Defining the key pharmacophore elements of PF-04620110: Discovery of a potent, orally-active, neutral DGAT-1 inhibitor

Article abstract of DOI:10.1016/j.bmc.2013.06.045

DGAT-1 is an enzyme that catalyzes the final step in triglyceride synthesis. mRNA knockout experiments in rodent models suggest that inhibitors of this enzyme could be of value in the treatment of obesity and type II diabetes. The carboxylic acid-based DGAT-1 inhibitor 1 was advanced to clinical trials for the treatment of type 2 diabetes, despite of the low passive permeability of 1. Because of questions relating to the potential attenuation of distribution and efficacy of a poorly permeable agent, efforts were initiated to identify compounds with improved permeability. Replacement of the acid moiety in 1 with an oxadiazole led to the discovery of 52, which possesses substantially improved passive permeability. The resulting pharmacodynamic profile of this neutral DGAT-1 inhibitor was found to be similar to 1 at comparable plasma exposures.

Full text of DOI:10.1016/j.bmc.2013.06.045

Bioorganic & Medicinal Chemistry 21 (2013) 5081–5097
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Defining the key pharmacophore elements of PF-04620110:
Discovery of a potent, orally-active, neutral DGAT-1 inhibitor
⇑
Robert L. Dow , Melissa P. Andrews, Jian-Cheng Li, E. Michael Gibbs, Angel Guzman-Perez,
Jennifer L. LaPerle, Qifang Li, Dawn Mather, Michael J. Munchhof, Mark Niosi, Leena Patel,
Christian Perreault, Susan Tapley, William J. Zavadoski
PharmaTherapeutics Research & Development, Pfizer Inc., 620 Memorial Drive, Cambridge, MA 02139, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
DGAT-1 is an enzyme that catalyzes the final step in triglyceride synthesis. mRNA knockout experiments
in rodent models suggest that inhibitors of this enzyme could be of value in the treatment of obesity and
type II diabetes. The carboxylic acid-based DGAT-1 inhibitor 1 was advanced to clinical trials for the
treatment of type 2 diabetes, despite of the low passive permeability of 1. Because of questions relating
to the potential attenuation of distribution and efficacy of a poorly permeable agent, efforts were initiated
to identify compounds with improved permeability. Replacement of the acid moiety in 1 with an oxadi-
azole led to the discovery of 52, which possesses substantially improved passive permeability. The result-
ing pharmacodynamic profile of this neutral DGAT-1 inhibitor was found to be similar to 1 at comparable
plasma exposures.
Received 24 April 2013
Revised 10 June 2013
Accepted 19 June 2013
Available online 1 July 2013
Keywords:
DGAT-1
Diabetes
Obesity
Passive permeability
Carboxylic acid bioisotere
Polar surface area
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
insulin-resistant state. In animal models of lipodystrophy, treat-
ment with the adipose regulatory hormone leptin reduces intracel-
Obesity and type 2 diabetes have been on a dramatic rise in
Westernized countries over the last few decades, resulting in sub-
stantial societal burdens. The increased prevalence of these condi-
tions is largely driven by a net imbalance between energy intake
and expenditure. This in turn results in a net increase in fat dispo-
sition resulting from excess storage of triglycerides. The resulting
increases in triglyceride stores in peripheral tissues, especially
skeletal muscle, have been implicated in the development of insu-
lin resistance1 and the constellation of lipotoxic disorders associ-
ated with metabolic syndrome X.2 Intracellular concentrations of
lipids in muscle have been shown to be tightly, negatively corre-
lated with insulin-stimulated glucose disposal in healthy males.3
This relationship is also found in humans with impaired glucose
tolerance and type 2 diabetes.4 An additional line of evidence sup-
porting the lipotoxicity hypothesis is the metabolic state of indi-
viduals suffering from the genetic disorders leading to
lipodystrophy.5 Lipodystrophy is characterized by the absence of
fat tissue, however, these individuals have excessive accumulation
of lipids in metabolic tissues such as skeletal muscle and the liver.
This aberrant distribution of lipids leads to an extreme
lular lipid levels in skeletal muscle and other tissues, leading to
improved insulin sensitivity.6,7 Given this critical role of lipid bur-
den, oral therapies that have the potential to reverse this state hold
significant potential in the treatment of type 2 diabetes, obesity
and metabolic syndrome X.
The enzymes involved in lipid biosynthesis pathways have at-
tracted considerable interest as potential targets for disruption of
aberrant lipid accumulation and its resulting impact on a range
of disease states.8,9 Triglyceride biosynthesis and the resulting lipid
burden placed on tissues are largely controlled by two major path-
ways in humans. The monoacylglyceride pathway is typically oper-
ative in tissues where dietary monoacylglycerides are reesterified,
such as small intestine, liver and adipose.10 Fatty acids that enter
this pathway come from dietary absorption or via de novo fatty
acid synthesis from acetyl CoA, via a series of enzyme-catalyzed
homologation reactions. The second pathway, found in most cell
types, is the glycerol phosphate pathway which sequentially adds
two fatty acyl chains to glycerol-3-phosphate generating a phos-
phatidic acid intermediate that is subsequently converted to tri-
glycerides.11 Both of these pathways converge at intermediate
diacylglycerols which are then converted to triglycerides through
acylation by a fatty acid acyl-CoA, catalyzed by acyl-CoA:diacyl-
glycerol acyltransferases (DGAT). This family of enzymes is
⇑
Corresponding author. Tel.: +1 (617) 551 3254; fax: +1 (845) 474 3821.
E-mail address: robert.l.dow@pfizer.com (R.L. Dow).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.045

5082
R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
composed of DGAT-1 and DGAT-2, which while they carry out the
same biotransformation, arise from divergent gene families.12–14
Consistent with the lipotoxicity hypothesis, mice lacking DGAT-1
(DGAT-1À/À) are resistant to diet-induced obesity and have in-
creased insulin sensitivity and energy expenditure.15,16 In addition,
transplantation of white adipose tissue from these mice into the
wild-type strain confers the enhanced metabolic profile observed
in the DGAT-1 knockout mice.17 These studies have spurred re-
search efforts to determine whether selective, small molecule
inhibitors of DGAT-1 can produce the same improved metabolic
profile observed in the DGAT-1À/À animals.
Several research groups have disclosed potent and selective
DGAT-1 inhibitors from several chemically-distinct series.18 Pre-
clinical studies with these compounds have confirmed that small
molecule DGAT-1 inhibitors can elicit metabolic outcomes compa-
rable to those observed in DGAT-1À/À mice.19–21 We have recently
disclosed an orally-active, novel DGAT-1 inhibitor PF-04620110 (1)
which was advanced to human clinical trials for the treatment of
type 2 diabetes.21 This pyrimidooxazepinone-based series arose
from specific design criteria aimed at eliminating the potential
photochemical and reactive metabolite risks associated with the
pyrimidinooxazine-based DGAT-1 inhibitor 2.22 As compound 1
advanced into human clinical studies there were questions as to
whether the poor passive permeability associated with agent
would result in limited distribution into key in vivo compartments,
potentially leading to a reduced efficacy maximum. This report de-
tails the discovery of neutral, potent and orally-active DGAT-1
inhibitors with high passive permeabilities and their resulting effi-
cacy in preclinical rodent models. Analysis of the key pharmaco-
phore elements of 1 will also be discussed as part of a broader
structure–activity-relationship (SAR) analysis.
responsible for the photochemical instability associated with this
lead. While our design resulted in a potent DGAT-1 inhibitor, with
a pharmacokinetic/safety profile supporting its advancement to
human clinical trials, there were a number of unanswered SAR
questions relating to this novel chemotype. The combination of
limited bicyclic core SAR disclosed around 222 and the inherent
chemical differences between the two core structures, led us to
pursue a systematic investigation of the key pharmacophore ele-
ments present in 1.
While the in-plane orientation of the phenycyclohexyl side-
chain relative to the heterobicylic cores of 1 and 2 (Fig. 1) sug-
gested this to be a necessary requirement for potent DGAT-1
inhibition, alternative bicyclic cores that led to alternative spatial
arrangements of these two pharmacophore elements were evalu-
ated prior to further optimization of 1. An additional goal was to
improve on its low passive permeability (apparent passive perme-
ability (Papp) = 1 mm 10À6 cm/s) associated with 1. This property
became the focus of our back-up effort since there was a concern
that this low passive permeability might limit exposure to key tar-
get tissues in vivo. Computational models suggested that conver-
sion of the lactam functionality in 1 to the corresponding bicyclic
amine-based core could lead to a substantial increase in passive
permeability (Papp = 7 Â 10À6 cm/s).23 Toward this end, global
reduction of 1 afforded amine 3. Modeling of the predicted low en-
ergy conformation of 3 (Fig. 1) suggested a significantly altered
relationship between the core and sidechain relative to 1. This
modification led to a loss in DGAT-1 potency of >110-fold relative
to 1 (Table 1). It is important to note that the loss in potency of 3 is
not driven by reduction of the carboxylic acid functionality, given
that a propyl alcohol analog of 1 is a potent inhibitor of DGAT-1
(unpublished data). An alternative lactam motif evaluated was
CO2H
CO2H
O
NH2
NH2
N
N
O
N
N
N
Me
Me
N
O
1
2
the regioisomeric lactam 4. Modeling of this lactam suggested its
low energy conformation would be similar to amine 3, while
retaining a neutral bicyclic core (Fig. 1). The lack of DGAT-1 inhib-
itory activity associated with 4 was consistent with the hypothesis
that an in-plane relationship between the bicyclic core and phen-
ycyclohexyl sidechain found in 1 was critical. These results led to
a focusing of efforts on analogs containing the pyrimidooxazepi-
2. Results and discussion
As previously reported,21 the bicyclic core of 1 arose from anal-
yses of alternative cores that retained what were assumed to be
the key pharmacophore elements of 2 and the spatial orientation
of these in three-dimensional space. Incorporation of the fused, se-
ven-membered lactam moiety in 1 properly orients the cyclohexyl-
acetic acid sidechain relative to 2 (Fig. 1). In addition, it eliminates
the conjugated imine-based bicyclic present in 2, which is likely
none core present in 1, with
a
goal of improving passive
permeability.
OH
CO2H
CO2H
NH2
N
NH2
N
O
NH2
N
N
N
N
N
N
N
O
O
Me
O
O
3
4
5

R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
5083
Figure 1. Overlay of crystal structure of 1 (green),21 minimized conformations of 2 (blue), 3 (yellow) and 4 (cyan).
Table 1
In vitro profiles of bicyclic core modifications of 1
CO2H
O
X
Z
N
Y
O
R2
R1
a
b
X
Y
Z
R1,R2
H,H
hDGAT-1 IC50 (nM)
TG synthesis IC50 (nM)
HLM CLapp (mL/min/kg)
PSA
1
3
4
5
6
7
8
9
10
11
12
13
14
NH2
N
N
19
8
1380
—
44
—
—
—
—
—
<8
11
—
<8
—
—
—
—
—
119
84
2960
>3000
59
>3000
>3000
>3000
>3000
1200
23
119
119
113
102
96
105
107
107
119
119
119
OH
N
N
N
N
N
CH
N
N
N
N
N
N
N
CH
N
N
N
N
H,H
H,H
H,H
H,H
H,H
H,H
Me,Me
H,Me
Me,H
OMe
NHMe
NMe2
NH2
NH2
NH2
NH2
NH2
99
32
—
8.4
—
—
146
625
19
8
—
a
Average of >2 determinations run in triplicate.
Inhibition of triglyceride synthesis determined in HT-29 cells. Average of >2 determinations run in triplicate.
b
As part of a program to explore the key binding interactions be-
tween the core of 1 and DGAT-1 a systematic analysis of the amino-
pyrimidine subunit was initiated. potential approach to
These results demonstrate that the 4-amino substituent is critical
for DGAT-1 inhibition. With a reduced PSA relative to 1, the regio-
isomeric aminopyridines were prepared to determine if they of-
fered an advantage. 2-Aminopyridine 10 had substantially
reduced potency, though the 4-amino analog 11 was determined
to have an IC50 value of 23 nM against hDGAT-1 and inhibited tri-
glyceride synthesis with an IC50 value of 99 nM. While 11 exhibited
a projected clearance profile comparable to 1, its passive perme-
ability (Papp = 1 Â10À6 cm/s) is not improved relative to the lead.
The structural core of 1 is similar to that in the cannabinoid
receptor-1 receptor antagonist 15,24 which showed a substantial
erosion of the therapeutic index in canine safety studies. Based
on mechanism work it was hypothesized that formation of 16
was responsible for the observed toxicology (Scheme 1). Based
on the structural overlap between 15 and 1 a two-pronged strategy
to address this potential reactive metabolite issue was pursued.
The first of these was a design approach based on steric blocking
of the ether bearing carbon in 1 was pursued. If tolerated from a
potency standpoint, incorporation of lipophilicity at this position
A
improving passive permeability within this series would be to in-
crease lipophilicity via incorporation of substituents in the 3-posi-
tion of the pyrimidine ring. Towards this end a 3-methyl analog 5
was prepared and shown to be a potent inhibitor of DGAT-1 activity
and triglyceride synthesis in HT-29 cells (Table 1). While low pro-
jected oxidative clearance was observed for this analog, the reduc-
tion in potency relative to 1 suggested that further efforts based on
modification of the 3-position were unlikely to provide an im-
proved balance of potency, permeability and microsomal stability.
An alternative approach to improving permeability was to alter
the amino group of 1 by replacement with less polar isosteric
replacements or masking as a secondary/tertiary amine, which
would result in reduced polar surface area (PSA). The corresponding
hydroxy (6) or methoxy (7) analogs were devoid of DGAT-1 inhibi-
tory activity. Conversion of the primary amine in 1 into the N-
methyl (8) or N,N-dimethylamines (9) also led to a loss of activity.

5084
R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
OHC
HO
N
N
N
F
F
F
F
O
O
O
N
N
N
Cl
Cl
Cl
Cl
15
16
(Canine-specific metabolite)
(PF-514273)
Scheme 1. Canine-specific metabolism of CB1-receptor antagonist PF-514273 (15).
would also have the potential to improve passive permeability.
Substituents in this position would roughly overlap space occupied
by the methyl groups on the quaternary carbon of 2. The direct
homolog 12 was less active than the 1, however, preparation of
the homochiral monomethyl derivatives revealed that each of
these methyl groups was having a differential impact on DGAT-1
inhibitory activity. S-Isomer 13 had substantially reduced activity
(IC50 = 625 nM), while its enantiomer 13 was equipotent to 1.
These results are consistent with the good overlap of the methyl
group of 13 with the pro-R methyl of 2 while the methyl of 12
occupies novel space, likely resulting in a negative steric interac-
tion with DGAT-1.
Monomethyl derivative 14 would be expected to dramatically
reduce the potential for metabolic activation of the substituted car-
bon center, as well as any metabolic activation leading to a much
less reactive keto-metabolite. In parallel to this design effort, a
more detailed evaluation of the metabolic fate of 1 in human
microsomal preparations was initiated. Acid 1 is very stable in hu-
man liver microsomes, with 93% of parent remaining after 1 h. Of
the portion that is turned over there was no evidence of the puta-
tive aldehyde metabolite being formed based on trapping experi-
ments with methoxymethylamine. This result gave us confidence
in advancing 1 to human clinical studies.
core, a key goal was to drive designs into physiochemical space
that would be predicted to lead to improved passive permeability.
In order to gain an understanding of the minimum pharmaco-
phore required to maintain potency in this system a series of
truncated analogs of 1 were prepared. Both the parent bicyclic
heterocycle 17 and N-methyl derivative 18 exhibited weak inhibi-
tion at 30 lM (Table 2). Reestablishment of the N-aryl motif affor-
ded substantial improvements in DGAT-1 inhibitory activity, with
4-alkyl functionalized analogs 19 and 20 possessing ligand effi-
ciencies modestly improved relative to 1.26 Attempts to incorpo-
rate ionizable/polar functionality in the vicinity of the aryl ring
(21) resulted in loss of DGAT-1 inhibitory activity. In order to
understand whether general lipophilicity in this region of the
inhibitor structure was sufficient for potency, a series of N-cyclo-
alkyl analogs were evaluated. Cyclopentyl derivative 22 was found
to have a reduced potency and ligand efficiency relative to neutral
N-aryl analogs. For a more direct comparison the aromatic ring
saturated analogs of 20 were prepared. The more active N-4-t-
butylcyclohexyl isomer 24 was 20-fold less active than the corre-
sponding aryl homolog. These results support a conclusion that
the N-aryl motif present in 1 is optimal for DGAT-1 inhibitory
activity.
In anticipation of an approach based on modification of the
cyclohexylacetic acid subunit of 1 as a means of improving perme-
ability, a set of analogs were prepared to map out the optimum dis-
play of the acidic functionality. Homologated acids 25 and 26 were
found to be equipotent to 1, although they had substantially re-
duced whole cell activities (Table 3). Tetrazole 27 was also a potent
inhibitor of DGAT-1 but was right-shifted around 10-fold in the cell
assay, due to poor permeability (Papp < 0.1 Â 10À6 cm/s) resulting
from increased PSA. These results led us to retain the core structure
present in 1 in efforts to optimize permeability. From the very
With the abosve studies providing confidence that the pyrimi-
dooxazepinone core of 1 represented a highly optimized scaffold,
attention turned to expanding the SAR for the phenylcyclohexyl-
acetic acid sidechain. With 1 possessing a lipophilic carboxylic
acid, it is reasonable to propose a binding site overlap with that
occupied by the acyl CoA substrate in DGAT-1. However, kinetic
analysis of inhibition of hDGAT-1 utilizing decanoyl CoA as the
acyl donor revealed 1 to be a noncompetitive inhibitor with re-
spect to this substrate (R2 = 0.98). As for efforts on the bicyclic
Table 2
In vitro profiles of truncated analogs 17–24
O
NH2
R1
N
N
N
O
R2
LE25
Permeability27 (Â10À6 cm/s)
a
b
R1
R2
hDGAT-1 IC50 (nM)
TG synthesis IC50 (nM)
1
19
8
—
0.38
—
—
0.41
0.43
—
0.32
—
1
17
18
19
20
21
22
23
24
H
Me
H
H
H
H
Me
H
H
20% inhibition @ 30
36% inhibition @ 30
2210
l
l
M
M
—
—
33
28
—
—
—
—
25% inhibition @ 30
681
43
14% inhibition @ 30
9% inhibition @ 30
—
—
l
l
M
M
4-Methylphenyl
4-t-Butylphenyl
4-Carboxyphenyl
Cyclopentyl
cis-4-t-Butylcyclohexyl
trans-4-t-Butylcyclohexyl
134
36% inhibition @ 30
92,100
27% inhibition @ 3
2950
l
M
lM
l
M
H
0.34
a
Average of >2 determinations run in triplicate.
Inhibition of triglyceride synthesis determined in HT-29 cells. Average of >2 determinations run in triplicate.
b

R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
5085
Table 3
In vitro profiles of carboxylic acid replacements 25–38
R
O
NH2
N
N
N
O
a
b
R
hDGAT-1 IC50 (nM)
TG synthesis IC50 (nM)
8
HLM CLapp (mL/min/kg)
<8
PSA
119
Permeability27 (Â10À6 cm/s)
OH
O
1
19
1
4
O
O
25
31
26
131
421
<8
<8
119
139
OH
Me
N
CO2H
26
<0.1
N
NH
27
28
29
76
34
32
774
—
<8
—
136
108
124
<0.1
17
4
N
N
OMe
O
O
NH2
34
<8
H
N
30
31
94
64
—
<8
39
110
110
13
22
O
O
O
H
N
13
H
N
OH
32
85
68
<8
131
4
Me Me
N
33
34
45
110
55
15
<8
102
122
24
2
O
O
OH
N
148
O
N
35
36
94
60
<8
9
111
122
11
2
O
O
NMe
N
170
180
O
OMe
N
N
37
38
50
66
79
107
<8
111
122
22
—
O
O
OH
249
a
Average of >2 determinations run in triplicate.
Inhibition of triglyceride synthesis determined in HT-29 cells. Average of >2 determinations run in triplicate.
b

5086
R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
Table 4
with neutral moieties were pursued. A number of design criteria
Rat pharmacokinetic profiles of key DGAT-1 inhibitors
were employed in selection of targets, including calculated proper-
ties cutoffs for polar surface area (PSA <140), molecular weight
(<500), predictive models of human microsomal clearance
(<30 mL/min/kg) and passive permeability (Papp >5 Â 10À6 cm/s).
Based on the excellent DGAT-1 inhibitory profile of primary amide
29, a library of substituted amides was pursued. The primary goals
for this set of compounds were to improve passive permeability
relative to 1, while also increasing in vivo half-life relative to 29.
Representative examples detailed in Table 3 (30–38) show that a
range of secondary/tertiary amides with or without polar function-
alities retain good to excellent levels of DGAT-1 enzyme and cell
inhibitory activities. While introduction of modest lipophilicity
(31/33) resulted in good passive permeability, there was an associ-
ated increase in oxidative clearance. Attempts to rebalance these
two properties through incorporation of additional polarity was
challenging given the high PSA starting point of the core structure.
Incorporation of hydroxyl (32/34/38) or amide (36) functionalities
led to enhanced microsomal stability, but decreased permeability
and DGAT-1 potency. These results led us to focus efforts on
amides with a PSA value intermediate between 33 and 34, such
as the morpholine and 4-methoxypiperidine amides, 35 and 37.
While the latter of these had high human microsomal clearance,
35 had a good balance of DGAT-1 activity, low clearance and good
permeability. Rat pharmacokinetic analysis revealed this amide to
have a half-life reduced relative to 1 and primary amide 29. During
b,c
CLpa
(mL/min/kg)
Vdssa
(L/kg)
Cmax
AUCb,c
(ng h/mL)
T1/2
(h)
%F
(ng/mL)
1
6.7
11.7
17
8.4
7.2
5.3
1.8
1.6
1.2
2.0
1.3
1.2
225
273
425
174
171
118
565
810
838
801
1260
727
7.3
4.3
2.0
3.1
2.6
2.7
100
88
77
85
100
53
29
35
49
50
52
a
b
c
Intravenous dose of 1 mg/kg.
Oral dose of 5 mg/kg dosed in 0.5% methylcellulose.
Free drug concentrations.
early stages of investigations on 1, the potent DGAT-1 inhibitory
activity of neutral analogs such as methyl ester 28 and primary
amide 29 demonstrated that an acidic functionality in the cyclo-
hexylphenyl pharmacophore was not obligate for activity. Because
29 had improved passive permeability relative to 1 it was ad-
vanced to a rat pharmacokinetics. Though it had low/no detectable
turnover in rat liver microsomes, 29 did have moderate clearance
in the rat (Table 4). This enhanced turnover in vivo was consistent
with the observation that 29 was unstable in plasma, presumably
through conversion to 1 by plasma amidases/esterases.
To more fully explore the opportunity in this chemical space a
series of compounds in which the carboxylic acid was replaced
Table 5
In vitro profiles of N-acylpiperidines
O
R
N
O
NH2
N
N
N
O
Permeability27 (Â10À6 cm/s)
a
b
R
hDGAT-1 IC50 (nM)
TG synthesis IC50 (nM)
HLM CLapp (mL/min/kg)
PSA
39
40
Methyl
i-Propyl
194
174
609
601
<8
11
102
102
5
16
OEt
41
274
393
—
111
6
42
85
514
41
102
21
43
44
215
113
470
574
<8
<8
111
122
3
1
O
N
OH
45
46
47
7
6750
276
<8
<8
13
141
102
128
-
N
O
88
79
19
14
1310
O
N
N
O
48
174
830
12
128
16
a
Average of >2 determinations run in triplicate.
Inhibition of triglyceride synthesis determined in HT-29 cells. Average of >2 determinations run in triplicate.
b

R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
5087
the course of this work both 29 and 35 were found to be positive in
an in vitro micronucleus assay performed in the absence of meta-
bolic activation. Based on these results efforts were shifted away
from this series of amides.
Based on their predicted improved PSA and passive permeability
a series of related oxazole and isoxazoles were prepared. While this
approach did restore high passive permeability (e.g., 47 and 48), all
of these analogs had reduced potency and microsomal stability.
These results suggest that it would be very difficult to generate a
balanced pharmacology/ADME profile within this series.
In parallel with the above efforts, a library of amides in which
the cyclohexylacetic acid moiety was replaced with a N-acylpiperi-
zine were evaluated. Data for representative members of this li-
brary are detailed in Table 5. Compounds possessing small N-acyl
substituents (e.g., 39–41) had modest DGAT-1 inhibitory activity.
Attempts to increase potency through the incorporation of more
lipophilic substituents, such as in 42, led to modest improvements
in potency, but were accompanied by significant metabolic insta-
bility. Attenuating this lipophilicity through addition of polarity
(43 and 44) were successful in reducing clearance potential (HLM
<8 mL/min/kg), but resulted in poor passive permeability. A set
of five-membered heterocycle-based amides were included in the
initial library and of these oxadiazole 45 was identified as a potent
inhibitor of DGAT-1 (IC50 = 7 nM). While this analog was 10-fold
more potent than benzoyl derivative 46, it suffered from a
ꢀ1000-fold disconnect with respect to inhibition of triglyceride
synthesis in HT-29 cells. This poor cellular activity was likely the
result of poor cellular penetration driven by the high PSA of 46.
Given the excellent in vitro pharmacology profiles of methyl es-
ter 28 and primary amide 29 efforts were reengaged on neutral
analogs of these leads, with a focus on non-amide replacements
for the carboxylic acid functionality of 1. Acetonitrile 49 was found
to have a good balance of DGAT-1 inhibitory activity, microsomal
stability and passive permeability (Table 6). Incorporation of a ter-
tiary alcohol (50) is also well tolerated in terms of suppression of
DGAT-1 activity, microsomal clearance and passive permeability.
Though not neutral under physiological conditions, it is worth not-
ing that the corresponding tertiary basic amine 51 retains good
DGAT-1 potency, however, it suffers from poor passive permeabil-
ity. Both 49 and 50 were evaluated in rat pharmacokinetic analyses
and based on the relative profiles, the latter was advanced to a rat
safety evaluation. In-life and clinical pathology analyses of Spra-
gue-Dawley rats treated for four days with oral doses (5, 50 and
500 mg/kg) of 50 revealed a number of side effects at the mid
Table 6
In vitro profiles of neutral analogs of 1
R
O
NH2
N
N
N
O
R
hDGAT-1 IC50 (nM)a
64
TG Synthesis IC50 (nM)b
41
HLM CLapp (mL/min/kg)
<8
PSA
105
Permeability27(x10À6 cm/sec)
27
49
50
N
OH
25
40
54
70
<8
12
102
107
23
2
Me
Me
Me
NH2
N
51
52
Me
O
60
23
<8
120
39
N
Me
O
Me
53
54
210
185
—
—
<8
<8
120
107
12
11
N
N
N
S
Me
Me
N
H
N
55
56
391
312
253
—
<8
<8
123
112
6
N
N
N
Me
N
0.6
Me
N
N
O
57
37
70
82
120
22
N
Me
a
Average of >2 determinations run in triplicate.
Inhibition of triglyceride synthesis determined in HT-29 cells. Average of >2 determinations run in triplicate.
b

5088
R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
Table 7
In vitro safety profile of 52
in combination with the low turnover in human microsomes pre-
dict an attractive pharmacokinetic profile in humans.
Compound 52 was very selective versus the closet DGAT-1
enzymatic family members hACAT-1 and hDGAT-2 (Fig. 7). The risk
of off-target pharmacology presenting a safety issue for 52 ap-
peared to be minimal based on selectivity indices of >150-fold
for a panel of 140 human receptors and channels. Negative results
in Ames and in vitro micronucleus analyses suggested low genetic
toxicology risk for this compound. No detectable inhibition against
a panel of human cytochrome P-450 isoforms indicated low prob-
ability of drug–drug interactions in the human clinical setting.
Patch clamp analysis of the potential hERG liability 52 revealed
Endpoint
Result
hACAT-1 inhibition
hDGAT-2 inhibition
Broad ligand/enzyme profiling
hERG inhibition
Ames assay
In vitro micronucleus
IC50 >10
IC50 >30
IC50 >10
23% Inhibition @ 30
Negative
l
l
l
M
M
M for All
lM
Negative
300
250
200
150
100
50
an IC50 >30 lM. Based on this in vitro safety profile, 52 was ad-
Vehicle
52 @ 0.1 mpk
52 @ 0.3 mpk
52 @ 1 mpk
1 @ 0.3 mpk
vanced to a four day rat safety evaluation at doses of 5, 50 and
500 mg/kg (po). No significant clinical signs, changes in body/liver
weights or histology/hematological endpoints were observed at all
doses. The only findings were slight elevations in total cholesterol
(1.5-fold), total triiodothyronine levels (1.3-fold, with no impact on
thyroxine or thyroid-stimulating hormone) and liver enzymes
(ALT, 1.5-fold) at 500 mg/kg. Based on this encouraging safety pro-
file, compound 52 was advanced to acute and chronic in vivo effi-
cacy evaluations (Table 7).
0
60
120
180
240
The pharmacodynamic potential of compound 52 was initially
evaluated in an oral triglyceride tolerance test. C57BL/6 mice were
treated with vehicle, 0.1, 0.3 or 1 mg/kg of 52 30 min prior to dos-
ing with a corn oil bolus. Plasma triglyceride (TG) levels were mon-
itored every hour over the course of the 4 h test period. Compound
52 inhibited the increase in plasma TG levels in a dose dependent
manner, with complete suppression of TG excursion at a dose of
0.3 mg/kg (Fig. 2). These results compared favorably with what is
seen for the lead clinical candidate 1, which exhibits a comparable
potency in this assay.
Minutes post corn oil challenge
Figure 2. Acute plasma triglyceride lowering effects of 52 in C57BL/6 mice.
and high doses. These included skin turgor, reductions in body
weight, increases in serum cholesterol and decreased T4 hormone
levels. These results led us to consider other options for neutral
replacements of the carboxylic acid moiety of 1.
Given the well established ability of azoles to serve as
bioisosteric replacements for amides and esters,
a series of
Compound 52 was advanced to a chronic efficacy study in db/db
mice maintained on a high fat diet. Animals were dosed with 0.5%
methylcellulose vehicle, compound 52 (1, 3 or 15 mg/kg, po, Q.D.)
five-membered heterocycle-based analogs were pursued.28 1,2,4-
Oxadiazole 52 was found to be a potent inhibitor of DGAT-1 and
cellular triglyceride synthesis. Its balanced physicochemical prop-
erties (LogD = 2.3, PSA = 120) resulted in an excellent ADME profile
with low microsomal clearance (<8 mL/min/kg) and high passive
permeability (39 Â 10À6 cm/s). Alternative oxadiazole, thiadiazole
and triazole motifs (53–56) were found to have both reduced
DGAT-1 inhibitory activities and passive permeabilities. Given
the profile of oxadiazole 52 the corresponding homologated analog
57 was evaluated. While incorporation of an additional methylene
unit had no significant impact on DGAT-1 inhibitory activity rela-
tive to 52, it was found to have substantially reduced metabolic
stability (HLM CLapp = 82 mL/min/kg). From this set of neutral het-
erocyclic-based analogs, oxadiazole 52 was selected for further
pharmacokinetic, safety and efficacy profiling. Pharmacokinetic
profiling in rat revealed 52 to have low clearance (5.3 mL/min/
kg) and moderate oral bioavailability of 53% (Fig. 4). These data
52 @ 1 mpk
10000
1000
100
10
52 @ 3 mpk
52 @ 15 mpk
1 @ 0.3 mpk
52: hDGAT-1 IC50 = 138 ng/mL
1: hDGAT-1 IC50 = 47 ng/mL
1
0
10
20
30
Time (h)
Figure 4. Day 14 free plasma exposures of 52 in mouse db/db chronic study.
100
80
60
40
20
0
20
Vehicle
15
10
5
52 @ 1 mpk
52 @ 3 mpk
52 @ 15 mpk
1 @ 0.3 mpk
0
2
4
6
8
10 12 14
-5
Day of treatment
0
2
4
6
8
10 12 14
Day of treatment
Figure 3. Body weight gain and food intake changes induced by chronic treatment of db/db mice with 52.

R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
5089
or 1 (0.3 mg/kg) for 14 days. Over the course of the study body
weight and food intake were measured daily for each animal
(n = 8/dose). Plasma endpoints, TG and non-esterified fatty acids
(NEFA) were measured on days 0, 7 and 14. On day 14, liver tissue
levels of TG and glycogen were evaluated. Separate satellite groups
(n = 4/dose) was utilized for drug exposure measurements. Over the
course of the 14 day treatment, compound 52 produced a dose–
responsive reduction in body weight (Fig. 3). This effect on body
weight was sustained throughout the course of the study, with no
rebound in weight gain observed. While there is an initial relative
reduction in food intake (FI) relative to controls in the first few days
of the study, FI rebounded at the mid-point in the study to then
parallel the vehicle treatment group. This suggests that the weight
loss observed in these animals was not driven entirely by the reduc-
tion in FI. The magnitude of weight loss at 1 mg/kg of 52 is approx-
imately equivalent to that observed for 1 at a comparable dose
(0.3 mg/kg). Free drug plasma exposures generated in satellite ani-
mals are plotted in Figure 4 (murine plasmafu = 0.09 and 0.35 for 52
and 1, respectively). At the highest dose of 52, plasma free drug
exposures were greater than the hDGAT-1 IC50 (murine data not
generated) over the duration of the study. At lower doses, expo-
sures were maintained at or near the IC50 for several hours during
the course of a 24 h period. The disconnect between IC50 coverage
and observed efficacy for the lowest dose of 52 and 1, could be
Postprandial Plasma FFA
Postprandial Plasma TG
1500
1000
* p ≤ 0.05
* p ≤ 0.05
*****
1000
*
*
*
Vehicle
*
500
0
52 @ 1 mpk
52 @ 3 mpk
52 @ 15 mpk
1 @ 0.3 mpk
*
** *
*
500
0
Day 0
Day 7
Day 14
Day 0
Day 7
Day 14
Day 14 Liver TG
Day 14 Liver Glycogen
250
200
150
100
50
400
300
200
100
0
* p ≤ 0.01
*
*
*
*
*
*
*
0
Figure 5. Triglyceride and free fatty acid changes induced by chronic treatment of db/db mice with 52.
NH2
O
OMe
NH2
N
O
O
OMe
(a)
N
H
O
N
H
+
HO
O
N
Cl
O
O
60
58
59
CO2Me
(c)
(b)
61
H2N
CO2H
CO2Me
NH2
N
(d) - (f)
OMe
NH2
N
N
N
H
O
N
N
O
O
O
O
62
4
Scheme 2. Synthesis of pyrimidooxazepinone 3. Reagents and conditions: (a) NaH, THF; (b) 1,2-dichloroethane, 60 °C; (c) sodium triacetoxyborohydride, 1,2-dichloroethane;
(d) trifluoroacetic acid, 1,2-dichloroethane; (e) 1,1-carbonyldiimidazole, DMAP, DMF, 60 °C; (f) lithium hydroxide, 1,4-dioxane, H2O.

5090
R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
CO2Me
CO2Me
O
Si
Cl
O
N
H
(a)
63
N
N
+
Cl
Cl
O
O
Si
65
N
Cl
Cl
(c)
(b)
64
CO2Me
CO2Me
O
Cl
O
O
Cl
+
N
N
N
N
O
67
66
(d)- (f)
(d)- (f)
CO2H
CO2H
O
O
NH2
NH2
N
N
N
N
O
O
10
11
Scheme 3. Syntheses of pyridooxazepinones 10 and 11. Reagents and conditions: (a) Et3N, THF, 0 °C; (b) MeOH, aq HCl I, 25 °C; (c) Cs2CO3, acetonitrile, reflux; (d) 4-
methoxybenzylamine, Et3N, DMA, sealed tube, 140 °C; (e) TFA, 50 °C; (f) LiOH, H2O, p-dioxane.
the result of difference in species pharmacology or efficacy being
driven in part by DGAT-1 inhibition in the liver/intestine, where
drug concentrations are likely elevated relative to plasma.
observed for 52 is consistent with DGAT-1 inhibition leading to
an improvement in insulin resistance.
Postprandial plasma triglycerides were significantly reduced
relative to vehicle control at days 7 and 14 for all doses of 52
and 1 (Fig. 5). There was no dose response to this effect since plas-
ma TG levels were essentially restored to basal levels at all doses of
52. As expected with treatment with a high-fat diet the control
mice had increased levels of plasma free fatty acids (FFA) over
the course of the study. With DGAT-1 being responsible for not
only synthesis, but also hydrolysis of TG, it was not clear what im-
pact inhibiting this enzyme would have on FFA in this study. While
no significant changes in plasma FFA levels for the treatment
groups were observed on day 7, by the end of the study levels were
substantially reduced at all doses of DGAT-1 inhibitor treatment
relative to the control animals. Similar effects were also observed
for liver TG levels after 14 days of treatment with 52. This reduc-
tion in liver TG content is consistent with what is observed in
DGAT1À/À mice.29 In high fat fed rodent models an inverse rela-
tionship between increased liver TG and reduced glycogen levels
has been described as a hallmark of the insulin resistant state.30
In the current study, day 14 liver glycogen levels were increased
relative to control animals in a dose–responsive manner (Fig. 5).
This profile of increased liver glycogen and reduced TG levels
3. Chemistry
Preparation of analogs 5, 12–25 were accomplished utilizing the
general procedures previously described for 1.21 Given the rela-
tively chemical inert nature of the 4-aminopyrimidooxazepinone
core, a wide range of chemistries could be carried out on advanced
intermediates.
The regioisomeric 4-aminopyrimidooxazepinone 3 was pre-
pared starting with condensation of chloroaldehyde 58 with the
sodium salt of
imine prepared from 60 and aniline 61 afforded benzylic amine
62. Selective deprotection of the benzyl ester protecting group fol-
lowed by CDI-mediated lactamization and hydrolysis of the methyl
ester afforded 3.
Preparation of the regioisomeric pyridooxazepinones 10 and 11
was accomplished utilizing the synthetic sequence depicted in
Scheme 3. Amide coupling of 6321 and acid chloride 64 afforded
amide 65. Removal of the alcohol protecting group, followed by base
catalyzed cyclization afforded a 1:2.4 mixture of regioisomeric chlo-
ropyridines 66 and 67, which were separated by chromatography.
a-hydroxyester 59 (Scheme 2). Reduction of the

R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
5091
The primary amine functionality was incorporated via condensation
with 4-methoxybenzylamine and deprotection with trifluoroacetic
acid. Base-catalyzed hydrolysis afforded acids 10 and 11.
Syntheses of the neutral heterocycle-based analogs of 1 are de-
picted in Scheme 4. Condensation of the acid chloride of 1 with N-
hydroxyacetamidine afforded 68 which was thermally cyclized to
afford oxadiazole 52. Regioisomeric oxadiazole 53 was generated
by dehydrative cyclization of bis-acetylated hydrazine 69. This
intermediate was also utilized to prepare the corresponding thiadi-
azole 54. Condensation of the acid chloride of 1 with acetamidraz-
one, followed by thermal cyclization provided triazole analog 55.
The corresponding N-methyl homolog 56 was prepared via con-
densation of thioamide 71 with acetyl hydrazide.
N
H
N
OH
4. Conclusion
Me
O
During preclinical evaluation of the prototypical DGAT-1 inhib-
itor 1 there were concerns around potential restriction of tissue dis-
tribution due to its associated low passive permeability, which in
turn could limit efficacy. This led to initiation of a back-up program
focused on the identification of analogs with enhanced passive per-
meability, while retaining the positive features of 1. The high polar
surface area associated with this carboxylic acid-based structure
limited the options available for tuning the properties to achieve
acceptable passive permeability. Extensive SAR studies led to the
identification of the neutral, oxadiazole 52 which is a potent
hDGAT-1 inhibitor, with a balanced ADME profile. Off-target phar-
macology and in vivo toleration analyses suggest that this com-
pound is a selective DGAT-1 inhibitor, with an encouraging safety
profile. Pharmacokinetic and pharmacodynamic evaluations re-
vealed this neutral compound possessed comparable maximal effi-
cacy relative to 1, suggesting that the polar nature of 1 did not
inhibit exposure to key efficacy tissues in preclinical models. How-
ever, if the rodent PK/PD models do not translate into low doses in
humans, there is the risk that the maximal absorbable dose of 1 will
likely be capped due to its poor permeability. Thus, the enhanced
permeability of 52 will likely allow a higher dosing paradigm in hu-
mans and that will allow for greater flexibility in addressing the po-
tential of DGAT-1 inhibition in treating obesity and diabetes.
O
NH2
N
(c)
(a)
(b)
N
52
N
1
O
68
O
H
N
Me
N
H
O
53
54
(e)
O
NH2
N
(a)
(d)
N
N
1
(f)
O
69
Me
N
H
N
5. Experimental
5.1. Chemistry
NH2
O
NMR spectra were recorded on a Varian Unity™ 400 or 500
(Varian Inc., Palo Alto, CA) at room temperature at 400 and
500 MHz 1H, respectively. Chemical shifts are expressed in parts
per million (d) relative to residual solvent as an internal reference.
The peak shapes are denoted as follows: s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; br s, broad singlet; v br s, very
broad singlet; br m, broad multiplet; 2s, two singlets.
Mass spectrometry analysis was also obtained by reversed
phase HPLC gradient method for chomatographic separation.
Molecular weight identification was recorded by positive and neg-
ative electrospray ionization (ESI) scan modes. A Waters/Micro-
mass ESI/MS model ZMD or LCZ mass spectrometer equipped
with Gilson 215 liquid handling system and HP 1100 DAD was
used to carry out the experiments.
O
NH2
N
(c)
(a)
(g)
N
55
N
1
O
70
H
N
Me
S
Purity determinations on all test compounds were determined
HPLC analysis. All compounds for which biochemical data is re-
ported are of >95% purity.
Silica gel column chromatography was performed with Bio-
tage™ columns (ISC, Inc., Shelton, CT) under low nitrogen pressure.
PF-04620110 was initially synthesized at Pfizer, but is now
commercially available through Sigma Aldrich.
O
NH2
N
(j)
(a)
56
N
N
1
(h)(i)
O
71
Scheme 4. Synthesis of heterocycles 52–56. Reagents and conditions: (a) oxalyl
chloride, dichloroethane; (b) hydroxyacetamidine, p-dioxane; (c) DMF, sealed tube,
120 °C; (d) acetyl hydrazide, p-dioxane; (e) triphenylphosphine, iodine, triethyl-
amine, dichloromethane; (f) Lawesson’s reagent, tetrahydrofuran, sealed tube,
120 °C; (g) acetamidrazone hydrochloride, tetrahydrofuran, triethylamine; (h)
methylamine hydrochloride, triethylamine, dichloromethane; (i) Lawesson’s
reagent, tetrahydrofuran, reflux; (j) mercury oxide, acetyl hydrazide, tetrahydrofu-
ran, microwave.
5.1.1. 2-{trans-4-[4-(4-Amino-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-6(5H)-yl)phenyl]cyclohexyl}ethanol (3)
To a stirred, cooled (0 °C) solution of 28 (103 mg, 0.25 mmol) in
tetrahydrofuran (20 mL) was added a 1 M tetrahydrofuran solution
of lithium aluminum hydride (0.50 mL, 0.5 mmol). After 3 h, the

5092
R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
reaction was quenched by sequential addition of water (20
15% aqueous sodium hydroxide (20 L) and then additional water
(60 L) and the resulting slurry was stirred for 1 h at room temper-
l
L),
at 50 °C for 1 h, cooled and acidified to pH ꢀ3 with 1 M aqueous
hydrogen chloride. 1,4-Dioxane was removed in vacuo and the
aqueous mixture was extracted with 10% i-propanol/dichloro-
methane, the organic layer dried over sodium sulfate and concen-
trated in vacuo. Chromatography on silica gel (0–10% methanol/
dichloromethane, 4 g column) afforded the title compound as an
off-white solid, 3.3 mg. 1H NMR (400 MHz, methanol-d4) d 1.17–
1.25 (m, 2H) 1.43–1.61 (m, 2H) 1.76–2.00 (m, 5H) 2.21 (d,
J = 6.83 Hz, 2H) 2.42–2.59 (m, 1H) 4.88 (s, 2H) 5.09 (s, 2H) 7.15
(d, J = 8.40 Hz, 2H) 7.27 (d, J = 8.40 Hz, 2H) 7.83 (s, 1H) 8.03 (s,
1H). LCMS (ESI) m/z: 397.1 (M+H).
l
l
ature. Filtration through a pad of CeliteÒ, washing with 10% isopro-
panol/dichloromethane and concentration in vacuo afforded a
sticky white solid. Chromatography on silica gel (4 g column, 1–
8% methanol/chloroform) afforded the title compound as a white
solid, 4 mg. 1H NMR (400 MHz, methanol-d4) d 7.90 (s, 1H), 6.96
(d, 2H), 6.58 (d, 2H), 4.52 (dd, 2H), 4.40 (s, 2H), 3.77 (dd, 2H),
3.55 (t, 2H), 2.30 (br t, 1H), 1.84–1.73 (m, 4H), 1.44–1.00 (m, 7H).
LCMS (ESI) m/z: 383.4 (M+H).
5.1.2. {trans-4-[4-(4-Amino-7-oxo-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-6(5H)-yl)phenyl]cyclohexyl}acetic acid (4)
To a stirred solution of 59 (2.44 g, 12.4 mmol) in tetrahydrofu-
ran (42 mL) was added sodium hydride (0.6 g of 60% in oil,
14.9 mmol). After 15 min, 58 (1.96 g, 12.4 mmol) was added and
the resulting yellow slurry was stirred for 17 h. The reaction mix-
ture was quenched with aqueous ammonium chloride, tetrahydro-
furan was removed in vacuo and the resulting slurry extracted
with ethyl acetate. The organic phase was dried over sodium sul-
fate and concentrated in vacuo to afford 60 as a yellow solid,
3.71 g. 1H NMR (400 MHz, chloroform-d) d 10.28 (s, 1H), 8.62 (br
s, 1H), 8.15 (s, 1H), 7.07–7.40 (m, 2H), 6.64–7.00 (m, 2H), 5.81
(br s, 1H), 5.13 (s, 2H), 4.85–5.04 (m, 2H), 3.78 (s, 3H). LCMS
(ESI) m/z: 318.3 (M+H).
A stirred solution of 60 (3.58 g, 5.60 mmol) and 61 (1.81 g,
7.32 mmol) in 1,2-dichloroethane (56 mL) was heated at 60 °C for
24 h. An additional portion of 61 (0.42 g) was added and heating
was continued for 4 h and then allowed to cool to ambient temper-
ature. The resulting solution was used as is in the next step.
The above solution was treated with sodium triacetoxyborohy-
dride (3.16 g, 14.2 mmol). After 17 h, saturated aqueous ammo-
nium chloride was added (10 mL), the organic layer was washed
with water, dried over sodium sulfate and concentrated in vacuo
to afford 62 as a white solid, 2.93 g. 1H NMR (400 MHz, chloro-
5.1.3. {trans-4-[4-(4-Amino-2-methyl-5-oxo-7,8-
dihydropyrimido[5,4-f][1,4]oxazepin-6(5H)-
yl)phenyl]cyclohexyl}acetic acid (5)
Prepared in analogy to compound 1.21 1H NMR (400 MHz, chlo-
roform-d) d 8.25 (br s, 1H), 7.35 (d, 2H), 7.18 (d, 2H), 6.64 (br s, 1H),
4.70–4.64 (m, 2H), 4.03–3.96 (m, 2H), 2.50 (dd, 1H), 2.43 (s, 3H),
2.31–2.24 (m, 2H), 1.99–1.85 (m, 5H), 1.59–1.43 (m, 2H), 1.24–
1.16 (m, 2H). LCMS (ESI) m/z: 411.4 (M+H).
5.1.4. {trans-4-[4-(4-Hydroxy-5-oxo-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-6(5H)-yl)phenyl]cyclohexyl}acetic acid (6)/
{trans-4-[4-(4-methoxy-5-oxo-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-6(5H)-yl)phenyl]cyclohexyl}acetic acid (7)
A stirred solution of methyl (trans-4-{4-[[4,6-dichloropyrimi-
din-5-yl)carbonyl](2-hydroxyethyl)amino]phenyl}cyclohexyl) ace-
tate21 (5.69 g, 12.2 mmol) and potassium carbonate (5.06 g,
36.6 mmol) in methanol (24.4 mL) was heated at reflux for 2 h.
After cooling, the methanol was removed in vacuo, the residue par-
titioned between ethyl acetate/water, the organic phase was dried
over sodium sulfate and concentrated in vacuo to afford a white so-
lid, 5.19 g. LCMS (ESI) m/z: 426.2 (M+H).
To a heated (50 °C), stirred solution of the above solid (1.50 g,
3.52 mmol) in p-dioxane (10 mL)/water (1 mL) was added an aque-
ous solution of lithium hydroxide (5 mL, 1 M). After 3 h, the reac-
tion was cooled and concentrated in vacuo. The residue was
suspended in water, acidified to pH ꢀ3 with 1 N aqueous hydro-
chloric acid and stirred for 5 min. The solids were filtered and air
dried. A 50 mg portion of these solids were purified by reverse
phase HPLC (PhenominixÒ C18 30 Â 50 mm column, 15–100% ace-
tonitrile/water (1% formic acid) gradient) to afford 6 (7.5 mg) and 7
(31 mg). Compound 6: 1H NMR (400 MHz, DMSO-d6) d 8.15 (s, 1H),
7.23 (AB q, 4H), 4.58 (t, 2H), 4.02 (t, 2), 2.50–2.42 (m, 1H), 2.13 (d,
2H), 1.85–1.64 (m, 5H), 1.53–1.40 (m, 2H), 1.18–1.03 (m, 2H).
LCMS (ESI) m/z: 396.1 (MÀH). Compound 7: LCMS (ESI) m/z:
412.2 (M+H).
form-d)
d 1.00–1.20 (m, 2H) 1.34–1.49 (m, 2H) 1.83 (d,
J = 11.72 Hz, 5H) 2.21 (d, J = 6.64 Hz, 2H) 2.34 (t, J = 12.20 Hz, 1H)
3.46 (br s, 1H) 3.64 (s, 3H) 3.77 (s, 3H) 4.20 (d, J = 3.32 Hz, 2H)
4.87 (s, 2H) 5.10 (s, 2H) 5.37 (s, 2H) 6.65 (d, J = 8.40 Hz, 2H)
6.77–6.89 (m, 2H) 7.02 (d, J = 8.40 Hz, 2H) 7.13–7.29 (m, 2H) 8.09
(s, 1H). LCMS (ESI) m/z: 549.5 (M+H).
To a stirred solution of 62 (2.90 g, 5.29 mmol) in dichlorometh-
ane (5 mL) was added trifluoroacetic acid (1.22 g, 10.6 mmol). After
1 h, the solution was concentrated in vacuo, the residue was
recrystallized from ethyl acetate/heptanes to afford an off-white
solid, 2.40 g. 1H NMR (400 MHz, DMSO-d6) d 0.89–1.11 (m, 2H)
1.18–1.40 (m, 2H) 1.52–1.78 (m, 5H) 2.16 (d, J = 6.83 Hz, 2H)
2.18–2.27 (m, 1H) 3.53 (s, 3H) 4.03 (s, 2H) 4.80 (s, 2H) 6.54 (d,
J = 8.20 Hz, 2H) 6.61 (br s, 2H) 6.85 (d, J = 8.40 Hz, 2H) 7.94 (s,
1H). LCMS (ESI) m/z: 429.1 (M+H).
5.1.5. (trans-4-{4-[4-(Methylamino)-5-oxo-7,8-
dihydropyrimido[5,4-f][1,4]oxazepin-6(5H)-
yl]phenyl}cyclohexyl)acetic acid (8)
A solution of the solid obtained above (39 mg, 0.09 mmol), 1,1-
carbonyldiimidazole (30 mg, 0.18 mmol) and 4-N,N-dimethylami-
nopyridine (0.1 mg, 0.001 mmol) in dimethylformamide (1 mL)
was stirred at 60 °C for 24 h. The solution was diluted into ethyl
acetate, washed with water, the organic layer dried over sodium
sulfate and concentrated in vacuo. Chromatography of the residue
on silica gel (0–10% methanol/dichloromethane, 4 g column) affor-
ded an off-white solid, 7.7 mg. 1H NMR (400 MHz, DMSO-d6) d
1.00–1.15 (m, 2H) 1.31–1.46 (m, 2H) 1.60–1.83 (m, 5H) 2.19 (d,
J = 6.64 Hz, 2H) 2.34–2.43 (m, 1H) 3.54 (s, 3H) 4.80 (s, 2H) 4.96
(s, 2H) 6.84 (s, 2H) 7.06 (d, 2H) 7.20 (d, 2H) 7.93 (s, 1H). LCMS
(ESI) m/z: 411.3 (M+H).
A solution of methyl {trans-4-[4-(4-chloro-5-oxo-7,8-dihydro-
pyrimido[5,4-f][1,4]oxazepin-6-(5H)-yl)phenyl]cyclohexyl}
ace-
tate21 (352 mg, 0.82 mmol) and a 2 M solution of methylamine in
THF (1.5 mL, 3 mmol) were stirred a ambient temperature for
65 h. The solution was concentrated in vacuo to afford a white so-
lid, 340 mg. LCMS (ESI) m/z: 425.2 (M+H).
A solution of the above solids and 1 M aqueous lithium hydrox-
ide (2.5 mL, 2.5 mmol) in p-dioxane (5 mL) was heated at 50 °C for
1.5 h. The reaction mixture was cooled, concentrated in vacuo, the
solids were suspended in water and the pH adjusted to ꢀ3 with 6 N
aqueous hydrochloric acid. The resulting solids were filtered and
dried at 50 °C in a vacuum oven for 17 h to afford the title com-
pound as an off-white solid, 314 mg. 1H NMR (400 MHz, chloro-
form-d) d 11.97 (br s, 1H), 8.61 (br s, 1H), 8.38 (s, 1H), 7.24 (d,
2H), 7.08 (d, 2H), 4.62 (dd, 2H), 3.97 (dd, 2H), 2.98 (s, 3H), 2.57–
To a stirred solution of the above solid (5 mg, 0.01 mmol) 1,4-
dioxane (0.1 mL) and water (0.025 mL) was added a 1 M solution
of aqueous lithium hydroxide (0.036 mL). This solution was heated

R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
5093
2.43 (m, 1H), 2.29 (d, 2H), 1.97–1.82 (m, 5H), 1.67–1.43 (m, 2H),
1.24–1.10 (m, 2H). LCMS (ESI) m/z: 411.1 (M+H).
vacuo to afford an oil. Chromatography on silica gel (10 g column,
20-50% ethyl acetate) afforded a white solid, 90 mg. 1H NMR
(400 MHz, chloroform-d) d 1.02–1.25 (m, 2H) 1.35–1.60 (m, 2H)
1.87 (d, J = 11.22 Hz, 5H) 2.24 (d, J = 6.65 Hz, 2H) 2.38–2.57 (m,
1H) 3.67 (s, 3H) 3.75 (s, 3H) 3.90 (t, J = 5.19 Hz, 2H) 4.44 (t,
J = 5.19 Hz, 2H) 4.54 (d, J = 4.98 Hz, 2H) 6.27 (d, J = 5.40 Hz, 1H)
6.80 (d, J = 8.72 Hz, 2H) 7.15–7.35 (m, 6H) 8.12 (d, J = 5.82 Hz,
1H). LCMS (ESI) m/z: 530.0 (M+H).
5.1.6. (trans-4-{4-[4-(Dimethylamino)-5-oxo-7,8-
dihydropyrimido[5,4-f][1,4]oxazepin-6(5H)-
yl]phenyl}cyclohexyl)acetic acid (9)
Prepared in analogy to compound 8. 1H NMR (400 MHz, DMSO-
d6) d 12.00 (br s, 1H), 8.23 (s, 1H), 7.26 (s, 4H), 4.47 (dd, 2H), 4.10
(br s, 2H), 3.00 (s, 6H), 2.50–2.41 (m, 1H), 2.10 (d, 2H), 1.82–1.63
(m, 5H), 1.53–1.38 (m, 2H), 1.18–1.03 (m, 2H). LCMS (ESI) m/z:
425.2 (M+H).
A
solution of the solid from the previous step (90 mg,
0.17 mmol) in trifluoroacetic acid (2.5 mL) was heated at 50 °C in
a sealed tube for 3 h. The reaction solution was concentrated in va-
cuo, the residue diluted into ethyl acetate, washed with saturated
aqueous sodium bicarbonate, dried over sodium sulfate and con-
centrated in vacuo to afford a white solid, 70 mg. 1H NMR
(400 MHz, chloroform-d) d 1.07–1.23 (m, 2H) 1.42–1.57 (m, 2H)
1.78–1.97 (m, 5H) 2.25 (d, J = 7.06 Hz, 2H) 2.49 (t, J = 12.25 Hz,
1H) 3.67 (s, 3H) 3.91 (t, J = 5.19 Hz, 2H) 4.47 (t, J = 4.98 Hz, 2H)
6.33 (d, J = 5.82 Hz, 1H) 7.20–7.30 (m, 4H) 8.02 (d, J = 5.40 Hz,
1H). LCMS (ESI) m/z: 410.0 (M+H).
5.1.7. {trans-4-[4-(6-Amino-5-oxo-2,3-dihydropyrido[3,4-
f][1,4]oxazepin-4(5H)-yl)phenyl]cyclohexyl}acetic acid (10)
A
stirred solution of 2-chloro-4-iodonicotinic acid (2.0 g,
7.0 mmol), one drop of dimethylformamide and thionyl chloride
(4.36 g, 36.7 mmol) was heated at 65 °C for 17 h. After cooling,
the reaction solution was azeotroped with toluene twice to afford
64 as a dark liquid.
To a stirred, cooled (0 °C) solution 63 (2.24 g, 5.52 mmol) and
diisopropylethylamine (2.8 g, 3.8 mmol) in tetrahydrofuran
(6 mL) was a solution of 64 (2.00 g, 10.0 mmol) in tetrahydrofuran
(6 mL) dropwise over a 15-min period. The reaction was then al-
lowed to stir at ambient temperature for 17 h, diluted into ethyl
acetate, washed with water and saturated aqueous sodium bicar-
bonate. The organic layer was dried over sodium sulfate, concen-
trated in vacuo and chromatographed on silica gel (100 g
column, 10–60% ethyl acetate/heptanes) to afford 65 as a red oil,
2.45 g. 1H NMR (400 MHz, chloroform-d) d 0.02 (s, 6H) 0.83 (s,
9H) 1.09 (t, J = 13.92 Hz, 2H) 1.29–1.43 (m, 2H) 1.72–1.89 (m,
5H) 2.21 (d, J = 6.65 Hz, 2H) 2.34 (t, J = 12.05 Hz, 1H) 3.65 (s, 3H)
3.84–3.92 (m, 2H) 3.95–4.03 (m, 2H) 6.99 (d, J = 8.31 Hz, 2H)
7.07 (d, J = 5.40 Hz, 1H) 7.27 (d, J = 8.31 Hz, 2H) 8.05 (d,
J = 5.40 Hz, 1H). LCMS (ESI) m/z: 579.2 (M+H).
A solution of 65 (2.45 g, 4.23 mmol) in 3% concd HCl/methanol
(17 mL) was stirred at ambient temperature for 1 h. The methanol
was removed in vacuo, the residue diluted into ethyl acetate,
washed with saturated aqueous sodium bicarbonate, water, dried
over sodium sulfate and concentrated in vacuo to afford a dark
red oil, 2.15 g. 1H NMR (400 MHz, chloroform-d) d 0.96–1.18 (m,
2H) 1.29–1.46 (m, 2H) 1.80 (dd, J = 22.22, 11.42 Hz, 5H) 2.21 (d,
J = 6.65 Hz, 2H) 2.35 (t, J = 12.05 Hz, 1H) 3.65 (s, 3H) 3.82–3.96
(m, 2H) 4.00–4.11 (m, 2H) 7.03 (d, J = 8.31 Hz, 2H) 7.10 (d,
J = 5.40 Hz, 1H) 7.29 (d, J = 8.72 Hz, 2H) 8.08 (d, J = 5.40 Hz, 1H).
LCMS (ESI) m/z: 465.0 (M+H).
A solution of the solid from the previous step (50 mg,
0.12 mmol) and lithium hydroxide (8.8 mg, 0.34 mmol) in a solu-
tion of tetrahydrofuran/methanol/water (3:2:1, 3 mL) was stirred
at ambient temperature for 20 h. The reaction was acidified to
pH 4 with 1 N aqueous hydrochloric acid and the resulting solids
were filtered and dried in vacuo to afford the title compound as
a white powder, 5.8 mg. 1H NMR (400 MHz, DMSO-d6) d 1.02–
1.18 (m, 2H) 1.30–1.53 (m, 2H) 1.63–1.88 (m, 5H) 2.11 (d,
J = 7.06 Hz, 2H) 2.38–2.55 (m, 1H) 3.85 (t, J = 5.19 Hz, 2H) 4.39 (t,
J = 4.98 Hz, 2H) 6.27 (d, J = 5.40 Hz, 1H) 7.26 (s, 4H) 7.95 (d,
J = 5.40 Hz, 1H). LCMS (ESI) m/z: 396.0 (M+H).
5.1.8. {trans-4-[4-(6-Amino-5-oxo-2,3-dihydropyrido[3,2-
f][1,4]oxazepin-4(5H)-yl)phenyl]cyclohexyl}acetic acid (11)
Employing intermediate 67 the title compound was prepared in
analogy to 10. 1H NMR (400 MHz, DMSO-d6) d 0.94–1.18 (m, 2H)
1.27–1.55 (m, 2H) 1.64–1.87 (m, 5H) 2.12 (d, J = 6.64 Hz, 2H)
2.36–2.55 (m, 1H) 4.07 (t, 2H) 4.70 (t, J = 5.19 Hz, 2H) 6.68 (d,
J = 7.06 Hz, 1H) 7.29 (s, 4H) 7.86 (d, J = 6.64 Hz, 1H). LCMS (ESI)
m/z: 396.0 (M+H).
5.1.9. {trans-4-[4-(4-Amino-8,8-dimethyl-5-oxo-7,8-
dihydropyrimido[5,4-f][1,4]oxazepin-6(5H)-
yl)phenyl]cyclohexyl}acetic acid (12)
Prepared in analogy to compound 1.21 1H NMR (400 MHz,
DMSO-d6): d 8.18 (s, 1H), 7.59 (br s, 1H) 7.20 (m, 4H), 3.79 (m,
2H), 2.42 (m, 1H), 2.08 (m, 2H), 1.78 (m, 4H), 1.67 (m, 1H), 1.43
(m, 2H), 1.24 (s, 6H), 166 1.05 (m, 2H). LCMS (ESI) m/z: 425.3 (M+H).
A slurry of the above oil (402 mg, 0.86 mmol) and cesium car-
bonate (1.13 g, 3.46 mmol) in acetonitrile (4.8 mL) was stirred at
reflux for 20 h. The reaction mixture was cooled, diluted into ethyl
acetate, washed with water, dried over sodium sulfate and concen-
trated in vacuo to afford an oil. Chromatography on silica gel (25 g
column, 10-70% ethyl acetate/heptane) afforded 66 (67 mg) and 67
(162 mg) as white solids. Compound 66: 1H NMR (400 MHz, chlo-
5.1.10. (trans-4-{4-[(8S)-4-Amino-8-methyl-5-oxo-7,8-
dihydropyrimido[5,4-f][1,4]oxazepin-6(5H)-
yl]phenyl}cyclohexyl)acetic acid (13)
Prepared in analogy to compound 1.21 1H NMR (400 MHz, meth-
anol-d4): d 8.19 (s, 1H), 7.33 (d, 2H), 7.24 (d, 2H), 5.04–4.94 (m,
1H), 3.92–3.87 (m, 2H), 2.58–2.44 (m, 1H), 2.25–2.18 (m, 2H),
1.93–1.86 (m, 5H), 1.60–1.46 (m, 2H), 1.35 (d, 3H), 1.28–1.10 (m,
2H). LCMS (ESI) m/z: 411.2 (M+H).
roform-d)
d 1.06–1.25 (m, 2H) 1.34–1.57 (m, 2H) 1.88 (d,
J = 10.39 Hz, 5H) 2.24 (d, J = 5.40 Hz, 2H) 2.49 (t, J = 12.25 Hz, 1H)
3.67 (s, 3H) 3.89 (t, J = 4.78 Hz, 2H) 4.55 (t, J = 4.78 Hz, 2H) 7.15–
7.44 (m, 5H) 8.29 (d, J = 7.06 Hz, 1H). LCMS (ESI) m/z: 429.0
(M+H). 67: 1H NMR (400 MHz, chloroform-d) d 1.03–1.22 (m, 2H)
1.37–1.57 (m, 2H) 1.88 (d, J = 10.80 Hz, 5H) 2.25 (d, J = 6.65 Hz,
2H) 2.38–2.59 (m, 1H) 3.67 (s, 3H) 3.92 (t, J = 5.19 Hz, 2H) 4.49
(t, J = 5.19 Hz, 2H) 6.96 (d, J = 5.40 Hz, 1H) 7.17–7.40 (m, 4H) 8.36
(d, J = 5.40 Hz, 1H). LCMS (ESI) m/z: 429.0 (M+H).
A solution of 66 (89 mg, 0.21 mmol), 4-methoxybenzylamine
(85 mg, 0.62 mmol) and triethylamine (105 mg, 1.0 mmol) in
dimethylacetamide (2.3 mL) was heated at 140 °C in a sealed tube
for 3 h. The cooled reaction solution was diluted into ethyl acetate,
washed with water, dried over sodium sulfate and concentrated in
5.1.11. trans-4-{4-[(8R)-4-Amino-8-methyl-5-oxo-7,8-
dihydropyrimido[5,4-f][1,4]oxazepin-6(5H)-
yl]phenyl}cyclohexyl)acetic acid (14)
Prepared in analogy to compound 1.21 1H NMR (400 MHz, meth-
anol-d4) d 1.11–1.25 (m, 2H) 1.36 (d, J = 6.64 Hz, 3H) 1.53 (q,
J = 12.88 Hz, 2H) 1.75–1.96 (m, 5H) 2.21 (d, J = 7.03 Hz, 2H) 2.46–
2.58 (m, 1H) 3.80–3.96 (m, 2H) 4.92–5.03 (m, 1H) 7.25 (d, 2H)
7.31 (d, 2H) 8.17 (s, 1H). LCMS (ESI) m/z: 411.1(M+H).
Compounds 17–24 were prepared in analogy to 1.21

5094
R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
5.1.12. 4-Amino-7,8-dihydropyrimido[5,4-f][1,4]oxazepin-
5(6H)-one (17)
1H NMR (400 MHz, DMSO-d6): d 8.10 (s, 1H), 7.55 (br s, 2H),
7.29–7.13 (m, 4H), 4.57–4.45 (m, 2H), 3.95–3.85 (m, 2H), 2.76–
2.62 (m, 2H), 2.50–2.31 (m, 1H), 1.80–1.62 (m, 5H), 1.45–1.25
(m, 2H), 1.28–0.96 (m, 2H). LCMS (ESI) m/z: 421.3 (M+H).
Prepared in analogy to compound 1.21 1H NMR (400 MHz,
DMSO-d6) d 9.08 (br s, 1H), 8.65 (br s, 1H), 8.35 (br s, 1H), 8.23
(s, 1H), 4.53 (m, 2H), 3.45 (m, 2H). LCMS (ESI) m/z: 181.4 (M+H).
5.1.22. 4-Amino-6-{4-[trans-4-(2-oxo-2-pyrrolidin-1-
ylethyl)cyclohexyl]phenyl}-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (33)
5.1.13. 4-Amino-6-methyl-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (18)
Prepared in analogy to 1.21 1H NMR (400 MHz, methanol-d4) d
8.08 (s, 1H), 4.57–4.52 (m, 2H), 3.67–3.63 (m, 2H), 3.12 (s, 3H).
LCMS (ESI) m/z: 195.3 (M+H).
A solution of 1 (12 mg, 0.03 mmol), pyrrolidine (5 mg,
0.08 mmol) and O-benzotriazole-N,N,N’,N’-tetramethyl-uronium-
hexafluoro-phosphate (12 mg, 0.04 mmol) in dimethylformamide
(0.4 mL) was heated at 55 °C for 18 h. Chromatography on silica
gel (4 g, 1–5% methanol/dichloromethane) afforded the title com-
pound as a white solid, 7 mg. 1H NMR (400 MHz, chloroform-d) d
8.24 (s, 1H) 8.15 (br s, 1H) 7.10–7.35 (m, 4H) 5.63 (br s, 1H)
4.60–4.70 (m, 2H) 3.91–4.03 (m, 2H) 3.35–3.49 (m, 4H) 2.39–
2.53 (m, 1H) 2.13–2.21 (m, 2H) 1.76–1.98 (m, 9H) 1.38–1.55 (m,
2H) 1.04–1.18 (m, 2H). LCMS (ESI) m/z: 450.4 (M+H).
5.1.14. 4-Amino-6-(4-methylphenyl)-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (19)
Prepared in analogy to 1.21 1H NMR (400 MHz, chloroform-d) d
8.23 (s, 1H), 7.22 (d, 2H), 7.11 (d, 2H), 4.64–4.62 (m, 2H), 3.97–3.94
(m, 2H), 2.36 (s, 3H). LCMS (ESI) m/z: 271.3 (M+H).
5.1.15. 4-Amino-6-(4-tert-butylphenyl)-7,8-
The following amides were prepared in analogy to 33.
dihydropyrimido[5,4-f][1,4]oxazepin-5(6H)-one (20)
Prepared in analogy to 1.21 1H NMR (400 MHz, methanol-d4) d
8.3 (s, 1H), 7.5 (d, 2H), 7.2 (d, 2H), 4.7 (t, 2H), 4.0(t, 2H), 1.3(s,
9H). LCMS (ESI) m/z: 313.5 (M+H).
5.1.23. N-({trans-4-[4-(4-Amino-5-oxo-7,8-
dihydropyrimido[5,4-f][1,4]oxazepin-6(5H)-
yl)phenyl]cyclohexyl}acetyl)-N-methylglycine (26)
1H NMR (400 MHz, DMSO-d6) d 8.17 (s, 1H), 7.59 (br s, 2H), 7.28
(s, 4H), 4.58 (d, 2H), 3.96 (d, 2H), 3.33 (s, 3H), 3.00 (s, 2H), 2.50–
2.43 (m, 1H), 2.24–2.10 (m, 2H), 1.87–1.69 (m, 5H), 1.50–1.40
(m, 2H), 1.18–1.03 (m, 2H). LCMS (ESI) m/z: 468.4 (M+H)
5.1.16. 4-[(8R)-4-Amino-8-methyl-5-oxo-7,8-
dihydropyrimido[5,4-f][1,4]oxazepin-6(5H)-yl]benzoic acid (21)
Prepared in analogy to 1.21 1H NMR (400 MHz, methanol-d4) d
8.20 (s, 1H), 8.12-8.07 (m, 2H), 7.52–7.46 (m, 2H), 5.05–4.95 (m,
1H), 3.98 (d, 2H), 1.39 (d, 3H). LCMS (ESI) m/z: 315.3 (M+H).
5.1.24. 2-trans-4-[4-(4-Amino-5-oxo-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-6(5H)-yl)phenyl]cyclohexyl}acetamide (29)
1H NMR (400 MHz, DMSO-d6) d 1.05 (br s, 2H) 1.26–1.50 (m,
2H) 1.59–1.71 (m, 1H) 1.75 (d, J = 11.32 Hz, 4H) 1.92 (d,
J = 7.03 Hz, 2H) 2.34–2.54 (m, 1H) 3.91 (t, J = 4.49 Hz, 2H) 4.41–
4.63 (m, 2H) 6.66 (br s, 1H) 7.13 (br s, 2H) 7.17–7.32 (m, 4H)
7.54 (s, 1H) 8.11 (s, 1H). LCMS (ESI) m/z: 396.3 (M+H).
5.1.17. 4-Amino-6-cyclopentyl-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (22)
Prepared in analogy to 1.21 1H NMR (400 MHz, methanol-d4) d
8.12 (s, 1H), 4.50–4.48 (m, 2H), 3.62–3.58 (m, 2H), 1.96–1.44 (m,
9H). LCMS (ESI) m/z: 249.3 (M+H).
5.1.18. 4-Amino-6-(cis-4-tert-butylcyclohexyl)-7,8-
dihydropyrimido[5,4-f][1,4]oxazepin-5(6H)-one (23)
Prepared in analogy to 1.21 1H NMR (400 MHz, chloroform-d) d
8.25 (s, 1H), 4.78 (m, 1H), 4.49 (t, 2H), 3.3.53 (t, 2H), 1.88 (m, 4H),
1.27 (m, 4H), 1.00 (m, 1H), 0.86 (s, 9H). LCMS (ESI) m/z: 319.5
(M+H).
5.1.25. 2-{trans-4-[4-(4-Amino-5-oxo-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-6(5H)-yl)phenyl]cyclohexyl}-N-ethylacetamide
(30)
1H NMR (400 MHz, DMSO-d6) d 8.17 (s, 1H), 7.75 (br s, 1H), 7.57
(br s, 2H), 7.12 (s, 4H), 4.53 (dd, 2H), 3.90 (dd, 2H), 3.00 (q, 2H),
2.47–2.39 (m, 1H), 1.97–1.92 (m, 2H), 1.79–1.65 (m, 5H), 1.44–
1.36 (m, 2H), 1.08–0.94 (m, 5H). LCMS (ESI) m/z: 424.5 (M+H).
5.1.19. 4-Amino-6-(trans-4-tert-butylcyclohexyl)-7,8-
dihydropyrimido[5,4-f][1,4]oxazepin-5(6H)-one (24)
5.1.26. 2-{trans-4-[4-(4-Amino-5-oxo-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-6(5H)-yl)phenyl]cyclohexyl}-N-
cyclopentylacetamide (31)
Prepared in analogy to 1.21 1H NMR (400 MHz, chloroform-d3) d
8.3 (s, 1H), 4.6 (m, 1H), 4.5 (t, 2H), 3.5 (t, 2H), 1.9 (m, 4H), 1.4–1.2
(m, 4H), 1 (m, 1H), 0.9 (s, 9H). LCMS (ESI) m/z: 319.5 (M+H).
LCMS (ESI) m/z: 464.2 (M+H).
5.1.20. 3-{trans-4-[4-(4-Amino-5-oxo-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-6(5H)-yl)phenyl]cyclohexyl}propanoic acid (25)
Prepared in analogy to 1.21 1H NMR (400 MHz, DMSO-d6) d
0.93–1.08 (m, 2H) 1.18–1.30 (m, 1H) 1.32–1.46 (m, 4H) 1.69–
1.82 (m, 4H) 2.14–2.23 (m, 2H) 3.22–3.30 (m, 1H) 3.85–3.95 (m,
2H) 4.49–4.56 (m, 2H) 7.15–7.26 (m, 4H) 7.54 (s, 2H) 8.11 (s, 1H)
11.93 (s, 1H). LCMS (ESI) m/z: 411.1(M+H).
5.1.27. 2-{trans-4-[4-(4-Amino-5-oxo-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-6(5H)-yl)phenyl]cyclohexyl}-N-[(1R)-1-
(hydroxymethyl)-2-methylpropyl]acetamide (32)
LCMS (ESI) m/z: 482.1 (M+H).
5.1.28. 4-Amino-6-[4-(trans-4-{2-[(3R)-3-hydroxypyrrolidin-1-
yl]-2-oxoethyl}cyclohexyl)phenyl]-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (34)
5.1.21. 6-(4-(trans-4-((2H-Tetrazol-5-
LCMS (ESI) m/z: 466.1 (M+H).
yl)methyl)cyclohexyl)phenyl)-4-amino-7,8-
dihydropyrimido[5,4-f][1,4]oxazepin-5(6H)-one (27)
To a stirred, cooled (0 °C) solution of trimethylaluminum (2 M
in toluene, 0.33 mL) were added trimethylsilyazide (38 mg,
0.33 mmol) and 49 (25 mg, 0.07 mmol). The reaction was heated
at 80 °C for 40 h, cooled and concentrated in vacuo. The residue
was chromatographed on silica gel (0–10% methanol/chloroform,
4 g column) to afford the title compound as a white solid, 2.8 mg.
5.1.29. 4-Amino-6-{4-[trans-4-(2-morpholin-4-yl-2-
oxoethyl)cyclohexyl]phenyl}-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (35)
1H NMR (400 MHz, chloroform-d) d 0.98–1.15 (m, 2H) 1.25–
1.37 (m, 1H) 1.36–1.50 (m, 2H) 1.50–1.61 (m, 2H) 1.79–1.92 (m,
4H) 2.24–2.38 (m, 2H) 2.40–2.56 (m, 1H) 3.37–3.51 (m, 2H)
3.52–3.68 (m, 6H) 3.90–4.01 (m, 2H) 4.65 (dt, J = 4.98, 3.81H z,

R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
5095
2H) 5.63 (br s, 1H) 7.10–7.32 (m, 4H) 8.13 (s, 1H) 8.24 (s, 1H). LCMS
(ESI) m/z: 480.5 (M+H).
5.1.40. 4-Amino-6-[4-(1-benzoylpiperidin-4-yl)phenyl]-7,8-
dihydropyrimido[5,4-f][1,4]oxazepin-5(6H)-one (46)
LCMS (ESI) m/z: 444.1 (M+H).
5.1.30. 4-Amino-6-(4-{trans-4-[2-(4-methyl-3-oxopiperazin-1-
yl)-2-oxoethyl]cyclohexyl}phenyl)-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (36)
5.1.41. 4-Amino-6-{4-[1-(isoxazol-5-ylcarbonyl)piperidin-4-
yl]phenyl}-7,8-dihydropyrimido[5,4-f][1,4]oxazepin-5(6H)-one
LCMS (ESI) m/z: 493 (M+H).
(47)
LCMS (ESI) m/z: 435.1 (M+H).
5.1.31. 4-Amino-6-(4-{trans-4-[2-(4-methoxypiperidin-1-yl)-2-
oxoethyl]cyclohexyl}phenyl)-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (37)
5.1.42. 4-Amino-6-{4-[1-(isoxazol-3-ylcarbonyl)piperidin-4-
yl]phenyl}-7,8-dihydropyrimido[5,4-f][1,4]oxazepin-5(6H)-one
(48)
1H NMR (400 MHz, chloroform-d) d 8.23 (s, 1H) 8.14 (br s, 1H)
7.11–7.30 (m, 4H) 4.60–4.68 (m, 2H) 3.88–4.00 (m, 3H) 3.61–
3.74 (m, 1H) 3.15–3.46 (m, 5H) 2.39–2.52 (m, 1H) 2.23 (dd,
J = 6.64, 3.73 Hz, 2H) 1.76–1.96 (m, 8H) 1.39–1.60 (m, 5H) 1.03–
1.17 (m, 2H). LCMS (ESI) m/z: 494.5 (M+H).
LCMS (ESI) m/z: 435.1 (M+H).
5.1.43. {trans-4-[4-(4-Amino-5-oxo-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-6(5H)-yl)phenyl]cyclohexyl}acetonitrile (49)
To a stirred solution of 29 (45 mg, 0.11 mmol) in tetrahydrofu-
ran (1 mL) were added oxalyl chloride (0.05 mL, 0.6 mmol) and
dimethylformamide (0.002 mL). After 2 h, saturated aqueous so-
dium bicarbonate was added and the mixture was diluted with
ethyl acetate, washed with water, dried over sodium sulfate and
concentrated in vacuo to afford the title compound as a light-yel-
low colored solid, 34 mg. 1H NMR (400 MHz, DMSO-d6): d 8.17 (s,
1H), 7.63 (br s, 2H) 7.22 (m, 4H), 4.57 (m, 2H), 3.90 (m, 2H), 2.43
(m, 3H), 1.78 (m, 4H), 1.63 (m, 1H), 1.43 (m, 2H), 1.08 (m, 2H).
LCMS (ESI) m/z: 378.3 (M+H).
5.1.32. 4-Amino-6-(4-{trans-4-[2-(3-hydroxyazetidin-1-yl)-2-
oxoethyl]cyclohexyl}phenyl)-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (38)
LCMS (ESI) m/z: 452.5 (M+H).
5.1.33. 6-[4-(1-Acetylpiperidin-4-yl)phenyl]-4-amino-7,8-
dihydropyrimido[5,4-f][1,4]oxazepin-5(6H)-one (39)
A solution of 4-amino-6-(4-piperidin-4-ylphenyl)-7,8-dihydro-
pyrimido[5,4-f][1,4]oxazepin-5(6H)-one (prepared in analogy
to 121
,
starting with commercially-available tert-butyl
4-(4-chlorophenyl)piperidine-1-carboxylate, 17 mg, 0.05 mmol),
(2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate) (38 mg, 0.1 mmol), triethylamine (20 mg,
0.2 mmol) and acetic acid (12 mg, 0.1 mmol) were stirred at ambi-
ent temperature for 17 h. Solvents were removed in vacuo and the
residue purified by reverse phase chromatography to afford the ti-
tle compound as white solid, 11 mg. LCMS (ESI) m/z: 382.1 (M+H).
The following compounds were made in analogy to 39.
5.1.44. 4-Amino-6-{4-[trans-4-(2-hydroxy-2-
methylpropyl)cyclohexyl]phenyl}-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (50)
To an ice cooled solution of 28 (40 mg, 0.10 mmol) was added
methyl magnesium bromide (1.4 M in toluene, 0.83 mL,
1.16 mmol), the cooling bath was allowed to expire and the reac-
tion mixture was stirred for 24 h. The reaction was partitioned be-
tween water and ethyl acetate, the organic phase dried over
sodium sulfate and concentrated in vacuo. Chromatography on sil-
ica gel (4 g, 1–5% methanol/dichloromethane) afforded the title
compound as a white solid, 10 mg. 1H NMR (400 MHz, chloro-
form-d) d 8.25 (s, 1H) 8.15 (br s, 1H) 7.22–7.30 (m, 2H) 7.12–
7.19 (m, 2H) 5.71 (br s, 1H) 4.63–4.69 (m, 2H) 3.94–4.03 (m, 2H)
2.41–2.53 (m, 1H) 1.82–1.98 (m, 4H) 1.38–1.56 (m, 5H) 1.04–
1.28 (m, 8H). LCMS (ESI) m/z: 411.4 (M+H).
5.1.34. 4-Amino-6-[4-(1-isobutyrylpiperidin-4-yl)phenyl]-7,8-
dihydropyrimido[5,4-f][1,4]oxazepin-5(6H)-one (40)
LCMS (ESI) m/z: 410.2 (M+H).
5.1.35. 4-Amino-6-{4-[1-(ethoxyacetyl)piperidin-4-yl]phenyl}-
7,8-dihydropyrimido[5,4-f][1,4]oxazepin-5(6H)-one (41)
LCMS (ESI) m/z: 426.1 (M+H).
5.1.45. 4-Amino-6-{4-[trans-4-(2-amino-2-
5.1.36. 4-Amino-6-{4-[1-(cyclohexylcarbonyl)piperidin-4-
yl]phenyl}-7,8-dihydropyrimido[5,4-f][1,4]oxazepin-5(6H)-one
(42)
methylpropyl)cyclohexyl]phenyl}-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (51)
To a stirred solution of 50 (100 mg, 0.24 mmol) and trimethyl-
silylazide (42 mg, 0.37 mmol) was added boron trifluoride etherate
(54 mg, 0.37 mmol) dropwise. After 30 h the reaction mixture was
partitioned between ethyl acetate and saturated aqueous sodium
bicarbonate. The organic layer was separated, dried over sodium
sulfate to afford a white solid (106 mg), which was taken onto
the next step without further purification.
LCMS (ESI) m/z: 450.2 (M+H).
5.1.37. 4-Amino-6-{4-[1-(tetrahydro-2H-pyran-4-
ylcarbonyl)piperidin-4-yl]phenyl}-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (43)
LCMS (ESI) m/z: 452.1 (M+H).
The material prepared in the previous step was dissolved in
ethyl acetate (10 mL)/ethanol (10 mL), 10% palladium-on-carbon
(25 mg) was added and the slurry was shaken under an atmo-
sphere of hydrogen gas (50 psi) for 20 h. The reaction mixture
was filtered through a pad of Celite, washing with ethyl acetate
and the combined filtrates were concentrated in vacuo. Chroma-
tography on silica gel utilizing a gradient of 3–10% of 10% ammo-
nium hydroxide in methanol/dichloromethane afforded the title
compound as a white solid, 21 mg. 1H NMR (400 MHz, methanol-
d4) d 1.14 (s, 6H) 1.16–1.30 (m, 3H) 1.36 (d, J = 4.98 Hz, 2H)
5.1.38. 4-Amino-6-(4-{1-[(cis-4-
hydroxycyclohexyl)carbonyl]piperidin-4-yl}phenyl)-7,8-
dihydropyrimido[5,4-f][1,4]oxazepin-5(6H)-one (44)
LCMS (ESI) m/z: 466.2 (M+H).
5.1.39. 4-Amino-6-{4-[1-(1,2,5-oxadiazol-3-
ylcarbonyl)piperidin-4-yl]phenyl}-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (45)
LCMS (ESI) m/z: 436.1 (M+H).

5096
R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
1.40–1.62 (m, 3H) 1.88 (dd, J = 23.06, 12.25 Hz, 3H) 2.43–2.57 (m,
1H) 3.94–4.04 (m, 2H) 4.61–4.72 (m, 2H) 7.17–7.26 (m, 2H)
7.26–7.33 (m, 2H) 8.14 (s, 1H). LCMS (ESI) m/z: 410.0 (M+H).
5.1.49. 4-Amino-6-(4-{trans-4-[(5-methyl-4H-1,2,4-triazol-3-
yl)methyl]cyclohexyl}phenyl)-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (55)
To an ice cooled, stirred mixture of 1 (50 mg, 0.13 mmol) in 1,2-
dichloroethane (0.42 mL) was added oxalyl chloride (0.11 mL,
1.26 mmol) and the resulting thick slurry was stirred at room tem-
perature for 2 h. The mixture was concentrated in vacuo, azeotr-
oped with toluene and the resulting solids dissolved in
tetrahydrofuran (3 mL). Acetamidrazone hydrochloride (41 mg,
0.38 mmol) and triethylamine (39 mg, 0.38 mmol) were added
and the mixture was stirred for 3 h. The reaction was concentrated
in vacuo and purified via medium pressure silica gel chromatogra-
phy (10 g column, 15 mL/min, 5–10% methanol/dichloromethane)
to afford 70 as a white solid, 18 mg.
A stirred solution of 70 (18 mg, 0.04 mmol) in DMF (1 mL) was
heated at 140 °C for 4 h, concentrated in vacuo and purified via
medium pressure silica gel chromatography (10 g column, 15 mL/
min, 5–10% methanol/dichloromethane) to afford the title com-
pound as a yellow solid, 0.5 mg. 1H NMR (400 MHz, chloroform-
d) d 8.25 (s, 1H), 7.23–7.28 (m, 2H), 7.12–7.18 (m, 2H), 4.65 (dd,
J = 4.98, 3.61 Hz, 2H), 3.90–4.02 (m, 2H), 2.34–2.42 (m, 6H), 2.12
(d, J = 6.83 Hz, 1H), 1.90 (br s, 5H), 1.40–1.60 (m, 3H). LCMS (ESI)
m/z: 432 (M+H).
5.1.46. 4-Amino-6-(4-{trans-4-[(3-methyl-1,2,4-oxadiazol-5-
yl)methyl]cyclohexyl}phenyl)-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (52)
To an ice cooled, stirred mixture of 1 (75 mg, 0.19 mmol) in 1,2-
dichloroethane (0.63 mL) was added oxalyl chloride (0.165 mL,
1.89 mmol) and the resulting thick slurry was stirred at room tem-
perature for 2 h. The mixture was concentrated in vacuo, azeotr-
oped with toluene and the resulting solids dissolved in p-dioxane
(1.5 mL), N-hydroxyacetamidine (140 mg, 1.9 mmol) added and
the mixture stirred at room temperature overnight. The reaction
mixture was concentrated in vacuo and chromatographed on silica
gel (12 g column, 5–10% methanol/dichloromethane over 30 min)
to afford 68 as a white solid, 86 mg.
To a stirred solution of 68 (37 mg, 0.082 mmol) in dimethyl-
formamide (1.0 mL) was heated under microwave conditions at
120 °C for 5 h. The reaction mixture was concentrated in vacuo
and chromatographed on silica gel (12 g column, 2.5–10% metha-
nol/dichloromethane over 30 min) to afford the title compound
as a white solid, 23 mg. 1H NMR (400 MHz, chloroform-d) d 8.24
(s, 1H) 8.12 (br s, 1H) 7.20–7.28 (m, 2H) 7.11–7.19 (m, 2H) 5.67
(br s, 1H) 4.59–4.70 (m, 2H) 3.91–4.01 (m, 2H) 2.76 (d, 2H) 2.43–
2.56 (m, 1H) 2.35 (s, 3H) 1.78–1.99 (m, 5H) 1.38–1.56 (m, 2H)
1.13–1.29 (m, 2H). LCMS (ESI) m/z: 435.1 (M+H).
5.1.50. 4-Amino-6-(4-{trans-4-[(4,5-dimethyl-4H-1,2,4-triazol-
3-yl)methyl]cyclohexyl}phenyl)-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (56)
To an ice cooled, stirred mixture of 1 (50 mg, 0.13 mmol) in 1,2-
dichloroethane (0.42 mL) was added oxalyl chloride (0.11 mL,
1.26 mmol) and the resulting thick slurry was stirred at room tem-
perature for 2 h. The mixture was concentrated in vacuo, azeotr-
oped with toluene and the resulting solids dissolved in 2 M
methylamine in tetrahydrofuran (0.63 mL, 1.26 mmol) and stirred
for 24 h. The solids were filtered, washed with ethyl ether and
dried in vacuo to afford 2-{4-[4-(4-amino-5-oxo-7,8-dihydro-5H-
9-oxa-1,3,6-triaza-benzocyclohepten-6-yl)-phenyl]-cyclohexyl}-
N-methylacetamide as a white solid, 50 mg.
A solution of 2-{4-[4-(4-amino-5-oxo-7,8-dihydro-5H-9-oxa-
1,3,6-triaza-benzocyclohepten-6-yl)-phenyl]-cyclohexyl}-N-
methylacetamide (35 mg, 0.085 mmol) and Lawesson’s reagent
(21 mg, 0.051 mmol) in tetrahydrofuran (0.57 mL) was heated at
reflux for 3 h. The reaction was cooled, concentrated in vacuo
and chromatographed on silica gel (4 g, 2–8% methanol/dichloro-
methane, 30 min) to afford 71 as a yellow solid, 11 mg.
A stirred slurry of 71 (11 mg, 0.026 mmol), mercury oxide
(6.4 mg, 0.029 mmol) and acetic hydrazide (4 mg, 0.052 mmol) in
tetrahydrofuran was stirred at room temperature for 16 h and then
heated at 80 °C under microwave conditions. The reaction mixture
was filtered through CeliteÒ, washing with methanol and then
chromatographed via prep HPLC (C18, 20–50% acetonitrile/water,
10 mL/min) to afford the title compound as a white solid, 4 mg.
1H NMR (400 MHz, chloroform-d) d 8.22 (s, 1H) 8.16 (br s, 1H)
7.24 (d, 2H) 7.14 (d, 2H) 6.02 (br s, 1H) 4.61–4.68 (m, 2H) 3.94–
4.01 (m, 2H) 3.46 (s, 3H) 2.65 (d, 2H) 2.45–2.54 (m, 1H) 2.41 (s,
3H) 1.77–1.94 (m, 5H) 1.36–1.51 (m, 2H) 1.13–1.29 (m, 2H). LCMS
(ESI) m/z: 448.2 (M+H).
5.1.47. 4-Amino-6-(4-{trans-4-[(5-methyl-1,3,4-oxadiazol-2-
yl)methyl]cyclohexyl}phenyl)-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (53)
To an ice cooled, stirred mixture of 1 (100 mg, 0.252 mmol) in
1,2-dichloroethane (0.84 mL) was added oxalyl chloride (0.221 mL,
2.52 mmol) and the resulting thick slurry was stirred at room tem-
perature for 2 h. The mixture was concentrated in vacuo, azeotroped
with toluene and the resulting solids dissolved in p-dioxane
(1.5 mL), acetic hydrazide (192 mg, 2.52 mmol) added and the mix-
ture stirred at room temperature for 96 h. The reaction mixture was
partitioned between dichloromethane and saturated aqueous so-
dium bicarbonate. The insoluble solids were filtered, washed with
water and dried in vacuo to afford 69 as a white solid, 83 mg.
To a stirred solution of triphenylphosphine (23 mg, 0.021 mmol),
iodine (21 mg, 0.084 mmol) and triethylamine (18 mg, 0.176 mmol)
was added 69 (20 mg, 0.044) and the resulting mixturewas stirredat
room temperature for 3.5 h. The reaction mixture was concentrated
in vacuo and chromatographed via prep HPLC (C18 column, 20–50%
acetonitrile/water, 10 mL/min) to afford the title compound as a
white solid, 6 mg. 1H NMR (400 MHz, chloroform-d) d 8.24 (s, 1H)
8.15 (br s, 1H) 7.20–7.30 (m, 2H) 7.11–7.19 (m, 2H) 5.72 (br s, 1H)
4.61–4.68 (m, 2H) 3.93–4.02 (m, 2H) 2.68–2.76 (m, 2H) 2.41–2.55
(m, 4H) 1.80–1.95 (m, 5H) 1.37–1.54 (m, 2H) 1.13–1.28 (m, 2H).
LCMS (ESI) m/z: 435.3 (M+H).
5.1.48. 4-Amino-6-(4-{trans-4-[(5-methyl-1,3,4-thiadiazol-2-
yl)methyl]cyclohexyl}phenyl)-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (54)
A solution of 69 (26 mg, 0.057 mmol) and Lawesson’s reagent
(14 mg, 0.034 mmol) in 1:1 p-dioxane:tetrahydrofuran (0.8 mL)
was heated in a sealed tube at 120 °C for 18 h. The reaction was
cooled, concentrated and chromatographed via prep HPLC (C18
column, 20–50% acetonitrile/water, 10 mL/min) to afford the title
compound as a white solid, 2.5 mg. 1H NMR (400 MHz, metha-
nol-d4) d 8.12 (s, 1H) 7.26–7.32 (m, 2H) 7.19–7.25 (m, 2H) 4.63–
4.69 (m, 2H) 3.96–4.02 (m, 2H) 2.97–3.01 (m, 2H) 2.70 (s, 3H)
2.48–2.58 (m, 1H) 1.83–1.92 (m, 5H) 1.42–1.57 (m, 2H) 1.18–
1.30 (m, 2H). LCMS (ESI) m/z: 451.1 (M+H).
5.1.51. 4-Amino-6-(4-{trans-4-[2-(3-methyl-1,2,4-oxadiazol-5-
yl)ethyl]cyclohexyl}phenyl)-7,8-dihydropyrimido[5,4-
f][1,4]oxazepin-5(6H)-one (57)
Prepared in analogy to 52, employing 25 as the starting material.
1H NMR (400 MHz, chloroform-d) d 8.23 (s, 1H), 8.21–8.02 (m, 1H),
7.27–7.09 (m, 4H), 5.80–5.55 (m, 1H), 4.68–4.57 (m, 2H), 4.00–3.91
(m, 2H), 2.90–2.79 (m, 2H), 2.52–2.41 (m, 1H), 2.37–2.31 (m, 3H),
1.8 (d, J = 12.30 Hz, 4H), 1.77–1.61 (m, 2H), 1.49–1.29 (m, 3H),
1.16–1.01 (m, 2H). LCMS (ESI) m/z: 449.3 (M+H).

R. L. Dow et al. / Bioorg. Med. Chem. 21 (2013) 5081–5097
5097
6. Unger, R. H.; Zhou, Y.-T.; Orci, L. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 2327.
7. Shimonura, I.; Hammer, R. E.; Ikemoto, S.; Brown, M. S.; Goldstein, J. L. Nature
1999, 401, 73.
8. Coleman, R. A.; Lee, D. P. Prog. Lipid Synth. 2004, 43, 134.
9. Koltun, D. O.; Zablocki, J. Enzymatic targets in the triglyceride synthesis
pathway. In Annual Reports in Medicinal Chemistry, volume 45; Macor, J. E.,
Ed.: Academic Press, New York, 2010; pp 109–122.
10. Kayden, H. J.; Senior, J. R.; Mattson, F. H. J. Clin. Invest. 1967, 46, 1695.
11. Kennedy, E. P. Annu. Rev. Biochem. 1957, 26, 119.
5.2. Molecular modeling
The conformational analysis was performed using OPLS_2005
force field.31,32
5.3. Biology
12. Cases, S.; Smith, S. J.; Zheng, Y. W.; Myer, H. M.; Lear, S. R.; Sande, E.; Novak, S.;
Collins, C.; Welch, C. B.; Lusis, A. J.; Erickson, S. K.; Farese, R. V., Jr. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 13018.
13. Oelkers, P.; Behari, A.; Cromley, D.; Billheimer, J. T.; Sturley, S. L. J. Biol. Chem.
1998, 273, 26765.
5.3.1. Enzyme and whole-cell assays
Experimental procedures for DGAT-1 enzymatic and HT-29
whole cell assays, as well as the triglyceride tolerance test in rats
can be found in the supporting information section of Ref. 21.
14. Cases, S.; Stone, S. J.; Zhou, P.; Yen, E.; Tow, B.; Lardizabal, K. D.; Voelker, T.;
Farese, R. V., Jr. J. Biol. Chem. 2001, 276, 38870.
5.3.2. 14-Day efficacy study in db/db mice
15. Smith, S. J.; Cases, S.; Jensen, D. R.; Chen, H. C.; Sande, E.; Tow, B.; Sanan, D. A.;
Raber, J.; Eckel, R. H.; Farese, R. V. Nat. Genet. 2000, 25, 87.
16. Chen, H. C.; Smith, S. J.; Ladha, Z.; Jensen, D. R.; Ferreira, L. D.; Pulawa, L. K.;
McGuire, J. G.; Pitas, R. E.; Eckel, R. H.; Farese, R. V. J. Clin. Invest. 2002, 109,
1049.
17. Chen, H. C.; Jensen, D. R.; Myers, H. M.; Eckel, R. H.; Farese, R. V. J. Clin. Invest.
2003, 111, 1715.
18. Dow, R. L. Acyl-CoA: Diacylglycerol Acyltransferase-1 Inhibition as an
Approach to the Treatment of Type 2 Diabetes. In New Therapeutic Strategies
for Type 2 Diabetes; Jones, R. M., Ed.; RSC Publishing: Cambridge, UK, 2012; pp
215–248.
19. King, A. J.; Judd, A. S.; Souers, A. J. Expert Opin. Ther. Patents 2010, 20, 19.
20. Birch, A. M.; Buckett, L. K.; Turnbull, A. V. Curr. Opin. Drug Disc. Dev. 2010, 13,
489.
21. Dow, R. L. Li, J-C.; Pence, M. P.; Gibbs, E. M.; LaPerle, J. L.; Litchfield, J.;
Piotrowski, D. W.; Munchhof, M. J.; Manion, T. B.; Zavadoski, W. J.; Walker, G.
S.; McPherson, R. K.; Tapley, S.; Sugarman, E.; Guzman-Perez, A.; DaSilva-
Jardine, P. ACS Med. Chem. Lett. 2011, 2, 407.
Male db/db mice (Charles River, Wilmington, MA) were bled,
randomized for body weight and housed four per cage in hanging
wire cages (4 per/cage/dose) at 21 + 2 °C with a 12:12 h light:dark
cycle, fed a pelleted high fat Western diet (Research Diets, New
Brunswick, NJ) and had ad libitum access to water. All procedures
were approved by and conducted in accordance with Institutional
Animal Care and Use Committee guidelines and regulations. At
4:00 pm of day 0, animals were dosed with 1, 3 or 15 mg/kg of
52 or 0.5% methylcellulose vehicle (n = 8). Food, water and body
weight measurements were collected daily at 4:00 pm immedi-
ately prior to dosing. Fed plasma triglyceride and non-esterified
free fatty acid were collected from orbital sinus bleeds 16 h post
dose on days 0, 7 and 14. Liver biopsies were collected on day
14, 16 h after final dose. Satellite PK groups (n = 4) were dosed uti-
lizing the protocols described above.
22. Fox, B. M.; Furukawa, N. H.; Hao, X.; Lio, K.; Inaba, T.; Jackson, S. M.; Kayser, F.;
Labelle, M.; Kexue, M.; Matsui, T.; McMinn, D. L.; Ogawa, N.; Rubenstein, S. M.;
Sagawa, S.; Sugimoto, K.; Suzuki, M.; Tanaka, M.; Ye, G. Yoshida, A.; Zhang, J. A.
Preparation of fused bicyclic nitrogen-containing heterocycles, useful in the
treatment or prevention of metabolic and cell proliferative diseases. WO 2004/
047756A2, Chem. Abstr. 141:38623.
6. Author contributions
23. Calculated passive permeability of 5 Â 10À6 cm/s. The calculated passive
permeability is based on a Pfizer training set of 126,024 measured passive
permeabilities.
The manuscript was written through contributions of all
authors. All authors have reviewed the final version of the
manuscript
24. Dow, R. L.; Carpino, P. A.; Hadcock, J. R.; Black, S. C.; Iredale, P. A.; DaSilva-
Jardine, P.; Schneider, S. R.; Paight, E. S.; Griffith, D. A.; Scott, D. O.; O’Connor, R.
E.; Nduaka, C. I. J. Med. Chem. 2009, 52, 2652.
25. Brabec, M. J. Aldehydes and acetals. In Patty’s Industrial Hygiene and
Toxicology, Clayton, G. D.; Clayton, F. E., Eds.; John Wiley & Sons, Inc: New
York, 1993; vol. IIB, 2629.
26. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430.
27. Di, L.; Whitney-Pickett, C.; Umland, J. P.; Zhang, H.; Zhang, X.; Gebhard, D. F.;
Lai, Y.; Federico, J. J., III; Davidson, R. F.; Smith, R.; Reyner, E. L.; Lee, C.; Feng, B.;
Rotter, C.; Varma, M. V.; Kempshall, S.; Fenner, K.; El-Kattan, A. F.; Liston, T. E.;
Troutman, M. D. J. Pharm. Sci. 2011, 100, 4974.
Acknowledgement
The authors would like to thank Philip Carpino for editorial
comments on this manuscript.
References and notes
28. Meanwell, N. A. J. Med. Chem. 2011, 54, 2529.
29. Villaneuva, C. J.; Monetti, M.; Shih, M.; Zhou, P.; Watkins, S. M.; Bhanot, S.;
Farese, R. V., Jr. Hepatology 2009, 50, 434.
30. Kusunoki, M.; Tsutsumi, K.; Hara, T.; Ogawa, H.; Nakamura, T.; Sakakibara, F.;
Fukuzawa, Y.; Suga, T.; Kakumu, S.; Nakaya, Y. Metabolism 2002, 51, 792.
31. Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. J. Am. Chem. Soc. 1996, 118,
11225.
1. McGarry, J. D. Diabetes 2002, 51, 7.
2. Alberti, K. G.; Zimmet, P.; Shaw, J. Lancet 2005, 366, 1059.
3. Stein, D. T.; Dobbins, R.; Szczepaniak, L.; Malloy, C.; McGarry, J. D. Diabetes
1997, 46, 23A.
4. Szsczepaniak, L. S.; Babcock, E. E.; Schick, F.; Dobbins, R. L.; Garg, A.; Burns, D.
K.; McGarry, J. D. Am. J. Physiol. 1999, 276, E977.
32. Kaminski, G. A.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen, W. J. J. Phys. Chem. B
2001, 105, 6474.
5. Huang-Doran, I.; Sleigh, A.; Rochford, J. J.; O’Rahilly, S.; Savage, D. B. J.
Endocrinol. 2010, 207, 245.