Exploiting the 4-Phenylquinazoline Scaffold for the Development of High Affinity Fluorescent Probes for the Translocator Protein (TSPO)

Article abstract of DOI:10.1021/acs.jmedchem.7b01031

The quinazoline class was exploited to search for a new translocator protein (TSPO) fluorescent probe endowed with improved affinity and residence time (RT). Computational studies on an "in-house" collection of quinazoline derivatives, featuring highly st

Full text of DOI:10.1021/acs.jmedchem.7b01031

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Article
Exploiting the 4-Phenylquinazoline Scaffold for the Development of
High Affinity Fluorescent Probes for the Translocator Protein (TSPO)
Ciro Milite, Elisabetta Barresi, Eleonora Da Pozzo, Barbara Costa, Monica Viviano,
Amalia Porta, Anna Messere, Gianluca Sbardella, Federico Da Settimo, Ettore
Novellino, Sandro Cosconati, Sabrina Castellano, Sabrina Taliani, and Claudia Martini
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01031 • Publication Date (Web): 31 Aug 2017
Downloaded from http://pubs.acs.org on September 4, 2017
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Exploiting the 4ꢀPhenylquinazoline Scaffold for the
Development of High Affinity Fluorescent Probes
for the Translocator Protein (TSPO)
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Ciro Milite,†,‡ Elisabetta Barresi, ¥,‡ Eleonora Da Pozzo, ¥ Barbara Costa, ¥ Monica Viviano,†
Amalia Porta,† Anna Messere,∆ Gianluca Sbardella,† Federico Da Settimo, ¥ Ettore Novellino,§
Sandro Cosconati,∆,* Sabrina Castellano,†, ,* Sabrina Taliani, ¥,* Claudia Martini. ¥
∥
†
¥Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy. Dipartimento di
Farmacia, Università di Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Salerno, Italy.
∥Dipartimento di Medicina, Chirurgia e Odontoiatria "Scuola Medica Salernitana", Università di
§
Salerno, Via Salvador Allende, Baronissi, Iꢀ84081 Salerno, Italy. Dipartimento di Farmacia,
∆
Università di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy. DiSTABiF,
University of Campania Luigi Vanvitelli, 81100 Caserta, Italy.
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Abstract
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The quinazoline class was exploited to search for a new Translocator Protein (TSPO) fluorescent
probe endowed with improved affinity and Residence Time (RT). Computational studies on an “inꢀ
house” collection of quinazoline derivatives, featuring highly steric demanding groups at the amide
nitrogen, suggested that, despite their molecular extension, these ligands are still easily lodged in
the TSPO binding site. Binding assays supported this hypothesis, highlighting a low
nanomolar/subnanomolar affinity of these ligands, together with a higher RT of the representative
compound 11 with respect to our previously reported indoleꢀbased fluorescent probe. Thanks to the
amenability of the amide nitrogen atom to be substituted with bulky groups, we developed
quinazolineꢀbased imaging tools by fluorescently labeling the scaffold at this position. Probes with
relevant TSPO affinity, favorable spectroscopic properties, and improved RT were identified.
Results from fluorescence microscopy showed that these probes specifically labeled the TSPO at
mitochondrial level in U343 cell line.
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Introduction
Neuroinflammation and activation of glial cells are important pathological processes that are
involved in the progression of many neurological disorders, including multiple sclerosis,
amyotrophic lateral sclerosis, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and
stroke. In particular, the activation of microglial cells involves changes in their morphology and
acquisition of new functions, including the expression and release of diverse proinflammatory
molecules and the phagocytosis of dead cells.1
During the past decade, several studies have validated the Translocator Protein 18 kDa (TSPO) as
a biomarker of neuroinflammation as well as of brain injury.2ꢀ4 This is a highly conserved protein
throughout evolution, from bacteria to humans, mainly located on the outer mitochondrial
membrane of steroidꢀproducing tissues (testis, adrenal cortical, ovarian granulose, and luteal cells)
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and it has been proposed to be an important player in the transport of cholesterol into mitochondria,
the first and rateꢀlimiting step for steroid hormone synthesis.5 Furthermore, considerable evidence
supports its regulatory role in a number of other cellular processes, including apoptosis, cell
proliferation, cellular respiration and immunomodulation.6
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Recently, a controversy surrounding the exact physiological role of this protein7 emerged
following reports proposing its catalytic activity,8 as well as challenging its essentiality for
steroidogenesis.9ꢀ11 Nevertheless, the reproducibly high level of expression in areas of inflammation
in the brain still validate TSPO as a sensitive imaging biomarker to evaluate neuroinflammation.4
Indeed, in the central nervous system (CNS), the expression level of TSPO is low in healthy brains,
but the protein is overexpressed in activated glial cells following CNS pathological conditions,
including gliomas, multiple sclerosis, as well as Alzheimer’s disease.2,4,12
Since its identification by means of the benzodiazepines diazepam and 4'ꢀchlorodiazepam (Ro5ꢀ
4864),13 a number of high affinity and selectivity TSPO ligands have been developed from different
structural classes, including isoquinoline carboxamides (e.g., 1ꢀ(2ꢀchlorophenyl)ꢀNꢀmethylꢀNꢀ(1ꢀ
methylpropyl)ꢀ1ꢀisoquinolinecarboxamide (PK11195, 1, Chart 1) widely considered as a
prototypical TSPO ligand and used as a reference compound in biological experiments),14
aryloxyanilides
(e.g.,
Nꢀ(2,5ꢀdimethoxybenzyl)ꢀNꢀ(5ꢀfluoroꢀ2ꢀphenoxyphenyl)acetamide
(DAA1106) and NꢀacetylꢀNꢀ(2ꢀmethoxybenzyl)ꢀ2ꢀphenoxyꢀ5ꢀpyridinamine (PBR28)),15,16 2ꢀ
phenylꢀimidazo[1,2ꢀa]pyridine acetamides (e.g., alpidem).17 Many of these ligands have been
developed to visualize activated microglia via several imaging techniques.18
In this context, we recently reported two class of highly potent and selective TSPO ligands, the 2ꢀ
arylindolꢀ3ꢀglyoxylamides A19ꢀ22 and the 4ꢀphenylquinazolineꢀ2ꢀcarboxamides B, designed as
azaisosteres of 1 (Chart 1).23,24 Ligands from class A were developed to fluorescent probes (see
compound 2 as an example, Chart 1) useful for investigating the localization and the expression
level of TSPO,25,26 and to a novel positron emission tomography (PET) radioligand labeled with
carbonꢀ11 (3, Chart 1) able to enter monkey’s brain and give a high proportion of TSPOꢀspecific
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binding.27 Similarly, ligands from 4ꢀphenylquinazolineꢀ2ꢀcarboxamide class have been developed
to visualize activated microglia via PET; a useful radioligand (4, Chart 1), the direct 4ꢀazaisostere
of 1, was identified and demonstrated to possess an array of properties that are superior to those
reported for [11C]ꢀ1, including higher affinity, lower lipophilicity, higher TSPOꢀspecific signal, and,
most importantly, very low genotype sensitivity in vitro.28 On these premises, compound 4 was
further evaluated for its in vivo sensitivity in the brain and peripheral organs in human subjects,
showing adequate sensitivity to robustly image all three affinity genotypes in the human brain.29
Taken together, all these findings corroborate the worth of the 4ꢀphenylquinazolineꢀ2ꢀcarboxamide
chemotype in the development of specific TSPO ligands, as well as molecular probes suitable for
TSPO imaging.
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Chart 1. General structure of 2ꢀarylindolꢀ3ꢀglyoxylamides (A)19ꢀ21 and 4ꢀphenylquinazolineꢀ2ꢀ
carboxamides (B),23,24 together with 1 and fluorescent (2)25,26 and radiolabeled (3, 4)27ꢀ29 probes
belonging from classes A and B.
In the present study, we evaluated the feasibility of developing novel fluorescently labeled TSPO
ligands with high specificity and attractive spectroscopic properties, featuring the 4ꢀ
phenylquinazolineꢀ2ꢀcarboxamide scaffold. In this respect, ligands incorporating fluorescent probes
may represent a safer, faster, and less expensive tool to achieve TSPO imaging if compared to the
classic radiolabeled ligand. In general, a fluorescent ligand can be rationally designed to retain the
pharmacological profile of the parent unlabeled ligand, thus allowing for the localization and realꢀ
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time monitoring of processes triggered by ligandꢀreceptor interactions (i.e internalization,
trafficking, sequestration, and recycling) within a living cell. In addition, the displacement of
fluorescent probes from their binding sites of a target protein by nonꢀfluorescent ligands allows for
the identification of the sites recognized by the ligands.30
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Results and discussion
Basis of the project.
The starting point for the probe development was our previously published twoꢀdimensional (2D)
pharmacophore/topological model,24 according to which the NꢀbenzylꢀNꢀethylꢀdisubstituted
quinazolines 5ꢀ6 (Chart 2 and Figure 1A)24 place their Nꢀbenzyl substituent towards the external
part of the receptor, herein referred to as L4 (see compound 6 as an example in Figure 1A). In
principle, this would outline that the aforementioned group could be functionalized with a steric
demanding fluorescent tracer without affecting the binding affinity for the target TSPO.
In this respect, the availability of an “inꢀhouse” library of quinazoline compounds encouraged us
to pursue derivatives of compounds 5 and 6 for the development of our probe. Considering the
elevated costs connected with the radioligand binding experiments, rather than directly screening all
of them, a fast, and costꢀeffective, in silico preꢀevaluation was firstly performed. Therefore, a threeꢀ
dimensional (3D) model of interaction between the rTSPO and a selection of quinazoline
derivatives (compounds 7ꢀ14), featuring highly steric demanding groups at the amide nitrogen (see
Chart 2 and Figure 1B) was generated. In particular, compounds 7ꢀ12 present a single bulky
substituent on amide nitrogen, whereas compounds 13 and 14 were characterized by double
substitution with steric demanding groups.
According to these calculations, all the selected compounds would interact within the TSPO
binding cleft by placing the pꢀsubstitutedꢀbenzyl group in the wide L4 cleft; differently from what
happens for compounds 5 and 6, now the 4ꢀphenyl substituent is lodged in the L1 cleft, while the
quinazoline core resides in the L3 one. Nevertheless, when analyzing the scores calculated by Glide
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docking software, the inspected compounds (6 and 7ꢀ12) are predicted to have a similar affinity for
the TSPO. On the other hand, Glide was not able to dock 13 and 14 in the TSPO site most probably
due to the steric hindrance of the double substitution at the amide nitrogen.
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Figure 1. Binding mode of compound 6 (panel A, orange sticks) and 7ꢀ12 (panel B, purple sticks)
into the rTSPO binding site (cyan ribbons and surface). In panel (A) the three receptor clefts
identified from the topological model are also outlined.
Encouraged by molecular modeling results, the affinity of the 4ꢀphenylquinazoline compounds 7ꢀ
14 at the TSPO was determined by binding competition experiments against [3H]ꢀ1, performed on
rat kidney mitochondrial membranes (Table 1). As predicted by computational studies, all the
compounds show high affinity for TSPO in the nanomolar/subnanomolar range, the only exception
being compounds 13 and 14, which feature two bulky substituents at the amide nitrogen,
substantiating our work hypothesis. In term of structureꢀaffinity relationships, the insertion at the
benzyl paraꢀposition of a highly steric demanding group such as pꢀtoluensulfonyloxy (7, 8), pꢀ
toluencarbonyloxy (9, 10), pꢀtoluencarbonylamino (11, 12) is well tolerated in terms of TSPO
binding affinity since all derivatives show low nanomolar/subnanomolar Ki values. The cooperative
effect of the chlorine at 2′ꢀposition of the 4ꢀphenyl ring seemed necessary for subnanomolar affinity
only when R1 is a pꢀtoluencarbonyloxy or pꢀtoluencarbonylamino group.
Reaching a high binding affinity for the biological target is not the only demand to develop
efficient pharmacological tools and drugs. In fact, emerging evidence is demonstrating that one of
the key factors characterizing the binding performances of a ligand is the Residence Time (RT),
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which is the time for which the ligand interacts with its target.31 In this respect, a kinetic radioligand
binding assay was recently set up to determine the RT of ligands in their interaction with TSPO by
using a theoretical mathematical model successfully applied to other ligandꢀtarget systems.32ꢀ35 In
principle, this kinetic parameter could represent a crucial key requirement for the suitability of a
novel fluorescent probe, as it is reasonable to speculate that a relatively high RT may be
advantageous for imaging studies. Thus, experiments were performed to measure the Kon and Koff
rate constants of our previously published indoleꢀbased fluorescent probe 2, together with that of
the highest affinity compound 8; then, RTs for 2 and 8 were derived from the values of the
corresponding Koff and compared with that of 132 (Table 2). Results of these inspections
demonstrated that 2 and 1 are rapid dissociating competitors of the [3H]ꢀ1 binding site, with RT
values of 36 ± 2 min and 34 ± 3 min, respectively. In contrast, derivative 8 shows a longer value
(RT = 55 ± 4 min), prompting us to develop a fluorescent phenylquinazolineꢀbased fluorescent
probe with improved RT.
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Chart 2. General structure of 4ꢀphenylquinazoline TSPO ligands (5ꢀ14) and fluorescent probes
(15, 16).
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Design of 4ꢀphenylquinazolineꢀbased fluorescent probes, TSPO Affinity and Kinetic Assays.
The inspection of the molecular alignment shown in Figure 1B, the high
nanomolar/subnanomolar TSPO affinity of the bulky phenylquinazoline ligands (Table 1), along
with the RT value of compound 8 strongly supported the design of fluorescent probes from this
chemotype, by inserting the fluorogenic 7ꢀnitroꢀ2,1,3ꢀbenzoxadiazolꢀ4ꢀyl (NBD) moiety at the 4ꢀ
phenyquinazolineꢀ2ꢀcarboxylic acid through an alkylenediamine spacer (tetramethylene, n=4, and
hexamethylene, n=6, diamino spacer for compounds 15 and 16, respectively, Chart 2). The length
of the spacer alkyl chain was modified to introduce some flexibility that would favor selfꢀadaptation
of the ligands into the L4 pocket of the receptor binding site, so as to maintain high TSPO affinity.
The wellꢀknown NBD group was selected as the fluorophore because its small size does not
generally affect the affinity of the parent ligand for the target protein. Furthermore, NBD is
solvatochromic, in that it exhibits low fluorescent activity in polar and protic environments but
becomes highly fluorescent in nonpolar solvents or when bound to membranes or to hydrophobic
clefts such as protein pockets.36 These properties suggest that an NBD fluorescent tracer may
possess reduced background fluorescence from nonbound fluorescent tracer allowing for both
homogeneous detection and use in imaging applications.
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As reported in Table 1, the fluorescent probes 15 and 16 showed significant TSPO affinity, with
comparable Ki values in the nanomolar range, suggesting that the two linkers (4 or 6 methylene
units, 15 and 16, respectively) are equally effective for a successful accommodation of the molecule
in the receptor L4 cleft of the TSPO binding site.
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Table 1. TSPO Binding Affinity of Quinazoline Derivatives 5ꢀ16.
8
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I% (1 µM)
or
i (nM)a
1.1 ± 0.1
0.23 ± 0.02
0.51 ± 0.05
0.47 ± 0.05
3.5 ± 0.4
0.50 ± 0.05
3.5 ± 0.4
0.61 ± 0.05
23.5 ± 2.5%
40.5 ± 2.0%
19.2 ± 2.9
25.3 ± 4.0
3.39 ± 0.34c
cmpd
R1
R2
R3
n
K
5b
6b
7
8
9
10
11
12
13
14
15
16
1
H
H
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
H
Cl
H
Cl
H
Cl
H
Cl
H
H
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
4
6
ꢀ
OSO2ꢀC6H4ꢀ4ꢀCH3
OSO2ꢀC6H4ꢀ4ꢀCH3
OCOꢀC6H4ꢀ4ꢀCH3
OCOꢀC6H4ꢀ4ꢀCH3
NHCOꢀC6H4ꢀ4ꢀCH3 CH2CH3
NHCOꢀC6H4ꢀ4ꢀCH3 CH2CH3
OSO2ꢀC6H4ꢀ4ꢀCH3
OCOꢀC6H4ꢀ4ꢀCH3
CH2C6H4ꢀ4ꢀOOSO2ꢀC6H4ꢀ4ꢀCH3
CH2C6H4ꢀ4ꢀOCOꢀC6H4ꢀ4′ꢀCH3
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
aThe concentration of test molecules that inhibited [3H]ꢀ1 binding to rat kidney mitochondrial
membranes by 50% (IC50) was determined with six concentrations of the compounds, each
performed in triplicate. Ki values and inhibition percentages at 1 ꢁM are the mean ± SEM of three
determinations. bData taken from ref. 25. cData taken from ref. 32.
Compound 15 was selected as representative and evaluated in competition kinetic association
assays, revealing Kon= 7.10 ± 2.00 x 105 Mꢀ1 minꢀ1, and Koff= 0.019 ± 0.005 minꢀ1 (Table 2). The RT
was calculated to be 53 ± 7 min (Table 2), which is comparable to that of 8. Since 15 is a slower
dissociation competitor than both fluorescent indoleꢀbased probe 2 and 1, this ligand was used for
further evaluation in experiments aimed at labeling TSPO in U343 glioma cells.
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Table 2. TSPO binding affinity and kinetic parameters of compounds 1, 2, 10, and 17.
7
8
9
TSPO ligand
Equilibrium Ki
(nM)
Kon (Mꢀ1, minꢀ1)
Koff (minꢀ1)
RT = 1/Koff
(min)
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12.1 ± 1.0a
0.51 ± 0.05
19.2 ± 2.9
2.09 ± 0.28 x 106
6.23 ± 0.56 x 107
7.10 ± 2.00 x 105
0.028 ± 0.001
0.018 ± 0.001
0.019 ± 0.005
36 ± 2
55 ± 4
53 ± 7
34 ± 3b
2
8
15
3.39 ± 0.34b
9.30 ± 0.94 x 106b 0.029 ± 0.003b
1
Ki were obtained from [3H]ꢀ1 competition binding experiments at equilibrium. Kon and Koff values
were obtained from competitive association kinetics assays using [3H]ꢀ1 as a probe. Values are
means ± SEM of three independent experiments, each performed in duplicate. aData taken from ref.
25; bData taken from ref. 32.
Chemistry
The synthetic protocols for the preparation of compounds 7ꢀ14 are outlined in Scheme 1.
Carboxylic acids 17 and 18, key intermediates in the synthesis of target compounds 7ꢀ14, were
prepared by condensation of 2ꢀaminobenzophenone or 2ꢀaminoꢀ2′ꢀchloroꢀbenzophenone with
glyoxylic acid in the presence of ammonium acetate, followed by light irradiation with 20 W
halogen tungsten lamp, as previously reported.23,24
Compounds 7ꢀ14 were obtained by direct coupling reaction of acids 17 and 18 and the
appropriate secondary amines 19ꢀ23, by carboxylate activation with using hydroxybenzotriazole
(HOBt) and OꢀbenzotriazoleꢀN,N,N′,N′ꢀtetramethylꢀuroniumꢀhexafluoroꢀphosphate (HBTU) in the
presence of Hünig’s base (N,Nꢀdiisopropylethylamine, DIEA) in dry THFꢀDMF.
Scheme 1
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Reagents and conditions: (a) DIEA, HOBt, HBTU, THF/DMF 7:2, room temperature, overnight,
58−85%.
Secondary amines 19 and 20 were promptly prepared by reductive alkylation of the appropriate
aldehyde (28 and 29, respectively) with ethylamine using sodium borohydride (NaBH4) in EtOH.
Conversely, the preparation of amines 21ꢀ23 was not immediate even if straightforward (Scheme 2).
Reductive alkylation of nitrobenzaldehyde with ethylamine furnished the Nꢀ(4ꢀ
nitrobenzyl)ethanamine 24 which, after classical Boc protection (compound 25), was reduced with
zinc powder in acetic acid to give the corresponding amino derivative 26. Subsequent coupling with
4ꢀmethylbenzoic acid, using HOBt and HBTU in the presence of DIEA, furnished amide 27, which,
after Boc deprotection with TFA in dry CH2Cl2, provided the desired secondary amine 21.
Scheme 2
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Reagents and conditions: (a) ethylamine, EtOH, room temperature, 12 h, then NaBH4, 0 °C,
71−97%; (b) Boc2O, EtOAc, room temperature, 1h, 92%; (c) Zn, AcOH, room temperature, 10 min,
90%; (d) 4ꢀmethylbenzoic acid, DIEA, HOBt, HBTU, THF/DMF 7:2, room temperature, overnight,
72%; (e) TFA/CH2Cl2 3:7, room temperature, 30 min, 99%; (f) NaBH4, MeOH, 0 °C, 93−94%; (g)
MsCl, TEA, CH2Cl2, 0 °C, 45 min; (h) NaN3, DMF, 80 °C, 1 h, 88−91%; (i) HꢀCube Pro™, fullꢀH2
mode, 30 °C, 10 bar, 1 mL minꢀ1 flow rate. 10% Pd/C CatCart cartridge, MeOH, 90−96%.
The preparation of secondary amines 22 and 23 started with the reduction of 4ꢀ(4ꢀ
toluenesulfonyloxy)benzaldehyde 28 and 4ꢀ(4ꢀmethylbenzoyloxy)benzaldehyde 29, to the
corresponding alcohols 30 and 31, respectively. The transformation of the latter into amines 36 and
37 was performed by mesylation (compound 32 and 33), subsequent substitution reaction with
sodium azide (compound 34 and 35), and reduction by means of a continuousꢀflow hydrogenation
protocol employing a fixedꢀbed catalyst (HꢀCube Pro™, Thales Nanotechnology Inc.) and using
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catalyst cartridges filled with 10% Pd/C. Finally, reductive alkylation with benzaldehydes 28 and 29
furnished the desired secondary amines 22 and 23.
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The general synthetic pathway yielding the novel phenylquinazolineꢀ2ꢀcarboxamides 15 and 16 in
the best yields is outlined in Scheme 3. After activation with thionyl chloride, the carboxylic acid 18
was coupled with NꢀBocꢀ1,4ꢀbutanediamine or NꢀBocꢀ1,6ꢀhexanediamine in the presence of TEA at
room temperature to yield derivatives 38 and 39, respectively. Boc deprotection with TFA in dry
CH2Cl2 gave the corresponding amine 40 and 41, which were then reacted with the fluorescent 4ꢀ
chloroꢀ7ꢀnitrobenzofurazan in dry DMF, at room temperature and in the presence of TEA, yielding
the desired fluorescent compounds 15 and 16.
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Scheme 3.
Reagents and conditions: (a) SOCl , reflux, 2 h, then NꢀBocꢀ1,4ꢀbutanediamine or NꢀBocꢀ1,6ꢀ
2
hexanediamine, TEA, THF, room temperature, 48 h, 70−71%; (b) TFA/CH2Cl2 1:2, room
temperature, 3 h, 74−86%; (c) 4ꢀchloroꢀ7ꢀnitrobenzofurazan, DMF, TEA, room temperature,
overnight, 55−72%.
Spectroscopic Properties of Fluorescent Ligand 15
The ultraviolet absorption and the emission spectra of compound 15 were measured to investigate
the spectroscopic properties of the ligand and how these were affected by the environment.
Compound 15, dissolved in dimethyl sulfoxide (DMSO), was diluted to a final concentration of 10
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ꢁM in different assay solutions (from aqueous to 60% v/v dioxaneꢀwater phosphate buffered saline
(PBS)). The percentage of DMSO did not exceed 1% of the final assay volume solution.
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Figure 2 shows the absorption spectra of compound 15. In an aqueous solution, the absorption
spectra of compound 15 is characterized by two absorption maxima at 335 nm and 485 nm with
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extinction coefficients of ε ε
= 5118 Mꢀ1 cmꢀ1 and = 9210 Mꢀ1 cmꢀ1, respectively.
0.16
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-0.02
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Figure 2. Absorption spectra of compound 15 at 10 ꢁM in solutions varying from aqueous to 60%
dioxaneꢀwater (v/v) PBS.
When 15 was dissolved in PBS solution containing 20% of dioxane, the wavelengths of the
absorption maxima remained almost constant, but the spectrum was characterized by an increase in
the extinction coefficients and a small hypsochromic shift of the 485 nm band. A further increase in
hydrophobicity of the medium (40% dioxane water (v/v) PBS) caused a slight increase in the
extinction coefficients. A decrease in the extinction coefficients was obtained further increasing the
hydrophobicity of the medium (60% dioxane water (v/v) PBS), even if these values remained
higher with respect to those observed in aqueous solution.
Figure 3 shows the emission spectra of compound 15. As expected, the decrease in polarity of the
environment (from the aqueous buffer to 60% dioxane in PBS) induced an approximately 3ꢀfold
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increase in quantum yield. This phenomenon was accompanied by a slight blueꢀshift of the
emission maximum from 542 to 538 nm.
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PBS/Dioxane=40/60
Figure 3. The influence of the polarity of the medium on the fluorescence of compound 15 was
investigated by the addition of dioxane (indicated in % v/v) in PBS measured at a λmax of 470 nm.
Fluorescent labeling of human glioblastoma cell line U343
Compounds 15 and 16 were then evaluated for their ability to specifically label TSPO in human
glioblastoma cell line U343. Representative confocal images in Figure 4 demonstrate uniform
cytoplasm labeling of cells with a granular structure resembling a mitochondrial expression pattern.
Labeling was clearly detected using compound 15 at 3 µM (Figure 4A) and no significant
difference was observed with higher ligand concentrations (Figure 4B). Similar results were
obtained with 16 (data not shown).
Specific mitochondrial labeling was confirmed by the coꢀlocalization of our TSPO affinity probes
with the mitochondrionꢀspecific probe MitoTracker Red. As shown in Figure 4C ̶ E, merged images
revealed a clearly overlap between the MitoTracker Red signal and the green one of compound 15.
Similar results were obtained with 16 (data not shown).
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Figure 4. Compound 15 specifically labels mitochondria of human glioblastoma cell line U343.
Panels A–C: U343 cells stained with 15 (3, 4 and 5 ꢁM); panels D–F: comparison of U343 cells
stained with mitochondria specific dye MitoTracker Red (D) and with compound 15 (E) and
merged image (F); panels G, H: human glioblastoma cell line U343 stained with 15 (3 ꢁM) in the
presence of 1 (10 and 50 ꢁM, respectively).
Moreover, displacement assays using the nonꢀfluorescent ligand 1 (Figure 4G, and H) indicate
that the tested compounds specifically label TSPO. The fluorescent staining obtained using 3 µM 15
was displaced when 1 was used at concentrations higher than 10 µM and the complete displacement
staining was achieved using 50 µM 1.
Under this experimental conditions, 15 and 16 did not show significant cytotoxic activity, as
determined by MTT method (see Figure S1 in Supporting Information).
Conclusion
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Molecular modeling studies and results from a biological evaluation of a small “inꢀhouse”
collection of bulky phenylquinazoline ligands strongly supported the design of fluorescent probes
from this chemotype. The fluorogenic 7ꢀnitroꢀ2,1,3ꢀbenzoxadiazolꢀ4ꢀyl (NBD) moiety was then
inserted at the 4ꢀphenyquinazolineꢀ2ꢀcarboxylic acid through an alkylenediamine spacer yielding
novel fluorescently tagged TSPO ligands endowed with high affinity, attractive spectroscopic
properties and improved RT with respect to our previously developed fluorescent indoleꢀbased
probe. Finally, results from fluorescence microscopy showed that these probes specifically label the
TSPO at the mitochondrial level U343 glioma cells, thereby representing potential new imaging
biomarkers for this protein.
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Experimental Section
Chemistry. All chemicals were purchased from SigmaꢀAldrich (Milan, Italy) or Fluorochem Ltd
(Hadfield, United Kingdom) and were of the highest purity. All solvents were reagent grade and,
when necessary, were purified and dried by standard methods. All reactions requiring anhydrous
conditions were conducted under a positive atmosphere of nitrogen in ovenꢀdried glassware.
Standard syringe techniques were used for anhydrous addition of liquids. Reactions were routinely
monitored by TLC performed on aluminumꢀbacked silica gel plates (Merck DC, Alufolien
Kieselgel 60 F254) with spots visualized by UV light (λ = 254, 365 nm) or using a KMnO4 alkaline
solution. Solvents were removed using a rotary evaporator operating at a reduced pressure of
∼10
Torr. Organic solutions were dried over anhydrous Na2SO4. Chromatographic separations were
performed on silica gel (silica gel 60, 0.015−0.040 mm; Merck DC) columns. Melting points were
determined on a Stuart SMP30 melting point apparatus in open capillary tubes and are uncorrected.
1H and 13C NMR spectra were recorded at 400 and 100 MHz respectively on a Bruker AscendTM
400 spectrometer. Chemical shifts are reported in δ (ppm) relative to the internal reference
1
tetramethylsilane (TMS). When the amide nitrogen bears two substituents, the H and 13C NMR
1
spectra show the presence of two different rotamers in equilibrium. Copies of H and 13C NMR
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spectra are included in the Supporting Information. Lowꢀresolution mass spectra were recorded on a
Finnigan LCQ DECA TermoQuest mass spectrometer in electrospray positive and negative
ionization modes (ESIꢀMS). Highꢀresolution mass spectra were recorded on a ThermoFisher
scientific Orbitrap XLTM mass spectrometer in electrospray positive ionization modes (ESIꢀMS).
Ultraviolet absorption spectra were recorded on a PerkinElmer EnSightTM Multimode Plate
Reader. Fluorescence spectra were recorded with a Gemini XS Molecular Device Spectramax
fluorescence Spectrophotometer. High performance liquid chromatography (HPLC) was performed
on a Shimadzu SPD 20A UV/VIS detector (λ = 220 nm) using Cꢀ18 column Phenomenex Synergi
Fusion – RP 80A (75 x 4.60 mm; 4 µm) at 25 °C using a mobile phase A (water + 0.1%
trifuoracetic acid (TFA)) and B (MeCN + 0.1% TFA) at a flow rate of 1 mL minꢀ1. The following
gradient was applied: isocratic elution for 1 min at 10% of solvent B, linear increase from 10% to
95% of solvent B over 10 min, hold at 95% solvent B for 3 min. The purity of all final compounds
was determined by HPLC, and it was found to be higher than 95%. Aldehydes 28 and 29 were
commercially available. The quinazolineꢀ2ꢀcarboxylic acids 17 and 18, the Nꢀ(4ꢀ
nitrobenzyl)ethanamine 24 and 4ꢀ((Nꢀethylꢀ4ꢀphenylquinazolineꢀ2ꢀcarboxamido)methyl)ꢀphenyl 4ꢀ
methylbenzenesulfonate 7 were prepared as previously reported.23
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4ꢀ((4ꢀ(2ꢀChlorophenyl)ꢀNꢀethylquinazolineꢀ2ꢀcarboxamido)methyl)ꢀphenyl
4ꢀ
Methylbenzenesulfonate (8). A solution of carboxylic acid 18 (305 mg, 1.07 mmol), HOBt (328
mg, 2.14 mmol), HBTU (812 mg, 2.14 mmol), and DIEA (0.76 mL, 4.28 mmol) in dry THF−DMF
(7:2, 9 mL) was added to a solution of amine 19 (327 mg, 1.07 mmol) in dry THF (3 mL) under N2
atmosphere. The reaction was stirred at room temperature overnight and then concentrated in vacuo.
The residue was dissolved in EtOAc (100 mL) and washed with NaHCO3, saturated aqueous
solution (3 × 30 mL) and brine. The organic solution was dried (Na2SO4), filtered, and concentrated
in vacuo. Purification by column chromatography on silica gel (EtOAc/ hexane, 7:3) provided
compound 8 as a white solid (520 mg, 85%); mp 68−71 °C. 1H NMR (400 MHz, CDCl3): δ = 8.17–
8.09 (m, 1H), 7.96–7.90 (m, 1H), 77.68–7.53 (m, 4H), 7.53–7.39 (m, 3H), 7.35–7.23 (m, 5H), 6.93,
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6.87 (2d, J = 8.5 Hz, 2H), 4.82–4.63, 4.45 (m, s, 2H), 3.56–3.44, 3.23 (m, q, J = 7.1 Hz, 2H), 2.41
(s, 3H), 1.15, 1.07 ppm (2t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ = 168.04, 167.99,
167.69, 167.09, 158.20, 158.10, 150.61, 150.51, 149.25, 149.09, 145.54, 145.50, 136.31, 135.79,
135.73, 135.59, 134.86, 134.82, 132.89, 132.84, 132.48, 131.09, 131.06, 131.00, 130.11, 130.07,
129.92, 129.88, 129.60, 129.42, 129.27, 129.20, 128.85, 128.81, 128.68, 128.63, 128.14, 127.14,
127.12, 127.07, 123.32, 123.22, 122.69, 122.61, 51.50, 47.17, 43.17, 40.19, 27.06, 21.86, 13.88,
12.50 ppm. HRMS (ESI): m/z calculated for C31H27ClN3O4S + H+ [M + H+]: 572.1405. Found
572.1384.
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4ꢀ((NꢀEthylꢀ4ꢀphenylquinazolineꢀ2ꢀcarboxamido)methyl)phenyl
4ꢀmethylbenzoate
(9).
Compound 9 was obtained as a white solid (291 mg, 58%) from acid 17 (250 mg, 1.00 mmol) and
amine 20 (269 mg, 1.00 mmol), according to the procedure described for amide 8; mp 70−72 °C. 1H
NMR (400 MHz, CDCl3): δ = 8.20–8.13 (m, 2H), 8.11–8.07 (m, 2H), 7.96–7.93 (m, 1H), 7.84–7.81
(m, 1H), 7.73–7.71 (m, 1H), 7.66–7.64 (m, 1H), 7.59–7.53 (m, 5H), 7.31 (d, J = 8.0 Hz, 2H), 7.22,
7.17 (2d, J = 8.4 Hz, 2H), 4.89, 4.55 (2s, 2H), 3.61, 3.30 (2q, J = 7.1 Hz, 2H), 2.46 (s, 3H), 1.27,
1.19 ppm (2t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ = 169.30, 167.92, 167.51, 165.38,
165.31, 158.40, 158.29, 151.21, 151.17, 150.73, 150.52, 144.65, 144.55, 136.87, 136.74, 134.79,
134.35, 134.29, 130.41, 130.38, 130.35, 129.71, 129.45, 129.43, 129.37, 129.27, 128.76, 128.73,
128.53, 128.46, 127.30, 126.95, 126.84, 122.61, 122.55, 122.07, 121.98, 51.55, 46.91, 42.84, 39.91,
21.91, 13.83, 12.48 ppm. HRMS (ESI): m/z calculated for C32H28N3O3 + H+ [M + H+]: 502.2125.
Found 502.2109.
4ꢀ((4ꢀ(2ꢀChlorophenyl)ꢀNꢀethylquinazolineꢀ2ꢀcarboxamido)methyl)phenyl 4ꢀmethylbenzoate
(10). Compound 10 was obtained as a white solid (322 mg, 60%) from acid 18 (285 mg, 1.00
mmol) and amine 20 (270 mg, 1.00 mmol), according to the procedure described for amide 8; mp
1
80−82 °C. H NMR (400 MHz, CDCl3): δ = 8.21–8.16 (m, 1H), 8.10–8.06 (m, 2H), 7.99–7.92 (m,
1H), 7.70–7.58 (m, 2H), 7.56–7.39 (m, 6H), 7.31 (d, J = 7.9 Hz, 2H), 7.21, 7.14 (2d, J = 8.4 Hz,
2H), 4.96–4.79, 4.59–4.51 (2m, 2H), 3.67–3.54, 3.30 (m, q, J = 7.1 Hz, 2H), 2.45 (s, 3H), 1.26, 1.15
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ppm (2t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ = 168.04, 167.66, 167.20, 165.37,
165.28, 158.40, 158.31, 150.72, 150.65, 150.56, 150.53, 144.64, 144.55, 135.81, 135.71, 134.80,
134.73, 134.18, 132.94, 131.17, 131.06, 131.03, 131.01, 130.37, 130.34, 130.08, 129.72 129.45,
129.43, 129.33, 129.27, 128.76, 128.70, 127.12, 127.09, 127.05, 126.96, 126.84, 123.31, 123.22,
122.07, 121.98, 51.54, 47.06, 42.85, 40.01, 21.91, 13.82, 12.50 ppm. HRMS (ESI): m/z calculated
for C32H27ClN3O3 + H+ [M + H+]: 536.1735. Found 536.1723.
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N
ꢀEthylꢀ
N
ꢀ(4ꢀ(4ꢀmethylbenzamido)benzyl)ꢀ4ꢀphenylquinazolineꢀ2ꢀcarboxamide
(11).
Compound 11 was obtained as a yellow solid (340 mg, 68%) from acid 17 (250 mg, 1.00 mmol)
and amine 21 (382 mg, 1.00 mmol), according to the procedure described for amide 8; mp 100–102
1
°C. H NMR (400 MHz, CDCl3): δ = 8.32, 8.05 (2br s, 1H, exchangeable with deuterium oxide),
8.15–8.11 (m, 2H), 7.95–7.90 (m, 1H), 7.78–7.70 (m, 5H), 7.66–7.61 (m, 2H), 7.58–7.51 (m, 3H),
7.47–7.4 (m, 2H), 7.22 ,7.15 (2d, J = 7.9 Hz, 2H), 4.85, 4.50 (2s, 2H), 3.58, 3.27 (2q, J = 7.1 Hz,
2H), 2.38, 2.33 (2s, 3H), 1.24, 1.19 ppm (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ =
169.27, 169.20, 168.03, 167.52, 165.84, 165.75, 158.32, 151.13, 142.45, 142.14, 137.95, 137.83,
136.75, 136.73, 134.33, 134.27, 132.98, 132.54, 132.16, 132.10, 130.43, 130.37, 129.49, 129.32,
129.27, 128.93, 128.74, 128.70, 128.50, 128.43, 127.28, 127.22, 127.19, 122.49, 120.78, 120.34,
51.72, 47.09, 42.8, 39.88, 21.61, 21.58, 13.82, 12.50 ppm. HRMS (ESI): m/z calculated for
C32H29N4O2 + H+ [M + H+]: 501.2285. Found 501.2275.
4ꢀ(2ꢀChlorophenyl)ꢀNꢀethylꢀN-(4ꢀ(4ꢀmethylbenzamido)benzyl)quinazolineꢀ2ꢀcarboxamide
(12). Compound 12 was obtained as a yellow solid (396 mg, 74%) from acid 18 (285 mg, 1.00
mmol) and amine 21 (382 mg, 1.00 mmol), according to the procedure described for amide 8; mp
1
110–112 °C. H NMR (400 MHz, CDCl3): δ = 8.18–8.15 (m, 1H), 8.14, 7.99 (br s, 1H,
exchangeable with deuterium oxide), 7.96–7.93 (m, 1H), 7.78–7.75 (m, 2H), 7.68–7.56 (m, 4H),
7.56–7.48 (m, 2H), 7.46–7.38 (m, 4H), 7.26–7.21 (m, 2H), 4.90–4.76, 4.51 (m, s, 2H), 3.62–3.52,
3.27 (m, q, J = 7.1 Hz, 2H), 2.39, 2.38 (2s, 3H), 1.22, 1.14 ppm (2t, J = 7.1 Hz, 3H). 13C NMR (100
MHz, CDCl3): δ = 168.02, 167.94, 167.70, 165.80, 165.73, 158.37, 158.33, 150.60, 150.53, 142.49,
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142.31, 137.91, 137.69, 135.76, 135.69, 134.78, 134.72, 133.07, 132.91, 132.48, 132.19, 132.10,
131.14, 131.03, 131.01, 130.07, 130.05, 129.52, 129.45, 129.31, 129.28, 129.24, 128.96, 128.74,
128.69, 127.24, 127.19, 127.12, 127.09, 127.07, 127.03, 123.25, 123.18, 120.69, 120.32, 51.69,
47.22, 42.83 , 39.95, 21.61, 13.82, 12.51 ppm. HRMS (ESI): m/z calculated for C32H28ClN4O2 + H+
[M + H+]: 535.1895. Found 535.1889.
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(((4ꢀPhenylquinazolineꢀ2ꢀcarbonyl)azanediyl)bis(methylene))bis(4,1ꢀphenylene)
bis(4ꢀ
methylbenzenesulfonate) (13). Compound 13 was obtained as a yellow solid (539 mg, 70%) from
acid 17 (250 mg, 1.00 mmol) and amine 22 (538 mg, 1.00 mmol), according to the procedure
described for amide 8; mp 115–117 °C. 1H NMR (400 MHz, CDCl3): δ = 8.17–8.12 (m, 2H), 7.97–
7.93 (m, 1H), 7.73–7.66 (m, 7H), 7.58–7.55 (m, 3H), 7.33–7.26 (m, 8H), 6.96, 6.92 (2d, J = 8.5 Hz,
4H), 4.64, 4.37 (2s, 4H), 2.46 ppm (s, 6H).13C NMR (100 MHz, CDCl3): δ = 169.37, 167.99,
157.65, 151.15, 149.35, 149.22, 145.62, 145.59, 136.58, 135.42, 134.99, 134.51, 132.66, 132.56,
130.59, 130.35, 130.05, 129.98, 129.95, 129.59, 129.34, 128.78, 128.67, 128.60, 127.36, 122.77,
122.70, 122.62, 50.91, 46.67, 21.89 ppm. HRMS (ESI): m/z calculated for C43H36N3O7S2 + H+ [M +
H+]: 770.1989. Found 770.1976.
(((4ꢀPhenylquinazolineꢀ2ꢀcarbonyl)azanediyl)bis(methylene))bis(4,1ꢀphenylene)
bis(4ꢀ
methylbenzoate) (14). Compound 14 was obtained as a yellow solid (467 mg, 67%) from acid 17
(250 mg, 1.00 mmol) and amine 23 (465 mg, 1.00 mmol), according to the procedure described for
amide 8; mp 120–122 °C. 1H NMR (400 MHz, CDCl3): δ = 8.19–8.15 (m, 2H), 8.11–8.08 (m, 4H),
7.97–7.93 (m, 1H), 7.76–7.73 (m, 2H), 7.67–7.63 (m, 1H), 7.58–7.55 (m, 3H), 7.53–7.48 (m, 4H),
7.32 (d, J = 8.0 Hz, 4H), 7.23, 7.18 (2d, J = 8.4 Hz, 4H), 4.79, 4.47 (2s, 4H), 2.46 ppm (s, 6H). 13C
NMR (100 MHz, CDCl3): δ = 169.17, 167.92, 165.24, 165.16, 157.88, 151.09, 150.67, 150.51,
144.51, 144.42, 136.57, 134.22, 133.98, 133.49, 130.31, 130.25, 130.22, 130.02, 129.32, 129.29,
128.59, 128.48, 127.17, 126.81, 126.72, 122.47, 122.01, 121.96, 50.59, 46.30, 21.77 ppm. HRMS
(ESI): m/z calculated for C45H36N3O5 + H+ [M + H+]: 698.2649. Found 698.2640.
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Nꢀ(4ꢀ(4ꢀNitrobenzo[c][1,2,5]oxadiazolꢀ7ꢀylamino)butyl)ꢀ4ꢀ(2ꢀchlorophenyl)quinazolineꢀ2ꢀ
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carboxamide (15). To a solution of amine 40 (355 mg, 0.28 mmol) in dry DMF (10 mL), a solution
of 4ꢀchloroꢀ7ꢀnitrobenzofurazan (55 mg, 0.28 mmol) in dry DMF (1 mL) was added dropwise.
After addition of a CH2Cl2 solution (1 mL) of TEA (0.04 mL, 0.30 mmol), the reaction mixture was
stirred at room temperature overnight and then concentrated in vacuo. The residue was taken up
with 0 °C cooled water (30 mL) and extracted with CH2Cl2 (3 × 30 mL). The combined organic
phases were washed with brine (10 mL), dried (Na2SO4), filtered, and concentrated in vacuo.
Purification by column chromatography on silica gel (EtOAc) provided compound 15 as a yellowꢀ
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brownish solid (104 mg, 72%); mp 57−59 °C. H NMR (400 MHz, DMSOꢀd6): δ = 9.58 (t, J = 5.1
Hz, 1H, exchangeable with deuterium oxide), 9.05 (t, J = 6.0 Hz, 1H, exchangeable with deuterium
oxide), 8.46 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H), 8.17–8.12 (m, 1H), 7.84–7.80 (m, 1H),
7.74–7.72 (m, 1H), 7.69–7.59 (m, 4H), 6.45 (d, J = 9.2 Hz, 1H), 3.54–3.53 (m, 2H), 3.42 (q, J = 9.6
Hz, 2H), 1.76–1.71 ppm (m, 4H). 13C NMR (100 MHz, DMSOꢀd6): δ = 167.73, 163.14, 154.54,
150.17, 135.85, 135.70, 132.01, 131.93, 131.69, 130.34, 130.10, 129.41, 127.97, 127.06, 123.42,
26.99, 21.22 ppm. HRMS (ESI): m/z calculated for C25H20ClN7O4 + H+ [M + H+]: 517.1265. Found
517.1272.
Nꢀ(6ꢀ(4ꢀNitrobenzo[c][1,2,5]oxadiazolꢀ7ꢀylamino)hexyl)ꢀ4ꢀ(2ꢀchlorophenyl)quinazolineꢀ2ꢀ
carboxamide (16). Compound 16 was obtained as a yellowꢀbrownish solid (114 mg, 75%) from
amine 41 (107 mg, 0.28 mmol), according to the procedure described for compound 15; mp 57−58
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°C. H NMR (400 MHz, DMSOꢀd6): δ = 9.57 (br s, 1H, exchangeable with deuterium oxide), 9.00
(t, J = 6.0 Hz, 1H, exchangeable with deuterium oxide), 8.49 (d, J = 8.8 Hz, 1H), 8.24 (d, J = 8.4
Hz, 1H), 8.16–8.12 (m, 1H), 7.84–7.80 (m, 1H), 7.74–7.72 (m, 1H), 7.69–7.59 (m, 4H), 6.41 (d, J =
8.8 Hz, 1H), 3.47–3.45 (m, 2H), 3.39–3.34 (m, 2H), 1.72–1.69 (m, 2H), 1.62–1.58 (m, 2H), 1.41–
1.39 ppm (m, 4H). 13C NMR (100 MHz, DMSOꢀd6): δ = 167.72, 163.04, 154.64, 150.19, 135.82,
135.72, 132.01, 131.93, 131.71, 130.31, 130.10, 129.44, 127.98, 127.05, 123.42, 29.47, 26.60 ppm.
HRMS (ESI): m/z calculated for C27H24ClN7O4 + H+ [M + H+]: 545.1578. Found 545.1572.
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4ꢀ((Ethylamino)methyl)phenyl 4ꢀmethylbenzenesulfonate (19). To a solution of 4ꢀ
formylphenyl 4ꢀmethylbenzenesulfonate 28 (914 mg, 3.31 mmol) in dry ethanol (10 mL) was added
ethylamine (2 M solution in THF, 1.80 mL, 3.64 mmol) under a nitrogen atmosphere. After stirring
at room temperature for 12 h, the mixture was cooled at 0 °C, and NaBH4 was added portion wise
until disappearance of the intermediate imine (TLC analysis). The reaction mixture, after addition
of water (20 mL), was concentrated in vacuo and extracted with EtOAc (3 × 40 mL). The combined
organic phases were washed with a saturated solution of NaHCO3 (3 × 20 mL) and brine (10 mL),
dried (Na2SO4), filtered, and concentrated in vacuo to yield the title compound as a paleꢀyellow oil
(970 mg, 96%). 1H NMR (400 MHz, CDCl3): δ = 7.69 (d, J = 8.1 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H),
7.23 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 8.4 Hz, 2H), 3.74 (s, 2H), 2.65 (q, J = 7.1 Hz, 2H), 2.53 (s,
3H), 1.62 (br s, 1H, exchangeable with deuterium oxide), 1.12 ppm (t, J = 7.1 Hz, 3H). MS (ESI)
m/z (%): 306 (100) [M + H]+.
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4ꢀ((Ethylamino)methyl)phenyl 4ꢀmethylbenzoate (20). Compound 20 was obtained as a paleꢀ
yellow oil (637 mg, 71%) from aldehyde 29 (800 mg, 3.33 mmol) and ethylamine (1.83 mL, 3.66
1
mmol), according to the procedure described for amine 19. H NMR (400 MHz, CDCl3): δ = 8.09
(d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H),
5.42–5.17 (m, 1H, exchangeable with deuterium oxide), 3.82 (s, 2H), 2.71 (q, J = 7.1 Hz, 2H), 2.45
(s, 3H), 1.15 ppm (t, J = 7.1 Hz, 3H). MS (ESI) m/z (%): 270 (100) [M + H]+.
mꢀ(4ꢀ((Ethylamino)methyl)phenyl)ꢀ4ꢀmethylbenzamide trifluoroacetate (21). Compound 27
(800 mg, 2.17 mmol) was treated with a mixture of TFA and CH2Cl2 (3:7, 14 mL). After stirring at
room temperature for 30 min the mixture was concentrated in vacuo to give the title compound as a
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hygroscopic solid (822 mg, 99%). H NMR (400 MHz, DMSOꢀd6): δ = 10.28 (br s, 1H,
exchangeable with deuterium oxide), 8.70 (br s, 2H, exchangeable with deuterium oxide), 7.88 (d, J
= 8.2 Hz, 2H), 7.83 (d, J = 7.9 Hz, 2H), 7.45 (d, J = 8.2 Hz, 2H), 7.35 (d, J = 7.9 Hz, 2H), 4.10 (s,
2H), 2.97 (q, J = 7.1 Hz, 2H), 2.39 (s, 3H), 1.21 ppm (t, J = 7.1 Hz, 3H). MS (ESI) m/z (%): 269
(100) [M + H]+.
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(Azanediylbis(methylene))bis(4,1ꢀphenylene) bis(4ꢀmethylbenzenesulfonate) (22). Compound
22 was obtained as a paleꢀyellow oil (944 mg, 97%) from aldehyde 28 (500 mg, 1.81 mmol) and
amine 36 (552 mg, 1.99 mmol) according to the procedure described for compound 19. 1H NMR
(400 MHz, CDCl3): δ = 7.71 (d, J = 8.0 Hz, 4H), 7.30 (d, J = 8.2 Hz, 4H), 7.24 (d, J = 8.2 Hz, 4H),
6.93 (d, J = 8.0 Hz, 4H), 3.73 (s, 4H), 2.45 ppm (s, 6H). MS (ESI) m/z (%): 538 (100) [M + H]+.
(Azanediylbis(methylene))bis(4,1ꢀphenylene) bis(4ꢀmethylbenzoate) (23). Compound 23 was
obtained as a colourless oil (881 mg, 91%) from aldehyde 29 (500 mg, 2.08 mmol) and amine 36
(550 mg, 2.29 mmol) according to the procedure described for compound 19. 1H NMR (400 MHz,
CDCl3): δ = 8.09 (d, J = 8.0 Hz, 4H), 7.41 (d, J = 7.9 Hz, 4H), 7.31 (d, J = 8.0 Hz, 4H), 7.18 (d, J =
7.9 Hz, 4H), 3.85 (s, 4H), 2.45 ppm (s, 6H). MS (ESI) m/z (%): 466 (100) [M + H]+.
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tertꢀButyl ethyl(4ꢀnitrobenzyl)carbamate (25). Nꢀ(4ꢀNitrobenzyl)ethanamine 2423 (1.09 g, 6.05
mmol) was dissolved in ethyl acetate (20 mL) and diꢀtertꢀbutyl dicarbonate (1.32 g, 6.05 mmol) was
added. After stirring at room temperature for 1 h, to the mixture water (50 mL) was added. The
organic layer was separated, and the aqueous phase was extracted with ethyl acetate (3 × 70 mL).
The combined organic phases were washed with brine (10 mL), dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc/hexanes, 1:9)
to give the title compound as an orange oil (1.56 g, 92%). 1H NMR (400 MHz, CDCl3): δ = 8.21 (d,
J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 4.52 (s, 2H), 3.53–3.11 (m, 2H), 1.47 (br s, 9H), 1.12 ppm
(t, J = 7.1 Hz, 3H). MS (ESI) m/z (%): 281 (100) [M + H]+.
tertꢀButyl (4ꢀaminobenzyl)(ethyl)carbamate (26). To an ice cooled solution of tertꢀbutyl
ethyl(4ꢀnitrobenzyl)carbamate 25 (1.00 g, 3.57 mmol) in AcOH (70 mL) zinc powder (2.33 g, 35.7
mmol) was added. After stirring at room temperature for 10 min, the solid was filtered off, and the
solvent was evaporated. The residue was taken up with EtOAc (200 mL) and washed with NaHCO3
saturated aqueous solution twice. The organic phase was dried (Na2SO4), filtered, and concentrated
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in vacuo to give the title compound as a yellow oil (805 mg, 90%). H NMR (400 MHz, CDCl3): δ
= 6.92 (d, J = 8.0 Hz, 2H), 6.50 (d, J = 8.0 Hz, 2H), 4.98 (s, 2H), 4.16 (s, 2H), 3.10 (br s, 2H,
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exchangeable with deuterium oxide), 1.42 (s, 9H), 0.94 ppm (t, J = 7.7 Hz, 3H). MS (ESI) m/z (%):
273 (100) [M + Na]+.
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tertꢀButyl ethyl(4ꢀ(4ꢀmethylbenzamido)benzyl)carbamate (27). Compound 27 was obtained as
a yellow oil (846 mg, 72%) from 4ꢀmethylbenzoic acid (434 mg, 3.19 mmol) and amine 26 (800
mg, 3.19 mmol), according to the procedure described for amide 8. 1H NMR (400 MHz, CDCl3): δ
= 7.98 (br s, 1H, exchangeable with deuterium oxide), 7.78 (d, J = 7.7 Hz, 2H), 7.60 (d, J = 7.9 Hz,
2H), 7.32–7.19 (m, 4H), 4.41 (s, 2H), 3.37–3.11 (m, 2H), 2.43 (s, 3H), 1.49 (s, 9H), 1.07 ppm (t, J =
6.8 Hz, 3H). MS (ESI) m/z (%): 369 (100) [M + H]+.
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4ꢀ(Hydroxymethyl)phenyl 4ꢀmethylbenzenesulfonate (30).37 A solution of 4ꢀformylphenyl 4ꢀ
methylbenzenesulfonate 28 (914 mg, 3.31 mmol) in methanol (10 mL) was refrigerated to 0 °C, and
NaBH4 was added portion wise until disappearance of the starting material (TLC analysis). The
reaction mixture, after addition of water (20 mL), was concentrated in vacuo and extracted with
EtOAc (3 × 40 mL). The combined organic phases were washed with a saturated solution of
NaHCO3 (3 × 20 mL) and brine (10 mL), dried (Na2SO4), filtered, and concentrated in vacuo to
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yield the title compound as a colourless oil (857 mg, 93%). H NMR (400 MHz, CDCl3): δ = 7.71
(d, J = 8.5 Hz, 2H), 7.36–7.24 (m, 4H), 6.97 (d, J = 8.5 Hz, 2H), 4.66 (s, 2H), 2.45 ppm (s, 3H). MS
(ESI) m/z (%): 279 (100) [M + H]+.
4ꢀ(Hydroxymethyl)phenyl 4ꢀmethylbenzoate (31).38 Compound 31 was obtained as a colourless
oil (754 mg, 94%) from aldehyde 29 (795 mg, 3.31 mmol), according to the procedure described for
alcohol 30. 1H NMR (400 MHz, CDCl3): δ = 8.09 (d, J = 7.9 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.31
(d, J = 7.9 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 4.73 (d, J = 5.8 Hz, 2H), 2.46 ppm (s, 3H). MS (ESI)
m/z (%): 243 (100) [M + H]+.
4ꢀ(((Methylsulfonyl)oxy)methyl)phenyl 4ꢀmethylbenzenesulfonate (32). To a solution of 4ꢀ
(hydroxymethyl)phenyl 4ꢀmethylbenzenesulfonate 31 (800 mg, 2.87 mmol) and trimethylamine
(0.48 mL, 3.44 mmol) in dry CH2Cl2 (23 mL), methanesulfonyl chloride (0.27 mL, 3.44 mmol) was
added at 0 °C. After stirring at room temperature for 45 min, the solvent was evaporated and the
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residue was taken up with water (20 mL) and extracted with CH2Cl2 (3 x 20 mL). The combined
organic phases were washed with 1N HCl (2 x 10 mL), NaHCO3, saturated aqueous solution (2 x 10
mL), and brine (10 mL), dried (Na2SO4), filtered, and concentrated in vacuo to give mesylate 38,
which was used without further purification in the subsequent reaction.
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4ꢀ(((Methylsulfonyl)oxy)methyl)phenyl 4ꢀmethylbenzoate (33). Compound 33 was obtained
from alcohol 31, according to the procedure described for mesylate 32 and used without further
purification in the subsequent reaction.
4ꢀ(Azidomethyl)phenyl 4ꢀmethylbenzenesulfonate (34). To a solution of 32 (1.02 g, 2.87
mmol) in DMF (10 mL) sodium azide (560 mg, 8.61 mmol) was added. After stirring at 80 °C for
1h, water (20 mL) was added and the mixture was extracted with CHCl3 (3 × 20 mL). The
combined organic phases were washed with NaHCO3 saturated aqueous solution (20 mL) and brine
(20 mL), dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by silica gel
chromatography (EtOAc/ hexanes, 7:3) to give the title compounds as a paleꢀyellow solid (766 mg,
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88%); mp 100–102 °C. H NMR (400 MHz, CDCl3): δ = 7.70 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.1
Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 7.00 (d, J = 8.4 Hz, 2H), 4.32 (s, 2H), 2.45 ppm (s, 3H). MS
(ESI) m/z (%): 304 (100) [M + H]+.
4ꢀ(Azidomethyl)phenyl 4ꢀmethylbenzoate (35). Compound 35 was obtained as a paleꢀyellow
solid (698 mg, 91%) from mesylate 33 (919 mg, 2.87 mmol), according to the procedure described
for azide 34. Mp 107–109 °C. 1H NMR (400 MHz, CDCl3): δ = 8.09 (d, J = 7.9 Hz, 2H), 7.39 (d, J
= 8.4 Hz, 2H), 7.32 (d, J = 7.9 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 4.38 (s, 2H), 2.46 ppm (s, 3H).
MS (ESI) m/z (%): 268 (100) [M + H]+.
4ꢀ(Aminomethyl)phenyl 4ꢀmethylbenzenesulfonate (36). A suitable glass vial was charged
with 50 mL of an azide 34 solution (0.05 M in MeOH). The reaction parameters (fullꢀH2 mode, 30
°C, 10 bar and 1 mL minꢀ1 flow rate) were selected on the HꢀCubeꢀPro™ hydrogenator fitted with a
30 mm 10% Pd/C CatCart. Firstly, MeOH was pumped through the system until the instrument had
achieved the desired reaction parameters and then the sample inlet line was switched to the vial
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containing the substrate. A total reaction volume of 55 mL was collected and the cartridge was
washed with MeOH for 5 min. Evaporation of the solvent afforded the desired amine as a colouless
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oil (624 mg, 90%). H NMR (400 MHz, CDCl3): δ = 7.71 (d, J = 7.9 Hz, 2H), 7.31 (d, J = 8.2 Hz,
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2H), 7.23 (d, J = 8.2 Hz, 2H), 6.94 (d, J = 7.9 Hz, 2H), 3.84 (s, 2H), 2.45 ppm (s, 3H). MS (ESI)
m/z (%): 278 (100) [M + H]+.
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4ꢀ(Aminomethyl)phenyl 4ꢀmethylbenzoate (37). Compound 37 was obtained as a colourless oil
(563 mg, 96%) from azide 35 (650 mg, 2.43 mmol), according to the procedure described for amine
36. 1H NMR (400 MHz, CDCl3): δ = 8.09 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 7.31 (d, J =
8.1 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 3.90 (s, 2H), 2.45 ppm (s, 3H). MS (ESI) m/z (%): 242 (100)
[M + H]+.
tertꢀButyl 4ꢀ(4ꢀ(2ꢀchlorophenyl)quinazolineꢀ2ꢀcarboxamido)butylcarbamate (38). A solution
of 4ꢀphenylquinazolineꢀ2ꢀcarboxylic acid 18 (568 mg, 2.00 mmol) in excess thionyl chloride (25
mL) was refluxed for 2 h under a nitrogen atmosphere. After the solution was cooled 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 (30 mL) and cooled to 0 °C, was added
dropwise a mixture of NꢀBocꢀ1,4ꢀbutanediamine (377 mg, 2.00 mmol) and triethylamine (0.28 mL,
2.00 mmol) in dry THF (10 mL). The mixture was stirred at room temperature for 48 h, filtered, and
evaporated. The crude residue was dissolved in DCM (20 mL), washed with 1N HCl, saturated
NaHCO3, and water, dried, and concentrated in vacuum. Purification by column chromatography on
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silica gel (hexane−EtOAc: 3ꢀ7) provided the title compound as a white solid; mp 68–70 °C. H
NMR (400 MHz, CDCl3): δ = 8.35–8.32 (m, 2H, 1H exchangeable with deuterium oxide), 8.02–
7.98 (m, 1H), 7.75–7.66 (m, 2H), 7.60 (d, J = 8.0 Hz, 1H), 7.57–7.28 (m, 3H), 4.64 (s, 1H,
exchangeable with deuterium oxide), 3.60 (q, J = 9.8 Hz, 2H), 3.20–3.18 (m, 2H), 1.75–1.71 (m,
2H), 1.64–1.60 (m, 2H), 1.44 ppm (s, 9H). MS (ESI) m/z (%): 455 (100) [M + H]+, 456 (36) [M + H
+ 1]+.
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tertꢀButyl 6ꢀ(4ꢀ(2ꢀchlorophenyl)quinazolineꢀ2ꢀcarboxamido)hexylcarbamate (39). Compound
39 was obtained as a white solid (676 mg, 70%) from acid 18 (568 mg, 2.00 mmol) and NꢀBocꢀ1,6ꢀ
hexanediamine (433 mg, 2.00 mmol), according to the procedure described for amide 38; mp 51–54
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°C. H NMR (400 MHz, CDCl3): δ = 8.35–8.31 (m, 2H, 1H exchangeable with deuterium oxide),
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7.99–7.96 (m, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.67– 7.63 (m, 1H), 7.58 (d, J=2.4 Hz, 1H), 7.53–7.47
(m, 3H), 4.60 (s, 1H, exchangeable with deuterium oxide), 3.55 (q, J = 9.8 Hz, 2H), 3.10–3.09 (m,
2H), 1.69–1.64 (m, 2H), 1.50–1.33 ppm (m, 15H). MS (ESI) m/z (%): 483 (100) [M + H]+, 484 (36)
[M + H + 1]+.
Nꢀ(4ꢀAminobutyl)ꢀ4ꢀ(2ꢀchlorophenyl)quinazolineꢀ2ꢀcarboxamide (40). A stirred solution of
compound 38 (600 mg, 1.32 mmol) in dry CH2Cl2 (10 mL) was treated dropwise with TFA (20
mL). After stirring at room temperature for 3 h, the reaction mixture was concentrated in vacuo.
The solid residue was taken up with water, cooled in an ice bath, treated with a 3M solution of
NaOH (20 mL) and extracted with CH2Cl2 (3 × 40 mL). The combined organic phases were washed
with brine (10 mL), dried (Na2SO4), filtered, and concentrated in vacuo to yield the title compound
as a colorless oil (347 mg, 74%). 1H NMR (400 MHz, CDCl3): δ = 8.47–8.44 (m, 1H, exchangeable
with deuterium oxide), 8.35 (d, J = 8.4 Hz, 1H), 8.01–7.98 (m, 1H), 7.74–7.66 (m, 2H), 7.61–7.49
(m, 4H), 3.60 (q, J = 9.4 Hz, 2H), 2.90–2.81 (m, 4H, 2H exchangeable with deuterium oxide), 1.76–
1.62 ppm (m, 4H). MS (ESI) m/z (%): 355 (100) [M + H]+, 356 (34) [M + H + 1]+.
Nꢀ(6ꢀAminohexyl)ꢀ4ꢀ(2ꢀchlorophenyl)quinazolineꢀ2ꢀcarboxamide (41). Compound 41 was
obtained as a colorless oil (408 mg, 86%) from derivative 39 (600 mg, 1.24 mmol), according to the
1
procedure described for amine 40. H NMR (400 MHz, CDCl3): δ = 8.35–8.33 (m, 2H, 1H
exchangeable with deuterium oxide), 8.03–7.98 (m, 1H), 7.75–7.68 (m, 2H), 7.62–7.50 (m, 4H),
3.58–3.55 (m, 2H), 3.38 (br s, 2H, exchangeable with deuterium oxide), 2.81–2.77 (m, 2H), 1.71–
1.67 (m, 2H), 1.59–1.55 (m, 2H), 1.42–1.35 ppm (m, 4H). MS (ESI) m/z (%): 383 (100) [M + H]+,
384 (34) [M + H + 1]+.
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Page 29 of 39
Journal of Medicinal Chemistry
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Docking Studies
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The previously developed rTSPO model24, generated based on the published mouse solution
structure of TSPO (PDB code 2MGY),39 was used to dock compound 6 and 7ꢀ12. These studies
were performed employing the Glide tool implemented in Maestro 9.8.40 The 3D structures of
compound 6 and 7ꢀ12 were generated with the Maestro fragment Build tool and then geometrically
optimized with Macromodel.40 Herein we used the rTSPO structure obtained in our previous work21
and prepared through the Protein Preparation Wizard of the Maestro 9.840 graphical user interface
which assigns bond orders, adds hydrogen atoms, and generates appropriate protonation states.
The docking grid box was centered on the residues lining the binding pocket of 1, with a grid box
dimension equal to 24 Å × 24 Å × 24 Å. The residues considered to center the docking grid are
A23, V26, L49, A50, I52, W107, V110, L114, A147, and L150 (which are conserved between the
mTSPO and rTSPO, apart from A110V). Finally, docking runs were carried out using the standard
precision (SP) method. Pictures were rendered employing the UCSF Chimera software.41
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Biological Evaluation.
[3H]ꢀ1 Displacement and Competition Kinetic Association Assays. Both the radioligand
binding assays were performed on rat kidneys membranes prepared as previously described.42 The
resulting membrane pellets were frozen at −20 °C. All the experimental procedures were carried out
following the guidelines of the European Community Council Directive 86ꢀ609 and have been
approved by the Committee for animal experimentation of the University of Pisa. For both
radioligand binding assays, an aliquot of membranes was thawed, suspended in Assay Buffer (AB,
TrisꢀHCl 50 mM, pH 7.4), and homogenized using Ultraturrax. Protein content in cell homogenate
was measured by the Bradford method.43
[3H]ꢀ1 displacement assay was performed as previously reported.32 Briefly, membrane
homogenates (20 ꢁg of proteins) were incubated with increasing concentrations of each tested
TSPO ligand and 1 nM [3H]ꢀ1 (85.7 ꢁCi/nmol specific activity, PerkinꢀElmer Life Sciences) in AB
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