Structure-guided development of dual β2 adrenergic/dopamine D2 receptor agonists

Article abstract of DOI:10.1016/j.bmc.2016.04.028

Aiming to discover dual-acting β₂ adrenergic/dopamine D₂ receptor ligands, a structure-guided approach for the evolution of GPCR agonists that address multiple targets was elaborated. Starting from GPCR crystal structures, we describ

Full text of DOI:10.1016/j.bmc.2016.04.028

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-guided development of dual b2 adrenergic/dopamine D2
receptor agonists
y
⇑
Dietmar Weichert , Markus Stanek, Harald Hübner, Peter Gmeiner
Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center, Friedrich-Alexander University, Schuhstraße 19, 91052 Erlangen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Aiming to discover dual-acting b2 adrenergic/dopamine D2 receptor ligands, a structure-guided approach
for the evolution of GPCR agonists that address multiple targets was elaborated. Starting from GPCR crys-
tal structures, we describe the design, synthesis and biological investigation of a defined set of com-
pounds leading to the identification of the benzoxazinone (R)-3, which shows agonist properties at the
adrenergic b2 receptor and substantial G protein-promoted activation at the D2 receptor. This directed
approach yielded molecular probes with tuned dual activity. The congener desOH-3 devoid of the ben-
zylic hydroxyl function was shown to be a b2 adrenergic antagonist/D2 receptor agonist with Ki values
in the low nanomolar range. The compounds may serve as a promising starting point for the investigation
and treatment of neurological disorders.
Received 19 February 2016
Revised 11 April 2016
Accepted 15 April 2016
Available online xxxx
Keywords:
G protein-coupled receptors
Dopamine receptor
b2 adrenergic receptor
Neurodegenerative disorders
Parkinson’s disease
Molecular probes
Ó 2016 Elsevier Ltd. All rights reserved.
Polypharmacology
1. Introduction
neuroinflammation caused by microglia cells, which are described
to mediate neuronal cell death in PD.6,7 Furthermore, studies found
Parkinson’s disease (PD) is a neurodegenerative disorder that is
characterized by a loss of dopaminergic neurons in areas of the
central nervous system (CNS) responsible for the control of motor
function.1 The lack of dopamine results in an imbalance of a range
of central neurotransmitters and thus in motor symptoms like
hypo- and dyskinesia, along with neuropsychiatric dysfunctions
like cognitive impairment, depression and dementia. To date, the
treatment primarily relies on the administration of levodopa,
which is converted to dopamine in the CNS, or dopamine D2 recep-
tor (D2R) agonists.2 However, this symptomatic therapy does not
effectively prevent the degenerative progression of this disease. It
has now been accepted that inflammatory processes in the brain
are responsible for its progressive aggravation, although multiple
factors can evoke neurodegeneration.1,3
Despite their therapeutically highly appreciated role in the
peripheral regulation of smooth muscle contraction, for instance
in asthma, b2 adrenergic receptors (b2ARs) are also widely
expressed within the CNS, where they mediate a number of
immunomodulatory, neuroprotective and cognitive enhancing
effects.4,5 Interestingly, stimulation of these receptors reduces
an association between up-regulation of b2ARs and astrocyte acti-
vation leading to neuroprotection and release of neurotrophic fac-
tors.8 Long-acting b2 agonists were shown to have beneficial effects
on dopaminergic neurons in PD mouse models that evaluated
inflammation-induced neurotoxicity.9,10 As an addition to
dopaminergic effects to improve motor function of PD patients, it
has been reasoned that concomitant activation of central b2ARs
may constitute a promising approach to alleviate the progressive
degeneration of dopaminergic neurons in PD.5 To evaluate whether
such a polypharmacological approach11 may leverage the develop-
ment of better drugs, we envisioned the design of pharmacological
probes displaying a dual activation profile at b2 adrenergic and
dopamine D2 receptors. Apart from potentially providing a more
effective therapy, such compounds may have advantages over
the administration of two separate chemical entities, like improved
patient compliance or less complex pharmacokinetic and pharma-
codynamic relationships.12
The design of compounds acting at more than one target, how-
ever, provides a major challenge, since it requires simultaneous
adjustment of both activities. The well-described ‘designed multi-
ple ligand’ strategy typically encompasses the combination of two
pharmacophores, each displaying affinity for one of the receptors,
in one molecule.13,14 As both the b2 adrenergic and the dopamine
D2 receptor belong to the family of G protein-coupled receptors
(GPCRs), which represent a class of therapeutically highly relevant
⇑
Corresponding author.
E-mail address: peter.gmeiner@fau.de (P. Gmeiner).
Present address: School of Biochemistry and Immunology, Trinity College Dublin,
y
152-160 Pearse Street, Dublin 2, Dublin, Ireland.
http://dx.doi.org/10.1016/j.bmc.2016.04.028
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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D. Weichert et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
membrane proteins, the design of dual acting compounds has pre-
viously been restricted to such ligand-based approaches due to a
lack of structural information. Recent advances in structural biol-
ogy, however, facilitated the solution of a growing number of
GPCR-crystal structures, co-crystallized in complex with high-
affinity ligands, thus providing crucial insights into ligand–recep-
tor interactions at the molecular level.15,16 We recently obtained
two individual crystal structures of the b2ARH93C mutant covalently
bound to disulfide-based agonists.17,18 Furthermore, the structure
of the D3 receptor, a D2-like subtype, in complex with the antago-
nist eticlopride has been published.19 We took advantage of this
valuable information for the structure-guided design, synthesis
and biological evaluation of dual acting ligands.
the D2-like family and share a high sequence homology of 78%
within the transmembrane helices. Most importantly, 17 of the
18 amino acids that form ligand–receptor interactions in the D3
structure can also be found within the D2 binding pocket.19
The orthosteric binding sites of the b2AR and the D2 receptor
share a certain degree of homology (Fig. 1), which is illustrated
by structural similarity between the neurotransmitters (nor)adre-
naline and dopamine, respectively. However, initial investigation
of representative and structurally diverse b2 adrenergic and D2
dopaminergic ligands at both receptors revealed poor dual binding
properties of the compounds (Supporting information, Supplemen-
tary Table S2), indicating that the development of dual acting
ligands for these receptors might not be straightforward. There-
fore, we performed a detailed analysis of the ligand–receptor inter-
actions within the orthosteric and the extended binding pocket
using the structure of the b2ARH93C bound to the covalent disul-
fide-based agonist FAUC50 and the D3 structure bound to eticlo-
pride to guide the structure-based rational design of dual acting
ligands, rather than applying a ‘designed multiple ligand’ strategy.
A direct comparison of both structures established the notion
that the reversible FAUC50-analog (R)-1 and bioisosteres thereof
should perfectly fit into the binding pocket of the dopamine recep-
tors (Fig. 1, Supplementary Fig. S3).17 Thus, the catechol-mimicking
8-hydroxyquinolin-2(1H)-one head group accommodates within
the orthosteric pocket, where it may form hydrogen bonds with
residues Ser5.42 and Ser5.46 in transmembrane helix 5, which are
conserved between b2ARs and D2Rs. These attractive interactions
were found to be crucial for catecholamine efficacy.21 Both recep-
tors should bind the protonated amino group of the ligands via a
salt-bridge to the conserved residue Asp3.32, while the stereogenic
2. Results and discussion
2.1. Design
GPCR crystal structures may leverage an effective development
of novel drug candidates20 because they can be used for structure-
based in silico docking screens, giving access to new chemotypes
and, as a consequence, to new biological profiles. Furthermore,
they can guide lead modification and optimization programs by
providing insights into crucial ligand–receptor interactions and
the bioactive ligand conformation. Both strategies can be per-
formed based on either the crystal structure of a given GPCR or
from a homology model of a structurally highly similar congener.
Although, to our knowledge, the D2 receptor has not yet been
crystallized, the high resolution structure of the D3 receptor pro-
vided a suitable template, since both receptor subtypes belong to
Figure 1. Surface representation of the binding pocket of the FAUC50 (yellow)-bound structure of the b2ARH93C (green, PDB ID: 3PDS) and eticlopride (blue) bound to the
dopamine D3 receptor (orange, PDB ID: 3PBL) on the left side. The right-hand panel presents a manual comparison of the binding pockets of the b2AR bound to the reversible
ligand (R)-1 and eticlopride bound to D3 and representative amino acids that mediate ligand–receptor interactions.
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D. Weichert et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
b-hydroxyl group in (R)-1 forms hydrogen bonds to Asp3.32 and
Asn7.39, thus anchoring the ligand within the binding pocket. For
the b2AR, it has been described that the (S)-configuration of the
b-hydroxyl group leads to agonists with reduced b2AR potency,
most likely, because it is still proximate to Asn7.39 but positioned
tially resulting in differential functional responses. Recently, the
benzoxazinone scaffold without the aromatic hydroxyl group was
employed as an N-substituted heterocyclic appendage of dopamine
receptor ligands, engaging in ligand–receptor interactions distinct
from the test compounds of type 1.14
away from Asp3.32 22
.
Interestingly, the residue 7.39 of the D2 and
To realize a high structural similarity with the native neuro-
transmitters noradrenaline and dopamine, we also considered a
catechol head group, resulting in molecular probes of type 2. As a
lipophilic appendage exhibiting attractive interactions with the
extended binding pocket, a methoxyphenyloxyethane fragment
was employed, because this moiety has been shown to be suitable
for b2AR agonists and it should fit into the binding pocket of D2R, as
indicated by comparison of the D3 receptor structure manually
aligned with a structure of the functionally selective b-blocker car-
vedilol, containing the lipophilic (2-methoxyphenoxy)ethyl moi-
ety, bound to the b1AR (Supplementary Fig. S3).30 To probe the
influence of this lipophilic appendage, we synthesized control
compounds of type 4.
the D3 receptor is a threonine. Manual alignment of the b2AR and
the D3 structure revealed distances of >6 Å between Thr7.39 and
the b-hydroxyl group of FAUC50, indicating that the binding pocket
of the D2-like receptors should tolerate the hydroxyl group in any
configuration enabling the development of dually binding b2AR/
D2R agonists. In addition, the alignment indicates that the hydroxyl
group of adrenergic (S)-amino alcohols should direct towards
His6.55 at D2-like receptors—a residue that has been implicated to
play a key role in D2 receptor activation (Supplementary Figs. S1
and S2).23,24
A detailed description of potential interactions
between the lipophilic appendage of (R)-1 and the D2 receptor is
provided in Supplementary Figure S3.
Structural surrogates devoid of the hydroxyl group, referred to
as desOH-analogs should also be investigated. Because of their sim-
ilarity to the phenethylamine partial structure of dopamine, the
desOH-analogs were expected to exhibit D2R binding and activa-
tion. The benzylic hydroxyl group is not necessarily crucial for
b2AR efficacy25 and, therefore, the desOH-analogs could also be
promising candidates for dually acting ligands.
As promising target compounds, we chose the 8-hydroxyquino-
lin-2(1H)-one-derived amino alcohols (R)- and (S)-117 and the
respective achiral amine desOH-1, devoid of the b-hydroxyl group.
Furthermore, oxa-analogs of type 3 were developed incorporating
2.2. Synthesis
The noradrenaline-derived molecular probes (R)- and (S)-2
were constructed by employing an expeditious synthetic route that
comprised chemoselective alkylation of unprotected (R)- and (S)-
noradrenaline using the electrophilic building block 6.18 The
corresponding mesylate 7 featuring a (2-methoxyphenoxy)ethyl
substituent was employed to synthesize the analogs (R)- and (S)-
4. To access desOH-2 and desOH-4, the bis-benzylether of dopa-
mine 5 was subjected to a reductive alkylation reaction in the pres-
ence of the aldehyde 8 or to alkylation using mesylate 7, furnishing
the target compounds after hydrogenolytic O-deprotection of
intermediates 9 and 10, respectively (Scheme 1).
a
catechol-mimicking 5-hydroxy-2H-benzo[b][1,4]oxazin-3(4H)-
one head group (Chart 1). This group has proven to be an excellent
surrogate for adrenaline in b2AR agonists,26–28 however, the
hydroxybenzoxazinone moiety has been sparsely investigated as
a catechol-substitute in dopaminergic ligands. The properties this
particular heterocycle are considerably different to the 8-hydrox-
yquinolin-2(1H)-one system. In a nanobody-stabilized structure
of the b2AR bound to a hydroxybenzoxazinone-derived agonist,
the ring oxygen atom of the benzoxazinone moiety may form an
H-bond with Asn6.55 in the b2AR.29 Furthermore, this group is not
planar, compared to the 8-hydroxyquinolin-2(1H)-one heterocycle
(Supplementary Fig. S4). Collectively, the catechol-mimicking
hydroxybenzoxazinone group should facilitate the binding of type
3 compounds at both, b2ARs and D2 receptors, similar to the
8-hydroxyquinolin-2(1H)-one-derived compounds of type 1. The
distinct properties of the hydroxybenzoxazinone moiety, however,
may lead to an individual interaction profile for ligands of type 3
compared to compounds of type 1 at dopamine receptors, poten-
The
5-hydroxy-2H-benzo[b][1,4]oxazin-3(4H)-one-derived
compounds (R)- and (S)-3 and desOH-3 were synthesized employ-
ing the heterocyclic intermediate 11 (Scheme 2), which was
generated from 2-nitroresorcinol.31 Benzyl protection and regios-
elective bromination in position 8 gave access to intermediate 12.
A Stille coupling enabled the introduction of a vinyl moiety to
yield 13, thus providing the starting material for a subsequent
Sharpless asymmetric dihydroxylation reaction that served as a
key step to access the isomers (R)- and (S)-14 in an enantioselec-
tive manner.17,32 Regioselective tosylation of the primary alcohol
furnished the derivative (R)-15.33 Intermediate O-silyl protection
and subsequent nucleophilic displacement of the tosylate group
using azide gave (R)-16. O-desilylation and chemoselective reduc-
tion of the azide yielded the chiral amino alcohol (R)-17. The
lipophilic appendage was introduced reductively using the
O
X
H
X
H
HN
N
O
O
HO
HO
N
O
O
R
HO
2
2
FAUC50: X = OH, R = SSCH2CH2NH2
(R)- 2: X = OH
(S)- 2: X = OH
desOH- 2: X = H
(R)- 1: X = OH, R = Ph
(S)- 1: X = OH, R = Ph
desOH- 1: X = H, R = Ph
O
O
O
O
X
X
H
H
N
HN
HO
O
O
HO
HO
N
2
(R)- 3: X = OH
(S)- 3: X = OH
desOH- 3: X = H
(R)- 4: X = OH
(S)- 4: X = OH
desOH- 4: X = H
Chart 1. Test compounds of type 1–4.
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D. Weichert et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
MsO
O
O
2
6
OH
(R)- 2
(R)- 4
HO
HO
NH2
MsO
O
a
2
O
7
( R)-noradrenaline
O
O
O
8
H
N
XO
XO
O
O
2
2
9: X = Bn
desOH- 2: X = H
BnO
BnO
b
a
c
c
NH2
MsO
O
2
H
O
XO
XO
N
O
5
7
2
O
10: X = Bn
desOH- 4: X = H
Scheme 1. Synthesis of the catechol-derived ligands. Reagents and conditions: (a) 6 or 7, DMSO or DMF, 70 °C, argon, 6 h (18–46% yield); (b) 8, NaBH(OAc)3, THF, rt, N2, 16 h
(69% yield); (c) H2, Pd/C, MeOH, rt, 1 h (70% yield).
O
O
O
O
O
OH
O
OH
H
N
HN
O
O
HN
HN
Y
g
c
b
O
X
O
Bn
O
Bn
2
(R)- 14: Y = OH
(R)- 15: Y = OTs
(R)- 16: Y = N 3
(R)- 17: Y = NH 2
(R)- 18: X = Bn
(R)- 3: X = H
13
O
O
d
e
f
h
O
O
HN
HN
Br
a
HO
O
Bn
11
12
i
O
O
O
O
O
O
H
N
O
k
HN
HN
HN
O
O
j
O
O
Bn
O
Bn
O
X
2
20
21: X = Bn
19
h
desOH- 3: X = H
Scheme 2. Synthesis of the 5-hydroxy-2H-benzo[b][1,4]oxazin-3(4H)-one-type ligands. Reagents and conditions: (a) (1) BnBr, K2CO3, acetone, reflux, 3 h; (2) Br2, THF/
CH3COOH, rt, 2 h (70% yield); (b) Bu3(vinyl)Sn, Pd(Ph3P)4, toluene, reflux, N2, 4 h (87% yield); (c) AD mix-b, tert-BuOH/water 1:1, 0 °C, 16 h, 65%; (d) TsCl, Bu2SnO, NEt3, MeCN/
THF 1:1, rt, 16 h (79% yield); (e) (1) tert-butyldimethylsilyl triflate, imidazole, DMF, N2, 0 °C–rt, 1 h (crude); (2) NaN3, DMSO, 65 °C, N2, 24 h (crude); (3) Bu4NF, THF, rt, 3 h
(76% yield over 3 steps); (f) SnCl2ꢀ2H2O, MeOH, rt 20 h (79% yield); (g) aldehyde 8, NaBH(OAc)3, THF, rt, N2, 18 h (45% yield); (h) H2, Pd/C, MeOH, rt, 5 h (35–67% yield): (i)
Bu3(cis-2-ethoxyethenyl)Sn, Pd(Ph3P)4, toluene, reflux, N2, 8 h (crude); (j) 2 N HCl/acetone 15:85, reflux, 1 h (crude); (k) 2-[3-methoxy-4-(3-phenylpropoxy)phenyl]
ethanamine, NaBH(OAc)3, THF, rt, N2, 20 h (25% yield over 3 steps).
aldehyde 8, to furnish compound (R)-18. Finally, hydrogenolysis
of the benzyl protecting group gave access to the molecular probe
(R)-3. The synthesis of the enantiomer (S)-3 was conducted from
(S)-14 following an identical reaction sequence. The evaluation of
the enantiomeric excess (ee) of the target compounds (R)- and
(S)-3 employing chiral HPLC revealed excellent ee values of
96.3% and 93.3%, respectively, and is described in more detail in
Supplementary Figure S5.
The target compound desOH-3 was synthesized starting from
the heterocyclic building block 12 (Scheme 2). A Stille coupling
reaction was used to introduce a cis-ethoxyethenyl moiety to
afford the synthetic intermediate 19.34 Subsequent hydrolysis of
the enol ether gave the substituted acetic aldehyde derivative 20
that was subjected to a reductive amination reaction with the
amine-substituted building block (2-[3-methoxy-4-(3-phenyl-
propoxy)phenyl]ethanamine).17 Hydrogenolytic cleavage of the
benzyl group in intermediate 21 provided a practical access to
the final product desOH-3. Compound desOH-1 was synthesized
accordingly from 8-hydroxyquinolin-2(1H)-one (Supplementary
Scheme S1).
2.3. Pharmacology
To investigate the pharmacological profile of the compounds at
the family of b adrenergic and dopamine receptors, we conducted
radioligand binding assays and compared the data with those
obtained for the reference agonists isoprenaline and quinpirole,
respectively (Table 1). Competition binding assays were performed
employing [3H]spiperone and cloned human dopamine receptor
subtypes D2L, D2S, D3, and D4.4 stably expressed in Chinese hamster
ovary (CHO) cells. D1 receptor affinities were determined using the
D1-selective radioligand [3H]SCH23390 and cloned human D1
receptors transiently expressed in HEK 293 cells.34 To evaluate b1
and b2 adrenergic receptor affinities, competition binding experi-
ments were conducted by using membranes from HEK 293 cells
transiently expressing murine b1 and homogenates from CHO cells
stably expressing human b2 and the radioligand [3H]CGP12177.
The parent compound (R)-1 showed high affinity to the b adren-
ergic receptor subtypes b1AR and b2AR resulting in Ki values at
5.8 nM and 0.48 nM, respectively.17 Compared to isoprenaline,
binding affinities were about 40–100 fold higher. Accordingly,
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D. Weichert et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Table 1
Radioligand binding data obtained for the compounds (R) and (S)-2, 3 and (R) and (S)-4 compared to the control compounds (R) and (S)-1 and the references isoprenaline and
quinpirole, employing murine b1 and human b2, D1, D2L, D2S, D3 and D4.4, receptors
Compound
Ki (SD)a (nM)
[3H]CGP12177
[3H]SCH23390
hD1
[3H]spiperone
hD3
mb1
hb2
hD2L
hD2S
3200 3200
hD4.4
(R)-1
(S)-1
desOH-1
(R)-2
5.8 3.3
0.48 0.082b
23 2.5b
6.3 1.8b
18 4.4b
47 12b
53 26b
0.25 0.031b
13 1.5b
2.8 0.86b
97 33
11,000 4800
6800 2100
2800 1200
430 110
5700 5000
2900 1700
64 27b
1700 1100
790 240
18 1.3b
62 20
3300 2900
9200 5400
900 210b
140 82
300 110b
150 28
85 21b
2300 1700
160 65b
67 16
110 21
(S)-2
desOH-2
(R)-3
(S)-3
desOH-3
(R)-4
530 210
2300 1100
5.1 1.2b
180 64
79 27b
2900 1600
1900 70
1700 350
120 64b
65 8.5
660 180
12 4.9b
690 400
400 42
1000 7.1
860
0
63 24b
130 14
38 11b
4500 4200
2800 350
960 340
890 300
430 110
210 110
8700 6000b
>50,000
1400 490
5400 850
55 11b
310 35
96 21
12 2.0b
17 0.95b
430 64
3.5 0.62b
2200 280
610 140
25 11
160 130
1500 420
5000 210
nd
640 14
300 14
(S)-4
850 490
610 230
nd
350 7.1
200 49
240 28
desOH-4
Quinpirole
Isoprenaline
85 8.8b
48 11
4.5 1.3
260 38b
48,000 8300
70 18b
15 3.7b
4700 830
8.5 1.4b
2900 900
220 48b
46 10b
12,000 4500
nd: not determined.
a
Ki values in nM standard deviation (SD) derived from two individual experiments each performed in triplicate.
Ki values in nM standard error of mean (SEM) derived from 3 to 8 individual experiments each performed in triplicate.
b
the enantiomer (S)-1 revealed Ki values in the nanomolar range but
Encouraged by these results, we evaluated the most promising
target compounds for their ability to modulate the function of b2
adrenergic and dopamine D2 receptors in comparison to the refer-
ence agonists isoprenaline and quinpirole, respectively. Thus, we
tested G protein-mediated signaling by employing an inositol
phosphate (IP) formation assay,35 using HEK293 cells transiently
transfected with a chimeric Gaqs5 or Gaqi5 protein and human
b2AR and D2S receptor, respectively. In addition, b-arrestin recruit-
ment was investigated using HEK293 cells transfected with b2AR or
D2S receptors and b-arrestin-2, tagged with complementary frag-
ments of the enzyme b-galactosidase.34
approximately 50-fold lower affinities when compared to its opti-
cal antipode. Interestingly, desOH-1 displayed substantial b2AR
affinity (Ki = 6.3 nM) indicating that removal of the b-hydroxyl
group is largely tolerated by the b adrenergic binding pocket. Radi-
oligand binding studies employing (R)- 1 and (S)-1 at D2-like recep-
tors showed low binding affinity with Ki values in the micromolar
range for both amino alcohols. However, Ki values between 18 nM
and 160 nM were determined for desOH-1 at D2/D3 receptors, indi-
cating dual ligand binding.
The benzoxazinones of type 3 showed affinities at b2AR that
were very similar to the type 1 bioisosteres with Ki values of
0.25, 13 and 2.8 nM for (R)-3, (S)-3 and desOH-3, respectively.
Interestingly, the compound family displayed significantly stron-
ger D2R binding. For the D2S subtype, (R)-3, (S)-3 and desOH-3 gave
Ki values of 65, 96 and 17 nM, suggesting that the benzoxazinone
head group was highly beneficial for a dual binding profile. As an
example, desOH-3 shows Ki values of 2.8, 12 and 17 nM at b2AR,
D2L and D2SRs, respectively.
Exchange of the heterocyclic benzoxazinone head groups by the
catechol moiety led to a significant attenuation of b2 adrenergic
receptor binding. In contrast, D2 dopaminergic affinities remained
largely unchanged for (R)-2 (Ki = 67 nM at D2SRs), and were only
moderately reduced for desOH-2.
The catechol derivatives of type 4 showed Ki values in the dou-
ble and triple digit nanomolar range for b2AR, D2L and D2SR with
analogous receptor-specific profiles. Again, the (R)-configured
amino alcohol isomer ((R)-4) proved to be optimal for b2 adrener-
gic receptor binding and the removal of the OH-group (desOH-4)
led to an improvement of D2-like receptor binding.
Our initial functional experiments were directed to the 5-
hydroxy-2H-benzo[b][1,4]oxazin-3(4H)-one-analogs of type 3,
which showed a promising dual binding profile at both, the b2AR
and D2R receptor (Fig. 2, Supplementary Table S1). The IP accumu-
lation assay revealed strong partial agonist activity at b2ARs (61%)
and excellent potency (EC50 = 7 nM) for the heterocyclic adrenaline
surrogate (R)-3. Similarly, substantial IP accumulation was
observed at the D2S receptor (Emax = 85%, EC50 = 98 nM), indicating
strong dopaminergic activity for (R)-3. Notably, the agonist proper-
ties of (R)-3 at D2S receptors revealed to be strongly biased, because
b-arrestin recruitment could only be observed at very high concen-
trations (EC50 = 13 000 nM). On the other hand, investigation of
b2AR-promoted b-arrestin recruitment induced by (R)-3 showed
a partial response at low nanomolar concentrations (EC50 = 13 nM,
Emax = 51%). Taken together, (R)-3 was identified as a dual b2AR/
D2R agonist largely devoid of dopamine receptor-promoted b-
arrestin recruitment. The enantiomer (S)-3 showed highly similar
intrinsic activities in all four ternary complexes, however, compar-
ison of the EC50 values reveals that the compound has significantly
higher dopaminergic activity but lower potency at b2ARs. These
functional results corroborate our hypotheses that guided the
design of the target compounds. The increase of binding affinity
and potency of the desOH-derivatives of type 1–3 compared to
the hydroxyl-substituted analogs confers that the catechol-mim-
icking head groups accommodate within the orthosteric pocket,
while the lipophilic appendage is positioned in the extended cavity
of the b2AR and D2 receptor. An identical binding mode at b2AR and
D2R rather than a hybrid structure with two slightly overlapping
pharmacophores is also supported by previous SARs of dopamine
receptor agonists,36 indicating that substituents at the catechol-
mimicking moiety lead to a significant reduction of binding affinity
and potency. Thus, the D2 receptor tolerates both stereochemical
To evaluate the effects of the test compounds of type 1–4 at clo-
sely related
a-adrenoceptor subtypes, we conducted radioligand
binding studies using porcine cortical membranes and the radioli-
gands [3H]prazosine and [3H]RX821002 that are selective for a1
and a2, respectively.
The results are summarized in Supplementary Table S3 and
indicate poor affinities in the micromolar range for the test com-
pounds (R)- and (S)-1, (S)-2 as well as (R)- and (S)-3 at both
a-
adrenoceptor subtypes. The catechol-derived compounds (R)-2 as
well as (R)- and (S)-4 and desOH-4 showed low nanomolar binding
affinity (4.7–50 nM) at the a1 and submicromolar affinity at the a2
subtype. Binding affinities in the submicromolar range were also
observed for the desOH-derivatives of type 2 and 3.
Please cite this article in press as: Weichert, D.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.028

6
D. Weichert et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
G-protein mediated signaling
β-arrestin-2 recruitment
A
B
β2 receptor
D2s receptor
β2 receptor
D2s receptor
100
80
60
40
20
0
100
100
80
60
40
20
0
100
80
60
40
20
0
80
60
40
20
0
-12 -11 -10 -9
-8
-7
-6
-5
-4
-12 -11 -10 -9
-8
-7
-6
-5
-4
-12 -11 -10 -9
-8
-7
-6
-5
-4
-12 -11 -10 -9
-8
-7
-6
-5
-4
(R)-3 [logM]
(R)-3 [logM]
(R)-3 [logM]
(R)-3 [logM]
100
80
60
40
20
0
100
100
80
60
40
20
0
100
80
60
40
20
0
80
60
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20
0
-12 -11 -10 -9
-8
-7
-6
-5
-4
-12 -11 -10 -9
-8
-7
-6
-5
-4
-12 -11 -10 -9
-8
-7
-6
-5
-4
-12 -11 -10 -9
-8
-7
-6
-5
-4
(S)-3 [logM]
(S)-3 [logM]
(S)-3 [logM]
(S)-3 [logM]
C
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
-12 -11 -10 -9
-8
-7
-6
-5
-4
-12 -11 -10 -9
-8
-7
-6
-5
-4
-12 -11 -10 -9
-8
-7
-6
-5
-4
-12 -11 -10 -9
-8
-7
-6
-5
-4
desOH-3 [logM]
desOH-3 [logM]
desOH-3 [logM]
desOH-3 [logM]
Figure 2. Receptor activation for the benzoxazinones (R)-3 (A), (S)-3 (B) and desOH-3 (C) at the b2 adrenergic and the dopamine D2s receptor in comparison to the reference
compounds isoprenaline (gray squares) or quinpirole (gray diamonds). Dose–response curves for G protein-mediated signaling (left side) were determined in a [3H]IP
accumulation assay with the human b2 receptor and the hybrid G protein Gaqs5 (blue) or with the human D2s receptor and the G protein Gaqi5 (red). b-Arrestin recruitment
was determined employing the PathHunterÒ assay using the appropriate b2 or D2S receptor and b-arrestin-2, tagged with complementary fragments of the enzyme b-
galactosidase. Graphs display pooled curves of 3–8 individual experiments.
orientations of the b-hydroxyl group, as illustrated by robust D2
activation by (R)- and (S)-3. Furthermore, the enantiomer (S)-3
revealed a 14-fold better potency for G protein activation at D2S
receptors than its optical antipode, providing evidence that it
may form stabilizing H-bond interactions with His6.55 (Supplemen-
tary Figs. S1 and S2). It is interesting to note that the arylethy-
lamine desOH-3 showed excellent ligand efficacy and potency at
compound has the potential to combine an improvement of motor
control with neuroprotective effects. The symptomatic treatment
of PD employing levodopa or dopaminergic agonists is well docu-
mented.2 In addition, several studies employing animal models of
PD and other neurological disorders acknowledged the neuropro-
tection harnessed by b2-agonists. Thus, the long-acting partial
b2-agonist salmeterol significantly reduced neurotoxic effects by
inhibition of microglia-mediated inflammatory responses in
mouse models of Parkinson‘s disease.9 Furthermore, b2-agonists
have been shown to prevent neuronal cell death in a rat model
of traumatic brain injury37 and in a murine model of amyotrophic
lateral sclerosis.38
These studies and, in particular, the notable neuroprotective
effect of the partial b2-agonist salmeterol suggest that the dual act-
ing test compound (R)-3, exhibiting a potent sub-maximal b2-acti-
vation profile similar to salmeterol,39 might provide a useful
strategy for the treatment of neurological disorders. Interestingly,
several clinical trials confirmed that partial b2-agonists mediated
reduced systemic side effects in asthmatic patients when com-
pared with full agonists,40–42 indicating that the lead compound
(R)-3 might also feature an advantageous safety profile.
D2SR with strong bias over b-arrestin recruitment. However, the
test compound behaved as a neutral antagonist at b2ARs, thus
demonstrating dual D2SR activating/b2AR blocking properties (Ki
from binding experiments = 2.8 nM). An analogous activation pat-
tern was observed for the structural analogs desOH-1, desOH-2
and desOH-4 (Supplementary Table S1).
3. Conclusion
In our search for novel molecular probes and lead compounds
that may ameliorate motor symptoms, while mediating neuropro-
tective effects in Parkinson’s disease, we performed a structure-
guided rational design to evolve b2 adrenergic agonists toward
dual acting b2 adrenergic/D2 dopaminergic agonists. Our assump-
tions were deduced from the comparison of available crystal struc-
tures for the b2AR and the D3 receptor and led to the development
of a narrow set of target compounds that efficiently yielded dual
binding ligands with varying efficacy.
Moreover, we identified the arylethylamine derivatives desOH-1
and desOH-3 to be antagonists at the b2AR, displaying single-digit
nanomolar affinity, but D2 receptor agonists with excellent potency.
Such dual acting compounds might be effective in the treatment of
Alzheimer’s patients that frequently suffer from apathy and depres-
sion, which are caused by a diminished dopaminergic activity in the
CNS, along with extrapyramidal effects including parkinsonism.43,44
Because a blockade of b2AR signaling has been shown to reduce
We found that the benzoxazinone-derived ligand (R)-3 displays
almost full G protein activation at D2S receptors with low nanomo-
lar potency and excellent partial agonist activity at b2ARs, along
with low off-target affinities at
a-adrenoceptors. Thus, this
Please cite this article in press as: Weichert, D.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.028

D. Weichert et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
c
-secretase activity and plaque formation in Alzheimer’s disease,
8.03 (d, J = 9.8 Hz, 1H), 8.62 (s, 2H), 10.35 (s, 1H), 10.46 (s, 1H).
13C NMR (150 MHz, (CD3)2SO) d (ppm) 27.77, 30.28, 31.18, 31.28,
47.80, 47.79, 55.49, 67.49, 112.79, 113.61, 114.18, 117.80, 120.58,
122.10, 122.83, 124.04, 125.69, 128.19, 128.75, 129.46, 136.47,
141.26, 142.76, 146.91, 149.11, 157.68 (TFA), 157.88 (TFA),
160.66. HR-MS calcd 472.2364; found 472.2364. HPLC: System 1:
tR = 13.1 min, purity: 98.7%; System 2: tR = 15.0 min, purity:>99%.
desOH-1 and desOH-3 might serve as promising lead com-
pounds.45,46 Interestingly, in contrast to its congener desOH-3, com-
pound desOH-1 displays micromolar affinities at a-adrenoceptors,
indicating a lower potential to induce off-target effects.
The structure-guided evolution approach described herein pro-
vides promising lead structures for the investigation of dual acting
ligands for neuropsychiatric disorders and further highlights that a
structure-based approach may leverage the efficient development
of molecular probes and therapeutics addressing GPCRs.
4.1.3. (R)-4-(1-Hydroxy-2-((3-methoxy-4-(3-phenylpropoxy)
phenethyl)amino)ethyl)benzene-1,2-diol trifluoroacetate ((R)-
2)
A solution of (R)-noradrenaline (41.8 mg, 0.247 mmol) and 6
(30.0 mg, 0.082 mmol) in anhydrous DMSO (2 mL) was heated to
70 °C for 6 h under an argon atmosphere. The reaction mixture
was allowed to cool to room temperature, degassed 0.1% aq triflu-
oroacetic acid solution (20 mL) was added and the resulting mix-
ture was frozen and lyophilized. The residue was dissolved in
MeOH (1.5 mL) and subsequently purified by preparative HPLC
(10–60% acetonitrile/0.1% aq trifluoroacetic acid) to give (R)-2 as
a light brown solid (17.3 mg, 38% yield). 1H NMR (600 MHz, CD3-
OD) d (ppm) 2.02–2.08 (m, 2H), 2.79 (t, J = 7.5 Hz, 2H), 2.91–3.01
(m, 2H), 3.1 (dd, J = 12.5, 9.8 Hz, 1H), 3.15 (dd, J = 12.5, 3.5 Hz,
1H), 3.22–3.28 (m, 2H), 3.85 (s, 3H), 3.95 (t, J = 6.3 Hz, 2H), 4.81
(dd, J = 9.8, 3.5 Hz, 1H), 6.72 (dd, J = 8.1, 2.1 Hz, 1H), 6.76 (d,
J = 8.1 Hz, 1H), 6.78 (dd, J = 8.2, 2 Hz, 1H), 6.84–6.87 (m, 2H), 6.89
(d, J = 2 Hz, 1H), 7.13–7.26 (m, 5H). 13C NMR (600 MHz, CD3OD) d
(ppm) 32.17, 32.74, 33.05, 50.08, 55.27, 56.62, 69.39, 69.97,
114.05, 114.13, 115.36, 116.38, 118.46, 122.19, 126.9, 129.38,
129.53, 130.71, 133.66, 142.95, 146.59, 146.68, 149.17, 151.36. .
HR-ESI-MS m/z [M+1]+ calcd 438.2275; found: 438.2286. HPLC Sys-
tem 1: tR = 17 min, purity: 100%; System 3: tR = 11.5 min purity:
4. Experimental section
4.1. Chemistry
4.1.1. General methods
Dry solvents and reagents were of commercial quality and were
used as purchased. MS were run on a Finnigan MAT TSQ 700 spec-
trometer by EI (70 eV) with solid inlet or a Bruker Esquire 2000 by
APC ionization. HRMS were run on a JEOL JMS-GC Mate II using
peak-matching (M/DM >5000) and on a Bruker Daltonics maXis
instrument employing an ESI source. NMR spectra were obtained
on a Bruker Avance 360 (1H at 360 MHz, 13C at 90 MHz) or a Bruker
Avance 600 (1H at 600 MHz, 13C at 150 MHz) spectrometer in the
solvents indicated. Chemical shifts are reported relative to TMS
or to the residual solvent peak. Melting points were determined
with a MEL-TEMP II melting point apparatus (Laboratory Devices,
USA) in open capillaries and are given uncorrected. IR spectra were
performed on a Jasco FT/IR 410 spectrometer (film of substance on
a NaCl crystal) or on a Perkin Elmer Spectrum BX FT-IR (neat).
Purification by flash chromatography was performed using Silica
Gel 60; TLC analyses were performed using Merck 60 F254 alu-
minum sheets and the spots were visualized using UV light
(254 nm) and reagents such as ninhydrin. Analytical HPLC/(APCI,
or ESI-)MS was performed on Agilent 1100 HPLC systems employ-
ing a VWL detector using 254 nm or 220 nm connected to a Bruker
Esquire 2000. The purity of all test compounds and key intermedi-
ates was determined by analytical HPLC to be >95% on Agilent
1100 HPLC systems employing a VWL detector using 254 nm or
99%. [
a]
25 = ꢂ12.2° (c 0.4, MeOH).
D
4.1.4. (S)-4-(1-Hydroxy-2-((3-methoxy-4-(3-phenylpropoxy)
phenethyl)amino)ethyl)benzene-1,2-diol trifluoroacetate ((S)-2)
(S)-2 (22.7 mg, 18% yield, [a]
25 = +9.8° (c 0.5, MeOH)) was pre-
D
pared from (S)-noradrenaline (106.5 mg, 0.63 mmol) as described
above for (R)-2. Analytical data are consistent with those reported
for (R)-2.
220 nm and an Agilent Zorbax SB-C8 (4.6 mm ꢁ 150 mm, 5
lm)
4.1.5. 4-(2-((3-Methoxy-4-(3-phenylpropoxy)phenethyl)amino)
ethyl)benzene-1,2-diol trifluoro-acetate (desOH-2)
with a flow rate of 0.5 mL/min (System 1: eluent, methanol/0.1%
aq formic acid, 10% methanol for 3 min to 100% methanol in
15 min, 100% for 6 min to 10% in 3 min, 10% for 3 min. System 2:
eluent, CH3CN/0.1% aq formic acid, 5% CH3CN to 80% in 15 min,
to 95% in 1 min, 95% for 3 min, to 5% in 1 min and 5% for 3 min. Sys-
tem 3: System 3: eluent, CH3CN/0.1% aq trifluoroacetic acid, 5%
CH3CN to 80% in 18 min, to 95% in 2 min, 95% for 2 min, to 5% in
3 min and 5% for 3 min). Preparative HPLC was conducted on an
To a solution of 9 (25 mg, 0.04 mmol) in methanol (2 mL) was
added 10% Pd/C (3 mg). The resulting suspension was stirred for
1 h using H2 gas under atmospheric pressure and was subsequently
filtered through celite. The filtrate was purified by preparative HPLC
(10–60% acetonitrile/0.1% aq trifluoroacetic acid) to give desOH-2 as
a white solid (15.4 mg, 70% yield). 1H NMR (600 MHz, CD3OD) d
(ppm) 2.03–2.11 (m, 2H), 2.8 (t, J = 7.5 Hz, 2H), 2.84 (t, J = 7.8 Hz,
2H), 2.92 (t, J = 8 Hz, 2H), 3.18–3.26 (m, 4H), 3.86 (s, 3H), 3.96 (t,
J = 6.3 Hz, 2H), 6.58 (dd, J = 8, 2.1 Hz, 1H), 6.69 (d, J = 2.1 Hz, 1H),
6.74 (d, J = 8 Hz, 1H), 6.77 (dd, J = 8.2, 2 Hz, 1H), 6.85–6.88 (m, 2H),
7.13–7.28 (m, 5H). 13C NMR (600 MHz, CD3OD) d (ppm) 30.69,
32.13, 34, 34.29, 49.81, 49.93, 55.92, 68.23, 112.37, 113.41, 115.05,
115.61, 118.73, 120.41, 125.88, 128.37, 128.47, 129.81, 130.99,
141.56, 144.04, 145.06, 147.15, 149.54. HR-ESI-MS m/z [M+1]+ calcd.
422.2324; found: 422.2326. HPLC System 1: tR = 17.1 min, purity:
98%; System 3: tR = 13.5 min, purity: 97%.
Agilent 1100 Preparative Series, using
a
MACHEREY-NAGEL
NUCLEODUR C18 HTec (32 mm ꢁ 250 mm, 5
lm) column, at a flow
of 32 mL/min, with the solvent system indicated.
4.1.2. 8-Hydroxy-5-[2-[[3-methoxy-4-(3-phenylpropoxy)phene-
thyl]amino]ethyl]quinolin-2(1H)-one trifluoroacetate (desOH-1)
A solution of 26 (45 mg, 0.08 mmol) and 10% Pd/C (5 mg) in
anhydrous MeOH (4 mL) was stirred for 6 h under H2 atmosphere
at room temperature. The suspension was filtered through celite
and the filtrate was concentrated in vacuo. The residue was puri-
fied by preparative HPLC (3–50% acetonitrile/0.1% aq trifluo-
roacetic acid) to give desOH-1 as a colorless oil (14 mg, 29%
yield). 1H NMR (600 MHz, (CD3)2SO) d (ppm) 1.97–2.07 (m, 2H),
2.73–2.75 (m, 2H), 2.84–2.86 (m, 2H), 3.09–3.13 (m, 4H), 3.18–
3.21 (m, 2H, HNCH2CH2Ph), 3.78 (s, 3H), 3.92 (t, J = 6.4 Hz, 2H),
6.58 (d, J = 9.8 Hz, 1H), 6.75 (dd, J = 8.2, 2.0 Hz, 1H), 6.87–6.93 (m,
4H), 7.17–7.20 (m, 1H), 7.22–7.23 (m, 2H), 7.27–7.30 (m, 2H),
4.1.6. (R)-5-Hydroxy-8-[1-hydroxy-2-[[3-methoxy-4-(3-phenyl-
propoxy)phenethyl]amino]ethyl]-2H-benzo[b][1,4]oxazin-3(4H)-
one trifluoroacetate ((R)-3)
A
solution of (R)-18 (8.0 mg, 0.014 mmol) and 10% Pd/C
(1.0 mg) in anhydrous MeOH (1 mL) was stirred for 5 h using H2
gas under atmospheric pressure at room temperature. The suspen-
Please cite this article in press as: Weichert, D.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.028

8
D. Weichert et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
sion was filtered through celite and the filtrate was concentrated in
vacuo. The residue was purified by preparative HPLC (5–55% ace-
tonitrile/0.1% aq trifluoroacetic acid) to give (R)-3 as a colorless
oil (5.6 mg, 67% yield). 1H NMR (600 MHz, CD3OD) d (ppm) 2.04–
2.09 (m, 2H), 2.78–2.83 (m, 2H), 2.91–3.00 (m, 2H), 3.07–3.11
(m, 1H), 3.22–3.29 (m, 3H), 3.86 (s, 3H), 3.96 (t, J = 6.3 Hz, 2H),
4.54–4.62 (m, 2H), 5.16–5.18 (m, 1H), 6.56 (d, J = 8.5 Hz, 1H),
6.78–6.79 (m, 1H), 6.86–6.92 (m, 2H), 7.02 (d, J = 8.6 Hz, 1H),
7.13–7.16 (m, 1H), 7.19–7.21 (m, 2H), 7.23–7.26 (m, 2H). 13C
NMR (150 MHz, CD3OD) d (ppm) 32.21, 32.74, 33.1, 50.14, 53.9,
56.7, 64.76, 68.19, 69.46, 110.21, 114.17, 115.46, 116.35, 120.75,
121.74, 122.24, 126.95, 129.42, 129.57, 130.69, 142.66, 142.99,
146.72, 149.3, 151.45, 166.63. HR-MS calcd 493.2330; found
493233.1. HPLC: System 1: tR = 11.3 min, purity: >99%; System 2:
4.1.10. (S)-4-(1-Hydroxy-2-((2-(2-methoxyphenoxy)ethyl)
amino)ethyl)benzene-1,2-diol trifluoro-acetate ((S)-4)
(S)-4 (20 mg, 20% yield, [a]
25 = +15.0° (c 0.3, MeOH)) was pre-
D
pared from (S)-noradrenaline (115 mg, 0.679 mmol) as described
above for (R)-4. Analytical data are consistent with those reported
for (R)-3.
4.1.11. 4-(2-((2-(2-Methoxyphenoxy)ethyl)amino)ethyl)benzene-
1,2-diol trifluoroacetate (desOH-4)
To a solution of 10 (16 mg, 0.033 mmol) in methanol (2 mL) was
added 10% Pd/C (2 mg). The resulting suspension was stirred using
H2 gas under atmospheric pressure for 1 h and was subsequently
filtered through celite. The filtrate was purified by preparative
HPLC (5–80% acetonitrile/0.1% aq trifluoroacetic acid) to give
desOH-4 as a gray solid (6.7 mg, 48% yield). 1H NMR (600 MHz,
CD3OD) d (ppm) 2.88 (t, J = 8 Hz, 2H), 3.32 (t, J = 8 Hz, 2H), 3.44
(t, J = 4.8 Hz, 2H), 3.84 (s, 3H), 4.23 (t, J = 4.8 Hz, 2H), 6.59 (dd,
J = 8.0, 2.0 Hz, 1H) 6.7 (d, J = 2 Hz, 1H), 6.72 (d, J = 8.0 Hz, 1H),
6.88–6.93 (m, 1H), 6.98–7.03 (m, 3H). 13C NMR (150 MHz, CD3OD)
d (ppm) 32.79, 48.26, 50.45, 56.39, 66.21, 113.32, 116.47, 116.74,
116.79, 120.95, 122.31, 124.19, 128.84, 145.71, 146.86, 148.44,
151.09. HR-ESI-MS m/z [M+1]+ calcd 304.1543; found: 304.1538.
HPLC System 1: tR = 12.6 min, purity: 98%; System 3: tR = 11.9 min
purity: 97%.
tR = 14.5 min, purity: >99%. [
a]
25 = ꢂ34.5° (c 0.46, MeOH).
D
4.1.7. (S)-5-Hydroxy-8-[1-hydroxy-2-[[3-methoxy-4-(3-phenyl-
propoxy)phenethyl]amino]ethyl]-2H-benzo[b][1,4]oxazin-3
(4H)-one trifluoroacetate ((S)-3)
(S)-3 (6.6 mg, 52% yield, [a]
25 = +37.2° (c 0.43, MeOH)) was pre-
D
pared from (S)-18 (12 mg, 0.021 mmol) as described above for (R)-
3. Analytical data are consistent with those reported for (R)-3.
4.1.8. 5-Hydroxy-8-[2-[[3-methoxy-4-(3-phenylpropoxy)phen-
ethyl]amino]ethyl]-2H-benzo[b][1,4]oxazin-3(4H)-one
trifluoroacetate (desOH-3)
4.1.12. 3-Methoxy-4-(3-phenylpropoxy)phenethyl methanesul-
fonate (6)
A solution of 21 (20 mg, 0.035 mmol) and 10% Pd/C (2 mg) in
anhydrous MeOH (2 mL) was stirred for 5 h using H2 gas under
atmospheric pressure at room temperature. The suspension was
filtered through celite and the filtrate was concentrated in vacuo.
The residue was purified by preparative HPLC (3–50% acetoni-
trile/0.1% aq trifluoroacetic acid) to give desOH-3 as a colorless
oil (7 mg, 35% yield). 1H NMR (600 MHz, (CD3)2SO) d (ppm) 1.97–
2.02 (m, 2H), 2.72–2.74 (m, 2H) 2.79–2.84 (m, 4H), 3.03–3.08 (m,
2H), 3.14–3.18 (m, 2H), 3.78 (s, 3H), 3.92 (t, J = 6.4 Hz, 2H), 4.53
(s, 2H), 6.48 (d, J = 8.3 Hz, 1H), 6.66 (d, J = 8.3 Hz, 2H), 6.73 (dd,
J = 8.2, 2.0 Hz), 6.86–6.89 (m, 2H), 7.17–7.20 (m, 1H), 7.22–7.23
(m, 2H), 7.27–7.30 (m, 2H), 8.51 (s, 2H), 9.86 (s, 1H), 9.93 (s, 1H).
13C NMR (150 MHz, (CD3)2SO) d (ppm) 28.02, 32.24, 33.12, 33.50,
50.44, 56.67, 60.20, 68.19, 69.44, 102.25, 110.22, 114.07, 115.33,
117.58, 122.20, 125.38, 126.95, 129.43, 129.59, 143.02, 144.07,
146.17, 149.18 151.39, 166.75. HR-ESI-MS [M++1]: calcd.
477.2384; found 477.2395. HPLC: System 1: tR = 15.2 min, purity:
>99%; System 2: tR = 17.4 min, purity: 97.6%.
A solution of 3-methoxy-4-(3-phenylpropoxy)phenylethanol
(500 mg, 1.75 mmol) and triethylamine (0.29 mL, 2.10 mmol) in
CH2Cl2 (25 mL) was cooled to 0 °C under argon atmosphere.
Methanesulfonyl chloride (0.14 mL, 1.75 mmol) was added drop-
wise and the mixture was stirred for 1 h at 0 °C. Water was added
and the aqueous layer was extracted with CH2Cl2. The combined
organic layers were washed with brine, dried over Na2SO4 and
evaporated to yield 5 as clear colorless oil. The crude product
was used for the next reaction without further purification.
4.1.13. 2-(2-Methoxyphenoxy)ethyl methanesulfonate (7)
A
solution of 2-(2-methoxyphenoxy)ethanol (160 mg,
0.95 mmol) and triethylamine (0.16 mL, 1.14 mmol) in CH2Cl2
(8 mL) was cooled to 0 °C under argon atmosphere. Methanesul-
fonyl chloride (73 lL, 0.95 mmol) was added dropwise and the
mixture was stirred for 1 h at 0 °C. Water was added and the aque-
ous layer was extracted with CH2Cl2. The combined organic layers
were washed with brine, dried over Na2SO4 and evaporated to
yield 6 as colorless oil. The crude product was used for the next
reaction without further purification.
4.1.9. (R)-4-(1-Hydroxy-2-((2-(2-methoxyphenoxy)ethyl)amino)
ethyl)benzene-1,2-diol trifluoro-acetate ((R)-4)
A solution of (R)-noradrenaline (115 mg, 0.679 mmol) and 7
(56 mg, 0.226 mmol) in anhydrous DMSO (2 mL) was heated to
70 °C for 6 h under an argon atmosphere. The reaction mixture
was allowed to cool to room temperature, degassed 0.1% aq triflu-
oroacetic acid solution (20 mL) was added and the resulting mix-
ture was frozen and lyophilized. The residue was dissolved in
MeOH (1.5 mL) and subsequently purified by preparative HPLC
(5–80% acetonitrile/0.1% aq trifluoroacetic acid) to give (R)-4 as a
light brown solid (45 mg, 46% yield). 1H NMR (600 MHz, CD3OD)
d (ppm) 3.24 (dd, J = 12.5, 10.3 Hz, 1H), 3.36 (dd, J = 12.5, 3.3 Hz,
1H), 3.46–3.53 (m, 2H), 3.85 (s, 3H), 4.29 (t, J = 4.9 Hz, 2H), 4.87
(dd, J = 10.3, 3.3 Hz, 1H), 6.76 (dd, J = 8.2, 1.9 Hz, 1H), 6.79 (d,
J = 8.2 Hz, 1H), 6.9 (d, J = 1.9 Hz, 1H), 6.91–6.95 (m, 1H), 7.01–
7.05 (m, 3H). 13C NMR (150 MHz, CD3OD) d (ppm) 47.99, 55.33,
56.29, 66.32, 69.9, 113.19, 114.07, 116.29, 116.58, 118.4, 122.22,
124.11, 133.59, 146.49, 146.58, 148.35, 151.06. HR-ESI-MS m/z
[M+1]+ calcd 320.1493; found: 320.1498. HPLC System 1:
tR = 12.5 min, purity: 99%; System 3: tR = 9.6 min purity: 99%.
4.1.14. 3-Methoxy-4-(3-phenylpropoxy)acetaldehyde (8)
A solution of 3-methoxy-4-(3-phenylpropoxy)phenylethanol
(19 mg, 0.07 mmol) and 2-iodoxybenzoic acid (56 mg, 0.08 mmol),
in acetonitrile (0.4 mL) was heated to 80 °C under argon atmo-
sphere and was stirred for 1 h. Water was added and the aqueous
phase was extracted with CH2Cl2. The combined organic layers
were dried (Na2SO4), filtered and evaporated. The crude material
was used for the next reaction.
4.1.15. N-(3,4-Bis(benzyloxy)phenethyl)-2-(3-methoxy-4-(3-
phenylpropoxy)phenyl)ethan-1-amine (9)
To a solution of 3,4-dibenzyloxyphenylethylammonium trifluo-
roacetate47 (670 mg, 1.497 mmol) and sodium triacetoxyborohy-
dride (1190 mg, 5.240 mmol) in THF (26 mL) a solution of 8
(255 mg, 0.943 mmol) in THF (26 mL) was added and the reaction
was stirred at room temperature for 3 h under nitrogen atmosphere.
The mixture was filtered over celite and the solvent was evaporated.
The residue was dissolved in CH2Cl2, washed with saturated NaHCO3
[a
]
D
25 = ꢂ15.8° (c 0.5, MeOH).
Please cite this article in press as: Weichert, D.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.028

D. Weichert et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
solution and brine. The organic phase was dried over Na2SO4 and
evaporated. The crude product was purified by flash chromatogra-
phy (CH2Cl2/MeOH 99:1 + 0.2% NH3) to give 9 as yellow oil
(393 mg, 69% yield). IR (NaCl) 3435, 3061, 3028, 2934, 1589, 1514,
THF/glacial acetic acid 9:1(70 mL) and bromine(680
l
L, 13.3 mmol)
was added dropwise. The reaction was stirred at room temperature
for 2 h. Then, 1% Na2S2O3 solution (200 mL) was added and the mix-
ture was kept at 0 °C for 18 h. The suspension was filtered and the
solid was dissolved in EtOAc. After the solvent was evaporated, the
residue was purified by flash chromatography (cyclohexane/EtOAc
4:1) to give 12 as yellowish solid (2.8 g, 70% yield). IR: 3316, 3132,
3079, 2955, 2842, 1660, 1610, 1565, 1485, 1455, 1377, 1266, 1169,
1454, 1422, 1263, 1027 cmꢂ1 1H NMR (360 MHz, CDCl3) d (ppm)
.
2.06–2.16 (m, 2H), 2.62–2.92 (m, 10H), 3.82 (s, 3H), 3.96 (t,
J = 6.6 Hz, 2H), 5.08–5.13 (m, 4H), 6.63 (dd, J = 8.2, 2 Hz, 1H) 6.66
(dd, J = 8.2, 2 Hz, 1H), 6.69 (d, J = 2 Hz, 1H), 6.73 (d, J = 8.2 Hz, 1H),
6.78 (d, J = 2 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 7.13–7.46 (m, 15H).
13C NMR (90 MHz, CDCl3) d (ppm) 30.72, 32.11, 35.25, 35.32,
50.81, 50.9, 56.01, 68.32, 71.37, 71.51, 112.6, 113.6, 115.43,
115.87, 120.61, 121.54, 125.86, 127.31, 127.37, 127.73, 127.76,
128.35, 128.43, 128.46, 132.21, 132.86, 137.32, 137.45, 141.56,
147.04, 147.62, 149.02, 149.57.ESI-MS m/z 603 [M+1]+.
835 cmꢂ1 1H NMR (360 MHz, CDCl3) d (ppm) 4.69 (s, 2H), 5.08 (s,
.
2H), 6.55 (d, J = 8.9 Hz, 1H), 7.11 (d, J = 8.9 Hz, 1H), 7.36–7.42 (m,
5H), 7.86 (s, 1H). 13C NMR (90 MHz, CDCl3) d (ppm) 67.42, 71.30,
102.09, 107.07, 116.86, 126.26, 127.81, 128.27, 128.85, 135.42,
140.91, 144.96, 163.48. HR-EIMS calcd. 333.0001; found: 333.001.
HPLC: System1: tR = 20.9 min, purity: 96.4%
4.1.16. N-(3,4-Bis(benzyloxy)phenethyl)-2-(2-methoxyphenoxy)
ethan-1-amine (10)
4.1.19. 5-(Benzyloxy)-8-vinyl-2H-benzo[b][1,4]oxazin-3(4H)-
one (13)
A
solution of 3,4-dibenzyloxyphenylethylamine (92 mg,
To a suspension of 12 (1.50 g, 4.50 mmol) and tetrakis(triph-
enylphosphine)palladium(0) (260 mg, 0.23 mmol) in anhydrous
toluene (60 mL), Z-tributyl(ethenyl)stannane (160 lL, 5.40 mmol)
0.276 mmol) and 7 (34 mg, 0.138 mmol) in dimethyl formamide
(2 mL) was heated at 80 °C for 20 h under an argon atmosphere.
After cooling to room temperature, water was added and the mix-
ture was extracted with CH2Cl2. The combined organic layers were
washed with brine, dried over Na2SO4 and evaporated. The crude
product was purified by flash chromatography (CH2Cl2/MeOH
95:5 + 0.2% NH3) to give 10 as a dark yellow oil (21 mg, 32% yield).
IR (NaCl) 3374, 3033, 2925, 1591, 1506, 1454, 1254, 1224, 1124,
was added and the resulting mixture was heated and stirredat reflux
for 4 h under N2 atmosphere. The reaction mixture was cooled to
room temperature, filtered over celite and evaporated. The residue
was purified by flash chromatography (hexane/EtOAc 4:1) to give
13 as yellow oil (1.09 g, 87% yield). IR: 3226, 3093, 2916, 1688,
1618, 1514, 1459, 1455, 1410, 1278, 1102, 911, 732 cmꢂ1. 1H NMR
(360 MHz, CDCl3) d (ppm) 4.69 (s, 2H,), 5.10 (s, 2H), 5.24 (dd,
J = 11.2, 1.3 Hz), 5.70 (dd, J = 17.7, 1.4 Hz, 1H), 6.63 (d, J = 8.7 Hz,
1H,), 6.82–6.84 (m, 1H,), 7.10 (d, J = 8.7 Hz, 1H), 7.36–7.42 (m, 5H),
7.80 (s, 1H).). 13C NMR (90 MHz, CDCl3) d (ppm) 67.11, 71.06,
106.00, 114.15, 115.80, 119.17, 120.58, 127.78, 128.57, 128.79,
129.99, 135.78, 141.44, 145.16, 164.03. APCI-MS m/z 282.1 [M++1].
HPLC: System 1: tR = 21.0 min, purity: 97.7%.
1025 cmꢂ1 1H NMR (600 MHz, CDCl3) d (ppm) 2.72 (t, J = 7.3 Hz,
.
2H), 2.87 (t, J = 7.3 Hz, 2H), 3.01 (t, J = 5.3 Hz, 2H), 3.8 (s, 3H),
4.08 (t, J = 5.3 Hz, 2H), 5.12–5.14 (m, 4H), 6.71 (dd, J = 8.1, 1.8 Hz,
1H) 6.81 (d, J = 1.8 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 6.87 .6.96 (m,
4H), 7.26–7.45 (m, 10H); 13C NMR (90 MHz, CDCl3) d (ppm)
35.89, 48.67, 51.09, 55.83, 68.73, 71.44, 71.55, 111.99, 114.17,
115.48, 115.98, 120.89, 121.53, 121.55, 127.29, 127.34, 127.7,
128.19, 128.41, 128.46, 133.45, 137.37, 137.51, 147.45, 148.33,
148.98, 149.72. ESI-MS m/z 484.8 [M+1]+.
4.1.20. (R)-5-(Benzyloxy)-8-(1,2-dihydroxyethyl)-2H-benzo[b]
[1,4]oxazin-3(4H)-one ((R)-14)
4.1.17. 5-Hydroxy-2H-benzo[b][1,4]oxazin-3(4H)-one (11)
A solution of 2-nitroresorcinol (5.0 g, 32.0 mmol) and 10% Pd/C
(500 mg) in anhydrous MeOH (150 mL) was stirred for 16 h using
AD-mix-b ((DHQ)2PHAL, 16 mmol; potassium carbonate,
498.8 mmol; potassium ferricyanide, 498.8 mmol; potassium
osmate dehydrate, 0.7 mmol) (2.56 g) was added to a mixture of
tert-butyl alcohol (11 mL) and water (11 mL). The mixture was
cooled to 0 °C, 13 (0.50 g, 1.78 mmol) was added and the reaction
mixture was stirred at 0 °C for 16 h. Then, sodium sulfite (2.73 g)
was added and the mixture was stirred at room temperature for
30 min. Ethyl acetate was added to the reaction mixture and the
aqueous phase was further extracted with ethyl acetate. The com-
bined organic layers were dried (Na2SO4), filtered and the solvent
was evaporated. The crude product was purified by column chro-
matography on silica gel (hexane/EtOAc 9:1) to give (R)-14 as col-
orless solid (0.36 g, 65% yield). IR: 3360, 3063, 2935, 1685, 1624,
H2 gas under atmospheric pressure at room temperature. The sus-
pension was filtered through celite and the solvent was evapo-
rated. The crude residue (3.5 g, 28.0 mmol) was dissolved in
anhydrous EtOAc (160 mL), anhydrous pyridine (3.4 mL,
42.0 mmol) was added and the solution was cooled to 0 °C under
N2 atmosphere. Then, chloroacetyl chloride (2.0 mL, 25.2 mmol)
was added dropwise and the mixture was stirred at 0 °C for 3 h.
The reaction mixture was diluted with EtOAc (300 mL) and was
washed with water, 0.1 N HCl and water (300 mL each). The
organic layer was dried, filtered and the solvent was removed in
vacuo. Next, the crude material was suspended in acetonitrile/
water 9:1 (150 mL) and K2CO3 (3.9 g, 28 mmol) was added. After
the suspension was stirred for 5 h, the pH was adjusted to 5–6
by using 2 N HCl and the acetonitrile was removed in vacuo. Satu-
rated NaCl (200 mL) solution was added and the mixture was
extracted 3ꢁ with EtOAc (200 mL each). The combined organic lay-
ers were dried, filtered and the solvent was removed to give 11 as a
brown solid (2.4 g, 52% yield). Analytical data are consistent with
those reported in literature.31
1559, 1516, 1471, 1387, 1274, 1096, 1027, 744 cmꢂ1 1H NMR
.
(600 MHz, CD3OD) d (ppm) 3.51–3.54 (m, 1H), 3.63–3.65 (m, 1H),
4.52–4.58 (m, 2H), 4.95–4.97 (m, 1H), 5.19 (s, 2H), 6.72 (d,
J = 8.7 Hz, 1H), 7.04 (d, J = 8.6 Hz, 1H,), 7.29–7.31 (m, 1H), 7.35–
7.37 (m, 2H), 7.44–7.45 (m, 2H). 13C NMR (150 MHz, CDCl3) d
(ppm) 67.45, 68.07, 69.99, 71.80, 107.82, 117.37, 122.23, 123.94,
128.75, 129.12, 129.60, 138.22, 142.88, 146.92, 167.02. APCI-MS
m/z 298.1 [M++1, ꢂH2O]. HPLC: System 1: tR = 17.1 min, purity:
>99%. [
a
]
D
25 = ꢂ32.1° (c 0.53, MeOH).
4.1.18. 5-(Benzyloxy)-8-bromo-2H-benzo[b][1,4]oxazin-3(4H)-
one (12)
A suspension of 11 (2.00 g, 12.1 mmol), benzyl bromide (1.6 mL,
13.3 mmol) and K2CO3 (2.35 g, 17.0 mmol) in acetone (60 mL) was
stirredatrefluxfor3 h.Themixturewascooledtoroomtemperature,
filteredandthesolventwasevaporated.Theresiduewasdissolvedin
4.1.21. (S)-5-(Benzyloxy)-8-(1,2-dihydroxyethyl)-2H-benzo[b]
[1,4]oxazin-3(4H)-one ((S)-14)
(S)-14 (0.39 mg, 70% yield, [a]
25 = +33.5° (c 0.96, MeOH))
D
was prepared from 13 (0.50 g, 1.78 mmol) as described above for
(R)-14. Analytical data are consistent with those reported for
(R)-14.
Please cite this article in press as: Weichert, D.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.028

10
D. Weichert et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4.1.22. (R)-2-[5-(Benzyloxy)-3-oxo-3,4-dihydro-2H-benzo[b]
[1,4]oxazin-8-yl]-2-hydroxyethyl 4-methyl-benzene sulfonate
((R)-15)
To a solution of (R)-14 (350 mg, 1.11 mmol) in a mixture of THF
(44 mL) and acetonitrile (44 mL), dibutyltin(IV) oxide (15 mg,
0.06 mmol), p-toluenesulfonyl chloride (222 mg, 1.16 mmol) and
4.1.26. (R)-8-(2-Amino-1-hydroxyethyl)-5-(benzyloxy)-2H-
benzo[b][1,4]oxazin-3(4H)-one ((R)-17)
A
suspension of (R)-16 (81 mg, 0.24 mmol), SnCl2ꢀ2H2O
(271 mg, 1.20 mmol) in MeOH (1.5 mL) was stirred at room tem-
perature for 20 h. NaHCO3 solution was added carefully and the
aqueous layer was extracted with CH2Cl2. The combined organic
layers were dried (Na2SO4), filtered and evaporated. The residue
was purified by flash chromatography (CH2Cl2/MeOH 9:1 + 0.2%
NH4OH) to give (R)-17 as brownish oil (59 mg, 79% yield). 1H
NMR (360 MHz, CD3OD) d (ppm) 2.82–2.87 (m, 1H), 3.00–3.02
(m, 1H), 4.54–4.61 (m, 2H), 5.00–5.02 (m, 1H), 5.21 (s, 2H), 6.75
(d, J = 8.7 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H), 7.29–7.32 (m, 1H),
7.35–7.38 (m, 2H), 7.45–7.47 (m, 2H). 13C NMR (150 MHz, CD3OD)
d (ppm) 47.11, 67.41, 68.08, 71.83, 108.01, 117.58, 121.71, 123.65,
127.76, 129.17, 129.62, 138.13, 142.55, 147.25, 166.78. APCI-MS
m/z 297.2 [M++1, ꢂH2O]. HPLC: System 1: tR = 13.8 min, purity:
triethylamine (170 lL, 1.22 mmol) were added. The reaction mix-
ture was stirred at room temperature for 16 h. Then, water was
added and the mixture was extracted with CH2Cl2. The combined
organic layers were dried (Na2SO4), filtered and the solvent was
evaporated. The residue was purified by column chromatography
on silica gel (hexane/EtOAc 1:1) to give (R)-15 as colorless oil
(414 mg, 80% yield). 1H NMR (600 MHz, CD3OD) d (ppm) 2.41 (s,
3H) 3.97–4.00 (m, 1H), 4.13–4.15 (m, 1H), 4.25 (d, J = 15.2 Hz,
1H), 4.38 (d, J = 15.2 Hz, 1H), 4.97–4.98 (m, 1H), 5.19–5.23 (m,
2H), 6.71 (d, J = 8.7 Hz, 1H), 6.97 (d, J = 8.6 Hz, 1H), 7.27–7.28 (m,
2H), 7.30–7.33 (m, 1H), 7.37–7.40 (m, 4H), 7.47–7.49 (m, 2H),
7.53–7.56 (m, 2H). 13C NMR (150 MHz, CDCl3) d (ppm) 21.58,
67.13, 67.76, 71.76, 73.95, 107.67, 117.25, 121.64, 122.43, 128.93,
129.06, 129.24, 129.66, 130.85, 133.96, 138.14, 142.14, 146.36,
147.24, 166.50. APCI-MS m/z 452.2 [M++1, ꢂH2O]. HPLC: System
>99%. [a]
25 = ꢂ38.0° (c 0.50, MeOH).
D
4.1.27. (S)-8-(2-Amino-1-hydroxyethyl)-5-(benzyloxy)-2H-benzo
[b][1,4]oxazin-3(4H)-one ((S)-17)
(S)-17 (60 mg, 67% yield, [a]
25 = +37.9° (c 0.52, MeOH)) was pre-
D
1: tR = 19.5 min, purity: 98.6%. [
a]
25 = ꢂ52.2° (c 0.51, MeOH).
D
pared from (R)-16 (100 mg, 0.29 mmol) as described above for (R)-
17. Analytical data are consistent with those reported for (R)-17.
4.1.23. (S)-2-[5-(Benzyloxy)-3-oxo-3,4-dihydro-2H-benzo[b]
[1,4]oxazin-8-yl]-2-hydroxyethyl 4-methyl-benzene sulfonate
((S)-15)
4.1.28. (R)-5-(Benzyloxy)-8-[1-hydroxy-2-[[3-methoxy-4-(3-
phenylpropoxy)phenyl-ethyl]amino]ethyl]-2H-benzo[b][1,4]
oxazin-3(4H)-one ((R)-18)
(S)-15 (453 mg, 76% yield, [a]
25 = +50.7° (c 0.81, MeOH)) was pre-
D
pared from (S)-14 (400 mg, 1.27 mmol) as described above for (R)-
15. Analytical data are consistent with those reported for (R)-15.
To a solution of (R)-17 (44 mg, 0.14 mmol) in THF (5 mL) and
NaBH(OAc)3 (38 mg, 0.18 mmol) a solution of crude 8 (0.07 mmol)
in THF (5 mL) was added and the reaction mixture was stirred for
18 h at room temperature under N2 atmosphere. The solvent was
evaporated and saturated NaHCO3 solution was added. The aque-
ous phase was extracted with CH2Cl2 and the combined organic
layers were dried (Na2SO4), filtered and evaporated. The residue
was purified by flash chromatography (CH2Cl2/MeOH 95:5 + 0.2%
NH4OH) to give (R)-18 as yellow oil (18 mg, 45% yield). 1H NMR
(360 MHz, CD3OD) d (ppm) 2.03–2.08 (m, 2H), 2.78–2.80 (m, 2H),
2.90–2.99 (m, 2H), 3.05–3.09 (m, 1H), 3.22–3.29 (m, 3H), 3.85 (s,
3H), 3.95 (t, J = 6.3 Hz, 2H), 4.56–4.62 (m, 2H), 5.17–5.19 (m, 1H),
5.22 (s, 2H), 6.76–6.79 (m, 2H), 6.85–6.91 (m, 2H), 7.10 (d,
J = 8.7 Hz, 1H), 7.14–7.16 (m, 1H), 7.19–7.21 (m, 2H), 7.23–7.26
(m, 2H), 7.29–7.31 (m, 1H), 7.34–7.37 (m, 2H), 7.44–7.45 (m,
2H). 13C NMR (90 MHz, CD3OD) d (ppm) 32.18, 32.68, 33.07,
50.11, 53.73, 56.67, 64.67, 68.12, 69.43, 71.82, 108.17, 114.14,
115.44, 117.67, 121.58, 122.21, 122.59, 126.91, 128.76, 129.20,
129.39, 129.54, 129.63, 130.63, 138.05, 142.37, 142.95, 147.53,
149.27, 151.43, 166.58. ESI-MS m/z 583.7 [M++1]. HPLC: System
4.1.24. (R)-8-(2-Azido-1-hydroxyethyl)-5-(benzyloxy)-2H-benzo
[b][1,4]oxazin-3(4H)-one ((R)-16)
A solution of (R)-15 (390 mg, 0.87 mmol) and imidazole (113 mg,
1.66 mmol) in anhydrous DMF (12 mL) was cooled to 0 °C under N2
atmosphere. tert-Butyltrimethylsilyl triflate (382 lL, 1.66 mmol)
was added dropwise and the reaction was allowed to warm to room
temperature and was stirred for 1 h. Then, water was added and the
mixture was extracted with CH2Cl2. The combined organic layers
were dried (Na2SO4), filtered and the solvent was evaporated. The
crude material was dissolved in anhydrous DMSO (10 mL). NaN3
(270 mg, 4.15 mmol) and NaI (137 mg, 0.91 mmol) were added
and the reaction mixture was heated to 65 °C for 24 h under N2
atmosphere. The reaction was allowed to cool to room temperature,
water (100 mL) was added and the mixture was extracted with CH2
Cl2. The combined organic layers were dried (Na2SO4), filtered and
the solvent was evaporated. The crude material was further dis-
solved in THF (10 mL) and a 1 M solution of TBAF in THF (1.74 mL,
1.74 mmol) was added at room temperature. The reaction was stir-
red at room temperature for 3 h. Then, the solvent was evaporated
and the residue was purified by column chromatography on silica
gel (hexane/EtOAc 3:2) to give (R)-16 as yellow oil (207 mg, 76%
yield). IR: 3403, 3056, 2916, 2102, 1684, 1627, 1558, 1514, 1464,
1: tR = 18.6 min, purity: >99%. [
a
]
D
25 = ꢂ29.5° (c 0.41, MeOH).
4.1.29. (S)-5-(Benzyloxy)-8-[1-hydroxy-2-[[3-methoxy-4-(3-
phenylpropoxy)phenyl-ethyl]amino] ethyl]-2H-benzo[b][1,4]
oxazin-3(4H)-one ((S)-18)
1386, 1273, 1096, 1029, 956, 905, 738 cmꢂ1 1H NMR (360 MHz,
.
(S)-18 (14 mg, 61% yield, [a]
25 = +28.7° (c 0.52, MeOH)) was pre-
D
CDCl3) d (ppm) 2.47 (s, 1H), 3.41–3.51 (m, 2H), 4.57–4.68 (m, 2H),
5.05–5.10 (m, 3H), 6.68 (d, J = 8.7 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H),
7.35–7.44 (m, 5H), 7.82 (s, 1H). 13C NMR (150 MHz, CDCl3) d (ppm)
56.67, 67.14, 68.43, 71.09, 106.02, 115.66, 120.86, 121.51, 127.80,
128.61, 128.80, 135.63, 140.65, 145.15, 163.57. . APCI-MS m/z
341.2 [M++1]. HPLC: System 1: tR = 19.1 min, purity: 98.8%.
pared from (R)-17 (31 mg, 0.10 mmol) as described above for (R)-
18. Analytical data are consistent with those reported for (R)-18.
4.1.30. (Z)-5-(Benzyloxy)-8-(2-ethoxyvinyl)-2H-benzo[b][1,4]
oxazin-3(4H)-one (19)
To a suspension of 12 (800 mg, 2.40 mmol) and tetrakis(triph-
enylphosphine)palladium(0) (139 mg, 0.12 mmol) in anhydrous
[a
]
D
25 = ꢂ61.7° (c 0.94, EtOAc).
toluene (25 mL), Z-tributyl(2-ethoxyethenyl)stannane (840 lL,
4.1.25. (S)-8-(2-Azido-1-hydroxyethyl)-5-(benzyloxy)-2H-benzo
[b][1,4]oxazin-3(4H)-one ((S)-16)
2.88 mmol) was added and the resulting mixture was heated and
stirred at 100 °C for 8 h under N2 atmosphere. The reaction
was cooled to room temperature, filtered over celite and
evaporated. The residue separated from most impurities by flash
(S)-16 (130 mg, 72% yield, [a]
25 = +66.9° (c 0.88, EtOAc)) was pre-
D
pared from (R)-15 (250 mg, 0.53 mmol) as described above for (R)-
16. Analytical data are consistent with those reported for (R)-16.
Please cite this article in press as: Weichert, D.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.028

D. Weichert et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
11
chromatography (hexane/EtOAc 3:2) to give 300 mg of 19, which
were directly employed in the following reaction.
cooled to room temperature, filtered over celite and evaporated.
The residue was purified by flash chromatography (hexane/EtOAc
3:2) to give 24 as yellow oil (364 mg, 62% yield). Analytical data
are consistent with those reported in literature.34
4.1.31. 2-[5-(Benzyloxy)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]
oxazin-8-yl]acetaldehyde (20)
To a solution of 19 (50 mg, 0.154 mmol) in acetone (1.00 mL),
2 N HCl (0.15 mL) was added and the resulting mixture was heated
and stirred at reflux for 1 h. The reaction was cooled to room tem-
perature and the solvent was evaporated. The residue was dis-
solved in H2O and extracted with CH2Cl2. The combined organic
layers were dried (Na2SO4), filtered and evaporated. The crude
material was used without further purification.
4.1.36. 2-[8-(Benzyloxy)-2-oxo-1,2-dihydroquinolin-5-yl]
acetaldehyde (25)
To a solution of 24 (348 mg, 1.08 mmol) in acetone (2 mL), 2 N
HCl (0.3 mL) was added and the resulting mixture was heated and
stirred at reflux for 1 h. The reaction was cooled to room tempera-
ture and solvent was evaporated. The residue was dissolved in H2O
and extracted with CH2Cl2. The combined organic layers were
dried (Na2SO4), filtered and evaporated. The crude material was
used without further purification.34
4.1.32. 5-(Benzyloxy)-8-[2-[[3-methoxy-4-(3-phenylpropoxy)
phenethyl]amino]ethyl]-2H-benzo[b][1,4]oxazin-3(4H)-one (21)
4.1.37. 8-(Benzyloxy)-5-[2-[[3-methoxy-4-(3-phenylpropoxy)
phenethyl]amino]ethyl]quinolin-2(1H)-one (26)
To
a solution of crude 20 (50 mg, 0.15 mmol) and 2-
[3-methoxy-4-(3-phenylpropoxy)phenyl]ethanamine17 (120 mg,
0.42 mmol) in THF (4 mL), NaBH(OAc)3 (52 mg, 0.25 mmol) was
added and the reaction mixture was stirred for 20 h at room tem-
perature under N2 atmosphere. The solvent was evaporated and
saturated NaHCO3 was added. The aqueous phase was extracted
with CH2Cl2 and the combined organic layers were dried (Na2SO4),
filtered and evaporated. The residue was purified by flash chro-
matography (CH2Cl2/MeOH 92.5:7.5 + 0.2% NH4OH) to give 21 as
yellow oil (22 mg, 25% yield).1H NMR (600 MHz, CD3OD) d (ppm)
1.99–2.05 (m, 2H), 2.68–2.84 (m, 10H), 3.78 (s, 3H), 3.90–3.93
(m, 2H), 4.44 (s, 2H), 5.13 (s, 2H), 6.57 (d, J = 8.5 Hz, 1H), 6.61–
6.65 (m, 2H), 6.73–6.76 (m, 2H), 7.12–7.24 (m, 5H), 7.27–7.30
(m, 1H), 7.34–7.36 (m, 2H), 7.44–7.48 (m, 2H). 13C NMR
(150 MHz, CD3OD) d (ppm) 29.73, 32.27, 33.11, 35.62, 50.14,
51.31, 56.52, 68.02, 69.47, 71.85, 107.74, 113.88, 115.14, 117.61,
121.03, 121.88, 125.17, 126.87, 128.67, 128.82, 129.14, 129.22,
129.37, 129.54, 129.62, 133.48, 138.19, 143.03, 143.77, 146.48,
148.54, 151.12, 166.86. APCI-MS m/z 567.7 [M++1]. HPLC: System
1: tR = 18.4 min, purity: 98.1%.
To a solution of crude 25 (72 mg, 0.25 mmol) and 2-[3-meth-
oxy-4-(3-phenylpropoxy)
phenyl]ethanamine17
(214 mg,
0.75 mmol) in THF (7 mL), NaBH(OAc)3 (160 mg, 0.50 mmol) was
added and the reaction mixture was stirred for 18 h at room tem-
perature under N2 atmosphere. The solvent was evaporated and
saturated NaHCO3 was added. The aqueous phase was extracted
with CH2Cl2 and the combined organic layers were dried (Na2SO4),
filtered and evaporated. The residue was purified by flash chro-
matography (CH2Cl2/MeOH 97.5:2.5 + 0.2% NH4OH) to give 26 as
yellow oil (62 mg, 44% yield). 1H NMR (600 MHz, CD3OD) d
(ppm) 1.98–2.02 (m, 2H), 2.68–2.70 (m, 2H), 2.73–2.84 (m, 6H),
2.98–3.00 (m, 2H, HNCH2CH2Ph), 3.76 (s, 3H), 3.89–3.91 (m, 2H),
5.24 (s, 2H), 6.58–6.59 (m, 1H), 6.64 (d, J = 9.8 Hz, 1H), 6.70–6.72
(m, 2H), 6.87 (d, J = 8.2 Hz, 1H), 7.01 (d, J = 8.2 Hz, 1H), 7.11–7.24
(m, 5H), 7.30–7.32 (m, 1H), 7.35–7.38 (m, 2H), 7.48–7.50 (m,
2H), 8.11 (d, J = 9.8 Hz, 1H). 13C NMR (150 MHz, CD3OD) d (ppm)
32.14, 32.25, 33.10, 35.88, 51.25, 51.44, 56.49, 69.49, 71.92,
113.41, 113.82, 115.13, 119.90, 122.01, 122.04, 124.62, 126.86,
128.92, 129.33, 129.36, 129.53, 129.72, 129.99, 130.40, 133.60,
137.80, 139.29, 142.99, 145.11, 148.46, 151.08, 164.14. APCI-MS
m/z 563.6 [M++1]. HPLC: System 1: tR = 15.2 min, purity: 96.6%.
4.1.33. 8-(Benzyloxy)quinolin-2(1H)-one (22)
Benzyl bromide (810 lL, 6.8 mmol) was added to a suspension
of 8-hydroxyquinolin-2(1H)-one (1.0 g, 6.2 mmol) and K2CO3
(1.2 g, 8.7 mmol) in acetone (30 mL). After being stirred at reflux
for 5 h, the mixture was allowed to cool to room temperature
and the solvent was evaporated. The residue was suspended in
Hꢂ2O and extracted with CH2Cl2. The combined organic layers
were dried (Na2SO4), filtered and evaporated. The crude material
was purified by flash chromatography (hexane/EtOAc 1:1) to give
22 as a white solid (1.4 g, 88% yield). Analytical data are consistent
with those reported in literature.17
4.1.38. 2-[3-Methoxy-4-(3-phenylpropoxy)phenyl]ethanol
A suspension of homovanillyl alcohol (400 mg, 2.38 mmol), (3-
bromopropyl)benzene (1.8 mL, 11.90 mmol) and K2CO3 (657 mg,
4.76 mmol) in acetonitrile was stirred at reflux temperature for
7 h. The reaction mixture was allowed to cool to room temperature
and the solvent was evaporated. The residue was suspended in H2O
and extracted with CH2Cl2. The combined organic layers were
dried (Na2SO4), filtered and evaporated. The residue was purified
by flash chromatography (hexane/EtOAc 3:2) to give pure product
as a colorless oil (649 mg, 95% yield). IR: 3418, 2938, 1645, 1590,
4.1.34. 8-(Benzyloxy)-5-bromoquinolin-2(1H)-one (23)
1514, 1466, 1454, 1418, 1261, 1232, 1158, 1139, 1034, 802 cmꢂ1
.
A solution of bromine (280 lL, 5.44 mmol) in glacial acetic acid
1H NMR (360 MHz, CDCl3) d (ppm) 2.13–2.18 (m, 2H), 2.80–2.83
(m, 4H), 3.83–3.85 (m, 2H), 3.87 (s, 3H), 4.01 (t, J = 6.6 Hz, 2H),
6.72–6.80 (m, 3H), 7.17–7.22 (m, 3H), 7.27–7.29 (m, 2H). 13C
NMR (90 MHz, CDCl3) d (ppm) 30.72, 32.12, 38.76, 56.03, 63.72,
68.31, 112.89, 113.68, 121.02, 125.88, 128.37, 128.48, 131.20,
141.55, 147.22, 149.66. ESI-MS m/z 309.2 [M++Na+]. HPLC: System
1: tR = 20.6 min, purity: 96.3%.
(6 mL) was added dropwise to a solution of 22 (1.37 g, 5.44 mmol)
and sodium acetate (1.48 g, 18.00 mmol) in glacial acetic acid
(25 mL). The reaction mixture was stirred at room temperature
for 3 h. Then, the solvent was evaporated and the residue was puri-
fied by flash chromatography (cyclohexane/EtOAc 7:3) to give 23
as white solid (1.31 g, 73% yield). Analytical data are consistent
with those reported in literature.17
4.1.39. 2-(2-Methoxyphenoxy)ethan-1-ol
4.1.35. (Z)-8-(Benzyloxy)-5-(2-ethoxyvinyl)quinolin-2(1H)-one
(24)
To a suspension of 23 (600 mg, 1.82 mmol) and tetrakis(triph-
enylphosphine)palladium(0) (115 mg, 0.10 mmol) in anhydrous
toluene (20 mL), Z-tributyl(2-ethoxyethenyl)stannane (730 lL,
2.18 mmol) was added and the resulting mixture was heated and
To a solution of NaOH (64.4 mg, 1.61 mmol) in ethanol (0.90 mL),
2-methoxyphenol (0.18 mL, 1.61 mmol) and 2-bromoethanol
(0.11 mL, 1.61 mmol) were added and the mixture was heated to
reflux for 3 h. After the reaction has cooled to room temperature,
water was added and the mixture was extracted with diethyl ether.
The combined organic layers were washed with 1 M NaOH and
brine, dried over Na2SO4 and evaporated. The crude product was
stirred at 100 °C for 8 h under N2 atmosphere. The reaction was
Please cite this article in press as: Weichert, D.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.028

12
D. Weichert et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
purified by flash chromatography (hexane/ethylacetate 2:1) to give
pure product as a clear yellow oil (189.0 mg, 70% yield). IR (NaCl)
cells were transiently co-transfected with cDNA of the b2 recep-
tor and the hybrid G protein Gaqs5 (Gaq protein with the last five
amino acids at the C-terminus replaced by the corresponding
3389, 3064, 2936, 1593, 1506, 1455, 1253, 1125, 1025, 745 cmꢂ1
.
1H NMR (600 MHz, CDCl3) d (ppm) 2.83 (s, 1H), 3.85 (s, 3H), 3.89–
3.93 (m, 2H), 4.11 (t, J = 4.5 Hz, 2H), 6.88–6.98 (m, 4H). 13C NMR
(600 MHz, CDCl3) d (ppm) 55.79, 61.27, 71.51, 111.87, 115.17,
121.06, 122.12, 148.03, 149.92 ESI-MS m/z 191.3 [M+1+Na]+.
sequence of Gas; gift from The J. David Gladstone Institutes, San
Francisco, CA).56 Functional activity at the D2S receptor was deter-
mined, similarly. Therefore, the human D2s receptor and the
hybrid G protein Gaqi5 (Gaq protein with the last five amino acids
at the C-terminus replaced by the corresponding sequence of Gai
;
4.1.40. Structure-guided design
gift from The J. David Gladstone Institutes, San Francisco, CA)
were transiently co-transfected in HEK 293 cells.56 In brief, 24 h
after transfection cells were transferred into 24 well plates,
myo-[3H]inositol (specific activity = 22.5 Ci/mmol, PerkinElmer,
Rodgau, Germany) was added and incubation was continued for
15 h. Cells were washed and test compounds were added for
120 min at 37 °C in the presence of 10 mM LiCl. After cell-lysis,
the corresponding extract was subjected to anion-exchange chro-
matography to yield total IP. Radioactivity was measured by scin-
tillation counting after adding 2.5 mL of Emulsifier-Safe
(PerkinElmer, Rodgau, Germany).
Data were analyzed and specific counts were normalized to 0%
for the non-stimulated receptor and 100% corresponding to the
maximal effect of the reference isoprenaline and quinpirole, for
the b2AR and D2SR, respectively. Dose–response curves were
calculated by non-linear regression using the algorithms of
PRISM 6.0.
Structures of the b2AR bound to FAUC50 (PDB ID: 3PDS) and the
dopamine D3 receptor bound to eticlopride (PDB ID: 3PBL) were
manually aligned in Pymol (Schrödinger, Inc., New York, NY).
Figures were prepared using Pymol.
4.1.41. Receptor binding studies
Receptor binding studies were carried out as described previ-
ously.48,49 In brief, human D1 binding measured using homoge-
nates of membranes from HEK 293 cells, which were transiently
transfected with the pcDNA3.1 vector containing the human D1
gene (from Missouri S&T cDNA Resource Center (UMR), Rolla,
MO) by the calcium phosphate method.50 Membranes were incu-
bated at final concentrations of 2–4 lg/well with receptor densi-
ties (Bmax values) of 3500–6000 fmol/mg protein and KD values of
0.23–0.64 nM with the radioligand [3H]SCH23390 (specific activity
80 Ci/mmol; Biotrend, Cologne, Germany) at 0.30–0.50 nM. For
51
52
competition binding experiments, human D2L, D2S
,
D3 and
53
D4.4 receptor membrane preparations from CHO cells stably
expressing the corresponding receptor were used together with
[3H]spiperone (specific activity = 81 Ci/mmol, PerkinElmer, Rod-
gau, Germany) at a final concentrations of 0.10–0.30 nM. The
4.1.43. b-Arrestin recruitment assay
b-Arrestin-2 recruitment stimulated by receptor activation
was performed utilizing the PathHunterÒ assay purchased from
DiscoveRx (DiscoveRx, Birmingham, UK) according to the manu-
facturer’s protocol and as described previously.34 In brief, HEK-
239 cells stably expressing the enzyme acceptor (EA)-tagged b-
arrestin fusion protein were transiently transfected with the Pro-
Link PK-tagged b2 adrenergic receptor (pCMV-ADRB2-PK from
DiscoveRx, Birmingham, UK) or the (ARMS2-PK2)-tagged dopa-
mine D2S receptor using the TransIT-293 transfection reagent
from Mirus (purchased from MoBiTec, Goettingen, Germany).
Cells (5000 cells per well) were seeded in 384-well plates and
maintained for 24 h. After incubation with different concentra-
tions of the test compounds for 90 min and 5 h for the b2AR
and D2SR, respectively, the detection mix was added and incuba-
tion was continued for further 60 min. Chemiluminescence was
measured using a plate reader for microplates (Clariostar, BMG
Labtech, Ortenberg, Germany). Experiments were performed in
duplicates.
assays were carried out at a protein concentration of 1–10 lg/
assay tube, KD values of 0.035–0.091 nM, 0.032–0.090 nM, 0.10–
0.17 nM, and 0.15–0.25 nM and corresponding Bmax values of
6600–1300 fmol/mg, 2100–8500 fmol/mg, 1500–8500 fmol/mg,
and 850–2100 fmol/mg for the D2L, D2S, D3 and D4.4 receptors,
respectively. Adrenergic binding data were generated using mem-
brane preparations from HEK 293 cells transiently expressing mur-
ine b1 (the cDNA was a generous gift from Brian Kobilka, Stanford
University, CA) and homogenates from CHO cells stably expressing
human b2 (cDNA from Missouri S&T cDNA Resource Center (UMR),
Rolla, MO) and the radioligand [3H]CGP12177 (specific activ-
ity = 38 Ci/mmol, PerkinElmer, Rodgau, Germany). Membranes
were incubated in HEPES buffer (25 mM HEPES, 5 mM MgCl2,
1 mM EDTA, pH 7.4) at final concentrations of 1–3
lg/well or 2–
5
lg/well with receptor densities (Bmax values) of 4000–
14,000 fmol/mg and 1800–3500 fmol/mg protein and specific
binding affinities (KD values) of 0.080–0.11 nM and 0.045–
0.070 nM for b1 and b2, respectively. Non-specific binding was
The resulting curves were analyzed and normalized to RLU val-
ues of 0% for the non-stimulated receptor and to 100% correspond-
ing to the maximal effect of the reference isoprenaline and
quinpirole for the b2AR and D2SR, respectively. Dose–response
curves were calculated by non-linear regression using the algo-
rithms of PRISM 6.0.
determined in the presence of 10 lM haloperidol and 10 lM
CGP12177 for dopamine receptors and adrenergic receptors,
respectively. Protein concentrations were determined according
to the method of Lowry using bovine serum albumin as standard.54
The resulting competition curves of the receptor binding exper-
iments were analyzed by nonlinear regression using the algorithms
in PRISM 6.0 (GraphPad Software, San Diego, CA). The data were
initially fit using a sigmoid model to provide an IC50 value, repre-
senting the concentration corresponding to 50% of maximal inhibi-
tion. IC50 values were transformed to Ki values according to the
equation of Cheng and Prusoff.55
Acknowledgments
This work was supported by grants from the German Research
Foundation (Deutsche Forschungsgemeinschaft Gm 13/10, GRK
1910). We thank Ralf Kling for helpful discussion on the structural
design and Martina Schaper for assistance during manuscript
preparation.
Supplementary data
4.1.42. IP accumulation assay
G protein-mediated signaling for the human b2 adrenergic and
the dopamine D2s receptors was determined by measuring ligand
stimulated IP accumulation as described previously.35 HEK 293
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.04.027.
Please cite this article in press as: Weichert, D.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.028

D. Weichert et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
13
Konetzki, I.; Sunahara, R. K.; Gellman, S. H.; Pautsch, A.; Steyaert, J.; Weis, W. I.;
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Please cite this article in press as: Weichert, D.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.028