p75

European Journal of Medicinal Chemistry 101 (2015) 288e294

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Short communication

Discovery of N-aryl-naphthylamines as in vitro inhibitors of the interaction between HIV integrase and the cofactor LEDGF/p75 Giuliana Cuzzucoli Crucitti a, Luca Pescatori a, Antonella Messore a, Valentina Noemi Madia a, Giovanni Pupo a, Francesco Saccoliti a, Luigi Scipione a, Silvano Tortorella a, Francesco Saverio Di Leva c, Sandro Cosconati b, Ettore Novellino c, Zeger Debyser d, Frauke Christ d, Roberta Costi a, *, Roberto Di Santo a a
di Roma, P-le Aldo Moro 5, 00185 Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione Cenci Bolognetti, “Sapienza” Universita Roma, Italy b
di Napoli, Via Vivaldi 43, 81100 Caserta, Italy DiSTABiF, Seconda Universita c
di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy Dipartimento di Farmacia, Universita d Molecular Virology and Gene Therapy, Molecular Medicine Katholieke Universiteit Leuven, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 March 2015 Received in revised form 18 June 2015 Accepted 19 June 2015 Available online 22 June 2015

A series of N-aryl-naphthylamines, exemplified by the structures 11e16, were chosen for an in-house library screening to assay their ability to disrupt the interaction between the LEDGF cofactor and the HIV integrase. Structure modification led also to design and synthesize new compounds 17aef. Compounds 11e,h,k,n, 13b, and 14 showed good activity in AlphaScreen assay. The most active compound 11e (IC50 ¼ 2.5 mM) was selected for molecular modeling studies and showed a binding mode similar to the one of the known LEDGIN 8. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: Integrase LEDGF/p75 LEDGINs N-aryl-naphthylamine

1. Introduction HIV integrase (IN) catalyzes the insertion of the retrotranscribed viral DNA (vDNA) into the genome of the host cell. It is well established that the integration process consists of two catalytic steps: the first is a hydrolytic reaction named 30 -processing (30 -P), followed by a transesterification called strand transfer (ST) [1,2]. Over the last two decades, IN drug design and discovery has been mainly focused on the direct inhibition of enzyme catalytic activities, leading to clinically approved IN inhibitors, like raltegravir [3], elvitegravir [4] and dolutegravir [5]. These compounds share a similar mode of action at the IN active site: they chelate the metals

Abbreviations: IN, integrase; vDNA, viral DNA; 30 -P, 30 -processing; ST, strand transfer; LEDGF, lens epithelium-derived growth factor; IBD, integrase binding domain; CCD, catalytic core domain; PPI, proteineprotein interaction; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CCID, cell culture infective dose. * Corresponding author. E-mail address: [email protected] (R. Costi). http://dx.doi.org/10.1016/j.ejmech.2015.06.036 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

coordinated by the three catalytic residues (DDE triad) and interact with vDNA in the complex, inhibiting the ST step. However, resistance against these inhibitors readily emerges in patients [6]. Therefore, development of next-generation IN inhibitors preferably targeting alternative sites of the enzyme is a major priority in the field of antiviral research. Recently, research interests have moved toward the design of inhibitors with an allosteric mechanism of action or inhibitors of the interactions with cellular cofactors that are essential for integration. Integration of lentiviruses including HIV is dictated by the specific interaction between IN and the cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) that acts as a molecular tether linking IN to the chromatin [7e12]. LEDGF is a transcriptional co-activator that is strongly associated with chromatin throughout the cell cycle. It is expressed as two spliced variants: the LEDGF/p52 and LEDGF/p75 proteins [13]. LEDGF/p75 shows a more extended C-terminal domain that includes the IN-binding domain (IBD), which is crucial for specific interaction with HIV-1 IN and other cellular binding partners [14]. This protein has been shown to stimulate the in vitro integration activity of IN Ref. [15] and is an

G. Cuzzucoli Crucitti et al. / European Journal of Medicinal Chemistry 101 (2015) 288e294

interaction partner of HIV-1 IN in human cells [16]. A crystal structure of a dimer of the IN catalytic core domain (CCD) bound to IBD has been reported [17]. These data, along with mutagenesis experiments [17e19], demonstrated that the proteineprotein interaction (PPI) surface between LEDGF/p75 and IN provides a well-defined pocket with multiple hydrophobic and hydrogen bond interactions. Rationally, this pocket is a good target for the design of small molecules for the purpose of inhibiting LEDGF/p75IN PPI. To date, only few compounds were discovered as inhibitors of the LEDGF/IN interaction: the most relevant ones (1e10) are reported in Chart 1. Among them, the 2-(quinolin-3-yl)acetic acid derivatives were discovered by a rational drug design approach [20]. These compounds were called “LEDGINs” because they bind to the LEDGF/p75 binding pocket in IN and they represent the first class of authentic small-molecule allosteric inhibitors to display antiretroviral activity tied to a specific disruption of the INeLEDGF/ p75 interaction [20]. Compound 8 (CX0516) [18] (Chart 1) inhibited the INeLEDGF/p75 interaction in vitro at micromolar concentration and the HIV replication in a cell assay, too. A co-crystal of IN CCD with 8 was also reported; the LEDGIN carboxyl moiety forms hydrogen bonds with both main-chain nitrogen atoms of IN E170 and H171, which mimics the IN protein contacts of the LEDGF/p75 residue D366. The IN residue A128 occupies a space adjacent to the chlorine atom between the phenyl and conjugated ring system of the 2-(quinolin-3-yl)acetic acid derivative.

289

The above structural information helped to design and synthesize more potent LEDGINs with improved biological activities, such as 9 (CX14442) [21] (Chart 1). Compound 9 is the first LEDGIN reported to display antiviral activity in the low nanomolar range, with an EC50 of 69 nM [21]. To date, the 2-(tert-butoxy)-2-substituted acetic acid derivatives are best studied LEDGINs; congeners of these compounds are in advanced preclinical development. A series of 2-(quinolin-3-yl)acetic acid derivatives, including the prototype 10 (BI-1001) [22], have also been disclosed in an international patent application by Boehringer Ingelheim Pharmaceuticals Inc (Chart 1) [22]. All LEDGINs have in common that they bind to an allosteric region of IN, disrupt the INeLEDGF/p75 proteineprotein interaction but also inhibit the LEDGF-independent IN catalytic function [23]. In an effort to discover novel scaffolds to obtain new inhibitors of the LEDGF/IN interaction, we decided to screen part of our in house library of compounds with the AlphaScreen assay [24]. A first selection within our library was based on the chemical similarity with the reported inhibitors of LEDGF/p75-IN interaction reported so far. In fact, a number of inhibitors reported in literature to date are characterized by an aromatic portion and a carboxylate function (Chart 1, compounds 1e3 and 5e10). Interestingly, among the 40 tested compounds, the N-aryl-naphthylamine 11k was found as a hit (Chart 2). Thus, compounds 11e16 related to 11k and already reported as inhibitors of Ab aggregation [25], were re-synthesized and tested in the AlphaScreen assay. Moreover, the novel derivatives 17 of 11k were designed, synthesized and tested to better understand the structure-activity relationships (SAR) within this new class of LEDGINs (Chart 2). 2. Results and discussion 2.1. Chemistry Compounds 11e16 were synthesized as reported previously [25]. The new quinolinone derivatives 17aef were prepared as described in Scheme 1. The 4-hydroxyquinoline 18 and the quinolinones 19 [26] and 20 [27], were alkylated with methyl 4-(bromomethyl)benzoate using NaH as a base, to obtain the alkylated derivatives 17b, 17d and 17f, that were in turn hydrolyzed with 1N NaOH in methanol to afford the required acids 17a, 17c and 17e. 2.2. Evaluation of biological activity

Chart 1. Small molecules inhibitors of the LEDGF/p75-IN interaction.

Compounds 11e17 were tested in the AlphaScreen assay as described previously [24] (Table 1). First the inhibition of the interaction between HIV-1 IN and LEDGF/p75 was determined at 100 mM of each compound. For those compounds that inhibited the interaction more than 50%, the IC50 value was determined (Table 1). Among them, compounds 11c and 17f proven inactive up to 100 mM, while 11e, 11h, 11k, 11n, 13b, and 14 were active at concentrations between 70.07 and 2.5 mM. The highest activity was found for acid derivative 11e, showing IC50 value 2.5 mM. Interestingly three of the most active compounds are carboxylic acids (11e, 11h and 13b) confirming a relevant role of this function for the binding with IN pocket [8]. A comparison of the activity of 11e and the methylated counterpart 11h also seems to confirm this hypothesis. However, the carboxylic function either free or esterified with a methyl group, although very important to increase the activity, seems to be not vital, considering the moderate activity of methoxy derivatives 11n and 14. An increase of the lipophylicity of the molecule seems to play a further role, considering the activity of compounds 11k, 11n and 14. Next, the effective concentration required to reduce HIV-1IIIB induced cytopathic effect by 50% in MT4 cells was determined (EC50) and in parallel the 50% cytotoxic

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Table 1 Biological activities of 11aep, 12aec, 13aeg, 14, 15a,b, 16a,b, 17aef and reference compound 9.

cpd

11a 11b 11c 11d 11e 11f 11g 11h 11i 11j 11k 11l 11m 11n 11o 11p 12a 12b 12c 13a 13b 13c 13d 13e 13f 13g 14 15a 15b 16a 16b 17a 17b 17c 17d 17e 17f 9 a b c d e f

R

H H F F OH OH OH OH OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OH OH OCH3 OH OH OH OCH3 OCH3 OCH3 OCH3 e OH OCH3 OH OCH3 H H COCH3 COCH3 COOH COOC2H5

R1

4-COOH 4-COOCH3 4-F 4-SCH3 4-COOH 4-COOCH3 4-OH 3-COOH-4-OH H 4-COOH 4-COOCH3 2-OCH3 4-OCH3 2-OCH3-5-I 3-COOH-4-OCH3 3-COOCH3-4-OCH3 COOH COOCH3 COOCH3 H COOH COOCH3 H COOH COOCH3 I e e e e e COOH COOCH3 COOH COOCH3 COOH COOCH3

Quenching %inhibitiona at 100 mM

16 84 52

no

92

74

Alphascreen %inhibitionb at 100 mM 39 62 63 nef 95 49 95 88 ne 33 59 ne 57 61 41 ne 56 28 ne 61 66 58 ne ne ne ne 100 38 ne 30 ne ne 25 ne 46 16 60

IN-LEDGF/p75 IC50c (mM)

MTT/MT-4 EC50d

CC50e

>100

>27.54 >38.40

27.54 38.40

2.5 ± 2.05

>1.07

1.07

19.12 ± 7.5

>125.6

125.6

28.93 ± 0.34

>12.48

12.48

70.07 ± 3.78

>23.67

23.67

13.76 ± 1.78

>3.41

3.41

>100 0.046 ± 0012

>5.63 0.069 ± 0.003

5.63 96.0 ± 16.0

34.8 ± 0.73

Percent inhibition exerted by compounds in a quench counter-screen when tested at 100 mM. Percent inhibition exerted by compounds in IN-LEDGF/p75 AlphaScreen when tested at 100 mM. Concentration required to inhibit the IN-LEDGF/p75 proteineprotein interaction by 50%. Cytotoxic concentration reducing MT-4 cell viability by 50%. Effective concentration required to reduce HIV-1 induced cytopathic effect by 50% in MT-4 cells. No effect.

concentration (CC50) was determined (Table 1). Antiviral activities of a few compounds (11b,c,e,h,n, 13b, 14 and 17f) that demonstrated the highest activity in AlphaScreen assay were tested, but unfortunately, have been found not higher than the concentrations inducing cellular toxicity. Thus, further medicinal chemistry optimization will be required in the future to improve the anti-HIV activity while reducing the cellular toxicity. 2.3. Molecular modeling Molecular docking studies were performed to investigate at atomic level the inhibitory properties of the newly identified

LEDGF-IN interaction inhibitors. In particular, the most potent derivative 11e was docked at the LEDGF/p75 binding site that is formed at the interface of a single IN CCD dimer (PDB code 2B4J) [17]. According to docking results, 11e perfectly fits into the crevice formed at the CCD dimer interface interacting with both CCD monomers, as shown in Fig. 1a. Here the ligand establishes hydrogen bonds with both the Glu170 and His171 backbone NH and with the His171 side-chain through its carboxylate moiety. On the other side, the ligand naphthyl ring is predicted to seep into the lipophilic pocket shaped by residues Leu102, Ala128, Ala129, Trp131, Trp132 (on the same IN CCD monomer), and Met178 (on the other monomer), where it can establish favorable hydrophobic

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Chart 2. Hit 11k its N-aryl-naphthylamine analogues 11e14 and related derivatives 15e17 as inhibitors of the LEDGF/p75-IN interaction (for substituents R and R1 see Table 1).

Scheme 1. i: methyl 4-(bromomethyl)benzoate, NaH, THF, reflux 5 h, 75e88% yield; ii: 1 N NaOH, CH3OH, 90  C, 6 h, 97e98% yield.

Fig. 1. a) Docking pose of compound 11e (magenta sticks) in the LEDGF/p75 binding site at the HIV-1 CCD dimer interface (PDB code 2B4J). Superimposition between the predicted binding mode of 11e and (b) the crystallographic poses Ile365-Asp366 dipeptide of LEDGF/p75 (PDB code 2B4J) and (c) compound 8 (PDB code 3LPU). Compound 11e and 8 are represented as magenta and brown sticks, respectively. The two CCD monomers are depicted as cyan and green surface/cartoons, respectively. LEDGF/p75 is shown as yellow cartoons. Amino acids involved in ligand binding are highlighted as sticks. H-bonds are depicted as dashed line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

contacts. As shown in Fig. 1b, this interaction pattern closely resembles that observed for the Ile365-Asp366 dipeptide of the LEDGF IBD at the IN CCD dimer interface, although the Ile365 backbone amide group of LEDGF can establish an additional Hbond with the Gln168 backbone carbonyl oxygen [17]. Furthermore, a binding mode similar to that predicted for 11e has been already experimentally found for some potent disruptors of the LEDGF-IN interaction such as 8 and its congeners [21], as shown in Fig. 1c. Nonetheless, these comparisons provide valuable hints for the future lead-optimization efforts suggesting that the potency of this series might be improved by introducing i) a methylene bridge between the phenyl ring and the carboxylate moiety to increase the flexibility of the latter group, which in turn could strengthen the Hbonds with Glu169 and His170; ii) an alkyl substituent close to the

carboxylate group to form further lipophilic interactions within the LEDGF binding cavity; and iii) an additional H-bond donor group able to interact with the Gln168 backbone CO similarly to the Ile365 backbone NH of LEDGF/p75. We are now testing these design hypotheses and results will be reported in due of course. 3. Conclusion In summary, from the screening of our in-house chemical library, compound 11e was identified as in vitro inhibitor of the interaction between HIV IN and the cofactor LEDGF/p75. Molecular modeling studies performed on 11e revealed a binding pose within the LEDGF/IN binding pocket similar to the one shown by 8, the first LEDGIN reported in literature. The result of this study can be useful

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for the development of new derivatives of 11e and for the further hit-to-lead optimization. 4. Experimental section 4.1. Chemistry Melting points were determined on a Bibby Stuart Scientific SMP1 melting point apparatus and are uncorrected. Compounds' purity was always >95% as determined by HPLC carried out with a pump/autosampler Waters (2695eAlliance model), a UV photo diode array detector Waters (2996 model) and a system data management Waters (Empower 2). Column used was generally Suplex pkb-100 (250  4.6 mm, 5 mm). IR spectra (Nujol mulls) were recorded on a PerkineElmer Spectrum-one spectrophotometer. 1H NMR spectra were recorded at 400 MHz on a Bruker AC 400 Ultrashield 10 spectrophotometer (400 MHz). Dimethylsulfoxided6 99.9% (code 44,139-2) and deuterochloroform 98.8% (code 41,675-4) of isotopic purity (Aldrich) were used as NMR solvent. The column chromatographies were performed on silica gel (Merck; 70e230 mesh). All compounds were routinely checked by TLC by using aluminium-baked silica gel plates (Fluka DC-Alufolien Kieselgel 60 F254). Plates were visualized by UV light. Solvents were reagent grade and, when necessary purified and dried by standard methods. Concentration of solutions after reactions and extractions involved the use of rotary evaporator (Büchi) operating at a reduced pressure (ca. 20 Torr). Organic solutions were dried over anhydrous sodium sulfate (Merck). Analyses indicated by the symbols of the elements or functions were within ±0.4% of the theoretical values. The 4-hydroxyquinoline 18 and methyl 4-(bromomethyl)benzoate were purchased from Sigma-Aldrich while quinolinones 19 [26] and 20 [27] were synthesized following the reported procedures in literature. 4.1.1. General procedure for the synthesis of derivatives 17b, 17d, 17f A well stirred solution of the appropriate 4-hydroxy-quinoline (4.6 mmol) in anhydrous THF (60 mL) was cooled at 0  C and treated with NaH (9.2 mmol, 0.36 g) under argon atmosphere. Methyl 4-(bromomethyl)benzoate (9.2 mmol, 2.1 g) was then added into the suspension and the mixture was refluxed for 5 h and monitored by TLC (SiO2) using ethyl acetate/ethanol 10:1 as eluent. When the starting material disappeared, the reaction was cooled at 0  C and saturated solution of NH4Cl was added till all the excess of NaH was neutralized. The organic solvent was then removed under reduced pressure, and the aqueous residue was extracted with ethyl acetate (3 x 25 mL). The organic phase was then washed with brine, dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. The raw material was then purified with a column chromatography using ethyl acetate/ ethanol 10:1 as eluent, to obtain the required alkylated quinolinone. 4.1.2. Methyl 4-((4-oxoquinolin-1(4H)-yl)methyl)benzoate (17b) Starting material, 4-hydroxyquinoline (18, 0.7 g); 17b was obtained as a white solid (75% yield); 153e154  C; crystallized from ethanol; IR n 1715 (C]O quinolinone), 1623 (ester) cm1. 1H NMR (DMSO-d6) d 3.82 (s, 3H, OCH3), 5.63 (s, 2H, CH2 benzyl), 6.17 (d, 1H, J2-3 ¼ 7.6 Hz, C3eH), 7.30e7.40 (m, 3H, quinolinone C6eH and benzene H), 7.49 (d, 1H, J7-8 ¼ 8.4 Hz, quinolinone C8eH), 7.61 (m, 1H, J7-8 ¼ J6-7 ¼ 8.4 Hz, J7-5 ¼ 1.2 Hz, quinolinone C7eH), 7.93 (d, 2H, Jo ¼ 8.4 Hz, benzene H), 8.19 (dd, 1H, J5-6 ¼ 8.4 Hz, J7-5 ¼ 1.2 Hz, quinolinone C5eH), 8.22 (d, 1H, J2-3 ¼ 7.6 Hz, quinolinone C2eH). Anal. (C18H15NO3) calc. C, 73.71; H, 5.15; N, 4.78; found C, 73.54; H,

5.25; N, 4.89. 4.1.3. Methyl 4-((3-acetyl-4-oxoquinolin-1(4H)-yl)methyl)benzoate (17d) Starting material, 3-acetylquinolin-4(1H)-one (19, 1.0 g); 17d was obtained as white solid (84% yield); 210e211  C; crystallized from ethanol; IR n 1719 (C]O quinolinone), 1660 (C]O acetyl), 1632 (ester) cm1. 1H NMR (DMSO-d6) d 2.67 (s, 3H, COCH3), 3.83 (s, 3H, OCH3), 5.83 (s, 2H, CH2 benzyl), 7.37 (d, 2H, Jo ¼ 8.0 Hz, benzene H), 7.47 (t, 1H, J5-6 ¼ J6-7 ¼ 8.0 Hz, C6eH), 7.58 (d, 1H, J7-8 ¼ 8.0 Hz, quinolinone C8eH), 7.68 (t, 1H, J7-8 ¼ J6-7 ¼ 8.0 Hz, quinolinone C7eH), 7.93 (d, 2H, Jo ¼ 8.0 Hz, benzene H), 8.33 (d, 1H, J5-6 ¼ 8.0 Hz, quinolinone C5eH), 8.90 (s, 1H, quinolinone C2eH). Anal. (C20H17NO4) calc. C, 71.63; H, 5.11; N, 4.18; found C, 71.45; H, 4.95; N, 4.31. 4.1.4. Ethyl 1-(4-(methoxycarbonyl)benzyl)-4-oxo-1,4dihydroquinoline-3-carboxylate (17f) Starting material, ethyl 4-oxo-1,4-dihydroquinoline-3carboxylate (20, 1.2 g); 17f was obtained as a white solid (88% yield); 176e177  C; crystallized from ethanol; IR n 1717 (C]O quinolinone), 1678 (C]O ester), 1645 (ester) cm1. 1H NMR (DMSOd6) d 1.30 (t, 3H, J ¼ 7.2 Hz, CH3CH2), 3.82 (s, 3H, OCH3), 4.27 (q, 4H, J ¼ 7.2 Hz, CH2CH3), 5.79 (s, 2H, CH2 benzyl), 7.37 (d, 2H, Jo ¼ 8.0 Hz, benzene H), 7.42 (t, 1H, J5-6 ¼ J6-7 ¼ 8.0 Hz, quinolinone C6eH), 7.53 (d, 1H, J7-8 ¼ 8.4 Hz, quinolinone C8eH), 7.66 (t, 1H, J7-8 ¼ J67 ¼ 8.0 Hz, quinolinone C7eH), 7.94 (d, 2H, Jo ¼ 8.0 Hz, benzene H), 8.26 (d, 1H, J5-6 ¼ 8.0 Hz, quinolinone C5eH), 8.97 (s, 1H, quinolinone C2eH). Anal. (C21H19NO5) calc. C, 69.03; H, 5.24; N, 3.83; found C, 69.21; H, 5.13; N, 3.70. 4.1.5. General procedure for the synthesis of derivatives 17a, 17c, 17e A solution of 1N NaOH (10 mL) was added into a well stirred solution of quinolinone derivative (2.8 mmol) in methanol (40 mL). The mixture was stirred at 90  C for 6 h and monitored through TLC (SiO2) using ethyl acetate/ethanol 10:1 as eluent. When the starting material disappeared, the reaction was cooled at 0  C, the organic solvent was then removed under reduced pressure, and the aqueous residue was treated with 1N HCl untill pH 2. The white solid that precipitated was filtered under vacuum, washed with water (3 x 10 mL) and dried. The product was characterized after crystallization. 4.1.5.1. 4-((4-Oxoquinolin-1(4H)-yl)methyl)benzoic acid (17a). Starting material, methyl 4-((4-oxoquinolin-1(4H)-yl)methyl)benzoate (17b, 0.85 g); 17a was obtained as white solid (98% yield); >300  C; crystallized from ethanol; IR n 3396 (OH), 1686 (C]O quinolinone), 1606 (C]O acid) cm1. 1H NMR (DMSO-d6) d 5.62 (s, 2H, CH2 benzyl), 6.16 (d, 1H, J2-3 ¼ 7.6 Hz, C3eH), 7.29e7.36 (m, 3H, quinolinone C6eH and benzene H), 7.50 (d, 1H, J7-8 ¼ 8.4 Hz, quinolinone C8eH), 7.59 (t, 1H, J7-8 ¼ J6-7 ¼ 8.0 Hz, quinolinone C7eH), 7.89 (d, 2H, Jo ¼ 8.0 Hz, benzene H), 8.17 (d, 1H, J5-6 ¼ 8.0 Hz, quinolinone C5eH), 8.24 (d, 1H, J2-3 ¼ 7.6 Hz, quinolinone C2eH), 13.00 (bs, 1H, COOH). Anal. (C17H13NO3) calc. C, 73.11; H, 4.69; N, 5.02; found C, 73.34; H, 4.75; N, 4.95. 4.1.5.2. 4-((3-Acetyl-4-oxoquinolin-1(4H)-yl)methyl)benzoic acid (17c). Starting material methyl 4-((3-acetyl-4-oxoquinolin-1(4H)yl)methyl)benzoate (17d, 1.0 g); 17c was obtained as white solid (97% yield); 252e253  C; crystallized from ethanol; IR n 3295 (OH), 1733 (C]O quinolinone), 1682 (C]O acetyl), 1661 (C]O acid) cm1. 1H NMR (DMSO-d6) d 2.65 (s, 3H, COCH3), 5.80 (s, 2H, CH2 benzyl), 7.33 (2, 2H, Jo ¼ 8.0 Hz, benzene H), 7.46 (t, 1H, J5-6 ¼ J67 ¼ 8.0 Hz, C6eH), 7.58 (d, 1H, J7-8 ¼ 8.4 Hz, quinolinone C8eH), 7.66

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(t, 1H, J7-8 ¼ J6-7 ¼ 8.4 Hz, quinolinone C7eH), 7.89 (d, 2H, Jo ¼ 8.0 Hz, benzene H), 8.32 (d, 1H, J5-6 ¼ 8.0 Hz, quinolinone C5eH), 8.89 (s, 1H, quinolinone C2eH), 14.00 (bs, 1H, COOH). Anal. (C19H15NO4) calc. C, 71.02; H, 4.71; N, 4.36; found C, 71.25; H, 4.56; N, 4.19.

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protection against the CPE of HIV, which is defined as the 50% effective concentration (IC50), was determined. The concentration of the compound killing 50% of the MT-4 cells, which is defined as the 50% cytotoxic concentration (CC50), was determined as well. 4.3. Molecular modeling

4.1.5.3. 1-(4-Carboxybenzyl)-4-oxo-1,4-dihydroquinoline-3carboxylic acid (17e). Starting material, ethyl 1-(4-(methoxycarbonyl)benzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (17f, 1.0 g); product 17e was obtained as white solid (98% yield); >300  C; crystallized from ethanol; IR n 3051 (OH), 1721 (C]O quinolinone), 1692 (C]O acid), 1614 (C]O acid) cm1. 1H NMR (DMSO-d6) d 5.98 (s, 2H, CH2 benzyl), 7.38 (d, 2H, Jo ¼ 8.0 Hz, benzene H), 7.63 (t, 1H, J5-6 ¼ J6-7 ¼ 7.6 Hz, quinolinone C6eH), 7.79 (d, 1H, J7-8 ¼ 8.0 Hz, quinolinone C8eH), 7.86 (t, 1H, J7-8 ¼ J67 ¼ 7.6 Hz, quinolinone C7eH), 7.91 (d, 2H, Jo ¼ 8.4 Hz, benzene H), 8.41 (d, 1H, J5-6 ¼ 7.6 Hz, quinolinone C5eH), 9.33 (s, 1H, quinolinone C2eH), 15.00 (bs, 2H, COOH). Anal. (C18H13NO5) calc. C, 66.87; H, 4.05; N, 4.33; found C, 66.76; H, 4.21; N, 4.20. 4.2. Biological evaluation 4.2.1. Expression and purification of recombinant HIV-1 integrase and LEDGF/p75 proteins His6-tagged HIV-1 integrase and 3xflag-tagged LEDGF/p75 were purified for AlphaScreen applications as described previously [28,29]. 4.2.2. IN-LEDGF/p75 AlphaScreen assay and emission quench AlphaScreen assay The AlphaScreen assay was performed according to the manufacturer's protocol (PerkinElmer, Benelux). Reactions were performed in a 25 mL final volume in 384-well Optiwell™ microtiter plates (PerkinElmer). The reaction buffer contained 25 mM TriseHCl (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 0.01% (v/v) Tween-20 and 0.1% (w/v) bovine serum albumin. His6-tagged integrase (300 nM final concentration) was incubated with compound at a final concentration of 20 mM for 30 min at 4  C. FLAG-tagged LEDGF/ p75 protein was then added at 300 nM final concentration, and the reaction was incubated for an additional 60 min at 4  C. Subsequently, 5 mL Ni-chelate-coated donor beads and 5 mL anti-FLAG antibody coated acceptor beads were added to a final concentration of 20 mg/mL of both beads. Proteins and beads were incubated for 1 h at 30  C in order to allow association to occur. Exposure of the reaction to direct light was omitted as much as possible and the emission of light from the acceptor beads was measured in the EnVision plate reader (PerkinElmer) and analyzed using the EnVision manager software. The emission quenching AlphaScreen assay was carried out identically to the IN-LEDGF/p75 AlphaScreen assay, except instead of the IN and LEDGF/p75 proteins, a FLAG-tagged GST construct was added at a final concentration of 500 nM. 4.2.3. Drug susceptibility assays The inhibitory effect of antiviral drugs on the HIV-induced cytopathic effect in MT-4 cell culture was determined by the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay [30]. This assay is based on the reduction of the yellow colored MTT by mitochondrial dehydrogenase of metabolically active cells to a blue formazan derivative, which can be measured spectrophotometrically. The 50% cell culture infective dose of the HIV strains was determined by titration of the virus stock using MT4 cells. For the drug susceptibility assays, MT-4 cells were infected with 100e300 50% cell culture infective doses (CCID50) of HIVIIIB [31] in the presence of five-fold serial dilutions of the antiviral drugs. The concentration of the compound achieving 50%

The tridimensional structure of compound 11e was generated using the Maestro Build Panel [32] and then submitted to PolakeRibiere conjugate gradient minimization (0.0005 kJ/(Å mol) convergence) using MacroModel [33]. Ligand protonation state was then assigned using Epik [34]. The crystal structure of the dimeric HIV-1 IN CCD in complex with the LEDGF/p75 IBD (PDB code 2B4J) [17] was used for docking calculations. The receptor structure was prepared using the “Pro€dinger 2012 molecular tein Preparation Wizard” panel of Schro modeling package [32]. In particular, the bond orders and disulfide bonds were assigned, water molecules were removed and hydrogen atoms were added and minimized using the OPLS-AA force field [35]. The LEDGF structure was removed prior to receptor grid generation. Docking studies were carried out with Glide v. 5.8 [36]. For the grid generation, a box centered on the LEDGF binding site at the IN CCD dimer interface was created. The Cartesian coordinates of the outer box, X, Y, and Z length were set to 25 Å. The default value (1.00) for the van der Waals radii scaling factor was chosen, which means no scaling for the nonpolar atoms was performed (no flexibility was simulated for the receptor). The standard precision (SP) mode of GlideScore function was used to score the obtained binding poses. The force field used for the docking was the OPLS2005 [35]. All figures were rendered using PyMOL (www.pymol.org). Author contributions R.C. and R.D.S. contributed equally. The manuscript was written through contribution of all authors. All authors have given approval to the final version of the manuscript. Conflict of interest The authors declare no competing financial interest. Acknowledgment The authors thank the Italian MIUR for financial support, PRIN 2010-2011 (2010W2KM5L_002). R. Di Santo, R. Costi, F. Christ and Z. Debyser thank the FP7 CHAARM project for support. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2015.06.036. References [1] A. Engelman, K. Mizuuchi, R. Craigie, HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer, Cell 67 (1991) 1211e1221. [2] (a) J.L. Gerton, D. Herschlag, P.O. Brown, Stereospecificity of reactions catalyzed by HIV-1 integrase, J. Biol. Chem. 274 (1999) 33480e33487; (b) A. Mazumder, N. Neamati, A.A. Pilon, S. Sunder, Y. Pommier, Chemical trapping of ternary complexes of human immunodeficiency virus type 1 integrase, divalent metal, and DNA substrates containing an abasic site. Implications for the role of lysine 136 in DNA binding, J. Biol. Chem. 271 (1996) 27330e27338. [3] V. Summa, A. Petrocchi, F. Bonelli, B. Crescenzi, M. Donghi, M. Ferrara, F. Fiore, C. Gardelli, O.G. Paz, D.J. Hazuda, P. Jones, O. Kinzel, R. Laufer, E. Monteagudo,

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