Synthesis and structure-activity relationship studies in peripheral benzodiazepine receptor ligands related to alpidem

Article abstract of DOI:10.1016/j.bmc.2007.06.044

The exploration of the structure-affinity relationships concerning a new class of peripheral benzodiazepine receptor (PBR) ligands related to alpidem has been pursued in order to evaluate the consistency of the structure-affinity relationships among diffe

Full text of DOI:10.1016/j.bmc.2007.06.044

Bioorganic & Medicinal Chemistry 16 (2008) 3428–3437
Synthesis and structure–activity relationship studies
in peripheral benzodiazepine receptor ligands related to alpidem
Andrea Cappelli,a,* Germano Giuliani,a Salvatore Valenti,a Maurizio Anzini,a
Salvatore Vomero,a Gianluca Giorgi,b Cristiana Sogliano,c Elisabetta Maciocco,c
Giovanni Biggioc and Alessandra Concasc
aDipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug Discovery and Development,
`
Universita degli Studi di Siena, Via A. Moro, 53100 Siena, Italy
`
b
Dipartimento di Chimica, Universita degli Studi di Siena, Via A. Moro, 53100 Siena, Italy
`
c
Dipartimento di Biologia Sperimentale ‘‘B. Loddo’’, Universita degli Studi di Cagliari, Cittadella Universitaria,
SS 554 (km 4.500), 09042 Monserrato (Cagliari), Italy
Received 8 May 2007; revised 18 June 2007; accepted 22 June 2007
Available online 27 June 2007
Abstract—The exploration of the structure–affinity relationships concerning a new class of peripheral benzodiazepine receptor
(PBR) ligands related to alpidem has been pursued in order to evaluate the consistency of the structure–affinity relationships among
different classes (and subclasses) of PBR ligands. The target amide derivatives were prepared following a previously published pro-
cedure based on the condensation of pyrrolo[3,4-b]quinoline derivatives 11a,b with glyoxylic acid mono-hydrate and the subsequent
amidation of the acids obtained via mixed anhydride. On the other hand, the preparation of compound 9g lacking the pharmaco-
phoric (d1) carbonyl group involved: (a) the double sequential attack of the dimethylmethyleneammonium salt obtained from
bis(dimethylamino)methane and acetyl chloride to pyrrolo[3,4-b]quinoline derivative 11b, (b) the quaternization of the obtained
allylamine derivative 13 with methyl iodide, and (c) the palladium-catalyzed allylation of N-methyl-p-anisidine by quaternary ally-
lammonium cation 14. The structure–affinity relationship trends observed in this subclass of tricyclic alpidem-related PBR ligands
find correlations in other classes (or subclasses) of PBR ligands. This result supports the initial pharmacophoric hypothesis and sug-
gests a common mode of interaction at the PBR binding site.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Tissue distribution analysis revealed an ubiquitous
expression of PBR with particularly high densities in ste-
roidogenic tissues such as adrenal gland, but also in kid-
ney, heart, testis, and at a lower level in the brain
parenchyma, ependyma, choroid plexus, and olfactory
neurons. Furthermore, PBR is overexpressed in a vari-
ety of tumors and the expression appears to be related
to the tumor malignancy degree.2 Augmented concen-
trations of PBR were observed in lesioned brain areas
in a variety of neuropathologies such as multiple sclero-
sis, Alzheimer’s disease, and Huntington’s disease.3
The peripheral benzodiazepine receptor (PBR), so called
because it was originally discovered in the periphery by
means of tritium-labeled diazepam,1 is a 169-amino acid
protein (18 kDa) with five trans-membrane domains
localized on the mitochondrial outer membrane,
although nonmitochondrial (nuclear or microsomal)
localizations in some cells have been suggested. Photola-
beling studies indicated that this receptor is functionally
linked to the voltage-dependent anion channel (VDAC)
and to the adenine nucleotide translocase (ANT) and
might be implicated in the regulation of the opening of
the mitochondrial permeability transition pore (MPTP).
The exact physiological function of PBR is not yet fully
understood, but a wide range of pharmacological activi-
ties, such as anticonvulsant, anxiolytic, immunomodulat-
ing, and cardiovascular, has been related to its
activation.4 In particular, this receptor appears to be
involved in steroidogenesis, the regulation of which repre-
sents a potential clinical application of PBR ligands.5
Keywords: PBR; Peripheral benzodiazepine receptor; Palladium-cata-
lyzed allylation; X-ray crystallography.
*
Corresponding author. Tel.: +39 0577 234320; fax: +39 0577
234333; e-mail: cappelli@unisi.it
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.044

A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 3428–3437
3429
The isoquinolinecarboxamide PK11195 (1, Fig. 1) is the
most widely used pharmacological tool for the study of
the expression and the function of PBR. Compound 1
labeled with positron emitter carbon-11 was used in the
imagingofbraintumors, multiplesclerosis, cerebralinfarc-
tion, and abnormalities of calcium channels in heart dis-
eases by means of positron emission tomography (PET).6
The design and synthesis of PBR ligands led to develop-
ment of potent compounds both in the class of alpidem-
related substances (8)12 and in the class of PK11195
derivatives13 as well as to the development of theoretical
models for the interaction of PBR with the best known
ligands.14 Some 2-quinolinecarboxamide derivatives
related to PK1195 were 11C-labeled and their prelimin-
ary evaluation suggested that these compounds are
promising PET tracers15 for the in vivo PET monitoring
of neurodegenerative processes.16 Moreover, the struc-
ture–affinity relationship (SAFIR) data obtained with
PK11195-related compounds were used in the design
of both the first PBR ligand bearing a carborane cage
potentially useful in boron neutron capture therapy
(BNCT)17 and one of the most potent PET radiotracers
for the in vivo imaging of the PBR so far described.18
The imidazopyridine alpidem binds with nanomolar
affinity both CBR and PBR and is capable of stimulat-
ing pregnenolone formation from the mitochondria of
C6-2B glioma cells.7
In recent years, a number of alpidem-related PBR ligands
have been described. Some examples include indol-3-
acetamides 2,7 benzofuran-3-acetamides 3,7 imidazo[1,2-
a]pyridineacetamides 4,8 pyrazolo[1,5-a]pyrimidineace-
tamides 5,9 and indol-3-glyoxylamides 6.10 Another
PBR ligand which may be included in the alpidem-related
compounds is pyridazino[4,5-b]indole-1-acetamide
SSR180575 (7). This compound shows high affinity and
selectivity for the rat and human PBR, and increased
pregnenolone accumulation in the brain and sciatic nerve
suggests that its neuroprotective effects could be mediated
via PBR stimulation of neurosteroid biosynthesis.11
The structural analysis of PBR ligands 1–8 suggests that
they may share the common four component pharmaco-
phore (d1, LA, PAR, FRA, see Ref. 12) in which a single
H-bonding acceptor moiety (d1, usually a suitably ori-
ented amide carbonyl group) plays a key role in the
interaction with PBR. However, alpidem and com-
pounds 1–6 show in their structure a bicyclic planar aro-
matic region (PAR), which is instead tricyclic in
SSR18057511 and in compounds 8.12
O
Within the large program focused on the medicinal
chemistry of PBR ligands, compounds 8 were subjected
to structural modification in the three of the four postu-
lated pharmacophoric groups (d1, LA, and FRA) to
give compounds 9 in order to evaluate the consistency
of the structure–affinity relationship trends among dif-
ferent classes (and subclasses) of PBR ligands (Fig. 2).
N
N
Cl
N
N
CH3
Cl
Cl
O
alpidem
N
PK11195 (1)
In this paper, we describe the synthesis, the preliminary
pharmacological evaluation, and the structure–affinity
relationship analysis of a short series of PBR ligands 9
based on the tricyclic system pyrrolo[3,4-b]quinoline.
R4
N
X
R5
R4
N
R3
R3
4
O
2 (X = NH)
3 (X = O)
O
2. Results
N
R2
R1
N
R2
R1
2.1. Chemistry
R5
The previously described target amide derivatives 8a–e
were re-prepared following the reported procedure
based on the condensation of pyrrolo[3,4-b]quinoline
derivative 11b with glyoxylic acid mono-hydrate and
H
N
R4
R3
N
N
R6
R4
N
O
R3
O
5
O
6
N
R2
N
R1
R1
R2
PAR
N
O
FRA
R3
CH3
N
O
O
N
Cl
H
9
N
Cl
X
N
N
δ1
N
N
R1
R2
LA
O
H
O
8
SSR180575 (7)
CH3
N
N
R2
H3C
R1
Figure 2. PBR ligands 9. The pharmacophoric groups subjected to
modification are highlighted.
Figure 1. Structures of some PBR ligands.

3430
A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 3428–3437
the subsequent transformation of the ylideneacetic acid
12b via mixed anhydride (Scheme 1).12
reaction mixture obtained in the absence of N-methyl-
p-anisidine and with increased amounts (with respect
to the catalytic ones) of palladium acetate and triphenyl-
phosphine (Scheme 3).
The newly designed compounds 9a–f were synthesized
by means of the same chemistry as shown in Scheme
1. Moreover, tertiary amide 8a was obtained by N-meth-
ylation of the corresponding secondary amide 9b with
methyl iodide in the presence of sodium hydride as the
base. This result demonstrates the feasibility of the label-
ing of 8a with [11C]CH3I.
The structure of intermediate allylpalladium complex 15
was characterized by crystallography, which demon-
strated the existence of two conformational polymorphs
of this compound (Fig. 4).
2.2. Binding studies
The preparation of compound 9g lacking the postulated
pharmacophoric (d1) carbonyl group was based on the
results of the studies performed in our laboratories on
the chemistry of the pyrrolo[3,4-b]quinoline system. In
fact, the access to target 9g was envisioned when it
was found that the dimethylmethyleneammonium salt19
obtained from bis(dimethylamino)methane and acetyl
chloride performed a double sequential attack (with
intermediate elimination) to pyrrolo[3,4-b]quinoline
derivative 11b to give compound 13 (Scheme 2). Thus,
dimethylamine derivative 13 was quaternized with
methyl iodide to obtain quaternary allylammonium cat-
ion 14. This compound was demonstrated to behave as
an allylating agent under Tsuji-Trost conditions and
reacted with N-methyl-p-anisidine to give allylamine
derivative 9g, the structure of which was confirmed by
crystallography (Fig. 3).
Compounds 8a–e and 9a–g were tested for their poten-
tial activity in inhibiting the specific binding of [3H]1
to rat cortical membrane in comparison with reference
compound 1 and the results of the binding studies are
shown in Table 1. Almost all the compounds tested
show PBR affinities in the nanomolar range with a mod-
ulation suitable for structure affinity relationship analy-
sis. Amides 8a,b show IC50 values very similar to those
previously reported, while benzyl derivatives 8c,d and
dipropylamide 8e are significantly more potent than for-
merly reported.12 We have no specific explanation for
this discrepancy, but the final results fit better with the
known SAFIR trends.9,13,17a
Comparison of the results obtained with compounds 8a
and 9b confirms the previously observed difference in
affinity between secondary and tertiary amides.13 In fact,
secondary amide 9b shows a significantly lower PBR affin-
ity when compared to its N-methylated counterpart 8a. It
In the setting-up of the palladium-catalyzed allylation
reaction, compounds 15 and 16 were isolated from the
O
COOC2H5
i
N
R3
N
CH2Cl
N
11a,b
10
ii
O
N
O
iii
R3
N
R3
N
H
N
H
O
H
O
O
12a,b
8a-e, 9a-f
N
R1
R2
O
N
O
iv
Cl
N
Cl
N
H
N
H
O
H
O
8a
9b
N
N
H3C
OCH3
H3CO
Scheme 1. Reagents: (i) R3C6H4NH2, C2H5OH; (ii) CHOCOOHÆH2O, (CH3CO)2O, CH3COOH; (iii) i-C4H9OCOCl, TEA, R1NHR2, CH2Cl2; (iv)
CH3I, NaH, DMF.

A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 3428–3437
3431
O
O
i
N
Cl
N
Cl
N
N
H
13
11b
N
ii
CH3
H3C
O
O
iii
N
Cl
N
Cl
N
N
H
CH3
H
14
9g
I
N
N
CH3
H3C
H3C
OCH3
Scheme 2. Reagents: (i) (CH3)2HCH2N(CH3)2, CH3COCl, K2CO3, DMF; (ii) CH3I, C2H5OC2H5, C2H5OH; (iii) CH3NH–C6H4OCH3, PPh3,
Pd(CH3COO)2, DMF.
is noteworthy that secondary amide 9b shows the same
IC50 value as its positional isomer 9c, which is a tertiary
amide with a hydrophilic substituent on the amide phenyl
group. The comparison of the most potent compounds
8a,b with 9a,c demonstrates the importance of a lipophilic
substituent in para-position of the amide phenyl.
The replacement of the amide phenyl group of compound
9a with the benzyl of 8c appears to be well tolerated by
PBR, whereas the same substitution with a propargyl
moiety is not accepted equally well (compare 9d vs 9a).
The removal of the lipophilic chlorine atom in the pen-
dant phenyl ring of the most active compounds 8a,b
leads to a decrease in PBR affinity of about one order
of magnitude (compounds 9e,f).
The transformation of the amide carbonyl of 8a into the
Figure 3. Crystallographic structure of 9g. Ellipsoids enclose 50%
methylene group of 9g produces a dramatic decrease
probability.
(2600 times) in the receptor affinity. This result can be
N(CH3)3
O
O
N
Cl
N
Cl
N
Pd(PPh3)n
N
(II)
Pd
H
H
CH3
Ph3P
14
15
I
I
N
H
H
CH3
H3C
CH3COO-
O
N
Cl
N
H
16
O
O
CH3
Scheme 3. Formation of the intermediate allylpalladium complex 15 and reaction with acetate anion to give 16.

3432
A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 3428–3437
Table 1. PBR binding affinities of compounds 8a–e and 9a–g
O
N
R3
N
H
X
8,9
N
R1
R2
Compound R1
R2
R3
X
IC50
(nM) SEMa
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
9f
4-(CH3O)C6H4 CH3
Cl @O 0.90 0.08
Cl @O 0.62 0.05
4-ClC6H4
CH3
CH3
CH2C6H5
CH2C6H5
(CH2)5CH3
C6H5
Cl @O
Cl @O
26 3.1
15 1.3
21 2.1
44 3.9
76 8.6
74 8.0
C2H5
(CH2)5CH3 Cl @O
CH3
H
Cl @O
Cl @O
Cl @O
4-(CH3O)C6H4
4-(OH)C6H4
CH2C„CH
CH3
CH3
Cl @O 320 29
4-(CH3O)C6H4 CH3
4-ClC6H4 CH3
4-(CH3O)C6H4 CH3
H
@O
@O
12 1.4
10 1.2
H
9g
1
Cl @H2 2360 274
2.1 0.2
a Each value is the mean SEM of three determinations and represents
the concentration giving half the maximum inhibition of [3H]1 (final
concentration 1 nM) specific binding to rat cortical membranes.
Figure 4. Crystallographic structures of the two polymorphic forms of
allylpalladium complex 15 ethyl acetate solvate. Ellipsoids enclose 50%
probability. Top: polymorph 1; 15Æ0.5 EtOAc; symmetry op. $1 = 3-x,-
y,-z; EtOAc moiety: spheres have arbitrary dimensions and hydrogen
atoms are omitted for clarity. Bottom: polymorph 2; 15ÆEtOAc.
considered the first direct evidence of the key role played
by the amide carbonyl in the interaction of these tricyclic
compounds with PBR.
Figure 5. Superposition of 8a crystallographic structure with a low
energy conformer of 9g. This result suggests that 9g is able to populate
the solid state conformation of 8a, which is a very rigid and highly
potent PBR ligand. Thus, the difference in PBR affinity between 8a and
9g is related to the key role played by the amide carbonyl in the
interaction with PBR.
In conclusion, the structure–affinity relationship trends
observed in this subclass of tricyclic alpidem-related
PBR ligandsfind correlationsin other classes (or subclass-
es) of PBR ligands (see Refs. 8–10,13,14,17a). This result
supports the initial pharmacophoric hypothesis (Fig. 5)
and suggests a common mode of interaction at PBR bind-
ing site.20
ried out by means of a Perkin-Elmer Series II CHNS/O
Analyzer 2400. Merck silica gel 60 (230–400 mesh) was
used for column chromatography. Merck TLC plates,
3. Experimental
3.1. Chemistry
1
silica gel 60 F254 were used for TLC. H NMR spectra
were recorded with a Bruker AC 200 spectrometer in
the indicated solvents (TMS as internal standard); the
values of the chemical shifts are expressed in ppm and
the coupling constants (J) in Hz. Mass spectra were
recorded on either a Varian Saturn 3 spectrometer or
a ThermoFinnigan LCQ-Deca.
All chemicals used were of reagent grade. Yields refer to
purified products and are not optimized. Melting points
were determined in open capillaries on a Gallenkamp
apparatus and are uncorrected. Microanalyses were car-

A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 3428–3437
3433
3.2. General procedure for the synthesis of compounds
11a,b
8.19 (d, J = 8.4, 1H), 8.32 (d, J = 8.5, 1H), 8.96 (s,
1H), 16.13 (s, 1H).
A mixture of 10 (2.2 g, 8.8 mmol) in 15 mL ofethanol with
aniline (or 4-chloroaniline in the case of 11b) (27 mmol)
was refluxed for 52 h. The reaction mixture was cooled
to room temperature, and the precipitate was collected
by filtration, washed with ethanol and petroleum ether,
and dried under reduced pressure to obtain 11a,b.
3.8. General procedure for the synthesis of compounds 9a–f
A mixture of the appropriate carboxylic acid (12a,b,
0.40 mmol) in dichloromethane (10 mL) and TEA
(0.19 mL, 1.4 mmol) was cooled at ꢀ10 °C, and isobutyl
chloroformate (0.057 mL, 0.44 mmol) was added with
stirring under argon for 30 min. Subsequently, the suit-
able amine (0.44 mmol) was added to the solution and
the reaction mixture was allowed to reach room temper-
ature and stirred for 1 h (for compound 9d the reaction
time was 17 h). The reaction mixture was then diluted
with dichloromethane, washed with brine, dried over so-
dium sulfate, and concentrated under reduced pressure.
Crude amides 9a–f were purified by flash-chromatogra-
phy with the appropriate eluent.
3.3. 2,3-Dihydro-2-phenyl-1H-pyrrolo[3,4-b]quinolin-1-
one (11a)
The title compound was obtained as white crystals (yield
1
72%, mp 240–241 °C). H NMR (CDCl3): 5.02 (s, 2H),
7.21 (t, J = 7.3, 1H), 7.41–7.49 (m, 2H), 7.62 (t, J = 7.0,
1H), 7.80–7.92 (m, 3H), 8.00 (d, J = 8.1, 1H), 8.16 (d,
J = 8.5, 1H), 8.67 (s, 1H). MS(ESI): m/z 261 (M+H+).
3.4. 2-(4-Chlorophenyl)-2,3-dihydro-1H-pyrrolo[3,4-b]-
quinolin-1-one (11b)
3.9. (E)-2-[2-(4-Chlorophenyl)-1,2-dihydro-1-oxo-3H-
pyrrolo[3,4-b]quinolin-3-ylidene]-N- methyl-N-phenylace-
tamide (9a)
The title compound was obtained as white crystals (yield
85%, mp 268–270 °C, lit.12 268–270 °C). 1H NMR
(CDCl3): 5.03 (s, 2H), 7.41–7.46 (m, 2H), 7.66 (t,
J = 7.3, 1H), 7.83–7.92 (m, 3H), 8.04 (d, J = 8.3, 1H),
8.19 (d, J = 8.5, 1H), 8.71 (s, 1H).
The title compound was purified by flash-chromatogra-
phy with dichloromethane/ethyl acetate (95:5) as the elu-
ent to give 9a as a white crystalline solid (yield 64%, mp
138–140 °C). H NMR (CDCl3): 3.59 (s, 3H), 5.58 (s,
1
1H), 6.98 (m, 2H), 7.10–7.17 (m, 5H), 7.42 (m, 2H),
7.68 (t, J = 7.6, 1H), 7.87–8.03 (m, 2H), 8.33 (d,
J = 8.4, 1H), 8.62 (s, 1H). MS(ESI): m/z 440 (M+H+).
Anal. Calcd for (C26H18ClN3O2Æ0.25 CH3COOC2H5):
C, 70.20; H, 4.36; N, 9.10. Found: C, 70.45; H, 4.32;
N, 8.93.
3.5. General procedure for the synthesis of compounds 12a,b
A mixture of the appropriate pyrroloquinoline deriva-
tive 11a,b (1.0 mmol) in glacial acetic acid (5 mL) and
acetic anhydride (5 mL) with glyoxylic acid monohy-
drate (0.30 g, 3.26 mmol) was heated at 110 °C for 5 h.
Then 0.3 g of glyoxylic acid monohydrate was added,
and the mixture was stirred at 110 °C for one additional
hour. Afterwards, the cooled reaction mixture was
poured into ice-water and allowed to stand at room
temperature overnight. The precipitate was extracted
with chloroform and the organic layer was extracted
with diluted NaOH. The organic layer was discarded,
the aqueous phase was acidified (pH 4–5) with 3 N
HCl and extracted with chloroform. The combined ex-
tracts were dried over sodium sulfate and concentrated
under reduced pressure to give the corresponding acids
12a,b.
3.10. (E)-2-[2-(4-Chlorophenyl)-1,2-dihydro-1-oxo-3H-
pyrrolo[3,4-b]quinolin-3-ylidene]-N-(4-methoxyphenyl)-
acetamide (9b)
The title compound was purified by flash-chromatogra-
phy with dichloromethane/ethyl acetate (9:1) as the elu-
ent to give 9b as a yellow solid (yield 90%). An analytical
sample recrystallized from n-hexane–ethyl acetate
1
melted at 272–274 °C. H NMR (CDCl3): 3.84 (s, 3H),
5.88 (s, 1H), 6.99 (m, 2H), 7.32 (m, 2H), 7.56 (m, 2H),
7.73–7.84 (m, 3H), 8.02 (t, J = 7.7, 1H), 8.15 (d,
J = 8.0, 1H), 8.31 (d, J = 8.3, 1H), 8.90 (s, 1H), 13.45
(s, 1H). MS(ESI, negative ions): m/z 454 (MꢀH+). Anal.
Calcd for (C26H18ClN3O3Æ0.25 CH3COOC2H5): C,
67.85; H, 4.22; N, 8.79. Found: C, 68.19; H, 3.90; N,
8.78.
3.6. (E)-(1,2-Dihydro-1-oxo-2-phenyl-3H-pyrrolo[3,4- b]-
quinolin-3-ylidene)acetic acid (12a)
The title compound was obtained as a light-brown solid
(yield 50%, mp 298–300 °C). 1H NMR (CDCl3): 5.89 (s,
1H), 7.33–7.63 (m, 5H), 7.84 (t, J = 7.3, 1H), 8.04 (m,
1H), 8.17 (d, J = 7.9, 1H), 8.30 (d, J = 8.4, 1H), 8.95
(s, 1H), 16.22 (br s, 1H). MS(ESI): m/z 317 (M+H+).
3.11. (E)-2-[2-(4-Chlorophenyl)-1,2-dihydro-1-oxo-3H-
pyrrolo[3,4-b]quinolin-3-ylidene]-N-(4-hydroxyphenyl)-N-
methylacetamide (9c)
The title compound was purified by flash-chromatogra-
phy with chloroform/ethyl acetate (6:4) as the eluent to
give 9c as a white solid (yield 60%). An analytical sample
recrystallized from dichloromethane melted at 205–
3.7. (E)-[2-(4-Chlorophenyl)-1,2-dihydro-1-oxo-3H-pyr-
rolo[3,4-b]quinolin-3-ylidene]acetic acid (12b)
1
The title compound was obtained as an off-white solid
(yield 55%, mp 271–272 °C, lit.12 272–273 °C). 1H
NMR (CDCl3): 5.89 (s, 1H), 7.27–7.35 (m, 2H), 7.54–
7.61 (m, 2H), 7.86 (t, J = 8.0, 1H), 8.03–8.11 (m, 1H),
207 °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. H NMR (CDCl3): 3.36 (s), 3.48
1

3434
A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 3428–3437
(s), 5.51 (s), 5.88 (s), 6.56 (m), 6.81–6.94 (m), 7.32–7.41
(m), 7.52 (m), 7.65 (t, J = 7.4), 7.78–7.90 (m), 7.98 (d,
J = 8.1), 8.15 (d, J = 8.1), 8.27 (d, J = 8.4), 8.60 (s),
8.71 (s). MS(ESI, negative ions): m/z 454 (MꢀH+).
Anal. Calcd for (C26H18ClN3O3): C, 68.50; H, 3.98; N,
9.22. Found: C, 68.33; H, 3.85; N, 9.10.
3.15. (E)-2-[2-(4-Chlorophenyl)-1,2-dihydro-1-oxo-3H-
pyrrolo[3,4-b]quinolin-3-ylidene]-N-(4-methoxyphenyl)-
N-methylacetamide (8a)
To a solution of 9b (0.030 g, 0.066 mmol) in 3 mL of dry
DMF and methyl iodide (0.50 mL, 8.0 mmol) cooled at
0–5 °C was added NaH (1.6 mg, 0.067 mmol). The reac-
tion mixture was stirred at room temperature for 30 min
and then poured into ice-water. The precipitate was ex-
tracted with dichloromethane and the organic layer was
dried over sodium sulfate and concentrated under re-
duced pressure. Purification of the residue by flash-chro-
matography with dichloromethane/ethyl acetate (95:5)
as the eluent gave 8a as a pale yellow oil which crystal-
lized on standing (0.022 g, yield 71%). Recrystallization
from diethyl ether gave an analytical sample of 8a mp at
3.12. (E)-2-[2-(4-Chlorophenyl)-1,2-dihydro-1-oxo-3H-
pyrrolo[3,4-b]quinolin-3-ylidene)-N-methyl-N-(prop-2-
ynyl)acetamide (9d)
Crude amide 9d was purified by flash-chromatography
with chloroform/ethyl acetate (6:4) as the eluent to ob-
1
tain a colorless glassy solid (yield 54%). The H NMR
spectrum of this amide shows the presence of two differ-
ent rotamers in equilibrium. For the sake of simplifica-
tion the integral intensities have not been given. 1H
NMR (CDCl3): 2.12 (t, J = 2.3), 2.32 (t, J = 2.5), 3.11
(s), 3.27 (s), 4.19 (d, J = 2.4), 4.53 (d, J = 2.4), 5.71 (s),
5.73 (s), 7.36–7.66 (m), 7.82 (m), 7.98 (d, J = 8.1), 8.10
(d, J = 8.5), 8.21 (d, J = 8.6), 8.66 (s). MS(ESI): m/z
402 (M+H+). Anal. Calcd for (C23H16ClN3O2Æ0.2
CHCl3): C, 65.45; H, 3.84; N, 9.87. Found: C, 65.28;
H, 3.91; N, 9.87.
1
158–160 °C (lit.12 159–160 °C). The H NMR spectrum
of this amide shows the presence of two different rota-
mers in equilibrium. For the sake of simplification the
integral intensities have not been given. 1H NMR
(CDCl3): 3.41 (s), 3.55 (s), 3.72 (s), 3.88 (s), 5.60 (s),
5.92 (s), 6.64–6.69 (m), 6.99–7.04 (m), 7.39–7.47 (m),
7.55–7.72 (m), 7.88–8.04 (m), 8.22–8.35 (m), 8.64 (s),
8.74 (s).
3.13. (E)-2-(1,2-Dihydro-1-oxo-2-phenyl-3H-pyrrolo[3,4-
b]quinolin-3-ylidene)-N-(4-methoxyphenyl)-N-methylace-
tamide (9e)
3.16. (E)-2-(4-Chlorophenyl)-2,3-dihydro-3-[2-(dimeth-
ylamino)ethylidene]-1H-pyrrolo[3,4-b]quinolin-1-one (13)
To a solution of bis(dimethylamino)methane (0.51 mL,
3.7 mmol) in 5 mL of dry DMF cooled at ꢀ5 °C acetyl
chloride (0.46 mL, 6.5 mmol) was slowly added and
the resulting mixture was stirred at room temperature
under argon for 10 min. The reaction mixture was added
with potassium carbonate (1.2 g, 8.8 mmol) and of pyr-
roloquinoline derivative 11a (0.50 g, 1.7 mmol), and the
resulting mixture was heated at 60 °C for 2.5 h and
poured into ice-water. The precipitate was extracted
with dichloromethane and the organic layer was dried
over sodium sulfate and concentrated under reduced
pressure. Purification of the residue by flash-chromatog-
raphy with ethyl acetate/triethylamine (8:2) as eluent
gave 13 as a pale yellow oil (0.51 g, yield 82%). 1H
NMR (CDCl3): 2.36 (s, 6 H), 4.14 (d, J = 7.0, 2H),
5.57 (t, J = 7.4, 1H), 7.33 (m, 2H), 7.51 (m, 2H), 7.64
(t, J = 7.3, 1H), 7.85 (m, 1H), 8.01 (d, J = 9.0, 1H),
8.25 (d, J = 8.5, 1H), 8.71 (s, 1H).
Crude amide 9e was purified by flash-chromatography
with dichloromethane/ethyl acetate (9:1) as the eluent
to obtain a pale-yellow solid (yield 63%). An analyt-
ical sample recrystallized from petroleum ether–ethyl
1
acetate melted at 193–195 °C. The H NMR spectrum
of this amide shows the presence of two different
rotamers in equilibrium. For the sake of simplifica-
tion the integral intensities have not been given. 1H
NMR (CDCl3): 3.41 (s), 3.54 (s), 3.71 (s), 3.87 (s),
5.60 (s), 5.92 (s), 6.66 (m), 6.98–7.09 (m), 7.39–7.71
(m), 7.90 (t, J = 7.9), 8.01 (d, J = 7.8), 8.25 (d,
J = 8.4), 8.32 (d, J = 8.4), 8.63 (s), 8.75 (s). MS(ESI):
m/z 436 (M+H+). Anal. Calcd for (C27H21N3O3): C,
74.47; H, 4.86; N, 9.65. Found: C, 74.42; H, 4.55;
N, 9.52.
3.14. (E)-N-(4-Chlorophenyl)-2-(1,2-dihydro-1-oxo-2-
phenyl-3H-pyrrolo[3,4-b]quinolin-3-ylidene)-N-methylac-
etamide (9f)
3.17. (E)-2-(4-Chlorophenyl)-2,3-dihydro-3-[2-(trimethy-
lammonium)ethylidene]-1H-pyrrolo[3,4-b]quinolin-1-one
iodide (14)
Crude amide 9f was purified by flash-chromatography
with dichloromethane/ethyl acetate (9:1) as the eluent
to obtain a colorless crystalline solid (yield 50%). An
analytical sample recrystallized from petroleum ether–
To a solution of 13 (0.18 g, 0.49 mmol) in 5 mL of abso-
lute ethanol and 5 mL of dry diethyl ether was added
methyl iodide (0.034 mL, 0.55 mmol) and the resulting
mixture was stirred at room temperature for 48 h. The
precipitate was collected by filtration, washed with
diethyl ether, and dried under reduced pressure to give
14 as a white solid (0.175 g, yield 70%, mp 219–
1
ethyl acetate melted at 191–193 °C. The H NMR spec-
trum of this amide shows the presence of two different
rotamers in equilibrium. For the sake of simplification
the integral intensities have not been given. H NMR
1
(CDCl3): 3.41 (s), 3.56 (s), 5.58 (s), 5.88 (s), 7.00–7.12
(m), 7.33–7.70 (m), 7.89 (t, J = 7.5), 8.00 (d, J = 8.0),
8.15 (d, J = 7.9), 8.28 (d, J = 8.3), 8.62 (s), 8.72 (s).
MS(ESI): m/z 440 (M+H+). Anal. Calcd for
(C26H18ClN3O2): C, 70.99; H, 4.12; N, 9.55. Found: C,
71.05; H, 3.92; N, 9.50.
1
222 °C). H NMR (DMSO-d6): 3.14 (s, 9H), 5.08 (d,
J = 8.7, 2H), 5.36 (t, J = 8.7, 1H), 7.63 (m, 4H), 7.80
(t, J = 7.5, 1H), 8.01 (t, J = 7.4, 1H), 8.29 (d, J = 8.1,
1H), 8.43 (d, J = 8.4, 1H), 9.06 (s, 1H). MS(ESI): m/z
378 (M+).

A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 3428–3437
3435
3.18. (E)-2-(4-Chlorophenyl)-2,3-dihydro-3-[2-[N-(4-
methoxyphenyl)methylamino]ethylidene]-1H-pyrrolo[3,4-
b]quinolin-1-one (9g)
collection on a Siemens P4 four-circle diffractometer
with graphite monochromated MoKa radiation
(k = 0.71069 A). The x/2H scan technique was used.
˚
The structures were solved by direct methods and the
refinements were carried out by full-matrix anisotropic
least-squares of F2 against all reflections. The hydrogen
atoms were located on Fourier difference maps or placed
in calculated positions and refined as riding atoms on
their heavy atoms with isotropic temperature factor.
Atomic scattering factors including f0 and f00 were taken
from Ref. 21. Structure solution was carried out by
SHELXS-97.22 Structure refinement and molecular
graphics were performed by SHELX-9721 and the
WinGX package,23 respectively.
A mixture of 14 (0.090 g, 0.18 mmol) in 9 mL of dry
DMF with triphenylphosphine (0.023 g, 0.088 mmol)
was stirred at room temperature under a stream of ar-
gon for 10 min, and then treated with palladium acetate
(0.010 g, 0.044 mmol) and N-methyl-p-anisidine
(0.024 g, 0.175 mmol). The reaction mixture was stirred
at room temperature for 20 h, poured into ice-water,
and extracted with diethyl ether. The organic layer was
washed with water, dried over sodium sulfate, and
concentrated under reduced pressure. Purification of
the residue by flash-chromatography with dichlorometh-
ane/ethyl acetate (95:5) as the eluent gave pure 9g as
a pale yellow oil which crystallized on standing. Recrys-
tallization from diethyl ether–dichloromethane gave
yellow prisms suitable for crystallographic studies
(0.060 g, yield 73%, mp 160–163 °C). 1H NMR (CDCl3):
2.93 (s, 3 H), 3.72 (s, 3H), 5.10 (d, J = 6.9, 2H), 5.54 (t,
J = 6.9, 1H), 6.81 (m, 4H), 7.27 (m, 2H), 7.46 (m, 2H),
7.67 (t, J = 7.5, 1H), 7.89 (t, J = 7.7, 1H), 8.04 (d,
J = 8.0, 1H), 8.28 (d, J = 8.5, 1H), 8.74 (s, 1H). MS(E-
SI): m/z 456 (M+H+). Anal. Calcd for (C27H22ClN3O2):
C, 71.13; H, 4.86; N, 9.22. Found: C, 70.89; H, 4.78; N,
9.37.
CCDC 646033 (9g), 644752 (15-Polymorph 1), and
644753 (15-Polymorph 2) contain the supplementary
crystallographic data for this paper. These data can be
obtained free of charge via www.ccdc.cam.ac.uk/conts/
retrieving.html (or from the Cambridge Crystallo-
graphic Data Centre, 12 Union Road, Cambridge CB2
1EZ, UK; fax: +44 1223 336 033; or e-mail:
deposit@ccdc.cam.ac.uk).
3.22. In vitro binding assays
Male Sprague–Dawley CD rats (Charles River Italia,
Calco, CO, Italy) with body masses of 200–250 g were
used. Rats were acclimatized to the new housing condi-
tions for at least 1 week. They were housed six per cage
under an artificial 12-h-light, 12-h-dark cycle at a con-
stant temperature of 22 2 °C and a relative humidity
of 65%. They had free access to water and standard lab-
oratory food at all times. Animal care and handling
throughout the experimental procedures were in accor-
dance with the European Communities Council Direc-
tive of 24 November 1986 (86/609/EEC).
3.19. Palladium complex (15)
To a solution of 14 (0.075 g, 0.15 mmol) in 8 mL of
anhydrous DMF with triphenylphosphine (0.040 g,
0.15 mmol) was added palladium acetate (0.017 g,
0.075 mmol) and the resulting mixture was stirred at
room temperature for 20 h. The reaction mixture was
then poured into ice-water and extracted with dichloro-
methane; the organic layer was dried over sodium sul-
fate and concentrated under reduced pressure. The
residue obtained was purified by flash-chromatography
with dichloromethane/ethyl acetate (95:5) as eluent to
give 0.020 g of 15 as a yellow solid which crystallized
from diethyl ether–dichloromethane–ethyl acetate in
two different polymorphic forms ethyl acetate solvate.
1H NMR (CDCl3): 3.41 (d, J = 8.5, 1H), 5.02 (d,
J = 12.8, 1H), 5.39 (dd, J = 8.9, 12.6, 1H), 7.26–7.69
(m, 18H), 7.81–7.90 (m, 3H), 8.01–8.13 (m, 2H), 8.80
(s, 1H).
Rats were sacrificed by decapitation and their brains
were rapidly dissected into the various areas which were
stored at ꢀ80 °C until the day of the assay. The binding
assays were performed as described in the lit.8 The cere-
bral cortex was subsequently thawed and then homoge-
nized in 50 volumes of ice-cold Dulbecco’s phosphate-
buffered saline (PBS), pH 7.4, at 4 °C with a Polytron
PT 10 disrupter (setting 5 for 20 s). The homogenate
was centrifuged at 40,000g and 4 °C for 30 min and
the resulting pellet was resuspended in the same volume
of fresh buffer and recentrifuged. The new pellet was
resuspended in 10 volumes of the incubation buffer
(PBS) and used for the binding assay.
3.20. (E)-2-[2-(4-Chlorophenyl)-1,2-dihydro-1-oxo-3H-
pyrrolo[3,4-b]quinolin-3-ylidene]ethyl acetate (16)
Compound 16 was obtained as a white solid (0.010 g, mp
171–173 °C) from the purification of the reaction mixture
of 15 by flash-chromatography (more polar fractions). 1H
NMR (CDCl3): 2.07 (s, 3H), 5.50 (t, J = 6.9, 1H), 5.80 (d,
J = 6.9, 2H), 7.34 (m, 2H), 7.49–7.68 (m, 3H), 7.85 (m,
1H), 8.00 (d, J = 8.3, 1H), 8.25 (d, J = 8.5, 1H), 8.68 (s,
1H). MS(ESI): m/z 401 (M+Na+).
[3H]PK11195 binding was measured in a final volume of
500 lL, consisting of 50 lL of membrane suspension
(0.15–0.20 mg of protein), 50 lL of [3H]PK11195 (sa
85.5 Ci/mmol, New England Nuclear; final assay con-
centration of 1 nM), 5 lL of drug solution or solvent,
395 lL of PBS. The binding reaction was performed at
25 °C for 90 min and began with the addition of mem-
branes. The incubation was terminated by rapid filtra-
tion through glass-fiber filters (Whatman GF/B) which
had been presoaked with 0.3% polyethyleneimine and
placed in a cell harvester filtration manifold (Brandel).
3.21. X-ray crystallography
Single crystals of 9g and the two polymorphic forms
ethyl acetate solvate of 15 were submitted to X-ray data

3436
A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 3428–3437
`
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Acknowledgments
The authors thank Prof. Stefania D’Agata D’Ottavi for
the careful reading of the manuscript, Dr. Roberto Be-
retta (Rottapharm, Monza, Italy) for the combustion
analyses, and INSTM (Consorzio Interuniversitario
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cially supported by MUR (Ministero dell’Universita e
della Ricerca)—PRIN (Programmi di ricerca di Rilevan-
te Interesse Nazionale).
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