Synthesis, Biological Evaluation, and Docking Studies of N

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Synthesis, Biological Evaluation, and Docking Studies of N-Substituted Acetamidines as Selective Inhibitors of Inducible Nitric Oxide Synthase Cristina Maccallini,†,| Antonia Patruno,†,| Neva Besˇker,†,| Jamila Isabella Alı`,† Alessandra Ammazzalorso,† Barbara De Filippis,† Sara Franceschelli,‡ Letizia Giampietro,† Mirko Pesce,‡ Marcella Reale,§ Maria L. Tricca,† Nazzareno Re,† Mario Felaco,‡ and Rosa Amoroso*,† Dipartimento di Scienze del Farmaco, Dipartimento di Scienze del MoVimento Umano, and Dipartimento di Oncologia e Neuroscienze, UniVersita` “G. d’Annunzio”, Via dei Vestini, 66100 Chieti, Italy ReceiVed July 10, 2008

New acetamidines structurally related to N-(3-(aminomethyl)benzyl)acetamidine (1, W1400) were designed as inhibitors of inducible nitric oxide synthase (iNOS). Six compounds were found to be selective for iNOS over endothelial nitric oxide synthase (eNOS), and among them, the most active and selective compound was the N-benzylacetamidine 2. A docking study was also performed to shed light on the effects of the structural modifications on the interaction of the designed inhibitors with the NOS. Chart 1. 1 and Designed NOS Inhibitors

Introduction a

Nitric oxide (NO ) is an important mediator involved in the regulation of many physiological and pathological processes, including neurotransmission and smooth muscle relaxation. The formation of NO is catalyzed by the enzyme nitric oxide synthase (NOS) via the NADPH- and O2-dependent oxidation of L-arginine.1 Three distinct isoforms of NOS have been identified: the constitutive endothelial (eNOS) and neuronal (nNOS), predominantly expressed in the vascular endothelium and in the nervous system, respectively, and the inducible (iNOS), which generates high levels of NO that modulates inflammation through multiple pathways and plays an important role in the regulation of immune reactions.2 Overproduction of NO by iNOS has been implicated in various pathological processes, including tissue damage and cell apoptosis following inflammation and ischemia, rheumatoid arthritis, and onset of colitis.3 Blocking the localized excess production of NO through inhibition of iNOS has been identified as a potential means of treating these diseases.4 Different types of NOS inhibitors have been described in the past few years, and their effects are caused by competition with the natural substrate of NOS, L-arginine, in the binding site and/ or in the oxidizing center of the enzyme (heme) or by the interaction with peptide motifs of the enzyme that influence its dimerization, affinity for cofactors, and interaction with associated proteins.5 Typical NOS inhibitors described to date include guanidines, amidines, isothioureas, and thiazines. Moreover, heterocyclic compounds (such as indazoles, benzoxazoles, and imidazoles) that incorporate a guanidine-like group into a cyclic structure have been reported as NOS inhibitors.6 N-(3-(Aminomethyl)benzyl)acetamidine (1, W1400) is the most selective inhibitor of purified human iNOS reported to * To whom correspondence should be addressed. Phone: +39-08713554686. Fax: +39-0871-3554911. E-mail: [email protected]. † Dipartimento di Scienze del Farmaco. | These authors contributed equally to this work. ‡ Dipartimento di Scienze del Movimento Umano. § Dipartimento di Oncologia e Neuroscienze. a Abbreviations: NO, nitric oxide; NOS, nitric oxide synthase; iNOS, inducible NOS; eNOS, endothelial NOS; NADPH, nicotinamide adenine dinucleotide phosphate; rmsd, root-mean-square deviation; MCMM, Monte Carlo multiple minimum; HEPES, 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid; Ts, tosylate; HPLC, high performance liquid chromatography.

date, 5000- and 2000-fold more potent against purified human iNOS than eNOS and nNOS, respectively.7 This compound is a time-, concentration-, and NADPH-dependent inactivator of iNOS and appears to be the first example of an enzyme inhibitor that acts without being modified in any way.8 In this paper, we report the synthesis and in vitro evaluation (activity and selectivity for iNOS vs eNOS) of novel acetamidines 2-11 (Chart 1) that are structurally related to the iNOS selective inhibitor 1. A docking study was also performed to shed light on the effects of the structural modifications on the interaction of the designed inhibitors with the NOS. Chemistry Compounds 2-4, 9, and 10 were prepared under mild conditions by the addition of the appropriate amine to S-2naphthylmethyl thioacetimidate hydrobromide9 (see Scheme 1). Acetamidine 3 was obtained in racemic and chiral forms, while 4 was prepared as its racemate. The treatment of BOC-protected m-xylylenediamine10 (12) with benzyl 4-methylbenzenesulfonate afforded a mixture of N-benzyl (13) and N-dibenzyl (14) derivatives that were separated by chromatography. Treatment of 13 and 14 with CF3COOH followed by NaOH afforded the deprotected intermediates 15 and 16. Reaction of 15 with S-2-naphthylmethyl

10.1021/jm800846u CCC: $40.75  2009 American Chemical Society Published on Web 02/09/2009

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Scheme 1a

a

Reagents and conditions: (a) RNH2, absolute EtOH, 0 °C, 1-3 h.

Scheme 2a

Figure 1. Percent of eNOS and iNOS activities compared with those of untreated samples (C) and 1. iNOS and eNOS activity was detected in the 11000g supernatant of THP-1 cell homogenates. Cells were induced to express iNOS with 10 µg/mL of bacterial LPS (Sigma) for 24 h after a 1 h preincubation of the inhibitors (150 µM). The concentrations of drugs was selected on the basis of the results from pilot studies using three different concentration of the inhibitor (50, 100, 150 µM) (data not shown).

aqueous solution of NaOH, as outlined in Scheme 4 (see Supporting Information). Results and Discussion

a Reagents and conditions: (a) BzOTs, dry CH3CN, N2, reflux, 5 days; (b) CF3COOH, 0 °C, 1 h; (c) NaOH, room temp; (d) S-2-naphthylmethyl thioacetimidate hydrobromide or ethylacetamidate hydrochloride, absolute EtOH, room temp, 7 h.

Scheme 3a

a Reagents and conditions: (a) 4-chloromethylpyridine or TsCl, dry DMSO, room temp, 24 h; (b) CF3COOH, 0 °C, 1 h; (c) NaOH, room temp; (d) S-2-naphthylmethyl thioacetimidate hydrobromide or ethylacetamidate hydrochloride, absolute EtOH, room temp, 24 h.

thioacetimidate hydrobromide or 16 with ethylacetamidate hydrochloride led to the desired acetamidines 5 and 6 as their hydrobromide or hydrochloride salts, respectively (Scheme 2). Condensation of 12 with 4-(chloromethyl)pyridine or ptoluensulphonyl chloride provided the Boc-protected intermediates 17 and 18, respectively. Submitting intermediates 17 and 18 to deprotection conditions (CF3COOH followed by NaOH) afforded amines 19 and 20, which were subsequently condensed with S-2-naphthylmethyl thioacetimidate hydrobromide or ethylacetamidate hydrochloride, respectively, to provide the desired 7 and 8 (Scheme 3). Acetamidine 11 was prepared as free base by reaction of acetamidine hydrochloride with benzenesulfonyl chloride in an

To assess the ability of test compounds to inhibit iNOS and eNOS, a preliminary biological evaluation was performed in vitro by using a whole-cell assay on THP-1 cells, a human myelomonocytic leukemia cell line. Figure 1 illustrates the iNOS and eNOS inhibition in the presence of a 150 µM 2-11; results are expressed as percentage of NOS activity and compared with those of 1. Among the amidines 2-4, differing from 1 by the absence of the 3-aminomethyl group and the addition of a methyl or an ethyl group on the benzylic carbon connected to the acetamidine nitrogen (2 and 4, respectively), 2, (S)-3, and rac-4 showed good iNOS and almost negligible eNOS inhibition. Amidines 5-8 were synthesized to investigate the effects of amine substitution at the 3-aminomethyl group. The addition of one or two benzyl groups to give 5 or 6 did not seem to influence iNOS activity with respect to 1 but resulted in an increased eNOS inhibition and thus in a lower selectivity. The introduction of 4-methylpyridine or tosylate group (Ts) as N-substituents (7 and 8, respectively) led to inhibition of iNOS but not of eNOS. Compounds 9 and 10 were designed to develop a new series of acetamidines in which the aromatic ring of 1 was replaced by a N-substituted piperidine linked to the acetamidine function in the 4-position. Both molecules showed good iNOS inhibition, but only 9 seemed to be somewhat selective. Finally, 11, with a phenyl separated from acetamidinic group by a sulfone, showed inhibition of iNOS and eNOS. On the basis of this preliminary study, acetamidines that did not significantly affect eNOS activity, i.e., 2, (S)-3, rac-4, 7-9, were selected for determination of IC50 and selectivities (ratio of eNOS IC50/iNOS IC50), performing an enzymatic activity assay (Table 1). The IC50 values obtained for 1 are in line with those found in literature.4 All compounds showed good iNOS IC50 and selectivities ranging from 57 to 1750. Amidines 2, rac-4, and 9 resulted in more potent iNOS inhibitors than 1, while (S)-3, 7, and 8 are less potent. These results seem to indicate that the removal of the aminomethyl group, rather than its substitution, improves the iNOS inhibition. Concerning the selectivity, the synthesized compounds can be considered very promising. With the exception of 7, the above amidines are very selective for iNOS over eNOS, according to the literature4a defining as “highly selective” agents showing 50- to 100-fold

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Table 1. Inhibition of NOS by Selected Acetamidinesa IC50 (µM) compd

iNOS

eNOS

selectivity eNOS/iNOS

2 (S)-3 rac-4 7 8 9 1

0.20 ( 0.03 0.45 ( 0.02 0.25 ( 0.02 0.35 ( 0.03 0.5 ( 0.05 0.25 ( 0.03 0.33 ( 0.06

350 ( 70 300 ( 60 80 ( 22 20 ( 2 300 ( 30 90 ( 6 1100 ( 104

1750 666 320 57 600 360 3300

a NOS inhibition at 20 µM arginine. Experiments were independently performed at least three times.

Figure 3. Top-ranked docked conformation of 4 (a), 7 (b), and 9 (c) to iNOS. Figure 2. Schematic drawing of the active site of iNOS. The main interactions of 1 with residues and cofactors important for binding are illustrated.

selectivity. One of the considered compound, 2, displayed a high iNOS to eNOS selectivity (1750), though lower than the 3300-fold selectivity observed with 1. Molecular Docking. Docking simulations were performed to predict the binding mode of all the designed inhibitors in the human iNOS and eNOS active sites and to evaluate their binding affinities and isoform selectivity. To evaluate the effectiveness of the Glide program in the docking of the considered iNOS ligands, we first docked 1 to its natural iNOS substrate and compared the resulting geometry with the corresponding crystal structure. The Glide predicted geometry is shown in Figure 2 (see Supporting Information) superimposed with the X-ray structure of the 1-iNOS complex. The root-mean-square deviation (rmsd) of these two conformations is 0.341 Å, indicating a good agreement with the crystal structure.7b The schematic drawing in Figure 2 (above) shows the main features reproduced by the docking procedure that have been considered responsible for the high potency of this inhibitor. In particular, we notice (i) the hydrogen bond interactions between the amidinic group and the carboxylate oxygen atoms of the catalytic Glu371 and the Trp366 residues, (ii) the interaction of the 3-aminomethyl group with both propionate arms of the heme cofactor, (iii) the position of the benzene ring atop one of the heme pyrrole rings. All the docked structures of 2-11 to iNOS share a strong interaction between the common amidine group and the carboxylate moiety of the Glu371 and Trp366 residues, and 2-8 also show the same orientation of the benzylic backbone and 5-8 the interaction of the 3-aminomethyl group with the heme propionate arms. Figure 3 displays the results of the docking of 4, 7, and 9. A comparison of the binding geometries of these compounds with that of 1 allows us to shed light on the main effects of the considered structural modifications on its interaction with the iNOS isoform, i.e., the elimination of the aminomethyl group, 2-4, the addition of a bulky benzyl or tosyl substituent on the nitrogen of the aminomethyl group, 5-8, and the replacement

of the benzyl with a piperidyl, 9 and 10, or a benzenesulphonyl core, 11. The most stable docking conformation of 4 (Figure 3a) shows essentially the same position of the benzylic backbone as that observed in the X-ray structure of 1, with the phenyl ring on one of the heme pyrrole rings but differently oriented, probably because of the lack of the 3-aminomethyl group and its interaction with the propionate arms of the heme cofactor. The ethyl group shows favorable hydrophobic contacts with the Pro344 and Val346 residues. Ligands 5-8, differing from 1 for the presence on the 3-aminomethyl group of aromatic substituents, show the same interaction of the latter group with the heme propionate arms and the same orientation of the benzylic core observed for 1, as exemplified by Figure 3b for 7. The presence of the aromatic substituents, Bz, CH2Pyr, or Ts, does not lead to significant steric clashes because of the presence of a sufficiently wide pocket or to significant interactions. However, the pyridine nitrogen in 7 is directed toward the amine group of Asn348, although the N · · · N distance of 2.30 Å indicates a weak electrostatic interaction rather than a hydrogen bond. In fact, 7 is the most potent inhibitor among 5-8. In ligands 9-10 the benzyl core of 1 is replaced by a piperidyl moiety, which assumes a similar position and is oriented in such a way to find favorable hydrophobic contacts with the Pro344 and Val346 residues; the piperidyl nitrogen does not show any interaction (Figure 3c for 9). From the docking calculations, a Gscore value for each inhibitor was calculated and compared with the experimental inhibitory activities, IC50, measured for 2-4 and 5-7. The experimental pIC50 values were plotted against the calculated docking scores and show a poor correlation (R2 ) 0.36). As the considered inhibitors show polar atoms or charged groups that could give rise to binding conformations significantly higher in energy than the lowest conformations in their unbound state, we have corrected the calculated docking scores for the conformational energies required for the ligands to adopt their binding conformations. For each ligand the conformational space was searched by using the Monte Carlo multiple minimum (MCMM) method in aqueous solution. The conformational energy penalty, ∆Econf, for each ligand was calculated by subtracting the internal

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energy of the lowest conformation in aqueous solution, excluding the solvation energy, from the energy of the protein bound conformation after constrained structure optimization. A multiple linear regression was carried out based on eq 1: pIC50 ) R + βGscore + γ∆Econf

(1)

This two-parameter equation showed a reasonably good correlation (Figure 5 in Supporting Information) with promising statistical characteristics [n ) 7 samples, correlation coefficient R2 ) 0.831, standard error σ ) 0.105, F-test statistic F ) 4.45, confidence level of 90%] and the best coefficients values R ) 7.791, β ) 0.200, and γ ) 0.147. The simulated geometry of 1 docked to the eNOS substrate is in good agreement with the crystal structure,11 with a rmsd of 0.242 Å (excluding the aminomethyl group atoms, affected by disorder), and shows essentially the same interactions discussed for the structure of the complex between 1 and iNOS. The docked structure explicitly shows the interaction of the 3-aminomethyl group with both propionate arms of the heme cofactor which cannot be unambiguously observed in the X-ray structure, since there is no clearly defined electron density corresponding to the 3-aminomethyl group.11 However, this result supports the assertion in ref 11 that “some residual electron density indicates that the amino nitrogen might interact with the propionate group from one pyrrole ring”. The docked structures of 2-11 to eNOS also share a strong interaction between the common amidinic group and the carboxylate moiety of the Glu363 and Trp358 residues and, whenever possible, the interaction of the 3-aminomethyl group with propionate arms above interaction. It is worth noting that 7 shows an hydrogen bond interaction between the pyridine nitrogen and the amine group of Asn340, with a N · · · N distance of 2.00 Å, stronger than the more labile electrostatic interaction observed with iNOS. This probably is why 7 is by far the least selective among the considered inhibitors, showing the lowest selectivity (57) and the highest eNOS IC50 (20 µM). The plot of the calculated docking scores against the experimental eNOS inhibitory activities shows a reasonable correlation between the calculated and experimental values (R2 ) 0.54) which, however, could be significantly improved (R2 ) 0.83) through the inclusion of the conformational energies through the same multiple linear approach employed for the docking to iNOS. The statistic for this two-parameter correlation on eNOS inhibition is slightly better than that for iNOS [correlation coefficient R2 ) 0.843, standard error σ ) 0.275, F-test statistic F ) 10.8, confidence level of 98%] although the correlation is mainly determined by the ∆Econf contribution. A comparative analysis of the calculated docking scores for the considered inhibitors of the two NOS isoforms shows better docking scores for the iNOS isoform, consistent with the observed selectivity. An analysis of the various energy contributions to the docking score of each ligand to the eNOS isoform shows the presence of bad contacts between the methyl on the amidine group and the Pro336 residue, which could be responsible for lower scores and therefore for the observed selectivity. Prompted by this result, we performed a quantitative analysis of the logarithm of the IC50 ratio of the two isoforms, the selectivity, i.e., -log(IC50iNOS/IC50eNOS). A reasonably good correlation was found for the calculated selectivity logarithm, estimated as differences of the docking scores ∆Gscore ) GscoreiNOS - GscoreeNOS, as a function of the experimental selectivity logarithm or ∆pIC50 ) -log(IC50iNOS/IC50eNOS) with a correlation coefficient R2 ) 0.852 (Figure 6 in Supporting Information).

Although based on a reduced set of seven experimental data, the above multiple linear regressions are promising, and our docking protocol can be employed not only for a rough prediction of binding geometries but also as an exploratory guide for a qualitative estimation of iNOS binding affinities and iNOS/ eNOS selectivities of this class of N-substituted acetamidine inhibitors. The higher inhibitory potency toward iNOS of 2-4 compared with that of the lead 1 indicates that the removal of the 3-aminomethyl group improves the iNOS inhibition, thus suggesting that the interaction of the latter group with the propionate arms of the heme cofactors does not contribute significantly to the binding energy of this class of aminidine inhibitors. On the other hand, the slightly lower selectivity over eNOS of 2-4 indicates the importance of this group in determining the high isoform specificity of 1, as suggested by the comparison of the X-ray structures with iNOS and eNOS, which points out the different position of the heme group in these two isoforms leading to different interactions of its propionate arms with the 3-aminomethyl group.11 Conclusions In this paper, we report the synthesis of novel acetamidines structurally related to 1 and the in vitro evaluation of their activities and selectivities toward iNOS. Among the synthesized compounds, 2, rac-4, and 9 exhibited inhibitory potency higher than the lead 1, maintaining a high selectivity over eNOS. In particular the best inhibition potency (0.20 µM) and selectivity (1750) were achieved with 1-(benzylamino)ethaniminium bromide (2). A docking study was also performed to shed light on the effects of the structural modifications on the interaction of the designed inhibitors with the NOS. The proposed docking protocol is promising and can be employed for a rough prediction of binding geometries and as an exploratory guide for a qualitative estimation of iNOS binding affinities and iNOS/ eNOS selectivities of this class of N-substituted acetamidine inhibitors. Experimental Section General Procedure for the Synthesis of 2-4, 9, and 10. To a stirred, cooled (0 °C) solution of the appropriate amine (7.1 mmol) in EtOH (25 mL) four equal portions of S-2-naphthylmethyl thioacetamidate hydrobromide (7.1 mmol), prepared according to literature procedure,9 were added. Reactions were allowed to warm to room temperature to facilitate complete conversion. The mixture was filtered. The filtrate was concentrated and partitioned between H2O (15 mL) and Et2O or CH2Cl2 (20 mL). The aqueous layer was separated, washed with Et2O (2 × 20 mL), and lyophilized to afford the desired acetamidine hydrobromide. General Procedure for the Synthesis of 5 and 6. To a stirred solution of tert-butyl 3-(aminomethyl)benzylcarbamate10 12 (7.9 mmol) in CH3CN dry (20 mL) a solution of benzyl 4-methylbenzenesulfonate in CH3CN dry (20 mL) was added, under N2, over a period of 45 min. The solution was stirred at 35 °C for 5 days and then concentrated. Purification of the residue by column chromatography (cyclohexane/EtOAc 5:5) furnished 13 and 14. Each compound was solubilized in CH2Cl2 (5 mL) and treated with CF3COOH (2.5 mL) at 0 °C for 2 h. The mixture was concentrated, and the residue, solubilized in a mixture of H2O and EtOH (1:1, 1.5 mL), was treated with 2 N NaOH. After evaporation of the solvent, 15 or 16 was obtained. Finally, ethylacetimidate hydrochloride (7.3 mmol) in EtOH (5 mL) was added to a solution of 15 or 16 (8.8 mmol) in EtOH (2 mL) at room temperature. After 7 h the mixture was concentrated and the residue suspended in H2O (15 mL) and washed with Et2O (3 × 15 mL) and AcOEt (2 × 15 mL). The aqueous layer was lyophilized to give the product 5 or 6.

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General Procedure for the Synthesis of 7 and 8. A solution of 12 (6.5 mmol) in DMSO dry (3 mL) was added to 4-(chloromethyl)pyridine or p-toluensulphonyl chloride (4.5 mmol) in DMSO dry (7 mL) at 0 °C under N2 over 40 min. The mixture was heated at room temperature and stirred for 24 h. The solvent was removed and the residue was dissolved in H2O (15 mL) and extracted with AcOEt (3 × 15 mL). The combined organic extracts were concentrated to give a residue that was purified by silica gel chromatography (CH2Cl2/MeOH 9:1) or by crystallization (toluene/ petroleum ether) to afford 17 or 18. Each compound was solubilized in CH2Cl2 (5 mL) and treated with CF3COOH (2.5 mL) at 0 °C for 2 h. The mixture was concentrated, and the residue, solubilized in a mixture of H2O and EtOH (1:1, 1.5 mL), was treated with 2 N NaOH. After evaporation of the solvent, 19 or 20 was obtained. Finally, S-2-naphtylmethyl thioacetamidate hydrobromide or ethylacetamidate hydrochloride (4 mmol), respectively, was added to a stirred solution of 19 or 20 (4 mmol) in EtOH (30 mL) at 0 °C under N2. After 72 h at room temperature, the solvent was removed and the residue was partitioned between H2O (10 mL) and Et2O (15 mL). The aqueous phase was washed with AcOEt (3 × 10 mL) and lyophilized to give the desired product 7 or 8. Synthesis of 11. To a stirred solution of acetamidine hydrochloride (10 mmol) and benzenesulfonyl chloride (10 mmol) in CH2Cl2 (20 mL), an aqueous solution of NaOH (20 mmol, 50% w/w) was added dropwise. After 24 h at room temperature, the mixture was filtered and H2O (10 mL) was added. The aqueous layer was separated and lyophilized to give a solid residue that was purified on semipreparative HPLC (H2O/MeOH 95:5, 1.5 mL/min) to give the amidine 11.

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(5)

(6)

(7)

Acknowledgment. The Italian Ministry for the University and the Research is acknowledged for financial support (Contracts 2006038520 and 2005033023). Supporting Information Available: General information on instrumentation; spectral data and elemental analysis results of 2, (S)-3, rac-4, 7-9; experimental procedures for biology and modeling; Scheme 4, Figures 2, 5 and 6. This material is available free of charge via the Internet at http://pubs.acs.org.

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