Design, synthesis, and functional assessment of Cmpd-15 derivatives as negative allosteric modulators for the β2-adrenergic receptor

Article abstract of DOI:10.1016/j.bmc.2018.03.023

The β₂-adrenergic receptor (β₂AR), a G protein-coupled receptor, is an important therapeutic target. We recently described Cmpd-15, the first small molecule negative allosteric modulator (NAM) for the β₂AR. Herein we report in details the design, synthesis and structure-activity relationships (SAR) of seven Cmpd-15 derivatives. Furthermore, we provide in a dose-response paradigm, the details of the effects of these derivatives in modulating agonist-induced β₂AR activities (G-protein-mediated cAMP production and β-arrestin recruitment to the receptor) as well as the binding affinity of an orthosteric agonist in radio-ligand competition binding assay. Our results show that some modifications, including removal of the formamide group in the para-formamido phenylalanine region and bromine in the meta-bromobenzyl methylbenzamide region caused dramatic reduction in the functional activity of Cmpd-15. These SAR results provide valuable insights into the mechanism of action of the NAM Cmpd-15 as well as the basis for future development of more potent and selective modulators for the β₂AR based on the chemical scaffold of Cmpd-15.

Full text of DOI:10.1016/j.bmc.2018.03.023

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and functional assessment of Cmpd-15 derivatives as
negative allosteric modulators for the b2-adrenergic receptor
Kaicheng Meng a,c, Paul Shim b,c, Qingtin Wang a, Shuai Zhao a, Ting Gu a, Alem W. Kahsai b, Seungkirl Ahn b,
Xin Chen a,
⇑
a School of Pharmaceutical and Life Sciences, Changzhou University, Jiangsu 213164, China
b Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The b2-adrenergic receptor (b2AR), a G protein-coupled receptor, is an important therapeutic target. We
recently described Cmpd-15, the first small molecule negative allosteric modulator (NAM) for the b2AR.
Herein we report in details the design, synthesis and structure-activity relationships (SAR) of seven
Cmpd-15 derivatives. Furthermore, we provide in a dose-response paradigm, the details of the effects
of these derivatives in modulating agonist-induced b2AR activities (G-protein-mediated cAMP production
and b-arrestin recruitment to the receptor) as well as the binding affinity of an orthosteric agonist in
radio-ligand competition binding assay. Our results show that some modifications, including removal
of the formamide group in the para-formamido phenylalanine region and bromine in the meta-bro-
mobenzyl methylbenzamide region caused dramatic reduction in the functional activity of Cmpd-15.
These SAR results provide valuable insights into the mechanism of action of the NAM Cmpd-15 as well
as the basis for future development of more potent and selective modulators for the b2AR based on
the chemical scaffold of Cmpd-15.
Received 8 February 2018
Revised 12 March 2018
Accepted 14 March 2018
Available online xxxx
Keywords:
b2-Adrenergic receptor (b2AR)
Negative allosteric modulator
Synthesis
Structure-activity relationships
Binding competition assays
Ó 2018 Published by Elsevier Ltd.
1. Introduction
agonist-b2AR induced G-protein activities (as measured via cAMP
accumulation) and binds to inactive state form of the receptor with
The b2-adrenergic receptor (b2AR) is a prototypical G protein-
coupled receptor (GPCR) and is also an important therapeutic tar-
get for diseases such as cardiac arrhythmias, hypertension, and
other cardiovascular diseases.1,2 Drugs that target the b2AR, such
as b-blockers, are orthosteric b-adrenergic receptor antagonists
that bind to the endogenous adrenaline binding site.3–6 To date,
all known b-adrenergic agonists and antagonists are orthosteric
ligands. However, the possibility that allosteric modulators that
potentiate or attenuate the activity of orthosteric ligands might
possess enhanced therapeutic efficacy, selectivity, or other novel
therapeutic properties has raised interest in identifying and char-
acterizing such modulators for the b2AR.
We recently carried out high throughput screening of DNA-
encoded small-molecule libraries (DEL), comprising 190 million
different unique compounds, against purified human b2AR. This
in vitro affinity based screening approach yielded the first allosteric
b-blocker, named as Cmpd-157 (Scheme 1). The compound inhibits
low micro-molar binding affinity. More recently, we co-crystallized
and solved the structure of inactive b2AR in complex with Cmpd-
15, in the form of a polyethylene glycol-carboxylic acid derivative
(Cmpd-15PA). The structure reveals that the b2AR NAM Cmpd-15
(‘allosteric b-blocker’) binds to a pocket which is composed of
the cytoplasmic ends of transmembrane segments 1, 2, 6 and 7
(TM1, TM2, TM6 and TM7) as well as intracellular loop 1 (ICL1)
and helix 8 (H8). A proposed mechanism of allosteric antagonism
of Cmpd-15 is that this modulator prevents b2AR from coupling
to Gs, and blocks the interactions with arrestins.8 Herein we report
in details the design and synthesis of seven Cmpd-15 derivatives,
and present a more detailed dose dependent assessment for their
ability to modulate agonist-mediated b2AR activities in down-
stream functional and radioligand binding competition assays.
2. Design and synthesis of Cmpd-15 derivatives
The structure of Cmpd-15 is shown in Scheme 1. Guided by
knowledge of the initial hits from our in vitro affinity-based selec-
tion with DEL described in our previous paper7, we designed 7
derivatives, focused on strategic points on the chemical structure
⇑
Corresponding author.
E-mail address: xinchen@cczu.edu.cn (X. Chen).
These authors contributed equally to this work.
c
https://doi.org/10.1016/j.bmc.2018.03.023
0968-0896/Ó 2018 Published by Elsevier Ltd.
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K. Meng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
R4 = 4-OMe), and 2-cyclohexyl-2-(4-hydroxyphenyl)acetic acid
O
(14c, R4 = 4-OH), respectively, affording the desired final products
(15A1-15A7). While 14a is commercial available, 14b was pre-
pared by alkylation of 2-(4-methoxyphenyl)acetic acid with cyclo-
hexyl 4-methylbenzenesulfonate in the presence of n-butyllithium
(Scheme 3). Upon treatment with boron tribromide, 14b was
demethylated to give 14c in 55% yield. The identity of all the prod-
ucts and the intermediates was confirmed by their 1H NMR, 13C
NMR and high-resolution mass spectrometry (HRMS) properties.
NH2
O
O
H
N
N
H
N
H
M1
M3
M2
O
Br
Cmpd-15
Scheme 1. Chemical structure of Cmpd-15.
3. Pharmacological activity assays of Cmpd-15 derivatives
We conducted both cell-based assays and radioligand binding
assays to characterize the pharmacological activity of seven deriva-
tives of Cmpd-15 at the b2AR in a more systematic way than the
previous report.7 The cell-based assays include the Promega
GloSensor assay and the DiscoverX PathHunter assay. The Promega
GloSensor assay was performed to measure agonist-induced cAMP
production as a means of quantifying G-protein activation, and the
DiscoverX PathHunter assay was performed to measure agonist-
induced b-arrestin recruitment to the receptor. For the highly
amplified GloSensor assay, HEK-293 cells endogenously expressing
the b2AR were pretreated with the derivatives and then read after
the stimulation with the orthosteric agonist, isoproterenol (ISO).
For the stoichiometric DiscoverX PathHunter assay, U2OS cells sta-
bly expressing the chimeric b2V2R, which has increased phospho-
rylation on C-terminus tail, leading to more stable interaction
with b-arrestin than the native receptor, were pretreated with
the derivatives, and then stimulated with ISO. Competition binding
with 125I-cyanopindolol (CYP) was performed to determine the
derivatives’ ability to affect the binding of an orthosteric agonist
to the b2AR. Both the allosteric derivatives and the orthosteric ago-
nist isoproterenol (ISO) were introduced to reconstituted in High-
Density-Lipoparticles (HDLs, or nanodiscs). The orthosteric antago-
nist 125I-CYP was then introduced and allowed to compete with ISO
in a dose-wise manner.
of Cmpd-15; with the aim of investigating the influence of steric,
hydrophilic, and hydrophobic features on NAM activity of the
derivatives. For the convenience of our SAR analyses, the structure
of Cmpd-15 is divided into three subunits (Scheme 1) similar to as
previously described7: the (meta-bromobenzyl)methylbenzamide
(M1), (para-formamido)phenyl-alanine (M2), and cyclohexyl-2-
phenylacetamido (M3) regions. We made certain modifications in
each region. For example, in region M1, an additional meta-bro-
mine was introduced to the phenyl ring (derivative 15A5), the
meta-bromine was replaced with meta-fluorine (derivative 15A4),
and the meta-bromine was removed (15A3); in region M2, the
para-formamide group was moved to the meta-position (15A2),
and completely removed (15A1); in region M3, a methoxy group
(15A7) and a hydroxyl group (15A6) were introduced to the
para-position of the phenyl ring, respectively.
The synthetic route for the designed Cmpd-15 derivatives is
outlined in Scheme 2. The chiral building block, substituted (S)-
phenyl alanine 5a–5c, was prepared through asymmetric phase
transfer catalytic alkylation9–11 of substituted benzyl bromide with
diphenylamine glycine tert-butyl ester 3. The latter was obtained
by condensation of benzophenone with glycine tert-butyl ester in
refluxing toluene and in the presence of boron trifluoride diethyl
etherate. Under the catalysis of O-allyl-N-9-anthracene methyl
bromide cinchonine (3A), 3 was alkylated by substituted benzyl
bromide in toluene/chloroform (2:1) and at ꢀ40 °C, affording (S)-
3-halobenzyl-2-diphenylimine glycine tert-butyl ester (4a–4c) in
highly stereochemistry-controlled manner. For example, (S)-3-
(3,5-dibromobenzyl)-2-diphenylimine glycine tert-butyl ester
(4a) was obtained with 94.9% ee. After acidic hydrolysis in
hydrochloric acid, 4a–4c were converted into the corresponding
L-phenyl alanines 5a–5c, and then the amino group was protected
with Fmoc, affording Fmoc-protected L-phenyl alanines 6a–6c. The
acidic hydrolysis didn’t racemize the amino acids.12 For example,
the ee value of (9H-fluoren-9-yl)methyl (S)-(3,5-dibromo)-pheny-
lalanine (6a) is 94.7%. In the next step, Fmoc-protected L-pheny-
lalanine methylamides 7a-7d were obtained by condensation of
methylamine with the corresponding L-phenylalanines in the pres-
ence of O-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium hex-
afluorophosphate (HBTU) and hydroxybenzotriazole (HOBt).13
Upon treatment with piperidine in DMF,13 the Fmoc group in 7a–
7d was removed smoothly, giving L-phenylalanine methylamides
8a–8d with satisfactory yields.
4. Results and discussion
All the Cmpd-15 derivatives were systematically evaluated for
their ability to modulate agonist-induced b2AR activities in a con-
centration-dependent manner by using two cellular functional
assays (Gas signaling via cAMP accumulation measurement and
b-arrestin recruitment) as well as a competition radio-ligand bind-
ing assay. The results are summarized in Tables 1 and 2.
Table 1 is the summary of the data showing the maximum
degree of inhibition of Emax induced by a derivative relative to that
of Cmpd-15. The data are consistent with the results in our previ-
ous report, in which we evaluated the inhibitory activity of these
derivatives at a single concentration (50
maximum fold shift of ISO IC50 or EC50 values induced by the
derivatives at 50
M in 125I-CYP competition binding (IC50) as well
l
M).7 Table 2 shows the
l
as both the GloSensor and PathHunter cell-based assays (EC50).
The effect of Cmpd-15 and its derivatives 15A1–15A7 on b2AR-
mediated functional activities is presented in Figs. 1–3. After pre-
treatment with Cmpd-15 or its derivatives at various concentra-
tions, b2AR-mediated activity were measured in cells upon
stimulation with isoproterenol (ISO) in a dose-dependent manner:
cAMP production by the endogenously expressed b2AR (Fig. 1) and
b-arrestin recruitment to the exogenously expressed b2V2R (Fig. 2).
A dose–response curve of ISO competition binding to the b2AR
reconstituted in HDL particles (nanodiscs) with radiolabeled 125I-
CYP and ISO was obtained in the presence of 50 mM of Cmpd-15
or its derivatives (Fig. 3). For Cmpd-15, there were both a decrease
in Emax and a curve shift of the EC50 to the right in both the cAMP
With amines 8a–8d in hand, we turn our attention to the
remaining steps in the synthetic sequence. By employing HBTU/
HOBt as the amide coupling agents, 8a–8d were coupled with
Fmoc-L-4-carbamoylphenylalanine (9a, R3 = 4-formamide), Fmoc-
L-3-carbamoylphenylalanine (9b, R3 = 3-formamide) or Fmoc-L-
phenylalanine (9c, R3 = H), respectively, and dipeptides 10a–10f
were generated. Under the same reaction conditions used for
preparation of 5a–5c, the Fmoc group in 10a–10f was taken off
by piperidine and resulted in amines 11a–11f. In the final step,
11a–11f were reacted with 2-cyclohexyl-2-phenyl acetic acid
(14a, R4 = H), 2-cyclohexyl-2-(4-methoxyphenyl)acetic acid (14b,
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K. Meng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3
O
Ph
Ph
But
CO2
a
Ph
N
N
Br
But
Ph
+
H2N
CO2
O
N
Ph
3A
2
1
3
R1
R1
R1
Fmoc
c
e
b
d
N
Ph
But
NH2
CO2
HN
R2
CO2
R2
H
R2
H
CO2
:
:
:
=R
:
=R
6a R1 2=Br
:
=R
5a
R1 2=Br
R1=Br, R2
4a
R1 2=Br
R1=Br, R2
=H
:
=H
=H
6b R1=Br, R2
6c R1=F, R2
:
=H
=H
5b
4b
=H
:
:
5c
R1=F, R2
4c
R1=F, R2
Fmoc
HN
R1
Fmoc
R1
HN
R1
R3
R3
Fmoc
CO2H
HN
NH2
O
HN
9a-9c
g
O
H
f
H
N
N
O
R2
R2
R2
O
HN
:
=R
=R
10a R1 2=Br, R
:
:
:
:
3=4-formamide
R1=Br, R2=H, R3=3-formamide
:
:
=R
8a R1 2=Br
7a
7b
R1 2=Br
R1=Br, R2
:
:
=H
10b
=H
=H
8b R1=Br, R2
=H
10c R1=F, R2=H, R
3=4-formamide
3=4-formamide
:
:
8c R1=F, R2
7c
R1=F, R2
:
:
:
=R
=R2=H
8d R1
10d R1 2=H, R
=R2=H
R1
7d
=R3=H
10e R1=Br, R2
10f R1=Br, R2=H, R
3=4-formamide
R3
CO2H
H2N
HN
R4
R1
O
O
H
N
R3
R4
h
14a-14c
N
H
N
O
O
H
i
O
R1
R2
HN
R2
=R
R1 2=Br, R
:
=R3=R4=H
=R
:
:
:
:
:
11a
11b
11c
11d
11e
15A1 R1=Br, R2
3=4-formamide
R1=Br, R2=H, R
:
15A2 R1=Br, R2 4=H, R
3=3-formamide
3=4-formamide
3=4-formamide
3=4-formamide
R
4=OH, 3=4-formamide
3=3-formamide
:
=R2=R
15A3 R1
4=H, R
=R
R1=F, R2=H, R
3=4-formamide
3=4-formamide
=R
R1=Br, R2
:
:
:
:
15A4 R1=F, R2 4=H, R
R1 2=H, R
=R
=R3=H
15A5 R1 2=Br, R4=H, R
:
11f
15A6 R1=Br, R2=H, R
R1=Br, R2=H, R
3=4-formamide
15A7
R1=Br, R2=H, R
R
4=OMe, 3=4-formamide
Scheme 2. Synthetic route for Cmpd-15 derivatives 15A1–15A7. Reagents and condition: (a) BF3.Et2O, toluene, reflux, 8 h; (b) 3A, substituted benzyl bromide, 50% aq. KOH,
toluene/CHCl3, ꢀ40 °C, 72 h; (c) 6 N HCl, 100 °C, 3 h; (d) Fmoc-Cl, Na2CO3, dioxane, 0 °C-rt, 22 h; (e) MeNH2.HCl, HBTU, HOBt, DIEA, DMF, rt, 12 h; (f) piperidine, DMF, rt, 2 h;
(g) 9, HBTU, HOBt, DIEA, DMF, rt; (h) piperidine, DMF, rt; (i) 14a–14c, HBTU, HOBt, DIEA, DMF, rt.
H
CO2
H
CO2
OH
OTs
a
c
b
MeO
HO
14c
12
14b
13
Scheme 3. Synthetic routes for 14b and 14c. Reagents and condition: (a) TsCl, pyridine, rt, 12 h, 79%; (b) 2-(4-methoxyphenyl)acetic acid, n-BuLi, ꢀ78 °C–25 °C, 13 h, 43%; (c)
BBr3, DCM, 0 °C–25 °C, 40 h, 55%.
production and b-arrestin recruitment to the active receptor (Figs.
1A and 2A). There was a right curve shift in the 125I-CYP competi-
tion-binding assay (Fig. 3A). Overall, we recapitulated the inhibi-
tory activity of Cmpd-15 with the similar extent to what we
obtained in our previous report.7
For derivative 15A5 which contains a second meta-bromine
group in the region M1, there was both a decrease in Emax and a
curve shift of the EC50 to the right in both the cAMP production
and b-arrestin recruitment assays (Figs. 1F and 2F), although to a
lesser degree compared to Cmpd-15. We observed a greater degree
of inhibition mediated by 15A5 in b-arrestin recruitment than in
cAMP production. There was a right curve shift in the 125I-CYP
competition-binding assay (Fig. 3F). It is possible that this second
bromine group may reduce 15A5 functional activity by decreasing
the affinity of the compound to its binding pocket due to increased
steric effects.
When the bromine was replaced with fluorine in the region M1
(15A4), there was no significant inhibition of Emax or a curve shift
of the EC50 in both the cAMP production and b-arrestin recruitment
assays (Figs. 1E and 2E). There was a small curve shift of the IC50 to
Please cite this article in press as: Meng K., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.023

4
K. Meng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Table 1
robustly decreasing the affinity of the compound to its binding
pocket due to increased steric effects.
Comparison of the allosteric modulation activity of 15A1–15A7 to Cmpd-15.
After the formamide group was removed from the phenyl ring
in the region M2, there was no significant inhibition of Emax or a
curve shift of the EC50 for the resulting derivative 15A1 in both
of the cell-based functional assays (Figs. 1B and 2B). There was also
no curve shift of the IC50 in the 125I-CYP competition-binding assay
with 15A1 (Fig. 3B). These results indicate that the formamide
group also has a critical role in Cmpd-150s functional activity, as
supported by the previous X-ray crystallographic study.8
In the region M3, when a hydroxy group was introduced to the
para-position of the phenyl group, there was no significant inhibi-
tion of Emax or a curve shift of the EC50 for the resulting derivative
15A6 in either the Glosensor and the PathHunter assays, and there
was no curve shift of the IC50 in the 125I-CYP competition-binding
assay (Figs. 1G–3G). This suggests that the hydrophobic nature of
the M3 region is important for Cmpd-150s functional activity as
suggested by our previous X-ray crystallographic study.8
Compound (50 mM)
Inhibitory Activity (%)a
Cell-based activity
cAMP
b-Arr
Cmpd-15
15A1
15A2
15A3
15A4
15A5
15A6
15A7
100
100
11 10
46 10
12 11
21
50
15
23
65
7
4
4
2
5
4
10
38
-2
8
6
5
4
13
5
47
6
a
Values are expressed as percentages of the Cmpd-15 blockade level in each
assay and represent mean SEM obtained from at least three independent exper-
iments. Statistical analysis was performed as described in 6.4 Data Analysis of
Experimental Section. All of the values obtained with analogs are significantly
different (P < 0.001) compared to the inhibition obtained by Cmpd-15, which was
normalized to 100%.
For derivative 15A7 which contains a para-methoxy group in
the region M3, there was no significant inhibition of Emax or a curve
shift of the EC50 in the cAMP production assay (Fig. 1H) while there
was both a small decrease in Emax and a curve shift of the EC50 to
the right in the b-arrestin recruitment assays (Fig. 2H). We also
observed no curve shift of the IC50 in the 125I-CYP competition-
binding assay with 15A7 (Fig. 3H). The presence of b-arrestin
recruitment inhibition strongly supports the claim that the
hydrophobic nature of the region M3 is important for Cmpd-150s
functional activity, as a methoxy group is less hydrophilic than a
hydroxyl group.
Table 2
Comparison of the EC50 fold shift for derivatives 15A1–15A7 in the 125I-CYP
competition binding and cell-based assays.
Compound (50
mM)
IC50 or EC50 Fold Shifta
125I-CYP competition
binding
Cell-based activity
cAMP
b-Arr
Cmpd-15
15A1
5.4 0.68
0.9 0.03***
3.6 0.62
0.7
7.0 1.32
1.5
0.09***
0.14***
Our SAR analysis here in this study is also consistent with
molecular interactions observed between Cmpd-15 and the resi-
dues of the binding site in the recently solved co-crystal structure
of b2AR–Cmpd-15 PA (Fig. 4). According to the co-crystal structure
of Cmpd-15PA with b2AR,8 the meta-bromobenzyl ring in region
15A2
15A3
1.3 0.06***
1.1 0.30***
1.4 0.27** 1.4 0.29**
1.1 0.28** 1.3
0.07***
15A4
2.1 0.06***
0.9
0.8
0.12***
2.3 0.10
0.09***
2.6 0.28**
15A5
15A6
4.6 0.37
1.2 0.44***
M1 of Cmpd-15PA forms a cation-
p
interaction with Arg63ICL1 in
1.2 0.22** 1.1
0.09***
1.1
0.25***
ICL1 of the b2AR; the para-formamido group in region M2 of
Cmpd-15PA forms polar interactions with side chains of
Thr2746.36 in TM6 and Arg3287.55 in TM7 of the b2AR. For region
M3, the cyclohexyl and phenyl rings form hydrophobic interac-
tions with the hydrophobic residues from b2AR, composing of
Val541.53 and Ile581.57 at the end of TM1, Leu64ICL1 in ICL1,
Ile722.43 in TM2, Leu2756.37 in TM6, Tyr3267.53 in TM7, and
15A7
0.8 0.07***
1.2
0.18***
a
Values are expressed as the fold-shift of either the EC50 value (cell-based
assays) or the IC50 value (competition binding assay) of the ISO curve in the pres-
ence of each derivative compared to that obtained DMSO-treated control curve.
They represent mean SEM obtained from at least three independent experiments.
Statistical analysis was performed as described in 6.4 Data Analysis of Experimental
Section. **P < 0.01; ***P < 0.001 compared to the fold shift of the EC50 obtained by
Cmpd-15.
8
Phe3328.50 in H8.
5. Conclusion
the right in the 125I-CYP competition-binding assay (Fig. 3E). It
appears to be the smaller size of the fluorine group which causes
the lack of allosteric activity of the analog since bromine and fluo-
rine are comparable in electronegativity.
After the bromine group was removed from the phenyl ring in
the region M1, the resulting derivative 15A3 has no significant
inhibition of Emax or a curve shift of the EC50 in both of the cell-
based functional assays (Figs. 1D and 2D). Also, there was no curve
shift of the IC50 in the 125I-CYP competition-binding assay (Fig. 3D).
This data shows that the bromine group has a critical role in Cmpd-
150s functional activity, also supported by the previous X-ray crys-
tallographic study.8
In the region M2, when the formamide group is in the meta-
position compared to the para-position of Cmpd-15 (15A2), there
was both a decrease in Emax and a curve shift of the EC50 to the
right in both the cAMP production and b-arrestin recruitment
assays, although to a lesser degree compared to Cmpd-15 (Figs.
1C and 2C). There was no curve shift of the IC50 in the 125I-CYP
competition-binding assay (Fig. 3C). It is possible that this meta-
position of the formamide reduces 15A20s functional activity by
We have designed and synthesized seven derivatives of Cmpd-
15 and carried out a structure-activity relationship study to exam-
ine their effects on agonist-induced b2AR activities. The pharmaco-
logical profiles of these analogs were evaluated for their effects at
agonist-mediated Gas signaling (via cAMP accumulation measure-
ment) and b-arrestins recruitment to active receptor as well as
competitive radio-ligand agonist binding experiments, all with a
dose-response paradigm. The present study demonstrates that
Cmpd-15 derivatives with formamide group in the region M2 as
well as bromine group in the region M1 are critical in its ability
for modulating the agonist-induced activity of b2AR. These were
observed by the fact that complete deletions of the two groups dra-
matically decreased the agonist-induced b2AR G-protein activity
and recruitment of b-arrestin compared to the parent compound.
It was also found that the increased polarity in region M3 is closely
associated with the loss of functional effects of Cmpd-15 at the
agonist induced b2AR activities. Hence, this further ascertains the
M3 region of Cmpd-150s interaction with the core hydrophobic
residues within the b2AR allosteric site, consistent with the pro-
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K. Meng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5
Fig. 1. Pharmacological activity of Cmpd-15 and its derivatives 15A1–15A7: cAMP production by the endogenously expressed b2AR. (A) Cmpd-15, (B) 15A1, (C) 15A2, (D)
15A3, (E) 15A4, (F) 15A5, (G) 15A6, (H) 15A7. Values were expressed as percentages of the maximal level of the isoproterenol-induced activity in the vehicle (DMSO) control.
Points on curves represent mean SEM obtained from at least three independent experiments done in duplicate.
Fig. 2. Pharmacological activity assays for Cmpd-15 and its derivatives 15A1–15A7: b-arrestin recruitment to the stably over-expressed b2V2R. (A) Cmpd-15, (B) 15A1, (C)
15A2, (D) 15A3, (E) 15A4, (F) 15A5, (G) 15A6, (H) 15A7. Values were expressed as percentages of the maximal level of the isoproterenol-induced activity in the vehicle
(DMSO) control. Points on curves represent mean SEM obtained from at least three independent experiments done in duplicate.
posed modes of binding of the compound in the recently solved X-
ray crystal structure of Cmpd-15 in complex with b2AR. Together,
the identification of the Cmpd-15 type chemical preferences and
detailed characterization of the novel Cmpd-15 binding allosteric
site at the b2AR should provide insight into designing and develop-
ing specific therapies in blocking b-receptor activities.
All moisture-sensitive reactions were carried out using anhydrous
solvents and under nitrogen atmosphere. 1H and 13C NMR spectra
were recorded on a Bruker Avance III spectrometer (300 MHz and
400 MHz) using TMS as the internal standard. High resolution mass
spectrometry (HRMS) was performed on Agilent Technologies
6540 UHD Accurate-Mass Q-TOF. Flash column chromatography
was performed with silica gel (300–400 mesh).
6. Experimental Section
6.1.1. (9H-Fluoren-9-yl)methyl (S)-(3,5-dibromo)phenylalanine (6a)
To a solution of benzophenone (3g, 16.4 mmol) and glycine tert-
butyl ester (1.8 g, 13.7 mmol) in anhydrous toluene (18 mL) was
added boron trifluoride diethyl etherate (0.12 mL), and the mixture
was refluxed at 130 °C for 8 h. After removal of the solvent in
6.1. Chemistry
All chemical reagents were purchased from Energy Chemicals
and Aladdin Chemicals (Shanghai, China) and used as received.
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6
K. Meng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Fig. 3. Pharmacological activity assays for Cmpd-15 and its derivatives 15A1-15A7: ISO competition binding to the b2AR reconstituted in nanodiscs with 125I-CYP. (A) Cmpd-
15, (B) 15A1, (C) 15A2, (D) 15A3, (E) 15A4, (F) 15A5, (G) 15A6, (H) 15A7. Values were expressed as percentages of the maximal level of 125I-CYP binding in the vehicle (DMSO)
control. Points on curves represent mean SEM obtained from at least three independent experiments done in duplicate.
Fig. 4. 2D projection of the interactions of Cmpd-15 with the allosteric binding site at the b2AR. Schematic representations of the favored interactions (polar and nonpolar) of
Cmpd-15 on the b2AR (PDB: 5X7D).
vacuo, the residue was dissolved in EtOAc (100 mL), and washed
with H2O (2 ꢁ 30 mL) and brine (2 ꢁ 30 mL), and dried (anhydrous
Na2SO4). The crude product was purified by flash column chro-
matography (eluting with hexane/EtOAc, 20:1) to afford N-
(diphenylamine)glycine tert-butyl ester (3) (1.4 g, 40% yield) as
white solid. M.p. 112–113 °C; 1H NMR (300 MHz, CDCl3): d 1.46
(s, 9H), 4.12 (s, 2H), 7.17–7.20 (m, 2H), 7.30–7.48 (m, 6H), 7.65–
7.68 (m, 2H); 13C NMR (75 MHz, CDCl3): d 28.2, 56.4, 81.2, 128.1,
128.9, 130.5, 139.5, 170.0, 171.6; HRMS (ESI, positive): Calcd. for
50% KOH aqueous solution (0.44 mL, 7.8 mmol) was added to a
stirred solution of 3 (458 mg, 1.55 mmol), 3,5-dibromobenzyl bro-
mide (2.55 g, 7.76 mmol, 5 equip) and O-allyl-N-9-anthracene
methyl bromide cinchonine (3A) (94 mg, 0.15 mmol) in toluene/
chloroform (22 mL, 2:1 v/v) at ꢀ40 °C, and the whole mixture
was stirred at the same temperature for 72 h. After being warmed
to ambient temperature, the reaction mixture was diluted with
H2O (100 mL), and the resulting mixture was extracted with EtOAc
(3 ꢁ 80 mL). The combined EtOAc extracts were washed with brine
(2 ꢁ 50 mL), and dried (anhydrous Na2SO4). The crude product was
C
19H21NO2Na [M+Na]+ 318.1465; observed: 318.1463.
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K. Meng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
7
purified by flash column chromatography (eluting with hexane/
EtOAc, 20:1) to give (S)-3-(3,5-dibromobenzyl)-2-diphenylimine
glycine tert-butyl ester (4a) as light yellow oil (0.7 g, 83% yield).
6.1.4. General procedure for preparation of compounds 7a–7d
Methylamine hydrochloride (87 mg, 1.3 mmol) and N,N-diiso-
propylethylamine (DIEA, 250 mg, 2 mmol) was added successively
to an ice-cold stirred solution of the substituted (S)-phenylalanine
6a–6c (0.64 mmol), HOBt (174 mg, 1.3 mmol) and HBTU (488 mg,
1.3 mmol) in DMF (6 mL) at 0 °C. The reaction mixture was stirred
for 30 min at the same temperature, and then allowed to warm to
ambient temperature while the stirring was continued for addi-
tional 12 h. The solvents and volatiles were removed under the
reduced pressure, and the residue was dissolved in EtOAc (100
mL), and then washed with saturated NaHCO3 solution (30 mL)
and brine (2 ꢁ 30 mL), respectively, and dried (anhydrous Na2SO4).
After the solvent was concentrated, the crude product was crystal-
lized from EtOAc to give the desired product as white fluffy solid.
½
a 2D0
ꢂ
ꢀ 89:9 (c = 1.38, CH2Cl2), 94.9% ee; 1H NMR (300 MHz, CDCl3):
d 1.37 (s, 9H), 3.01–3.15 (m, 2H), 4.05 (q, J = 4.5 Hz, 1H), 6.51–6.57
(m, 3H), 6.70 (d, J = 6.0 Hz, 2H), 7.19–7.26 (m, 6H), 7.27–7.33 (m,
2H); 13C NMR (75 MHz, CDCl3): d 28.2, 39.8, 65.3, 81.4, 125.4,
128.4, 128.9, 130.3, 132.7, 132.9, 137.7, 139.6, 170.8, 170.9; HRMS
(ESI, positive): Calcd. for
C
26H25Br2NO2 [M+H]+ 542.0252,
544.0236; observed: 542.0326, 544.0310.
A mixture of 4a (132 mg, 0.36 mmol) and 6 M HCl (5 mL) was
heated at 100 °C for 3 h, and then the solvent and volatiles were
removed by a rotary evaporator to give (S)-3,5-dibromo phenylala-
nine hydrochloride (5a) (100 mg, 78% yield) as white solid. The
product was used for the next step without further purification.
M.p. 245–246 °C; 1H NMR (300 MHz, DMSO-d6): d 3.16 (q, J = 4.5
Hz, 2H), 4.18 (t, J = 4.5 Hz, 1H), 7.56 (s, 2H), 7.74 (s, 1H); 13C
NMR (75 MHz, DMSO-d6): d 34.6, 52.7, 122.5, 131.8, 132.2, 140.1,
170.0; HRMS (ESI, positive): Calcd. for C9H9Br2NO2 [M+H]+
321.9000, 323.8980; observed: 321.9073, 323.9053.
6.1.4.1.
(9H-Fluoren-9-yl)methyl
(S)-(3-(3,5-dibromophenyl)-1-
(methylamino)-1-oxopropan-2-yl)carbamate (7a). 56% yield; 1H
NMR (400 MHz, DMSO-d6): d 2.59 (d, J = 6.0 Hz, 3H), 2.71–2.76
(m, 1H), 2.95 (dd, J = 18.2, 5.2 Hz, 1H), 4.09–4.21 (m, 4H), 7.26–
7.43 (m, 4H), 7.56–7.87 (m, 6H), 7.87 (d, J = 10.0 Hz, 2H), 8.01 (d,
J = 6.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): d 26.0, 37.2, 47.0,
56.5, 66.2, 120.6, 122.5, 125.8, 127.5, 128.1, 131.8, 141.0, 141.1,
To an ice-cold solution of 5a (135 mg, 0.37 mmol), dioxane (0.5
mL) and 10% Na2CO3 aqueous solution (1 mL) was added dropwise
a solution of Fmoc-Cl (97 mg, 0.37 mmol) in dioxane (1 mL). The
mixture was stirred at 0 °C for 4 h, and then warmed to ambient
temperature with the stirring continued for additional 18 h. The
reaction was quenched by adding 2 M HCl (2 mL) and H2O (40
mL). The resulting mixture was extracted with EtOAc (2 ꢁ 60
mL), and the combined extracts were washed with brine (2 ꢁ 30
mL), and dried. The crude product was purified by flash column
chromatography (eluting with hexane/EtOAc, 20:1) to give (9H-flu-
oren-9-yl)methyl (S)-(3,5-dibromo)phenylalanine (6a) (105 mg,
51% yield) as white solid. M.p. 238–239 °C; 94.7% ee. 1H NMR
(400 MHz, DMSO-d6): d 2.84 (q, J = 10.8 Hz, 1H), 3.10 (dd, J = 13.7,
3.8 Hz, 1H), 4.15–4.22 (m, 4H), 7.29 (q, J = 8.0 Hz, 2H), 7.40 (t, J =
7.2 Hz, 2H), 7.53 (d, J = 1.2 Hz, 2H), 7.60–7.63 (m, 2H), 7.68 (s,
1H), 7.76 (d, J = 6.3 Hz, 1H), 7.86 (d, J = 5.7 Hz, 2H); 13C NMR
(DMSO-d6, 100 MHz): d 35.6, 46.6, 55.0, 65.8, 120.1, 122.1, 125.2,
125.3, 127.1, 127.7, 131.5, 140.7, 143.0, 143.8, 156.0, 172.9; HRMS
143.8, 144.2, 156.3, 171.9; HRMS (ESI, positive): Calcd. for C25H22
-
Br2N2O3Na [M+Na]+ 578.9889, 580.9869; observed: 578.9886,
580.9869.
6.1.4.2. (9H-Fluoren-9-yl)methyl (S)-(3-(3-bromophenyl)-1-(methy-
lamino)-1-oxopropan-2-yl)carbamate (7b). 94% yield; 1H NMR (300
MHz, DMSO-d6): d 2.56 (d, J = 4.8 Hz, 3H), 2.79–2.98 (m, 4H), 3.16
(s, 1H), 4.45 (d, J = 6.8 Hz, 1H), 7.14–7.44 (m, 7H), 7.75 (d, J = 8.4
Hz, 2H), 7.90 (d, J = 4.2 Hz, 2H), 8.11 (d, J = 7.8 Hz, 1H); 13C NMR
(75 MHz, DMSO-d6): d 25.6, 37.2, 46.6, 56.2, 65.7, 120.1, 121.4,
125.4, 126.3, 127.7, 128.0, 129.1, 129.9, 132.7, 140.7, 141.0,
143.9, 155.9, 171.6; HRMS (ESI, positive): Calcd. for C25H23BrN2O3
[M+H]+ 501.0784, 503.0764; observed: 501.0779, 503.0763.
6.1.4.3. (9H-Fluoren-9-yl)methyl (S)-(3-(3-fluorophenyl)-1-(methy-
lamino)-1-oxopropan-2-yl)carbamate (7c). 64% yield; 1H NMR
(300 MHz, DMSO-d6): d 2.60 (d, J = 6.0 Hz, 3H), 2.79 (t, J = 14.0
Hz, 1H), 2.98 (dd, J = 13.5, 5.6 Hz, 1H), 4.12–4.19 (m, 4H), 7.02 (t,
J = 9.0 Hz, 1H), 7.12–7.43 (m, 7H), 7.63 (t, J = 6.0 Hz, 2H), 7.71 (d,
J = 8.1 Hz, 1H), 7.86–7.99 (m, 3H); 13C NMR (75 MHz, DMSO-d6):
d 25.7, 37.3, 46.7, 56.2, 65.8, 113.3, 115.9, 116.2, 120.2, 125.5,
127.2, 127.8, 130.1, 140.8, 141.4, 143.8, 143.9, 155.9, 171.7; HRMS
(ESI, negative): Calcd. for
543.9588; observed: 541.9600, 543.9581.
C
24H19Br2NO4 [MꢀH]ꢀ 541.9608,
6.1.2. (9H-Fluoren-9-yl)methyl (S)-(3-bromo)phenylalanine (6b)
By following the procedure used for preparing 6a, (9H-fluoren-
9-yl)methyl (S)-(3-bromo)phenylalanine (6b) was obtained by
replacing 3,5-dibromobenzyl bromide with 3-bromobenzyl bro-
mide. 1H NMR (300 MHz, DMSO-d6): d 2.82–2.90 (m, 1H), 3.09
(dd, J = 13.8, 4.2 Hz, 1H), 4.14–4.22 (m, 4H), 7.21–7.34 (m, 4H),
7.41 (dd, J = 7.5, 1.2 Hz, 3H), 7.52 (s, 1H), 7.61 (t, J = 6.6 Hz, 2H),
7.80 (d, J = 8.4 Hz, 3H); 13C NMR (75 MHz, DMSO-d6): d 36.6,
46.7, 56.0, 65.7, 120.2, 125.3, 127.2, 127.8, 128.1, 129.3, 130.0,
132.8, 140.8, 141.3, 143.0, 143.9, 156.0, 172.9; HRMS (ESI, posi-
tive): Calcd. for C24H20BrNO4Na [M+Na]+ 488.0468, 490.0447;
observed: 488.0466, 490.0447.
(ESI, positive): Calcd. for
observed: 441.1580.
C
25H23FN2O3Na [M+Na]+ 441.1585;
6.1.4.4. (9H-Fluoren-9-yl)methyl (S)-(1-(methylamino)-1-oxo-3-
phenylpropan-2-yl)carbamate (7d). 88% yield; 1H NMR (300 MHz,
DMSO-d6): d 2.58 (d, J = 6.0 Hz, 3H), 2.78 (dd, J = 18.0, 8.4 Hz,
1H), 2.93–2.99 (m, 1H), 4.10–4.18 (m, 4H), 7.17–7.44 (m, 10H),
7.62–7.67 (m, 3H), 7.87 (d, J = 9.6 Hz, 2H), 7.95 (s, 1H); 13C NMR
(75 MHz, DMSO-d6): d 26.0, 37.7, 47.0, 56.5, 66.1, 113.4, 116.5,
120.6, 125.8, 127.5, 128.1, 130.3, 130.4, 141.1, 141.8, 156.3,
172.1; HRMS (ESI, positive): Calcd. for C25H24N2O3Na [M+Na]+
423.1679; observed: 423.1679.
6.1.3. (9H-Fluoren-9-yl)methyl (S)-(3-fluoro)phenylalanine (6c)
By following the procedure used for preparing 6a, (9H-fluoren-
9-yl)methyl (S)-(3-fluoro)phenylalanine (6c) was obtained by
replacing 3,5-dibromobenzyl bromide with 3-fluorobenzyl bro-
mide. M.p. 145–148 °C; 1H NMR (300 MHz, DMSO-d6): d 2.88–
2.96 (m, 1H), 3.15 (dd, J = 17.9, 6.4 Hz, 1H), 4.15–4.29 (m, 4H),
7.01–7.18 (m, 3H), 7.26–7.42 (m, 8H), 7.62 (dd, J = 9.6, 5.6 Hz,
3H); 13C NMR (75 MHz, DMSO-d6): d 36.6, 47.0, 55.7, 66.1, 113.8,
116.6, 120.6, 125.5, 125.7, 127.5, 128.1, 130.4, 141.1, 141.3,
144.2, 156.4, 173.7; HRMS (ESI, negative): Calcd. for C24H20FNO4
[MꢀH]ꢀ 404.1304; observed: 404.1300.
6.1.5. General procedure for preparation of compounds 8a–8d
To a stirred solution of 7a–7d (0.21 mmol) in DMF (4 mL) was
added piperidine (2 mL) at room temperature. The reaction mix-
ture was stirred at ambient temperature and under nitrogen atmo-
sphere for 2 h. The solvent and volatiles were removed under
reduced pressure, and the residue was purified by flash column
chromatography (eluting with DCM/MeOH, 20:1) to afford (S)-2-
amino-3-aryl-N-methylpropanamides (8a–8d) as light yellow
solid.
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K. Meng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
6.1.5.1. (S)-2-Amino-3-(3,5-dibromophenyl)-N-methylpropanamide
(8a). 90% yield; 1H NMR (300 MHz, DMSO-d6): d 2.49 (d, J = 1.8
Hz, 3H), 2.51–2.63 (m, 1H), 2.85 (dd, J = 13.8, 6.0 Hz, 1H), 3.31
(dd, J = 8.4, 5.1 Hz, 1H), 7.18–7.26 (m, 2H), 7.36–7.41 (m, 1H),
7.81 (d, J = 4.5 Hz, 1H); 13C NMR (75 MHz, DMSO-d6): d 25.8,
40.5, 56.4, 122.4, 131.4, 131.8, 144.5, 174.8; HRMS (ESI, positive):
Calcd. for C10H12Br2N2ONa [M+Na]+ 356.9209, 358.9188; observed:
356.9208, 358.9192.
(m, 4H), 4.42–4.50 (m, 1H), 7.18–7.43 (m, 11H), 7.57–7.62 (m,
2H), 7.76 (d, J = 8.1 Hz, 2H), 7.86–7.91 (m, 3H), 8.20 (d, J = 8.1 Hz,
1H); 13C NMR (75 MHz, DMSO-d6): d 25.6, 35.9, 46.6, 53.9, 54.2,
56.0, 65.8, 120.2, 121.5, 125.4, 126.4, 127.4, 127.8, 128.2, 129.3,
130.3, 132.4, 137.7, 140.6, 140.8, 141.6, 141.7, 143.8, 143.9,
155.8, 167.9, 171.0, 171.2, 171.3; HRMS (ESI, positive): Calcd. for
C
35H33BrN4O5Na [M+Na]+ 691.1527, 693.1506; observed:
691.1522, 693.1496.
6.1.5.2.
(S)-2-Amino-3-(3-bromophenyl)-N-methylpropanamide
6.1.6.3. (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-3-(3-fluorophenyl)-1-
(methylamino)-1-oxopropan-2-yl)amino)-3-(4-carbamoylphenyl)-1-
oxopropan-2-yl)carbamate (10c). (S)-2-Amino-3-(3-fluorophenyl)-
N-methylpropanamide was used for replacing (S)-2-amino-3-
(3,5-dibromophenyl)-N-methylpropanamide in the described pro-
cedure, and 10c was obtained in 64% yield. 1H NMR (300 MHz,
DMSO-d6): d 2.56 (d, J = 6.0 Hz, 3H), 2.68–2.88 (m, 2H), 2.96–3.01
(m, 2H), 4.11–4.32 (m, 4H), 4.52 (dd, J = 10.8, 6.0 Hz, 1H), 6.95–
7.06 (m, 3H), 7.22–7.42 (m, 8H), 7.61 (t, J = 6.4 Hz, 3H), 7.77–7.88
(m, 8H), 7.86 (d, J = 7.5 Hz, 1H); 13C NMR (75 MHz, DMSO-d6): d
25.7, 37.9, 38.3, 46.6, 53.9, 56.0, 65.8, 113.4, 116.2, 120.2, 125.4,
127.2, 127.8, 129.9, 130.0, 132.4, 140.7, 140.8, 141.7, 143.8,
143.9, 155.8, 160.5, 163.7, 167.9, 171.0, 171.3; HRMS (ESI, posi-
tive): Calcd. for C35H33FN4O5Na [M+Na]+ 631.2327; observed:
631.2322.
(8b). 75% yield; 1H NMR (300 MHz, DMSO-d6): d 2.49 (d, J = 4.8
Hz, 3H), 2.51–2.63 (m, 1H), 2.88 (dd, J = 18.6, 6.8 Hz, 1H), 3.31
(dd, J = 11.2, 6.4 Hz, 1H), 7.18–7.26 (m, 2H), 7.36–7.41 (m, 1H),
7.81 (d, J = 6.0 Hz, 1H); 13C NMR (75 MHz, DMSO-d6): d 25.5,
40.2, 56.3, 121.5, 128.5, 129.0, 130.2, 132.1, 142.1, 174.7; HRMS
(ESI, positive): Calcd. for
259.0264; observed: 257.0285, 259.0265.
C
10H13BrN2ONa [M+Na]+ 257.0284,
6.1.5.3.
(S)-2-Amino-3-(3-fluorophenyl)-N-methylpropanamide
(8c). 91% yield; 1H NMR (400 MHz, DMSO-d6): d 2.53 (d, J = 2.0
Hz, 2H), 2.58–2.68 (m, 1H), 2.93 (dd, J = 17.6, 6.4 Hz, 1H), 6.99–
7.04 (m, 3H), 7.27 (dd, J = 10.4, 9.6 Hz, 1H), 7.86 (d, J = 4.4 Hz,
1H); 13C NMR (100 MHz, DMSO-d6): d 25.4, 40.1, 56.3, 121.4,
128.5, 128.9, 130.2, 132.0, 142.0, 174.7; HRMS (ESI, positive):
Calcd. for C10H13FN2O [M+H]+ 197.1085; observed:197.1090.
6.1.6.4. (9H-Fluoren-9-yl)methyl ((S)-3-(4-carbamoylphenyl)-1-(((S)-
1-(methylamino)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-
2-yl)carbamate (10d). (S)-2-Amino-3-phenyl-N-methylpropana-
mide was used for replacing (S)-2-amino-3-(3,5-dibromophenyl)-
N-methylpropanamide in the described procedure, and 10d was
obtained in 93% yield. 1H NMR (300 MHz, DMSO-d6): d 2.56 (d,
J = 6.0 Hz, 3H), 2.73–2.84 (m, 2H), 2.94 (dd, J = 10.8, 7.2 Hz, 2H),
4.12–4.29 (m, 4H), 4.52 (dd, J = 10.8, 6.2 Hz, 1H), 7.18–7.43 (m,
12H), 7.57 (t, J = 9.2 Hz, 3H), 7.76 (6, J = 10.8 Hz, 2H), 7.86–8.22
(m, 5H); 13C NMR (75 MHz, DMSO-d6): d 25.6, 37.3, 37.6, 46.6,
53.8, 55.9, 65.7, 120.1, 125.4, 126.4, 127.1, 127.7, 128.0, 129.1,
129.9, 132.7, 140.3, 140.7, 141.6, 143.7, 143.8, 155.7, 167.8,
170.9, 171.2; HRMS (ESI, positive): Calcd. for C35H34N4O5Na [M
+Na]+ 613.2421; observed: 631.2322.
6.1.5.4.
(S)-2-Amino-N-methyl-3-phenylpropanamide
(8d). 70%
yield; m.p. 58–60 °C; 1H NMR (300 MHz, DMSO-d6): d 2.66 (d, J =
4.8 Hz, 3H), 2.88–2.95 (m, 1H), 3.07 (dd, J = 13.2, 4.2 Hz, 1H), 4.56
(s, 1H), 7.28–7.57 (m, 3H), 7.74–8.23 (m, 1H), 8.39 (d, J = 5.2 Hz,
1H); 13C NMR (75 MHz, DMSO-d6): d 34.5, 40.1, 52.7, 122.5,
131.8, 132.2, 140.1, 170.0; HRMS (ESI, positive): Calcd. for
C
10H14N2O [M+H]+ 179.1179; observed: 179.1180.
6.1.6. General procedure for preparation of compounds 10a–10f
HOBt (71 mg, 0.52 mmol) and HBTU (199 mg, 0.52 mmol) was
added to a stirred solution of phenylalanine derivatives 9a–9c
(0.52 mmol) in DMF (6 mL) at rt. After the mixture was cooled to
0 °C, amines 8a–8d (0.7 mmol) and DIEA (1 mmol) were intro-
duced, respectively. The whole reaction mixture was stirred at rt
for 12 h, the solvents and volatiles were removed under the
reduced pressure. The solid residue was crystallized from dichlor-
omethane to give the desired products as white solid.
6.1.6.5. (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-3-(3-bromophenyl)-1-
(methylamino)-1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-
yl)carbamate (10e). Fmoc-L-phenylalanine was used for replacing
Fmoc-L-4-carbamoylphenylalanine, as well as (S)-2-amino-3-(3-
bromophenyl)-N-methylpropanamide replacing of (S)-2-amino-3-
(3,5-dibromophenyl)-N-methylpropanamide, in the described pro-
cedure, and 10e was obtained in 67% yield. 1H NMR (400 MHz,
DMSO-d6): d 2.56 (d, J = 5.6 Hz, 3H), 2.68–2.88 (m, 2H), 2.95 (dd,
J = 11.6, 6.8 Hz, 2H), 4.01–4.51 (m, 5H), 7.16–7.42 (m, 10H), 7.57
(t, J = 10.0 Hz, 3H), 7.78–8.25 (m, 7H); 13C NMR (100 MHz,
DMSO-d6): d 25.6, 37.7, 46.6, 53.9, 56.0, 65.8, 120.2, 125.3, 125.4,
126.5, 127.2, 127.4, 127.7, 128.1, 129.1, 130.0, 132.4, 132.8,
140.3, 140.8, 141.6, 143.8, 143.9, 167.9, 171.0, 171.3; HRMS (ESI,
6.1.6.1. (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-3-(3,5-dibromophenyl)-
1-(methylamino)-1-oxopropan-2-yl)amino)-3-(4-carbamoylphenyl)-
1-oxopropan-2-yl)carbamate (10a). 76% yield; 1H NMR (300 MHz,
DMSO-d6): d 2.57 (d, J = 4.5 Hz, 3H), 2.73–2.85 (m, 2H), 2.94–3.01
(m, 2H), 3.40–4.47 (m, 5H), 7.19–7.43 (m, 11H), 7.58 (t, J = 3.6
Hz, 2H), 7.77–7.92 (m, 2H), 8.21 (d, J = 8.4 Hz, 1H); 13C NMR (75
MHz, DMSO-d6): d 26.0, 37.7, 47.0, 54.2, 56.3, 66.1, 113.4, 113.7,
116.5, 120.6, 125.7, 127.8, 128.1, 129.5, 130.4, 132.7, 140.9,
141.0, 141.1, 142.0, 144.2, 156.1, 168.2, 171.3, 171.6; HRMS (ESI,
positive): Calcd. for
C
35H32Br2N4O5Na [M+Na]+ 769.0632,
positive): Calcd. for
C
34H32BrN3O5Na [M+Na]+ 648.1468,
771.0611; observed: 769.0630, 771.0603.
650.1448; observed: 648.1463, 650.1450.
6.1.6.2. (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-3-(3-bromophenyl)-1-
(methylamino)-1-oxopropan-2-yl)amino)-3-(3-carbamoylphenyl)-1-
oxopropan-2-yl)carbamate (10b). Fmoc-L-3-carbamoylphenylala-
nine was used for replacing Fmoc-L-4-carbamoylphenylalanine,
6.1.6.6. (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-3-(3-bromophenyl)-1-
(methylamino)-1-oxopropan-2-yl)amino)-3-(4-carbamoylphenyl)-1-
oxopropan-2-yl)carbamate (10f). (S)-2-Amino-3-(3-bromophenyl)-
N-methylpropanamide was used for replacing (S)-2-amino-3-
(3,5-dibromophenyl)-N-methylpropanamide in the described pro-
cedure, and 10f was obtained in 78% yield. 1H NMR (300 MHz,
DMSO-d6): d 2.56 (d, J = 6.0 Hz, 3H), 2.73–2.84 (m, 2H), 2.94 (dd,
J = 10.8, 7.2 Hz, 2H), 3.99–4.50 (m, 5H), 7.18–7.43 (m, 10H), 7.58
(t, J = 4.8 Hz, 2H), 7.76–8.22 (m, 6H); 13C NMR (75 MHz, DMSO-
as
well
as
(S)-2-amino-3-(3-bromo-phenyl)-N-methyl-
propanamide replacing of (S)-2-amino-3-(3,5-dibromophenyl)-N-
methylpropanamide, in the described procedure, and 10b was
obtained in 78% yield. 1H NMR (300 MHz, DMSO-d6): d 2.56 (d,
J = 4.5 Hz, 3H), 2.73–2.85 (m, 2H), 2.94–3.00 (m, 2H), 3.99–4.29
Please cite this article in press as: Meng K., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.023

K. Meng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
9
d6): d 25.6, 37.8, 46.6, 53.9, 54.2, 56.0, 65.8, 120.2, 121.5, 125.4,
126.4, 127.2, 127.4, 127.8, 128.2, 128.5, 129.3, 130.3, 137.7,
140.8, 141.6, 143.8, 143.9; HRMS (ESI, positive): Calcd. for
6.1.7.6. 4-((S)-2-Amino-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-
1-oxopropan-2-yl)amino)-3-oxopropyl)benzamide (11f). 95% yield;
1H NMR (300 MHz, DMSO-d6): d 2.60 (d, J = 4.5 Hz, 3H), 2.77–2.85
(m, 1H), 2.97 (dd, J = 13.5, 4.2 Hz, 1H), 4.09–4.22 (m, 4H), 6.99–
7.17 (m, 3H), 7.25–7.43 (m, 5H), 7.61–7.89 (m, 6H); 13C NMR (75
MHz, DMSO-d6): d 25.7, 37.7, 44.1, 53.4, 56.3, 126.5, 127.5, 128.2,
129.2, 129.3, 130.0, 132.2, 132.7, 140.5, 142.3, 167.9, 171.1,
174.1; HRMS (ESI, positive): Calcd. for C20H23BrN4O3Na [M+Na]+
469.0846, 471.0825; observed: 469.0845, 471.0827.
C
34H32BrN3O5Na [M+Na]+ 691.1527, 693.1506; observed:
691.1506, 693.1493.
6.1.7. General procedure for preparation of compounds 11a-11f
Piperidine (2 mL) was added to a stirred solution of 10a–10f
(0.4 mmol) dissolved in DMF (4 mL), and the mixture was stirred
at rt for 2 h. After the completion of the reaction, the solvent and
volatiles were removed under reduced pressure, and the solid resi-
due was crystallized from EtOAc to give the desired product as
light brown solid.
6.1.8. Procedure for preparation of 2-cyclohexyl-2-(4-methoxyphenyl)
acetic acid (14b, R4 = OMe) and 2-cyclohexyl-2-(4-hydroxyphenyl)
acetic acid (14c, R4 = OH)
To a stirred solution of cyclohexanol (5g, 50 mmol) in pyridine
(180 mL) was added 4-methylbenzenesulfonyl chloride (14.3 g, 75
mmol) at 0 °C. The reaction mixture was stirred at rt for 12 h or
until the material completely disappeared as checked by TLC. After
removal of solvent by a rotary evaporator, the crude product was
purified by flash column chromatograph (eluting with hexane:
EtOAc, 50:1) to afford 4-methylbenzenesulfonate (13) (10.1 g,
79% yield) as whit solid. 1H NMR (300 MHz, CDCl3): d 1.24–1.31
(m, 3H), 1.45–1.57 (m, 3H), 1.66–1.80 (m, 4H), 2.44 (s, 3H), 4.45
(m, 1H), 7.32–7.34 (m, 2H), 7.78–7.81 (m, 2H); 13C NMR (75
MHz, CDCl3): d 21.7, 23.5, 24.9, 32.4, 81.8, 127.6, 129.8, 134.8,
6.1.7.1.
4-((S)-2-Amino-3-(((S)-3-(3,5-dibromophenyl)-1-(methy-
lamino)-1-oxopropan-2-yl)amino)-3-oxopropyl)benzamide (11a). 76%
yield; 1H NMR (300 MHz, DMSO-d6): d 2.57 (d, J = 4.2 Hz, 3H),
2.78–2.84 (m, 2H), 2.99 (dd, J = 13.2, 4.2 Hz, 3H), 4.46 (s, 1H), 7.18
(d, J = 8.1 Hz, 2H), 7.30–7.97 (m, 6H), 8.12 (d, J = 3.9 Hz, 1H),
8.29–8.30 (m, 2H); 13C NMR (75 MHz, DMSO-d6): d 25.7, 37.2,
40.1, 53.2, 56.3, 122.1, 127.5, 129.1, 131.4, 131.5, 132.2, 142.2,
143.0, 167.9, 170.8, 174.0; HRMS (ESI, positive): Calcd. for
C
20H22Br2N4O3Na [M+Na]+ 546.9951, 548.9930; observed:
546.9947, 548.9930.
144.4; HRMS (ESI, negative): Calcd. for
253.0904; observed: 253.0901.
C
13H18O3S[Mꢀ1]ꢀ
6.1.7.2. 3-((S)-2-Amino-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-
1-oxopropan-2-yl)amino)-3-oxopropyl)benzamide (11b). 75% yield;
1H NMR (300 MHz, DMSO-d6): d 2.59 (d, J = 6.0 Hz, 3H), 2.73–2.81
(m, 1H), 2.95 (dd, J = 13.8, 4.2 Hz, 1H), 4.09–4.19 (m, 4H), 7.23–
7.43 (m, 7H), 7.54 (s, 1H), 7.64 (t, J = 6.6 Hz, 2H), 7.70 (d, J = 8.7
Hz, 1H), 7.87–8.01 (m, 3H); 13C NMR (75 MHz, DMSO-d6): d 26.0,
38.0, 44.6, 53.6, 56.6, 121.8, 126.9, 127.8, 128.8, 129.5, 129.7,
130.7, 132.5, 132.6, 141.1, 142.3, 142.6, 168.2, 171.4, 174.3; HRMS
To a stirred solution of 2-(4-methoxyphenyl)acetic acid (2.5 g,
15 mmol) in THF (125 mL) was added slowly n-BuLi (18 mL,
2.5 M hexane solution, 45 mmol) at ꢀ78 °C. 30 min later, 13 (3.8
g, 15 mmol) was added, and the mixture was stirred for an addi-
tional 1 h at -78 °C, and then allowed to warm to ambient temper-
ature with the stirring was continued for 12 h. The reaction was
quenched by adding H2O (10 mL). After removal of solvents by a
rotary evaporator, the crude product was purified by flash column
chromatograph (eluting with DCM:MeOH, 100:1) to afford 14b
(1.7 g, 43% yield) as white solid. M.p. 177–178 °C; 1H NMR (300
MHz, CDCl3): d 0.68–0.77 (m, 4H), 1.02–1.36 (m, 2H), 1.61–2.05
(m, 5H), 3.15 (d, J = 10.5 Hz, 1H), 3.78 (s, 3H), 6.82 (dd, J = 6.6,
1.3 Hz, 2H), 7.22 (dd, J = 6.9, 2.1 Hz, 2H); 13C NMR (75 MHz, CDCl3):
d 26.1, 26.4, 30.4, 32.1, 40.8, 55.4, 58.1, 114.0, 129.4, 129.8, 159.0,
180.7; HRMS (ESI, negative): Calcd. for C15H20O3 [Mꢀ1]ꢀ 247.1340;
observed: 247.1343.
BBr3 (5.4 mL, 58 mmol) was added dropwise to an ice-cold stir-
red solution of 14b (1.2 g, 4.8 mmol) in DCM (20 mL). The mixture
was stirred at 0 °C for 4 h, and then allowed to warm to ambient
temperature with the stirring continued for additional 36 h. The
reaction was quenched by adding H2O (30 mL), and the resulting
mixture was extracted with DCM (3 ꢁ 120 mL). The combined
extracts were washed with brine (2 ꢁ 80 mL), and dried. The crude
product was purified by flash column chromatograph (DCM:MeOH,
40:1) to afford 14c (600 mg, 55% yield) as white solid. 1H NMR
(300 MHz, CDCl3): d 1.06–1.23 (m, 4H), 1.27–1.35 (m, 2H), 1.61–
1.95 (m, 5H), 3.06 (d, J = 8.1 Hz, 1H), 6.71 (d, J = 6.6 Hz, 2H), 7.12
(d, J = 6.6 Hz, 2H); 13C NMR (75 MHz, CDCl3): d 27.1, 27.5, 31.4,
33.1, 42.0, 59.4, 116.1, 130.3, 130.5, 157.6, 178.2; HRMS (ESI, neg-
ative): Calcd. for C14H18O3 [Mꢀ1]ꢀ 233.1183; observed: 233.1187.
(ESI, positive): Calcd. for
471.0825; observed: 469.0841, 471.0825.
C
20H23BrN4O3Na [M+Na]+ 469.084,
6.1.7.3. 4-((S)-2-Amino-3-(((S)-3-(3-fluorophenyl)-1-(methylamino)-
1-oxopropan-2-yl)amino)-3-oxopropyl)benzamide (11c). 79% yield;
1H NMR (300 MHz, DMSO-d6): d 2.57 (d, J = 3.6 Hz, 3H), 2.72–
2.95 (m, 4H), 3.38 (d, J = 4.2 Hz, 1H), 6.97 (d, J = 10.4 Hz, 2H),
7.18–7.31 (m, 3H), 7.75 (d, J = 7.8 Hz, 2H), 7.93–8.16 (m, 3H); 13C
NMR (75 MHz, DMSO-d6): d 25.6, 37.6, 43.8, 53.3, 56.7, 121.4,
127.4, 128.4, 129.1, 129.3, 130.3, 132.1, 132.2, 140.7, 142.1,
167.8, 170.9, 173.8; HRMS (ESI, positive): Calcd. for C20H23FN4O3Na
[M+Na]+ 409.1646; observed: 409.1646.
6.1.7.4.
4-((S)-2-Amino-3-(((S)-1-(methylamino)-1-oxo-3-phenyl-
propan-2-yl)amino)-3-oxopropyl)benzamide (11d). 90% yield; 1H
NMR (300 MHz, DMSO-d6): d 2.56 (d, J = 4.8 Hz, 3H), 2.76–2.98
(m, 4H), 3.16 (s, 1H), 4.45 (d, J = 5.1 Hz, 1H), 7.14–7.39 (m, 7H),
7.75 (d, J = 8.4 Hz, 2H), 7.90–8.13 (m, 3H); 13C NMR (75 MHz,
DMSO-d6): d 25.7, 37.7, 40.1, 53.3, 56.2, 121.5, 127.5, 128.5,
129.4, 130.4, 132.3, 140.7, 142.3, 167.9, 171.0, 174.0; HRMS (ESI,
positive): Calcd. for C20H24N4O3Na [M + Na]+ 391.1741; observed:
391.1741.
6.1.7.5. (S)-2-Amino-N-((S)-3-(3-bromophenyl)-1-(methylamino)-1-
oxopropan-2-yl)-3-phenylpropanamide (11e). 90% yield; 1H NMR
(400 MHz, DMSO-d6): d 2.59 (d, J = 6.0 Hz, 3H), 2.73–2.81 (m,
1H), 2.96 (dd, J = 14.8, 5.6 Hz, 1H), 4.09–4.20 (m, 4H), 7.19–7.33
(m, 5H), 7.38–7.45 (m, 4H), 7.53 (s, 1H), 7.61–7.71 (m, 3H), 7.86
(d, J = 10.0 Hz, 2H), 7.99 (d, J = 6.0 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6): d 5.7, 37.7, 40.4, 53.3, 56.2, 121.5, 127.5, 128.5, 129.2,
129.4, 130.4, 132.1, 132.3, 140.7, 142.3, 167.9, 174.0; HRMS (ESI,
6.1.9. General procedure for preparation of the title compounds
(15A1–15A7)
HOBt (75 mg, 0.56 mmol) and HBTU (211 mg, 0.56 mmol) were
added to a stirred solution of substituted 2-phenylacetic acid 14a–
14c (0.56 mmol) in DMF (5 mL) at rt. After the mixture was cooled
to 0 °C, dipeptide amine 11a–11e (0.28 mmol) was added, respec-
tively, followed by addition of DIEA (0.84 mmol). The reaction mix-
ture was stirred at ambient temperature for 12 h, and the solvent
and volatiles were evaporated under the reduced pressure, and
positive): Calcd. for
428.0764; observed: 426.0783, 428.0764.
C
19H22BrN3O2Na [M+Na]+ 426.0788,
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10
K. Meng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
then the solid residue was crystallized from EtOAc to generate the
desired product as white solid.
6.1.9.6. 4-((2S)-3-(((S)-3-(3-Bromophenyl)-1-(methylamino)-1-oxo-
propan-2-yl)amino)-2-(2-cyclohexyl-2-(4-hydroxyphenyl)acetamido)-
3-oxopropyl)benzamide (15A6). 74% yield; 1H NMR (300 MHz,
DMSO-d6): d 0.40–0.63 (m, 2H), 0.79–1.23 (m, 4H), 1.35–1.56 (m,
4H), 1.75–1.83 (m, 1H), 2.55 (d, J = 4.2 Hz, 3H), 2.67–3.10 (m, 5H),
4.31–4.63 (m, 2H), 6.63 (d, J = 6.2 Hz, 2H), 7.00–7.43 (m, 11H),
7.58–7.92 (m, 4H), 8.04–8.18 (m, 2H), 9.25 (s, 1H); 13C NMR (7.5
MHz, DMSO-d6): d 26.0, 26.6, 30.7, 31.7, 37.5, 37.9, 53.9, 54.0,
54.3, 57.5, 115.2, 119.0, 121.9, 127.7, 128.7, 129.5, 130.0, 130.7,
132.3, 132.5, 140.8, 141.0, 141.8, 156.4, 168.2, 171.1, 171.4,
173.4; HRMS (ESI, positive): Calcd for C34H39BrN4O5Na [M+Na]+
685.1996, 687.1976; observed: 685.2000, 687.1989.
6.1.9.1. (2S)-3-(3-Bromophenyl)-2-((2S)-2-(2-cyclohexyl-2-phenylac-
etamido)-3-phenylpropan-amido)-N-methylpropanamide (15A1). 70%
yield; 1H NMR (300 MHz, DMSO-d6): d 0.61–0.90 (m, 2H), 1.08–
1.56 (m, 7H), 2.21–2.47 (m, 2H), 2.55 (d, J = 3.3 Hz, 3H), 2.66–3.02
(m, 4H), 3.22 (d, J = 8.1 Hz, 1H), 6.98–7.04 (m, 2H), 7.13–7.43 (m,
9H), 7.55 (d, J = 6.0 Hz, 1H), 7.77–7.90 (m, 3H), 8.15 (t, J = 7.8 Hz,
2H); 13C NMR (75 MHz, DMSO-d6): d 25.6, 26.2, 30.3, 31.3, 37.6,
53.4, 53.5, 53.8, 53.9, 58.0, 110.3, 118.9, 121.5, 123.9, 126.4, 127.2,
128.0, 128.3, 128.5, 129.4, 130.2, 131.9, 139.4, 140.6, 167.8, 171.0,
172.5; HRMS (ESI, positive): Calcd. for C33H38BrN3O3Na [M+Na]+
626.1989, 628.1968; observed: 626.1985, 628.1971.
6.1.9.7. 4-((2S)-3-(((S)-3-(3-Bromophenyl)-1-(methylamino)-1-oxo-
propan-2-yl)amino)-2-(2-cyclohexyl-2-(4-methoxyphenyl)acetamido)-
3-oxopropyl)benzamide (15A7). 70% yield; 1H NMR (300 MHz,
DMSO-d6): d 0.40–0.63 (m, 2H), 0.85–0.95 (m, 2H), 1.07–1.23 (m,
3H), 1.34–1.87 (m, 4H), 2.55 (d, J = 3.6 Hz, 3H), 2.64–3.01 (m,
4H), 3.09 (d, J = 8.1 Hz, 1H), 3.68 (d, J = 10 Hz, 3H), 4.27–4.65 (m,
2H), 6.75–6.81 (m, 2H), 7.00–7.08 (m, 5H), 7.15–7.41 (m, 5H),
7.54 (d, J = 6.0 Hz, 1H), 7.77–8.15 (m, 4H); 13C NMR (75 MHz,
DMSO-d6): d 26.0, 26.5, 30.6, 31.0, 31.6, 37.6, 37.9, 53.9, 54.2,
55.4, 57.4, 113.7, 121.8, 127.7, 128.7, 129.3, 129.5, 129.7, 130.7,
131.9, 132.4, 140.8, 141.3, 141.8, 158.3, 168.1, 171.1, 172.9,
173.2; HRMS (ESI, positive): Calcd for C35H41BrN4O5Na [M+Na]+
699.2153, 701.2132; observed: 699.2149, 701.2144.
6.1.9.2. 3-((2S)-3-(((S)-3-(3-Bromophenyl)-1-(methylamino)-1-oxo-
propan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)
benzamide (15A2). 81% yield; 1H NMR (300 MHz, DMSO-d6): d
0.41–0.64 (m, 2H), 0.79–1.23 (m, 5H), 1.34–1.59 (m, 3H), 1.88 (d,
J = 13.6 Hz, 1H), 2.55 (d, J = 6.0 Hz, 3H), 2.63–3.05 (m, 4H), 3.16
(d, J = 14.4 Hz, 1H), 4.26–4.64 (m, 2H), 6.95 (t, J = 10.0 Hz, 3H),
7.15–7.33 (m, 9H), 7.38–7.41 (m, 1H), 7.51 (d, J = 10.8 Hz, 1H),
7.77–7.79 (m, 2H), 8.09–8.22 (m, 2H); 13C NMR (75 MHz, DMSO-
d6): d 25.9, 26.0, 26.5, 30.7, 31.6, 37.6, 37.9, 53.9, 54.2, 58.3,
121.8, 126.8, 127.7, 128.7, 128.8, 139.6, 130.6, 132.3, 132.4,
132.5, 139.8, 140.0, 140.7, 140.9, 141.2, 141.8, 168.0, 168.1,
171.1, 172.8; HRMS (ESI, positive): Calcd. For C34H39BrN4O4Na
[M + Na]+ 669.2047, 671.2026; observed: 669.2047, 671.2034.
6.2. Cell-based activity assays
6.1.9.3.
4-((2S)-2-(2-Cyclohexyl-2-phenylacetamido)-3-(((S)-1-
6.2.1. Measurements of cAMP production
(methylamino)-1-oxo-3-phenylpropan-2-yl)amino)-3-oxopropyl)ben-
zamide (15A3). 81% yield; 1H NMR (300 MHz, DMSO-d6): d 0.84–
1.07 (m, 2H), 1.10–1.20 (m, 3H), 1.55–1.90 (m, 6H), 2.50 (d, J =
0.9 Hz, 3H), 2.52–2.99 (m, 4H), 3.17 (d, J = 10.8 Hz, 1H), 4.41–4.44
(m, 2H), 6.96–7.43 (m, 14H), 7.35 (d, J = 8.1 Hz, 1H), 7.78–8.17 (m,
4H); 13C NMR (75 MHz, DMSO-d6): d 25.7, 26.0, 26.5, 30.7, 31.6,
37.6, 37.9, 53.9, 54.2, 58.3, 121.8, 126.8, 127.7, 128.6, 128.8,
129.7, 130.7, 132.3, 132.5, 140.0, 140.9, 141.8, 168.0, 168.1,
171.3, 172.8; HRMS (ESI, positive): Calcd. for C34H40N4O4Na [M
+Na]+ 591.2942; observed: 591.2937.
Glosensor (Promega) was used to monitor the level of cAMP as
described previously14 with slight modifications. HEK-293 cells
expressing a chemiluminescence-based cAMP biosensor were pla-
ted in 96-well plates at a density of 80,000 cells per well. On the
following day, the cells were incubated in an incubator at 27 °C
for 1 h after adding the Glosensor reagent. Subsequently, cells were
treated with either 0.5% DMSO or Cmpd-15 derivatives at different
concentrations in HANK’s balanced solution (Sigma), which also
contained 20 mM HEPES, pH 7.4, 0.05% bovine serum albumin
(BSA) and 3-isobutyl-1-methylxanthine (IBMX; Sigma) at 100 lM
as the final concentration. The cells were further incubated for
20 min, and then stimulated with a serial dilution of ISO for 5
min at ambient temperature. A NOVOstar microplate reader
(BMG Labtech) was used to read the luminescence signals.
6.1.9.4. 4-((2S)-3-(((S)-3-(3-Fluorophenyl)-1-(methylamino)-1-oxo-
propan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)
benzamide (15A4). 41% yield; 1H NMR (300 MHz, DMSO-d6):
d0.41–0.64 (m, 2H), 0.85–1.23 (m, 4H), 1.34–1.58 (m, 4H), 1.72–
1.90 (m, 1H), 2.54 (d, J = 4.2 Hz, 3H), 2.68–3.05 (m, 4H), 3.23 (d, J
= 1.8 Hz, 1H), 4.30–4.64 (m, 2H), 6.86–7.33 (m, 14H), 7.52 (d, J =
8.1 Hz, 1H), 7.77–8.28 (m, 3H); 13C NMR (75 MHz, DMSO-d6): d
24.9, 25.2, 30.6, 30.8, 31.1, 37.6, 37.9, 43.3, 53.9, 37.4, 121.8,
126.8, 127.7, 128.4, 128.6, 129.3, 129.7, 130.6, 132.3, 132.6,
140.8, 141.0, 141.3, 141.7, 168.1, 171.0, 171.2, 173.1; HRMS (ESI,
positive): Calcd. for C34H39FN4O4Na [M+Na]+ 609.2848; observed:
609.2845.
6.2.2. Measurements of b-arrestin recruitment
PathHunter, an enzyme fragment complementation assay (Dis-
coveRx),15 was used to monitor the extent of b-arrestin recruit-
ment as described previously14 with slight modifications. U2OS
cells, stably expressing enzyme acceptor-tagged b-arrestin2 and
the ProLink-tagged b2V2R were plated in 96-well plates at a density
of 25,000 cells per well. Similar to the Glosensor assay, the cells
were pre-treated with 0.5% DMSO or Cmpd-15 derivatives. Subse-
quently, cells were stimulated with agonists at 37 °C for 45 min,
and were then terminated by adding the PathHunter detection
reagents (DiscoveRx). After further incubation at 27 °C for an addi-
tional hour, a NOVOstar microplate reader (BMG Labtech) was
used to read the luminescence signals.
6.1.9.5. 4-((2S)-3-(((S)-3-(3,5-Dibromophenyl)-1-(methylamino)-1-
oxopropan-2-yl)amino-2-(2-cyclohexyl-2-phenylacetamido)-3-oxo-
propyl)benzamide (15A5). 50% yield; 1H NMR (300 MHz, DMSO-
d6): d 0.46–0.65 (m, 1H), 0.83–1.20 (m, 5H), 1.43–1.91 (m, 5H),
2.49 (d, J = 4.5 Hz, 3H), 2.59–2.95 (m, 3H), 3.02 (d, J = 10.8 Hz,
2H), 4.22–4.60 (m, 2H), 6.87–7.27 (m, 14H), 7.64–7.73 (m, 1H),
7.97–8.21 (m, 2H); 13C NMR (75 MHz, DMSO-d6): d 25.6, 25.7,
26.2, 30.3, 31.3, 37.2, 37.4, 53.8, 54.0, 58.0, 122.2, 126.5, 127.4,
128.6, 129.3, 131.5, 132.0, 139.6, 141.0, 141.5, 142.7, 142.9,
167.9, 170.8, 171.2, 172.5; HRMS (ESI, positive): Calcd for
6.3. Radioligand binding assays
The reaction mixture (250
l
L) was composed of ꢃ0.7 ng the
b2AR in nanodiscs, 60 pM [125I]-cyanopindolol (CYP) (2200 Ci/
mmol; PerkinElmer), Cmpd-15 derivatives and ISO (at different
concentrations). Nonspecific binding was determined using 20
C
34H38Br2N4O4Na [M+Na]+ 747.1152, 749.1132; observed:
747.1151, 749.1138.
lM propranolol. Every component in the mixture was diluted in
Please cite this article in press as: Meng K., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.023

K. Meng et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
11
the binding buffer (20 mM Hepes, pH 7.4, 100 mM NaCl) supple-
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the binding buffer using a harvester (Brandel). A Packard Cobra
Quantum gamma counter (Packard) was used to quantify the
bound [125I].
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