Design, Synthesis, and Biological Activity of New N-(Phenylmethyl

Article Cite This: J. Med. Chem. 2018, 61, 2725−2736

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Design, Synthesis, and Biological Activity of New N‑(Phenylmethyl)benzoxazol-2-thiones as Macrophage Migration Inhibitory Factor (MIF) Antagonists: Efficacies in Experimental Pulmonary Hypertension Morane Le Hiress,†,‡ Bernardin Akagah,⊥ Guillaume Bernadat,∥ Ly Tu,†,‡ Raphael̈ Thuillet,†,‡ Alice Huertas,†,‡,§ Carole Phan,†,‡ Elie Fadel,†,‡ Gérald Simonneau,†,‡,§ Marc Humbert,†,‡,§ Gael̈ Jalce,*,⊥ and Christophe Guignabert*,†,‡ †

INSERM UMR_S 999, Hôpital Marie Lannelongue, 92350 Le Plessis-Robinson, France Université Paris-Sud et Université Paris-Saclay, 94270 Le Kremlin-Bicêtre, France § AP-HP, Service de Pneumologie, Centre de Référence de l’Hypertension Pulmonaire Sévère, DHU Thorax Innovation, Hôpital Bicêtre, 94270 Le Kremlin-Bicêtre, France ∥ BioCIS, Université Paris-Sud, CNRS, Université Paris-Saclay, 92290 Châtenay-Malabry, France ⊥ MIFCARE, 24 rue du Faubourg Saint-Jacques, 75014 Paris, France ‡

S Supporting Information *

ABSTRACT: Macrophage migration inhibitory factor (MIF) is a key pleiotropic mediator and a promising therapeutic target in cancer as well as in several inflammatory and cardiovascular diseases including pulmonary arterial hypertension (PAH). Here, a novel series of N-(phenylmethyl)-benzoxazol-2-thiones 5−32 designed to target the MIF tautomerase active site was synthesized and evaluated for its effects on cell survival. Investigation of structure−activity relationship (SAR) particularly at the 5-position of the benzoxazole core led to the identification of 31 that potently inhibits cell survival in DU-145 prostate cancer cells and pulmonary endothelial cells derived from patients with idiopathic PAH (iPAH-ECs), two cell lines for which survival is MIFdependent. Molecular docking studies helped to interpret initial SAR related to MIF tautomerase inhibition and propose preferred binding mode for 31 within the MIF tautomerase active site. Interestingly, daily treatment with 31 started 2 weeks after a subcutaneous monocrotaline injection regressed established pulmonary hypertension in rats.

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MIF is a 114 amino-acid protein characterized by a β−α−β building block and a catalytic N-terminal proline residue (Pro1) that confers it a keto−enol tautomerase enzymatic activity. Although the biological relevance of the MIF tautomerase activity is not fully understood yet, partly because of the lack of bona fide substrates, the tautomerase active site still constitutes an attractive entry point for the design of small molecules that inhibit MIF biological functions.5,18,20−26 Despite identification of several different selective MIF inhibitors, none have been approved yet for clinical use.27−29

INTRODUCTION

Macrophage migration inhibitory factor (MIF) is a pleiotropic mediator released from cells such as macrophages, T-cells, and endothelial cells upon activation by stress, endotoxins, and inflammatory and immune stimuli. MIF plays an important role in the inflammatory response by counteracting the action of glucocorticoids1 and promoting the release of other inflammatory cytokines such as TNF-α and IL-6.2−7 MIF is also overexpressed in various cancer cells and promotes tumorigenesis.2,5,8−18 Upon binding to CD74, MIF initiates a signaling cascade through the sustained and transient activation of MAPK/ERK1/2, AKT (protein kinase B) and NF-κB signaling pathways, leading to cell proliferation and survival.2,5,8−17,19 © 2018 American Chemical Society

Received: September 5, 2017 Published: March 10, 2018 2725

DOI: 10.1021/acs.jmedchem.7b01312 J. Med. Chem. 2018, 61, 2725−2736

Journal of Medicinal Chemistry

Article

Figure 1. Synthesis of N-(phenylmethyl)-benzoxazol-2-thiones 5−32.

benzoxazol-2-thiones 5−32 in good overall yield with carbon disulfide and potassium carbonate in refluxing ethanol/water mixture.

Here, we report the design, synthesis, and biological evaluation of a novel series of N-(phenylmethyl)-benzoxazol2-thiones 5−32 as thiocarbonyl analogues of the MIF-CD74 antagonist N-(5-methylphenyl)-benzoxazol-2-one 33.21,24 First, our molecular docking studies showed that compounds 5−32 could bind to the MIF tautomerase pocket. Since the biological relevance of the MIF tautomerase active site is still controversial and that several reliability issues are associated with the MIF tautomerase assay,27,28 we then screened the antisurvival activity of 5−32 in a cell-based assay using DU-145, a cell line that relies on MIF for survival.17 SAR investigation, particularly at the 5-position (R1) of the benzoxazole core, led us to identify N-(3-hydroxy-4-fluorobenzyl)-5-trifluoromethylbenzoxazol-2-thione 31 as the most potent inhibitor of DU-145 cell survival. Because MIF and its signaling through CD74 are key players in the pathogenesis of pulmonary arterial hypertension (PAH),5 we next evaluated the antisurvival effect of 31 against primary cultures of pulmonary endothelial cells derived from patients with idiopathic PAH (iPAH-ECs). Consistent with our findings obtained with the DU-145 cells, 31 exhibited a similar antisurvival activity against iPAH-ECs. To assess the binding affinity of 31 for MIF, we used the MIF tautomerase assay and found a Ki value in the sub-micromolar range (0.3−1 μM). Then, we determined the inhibitory activity of 31 against rhMIF-dependent ERK and AKT phosphorylation in iPAH-ECs, and found that 31 acts as a MIF antagonist. Finally, 31 has been shown to reverse monocrotaline (MCT)induced pulmonary hypertension in rats, in a curative approach.

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RESULTS AND DISCUSSION All the prepared N-(phenylmethyl)-benzoxazol-2-thione derivatives 5−32 designed to bind to the MIF tautomerase active site were screened at 100 μM for their inhibition of DU-145 cell survival, a prostate cancer cell line that relies on MIF for survival.17 N-(Benzyl)-5-methylbenzoxazol-2-thione 7 was the first compound synthesized then tested. When added to the media of DU-145 cells maintained in serum-free medium for 24 h, compound 7 inhibited their cell survival by 36 ± 1%, which represents a 4.5-fold increase in potency compared to the prototypical MIF antagonist (S,R)-3-(4-hydroxyphenyl)-4,5dihydro-5-isoxazole acetic acid methyl ester34 (ISO-1, 35) (Table 1). Surprisingly, its oxygen analogue 33 did not affect DU-145 cell survival. These encouraging results prompted us to explore the SAR of the N-(phenylmethyl)-benzoxazol-2-thiones 5−32 by first varying substituents on the benzoxazole moiety (R1) and then on the N-benzyl part (R2), with the aim of improving inhibitory potency of hit compound 7 against DU145 cell survival. We started our SAR studies by replacing the methyl group at the 5-position of the benzoxazole ring by hydrogen, fluorine, and chlorine substituents (5, 11−12) while holding the Nbenzyl part unsubstituted. 5-Hydrogen substituent (5) was not tolerated, whereas 5-fluoro and 5-chloro derivatives (11−12) almost retained activity compared to hit compound 7. These preliminary results seem to indicate that hydrophobic substituents (7, 11−12) at the 5-position of the benzoxazole ring are preferred since the hydrogen (5) led to a total loss of activity. To evaluate the effect of the substituent size, we have replaced the methyl (7) by larger alkyl groups such as isopropyl (8) and tert-butyl (9). None of the corresponding compounds inhibited survival of DU-145 cells, indicating that isopropyl and tert-butyl substituents are too large to be tolerated. Only the bulky and electron-withdrawing 5-trifluoromethyl substituent (10) has substantially improved antisurvival activity compared

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CHEMISTRY All N-(phenylmethyl)-benzoxazol-2-thiones 5−32 were synthesized according to a three-step synthetic pathway allowing rapid access to analogues (Figure 1). The first step consists of a condensation of commercially available 2-aminophenol 1 and benzaldehyde 2 derivatives in tetrahydrofuran at room temperature, in the presence of magnesium sulfate. Subsequent reduction of the resulting Schiff bases 3 with sodium borohydride in methanol at 0 °C led to the amino alcohols 4 which are cyclized into their corresponding N-(phenylmethyl)2726



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± 1%, 24 ± 2% vs 36 ± 1%, Table 1). Replacing the methyl group in compound 20 by an electron-donating substituent such as methoxy (32) significantly decreased activity (46 ± 3% vs 16 ± 4%, Table 1), reinforcing the fact that hydrophobic electron-withdrawing groups are preferred at the 5-position of the benzoxazole ring. All substitutions at the 4-position were less tolerated, except for 4-methoxy compound (25) that was found as active as hit compound 7. N-(3-Hydroxybenzyl)-5-methylbenzoxazol-2-thione 20 was chosen as a prototype for further activity optimization. The addition of a fluorine atom (30) at the 4-position on the phenolic group in 20 with the aim of increasing acidity of the phenolic proton did not improve activity compared to hit compound 7. This result, and the fact that 3-methoxy compound 21 was found as active as its hydroxy derivative 20, indicate that the phenolic group is not essential for activity. Replacing the methyl in compound 30 by the bulky and electron-withdrawing trifluoromethyl group led to the most potent inhibitor 31, which displays an apparent EC50 value of 60.5 ± 11.9 μM against DU-145 cell survival (Figure 2), a value

Table 1. Effects of N-(Phenylmethyl)-benzoxazol-2-thiones 5−32 on DU-145 Cell Survival compound

R1

R2

X

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

H 4-CH3 5-CH3 5-CH(CH3)2 5-C(CH3)3 5-CF3 5-F 5-Cl 6-CH3 5-CH3 5-CH3 5-CH3 5-CH3 5-CH3 5-CH3 5-CH3 5-CH3 5-CH3 5-CH3 5-CH3 5-CH3 5-CH3 5-CH3 5-CH3 5-CH3 5-CH3 5-CF3 5-OMe 5-CH3 5-CF3

H H H H H H H H H 2-OH 2-OCH3 2-F 2-Cl 2-NO2 2-CH3 3-OH 3-OCH3 3-F 3-NO2 3-Me 4-OCH3 4-F 4-Cl 4-NO2 4-CH3 3-OH, 4-F 3-OH, 4-F 3-OH H 3-OH, 4-F

S S S S S S S S S S S S S S S S S S S S S S S S S S S S O O

a

inhibition of DU-145 cell survival at 100 μM (%) NAa NA 36 ± 1 NA NA 48 ± 2 31 ± 1 34 ± 2 24 ± 2 NA 29 ± 3 28 ± 3 NA 14 ± 2 NA 46 ± 3 47 ± 2 46 ± 2 24 ± 2 32 ± 1 36 ± 1 25 ± 2 22 ± 2 13 ± 2 NA 31 ± 5 84 ± 2 16 ± 4 NA NA 8±7

No activity observed at the tested concentration.

to hit compound 7 (48 ± 2% versus 36 ± 1%, Table 1). This result highlights the importance of the electron-withdrawing property of the hydrophobic substituent at the 5-position of the benzoxazole core since the trifluoromethyl (10) and fluorine (11), which are respectively similar in size to the isopropyl (8) and hydrogen (5), were found to be active, and the last two, not. Moving the methyl group from the 5- to the 4-position (6) resulted in total loss of activity, and placing it at the 6-position (13) led to decreased activity compared to hit compound 7 (24 ± 2% vs 36 ± 1%, Table 1). While maintaining the methyl group at the 5-position of the benzoxazole moiety constant, we next investigated the SAR of the N-benzyl moiety (R2) by introducing atoms or groups such as hydroxyl, methoxy, fluoro, chloro, nitro, or methyl at 2-, 3-, or 4-position. All substituents introduced at the 2-position (14−19) led to decreased activity compared to hit compound 7. Compounds 14, 17, and 19 were even found to be inactive, indicating that hydroxy, chlorine, and methyl substituents were not tolerated at this position. In contrast, the 3-position proved to be a key position to substitute for improving inhibitory potency since hydroxyl, methoxy, and fluoro derivatives 20−22 were almost 1.3-fold more potent than hit compound 7 (46 ± 3%, 47 ± 2%, 46 ± 2% versus 36 ± 1%, Table 1). 3-Nitro (23) and 3-methyl (24) derivatives led respectively to a 1.5-fold decrease and similar activity compared to hit compound 7 (32

Figure 2. Effect of compound 31 on DU-145 cell survival. Horizontal lines display the mean ± SEM (n = 4).

that is certainly underestimated because of the low aqueous solubility of this compound. Indeed, at the apparent EC50 value, the concentration of 31 was found to be in the range of 3−4 μM in PBS at room temperature at pH 7,4 (incubation time: 24 h, final DMSO concentration: 1%). To assess if these N-(phenylmethyl)-benzoxazol-2-thione derivatives 5−32 designed to bind to the MIF tautomerase active site show affinity for MIF, we performed tautomerase inhibition kinetic studies using 4-HPP as substrate for a selection of five compounds (7, 8, 20, 30, and 31). 7, the first compound tested in the DU145 cell-based assay, showed a Ki value in the range of 1−3 μM. This activity was lost when the methyl was replaced by an isopropyl (8), but improved with 20 (0.1−0.6 μM) that bears a hydroxyl group at the 3-position on the N-benzyl moiety (R2). The addition of a fluorine atom (30) at the 4-position on the phenolic group in 20 led to a slight decrease of the affinity for MIF (2−5 μM versus 0.1−0.6 μM, respectively). Interestingly, when the methyl in 30 was 2727



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replaced by a trifluoromethyl (31) the Ki value was also found in the sub-micromolar range (0.3−1 μM). To better understand these results, we studied the binding modes of the N-(phenylmethyl)-benzoxazol-2-thione derivatives 5−32 within the MIF tautomerase active site by performing protein−ligand docking against three different Xray cocrystal structures30−32 available from the PDB.33 Figure 3

the negatively charged trifluoromethyl group could also be engaged in electrostatic interactions, thus explaining the better Ki value observed for 31 compared to its methyl derivatives 20 and 30. The loss of activity observed for 8 could be partly explained by unfavorable steric contacts with residues at the back pocket of the tautomerase site in the “in” mode (Figure 3A). In the “out” mode, however, it is more difficult to explain as the 5-position of the benzoxazole core is exposed to solvent (Figure 3B). To gain additional insights into the ability of compound 31 to inhibit cell survival, DU-145 cells were treated with increasing doses of 31 and harvested for direct determination of cell proliferation and apoptosis. On one hand, we found that 31 decreases cell proliferation in a dose-dependent manner using a BrdU (5-bromo-2′-deoxyuridine) incorporation assay (Figure 4A). On the other hand, we found that 31 increases cell apoptosis as reflected by the dose-dependent inductions of the caspase-3/7 activity (Figure 4B). Consistent with these data, we found that a dose of 100 μM led to a marked increase in the percentages of Annexin V-FITC positive and PI negative cells (early apoptosis; 16.7 ± 2.1 versus 2.5 ± 1.2) assessed by flow cytometry (Figure 4C), and TUNEL positive cells (Figure 4D). To investigate the antisurvival effect of 31 in a PAH cellbased model in vitro, we used primary cultures of human pulmonary endothelial cells derived from patients with idiopathic pulmonary arterial hypertension (iPAH-ECs), which also overexpress CD74.5 At a dose of 100 μM, compound 31 led to a 70 ± 1% and 84 ± 2% decrease in cell survival respectively in the iPAH-ECs and DU-145 cells (Figure 5). In addition, we found that 31 dose-dependently attenuated rhMIF-induced AKT and ERK activation states in these human primary cells, supporting that 31 acts as a MIF antagonist (Figure 6). These interesting results obtained with 31 encouraged us to evaluate its pharmacology efficacy in the MCT-induced PH model, which is among the most widely used experimental model of PH. Although this model does not reproduce the full spectrum of changes seen in lung specimens from PAH patients, MCT induced progressive changes in mean pulmonary artery pressure (mPAP), right ventricular hypertrophy (RVH), and muscularization of pulmonary arterioles. Daily treatment with compound 31 started 2 weeks after a subcutaneous MCT injection substantially regressed the degree of established PH (Figure 7). On day 28, in MCT-injected rats treated with vehicle, a marked increase in mPAP and RVH was found compared with controls (Figure 7). However, there was a substantial reduction in values of mPAP between MCT-injected rats treated with 31 at the dose of 30 mg/kg/d when compared with MCT-injected rats treated with vehicle (Figure 7). No change in body weight, heart rate, and systemic blood pressures were observed between rats treated with 31 or vehicle. In order to verify the translational potential of 31, we assessed its role on potential cardiac effects in our in vivo studies. Indeed, it has been recently reported that 35 can lead to cardiac fibrosis.5 In contrast, we found that 31 did not induce cardiac fibrosis in MCT-injected rats. Indeed, Picrosirius red staining of tissue sections of right ventricle myocardium showed increased collagen fibers in endomysium in MCT-injected rats when compared with control rats (Figure 8). We found that this increased right ventricle fibrosis was reversed in MCT-injected rats receiving 31 at the dose of 30 mg/kg when compared with vehicle-treated MCT-injected rats. Notably, there was no

Figure 3. Calculated binding mode of compound 30 (A) and compound 31 (B) within the MIF tautomerase active site (PDB: 1GCZ).

shows the calculated binding mode of compound 30 (“in” mode, Figure 3A) and that of the most potent derivative 31 (“out” mode, Figure 3B). In the MIF-30 complex, the rigid benzoxazole moiety was found buried into the tautomerase active site, allowing hydrophobic interactions with Val106 and Met2 residues, both located at the back of the tautomerase binding site. Additional binding interactions of the benzoxazole core with the backbone N−H of Ile64 and the terminal NH3+ group of Lys32 side chain via hydrogen bonds are observed. The fluorophenol moiety is oriented outward at the entrance of the active site cavity and establishes aryl−aryl interactions with the almost parallel Tyr36 residue. In the MIF-31 complex, interestingly, the benzoxazole ring was turned outward the active site cavity allowing hydrophobic interactions between the trifluoromethyl group and surface residues Phe113 and Ile64. Performing the docking on a series of conformational samples taken from molecular dynamics of MIF cocrystals as well as with a different program (Autodock Vina) also led to this “out” binding mode as the preferred one for compound 31. In addition to its hydrophobic interactions with surface residues, 2728



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Figure 4. Effects of compound 31 on the balance between cell proliferation and apoptosis in DU-145 cells. BrdU incorporation (A), caspase 3/7 activity (B), Annexin V-FITC assay (C), and representative terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) images (D) in DU-145 cells compound 31 treatment. Horizontal lines display the mean ± SEM (n = 5). Scale bar = 50 μm in all sections. ***p-value < 0.001 versus DU-145 cells treated with 0 μM. RFU = relative fluorescence units. DAPI = 4′,6-diamidino-2-phenylindole; FITC = fluorescein isothiocyanate; NS = not significant.

significant difference in collagen area fraction in left ventricle myocardium rats treated or not with 31 (Figure 8).

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CONCLUSION

In conclusion, we have designed, synthesized, and evaluated a new series of N-(phenylmethyl)-benzoxazol-2-thiones 5−32 as MIF antagonists. SAR studies revealed that the 5-position of the benzoxazole core plays a key role in inhibition of DU-145 cell survival, a prostate cancer cell line for which survival is MIF-dependent. Among the tested compounds, 31 which bears a trifluoromethyl group at the 5-position of the benzoxazole ring was shown to be the most active against DU-145 cell survival. This compound exhibited similar efficacy against primary cultures of iPAH-ECs and seems to act via inhibition of MIF-dependent pro-survival pathways. Furthermore, 31 was found to inhibit MIF tautomerase activity in a dose-dependent manner with a Ki value in the sub-micromolar range. Molecular docking studies showed for 31 a flip-over of the benzoxazole core within the MIF tautomerase active site due to the presence

Figure 5. Comparison of the effects of compound 31 on cell survival of DU-145 and of pulmonary endothelial cells derived from patients with idiopathic pulmonary arterial hypertension (iPAH-ECs). Horizontal lines display the mean ± SEM (n = 4). ***p-value

Design, Synthesis, and Biological Activity of New N-(Phenylmethyl

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