Discovery and pharmacological profile of new 1H-indazole-3-carboxamide and 2H-pyrrolo[3,4-c]quinoline derivatives as selective serotonin 4 receptor ligands

Article abstract of DOI:10.1021/jm300573d

Since the discovery of the serotonin 4 receptor (5-HT₄R), a large number of receptor ligands have been studied. The safety concerns and the lack of market success of these ligands have mainly been attributed to their lack of selectivity. In thi

Full text of DOI:10.1021/jm300573d

Article
pubs.acs.org/jmc
Discovery and Pharmacological Profile of New 1H‑Indazole-3-
carboxamide and 2H‑Pyrrolo[3,4‑c]quinoline Derivatives as Selective
Serotonin 4 Receptor Ligands
Guido Furlotti,*,† Maria Alessandra Alisi,† Claudia Apicella,§ Alessandra Capezzone de Joannon,§
Nicola Cazzolla,† Roberta Costi,‡ Giuliana Cuzzucoli Crucitti,‡ Beatrice Garrone,§ Alberto Iacovo,‡
Gabriele Magaro,† Giorgina Mangano,§ Gaetano Miele,‡ Rosella Ombrato,† Luca Pescatori,‡
̀
Lorenzo Polenzani,§ Federica Rosi,‡ Marco Vitiello,§ and Roberto Di Santo*,‡
†Department of Chemistry and § Department of Pharmacology, Angelini Santa Palomba Research Center, Piazzale della Stazione snc,
00040 Pomezia, Italy
‡Istituto PasteurFondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” Universita
̀
di Roma,
Piazzale Aldo Moro 5, 00185 Rome, Italy
S
* Supporting Information
ABSTRACT: Since the discovery of the serotonin 4 receptor
(5-HT4R), a large number of receptor ligands have been
studied. The safety concerns and the lack of market success of
these ligands have mainly been attributed to their lack of
selectivity. In this study we describe the discovery of N-[(4-
piperidinyl)methyl]-1H-indazole-3-carboxamide and 4-[(4-
piperidinyl)methoxy]-2H-pyrrolo[3,4-c]quinoline derivatives
as new 5-HT4R ligands endowed with high selectivity over
the serotonin 2A receptor and human ether-a-go-go-related
gene potassium ion channel. Within these series, two
molecules (11ab and 12g) were identified as potent and selective 5-HT4R antagonists with good in vitro pharmacokinetic
properties. These compounds were evaluated for their antinociceptive action in two analgesia animal models. 12g showed a
significant antinociceptive effect in both models and is proposed as an interesting lead compound as a 5-HT4R antagonist with
analgesic action.
irritable bowel syndrome (IBS) (cisapride (1), tegaserod (2),
and prucalopride (3)) and in the treatment of heart failure, with
piboserod (4) producing significant improvement in left
ventricular function during clinical trials on patients with
symptomatic heart failure (Chart 1).10−13 Further studies led to
the discovery of 5-HT4R antagonists such as compounds 5−9
(Chart 1).9,14,15 Moreover, the interest in 5-HT4R ligands
increased when the 5-HT4R was proposed to be involved in the
mechanism of nociception. The first example of this
involvement was demonstrated when three nonselective 5-
HT4R agonists, 1, endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-
yl)-2,3-dihydro-3-ethyl-2-oxo-1H-benzimidazole-1-carboxamide
hydrochloride (BIMU 1), and endo-N-(8-methyl-8-azabicyclo-
[3.2.1]oct-3-yl)-2,3-dihydro-3-(1-methylethyl)-2-oxo-1H-benzi-
midazole-1-carboxamide hydrochloride (BIMU 8), showed an
antinociceptive effect in animal models.16 Afterward, several
studies on 5-HT4R antagonists such as 5, 6, and SDZ-205557
(10) or partial agonists such as 2 have suggested that the
INTRODUCTION
■
Serotonin (5-hydroxytryptamine, 5-HT) is a major neuro-
transmitter involved in a vast number of processes in both the
central and peripheral nervous systems. Its pharmacological
action is mediated by a set of specific receptors. Seven families
of 5-HT receptors (5-HTRs) have been identified so far that
have been cloned and coded from 5-HT1 to 5-HT7.1−3 With
the exception of 5-HT3, all 5-HTRs belong to the superfamily
of G-protein-coupled receptors and have the typical heptahel-
ical structure of a transmembrane protein monomer. 5-HT4
receptor (5-HT4R) is positively coupled to adenylate cyclase by
Gs protein. After its activation, 5-HT4R induces an increase in
intracellular levels of cyclic adenosine monophosphate (cAMP),
activating protein kinase A (PKA), which in turn results in the
modulation of a series of ionic cellular currents.4,5 5-HT4Rs are
widely distributed in both the central and peripheral tissues and
are involved in several neuronal functions.6,7 Thus, since its
discovery, 5-HT4R has been considered as a potential
therapeutic target, and a large number of 5-HT4R ligands
have been described and studied for the treatment of a number
of disease indications in the past 15 years.8,9 The most
significant results have been obtained for the treatment of
Received: April 24, 2012
Published: October 9, 2012
© 2012 American Chemical Society
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Chart 1. 5-HT4R Ligands Currently Used in Clinical Practice or Trials, 1−10, Including Compounds 4−9 Used To Develop
Pharmacophore Models, and Hit Compounds 11b and 12a Found in the Present Study by a VS Approach
inactivation of this receptor may be an effective method to treat
some types of pain (Chart 1).17−21
program. Thus, we decided to apply a virtual screening (VS)
approach on our in-house chemical library including around
9500 heterocyclic compounds.
Unfortunately, most drugs active on the serotoninergic
system show side effects consistent with low selectivity toward
other serotoninergic receptors or interaction with ion channels
such as the human ether-a-go-go-related gene (hERG)
potassium channel.22−26
The interaction with these channels is associated with an
adverse cardiac QT-interval prolongation and caused the
withdrawal of 1 from the market in 2000.27 Nowadays, hERG
inhibition is routinely investigated in the early phase of the drug
discovery process to decrease the risk of subsequent cardiac
safety concerns.28
Studies on 5-HT4R antagonists described in the literature in
the past two decades have provided crucial insights into the
structural motifs required for a ligand to have a strong
interaction with this receptor.9,14,15,29,30 Therefore, we built a
3D pharmacophore model based on the literature antagonists
and used it for the VS of our in-house library. A total of 71
virtual hits were identified that were then tested in vitro in a
human recombinant 5-HT4R binding assay using 4 as a
reference compound. Two hit compounds were identified, N-
[(1-butyl-4-piperidinyl)methyl]-1H-indazole-3-carboxamide
(11b) and 4-(4-piperidinylmethoxy)-2H-pyrrolo[3,4-c]-
quinoline (12a), and derivatives of 11b and 12a were
Because of the lack of structural data on 5-HT4R, there is
insufficient information to perform a target-based drug design
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synthesized to define the structure−activity relationships
(SARs) in these series. In this paper we report the activities
of the newly synthesized compounds (11a−ab and 12a−k) on
5-HT4R, as well as on relevant off-target proteins such as 5-
HT2A receptors, and inhibition of the hERG potassium ion
channel. In vitro absorption, distribution, metabolism, and
excretion (ADME) properties and antinociceptive efficacy of
the most promising compounds 11ab and 12g in two different
animal models are also described.
APR29 contained three structural features: a hydrogen bond
acceptor, an aromatic ring, and a positively charged group
distributed as shown in Figure 2 overlaid with reference
compounds 7 and 8 (Chart 1, Figure 2).
RESULTS AND DISCUSSION
■
Ligand Design. A collection of 16 5-HT4 antagonists
characterized by different chemical scaffolds were taken from
the literature,9,14,15 and among them, six compounds (4−9)
were used to define a data set to generate several 3D
pharmacophore models (Chart 1). Only three models (referred
to as APR29, HPR45, and AAP29) were selected for the
validation step on the basis of (i) the highest score obtained
and (ii) the largest diversity of electronic features to match
chemical groups with different properties (e.g., hydrogen bond
acceptor, hydrophobic, positive charge, and aromatic ring) in
different combinations. Each model was used to perform a hit
search on a database of 1000 druglike ligand decoys from
Figure 2. Pharmacophore model APR29 for 5-HT4 antagonists 7 and
8: aromatic ring (R12, brown), positive ionizable group (P11, blue),
and acceptor H-bond group (A4, red).
APR29 was then used to carry out a virtual hit search in a
multiconformer version of our proprietary database (Angelini
corporate database). The hits retrieved from the pharmaco-
phore search were filtered for non-drug-like groups and
properties, giving 71 compounds characterized by several
different chemical scaffolds. These molecules were submitted to
a single concentration binding assay, using the human
recombinant 5-HT4R and 4 as a reference compound (data
not shown). From this, compounds 11b and 12a (Chart 1)
emerged as promising hits, showing good 5-HT4R binding
affinity, with binding inhibition values of 98% and 48%,
respectively, at a concentration of 1 μM compared to 4, which
showed 100% inhibition at the same concentration. The two hit
compounds matched all three pharmacophoric features of
APR29, proving the validity of this pharmacophore approach
for identifying novel scaffold hits (Figure 3).
Schrodinger and 10 known active compounds, seeded to have a
̈
random hit rate of 1%. The enrichment factor (EF) was
calculated after each VS process, and these values were
compared to determine the best performing model. The EF
is the measure of how many active compounds are found within
a defined “early recognition” fraction of the ordered list relative
to a random distribution and is calculated as follows:
EF = Nx%exptl/(N
× x%)
active
(1)
where Nx%
is the number of experimentally found active
exptl
structures in the top x% of the sorted database and Nactive is the
total number of active structures in the database.
The best outcome for each VS protocol is 100% (10 out of
10) at the top 1%. Figure 1 shows that the APR29
pharmacophore gave the best result compared to the other
pharmacophores, showing the maximum EFs at the top 5% of
the data set.
Figure 3. Match of compounds 11b (orange) and 12a (green) with
the three pharmacophore features of the APR29 hypothesis.
On the basis of its ease of chemical manipulation, we first
decided to study the chemical space around hit compound 11b
(N-[(1-butyl-4-piperidinyl)methyl]-1H-indazole-3-carboxa-
mide), focusing our initial attention on the following
substitution sites: (i) the piperidine nitrogen, (ii) the indazole
nitrogens, and (iii) the 5-position of the indazole ring. We
subsequently applied the SAR from this series to design and
synthesize a focused library of 2H-pyrrolo[3,4-c]quinoline
derivatives based on 12a.
Figure 1. Enrichment for the top 2%, 5%, and 10% of the data set for
the AAP29, APR29, and HPR32 hypotheses.
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a
Scheme 1. Synthetic Route to Compounds 11a−g,m,q−y
a
Reagents and conditions: (a) toluene, room temperature; (b) H2, 10% Pd/C, CH3CO2H; (c) alkyl bromide, K2CO3, EtOH, reflux (or DMF, 80
°C); (d) 2-vinylpyridine, CH3CO2H, H2O, reflux → room temperature; (e) CH3I, CH3COCH3, room temperature; (f) CH3I, acetone, room
temperature.
Synthesis. The N-(4-piperidinylmethyl)-1H-indazole-3-car-
boxamides 11a−g,m,q−y were prepared as shown in Scheme 1
(see Tables 1−3 for the actual structures). The appropriate N-
alkyl-1H-indazole-3-carbonyl chlorides (13 or 1431) were
reacted with 1-[1-(phenylmethyl)-4-piperidinyl]methylamine
to afford compounds 11q and 15, which were then hydro-
genated to give N-unsubstituted derivatives 11a and 16,
respectively. Compounds 11b−d,f,g,m,t−y were obtained by
nucleophilic substitution of 11a and 16 with the appropriate
alkyl halide or by reaction with 2-vinylpyridine for 11e and 11r
(Scheme 1). Finally, 11s was obtained by methylation of
piperidine 11m.
The 5-substituted N-[(1-butyl-4-piperidinyl)methyl]-1(2)-
methylindazole-3-carboxamides 11h−k were obtained by
transformation of the 5-substituted-1(2)-methylindazole-3-
carboxylic acids 17−2032,33 into the corresponding acyl
chlorides followed by amidation with 1-(1-butyl-4-piperidinyl)-
methylamine (Scheme 2) (see Table 1 for the actual
structures).
anhydride afforded indazole-3-carboxamides 11n−p (Scheme
3).
Derivatives 11z−ab were obtained as shown in Scheme 4
(see Table 3 for the actual structures). Compound 16 was
alkylated under basic conditions with 1-(2-bromoethyl)-4-
nitrobenzene or ethyl 4-(2-bromoethyl)benzoate34 to afford
11z and 21, respectively. Then 11z was reduced with hydrogen
in the presence of Pd to afford amine 11aa, while basic
hydrolysis of 21 gave the corresponding carboxylic acid 11ab
(Scheme 4).
4-(4-Piperidinylmethoxy)-2H-pyrrolo[3,4-c]quinolines 12a−
e,i−k were prepared starting from nitrocinnamate 22, which
underwent annulation with (4-tolylsulfonyl)methyl isocyanide
(TosMIC) in the presence of sodium hydride to afford pyrrole
23 (Scheme 5) (see Table 4 for the actual structures).35
The latter compound was transformed into 2H-pyrrolo[3,4-
c]quinolin-4(5H)-one (24) in a two-step, one-pot reaction
carried out with iron powder in glacial acetic acid at 85 °C.35
Substitution at the 2-position of the pyrroloquinoline ring was
easily achieved by alkylation of intermediate 24 with the
appropriate alkyl halide in the presence of K2CO3 to achieve 25
and 26.
Scheme 3 shows the synthetic pathway to obtain compounds
11l,n−p (see Table 2 for the actual structures). Indazole-3-
carbonyl chloride was first reacted with 1-[1-(2-phenylethyl)-4-
piperidinyl]methylamine to furnish 11l. Subsequent alkylation
with the appropriate alkyl bromide or acetylation with acetic
Substitution at the 4-position was achieved via a two-step
process involving a POCl3 chlorination and subsequent
nucleophilic displacement of the resultant chlorides 27 and
28, with the appropriate (1-alkyl-4-piperidinyl)methanol
derivative to afford 12b−d and 29. Benzyl derivative 29 was
deprotected by catalytic hydrogenation to give intermediate
12a, which was alkylated under basic conditions to give 12e,i−
k.
a
Scheme 2. Synthetic Route to Compounds 11h−k
Compounds 12f−h were obtained as shown in Scheme 6
(see Table 4 for the actual structures). Alkylation of piperidine
derivative 12a with 1-(2-bromoethyl)-4-nitrobenzene gave
nitro derivative 30, which was reduced to amino compound
12f. Esters 31 and 32 were obtained by alkylation of 12a with
the appropriate ethyl36 or methyl (35) (haloethyl)benzoate and
were subsequently hydrolyzed to generate the final products
12g and 12h (Scheme 6).
a
Reagents and conditions: (a) (i) SOCl2, toluene, reflux; (ii) 1-(1-
butyl-4-piperidinyl)methylamine, toluene, room temperature.
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Table 1. Binding Properties of Derivatives 11a−k for the Human 5-HT4R and Functional Inhibition of the hERG Potassium Ion
Channel
a
b
c
Percent displacement of the [3H]5 ligand from the recombinant human 5-HT4R, mean values of duplicate measurements. See ref 38. Tested as
d
e
maleic salt. Tested as hydrochloride salt. Tested as oxalic salt.
a
Biological Activities. 5-HTR Affinity in Vitro Assays. The
newly synthesized compounds 11a−ab and 12a−k were tested
for their activity on 5-HT4R in competition binding assay, at
different concentrations, using [3H]5 as a radioligand and 4 as a
reference compound. Preliminary data on the binding profile of
several hits (11b,d,m) suggested low selectivity over all the 5-
HT subtype receptors and transporter (Supporting Informa-
tion); therefore, the affinities of the above compounds versus
some off-target proteins were also tested. The screening panel
demonstrated significant activity on the 5-HT2AR, so we also
decided to screen the most interesting compounds in a
multiconcentration binding assay for the 5-HT2AR to determine
the activity at this target. These assays were performed in vitro
using [3H]ketanserin as a radiologand and methysergide as a
reference compound (Tables 1−4).
Scheme 3. Synthetic Route to Compounds 11l,n−p
a
Reagents and conditions: (a) 1-[1-(2-phenylethyl)-4-piperidinyl]-
methylamine, toluene, reflux; (b) alkyl bromide, DMF, NaH, 0 °C →
room temperature; (c) (CH3CO)2O, CH2Cl2, room temperature.
5-HT4R Affinity in Vitro Assays. 1H-Indazole-3-carbox-
amide Series. The effect of substituents at the piperidinyl
nitrogen (R1) was studied by replacement of the butyl chain
(11b) with several alkyl-, aryl-, and heteroarylalkyl groups
(Table 1). The unsubstituted derivative 11a showed reduced
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Table 2. Binding Properties of Derivatives 11d,l−p for the Human 5-HT4 and 5-HT2A Receptors and Functional Inhibition of
the hERG Potassium Ion Channel
a
inhibition (%) of 5-HT4
b
c
compd
R
10−8
84
M
10−9
M
inhibition (%) of 5-HT2A (10−7 M)
inhibition (%) of hERG (10−6 M)
d
11d
CH3
H
29
15
88
49
79
42
d
11l
56
108
88
d
11m
CH(CH3)2
80
85
54
d
11n
11o
11p
4
CH(CH3)CH2CH3
CH2CH2CH(CH3)2
COCH3
d
14
d
90
46
73
95
67
methysergide
97
a
b
Precentage of displacement of the [3H]5 ligand from the recombinant human 5-HT4R, mean values of duplicate measurements. Precentage of
c
d
displacement of [3H]ketanserin ligand from the recombinant human 5-HT4R, mean values of duplicate measurements. See ref 38. Tested as
hydrochloride salt.
a
Scheme 4. Synthetic Route to Compounds 11z,aa,ab
a
Reagents and conditions: (a) 4-(2-bromoethyl)-1-nitrobenzene, EtOH, K2CO3, reflux; (b) ethyl 4-(2-bromoethyl)benzoate,34 KI, triethylamine, 2-
butanone, reflux; (c) 10% Pd/C, H2, EtOH, room temperature; (d) 1 N NaOH, THF, EtOH, room temperature.
affinity compared to 11b, and binding was markedly increased
by larger groups such as cyclohexylethyl, phenylethyl, 2-
pyridinylethyl, (4-chlorophenyl)ethyl, and phenylbutyl (com-
pounds 11c−g, 64−84% inhibition at 10−8 M). Elongation of
the alkyl linker between the piperidine nitrogen and the phenyl
group from two to four carbon atoms gave a slight reduction of
binding inhibition (compare 11d and 11g).
The effect of substitution on the indazole ring was also
studied; in particular, we chose to study the 5-position due to
its structural similarity with 5-HT. Chloro, methoxy, and
methyl groups were selected as substituents as they have
different sizes and electronics. The influence of these
substituents on the 5-HTR affinity is usually dependent on
the 5-HTR subtype, and in particular, 5-methoxytriptamine has
been reported as a 5-HT4R agonist comparable to 5-HT.37
Introduction of a methyl group (11h) or a chlorine atom (11j)
was detrimental for binding inhibition, with values of 43% and
40%, respectively, at 0.1 μM compared to 88% for the
unsubstituted compound 11b at the same concentration. A
methoxy group in the same position (11i) totally depleted any
activity even at the highest concentration tested (−9% at 0.1
μM). This drop in binding in the indazole series has not
previously been shown for the 5-HT4 receptor.29 The same
drop of activity was also observed when the methyl group of
11b was shifted from the 1- to 2-position of the indazole ring
(11b and 11k).
A small series of derivatives were also designed to study the
influence of substitution at the 1-position of the indazole ring
on binding. Thus, we synthesized and tested a few derivatives of
compound 11d, in which the methyl group on N1 was removed
or replaced with larger alkyl groups or with an acetyl (Table 2).
Desmethyl compound 11l showed a lower inhibition for 5-
HT4R, while the larger isopropyl derivative 11m was more
active than the parent compound 11b (88% at 1 nM). A further
increase in the size of the alkyl group, such as for 1-
methylpropyl (11n) and isopentyl (11o) derivatives, had a
detrimental effect on binding inhibition. Interestingly, the 1-
acetyl analogue 11p showed better activity than methyl
derivative 11d. Unfortunately, its chemical instability possibly
due to hydrolytic degradation prevented further development.
Finally, the SAR around the basic site of the molecule was
investigated, keeping the isopropyl group on the 1-position and
the unsubstituted benzene of the indazole ring constant as
these substitution patterns both gave good affinity. Therefore,
the N-[(1-substituted-4-piperidinyl)methyl]-1-isopropyl-1H-in-
dazole-3-carboxamides 11m,q−ab were prepared and assayed
for their ability to bind 5-HT4R (Table 3).
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Table 3. Binding Properties of Derivatives 11m,q−ab for the Human 5-HT4 and 5-HT2A Receptors, Functional Inhibition of the
hERG Potassium Ion Channel, and Calculated log D at pH 7.4
a
Binding affinity for the human recombinant 5-HT4 receptor, displacement of the [3H]5 ligand expressed as pKi, mean values of duplicate
b
measurements. Confidence intervals of 95% for Ki are not greater than 10% of Ki. Binding affinity for the human recombinant 5-HT2A receptor,
displacement of the [3H]ketanserin ligand expressed as pKi, mean values of duplicate measurements. Confidence intervals of 95% for Ki are not
c
d
e
f
greater than 10% of Ki. See ref 37. log D(7.4) values calculated by ACDLabs 12.0. Tested as hydrochloride salt. Extrapolated values from three-
concentration assay. Tested as iodide salt. Tested as dihydrochloride salt. Tested as dimaleic salt. Tested as oxalic salt.
g
h
i
j
When 2-pyridinylethyl or cyclohexylethyl groups were linked
to the piperidine nitrogen, excellent affinities were obtained,
with compounds 11r and 11t showing pKi values comparable to
that of compound 11m (10.0, 9.8, and 10.1, respectively).
Shortening of the linker to one carbon atom between the
phenyl and the basic site of 11m led to benzyl derivative 11q,
which was about 10 times less potent than the parent
compound.
400 times lower than that of the desmethylated counterpart
11m. This drop in activity demonstrates the importance of
steric and electronic requirements for the basic site of the
molecule for its efficient binding with the receptor.39
Reasonably potent 5-HT4 receptor ligands were obtained
when polar alkyl chains were introduced as substituents on the
piperidine nitrogen. Morpholine 11u, amine 11v, sulfonamide
11w, and amide 11x all showed pKi values ranging from 9.1 to
9.5.
Quaternarization of the basic tertiary nitrogen atom (11s)
was totally detrimental to activity, showing a pKi value about
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a
Scheme 5. Synthetic Route to Compounds 12a−e,i−k
a
Reagents and conditions: (a) TosMIC, NaH, DMSO, Et2O, room temperature; (b) Fe, CH3CO2H, 85 °C; (c) XR2, K2CO3, DMF, 90 °C; (d)
POCl3, Et3N, 120 °C; (e) (1-alkyl-4-piperidinyl)methanol, NaH, DMF, 146 °C; (f) H2, 10% Pd/C, EtOH; (g) XR1, K2CO3, EtOH, reflux; (h) XR1,
EtOH, NaHCO3, reflux; (i) XR1, NaI, Et3N, 2-butanone, reflux; (j) 4-(2-bromoethyl)benzyl alcohol (33) or [4-(methoxymethyl)phenyl]ethyl
bromide (34), Et3N, 2-butanone, reflux.
A study of the effect of the substitution on the 4-position of
the phenyl ring of 11m was also performed. Binding affinity
values in the subnanomolar range were found for 4-hydroxy
(11y) and 4-amino (11aa) derivatives (pKi = 9.6 and 9.7,
respectively). A slight decrease in affinity was found when the
hydroxyl or amino groups were replaced by nitro or carboxylic
moieties (11z and 11ab have pKi = 8.9 and 9.1, respectively).
All these results support the hypothesis that the 5-HT4R has a
large pocket around the interaction point with the basic site of
the ligand, which can accommodate both large hydrophobic
moieties and polar terminal chains.40
2H-Pyrrolo[3,4-c]quinoline Series. A focused series of 2H-
pyrrolo[3,4-c]quinoline derivatives related to hit 12a (Table 4)
were also synthesized, taking into account the SAR around the
indazole-3-carboxamide series. Due to their cumbersome
syntheses, substitution on the benzene of the pyrroloquinoline
ring was not considered in this study. Contrary to that observed
in the previous series, the methyl derivative 12c showed 5-
HT4R binding affinity 2 orders of magnitude higher than that of
the isopropyl counterpart 12d (Table 4, pKi = 8.7 and 6.9,
respectively). This could be due to the steric clash caused by
the isopropyl group, on the rigid tricyclic pyrroloquinoline ring,
with the receptor. This negative interaction may not occur for
the more conformationally free indazolecarboxamide moiety.
Thus, the methyl group was selected for the remainder of the
SAR studies.
Finally, a few derivatives of 12a were designed, in which the
butyl group on the piperidine ring was removed or substituted
with the groups that gave the most potent and selective ligands
within the indazolecarboxamide series (see the sections
“Selectivity for 5-HT4R vs 5-HT2AR” and “Effect on the
hERG Ion Channel”). These included the morpholinylethyl or
(un)substituted phenylethyl moieties.
Four substituted phenylethyl derivatives (12h−k) were
synthesized to further explore the chemical space around the
phenyl ring that seemed to be relevant for both affinity and
selectivity in the previous series.
Substitution on the nitrogen of the piperidine ring gave
similar results in both series. The phenylethyl derivative 12c
showed the highest affinity (pKi = 8.7), and an increase in
affinity was also observed for the 4-carboxylic derivative 12g
and the amide 12k. Removal of the acetyl group of 12k gave
amine 12f, which showed a slight decrease in affinity (pKi =
8.3) compared to the parent compound. Replacement of the
carboxylic group of 12g with a hydroxymethyl or a
methoxymethyl moiety gave compounds that showed a
moderate decrease in activity (12i and 12j, pKi = 8.0 and 7.7,
respectively), while moving the carboxylic group from the 4- to
the 2-position of the phenyl ring gave compound 12h, which
showed a 2 orders of magnitude drop in binding affinity.
Unsubstituted piperidine derivative 12a was 200 times less
active than phenylethyl derivative 12c, while the butyl analogue
12b had pKi = 8.1. Finally, the morpholine derivative 12e and
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Table 4. Binding Properties of Derivatives 12a−k for the Human 5-HT4 and 5-HT2A Receptors, Functional Inhibition of the
hERG Potassium Ion Channel, and Calculated log D at pH 7.4
a
Binding affinity for the human recombinant 5-HT4 receptor, displacement of the [3H]5 ligand expressed as pKi, mean values of duplicate
b
measurements. Confidence intervals of 95% for Ki are not greater than 10% of Ki. Binding affinity for the human recombinant 5-HT2A receptor,
displacement of the [3H]ketanserin ligand expressed as pKi, mean values of duplicate measurements. Confidence intervals of 95% for Ki are not
c
d
e
f
greater than 10% of Ki. See ref 37. log D(7.4) values calculated by ACDLabs 12.0. Percent inhibition at a concentration of 30 μM. Tested as
hydrochloride salt.
the phenyl derivative 12c showed comparable binding activities
(pKi = 8.6 and 8.7, respectively).
and 11m) for all the 5-HT subtype receptors and transporter
(see the Supporting Information), which confirmed the
presence of significant activity for the off-target 5-HT2AR. We
therefore decided to screen the most interesting compounds
against 5-HT2AR (Tables 1−4).
Low selectivity between 5-HT4 and 5-HT2A receptors was
confirmed for a number of compounds within the indazole-
carboxamide series, which show pKi values for 5-HT2AR
between 7 and 8 (11m,r,y,aa). However, small chemical
Selectivity over 5-HT4R vs 5-HT2AR. Throughout the SAR
study directed toward obtaining potent 5-HT4R ligands,
attention was paid to the selectivity of our compounds versus
some off-target proteins. Our concern for a potential lack of
selectivity arose from (i) literature data showing that 5-HT4R
has structural features partially overlapping those of 5-HT2AR41
and (ii) preliminary binding data of a few initial hits (11b, 11d,
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a
found in this chemical series could be attributed to the higher
lipophilicity of these compounds, which have log D(7.4) values
almost 1 unit higher than those of their N-(4-piperidinylmeth-
yl)-1-isopropyl-1H-indazole-3-carboxamide counterparts (com-
pare log D(7.4) values of 12c,e−g with those of 11m,u,aa,ab,
respectively). Only the introduction of the carboxylic acid
group (12g) significantly decreased the affinity for the hERG
ion channel.
Scheme 6. Synthetic Route to Compounds 12f−h
Taking into account the interesting in vitro properties of the
carboxylates 11ab and 12g (high affinity for 5-HT4R and good
selectivity toward the 5-HT2AR and hERG channel), we
decided to further investigate their pharmacological profile
with several biologically relevant receptors and enzymes.45
Compound 11ab did not show any significant interaction
(<50% inhibition at 100 μM) for all the biological targets
assayed, whereas compound 12g shows only very weak binding
affinity for σ1 and σ2 receptors, with 58% and 68% inhibition at
10 μM, respectively (Table 2 in the Supporting Information).
5-HT4R Antagonism. The 5-HT4R antagonism was tested
for a few of the most interesting derivatives. Compounds
11b,v,x,y,aa lacked any intrinsic activity on intracellular basal
Ca2+ levels in the HEK293 cellular functional assay, whereas
they antagonized (1 μM) 5-HT-induced intracellular ion levels
(Supporting Information). The same result was also obtained
for 11y and 11aa in an intracellular cAMP level functional assay
for human 5-HT4R (Supporting Information). The most
interesting compounds 11ab and 12g showed full receptor
antagonism in the cAMP-related functional assay on human
recombinant 5-HT4(e) receptor (Chinese hamster ovarian,
CHO; 5 as reference antagonist compound), with IC50 values
of 9.8 and 2.8 nM and Kb values of 1.4 and 0.41 nM,
respectively (5, IC50 = 0.21 nM, Kb = 0.03 nM) (Supporting
Information).
Pharmacokinetic Studies. 11ab and 12g were tested in an
in vitro liver microsome assay from five different species (rat,
dog, miniature pig, monkey, and human) (Table 5). Intrinsic
clearance was also calculated for rat and human microsomes
(Table 6). Both compounds showed high metabolic stability in
all the tested species, with the exception of 12g in cynomolgus
monkey liver microsomes, where it showed low and moderate
stability at 1 and 10 μM, respectively. This metabolic behavior
could possibly be attributed to the presence of a specific
cytochrome P450 in monkey (CYP2C76), where compound
12g could be metabolized, as observed for other compounds
such as tolbutamide and testoterone.46 The low values of
intrinsic clearance for 11ab (3.9 and 2.5 mL/min/kg,
respectively) and 12g (8.32 and 0.65 mL/min/kg, respectively)
supported the selection of these compounds for further in vivo
evaluation.47
a
Reagents and conditions: (a) ethyl 4-(2-chloroethyl)benzoate36 or
methyl 2-(2-bromoethyl)benzoate (35), NaI, Et3N, 2-butanone, reflux;
(b) 4-(2-bromoethyl)-1-nitrobenzene, K2CO3, DMF, 70 °C; (c) H2,
10% Pd/C, EtOAc (d) 1 N NaOH, THF, EtOH.
modifications led to significant changes in activity. Among the
most potent 5-HT4 ligands, low or very low 5-HT2AR binding
affinity (pKi < 6) was obtained with derivatives having polar
terminal moieties (11u−w) or electron-withdrawing substitu-
ents on the phenyl moiety (11z and 11ab) (Table 3). These
results gave us useful information about the electronic and
structural requirements needed to obtain selectivity toward the
5-HT2AR, particularly in the region “beyond” the basic site of
the molecules, where the 5-HT4R shows more tolerance.
Interestingly, the 4-(4-piperidinylmethoxy)-2H-pyrrolo[3,4-
c]quinolines display lower affinity for the 5-HT4 and 5-HT2A
receptors compared to their indazolecarboxamide counterparts.
Fortunately, the positive effect on 5-HT4 selectivity shown by
the morpholinylethyl and (4-carboxyphenyl)ethyl moieties in
the indazolecarboxamide series was retained in the pyrroloqui-
nolines, with compounds 12e and 12g having pKi < 5 on 5-
HT2AR. The 4-acetamido derivative 12k also showed high
selectivity (5-HT2A, pKi = 5.5).
Effect on the hERG Ion Channel. Our ligands are
lipophilic molecules with a basic site, and these structural
features are often associated with hERG inhibition.42 We
therefore carried out a functional hERG/Kv11.1 cellular assay
on selected compounds using 1 as a reference compound
(100% at 10 μM).38 The hERG inhibitions shown as percent
inhibition at 1 μM or IC50 values are reported in Tables 1−4,
together with the calculated values of log D (pH 7.4) (Tables 3
and 4). log D(7.4) (octanol/water) was selected as a measure of
hydrophobicity since it considers the predominance of
protonated species or zwitterions at pH 7.4. The results show
that in general there is a correlation between the lipophilicity
and the hERG activity.42 Within the indazole series, neither the
Me2N− or MeSO2NH− groups on the alkyl chain terminal
moiety nor the −OH and −NO2 groups on the 4-position of
the phenyl ring led to a significant reduction of hERG
inhibition activity (see compounds 11v,w,y,z). On the other
hand, the morpholine and aniline derivatives 11u and 11aa
(IC50 = 32.5 and 22.6 μM) showed a reduction in inhibition.
The best result was obtained with carboxylic acid derivative
11ab, which was inactive in the hERG assay at the highest
concentration tested (100 μM). The use of carboxylic acid
groups to mitigate hERG has been previously reported.43,44
Unfortunately, the newly synthesized 2H-pyrrolo[3,4-c]-
quinoline derivatives generally exhibited high affinity for the
hERG channel. All tested compounds showed IC50 values
ranging from 0.01 to 1.22 μM, including morpholine and
aniline derivatives 12e,f. One notable exception was carboxylic
acid derivative 12g (IC50 = 9.96 μM). The high hERG affinity
Antinociceptive Effect. The results reported in the
literature for 5-HT4R antagonists in animal models of
analgesia16−21 prompted us to assay 11ab and 12g in two
standard antinociceptive assays in rats: the hot plate test and
the formalin test.48,49 The maximal dose for in vivo studies (10
mg/kg) was selected according to preliminary behavioral and
toxicological results obtained in the Irwin test on male rats after
a single oral dose administration of compound (data not
shown).50,51
Oral administration of 11ab in the hot plate test produced a
weak, but significant nociceptive response at the highest dose
tested (20% increase of latency at 10 mg/kg, p < 0.05 vs vehicle
group). Rats treated with 12g in the same experiment showed a
significant latency increase of antinociceptive response at 5 and
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Table 5. Metabolic Stability of 11ab and 12g in Pooled Liver
Microsomes from Different Species
Table 7. Antinociceptive Activity of 11ab, 12g, and
Morphine in the Hot Plate Test after Oral Administration in
Rats
percentage of compd remaining
a
a
a
dose (mg/kg, po)
latency (s)
11ab
12g
vehicle
3.3 0.15
3.8 0.13
3.7 0.15
3.7 0.20
conc (μM)
30 min
60 min
30 min
60 min
11ab
0.5
1
Human
103
1
101
99
104
106
99
99
5
10
99
b
10
0.5
1
4.1 0.21
Mini-Pig
109
12g
3.3 0.15
3.6 0.28
1
110
106
101
102
96
10
99
102
b
5
4.4 0.22
Cynomolgus Monkey
b
10
4.0 0.22
1
91
93
86
9
0.4
61
b
morphine
6
5.3 0.34
10
87
80
a
b
Latency was measured 1 h postdosing. p < 0.05 vs vehicle group by
analysis of variance (ANOVA) followed by Dunnet’s test for multiple
comparisons.
Beagle Dog
1
97
93
97
115
98
107
94
10
101
Rat
vitro assays on human 5-HT4R. A number of derivatives of both
11b and 12a were designed, synthesized, and tested to define
the SAR in these new classes of ligands. Potent ligands were
found in both series. Selectivity over undesirable biological
targets such as 5-HT2AR and hERG was required, so screening
on these off-target proteins was performed to find compounds
with the highest 5-HT4R binding affinity and the lowest
interaction with the 5-HT2AR and hERG potassium ion
channel. Introduction of large lipophilic or weak polar terminal
groups on the basic site of the hit compounds of both series led
to ligands with high 5-HT4R affinities. However, among these,
only derivatives with a carboxylate function on the phenethyl
moiety (compounds 11ab and 12g) showed good selectivity
over 5-HT2A and the hERG potassium ion channel. Therefore,
11ab and 12g were further studied in vitro (i) in a functional
test of 5-HT4 antagonism, (ii) for their selectivity toward a large
panel of biological targets, and (iii) to assess their metabolic
stability in several species. Both compounds were potent
antagonists with a good selectivity profile and metabolic
stability. Finally, 12g showed promising pharmacological
properties in in vivo models of analgesia (hot plate and
formalin tests), demonstrating significant antinociceptive
activity after oral administration. Therefore, we considered
12g as a candidate for preclinical studies as a potent and
selective 5-HT4 antagonist with excellent analgesic properties.
1
86
87
85
87
93
79
89
10
91
Mouse
97
1
92
85
96
79
97
10
100
104
a
Mean values from three experiments. Warfarin, propranolol, and
testosterone incubated as a cocktail were used as a positive control.
Table 6. Evaluation of the in Vitro Cross-Species Intrinsic
Clearance with Rat and Human Liver Microsomes for 11ab
and 12g
a
b
CLint (μL/min/mg)
CLint (μL/min/kg)
compd
rat
human
rat
human
11ab
12g
2.0
4.2
2.3
0.6
3.9
8.3
2.5
0.7
a
Mean values from three experiments. Midazolam and propranolol
b
were used as a positive control. Scale-up factors for microsomes used
are 45 mg of liver/g of body mass and 44 and 24 g of liver/kg of body
mass for rat and human, respectively.
10 mg/kg (p < 0.05 vs vehicle group) with an improvement of
39% and 26%, respectively. Morphine, which was used as a
reference drug, produced a significant antinociceptive response
(p < 0.05 vs vehicle group) at 6 mg/kg with a 67% latency
increase (Table 7).
The results obtained in the formalin test are reported in
Figure 4. Compounds 11ab and 12g orally administered at 10
mg/kg did not inhibit the paw licking in mice during the first
phase of the model (0−10 min), when there is a direct effect on
nociceptors, whereas a significant analgesic effect was observed
in the second phase of the experiment (panel A, 10−40 min, p
< 0.05 vs vehicle group), when an inflammatory response is
EXPERIMENTAL SECTION
Computational Studies. The computational study was performed
■
using the Schrodinger suite (www.schrodinger.com). Active com-
̈
pounds were extracted from the literature9,14,15 and used as a basic set
for pharmacophore modeling. 3D structures of the ligands were first
generated by means of Macromodel (version 9.1, 2006, Schrodinger
̈
LLC) and then minimized using the OPLS-2005 force field (version
2.0, 2006, Schrodinger LLC) in a continuum dielectric model with a
̈
dielectric constant of 1.0. Molecules were kept in their neutral form
present. Analysis of the area under the curve (AUC0−40′
)
throughout the process. LigPrep (version 2.0, 2006, Schrodinger LLC)
̈
confirmed a statistically significant analgesic activity following
administration of 12g (Figure 4B, p < 0.05 vs vehicle group). As
expected, morphine at 3 mg/kg significantly inhibited the
licking time in both the early and late phases (data not shown).
was then used to generate stereoisomers and the most probable
ionization states at pH 7
2. Conformers were generated using a
maximum of 1000 steps of rapid ConfGen sampling, followed by up to
5000 iterations of truncated Newton conjugate gradient minimization.
The OPLS-2005 force field with distance-dependent dielectric
solvation treatment was employed. Each minimized conformer was
filtered through a relative energy window of 10 kcal/mol and a
redundancy check of 1 Å in the heavy atom positions. Pharmacophore
development was carried out using PHASE,52 where each ligand
structure is represented by a series of chemical features (points) in the
CONCLUSIONS
■
A VS computational approach performed on our in-house
library led us to identify hit compounds 11b and 12a as new 5-
HT4R ligands. The activity of these hits was confirmed in in
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Figure 4. Effect of 11ab and 12g oral administration in the formalin test in mice: (A) time-course curve, (B) area under time-course curve (0−40
min). The asterisk indicates p < 0.05 vs vehicle group by ANOVA followed by Dunnet’s test for multiple comparisons.
3D space, which set the noncovalent binding properties between the
ligand and its target receptor. After applying default feature definitions
to each ligand, we performed the common pharmacophore search
using a terminal box size of 1 Å, and we required that all the actives
should be matched. These pharmacophore sites are characterized by
type, location, and, if applicable, directionality. PHASE provides six
built-in types of pharmacophore features: hydrogen bond acceptor
(A), hydrogen bond donor (D), hydrophobic (H), negative ionizable
(N), positive ionizable (P), and aromatic ring (R).
spectrophotometer. The UV measurement was carried out using
matched 1 cm quartz cells and substance dissolved in 95° ethanol.
Vibration infrared (IR) spectroscopy was performed using a Perkin-
Elmer model FT-2000 infrared spectrophotometer equipped with a
universal attenuated total reflectance (UATR) accessory. Elemental
analysis was conducted by means of a CHNS-O EA1108 elemental
analyzer, Carlo Erba Instruments, and the results were within 0.4% of
the theoretical values, unless otherwise noted. Ultraperformance liquid
chromatography/quadrupole time-of-flight (UPLC/QToF) exact mass
data were obtained by means of a SYNAPT MS−ACQUITY UPLC
system, Waters. The system was operated in positive ion mode in the
“V-Optics” configuration. Leucine enkephalin (200 pg/μL) was
employed as the lock mass to provide authenticated exact mass
measurement in MS and MS/MS modes within 5 ppm rms mass
accuracy. The column was an Acquity BEH C18 (2.1 × 50 mm, 1.7
μm). Differential scanning calorimetry data were obtained on a Perkin-
Elmer DSC7 differential scanning calorimeter.
1-[1-(Phenylmethyl)-4-piperidinyl]methylamine, 1-methyl-1H-in-
dazole-3-carbonyl chloride (13), 1,5-dimethyl-1H-indazole-3-carbox-
ylic acid (17), 2-methyl-2H-indazole-3-carboxylic acid (20), methyl 5-
chloro-1H-indazole-3-carboxylate, 1-(1-butyl-4-piperidinyl)-
methylamine, 1-[(2-phenylethyl)-4-piperidinyl]methylamine, 1H-inda-
zole-3-carbonyl chloride, (2-bromoethyl)cyclohexane, N-(2-
bromoethyl)methansulfonamide, 2-(4-hydroxyphenyl)ethyl bromide,
ethyl 3-(2-nitrophenyl)propenoate, 1-butyl-4-piperidinemethanol, 1-
(2-phenylethyl)-4-piperidinemethanol, 4-piperidinemethanol, 1-(phe-
nylmethyl)-4-piperidinemethanol, 1-(2-bromoethyl)-4-nitrobenzene,
N-[4-(2-bromoethyl)phenyl]acetamide, and 2-(4-chlorophenyl)ethyl
bromide are commercially available. 1-Isopropyl-1H-indazole-3-
carbonyl chloride (14),31 ethyl 4-(2-bromoethyl)benzoate,34 ethyl 4-
(2-chloroethyl)benzoate,36 5-methoxy-1-methyl-1H-indazole-3-carbox-
ylic acid (18),32 and ethyl 3-(2-nitrophenyl)propanoate (22)35 were
prepared as previously described.
Pharmacophores with features common to all training set active
compounds were identified and scored according to superposition of
pharmacophore site points, alignment of vector characteristics, overlap
of molecular volumes, and penalization of matches to the set of
inactive compounds. Different 3D queries were generated and then
used to discriminate true positives. The model was validated by means
of the search of 5-HT4 active compounds in a database composed of
known actives (not used in the development of the model) and decoy
ligands from Schrodinger.53 The validated pharmacophoric model was
̈
then used to identify hits from the proprietary database. The
proprietary database was first prepared using LigPrep to generate
3D structures, stereoisomers, tautomers, and ionization states. Then
the structures were used to create a PHASE database, which entails the
following steps: expanding structures into conformational ensembles
and mapping pharmacophore features to each molecule. The phase
database was the starting point for the ligand-based VS. When the
pharmacophore hypothesis was used to filter the phase database, all
features of the pharmacophore hypothesis were required to match.
The distance tolerance was set to its default value of 2.0 Å in all
pharmacophore searches. The pharmacophore matching score of
PHASE was used in the VS procedure. The identified virtual hits were
then submitted to the in vitro assay.
Chemistry. General Procedures. Reagents were purchased from
Sigma-Aldrich and were used as received. Reaction progress was
monitored by TLC using Merck silica gel 60 F254 (0.04−0.063 mm)
with detection by UV (214 or 254 nm). Merck silica gel 60 or
aluminum oxide 90 (active neutral) was used for column
chromatography. Melting points (uncorrected) were determined in
open Pyrex capillary tubes using a Buchi 510 melting point apparatus.
The compounds' purities were always ≥95% determined by high-
pressure liquid chromatography (HPLC). HPLC analysis was carried
out with a pump/autosampler from Waters (2695-Alliance model), a
UV photodiode array detector from Waters (2996 model), and a data
management system from Waters (Empower 2). The column used was
generally Suplex pkb-100 (250 × 4.6 mm, 5 μm). Proton nuclear
magnetic resonance (1H NMR) spectra were obtained using a Bruker
Avance system, operating at 300 or 400 MHz. All resonance bands
were referenced to tetramethylsilane (internal standard). UV−vis
spectra were recorded using a Perkin-Elmer (UV/vis) Lambda 25
Syntheses. Specific examples presented below illustrate general
synthetic procedures.
1-Isopropyl-N-[[1-(phenylmethyl)-4-piperidinyl]methyl]-1H-inda-
zole-3-carboxamide (11q). 1-Isopropyl-1H-indazole-3-carbonyl
chloride (14;31 9.3 g, 42 mmol) was added in portions into a well-
stirred suspension of 1-[1-(phenylmethyl)-4-piperidinyl]methylamine
(8.6 g, 42 mmol) in toluene (100 mL) at room temperature. After
being stirred for 12 h, the mixture was filtered, and the crude product
obtained was recrystallized from isopropyl alcohol/water (50:1) to
achieve 11q. This compound was treated with 2.5 N HCl in ethanol to
furnish the corresponding hydrochloride, which was recrystallized
from n-hexane/ethyl acetate (7:3; 31%, 109−111 °C). IR (KBr): ν
3498.24, 2931.81, 2538.38, 1630.07, 1543.92, 1455.10, 1203.76,
1
947.24, 763.94 cm−1. H NMR (DMSO-d6): δ 1.55 (d, J = 7 Hz,
6H), 1.10−2.25 (m, 5H), 2.80−3.80 (m, 10H), 4.0−4.6 (m, 2H), 5.08
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(sept, J = 7 Hz, 1H), 7.0−8.0 (m, 8H), 8.05−8.70 (m, 2H), 11.04 (br
s, 1H). 13C NMR (DMSO-d6): δ 21.84, 26.61, 33.98, 43.21, 50.20,
51.13, 58.88, 110.23, 121.77, 122.18, 126.22, 128.58, 129.22, 129.89,
131.37, 136.67, 139.49, 162.21. Anal. (C24H30N4O·HCl·2H2O) C, H,
N, Cl.
(DMSO-d6): δ 13.43, 19.45, 25.31, 26.64, 33.63, 35.99, 42.77, 51.09,
55.27, 100.42, 111.46, 118.68, 122.82, 135.85, 136.82, 155.34, 162.26,
164.51. Anal. (C20H30N4O2·C2H2O4·1/2H2O) C, H, N, Cl.
N-[(1-Butyl-4-piperidinyl)methyl]-5-chloro-1-methyl-1H-inda-
zole-3-carboxamide (11j). 11j was prepared according to the
procedure described for 11h starting from 19. Crude 11j was then
treated with 2.5 N HCl in ethanol to obtain the corresponding
hydrochloride (22%, 250−251 °C). IR (KBr): ν 3418.18, 2933.70,
N-[[1-(2-Phenylethyl)-4-piperidinyl]methyl]-1H-indazole-3-car-
boxamide (11l). 11l was prepared according to the procedure used for
compound 11q starting from commercially available 1H-indazole-3-
carbonyl chloride using 1-[(2-phenylethyl)-4-piperidinyl]methylamine
as reactant. 11l·HCl was recrystallized from ethyl acetate/ethanol
(33%, 192−194 °C). IR (KBr): ν 2927.12, 2552.32, 1650.83, 1549.71,
1
2496.57, 1656.26, 1536.17, 1480.92, 1213.02, 806.84 cm−1. H NMR
(DMSO-d6): δ 10.35 (br s, 1H), 8.58 (t, J = 5.94 Hz, 1H), 8.15 (d, J =
1.98 Hz, 1H), 7.82 (d, J = 8.92 Hz, 1H), 7.50 (dd, J = 1.98, 8.92 Hz,
1H), 4.15 (s, 3H), 3.44 (d, J = 11.56 Hz, 2H), 3.21 (t, J = 6.28 Hz,
2H), 2.71−3.12 (m, 4H), 1.76−2.05 (m, 3H), 1.47−1.75 (m, 4H),
1.31 (m, J = 7.30 Hz, 2H), 0.90 (t, J = 7.30 Hz, 3H). 13C NMR
(DMSO-d6): δ 13.42, 19.48, 23.14, 25.01, 26.76, 31.15, 33.87, 36.14,
43.18, 47.72, 51.31, 55.58, 112.36, 120.48, 122.88, 126.84, 136.30,
139.47, 161.59. Anal. (C19H27N4OCl·HCl) C, H, N, Cl.
1
1469.81, 1232.18, 1154.07, 954.45, 763.04 cm−1. H NMR (DMSO-
d6): δ 13.83 (s, 1H), 10.93 (br s, 1H), 8.60 (t, J = 5.72 Hz, 1H), 8.21
(d, J = 7.63 Hz, 1H), 7.65 (d, J = 8.00 Hz, 1H), 7.02−7.54 (m, 7H),
2.64−4.04 (m, 11H), 1.26−2.29 (m, 5H). 13C NMR (DMSO-d6): δ
26.87, 29.31, 33.94, 43.13, 51.42, 56.58, 110.64, 121.50, 121.88,
126.36, 126.66, 128.56, 137.25, 141.05, 162.50. Anal.
(C22H26N4O·HCl·1/3H2O) C, H, N, Cl.
N-[(1-Butyl-4-piperidinyl)methyl]-2-methyl-2H-indazole-3-car-
boxamide (11k). 11k was prepared according to the procedure
described for 11h starting from 20. 11k (5.8 g, 17.6 mmol) was
converted to the maleate salt by treatment with maleic acid (2.1 g, 17.6
mmol) in ethanol (15 mL), which was recrystallized from ethyl acetate
(40%, 136−137 °C). IR (KBr): ν 3318.37, 2958.21, 2687.75, 1654.57,
N-[[1-(Phenylmethyl)-4-piperidinyl]methyl]-1-methyl-1H-inda-
zole-3-carboxamide (15). 15 was prepared according to the
procedure used for compound 11q starting from commercially
available 1-methyl-1H-indazole-3-carbonyl chloride (13) using 1-[1-
(phenylmethyl)-4-piperidinyl]methylamine as reactant. 15·HCl was
obtained (71%, 247−248 °C). IR (KBr): ν 3398.6, 2918.29, 2488.17,
1658.98, 1540.34, 1495.21, 1432.32, 1227.29, 1168.33, 747.70, 700.79
1
1533.95, 1362.15, 1227.22, 1047.89, 872.64, 760.12 cm−1. H NMR
(DMSO-d6): δ 19.99 (br s, 1H), 9.17 (br s, 1H), 8.67 (t, J = 5.44 Hz,
1H), 7.77 (dd, J = 7.42, 13.69 Hz, 2H), 7.00−7.49 (m, 2H), 6.06 (s,
2H), 4.31 (s, 3H), 2.67−3.72 (m, 8H), 1.06−2.22 (m, 9H), 0.91 (t, J =
7.00 Hz, 3H). 13C NMR (DMSO-d6): δ 13.42, 19.37, 25.36, 27.13,
33.62, 39.92, 43.74, 51.62, 55.70, 117.22, 119.97, 120.32, 122.73,
125.76, 128.79, 136.00, 146.40, 159.86, 167.19. Anal.
(C19H28N4O·C4H4O4) C, H, N, Cl.
1
cm−1. H NMR (DMSO-d6): δ 1.20−2.20 (m, 5H), 2.63−3.68 (m,
6H), 4.13 (s, 3H), 4.12−4.36 (m, 2H), 7.26−7.81 (m, 8H), 8.15−8.25
(m, 1H), 8.40−8.75 (m, 1H), 11.10 (br s, 1H). 13C NMR (DMSO-
d6): δ 23.01, 26.56, 33.89, 35.79, 43.13, 47.37, 51.13, 58.90, 110.24,
121.68, 122.17, 126.46, 128.60, 129.23, 129.87, 131.35, 136.77, 140.84,
162.00. Anal. (C22H26N4O·HCl) C, H, N, Cl.
N-[(1-Butyl-4-piperidinyl)methyl]-1,5-dimethyl-1H-indazole-3-
carboxamide (11h). Thionyl chloride (4 mL, 54 mmol) was added to
a stirred solution of the commercially available 17 (5.1 g, 29.6 mmol)
in toluene, and the mixture was stirred under reflux for 2 h. After
removal of the solvent under vacuum, the residue was recrystallized
from n-hexane to give 3.5 g of 1,5-dimethyl-1H-indazol-3-carbonyl
chloride. 1-(1-Butyl-4-piperidinyl)methylamine (2.4 g, 14 mmol) in
toluene (30 mL) was added dropwise to a suspension in toluene (30
mL) of 2.9 g of the intermediate (14 mmol). The mixture was stirred
for 3 h at room temperature. The solid was filtered and dissolved in
H2O. The solution was treated with 6 N NaOH solution until pH 8
and extracted with CH2Cl2 (2 × 200 mL). The organic layer was
washed with brine, dried over Na2SO4, filtered, and evaporated under
vacuum. The residue was purified by column chromatography on silica
gel (CHCl3/MeOH (95:5) as eluent) and then treated with 2.5 N HCl
in ethanol to give the corresponding hydrochloride. 11h·HCl was
recrystallized from isopropyl ether/isopropyl alcohol (4 g, 75%, 212−
213 °C). IR (KBr): ν 3421.06, 2957.55, 2496.59, 1659.87, 1538.80,
1-Methyl-N-(4-piperidinylmethyl)-1H-indazole-3-carboxamide
(11a). Compound 15 (6 g, 16.6 mmol) was dissolved in 312 mL of
ethanol/acetic acid (30:1.2), and the resulting solution was hydro-
genated by a Parr apparatus (28 psi) in the presence of 10% Pd/C (2.4
g) for 24 h at room temperature. The catalyst was filtered off, the
solvent was evaporated, and the residue was portioned between 2 N
NaOH (100 mL) and CH2Cl2 (100 mL). The organic layer was
washed with brine, dried (Na2SO4), filtered, and concentrated in
vacuum to obtain the crude product 11a, which was directly converted
to maleic salt by treatment with 0.66 g of maleic acid (5.7 mmol)
dissolved in 25 mL of absolute ethanol, which was recrystallized from
ethyl acetate/ethanol (1:1) to provide 1.0 g of salt (16%, 153−154
°C). IR (KBr): ν 3389.42, 2929.19, 1659.66, 1538.79, 1362.74,
1214.19, 1164.27, 867.19, 752.18 cm−1. 1H NMR (DMSO-d6): δ 1.1−
1.65 (m, 5H), 1.65−2.15 (m, 6H), 4.14 (s, 3H) 6.08 (s, 2H), 7.1−8.7
(m, 7H), 20.12 (br s, 1H). 13C NMR (DMSO-d6): δ 26.30, 33.66,
35.81, 42.98, 110.27, 121.68, 122.20, 126.49, 136.09, 136.77, 140.86,
162.06, 167.19. Anal. (C15H20N4O·C4H4O4) C, H, N, Cl.
1-Isopropyl-N-(4-piperidinylmethyl)-1H-indazole-3-carboxamide
(16). 16 was prepared according to the procedure described for 11a,
starting from 11q·HCl salt, 35 psi. Compound 16 was treated with 2.5
N HCl in ethanol solution to afford the corresponding hydrochloride,
which was recrystallized from ethanol/ethyl acetate (42%, 211−214
°C). IR (KBr): ν 3315.32, 2912.86, 2784.55, 1658.70, 1541.40,
1279.82, 1203.35, 750.09, 691.72, 619.88 cm−1. 1H NMR (DMSO-d6):
δ 1.55 (d, J = 7 Hz, 6H), 1.31−2.18 (m, 5H), 2.58−3.64 (m, 6H), 5.09
(m, J = 7 Hz, 1H), 7.12−7.60 (m, 2H), 7.80 (d, J = 8 Hz, 1H), 8.20 (d,
J = 8 Hz, 1H), 8.41 (t, J = 6 Hz, 1H), 8.82−9.60 (2 br s, 2H). Anal.
(C17H24N4O·HCl) C, H, N, Cl.
1
1500.34, 1459.45, 1181.06, 1163.97, 804.76 cm−1. H NMR (DMSO-
d6): δ 10.54 (br s, 1H), 8.43 (t, J = 6.11 Hz, 1H), 7.89−7.99 (m, 1H),
7.62 (d, J = 8.59 Hz, 1H), 7.30 (dd, J = 1.49, 8.75 Hz, 1H), 4.10 (s,
3H), 3.29−3.52 (m, 2H), 3.21 (t, J = 6.28 Hz, 2H), 2.70−3.11 (m,
4H), 2.44 (s, 3H), 1.83 (d, J = 12.88 Hz, 3H), 1.48−1.76 (m, 4H),
1.30 (m, J = 7.40 Hz, 2H), 0.90 (t, J = 7.20 Hz, 3H). 13C NMR
(DMSO-d6): δ 13.42, 19.48, 20.97, 23.17, 25.01, 26.78, 31.18, 33.97,
35.82, 43.10, 47.72, 51.33, 55.58, 109.94, 120.58, 122.49, 128.46,
131.26, 136.13, 139.64, 162.10. Anal. (C20H30N4O·HCl) C, H, N, Cl.
N-[(1-Butyl-4-piperidinyl)methyl]-5-methoxy-1-methyl-1H-inda-
zole-3-carboxamide (11i). 11i was prepared according to the
procedure described for 11h starting from 18.32 Crude 11i was then
converted to the corresponding oxalic salt by treatment with oxalic
acid in ethanol (29%, 191−192 °C). IR (KBr): ν 3410.07, 2944.72,
2677.99, 1720.71, 1655.28, 1541.25, 1497.68, 1271.15, 1206.59,
2-Methyl-4-(4-piperidinylmethoxy)-2H-pyrrolo[3,4-c]quinoline
(12a). Pd/C (10%, 200 mg) was added into a solution of 29 (3.5
mmol, 1.34 g) in methanol (150 mL). The mixture was stirred for 4
days under a H2 atmosphere at room temperature and pressure. A H2
stream was passed through every 3 h, and Pd/C (10%, 200 mg) was
added every 24 h. After the palladium was filtered off, the solvent was
removed under reduced pressure to obtain a residue, which was
purified by column chromatography on aluminum oxide (chloroform/
methanol (2:1) as eluent) to obtain 410 mg of pure 12a as an oil
(40%). 1H NMR (CDCl3): δ 1.47−1.57 (m, 2H), 1.91−2.29 (m, 5H),
1
814.91, 707.69 cm−1. H NMR (DMSO-d6): δ 9.21 (br s, 2H), 8.41
(t, J = 6.14 Hz, 1H), 7.65 (d, J = 9.35 Hz, 1H), 7.56 (d, J = 2.05 Hz,
1H), 7.11 (dd, J = 2.48, 9.21 Hz, 1H), 4.10 (s, 3H), 3.81 (s, 3H), 3.41
(d, J = 11.98 Hz, 2H), 3.23 (t, J = 6.28 Hz, 2H), 2.91−3.03 (m, 2H),
2.84 (t, J = 11.55 Hz, 2H), 1.84 (d, J = 12.57 Hz, 3H), 1.38−1.70 (m,
4H), 1.30 (m, J = 7.50 Hz, 2H), 0.90 (t, J = 7.20 Hz, 3H). 13C NMR
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2.76 (m, 1H), 3.12 (m, 1H), 3.26 (m, 1H), 4.03 (s, 3H), 4.49 (d, 2H),
7.30−7.47 (m, 4H), 7.78 (m, 1H), 7.94 (m, 1H). Anal. (C18H21N3O)
C, H, N.
from ethyl acetate/ethanol (18%, 170−171 °C). IR (KBr): ν 3407.39,
2926.15, 1653.92, 1542.02, 1494.36, 1404.81, 1222.82, 1166.49,
1
753.52, 720.00 cm−1. H NMR (DMSO-d6): δ 9.36 (br s, 2H), 8.54
N-[(1-Butyl-4-piperidinyl)methyl]-1-methyl-1H-indazole-3-car-
boxamide (11b). 1-Bromobutane (3.75 g, 27 mmol) and K2CO3 (3.76
g, 27 mmol) were added to a solution of 11a (3.6 g, 13 mmol) in 15
mL of ethanol. The reaction mixture was stirred under reflux for 2 h.
The solid was filtered off, and the solution was concentrated under
vacuum. The residue was diluted with ethyl acetate and treated with 1
N HCl solution. The aqueous layer was neutralized with 1 N NaOH
solution until pH 8 was reached and then extracted with CH2Cl2. The
organic layer was dried over Na2SO4, filtered, and concentrated under
vacuum. The residue was filtered through a silica gel pad that was
washed with CHCl3. The crude product was converted to the
corresponding hydrochloride in the presence of 2.5 N HCl in ethanol.
The salt that was obtained was recrystallized from isopropyl alcohol
(4.3 g, 91%, 194−195 °C). IR (KBr): ν 3343.52, 2931.41, 2486.99,
1650.42, 1546.64, 1433.36, 1227.18, 1173.28, 953.87, 754.06 cm−1. 1H
NMR (DMSO-d6): δ 0.94 (t, J = 7 Hz, 3H), 1.2−2.2 (m, 9H), 2.7−3.6
(m, 8H), 4.08 (s, 3H), 7.2−7.5 (m, 4H), 8.3 (d, J = 7 Hz, 1H), 10.5
(br s, 1H). 13C NMR (DMSO-d6): δ 13.42, 19.48, 25.00, 26.78, 33.92,
35.81, 43.17, 51.31, 55.58, 110.26, 121.68, 122.17, 126.46, 136.79,
140.84, 162.01. Anal. (C19H28N4O·HCl) C, H, N, Cl.
(t, J = 5.62 Hz, 1H), 8.19 (d, J = 7.81 Hz, 1H), 7.74 (d, J = 8.00 Hz,
1H), 7.47 (dt, J = 1.22, J = 7.57 Hz, 1H), 7.04−7.35 (m, 6H), 4.13 (s,
3H), 2.33−3.63 (m, 10H), 1.07−2.07 (m, 9H). 13C NMR (DMSO-
d6): δ 22.98, 26.59, 28.01, 33.55, 34.45, 35.79, 42.77, 51.09, 55.40,
110.26, 121.68, 122.17, 125.73, 126.46, 128.21, 136.79, 140.84, 141.54,
162.01, 164.46. Anal. (C25H32N4O·C2H2O4) C, H, N, Cl.
1-Isopropyl-N-[[1-(2-phenylethyl)-4-piperidinyl]methyl]-1H-inda-
zole-3-carboxamide (11m). 11m was prepared according to the
procedure described for 11b starting from 16 using 1-[1-(2-
phenylethyl)-4-piperidinyl]methylamine as reactant. 11m was purified
by flash chromatography on silica gel (CHCl3/MeOH (95:5) as
eluent) and then treated with 2.5 N HCl in EtOH solution to afford
the corresponding hydrochloride (72%, 211−212 °C). IR (KBr): ν
3331.53, 2945.48, 2558.39, 1652.94, 1546.02, 1206.58, 942.45, 743.17
cm−1. 1H NMR (DMSO-d6): δ 1.56 (d, J = 7 Hz, 6H), 1.50−2.30 (m,
5H), 2.70−3.90 (m, 10H), 5.10 (m, J = 7 Hz, 1H), 7.05−7.63 (m,
7H), 7.81 (d, J = 8 Hz, 1H), 8.21 (d, J = 8 Hz, 1 H), 8.47 (t, J = 6 Hz,
1H), 11.05 (br s, 1H). 13C NMR (DMSO-d6): δ 21.86, 26.90, 29.27,
33.98, 43.24, 50.22, 51.41, 56.57, 110.26, 121.79, 122.20, 126.23,
126.68, 128.56, 136.67, 137.26, 139.52, 162.22. Anal.
(C25H32N4O·HCl) C, H, N, Cl.
N-[[1-(2-Cyclohexylethyl)-4-piperidinyl]methyl]-1-isopropyl-1H-
indazole-3-carboxamide (11t). 11t was prepared according to the
procedure described for 11b starting from 16 using (2-bromoethyl)-
cyclohexane as reactant. The crude 11t was treated with 2.5 N HCl in
ethanol to afford the corresponding hydrochloride, which was
recrystallized from ethyl acetate/ethanol (36%, 244−246 °C dec).
IR (KBr): ν 3329.19, 2922.70, 2552.33, 1655.91, 1544.81, 1441.61,
1207.91, 942.82, 738.08 cm−1. 1H NMR (DMSO-d6): δ 1.55 (d, J = 7
Hz, 6H), 0.68−2.18 (m, 17H), 2.63−3.70 (m, 10H), 5.09 (m, J = 7
Hz, 1H), 7.12−7.60 (m, 2H), 7.80 (d, J = 8 Hz, 1H), 8.20 (d, J = 8 Hz,
1H), 8.41 (t, J = 6 Hz, 1H), 10.70 (br s, 1H). 13C NMR (DMSO-d6):
δ 21.83, 25.42, 25.82, 26.82, 30.11, 32.32, 33.98, 35.00, 43.23, 50.19,
51.31, 54.13, 110.23, 121.76, 122.15, 126.20, 136.65, 139.49, 162.18.
Anal. (C25H38N4O·HCl·1/2H2O) C, H, N, Cl.
1-Isopropyl-N-[[1-(2-morpholin-4-ylethyl]-4-piperidinyl]methyl]-
1H-indazole-3-carboxamide (11u). 11u was prepared according to
the procedure described for 11b starting from 16 using 4-(2-
chloroethyl)morpholine as reactant. The crude 11u was treated with
2.5 N HCl in ethanol to give the corresponding dihydrochloride,
which was recrystallized from ethanol (63%, 266−267 °C dec). IR
(KBr): ν 3375.90, 2934.70, 2448.48, 1652.35, 1539.86, 1455.66,
1203.01, 1133.42, 1100.85, 754.59 cm−1. 1H NMR (DMSO-d6): δ 1.55
(d, J = 7 Hz, 6H), 1.30−2.25 (m, 5H), 2.75−4.30 (m, 19H), 5.09 (m, J
= 7 Hz, 1H), 7.12−7.60 (m, 2H), 7.81 (d, J = 8 Hz, 1H), 8.20 (d, J = 8
Hz, 1H), 8.45 (t, J = 6 Hz, 1H), 10.80 (br s, 1H), 10.60 (br s, 1H). 13C
NMR (DMSO-d6): δ 21.86, 26.84, 33.68, 43.17, 49.17, 49.65, 50.20,
51.33, 51.95, 63.13, 110.26, 121.77, 122.20, 126.23, 136.64, 139.50,
162.22. Anal. (C23H35N5O2·2HCl·1/2H2O) C, H, N, Cl.
N-[[1-(2-Cyclohexylethyl)-4-piperidinyl]methyl]-1-methyl-1H-in-
dazole-3-carboxamide (11c). 11c was prepared according to the
procedure described for 11b using (2-bromoethyl)cyclohexane as
reactant. The crude product was treated with 2.5 N HCl in ethanol to
furnish the corresponding hydrochloride, which was recrystallized
from ethyl acetate/ethanol (8:2; 78%, 227−230 °C). IR (KBr): ν
3370.73, 2927.48, 2850.47, 2497.79, 1650.41, 1536.21, 1493.85,
1
1217.49, 1164.54, 945.18, 753.33 cm−1. H NMR (CDCl3): δ 10.66
(br s, 1H), 8.57 (t, J = 5.50 Hz, 1H), 8.19 (d, J = 7.57 Hz, 1H), 7.75
(d, J = 8.00 Hz, 1H), 7.37−7.59 (m, 1H), 7.16−7.36 (m, 1H), 4.15 (s,
3H), 2.67−3.66 (m, 8H), 0.59−2.16 (m, 18H). 13C NMR (DMSO-
d6): δ 23.15, 25.44, 25.83, 26.81, 30.14, 32.34, 33.92, 35.02, 35.81,
43.15, 47.72, 51.33, 54.15, 110.26, 121.68, 122.17, 126.46, 136.79,
140.84, 162.00. Anal. (C23H34N4O·HCl) C, H, N, Cl.
1-Methyl-N-[[1-(2-phenylethyl)-4-piperidinyl]methyl]-1H-inda-
zole-3-carboxamide (11d). 11d was prepared according to the
procedure used for compound 11b using (2-bromoethyl)benzene as
reactant. The crude product was treated with 2.5 N HCl in ethanol to
obtain the corresponding hydrochloride, which was recrystallized from
ethyl acetate/ethanol (8:2; 82%, 219−220 °C). IR (KBr): ν 3412.96,
2938.59, 2511.29, 1676.74, 1540.82, 1496.40, 1545.13, 1219.78,
1170.78, 949.11, 746.37, 698.47 cm−1. 1H NMR (DMSO-d6): δ
1.5−2.1 (m, 5H), 2.8−3.7 (m, 10H), 4.1 (s, 3H), 7.2−7.9 (m, 9H), 8.2
(d, J = 7 Hz, 1H), 8.6 (t, 1H). 13C NMR (DMSO-d6): δ 23.21, 26.85,
29.27, 33.91, 35.81, 43.15, 51.41, 56.55, 110.26, 121.70, 122.18,
126.48, 126.68, 128.56, 136.79, 137.23, 140.84, 162.03. Anal.
(C23H28N4O·HCl) C, H, N, Cl.
N-[[1-[2-(4-Chlorophenyl)ethyl]-4-piperidinyl]methyl]-1-methyl-
1H-indazole-3-carboxamide (11f). 11f was prepared according to the
procedure used for compound 11b using 2-(4-chlorophenyl)ethyl
bromide as reactant. The crude product was treated with 2.5 N HCl in
ethanol to furnish the corresponding hydrochloride, which was
recrystallized from ethanol/water (58%, 245−246 °C). IR (KBr): ν
3402.56, 2945.21, 2332.05, 1661.10, 1534.41, 1493.17, 1215.08,
858.67, 771.02 cm−1. 1H NMR (DMSO-d6): δ 11.08 (br s, 1H),
8.59 (t, J = 5.69 Hz, 1H), 8.20 (d, J = 7.66 Hz, 1H), 7.75 (d, J = 8.00
Hz, 1H), 7.14−7.59 (m, J = 3.00 Hz, 6H), 4.15 (s, 3H), 2.63−3.74 (m,
10H), 1.33−2.23 (m, 5H). 13C NMR (DMSO-d6): δ 26.94, 28.68,
33.98, 35.91, 38.79, 39.06, 39.34, 39.61, 39.89, 40.18, 40.45, 43.24,
51.54, 56.34, 110.36, 121.78, 122.29, 126.58, 128.61, 130.60, 131.45,
136.37, 136.89, 140.94, 162.13. Anal. (C23H27N4ClO·HCl) C, H, N,
Cl.
N-[[1-[3-(Dimethylamino)propyl]-4-piperidinyl]methyl]-1-iso-
propyl-1H-indazole-3-carboxamide (11v). 11v was prepared accord-
ing to the procedure described for 11b starting from 16 (480 mg, 1.6
mmol) using N-(3-chloropropyl)-N,N-dimethylamine hydrochloride
(580 mg, 3.7 mmol) as reactant. Compound 11v was treated with
maleic acid in ethanol to afford the corresponding dimaleate, which
was recrystallized from ethanol (840 mg, 84%, 155−156 °C). IR
(KBr): ν 3433.88, 2934.55, 2697.84, 1572.28, 1385.77, 1200.30,
1
1005.05, 875.99, 864.97, 751.10 cm−1. H NMR (DMSO-d6): δ 1.55
(d, J = 7 Hz, 6H), 1.20−1.60 (m, 2H), 1.68−2.28 (m, 5H), 2.81 (s,
6H), 2.75−3.75 (m, 13H), 5.09 (sept, J = 7 Hz, 1H), 6.09 (s, 4H),
7.12−7.60 (m, 2H), 7.81 (d, J = 8 Hz, 1H), 8.20 (d, J = 8 Hz, 1H),
8.45 (t, J = 6 Hz, 1H), 19 (br s, 2H). 13C NMR (DMSO-d6): δ 19.13,
21.86, 26.82, 33.54, 42.36, 42.71, 50.20, 51.45, 52.67, 53.92, 110.26,
121.74, 122.21, 126.26, 135.75, 136.64, 139.52, 162.27, 167.17. Anal.
(C22H35N5O·C8H8O8·1/2H2O) C, H, N.
1-Methyl-N-[[1-(4-phenylbutyl)-4-piperidinyl]methyl]-1H-inda-
zole-3-carboxamide (11g). 11g was prepared according to the
procedure used for compound 11b using 4-phenylbutyl bromide as
reactant. The obtained crude product was reacted with oxalic acid in
ethanol to give the corresponding oxalic salt, which was recrystallized
1-Isopropyl-N-[[1-[2-[(methylsulfonyl)amino]ethyl]-4-
piperidinyl]methyl]-1H-indazole-3-carboxamide (11w). 11w was
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prepared according to the procedure described for 11b starting from
16 (4.8 g, 16 mmol) using N-(2-bromoethyl)methanesulfonamide (3.4
g, 16.8 mmol) as reactant. The compound was treated with 2.5 N HCl
in ethanol solution to furnish the corresponding hydrochloride, which
was recrystallized with ethyl acetate/ethanol (1.5 g, 20%, 186−187 °C
dec). IR (KBr): ν 3341.98, 2938.43, 2543.75, 1651.59, 1542.67,
solution of 12a (2.2 mmol, 0.65 g) in DMF (5 mL). The mixture was
stirred at 70 °C for 2 h and 45 min. After cooling, the mixture was
diluted with water (20 mL) and extracted with ethyl acetate (3 × 30
mL). The combined organic layers were washed with brine (3 × 50
mL), dried over Na2SO4, filtered, and evaporated under reduced
pressure to give a crude product, which was purified by column
chromatography on silica gel (chloroform/methanol (10:1) as eluent)
to give 0.62 g of pure product (62%, 157−159 °C, recrystallized from
1
1325.86, 1208.22, 1157.42, 967.42, 750.30 cm−1. H NMR (DMSO-
d6): δ 1.55 (d, J = 7 Hz, 6H), 1.40−2.30 (m, 5H), 3.00 (s, 3H), 2.75−
3.80 (m, 10H), 5.09 (m, J = 7 Hz, 1H), 7.12−7.70 (m, 3H), 7.80 (d, J
= 8 Hz, 1H), 8.20 (d, J = 8 Hz, 1H), 8.45 (t, J = 6 Hz, 1H), 10.73 (br s,
1H). 13C NMR (DMSO-d6): δ 21.86, 26.78, 33.75, 36.98, 39.30, 43.20,
50.20, 51.79, 55.53, 110.24, 121.77, 122.18, 122.24, 126.23, 136.65,
139.50, 162.21. Anal. (C20H31N503S·HCI) C, H, N, Cl, S.
1
toluene/cyclohexane). H NMR (CDCl3): δ 1.65 (m, 2H), 2.00 (m,
3H), 2.19 (m, 2H), 2.72 (t, J = 8.2 Hz, 2H), 3.02 (t, J = 8.2 Hz, 2H),
3.12 (m, 2H), 4.03 (s, 3H), 4.53 (d, 2H), 7.32−7.47 (m, 5H), 7.78 (m,
1H), 7.93 (m, J = 8.1 Hz, J = 1.1 Hz, 2H), 8.20 (d, J = 8.7 Hz, 2H).
Anal. (C26H28N4O3) C, H, N.
1-Isopropyl-N-[[1-[2-(4-hydroxyphenyl)ethyl]-4-piperidinyl]-
methyl]-1H-indazole-3-carboxamide (11y). 11y was prepared
according to the procedure described for 11b starting from 16 using
2-(4-hydroxyphenyl)ethyl bromide as reactant. Crude 11y was treated
with 2.5 N HCl in ethanol to give the corresponding hydrochloride,
which was crystallized from absolute ethanol (29%, 218−220 °C). IR
(KBr): ν 3167.62, 2511.31, 1632.00, 1557.96, 1515.87, 1457.54,
1206.14, 833.57, 746.65 cm−1. 1H NMR (DMSO-d6): δ 1. 55 (d, J = 7
Hz, 6H), 1. 63−2.15 (m, 5H), 2.70−3.75 (m, 10H), 5.09 (m, J = 7 Hz,
1H), 6.75 (d, J = 8 Hz, 2H), 7.06 (d, J = 8 Hz, 2H), 7.21−7. Thirty
(m, 1 H), 7.40−7.50 (m, 1 H), 7.8 (d, J = 8 Hz, 1H), 8.21 (d, J = 8 Hz,
1H), 8.46 (m, 1H), 9.40 (s, 1H), 10.80 (br s, 1H). 13C NMR (DMSO-
d6): δ 21.86, 26.91, 28.50, 33.97, 43.21, 50.22, 51.44, 57.00, 110.24,
115.36, 121.77, 122.20, 126.23, 126.98, 129.48, 136.67, 139.50, 156.15,
162.22. Anal. (C25H32N4O2·HCl) C, H, N, Cl.
1-Methyl-N-[[1-(2-pyridin-2-ylethyl)-4-piperidinyl]methyl]-1H-in-
dazole-3-carboxamide (11e). 2-Vinylpyridine (14.8 mmol, 1.6 mL)
was added to a solution of 11a (4 g, 14.7 mmol) in 1.8 mL of water/
glacial acetic acid (4:5). The mixture was stirred under reflux for 5 h
and then at room temperature overnight. The solution was treated
with water and then with solid NaOH until pH 8 was reached. The
mixture was extracted with CH2Cl2, dried over Na2SO4, filtered, and
concentrated under vacuum. The residue was filtered through a silica
gel pad that was washed with CHCl3/MeOH (95:5). The crude 11e
was recrystallized from n-hexane/ethyl acetate to give 3.8 g of the title
product. The dihydrochloride salt was prepared by treating 11e with
0.1 N HCl in diethyl ether (3.3 mL) and was recrystallized from ethyl
acetate/ethanol (95:5; 2.5 g, 35.5%, 214−215 °C). IR (KBr): ν
3386.36, 2598.25, 1655.28, 1619.82, 1538.55, 1232.88, 939.72, 777.15,
1
751.34 cm−1. H NMR (DMSO-d6): δ 11.36 (br s, 1H), 8.82 (d, J =
1-Isopropyl-N-[[1-[2-(4-nitrophenyl)ethyl]-4-piperidinyl]methyl]-
1H-indazole-3-carboxamide (11z). 11z was prepared according to
the procedure described for 11b starting from 16 using 1-(2-
bromoethyl)-4-nitrobenzene as reactant. The product was purified
by flash chromatography on silica gel (ethyl acetate as eluent). Pure
11z was treated with a stoichiometric amount of oxalic acid in ethyl
acetate to generate the corresponding oxalate, which was recrystallized
twice from ethyl acetate/ethanol (9:1; 39%, 98 °C dec). IR (diffuse
reflectance with KBr): ν 3325.5, 2981.0, 2937.6, 1654.7, 1520.1,
4.64 Hz, 1H), 8.32−8.70 (m, 2H), 7.67−8.26 (m, 4H), 7.48 (dt, J =
1.22, 7.57 Hz, 1H), 7.17−7.36 (m, 1H), 4.15 (s, 3H), 2.70−3.95 (m,
11H), 1.30−2.45 (m, 5H). 13C NMR (DMSO-d6): δ 26.83, 28.19,
33.67, 35.91, 38.77, 39.05, 39.34, 39.61, 39.89, 40.16, 40.43, 43.15,
51.58, 54.16, 110.36, 121.78, 122.29, 124.72, 126.57, 136.88, 140.94,
143.55, 143.85, 153.48, 162.13. Anal. (C22H27N5O·2HCl) C, H, N, Cl.
1-Isopropyl-N-[[1-(2-pyridin-2-ylethyl)-4-piperidinyl]methyl]-1H-
indazole-3-carboxamide (11r). 11r was prepared according to the
procedure described for 11e starting from 16. The residue was purified
by flash chromatography on silica gel (CHCl3/MeOH (97:3) as
eluent) to yield 11r, which was treated with 2.5 N HCl in ethanol
solution to give the hydrochloride, which was recrystallized from ethyl
acetate/ethanol (33%, 122−123 °C). IR (KBr): ν 3322.07, 2934.88,
2635.47, 1643.93, 1548.14, 1439.24, 1206.50, 941.32, 753.85 cm−1. 1H
NMR (DMSO-d6): δ 1.55 (d, J = 7 Hz, 6H), 1.68−2.30 (m, 5H),
2.80−3.78 (m, 12H), 5.10 (m, J = 7 Hz, 1H), 7.12−7.60 (m, 4H),
7.68−8.00 (m, 2H), 8.21 (d, J = 7 Hz, 1H), 8.33−8.70 (m, 2H), 11.05
(br s, 1H). 13C NMR (DMSO-d6): δ 21.86, 31.16, 50.20, 110.24,
121.77, 122.00, 122.18, 123.34, 126.23, 136.87, 139.50, 149.02, 157.00,
162.22. Anal. (C24H31N5O·HCl·H2O) C, H, N Cl.
4-[[[(1-Isopropyl-1H-indazol-3-yl)carbonyl]amino]methyl]-1-
methyl-1-(2-phenylethyl)piperidinium Iodide (11s). Methyl iodide
(0.6 mL, 10 mmol) was slowly added into a solution of 11m (4 g, 9.9
mmol) in acetone (40 mL). The mixture was stirred at room
temperature for 3 h and then filtered, and the solid that was obtained
was recrystallized from ethyl acetate/ethanol (1:1; 1.4 g, 26%, 209−
210 °C). 1H NMR (DMSO-d6): δ 8.48 (t, J = 5.67 Hz, 1H), 8.20 (d, J
= 7.43 Hz, 1H), 7.82 (d, J = 8.20 Hz, 1H), 7.12−7.58 (m, 7H), 5.11
(m, J = 6.68 Hz, 1H), 2.87−3.93 (m, 13H), 1.36−2.23 (m, 5H), 1.56
(d, J = 6.65 Hz, 6H). 13C NMR (DMSO-d6): δ 21.86, 23.56,27.51,
33.46, 42.63, 43.93, 50.23, 59.51, 66.91, 110.26, 121.73, 122.20,
126.25, 126.87, 128.61, 128.96, 136.39, 136.67, 139.50, 162.29. IR
(KBr): ν 3314.16, 2976.00, 1646.96, 1550.21, 1487.41, 1204.03,
1130.61, 930.35, 753.43, 710.40, 640.40 cm−1. Anal. (C26H35IN4O) C,
H, N, I.
1
1347.6, 1203.8, 855.8, 751.0, 707. 0 cm−1. H NMR (DMSO-d6 +
D2O): δ 1.55 (d, J = 7 Hz, 6H), 1.44−1.66 (m, 2H), 1.83−2.02 (m,
3H), 2.98 (t, J = 12 Hz, 2H), 3.10−3. 40 (m, 6H), 3.55 (d, J = 12 Hz,
2H), 5.07 (m, J = 7 Hz, 1H), 7.28 (t, J = 8 Hz, 1H), 7.46 (t, J = 7 Hz,
1H), 7.59 (d, J = 9 Hz, 2H), 7.79 (d, J = 8 Hz, 1H), 8.11−8.26 (m,
3H), 8.42 (t, J = 6 Hz, 1H). 13C NMR (DMSO-d6): δ 21.86, 26.81,
29.58, 33.78, 42.92, 50.22, 51.25, 55.73, 110.24, 121.80, 122.20,
122.26, 123.58, 126.25, 130.06, 136.68, 139.52, 145.84, 146.38, 162.22,
164.60. Anal. (C25H31N5O3·C2H2O4·1/2H2O) C, H, N.
1-Isopropyl-N-[[1-[3-(methylamino)-3-oxo-1-propyl]-4-
piperidinyl]methyl]-1H-indazole-3-carboxamide (11x). A mixture of
16 (6.9 g, 23 mmol), 3-chloro-N-methylpropanamide (4.8 g, 35.4
mmol), and K2CO3 (10 g, 72 mmol) in DMF (100 mL) was stirred at
80 °C overnight. The cooled solution was poured into water and
extracted with ethyl acetate. The organic layer was washed with 0.5 N
NaOH, dried over Na2SO4, filtered, and concentrated under vacuum.
Crude 11x was treated with Et2O/HCl to afford the corresponding
hydrochloride, which was recrystallized from isopropyl alcohol/
diisopropyl ether (5.2 g, 52%, 190−191.5 °C). IR (diffuse reflectance
with KBr): ν 3474, 3352, 3203, 1638, 1541, 1383, 1263, 1207, 948,
832, 760, 642 cm−1. 1H NMR (DMSO-d6): δ 10.31 (br s, 1H), 8.36 (t,
J = 6.19 Hz, 1H), 8.17 (m, J = 0.93, 8.22 Hz, 1H), 8.10 (q, J = 4.30 Hz,
1H), 7.79 (d, J = 8.59 Hz, 1H), 7.43 (m, J = 1.16 Hz, J = 6.98 Hz, J =
8.38 Hz, 1H), 7.25 (m, J = 0.66 Hz, J = 6.98 Hz, J = 8.05 Hz, 1H), 5.08
(m, J = 6.61 Hz, 1H), 3.12−3.58 (m, 8H), 2.77−2.98 (m, 2H), 2.65 (t,
J = 8.17 Hz, 2H), 2.59 (d, J = 4.46 Hz, 3H), 1.78−2.12 (m, 3H), 1.46−
1.74 (m, 2H), 1.55 (d, J = 6.61 Hz, 6H). 13C NMR (DMSO-d6): δ
21.84, 25.47, 26.88, 29.29, 33.84, 43.17, 50.20, 51.50, 52.08, 110.24,
121.77, 122.18, 126.23, 136.65, 139.50, 162.21, 169.05. Anal.
(C21H31N5O2·HCl·3/4H2O) C, H, N, Cl.
2-Methyl-4-[[1-(2-morpholin-4-ylethyl)-4-piperidinyl]methoxy]-
2H-pyrrolo[3,4-c]quinoline (12e). 4-(2-Chloroethyl)morpholine hy-
drochloride (1.3 mmol, 240 mg) and sodium hydrogen carbonate (3.7
mmol, 310 mg) were added into a solution of 12a (1.3 mmol, 380 mg)
in absolute ethanol (12 mL). The mixture was stirred at reflux for 3 h
and 15 min. After cooling, the solvent was removed under reduced
pressure and the residue was taken up in water (50 mL) and extracted
2-Methyl-4-[[1-[2-(4-nitrophenyl)ethyl]-4-piperidinyl]methoxy]-
2H-pyrrolo[3,4-c]quinoline (30). 2-(4-Nitrophenyl)ethyl bromide (2.6
mmol, 0.61 g) and K2CO3 (6.6 mmol, 0.91 g) were added into a
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with ethyl acetate (3 × 50 mL). The combined organic layers were
washed with brine, dried over Na2SO4, filtered, and evaporated under
reduced pressure to give 480 mg of pure 12e (90%, oil). H NMR
Methyl 2-[2-[4-[[(2-Methyl-2H-pyrrolo[3,4-c]-4-quinolinyl)oxy]-
methyl]-1-piperidinyl]ethyl]benzoate (32). 32 was obtained accord-
ing to the procedure described for 12k using 35 as alkylating agent.
Crude 32 thus obtained was purified by column chromatography on
silica gel (ethyl acetate as eluent) to give pure 32 (30%, yellow oil). 1H
NMR (CDCl3): δ 1.28 (m, 1H), 1.65−1.68 (m, 1H), 1.96−1.98 (m,
3H), 2.26 (m, 2H), 2.70−2.74 (m, 2H), 3.17−3.29 (m, 4H), 3.92 (s,
3H), 3.99 (s, 3H), 4.50 (dd, 2H), 7.27−7.36 (m, 5H), 7.39−7.48 (m,
2H), 7.75 (dd, 1H), 7.88−7.92 (m, 2H). Anal. (C28H31N3O3) C, H, N.
Ethyl 4-[2-[4-[[[(1-Isopropyl-1H-indazol-3-yl)carbonyl]amino]-
methyl]-1-piperidinyl]ethyl]benzoate (21). A solution of 16 (5.3 g,
17.6 mmol), ethyl 4-(2-bromoethyl)benzoate34 (19 g, 74 mmol),
potassium iodide (15.6 g, 94 mmol), and triethylamine (13.1 mL, 94
mmol) in 2-butanone (200 mL) was stirred under reflux for 48 h. The
mixture was cooled to room temperature, poured into water (1.7 L),
and extracted with ethyl acetate (3 × 350 mL). The organic phases
were washed with brine, dried over Na2SO4, filtered, and concentrated
under vacuum. The crude product was purified by column
chromatography on aluminum oxide (Et2O/n-hexane (1:1 → 7:3) as
eluent) to obtain 3.5 g (42%) of the title compound, which was used
1
(CDCl3): δ 1.61 (m, 2H), 1.89−1.97 (m, 3H), 2.13 (m, 2H), 2.56 (m,
4H), 2.63 (m, 4H), 3.09 (m, 2H), 3.78 (m, 4H), 4.03 (s, 3H), 4.49 (d,
2H), 7.32−7.46 (m, 4H), 7.78 (m, 1H), 7.93 (m, 1H). Anal.
(C24H32N4O2) C, H, N. The product was converted to the
hydrochloride as described for the compound 12d (41%, 190−192
°C, from isopropyl ether/isopropyl alcohol). 1H NMR (DMSO-d6): δ
1.71−2.01 (m, 2H), 2.16−2.48 (m, 3H), 2.94−3.14 (m, 4H), 3.19−
3.39 (m, 4H), 3.57 (t, J = 7.31 Hz, 2H), 3.81 (d, J = 12.57 Hz, 2H),
3.97 (t, J = 4.38 Hz, 3H), 4.01 (s, 3H), 4.42 (d, J = 5.70 Hz, 2H), 7.42
(d, J = 1.75 Hz, 1H), 7.45−7.58 (m, 4H), 7.61 (s, 1H), 7.78 (d, J =
7.75 Hz, 1H).
4-[2-[4-[[(2-Methyl-2H-pyrrolo[3,4-c]-4-quinolinyl)oxy]methyl]-1-
piperidinyl]ethyl]benzenemethanol (12i). Compound 33 (800 mg,
3.7 mmol) was added into a solution of 12a (3.3 mmol, 990 mg) in 2-
butanone (33.5 mL). The mixture was stirred at reflux for 30 min, and
then triethylamine (130 mg, 1.29 mmol, 0.2 mL) was added. After the
mixture was stirred for 2 h at the same temperature, a further portion
of triethylamine (3.3 mmol, 0.33 g) was added. After 2 h the mixture
was cooled at room temperature, diluted with water, and extracted
with 2-butanone. The organic layer was dried over anhydrous Na2SO4,
filtered, and evaporated under reduced pressure to give crude 12i,
which was purified by column chromatography on aluminum oxide
(ethyl acetate as eluent) to obtain 0.67 g of pure 12i (48.5%, 145−146
1
for the next step without further purification. H NMR (CDCl3): δ
1.32−1.51 (m, 2H), 1.38 (t, J = 7.16 Hz, 3H), 1.61 (d, J = 6.72 Hz,
6H), 1.64−1.91 (m 3H), 2.05 (m, J = 11.55 Hz, J = 2.34 Hz, 2H),
2.54−2.66 (m, 2H), 2.81−2.91 (m, 2H), 3.02 (d, J = 11.69 Hz, 2H),
3.42 (t, J = 6.43 Hz, 2H), 4.36 (q, J = 7.16 Hz, 2H), 4.87 (m, J = 6.72
Hz, 1H), 7.15 (br t, J = 6.28 Hz, 1H), 7.22−7.31 (m, 3H), 7.35−7.49
(m, 2H), 7.96 (d, J = 7.92 Hz, 2H), 8.39 (m, J = 8.18 Hz, J = 1.02 Hz,
1H). Anal. (C28H36N4O3) C, H, N.
1
°C, from benzene/cyclohexane). H NMR (CDCl3): δ 1.31 (s, 1H),
1.64 (m, 2H), 1.84 (s, 1H), 1.99 (m, 3H), 2.11 (m, 1H), 2.64−2.68
(m, 2H), 2.87−2.92 (m, 2H), 3.14 (d, 2H), 4.03 (s, 3H), 4.53 (d, 2H),
4.72 (d, 2H), 7.26−7.28 (m, 2H), 7.32−7.38 (m, 5H), 7.44 (m, 1H),
7.78−7.80 (dd, 1H), 7.93 (dd, 1H). Anal. (C27H31N3O2) C, H, N.
4-[[1-[2-[4-(Methoxymethyl)phenyl]ethyl]-4-piperidinyl]-
methoxy]-2-methyl-2H-pyrrolo[3,4-c]quinoline (12j). 12j was pre-
pared according to the procedure described for 12i using 34 as
alkylating agent (48.5%, 102−105 °C, from 2-propanol/n-hexane). 1H
NMR (CDCl3: δ 1.61−1.68 (m, 2H), 1.96−1.99 (m, 3H), 2.11 (m,
2H), 2.66 (m, 2H), 2.89 (m, 2H), 3.13 (m, 2H), 3.44 (s, 3H), 4.03 (s,
3H), 4.48 (s, 2H), 4.51−4.54 (d, 2H), 7.25 (m, 2H), 7.32−7.34 (m,
3H), 7.36−7.39 (m, 2H), 7.42−7.45 (dd, 1H), 7.78−7.80 (dd, 1H),
7.92−7.94 (dd, 1H). Anal. (C28H33N3O2) C, H, N.
1-sec-Butyl-N-[[1-(2-phenylethyl)-4-piperidinyl]methyl]-1H-inda-
zole-3-carboxamide (11n). 11l (15.8 g, 44 mmol) was added into a
stirred suspension of NaH (1.76 g, 60% suspension in mineral oil) in
DMF (100 mL) cooled at 0 °C. The mixture was stirred at room
temperature for 1 h, and then 2-bromobutane (6 mL, 44 mmol) was
added. After 24 h at the same temperature, water (200 mL) was added,
the solution was filtered, and the solid was dissolved in ethyl acetate.
The residual inorganic material was filtered off, and the solution was
concentrated under vacuum and treated with Et2O/HCl. The
hydrochloride that formed was recrystallized from ethanol/ethyl
acetate (11.6 g, 65%, 235−237 °C). IR (KBr): ν 3327.21, 2930.72,
2545.81, 1650.95, 1545.41, 1489.88, 1200.85, 941.16, 748.85, 702.42
cm−1. 1H NMR (CDCl3): δ 12.27 (br s, 1H), 8.33 (m, J = 0.98 Hz, J =
1.10 Hz, J = 7.93 Hz, 1H), 6.98−7.61 (m, 9H), 4.37−4.82 (m, 1H),
1.69−3.94 (m, 18H), 1.58 (d, J = 6.84 Hz, 3H), 0.79 (t, J = 7.30 Hz,
3H). 13C NMR (DMSO-d6): δ 10.68, 20.18, 26.90, 29.03, 29.27, 33.98,
43.27, 51.39, 55.91, 56.57, 110.23, 121.77, 122.00, 122.14, 126.25,
1 2 6 . 6 6 , 1 2 8 . 5 6 , 1 3 7 . 2 6 , 1 4 0 . 4 3 , 1 6 2 . 2 6 . A n a l .
(C26H34N4O·HCl·1/2H2O) C, H, N, Cl.
4-[2-[4-[[(2-Methyl-2H-pyrrolo[3,4-c]-4-quinolinyl)oxy]methyl]-1-
piperidinyl]ethyl]phenylacetamide (12k). A mixture of 12a (0.5 g,
1.7 mmol), 4-(2-bromoethyl)phenylacetamide (2.2 g, 9.0 mmol), NaI
(9.0 mmol, 1.34 g), and triethylamine (0.9 g, 9.0 mmol) in 2-butanone
(22 mL) was stirred at reflux for 12 h. After cooling, the mixture was
poured into water (200 mL) and extracted with ethyl acetate (2 × 50
mL). The combined organic layers were extracted with 1 N HCl (3 ×
50 mL) to extract the final amine as a hydrochloride. The solid that
formed and the acidic phases were combined and treated with sodium
carbonate until pH 8 and then extracted again with ethyl acetate (3 ×
50 mL). The organic layers were collected, dried over anhydrous
Na2SO4, filtered, and evaporated under reduced pressure. The crude
product was purified by column chromatography on aluminum oxide
(ethyl acetate as eluent) to give 330 mg of pure 12k (42.5%, 145−146
1-Isopentyl-N-[[1-(2-phenylethyl)-4-piperidinyl]methyl]-1H-inda-
zole-3-carboxamide (11o). 11o was prepared according to the
procedure described for 11n using isopentyl bromide as reactant.
Crude 11o was treated with 2.5 N HCl in ethanol solution to give the
corresponding hydrochloride, which was recrystallized from isopropyl
alcohol (25%, 217−218 °C). IR (KBr): ν 3369.1, 2924.6, 2553.2,
1
1655.4, 1542.4, 1440.0, 1183.1, 943.3, 754.9, 708.8 cm−1. H NMR
1
(100 MHz, DMSO-d6): δ 11.03 (br s, 1H), 8.51 (t, J = 5.66 Hz, 1H),
8.21 (d, J = 7.68 Hz, 1H), 7.77 (d, J = 8.20 Hz, 1H), 7.08−7.60 (m,
7H), 4.50 (t, J = 7.31 Hz, 2H), 2.63−4.04 (m, 10H), 1.26−2.27 (m,
8H), 0.94 (d, J = 6.21 Hz, 6H). 13C NMR (DMSO-d6): δ 22.19, 25.24,
26.85, 29.26, 33.91, 38.02, 43.21, 47.09, 51.36, 56.55, 110.17, 121.80,
122.15, 126.43, 126.65, 128.55, 136.88, 137.25, 140.25, 162.09. Anal.
(C27H36N4O·HCl) C, H, N, Cl.
°C, from ethanol/hexane). H NMR (CDCl3): δ 1.64−1.76 (m, 6H),
1.99−2.02 (m, 2H), 2.23−2.24 (s, 3H), 2.93−2.95 (m, 2H), 2.97 (m,
2H), 3.26 (m, 2H), 4.04 (s, 3H), 4.53−4.54 (d, 2H), 7.18 (m, 1H),
7.22−7.24 (m, 2H), 7.35 (m, 1H), 7.39 (m, 4H), 7.77−7.79 (m, 1H),
7.93 (dd, 1H). Anal. (C28H32N4O2) C, H, N.
Ethyl 4-[2-[4-[[(2-Methyl-2H-pyrrolo[3,4-c]-4-quinolinyl)oxy]-
methyl]-1-piperidinyl]ethyl]benzoate (31). 31 was obtained accord-
ing to the procedure described for 12k using ethyl 4-(2-chloroethyl)-
benzoate36 as reactant. The crude product was purified by column
chromatography on aluminum oxide (chloroform as eluent) to give
pure 31 (51%, brown oil). 1H NMR (CDCl3): δ 1.44 (t, 3H), 1.65 (m,
2H), 1.98 (m, 3H), 2.18 (m, 2H), 2.70 (t, 2H), 2.97 (t, 2H), 3.14 (m,
2H), 4.01 (s, 3H), 4.42 (q, 2H), 4.52 (d, 2H), 7.32−7.46 (m, 6H),
7.78 (dd, J = 8.1 Hz, J = 1.1 Hz, 1H), 7.92 (dd, J = 8.1 Hz, J = 1.1 Hz,
1H), 8.02 (d, J = 8.4 Hz, 2H). Anal. (C29H33N3O3) C, H, N.
2-Methyl-2H-pyrrolo[3,4-c]quinolin-4(5H)-one (25). Methyl iodide
(1.5 g, 11 mmol) and anhydrous potassium carbonate (1.5 g, 11
mmol) were added into a solution of 24 (2.0 g, 11 mmol) in
anhydrous DMF (10 mL). The mixture was stirred at 90 °C for 15 h.
After cooling, the reaction mixture was treated with water (30 mL)
and filtered. The solid obtained was purified by column chromatog-
raphy on silica gel (chloroform/methanol mixture (10:1) as eluent) to
1
give 1.0 g of 25 (46%, sublimes at 225 °C, from toluene). H NMR
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(DMSO-d6): δ 3.91 (s, 3H), 7.09−7.25 (m, 3H), 7.58−7.81 (m, 3H),
10.73 (s, 1H). Anal. (C12H10N2O) C, H, N.
δ 1.66−1.94 (m, 2H), 1.96−2.29 (m, 3H), 2.86−3.89 (m, 8H), 4.00 (s,
3H), 4.49 (d, J = 6.04 Hz, 2H), 7.22−7.46 (m, 6H), 7.63−7.76 (m,
2H), 7.80 (d, J = 1.83 Hz, 1H), 8.01 (dd, J = 7.50, J = 1.65 Hz, 1H),
10.00 (br s, 3H), 10.68 (br s, 1H).
2-Isopropyl-2H-pyrrolo[3,4-c]quinolin-4(5H)-one (26). To a sol-
ution of 24 (3.0 g, 16 mmol) in dioxane (150 mL) brought to reflux
was added potassium metal (580 mg, 15 mmol), and the mixture was
stirred at reflux until total disappearance of the metal was observed (2
h). After cooling, 2-iodopropane (2.8 g, 16 mmol) and 18-crown-6
ether (3.9 g, 15 mmol) were added, and the mixture was refluxed for
5.5 h. A further portion of 2-iodopropane (1.4 g, 8.1 mmol) was then
added, and the reaction mixture was stirred at reflux for a further 15 h.
After cooling, the dioxane was removed under reduced pressure, and
the residue was taken up in ethyl acetate (100 mL) and washed with
brine (3 × 50 mL). The organic solution was dried over anhydrous
sodium sulfate, and the solvent was removed under reduced pressure
to obtain a crude product, which was purified by column
chromatography of aluminum oxide (ethyl acetate as eluent, 0.9 g,
4-[2-[4-[[[(1-Isopropyl-1H-indazol-3-yl)carbonyl]amino]methyl]-
1-piperidinyl]ethyl]benzoic Acid (11ab). 21 (3.5 g, 7.3 mmol) was
dissolved in THF/EtOH (1:1; 34 mL), and 1 N NaOH (15.8 mL) was
added. The reaction was stirred at room temperature overnight. HCl
(1 N; 15.8 mL) was added until precipitation, the mixture was cooled
in an ice bath, and the resulting solid was filtered and recrystallized
twice from ethanol/ethyl acetate (35:25; 1.9 g, 58%, 180−182 °C). IR
(UATR): ν 3314, 2980, 2934, 2862, 1659, 1535, 1487, 1463, 1357,
1
1206, 753, 650 cm−1. H NMR (DMSO-d6): δ 15−9 (very br s, 1H),
8.11−8.24 (m, 2H), 7.85 (d, J = 8.26 Hz, 2H), 7.77 (d, J = 8.59 Hz,
1H), 7.43 (m, J = 0.99, 7.10, 8.42 Hz, 1H), 7.33 (d, J = 8.26 Hz, 2H),
7.21−7.29 (m, 1H), 5.07 (m, J = 6.61 Hz, 1H), 3.21 (t, J = 6.28 Hz,
2H), 2.95 (d, J = 11.23 Hz, 2H), 2.74−2.86 (m, 2H), 2.53−2.61 (m,
2H), 1.99 (t, J = 10.73 Hz, 2H), 1.58−1.75 (m, 3H), 1.54 (d, J = 6.61
Hz, 6H), 1.08−1.32 (m, 2H). 13C NMR (DMSO-d6): δ 21.84, 29.26,
32.18, 35.60, 43.71, 50.19, 52.50, 58.88, 110.20, 121.85, 122.12,
122.26, 126.20, 128.60, 129.25, 129.69, 136.80, 139.52, 144.89, 162.06,
167.66. Anal. (C26H32N4O3) C, H, N.
1
25%, 189−190 °C, from toluene). H NMR (DMSO-d6): δ 1.48 (d,
6H), 4.52 (m, 1H), 7.02−7.20 (m, 3H), 7.63−7.70 (m, 2H), 7.77 (m,
1H), 10.62 (s, 1H). Anal. (C14H14N2O) C, H, N.
1-Acetyl-N-[[1-(2-phenylethyl)-4-piperidinyl]methyl]-1H-inda-
zole-3-carboxamide (11p). Acetic anhydride (0.26 mL, 2.75 mmol)
was added into a suspension of 11l (500 mg, 1.4 mmol) in CH2Cl2 (5
mL), and the mixture was stirred at room temperature overnight. The
solution was treated with 1 N NaOH until pH 8 and extracted with
CH2Cl2. The organic layer was washed with water, dried over Na2SO4,
filtered, and concentrated under vacuum. The residue was treated with
Et2O/HCl, and the hydrochloride that formed was recrystallized from
n-hexane/ethyl acetate (280 mg, 42%, 246−247 °C). IR (KBr): ν
3431.82, 2937.44, 2547.28, 1727.70, 1676.48, 1536.32, 1375.93,
1325.32, 1375.93, 1325.45, 1197.07, 1147.76, 953.51, 767.44 cm−1.
1H NMR (DMSO-d6): δ 11.13 (br s, 1H), 8.96 (t, J = 5.82 Hz, 1H),
4-[2-[4-[[(2-Methyl-2H-pyrrolo[3,4-c]-4-quinolinyl)oxy]methyl]-1-
piperidinyl]ethyl]benzoic Acid (12g). 12g was prepared according to
the procedure described for 11ab using ethanol as solvent (27%, 155−
166 °C, from ethanol). 1H NMR (DMF-d7): δ 1.53 (m, 2H), 1.93 (m,
3H), 2.23 (m, 2H), 2.74 (m, 2H), 2.97 (m, 2H), 3.17 (m, 2H), 3.25
(m, 2H), 4.13 (s, 3H), 4.48 (d, 2H), 7.36 (m, 1H), 7.43 (m, 1H), 7.49
(d, J = 8.1 Hz, 2H), 7.62 (d, J = 1.9 Hz, 1H), 7.83 (d, J = 1.9 Hz, 1H),
7.70 (d, 1 H), 8.01 (d, J = 8.1 Hz, 2H), 8.08 (m, 1H), 14−16 (br s,
1H). Anal. (C27H29N3O3) C, H, N.
2-[2-[4-[[(2-Methyl-2H-pyrrolo[3,4-c]-4-quinolinyl)oxy]methyl]-1-
piperidinyl]ethyl]benzoic Acid (12h). 12h was prepared according to
the procedure described for 11ab using ethanol as solvent (50%, 165
°C, from ethyl acetate). 1H NMR (DMF-d7): δ 1.30 (m, 2H), 1.51 (m,
2H), 1.79−1.82 (m, 1 H), 2.51 (m, 5H), 2.87−2.92 (m, 2H), 3.07−
3.10 (m, 2H), 3.98 (s, 3H), 4.35 (d, 2H), 7.23−7.37 (m, 5H), 7.57−
7.62 (m, 3H), 7.72 (d, 1 H), 7.96 (dd, 1 H). Anal. (C27H29N3O3) C,
H, N.
8.31 (t, J = 7.35 Hz, 2H), 7.06−7.87 (m, 7H), 2.68−3.90 (m, 13H),
1.37−2.31 (m, 5H). 13C NMR (DMSO-d6): δ 22.59, 26.82, 29.26,
33.80, 43.45, 51.34, 56.54, 114.75, 122.40, 124.00, 125.34, 126.66,
128.55, 129.86, 137.23, 139.44, 161.01, 171.09. Anal.
(C24H28N4O2·HCl) C, H, N, Cl.
N-[[1-[2-(4-Aminophenyl)ethyl]-4-piperidinyl]methyl]-1-isoprop-
yl-1H-indazole-3-carboxamide (11aa). A solution of 11z (2.7 g, 6
mmol) in ethanol (30 mL) was hydrogenated on 10% Pd/C (270 mg)
by a Parr apparatus at 40 psi for 5 h at room temperature. The mixture
was then filtered, and the filtrate was concentrated at reduced pressure.
The crude 11aa was treated with 2.5 N HCl in ethanol to afford the
corresponding hydrochloride, which was recrystallized from ethyl
acetate/ethanol (8:2; 1.4 g, 45%, 278 °C dec). IR (diffuse reflectance
with KBr): ν 3504, 3413, 3237, 2634, 2053, 1630, 1547, 1514, 1287,
1210, 941, 762, 642 cm−1. 1H NMR (DMSO-d6): δ 1.55 (d, J = 7 Hz,
6H), 1.45−2.13 (m, 5H), 2.80−3.84 (m, 12H), 5.08 (sept, J = 7 Hz,
1H), 7.20−7.49 (m, 6H), 7.79 (d, J = 9 Hz, 1H), 8.18 (d, J = 9 Hz,
1H), 8.39 (t, J = 6 Hz, 1H), 9.15−11.18 (m, 4H). 13C NMR (DMSO-
d6): δ 21.86, 26.90, 28.74, 33.95, 43.21, 50.22, 51.44, 56.32, 110.26,
121.77, 122.20, 122.82, 126.23, 129.77, 131.66, 136.26, 136.65, 139.50,
162.22. Anal. (C25H33N5O·2HCl·H2O) C, H, N, Cl.
Ethyl 4-(2-Nitrophenyl)-1H-pyrrole-3-carboxylate (23). A solution
of ethyl 3-(2-nitrophenyl)propanoate (22)35 (26.6 g, 120 mmol) and
TosMIC (25.4 g, 130 mmol) in anhydrous dimethyl sulfoxide/ethyl
ether (1:2; 450 mL) was added dropwise to a well-stirred suspension
of 60% sodium hydride in paraffin (10.4 g, 260 mmol) in anhydrous
ethyl ether (300 mL) under a stream of argon. After the addition, the
mixture was stirred at room temperature for 25 min, water (500 mL)
was then added, and the resulting solution was extracted with ethyl
acetate (3 × 600 mL). The combined organic phases were washed
with brine (3 × 300 mL), dried over anhydrous sodium sulfate, and
evaporated under reduced pressure. The crude product obtained was
purified by column chromatography on aluminum oxide (chloroform/
ethyl acetate (1:1) as eluent) to give 12.8 g of 23 (41%, 159−161 °C,
from ethanol). 1H NMR (CDCl3): δ 2.51 (m, 3H), 4.01 (q, 2H), 6.51
(m, 1H), 7.31−7.33 (m, 1H), 7.39−7.41 (m, 2H), 7.49−7.53 (m, 1H),
7.92−7.94 (m, 1H), 12.0 (s, 1H). Anal. (C13H12N2O4) C, H, N.
2H-Pyrrolo[3,4-c]quinolin-4(5H)-one (24). Iron powder (6.7 g, 120
mmol) was added over 15 min into a solution of 23 (2.0 g, 7.7 mmol)
in glacial acetic acid (100 mL) mechanically stirred at 85 °C. The
mixture was stirring at the same temperature for 45 min. After cooling,
the iron was removed by filtration and washed several times with
tetrahydrofuran, and the filtrate was evaporated under reduced
pressure. The crude product was purified by column chromatography
on aluminum oxide (ethyl acetate as eluent) to give 1.1 g of 24 (77%,
sublimes at 280 °C from ethanol). 1H NMR (DMSO-d6): δ 7.05−7.28
(m, 3H), 7.57−7.63 (m, 2H), 7.84−7.88 (m, 1H), 10.7 (s, 1H), 12.1
(s, 1H). Anal. (C11H8N2O) C, H, N.
4-[[1-[2-(4-Aminophenyl)ethyl]-4-piperidinyl]methoxy]-2-methyl-
2H-pyrrolo[3,4-c]quinoline (12f). To a solution of the product 30 (1.4
mmol, 0.61 g) in ethyl acetate (100 mL) was added 10% Pd/C (200
mg). The mixture was stirred under a H2 atmosphere at room
temperature and pressure for 4 h. A further portion of 10% Pd/C (100
mg) was then added, and the mixture was left under a H2 atmosphere
at room temperature and pressure for 19 h. A stream of H2 was passed
through every 3 h. Then the mixture was filtered under vacuum on a
Merck RP18 cartridge to remove the palladium, and the solvent was
removed under reduced pressure to give 0.57 g of pure 12f (99%,
1
150−152 °C, from toluene/cyclohexane). H NMR (CDCl3): δ 1.69
(m, 2H), 1.98 (m, 3H), 2.21 (m, 2H), 2.69 (m, 2H), 2.87 (m, 2H),
3.20 (m, 2H), 3.64 (br s, 2H), 4.04 (s, 3H), 4.53 (d, 2H), 6.69 (d, J =
8.4 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H), 7.32−7.47 (m, 4H), 7.79 (dd, J
= 7.7 Hz, J = 1.2 Hz, 1H), 7.93 (m, J = 7.7 Hz, J = 1.2 Hz, 1H). Anal.
(C26H30N4O) C, H, N. The product was converted to the
hydrochloride as described for the compound 12e (95%, 165−167
4-Chloro-2-methyl-2H-pyrrolo[3,4-c]quinoline (27). A mixture of
product 25 (1.0 g, 5.0 mmol), phosphorus oxychloride (16.2 mL), and
triethylamine (1.2 mL) was stirred at 120 °C for 6 h. After cooling, the
reaction mixture was poured cautiously into ice and extracted with
1
°C, from diisopropyl ether/isopropyl alcohol). H NMR (DMSO-d6):
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Journal of Medicinal Chemistry
Article
ethyl acetate (3 × 100 mL). The combined organic phases were
washed with brine (1 × 50 mL), with saturated sodium bicarbonate
solution (3 × 50 mL), and then again with brine (3 × 50 mL). The
organic solution was dried over anhydrous Na2SO4, filtered, and
evaporated under reduced pressure. The crude product obtained was
purified by column chromatography on silica gel (n-hexane/ethyl
acetate (1:1) as eluent) to give 1.0 g of 27 (92%, 123−124 °C, from
4-[[1-(Phenylmethyl)-4-piperidyl]methoxy]-2-methyl-2H-pyrrolo-
[3,4-c]quinoline (29). Compound 29 was prepared according to the
procedure described for 12b using 1-(phenylmethyl)-4-piperidineme-
thanol as reactant. The residue was purified by column chromatog-
raphy on aluminum oxide (chloroform/petroleum ether (1:1) as
1
eluent) to give pure 29 as an oil (81%). H NMR (CDCl3): δ 1.58−
1.59 (m, 2H), 1.91−2.11 (m, 5H), 2.94−3.02 (m, 2H), 3.59 (s, 2H),
4.02 (s, 3H), 4.50 (d, 2H), 7.31−7.39 (m, 8H), 7.44 (m, 1H), 7.77 (m,
1H), 7.92 (m, 1H). Anal. (C25H27N3O) C, H, N.
1
benzene). H NMR (DMSO-d6): δ 4.09 (s, 3H), 7.51−7.58 (m, 2H),
7.76 (m, 1H), 7.82−7.85 (m, 1H), 7.97 (m, 1H), 8.15−8.17 (m, 1H).
Anal. (C12H9N2Cl) C, H, N, Cl.
Biological Assays. In Vitro Binding Assay for 5-HT4R. The 5-
HT4R binding assay was performed on homogenates from human
embryonic kidney (HEK-293) cells transfected with the human 5-
HT4R, using 0.2 nM [3H]5 as radioligand. Nonspecific binding was
measured in the presence of 10 μM 4·HCl. Assays were conducted in
25 mM Tris−HCl (pH 7.4), 0.5 mM EDTA, and 10 mM MgSO4.
Binding was initiated by addition of 200 μL of membrane
homogenates (43−61 μg of protein/mL); after 60 min of incubation
at 27 °C, the membranes were harvested onto glass fiber (GF/B)
filters (Unifilter, Packard) (treated with 0.3% poly(ethylenimine))
using a Filtermate cell harvester (Packard). Subsequently, the filters
were washed with 2.5 mL of ice-cold buffer and dried for 30 min in an
oven at 45 °C, and 30−35 μL of Microscint 20 (Packard) was added
to each well. At least 10 h later the radioactivity was measured using a
TopCount (Packard) for 1 min. The compounds were tested in
duplicate at eight concentrations ranging from 10−12 to 10−5 M in
duplicate competition curves. The compounds were dissolved in
dimethyl sulfoxide (DMSO), serially diluted 1:10 in DMSO, and
further diluted in incubation buffer (1% vehicle DMSO final
concentration).
The protein concentration was determined by the bicinchoninic
acid (BCA) method (Pierce) using bovine serum albumin (BSA) as
the standard.
Results at a single concentration are expressed as percent inhibition
vs a positive control. The IC50 values (concentration causing a half-
maximal inhibition of control specific binding) with Hill coefficients
(nH) were determined by computer-assisted nonlinear regression
analysis of eight concentrations in duplicate competition curves. The
inhibition constants pKi (defined as the negative log of Ki) were
calculated from the Cheng−Prusoff equation.
In Vitro Binding Assay for 5-HT2AR. The 5-HT2AR binding assay
was performed on cell homogenates obtained from a stable
recombinant CHO-K1 cell line expressing the human 5-HT2AR,
using 0.7 nM [3H]ketanserin as ligand. Nonspecific binding was
measured in the presence of 20 μM mianserin. Assays were conducted
in buffer containing 50 mM Tris (pH 7.4), 5 mM CaCl2, 0.1% ascorbic
acid, and 10 μg/mL saponin. Binding was initiated by addition of 20
μL of membrane homogenates (750 μg/mL protein); after 60 min of
incubation at 25 °C, the membranes were harvested onto glass fiber
(GF/B) filters (Unifilter, Packard) (treated with 0.3% poly-
(ethylenimine)) using a Filtermate cell harvester (Packard).
Subsequently, the filters were washed with 2.5 mL of ice-cold buffer
and dried for 30 min in an oven at 45 °C, and 30−35 μL of Microscint
20 (Packard) was added to each well. At least 10 h later the
radioactivity was measured using a TopCount (Packard) for 1 min.
The compounds were tested in duplicate at eight concentrations
ranging from 10−12 to 10−5 M. The compounds were dissolved in
DMSO and serially diluted 1:10 in DMSO, and each dilution was
further diluted in incubation buffer (final DMSO concentration 1%).
The protein concentration was determined by the BCA method
(Pierce) using BSA as the standard.
Results at a single concentration are expressed as percent inhibition
vs a positive control. The IC50 values (concentration causing a half-
maximal inhibition of control specific binding) with Hill coefficients
(nH) were determined by computer-assisted nonlinear regression
analysis of eight concentrations in duplicate competition curves. The
inhibition constants pKi (defined as the negative log of Ki) were
calculated from the Cheng−Prusoff equation.
In Vitro Cross-Species Intrinsic Clearance with Rat and
Human Liver Microsomes. Compounds were incubated at 37 °C at
a concentration of 1 μM in Dulbecco’s buffer at pH 7.4 with rat and
4-Chloro-2-isopropyl-2H-pyrrolo[3,4-c]quinoline (28). 28 was
obtained according to the procedure used for the preparation of
compound 27 starting from 26. The crude product obtained was
purified by column chromatography on silica gel (chloroform as
eluent) to give pure 28 (91%, 70−72 °C, from cyclohexane). 1H NMR
(CDCl3): δ 1.64 (d, 6H), 4.59 (m, 1H), 7.46−7.49 (m, 3H); 7.55 (m,
1H), 7.93−7.98 (m, 2H). Anal. (C14H13N2Cl) C, H, N, Cl.
4-[(1-Butyl-4-piperidinyl)methoxy]-2-methyl-2H-pyrrolo[3,4-c]-
quinoline (12b). A solution of 1-butyl-4-piperidinemethanol (1.14 g,
6.72 mmol) in anhydrous DMF (14 mL) was added dropwise into a
suspension of 60% NaH in paraffin (270 mg, 6.72 mmol) in the same
solvent (14 mL). The reaction mixture was stirred at room
temperature for 10 min, and then the alkoxide that formed was
added into a solution of 27 (470 mg, 2.17 mmol) in DMF (14 mL)
preheated to 146 °C. The reaction mixture was stirred at 146 °C for 1
h. After cooling, it was poured onto ice and extracted with ethyl acetate
(3 × 50 mL). The combined organic extracts were washed with brine
(3 × 20 mL), dried over anhydrous Na2SO4, filtered, and evaporated
under reduced pressure. The residue was purified by column
chromatography on aluminum oxide (chloroform as eluent) to give
520 mg of pure 12b (68%, 83−85 °C, from benzene/cyclohexane). 1H
NMR (CDCl3): δ 0.97 (t, 3H), 1.35−1.59 (m, 6H), 1.90−2.07 (m,
5H), 2.37 (m, 2H), 3.03 (m, 2H), 3.99 (s, 3H), 4.49 (d, 2H), 7.28-
7.49 (m, 4H), 7.75 (m, 1 H), 7.88 (m, 1 H). Anal. (C22H29N3O) C, H,
N.
2-Methyl-4-[[1-(2-phenylethyl)-4-piperidinyl]methoxy]-2H-
pyrrolo[3,4-c]quinoline (12c). 12c was prepared according to the
procedure described for 12b starting from 27 (0.50 g, 2.3 mmol) using
1-(2-phenylethyl)-4-piperidinemethanol (1.77 g, 8.05 mmol) as
reactant. The crude product was purified by column chromatography
on aluminum oxide (n-hexane/ethyl acetate (4:1) as eluent) to give
620 mg of pure 12c (67%, 90−92 °C, recrystallized from benzene/
cyclohexane). 1H NMR (CDCl3): δ 1.67 (m, 2H), 1.94−1.97 (m, 3H),
2.17 (m, 2H), 2.69 (m, 2H), 2.90 (m, 2H), 3.15 (m, 2H), 3.98 (s, 3H),
4.49 (d, 2H), 7.20−7.42 (m, 9H), 7.74 (m, 1H), 7.88 (m, 1H). Anal.
(C26H29N3O) C, H, N.
2-Isopropyl-4-[[1-(2-phenylethyl)-4-piperidinyl]methoxy]-2H-
pyrrolo[3,4-c]quinoline (12d). Compound 12d was prepared
according to the procedure described for 12b starting from 28 using
1-(2-phenylethyl)-4-piperidinemethanol as reactant. The residue was
purified by column chromatography on aluminum oxide (n-hexane/
ethyl acetate (4:1) as eluent) to give pure 12d (73%, 90−93 °C, from
1
n-hexane). H NMR (CDCl3): δ 1.61−1.66 (m, 8H), 1.94 (m, 3H),
2.12 (m, 2H), 2.65 (m, 2H), 2.86 (m, 2H), 3.12 (m, 2H), 4.49 (d,
2H), 4.54 (m, 1H), 7.21−7.45 (m, 9H), 7.74 (m, 1H), 7.90 (m, 1H).
Anal. (C28H33N3O) C, H, N. A solution of HCl in methanol was
prepared by addition dropwise of acetyl chloride (2.67 mmol, 200 mg)
to 10 mL of methanol cooled at 0 °C. The solution was gently stirred
for a few minutes, followed by addition dropwise of a solution of 12d
(1.0 g, 2.34 mmol) in methanol (5.0 mL). When the addition was
complete, the mixture was stirred at 0 °C for 45 min, followed by
addition of anhydrous ethyl ether (about 200 mL) until precipitation
of the salt was observed. The obtained salt was filtered, washed with
anhydrous ethyl ether (3 × 2 mL), and dried under vacuum at 45 °C
for 6 h (630 mg, 63%, 138−140 °C, from diisopropyl ether/isopropyl
1
alcohol). H NMR (DMSO-d6): δ 1.57 (d, J = 6.59 Hz, 6H), 1.79−
2.29 (m, 5H), 2.86−3.47 (m, 6H), 3.63 (d, J = 11.71 Hz, 2H), 4.55 (d,
J = 4.03 Hz, 2H), 4.68 (m, J = 6.59 Hz, 1H), 7.20−7.48 (m, 7H), 7.80
(br s, 1H), 7.95−8.16 (m, 3H), 10.93 (br s, 1H).
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Journal of Medicinal Chemistry
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human liver microsomes for up to 60 min to determine their metabolic
stability and intrinsic clearance. Samples (25 μL) were taken at time 0
and after 5, 10, 20, 30, and 60 min and added to 50 μL of cold
acetonitrile to stop the reaction and to 20 μL of cold acetonitrile
containing the internal standard warfarin. The samples were then
centrifuged, and the supernatant was analyzed immediately. Mid-
azolam and propranolol were used as positive controls and incubated
under the same conditions as the test compounds. Control tests were
performed by incubating the compounds with rat and human liver
microsomes in Dulbecco’s buffer in the absence of NADPH for 60
min. The single time point metabolic stability of the test compounds
was evaluated by calculating the percentage of parent compound
remaining after 60 min of incubation. The intrinsic clearance (CLint) of
the test compounds was calculated using the half-life approach. The
half-life and CLint were determined from the concentration remaining
at the different sampling points using the LC−MS/MS method.
Cross-Species Metabolic Stability in Liver Microsomes from
Mouse, Rat, Dog, Miniature Pig, Monkey, and Human. The
samples consisted of the test articles at 1 and 10 μM, pooled
microsomes at 0.5 mg/mL final protein concentration, and NADPH
regenerating system in a final volume of 200 μL. DMSO was the test
article solvent, and the final DMSO concentration was 0.5%. The assay
was standardized for both phosphate buffer (75 mM, pH 7.4) and the
NADPH regenerating system (MgCl2, 3.3 mM; G6P, 3.3 mM; G6PD,
0.4 U/mL; NADP+, 1.3 mM). Positive controls (warfarin, propranolol,
and testosterone, incubated as a cocktail) were treated as the test
articles. The samples were incubated at 37 °C in a 96-well plate in a
humidified incubator. At t = 0, 30, and 60 min, 100 μL of acetonitrile
containing the internal standards (0.2 μM metoprolol and 0.4 μM
diclofenac) for LC−MS analysis was added to stop the reaction. The
samples were diluted 10-fold to bring them within the linear range of
the instrumental measures. The samples were centrifuged prior to
analyses by LC−MS/MS using positive electronspray ionization (ESI)
and selected reaction monitoring (SRM) using a gradient LC method.
The LC conditions included a 5−91% acetonitrile gradient in water
containing 0.1% formic acid with a total run time of 6.5 min. The LC
was isocratic for 0.5 min followed by a linear gradient of acetonitrile
from 5% to 91% over 1 min, with a 2.5 min hold at 91% acetonitrile.
The eluent flow rate was 0.5 mL/min, and the column was an
XDBC18 (2.1 × 50 mm, Agilent). The re-equilibration period was 1.5
min at 1.4 mL/min followed by 0.5 min at 0.5 mL/min. The internal
standard for positive mode ESI was metoprolol.
Hot Plate Test. The method originally described by Woolfe and
Mac Donald was used with some modifications.54 The hot plate
consists of an electrically heated surface with the temperature fixed at
56 0.2 °C. The animals are placed on the hot plate, and the time
until a nocifensive response (i.e., licking, flinching, jumping) occurs is
recorded by a stopwatch. During the test, latency for nocifensive
response was recorded 1 h after drug administration. The maximum
latency time was 30 s to minimize tissue damage. The effect on latency
was also calculated as a percent response increase with respect to
vehicle-treated rats.
Formalin Test. The formalin test in mice is a valid and reliable
model of nociception. The noxious stimulus is a subcutaneous
injection of 20 μL of a 1% solution of formalin in saline into the dorsal
surface of the right hind paw of the mouse.55 The formalin injection
produces a distinct biphasic response consisting of licking or biting the
paw. The early phase, occurring from 0 to 10 min after the formalin
injection, is due to a direct effect on nociceptors, while the late phase
from 15 to 40 min seems to be an inflammatory response where pain is
responsive to anti-inflammatory drugs. Mice were placed in a Plexiglas
cage, utilized as an observation chamber, 1 h before administration of
the formalin injections. The tested drugs were orally given 30 min
before formalin. The total amount of time (s) that the animal spent
licking or biting the paw after the formalin injection was recorded for a
period of 40 min within 5 min intervals.
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional experimental procedures regarding the pharmaco-
phore approach, synthetic methods for compounds 29 and 33−
35, analyses, 5-HT4R functional assay, and in vitro pharmacol-
ogy profiles. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +39-0691045265 (G.F.); +39-0649913965 (R.D.S.).
Fax: +39-0691984597 (G.F.); +39-0649913133 (R.D.S.). E-
mail: g.furlotti@angelini.it (G.F.); roberto.disanto@uniroma1.it
(R.D.S.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Claudio Dagostin and Katie Ellard for their help in
reviewing the manuscript.
ABBREVIATIONS USED
■
5-HT, 5-hydroxytryptamine or serotonin; 5-HTR, 5-HT
receptor; 5-HT4R, serotonin 4 receptor; 5-HT2AR, serotonin
2A receptor; VS, virtual screening; EF, enrichment factor;
TosMIC, (4-tolylsulfonyl)methyl isocyanide; CHO, Chinese
hamster ovarian
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