Synthesis and structure-activity relationship studies in translocator protein ligands based on a pyrazolo[3,4-b]quinoline scaffold

Article abstract of DOI:10.1021/jm200770f

As a further development of our large program focused on the medicinal chemistry of translocator protein [TSPO (18 kDa)] ligands, a new class of compounds related to alpidem has been designed using SSR180575, emapunil, and previously published pyrrolo[3,4-b]quinoline derivatives 9 as templates. The designed compounds were synthesized by alkylation of the easily accessible 4-methyl-2-phenyl-1H-pyrazolo[3,4-b]quinolin-3(2H)-one derivatives 13-15 with the required bromoacetamides. Along with the expected 2-(4-methyl-3-oxo-2- phenyl-2,3-dihydro-1H-pyrazolo[3,4-b]quinolin-1-yl)acetamide derivatives 10, 2-(4-methyl-3-oxo-2-phenyl-2H-pyrazolo[3,4-b]quinolin-9(3H)-yl)acetamide isomers 11 were isolated and characterized. The high TSPO affinity shown by new pyrazolo[3,4-b]quinoline derivatives 10 and especially 11 leads the way to further expand the chemical diversity in TSPO ligands and provides new templates and structure-affinity relationship data potentially useful in the design of new anxiolytic and neuroprotective agents.

Full text of DOI:10.1021/jm200770f

ARTICLE
pubs.acs.org/jmc
Synthesis and StructureꢀActivity Relationship Studies in Translocator
Protein Ligands Based on a Pyrazolo[3,4-b]quinoline Scaffold
Andrea Cappelli,*,† Giulia Bini,† Salvatore Valenti,† Germano Giuliani,† Marco Paolino,† Maurizio Anzini,†
Salvatore Vomero,† Gianluca Giorgi,‡ Antonio Giordani,§ Luigi Piero Stasi,§ Francesco Makovec,§
Carla Ghelardini,|| Lorenzo Di Cesare Mannelli,|| Alessandra Concas,^ Patrizia Porcu,^ and Giovanni Biggio^
†Dipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug Discovery and Development,
Universitꢀa degli Studi di Siena, Via A. Moro, 53100 Siena, Italy
‡Dipartimento di Chimica, Universitꢀa degli Studi di Siena, Via A. Moro, 53100 Siena, Italy
§Rottapharm SpA, Via Valosa di Sopra 7, 20052 Monza, Italy
Dipartimento di Farmacologia Preclinica e Clinica “M. Aiazzi Mancini”, Universitꢀa degli Studi di Firenze,Viale G. Pieraccini 6,
50139 Firenze, Italy
^Dipartimento di Biologia Sperimentale “B. Loddo”, Universitꢀa degli Studi di Cagliari, Cittadella Universitaria, SS 554 (km 4.500),
09042 Monserrato (Cagliari), Italy
S Supporting Information
b
ABSTRACT: As a further development of our large program
focused on the medicinal chemistry of translocator protein
[TSPO (18 kDa)] ligands, a new class of compounds related
to alpidem has been designed using SSR180575, emapunil,
and previously published pyrrolo[3,4-b]quinoline derivatives
9 as templates. The designed compounds were synthesized
by alkylation of the easily accessible 4-methyl-2-phenyl-1H-
pyrazolo[3,4-b]quinolin-3(2H)-one derivatives 13ꢀ15 with
the required bromoacetamides. Along with the expected 2-(4-
methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazolo[3,4-b]quinolin-1-yl)acetamide derivatives 10, 2-(4-methyl-3-oxo-2-phenyl-
2H-pyrazolo[3,4-b]quinolin-9(3H)-yl)acetamide isomers 11 were isolated and characterized. The high TSPO affinity shown by
new pyrazolo[3,4-b]quinoline derivatives 10 and especially 11 leads the way to further expand the chemical diversity in TSPO
ligands and provides new templates and structureꢀaffinity relationship data potentially useful in the design of new anxiolytic
and neuroprotective agents.
’ INTRODUCTION
MPTP opening, and apoptosis as well as cell proliferation and
differentiation. It has been suggested that TSPO functions are
related to various aspects of the host-defense response.5
The peripheral benzodiazepine receptor (PBR) is a protein of
18 kDa showing five transmembrane domains, pharmacologi-
cally distinct from the central benzodiazepine receptor (CBR),
originally discovered in the periphery as an alternative binding
site of diazepam, and recently renamed “translocator protein
(18 kDa)” (TSPO).1 TSPO is primarily localized at the outer
mitochondrial membrane, in particular, at the outer/inner mi-
tochondrial membrane contact sites,2 but it is also localized at the
nucleus and the perinuclear region in breast cancer cells3 as well
as at the plasma membrane of erythrocytes.4 TSPO interacts with
various other proteins, including the 32 kDa voltage-dependent
anion channel (VDAC) and the 30 kDa adenine nucleotide
transporter (ANT), which are components of the mitochondrial
permeability transition pore (MPTP).
TSPO can be found in the whole body: glandular and secretory
tissues are particularly rich in TSPO, but it is also present in renal
and myocardial tissue, as well as in hemopoietic and lymphatic
cells. Low levels of TSPO are expressed in liver and brain, where
it is limited to glial cells (astrocites and microglia). A dramatic
increase in TSPO density occurs in glial cells in response to brain
injury or inflammation as well as following a number of neuro-
pathological conditions.6 These include gliomas, stroke, herpes
and HIV encephalitis, and neurodegenerative disorders such as
Alzheimer’s disease, multiple sclerosis, amyotrophic lateral sclero-
sis, Parkinson’s disease, and Huntington’s disease. Moreover,
changes in TSPO expression levels have been observed in patients
suffering from generalized and separation anxiety, panic attacks,
Although the exact physiological role of TSPO is not yet com-
pletely defined, evidence has been collected about its involve-
ment in cholesterol transport across the mitochondrial mem-
brane, regulation of steroid biosynthesis, mitochondrial respiration,
Received: June 14, 2011
Published: September 14, 2011
r
2011 American Chemical Society
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Figure 2. Design of TSPO ligands 10.
Our interest in the design and synthesis of TSPO ligands led to
development of potent compounds 9 structurally related to
alpidem.13 Thus, in the progress of our large program focused
on the medicinal chemistry of TSPO ligands, pyrrolo[3,4-b]-
quinoline derivatives 9, pyridazino[4,5-b]indole-1-acetamide 7,
and emapunil (8) were considered as structural templates in the
design of new tricyclic derivatives 10 (Figure 2).
In particular, the pyrrolo[3,4-b]quinoline tricyclic system of
compounds 9 was transformed into the pyrazolo[3,4-b]quinoline
ring system of compounds 10 that can be considered as a structural
hybrid between the pyridazino[4,5-b]indole moiety of 7 and the
pyrrolo[3,4-b]quinoline system of compounds 9. Moreover, the
rigid ylideneacetamide moiety of compounds 9 was converted into
an acetamide side chain attached to the heteroaromatic system
through a heterocyclic nitrogen atom as was the case with emapunil.
This paper describes the synthesis and the preliminary pharmaco-
logical (in vitro and in vivo) characterization of TSPO ligands 10
based on the pyrazolo[3,4-b]quinoline scaffold and the serendipi-
tous discovery of isomers 11 with subnanomolar TSPO affinity.
Figure 1. Structure of some relevant TSPO ligands.
post-traumatic stress, and obsessiveꢀcompulsive disorders.7 There-
fore, TSPO can be exploited as a diagnostic marker to follow disease
progression and therapy efficacy by means of the biomedical imaging
technique PET (positron emission tomography) but also as a thera-
peutic target.
’ RESULTS
Chemistry. Target pyrazolo[3,4-b]quinoline derivatives 10
were synthesized as depicted in Scheme 1.
In addition to classical synthetic TSPO ligands PK11195 (1,
Figure 1) and Ro5ꢀ4864 (2), several other chemically different
ligands have been developed, especially in the family of molecules
related to alpidem (3). Among them, indoleacetamides 4,8 indo-
lylglyoxylamides 5,9 pyrazolopyrimidine derivatives 6,10 and SSR1-
8057511 (7, Figure 1) stimulate the biosynthesis of pregnenolone
metabolites acting as positive allosteric modulators of GABA neuro-
transmission. Thus, TSPO ligands capable of stimulating neuroster-
oid biosynthesis can represent promising candidates for fast-acting
anxiolytic agents lacking the unwanted benzodiazepine-like side
effects. For instance, emapunil (AC5216, XBD-173, 8) enhances
GABA-mediated neurotransmission and exerts antipanic activity in
humans without causing sedation or withdrawal symptoms.12 On
the other hand, 7 was reported to promote neuronal survival and
repair in axotomy and neuropathy models and is potentially useful in
the treatment of neurodegenerative diseases. The increase in
pregnenolone accumulation in the brain and sciatic nerve suggests
that the neuroprotective effects shown by 7 are steroid-mediated.11
Reaction of 2-chloroquinoline derivative 1214 with the suitable
phenylhydrazines gave the corresponding tricyclic compounds
13ꢀ15 as dark-red crystalline solids. The structure of compound
13 was characterized by crystallographic studies (Figure 3).
Compounds 13ꢀ15 were alkylated in the presence of sodium
carbonate with the required bromoacetamide derivatives 16aꢀm
(see Supporting Information) to obtain substantial amounts of
alkylation products 10 (yellow crystals) along with small amounts of
intensely dark-red-colored compounds 11. In some instances the
purification of compounds 11 proved to be difficult. How-
ever, compounds 11a,b,e,g,n,q were isolated in acceptable yield
(6ꢀ30%) and good purity after repeated chromatographic
purifications and subsequent recrystallization and were appro-
priately characterized.
The structure of some representative target pyrazolo[3,4-b]-
quinoline derivatives (10b,e,h,q,r,t) was studied by crystallogra-
phy, which confirmed that (in the experimental conditions) the
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Journal of Medicinal Chemistry
ARTICLE
Scheme 1. Synthesis of Target Compounds 10 and 11a
Figure 4. Crystallographic structure of compound 10h. Ellipsoids
enclose 50% probability.
a Reagents: (i) C6H4NHNH2, 1-pentanol, (or X-C6H4NHNH2 HCl,
3
Na2CO3, 1-pentanol); (ii) Na2CO3, DMF.
Figure 5. Comparison of the 1H NMR features of compounds 10b and
11b. The spectra were performed at 400 MHz in CDCl3 at room
temperature, and the chemical shift values are expressed in ppm. The
double arrows indicate significant NOESY contacts.
signals attributed to H-20/60 in the couple 10b/11b. In com-
pound 10b, the proximity between acetamide substituent and
FRA leads the phenyl substituent to adopt an orientation almost
perpendicular with respect to the plane of the tricyclic system
(Figure 4) and the signal attributed to H-20/60 resonates at 7.61 ppm.
On the other hand, the phenyl substituent of compound 11b can
be considered a true freely rotating aromatic ring when potential
nonconventional H-bonds are neglected. However, the low-field
chemical shift (8.21 ppm, Figure 5) of the signal attributed to
H-20/60 of 11b suggests that the FRA of this compound spends a
significant time in the coplanar conformation.
Figure 3. Structure of compound 13 asymmetric unit found by crystal-
lography. Ellipsoids enclose 50% probability.
In Vitro Binding Studies. Compounds 10aꢀt and 11a,b,e,g,n,q
were tested for their potential activity in inhibiting the specific
binding of [3H]1 to rat cerebral cortex membranes in compar-
ison with unlabeled 1, and the results of the binding studies are
shown in Table 1. The compounds tested show TSPO affinities
in the micromolar to the subnanomolar range with a modulation
suitable for structureꢀaffinity relationship analysis.
preferred alkylation site is the free pyrazole nitrogen and it shows
for this atom a pyramidal character in all the compounds studied
(Figure 4).
Mass spectrometry confirmed that compounds 11a,b,e,g,n,q
are isomers of compounds 10a,b,e,g,n,q, and the comparison of
NOESY spectrum of compound 11b with that of its isomer 10b
showed that the acetamide methylene of the former is in dipolar
contact with H-8, while in the latter a weak cross peak was
observed between H-20/60 and acetic CH2 protons (Figure 5).
The NMR studies performed with the couple 10b/11b led to the
hypothesis that the freely rotating aromatic ring (FRA)13 of
compound 11b is at least partially constrained in a coplanar
position with respect to the tricyclic system. This hypothesis is
supported by the difference in the chemical shift observed for the
In particular, the most striking result is that compounds 11 are
very potent TSPO ligands showing subnanomolar IC50 values,
whereas the corresponding isomers 10 are around 1ꢀ2 orders of
magnitude less potent. The difference suggests a better spatial
arrangement for the acetamide side chain with respect to the
planar aromatic region (PAR)13 in compounds 11 with respect to
compounds 10 (Figure 6).
It is noteworthy that the structureꢀaffinity relationships are
quite similar in the two different series of isomers 10a,b,e,g and
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Table 1. TSPO Binding Affinities of Compounds 10 and 11
of compounds 10 shows a complex trend in which the above-
cited dramatic effects coexist with a subtle affinity modulation
resulting from significant structural changes. As a matter of fact,
some compounds 10 with different substituents display very
similar affinities in the submicromolar range (e.g., 10b,e,f,j,k).
The most significant structureꢀaffinity relationships concern the
negative effect of the presence of branched substituents in
compound 10c (compare 10c with 10d) and of the benzyl
substituent in 10i (compare 10i with 10a). These negative effects
are less significant when a single branched substituent is present,
as it occurs in compound 10k (compare 10k with 10j), and when
the benzyl substituent is combined with a small alkyl substituent.
In fact, benzyl derivatives 10j,k,l show TSPO affinities higher
with respect to both dibenzyl derivative 10m and N-methylbenzyl
congener 10i. Finally, the presence of a fluorine atom in the freely
rotating aromatic ring (FRA) is tolerated in N-methyl-p-chloroa-
cetanilide derivatives 10q, 11q, and N-methyl-N-butylacetamide
derivative 11n.
compd
10a
R1
R2
X
IC50 (nM) ( SEMa
ꢀCH3
ꢀ(CH2)3CH3
ꢀC2H5
ꢀCH(CH3)2
H
H
H
H
H
H
H
H
H
H
H
H
H
F
65 ( 10
180 ( 27
10b
10c
10d
10e
10f
ꢀC2H5
ꢀCH(CH3)2
1392 ( 153
90 ( 9.9
ꢀ(CH2)2CH3 ꢀ(CH2)2CH3
ꢀ(CH2)3CH3 ꢀ(CH2)3CH3
152 ( 21
ꢀCH3
ꢀC6H5
117 ( 7.0
1.8 ( 0.24
17 ( 1.6
10g
10h
10i
ꢀCH3
p-Cl-C6H4ꢀ
p-CH3OC6H4ꢀ
ꢀCH2C6H5
ꢀC2H5
In Vivo Studies. On the basis of the intriguing results described
for emapunil and pyridazino[4,5-b]indole-1-acetamide 7,11,12 some
compounds showing TSPO affinities in the nanomolar range were
selected for a suitable in vivo pharmacological characterization in
anxiety and neuropathy animal models.
ꢀCH3
ꢀCH3
881 ( 70
10j
ꢀCH2C6H5
ꢀCH2C6H5
ꢀCH2C6H5
ꢀCH2C6H5
ꢀCH3
220 ( 37
10k
10l
ꢀCH(CH3)2
ꢀ(CH2)3CH3
ꢀCH2C6H5
ꢀ(CH2)3CH3
ꢀC2H5
304 ( 36
Thus, the potential antianxiety activity of compounds 10g,q,t
and 11a was evaluated in mice by means of the light/dark box test.
In this experimental paradigm, the activity of the test compounds is
indicated by the increase in the time spent in the aversive compart-
ment (illumined area) with respect to that spent in the dark.
Compound 10t, at oral doses of 3.0 and 10 mg/kg, increased
significantly the time spent in the aversive compartment (Table 2).
Similar results were obtained with emapunil at the same doses,
but some discrepancies were observed with respect to the results
described in the literature for this reference compound, which
was reported to show significant effects at oral doses of 0.003 and
0.01 mg/kg.12b The other reference compound used in this test,
diazepam at an oral dose of 1.0 mg/kg, produced an effect very
similar to that shown by 10t at the maximal dose tested (10 mg/kg).
These results for compound 10t indicate its potential utility for the
treatment of anxiety disorders. On the other hand, compound 11a
was significantly active only at the maximal dose (10 mg/kg) and the
remaining compounds 10g,q were inactive even at 30 mg/kg po.
These results demonstrate that very minor structural modifications
may alter the physicochemical features responsible for the pharma-
cokinetic and in vivo pharmacological properties without altering
the binding to the receptor. In fact, the variation of the atom in para-
position of FRA in the series of compounds 10g,q,t produces
significant changes in the in vivo activity different from those
observed in the in vitro binding to TSPO.
697 ( 91
10m
10n
10o
10p
10q
10r
3464 ( 416
110 ( 14
ꢀC2H5
F
2271 ( 204
87 ( 6.1
ꢀ(CH2)2CH3 ꢀ(CH2)2CH3
F
ꢀCH3
ꢀCH3
p-Cl-C6H4ꢀ
ꢀ(CH2)3CH3
F
6.8 ( 0.81
144 ( 23
Cl
Cl
Cl
H
H
H
H
F
10s
ꢀ(CH2)2CH3 ꢀ(CH2)2CH3
90 ( 8.1
10t
ꢀCH3
ꢀCH3
ꢀC2H5
p-Cl-C6H4ꢀ
ꢀ(CH2)3CH3
ꢀC2H5
16 ( 1.3
11a
11b
11e
11g
11n
11q
PK11195
0.14 ( 0.002
0.83 ( 0.074
0.34 ( 0.049
0.13 ( 0.067
0.13 ( 0.016
0.28 ( 0.064
1.3 ( 0.15
ꢀ(CH2)3CH3 ꢀ(CH2)3CH3
ꢀCH3
ꢀCH3
ꢀCH3
p-Cl-C6H4ꢀ
ꢀ(CH2)3CH3
p-Cl-C6H4ꢀ
F
a Each value is the mean ( SEM of 3 independent determinations
performed in duplicate and represents the concentration giving half the
maximum inhibition of [3H]1 (final concentration 1.0 nM) specific
binding to rat cerebral cortex membranes.
11a,b,e,g. As a matter of fact, N-methyl-p-chloroacetanilide
derivatives 10g and 11g are the most potent ligands of the
respective series and the affinity is lower in the compounds
bearing two n-butyl substituents (10e and 11e) or two ethyl
substituents (10b and 11b). However, in the subseries of
compounds 10b,e,g, the affinity modulation is more dramatic,
with variations of 2 orders of magnitude, while in the subseries
11b,e,g, the affinity variations are within 1 order of magnitude.
Moreover, in the series of compounds 10, the replacement of
p-chlorophenyl substituent with a n-butyl one produces a signifi-
cant decrease in TSPO affinity (compare 10a vs 10g), while
N-methyl-N-butylacetamide derivative 11a and N-methyl-p-chlor-
oacetanilide derivatives 11g are equipotent. These results suggest
the existence of both analogies and differences in the interac-
tion of compounds 10 and 11 with TSPO recognition site. In
general, the structureꢀaffinity relationship analysis in the series
It is noteworthy that compound 10t was found to be more
active than its analogues 10g,q also in a rat model of mono-
neuropathy (Table 3). In fact, compound 10t, at oral doses of 5.0
and 50 mg/kg, decreased the paw pressure significantly, while
10g,q showed significant effects only at the highest dose tested
(50 mg/kg). The duration of the action of the compounds was
rather short because after 45 min from the administration a
significant effect was observed only for p-chloroderivative 10t at
the highest oral dose of 50 mg/kg.
’ CONCLUSION
The straightforward synthesis and the high TSPO affinity shown
by new pyrazolo[3,4-b]quinoline derivatives 10 and especially 11
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Figure 6. Comparison between the electrostatic potentials mapped on the electron density surface of isomers 10q (left, IC50 = 6.8 nM) and 11q (right,
IC50 = 0.28 nM). The calculations were performed at HartreeꢀFock level of theory and using 6-31G* as basis set. The scale of the electrostatic potential
is negative (red) to positive (blue). The difference in TSPO affinity between compounds 10 and 11 is probably related to the spatial arrangement of the
amide carbonyl playing a key role in the interaction with TSPO.
TLC. 1H NMR spectra were recorded by means of a Bruker AC 200 or a
Bruker DRX 400 AVANCE spectrometers in the indicated solvents
(TMS as internal standard); the values of the chemical shifts are
Table 2. Effect of Compounds 10g,q,t, 11a, Emapunil, and
Diazepam in the Light/Dark Box Testa
compd
10g
dose (mg/kg) po
time in light box (s)
expressed in ppm, and the coupling constants (J) in Hz. Mass spectra
were recorded on a ThermoFinnigan LCQ-Deca. The purity of com-
pounds 10aꢀt and 11a,b,e,g,n,q was assessed by RP-HPLC by using
two different eluent systems and was found to be higher than 98%.
An Agilent 1100 series system equipped with a Zorbax Eclipse XDB-C8
(4.6 mm ꢁ 150 mm) column was used in the HPLC analysis with
acetonitrileꢀwaterꢀmethanol (60:35:5) or acetonitrileꢀmethanol (70:30)
as the mobile phase at a flow rate of 0.5 mL/min. UV detection was
achieved at 254 nm.
3
10
30
3
103.8 ( 7.1
112.3 ( 8.0
107.6 ( 7.7
106.8 ( 9.3
117.5 ( 8.7
111.3 ( 7.1
108.9 ( 8.3
135.2 ( 7.6*
169.0 ( 6.4*
103.7 ( 7.4
117.4 ( 9.2
151.5 ( 8.1*
110.8 ( 6.9
137.3 ( 8.6∧
162.3 ( 7.7*
171.6 ( 9.3*
116.4 ( 7.5
10q
10
30
1
10t
N-Butyl-N-methyl-2-(4-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazolo-
[3,4-b]quinolin-1-yl)acetamide (10a). A mixture of compound 13 (0.20 g,
0.73 mmol) in DMF (10 mL) with Na2CO3 (0.10 g, 0.94 mmol) was
stirred at room temperature for 1 h. Then, a solution of bromoacetamide
16a (0.31 g, 1.5 mmol) in DMF (3.0 mL) was added, and the resulting
mixture was stirred overnight at room temperature. The reaction mixture
was then concentrated under reduced pressure, and the residue was
partitioned between dichloromethane and water. The organic layer was
washed with water to neutrality, dried over sodium sulfate, and concen-
trated under reduced pressure. Purification of the residue by flash-
chromatography with diethyl etherꢀethyl acetate (8:2) as the eluent
gave pure 10a as a yellow crystalline solid (0.19 g, yield 65%, mp
133ꢀ134 ꢀC). The 1H NMR spectrum of this amide shows the presence
of two different rotamers in equilibrium. For the sake of simplification,
the integral intensities have not been given. 1H NMR (200 MHz,
CDCl3): 0.76 (m), 0.94ꢀ1.38 (m), 2.65 (s), 2.73 (s), 2.97ꢀ3.09 (m),
3.15 (s), 4.76 (s), 4.81 (s), 7.27 (m), 7.38ꢀ7.68 (m), 7.71 (m), 7.91
(d, J = 8.3), 8.08 (d, J = 8. 4). MS (ESI): m/z 425 (M + Na+).
N-Butyl-N-methyl-2-(4-methyl-3-oxo-2-phenyl-2H-pyrazolo[3,4-b]-
quinolin-9(3H)-yl)acetamide (11a). The title compound was separated
by flash chromatography [diethyl etherꢀethyl acetate (8:2)] as the more
polar fraction during the purification of isomer 10a. Compound 11a was
further purified by precipitation with n-hexane from a dichloromethane
solution to obtain a dark-red solid (0.045 g, yield 15%, mp 245ꢀ246 ꢀC).
The 1H NMR spectrum of this amide shows the presence of two different
rotamers in equilibrium. For the sake of simplification, the integral
intensities have not been given. 1H NMR (200Mz, CDCl3): 0.89 (t, J =
7.2), 1.04 (t, J = 7.2), 1.24ꢀ1.82 (m), 2.99 (s), 3.04 (s), 3.21 (s),
3.39ꢀ3.52 (m), 5.18 (s), 5.23 (s), 7.13 (t, J = 7.4), 7.21ꢀ7.31(m), 7.39
(t, J = 7.2), 7.64 (t, J = 7.7), 7.97 (d, J = 8.1), 8.17 (m). MS (ESI): m/z
425 (M + Na+).
3
10
1
11a
3
10
1
emapunil
3
10
1
diazepam
vehicle
a All drugs were administered 30 min before the test. Each value is the
mean ( SEM of at least 8 mice per group. ∧P < 0.05; *P < 0.01 versus
vehicle-treated mice.
leads the way to further expand the chemical diversity in TSPO
ligands andprovidesnew templates and structureꢀaffinity relation-
ship data for the design of new TSPO modulators. Moreover,
the oral activity of compound 10t in both the light/dark box test
and in a model of mononeuropathy offers additional evidence
of the potential role of TSPO as a therapeutic target.
’ EXPERIMENTAL SECTION
Chemistry. All chemicals used were of reagent grade. Yields refer to
purified products and are not optimized. Melting points were deter-
mined in open capillaries on a Gallenkamp apparatus and are uncor-
rected. Merck silica gel 60 (230ꢀ400 mesh) was used for column
chromatography. Merck TLC plates, silica gel 60 F254, were used for
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Table 3. Effect of Compounds 10g,q,t in a Rat Model of Mononeuropathy (Right) Evaluated in the Paw Pressure Testa
paw pressure (g)
compd
dose (mg/kg) po
before treatment
after 15 min
after 30 min
after 45 min
10g
5
5
left
61.6 ( 2.5
32.5 ( 2.7
62.1 ( 2.4
34.0 ( 2.9
62.7 ( 2.3
33.1 ( 2.6
65.6 ( 2.2
32.5 ( 2.4
58.9 ( 2.6
33.5 ( 3.2
63.1 ( 2.6
31.8 ( 3.0
61.6 ( 2.7
32.3 ( 2.5
57.8 ( 3.4
39.2 ( 2.7
61.0 ( 2.5
48.1 ( 3.2*
58.7 ( 4.1
35.6 ( 3.5
60.7 ( 2.3
49.4 ( 2.8*
61.4 ( 3.5
44.8 ( 2.3∧
62.2 ( 2.8
56.3 ( 2.2*
63.5 ( 2.8
34.8 ( 3.7
63.6 ( 2.8
35.9 ( 2.5
58.0 ( 2.0
44.0 ( 2.3∧
57.7 ( 3.8
36.4 ( 2.6
59.3 ( 1.8
47.2 ( 2.5*
58.4 ( 3.3
35.8 ( 2.7
61.8 ( 3.2
49.4 ( 2.9*
59.6 ( 2.6
31.3 ( 2.8
61.4 ( 3.3
33.4 ( 2.8
59.0 ( 2.9
37.0 ( 2.2
64.1 ( 3.9
36.3 ( 3.7
63.1 ( 3.3
36.3 ( 2.6
63.8 ( 3.2
32.7 ( 2.8
64.8 ( 3.5
42.5 ( 2.5∧
63.0 ( 2.4
32.9 ( 2.6
right
left
50
50
5
right
left
10q
5
right
left
50
50
5
right
left
10t
5
right
left
50
50
right
left
vehicle
right
a Each value is the mean ( SEM of at least 4-5 rats per group. ∧P < 0.05; *P < 0.01 versus vehicle-treated rats.
N,N-Diisopropyl-2-(4-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazolo-
[3,4-b]quinolin-1-yl)acetamide (10c). The title compound was prepared
from 13 (0.15 g, 0.54 mmol) and 16c (0.25 g, 1.1 mmol) following the
procedure described for compound 10a and was purified by flash
chromatography with n-hexaneꢀethyl acetate (65:35) as the eluent to
obtain a yellow crystalline solid (0.056 g, yield 25%). An analytical sample
was obtained by recrystallization from ethyl acetate by slow evaporation
(mp 215ꢀ216 ꢀC). 1H NMR (200 MHz, CDCl3): 0.91 (d, J = 6.2, 6H),
1.10 (d, J = 6.2, 6H), 3.17 (s, 3H), 3.37 (m, 1H), 3.62 (m, 1H), 4.78
(s, 2H), 7.29 (m, 1H), 7.40ꢀ7.56 (m, 5H), 7.70 (m, 1H), 7.92 (d, J = 8.7,
1H), 8.10 (d, J = 8.8, 1H). MS (ESI): m/z 417 (M + H+)
N,N-Dipropyl-2-(4-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazolo-
[3,4-b]quinolin-1-yl)- acetamide (10d). The title compound was pre-
pared from 13 (0.15 g, 0.54 mmol) and 16d (0.25 g, 1.1 mmol) following
the procedure described for compound 10a and was purified by flash
chromatography with n-hexaneꢀethyl acetate (65:35) as the eluent to
obtain a yellow oil which crystallized on standing (0.090 g, yield 40%,
mp 141ꢀ142 ꢀC). 1H NMR (200 MHz, CDCl3): 0.71 (m, 6H), 1.32
(m, 4H), 2.99 (m, 4H), 3.16 (s, 3H), 4.79 (s, 2H), 7.28 (m, 1H),
7.40ꢀ7.58 (m, 5H), 7.69 (t, J = 7.2, 1H), 7.91 (d, J = 8.5, 1H), 8.11 (d,
J = 8.5, 1H). MS (ESI): m/z 417 (M + H+).
Figure 7. Crystallographic structure of compound 10b. Ellipsoids enclose
50% probability.
N,N-Diethyl-2-(4-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazolo-
[3,4-b]quinolin-1-yl)acetamide (10b). The title compound was pre-
pared from 13 (0.17 g, 0.62 mmol) and 16b (0.31 g, 1.6 mmol) following
the procedure described for compound 10a and was purified by flash
chromatography with diethyl etherꢀethyl acetate (8:2) as the eluent to
obtain a yellow crystalline solid (0.12 g, yield 50%) (Figure 7). An
analytical sample was obtained by recrystallization from ethyl acetate by
N,N-Dibutyl-2-(4-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazolo-
[3,4-b]quinolin-1-yl)acetamide (10e). The title compound was pre-
pared from 13 (0.15 g, 0.54 mmol) and 16e (0.27 g, 1.1 mmol) following
the procedure described for compound 10a and was purified by flash
chromatography with n-hexaneꢀethyl acetate (65:35) as the eluent to
obtain a yellow crystalline solid (0.13 g, yield 54%) (Figure 8). An
analytical sample was obtained by recrystallization from ethyl acetate by
1
slow evaporation (mp 171ꢀ172 ꢀC). H NMR (200 MHz, CDCl3):
1
slow evaporation (mp 173ꢀ174 ꢀC). H NMR (200 MHz, CDCl3):
0.82ꢀ0.94 (m, 6H), 2.97ꢀ3.20 (m, 7H), 4.78 (s, 2H), 7.28 (m, 1H),
7.38ꢀ7.60 (m, 5H), 7.70 (m, 1H), 7.92 (d, J = 8.7, 1H), 8.10 (d, J = 8.6,
1H). MS (ESI): 389 (M + H+).
0.74ꢀ0.85 (m, 6H), 1.02ꢀ1.37 (m, 8H), 2.92ꢀ3.10 (m, 4H), 3.17
(s, 3H), 4.80 (s, 2H), 7.30 (m, 1H), 7.40ꢀ7.59 (m, 5H), 7.70 (t, J = 8.0,
1H), 7.92 (d, J = 8.5, 1H), 8.12 (d, J = 8.2, 1H). MS (ESI): 445 (M + H+).
N,N-Dibutyl-2-(4-methyl-3-oxo-2-phenyl-2H-pyrazolo[3,4-b]quinolin-
9(3H)-yl)acetamide (11e). The title compound was separated by flash
chromatography [n-hexaneꢀethyl acetate (65:35)] as the more polar
fraction during the purification of isomer 10e. Compound 11e was
further purified by recrystallization from ethyl acetate to obtain a dark-
red solid (0.052 g, yield 22%, mp 193ꢀ194 ꢀC). 1H NMR (200 MHz,
CDCl3): 0.88 (t, J = 7.1, 3H), 1.03 (t, J = 7.3, 3H), 1.19ꢀ1.84 (m, 8H),
3.04 (s, 3H), 3.33ꢀ3.50 (m, 4H), 5.23 (s, 2H), 7.13 (t, J = 7.4, 1H),
N,N-Diethyl-2-(4-methyl-3-oxo-2-phenyl-2H-pyrazolo[3,4-b]quinolin-
9(3H)-yl)acetamide (11b). The title compound was separated by flash
chromatography [diethyl etherꢀethyl acetate (8:2)] as the more polar frac-
tion during the purification of isomer 10b. Compound 11b was further
purified by washing with ethanol to obtain a dark-red solid (0.058 g, yield
24%, mp 260ꢀ261 ꢀC). 1H NMR (200 MHz, CDCl3): 1.15 (t, J = 7.2, 3H),
1.37 (t, J = 7.0, 3H), 3.01 (s, 3H), 3.49 (m, 4H), 5.18 (s, 2H), 7.14 (t, J = 7.4,
1H), 7.23- 7.30 (m, 2H), 7.39 (t, J= 7.8, 2H), 7.64 (t, J= 7.6, 1H), 7.95 (d, J=
8.0, 1H), 8.17 (d, J = 7.9, 2H). MS (ESI, negative ions): m/z 387 (M ꢀ H+).
7170
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Journal of Medicinal Chemistry
ARTICLE
(m, 4H), 7.29ꢀ7.54 (m, 6H), 7.73 (m, 1H), 7.96 (d, J = 8.4, 1H), 8.14
(d, J = 8.6, 1H). MS (ESI): m/z 453 (M + H+).
N-Benzyl-N-methyl-2-(4-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazolo[3,4-b]quinolin-1-yl)acetamide (10i). The title compound
was prepared from 13 (0.10 g, 0.36 mmol) and 16i (0.18 g, 0.74 mmol)
following the procedure described for compound 10a and was purified
by flash chromatography with n-hexaneꢀethyl acetate (65:35) as the
eluent to obtain a yellow crystalline solid (0.097 g, yield 62%, mp
181ꢀ182 ꢀC). The 1H NMR spectrum of this amide shows the presence
of two different rotamers in equilibrium. For the sake of simplification,
the integral intensities have not been given. 1H NMR (200 MHz,
CDCl3): 2.70 (s), 2.72 (s), 3.18 (s), 4.32 (s), 4.34 (s), 4.85 (s), 4.90
(s), 6.88 (m), 7.00 (m), 7.19ꢀ7.33 (m), 7.42ꢀ7.62 (m), 7.73 (t, J = 7.5),
7.95 (d, J = 8.4), 8.13 (d, J = 8.4). MS (ESI): m/z 437 (M + H+).
N-Benzyl-N-ethyl-2-(4-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazolo-
[3,4-b]quinolin-1-yl)acetamide (10j). The title compound was prepared
from 13 (0.15 g, 0.54 mmol) and 16j (0.29 g, 1.1 mmol) following the
procedure described for compound 10a and was purified by flash
chromatography with n-hexaneꢀethyl acetate (65:35) as the eluent to
obtain a yellow crystalline solid (0.11 g, yield 45%). An analytical sample
was obtained by recrystallization from ethyl acetate by slow evaporation
Figure 8. Crystallographic structure of compound 10e. Ellipsoids
enclose 50% probability.
7.22ꢀ7.42 (m, 4H), 7.65 (m, 1H), 7.97 (d, J = 8.7, 1H), 8.16 (d, J = 8.2,
2H). MS (ESI): 445 (M + H+).
1
(mp 170ꢀ171 ꢀC). The H NMR spectrum of this amide shows the
presence of two different rotamers in equilibrium. For the sake of
N-Methyl-2-(4-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazolo-
[3,4-b]quinolin-1-yl)-N-phenylacetamide (10f). The title compound
was prepared from 13 (0.10 g, 0.36 mmol) and 16f (0.18 g, 0.79 mmol)
following the procedure described for compound 10a and was purified
by flash chromatography with n-hexaneꢀethyl acetate (65:35) as the
eluent to obtain a yellow crystalline solid (0.094 g, yield 62%, mp
209ꢀ210 ꢀC). The 1H NMR spectrum of this amide shows the presence
of two different rotamers in equilibrium. For the sake of simplification,
the integral intensities have not been given. 1H NMR (200 MHz,
CDCl3): 3.03 (s, 3H), 3.18 (s, 3H), 4.52 (s, 2H), 6.75 (m, 2H),
7.26ꢀ7.53 (m, 9H), 7.73 (t, J = 8.2, 1H), 7.96 (d, J = 8.0, 1H), 8.13
(d, J = 7.6, 1H). MS (ESI): m/z 423 (M + H+).
1
simplification, the integral intensities have not been given. H NMR
(200 MHz, CDCl3): 0.89 (m), 3.02ꢀ3.22 (m), 4.28 (s), 4.36 (s), 4.83
(s), 4.88 (s), 6.88 (m), 7.00 (m), 7.16ꢀ7.35(m), 7.43ꢀ7.62 (m), 7.73 (t,
J = 7.0), 7.95 (d, J = 8.6), 8.14 (d, J = 8.1). MS (ESI): m/z 451 (M + H+).
N-Benzyl-N-isopropyl-2-(4-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazolo[3,4-b]quinolin-1-yl)acetamide (10k). The title compound
was prepared from 13 (0.15 g, 0.54 mmol) and 16k (0.30 g, 1.1 mmol)
following the procedure described for compound 10a and was purified
by flash chromatography with n-hexaneꢀethyl acetate (65:35) as the
eluent to obtain a yellow crystalline solid (0.098 g, yield 39%). An
analytical sample was obtained by recrystallization from ethyl acetate by
1
N-(4-Chlorophenyl)-N-methyl-2-(4-methyl-3-oxo-2-phenyl-2,3-dihydro-
1H-pyrazolo[3,4-b]quinolin-1-yl)acetamide (10g). The title compound
was prepared from 13 (0.10 g, 0.36 mmol) and 16g (0.19 g, 0.72 mmol)
following the procedure described for compound 10a and was purified by
flash chromatography with diethyl etherꢀethyl acetate (7:3) as the eluent
to obtain a yellow crystalline solid (0.086 g, yield 52%). An analytical
sample was obtained by recrystallization from ethyl acetate by slow
slow evaporation (mp 156ꢀ157 ꢀC). The H NMR spectrum of this
amide shows the presence of two different rotamers in equilibrium. For
the sake of simplification, the integral intensities have not been given. 1H
NMR (200 MHz, CDCl3): 0.92 (m), 3.18 (s), 3.96 (m), 4.20 (s), 4.34
(s), 4.51 (m), 4.70 (s), 4.97 (s), 6.92 (m), 7.10ꢀ7.34 (m), 7.42ꢀ7.57
(m), 7.73 (m), 7.95 (m), 8.13 (d, J = 8.3). MS (ESI): m/z 465 (M + H+).
N-Benzyl-N-butyl-2-(4-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazolo[3,4-b]quinolin-1-yl)acetamide (10l). The title compound
was prepared from 13 (0.15 g, 0.54 mmol) and 16l (0.31 g, 1.1 mmol)
following the procedure described for compound 10a and was purified
by flash chromatography with n-hexaneꢀethyl acetate (65:35) as the
eluent to obtain a yellow crystalline solid (0.083 g, yield 32%, mp
151ꢀ152 ꢀC). The 1H NMR spectrum of this amide shows the
presence of two different rotamers in equilibrium. For the sake of
simplification, the integral intensities have not been given. 1H NMR
(200 MHz, CDCl3): 0.75 (m), 1.02ꢀ1.37 (m), 2.95 (t, J = 7.2), 3.10
(t, J = 7.8), 3.17 (s), 4.27 (s), 4.34 (s), 4.82 (s), 4.89 (s), 6.88 (m), 6.99
(m), 7.15ꢀ7.31 (m), 7.39ꢀ7.58 (m), 7.71 (t, J = 7.8), 7.95 (d, J = 8.4),
8.13 (d, J = 8.2). MS (ESI): m/z 479 (M+H+).
1
evaporation (mp 180ꢀ181 ꢀC). H NMR (200 MHz, CDCl3): 3.01
(s, 3H), 3.18 (s, 3H), 4.52 (s, 2H), 6.70 (m, 2H), 7.22ꢀ7.37 (m, 3H),
7.43ꢀ7.54 (m, 5H), 7.73 (m, 1H), 7.95 (d, J = 8.5, 1H), 8.14 (d, J = 8.7,
1H). MS (ESI): m/z 457 (M + H+).
N-(4-Chlorophenyl)-N-methyl-2-(4-methyl-3-oxo-2-phenyl-2H-
pyrazolo[3,4-b]quinolin-9(3H)-yl)acetamide (11g). The title com-
pound was separated by flash chromatography [diethyl etherꢀethyl
acetate (7:3)] as the more polar fraction during the purification of
isomer 10g. Compound 11g was further purified by precipitation
with n-hexane from a dichloromethane solution to obtain a dark-red
1
solid (0.049 g, yield 30%, mp 239ꢀ240 ꢀC). H NMR (400 MHz,
CDCl3): 3.04 (s, 3H), 3.30 (s, 3H), 4.95 (s, 2H), 7.15ꢀ7.46 (m, 9H),
7.66 (t, J = 7.8, 1H), 7.97 (d, J = 8.0, 1H), 8.15 (d, J = 7.8, 2H). MS
(ESI): m/z 479 (M + Na+).
N,N-Dibenzyl-2-(4-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazolo-
[3,4-b]quinolin-1-yl)acetamide (10m). The title compound was pre-
pared from 13 (0.15 g, 0.54 mmol) and 16m (0.32 g, 1.1 mmol) following
the procedure described for compound 10a and was purified by flash
chromatography with n-hexaneꢀethyl acetate (65:35) as the eluent to
obtain a yellow crystalline solid (0.068 g, yield 25%). An analytical sample
was obtained by recrystallization from ethyl acetate by slow evaporation
N-(4-Methoxyphenyl)-N-methyl-2-(4-methyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazolo[3,4-b]quinolin-1-yl)acetamide (10h). The title
compound was prepared from 13 (0.10 g, 0.36 mmol) and 16h (0.19 g,
0.74 mmol) following the procedure described for compound 10a and
was purified by flash chromatography with acetateꢀpetroleum ether
(7:3) as the eluent to obtain a yellow crystalline solid (0.068 g, yield
42%). An analytical sample was obtained by recrystallization from ethyl
1
(mp 185ꢀ187 ꢀC). H NMR (200 MHz, CDCl3): 3.20 (s, 3H), 4.22
(s, 2H), 4.35 (s, 2H), 4.86(s, 2H), 6.91 (m, 2H), 6.99 (m, 2H), 7.22ꢀ7.60
(m, 12H), 7.76 (t, J = 7.7, 1H), 7.97 (d, J = 8.5, 1H), 8.15 (d, J = 8.3, 1H).
MS (ESI): m/z 513 (M + H+).
1
acetate by slow evaporation (mp 205ꢀ207 ꢀC). H NMR (200 MHz,
CDCl3): 2.99 (s, 3H), 3.19 (s, 3H), 3.81 (s, 3H), 4.52 (s, 2H), 6.64ꢀ6.80
7171
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Journal of Medicinal Chemistry
ARTICLE
N-Butyl-2-[2-(4-fluorophenyl)-4-methyl-3-oxo-2,3-dihydro-1H-
pyrazolo[3,4-b]quinolin-1-yl]-N-methylacetamide (10n). The title com-
pound was prepared from 14 (0.25 g, 0.85 mmol) and 16a (0.38 g,
1.8 mmol) following the procedure described for compound 10a and was
purified by flash chromatography with diethyl etherꢀethyl acetate (8:2)
as the eluent to obtain a yellow crystalline solid (0.25 g, yield 70%). An
analytical sample was obtained by recrystallization from ethyl acetate by
1
slow evaporation (mp 161ꢀ162 ꢀC). The H NMR spectrum of this
amide shows the presence of two different rotamers in equilibrium. For
the sake of simplification, the integral intensities have not been given. 1H
NMR (200 MHz, CDCl3): 0.75ꢀ0.87 (m), 1.01ꢀ1.38 (m), 2.70 (s), 2.82
(s), 3.05ꢀ3.13 (m), 3.16 (s), 4.73 (s), 4.79 (s), 7.18 (m), 7.42ꢀ7.58 (m),
7.72 (m), 7.92 (d, J = 8.5), 8.12 (d, J = 8.3). MS (ESI): m/z 421 (M + H+).
N-Butyl-2-[2-(4-fluorophenyl)-4-methyl-3-oxo-2H-pyrazolo[3,4-b]-
quinolin-9(3H)-yl]-N-methylacetamide (11n). The title compound
was separated by flash chromatography [diethyl etherꢀethyl acetate
(8:2)] as the more polar fraction during the purification of isomer 10n.
Compound 11n was further purified by washing with ethanol to obtain a
Figure 9. Crystallographic structure of compound 10q. Ellipsoids
enclose 50% probability.
1
dark-red solid (0.030 g, yield 8.4%, mp 259ꢀ260 ꢀC). The H NMR
spectrum of this amide shows the presence of two different rotamers in
equilibrium. For the sake of simplification, the integral intensities have
not been given. 1H NMR (200 MHz, CDCl3): 0.88 (t, J = 7.3), 1.05 (t,
J = 7.3), 1.23ꢀ1.83 (m), 2.99 (s), 3.04 (s), 3.22 (s), 3.39ꢀ3.52 (m), 5.20
(s), 5.23 (s), 7.07 (m), 7.20ꢀ7.32 (m), 7.65 (m), 7.98 (d, J = 7.7), 8.13
(m). MS (ESI): m/z 443 (M + Na+).
N,N-Diethyl-2-[2-(4-fluorophenyl)-4-methyl-3-oxo-2,3-dihydro-1H-
pyrazolo[3,4-b]quinolin-1-yl]acetamide (10o). The title compound
was prepared from 14 (0.25 g, 0.85 mmol) and 16b (0.33 g, 1.7 mmol)
following the procedure described for compound 10a and was purified by
flash chromatography with diethyl etherꢀethyl acetateꢀdichloromethane
(75:20:5) as the eluent to obtain a yellow crystalline solid (0.16 g, yield
46%). An analytical sample was obtained by recrystallization from ethyl
1
acetate by slow evaporation (mp 225ꢀ226 ꢀC). H NMR (200 MHz,
CDCl3): 0.93 (t, J = 7.2, 6H), 3.04ꢀ3.20 (m, 7H), 4.80 (s, 2H), 7.18 (t, J =
8.6, 2H), 7.43ꢀ7.57 (m, 3H), 7.73 (t, J= 7.3, 1H), 7.96 (d, J= 8.5, 1H), 8.12
(d, J = 8.1, 1H). MS (ESI): m/z 407 (M + H+).
N,N-Dipropyl-2-[2-(4-fluorophenyl)-4-methyl-3-oxo-2,3-dihydro-1H-
pyrazolo[3,4-b]quinolin-1-yl]acetamide (10p). The title compound was
prepared from 14 (0.18 g, 0.61 mmol) and 16d (0.29 g, 1.3 mmol)
following the procedure described for compound 10a and was purified by
flash chromatography with n-hexaneꢀethyl acetate (65:35) as the eluent
to obtain a yellow crystalline solid (0.144 g, yield 54%). An analytical
sample was obtained by recrystallization from ethyl acetate by slow
Figure 10. Crystallographic structure of compound 10r. Ellipsoids
enclose 50% probability.
obtain a dark-red solid (0.021 g, yield 6.5%, mp 232ꢀ234 ꢀC). 1H NMR
(400 MHz, CDCl3): 3.04 (s, 3H), 3.31 (s, 3H), 4.94 (s, 2H), 7.12 (t, J =
8.8, 2H), 7.19ꢀ7.36 (m, 4H), 7.46 (d, J = 8.3, 2H), 7.67 (t, J = 7.2, 1H),
7.98 (d, J = 7.2, 1H), 8.11 (m, 2H). MS (ESI): m/z 497 (M + Na+).
N-Butyl-2-[2-(4-chlorophenyl)-4-methyl-3-oxo-2,3-dihydro-1H-
pyrazolo[3,4-b]quinolin-1-yl]-N-methylacetamide (10r). The title
compound was prepared from 15 (0.20 g, 0.65 mmol) and 16a (0.27 g,
1.3 mmol) following the procedure described for compound 10a and was
purifiedbyflash chromatography withn-hexaneꢀethyl acetate(65:35) as
the eluent to obtain a yellow crystalline solid (0.19 g, yield 67%) (Figure 10).
An analytical sample was obtained by recrystallization from ethyl
acetate by slow evaporation (mp 186ꢀ187 ꢀC). The 1H NMR
spectrum of this amide shows the presence of two different rotamers
in equilibrium. For the sake of simplification, the integral intensities
1
evaporation (mp 179ꢀ180 ꢀC). H NMR (CDCl3): 0.66ꢀ0.80 (m,
6H), 1.34 (m, 4H), 3.01 (m, 4H), 3.16 (s, 3H), 4.77 (s, 2H), 7.18 (t, J =
8.4, 2H), 7.42ꢀ7.57 (m, 3H), 7.72 (t, J = 7.7, 1H), 7.92 (d, J = 8.4, 1H),
8.12 (d, J = 8.1, 1H). MS (ESI): m/z 435 (M + H+).
N-(4-Chlorophenyl)-2-[2-(4-fluorophenyl)-4-methyl-3-oxo-2,3-dihydro-
1H-pyrazolo[3,4-b]quinolin-1-yl)-N-methylacetamide (10q). The title
compound was prepared from 14 (0.20 g, 0.68 mmol) and 16g (0.40 g,
1.5 mmol) following the procedure described for compound 10a and was
purified by flash chromatography with diethyl etherꢀethyl acetate (7:3)
as the eluent to obtain a yellow crystalline solid (0.20 g, yield 62%)
(Figure 9). An analytical sample was obtained by recrystallization from
1
1
ethyl acetate by slow evaporation (mp 199ꢀ200 ꢀC). H NMR (200
have not been given. H NMR (200 MHz, CDCl3): 0.81 (m), 1.16
MHz, CDCl3): 3.01 (s, 3H), 3.17 (s, 3H), 4.47 (s, 2H), 6.80 (d, J = 8.4,
2H), 7.13ꢀ7.17 (m, 2H), 7.30 (d, J = 8.5, 2H), 7.47 (m, 3H), 7.74 (t, J =
7.1, 1H), 7.95 (d, J = 8.5, 1H), 8.13 (d, J = 8.7, 1H). MS (ESI): m/z 475
(M + H+).
(m), 1.30 (m), 2.68 (s), 2.83 (s), 3.05ꢀ3.16 (m), 4.75 (s), 4.80 (s),
7.42ꢀ7.56 (m), 7.70 (m), 7.91 (d, J = 8.6), 8.12 (d, J = 8.0). MS
(ESI): m/z 437 (M + H+).
2-[2-(4-Chlorophenyl)-4-methyl-3-oxo-2,3-dihydro-1H-pyrazolo-
[3,4-b]quinolin-1-yl]-N,N-dipropylacetamide (10s). The title com-
pound was prepared from 15 (0.20 g, 0.65 mmol) and 16d (0.29 g,
1.3 mmol) following the procedure described for compound 10a and
was purified by flash chromatography with n-hexaneꢀethyl acetate (65:35)
as the eluent to obtain a yellow crystalline solid (0.154 g, yield 53%).
N-(4-Chlorophenyl)-2-[2-(4-fluorophenyl)-4-methyl-3-oxo-2H-
pyrazolo[3,4-b]quinolin-9(3H)-yl]-N-methylacetamide (11q). The title
compound was isolated by flash chromatography [diethyl etherꢀethyl
acetate (7:3)]as the more polar fraction during the purification of isomer
10q. Compound 11q was further purified by washing with ethanol to
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temperature, the precipitate was collected by filtration, washed with water,
and dried under reduced pressure to give pure compound 15 as dark-
brownplates (1.1g, yield 81%, mp >300 ꢀC decꢀC). 1H NMR (200 MHz,
DMSO-d6): 2.98 (s, 3H), 7.25 (t, J = 7.7, 1H), 7.45 (m, 3H), 7.67 (t, J =
7.6, 1H), 8.01 (d, J = 8.3, 1H), 8.14 (d, J = 8.8, 2H), 12.69 (br s, 1H). MS
(ESI, negative ions): m/z 308 (M ꢀ H+).
X-ray Crystallography. Single crystals of 10b,e,h,q,r,t and 13 were
submitted to X-ray data collection on an Oxford-Diffraction Xcalibur
Sapphire 3 diffractometer with a graphite monochromated Mo Kα
radiation (λ = 0.71073 Å) at 293 K. The structures were solved by direct
methods implemented in SHELXS-97 program.15 The refinements were
carried out by full-matrix anisotropic least-squares on F2 for all reflec-
tions for non-H atoms by means of the SHELXL-97 program.16
Crystallographic data (excluding structure factors) for the structures
solved in this paper have been deposited with the Cambridge Crystal-
lographic Data Centre as supplementary publication no. CCDC 817653,
817658, 817652, 817661, 817660, 817659, and 817651. Copies of the
data can be obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK; (fax: + 44 (0) 1223 336 033; or e-mail:
deposit@ccdc.cam.ac.uk).
In Vitro Binding Assays. Male SpragueꢀDawley CD rats (Charles
River Italia, Calco, CO, Italy) with body masses of 200ꢀ250 g were used.
Rats were acclimatized to the new housing conditions for at least one week.
They were housed six per cage under an artificial 12 h light/12 h dark cycle
(lights on at 08:00 h), a constant temperature of 22 ( 2 ꢀC and a relative
humidity of 65%. They had free access to water and standard laboratory
food at all times. Animal care and handling throughout the experimental
procedures were in accordance with the European Communities Council
Directive of 24 November 1986 (86/609/EEC).
Rats were sacrificed by decapitation, their brains were rapidly dissected,
and the cerebral cortex was immediately frozen on dry ice and stored
at ꢀ80 ꢀC until the assay was performed. The binding assays were
performed as described in the literature.13b On the day of the assay, the
rat cerebral cortex was thawed and homogenized in 50 volumes of ice-
cold Dulbecco’s phosphate-buffered saline (PBS, pH 7.4) with a Polytron
PT 10 homogenizer (setting 5 for 20 s). The homogenate was centri-
fuged at 40000g, 4 ꢀC for 30 min; the resulting pellet was resuspended in
the same volume of ice-cold buffer and recentrifuged. The new pellet was
resuspended in 10 volumes of ice-cold incubation buffer (PBS) and used
for the binding assay.
Figure 11. Crystallographic structure of compound 10t. For clarity,
only one molecule of the asymmetric unit is depicted. Ellipsoids enclose
50% probability.
An analytical sample was obtained by recrystallization from ethyl
acetate by slow evaporation (mp 197ꢀ198 ꢀC). 1H NMR (200 MHz,
CDCl3): 0.71 (m, 6H), 1.32 (m, 4H), 2.99 (m, 4H), 3.12 (s, 3H), 4.76
(s, 2H), 7.38ꢀ7.54 (m, 5H), 7.68 (t, J = 8.1, 1H), 7.90 (d, J = 8.2, 1H),
8.07 (d, J = 8.3, 1H). MS (ESI): m/z 451 (M + H+).
N-(4-Chlorophenyl)-2-[2-(4-chlorophenyl)-4-methyl-3-oxo-2,3-di-
hydro-1H-pyrazolo[3,4-b]quinolin-1-yl]-N-methylacetamide (10t).
The title compound was prepared from 15 (0.20 g, 0.65 mmol) and 16g
(0.34 g, 1.3 mmol) following the procedure described for compound
10a and was purified by flash chromatography with n-hexaneꢀethyl
acetate (65:35) as the eluent to obtain a yellow crystalline solid (0.184 g,
yield 58%) (Figure 11). An analytical sample was obtained by recrys-
tallization from ethyl acetate by slow evaporation (mp 197ꢀ198 ꢀC). 1H
NMR (200 MHz, CDCl3): 3.01 (s, 3H), 3.17 (s, 3H), 4.50 (s, 2H),
6.79 (d, J = 8.4, 2H), 7.31 (d, J = 8.3, 2H), 7.48 (m, 5H), 7.76 (t, J = 7.9,
1H), 7.96 (d, J = 8.6, 1H), 8.14 (d, J = 8.2, 1H). MS (ESI): m/z 491
(M + H+).
[3H]1 binding was measured in a final volume of 1000 μL, consisting
of 100 μL of membrane suspension (0.15ꢀ0.20 mg of protein), 100 μL
of [3H]1 (specific activity 83.4 Ci/mmol, New England Nuclear, Perkin-
Elmer, Monza, MI, Italy; final assay concentration of 1 nM), 5 μL of drug
solution or solvent (dimethyl sulfoxide), and 795 μL of PBS. The
binding reaction was performed at 25 ꢀC for 90 min and started with the
addition of membranes. The incubation was terminated by rapid
filtration through glass-fiber filters (Whatman GF/B, GE Healthcare,
Milan, Italy) which had been presoaked with 0.3% polyethyleneimine
and placed in a cell harvester filtration manifold (Brandel). The filters
were washed five times with 4 mL of ice-cold PBS, and filter-bound
radioactivity was quantified by liquid scintillation spectrometry. Non-
specific binding was defined as binding in the presence of 10 μM of
unlabeled compound 1 (Sigma-Aldrich, Milan, Italy). Specific binding
was determined by subtracting the nonspecific from the total binding
and was about 75ꢀ80% of the total binding. Sigmaplot 10 software
(Systat Software, Inc., San Jose, CA, USA) was used to determine the
concentration of the test compounds that inhibited [3H]ligand binding
by 50% (IC50). Ten concentrations of each compound were assayed,
each performed in duplicate.
4-Methyl-2-phenyl-1H-pyrazolo[3,4-b]quinolin-3(2H)-one (13). A
mixture of 1214 (0.89 g, 3.56 mmol) in 20 mL of 1-pentanol with
phenylhydrazine (1.4 mL, 14.2 mmol) was refluxed for 5 h. After cooling
to room temperature, the precipitate was collected by filtration, washed
with water, and dried under reduced pressure to give pure compound 13
as a dark solid (0.85 g, yield 87%, mp >300 ꢀC dec). An analytical sample
was obtained by recrystallization from CHCl3 by slow evaporation. 1H
NMR (DMSO-d6): 2.97 (s, 3H), 7.11 (t, J = 7.3, 1H), 7.24 (t, J = 7.4,
1H), 7.36ꢀ7.44 (m, 3H), 7.66 (t, J = 7.3, 1H), 8.00 (d, J = 8.3, 1H), 8.10
(d, J = 8.1, 2H), 12.61 (br s, 1H). MS (ESI): m/z 276 (M + H+).
2-(4-Fluorophenyl)-4-methyl-1H-pyrazolo[3,4-b]quinolin-3(2H)-one
(14). A mixture of 1214 (1.2 g, 4.8 mmol) in 50 mL of 1-pentanol with
(4-fluorophenyl)hydrazine hydrochloride (3.0 g, 18.5 mmol) and sodium
carbonate (1.1 g, 10.4 mmol) was refluxed for 10 h. After cooling to room
temperature, the precipitate was collected by filtration, washed with water,
anddriedunder reducedpressure togivepurecompound14 asdark plates
(1.3 g, yield 92%, mp >300 ꢀC dec ꢀC). 1H NMR (200 MHz, DMSO-d6):
2.98 (s, 3H), 7.25 (m, 3H), 7.43 (d, J = 8.3, 1H), 7.67 (t, J = 7.6, 1H), 8.01
(d, J = 8.1, 1H), 8.11 (m, 2H), 12.69 (br s, 1H). MS (ESI, negative ions):
m/z 292 (M ꢀ H+).
2-(4-Chlorophenyl)-4-methyl-1H-pyrazolo[3,4-b]quinolin-3(2H)-one
(15). A mixture of 1214 (1.1 g, 4.4 mmol) in 50 mL of 1-pentanol with
(4-chlorophenyl)hydrazine hydrochloride (3.2 g, 18 mmol) and sodium
carbonate (1.0 g, 9.4 mmol) was refluxed for 5 h. After cooling to room
In Vivo Studies. Animals. Male mice and rats from Harlan-Nossan
(Comerio, Varese, Italy) breeding farm were used. Fifteen mice or four
rats were housed per cage. The cages were placed in the experimental
room 24 h before the test for acclimatization. The animals were kept at
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23 ( 1 ꢀC with a 12 h light/dark cycle, light at 7 a.m., with food and
water ad libitum. All experiments were carried out according to the
guidelines of the European Community Council.
’ ACKNOWLEDGMENT
We are grateful to Prof. Stefania D’Agata D’Ottavi for the
careful reading of the manuscript and to Dr. Francesco Berrettini
(CIADS, Universitꢀa di Siena) for the X-ray data collection. This
work was financially supported by Ministero dell’Istruzione,
dell’Universitꢀa e della Ricerca (MIUR)ꢀProgrammi di ricerca
di Rilevante Interesse Nazionale (PRIN).
Chronic Constriction Injury. A peripheral mononeuropathy was
produced in adult rats by placing loosely constrictive ligatures around
the common sciatic nerve according to the method described by
Bennett.17 Rats were anaesthetised with chloral hydrate. The common
sciatic nerve was exposed at the level of the middle of the thigh by blunt
dissection through biceps femoris. Proximal to sciatica’s trifurcation,
about 1 cm of the nerve was freed of adhering tissue and four ligatures
(3/0 silk tread) were tied loosely around it with about 1 mm spacing.
The length of the nerve thus affected was 4ꢀ5 mm. Great care was taken
to tie the ligatures so that the diameter of the nerve was seen to be just
barely constricted when viewed with 40ꢁ magnification. In every
animal, an identical dissection was performed on the opposite side
except that the sciatic nerve was not ligated. The left paw was untouched.
Paw Pressure Test. The nociceptive threshold in the rat was deter-
mined with an analgesimeter (Ugo Basile, Varese, Italy) according to the
method described by Leighton.18 The instrument exerts a force, which is
applied at a constant rate (32 g per second) with a cone-shaped pusher
on the upper surface of the rat hind paw. The force is continuously
monitored by a pointer moving along a linear scale. The pain threshold is
given by the force, which induces the first struggling movements from
the rat. Pretested rats, which scored below 40 g or over 75 g during the
test before drug administration (25%) were rejected. An arbitrary cut off
value of 250 g was adopted.
Light/Dark Box Test. The apparatus (50 cm long, 20 cm wide, and
20 cm high) consisted of two equal acrylic compartments, one dark and
one light, illuminated by a 60 W bulb lamp and separated by a divider with a
10 cm ꢁ 3 cm opening at floor level. Each mouse was tested by placing it in
the center of the lighted area, away from the dark one, and allowing it to
explore the novel environment for 5 min. The time spent in the illuminated
side was measured. This test exploited the conflict between the animal’s
tendency to explore a new environment and its fear of bright light.19
Drugs. The new compounds and Emapunil were administered by the
po route and were suspended in 1% carboxymethylcellulose (CMC)
sodium salt and sonicated immediately before use. Drug concentrations
were prepared in such a way that the necessary dose could be administered
in a 10 mL/kg volume of carboxymethylcellulose 1% by the po route.
Statistical Analysis. All experimental result are given as the mean (
SEM. Each value represents the mean of 25 mice. An analysis of variance,
ANOVA, followed by Fisher’s protected least significant difference
procedure for post hoc comparison, was used to verify significance
between two means of behavioral results. The data were analyzed with
the StatView software for Macintosh (1992). P values of less than 0.05
were considered significant.
’ ABBREVIATIONS USED
TSPO, translocator protein (18 kDa); PBR, peripheral benzo-
diazepine receptor; CBR, central benzodiazepine receptor; VDAC,
voltage-dependent anion channel; ANT, adenine nucleotide
transporter; MPTP, mitochondrial permeability transition pore;
PET, positron emission tomography; GABA, γ-aminobutyric acid;
FRA, freely rotating aromatic ring; PAR, planar aromatic region;
PBS, phosphate-buffered saline; CMC, carboxymethylcellulose
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: +39 0577 234320. Fax: +39 0577 234333. E-mail:
cappelli@unisi.it.
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