Synthesis and characterization of high-affinity 4,4-difluoro-4-bora-3a,4a- diaza-s-indacene-labeled fluorescent ligands for human β-adrenoceptors

Article abstract of DOI:10.1021/jm2008562

The growing practice of exploiting noninvasive fluorescence-based techniques to study G protein-coupled receptor pharmacology at the single cell and single molecule level demands the availability of high-quality fluorescent ligands. To this end, this study evaluated a new series of red-emitting ligands for the human β-adrenoceptor family. Upon the basis of the orthosteric ligands propranolol, alprenolol, and pindolol, the synthesized linker-modified congeners were coupled to the commercially available fluorophore BODIPY 630/650-X. This yielded high-affinity β-adrenoceptor fluorescent ligands for both the propranolol and alprenolol derivatives; however, the pindolol-based products displayed lower affinity. A fluorescent diethylene glycol linked propranolol derivative (18a) had the highest affinity (log K_D of -9.53 and -8.46 as an antagonist of functional β2- and β1-mediated responses, respectively). Imaging studies with this compound further confirmed that it can be employed to selectively label the human β2-adrenoceptor in single living cells, with receptor-associated binding prevented by preincubation with the nonfluorescent β2-selective antagonist 3-(isopropylamino)-1-[(7- methyl-4-indanyl)oxy]butan-2-ol (ICI 118551) (J. Cardiovasc. Pharmacol.1983, 5, 430-437.)

Full text of DOI:10.1021/jm2008562

ARTICLE
pubs.acs.org/jmc
Synthesis and Characterization of High-Affinity 4,
4-Difluoro-4-bora-3a,4a-diaza-s-indacene-Labeled Fluorescent
Ligands for Human β-Adrenoceptors
||
||
Jillian G. Baker,‡, Luke A. Adams,†, Karolina Salchow,‡ Shailesh N. Mistry,† Richard J. Middleton,†,§
Stephen J. Hill,*,‡ and Barrie Kellam*,†
†School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD,
United Kingdom
‡Institute of Cell Signalling, Medical School, University of Nottingham, Nottingham NG7 2UH, United Kingdom
§CellAura Technologies Ltd., BioCity, Pennyfoot Street, Nottingham NG1 1GF, United Kingdom
S Supporting Information
b
ABSTRACT: The growing practice of exploiting noninvasive fluor-
escence-based techniques to study G protein-coupled receptor
pharmacology at the single cell and single molecule level demands
the availability of high-quality fluorescent ligands. To this end, this
study evaluated a new series of red-emitting ligands for the human
β-adrenoceptor family. Upon the basis of the orthosteric ligands
propranolol, alprenolol, and pindolol, the synthesized linker-mod-
ified congeners were coupled to the commercially available fluor-
ophore BODIPY 630/650-X. This yielded high-affinity β-
adrenoceptor fluorescent ligands for both the propranolol and
alprenolol derivatives; however, the pindolol-based products dis-
played lower affinity. A fluorescent diethylene glycol linked propra-
nolol derivative (18a) had the highest affinity (log KD of ꢀ9.53 and
ꢀ8.46 as an antagonist of functional β2- and β1-mediated responses, respectively). Imaging studies with this compound further
confirmed that it can be employed to selectively label the human β2-adrenoceptor in single living cells, with receptor-associated
binding prevented by preincubation with the nonfluorescent β2-selective antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]-
butan-2-ol (ICI 118551) (J. Cardiovasc. Pharmacol. 1983, 5, 430ꢀ437.)
’ INTRODUCTION
data generated represents an average for the entire cell popula-
tion. Fluorescent techniques, however, offer a wider spectrum of
more detailed pharmacological investigations to be undertaken,
especially the quantification and visualization of specific ligandꢀ
receptor complexes in intact cells.7
G-protein coupled receptors (GPCRs) remain a prime target
for potential disease modulation, and therefore, research focused
upon them persists as a significant component of the modern day
drug discovery arena.2 The β-adrenoceptors (β-AR) represent
just one subset of this broad receptor family and are themselves
further classified into three specific subtypes, β1-, β2-, and β3-
AR.3 For several decades these subtypes have been the molecular
target for numerous drug discovery projects, and the drugs
resulting from these endeavors have established themselves as
key therapeutic agents in the treatment of numerous chronic
cardiovascular and respiratory diseases.4 More recently, their
potential involvement in metastatic cancer progression has
become apparent and rekindled further the therapeutic impor-
tance of this receptor class.5,6 Our current understanding of the
molecular pharmacology of the β-AR has been derived primarily
from the use of radioligand binding techniques for the elucida-
tion of ligandꢀreceptor interactions and the quantification of
intracellular second messenger generation. As such, this type of
experiment requires the use of large numbers of cells, and the
The recent discovery that GPCRs can regulate signaling path-
ways that are independent of G proteins has led to a realization
that different signal transduction pathways can be selectively
stimulated by agonists acting at a common cell surface receptor.8ꢀ11
GPCRs are not uniformly distributed at the cell surface but
instead are organized within membrane compartments and
microdomains,12,13 providing a mechanism by which intracellular
signaling can be orchestrated in different areas of an individual
cell and behave differently in specific cell types.14 This new
mechanistic information has focused attention on the need to
develop fluorescent ligands to study the spatial and temporal
aspects of ligandꢀreceptor interactions.15,16 With their established
Received: July 1, 2011
Published: August 29, 2011
r
2011 American Chemical Society
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Figure 1. Previously reported fluorescent β-AR ligands.
clinical and therapeutic importance,17 it is therefore not surpris-
ing that β-AR have been investigated within this area of pharma-
cological imaging.
(BODIPY TMRꢀCGP 12177) (7) has also been reported and
shown to be a long-acting β2-AR partial agonist capable of labeling
β2-AR in the plasma membrane of living cells.32 More recently, the
first description of a fluorescently labeled arterenol derivative
(Alexa-NA) (8) and its use in fluorescence correlation spectroscopy
(FCS) based studies to investigate β2-AR in living cells was
published.33 In addition, a europium-chelated fluorescent deri-
vative of pindolol for use in time-resolved receptorꢀligand
binding assays has also been described, but its full structure was
not revealed.34
Within this rapidly evolving arena of pharmacological imaging,
we have previously reported the synthesis of red-shifted BOD-
IPY-labeled fluorescent adenosine receptor agonists16,35,36,38 and
antagonists37,38 and demonstrated their utility in measuring the
diffusion of receptor species within defined membrane micro-
domains using the technique of fluorescence correlation spec-
troscopy (FCS).39 Avoiding the issues of autofluorescence, high-
lighted as being severely problematic with many of the derivatives
discussed above, was one of our primary objectives in designing
pharmacologically useful fluorescent ligands. Clearly, fluorescent
ligands that emit light in the red region of the electromagnetic
spectrum meet this requirement and, in addition, often possess
excellent photophysical properties suited for techniques such as
LSCM and FCS. With the previously described fluorescent β-AR
ligands either not meeting these requirements for fluorescence
microscopy or, as in the case of BODIPY TMRꢀCGP 12177, no
longer commercially available, we were keen to generate a new
suite of red-shifted fluorescent β-AR ligands for use in LSCM-
and FCS-based experiments. Three families of fluorescent ligands
based on propranolol, alprenolol, and pindolol were synthesized,
To-date, however, only a handful of reports have been
published detailing limited success in the synthesis of fluorescent
β-AR ligands (Figure 1). The earliest studies detailed the use of
fluorescent propranolol derivatives 9-aminoacridinylproprano-
lol (9-AAP) (1)18ꢀ22 and dansyl-propanolol (DAPN) (2)23 to
investigate receptor mapping and clustering. Following this,
synthesis of an NBDꢀalprenolol derivative (3) was reported
and utilized in further clustering studies.24 However, it was later
demonstrated that 3 and NBDꢀpindolol (4) were in fact both
nonselective and noncompetitive.25,26 Additionally, two reports
also detailed that during their use of 1 and 2 they were unable to
distinguish between the binding of these ligands to cellular
receptors and autofluorescent granules, concluding that the
fluorescent output from these ligands was therefore of too low
an intensity to be detected by fluorescence microscopy.27,28
A decade later, a significant advance was achieved through the
synthesis of bordifluoropyrromethene- (BODIPY) (5) and
fluorescein-labeled (6) β-AR ligands.29 On the basis of the
hydrophilic β-AR ligand 4-[3-[(1,1-dimethylethyl)amino]-2-hy-
droxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one (CGP 12177),30
it was demonstrated that the BODIPY conjugate (5) displayed
similar binding characteristics when compared with the standard
ligand. The fluorescein conjugate’s (6) receptor-specific signal
was not sufficiently strong enough for subsequent pharmacolo-
gical measurement and has indeed since been demonstrated
to suffer from significant photobleaching during fluores-
cence microscopy experiments.31 A variant of CGP 12177
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Scheme 1. Synthetic Route to BODIPY 630/650-X-Labeled Propranolol Derivativesa
a Reagents and conditions: (i) for 10a, (2S)-glycidyl-3-nitrobenzenesulfonate, NaH, DMF, 56%; for 10b, epichlorohydrin, K2CO3, 2-butanone, Δ.
(ii) 11aꢀc, DMF/H2O (9:1), 85 °C, 16 h, 35ꢀ70%. (iii) Pd/C, H2, MeOH, rt, 3 h, quantitative. (iv) BODIPY-X-630/650-OSu, DMF, rt, 4 h. (v) Benzyl
2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (15), DMF/H2O (9:1), 85 °C, 16 h, 35ꢀ67%.
characterized, and pharmacologically assessed across the human
forms of the β1-, β2-, and β3-ARs. Our choices of β-AR ligands
were based primarily upon synthetic tractability, a desire to
improve upon those ligands previously described, and to address
three key questions surrounding ligand design: (i) Was the effect
of linker length and composition similar to what we had observed
for the corresponding adenosine ligands?36 (ii) With structural
extension of the pharmacophore to include both a linker and
fluorophore, did the stereochemistry of the crucial carbinol center
exert the same effect on affinity as that previously established for
β-AR antagonists? (iii) Could the best fluorophoreꢀlinker combi-
nation from the initial “propranolol” library be transposed onto
alternative orthosteric head-groups (alprenolol and pindolol) without
deleterious effects on pharmacology.
chemoselective acylation of the terminal primary amine with
commercially available 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-
3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoic acid
succinimidyl ester (BODIPY 630/650-X, SE; Invitrogen).
Aminolysis of the fluorophore active ester proceeded smoothly
and the fluorescently labeled ligands 14aꢀf, 18a, and 18b were
isolated and purified using preparative thin-layer chromatogra-
phy (PTLC); their purity was confirmed using RP-HPLC with
photodiode array detection between 190 and 800 nm. Evaluation
of these ligands highlighted that linker length exerted minimal
overall influence on receptor affinity, and for expeditious reasons,
the alprenolol and pindolol conjugates were therefore synthe-
sized using only the ethylamino (short hydrocarbon) and
ethoxyethoxyethylamino (long polyether) linker variants.
The S-enantiomer (23a and 26a) and racemic (23b and 26b)
alprenolol-BODIPY fluorescent ligands were therefore synthe-
sized using an identical strategy to that of the propranolol
derivatives, but starting from commercially available 2-allylphe-
nol and replacing the Cbz-protected diamines with their tert-
butoxycarbonyl (Boc) equivalents (Scheme 2).
Finally, having also established with these initial two series that
the (S)-enantiomer exerted the expected shift in log KD values,
we generated the final pindolol-based molecules in their
inexpensive racemic form. Therefore, (()-4-[(oxiran-2-yloxy)-
methyl]-1H-indole (28) was ring-opened using either 11a or 15
to afford the Cbz-protected carbinolamine congeners 29 and 32,
respectively. Cbz hydrogenolysis and acylation of the relin-
quished amines with BODIPY 630/650-X, SE ultimately
’ RESULTS AND DISCUSSION
Synthesis. We initially embarked upon the synthesis of a
focused library of fluorophore-conjugated single enantiomer and
racemic propranolol derivatives (Scheme 1). Commercially avail-
able 1-napthol (9) was therefore alkylated with either (2S)-glycidyl-
3-nitrobenzenesulfonate or racemic epichlorohydrin as previously
described to afford epoxides 10a and 10b, respectively.40
Nucleophilic ring-opening of the epoxides was effected using a
range of appropriately mono-Cbz-protected diamines (11aꢀc
and 15) to afford carbinolamines 12aꢀf, 16a, and 16b. Subse-
quent hydrogenolysis of the Cbz protecting groups afforded the
linker-modified congeners 13aꢀf, 17a, and 17b primed for
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Scheme 2. Synthetic Route to BODIPY 630/650-X-Labeled Alprenolol Derivativesa
a Reagents and conditions: (i) for 20a, (2S)-glycidyl-3-nitrobenzenesulfonate, NaH, DMF, 56%; for 20b, epichlorohydrin, K2CO3, 2-butanone, Δ, 54%.
(ii) tert-Butyl 2-aminoethylcarbamate, DMF/H2O (9:1), 85 °C, 16 h, 35ꢀ70%. (iii) 2 M HCl in dioxane, 89ꢀ94%. (iv) BODIPY-X-630/650-OSu,
DMF, rt, 4 h, 68ꢀ98%. (v) tert-Butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate, DMF/H2O (9:1), 85 °C, 16 h, 77%.
afforded the racemic fluorescent pindolol derivatives 31 and
33 (Scheme 3).
KD = ꢀ7.76) and β3-AR (log KD = ꢀ7.09). The equivalent
molecule from the alprenolol series 26a also displayed high
affinity for the β1-AR (log KD = ꢀ7.65) and β2-AR (log KD =
ꢀ9.03; Figure 3). However, 34, from the pindolol series, was an
order of magnitude less potent compared to its parent molecule
(Table 1).
It was notable that the addition of fluorophore and linker did
not markedly change the affinities of the parent compounds for
the β3-AR. In the case of both propranolol and alprenolol, the
C-2 linker derivatives had the highest affinity for this particular
receptor (Table 1).
Functional Reporter Gene Studies. To gain some insight into
the ability of these ligands to antagonize agonist-stimulated
responses at each receptor, we evaluated their ability to attenuate
functional activity in the CHO cells as they also expressed the
target receptor and a six cyclic AMP response element (6 ꢁ
CRE) reporter gene driving the expression of a human-secreted
placental alkaline phosphatase reporter gene.41,42 Agonist re-
sponses were elicited by cimaterol (β1, site 1), salbutamol (β2),
and fenoterol (β3). For the β1-AR site 2, agonist responses were
elicited by CGP 12177. All of the propranolol derivatives were
potent competitive antagonists of this response, with 18a once
again displaying the highest affinity (Table 2; Figure 4).
The alprenolol and pindolol series showed partial agonist
activity (Figure 5; Table 3) on all three β-AR subtypes. These
Pharmacology. 3H-CGP 12177 Whole Cell Binding Studies.
Measurement of 3H-CGP 12177 binding to CHO cells expres-
sing the human β1-, β2-, or β3-AR indicated that all compounds
displayed the highest affinity for the human β2-AR (Table 1).
The lowest affinity compounds were the pindolol derivatives 31
and 34. Increasing the linker chain length from C-2 to C-8 for the
propranolol series caused a modest increase in affinity from C-2
(14a, 14d) to C4 (14b, 14e) but then a 10-fold reduction in
affinity with a further doubling in linker chain length to C-8 (14c,
14f) (Table 1). It was notable that the C-8 derivative 14f did not
completely displace the specific binding of 3H-CGP 12177 to the
human β1-AR (maximal displacement of specific binding =65.9 (
2.0%; Table 1; Figure 2a).
This was seen to a lesser extent with the pure S-enantiomer
14c (85.3 ( 2.4% maximal inhibition; Figure 2a), suggesting that
the R-enantiomer may be contributing an allosteric influence on
the orthosteric binding site. It was also striking that 14f did not
produce an effect to the same degree in cells expressing the
human β2-AR (Figure 2b).
Substitution of a polyethylene glycol linker in place of the C-8
hydrocarbon chain in 14c provided the highest affinity ligand 18a
at the human β2-AR (log KD = ꢀ9.21; Table 1). This compound
also had lower but respectable affinities at the human β1-AR (log
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Scheme 3. Synthetic Route to BODIPY 630/650-X-Labeled Pindolol Derivativesa
a Reagents and conditions: (i) epichlorohydrin, Cs2CO3, microwave (MW) 120 °C, 30 min, 97%. (ii) 11a, DIPEA, iPrOH/MeCN/H2O (7:2:1), MW
90 °C, 60 min, 48%. (iii) Pd/C, H2, MeOH, rt, 3 h, 88%. (iv) BODIPY-X-630/650-OSu, DMF, rt, 4 h. (v) Benzyl 2-(2-(2-aminoethoxy)-
ethoxy)ethylcarbamate (15), iPrOH/MeCN/H2O (7:2:1), MW 90 °C, 60 min, 40%.
Table 1. Ligand-Binding Parameters for Fluorescent Antagonists Acting at the Human β-ARa
chirality
linkerb
β1 log KD
n
β2 log KD
n
β3 log KD
n
propranolol
14a
ꢀ8.22 ( 0.04
ꢀ7.44 ( 0.05
ꢀ7.48 ( 0.07
ꢀ6.73 ( 0.09c
ꢀ7.05 ( 0.07
ꢀ7.25 ( 0.07
ꢀ6.72 ( 0.05d
ꢀ7.76 ( 0.06
ꢀ7.14 ( 0.08
ꢀ7.95 ( 0.03
ꢀ7.04 ( 0.05
ꢀ7.26 ( 0.07
ꢀ7.65 ( 0.07
ꢀ7.50 ( 0.05
ꢀ8.58 ( 0.04
ꢀ6.06 ( 0.07
ꢀ7.01 ( 0.09
9
ꢀ9.22 ( 0.03
ꢀ8.86 ( 0.03
ꢀ9.14 ( 0.06
ꢀ8.15 ( 0.06
ꢀ8.56 ( 0.04
ꢀ8.87 ( 0.04
ꢀ8.24 ( 0.05
ꢀ9.21 ( 0.06
ꢀ8.48 ( 0.06
ꢀ9.30 ( 0.02
ꢀ8.54 ( 0.07
ꢀ8.63 ( 0.05
ꢀ9.03 ( 0.06
ꢀ8.91 ( 0.06
ꢀ9.27 ( 0.07
ꢀ7.33 ( 0.09
ꢀ7.96 ( 0.10
10
8
ꢀ6.67 ( 0.10
ꢀ7.35 ( 0.06
ꢀ6.91 ( 0.08
ꢀ7.17 ( 0.05
ꢀ7.03 ( 0.07
ꢀ6.98 ( 0.13
ꢀ7.11 ( 0.09
ꢀ7.09 ( 0.08
ꢀ6.53 ( 0.07
ꢀ6.86 ( 0.08
ꢀ7.16 ( 0.09
ꢀ7.33 ( 0.12
ꢀ7.00 ( 0.06
ꢀ7.09 ( 0.04
ꢀ6.78 ( 0.10
ꢀ6.48 ( 0.06
ꢀ6.42 ( 0.12
4
S-enantiomer
S-enantiomer
S-enantiomer
racemate
C-2
8
9
4
7
14b
C-4
9
14c
C-8
9
9
4
14d
C-2
6
6
6
14e
racemate
C-4
6
6
6
14f
racemate
C-8
6
6
6
18a
S-enantiomer
racemate
PEG-8
PEG-8
8
8
4
18b
6
6
6
alprenolol
23a
6
6
6
S-enantiomer
racemate
C-2
7
7
7
23b
C-2
6
6
6
26a
S-enantiomer
racemate
PEG-8
PEG-8
6
6
5
26b
12
7
12
7
10
7
pindolol
31
racemate
racemate
C2
7
7
7
34
PEG-8
6
6
6
a log KD values obtained from 3H-CGP 12177 binding in whole CHO cells expressing either the human β1-, β2-, or β3-AR. Values are mean ( SE from n
separate experiments. b The linker composition describes the atoms between the amino group of the carbinolamine and the nearest amide nitrogen: C-2,
ethyl; C-4, butyl, C-8, octyl; PEG-8, ethoxyethoxyethyl. c Apparent log KD value (85.3 ( 2.4% maximal inhibition of specific binding). d Apparent log KD
value (65.9 ( 2.0% maximal inhibition of specific binding).
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responses. The first site has high affinity for the majority of
β-antagonists and is the site at which isoprenaline and other
catecholamines elicit their agonist responses. The second site can
be activated by high concentrations of CGP 12177 and is generally
relatively resistant to antagonism by standard β-blockers.46 In
order to evaluate the ability of fluorescent ligands to antagonize
functional responses via these two sites, we used cimaterol as an
agonist of site 1 and CGP 12177 as an agonist of site 2.47,10 All
fluorescent ligands were effective antagonists of site 1 of the
β1-AR with the PEG-8 derivatives 18a (Table 2), 26a, and 34
being the most potent. The inhibition of agonist responses to
CGP 12177 provided the first indication of the affinity of these
fluorescent ligands for site 2 of the β1-AR. All compounds tested
had a lower affinity for site 2 than for site 1 (Table 2). However, it
was striking that linker length and the presence of the fluoro-
phore did not markedly change the affinity of the parent ligand
for site 2 (Table 2). As noted from binding studies, this was also
true of the β3-AR, and the functional studies confirmed the
higher affinity of C2-linked propranolol and alprenolol deriva-
tives for this receptor (Table 2).
Confocal Imaging. An essential requirement of a fluorescent
ligand for any GPCR is that its binding to cell surface receptors
can be visualized and that this binding displays the pharmacology
of the target receptor. We have previously shown from our work
on adenosine A1 and A3 receptors that the BODIPY 630/650
fluorophore is heavily quenched in aqueous environments but
markedly increases its quantum yield when bound to cell surface
receptors because of its location in a more lipid environment.38
This property has proved highly beneficial in studying ligand-
binding kinetics at the single cell level.15 Figure 6 shows the
binding of 3 nM 18a to CHO cells expressing the β2-AR. Bright
labeling of cell surface β2-AR can be detected even in the con-
tinued presence of 18a.
Quenching of the ligand present in the extracellular fluid is
evident from the lack of fluorescence between the individual cells.
Cell surface binding of 18a can be markedly inhibited by increas-
ing concentrations (1ꢀ100 nM) of the β2-selective antagonist
ICI 118551, consistent with its high affinity for the β2-AR. In
contrast, the selective β1-AR antagonist 1-[2-((3-carbamoyl-
4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-
2-imidazolyl)phenoxy]-2-propanol (CGP 20712A)48 only began
to displace at concentrations above 10 μM, which is consistent
with its low affinity for this receptor.49 Finally, 3nM 18a
produced only a low level of binding to the cell surface of control
CHO cells not expressing the β2-AR (Figure 6). This low level of
binding was entirely consistent with the nonspecific binding
observed in receptor-expressing cells incubated with 100 nM ICI
118551 (Figure 6).
3
Figure 2. Fluorescent ligand displacement of H-CGP 12177 specific
binding at the human β-AR in whole cells. Inhibition of 3H-CGP 12177
specific binding to whole cells by compounds 18a, 18b, 14c, and 14f in
(a) β1 cells, (b) β2 cells, and (c) β3 cells. Nonspecific binding was
determined by (a) 10 μM CGP 20712A and (b and c) 10 μM ICI
118551.1 The concentrations of 3H-CGP 12177 present in each case are
(a and b) 1.3 nM and (c) 18 nM. Data points are mean ( SE of triplicate
determinations. These single experiments are representative of (a and b)
six and (c) four separate experiments.
observations are consistent with the agonist nature of the parent
compounds on all three receptors (Table 3).41ꢀ43 Interestingly,
the presence of the fluorophore/linker combination reduced the
agonist efficacy of both alprenolol and pindolol at the β2-AR,
particularly, but had a much smaller influence on agonist efficacy
at the other two β-ARs (Table 3). This suggests that the presence of
the BODIPY unit or the associated linker chemistry can modify
agonist efficacy in a receptor subtype specific manner and implies
that the design of ligands with receptor selectivity based on efficacy
rather than ligand-binding affinity may be possible.44 As a conse-
quence of the partial agonist activity, the log KD values for alprenolol,
pindolol, and their derivatives shown in Table 2 were calculated
according to the partial agonist method of Stephenson.45
’ CONCLUSIONS
A key feature of the current state of research into GPCRs is a
growing awareness of the need to investigate their cellular
location and involvement in proteinꢀprotein interactions. This
requires the development of methodologies to investigate the
allosteric regulation of the ligandꢀreceptor binding by small
molecules or associated signaling proteins. We have shown
previously that fluorescent ligands can be used to provide insights
into the membrane organization of GPCRs using fluorescence
correlation spectroscopy37,16 and also to reveal novel allosteric
interactions using kinetic analysis of ligand association and
dissociation in single living cells.15,50 A key prerequisite for these
Previous work on the human β1-AR has shown that this receptor
can exist in two conformations that can both elicit functional
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Figure 3. Fluorescent ligand 26a displacement of 3H-CGP 12177 specific binding at the human β-adrenoceptors in whole cells. Inhibition of 3H-CGP
12177 specific binding to whole cells by compound 26a in (a) β1 and β2 cells and (b) β3 cells. Nonspecific binding was determined by (a) 10 μM CGP
20712A (β1) and (b) 10 μM ICI 118551 (β2 and β3). The concentrations of 3H-CGP 12177 present in each case are (a) 1.7 nM and (b) 6.4 nM. Data
points are mean ( SE of triplicate determinations. These single experiments are representative of (a) six and (b) five separate experiments.
Table 2. Fluorescent Ligand log KD Values from Antagonism of Agonist-Mediated Enhancement of CRE Reporter Gene
Responses at the Human β-ARa
β1 (site 1) log KD
n
β1 (site 2) log KD
n
β2 log KD
n
β3 log KD
n
propranolol
14a
ꢀ8.74 ( 0.03
ꢀ8.08 ( 0.06
ꢀ7.87 ( 0.10
ꢀ7.28 ( 0.08
ꢀ7.12 ( 0.08
ꢀ7.51 ( 0.06
ꢀ6.66 ( 0.05
ꢀ8.46 ( 0.07
ꢀ7.38 ( 0.08
ꢀ8.37 ( 0.07
ꢀ7.33 ( 0.08
ꢀ7.36 ( 0.06
ꢀ8.12 ( 0.09
ꢀ7.76 ( 0.08
ꢀ8.62 ( 0.07
ꢀ6.52 ( 0.06
ꢀ7.42 ( 0.05
10
8
ꢀ6.70 ( 0.10
ꢀ7.04 ( 0.09
ꢀ6.74 ( 0.09
ꢀ6.10 ( 0.10
ꢀ6.45 ( 0.10
ꢀ6.62 ( 0.08
>ꢀ6
21
9
ꢀ9.62 ( 0.05
ꢀ9.37 ( 0.04
ꢀ9.56 ( 0.05
ꢀ8.76 ( 0.10
ꢀ8.59 ( 0.08
ꢀ9.13 ( 0.06
ꢀ8.60 ( 0.05
ꢀ9.53 ( 0.11
ꢀ9.36 ( 0.11
ꢀ9.53 ( 0.07
ꢀ8.74 ( 0.06
ꢀ8.80 ( 0.08
ꢀ9.37 ( 0.08
ꢀ9.21 ( 0.09
ꢀ9.18 ( 0.04
ꢀ7.52 ( 0.05
ꢀ8.14 ( 0.07
13
15
13
16
15
18
9
ꢀ6.79 ( 0.02
ꢀ7.58 ( 0.07
ꢀ6.82 ( 0.09
ꢀ6.85 ( 0.05
ꢀ7.20 ( 0.09
ꢀ7.10 ( 0.05
ꢀ6.94 ( 0.09
ꢀ6.97 ( 0.08
ꢀ7.24 ( 0.09
ꢀ7.12 ( 0.08
ꢀ7.38 ( 0.05
ꢀ7.63 ( 0.19
ꢀ7.21 ( 0.07
ꢀ7.24 ( 0.10
>ꢀ6
6
13
6
14b
11
9
7
14c
13
10
8
7
14d
10
9
12
10
12
6
14e
14f
10
7
5
18a
ꢀ7.17 ( 0.06
ꢀ6.95 ( 0.10
ꢀ6.75 ( 0.12
ꢀ7.13 ( 0.11
ꢀ6.92 ( 0.09
ꢀ7.38 ( 0.08
ꢀ7.11 ( 0.11
>ꢀ5
7
17
18
13
12
9
18b
8
5
9
alprenolol
23a
12
9
7
12
8
9
23b
4
4
9
26a
7
3
6
7
26b
13
9
11
8
15
6
17
7
pindolol
31
4
>ꢀ6
>ꢀ6
4
8
ꢀ6.75 ( 0.02
ꢀ6.54 ( 0.11
4
34
8
4
12
6
a log KD values were obtained from inhibition of functional CRE-SPAP responses to cimaterol (β1, site 1), CGP 12177 (β1, site 2), salbutamol (β2), and
fenoterol (β3) in CHO cells expressing either the human β1-, β2- or β3-AR. Values are the mean ( SE from n separate experiments.
approaches is the design and synthesis of novel fluorescent
ligands with the correct pharmacological and biophysical proper-
ties. In the present study, we have designed, synthesized, and
characterized 14 new red-emitting fluorescent ligands (14aꢀf,
18a,b, 23a,b, 26a,b, 31, and 34) for the human β-AR. Using
alkyl- or polyether-based linker extension from the nitrogen atom
of the conserved aryloxypropanolamine portion of propranolol,
alprenolol, and pindolol generated a range of fully deprotected
congeners, functionally equipped to undergo a high yielding
chemoselective acylation with the commercially available fluor-
ochrome BODIPY 630/650-X, SE (Molecular Probes). No
protecting group manipulations were required following fluor-
ophore attachment. This new range of fluorescent β-blockers
displayed high affinity for the β2-AR, with some (18a and 26a)
also possessing moderate affinity for the β1-AR. An evaluation
of the pharmacology of the new fluorescent derivatives was
undertaken. Compound 18a, in particular, displayed a log KD
of ꢀ9.53 as an antagonist of functional responses mediated by
the human β2-AR and a log KD of ꢀ8.46 as an antagonist of the
high-affinity catecholamine site of the β1-AR.
Critically, we were further able to utilize these molecules to
visualize ligandꢀreceptor interactions using confocal micro-
scopy. Confirmation of selective fluorescent labeling of, for
example, the human β2-AR in single living cells was achieved
by incubating CHO-β2 cells expressing the human β2-AR with a
3 nM concentration of 18a. This resulted in clear labeling of the
cell membrane, with the receptor specific proportion of this
binding blocked by preincubation with the nonfluorescent
β2-selective antagonist ICI 118551. This fluorescent propranolol
derivative should therefore be invaluable in future mechanistic
studies aimed at unraveling the intricacies of cell signaling via this
prototypical GPCR.
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Journal of Medicinal Chemistry
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Figure 4. Fluorescent ligand 18a is a competitive antagonist at all three human β-AR as measured via a whole cell reporter gene assay. CRE-SPAP production
in the absence and presence of 10, 100, and 1000 nM compound 18a in (a) β1 cells following stimulation by cimaterol, (b) β1 cells following stimulation by
CGP 12177, (c) β2 cells following stimulation by salbutamol, and (d) β3 cells following stimulation by fenoterol. Bars represent basal CRE-SPAP production,
that in response to 10 μM isoprenaline, and that in response to 10, 100, or 1000 nM compound 18a alone. Data points are mean ( SE of triplicate deter-
minations. These single experiments are representative of (a) five, (b) seven, (c) 10, and (d) four separate experiments. The Schild slope for part (a) is 1.08.
Figure 5. Partial agonism effects of compound 26a are competitively antagonized at all three human β-AR. CRE-SPAP production in the absence and
presence of 10, 100, and 1000 nM compound 26a in (a) β1 cells following stimulation by cimaterol, (b) β1 cells following stimulation by CGP 12177, (c)
β2 cells following stimulation by salbutamol, and (d) β3 cells following stimulation by fenoterol. Bars represent basal CRE-SPAP production, that in
response to 10 μM isoprenaline, and that in response to 10, 100, or 1000 nM compound 26a alone. Data points are mean ( SE of triplicate
determinations. These single experiments are representative of (a) five, (b) eight, (c) four, and (d) five separate experiments.
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Table 3. Partial Agonist Actions of Alprenolol and Pindolola
biological systems were therefore assessed as being ∼95%. Mono-
protected diamines [benzyl 2-aminoethylcarbamate, tert-butyl 2-ami-
noethylcarbamate, tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate]
were purchased (Sigma-Aldrich) or were synthesized [benzyl 4-aminobu-
tylcarbamate, benzyl 8-aminooctylcarbamate, benzyl 2-(2-(2-aminoethoxy)-
ethoxy)ethylcarbamate] as previously described.36,51
Derivatives at the Three Human β-ARb
β1 %c
n
β2 %c
n
β3 %c
n
alprenolol
23a
46.4 ( 3.1
40.8 ( 2.6
33.1 ( 2.5
28.9 ( 2.0
30.9 ( 1.8
64.3 ( 2.6
31.1 ( 2.0
20.8 ( 1.8
13
10
4
52.8 ( 1.8
12.2 ( 1.4
11.7 ( 2.3
26.9 ( 2.1
22.9 ( 2.3
52.3 ( 1.9
10.8 ( 1.0
11.0 ( 1.5
7
4
3
5
6
3
4
4
62.4 ( 3.2
56.6 ( 3.4
60.6 ( 5.0
63.5 ( 3.6
49.6 ( 2.3
85.2 ( 3.2
49.4 ( 4.9
53.2 ( 3.5
12
9
(S)-2-((Naphthalen-1-yloxy)methyl)oxirane (10a). A portion
of 60% NaH in mineral oil (610 mg, equivalent to 366 mg of NaH,
15.26 mmol, 1.1 equiv) was dispersed in anhydrous DMF (2 mL)
under an atmosphere of nitrogen at room temperature. To this
vigorously stirred suspension was added a solution of 1-naphthol
(2.00 g, 13.87 mmol) in anhydrous DMF (8 mL). The resulting pale
green suspension was stirred at room temperature for 30 min. A
solution of (S)-glycidyl nosylate (3.632 g, 14.01 mmol, 1.01 equiv) in
anhydrous DMF (5 mL) was added dropwise to the reaction mixture.
Once addition was complete, the mixture was heated at 60 °C over-
night before cooling and quenching with aqueous saturated NH4Cl.
The mixture was concentrated to dryness under reduced pressure and
the resulting residue dispersed in water (100 mL). The aqueous slurry
was extracted with Et2O (3 ꢁ 50 mL), and the combined organic
extracts were washed with aqueous 1 M NaOH (50 mL) and brine
(50 mL). After drying over anhydrous MgSO4, the organic extracts
were concentrated under reduced pressure. The crude product was
further purified by flash column chromatography (eluent EtOAc/
petroleum ether 40ꢀ60 0:100 to 30:70 over 10 column volumes) to
afford a colorless oil (2.44 g, 88%). [α]2D6 = +16.7o (c 1.65, CHCl3);
lit.52 [α]D20 = +17.3o (c 2.0, CHCl3). 1H NMR: δ 2.86 (1H, dd, J = 4.9/2.7
Hz), 2.98 (1H, dd, J = 4.8/4.2 Hz), 3.48ꢀ3.54 (1H, m), 4.16 (1H, dd, J =
11.0/5.6 Hz), 4.41 (1H, dd, J = 11.0/3.2 Hz), 6.82 (1H, d, J =
7.0 Hz), 7.37 (1H, dd, J = 7.9 Hz), 7.43ꢀ7.54 (3H, m), 7.76ꢀ7.85
(1H, m), 8.27ꢀ8.35 (m, 1H). 13C NMR: δ 44.91, 50.39, 69.10, 105.12,
121.00, 122.16, 125.47, 125.73, 125.84, 126.65, 127.59, 134.66, 154.38.
HRMS (TOF ES+): calcd for C13H13O2 201.0910, found 201.0904 (MH+).
(S)-2-((2-Allylphenoxy)methyl)oxirane (20a). A portion of
60% NaH in mineral oil (656 mg, equivalent to 394 mg NaH, 16.40
mmol, 1.1 equiv) was dispersed in anhydrous DMF (2 mL) under an
atmosphere of nitrogen at room temperature. To this vigorously stirred
suspension was added a solution of 2-allylphenol (2.00 g, 14.91 mmol)
in anhydrous DMF (8 mL). The resulting pale green suspension was
stirred at room temperature for 30 min. A solution of (S)-glycidyl
nosylate (3.903 g, 15.05 mmol, 1.01 equiv) in anhydrous DMF (5 mL)
was added dropwise to the reaction mixture. Once addition was
complete, the mixture was heated at 60 °C overnight before cooling
and quenching with aqueous saturated NH4Cl. The mixture was
concentrated to dryness under reduced pressure and the resulting
residue dispersed in water (100 mL). The aqueous slurry was extracted
with Et2O (3 ꢁ 50 mL), and the combined organic extracts were washed
with aqueous 1 M NaOH (50 mL) and brine (50 mL). After drying over
anhydrous MgSO4, the organic extracts were concentrated under reduced
pressure. The crude product was further purified by flash column chromato-
graphy (eluent EtOAc/petroleum-ether 40ꢀ60 0:100 to 30:70 over
10 column volumes) to afford a clear yellow oil (2.97 g, quantitative).
[α]2D1 = +8.97° (c 4.18, DCM). 1H NMR: δ 2.78 (1H, dd, J = 5.0/2.7
Hz), 2.90 (1H, dd, J = 4.9/4.2 Hz), 3.33ꢀ3.39 (1H, m), 3.41 (2H, br d,
J = 6.6 Hz), 3.99 (1H, m, J = 11.0/5.4 Hz), 4.23 (1H, dd, J = 11.0/3.1
Hz), 5.00ꢀ5.11 (2H, m), 5.93ꢀ6.06 (1H, m), 6.84 (1H, d, J = 8.2 Hz),
6.92 (1H, ddd, J = 7.4/7.4/1.1 Hz), 7.10ꢀ7.23 (2H, m). 13C NMR: δ
34.50, 44.80, 50.44, 68.88, 111.73, 115.62, 121.31, 127.46, 129.19,
130.11, 137.03, 156.28. HRMS (TOF ES+): calcd for C12H15O2
191.1067, found 191.1060 (MH+).
23b
5
26a
12
14
8
5
26b
13
7
pindolol
31
4
4
34
8
8
a The propranolol derivatives did not stimulate significant agonist
responses. b Values are mean ( SE from n separate experiments.
c Agonist action is expressed as a percentage of the CRE-SPAP responses
to 10 μM isoprenaline in each experiment. As the top of the concentra-
tion response curve was not achieved by many fluorescent ligands at the
maximum possible experimental concentration of 1 μM, the response to
alprenolol, pindolol, and their derivatives at 1 μM is given.
’ EXPERIMENTAL SECTION
General Chemistry Methods. Chemicals and solvents (HPLC
grade) were purchased from the standard suppliers and were used
without further purification. BODIPY fluorophores were obtained from
Molecular Probes, Cambridge Bioscience, Cambridge, UK. Merck
Kieselgel 60 (230ꢀ400 mesh) for flash chromatography was supplied
by Merck KgaA (Darmstadt, Germany), and deuterated solvents were
purchased from Goss International Limited. 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 Kieselgel 60 F254). Visua-
lization was by examination under UV light (254 and 366 nm). General
staining employed KMnO4. A solution of ninhydrin (EtOH) was used
for the visualization of primary amines. All organic extracts collected
after aqueous workup procedures were dried over anhydrous magne-
sium sulfate, filtered under gravity, and evaporated to dryness. Organic
solvents were evaporated in vacuo at a temperature of <35 °C (water
bath). Purification using preparative layer chromatography (PLC) was
carried out using Fluka silica gel GF254 on glass plates (200 mm ꢁ 200
mm ꢁ 1 mm).
Melting points were recorded on a Gallenkamp 3A 3790 apparatus
and are uncorrected. FT-infrared spectra were recorded as thin films or
KBr discs in the range of 4000ꢀ600 cmꢀ1 using an Avatar 360 Nicolet
FT-IR spectrophotometer. Optical rotation was measured on a Belling-
ham-Stanley ADP220 polarimeter. Mass spectra and HRMS TOF-ES
mass spectra were recorded on a Waters 2795 Separation Module/
Micromass LCT platform. Proton nuclear magnetic resonance spectra
were recorded on a Bruker-AV 400 (400 MHz) spectrometer. Carbon
nuclear magnetic resonance spectra were recorded at 100.6 MHz. Unless
otherwise stated, spectra were recorded in CDCl3. Chemical shifts (δ)
are recorded in ppm with reference to the chemical shift of the
deuterated solvent/an internal tetramethylsilane standard. Coupling
constants (J) are recorded in hertz and the signal multiplicities described
by s, singlet; d, doublet; t, triplet; q, quartet; br, broad; m, multiplet; dd,
doublet of doublets. Spectra were assigned using appropriate COSY,
DEPT, and HMQC sequences. Analytical reverse-phase high-perfor-
mance liquid chromatography (RP-HPLC) was performed on a Waters
Millennium 995 LC system using columns, gradients, and flow rates as
described. The eluent was monitored using photodiode array detection.
Mobile phases were solvent A, water; solvent B, acetonitrile (both
containing 0.06% v/v trifluoroacetic acid) degassed by helium bubble
and sonication, respectively. The purities of all compounds tested in
(()-4-(Oxiran-2-ylmethoxy)-1H-indole (28). 4-Hydroxy-1H-
indole (100 mg, 0.75 mmol) and Cs2CO3 (257 mg, 0.79 mmol, 1.05
equiv) were placed in a 10 mL MW vial, prior to the addition of (()-
epichlorohydrin (3.0 mL). The mixture was heated in the MW reactor at
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Figure 6. Confocal visualization of the effect of ICI 118551 or CGP 20712A on the binding of the BODIPY-630/650 derivative of propranolol 18a to
CHO K1 cells expressing the β2-AR. Antagonists were incubated with cells for 30 min prior to addition of 3nM 18a for 10 min. Imaging of cells was
undertaken in the continued presence of fluorescent ligand. The lower right-hand panel shows the binding of 18a to control CHO-K1 cells.
120 °C (dynamic program, max. pressure 250 psi, max. power 300 W)
for 30 min. The reaction mixture was diluted with water (20 mL) before
extracting with DCM (3 ꢁ 10 mL). The combined organic extracts were
dried over anhydrous Na2SO4 before concentration under reduced
pressure. The crude product was further purified by flash column
chromatography (eluent EtOAc/petroleum ether 40ꢀ60 0:100 to
30:70 over 16 column volumes), to afford a pale blue solid (0.138 g,
97%). Mp: 56ꢀ58 °C. 1H NMR: δ 2.83 (1H, dd, J = 4.9/2.7 Hz), 2.94
(1H, dd, J = 4.9/4.2 Hz), 3.44ꢀ3.52 (1H, m), 4.14 (1H, dd, J = 11.2/5.6
Hz), 4.38 (1H, dd, J = 11.2/3.2 Hz), 6.54 (1H, d, J = 7.5 Hz), 6.68ꢀ6.75
(1H, m), 7.03 (1H, d, J = 8.2 Hz), 7.08 (1H, dd, J = 2.8 Hz), 7.12 (1H,
dd, J = 7.9 Hz), 8.30 (1H, br s). 13C NMR: δ 44.97, 50.48, 68.90, 99.90,
100.85, 105.11, 118.87, 122.63, 122.95, 137.50, 152.28. HRMS (TOF
ES+): calcd for C11H12NO2 190.0863, found 190.0856 (MH+).
2-aminoethylcarbamate (0.221 g, 1.38 mmol) in DMF/H2O 9:1
(3 mL) was heated at 85 °C for 16 h. The solvent was evaporated
under reduced pressure and the target compound purified by flash
column chromatography on silica (10% MeOH in CH2Cl2) to afford the
title compound as a colorless oil (0.102 g, 53%). [α]2D6 = +3.1° (c 0.8,
15:85 MeOH/CH2Cl2). 1H NMR: 1.44 (9H, s), 2.69ꢀ2.85 (2H, br s),
2.79 (2H, t, J = 5.8), 2.82 (1H, dd, J = 12.3, 7.7), 2.89 (1H, dd, J = 12.3, 4.1),
3.20ꢀ3.31 (2H, m), 3.38 (2H, d, J = 6.1), 3.98 (2H, d, J = 5.4), 4.06ꢀ4.14
(1H, m), 4.96ꢀ5.08 (3H, m), 5.97 (1H, ddt, J = 16.7, 10.4, 6.4), 6.83 (1H,
d, J = 7.7), 6.91 (1H, t, J = 7.7), 7.13 (1H, dd, J = 7.7, 1.5), 7.17 (1H, td, J =
7.7, 1.5). 13C NMR: 28.5 (3 ꢁ CH3), 34.8, 40.3, 49.4, 51.8, 68.6, 70.5, 79.5,
111.5, 115.4, 121.1, 127.6, 128.6, 130.2, 137.4 156.4. HRMS (TOF ES+):
calcd for C19H31N2O4 351.2284, found 351.2319 (MH+).
(()-Benzyl 2-(2-(2-(3-(1H-Indol-4-yloxy)-2-hydroxypropy-
lamino)ethoxy)ethoxy)ethylcarbamate (32). (()-4-(Oxiran-2-
ylmethoxy)-1H-indole (28) (100 mg, 0.53 mmol) and benzyl 2-(2-(2-
aminoethoxy)ethoxy)ethylcarbamate (15) (224 mg, 0.79 mmol, 1.5
equiv) were dissolved in propan-2-ol/acetonitrile/water (7:2:1, 5 mL)
and heated in a MW reactor at 90 °C (dynamic program, max. pressure
250 psi, max. power 300 W) for 60 min. The reaction mixture was
concentrated under reduced pressure and the residue purified by flash
column chromatography (eluent 1 M methanolic NH3/DCM 0:100 to
10:90 over 12 column volumes), to give 100 mg of a pale yellow viscous
oil (40%). 1H NMR: δ 8.72 (1H, br s), 7.23ꢀ7.41 (5H, m), 7.03ꢀ7.10
(2H, m), 7.00 (1H, d, J = 8.1 Hz), 6.61 (1H, br s), 6.46 (1H, d, J = 7.4 Hz),
5.67ꢀ5.88 (1H, br m), 5.07 (2H, s), 3.98ꢀ4.27 (3H, m), 3.53ꢀ3.65
(6H, m), 3.50 (2H, t, J = 4.9 Hz), 3.35 (2H, dt, J = 5.1/5.1 Hz),
2.72ꢀ2.95 (5H, m). 13C NMR: δ 156.73, 152.32, 137.46, 136.66,
128.55, 128.16, 128.12, 122.94, 122.60, 118.77, 104.97, 100.70, 99.62,
70.51, 70.20, 70.11, 68.46, 66.69, 51.97, 48.98, 40.91. IR (NaCl, film):
3319, 1707, 1253, 1090, 727 cmꢀ1. HRMS (TOF ESꢀ): calcd for
C25H31N3O6 470.2297, found 470.2283 (MHꢀ).
(()-Benzyl 4-(2-Hydroxy-3-(naphthalen-1-yloxy)propyl-
amino)butylcarbamate (12e). A solution of 2-(napthalen-1-ylox-
ymethyl)oxirane (10b) (0.20 g, 1 mmol) and benzyl 4-aminobutylcar-
bamate (11b) (0.56 g, 2.5 mmol) in DMF/H2O 9:1 (5 mL) was heated
at 85 °C for 16 h. The solvent was evaporated under reduced pressure
and the crude material was purified by flash column chromatography on
silica employing a gradient from 0 to 10% MeOH in CH2Cl2 as eluent to
afford the title compound as a yellow solid (0.245 g, 58%). Mp
1
90ꢀ91 °C. H NMR: 1.49ꢀ1.61 (4H, m), 2.45ꢀ2.74 (4H, m), 2.86
(1H, dd, J = 12.1, 7.5), 2.95 (1H, dd, J = 12.1, 3.7), 3.12ꢀ3.25 (2H, m),
4.09ꢀ4.25 (3H, m), 5.09 (2H, s), 5.19 (1H, br s), 6.81 (1H, d, J = 7.5),
7.27ꢀ7.40 (6H, m), 7.42ꢀ7.52 (3H, m), 7.76ꢀ7.84 (1H, m). 13C
NMR: 27.5, 27.9, 41.0, 49.5, 52.2, 66.7, 68.5, 70.8, 105.1, 120.8, 121.9,
125.4, 125.7, 126.0, 126.6, 127.7, 128.2, 128.2, 128.6 (3 ꢁ CH), 134.6,
136.8, 154.4, 156.6. IR (NaCl, film): 2933, 1701, 1269, 1242, 1102,
772 cm-1. MS m/z (TOF ES+): 423 (MH+, 100%). HRMS (TOF ES+):
calcd for C25H30N2O4 423.2284, found 423.2247.
(S)-tert-Butyl 2-(3-(2-Allylphenoxy)-2-hydroxypropylamino)-
ethylcarbamate (21a). A solution of (S)-2-((2-allylphenoxy)-
methyl)oxirane (20a) (0.105 g, 0.553 mmol) and tert-butyl
Method A. Cbz-Group Hydrogenolysis. To a solution of the
Cbz-protected amine (100ꢀ130 μM) in MeOH/H2O (9:1) was added
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10% Pd/C (1:1 w/w with protected amine). The solvent was briefly
degassed and hydrogen was added via balloon; the mixture was then
stirred at room temperature for 1 h. The reaction mixture was filtered
through Celite and the pad was washed with MeOH (ꢁ2). The solvent
was evaporated under reduced pressure to afford the deprotected amine,
which was used without further purification.
(()-1-(4-Aminobutylamino)-3-(napthalenyl-1-yloxy)propan-
2-ol (13e). The compound was prepared via method A to yield a
colorless oil (quant.). 1H NMR (CD3OD): 1.63ꢀ1.79 (4H, m),
2.71ꢀ2.84 (2H, m), 2.89 (2H, t), 2.92 (1H, dd, J = 12.3, 8.7), 3.03
(1H, dd, J = 12.3, 3.6), 4.14 (1H, dd, J = 9.8, 5.3), 4.18 (1H, dd, J = 9.8,
5.3), 4.28 (1H, dtd, J = 8.7, 5.3, 3.6), 6.91 (1H, dd, J = 7.6, 0.9),
7.34ꢀ7.50 (4H, m), 7.76ꢀ7.82 (1H, m), 8.26ꢀ8.34 (1H, m). 13C NMR
(CD3OD): 27.5, 27.7, 40.7, 49.7, 52.9, 69.2, 71.8, 106.0, 121.6, 122.9,
126.1, 126.9, 127.0, 127.4, 128.5, 136.0, 155.7. IR (NaCl, film): 3354,
2939, 1579, 1398, 1270, 1241, 1102, 795, 772 cmꢀ1. HRMS (TOF ES+):
calcd for C17H25N2O2 289.1916, found 289.1895 (MH+).
(S)-1-(2-Allylphenoxy)-3-(2-aminoethylamino)propan-2-
ol (22a). (S)-tert-Butyl 2-(3-(2-allylphenoxy)-2-hydroxypropylamino)-
ethylcarbamate (21a) (0.04 g, 0.114 mmol) was dissolved in a 2 M
solution of HCl in dioxane (2 mL) and the reaction mixture was stirred at
room temperature for 18 h. The solvent was evaporated under reduced
pressure and the resultant residue was azeotroped sequentially with toluene
and EtOAc to afford the dihydrochloride salt of the title compound as a
white solid (0.036 g, 98%), which was used without further purification.
Mp: 142ꢀ145 °C. [α]D26 = ꢀ2.2° (c 0.46, 15:85 MeOH/CH2Cl2). 1H
NMR (CD3OD): 3.28 (1H, dd, J = 12.5, 9.8), 3.36ꢀ3.49 (7H, m), 4.01
(1H, dd, J = 10.0, 5.7), 4.09 (1H, dd, J = 10.0, 4.8), 4.32ꢀ4.40 (1H, m),
4.98ꢀ5.06 (2H, m), 5.94ꢀ6.05 (1H, m), 6.91 (1H, td, J = 7.7, 1.0), 6.95
(1H, d, J = 7.7), 7.13 (1H, dd, J = 7.7, 1.7), 7.18 (1H, td, J = 7.7, 1.7). 13C
NMR (CD3OD): 35.3, 36.8, 45.9, 52.0, 66.8, 71.0, 112.7, 115.7, 122.3,
128.5, 129.8, 131.0, 138.5, 157.4. IR (KBr): 3299, 2956, 1600, 1491,
1455, 1258, 1214, 764 cmꢀ1. HRMS (TOF ES+): calcd for C14H23N2O2
251.1760, found 251.1773 (MH+).
(()-13-Amino-1-(1H-indol-4-yl)-1,8,11-trioxa-5-azatridecan-
3-ol (33). The compound was prepared via method A to yield a colorless oil
(88%). 1HNMR(CD3OD): 2.86ꢀ3.02(6H, m), 3.58ꢀ3.69(8H, m), 4.12
(1H, dd, J = 9.9, 6.0), 4.15 (1H, dd, J = 9.9, 4.7), 4.16ꢀ4.24 (1H, m), 6.41
(1H, d, J = 3.0), 6.64 (1H, d, J = 7.6), 6.91 (1H, t, J = 7.8), 7.16 (1H, dd, J =
7.8, 0.7), 7.20 (1H, d, J = 3.3). 13C NMR (CD3OD): 41.1, 49.8, 52.9, 69.6,
69.6, 70.4, 71.3, 71.3, 71.7, 102.7, 103.6, 114.6, 120.4, 125.2, 128.2, 131.1,
146.7. IR (NaCl, film): 2919, 1576, 1253, 1091, 728 cmꢀ1. HRMS (TOF
ES+): calcd for C17H27N3O4 338.2080, found 338.2062 (MH+).
Method B. Coupling of Amines with BODIPY 630/650-X,
SE. BODIPY 630/650-X, SE (1ꢀ2.5 mg, 1 equiv) and amine (2ꢀ4
equiv) were dissolved in anhydrous DMF (1 mL) [in the case of the
alprenolol congener HCl salts, 2 equiv (with respect to the congener) of
N,N-diisoproylethylamine (DIPEA) was also added (method B1)] and
stirred under a nitrogen atmosphere, with the exclusion of light, for 2 h.
The solvent was evaporated under reduced pressure and this crude
mixture was purified by PTLC on silica using (10:90 MeOH/CH2Cl2)
as eluent to give compounds 14aꢀf, 18a,b, 23a,b, 26a,b, 31, and 34 as
blue amorphous solids. When submitted to analytical HPLC [YMC C8,
150 ꢁ 4.6 mm, 1 mL minꢀ1, 35ꢀ100% B or 5ꢀ100% B, 30 min,
monitored using photodiode array detection between 190 and 800 nm;
mobile phases, solvent A, H2O; solvent B, MeCN (both A and B
containing 0.06% TFA as additive)] all fluorescent conjugates were
observed to elute as single and symmetrical peaks at the retention time tR
given below for each compound. For the initial conjugates synthesized
within the racemic propranolol series, we further analyzed compound
purity by performing a second analytical HPLC [Jones C4, 150 ꢁ 4.6
mm, 1 mL minꢀ1, 35ꢀ100% B, 30 min, monitored using photodiode
array detection between 190 and 800 nm; solvent A, H2O; solvent B,
MeCN (both A and B containing 0.06% TFA as additive)]. Again, all
target compounds elutedas homogeneoussingle peaks (with the expected
reduction in retention time). The identities of all the compounds were
further analyzed by HRMS (TOF ES+). The purities of all compounds
tested in biological systems were determined as being ∼95%.
(S)-N-[2-(2-Hydroxy-3-(naphthalen-1-yloxy)propylamino)-
ethyl]-6-(2-(2-(4,4-difluoro-4,4a-dihydro-5-(thiophen-2-yl)-
4-bora-3a,4a-diaza-s-indacene-3-yl)vinyl)phenoxy)acetamido)-
hexanamide (14a). The compound was prepared via method B
(78%). tR 9.61 min (YMC C8, 150 ꢁ 4.6 mm, 1 mL minꢀ1, 35ꢀ100% B, 30
min; solvent A, H2O; solvent B, MeCN, both A and B contain 0.06% TFA
as additive). HRMS (TOF ES+): calcd for C44H47BF2N5O5S 806.3359,
found 806.3425 (MH+).
(S)-N-[2-(2-Hydroxy-3-(naphthalen-1-yloxy)propylamino)-
butyl]-6-(2-(2-(4,4-difluoro-4,4a-dihydro-5-(thiophen-2-yl)-
4-bora-3a,4a-diaza-s-indacene-3-yl)vinyl)phenoxy)acetamido)-
hexanamide (14b). The compound was prepared via method B (80%).
tR 15.68 min (YMC C8, 150 ꢁ 4.6 mm, 1 mL minꢀ1, 5ꢀ100% B, 30 min;
solvent A, H2O; solvent B, MeCN, both A and B contain 0.06% TFA as
additive). HRMS (TOF ES+): calcd for C46H51BF2N5O5S 834.3672, found
834.3690 (MH+).
(S)-N-[2-(2-Hydroxy-3-(naphthalen-1-yloxy)propylamino)-
octyl]-6-(2-(2-(4,4-difluoro-4,4a-dihydro-5-(thiophen-2-yl)-
4-bora-3a,4a-diaza-s-indacene-3-yl)vinyl)phenoxy)acetamido)-
hexanamide (14c). The compound was prepared via method B (71%).
tR 10.22 min (YMC C8, 150 ꢁ 4.6 mm, 1 mL minꢀ1, 35ꢀ100% B, 30 min;
solvent A, H2O; solvent B, MeCN, both A and B contain 0.06% TFA as
additive). HRMS (TOF ES+): calcd for C50H59BF2N5O5S 890.4298, found
890.4278 (MH+).
(()-N-[2-(2-Hydroxy-3-(naphthalen-1-yloxy)propylamino)-
ethyl]-6-(2-(2-(4,4-difluoro-4,4a-dihydro-5-(thiophen-2-yl)-4-
bora-3a,4a-diaza-s-indacene-3-yl)vinyl)phenoxy)acetamido)-
hexanamide (14d). The compound was prepared via method B (98%).
tR1 10.37 min (YMC C8, 150 ꢁ 4.6 mm, 1 mL minꢀ1, 35ꢀ100% B,
30 min). tR2 7.45 min (Jones C4, 150 ꢁ 4.6mm, 1 mLminꢀ1, 35ꢀ100% B,
30 min; solvent A, H2O; solvent B, MeCN, both A and B contain 0.06%
TFA as additive). HRMS (TOF ES+): calcd for C44H47BF2N5O5S
806.3359, found 806.3279 (MH+).
(()-N-[4-(2-Hydroxy-3-(naphthalen-1-yloxy)propylamino)-
butyl]-6-(2-(2-(4,4-difluoro-4,4a-dihydro-5-(thiophen-2-yl)-4-
bora-3a,4a-diaza-s-indacene-3-yl)vinyl)phenoxy)acetamido)-
hexanamide (14e). The compound was prepared via method B (77%).
tR1 9.48 min (YMCC8, 150ꢁ 4.6 mm, 1 mL minꢀ1, 35ꢀ100% B, 30min).
tR2 7.54 min (Jones C4, 150 ꢁ 4.6 mm, 1 mL minꢀ1, 35ꢀ100%B, 30 min).
1H NMR (CD3OD): 1.22ꢀ1.33 (2H, m), 1.47ꢀ1.64 (6H, m), 1.66ꢀ1.76
(2H, m), 2.16 (2H, t, J = 7.3), 3.06 (2H, t, J = 8.0), 3.16ꢀ3.33 (6H, m),
4.13 (1H, dd, J = 9.9, 5.7), 4.20 (1H, dd, J = 9.9, 5.0), 4.34ꢀ4.41 (1H, m),
4.55 (2H, s), 6.83 (1H, d, J = 4.2), 6.90(1H, d, J = 7.1), 7.02 (2H, app d, J =
8.8), 7.11 (1H, d, J = 4.2), 7.11 (1H, d, J = 4.2), 7.18 (1H, d, J = 4.4), 7.19
(1H, dd, J = 5.0, 3.8), 7.33 (1H, s), 7.37 (1H, t, J = 7.9), 7.43ꢀ7.56 (4H,
m), 7.57ꢀ7.63 (3H, m), 7.81 (1H, dd, J = 7.1, 1.5), 8.12 (1H, dd, J = 3.9,
1.0), 8.22 (1H, br t, J = 6.0), 8.24ꢀ8.28 (1H, m). HRMS (TOF ES+):
calcd for C46H51BF2N5O5S 834.3672, found 834.3622 (MH+).
(()-N2-(2-Hydroxy-3-(naphthalen-1-yloxy)propylamino)-
octyl]-6-(2-(2-(4,4-difluoro-4,4a-dihydro-5-(thiophen-2-yl)-
4-bora-3a,4a-diaza-s-indacene-3-yl)vinyl)phenoxy)acetamido)-
hexanamide (14f). The compound was prepared via method B (69%). tR
14.21 min (YMC C8, 150 ꢁ 4.6 mm, 1 mL minꢀ1, 5ꢀ100% B, 30 min;
solvent A, H2O; solvent B, MeCN, both A and B contain 0.06% TFA as
additive). HRMS (TOF ES+): calcd for C50H59BF2N5O5S 890.4298, found
890.4321 (MH+).
(S)-N-[3-Hydroxy-1-(naphthalen-1-yl)-1,8,11-trioxa-5-aza-
tridecan-13-yl]-6-(2-(2-(4,4-difluoro-4,4a-dihydro-5-(thiophen-
2-yl)-4-bora-3a,4a-diaza-s-indacene-3-yl)vinyl)phenoxy)-
acetamido)hexanamide (18a). The compound was prepared
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Journal of Medicinal Chemistry
ARTICLE
via method B (87%). tR 10.75 min (YMC C8, 150 ꢁ 4.6 mm, 1 mL
minꢀ1, 35ꢀ100% B, 30 min; solvent A, H2O; solvent B, MeCN, both
A and B contain 0.06% TFA as additive). HRMS (TOF ES+): calcd
for C48H55BF2N5O7S 894.3883, found 894.3849 (MH+).
(2H, tt, J = 7.6, 7.6), 1.57 (2H, tt, J = 7.6, 7.6), 2.14 (2H, t, J = 7.4),
3.11ꢀ3.22 (3H, m), 3.25 (2H, t, J = 6.9), 3.28ꢀ3.37 (2H, m),
3.44ꢀ3.50 (1H, m), 3.47 (2H, t, J = 5.6), 3.53ꢀ3.58 (2H, m),
3.58ꢀ3.63 (2H, m), 3.72 (2H, t, J = 4.8), 4.13 (1H, dd, J = 9.8,
5.4), 4.18 (1H, dd, J = 9.8, 5.0), 4.26ꢀ4.35 (1H, m), 4.55 (2H, s), 6.43
(1H, d, J = 3.2), 6.63 (1H, d, J = 7.7), 6.85 (1H, d, J = 4.5), 6.92 (1H, t,
J = 7.8), 7.03 (2H, app d, J = 9.0), 7.11ꢀ7.15 (2H, m), 7.17ꢀ7.22
(4H, m), 7.35 (1H, s), 7.55 (2H, br d, J = 8.3), 7.58ꢀ7.63 (3H, m),
8.12 (1H, dd, J = 3.8, 1.0). HRMS (TOF ES+): calcd for C46H54-
BF2N6O7S 883.3836, found 883.3886 (MH+).
(()-N-[3-Hydroxy-1-(naphthalen-1-yl)-1,8,11-trioxa-5-aza-
tridecan-13-yl]-6-(2-(2-(4,4-difluoro-4,4a-dihydro-5-(thiophen-
2-yl)-4-bora-3a,4a-diaza-s-indacene-3-yl)vinyl)phenoxy)-
acetamido)hexanamide (18b). The compound was prepared via
method B (81%). tR 9.52 min (YMC C8, 150 ꢁ 4.6 mm, 1 mL minꢀ1
,
35ꢀ100% B, 30 min; solvent A, H2O; solvent B, MeCN, both A and B
contain 0.06% TFA as additive). HRMS (TOF ES+): calcd for
C48H55BF2N5O7S 894.3883, found 894.3926 (MH+).
General Pharmacology Methods: Cell Lines and Cell Culture.
CHO-K1 stably expressing a six cyclic AMP response (CRE) secreted
placental alkaline phosphatase (SPAP) reporter gene and either
human β1 (1146 fmol/mg of protein), human β2 (466 fmol/mg of
protein), or human β3-AR (790 fmol/mg of protein) were used
throughout this study.44 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.
(S)-N-[2-(3-(2-Allylphenoxy)-2-hydroxypropylamino)ethyl]-
6-(2-(2-(4,4-difluoro-4,4a-dihydro-5-(thiophen-2-yl)-4-bora-3a,
4a-diaza-s-indacene-3-yl)vinyl)phenoxy)acetamido)hexan-
amide (23a). The compound was prepared via method B1 (79%). tR
9.33 min (YMC C8, 150 ꢁ 4.6 mm, 1 mL minꢀ1, 35ꢀ100% B, 30 min).
1H NMR (CD3OD): 1.24ꢀ1.35 (2H, m), 1.54 (2H, tt, J = 7.6, 7.6), 1.60
(2H, tt, J = 7.6, 7.6), 2.17 (2H, t, J = 7.5), 2.82ꢀ2.94 (3H, m), 3.00 (1H,
dd, J = 12.5, 3.5), 3.23ꢀ3.31 (2H, m), 3.33ꢀ3.40 (4H, m), 3.92 (1H,
dd, J = 9.6, 5.4), 3.97 (1H, dd, J = 9.6, 5.1), 4.07ꢀ4.16 (1H, m), 4.57
(2H, s), 4.96ꢀ5.04 (2H, m), 5.97 (1H, ddt, J = 16.9, 10.3, 6.5),
6.82ꢀ6.92 (3H, m), 7.05 (2H, app d, J = 9.0), 7.08ꢀ7.19 (4H, m),
7.20 (1H, d, J = 3.8), 7.21 (1H, d, J = 3.8), 7.35 (1H, s), 7.55 (2H, br d,
J = 7.7), 7.59ꢀ7.65 (3H, m), 8.12 (1H, dd, J = 3.8, 1.0). HRMS (TOF
ES+): calcd for C43H49BF2N5O5S 796.3516, found 796.3474 (MH+).
(()-N-[2-(3-(2-Allylphenoxy)-2-hydroxypropylamino)-
ethyl]-6-(2-(2-(4,4-difluoro-4,4a-dihydro-5-(thiophen-2-yl)-
4-bora-3a,4a-diaza-s-indacene-3-yl)vinyl)phenoxy)acetamido)-
hexanamide (23b). The compound was prepared via method B1 (29%).
tR 13.33 min (YMC C8, 150 ꢁ 4.6 mm, 1 mL minꢀ1, 5ꢀ100% B, 30 min;
solvent A, H2O; solvent B, MeCN, both A and B contain 0.06% TFA as
additive). HRMS (TOF ES+): calcd for C43H49BF2N5O5S 796.3516, found
795.3345 (MH+).
(S)-N-[3-Hydroxy-1-(2-(prop-2-en-1-yl)phenyl)-1,8,11-trioxa-
5-azatridecan-13-yl]-6-(2-(2-(4,4-difluoro-4,4a-dihydro-
5-(thiophen-2-yl)-4-bora-3a,4a-diaza-s-indacene-3-yl)vinyl)-
phenoxy)acetamido)hexanamide (26a). The compound was
prepared via method B1 (53%). tR 13.35 min (YMC C8, 150 ꢁ 4.6 mm,
1 mL minꢀ1, 5ꢀ100% B, 30 min; solvent A, H2O; solvent B, MeCN,
both A and B contain 0.06% TFA as additive). HRMS (TOF ES+): calcd
for C47H57BF2N5O7S 884.4040, found 884.4086 (MH+).
(()-N-[3-Hydroxy-1-(2-(prop-2-en-1-yl)phenyl)-1,8,11-
trioxa-5-azatridecan-13-yl]-6-(2-(2-(4,4-difluoro-4,4a-dihydro-
5-(thiophen-2-yl)-4-bora-3a,4a-diaza-s-indacene-3-yl)vinyl)-
phenoxy)acetamido)hexanamide (26b). The compound was
prepared via method B1 (68%). tR 9.57 min (YMC C8, 150 ꢁ 4.6 mm,
1 mL minꢀ1, 35ꢀ100% B, 30 min; solvent A, H2O; solvent B, MeCN,
both A and B contain 0.06% TFA as additive). HRMS (TOF ES+): calcd
for C47H57BF2N5O7S 884.4040, found 884.4045 (MH+).
3H-CGP 12177 Whole Cell Binding. Cells were grown to con-
fluence in white-sided tissue culture treated 96-well view plates. 3H-CGP
12177 whole cell competition binding was performed as previously
3
described,45 using H-CGP 12177 in the ranges of 0.71ꢀ3.14 nM (for
β1 and β2) and 4.64ꢀ63.0 nM (β3) (total volume 200 μL per well).
CRE-SPAP Production. Cells were grown to confluence in clear
plastic tissue culture treated 96-well plates, and CRE-SPAP secretion
into the media was measured between 5 and 6 h after the addition of
agonist as previously described.36
Confocal Imaging. Cells were grown in Labtek eight-well plates
(Nunc Nalgene, Rochester, NY) for at least 18 h and grown to 80ꢀ100%
confluence before imaging. Following washing of the cells in HEPES-
buffered saline solution (HBSS; 25 mM HEPES, 10 mM glucose,
146 mM NaCl, 5 mM KCl, 1 mM MgSO4, 2 mM sodium pyruvate,
1.3 mM CaCl2), live cell imaging was performed at room temperature
using a Zeiss LSM 710 laser scanning confocal microscope and a Zeiss
Plan-Neofluar 40 ꢁ 1.3 NA oil-immersion objective. A 633 nm HeNe
laser was used for the excitation of the BODIPY630/650ꢀ18a conjugate
with emission being detected using a 650 nm long-pass filter. The
pinhole diameter (1 Airy Unit; 1.1 μm optical slice), laser power, and
gain remained constant between experiments.
Data Analysis: Whole Cell Binding. All data points on each
competition binding curve were performed in triplicate and each 96-well
plate also contained six determinations of total and nonspecific binding. In all
cases where a KD value is stated, the competing ligand completely inhibited
the specific binding of 3H-CGP 12177. For compounds 14c and 14f, where
inhibition of binding was only 66% and 85%, respectively, an apparent KD
value is stated. A one-site sigmoidal response curve 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.
ð100 ꢁ AÞ
%uninhibited binding ¼ 100 ꢀ
þ NS
ð1Þ
(()-N-[2-(2-Hydroxy-3-(1H-indol-4-yloxy)propylamino)-
ethyl]-6-(2-(2-(4,4-difluoro-4,4a-dihydro-5-(thiophen-2-yl)-
4-bora-3a,4a-diaza-s-indacene-3-yl)vinyl)phenoxy)acetamido)-
hexanamide (31). The compound was prepared via method B (39%). tR
9.19 min (YMC C8, 150 ꢁ 4.6 mm, 1 mL minꢀ1, 35ꢀ100% B, 30 min;
solvent A, H2O; solvent B, MeCN, both A and B contain 0.06% TFA as
additive). HRMS (TOF ES+): calcd for C42H46BF2N6O5S 795.3312, found
795.3345 (MH+).
ðA þ IC50Þ
where A in the concentration of the competing ligand, IC50 is the con-
centration at which half of the specific binding of 3H-CGP 12177 has been
inhibited, and NS is the nonspecific binding.
From the IC50 value and the known concentration of 3H-CGP 12177,
a KD value was calculated using the equation
IC50
(()-N-[3-Hydroxy-1-(1H-indol-4-yl)-1,8,11-trioxa-5-azatri-
decan-13-yl]-6-(2-(2-(4,4-difluoro-4,4a-dihydro-5-(thiophen-
2-yl)-4-bora-3a,4a-diaza-s-indacene-3-yl)vinyl)phenoxy)-
acetamido)hexanamide (34). The compound was prepared via
KD
¼
ð2Þ
1 þ f½3H-CGP 12177ꢂ=KDð H-CGP 12177Þg
3
method B (68%). tR 9.40 min (YMC C8, 150 ꢁ 4.6 mm, 1 mL minꢀ1
,
Inhibition of Agonist-Stimulated CRE-SPAP Production.
Agonist responses were best described by a one-site sigmoidal
35ꢀ100% B, 30 min). 1H NMR (CD3OD): 1.21ꢀ1.32 (2H, m), 1.51
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Journal of Medicinal Chemistry
ARTICLE
concentration response curve (eq 3)
styryloxy)acetyl)aminohexanoic acid succinimidyl ester; Cbz, ben-
zyloxycarbonyl; CHO, chinese hamster ovary cell ; CGP 20712A,
1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-
4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol; CGP 12177,
4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-
2H-benzimidazol-2-one; FCS, fluorescence correlation spectros-
copy; GPCR, G-protein coupled receptor; ICI 118551, 3-
(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol; LSCM,
laser scanning confocal microscopy; MW, microwave; NBD, 7-
nitrobenzo-2-oxa-1,3-diazole.
Emax ꢁ ½Aꢂ
response ¼
ð3Þ
IC50 þ ½Aꢂ
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
The affinities of antagonist drugs were calculated (KD values) from
the shift of the agonist concentration responses in the presence of a fixed
concentration of antagonist using eq 4:
½Bꢂ
DR ¼ 1 þ
ð4Þ
KD
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G-protein coupled receptor target family. ChemMedChem 2006, 1,
760–782.
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].
Where clear partial agonism was observed (e.g., Figure 5), the affinity
was calculated according to the method45 of Stephenson using eq 5:
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Y ꢁ ½Pꢂ
1 ꢀ Y
½A2ꢂ ꢀ ½A1ꢂ
½A3ꢂ
KDðpartial agonistÞ ¼
where Y ¼
ð5Þ
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where [P] in the concentration of the partial agonist, [A1] in the
concentration of the agonist at the point where the partial agonist alone
agonist causes the same response, [A2] is the concentration of agonist
causing a given response above that achieved by the partial agonist, and
[A3] the concentration of the agonist, in the presence partial agonist,
causing the same stimulation as [A2].
(6) Sloan, E. K.; Priceman, S. J.; Cox, B. F.; Yu, S.; Pimente, M. A.;
Tangkanangnukul, V.; Arevalo, J. M. C.; Morizono, K.; Karanikolas,
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’ ASSOCIATED CONTENT
S
Supporting Information. Full experimental procedures
b
for compounds 12aꢀd, 12f, 13aꢀd, 13f, 14aꢀd, 14f, 16a,b, 17a,
b, 18a,b, 21b, 22b, 23b, 24a,b, 25a,b, 26a,b, 29ꢀ31. Thismaterialis
available free of charge via the Internet at http://pubs.acs.org
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’ AUTHOR INFORMATION
Corresponding Author
*B.K.: tel, +44-115-9513026; fax, +44-115-9513412; e-mail, barrie.
kellam@nottingham.ac.uk. S.J.H.: tel, +44-115-8230082; fax: +44-
115-8230081; e-mail, stephen.hill@nottingham.ac.uk.
(9) Kenakin, T.; Miller, L. J. Seven transmembrane receptors as
shapeshifting proteins: The impact of allosteric modulation and
functional selectivity on new drug discovery. Pharmacol. Rev. 2010,
62, 265–304.
Author Contributions
These authors contributed equally to this work
(10) Baker, J. G.; Hill, S. J. Multiple GPCR conformations and
signalling pathways: Implications for antagonist affinity estimates.
Trends Pharmacol. Sci. 2007, 28, 374–381.
(11) Williams, C.; Hill, S. J. GPCR signalling: Understanding the
pathway to successful drug discovery. Methods Mol. Biol. 2009, 552, 39–50.
(12) Hill, S. J. G-protein-coupled receptors: Past, present and future.
Br. J. Pharmacol. 2006, 147, S27–S37.
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pharmacologically relevant signal transduction molecules. Annu. Rev.
Pharmacol. Toxicol. 2008, 48, 359–391.
Notes
S.J.H. and B.K. are founding directors of the University of
Nottingham spin-off company CellAura Technologies Ltd.
R.M. is currently employed by CellAura Technologies Ltd.
’ ACKNOWLEDGMENT
(14) Tobin, A. B.; Butcher, A. J.; Kong, K. C. Location, location,
location. Site-specific GPCR phosphorylation offers a mechanism for
cell-type specific signalling. Trends Pharmacol. Sci. 2008, 29, 413–420.
(15) May, L. T.;Self, T. J.;Briddon, S. J.; Hill, S. J. The effect of allosteric
modulators on the kinetics of agonist-G protein-coupled receptor interac-
tions in single living cells. Mol. Pharmacol. 2010, 78, 511–523.
(16) Cordeaux, Y.; Briddon, S. J.; Alexander, S. P. H.; Kellam, B.;
Hill, S. J. Agonist-occupied A3 adenosine receptors exist within hetero-
geneous complexes in membrane microdomains of individual living
cells. FASEB J. 2008, 22, 850–860.
J.G.B. is a Wellcome Trust Clinician Scientist Fellow
(073377/Z/03/Z). This work was supported by The BBSRC
(Grant Number BB/0521581/1) and The University of Not-
tingham. The molecules described in this paper are the subject of
an international patent application, WO 2004/088312.
’ ABBREVIATIONS USED
β-AR, β-adrenoceptor; BODIPY 630/650 or BY630, 6-(((4,
4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)-
styryloxy)acetyl)aminohexanoic acid; BODIPY 630/650-X, SE, 6-
(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)-
(17) Baker, J. G.; Hill, S. J.; Summers, R. J. Evolution of β-blockers:
From anti-anginal drugs to ligand-directed signalling. Trends Pharmacol.
Sci. 2011, 32, 227–234.
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