Synthesis and structure-affinity relationships of selective high-affinity 5-HT4 receptor antagonists: Application to the design of new potential single photon emission computed tomography tracers

Article abstract of DOI:10.1021/jm300943r

The work described herein aims at finding new potential ligands for the brain imaging of 5-HT₄ receptors (5-HT₄Rs) using single-photon emission computed tomography (SPECT). Starting from the nonsubstituted phenanthridine compound 4a,

Full text of DOI:10.1021/jm300943r

Article
pubs.acs.org/jmc
Synthesis and Structure−Affinity Relationships of Selective High-
Affinity 5‑HT4 Receptor Antagonists: Application to the Design of
New Potential Single Photon Emission Computed Tomography
Tracers
Emmanuelle Dubost,† Noe Dumas,‡ Christine Fossey,† Rosa Magnelli,† Sabrina Butt-Gueulle,†
́
Celine Ballandonne,† Daniel H. Caignard,§ Fabienne Dulin,† Jana Sopkova de-Oliveira Santos,†
́
Philippe Millet,‡ Yves Charnay,‡ Sylvain Rault,† Thomas Cailly,† and Frederic Fabis*,†
†UFR des Sciences Pharmaceutiques, Universite
ICORE, Boulevard Becquerel, F-14032 Caen, France
‡Unite
de Morphologie et Unite de Neuroimagerie, Service de Neuropsychiatrie, Ho
Petit-Bel-Air, 2, CH1225 Chene-Bourg/Genev
́
de Caen Basse-Normandie, EA 4258 CERMN, FR CNRS 3038 INC3M, SF-4206
́
́
̂ ̀
pitaux Universitaires de Geneve, Ch. du
̂
̀
e, Switzerland
§Institut de Recherches Servier, 125 Chemin de Ronde, F-78290 Croissy sur Seine, France
S
* Supporting Information
ABSTRACT: The work described herein aims at finding new
potential ligands for the brain imaging of 5-HT4 receptors (5-
HT4Rs) using single-photon emission computed tomography
(SPECT). Starting from the nonsubstituted phenanthridine
compound 4a, exhibiting a Ki value of 51 nM on the 5-HT4R,
we explored the structure−affinity in this series. We found that
substitution in position 4 of the tricycle with a fluorine atom gave the best result. Introduction of an additional nitrogen atom
inside the tricyclic framework led to an increase of both the affinity and selectivity for 5-HT4R, suggesting the design of the
antagonist 4v, exhibiting a high affinity of 0.04 nM. Several iodinated analogues were then synthesized as potential SPECT
tracers. The iodinated compound 11d was able to displace the reference radioiodinated 5-HT4R antagonist (1-butylpiperidin-4-
yl)methyl-8-amino-7-iodo[123I]-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate {[123I]1, [123I]SB 207710} both in vitro and in
vivo in brain. Compound 11d was radiolabeled with [125I]iodine, providing a potential SPECT candidate for brain imaging of 5-
HT4R.
IIb.15,16 Discovery of active 5-HT4Rs agonists and antagonists
INTRODUCTION
■
remains of great interest in clinical research. To this end,
molecular imaging techniques using positron emission
tomography (PET) or single photon emission computed
tomography (SPECT) have emerged as valuable tools, both
in clinical studies and drug discovery programs.17,18 These
noninvasive techniques have found broad applications,
including diagnosis, imaging of neurotransmitter receptors, in
vivo binding studies of new ligands, and establishing treatment
strategies. The bottleneck of these techniques remains the
limited availability of suitable radioligands.
Since its discovery more than 2 decades ago,1,2 the serotonin 4
receptor subtype (5-HT4R) has emerged as a promising target
for drug discovery, development, and medical applications.3,4
Pharmacological investigations coupled to the discovery of
ligands exhibiting high affinity and selectivity for 5-HT4Rs have
led to knowing their anatomical distribution and functional
roles. 5-HT4Rs are found in the peripheral system, where they
are implicated in gastrointestinal disorders5 and heart failure.6
Brain 5-HT4Rs are mainly expressed in striatum, globus
pallidus, nucleus accumbens, and substantia nigra.7 Their
distribution in the central nervous system and pharmacological
studies using selective agonists and/or antagonists has shown
that 5-HT4Rs are implicated in cognition,8 learning and
memory processes,9 and more recently in neuropsychiatric
disorders such as Alzheimer’s disease,10,11 food intake,12 and
depression.13 Although efforts have been made by both
academics and pharmaceutical companies to develop 5-HT4R
ligands with potential medical applications, only peripheral
agonists have yet reached the market for gastrointestinal
disorders.14 Brain 5-HT4 receptor ligands have entered clinical
trials for the treatment of Alzheimer disease but failed in phase
(1-Butylpiperidin-4-yl)methyl-8-amino-7-iodo[123I]-2,3-
dihydrobenzo[b][1,4]dioxine-5-carboxylate {[123I]1, [123I]SB
207710}19 and (1-methyl[11C]piperidin-4-yl)methyl-8-amino-
7-chloro-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate {[11C]
2, [11C]SB 207145}20 have been described as potential
radiotracers for respectively PET or SPECT imaging (Chart
1). [11C]2 has been successfully used in minipig for the
determination of radioligand metabolism and binding ki-
Received: July 3, 2012
Published: October 26, 2012
© 2012 American Chemical Society
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Chart 1. Literature and Prospective 5-HT4R Radiotracers
netics.21 Further studies have shown that this radiotracer can be
used for quantitative PET measurements of 5-HT4R in the
human brain.22,23 Nevertheless, its short half-life (20.9 min)
limits its use in facilities where both a cyclotron and a PET
camera are in proximity. Other analogs of 1 have recently been
shown to exhibit pharmacological properties that could make
them new promising 5-HT4R PET radiotracers.24 Among them,
fluorinated compounds could serve as PET radiotracers of
longer half-life. [123I]1 is to date the only SPECT tracer
reported for brain imaging of 5-HT4R, but due to low brain
penetration and rapid metabolism, no further investigations
with this product have been reported. We report here work
aimed at developing new 5-HT4R radioligands for SPECT
imaging. On the basis of previous works in the lab concerning
the synthesis and evaluation of 5-HT4R ligands,25,26 we
synthesized and evaluated new diversely substituted phenan-
thridine derivatives. As a result, we were able to design several
selective and high-affinity 5-HT4R antagonists among which
some iodinated compounds were identified and successfully
radiolabeled with 125I, representing new potential radioligands
for SPECT imaging studies of the 5-HT4R (Chart 1).
a
Table 1. Synthesis of (Aza)Phenanthridines 4a−v
b
starting material
R
X
Y
product
yield (%)
3a
3b
3c
3d
3e
3f
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
4a
4b
4c
4d
4e
4f
76
57
70
49
91
87
71
59
65
47
63
62
67
2-F
2-Cl
2-Me
2-MeO
3-Cl
3-MeO
3-Me
3-F
3g
3h
3i
4g
4h
4i
3j
4-Me
4-MeO
4-Cl
4-F
4j
3k
3l
4k
4l
3m
3n
3o
3p
3q
3r
3s
3t
4m
4n
4o
4p
4q
4r
4s
4t
RESULTS
c
7-F
9
■
Chemistry. The starting (aza)phenanthridinones 3a−v
were prepared according to the general route as previously
described.27 Compounds 4a−v were obtained using a two-step
procedure involving the formation of an imidoyl chloride
intermediate in phosphorus oxychloride at 80 °C and the
subsequent nucleophilic aromatic substitution of the chlorine
atom with (1-propylpiperidin-4-yl)methanol, using conditions
based on previous results.25,26 Compounds 4a−v were obtained
in 42−91% overall yields, except compound 4n, which was
prepared in 9% overall yield in a three-step procedure28 (Table
1).
8-NO2
8-MeO
8-F
57
70
44
71
62
42
54
83
9-Me
9-F
H
3u
3v
H
CH
CH
4u
4v
4-F
N
a
Reagents and conditions: (i) POCl3, 80 °C, overnight; (ii) (1-
propylpiperidin-4-yl)methanol, NaH, DMF, 0 °C to rt, overnight.
Isolated yields. Yield over three steps; see experimental data for
details.
b
c
For the design of potential SPECT ligands, we were
interested in the introduction of iodine atoms either on the
tricyclic framework or on the lateral chain. Iodinated
phenanthridinone 8a and benzonaphthyridinone 8b were
obtained in good overall yields starting from 2-trimethylsilyl-
fluorobenzene by a four-step procedure involving borylation,
Suzuki cross-coupling reaction, iododesilylation, and anionic
ring closure (Scheme 1). For the iodinated side chain, we chose
to add a terminal iodoaryl group to the propyl chain.25,26 Thus,
10 was prepared starting from 4-iododihydrocinnamic acid
involving amidation with ethyl isonipecotate and reduction of
both the carboxamide and ester function with diisobutylalumi-
nium hydride (Scheme 2).
Synthesis of Radioligands. Stannylated precursors for
radioiodination were prepared from iodinated compounds
11a,b,d using palladium-catalyzed tin−iodine exchange in the
presence of PPh3 in toluene.29 Radioiodination from the
stannylated compounds 12a,b,d was performed using Na125I as
the source of radioactive iodine H2O2 (30%) as the oxidant in
acidic medium (Scheme 3). After HPLC purification, the
radioiodinated compounds 13a,b,d were obtained in 56−85%
radiochemical yields. The products 13a,b,d were found to be all
carrier-free, exhibiting apparent specific radioactivities of
respectively 240, 110, and 280 Ci/mmol (11%, 5% and 13%
of the carrier free specific activity).
The iodinated compounds 11a−d were obtained using the
two-step chlorodehydroxylation/SNAr sequence starting from
phenanthridinones 3a and 8a, benzonaphthyridinones 3v and
8b, and the appropriate piperidine derivative (Table 2).
5-HT4R Binding Affinity and Functional Assays.
Twenty-six compounds (4a−v, 11a−d) were initially screened
for their affinity toward 5-HT4R in guinea pig striatal
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Scheme 1. Synthesis of 4-Iodo(aza)phenanthridin-6(5H)-
ones 8a,b
Table 2. Synthesis of Iodinated (Aza)Phenanthridines 11a−
a
a
d
b
starting material
R
X
Z
product yield (%)
8a
8b
3a
3v
I
CH
N
H
H
11a
11b
11c
11d
76
71
39
41
I
H
F
CH
N
4-iodophenyl
4-iodophenyl
a
Reagents and conditions: (i) POCl3, 80 °C, overnight; (ii) 1-
propylpiperidin-4-ylmethanol or 10, NaH, DMF, 0 °C to rt, overnight.
Isolated yields.
b
a
Reagents and conditions: (i) s-BuLi 0.75 h, B(OMe)3 0.75 h, THF,
−78 °C to rt; (ii) 2-BrPhCN or 2-Cl-3-CN-pyridine, Na2CO3,
Pd(OAc)2, PPh3, DME/H2O, 14 h, 90 °C; (iii) ICl, DCM, 4.5 h, rt;
(iv) KOH, t-BuOH, sealed tube, 1 h, 150 °C.
Scheme 3. Preparation of Stannylated Precursors 12a,b,d
a
and 125I Labeling
membranes at 10−6 and 10−8 M. Twenty compounds were
selected for Ki determination in 5-HT4R guinea pig striatal
membranes. These ligands show Ki values between 2.2 and 691
nM. Among them, 10 were chosen for human 5-HT4R Ki
determination and, except 4t, all ligands showed better affinity
for human 5-HT4R compared to guinea pig 5-HT4R, exhibiting
Ki values between 0.04 and 33 nM (Table 3). All compounds
were evaluated for their intrinsic activity and showed either an
inverse agonist (4k−m,u) or a full antagonist profile (4v, 11a,b,
and 11d), as shown in Table 4.
5-HTRs Binding Profile. Compounds 4k, 4l, 4m, 4u, 4v,
11a, 11b, and 11d with the highest affinities for 5-HT4R were
screened toward other 5-HTR subtypes. Results are shown in
Table 5.
DISCUSSION
■
a
Unlike PET imaging using 18F, for which both a suitable
fluorinated ligand and a precursor for radiofluorination have to
be designed, for SPECT imaging an iodinated ligand can serve
as both the nonradioactive ligand for pharmacological studies
and the precursor for the introduction of 123I via successive
stannylation−iododestannylation reactions. A major challenge
with SPECT imaging is to find a suitable iodinated ligand
exhibiting high affinity and receptor selectivity. Whereas a
hydrogen atom often can be replaced by a fluorine atom
without significant decrease in biological activities, introduction
of an iodine atom can lead to a dramatic decrease in affinity due
to its large atomic radius.
Reagents and conditions: (i) (Bu3Sn)2, Pd(OAc)2, PPh3, PhMe/H2O,
16 h, 90 °C. (ii) Na125I, 30% H2O2, EtOH/AcOH, rt, 20 min.
introduction of substituents, including an iodine atom. The
chemical routes we have developed allowed the introduction of
substituents in position 2, 3, 4, 7, 8, and 9 (Table 3). The
results show that substitution of the phenanthridine in position
2, 3, 8, or 9 was detrimental for affinity compared to
unsubstituted compound 4a; all compounds show Ki values
above 100 nM, even with the small fluorine group. Better
results were obtained when substituents were placed in
positions 4 and 7. The 7-fluoro compound 4n showed a slight
increase in affinity (Ki = 22 nM), whereas substituents in
position 4 led to significant improvements in affinity
(compounds 4k−m), except the 4-methyl-substituted com-
pound 4j. Interestingly, while compounds 4k−m exhibited 35,
Starting from the unsubstituted phenanthridine 4a, which
exhibited good 5-HT4R binding affinity (Ki = 51 nM), we
explored the influence of substitution of the tricyclic ring
system in this series to find the best positions for the
a
Scheme 2. Synthesis of {1-[3-(4-Iodophenyl)propyl]piperidin-4-yl}methanol 10
a
Reagents and conditions: (i) Ethyl isonipecotate, HOBt, EDCI, NEt3, 24 h, rt; (ii) DIBALH, THF, 3 h, −10 °C to rt.
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Table 3. Binding Affinities of New 5-HT4R Ligands
5-HT4 Ki (nM)
a
b
c
compd
R
X
Y
Z
% inhbn (10−6 M/10−8 M)
guinea pig
human
d
4a
4b
4c
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
100/11
47/0
51.5
NM
NM
246
233
NM
1900
691
101
100
35.0
21.9
20.1
21.6
NM
NM
154
NM
209
22.8
13.1
2.2
NM
NM
NM
NM
NM
NM
NM
NM
2-F
2-Cl
2-Me
2-MeO
3-Cl
3-MeO
3-Me
3-F
27/0
4d
4e
4f
88/7
83/9
85/0
4g
4h
4i
NM
65/30
100/14
100/0
100/22
100/24
100/63
100/0
54/0
e
33.0
NM
17.0
4j
4-Me
4-MeO
4-Cl
4-F
e
4k
4l
e
5.0
e
4m
4n
4o
4p
4q
4r
3.1
7-F
NM
NM
NM
NM
NM
NM
8-NO2
8-MeO
8-F
85/0
90/4
9-Me
9-F
28/0
4s
84/0
e
4t
H
100/50
96/64
100/98
100/0
100/20
100/3
403
e
4u
4v
11a
11b
11c
11d
H
CH
CH
CH
CH
CH
CH
7.5
f
f
f
4-F
N
0.04
1.20
0.26
NM
0.23
4-I
CH
N
13.5
4.6
4-I
H
CH
N
4-iodophenyl
4-iodophenyl
115
2.5
f
4-F
100/94
a
b
c
Inhibition percentages were determined by using guinea pig striatal membrane 5-HT4R. Guinea pig striatal membrane 5-HT4R (n = 3). Human 5-
d
e
f
HT4R (n = 3). NM = not measured. Ki determinations were performed at NIMH PDSP. Ki determinations were performed at CEREP. See
Experimental Section for details.
Table 4. Intrinsic Activity and cLogD of 5-HT4R Ligands 4k, 4l, 4m, 4u, 4v, 11a, 11b, and 11d
4k
17
4l
5.0
4m
3.1
4u
7.5
4v
11a
11b
0.26
11d
0.23
human 5-HT4 Ki(nM)
efficacy
0.04
antag
−
1.20
antag
−
a
a
a
a
b
b
b
b
inv ag
79.4
−
inv ag
251
−
inv ag
63.1
−
inv ag
10
antag
antag
EC50 (nM)
−
0.5
−
6.3
KB (nM)
−
1.84
0.025
5.0
c
cLogD
2.48
3.25
2.79
1.98
3.58
2.77
4.67
a
b
Functional assays performed at NIMH PDSP; inv ag = inverse agonist. Functional assays performed at CEREP; antag = antagonist. See
c
Experimental Section for details. Calculated log D (pH = 7.4) using MarvinSketch 5.2.6.
22, and 20 nM Ki values respectively for guinea pig 5-HT4R,
against human 5-HT4R they showed significantly higher
binding affinities with Ki values of respectively 17, 5, and 3
nM. Introduction of a nitrogen atom in position 9 or 10 led to
benzonaphthyridines 4t and 4u, which exhibited increased
activity compared to 4a with Ki = 23 and 13 nM, respectively.
When fluorine was introduced in position 4 to give
benzonaphthyridine 4v, the affinity was significantly increased
to 2.2 nM on guinea pig 5-HT4R, and remarkably, this
compound exhibited a very high affinity on human 5-HT4R
with a Ki value of 0.04 nM (pKi = 10.4, pKB = 10.6). This
compound represents one of the most active 5-HT4R
antagonist reported to date, being in the same range as the
reference antagonists [1-(2-methanesulfonamidoethyl)-
piperidin-4-yl]methyl 1-methylindole-3-carboxylate (GR
113808)30 or 1.31
With these structure−affinity results in hand, we investigated
the design of iodinated compounds for potential development
of SPECT radiotracers. As the position 4 seemed to give the
best results in both the phenanthridine and benzonaphthyridine
series, we chose to introduce an iodine atom in this position.
Iodinated compounds 11a and 11b were synthesized and
evaluated for their 5-HT4R binding affinity. Despite differences
between fluorine and iodine in terms of size and electronic
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a
Table 5. Binding Affinities of Compounds 4k, 4l, 4m, 4u, 4v, 11a, 11b, and 11d toward 5-HT Receptors
human 5-HTR
4k
17
4l
4m
3.1
4u
7.5
4v
11a
11b
11d
5-HT4
5.0
0.04
4987
>104
>104
>104
>104
136
1.20
>104
>104
>104
>104
1019
162
0.26
0.23
680
>104
>104
NM
810
5-HT1A
5-HT1B
5-HT1D
5-HT1E
5-HT2A
5-HT2B
5-HT2C
5-HT3
>104
>104
2569
>104
5962
29.3
>104
>104
>104
>104
3473
8836
>104
2360
>104
1780
98.0
729
>104
>104
>104
>104
>104
40.4
1049
710
>104
>104
>104
>104
>104
627
5629
>104
>104
>104
1825
>104
341
>104
>104
>104
1018
340
2793
492
772
400
2470
>104
6256
1914
680
641
1314
5805
>104
153
410
5-HT5A
5-HT6
>104
>104
384
>104
>104
>104
>104
>104
1945
1700
>104
150
5-HT7
a
Selectivity was performed at NIMH PDSP, except for compound 11d, for which the selectivity was performed at CEREP.
Figure 1. Docking poses of compounds 4m (A), 4v (B and C), and 11d (D).
properties, 11a and 11b exhibited Ki values of 13 and 4.6 nM
for guinea pig 5-HT4R and respectively, 1.2 and 0.26 nM for
human 5-HT4R, in the same range as their fluorinated
analogues 4m and 4v. Further, we investigated compounds
iodinated on the N-propyl group. In order to avoid the
expected elimination of the iodine atom if bonded to a
saturated carbon, it was not directly attached to the N-propyl
chain but to an aromatic group using the N-3-(4-iodophenyl)-
propyl chain. Previous results have shown that some 5-HT4R
ligands bearing bulky aromatic group bonded to the lateral
chain can exhibit high affinities.3 Accordingly, compounds 11c
and 11d were synthesized. Despite the introduction of the
bulky iodoaryl group, the iodinated 4-fluorobenzonaphthyr-
idine 11d was found to exhibit high affinity for both guinea pig
5-HT4R (Ki = 2.5 nM) and human 5-HT4R (Ki = 0.23 nM) on
the similar order of magnitude as compound 4v. Along with
high binding affinities, intrinsic activity and binding selectivity
are other key parameters for the development of suitable
ligands for imaging. The best 5-HT4R ligands 4k−m, 4u−v,
11a,b, and 11d were evaluated for both intrinsic activity and
selectivity toward other 5-HTRs (Tables 4 and 5). Compounds
4k−m and 4u appeared to be inverse agonists with pEC50
ranging from 6.6 to 8.0, whereas compounds 4v, 11a,b, and 11d
were found to be full antagonists with pKB values ranging from
8.2 to 10.6, in accordance with their Ki values. The difference
observed in intrinsic activity could be due to high constitutive
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activity of the cloned receptors used in the functional tests.
Some ligands have been found to behave either as antagonists
or as inverse agonists, depending on the functional models
used.32 These compounds were then evaluated for their
selectivity toward other 5-HT receptors. All compounds
showed high selectivity toward other 5-HT receptors except
5-HT2BR, for which compounds 4k−m exhibited almost the
same potency as for 5-HT4R. Interestingly, the additional
nitrogen atom in position 10 not only led to increased affinities
for 5-HT4R but also contributes to decreased affinity for 5-
HT2BR, resulting in compounds with higher selectivity (>100).
In order to try to rationalize this result we performed docking
studies using a model of 5-HT4R based on the crystal structures
of compounds 4m, 4v, and 11d assuming that the crystalline
conformations are the most energetically stable (see Supporting
Information). The homology model of the human 5-HT4
receptor was constructed using the β2-adrenergic receptor
crystal structure, one of the available GPCR structures that
exhibits a sequence identity of about 40% with the 5-HT4R.33
The two crystallographic conformers of compound 4m were
docked into the 5-HT4R model (see Supporting Information).
In the selected pose (Figure 1A, mean ChemScore fit = 31.18),
the basic piperidine nitrogen interacted with Asp100 (consistent
with the constraint used during the docking), and an additional
polar interaction could form through this basic nitrogen and the
Tyr302 hydroxyl group.34 The tricyclic framework was oriented
toward the transmembrane helix 5 (TM5, colored in yellow in
Figure 1), in a position analogous to that observed for β-
adrenergic receptor ligands in solved X-ray structures.33,35 In
the 4m docking position, the tricycle is oriented parallel to the
TM5 helix axis and is surrounded by several aromatic residues
(Phe186, Tyr192, Phe275, Phe276), oriented approximately
perpendicularly to the tricyclic framework, indicating possible
π stacking interactions. The fluorine atom pointed inside the
receptor. The addition of a supplementary nitrogen atom in
derivative 4v could lead to a new hydrogen bond with the Ser197
hydroxyl group as seen in Figure 1B (mean ChemScore fit =
33.10). While this additional interaction could explain the
increased affinity for the 5-HT4R of 4v versus 4m, it does not
explain its lower affinity for the 5-HT2BR. Indeed, a careful
comparison of these receptors sequences showed that Ser197 is
present in both the 5-HT4 and 5-HT2B receptors; therefore, it is
not probable that the additional nitrogen interacts with this
serine. Comparison of amino acid sequences in the TM5 part of
the binding cavity shows three differences: Tyr192(5-HT4R)/
Phe217(5-HT2BR), Cys196(5-HT4R)/Gly221(5-HT2BR), and
Ala193(5-HT4R)/Met218(5-HT2BR). We reasoned that the
supplementary nitrogen atom in 4v could interact with the
hydroxyl group of the 5-HT4R Tyr192. Therefore, the docking
studies of 4v were carried out by taking into account a
supplementary hydrogen-bonding constraint between this
additional nitrogen atom and the Tyr192 hydroxyl group. In
the best scoring docking pose of this study (mean ChemScore
fit = 41.55), the tricycle was oriented perpendicularly to the
TM5 helix axis (Figure 1C), the N-1 nitrogen atom formed the
H-bond with Tyr192 as set during the docking and was located
far from the Ser197. Furthermore, in this docking pose, the
fluorine, the N-6 nitrogen atom, and the oxygen atom were
placed near to the 5-HT4R Trp294 (TM7), so that electrostatic
interactions can occur with this amino acid. Binding
interactions with this Trp294 have been previously shown to
be essential for some 5-HT4R ligands.36 The docking of
compound 11d showed that it can be positioned in the same
manner as 4v (mean ChemScore fit = 33.54), the 4-iodophenyl
group going up toward the extracellular entrance (Figure 1D),
as it was observed for some cocrystallized extended ligands such
as the full agonist carmoterol in β1-adrenergic receptor37 or for
the antagonist JDTic in κ-opioid receptor.38
Among the iodinated compounds, 11a, 11b, and 11d could
be considered as promising candidates for the development of a
SPECT tracer owing to their subnanomolar binding affinities,
high selectivity over other 5-HTRs including 5-HT2BR, and
computed lipophilicities within a range adequate for brain
penetration.39 Compound 11d, which exhibits the highest Ki
value and selectivity, was chosen for further evaluation. In order
to address its 5-HT4R specific binding capacity, in vitro
competition experiments with the selective and specific
antagonist radioligand [125I]1 were performed. Increasing
concentrations of 11d coadministered with [125I]1 show a
decrease in the 5-HT4R-specific radioactivity at 10 pM of 11d,
while increasing the concentration to 1 nM led to the almost
complete abolishment of the signal (Figure 2). The same
Figure 2. Autoradiograms obtained by incubation of sections with 100
pM of [125I]1 in the presence of growing concentrations of 11d (1, 10,
100, and 1000 pM from left to right).
experiment was performed ex vivo. The specific binding of
[
125I]1 is slightly noticeable in the olfactory tubercles but is
markedly reduced compared to a reference experiment (Figure
3). The activity between the hemispheres in the coinjection
Figure 3. Ex vivo autoradiogram after intravenous injection of [125I]1
alone (left) and after coinjection with 50 μg/kg of 11d to a mouse
(right).
experiment is due to the presence of blood in the brain
sections. Taken together, these two experiments show that (1)
11d is able to compete with the specific radioligand [125I]1
both in vitro and in vivo at very low concentration and (2) 11d
is able to cross the blood−brain barrier, making 11d a suitable
candidate for use in SPECT imaging studies.
The radioiodinated compound 13d was successfully prepared
from 11d by a two-step sequence involving stannylation and
Sn−125I exchange (Scheme 3). 13d was obtained in a 85%
radiochemical yield, and the specific radioactivity was measured
at 280 Ci/mmol (13% of the carrier free specific activity). The
two other iodinated compounds 11a and 11b were radio-
iodinated following the same strategy affording 13a and 13b in
respectively 56 and 70% RCY and with a specific activity of
9698
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Journal of Medicinal Chemistry
Article
difluorophenyl)nicotinonitrile40 (1.5 g, 6.9 mmol), and t-BuOH (40
mL). The tube was heated to 150 °C for 0.5 h. Water (30 mL) was
added and the obtained precipitate was filtered. The solid was then
dried under vacuum to afford 3v (1.3 g) as a white powder. Yield: 87%.
Mp: >260 °C. IR (KBr): ν (cm−1) 3022, 1675 (CO), 1587, 1419, 763.
1H NMR (400 MHz, DMSO-d6): δ 7.28 (m, 1H), 7.49 (m, 1H), 7.70
(dd, 1H, 3J = 7.8 Hz, 3J = 3.9 Hz), 8.43 (d, 1H, 3J = 7.8 Hz), 8.68 (dd,
respectively 240 and 110 Ci/mmol (11 and 5% of the carrier
free specific activity).
CONCLUSION
■
Our studies aimed at the development of new potential
radiotracers for SPECT imaging of brain 5-HT4 receptors.
Starting from a phenanthridine scaffold we designed new
antagonists exhibiting high affinity and selectivity toward this
receptor. The fluorinated compound 4v has shown the best
profile with a low subnanomolar Ki value and a high selectivity
toward other 5-HT receptor subtypes. Compared to the
phenanthridine analog 4m, the additional nitrogen atom in
compound 4v led to a significant improvement of both affinity
and selectivity for the 5-HT4R. Having established the
structure−affinity relationships for this series, we have
successfully introduced iodine atoms without negatively
affecting either affinities or selectivities. Among these iodinated
compounds, 11d was chosen for further evaluation as a
potential radiotracer. This compound was able to displace a
specific 5-HT4R ligand both in vitro and in vivo at
subnanomolar concentrations, thereby demonstrating receptor
specificity and capacity to cross the blood−brain barrier. Three
iodinated compounds were successfully radiolabeled with [125I]
and owing to their favorable pharmacological properties
represent novel candidates for further evaluation as SPECT
radiotracers.
3
4
3
1H, J = 7.8 Hz, J = 1.9 Hz), 9.07 (d, 1H, J = 3.9 Hz), 11.87 (brs,
1H). 13C NMR (100 MHz, DMSO-d6): δ 116.5 (d, 2J = 17 Hz), 119.7
(d, J = 3 Hz), 121.0 (d, J = 2 Hz), 121.6, 122.2 (d, J = 7 Hz), 123.9,
2
1
126.5 (d, J = 14 Hz), 135.9, 149.4 (d, J = 243 Hz), 149.9 (d, J = 3
Hz), 154.3, 160.7. HRMS/EI: calcd for C12H7FN2O 214.0542, found
214.0548.
General Procedure A for the Synthesis of Compounds 4a−v
and 11a−d. The chosen (aza)phenanthridin-6(5H)-one (3a−v, 8a−
b) and POCl3 (5 mL mmol−1) were heated to 90 °C overnight in a
round-bottom flask. After cooling, the mixture was poured carefully on
cold water and crushed ice. The pH was carefully adjusted to 12 using
a 28% ammonia solution. The product was extracted using EtOAc
(three times). The organic phase was dried with MgSO4, filtered, and
evaporated. The crude material was added at 0 °C to a solution of
either (1-propylpiperidin-4-yl)methanol or {1-[3-(4-iodophenyl)-
propyl]piperidin-4-yl}methanol 10 (1 equiv) and NaH (4 equiv) in
anhydrous DMF (10 mL mmol−1). The solution was allowed to reach
room temperature, stirred overnight, hydrolyzed with water, and
extracted with AcOEt (3 times). The combined organic phases were
washed with water (three times), dried over MgSO4, filtered,
evaporated, and purified by silica gel chromatography.
6-(1-Propylpiperidin-4-yl)methyloxyphenanthridine (4a).
Starting from 3a (213 mg, 1.1 mmol) using general procedure A
and cyclohexane/ethyl acetate 8/2 with 5% of NEt3 as the eluent for
the chromatography, 4a was obtained as a white powder (277 mg).
Yield: 76%. Mp: 74−76 °C. IR (KBr): ν (cm−1) 2924, 1590, 1461,
1343, 1319. 1H NMR (400 MHz, CDCl3): δ 0.91 (t, 3H, 3J = 7.6 Hz),
1.50−1.62 (m, 4H), 1.91−2.03 (m, 5H), 2.30−2.34 (m, 2H), 3.00−
3.03 (m,2H), 4.50 (d, 2H, 3J = 6.0 Hz), 7.47 (ddd, 1H, 3J = 8.0 Hz, 3J
= 6.8 Hz, 4J = 1.2 Hz), 7.59−7.64 (m, 2H), 7.80 (ddd, 1H, 3J = 8.4 Hz,
3J = 7.2 Hz, 4J = 1.6 Hz), 7.86 (dd, 1H, 3J = 8.4 Hz, 4J = 1.6 Hz), 8.38
EXPERIMENTAL SECTION
■
All chemical reagents and solvents were purchased from commercial
sources and used without further purification except THF, which was
distilled from Na/benzophenone. Thin-layer chromatography (TLC)
was performed on silica gel plates. Silica gel 0.06−0.2 mm, 60 Å was
used for all column chromatography. Melting points were determined
on a Kofler melting point apparatus. IR spectra were recorded as neat
films on a Nicolet 380 FT-IR or on KBr disks using a PerkinElmer BX-
(dd, 1H, 3J = 8.0 Hz, 4J = 1.2 Hz), 8.40−8.42 (m, 1H), 8.48−8.51 (m,
1H). 13C NMR (100 MHz, CDCl3): δ 12.3, 20.4, 29.5 (2C), 36.1, 53.8
(2C), 61.4, 70.6, 120.4, 122.0, 122.2, 122.6, 124.4, 125.2, 127.3, 127.9,
128.9, 130.9, 134.9, 143.5, 159.1. LC−MS (ESI): tR = 5.02 min; [M +
H]+ 335.58. HRMS/EI: calcd for C22H26N2O 334.2046, found
334.2046.
1
FT-IR. H and 13C NMR spectra were recorded on a JEOL Lambda
400 spectrometer with chemical shifts expressed in parts per million
(in DMSO-d6 or CDCl3). High-resolution mass spectra (EI) were
performed on a JEOL GC-Mate spectrometer. High-resolution mass
spectra (ESI) were performed on a Bruker APEX III FT-ICR-MS
system. Elemental analyses were performed at the “Institut de
Recherche en Chimie Organique Fine” (Rouen, France). The purities
of all tested compounds were analyzed by LC−MS, with the purity all
being higher than 95%. Analyses were performed using a Waters
alliance 2695 using the following gradient: A (95%)/B (5%) to A
(5%)/B (95%) in 10 min. This ratio was held for 3 min before
returning to initial conditions in 1 min. Initial conditions were then
maintained for 5 min (A, H2O; B, MeCN; each containing 0.1%
HCOOH; column, C18 Xterra MSC118/2.1_50 mm). MS detection
was performed with a Micromass ZMD 2000. Suitable crystals of
solved structures were obtained by slow evaporation from MeCN
solution. Data for crystal structures analysis were collected at 296 K
with a Bruker−Nonius Kappa CCD area detector diffractometer with
graphite−monochromatized Mo Kλ radiation (λ = 0.710 73 Å). The
structures were solved using direct methods and refined by full-matrix
least-squares analysis on F2. SHELXS−97 (Sheldrick) was used to
solve structures, to refine structures, and to prepare material for
publication. Crystallographic data for compounds 4c, 4d, 4g, 4h, 4j,
4m, 4p, 4q, 4r, 4v, and 11d have been deposited at the Cambridge
Crystallographic Data Centre, CCDC No 889427, 889429−889437,
and 889548. Copies of this information may be obtained free of charge
from the Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK
(+44−1223−336408; E-mail, deposit@ccdc.cam.ac.uk; Web site,
http://www.ccdc.cam.ac.uk).
2 - F l u o r o - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4b). Starting from 3b (180 mg, 0.84
mmol), using general procedure A and cyclohexane/ethyl acetate 7/3
with 5% of NEt3 as the eluent for the chromatography, 4b was
obtained as a white powder (169 mg). Yield: 57%. Mp: 115−116 °C.
IR (KBr): ν (cm−1) 3076, 2927, 2764, 1618, 1591, 1495, 1453, 1436,
1348, 1315, 1243. 1H NMR (400 MHz, CDCl3): δ 0.91 (t, 3H), 1.53−
1.59 (m, 4H), 1.90−2.02 (m, 5H), 2.29−2.33 (m, 2H), 2.99−3.02 (m,
2H), 4.45 (d, 2H, 3J = 6.1 Hz), 7.33 (ddd, 1H, 3J = 8.8 Hz, 3J = 8.0 Hz,
4J = 2.8 Hz), 7.64 (ddd, 1H, 3J = 8.0 Hz, 3J = 6.8 Hz, 4J = 1.2 Hz), 7.77
(ddd, 1H, 3J = 8.0 Hz, 3J = 7.2 Hz, 4J = 1.6 Hz), 7.81 (dd, 1H, 3J = 8.8
Hz, 3J = 5.2 Hz), 7.98 (dd, 1H, 3J = 10.0 Hz, 4J = 2.8 Hz), 8.33 (d, 1H,
3
4
3J = 8.4 Hz), 8.36 (dd, 1H, J = 8.0 Hz, J = 1.2 Hz). 13C NMR (100
MHz, CDCl3): δ 12.1, 20.2, 29.3 (2C), 35.9, 53.6 (2C), 61.2, 70.5,
107.1 (d, 2J = 23 Hz), 116.9 (d, 2J = 24 Hz), 120.2, 121.9, 123.3 (d, J =
9 Hz), 125.1, 127.7, 129.4 (d, J = 5 Hz), 130.8, 134.0 (d, J = 4 Hz),
139.8, 158.3 (d, J = 2 Hz), 159.7 (d, 1J = 241 Hz). LC−MS (ESI): tR =
5.23 min; [M + H]+ 353.34. HRMS/EI: calcd for C22H25FN2O
352.1950, found 352.1938.
2 - C h l o r o - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4c). Starting from 3c (670 mg, 2.9
mmol), using general procedure A and cyclohexane/ethyl acetate 8/2
with 5% of NEt3 as the eluent for the chromatography, 4c was
obtained as a white powder (753 mg). Yield: 70%. Mp: 110−111 °C.
IR (KBr): ν (cm−1) 2930, 1589, 1345, 1317, 1096, 820. 1H NMR (400
MHz, CDCl3): δ 0.92 (t, 3H, 3J = 6.8 Hz), 1.50−1.61 (m, 4H), 1.91−
7-Fluorobenzo[h]-1,6-naphthyridin-5(6H)-one (3v). In a
sealed tube were introduced KOH (1.94 g, 34.6 mmol), 2-(2,3-
9699
dx.doi.org/10.1021/jm300943r | J. Med. Chem. 2012, 55, 9693−9707

Journal of Medicinal Chemistry
Article
2.02 (m, 5H), 2.30−2.34 (m, 2H), 3.00−3.03 (m, 2H), 4.47 (d, 2H, 3J
LC−MS (ESI): tR = 5.25 min; [M + H]+ 365.38. HRMS/EI: calcd for
C23H28N2O2 364.2150, found 364.2140.
= 5.9 Hz), 7.54 (dd, 1H, 3J = 8.8 Hz, 4J = 1.9 Hz), 7.65 (t, 1H, 3J = 6.8
4
3 - M e t h y l - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4h). Starting from 3h (120 mg, 0.57
mmol), using general procedure A and cyclohexane/ethyl acetate 8/2
with 5% of NEt3 as the eluent for the chromatography, 4h was
obtained as a white powder (118 mg). Yield: 59%. Mp: 107−108 °C.
Hz), 7.76−7.81 (m, 2H), 8.33 (d, 1H, J = 2.0 Hz), 8.35−8.39 (m,
2H). 13C NMR (100 MHz, CDCl3): δ 12.1, 20.2, 29.3 (2C), 35.9, 53.6
(2C), 61.2, 70.6, 120.3, 121.8, 121.9, 123.5, 125.1, 127.8, 129.0, 129.1,
129.8, 131.0, 133.7, 141.8, 159.1. LC−MS (ESI): tR = 5.58 min; [M +
H]+ 369.26, 371.26. HRMS/EI: calcd for C22H25ClN2O 368.1655,
found 368.1659.
1
IR (KBr): ν (cm−1) 3430, 2939, 1590, 1344, 1314, 1090, 774. H
NMR (400 MHz, CDCl3): δ 0.91 (t, 3H, 3J = 6.8 Hz), 1.53−1.60 (m,
4H), 1.92−2.03 (m, 5H), 2.30−2.34 (m, 2H), 2.53 (s, 3H), 3.00−3.03
(m, 2H), 4.49 (d, 2H, 3J = 5.8 Hz), 7.30 (d, 1H, 3J = 8.8 Hz), 7.59 (t,
1H, 3J = 7.8 Hz), 7.68 (s, 1H), 7.78 (t, 1H, 3J = 7.8 Hz), 8.29 (d, 1H,
2 - M e t h y l - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4d). Starting from 3d (400 mg, 1.9
mmol), using general procedure A and cyclohexane/ethyl acetate 8/2
with 5% of NEt3 as the eluent for the chromatography, 4d was
obtained as a white powder (326 mg). Yield: 49%. Mp: 87−89 °C. IR
(KBr): ν (cm−1) 2951, 2928, 2798, 2761, 1589, 1344, 1317, 1302,
1148, 1088, 820, 772. 1H NMR (400 MHz, CDCl3): δ 0.91 (t, 3H, 3J =
7.8 Hz), 1.52−1.60 (m, 4H), 1.92−2.02 (m, 5H), 2.30−2.34 (m, 2H),
3J = 7.8 Hz), 8.36 (d, 1H, 3J = 8.8 Hz), 8.46 (d, 1H, 3J = 7.8 Hz). 13
C
NMR (100 MHz, CDCl3): δ 12.1, 20.2, 21.5, 29.4 (2C), 36.0, 53.7
(2C), 61.3, 70.4, 119.9, 120.0, 121.6, 121.9, 125.0, 125.9, 126.7, 127.6,
130.7, 134.9, 138.9, 143.4, 159.0. LC−MS (ESI): tR = 5.64 min; [M +
H]+ 349.33. HRMS/ESI: calcd for C23H29N2O [M + H]+ 349.2280,
found 349.2266.
3
2.56 (s, 3H), 3.00−3.03 (m, 2H), 4.48 (d, 2H, J = 5.9 Hz), 7.43 (d,
3
3
1H, J = 7.8 Hz), 7.61 (t, 1H, J = 6.8 Hz), 7.75−7.80 (m, 2H), 8.20
(s, 1H), 8.36 (d, 1H, 3J = 7.8 Hz), 8.48 (d, 1H, 3J = 7.8 Hz). 13C NMR
(100 MHz, CDCl3): δ 12.1, 20.2, 21.7, 29.3 (2C), 36.0, 53.7 (2C),
61.3, 70.4, 120.2, 121.8, 121.9, 122.2, 125.0, 127.0, 127.5, 130.3, 130.6,
133.8, 134.6, 141.4, 158.4. LC−MS (ESI): tR = 5.53 min; [M + H]+
349.35. Anal. Calcd for C22H26N2O: C, 79.01; H, 7.84; N, 8.38.
Found: C, 79.27; H, 7.91; N, 8.01.
3 - F l u o r o - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4i). Starting from 3i (100 mg, 0.47
mmol), using general procedure A and cyclohexane/ethyl acetate 7/3
with 5% of NEt3 as the eluent for the chromatography, 4i was obtained
as a colorless oil (107 mg). Yield: 65%. IR (KBr): ν (cm−1) 2995,
1
2852, 1588, 1463, 1260, 1088, 1019, 799, 770. H NMR (400 MHz,
3
CDCl3): δ 0.91 (t, 3H, J = 7.8 Hz), 1.54−1.64 (m, 9H), 1.90−2.02
2 - M e t h o x y - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4e). Starting from 3e (300 mg, 1.33
mmol), using general procedure A and cyclohexane/ethyl acetate 9/1
with 5% of NEt3 as the eluent for the chromatography, 4e was
obtained as a colorless oil (440 mg). Yield: 91%. IR (KBr): ν (cm−1)
2935, 1620, 1591, 1498, 1345, 1316, 1243. 1H NMR (400 MHz,
DMSO-d6): δ 0.84 (t, 3H, 3J = 7.8 Hz), 1.38−1.45 (m, 4H), 1.81−1.91
(m, 5H), 2.19−2.22 (m, 2H), 2.86−2.89 (m, 2H), 3.94 (s, 3H), 4.36
(d, 2H, 3J = 5.9 Hz), 7.27 (dd, 1H, 3J = 8.8 Hz, 4J = 2.9 Hz), 7.70−7.76
(m, 2H), 7.90 (t, 1H, 3J = 6.8 Hz), 8.04 (d, 1H, 4J = 2.9 Hz), 8.28 (d,
(m, 5H), 2.30−2.34 (m, 2H), 3.01−3.04 (m, 2H), 4.48 (d, 2H, 3J = 5.8
Hz), 7.19−7.28 (m, 1H), 7.52 (dd, 1H, 3J = 10.8 Hz, 3J = 2.9 Hz), 7.62
(t, 1H, 3J = 7.8 Hz), 7.80 (t, 1H, 3J = 6.8 Hz), 8.34−8.43 (m, 3H). 13
C
NMR (100 MHz, CDCl3): δ 12.1, 20.2, 29.3 (2C), 35.9, 53.7 (2C),
61.3, 70.3, 112.7 (d, 2J = 22 Hz), 112,9 (d, 2J = 23 Hz), 119.0 (d, J = 1
Hz), 119.6, 121.7, 123.7 (d, J = 10 Hz), 125.2, 127.0, 131.1, 134.5,
144.8 (d, J = 12 Hz), 159.7, 163.0 (d, 1J = 246 Hz). LC−MS (ESI): tR
= 5.47 min; [M + H]+ 353.28. HRMS/EI: calcd for C22H25FN2O
352.1950, found 352.1941.
4 - M e t h y l - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4j). Starting from 3j (217 mg, 1.03
mmol), using general procedure A and cyclohexane/ethyl acetate 9/1
with 5% of NEt3 as the eluent for the chromatography, 4j was obtained
as a white powder (170 mg). Yield: 47%. Mp: 93−95 °C. IR (KBr): ν
(cm−1) 2938, 2764, 1589, 1312, 760. 1H NMR (400 MHz, CDCl3): δ
3
3
1H, J = 7.8 Hz), 8.76 (d, 1H, J = 8.8 Hz). 13C NMR (100 MHz,
DMSO-d6): δ 11.9, 19.7, 28.8 (2C), 35.5, 53.1 (2C), 55.6, 60.3, 70.0,
104.3, 118.3, 119.2, 122.8, 122.9, 124.3, 127.9, 128.6, 131.1, 133.9,
137.1, 156.5, 156.7. LC−MS (ESI): tR = 5.31 min; [M + H]+ 365.38.
HRMS/ESI: calcd for C23H29N2O2 [M + H]+ 365.2229, found
365.2211.
3
0.91 (t, 3H, J = 7.5 Hz,), 1.51−1.62 (m, 4H), 1.92−2.04 (m, 5H),
3 - C h l o r o - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4f). Starting from 3f (124 mg, 0.54
mmol), using general procedure A and cyclohexane/ethyl acetate 95/5
with 5% of NEt3 as the eluent for the chromatography, 4f was obtained
as a white powder (173 mg). Yield: 87%. Mp: 84−86 °C. IR (KBr): ν
(cm−1) 3400, 2939, 1590, 1482, 1352, 1317, 1081, 765. 1H NMR (400
3
2.30−2.33 (m, 2H), 2.73 (s, 3H), 3.01 (m, 2H), 4.52 (d, 2H, J = 6.5
Hz), 7.37 (dd, 1H, 3J = 8.0 Hz, 3J = 7.0 Hz), 7.48−7.50 (m, 1H), 7.61
(ddd, 1H, 3J = 8.0 Hz, 3J = 7.5 Hz, 4J = 1.5 Hz), 7.78 (ddd, 1H, 3J = 8.0
3
4
3
Hz, J = 7.0 Hz, J = 1.5 Hz), 8.27−8.29 (m, 1H), 8.37 (dd, 1H, J =
8.1 Hz, 4J = 1.4 Hz), 8.49−8.51 (m, 1H). 13C NMR (100 MHz,
CDCl3): δ 12.1, 18.3, 20.2, 29.4 (2C), 35.9, 53.7 (2C), 61.2, 70.3,
119.8, 119.9, 122.0, 122.1, 123.7, 124.9, 126.9, 129.4, 130.6, 135.2,
135.7, 141.8, 157.6. LC−MS (ESI): tR = 5.65 min; [M + H]+ 349.57.
HRMS/EI: calcd for C23H28N2O 348.2201, found 348.2215.
3
MHz, DMSO-d6): δ 0.90 (t, 3H, J = 6.4 Hz), 1.70−1.80 (m, 4H),
2.02−2.04 (m, 5H), 2.31−2.34 (m, 2H), 2.93−3.00 (m, 2H), 4.48 (s,
3
3
2H), 7.57 (d, 1H, J = 8.8 Hz), 7.76−7.81 (m, 2H), 7.96 (t, 1H, J =
6.8 Hz), 8.37 (d, 1H, 3J = 6.8 Hz), 8.67 (d, 1H, 3J = 8.8 Hz), 8.75 (d,
4 - M e t h o x y - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4k). Starting from 3k (150 mg, 0.66
mmol), using general procedure A and cyclohexane/ethyl acetate 9/1
with 5% of NEt3 as the eluent for the chromatography, 4k was
obtained as a white powder (153 mg). Yield: 47%. Mp: 99−100 °C. IR
1H, J = 7.8 Hz). 13C NMR (100 MHz, CDCl3): δ 12.0, 20.5, 28.9
3
(2C), 36.2, 53.1 (2C), 61.2, 70.6, 119.7, 121.8, 122.2, 122.9, 125.1,
127.8, 128.7, 129.1, 129.8, 131.0, 133.7, 141.8, 159.1. LC−MS (ESI):
tR = 5.79 min; [M + H]+ 369.32, 371.33. HRMS/EI: calcd for
C22H25ClN2O 368.1655, found 368.1666.
1
(KBr): ν (cm−1) 3433, 2951, 2768, 1590, 1321, 1256, 748. H NMR
3
3 - M e t h o x y - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4g). Starting from 3g (291 mg, 1.3
mmol), using general procedure A and cyclohexane/ethyl acetate 9/1
with 5% of NEt3 as the eluent for the chromatography, 4g was
obtained as a white powder (334 mg). Yield: 71%. Mp: 76−77 °C. IR
(KBr): ν (cm−1) 2932, 1615, 1587, 1484, 1350, 1315, 1174, 1087, 983,
770. 1H NMR (400 MHz, CDCl3): δ 0.91 (t, 3H, 3J = 7.8 Hz), 1.50−
1.62 (m, 4H), 1.92−2.03 (m, 5H), 2.30−2.34 (m, 2H), 3.01−3.03 (m,
2H), 3.96 (s, 3H), 4.48 (d, 2H, 3J = 6.8 Hz), 7.09 (dd, 1H, 3J = 8.8 Hz,
4J = 2.0 Hz), 7.30 (d, 1H, 4J = 2.0 Hz), 7.55 (t, 1H, 3J = 6.8 Hz), 7.75
(400 MHz, CDCl3): δ 0.92 (t, 3H, J = 7.8 Hz), 1.52−1.67 (m, 4H),
1.94−2.04 (m, 5H), 2.31−2.35 (m, 2H), 3.01−3.04 (m, 2H), 4.08 (s,
3H), 4.56 (d, 2H, 3J = 5.9 Hz), 7.10 (d, 1H, 3J = 7.8 Hz), 7.42 (t, 1H,
3J = 8.8 Hz), 7.64 (t, 1H, 3J = 7.8 Hz), 7.80 (t, 1H, 3J = 8.8 Hz), 8.03
3
3
3
(d, 1H, J = 7.8 Hz), 8.40 (d, 1H, J = 7.8 Hz), 8.49 (d, 1H, J = 7.8
Hz). 13C NMR (100 MHz, CDCl3): δ 12.1, 20.2, 29.3 (2C), 36.1, 53.7
(2C), 56.5, 61.3, 70.4, 109.4, 114.4, 120.2, 122.4, 123.6, 124.3, 125.1,
127.3, 130.8, 133.8, 134.9, 154.5, 158.4. LC−MS (ESI): tR = 5.24 min;
[M + H]+ 365.38. HRMS/EI: calcd for C23H28N2O2 364.2150, found
364.2139.
3
3
3
(t, 1H, J = 8.8 Hz), 8.28 (d, 1H, J = 8.8 Hz), 8.34 (d, 1H, J = 7.8
Hz), 8.37 (d, 1H, 3J = 7.8 Hz). 13C NMR (100 MHz, CDCl3): δ 12.1,
20.2, 29.3 (2C), 36.0, 53.7 (2C), 55.5, 61.3, 70.5, 108.5, 114.6, 116.2,
119.1, 121.3, 123.3, 125.1, 126.0, 130.9, 134.9, 145.0, 159.5, 160.3.
4 - C h l o r o - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4l). Starting from 3l (150 mg, 0.65
mmol), using general procedure A and cyclohexane/ethyl acetate 9/1
with 5% of NEt3 as the eluent for the chromatography, 4l was obtained
9700
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Journal of Medicinal Chemistry
Article
3
as a white powder (149 mg). Yield: 62%. Mp: 75−77 °C. IR (KBr): ν
(cm−1) 3434, 2919, 2852, 1591, 1458, 1399, 1342, 1097, 747. 1H
NMR (400 MHz, CDCl3): δ 0.91 (t, 3H, 3J = 6.8 Hz), 1.52−1.62 (m,
4H), 1.92−2.03 (m, 5H), 2.30−2.34 (m, 2H), 3.00−3.03 (m, 2H),
CDCl3): δ 0.86 (t, 3H, J = 6.8 Hz), 1.51−1.57 (m, 4H), 1.93−2.03
(m, 5H), 2.30−2.34 (m, 2H), 3.00−3.03 (m, 2H), 3.98 (s, 3H), 4.51
(d, 2H, 3J = 6.8 Hz), 7.43 (m, 2H), 7.45 (t, 1H, 3J = 6.8 Hz), 7.71 (d,
1H, 3J = 2.9 Hz), 7.84 (d, 1H, 3J = 8.8 Hz), 8.33 (d, 1H, 3J = 7.8 Hz),
8.42 (d, 1H, 3J = 8.8 Hz). 13C NMR (100 MHz, CDCl3): δ 12.1, 20.2,
29.3 (2C), 35.8, 53.7 (2C), 55.5, 61.3, 70.3, 105.3, 120.8, 121.3, 121.4,
122.4, 123.5, 124.3, 127.6, 127.7, 128.9, 142.2, 158.2, 158.7. LC−MS
(ESI): tR = 5.47 min; [M + H]+ 365.33. HRMS/EI: calcd for
C23H28N2O2 364.2150, found 364.2135.
3
3
3
4.60 (d, 2H, J = 6.8 Hz), 7.38 (t, 1H, J = 7.8 Hz), 7.67 (t, 1H, J =
3
3
6.8 Hz), 7.73 (d, 1H, J = 7.8 Hz), 7.83 (t, 1H, J = 6.8 Hz), 8.33 (d,
1H, 3J = 7.8 Hz), 8.40 (d, 1H, 3J = 7.8 Hz), 8.49 (d, 1H, 3J = 8.8 Hz).
13C NMR (100 MHz, CDCl3): δ 12.1, 20.2, 29.3 (2C), 35.9, 53.7
(2C), 61.2, 70.8, 120.1, 120.8, 122.2, 124.0, 124.1, 125.2, 127.8, 129.1,
131.2, 132.0, 134.7, 139.8, 159.2. LC−MS (ESI): tR = 5.64 min; [M +
H]+ 369.35, 371.30. HRMS/ESI: calcd for C23H26ClN2O [M + H]+
369.1734, found 365.1729.
8 - F l u o r o - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4q). Starting from 3q (0.26 g, 1.22
mmol), following general procedure A and using cyclohexane/ethyl
acetate (9/1) as the eluent for the chromatography, 4q was obtained
as a white powder (0.19 g). Yield: 44%. Mp: 70−71 °C. IR (KBr): ν
4 - F l u o r o - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4m). Starting from 3m (260 mg, 1.22
mmol), using general procedure A and cyclohexane/ethyl acetate 8/2
with 5% of NEt3 as the eluent for the chromatography, 4m was
obtained as a white powder (288 mg). Yield: 67%. Mp: 105−106 °C.
IR (KBr): ν (cm−1) 2925, 1591, 1348, 1317, 1234, 753. 1H NMR (400
MHz, CDCl3): δ 0.91 (t, 3H, 3J = 7.5 Hz), 1.51−1.64 (m, 4H), 1.93−
2.03 (m, 5H), 2.31−2.34 (m, 2H), 3.02−3.04 (m, 2H), 4.55 (d, 2H, 3J
= 6.0 Hz), 7.33−7.41 (m, 2H), 7.67 (ddd, 1H, 3J = 8.0 Hz, 3J = 7.0 Hz,
(cm−1) 2939, 755. H NMR (400 MHz, CDCl3): δ 0.92 (t, 3H, J =
1
3
7.0 Hz), 1.53−1.60 (m, 4H), 1.92−2.02 (m, 5H), 2.31−2.34 (m, 2H),
3
3
3.01−3.03 (m, 2H), 4.47 (d, 2H, J = 6.8 Hz), 7.48 (t, 1H, J = 7.5
Hz), 7.52 (dt, 3J = 8.5 Hz, 4J = 2.5 Hz, 1H), 7.61 (t, 1H, 3J = 7.5 Hz),
7.86 (d, 1H, 3J = 8.0 Hz), 7.97 (dd, 1H, 3J = 9.0 Hz, 4J = 2.5 Hz), 8.34
(d, 1H, 3J = 8.0 Hz), 8.48 (dd, 1H, 3J = 9.0 Hz, 4J = 5.5 Hz). 13C NMR
(100 MHz, CDCl3): δ 12.1, 20.2, 29.3 (2C), 35.9, 53.7 (2C), 61.3,
70.3, 110.1 (d, 2J = 22 Hz), 119.8 (d, 2J = 22 Hz), 121.6 (d, J = 7 Hz),
121.9 (d, J = 11 Hz), 122.0, 124.5 (d, J = 7 Hz), 124.7, 128.0, 128.7,
131.5 (d, J = 2 Hz), 143.0, 158.2 (d, J = 2 Hz), 161.7 (d,1J = 246 Hz).
LC−MS (ESI): tR = 5.41 min; [M + H]+ 353.31. HRMS/EI: calcd for
C22H25FN2O 352.1950, found 352.1958.
3
3
4
4J = 1.0 Hz), 7.82 (ddd, 1H, J = 8.2 Hz, J = 7.2 Hz, J = 1.4 Hz),
8.17−8.18 (m, 1H), 8.39−8.41 (m, 1H), 8.46 (d, 1H, 3J = 8.5 Hz). 13
NMR (100 MHz, CDCl3): δ 12.1, 20.2, 29.3 (2C), 36.0, 53.6 (2C),
61.2, 70.7, 113.9 (d, J = 20 Hz), 117.5 (d, J = 4 Hz), 120.3, 122.2,
C
2
123.8 (d, J = 8 Hz), 124.5 (d, J = 2 Hz), 125.2, 127.7, 131.2, 132.7 (d,
1
2J = 11 Hz), 134.2 (d, J = 3 Hz), 157.5 (d, J = 250 Hz), 159.1. LC−
9 - M e t h y l - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4r). Starting from 3r (1.00 g, 4.77
mmol), following general procedure A and using cyclohexane/ethyl
acetate (9/1) as the eluent for the chromatography 4r was obtained as
a white powder (1.18 g). Yield: 71%. Mp: 90−91 °C. IR (KBr): ν
(cm−1) 3411, 3064, 2933, 1596, 1336, 758. 1H NMR (400 MHz,
MS (ESI): tR = 5.32 min; [M + H]+ 353.30. HRMS/EI: calcd for
C22H25FN2O 352.1951, found 352.1943.
7 - F l u o r o - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4n). In sealed tube were introduced
KOH (1.07 g, 19 mmol), 2′-fluoro-3-fluorobiphenyl-2-carbonitrile41
(0.82 g, 3.8 mmol), and t-BuOH (30 mL). The tube was heated to 150
°C for 0.5 h. Water (20 mL) was added and the resulting precipitate
was filtered. The solid was then dried under vacuum to afford a
mixture of the expected 7-fluorophenanthridin-6(5H)-one along with
side products. The mixture was then subjected to general procedure A
using cyclohexane/ethyl acetate 9/1 as the eluent for the
chromatography. 4n was obtained as yellow oil (0.12 g). Yield: 9%.
IR (KBr): ν (cm−1) 3432, 2931, 2765, 1595, 1338, 758. 1H NMR (400
MHz, CDCl3): δ 0.91 (t, 3H, 3J = 6.8 Hz), 1.52−1.54 (m, 4H), 1.99−
2.02 (m, 5H), 2.30−2.34 (m, 2H), 3.00−3.04 (m, 2H), 4.47 (d, 2H, 3J
= 6.8 Hz), 7.28−7.31 (m, 1H), 7.47 (t, 1H, 3J = 6.8 Hz), 7.63 (t, 1H, 3J
3
CDCl3): δ 0.90 (t, 3H, J = 6.8 Hz), 1.50−1.61 (m, 4H), 1.92−2.01
(m, 5H), 2.30−2.32 (m, 2H), 2.60 (s, 3H), 2.99−3.02 (m, 2H), 4.48
3
3
(d, 2H, J = 6.8 Hz), 7.42−7.46 (m, 2H), 7.59 (t, 1H, J = 6.8 Hz),
7.83 (d, 1H, J = 8.8 Hz’), 8.23−8.26 (m, 2H), 8.37 (d, 1H, J = 7.8
Hz). 13C NMR (100 MHz, CDCl3): δ 12.1, 20.2, 22.3, 29.3 (2C), 35.9,
53.7 (2C), 61.3, 70.3, 118.1, 121.6, 122.0, 122.3, 124.0, 124.9, 127.6,
128.6, 128.7, 134.8, 141.1, 143.6, 159.0. LC−MS (ESI): tR = 5.61 min;
[M + H]+ 349.32. HRMS/EI: calcd for C23H28N2O 348.2201, found
348.2202.
3
3
9 - F l u o r o - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4s). Starting from 3s (0.20 g, 0.94
mmol), following general procedure A and using cyclohexane/ethyl
acetate (9/1) as the eluent for the chromatography, 4s was obtained,
after recrystalisation in MeCN, as a white powder (0.20 g). Yield: 62%.
Mp: 79−80 °C. IR (KBr): ν (cm−1) 3399, 2944, 2765, 1593, 1336,
762. 1H NMR (400 MHz, CDCl3): δ 0.90 (t, 3H, 3J = 7.2 Hz), 1.52−
1.59 (m, 4H), 1.89−1.99 (m, 5H), 2.30−2.33 (m, 2H), 3.00 (m, 2H),
4.48 (d, 2H, 3J = 6.8 Hz), 7.33 (ddd, 1H, 3J = 8.9 Hz, 3J = 8.2 Hz, 4J =
3
3
= 6.8 Hz), 7.72 (m, 1H), 7.83 (d, 1H, J = 9.8 Hz), 8.29 (d, 1H, J =
8.7 Hz), 8.36 (d, 1H, J = 6.8 Hz). 13C NMR (100 MHz, CDCl3): δ
3
12.1, 20.2, 29.3 (2C), 35.9, 53.7 (2C), 61.3, 70.3, 109.7 (d, J = 9 Hz),
2
111.7 (d, J = 5 Hz), 114.1 (d, J = 23 Hz), 121.2, 122.5, 124.5, 127.6,
129.4, 131.4 (d, 2J = 10 Hz), 137.7, 143.4, 157.6 (d, J = 7 Hz), 160.23
1
(d, J = 262 Hz). LC−MS (ESI): tR = 5.38 min; [M + H]+ 353.47.
HRMS/EI: calcd for C22H25FN2O 352.1950, found 352.1935.
8-Nitro-6-(1-propylpiperidin-4-yl)methyloxyphenanthridine
(4o). Starting from 3o (0.80 g, 3.3 mmol), following general procedure
A and using cyclohexane/ethyl acetate (9/1) as eluent for the
chromatography, 4o was obtained as a white powder (0.72 g). Yield:
57%. Mp: 123−125 °C. IR (KBr): ν (cm−1) 2940, 2765, 1604, 1344.
3
3
4
2.5 Hz), 7.47 (ddd, 1H, J = 8.1 Hz, J = 7.1 Hz, J = 1.3 Hz), 7.64
(ddd, 1H, 3J = 8.1 Hz, 3J = 7.1 Hz, 4J = 1.4 Hz), 7.86 (dd, 1H, 3J = 8.1
4
3
4
Hz, J = 1.4 Hz), 8.04 (dd, 1H, J = 10.7 Hz, J = 2.9 Hz), 8.28 (dd,
1H, 3J = 8.3 Hz, 4J = 1.4 Hz), 8.39 (dd, 1H, 3J = 8.8 Hz, 3J = 5.8 Hz).
13C NMR (100 MHz, CDCl3): δ 12.1, 20.2, 29.3 (2C), 35.9, 53.7
(2C), 61.3, 70.3, 107.2 (d, 2J = 22 Hz), 115.8 (d, 2J = 23 Hz), 116.9 (d,
4J = 1 Hz), 121.9 (d, 4J = 3 Hz), 122.3, 124.3, 127.8, 127.9 (d, 3J = 10
3
1H NMR (400 MHz, CDCl3): δ 0.92 (t, 3H, J = 7.8 Hz), 1.53−1.58
(m, 4H), 1.95−2.05 (m, 5H), 2.31−2.35 (m, 2H), 3.02−3.05 (m, 2H),
Hz), 129.3, 137.1 (d, 3J = 11 Hz), 143.8, 158.5, 164.3 (d, 1J = 249 Hz).
LC−MS (ESI): tR = 5.45 min; [M + H]+ 353.28. HRMS/EI: calcd for
C22H25FN2O 352.1950, found 352.1938.
3
3
3
4.52 (d, 2H, J = 6.8 Hz), 7.54 (t, 1H, J = 6.8 Hz), 7.73 (t, 1H, J =
6.8 Hz), 7.90 (d, 1H, 3J = 7.8 Hz), 8.42 (d, 1H, 3J = 7.8 Hz), 8.55 (m,
2H), 9.19 (d, 1H, 4J = 1.9 Hz). 13C NMR (100 MHz, CDCl3): δ 12.1,
20.2, 29.3 (2C), 35.9, 53.5 (2C), 61.3, 70.3, 119.8, 121.0, 121.4, 123.0,
123.5, 124.5, 125.1, 128.2, 130.9, 138.9, 144.7, 146.2, 158.4. LC−MS
(ESI): tR = 5.39 min; [M + H]+ 380.36. HRMS/EI: calcd for
C22H25N3O3 379.1895, found 379.1913.
5-(1-Propylpiperidin-4-yl)methyloxybenzo[c]-2,6-naphthyri-
dine (4t). Starting from 3t (0.1 g, 0.5 mmol), following general
procedure A and using cyclohexane/ethyl acetate (4/1) and
cyclohexane/ethyl acetate (1/1) as the eluents for the chromatog-
raphy, 4t was obtained as a white powder (72 mg). Yield: 42%. Mp:
113−115 °C. IR (KBr): ν (cm−1) 2953, 2932, 1613, 1585, 1459, 1337,
1235, 1128, 1985, 844, 768, 673. 1H NMR (400 MHz, CDCl3): δ 0.92
(t, 3H, 3J = 7.4 Hz), 1.50−1.61 (m, 4H), 1.91−2.03 (m, 5H), 2.32 (m,
2H), 3.02 (d, 2H, J = 11.7 Hz), 4.51 (d, 2H, 3J = 6.5 Hz), 7.55 (t, 1H, J
= 7.6 Hz), 7.69 (t, 1H, J = 7.5 Hz), 7.90 (d, 1H, 3J = 8.3 Hz), 8.12 (d,
8 - M e t h o x y - 6 - ( 1 - p r o p y l p i p e r i d i n - 4 - y l ) -
methyloxyphenanthridine (4p). Starting from 3p (0.80 g, 3.55
mmol), following general procedure A and using cyclohexane/ethyl
acetate (9/1) as the eluent for the chromatography, 4p was obtained
as a white powder (0.90 g). Yield: 70%. Mp: 92−94 °C. IR (KBr): ν
1
(cm−1) 3430, 2765, 2940, 1590, 1462, 1219. H NMR (400 MHz,
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Journal of Medicinal Chemistry
Article
1H, 3J = 5.3 Hz), 8.54 (d, 1H, 3J = 8.3 Hz), 8.83 (d, 1H, 3J = 5.3 Hz),
9.92 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 12.0, 20.1, 29.1 (2C),
35.7, 53.5 (2C), 61.1, 70.8, 117.1, 120.2, 121.4, 124.1, 125.2, 127.9,
128.6, 129.5, 143.5, 146.1, 143.4, 157.5. LC−MS (ESI): tR = 4.59 min;
[M + H]+ 336.25. HRMS/ESI: calcd for C21H26N3O [M + H]+
336.2076, found 336.2067.
CDCl3): δ 0.35 (s, 9H), 7.25 (t, 1H, 3J = 7.8 Hz), 7.40−7.53 (m, 4H),
3
3
7.65 (t, 1H, J = 7.8 Hz), 7.77 (d, 1H, J = 7.8 Hz). 13C NMR (100
MHz, CDCl3): δ −1.0, 112.9, 118.1, 124.1 (d, J = 3 Hz), 125.0 (d, 2J =
19 Hz), 127.5 (d, 3J = 36 Hz), 127.9, 130.9, 132.4, 132.4, 133.2, 136.1
1
(d, J = 13 Hz), 140.1, 163.5 (d, J = 242 Hz). HRMS/EI: calcd for
C16H16FNSi 269.1036, found 269.1034.
2-(2-Fluoro-3-trimethylsilanylphenyl)nicotinonitrile (6b).
The same procedure as for 6a, starting from 2-chloronicotinonitrile
(1 g, 7.2 mmol), Pd(OAc)2 (81 mg, 0.36 mmol), PPh3 (189 mg, 0.72
mmol), 5 (2.29 g, 10.8 mmol) and Na2CO3 (3.06 g, 28.8 mmol).
Cyclohexane/AcOEt 8/2 was used as the eluent for chromatography
to afford 6b (1.85 g) as a colorless oil. Yield: 95%. IR (KBr): ν (cm−1)
5-(1-Propylpiperidin-4-yl)methyloxybenzo[h]-1,6-naphthyri-
dine (4u). Starting from 3u (0.1 g, 0.5 mmol), following general
procedure A and using cyclohexane/ethyl acetate (1/1) and ethyl
acetate with 5% of NEt3 as the eluents for the chromatography, 4u was
obtained as a white powder (92 mg). Yield: 54%. Mp: 114−115 °C. IR
1
(KBr): ν (cm−1) 2931, 1605, 1590, 1458, 1328, 767, 733. H NMR
(400 MHz, CDCl3): δ 0.84 (t, 3H, 3J = 7.3 Hz), 1.18 (brs, 1H), 1.43−
1.56 (m, 3H), 1.18−1.96 (m, 5H), 2.23−2.27 (m, 2H), 2.95 (d, 2H, J
= 11.2 Hz), 4.44 (d, 2H, 3J = 6.3 Hz), 7.45−7.50 (m, 2H), 7.62−7.65
(m, 1H), 7.80 (d, 1H, 3J = 7.5 Hz), 8.55 (dd, 1H, 3J = 8.2 Hz, 4J = 1.4
Hz), 8.88 (dd, 1H, 3J = 8.1 Hz, 4J = 1.4 Hz), 9.03 (dd, 1H, 3J = 4.4 Hz,
4J = 1.2 Hz). 13C NMR (100 MHz, CDCl3): δ 12.1, 20.2, 29.3 (2C),
1
2957, 2231 (CN), 1604, 1428, 1414, 1251, 862, 842, 762. H NMR
3
(400 MHz, CDCl3): δ 0.36 (s, 9H), 7.29 (m, 1H), 7.43 (dd, 1H, J =
3
3
8.3 Hz, J = 4.9 Hz), 7.53−7.60 (m, 2H), 8.08 (d, 1H, J = 7.8 Hz),
8.90 (dd, 1H, 3J = 4.9 Hz, 4J = 1.9 Hz). 13C NMR (100 MHz, CDCl3):
δ −1.0, 110.6, 116.5, 122.0, 124.2 (d, J = 3 Hz), 124.7 (d, 2J = 19 Hz),
127.5 (d, 2J = 31 Hz), 132.3 (d, J = 2 Hz), 137.2 (d, J = 11 Hz), 140.6,
152.5, 157.9, 163.8 (d, 1J = 243 Hz). HRMS/EI: calcd for
C15H15FN2Si 270.0988, found 270.0979.
35.9, 53.6 (2C), 61.2, 70.8, 115.4, 122.3, 123.5, 123.7, 124.9, 127.2,
130.4, 133.0, 144.9, 150.8, 152.9, 158.4. LC−MS (ESI): tR = 4.84 min;
[M + H]+ 336.28. HRMS/EI: calcd for C21H25N3O 335.1997, found
335.1998.
2′-Fluoro-3′-iodobiphenyl-2-carbonitrile 7a. In a round-
bottom flask were introduced 6a (1.05 g, 3.7 mmol), DCM (30
mL) and ICl (279 μL, 5.6 mmol). The solution was allowed to stir for
4.5 h and a saturated Na2S2O3 solution in water (40 mL) was added.
The aqueous layer was then extracted with DCM (3 × 30 mL) and the
combined organic layers were dried MgSO4, filtered and evaporated in
vacuo to afford 7a (1.11 g) as a white powder. Yield: 88%. Mp: 123−
7-Fluoro-5-(1-propylpiperidin-4-yl)methyloxybenzo[h]-1,6-
naphthyridine (4v). Starting from 3v (1 g, 4.6 mmol), following
general procedure A and using ethyl acetate and ethyl acetate/NEt3
98/2 as the eluents for the chromatography, 4v was obtained as a
white powder (1.37 g). Yield: 83%. Mp: 119−120 °C. IR (KBr): ν
(cm−1) 2952, 2932, 1605, 1589, 1329, 1235, 1152, 774. 1H NMR (400
1
125 °C. IR (KBr): ν (cm−1) 2222 (CN), 1443, 1425, 788, 759. H
3
NMR (400 MHz, CDCl3): δ 7.03 (t, 1H, 3J = 7.8 Hz), 7.40 (m, 1H),
7.49−7.53 (m, 2H), 7.67 (td, 1H, J = 7.8 Hz, J = 1.9 Hz), 7.67 (d, 1H,
3J = 7.8 Hz), 7.84 (m, 1H). 13C NMR (100 MHz, CDCl3): δ 82.2 (d,
MHz, CDCl3): δ 0.91 (t, 3H, J = 6.8 Hz), 1.50−1.64 (m, 4H), 1.77
(brs, 1H), 1.90−2.04 (m, 4H), 2.30−2.34 (m, 2H), 3.02 (d, 2H, J =
11.7 Hz), 4.56 (d, 2H, 3J = 6.8 Hz), 7.41−7.49 (m, 2H), 7.59 (dd, 1H,
3J = 8.3 Hz, 3J = 4.8 Hz), 8.64 (dd, 1H, 3J = 7.8 Hz, 4J = 1.9 Hz), 8.72
(dd, 1H, 3J = 8.3 Hz, 4J = 1.9 Hz), 9.11 (dd, 1H, 3J = 4.8 Hz, 4J = 1.9
Hz). 13C NMR (100 MHz, CDCl3): δ 12.1, 20.2, 29.2 (2C), 35.8, 53.6
2
2J = 25 Hz), 112.6, 117.7, 125.8 (d, J = 5 Hz), 126.3 (d, J = 17 Hz),
128.6, 130.8 (d, J = 2 Hz), 131.4 (d, J = 2 Hz), 132.6, 133.3, 138.6,
1
140.2 (d, J = 2 Hz), 158.2 (d, J = 246 Hz). HRMS/EI: calcd for
2
(2C), 61.2, 71.1, 115.6, 115.6 (d, J = 19 Hz), 119.2 (d, J = 4 Hz),
C13H7FIN 322.9607, found 322.9601.
122.8, 124.4 (d, J = 7 Hz), 125.5 (d, J = 2 Hz), 133.2, 134.1 (d, 2J = 11
Hz), 150.3 (d, J = 3 Hz), 153.3, 157.0 (d, 1J = 250 Hz), 158.6. LC−MS
(ESI): tR = 6.00 min; [M + H]+ 354.37. HRMS/EI: calcd for
C21H24FN3O 353.1903, found 353.1915.
2-(2-Fluoro-3-iodophenyl)nicotinonitrile (7b). The same
procedure as for 7a was used, starting from 6b (0.82 g, 3 mmol)
and ICl (228 μL, 4.5 mmol). The crude was purified by flash
chromatography using cyclohexane/AcOEt 8/2 as the eluent to afford
7b (0.82 g) as a white powder. Yield: 84%. Mp: 100−101 °C. IR
2-Fluoro-3-(trimethylsilanyl)phenylboronic Acid (5). In a
three necks round-bottom flask under N2 at −80 °C were introduced
(2-fluorophenyl)trimethylsilane42 (7.4 g, 44 mmol), THF (90 mL),
and 1.3 M s-BuLi in n-hexane/cyclohexane 98/2 (33.82 mL, 44
mmol). The yellow solution was stirred for 0.75 h, trimethyl borate
(5.49 mL, 48.4 mmol) was added, and the solution was stirred again
for 0.75 h. The mixture was allowed to reach room temperature,
hydrolyzed with water (200 mL), washed with Et2O (3 × 150 mL),
acidified to pH = 1 using 1 M HCl and extracted with Et2O (3 × 150
mL). The combined organic layers were dried over MgSO4, filtered,
and evaporated in vacuo to afford 5 (6.4 g) as white crystals. Yield:
69%. Mp: 93−94 °C. IR (KBr): ν (cm−1) 3512, 3351, 2952, 1423,
1
(KBr): ν (cm−1) 2228 (CN), 1424, 1227, 805, 761, 725. H NMR
(400 MHz, CDCl3): δ 7.08 (t, 1H, 3J = 7.8 Hz), 7.48 (dd, 1H, 3J = 7.8
3
3
Hz, J = 4.9 Hz), 7.56 (m, 1H), 7.92 (m, 1H), 8.11 (dd, 1H, J = 7.8
4
3
4
Hz, J = 1.9 Hz), 8.92 (dd, 1H, J = 4.9 Hz, J = 1.9 Hz). 13C NMR
(100 MHz, CDCl3): δ 82.3 (d, 2J = 26 Hz), 110.4, 116.1, 122.6, 126.0
(d, J = 5 Hz), 126.1 (d, 2J = 16 Hz), 131.4 (d, J = 2 Hz), 140.8, 141.4
1
(d, J = 2 Hz), 152.6, 156.5, 158.5 (d, J = 247 Hz). HRMS/EI: calcd
for C12H6FIN2 323.9568, found 323.9559.
4-Iodophenanthridin-6(5H)-one (8a). The same procedure as
for compound 3v was used, with 7a (1.11 g, 3.4 mmol), KOH (0.96 g,
14 mmol), and t-BuOH (30 mL) and heating at 150 °C for 1 h. 8a
(0.73 g) was obtained as a white powder. Yield: 67%. Mp: 240−242
°C. IR (KBr): ν (cm−1) 3274, 1654 (CO), 1603, 1351, 752, 709, 625.
1H NMR (400 MHz, DMSO-d6): δ 7.04 (t, 1H, 3J = 7.8 Hz), 7.66 (t,
1H, 3J = 7.8 Hz), 7.86 (t, 1H, 3J = 7.8 Hz), 8.00 (d, 1H, 3J = 6.8 Hz),
8.33 (d, 1H, 3J = 6.8 Hz), 8.42 (d, 1H, 3J = 7.8 Hz), 8.50 (d, 1H, 3J =
8.8 Hz), 9.29 (brs, 1H). 13C NMR (100 MHz, DMSO-d6): δ 105.1,
119.0, 122.6, 123.6, 125.9, 127.5, 128.2, 132.5, 134.0, 137.3, 137.7,
139.3, 172.9. HRMS/EI: calcd for C13H8INO 320.9651, found
320.9665.
1
1351, 844, 760, 605. H NMR (400 MHz, CDCl3): δ 0.32 (s, 9H),
5.29 (s, 1H), 5.31 (s, 1H), 7.19 (td, 1H, 3J = 7.2 Hz, J = 1.5 Hz), 7.52
(m, 1H), 7.84 (td, 1H, 3J = 7.2 Hz, J = 1.6 Hz). 13C NMR (100 MHz,
CDCl3): δ −1.0, 124.3 (d, 4J = 2 Hz), 125.6 (d, 2J = 36 Hz), 138.2 (d,
3J = 7 Hz), 138.8 (d, 3J = 13 Hz), 172.7 (d, 1J = 235 Hz), signal of the
carbon bonded to the boron is missing.
2′-Fluoro-3′-(trimethylsilanyl)biphenyl-2-carbonitrile (6a).
In a round-bottom flask under N2 were introduced DME (15 mL)
and water (15 mL). The solution was degassed by bubbling N2 for 15
min, and Pd(OAc)2 (62 mg, 0.27 mmol) and PPh3 (144 mg, 0.55
mmol) were added. The solution was heated to 50 °C for 10 min, and
2-bromobenzonitrile (1 g, 5.5 mmol), 5 (1.75 g, 8.25 mmol), and
Na2CO3 (2.33 g, 22 mmol) were added. The solution was heated to 90
°C for 14 h, cooled to room temperature, filtered over a pad of Celite,
and extracted with EtOAc (3 × 50 mL). The combined organic layers
were dried over MgSO4, filtered, evaporated in vacuo, and purified by
flash chromatography using cyclohexane/AcOEt 9/1 as the eluent to
afford 6a (1.45 g) as a colorless oil. Yield: 98%. IR (KBr): ν (cm−1)
7-Iodobenzo[h]-1,6-naphthyridin-5(6H)-one (8b). The same
procedure as for compound 3v was used, with 7b (0.7 g, 2.16 mmol),
KOH (0.6 g, 10.8 mmol), and t-BuOH (35 mL) and heating at 150 °C
for 1 h. 8b (0.51 g) was obtained as a white powder. Yield: 73%. Mp:
230−231 °C. IR (KBr): ν (cm−1) 3325, 1660 (CO), 1594, 1424, 765,
1
3
621. H NMR (400 MHz, CDCl3): δ 7.11 (t, 1H, J = 7.8 Hz), 7.56
(dd, 1H, 3J = 7.8 Hz, 3J = 4.8 Hz), 8.01 (d, 1H, 3J = 7.8 Hz), 8.74 (dd,
1H, 3J = 7.8 Hz, 4J = 1.9 Hz), 8.80 (d, 1H, 3J = 6.8 Hz), 8.90 (brs, 1H),
9.04 (dd, 1H, 3J = 4.8 Hz, 4J = 1.9 Hz). 13C NMR (100 MHz, CDCl3):
δ 84.4, 121.1, 121.3, 123.5, 124.6, 125.6, 136.4, 136.8, 141.0, 150.8,
1
2957, 2227 (CN), 1637, 1412, 585, 842, 761. H NMR (400 MHz,
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154.4, 161.4. HRMS/EI: calcd for C12H7IN2O 321.9603, found
321.9592.
2.05 (m, 7H), 2.37 (t, 2H, 3J = 7.8 Hz), 2.59 (t, 2H, 3J = 6.7 Hz), 3.00
(d, 2H, 3J = 10.7 Hz), 4.50 (d, 2H, 3J = 5.8 Hz), 6.95 (d, 2H, 3J = 8.8
3
3
Ethyl 1-[3-(4-Iodophenyl)propanoyl]piperidine-4-carboxy-
late (9). To a solution of 4-iododihydrocinamic acid43 (5 g, 18.11
mmol) in DCM (50 mL) were added at room temperatue HOBt (3.67
g, 27.17 mmol), EDCI (5.21 g, 27.17 mmol), NEt3 (3.79 mL, 27.17
mmol), and ethyl isonipecotate (3.07 mL, 19.92 mmol). The solution
was allowed to stir at room temperature for 24 h, evaporated to
dryness, and purified by silica gel chromatography using cyclohexane/
EtOAc 9/1 and 7/3 as the eluents to afford 9 (6.31 g) as white
powder. Yield: 84%. Mp: 73−74 °C. IR (KBr): ν (cm−1) 2953, 2930,
1729 (CO), 1644 (CO), 1447, 1178, 1040, 1006, 811. 1H NMR (400
Hz), 7.48 (d, 1H, J = 6.8 Hz), 7.59 (d, 2H, J = 8.8 Hz), 7.63 (m,
2H), 7.82 (d, 1H, 3J = 6.8 Hz), 7.86 (d, 1H, J = 8.8 Hz), 8.38 (d, 1H,
3
3
3J = 6.8 Hz), 8.42 (d, 1H, J = 7.8 Hz), 8.51(d, 1H, J = 7.8 Hz). 13C
NMR (100 MHz, CDCl3): δ 28.6, 29.3 (2C), 33.3, 35.8, 53.6 (2C),
58.3, 70.4, 90.7, 120.1, 121.8, 122.1, 122.3, 124.3, 125.0, 127.1, 127.7,
128.7, 130.5 (2C), 130.8, 134.7, 137.3 (2C), 141.8, 143.3, 158.9. LC−
MS (ESI): tR = 6.33 min; [M + H]+ 537.25. HRMS/ESI: calcd for
C28H30IN2O 537.1397, found 537.1401.
7-Fluoro-5-{1-[3-(4-iodophenyl)propyl]piperidin-4-yl}-
methyloxybenzo[h]-1,6-naphthyridine (11d). Starting from 3v
(0.5 g, 2.33 mmol), using general procedure A and cyclohexane/ethyl
acetate 8/2 as the eluent for the chromatography, 11d was obtained as
a white powder (0.53 g). Yield: 41%. Mp: 142−144 °C. IR (KBr): ν
(cm−1) 2925, 1603, 1589, 1354, 1330, 1315, 1106, 794, 770. 1H NMR
(400 MHz, CDCl3): δ 1.59 (m, 2H), 1.78−2.05 (m, 7H), 2.37 (t, 2H,
3J = 7.8 Hz), 2.59 (t, 2H, 3J = 6.7 Hz), 2.99 (d, 2H, 3J = 10.7 Hz), 4.56
3
MHz, CDCl3): δ 1.26 (t, 3H, J = 6.8 Hz), 1.55−1.65 (m, 2H), 1.90
(m, 2H), 2.49 (m, 1H), 2.58 (d, 2H, 3J = 7.8 Hz), 2.79 (m, 1H), 2.91
(d, 2H, 3J = 7.8 Hz), 3.03 (m, 1H), 3.74 (m, 1H), 4.14 (t, 2H, 3J = 6.8
3
3
Hz), 4.42 (m, 1H), 6.97 (d, 2H, J = 8.3 Hz), 7.60 (d, 2H, J = 8.3
Hz). 13C NMR (100 MHz, CDCl3): δ 14.2, 27.8, 28.3, 30.8, 34.7, 40.9,
41.0, 44.7, 60.6, 91.2, 130.5 (2C), 137.5 (2C), 140.9, 170.1, 174.1.
HRMS/EI: calcd for C17H22INO3 415.0644, found 415.0634.
3
3
(d, 2H, J = 5.8 Hz), 6.95 (d, 2H, J = 8.8 Hz), 7.41−7.49 (m, 2H),
7.56−7.62 (m, 3H), 8.64 (dd, 1H, J = 7.8 Hz, J = 1.9 Hz), 8.73 (dd,
1H, 3J = 8.3 Hz, 4J = 1.9 Hz), 9.11 (dd, 1H, 3J = 4.9 Hz, 4J = 1.9 Hz).
13C NMR (100 MHz, CDCl3): δ 28.5, 29.2 (2C), 33.3, 35.8, 53.5
{1-[3-(4-Iodophenyl)propyl]piperidin-4-yl}methanol (10). To
a solution of 9 (1.31 g, 3.15 mmol) in anhydrous THF (20 mL) at −5
°C was added 1 M DIBALH solution in n-hexane (12.62 mL, 12.62
mmol). The mixture was allowed to stir at room temperature for 15 h,
carefully hydrolyzed with water (30 mL), extracted with EtOAc (3 ×
50 mL), dried with MgSO4, filtered, and evaporated. The crude
product was then purified by silica gel chromatography using
cyclohexane/EtOAc 9/1 and 7/3 as the eluents to afford 10 (0.96g)
as a colorless oil. Yield: 85%. IR (KBr): ν (cm−1) 3400 (OH), 2922,
1728, 1483, 1042, 1006, 797. 1H NMR (400 MHz, CDCl3): δ 1.26 (d,
(2C), 58.2, 71.0, 90.7, 115.6, 115.7 (d, 2J = 19 Hz), 119.2, 122.7, 124.4
(d, J = 7 Hz), 125.4 (d, J = 1 Hz), 130.5 (2C), 133.1, 134.1 (d, 2J = 11
Hz), 137.2 (2C), 141.8, 150.2 (d, J = 3 Hz), 153.2, 157.0 (d, 1J = 250
Hz), 158.5. LC−MS (ESI): tR = 6.04 min; [M + H]+ 556.38. HRMS/
ESI: calcd for C27H28FIN3O 556.1256, found 556.1260.
General Procedure B for the Synthesis of Stannylated
Compounds 12a,b,d. In a Schlenk flask under nitrogen were
introduced toluene (4 mL), water (0.4 mL), Pd(OAc)2 (0.025 mmol),
and PPh3 (0.05 mmol). The mixture was heated at 50 °C for 0.3 h, and
the iodinated derivative (11a, 11b, or 11d; 0.5 mmol in 7 mL of
toluene) and hexa-n-butylditin (0.75 mmol) were added. The mixture
was heated at 90 °C for 16 h, filtered on Celite, and evaporated in
vacuo. The obtained crude oil was then purified by chromatography on
silica gel using Et2O and Et2O/NEt3 (99/1) as the eluents.
3
1H, J = 5.9 Hz), 1.26 (m, 2H), 1.70−1.80 (m, 5H), 1.92 (m, 2H),
2.32 (t, 2H, 3J = 7.8 Hz), 2.56 (t, 2H, 3J = 7.8 Hz), 2.92 (m, 2H), 3.49
3
3
3
(d, 2H, J = 6.8 Hz), 6.93 (d, 2H, J = 8.3 Hz), 7.58 (d, 2H, J = 8.3
Hz). 13C NMR (100 MHz, CDCl3): δ 28.6, 28.8 (2C), 33.3, 38.6, 53.5
(2C), 58.2, 67.9, 92.0, 130.5 (2C), 137.3 (2C), 141.9. HRMS/EI:
calcd for C15H22INO 359.0746, found 359.0759.
4-Iodo-6-(1-propylpiperidin-4-yl)methyloxyphenanthridine
(11a). Starting from 8a (0.47 g, 1.46 mmol), using general procedure
A and cyclohexane/ethyl acetate 8/2 as the eluent for the
chromatography, 11a was obtained as a yellow powder (0.51 g).
Yield: 76%. Mp: 87−89 °C. IR (KBr): ν (cm−1) 2926, 1591, 1396,
1339, 1136, 751. 1H NMR (400 MHz, CDCl3): δ 0.91 (t, 3H, 3J = 7.3
Hz), 1.51−1.64 (m, 4H), 1.93−2.09 (m, 5H), 2.31 (t, 2H, J = 7.8 Hz),
6-(1-Propylpiperidin-4-yl)methyloxy-4-(tributylstannyl)-
phenanthridine (12a). Starting from 11a and following general
procedure B, 12a was obtained as a colorless oil (140 mg). Yield: 45%.
IR (KBr): ν (cm−1) 2955, 2922, 2870, 2851, 1589, 1459, 1339, 1311,
761. 1H NMR (400 MHz, CDCl3): δ 0.85 (t, 6H, J = 6.3 Hz), 0.92 (t,
6H, J = 6.8 Hz), 1.16−1.20 (m, 4H), 1.27−1.43 (m, 10H), 1.50−1.62
(m, 10H), 1.97 (d, 2H, J = 9.7 Hz), 2.06 (t, 1H, J = 11.2 Hz), 2.36 (t,
2H, J = 7.8 Hz), 3.05 (d, 2H, J = 10.7 Hz), 4.48 (d, 2H, J = 5.8 Hz),
7.46 (t, 1H, 3J = 7.8 Hz), 7.61 (t, 1H, 3J = 7.8 Hz), 7.78 (m, 2H), 8.38
3
3.02 (d, 2H, J = 10.7 Hz), 4.63 (d, 2H, J = 6.8 Hz), 7.19 (t, 1H, J =
3
3
6.8 Hz), 7.67 (t, 1H, J = 7.8 Hz), 7.82 (t, 1H, J = 7.8 Hz), 8.20 (d,
1H, J = 7.8 Hz), (t, 2H, J = 7.3 Hz), 8.49 (d, 1H, J = 8.8 Hz). 13C
NMR (100 MHz, CDCl3): δ 12.1, 20.2, 29.2 (2C), 35.6, 53.5 (2C),
61.1, 71.2, 101.8, 120.2, 122.0, 122.6, 123.0, 125.2, 125.5, 127.8, 131.1,
132.9, 134.9, 138.9, 159.5. LC−MS (ESI): tR = 5.84 min; [M + H]+
461.28. HRMS/EI: calcd for C22H25IN2O 460.1012, found 460.1008.
7-Iodo-5-(1-propylpiperidin-4-yl)methyloxybenzo[h]-1,6-
naphthyridine (11b). Starting from 8b (0.16 g, 0.5 mmol), using
general procedure A and cyclohexane/ethyl acetate 8/2 with 5% of
NEt3 as the eluent for the chromatography, 11b was obtained as a
yellow powder (0.16 g). Yield: 71%. Mp: 120−122 °C. IR (KBr): ν
(cm−1) 2917, 1603, 1588, 1341, 1093, 762. 1H NMR (400 MHz,
3
3
3
3
(t, 2H, J = 7.8 Hz), 8.50 (d, 1H, J = 8.7 Hz). 13C NMR (100 MHz,
CDCl3): δ 10.2 (3C), 12.0, 13.7 (3C), 20.0, 27.4 (3C), 29.1 (2C), 29.4
(3C), 35.7, 53.5 (2C), 61.1, 70.6, 119.9, 121.4, 121.9, 122.3, 124.1,
124.9, 126.9, 130.6, 135.4, 136.9, 143.6, 148.0, 157.7. HRMS/ESI:
calcd for C34H53N2OSn 625.3181, found 625.3186.
7-(1-Propylpiperidin-4-yl)methyloxy-5-(tributylstannyl)-
benzo[h]-1,6-naphthyridine (12b). Starting from 11b and
following general procedure B, 12b was obtained as a colorless oil
(139 mg). Yield: 44%. IR (KBr): ν (cm−1) 2956, 2924, 2871, 2852,
1603, 1460, 1328, 1153, 773. 1H NMR (400 MHz, CDCl3): δ 0.85 (t,
6H, J = 7.3 Hz), 0.92 (t, 6H, J = 6.8 Hz), 1.16−1.21 (m, 4H), 1.25−
1.43 (m, 10H), 1.51−1.63 (m, 10H), 1.95 (d, 2H, J = 10.7 Hz), 2.06
(t, 1H, J = 11.2 Hz), 2.36 (t, 2H, J = 7.8 Hz), 3.06 (d, 2H, J = 10.7
Hz), 4.48 (d, 2H, J = 5.8 Hz), 7.51−7.56 (m, 2H), 7.86 (dd, 1H, 3J =
6.8 Hz, 4J = 1.9 Hz), 8.62 (dd, 1H, 3J = 7.8 Hz, 4J = 1.9 Hz), 8.92 (d,
3
CDCl3): δ 0.91 (t, 3H, J = 7.3 Hz), 1.51−1.59 (m, 4H), 1.91−2.08
(m, 5H), 2.32 (m, 2H), 3.02 (d, 2H, J = 8.7 Hz), 4.63 (d, 2H, 3J = 5.8
Hz), 7.27 (t, 1H, J = 7.8 Hz), 7.59 (dd, 1H, 3J = 7.8 Hz, 3J = 4.9 Hz),
8.28 (d, 1H, 3J = 8.7 Hz), 8.64 (dd, 1H, 3J = 7.8 Hz, 4J = 1.9 Hz), 8.96
(m, 1H), 9.11 (dd, 1H, 3J = 4.9 Hz, 4J = 1.9 Hz). 13C NMR (100 MHz,
CDCl3): δ 12.0, 20.0, 29.1 (2C), 35.5, 53.5 (2C), 61.0, 71.5, 100.6,
115.2, 122.7, 124.1, 124.3, 125.9, 133.1, 140.4, 144.1, 150.7, 153.2,
158.8. LC−MS (ESI): tR = 5.22 min; [M + H]+ 462.22. HRMS/EI:
calcd for C21H24IN3O 461.0964, found 461.0970.
3
3
4
1H, J = 7.8 Hz), 9.09 (dd, 1H, J = 4.9 Hz, J = 1.9 Hz). 13C NMR
(100 MHz, CDCl3): δ 10.2 (3C), 12.0, 13.5 (3C), 20.0, 27.4 (3C),
29.1 (3C), 29.3 (2C), 35.6, 53.4 (2C), 61.1, 70.9, 115.1, 122.0, 122.7,
123.9, 124.7, 132.9, 138.6, 142.9, 149.7, 151.3, 152.8, 157.3. HRMS/
ESI: calcd for C33H52N3OSn 626.3133, found 626.3136.
6 -{ 1- [ 3 - ( 4 - I o d o ph e n yl ) p r o p y l ] p i p e r i d i n - 4 - y l } -
methyloxyphenanthridine (11c). Starting from 3a (0.5 g, 2.56
mmol), using general procedure A and cyclohexane/ethyl acetate 8/2
as the eluent for the chromatography, 11c was obtained as a colorless
oil (0.54 g). Yield: 39%. IR (KBr): ν (cm−1) 2925, 1637, 1620, 1486,
1317, 759, 727. 1H NMR (400 MHz, CDCl3): δ 1.58 (m, 2H), 1.79−
7-Fluoro-5-{1-[3-(4-tributylstannylphenyl)propyl]piperidin-
4-yl}methyloxybenzo[h]-1,6-naphthyridine (12d). Starting from
11d and following general procedure B, 12d was obtained as a
colorless oil (147 mg). Yield: 41%. IR (KBr): ν (cm−1) 2954, 2925,
1
1608, 1591, 1462, 1331, 1314, 1240, 1151, 1083, 769. H NMR (400
9703
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MHz, CDCl3): δ 0.85 (t, 9H, J = 7.3 Hz), 0.92 (t, 6H, J = 8.1 Hz),
1.18−1.29 (m, 8H), 1.42−1.52 (m, 6H), 1.79−1.96 (m, 7H), 2.34 (t,
2H, J = 7.8 Hz), 2.06 (t, 2H, J = 7.8 Hz), 2.94 (d, 2H, J = 11.2 Hz),
isocratic HPLC using a Bondclone C18 (10 μm, 300 × 7.8 mm,
Phenomenex) column at 3 mL min−1. The apparent specific activity
was determined thanks to a calibration curve measured using growing
amounts of the cold reference compound and to UV monitoring
during the HPLC purification step. The overall radiochemical yield
was 56%. The apparent specific radioactivity was 240 Ci/mmol (11%
of the carrier-free specific activity).
Radiosynthesis of 13b. A 0.05 M NaOH solution containing 128
μCi of carrier-free Na125I (PerkinElmer, 1 μL) was added to a mixture
made of 12b (3 μg) in EtOH (1.5 μL), glacial acetic acid (5 μL), and
30% hydrogen peroxide solution (5 μL). After incubation at room
temperature for 20 min, HPLC phase (MeCN/water 80/20, 10 mM
AcOH buffer, pH 5, 350 μL) was added and 13b was isolated by an
isocratic HPLC using a Bondclone C18 (10 μm, 300 × 7.8 mm,
Phenomenex) column at 3 mL min−1. The apparent specific activity
was determined thanks to a calibration curve measured using growing
amounts of the cold reference compound and to UV monitoring
during the HPLC purification step. The overall radiochemical yield
was 70%. The apparent specific radioactivity was 110 Ci/mmol (5% of
the carrier-free specific activity).
Radiosynthesis of 13d. A 0.05 M NaOH solution containing 210
μCi of carrier-free Na125I (PerkinElmer, 1 μL) was added to a mixture
made of 12b (10 μg) in EtOH (1 μL), glacial acetic acid (5 μL), and
30% hydrogen peroxide solution (5 μL). After incubation at room
temperature for 20 min, HPLC phase (MeCN/water 80/20, 10 mM
AcOH buffer, pH 5, 350 μL) was added and 13d was isolated by an
isocratic HPLC using a Bondclone C18 (10 μm, 300 × 7.8 mm,
Phenomenex) column at 3 mL min−1. The apparent specific activity
was determined thanks to a calibration curve measured using growing
amounts of the cold reference compound and to UV monitoring
during the HPLC purification step. The overall radiochemical yield
was 85%. The apparent specific radioactivity was 280 Ci/mmol (13%
of the carrier-free specific activity).
3
3
4.48 (d, 2H, J = 6.1 Hz), 7.10 (d, 2H, J = 7.8 Hz), 7.30 (d, 2H, J =
7.8 Hz), 7.34−7.41 (m, 2H), 7.52 (dd, 1H, 3J = 8.2 Hz, 3J = 4.58 Hz),
8.57 (dd, 1H, 3J = 8.2 Hz, 4J = 1.8 Hz), 8.64 (m, 1H), 9.04 (dd, 1H, 3J
= 4.5 Hz, 4J = 1.8 Hz). 13C NMR (100 MHz, CDCl3): δ 9.5 (3C), 13.6
(3C), 27.3 (3C), 28.7, 29.1 (3C), 29.2 (2C), 33.8, 35.8, 53.5 (2C),
2
58.6, 71.0, 115.6, 115.6 (d, J = 20 Hz), 119.2 (d, J = 4 Hz), 122.8,
124.4 (d, J = 7 Hz), 125.5 (d, J = 2 Hz), 128.1 (2C), 133.2, 134.1 (d, 2J
= 11 Hz), 136.4 (2C), 138.5, 141.8, 150.3 (d, J = 4 Hz), 153.3, 157.0
(d, 1J = 250 Hz), 158.6. HRMS/ESI: calcd for C39H55FN3OSn
720.3353, found 720.3356.
Pharmacological Assay and Screen. Binding of all compounds
to native 5-HT4R from guinea pig was determined using the method of
Grossman.44 For membrane preparations, male guinea pigs (300−350
g, Charles River) were subjected to euthanasia by cervical dislocation
and decapitated. Brains were rapidly removed at 4 °C and striatal
regions carefully dissected and pooled. The tissues were then
suspended in 10 volumes of HEPES buffer (50 mM, pH 7.4) at 4
°C. After homogenization at 4 °C (Ultra-Turrax, maximal speed, 15 s),
and ultracentrifugation (23 000g, 60 min, 4 °C), the pellet was
resuspended in 10 volumes of HEPES buffer (50 mM, pH 7.4) at 4 °C
in order to obtain a tissue concentration of about 100 mg protein/mL.
The protein concentration was determined by the method of Lowry45
using bovine serum albumin as standard. For radioligand binding
studies, 600 μg of membrane was incubated in duplicate at 37 °C for
30 min with [3H]GR 113808 (Perkin-Elmer), a fixed concentration of
compound, and HEPES buffer (50 mM, pH 7.4) at 37 °C. Incubation
was terminated by rapid vacuum filtration through 0.5% polyethyle-
nimine-presoaked Whatman GF/B filters (Alpha Biotech) using a
Brandel cell harvester. Filters were subsequently washed three times
with 4 mL of HEPES buffer (50 mM, pH 7.4) at 4 °C. The method
was validated from saturation studies: six concentrations of [3H]GR
113808 were used to give final concentrations of 0.02−0.8 nM, and
nonspecific binding of [3H]GR 113808 was defined in the presence of
30 μM serotonin to determine the Kd and the Bmax. For competition
studies, [3H]GR 113808 was used to give a final concentration of 0.1
nM. Percentages of inhibition of the binding of [3H]GR 113808 were
obtained for concentrations of 10−6 and 10−8 M of the ligands tested.
For some of these compounds, affinity constants were calculated from
five-point inhibition curves using the EBDA-Ligand software and
expressed as Ki SD.
Ligands 4i, 4k, 4l, 4m, 4t, and 4u were submitted to the National
Institute of Mental Health Psychoactive Drug Screening Program
(NIMH PDSP) for assessment of binding affinity to human
recombinant 5-HT4 receptors and to other serotonin receptors (5-
HT1A‑E, 5-HT2A‑C, 5-HT3, 5-HT5A, 5-HT6, 5-HT7). For human 5-
HT4R Ki determinations, [3H]GR 113808 was used as hot ligand and
GR 113808 as reference. 5-HT4R membrane was made with HEK T
cells transiently transfected with human 5-HT4 DNA. The binding
protocol is the same as for other 5-HT subtypes in the PDSP assay
protocol book. Selected ligands 4k, 4l, 4m, and 4u were also assessed
for agonist/partial agonist activity and for antagonist activity. For 5-
HT4R-mediated Gs activation, cAMP was measured using GloSensor
tech from Promega, with serotonin as a reference agonist according to
a literature procedure.46 For other experimental details, please refer to
the PDSP Web site http://pdsp.med.unc.edu/ and click on “Binding
Assay”.
Radiosynthesis of [125I]1. A 0.05 M NaOH (1 μL) solution
containing 210 μCi of carrier -free Na125I (PerkinElmer) was added to
a mixture made of 1 trimethylstannyl precursor (ERAS Labo, 100 μg)
in a mixture containing EtOH (10 μL), glacial acetic acid (5 μL), and
30% hydrogen peroxide solution (5 μL). After an incubation at room
temperature for 20 min, HPLC phase (MeCN/water 30/70, 10 mM
H3PO4 350 μL) was added and [125I]1 was isolated by an isocratic
HPLC using a Bondclone C18 (10 μm, 300 × 7.8 mm, Phenomenex)
column at 3 mL min−1. The apparent specific activity was determined
thanks to a calibration curve measured using growing amounts of the
cold reference compound and to UV monitoring during the HPLC
purification step. After collection, pooling, and evaporation of the
corresponding fractions, the overall radiochemical yield of iodination
with 125I was 70%. The apparent specific radioactivity was 146 Ci/
mmol (6.6% of the carrier-free specific activity).
In Vitro Competition Experiments. For each in vitro autoradio-
graphic experiment, frozen rat brain coronal sections at the level of the
striatum and accumbens nucleus were thawed and dried, preincubated
in 50 mM Tris-HCl pH = 7.4 for 5 min at room temperature, and then
incubated in the same buffer with given concentrations of the
radioligand for 30 min at room temperature. During this step,
competition experiments were done by incubating the sections with
100 pM of [125I]1 and various concentrations of 11d. After incubation,
sections were washed three times for 5 min in ice-cold buffer, dipped
in ice-cold deionized water, and rapidly dried under a cold air stream.
The labeled sections were exposed on phosphorimaging plates
overnight before detection with a Fujifilm BAS scanner.
Other selected ligands 4v, 11a, 11b, and 11d were evaluated for
binding to human 5-HT4 and other serotonin receptors (5-HT1A‑E, 5-
HT2A‑C, 5-HT3, 5-HT5A, 5-HT6, 5-HT7) as well as for intrinsic activity
at CEREP. Detailed assay protocols are available at the CEREP Web
site (http://www.cerep.com).
Radiosynthesis of 13a. A 0.05 M NaOH solution containing 165
μCi of carrier-free Na125I (PerkinElmer, 1 μL) was added to a mixture
made of 12a (3 μg) in EtOH (1.5 μL), glacial acetic acid (5 μL), and
30% hydrogen peroxide solution (5 μL). After incubation at room
temperature for 20 min, HPLC phase (MeCN/water 80/20, 10 mM
AcOH buffer, pH 5, 350 μL) was added, and 13a was isolated by an
In Vivo Competition Experiments. For the in vivo competition
experiment, a mouse was injected intravenously with a saline solution
containing a dose of 50 μg/kg of 11d and 30 μCi [125I]1 (146 Ci/
mmol specific activity, 6.6% of the carrier free). The animal was then
sacrificed with CO2, and 20-μm-thin cryosections were collected in
5HT4R-rich regions (striatum, accumbens nucleus, olfactive tubercles),
and finally exposed overnight on phosphorimaging plates before
detection with a Fujifilm BAS scanner.
Molecular Modeling Study. Receptor Model. First, the sequence
of the human 5-HT4R was retrieved from the UniProt Knowledgebase
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Journal of Medicinal Chemistry
Article
(UniProtKB)47 (ID: Q712M9_HUMAN). Using screening methods
like FUGUE,48 SP3,49 PSIBLAST,50,51 HHSEARCH,52 and the @
tome-2 server,53 the β2 adrenergic receptor has been identified as the
best 3D experimental template for the homology modeling of the 5-
HT4R (sequence identity = 40%). The high-resolution (2.4 Å) crystal
structure of the human β2 adrenergic receptor (β2AR)-T4 lysozyme
fusion protein bound to the carazolol (PDB ID: 2RH1)33 was used as
the 3D template. The alignment between the two sequences was
manually optimized to avoid insertions and deletions in secondary
structure elements (see Supporting Information for final sequence
alignment). The disulfide bond C93−C184 between the trans-
membrane helix 3 (TM3) and the extracellular loop (ECL2) was
conserved. This alignment was used as the basis for the homology
modeling with the Modeler software.54 The resulting model was then
evaluated by methods like verify3D55 and Eval23D.56
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non-classical 5-hydroxytryptamine receptor positively coupled with
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Docking Studies. The docking of the compounds into the 5-HT4R
was carried out with the GOLD program (v 5.0) using the default
parameters.57,58 This program applies a genetic algorithm to explore
conformational spaces and ligand binding modes. To evaluate the
proposed ligand positions, the ChemScore fitness function was applied
in these docking studies. The binding site in the 5-HT4R model was
defined as a 10 Å sphere centered on the aspartic acid residue Asp100
.
Because the mutagenesis studies have shown that the interaction
between the positively ionizable amine of ligands and Asp100 of 5-
HT4R is crucial for ligand binding, a hydrogen bond constraint
between positively ionizable amine ligand and OD atom of Asp100 was
used during the docking.36 Furthermore, special attention was paid
during the docking procedure to the following amino acids in the
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2007, 203, 274−278.
binding site, which were kept flexible: Arg96, Asp100, Thr104, Tyr192
,
Ser197, and Trp294. For 4v docking, a second hydrogen bond constraint
between the tricycle nitrogen and the hydroxyl group of Tyr192 was
added. For the two crystal conformers available for each compound, a
dozen separate docking procedures were carried out and analyzed.
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Hen, R.; Charnay, Y.; Bockaert, J.; Compan, V. Anorexia induced by
activation of serotonin 5-HT4 receptors is mediated by increases in
CART in the nucleus accumbens. Proc. Natl. Acad. Sci. U. S. A. 2007,
104, 16335−16340.
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Manta, S.; Lambas-Senas, L.; Wiborg, O.; Haddjeri, N.; Pineyro, G.;
Sadikot, A. F.; Debonnel, G. Serotonin(4) (5-HT(4)) receptor
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ASSOCIATED CONTENT
■
S
* Supporting Information
X-ray crystallographic data of compounds 4c, 4d, 4g, 4h, 4j,
4m, 4p, 4q, 4r, 4v, and 11d and amino acid sequences
alignment of 5-HT4R and human β2-adrenergic receptor. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(14) Manabe, N.; Wong, B. S.; Camilleri, M. New generation 5-HT4
receptor agonists: Potential for for treatment of gastrointestinal
motility disorders. Expert Opin. Invest. Drugs 2010, 19, 765−775.
(15) Moser, P. C.;, E.; Bergis, O.; Jegham, S.; Lochead, A.;
Duconseille, E.; Terranova, J.-P.; Caille, D.; Berque-Bestel, I.;
Lezoualc’h, F.; Fischmeister, R.; Dumuis, A.; Bockaert, J.; George,
P.; Soubrie, P.; Scatton, B. SL65.0155, a novel 5-hydroxytryptamine4
receptor partial agonist with potent cognition-enhancing properties. J.
Pharmacol. Exp. Ther. 2002, 302, 731−742.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +33(0)231566808. E-mail: frederic.fabis@unicaen.fr.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the National Institute of Mental Health’s Psycho-
active Drug Screening Program, Contract #HHSN-271-2008-
00025-C (NIMH PDSP) which generously provided Ki
determinations and functional assays. The NIMH PDSP is
Directed by Bryan L. Roth MD, Ph.D. at the University of
North Carolina at Chapel Hill and Project Officer Jamie Driscol
(16) Mohler, E. G.; Shacham, S.; Noiman, S.; Lezoualc’h, F.; Robert,
S.; Gastineau, M.; Rutkowski, J.; Marantz, Y.; Dumuis, A.; Bockaert, J.;
Gold, P. E.; Ragozzino, M. E. VRX-03011, a novel 5-HT4 agonist,
enhances memory and hippocampal acetylcholine efflux. Neuro-
pharmacology 2007, 53, 563−573.
́
(17) Wong, D. F.; Grunder, G.; Brasic, J. R. Brain imaging research:
̈
Does the science serve clinical practice? Int. Rev. Psychiatry 2007, 19,
541−558.
́
at NIMH, Bethesda, MD. We thank Aurelien Lesnard for
helpful management of biological results. This work was in part
supported by the Swiss National Science Foundation (Grant
No. 310030_120369).
(18) Gibson, R. E.; Burns, H. D.; Hamill, T. G.; Eng, W. S.; Francis,
B. E.; Ryan, C. Non-invasive radiotracer imaging as a tool for drug
development. Curr. Radiopharm. Des. 2000, 6, 973−989.
(19) Pike, V. W.; Halldin, C.; Nobuhara, K.; Hiltunen, J.; Mulligan, R.
S.; Swahn, C.-G.; Karlsson, P.; Olsson, H.; Hume, S. P.; Hirani, E.;
Whalley, J.; Pilowsky, L. S.; Larson, S.; Schnell, P.-O.; Ell, P. J.; Farde,
L. Radioiodinated SB 207710 as a radioligand in vivo: Imaging of brain
5-HT4 receptors with SPET. Eur. J. Nucl. Med. Mol. Imaging 2003, 30,
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