Potent haloperidol derivatives covalently binding to the dopamine D2 receptor

Article abstract of DOI:10.1016/j.bmc.2017.06.034

The dopamine D₂ receptor (D₂R) is a common drug target for the treatment of a variety of neurological disorders including schizophrenia. Structure based design of subtype selective D₂R antagonists requires high resolution crystal structures of the receptor and pharmacological tools promoting a better understanding of the protein-ligand interactions. Recently, we reported the development of a chemically activated dopamine derivative (FAUC150) designed to covalently bind the L94C mutant of the dopamine D₂ receptor. Using FAUC150 as a template, we elaborated the design and synthesis of irreversible analogs of the potent antipsychotic drug haloperidol forming covalent D₂R-ligand complexes. The disulfide- and Michael acceptor-functionalized compounds showed significant receptor affinity and an irreversible binding profile in radioligand depletion experiments.

Full text of DOI:10.1016/j.bmc.2017.06.034

Accepted Manuscript
Potent haloperidol derivatives covalently binding to the dopamine D2 receptor
Tobias Schwalbe, Jonas Kaindl, Harald Hübner, Peter Gmeiner
PII:
S0968-0896(17)31267-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.06.034
BMC 13817
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 April 2017
16 June 2017
19 June 2017
Please cite this article as: Schwalbe, T., Kaindl, J., Hübner, H., Gmeiner, P., Potent haloperidol derivatives covalently
binding to the dopamine D2 receptor, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/
j.bmc.2017.06.034
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Potent haloperidol derivatives covalently binding to the
dopamine D2 receptor
Tobias Schwalbe, Jonas Kaindl, Harald Hübner, Peter Gmeiner*
Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich-Alexander
University Erlangen-Nürnberg, Schuhstraβe 19, D-91052 Erlangen, Germany
*Corresponding Author. Phone: +49-(0)9131-85-24116. Email:
peter.gmeiner@fau.de
Keywords
G protein-coupled receptor, dopamine D2 receptor, haloperidol, covalent ligand,
disulfide tethering, chemical probe
TOC Graphic
Abstract
The dopamine D2 receptor (D2R) is a common drug target for the treatment of a
variety of neurological disorders including schizophrenia. Structure based design of
subtype selective D2R antagonists requires high resolution crystal structures of the
receptor and pharmacological tools promoting a better understanding of the protein-
ligand interactions. Recently, we reported the development of a chemically activated
dopamine derivative (FAUC150) designed to covalently bind the L94C mutant of the
dopamine D2 receptor. Using FAUC150 as a template, we elaborated the design and
synthesis of irreversible analogs of the potent antipsychotic drug haloperidol forming
covalent D2R-ligand complexes. The disulfide- and Michael acceptor-functionalized
compounds showed significant receptor affinity and an irreversible binding profile in

radioligand depletion experiments.
1. Introduction
The selective covalent ligation of small molecules to pharmacological target proteins
has gained of remarkable importance involving the application as irreversible drugs1, 2
or as biochemical tools used for protein labelling (e.g. with fluorescent dyes)3, 4 or
protein crystallization.5-7 Especially, the covalent tethering of drugs or
neurotransmitters to specific receptor binding sites has gained in importance. The
required covalent ligand is usually composed of a target-specific pharmacophore and
a chemo- or photoreactive probe, which is designed to form a covalent bond with an
adjacent amino acid residue of a wild-type receptor or a mutated variant.8-10
The structural elucidation of membrane proteins, in particular the class A of G-protein
coupled receptors (GPCRs), has substantially benefited from covalent ligands.
Irreversible binding allows the stabilization of particular functional states enabling the
selection of state-specific nanobodies and homogenous crystallogenesis of GPCR
complexes and, thus, facilitating X-ray crystallography. 6, 7, 11-13
Very recently, we could synthesize and functionally investigate stable pairs of
aminergic GPCRs bound to covalent neurotransmitter analogs. Hence, a covalent
derivative of the endogenous ligand dopamine (FAUC150) could be tethered to a
cysteine mutant of the dopamine D2 receptor (D2RL2.64C).7 D2R is involved in multiple
neurological and psychiatric disorders including Parkinson’s disease, schizophrenia
and depression.14 The potent D2R antagonist haloperidol has been used as a typical
antipsychotic drug for decades, although the compound exhibits severe neurological
15
side effects.14,
To design safer and more efficacious antipsychotics, a better
understanding of the molecular origins of D2R - haloperidol interactions is required.
An X-ray crystal structure of the related D3 dopamine receptor subtype in complex
with the antagonist eticlopride was determined.16 However, a high resolution
structure of D2R bound to an antagonist could not be resolved, yet. This illustrates
that the structure-based design of novel drugs urgently requires powerful biochemical
tools.
Taking advantage of our experience with the covalent attachment of the endogenous

agonist dopamine to the D2RL2.64C receptor mutant, we were encouraged to apply this
concept on the selective D2R antagonist haloperidol (Fig. 1). Based on the chemical
structure of haloperidol, we designed a small set of functionalized analogs 1-5
sharing the identical orthosteric head group of the parent compound. To facilitate
covalent tethering, we intended to address the cysteine mutant in the position
TM2.64 by functionalization with four different chemoreactive appendages. Here, we
present the synthesis of the ligands 1-5. Furthermore, we investigated the
compounds for D2R binding and tested their ability to irreversibly occupy the receptor
binding site indicating covalent tethering.
Figure 1: Ligand-based design of covalent D2R antagonists derived from the lead compound
haloperidol.
2. Results and discussion
2.1.Design
Due to its structural similarity to the endogenous ligand dopamine, we chose the
para-chlorophenyl-substituted 4-hydroxypiperidine (6) substructure of haloperidol to
be a recurring element throughout our ligand-based design (Fig. 1). We intended to

attach this orthosteric pharmacophore to a covalently binding appendage subdivided
into three interconnected key elements mimicking the haloperidol tail: 1) a pi-system,
2) an appropriate linker to overcome the distance between the basic center and the
nucleophilic residue of cysteine at position 2.64 of the receptor and 3) a
chemoreactive probe.
In order to preserve the majority of the haloperidol pharmacophore, our initial
investigations were directed to a formal replacement of the fluorine atom of the 4’-
fluoro-butyrophenone by an ethoxy linker attached to a disulfide based covalent
function leading to compound 1. To investigate a heterocyclic bioisostere, the
benzene ring was replaced by a 1,2,3-triazole (compound 4). Our previous
investigations on the covalent dopamine ligand FAUC150, indicated that 1 might
exceed the ideal spacer length between the basic amine center and the disulfide
function, we considered to formally replace the dihydroxyphenethylamine scaffold of
FAUC150 by the respective haloperidol substructure and to vary the linker length (C2
or C3 linker) towards the covalent tag (compounds 2 and 3). All of our disulfide-based
haloperidol derivatives are expected to bind the cysteine by a thiol-disulfide-
interchange reaction, which results in a mainly affinity-driven ligation to the
receptor.17-19 To use an alternative cross-linking reaction, an irreversible analog of
haloperidol replacing the disulfide group by a rather cysteine selective Michael
acceptor was approached.8, 20 Thus, we envisaged to link a maleimide chemical
probe via a 1,3-diethyl urea group.
2.2.Chemistry
The synthesis of the haloperidol derivative 1 started from the 4’-chlorophenyl-4-
hydroxypiperidine 6, which was N-alkylated by the commercially available compound
7 to give the intermediate 8 (Scheme 1). Subsequent alkylation with different pre-
functionalized 2-mercaptoethyl methane sulfonates gave undesired side products or
failed. Therefore, we followed an alternative route by a stepwise integration of the
ethylene linker and the disulfide tag. Alkylation of 8 with 1,2-dibromoethane afforded
the intermediate 9. Subsequent nucleophilic substitution by potassium thioacetate
provided 10 in quantitative yield. Reaction of thioester 10 with sodium methoxide
resulted in the deprotection of the aliphatic thiol, which was scavenged by the
addition of bis(hydroxyethylene)disulfide to obtain the disulfide-functionalized ligand
1.

Scheme 1. Synthesis of covalent haloperidol derivative 1. Reagents and conditions: (a) KI,
DIPEA, DMF, 70°C, 5h, 23% (b) 1,2-dibromoethane, K2CO3, CH3CN, 70°C, 46h, 28% (c),
potassium thioacetate, acetone, 60°C, 90min, 90% (d) NaOMe (0.5 M), MeOH, 2min, room
temperature (e) bis(hydroxyethylene)disulfide, MeOH, 2h, room temperature, 20%
The haloperidol derivatives 2 and 3 (Scheme 2) were prepared in a similar manner as
described for the covalent dopamine derivative DW150.7 The methane sulfonates 11
and 12 consist of a thiopyridyl disulfide and an aromatic scaffold linked by an
ethylene or propylene spacer, respectively, and were prepared as previously
reported.7 Compound 6 was alkylated with the electrophilic building blocks 11 and 12.
To initiate a disulfide interchange reaction, the resulting thiopyridyl disulfide precursor
13 and 14 were reacted with cysteamine hydrochloride leading to formation of the
final products 2 and 3, respectively.
Scheme 2. Synthesis of the covalent haloperidol derivatives 2 (n = 1) and 3 (n = 2).

Reagents and conditions: (a) DMSO, 70°C, 16h (b) MeOH, cysteamine hydrochloride, rt, 1h,
8-27%
The bioisosteric replacement of the butyrophenone scaffold of haloperidol by a
butyryl substituted 1,2,3-triazole required the preparation of the disulfide-
functionalized ligand 4, which is outlined in Scheme 3. The synthesis began with the
acylation of double TMS-protected acetylene 15 with 4-bromobutyryl chloride giving
compound 16, according to a protocol described by Gronnier et al.21 Unfortunately,
the subsequent reaction of 16 with the 4-hydroxypiperidine 6 led to the precipitation
1
of the side product 17. H-NMR and LCMS analysis suggested hetero-Michael
addition and cyclopropane formation. Modification of solvent (DMF, DMSO, toluene),
base (K2CO3, NaHCO3, w/ or w/o KI additive), reaction time (6h to overnight) and
temperature (room temperature to 70°C) did not lead to formation of the desired
product 20. Masking the ketone of 16 with 1,2-ethanediol to form the intermediate 18
induced deactivation of the Michael system and reduction of the CH-acidity of ketone
16. The following alkylation of 6 with 18 afforded the tertiary amine 19 almost
quantitatively. Acidic cleavage of the acetal group provided the ketone 20. Finally, the
chemoreactive disulfide probe was attached by copper assisted click chemistry
(CuAAC) of the TMS-protected alkyne22 with the symmetric bis(2-
azidoethylene)disulfide 23, which was prepared according to literature.23 The TMS-
protective group of 20 was cleaved under aqueous conditions and the resulting
intermediate underwent a triazole cycloaddition affording the trans-isomer of 4, which
was structurally confirmed by 2D-NMR (Supplementary Data).

Scheme 3. Synthesis of covalent haloperidol derivative 4. Reagents and conditions: (a) 4-
Bromobutyryl chloride, AlCl3, CH2Cl2, 0°C to room temperature, 57% (b) 1,2-Dihydroxyethan,
pTsOH, toluene, 100°C, 62% (c) 4-(4-Chlorophenyl)piperidin-4-ol, DIPEA, KI, DMF, room
temperature, 97% (d) HCl (2M) in MeOH, room temperature, 19h, 60% (e) 23, sodium L-
ascorbate, CuCl2 x H4O2, tBuOH/H2O, N2, 16h, room temperature, 19% (f) H2SO4 (conc), HBr
(wt 48%), 100°C, 3h, 69% (g) NaN3, DMF, 16h, 80°C, 82%.
In order to obtain a maleimide-based covalent derivative of haloperidol, the synthesis
started from the building block 6, which was treated with the commercially available
2-(Boc-amino)ethyl bromide providing compound 24 (Scheme 4). Subsequently, 24
converted to 25 by acidic Boc-deprotection following a described protocol.24, 25 The
maleimide building block 26 was transformed to the corresponding isocyanate 27 via
Curtius rearrangement using sodium azide in combination with ethyl chloroformiate.26
Reaction of the crude isocyanate 27 with the amine 25 yielded the urea derivative 5.

Scheme 4. Synthesis of covalent haloperidol derivative 5. Reagents and conditions: (a) 2-
(Boc-amino)ethyl bromide, NaI, DIPEA, acetonitrile, N2, 14h, reflux, 50% (b) HCl in dioxane
(4M), 15h, room temperature, 98% (c) NaN3, ethyl chloroformate, TEA, acetone 60min, 0°C
(d) 27, TEA, DMF, 50h, room temperature, 7%
2.3.Receptor binding studies
2.3.1. Reversible binding affinity to D2-like receptor subtypes
To assess binding affinity of our designed haloperidol derivatives 1-5, we conducted
radioligand binding assays using CHO cells stably expressing the subtypes of the
human dopamine D2-like receptor family including the isoforms D2ShortR and D2Long
R
as well as the closely related D3R and D4.4R subtypes. Additionally, we tested these
compounds on the dopamine receptor mutant D2shortRL.2.64C transiently expressed in
HEK 293T cells. Binding assays were performed with the radioligand [3H]spiperone
and the resulting binding affinities of the covalent ligands were compared to that of
the reference antagonist haloperidol (Table 1).
The D2-type receptor affinities of the test compounds 1–5 were significantly lower
than for the parent compound haloperidol. Nevertheless, the Ki values in the
nanomolar or low micromolar range clearly indicated significant specific binding. The
disulfide derivatives 1 and 4 showed the highest affinities (1: Ki (D2longR) = 240 nM, Ki
(D2shortR) = 73 nM; 4: Ki (D2longR) = 260 nM, Ki (D2shortR) = 140 nM). This indicates

that subtle structural modifications such as the substitution of the fluorine atom or the
bioisosteric replacement of the fluoro-phenyl ring were well tolerated by the
receptors. Additionally, the subtype selectivity for D2R over D3R showed a 4-6-fold
reduction when compared to haloperidol (D3R/D2shortR = 1.9 for 1 and 1.5 for 4,
respectively versus D3R/D2shortR = 9.2 for haloperidol), whereas the distinct D2R/D4R-
selectivity of haloperidol (D4R/D2shortR = 23) was retained for both ligands
(D4R/D2shortR = 13 for 1 and 23 for 4, respectively). Binding experiments with the
D2shortRL2.64C mutant with the compounds 1 and 4 led only to a minor loss in affinity
relative to the wildtype receptor D2shortR (0.22- to 0.44-fold), though, compounds 2, 3,
and 5 slightly gained in affinity (1.1- to 2.7-fold).

Table 1. Receptor binding affinities of the covalent ligands 1-5 in comparison to the reference haloperidol for the human receptor subtypes of the
D2 family (D2longR, D2shortR, D3R, and D4R) and for the mutant D2shortRL2.64C
.
Ki values(nM SD)a
selectivity pattern
compd.
hD2long
R
hD2short
R
hD2shortRL2.64C
330 21
hD3R
hD4.4R
D3R / D2shortRb
1.9
D4R/ D2shortRc
13
D2shortR/ D2shortRL2.64Cd
1
240 250
1500 140
1000 120
260 84
73 52
1500 0
140 100
970 190
0.22
2.7
2
550 91
850 220
320 130
860 84
n.d.
390 50
600 430
210 49
1100 140
1500 280
3200 2400
>50000
0.26
0.63
1.5
0.73
1.6
23
3
940 230
140 79
1.1
4
0.44
1.7
5
2200 0
1500 280
0.25 0.027
690 330
2.3 0.11
0.46
9.2
>33
29
haloperidole
0.42 0.087
7.2 2.2
n.d.
a Ki values as means of two independent experiments each done in triplicate and displayed in nM SD. b Selectivity for D2shortR over D3R calculated by the ratio of
Ki(D3R)/Ki(D2shortR). c Selectivity for D2shortR over D4R calculated by the ratio of Ki(D4R)/Ki(D2shortR). d Selectivity for D2shortRL2.64C mutant over the D2shortR calculated
e
by the ratio of Ki(D2shortR)/Ki(D2shortRL2.64C). Ki values as means of 4-6 independent experiments each done in triplicate and displayed in nM S.E.M.. n.d. = not
determined

2.3.2. Irreversible radioligand depletion
We designed the haloperidol derivatives 1-5 to form a covalent bond with the thiol
residue of a cysteine amino acid introduced by a single L94C mutation in the
transmembrane helix 2 of the dopamine D2 receptor. To investigate the reactivity of
our covalently binding probes, we performed a radioligand depletion assay. In a
screening approach HEK293T cells transiently expressing the D2shortRL2.64C mutant
were incubated for 120 min with compounds 1-5 at two different concentrations both
representing the 5-fold and 20-fold concentration according to the corresponding Ki
values for binding at D2shortR. Once a ligand is covalently bound to the receptor
mutant, the binding crevice of the receptor remains blocked even after several steps
of careful washing, which causes a complete removal of reversibly bound ligand.
Subsequent radioligand binding experiments reveal a reduced binding of the
radioligand [3H]spiperone relative to the amount of blocked binding site. Figure 2
shows the screening results for the covalent ligands 1-5 relative to the reversible
effect of the reference haloperidol. All test compounds display a dose-dependent
blocking of D2shortRL2.64C within a range of 20 to 95%. For the butyrophenone
derivative 1, only minor blocking (20%) was determined at low concentration
revealing a weaker tendency to form a stable covalent bond, while at high
concentration blocking was improved up to 70%. Compounds 2-5 exhibited
substantial irreversible binding at low concentration in the range of 60% to 85%, while
increasing the concentration of active reagent led to an inhibition of radioligand
binding in the range of 70% to 95%. Among the tested ligands, the disulfide 3 and the
maleimide 5 showed superior covalent binding, leading to a remaining radioligand
binding of only 10% and 5%, respectively. Despite their weak affinity for all D2-
subtypes, compound 3 and 5 exhibit strong covalent binding of >90% conferring an
optimized orientation of the reactive linker to the mutated amino acid and an effective
reaction with the cysteine in position 2.64. To learn more about the cross-linking
process, we chose to examine the covalent binding kinetics of the most promising
disulfide-based ligands 2-4 as well as those of the maleimide-based derivative 5
(Figure 3). Therefore, we performed time-dependent radioligand depletion
experiments incubating the receptor together with the covalent ligand for four
different time periods. Interestingly, all haloperidol analogs 2-5 showed a very fast

onset of covalent ligation with an amount of blocking in the range of 70% to 85% after
30 minutes with the oxypropyldisulfide 3 exhibiting best blocking yield (87% at 30
min). Extending the time of incubation for up to 240 min did not facilitate any distinct
increase of blocking for the disulfide derivatives 2-4 (for 2: 72% at 30 min to 82% at
240 min, for 3: 87% at 30 min to 85% at 240 min, for 4: 80% at 30 min to 86% at 120
min) (Figure 3A-C). By contrast a continuous increase of blocking could be observed
for the maleimide 5 revealing an inhibition of the receptor of more than 90% after 120
min (Figure 3D). This may be due the fact that the disulfides 2-4 show some reactivity
with constituents within the assay resulting in a partial removal of reactive compound
from the receptor during extended incubation. This may not be the case for the
maleimide 5 leading to nearly complete blocking of the receptor binding site.
Figure 2. Screening of the blocking behavior of 1-5 in comparison to haloperidol at the
mutant receptor D2shortRL2.64C
.
Membranes were preincubated with two different
concentrations of compounds for 120 min. After washing specific binding of [3H]spiperone
was measured indicating the amount of blocking of the receptor binding site. Incubation was
run at 20 nM for haloperidol (striped bar), 1 µM (light grey) and 10 µM (black) for 1, 5 µM
(light grey) and 20 µM (black) for 2, 3 and 5, and 1 µM (light grey) and 5 µM (black) for 4,
respectively. Bars represent the average values ( SD) of two to six individual experiments
each done in quadruplicate.

A
B
D
C
Figure 3. Kinetics of blocking behavior of the test compounds 2-5 at D2shortRL2.64C
.
Radioligand depletion was measured time dependently at two different concentrations for
compound 2 (A), 3 (B), 4 (C), and 5 (D) indicating a fast reaction of all test ligands to
covalently block the receptor binding site. Graphs show the average data of pooled curves
derived from two to four independent experiments each done in quadruplicate.
2.1.Ligand-Receptor Interactions
To investigate the binding mode of compound 3 and 4, docking studies were
performed using a recently established homology model of D2R27 in which we
introduced the L2.64C mutation. The consistent para-chlorophenyl-substituted 4-
hydroxypiperidine moiety occupies the orthosteric binding pocket formed by residues
of transmembrane helices (TMs) 3, 5, 6, 7 and extracellular loop 2 (EL2) showing
hydrophobic interactions to Phe6.51, Phe6.52, His6.55 and Phe7.38 as well as
Ile183 and Ile184 of EL2 (Figure 4). The canonical salt bridge between their
positively charged nitrogen and the carboxylate of Asp3.32 is present for the two
ligands. Additionally, a hydrogen bond is formed between their hydroxyl group and

the backbone oxygen of Asp3.32. As expected, further interactions of compounds 3
and 4 to residues located in TM2 and TM7 (Glu2.65 and Tyr7.35, respectively)
position the disulfide-based cysteine binding probe in close proximity to Cys2.64,
allowing the covalent binding to the mutant receptor.
Figure 4. Ligand-receptor interactions of compounds 3 and 4 at D2R-L2.64C (light gray and
dark gray ribbons, respectively). Amino acids expected to interact with the two compounds
are displayed as light gray and dark gray sticks. Compound 3 within D2R is shown by green
sticks (A), while compound 4 within D2R is shown by blue sticks (B). The Ballesteros-
Weinstein numbering has been used for transmembrane (TM) residues, whereas sequence
numbers are given for residues of extracellular loops (EL).
3. Conclusions
Despite the apparent structural similarity of the endogenous neurotransmitter
dopamine and the 4-(4-chlorophenyl)-piperidin-4-ol pharmacophore of haloperidol,
the exact binding mode of haloperidol and structurally similar antipsychotic drugs is
29
an open question.28,
Due to the current absence of crystal structures of an
antagonist-bound dopamine D2 receptor, substantial efforts have to be invested to
learn more about the receptor-ligand interactions responsible for the stabilization of
the receptor in its inactive state. To our knowledge, only a few covalent ligands have
been reported to irreversibly antagonize the D2-receptors.30-32 In this work, we
synthesized a set of covalent ligands derived from the classical antipsychotic drug
haloperidol and the covalent dopamine analog FAUC150 irreversibly binding to a
L94C cysteine mutant of the dopamine D2 receptor.7 We envisaged to apply different

disulfide-based cysteine binding probes as well as a maleimide-based Michael
acceptor together with structural modifications including a partial shortening of the
haloperidol lead structure. The formal replacement of the single fluorine atom of
haloperidol by an ethoxy linker and a disulfide appendage led to moderate binding
affinity of the ligand (1), however, the rate of covalent binding was comparatively low.
Bioisosteric replacement of the butyrophenone moiety to an acylated 1,2,3-triazole or
transferring the homovanillyl appendage of FAUC150 increased the efficiency of
covalent binding significantly (2 - 4). The shortest haloperidol derivative modified with
a maleimide covalent probe (5) clearly lost in binding affinity, however, it exhibited
high covalent binding efficiency. In summary, we were able to show that our
L2.64C
haloperidol derivatives covalently bind the dopamine receptor mutant D2
and
that these ligands compete with the radioligand [3H]-spiperone for the same binding
site since the binding of most of our derivatives could not be reversed by adding an
excess of radioligand. Future studies will aim for the structural optimization of the
reported covalent analogs. Moreover, we plan to implement these ligands in
structural and functional investigations of dopamine receptors.
4. Experimental section
4.1.General
Unless otherwise noted, reactions were performed under nitrogen atmosphere and
reagents as well as dry solvents were of commercial quality as purchased. MS was
run on a BRUKER ESQUIRE 2000 using electrospray ionization (ESI). HRMS-ESI
was run on an AB Sciex Triple TOF660 SCiex, source type ESI or at the Chair of
Organic Chemistry, Friedrich Alexander University Erlangen-Nürnberg on a Bruker
Daltonik micrOTOF II focus or Bruker Daltonik maXis 4G, source type ESI or APPI.
1
NMR spectra were obtained on a Bruker Avance 400 (400 MHz for H and 101 MHz
for 13C) or a Bruker Avance 600 (600 MHz for 1H and 151 MHz for 13C) spectrometer
at 300 K in the solvents indicated. The chemical shifts are reported in ppm (δ) relative
to TMS. Purification via column chromatography was performed using silica gel 60.
TLC analyses were performed on Merck 60 F254 aluminum plates in combination
with UV detection (254 nm) and/or TLC staining (Ninhydrin, KMnO4, Vanillin, FeCl3).
Analytical HPLC was conducted on an Agilent 1200 HPLC system employing a DAD
detector and with a ZORBAX ECLIPSE XDB-C8 (4.6 x 150 mm, 5 µm) column and

using one of the following binary solvent systems. System 1: eluent, methanol/0.1%
aq formic acid, 10% methanol for 3 min, to 100% in 15 min, 100% for 6 min, to 10%
in 3min, then 10% for 3min, flow rate 0.5 mL/min; System 2: CH3CN/0.1% aq TFA,
10% CH3CN for 3 min, to 100% in 15 min, 100% for 6 min, to 10% in 3min, then 10%
for 3min, flow rate 0.5 mL/min; System 3: methanol/0.1% aq TFA, 10% methanol for
3 min, to 100% in 15 min, 100% for 6 min, to 10% in 3min, then 10% for 3min, flow
rate 0.5 mL/min; Preparative HPLC was performed on an Agilent 1100 Preparative
Series, using a ZORBAX ECLIPSE XDB-C8 PrepHT column (21.5 x 150 mm, 5 µm,
flow rate 10 mL/min) , a VP 250/32 NUCLEODUR C18 HTec column (5µm, flow rate
32 mL/min) or a VP 250/10 NUCLEODUR C18 HTec column (5µm, flow rate 2-4
mL/min) with the solvent systems indicated and a detection wavelength of 220 nm or
254 nm.
4.2.Synthesis
4.2.1. 4-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-1-(4-hydroxyphenyl)butan-
1-one (8)
4(4-chlorophenyl)-piperidin-4-ol (6) and 4-Chloro-4’-hydroxybutyrophenone (7) were
commercially available. The amine 6 (2.0g, 9.5 mmol), compound 7 (2.1 g, 10 mmol),
DIPEA (4.1 mL, 24 mmol) and potassium iodide (94 mg, 0.57 mmol) were placed in a
Schlenk flask and dissolved in 60 mL anhydrous DMF. After stirring for 16 hours at
70°C the solvent was evaporated under reduced pressure and the residue was taken
up in ethyl acetate and acidified with 2N HCl (pH = 3). The resulting precipitate was
filtered and washed with CH2Cl2 /methanol (2/1) and dried in vacuum to give 8 as a
pale yellow solid. (0.80 g, 23%). 1H NMR (600 MHz, DMSO-d6) δ 7.82 (d, J = 8.7 Hz,
2H), 7.40 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.4 Hz, 2H), 2.87
(t, J = 6.9 Hz, 2H), 2.64 – 2.55 (m, 2H), 2.36 – 2.28 (m, 4H), 1.84 – 1.70 (m, 3H),
1.53 – 1.42 (m, 2H). 13C NMR (150 MHz, DMSO-d6) δ 197.91, 163.05, 149.26,
130.66, 130.35, 127.90, 127.65, 126.81, 115.35, 69.59, 57.43, 48.94, 37.87, 35.10,
22.08; ESI-MS m/z 374.15 [M+H]+. HPLC (system 1, 220 nm): tR = 15.3 min, purity:
96%, (system 2, 220 nm): tR = 14.9 min, purity: 98%. HR-ESI-MS m/z [M+H]+ calcd
for C21H24ClNO3, 374.1517; found, 374.1515
4.2.2. 1-(4-(2-bromoethoxy)phenyl)-4-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-
yl)butan-1-one (9)

Compound 8 (0.20 g, 0.54 mmol), 1,2-dibromoethane (0.23 mL, 2.7 mmol) and
K2CO3 (0.15 g, 1.1 mmol) were dissolved in acetonitrile (20mL) and stirred at 70°C
for 41 hours. An additional portion of 1,2-dibromoethane (0.09 mL, 1.1 mmol) was
added to the mixture and stirring/heating was continued for 5 hours. The reaction was
allowed to cool to room temperature and the solvent was removed under reduced
pressure. The residue was taken up in H2O and extracted multiple times with CH2Cl2.
After drying (Na2SO4) the solvent was evaporated and the crude product was purified
via flash column chromatography (eluents: CH2Cl2/methanol 9/1) to afford compound
9 (74 mg, 28%). 1H NMR (600 MHz, DMSO-d6) δ 7.97 (d, J = 8.8 Hz, 2H), 7.39 (d, J
= 8.3 Hz, 2H), 7.35 (d, J = 8.3 Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 4.91 (s, 1H), 4.42 (t,
J = 5.4 Hz, 2H), 3.83 (t, J = 5.3 Hz, 2H), 2.96 (t, J = 6.9 Hz, 2H), 2.81 – 2.58 (m, 2H),
2.58 – 2.24 (m, 4H), 1.89 – 1.81 (m, 2H), 1.77 (s, 2H), 1.58 – 1.47 (m, 2H). 13C NMR
(150 MHz, DMSO-d6) δ 198.05, 161.54, 149.43, 130.81, 130.40, 130.24, 127.72,
126.74, 114.41, 69.27, 68.00, 62.79, 48.82, 37.32, 35.24, 31.21, 21.55; ESI-MS m/z
480.1/482.1 [M+H]+ HR-ESI-MS m/z [M+H]+ calcd for C23H27BrNO3, 480.0936; found,
,
480.0936
4.2.3. S-(2-(4-(4-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)butanoyl)phenoxy)-
ethyl)ethanethioate (10)
Compound 8 (67 mg, 0.14 mmol) and potassium thioacetate (48 mg, 0.42 mmol)
were dissolved in 4.0 mL dry acetone and heated at 60°C for 90 minutes. The
reaction was allowed to cool to room temperature and the solvent was removed
under reduced pressure. The residue was taken up in H2O and extracted with
CH2Cl2. The combined organic fractions were dried (Na2SO4) and the solvent was
1
evaporated to give compound 10 without further purification step (60 mg, 90%). H
NMR (600 MHz, DMSO-d6) δ 8.00 – 7.92 (m, 2H), 7.45 – 7.37 (m, 2H), 7.35 – 7.28
(m, 2H), 7.00 – 6.91 (m, 2H), 4.16 (t, J = 6.5 Hz, 2H), 3.29 (t, J = 6.5 Hz, 2H), 3.00 (t,
J = 6.9 Hz, 2H), 2.99 – 2.94 (m, 3H), 2.68 – 2.56 (m, 4H), 2.38 (s, 3H), 2.22 – 2.13
(m, 2H), 2.09 – 2.01 (m, 2H), 1.79 – 1.68 (m, 2H) 13C NMR (100 MHz, CDCl3)
198.58, 195.45, 162.25, 146.85, 132.94, 130.73, 130.51, 128.54, 126.24, 114.37,
71.16, 66.79, 57.97, 49.41, 38.31, 36.10, 30.73, 28.37, 21.86; HPLC (system 1, 254
nm): tR = 17.6 min, purity: 93%; (system 2, 254 nm) tR = 16.3 min, purity: 92%; ESI-
MS m/z 476.2 [M+H]+; HR-ESI-MS m/z [M+H]+ calcd for C25H30ClNO4S, 476.1657;
found, 476.1657

4.2.4. 4-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-1-(4-(2-((2-hydroxyethyl)
disulfanyl)ethoxy)phenyl)butan-1-one x HCOOH (1)
Compound 10 (51 mg, 0.11 mmol) was dissolved in 2.0 mL anhydrous MeOH and a
0.5M solution of NaOMe (0.26 mL, 0.13 mmol) was added while stirring. After 2
minutes Bis(hydroxyethylene)disulfide (30µL, 0.21 mmol) was added with a
microsyringe and the reaction was stirred for additional 2 hours at room temperature.
Purification by preparative HPLC (C8 eluents: MeOH/0.1% aq. HCOOH 25-100%)
1
yielded the formic acid salt of product 1 (12 mg, 20%) as a colorless solid. H NMR
(400 MHz, DMSO-d6) δ 8.18 (s, 1H), 8.03 – 7.88 (m, 2H), 7.49 – 7.38 (m, 2H), 7.38 –
7.30 (m, 2H), 7.16 – 7.01 (m, 2H), 4.32 (t, J = 6.2 Hz, 2H), 3.68 – 3.57 (m, 2H), 3.20
– 3.08 (m, 2H), 3.04 – 2.92 (m, 2H), 2.86 – 2.72 (m, 4H), 2.62 – 2.52 (m, 3H), 1.94 –
1.73 (m, 4H), 1.63 – 1.47 (m, 2H) 13C NMR (101 MHz, DMSO-d6) δ 197.91, 161.84,
148.63, 130.90, 130.25, 130.15, 127.77, 126.75, 114.35, 69.13, 66.02, 59.55, 59.36,
56.78, 48.70, 41.11, 37.04, 35.17, 21.15; HPLC (system 1, 220 nm): tR = 14.0 min,
purity: >99%, (system 2, 220 nm): tR = 16.0 min, purity: 96%; ESI-MS m/z 510.2
[M+H]+; HR-ESI-MS m/z [M+H]+ calcd for C25H32ClNO4S2, 510.1534; found, 510.1534
4.2.5. 1-(4-(2-((2-aminoethyl)disulfanyl)ethoxy)-3-methoxyphenethyl)-4-(4-
chlorophenyl)piperidin-4-ol x 2·TFA (2)
Compounds 6 (153 mg, 722 µmol) and 11 (100 mg, 241 µmol) were placed in a
sealed tube and dissolved in 2 mL of anhydrous DMSO. The solution was heated at
70°C for 16 hours. The reaction mixture was allowed to cool to room temperature
followed by the addition of H2O and subsequent lyophilization. After a first purification
step via flash column chromatography on silica gel (eluents: CH2Cl2/MeOH 98/2 to
90/10) the resulting oil was redissolved in dry MeOH (1mL) and cysteamine
hydrochloride (10 mg, 88 µmol) was added. The mixture was stirred for 1 hour at
room temperature and the product 2 was isolated by preparative HPLC (C8 eluents:
CH3CN /aq. TFA 0.1%; 5-45%) to give the corresponding TFA salt (12 mg, 8% yield).
1H NMR (600 MHz, CD3OD) δ 7.53 – 7.47 (m, 2H), 7.41 – 7.35 (m, 2H), 6.97 (d, J =
2.1 Hz, 1H), 6.96 (d, J = 8.2 Hz, 1H), 6.86 (dd, J = 8.2, 2.0 Hz, 1H), 4.26 (t, J = 6.1
Hz, 2H), 3.86 (s, 3H), 3.64 – 3.57 (m, 2H), 3.52 – 3.44 (m, 2H), 3.44 – 3.38 (m, 2H),
3.32 – 3.28 (m, 2H), 3.14 (t, J = 6.1 Hz, 2H), 3.10 – 3.04 (m, 2H), 3.00 (t, J = 6.6 Hz,
2H), 2.34 (td, J = 14.1, 4.3 Hz, 2H), 2.02 – 1.95 (m, 2H) 13C NMR (151 MHz,
CD3OD) δ 151.42, 148.48, 146.99, 134.30, 131.35, 129.53, 127.41, 122.28, 115.93,

114.22, 69.21, 68.68, 59.28, 56.57, 50.33, 39.09, 38.96, 36.67, 35.41, 31.09. HPLC
(system 2, 220 nm): tR = 14.0 min, purity: >99%; (system 3, 220 nm): tR = 16.2 min,
purity: >99%; ESI-MS m/z 497.2 [M+H]+ HR-ESI-MS m/z [M+1]+ calcd for
;
C24H34ClN2O3S2, 497.169389; found, 497.168036.
4.2.6. 1-(4-(3-((2-aminoethyl)disulfanyl)propoxy)-3-methoxyphenethyl)-4-(4-
chlorophenyl)piperidin-4-ol x 2·TFA (3)
Compounds 6 (0.15g, 0.70 mmol) and 12 (0.10 g, 0.23 mmol) were placed in a
sealed tube and dissolved in 2 mL of anhydrous DMSO. The solution was heated at
70°C for 20 hours. The reaction mixture was allowed to cool to room temperature
followed by the addition of H2O and subsequent lyophilization. After a first purification
step via flash chromatography on silica gel (eluents: CH2Cl2/MeOH; 98/2 to 90/10)
the resulting oil was redissolved in dry MeOH (1mL) and cysteamine hydrochloride
(10 mg, 88 µmol) was added. The mixture was stirred for 1 hour at room temperature
and the product was isolated by preparative HPLC (C8 eluents: CH3CN /aq. TFA
0.1%; 5-45%) as the corresponding TFA salt (39 mg, 27% yield). 1H NMR (600 MHz,
CD3OD) δ 7.50 (d, J = 8.6 Hz, 2H), 7.38 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 1.9 Hz, 1H),
6.93 (d, J = 8.2 Hz, 1H), 6.85 (dd, J = 8.2, 2.0 Hz, 1H), 4.09 (t, J = 6.0 Hz, 2H), 3.85
(s, 3H), 3.64 – 3.57 (m, 2H), 3.52 – 3.43 (m, 2H), 3.44 – 3.38 (m, 2H), 3.32 – 3.26 (m,
2H), 3.10 – 3.03 (m, 2H), 2.99 – 2.90 (m, 4H), 2.38 – 2.28 (m, 2H), 2.23 – 2.13 (m,
2H), 2.05 – 1.92 (m, 2H). 13C NMR (151 MHz, CD3OD) δ 151.37, 148.93, 146.98,
134.31, 130.85, 129.53, 127.41, 122.25, 115.54, 114.04, 69.20, 68.48, 59.32, 56.57,
50.33, 39.28, 36.67, 35.49, 35.15, 31.10, 29.93; HPLC (system 2, 220 nm): tR = 14.2
min, purity: >99%, (system 3, 220 nm): tR = 16.5 min, purity: >99%; ESI-MS m/z
511.2 [M+H]+ HR-ESI-MS m/z [M+H]+ calcd for C25H36ClN2O3S2, 511.185039; found,
511.185663.
4.2.7. 6-bromo-1-(trimethylsilyl)hex-1-yn-3-one (16)
Compound 16 was synthesized following the literature.33 Anhydrous AlCl3 (1.88 g,
14.1 mmol) was placed in a Schlenk flask and stirred in 16 mL methylene chloride at
0°C under argon atmosphere. 4-Bromobutyryl chloride (1.49 mL, 12.9 mmol) was
added to the cooled solution followed by the addition of bis(trimethylsilyl)acetylene
(2.00 g, 11.7 mmol) in small portions. After stirring for 1 hour at 0°C the reaction
mixture was quenched with cold H2O and extracted with methylene chloride. The

combined organic layers were washed with H2O and Brine, dried over Na2SO4 and
the solvent was removed under reduced pressure. Flash column chromatography
(eluent: n-hexane/EtOAc 100/1, KMnO4 staining) yielded the product 16 as a
colorless oil (1.65 g, 57%). 1H NMR (600 MHz, CDCl3) δ 3.45 (t, J = 6.4 Hz, 2H), 2.78
(t, J = 7.0 Hz, 2H), 2.25 – 2.16 (m, 2H), 0.25 (s, 9H). 13C NMR (151 MHz, CDCl3) δ
186.14, 101.87, 98.66, 43.53, 32.64, 26.70, -0.64. HPLC (system 2, 254 nm): tR =
21.4 min, purity: 96%; ESI-MS m/z 246.9/248.9 [M+H]+
4.2.8. ((2-(3-bromopropyl)-1,3-dioxolan-2-yl)ethynyl)trimethylsilane (18)
Compound 16 (0.50 g, 2.0 mmol) 1,2-ethanediol (1.3 g, 20 mmol) and para-
toluenesulfonic acid (77 mg, 0.40 mmol) were dissolved in 50 mL toluene and stirred
at 100°C for 16 hours. The reaction mixture was allowed to cool to room temperature
and the solvent was evaporated under reduced pressure. The crude product was
purified via flash column chromatography on silica gel (eluent: n-hexane/ethyl acetate
1
20/1, KMnO4 staining) to yield compound 18 (0.37 g, 62%) as colorless oil. H NMR
(600 MHz, CDCl3) δ 4.14 – 4.05 (m, 2H), 4.01 – 3.94 (m, 2H), 3.48 (t, J = 6.7 Hz,
2H), 2.16 – 2.09 (m, 2H), 2.06 – 2.02 (m, 2H), 0.18 (s, 9H). 13C NMR (151 MHz,
CDCl3) δ 102.56, 102.34, 89.21, 64.83, 37.75, 33.59, 27.57, -0.08 ESI-MS m/z
290.6/292.6 [M+H]+
4.2.9. 4-(4-chlorophenyl)-1-(3-(2-((trimethylsilyl)ethynyl)-1,3-dioxolan-2-
yl)propyl)piperidin-4-ol (19)
Compound 18 (0.20 g, 0.69 mmol) and amine 6 (0.73 g, 3.4 mmol) were dissolved in
2.0 mL anhydrous DMF. Potassium iodide (23 mg, 0.14 mmol) and DIPEA (0.60 mL,
3.4 mmol) were added to the reaction mixture and stirred for 13 hours at room
temperature. The solvent was evaporated under reduced pressure and the crude
product was purified by flash column chromatography on silica gel (eluents: CH2Cl2 /
MeOH 95/5) to give product 19 (0.28 g, 97%) as a pale yellow oil. 1H NMR (600 MHz,
CDCl3) δ 7.51 – 7.44 (m, 2H), 7.35 – 7.30 (m, 2H), 4.12 – 4.03 (m, 2H), 4.06 – 3.97
(m, 2H), 3.38 – 3.29 (m, 2H), 3.18 (t, J = 12.4 Hz, 2H), 3.04 – 2.97 (m, 2H), 2.77 (t, J
= 14.4 Hz, 2H), 2.16 – 2.06 (m, 2H), 1.99 (t, J = 7.1 Hz, 2H), 1.95 – 1.87 (m, 2H),
0.19 (s, 9H) 13C NMR (151 MHz, CDCl3) δ 144.77, 133.73, 128.90, 126.16, 102.32,
101.94, 89.65, 69.86, 64.88, 57.33, 48.95, 36.07, 35.83, 19.12, -0.04. HPLC (system
1, 210 nm): tR = 18.2 min, purity: 86%, (system 2, 210 nm): tR = 17.4 min, purity:

86%; ESI-MS m/z 422.17 [M+H]+; HR-ESI-MS m/z [M+H]+ calcd for C22H33ClNO3Si,
422.191275; found, 422.190363.
4.2.10. 6-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-1-(trimethylsilyl)-hex-1-yn-
3-one trifluoroacetate (20)
19 (0.10 g, 0.24 mmol) was dissolved in 0.5 mL MeOH and treated with 2N aq. HCl
(1.0mL, 2.0 mmol) for 2 hours at room temperature. The mixture was diluted with
5mL H2O and lyophilized to dryness. The crude product was purified via preparative
HPLC (C18, eluents: CH3CN / 0.1% aq. TFA, 10-45%) to give 20 as a white solid (70
1
mg, 60%). H NMR (600 MHz, CDCl3) δ 12.40 (s, 1H), 7.44 – 7.38 (m, 2H), 7.37 –
7.31 (m, 2H), 3.54 – 3.46 (m, 2H), 3.30 – 3.21 (m, 3H), 3.04 (t, J = 8.0 Hz, 2H), 2.77
(t, J = 6.5 Hz, 2H), 2.57 (td, J = 14.0, 4.1 Hz, 2H), 2.36 (s, 3H), 2.15 – 2.06 (m, 2H),
1.97 – 1.84 (m, 2H), 0.25 (s, 9H) 13C NMR (91 MHz, CDCl3) δ 185.36, 144.29,
134.09, 129.06, 125.96, 101.46, 99.95, 69.39, 56.25, 48.81, 41.93, 35.52, 17.98, -
0.75. ESI-MS m/z 378.18 [M+H]+; HPLC (system 1, 230nm) : tR = 17.9 min, purity:
98%, (system 2, 230 nm): tR = 17.1 min, purity: 73%; HR-ESI-MS m/z [M+H]+ calcd
for C20H28ClNO2Si, 377.1578; found, 377.1578.
4.2.11. Bis(2-bromoethyl)disulfide (22)
Bis(2-bromoethyl)disulfide (22) was synthesized following the literature23. To an ice-
cooled solution of conc. H2SO4 (20 mL) was slowly added HBr (48% wt, 28 mL)
under stirring. Subsequently, Bis(2-hydroxyethyl)disulfide 21 (0.52 mL, 4.3 mmol)
was added dropwise and the solution was stirred for 16 hours at room temperature
followed by stirring at 100°C for 4 hours. The mixture was allowed to cool to room
temperature and extracted with methylene chloride. The combined organic solvents
were washed with H2O, 10% Na2CO3 solution and dried (Na2SO4). Evaporation of the
solvent under reduced pressure gave compound 21 (0.83 g, 69%) which was used in
the next step without further purification. 1H NMR (600 MHz, CDCl3) δ 3.67 – 3.58 (m,
4H), 3.15 – 3.07 (m, 4H). Analytical data were in agreement with the literature.
4.2.12. Bis(2-azidoethyl)disulfide (23)
Bis(2-azidoethyl)disulfide (23) was synthesized following the literature23. Compound
22 (0.15 g, 0.54 mmol) and sodium azide (0.17 g, 2.7 mmol) were dissolved in 8.0
mL DMF and heated at 80°C for 16 hours. After cooling to room temperature the

reaction mixture was extracted with diethyl ether, washed with brine and dried over
Na2SO4. Careful evaporation of the solvent (no heating, slow rotation, potentially
explosive!) gave the crude product 23 which was used in the next step without further
1
purification (90 mg, 82%). H NMR (400 MHz, CDCl3) δ 3.6 (t, J = 6.7 Hz, 4H), 2.88
(t, J = 6.7 Hz, 4H). Analytical data were in agreement with the literature.
4.2.13. 1-(1-(2-((2-azidoethyl)disulfanyl)ethyl)-1H-1,2,3-triazol-4-yl)-4-(4-(4-
chlorophenyl)-4-hydroxypiperidin-1-yl)butan-1-one x TFA (4)
Compound 20 (62 mg, 0.13 mmol), sodium L-ascorbate (5.0 mg, 25 µmol), CuCl2 x
H4O2 (2.5 mg, 13 µmol) and bis(azidoethylene)disulfide (31 mg, 0.15 mmol) were
placed in a Schlenk flask and dissolved In 1.0 mL CH3CN/H2O (4/1) under argon
atmosphere. After stirring for 2 hours at 70°C the reaction mixture was allowed to
cool to room temperature and the desired product 4 was purified via preparative
1
HPLC (C8, eluents: CH3CN / 0.1% aq. TFA; 16 mg, 19%). H NMR (600 MHz,
DMSO-d6) δ 9.37 (s, 1H), 8.85 (s, 1H), 7.51 – 7.45 (m, 2H), 7.48 – 7.42 (m, 2H), 5.62
(s, 1H), 4.77 (t, J = 6.5 Hz, 2H), 3.60 (t, J = 6.4 Hz, 2H), 3.51 – 3.49 (m, 4H), 3.32 (t,
J = 6.5 Hz, 2H), 3.29 – 3.21 (m, 2H), 3.18 (t, J = 7.2 Hz, 2H), 2.97 (t, J = 6.4 Hz, 2H),
2.18 (m, J = 14.1, 4.4 Hz, 2H), 2.11 – 2.02 (m, 2H), 1.84 (d, J = 14.2 Hz, 2H). 13C
NMR (91 MHz, DMSO-d6) δ 192.65, 146.70, 146.54, 131.61, 128.18, 127.54,
126.61, 67.68, 55.36, 49.13, 48.42, 48.38, 36.76, 36.66, 35.87, 35.03, 17.77; HPLC
(system 1, 254 nm): tR = 16.6 min, purity: 97%, (system 2, 254 nm): tR = 16.4 min,
purity: 98%; ESI-MS m/z 510.20 [M+H]+
HR-ESI-MS m/z [M+H]+ calcd for
;
C21H28ClN7O2S2, 510.150; found, 510.1507
4.2.14. tert-butyl-(2-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-ethyl)-
carbamate (24)
Compound 24 was prepared as described in the literature.24 Amine 6 (0.40 mg, 1.9
mmol), sodium iodide (0.28 g, 1.9 mmol) and DIPEA (0.31 mL, 1.9 mmol) were
dissolved in 40mL acetonitrile. A solution of 2-(Boc-amino)ethylbromide (0.55 g, 2.5
mmol) in 2.5 mL acetonitrile was added dropwise and the mixture was heated to
reflux for 14 hours. After the reaction was allowed to cool to room temperature the

solvent was removed under reduced pressure. The residue was taken up in K2CO3
solution (1M, 50 mL) and ethyl acetate. The combined organic fractions were washed
with H2O, Brine and dried (Na2SO4). Evaporation of the solvent under reduced
pressure gave the crude product which was purified via flash column chromatography
on silica gel (eluents: n-hexane/ethyl acetate 2/3 to 1/4) to yield compound 24 (338
mg, 50%). 1H NMR (600 MHz, DMSO-d6) δ 7.51 – 7.45 (m, 2H), 7.38 – 7.32 (m, 2H),
6.62 (t, J = 5.8 Hz, 1H), 4.87 (s, 1H), 3.08 – 3.01 (m, 2H), 2.67 – 2.60 (m, 2H), 2.42 –
2.35 (m, 2H), 2.35 (t, J = 7.1 Hz, 2H), 1.88 (td, J = 12.8, 4.3 Hz, 2H), 1.59 – 1.51 (m,
2H), 1.38 (s, 9H) 13C NMR (101 MHz, DMSO-d6) δ 155.53, 149.19, 130.73, 127.71,
126.82, 77.49, 69.45, 57.61, 49.10, 37.90, 37.67, 28.26; HPLC (system 1, 210 nm):
tR = 16.9 min, purity: 88%; ESI-MS m/z 355.0 [M+H]+ Analytical data were in
agreement with the literature.
4.2.15. 1-(2-aminoethyl)-4-(4-chlorophenyl)piperidin-4-ol hydrochloride (25)
The Boc-protected amine 24 (0.31 g, 0.87 mmol) was stirred in a 4M solution of HCl
in 1,4-dioxane (2.0 mL, 7.9 mmol) for 15 hours at room temperature. Evaporation of
the solvent under reduced pressure gave the hydrochloric salt of 25 (0.28 g, 98%). 1H
NMR (600 MHz, DMSO-d6) δ 7.52 (d, J = 8.5 Hz, 2H), 7.38 (d, J = 8.5 Hz, 2H), 3.62
(d, J = 12.0 Hz, 2H), 3.51 (m, 6H), 2.53 (td, J = 14.1, 4.3 Hz, 2H), 2.02 – 1.94 (m, 2H)
13C NMR (101 MHz, DMSO-d6) δ 146.87, 134.32, 129.52, 127.47, 69.15, 68.14,
54.75, 51.03, 36.58, 35.33; ESI-MS m/z 255.0 [M+H]+ Analytical data were in
agreement with the literature.25
4.2.16. 1-(2-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)ethyl)-3-(2-(2,5-dioxo-2,5-
dihydro-1H-pyrrol-1-yl)ethyl)urea (5)
3-maleimidepropionic acid 26 (0.10 g, 0.59 mmol) and TEA (90 µL, 0.65 mmol) were
dissolved in 1.2 mL acetone and cooled to -5°C. After 2 minutes ethyl chloroformate
(60µL, 0.65 mmol) was added dropwise and stirred for additional 10 minutes. Sodium
azide (38 mg, 0.59 mmol) was added and the reaction was stirred for 45 minutes.
Subsequently, the mixture was diluted with H2O, extracted with toluene (30 mL) and
dried with Na2SO4. The organic phase was heated at 130°C for 100 minutes and
allowed to cool to room temperature afterwards. The solvent was removed under
reduced pressure and the crude oil was taken up in DMF (4 mL) and used in the next
step without further purification. The hydrochloric salt of amine 25 (0.12 mg, 0.37

mmol) was added to the solution and stirred for 50 hours at room temperature.
Evaporation of the solvent and purification by preparative HPLC (C8, eluents:
CH3CN/0.1% aq. TFA) gave the TFA salt of 5 as white solid (13 mg, 7%). 1H NMR
(600 MHz, DMSO-d6) δ 8.21 (br s, 1H), 7.53 – 7.45 (m, 2H), 7.44 – 7.35 (m, 2H),
7.00 (s, 2H), 6.25 (t, J = 6.1 Hz, 1H), 5.99 (d, J = 7.5 Hz, 1H), 5.18 (s, 1H), 3.43 (t, J
= 5.9 Hz, 2H), 3.21 – 3.11 (m, 4H), 3.00 – 2.89 (m, 2H), 2.80 – 2.70 (m, 2H), 2.69 –
2.61 (m, 2H), 2.08 – 1.96 (m, 2H), 1.76 – 1.61 (m, 2H) 13C NMR (101 MHz, DMSO-
d6) δ 171.06, 158.14, 148.18, 134.50, 131.08, 127.89, 126.74, 68.75, 57.19, 48.81,
37.95, 37.58, 36.59, 36.09; HPLC (system 1, 210 nm): tR = 15.4 min, purity: >99%;
(system 2, 210 nm): tR = 13.8 min, purity: >99%; ESI-MS m/z 421.2 [M+H]+; HR-ESI-
MS m/z [M+H]+ calcd for C20H26ClN4O4, 421.1637; found, 421.1637.
4.3.Biological Investigation
4.3.1. Receptor Binding Experiments
35
Receptor binding studies were carried out as described previously.34,
In brief,
competition binding experiments with the human D2longR, D2shortR,36 D3R37 and D4.4R38
receptors were perform when using preparations of membranes from CHO cells
stably expressing the corresponding receptor together with [3H]spiperone (specific
activity = 81 Ci/mmol, PerkinElmer, Rodgau, Germany) at a final concentration of
0.15-0.30 nM. The assays were carried out at protein concentrations of 5-6 µg/assay
tube, Bmax values of 800-1500 fmol/µg and KD values of 0.060-0.10 nM for D2longR,
concentrations of 1-4 µg/assay tube, Bmax values of 2600-8500 fmol/µg and KD
values of 0.050-0.090 nM for D2shortR, concentrations of 1-3 µg/assay tube, Bmax
values of 2300-8500 fmol/µg and KD values of 0.080-0.15 nM for D3R, and
concentrations of 4-10 µg/assay tube, Bmax values of 1000-1700 fmol/µg and KD
values of 0.14-0.25 nM for D4.4R, respectively. Radioligand binding assays with the
mutant receptor D2shortRL2.64C were performed using homogenates of membranes
from HEK 293T cells, which were transiently transfected with the pcDNA3.1 vector
containing the appropriate mutation using the Mirus TransIT-293 transfection reagent
(MoBiTec, Goettingen, Germany).7 Unspecific binding was determined in the
presence of 10 µM haloperidol, protein concentration was established by the method
of Lowry using bovine serum albumin as standard.39 The resulting competition curves
of the receptor binding experiments were analyzed by nonlinear regression using the
algorithms in PRISM 6.0 (GraphPad Software, San Diego, CA). The data were

initially fit using a sigmoid model to provide an IC50 value, representing the
concentration corresponding to 50% of maximal inhibition. IC50 values were
transformed to Ki values according to the equation of Cheng and Prusoff.40
4.3.2. Radioliogand Depletion Assays.
Tests on covalent blocking of the receptor were carried out as described previously.7
Membranes from HEK 293T cells transiently expressing the human D2shortRL2.64C
were preincubated in binding buffer (50 mM Tris, 1 mM EDTA, 5 mM MgCl2, 100
µg/mL bacitracin, 5 µg/mL soybean trypsin inhibitor at pH 7.4) at 37°C at a protein
concentration of 50 µg/mL and the test compounds applying two different
concentrations roughly representing the 5-fold or 20-fold Ki value derived from the
binding experiment with the wild-type receptor. In particular the compounds 2, 3 and
5 were tested at concentrations of 5 µM and 20 µM, ligand 1 at 1 µM and 10 µM, and
compound 4 at 1 µM and 5 µM, respectively. As a reference haloperidol was used at
20 nM (100-fold). For screening of the test compounds preincubation was run for 120
min, while kinetic studies were performed with incubation times from 15 min to 240
min. Generally incubation was stopped by centrifugation and the amount of reversibly
bound ligand was washed out for four times (resuspension of the memebranes in
buffer for 30 min followed by centrifugation). Washed membranes were used for
radioligand binding experiments with [3H]spiperone to determine the remaining
specific binding at the mutant receptor according to the standard protocols for
radioligand binding. Non-specific binding was determined in the presence of 10 µM
haloperidol. Data analysis was performed by normalizing the receptor bound
radioactivity derived from unspecific binding equal to 0% and total binding equal to
100%. Average values were calculated from two to four individual experiments each
done in quadruplicate and displayed in % SD.
4.1.Molecular Docking
Docking studies were performed using a recently published homology model of
D2R.27 We introduced the L2.64C mutation utilizing UCSF Chimera41 at which we
selected the most probable rotamer of the dunbrack rotamer library.42 The receptor

structure
was
protonated
by
means
of
the
H++
server
(http://biophysics.cs.vt.edu/H++)43 for pH 7 applying the default parameters. The
investigated compounds 3 and 4 were geometry-optimized by means of Gaussian
0944 at the B3LYP/6-31G(d) level (attributing a formal charge of +2 and +1,
respectively) and subsequently docked into the modified D2R model using AutoDock
Vina.45 We applied a search space of 26 Å × 24 Å × 30 Å to ensure a complete
coverage of the binding pocket. The ligands were subjected to the docking procedure
using an exhaustiveness value of 32 and a randomly selected starting position.
Twenty conformations of each ligand were obtained and inspected manually. On the
basis of the scoring function of AutoDock Vina and the presence of the canonical salt
bridge to Asp3.32, we selected one final conformation for each ligand. The ligand-
receptor complexes were submitted to an energy minimization procedure as
described.27 Figures were prepared using UCSF Chimera.
Acknowledgements
This work has been supported by the International Graduate Program
(Internationales Doktorandekolleg, IDK) “Receptor Dynamics: Emerging Paradigms
for Novel Drugs” of the Elite network of Bavaria (Elitenetzwerk Bayern) and the
Deutsche Forschungsgemeinschaft (DFG GRK1910, DFG Gm 13/10). We thank Ralf
for helpful discussions.
Supplementary data
Supplementary data associated with this article can be found in the online version.
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