Regiospecific Introduction of Halogens on the 2-Aminobiphenyl Subunit Leading to Highly Potent and Selective M3 Muscarinic Acetylcholine Receptor Antagonists and Weak Inverse Agonists

Article abstract of DOI:10.1021/acs.jmedchem.0c00297

Muscarinic M₃ receptor antagonists and inverse agonists displaying high affinity and subtype selectivity over the antitarget M₂ are valuable pharmacological tools and may enable improved treatment of chronic obstructive pulmonary disease (COPD), asthma, or urinary incontinence. On the basis of known M₃ antagonists comprising a piperidine or quinuclidine unit attached to a biphenyl carbamate, 5-fluoro substitution was responsible for M₃ subtype selectivity over M₂, while 3′-chloro substitution substantially increased affinity through a σ-hole interaction. Resultantly, two piperidinyl-and two quinuclidinium-substituted biphenyl carbamates OFH243 (13n), OFH244 (13m), OFH3911 (14n), and OFH3912 (14m) were discovered, which display two-digit picomolar affinities with K_i values from 0.069 to 0.084 nM, as well as high selectivity over the M₂ subtype (46-to 68-fold). While weak inverse agonistic properties were determined for the biphenyl carbamates 13m and 13n, neutral antagonism was observed for 14m and 14n and tiotropium under identical assay conditions.

Full text of DOI:10.1021/acs.jmedchem.0c00297

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Article
Regiospecific introduction of halogens on the 2-aminobiphenyl
subunit leads to highly potent and selective M3 muscarinic
acetylcholine receptor antagonists and weak inverse agonists
Oliver Fischer, Josefa Hofmann, Hannelore Rampp, Jonas Kaindl, Gerald Pratsch, Amelie Bartuschat,
Regina Verena Taudte, Martin Friedrich Fromm, Harald Hübner, Peter Gmeiner, and Markus Rolf Heinrich
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00297 • Publication Date (Web): 23 Mar 2020
Downloaded from pubs.acs.org on March 29, 2020
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Regiospecific introduction of halogens on the 2-aminobiphenyl subunit leads to highly
potent and selective M3 muscarinic acetylcholine receptor antagonists and weak inverse
agonists
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a
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a
Oliver Fischer, Josefa Hofmann, Hannelore Rampp, Jonas Kaindl, Gerald Pratsch, Amelie
a
b
b
a
a
Bartuschat, R. Verena Taudte, Martin F. Fromm, Harald Hübner, Peter Gmeiner, Markus R.
Heinrich a,*
aDepartment of Chemistry and Pharmacy, Pharmaceutical Chemistry, Friedrich-Alexander-
Universität Erlangen-Nürnberg, Nikolaus-Fiebiger-Str. 10, 91058 Erlangen, Germany
bInstitute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-
Universität Erlangen-Nürnberg, Fahrstr. 17, 91054 Erlangen, Germany
markus.heinrich@fau.de
Abstract
Muscarinic M receptor antagonists and inverse agonists displaying high affinity and subtype
3
selectivity over the anti-target M are valuable pharmacological tools and may enable improved
2
treatment of chronic obstructive pulmonary disease (COPD), asthma or urinary incontinence.
Based on known M antagonists comprising a piperidine or quinuclidine unit attached to a biphenyl
3
carbamate, 5-fluoro substitution was responsible for M subtype selectivity over M , while 3'-
3
2
chloro substitution substantially increased affinity through a σ hole interaction. Resultantly, two
piperidinyl- and two quinuclidinium-substituted biphenyl carbamates OFH243 (13n), OFH244
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(
13m), OFH3911 (14n) and OFH3912 (14m) were discovered, which display two-digit picomolar
affinities with K values from 0.069 nM to 0.084 nM, as well as high selectivity over the M subtype
i
2
(46- to 68-fold). While weak inverse agonistic properties were determined for the biphenyl
carbamates 13m and 13n, neutral antagonism was observed for 14m and 14n and tiotropium under
identical assay conditions.
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Introduction
Around thirty percent of the drugs marketed today exhibit their effects via G protein-coupled
receptors. Among this family of membrane proteins, muscarinic acetylcholine receptors (mAChRs)
1
play an important role in various physiological processes. The group of mAChRs is known to
comprise five receptor subtypes M to M , which signal either via the G (for M M M ) or G
i
1
5
q
1,
3,
5
pathway (for M and M ). While the M subtype has been shown to be involved in CNS functions
2
4
1
including cognition, the M receptor is mainly located at the heart and is further associated with
2
the regulation of gastrointestinal motility. For the M subtype, a predominant occurrence has been
3
found at the lung and the bladder. The M and M receptor subtypes mainly occur in the central
4
5
2
nervous system. While the M receptor has been investigated with regard to the treatment of
4
3
4
5
Alzheimer , schizophrenia and Parkinson’s disease , little is so far known about the M subtype
5
and its biological relevance.6
Against this background and the multitude of diverse pharmacological functions mediated by
mAChRs, high subtype selectivity is undoubtedly an important aspect for therapeutic efficacy and
7
limitation of side effects. Nevertheless, compounds with low subtype selectivity may also serve
as useful pharmaceuticals for two reasons: At first, the low selectivity among the subtypes may be,
to a certain extent, counterbalanced by kinetic selectivity, meaning that the drug’s residence time
8
in the desired receptor subtype is much longer than in the competing subtypes. Secondly, selective
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activity can be also a result of local application taking advantage of tissue-specific expression. A
prominent example, in which the drug benefits from both effects, is tiotropium bromide (1) a non-
selective muscarinic M receptor antagonist to treat chronic obstructive pulmonary disease (COPD)
3
(
Figure 1).9-10
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O
O
O
N
S
S
OH
O
N
N
H2N
O
H
N
O
Br
O
S
HCOO
O
F
1
2
3
Figure 1. Tiotropium bromide (1), darifenacin (2), and BS46 (3).
While a number of recent studies strongly support the cardiovascular safety of tiotropium
bromide,11-12 other clinical studies still give hints to M receptor mediated side effects,13-15 thereby
2
indicating that kinetic selectivity and local application might not fully be able to overcome a lack
of subtype selectivity. An orally applied M receptor antagonist showing much higher subtype
3
selectivity is darifenacin (2) (Figure 1), which is less potent in respiratory disease but widely used
for the treatment of urinary incontinence.16-17 Despite its selectivity towards the M subtype (6.9-
3
fold over M , 56-fold over M ), darifenacin (2) may still lead to adverse effects, including dry
1
2
1
8
mouth, constipation and abnormal vision. In addition, its in-vivo activity suffers from problematic
19
metabolism and fast degradation. Against this background, and despite the existence of broadly
established and well-studied M antagonistic drugs, such as 1 and 2, more detailed insight about
3
the activity, pharmacology and structural recognition of the related pharmaceutically active ligands
would be highly desirable.
Today, it is possible to obtain valuable information from crystal structures leading to an increased
number of GPCR ligands being designed and optimized by structure-based drug design, thereby
20
demonstrating the vast potential of this rationale. Recent crystal structures of the human M and
2
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M receptor binding the antagonists R-(-)-3-quinuclidinyl benzilate and tiotropium bromide,
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respectively, showed that both ligands are located in a deeply buried binding pocket within the
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1
highly conserved transmembrane receptor core adopting a similar binding pose. One significant
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difference between the M and the M subtype derives from the exchange of Phe181 (M ) for a
2
3
2
sterically less demanding Leu225 (M ) in the extracellular loop ECL2. This modification offers
3
additional space for ligands binding to the M receptor, which is not available in the M receptor,
3
2
and it can thus be exploited to achieve high affinity and good subtype selectivity over the M2
subtype, as we have shown in our very recent development of the M receptor antagonist BS46 (3)
3
(
Figure 1). 21-22
Having shown that the Phe181/Leu225 distinction between the M and the M receptor subtype
2
3
represents an eligible starting point for the optimization of M subtype-selective ligands, we
3
continued our research on the lipophilic aromatic moieties pointing at the critical amino acids in
ECL2 thereby particularly focusing on non-heterocyclic biphenyl carbamates. The biphenyl motif
already was the focus of previous studies23-26 and patents,27-29 however none of those focused on a
more detailed exploration of substituent effects on the ring systems A and B of the biphenyl moiety
2
3
(
Figure 2). Biphenyl carbamates such as 4a and 4b were for example studied by Naito, and the
pharmacophoric moiety was also incorporated into bivalent ligands25-26 addressing muscarinic and
30
adrenergic receptors. For the most prominent example of this approach, batefenterol (4c), a
dosage for phase III clinical trials was selected in 2019.31
O
N
OH
N
O
NH
O
O
N
O
H
HN
O
HN
O
O
N
N
HN
A
O
B
B
H
B
Cl
HN
O
N
O
A
A
B
H
OH
4a
4b
4c
5
A
F
Naito et al. (1998)
Ki (M3) = 27 nM
M /M = 10
Naito et al. (1998)
Ki (M3) = 0.47 nM
M /M = 16
Hughes et al. (2015)
K (M ) = 2.0 nM
i
3
K (M ) = 0.34 nM
i
3
2
3
2
3
EC50 ( ) = 0.25 nM
2
M /M = 91
2
3
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Figure 2. Examples of M receptor antagonists 4a, 4b, batefenterol (4c) and ABH423 (5) with a
3
biphenyl carbamate unit as common structural motif.
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In this work, we provide insights into effects of substitution on the biphenyl unit combined with
structural changes in the basic alkyl amine moiety using our previously developed, highly subtype
2
2
selective compound ABH423 (5) as a lead structure. Besides its M receptor affinity and subtype
3
selectivity over M , this compound was chosen due to its substantial reduction of airway resistance
2
22
in mice while leaving the heart rate unaffected. Important aspects addressed in this study are
whether selectivity without 5-fluorine substitution on ring A can be achieved, and whether further
substituents on ring B may increase affinity and selectivity. Generally, it was our goal to not only
develop a ligand with high affinity for the M receptor and substantial selectivity for the M over
3
3
the M subtype, but one which would also be synthetically accessible in larger quantities to enable
2
broad pharmacological investigations in the future. In a chemistry-driven approach using
regioselective radical arylation, these multiple requirements could be met by carefully optimizing
the biphenyl carbamate lead structure and by exploiting an uncommon interaction by mediated by
regioselective halogen substitution.
Results
Chemistry
The synthesis of the 30 ligands 8a,d,k,m,n 11, 13a-o, 14a,d,m,n, 15, 16m,n, 17 and 18 was based
on three novel radical chemistry methods for the preparation of 2-aminobiphenyls (Scheme 1).32-34
These reactions exploit the comparably high regioselectivity that can be achieved in radical
3
5
arylations of anilines under neutral or basic conditions. Since the methods start from simple
precursors such as aryldiazonium salts or arylhydrazines, whereat the aniline can be employed
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without additional functionalization or protecting group, these reactions represent an interesting
alternative to well-established Suzuki-type cross couplings. Using triphosgene, all 2-
aminobiphenyls 6a-o were then further converted to the corresponding isocyanates, which provided
the carbamates 7a-o and 8a,d,k,m,n upon reaction with either N-Boc 4-piperidinemethanol (9) or
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(
R)-quinuclidin-3-ol (10). The carbonyl diamide (urea) derivative 11 was obtained from 6d and
R)-quinuclidin-3-amine (12). Trifluoroacetic acid mediated deprotection of 7a-o gave the ligands
(
13a-o, and methylation of 8a,d,m,n, 11 and 13m,n using methyl iodide furnished the quaternary
ammonium salts 14a,d,m,n, 15 and 16m,n after exchange of the counterion to trifluoroacetate. A
slightly modified strategy turned out as beneficial for the tropane and scopine derivatives 17 and
18, whereat 3α-tropanol (19) or scopine (20) were at first converted by triphosgene to their
corresponding chloroformates, which were finally coupled to 2-amino-5-fluorobiphenyl (6d).
NH2
NH2
NH2
+
N2 Cl-
N
R2
R2
H
R1
R1
a
b
N
O
O
3'
2
'
HN
O
NH2
4'
5'
HN
X
c
e
1'
2
NBoc
1
R2
3
R2
R2 6'
4
6
R1
a-o
f
R1
R1
5
7
6a-o
a: R1 = H ; R = H
2
8
; X = O
; X = O
1
2
8d: R = 5-F; R2 = H
8
k: R1 = 5-F; R = 3',4'-Cl2 ; X = O
8m: R = 5-F; R2 = 3'-Cl
1
O
O
; X = O
3'
3'
8
n: R1 = 5-F; R = 3'-Cl, 4'-F ; X = O
1
2
2
'
2'
1'
11: R = 5-F; R2 = H
4
'
HN
4'
5'
HN
; X = NH
O
O
g
1
'
2
2
d
5'
1
N
1
NH
3
3
R2 6'
R2 6'
4
R1
HCO2
4
R1
6
6
g
5
5
6m: R1 = 5-F ; R2 = 3'-Cl
13a: R1 = H ; R2 = H
1
1
1
2
1
2
6n:
R
= 5-F ; R = 3'-Cl, 4'-F
13b:
13c:
R
R
R
= H ; R = 4'-Cl
1
1
2
N
R3
= H ; R = 3',4',5'-F3
O
N
2
O
1
1
3d:
3e:
= 5-F ; R = H
3'
4
1
1
= 5-F ; R2 = 2'-Cl
R
R
HN
O
2'
1
4
'
X
HN
2
13f:
R
R
R
= 5-F ; R = 4'-F
'
2
CF3CO2
1
1
= 5-F ; R2 = 4'-Cl
5'
1
1
1
3g:
3h:
3
2 6'
2
R
or
= 5-F ; R = 4'-Br
1
1
1
1
1
= 5-F ; R2 = 4'-CN
6
4
HCO2
13i:
R
R
R
R
2
5
R1
13j:
= 5-F ; R2 = 4'-OMe
F
3
13k:
= 5-F ; R = 3',4'-Cl2
= 5-F ; R2 = 3',4',5'-F3
4
14a: R1 = H ; R = H
2
17: R , R
18: R , R
= H
; X = O
; X = O
; X = O
13l:
3
4
1
2
= -O-
14d: R = 5-F; R2 = H
1
3m: R = 5-F ; R2 = 3'-Cl
4m: R1 = 5-F; R = 3'-Cl
1
1
1
1
2
1
13o:
3n:
R
R
= 5-F ; R = 3'-Cl, 4'-F
1
2
4n: R1 = 5-F; R = 3'-Cl, 4'-F ; X = O
= 5-F; R2 = H
; X = NH
= 5-F ; R2 = 3',5'-Cl2
15:
R
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Scheme 1. Synthetic routes to ligands 8a,d,k,m,n 11, 13a-o, 14a,d,m,n, 15, 16m,n, 17 and 18
a
based on the radical arylation of anilines. Reagents and conditions: a) NaOH, 75-95 °C; b) O
2
(air), 60 °C or MnO , air, rt; c) triphosgene, CH Cl , 0 °C, then N-boc-4-piperidinemethanol,
2
2
2
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
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4
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5
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5
5
5
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5
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0
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0
DIPEA, CH Cl , 0 °C; d) triphosgene, CH CN, 0 °C, 3α-tropanol (19) or scopine (20); then 6d,
2
2
3
pyridine, 80 °C or 60 °C; e) triphosgene, CH Cl , 0 °C, then (R)-quinuclidin-3-ol (10) or (R)-
2
2
quinuclidin-3-amine (12), toluene, 110 °C f) 10% TFA in CH Cl , rt; g) MeI, K CO , CH Cl , rt.
2
2
2
3
2
2
1
The identity of all prepared ligands was verified by H NMR spectroscopy and their purity was
established by analytical HPLC.
Ligand binding and functional properties
Ligand binding. Radioligand competition binding assays were performed to determine binding
affinities and the selectivity profiles of the new muscarinic receptor ligands 8a,d,k,m,n, 11, 13a-
o, 14a,d,m,n, 15, 16m,n, 17 and 18, in comparison with the reference drug darifenacin (2). The
3
binding data were generated by measuring the ligands’ ability to compete with [ H]N-
methylscopolamine using membranes of HEK293T cells transiently transfected with the cDNA of
the human M , M or M receptor.
1
2
3
22
In the initial stage, the lead structure ABH423 (5) was modified at diverse positions to explore
starting points for further variations (Figure 3, variations shown in blue). Replacement of the basic
quinuclidine moiety by a tropane and or a scopine unit, such as in 17 and 18, partially reduced M3
receptor affinity and selectivity. Variation of carbamate to give the urea derivative 11 led to a
significant loss in affinity, which became even stronger after quaternization for the ammonium salt
1
5. In contrast, quaternization of ABH423 (5) increased affinity but slightly reduced selectivity,
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9
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
as determined for ligand 14d. Further substituents at the non-fluorinated phenyl ring B, as they
were introduced in the 3',4'-dichloro derivative 8k, resulted in a minor loss of affinity and
selectivity, thus showing that there could be possible room for improvements associated with the
optimization of the substitution pattern on ring B. Among the diverse variations shown in Figure
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
3
, the piperdin-4-ylmethyl derivative 13d retained the highest M subtype selectivity relative to the
3
M receptor at an acceptable loss of M receptor affinity. Against this background, we decided to
2
3
continue our studies with further variations on the biphenyl core and in combination with the (R)-
quinuclidin-3-yl and the piperidin-4-ylmethyl units as basic side chains.
N
O
N
O
HN
F
O
O
Ki (M1) = 0.93 ± 0.19
Ki (M2) = 30 ± 3.7
Ki (M3) = 1.1 ± 0.24
M2/M3 = 27
O
HN
F
O
HN
F
O
NH
Ki (M1) = 1.1 ± 0.14
Ki (M2) = 47 ± 5.4
Ki (M3) = 0.65 ± 0.049
M2/M3 = 72
Ki (M1) = 2.1 ± 0.40
Ki (M2) = 52 ± 9.7
Ki (M3) = 0.63 ± 0.079
M2/M3 = 83
17
13d
18
N
O
N
N
Cl
Cl
O
HN
O
O
B
3
K (M ) = 0.53 ± 0.10
HN
O
i
1
HN
F
N
A
Ki (M2) = 31 ± 10
Ki (M3) = 0.34 ± 0.050
M2/M3 = 91
H
Ki (M1) = 5.3 ± 0.37
Ki (M2) = 130 ± 6.7
Ki (M3) = 2.3 ± 0.27
M2/M3 = 57
5
Ki (M1) = 0.73 ± 0.24
Ki (M2) = 13 ± 0
Ki (M3) = 0.53 ± 0
M2/M3 = 25
F
ABH423 (5)
F
8k
11
CF3COO
O
CF3COO
O
N
N
HN
O
HN
N
H
Ki (M1) = 45 ± 4.2
Ki (M2) = 370 ± 120
Ki (M3) = 19 ± 2.8
M2/M3 = 19
Ki (M1) = 0.75 ± 0.12
Ki (M2) = 14 ± 2.0
Ki (M3) = 0.23 ± 0.038
M2/M3 = 61
F
14d
F
15
Figure 3. Initial structural variations of the lead structure ABH423 (5). K values are the means
i
from 3-7 individual experiments each done in triplicate [nM±SEM]. For compound 8k, 13d and
15 binding affinity is displayed in K ±SD derived from two experiments.
i
Having observed that the piperidin-4-ylmethyl unit might be a useful replacement for the (R)-
quinuclidin-3-yl moiety, the question arose whether a certain subtype selectivity could even be
achieved without 5-fluorine substitution on the ring A. Table 1 gives an overview over the binding
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and selectivity data determined for darifenacin (2) and six ligands bearing unsubstituted 2-
aminobiphenyl moieties, whereat the biphenyl carbamates 4a and 8a (8a identical to 4b, see Figure
2
3
2) were introduced to enable a comparison to the previous studies by Naito. A comparison of the
3
6
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
data obtained for darifenacin (2) with those previously reported, shows that our assay conditions
lead to comparable results for binding to the M and M subtypes, but our biological evaluation
1
3
tends to be more sensitive for the M subtype. This deviation for the M subtype, which roughly
2
2
corresponds to a factor of 4, was also found for the carbamate 8a that was studied by us and
23
previously by Naito et al. As a result, the M /M subtype selectivities reported in this work may
2
3
to a certain extent be underestimated.
Notably, the best M over M subtype selectivities among our biphenyl carbamates were found for
3
2
the piperidin-4-ylmethyl derivatives 13a and 13c, and 13a also displayed a high affinity of K =
i
0
.47 nM towards the M receptor. The highest binding affinity to the M subtype was determined
3 3
for the quaternary ammonium salt 14a, which is in agreement with the data of the closely related
analogue 14d (Figure 3), albeit 14a showed a significantly lower M over M subtype selectivity
3
2
than 14d.
Table 1. Muscarinic acetylcholine M receptor ligands bearing an unsubstituted ring A in the 2-
3
aminobiphenyl moiety.
N
O
O
NH
O
HN
O
HN
O
HN
O
B
NH
A
8ae
4a
13a
N
O
F
O
O
Cl
F
F
HN
O
HN
O
HN
O
NH
NH
CF3COO
13b
13c
14a
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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
compound
K
i
(nM ± SEM)a
selectivityb
/M
12
M
8.6 ± 3.3
1
M
2
M
3
M
1
3
2 3
M /M
darifenacin (2)
12 ± 1.2
47c
270d
2.0 ± 0.33
7.6d
17 ±3.5
120 ± 5.8
28 ±1.7
1.7 ± 0.42
0.74 ± 0.14
0.8c
27d
0.36 ± 0.047
0.47d
0.47 ± 0.090
11 ± 3.5
0.90 ± 0.080
0.17 ± 0.040
16
56
10
5
.5c
6.9
1.2
1.6
1.4
3.8
3.0
3.9
2.4
4
8
a
32d
ae
0.56 ± 0.11
5.6
16
36
11
31
10
0
.65d
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
1
3a
1.8 ±0.33
34 ± 4.4
3.5 ± 0.75
0.40 ± 0.090
13b
13c
1
4a
aK
values in nM±SEM are the means of 3-5 individual experiments each done in triplicate. Selectivity was determined
b
i
c
by calculating the ratio of K
i
(M
1
)/K
i
(M
3
) or K
i
(M
2
)/K
i
(M
3
), respectively. Reported affinities according to Hirose et al.
23
(
2, Figure 1).36 dReported affinities according to Naito et al. (4a, Figure 2). eLigand 8a is identical to reference
2
3
compound 4b described by Naito et al. (Figure 2) .
The fact that a remarkable selectivity for the M over the M receptor can be achieved with
3
2
piperidin-4-ylmethyl ligands could be due to an increased distance between the basic amine, acting
as anchor unit, and the biphenyl carbamate. In this way, ring A might be pushed closer to the
differentiating amino acids Phe181 (M ) and Leu225 (M ) in the extracellular loop ECL2, which
2
3
would explain the increased subtype selectivity. Related results from computational studies are
presented below and support this assumption.
Having observed that substitution on ring B can be tolerated (Figure 3), we turned to investigate
the substituent effects in more detail (Table 2). At first, it appeared that, compared to the
unsubstituted biphenyl 13d, only the small fluorine atom of 13f in position 4' of ring B would be
beneficial, albeit a slight loss of affinity. Larger atoms or groups, or poly-substitution, as they are
present in the biphenyl carbamates 13e, 13g-j and 13l were not well tolerated. Then, however, the
high binding affinity and subtype selectivity of the 3',4'-dichloro derivative 13k (K = 0.25 nM,
i
M /M = 68) attracted our attention, which is even more remarkable since the 4'-chloro analogue
2
3
1
3g (K = 14 nM, M /M = 23) showed a significantly worse profile. The increase in M receptor
i 2 3 3
affinity (and similarly to the other subtypes M and M ) should thus be due to a favorable ligand-
1
2
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receptor interaction mediated by the chlorine atom in 3'-position. Notably, 3',4'-dichloro
substitution on ring B had previously led to a reduction of M receptor affinity and M over M
2
3
3
subtype selectivity (c.f. ligands 8d and 8k, Figure 3), thereby pointing out the highly sensitive
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
interplay of the basic amine and the substitution on the biphenyl.
Table 2. Substitution at ring B combined with 5-fluorine substitution on ring A
O
O
R
HN
A
O
HN
F
O
B
NH
NH
F
13d
13e-l
(nM ± SEM)a
K
i
selectivityb
compound
R =
M
1
M
2
M
3
M
1
/M
3
2 3
M /M
1
1
1
1
13h
13i
1
1
1
3d
3e
3f
3g
--
2´-Cl
4´-F
4´-Cl
4´-Br
4`-CN
4`-OMe
2.1 ± 0.40
53 ± 14
4.8 ± 0.95
34 ± 1.6
52 ± 9.7
980 ± 210
98 ± 29
320 ± 91
340 ± 40
28000 ± 6400c
2400 ± 1400c
17 ± 3.5
0.63 ± 0.080
30 ± 6.4
1.0 ± 0.24
14 ± 2.3
7.0 ± 0.41
2000 ± 0c
84 ± 7.1c
0.25 ± 0.020
6.6 ± 0.42
3.3
1.8
4.8
2.4
2.1
7.5
3.0
2.6
3.6
83
33
98
23
49
14
29
68
79
15 ± 1.8
15000 ± 710c
250 ± 21c
0.66 ± 0.25
24 ± 2.7
3j
3k
3l
3´,4´-Cl
3´,4´,5´-F
2
3
520 ± 43
aK
values in nM±SEM are the means of 3-7 individual experiments each done in triplicate. Selectivity was determined
by calculating the ratio of K
b
i
c
i
(M
1
)/K
i
(M
3
) or K
i
(M
2
)/K
i
(M
3
), respectively. Ki values in nM±SD are the means of two
individual experiments each done in triplicate.
The results from the more detailed investigation of the 3'-chloro substitution effect are summarized
in Table 3. Taking the 3',4'-dichloro derivatives 8k and 13k as reference compounds, whereat the
direct comparison reveals an advantage of the basic piperidin-4-ylmethyl over the quinuclidin-3-
yl moiety, the single chlorine atom in 3'-position in biphenyl carbamate 13m was able to increase
the binding affinity towards the M receptor by a factor of 3 compared to 13k while maintaining
3
subtype selectivity on a high level. As a σ hole interaction of the 3'-chloro substituent with a closely
located donor group in the receptor appeared to be a reasonable explanation for the stronger binding
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1
1
1
1
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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
of 13m, we tried to amplify this effect by adding a further fluorine atom in 4' position of ring B.
Carbamate 13n, possessing a larger σ hole at the 3'-chlorine atom than 13m (see also molecular
electrostatic potential (MEP) calculations below), then indeed displayed a further increased binding
affinity (K = 0.069 nM), albeit at a slightly decreased M /M subtype selectivity of 49. The effect
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
i
2
3
of a second meta substituent on ring B, which was investigated with the 3',5'-dichloro derivative
3o, turned out to be a significantly decreased binding to the M subtype, thus supporting the
1
3
assumption that the binding affinity is very sensitive to changes on the B ring.
Table 3. Ligands bearing a 3'-chloro substituent on ring B.
N
N
O
O
O
O
R
R
R
R
HN
F
O
HN
F
O
HN
F
O
HN
F
O
NH
HCO2
N
HCO2
8k,m,n
13k,m-o
14m,n
16m,n
compound
R =
K
i
(nM ± SEM)a
selectivityb
2 3
/M M /M
M
1
M
2
M
3
M
1
3
8
k
3´,4´-Cl
3´-Cl
3´-Cl, 4´-F
2
0.73 ± 0.24c
0.53 ± 0.11
0.36 ± 0.079
0.66 ± 0.25
0.34 ± 0.14
0.27 ± 0.11
3.3 ± 1.2
0.11 ± 0.022
0.12 ± 0.009
1.8 ± 0.14
13 ± 0c
31 ± 18
14 ± 2.7
17 ± 3.5
5.7 ± 1.4
3.4 ± 0.64
70 ± 12
3.3 ± 1.2
3.7 ± 0.71
82 ± 10
0.53 ± 0c
0.39 ± 0.081
0.35 ± 0.083
0.25 ± 0.020
0.084 ± 0.010
0.069 ± 0.010
1.5 ± 0.18
0.072 ± 0.004
0.070 ± 0.011
2.7 ± 0.44
1.4
1.4
1.0
2.6
4.0
3.9
2.2
1.5
1.7
0.67
0.65
25
79
40
68
68
49
47
46
53
30
23
8
m
8
n
1
3k
3m
3n
3o
3´,4´-Cl
3´-Cl
2
1
1
1
3´-Cl, 4´-F
3´,5´-Cl
3´-Cl
2
14m
14n
16m
3´-Cl, 4´-F
3´-Cl
3´-Cl, 4´-F
1
6n
1.1 ± 0.096
39 ± 2.3
1.7 ± 0.13
a
b
Ki values in nM±SEM are the means of 3-4 individual experiments each done in triplicate. Selectivity was determined
by calculating the ratio of K
c
i
(M
1
)/K
i
(M
3
) or K
i
(M
2
)/K
i
(M
3
), respectively. Ki values in nM±SD are the means of two
individual experiments each done in triplicate.
The favorable 3'-chloro and 3'-chloro,4'-fluoro substitution patterns on the biphenyl cores of 13m
and 13n were further evaluated in combination with the basic quinuclidine moiety to give ligands
8m and 8n. Through this modification, the M over M subtype selectivity was largely retained, or
3
2
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even improved for 8m, but the affinities of 8m and 8n towards the M receptor dropped
3
significantly. Finally, quaternization of 8m,n and 13m,n was carried out to yield the corresponding
ammonium salts 14m,n and 16m,n, which should show improved pharmacokinetic profiles as they
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
cannot pass the blood-brain barrier and are thus unable to interact with M receptors located in the
1
CNS. Again, the strong interplay of the amine moiety – now being present as ammonium ion – and
the biphenyl subunit became apparent. While the dimethylpiperidinium derivatives 16m,n
displayed relatively weak binding to the M subtype and below average M over M subtype
3
3
2
selectivities, the quinuclidinium salts 14m and 14n reached highly favorable binding profiles with
affinities of K = 0.072 nM and K = 0.070 nM, and M over M subtype selectivities of 46 and 53,
i
i
3
2
respectively.
In terms of binding affinity towards the M subtype and selectivity for the M over the M receptor
3
3
2
the biphenyl carbamates OFH244 (13m), OFH243 (13n), OFH 3912 (14m) and OFH3911 (14n)
range among the best ligands ever developed for this particular muscarinic receptor subtype. In this
context, it is interesting to note that 3'-chloro substitution on the B ring already appeared in earlier
studies, but this effect was so far mainly exploited for the development of highly potent M receptor
1
agonists and non-selective muscarinic antagonists.25,37-39 As briefly mentioned above, the
comparably high affinities of OFH 3912 (14m) and OFH3911 (14n) to the M subtype, which is
1
predominantly located in the CNS, postganglionic nerves and exocrine glands,40-42 are less
problematic for the quaternary ammonium salts 14m and 14n, as these compounds are unable to
pass the blood-brain barrier. For the amines OFH243 (13n) and OFH244 (13m), in contrast, brain
penetration can basically not be excluded. In this context, it was interesting to see that the mild
43
CNS related side effects of darifenacin (2) are not related to the presence of a protonatable,
aliphatic amine in the structure, but due to the fact that darifenacin (2) is a substrate for P-
4
4
glycoprotein (P-gp) and, therefore, is actively transported away from the brain. In agreement with
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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
that, it has been reported that darifenacin (2) at pharmacological doses does not decrease the
binding potential of a PET radioligand which can label muscarinic receptors in the rat cerebral
4
5
cortex and corpus striatum. Against this background, and to evaluate whether the amines OFH243
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
(13n) and OFH244 (13m) might also benefit from an interaction with P-gp, transcellular transport
studies have been carried out (see below).
Functional profile of target compounds. Functional data at the M receptor were obtained
3
®
applying an HTFR based IP accumulation assay (IP-One ) to measure the G coupled activation.
1
q
Arrestin recruitment was measured with an enzyme fragment complementation assay
®
(PathHunter ) (Figure 4). Relative to the reference agonist carbachol the test compounds OFH244
(13m) and OFH243 (13n) revealed weak inverse agonist profiles with efficacies of 23% under
basal activity, while OFH3912 (14m) and OFH3911 (14n) were found to be neutral antagonists for
G-protein coupled signaling (Figure 4A, Table 4). For the reference compound darifenacin (2) a
very weak inverse agonist effect (efficacy of -10%) was determined, while tiotropium bromide (1)
was found to act as a neutral antagonist (Figure S3A). Looking at arrestin recruitment all test
compounds show neutral antagonism at the M subtype (Figure 4C, Figure S3E,G, Table 4). This
3
overall observation was confirmed when looking at the inhibitory effect of the test compounds on
the EC concentration of carbachol. While the quinuclidinium ligands 14m and 14n showed
8
0
inhibitory potencies of 6.3 and 6.8 nM (Figure 4B), respectively, which is in the same range as the
inhibitory power of tiotropium (IC = 5.8 nM) (Figure S3B, Table 4), 13m and 13n revealed IC
50
5
0
values of 14 nM and 9.3 nM with an inverse efficacy of -14% and -13%, respectively. Together
with the piperidine derivatives 8m and 8n, which showed IC values of 17 nM and 19 nM, all
5
0
tested ligands revealed a 2.5- to 7-fold better M receptor inhibition when compared to the reference
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darifenacin (IC = 45 nM) (Figure S3B,C,D, Table 4). Similar results could be observed for
5
0
arrestin recruitment (Figure 4D, Figure S3E-H, Table 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
A
B
100
carbachol
13m
100
75
50
25
0
75
50
25
0
13n
14m
1
1
3m + carbachol
3n + carbachol
14n
14m + carbachol
4n + carbachol
1
-25
-25
-13 -12 -11 -10 -9
-8
-7
-6
-5
-13 -12 -11 -10 -9
-8
-7
-6
-5
compound [logM]
compound [logM]
C
D
1
00
carbachol
3m
13n
100
75
50
25
0
1
7
5
2
5
0
5
0
1
1
4m
4n
13m + carbachol
13n + carbachol
14m + carbachol
14n + carbachol
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2
-13 -12 -11 -10 -9
-8
-7
-6
-5
compound [logM]
compound [logM]
Figure 4. Functional properties of OFH243 (13n), OFH244 (13m), OFH3911 (14n) and OFH3912
®
(14m) applying an IP accumulation assay (IP-One ) as readout for G-protein-mediated signaling
1
®
(
A, B) and a -arrestin recruitment (PathHunter ) assay for arrestin signaling (C, D). A) The
piperidines 13m and 13n show inverse agonist activity, while neutral antagonism was determined
for the quaternized quinuclidines 14m and 14n when looking at G-protein signaling. B) Inhibition
of the agonist effect of 100 nM carbachol was measured for 13m, 13n, 14m, and 14n with inverse
agonist properties for 13m and 13n. C) Neutral antagonism of arrestin signaling was observed for
all test compounds. D) Inhibition of arrestin recruitment induced by 50 µM of carbachol show
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
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5
5
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inhibitory properties for 13m, 13n, 14m, and 14n, which are similar to that of the G-protein
pathway. (A-D) Pooled curves from 3-10 experiments each done in duplicates).
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
Table 4. Functional data for the test compounds 8m, 8n, 13m, 13n, 14m, 14n and the references
tiotropium (1), darifenacin (2) and carbachol derived from an IP accumulation test and an arrestin
1
recruitment assay with M R.
3
G-protein signalinga
agonist modec
antagonist moded
compound
carbachol
EC50 [nM]
eff [%]e
EC50 [nM]
-
eff [%]e
EC50 [µM]
eff [%]e
100
<3
EC50 [nM]
-
eff [%]e
25±3.1
100
-
22±2.2
-
8
m
-
<3
17±3.2
19±2.7
14±1.6
9.3±1.3
6.3±0.2
6.8±0.9
5.8±0.7
45±14
0.4±1.2
-0.2±0.9
-14±5.8
-13±3.8
1.6±2.3
-0.8±0.6
0.8±1.1
-6.2±3.4
-
-
-
-
-
-
-
-
27±4.9
25±4.7
26±4.8
4.1±1.4
2.2±0.3
2.2±0.3
0.4±0.1
11±3.2
-2.2±0.6
-1.8±1.2
-1.8±1.2
2.4±0.8
1.4±0.6
1.0±0.5
-1.2±0.7
2.0±1.9
8n
-
<3
<3
13m
6.9±1.6
-24±2.5
-23±3.2
<3
<3
1
3n
4m
4n
4.1±1.5
<3
1
-
<3
1
-
-
<3
<3
tiotropium
darifenacin
<3
<3
32±13
-9.9±2.2
<3
a
®
IP-One assay (Cisbio) measuring the receptor stimulated accumulation of IP
1
in HEK293T cells transiently
transfected with M receptors. Values are the means±SEM derived from 4-10 individual experiments each done in
3
b
duplicate. Test on arrestin-2 recruitment (DiscoverX) applying an arrestin complementation assay. Values are the
c
means±SEM derived from 3-11 single experiments each done in duplicate. Agonist mode displaying the stimulating
d
effect of a test compound. Antagonist mode expressing the inhibitory effect of an antagonist on the EC80 concentration
e
of carbachol. Efficacy determined relative to the full effect of quinpirole (100%) and the basal effect of buffer (0%).
Regarding the functional data of established M receptor ligands, it is worth to note that tiotropium
3
(1) is typically referred to as an antagonist although inverse agonistic properties have also been
4
6
reported. Since tiotropium (1) appears as a neutral antagonist under our assay conditions, while
OFH243 (13n) and OFH244 (13m) show a weak inverse agonistic profile, the actual decrease of
activation attainable by 13m and 13n may even be underestimated in our present study. For the
biphenyl carbamates 4a and 4b (Figure 2), no functional data has been reported that would give a
hint towards inverse antagonism.23 The same is true for batefenterol (4c) (Figure 2), which
currently enters clinical phase III trials.30
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Transcellular transport studies
The data obtained from the transcellular transport studies clearly indicate that both OFH243 (13n)
and OFH244 (13m) are substrates of P-glycoprotein. OFH243 (13n) basal-to-apical translocation
at 1 µM was considerably higher compared to and apical-to-basal translocation (9.2 ± 1.7 vs 3.5 ±
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
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2
2
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3
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3
4
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4
4
4
5
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5
5
5
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5
5
6
0
1
2
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9
0
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9
0
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2
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8
9
0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
.0 a.u., P < 0.001) yielding an efflux ratio (i.e. ratio of basal-to-apical and apical-to-basal
translocation) of 2.7. Similarly, OFH243 (13n) at 10 µM showed a pronounced polarized transport
in the basal to apical direction (99.5 ± 13.7 vs 46.2 ± 23.2 a.u., efflux ratio 2.2, P < 0.05).
OFH244 (13m) basal-to-apical translocation at 1 µM was also considerably higher compared to
apical-to-basal translocation (9.0 ± 3.4 vs 3.7 ± 1.1 a.u., P < 0.001) yielding an efflux ratio of 2.4.
Moreover, OFH244 (13m) at 10 µM showed a pronounced polarized transport in the basal to apical
direction (79.3 ± 7.0 vs 46.5 ± 17.2 a.u., efflux ratio 1.7, P < 0.01). Polarized, basal to apical net
transport was abolished by adding the P-gp inhibitor valspodar to the apical compartment for both
OFH243 (13n) and OFH244 (13m) (basal to apical vs apical to basal, n.s., for both compounds and
both concentrations).
The control experiments using the P-glycoprotein substrate digoxin showed the expected results.
In the absence of valspodar, basal-to-apical translocation of digoxin was considerably higher
compared to apical-to-basal translocation (19.9 ± 2.4% vs 6.7 ± 0.3% of the administered dose, P
<
0.001). This pronounced polarized digoxin transport was completely abolished with apical
administration of valspodar: basal to apical and apical to basal digoxin translocation were now
nearly identical (10.2 ± 0.6 vs 10.5 ± 0.5, n.s.).
Metabolic stability
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6
The metabolic stability of the secondary amines OFH243 (13n) and OFH244 (13m), as well as the
quaternary ammonium salts OFH3911 (14n) and OFH3912 (14m) was studied using male rat liver
microsomes and imipramine as a positive control under established conditions.47,48 The results for
OFH243 (13n), OFH244 (13m), OFH3911 (14n), OFH3912 (14m), and the reference compound
imipramine are shown in Figure 5.
0
1
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8
9
0
1
2
3
4
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8
9
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9
0
1
00.0
80.0
6
4
0.0
0.0
20.0
0
.0
0
20
40
60
80
100
120
time [min]
OFH243 (13n)
OFH3911 (14n)
Imipramine
OFH244 (13m)
Figure 5. Oxidative metabolism of OFH243 (13n), OFH244 (13m), OFH3911 (14n), OFH3912
14m), and imipramine after exposure to male rat liver microsomes. Results from LC−MS analysis
(
are depicted in percentage of non-metabolized 13m, 13n, 14m, 14n and imipramine.
In comparison to imipramine, which is typically classified as a moderately stable positive control,
all tested substances 13m, 13n, 14m, and 14n show substantially greater metabolic stability. After
1
20 min of incubation time, LC/MS analysis showed ca. 74% of unchanged amine 13n, 53% of
unchanged amine 13m, 63% of unchanged ammonium salt 14n, and 65% of unchanged ammonium
salt 14m, respectively, whereas imipramine was fully converted after 60 min.
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Docking and molecular electrostatic potential calculations
Docking studies49 and calculations of the molecular electrostatic potential (MEP)50-51 were
performed to provide possible explanations for the effects observed in the ligand binding studies.
In the first stage of our investigations, we evaluated whether subtype selectivity towards the M3
1
1
1
1
1
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1
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2
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3
4
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4
4
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4
5
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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
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
over the M subtype could also be achieved without the 5-fluoro substitution on the A ring of the
2
biphenyl. Taking into account the results summarized in Table 1, it became obvious that small
structural changes in the basic aliphatic side chain can lead to a remarkable selectivity as observed
for ligand 13a (K = 0.47 nM, M /M = 36). Since the underlying effect on subtype selectivity is
i
2
3
probably the chain length between the hydrogen-bond accepting carbamate, which undergoes
hydrogen bonding with Asn507 (M ) and the positively charged, protonated amine, which forms
3
an ionic interaction with Asp147 (M ), three representative ligands 4a, 8a, 13a were docked into
3
5
2
the M receptor (PDB entry 4U15) (see Figure 6, A-C). Indeed, changing the chain from
3
piperidin-4-yl to 3-quinuclidinyl and piperidin-4-ylmethyl for 4a, 8a and 13a, respectively, leads
to slight changes in relative orientation and distance of the biphenyl unit with respect to the critical
side chains of Leu225 (M ) or Phe181 (M ) in ECL2, which is likely to be the reason for the
3
2
experimentally observed differentiation between the M and M subtypes. Comparing the small
2
3
effects for ligands 4a, 8a, 13a (Figure 6, A-C), the 5-fluorine substitution in ligand 13d (Figure 6,
D) undoubtedly is an optimal structural requirement to achieve subtype selectivity. Regarding the
recently published crystal structure of BS46 (3) in the M receptor subtype, the docking poses
3
shown in Figure 6 are in good agreement.22
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A
B
D
0
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1
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9
0
1
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3
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9
0
C
Figure 6. Docking results of 4a (light brown, A), 8a (orange, B), 13a (brown, C) and 13d (blue,
52
D) at the M receptor crystal structure (PDB entry 4U15, white) superimposed with the M
3
2
5
3
receptor crystal structure (PDB entry 3UON, black) . Shortest distance of the 5-substituent as well
as the relative orientation angle of the biphenyl towards Phe181 (M ) are shown.
2
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Further docking experiments showed a possible interaction between a chloro substituent in
3
'-position of ring B and the backbone carbonyl unit of Tyr148 from TM3 in the M subtype (Figure
3
7). This so called halogen bonding is an important interaction seen in several receptor-ligand
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
4
4
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4
4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
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8
9
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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0
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2
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8
9
0
54
complexes. Basically, halogens bond may be formed between the positively polarized  hole of
a halogen atom (F << Cl < Br < I) and a proximate hydrogen-bond acceptor. The halogen bond
interaction typically follows strict geometric rules, whereas the halogen atom should ideally point
at the acceptor at an angle of 180°. Deviations of 25-30° already lead to an approximately 50%
decrease of bond strength. The distance should be smaller than the sum of the individual van der
Waals radii of the respective atoms (< 3.27 Å for a chlorine-oxygen interaction), and in case that a
carbonyl unit is involved, the C-O-X angle should be close to 120° so that the halogen atom points
directly towards one of the lone pairs of the carbonyl oxygen.55
The favorable effect of a 3'-chloro substitution on ring B, first observed for ligand 13k in
comparison with 13g (Table 2), provided the rationale of synthesizing further 3'-chloro substituted
derivatives (Table 3) to test the hypothesis. In this context, it should be noted that the used docking
49
software (AutoDock Vina) does not include halogen bonding in its scoring function. Docked poses
that would allow halogen bond interactions could only be observed for 13k and OFH243 (13n).
The high binding affinity for OFH244 (13m) suggests that similar contacts are very likely to be
present, even so the  hole is less pronounced than for 13k and 13n. Interestingly, performing the
5
6
docking with AutoDock VinaXB , a newer version including halogen interactions in its scoring
function, did not show any relevant differences for the binding poses or their scoring.
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0
1
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Docking poses of 13k (left) and OFH243 (13n, right) in the M receptor subtype (PDB
3
52
entry 4U15, white) suggesting halogen bonding between 3'-chloro substituent and the carbonyl
unit of Tyr148.
Additional support for the halogen bond hypothesis was obtained by molecular electrostatic
potential (MEP) calculations50-51 of the 3'-chlorinated ligands 13g,k,m,n, and 13o (Figure 8). These
show a comparatively large  hole for all 3'-chloro substituted ligands due to the electronic
properties of the aromatic biphenyl moiety. As predicted, this  hole can be increased by the
introduction of further inductively electron-withdrawing substituents on the B ring such as 4'-fluoro
(as in ligand OFH243 (13n)), 4'-chloro (as in ligand 13k) or 5'-chloro (as in ligand 13o).
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R
R
R
5
'
5'
4'
5'
4'
4'
Cl 3'
Cl
Cl 3'
3'
Cl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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4
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5
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5
5
5
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5
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0
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9
0
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9
0
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0
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8
9
0
1
3k
13m
13g
R
R
5
4
F
'
'
5'
4
for R is:
Cl
'
Cl 3'
3'
Cl
O
NH
O
F
NH
-10
0
+
10 kcal
1
3n
13o
Figure 8. The molecular electrostatic potential (MEP) of ligands 13k, 13m, 13g, 13n and 13o
bearing at least one chloro substituent on ring B is mapped onto isodensity surface (0.001 au)
calculated at the HF/6-31G* level.
Discussion and Conclusions
In summary, it has been shown that biphenyl carbamates can be developed towards highly potent
and subtype selective ligands for the muscarinic M receptor. Besides 5-fluoro substitution, which
3
enables subtype selectivity for the M over the M receptor, the introduction of a chlorine atom in
3
2
3
’ position leads to an increased binding affinity to all three subtypes. This effect, which can be
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2
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5
5
5
6
rationalized by a halogen bond interaction between the  hole on the chlorine atom and the carbonyl
of Tyr148, may however only be exploited if the amine or ammonium moiety of the ligand is
correctly chosen. Whereas 3',4'-dichloro substitution on the lead compound ABH423 (5) led to a
decrease of both M receptor affinity and M /M subtype selectivity in ligand 8k, the identical
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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3
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9
0
1
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9
0
1
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3
4
5
6
7
8
9
0
3
2
3
modification of 13d, now bearing a basic 4-piperidinylmethyl instead of a quinuclidinyl moiety,
results in an increased M receptor affinity and a more or less unchanged M /M selectivity
3
2
3
observed for ligand 13k. Further optimization of 13k revealed that a  hole on the 3'-chlorine atom,
as it is also present in OFH243 (13n), OFH244 (13m), OFH3911 (14n) and OFH3912 (14m) can
translate into even stronger binding to the M subtype, with at the same time good selectivities
3
towards the M over the M subtype ranging from 46 to 68. The comparably high affinities of 14n
3
2
and 14m to the M subtype can be considered as less problematic due to the inability of permanently
1
charged ammonium compounds to pass the blood-brain barrier. For the secondary amines 13n and
1
3m, which also display high affinities to the M receptor, it has been shown that these compounds
1
are substrates of P-gp, so that they are likely to be actively transported away from the brain as it
has been reported for darifenacin (2). Since the efflux ratios calculated from monolayer
experiments with P-gp expressing cell lines are higher than 2 for the reference compound
darifenacin (2) as well as for OFH243 (13n) and OFH244 (13m) at low micromolar concentrations,
we expect a qualitatively similar effect of P-gp expressed in the blood-brain barrier on CNS
accumulation of these compounds.57
Related to the  hole effect, which can explain the high affinities of 3'-chloro substituted biphenyl
carbamates to the M subtype, the size of the  hole was estimated by molecular electrostatic
3
potential (MEP) calculations. Docking studies using recently published crystal structures further
showed that the subtype selectivity for the M over the M receptor can to a certain extent be
3
2
achieved without 5-fluoro substitution, but that 5-fluorination remains the optimal structural
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requirement. All in all, OFH243 (13n), OFH244 (13m), OFH 3911 (14n) and OFH 3912 (14m)
range among the top ligands developed in this field so far, which are moreover accessible via a
straightforward and scalable synthetic route.
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Given the favorable properties of 13m,n and 14m,n, whereat the amines 13m,n were found to be
weak inverse agonists and the ammonium salts 14m,n to be neutral antagonists in the G pathway,
q
these compounds represent valuable starting points for future developments towards highly potent
and selective blockers of M receptor signaling with potential applications in the fields of COPD
3
and overactive bladder treatment. Studies on the incorporation of 13m,n and 14m,n into bivalent
ligands, such as the clinical candidate batefenterol (4c), are currently underway in our laboratory.
For research groups interested in a deeper investigation of the pharmacology of OFH243 (13n),
OFH244 (13m), OFH3911 (14n) and OFH3912 (14m), we would be happy to provide samples –
also on a larger scale – within a future collaboration.
Experimental Section
Synthesis.
1
Solvents and reagents are obtained from commercial sources and used as received. H-NMR and
1
3
1
13
C-NMR spectra are recorded on Bruker Avance 600 ( H: 600 MHz; C: 151 MHz), Bruker
1
13
1
13
Avance 360 ( H: 360 MHz; C: 91 MHz), and Bruker Avance 400 ( H: 400 MHz; C: 101 MHz)
1
spectrometers. For H-NMR spectra CDCl is used as solvent referenced to TMS (0 ppm): CHCl
3
3
(
7.26 ppm), (CD ) SO (2.50 ppm), CD OD (3.31 ppm). Chemical shifts are reported in parts per
3 2 3
million (ppm). Coupling constants are in Hertz (Hz). The following abbreviations are used for the
description of signals: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), bs (broad
13
singlet). C-NMR (DEPTQ) spectra are recorded in CDCl using CDCl (77.16 ppm) and in
3
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(
CD ) SO using (CD ) SO (39.52 ppm) as standard, respectively. Chemical shifts are given in parts
3
2
3 2
per million (ppm). Mass spectra are recorded using electron spray ionization (ESI) and
Atomospheric Pressure Photoionization (APPI) and a sector field mass analyzer for HRMS
measurements. Analytical TLC is carried out on Merck silica gel plates using short wave (254 nm)
UV light to visualize components. Silica gel (Kieselgel 60, 40-63mm, Merck) is used for flash
column chromatography. Solvent ratios of the liquid phase are given in v/v. The purity of the
substances was determined using an analytical HPLC: System A: Phenomenex Gemini NX-C18
analytical column, 4.6 mm x 250 mm, 5 μm, flow rate: 0.75 mL/min, Eluent: CH CN in H O +
0
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.1% HCO H (0-15 min 5%-90%, 15-20 min 90%). System B: Waters XBridge C18 analytical
2
column, 4.6 mm x 50 mm, 3.5 µm, flow rate: 0.5 mL/min, Eluent: CH CN in H O + 0.1% HCO H
3
2
2
(0-15 min 10%-90%, 15-20 min 90%). The purity of all target compounds 8a-n, 11, 13a-o, 14a-n,
1
5, 16m-n, 17, and 18 was determined as >95%.
General Procedure A: Synthesis of aminobiphenyls from arylhydrazine derivatives with
MnO2
To a stirred suspension of the aniline derivative (20.0 mmol, 20 eq.) and MnO (435 mg, 5 mmol,
2
5
eq.) in acetonitrile (5.0 mL), a solution of the arylhydrazine derivative (1.00 mmol, 1.0 eq.) in
acetonitrile (2.0 mL) was added over a course of 1 h. After termination of the reaction, the reaction
®
mixture was filtered over Celite and washed with ethyl acetate. Afterwards, the solvent was
removed under reduced pressure and further purification of the biphenyl-2-amines was achieved
using Kugelrohr distillation and subsequent flash column chromatography.
General Procedure B: Synthesis of aminobiphenyls from arylhydrazine hydrochlorides with
NaOH
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To the neat aniline derivate (20.0 mmol) an aqueous solution of 1 M sodium hydroxide (1.0 ml)
was added. After heating to 60 °C - 90 °C the arylhydrazine hydrochloride was added portionwise
over a period of 9 hours. The reaction is completed after 24 hours at the given temperature,
monitored by TLC. The remaining aniline was recovered by Kugelrohr distillation, and the
products were purified by column chromatography on silica gel as described in the respective
reaction procedure.
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Biphenyl-2-amine (6a) was prepared from phenyl hydrazine (0.098 mL, 1.00 mmol), MnO (435
2
mg, 5.00 mmol) and aniline (20.0 mmol, 1.83 mL) according to General Procedure A. The excess
of aniline was removed by Kugelrohr distillation in vacuo at 85 °C. The desired product 6a was
obtained by flash column chromatography (hexane / EtOAc = 8 : 1) as a brown oil (105 mg, 0.62
1
mmol, 62% yield). H-NMR (600 MHz, CDCl )  6.70 (d, J = 8.0 Hz, 1H), 6.82 (dt, J = 1.2,
3
7
.5 Hz, 1H), 7.08 - 7.15 (m, 2H), 7.28 - 7.32 (m, 1H), 7.37 - 7.43 (m, 4H). Nitrogen-bound proton
signals missing.
4’-Chlorobiphenyl-2-amine (6b) was prepared from aniline (20.0 mmol, 1.82 mL) and 4’-
chlorophenylhydrazine hydrochloride (1.00 mmol, 179 mg) according to General Procedure B at
6
0 °C. The excess of aniline was removed by Kugelrohr distillation in vacuo at 75 °C. The crude
products were purified by column chromatography (hexane / EtOAc = 8:1) to give 6b as a brown
1
oil (0.48 mmol, 99.0 mg, 48% yield). H-NMR (600 MHz, CDCl ) δ 6.76 (dd, J = 1.2, J = 8.6 Hz,
3
1
1
H), 6.86 (dt, J = 1.2, 7.5 Hz, 1H), 7.08 (dd, J = 1.6, 7.6 Hz, 1H), 7.16 (ddd, J = 1.6, 7.4, 8.0 Hz,
H), 7.38 - 7.42 (m, 4H). Nitrogen-bound proton signals missing.
3
’,4’,5’-Trifluorobiphenyl-2-amine (6c) was prepared from 3,4,5-trifluorophenyl hydrazine
(0.90 mmol, 146 mg), MnO (392 mg, 4.50 mmol) and aniline (18.0 mmol, 1.64 mL) according to
2
General Procedure A. The excess of aniline was removed by Kugelrohr distillation in vacuo at
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5 °C. The desired product 6c was obtained by flash column chromatography (hexane / EtOAc =
1
1
0:1  4:1) as a brown oil (0.53 mmol, 118 mg, 59% yield). H-NMR (360 MHz, CDCl )  3.79
3
(bs, 2H), 6.77 (dd, J = 0.8, 8.0 Hz, 1H), 6.83 (dt, J = 1.0, 7.5 Hz, 1H), 7.06 (dd, J = 1.6, 7.6 Hz,
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H), 7.07-7.13 (m, 2H), 7.19 (ddd, J = 1.6, 7.4, 8.0 Hz, 1H).
5
-Fluorobiphenyl-2-amine (6d) was prepared from phenyl hydrazine (0.10 mL, 1.00 mmol),
MnO (435 mg, 5.00 mmol) and 4-fluoroaniline (20.0 mmol, 1.92 mL) according to General
2
Procedure A. The excess of 4-fluoroaniline was removed by Kugelrohr distillation in vacuo at 85
°
:
C. The desired product 6d was obtained by flash column chromatography (hexane / EtOAc = 10
1
1) as a brown oil (559 µmol, 138 mg, 56% yield). H-NMR (600 MHz, CDCl )  3.54 (bs, 2H),
3
6.73 - 6.75 (m, 1H), 6.86 - 6.90 (m, 1H), 7.34 - 7.47 (m, 1H), 7.42 - 7.47 (m, 5H).
2’-Chloro-5-fluorobiphenyl-2-amine (6e) was prepared from 2-chlorophenyl hydrazine
(1.00 mmol, 143 mg), MnO (435 mg, 5.00 mmol) and 4-fluoroaniline (20.0 mmol, 1.92 mL)
2
according to General Procedure A. The excess of 4-fluoroaniline was removed by Kugelrohr
distillation in vacuo at 75 °C. The desired product 6e was obtained by flash column
1
chromatography (hexane/EtOAc = 10:1) as a brown oil (0.56 mmol, 124 mg, 56% yield). H-NMR
(600 MHz, CDCl )  3.47 (bs, 2H), 6.73 (dd, J = 4.8, 8.8 Hz, 1H), 6.81 (dd, J = 3.0, 8.9 Hz, 1H),
3
6
.93 (dt, J = 3.0, 8.4, 8.5 Hz, 1H), 7.30-7.36 (m, 3H), 7.48-7.52 (m, 1H).
4
’,5-Difluorobiphenyl-2-amine (6f) prepared from 4-fluoroaniline (20.0 mmol, 1.92 mL) and 4-
fluorophenylhydrazine hydrochloride (1.00 mmol, 163 mg) according to General Procedure B at
0 °C. The excess of 4-fluoroaniline was removed by Kugelrohr distillation in vacuo at 80 °C. The
6
crude product was purified by column chromatography (hexane / EtOAc = 8:1) to give 6f as black
1
crystals (0.53 mmol, 108 mg, 53% yield). H-NMR (600 MHz, CDCl ) δ 6.69 (dd, J = 4.8, 8.7 Hz,
3
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H), 6.83 (dd, J = 3.0, 9.2 Hz, 1H), 6.84 - 6.89 (m, 1H), 7.13 (t, J = 8.7 Hz, 2H), 7.40 (dd, J = 5.4,
.8 Hz, 2H). Nitrogen-bound proton signals missing.
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’-Chloro-5-fluorobiphenyl-2-amine (6g) was prepared from 4-fluoroaniline (20.0 mmol,
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.92 mL) and 4-chlorophenylhydrazine hydrochloride (1.00 mmol, 179 mg) according to General
Procedure B at 60 °C. The excess of 4-fluoroaniline was removed by Kugelrohr distillation in vacuo
at 80 °C. The crude product was purified by column chromatography (hexane / EtOAc = 8:1) to
1
give 6g as a dark brown oil (0.55 mmol, 121 mg, 55% yield). H-NMR (360 MHz, CDCl ) δ 6.70
3
(dd, J = 4.8, 8.7 Hz, 1H), 6.83 (dd, J = 3.0, 9.2 Hz, 1H), 6.85 (ddd, J = 3.0, 8.2, 8.3 Hz, 1H), 7.38
(
d, J = 8.8 Hz, 2H), 7.43 (d, J = 8.8 Hz, 2H). Nitrogen-bound proton signals missing.
’-Bromo-5-fluorobiphenyl-2-amine (6h) was prepared from 4-Bromophenyl hydrazine
2.00 mmol, 374 mg), MnO (870 mg, 10.00 mmol) and 4-fluoroaniline (40.0 mmol, 3.84 mL)
4
(
2
according to General Procedure A. The excess of aniline was removed by Kugelrohr distillation in
vacuo at 90 °C. The desired product 6h was obtained by flash column chromatography
1
(hexane/EtOAc = 20:1) as a brown oil (0.50 mmol, 134 mg, 25% yield). H NMR (400 MHz,
CDCl ) δ 3.8 (s, 2H), 6.7 – 6.8 (m, 1H), 6.8 – 6.9 (m, 1H), 6.9 (ddd, J = 3.0, 8.1, 8.7 Hz, 1H), 7.3
3
– 7.4 (m, 2H), 7.6 – 7.7 (m, 2H).
4’-Cyano-5-fluorobiphenyl-2-amine (6i) was prepared from 4-cyanophenyl hydrazine
(1.00 mmol, 133 mg), MnO (435 mg, 5.00 mmol) and 4-fluoroaniline (20.0 mmol, 1.92 mL)
2
according to General Procedure A. The excess of aniline was removed by Kugelrohr distillation in
vacuo at 75 °C. The desired product 6i was obtained by flash column chromatography
1
(hexane/EtOAc = 10:1) as a brown oil (0.58 mmol, 123 mg, 58% yield). H-NMR (600 MHz,
CDCl )  3.68 (bs, 2H), 6.73 (dd, J = 4.7, 8.7 Hz, 1H), 6.84 (dd, J = 2.9, 9.0 Hz, 1H), 6.93 (dt,
3
J = 2.9, 8.4, 8.4 Hz, 1H), 7.59 (d, J = 8.2 Hz, 2H), 7.75 (d, J = 8.3 Hz, 2H).
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