4.6, MeOH); ¹H NMR (400 MHz, DMSO-d₆) d 0.93

(t, J = 7.5 Hz, 3H), 1.24 (d, J = 6.2 Hz, 3H), 1.67 (quin-

tet, J = 6.6 Hz, 2H), 2.09 (q, J = 7.4 Hz, 2H), 2.97 (m,

1H), 3.14 (s, 3H), 3.25 (m, 4H), 3.29 (t, J = 6.0 Hz,

2H), 4.13 (br s, 1H), 5.30 (br s, 1H), 5.71 (s, 2H), 6.24

(dd, J = 7.9, 1.7 Hz, 1H), 6.38 (d, J = 1.4 Hz, 1H), 6.65

(d, J = 8.0 Hz, 1H); MS(ESI+): m/z 358.1 (M+1). Elem.

Anal. Calcd for C₁₉H₂₇N₅O₂: C, 63.84; H, 7.61; N,

19.59. Found: C, 63.61; H, 7.37; N, 19.07.

6.1.8. (2R)-6-(2,4-diamino-6-ethyl-pyrimidin-5-yl)-4-(3-

methoxypropyl)-2-methyl-2H-benzo[b][1,4]oxazin-3(4H)-

one (11). Step 1. A solution of 1.73 g (7.93 mmol) of 4-

bromo-2-nitrophenol (48) and 0.75 (6.61 mmol) of ethyl

(S)-lactate in 100 mL of anhydrous CH₂Cl₂was treated

with 6.82 g (10.6 mmol, 1.55 mmol/g) of polymer sup-

ported-triphenylphosphine. The resulting suspension

was stirred at room temperature for 10 min and then

cooled in an ice bath. Diisopropyl azodicarboxylate

(1.56 mL, 7.93 mmol) was added to the suspension in a

dropwise fashion. The dark yellow suspension was al-

lowed to warm to room temperature while stirring over

19 hours. The resin was collected on a medium frit and

rinsed with CH₂Cl₂(3·). The ﬁltrate was concentrated

and the crude residue was puriﬁed by ﬂash chromatog-

raphy (SiO₂, 5% EtOAc/hexanes then gradient to 15%

EtOAc/hexanes) to yield 1.033 g (49%) of (R)-ethyl 2-

(4-bromo-2-nitrophenoxy)propanoate as a viscous oil.

¹H NMR (400 MHz, CDCl₃) d 1.24 (t, J = 7.1 Hz,

3H), 1.68 (d, J = 6.8 Hz, 3H), 4.21 (dq, J = 7.3, 1.7 Hz,

2H), 4.80 (q, J = 6.9 Hz, 1H), 6.86 (d, J = 9.1 Hz, 1H),

7.57 (dd, J = 8.8, 2.5 Hz, 1H), 7.95 (d, J = 2.4 Hz, 1H).

6.1.7. (rac)-6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-4-(3-

methoxy-propyl)-2-methyl-4H-benzo[1,4]oxazin-3-one (10).

Step 1. A solution of 2.8 g (9.3 mmol) of 50 in 100 mL of

THF was cooled in a dry ice/acetone bath. LiHMDS

(10 mL, 10 mmol, 1.0 M in THF) was added in a drop-

wise fashion, and the reaction mixture was stirred at

ꢁ78 ꢂC for 15 min. Iodomethane (0.70 mL, 11.2 mmol)

was added slowly, and the mixture was stirred at

ꢁ78 ꢂC for 2 h. The cold bath was removed, and the

mixture was allowed to warm to room temperature

and stirred for 16 h. The mixture was partially concen-

trated, diluted with H₂O, and extracted with EtOAc

(3·). The combined organic layers were washed with

brine, dried over MgSO₄, ﬁltered, and concentrated.

Puriﬁcation by ﬂash column chromatography (SiO₂,

100% hexanes then gradient to 30% EtOAc/hexanes)

yielded 1.12 (38%) of 71 as an oil. ¹H NMR

(400 MHz, CDCl₃) d 1.53 (d, J = 6.8 Hz, 3H), 1.92

(ddd, J = 13.4, 6.2, 6.0 Hz, 2H), 3.34 (s, 3H), 3.40 (t,

J = 5.9 Hz, 2H), 3.97 (t, J = 7.0 Hz, 2H), 4.58 (q,

J = 6.8 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 7.08 (dd,

J = 8.6, 2.2 Hz, 1H), 7.24 (d, J = 2.2 Hz, 1H); MS(E-

SI+): m/z 314.0, 316.0 ([M+1], 1:1 ratio of Br isotopes).

Step 2. A solution of 1.03 g (3.25 mmol) of ethyl (R)-2-

(4-bromo-2-nitrophenoxy)propanoate in 20 mL of gla-

cial acetic acid was treated with 3.63 g (64.9 mmol) of

iron dust and heated in a 50 ꢂC oil bath for 4 h. The

resulting gray suspension was cooled to room tempera-

ture, diluted with EtOAc, and ﬁltered through a Celite

plug. The ﬁltrate was washed with deionized H₂O and

saturated aqueous NaHCO₃, dried over MgSO₄, ﬁl-

tered, and concentrated to aﬀord 1.38 g of (R)-6-bro-

mo-2-methyl-2H-benzo[b][1,4]oxazin-3(4H)-one (63) as

a solid which was used without further puriﬁcation.

[a]_Dꢁ21.2ꢂ (c 4.9, CH₂Cl₂); ¹H NMR (400 MHz,

CDCl₃) d 1.59 (d, J = 6.8 Hz, 3H), 4.66 (q, J = 6.8 Hz,

1H), 6.87 (d, J = 8.6 Hz, 1H), 6.97 (d, J = 2.2 Hz, 1H),

7.09 (dd, J = 8.6, 2.2 Hz, 1H), 8.46 (s, 1H). MS(ESI+):

m/z 241.9, 243.9 (1:1 ratio, Br isotopes).

Step 2. A suspension of 785 mg (2.50 mmol) of 71,

700 mg (2.8 mmol) of bis(pinacolato)diboron, and

700 mg (8.0 mmol) of KOAc in 20 mL of anhydrous

1,4-dioxane was degassed by an Ar sparge for 10 min.

PdCl₂(dppf)–CH₂Cl₂complex (200 mg (0.25 mmol))

was added, and the resulting dark red suspension was

heated in a 110 ꢂC oil bath for 16 h. The mixture was ﬁl-

tered through Celite, concentrated, and partitioned be-

tween EtOAc and H₂O. The organic layer was washed

with brine, dried over MgSO₄, ﬁltered, and concen-

trated. Puriﬁcation by ﬂash column chromatography

(SiO₂, 100% hexanes then gradient to 50% EtOAc/hex-

anes) yielded 600 mg (66%) of 81 as an oil. MS(ESI+):

m/z 362.1 (M+1).

Step 3. A solution of 600 mg (2.48 mmol) of (R)-6-bro-

mo-2-methyl-2H-benzo[b][1,4]oxazin-3(4H)-one (63) in

20 mL of anhydrous DMF under an Ar atmosphere

was cooled in an ice bath and treated with 455 mg

(2.97 mmol, 60% dispersion in mineral oil) of NaH in

a single portion. The resulting gray suspension was stir-

red in an ice bath for 10 min and treated with 2–3 drops

of 15-crown-5, followed by 455 mg (2.97 mmol) of 1-

bromo-3-methoxypropane. The reaction mixture was

Step 3. A mixture of 0.58 g (1.61 mmol) of 81, 349 mg

(1.61 mmol) of 54, and 56 mg (0.05 mmol) of Pd(PPh₃)₄

in 15 mL of anhydrous DMF and 7 mL of anhydrous

toluene was degassed by Ar sparge for 10 min. K₃PO₄

(0.51 g, 2.4 mmol) was added, and the yellow suspension

was heated at 100 ꢂC for 18 h. The mixture was diluted

5930

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

stirred while warming to room temperature over 1 h. Ex-

cess hydride was quenched by the careful addition of

H₂O, and the reaction mixture was poured into

300 mL of H₂O. The aqueous layer was extracted with

EtOAc (3·). The combined organic layers were washed

with H₂O and brine, dried over MgSO₄, ﬁltered, and

concentrated. Puriﬁcation of the crude residue by ﬂash

column chromatography (SiO₂, 5% EtOAc/hexanes then

gradient to 20% EtOAc/hexanes) provided 528 mg

(68%) of (R)-6-bromo-4-(3-methoxypropyl)-2-methyl-

2H-benzo[b][1,4]oxazin- 3(4H)-one (72) as a clear vis-

cous oil. [a]_Dꢁ31.6ꢂ (c 8.1, CH₂Cl₂); ¹H NMR

(400 MHz, CDCl₃) d 1.55 (d, J = 6.6 Hz, 3H), 1.94

(quintet, J = 6.2 Hz, 2H), 3.36 (s, 3H), 3.42 (t,

J = 6.0 Hz, 2H), 3.96–4.01 (t, J = 6.8 Hz, 2H), 4.60 (q,

J = 6.8 Hz, 1H), 6.86 (d, J = 8.3 Hz, 1H), 7.10 (dd,

J = 8.6, 2.2 Hz, 1H), 7.26 (d, J = 2.2 Hz, 1H). MS

(ESI+): m/z 314.0, 316.0 (1:1 ratio, Br isotopes).

5.5, CH₃OH); IR(ATR) 3450, 3309, 3149, 2975, 2876,

1665, 1628, 1558, 1448, 1384, 1269, 1112 cm^ꢁ^{1 1}H

;

NMR (400 MHz, DMSO-d₆) d 0.93 (t, J = 7.5 Hz,

3H), 1.40 (d, J = 6.6 Hz, 3H), 1.72 (m, 2H), 2.09 (q,

J = 7.6 Hz, 2H), 3.12 (s, 3H), 3.28 (t, J = 6.1 Hz, 2H),

3.88 (m, 2H), 4.62–4.71 (m, 1H), 5.51 (m, 2H), 5.78

(br s, 2H), 6.74 (d, J = 7.8 Hz, 1H), 6.90 (d,

J = 5.8 Hz, 1H), 6.99 (d, J = 8.1 Hz, 1H); ¹³C NMR

(100 MHz, DMSO-d₆) d 13.9, 16.9, 28.2, 27.1, 38.9,

58.5, 69.4, 73.4, 106.69, 117.9, 126.3, 129.6, 131.1,

143.8, 162.7, 166.6, 167.1. HRMS (ESI) m/z Calcd for

C₁₉H₂₆N₅O₃(M+H)⁺372.2035. Found: 372.2039. Elem.

Anal. Calcd for C₁₉H₂₅N₅O₃: C, 61.44; H, 6.78; N,

18.69. Found: C, 61.23; H, 6.78; N, 18.69.

6.1.9. (2S)-6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-4-(3-

methoxypropyl)-2-methyl-4H-benzo[1,4]oxazin-3-one (12).

The title compound was prepared via the identical route

as described above for analog 11, utilizing methyl (R)-

lactate to aﬀord a white solid foam. ½aꢂ

24:7

D

Step 4. An oven-dried 50 mL round bottom ﬂask was

ﬂushed with Ar and charged with 525 mg (1.67 mmol)

of (R)-6-bromo-4-(3-methoxypropyl)-2-methyl-2H-ben-

zo[b] [1,4]oxazin- 3(4H)-one (72), 509 mg (2.01 mmol)

of bis(pinnocolato)diborane, 492 mg (5.01 mmol) of

potassium acetate, and 9 mL on anhydrous 1,4-dioxane.

The reaction mixture was degassed by bubbling Ar gas

through the white suspension for 15 min. PdCl₂(dppf)–

dichloromethane complex (68 mg, 80 mmol) was added

in a single portion, and the resulting orange suspension

was heated to reﬂux for 8 h. The reaction mixture was

cooled to room temperature, diluted with EtOAc, ﬁl-

tered through Celite, and concentrated. The residue

was puriﬁed by ﬂash chromatography (SiO₂, 5%

EtOAc/hexanes then gradient to 20% EtOAc/hexanes)

to yield 441 mg (73%) of (R)-4-(3-methoxypropyl)-2-

methyl-6-(4,4,5,5-tetramethyl-1, 3,2-dioxaborolan-2-yl)-

2H-benzo[b][1,4]oxazin-3(4H)-one (82) as a viscous

+23.8 (c 3.7,

CH₃OH), IR(ATR) 3448, 3429, 3405, 3314, 3174,

2973, 2927, 1669, 1623, 1554, 1440, 1373, 1260, 1106,

1

872, 665 cm^ꢁ1; H NMR (400 MHz, DMSO-d₆)d 0.93

(t, J = 7.6 Hz, 3H), 1.40 (d, J = 6.6 Hz, 3H), 1.70 (quin-

tet, J = 6.7 Hz, 2H), 2.09 (q, J = 7.5 Hz, 2H), 3.12

(s, 3H), 3.28 (t, J = 7.5 Hz, 2H), 3.87 (m, 2H), 4.67

(m, 1H), 5.50 (br s, 2H), 5.77 (s, 2H), 6.74 (d,

J = 8.6 Hz, 1H), 6.90 (d, J = 5.9 Hz, 1H), 6.99 (d,

J = 8.1 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) d

13.9, 16.9, 27.6, 28.2, 38.9, 58.5, 69.9, 73.4, 106.7,

117.9, 126.3, 129.6, 131.1, 162.7, 162.9. HRMS (ESI)

m/z Calcd for C₁₉H₂₆N₅O₃(M+H)⁺: 372.2035. Found:

372.2025. Elem. Anal. Calcd for C₁₉H₂₅N₅O₃Æ0.3H₂O:

C, 60.67; H, 6.65; N, 18.62. Found: C, 60.67; H, 6.84;

N, 18.40.

6.1.10. 6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-4-(3-meth-

oxy-propyl)-2-propyl-4H-benzo[1,4]oxazin-3-one (13).

Step 1. A solution of 2.5 g (11 mmol) of 4-bromo-2-

nitrophenol (48) and 1.4 g (9.08 mmol) of 2-hydroxyva-

leric acid ethyl ester 57 in 50 mL of anhydrous CH₂Cl₂

was treated with 5.1 g (15 mmol, 3 mmol/g) of poly-

mer-supported PPh₃, and the resulting suspension was

stirred at room temperature for 10 min. The mixture

was cooled in an ice bath, and 2.23 mL (11.5 mmol) of

DIAD was added in a dropwise fashion. The resulting

yellow suspension was stirred for 18 h while slowly

warming to room temperature. The resin was collected

on a coarse frit and rinsed with CH₂Cl₂(3·). The com-

bined ﬁltrates were concentrated. Puriﬁcation by ﬂash

column chromatography (SiO₂, 5% EtOAc/hexanes then

gradient to 15% EtOAc/hexanes) gave 3.2 g (97%) of

1

oil. H NMR (400 MHz, CDCl₃) d 1.34 (s, 12H), 1.54

(d, J = 6.8 Hz, 3H), 1.97 (quintet, J = 6.4 Hz, 2H), 3.35

(s, 3H), 3.43 (t, J = 6.1 Hz, 2H), 4.08 (t, J = 7.0 Hz,

2H), 4.63 (q, J = 6.8 Hz, 1H), 6.98 (d, J = 7.8 Hz, 1H),

7.47 (dd, J = 7.8, 1.2 Hz, 1H), 7.52 (d, J = 1.2 Hz, 1H).

MS(ESI+): m/z 362.2 [M+1].

Step 5. An oven-dried ﬂask was ﬂushed with N₂and

charged with 440 mg (1.22 mmol) of (R)-4-(3-methoxy-

propyl)-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxa-

borolan-2-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (82),

397 mg (1.83 mmol) of 5-bromo-6-ethyl-pyrimidine-

2,4-diamine 54,¹⁷155 mg (3.65 mmol) of LiCl, 1.19 g

(3.65 mmol) of Cs₂CO₃, 8 mL of 1,4-dioxane, and

1 mL of deionized H₂O. The reaction mixture was de-

gassed by bubbling N₂through the white suspension

for 15 min. Pd(PPh₃)₄(141 mg, 0.122 mmol) was added,

and the light yellow suspension was heated to reﬂux for

20 h. The reaction mixture was cooled, diluted with

EtOAc, and washed with H₂O. The organic layer was

dried over MgSO₄, ﬁltered, and concentrated. Puriﬁca-

tion by ﬂash column chromatography (SiO₂, 100%

CH₂Cl₂then gradient to 85:15 CH₂Cl₂/CH₃OH) to

provide 207 mg (46%) of (2R)-6-(2,4-diamino-6-ethyl-

pyrimidin-5-yl)-4-(3-methoxypropyl)-2-methyl-2H-ben-

zo [b][1,4]oxazin-3(4H)-one (11) as a solid. [a]_Dꢁ25.3ꢂ (c

ethyl

MS(ESI+): m/z 348.0, 350.0 ([M+1], 1:1 ratio of Br

2-(4-bromo-2-nitro-phenoxy)-butyrate

(57).

isotopes).

Step 2. A solution of 3.1 g (9.0 mmol) of 57 in 125 mL of

glacial acetic acid was treated with 5.0 g (90 mmol) of Fe

dust, and the resulting black suspension was heated in a

60 ꢂC oil bath for 18 h. After cooling to room tempera-

ture, the gray suspension was ﬁltered through a Celite

pad, rinsing with EtOAc (200 mL). The ﬁltrate was con-

centrated, and the residue was dissolved in EtOAc and

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

5931

washed with H₂O (2·). The organic layer was dried over

MgSO₄, ﬁltered, and concentrated to 2.36 g (98%) of 65

as an opaque oil that was used without further puriﬁca-

6.1.11. 6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-4-(3-meth-

oxy-propyl)-2-phenyl-4H-benzo[1,4]oxazin-3-one (14).

Step 1. A solution of 1.09 g (4.99 mmol) of 4-bromo-2-

nitrophenol 48 and 0.75 g (4.16 mmol) of ethyl R-(ꢁ)-

mandelate 58 in 50 mL of anhydrous CH₂Cl₂was

treated with 1.84 g (6.66 mmol) of polymer-supported

PPh₃, and the resulting suspension was stirred at room

temperature for 10 min. The mixture was cooled in an

ice bath, and 0.98 mL (4.99 mmol) of diisopropyl azadi-

carboxylate was added in a dropwise fashion. The

resulting yellow suspension was stirred for 18 h while

slowly warming to room temperature. The resin was col-

lected on a coarse frit and rinsed with CH₂Cl₂(3·). The

combined ﬁltrates were concentrated. Puriﬁcation by

ﬂash column chromatography (SiO₂, 5% EtOAc/hex-

anes then gradient to 15% EtOAc/hexanes) gave 1.41 g

(89%) of ethyl (2S)-2-(4-bromo-2-nitro-phenoxy)-man-

delate as a viscous yellow oil. ¹H NMR (400 MHz,

CDCl₃)d 1.20 (t, J = 7.2 Hz, 3H), 4.12–4.23 (m, Hz,

2H), 5.72 (s, 1H), 6.91 (d, J = 9.0 Hz, 1H), 7.38–7.46

(m, 3H), 7.57–7.64 (m, 3H), 8.04 (d, J = 2.4 Hz, 1H).

1

tion. H NMR (400 MHz, CDCl₃) d 0.99 (t, J = 7.5 Hz,

3H), 1.52–1.63 (m, 2H), 1.84–1.92 (m, 2H), 4.59 (dd,

J = 8.1, 5.1 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 6.97 (d,

J = 2.2 Hz, 1H), 7.09 (dd, J = 8.6, 2.2 Hz, 1H), 8.90

(br s, 1H); MS(ESI+): m/z 270.0, 272.0 ([M+1], 1:1 ratio

of Br isotopes).

Step 3. The residue from Step 2 was dissolved in 60 mL

of anhydrous DMF and cooled in an ice bath. A 60%

dispersion of NaH in mineral oil (426 mg, 10.6 mmol)

was added in portions, and the resulting suspension

was stirred for 30 min. 1-Bromo-3-methoxypropane

(1.63 g, 10.6 mmol) was added, the ice bath was re-

moved, and the mixture was stirred at ambient temper-

ature for 18 h. The reaction mixture was diluted with

H₂O and extracted with EtOAc. The organic layer was

dried over MgSO₄and concentrated to give 2.53 g

(87%) of 74 that was used without further puriﬁcation.

¹H NMR (400 MHz, CDCl₃)d 0.93 (t, J = 7.3 Hz, 3

H), 1.44–1.56 (m, 2H), 1.71–1.81 (m, 2H), 1.87–1.94

(m, 2H), 3.33 (s, 3H), 3.38 (t, J = 5.9 Hz, 2H), 3.90–

3.99 (m, 2H), 4.51 (dd, J = 8.0, 4.0 Hz, 1H), 6.82 (d,

J = 8.0 Hz, 1H), 7.06 (dd, J = 8.0, 2.0 Hz, 1H), 7.22 (d,

J = 2.20 Hz, 1H); MS(ESI+): m/z 344.1, 346.1 ([M+1],

1:1 ratio of Br isotopes).

Step 2. Ethyl (2S)-2-(4-bromo-2-nitro-phenoxy)-man-

delate (1.41 g, 3.71 mmol) was dissolved in 205 mL of

glacial acetic acid. Fe dust (4.15 g, 74 mmol) was added,

and the resulting black suspension was heated in a 60 ꢂC

oil bath for 18 h. After cooling to room temperature, the

gray suspension was diluted with EtOAc and ﬁltered

through a Celite pad. The ﬁltrate was washed with

H₂O (2·) and saturated aqueous NaHCO₃, dried over

MgSO₄, and concentrated to 1.071 g (95%) of 66 as a

Step 4. A suspension of 2.2 g (6.4 mmol) of 74, 1.8 mg

(7.1 mmol) of bis(pinacolato)diboron, and 2.0 g

(19 mmol) of KOAc in 40 mL of anhydrous 1,4-dioxane

was degassed by an Ar sparge for 10 min. PdCl₂(dppf)–

CH₂Cl₂complex (520 mg (0.64 mmol)) was added, and

the resulting dark red suspension was heated in a

110 ꢂC oil bath for 16 h. The mixture was ﬁltered

through Celite, concentrated, and partitioned between

EtOAc and H₂O. The organic layer was washed with

brine, dried over MgSO₄, ﬁltered, and concentrated.

Puriﬁcation by ﬂash column chromatography (SiO₂,

100% hexanes then gradient to 50% EtOAc/hexanes)

yielded 1.8 g (72%) of 84 as an oil MS(ESI+): m/z

391.0 (M+1).

white solid that was used without further puriﬁcation.

24

D

½aꢂ ꢁ79.8ꢂ (c 5.6, CHCl₃); ¹H NMR (400 MHz,

CDCl₃) d 5.71 (s, 1H), 6.92 (d, J = 8.6 Hz, 1H), 6.96

(d, J = 2.2 Hz, 1H), 7.10 (dd, J = 8.6, 2.2 Hz, 1H),

7.35–7.40 (m, 3H), 7.41–7.45 (m, 2H), 8.60 (br s, 1H);

MS(ESI+): m/z 303.9, 305.9 ([M+1], 1:1 ratio of Br

isotopes).

Step 3. A solution of 750 mg (2.47 mmol) of 66 in 20 mL

of anhydrous DMF was cooled in an ice bath. A 60%

dispersion of NaH in mineral oil (108 mg, 2.71 mmol)

was added, and the resulting suspension was stirred

for 15 min. 1-Bromo-3-methoxypropane (453 mg,

2.96 mmol) and 0.1 mL of 15-crown-5 was added, the

ice bath was removed, and the mixture was stirred at

ambient temperature for 4 h. The reaction mixture was

diluted with H₂O and extracted with EtOAc (3·). The

combined organic layers were washed with H₂O and

brine, dried over MgSO₄and concentrated. Puriﬁcation

by ﬂash column chromatography (SiO₂, 5% EtOAc/hex-

anes then gradient to 15% EtOAc/hexanes) gave 524 mg

(56%) of 75 as a white solid that was racemic by chiral

Step 5. A mixture of 1.68 g (4.32 mmol) of 84, 1.1 g

(5.2 mmol) of 54, 500 mg (13 mmol) of LiCl, and

500 mg (0.43 mmol) of Pd(PPh₃)₄in 40 mL of anhy-

drous 1,4-dioxane and 10 mL of H₂O was degassed by

Ar sparge for 10 min. Cs₂CO₃(4.0 g, 13 mmol) was

added, and the mixture was heated at reﬂux for 18 h.

The black mixture was diluted with EtOAc and ﬁltered

through Celite. The ﬁltrate was washed with H₂O, dried

over Na₂SO₄, ﬁltered, and concentrated. Puriﬁcation by

ﬂash column chromatography (SiO₂, 100% EtOAc then

gradient to 40% MeOH/EtOAc) gave 356 mg (21%) of

1

HPLC analysis. H NMR (400 MHz, CDCl₃) d 1.92–

2.04 (m, 2H), 3.35 (s, 3H), 3.37–3.47 (m, 2H), 4.01 (quin-

tet, J = 7.1 Hz, 1H), 4.09 (quintet, J = 6.8 Hz, 1H), 5.71

(s, 2H), 6.92 (d, J = 8.6 Hz, 1H), 7.09 (dd, J = 8.6,

2.2 Hz, 1H), 7.25 (d, J = 2.2 Hz, 1H), 7.30–7.39 (m, 5

H); MS(ESI+): m/z 376.0, 378.0 ([M+1], 1:1 ratio of

Br isotopes).

13. ¹H NMR (400 MHz, DMSO-d₆)

d 0.93 (t,

J = 7.5 Hz, 3H), 0.98 (t, J = 7.5 Hz, 3H), 1.48 (m, 2H),

1.76 (m, 4H), 2.12 (m, 2H), 3.17 (s, 3H), 3.33 (t,

J = 6.1 Hz, 2H), 3.92 (m, 2H), 4.61 (m, 1H), 5.57 (br s,

2 H), 5.83 (s, 2H), 6.78 (m, 1H), 6.94 (m, 1H), 7.04

(m, 1H). MS: m/z 400.2 [M+1]. Elem. Anal. Calcd for

C₂₁H₂₉N₅O₃: C, 63.14; H, 7.32; N, 17.53. Found: C,

63.08; H, 7.38; N, 17.27.

Step 4. A suspension of 524 mg (1.39 mmol) of 75,

424 mg (1.67 mmol) of bis(pinacolato)diboron, and

5932

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

410 mg (4.18 mmol) of KOAc in 14 mL of anhydrous

1,4-dioxane was degassed by an Ar sparge for 10 min.

Pd–Cl₂(dppf)–CH₂Cl₂complex (57 mg (0.07 mmol))

was added, and the resulting dark red suspension was

heated in a 110 ꢂC oil bath for 16 h. The mixture was ﬁl-

tered through Celite, concentrated, and partitioned be-

tween EtOAc:H₂O. The organic layer was washed with

brine, dried over MgSO₄, ﬁltered, and concentrated.

Puriﬁcation by ﬂash column chromatography (SiO₂,

5% EtOAc/hexanes then gradient to 30% EtOAc/hex-

anes) yielded 412 mg (70%) of 85 as a white solid. MS(E-

SI+): m/z 424.0 (M+1).

(18.7 mmol) of (4-bromo-2-nitrophenoxy)-(3,4-diﬂuoro-

phenyl)acetic acid methyl ester and the mixture was

heated at 60 ꢂC for 3 h. The reaction mixture was diluted

with EtOAc and ﬁltered through Celite. The ﬁltrate was

washed with H₂O, saturated aqueous NaHCO₃, and

brine, dried over MgSO₄, ﬁltered, and concentrated to

a light yellow solid. Trituration with CH₂Cl₂and hex-

anes aﬀorded 4.77 g (75%) of 6-bromo-2-(3,4-diﬂuoro-

phenyl)-4H-benzo[1,4]oxazin-3-one 67 as

yellow-orange solid. MS(ESI+): m/z 340.0, 341.9

a

light

([M+1], 1:1 ratio of Br isotopes).

Step 4. A mixture of 4.76 g (14.0 mmol) of 67, 2.03 g

(14.7 mmol) of K₂CO₃, and 2.25 g (14.7 mmol) of 1-bro-

mo-3-methoxypropane in 100 mL of anhydrous CH₃CN

was heated to reﬂux for 16 h, cooled to room tempera-

ture, and ﬁltered through a pad of Celite. The ﬁltrate

was concentrated to aﬀord 5.84 g (100%) of 76 as a vis-

Step 5. A mixture of 412 mg (0.97 mmol) of 85, 254 mg

(1.17 mmol) of 54, 124 mg (2.92 mmol) of LiCl, and

952 mg (2.92 mmol) of Cs₂CO₃in 8 mL of anhydrous

1,4-dioxane was degassed by freeze–pump–thaw tech-

niques (2·). Pd(PPh₃)₄(113 mg, 0.097 mmol) was added,

and the mixture was heated at 100 ꢂC for 18 h. The

black mixture was diluted with EtOAc and ﬁltered

through Celite. Puriﬁcation by ﬂash column chromato-

graphy (SiO₂, 100% CH₂Cl₂then gradient to 85:15

CH₂Cl₂:MeOH) gave 117 mg (28%) of 14 as a brown-

white solid that exists as a 1:1 mixture of restricted rota-

1

cous orange oil that solidiﬁed upon standing. H NMR

(400 MHz, CDCl₃) d 1.91–1.99 (m, 2H), 3.34 (s, 3H),

3.35–3.44 (m, 2H), 3.97–4.09 (m, 2H), 6.92 (d,

J = 8.6 Hz, 1H), 7.11 (dd, J = 6.6, 2.0 Hz, 1H), 7.25 (d,

J = 2.0 Hz, 1 H); MS(ESI+): m/z 412.0, 414.0 ([M+1],

1:1 ratio of Br isotopes).

1

tional isomers. H NMR (400 MHz, DMSO-d₆)d 0.88–

1.06 (m, 3H), 1.80 (quintet, J = 7.0 Hz, 2H), 2.12 (m,

2H), 3.18 (s, 3H), 3.36 (t, J = 6.2 Hz, 2H), 3.89–4.12

(m, 2H), 5.52 (br s, 2H), 5.77–5.90 (m, 3H), 6.81 (d,

J = 8.0 Hz, 1H), 6.99 (s, 1H), 7.09 (d, J = 9.0 Hz, 1H),

7.32–7.47 (m, 5H); MS(ESI+): m/z 434.2 [M+1]. Elem.

Anal. Calcd for C₂₄H₂₇N₅O₃Æ 0.3CH₂Cl₂: C, 63.71; H,

6.00; N, 15.08. Found: C, 63.74; H, 6.25; N, 14.85.

Step 5. A solution of 500 mg (1.21 mmol) of 76 in 15 mL

of anhydrous 1,4-dioxane was treated with 461 mg

(1.82 mmol) of bis(pinacolato)diboron and 356 mg

(3.63 mmol) of KOAc, and degassed by N₂sparge

for 15 min. PdCl₂(dppf)–CH₂Cl₂complex (49 mg

(0.06 mmol)) was added, and the resulting dark red sus-

pension was heated in a 110 ꢂC oil bath for 16 h. The

mixture was cooled to room temperature and treated

with 591 mg (2.72 mmol) of 54, 154 mg (3.63 mmol) of

LiCl, 1.18 g (3.62 mmol) of Cs₂CO₃, and 4 mL of

H₂O. The resulting biphasic mixture was degassed by

N₂sparge for 15 min. Pd(PPh₃)₄(386 mg, 0.33 mmol)

was added, and the mixture was heated at reﬂux for

20 h. The black mixture was diluted with CH₂Cl₂and ﬁl-

tered through a SiO₂pad, rinsing with MeOH. Puriﬁca-

tion by ﬂash column chromatography (SiO₂, 100%

CH₂Cl₂then gradient to 90:10 CH₂Cl₂/MeOH) then

(SiO₂, 100% EtOAc then gradient to 90:10 EtOAc/

MeOH) gave 266 mg (39%) of 15 as a light yellow solid

that exists as a 1:1 mixture of restricted rotational

isomers. ¹H NMR (400 MHz, CDCl₃) d 1.05 (t,

J = 7.6 Hz, 3H), 1.91 (quintet, J = 6.1 Hz, 2H), 2.25 (q,

J = 8.0 Hz, 2H), 3.26 (s, 3H), 3.36–3.44 (m, 2H), 3.99–

4.09 (m, 2H), 4.51 (br s, 1H), 4.57 (br s, 1H), 4.85 (br

s, 2H), 5.63 (s, 1H), 6.89 (dd, J = 8.0, 2.0 Hz, 1H),

6.93 (m, 1H), 7.12 (dd, J = 8.1, 2.2 Hz, 1H) 7.14–7.19

(m, 1H), 7.29 (m, 2 H). MS(ESI+): m/z 470.2 [M+1].

6.1.12. 6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-4-(3-meth-

oxy-propyl)-2-(3,4-diﬂuoro-phenyl)-4H-benzo[1,4]oxazin-

3-one (15). Step 1. A solution of 4.10 g (21.1 mmol) of

(3,4-diﬂuorophenyl)-hydroxyacetic acid 131 and

0.90 mL of 37% aqueous HCl in 90 mL of MeOH was

heated to reﬂux for 18 h, cooled to room temperature,

and concentrated. The residue was partitioned between

saturated NaHCO₃and EtOAc. The aqueous layer was

extracted with EtOAc. The combined organic extracts

were washed with brine, dried over MgSO₄, ﬁltered, and

concentrated to give 3.87 g (91%) of methyl (3,4-diﬂuor-

ophenyl)hydroxyacetate (59), which was used without

further puriﬁcation. MS(ESI+): m/z 201.0 (Mꢁ1).

Step 2. A suspension of 3.79 g (18.7 mmol) of 59, 4.16 g

(18.7 mmol) of 4-bromo-2-nitrophenol 48, and (18.1 g,

28.1 mmol) of polymer-supported PPh₃in 200 mL of

anhydrous CH₂Cl₂at 0 ꢂC was treated with 4.26 mL

(20.6 mmol) of DIAD in a dropwise fashion. The reac-

tion mixture was stirred at 0 ꢂC for 1 h, allowed to warm

to room temperature, and stirred for 48 h. The mixture

was ﬁltered through a medium frit, and the ﬁltrate was

washed with 1 N NaOH, H₂O, and brine. The organic

layer was dried over MgSO₄, ﬁltered, and concentrated

to give 10.41 g (100%) of (4-bromo-2-nitrophenoxy)-

(3,4-diﬂuorophenyl)acetic acid methyl ester, which was

used directly without further puriﬁcation.

Diagnostic peaks for the restricted rotational isomer:

1.08 (t, J = 7.6 Hz, 3H), 2.29 (q, J = 8.0 Hz, 2H), 5.68

(s, 1H).

6.1.13. 6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-4-(3-meth-

oxy-propyl)-2-(4-ﬂuoro-phenyl)-4H-benzo[1,4]oxazin-3-

one (16). Step 1. A solution of 2.00 g (11.8 mmol) of (4-

ﬂuorophenyl)-hydroxyacetic acid and 0.5 mL of 37%

aqueous HCl in 50 mL of MeOH was heated to reﬂux

for 6 h, cooled to room temperature, and concentrated.

Step 3. Glacial AcOH (100 mL) and 10.47 g

(187.5 mmol) of iron dust were added to 10.41 g

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

5933

The residue was partitioned between saturated NaHCO₃

and EtOAc. The aqueous layer was extracted with

EtOAc. The combined organic extracts were washed

with brine, dried over MgSO₄, ﬁltered, and concentrated

to give 2.04 g (94%) of methyl (4-ﬂuorophenyl)-hydrox-

yacetate (60), which was used without further puriﬁca-

tion. MS(ESI+): m/z 201.0 (Mꢁ1).

(s, 1H), 6.95–7.07 (m, 3H), 7.36 (dd, J = 8.8, 5.1 Hz,

2H), 7.47 (dd, J = 7.9, 1.1 Hz, 1H), 7.51 (s, 1H). MS(E-

SI+): m/z 360.2 [M+1].

Step 6. A solution of 313 mg (0.709 mmol) of 87 in 3 mL

of anhydrous 1,4-dioxane and 1 mL of H₂O was treated

with 346 mg (1.59 mmol) of 54, 90 mg (2.12 mmol) of

LiCl, and 0.69 g (2.12 mmol) of Cs₂CO₃. The resulting

mixture was degassed by Ar sparge for 25 min.

Pd(PPh₃)₄(410 mg, 0.36 mmol) was added, and the mix-

ture was heated at 150 ꢂC by microwave heating for 2 h.

The black mixture was diluted with MeOH and ﬁltered

through a Celite pad, rinsing with MeOH. The resulting

solution was concentrated, dissolved in CH₂Cl₂, and

washed with 1 N HCl. The organic layer was washed

with saturated aqueous NaHCO₃and brine, dried over

MgSO₄, ﬁltered, and concentrated. Puriﬁcation by ﬂash

column chromatography (SiO₂, 100% CH₂Cl₂then gra-

dient to 90:10 CH₂Cl₂/MeOH), then (SiO₂, 100% EtOAc

then gradient to 90:10 EtOAc/MeOH) gave 134 mg

(42%) of 16 as a light-yellow solid that exists as a 1:1

mixture of restricted rotational isomers. ¹H NMR

(400 MHz, CDCl₃) d 1.08 (t, J = 7.6 Hz, 3H), 1.95 (quin-

tet, J = 7.1 Hz, 2H), 2.28 (q, J = 7.7 Hz, 2H), 3.28 (s,

3H), 3.43 (m, 2H), 4.06 (ddd, J = 7.3, 7.3, 3.3 Hz, 2H),

4.48 (br s, 1H), 4.55 (br s, 1H), 4.84 (br s, 2H), 5.73

(s, 1H), 6.89 (dd, J = 8.1, 1.7 Hz, 1H), 6.94 (dd,

J = 4.8, 1.6 Hz, 1H), 7.06 (ddd, J = 8.7, 8.7, 3.2 Hz,

2H), 7.10 (dd, J = 8.1, 2.0 Hz, 1H), 7.42 (dd, J = 7.32,

5.37 Hz, 2 H); MS(ESI+): m/z 452.2 [M+1].

Step 2. A suspension of 2.01 g (10.93 mmol) of 60, 2.43 g

(10.9 mmol) of 4-bromo-2-nitrophenol 48, and (10.6 g,

16.38 mmol) of polymer-supported PPh₃in 100 mL of

anhydrous CH₂Cl₂was cooled in an ice bath and treated

with 2.5 mL (12.06 mmol) of DIAD in a dropwise fash-

ion. The reaction mixture was stirred at 0 ꢂC to 16 h, al-

lowed to warm to room temperature, and stirred for

35 h. The mixture was ﬁltered through a medium frit,

and the ﬁltrate was washed with 1 N NaOH, 1 N HCl,

saturated aqueous NaHCO₃, and brine. The organic

layer was dried over MgSO₄, ﬁltered, and concentrated

to give 6.03 g of a sticky, light-yellow solid, which was

used directly without further puriﬁcation.

Step 3. The residue from Step 2 was dissolved in 100 mL

of glacial AcOH and treated with 11.05 g (198 mmol) of

iron dust. The mixture was heated at 60 ꢂC for 45 min.

The reaction mixture was diluted with EtOAc and ﬁl-

tered through Celite. The ﬁltrate was washed with

H₂O, saturated aqueous NaHCO₃, and brine, dried over

MgSO₄, ﬁltered, and concentrated to a light-yellow

solid. Trituration with CH₂Cl₂aﬀorded 4.96 g (78%)

of 6-bromo-2-(3,4-diﬂuorophenyl)-4H-benzo[1,4]oxa-

zin-3-one 68 as a light-yellow-tan solid that was used

without further puriﬁcation.

Diagnostic peaks for the restricted rotational isomer:

1.13 (t, J = 7.6 Hz, 3H), 2.33 (q, J = 7.57 Hz, 2H), 5.67

(s, 1H).

Step 4. A mixture of 2.47 g (7.66 mmol) of 68, 1.06 g

(7.67 mmol) of K₂CO₃, and 1.17 g (7.65 mmol) of 1-bro-

mo-3-methoxypropane in 100 mL of anhydrous CH₃CN

was heated to reﬂux for 14 h, cooled to room tempera-

ture, and ﬁltered through a pad of Celite. The ﬁltrate

was concentrated to give 3.08 g (100%) of 77 as a

6.1.14. 6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-2-(3,5-

diﬂuoro-phenyl)-4-(3-methoxy-propyl)-4H-benzo[1,4]oxa-

zin-3-one (17). Step 1. A solution of 2.00 g (10.6 mmol)

of 3,5-diﬂuoromandelic acid in 50 mL of MeOH was

treated with 0.5 mL concd HCl and heated to reﬂux

for 6 h. After cooling to room temperature, the solution

was concentrated under rotary evaporation. The residue

was dissolved in EtOAc and washed with H₂O and sat-

urated aqueous NaHCO₃. The organic layer was dried

over MgSO₄, ﬁltered, and concentrated to yield 1.84 g

(85%) of methyl 3,5-diﬂuoromandelate as a white solid

1

light-yellow oil that solidiﬁed upon standing. H NMR

(400 MHz, CDCl₃)d 1.94–2.01 (m, 2H), 3.35 (s, 3H),

3.38–3.46 (m, 2H), 4.05 (dq, J = 19.3, 7.2 Hz, 2H),

5.66 (s, 1H), 6.91 (d, J = 8.6 Hz, 1H), 6.99–7.06 (m,

2H), 7.10 (dd, J = 8.6, 2.2 Hz, 1H), 7.26 (d, J = 2.2 Hz,

1H), 7.32–7.37 (m, 2H); MS(ESI+): m/z 394.0, 396.0

([M+1], 1:1 ratio of Br isotopes).

1

that was used without further puriﬁcation. H NMR

(400 MHz, CDCl₃) d 3.50 (d, J = 5.4 Hz, 1H), 3.82 (s,

3H), 5.17 (d, J = 5.4 Hz, 1H), 6.78 (tt, J = 8.8, 2.3 Hz,

1H), 6.97–7.05 (m, 2 H).

Step 5. A solution of 1.29 g (3.27 mmol) of 77 in 15 mL

of anhydrous 1,4-dioxane was treated with 1.22 g

(4.80 mmol) of bis(pinacolato)diboron and 1.18 g

(3.68 mmol) of KOAc, and degassed by N₂sparge for

Step 2. To a solution of 2.4 g (11 mmol) of 4-bromo-2-

nitrophenol (48) and 1.84 g (9.08 mmol) of methyl 3,5-

diﬂuoromandelate in 50 mL of anhydrous CH₂Cl₂

under an Ar atmosphere, polymer-supported PPh₃

(4.8 g, 3 mmol/g) was added and the resulting suspen-

sion was stirred at room temperature for 10 min. The

mixture was cooled in an ice bath, and 2.10 ml

(10.86 mmol) of diisopropyl azadicarboxylate was

added in a dropwise fashion. The resulting yellow sus-

pension was stirred for 18 h while slowly warming to

room temperature. The resin was collected on a coarse

frit and rinsed with CH₂Cl₂(3·). The combined ﬁltrates

15 min.

PdCl₂(dppf)–CH₂Cl₂

complex

(163 mg

(0.20 mmol)) was added, and the resulting dark red sus-

pension was heated in a 110 ꢂC oil bath for 15 h. The

mixture was cooled to room temperature and ﬁltered

through a SiO₂pad, rinsing with EtOAc. The ﬁltrate

was concentrated and puriﬁed by ﬂash column chroma-

tography (SiO₂, 100% CH₂Cl₂then gradient to 10%

EtOAc/CH₂Cl₂) to yield 1.14 g (79%) of 87 as a viscous

yellow oil. ¹H NMR (400 MHz, CDCl₃) d 1.33 (s, 12 H)

2.00 (quintet, J = 6.4 Hz, 2H), 3.34 (s, 3H), 3.43 (ddd,

J = 6.0, 2.3 Hz, 3H), 4.13 (t, J = 7.8 Hz, 3H), 5.69

5934

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

were concentrated. Puriﬁcation by ﬂash column

chromatography (SiO₂, 5% EtOAc/hexanes then gradi-

ent to 15% EtOAc/hexanes) gave 3.3 g of ethyl (4-bro-

mo-2-nitro-phenoxy)-(3,5-diﬂuoro-phenyl)-acetate. ¹H

NMR (400 MHz, CDCl₃)d 3.77 (s, 3H), 5.70 (s, 1H),

6.86 (tt, J = 8.8, 2.4 Hz, 1H), 6.89 (d, J = 9.0 Hz, 1H),

7.14–7.22 (m, 2H), 7.63 (dd, J = 8.8, 2.4 Hz, 1H), 8.08

(d, J = 2.4 Hz, 1H).

(m, 21H), 7.08 (d, J = 4.0 Hz, 1H), 7.49 (dd, J = 8.0,

2.0 Hz, 1H), 7.53 (d, J = 2.0 Hz, 1H); MS(ESI+): m/z

414.0 [M+1].

Step 6. A mixture of 181 mg (0.836 mmol) of 5-bromo-6-

ethyl-2,4-diamine 54,¹⁷320 mg (0.697 mmol) 88, 89 mg

(0.089 mmol) LiCl, and 81 mg (0.07 mmol) Pd(PPh₃)₄

in 30 mL of 1,4-dioxane and 4 mL H₂O was degassed

by sparging with Ar for 10 min. Cs₂CO₃(681 mg,

2.09 mmol) was added, and the mixture was heated to

reﬂux for 18 h. The black suspension was diluted with

EtOAc and ﬁltered through a Celite plug. The ﬁltrate

was washed with H₂O, dried over Na₂SO₄, ﬁltered,

and concentrated. Puriﬁcation by ﬂash column chroma-

tography (SiO₂, 100% EtOAc then gradient to 40%

MeOH/EtOAc) provided 327 mg (22%) of 6-(2,4-diami-

no-6-ethyl-pyrimidin-5-yl)-2-(3,5-diﬂuoro-phenyl)-4-(3-

methoxy-propyl)-4H-benzo[1,4]oxazin-3-one 17 as a

glassy solid. IR(ATR) 3322, 3172, 2974, 1678, 1597,

Step 3. Ethyl (4-bromo-2-nitro-phenoxy)-(3,5-diﬂuoro-

phenyl)-acetate (3.3 g, 7.74 mmol) was dissolved in

125 mL of glacial acetic acid. Fe dust (4.3 g, 77 mmol)

was added, and the resulting black suspension was

heated in a 60 ꢂC oil bath for 18 h. After cooling to

room temperature, the gray suspension was ﬁltered

through a Celite pad, rinsing with EtOAc (200 mL).

The ﬁltrate was concentrated, and the residue was dis-

solved in EtOAc and washed with H₂O (2·). The organ-

ic layer was dried over MgSO₄, ﬁltered, and

concentrated to a pale yellow solid that was used with-

1555, 1442, 1262, 1119 cm^ꢁ^{1 1}H NMR (400 MHz,

;

1

out further puriﬁcation. H NMR (400 MHz, CDCl₃)

DMSO-d₆) d 0.94 (m, 3 H), 1.76 (m, 2H), 2.09 (m,

2H), 3.12 (s, 3H), 3.32 (t, J = 6.1 Hz, 2H), 3.95 (m,

2H), 5.53 (br s, 2H), 5.83 (m, 2H), 5.92 (s, 1H), 6.81

(dd, J = 8.1, 1.7 Hz, 1H), 6.99 (s, 1H), 7.10 (d,

J = 6.6 Hz, 2H), 7.14 (d, J = 8.3 Hz, 1H), 7.27 (tt,

J = 9.3, 2.2 Hz, 2 H). HRMS (ESI) m/z Calcd for

C₂₄H₂₆F₂N₅O₃(M+H)⁺470.2003. Found: 470.2018.

Elem. Anal. Calcd for C₂₄H₂₅F₂N₅O₃Æ0.3EtOAc: C,

62.22; H, 5.92; N, 13.95. Found: C, 62.20; H, 5.88; N,

14.20.

d 5.67 (s, 1H), 6.80 (tt, J = 8.8, 2.3 Hz, 1H), 6.96–6.99

(m, 2H), 7.00–7.03 (m, 2H), 7.14 (dd, J = 8.6, 2.2 Hz,

1H), 8.76 (br s, 1H). MS(ESI+): m/z 339.9, 340.9

[M+1, 1:1 ratio of Br isotopes].

Step 4. The solid residue was dissolved in 60 mL of

anhydrous DMF, and the solution was cooled in a ice

bath. NaH (263 mg, 6.59 mmol, 60% dispersion in min-

eral oil) was added in portions, and the reaction mixture

was stirred at 0 ꢂC for 30 min. 1-Bromo-3-methoxypro-

pane (1.1 g, 7.1 mmol) was added, and the mixture was

stirred at room temperature for 18 h. Excess hydride

was quenched by the slow addition of H₂O. The result-

ing mixture was diluted with H₂O and extracted with

EtOAc (2·). The combined organic layers were dried

over MgSO₄, ﬁltered, and concentrated. Puriﬁcation

by ﬂash column chromatography (SiO₂, 5% EtOAc/hex-

anes then gradient to 15% EtOAc/hexanes) aﬀorded

6.1.15. 6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-4-(3-meth-

oxy-propyl)-2-(2,3-diﬂuoro-phenyl)-4H-benzo[1,4]oxazin-

3-one (18). Step 1. A solution of 4.48 g (22.2 mmol) of

methyl (2,3-diﬂuorophenyl)-hydroxyacetate 62 and

4.94 g (22.2 mmol) of 4-bromo-2-nitrophenol 48 in

225 mL of anhydrous CH₂Cl₂was treated with 21.5 g

(33.3 mmol, 1.55 mmol/g) of polymer-supported PPh₃

and cooled in an ice bath. DIAD (5.06 mL, 24.4 mmol)

was added dropwise, and the resulting bright-orange

suspension was stirred at 0 ꢂC for 1 h, warmed to room

temperature, and stirred for an additional 45 h. The re-

sin was removed by ﬁltration through a coarse frit, rins-

ing with CH₂Cl₂. The ﬁltrate was washed with 1 N

NaOH, H₂O, and brine, dried over MgSO₄, ﬁltered,

and concentrated to give 12.74 g of a moist yellow solid

that was used without puriﬁcation in the following step.

1

0.96 g (50%) of 78 as a clear oil. H NMR (400 MHz,

CDCl₃) d 1.91–1.99 (m, 2H), 3.33 (s, 3H), 3.35–3.44

(m, 2H), 3.95–4.10 (m, 2H), 5.65 (s, 1H), 6.74 (tt,

J = 8.8, 2.1 Hz, 1H), 6.94 (m, 2H), 6.95 (d, J = 8.6 Hz,

1H), 7.12 (dd, J = 8.4, 2.1 Hz, 1H), 7.26 (d, J = 2.2 Hz,

1H); MS(ESI+): m/z 414.0 [M+1].

Step 5. A 250 mL round bottom ﬂask was ﬂushed with

Ar and charged with 610 mg (2.40 mmol) of bis(pinaco-

lato)diboron, 643 mg (6.55 mmol) KOAc, and 357 mg

(0.437 mmol) of PdCl₂(dppf)–CH₂Cl₂complex. A solu-

tion of 900 mg (2.18 mmol) of 78 in 50 mL of anhydrous

1,4-dioxane was added, and the resulting dark orange

suspension was heated at 110 ꢂC for 16 h. The resulting

black mixture was ﬁltered through a Celite plug and

concentrated. The residue was partitioned between

EtOAc and H₂O. The organic layer was washed with

brine, dried over MgSO₄, ﬁltered, and concentrated.

Puriﬁcation by ﬂash column chromatography (SiO₂,

100% hexanes then gradient to 50% EtOAc/hexanes)

gave 337 mg (34%) of 88 as a solid foam. ¹H NMR

(400 MHz, CDCl₃) d 1.33 (s, 12H), 1.99 (quintet,

J = 6.84 Hz, 2H), 3.33 (s, 3H), 3.36–3.46 (m, 2H), 4.12

(m, 2H), 6.71 (tt, J = 8.0, 4.0 Hz, 1H), 6.90–7.00

Step 2. The residue from the preceding step was dis-

solved in 100 mL of glacial acetic acid and treated with

12.25 g (219 mmol) of iron dust. The resulting black sus-

pension was heated at 60 ꢂC for 1 h. After cooling to

room temperature, the gray suspension was ﬁltered

through Celite, rinsing with EtOAc. The ﬁltrate was

washed with H₂O (3·), saturated aqueous NaHCO₃

(2·), and brine, dried over MgSO₄, ﬁltered, and concen-

trated. The residue was dissolved in 50 mL of CH₂Cl₂

and precipitated by addition of 50 mL of hexanes and

cooling in an ice bath. The solid was collected on a med-

ium frit to give 2.347 g (32%) of 6-bromo-2-(3,4-diﬂu-

orophenyl)-4H-benzo[1,4]oxazin-3-one 70 as a white

1

solid. H NMR (400 MHz, DMSO-d₆) d 6.01 (s, 1H),

6.92–6.98 (m, 1H), 7.10 (s, 1H), 7.11 (dd, J = 7.6,

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

5935

2.4 Hz, 2H), 7.23–7.32 (m, 2H), 7.52 (dddd, J = 10.2,

7.8, 7.1, 3.12 Hz, 1H), 11.16 (s, 1H); MS(ESI+): m/z

339.9, 341.9 ([M⁺], 1:1 ratio of Br isotopes).

(125 mmol) of ethyl 2-bromoisobutyrate, and heated to

reﬂux for 6 h. An additional 18.5 mL of ethyl 2-bromo-

isobutyrate was added and the suspension was heated at

reﬂux for an additional 18 h. After cooling to room

temperature, the mixture was diluted with EtOAc and

ﬁltered through Celite. The ﬁltrate was concentrated

to an orange oil that was used without further

puriﬁcation.

Step 3. A solution of 585 mg (1.72 mmol) of 70 in 9 mL

of anhydrous CH₃CN was treated with 316 mg

(3.06 mmol) of 1-bromo-3-methoxypropane and

285 mg (2.06 mmol) of K₂CO₃and heated to reﬂux for

18 h. The mixture was cooled to room temperature, di-

luted with EtOAc, ﬁltered through a Celite plug, and

concentrated. Puriﬁcation by ﬂash column chromato-

graphy (SiO₂. 5% EtOAc/hexanes then gradient to 20%

EtOAc/hexanes) aﬀorded 289 mg (38%) of 79 as a vis-

Step 2. The oil was dissolved in 500 mL of glacial acetic

acid and treated with 58.1 g (1040 mmol) of Fe powder.

The black suspension was heated at 60 ꢂC for 3 h. After

cooling to room temperature, the gray suspension was

ﬁltered through Celite, which was rinsed with 200 mL

of EtOAc (4·). The combined ﬁltrates were concen-

trated to dryness. The solid residue was triturated with

EtOAc and ﬁltered through Celite to remove inorganic

salts. The ﬁltrate was washed with H₂O (3·) and satu-

rated aqueous NaHCO₃, dried over MgSO₄, ﬁltered,

and concentrated. The solid residue was recrystallized

from hexanes to yield 14.72 g of 6-bromo-2,2-di-

methyl-4H-benzo[1,4]oxazin-3-one 93 as a tan-white so-

lid. The mother liquor was concentrated and the residue

recrystallized from hexanes to yield an additional 4.46 g

1

cous oil. H NMR (400 MHz, CDCl₃) d 2.01 (quintent,

J = 6.50 Hz, 2H), 3.37 (s, 3H), 3.46 (t, J = 5.8 Hz, 2H),

4.08 (ddd, J = 7.0, 7.0, 3.3 Hz, 2H), 5.80 (s, 1H), 6.87

(d, J = 8.4 Hz, 1H), 7.07–7.11 (m, 2H), 7.12 (dd,

J = 8.6, 2.1 Hz, 1H), 7.17–7.25 (m, 1H), 7.34 (d,

J = 2.1 Hz, 1 H); MS(ESI+): m/z 411.9, 413.9 ([M⁺],

1:1 ratio of Br isotopes).

Step 4. A solution of 289 mg (0.70 mmol) of 79 in 20 mL

of anhydrous 1,4-dioxane was treated with 267 mg

(1.05 mmol) of bis(pinacolato)diboron and 206 mg

(2.10 mmol) of KOAc, and degassed by N₂sparge for

1

of 93 (72% total yield). H NMR (400 MHz, CDCl₃) d

15 min.

PdCl₂(dppf)–CH₂Cl₂

complex

(29 mg

1.55 (s, 6H), 6.83 (d, J = 8.6 Hz, 1H), 7.01 (d,

J = 2.2 Hz, 1H), 7.08 (dd, J = 8.3, 2.2 Hz, 1H), 9.48

(br s, 1H); MS(ESI+): m/z 255.9, 257.9 [M+1, 1:1 ratio

of Br isotopes].

(0.036 mmol)) was added, and the resulting dark red

suspension was heated at reﬂux for 14 h. The mixture

was cooled to room temperature and treated with

342 mg (1.58 mmol) of 54, 89 mg (2.10 mmol) of LiCl,

685 mg (2.10 mmol) of Cs₂CO₃, and 5 mL of H₂O.

The resulting mixture was degassed by N₂sparge for

15 min. Pd(PPh₃)₄(203 mg, 0.18 mmol) was added,

and the mixture was heated at reﬂux for 4 h. The black

mixture was diluted with CH₂Cl₂and washed with H₂O,

1 N HCl, saturated aqueous NaHCO₃, and brine, dried

over MgSO₄, and ﬁltered. The solution was partially

concentrated and loaded onto a 20 g pad of SiO₂. The

column was washed with CH₂Cl₂until the ﬁltrates were

colorless. The product was then eluted from the column

by washing with MeOH. The ﬁltrate was concentrated,

and puriﬁcation by ﬂash column chromatography

(SiO₂, 100% EtOAc then gradient to 10% MeOH/

EtOAc), gave 86 mg (26%) of 18 as an amber solid that

exists as a 1:1 mixture of restricted rotational isomers.

¹H NMR (400 MHz, CDCl₃) d 1.11 (t, J = 7.6 Hz,

3H), 1.88–2.05 (m, 2H), 2.33 (q, J = 7.6 Hz, 2H), 3.29

(s, 3H), 3.45 (t, J = 6.0 Hz, 2H), 4.00–4.17 (m, 2H),

4.54 (bs, 2H), 4.82 (br s, 2H), 5.31 (s, 1H), 6.90 (ddd,

J = 8.0, 2.0, 2.0 Hz, 1H), 02 (d, J = 2.0 Hz, 1H), 7.09

(dd, J = 8.0, 2.0 Hz, 1H), 7.12–7.17 (m, 2H), 7.19–7.26

(ddd, J = 9.9, 7.8, 7.8, 2.0 Hz, 1H); MS(ESI+): m/z

470.1 [M+1].

Step 3. A solution of 14.9 g (58.2 mmol) of 6-bromo-2,2-

dimethyl-4H-benzo[1,4]oxazin-3-one 93 in 150 mL of

anhydrous DMF was cooled in an ice bath and treated

with 2.8 g (70.0 mmol) of a 60% dispersion of NaH in

mineral oil in a single portion. The resulting mixture

was stirred at 0 ꢂC for 15 min. 1-Bromo-3-methoxypro-

pane (11.0 g, 70.0 mmol) was then added in a dropwise

fashion over 5 min. The reaction mixture was stirred at

0 ꢂC for 1 h, warmed to room temperature, and stirred

for 16 h. The mixture was diluted with 600 mL of H₂O

and extracted with EtOAc (3· 200 mL). The combined

organics were washed with H₂O (2· 50 mL) and brine,

dried over MgSO₄, ﬁltered, and concentrated. Puriﬁca-

tion by ﬂash column chromatography (SiO₂, 100% hex-

anes then gradient to 40% EtOAc/hexanes) aﬀorded

1

15.8 g (80%) of 94 as a clear oil. H NMR (400 MHz,

DMSO-d₆) d 1.39 (s, 6H), 1.76 (quintet, J = 7.1 Hz,

2H), 3.22 (s, 3H), 3.33 (t, J = 6.0 Hz, 2H), 3.92 (t,

J = 7.1 Hz, 2H), 6.94 (d, J = 8.6 Hz, 1H), 7.17 (dd,

J = 8.4, 2.1 Hz, 1H), 7.36 (d, J = 2.2 Hz, 1H); MS(ESI+):

m/z 328.0 330.0 [M+1, 1:1 ratio of Br isotopes].

Step 4. A mixture of 573 mg (1.75 mmol) of 94, 532 mg

(2.10 mmol) of bis(pinacolato)diboron, and 514 mg

(5.24 mmol) of KOAc in 9 mL of anhydrous 1,4-diox-

ane was degassed by Ar sparge for 20 min.

PdCl₂(dppf)–CH₂Cl₂complex (71 mg, 0.087 mmol)

was added, and the resulting deep orange suspension

was heated in a 100 ꢂC oil bath for 2.5 h. After cooling

to room temperature, 568 mg (2.62 mmol) of 54, 1.71 g

(5.24 mmol) of Cs₂CO₃, 222 mg (5.24 mmol) of LiCl,

and 1.1 mL of H₂O were added. The mixture was de-

gassed by Ar sparge for 10 min, and 202 mg

Diagnostic peaks for the restricted rotational isomer:

1.12 (t, J = 7.6 Hz, 3H), 2.34 (q, J = 7.6 Hz, 2H), 5.82

(br s, 2H), 5.85 (br s, 2 H).

6.1.16. 6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-4-(3-meth-

oxypropyl)-2,2-dimethyl-4H-benzo[1,4]oxazin-3-one (19).

Step 1. A solution of 22.7 g (104 mmol) of 4-bromo-2-

nitrophenol 48 in 200 mL of anhydrous CH₃CN was

treated with 17.2 g (125 mmol) of K₂CO₃and 18.3 mL

5936

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

(0.175 mmol) Pd(PPh₃)₄was added. The mixture was

heated in a 100 ꢂC oil bath for 18 h. After cooling to

room temperature, the mixture was diluted with EtOAc

and washed with H₂O and brine. The organic layer was

dried over MgSO₄, ﬁltered, and concentrated. Puriﬁca-

tion by ﬂash column chromatography (SiO₂, 100%

CH₂Cl₂then gradient to 85:15 CH₂Cl₂/MeOH) gave

115 mg (17%) of 19 as a gray solid. IR (ATR) 3427,

3153, 2976, 1666, 1631, 1555, 1449, 1385, 1361, 1263,

Step 3. A solution of 885 mg (2.59 mmol) of 96, 790 mg

(3.11 mmol) bis(pinacolato)diboron, and 764 mg

(7.78 mmol) of KOAc in 15 mL of anhydrous 1,4-diox-

ane was degassed by N₂sparge for 20 min.

PdCl₂(dppf)–CH₂Cl₂complex (106 mg, 0.13 mmol) was

added, and the mixture was heated in an 85 ꢂC oil bath

for 18 h. After cooling to room temperature, the mixture

was diluted with EtOAc, ﬁltered through a Celite plug,

and concentrated. Puriﬁcation by ﬂash column chroma-

tography (SiO₂,75% EtOAc/hexanes then gradient to

100% EtOAc) gave 1.04 g (100%) of N-{2-[2,2-dimethyl-

3-oxo-6-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-

2,3-dihydro-benzo[1,4]oxazin-4-yl]-ethyl}-acetamide as a

dark solid foam. MS(ESI+): m/z 389.2 [M+1].

1122, 811 cm^ꢁ1

;

¹H NMR (400 MHz, DMSO-d₆)d

0.98 (t, J = 7.6 Hz, 3H), 1.42 (s, 3H), 1.43 (s, 3H),

1.75 (quintet, J = 6.6 Hz, 2H), 2.14 (qd, J = 7.5,

1.3 Hz, 2H), 3.17 (s, 3H), 3.32 (m, 2H), 3.91 (m, 2H),

5.59 (bs, 2H), 5.83 (s, 2H), 6.79 (dd, J = 8.0, 4.0 Hz,

1H), 6.92 (d, J = 4.0 Hz, 1H), 7.00 (d, J = 8.0 Hz,

1H); ¹³C NMR (100 MHz, DMSO-d₆) d 13.2, 27.5,

39.6, 39.8, 57.9, 69.3, 77.2, 128.8, 130.2, 141.8, 162.0,

162.3, 166.5, 167.6. HRMS (ESI) m/z Calcd for

C₂₀H₂₈N₅O₃(M+H)⁺386.2191. Found 386.2174. Elem.

Anal. Calcd for C₂₀H₂₇N₅O₃: C, 62.32; H, 7.06; N,

18.17. Found: C, 62.04; H, 7.10; N, 17.88.

Step 4. A mixture of 397 mg (1.02 mmol) of N-{2-

[2,2-dimethyl-3-oxo-6-(4,4,5,5-tetramethyl-[1,3,2]dioxaboro-

lan-2-yl)-2,3-dihydro-benzo[1,4]oxazin-4-yl]-ethyl}-acet-

amide, 266 mg (1.23 mmol) of 54, 515 mg (3.07 mmol)

of CsOHÆH₂O, and 130 mg (3.07 mmol) of LiCl was sus-

pended in 6 mL of anhydrous 1,4-dioxane and 0.75 mL

of H₂O. After degassing by N₂sparge for 20 min,

118 mg (0.10 mmol) of Pd(PPh₃)₄was added, and the

resulting yellow suspension was heated in a 100 ꢂC oil

bath for 18 h. The reaction mixture was diluted with

EtOAc, dried over MgSO₄, and ﬁltered through a Celite

plug. The ﬁltrate was concentrated, and the residue puri-

ﬁed by ﬂash column chromatography (SiO₂, 95:5

CH₂Cl₂/MeOH then gradient to 80:20 CH₂Cl₂/MeOH)

to aﬀord 149 mg (37%) of 20 as a solid white foam.

IR(ATR) 3441, 3339, 1664, 1618, 1549, 1441, 1384,

6.1.17. N-(2-(6-(2,4-diamino-6-ethyl-pyrimidin-5-yl)-2,2-

dimethyl-3-oxo-2,3-dihydrobenzo[b][1,4]-oxazin-4-yl)-

ethyl)-acetamide (20). Step 1. A solution of 2.50 g

(9.76 mmol) of 6-bromo-2,2-dimethyl-4H-benzo[1,4]

oxazin-3-one 93 in 50 mL of anhydrous DMF was

cooled in an ice bath under an Ar atmosphere and trea-

ted with 429 mg (10.7 mmol) of 60% NaH in mineral oil.

The resulting gray suspension was stirred at 0 ꢂC for

15 min. 15-Crown-5 (0.1 mL) and 1.41 g (11.71 mmol)

of bromoacetonitrile were added. The ice bath was re-

moved, and the reaction mixture was stirred at room

temperature for 18 h. Excess hydride was quenched by

the slow addition of H₂O, and the resulting slurry was

poured into 1 L of H₂O. The aqueous layer was ex-

tracted with EtOAc (3·). The combined organics were

washed with H₂O (2·) and brine, dried over MgSO₄, ﬁl-

tered, and concentrated. Puriﬁcation by ﬂash column

chromatography (SiO₂, 5% EtOAc/hexanes then gradi-

ent to 20% EtOAc/hexanes) aﬀorded 3.00 g (98%) of 2-

(6-bromo-2,2-dimethyl-3-oxo-2,3-dihydrobenzo-[b][1,4]-

1358, 1263 cm^ꢁ^{1 1}H NMR (400 MHz, DMSO-d₆) d

;

0.93 (t, J = 7.5 Hz, 3H), 1.38 (s, 6H), 1.66 (s, 3H), 2.10

(q, J = 7.6 Hz, 3H), 3.12 (m, 2H), 3.83 (dd, J = 6.9,

6.9 Hz, 2H), 5.52 (br s, 2H), 5.78 (br s, 2H), 6.73 (dd,

J = 8.0, 1.7 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 7.00 (d,

J = 1.8 Hz, 1H), 7.95 (t, J = 6.0 Hz, 1H); ¹³C NMR

(100 MHz, DMSO-d₆) d 13.8, 23.1, 24.2, 28.1, 36.3,

77.8, 106.8, 117,6, 118.3, 126.4, 129.7, 130.7, 142.4,

162.5, 163.0, 168.5, 170.2. HRMS (ESI+) Calcd for

C₂₀H₂₇N₆O₃(M+H)⁺: 399.2144. Found: 399.2138.

1

oxazin-4-yl)- acetonitrile 95 as a clear viscous oil. H

6.1.18. N-{2-[6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-2-

methyl-3-oxo-2-triﬂuoromethyl-2,3-dihydro-benzo[1,4]-

oxazin-4-yl]-ethyl}-acetamide (21). Step 1. A solution of

1.16 g (6.74 mmol) of methyl 3,3,3-triﬂuoro-2-methyl-

propionate in 20 mL of anhydrous THF was treated

with 308 mg (7.70 mmol) of a 60% NaH dispersion in

mineral oil. The resulting orange solution was stirred

at room temperature for 10 min. Then, 5 drops of 15-

crown-5 were added via syringe, followed by 1.41 g

(6.42 mmol) of 4-bromo-2-nitroﬂuorobenzene 97. The

resulting orange solution was stirred at room tempera-

ture for 17 h and then heated in a 45 ꢂC oil bath for

8 h. Excess hydride was quenched by the careful addi-

tion of MeOH. The solution was diluted with EtOAc,

and then washed with brine, dried over MgSO₄, ﬁltered,

and concentrated to dryness. Puriﬁcation by ﬂash col-

umn chromatography (SiO₂, 100% hexanes then gradi-

ent to 10% EtOAc/hexanes) yielded 1.60 g of 98.

NMR (400 MHz, CDCl₃) d 1.51 (s, 6H), 4.79 (s, 2H),

6.89 (d, J = 8.5 Hz, 1H), 7.12 (d, J = 2.2 Hz, 1H), 7.20

(dd, J = 8.6, 2.2 Hz, 1H). MS(ESI+): m/z 296.9, 298.9

([M+1], Br isotopes, 1:1 ratio).

Step 2. A solution of 1.03 g (3.49 mmol) of 95, 0.49 mL

(5.24 mmol) of Ac₂O, and 50 mL of THF was treated

with 1.0 g of Raney nickel. The reaction mixture was

shaken under 50 psi of H₂pressure for 12 h. The catalyst

was removed by ﬁltration through a Celite plug, and the

ﬁltrate was concentrated. Puriﬁcation by ﬂash column

chromatography (SiO₂, 5% EtOAc/hexanes then gradi-

ent to 100% EtOAc) yielded 886 mg (74%) of N-(2-(6-

bromo-2,2-dimethyl-3-oxo-2,3-dihydrobenzo[b][1,4]

1

oxazin-4-yl)- ethyl)-acetamide 96 as a white solid. H

NMR (400 MHz, CDCl₃) d 1.49 (s, 6H), 1.97 (s, 3H),

3.54 (q, J = 5.6 Hz, 2H), 4.05 (t, J = 6.4 Hz, 2H), 5.97

(br s, 1H), 6.84 (d, J = 8.6 Hz, 1H), 7.12 (dd, J = 8.6,

2.2 Hz, 1H), 7.34 (d, J = 2.2 Hz, 1H). MS(ESI+): m/z

341.0, 343.0 ([M+1], Br isotopes, 1:1 ratio).

Step 2. A solution of 1.60 g (4.14 mmol) of 98 in 20 mL

of glacial acetic acid was treated with 2.31 g (41.4 mmol)

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

5937

of iron dust and heated in a 60 ꢂC oil bath for 4 h. The

resulting gray suspension was cooled to room tempera-

ture, diluted with EtOAc, and ﬁltered through a Celite

pad. The ﬁltrate was concentrated to dryness. The solid

residue was dissolved in EtOAc and washed with H₂O

(3·) and saturated aqueous NaHCO₃. The organic layer

was dried over MgSO₄, ﬁltered, and concentrated. Puri-

ﬁcation by ﬂash column chromatography (SiO₂, 100%

hexanes then gradient to 10% EtOAc/hexanes) gave

1.105 g (86%) of 105 as a clear oil. MS(ESI+): m/z

307.9, 309.9 [M+1], Br isotopes, 1:1 ratio.

60% CH₃CN/H₂O/0.1% TFA) yielded 145 mg of the

TFA salt of 21 as a white solid. IR(ATR) 3398, 3124,

1675, 1638, 1532, 1452, 1389, 1373, 1194, 1157,

1

1140 cm^ꢁ1; H NMR (400 MHz, DMSO-d₆) d 0.99 (t,

J = 7.6 Hz, 3H), 1.63 (s, 3H), 1.65 (s, 3H), 2.22 (q,

J = 7.42 Hz, 2H), 3.19 (m, 2H), 3.91 (m, 2 H) 6.82 (d,

J = 8.6 Hz, 1H), 6.91 (dt, J = 8.2, 2.1 Hz, 1H), 7.14 (t,

J = 7.8 Hz, 1H), 7.26 (dd, J = 8.4, 1.4 Hz, 1H), 7.56

(bs, 2 H) 7.98 (t, J = 5.9 Hz, 1H), 8.15 (d, J = 10.6 Hz,

1H)

12.36

(s,

1H).

HRMS

Calcd

[C₂₀H₂₃F₃N₆O₃+H]: 453.1862. Found: 453.1858.

for

1

Step 3. A solution of 1.11 g (3.56 mmol) of 105 and

0.30 mL (4.28 mmol) of bromoacetonitrile in 20 mL of

anhydrous CH₃CN was treated with 0.59 g (4.28 mmol)

of K₂CO₃, and heated to reﬂux for 18 h. The mixture

was diluted with EtOAc, ﬁltered through a Celite plug,

and concentrated under reduced pressure. Puriﬁcation

by ﬂash column chromatography (SiO₂, 5% EtOAc/hex-

anes then gradient to 15% EtOAc/hexanes) yielded

0.96 g (77%) of 115 as a white solid. ¹H NMR

(400 MHz, CDCl₃)d 1.79 (s, 3H), 4.78 (d,J = 17.6 Hz,

1H), 4.94 (d, J = 17.6 Hz, 1H), 6.99 (d, J = 8.6 Hz,

1H), 7.15 (d, J = 2.1 Hz, 1H), 7.27 (dd, J = 8.8, 2.2 Hz,

1H); MS(ESI+): m/z 347.9, 349.9 ([Mꢁ1], 1:1 ratio of

Br isotopes).

Diagnostic peaks for the minor rotational isomer: H

NMR (400 MHz, DMSO-d₆) d 0.99 (t, J = 7.6 Hz,

3H), 1.67 (s, 2H), 3.14 (m, 1H), 3.32 (m, 1H), 3.83 (m,

1H), 3.98 (m, 1H).

6.1.19. 6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-2-ethyl-4-

(3-methoxy-propyl)-2-methyl-4H-benzo[1,4]oxazin-3-one

(22). Step 1. A solution of 2.52 g (19.1 mmol) of methyl

2-methyl-2-hydroxy-butanate in 80 mL of anhydrous

THF was treated with 873 mg (21.8 mmol) of a 60%

NaH dispersion in mineral oil. The resulting gray sus-

pension was stirred at room temperature for 10 min.

Then, 5 drops of 15-crown-5 were added via syringe, fol-

lowed by 2.24 mL (18.2 mmol) of 4-bromo-2-nitroﬂu-

orobenzene 97. The resulting yellow suspension was

stirred at room temperature for 17 h. Excess hydride

was quenched by the careful addition of MeOH. The

solution was diluted with EtOAc, washed with brine,

dried over MgSO₄, ﬁltered, and concentrated to dryness.

Puriﬁcation by ﬂash column chromatography (SiO₂,

100% hexanes then gradient to 20% EtOAc/hexanes)

aﬀorded 3.89 g (64%) of 99 as a yellow oil.

Step 4. A solution of 0.96 g (2.74 mmol) of 115 and

1 mL of Ac₂O in 50 mL of THF was treated with 1.0 g

Raney nickel. The resulting suspension was shaken un-

der a 22 psi H₂atmosphere for 1 h. The mixture was ﬁl-

tered through Celite and concentrated. Puriﬁcation by

ﬂash column chromatography (SiO₂, 15% EtOAc/hex-

anes then gradient to 70% EtOAc/hexanes) yielded

300 mg (28%) of 120 as a white solid. ¹H NMR

(400 MHz, CDCl₃) d 1.74 (s, 3H); 1.95 (s, 3H), 3.52

(q, J = 6.5 Hz, 2H), 3.99 (ddd, J = 20.5, 12.7, 6.5 Hz,

1H); 4.18 (ddd, J = 21.1, 14.1, 6.8 Hz, 1H), 5.79 (br s,

1H), 6.89 (d, J = 8.6 Hz, 1H), 7.15 (dd, J = 8.6, 2.2 Hz,

1H), 7.41 (d, J = 2.15 Hz, 1H); MS(ESI+): m/z 394.9,

396.9 ([Mꢁ1], 1:1 ratio of Br isotopes).

Step 2. A solution of 3.55 g (10.7 mmol) of 99 in 150 mL

of glacial acetic acid was treated with 5.97 g (107 mmol)

of iron dust and heated in a 60 ꢂC oil bath for 18 h. The

resulting gray suspension was cooled to room tempera-

ture, diluted with EtOAc, and ﬁltered through a Celite

pad. The ﬁltrate was concentrated to dryness. The solid

residue was dissolved in EtOAc and washed with H₂O

(3·) and saturated aqueous NaHCO₃. The organic layer

was dried over MgSO₄, ﬁltered, and concentrated to

yield 2.83 g (98%) of 106 as an opaque oil that was used

without further puriﬁcation. MS(ESI+): m/z 272.0,

274.0 [M+1], Br isotopes, 1:1 ratio.

Step 5. A solution of 295 mg (0.747 mmol) of 120,

227 mg (0.89 mmol) of bis(pinacolato)diboron, and

220 mg (2.24 mmol) of KOAc in 10 mL of anhydrous

1,4-dioxane was degassed by Ar sparge for 15 min.

PdCl₂(dppf)–CH₂Cl₂complex (31 mg, 0.037 mmol)

was added, and the resulting red-orange mixture was

heated at 90 ꢂC for 18 h. After cooling to room temper-

ature, the black mixture was diluted with EtOAc, ﬁltered

through a Celite plug, and concentrated. The crude res-

idue was dissolved in 12 mL of anhydrous 1,4-dioxane

and 1.5 mL of H₂O. LiCl (95 mg, 2.24 mmol), 194 mg

(0.90 mmol) of 54, and 376 mg (2.24 mmol) of CsOHÆ-

H₂O was added, and the resulting suspension was de-

gassed by Ar sparge for 20 min. Pd(PPh₃)₄(86 mg,

0.075 mmol) were added, and the mixture was heated

in a 100 ꢂC oil bath for 23 h. The mixture was diluted

with EtOAc, ﬁltered through a Celite plug, and concen-

trated. Puriﬁcation by ﬂash column chromatography

(SiO₂, 100:0 CH₂Cl₂/MeOH then gradient to 80:20

CH₂Cl₂/MeOH) followed by preparatory reverse phase

HPLC (10% CH₃CN/H₂O/0.1% TFA then gradient to

Step 3. A solution of 1.50 g (5.6 mmol) of 106 in 25 mL

of anhydrous DMF was cooled in an ice bath and trea-

ted with 290 mg (7.2 mmol) of a 60% dispersion of NaH

in mineral oil in portions. The resulting mixture was stir-

red at 0 ꢂC for 30 min and then treated with 1.0 g

(6.7 mmol) of 1-bromo-3-methoxypropane. The ice bath

was removed, and the reaction mixture was stirred at

room temperature for 16 h. Excess NaH was carefully

quenched by the dropwise addition of H₂O. The mixture

was diluted with H₂O and extracted with EtOAc (3·).

The combined organics were dried over MgSO₄, ﬁltered,

and concentrated. Puriﬁcation by ﬂash column chroma-

tography (SiO₂. 5% EtOAc/hexanes then gradient to

20% EtOAc/hexanes) aﬀorded 1.65 mg (87%) of 112.

¹H NMR (400 MHz, CDCl₃) d 0.94 (t, J = 7.5 Hz,

5938

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

3H), 1.44 (s, 3H), 1.69 (q, J = 14.34, 7.4 Hz, 2H), 1.83–

1.97 (m, 4H), 3.34 (s, 3H), 3.39 (t, J = 6.0 Hz, 2H), 3.87–

4.05 (m, 2H), 6.81 (d, J = 8.6 Hz, 1 H), 7.06 (dd, J = 8.6,

2.2 Hz, 1H), 7.19 (d, J = 2.2 Hz, 1H); MS(ESI+): m/z

342.1, 344.1 ([M⁺], 1:1 ratio of Br isotopes).

dryness. The solid residue was dissolved in EtOAc and

washed with H₂O (3·) and saturated aqueous NaHCO₃.

The organic layer was dried over MgSO₄, ﬁltered, and

concentrated. Puriﬁcation by ﬂash column chromatog-

raphy (SiO₂, 5% EtOAc/hexanes then gradient to 20%

EtOAc/hexanes) aﬀorded 821 mg (35%) of 107 as a yel-

low oil to yield 531 mg (82%) of 107 as a tan-white solid.

MS(ESI+): m/z 284.0, 286.0 [M+1], Br isotopes, 1:1

ratio.

Step 4. A solution of 1.2 g (5.6 mmol) of 112 in 40 mL of

anhydrous 1,4-dioxane was treated with 1.2 g

(4.6 mmol) of bis(pinacolato)diboron, 1.0 g (11.0 mmol)

of KOAc, and 290 mg (0.35 mmol) of PdCl₂(dppf)–

CH₂Cl₂complex. The resulting dark red suspension

was heated in a 110 ꢂC oil bath for 16 h. The mixture

was cooled to room temperature and ﬁltered through

Celite, rinsing with EtOAc. The ﬁltrate was concen-

trated to an oil, which was used without puriﬁcation

in the following reaction. MS(ESI+): m/z 390.3 [M⁺].

Step 3. A solution of 530 mg (1.87 mmol) of 107 in

10 mL of anhydrous DMF was cooled in an ice bath

and treated with 82 mg (2.06 mmol) of a 60% dispersion

of NaH in mineral oil. The resulting gray suspension

was stirred at 0 ꢂC for 15 min, and then treated with

0.1 mL of 15-crown-5 and 343 mg (2.24 mmol) of 1-bro-

mo-4-methoxypropane. The reaction mixture was stir-

red at room temperature for 16 h, and then poured

into 250 mL of H₂O and extracted with EtOAc (3·).

The combined organic layers were washed with H₂O

(2·) and brine, dried over MgSO₄, ﬁltered, and concen-

trated. Puriﬁcation by ﬂash column chromatography

(SiO₂, 5% EtOAc/hexanes, then gradient to 20%

EtOAc/hexanes) gives 527 mg of 113 as a clear viscous

Step 5. The residue was dissolved in 40 mL of anhydrous

1,4-dioxane and 8 mL of H₂O, and treated with 0.91 g

(4.2 mmol) of 54, 400 mg (11 mmol) of LiCl, 2.0 g

(11 mmol) of CsOHÆH₂O, and 410 mg (0.35 mmol) of

Pd(PPh₃)₄. The resulting mixture was degassed by Ar

sparge for 15 min and then heated at reﬂux for 16 h.

The resulting black mixture was diluted with EtOAc

and ﬁltered through Celite. The ﬁltrate was washed with

H₂O, dried over Na²SO₄, ﬁltered, and concentrated.

Puriﬁcation by ﬂash column chromatography (SiO₂,

100% EtOAc then gradient to 40% MeOH/EtOAc)

and precipitation from EtOAc yielded 334 mg (24%) of

1

oil: H NMR (400 MHz, CDCl₃) d 0.88 (t, J = 7.9 Hz,

6H), 2.06 (q, J = 7.6 Hz, 4H), 3.75 (s, 3H), 6.75 (d,

J = 8.8 Hz, 1H), 7.48 (dd, J = 9.0, 2.7 Hz, 1H).

MS(ESIꢁ): m/z 356.0, 358.0 (M⁺, 1:1 ratio of Br

isotopes).

1

22 as a white solid. H NMR (400 MHz, DMSO-d₆) d

0.86 (m, 3H), 0.93 (t, J = 7.8 Hz, 3H), 1.39 (s, 3H),

1.63 (m, 1H), 1.70 (m, 2H), 1.84 (m, 1H), 2.09 (q,

J = 6.6 Hz, 2H), 3.12 (s, 3H), 3.28 (m, 2H), 3.87 (m,

2H), 5.51 (br s, 2H), 5.74 (br s, 2H), 6.72 (d,

J = 7.3 Hz, 1H), 6.85 (s, 1H), 6.95 (d, J = 7.3 Hz, 1H);

MS(ESI+): m/z 400.6 (M+1). Elem. Anal. Calcd for

C₂₁H₂₉N₅O₃Æ0.2H₂O: C, 62.66; H, 7.35; N, 17.40.

Found: C, 62.65; H, 7.17; N, 17.07.

Step 4. A 50 mL oven-dried Schlenk ﬂask was charged

with 525 mg (1.47 mmol) of 113, 449 mg (1.77 mmol)

of bis(pinacolato)diboron, 434 mg (4.42 mmol) of

KOAc, and 8 mL of anhydrous 1,4-dioxane under an

Ar atmosphere. The clear suspension was degassed by

Ar sparge through the suspension for 15 min.

PdCl₂(dppf)–CH₂Cl₂1:1 complex (60 mg, 0.07 mmol)

was added in a single portion, and the resulting brick-

red suspension was heated in a 90 ꢂC oil bath for 3 h.

After cooling to r.t., the reaction mixture was diluted

with EtOAc, ﬁltered through a Celite plug, and concen-

trated. The residue was used without further puriﬁcation

in the following reaction. MS(ESI+): m/z 404.3 (M+1).

6.1.20. 6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-2,2-

diethyl-4-(3-methoxy-propyl)-4H-benzo[1,4]oxazin-3-one

(23). Step 1. A solution of 1.05 g (7.16 mmol) of methyl

2-ethyl-2-hydroxy-butanate in 35 mL of anhydrous

THF was treated with 327 mg (8.18 mmol) of a 60%

NaH dispersion in mineral oil. The resulting gray sus-

pension was stirred at room temperature for 10 min.

Then, 5 drops of 15-crown-5 were added via syringe, fol-

lowed by 0.84 mL (6.82 mmol) of 4-bromo-2-nitroﬂu-

orobenzene 97. The resulting yellow suspension was

stirred at room temperature for 17 h. Excess hydride

was quenched by the careful addition of MeOH. The

solution was diluted with EtOAc, and then washed with

brine, dried over MgSO₄, ﬁltered, and concentrated to

dryness. Puriﬁcation by ﬂash column chromatography

(SiO₂, 100% hexanes then gradient to 10% EtOAc/hex-

anes) aﬀorded 821 mg (35%) of 100 as a yellow oil.

Step 5. The residue was dissolved in 8 mL of 1,4-dioxane

and 1 mL of H₂O under an Ar atmosphere. Cs₂CO₃

(1.44 g, 4.42 mmol), 187 mg (4.42 mmol) of LiCl, and

480 mg (2.21 mmol) of 54 were added, and the resulting

dark suspension was degassed by Ar sparge for 20 min.

Pd(PPh₃)₄(170 mg, 0.154 mmol) was added, and the

mixture was heated in a 100 ꢂC oil bath for 24 h. After

cooling to room temperature, the mixture was diluted

with EtOAc, ﬁltered through a Celite plug, and concen-

trated. Puriﬁcation by ﬂash column chromatography

(SiO₂, 100:0 CH₂Cl₂/MeOH, then gradient to 90:10

CH₂Cl₂/MeOH) yielded 56 mg (12%) of 23 as a green-

white solid foam. ¹H NMR (400 MHz, DMSO-d₆) d

0.84 (t, J = 6.8 Hz, 6H), 0.93 (t, J = 7.3 Hz, 3H), 1.62–

1.73 (m, 4H), 1.83 (quintet, J = 7.3 Hz, 2H), 2.09 (q,

J = 7.2 Hz, 2H), 3.12 (s, 3H), 3.28 (q, J = 4.0 Hz, 2H),

3.828 (m, 2H), 5.52 (br s, 2 H) 5.77 (bs, 2H), 6.72 (dd,

J = 8.0, 2.0 Hz, 1 H), 6.83 (d, J = 4.0 Hz, 1H), 6.95 (d,

J = 8.0 Hz, 1H); MS(ESI) 414.2 (M+H).

Step 2. A solution of 820 mg (2.28 mmol) of 100 in

10 mL of glacial acetic acid was treated with 1.27 g

(22.8 mmol) of iron dust and heated in a 60 ꢂC oil bath

for 3 h. The resulting gray suspension was cooled to

room temperature, diluted with EtOAc, and ﬁltered

through a Celite pad. The ﬁltrate was concentrated to

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

5939

6.1.21. N-{2-[6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-2,2-

[cyclopropyl]-3-oxo-benzo[1,4]oxazin-4-yl]-ethyl}-acetam-

ide (24). Step 1. A solution of 932 mg (7.16 mmol) of

ethyl 2-hydroxy-cyclopropylacetate in 20 mL of anhy-

drous THF was treated with 327 mg (8.18 mmol) of a

60% NaH dispersion in mineral oil. The resulting gray

suspension was stirred at room temperature for

10 min. Then, 5 drops of 15-crown-5 were added via syr-

inge, followed by 0.84 mL (6.82 mmol) of 4-bromo-2-

nitroﬂuorobenzene 97. The resulting yellow suspension

was stirred at room temperature for 17 h. Excess hydride

was quenched by the careful addition of MeOH. The

solution was diluted with EtOAc, and then washed with

brine, dried over MgSO₄, ﬁltered, and concentrated to

dryness. Puriﬁcation by ﬂash column chromatography

(SiO₂, 100% hexanes then gradient to 10% EtOAc/hex-

anes) aﬀorded 1.60 g (71%) of 101 as a yellow oil.

ane was degassed by Ar sparge for 15 min. PdCl₂(dppf)–

CH₂Cl₂complex (90 mg, 0.111 mmol) was added, and

the resulting red-orange mixture was heated at 90 ꢂC

for 18 h. After cooling to room temperature, the black

mixture was diluted with EtOAc, ﬁltered through a Cel-

ite plug, and concentrated. The crude residue was dis-

solved in 7 mL of anhydrous 1,4-dioxane and 0.9 mL

of H₂O. LiCl (281 mg, 6.63 mmol), 576 mg (2.65 mmol)

of 54, and 1.11 g (6.63 mmol) of CsOHÆH₂O were added,

and the resulting suspension was degassed by Ar sparge

for 20 min. Pd(PPh₃)₄(255 mg, 0.22 mmol) was added,

and the mixture was heated in a 100 ꢂC oil bath for

18 h. The mixture was diluted with EtOAc, ﬁltered

through a Celite plug, and concentrated. Puriﬁcation

by ﬂash column chromatography (SiO₂, 95:5 CH₂Cl₂/

MeOH then gradient to 70:30 CH₂Cl₂/MeOH) followed

by preparatory reverse phase HPLC (10% CH₃CN/H₂O/

0.1% TFA then gradient to 60% CH₃CN/H₂O/0.1%

TFA) yielded 280 mg of the TFA salt of 24 as a white

solid. IR(ATR) 3365, 3103, 1674, 1629, 1399, 1193,

Step 2. A solution of 1.60 g (4.84 mmol) of 101 in 20 mL

of glacial acetic acid was treated with 2.70 g (48.4 mmol)

of iron dust and heated in a 60 ꢂC oil bath for 3 h. The

resulting gray suspension was cooled to room tempera-

ture, diluted with EtOAc, and ﬁltered through a Celite

pad. The ﬁltrate was concentrated to dryness. The solid

residue was dissolved in EtOAc and washed with H₂O

(3·) and saturated aqueous NaHCO₃. The organic layer

was dried over MgSO₄, ﬁltered, and concentrated to

yield 1.24 g (100%) of 108 as a white solid that was used

without further puriﬁcation. MS(ESI+): m/z 253.9,

256.0 [M+1], Br isotopes, 1:1 ratio.

1169, 1143 cm^ꢁ^{1 1}H NMR (400 MHz, DMSO-d₆) d

;

1.02 (t, J = 7.6 Hz, 3H), 1.19 (ddd, J = 10.0, 5.5,

3.6 Hz, 2H), 1.26 (ddd, J = 10.4, 7.3, 2.2 Hz, 2H), 1.70

(s, 3H), 2.24 (q, J = 7.5 Hz, 2H), 3.11–3.29 (m, 2H),

3.81–3.91 (m, 2H), 6.84 (dd, J = 8.1, 1.9 Hz, 1H), 6.91

(br s, 1H), 6.98 (d, J = 8.2 Hz, 1H), 7.45 (br s, 2H),

8.01 (t, J = 5.9 Hz, 1H), 8.13 (bs, 1H), 12.08 (bs, 1H);

HRMS Calcd for [C₂₀H₂₄N₆O₃+H]: 397.1988. Found:

397.2006. Elem. Anal. Calcd for C₂₀H₂₄N₆O₃Æ1.8T-

FAÆ0.5H₂O: C, 46.35; H, 4.41; N, 13.72; F, 16.93.

Found: C, 46.65; H, 4.41; N, 13.72.

Step 3. A solution of 1.24 g (4.87 mmol) of 108 and

0.41 ml (5.85 mmol) of bromoacetonitrile in 25 mL of

anhydrous CH₃CN was treated with 0.74 g (5.36 mmol)

of K₂CO₃and heated to reﬂux for 18 h. The mixture was

diluted with EtOAc, ﬁltered through a Celite plug, and

concentrated under reduced pressure. Puriﬁcation by

ﬂash column chromatography (SiO₂, 100% hexanes then

gradient to 15% EtOAc/hexanes) yielded 1.07 g (75%) of

6.1.22. 6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-2-(3,5-

diﬂuoro-phenyl)-4-(3-methoxy-propyl)-2-methyl-4H-

benzo[1,4]oxazin-3-one (25). Step 1. A solution of 1.50 g

(6.50 mmol) of 2-(3,5-diﬂuorophenyl)-2-hydroxy-propi-

onic acid ethyl ester in 40 mL of anhydrous THF was

treated with 312 mg (7.80 mmol) of a 60% NaH disper-

sion in mineral oil. The resulting gray suspension was

stirred at room temperature for 10 min. Then, 30 lL

of 15-crown-5 was added via syringe, followed by

1.50 g (6.83 mmol) of 4-bromo-2-nitroﬂuorobenzene

97. The resulting orange solution was stirred at room

temperature for 17 h. Excess hydride was quenched by

the careful addition of MeOH. The solution was diluted

with EtOAc, and then washed with brine, dried over

MgSO₄, ﬁltered, and concentrated to dryness.

1

116 as a white solid. H NMR (400 MHz, CDCl₃) 1.31

(dd, J = 8.4, 5.7 Hz, 2H), 1.48 (dd, J = 8.7, 5.4 Hz,

2H), 4.81 (s, 2H), 6.83 (d, J = 8.4 Hz, 1H), 7.15 (d,

J = 2.0 Hz, 1H), 7.20 (dd, J = 8.6, 2.0 Hz, 1H); MS(E-

SI+): m/z 293.0, 294.0 ([Mꢁ1], 1:1 ratio of Br isotopes).

Step 4. A solution of 1.07 g (3.64 mmol) of 116 and

0.51 mL (5.47 mmol) of Ac₂O in 50 mL of THF was

treated with 1.0 g Raney nickel. The resulting suspen-

sion was shaken under a 51 psi H₂atmosphere for

17 h. The mixture was ﬁltered through Celite and con-

centrated. Puriﬁcation by ﬂash column chromatography

(SiO₂, 10% EtOAc/hexanes then gradient to 70%

EtOAc/hexanes) yielded 0.802 g (65%) of 121 as a white

Step 2. The orange residue was dissolved in 20 mL of

glacial acetic acid, treated with 3.63 g (65.0 mmol) of

iron dust, and heated in a 60 ꢂC oil bath for 4 h. The

resulting gray suspension was cooled to room tempera-

ture, diluted with EtOAc, and ﬁltered through a Celite

pad. The ﬁltrate was concentrated to dryness. The solid

residue was dissolved in EtOAc and washed with H₂O

(3·) and saturated aqueous NaHCO₃. The organic layer

was dried over MgSO₄, ﬁltered, and concentrated. Puri-

ﬁcation by ﬂash column chromatography (SiO₂, 5%

EtOAc/hexanes then gradient to 15% EtOAc/hexanes)

gave 1.87 g (5.28 mmol) 109 as a white semisolid (81%,

1

solid. H NMR (400 MHz, CDCl₃) d 1.23 (dd, J = 8.0,

4.9 Hz, 2H), 1.40 (dd, J = 8.2, 5.5 Hz, 2H), 1.98 (s,

3H), 3.55 (q, J = 6.1 Hz, 2H), 4.06 (t, J = 6.5 Hz, 2H),

6.05 (bs, 1H), 6.75 (d, J = 8.4 Hz, 1H), 7.11 (dd,

J = 8.4, 2.1 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H); MS(E-

SI+): m/z 339.0, 341.0 ([Mꢁ1], 1:1 ratio of Br isotopes).

1

Step 5. A solution of 750 mg (2.21 mmol) of 121, 674 mg

(2.65 mmol) of bis(pinacolato)diboron, and 651 mg

(6.63 mmol) of KOAc in 11 mL of anhydrous 1,4-diox-

2 steps). H NMR (400 MHz, CDCl3) d 1.89 (s, 3H),

6.71 (tt, J = 8.6, 2.4 Hz, 1H), 6.89 (d, J = 2.2 Hz, 1H),

6.96–7.01 (m, 3H), 7.11 (dd, J = 8.6, 2.2 Hz, 1H), 8.28

5940

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

1

Diagnostic peaks for the minor rotational isomer: H

NMR (400 MHz, DMSO-d₆) d 0.99 (t, J = 8.0 Hz,

3H), 1.36 (s, 3H), 2.17 (q, J = 4 Hz, 2H), 8.03, (br s,

1H), 12.75 (br s, 1H).

(br s, 1H); MS(ESI+): m/z 353.8, 355.8 [M+1], Br iso-

topes, 1:1 ratio.

Step 3. A solution of 850 mg (2.4 mmol) of rac-6-bromo-

2-(3,5-diﬂuoro-phenyl)-2-methyl-4H-benzo[1,4]oxazin-3-

one 109 in 10 mL of anhydrous CH₃CN was treated

with 460 mg (3.0 mmol) of 1-bromo-3-methoxypropane

and 365 mg (2.64 mmol) of K₂CO₃, and heated at reﬂux

for 18 h. The resulting suspension was cooled to room

temperature, diluted with EtOAc, and ﬁltered through

a Celite plug. The ﬁltrate was concentrated under re-

duced pressure. Puriﬁcation by ﬂash column chromato-

graphy (SiO₂, 100% hexanes then gradient to 10%

EtOAc/hexanes) gave 752 mg (73%) of 114 as a light-yel-

low low-melting solid. ¹H NMR (400 MHz, CDCl₃)d

1.82 (s, 3H), 1.87–1.97 (m, 2H), 3.29 (s, 3H), 3.33–3.28

(m, 1H), 3.35-3.40 (m, 1H), 3.91 (ddd, J = 14.3, 7.3,

7.2 Hz, 1H), 4.05 (ddd, J = 14.3, 7.3, 7.2 Hz, 1H), 6.61

(tt, J = 8.7, 2.4 Hz, 1H), 6.85–6.90 (m, 2H), 6.91 (d,

J = 8.56 Hz, 1H), 7.04 (dd, J = 8.6, 2.2 Hz, 1H), 7.15

(d, J = 2.2 Hz, 1H); MS(ESI+): m/z 426.0, 427.9

([M+1], 1:1 ratio of Br isotopes).

6.1.23. N-{2-[6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-2-

(3,5-diﬂuoro-phenyl)-2-methyl-3-oxo-2,3-dihydro-benzo-

[1,4]oxazin-4-yl]-ethyl}-acetamide (26). Step 1. A solu-

tion of 5.00 g (14.1 mmol) of 109 in 50 mL of anhydrous

CH₃CN was treated with 1.97 mL (28.24 mmol) of

bromoacetonitrile and 2.34 g (16.9 mmol) of K₂CO₃,

and heated at reﬂux for 18 h. The resulting suspension

was cooled to room temperature, diluted with EtOAc,

and ﬁltered through a Celite plug. The ﬁltrate was

washed with saturated aqueous NaHCO₃and brine,

dried over MgSO₄, and concentrated under reduced

pressure. Puriﬁcation by ﬂash column chromatography

(SiO₂, 5% EtOAc/hexanes then gradient to 20%

EtOAc/hexanes) gave 3.17 g (57%) of 117 as a white so-

lid. MS(ESI+): m/z 391.9, 393.9 ([M+1], 1:1 ratio of Br

isotopes).

Step 2. A solution of 3.17 g (8.07 mmol) of 117 and

1.00 g (12.2 mmol) of NaOAc in 10 mL of Ac₂O and

90 mL of AcOH was treated with 1.5 g of Raney nickel

and hydrogenated under a 50 psi H₂atmosphere at

50 ꢂC for 24 h. The mixture was ﬁltered through Celite

and concentrated to dryness. Puriﬁcation by ﬂash col-

umn chromatography (SiO₂, 60% EtOAc/hexanes then

gradient to 100% EtOAc) aﬀorded 2.48 g (70%) of 122

Step 4. A 100 mL round bottom ﬂask was ﬂushed with

Ar and charged with 560 mg (1.31 mmol) of 114,

430 mg (1.70 mmol) of bis(pinacolato)diboron, 400 mg

(4.0 mmol) of KOAc, 110 mg (0.13 mmol) of

PdCl₂(dppf)–CH₂Cl₂complex, and 20 mL of anhydrous

1,4-dioxane. The resulting dark orange suspension was

heated at 110 ꢂC for 16 h. After cooling to room temper-

ature, the resulting black mixture was ﬁltered through a

Celite plug and concentrated to a viscous oil that was

used without further puriﬁcation. MS(ESI+): m/z

474.1 [M+1].

1

as a white solid foam. H NMR (400 MHz, CDCl₃) d

1.87 (s, 3H), 1.99 (s, 3H), 3.51 (m, 2H), 3.98 (dt,

J = 14.2, 6.2, 6.2 Hz, 1H), 4.20 (ddd, J = 14.1, 67.0,

6.8 Hz, 1H), 5.94 (br s, 1H), 6.69 (tt, J = 8.67, 2.3 Hz,

1H), 6.86–6.93 (m, 2H), 6.97 (d, J = 8.6 Hz, 1H), 7.14

(dd, J = 8.6, 2.1 Hz, 1H), 7.31 (d, J = 2.1 Hz, 1H);

MS(ESI+): m/z 439.0, 441.0 ([M+1], 1:1 ratio of Br

isotopes).

Step 5. The residue from Step 4 was dissolved in 20 mL

of 1,4-dioxane and 5 mL of H₂O. 5-bromo-6-ethyl-2,4-

diaminopyrimidine 54 (340 mg, 1.60 mmol), 200 mg

(4.0 mmol) LiCl, and 700 mg (4.0 mmol) CsOHÆH₂O

were added, and the resulting suspension was degassed

with Ar sparge for 10 min. Pd(PPh₃)₄(150 mg,

0.13 mmol) was added and the reaction mixture was

heated at reﬂux for 18 h. The black suspension was di-

luted with EtOAc and ﬁltered through a Celite plug.

The ﬁltrate was washed with H₂O, dried over Na₂SO₄,

ﬁltered, and concentrated. Puriﬁcation by ﬂash column

chromatography (SiO₂, 100% EtOAc then gradient to

40% MeOH/EtOAc) provided 327 mg (22%) of 25 as a

glassy solid. Puriﬁcation by preparative reverse-phase

HPLC (CH₃CN/H₂O with 0.1% TFA) followed by

lyophilization aﬀorded the TFA salt of 25 as a white

amorphous powder that exists as a mixture of restricted

Step 3. A suspension of 3.07 g (6.99 mmol) of 122, 1.7 g

(2.1 mmol) of PdCl₂(dppf)–CH₂Cl₂complex, 2.3 g

(9.1 mmol) of bis(pinacolato)diboron, and 2.1 g

(21 mmol) of KOAc in 35 mL of anhydrous 1,4-dioxane

was degassed by Ar sparge for 20 min and then heated at

110 ꢂC for 2.5 h. After cooling to room temperature, the

black mixture was diluted with EtOAc and washed with

H₂O and brine, dried over MgSO₄, ﬁltered, and concen-

trated. Puriﬁcation by ﬂash column chromatography

(SiO₂, 100% EtOAc then gradient to 25% MeOH/

EtOAc) gave 3.9 g of an impure black solid that was

used without further puriﬁcation. MS(ESI+): m/z

487.2 [M+1].

1

rotational isomers. H NMR (400 MHz, DMSO-d₆) d

0.85 (t, J = 7.6 Hz, 3H), 1.74 (m, 2H), 1.77 (s, 3H),

2.06 (q, (J = 8.0 Hz, 2H)), 3.13 (s, 3H), 3.28 (t,

J = 5.9 Hz, 2H), 3.92 (m, 2H), 6.75 (br s, 1H), 6.88

(ddd, J = 8.0, 4.0, 4.0 Hz, 1H), 6.99–7.06 (m, 3H), 7.15

(m, 1H), 7.25 (d, J = 8.3 Hz, 1H), 7.72 (br s, 2H), 8.05

(br s, 1H), 12.67 (br s, 1H). HRMS (ESI+) Calcd for

C₂₅H₂₇F₂N₅O₃(M+H)⁺: 483.2082. Found: 484.2148

(M+1). Elem. Anal. Calcd for C₂₅H₂₇F₂N₅O₃Æ1.5TFA:

C, 51.49; H, 4.40; N, 10.74. Found: C, 51.46; H, 4.56;

N, 10.95.

Step 4. A suspension of 2.9 g (6 mmol) of the residue

from Step 3, 1.29 g (5.96 mmol) of 54, 0.76 g (18 mmol)

of LiCl, and 3.0 g (18 mmol) of CsOHÆH₂O in 40 mL of

1,4-dioxane and 4 mL of H₂O was degassed by Ar

sparge for 20 min. Pd(PPh₃)₄(0.69 g, 0.6 mmol) was

added, and the mixture was heated to reﬂux for 2 h.

After cooling to room temperature, the mixture was di-

luted with EtOAc and washed with H₂O and brine,

dried over MgSO₄, ﬁltered, and concentrated. Puriﬁca-

tion by ﬂash column chromatography (SiO₂, 18%

N. A. Powell et al. / Bioorg. Med. Chem. 15 (2007) 5912–5949

5941

MeOH/EtOAc isocratic) yielded 0.67 g (23%) of 26 as an

oﬀ-white powder. An additional 0.36 mg (35% total

yield) of 26 could be obtained by preparatory reverse-

phase HPLC (10% CH₃CN/H₂O/0.1% TFA then gradi-

ent to 70% CH₃CN/H₂O/0.1% TFA) of the impure frac-

800 mg (5.0 mmol) of CsOHÆH₂O and heated at reﬂux

for 18 h. The mixture was diluted with EtOAc and ﬁl-

tered through a Celite plug. The ﬁltrate was washed with

H₂O, dried over Na₂SO₄, ﬁltered, and concentrated.

Puriﬁcation by ﬂash column chromatography (SiO₂,

0:100 MeOH/EtOAc then gradient to 20:80 MeOH/

EtOAc) and precipitation from CH₃CN aﬀorded

146 mg (19%) of 27 as a white powder that exists as a

1

tions and neutralization of the resulting TFA salt. H

NMR (400 MHz, DMSO-d₆) d 0.86 (t, J = 7.4 Hz,

3H), 1.71 (s, 3H), 1.81 (s, 3H), 2.02 (q, J = 7.3 Hz,

2H), 3.12–3.25 (m, 1H), 3.27–3.37 (m, 1H), 3.85–4.08

(m, 2H), 5.58 (br s, 1H), 5.76 (br s, 1H), 5.97 (br s,

2H), 6.83 (t, J = 7.2 Hz, 1H), 7.06 (s, 1H), 7.10 (d,

J = 6.3 Hz, 2H), 7.16–7.27 (q, J = 8.6 Hz, 1H), 8.02 (t,

J = 6.0 Hz, 1H). MS(ESI+): m/z 497.1 (M+1). Elem.

Anal. Calcd for C₂₅H₂₆F₂N₆O₃Æ1.1H₂O: C, 57.93; H,

5.53; N, 16.21; H₂O, 4.21. Found: C, 58.15; H, 5.52;

N, 16.14; H₂O, 4.38.

1

1:1 mixture of restricted rotational isomers. H NMR

(400 MHz, DMSO-d₆) d 0.85 (t, J = 7.6 Hz, 3H), 0.93

(m, 3H), 1.70 (m, 2H), 1.95 (m, 2H), 2.07 (m, 1H),

2.28 (m, 1H), 3.12 (s, 3H), 3.26 (m, 2H), 3.93 (m, 2H),

5.36 (br s, 1H), 5.57 (br s, 1H), 5.79 (s, 2H), 6.85 (m,

2H), 7.04 (s, 2H), 7.15 (m, 1H), 7.26 (d, J = 8.1 Hz,

1H). MS(ESI+): m/z 498.2 (M+1). Elem. Anal. Calcd

for C₂₆H₂₉F₂N₅O₃0.25H₂O: C, 66.22; H, 5.92; N,

13.95. Found: C, 62.20; H, 5.88; N, 14.20.

1

Diagnostic peaks for the minor rotational isomer: H

NMR (400 MHz, DMSO-d₆)d 0.98 (t, J = 7.4 Hz, 3H),

2.16 (q, J = 4 Hz, 2H).

Diagnostic peaks for the rotational isomer: 0.86

(t, J = 7.6 Hz, 3H).

6.1.24. 6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-2-(3,5-

diﬂuoro-phenyl)-2-ethyl-4-(3-methoxy-propyl)-4H-benzo-

[1,4]oxazin-3-one (27). Step 1. A solution of 900 mg

(2.18 mmol) of 78 in 25 mL of anhydrous DMF was

cooled in an ice bath and treated with 120 mg

(3.1 mmol) of a 60% dispersion of NaH in mineral oil

in portions. The resulting orange solution was stirred

at 0 ꢂC for 30 min. Iodoethane (0.23 mL, 2.8 mmol)

was added, the ice bath was removed, and the mixture

was stirred at room temperature for 16 h. Excess hydride

was quenched by the dropwise addition of H₂O, and the

mixture was poured into H₂O and extracted with

EtOAc. The organic layer was washed with brine, dried

over MgSO₄, ﬁltered, and concentrated. Puriﬁcation by

ﬂash column chromatography (SiO₂, 100% hexanes then

gradient to 20% EtOAc/hexanes) yielded 790 mg (82%)

6.1.25. 6-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-2-(3,4-

diﬂuoro-phenyl)-4-(3-methoxy-propyl)-2-methyl-4H-

benzo[1,4]oxazin-3-one (28). Step 1. A solution of

500 mg (1.21 mmol) of 76 in 15 mL of anhydrous THF

was cooled in an ice bath and treated with 73 mg

(3.04 mmol) of a 60% dispersion of NaH in mineral

oil. The reaction mixture was stirred at 0 ꢂC for

30 min and then 94 lL (1.5 mmol) of MeI was added.

The reaction mixture was allowed to warm to room tem-

perature while stirring for 17 h. Excess hydride was

quenched by the dropwise addition of H₂O, and the

mixture was concentrated and diluted with EtOAc and

H₂O. The aqueous layer was extracted with EtOAc,

and the combined organic extracts are washed with

brine, dried over MgSO₄, ﬁltered, and concentrated to

give 530 mg (quantitative) of 126, which was used di-

1

of 125 as an opaque oil. H NMR (400 MHz, CDCl₃)d

rectly in the next step. H NMR (CDCl₃, 400 MHz) d

1.01 (t, J = 7.3 Hz, 3H), 1.84–1.98 (m, 2H), 2.05 (dq,

J = 14.7, 7.3 Hz, 1H), 2.37 (dq, J = 14.5, 7.3 Hz, 1H)

3.32 (ddd, J = 9.5, 6.8, 4.9 Hz, 1H), 3.33 (s, 3H), 3.39

(ddd, J = 9.5, 6.4, 4.9 Hz, 2H), 3.95 (ddd, J = 14.4, 7.2,

7.2 Hz, 1H,) 4.05 (ddd, J = 14.0, 7.6, 6.0 Hz, 1H), 6.66

(tt, J = 8.7, 2.4, 2.3 Hz, 1H), 6.87–6.95 (m, 2H), 6.98

(d, J = 8.6 Hz, 1 H), 7.10 (dd, J = 8.3, 2.0 Hz, 1H),

7.14 (d, J = 2.2 Hz, 1H); MS(ESI+): m/z 439.0, 441.0

([Mꢁ1], 1:1 ratio of Br isotopes).

7.13 (m, 2H), 7.05 (m, 3H), 6.91 (d, J = 8.4 Hz, 1H),

4.05 (m, 1H), 3.93 (m, 1H), 3.34 (m, 2H), 3.32 (s, 3H),

1.92 (m, 2H), 1.82 (s, 3H).

Step 2. A mixture of 0.53 g (1.21 mmol) of 126, 357 mg

(3.64 mmol) of KOAc, and 462 mg (1.82 mmol) of

bis(pinacolato)diboron in 20 mL of anhydrous 1,4-diox-

ane was degassed by Ar sparge for 20 min. PdCl₂(dppf)–

CH₂Cl₂complex (50 mg, 0.061 mmol) was added, and

the reaction mixture was heated to reﬂux for 13 h and

then cooled to room temperature. LiCl (154 mg,

3.63 mmol), 592 mg (2.73 mmol) of 54, 1.18 g

(3.62 mmol) of Cs₂CO₃, and 5 mL of H₂O were added.

The resulting suspension was degassed by N₂sparge

for 20 min. Pd(PPh₃)₄(350 mg, 0.303 mmol) was added,

and the mixture was heated to reﬂux for 4.5 h, cooled to

room temperature, and stirred for 93 h. The solvent was

evaporated, and the resulting residue was partitioned

between CH₂Cl₂and H₂O. The biphasic mixture was

ﬁltered through a pad of Celite. The aqueous layer

was extracted with CH₂Cl₂, and the combined organic

layers were washed with 1 N HCl, saturated aqueous

NaHCO₃, and brine, dried over MgSO₄for 14 h. Poly-

mer-supported PPh₃was added and the suspension

was stirred at room temperature for 1 h. The mixture

Step 2. A suspension of 680 mg (1.54 mmol) of 125,

510 mg (2.00 mmol) of bis(pinacolato)diboron, 500 mg

(5.0 mmol) of KOAc, and 130 mg (0.15 mmol) of

PdCl₂(dppf)–CH₂Cl₂complex in 40 mL of anhydrous

1,4-dioxane was heated at 110 ꢂC for 16 h. After cooling

to room temperature, the black mixture was diluted with

EtOAc, ﬁltered through a Celite plug, and concentrated

to a black oil that was used without further puriﬁcation.

MS(ESI+): m/z 488.3 [M+1].

Step 3. The residue was dissolved in 30 mL of anhydrous

1,4-dioxane and 6 mL of H₂O and treated with 200 mg

(5 mmol) of LiCl, 400 mg (1.8 mmol) of 54, and

255 mg (0.15 mmol) of Pd(PPh₃)₄. The mixture was de-

gassed by Ar sparge for 10 min, and then treated with

Article Doi

Rational design of 6-(2,4-diaminopyrimidinyl)-1,4-benzoxazin-3-ones as small molecule renin inhibitors

DOI: 10.1016/j.bmc.2007.05.069

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Authors:

Article abstract of DOI:10.1016/j.bmc.2007.05.069

Full text of DOI:10.1016/j.bmc.2007.05.069

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