Use of 5-hydroxy-4H-benzo[1,4]oxazin-3-ones as β2-adrenoceptor agonists

Article abstract of DOI:10.1016/j.bmcl.2009.10.013

Novel β₂-agonists with a 5-hydroxy-4H-benzo[1,4]oxazin-3-one moiety as head group are described. Systematic chemical variations at the phenethylamine residue of these compounds lead to the discovery of compound 6m as potent, full agonist of the β₂-adrenoceptor with a high β₁/β₂-selectivity. Molecular modeling revealed an interaction between the carboxylic acid group of 6m and a lysine residue (K305) of the β₂-receptor as putative explanation for the high observed selectivity. Further, compound 6m displayed in a guinea pig in vivo model a complete reversal of acetylcholine induced bronchoconstriction which lasted over the complete study time of 5 h.

Full text of DOI:10.1016/j.bmcl.2009.10.013

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6640–6644
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Use of 5-hydroxy-4H-benzo[1,4]oxazin-3-ones as b2-adrenoceptor agonists
Christoph Hoenke a, Thierry Bouyssou b, Christofer S. Tautermann c, Klaus Rudolf a, Andreas Schnapp b,
Ingo Konetzki a,
*
a Department of Chemical Research, Boehringer Ingelheim Pharma GmbH & Co. KG, 88397 Biberach, Germany
b Department of Pulmonary Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG, 88397 Biberach, Germany
c Department of Lead Discovery, Boehringer Ingelheim Pharma GmbH & Co. KG, 88397 Biberach, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel b2-agonists with a 5-hydroxy-4H-benzo[1,4]oxazin-3-one moiety as head group are described. Sys-
tematic chemical variations at the phenethylamine residue of these compounds lead to the discovery of
compound 6m as potent, full agonist of the b2-adrenoceptor with a high b1/b2-selectivity. Molecular mod-
eling revealed an interaction between the carboxylic acid group of 6m and a lysine residue (K305) of the
b2-receptor as putative explanation for the high observed selectivity. Further, compound 6m displayed in
a guinea pig in vivo model a complete reversal of acetylcholine induced bronchoconstriction which lasted
over the complete study time of 5 h.
Received 3 September 2009
Revised 2 October 2009
Accepted 3 October 2009
Available online 12 October 2009
Keywords:
LABA
b2
Ó 2009 Elsevier Ltd. All rights reserved.
Adrenoceptor agonist
Asthma
COPD
Benzoxazinone
O
b2-Adrenoceptor agonists have been used as bronchodilating
agents for the last decades for the treatment of pulmonary diseases
like asthma.1 The first truly b2-selective agonists, salbutamol and
terbutaline, were discovered some 40 years ago and since then ef-
forts continued to develop drugs with an extended duration of ac-
tion and a superior safety profile devoid of undesired effects like
tachycardia and muscle tremor associated with the activation of
b-adrenoceptors outside the lung tissue. This endeavor culminated
in the development of the so-called third generation of b2-agonists
suitable for a once a day regimen.2
O
OH
H
N
HN
HO
OH
1
2
OH
H
N
H
N
O
HO
O
A screening of betamimetic-like structures from our compound
collection revealed phenyl ethanolamine 1 as potent b2-agonist
(Fig. 1 and Table 1).3 Compound 1 contains at its left-hand side a
5-hydroxy-4H-benzo[1,4]oxazin-3-one moiety. This heterocycle
has been previously used only by our company in the design of
b2-adrenoceptor agonists and offered a more advantageous intel-
lectual property position than other head groups from known b2-
agonists. The right-hand side of compound 1 is described by a
phenethylamine residue with two geminal methyl groups at the
carbon atom next to the amine, a structure that closely resembles
the amine portion of the marketed b.i.d. (twice-daily)4 b2-agonist
formoterol 2.
OH
H
Cl
N
O
N
H2N
Cl
3
OH
H
N
HO
HO
O
We concluded from the in vitro characteristics of formoterol
(compound 2, Fig. 1 and Table 1) that a high b1/b2-selectivity is
4
* Corresponding author. Tel.: +49 7351 5498716.
E-mail address: ingo.konetzki@boehringer-ingelheim.com (I. Konetzki).
Figure 1. Structures of the 5-hydroxy-4H-benzo[1,4]oxazin-3-one 1, the investi-
gational drug picumeterol 3 and the b.i.d. LABAs formoterol 2 and salmeterol 4.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.013

C. Hoenke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6640–6644
6641
Table 1
Potency and intrinsic activity at the human b1- and b2-adrenoceptors for formoterol fumarate 2 and the compounds shown in Scheme 1
Compounds
R
hb1
EC50 (nM)
hb1
hb1
n
hb2
EC50 (nM)
hb2
hb2
n
b1/b2
IAa (%)
IAa (%)
2
1
86 23
0.9
0.8
0.6
5.0 1.9
5.6
6.7 4.7
26.5 19.1
5.0
7.2 1.1
3.7 0.6
3.7 1.4
3.6 0.8
33.0 7.8
253 124
>10,000
294
122
154
107
92
73
140
95 16
131 98
76
66 13
112 77
129 51
124 35
75 11
51
19
73
15
8
7
2
1
1
1
2
1
2
2
1
2
2
2
2
3
3
2
1
1
1
1
1
1
0.4 0.2
0.1 0.1
0.2
0.1
0.4 0.0
0.6
1.5 1.3
13.4 0.9
1.7
0.5 0.2
0.4 0.1
0.6 0.6
0.7 0.5
2.8 0.1
2.9 0.6
70 38
20.0
170
131
>10,000
>10,000
>10,000
133 48
92 21
139
118
144 13
170
3
2
1
1
2
1
2
2
1
2
2
2
2
2
3
2
1
1
1
1
1
1
215
9
4
6
13
9
4
2
3
14
9
4-OH
H
2-Me
3-Me
6a
6b
6c
6d
6e
6f
6g
6h
6i
7
4-Et
4-i-Pr
4-t-Bu
4-Ph
3,5-Di-fluoro
4-CF3
3-CF3
4-OCF3
4-CO2H
3-CO2H
H
2-Me
4-Et
3-CF3
4-Et
4-i-Pr
3,5-Di-fluoro
108
1
84 30
117
121
6
125 34
163 42
132 14
130 11
116 12
6j
6k
6l
6
5
12
87
>144
15
>59
14
n.m.
n.m.
n.m.
6m
8a
8b
8d
8j
10d
10e
10h
4
4
44
76
21
44
5
6
>10,000
ꢀ1869
ꢀ695
9
9
7
ꢀ1004
ꢀ645
2
1
a
Cloned human b1- and b2-adrenoceptors are expressed in CHO-K1 cell lines and the intracellular accumulation of cAMP after addition of various test compounds is
measured. The intrinsic activity (IA) is reported as percentage of the maximal effect of isoprenaline (=100%). The standard deviation is given; n.m. = not meaningful.
required to minimize b1-adrenoceptor mediated cardiovascular
O
O
O
O
O
O
effects. A high selectivity was therefore defined as an important
optimization goal in the development of a long acting beta2-
agonist (LABA) and consequently an improvement of the modest
b1/b2-selectivity observed for compound 1 was imperative. This
study focuses on the introduction of substituents at the right phe-
nyl group of 1 and their influence on the molecule’s potency and
efficacy towards the b1- and b2-adrenoceptor. Mainly lipophilic
substituents were selected for this purpose due to reports claiming
that the duration of action is increasing with the lipophilicity of the
b2-agonist.5,6
During our early optimization phase, analogs of the 5-hydroxy-
4H-benzo[1,4]oxazin-3-one containing compounds were synthe-
sized with head groups previously used in b2-agonists. To evaluate
the influence of these head groups, the analogs were investigated
with respect to their agonistic activity and sub-type selectivity to-
wards the b-adrenoceptors. The outcome of such a comparison
with two aryl moieties, notably the 2,6-dichloro-phenylamine
and the 2-hydroxymethyl-phenol (saligenin), is also reported in
this publication. Both head groups are well established for use in
b2-agonists. The 2,6-dichloro-phenylamine moiety can be found
O
OH
a, b, c
H
N
HN
OH
OH
HN
HO
R
BnO
6
5
O
O
OH
d, e
H
N
O
HO
R
OH
BnO
HO
7
8
OH
f, g
H
N
Cl
OH
OH
Cl
R
H2N
H2N
for instance in the investigational b2-agonist picumeterol
3
Cl
Cl
whereas the saligenin is present in salmeterol 4 or salbutamol
(Fig. 1).
9
10
The target compounds described in this study are listed in
Table 1 and the general preparative methods used to access the
compounds are outlined in Scheme 1.7 Reaction of the glyoxal
hydrate 53 with the phenethylamines 15 in the presence of a
reducing agent afforded benzyl protected phenyl ethanolamines.
This one-pot reaction comprises a reductive amination and a
reduction of the keto functionality. The subsequent removal of
the protecting group yielded the 5-hydroxy-4H-benzo[1,4]oxa-
zin-3-ones 6. The saligenines 8 and 2,6-dichloro-phenylamines
10 were obtained in an analogous fashion from the coupling of
the hemi-acetal 78 or glyoxal hydrate 99 with the amines 15. Raney
nickel was preferably used as catalyst in the final debenzylation
step required for the preparation of the saligenines since palladium
on carbon resulted in some cases in the formation of the 2-methyl
phenol derivative as side product.
Scheme 1. Reagents and conditions: (a) 1 equiv of amine 15, THF, 30 min, then
LiBH4, 0 °C to rt; (b) Pd/C, H2, MeOH; (c) only for 6l and 6m: aqueous 1 N NaOH, rt;
(d) 1 equiv of amine 15, THF, rt, 30 min, then LiBH4, 0–50 °C; (e) Pd/C, H2, MeOH or
Raney-Ni, H2, EtOH; (f) amine 15, EtOH, 50–60 °C; (g) NaBH4, EtOH, rt.
The phenethylamines 15 which were used as substrates in the
above mentioned coupling reactions are shown in Table 2 and their
synthesis is presented in Scheme 2. A common key step in the
preparation of all amines is a Ritter reaction with the (2-methyl-
propenyl)-benzenes 13 or the tertiary alcohols 17 as substrate
and sodium cyanide or acetonitrile as reagent. Both substrates
can be derived by multiple ways. The (2-methyl-propenyl)-ben-
zenes 13 can be either obtained from a nucleophilic attack of phen-
ylmagnesiumbromide to isobutyraldehyde followed by an
elimination reaction (Scheme 1, method A) or from benzaldehydes

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C. Hoenke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6640–6644
Table 2
Method A
Method B
Phenethylamines 15 prepared according to Scheme 2
Br
Educt
Product
Method
E
O
R
R
R
HO
H2N
O
15a
15b
11
16
e
a
H2N
H2N
H2N
H2N
Ea
OH
f
O
HO
Br/Cl
R
R
R
R
R
O
12
13
19
17
18
15c
15d
B
Method C
O
c
b
H
N
c
Ea
O
R
O
14
Cl
g
15e
C
d
H2N
O
R
O
H2N
15
15f
B
B
O
O
Method D
i
H2N
H2N
H
h
N
HO
15g
O
R
R
Ph
F
Ph
O
17
20
F
Method E
Br
15h
C
Scheme 2. Reagents and conditions: (a) Mg-Grignard preparation in Et2O, then
isobutyraldehyde; (b) p-toluenesulfonic acid monohydrate, toluene, reflux; (c)
NaCN, AcOH, concd H2SO4; (d) concd HCl, EtOH or MeOH, reflux; for 15k: potassium
hydroxide, ethylene glycol, 140 °C; (e) MeMgBr, THF; (f) Mg-Grignard preparation
in Et2O, then acetone; (g) isopropyl triphenylphosphonium iodide, n-BuLi, THF,
Et2O; (h) MeCN, AcOH, concd H2SO4; (i) potassium hydroxide, ethylene glycol,
reflux.
F
F
O
O
H2N
15i
15j
15k
B
B
A
O
O
CF3
CF3
CF3
via a Wittig olefination (Scheme 1, method D). Reaction of phenyl
acetic acid esters or acetone with the appropriate Grignard re-
agents delivers the tertiary alcohols 17 (Scheme 1, methods B
and C, respectively).
All examples from this study were tested in cellular functional
assays to determine their potency and efficacy (here, intrinsic
activity compared to the full agonist isoprenaline) for the human
b1- and b2-adrenoceptor. In these assays, the intracellular rise of
cAMP induced by a b-agonist is quantified in CHO cell lines stably
expressing either the human b1- or b2-adrenoceptor. Marketed for-
moterol fumarate was measured for comparison and the results
are listed in Table 1.
CF3 H2N
Br
O
H2N
OCF3
OCF3
H2N
O
O
O
15l
D
O
O
All 5-hydroxy-4H-benzo[1,4]oxazin-3-ones 6a–m from this
study can be classified as full or even super agonists of the b2-
adrenoceptor based on their intrinsic activities (>>100% for super
agonists). The examples 6a–d and 6h–k with sterically less
demanding lipophilic substituents R showed subnanomolar EC50
values at this receptor. These compounds were roughly equipotent
to formoterol 2 and more potent than their analogs 6e–g with lar-
ger lipophilic groups. Unfortunately, only a modest b1/b2-selectiv-
ity was observed for all 5-hydroxy-4H-benzo[1,4]oxazin-3-ones
O
O
H2N
15m
D
O
O
a
The 1-phenyl-propan-2-one derivative was converted into the tertiary alcohol
17 through reaction with MeMgBr.

C. Hoenke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6640–6644
6643
3 µg/kg
10 µg/kg
120
100
80
60
40
20
0
time [minutes]
Figure 3. Proposed binding mode of compound 6m into the b2-receptor. The
carboxylic acid group of the ligand coordinates with a lysine residue (K305, cyan) at
the extracellular end of the transmembrane domain (TM) 7 as indicated by the
dotted line. In the b1-receptor, an aspartate residue is found at the corresponding
position (D356, pink), unable to form a favorable interaction with the acid. The
aspartate residue which is identical in the b1- and b2-adrenoceptors is also shown
(gray).
Figure 2. Bronchoprotective effect of example 6m in the Konzett–Roessler model.
Bronchospams were induced in guinea pigs by iv injections of acetylcholine every
10 min. Bronchoprotection is expressed as the percentage of inhibition of the
increase in pulmonary resistance induced by acetylcholine (N = 2 animals per dose).
with lipophilic groups and it became evident from these data that
the position of the substituent at the phenyl ring does not have any
substantial influence on the selectivity. EC50 values around 3 nM
were measured for the carboxylic acid containing compounds 6l–
m at the b2-receptor. Only the meta-substituted example 6m dis-
played a high b1/b2-selectivity of 87 and a reduced intrinsic activity
of 51% at the b1-adrenoceptor warranting a further investigation of
this compound.
For this purpose, compound 6m was tested in a guinea pig
in vivo model10 of acetylcholine induced bronchoconstriction as
previously reported.11 In this model, the ability of a test compound
to reverse the acetylcholine induced bronchoconstriction is deter-
mined. Additionally, the heart rate is recorded as an indicator of
the systemic availability of the b2-agonist. Example 6m showed a
dose-dependent bronchodilation in this model which lasted for
b2-Agonists with a saligenin16 or a 2,6-dichloro-phenylamine17
head group and a phenethylamine residue with two geminal
methyl groups have been already reported in the literature. How-
ever, functional cellular assays were not available for the human
b-adrenoceptors at the time of their first publication and an
in vitro characterization of such compounds was therefore of
interest.
The saligenines 8 displayed a significant lower in vitro potency
at the b2-adrenoceptor compared to the corresponding 5-hydroxy-
4H-benzo[1,4]oxazin-3-ones. The most potent example, compound
8b, had an EC50 value of 20 nM at the b2-receptor, which was con-
sidered as insufficient to justify a further profiling.
Only a marginal activity was observed in the in vitro assays for
the three 2,6-dichloro-phenylamines 10. These findings are in line
with results obtained for other 2,6-dichloro-phenylamines synthe-
sized in our laboratories (data not shown) and consequently the
use of this head group was discontinued.
In conclusion, example 6m was identified from a series of com-
pounds with a 5-hydroxy-4H-benzo[1,4]oxazin-3-one head group
as potent and selective b2-agonist. A duration of action over the
complete study period of 5 h was demonstrated for this compound
in a guinea pig in vivo model. Unfortunately, the tested 5-hydroxy-
4H-benzo[1,4]oxazin-3-one displayed an inferior therapeutic win-
dow in this model compared to the marketed drug formoterol.
These results prompted us to focus our optimization efforts on a
different part of the phenyl ethanolamine structure and the results
will be reported in due course.
the applied doses of 3 and 10 lg/kg over the complete study period
of 5 h (Fig. 2). A full reversal of the induced bronchospasm was ob-
served at the highest tested dose which was unfortunately accom-
panied by a significant increase (>10%) in heart rate.12 Formoterol
which was tested as comparator in this model exhibited also a
dose-dependent bronchoprotection and exerted a full bronchopro-
tection of 100% at doses P1
significant increase in heart rate was only evident at a dose of
10 g/kg which was 10-fold above the first dose reaching 100%
l
g/kg.12 In contrast to example 6m, a
l
bronchoprotection. Consequently, the profiling of compound 6m
was stopped, since it did show an inferior therapeutic window
compared to formoterol.
A receptor model was established based on the recently pub-
lished structure of the b2-adrenoceptor co-crystallized with the in-
verse agonist carazolol with the aim to locate the amino acid
residues responsible for the high selectivity of compound 6m.13–
Supplementary data
15
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.10.013.
The model revealed an interaction between the carboxylic acid
group of the ligand and a lysine residue (K305) within helix7 of
the loop area. In the b1-receptor, an aspartate residue is found at
the corresponding position that should reduce the overall binding
energy of the ligand through electrostatic repulsion of the two car-
boxylic acid groups (Fig. 3). This interaction has been previously
not reported in the literature and should be useful in the future de-
sign of selective b2-agonists.
References and notes
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6644
C. Hoenke et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6640–6644
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Analytical data of compound 6c: Mass spectroscopy: [M+H]+ = 371. 1H NMR
(400 MHz, DMSO-d6) d = 9.97 (s, 1H), 9.95 (s, 1H), 8.59 (m, 1H), 8.44 (m, 1H), 7.22
(1H, m), 7.01 (d, 1H, J = 7.6 Hz), 7.00 (m, 2H), 6.97 (d, 1H, J = 8.6 Hz), 6.58 (d, 1H,
J = 8.6 Hz), 5.94 (m, 1H), 5.08 (d, 1H, J = 10.0 Hz), 4.62 (d, 1H, J = 14.9), 4.56 (d, 1H,
J = 14.9 Hz), 3.16 (m, 1H), 2.91 (m, 3H), 2.30 (s, 3H), 1.19 (s, 6H). 13C NMR
(100 MHz, DMSO-d6) d = 164.2, 157.7 (2JC,F = 31 Hz), 144.9, 141.2, 137.3, 135.2,
131.2, 128.1, 127.7, 127.6, 120.0, 120.0, 117.2 (1JC,F = 300 Hz), 115.3, 109.0, 66.9,
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7. Synthesis protocol of a representative example (compound 6c): A solution of 329 mg
(1.00 mmol) 5-benzyloxy-8-(2,2-dihydroxy-acetyl)-4H-benzo[1,4]oxazin-3-one
and 163 mg (1.00 mmol) 1,1-dimethyl-2-m-tolyl-ethylamine in 5 mL THF was
stirred for 30 min at ambient temperature and then cooled to 0 °C.
Lithiumborohydride (1.5 mL of a 2 M solution in THF) was slowly added and
stirring was continued over 30 min at ambient temperature. The solution was
diluted with10 mLdichloromethaneand3 mLwater, stirred foradditional1 hand
then filtered through a short column of ExtrelutÒ. The column was washed with
dichloromethane (3 Â 10 mL) and the combined filtrates were evaporated. The
residue was dissolved in methanol and hydrogenated in the presence of 100 mg
palladium on carbon at ambient temperature and a hydrogen pressure of 50 psi.
The catalyst was filtered off, the solvent removed in vacuo and the remainder
15. More detailed information regarding the receptor model are compiled in
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