Conjugation of Short Peptides to Dibenzodiazepinone-Type Muscarinic Acetylcholine Receptor Ligands Determines M2R Selectivity

Article abstract of DOI:10.1021/acs.jmedchem.8b01967

Muscarinic acetylcholine receptors (MRs), comprising five subtypes (M₁R-M₅R) in humans, exhibit a high degree of structural similarity. Therefore, subtype-selective MR agonists and antagonists are lacking. We present an approach to highly M₂R-selective MR antagonists based on the conjugation of di- or tripeptides to M₂R-preferring dibenzodiazepinone-type MR antagonists. M₂R selectivity was dependent on the peptide sequence and on the type of linker. The introduction of basic amino acids resulted in improved M₂R selectivity (e.g., UR-AP148 (48): pK_i (hM₂R) of 8.97, ratio of K_i M₁R/M₂R/M₃R/M₄R/M₅R of 49:1:6500:60:400) compared to reported pyridobenzo- and dibenzodiazepinone-type MR ligands. A supposed dualsteric binding mode of the DIBA-peptide conjugates, such as 48, at MRs was supported by molecular dynamics simulations.

Full text of DOI:10.1021/acs.jmedchem.8b01967

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Article
Conjugation of short peptides to dibenzodiazepinone-type
2
muscarinic acetylcholine receptor ligands determines MR selectivity
Andrea Pegoli, David Wifling, Corinna G. Gruber, Xueke She,
Harald Hübner, Günther Bernhardt, Peter Gmeiner, and Max Keller
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01967 • Publication Date (Web): 10 May 2019
Downloaded from http://pubs.acs.org on May 11, 2019
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Conjugation of short peptides to
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dibenzodiazepinone-type muscarinic acetylcholine
receptor ligands determines M R selectivity
2
Andrea Pegoli,† David Wifling, Corinna G. Gruber, Xueke She, Harald Hübner,
,∥,#
†,#
†,#
†,§
‡
†
‡
,†
Günther Bernhardt, Peter Gmeiner, and Max Keller*
†Institute of Pharmacy, Faculty of Chemistry and Pharmacy, University of Regensburg,
Universitätsstr. 31, D-93053 Regensburg, Germany
‡Department of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich Alexander
University, Nikolaus-Fiebiger-Straße 10, D-91058 Erlangen, Germany
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ABSTRACT
Muscarinic acetylcholine receptors (MRs), comprising five subtypes (M R-M R) in humans,
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5
exhibit a high degree of structural similarity. Therefore, subtype-selective MR agonists and
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antagonists are lacking. We present an approach to highly M R-selective MR antagonists
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based on the conjugation of di- or tripeptides to M R-preferring dibenzodiazepinone-type MR
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antagonists. M R selectivity was dependent on the peptide sequence and on the type of linker.
2
The introduction of basic amino acids resulted in improved M R selectivity (e.g. UR-AP148
2
(
48): pK (hM R) 8.97, ratio of K M R/M R/M R/M R/M R 49:1:6500:60:400) compared to
i 2 i 1 2 3 4 5
reported pyridobenzo- and dibenzodiazepinone-type MR ligands. A supposed dualsteric
binding mode of the DIBA-peptide conjugates, such as 48, at MRs was supported by
molecular dynamics simulations.
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INTRODUCTION
In humans, muscarinic acetylcholine receptors (MRs) comprise five receptor subtypes (M1-
M ), all belonging to the class A of G-protein coupled receptors. MRs are expressed in the
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CNS as well as in the periphery being involved in the regulation of, e.g., cognition and
1
smooth muscle contraction, respectively. Due to the high structural similarity of MRs, most
pronounced in the region of the orthosteric binding site,2, 3 subtype-selective MR agonists and
antagonists are lacking. Such compounds are considered to improve pharmacotherapy. For
example, in the CNS, antagonism at presynaptic M Rs has been suggested as an approach to
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enhance cholinergic function in Alzheimer’s disease patients.4-7 However, for that purpose,
highly selective M R antagonists are needed, because concomitant blocking of postsynaptic
2
M Rs must be strictly prevented.
1
To date, to the best of our knowledge, only one compound class (diaryl sulfones linked to a
piperidinyl moiety) has afforded M R ligands with pronounced selectivity (> 100fold in
2
8
binding affinity) over the other MR subtypes (compound 1a, Figure 1). Strikingly, the amino
group of the anthranoyl moiety in 1a seems to be crucial for high M R selectivity, which
2
8
becomes obvious from the considerably lower M R selectivity of congener 1b, bearing a
2
methyl instead of the amino group in 1a (Figure 1). Another well studied class of M subtype-
2
preferring MR antagonists are pyridobenzodiazepinone-type (e.g. 2-4,9-12 Figure 1) and
dibenzodiazepinone-type (e.g. 5-10,10, 13-16 Figure 1) tricyclic MR ligands. The structures and
MR binding data of selected tricyclic M R antagonists with high M R affinity (pK > 7.5) (2-
2
2
i
1
0), including the most M R-selective dibenzodiazepinone-type MR ligand reported in
2
1
6
literature to date (8), are shown in Figure 1.
Recently, investigations using tritiated analogs of the dibenzodiazepinone-type MR
antagonists 7, 9 and 10, showing low to moderate M R selectivity (Figure 1), revealed that
2
these compounds bind to the M R in a dualsteric manner with the tricyclic moiety residing in
2
the orthosteric binding pocket and the ‘side chains’ reaching the vestibule (common allosteric
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site) of the receptor.15, 16 Moreover, these studies demonstrated that the attachment of various
bulky moieties to the dibenzodiazepinone core structure ‘A’ (cf. box in Figure 1) was well
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5, 16
tolerated with respect to M R binding (e.g. 8-10).
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diarylsulfones:
pyridobenzodiazepinones:
O
O
O
O
O
O
O
O
O
S
NH
NH
O
NH
O
HN
NH
N
Cl
N
N
N
N
N
N
N
N
N
N
Cl
HN
O
N
O
N
N
N
HN
N
N
R
N
8N
tripitramine (4)
N
5
3
O
1
a R = NH2
N

N
NH
1b R = CH3
BIBN 99 (3b)
O
N
O
AF-DX 384 (2)
AQ-RA 741 (3a)
dibenzodiazepinones:
N
O
O
H
N
A =
N
N
A
A
N
N
H
A
A
N
N
A
N
N
S
NH
O
N
O
N
N
O
DIBA (5)
BIBN 140 (6)
UNSW-MK259 (7)
UR-SK75 (8)
O
O
A
N
O
O
O
N
O
NH
H
N
N
H
N
N
N
O
N
HN
N
N
N
N
H
H
O
O
NH
N
A
UR-AP060 (10)
UR-SK-59 (9)
N
H
HN
M1
O
M2R selectivity
ratios Ki (M1,3,4,5R)/Ki (M2R)
pKi
Compd.
Ref.
M2
M3
M4
M5
M1
M3
M4
M5
1
a
a
a
-
9.70
8.57
8.22
8.71
8.37
8.3
-
-
-
-
2926
230
2722 148
186
48
1b
2, AF-DX 384
-
-
-
172
11
14
1.7
b
c
b
d
e
f
7.51
7.79
7.54
7.3
7.18
6.75
7.20
7.0
8.00
8.30
8.19
8.1
6.27
6.04
6.08
5.9
5.1
89
8.3
6.8
91
2.6
1.5
1.6
5.8
470
200
250
48
3a, AQ-RA 741
15
10
35
5.9
20
3
4
5
6
b, BIBN 99
, tripitramine
, DIBA
5.97
8.80
8.4
7.52
6.11
7.42
8.0
6.76
8.19
8.7
5.84
7.47
n.a.
6.5
26
9.57
9.5
140
37
24
130
-
g
d
c
h
h
13
40
11
20
7.0
6.3
3.0
, BIBN 140
6.5
8.1
6.4
7.3
50
40
230
470
300
55
7, UNSW-MK259
8, UR-SK75
8.07
8.84
8.56
9.12
7.22
7.88
7.55
8.63
8.59
8.57
6.75
7.47
6.96
79
10.1
180
78
35
9, UR-SK59
9.44
9.39
7.6
3.6
7.4
1.1
10, UR-AP060
i
8.82
7.81
9.33
7.64
37
Figure 1. Structures and MR binding data of two described diarylsulfone-type M R-selective
2
MR ligands (1a, 1b) and of a selection of reported M R-preferring tricyclic MR antagonists
2
(
compounds 2-10). References: a) Clader et al. (reported K values (M R) were converted to
i 2
pK values; IC , pIC , K or pK values for the MR subtypes M , M , M and M were not
i
50
50
i
i
1
3
4
5
8
9
given, instead, only the ratios K (M
R)/K (M R) were provided), b) Dörje et al., c) Keller
i 2
i
1,3,4,5
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et al. (data reported as pIC ; data were re-analyzed to obtain pK values), d) Doods et al.,
5
0
i
1
1
12
e) Doods et al., f) Maggio et al. (reported K values were converted to pK values), g)
i
i
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3
16
Gitler et al. (reported K values were converted to pK values), h) She et al., i) Pegoli et
i
i
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al. . Except for 5, data refer to human receptors. In case of 5, M and M were rat receptors,
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4
whereas M and M receptors were not specified.
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As the dualsteric ligand approach is considered useful to develop MR ligands with improved
subtype selectivity,17-20 we wanted to explore, how the attachment of putatively allosterically
interacting peptide moieties to the dibenzodiazepinone core structure influences M R affinity
2
and selectivity (Figure 2). For this purpose, various di- and tripeptides were conjugated to the
dibenzodiazepinone pharmacophore via two linkers, differing in length and polarity, resulting
in ‘DIBA-peptide conjugates’, which were characterized with respect to binding at MRs M1-
M5.
·
preparation of 'DIBA-peptide conjugates':
di/tripeptide
linker
N
NH
N
O
containing aa:
use of two spacers
O
Gly, Ala, Leu, Tyr, differing in length
Trp, Gln, Lys, Arg and polarity
dibenzodiazepinone
pharmacophore
·
·
determination of M -M receptor binding data
1 5
MD simulations of M Rs (x = 1-5) bound to the most M R-
x
2
selective compounds
Figure 2. Rationale of the present study.
RESULTS AND DISCUSSION
The selection of amino acids, used to prepare the di- and tripeptide moieties of the DIBA-
peptide conjugates, was driven by the fact that acidic amino acids, present at the extracellular
surface of MRs, vary in number and position (see Figure S8, Supporting Information).3, 21, 22
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Therefore, Lys and Arg were chosen as potential interaction partners of these acidic amino
acids of the receptor. Gln was used as a non-basic, polar amino acid, capable of forming
hydrogen bonds, as well as the aromatic amino acids Tyr and Trp to potentially address
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Tyr832.64, Tyr177ECL2.51 and Trp4227.35 located within the vestibule of the M R (cf. Figure
2
S8A, Supporting Information). Additionally, Ala and Leu were incorporated as nonpolar
amino acids. Neither the selection of individual amino acids nor their combination were
guided by computational chemistry (docking, simulations) for the following reasons: docking
studies alone, requiring considerably less computing time compared to simulations, have
limited predictive value; docking studies with subsequent time-consuming simulations were at
that stage of the project not considered reasonable to be performed for numerous potential
DIBA-peptide conjugates. We rather selected amino acid sequences covering the following
combinations of physicochemically different types of amino acid residues: aromatic/aromatic
(Trp/Tyr), polar/hydrophobic (Gln/Leu), basic/aromatic (Lys/Tyr) and basic/basic (Lys/Arg or
Arg/Arg) (cf. Scheme 1). In the tripeptides, Ala was chosen as a ‘spacer’ amino acid to
increase the distance between the aforementioned amino acid pairs.
The estimation of the appropriate length of the linker, by which the dibenzodiazepinone
scaffold and the peptide moiety are held together, was supported by the reported crystal
structure of the M R bound to the non-selective MR antagonist 3-quinuclidinyl benzilate
2
2
1
(
QNB) (PDB ID: 3UON ): recently, induced fit docking of the dibenzodiazepinone
derivative 7 (cf. Figure 1) to this crystal structure, followed by MD simulation (2 µs), yielded
15
a binding pose of 7 (included in Figure S14, Supporting Information), which was used to
identify the putative position of the tricyclic dibenzodiazepinone moiety of the DIBA-peptide
conjugates in the orthosteric binding site of the M R. The chosen lengths of the two linkers
2
were supposed to allow for a dualsteric binding mode of the DIBA-peptide conjugates, i.e. the
dibenzodiazepinone pharmacophore presumably residing in the orthosteric binding site, and
the peptide moiety being located in the vestibule or at the surface of the receptor.
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The N-terminally acetylated peptides 11-25 were synthesized on a chlorotrityl resin according
to the Fmoc strategy. After coupling and Fmoc-deprotection of the final amino acids, the N-
termini were acetylated by treatment of the resins with acetic anhydride. The peptides were
cleaved off the resin under mild acidic conditions to maintain protecting groups at the side
chains (Scheme 1). Coupling of the C-termini of 11-21 to amines 26 and 27, followed by
cleavage of protecting groups, afforded the DIBA-peptide conjugates 28-49 (Scheme 1).
Likewise, conjugation of peptides 22-25 to amine 27, followed by deprotection, yielded
compounds 50-53. Whereas pronounced epimerization occurred during amide bond formation
between the peptides and amine 26 or 27 when using HBTU/HOBt as coupling reagent and an
excess of DIPEA (data not shown), epimerization was markedly reduced or precluded by
using EDC/HOBt as coupling reagent and avoiding excessive DIPEA (in case of amine 26) or
omitting DIPEA (in case of amine 27). Coupling of Boc-Arg(Pbf)-OH (54) to amine 27,
followed by deprotection, afforded 55, which was acetylated to give 56 (Scheme 1).
Propionylation of amine 27 yielded the reference compound 58 (Scheme 1).
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Scheme 1. Synthesis of DIBA-peptide conjugates 28-53 and dibenzodiazepinone
derivatives 55, 56 and 58.a
Ac-Ala-Ala-OH
(11)
(12)
Ac-Ala-Ala-NH-X
Ac-Trp-Tyr-NH-X
× 3 TFA (28) Ac-Lys-Ala-Tyr-NH-X × 4 TFA (34)
× 3 TFA (29) Ac-Tyr-Lys-NH-X × 4 TFA (35)
b
'
long linker'
series
Ac-Trp(Boc)-Tyr(tBu)-OH
Ac-Trp(Boc)-Ala-Tyr(tBu)-OH (13)
H2N
4 TFA
26
X
Ac-Trp-Ala-Tyr-NH-X × 3 TFA (30) Ac-Tyr-Ala-Lys-NH-X × 4 TFA (36)
Ac-Gln-Leu-NH-X
× 3 TFA (31) Ac-Lys-Arg-NH-X × 5 TFA (37)
Ac-Gln-Ala-Leu-NH-X × 3 TFA (32) Ac-Lys-Ala-Arg-NH-X × 5 TFA (38)
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×
Ac-Gln(Trt)-Leu-OH
(14)
(15)
(16)
Ac-Gln(Trt)-Ala-Leu-OH
Ac-Lys(Boc)-Tyr(tBu)-OH
Ac-Lys-Tyr-NH-X
× 4 TFA (33)
Ac-Lys(Boc)-Ala-Tyr(tBu)-OH (17)
Ac-Tyr(tBu)-Lys(Boc)-OH
(18)
Ac-Tyr(tBu)-Ala-Lys(Boc)-OH (19)
20)
Ac-Lys(Boc)-Ala-Arg(Pbf)-OH (21)
H
N
a
aa
Ac-Ala-Ala-NH-Y
Ac-Trp-Tyr-NH-Y
× 1 TFA (39) Ac-Tyr-Ala-Lys-NH-Y × 2 TFA (47)
H2N
27
Y
2
resin
-ClTrt
× 1 TFA (40) Ac-Lys-Arg-NH-Y
× 3 TFA (48)
Ac-Lys(Boc)-Arg(Pbf)-OH
(
'short linker'
series
Ac-Trp-Ala-Tyr-NH-Y × 1 TFA (41) Ac-Lys-Ala-Arg-NH-Y × 3 TFA (49)
preloaded
with 1 aa
c
Ac-Gln-Leu-NH-Y
Ac-Gln-Ala-Leu-NH-Y × 1 TFA (43) Ac-Arg-Arg-NH-Y
Ac-Lys-Tyr-NH-Y
Ac-Lys-Ala-Tyr-NH-Y × 2 TFA (45) Ac-Lys-Arg-Gly-NH-Y × 3 TFA (53)
× 1 TFA (42) Ac-Arg-Lys-NH-Y
× 3 TFA (50)
Ac-Arg(Pbf)-Lys(Boc)-OH
Ac-Arg(Pbf)-Arg(Pbf)-OH
(22)
(23)
× 3 TFA (51)
× 2 TFA (44) Ac-Arg-Lys-Gly-NH-Y × 3 TFA (52)
Ac-Arg(Pbf)-Lys(Boc)-Gly-OH (24)
Ac-Lys(Boc)-Arg(Pbf)-Gly-OH (25)
Ac-Tyr-Lys-NH-Y
× 2 TFA (46)
c
d
2
7
7
H-Arg-NH-Y × 3 TFA
Ac-Arg-NH-Y × 2 TFA
Boc-Arg(Pbf)-OH (54)
55
Ac2O
56
O
× 1 TFA
Y
e
2
X =
Y =
NH
O
O
N
N
N
NH
58
H
N
N
N
O
O
N
N
O
O
O
57
O
aReagents and conditions: (a) Fmoc-strategy SPPS, amino acid coupling: Fmoc-aa, HBTU,
HOBt, DIPEA, DMF/NMP (8:2 v/v), rt or 35 °C, 2 × 60 min (‘double coupling’), Fmoc
deprotection: 20% piperidine in DMF/NMP (8:2 v/v), rt, 2 × 10 min; (b) (1) EDC, HOBt,
DIPEA, DMF, 5 °C, 3 h, (2) TFA/H O (95:5 v/v), rt, 3 h, 27-66%; (c) (1) EDC, HOBt, DMF,
2
5 °C, 3 h, (2) TFA/H O (95:5 v/v), rt, 3 h, 30-75%; (d) acetic anhydride, DIPEA, DMF, rt, 2
2
h, 78%; (e) DIPEA, DMF, rt, 30 min, 80%.
M -M receptor affinities of 28-53, 55, 56 and 58 were determined by competition binding
1
5
3
with the orthosteric MR antagonist [ H]NMS using genetically engineered CHO cells (Table
1
) (displacement curves are shown in Figures S1-S5, curve slopes are presented in Table S1,
Supporting Information). The subset of compounds with the longer linker, containing a basic
piperazine moiety (28-38), exhibited higher affinities at all MR subtypes compared to the
subset of derivatives containing the shorter linker (39-53, 55, 56).
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Strikingly, within the ‘long-linker’ subset of compounds (28-38), the ranges of pK values
i
determined for M R, M R and M R binding were very narrow (pK : M R 8.50-9.06, M R
1
2
4
i
1
2
9.37-10.2, M R 8.45-9.04; Table 1). By contrast, the ‘short-linker’ DIBA-peptide conjugates
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
39-53) exhibited wider ranges of MR affinities (e.g. pK M R: 6.4-8.97), suggesting that, in
i 2
case of the M , M and M receptor, the SAR of the ‘long-linker’ series of compounds did not
1
2
4
correlate with the SAR of the respective ‘short-linker’ counterparts. Indeed, this became
obvious by plotting the pK values (M R, M R, M R) of compounds 28-38 (‘long-linker’
i
1
2
4
DIBA-peptide conjugates) against the pK values of 39-49 (corresponding ‘short-linker’
i
2
DIBA-peptide conjugates) and linear regression analysis (R < 0.44, Figure S6, Supporting
Information). Likewise, the SARs of both linker series were also nonparallel in case of the
2
M R (R = 0.061, Figure S6, Supporting Information). Merely the M R binding data revealed
3
5
2
a slight linear correlation between the two linker series (R = 0.71, Figure S6, Supporting
Information). Consequently, the missing correlation between the SARs of the two series of
compounds (long vs. short linker) is also reflected by disagreeing MR selectivity profiles of
individual pairs of compounds differing only in the type of the linker, i.e. bearing identical
peptide and dibenzodiazepinone moieties (e.g. 30 vs. 41, selectivity profiles: Ki
M R/M R/M R/M R/M R 27:1:410:19:390 vs. 4.7:1:56:1.9:6.2; cf. Table 1). The degree of
1
2
3
4
5
correlation between the selectivity profiles of such pairs of DIBA peptide conjugates became
also obvious by plotting the pK values (M R-M R) of the ‘short-linker’ compound against the
i
1
5
pK values (M R-M R) of the ‘long-linker’ counterpart (Figure S7, Supporting Information):
i
1
5
for the compound pairs 29/40, 30/41, 33/44, 35/46 and 36/47 the correlation was rather low
2
(
R < 0.71), and for the pairs 28/39, 34/45, 37/48 and 38/49 a moderate linear correlation was
2
found (R = 0.83-0.89).
The DIBA-peptide derivative with the lowest pronounced M R selectivity, containing Gln,
2
Ala and Leu, was found in the ‘short-linker’ subset of compounds (43, cf. Table 1). The ‘long-
linker’ derivative 38 (UR-AP158) and the ‘short-linker’ derivative 48 (UR-AP148) exhibited
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the highest M R affinities within the respective compound subsets as well as higher M R
2
2
selectivities (Table 1) compared to reported, so-called ‘M R-selective’ pyridobenzo- and
2
dibenzodiazepinone-type MR ligands (e.g. 2 and 5, cf. Figure 1).9-16 As both, 38 and 48,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
contain two basic amino acids (Lys, Arg), these results suggest that positively charged
residues are advantageous for M R binding, being in agreement with reports on orthosteric
2
1
9, 21, 23
and allosteric M R ligands.
2
For the majority of the presented compounds, the M R selectivity towards the M R and M R
2
1
4
was less pronounced than towards the M R and M R (Table 1), being consistent with the
3
5
selectivity profile of previously described, tricyclic M R-preferring MR antagonists (cf.
2
selectivity profiles of 2, 3a, 4 and 7-10 given in Figure 1). The modulatory effect of the
peptidyl moiety on M R selectivity was confirmed by the low M R selectivity of reference
2
2
compound 58, which contains a small propionyl group instead of the peptide moiety (Scheme
1, Table 1).
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Table 1. M -M receptor binding data (pK values) of 28-53, 55, 56 and 58 obtained from
1
5
i
3
equilibrium competition binding experiments with [ H]NMS at intact CHO-hM R cells
x
(x = 1-5).a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
selectivity toward M R
(
ratios K
i i
(M1,3,4,5R)/K
cmpd.
structure
for X and Y cf. Scheme 1)
pK
i
(M
2
R))
(
M
1
R
M
2
R
M
3
R
M
4
R
M
5
R
M
1
M
3
M
4
M
5
8
0
.50 ± 9.37 ± 6.81 ± 8.61 ± 6.34 ±
2
2
8
9
Ac-Ala-Ala-NH-X
Ac-Trp-Tyr-NH-X
7.5 370 7.5 1100
6.3 79 7.0 220
.05
0.05
0.09
0.10
0.07
9
0.17
.06 ± 9.83 ± 7.87 ± 8.97 ± 7.41 ±
0.12
0.09
0.13
0.04
8
0
.64 ± 10.1 ± 7.45 ± 8.79 ± 7.51 ±
.05 0.03 0.06 0.04 0.10
30
Ac-Trp-Ala-Tyr-NH-X
Ac-Gln-Leu-NH-X
27 410 19 390
9.6 270 5.4 1300
8.4 440 11 710
14 260 12 780
8.3 240 8.1 560
32 220 13 1900
22 290 28 260
20 800 37 1900
8.62 ± 9.62 ± 7.21 ± 8.92 ± 6.53 ±
.04 0.10 0.13 0.15 0.13
3
3
3
3
1
2
3
4
0
8
0
.53 ± 9.45 ± 6.82 ± 8.45 ± 6.62 ±
Ac-Gln-Ala-Leu-NH-X
Ac-Lys-Tyr-NH-X
.07
0.05
0.20
0.09
0.01
0.11
0.16
0.10
0.15
8
0
.85 ± 10.1 ± 7.56 ± 8.95 ± 7.15 ±
.02
8.90 ± 9.76 ± 7.35 ± 8.90 ± 7.02 ±
0.15 0.08 0.07 0.13 0.14
Ac-Lys-Ala-Tyr-NH-X
Ac-Tyr-Lys-NH-X
8
0
.67 ± 10.1 ± 7.79 ± 9.04 ± 6.85 ±
35
.17
0.03
0.02
0.06
0.13
0.12
0.03
0.05
0.08
8
0
.63 ± 9.94 ± 7.55 ± 8.49 ± 7.53 ±
.10
3
6
Ac-Tyr-Ala-Lys-NH-X
Ac-Lys-Arg-NH-X
8
0
.91 ± 10.2 ± 7.31 ± 8.69 ± 6.95 ±
3
3
7
8
.10
0.06
0.06
0.17
0.11
8
0
.64 ± 10.2 ± 6.96 ± 8.68 ± 6.53 ±
(UR-
Ac-Lys-Ala-Arg-NH-X
45 1800 40 4900
.03
0.03
0.07
0.12
0.09
AP158)
6
0
.52 ± 7.64 ± 5.86 ± 7.10 ± 5.87 ±
3
9
Ac-Ala-Ala-NH-Y
Ac-Trp-Tyr-NH-Y
14
5.0
4.7
59
46
56
3.7
62
.09
0.04
0.03
0.09
0.11
6
0
.81 ± 7.48 ± 5.80 ± 7.03 ± 7.23 ±
40
2.8 1.8
1.9 6.2
.12
0.05
0.04
0.05
0.12
7.03 ± 7.70 ± 5.94 ± 7.43 ± 6.92 ±
0.06
4
4
4
4
1
2
3
4
Ac-Trp-Ala-Tyr-NH-Y
Ac-Gln-Leu-NH-Y
0.05
0.04
0.11
5.97 ± 5.82 ±
0.08 0.04
6.38 ± 5.92 ±
0.11 0.04
0.09
5
0
.98 ± 6.51 ±
< 5
3.4 >27 3.6 4.9
2.2 >36 1.7 4.4
.07
0.02
0.10
6
7
.27 ± 6.62 ±
Ac-Gln-Ala-Leu-NH-Y
Ac-Lys-Tyr-NH-Y
< 5
0
.12
.52 ± 8.74 ± 5.79 ± 7.97 ± 7.13 ±
0.08 0.09 0.08 0.12
17 720 5.9
14 160 5.8
1.8 210 6.6
43
53
15
0.10
7
0
.23 ± 8.37 ± 6.18 ± 7.63 ± 6.65 ±
.05 0.02 0.05 0.10 0.05
45
Ac-Lys-Ala-Tyr-NH-Y
Ac-Tyr-Lys-NH-Y
7.06 ± 7.33 ± 5.00 ± 6.53 ± 6.15 ±
.01 0.04 0.01 0.08 0.02
4
6
0
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
0
.75 ± 8.03 ± 5.30 ± 7.24 ± 6.68 ±
4
4
7
8
Ac-Tyr-Ala-Lys-NH-Y
Ac-Lys-Arg-NH-Y
19 550 5.9
23
.08
0.10
0.04
0.05
0.09
7
0
.38 ± 8.97 ± 5.17 ± 7.21 ± 6.37 ±
(UR-
49 6500 60 400
.14
0.02
0.07
0.06
0.07
AP148)
6
0
.94 ± 8.11 ± 5.57 ± 7.05 ± 6.37 ±
4
5
5
9
0
1
Ac-Lys-Ala-Arg-NH-Y
Ac-Arg-Lys-NH-Y
Ac-Arg-Arg-NH-Y
Ac-Arg-Lys-Gly-NH-Y
Ac-Lys-Arg-Gly-NH-Y
H-Arg-NH-Y
15 350 12
6.5 400 17
12 710 19
56
39
97
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
.03
0.02
0.06
0.11
0.08
7
0
.02 ± 7.92 ± 5.26 ± 6.59 ± 6.30 ±
.08
0.18
0.13
0.06
0.19
7.70 ± 8.77 ± 6.02 ± 7.53 ± 6.79 ±
0.11 0.03 0.19 0.16 0.05
7
0
.64 ± 8.83 ± 6.11 ± 7.76 ± 6.44 ±
52
15 620 11 260
8.2 180 6.9 49
18 260 9.8 410
.05
0.10
0.05
0.16
0.15
0.03
0.10
0.13
0.10
7
6
.49 ± 8.38 ± 6.19 ± 7.56 ± 6.70 ±
.12
5
5
3
5
0
.97 ± 8.27 ± 5.90 ± 7.25 ± 5.62 ±
0
.07
0.12
0.17
0.11
0.11
7
0
.01 ± 7.95 ± 5.83 ± 6.83 ± 6.01 ±
56
Ac-Arg-NH-Y
8.4 140 13
1.1 69 2.7
86
98
.09
0.09
0.14
0.10
0.11
7.07 ± 7.10 ± 5.27 ± 6.68 ± 5.15 ±
0.05 0.05 0.08 0.08 0.14
5
8
propanoyl-NH-Y
a
Presented are means ± SEM from at least three independent experiments (each performed in triplicate). K
d
values (reported
, 0.035/0.1 nM; M
2
R selective members of the presented series of compounds are highlighted (38, green; 48, purple).
1
4
3
previously )/concentrations of [ H]NMS: M
.24/0.3 nM. The most M
1
, 0.15/0.2 nM; M
2
, 0.090/0.2 nM; M
3
, 0.089/0.2 nM; M
4
5
,
0
For 38 and 48, showing the highest M R selectivity, M R antagonism was investigated and
2
2
confirmed in an IP1 accumulation assay15, 24, 25 (Figure 3). The pIC values of 38 and 48
50
amounted to 7.98 and 7.03, respectively. Moreover, plasma stabilities were studied for 38, 48
and 52, which proved to be highly stable (> 99% intact compound after 24 h, Table 2).
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A
100
80
CCh
38
60
48
40
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
20
0
-12
-10
-8
-6
-4
log(c)
B
100
80
60
atropine
40
20
0
38
48
-12
-10
-8
-6
-4
log(c)
Figure 3. Investigation of M R agonism and antagonism of compounds 38 and 48 in an IP1
2
accumulation assay using HEK-hM -Gα - cells. (A) Concentration-dependent effects of
2
qi5 HA
CCh, 38 and 48 on the accumulation of IP1. 38 and 48 elicited no response. pEC of CCh
5
0
(mean ± SEM): 6.97 ± 0.03. Data represent means ± SEM from 14 (CCh) or four (38, 48)
independent experiments (performed in duplicate). (B) Concentration-dependent inhibition of
the CCh (0.3 μM) induced IP1 accumulation by the MR antagonists atropine, 38 and 48. pIC50
values (means ± SEM): 38, 7.98 ± 0.19; 48, 7.03 ± 0.06; atropine, 8.09 ± 0.03. Data represent
means ± SEM from four (38), five (atropine) or six (48) independent experiments (performed
in duplicate).
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Stabilities of 38, 48 and 52 in human plasma/PBS (1:2 v/v) at 37 °C.a
%
intact peptide in plasma after the specified
incubation times
compd.
1 h
6 h
24 h
> 99
> 99
> 99
48 h
> 99
3
4
5
8
8
2
> 99
99 ± 3
> 99
> 99
> 99
> 99
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
> 99
83 ± 6
aThe initial concentrations of compounds 38, 48 and 52 in plasma/PBS
1:2 v/v) were 100 µM. Data represent the mean (± SEM) of three
(
independent experiments (SEM not given when no decomposition was
detected). LC-MS analysis revealed the Lys-Gly amide bond in 52 as
the major cleavage site (structure of 52 cf. Scheme 1 or Table 1).
In order to visualize the interaction of 38 and 48 with MRs by computational chemistry, MD
simulations (20 µs) were performed with all MR subtypes bound to 38 or 48 (except for the
M R bound to 38). The lowest free energy (MM-PBSA) binding poses obtained from the MD
3
simulations, and the time courses over the simulations are depicted in Figure 4 (M R) and
2
Figures S9-S12 (Supporting Information) (M R, M R-M R). The simulations revealed a
1
3
5
dualsteric binding mode of 38 and 48 at the M R, M R, M R and M R with the tricyclic
1
2
4
5
dibenzodiazepinone moiety residing in the orthosteric binding pocket (Figure 4, Figures S9,
S11 and S12, Supporting Information).
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
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7
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9
0
1
2
3
4
5
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0
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2
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7
8
9
0
2
1
Figure 4. MD simulations (20 µs) of the hM R (inactive state, PDB ID: 3UON ) bound to 38
2
(A, C) or 48 (B, D). (A, B) Lowest free energy (MM-PBSA) poses of 38 (green) and 48
(purple), respectively, obtained from analysis of the MD simulation trajectories. Amino acids,
involved in H-bonding, salt bridges (both indicated as yellow dashed lines) or - interactions
(green dashed lines) with the ligands are labeled: in A, D1033.32 (HB, SB), E172ECL2 (HB,
SB), E175ECL2 (HB, SB), C176ECL2 (HB), I178ECL2 (HB), Y4036.51 (-), N4046.52 (HB),
N419ECL3 (HB) and Y4267.39 (HB), in B, Y802.61 (HB), D1033.32 (HB, SB), Y1043.33 (HB),
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E172ECL2 (HB, SB), E175ECL2 (HB, SB), I178ECL2 (HB). (C, D) Time courses of the 20-μs MD
simulations of the hM R bound to 38 (C) or 48 (D) showing superimposed snap shots
2
collected every 1 µs. HB = hydrogen bond. SB = salt bridge.
0
1
2
3
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5
6
7
8
9
0
1
2
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0
This was consistent with previously reported investigations on the binding mode of 7, 9 and
1
5, 16
1
0 at the M R,
and in agreement with the fact that 38 and 48 were capable of fully
2
3
displacing the orthosteric ligand [ H]NMS from M Rs when using a high radioligand
2
3
concentration (i.e. receptor occupancy by [ H]NMS > 95%) (Figure 5), indicating a
3
competitive mechanism between the orthosteric radioligand [ H]NMS and the DIBA-peptide
conjugates 38 and 48. An MD simulation of the M R, bound to 38, was not performed as
3
preceding induced-fit docking afforded no reasonable binding pose for 38. In case of 48, the
initial dualsteric binding pose of 48 at the M R started to disappear during the simulation due
3
to an upward movement of the dibenzodiazepinone moiety (‘unbinding’ process, cf. Figure
S10B, Supporting Information), being in agreement with the low binding affinity of 48 at the
M R (pK = 5.17, cf. Table 1).
3
i
A detailed discussion of the binding modes of 38 and 48, suggested by MD simulations, is
provided in the Supporting Information. It should be mentioned that the coordinates of the
26
recently reported crystal structure of the M R in complex with 2 (PDB ID: 5ZKB ) were not
2
available at the time the presented MD simulations with the M R were performed. As the
2
dibenzodiazepinone substructure of the investigated DIBA-peptide conjugates is structurally
26
similar to compound 2 (structure shown Figure 1), the recently reported M R structure
2
might be considered as template for future computational studies of M R binding of tricyclic
2
muscarinic receptor antagonists.
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Figure 5. Effect of the DIBA-peptide conjugates 38 and 48 on M R equilibrium binding of
2
3
[ H]NMS (K = 0.09 nM) using a radioligand concentration of 0.2 nM (filled symbols, data
d
taken from Figures S2 and S4) or 2 nM (open symbols). Experiments were performed at intact
CHO-hM R cells at 22 °C. Unspecific binding was determined in the presence of atropine
2
3
(500-fold excess to [ H]NMS). Data, representing mean values ± SEM from at least four
independent experiments (each performed in triplicate), were analyzed by four-parameter
logistic fits. The use of a high radioligand concentration, resulting in a high receptor
3
occupancy by [ H]NMS, can ‘unmask’ a potential non-competitive, negative allosteric
mechanism as previously demonstrated, e.g. for the negative allosteric M R modulator W84
2
(indicated by an elevated lower curve plateau and a missing rightward shift of the curves; cf.
1
5
Figure 8C in Pegoli et al. 2017 ). Here, the determination of the effects of 38 and 48 on M R
2
3
equilibrium binding of [ H]NMS yielded curves, which were rightward shifted and exhibited
lower curve plateaus not different from zero. This indicates a competitive mechanism between
the orthosteric MR antagonist NMS and 38 as well as NMS and 48.
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2
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CONCLUSION
Attachment of di- and tripeptides to dibenzodiazepinone-type MR ligands via two different
linkers yielded DIBA-peptide conjugates (‘long-linker’ series: 28-38, ‘short-linker’ series: 39-
0
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9
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9
0
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2
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5
6
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9
0
5
3) with medium to high M R affinity. M R-M R binding data of these compounds revealed
2 1 5
that M R selectivity is dependent on the linker type and can be tuned by the peptide sequence.
2
The study suggests basic residues to be favorable with respect to M R selectivity. The
2
following conclusions can be drawn from MR affinities and MD simulations, the latter
confirming a dualsteric binding mode at MRs: whereas the longer linker, containing a basic
piperazine moiety, is favorable with respect to high M R affinity, the shorter aliphatic linker
2
is favorable in terms of tuning MR selectivity by the peptide moiety.
The presented type of selective M R antagonists (compounds 38 and 48) requires, in contrast
2
to M R ligands such as 1a (cf. Figure 1), structural optimization with respect to a potential
2
application as therapeutics acting in the CNS. This may be achieved, e.g., by a bioisosteric
replacement of strongly basic moieties, such as guanidine groups, by moderately basic
surrogates. As MR antagonists such 38 or 48 represent the cold forms of potential tritiated
selective M R ligands, accessible by replacement of the acetyl residue by a commercially
2
available tritiated acetyl moiety, the presented compound class is superior to 1a in terms of
providing convenient access to radiolabeled M R-selective ligands, which represent useful
2
molecular tools.
To the best of our knowledge, the presented approach, i.e. triggering receptor subtype
selectivity of small-molecule, nonpeptide receptor ligands by attachment of short peptides,
has not been described before. This concept can potentially be applied to other receptor
families with respect to the identification of lead structures for the development of dualsteric
ligands with improved subtype selectivity.
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EXPERIMENTAL SECTION
General experimental conditions. Acetonitrile (HPLC gradient grade) was obtained from
Honeywell (Seelze, Germany) or Sigma-Aldrich (Taufkirchen, Germany). DMF, atropine,
EDC, DIPEA, HFIP, 1-methyl-D-tryptophan (59) and TFA were purchased from Sigma-
Aldrich. Absolute EtOH was obtained from Honeywell. CH Cl , HOBt, DMF for peptide
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
synthesis and NMP for peptide synthesis were obtained from Acros Organics/Fisher Scientific
Nidderau, Germany). Boc-Arg(Pbf)-OH (54) was purchased from Carbolution Chemicals
(
(St. Ingbert, Germany). Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln-(Trt)-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH), acetic anhydride and solid supports
for SPPS (H-Ala-, H-Arg(Pbf)-, H-Leu-, H-Lys(Boc)-, H-Tyr(tBu)- and H-Gly-2-ClTrt PS
resin) were purchased from Merck Chemicals (Darmstadt, Germany). Deuterated solvents
were from Deutero (Kastellaun, Germany). HBTU and piperidine were from Iris Biotech
1
6
14
27
(
Marktredwitz, Germany). The syntheses of compounds 26, 27 and 57 were described
elsewhere. Millipore water was used for the preparation of buffers and HPLC eluents. 1.5- or
-mL polypropylene reaction vessels with screw cap (Süd-Laborbedarf, Gauting, Germany)
2
were used for small scale reactions, for the investigation of plasma stabilities and for the
preparation and storage of stock solutions. Temperature-controlled reactions and plasma
stabilities were performed in 1.5-mL reaction vessels using a Thermocell Mixing Block MB-
1
6
02 from Bioer (Hangzhou, China). NMR spectra were recorded on a Bruker Avance III HD
1
13
00 equipped with a cryogenic probe (14.1 T, H: 600.1 MHz, C: 150.9 MHz) (Bruker,
Karlsruhe, Germany). Abbreviations for the multiplicities of the signals are s (singlet), d
doublet), t (triplet), m (multiplet) and brs (broad-singlet). HRMS was performed on an
(
Agilent 6540 UHD Accurate-Mass Q-TOF LC/MS system (Agilent Technologies, Santa
Clara, CA USA) using an ESI source. Optical rotations at 589 nm (Na-D line) were measured
on a Polarimeter P8000-T equipped with an electronic Peltier thermostat PT31 (A. KRÜSS
Optronic, Hamburg, Germany) using a thermostated (20 °C) micro-cuvette (layer thickness: 1
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9
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2
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dm, volume: 0.9 mL) and acetonitrile/water (3:7 v/v) as solvent. Preparative HPLC was
performed with a system from Knauer (Berlin, Germany), composed of two K-1800 pumps
and a K-2001 detector, or with a Prep 150 LC System from Waters (Eschborn, Germany)
consisting of a 2545 Binary Gradient Module, a 2489 UV/Visible Detector and a Waters
Fraction Collector III. When using acetonitrile as organic solvent, a Kinetex-XB C18 (5 μm,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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7
8
9
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8
9
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1
2
3
4
5
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7
8
9
0
2
50 mm × 21 mm; Phenomenex, Aschaffenburg, Germany) was used as stationary phase at a
flow rate of 18 mL/min (30-33, 35-38, 41, 42, 44-53, 55, 56) or 20 mL/min (28, 29, 34, 39,
0, 43, 58). When using MeOH as organic solvent, a Gemini-NX C18 (5 μm, 250 mm × 21
4
mm; Phenomenex) served as stationary phase at a flow rate of 15 mL/min (compounds 28 and
39). Mixtures of 0.1% aq TFA and acetonitrile or mixtures of 0.1% aq TFA and MeOH were
used as mobile phase. The detection wavelength was 220 nm. The solvent of collected
fractions was removed by lyophilization. Analytical HPLC analysis was performed with a
system from Agilent Technologies composed of a 1290 Infinity binary pump equipped with a
degasser, a 1290 Infinity autosampler, a 1290 Infinity thermostated column compartment, a
1
260 Infinity diode array detector, and a 1260 Infinity fluorescence detector. A Kinetex-XB
C18 (2.6 μm, 100 × 3 mm; Phenomenex) was used as stationary phase at a flow rate of 0.5
mL/min (28-49) or 0.6 mL/min (50-53, 55, 56, 58). Mixtures of 0.04% aq TFA (A) and
acetonitrile (B) or A and MeOH (C) were used as mobile phase. The following linear
gradients were applied: compounds 28-48, 50-53, 55 and 56: 0-20 min: A/B 90:10-70:30, 20-
22 min: 70:30-5:95, 22-26 min: 5:95; compound 58: 0-12 min: A/B 90:10-70:30, 12-16 min:
7
0:30-5:95, 16-20 min: 5:95; compounds 28, 29, 31, 32, 34, 39, 42, 43 and 45: 0-20 min: A/C
5:15-52:48, 20-23 min: 52:48-5:95, 23-27 min: 5:95; compound 40: 0-20 min: A/C 85:15-
8
35:65, 20-23 min: 35:65-5:95, 23-27 min: 5:95. For all analytical HPLC runs the oven
temperature was set to 25 °C, the injection volume was 20 μL and detection was performed at
2
20 nm. Compound concentrations were between 40 and 150 μM.
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Compound characterization. Target compounds (28-53, 55, 56, 58) were characterized by
1
13
1
H-NMR, C-NMR and 2D-NMR ( H-COSY, HSQC, HMBC) spectroscopy, HRMS and RP-
HPLC analysis. Additionally, specific optical rotation was determined for compounds 38, 48
and 52. The purity (HPLC, 220 nm) of all target compounds amounted to ≥97%
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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7
8
9
0
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9
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(chromatograms shown in the Supporting Information).
1
13
Annotation concerning the H- and C-NMR spectra of 28-53, 55, 56, 58: due to a slow
rotation about the exocyclic dibenzodiazepinone amide group on the NMR time scale, two
1
13
isomers (ratios provided in the experimental protocols) were evident in the H- and C-NMR
spectra.
1
Annotation concerning the H-NMR spectra (solvent: DMSO-d ) of 50-53, 55 and 56: in
6
order to integrate signals interfering with the broad water signal at ca 3.5 ppm, spectra were
additionally recorded in DMSO-d /D O (4:1 v/v) (spectra shown in the Supporting
6
2
Information).
Screening for pan-assay interference compounds (PAINS). Screening of all target
compounds
(28-53,
55,
56,
58)
for
PAINS
via
the
public
tool
2
8
http://zinc15.docking.org/patterns/home gave no hits.
Solid-phase peptide synthesis (Fmoc strategy) of N-terminally acetylated, side chain-
protected peptides 11-25. 5-mL BD Discardit II syringes (Becton Dickinson, Heidelberg,
Germany) or 5-mL B.Braun syringes (ALMO-Erzeugnisse Erwin Busch, Bad Arolsen,
Germany) equipped with a polyethylene frit (average pore size 35 µm; Roland Vetter
Laborbedarf, Ammerbuch, Germany) were used as reaction vessels. DMF/NMP (8:2 v/v) was
used as solvent throughout (ca 2.2 mL per 1 mmol Fmoc-aa). Protected amino acids (5-fold
excess) were preactivated with HBTU/HOBt/DIPEA (5/5/10 equiv.) in DMF/NMP (8:2) at rt
for 5 min and added to the resin. The vessel was shaken at rt or 35 °C for 60 min. The
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coupling procedure was repeated (‘double coupling’). After completed coupling of an Fmoc-
aa, the resin was washed with DMF/NMP (8:2) (4×) followed by Fmoc deprotection with
20% piperidine in DMF/NMP (8:2) at rt (2 × 10 min) and washing of the resin with
DMF/NMP (8:2) (5×). After coupling and Fmoc-deprotection of the final Fmoc-aa, a mixture
formed by acetic anhydride (5 equiv.) and DIPEA (10 equiv.) in DMF/NMP (8:2) was added
and the vessel was shaken at rt for 30 min followed by washing with DMF/NMP (8:2) (4×)
and CH Cl (treated with K CO ) (6×). The side-chain protected peptides were cleaved off by
0
1
2
3
4
5
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7
8
9
0
1
2
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3
treatment of the resin with CH Cl /HFIP (4:1 v/v) at rt (2 × 20 min). The resin was separated
2
2
by filtration and washed once with CH Cl /HFIP (4:1) (ca 1 mL). The filtrates were
2
2
combined, the volatiles evaporated and the residue was dissolved in acetonitrile/water (2:8 or
:7 v/v) (ca 2 mL) followed by the addition of water (ca 50 mL) and lyophilization.
3
Compounds 11-25 were used in the next step without purification. The chemical identities and
purities of 11-25 were assessed by HRMS and analytical HPLC (data not shown).
General procedure for the synthesis of compounds 28-53. The coupling of the side chain-
protected peptides 11-25 to amines 26 or 27 was performed in 1.5- or 2-mL polypropylene
reaction vessels with screw cap. HOBt (1.2 equiv.) and EDC (1.2 equiv.) were added to a
cooled (5 °C) solution of the respective side chain-protected peptide (1 equiv.) in DMF (0.1-
0
.4 mL). In the case of compounds derived from 26, a solution of 26
(tetrakis(hydrotrifluoroacetate), 1-1.2 equiv.) and DIPEA (3 equiv.) in DMF (0.1 mL) was
immediately added and stirring was continued at 5 °C for 3 h. For the synthesis of compounds
derived from 27 a solution of 27 (free base, 1-1.2 equiv.) in DMF (0.1 mL) was immediately
added and stirring was continued at 5 °C for 3 h. 1% aq TFA (0.1-0.4 mL, depending on the
amount of DMF used to dissolve the peptide) was added and the mixture was subjected to
preparative HPLC to isolate the protected intermediates (conditions not given). The latter
were dissolved in TFA/CH Cl /H O (10:10:1 v/v/v) (products without Arg) or TFA/H O
2
2
2
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(
95:5 v/v) (products containing Arg). The mixtures were stirred at rt for 3 h, CH Cl (ca 20
2
2
mL) was added and the volatiles were removed by evaporation. The final products were
purified by preparative HPLC.
1
1
1
1
1
1
1
1
1
1
2
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Experimental synthetic protocols and analytical data of compounds 55, 56 and 58. (S)-2-
Amino-5-guanidino-N-(4-(1-(2-oxo-2-(11-oxo-10,11-dihydro-5H-
dibenzo[b,e][1,4]diazepin-5-yl)ethyl)piperidin-4-yl)butyl)pentanamide
tris(hydrotrifluoroacetate) (55) Compound 55 was prepared from amine 27 (55.6 mg, 0.137
mmol) and compound 54 (60 mg, 0.114 mmol) according to the general procedure for the
synthesis of 28-53. Purification by preparative HPLC (gradient: 0-25 min: 0.1% aq
TFA/acetonitrile 81:19-72:28, t = 7 min) yielded 55 as a white fluffy solid (67 mg, 0.074
R
1
mmol, 65%). Ratio of configurational isomers evident in the NMR spectra: ca 1.5:1. H-NMR
(600 MHz, DMSO-d ): δ (ppm) 1.13-1.29 (m, 4H), 1.30-1.52 (m, 7H), 1.64-1.71 (m, 2H),
6
1
0
.71-1.83 (m, 2H), 2.83-2.96 (m, 1H), 2.96-3.04 (m, 1H), 3.04-3.14 (m, 4H), 3.28-3.35 (m,
.8H), 3.55-3-63 (m, 1.2H), 3.65-3.73 (m, 1H), 3.75 (d, 0.6H, J 17 Hz), 3.90 (d, 0.4H, J 17
Hz), 4.32-4.48 (m, 1H), 6.95-7.49 (m, 7H), 7.49-7.56 (m, 1.4H), 7.56-7.63 (m, 0.6H), 7.63-
7.79 (m, 2H), 7.79-7.92 (m, 2H), 8.11-8.24 (m, 3H), 8.41-8.48 (m, 1H), 9.57 (brs, 1H), 10.73
13
(s, 0.4H), 10.78 (s, 0.6H). C-NMR (150.9 MHz, MeOH-d ): δ (ppm) 22.32, 24.20, 28.31,
4
2
8.74, 28.98, 32.81, 34.95, 38.67, 40.08, 51.94, 52.80, 53.24, 55.96, 116.05 (TFA), 118.04
(TFA), 121.91, 122.34, 124.93, 125.51, 126.75, 127.27, 127.71, 128.75, 129.00, 129.71,
1
3
30.04, 130.38, 130.97, 131.63, 133.07, 133.80, 134.68, 135.77, 140.99, 156.84, 158.3 (q, J
2 Hz, carbonyl group of TFA), 163.70, 164.35, 165.78, 166.07, 168.05, 171.50. HRMS
+
+
(ESI): m/z [M+H] calcd. for [C H N O ] 563.3453, found 563.3462. RP-HPLC (220 nm):
3
0
43
8
3
9
9% (t = 6.2 min, k =7.1). C H N O · C H F O (562.72 + 342.07).
R 30 42 8 3 6 3 9 6
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(
S)-2-Acetamido-5-guanidino-N-(4-(1-(2-oxo-2-(11-oxo-10,11-dihydro-5H-
dibenzo[b,e][1,4]diazepin-5-yl)ethyl)piperidin-4-yl)butyl)pentanamide
bis(hydrotrifluoroacetate) (56) Acetyl anhydride (3.6 µL, 0.038 mmol) was added to a
solution of 55 (25 mg, 0.032 mmol) and DIPEA (28 µL, 0.16 mmol) in DMF (0.3 mL) and
the mixture was stirred at rt for 2 h. Isolation of the product by preparative HPLC (gradient:
0
1
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3
4
5
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7
8
9
0
1
2
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9
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9
0
0
-25 min: 0.1% aq TFA/acetonitrile 81:19-72:28, t = 9 min) afforded 56 as a white fluffy
R
solid (20.8 mg, 0.025 mmol, 78%). Ratio of configurational isomers evident in the NMR
1
spectra: ca 1.5:1. H-NMR (600 MHz, DMSO-d ): δ (ppm) 1.13-1.26 (m, 4H), 1.30-1.50 (m,
6
8
H), 1.57-1.66 (m, 1H), 1.69-1.82 (m, 2H), 1.83 (s, 3H), 2.85-2.96 (m, 1H), 2.96-3.14 (m,
5H), 3.28-3.35 (m, 1H), 3.57-3.61 (m, 1H), 3.76 (d, 0.6H, J 16 Hz), 3.91 (d, 0.4H, J 16 Hz),
4
7
.13-4.21 (m, 1H), 4.33-4.48 (m, 1H), 7.03 (brs, 2H), 7.20-7.31 (m, 2H), 7.31-7.39 (m, 1.6H),
.39-7.49 (m, 1.4H), 7.49-7.55 (m, 1.4H), 7.55-7.62 (m, 0.6H), 7.62-7.79 (m, 3H), 7.79-7.85
(m, 0.6H), 7.85-7.93 (m, 1.4H), 7.95-8.02 (m, 1H), 9.55 (brs, 1H), 10.73 (s, 0.4H), 10.78 (s,
13
0
3
1
.6H). C-NMR (150.9 MHz, MeOH-d ): δ (ppm) 22.49, 25.16, 28.69, 29.09, 29.26, 32.74,
4
4.90, 38.31, 40.33, 52.13, 52.65, 52.78, 53.23, 55.93, 116.05 (TFA), 118.10 (TFA), 121.88,
22.30, 124.89, 125.47, 126.74, 127.25, 127.70, 128.25, 128.70, 128.95, 129.69, 130.00,
130.36, 131.61, 133.03, 133.76, 134.68, 135.77, 140.97, 156.77, 158.2 (broad signal, carbonyl
group of TFA), 163.67, 164.17, 165.73, 166.03, 169.19, 171.22. HRMS (ESI): m/z [M+H]+
+
calcd. for [C H N O ] 605.3558, found 605.3560. RP-HPLC (220 nm): 98% (t = 8.0 min,
3
2
45
8
4
R
k = 9.5). C H N O · C H F O (604.76 + 228.05).
3
2
44
8
4
4
2
6
4
N-(4-(1-(2-Oxo-2-(11-oxo-10,11-dihydro-5H-dibenzo[b,e][1,4]diazepin-5-
yl)ethyl)piperidin-4-yl)butyl)propionamide hydrotrifluoroacetate (58) DIPEA (12 µL,
.071 mmol) and a solution of 57 (12 mg, 0.071 mmol) in DMF (30 µL) were added to a
0
solution of 27 (28 mg, 0.071 mmol) in DMF (0.25 mL) and the mixture was stirred at rt for 30
min followed by the addition of 10% aq TFA (0.14 mL). Purification by preparative HPLC
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(
gradient: 0-30 min: 0.1% aq TFA/acetonitrile 90:10-57:43, t = 18 min) yielded 58 as a white
R
fluffy solid (33 mg, 0.057 mmol, 80%). Ratio of configurational isomers evident in the NMR
1
spectra: ca 1.5:1. H-NMR (600 MHz, MeOH-d ): δ (ppm) 1.11 (t, 3H, J 7.7 Hz), 1.27-1.37
4
1
1
1
1
1
1
1
1
1
1
2
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0
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0
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0
(m, 4H), 1.38-1.56 (m, 5H), 1.85-2.00 (m, 2H), 2.17 (q, 2H, J 7.7 Hz), 2.87-2.96 (m, 1H),
2
0
.99-3.08 (m, 1H), 3.15 (t, 2H, J 7.1 Hz), 3.39-3.49 (m, 1H), 3.67-3.83 (m, 2H), 4.39 (d,
.6H, J 17 Hz), 4.43 (d, 0.4H, J 17 Hz), 7.23-7.31 (m, 0.8H), 7.31-7.36 (m, 1.2H), 7.36-7.41
(m, 0.4H), 7.44-7.55 (m, 2.2H), 7.59-7.71 (m, 2H), 7.73-7.78 (m, 0.4H), 7.90 (d, 0.6H, J 8.1
13
Hz), 7.97 (d, 0.4H, J 7.8 Hz). C-NMR (150.9 MHz, MeOH-d ): δ (ppm) 10.59, 24.66,
4
3
0.22, 30.41, 30.47, 34.39, 36.31, 40.07, 54.95, 55.01, 55.37, 57.94, 58.04, 123.08, 123.64,
126.84, 127.51, 127.87, 128.49, 128.89, 129.45, 130.10, 130.54, 130.88, 131.20, 131.70,
1
1
31.97, 132.34, 133.01, 133.43, 134.57, 134.94, 135.45, 135.73, 137.04, 141.02, 142.68,
62.1 (broad signal, carbonyl group of TFA), 164.96, 165.45, 168.56, 168.81, 177.02. HRMS
+
+
(ESI): m/z [M+H] calcd. for [C H N O ] 463.2709, found 463.2729. RP-HPLC (220 nm):
2
7
35
4
3
9
9% (t = 8.3 min, k = 8.0). C H N O · C HF O (462.59 + 114.02).
R 27 34 4 3 2 3 2
Cell culture. CHO-K9 cells, stably transfected with the DNA of human muscarinic receptors
M -M (obtained from Missouri S&T cDNA Resource Center; Rolla, MO) were cultured in
1
5
HAM’s F12 medium supplemented with fetal calf serum (Biochrom, Berlin, Germany) (10%)
and G418 (Biochrom) (750 μg/mL). HEK-293 cells were maintained in DMEM/HAM’s F12
(Thermo Scientific) supplemented with fetal bovine serum (Thermo Scientific) (10%) and
Penicillin/Streptomycin (Thermo Scientific) (10,000 U/mL).
IP1 accumulation assay. The measurement of M R stimulated activation of the G-protein
2
mediated pathway was performed as previously described applying the IP-One HTRF assay
1
5
(Cisbio, Codolet, France) according to the manufacturer’s protocol. In brief, HEK-293 cells
were grown to a confluency of approximately 70% and transiently cotransfected with the
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cDNAs of the human M receptor (Missouri S&T cDNA Rescourse Center) and the hybrid G-
2
protein Gαqi5‑HA (Gα protein with the last five amino acids at the C-terminus replaced by the
q
corresponding sequence of Gα ; gift from the J. David Gladstone Institutes, San Francisco,
i
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CA),19, 29 using TransIT-293 Mirus transfection reagent (MoBiTec, Goettingen, Germany).
After 1 day, cells were detached from the culture dish with Versene (Life Technologies,
Darmstadt, Germany), seeded into black 384-well plates (10,000 cells/well) (Greiner Bio-
One, Frickenhausen, Germany) and maintained for 24 h at 37 °C. After incubation with the
test compounds, dissolved in stimulation buffer, at 37 °C for 1 h the detection reagents were
added (IP1-d2 conjugate and Anti IP1cryptate TB conjugate, each dissolved in lysis buffer),
and incubation was continued at rt for 60 min. Time resolved fluorescence resonance energy
transfer (HTRF) was determined using a Clariostar plate reader (BMG, Ortenberg, Germany)
measuring fluorescence at 620 (±10) nm and 670 (±10) nm (excitation at 330 nm).
Antagonistic activities of 38 and 48 were determined after preincubation of the cells with 38
or 48 for 30 min, followed by addition of CCh (final concentration: 0.3 μM) and continued
incubation at 37 °C for 1 h.
3
Radioligand competition binding. Competition binding experiments with [ H]NMS
(specific activity: 80 Ci/mmol; purchased from American Radiolabeled Chemicals (St. Louis,
MO) via Hartman Analytics (Braunschweig, Germany) were performed at intact CHO-hM R
x
14
cells (x = 1-5) as described previously with minor modifications. Cells were seeded in tissue
culture treated white 96-well plates with clear bottom (Corning Incorporated Life Sciences,
Tewksbury, MA; Corning cat. no. 3610) one or two days prior to the experiment. Depending
14
on the level of receptor expression, the confluency of the cells was 30–50% (M R), 50-70%
1
(
M R, M R, M R) or 90-100% (M R) on the day of the experiment. All experiments were
3 4 5 2
performed at 22 ± 1 °C. The culture medium was removed by suction, the cells were washed
with phosphate buffered saline (PBS) (200 µL) and covered with 160 µL of Leibovitz’s L15
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medium (Life Technologies) supplemented with 1% bovine serum albumin (Serva,
Heidelberg, Germany), in the following referred to as L15 medium. For total binding L15
3
medium (20 µL) and L15 medium (20 µL) containing [ H]NMS (10-fold concentrated) were
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added. To determine unspecific binding and the effect of a compound of interest on [ H]NMS
equilibrium binding, L15 medium (20 µL) containing atropine or the compound of interest
3
(
10-fold concentrated) and L15 medium (20 µL) containing [ H]NMS (10-fold concentrated)
were added. Samples were incubated for 3 h under gentle shaking. After incubation, the liquid
was removed by suction, the cells were washed twice with ice-cold PBS (200 µL) and lysis
solution (urea (8 M), acetic acid (3 M) and Triton-X-100 (1%) in water) (25 µL) was added.
The plates were shaken for 30 min, liquid scintillator (Optiphase Supermix, PerkinElmer,
Rodgau, Germany) (200 µL) was added, and the plates were sealed with a transparent sealing
tape (permanent seal for microplates, PerkinElmer, prod. no. 1450–461). The plates were
turned up-side down several times in order to achieve complete mixing of scintillator and
lysis solution. The samples were kept in the dark for at least 1 h prior to the measurement of
radioactivity (dpm) with a MicroBeta2 plate counter (PerkinElmer). Applied concentrations
3
of [ H]NMS: 0.2 nM (M R, M R, M R), 0.1 nM (M R), 0.3 nM (M R) or 2 nM (M R).
1
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4
5
2
Unspecific binding was determined in the presence of atropine (1000-fold excess to
[3
H]NMS) and amounted to < 15% of total binding.
Investigation of the stability of 38, 48 and 52 in human plasma. The metabolic stabilities
of 38, 48 and 52 were investigated in human blood plasma/PBS pH 7.4 (1:2 v/v) (in the
following referred to as plasma/PBS) at 37 °C. 1-Methyl-D-tryptophan (59) was used as
internal standard. As the purity (RP-HPLC, 220 nm) of 59 was < 95% (data not shown) it was
purified by preparative HPLC to give a purity of > 99%. Plasma was obtained by the
collection of human blood from a healthy donor in 5.5-mL heparinized plasma-monovettes,
followed by centrifugation at 1200 g at 4 °C for 10 min. The supernatants were pooled in two
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0-mL Falcon tubes and centrifuged again at 1200 g at 4 °C for 10 min. The plasma was
aliquoted and stored at −80 °C.
For the addition of the compounds to plasma/PBS, 5 mM stock solutions in acetonitrile/0.04%
aqueous TFA (3:7 v/v) were used. Recoveries were determined for concentrations of 80 μM
and 4 μM, and an internal standard concentration of 10 μM. For this purpose, 59 and the
DIBA-peptide conjugates were added to plasma/PBS (total volume: 70 μL), immediately
followed by vortexing (ca 10 s) and precipitation of protein by the addition of
EtOH/acetonitrile (1:1 v/v) (140 μL). The mixture was vortexed for 5 min und centrifuged at
0
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6,100 g at 4 °C for 10 min. Aliquots (180 μL) of the supernatant were transferred into 1.5-
mL polypropylene reaction vessels containing 10% aqueous TFA (5 μL). The volatiles were
removed in a vacuum concentrator under reduced pressure at 40 °C (ca 60 min), and the
residue was taken up in acetonitrile/0.04% aqueous TFA (1:9 v/v) (90 μL) by vortexing (2
min). The samples were filtrated using a 0.2-μm RC-membrane filter (Phenomenex,
Aschaffenburg, Germany) and analyzed by RP-HPLC using the analytical HPLC system and
the conditions as described under general experimental conditions, but applying the following
gradient: 0–12 min: A/B 90:10–70:30, 12–16 min: 70:30–5:95, 16–20 min: 5:95. On the day
of an experiment four-point calibrations were performed for the respective DIBA-peptide
conjugates and the internal standard. Peak areas representing 100% recovery were obtained by
analyzing 53.3 μM and 2.67 μM DIBA-peptide solutions as well as a 6.67 μM solution of 59
(in duplicate each). All peak areas were transformed into concentrations (μM) and percent
recoveries of 38, 48, 52 and 59 were calculated based on the average values of the 100%
reference samples (see Table S2, Supporting Information). Recovery ratios were obtained by
dividing the recovery of the DIBA-peptide conjugate by the recovery of 59 for each
individual sample (n = 3 or 4, cf. Table S2, Supporting Information).
For the investigation of the stabilities in plasma, the DIBA-peptide conjugates (38, 48, 52)
and 59 were added to plasma/PBS at a concentration of 100 μM and 10 μM, respectively (in
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triplicate each). After 1, 6, 24 and 48 h of incubation under shaking at 37 °C, aliquots (70 μL)
were taken and processed as described above for the determination of recoveries. On the day
of an experiment four-point calibrations were performed for the respective DIBA-peptide
conjugates and 59. Peak areas representing 100% recovery were obtained by analyzing 66.7
μM ‘DIBA-peptide’ solutions and a 6.67 μM solution of 59 (in duplicate). Based on the
calibration, the peak areas of the 100% references and of the samples were transformed into
concentrations (μM). Recoveries of 38, 48 and 52 were calculated by multiplying the recovery
of 59, obtained for each individual sample, with the recovery ratio obtained for the
concentration of 80 µM (see above). The plasma concentrations of the DIBA-peptide
conjugates were obtained by dividing the determined ‘DIBA-peptide’ concentration by the
respective recovery. Degradation (%) of 38, 48 and 52 was calculated based on the average
values of the 100% reference samples.
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Note: recovery ratios obtained for a concentration of 4 µM were not used as 38, 48 and 52
exhibited high stabilities (lowest concentration in plasma ca 80 µM (52, 48 h)).
Molecular dynamics simulations
MR models. In case of the hM R, a described model derived from the M R crystal structure
2
2
21
15
(
PDB ID: 3UON ) was used. Models of the hM R, hM R and hM R were obtained by
1
3
4
3
22
refinement of reported crystal structures (PDB ID 5CXV, PDB ID 4DAJ and PDB ID
3
30-32
5DSG, respectively) using Modeller 9.18
The homology model of the hM R was
5
21
prepared based on the M R crystal structure (PDB ID: 3UON ) using Modeller 9.18.
2
Replacement of ICL3 of the hM R, hM R, hM R and hM R by eight alanines was performed
1
3
4
5
1
5
using the respective coordinates of the described hM R model. The receptor models (M R,
2
1
M R, M R, M R, M R) contained a disulfide bridge between Cys and CysECL2.50 as well as
3
.25
2
3
4
5
between the two cysteines in ECL3. Coordinates other than ligand and receptor molecules
were removed. The protein preparation wizard (Schrödinger LLC, Portland, OR USA) was
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