Design of iodinated radioligands for SPECT imaging of central human 5-HT4R using a ligand lipophilicity efficiency approach

Article abstract of DOI:10.1016/j.bioorg.2020.103582

A series of iodinated ligands for the SPECT imaging of 5-HT₄ receptors was designed starting from the previously reported hit MR-26132. We focused on the modulation of the piperidine-containing lateral chain by introducing hydrophilic groups in order to decrease the liphophilicity of the new ligands. All the synthesized compounds were tested for their binding affinities on 5-HT₄Rs and based on the Ligand Lipophilicity Efficiency approach, compound 13 was further selected for radioiodination with iodine-125 and imaging experiments. Compound 13 showed its ability to displace the specific signal of the reference compound [¹²⁵I]SB-207710 but no significant detection of [¹²⁵I]13 was observed in vivo in SPECT experiments.

Full text of DOI:10.1016/j.bioorg.2020.103582

Bioorganic Chemistry 96 (2020) 103582
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design of iodinated radioligands for SPECT imaging of central human 5-
HT R using a ligand lipophilicity efficiency approach
4
a
b
a
a
c
Victor Babin , Benjamin B. Tournier , Audrey Davis , Emmanuelle Dubost , Gilbert Pigrée ,
d
d
a,c,e
f
Jean-François Lohier , Vincent Reboul , Thomas Cailly
, Jean-Philippe Bouillon ,
b,g
a,⁎
Philippe Millet , Frédéric Fabis
a
b
c
Normandie Univ, UNICAEN, Centre d’Etudes et de Recherche sur le Médicament de Normandie (CERMN), 14000 Caen, France
Division of Adult Psychiatry, Department of Psychiatry, University Hospitals of Geneva, Switzerland
Normandie Univ, UNICAEN, IMOGERE, 14000 Caen, France
d
Normandie Univ, Laboratoire de Chimie Moléculaire et Thioorganique, UMR CNRS 6507, INC3M, FR 3038, ENSICAEN & Université de Caen-Normandie, 14050 Caen,
France
e
Department of Nuclear Medicine, CHU Côte de Nacre, 14000 Caen, France
f
Normandie Univ, INSA Rouen, UNIROUEN, CNRS, COBRA (UMR 6014), 76000 Rouen, France
g
Department of Psychiatry, University of Geneva, Switzerland
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of iodinated ligands for the SPECT imaging of 5-HT receptors was designed starting from the previously
4
Serotonin
reported hit MR-26132. We focused on the modulation of the piperidine-containing lateral chain by introducing
SPECT imaging
hydrophilic groups in order to decrease the liphophilicity of the new ligands. All the synthesized compounds
5
-HT
4
were tested for their binding affinities on 5-HT Rs and based on the Ligand Lipophilicity Efficiency approach,
4
Radio-iodination
LLE
compound 13 was further selected for radioiodination with iodine-125 and imaging experiments. Compound 13
1
25
showed its ability to displace the specific signal of the reference compound [ I]SB-207710 but no significant
detection of [1 I]13 was observed in vivo in SPECT experiments.
25
1
. Introduction
Serotonin (5-hydroxytryptamine, 5-HT) plays a pivotal role in the
psychiatric disorders such as depression [13], anorexia [14] or age
associated memory impairment such as in Alzheimer’s Disease [15,16].
In this context, design of radioligands able to target the 5-HT R can
4
control of physiological functions by interacting with seven 5-HT re-
ceptors families (5-HT1-7Rs) [1]. Dysregulation of 5-HT neuro-
transmission results in a number of disorders, and the better knowl-
edge of these pathophysiological ways has led to the development of
effective and selective ligands of 5-HTRs to study and modulate ser-
otonin functions [2]. Beyond these pharmacologic and therapeutic
strategies, there is a growing interest in using molecular imaging tools
such as radiotracers allowing to study in-vivo in a non-invasive way
the physiological and pathological role of the serotonergic system
provide effective tools to detect and monitor related dysfunctions using
non-invasive techniques such as Positron Emission Tomography (PET)
or Single Photon Emission Computed Tomography (SPECT). To date,
three radioligands targeting 5-HT Rs were described in the literature
4
1
23
(Fig. 1). [ I]SB-207710, developed for SPECT imaging, was the first
radioligand able to label in vivo the 5-HT R rich areas in monkey but,
4
due to low brain penetration, no further investigations were reported
1
1
[17]. The PET antagonist radioligand [ C]SB-207145, structurally
related to SB-207710, has been successfully used for in vivo studies in
[
3,4].
Among these receptors, the 5-HT
pressed in both peripheral and central nervous system. In Peripheral
human [18,19]. However, the short half-life of the radioisotope (t =
½
4
R, discovered in 1988 [5], is ex-
20.4 min) significantly restricted its use for more advanced works in
18
clinic. More recently, [ F]MNI-698 [20–22], the fluorinated analogue
regions, the 5-HT
4
R can be found in the heart, the gastrointestinal tract,
of SB-207145 also exhibited its abilities to label in vivo the 5-HT R rich
4
the adrenal glands and the urinary bladder [6] and have been related to
gastrointestinal disorders [7,8], heart failures [9–11] and hyper-
areas in monkey with a suitable brain penetration but its use remains
limited due to the short metabolic stability as all compounds of this
class caused by esterases.
aldosteronism [12]. Central 5-HT Rs have been found to be involved in
4
⁎
Corresponding author.
E-mail address: frederic.fabis@unicaen.fr (F. Fabis).
https://doi.org/10.1016/j.bioorg.2020.103582
Received 8 July 2019; Received in revised form 3 December 2019; Accepted 11 January 2020
Available online 13 January 2020
0045-2068/ © 2020 Elsevier Inc. All rights reserved.

V. Babin, et al.
Bioorganic Chemistry 96 (2020) 103582
Fig. 1. Previously described central 5-HT R radiotracers.
4
Fig. 2. Previous work concerning (diaza)phenanthridines 5-HT R radioligands and targeted structures.
4
Previously, our group reported the synthesis of radioiodinated li-
gands of the 5-HT R based on a iodophenanthridine scaffold [23]
Fig. 2). Even if these compounds demonstrated high affinity and se-
lectivity toward the 5-HT R, none of the synthesized radioligands in
this series were able to label 5-HT R-containing regions in rodent’s
brain. In a second study [24], our group pointed out that 5-HT R li-
gands with reduced lipophilicities in the diazaphenanthridine series
could also act as potent and selective 5-HT R ligands. Among the
evaluated radioligands in this work, compound MR-26132, demon-
strated, in SPECT in vivo studies, its ability to label 5-HT R-containing
regions in rat brain along with off-target labelling. Considering the high
affinity for the 5-HT R of MR-26132 and its selectivity toward the
Thus, aminoalkyl chains containing 2–4 carbons were introduced by
reacting the amine 3 and commercial N-bromoalkyl-phthalimides. The
corresponding phthalimides 4 and 5 were deprotected with hydrazine
to give the primary amines 7 and 11, which were reacted with one
equivalent of methanesulfonyl chloride to led to the sulfonamides 8 and
12. The 4-carbons sulfonamide 15 was obtained from 6 without iso-
lating the corresponding amine. Using two equivalents of methane-
sulfonyl chloride from 11 gave the disulfonimide 14. Acetamide ligands
9 and 13 were prepared from 7 and 11 using acetic anhydride whereas
the sulfamate ligand 10 was synthesized using N,N-dimethylsulfamoyl
chloride as reactant from 7. A hydroxypropyl chain was introduced by
reacting the commercially available 3-bromopropoxy-tert-butyldi-
methylsilane with 3 to afford compound 16 which was subsequently
deprotected by treatment with TBAF to afford the expected alcohol 17.
Finally, sulfur derivatives were investigated. Reaction of 3-(methylthio)
4
(
4
4
4
4
4
4
other 5-HTRs, we hypothesize that the lipophilicity of MR-26132 could
be even more finely tuned to abolish the off-target labelling probably
due to unspecific interactions. In the present manuscript, a new gen-
eration of iodinated diazaphenanthridine ligands with reduced lipo-
philicity were designed. We chose to introduce nitrogen-, oxygen- and
sulfur-based hydrophilic groups on the piperidine moiety of our scaffold
to decrease the LogP within a 1–3 range. Here, we report the synthesis
and evaluation of twenty-five new iodinated compounds following this
strategy. To better address the problem of candidate selection for the
imaging experiments, the Ligand Lipophilicity Efficiency (LLE) ap-
propionaldehyde with 3 in the presence of NaBH(OAc)
3
gave the
thioether 18. Thus, oxidation of the sulfur atom gave sulfone 19 using
m-CPBA and sulfoximine 20 using PhI(OAc) in the presence of am-
2
monium carbamate [27].
Oxane and piperidine groups were then added on the piperidine
moiety of 3 (Scheme 3). Ligand 21, containing an oxane scaffold, was
synthesized from 3 using 1,6-dioxaspiro[2.5]octane, previously pre-
pared according to a literature procedure [28]. Reaction of tert-butyl 4-
(iodomethyl)piperidine-1-carboxylate [29], with amine 3 followed by
Boc deprotection provided the bis-piperidyl ligand 22. The NH terminal
piperidine of 22 was then functionalized as sulfonamide 23, amide 24
and phosphoramidate 25 moieties through reactions with respectively
mesyl chloride, acetic anhydride and diethyl chlorophosphate.
proach [25] has been used by correlating lipophilicity and 5-HT
for each compound.
4
R pKi
2
. Results and discussion
.1. Chemistry
All the ligands were synthesized from the common precursor 3,
2
2.2. Binding and LLE studies
which was obtained following a previously established synthetic route
[
24]. Starting from iodopyrazino[2,3-c]quinolin-5(6H)-one 1 [26], the
Binding affinities of the synthesized compounds were evaluated
−8
−
6
expected compound was isolated after successive chlorodehydroxyla-
tion using the Vilsmeier-Haack reagent and nucleophilic substitution
with tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate (Scheme 1).
Boc deprotection with TFA afforded the amine 3.
toward human 5-HT
4
R as inhibition percentages at 10
M and 10
M, followed by K
i
determination. Lipophilicity (clogP) was simulated
based on MarvinSketch® software (version 5.2.6) and LLE was calcu-
lated for each compound by substracting cLogP to pK . Results are
i
We first designed several ligands where hydrophilic groups were
introduced by means of a 2–4 carbons alkyl spacer moiety (Scheme 2).
summarized in Table 1 and compared to MR-26132, the 5-HT R control
4
ligand for this study.
2

V. Babin, et al.
Bioorganic Chemistry 96 (2020) 103582
Scheme 1. Synthesis of precursor 3.
Reagents and conditions: (i) (COCl) ,
2
DMF, CHCl , reflux, 2 h; (ii) tert-butyl
3
4
-(hydroxymethyl)piperidine-1-carbox-
ylate, NaH, DMF, rt, 2 h, 64% over 2
steps; (iii) (a) TFA, DCM, 2 h, rt; (b)
K
3
PO , rt, 30 min; 98%.
4
In vitro results showed that most of the synthesized ligands dis-
conditions while fitting in the dashed area. Among these compounds,
ligand 13 exhibiting the highest pKi value was selected as a candidate
for radiolabelling.
played a binding affinity toward the 5-HT
4
R below 30 nM (pK > 7.6)
i
excluding the bulky phthalimide derivatives 4–6. Among them, eleven
compounds such as amine derivatives 8, 9, 11, 13, 15 and 22 as well as
sulfur derivatives 19 and 20 and alcohol derivatives 17 and 21 ex-
hibited higher affinities than the previous reference (MR-26132,
Prior to radiolabeling and imaging experiments, selectivity toward
other 5-HT receptors and functional profile of compound 13 were as-
sessed (Table 2). Binding affinities of 13 were evaluated toward a panel
−6
K
i
= 17.7 nM). The highest affinity was obtained with an acetamide
of 5-HT receptors as inhibition percentages at 10 M and results show
a slight affinity toward 5-HT2b and 5-HT2c receptors. Inhibition per-
hydrophilic group linked by a propyl spacer (13, K = 3.9 nM).
i
Moreover, the length of the carbon chain does not seem to have a
centage of agonist and antagonist response toward 5-HT R were also
4
crucial effect on binding results. The Ki of the sulfonamides were rather
measured and revealed that 13 is 5-HT R antagonist.
4
identical with an ethyl spacer (8, K = 8.5 nM), a propyl spacer (12,
i
K
i
= 17.7 nM) or a butyl spacer (15, K
i
= 11.2 nM). Interestingly,
2.3. Radiochemistry
ligands with bulky groups, as described in a recent study [30–32], such
The preparation of [1 I]13 started from 13, which was first stan-
nylated using a palladium-catalyzed stannylation procedure to give the
tin precursor 26 [34] (Scheme 4). Then, iodine-125 was introduced
25
as piperidine 22–24 or oxane 21 also exhibited high affinity toward 5-
HT . As expected, lipophilicity simulation showed that introduction of
4
heteroatoms significantly decreases the lipophilicity. Compared to MR-
1
25
2
6132 (clogP
=
4.19), introduction of
a
hydroxy group (17,
after a tin-iodine exchange using [ I]NaI as the source of the radio-
isotope and hydrogen peroxide as the oxidant in acidic medium. Fi-
clogP = 2.68) or amino group (11, clogP = 2.57) considerably de-
creases the hydrophobicity of the ligand. Chemical modulations of the
amino group lead to the lowest values of clogP with a minimum for the
disulfonimide group (14, clogP = 1.37). Interestingly, sulfur deriva-
tives such as sulfone (19, clogP = 1.79) turned out to be an attractive
function to decrease lipophilicity.
1
25
nally, HPLC purification afforded the radio-iodinated compound [ I]
13 with a radiochemical yield of 81% and a molar activity greater than
100 GBq/μmol.
2.4. In vitro competition experiments 13 versus SB-207710
In order to select the best candidate for radioiodination and SPECT
imaging experiments, we choose to use the Ligand Lipophiliy Efficiency
To determine the specificity of compounds 13 and MR-26132 to
(
LLE), a valuable decision aid tool for medicinal chemistry [33]. In vitro
bind to the 5-HT receptor, a competition experiment with the re-
4
binding results toward 5-HT
4
R (pK
i
), calculated lipophilicity (clogP)
ference ligand SB-207710 was performed on human brain hippocampal
sections (Fig. 4). A displacement experiment was performed and was
analyzed by the measurement of the SBR (Specific Binding Ratio) which
corresponds to the ratio minus one between the radioactivity measured
on a section in the presence of the radioactive compound alone and the
radioactivity measured on a section in the presence of the radioactive
compound and the tested compound. An example of displacement of
and ligand lipophilicity efficiency defined as LLE = pKi - cLogP were
reported on the plot (Fig. 3). The dashed area was defined on the plot to
select the more convenient structure for the radiolabeling using three
parameters: an adequate lipophilicity (1 < clogP < 3), a suitable
binding affinity toward the receptor (pK > 8) and an appropriate
i
Ligand Lipophilicity Efficiency (5 < LLE < 7). For comparison, MR-
1
25
2
6132 and SB-207710 (pK
i
= 8.66 and clogP = 3.2 [9]) were also
the binding of [ I]SB-207710 by 13 and MR-26132 is given in
Fig. 4A–C. The SBR in the different areas of the hippocampus shows
that compounds 13 and MR-26132 induce a shift in [1 I]SB-207710
represented on the plot. Among the twenty-two compounds represented
25
on the plot, 8, 11, 13, 17 and 20 are within the three required
Scheme 2. Synthesis of ligands 4–20. Reagents and
conditions: (i) Br(CH
2
)
n
NPhth, NEt , MeCN, reflux,
3
2
4 h, 68–85%; (ii) (a) N
2
H
4
, EtOH, reflux, 2 h; (b)
, DCM, 0 °C to rt or Ac O, DCM, 0 °C to rt
Cl, NEt , DCM, 0 °C to rt; for amine 7
MsCl, NEt
3
2
or Me
2
NSO
2
3
and 11, a fumarate salt was prepared using fumaric
acid, iPrOH, 40 °C,
1
h; 47–81%; (iii) Br
, MeCN, reflux, 16 h, 63%;
iv) TBAF, THF, rt, 16 h, 49%; (v) 3-(methylthio)
, DCM, rt,
h, 57%; (vi) mCPBA, DCM, rt, 2 h, 66%; (vii)
NH COONH , PhI(OAc) , MeOH, rt, 16 h, 57%.
(
CH
2
)
3
OTBDMS, NEt
3
(
propionaldehyde, AcOH, NaHB(OAc)
3
3
2
4
2
3

V. Babin, et al.
Bioorganic Chemistry 96 (2020) 103582
Scheme 3. Synthesis of ligands 21–25. Reagents
and conditions: (i) 1,6-dioxaspiro[2.5]octane, NEt ,
3
MeOH, reflux, 16 h, 81%; (ii) tert-butyl 4-(iodo-
methyl)piperidine-1-carboxylate, NEt , MeCN, re-
PO , rt,
, DCM,
O, DCM, 0 °C to RT,
, NEt , DCM, RT,
3
flux, 16 h, (iii) (a) TFA, DCM, 2 h, rt; (b) K
3
4
3
0
1
1
0 min, 48% over 2 steps; (iv) MsCl, NEt
3
°C to RT, 10 min, 89%; (v) Ac
2
0 min, 93%; (vi) ClP(O)(OEt)
2
3
2 h, 29%.
Table 1
Binding Affinities toward human 5-HT R and LLE calculation for MR26132 compound 3–25.
4
Ligand
n
R
% inh (10−6 M/10−8 M)a
Ki (5-HT
4
)b
clogPc
LLEd
MR-26132
3
0
2
3
4
2
2
2
2
3
3
3
3
4
3
3
3
3
3
1
H
100/68
100/39
100/37
100/18
100/56
100/84
100/100
100/95
100/84
100/76
100/68
100/94
100/49
100/96
100/62
100/93
100/100
100/93
100/82
100/84
17.7 ± 4.1 nM
36.8 ± 4.7 nM
543 ± 195 nM
312 ± 57 nM
4.19
2.93
4.06
4.12
4.63
2.51
1.83
2.33
1.82
2.57
1.89
2.39
1.37
2.40
5.32
2.68
4.32
1.79
2.82
2.50
3.56
4.50
2.21
2.38
n.m.
5.23
6.24
5.48
5.83
5.44
5.86
6.02
5.87
5.86
2.36
5.40
3.39
6.11
5.20
5.27
3
4
5
6
7
8
9
H
Nphth
Nphth
Nphth
e
n.m.
NH
2
18.3 ± 9.4 nM
8.5 ± 6.8 nM
15.4 ± 2.3 nM
22.6 ± 4.7 nM
9.7 ± 1.7 nM
17.7 ± 2.4 nM
3.9 ± 1.3 nM
56.7 ± 10.8 nM
11.2 ± 4.6 nM
21.4 ± 8.8 nM
8.4 ± 3.9 nM
19.7 ± 2.3 nM
12.4 ± 10.3 nM
9.5 ± 1.5 nM
17.0 ± 2.0 nM
NHSO
NHAc
NHSO
2
2
2
Me
1
1
1
1
1
1
1
1
1
1
2
2
0
1
2
3
4
5
6
7
8
9
0
1
NMe
Me
2
NH
2
NHSO
NHAc
N(SO
2
2
Me)
2
NHSO
Me
OTBDMS
OH
SMe
2
SO Me
S(O)(NH)Me
2
2
2
3
1
1
100/94
100/76
10.5 ± 3.3 nM
28.0 ± 5.4 nM
3.41
2.52
4.57
5.03
2
2
4
5
1
1
100/93
100/53
22.6 ± 4.7 nM
n.m.
3.02
3.93
4.42
n.m.
a
b
c
d
e
Inhibition percentages were measured using human 5-HT
Ki were measured using human 5-HT R.
4
R.
4
Calculated logP were obtained using MarvinSketch® software (version 5.2.6).
LLE was obtained by subtracting cLogP to pK
i
for each compounds (LLE = pK – cLogP).
i
n.m. = not measured.
4

V. Babin, et al.
Bioorganic Chemistry 96 (2020) 103582
Nevertheless, the low SBR obtained in the mirror experiment could be
correlated to a high unspecific binding of these two compounds which
are not displaced by SB-207710 at high concentration.
2
.5. SPECT in vivo imaging with [125I]13
In order to determine the ability of compound 13 to bind in vivo to
the 5-HT receptor, a SPECT imaging experiment was performed. After
4
125
the intravenous injection of [ I]13, radioactivity levels were mon-
itored in the hippocampus and striatum, two 5-HT -rich regions
Fig. 5). An early peak corresponding to the passage of the blood in the
4
(
brain is measured. This peak is followed by an immediate return to
residual values, demonstrating the absence of a specific signal in the
brain. This result shows that compound 13 is either unable to cross the
blood brain barrier or strongly bound to plasma proteins leading to a
very low concentration in the brain. A quick metabolism cannot be
excluded nor a high excretion of the radioligand by Pgp (permeability
glycoprotein) [35].
3
. Conclusion
Starting from the previously reported iodinated 5-HT R ligand MR-
4
2
6132 which showed a too high unspecific binding in SPECT imaging
experiments, we designed new ligands by adding on the lateral chain of
piperidine diverse hydrophilic groups in order to decrease the lipo-
philicity of MR-26132. We were able to obtain new high affinities io-
Fig. 3. cLogP versus pKi plot (LLE = pKi-cLogP).
dinated 5-HT R ligand with decreased LogP. Compound 13 has been
4
Table 2
selected using the Ligand Lipophilicity Efficiency to be radioiodinated.
Selectivity and functional profile of compound 13.
Its ability to displace the specific signal of the reference radioligand
5
-HTR
% (10−6 M of 13)
[
125
I]-SB207710 has been demonstrated in vitro, but this compound
exhibits a too high off-target unspecific binding as proved by the dis-
placement experiments. Moreover, this compound exhibits a very low
signal to noise ratio in in vivo SPECT imaging experiments. Overall,
even if the LLE has allowed here the selection of a ligand able to dis-
place in vitro a specific radioligand, we think that this approach should
be adjusted and/or completed to better suit to the CNS radio-imaging
context.
-HT1aa
9.9
d
5
5
5
5
5
5
5
5
5
5
5
5
b
d
-HT1b
6.7
c
a
a
d
d
d
d
d
-HT1d
12.1
43.4
75.6
71.5
16.8
-HT2a
-HT2b
a
-HT2c
a
-HT
3
a
d
d
-HT5a
−3.8
−3.1
a
-HT
-HT
-HT
-HT
6
7
4
4
a
a
a
d
10.7
4. Experimental
e
agonist effect
antagonist effect
−4.1
f
102
4
.1. Chemistry
a
b
c
d
e
Test performed on human recombinant CHO cells.
Test performed on rat cerebral cortex.
4.1.1. General experimental information
Test performed on rat recombinant CHO cells.
All solvents and chemicals were used as purchased unless stated
otherwise. All NMR spectra were recorded on Bruker Avance III 400
spectrometer. Proton and carbon-13 NMR spectra are reported as che-
mical shifts (δ) in parts per million (ppm). Coupling constants (J) are
reported in units of hertz (Hz). The following abbreviations are used to
describe multiplets: s (singlet), d (doublet), t (triplet), q (quartet), m
Inhibition percentages were determined at CEREP (n = 2).
Percentage of agonist response was determined at CEREP
(
n = 2).
f
Inhibition percentage of agonist response was determined at
CEREP (n = 2).
(
multiplet), br (broad). High resolution mass spectra (HRMS, m/z) were
recorded on a Waters Acquity UPLC H-ClassXevo G2-XS spectrometer
using positive electrospray ionization (ESI). Infrared spectra were re-
which is even greater than when using SB-207710. The mirror ex-
1
25
125
periment using [ I]13 or [ I]MR-26132 with SB-207710 shows a
lower capacity of the reference compound to displace the signal due to
the compounds tested. Taken together, these results show that both 13
corded using
a
Perkin-Elmer Spectrum 65 FT-IR spectrometer.
−1
Absorptions are reported in wavenumbers (cm ) and only peaks of
interest are reported. Melting points of solids were measured on a
Stuart Automatic Melting point SMP-50 apparatus. Flash column
chromatography was performed over silica gel C60 (40–60 μm) or RP-
and MR-26132 bind to the 5-HT Rs and are more potent than SB-
4
125
2
07710 to displace the specific binding of
[
I]SB-207710.
Scheme 4. Radiolabeling of 13. Reagents and conditions: (i) Pd
2
dba
3
, (SnBu
3
)
2
, DIPEA, iPrOH, 70 °C, 48 h, 47%; (ii) [125I]NaI, H
2
O , AcOH, MeCN, rt, 40 min.
2
5

V. Babin, et al.
Bioorganic Chemistry 96 (2020) 103582
Fig. 4. Competition experiment between 13 and
MR-26132 and the reference compound SB-
2
07710. Representative examples of binding in
125
human brain slices when exposed to [ I]SB-
2
07710 alone (A) or in the presence of 13 (B) or
MR-26132 (C). (D) SBR were calculated in dif-
ferent brain areas and using different couples of
radioactive/cold molecule as indicated in the
figure.
1
8 (50 μm) using eluent systems as described for each experiment.
the filtrate was dried over MgSO . The resulting solution was filtered
4
Unless otherwise specified, all reagents were obtained from commercial
suppliers.
and evaporated to give the primary amine. 7-iodopyrazino[2,3-c]qui-
nolin-5(6H)-one was obtained according to a described procedure [26].
4
.1.2. General method 1: Removal of phthalimide protection
4.1.3. tert-Butyl
4-[((7-iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl]
In a round bottom flask to a suspension of phthalimide derivative
piperidine-1-carboxylate 2
(
1.00 equiv.) in ethanol (15 mL/mmol), hydrazine monohydrate (15.0
In a round bottom flask, oxalyl chloride (2.2 mL, 25.5 mmol) and
DMF (2.00 mL, 26.4 mmol) were added to chloroform (20 mL) at 0 °C
and the solution was stirred during 3 h at room temperature. 7-
Iodopyrazino[2,3-c]quinolin-5(6H)-one 1 (800 mg, 2.47 mmol) was
added to the mixture and the solution was refluxed for 2 h. The re-
sulting mixture was carefully hydrolysed with water and the pH was
adjusted to 10 using an aqueous ammonia solution (28%). The resulting
equiv.) was added and the solution was stirred under reflux until the
solubilisation of the mixture. The solution was cooled to room tem-
perature and filtered. The precipitate was washed with di-
chloromethane (10 mL/mmol of phthalimide derivative) and the fil-
trate was evaporated. The crude mixture was dissolved in
dichloromethane (10 mL/mmol of phthalimide derivative), filtered and
Fig. 5. SPECT experiment with [125I]13. Coronal (A), sagittal (B) and axial (C) planes of in vivo [ I]13 SPECT image (10–55 min post-injection) showing the
125
absence and the probable accumulation of [1 I]13 in the brain and the pituitary, respectively. (D) Time-activity-curves for the striatum and the hippocampus.
25
6

V. Babin, et al.
Bioorganic Chemistry 96 (2020) 103582
solution was extracted with dichloromethane (3 × 30 mL), dried over
4.1.6. 2-(3-(4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)
MgSO
4
, filtered and evaporated affording the expected chlorimine. In a
piperidin-1-yl)propyl)-2,3-dihydro-1H-isoindole-1,3-dione 5
round bottom flask, NaH (108 mg, 2.72 mmol) was added to a solution
In a round bottom flask, compound 3 (240 mg, 0.57 mmol) was
dissolved in acetonitrile (15 mL) and N-(3-bromopropyl)phthalimide
(185 mg, 0.70 mmol) and triethylamine (0.19 mL, 1.4 mmol) were
added. The solution was stirred at 80 °C for 12 h and the white pre-
cipitate formed was filtered, washed with acetonitrile (2 × 20 mL)
of tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate (585 mg,
2
.72 mmol) in dry DMF (30 mL) under N at 0 °C. The reaction mixture
2
was stirred for 15 min, warmed to room temperature and was stirred for
5 min. The chlorimine previously obtained above was added and the
reaction mixture was stirred at room temperature overnight. Water
30 mL) was carefully added and the resulting mixture was extracted
with ethyl acetate (3 × 30 mL), dried over MgSO , filtered and eva-
1
affording the expected product without further purification as a white
1
(
solid (284 mg, 81%). H NMR (400 MHz, CDCl
3
): δ = 9.07 (d,
3
3
3
4
4
J = 2.0 Hz, 1H), 9.00 (d, J = 2.0 Hz, 1H), 8.90 (dd, J = 1.4 Hz,
4
3
porated. The crude product was then purified by silica gel chromato-
J = 8.0 Hz, 1H), 8.34 (dd, J = 1.4 Hz, J = 7.6 Hz, 1H), 7.87–7.81
3
graphy using CH
2
Cl
2
/AcOEt (100/0 to 80/20) as eluent affording the
(m, 2H), 7.71–7.67 (m, 2H), 7.31 (t, J = 7.8 Hz, 1H), 4.59 (d,
1
3
3
3
expected product (835 mg, 64%) as a light orange solid. H NMR
J = 7.0 Hz, 2H), 3.76 (t, J = 6.9 Hz, 2H), 2.95–2.87 (m, 2H), 2.41 (t,
3
3
3
(
400 MHz, CDCl
3
): δ = 9.09 (d, J = 2.0 Hz, 1H), 9.01 (d, J = 2.0 Hz,
J = 6.9 Hz, 2H), 2.24–2.11 (m, 1H), 1.95–1.83 (m, 6H), 1.33–1.20 (m,
3
4
13
1
H), 8.91 (dd, J = 8.0 Hz, J = 1.4 Hz, 1H), 8.35 (dd, J = 7.6 Hz,
2H). C NMR (100 MHz, CDCl
3
): δ = 168.6 (2C), 158.6, 147.8, 145.9,
4
3
3
J = 1.4 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 4.72 (d, J = 6.8 Hz, 2H),
145.5, 143.9, 141.5, 134.0 (2C), 132.5 (2C), 131.1, 126.6, 124.7,
4
.26–4.07 (m, 2H), 2.85–2.70 (m, 2H), 2.49–2.36 (m, 1H), 2.00–1.93
13
123.4, 123.3 (2C), 100.7, 72.9, 56.7, 53.5 (2C), 37.0, 35.1, 29.4 (2C),
−1
(
m, 2H), 1.47 (s, 9H), 1.46–1.34 (m, 2H). C NMR (100 MHz, CDCl
3
):
25.6. IR (KBr): 2942, 1716, 1576, 1432, 1382, 1330, 1053 cm . mp:
+
δ = 158.6, 155.0, 147.9, 145.9, 145.5, 143.8, 141.6, 131.1, 126.7,
204–205 °C. HRMS/ESI: calculated for
608.1159, found 608.1164.
C
28
H
27IN
5
O
3
[M+H]
1
24.8, 123.4, 100.7, 79.5, 72.4, 43.8 (2C), 35.6, 29.2 (2C), 28.6 (3C).
−1
IR (KBr): 2969, 2936, 1692, 1423, 1139, 1047 cm . mp 174–176 °C.
+
HRMS/ESI: calculated for C22
H
25IN
4
NaO
3
[M+Na] 543.0869, found
4.1.7. 2-(3-(4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)
piperidin-1-yl)butyl)-2,3-dihydro-1H-isoindole-1,3-dione 6
5
43.0870.
In a round bottom flask, compound 3 (292 mg, 0.70 mmol) was
dissolved in acetonitrile (20 mL) and N-(4-bromopbutyl)phthalimide
4
.1.4. 4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)piperidine 3
(
240 mg, 0.85 mmol) and triethylamine (0.19 mL, 1.4 mmol) were
In a round bottom flask, 2 (500 mg, 0.96 mmol) was dissolved in
added. The solution was stirred at 80 °C for 24 h and the white pre-
cipitate formed was filtered, washed with acetonitrile (2 × 20 mL)
dichloromethane (20 mL). Trifluoroacetic acid (1.10 mL, 14.4 mmol)
was added and the solution was stirred at room temperature. After 2 h
the solvent was evaporated in vacuo and the crude product was dis-
affording the expected product without further purification as a white
1
solid (300 mg, 68%). H NMR (400 MHz, CDCl
3
): δ = 9.06 (d,
solved in a saturated solution of K
3
PO (20 mL). The solution was
4
3
3
3
4
4
J = 2.0 Hz, 1H), 8.99 (d, J = 2.0 Hz, 1H), 8.87 (dd, J = 8.0 Hz,
stirred 30 min at room temperature and the resulting mixture was ex-
3
J = 1.4 Hz, 1H), 8.31 (dd, J = 7.6 Hz, J = 1.4 Hz, 1H), 7.84–7.78
tracted with dichloromethane (3 × 15 mL). The combined organics
3
(
m, 2H), 7.72–7.65 (m, 2H), 7.26 (t, J = 7.8 Hz, 1H), 4.70 (d,
layers were dried over MgSO , filtered and evaporated to obtain the
4
3
3
J = 6.8 Hz, 2H), 3.70 (t, J = 7.1 Hz, 2H), 3.01–2.93 (m, 2H),
expected product without further purification as
a
yellow solid
2
.41–2.33 (m, 2H), 2.30–2.18 (m, 1H), 2.04–1.91 (m, 4H), 1.74–1.64
13
1
3
(
395 mg, 98%). H NMR (400 MHz, CDCl
3
): δ = 9.04 (d, J = 2.0 Hz,
(
m, 2H), 1.60–1.46 (m, 4H). C NMR (100 MHz, CDCl ): δ = 168.5
3
3
3
4
1
8
H), 8.97 (d, J = 2.0 Hz, 1H), 8.84 (dd, J = 8.0 Hz, J = 1.4 Hz, 1H),
(
2C), 158.6, 147.8, 145.8, 145.4, 143.8, 141.5, 134.0 (2C), 132.2 (2C),
3
4
3
.30 (dd, J = 7.6 Hz, J = 1.4 Hz, 1H), 7.26 (t, J = 7.8 Hz, 1H), 4.68
1
3
1
31.1, 126.6, 124.7, 123.3, 123.3 (2C), 100.7, 72.7, 58.6, 53.6 (2C),
8.0, 35.2, 29.3 (2C), 26.8, 24.5 IR (KBr): 2930, 1708, 1592, 1400,
3
(
(
d, J = 6.8 Hz, 2H), 3.18–3.09 (m, 2H), 2.74–2.60 (m, 2H), 2.42–2.30
1
3
m, 1H), 1.99–1.88 (m, 3H), 1.45–1.33 (m, 2H). C NMR (100 MHz,
−1
351, 1176, 719 cm . mp: 154–158 °C. HRMS/ESI: calculated for
CDCl
3
): δ = 158.6, 147.8, 145.8, 145.4, 143.7, 141.4, 131.1, 126.5,
C
29
H29IN
5
O
3
[M+H]+ 622.1323, found 622.1315.
1
3
24.6, 123.2, 100.7, 73.0, 46.4 (2C), 35.7 (2C), 30.5. IR (KBr): 3435,
−1
392, 2910, 1592, 1452, 1349, 1178, 1045 cm . mp: 178–180 °C.
4
.1.8. 2-(4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)piperidin-
-yl)ethan-1-amine, fumarate salt 7
+
HRMS/ESI: calculated for C17
H18IN
4
O [M+H] 421.0525, found
1
4
21.0525.
Starting from compound 4 (90 mg, 0.15 mmol) and using General
method 1, the resulting amine was dissolved in propan-2-ol (10 mL)
and fumaric acid (17 mg, 0.15 mmol) was added. The solution was
stirred at 40 °C for 1 h and the beige precipitate formed was filtered,
washed with propan-2-ol (3 × 5 mL) affording the expected product
4
.1.5. 2-(3-(4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)
piperidin-1-yl)ethyl)-2,3-dihydro-1H-isoindole-1,3-dione 4
In a round bottom flask, compound 3 (712 mg, 1.70 mmol) was
dissolved in acetonitrile (30 mL) and N-(2-bromoethyl)phthalimide
1
without further purification as a beige solid (71 mg, 81%). H NMR
3
(
526 mg, 2.08 mmol) and triethylamine (0.45 mL, 3.50 mmol) were
(400 MHz, DMSO‑d
6
): δ = 9.24 (d, J = 2.0 Hz, 1H), 9.12 (d,
3
4
3
added. The solution was stirred at 80 °C for 24 h and the white pre-
cipitate formed was filtered, washed with acetonitrile (2 × 20 mL)
J = 2.0 Hz, 1H), 8.80 (dd, J = 8.0 Hz, J = 1.4 Hz, 1H), 8.36 (dd,
3 3
4
J = 7.6 Hz, J = 1.4 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 6.41 (s, 2H),
3
affording the expected product without further purification as a white
4.57 (d, J = 6.4 Hz, 2H), 2.95–2.85 (m, 4H), 2.52–2.48 (m, 2H),
1
solid (875 mg, 85%). H NMR (400 MHz, CDCl
3
): δ = 9.07 (d,
2.09–1.94 (m, 3H), 1.88–1.78 (m, 2H), 1.51–1.39 (m, 2H) (Signals due
3
3
3
4
13
J = 2.0 Hz, 1H), 9.00 (d, J = 2.0 Hz, 1H), 8.90 (dd, J = 1.4 Hz,
to the OH and NH
2
are missing). C NMR (100 MHz, DMSO‑d
6
):
4
3
J = 8.0 Hz, 1H), 8.33 (dd, J = 1.4 Hz, J = 7.6 Hz, 1H), 7.87–7.81
δ = 168.2 (2C), 158.5, 148.8, 146.4, 144.6, 142.9, 141.0, 135.3 (2C),
130.3, 126.8, 124.1, 122.9, 100.9, 71.4, 55.0, 52.8 (2C), 35.9, 34.9,
28.6 (2C). IR (KBr): 3434, 2928, 1594, 1461, 1370, 1310, 1174,
3
(
m, 2H), 7.73–7.68 (m, 2H), 7.29 (t, J = 7.9 Hz, 1H), 4.68 (d,
3
3
J = 6.9 Hz, 2H), 3.84 (t, J = 7.0 Hz, 2H), 3.10–3.00 (m, 2H),
−1
2
.70–2.59 (m, 2H), 2.32–2.18 (m, 1H), 2.17–2.05 (m, 2H), 2.00–1.89
843 cm . mp: 173–175 °C. HRMS/ESI: calculated for C19
H
23IN O [M
5
1
3
+
(
m, 2H), 1.54–1.39 (m, 2H). C NMR (100 MHz, CDCl
3
): δ = 168.5
+H] 464.0952, found 464.0947.
(
2C), 158.6, 147.8, 145.9, 145.5, 143.9, 141.5, 134.0 (2C), 132.3 (2C),
1
3
1
31.1, 126.6, 124.7, 123.3, 123.3 (2C), 100.7, 72.9, 56.1, 53.5 (2C),
5.6, 35.1, 29.4 (2C). IR (KBr): 2939, 1714, 1592, 1431, 1393, 1329,
4.1.9. N-(2-(4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)
piperidine-1-yl)ethyl) Methanesulfonamide 8
−
1
177, 1046 cm
.
mp: 206–208 °C. HRMS/ESI: calculated for
Starting from compound 4 (280 mg, 0.47 mmol) and using General
method 1, the resulting amine was dissolved in dichloromethane
+
C
27
H
25IN
5
O [M+H] 594.1008, found 594.1002.
3
7

V. Babin, et al.
Bioorganic Chemistry 96 (2020) 103582
(
(
15 mL) and the solution was cooled down to 0 °C. Triethylamine
0.13 mL, 0.94 mmol) and methanesulfonyl chloride (0.03 mL,
4.1.12. 3-(4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)piperidin-
1-yl)propan-1-amine, fumarate salt 11
0
5
.47 mmol) were added to the solution and the mixture was stirred
min at 0 °C and 5 min at room temperature. The organic layer was
Starting from compound 5 (75 mg, 0.15 mmol) and using General
method 1, the resulting amine was dissolved in propan-2-ol (10 mL)
and fumaric acid (17 mg, 0.15 mmol) was added. The solution was
stirred at 40 °C for 1 h and the beige precipitate formed was filtered,
washed with propan-2-ol (3 × 5 mL) affording the expected product
washed with water (15 mL), dried over MgSO
4
, filtered and evaporated.
The residue was purified by silica gel chromatography using di-
chloromethane/methanol (100/0 to 90/10) as eluent affording the
1
1
expected product as a white solid (155 mg, 61%). H NMR (400 MHz,
without further purification as a beige solid (54 mg, 74%). H NMR
3
3
3
CDCl
3
): δ = 9.09 (d, J = 2.0 Hz, 1H), 9.02 (d, J = 2.0 Hz, 1H), 8.90
(400 MHz, DMSO‑d
6
): δ = 9.24 (d, J = 2.1 Hz, 1H), 9.12 (d,
4
3
4
3
3
4
3
(
dd, J = 8.0 Hz, J = 1.4 Hz, 1H), 8.34 (dd, J = 7.6 Hz, J = 1.4 Hz,
J = 2.1 Hz, 1H), 8.81 (dd, J = 1.4 Hz, J = 8.0 Hz, 1H), 8.37 (dd,
3
3
4
3
3
1
2
1
H), 7.31 (t, J = 7.8 Hz, 1H), 4.73 (d, J = 6.6 Hz, 2H), 3.22–3.19 (m,
H), 2.97 (s, 3H), 2.95–2.91 (m, 2H), 2.57–2.54 (m, 2H), 2.28–2.17 (m,
H), 2.14–2.11 (m, 2H), 2.09–1.96 (m, 2H), 1.57–1.49 (m, 2H) (Signal
J = 1.4 Hz, J = 7.6 Hz, 1H), 7.37 (t, J = 7.7 Hz, 1H), 6.43 (s, 2H),
3 3
4.57 (d, J = 6.6 Hz, 2H), 2.97–2.90 (m, 2H), 2.82 (d, J = 7.2 Hz,
3
2H), 2.38 (d, J = 6.7 Hz, 2H), 2.10–1.98 (m, 1H), 1.98–1.89 (m, 2H),
1
3
due to the NH is missing). C NMR (100 MHz, CDCl
3
): δ = 158.6,
1.89–1.81 (m, 2H), 1.77–1.66 (m, 2H), 1.49–1.35 (m, 2H) (Signals due
1
3
1
7
2
47.9, 145.9, 145.5, 143.8, 141.5, 131.1, 126.7, 124.7, 123.4, 100.7,
to the OH and the NH
2
are missing). C NMR (100 MHz, DMSO‑d ):
6
2.5, 57.0, 53.2 (2C), 40.2, 39.9, 35.2, 29.4 (2C). IR (KBr): 3265, 2924,
δ = 168.0 (2C), 158.5, 148.8, 146.4, 144.6, 142.9, 141.0, 135.3 (2C),
130.3, 126.8, 124.1, 122.9, 100.9, 71.4, 55.2, 52.7 (2C), 37.6, 35.0,
28.6 (2C), 24.1. IR (KBr): 3426, 2934, 1592, 1474, 1461, 1370,
−
1
826, 1596, 1314, 1112, 780 cm . mp: 147–149 °C. HRMS/ESI: cal-
+
culated for C20
H25IN
5
O
3
S [M+H] 542.0723, found 542.0723.
−
1
1
174 cm . mp: 177–180 °C. HRMS/ESI: calculated for C20
H
25IN O [M
5
+
+
H] 478.1098, found 478.1104.
4
.1.10. (2-(4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)
piperidin-1-yl)ethyl)acetamide 9
4
.1.13. N-(3-(4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)
Starting from compound 4 (190 mg, 0.32 mmol) and using General
method 1, the resulting amine was dissolved in dichloromethane
piperidin-1-yl)propyl) methanesulfonamide 12
Starting from compound 5 (150 mg, 0.25 mmol) and using General
method 1, the resulting amine was dissolved in dichloromethane
(
(
15 mL) and the solution was cooled down to 0 °C. Acetic anhydride
0.03 mL, 0.32 mmol) was added to the solution and the mixture was
(
(
20 mL) and the solution was cooled down to 0 °C. Triethylamine
0.07 mL, 0.50 mmol) and methanesulfonyl chloride (0.01 mL,
stirred 5 min at 0 °C and 5 min at room temperature. The organic layer
was washed with water (15 mL), extracted twice with dichloromethane
0
5
.25 mmol) were added to the solution and the mixture was stirred
min at 0 °C and 5 min at room temperature. The organic layer was
(
2 × 15 mL), dried over MgSO , filtered and evaporated. The residue
4
was purified by silica gel chromatography using dichloromethane/
washed with water (15 mL), dried over MgSO
4
, filtered and evaporated.
methanol (100/0 to 95/5) as eluent affording the expected product as a
The residue was purified by silica gel chromatography using di-
chloromethane/methanol (100/0 to 95/5) as eluent affording the ex-
1
3
4
white solid (100 mg, 71%). H NMR (400 MHz, CDCl
3
): δ = 9.06 (d,
3
3
3
4
J = 2.0 Hz, 1H), 8.99 (d, J = 2.0 Hz, 1H), 8.86 (dd, J = 8.0 Hz,
1
pected product as a beige solid (83 mg, 61%). H NMR (400 MHz,
3
J = 1.5 Hz, 1H), 8.31 (dd, J = 7.6 Hz, J = 1.5 Hz, 1H), 7.28 (t,
3
3
3
DMSO‑d
6
): δ = 9.19 (d, J = 1.9 Hz, 1H), 9.08 (d, J = 1.9 Hz, 1H),
3
J = 7.8 Hz, 1H), 6.21 (br s, 1H), 4.71 (d, J = 6.7 Hz, 2H), 3.38–3.31
4
4
8
.72 (dd, J = 1.3 Hz, J = 8.0 Hz, 1H), 8.31 (dd, J = 1.3 Hz,
3
(
m, 2H), 2.99–2.92 (m, 2H), 2.52–2.45 (m, 2H), 2.34–2.21 (m, 1H),
3
J = 7.6 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.02 (br s, 1H), 4.52 (d,
J = 6.5 Hz, 2H), 3.00–2.91 (m, 4H), 2.89 (s, 3H), 2.42–2.35 (m, 2H),
2
.11–2.02 (m, 2H), 1.99 (s, 3H), 1.99–1.94 (m, 2H), 1.59–1.47 (m, 2H).
3
1
3
C NMR (100 MHz, CDCl ): δ = 170.3, 158.5, 147.9, 145.8, 145.4,
3
2
.09–2.95 (m, 3H), 1.89–1.81 (m, 2H), 1.69–1.59 (m, 2H), 1.49–1.36
13
1
3
1
43.7, 141.5, 131.0, 126.6, 124.7, 123.3, 100.7, 72.6, 57.0, 53.3 (2C),
6.2, 35.1, 29.2 (2C), 23.5. IR (KBr): 3278, 2909, 1647, 1590, 1455,
(
m, 2H). C NMR (100 MHz, DMSO‑d
6
): δ = 158.3, 148.7, 146.3,
1
44.5, 142.8, 140.9, 130.2, 126.7, 124.0, 122.8, 100.9, 71.3, 55.3, 52.8
−
1
346, 1180 cm
. mp: 209–211 °C. HRMS/ESI: calculated for
+
(
2C), 40.9, 39.2, 34.8, 28.6 (2C), 26.5. IR (KBr): 3243, 2919, 1592,
C
21
H
25IN
5
O [M+H] 506.1061, found 506.1053.
2
−1
1
456, 1309, 1143, 781 cm . mp: 176–178 °C. HRMS/ESI: calculated
for C21
H
27IN
5
O
3
S [M+H]+ 556.0879, found 556.0881.
4
.1.11. ((2-(4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)
piperidin-1-yl)ethyl)sulfamoyl) dimethylamine 10
4.1.14. N-(3-(4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)
piperidin-1-yl)propyl)-acetamide 13
Starting from compound 4 (195 mg, 0.33 mmol) and using General
method 1, the resulting amine was dissolved in dichloromethane
Starting from compound 5 (250 mg, 0.41 mmol) and using General
method 1, the resulting amine was dissolved in dichloromethane
(20 mL) and the solution was cooled down to 0 °C. Acetic anhydride
(0.04 mL, 0.41 mmol) was added to the solution and the mixture was
stirred 5 min at 0 °C and 5 min at room temperature. The organic layer
was washed with water (15 mL), extracted with dichloromethane
(
15 mL) and the solution was cooled down to 0 °C. N,N-di-
methylsulfamoyl chloride (0.03 mL, 0.33 mmol) and triethylamine
(
0.09 mL, 0.66 mmol) were added to the solution and the mixture was
stirred 5 min at 0 °C and 5 min at room temperature. The organic layer
was washed with water (15 mL), extracted with dichloromethane
(
2 × 15 mL), dried over MgSO
4
, filtered and evaporated. The residue
(2 × 15 mL), dried over MgSO , filtered and evaporated. The residue
4
was purified by silica gel chromatography using dichloromethane/
was purified by silica gel chromatography using dichloromethane/
methanol (100/0 to 95/5) as eluent affording the expected product as a
methanol (100/0 to 90/10) as eluent affording the expected product as
1
3
4
1
white solid (105 mg, 55%). H NMR (400 MHz, CDCl
3
): δ = 9.08 (d,
a beige solid (151 mg, 61%). H NMR (400 MHz, CDCl
3
): δ = 9.07 (d,
3
3
3
4
3
3
3
4
J = 2.0 Hz, 1H), 9.01 (d, J = 2.0 Hz, 1H), 8.90 (dd, J = 8.0 Hz,
J = 2.0 Hz, 1H), 8.98 (d, J = 2.0 Hz, 1H), 8.88 (dd, J = 1.4 Hz,
3
4
3
J = 1.4 Hz, 1H), 8.34 (dd, J = 7.6 Hz, J = 1.4 Hz, 1H), 7.31 (t,
J = 8.0 Hz, 1H), 8.32 (dd, J = 1.4 Hz, J = 7.6 Hz, 1H), 7.39 (br s,
3 3
3
J = 7.8 Hz, 1H), 4.73 (d, J = 6.8 Hz, 2H), 3.15–3.08 (m, 2H),
1H), 7.30 (t, J = 7.8 Hz, 1H), 4.73 (d, J = 6.6 Hz, 2H), 3.36–3.30 (m,
3
2
2
.97–2.89 (m, 2H), 2.81 (s, 6H), 2.55–2.50 (m, 2H), 2.33–2.21 (m, 1H),
.13–2.03 (m, 2H), 2.02–1.93 (m, 2H), 1.57–1.44 (m, 2H) (Signal due
2H), 3.07–3.00 (m, 2H), 2.49 (t, J = 6.2 Hz, 2H) 2.35–2.22 (m, 1H),
2.07–1.98 (m, 4H), 1.95 (s, 3H), 1.72–1.64 (m, 2H), 1.58–1.46 (m, 2H).
1
3
13
to the NH is missing). C NMR (100 MHz, CDCl
3
): δ = 158.6, 147.9,
C NMR (100 MHz, CDCl ): δ = 170.0, 158.5, 147.8, 145.8, 145.4,
3
1
5
2
45.9, 145.5, 143.8, 141.6, 131.1, 126.7, 124.8, 123.4, 100.7, 72.5,
143.6, 141.4, 130.9, 126.6, 124.7, 123.3, 100.6, 72.3, 57.9, 53.5 (2C),
39.8, 35.1, 29.5 (2C), 25.1, 23.5. IR (KBr): 3273, 2913, 1642, 1592,
6.6, 53.2 (2C), 40.1, 38.2 (2C), 35.3, 29.4 (2C). IR (KBr): 3293, 2937,
−
1
−1
800, 1595, 1427, 1334, 1156, 945 cm . mp: 141–142 °C. HRMS/ESI:
1466, 1346, 1180, 853 cm . mp: 135–138 °C. HRMS/ESI: calculated
+
[M+H]+ 520.1212, found 520.1209.
calculated for C21
H
28IN
6
O
3
[M+H] 571.0985, found 571.0988.
for C22
H
27IN
O
5 2
8

V. Babin, et al.
Bioorganic Chemistry 96 (2020) 103582
4
.1.15. N-(3-(4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)
4.1.18. 4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)-1-(3-
piperidin-1-yl)propyl) methanesulfonylmethanesulfonamide 14
hydroxy)propyl)piperidine, formate salt 17
Starting from compound 5 (115 mg, 0.19 mmol) and using General
method 1, the resulting amine was dissolved in dichloromethane
In a round bottom flask, compound 16 (190 mg, 0.45 mmol) and
TBAF (1 M in THF, 0.75 mL, 0.75 mmol) were added in THF (10 mL).
The solution was stirred 16 h at room temperature and THF was eva-
porated. The residue was purified by reverse gel chromatography using
water/acetonitrile (80/20 to 50/50) and formic acid (2%) as eluent
(
15 mL) and the solution was cooled down to 0 °C. Methanesulfonyl
chloride (0.03 mL, 0.38 mmol) was added dropwise and the mixture
was stirred 15 min at 0 °C and 30 min at room temperature. The organic
1
layer was washed with water (15 mL), dried over MgSO
4
, filtered and
affording the expected product as a white solid (64 mg, 49%). H NMR
3
evaporated. The residue was purified by silica gel chromatography
using dichloromethane/methanol (100/0 to 95/5) as eluent affording
(400 MHz, DMSO‑d
6
): δ = 9.25 (d, J = 2.0 Hz, 1H), 9.13 (d,
3
4
3
J = 2.0 Hz, 1H), 8.83 (dd, J = 1.2 Hz, J = 8.0 Hz, 1H), 8.38 (dd,
1
4
3
3
the expected product as a white solid (59 mg, 47%). H NMR
J = 1.2 Hz, J = 7.6 Hz, 1H), 8.33 (br s, 1H), 7.39 (t, J = 7.8 Hz,
3 3
3
3
4
(
400 MHz, CDCl
3
): δ = 9.06 (d, J = 2.0 Hz, 1H), 8.99 (d, J = 2.0 Hz,
1H), 4.58 (d, J = 6.4 Hz, 2H), 3.40 (t, J = 6.3 Hz, 2H), 3.01–2.90 (m,
4
3
3
1
H), 8.87 (dd, J = 1.4 Hz, J = 8.0 Hz, 1H), 8.32 (dd, J = 1.4 Hz,
2H), 2.39 (t, J = 6.3 Hz, 2H), 2.01–1.92 (m, 3H), 1.90–1.81 (m, 2H),
3
3
3
J = 7.6 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H), 4.59 (d, J = 7.0 Hz, 2H),
1.64–1.54 (m, 2H), 1.49–1.37 (m, 2H) (Signals due to the OH are
3
13
3
.80 (t, J = 6.9 Hz, 2H), 3.29 (s, 6H), 3.05–2.94 (m, 2H), 2.47–2.36
missing). C NMR (100 MHz, DMSO‑d
6
): δ = 165.0, 158,4, 148,8,
1
3
(
m, 2H), 2.33–2.20 (m, 1H), 2.07–1.90 (m, 6H), 1.62–1.46 (m, 2H).
C
146.4, 144,6, 142,8, 141.0, 130.3, 126.8, 124.0, 122.9, 100.9, 71.2,
NMR (100 MHz, CDCl
3
): δ = 158.9, 147.8, 145.8, 146.6, 143.8, 141.5,
59.2, 55.0, 52.4 (2C), 34.4, 28.9 (2C), 27.9. IR (KBr): 3430, 2929, 1597,
−1
1
31.1, 126.6, 124.7, 123.3, 100.7, 72.6, 55.4, 53.4 (2C), 47.5, 43.8
1353, 1117 cm . mp: greater than 250 °C. HRMS/ESI: calculated for
(
2C), 35.1, 29.2 (2C), 27.8. IR (KBr): 2923, 1594, 1456, 1360, 1323,
C
20
H24IN
4
O
2
[M+H]+ 479.0944, found 479.0944.
−
1
1
151, 511 cm
.
mp: 176–178 °C. HRMS/ESI: calculated for
+
C
22
H
29IN
5
O
3
S
2
[M+H] 634.0655, found 634.0666.
4.1.19. 4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)-1-(3-
methylsulfanyl)propyl) piperidine 18
4
.1.16. N-(4-(4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)
In a round bottom flask, compound 3 (313 mg, 0.75 mmol) was
dissolved in dichloromethane (8 mL), 3-(methylthio)propionaldehyde
(0.07 mL, 0.91 mmol) and three drops of acetic acid were added. The
solution was stirred for 2 h at room temperature and sodium triace-
toxyborohydride (240 mg, 1.52 mmol) was added. The reaction was
stirred at room temperature and monitored by TLC. The solution was
carefully hydrolysed with an aqueous solution of NaOH (1 M, 10 mL),
piperidine-1-yl)butyl) methanesulfonamide 15
Starting from compound 6 (170 mg, 0.30 mmol) and using General
method 1, the resulting amine was dissolved in dichloromethane
(
(
15 mL) and the solution was cooled down to 0 °C. Triethylamine
0.08 mL, 0.60 mmol) and methanesulfonyl chloride (0.02 mL,
0
5
.30 mmol) were added to the solution and the mixture was stirred
min at 0 °C and 5 min at room temperature. The organic layer was
extracted with dichloromethane (3 × 15 mL) dried over MgSO , fil-
4
washed with water (15 mL), dried over MgSO
4
, filtered and evaporated.
tered and evaporated. The residue was purified by silica gel chroma-
tography using dichloromethane/methanol (100/0 to 95/5) as eluent
The residue was purified by silica gel chromatography using di-
chloromethane/methanol (100/0 to 90/10) as eluent affording the
1
affording the expected product as a beige solid (285 mg, 57%). H NMR
1
3
3
4
expected product as a beige solid (112 mg, 72%). H NMR (400 MHz,
(400 MHz, CDCl
3
): δ = 9.08 (d, J = 2.0 Hz, 1H), 9.00 (d, J = 2.0 Hz,
3
3
4
3
CDCl
3
): δ = 9.08 (d, J = 2.0 Hz, 1H), 9.00 (d, J = 2.0 Hz, 1H), 8.90
1H), 8.90 (dd, J = 1.4 Hz, J = 8.0 Hz, 1H), 8.33 (dd, J = 1.4 Hz,
4
3
4
3
3
3
3
(
dd, J = 8.0 Hz, J = 1.4 Hz, 1H), 8.34 (dd, J = 7.6 Hz, J = 1.4 Hz,
J = 7.6 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 4.73 (d, J = 6.8 Hz, 2H),
3.12–3.02 (m, 2H), 2.56–2.46 (m, 4H), 2.36–2.24 (m, 1H), 2.17–2.06
3
3
1
4
4
H), 7.31 (t, J = 7.8 Hz, 1H), 4.74 (d, J = 6.8 Hz, 2H), 3.13–3.02 (m,
1
3
H), 2.91 (s, 3H), 2.45–2.39 (m, 2H), 2.37–2.25 (m, 1H), 2.13–1.98 (m,
(m, 5H), 2.05–1.98 (m, 2H), 1.91–1.81 (m, 2H), 1.69–1.55 (m, 2H).
C
1
3
H), 1.75–1.60 (m, 6H) (Signal to the NH is missing). C NMR
NMR (100 MHz, CDCl ): δ = 158.5, 147.8, 145.8, 145.4, 143.7, 141.4,
3
(
100 MHz, CDCl
3
): δ = 158.6, 147.9, 145.9, 145.5, 143.8, 141.6,
131.0, 126.6, 124.7, 123.3, 100.7, 72.5, 57.8, 53.5 (2C), 35.0, 32.3
(2C), 29.0, 26.3, 15.6. IR (KBr): 2912, 1591, 1454, 1349, 1176,
1
3
1
31.1, 126.7, 124.7, 123.4, 100.7, 72.5, 58.4, 53.3 (2C), 43.4, 40.2,
−1
5.1, 29.1, 28.7 (2C), 25.0. IR (KBr): 3273, 2921, 2772, 1594, 1313,
1142 cm . mp: 108–110 °C. HRMS/ESI: calculated for C21
[M+H]+ 509.0874, found 509.0872.
H
26IN OS
4
−
1
137, 784 cm
.
mp: 139–140 °C. HRMS/ESI: calculated for
+
C
22
H
29IN
5
O
3
S [M+H] 570.1047, found 570.1036.
4
.1.20. 4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)-1-(3-
4
.1.17. 1-(3-((Tert-butyldimethylsilyl)oxy)propyl)-4-(((7-iodopyrazino
methylsulfonyl)propyl) piperidine 19
[
2,3-c]quinolin-5-yl)oxy)methyl)piperidine 16
In a round bottom flask, compound 18 (100 mg, 0.20 mmol) was
dissolved in dichloromethane (2 mL) at 0 °C. 3-Chloroperoxybenzoic
acid (85 mg, 0.49 mmol) was slowly added and the reaction mixture
was stirred at room temperature for two hours. The reaction mixture
Compound 3 (190 mg, 0.45 mmol) was dissolved in acetonitrile
(
25 mL), (3-bromopropoxy)-tert-butyldimethylsilane (88 mg,
0
.35 mmol) and triethylamine (0.08 mL, 0.58 mmol) were added. The
solution was stirred at reflux for 16 h. Water was added (20 mL) and the
organic layer was extracted with ethyl acetate (3 × 20 mL), dried over
was quenched with a saturated aqueous solution of Na
2
S
2
O (5 mL) and
3
extracted with dichloromethane (2 × 5 mL). The combined organic
MgSO
4
, filtered and evaporated. The residue was purified by silica gel
layers were dried over anhydrous MgSO , filtered and concentrated
4
chromatography using dichloromethane/methanol (100/0 to 90/10) as
under reduced pressure. The residue was purified by silica gel chro-
matography using dichloromethane/methanol (100/0 to 95/5) as
eluent affording the expected product as a white solid (170 mg, 63%).
1
3
4
3
3
3
1
H NMR (400 MHz, CDCl
3
): δ = 9.08 (d, J = 2.0 Hz, 1H), 9.00 (d,
eluent affording the expected product as a beige solid (71 mg, 66%). H
4
3
3
J = 2.0 Hz, 1H), 8.90 (dd, J = 1.4 Hz, J = 8.0 Hz, 1H), 8.34 (dd,
NMR (400 MHz, CDCl
3
): δ = 9.09 (d, J = 2.0 Hz, 1H), 9.01 (d,
3
3
3
4
J = 1.4 Hz, J = 7.6 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 4.74 (d,
J = 2.0 Hz, 1H), 8.91 (dd, J = 1.4 Hz, J = 8.0 Hz, 1H), 8.35 (dd,
3 3
3
4
3
J = 6.8 Hz, 2H), 3.67 (t, J = 6.1 Hz, 2H), 3.29–3.11 (m, 2H),
J = 1.7 Hz, J = 7.6 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 4.72 (d,
2
.72–2.55 (m, 2H), 2.43–2.19 (m, 3H), 2.14–2.01 (m, 2H), 1.95–1.68
J = 6.8 Hz, 2H), 3.15–3.08 (m, 2H), 3.00–2.93 (m, 2H), 2.92 (s, 3H),
1
3
3
(
m, 4H), 0.87 (s, 9H), 0.04 (s, 6H). C NMR (100 MHz, CDCl
3
):
2.50 (t, J = 6.8 Hz, 2H), 2.34–2.20 (m, 1H), 2.10–1.94 (m, 6H),
1
3
δ = 158.5, 147.9, 145.9, 145.5, 143.7, 141.6, 131.0, 126.7, 124.7,
1.58–1.46 (m, 2H). C NMR (100 MHz, CDCl ): δ = 158.5, 147.8,
3
1
23.4, 100.7, 72.2, 61.3, 55.8, 53.3 (2C), 34.6, 29.3, 28.4 (2C), 26.1
145.8, 145.4, 143.7, 141.5, 131.0, 126.5, 124.6, 123.2, 100.6, 72.5,
56.4, 53.2 (2C), 52.8, 40.7, 35.1, 29.2 (2C), 20.0. IR (KBr): 2919, 1637,
(
2C), 18.4, −5.2 (3C). IR (KBr): 2927, 2858, 1591, 1472, 1351, 1110,
−
1
−1
8
35, 778 cm
.
mp: 102–105 °C. HRMS/ESI: calculated for
1595, 1475, 1281, 1136 cm . mp: 115–116 °C. HRMS/ESI: calculated
+
S [M+H]+ 541.0768, found 541.0770.
C
26
H
38IN
4
O
2
Si [M+H] 593.1809, found 593.1813.
for C21
H
26IN
O
4 3
9

V. Babin, et al.
Bioorganic Chemistry 96 (2020) 103582
4
.1.21. Imino(3-(4-(((7-iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)
by silica gel chromatography using cyclohexane/EtOAc (95/5) as
6
piperidine-1-yl)propyl) methyl-λ -sulfanone 20
eluent affording the expected tert-butyl 4-(iodomethyl)piperidine-1-
In a round bottom flask, compound 18 (125 mg, 0.25 mmol) was
dissolved in methanol (1.5 mL), ammonium carbamate (29 mg,
carboxylate as a colorless oil (2.78 g, 92%). Data are consistent with
1
literature [36]. H NMR (400 MHz, CDCl
3
): δ = 4.25–4.00 (m, 2H),
3
0
.38 mmol) and PhI(OAc)
2
(165 mg, 0.51 mmol) were added. The so-
3.10 (d, J = 6.9 Hz, 2H), 2.76–2.60 (m, 2H), 1.87–1.78 (m, 2H),
1
3
lution was stirred at room temperature for 16 h. The reaction mixture
was evaporated under vacuum then the crude mixture was purified by
filtration on silica using dichloromethane/methanol (70/30) as eluent
1.67–1.54 (m, 1H), 1.45 (s, 9H), 1.20–1.07 (m, 2H).
C NMR
(100 MHz, CDCl
3
): δ = 154.8, 79.7, 43.3 (2C), 38.8, 32.7 (2C), 28.6
(3C), 13.7.
1
affording the expected product as a brown foam (77 mg, 57%). H NMR
3
3
4
(
400 MHz, CDCl
3
): δ = 9.09 (d, J = 2.0 Hz, 1H), 9.01 (d, J = 2.0 Hz,
4
3
1
H), 8.91 (dd, J = 1.4 Hz, J = 8.0 Hz, 1H), 8.35 (dd, J = 1.4 Hz,
4.1.25. 4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)-1-
((piperidin-4-yl)methyl)piperidine 22
3
3
3
J = 7.6 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 4.73 (d, J = 6.8 Hz, 2H),
3
.21–3.14 (m, 2H), 3.00 (s, 3H), 3.00–2.90 (m, 2H), 2.55–2.47 (m, 2H),
.35–2.22 (m, 1H), 2.18–1.90 (m, 7H), 1.60–1.45 (m, 2H) (Signal to the
In a round bottom flask, compound 3 (198 mg, 0.47 mmol) was
dissolved in acetonitrile (20 mL), triethylamine (0.14 mL, 0.96 mmol)
and tert-butyl 4-(iodomethyl)piperidine-1-carboxylate (190 mg,
0.58 mmol) were added. The solution was stirred under reflux for 16 h
and was cooled down to room temperature. The beige precipitate was
filtered, washed with acetonitrile (2 × 5 mL) affording the expected
intermediate without further purification. In a round bottom flask, tert-
butyl-4-((4-(((7-iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)piper-
idin-1-yl)methyl)-piperidine-1-carboxylate intermediate was dissolved
in dichloromethane (15 mL). Trifluoroacetic acid (0.35 mL, 2.5 mmol)
was added and the solution was stirred at room temperature. After 2 h,
the solvent was evaporated in vacuo and the crude product was dis-
2
1
3
NH is missing). C NMR (100 MHz, CDCl
3
): δ = 158.6, 147.9, 145.9,
1
5
1
45.5, 143.8, 141.6, 131.1, 126.7, 124.7, 123.4, 100.7, 72.6, 56.7,
5.5, 53.5, 53.3, 43.3, 35.2, 29.4, 29.3, 20.7. IR (KBr): 2912, 1591,
−
1
454, 1349, 1176, 1142 cm . HRMS/ESI: calculated for C21
H
27IN
5
O S
2
+
[
M+H] 540.0931, found 540.0930.
4.1.22. 1,6-dioxaspiro[2,5]octane
NaH (0.88 g, 23.26 mmol, 60% dispersion in mineral oil) was added
to a suspension of trimethylsulfonoxonium iodide (5.15 g, 23.26 mmol)
in THF (40 mL). The reaction mixture was heated at reflux for 3 h, then
tetrahydro-4H-pyran-4-one (1.82 mL, 20.0 mmol) was added, and the
mixture was held at reflux for a further 2 h. The mixture was filtered
and the filtrate was concentrated in vacuo. The residue was redissolved
in DCM (40 mL) and any solid material removed by filtration. The fil-
trate was concentrated in vacuo to give 1,6-dioxaspiro[2,5]octane as a
solved in a saturated solution of K
3
PO (15 mL). The solution was
4
stirred 30 min at room temperature and the resulting mixture was ex-
tracted with dichloromethane (3 × 20 mL). The combined organics
layers were dried over MgSO , filtered and evaporated affording the
4
expected product without further purification as a beige solid (142 mg,
1
1
3
colorless oil (1.60 g, 70%). Data are consistent with literature [28]. H
48%). H NMR (400 MHz, CDCl
3
): δ = 9.06 (d, J = 2.0 Hz, 1H), 9.00
3
4
3
NMR (400 MHz, CDCl
3
): δ = 3.89–3.76 (m, 4H), 2.68 (s, 2H),
(d, J = 2.0 Hz, 1H), 8.88 (dd, J = 1.4 Hz, J = 8.0 Hz, 1H), 8.32 (dd,
1
3
4
3
3
3
1
.90–1.81 (m, 2H), 1.56–1.48 (m, 2H). C NMR (100 MHz, CDCl
3
):
J = 1.4 Hz, J = 7.6 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H), 4.71 (d,
δ = 66.7 (2C), 56.6, 54.0, 34.0 (2C).
J = 6.8 Hz, 2H), 3.16–3.08 (m, 2H), 2.93–2.86 (m, 2H), 2,67–2.58 (m,
3
2
H), 2.29–2.18 (m, 1H), 2.16 (d, J = 7.1 Hz, 2H), 1.99–1.88 (m, 4H),
4
.1.23. 4-((4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)
1.81–1.74 (m, 2H), 1.70–1.58 (m, 1H), 1.57–1.43 (m, 2H), 1.22–1.10
13
piperidin-1-yl)methyl)oxan-4-ol 21
(m, 2H) (Signal due to the NH is missing). C NMR (100 MHz, CDCl ):
3
In a round bottom flask, compound 3 (103 mg, 0.25 mmol) was
δ = 158.7, 147.8, 145.9, 145.4, 143.8, 141.5, 131.1, 100.7, 126.6,
dissolved in methanol (25 mL), 1,6-dioxaspiro[2,5]octane (60.0 mg,
124.7, 123.3, 72.9, 65.6, 54.1 (2C), 46.3 (2C), 35.4, 33.8, 31.6 (2C),
−
+
1
0
.50 mmol) and triethylamine (0.07 mL, 0.50 mmol) were added. The
29.5 (2C). IR (KBr): 3422, 2927, 2898, 1610, 1354, 1173, 778 cm
.
solution was stirred under reflux for 16 h. Methanol was evaporated
and the resulting mixture was dissolved in ethyl acetate (25 mL). The
mp: 140–143 °C. HRMS/ESI: calculated for C23
518.1419, found 518.1417.
H
29IN O [M+H]
5
organic layer was washed with brine (25 mL), dried over MgSO , fil-
4
tered and evaporated. The residue was purified by silica gel chroma-
tography using dichloromethane/methanol (100/0 to 95/5) as eluent
4.1.26. 4-(((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)-1-((1-
1
affording the expected product as a white solid (108 mg, 81%). H NMR
methanesulfonylpiperidin-4-yl)methyl)piperidine 23
3
3
4
(
400 MHz, CDCl
3
): δ = 9.07 (d, J = 2.0 Hz, 1H), 9.00 (d, J = 2.0 Hz,
In a round bottom flask, compound 22 (90 mg, 0.17 mmol) was
dissolved in dichloromethane (10 mL) and the solution was cooled
down to 0 °C. Triethylamine (0.03 mL, 0.25 mmol) and methane-
sulfonyl chloride (0.01 mL, 0.17 mmol) were added and the solution
was stirred 5 min at 0 °C and 5 min at room temperature. The organic
4
3
1
H), 8.88 (dd, J = 1.4 Hz, J = 8.0 Hz, 1H), 8.32 (dd, J = 1.4 Hz,
3
3
3
J = 7.6 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H), 4.70 (d, J = 6.8 Hz, 2H),
3
.83–3.71 (m, 4H), 2.96–2.88 (m, 2H), 2.46–2.37 (m, 2H), 2.33 (s, 2H),
.30–2.18 (m, 1H), 1.98–1.89 (m, 2H), 1.62–1.42 (m, 6H) (Signal due
2
1
3
to the OH is missing). C NMR (100 MHz, CDCl
3
): δ = 158.6, 147.9,
layer was washed with water (20 mL), dried over MgSO , filtered and
4
1
6
3
1
5
45.9, 145.5, 143.8, 141.5, 131.1, 126.6, 124.7, 123.4, 100.7, 72.6,
evaporated. The residue was purified by silica gel chromatography
using dichloromethane/methanol (100/0 to 95/5) as eluent affording
9.6, 68.4, 67.4, 64.1, 56.3, 53.9, 37.2, 34.7, 31.9, 29.9, 29.4. IR (KBr):
−
1
1
442, 2936, 1677, 1591, 1425, 1349, 1177, 1111 cm
.
mp:
the expected product as a beige solid (100 mg, 89%). H NMR
+
3
3
4
69–170 °C. HRMS/ESI: calculated for
35.1206, found 535.1201.
C
23
H
28IN
4
O
3
[M+H]
(400 MHz, CDCl
3
): δ = 9.07 (d, J = 2.0 Hz, 1H), 8.99 (d, J = 2.0 Hz,
4
3
1H), 8.87 (dd, J = 1.4 Hz, J = 8.0 Hz, 1H), 8.32 (dd, J = 1.4 Hz,
3
3
3
J = 7.6 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H), 4.70 (d, J = 6.8 Hz, 2H),
4.1.24. tert-Butyl 4-(iodomethyl)piperidine-1-carboxylate
3.81–3.74 (m, 2H), 2.95–2.87 (m, 2H), 2.75 (s, 3H), 2.67–2.59 (m, 2H),
3
Under nitrogen at 0 °C, I (2.83 g, 11.16 mmoL) was added to a
2
2.30–2.20 (m, 1H), 2.21 (d, J = 7.1 Hz, 2H), 2.05–1.91 (m, 4H),
mixture of triphenylphosphine (2.93 g, 11.16 mmol), imidazole (0.76 g,
1.16 mmol) and tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate
2.00 g, 9.30 mmol) in anhydrous THF (200 mL). The resulting mixture
was allowed to stir for 18 h at room temperature and an aqueous sa-
turated solution of Na was added (200 mL). Extraction was per-
formed using EtOAc (3 × 200 mL) and the combined organic layers
were dried over MgSO , filtered and evaporated. The crude was purified
1.91–1.83 (m, 2H), 1.69–1.55 (m, 1H), 1.59–1.46 (m, 2H), 1.34–1.21
1
3
1
(m, 2H). C NMR (100 MHz, CDCl ): δ = 158.6, 147.9, 145.8, 145.4,
3
(
143.8, 141.5, 131.1, 126.6, 124.7, 123.3, 100.7, 72.7, 64.6, 54.1 (2C),
46.3 (2C), 35.2, 34.6, 33.3, 30.5 (2C), 29.3 (2C). IR (KBr): 2918, 2855,
−1
S
2 2
O
3
1592, 1318, 1147, 781 cm . mp: 173–175 °C. HRMS/ESI: calculated
for C24
H
31IN
5
O
3
S [M+H]+ 596.1192, found 596.1194.
4
10

V. Babin, et al.
Bioorganic Chemistry 96 (2020) 103582
4
.1.27. 1-(4-((4-((7-Iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)
through a pad of celite. The organic layer was washed with water
piperidin-1-yl)methyl) piperidin-1-yl)ethan-1-one 24
(3 mL), dried over MgSO , filtered and concentrated. The residue was
4
In a round bottom flask, compound 22 (100 mg, 0.19 mmol) was
dissolved in dichloromethane (10 mL) and the solution was cooled
down to 0 °C. Acetic anhydride (0.02 mL, 0.19 mmol) was added and
the solution was stirred 5 min at 0 °C and 5 min at room temperature.
The organic layer was washed with water (10 mL) and extracted with
purified by silica gel chromatography using dichloromethane/methanol
(100/0 to 90/10) as eluent affording the expected product as a light
1
brown oil (42 mg, 47%). H NMR (400 MHz, CDCl
3
): δ = 9.05 (d,
3
3
3
4
J = 2.0 Hz, 1H), 8.95 (d, J = 2.0 Hz, 1H), 8.88 (dd, J = 1.6 Hz,
J = 8.0 Hz, 1H), 7.99–7.84 (m, 1H), 7.61–7.54 (m, 1H), 7.38 (br s,
3
dichloromethane (2 × 10 mL), dried over MgSO
4
, filtered and evapo-
1H), 4.57 (d, J = 6.7 Hz, 2H), 3.39–3.31 (m, 2H), 3.15–3.07 (m, 2H),
2.60–2.52 (m, 2H), 2.22–2.04 (m, 5H), 1.96 (s, 3H), 1.78–1.70 (m, 2H),
1.61–1.48 (m, 8H), 1.37–1.27 (m, 6H), 1.26–1.16 (m, 6H), 0.85 (t,
rated. The residue was purified by silica gel chromatography using di-
chloromethane/methanol (100/0 to 95/5) as eluent affording the ex-
1
3
13
pected product (84 mg, 93%). H NMR (400 MHz, CDCl
3
): δ = 9.07 (d,
J = 7.3 Hz, 9H). C NMR (100 MHz, CDCl
3
): δ = 170.1, 157.2, 149.3,
3
4
3
3
3
J = 2.0 Hz, 1H), 9.00 (d, J = 2.0 Hz, 1H), 8.88 (dd, J = 8.0 Hz,
147.5, 146.6, 144.7, 143.4, 139.8, 131.0, 125.4, 124.4, 122.2, 71.6,
3
4
J = 1.4 Hz, 1H), 8.32 (dd, J = 7.6 Hz, J = 1.4 Hz, 1H), 7.29 (t,
57.7, 53.3 (2C), 39.6, 35.1, 29.4 (3C), 29.3 (2C), 27.6 (3C), 25.0, 23.5,
3
J = 7.8 Hz, 1H), 4.71 (d, J = 6.8 Hz, 2H), 4.62–4.54 (m, 1H),
13.9 (3C), 10.4 (3C). HRMS/ESI: calculated for C24
H
54IN
5
O Sn [M
2
+
3
.82–3.74 (m, 1H), 3.06–2.96 (m, 1H), 2.96–2.85 (m, 2H), 2.58–2.48
+H] 680.3308, found 680.3295.
(
m, 1H), 2.31–2.20 (m, 1H), 2.20–2.13 (m, 2H), 2.07 (s, 3H), 2.04–1.90
m, 4H), 1.88–1.80 (m, 1H), 1.79–1.68 (m, 2H), 1.60–1.45 (m, 2H),
(
4.2. Binding experiments on 5-HT R with MR-26132 and compound 3–25
4
1
3
1
1
7
2
9
.16–1.00 (m, 2H). C NMR (100 MHz, CDCl ): δ = 168.9, 158.6,
3
47.8, 145.9, 145.5, 143.8, 141.5, 131.1, 126.6, 124.7, 123.3, 100.7,
2.8, 64.9, 54.4, 53.9, 46.7, 41.8, 35.3, 34.0, 31.6, 30.7, 29.4 (2C),
1.7. IR (KBr): 2937, 1624, 1590, 1454, 1424, 1348, 1178, 1048,
For radioligand binding studies, 2.5 µg of proteins (5-HT4B mem-
brane preparations, HTS110M, Eurofins) were incubated in duplicate at
−
6
−8
25 °C for 60 min in the absence or the presence of 10
or 10 M of
−1
3
81 cm . mp: 165–167 °C. HRMS/ESI: calculated for C25
H31IN
5
O
2
[M
each ligands (MR-26132, 3–25) and 0.5 nM [ H]GR-113808 (NET
1152, Perkin Elmer) in 25 mM Tris buffer (pH 7.4). At the end of the
incubation, homogenates were filtered through Whatman GF/C filters
(FP-200, Alpha Biotech) presoaked with 0.5% polyethylenimine using a
Brandel cell harvester. Filters were subsequently washed three times
with 1 mL of ice-cold 25 mM Tris buffer (pH 7.4). Non-specific binding
was evaluated in parallel in the presence of 30 µM serotonin. For li-
gands MR-26132, 3–5 and 7–24, affinity constants were calculated
from five-point inhibition curves using the Prism 6 software and ex-
+
+
H] 560.1517, found 560.1522.
4
.1.28. Diethyl (4-((4-(((7-iodopyrazino[2,3-c]quinolin-5-yl)oxy)methyl)
piperidin-1-yl)methyl) piperidine-1-yl)phosphonate, fumarate salt 25
In a round bottom flask, compound 22 (150 mg, 0.29 mmol) was
dissolved in dichloromethane (15 mL) and the solution was cooled
down to 0 °C. Diethyl chlorophosphate (0.04 mL, 0.29 mmol) and
triethylamine (0.08 mL, 0.58 mmol) were added and the solution was
stirred 5 min at 0 °C and 12 h at room temperature. The organic layer
was washed with water (15 mL) and extracted with dichloromethane
pressed as K
i
±
SD.
(
2 × 15 mL), dried over MgSO
4
, filtered and evaporated. The residue
4.3. Selectivity and intrinsic activity of 13
was purified by silica gel chromatography using dichloromethane/
methanol (100/0 to 95/5) as eluent affording diethyl (4-((4-(((7-iodo-
pyrazino[2,3-c]quinolin-5-yl)oxy)methyl)piperidin-1-yl)methyl)piper-
idine-1-yl)phosphonate as a beige solid (50 mg, 29%). In a round
bottom flask, the solid (50 mg,) was dissolved in propan-2-ol (15 mL)
and fumaric acid (9.0 mg, 0.08 mmol) was added. The solution was
stirred at 40 °C for 30 min and the beige precipitate formed was filtered,
washed with propan-2-ol (3 × 5 mL) affording the expected product
Compound 13 was evaluated toward other serotonin receptors as
well as for intrinsic activity at CEREP. Detailed assay protocols are
available at the CEREP web site (http://www.cerep.com) under the
following reference numbers. Binding assays: 5-HT1aR (0131), 5-
HT1bR (0132), 5-HT1dR (1974), 5-HT2aR (0135), 5-HT2bR (1609), 5-
HT2cR (0137), 5-HT3R (0411), 5-HT5aR (0140), 5-HT6R (0142), 5-
HT7R (0144). Functional assay: 5-HT4eR (G049).
1
without further purification as a beige solid (65 mg). H NMR
3
(
400 MHz, CD
3
OD): δ = 9.16 (d, J = 2.0 Hz, 1H), 9.00 (d,
4.4. Radiochemistry
3
3
3
4
J = 2.0 Hz, 1H), 8.85 (dd, J = 8.0 Hz, J = 1.4 Hz, 1H), 8.33 (dd,
4
3
125
125
125
J = 7.6 Hz, J = 1.4 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 6.69 (s, 2H)
[
I]SB-207710, [ I]13 and [ I]MR-26132 were obtained
3
4
3
.73 (d, J = 6.1 Hz, 2H), 4.07–3.97 (m, 4H), 3.69–3.62 (m, 2H),
by incubation (30 min) of their respective tribulytin precursor (50 µg)
1
25
.59–3.49 (m, 2H), 3.09–2.99 (m, 4H), 2.86–2.76 (m, 2H), 2.54–2.42
in acid acetic (2 µl) with [ I]NaI (37 MBq) and hydrogen peroxide
(
m, 1H), 2.28–2.20 (m, 2H), 2.12–2.00 (m, 1H), 1.96–1.86 (m, 2H),
(1 µl). Reactions were injected onto a reversed-phase column
125 125
3
4
125
1
.85–1.76 (m, 2H), 1.31 (td,
J
H-H = 7.1 Hz,
J
H-P = 0.7 Hz, 6H),
(Bondclone C18) and [ I]SB-207710, [ I]13 and [ I]MR-
26132 were isolated by a linear gradient HPLC run (5–95% ACN in
1
3
1
.30–1.20 (m, 2H) (Signals due to the OH are missing). C NMR
(
100 MHz, CD
3
OD): δ = 171.2 (2C), 159.4, 150.0, 146.9, 144.7, 142.7,
7 mM H
3
PO , 10 min). Radiochemical yields were greater than 80%.
4
1
36.2 (2C), 131.4, 127.8, 125.6, 124.5, 101.1, 71.9, 63.9 (d, JC-
= 6.0 Hz, 2C), 63.3, 54.0 (2C), 45.1 (d, JC-P = 2.6 Hz, 2C), 34.1, 32.6,
1.2 (d, JC-P = 4.9 Hz, 2C), 27.3 (2C), 16.5 (d, JC-P = 6.8 Hz, 2C). IR
The molar activities were greater than 100 GBq/μmol, based on the
limit of detection of the ultraviolet absorbance and on the calibration
curves established with cold reference compounds.
P
3
−
1
(
KBr): 3429, 2944, 1710, 1594, 1458, 1353, 1177, 1137, 1035 cm
.
+
mp: 133–136 °C. HRMS/ESI: calculated for C27
H38IN
5
O
4
P [M+H]
4.5. Evaluation of 13 as SPECT radiotracer
6
54.1705, found 654.1706.
4
.5.1. Ethics
4
.1.29. N-(3-(4-(((7-(Tributylstannyl)pyrazino[2,3-c]quinolin-5-yl)oxy)
All experimental procedures were conducted with the agreement of
methyl)piperidin-1-yl) propyl)acetamide 26
the Ethics Committee for Animal Experimentation of the Canton of
Geneva and the General direction of health of the canton of Geneva,
Switzerland.
In a sealed tube under argon, compound 13 (70 mg, 0.13 mmol) and
tris(benzylideneacetone)dipalladium(0) (6 mg, 0.007 mmol) were dis-
solved in propan-2-ol (1 mL). Hexa-n-butylditin (0.08 mL, 0.16 mmol)
and N,N-diisopropylethylamine (0.05 mL, 0.33 mmol) were added and
the solution was stirred at 70 °C for 48 h. The solution was cooled down
to room temperature, diluted with ethyl acetate (2 mL) and filtered
4.5.2. In vitro competition experiments with 13
Hippocampal brain sections (26 µm, n = 8) were immersed in 1x
PBS (15 min), in radioactive buffer (90 min) and then rinsed twice in
11

V. Babin, et al.
Bioorganic Chemistry 96 (2020) 103582
4
°C Tris-MgCl
The radioactive buffer consists of Tris-MgCl
HCl, 50 mM MgCl
2
-EtOH buffer (3 min) and briefly washed in cold water.
-EtOH buffer (50 mM Tris
, 20% EtOH, pH = 7.4) contains either the radio-
ncbi.nlm.nih.gov/pubmed/11293643.
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2
active compounds alone or in presence of 10–50 μM of unlabeled
compound. Slides were air-dried before exposure onto gamma-sensitive
phosphor imaging plates (Fuji BAS-IP MS2325) for 30 min. Brain sec-
tions were then treated for Nissl staining in order to delineate the re-
gion of interests: cornu ammonis (1–4), entorhinal and temporal cortex
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1
25
Isotopenmessgerate GmbH) in presence of homemade [ I] calibration
curves. Specific binding ratio (SBR) was calculated as follows: (Average
radioactivity in ROI/radioactivity in ROI in the presence of unlabeled
radiotracer) – 1.
[
[
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4.5.3. In vivo SPECT imaging with 13
Animals were anesthetized with 3% isoflurane and placed in the U-
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1
25
(
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hippocampus and striatum.
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Declaration of Competing Interest
(
2003) 1520–1528, https://doi.org/10.1007/s00259-003-1307-x.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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R. Hopper, R. Gunn, Synthesis and evaluation of [11C]SB207145 as the first in vivo
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Acknowledgements
The authors are grateful for financial support provided by the
Regional Council of Normandy, FEDER, Crunch Network, la Ligue
Contre le Cancer, The ARC foundation, the University of Caen,
Tremplin Carnot I2C and the French Ministry of Research.
[20] F. Caillé, T.J. Morley, A.A.S. Tavares, C. Papin, N.M. Twardy, D. Alagille, H.S. Lee,
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[
21] A.A.S. Tavares, F. Caillé, O. Barret, C. Papin, H. Lee, T.J. Morley, K. Fowles,
D. Holden, J.P. Seibyl, D. Alagille, G.D. Tamagnan, Whole-body biodistribution and
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brain: [(18)F]MNI-698, Nucl. Med. Biol. 41 (2014) 432–439, https://doi.org/10.
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.bioorg.2020.103582.
[
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22] A.A.S. Tavares, F. Caillé, O. Barret, C. Papin, H. Lee, T.J. Morley, K. Fowles,
D. Holden, J.P. Seibyl, D. Alagille, G.D. Tamagnan, In vivo evaluation of 18F-
MNI698: an 18F-labeled radiotracer for imaging of serotonin 4 receptors in brain, J.
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