Synthesis and biological evaluation of 4-phenylquinazoline-2-carboxamides designed as a novel class of potent ligands of the translocator protein

Article abstract of DOI:10.1021/jm201703k

A series of novel 4-phenylquinazoline-2-carboxamides (1-58) were designed as aza-isosters of PK11195, the well-known 18 kDa translocator protein (TSPO) reference ligand, and synthesized by means of a very simple and efficient procedure. A number of these derivatives bind to the TSPO with K_i values in the nanomolar/subnanomolar range, show selectivity toward the central benzodiazepine receptor (BzR) and exhibit structure-affinity relationships consistent with a previously published pharmacophore/topological model of ligand-TSPO interaction.

Full text of DOI:10.1021/jm201703k

Brief Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of 4-Phenylquinazoline-
2-carboxamides Designed as a Novel Class of Potent Ligands
of the Translocator Protein
Sabrina Castellano,† Sabrina Taliani,*,‡ Ciro Milite,† Isabella Pugliesi,‡ Eleonora Da Pozzo,§
Elisa Rizzetto,∥ Sara Bendinelli,§ Barbara Costa,§ Sandro Cosconati,⊥ Giovanni Greco,# Ettore Novellino,#
Gianluca Sbardella,† Giorgio Stefancich,∥ Claudia Martini,§ and Federico Da Settimo‡
†Dipartimento di Scienze Farmaceutiche e Biomediche, Universita
‡Dipartimento di Scienze Farmaceutiche, Universita
di Pisa, Via Bonanno 6, 56126 Pisa, Italy
§Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita
di Pisa, Via Bonanno 6, 56126 Pisa, Italy
∥Dipartimento di Scienze Chimiche e Farmaceutiche, Universita
di Trieste, Piazzale Europa, 1, 34127 Trieste, Italy
di Napoli, Via Vivaldi 43, 81100 Caserta, Italy
̀
di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy
̀
̀
̀
⊥Dipartimento di Scienze Ambientali, Seconda Universita
̀
#Dipartimento di Chimica Farmaceutica e Tossicologica, Universita
80131 Napoli, Italy
̀
di Napoli “Federico II”, Via D. Montesano 49,
S
* Supporting Information
ABSTRACT: A series of novel 4-phenylquinazoline-2-carboxamides (1−58) were designed as aza-isosters of PK11195, the well-
known 18 kDa translocator protein (TSPO) reference ligand, and synthesized by means of a very simple and efficient procedure.
A number of these derivatives bind to the TSPO with Ki values in the nanomolar/subnanomolar range, show selectivity toward
the central benzodiazepine receptor (BzR) and exhibit structure−affinity relationships consistent with a previously published
pharmacophore/topological model of ligand−TSPO interaction.
INTRODUCTION
■
The human 18 kDa translocator protein (TSPO) is a highly
hydrophobic protein mainly located at the contact sites
between the outer and the inner mitochondrial membranes.
TSPO is expressed in many peripheral tissues, including heart,
kidney, and lung, with higher expression levels in steroid
producing tissues. Low levels are expressed in brain, where it is
limited to glial cells (astrocytes and microglia).1
The TSPO is involved in a variety of important physiological
processes, including cholesterol transport, steroidogenesis,
calcium homeostasis, lipid metabolism, mitochondrial oxida-
tion, cell growth and differentiation, apoptosis induction, and
regulation of immune functions. This makes TSPO an intrigu-
ing target to develop novel potential therapeutic tools for the
treatment of a variety of diseases.1,2 In addition, the alteration
of its expression levels in a number of human pathologies
involving a perturbation of cell survival/death processes, such
as cancer (particularly solid colon, breast, and prostate cancers),
cerebral injury, including neurodegenerative disorders (Hun-
tington’s and Alzheimer’s diseases), or inflammatory and
neoplastic brain disorders, highlights TSPO as a valid diag-
nostic marker for related-disease state and progression.3
However, the exact physiopathological role of TSPO is not
completely defined, encouraging a continued research in this
field to develop new potent and selective ligands as suitable
tools to deepen the knowledge about this protein functioning.
Since its identification by means of the benzodiazepines
diazepam and Ro5−4864 (Figure 1),4 structurally different
classes of high affinity and selectivity ligands have been reported,5,6
Figure 1. Structure of known TSPO ligands and newly synthesized
quinazoline derivatives within the framework of our pharmacophore/
topological model of ligand−TSPO interaction.13
including the isoquinolinecarboxamides, of which the 1-(2-
chlorophenyl)-N-methyl-N-(1-methylpropyl)-1-isoquinoline
carboxamide (PK11195, Figure 1) is widely considered as a proto-
typical TSPO ligand, used as reference compound in biological
experiments7 and radiolabeled for imaging studies.8,9 Nevertheless,
PK11195 shows several drawbacks, principally caused by its high
lipophilicity, such as, for example, high level of nonspecific binding
and a poor signal-to-noise ratio in PET studies.9
Received: December 19, 2011
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm201703k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
To pursue our interest in the medicinal chemistry of TSPO
ligands,7,10,11 and with the aim to identify novel chemotype
ligands, we now report the study of a series of quinazoline-2-
carboxamide derivatives (1−58, Figure 1), straightforwardly
obtained with a simple and efficient synthetic procedure.
Although structurally related to the isoquinolinecarboxamide
PK11195 (Figure 1), these quinazoline derivatives were as-
sumed to have a better drug-like character, possessing a higher
hydrophilicity and water solubility than their quite lipophilic
isoquinoline counterpart. Indeed, preliminary calculations of
several physicochemical and pharmacokinetic parameters con-
ducted on the structures of PK11195 and of its aza-isoster 14
supported our hypotheses (see Results and Discussion). In a 1985
patent,12 reporting on the synthesis and the affinity for TSPO
of arene- and heteroarene-carboxamides, a couple of quinazoline
compounds were described but without any investigation of their
pharmacological properties.
Taking into account the structural requirements needed for
high TSPO affinity and selectivity,6 compounds 1−58 featuring
different combinations of R1−R5 substituents were designed.
Specifically, symmetrically or asymmetrically N,N-disubstituted
quinazolines bearing linear, branched, or alicyclic alkyl chains
were synthesized. In addition, to investigate the role of a double
substitution on the amide nitrogen to gain high TSPO affinity
in this class, a number of N-monosubstituted derivatives were
studied. Finally, a chlorine atom was inserted at different posi-
tions (2′, 4′, 6) of the basic 4-phenylquinazoline scaffold. All novel
compounds (1−58) have been tested for their affinity at TSPO in
rat kidney membranes, and SARs were discussed in light of a
previously published pharmacophore/topological model of
ligand−TSPO interaction.10,13 In addition, a preliminary in vitro
biological characterization has been performed on compounds 9
and 14 using PK11195 as a standard reference.
activation of the acids 77−82 with thionyl chloride, direct
coupling with the proper commercially available N,N-dialkyl-
amine yielded N,N-dialkylquinazolinecarboxamides 2, 5−12,
16−20, 24−32, 42−46, 50−54, and 58 (method A, Scheme 1).
When the required N-methyl-N-alkylamine was not available,
the intermediate carboxylic acids 77−82 were activated with
thionyl chloride and reacted with the appropriate primary
amines to give the secondary amides 1, 13, 21, 39, 47, 55,
and 59−70, which were subsequently N-methylated with
methyl iodide in the presence of sodium hydride to furnish
compounds 3, 4, 14, 15, 22, 23, 33−38, 40, 41, 48, 49, 56, and
57 (method B, Scheme 1).
BIOLOGY
■
The TSPO binding affinities of compounds 1−58 were
determined by competition experiments against [3H]-
PK11195,7,10 carried out in rat kidney membranes (Table 1).
The TSPO/central benzodiazepine receptor (BzR) selectivity
of compounds 1−58 was evaluated by experiments against
[3H]flumazenil in rat cerebral cortex membranes (Table S6, SI).7,10
In addition, to explore the effect of this new class of compounds
on TSPO function, a representative subset of TSPO ligands
was examined for the ability to stimulate pregnenolone forma-
tion from rat C6 glioma cells (2, 7, 9, 14, 22, and 25)7,10 and
for the effect on the proliferation/viability of U87MG glioma
cells (9 and 14).15,16
RESULTS AND DISCUSSION
■
Many of the synthesized compounds show high affinity for
TSPO in the nanomolar/subnanomolar range, with a potency
higher than that of the lead PK11195 (Table 1) and a high
selectivity for TSPO over BzR, as they did not significantly
inhibit the binding of [3H]flumazenil in membranes from rat
brain tissues (inhibition percentages at 10 μM concentration
ranging from 0% to 48%, Table S6, SI). The SARs emerging
from this series are herein discussed in light of a previously
published pharmacophore/topological model of ligand−TSPO
interaction made up by three lipophilic pockets (L1, L3, and L4)
and an H-bond donor group (H1) (Figure 1).13
CHEMISTRY
■
The key intermediates in the synthesis of phenylquinazoline-2-
carboxamides 1−58 are the carboxylic acids 77−82, prepared
by condensation of the appropriate 2-aminobenzophenones
71−76 with glyoxylic acid in the presence of ammonium
acetate, followed by light irradiation with 20 W halogen tungsten
lamp (Scheme 1; see Supporting Information (SI) for details).
All the N-monosubstituted quinazolines tested (1, 13, 21, 39,
47, 55) are devoid of affinity. These compounds miss hydro-
phobic contacts which would otherwise take place between
an N-alkyl group and the L4 pocket. Moreover, it is hypothe-
sized that they adopt a conformation unsuited for optimal
interaction with the receptor. In fact, as detailed in the SI,
molecular mechanics (MM) calculations revealed that the
N-monosubstituted and the N,N-disubstituted derivatives are
predicted to adopt low energy conformations characterized by
the OC-N< moiety laying within the plane (torsion angle
N1−C2−CO about 0°) and out of the plane (torsion angle
N1−C2−CO about 30°) of the quinazoline ring (Figure 2),
respectively. Such a difference in the conformational properties
of the two subsets of compounds might depend on the fact that
each of the two quinazoline nitrogens attracts electrostatically
the N-hydrogen while repelling sterically any N-alkyl group.
We could speculate that only the carbonyl group of the N,N-
disubstituted derivatives is correctly oriented within the binding
cleft to make a strong H-bond with the H1 site. Our hypothesis
is consistent with the results of the excellent work by Cappelli and
co-workers, who mapped the TSPO binding site using conforma-
tionally restrained analogues of PK11195.17 According to these
authors, the TSPO-bound conformations of the most potent
a
Scheme 1. Synthesis of Quinazoline Derivatives 1−58
a
(a) CHOCOOH, NH4OAc, EtOH, RT, 0.5 h; (b) light irradiation,
DMF, RT, 12 h, 62−86% (two steps); (c) SOCl2, reflux, 2 h, then
monoalkyl- or dialkylamine, NEt3, THF, RT, 36−48 h, 43−88%; (d)
NaH, CH3I, DMF, 0 °C to RT, 0.5 h, 67−93%.
Noncommercially available 2-aminobenzophenones 72, 73, and
76 were synthesized according to literature procedures.14 After
B
dx.doi.org/10.1021/jm201703k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
Table 1. TSPO Affinity of Quinazoline Derivatives 1−58
Figure 2. Low energy conformations as calculated by MM simulations
of the phenyl-truncated N,N-disubstituted and the N-monosubstituted
quinazolines represented as green and cyan sticks, respectively. The
N1−C2−CO dihedral angle value is also labeled.
a
N
R1
R2
R5
R3
R4
I% (1 μM) or Ki (nM)
1
H
CH(CH3)CH2CH3
(CH2)3CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
0%
46.0 5.1
3.00 0.30
66.5 6.2
70.3 6.2
36.8 3.0
3.30 0.30
2.48 0.21
0.690 0.070
36%
2
CH3
CH3
CH3
CH3
3
CH(CH3)CH2CH3
CH2CH(CH3)2
CH(CH3)2
4
isoquinoline-3-carboxamides are characterized by the carbonyl
group oriented out of the plane of the isoquinoline ring system.
In an attempt to explain the total lack of affinity shown by
the N-monosubstituted derivatives compared with their potent
N,N-disubstituted counterparts, we also pairwise examined their
5
6
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)2CH3
7
(CH2)3CH3
8
(CH2)4CH3
9
(CH2)5CH3
10
11
12
13
14
15
16
17
18
19
20
21
22
(CH2)4
1
(CH2)5
(CH2)6
61%
IR and H NMR spectra to verify whether the OC-NH−
79%
moiety was switched to the corresponding HO-CN−
H
CH(CH3)CH2CH3
63%
tautomer. Yet, no data supported such a hypothesis.
CH3
CH(CH3)CH2CH3
CH2CH(CH3)2
CH(CH3)2
3.06 0.30
6.98 0.71
12.5 1.3
65.1 7.0
70%
Noticeably, the introduction of a second nitrogen atom in the
isoquinoline nucleus of PK11195 produces a favorable effect on
TSPO affinity, as product 14 shows a 3-fold gain in potency with
respect to its parent compound. Compound 9, with R1 = R2 =
n-hexyl, R3 = R4 = R5 = H, is the most potent one (Ki 0.7 nM)
and also slightly less potent compounds (3, 7, 8, 14, 15, 22, 25,
29, 33−38) display Ki values lower than PK11195. It is noteworthy
that all these molecules share the following features: (i) they are
N,N-disubstituted, (ii) one of the two N-alkyl groups contains a
number of carbon atoms comprised between 4 and 6. Taken
together, the above data suggest that an optimal ligand−TSPO
interaction requires full occupation of the L4 hydrophobic pocket
by at least one bulky N-alkyl group. The shape of such N-alkyl
group is, in some instances, a key determinant of affinity:
compare the N-s-butyl derivatives 3 (Ki 3.00 nM) and 22
(Ki 2.67 nM) with their much less potent N-i-butyl isomers 4 (Ki
66.5 nM) and 23 (Ki 20.7 nM), respectively. In contrast, the N-s-
butyl and the N-i-butyl derivatives 14 (Ki 3.06 nM) and 15 (Ki
6.98 nM) are practically equipotent, and the N-i-butyl derivatives
41 (Ki 842 nM) and 57 (Ki 60.7 nM) are more potent than their
N-s-butyl counterparts 40 (inhib 22%) and 56 (Ki 179 nM).
Chirality does not impact on the affinity within the subset of N-
s-butyl derivatives assayed as pure enantiomers (compare 33 (Ki
6.09 nM) vs 34 (Ki 6.41 nM), 35 (Ki 2.2 nM) vs 36 (Ki 1.55 nM)
and 37 (Ki 2.51 nM) vs 38 (Ki 1.57 nM)), differently from what
happens with PK11195, for which the R-enantiomer is reported to
show higher TSPO affinity than the racemic mixture in rats.18
Affinity is worsened to different extents by any of the following
features: (i) anellation of the side chain to give the cyclic
pyrrolidinyl- piperazinyl- and azepinyl-amides 10−12, 18−20,
26−28, 30−32, 44−46, and 52−54, (ii) insertion of a chlorine
at the 6-position of the quinazoline ring (29−32 and 39−58),
with a few exception (compare 51 and 17). These data sug-
gest that a rigid carboxamide side chain and a substituent at
6-position of the quinazoline ring cannot fit into the L4 pocket
or the L1 pocket for sterical reasons, respectively.
CH3
CH3
(CH2)5CH3
(CH2)5CH3
(CH2)4
(CH2)5
(CH2)6
46.0 5.3
8.77 0.82
37%
H
CH(CH3)CH2CH3
CH3
CH(CH3)CH2CH3
CH2CH(CH3)2
CH(CH3)2
2.67 0.30
23
24
25
26
27
28
29
30
31
32
33
34
35
36
CH3
H
H
H
H
H
H
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
20.7 2.3
50.4 4.9
3.42 0.29
644 60
493 50
84.3 8.5
4.31 0.50
64%
CH3
(CH2)5CH3
(CH2)5CH3
(CH2)4
(CH2)5
(CH2)6
(CH2)5CH3
(CH2)5CH3
(CH2)4
(CH2)5
(CH2)6
76%
77%
CH3
CH3
CH3
CH3
(S)-CH(CH3)CH2CH3
6.09 0.52
6.41 0.51
2.20 0.20
1.55 0.19
(R)-CH(CH3)CH2CH3
(S)-CH(CH3)CH2CH3
(R)-CH(CH3)CH2CH3
H
H
H
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
CH3
(S)-CH(CH3)CH2CH3
(R)-CH(CH3)CH2CH3
CH(CH3)CH2CH3
CH(CH3)CH2CH3
CH2CH(CH3)2
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
2.51 0.27
1.57 0.14
0%
CH3
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
CH3
22%
CH3
842 75
0%
CH3
CH(CH3)2
(CH2)5CH3
(CH2)5CH3
38.5 4.0
43%
(CH2)4
(CH2)5
(CH2)6
46%
38%
H
CH(CH3)CH2CH3
0%
CH3
CH(CH3)CH2CH3
CH2CH(CH3)2
CH(CH3)2
43%
CH3
77%
CH3
0%
(CH2)5CH3
(CH2)5CH3
30.4 3.1
73%
(CH2)4
(CH2)5
(CH2)6
273 30
415 40
49%
H
CH(CH3)CH2CH3
CH3
CH3
CH3
CH(CH3)CH2CH3
CH2CH(CH3)2
CH(CH3)2
179 18
60.7 5.7
39%
Mixed results are associated with the absence/presence of a
chlorine at the 2′- or 4′-position of the pendant phenyl: the N-s-
butyl derivatives 3 (Ki 3.00 nM), 14 (Ki 3.06 nM), and 22
(Ki 2.67 nM), differing in the above substitution pattern, are
essentially equipotent; the N-i-butyl and N-i-propyl deriva-
tives 4 (Ki 66.5 nM) and 5 (Ki 70.3 nM) are significantly less
potent than their 2′-chloro derivatives 15 (Ki 6.98 nM) and 16
(Ki 12.5 nM) and are slightly less potent than their 4′-chloro
derivatives 23 (Ki 20.7 nM) and 24 (Ki 50.4 nM). These data,
Ro5-4864
PK11195
Alpidem
23.0 3.0
9.30 0.5
0.5−70
a
The concentration of test molecules that inhibited [3H]PK11195
binding to rat kidney mitochondrial membranes by 50% (IC50) was
determined with six concentrations of the compounds, each per-
formed in triplicate. Ki values and inhibition percentages at 1 μM are
the mean SEM of three determinations.
C
dx.doi.org/10.1021/jm201703k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
characterized by interdependent (nonadditive) effects of the R2,
R3, and R4 substituents on affinity, are difficult to be rationalized
using a “static” pharmacophore/topological model. It is tempt-
ing to hypothesize that a strong hydrophobic interaction of
R2 = s-butyl with the L4 pocket may pose the ligand in the
binding cleft so as to weaken the beneficial effects of R3 or R4 = Cl
within the L3 pocket (e.g., 3, 14, and 22). Otherwise, a less strong
interaction of R2 = i-butyl or i-propyl with the L4 pocket may
allow a better fit of R3 and R4 into the L3 pocket (e.g., 4 and 15 or
5 and 16). When the interaction with the L4 pocket is optimized
(R1 = R2 = n-hexyl), the presence of one or even two chlorine
atoms is tolerated (e.g., 17, 25, 29, and 51).
U87MG cell survival (Figure S3, SI). On the basis of these
preliminary data, it may be speculated that these quinazoline
ligands possess the ability to bind the TSPO with high affinity,
leading to protein conformational changes that do not
significantly affect the steroidogenic TSPO function, whereas
they produce a slight modulation on cell proliferation/viability.
CONCLUSION
■
A simple and efficient synthetic procedure has been developed
to obtain quinazoline azaisosters of PK11195, a potent TSPO
ligand widely used as reference compound. This strategy allowed
us to easily prepare a great number of related derivatives (1−58),
many of which show nanomolar/subnanomolar Ki values for the
TSPO, with improved affinity with respect to the lead PK11195.
Compound 9 (N,N-di-n-hexyl-4-phenylquinazoline-2-carboxa-
mide) stands out as the most potent ligand of the series with a
Ki of 0.7 nM, and high selectivity toward BzR.
Some physicochemical and pharmacokinetic properties of 14
and its parent compound PK11195 were calculated and
compared as a rough assessment of the drug-like character of
our quinazoline derivatives. For such a purpose, we employed
the Qikprop program (Schrodinger. LLC New York). The
̈
results are summarized in Table S5, SI. QSAR studies on CNS
active drugs and their analogues, together with retrospective
analyses based on marketed CNS drugs, have suggested the
physical and chemical properties that CNS drugs must possess.
They are: molecular weight (MW) less than 450, ClogP less
than 5, number of H-bond acceptor atoms less than 7, polar
surface area (PSA) less than 90 Å, number of rotatable bonds
(RB) less or equal to 10. Thus, for CNS penetration, the
physical properties, usually, have a smaller range than general
therapeutics (the latter ranges are reported in Table S5, SI).
On the basis of these premises, compound 14 is predicted to
have a good probability of entering the CNS, as all its estimated
physicochemical parameters fall in the aforementioned ranges.
Furthermore, it is worth mentioning that, despite the structural
similarity between the two compounds resulting in comparable
structure-derived physicochemical properties, compound 14
scores better than PK11195 if we consider the Lipinski’s
Rule-of-Five and the Jorgensen’s Rule-of-Three, both aimed at
identifying drug-like molecules.
To explore the effect of the compounds on steroid bio-
synthesis, a representative subset of TSPO quinazoline ligands
(2, 7, 9, 14, 22, and 25) and PK11195 and Ro5−4864 as
standards, were examined for their ability to stimulate preg-
nenolone formation from rat C6 glioma cells.7,10 In this assay, all
tested derivatives, with the exception of the practically inactive 9,
exhibited low/medium percentage increase in pregnenolone pro-
duction vs control, with an efficacy lower than that of
PK11195 and Ro5−4864 (Table S7, SI), independently from
their Ki values. The lack of any correlation between affinity
and steroidogenic activity showed by these quinazoline ligands is
in line with data from other known classes of highly affine TSPO
ligands, including 2-phenylindolglyoxylamides, imidazo-pyridines,
and pyrazolo-pyrimidines.6 Furthermore, to investigate
whether the newly synthesized compounds affected another
well-known TSPO function, which is the regulation of life−
death cell processes, compounds 9 and 14 were preliminarily
evaluated for their effect on the proliferation/viability of U87MG
glioma cells. PK11195 was also tested as reference compound.
As detailed in the SI, the colorimetric viability MTS assay16
showed that PK11195 produced effects in agreement with
those reported in the literature19 on the same cell line, namely a
statistically significant reduction of U87MG cell proliferation/
viability (Figure S1, SI) only at the highest concentration tested
(100 μM). Compound 14 showed comparable results with those
obtained with PK11195 (Figure S2, SI), whereas compound 9 was
more effective than the reference standard at inhibiting the
SARs emerging from this series were rationalized in light of a
previously published pharmacophore/topological model of
ligand−TSPO interaction, allowing to define the main struc-
tural requirements for an optimal interaction with the target
protein, that is: (i) N,N-disubstitution on the carboxamide
moiety, (ii) at least one of the two N-alkyl groups with a number
of carbon atoms comprised between 4 and 6. The comparison of
some calculated physicochemical and pharmacokinetic properties
of compound 14 and of the lead PK11195, evidenced a better
drug-like character for our quinazoline derivative.
In a preliminary biological in vitro investigation, the effects
exerted by these new derivatives on steroidogenesis and life−
death cell processes, with respect to those produced by the lead
PK11195, suggested that even little structural modifications
may affect ligand-mediated modulation of TSPO functions.
Taken together, all these findings highlighted the quinazoline
nucleus as a suitable scaffold to further expand the chemical
diversity in TSPO ligands and provided SARs data for the
design of new TSPO modulators useful to deepen the know-
ledge about this protein, even by means of the development of
imaging tracers with improved specificity.
EXPERIMENTAL SECTION
Chemistry. General directions are in the SI. Purity of tested
compounds is ≥95% (combustion analysis).
■
General Procedure for the Synthesis of N-alkyl-4-phenyl-
quinazoline-2-carboxamides 1, 2, 5−13, 16−21, 24−32,
39, 42−47, 50−55, and 58−70. A solution of the appropriate
2-carboxylic acid 77−82 (2.0 mmol) in thionyl chloride (15 mL) was
refluxed for 2 h under nitrogen atmosphere. After cooling at room
temperature, the excess thionyl chloride was removed at reduced
pressure and the crude material dried under vacuum. To the residue,
dissolved in dry THF (10 mL) and cooled to 0 °C, a mixture of
the proper amine (2.0 mmol) and triethylamine (2.0 mmol) in dry
THF (5 mL) was added dropwise. The mixture was stirred at room
temperature for 36−48 h (TLC analysis), filtered, and evaporated. The
crude residue was dissolved in DCM (20 mL), washed with HCl 1N,
saturated NaHCO3, and water, dried, and concentrated in vacuum.
Purification by column chromatography on silica gel (DCM−EtOAc)
provided title compounds (yields, physical, and spectral data are
reported in Tables S2 and S3, SI).
General Procedure for the Synthesis of N-Alkyl-N-methyl-4-
phenylquinazoline-2-carboxamides 3, 4, 14, 15, 22, 23, 33−38,
40, 41, 48, 49, 56, and 57. Sodium hydride (5.5 mmol) was added
portionwise, under nitrogen atmosphere, to an ice-cooled solution of
the appropriate N-alkyl-4-phenylquinazoline-2-carboxamide (5.0 mmol)
in dry DMF (5 mL). The mixture was stirred for 30 min and treated
with an excess of methyl iodide (0.68 mL, 11.0 mmol). The mixture
D
dx.doi.org/10.1021/jm201703k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
was stirred for 1 h at 25 °C, and an ice cooled solution of HCl 1N and
chloroform were added. The organic layer was separated, washed with
brine, dried, and concentrated in vacuum. Purification by column
chromatography on silica gel (DCM−EtOAc) provided title compounds
(yields, physical, and spectral data are reported in Table S4, SI).
of potent and selective ligands at the peripheral benzodiazepine
receptor. J. Med. Chem. 2004, 47, 1852−1855.
(8) Matarrese, M.; Moresco, R. M.; Cappelli, A.; Anzini, M.; Vomero, S.;
Simonelli, P.; Verza, E.; Magni, F.; Sudati, F.; Soloviev, D.; Todde, S.;
Carpinelli, A.; Kienle, M. G.; Fazio, F. Labeling and evaluation of N-[11C]-
methylated quinoline-2-carboxamides as potential radioligands for visual-
ization of peripheral benzodiazepine receptors. J. Med. Chem. 2001, 44,
579−585.
ASSOCIATED CONTENT
■
S
* Supporting Information
(9) Chaveau, F.; Boutin, H.; Van Camp, N.; Dolle, F.; Tavitian, B. Nuclear
imaging of neuroinflammation: a comprehensive review of [11C]PK11195
challengers. Eur. J. Nucl. Med. Mol. Imaging 2008, 35, 2304−2319.
(10) Da Settimo, F.; Simorini, F.; Taliani, S.; La Motta, C.; Marini,
A. M.; Salerno, S.; Bellandi, M.; Novellino, E.; Greco, G.; Cosimelli, B.;
Da Pozzo, E.; Costa, B.; Simola, N.; Morelli, M.; Martini, C.
Anxiolytic-like effects of N,N-dialkyl-2-phenylindol-3-ylglyoxylamides
by modulation of translocator protein promoting neurosteroid
biosynthesis. J. Med. Chem. 2008, 51, 5798−5806.
(11) Cosimelli, B.; Simorini, F.; Taliani, S.; La Motta, C.; Da Settimo,
F.; Severi, E.; Greco, G.; Novellino, E.; Costa, B.; Da Pozzo, E.;
Bendinelli, S.; Martini, C. Tertiary amides with a five-membered
heteroaromatic ring as new probes for the translocator protein. Eur. J.
Med. Chem. 2011, 46, 4506−4520.
(12) Dubroeucq, M. C.; Renault, C.; Le Fur, G. Derivatives of arene
and hetero-arene carboxamides and their use as medicaments. US
1983/482082, US19830482082, 19830405, Feb 12, 1985.
General chemistry directions, general procedure for the
synthesis of 4-phenylquinazoline-2-carboxylic acids 77−82,
yields, physical, and spectral data of compounds 1−70, and
77−82, computational chemistry, calculated physicochemical
and pharmacokinetic properties of compound 14 and PK11195,
biological methods, BzR binding data of compounds 1−58,
increase in pregnenolone production of selected quinazoline
derivatives, effect on proliferation/viability of U87MG glioma
cells of compounds 9 and 14. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (+39)0502219547. Fax: (+39)0502219605. E-mail:
taliani@farm.unipi.it.
(13) Campiani, G.; Nacci, V.; Fiorini, I.; De Filippis, M. P.; Garofalo,
A.; Ciani, S. M.; Greco, G.; Novellino, E.; Williams, D. C.; Zisterer,
D. M.; Woods, M. J.; Mihai, C.; Manzoni, C.; Mennini, T. Synthesis,
biological activity, and SARs of pyrrolobenzoxazepine derivatives, a
new class of specific “peripheral-type” benzodiazepine receptor ligands.
J. Med. Chem. 1996, 39, 3435−3450.
Author Contributions
All authors have given approval to the final version of the
manuscript.
Funding
(14) Sugasawa, T.; Toyoda, T.; Adachi, M.; Sasakura, K. Amino-
haloborane in organic synthesis. 1. Specific ortho substitution reaction
of anilines. J. Am. Chem. Soc. 1978, 100, 4842−4845.
(15) Chelli, B.; Lena, A.; Vanacore, R.; Da Pozzo, E.; Costa, B.; Rossi.,
L.; Salvetti, A.; Scatena, F.; Ceruti, S.; Abbracchio, M. P.; Gremigni, V.;
Martini, C. Peripheral benzodiazepine receptor ligands: mitochondrial
transmembrane potential depolarization and apoptosis induction in rat C6
glioma cells. Biochem. Pharmacol. 2004, 68, 125−134.
(16) Ruan, S.; Okcu, M. F.; Pong, R. C.; Andreeff, M.; Levin, V.;
Hsieh, J. T.; Zhang, W. Attenuation of WAF1/Cip1 expression by an
antisense adenovirus expression vector sensitizes glioblastoma cells to
apoptosis induced by chemotherapeutic agents 1,3-bis(2-chloroethyl)-
1-nitrosourea and cisplatin. Clin. Cancer Res. 1999, 5, 197−202.
(17) Cappelli, A.; Anzini, M.; Vomero, S.; De Benedetti, P. G.;
Menziani, M. C.; Giorgi, G.; Manzoni, C. Mapping the peripheral benzo-
diazepine receptor binding site by conformationally restrained derivatives
of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3- isoquinolinecar-
boxamide (PK11195). J. Med. Chem. 1997, 40, 2910−2921.
(18) Shah, F.; Hume, S. P.; Pike, V. W.; Ashworth, S.; McDermott, J.
Synthesis of the enantiomers of [N-methyl-11C]PK11195 and
comparison of their behaviours as radioligands for PK binding sites
in rats. Nucl. Med. Biol. 1994, 21, 573−581.
This study was financially supported by grants from Istituto
Toscano Tumori (grant 2007), MIUR (PRIN 2009, Futuro in
Ricerca 2010), Universita di Salerno and Universita di Trieste.
̀ ̀
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
BBB, blood−brain barrier; BzR, central benzodiazepine receptor;
CNS, central nervous system; PBR, peripheral benzodiazepine
receptor; PET, positron emission tomography; SARs, structure−
activity relationships; TSPO, 18 kDa translocator protein
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