Discovery and optimization of efficacious neutral 4-amino-6-biphenyl-7,8- dihydropyrimido[5,4-f][1,4]oxazepin-5-one diacylglycerol acyl transferase-1 (DGAT1) inhibitors

Article abstract of DOI:10.1039/c2md20231j

A series of neutral DGAT1 inhibitors with good potency and pharmacokinetics (PK) has been designed by modification of an acidic startpoint. This was achieved by selecting the acid with the highest ligand lipophilicity efficiency (LLE) and replacing the ac

Full text of DOI:10.1039/c2md20231j

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CONCISE ARTICLE
Discovery and optimization of efficacious neutral 4-
amino-6-biphenyl-7,8-dihydropyrimido[5,4-f][1,4]
oxazepin-5-one diacylglycerol acyl transferase-1
(DGAT1) inhibitors†
Cite this: Med. Chem. Commun., 2013,
4, 165
*
Frederick W. Goldberg, Alan M. Birch, Andrew G. Leach, Sam D. Groombridge,
Wendy L. Snelson, Pablo Morentin Gutierrez, Clare D. Hammond, Susan Birtles
and Linda K. Buckett
A series of neutral DGAT1 inhibitors with good potency and pharmacokinetics (PK) has been designed by
modification of an acidic startpoint. This was achieved by selecting the acid with the highest ligand
lipophilicity efficiency (LLE) and replacing the acid with neutral isosteres. PK properties (Fabs) were then
improved by removing the sidechain to reduce molecular weight and polar surface area (PSA).
Compound 13 has shown good cross-species PK, with pre-clinical efficacy and PK/PD relationships
comparable to those previously described for acidic inhibitors.
Received 6th August 2012
Accepted 13th September 2012
DOI: 10.1039/c2md20231j
www.rsc.org/medchemcomm
One concern we had at the outset of the program was that
removing the carboxylic acid would be expected to increase log
Introduction
Diacylglycerol acyl transferase-1 (DGAT1) is a target of great
interest to the pharmaceutical industry, as inhibiting DGAT1
could be a suitable mechanism for treating diabetes, obesity
and other diseases that constitute metabolic syndrome.1 Small
molecule inhibitors of DGAT1 (Fig. 1) have progressed into the
clinic from Novartis (LCQ-908, phase II), Pzer (PF-04620110,
phase I – discontinued)2 and AstraZeneca (AZD7687, phase I –
discontinued, structure undisclosed) and preclinical
compounds have been disclosed by Roche (RO-6036),3 Abbott
(A-922500)4 and AstraZeneca (AZD3988),5 amongst others.
A recurring theme of the published DGAT1 inhibitors is the
presence of a carboxylic acid moiety. In general, addition of a
carboxylic acid has been used to combine good potency with
good physical and pharmacokinetic (PK) properties, as we
found with the recently published discovery of AZD3988.5
However, the carboxylic acid moiety can be associated with a
risk of idiosyncratic toxicity in the clinic, from the formation of
the corresponding acyl glucuronide.6 Despite the prevalence of
acidic DGAT series, neutral DGAT inhibitors such as RO-6036
are known. As there was both rationale and precedent for
discovering neutral DGAT inhibitors we initiated a program to
use in-house biphenyl acidic compounds such as 1 (Fig. 2) as
startpoints for the discovery of a neutral series.
D and decrease solubility. Acid 1 is very potent (pIC50 ¼ 8.2) with
a low log D7.4 (0.5), although we were still concerned that using 1
as a startpoint for a neutral series would lead to a compound
with high lipophilicity that would be difficult to optimise to a
clinical candidate. We therefore synthesised and proled 2, as
the prediction was that this would give us a lower log D start-
point, and there was precedent of the 4-amino-7,8-dihydropyr-
imido[5,4-f][1,4]oxazepin-5-one group for DGAT inhibition.2
Pleasingly, 2 was potent (DGAT pIC50 ¼ 7.6) at very low log D7.4
(ꢀ0.4). Although 2 is less potent than 1, it has an exceptionally
high ligand lipophilicity efficiency (LLE, dened in this article
as pIC50 ꢀ log D7.4) of 8.0, and was therefore our preferred
startpoint for developing a neutral series.
Initially the analogous primary amide was designed, as the
primary amide is a conservative isosteric neutral replacement
for an acid. Thus 3 was equipotent to 2, with a log D7.4 that was
in the desirable range for an oral drug (Table 1), although as
expected LLE decreases as a consequence of the increase in
log D. Amide 3 also retained good solubility and improved free
fraction in human plasma protein binding (PPB), but at the cost
of reduced in vitro metabolic stability. Nevertheless 3 was taken
forward as a neutral lead compound for further optimisation.
Chemistry
Synthesis of the initial hits 2 and 3 started from methyl 2-(3-
chloro-4-hydroxyphenyl)acetate 4 (Scheme 1), which was con-
verted to triate 5 with PhN(Tf)2 and then converted to the
boronate ester 6. Suzuki coupling of 6 with bromide 7 gave the
AstraZeneca R&D, Mereside, Alderley Park, Maccleseld, Cheshire, SK10 4TG, UK.
E-mail: frederick.goldberg@astrazeneca.com
† Electronic supplementary information (ESI) available: Experimental protocols
and references thereof are given. See DOI: 10.1039/c2md20231j
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Fig. 1 Structures of reported DGAT1 inhibitors.
biaryl ester 8, which could be saponied to give acid 2. This was potent, although only 14 was more potent than the primary
then converted with a HATU coupling to the primary amide 3. amide 3. We also investigated other polar functionality such as
The 4-amino-7,8-dihydropyrimido[5,4-f][1,4]oxazepin-5-one reversed amides, sulfones and sulfonamides (17–21). We used
core 7 was prepared using an analogous route to similar inter- the reversed amide to investigate the effects of cyclic groups (22
mediates that have been previously reported by Pzer.2 Thus 4- and 23) and spacing a base out with different lengths of linker
bromoaniline was alkylated with (2-bromoethoxy)(tert-butyl) (24, 25 and 26). These changes were all tolerated, and interest-
dimethylsilane 9 (Scheme 2) to give intermediate 10, which was ingly spacing the base out can drop log D signicantly and thus
then acylated with 4,6-dichloropyrimidine-5-carbonyl chloride improve LLE, although not surprisingly given the low log D and
to give 11. Compound 11 was deprotected with HCl and the ionisable centre the predicted absorptions of 24, 25 and 26 as
resulting alcohol was cyclised under basic conditions to give the assessed in vitro by Caco-2 A–B Papp were poor.
desired ring system 12. The resulting chloropyrimidine was
Through this approach of using isosteric polar groups to the
then displaced with ammonia to give 7, which could be coupled primary amide startpoint we identied a number of potent
up with a range of boronate esters to give the desired biaryl compounds with improved in vitro metabolic stability and rat
target compounds, e.g. with 2-uorobenzeneboronic acid as PK as shown in Table 2. However, none of them improved
shown to give 13.
signicantly upon the potency of the startpoint 3. Our conclu-
sion was that the SAR was surprisingly at; despite the diversity
of compounds 3–26 there is less than a log unit variation in
potency, suggesting that the acid/amide isosteres that we had
focussed on may not be necessary. In addition, although the
carefully designed examples succeeded in reducing clearance
vs. the primary amide, the molecular weight (MW) of these
compounds is relatively high (437–506) and many of them had
low Fabs in rat. Consequently we decided to change our strategy
Results and discussion
We initially aimed to improve the metabolic stability of 3 by
varying the amide, focussing on groups with additional polarity
to keep log D7.4 low (<2). Compounds 14, 15 and 16 (Table 3)
incorporate secondary hydroxyl or amide functionality
(designed for solubility and low metabolic liability) and were
Fig. 2 Structures of acidic startpoints.
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Table 1 Comparison of properties of acid 2 to primary amide 3
Solubility
(mM)
Hu PPB %
free
Rat Heps CLint
Compound
DGAT1 pIC50
log D7.4
LLE
(mL minꢀ1 ꢁ 106)
a
Acid 2
Amide 3
7.6
7.5
ꢀ0.4
8.0
6.2
349
201
5.6
17.2
14
35
1.3
a
All potency data in this communication is quoted as the mean pIC50 of at least 3 independent measurements.
Scheme 1 Synthesis of initial hits 2 and 3. Reagents and conditions: (i) PhN(Tf)2, Na2CO3, THF, 69%; (ii) bis(pinacolato)diboron, CH3CO2K, PdCl2(dppf)-DCM adduct,
1,4-dioxane, 42%; (iii) PdCl2(dppf)-DCM adduct, K3PO4, DME, MeOH, H2O, 46%; (iv) LiOH, 1,4-dioxane, H2O, 72%; (v) NH4Cl, HATU, i-Pr2NEt, DMF, 53%.
Scheme 2 Synthesis of 4-amino-7,8-dihydropyrimido[5,4-f][1,4]oxazepin-5-on core 7 and compound 13. Reagents and conditions: (i) Na2CO3, DMF, 51%; (ii) 4,6-
dichloropyrimidine-5-carbonyl chloride, Et3N, THF, 106%; (iii) HCl, MeOH, 104%; (iv) Et3N, MeCN, 76%; (v) NH3, 1,4-dioxane, 89%; (vi) 2-fluorobenzeneboronic acid,
PdCl2(dppf)-DCM adduct, K3PO4, DME, MeOH, H2O, 47%.
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Table 2 Exploration of sidechain SAR/SPR
Hu
PPB %
free
Rat CLc
mL minꢀ1
kgꢀ1
Caco-2
DGAT
pIC50
log
D7.4
Solubilitya
(mM)
Rat heps CLint
Rat
A to B Papp
Cpd
R
(mL minꢀ1 ꢁ 106)
F%b
(ꢁ10ꢀ6 cm sꢀ1
)
3
7.5
7.7
1.3
1.1
201
41
17.2
24.1
35
14
15
<4.6
15
12
1.1
7.4
7.3
1.1
0.9
1
22.3
30.2
<3.1
<4.6
16
34
3
25
5
17
18
7.4
7.3
21.2
9.4
<2.8
11
61
1.9
1.8
6
4
19.7
14.5
19
7.3
13.3
<2
100
7
20
21
7.5
7.5
2.1
1.8
1
2
8.6
5.9
4.1
28
21
5
3
18.8
11.3
12.2
22
23
24
7.1
7.2
7.4
0.9
1.1
0.1
148
19
22.5
12.3
>49
18.4
<2
2.0
6.4
0.2
200
4.9
25
7.3
ꢀ0.5
237
48.0
<2
0.3
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Table 2 (Contd. )
Hu
PPB %
free
Rat CLc
Caco-2
DGAT
pIC50
log
D7.4
Solubilitya
(mM)
Rat heps CLint
Rat
mL minꢀ1
kgꢀ1
A to B Papp
Cpd
R
(mL minꢀ1 ꢁ 106)
F%b
(ꢁ10ꢀ6 cm sꢀ1
)
26
7.3
ꢀ1
280
42.0
<2
0.2
27
28
29
30
7.8
7.3
8.3
7.6
2.9
2.4
3.5
5
3.6
9.3
1.2
11.1
4.8
65
14
21
33
32.5
48.2
16.6
16.8
NV
7
53.2
31
32
33
7.7
8.0
7.1
3.2
<1
1
2.2
0.7
1.2
9.1
6.4
9.3
150
87
16
6
>3.9
3.8
<1
150
24
34
6.5
7.8
2.5
3.7
<1
1
5.9
1.2
4.6
54.8
16.2
35
9.5
110
12
a
b
Solubility performed under thermodynamic conditions using semicrystalline or crystalline solid. Oral bioavailability observed when dosing
compound as a low dose (2.3–6.4 mmol kgꢀ1) solution to male Han Wistar rats. Intravenous clearance in male Han Wistar rats when dosing
c
compound as a low dose (1.4–2.9 mmol kgꢀ1) solution to male Han Wistar rats.
and cut down the sidechain and pursue simple, small MW methyl at C4 (29) gave a particularly potent compound (pIC50
¼
substituents to improve absorption.7 To our surprise, removing 8.3), albeit with high log D7.4 (3.5), low solubility and high
the sidechain altogether to give 27 (Table 2) maintained clearance.
potency. log D7.4 was higher (2.9) but despite moderately high in
Having identied new lower molecular weight neutral
vitro and in vivo clearance, 27 had improved Caco-2 absorption startpoints such as 27, we then aimed to identify structural
and high bioavailability in rat (65%), suggesting that removing changes elsewhere on the scaffold that might maintain or
the sidechain may improve absorption. We further explored this improve potency and improve properties. An interesting feature
by examining small substituents on the ring, which suggested of the biphenyl core is that ortho (2,20) substituents would be
that a range of substituents were well tolerated at C4 position expected to effect the conformational preference of the biphenyl
(28–32). Chloro was well tolerated at C5 (35) and chloro and (i.e. the dihedral angle between the phenyl rings), as well as
cyano were moderately tolerated at C3 (33 and 34). Addition of a placing the ortho group into a position where it may improve
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Table 3 Optimisation of 2,20 groups on biphenyl core
Solubility
(mM)
Hu
PPB % free
Rat F
%
Rat IV CL
cpd
A
X
Y
Z
DGAT pIC50
log D7.4
(mL minꢀ1 kgꢀ1
)
36
27
37
38
39
40
41
42
43
13
C
C
C
C
C
C
C
N
C
C
H
Cl
Me
F
Cl
Cl
F
Cl
Cl
F
H
H
H
F
H
Me
F
—
CN
H
H
H
H
H
F
H
F
H
H
H
6.6
7.8
7.4
7.8
8.0
7.7
7.8
7.1
7.5
7.4
2.4
2.9
2.7
2.5
3.0
3.4
2.9
1.5
2.1
2.8
3
5
6
140
3
1
9.6
3.6
6.9
9.5
5.6
4.1
4.0
18.2
20.6
8.0
65
14
21
18
77
287
68
93
20
7
12
binding. Thus removing the chlorine to give the unsubstituted Ames mutagenicity of the corresponding biphenyl fragments to
biphenyl signicantly decreases potency (Table 3, cf. 36 and 27). assess this risk – this work has been published separately.8 We
Other ortho substituents and 2,6 or 2,20-bis-ortho substituents found that electron withdrawing groups, particularly F or Cl, at
all improved potency relative to 36 as shown in Table 3. The the position meta to the amino group (Z) in the aminobiphenyl
2-chloro-20-uoro compound 39 was the most potent (pIC50
¼
fragment decrease the likelihood of the aniline being active in
8.0), although it demonstrated poor bioavailability (rat F ¼ the Ames test. The meta uorine alone is insufficient to elimi-
18%). Ortho-uoro compound 13 combined good potency nate activity. The addition of further electron withdrawing
(pIC50 ¼ 7.4) and attractive PK properties (rat F ¼ 93%, iv CL ¼ groups such as in 39 or 41 leads to aminobiphenyl fragments
7) and thus was taken forward to mouse PK and PK/PD studies that are not active in the Ames test. By contrast, the amino-
(vide infra). It is also noteworthy that replacing the phenyl ring biphenyl embedded in 13 is found to be active in the Ames test.
with a pyridine reduced potency (cf. 42 and 27) but also reduced To further prole compound 13 we tested it for enzyme
log D7.4 dramatically and consequently pyridine 42 has good activity against ACAT1 and DGAT2, but it was found to be
LLE for a neutral DGAT inhibitor (pIC50 ꢀ log D7.4 ¼ 5.6) and inactive (>100 mM) in both assays.
excellent physical properties (solubility ¼ 287 mM, Hu PPB ¼
18% free). All compounds in Table 3 showed good in vitro
absorption (Caco2 A–B Papp > 10 ꢁ 10ꢀ6 cm sꢀ1, data not
PK and PD of compound 13
shown), in marked contrast to early lead compounds such as 14
(Caco2 A–B Papp ¼ 1.1 ꢁ 10ꢀ6 cm sꢀ1, Table 2), presumably due
to the lower MW and polar surface area of these simplied
structures.
As there is an embedded biphenyl aniline in these
compounds, we investigated the potential for release of Ames
positive fragments. We did not observe the release of the cor-
responding aniline in either in vitro or in vivo rat metabolite
identication (Met ID) studies, but nevertheless we assessed the
We have previously described acidic DGAT1 inhibitors which
had attractive PK proles in pre-clinical species.5,9 Our goal in
this programme was to identify neutral compounds which had
similarly high bioavailability and low clearance in pre-clinical
species. In addition we sought a compound which would give
dose-proportional exposure at high doses in order to allow pre-
clinical toxicity studies to be performed with high exposure
margins compared to the IC50. Compound 13 was selected for
full pre-clinical PK proling at low dose (Table 4), where it gave
Table 4 Low dose PK properties of compound 13a
Species
CLp (mL minꢀ1 kgꢀ1
)
Vdss (L Kgꢀ1
)
iv t1/2 (h)
po t1/2 (h)
po Cmax (mM)
Bioavailability (%)
Mouse
Rat
Dog
2.1
7.3
14.8
0.6
1.9
2.4
3.5
6.7
1.2
3.1
3.3
1.7
10.2
3.1
0.4
>100
93
76
a
Compound was dosed iv at 2 mg kgꢀ1 (mouse), 1 mg kgꢀ1 (rat) and 0.5 mg kgꢀ1 (dog) and po at 2 mg kgꢀ1 (mouse), at 2 mg kgꢀ1 (rat) and 0.5 mg
kgꢀ1 (dog). Vehicle for all studies was 5 : 95 DMSO : hydroxypropyl betacyclodextrin.
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Fig. 3 Mean total plasma concentrations following po dosing of compound 13
to 2 male CD1 mice (300 mg kgꢀ1 suspension in PVT–AOT), with error bars
showing standard deviation.
Fig. 5 PK/PD analysis of rat OLTT (plasma triglycerides vs. free compound
concentrations in plasma) for compound 13.
good bioavailability in mouse, rat and dog. It’s also noteworthy
that 13 was stable to human hepatocytes (CLint < 2 mL minꢀ1
ꢁ
Based on this favourable PK prole 13 was tested in two
pharmacodynamic models in rats: the adipose triacylglycerol
(TAG) synthesis test and the oral lipid tolerance test (OLTT).
Methods for both these tests can be found in a previous
communication.9 Fig. 4 shows the relationship between adipose
TAG synthesis (expressed as TAG : DAG ratio in adipose tissue)
and free compound levels in plasma in the rat. A direct response
PK/PD model (Emax sigmoidal model, PK/PD parameters for the
model t are given in the ESI†) was used to successfully t the
PK/PD data showing a clear correlation between free compound
levels in plasma and an effect in the adipose TAG/DAG ratio.
The in vivo IC50 (7.1 nM) in the adipose tissue assay is in very
good agreement with the in vitro IC50 (7.7 nM).
106).
To investigate exposure at high dose compound 13 was given
to mice at 300 mg kgꢀ1 in a suspension formulation with pol-
yvinylpyrrolidone–dioctyl sodium sulfosuccinate (PVT–AOT).
PVT–AOT is known to stabilise drug nanoparticles, and is
considered suitable to use for efficacy/toxicity studies as both
PVT and AOT are common pharmaceutical excipients and are
listed on the FDA inactive ingredients guide. Excellent exposure
was achieved (Fig. 3) with free drug concentrations (adjusted for
mouse plasma protein binding) >1000 fold above the
compound’s human DGAT1 IC50
.
Fig. 5 shows the relationship between plasma triglycerides
and free compound levels in plasma in the rat OLTT assay. A
direct response PK/PD model (Emax sigmoidal model, PK/PD
parameters for the model t are given in the ESI†) was used to
successfully t the PK/PD data showing a clear correlation
between compound levels in plasma and an effect in the OLTT.
As for the adipose TAG synthesis assay, the in vivo IC50 (12.7 nM)
in OLTT assay is in very good agreement with the in vitro IC50
(7.7 nM).
Conclusions
In summary, we have successfully demonstrated that acidic
startpoints can be modied into neutral DGAT inhibitors,
which combine good potency, solubility and PK properties
despite the absence of an ionisable group. This was achieved by
selecting an acidic startpoint with high LLE (pIC50 ꢀ log D) and
converting the acid to a neutral amide. We then found that
removal of the amide to simplify the structure and reduce MW
led to improved PK properties whilst (unexpectedly) maintain-
ing potency. 13 was proled in two different in vivo models,
Fig.
4
PK/PD analysis of rat adipose TAG synthesis assay (adipose tissue
TAG : DAG ratio vs. free compound concentration in plasma) for compound 13.
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demonstrating similar relationships between plasma concen- EtOAc in isohexane to afford the title compound 6 (4.89 g, 42%)
1
trations and pharmacodynamic effects to those previously as a brown oil; H NMR (400 MHz, DMSO) 1.30 (12H, s), 3.61
reported for acidic compounds.
(3H, s), 3.73 (2H, s), 7.21–7.23 (1H, m), 7.34 (1H, d), 7.58 (1H, d).
4-bromo-N-[2-[tert-butyl(dimethyl)silyl]oxyethyl]aniline 10. (2-
Bromoethoxy)(tert-butyl)dimethylsilane (8.61 mL, 40.1 mmol)
was added dropwise to 4-bromoaniline (6.28 g, 36.5 mmol) and
sodium carbonate (7.73 g, 73.0 mmol) in DMF (60 mL) at rt over
a period of 3 min. The resulting suspension was stirred at 60 ꢂC
for 3 days. The reaction was partially concentrated and added
dropwise to cold water (500 mL), then extracted twice with
EtOAc. The combined organic layers were washed with sat.
brine, dried (MgSO4) and evaporated. The resulting crude
product was puried by ash silica chromatography, elution
gradient 5–100% DCM in isohexane, to afford the title
compound 10 (6.23 g, 52%) as an orange oil; 1H NMR (400 MHz,
DMSO) 0.00 (6H, s), 0.83 (9H, s), 3.07–3.12 (2H, m), 3.66 (2H, t),
5.69 (1H, t), 6.52 (2H, d), 7.15 (2H, d); m/z MH+ ¼ 330, 332. N-(4-
bromophenyl)-N-[2-[tert-butyl(dimethyl)silyl]oxyethyl]-4,
6-dichloro-pyrimidine-5-carboxamide 11. 4,6-Dichloropyrimidine-
5-carbonyl chloride (2.53 g, 11.95 mmol) as a solution in THF
(15 mL) was added dropwise to 10 (3.76 g, 11.38 mmol) and
triethylamine (1.71 mL, 12.29 mmol) in THF (30 mL) at 0 ꢂC over
a period of 1 min. The resulting solution was stirred at 0 ꢂC for 2
h. The reaction mixture was evaporated to dryness and redis-
solved in EtOAc, then washed sequentially with water and sat.
brine. The organic layer was dried over Na2SO4, ltered and
evaporated to afford the title compound 11 (6.07 g, 106%) as an
orange oil which was used without further purication; 1H NMR
(400 MHz, DMSO) 0.00 (6H, s), 0.81 (9H, s), 3.77 (2H, t), 3.97
(2H, t), 7.33 (2H, d), 7.54 (2H, d), 8.84 (1H, s). N-(4-Bromophenyl)-
Experimental section
General
All solvents and chemicals used were reagent grade. Flash
column chromatography was carried out using prepacked silica
cartridges from Redisep, Biotage, or Crawford and eluted using
an Isco Companion system. Purity and characterization of
compounds were established by a combination of liquid chro-
matography-mass spectroscopy (LC-MS) and NMR analytical
techniques and was >95% for all compounds. 1H NMR were
recorded on a Bruker Avance DPX400 (400 MHz) and were
determined in CDCl3 or DMSO-d6. 13C NMR spectra were
recorded at 101 or 175 MHz. Chemical shis are reported in
ppm relative to tetramethylsilane (TMS) (0 ppm) or solvent
peaks as the internal reference. Merck precoated thin layer
chromatography (TLC) plates (silica gel 60 F254, 0.25 mm, art.
5715) were used for TLC analysis. Preparative HPLC was per-
formed on C18 reversed-phase silica on a Waters or Phenom-
enex preparative reversed-phase column using decreasingly
polar mixtures of water (containing 1% formic acid or 1% aq.
NH4OH) and MeCN. All reactions were performed under
nitrogen unless otherwise stated. All in vivo experiments were
performed in compliance with the relevant laws and institu-
tional guidelines.
Synthesis of key compounds 2, 3 and 13 (see supporting info
for synthesis of other compounds)
4,6-dichloro-N-(2-hydroxyethyl)pyrimidine-5-carboxamide.
N-(4-
Bromophenyl)-N-(2-(tert-butyldimethylsilyloxy)ethyl)-4,6-dichlor-
Methyl 2-(3-chloro-4-(triuoromethylsulfonyloxy)phenyl)acetate 5. opyrimidine-5-carboxamide (6.06 g, 11.99 mmol) in methanol (8
Methyl 2-(3-chloro-4-hydroxyphenyl)acetate 4 (1.14 g, 5.68 mL) was added in one portion to conc. aq. HCl (1.55 mL) in
mmol),
1,1,1-triuoro-N-phenyl-N-(triuoromethylsulfonyl) methanol (50 mL) at rt. The resulting solution was stirred at rt
methanesulfonamide (2.03 g, 5.68 mmol) and potassium for 30 min. The reaction mixture was evaporated to dryness and
carbonate (2.36 g, 17.1 mmol) were suspended in THF (10 mL) redissolved in EtOAc, then washed sequentially with sat. aq.
and sealed into a microwave tube. The reaction was heated at NaHCO3 and sat. brine. The organic layer was dried over
120 ꢂC for 6 min and cooled to rt. The suspension was ltered, Na2SO4, ltered and evaporated. The resulting crude product
then the solid was washed with EtOAc (20 mL) and the ltrate was puried by ash silica chromatography, elution gradient
was evaporated to afford crude product. The crude product was 10–90% EtOAc in isohexane, to afford the title compound (4.88
1
puried by ash silica chromatography, elution gradient 0–10% g, 104%) as a colourless oil; H NMR (400 MHz, DMSO) 3.57–
EtOAc in isohexane, to afford the title compound 5 (1.31 g, 69%) 3.62 (2H, m), 3.90 (2H, t), 4.84 (1H, t), 7.37 (2H, d), 7.55 (2H, d),
as a colourless oil; 1H NMR (400 MHz, CDCl3) 3.63 (2H, s), 3.73 8.83 (1H, s); m/z MH+ ¼ 392. 6-(4-Bromophenyl)-4-chloro-7,8-
(3H, s), 7.25–7.28 (1H, m), 7.31 (1H, d), 7.47 (1H, s); [M ꢀ H]ꢀ
¼
dihydropyrimido[5,4-f][1,4]oxazepin-5(6H)-one 12. Triethylamine
331. Methyl 2-(3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan- (9.23 mL, 66.39 mmol) was added in one portion to N-(4-bro-
2-yl)phenyl)acetate 6. To a degassed solution of 5 (14.6 g, 37.2 mophenyl)-4,6-dichloro-N-(2-hydroxyethyl)pyrimidine-5-carbox-
mmol) in 1,4-dioxane (300 mL) was added potassium acetate amide (6.83 g, 17.5 mmol) in acetonitrile (78 mL) at rt. The
(10.2 g, 104.2 mmol), bis(pinacolato)diboron (11.8 g, 46.5 resulting solution was stirred at 80 ꢂC for 6 h. The reaction
mmol) and PdCl2(dppf)-DCM adduct (1.82 g, 2.23 mmol). The mixture was evaporated to dryness and redissolved in EtOAc,
suspension was degassed and then heated at reux overnight then washed with water and sat. brine. The organic layer was
before being allowed to cool to rt. The reaction mixture was dried over Na2SO4, ltered and evaporated. The resulting crude
ltered through a silica pad and washed through with EtOAc, solid was triturated with MeOH (50 mL) then washed
then evaporated. The residue was partitioned between EtOAc with MeOH (25 mL) and ether (50 mL) and dried under
and water, and the organic layer was separated and washed with vacuum to give the title compound 12 (4.73 g, 76%) as a white
sat. brine and evaporated. The resulting crude product was solid; 1H NMR (400 MHz, DMSO) 4.23 (2H, t), 4.79 (2H, t), 7.49
puried by ash silica chromatography, elution gradient 0–50% (2H, d), 7.73 (2H, d), 8.89 (1H, s); m/z MH+ ¼ 354, 356.
172 | Med. Chem. Commun., 2013, 4, 165–174
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4-Amino-6-(4-bromophenyl)-7,8-dihydropyrimido[5,4-f][1,4]oxaze- crude product was triturated with EtOAc. The resulting solid
pin-5(6H)-one 7. 0.5 M ammonia in 1,4-dioxane (300 mL) was was washed with water and dried under vacuum to afford the
1
added in one portion 12 (6.59 g, 14.87 mmol) at rt. The resulting title compound 3 (79 mg, 53%) as an off white solid; H NMR
ꢂ
solution was stirred at 55 C for 3 days. The reaction mixture (400 MHz, DMSO) 3.44 (2H, s), 4.05 (2H, t), 4.63 (2H, t), 6.94 (1H,
was allowed to cool and a precipitate was collected by ltration, s), 7.29–7.32 (1H, m), 7.37 (1H, d), 7.46–7.50 (5H, m), 7.52 (1H,
washed with water and methanol and dried under vacuum to s), 7.64 (2H, s), 8.18 (1H, s); 13C NMR (101 MHz, DMSO) 41.20,
1
afford the title compound 7 (4.44 g, 89%) as a white solid; H 49.06, 73.11, 96.33, 126.11 (2C), 128.31, 129.71 (2C), 130.30,
NMR (400 MHz, DMSO) 3.95–4.00 (2H, m), 4.57–4.62 (2H, m), 130.75, 131.10, 136.81, 136.96, 137.94, 141.99, 159.15, 165.25,
7.34–7.38 (2H, m), 7.57–7.63 (4H, m), 8.16 (1H, s); m/z MH+ ¼ 165.68, 166.01, 171.52; m/z MH+ ¼ 424; HRMS (ESI) calc.
335, 337. Methyl 2-(40-(4-amino-5-oxo-7,8-dihydropyrimido[5,4-f] for C21H19O3N5Cl(MH+) 424.1171, found 424.1173. 4-Amino-6-
[1,4]oxazepin-6(5H)-yl)-2-chlorobiphenyl-4-yl)acetate 8. 4-Amino- (20-uorobiphenyl-4-yl)-7,8-dihydropyrimido[5,4-f][1,4]oxazepin-
6-(4-bromophenyl)-7,8-dihydropyrimido[5,4-f][1,4]oxazepin- 5(6H)-one 13. 4-Amino-6-(4-bromophenyl)-7,8-dihydropyrimido
5(6H)-one 7 (214 mg, 0.64 mmol), methyl 2-(3-chloro-4-(4,4,5, [5,4-f][1,4]oxazepin-5(6H)-one 7 (0.25 g, 0.75 mmol), 2-uo-
5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetate 6 (198 mg, robenzeneboronic acid (0.136 g, 0.97 mmol), PdCl2(dppf)-DCM
0.64 mmol) and tripotassium phosphate (163 mg, 0.77 mmol) adduct (0.030 g, 0.04 mmol) and tripotassium phosphate (0.190
were suspended in DME (4 mL), methanol (2 mL) and water g, 0.90 mmol) were suspended in DME (3 mL), methanol (1.5
(1 mL) and sealed into a microwave tube. The mixture was mL) and water (0.7 mL) and sealed into a microwave tube. The
degassed under vacuum and the atmosphere replaced mixture was degassed under vacuum and the atmosphere
with nitrogen. (1,10-Bis(diphenylphosphino)ferrocene)-dichlor- replaced with nitrogen. The reaction was heated at 110 ꢂC for 40
opalladium(II) (DCM adduct) (26 mg, 0.03 mmol) was added and min in the microwave reactor and cooled to rt. The reaction
ꢂ
the reaction was heated at 110 C for 30 min and cooled to rt. mixture was evaporated to dryness and re-dissolved in methyl
The reaction mixture was evaporated to dryness, redissolved in THF, and washed sequentially with water and sat. brine. The
DCM and washed with water. The organic layer was dried over resulting solid was isolated and stirred with with MeOH/DCM,
Na2SO4, ltered and evaporated. The resulting crude product then ltered. The ltrate was combined with the organic layer
was puried by ash silica chromatography, elution gradient from above, ltered through a phase separation funnel and
10–100% EtOAc in iso-hexane then 0–30% MeOH in EtOAc to evaporated. The resulting crude product was puried by
afford the title compound 8 (129 mg, 46%) as a white solid; 1H preparative HPLC to afford the title compound 13 (0.123 g, 47%)
NMR (400 MHz, DMSO) 3.65 (3H, s), 3.78 (2H, s), 4.05 (2H, t), as a white solid; 1H NMR (400 MHz, CDCl3) 4.08 (2H, dd), 4.70–
4.63 (2H, t), 7.32–7.35 (1H, m), 7.36–7.39 (2H, m), 7.41–7.44 4.75 (2H, m), 5.64 (1H, s), 7.13–7.24 (2H, m), 7.31–7.40 (3H, m),
(1H, m), 7.49–7.52 (3H, m), 7.62 (2H, s), 8.18 (1H, s); m/z MH+ ¼ 7.44 (1H, td), 7.61–7.68 (2H, m), 8.09–8.26 (1H, m), 8.30 (1H, s);
439. 2-(40-(4-Amino-5-oxo-7,8-dihydropyrimido[5,4-f][1,4]oxazepin- 13C NMR (101 MHz, DMSO) 49.1, 73.0, 96.4, 116.1, 124.9, 126.5,
6(5H)-yl)-2-chlorobiphenyl-4-yl)acetic acid 2. 1 M aq. LiOH (0.86 126.5, 127.5, 129.3, 129.3, 129.6, 130.7, 133.3, 142.2, 159.1,
mL, 0.86 mmol) was added to methyl 2-(40-(4-amino-5-oxo-7,8- 159.3, 165.3, 165.8, 166.1; m/z MH+ ¼ 351; HRMS (ESI) calc. for
dihydropyrimido[5,4-f][1,4]oxazepin-6(5H)-yl)-2-chlorobiphenyl-
4-yl)acetate 8 (126 mg, 0.29 mmol) in 1,4-dioxane (2.23 mL) and
water (0.74 mL) at rt. The resulting solution was stirred at 50 ꢂC
for 45 min. The mixture was cooled to rt and the pH adjusted to
C
19H16O2N4F(MH+) 351.1252, found 351.1249.
Acknowledgements
3–4 using 2 M aq. HCl to give a thick precipitate. The resulting The authors would like to thank Usha Chauhan for generating
mixture was evaporated to remove the organic solvent and the high quality enzyme data and Andy Turnbull for all his support
residual suspension was diluted with water (10 mL) and stirred and leadership of the project.
vigorously for 1 h. The resulting solid was ltered off and
washed with water and ether and dried under vacuum to give a
Notes and references
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DMSO) 3.73 (2H, s), 4.12 (2H, t), 4.70 (2H, t), 7.37–7.40 (1H, m),
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This journal is ª The Royal Society of Chemistry 2013