Synthesis and in vitro and in vivo characterization of highly β1-selective β-adrenoceptor partial agonists

Article abstract of DOI:10.1021/jm400348g

β-Adrenoceptor antagonists boast a 50-year use for symptomatic control in numerous cardiovascular diseases. One might expect highly selective antagonists are available for the human β-adrenoceptor subtype involved in these diseases, yet few truly β₁-selective molecules exist. To address this clinical need, we re-evaluated LK 204-545 (1),(1) a selective β₁-adrenoceptor antagonist, and discovered it possessed significant partial agonism. Removal of 1's aromatic nitrile afforded 19, a ligand with similar β₁-adrenoceptor selectivity and partial agonism (log K_D of -7.75 and -5.15 as an antagonist of functional β₁- and β₂-mediated responses, respectively, and 34% of the maximal response of isoprenaline (β₁)). In vitro β-adrenoceptor selectivity and partial agonism of 19 were mirrored in vivo. We designed analogues of 19 to improve affinity, selectivity, and partial agonism. Although partial agonism could not be fully attenuated, SAR suggests that an extended alkoxyalkoxy side chain, alongside substituents at the meta- or para-positions of the phenylurea, increases ligand affinity and β₁-selectivity.

Full text of DOI:10.1021/jm400348g

Article
pubs.acs.org/jmc
Synthesis and in Vitro and in Vivo Characterization of Highly
β1‑Selective β‑Adrenoceptor Partial Agonists
Shailesh N. Mistry,†,∥ Jillian G Baker,*,‡,∥ Peter M Fischer,† Stephen J Hill,‡ Sheila M Gardiner,‡
and Barrie Kellam*,†
†School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, United
Kingdom
‡Institute of Cell Signalling, School of Biomedical Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham
NG7 2UH, United Kingdom
S
* Supporting Information
ABSTRACT: β-Adrenoceptor antagonists boast a 50-year use for
symptomatic control in numerous cardiovascular diseases. One might
expect highly selective antagonists are available for the human β-
adrenoceptor subtype involved in these diseases, yet few truly β1-selective
molecules exist. To address this clinical need, we re-evaluated LK 204-545
(1),1 a selective β1-adrenoceptor antagonist, and discovered it possessed
significant partial agonism. Removal of 1’s aromatic nitrile afforded 19, a
ligand with similar β1-adrenoceptor selectivity and partial agonism (log
KD of −7.75 and −5.15 as an antagonist of functional β1- and β2-mediated
responses, respectively, and 34% of the maximal response of isoprenaline
(β1)). In vitro β-adrenoceptor selectivity and partial agonism of 19 were
mirrored in vivo. We designed analogues of 19 to improve affinity,
selectivity, and partial agonism. Although partial agonism could not be fully attenuated, SAR suggests that an extended
alkoxyalkoxy side chain, alongside substituents at the meta- or para-positions of the phenylurea, increases ligand affinity and β1-
selectivity.
(LABAs, e.g., salmeterol, formoterol, indacaterol, and vilanter-
ol).
INTRODUCTION
■
β-Adrenoceptor ligands comprise one of the most commonly
used classes of drugs in clinical practice. β-Agonists have been
used since the 1940s for their beneficial effects in respiratory
disease2,3 and β-antagonists (β-blockers) since the 1960s for
the control of cardiovascular disease,4,5 and both classes remain
among the most commonly prescribed drugs today.
The major therapeutic aim of β-blockade is antagonism of
endogenous catecholamines at the cardiac β1-adrenoceptors to
reduce myocardial demand and workload.6 This reduces
mortality following myocardial infarction,7 and therefore β-
blockers are used for both symptomatic control and life
prolongation in ischemic heart disease and acute coronary
syndrome and are increasingly being used earlier in the disease
process.8 β-Blockers also reduce mortality in patients with heart
failure9−14 and are used in the management of arrhythmias,
hypertension, portal hypertension, benign essential tremor,
thyrotoxicosis, anxiety, migraine, and glaucoma.15
The major therapeutic aim of β-agonist administration is to
mimic adrenaline and stimulate the β2-adrenoceptors in
bronchial smooth muscle, thus increasing airway caliber and
relieving the breathlessness and wheeze of bronchospasm.16 β-
Agonists are used in the emergency setting for short-term
rescue of bronchospasm in asthma and COPD (e.g., salbutamol
inhalers and nebulizers) as well as in the daily prevention of
bronchospasm, particularly using longer acting molecules
As β-ligands have been used in clinical practice for around for
70 years, and a great many different agonist and antagonist
molecules have been generated, it would be expected that good
tool agonist and antagonist compounds for each of the human
β-adrenoceptor subtypes are available. However, it is surprising
that there are actually very few truly selective molecules,
especially for the β1-adrenoceptor. Drug discovery efforts
driven by the clinical need have yielded several highly selective
β2-adrenoceptor agonists for the treatment of asthma and
COPD (e.g., salmeterol, formoterol, indacaterol, and vilanterol)
and one highly selective β2-antagonist (ICI 118551) that has
become a very useful tool in pharmacological studies of β-
adrenoceptors. For the β1-adrenoceptor, despite the fact that
more β1-selective β-blockers would be preferable to minimize
the β2-mediated respiratory side effect of bronchospasm in the
clinical setting, the selectivity of the clinically available β-
blockers for the human β1-adrenoceptor over the human β2-
adrenoceptor is poor.17−21 For this reason, β-blockers remain
absolutely contraindicated in patients with asthma and
relatively contraindicated in people with COPD. Our desire
to address this significant unmet clinical need initially led us to
Received: December 9, 2012
© XXXX American Chemical Society
A
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Scheme 1. Synthesis of 1-(2-(3-(2-Cyano-4-(2-(cyclopropylmethoxy)ethoxy)phenoxy)-2-hydroxypropylamino)ethyl)-3-(4-
hydroxyphenyl)urea (1) and 1-(2-(3-(4-(2-(Cyclopropylmethoxy)ethoxy)phenoxy)-2-hydroxypropylamino)ethyl)-3-(4-
a
hydroxyphenyl)urea Hydroformate (19)
a
Reagents and conditions: (a) Et3N, pivaloyl chloride, DMF, 0 °C → rt, 72%; (b) (i) NaH, DMF; (ii) p-methoxybenzyl bromide, 0 °C → rt, 33%;
(c) 37% NH3 (aq), I2, THF, 98%; (d) sodium tert-butoxide, MeOH, 34%; (e) allyloxyethanol, DIAD, PPh3, DCM, 100%; (f) Et2Zn, CH2I2, toluene
0 °C → rt, 25% 8 and 29% 9; (g) CAN, H2O, MeCN; (h) Et3N, rac-epichlorohydrin, 80 °C, 100%; (i) 4-(benzyloxy)phenylisocyanate, DCM, 94%;
(j) (i) concd HCl; (ii) 2 M NaOH (aq), neutralization, 73%; (k) 13, propan-2-ol, reflux, 15%; (l) allyloxyethanol, DIAD, Ph3P, DCM, rt, 71%; (m)
Et2Zn, CH2I2, toluene, 0 °C → rt, 97%; (n) H2, 10% Pd/C, EtOH, 100%; (o) (i) 2 M NaOH(aq); (ii) rac-epichlorohydrin, 60 °C, 62%; (p) 13,
propan-2-ol, reflux, 21%.
assess reported ligands that displayed very high β1-adrenocep-
tor versus β2-adrenoceptor selectivity. From the literature, one
of the most highly β1-selective antagonists described to date
was 1 (LK 204-545).1 Although this ligand provided an ideal
structural template to investigate further, there was no
published synthetic route and, in reality, very little pharmaco-
logical data available for this compound. Furthermore, it was
not commercially available and therefore not readily accessible
for pharmacological and other studies. As we also observed,
there were also very few β1-selective β-agonists, with xamoterol
and ICI 89406 being the most selective β1-partial agonists
reported to date.17−22
benchmark any newer ligands against, we embarked upon a
new and more expeditious route to its synthesis (Scheme 1).
The new synthesis of 1 relied upon an alternative synthesis of
epoxide 10, with subsequent aminolysis of 10 using amine 13.
The availability of methods to easily convert an aryl aldehyde to
the corresponding nitrile24 allowed 2,5-dihydroxybenzaldehyde
(2) to be selected as an appropriate starting material in the
synthesis of epoxide 10.
A procedure reported by Chen et al.25 facilitated selective
monoprotection of 2 using pivaloyl chloride to generate
pivaloate ester 3 in good yield. The remaining hydroxy group
then required protection orthogonal with respect to the
pivaloate ester; hence, the p-methoxybenzyl (PMB) group
was selected for this purpose. The ability to cleave a PMB ether
under oxidative conditions, using reagents such as ceric(IV)
ammonium nitrate (CAN), was anticipated,26 because this
would allow selective removal of the PMB group in the
presence of the nitrile later in the synthesis.
Conversion of aldehyde 4 to the desired nitrile was achieved
cleanly by oxidative amination24,27 in nearly quantitative yield
to give 5. Alkoxide-mediated removal of the pivaloate ester then
revealed phenol 6, which was subsequently submitted to
Mitsunobu coupling with 2-(allyloxy)ethanol to generate ether
7. Cyclopropanation of 7 using Simmons−Smith methodology
gave a mixture of products.28 The desired PMB-protected
product 8 was isolated and subjected to CAN-mediated
oxidative PMB group cleavage conditions. Unfortunately, this
Here, we report a synthesis and the in vitro pharmacological
characterization of 1 and several novel, highly β1-selective β-
adrenoceptor ligands, most of which display significant agonist
activity.
RESULTS AND DISCUSSION
■
Synthesis. Whereas the synthesis of 1 is not described in
the literature, a synthetic route to the related compound, 1-(2-
cyano-4-(2-cyclopropylmethoxyethoxy)phenoxy)-3-(2-(3-
phenylureido)ethylamino)-2-propanol, is outlined as an exam-
ple of this class of compound in a patent.23 The introduction of
the cyano group employed an undesirable cyanation step,
alongside several protection and deprotection steps, complicat-
ing the synthesis and compromising overall yield. Conscious
that we would require appreciable quantities of this ligand to
B
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a
Table 1. Human β1- and β2-Adrenoceptor Binding Affinities for Established β-Adrenoceptor Ligands, 1 and 19
compd
log KD β1
n
log KD β2
n
selectivity ratio (β1/β2)
CGP 20712A
ICI 118551
xamoterol
ICI 89406
1
−8.79 0.07
−6.62 0.01
−7.09 0.04
−8.75 0.03
−8.04 0.03
−7.49 0.03
9
−5.82 0.04
−9.17 0.03
−5.76 0.04
−6.84 0.03
−5.29 0.04
b
8
933
9
8
8
8
354 β2 selective
21
9
9
81
9
11
11
562
19
11
a
Values represent the mean SEM of n separate experiments. Log KD values were obtained from 3H-CGP 12177 whole cell binding studies in CHO
cells stably expressing the human β1 or β2-adrenoceptors. Incomplete inhibition of specific H-CGP12177 binding meant that generation of an
b
3
affinity value (log KD value) by this method was not possible.
a
Scheme 2. Synthesis of Hydroxyphenylureas, Varied at the Alkoxyethyl Terminus
a
Reagents and conditions: (a) NaBH4, ZrCl4, THF 0−5 °C, 81%; (b) (i) NaH, DMF 60 °C; (ii) chloroacetic acid, 60 °C, 50%; (c) LiAlH4, THF, 0
°C, 67%; (d) Ph3P, 14, di-tert-butyl azodicarboxylate or diethyl azodicarboxylate, DCM, 35−85%; (e) H2, 10% Pd/C, EtOH, 68−100%; (f) (i) NaH,
DMF, 0 °C → rt; (ii) rac-epichlorohydrin, 71−84%; (g) (i) 2 M NaOH(aq); (ii) rac-epichlorohydrin, 60 °C, 84%; (h) 13, propan-2-ol, reflux, 21−
29%.
did not yield the desired phenol 8, possibly due to instability of
the molecule to CAN, for example, through dealkylation of the
aralkyl ether.29 Re-examination of crude reaction products from
the cyclopropanation step revealed that apart from the expected
PMB-protected product 9, the oxidative conditions of the
Simmons−Smith procedure had caused partial removal of the
PMB group to afford phenol 8 directly, although in modest
yield. Consequently, phenol 8 was recovered, albeit as a side
product and in relatively low yield. Alkylation of phenol 8 with
rac-epichlorohydrin in the presence of TEA afforded epoxide
10 in quantitative yield, with no further purification required. In
this preliminary study, although it was well understood that the
S-form was the more pharmacologically active of the two
aryloxypropanolamine enantiomers, we elected to synthesize
racemic derivatives throughout, as a more expeditious and cost-
effective route to explore early SAR, with the intention that
favorable pharmacology would prompt synthesis of individual
stereoisomers if warranted.
10 with 13 under neutral conditions, by reflux in propan-2-ol,
afforded 1 after purification by flash column chromatography.
Pharmacological analysis of 1 in our hands (cf. Table 1 and
pharmacology discussion) indicated a slightly lower β1/β2-
selectivity than previously reported.1 In addition to this, we
noted with interest that previous studies indicated that the
presence of substituents of the phenyl ring ortho to the
oxypropanolamine moiety did not improve β1/β2-selectivity but
led to either increased or decreased affinity at both receptors
simultaneously.30−35 Because our aim was to devise β1-selective
ligands, we were therefore encouraged to explore whether
removal of the cyano group was a worthwhile modification
because, if successful, it would allow a more expeditious route
into a range of new analogues. We therefore embarked upon
the more efficient synthesis of the decyanated analogue of 1
(19), which had not previously been described, to assess the
impact of the nitrile group on β1/β2-receptor pharmacology
(Scheme 1).
Construction of aminophenol 13 required initial addition of
1,2-ethanediamine (11) to 4-(benzyloxy)phenyl isocyanate.
Dropwise introduction of a solution of the isocyanate into a
solution containing an excess of 11 allowed selective mono-
addition to occur, as the product urea 12 was found to
precipitate on formation, allowing facile isolation and
purification by filtration. Conversely, the poor solubility of 12
in a variety of appropriate solvents meant that attempted
hydrogenolysis progressed slowly. Acidolytic O-benzyl depro-
tection was therefore chosen as an alternative to give
aminophenol 13 in good yield. Finally, aminolysis of epoxide
Mitsunobu coupling of 2-(allyloxy)ethanol with commer-
cially available 4-(benzyloxy)phenol (14) furnished the desired
phenylether 15, whose terminal allyl ether function was
efficiently converted to the corresponding cyclopropylmethyl
ether 16 using Simmons−Smith cyclopropanation.28 Subse-
quent benzyl ether hydrogenolysis of 16 over Pd(0) on
charcoal, using standard conditions, afforded phenol 17 in
quantitative yield. Alkylation of 17 was achieved in moderate
yield by initial deprotonation in aqueous 2 M NaOH solution,
followed by heating in excess rac-epichlorohydrin to afford
oxirane 18. Finally, oxirane 18 was subjected to aminolysis with
C
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Scheme 3. Synthesis of Aryl Monosubstituted 3-(2-((3-(4-(2-(Cyclopentyloxy)ethoxy]phenoxy)-2-hydroxypropyl)amino]ethyl)-
a
1-phenylureas
a
Reagents and conditions: (a) o-tolylisocyanate (30), DCM, 0 °C → rt, 64%; (b) di-tert-butyl dicarboxylate, DCM, 87%; (c) substituted
phenylisocyanate, DCM, 0 °C → rt, 46 − 95%; (d) MeOH/concd HCl, 80−100%; (e) phthalic anhydride, 150 °C, 94%; (f) (i) diphenylphosphoryl
azide, TEA, toluene; (ii) reflux; (iii) 2-aminophenol or 3-aminophenol, 75−76%; (g) hydrazine monohydrate, EtOH, reflux; (ii) acidic workup, 51−
60%; (h) 33a−r, 10 M NaOH(aq), propan-2-ol, reflux, 7−31%; (i) 33t−u, Et3N, propan-2-ol, reflux, 21−25%.
13 under basic conditions by refluxing in propan-2-ol. The
desired aryloxypropanolamine 19 was obtained as the hydro-
formate salt, after PLC and subsequent preparative RP-HPLC
purification.
Machin et al.38 was employed. This entailed initial alkylation of
4-fluorophenethyl alcohol (22) with chloroacetic acid to give
alkoxyacetic acid 23. Subsequent reduction was achieved with
LiAlH4, to give alcohol 24 in acceptable yield.
Pharmacological and in vivo analysis of our early lead ligand
19 gave mixed results. Whereas its selectivity was somewhat
diminished compared to that of 1 (cf. pharmacology results),
both compounds possessed significant partial agonism,
rendering them unsuitable for future therapeutic development
as a selective β1-adrenoceptor antagonist. Therefore, to explore
the SARs of this class of molecule further and to ascertain if
selectivity could be enhanced while decreasing partial agonism,
the more facile synthesis of 19 now allowed access to new
analogues in a more expeditious manner. Initially we tried to
ascertain the effect of the alkoxyalkylether and the phenylurea
termini on affinity, selectivity, and partial agonism. To explore
the influence of the alkoxyalkoxy terminus on pharmacology,
and on the basis of established literature precedent, we
synthesized oxiranes 28a−c in moderate yield (Scheme 2),36
essentially in the same manner as for 10. Although 2-
ethoxyethanol (25) was commercially available, the analogous
2-(cyclopentyloxy)ethanol (21) and 2-(4-fluorophenethyloxy)-
ethanol (24) starting materials required independent synthesis
(Scheme 2).
The alcohols 21, 24, and 25 were subjected to Mitsunobu
coupling with 4-(benzyloxyphenol) (14) in a similar manner as
previously described, to give O-benzyl ethers 26a−c. Hydro-
genolysis under standard conditions afforded phenols 27a−c.
Subsequent alkylation of phenol 27c was carried out using the
previously described method to give oxirane 28c. In the case of
phenols 27a,b, NaH was used as a base in anhydrous DMF,
with subsequent alkylation proceeding at room temperature to
furnish oxiranes 28a,b. The target aryloxypropanolamines 29a−
c were obtained in the free base form, from oxiranes 28a−c
through aminolysis with 13 as previously detailed.
Encouraged by the pharmacological data of the novel
compounds possessing the 2-(cyclopentyloxy)ethoxyphenyl
terminus (cf. Table 4 and pharmacology discussion), we kept
this group constant to investigate the pharmacological effects of
modifying the phenylurea substituent at the opposite pole of
the molecule. This required synthesis of a small set of phenyl-
substituted 1-(2-aminoethyl)-3-phenylureas (Scheme 3).
In accordance with the synthesis of 12, attempted
condensation of o-tolylisocyanate (30) with an excess of
ethylenediamine (11) afforded urea 33b in only modest yield,
alongside the undesirable bis-urea side product. To avoid this
complication with the remaining analogues, tert-butyl-2-amino-
ethylcarbamate (31) was employed instead of 11. Selective
2-(Cyclopentyloxy)ethanol (21) was obtained in good yield
from ketal 20, according to the NaBH4/ZrCl4 reductive
cleavage conditions reported by Chary et al.37 In the case of
2-(4-fluorophenethyloxy)ethanol (24), the route described by
D
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Figure 1. Inhibition of 3H-CGP 12177 binding to whole cells by (a, b) 1 and (c, d) 19 in (a, c) CHO β1 cells and (b, d) CHO β2 cells. Bars represent
total 3H-CGP 12177 binding, and nonspecific binding was determined in the presence of 10 μM propranolol. The concentration of 3H-CGP 12177
present in each case was 0.73 nM. Data points are mean SEM of triplicate determinations. These single experiments are representative of (a) 9 and
(b−d) 11 separate experiments.
Figure 2. 3H-cAMP accumulation in response to cimaterol in (a) CHO β1 cells, (b) CHO β2 cells, and (c) CHO β3 cells in the absence and presence
of 19. Bars show basal 3H-cAMP accumulation, that in response to 10 μM isoprenaline, and that in response to 30 nM, 100 nM, 300 nM, 1 μM, 3
μM, and 10 μM 19 alone. Data points are the mean SEM of triplicate values from single experiments that are representative of seven separate
experiments in each case.
mono-Boc protection of ethylenediamine (11) was accom-
plished in excellent yield using a literature method39 to give
amine 31 (Scheme 3). The stoichiometric condensation of 31
with corresponding substituted phenylisocyanates permitted
preparation of the Boc-protected intermediates 32a and 32c−r
in moderate to good yield, with the pure products being easily
isolated by filtration following precipitation with hexanes. Boc-
deprotection of these intermediates with concentrated HCl in
methanol proceeded smoothly and in excellent yields, affording
the desired amines 33a and 33c−r as their respective
hydrochloride salts. The remaining 1-(2-aminoethyl)-3-
(hydroxyphenyl)urea analogues (33t−u) required an alter-
native synthetic strategy, due to the lack of commercially
available phenylisocyanate starting materials. However, the
availability of 2-amino and 3-aminophenol allowed urea
synthesis via condensation with the appropriate alkylisocya-
nates (Scheme 3).
Using β-alanine (34) as a starting material, the conversion of
the primary amine to the phthalimide eliminated possible side
reactions during isocyanate formation; monoacylated amine
E
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a
Table 2. Antagonist Affinities of Established Ligands and 19 at the Human β1-, β2-, and β3-Adrenoceptors
ligand
log KD β1
n
log KDβ2
n
log KDβ3
n
selectivity β1 vs β2
CGP 20712A
xamoterol
ICI 89406
1
−9.48 0.06
−7.75 0.05
−9.48 0.04
−8.77 0.05
−7.75 0.06
12
11
12
11
21
−5.98 0.03
−6.18 0.05
−7.25 0.05
−5.54 0.05
−5.15 0.06
4
4
4
4
7
−5.26 0.04
−4.70 0.08
−5.72 0.05
>10 μM
4
4
4
4
7
3162
37
170
1698
398
19
>10 μM
a
Values represent the mean SEM of n separate experiments. Log KD values were obtained from 3H-cAMP accumulation assays following inhibition
of the cimaterol agonist response in CHO cells stably expressing the human β1-, β2-, or β3-adrenoceptors.
a
Table 3. Agonist Responses of Established Ligands and 19 at the Human β1-, β2-, and β3-Adrenoceptors
β1
β2
β3
% isop max
compd
log EC50 β1
% isop max
n
log EC50 β2
% isop max
n
log EC50 β3
n
b
CGP 20712A
xamoterol
ICI 89406
1
no response
−7.96 0.03
−9.09 0.03
−8.64 0.04
−8.01 0.04
0
4
5
5
5
6
10 μM
1.7 0.0
5.1 2.5
4.9 0.2
15.2 0.7
8.0 1.1
4
4
4
4
7
no response
0
4
4
4
4
7
b
47.6 1.7
27.3 0.05
37.1 2.2
33.6 2.4
−6.15 0.06
−7.22 0.08
100 μM
38.3 1.4
11.7 0.5
8.6 0.4
12.5 2.3
b
10 μM
b
b
10 μM
10 μM
b
b
19
10 μM
10 μM
a
Values represent the mean SEM of n separate experiments. Log EC50 values and % isoprenaline maximal responses were obtained from 3H-cAMP
accumulation for cells expressing the human β1-adrenoceptor. For the human β2- and β3-adrenoceptors, the top of the agonist concentration
response curve was not obtained even with the maximum concentration of ligand. In these instances, the percentage of isoprenaline response is given
for the response at 10 μM of ligand or, in the case of xamoterol, 100 μM.
b
3
protecting groups still have the propensity to attack the
isocyanate functionality.40 Phthalimide protection of 34 was
effected through a solvent-free method, by heating directly with
phthalic anhydride to give acid 35,41 which was converted to
the corresponding acyl azide with the aid of diphenylphos-
phoryl azide in the presence of TEA; careful reflux of this
intermediate promoted Curtius rearrangement to the iso-
cyanate. The isocyanate solution was divided into two equal
portions, prior to addition of either 2- or 3-aminophenol,
producing protected intermediates 36a and 36b. The desired
amines 33t and 33u were once again isolated as the
hydrochloride salts after standard hydrazinolytic cleavage of
the phthalimide group and subsequent acidic workup. Finally,
aminolysis of oxirane 28a with amines 33a−u and subsequent
PLC purification afforded the target aryloxypropanolamine
compounds 37a−u (Scheme 3).
Antagonist Affinity Measurements: H-cAMP Accumula-
tion. Because quantifiable antagonist affinity was difficult to
determine for 19 at the human β2- and β3-adrenoceptors, an
alternative assay was employed to establish accurate values via
measurement from the shift of an agonist response (Figure 2;
Table 2).
Cimaterol was chosen as the agonist as it is a chemically
stable nonselective agonist and thus could be used across all
three human β-adrenoceptors and because cimaterol agonist
responses are also readily inhibited by classical β-blockers,
suggesting that its primary action is agonism of the catechol-
amine conformation of the human β1-adrenoceptor.42 Cimater-
ol stimulated responses were 96.9 1.1% (log EC50 = −8.13
0.03, n = 9), 98.9 1.7% (log EC50 = −8.78 0.06, n = 9), and
89.6
1.9% (log EC50 = −6.62
0.06, n = 9) of the
isoprenaline maximum response at the human β1-, β2-, and β3-
adrenoceptors, respectively (Figure 2). These agonist responses
were antagonized by both 1 and 19 (Table 2). As both ligands
examined demonstrated clear partial agonist stimulatory effects
(e.g., Figure 2), the data were analyzed by the partial agonist
method of Stephenson.43
3
Pharmacology. Ligand Affinity Measurements: H-CGP
12177 Whole Cell Binding. We initially evaluated and
compared the affinity of 1 with that of the decyanated analogue
19 at the human β1-, β2-, and β3-adrenoceptors, each stably
expressed in Chinese hamster ovary cells. The KD values for β-
adrenoceptor binding of 3H-CGP 12177 in these cell lines have
previously been established and are 0.42, 0.17, and 109.2 nM
Agonist Responses: 3H-cAMP Accumulation. Given the
significant degree of agonist activity seen above, full
concentration response relationships were investigated for the
ligands. 1 caused an agonist response at the human β1-
adrenoceptor that was 37.1 2.2% of the maximum response
to isoprenaline (n = 5). It is thus a partial agonist at the human
β1-adrenoceptor. Agonist responses were also seen at the
human β2- and β3-adrenoceptors; however, the top of the
concentration response curve was not reached in each case. At
the maximum concentration of 10 μM, 1 stimulated responses
for the β1-, β2-, and β3-adrenoceptors, respectively.20
1
completely inhibited specific binding to the human β1-
adrenoceptor to yield a log KD of −8.04 0.03, n = 9, and
−5.29
0.04, n = 11, for the human β2-adrenoceptor, thus
giving a selectivity for the β1-adrenoceptor of 562-fold (Figure
1; Table 1). Likewise, 19 was also shown to inhibit specific
binding to the human β1-adrenoceptor to yield a log KD of
−7.49
0.03. However, its affinity for the human β2-
that were 15.2
0.7% (β2) and 8.6
0.4% (β3) of the
adrenoceptor was so low that measurement of complete
isoprenaline maximum at each receptor. Similar responses were
seen for 19 (Table 3).
3
inhibition of specific H-CGP 12177 binding was not possible
(Figure 1d), so that affinity (KD values) could not be calculated
by this method. The affinity of both ligands for the human β3-
adrenoceptor was also too low to establish reliable KD values
from this method.
In Vivo Selectivity and Agonist Actions of 19. To assess the
potential clinical effect of this degree of selectivity and partial
agonism, these parameters were investigated in a freely moving
conscious rat model. Previous studies with this model have
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shown that the heart rate response to isoprenaline is solely a β1-
mediated response and that the hindquarters vascular
conductance is solely a β2-mediated response.44 We employed
the hydrochloride salt of 19 (2 mg/kg iv bolus, 1 mg/kg/h iv
infusion) and observed that it significantly inhibited the β1-
mediated heart rate responses to isoprenaline (at 40 and 120
ng/kg/min, 30−90 min following administration) while having
no effect on the β2-mediated hindquarters response (n = 4
rats), consistent with this compound’s β1-adrenoceptor
selectivity (Figure 3). In addition, the ligand caused an increase
extremities of our lead compounds could be modified to
improve affinity and selectivity while attenuating partial
agonism, we employed an iterative screening cascade approach.
Initially we used a H-CGP 12177 whole cell binding assay to
ascertain antagonist affinity measurements (Table 4).
3
Informed from previously established SARs,36 we explored
three variants of the p-alkoxyalkoxy side chain present in 1 and
19, namely a para-positioned 2-(cyclopentyloxy)ethoxy- (29a),
a 2-(4-fluorophenethyloxy)ethoxy- (29b), and an 2-(ethyloxy)-
ethoxy- (29c) moiety. Because the 2-(cyclopentyloxy)ethoxy
side chain displayed the best combination of an increase in both
binding affinity and β1/β2-selectivity when compared to 19, we
retained this moiety to undertake a SAR evaluation of the
phenylurea portion of the molecule (Table 5). The
unsubstituted phenylurea (37a) displayed a loss in affinity at
both the β1- and β2-adrenoceptors yet maintained good β1-
selectivity. Insertion onto the phenyl ring of individual electron-
withdrawing or electron-donating groups at the ortho-, meta-, or
para-positions displayed a set of measurable trends. In general,
the ligand binding affinity remained within an order of
magnitude for the respective adrenoceptor subtypes (β1 log
KD between −6.94 and −8.17 M; β2 log KD between −5.52 and
−6.54 M) with all ligands showing selectivity for the β1-
subtype. In most cases (37b, 37e, 37k, 37n, 37q, and 37t) an
ortho-positioned substituent resulted in the weakest β1-
adrenoceptor binding affinity within each three-ligand subset
alongside the smallest β1/β2-selectivity. The only exception to
this observation was that of the o-fluoro derivative (37h),
which, while displaying the second highest affinity of the three
ligands (37h−j) for the β1-adrenoceptor, did possess the
greatest, albeit modest, β1/β2-selectivity (76-fold).
The optimal ligands in terms of both affinity and selectivity
were substituted in the meta- or para-positions with either a
chlorine atom (37l and 37m) or a hydroxyl group (29a and
37u) or, as mentioned above, were unsubstituted (37a). To
cross-validate these results and attempt to determine β3-
adrenoceptor affinity, we repeated the alternative assay for
determining affinity whereby the shift of a cimaterol-mediated
dose−response curve is measured, focusing upon the best
ligands from the phenylurea SAR study and re-evaluating the
original alkoxyalkoxy compounds (Table 6).
Interestingly, this assay gave values for ligand affinity (and
selectivity) slightly higher than that of the binding assay, for
example, with 29a β1-selectivity 478-fold (over β2) in the
binding assay but 513-fold in the cAMP assay and with 37l 331-
fold in the binding assay and 537-fold in the cAMP assay,
similar to what was observed with previous ligands (Tables 1
and 2). This method therefore provides a similar pattern of β1-
versus β2-selectivity, allowing comparison between different
chemical analogues. Even using this method, the affinity at the
Figure 3. Absolute values for heart rate (a) and hindquarters
conductance (b) in conscious freely moving rats (n = 4 per group)
before and at the end of 3 min infusions of isoprenaline (12, 40, and
120 ng/kg/min). Isoprenaline responses were measured before and at
intervals after saline or 19 (HCl salt) administration. 19 (HCl salt)
was given as a 2 mg/kg bolus followed by 1 mg/kg/h infusion for 90
min, and isoprenaline responses were measured during the infusion
(30−90 min) and after the 19 (HCl salt) infusion was turned off (4−5
and 24−25 h). Isoprenaline responses measured in saline-treated
animals at 30−90 min, 4−5 h, and 24−25 h were highly reproducible,
but for the sake of clarity only the 30−90 min time point is shown
here.
in basal heart rate (from 424 9 to 465 3 beats/min, an
increase that corresponds to 44% of the response to 120 ng/
kg/min isoprenaline, n = 4 animals), in keeping with the partial
agonist actions observed in the in vitro cell studies with 19.
β-Adrenoceptor Profiling of Second- and Third-Gener-
ation Ligands. To investigate whether the structural
Table 4. Human β1- and β2-Adrenoceptor Binding Affinity and Receptor Selectivity of 1-(2-(3-(4-(2-
a
(Alkyloxy)ethoxy)phenoxy)-2-hydroxypropylamino)ethyl)-3-((4-hydroxy)phenyl)ureas 28a−c
compd
R1
log KD β1
n
log KD β2
n
selectivity ratio (β1/β2)
19
−7.49 0.03
−8.13 0.05
−8.50 0.05
−7.04 0.04
11
12
9
b
11
7
29a
29b
29c
cyclopentyl
−5.45 0.07
−5.91 0.05
b
478
389
p-FC6H4CH2CH2
CH3CH2
9
16
16
a
Values represent the mean SEM of n separate experiments. Log KD values were obtained from 3H-CGP 12177 whole cell binding studies in CHO
b
3
cells stably expressing the human β1- or β2-adrenoceptors. Incomplete inhibition of specific H-CGP12177 binding meant that generation of an
affinity value (log KD value) by this method was not possible.
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a
Table 5. Human β1- and β2-Adrenoceptor Binding Affinities and Receptor Selectivity of Substituted Phenylureas (37a−37u)
compd
R2
log KD β1
n
log KD β2
n
selectivity ratio (β1/β2)
37a
37b
37c
37d
37e
37f
H
−7.91 0.03
−7.33 0.03
−8.04 0.04
−7.76 0.04
−7.02 0.04
−7.76 0.03
−7.80 0.04
−7.82 0.03
−8.17 0.03
−7.70 0.04
−7.11 0.03
−8.21 0.06
−7.95 0.05
−7.11 0.01
−7.92 0.05
−7.77 0.04
−6.94 0.01
−7.77 0.06
−7.70 0.04
−6.99 0.08
−7.89 0.06
10
6
6
6
7
7
7
6
6
5
7
10
7
5
6
6
6
6
6
6
6
−5.58 0.03
−6.27 0.02
−6.06 0.03
−5.80 0.03
−5.93 0.02
−6.05 0.03
−5.86 0.04
−5.94 0.04
−6.54 0.02
−5.92 0.09
−5.54 0.03
−5.69 0.07
−5.91 0.06
−6.10 0.03
−5.99 0.04
−5.86 0.04
−5.84 0.02
−5.86 0.06
−5.76 0.04
−5.96 0.05
−5.85 0.06
9
213
11
96
91
12
51
87
76
43
60
37
331
110
10
85
81
13
81
87
11
110
o-CH3
m-CH3
p-CH3
o-OCH3
m-OCH3
p-OCH3
o-F
6
6
6
8
10
5
37g
37h
37i
6
m-F
6
37j
p-F
6
37k
37l
o-Cl
5
m-Cl
12
9
37m
37n
37o
37p
37q
37r
37s
37t
p-Cl
o-Br
6
m-Br
6
p-Br
6
o-CF3
m-CF3
p-CF3
o−OH
m−OH
6
6
6
6
37u
6
a
Values represent the mean SEM of n separate experiments. Log KD values were obtained from 3H-CGP 12177 whole cell binding studies in CHO
cells stably expressing the human β1- or β2-adrenoceptors.
a
Table 6. Antagonist Affinities for Second- and Third-Generation Ligands at the Human β1-, β2-, and β3-Adrenoceptors
compd
log KD β1
n
log KDβ2
n
log KDβ3
>10 μM
n
selectivity β1 vs β2
19
−7.75 0.06
−8.55 0.04
−9.00 0.04
−7.57 0.05
−8.46 0.05
−8.83 0.06
21
12
9
−5.15 0.06
−5.84 0.06
−6.05 0.05
>10 μM
7
4
4
4
4
4
7
4
4
4
4
4
398
513
29a
29b
29c
37a
37l
>10 μM
−5.58 0.09
>10 μM
891
12
12
9
>372
347
−5.92 0.02
−6.10 0.10
>10 μM
−5.22 0.08
537
a
Values represent the mean SEM of n separate experiments. Log KD values were obtained from 3H-cAMP accumulation assays following inhibition
of the cimaterol agonist response in CHO cells stably expressing the human β1-, β2-, or β3-adrenoceptors.
a
Table 7. Agonist Responses of Second- and Third-Generation Ligands at the Human β1-, β2-, and β3-Adrenoceptors
β1
% isop max
β2
β3
b
b
compd
log EC50 β1
n
β2
% isop max at 10 μM
n
β3
% isop max at 10 μM
n
1
−8.64 0.04
−8.01 0.04
−8.44 0.04
−8.64 0.04
−7.64 0.04
−8.35 0.10
−8.52 0.08
37.1 2.2
33.6 2.4
25.5 1.1
23.8 1.5
35.9 1.7
28.3 1.9
29.1 1.7
5
6
5
5
5
5
4
10 μM
10 μM
10 μM
10 μM
10 μM
10 μM
10 μM
15.2 0.7
8.0 1.1
4
7
4
4
3
4
3
10 μM
10 μM
10 μM
10 μM
10 μM
10 μM
10 μM
8.6 0.4
12.5 2.3
4.0 0.6
7.9 0.1
1.3 0.1
4.0 0.3
2.0 0.1
4
7
3
4
3
4
3
19
29a
29b
29c
37a
37l
15.3 1.1
16.0 1.3
27.8 2.3
15.3 1.0
12.7 0.7
a
Values represent the mean SEM of n separate experiments. Log EC50 values and % isoprenaline maximal responses were obtained from 3H-cAMP
accumulation for cells expressing the human β1-adrenoceptor. For the human β2- and β3-adrenoceptors, the top of the agonist concentration
response curve was not obtained even with the maximum concentration of ligand. In these instances, the percentage of isoprenaline response is given
for the response at 10 μM of ligand.
b
human β3-adrenoceptor once again could not be assessed,
because the maximum concentration of test compounds did not
cause a sufficient shift of the cimaterol concentration response
curve (e.g., Tables 2 and 6; Figure 2).
Partial Agonism of Second- and Third-Generation
Ligands. Although the affinity and selectivity profiles for
many of the ligands were extremely encouraging, we were also
aware that the original lead compounds (19 and 1) also
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displayed significant partial agonism at the β-adrenoceptors. We
therefore investigated the full concentration response relation-
ships for the best ligands (Table 7).
decyanated version of the molecule, and the pharmacological
effect of this maneuver was to reduce affinity at both human β1-
and β2-adrenoceptors.
Whereas the affinities of the second- and third-generation
ligands were comparable to those of 1 and 19, there was a
modest drop in efficacy at the β1-adrenoceptor, suggesting a
marginal attenuation of partial agonism with these compounds.
Interestingly, the one exception to this observation was 29c,
which contained the linear 2-(ethyloxy)ethoxy side chain. In
this example, the affinity at the β1-adrenoceptor was an order of
magnitude less than that of 1 and the 2-(4-
fluorophenethyloxy)ethoxy derivative (29b), yet this com-
pound displayed a similar efficacy profile.
It was clear from the antagonist studies that 1 and its
derivative 19 had substantial agonist activity at the human β-
adrenoceptors. Given this observation, we investigated the
potential clinical impact of this degree of partial agonism in a
fully conscious rat model. Infusion of the hydrochloride salt of
19 resulted in a substantial increase in the basal (β1-mediated)
heart rate, in keeping with its β1-partial agonist effects, followed
by inhibition of the β1-heart rate response to the isoprenaline
(compare Figure 2a in CHO β1 cells and Figure 3a in conscious
rats). No effect on basal hindquarters vascular conductance or
on the β2-mediated hindquarters response to isoprenaline was
observed, consistent with high β1-selectivity, thereby demon-
strating the β1-selective partial agonist effects in vivo. This
agonist effect is significant because β-ligands with agonist
properties (e.g., xamoterol) have been shown to be less
beneficial and, at times, even detrimental if used long-term in
cardiovascular disease.44,50,51 Thus, although 19 maintained its
β1-selectivity in vivo, its partial agonist properties were too
great for it to be pursued as a potential clinical compound.
Given 19 had reduced affinity and selectivity compared to 1,
we designed a series of analogues of the molecule looking for
evidence of structure−activity relationships related to affinity,
selectivity, and partial agonism. Substitution of the alkoxyalkoxy
side chain afforded ligands with increased β1-adrenoceptor
affinity (29a and 29b). Because all molecules remained partial
agonists at the human β1-adrenoceptor (Table 7), we therefore
examined the phenylurea portion of the molecule in an attempt
to attenuate this pharmacology. Generally, regardless of the
substituent, substitution in the meta- and para-positions of the
phenyl ring afforded higher β1-affinity (and therefore higher β1-
selectivity) than substitutions at the ortho-position. Once again,
however, these ligands retained their β1-selective partial agonist
activity, and the agonist actions were demonstrated to occur via
the catecholamine conformation.
In summary, 1 and the new family of ligands that we have
generated are β1-selective partial agonists. Compared with
currently published ligands, for example, xamoterol and ICI
89406, 1 and some of our novel analogues are the most β1-
selective agonists to date and will prove to be useful
pharmacological tools; however, the observed clinically
deleterious effects of significant partial agonism in β-block-
ers44,50,51 preclude any of these ligands from further clinical
development. However, this preliminary body of work clearly
demonstrates that it is possible to generate more β1-selective
molecules than those currently available and in clinical use.
Molecules of higher selectivity, but devoid of partial agonist
effects, should prove to be a very powerful treatment for people
with concomitant cardiovascular and respiratory disease and is
the focus of ongoing work within our laboratories.
Assessing the Site of Agonist Action at the Human β1-
Adrenoceptor. As the β1-adrenoceptor is now accepted to exist
in at least two agonist conformational states,45−48 it was
important to assess at which state of the β1-adrenoceptor these
novel β-adrenoceptor ligands were exerting their partial agonist
actions. CGP 20712A (neutral high-affinity β1-adrenoceptor
antagonist, capable of inhibiting both conformational states of
the β1-adrenoceptor but with different affinities)42 measured in
the presence of cimaterol yielded a high affinity value of log KD
= −9.48 (Supporting Information Table 1) compatible with the
agonist activity occurring via the primary catecholamine site of
the receptor. CGP 12177 (log EC50 = −7.91 0.04, 58.2
2.6% isoprenaline maximum, n = 5) was antagonized by CGP
20712A to yield a much lower affinity (log KD = −7.22 0.03,
Supporting Information Table 1), demonstrating that a far
higher concentration of CGP 20712A is required to block the
CGP 12177 agonist response. The agonist activity of CGP
12177 was therefore occurring via the secondary site of the β1-
adrenocptor (Supporting Information Figure 1).42 The agonist
response of our compounds was therefore inhibited by CGP
20712A to determine the site of their agonist actions. The
affinity values for CGP 20712A were all high (log KD values
between −9.51 and −9.09, Supporting Information Table 1).
suggesting that, like the partial agonist xamoterol and ICI
89406 (and unlike CGP 12177), our new partial agonists were
indeed achieving their agonist actions via the catecholamine
conformation of the receptor. The human β3-adrenoceptor,
similar to the human β1-adrenoceptor, has also been
demonstrated to exist in two conformational states.49 However,
given the low affinity and efficacy of our novel compounds for
the human β3-adrenoceptor, it was not possible to determine at
which receptor conformational state the β3-agonist responses
were occurring.
CONCLUSIONS
■
Despite 70 years of experience and the availability of many
adrenoceptor ligands, there remain very few truly selective
ligands for the β1-adrenoceptor. To date, CGP 20712A is a
widely known and truly selective β1-antagonist, whereas ICI
118551 is a β2-selective antagonist.18−20 However, although
several very highly β2-selective agonists exist (e.g., salmeterol
and formoterol), there are no selective β1-agonist compounds
− noradrenaline, dobutamine, xamoterol, and ICI 89406 all
having relatively poor selectivity.18−20 Here we have charac-
terized the pharmacological properties of 1 and some novel
derivatives and thus present the most selective β1-partial
agonists reported to date.
EXPERIMENTAL SECTION
■
General Chemistry Methods. Chemicals and solvents were
purchased from standard suppliers and used without further
purification. Merck Kieselgel 60, 230−400 mesh, for flash column
chromatography (FCC), was supplied by Merck KgaA (Darmstadt,
Germany), and deuterated solvents were purchased from Goss
International Limited (England) and Sigma-Aldrich Co. Ltd.
(England).
1 was confirmed as being a truly selective β1-adrenoceptor
ligand with a β1 over β2 selectivity of 562-fold as measured by
whole-cell binding. The first analogue of 1 made was 19, a
Unless otherwise stated, reactions were carried out at ambient
temperature. Reactions were monitored by thin layer chromatography
on commercially available precoated aluminum-backed plates (Merck
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Kieselgel 60 F254). Visualization was by examination under UV light
(254 and 366 nm). General staining was carried out with KMnO4 or
phosphomolybdic acid. A solution of ninhydrin (in ethanol) was used
to visualize primary and secondary amines. All organic extracts
collected after aqueous workup procedures were dried over anhydrous
MgSO4 or Na2SO4 before gravity filtration and evaporation to dryness.
Organic solvents were evaporated in vacuo at ≤40 °C (water bath
temperature). Purification using preparative layer chromatography
(PLC) was carried out using Fluka silica gel 60 PF254 containing
gypsum (200 mm × 200 mm × 1 mm). Flash chromatography was
performed using Merck Kieselgel 60 (0.040−0.063 mm).
stirred overnight in a solution of concd HCl (10 mL). The mixture was
concentrated under reduced pressure and the residue redissolved in
water (10 mL) before neutralization with aqueous 0.5 M NaOH. After
reconcentration, the residue was dissolved in the minimum amount of
MeOH and filtered (gravity), before purification by PLC (eluent 37%
aqueous NH3/MeOH/DCM 2:25:73). This gave 56 mg (73%) of a
brown semisolid: FT-IR 3339 (1° amine N−H, str), 3118 (br, O−H,
str), 1643 (urea CO, str), 1615 (1° amine N−H, bend), 1570 (aryl,
str), 836 (aryl C−H, bend, para-disubstituted ring); 1H NMR
(MeOD-d4) δ 7.17 (d, J = 8.7 Hz, 2H, aryl 3-H and 5-H), 6.73 (d,
J = 8.7 Hz, 2H, aryl 2-H and 6-H), 3.45 (t, J = 5.6 Hz, 2H,
CH2NH(CO)NH), 3.05 (t, J = 6.0 Hz, 2H, CH2NH2); 13C NMR
(MeOD-d4) δ 159.38 (4° aryl 4-C), 154.70 (CO), 132.02 (4° aryl 1-
C), 123.61, 116.32 (aryl CH), 41.73 (CH2NH2), 38.79 (CH2NH(C
O)NH); m/z HRMS (TOF ES+) C9H14N3O2 [MH]+ calcd 196.1081;
found 196.1081.
Melting points (mp) were recorded on a Reichert 7905 apparatus or
Perkin-Elmer Pyris 1 differential scanning calorimeter and were
uncorrected. Fourier transform infrared (FT-IR) spectra were
recorded as thin films or KBr disks in the range of 4000−500 cm−1
using an Avatar 360 Nicolet FT-IR spectrophotometer. Hig- resolution
mass spectra (HRMS)−time-of-flight electrospray (TOF ES ) were
recorded on a Waters 2795 separation module/micromass LCT
1-(Benzyloxy)-4-[2-(cyclopentyloxy)ethoxy]benzene (26a).
2-(Cyclopentyloxy)ethanol (21) (3.751 g, 28.81 mmol), triphenyl-
phosphine (9.448 g, 36.02 mmol, 1.25 equiv), and 4-(benzyloxy)-
phenol (14) (5.769 g, 28.81 mmol, 1 equiv) were dissolved in DCM
(70 mL). Di-tert-butyl azodicarboxylate (8.294 g, 36.02 mmol, 1.25
equiv) in DCM (20 mL) was added dropwise to the reaction mixture,
which was allowed to stir overnight. After removal of approximately
half of the solvent from the reaction mixture under reduced pressure,
the resulting slurry was diluted with hexanes (100 mL) and washed
with aqueous 1 M HCl (2 × 50 mL), aqueous 1 M NaOH (2 × 50
mL), water (2 × 50 mL), and brine (1 × 50 mL). The organic layer
was concentrated and redissolved in DCM (30 mL). On addition of
hexanes a precipitate of triphenylphosphine oxide began to form. The
flask was left in the freezer for 1 h before filtration of the precipitate
and washing with hexanes and Et2O. After concentration of the filtrate,
purification was achieved via FCC (eluent Et2O/hexanes 10:90) to
give 6.75 g (75%) of a clear colorless oil: FT-IR 2870, 2954 (alkyl C−
H, str), 1507 (aryl, str), 1109 (C−O−C, str), 824 (aryl C−H, bend,
1
platform. H NMR spectra were recorded on a Bruker-AV 400 at
400.13 MHz. 13C NMR spectra were recorded at 101.62 MHz.
Chemical shifts (δ) are recorded in parts per million (ppm) with
reference to the chemical shift of the deuterated solvent/an internal
tetramethylsilane (TMS) standard. Coupling constants (J) and
carbon−fluorine coupling constants (JCF) are recorded in hertz and
the significant multiplicities described by singlet (s), doublet (d),
triplet (t), quadruplet (q), broad (br), multiplet (m), doublet of
doublets (dd), and doublet of triplets (dt). Spectra were assigned using
appropriate COSY, distortionless enhanced polarization transfer
(DEPT), HSQC, and HMBC sequences. Unless otherwise stated, all
spectra were recorded in CDCl3.
Analytical reverse-phase high-performance liquid chromatography
(RP-HPLC) was performed on a Waters Millenium 995 LC using both
system 1 and system 2 described below and was used to confirm that
all final products were ≥95% pure.
System 1: Phenomenex Onyx Monolithic reverse phase C18 column
(100 × 4.6 mm), a flow rate of 3.00 mL/min and UV detection at 287
nm. Linear gradient 5−95% solvent B over 10 min. Solvent A, 0.1%
formic acid (FA) in water; solvent B, 0.1% FA in MeCN.
1
para-disubstituted ring), 738, 696 (aryl C−H bend, phenyl ring); H
NMR δ 7.34 − 7.48 (m, 5H, aromatic benzyl CH), 6.91, 6.96 (d, J =
9.2 Hz, 2 × 2H, aryl-dioxy ring), 5.04 (s, 2H, PhCH2O), 4.09 (t, J =
c
4.9 Hz, 2H, CH2OArOBn), 4.02−4.06 (m, 1H, Pe CH), 3.76 (t, J =
c
System 2: Waters symmetry reverse phase C18 column (75 × 4.6
mm), a flow rate of 1.00 mL/min, and UV detection at 287 nm. Linear
gradient 5−95% solvent B over 20 min. Solvent A, 0.1% FA in water;
solvent B, 0.1% FA in MeOH. Preparative HPLC was performed using
a Phenomenex Onyx Monolithic reverse phase C18 column (100 × 10
mm), a flow rate of 14.10 mL/min, and UV detection at 287 nm.
Samples were run in 5−95% solvent B over 10 min. Solvent A, 0.1%
FA in water; solvent B, 0.1% FA in MeCN. All retention times (Rt) are
quoted in minutes.
1-(2-Aminoethyl)-3-(4-benzyloxy)phenylurea (12). A solution
of 4-(benzyloxy) phenylisocyanate (3.739 g, 16.61 mmol) in
anhydrous DCM (30 mL) was dripped into a flask containing
vigorously stirred 1,2-ethanediamine (11) (6 mL, 89.80 mmol, 5.4
equiv) under nitrogen. Instant precipitation of a white solid was noted,
and the reaction was allowed to stir for a further 3 h after addition of
isocyanate solution was complete. After removal of all volatiles under
reduced pressure, the crude solid was washed with Et2O, before drying
to give 4.472 g (94%) of white solid: mp 147−149 °C; FT-IR 3300
(primary (1°) amine N−H, str), 2932, 2864 (alkyl C−H, str), 1642
(urea CO, str), 1604 (1° amine N−H, bend), 1111 (C−O, str), 830
(aryl C−H, bend, para-disubstituted ring), 741, 697 (aryl C−H, bend,
phenyl ring); 1H NMR (DMSO-d6) δ 8.48 (s, 1H, NHAr), 7.31−7.44
(m, 5H, aromatic benzyl CH), 7.28, 6.88 (d, J = 9.0 Hz, 2 × 2H, para-
disubstituted ring), 6.24 (t, J = 5.2 Hz, 1H, NHCONHAr), 5.02 (s,
2H, PhCH2O), 4.27 (br s, 2H, NH2), 3.10−3.17 (m, 2H, CH2NH),
2.67 (t, J = 6 Hz, CH2NH2); 13C NMR (DMSO-d6) δ 155.72 (CO),
152.88, 137.37, 133.97 (4 °C), 128.37, 127.71, 127.63 (benzyl CH),
119.29, 114.87 (aryl CH), 69.37 (benzyl CH2), 40.87 (CH2NH2),
40.38 (CHNH); m/z HRMS (TOF ES+) C16H20N3O2 [MH]+ calcd
286.1550; found 286.1547.
5.3 Hz, 2H, CH2CH2OArOBn), 1.73−1.84 (m, 6H, Pe CH2), 1.56−
c
1.63 (m, 2H, Pe CH2); 13C NMR δ 153.02, 153.26 (4 °C, aryl-dioxy
ring), 137.29 (4° benzyl C), 127.44, 127.83, 128.50 (benzyl CH),
115.62, 115.68 (CH aryl-dioxy ring), 81.89 (cPe CH), 70.55 (benzyl
CH2), 68.16 (CH2OArOBn), 67.28 (CH2CH2OArOBn), 32.27 (2-C
c
c
and 5-C Pe ring), 23.55 (3-C and 4-C Pe ring); m/z HRMS (TOF
ES+) C20H25O3 [MH]+ calcd 313.1798; found 313.1766.
4-(2-[Cyclopentyloxy]ethoxy)phenol (27a). 1-(2-
(Cyclopentyloxy)ethoxy)-4-(benzyloxy)benzene (26a) (6.326 g,
20.25 mmol) was hydrogenated according to the general procedure
for O-benzyl deprotection to give the title compound in quantitative
yield as a clear colorless oil: FT-IR 3381 (br, O−H, str), 2960, 2871
(alkyl C−H, str), 1510 (aryl, str), 1104 (C−O−C, str), 827 (aryl C−
1
H, bend, para-disubstituted ring); H NMR δ 7.60 (br s, 1H, OH),
6.69, 6.73 (d, J = 9.2 Hz, 2 × 2H, para-disubstituted phenol), 3.96−
c
3.99 (m, 3H, CH, CH2OAr), 3.70 (t, J = 5.0 Hz, 2H, PeOCH2),
1.62−1.78 (m, 6H, cPe CH2), 1.45−1.53 (m, 2H, cPe CH2); 13C NMR
δ 150.05, 152.01 (4 °C), 115.53, 115.88 (CH phenolic ring), 82.05
(cPe CH), 67.82 (CH2OAr), 67.11 (CH2CH2OAr), 32.87 (2-C and 5-
c
c
C Pe ring), 23.20 (3-C and 4-C Pe ring); m/z HRMS (TOF ES−)
C13H17O3 [M − H]− calcd 221.1183; found 221.1191.
2-{4-[2-(Cyclopentyloxy)ethoxy]phenoxymethyl}oxirane
(28a). NaH 60% suspension in mineral oil (863 mg, equivalent to 518
mg of NaH, 21.58 mmol, 1.1 equiv) was suspended in anhydrous
DMF (20 mL) with stirring under a nitrogen atmosphere. After 5 min,
4-(2-(cyclopentyloxy)ethoxy)phenol (27a) (4.360 g, 19.61 mmol) in
anhydrous DMF (20 mL) was added dropwise, with the vessel cooled
over an ice bath. This mixture was then allowed to stir at rt for 20 min
before addition of rac-epichlorohydrin (15.34 mL, 196.10 mmol, 10
equiv). The mixture was stirred for 7 h and then quenched cautiously
with MeOH. After removal of all volatiles under reduced pressure, the
crude residue was partitioned between water (30 mL) and Et2O (30
1-(2-Aminoethyl)-3-(4-hydroxy)phenylurea (13). 1-(2-Amino-
ethyl)-3-(4-(benzyloxy)phenyl) urea (12) (113 mg, 0.40 mmol) was
J
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Journal of Medicinal Chemistry
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mL) and the aqueous layer washed again with Et2O (3 × 30 mL). The
combined organic extracts were concentrated before purification
through a silica plug (initial wash with hexanes, followed by EtOH/
DCM 5:95) to give 4.558 g (84%) of a clear yellow oil: FT-IR 3052
(epoxide C−H, str, weak), 2961, 2870 (alkyl C−H, str), 1508 (aryl,
str), 1110 (C−O−C, str), 828 (aryl C−H, bend, para-disubstituted
to the bottom of the plate, and a sealant top was applied to the top of
the plate. The plates were left at room temperature overnight in the
dark and then counted on a Topcount at 21 °C for 2 min/well.
(3) 3H-cAMP Accumulation. Cells were grown to confluence in 24-
3
well plates. The cells were prelabeled with H-adenine by incubation
for 2 h with 2 μCi/mL 3H-adenine in serum-free medium (0.5 mL per
1
3
ring); H NMR δ 6.80 (s, 4H, aryl C−H), 4.11 (dd, J = 11.1/3.1 Hz,
well). The H-adenine was removed, and each well washed by the
1H, ArOCH2CH), 3.99 (t, J = 4.9 Hz, 2H, CH2OAr), 3.92−3.96 (m,
1H, cPe CH), 3.82 (dd, J = 11.1/5.7, 1H, ArOCH2CH), 3.67 (t, J = 5.3
Hz, 2H, cPeOCH2), 3.25−3.29 (m, 1H, epoxide CH), 2.82 (d, J = 4.9/
4.9 Hz, 1H, epoxide CH2), 2.67 (dd, J = 5.0/2.7 Hz, 1H, epoxide
CH2), 1.58 − 1.77 (m, 6H, cPe CH2), 1.41 − 1.56 (m, 2H, cPe CH2);
13C NMR δ 152.65, 153.36 (4 °C), 115.46, 115.53 (aryl CH), 69.35
(ArOCH2CH), 68.08 (CH2OAr), 67.18 (CH2CH2OAr), 50.13
addition and removal of 1 mL of serum-free medium. One milliliter of
serum-free medium containing 1 mM IBMX was added to each well,
and the cells were incubated for 15 min. When used, antagonists were
added at a final concentration at this stage and thus had 15 min of
incubation. Agonist (in 10 μL of serum-free medium) was added to
each well, and the plates were incubated for 5 h. The reaction was
terminated by the addition of 50 μL of concentrated HCl per well. The
plates were then frozen and thawed, and 3H-cAMP was separated from
other 3H-nucleotides by sequential Dowex and alumina column
chromatography, as previously described.52
c
(epoxide CH), 44.49 (epoxide CH2), 32.17 (2-C and 5-C Pe ring),
c
23.45 (3-C and 4-C Pe ring); m/z HRMS (TOF ES+) C16H22NaO4
[MNa]+ calcd 301.1410; found 301.1414.
As measurements of partial agonism depend on many factors,
including particularly the receptor expression level in the cells being
examined, all cAMP experiments were performed in cells at the same
passage throughout this entire study. In addition, whenever possible,
experiments were performed with all ligands being investigated in
parallel studies on the same day. Thus, every ligand was examined on
the same split of cells on the same day, with each separate n number
being performed on different days.
(C) Pharmacological Data Analysis. (1) Whole Cell Binding. All
data points on each binding curve were performed in triplicate, and
each 96-well plate also contained three to six determinations of total
and nonspecific binding. Nonspecific binding was determined in the
presence of 10 μM propranolol.
1-{2-[(3-{4-[2-(Cyclopentyloxy)ethoxy]phenoxy}-2-
hydroxypropyl)amino]ethyl}-3-(4-hydroxyphenyl)urea (29a).
2-((4-(2-(Cyclopentyloxy)ethoxy)phenoxy)methyl)oxirane (28a) was
opened with 1-(2-aminoethyl)-3-(4-hydroxyphenyl)urea (13) as
described in the general procedure for aminolysis of oxiranes (cf.
Supporting Information). Yield: 29%, white amorphous solid; mp
135−138 °C; FT-IR 3311 (br, O−H, str), 2929, 2869 (alkyl C−H,
str), 1636 (urea CO, str), 1509, 1568 (aryl, str), 1111 (C−O−C,
str), 820 (aryl C−H, bend, para-disubstituted ring); 1H NMR
(DMSO-d6) δ 8.91 (br s, 1H, phenol), 8.20 (s, 1H, NH(C
O)NHAr), 7.13 (d, J = 8.8 Hz, 2H, aryl C−H ortho to urea), 6.84 (s,
4H, aryl-dioxy ring), 6.62 (d, J = 8.8 Hz, 2H, aryl C−H ortho to
phenol), 6.02 (t, J = 5.4 Hz, 1H, NH(CO)NHAr), 4.96 (br s, 1H,
NH), 3.79−3.97 (m, 6H, CH2OAr, cPe CH, CH(OH), ArOCH2), 3.61
(t, J = 4.8 Hz, 2H, cPeOCH2), 3.12−3.16 (m, 2H, NHCH2CH2), 2.56
− 2.71 (m, 4H, CH(OH)CH2NH, NHCH2CH2), 1.54−1.69 (m, 6H,
A one-site sigmoidal binding curve (eq 1) was then fitted to the data
using Graphpad Prism 2.01, and the IC50 was then determined as the
concentration required to inhibit 50% of the specific binding.
c
cPe CH2), 1.42−1.51 (m, 2H, Pe CH2); 13C NMR (DMSO-d6) δ
(100A)
(A + IC50
152.74, 152.49, 151.85, 132.14 (aryl 4 °C), 155.65 (CO), 119.72
(aryl C−H ortho to urea), 115.33 (CH aryl-dioxy ring), 115.04 (aryl
C−H ortho to phenol), 80.84 (cPe CH), 71.23 (ArOCH2), 68.08
(CH(OH)), 67.71 (CH2OAr), 66.72 (cPeOCH2), 52.20 (CH(OH)
CH2NH), 49.38 (NHCH2CH2), 39.15 (NHCH2CH2), 31.78 (2-C and
% uninhibited binding = 100 −
+ NS
)
(1)
In eq 1, A is the concentration of the competing ligand, IC50 is the
3
concentration at which half of the specific binding of H-CGP 12177
c
5-C cPe ring), 23.12 (3-C and 4-C Pe ring); m/z HRMS (TOF ES+)
has been inhibited, and NS is the nonspecific binding.
C25H36N3O6 [MH]+ calcd 474.2599; found 474.2578; RP-HPLC Rt
3.17 (system 1), 10.30 (system 3).
From the IC50 value and the known concentration of radioligand
[3H-CGP 12177], a KD (concentration at which half the receptors are
bound by the competing ligand) value was calculated using eq 2:
General Pharmacology Methods. (A) Pharmacology Materials.
Fetal calf serum was from PAA Laboratories (Teddington, Middlesex,
UK). White-sided view plates were supplied by Thermo Fisher
Scientific (Basingstoke, UK). Microscint 20 scintillation fluid and
Ultima Gold XR scintillation fluid were from PerkinElmer. Radio-
ligands (3H-CGP 12177, 3H-adenine, and 14C-cAMP) were from
Amersham International (Buckinghamshire, UK). ICI 89406, cimater-
ol, and CGP 20712A were from Tocris Life Sciences (Avonmouth,
UK). All other reagents were from Sigma Chemicals (Poole, Dorset,
UK).
IC50
KD
=
1 + ([3H‐CGP 12177]/KD 3H‐CGP 12177)
(2)
(2) 3H-cAMP Accumulation. Agonist responses were best described
by a one-site sigmoidal concentration response curve (eq 3)
Emax × [A]
response =
EC50 + [A]
(3)
(B) In Vitro Experiments. (1) Cell Culture. CHO-K1 stably
expressing either the human β1 (1146fmol/mg protein), the human
β2 (466fmol/mg protein), or the human β3-adrenoceptor (790fmol/
mg protein) was used throughout this study.20 Cells were grown in
Dulbecco’s modified Eagle’s medium nutrient mix F12 (DMEM/F12)
containing 10% fetal calf serum and 2 mM L-glutamine in a 37 °C
humidified 5% CO2/95% air atmosphere.
where Emax is the maximum response, [A] is the agonist concentration,
and EC50 is the concentration of agonist that produces 50% of the
maximal response
As several ligands have too low affinity at the human β2 and β3
receptors to assess accurately in the whole cell binding assay, the
affinity (log KD value) of these antagonists was calculated in functional
assays from the shift of the agonist concentration responses in the
presence of a fixed concentration of antagonist using eq 4
(2) 3H-CGP 12177 Whole Cell Binding. Cells were grown to
3
confluence in white-sided, flat-bottom 96-well view plates. H-CGP
12177 whole cell binding was performed as described.20 Briefly, the
medium was removed from each well, and 100 μL of serum-free
medium containing the competing ligand at twice the final required
concentration was added to each well. A fixed concentration of 100 μL
3H-CGP 12177 was then immediately added to each well (1:2 dilution
in well), and the cells were incubated for 2 h at 37 °C, 5% CO2. The
cells were washed twice by the addition and removal of 200 μL of 4 °C
phosphate-buffered saline per well. One hundred microliters of
Microscint 20 was added to each well, a white sticky base was applied
[B]
KD
DR = 1 +
(4)
where DR (dose ratio) is the ratio of the agonist concentration
required to stimulate an identical response in the presence and absence
of a fixed concentration of antagonist [B].
Several of the compounds, however, displaced clear partial agonist
activities; that is, they antagonized the more efficacious agonist
cimaterol while stimulating an agonist response of their own. When
K
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Journal of Medicinal Chemistry
Article
clear partial agonism was seen, the affinity (log KD) was calculated
(4) In Vivo Data Analysis. Data were analyzed offline using IDEEQ
software. Responses to isoprenaline were measured as the difference
between steady-state values immediately before the isoprenaline
infusion and during the third minute of infusion. Changes in baseline
were measured as the difference between the control values for the last
dose of isoprenaline before, and the first dose of isoprenaline after,
saline or 19 administration. Data were analyzed by t test or ANOVA
with Bonferroni correction as appropriate; P < 0.05 was taken as
significant (GraphPad Prism version 5.02).
according to the method of Stephenson using eq 5:43
[A2] − [A1]
Y × [P]
1 − Y
KD partial agonist =
where Y =
[A3]
(5)
In eq 5 [P] is the concentration of the partial agonist, [A1] is the
concentration of the agonist at the point where the partial agonist
alone causes the same response, [A2] is the concentration of agonist
causing a given response above that achieved by the partial agonist,
and [A3] is the concentration of the agonist in the presence the partial
agonist causing the same stimulation as [A2].
Isoprenaline (10 μM) was included in all experiments, and therefore
all maximal responses are expressed as a percentage of this maximum.
(D) In Vivo Experiments. (1) Animals and Surgery. Adult male
Sprague−Dawley rats (Charles River, Margate, Kent, UK), weighing
300−350 g, were housed in groups in a temperature-controlled (21−
23 °C) environment with a 12 h light−dark cycle (lights on at 6:00
a.m.) and free access to food (Teklad Global 18% Protein Rodent
Diet, Bicester, Oxon, U.K.) and water for at least 7 days after arrival
from the supplier before any surgical intervention.
ASSOCIATED CONTENT
■
S
* Supporting Information
Full experimental procedures and compound characterization
data for compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
Surgery was performed in two stages under general anesthesia
(fentanyl and medetomidine, 300 μg/kg of each ip, supplemented as
required), with reversal of anesthesia and postoperative analgesia
provided by atipamezole (1 mg/kg sc) and buprenorphine (0.02 mg/
kg sc). At the first surgical stage, a miniature pulsed Doppler flow
probe was sutured around the distal abdominal aorta to monitor
hindquarters hemodynamics. The wires from the probe were taped
and sutured at the nape of the neck, and the animals were returned to
the holding room. At the second surgical stage, which took place at
least 10 days after the surgery for probe implantation, and following a
satisfactory inspection from the Named Veterinary Surgeon, catheters
were implanted in the distal abdominal aorta via the caudal artery (for
arterial blood pressure (BP) monitoring and the derivation of heart
rate (HR)) and in the right jugular vein (for drug administration).
Three separate intravenous catheters were placed in the jugular vein to
enable concurrent administration of different substances. At this stage,
the wires from the probe were soldered into a miniature plug
(Microtech Inc., Boothwyn, PA, USA), which was mounted onto a
custom-designed harness worn by the rat. The catheters emerged from
the same point as the probe wires and were fed through a protective
spring secured to the harness and attached to a counterbalanced pivot
system. The arterial catheter was connected to a fluid-filled swivel for
overnight infusion of heparinized (15 units/mL) saline to maintain
patency.
*(B.K.) Phone: +44-115-9513026. Fax: +44-115-9513412. E-
mail: barrie.kellam@nottingham.ac.uk. (J.G.B.) Phone: +44-
115-8230085. Fax: +44-115-8230081. E-mail: jillian.baker@
nottingham.ac.uk.
Author Contributions
∥S.N.M. and J.G.B. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the BBSRC and the University of
Nottingham. J.G.B. is a Wellcome Trust Clinician Scientist
(Grant 073377/Z/.3/Z). We thank June McCulloch for
technical assistance in running the cAMP chromatography
columns and Philip Kemp and Julie March for technical
assistance in running the in vivo experiments.
ABBREVIATIONS USED
■
aq, aqueous solution; β-AR, beta-adrenoceptor; Bn, benzyl;
Boc, tert-butoxycarbonyl; Bz, benzoyl; calcd, calculated; cAMP,
cyclic adenosine monophosphate; CAN, ceric(IV) ammonium
nitrate; CGP 20712A, 2-hydroxy-5-(2-[{hydroxy-3-(4-[1-meth-
yl-4-trifluoromethyl-2-imidazolyl]phenoxy)propyl}amino]-
ethoxy)benzamide; CHO, Chinese hamster ovary; concd,
concentrated; COPD, chronic obstructive pulmonary disease;
cPr, cyclopropyl; CRE-SPAP, cAMP response element-secreted
placental alkaline phosphatase; CV, column volume; DCM,
dichloromethane; DIAD, diisopropyl azodicarboxylate; DMF,
N,N-dimethylformamide; FCC, flash column chromatography;
FSK, forskolin; GPCR, G protein-coupled receptor; ICI 118551
, (−)-1-(2,3-[dihydro-7-methyl-1H-inden-4-yl]oxy)-3-([1-
methylethyl]-amino)-2-butanol; LABA, long-acting β-2 agonist;
lit., literature; LK 204-545, 1-(2-(3-(2-cyano-4-(2-
(cyclopropylmethoxy)ethoxy)phenoxy)-2-hydroxypropyl-
amino)ethyl)-3-(4-hydroxyphenyl)urea; m-CPBA, meta-chlor-
operbenzoic acid; mp, melting point; MW, microwave; PE,
petroleum ether 40−60; Phth, phthalimide; PLC, preparative
layer chromatography; PMB, para-methoxybenzyl; rac, racemic;
RP-HPLC, reverse phase high-performance liquid chromatog-
raphy; rt, room temperature; SAR, structure−activity relation-
ship; TEA, triethylamine
Experiments began 24 h after surgery for catheter implantation, with
animals fully conscious and unrestrained in home cages, with free
access to food and water. All procedures were carried out with
approval of the University of Nottingham Local Ethical Review
Committee, under Home Office Project and Personal License
Authority.
(2) Cardiovascular Recordings. Cardiovascular variables were
recorded using a customized, computer-based system (Instrument
Development Engineering Evaluation (IDEEQ), Maastrich Instru-
ments Bv, The Netherlands) connected to a transducer amplifier
(Gould, USA; model 13-4615-50) and a Doppler flowmeter (Crystal
Biotech (Holliston, USA) VF-1 mainframe (pulse repetition frequency
= 125 kHz) fitted with high-velocity (HVPD-20) modules). Raw data
were sampled by IDEEQ every 2 ms, averaged, and stored to disc
every cardiac cycle. Hindquarters vascular conductance (HVC)
changes were calculated from the changes in BP and Doppler shift.
(3) Experimental Protocol. In all experiments, atropine methyl
nitrate (1 mg/kg/h; 0.4 mL/h) was infused continuously to remove
any parasympathetic influence on the control of heart rate. Starting 2 h
after the onset of the atropine infusion, rats were given 3 min infusions
(0.15 mL/min) of isoprenaline (12, 40, and 120 ng/kg/min) in
ascending order separated by at least 20 min. At least 45 min after the
last infusion of isoprenaline, saline or 19 was given as an iv bolus (0.1
mL) maintained by continuous infusion (0.4 mL/h), and the
isoprenaline infusions were repeated, starting 30 min thereafter.
L
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Journal of Medicinal Chemistry
Article
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