Structure–Activity Relationship Refinement and Further Assessment of

Article pubs.acs.org/jmc

Structure−Activity Relationship Refinement and Further Assessment of 4‑Phenylquinazoline-2-carboxamide Translocator Protein Ligands as Antiproliferative Agents in Human Glioblastoma Tumors Sabrina Castellano,† Sabrina Taliani,*,‡ Monica Viviano,† Ciro Milite,† Eleonora Da Pozzo,‡ Barbara Costa,‡ Elisabetta Barresi,‡ Agostino Bruno,§ Sandro Cosconati,∥ Luciana Marinelli,§ Giovanni Greco,§ Ettore Novellino,§ Gianluca Sbardella,† Federico Da Settimo,‡ and Claudia Martini‡ †

Dipartimento di Farmacia, Universitá di Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Salerno, Italy Dipartimento di Farmacia, Universitá di Pisa, Via Bonanno 6, 56126 Pisa, Italy § Dipartimento di Farmacia, Universitá di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy ∥ DiSTABiF, Seconda Universitá di Napoli, Via Vivaldi 43, 81100 Caserta, Italy ‡

S Supporting Information *

ABSTRACT: Structure−activity relationships (SARs) within the 4-phenylquinazoline-2-carboxamide series of translocator protein (TSPO) ligands have been explored further by the synthesis and TSPO binding affinity evaluation of N-benzyl-N-ethyl/methyl derivatives variously decorated at the 6-, 2′-, 4′-, and 4″-positions. Most of the compounds showed high affinity with Ki values in the nanomolar/subnanomolar range. A pharmacophore model was developed and employed to better address SAR data presented by the new TSPO ligands. A subset of the new compounds (5, 8, 12, and 19) were tested for their ability to inhibit the viability of human glioblastoma cell line U343. The observed antiproliferative effect was demonstrated to be specific for compound 19, endowed with the best combination of binding affinity and efficacy. Furthermore, the ability of 19 to induce mitochondrial membrane dissipation (Δψm) substantiated the intracellular pro-apoptotic mechanism activated by the binding of this class of ligands to TSPO.

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INTRODUCTION The translocator protein (TSPO), originally named the peripheral-type benzodiazepine receptor (PBR),1,2 is an 18 kDa [169 amino acid (aa)] mitochondrial protein typically localized on the outer mitochondrial membrane,3 although other expression locations are possible, including the nuclei, lysosome, Golgi apparatus, peroxisomes, plasma membrane, and mature erythrocytes.3−5 TSPO plays a key role in the regulation of numerous cellular processes, such as steroid biosynthesis, cholesterol metabolism, apoptosis, and cellular proliferation.3 With its five-α-helical transmembrane domain, the TSPO is believed to form a complex with several proteins [e.g., voltage-dependent anion channel (VDAC) and adenine nucleotide transporter (ANT)] of the outer and inner mitochondrial membranes collectively known as the mitochondrial permeability transition pore (MPTP), an important regulator of apoptotic and necrotic cell death during injury.3,6 In normal homeostatic conditions, the MPTP maintains the transmembrane potential (Δψm) through active pore opening and closing. The prolonged opening of the MPTP results in the release of apoptotic factors from the mitochondria (cytochrome c, apoptosis-inducing © 2014 American Chemical Society

factor, and Smac) into the cytosol, osmotic swelling of the mitochondrial matrix, and deregulation of ATP synthesis and oxidative phosphorylation, causing apoptotic and necrotic signaling cascade reactions and subsequent cell death.3,6,7 TSPO tends to be expressed in tissues that produce steroids and that are mitochondrially enriched such as the myocardium, skeletal muscle, and renal tissue, whereas tissues such as the liver and brain exhibit comparatively modest expression.3 Aberrant expression of TSPO has been linked to multiple diseases, including cancer, brain injury, neurodegeneration, and ischemia−reperfusion injury.8 Specifically, TSPO is highly expressed in cancerous tissues of the breast, ovary, colon, prostate, and brain, compared to normal human tissues, suggesting a role for TSPO in carcinogenesis.9−13 A positive correlation between TSPO levels and the metastatic potential of human breast and brain gliomas, as well as astrocytomas, has also been shown.8,14 Cancer cells are sensitive to apoptotic cell death induced by binding of ligands to this protein, thus suggesting TSPO as a potential anticancer drug target.15 Received: November 8, 2013 Published: February 28, 2014 2413

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Chart 1. Structures of Known TSPO Ligandsa and Newly Synthesized Quinazoline Derivatives 5−23

a

The N,N-dialkyl(2-phenylindol-3-yl)glyoxylamides I and the N,N-dialkyl-4-phenylquinazoline-2-carboxamides II are represented within the framework of a previously published pharmacophore/topological model of the ligand−TSPO interaction.19,24,30

isosteres of 1, the well-known TSPO reference ligand.31 Noticeably, the introduction of a second nitrogen atom in the isoquinoline nucleus of 1 produced a favorable effect on TSPO affinity, as a great number of derivatives II bind to the TSPO with Ki values in the nanomolar/subnanomolar range.30 Structure−activity relationships (SARs) of compounds from class II were rationalized in light of a previously published pharmacophore/topological model made up of three lipophilic pockets (L1, L3, and L4) and a H-bond donor group (H1) (Chart 1).30,32 The main structural requirement for an optimal interaction with the target protein resulted in the N,Ndisubstitution on the carboxamide moiety. Molecular mechanics calculations indicated that the N,N-disubstituted derivatives adopt low-energy conformations characterized by the OC N< moiety lying out of the plane (torsion angle N1C2 CO about 30°) of the quinazoline ring, making the carbonyl group correctly oriented within the binding cleft to engage a strong H-bond with the H1 site.30 This hypothesis was consistent with the results of the work on conformationally constrained analogues of 1 featuring the isoquinoline-3carboxamide core scaffold.33 Furthermore, among the N,N-dialkyl-4-phenylquinazoline-2carboxamides II, the most potent derivatives feature at least one of the two N-alkyl groups with the number of carbon atoms in the 4−6 range. This suggests that the optimal ligand−TSPO interaction requires the full occupancy of the L4 hydrophobic pocket with at least one bulky N-alkyl group as a second crucial feature.30 A preliminary in vitro biological characterization performed on selected compounds, chosen as representative of class II, showed that these quinazoline ligands possess the ability to bind the TSPO with high affinity, without significantly affecting

Actually, classic TSPO ligands such as the isoquinolinecarboxamide derivative 1 (PK11195),16,17 Chart 1, and the benzodiazepine 2 (Ro5-4864),18 Chart 1, as well as (2phenylindol-3-yl)glyoxylamide derivative PIGA, previously synthesized by us,19,20 are able to produce MPTP opening that leads to dissipation of the Δψm, one of the early events of the apoptotic cascade activation.7,21 In this view, TSPO ligands may offer new clinical options to oncologists dealing with tumors that fail to respond to conventional chemotherapies, such as glioblastoma multiforme (GBM). GBM is the most common and deadliest malignant primary brain tumor in adults. The standard treatment is surgery, followed by radiation therapy or combined radiation therapy and chemotherapy. Unfortunately, traditional treatments are unlikely to result in a prolonged remission, and GBM is one of the cancers with the worst prognosis and an average life expectancy of about 12 months after diagnosis.22,23 The ineffectiveness of the current standard chemotherapy, consisting in the use of DNA-damaging agents, is due, at least partially, to effective oncogenic blockade of those mechanisms which would activate cell death following genotoxic insult. In this view, the activation of the mitochondrion-mediated cell death machinery by the use of drugs inducing mitochondrial outer membrane permeabilization may represent a strategic approach for the development of an alternative therapeutic treatment of GBM. In the past decade, we identified two novel classes of potent and selective TSPO ligands, the N,N-dialkyl(2-phenylindol-3yl)glyoxylamides I (Chart 1),19,24−27 designed as conformationally constrained analogues of the indoleacetamide 3 (FGIN-1-27), Chart 1,28,29 and, more recently, the N,Ndialkyl-4-phenylquinazoline-2-carboxamides II (Chart 1),30 aza 2414

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Scheme 1. Synthesis of Quinazoline Derivatives 5−23a

a

Reagents and conditions: (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 monoalkylamine, NEt3, THF, rt, 36−48 h, 43−88%; (d) NaH, CH3I or CH3CH2I, DMF, 0 °C to rt, 0.5 h, 67−93%; (e) LiOH, THF/H2O (4:1), rt, 12 h, 74%; (f) SOCl2, reflux, 2 h, then N-(4-nitrobenzyl)ethanamine (52) or N-(4-(tosylhydroxy)benzyl)ethanamine (53), NEt3, THF, rt, 36−48 h, 43−88%; (g) 1 N NaOH, EtOH, reflux, 1 h, 76%.

agreement with the proposed pharmacophore/topological model.24 Moreover, the decoration at the C4 position of the phenyl ring with various substituents (CH3, OCH3, OH, COOCH2CH3, COOH) allowed us to probe the steric and electronic features of the L4 lipophilic pocket of the TSPO binding site. In this view, the insertion of a benzyl moiety in the 4phenylquinazoline-2-carboxamide scaffold has an added value in the SAR studies of TSPO ligands. The absence of X-ray structures, not only for TSPO but also for closely related proteins, makes the construction of a three-dimensional (3D) TSPO homology model an arduous attempt. This implies that the rational design of new TSPO ligands must necessarily rely on ligand-based methods. Thus, comprehensive SAR studies on different classes of TSPO ligands are of utmost importance for the design of new TSPO modulators. Actually, to the best of our knowledge, the insertion of a benzyl group at the amide moiety has never been studied in terms of SAR even in the countless series of 1 analogues reported in the literature. The synthesis and the biological evaluation of the novel 4phenylquinazoline derivatives 5−23, 39, and 40 reported in the present study allowed a new 3D pharmacophore model to be generated and validated, outlining its predictive power.

the steroidogenic TSPO function, but producing a modulation of cell viability on human glioblastoma−astrocytoma cell line U87MG.30 In addition, data from these experiments suggested that even small structural modifications on the 4-phenylquinazoline-2-carboxamide core may affect ligand-mediated modulation of TSPO functions.30 Starting from the observation that the presence of a benzyl moiety is a common feature of high-affinity TSPO ligands [e.g., 4 (AC-5216), N-benzyl-N-ethyl(2-phenylindol-3-yl)glyoxylamides I, Chart 1] endowed with a particularly enhanced functional profile,24,34,35 herein we designed and synthesized a novel series of N-benzyl-substituted 4-phenylquinazoline-2carboxamides (5−23). In particular, it was anticipated that the benzyl group would meet one of the critical structural requirements of 4phenylquinazoline-2-carboxamides for binding to TSPO, namely, the presence of a substituent with the number of carbon atoms between 4 and 6 on the carboxamide nitrogen. In fact, in the indolylglyoxylamide class I, the N-benzyl-N-ethyl derivatives showed affinities roughly similar to those exhibited by N,N-di-n-butyl compounds, suggesting that a benzyl group may establish hydrophobic interactions like an aliphatic moiety of similar size within the lipophilic pockets L3 and L4, in 2415

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Table 1. TSPO Affinity of Quinazoline Derivatives IIa−j, 5−23, 39, and 40

N b

IIa IIbb IIcb IIdb IIeb IIfb IIgb IIhb IIib IIjb 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 39 40 1 2

R1

R2

R3

R4

R5

I (%) (1 μM) or Ki (nM)a

(CH2)3CH3 (CH2)4CH3 (CH2)5CH3 (CH2)5CH3 (CH2)5CH3 (CH2)5CH3 CH(CH3)CH2CH3 CH(CH3)CH2CH3 CH(CH3)CH2CH3 CH(CH3)CH2CH3 CH2CH3 CH2CH3 CH2CH3 CH2CH3 CH2CH3 CH2CH3 CH2CH3 CH2CH3 CH2CH3 CH2CH3 CH2CH3 CH2CH3 CH2CH3 CH2CH3 CH2CH3 CH3 CH3 CH3 CH3 H H

(CH2)3CH3 (CH2)4CH3 (CH2)5CH3 (CH2)5CH3 (CH2)5CH3 (CH2)5CH3 CH3 CH3 CH3 CH3 H H H H H H H CH3 OCH3 OH NO2 COOC2H5 COOH CH3 CH3 H H H H H H

H H H Cl H H H Cl H H H H F Cl H H H H H H H H H F Cl H F Cl H H Cl

H H H H Cl H H H Cl H H H H H F Cl CH3 H H H H H H H H H H H Cl H H

H H H H H Cl H H H Cl H Cl H H H H H H H H H H H H H H H H H H H

3.30 ± 0.30 2.48 ± 0.21 0.690 ± 0.070 65.1 ± 7.0 3.42 ± 0.30 38.5 ± 4.0 3.00 ± 0.30 3.06 ± 0.30 2.67 ± 0.30 22% 1.13 ± 0.10 24.3 ± 2.43 0.888 ± 0.090 0.235 ± 0.020 1.68 ± 0.17 0.762 ± 0.080 1.49 ± 0.15 2.57 ± 0.26 2.77 ± 0.27 8.76 ± 0.88 4.20 ± 0.42 3.65 ± 0.37 719 ± 72 0.600 ± 0.060 0.489 ± 0.050 3.99 ± 0.40 1.11 ± 0.11 0.442 ± 0.040 6.12 ± 0.61 41% 16% 9.30 ± 0.50 23.0 ± 3.0

a

The 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 μM are the mean ± SEM of three determinations. bData taken from ref 30.

Furthermore, selected compounds, representative of the whole data set, were tested for their ability to inhibit the viability of human glioblastoma cell line U343.

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with methyl or ethyl iodide in the presence of sodium hydride to furnish compounds 5−13, 16, and 18−23 (Scheme 1). Unfortunately, the same strategy could not be exploited to obtain derivatives 14 and 15. In fact, treatment of the secondary amide 48 with ethyl iodide in the presence of sodium hydride gave only degradation products. Conversely, the desired tertiary amide 15 was obtained by direct coupling of the activated acid 31 and N-(4-nitrobenzyl)ethanamine (52). This method was also followed to obtain derivative 51, using N-(4-(tosyloxy)benzyl)ethanamine (53). The phenol derivative 14 was obtained by deprotecting the tosylate 51 with NaOH. Finally, hydrolysis of the ethyl ester 16 with lithium hydroxide at room temperature gave the carboxylic acid derivative 17. Non commercially available 2-aminobenzophenones 24−30 were synthesized according to literature procedures.36 Secon-

RESULTS AND DISCUSSION

Chemistry. The key intermediates in the synthesis of target compounds 5−23 were the carboxylic acids 31−37, which were prepared by condensation of the appropriate 2-aminobenzophenones 24−30 with glyoxylic acid in the presence of ammonium acetate, followed by light irradiation with a 20 W halogen tungsten lamp, as previously reported.30 After activation with thionyl chloride, acids 31−37 were coupled with the appropriate primary amine to give secondary amides 38−50, which were subsequently N-methylated or N-ethylated 2416

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Figure 1. (a) Representation of the pharmacophore features of the developed model, H-bond acceptor (red sphere A), aromatic rings (orange circles R1, R2, and R3), custom features (cyan spheres X1 and X2), and excluded volumes (yellow circles). (b) Representation of the developed pharmacophore model and some of the aligned ligands employed to build the model.

best performing element (IIc) of the previously reported series of N,N-dialkyl-4-phenylquinazolines.30 The favorable effect on affinity resulting from the simultaneous occupation of the L4 and L3 pockets by an N-benzyl group and a chlorinesubstituted phenyl ring, respectively, contrasts with the SARs exhibited by derivatives of series II, for which combining a large alkyl group into L4 and a chlorine on the phenyl into L3 did not improve the potency (compare IId and IIe with IIc, and IIh and IIi with IIg).30 Taken together, all the binding data so far obtained seem to confirm, as stated in our previous paper,30 that mixed results are associated with the presence and chemical nature of a substituent at the 2′-position of the pendant 4-phenyl, due to interdependent (nonadditive) effects of the R2, R3, and R4 substituents on the affinity of a series of congeneric ligands. To probe the L4 lipophilic pocket, a small number of substituents at the 4′-position of the benzyl moiety (R2 = CH3, OCH3, OH, NO2, COOCH2CH3, COOH) were inserted into the structure of 5 to yield compounds 12−17. These substitutions did not cause any significant improvement in affinity. In this subset it is worth outlining the dramatic drop in affinity caused by the ionizable carboxylic group featured by 17. These data suggest that the L4 pocket is mostly lipophilic and therefore does not tolerate the negative charge carried by a carboxylate. Moreover, this pocket is not sterically demanding as it is capable of accommodating the relatively large COOC2H5 group featured by 16. Furthermore, the insertion of an electron-withdrawing substituent at the 2′-position of compound 12 afforded compounds 18 (R3 = F) and 19 (R3 = Cl) with improved TSPO binding affinity [Ki = 0.600 nM (18) and Ki = 0.489 nM (19) vs Ki = 2.57 nM (12)], confirming the SAR trend displayed by derivatives 7 and 8 with respect to 5. The N-methyl-N-benzyl derivatives 20−23 showed high affinity in the low nanomolar range (Ki values from 0.442 to 6.12 nM). Also, within this subset, the presence of a chlorine atom at 2′-position yielded the most active compound 22 (Ki = 0.442 nM). Nonetheless, a pairwise comparison of derivatives 20−23 with their corresponding ethyl analogues highlighted that the replacement of the N-ethyl with an N-methyl substituent does not produce any significant improvement in affinity. Therefore, this class was not further investigated. Due to the well-established selectivity of N,N-dialkyl-4phenylquinazoline-2-carboxamides for TSPO over the central benzodiazepine receptor (BzR),30 only a few among the novel quinazoline derivatives (randomly selected) were evaluated for their BzR affinity by means of experiments against [3H]flumazenil in rat cerebral cortex membranes.30 None of the compounds tested showed appreciable binding properties in

dary amines 52 and 53 were promptly prepared by reductive alkylation of the appropriate aldehyde with ethylamine using sodium borohydride (NaBH4) in EtOH (see the Experimental Section). Binding Affinity Assays. The binding affinity of all the newly synthesized 4-phenylquinazoline derivatives 5−23, 39, and 40 at the TSPO was determined by competition experiments against [3H]1,30 carried out in rat kidney membranes. The results, expressed as Ki values, are given in Table 1, together with the Ki values of the standard TSPO ligands 1 and 2 (Chart 1). The binding data of some of the previously investigated quinazoline derivatives30 are included for comparison purposes at the top of Table 1 (IIa−j). From a general point of view, it should be noted that most of the newly synthesized compounds show high affinity for TSPO in the nanomolar/subnanomolar range, with a potency higher or comparable to that of previously reported N,N-dialkylsubstituted derivatives.30 These data suggest that the benzyl group is effective in engaging a lipophilic interaction within the L4 hydrophobic pocket similarly to an N-alkyl group with the number of carbon atoms comprised between 4 and 6. Furthermore, in agreement with previous findings,30 the ligand−TSPO interaction requires double substitution at the carboxamido nitrogen, as the N-monosubstituted quinazolines tested (39 and 40) are devoid of appreciable affinity. Compounds 5−19, featuring an ethyl group as the second substituent on the carboxamido nitrogen, were first investigated. The unsubstituted derivative 5 (R2 = R3 = R4 = R5 = H) shows a Ki value in the low nanomolar range (Ki = 1.13 nM), that is, an affinity slightly worse than that of the most potent compound of the previously described series II, the N,N-di-nhexyl derivative IIc (Ki = 0.69 nM). The lower affinity of the 6-chloro derivative 6 compared with the lead 5 is in agreement with our previous findings in series II, interpreted in terms of unfavorable steric interactions between the 6-position of the quinazoline ring and the L1 pocket (compare the low affinities of IIf and IIj with those of IIc and IIg, respectively). Insertion of small substituents at the 2′-position (F, Cl) or 4′-position (F, Cl, CH3) of the pendant 4-phenyl ring of 5 yielded 7−11, which are provided with a similar or improved affinity (Ki values ranging from 0.235 to 1.68 nM). The most beneficial effect in this subset was produced by the presence of a chlorine at the 2′-position (8) and, to a minor extent, at the 4′-position (10). Compound 8 (Ki = 0.235 nM) turned out to be the most potent of the whole series, with a 5-fold gain in affinity with respect to its unsubstituted derivative 5 (Ki = 1.13 nM). Such a compound is also slightly more potent than the 2417

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ranked compounds is reported in Table S3 in the Supporting Information). Test set 2 includes 3979 compounds (18 actives and 3961 putative inactives, i.e., decoys), with an active content equal to 0.45%, which mimics real-life screening situations.47,48 In this test, the 3.0−2.0 fitness range is only populated by active ligands (16 out of 18), while the promiscuous fitness region (range 2.0−1.0) is made up by 2 actives and 8 decoy ligands (Figure S1, Supporting Information). Finally, in the 1.0−0.0 range of the fitness score only one decoy is ranked, and all the other compounds do not match the pharmacophore model (the complete list of the ranked compounds is reported in Table S4 in the Supporting Information). This validation outlined the predictive power of the presented pharmacophore model, thus encouraging its employment to better address SAR data presented by the new TSPO ligands (Table 1). As depicted in Figure 2, the benzyl group of

this assay (data not shown), thus confirming their selectivity for TSPO. Computational Studies. The TSPO is an integral transmembrane (TM) domain which is organized in a fiveTM bundle.37,38 This protein still represents a challenging target for crystallographers, and to date, no X-ray crystal structures for TSPO have been disclosed. Also, the absence of X-ray structures for closely related proteins makes the construction of a 3D TSPO homology model an arduous attempt.39,40 On the contrary, in the literature different classes of TSPO ligands were reported,19,24,30,33,41,42 and in this context, ligand-based approaches have proven to be useful in shedding light on the structural requirements necessary to obtain strong TSPO binders and in rationalizing the activity profile of different TSPO ligand classes.43,44 Herein, with the aim of better clarifying the structural features underlying the activity profile of the newly synthesized compounds (Table 1), we generated a 3D pharmacophore model. The pharmacophore hypothesis and subsequent ligand alignment were carried out using the PHASE suite available within the Maestro package of Schroedinger 9.1,45 on the basis of the SARs highlighted in previously reported works for structurally related TSPO ligands tested under similar experimental conditions (Table S1, Supporting Information).30,33 As reported in Figure 1a, the resulting model consists of one H-bond acceptor (red sphere A), three aromatic rings (orange circles R1, R2, and R3), and two custom features accounting for the disubstituted amide (cyan spheres X1 and X2). Two excluded volumes were manually added (Figure 1, yellow spheres): (i) one excluded volume was added in close proximity to the 6-position of the quinazoline ring, since the insertion of substituents in this position is detrimental for TSPO ligand binding (Figure 1, yellow sphere);30 (ii) the second excluded volume was added in proximity to the disubstituted carboxamide to account for the low binding affinity of fiveand six-membered ring amides (Figure 1, yellow sphere).30 For the best pharmacophore variant, 1 is the reference ligand (Figure 1a; see the subsection “Computational Studies” in the Experimental Section for a full explanation). Interestingly, 1 adopts a conformation characterized by the OCN< group lying out of the plane of the quinazoline ring (Figure 1a, torsion angle ∼30°), in agreement with previous studies.30,46 Moreover, according to the same works, the N-methyl groups adopts a cisoid conformation with respect to the OC amide group. The above-described conformational features are shared by all the aligned active ligands used to generate the 3D pharmacophore (Figure 1b). Validation of this 3D pharmacophore model was achieved by screening two different test sets (Tables S2 and S3, Supporting Information). Both test sets were screened using the Find Matches tool available in PHASE.45 Test set 1 is made up of 57 compounds (37 actives and 20 inactives/poorly actives). In Figure S1 (Supporting Information) the performance of the 3D pharmacophore model is reported as percentages of the screened database falling into three calculated fitness score ranges. Interestingly, of the best 31 scoring ligands (range 3.0−2.0), 30 are true actives, while the 2.0−1.0 fitness score range represents a promiscuous scoring region including both actives (7) and inactives (8). No compounds are ranked in the 1.0−0.0 fitness range, while 11 compounds (all inactive) are not ranked as they do not match the pharmacophore hypothesis at all (the complete list of the

Figure 2. Alignment of compound 8 (green) to the pharmacophore model and to the reference ligand 1 (orange). It can be appreciated that the benzyl group extends beyond the region occupied by the less bulky sec-butyl chain of 1.

5−23, exemplified by compound 8, extends toward the region occupied by the less bulky sec-butyl chain of 1, suggesting that the hosting receptor L4 pocket is roomier than postulated from previous studies,30,33 thus accounting for the generally high affinity of this new series. Additionally, such a cleft should feature a rather lipophilic nature as demonstrated by the poor affinity of the negatively charged 17 compared to the neutral compounds (12, 13, and 16). To probe whether the different nature and position of the substituents at the pendant 4-phenyl ring might favor a specific conformation of these compounds, a QM energy scan for the N3−C4−C1′−C2′ dihedral angle (herein defined as ω) of 5, 7, 8, and 10 (unsubstituted and 2′-F-, 2′-Cl-, and 4′-Clsubstituted, respectively) was performed. Plotting ω vs the calculated energy (kcal/mol) allowed demonstration that, as expected, the 4′-substitution has a minor influence on the dihedral energy landscape with a calculated ω rotational energy barrier (occurring at ω = 180°) of 5.29 and 4.99 kcal/mol for 5 and 10, respectively, and a minimum energy conformer featuring an ω value of 220° for both compounds (see Figure 3a,b). In this respect, the slightly higher affinity of 10 compared to 5 should be ascribed to additional interactions induced by the chlorine substituent at the 4′-position rather than to conformational effects. On the contrary, the same calculations on 7 and 8 revealed that the 2′-substitution influences the conformational behavior of these compounds (Figure 3c,d). In particular, the presence of a 2′-Cl atom in 8 forces the lowest energy ω angle to have a value of 240°, with an energy difference from the highest energy angle (180°) of 13.28 kcal/ 2418

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Figure 3. Dihedral energy scan plot for compounds 5 (a), 10 (b), 8 (c), and 7 (d). For each compound the ligand maximum energy conformers (*) and the minimum energy conformers (1 and/or 2) are depicted as insets.

described in several cellular systems.50−53 Initially, the optimal membrane protein content of 30 μg was determined and used for all subsequent binding experiments. Then the specific [3H]1 binding to U343 cell membranes was detected, and the [3H]1 equilibrium binding parameters (dissociation constant, Kd; maximum number of binding sites, Bmax) were determined by Scatchard analysis of saturation binding data, a representative example of which is shown in Figure 4.

mol (Figure 3c). In this respect, we are tempted to postulate that the bulky 2′-Cl atom, at least for this N-benzyl-substituted 4-phenylquinazoline-2-carboxamide series, induces a specific conformation that is more similar to the putative bioactive one, thus accounting for the enhanced affinity at TSPO. As reported in Figure 3d, the influence of the 2′-F atom (7) on the conformational energy landscape of ω is somehow less evident than that of 2′-Cl. Most specifically, in this case both the ω values of 220° and 240° are equally stable (two minima), and the difference from the maximum energy value (0°) is 9.41 kcal/mol. According to this hypothesis, the less marked influence of the smaller 2′-F atom in 7 in inducing a specific conformation would explain why this compound features an affinity lower than that of 8 and slightly higher than that of 5. On the whole, molecular modeling studies seem to indicate that the coexistence of the 2′-substitution and the benzyl group on the carboxamide moiety might have a positive effect in favoring a specific binding pose in the TSPO binding site, resulting in an enhanced affinity. These observations should be further validated by experimental studies (i.e., X-ray) and/or docking calculations/molecular dynamic simulations, which are not viable at this time in the absence of a thoroughly validated theoretical receptor model. Biological Studies on the U343 Glioblastoma Multiforme Cell Line. In our previous paper, compound IIc was shown to be effective in inhibiting U87MG cell viability.30 However, as glioblastoma multiforme (GBM) tumors are molecularly heterogeneous and, at present, it is not known which cell line can be the best model for such a heterogeneity,49 we decided to turn our attention to investigate U343 GBM cells (U-343 MGa, CLS Cell Lines Service), another human GBM cell line derived from a cancer of a Caucasian male. This cell line is a monolayer with epithelial morphology and a doubling time of 1.5 days. First, the presence of TSPO in U343 GBM cells was investigated by radioligand binding assays using [3H]1, as

Figure 4. [3H]1 saturation curve and Scatchard plot in U343 cells. Equilibrium binding parameters (dissociation constant, Kd; maximum number of binding sites, Bmax) were determined by Scatchard analysis of saturation binding data, a representative example of which is shown in the figure.

Specific [3H]1 binding was found to be saturable, whereas nonspecific binding increased linearly with the radioligand concentration and was less than 10% of the total binding (data not shown). Scatchard analysis yielded a single straight-line plot indicating the presence of a homogeneous population of 2419

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binding sites. The mean Kd and Bmax values were 8.57 ± 0.25 nM and 6679 ± 280 fmol/mg of proteins, respectively. Then a representative subset of TSPO ligands (5, 8, 12, and 19) were examined for the effect on the viability of U343 GBM cells. Compounds IIc and 1 were also tested, for comparison purpose and as a reference standard, respectively. An initial screening was performed by exposition of U343 cells to a single concentration (50 μM) of each compound for 24 h, and cell viability was quantitatively determined using the colorimetric viability MTS assay. After the cell exposure with each compound, the percentage of viable cells was calculated with respect to control cells (100%), and the results are depicted in Figure 5. Among the tested compounds, IIc and 19 were

compounds, causing a cell viability reduction of barely 40% with respect to untreated controls (taken as 100%) at the maximum tested ligand concentration. In summary, compounds IIc and 19 were able to reduce U343 GBM cell viability in a dose-dependent manner, supporting the specificity of their effect (see Figure 6). Then the nature of cell death induced by these TSPO ligands in U343 cells was evaluated. Depolarization of the mitochondrial membrane being the early intracellular event of apoptosis activated by TSPO ligands, the efficacy of IIc and 19 to induce mitochondrial membrane potential (Δψm) dissipation was estimated in U343 GBM cells by the cell fluo-analyzer Muse (Merck Millipore). The results evidenced a statistically significant increase in depolarized cells after 5 h of treatment with 10 μM IIc or 19 (p < 0.005) with respect to the control (Figure 7). Cells treated with the uncoupling agent carbonyl

Figure 5. Drug inhibition of cell survival. The cell survival was evaluated after 24 h of cell treatments with 50 μM compounds. The percentages of viable cells were calculated with respect to the control and reported as the mean ± SEM of three determinations. The Bonferroni multiple comparison test showed a significant inhibition in cell survival for novel compounds.

endowed with the best combination of binding affinity value and efficacy in inhibiting the viability of U343 GBM cells, being much more effective than the reference standard 1; thus, they were further investigated to exclude no specific cytotoxic effects. To ascertain whether the observed effect was dose-dependent, cells were exposed to increasing compound concentrations (from 10 nM to 100 μM) at a single incubation time (24 h). The classical TSPO ligand 1 was used as a standard. As shown in Figure 6, the survival assay evidenced a concentration dependence for the effect produced by the tested compounds. In particular, this analysis revealed that the concentrations of IIc and 19 that gave a half-maximal response in survival inhibition (IC50) were 17.73 and 15.08 μM, respectively. 1 affected cell viability to a much lower extent than the novel

Figure 7. Mitochondrial potential (Δψm) dissipation analyses. The Δψm dissipation was assessed after 5 h of cell treatments. CCCP was used as a positive control. The percentages of live, depolarized, and dead cells for each sample were provided by the instrument and are reported in the histograms. The Bonferroni multiple comparison test showed a statistically significant difference in the number of live and depolarized cells for novel compounds with respect to the control.

cyanide (3-chlorophenyl)hydrazone (CCCP), used as a positive control, showed the expected increase in depolarization. In accordance with survival data, 1 affected mitochondrial depolarization to a lower extent than the novel compounds.

Figure 6. Concentration-dependent survival analyses. The inhibition of cell survival was evaluated after 24 h of cell treatments with increasing concentrations of compounds. The percentages of viable cells were calculated with respect to the control and reported as the mean ± SEM of three determinations. The Bonferroni multiple comparison test showed a significant inhibition in cell survival for novel compounds. 2420

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reported as the most involved in GBM, in U343 cells. These data strongly substantiated the specificity of the inhibition of U343 GBM cell growth and the intracellular pro-apoptotic mechanism activated by ligand binding to TSPO, highlighting TSPO as a potential strategic target for the development of an alternative therapeutic treatment of GBM.

These results indicate the intracellular pro-apoptotic mechanism activated following ligand−TSPO binding. The ability of 19 to interact with different receptor proteins or enzymes was assayed to substantiate that the effects elicited by the compound on U343 GBM cells were specifically due to its interaction with TSPO. [35S]GTPγS binding experiments were performed on U343 GBM cell membranes to assess the compound ability to positively affect G protein-coupled receptors (GPCRs). The assay measures the binding of the radiolabeled nonhydrolyzable analogue of GTP to the activated Gα subunit.54 The results (Figure S2 in the Supporting Information) showed that 19, tested between 0.1 nM and 100 μM, was not able to increase basal GTPγS binding, suggesting that this compound did not activate any GPCRs. Furthermore, the ability of compound 19 to target a set of 13 human kinases [Abl kinase, c-kit kinase, EGFR kinase, FLT-1 kinase (VEGFR1), FLT-3 kinase, HER2/ErbB2 kinase, KDR kinase (VEGFR2), PDGFRα kinase, PDGFRβ kinase, PKCβ1, PKCβ2, Ret kinase, mTOR kinase (FRAP1)], which has been reported to be mainly involved in GBM,55,56 was assessed. No inhibitory activity was detected for 19, when assayed at 1 and 10 μM concentrations (see Figure S3 and Table S5 in the Supporting Information), thus excluding any involvement of the tested kinases in the effects of 19 on the viability of U343 cells. Taken together, these biological results substantiate that the cytotoxic effects produced by compound 19 on U343 GBM cells are mediated by a specific interaction with TSPO. Further studies could be carried out to demonstrate the ability of these compounds in inhibiting tumor growth in vivo. Actually, current literature data report that TSPO compounds showing micromolar in vitro activity were also able to inhibit tumor growth in vivo.57−60

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EXPERIMENTAL SECTION

Chemistry. All chemicals were purchased from Aldrich Chimica (Milan, Italy) 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 oven-dried glassware. Standard syringe techniques were used for anhydrous addition of liquids. Reactions were routinely monitored by TLC performed on aluminum-backed 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. Infrared (IR) spectra were recorded neat on a Shimadzu IR Affinity-1 FTIR instrument, fitted with a MIRacle 10 singlereflection ATR accessory at room temperature. 1H NMR spectra were recorded at 300 MHz on a Bruker Avance 300 spectrometer. Chemical shifts are reported in δ (ppm) relative to the internal reference tetramethylsilane (TMS). When the amide nitrogen bears two substituents, the 1H NMR spectra show the presence of two different rotamers in equilibrium. Copies of these 1H NMR spectra are included in the Supporting Information. The presence of two different rotamers in equilibrium was demonstrated for compound 19, taken as representative of the whole series. 1H NMR spectra of 19 were recorded at 20 and 80 °C in DMSO-d6, evidencing, at the higher temperature, coalescence of peaks arising from the benzyl CH2 (data not shown). Mass spectra were recorded on a Finnigan LCQ DECA TermoQuest (San Jose, CA) mass spectrometer in electrospray positive and negative ionization modes (ESI-MS). The purity of the tested compounds was established by combustion analysis, confirming a purity ≥95%. Elemental analyses (C, H, N) were performed on a Perkin-Elmer 2400 CHN elemental analyzer at the laboratory of microanalysis of the Department of Chemistry and Biology, University of Salerno (Italy); the analytical results were within ±0.4% of the theoretical values. When the elemental analysis is not included, compounds were used in the next step without further purification. General Procedure for the Synthesis of N-Alkyl-N-benzyl-4phenylquinazoline-2-carboxamides 5−13, 16, and 18−23. Sodium hydride (5.50 mmol) was added portionwise, under a nitrogen atmosphere, to an ice-cooled solution of the appropriate Nbenzyl-4-phenylquinazoline-2-carboxamide 38−50 (5.00 mmol) in dry DMF (5 mL). The mixture was stirred for 30 min and treated with an excess of methyl iodide or ethyl iodide (11.0 mmol). The mixture was stirred for 1 h at 25 °C, and an ice-cooled solution of 1 N HCl and chloroform were added. The organic layer was separated, washed with brine, dried, and concentrated in vacuum. Purification by column chromatography on silica gel (hexane−EtOAc) provided the title compounds. N-Benzyl-N-ethyl-4-phenylquinazoline-2-carboxamide (5). Yield: 89%. Mp: 118−119 °C. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.20−8.13 (m, 2H), 7.96−7.90 (m, 1H), 7.83−7.81 (m, 1H), 7.73−7.26 (m, 10H), 4.88, 4.53 (2s, 2H), 3.60, 3.27 (2q, J = 7.2 Hz, 2H), 1.25, 1.17 (2t, J = 7.2 Hz, 3H) ppm. IR (neat): ν̅ = 1643, 1560, 1544, 1483, 1390 cm−1. MS (ESI): m/z 368 [M + H]+. Anal. Calcd for C24H21N3O: C, 74.85; H, 5.76; N, 11.44. Found: C, 74.80; H, 5.76; N, 11.47. N-Benzyl-N-ethyl-4-(2-fluorophenyl)quinazoline-2-carboxamide (6). Yield: 92%. Oil. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.22−8.14 (m, 1H), 8.00−7.92 (m, 1H), 7.90−7.82 (m,

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CONCLUSION A novel series of N-benzyl-N-ethyl/methyl-4-phenylquinazoline-2-carboxamides (5−23) variously decorated at the 6-, 2′-, 4′-, and 4″-positions were synthesized and biologically evaluated as selective TSPO ligands. Most of the newly synthesized compounds showed high TSPO binding affinity with Ki values in the nanomolar/subnanomolar range. SAR data were addressed by molecular modeling studies, indicating that the coexistence of the 2′-substitution and the benzyl group on the carboxamide moiety might have a positive effect in favoring a specific pose in the TSPO binding site, resulting in an enhanced affinity. Moreover, the observation that the L4 pocket is sterically less demanding compared with the L1 and L3 pockets may pave the way for the future development of novel 4-phenylquinazoline-2-carboxamides bearing in the paraposition of the N-benzyl group relatively lengthy R 2 substituents as potential fluorescent probes for TSPO, similarly to that done in series I of (2-phenylindol-3-yl)glyoxylamides.25,26 A subset of selected compounds (5, 8, 12, and 19) were assayed in vitro to determine their ability to induce death in the U343 GBM cell line. After an initial screening, ligand 19, showing the best combination of binding affinity and efficacy in inhibiting the viability of U343 GBM cells, was further investigated to exclude nonspecific cytotoxic effects. Noticeably, 19 was able to reduce U343 GBM cell viability in a dosedependent manner and to cause the dissipation of the mitochondrial membrane potential. In addition, 19 was ineffective in activating GPCRs and a set of 13 kinases, 2421

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1H), 7.70−7.25 (m, 10H), 4.88, 4.54 (2s, 2H), 3.59, 3.27 (2q, J = 7.0 Hz, 2H), 1.25, 1.16 (2t, J = 7.0 Hz, 3H) ppm. IR (neat): ν̅ = 1645, 1562, 1545, 1485, 1389 cm−1. MS (ESI): m/z 386 [M + H]+. Anal. Calcd for C24H20FN3O: C, 74.79; H, 5.23; N, 10.90. Found: C, 74.86; H, 5.23; N, 10.88. N-Benzyl-4-(2-chlorophenyl)-N-ethylquinazoline-2-carboxamide (7). Yield: 87%. Mp: 69−70 °C. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.25−8.15 (m, 1H), 8.05−7.95 (m, 1H), 7.70−7.30 (m, 11H), 4.95−4.78, 4.53 (m, s, 2H), 3.64−3.50, 3.27 (m, q, J = 7.1 Hz, 2H), 1.23, 1.13 (2t, J = 7.1 Hz, 3H) ppm. IR (neat): ν̅ = 1645, 1560, 1543, 1490, 1389 cm−1. MS (ESI): m/z 402 [M + H]+. Anal. Calcd for C24H20ClN3O: C, 71.73; H, 5.02; N, 10.46. Found: C, 71.73; H, 5.00; N, 10.44. N-Benzyl-N-ethyl-4-(4-fluorophenyl)quinazoline-2-carboxamide (8). Yield: 85%. Mp: 99−100 °C. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.21−8.07 (m, 2H), 7.99−7.91 (m, 1H), 7.88−7.82 (m, 1H), 7.72−7.62 (m, 2H), 7.48−7.42 (m, 2H), 7.40−7.35 (m, 1H), 7.34−7.27 (m, 3H), 7.24−7.19 (m, 1H), 4.87, 4.52 (2s, 2H), 3.60, 3.27 (2q, J = 6.8 Hz, 2H), 1.25, 1.16 (2t, J = 6.8 Hz, 3H) ppm. IR (neat): ν̅ = 1643, 1560, 1541, 1489, 1387 cm−1. MS (ESI): m/z 408 [M + Na]+. Anal. Calcd for C24H20FN3O: C, 74.79; H, 5.23; N, 10.90. Found: C, 74.58; H, 5.21; N, 10.94. N-Benzyl-4-(4-chlorophenyl)-N-ethylquinazoline-2-carboxamide (9). Yield: 95%. Mp: 117−118 °C. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.21−8.05 (m, 2H), 7.98−7.91 (m, 1H), 7.81−7.78 (m, 1H), 7.70−7.61 (m, 2H), 7.59−7.28 (m, 7H), 4.87, 4.52 (2s, 2H), 3.60, 3.26 (2q, J = 6.4 Hz, 2H), 1.26, 1.16 (2t, J = 6.4 Hz, 3H) ppm. IR (neat): ν̅ = 1636, 1558, 1541, 1478, 1379 cm−1. MS (ESI): m/z 424 [M + Na]+. Anal. Calcd for C24H20ClN3O: C, 71.73; H, 5.02; N, 10.46. Found: C, 71.56; H, 5.03; N, 10.49. N-Benzyl-N-ethyl-4-(4-methylphenyl)quinazoline-2-carboxamide (10). Yield: 88%. Mp: 108−109 °C. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.22−8.10 (m, 2H), 7.97−7.88 (m, 1H), 7.76−7.72 (m, 1H), 7.67−7.59 (m, 2H), 7.50−7.42 (m, 2H), 7.41− 7.35 (m, 2H), 7.35−7.27 (m, 3H), 4.87, 4.52 (2s, 2H), 3.59, 3.26 (2q, J = 6.5 Hz, 2H), 2.48, 2.47 (2s, 3H), 1.24, 1.16 (2t, J = 6.5 Hz, 3H) ppm. IR (neat): ν̅ = 1641, 1560, 1543, 1485, 1389 cm−1. MS (ESI): m/z 382 [M + H]+. Anal. Calcd for C25H23N3O: C, 78.71; H, 6.08; N, 11.02. Found: C, 78.47; H, 6.06; N, 11.06. N-Benzyl-6-chloro-N-ethyl-4-phenylquinazoline-2-carboxamide (11). Yield: 96%. Mp: 133−134 °C. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.15−8.08 (m, 2H), 7.91−7.78 (m, 2H), 7.70−7.65 (m, 1H), 7.63−7.52 (m, 3H), 7.49−7.27 (m, 5H), 4.87, 4.51 (2s, 2H), 3.59, 3.25 (2q, J = 7.2 Hz, 2H), 1.25, 1.17 (2t, J = 7.2 Hz, 3H) ppm. IR (neat): ν̅ = 1653, 1558, 1541, 1479, 1383 cm−1. MS (ESI): m/z 402 [M + H]+. Anal. Calcd for C24H20ClN3O: C, 71.73; H, 5.02; N, 10.46. Found: C, 71.94; H, 5.02; N, 10.45. N-Ethyl-N-(4-methylbenzyl)-4-phenylquinazoline-2-carboxamide (12). Yield: 96%. Oil. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.20−8.11 (m, 2H), 7.98−7.90 (m, 1H), 7.84−7.80 (m, 1H), 7.72−7.69 (m, 1H), 7.68−7.61 (m, 1H), 7.60−7.51 (m, 3H), 7.35, 7.33 (2d, J = 7.8 Hz, 2H), 7.18, 7.11 (2d, J = 7.8 Hz, 2H), 4.83, 4.48 (2s, 2H), 3.58, 3.24 (2q, J = 7.0 Hz, 2H), 2.35, 2.32 (2s, 3H), 1.24, 1.16 (2t, J = 7.0 Hz, 3H) ppm. IR (neat): ν̅ = 1645, 1560, 1541, 1485, 1389 cm−1. MS (ESI): m/z 382 [M + H]+. Anal. Calcd for C25H23N3O: C, 78.71; H, 6.08; N, 11.02. Found: C, 78.45; H, 6.09; N, 11.05. N-Ethyl-N-(4-methoxybenzyl)-4-phenylquinazoline-2-carboxamide (13). Yield: 95%. Oil. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.20−8.12 (m, 2H), 7.97−7.91 (m, 1H), 7.83−7.80 (m, 1H), 7.74−7.71 (m, 1H), 7.68−7.61 (m, 1H), 7.60−7.53 (m, 3H), 7.40, 7.37 (2d, J = 8.5 Hz, 2H), 6.90, 6.83 (2d, J = 8.5 Hz, 2H), 4.81, 4.45 (2s, 2H), 3.81, 3.79 (2s, 3H), 3.57, 3.24 (2q, J = 6.9 Hz, 2H), 1.23, 1.16 (2t, J = 6.9 Hz, 3H) ppm. IR (neat): ν̅ = 1643, 1562, 1543, 1485, 1389 cm−1. MS (ESI): m/z 398 [M + H]+. Anal. Calcd for C25H23N3O2: C, 75.54; H, 5.83; N, 10.57. Found: C, 75.60; H, 5.83; N, 10.58. Ethyl 4-((N-Ethyl-4-phenylquinazoline-2-carboxamido)methyl)benzoate (16). Yield: 89%. Oil. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.20−8.08 (m, 2H), 8.07−7.88 (m, 3H), 7.83−7.78 (m, 1H), 7.67−7.50 (m, 7H), 4.92, 4.59 (2s, 2H), 4.38, 4.39 (2q, J =

7.1 Hz, 2H), 3.60, 3.29 (2q, J = 6.9 Hz, 2H), 1.40, 1.39 (2t, J = 7.1 Hz, 3H), 1.26, 1.17 (2t, J = 6.9 Hz, 3H) ppm. IR (neat): ν̅ = 1715, 1647, 1560, 1541, 1485, 1387 cm−1. MS (ESI): m/z 440 [M + H]+. Anal. Calcd for C27H25N3O3: C, 73.78; H, 5.73; N, 9.56. Found: C, 74.01; H, 5.71; N, 9.60. N-Ethyl-4-(2-fluorophenyl)-N-(4-methylbenzyl)quinazoline-2-carboxamide (18). Yield: 94%. Mp: 50−51 °C. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.22−8.15 (m, 1H), 8.00−7.90 (m, 1H), 7.89−7.80 (m, 1H), 7.70−7.49 (m, 4H), 7.40−7.09 (m, 5H), 4.83, 4.48 (2s, 2H), 3.58, 3.24 (2q, J = 6.8 Hz, 2H), 2.35, 2.31 (2s, 3H), 1.24, 1.15 (2t, J = 6.8 Hz, 3H) ppm. IR (neat): ν̅ = 1647, 1560, 1543, 1485, 1389 cm−1. MS (ESI): m/z 422 [M + Na]+. Anal. Calcd for C25H22FN3O: C, 75.17; H, 5.55; N, 10.52. Found: C, 75.26; H, 5.56; N, 10.55. 4-(2-Chlorophenyl)-N-ethyl-N-(4-methylbenzyl)quinazoline-2carboxamide (19). Yield: 91%. Mp: 58−60 °C. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.23−8.14 (m, 1H), 8.00−7.89 (m, 1H), 7.71−7.05 (m, 10H), 4.84, 4.47 (q, s, J = 14.2 Hz, 2H), 3.60− 3.45, 3.26 (m, q, J = 6.8 Hz, 2H), 2.35, 2.31 (2s, 3H), 1.22, 1.12 (2t, J = 6.8 Hz, 3H) ppm. IR (neat): ν̅ = 1647, 1560, 1543, 1489, 1389 cm−1. MS (ESI): m/z 438 [M + Na]+. Anal. Calcd for C25H22ClN3O: C, 72.19; H, 5.33; N, 10.10. Found: C, 71.95; H, 5.31; N, 10.13. N-Benzyl-N-methyl-4-phenylquinazoline-2-carboxamide (20). Yield: 88%. Mp: 65−68 °C. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.22−8.13 (m, 2H), 7.99−7.91 (m, 1H), 7.86−7.80 (m, 1H), 7.76−7.53 (m, 5H), 7.49−7.26 (m, 5H), 4.85, 4.52 (2s, 2H), 3.09, 2.94 (2s, 3H) ppm. IR (neat): ν̅ = 1647, 1558, 1541, 1489, 1389 cm−1. MS (ESI): m/z 354 [M + H]+. Anal. Calcd for C23H19N3O: C, 78.16; H, 5.42; N, 11.89. Found: C, 78.36; H, 5.42; N, 11.85. N-Benzyl-4-(2-fluorophenyl)-N-methylquinazoline-2-carboxamide (21). Yield: 93%. Mp: 52−54 °C. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.25−8.13 (m, 1H), 8.02−7.91 (m, 1H), 7.89−7.83 (m, 1H), 7.71−7.25 (m, 10H), 4.85, 4.52 (2s, 2H), 3.08, 2.94 (2s, 3H) ppm. IR (neat): ν̅ = 1647, 1560, 1543, 1489, 1389 cm−1. MS (ESI): m/z 394 [M + Na]+. Anal. Calcd for C23H18FN3O: C, 74.38; H, 4.88; N, 11.31. Found: C, 74.19; H, 4.87; N, 11.33. N-Benzyl-4-(2-chlorophenyl)-N-methylquinazoline-2-carboxamide (22). Yield: 89%. Mp: 128−129 °C. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.24−8.15 (m, 1H), 8.01−7.92 (m, 1H), 7.73−7.27 (m, 11H), 4.98−4.71, 4.62−4.44 (2m, 2H), 3.07, 2.93 (2s, 3H) ppm. IR (neat): ν̅ = 1641, 1560, 1543, 1496, 1393 cm−1. MS (ESI): m/z 410 [M + Na]+. Anal. Calcd for C23H18ClN3O: C, 71.22; H, 4.68; N, 10.83. Found: C, 71.13; H, 4.68; N, 10.81. N-Benzyl-4-(4-chlorophenyl)-N-methylquinazoline-2-carboxamide (23). Yield: 95%. Mp: 61−63 °C. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.22−8.07 (m, 2H), 8.02−7.93 (m, 1H), 7.82−7.28 (m, 10H), 4.85, 4.51 (2s, 2H), 3.10, 2.94 (2s, 3H) ppm. IR (neat): ν̅ = 1647, 1560, 1541, 1483, 1398 cm−1. MS (ESI): m/z 410 [M + Na]+. Anal. Calcd for C23H18ClN3O: C, 71.22; H, 4.68; N, 10.83. Found: C, 71.07; H, 4.67; N, 10.83. N-Ethyl-N-(4-hydroxybenzyl)-4-phenylquinazoline-2-carboxamide (14). To a solution of 51 (200 mg, 0.37 mmol) in EtOH (5 mL) was added an aqueous solution of 1 N NaOH (2.00 mL, 2.00 mmol). The mixture was heated at reflux and stirred for 1 h. The reaction mixture was then cooled to room temperature, treated with 1 N HCl to pH 9, and concentrated in vacuo. The residue was treated with water (30 mL) and extracted with EtOAc (3 × 30 mL). The combined organic phases were washed with brine (10 mL), dried (Na2SO4), filtered, and concentrated in vacuo to yield the title compound. Yield: 96%. Mp: 80−81 °C. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.22−8.10 (m, 2H), 8.00−7.90 (m, 1H), 7.85−7.77 (m, 1H), 7.76−7.50 (m, 5H), 7.39−7.26 (m, 2H), 6.82, 6.77 (2d, J = 8.2 Hz, 2H), 5.37, 5.22 (2s, 1H, exchangeable with deuterium oxide), 4.78, 4.43 (2s, 2H), 3.56, 3.26 (2q, J = 7.0 Hz, 2H), 1.23, 1.17 (2t, J = 7.0 Hz, 3H) ppm. IR (neat): ν̅ = 3238, 1624, 1560, 1516, 1487, 1389 cm−1. MS (ESI): m/z 384 [M + H]+. Anal. Calcd for C24H21N3O2: C, 75.18; H, 5.52; N, 10.96. Found: C, 75.00; H, 5.50; N, 11.01. N-Ethyl-N-(4-nitrobenzyl)-4-phenylquinazoline-2-carboxamide (15). Reaction of carboxylic acid 31 and secondary amine 52, 2422

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Journal of Medicinal Chemistry

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2H), 7.46 (d, J = 7.7 Hz, 2H), 2.46 (s, 3H) ppm. IR (neat): ν̅ = 2928, 1718, 1558, 1489, 1387 cm−1. MS (ESI): m/z 263 [M − H]−. 6-Chloro-4-phenylquinazoline-2-carboxylic Acid (37).30 Yield: 86%. Mp: 213-215 °C (DCM/cyclohexane). 1H NMR (300 MHz, DMSO-d6): δ = 8.27 (d, J = 8.9 Hz, 1H), 8.17−8.14 (m, 1H), 8.09− 8.01 (m, 1H), 7.85−7.80 (m, 2H), 7.70−7.63 (m, 3H) ppm. IR (neat): ν̅ = 3527, 3404, 1718, 1560, 1483, 1381 cm−1. MS (ESI): m/z 283 [M − H]−. General Procedure for the Synthesis of N-Benzyl-4-phenylquinazoline-2-carboxamides 38−50. A solution of the appropriate 2-carboxylic acid 31−37 (2.00 mmol) in thionyl chloride (15 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 (10 mL) and cooled to 0 °C, was added dropwise a mixture of the appropriate primary amine (2.00 mmol) and triethylamine (2.00 mmol) in dry THF (5 mL). 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 1 N HCl, saturated NaHCO3, and water, dried, and concentrated in vacuum. Purification by column chromatography on silica gel (DCM−EtOAc) provided the title compounds. N-Benzyl-4-phenylquinazoline-2-carboxamide (38). Yield: 82%. Mp: 232−233 °C. 1H NMR (300 MHz, CDCl3): δ = 8.66 (br t, J = 5.6 Hz, 1H, exchangeable with deuterium oxide), 8.34 (d, J = 8.5 Hz, 1H), 8.18 (d, J = 8.5 Hz, 1H), 8.01−7.97 (m, 1H), 7.81−7.77 (m, 2H), 7.70−7.69 (m, 1H), 7.62−7.57 (m, 3H), 7.44−7.40 (m, 2H), 7.37− 7.32 (m, 2H), 7.31−7.27 (m, 1H), 4.79 (d, J = 5.6 Hz, 2H) ppm. IR (neat): ν̅ = 3273, 2926, 1668, 1558, 1522, 1489, 1389 cm−1. MS (ESI): m/z 340 [M + H]+. Anal. Calcd for C22H17N3O: C, 77.86; H, 5.05; N, 12.38. Found: C, 77.65; H, 5.07; N, 12.34. N-Benzyl-4-(2-fluorophenyl)quinazoline-2-carboxamide (39). Yield: 76%. Mp: 151−152 °C. 1H NMR (300 MHz, CDCl3): δ = 8.60 (br t, J = 6.6 Hz, 1H, exchangeable with deuterium oxide), 8.33 (d, J = 8.5 Hz, 1H), 8.04−7.97 (m, 1H), 7.92−7.86 (m, 1H), 7.75− 7.55 (m, 3H), 7.46−7.28 (m, 7H), 4.80 (d, J = 6.0 Hz, 2H) ppm. IR (neat): ν̅ = 3300, 2928, 1661, 1560, 1514, 1489, 1393 cm−1. MS (ESI): m/z 358 [M + H]+. Anal. Calcd for C22H16FN3O: C, 74.94; H, 4.51; N, 11.76. Found: C, 74.05; H, 4.49; N, 11.78. N-Benzyl-4-(2-chlorophenyl)quinazoline-2-carboxamide (40). Yield: 72%. Mp: 150−151 °C. 1H NMR (300 MHz, CDCl3): δ = 8.60 (br t, J = 5.7 Hz, 1H, exchangeable with deuterium oxide), 8.34 (d, J = 8.5 Hz, 1H), 8.10−7.89 (m, 1H), 7.76−7.63 (m, 2H), 7.61− 7.39 (m, 5H), 7.40−7.25 (m, 4H), 4.78 (d, J = 5.7 Hz, 2H) ppm. IR (neat): ν̅ = 3273, 2922, 1684, 1560, 1522, 1497, 1395 cm−1. MS (ESI): m/z 396 [M + Na]+. Anal. Calcd for C22H16ClN3O: C, 70.68; H, 4.31; N, 11.24. Found: C, 70.59; H, 4.32; N, 11.21. N-Benzyl-4-(4-fluorophenyl)quinazoline-2-carboxamide (41). Yield: 69%. Mp: 183−184 °C. 1H NMR (300 MHz, CDCl3): δ = 8.61 (br t, J = 6.0 Hz, 1H, exchangeable with deuterium oxide), 8.33 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 8.03−7.99 (m, 1H), 7.84−7.79 (m, 2H), 7.76−7.71 (m, 1H), 7.43 (d, J = 7.1 Hz, 2H), 7.38−7.34 (m, 2H), 7.31−7.26 (m, 3H), 4.80 (d, J = 6.0 Hz, 2H) ppm. IR (neat): ν̅ = 3302, 2930, 1670, 1560, 1514, 1489, 1391 cm−1. MS (ESI): m/z 358 [M + H]+. Anal. Calcd for C22H16FN3O: C, 73.94; H, 4.51; N, 11.76. Found: C, 74.09; H, 4.50; N, 11.76. N-Benzyl-4-(4-chlorophenyl)quinazoline-2-carboxamide (42). Yield: 70%. Mp: 181−182 °C. 1H NMR (300 MHz, CDCl3): δ = 8.60 (br t, J = 6.1 Hz, 1H, exchangeable with deuterium oxide), 8.33 (d, J = 8.4 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 8.01−7.98 (m, 1H), 7.78−7.69 (m, 3H), 7.56 (d, J = 7.8 Hz, 2H), 7.44−7.40 (m, 2H), 7.38−7.33 (m, 2H), 7.32−7.28 (m, 1H), 4.79 (d, J = 3.8 Hz, 2H) ppm. IR (neat): ν̅ = 3277, 2914, 1661, 1558, 1513, 1487, 1391 cm−1. MS (ESI): m/z 374 [M + H]+. Anal. Calcd for C22H16ClN3O: C, 70.68; H, 4.31; N, 11.24. Found: C, 70.86; H, 4.31; N, 11.21. N-Benzyl-4-(4-methylphenyl)quinazoline-2-carboxamide (43). Yield: 60%. Mp: 176−177 °C. 1H NMR (300 MHz, CDCl3): δ = 8.67 (br t, J = 6.1 Hz, 1H, exchangeable with deuterium oxide), 8.33 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 8.06−7.96 (m, 1H), 7.74−7.56 (m, 3H), 7.45−7.28 (m, 7H), 4.79 (d, J = 6.0 Hz, 2H), 2.49

according to the general procedure for the synthesis of N-benzyl-4phenylquinazoline-2-carboxamides 5−13, 16, and 18−23, yielded the title compound. Yield: 65%. Mp: 116−117 °C. 1H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.27−8.08 (m, 4H), 8.01−7.90 (m, 1H), 7.87−7.81 (m, 1H), 7.72−7.47 (m, 7H), 4.95, 4.65 (2s, 2H), 3.61, 3.35 (2q, J = 7.1 Hz, 2H), 1.29, 1.20 (2t, J = 7.1 Hz, 3H) ppm. IR (neat): ν̅ = 1647, 1558, 1548, 1508, 1489, 1394, 1339 cm−1. MS (ESI): m/z 413 [M + H]+. Anal. Calcd for C24H20N4O3: C, 69.89; H, 4.89; N, 13.58. Found: C, 70.02; H, 4.89; N, 13.60. 4-((N-Ethyl-4-phenylquinazoline-2-carboxamido)methyl)benzoic Acid (17). To a solution of 16 (200 mg, 0.46 mmol) in a THF/H2O (4:1) mixture (8 mL) was added LiOH (44.0 mg, 1.80 mmol). The mixture was stirred at room temperature for 12 h and then treated with HCl (1 N) to pH 7. The solution was extracted with EtOAc (3 × 30 mL), and the combined organic phases were washed with brine (10 mL), dried (Na2SO4), filtered, and concentrated in vacuo. Purification by column chromatography on silica gel (DCM− EtOAc) provided the title compound. Yield: 74%. Mp: 144−145 °C. 1 H NMR (300 MHz, CDCl3, mixture of rotamers): δ = 8.28−8.07 (m, 2H), 8.05−7.87 (m, 3H), 7.84−7.77 (m, 1H), 7.70−7.42 (m, 7H), 4.92, 4.61 (2s, 2H), 3.60, 3.31 (2q, J = 6.9 Hz, 2H), 1.24, 1.17 (2t, J = 6.9 Hz, 3H) ppm. IR (neat): ν̅ = 3433, 1709, 1643, 1562, 1543, 1485, 1389 cm−1. MS (ESI): m/z 410 [M − H]−. Anal. Calcd for C25H21N3O3: C, 72.98; H, 5.14; N, 10.21. Found: C, 72.98; H, 5.14; N, 10.19. General Procedure for the Synthesis of 4-Phenylquinazoline-2-carboxylic Acids 31−37. An aqueous solution of glyoxylic acid (10.0 mmol in 10 mL) was added dropwise to a solution of the appropriate 2-aminobenzophenone 24−30 (10.0 mmol), commercially available or prepared as previously reported,30 and ammonium acetate (30.0 mmol) in 96% ethanol (30 mL). The mixture was stirred at room temperature for 15−30 min. After addition of 20 mL of water, the orange solid was collected by filtration and washed with 96% ethanol and diethyl ether to give the corresponding 1,2-dihydro-4phenylquinazoline-2-carboxylic acids 31−37, which were dissolved in DMF (15 mL) and allowed to stand for 12 h under external light irradiation with a 20 W halogen tungsten lamp. After addition of 15 mL of water, the mixture was cooled in an ice bath, and the yellow precipitate formed was collected by filtration and washed with water and diethyl ether to give the 4-phenylquinazoline-2-carboxylic acids 31−37, which were recrystallized from the appropriate solvent. 4-Phenylquinazoline-2-carboxylic Acid (31).30 Yield: 62%. Mp: 98 °C (DCM/cyclohexane). 1H NMR (300 MHz, DMSO-d6): δ = 8.26− 8.23 (m, 1H), 8.16−8.11 (m, 2H), 7.89−7.81(m, 3H). 7.71−7.61 (m, 3H) ppm. IR (neat): ν̅ = 3169, 3057, 1751, 1541, 1490, 1346 cm−1. MS (ESI): m/z 249 [M − H]−. 4-(2-Fluorophenyl)quinazoline-2-carboxylic Acid (32). Yield: 65%. Mp: 203−204 °C (acetonitrile). 1H NMR (300 MHz, DMSO-d6): δ = 8.28−8.23 (m, 1H), 8.20−8.12 (m, 1H), 7.89−7.80 (m, 2H), 7.77− 7.68 (m, 2H), 7.57−7.45 (m, 2H) ppm. IR (neat): ν̅ = 2930, 2859, 1728, 1566, 1456, 1395 cm−1. MS (ESI): m/z 267 [M − H]−. 4-(2-Chlorophenyl)quinazoline-2-carboxylic Acid (33).30 Yield: 68%. Mp: 168−169 °C (acetonitrile). 1H NMR (300 MHz, DMSOd6): δ = 8.27−8.25 (m, 1H), 8.18−8.13 (m, 1H), 7.87−7.82 (m, 1H), 7.75−7.60 (m, 5H) ppm. IR (neat): ν̅ = 3169, 3066, 1753, 1560, 1473, 1394 cm−1. MS (ESI): m/z 283 [M − H]−. 4-(4-Fluorophenyl)quinazoline-2-carboxylic Acid (34). Yield: 73%. Mp: 186−187 °C (acetonitrile). 1H NMR (300 MHz, DMSO-d6): δ = 8.29−8.09 (m, 3H), 7.99−7.81 (m, 3H), 7.59−7.42 (m, 2H) ppm. IR (neat): ν̅ = 2926, 2855, 1728, 1564, 1491, 1391 cm−1. MS (ESI): m/z 267 [M − H]−. 4-(4-Chlorophenyl)quinazoline-2-carboxylic Acid (35).30 Yield: 81%. Mp: 197−198 °C (acetonitrile). 1H NMR (300 MHz, DMSOd6): δ = 8.24−8.22 (m, 1H), 8.17−8.12 (m, 2H), 7.90−7.83 (m, 3H), 7.72 (d, J = 7.8 Hz, 2H) ppm. IR (neat): ν̅ = 3523, 3367, 1708, 1593, 1487, 1404 cm−1. MS (ESI): m/z 283 [M − H]−. 4-(4-Methylphenyl)quinazoline-2-carboxylic Acid (36). Yield: 90%. Mp: 98−99 °C (acetonitrile). 1H NMR (300 MHz, DMSOd6): δ = 8.26−8.08 (m, 3H), 7.90−7.83 (m, 1H), 7.74 (d, J = 7.7 Hz, 2423

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Journal of Medicinal Chemistry

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(s, 3H) ppm. IR (neat): ν̅ = 3292, 2922, 1655, 1560, 1516, 1489, 1387 cm−1. MS (ESI): m/z 376 [M + Na]+. Anal. Calcd for C23H19N3O: C, 78.16; H, 5.42; N, 11.89. Found: C, 78.16; H, 5.44; N, 11.85. N-Benzyl-6-chloro-4-phenylquinazoline-2-carboxamide (44). Yield: 82%. Mp: 170−171 °C. 1H NMR (300 MHz, CDCl3): δ = 8.60 (br t, J = 6.1 Hz, 1H, exchangeable with deuterium oxide), 8.29 (d, J = 9.0 Hz, 1H), 8.14 (d, J = 2.3 Hz, 1H), 7.92 (dd, J = 9.0 Hz, 2.3 Hz, 1H), 7.80−7.74 (m, 2H), 7.63−7.58 (m, 3H), 7.45−7.28 (m, 5H), 4.79 (d, J = 6.1 Hz, 2H) ppm. IR (neat): ν̅ = 3287, 2918, 1693, 1560, 1518, 1479, 1385 cm−1. MS (ESI): m/z 374 [M + H]+. Anal. Calcd for C22H16ClN3O: C, 70.68; H, 4.31; N, 11.24. Found: C, 70.47; H, 4.30; N, 11.27. N-(4-Methylbenzyl)-4-phenylquinazoline-2-carboxamide (45). Yield: 72%. Mp: 212−213 °C. 1H NMR (300 MHz, CDCl3): δ = 8.61 (br t, J = 6.0 Hz, 1H, exchangeable with deuterium oxide), 8.34 (d, J = 8.5 Hz, 1H), 8.18 (d, J = 8.5 Hz, 1H), 8.00−7.96 (m, 1H), 7.80−7.77 (m, 2H), 7.71−7.68 (m, 1H), 7.60−7.56 (m, 3H), 7.31 (d, J = 7.8 Hz, 2H), 7.16 (d, J = 7.8 Hz, 2H), 4.75 (d, J = 6.0 Hz, 2H), 2.34 (s, 3H) ppm. IR (neat): ν̅ = 3264, 2926, 1659, 1558, 1516, 1489, 1391 cm−1. MS (ESI): m/z 354 [M + H]+. Anal. Calcd for C23H19N3O: C, 78.16; H, 5.42; N, 11.89. Found: C, 78.31; H, 5.43; N, 11.86. N-(4-Methoxybenzyl)-4-phenylquinazoline-2-carboxamide (46). Yield: 80%. Mp: 188−189 °C. 1H NMR (300 MHz, CDCl3): δ = 8.59 (br t, J = 6.0 Hz, 1H, exchangeable with deuterium oxide), 8.34 (d, J = 8.4 Hz, 1H), 8.17 (d, J = 8.4 Hz, 1H), 8.00−7.96 (m, 1H), 7.81−7.78 (m, 2H), 7.71−7.67 (m, 1H), 7.60−7.56 (m, 3H), 7.35 (d, J = 8.2 Hz, 2H), 6.88 (d, J = 8.2 Hz, 2H), 4.72 (d, J = 5.9 Hz, 2H), 3.80 (s, 3H) ppm. IR (neat): ν̅ = 3283, 2930, 1657, 1560, 1514, 1489, 1393 cm−1. MS (ESI): m/z 370 [M + H]+. Anal. Calcd for C23H19N3O2: C, 74.68; H, 5.18; N, 11.37. Found: C, 74.88; H, 5.18; N, 11.34. Ethyl 4-((4-Phenylquinazoline-2-carboxamido)methyl)benzoate (47). Yield: 81%. Mp: 114−115 °C. 1H NMR (300 MHz, CDCl3): δ = 8.73 (br t, J = 6.0 Hz, 1H, exchangeable with deuterium oxide), 8.35 (d, J = 8.0 Hz, 1H), 8.19 (d, J = 8.0 Hz, 1H), 8.05−7.95 (m, 3H), 7.84−7.76 (m, 2H), 7.75−7.67 (m, 1H), 7.64−7.55 (m, 3H), 7.47 (d, J = 8.5 Hz, 2H), 4.85 (d, J = 6.0, 2H), 4.37 (q, J = 7.1 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H) ppm. IR (neat): ν̅ = 3292, 2926, 1717, 1667, 1560, 1522, 1489, 1391 cm−1. MS (ESI): m/z 412 [M + H]+. Anal. Calcd for C25H21N3O3: C, 72.98; H, 5.14; N, 10.21. Found: C, 72.86; H, 5.14; N, 10.18. N-(4-Nitrobenzyl)-4-phenylquinazoline-2-carboxamide (48). Yield: 70%. Mp: 154−155 °C. 1H NMR (300 MHz, CDCl3): δ = 8.85 (br t, J = 6.0 Hz, 1H, exchangeable with deuterium oxide), 8.35 (d, J = 8.2 Hz, 1H), 8.25−8.15 (m, 3H), 8.05−7.95 (m, 1H), 7.90− 7.70 (m, 3H), 7.65−7.50 (m, 5H), 4.88 (d, J = 6.3 Hz, 2H) ppm. IR (neat): ν̅ = 3314, 2930, 1672, 1560, 1513, 1489, 1393, 1346 cm−1. MS (ESI): m/z 385 [M + H]+. Anal. Calcd for C22H16N4O3: C, 68.74; H, 4.20; N, 14.58. Found: C, 68.95; H, 4.21; N, 14.62. 4-(2-Fluorophenyl)-N-(4-methylbenzyl)quinazoline-2-carboxamide (49). Yield: 73%. Mp: 219−220 °C. 1H NMR (300 MHz, CDCl3): δ = 8.55 (br t, J = 6.0 Hz, 1H, exchangeable with deuterium oxide), 8.32 (d, J = 8.4 Hz, 1H), 8.02−7.96 (m, 1H), 7.94−7.81 (m, 1H), 7.77−7.52 (m, 3H), 7.40−7.27 (m, 4H), 7.21 (d, J = 7.8 Hz, 2H), 4.74 (d, J = 6.0 Hz, 2H), 2.34 (s, 3H) ppm. IR (neat): ν̅ = 3277, 2926, 1663, 1558, 1516, 1489, 1393 cm−1. MS (ESI): m/z 394 [M + Na]+. Anal. Calcd for C23H18FN3O: C, 74.38; H, 4.88; N, 11.31. Found: C, 74.22; H, 4.87; N, 11.30. 4-(2-Chlorophenyl)-N-(4-methylbenzyl)quinazoline-2-carboxamide (50). Yield: 77%. Mp: 150−152 °C. 1H NMR (300 MHz, CDCl3): δ = 8.55 (br t, J = 5.8 Hz, 1H, exchangeable with deuterium oxide), 8.33 (d, J = 8.5 Hz, 1H), 8.08−7.86 (m, 1H), 7.82−7.60 (m, 2H), 7.59−7.42 (m, 4H), 7.31 (d, J = 7.9 Hz, 2H), 7.16 (d, J = 7.9 Hz, 2H), 4.74 (d, J = 5.5 Hz, 2H), 2.34 (s, 3H) ppm. IR (neat): ν̅ = 3321, 2924, 1668, 1565, 1516, 1495, 1391 cm−1. MS (ESI): m/z 410 [M + Na]+. Anal. Calcd for C23H18ClN3O: C, 71.22; H, 4.68; N, 10.83. Found: C, 71.30; H, 4.68; N, 10.81. 4-((N-Ethyl-4-phenylquinazoline-2-carboxamido)methyl)phenyl 4-Methylbenzenesulfonate (51). Reaction of the carboxylic acid 31 and secondary amine 53, according to the general procedure for the synthesis of N-benzyl-4-phenylquinazoline-2-

carboxamides 5−13, 16, and 18−23, yielded the title compound. Yield: 95%. Mp: 85−87 °C. 1H NMR (300 MHz, CDCl3): δ = 8.20− 8.10 (m, 2H), 8.00−7.90 (m, 1H), 7.85−7.79 (m, 1H), 7.75−7.53 (m, 7H), 7.44−7.26 (m, 4H), 7.01−6.90 (m, 2H), 4.81, 4.48 (2s, 2H), 3.54, 3.26 (2q, J = 7.4 Hz, 2H), 2.45 (s, 3H), 1.20, 1.15 (2t, J = 7.4 Hz, 3H) ppm. IR (neat): ν̅ = 1647, 1558, 1541, 1487, 1373 cm−1. MS (ESI): m/z 538 [M + H]+. Anal. Calcd for C31H27N3O4S: C, 69.25; H, 5.06; N, 7.82. Found: C, 69.41; H, 5.07; N, 7.80. N-(4-Nitrobenzyl)ethanamine (52). To a solution of 4-nitrobenzaldehyde (500 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. The solution was stirred at room temperature for 12 h and then refrigerated to 0 °C, and NaBH4 was added portionwise 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 pale-yellow oil, which was used without further purification. Yield: 93%. 1H NMR (300 MHz, CDCl3): δ = 8.17 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.5 Hz, 2H), 3.89 (s, 2H), 2.67 (q, J = 7.1 Hz, 2H), 1.64 (br s, 1H, exchangeable with deuterium oxide), 1.14 (2t, J = 7.1 Hz, 3H) ppm. 4-((Ethylamino)methyl)phenyl 4-Methylbenzenesulfonate (53). Reductive alkylation of 4-formylphenyl 4-methylbenzenesulfonate, according to the procedure for the synthesis of 52, yielded the title compound as a pale yellow oil. Yield: 96%. 1H NMR (300 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 (t, J = 7.1 Hz, 3H) ppm. Computational Studies. Training Set. Twenty-two isoquinoline and quinazoline derivatives tested under similar conditions using rat kidney membrane were selected as the training set (Table S1, Supporting Information).30 The ligand set, thus generated, consists of 10 active and 12 inactive TSPO ligands. The compounds were built using the fragment builder tool available in the Maestro package of Schroedinger 9.145 and minimized employing Macromodel, MMFFs as the force field, and water as the implicit solvent until a convergence value of 0.05 kJ/(mol·Å). The computational protocol applied consists of the application of 500 steps of the Polak−Ribiére conjugate gradient (PRCG) for structure minimizations. Generation of the Pharmacophore. A relevant step, in generating a pharmacophore model, is represented by the exhaustive sampling of the conformational space of the ligands under investigation. This represents a crucial step in our study, since it has been hypothesized that both isoquinoline and quinazoline derivatives are characterized by peculiar bioactive conformations at the carboxamide region.30,46 Particularly relevant features related to the carboxamide moiety are (i) the OCN< moiety lying out of the plane of the aromatic system and (ii) the smaller substituent of the disubstituted amidic nitrogen in a cisoid conformation with respect to the OC amide group.46 Therefore, the 22 compounds were submitted to an extensive conformational search employing the advanced search method of the ConfGen tool available in the Maestro package of Schroedinger 9.1.45 Conformations were generated by the torsional search method, using implicit water as the solvent and MMFFs as the force field. For each molecule, the maximum number of conformations returned was set equal to 1000 and the search method was conducted applying the thorough method, with the number of rotatable bonds equal to 36 and sampling various amide bond conformations. The maximum number of ring conformations was set equal to 32, and the energy window for saving conformations was fixed equal to 200 kJ/mol. Finally, redundant conformations were eliminated if the heavy atom RMSD was lower than 0.8 Å. The conformations, thus obtained, were imported into the “Develop Pharmacophore Hypothesis” panel of the PHASE suite, available in the Maestro package of Schroedinger 9.1.45 The pharmacophore hypothesis and subsequent ligand alignment were carried out using the PHASE suite.45 On the basis of the ligand structural features, the following pharmacophore features were used: 2424

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Journal of Medicinal Chemistry

Article

Biological Evaluation. Materials. Cell culture media and growth supplements were obtained from Lonza (Milan, Italy). Nonessential amino acids, N-methyl-N-(1-methylpropyl)-1-(2-chlorophenyl)-3-isoquinolinecarboxamide (1), CCCP, and protease inhibitors were from Sigma-Aldrich srl (Milan, Italy). [3H]1 (specific activity 73.6 Ci/ mmol) was purchased from Perkin-Elmer Italia spa (Monza, Italy). The Cell Titer 96 Aqueous One Solution Cell Proliferation Assay was obtained from Promega (Milan, Italy). The Muse Mitopotential Assay Kit was from Millipore spa (Milan, Italy). All other reagents were from standard commercial sources. The experimental protocol for the [35S]GTPγS binding experiments is reported in the Supporting Information. The screening of compound 19 on a set of 13 human kinases [Abl kinase, c-kit kinase, EGFR kinase, FLT-1 kinase (VEGFR1), FLT-3 kinase, HER2/ErbB2 kinase, KDR kinase (VEGFR2), PDGFRα kinase, PDGFRβ kinase, PKCβ1, PKCβ2, Ret kinase, mTOR kinase (FRAP1)] was performed by Cerep (France), according to the company’s standard operating procedures. Compound Affinity Studies: [3H]1 Binding to Rat Kidney Mitochondrial Membranes. For affinity binding studies, crude mitochondrial membranes were incubated with 0.6 nM [3H]1 in the presence of a compound concentration range of 0.05 nM to 20 μM in 50 mM Tris−HCl, pH 7.4, as previously described.19 For the active compounds, the IC50 values were determined and Ki values were derived in accordance with the equation of Cheng and Prusoff.62 U343 GBM Cell culture Conditions. The human U343 GBM cell line was purchased by the National Institute for Cancer Research (ICLC, Genoa, Italy) and maintained in standard culture conditions (37 °C, 95% humidity, 5% CO2) in Eagle’s minimum essential medium (Earle’s BSS) supplemented with 2 mM L-glutamine, 100 U/ mL penicillin, 100 μg/mL streptomycin, 0.1 mM nonessential amino acids (NEAAs), 1.0 mM sodium pyruvate, and 10% fetal bovine serum (FBS). TSPO Characterization in U343 Cells: [3H]1 Binding Assays. As previously reported,52 for crude membrane preparation, confluent U343 cells derived from a 175 cm2 Petri dish were harvested using phosphate-buffered saline (PBS), pH 7.4, supplemented with 0.04% EDTA. After cell collection by centrifugation (200g for 5 min), the pellet was suspended in ∼10 mL of 5 mM ice-cold Tris−HCl buffer, pH 7.4, containing protease inhibitors (160 μg/mL benzamidine, 200 μg/mL bacitracin, and 20 μg/mL trypsin inhibitor) and homogenized with an Ultraturrax. Then the homogenate was centrifuged at 48000g for 15 min at 4 °C, and the supernatant was discarded. The obtained pellet was suspended in ∼10 mL of Tris−HCl, 50 mM, pH 7.4 (assay buffer), containing the same amounts of protease inhibitors as described above, and the homogenate was pelleted by centrifugation (48000g, 15 min, 4 °C). The pellet was washed once with assay buffer, and an additional centrifugation step followed (48000g, 15 min, 4 °C). The resulting cell membrane pellet was suspended at a final concentration of 1 mg of proteins/mL in assay buffer and used for binding assays. The protein content of 20 μL of membrane suspension was measured by the Bradford method63 using the Bio-Rad Protein Assay reagent, according to the manufacturer’s protocol, with bovine serum albumin (BSA) used as the standard. To determine the presence of specific [3H]1 binding to U343 cell membrane suspensions, equilibrium radioligand binding assays were performed. Briefly, different aliquots of U343 cell membranes (10− 100 μg of proteins) were incubated with [3H]1 (1.5 nM) in the presence (nonspecific binding) or in the absence (total binding) of unlabeled 1 (1 μM), in a final volume of 500 μL of assay buffer for 90 min at 0 °C. For saturation experiments, aliquots of U343 cell membranes (30 μg of proteins) were incubated in duplicates with eight increasing [3H]1 concentrations (0.5−30 nM) in the same above-described conditions. In each assay, the final vehicle concentration in the incubation buffer was less than 1% and did not interfere with specific [3H]1 binding. TSPO Functional Studies: Compound Cell Treatments. For functional experiments, U343 cells, grown in flasks or Petri dishes, were detached by mild trypsinization and counted using the Scepter 2.0 hand-held automated cell counter (http://www.millipore.com/

(i) hydrogen bond acceptor (A), (ii) hydrogen bond donor (D), (iii) hydrophobic group (H), (iv) aromatic ring (R), (v) a custom feature X accounting for substituted amides, and (vi) a custom feature Y, which corresponds to the combination of hydrophobic groups and aromatic rings. Common pharmacophores were identified by applying a treebased partitioning technique, which groups similar pharmacophores according to their intersite distances (distance between pairs of sites in the pharmacophore). The minimum intersite distance was set equal to 2 Å, with a maximum tree depth of 5, an initial box size of 32 Å, and a final box size of 1 Å. PHASE returns a list of pharmacophore variants, and for each one the representative ligand (the ligand exactly matching all the pharmacophore features of the 3D pharmacophore variant) is reported. The 3D pharmacophore models, thus obtained, were evaluated on the basis of a scoring procedure, which yielded the best alignment function of the active compounds. In detail, 40 common pharmacophoric variants were obtained, which were clustered using the complete linkage method. For this clustering algorithm the distance between clusters is the largest distance between any pair of objects (one object from each cluster). Finally, the best pharmacophore variant was chosen on the basis of the (i) post hoc scores (this score is the result of rescoring and is a weighted combination of the vector, site, volume, and selectivity scores) and (ii) size of the clusters (number of pharmacophore variants which belong to the cluster). Assessment of the Pharmacophore. The selected pharmacophore model was evaluated on the basis of its ability to discriminate between active and inactive compounds using the Find Matches tool available in PHASE45 and screening two different test sets. Find Matches is a panel where the users can specify the database to search and the pharmacophore hypothesis to use in the search. The search is performed in two steps: finding matches to the hypothesis and the scoring of the hits. Ligands matching the pharmacophore are ranked on the basis of the fitness score (0 ≤ F ≤ 3), which represents a measure of how well the matching pharmacophore site points align to those of the hypothesis, how well the matching vector features (acceptors, donors, aromatic rings, etc.) overlie those of the hypothesis, and how well the matching conformation superimposes, in an overall sense, with the reference ligand conformation. The fitness score is defined by

⎛ Salign ⎞ ⎟ + WvecSvec + WvolSvol + WivolSivol S = Wsite⎜⎜1 − Calign ⎟⎠ ⎝ where Salign is the alignment score, Calign the alignment cutoff (1), Wsite the weight of the site score (1), Svec the vector score, Wvec the weight of the vector score (1), Svol the volume score, Wvol the weight of the volume score, Sivol the included volume score, and Wivol the weight of the volume score. Two test sets were screened using the Find Matches tool and the selected pharmacophore model: test set 1, which is composed of 57 isoquinoline and quinazoline derivatives (37 actives and 20 inactives, Table S2 Supporting Information), and test set 2 (18 TSPO active ligands and 3961 compounds, retrieved from the whole DUD database).61 It is worth mentioning that test set 2 has an active content equal to 0.45% that mimics real-life screening situations.47,48 The compounds included in test sets 1 and 2 were prepared by applying the same protocol described for the training set compounds and were submitted to the same conformational search methods. QM Geometry Scan. The Jaguar tool within Schroedinger software suite 9.145 was used to perform a geometry scan for compounds 5, 7, 8, and 10. Geometry scans are a series of jobs run that vary only the value of one or more variables used to define an internal or Cartesian coordinate in the input structure. A preliminary relaxed geometry scan (without constraints on the ligand geometry) was conducted on compound 5. In this case the DFT/B3LYP 6-31G** level of theory was used in the gas phase, and the N3−C4−C1′−C2′ dihedral angle (herein defined as ω) was varied in 19 steps from 0 to 360°. The obtained geometrical minimum was used as the starting point to rebuild compounds 7, 8, and 10. Therefore, compounds 5, 7, 8, and 10 were submitted to a rigid geometry scan allowing the rotation of the ω dihedral angle. 2425

dx.doi.org/10.1021/jm401721h | J. Med. Chem. 2014, 57, 2413−2428

Journal of Medicinal Chemistry

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catalogue/item/phcc20060, Millipore, Milan, Italy). Then the cells were seeded in 96-well plates at a density of ∼4.5 × 103 cells per well for the proliferation assay or in 6-well plates at a density of ∼60 × 103 cells per well for the apoptosis measurement and allowed to proliferate for 24 h. Then the culture medium was replaced by complete medium supplemented with different TSPO ligands or by complete medium added with DMSO or EtOH (control cells). The cells were incubated for 24 h (proliferation assay) or 5 h (apotosis assay). In each assay, the vehicle in which compounds were dissolved never exceeded 1% of the final assay volume, and we verified that this amount did not affect cell survival. Notably for the apoptotic measurement, U343 cells were exposed for 15 min to the uncoupling agent CCCP (3 μM) as a depolarized positive control. Proliferation Analyses by MTS Conversion Assay. The number of living cells in U343 treated for 24 h was measured by using the quantitative colorimetric MTS conversion assay as previously described.52 All measurements were performed in duplicate, and the experiments were repeated at least three times. Mitochondrial Membrane Potential Assay. The mitochondrial depolarization state of treated cells was assessed using the Muse cell analyzer (Merck Millipore), which is a miniaturized cytometry packing three-parameter analysis using specific fluorescent dyes. We simultaneously measured changes in the mitochondrial membrane potential (using the red Muse MitoPotential dye) and in cellular plasma membrane permeabilization (using the Muse MitoPotential 7-AAD reagent). Briefly, both floating and adherent treated cells were collected, centrifuged at 300g for 5 min, and suspended in cell culture medium. Then a 100 μL aliquot of cell suspension (about 20 000 cells/mL) was first added to 95 μL of diluted Muse MitoPotential dye, and after 20 min at rt, it was added to 5 μL of 7-AAD reagent dye. After 5 min, the cell suspensions were acquired by a Muse cell analyzer, and the percentage of live, depolarized, and dead cells was analyzed in accordance with the Millipore guidelines. Data Analyses. Scatchard analyses of saturation binding data, displacement curves, graphic presentation, and statistical analyses were performed using the nonlinear multipurpose curve-fitting Graph-Pad Prism computer program (Graph Pad Software, version 5.0; San Diego, CA). In detail, for saturation binding studies one-site binding curve fitting was used. IC50 values were derived by semilog plots of ligand displacement experiment data. The Cheng and Prusoff equation was used to calculate Ki values.55 Statistical analyses were performed by one-way ANOVA with the Bonferroni post-test, as appropriately specified in the relevant figure legends. A p value of