Route to prolonged residence time at the histamine H1 receptor: Growing from desloratadine to rupatadine

Article abstract of DOI:10.1021/acs.jmedchem.9b00447

Drug-target binding kinetics are an important predictor of in vivo drug efficacy, yet the relationship between ligand structures and their binding kinetics is often poorly understood. We show that both rupatadine (1) and desloratadine (2) have a long residence time at the histamine H₁ receptor (H₁R). Through development of a [³H]levocetirizine radiolabel, we find that the residence time of 1 exceeds that of 2 more than 10-fold. This was further explored with 22 synthesized rupatadine and desloratadine analogues. Methylene-linked cycloaliphatic or β-branched substitutions of desloratadine increase the residence time at the H₁R, conveying a longer duration of receptor antagonism. However, cycloaliphatic substituents directly attached to the piperidine amine (i.e., lacking the spacer) have decreased binding affinity and residence time compared to their methylene-linked structural analogues. Guided by docking studies, steric constraints within the binding pocket are hypothesized to explain the observed differences in affinity and binding kinetics between analogues.

Full text of DOI:10.1021/acs.jmedchem.9b00447

Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC
Article
The route to prolonged residence time at the histamine
1
H receptor: growing from desloratadine to rupatadine
Reggie Bosma, Zhiyong Wang, Albert J. Kooistra, Nick Bushby, Sebastiaan
Kuhne, Jelle van den Bor, Michael J. Waring, Chris de Graaf, Iwan J. P. De
Esch, Henry F. Vischer, Robert J Sheppard, Maikel Wijtmans, and Rob Leurs
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00447 • Publication Date (Web): 20 Jun 2019
Downloaded from http://pubs.acs.org on June 24, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The route to prolonged residence time at the histamine H receptor: growing from desloratadine to
1
rupatadine
a1
a1
a
c
a
a
Reggie Bosma , Zhiyong Wang , Albert J. Kooistra , Nick Bushby , Sebastiaan Kuhne , Jelle van den Bor ,
d
a
a
a
b
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
Michael J. Waring , Chris de Graaf , Iwan J. de Esch , Henry F. Vischer , Robert J. Sheppard , Maikel
a
a
Wijtmans and Rob Leurs *
a
Amsterdam Institute for Molecules, Medicines and Systems; Division of Medicinal Chemistry, Faculty of
Science, VU University Amsterdam, De Boelelaan 1083, 1081 HV, Amsterdam, the Netherlands.
b
Medicinal Chemistry, Research and Early Development, Cardiovascular, Renal and Metabolism,
BioPharmaceuticals R&D, AstraZeneca, Gothenburg, 431 50, Sweden.
c
Operations, BioPharmaceuticals R&D, AstraZeneca, Alderley Park, Macclesfield, SK10 4TG, United
Kingdom.
d
Medicinal Chemistry, Research and Early Development, Oncology R&D, AstraZeneca, Alderley Park,
Macclesfield, SK10 4TG, United Kingdom.
1Authors contributed equally to this manuscript.
*Corresponding author: r.leurs@vu.nl
Abstract
Drug-target binding kinetics are an important predictor of in vivo drug efficacy, yet the relationship
between ligand structures and their binding kinetics is often poorly understood. We show that both
rupatadine (1) and desloratadine (2) have a long residence time at the histamine H receptor (H R).
1
1
3
Through development of a [ H]levocetirizine radiolabel, we find that the residence time of 1 exceeds
that of 2 more than 10-fold. This was further explored with 22 synthesized rupatadine and desloratadine
analogs. Methylene-linked cycloaliphatic or beta-branched substitutions of desloratadine increase the
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 59
1
2
3
4
5
6
7
8
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
residence time at the H R, conveying a longer duration of receptor antagonism. However, cycloaliphatic
1
substituents directly attached to the piperidine amine (i.e. lacking the spacer) have decreased binding
affinity and residence time compared to their methylene-linked structural analogs. Guided by docking
studies, steric constraints within the binding pocket are hypothesized to explain the observed
differences in affinity and binding kinetics between analogs.
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
ACS Paragon Plus Environment

Page 3 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Introduction
Drugs have to bind a therapeutically relevant target to exhibit a biological effect and, as such, target
binding is well characterized during the development process of many drugs. The binding affinity is an
often-used parameter to measure drug binding to a target (quantified as K or K values), implicitly
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
D
i
assuming ligand binding occurs under equilibrium conditions. However, drug pharmacodynamics can
also be characterized by the drug-target binding kinetics, which provide important details about the
mechanism of target binding, unexplained by solely the binding affinity.1–3 The drug-target residence
time, which is a measure for the lifetime of a drug-target complex, is currently discussed as one of the
important contributors to the biological efficacy of drugs in vivo.3–10 It has been postulated that a
suitably long drug-target residence time might increase the therapeutic window in vivo when clearance
of the drug is faster than the dissociation of the drug from the receptor.11,12 In such cases, drug action
would last longer than the presence of free drug plasma concentrations (i.e. hysteresis). Thus, duration
of therapeutic action may not only depend on drug absorption, distribution, metabolism, and excretion
(
and the nature of its metabolites), but can also be a direct effect of prolonged target binding.13–15
As the target of 33% of all small molecule drugs, the G protein-coupled receptors (GPCRs) are an
1
6
important class of proteins in drug discovery. The histamine H receptor (H R) is an archetypical GPCR
1
1
1
7
and is successfully targeted by antagonists for the treatment of, for example, allergic disorders. A long
duration of action has been observed in vivo for second generation H R antagonists, like levocetirizine
1
1
8,19
and fexofenadine, which have a long residence time at the H R.
Hysteresis was indeed observed for
1
levocetirizine and fexofenadine.18–20 A strong hysteresis of H R antagonism has also been shown for
1
rupatadine (1), which antagonizes the histamine-induced flare response up to 72 hours after oral
21
administration, whereas plasma levels could only be detected up to 12 hours after administration. This
might be explained by metabolism of rupatadine to metabolites such as desloratadine (2), which is a
known antihistamine itself with a long H R residence time (>1 h) and a long plasma half-life in vivo
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 59
1
2
3
4
5
6
7
8
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
(
human). 19,21–27 Yet, a potentially long drug-target residence time of rupatadine may also be a crucial
contributing factor to its observed long duration of action.
Here, we report the measurement of the residence times of rupatadine and desloratadine at the H R. It
1
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
was shown that rupatadine has a ≥ 10 fold longer residence time at the H R, relative to desloratadine. As
1
a consequence, rupatadine completely antagonized the histamine-induced calcium mobilization in HeLa
cells for > 2 hrs after removal of unbound antagonist, whereas inspected under the same conditions,
desloratadine allowed a time-dependent gradual recovery of the histamine-induced response. To
understand the structure-kinetics relationship (SKR) for rupatadine and desloratadine in more detail, the
binding kinetics at the H R were characterized for newly synthesized analogs (3-24) that retain the core
1
scaffold of 1 and 2 but contain a diverse set of aromatic and aliphatic N-substituents on the piperidine
ring. It was shown that relatively small aliphatic N-substitutions were sufficient for a prolonged H1R
residence time compared to desloratadine, unless this was negated by steric interference in the binding
pocket.
1
Figure 1-Structures of the investigated H R antagonists and synthesized structural analogs.
Results
Binding properties of rupatadine and desloratadine at the H R.
1
2
1
Based on the long duration of action of rupatadine in vivo, we hypothesized that it would exhibit a long
residence time at the H R. Therefore, binding of rupatadine and its structural analog desloratadine to
1
4
ACS Paragon Plus Environment

Page 5 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3
the human H R was investigated, initially using [ H]mepyramine and standardized competition binding
1
2
6
experiments. The H R binding affinity of desloratadine (pK 9.1 ± 0.1) determined in these experiments
1
i
2
4–26
was consistent with previously reported affinity values (pK = 8.8 – 10
). Rupatadine (pK 8.4 ± 0.1)
i
i
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
was shown to have a five-fold lower binding affinity for the H R than desloratadine. To the best of our
1
knowledge, the binding affinity of rupatadine on the human H R has not been reported in the literature.
1
2
8
Its H R activity on guinea pig ileum is known, as well as that for a series of derivatives.
1
Competitive association experiments were subsequently performed to examine the binding kinetics of
3
rupatadine and desloratadine at the H R. Initially, [ H]mepyramine was selected as radioligand and
1
experiments were performed at 25°C with an 80 min incubation time, in the manner described
2
6
3
previously. A clear initial overshoot in [ H]mepyramine binding was observed for both unlabeled
ligands (Figure 2A), which is indicative of the long residence times of the unlabeled ligands relative to
[3H]mepyramine.29,30 However, since the binding curves of rupatadine and desloratadine showed similar
overshoot patterns, it was difficult to discern differences in their binding kinetics using the Motulsky-
3
0
Mahan analysis. Desloratadine was found to have a residence time of 190 ± 40 min (similar to that
reported in the literature25,26), but for rupatadine the k value (and thus the residence time) could not
off
be accurately constrained by the model. To overcome this limitation, it was speculated that the
residence times of desloratadine and rupatadine at the H R might be better discriminated using a
1
radioligand with a longer residence time, and one more closely matched to desloratadine and
rupatadine than mepyramine. With this in mind, levocetirizine was considered a better alternative as it
2
5
is known to have a 100-fold longer residence time at the H R than mepyramine. Radiolabeled
1
2
5
levocetirizine has previously been disclosed but without synthetic details for its preparation. The
radiolabel was prepared by us using a six-step sequence progressing through intermediates 25-28.
Separation of the enantiomers of 28, followed by Pd-catalyzed dehalotritiation of the corresponding aryl
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 59
1
2
3
4
5
6
7
8
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
-1
iodide delivered the ligand with a specific activity of 956 GBq mmol (Scheme 1, and supplementary
information).
O
OH
Cl
BrMg
a)
b)
+
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
Cl
I
Cl
I
Cl
25
26
O
OH
O
OH
O
O
OH
OH
O
O
c)
d)
e)
f)
N
N
N
N
N
N
N
N
I
Cl
I
Cl
I
Cl
T
Cl
[3H]-levocitrizine
27
28
(S)-28
3
Scheme 1 - Synthesis of [ H]levocetirizine. Key: (a) Et
2
O, 0 °C to r.t., 16 h, 88 %; (b) SOCl
2
, DCM, r.t., 20 h, 95 %; (c) 2-(piperazin-
1
-yl)ethanol, PhMe, 80 °C, 20 h, 21 %; (d) (1) KOH, DMF, 0 °C, 90 min; (2) sodium 2-chloroacetate, DMF, 0 °C, 3 h, 57 %; (e)
Chiral separation; (f) T , Pd/C (10 %), Et N, EtOH.
2
3
Figure 2 – Radioligand association binding when co-incubated with rupatadine and desloratadine. A homogenate of HEK293T
3
cells expressing the H
1
R was incubated with: (A) [ H]mepyramine (3.8 nM) alone, or in the presence of either rupatadine (130
3
nM) or desloratadine (4 nM); or (B), - [ H]levocetirizine (6.6 nM) alone, or in the presence of either rupatadine (6 nM) or
6
ACS Paragon Plus Environment

Page 7 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
desloratadine (0.7 nM). Representative graphs of 3 experiments are shown depicting individual measurements with duplicate
values per time point.
[3H]levocetirizine was then employed in competitive association experiments to characterize the binding
kinetics of rupatadine and desloratadine using an incubation time of 6 hours (to ensure a steady state in
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
[3
H]levocetirizine binding). In the presence of desloratadine, [ H]levocetirizine binding to the H R
3
1
increased gradually over time, whereas in the presence of rupatadine a clear initial overshoot in
[3H]levocetirizine binding was observed (Figure 2). Based on these curve shapes, it is clear that
rupatadine has a longer residence time on the H R than desloratadine. Fitting the data to the Motulsky-
1
30
Mahan model did not result in a precise fit of the k values, but indicated the k of desloratadine at
off
off
-1
the H R to be > 0.03 min (P = 95% in all 3 experiments) corresponding to a residence time of < 33 min.
1
-1
In the case of rupatadine, the k value at the H R is < 0.0033 min (P = 95% in all 3 experiments), which
off
1
corresponds to a residence time of > 300 min. Thus, rupatadine has a very long residence time at the
H R, which is at least 10-fold longer than observed for desloratadine.
1
Design and synthesis of rupatadine analogs at the H1R
To identify the structural features that drive the longer residence time of rupatadine as compared to
desloratadine at the H R, various analogs were synthesized and pharmacologically characterized.
1
Rupatadine contains a 5-methylpyridin-3-yl group connected through a methylene to the basic amine of
desloratadine (Figure 1). To study the SKR, we synthesized analogs with the methyl group on different
positions of the pyridine ring (3-5), and the pyridine analog without the methyl group (6). Two positional
isomers of 6 (7, 8), and two pyrimidines (9-10) were also prepared. Additionally, the pyridine ring of
rupatadine was replaced by a phenyl ring with (11), or without (12), a 3-methyl group. Finally, to
gradually bridge the transition to 2, a set of analogs was synthesized in which the basic amine of
desloratadine was substituted with a range of alkyl groups (13-24), varying in size, level of
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 59
1
2
3
4
5
6
7
8
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
constrainment and point of attachment (with or without the one-carbon spacer). Of these, only 3-8, 12,
2
3 and 24 have been reported before.28,31–34
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
Cl
N
N
H
2
O O
Si
O
O
Br
a)
b)
c)
d)
H CO e)
2
Ar
R
R
R
4-8, 11, 12, 16
3, 9, 10
13-15, 17-20, 22, 23
24
21
Scheme 2 – Synthesis of rupatadine analogs. Key: (a) K
c) NaHB(OAc) , DCE, rt, 14 h, 52—71%; (d) NaHB(OAc)
AcOH, DCM, rt, 48 h, 17%.
2
CO
3
, DMF, rt, 18 h, 36—86%; (b) NaHB(OAc)
3
, DCE, rt, 14 h, 64—88%;
(
3
3 3
, MeOH, DCM, AcOH, rt, 1.5 h, 60% as fumarate salt; (e) NaHB(OAc) ,
All rupatidine analogues were efficiently obtained in one step from commercially available desloratidine
2), as depicted in Scheme 2. Compounds 4-8, 11-12 and 16 were obtained via nucleophilic substitution
(
of the corresponding alkyl bromides in moderate to good yields (36-86%). Reductive alkylation of 2 with
different aromatic aldehydes afforded 3, 9 and 10 (64-88% yield). Compounds 13-15, 17-20, 22 and 23
were synthesized by reductive alkylation using aliphatic carbonyl compounds in acceptable to good
yields (52-71%). Methyl-derivative 24 was obtained as the fumarate salt from aqueous formaldehyde
and NaBH(OAc) in 60% yield. Attempted synthesis of cyclopropyl-substituted analogue 21 via alkylation
3
of 2 with cyclopropylbromide failed. However, reductive alkylation of 2 with (1-
ethoxycyclopropoxy)triethylsilane delivered the desired product, albeit in low isolated yield (17%).35
Pharmacological characterization
H R binding affinity
1
8
ACS Paragon Plus Environment

Page 9 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
All rupatadine analogs containing an aromatic group (3-12) had comparable binding affinities at the H1R
(
pK 7.9 – 8.5) as rupatadine, which were 4-16 fold lower than the binding affinity of desloratadine (pK =
i
i
9
.1). Substituting the benzene of 12 for a cyclohexane (13) did not affect the binding affinity (<2-fold).
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
However, substituting the benzene of 12 for smaller methylene-linked cycloaliphatic N-substituents (14,
6 and 17) resulted in 2-6 fold higher binding affinities at the H R, similar to the binding affinity of
1
1
desloratadine. Likewise, analogs 22-24 with small acyclic aliphatic substituents had a high binding
affinity at the H R as well (pK 9.0-9.4), again similar to that of desloratadine (pK 9.1). Interestingly, the
1
i
i
one-carbon linker between the basic amine and the cyclic aliphatic substituents of 13, 14, 16 and 17 is
important for a high affinity binding, since a 2-8 fold reduced binding affinity is observed for analogs that
lack this spacer (18 -21, respectively).
1 i
Table 1 – H R binding of rupatadine and desloratadine analogs. Binding affinity (pK ) values were determined by competition
3
binding experiments using [ H]mepyramine and KRI-values were determined by dual-point competition association
3
experiments using [ H]levocetirizine. Depicted values represent the mean ± SEM of ≥ 3 experiments.
Cl
N
N
R
Cmpd#
1
Name
R
pKi
KRI
rupatadine
8.4 ± 0.1
2.3 ± 0.2
N
2
desloratadine
9.1 ± 0.1
0.82 ± 0.04
H
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 59
1
2
3
4
5
6
7
8
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
3b
4b
5b
6b
7b
8b
9
VUF15718
7.89 ± 0.05
2.4 ± 0.4
5.4 ± 3.5
5.8 ± 1.7
4.3 ± 1.5
3.8 ± 0.5
2.4 ± 0.5
1.3 ± 0.1
1.3 ± 0.1
3.3 ± 0.9
N
N
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
VUF15769
VUF15717
VUF15713
VUF15712
VUF15714
VUF15877
VUF15886
VUF15716
8.05 ± 0.04
8.12 ± 0.04
8.3 ± 0.1
8.1 ± 0.3
8.5 ± 0.1
8.5 ± 0.1
8.3 ± 0.1
8.1 ± 0.1
N
N
N
N
N
N
N
1
1
0
1
N
10
ACS Paragon Plus Environment

Page 11 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
12b
VUF15715
8.4 ± 0.1
1.9 ± 0.5
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
1
1
1
3
4
5
VUF16138
VUF16140
VUF16141
8.6 ± 0.2
8.9 ± 0.2
1.9 ± 0.3
1.6 ± 0.2
1.4 ± 0.2
8.75 ± 0.05
1
1
6
7
VUF16137
VUF16139
9.1 ± 0.2
9.2 ± 0.1
1.2 ± 0.1
1.7 ± 0.1
1
8
VUF16136
7.7 ± 0.1
0.9 ± 0.1
1
2
9
0
VUF16135
VUF16142
8.4 ± 0.2
0.7 ± 0.1
8.67 ± 0.04
0.81 ± 0.04
21
VUF 16219
VUF16143
8.5 ± 0.1
9.0 ± 0.1
0.76 ± 0.03
1.1 ± 0.3
2
2
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 59
1
2
3
4
5
6
7
8
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
23b
VUF16144
9.38 ± 0.03
9.4 ± 0.1
1.2 ± 0.1
1.1 ± 0.2
2
4a,b
VUF15007
a
b
fumarate salt. Previously reported (see text for references).
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
Analysis of binding kinetics
To explore the relative residence time of all analogs, a dual-point competition association was
29
performed to determine the kinetic rate index (KRI). This methodology is based on the initial overshoot
in radioligand binding when co-incubated with an unlabeled ligand, which is an indicator of a relatively
long residence time of the unlabeled ligand compared to that of the radioligand (Figure 2). The
overshoot is quantified by measuring the radioligand binding at two time points. The ratio in
[3
H]levocetirizine binding at both time points (1 and 6 hours) is > 1 for unlabeled ligands that cause an
3
initial overshoot in [ H]levocetirizine binding and hence have a relatively long residence time compared
3
to [ H]levocetirizine. Using this assay setup, a KRI value of 0.9 ± 0.1 was obtained for unlabeled
levocetirizine, demonstrating that, as expected, it has a residence time essentially the same as the
3
radioligand. Desloratadine does not cause an initial overshoot in [ H]levocetirizine binding (Figure 2B)
and has a KRI value of 0.82 ± 0.04. In contrast, rupatadine binds the H R with a much longer residence
1
time (Figure 2B), which is indeed reflected by its KRI value of 2.3 ± 0.2 (Table 1). The KRI values for all
analogs are given in Table 1. All analogs with an aromatic substituent (3-12) show KRI values > 1,
indicative of a consistently long residence time at the H R (Table 1). More notably, among the analogs
1
with aliphatic substitutions on the piperidine ring (1,2,13-24), large differences in the KRI values were
observed (Figure 3). This intriguing SKR in the aliphatic series, in combination with the lack thereof in the
aromatic series, led us to focus on the former series.
12
ACS Paragon Plus Environment

Page 13 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
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
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
Figure 3– Aliphatic substituents on the basic amine of desloratadine cause differential binding kinetics at the H R. A
3
homogenate of HEK293T cells expressing the H
1
R was incubated with [ H]levocetirizine and the respective ligands. Binding of
[3
H]levocetirizine was determined after 1 h and 6 h and the KRI value was determined as the ratio in [ H]levocetirizine binding
3
at both time points (6 h/1h). The bars depict the mean and SEM of ≥ 3 experiments. The upper and lower dotted lines represent
the KRI of reference ligands 1 and 2, respectively.
Analogs with cycloaliphatic groups and a one-carbon spacer (13, 14, 16 and 17) show high KRI values,
also indicating a long residence time on the H R. However, structural analogs with the same
1
cycloaliphatic group without the one-carbon spacer (18-21) show similar KRI values to desloratadine,
indicative of a shorter residence time at the H R. Additionally, analogs with small acyclic aliphatic
1
substituents (22-24) had an average KRI value slightly larger than 1, implying an increased residence
time at the H R compared to desloratadine. Correlation between affinity and residence time parameters
1
of GPCR ligands and physicochemical properties of the ligands has been investigated, including affinities
3
6,37
by various research groups.38–45 Therefore, we investigated whether
for H R receptor antagonists,
1
correlations exist between our pK /KRI values and key physicochemical parameters (log D7.4, Polar
i
Surface Area (PSA), van der Waals volume, pK value of the conjugate acid of the piperidine nitrogen
a
atom). However, Supplementary Fig 1, Table 1 and Table 2 show that no strong correlations are evident.
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 59
1
2
3
4
5
6
7
8
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
Duration of functional H R-antagonism
1
Since large differences were observed in the KRI values of the aliphatic rupatadine analogs, these
differences were explored in more detail by measuring the kinetics of functional H R-antagonism
1
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
following a pre-incubation with the selected analogs of interest. The functional recovery time of the H1R
46
was previously shown to be correlated with the residence time of antagonists. As such, HeLa cells, with
endogenous expression of the H R, were pre-incubated with 10 times the K -concentration of the
1
i
respective compound. Unbound ligands were then depleted by washing the cells, which were
subsequently stimulated after different incubation times with 10 µM histamine. The intracellular
calcium mobilization following administration of histamine was determined with the calcium sensitive
fluorescent-dye (Fluo4 NW).
Pre-incubating HeLa cells with desloratadine, which has a low KRI value (<1), resulted in functional
recovery of the H R over time (Figure 4A; Supplementary Table 1). However, cells pre-treated with
1
rupatadine were completely unresponsive to histamine, for at least for 2 hours after removing unbound
rupatadine, suggesting very persistent target-engagement by rupatadine. In Figure 4B, the functional
recovery of the H R is compared after pre-treating the cells with analogs containing cycloaliphatic N-
1
substituents on the piperidine with or without a one-carbon spacer. Analogs with a one-carbon spacer
(14, 16 and 17) completely abolished the histamine-induced calcium response for at least 2 hours,
similarly to rupatadine. In contrast, and in line with the measured KRI values, removing the one-carbon
14
ACS Paragon Plus Environment

Page 15 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
spacer (19-21), allowed a relatively fast functional recovery of the histamine response.
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
Figure 4– Functional recovery of histamine-induced calcium mobilization after a pre-incubation with ligands that bind the
H
1 1
R. HeLa cells were pre-incubated for 18-20 h with the respective H R-ligand, reaching stable and high (±90%) occupancy of
the endogenously expressed H R. Cells were then labeled with Fluo4NW in the presence of the respective ligands for 1 h. All
1
excess Fluo4NW and unbound ligands were removed by wash steps and cells were subsequently stimulated with histamine (10-
5
M) after different incubation times. Representative graphs of ≥ 3 experiments are shown, which depict the normalized calcium
1
mobilization that was measured at each time point after washout. (A) Cells were pre-incubated with the reference H R
antagonists: rupatadine (1) and desloratadine (2). (B) Cells were pre-incubated with compounds having various cycloalkyl
substituents on the basic amine with (14, 16, 17) or without (19, 20, 21) a one-carbon spacer.
The differences in the combined kinetic/affinity binding profiles of the compounds were further
explored on a structural level with docking studies. Using the X-ray crystal structure of the H R with the
1
4
7
structurally-related ligand doxepin bound, reference compounds desloratadine, rupatadine, as well as
4
8
all analogs (3-24) were docked using PLANTS. . Figure 5 shows the postulated binding modes of
desloratadine, rupatadine, and the representative pair 14/19, in comparison to the binding mode of the
co-crystallized ligand doxepin. Desloratadine likely adopts a similar binding pose to that observed for
doxepin in the H R crystal structure (Figure 5A). Rupatadine was also found to adopt a similar binding
1
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 59
1
2
3
4
5
6
7
8
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
mode to doxepin, but its (5-methylpyridin-3-yl)methyl moiety targets an additional area of the H1R
binding pocket towards the extracellular vestibule (Figure 5B). Since the available space in the H1R
pocket next to the amine-binding region is limited by I4547.39 and Y4587.43, it is postulated that the
cyclopentyl substituent of 19 encounters greater steric hindrance than the cyclopentlymethyl
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
substituent of 14 (Figure 5C), in line with the altered H R binding characteristics of 19. (Figure 3 and
1
Figure 4B). This steric hindrance results in a tilted binding mode compared to desloratadine, which is not
observed for optimal binding of analogs with a methylene spacer between the desloratadine scaffold
and the cyclopentyl group (14, Figure 5D). The spacer allows the aliphatic group to turn towards the
extracellular vestibule (in the direction of H4507.35) where more room is available, possibly preventing a
steric clash with I4547.39 and Y4587.43 (Figure 5D).
Figure 5– Proposed binding modes of A) Desloratadine (yellow), B) Rupatadine (salmon), and C/D) compound 19 (orange) in
4
8
47
comparison to compound 14 (blue) based on docking into the crystal structure (PDB-code 3RZE ) of the H
1
R in complex
1
with Doxepin (magenta, see A). The clipped molecular surface of H R highlights the limited space for growing from the amine
16
ACS Paragon Plus Environment

Page 17 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
of desloratadine due to I4547.39 and Y4587.43. To highlight the fit of the substituents of rupatadine (2, B), 19 (C), and 14 (D)
1
compared to desloratadine in the H R binding pocket, they are shown as transparent surfaces.
Discussion and conclusions
A long drug-target residence time has been postulated to benefit the in vivo efficacy of several drugs for
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
3
,4,7,49,50
a broad number of drug targets, among which is the H R.
Affinity-based optimization of drug
1
binding does not necessarily reflect differences in target residence time,51,52 and a discrepancy between
affinity and residence time at the H R was previously described. Moreover, in the case of ligands that do
1
not reach a binding equilibrium within the time-frame of a binding experiment, i.e. ligands with a very
53
slow off rate like rupatadine, the pK will be underestimated. The drug-target residence time can
i
therefore provide additional information for the optimization of drugs that would be lost by focusing on
only the binding affinity. Since the residence time is not routinely incorporated in drug development,
design strategies for optimizing the drug-target residence time of lead compounds are not widely
available. Since rupatadine is shown here to have a much longer residence time at the H R than its close
1
structural analog desloratadine, despite a reduced binding affinity, it provides an opportunity for a
3
detailed investigation of the SKR for this GPCR. Towards this end, we synthesized [ H]levocetirizine
which proved to be a useful tool to map the differences in the KRIs between the two antihistamines. It
was therefore employed to determine the relative residence times of structural analogs 3-24. Several
analogs of rupatadine were designed to replace the (5-methylpyridin-3-yl)methyl group with other
aromatic moieties (3-12). Interestingly, the K values of 3-12 are within 4-fold of the K value of
i
i
rupatadine. Additionally, all aromatic analogs have a long apparent residence time, as is reflected by the
KRI > 1. Removing the aromatic character of the functional group of 12 by replacing it with a cyclohexyl
group (13) does not affect the observed H R binding properties either. Hence, the strong effect on the
1
residence time by the (5-methylpyridin-3-yl)methyl group of rupatadine (compared to desloratadine)
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 59
1
2
3
4
5
6
7
8
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
cannot be explained by the aromatic character, nor by the pyridine nitrogen atom and the methyl
substituent.
To further probe the SKR between rupatadine and desloratadine, a series of analogs was characterized
that had different aliphatic substituents on the piperidine group (13-24). Strikingly, most aliphatic
moieties afford an increase in the KRI compared to desloratadine, whereas the binding affinity remains
similar or even decreases. For example, 13-15 contain relatively large aliphatic substituents (≥ 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
carbons) and have a slightly reduced binding affinity (pK 8.6 – 8.9) and a high KRI (> 1.4) as compared to
i
desloratadine. Moreover, analogs with small (≤ 3-carbons) acyclic aliphatic substituents (22-24) have a
similar binding affinity but still a slightly higher KRI compared to desloratadine. This suggests that
growing an aliphatic group from the piperidine increases the residence time at the H R. This trend is
1
disrupted, however, for analogs that contain cycloaliphatic groups directly substituted on the amine (18-
21) instead of being separated from the amine by a one-carbon spacer (13, 14, 16 and 17). Analogs
without the methylene spacer (18-21) are marked by a diminished KRI and binding affinity compared to
analogs with a methylene spacer, whereas the KRI values are of the same magnitude as desloratadine.
This cliff in the SKR trend was validated for a subset of analogs by studying the kinetics of functional H1R
4
6
antagonism, which is known to reflect differential residence times at the H R. Representative analogs
1
in which the cycloaliphatic group is substituted with a one-carbon spacer (14, 16 and 17) completely
inhibit the functional response of the H R for at least 2 hours after removal of unbound ligands, as was
1
observed for rupatadine. In contrast, analogs with the same cycloaliphatic groups without a one-carbon
spacer (19-21) allowed a clear recovery of the H R functional response, as was also observed for
1
desloratadine. Hence, the relevance of the methylene-spacer for the binding kinetics of analogs with
relatively large N-substituents was confirmed by the duration of functional H R inhibition.
1
The observed residence time/affinity cliff correlated with the binding poses of the representative pair 14
and 19 in the H R binding pocket. Our docking studies suggest that the reduced flexibility of the
1
18
ACS Paragon Plus Environment

Page 19 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
cycloaliphatic group without a spacer (19) might lead to a suboptimal fit due to steric hindrance (Figure
5
C, D). The increase in ligand residence time at the H R, as was observed for most analogs with an
1
aliphatic group on the basic amine of desloratadine (vide supra), seems therefore to be mitigated when
the shape of the H R binding pocket, i.e. the steric constraints imposed by residues I454 and Y4587.43
7.39
,
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
1
is interfering with the binding position of the desloratadine scaffold.
Recently, it was shown that N-methylation of H R ligands with a primary or secondary amine increased
1
7
.39 42
the binding affinity at the H R by displacing a water molecule near I454 . However, this effect on the
1
binding affinity was not observed for analogs with a chlorine moiety on the aromatic rings. Consistent
with this finding, N-methylation of desloratadine (which contains a chlorine group), affording 24, had
only modest effects on the H R binding affinity. Interestingly, 24 did have a higher KRI compared to
1
desloratadine but not to the same extent as was observed for larger aliphatic substituents (e.g. 13-17).
Substitution with aliphatic or aromatic groups on the piperidine possibly reduces the resolvation of both
the ligand and binding site during a dissociation event. For ligand dissociation from the CRF R, for
1
example, a low degree of ligand solvation during egress from the pocket was related to a long residence
4
0
time at the receptor. Moreover, hydrophobic shielding of H-bonds can increase the lifetime of such
3
9
interactions and consequently result in an increased residence time. This is corroborated by the fact
that ligands with a relatively high KRI (>1.5) were relatively lipophilic (logD7.4, Supplementary Figure 1).
Considering that N-substitution of H R ligands was shown to interfere with the water network in the
1
5
4
3.32
binding site and that the salt bridge between the basic amine of ligands and D107 is crucial for a
4
1
high binding affinity at the receptor, shielding this interaction pair might prevent a rapid egress of the
ligands from the binding site.
In conclusion, compared to desloratadine, rupatadine has an extremely long residence time at the H1R
despite an apparent loss in binding affinity, resulting in a longer duration of functional H R antagonism.
1
3
Development of a [ H]levocetirizine radiolabel allowed a detailed SKR study, which shows that aliphatic
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 59
1
2
3
4
5
6
7
8
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
N-substitution of the piperidine ring from desloratadine is enough to obtain antagonists with a long
residence time at the H R without increasing the observed binding affinity. Analogs with large flexible
1
cycloaliphatic or aromatic substituents, like the (5-methylpyridin-3-yl)methyl substituent of rupatadine,
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
have a long residence time at the H R. Notably, analogs with cycloaliphatic substituents required an
1
additional methylene spacer on the amine for an optimal binding of the H R. Modeling studies suggest
1
that the combined affinity/kinetics profiles of analogs without a methylene spacer are possibly linked to
the steric complementarity in the ligand-H R complex. Aliphatic N-substitution of H R antagonists is a
1
1
new potential strategy to optimize the residence time at the receptor. The presented SKR highlights that
subtle structural changes of small-molecule ligands can have a profound effect on the binding kinetics at
GPCRs.
Experimental section
Pharmacological assays
All compounds that were tested in pharmacological assays (1-24, 28) are confirmed to pass a publicly
available PAINS filter.55,56
Radioligand binding experiments
2
6
Radioligand binding experiments were performed as described before, with minor alterations. Cell
pellets were produced from HEK293T cells expressing the N-terminally HA-tagged H R, and pellets were
1
stored at -20 °C. Upon experimentation, cells were thawed, resuspended in radioligand binding buffer
[
Na HPO (50 mM) and KH PO (50 mM), pH 7.4] and homogenized with a Branson sonifier 250 (Branson
2
4
2
4
Ultrasonics, Danbury, CT, USA). Cell homogenates (0.5 – 3 µg/well) were then incubated with the
respective ligands under gentle agitation, as specified for the various assay formats below. After the
incubation time, binding reactions were terminated with the cell harvester (Perkin Elmer) using rapid
filtration and wash steps over PEI-coated GF/C filter plates. Filter bound radioligand was then quantified
by scintillation counting using Microscint-O and the Wallac Microbeta counter (Perkin Elmer).
20
ACS Paragon Plus Environment

Page 21 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
In competition binding experiments, cell homogenates were incubated for 4 h at 25 °C with a single
3
-5
concentration of [ H]mepyramine (1.5 – 4nM) and increasing concentrations unlabeled ligands (10 M –
1
0-13 M). IC values were obtained by analyzed the displacement curves with GraphPad Prism 7.03
50
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
(GraphPad Software, San Diego, USA) and were converted to K values using the Cheng-Prusoff
i
equation.57
3
The binding rate constants of [ H]levocetirizine were determined using the previously described
3
methodology, by using four different concentrations of [ H]levocetirizine (1 – 35 nM) for a total
2
6
incubation time of 360 min, with an incubation temperature of 37 °C (data not shown). This resulted in
6
-1
-1
-1
a k of 3.7 ± 0.4 10 ·min ·M and a k of 0.022 ± 0.003 min . In competitive association experiments
on
off
3
with [ H]levocetirizine as radioligand, cell homogenates were incubated at 37°C for various incubation
3
times with a single concentration of [ H]levocetirizine (5 – 8 nM) in the absence of unlabeled ligand as
well as with three different concentrations of either desloratadine (2 – 60 nM) or rupatadine (0.1 – 7
3
nM). The k and k values for the binding of [ H]levocetirizine are constrained during the analysis of
on
off
the H R binding kinetics of desloratadine and rupatadine. Kinetic binding rate constants as well as their
1
asymmetrical 95% confidence intervals (95% CI) were determined using GraphPad Prism 7.03. Since the
95% CI values were very broad, values are depicted to be higher or lower than the 95% CI boundary
value observed over all individually performed experiments. Graphs depict a representative graph with
mean and SD of duplicate values showing, for clarity, only a single concentration unlabeled ligand.
3
Competitive association experiments with [ H]mepyramine as radioligand were performed as described
2
6
before, with minor alterations. Briefly, cell homogenates were incubated at 25°C for various incubation
3
times with a single concentration of [ H]mepyramine (2.5 nM – 5.5 nM) in the absence or presence of a
single concentration unlabeled ligand (desloratadine [4 – 8 nM] or rupatadine [80 – 250 nM]).
In dual-point competition association experiments the kinetic rate index (KRI) value at the H R is
1
determined. Cell homogenates were incubated on a 96-well plate for 1 h and 6 h at 37°C, with a single
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 59
1
2
3
4
5
6
7
8
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
3
concentration of [ H]levocetirizine (4 – 11 nM) together with a single concentration of unlabeled ligand
that equals the respective K -value of that ligand at the H R. All conditions were measured in triplicate
i
1
3
per experiment (n=3). Additionally, for each 96-well plate, [ H]levocetirizine was incubated with a large
-5
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
excess of mianserin (10 M) to determine non-specific binding levels of the radioligand (n=6) and, as a
3
positive control, [ H]levocetirizine binding was determined in the absence of competitor (maximal
3
binding, n=6). [ H]levocetirizine binding levels were baseline corrected by subtracting non-specific
3
binding levels and KRI values were then calculated by the ratio of [ H]levocetirizine binding after a 1 h
3
incubation time over the [ H]levocetirizine binding after a 6 h incubation time. KRI-values are a
quantitative measure for the overshoot in radioligand binding, which results from incubating the
radioligand with an unlabeled ligand that has a relatively low koff.29,30 It is therefore crucial that the
concentrations of unlabeled ligands are comparable and lead to a sub-maximal inhibition of the
3
radioligand binding. Therefore KRI-values were only accepted when the %-inhibition of [ H]levocetirizine
3
binding (compared to the maximal [ H]levocetirizine binding) was (1) less than 80% after either 1 or 6 h
and (2) more than 20% inhibition after 6 h. In the case that data points had to be excluded, the
concentration unlabeled ligands were attenuated (ranging from 1 x K to 3 x K concentrations). All
i
i
experiments were performed in triplicate or more.
Intracellular calcium mobilization assay
46
The functional recovery of the H R following antagonism was measured as described before. In short,
1
4
HeLa cells, endogenously expressing the H R, were seeded 2·10 cells/well in a clear bottom 96-well
1
plate which were pre-incubated overnight with a concentration antagonist corresponding to 10 times
the respective K at the H R (24 wells per antagonist). After 18-20 h, cells were labeled with the Fluo-
i
1
4
NW dye in the presence of the respective concentration antagonist for an hour. Both the excess dye-
solution as well as the unbound antagonists were removed by washing the cells two times and cells
were then reconstituted in HBSS buffer supplemented with probenecid (2.5 mM) (t ). Following the
0
22
ACS Paragon Plus Environment

Page 23 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
wash step, cells were stimulated every 5 min by histamine injection, into a single well, using the
NOVOstar plate reader (BMG Labtech, Ortenberg, Germany), while simultaneously detecting the calcium
mediated Fluo4NW fluorescence (λexcitation 494 nm and λemission 516 nm). For each well stimulated with
histamine, a consecutive triton-x100 injection after 65 sec was used to lyse the cells leading to
saturation of the Fluo4 NW with calcium. The histamine-induced peak-response was then normalized to
basal levels of fluorescence (prior to histamine injection; 0) and saturated Fluo4 NW fluorescence
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
(following Triton X-100 injection; 1). This led to a reproducible histamine induced response over time for
HeLa cells pretreated with vehicle condition, which was set to a 100%. Histamine-induced peak-
responses were plotted against the difference in time between t and the subsequent histamine
0
injection. The recovery time (RecT) was determined for antagonists by non-linear regression using the
one-phase association model in GraphPad Prism 7.03.
Molecular modeling
SMILES for compounds 1-24 were obtained from ChemBioDraw Ultra (version 16.0.1.4), and were
subsequently used as input for ChemAxon’s calculator for protonation (pH = 7.4). A 3D conformation
was then generated using Molecular Networks’ CORINA (version 3.49) and stored in Tripos MOL2 format
(gold extension). The doxepin-bound H R structure was obtained from the protein data bank (PDB-code
1
3RZE) after which the fused T4-lysozyme was removed from the structure. The complex was further
prepared for docking using MOE (Chemical Computing Group, version 2016.0802). Using PLANTS
4
8
(
version 1.2) each compound was docked into the H R binding pocket 3-times with the following
1
settings: search speed 1, cluster rmsd 1.0, cluster structures 10, and scored using the ChemPLP scoring
function. The binding site was defined by the center of the co-crystallized ligand doxepin with a radius of
1
1Å. The resulting docking poses were visually inspected and the poses with the best overlap with each
other as well as the doxepin reference compound were selected, which were also the highest-ranking
5
8
poses for each compound. Moreover, using an interaction fingerprint similarity analysis all docking
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 59
1
2
3
4
5
6
7
8
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
poses were compared to the binding mode of the co-crystallized compound Doxepin. All selected
docking poses have an interaction fingerprint similarity of at least 0.72 compared to the binding mode of
Doxepin and are depicted in the supporting information (Figure S2-S3). The binding mode figures were
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
created with PyMol (version 1.8.0).
Chemistry
General procedures
Synthesis of rupatadine analogs 3-24
Anhydrous THF, DCM, DMF, and Et O were obtained by elution through an activated alumina column prior
2
to use. All other solvents and chemicals were acquired from commercial suppliers and were used as
received. ChemBioDraw Ultra 16.0.1.4 was used to generate systematic names for all molecules. All
reactions were performed under an inert atmosphere (N ). TLC analyses were carried out with alumina
2
silica plates (Merck F ) using staining and/or UV visualization. Column purifications were performed
2
54
1
manually using Silicycle Ultra Pure silica gel or automatically using Biotage equipment. NMR spectra ( H,
13C, and 2D) were recorded on a Bruker 300 (300 MHz), Bruker 500 (500 MHz) or a Bruker 600 (600 MHz)
spectrometer. Chemical shifts are reported in ppm (δ) and the residual solvent was used as internal
1
13
standard (δ H NMR: CDCl 7.26; DMSO-d6 2.50; CD OD 3.31; δ C NMR: CDCl 77.16; DMSO-d6 39.52;
3
3
3
CD OD 49.00). Data are reported as follows: chemical shift (integration, multiplicity (s = singlet, d =
3
doublet, t = triplet, q = quartet, br = broad signal, m = multiplet, app = apparent), and coupling constants
(Hz)). A Bruker microTOF mass spectrometer using ESI in positive ion mode was used to record HRMS
spectra. A Shimadzu LC-20AD liquid chromatograph pump system linked to a Shimadzu SPD-M20A diode
array detector with MS detection using a Shimadzu LC-MS-2010EV mass spectrometer was used to
perform LC-MS analyses. An Xbridge (C18) 5 µm column (50 mm, 4.6 mm) was used. The solvents that
were used were the following: solvent B (acetonitrile with 0.1% formic acid) and solvent A (water with
0
.1% formic acid), flow rate of 1.0 mL/min, start 5% B, linear gradient to 90% B in 4.5 min, then 1.5 min at
24
ACS Paragon Plus Environment

Page 25 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
9
0% B, then linear gradient to 5% B in 0.5 min, then 1.5 min at 5% B; total run time of 8 min. All compounds
have a purity of ≥95% (unless specified otherwise), calculated as the percentage peak area of the analyzed
compound by UV detection at 254 nm (values are rounded). Reverse-phase column chromatography
purifications were performed using Buchi PrepChem C-700 equipment with a discharge deuterium lamp
ranging from 200-600 nm to detect compounds using solvent B (acetonitrile with 0.1% formic acid),
solvent A (water with 0.1% formic acid), flow rate of 15.0 mL/min and a gradient (start 95% A for 3.36 min,
then linear gradient to 5% A in 30 min, then at 5% A for 3.36 min, then linear gradient to 95% A in 0.5 min,
then 1.5 min at 95% 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
1
13
The Supporting Information lists H-NMR and C-NMR spectroscopy data as well as high-resolution
mass spectroscopy and LC-MS chromatograms (Figure S4 – Figure S69).
3
Synthesis of [ H]levocetirizine (25-28)
Column chromatography was carried out using pre-packed silica gel cartridges (SiliCycle, Quebec, Canada)
1
on an Isco Companion (Teledyne Isco, NE, USA). H NMR spectra were recorded on a Bruker (600 MHz or
4
00 MHz) using the stated solvent. Chemical shifts (δ) in ppm are quoted relative to CDCl (δ 7.26 ppm)
3
and DMSO-d (δ 2.50 ppm). Liquid chromatography-mass spectrometry (LC-MS) data was collected using
6
a Waters Alliance LC (Waters Corporation, MA, USA) with Waters ZQ mass detector. Analytical HPLC data
was recorded using Agilent 1200 HPLC system with a β-Ram Flow Scintillation Analyser, using the following
conditions: Waters Sunfire C , 3.5 µm, 4.6 x 100 mm column at 40°C, eluting with 5% acetonitrile/water
1
8
+
0.1% TFA to 95% acetonitrile/water + 0.1% TFA over a 32 minute gradient. Specific activities were
determined gravimetrically with a Packard TriCarb 2100CA Liquid Scintillation Analyser (Packard
Instrument Company Inc., IL, USA) using Ultima GoldTM cocktail. Reactions with tritium gas were carried
out on a steel manifold obtained from RC Tritec AG (Teufen, Switzerland). Specific activity was calculated
by comparison of the ratio of tritium/hydrogen or carbon-14/carbon-12 for the tracer against the
3
unlabelled reference. [ H]Methyl nosylate was obtained from Quotient Bioresearch as a solution in
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 59
1
2
3
4
5
6
7
8
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
-1
toluene at 3150 GBq mmol . Tritium gas was supplied absorbed onto a depleted uranium bed by RC Tritec
AG (Teufen, Switzerland). All other reagents and solvents were obtained from Sigma-Aldrich and Fisher
and were used without further purification.
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
Detailed experimental procedures
8
-chloro-11-(1-((4-methylpyridin-3-yl)methyl)piperidin-4-ylidene)-6,11-dihydro-5H-
benzo[5,6]cyclohepta[1,2-b]pyridine (3)
A mixture of 4-methylnicotinaldehyde (93 mg, 0.77 mmol), desloratadine (200 mg, 0.643 mmol) and
NaBH(OAc) (218 mg, 1.027 mmol) in DCE (10 mL) was stirred at room temperature for 12 h. The resulting
3
solution was diluted with water and extracted with DCM. The organic phase was washed with satd. aq.
NaHCO -solution.The organic layer was dried over Na SO , filtered and concentrated under vacuum. The
3
2
4
crude mixture was purified by reverse-phase column chromatography (H O/MeCN/HCOOH). The product
2
fraction was evaporated, extracted with DCM/satd. aq. Na CO solution, dried (MgSO ) and concentrated
2
3
4
to yield the title compound as a pink foam (170 mg, 64% yield).
1
H NMR (500 MHz, CDCl ) δ 8.42 – 8.22 (m, 3H), 7.39 (dd, J = 7.6, 1.3 Hz, 1H), 7.15 – 6.97 (m, 5H), 3.48 –
3
1
3
3
.25 (m, 4H), 2.87 – 2.60 (m, 4H), 2.51 – 2.20 (m, 7H), 2.19 – 2.04 (m, 2H). C NMR (126 MHz, CDCl ) δ
3
157.46, 150.18, 148.22, 147.40, 146.49, 139.49, 138.86, 137.76, 137.35, 133.43, 132.62, 132.56, 132.41,
130.79, 128.94, 125.98, 125.43, 122.13, 57.98, 54.80, 54.71, 31.79, 31.40, 30.97, 30.76, 18.81. HRMS:
+
C H ClN (M+H) calcd: 416.1894, found: 416.1883. LC-MS: t = 3.0 min, purity>96% (254 nm), m/z: 416.2
2
6
27
3
R
(
M+H)+.
8-chloro-11-(1-((2-methylpyridin-3-yl)methyl)piperidin-4-ylidene)-6,11-dihydro-5H-
benzo[5,6]cyclohepta[1,2-b]pyridine (4)
A mixture of desloratadine (155 mg, 0.50 mmol), 3-(chloromethyl)-2-methylpyridine hydrochloride (116
mg, 0.65 mmol) and K CO (180 mg, 1.30 mmol) in DMF (10 mL) was stirred at room temperature for 18
2
3
26
ACS Paragon Plus Environment

Page 27 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
h. The mixture was diluted with EtOAc, washed with water (2x) and brine, dried over Na SO , filtered and
2
4
concentrated under vacuum. The crude mixture was purified by reverse-phase column chromatography
H O/MeCN/HCOOH) to yield the title compound as a pink foam (120 mg, 58% yield).
(
2
1
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
H NMR (500 MHz, CDCl ) δ 8.42 – 8.31 (m, 2H), 7.59 (d, J = 7.0 Hz, 1H), 7.42 (d, J = 7.6 Hz, 1H), 7.16 – 7.02
3
(m, 5H), 3.49 – 3.29 (m, 4H), 2.88 – 2.65 (m, 4H), 2.60 – 2.45 (m, 4H), 2.45 – 2.26 (m, 3H), 2.23 – 2.06 (m,
1
3
2
1
H). C NMR (126 MHz, CDCl ) δ 157.62, 157.44, 147.40, 146.61, 139.50, 138.78, 137.76, 137.32, 137.01,
3
33.37, 132.69, 132.59, 131.89, 130.78, 128.92, 125.98, 122.11, 121.00, 59.61, 54.92, 54.84, 31.77, 31.41,
+
30.95, 30.72, 22.26. HRMS: C H ClN (M+H) calcd: 416.1888, found: 416.1906. LC-MS: t = 2.9 min,
2
6
27
3
R
purity >98% (254 nm), m/z: 416.2 (M+H)+.
8
-chloro-11-(1-((6-methylpyridin-3-yl)methyl)piperidin-4-ylidene)-6,11-dihydro-5H-
benzo[5,6]cyclohepta[1,2-b]pyridine (5)
A mixture of desloratadine (200 mg, 0.643 mmol), 5-(bromomethyl)-2-methylpyridine hydrobromide (224
mg, 0.836 mmol) and K CO (231 mg, 1.67 mmol) in DMF (10 mL) was stirred at room temperature for 18
2
3
h. The mixture was diluted with EtOAc, washed with water (2x) and brine, dried over Na SO , filtered and
2
4
concentrated under vacuum. The crude mixture was purified by reverse-phase column chromatography
(H O/MeCN/HCOOH). The product fraction was evaporated, extracted with DCM/satd. aq. Na CO
2
2
3
solution, dried (MgSO ) and concentrated to yield the title compound as a pink foam (170 mg, 64% yield).
4
1
H NMR (500 MHz, CDCl ) δ 8.41 – 8.33 (m, 2H), 7.60 (d, J = 7.3 Hz, 1H), 7.41 (d, J = 7.6 Hz, 1H), 7.16 – 7.02
3
1
3
(
m, 5H), 3.50 (s, 2H), 3.42 – 3.28 (m, 2H), 2.86 – 2.68 (m, 4H), 2.57 – 2.48 (m, 4H), 2.47 – 2.12 (m, 5H). C
NMR (126 MHz, CDCl ) δ 157.41, 157.37, 149.71, 146.60, 139.51, 138.20, 137.70, 137.42, 137.32, 133.38,
3
1
3
33.00, 132.69, 130.70, 130.1, 128.95, 126.01, 123.01, 122.14, 59.62, 54.56, 54.45, 31.76, 31.42, 30.64,
+
0.39, 24.10. HRMS: C H ClN (M+H) calcd: 416.1894, found: 416.1886. LC-MS: t = 3.0 min, purity >98%
2
6
27
3
R
(
254 nm), m/z: 416.2 (M+H)+.
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 59
1
2
3
4
5
6
7
8
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
8
-chloro-11-(1-(pyridin-3-ylmethyl)piperidin-4-ylidene)-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-
b]pyridine (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 mixture of desloratadine (500mg, 1.61mmol), 3-(bromomethyl)-pyridine hydrobromide (529 mg, 2.09
mmol) and K CO (579 mg, 4.19 mmol) in DMF (10 mL) was stirred at room temperature for 18 h. The
2
3
mixture was diluted with EtOAc, washed with water (2x) and brine, dried over Na SO , filtered and
2
4
concentrated under vacuum. The crude mixture was purified by reverse-phase column chromatography
(H O/MeCN/HCOOH) to yield the title compound as a pink foam (430 mg, 66% yield).
2
1
H NMR (500 MHz, CDCl ) δ 8.54 – 8.43 (m, 2H), 8.37 (d, J = 4.6 Hz, 1H), 7.67 (d, J = 7.7 Hz, 1H), 7.41 (d, J
3
=
4
7.6 Hz, 1H), 7.25 – 7.20 (m, 1H), 7.15 – 7.02 (m, 4H), 3.49 (s, 2H), 3.43 – 3.27 (m, 2H), 2.88 – 2.64 (m,
1
3
H), 2.55 – 2.46 (m, 1H), 2.45 – 2.25 (m, 3H), 2.21 – 2.02 (m, 2H). C NMR (126 MHz, CDCl ) δ 157.44,
3
150.40, 148.60, 146.63, 139.51, 138.60, 137.73, 137.34, 136.81, 133.63, 133.40, 132.79, 132.63, 130.81,
+
1
28.96, 126.01, 123.37, 122.15, 60.04, 54.73, 54.66, 31.80, 31.42, 30.89, 30.65. HRMS: C H ClN (M+H)
2
5
25
3
+
calcd: 402.1737, found: 402.1733. LC-MS: t = 3.0 min, purity >99% (254 nm), m/z: 402.1 (M+H) .
R
8-chloro-11-(1-(pyridin-4-ylmethyl)piperidin-4-ylidene)-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-
b]pyridine (7)
A mixture of desloratadine (500 mg, 1.61 mmol), 4-(bromomethyl)-pyridine hydrobromide (529 mg, 2.09
mmol) and K CO (579 mg, 4.19 mmol) in DMF (10 mL) was stirred at room temperature for 18 h. The
2
3
mixture was diluted with EtOAc, washed with water (2x) and brine, dried over Na SO , filtered and
2
4
concentrated under vacuum. The crude mixture was purified by reverse-phase column chromatography
H O/MeCN/HCOOH) to yield the title compound as a pink foam (229 mg, 36% yield).
(
2
1
H NMR (500 MHz, CDCl ) δ 8.49 (d, J = 5.6 Hz, 2H), 8.35 (d, J = 4.6 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.24 (d,
3
J = 5.4 Hz, 2H), 7.16 – 7.00 (m, 4H), 3.46 (s, 2H), 3.42 – 3.24 (m, 2H), 2.85 – 2.60 (m, 4H), 2.57 – 2.23 (m,
28
ACS Paragon Plus Environment

Page 29 of 59
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
13
4
1
3
H), 2.20 – 2.04 (m, 2H). C NMR (126 MHz, CDCl ) δ 157.38, 149.64, 147.76, 146.58, 139.50, 138.46,
3
37.69, 137.38, 133.41, 132.82, 132.63, 130.78, 128.96, 126.00, 123.89, 122.18, 61.56, 54.85, 54.82,
+
1.77, 31.40, 30.89, 30.67. HRMS: C H ClN (M+H) calcd: 402.1737, found: 402.1741. LC-MS: t = 3.0
25 25
3
R
min, purity >99% (254 nm), m/z: 402.1 (M+H)+.
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
8
-chloro-11-(1-(pyridin-2-ylmethyl)piperidin-4-ylidene)-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-
b]pyridine (8)
A mixture of desloratadine (500 mg, 1.61mmol), 2-(bromomethyl)-pyridine hydrobromide (529 mg, 2.09
mmol) and K CO (579 mg, 4.19 mmol) in DMF (10 mL) was stirred at room temperature for 18 h. The
2
3
mixture was diluted with EtOAc, washed with water (2x) and brine, dried over Na SO , filtered and
2
4
concentrated under vacuum. The crude mixture was purified by reverse-phase column chromatography
(H O/MeCN/HCOOH) to yield the title compound as a pink foam (399 mg, 62% yield).
2
1
H NMR (500 MHz, CDCl ) δ 8.52 (d, J = 4.5 Hz, 1H), 8.36 (d, J = 4.6 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.40 (d,
3
J = 7.2 Hz, 2H), 7.18 – 6.98 (m, 5H), 3.63 (s, 2H), 3.45 – 3.27 (m, 2H), 2.87 – 2.68 (m, 4H), 2.59 – 2.40 (m,
1
3
2
H), 2.40 – 2.27 (m, 2H), 2.26 – 2.10 (m, 2H). C NMR (126 MHz, CDCl ) δ 158.54, 157.56, 149.24, 146.60,
3
139.52, 138.86, 137.75, 137.28, 136.44, 133.43, 132.62, 132.57, 130.87, 128.96, 125.97, 123.22, 122.11,
+
122.06, 64.46, 55.07, 55.05, 31.82, 31.40, 30.94, 30.71. HRMS: C H ClN (M+H) calcd: 402.1737, found:
2
5
25
3
+
4
02.1738. LC-MS: t = 3.1 min, purity >99% (254 nm), m/z: 402.1 (M+H) .
R
8
-chloro-11-(1-(pyrimidin-2-ylmethyl)piperidin-4-ylidene)-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-
b]pyridine (9)
A mixture of pyrimidine-2-carbaldehyde (91 mg, 0.84 mmol), desloratadine (218 mg, 0.7 mmol) and
NaBH(OAc) (237 mg, 1.12 mmol) in DCE (10 mL) was stirred at room temperature for 12 h. The resulting
3
solution was diluted with water and extracted with DCM. The organic phase was washed with satd. aq.
29
ACS Paragon Plus Environment