Design, Synthesis, and Biological Activity of New N -(Phenylmethyl)-benzoxazol-2-thiones as Macrophage Migration Inhibitory Factor (MIF) Antagonists: Efficacies in Experimental Pulmonary Hypertension

Article abstract of DOI:10.1021/acs.jmedchem.7b01312

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 MIF-dependent. 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.

Full text of DOI:10.1021/acs.jmedchem.7b01312

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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, Raphaël Thuillet, Alice Huertas,
Carole Phan, Elie Fadel, Gérald Simonneau, Marc Humbert, Gaël Jalce, and Christophe Guignabert
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01312 • Publication Date (Web): 10 Mar 2018
Downloaded from http://pubs.acs.org on March 11, 2018
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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
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Running title: Small molecule MIF antagonist reverses established PH
Morane Le Hiress†,‡, Bernardin Akagah⊥, Guillaume Bernadat∥, Ly Tu†,‡, Raphaël Thuillet†,‡,
Alice Huertas†,‡,§, Carole Phan†,‡, Elie Fadel†,‡, Gérald Simonneau†,‡,§, Marc Humbert†,‡,§
Gaël Jalce*,⊥, and Christophe Guignabert*,†,‡
,
† INSERM UMR_S 999, Hôpital Marie Lannelongue, 92350 Le PlessisꢀRobinson, France;
‡ Université ParisꢀSud et Université ParisꢀSaclay, 94270 Le KremlinꢀBicêtre, France;
§
APꢀHP, Service de Pneumologie, Centre de Référence de l’Hypertension Pulmonaire Sévère,
DHU Thorax Innovation, Hôpital Bicêtre, 94270 Le KremlinꢀBicêtre, France;
∥ BioCIS, Université
́ ParisꢀSud, CNRS, Université́ ParisꢀSaclay, 92290 ChatenayꢀMalabry,
France;
⊥ MIFCARE, 24 rue du Faubourg SaintꢀJacques, 75014 Paris, France.
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ABSTRACT
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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 Nꢀ(phenylmethyl)ꢀ
benzoxazolꢀ2ꢀthiones 5-32 designed to target the MIF tautomerase active site was synthesized
and evaluated for its effects on cell survival. Investigation of structureꢀactivity relationship
(SAR) particularly at the 5ꢀposition of the benzoxazole core led to the identification of 31 that
potently inhibits cell survival in DUꢀ145 prostate cancer cells and pulmonary endothelial cells
derived from patients with idiopathic PAH (iPAHꢀECs), two cell lines for which survival is MIFꢀ
dependent. 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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Word count: 149 words
Keywords: CD74 • MIF • Pulmonary vascular remodeling • Cell survival • Therapeutic
innovation
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INTRODUCTION
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Macrophage migration inhibitory factor (MIF) is a pleiotropic mediator released from cells such
as macrophages, Tꢀcells and endothelial cells upon activation by stress, endotoxins,
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 TNFꢀα and ILꢀ6.2ꢀ7 MIF is also overexpressed in various cancer cells and
promotes tumorigenesis.2, 5, 8ꢀ18 Upon binding to CD74, MIF initiates a signaling cascade through
the sustained and transient activation of MAPK/ERK1/2, AKT (protein kinase B) and NFꢀκB
signaling pathways, leading to cell proliferation and survival.2, 5, 8ꢀ17, 19
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MIF is a 114 aminoꢀacid protein characterized by a βꢀαꢀβ building block and a catalytic Nꢀ
terminal proline residue (Pro1) that confers it a ketoꢀenol 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,
20ꢀ26
Despite identification of several different selective MIF inhibitors, none have yet been
approved for clinical use.27ꢀ29
Here, we report the design, synthesis and biological evaluation of a novel series of Nꢀ
(phenylmethyl)ꢀbenzoxazolꢀ2ꢀthiones 5-32 as thiocarbonyl analogues of the MIFꢀCD74
antagonist Nꢀ(5ꢀmethylphenyl)ꢀbenzoxazolꢀ2ꢀone 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
27, 28
issues are associated with the MIF tautomerase assay,
we then screened the antiꢀsurvival
activity of 5-32 in a cellꢀbased assay using DUꢀ145, a cell line that relies on MIF for survival.17
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SAR investigation, particularly at the 5ꢀposition (R1) of the benzoxazole core, led us to identify
Nꢀ(3ꢀhydroxyꢀ4ꢀfluorobenzyl)ꢀ5ꢀtrifluoromethylbenzoxazolꢀ2ꢀthione 31 as the most potent
inhibitor of DUꢀ145 cell survival. Because MIF and its signaling through CD74 are key players
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in the pathogenesis of pulmonary arterial hypertension (PAH) , we next evaluated the antiꢀ
survival effect of 31 against primary cultures of pulmonary endothelial cells derived from
patients with idiopathic PAH (iPAHꢀECs). Consistent with our findings obtained with the DUꢀ
145 cells, 31 exhibited a similar antiꢀsurvival activity against iPAHꢀECs. To assess the binding
affinity of 31 for MIF, we used the MIF tautomerase assay and found a Ki value in the subꢀ
micromolar range (0.3ꢀ1µM). Then, we determined the inhibitory activity of 31 against rhMIFꢀ
dependent ERK and AKT phosphorylation in iPAHꢀECs, 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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CHEMISTRY
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All Nꢀ(phenylmethyl)ꢀbenzoxazolꢀ2ꢀthiones 5-32 were synthesized according to a threeꢀstep
synthetic pathway allowing a rapid access to analogues (Figure 1). The first step consists of a
condensation of commercially available 2ꢀaminophenol 1 and benzaldehyde 2 derivatives in
tetrahydrofuran at room temperature, in 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 Nꢀ(phenylmethyl)ꢀbenzoxazolꢀ2ꢀthiones
5-32 in good overall yield with carbon disulfide and potassium carbonate in refluxing
ethanol/water mixture.
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RESULTS AND DISCUSSION
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All the prepared Nꢀ(phenylmethyl)ꢀbenzoxazolꢀ2ꢀthione derivatives 5-32 designed to bind to the
MIF tautomerase active site were screened at 100µM for their inhibition of DUꢀ145 cell survival,
a prostate cancer cell line that relies on MIF for survival.17 Nꢀ(Benzyl)ꢀ5ꢀmethylbenzoxazolꢀ2ꢀ
thione 7 was the first compound synthesized then tested. When added to the media of DUꢀ145
cells maintained in serumꢀfree medium for 24 hours, compound 7 inhibited their cell survival by
36 ± 1 %, which represents a 4.5ꢀfold increase in potency compared to the prototypical MIF
antagonist (S,R)ꢀ3ꢀ(4ꢀhydroxyphenyl)ꢀ4,5ꢀdihydroꢀ5ꢀisoxazole acetic acid methyl ester34 (ISOꢀ1,
35) (table 1). Surprisingly, its oxygen analogue 33 did not affect DUꢀ145 cell survival. These
encouraging results prompted us to explore the SAR of the Nꢀ(phenylmethyl)ꢀbenzoxazolꢀ2ꢀ
thiones 5-32 by first varying substituents on the benzoxazole moiety (R1) and then on the Nꢀ
benzyl part (R2), with the aim of improving inhibitory potency of hit compound 7 against DUꢀ
145 cell survival.
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We started our SAR studies by replacing the methyl group at the 5ꢀposition of the benzoxazole
ring by hydrogen, fluorine, and chlorine substituents (5, 11-12) while holding the Nꢀbenzyl part
unsubstituted. 5ꢀhydrogen substituent (5) was not tolerated whereas 5ꢀfluoro and 5ꢀchloro
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 5ꢀposition of the
benzoxazole ring are preferred and that their electronꢀwithdrawing nature (11-12) is important
for activity since the fluoro substituent (11) has retained activity contrary to the quite similar in
size hydrogen (5) which 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 tertꢀbutyl
(9). None of the corresponding compounds inhibited survival of DUꢀ145 cells, indicating that
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isopropyl and tertꢀbutyl substituents are too large to be tolerated. Only the bulky and electronꢀ
withdrawing 5ꢀtrifluoromethyl substituent (10) has substantially improved antiꢀsurvival activity
compared to hit compound 7 (48 ± 2 % versus 36 ± 1 %, table 1). This result confirms the
importance of the electronꢀwithdrawing property of the hydrophobic substituent at the 5ꢀposition
of the benzoxazole core since the trifluoromethyl (10) which has similar bulkiness than the
isopropyl (8) was found active, and the later not. Moving the methyl group from the 5ꢀ to the 4ꢀ
position (6) resulted in total loss of activity, and placing it at the 6ꢀposition (13) led to decreased
activity compared to hit compound 7 (24 ± 2 % vs 36 ± 1 %, table 1).
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While maintaining the methyl group at the 5ꢀposition of the benzoxazole moiety constant, we
next investigated the SAR of the Nꢀbenzyl moiety (R2) by introducing atoms or groups such as
hydroxyl, methoxy, fluoro, chloro, nitro or methyl at 2ꢀ, 3ꢀ or 4ꢀposition. All substituents
introduced at the 2ꢀposition (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 3ꢀposition proved to be a
key position to substitute for improving inhibitory potency since hydroxyl, methoxy and fluoro
derivatives 20-22 were almost 1.3ꢀfold more potent than hit compound 7 (46 ± 3 %, 47 ± 2 %, 46
± 2 % versus 36 ± 1%, table 1). 3ꢀNitro (23) and 3ꢀmethyl (24) derivatives led respectively to
1.5ꢀfold decrease and similar activity compared to hit compound 7 (32 ± 1 %, 24 ± 2 % vs 36 ± 1
%, table 1). Replacing the methyl group in compound 20 by an electronꢀdonating substituent
such as methoxy (32) significantly decreased activity (46 ± 3 % vs 16 ± 4 %, table 1), reinforcing
the fact that hydrophobic electronꢀwithdrawing groups are preferred at the 5ꢀposition of the
benzoxazole ring. All substitutions at the 4ꢀposition were less tolerated, except for 4ꢀmethoxy
compound (25) that was found as active as hit compound 7.
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Nꢀ(3ꢀHydroxybenzyl)ꢀ5ꢀmethylbenzoxazolꢀ2ꢀthione 20 was chosen as prototype for further
activity optimization. The addition of a fluorine atom (30) at the 4ꢀposition 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 3ꢀmethoxy 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 electronꢀwithdrawing trifluoromethyl group led to
the most potent inhibitor 31 which displays an apparent EC50 value of 60.5 ± 11.9 µM against
DUꢀ145 cell survival (Figure 2), a value that is certainly underestimated because of the low
aqueous solubility of this compound. Indeed, at apparent EC50 value, concentration of 31 was
found to be in the range of 3ꢀ4 ꢁM in PBS at room temperature at pH 7,4 (incubation time: 24h,
final DMSO concentration: 1%).
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To assess if these Nꢀ(phenylmethyl)ꢀbenzoxazolꢀ2ꢀthione derivatives 5-32 designed to bind to the
MIF tautomerase active site show affinity for MIF, we performed tautomerase inhibition kinetic
studies using 4ꢀHPP as substrate for a selection of 5 compounds (7, 8, 20, 30 and 31). 7, the first
compound tested in the DU145 cellꢀbased assay, showed a Ki value in the range of 1ꢀ3µM. This
activity was lost when the methyl was replaced by an isopropyl (8), but improved with 20 (0.1ꢀ
0.6µM) that bears a hydroxyl group at the 3ꢀposition on the Nꢀbenzyl moiety (R2). The addition
of a fluorine atom (30) at the 4ꢀposition on the phenolic group in 20 led to a slight decrease of
the affinity for MIF (2ꢀ5µM versus 0.1ꢀ0.6µM, respectively). Interestingly, when the metyl in 30
was replaced by a trifluoromethyl (31) the Ki value was also found in the submicromolar range
(0.3ꢀ1µM).
To better understand these results, we studied the binding modes of the Nꢀ(phenylmethyl)ꢀ
benzoxazolꢀ2ꢀthione derivatives 5-32 within the MIF tautomerase active site by performing
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30ꢀ32
proteinꢀligand docking against three different Xꢀray cocrystal structures
available from the
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PDB.33 Figure 3 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 MIFꢀ30 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
arylꢀaryl interactions with the almost parallel Tyr36 residue. In the MIFꢀ31 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 coꢀcrystals 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, 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 30. The loss of activity observed for 8 could be partly
explained by unfavourable 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 5ꢀ
position of the benzoxazole core is exposed to solvent (Figure 3B).
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To gain additional insights into the ability of compound 31 to inhibit cell survival, DUꢀ145 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 doseꢀ
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dependent manner using a BrdU (5ꢀBromoꢀ2´ꢀDeoxyuridine) incorporation assay (Figure 4A).
On the other hand, we found that 31 increases cell apoptosis as reflected by the doseꢀdependent
inductions of the caspaseꢀ3/7 activity (Figure 4B). Consistent with these data, we found that a
dose of 100µM led to a marked increase in the percentages of Annexin VꢀFITC 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).
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To investigate the antiꢀsurvival effect of 31 in a PAH cellꢀbased model in vitro, we used primary
cultures of human pulmonary endothelial cells derived from patients with idiopathic pulmonary
arterial hypertension (iPAHꢀECs), which also overexpress CD74.5 At a dose of 100µM,
compound 31 led to a 70 ± 1 % and 84 ± 2 % decrease in cell survival respectively in the iPAHꢀ
ECs and DUꢀ145 cells (Figure 5). In addition, we found that 31 doseꢀdependently attenuated
rhMIFꢀinduced 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 MCTꢀinduced 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 MCTꢀinjected 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 MCTꢀinjected
rats treated with 31 at the dose of 30 mg/kg/d when compared with MCTꢀinjected rats treated
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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 MCTꢀinjected rats. Indeed, Picrosirius red staining of tissue sections of
right ventricle myocardium showed increased collagen fibers in endomysium in MCTꢀinjected
rats when compared with control rats (Figure 8). We found that this increased right ventricle
fibrosis was reversed in MCTꢀinjected rats receiving 31 at the dose of 30 mg/kg when compared
with vehicleꢀtreated MCTꢀinjected rats. Notably, there was no significant difference in collagen
area fraction in left ventricle myocardium rats treated or not with 31 (Figure 8).
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CONCLUSION
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In conclusion, we have designed, synthesized and evaluated a new series of Nꢀ(phenylmethyl)ꢀ
benzoxazolꢀ2ꢀthiones 5-32 as MIF antagonists. SAR studies revealed that the 5ꢀposition of the
benzoxazole core plays a key role in inhibition of DUꢀ145 cell survival, a prostate cancer cell
line for which survival is MIFꢀdependent. Among the tested compounds, 31 which bears a
trifluoromethyl group at the 5ꢀposition of the benzoxazole ring was shown to be the most active
against DUꢀ145 cell survival. This compound exhibited similar efficacy against primary cultures
of iPAHꢀECs and seems to act via inhibition of MIFꢀdependent proꢀsurvival pathways.
Furthermore, 31 was found to inhibit MIF tautomerase activity in a doseꢀdependent manner with
a Ki value in the subꢀmicromolar range. Molecular docking studies showed for 31 a flipꢀover of
the benzoxazole core within the MIF tautomerase active site due to the presence of the
trifluoromethyl group. Finally, compound 31 reversed established PH induced by monocrotaline
in rats by decreasing values of mean pulmonary arterial pressure, pulmonary vascular resistance,
degree of pulmonary vascular remodeling and right ventricular hypertrophy. Additional
optimization of activity and drug properties of these Nꢀ(phenylmethyl)ꢀbenzoxazolꢀ2ꢀthiones 5-
32 are ongoing.
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EXPERIMENTAL SECTION
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Chemistry: General information.
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Tetrahydrofuran was distilled over sodium/benzophenone under nitrogen atmosphere. Methanol
was distilled from magnesium turnings under nitrogen atmosphere. Analytical TLC was
performed on silica gel plates and visualized by exposure to ultraviolet light (254 nm) or by
using a solution of 5% phosphomolybdic acid in EtOH or a solution of vanillin in EtOH/H2SO4
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(95:5). H NMR spectra were recorded in CDCl3, DMSOꢀd6, CD3OD or (CD3)2CO on Bruker
Avance 300 (300 MHz). Flash chromatography was carried out on silica gel (230ꢀ400 mesh).
Chemical shifts are reported in parts per million (ppm, δ) downfield from tetramethylsilane
(TMS) and are referenced to CHCl3 (7.26 ppm), DMSOꢀd6 (2.50 ppm), CD3OD (3.35 ppm) or
(CD3)2CO (2.04 ppm) as internal standard. All coupling constants are absolute values and J
values are expressed in Hertz (Hz). The following abbreviations are used to indicate signal
multiplicity: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), brs (broad singlet), dd
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(doublet of doublets), dq (doublet of quartets), app t (apparent triplet). C NMR spectra were
recorded in CDCl3, DMSOꢀd6, CD3OD or (CD3)2CO on Bruker Avance 300 (75 MHz). Chemical
shifts are reported in parts per million (ppm, δ) downfield from tetramethylsilane (TMS) and are
referenced to CDCl3 (77.0 ppm), DMSOꢀd6 (39.5 ppm), CD3OD (49.3 ppm) or (CD3)2CO (29.8
ppm) as internal standard. Mass spectra (MS) were measured on a NermagꢀSidar R10ꢀ10C
spectrometer with a quadrupole filter. High resolution mass spectra (HRMS) were measured on a
Xevo QꢀTof Waters spectrometer. Analytical HPLC was performed for compound purity
determination using Xbridge C18, 3.5 ꢁm, 2.1 mm × 150 mm columns and UV detection. The
solvent system used for HPLC analyses was water + 0.1% formic acid:acetonitrile with a
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gradient from 99:1 to 100:0 over 20 min at 1.0 mL/min. The purity of all the tested compounds is
greater than or equal to 95% unless noted.
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General procedure for the synthesis of N-(phenylmethyl)-benzoxazol-2-thiones 5-32 is
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exemplified by the synthesis of compound 5.
To a solution of 2ꢀaminophenol 1 (0.46 g, 3.78 mmol, 1 equiv) in THF (50 ml) at rt were added
benzaldehyde 2 (0.40 g, 3.78 mmol, 1 equiv) and magnesium sulfate (1.82 g, 15.12 mmol, 4
equiv). The mixture was stirred for 12h, then filtered and concentrated in vacuo. The crude
residue obtained was dissolved in MeOH (50 mL), then NaBH4 (0.17 g, 4.53 mmol, 1.2 equiv)
was gradually added in small portions at 0°C. The reaction mixture was stirred at this
temperature during 1h, then quenched with cold water. The crude residue was extracted with
EtOAc (3 x 75 ml) and dried over anhydrous MgSO4. After removal of the solvent in vacuo, the
crude residue was dissolved in a mixture of EtOH/H2O, then KOH (0.11 g, 1.89 mmol, 0.5
equiv) and CS2 (0.91 ml, 15.12 mmol, 4 equiv) were added and the reaction mixture was heated
under reflux during 16h, then cooled at rt. Addition of cold water formed a precipitate which was
filtered off, then purified by flash chromatography on silica gel (Cyclohexane:EtOAc, 70:30) to
afford compound 5 (0.87 g, 90% yield) as a solid.
General procedure for the synthesis of N-(phenylmethyl)-benzoxazol-2-ones 33-34 is
exemplified by the synthesis of compound 33.
To a solution of 4ꢀmethylꢀ2ꢀaminophenol 1 (0.49 g, 4.00 mmol, 1 equiv) in THF (50 ml) at rt
were added benzaldehyde 2 (0.42 g, 4.00 mmol, 1 equiv) and magnesium sulfate (1.92 g, 16.00
mmol, 4 equiv). The mixture was stirred for 12h, then filtered and concentrated in vacuo. The
crude residue obtained was dissolved in MeOH (50 mL), then NaBH4 (0.18 g, 4.80 mmol, 1.2
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equiv) was gradually added in small portions at 0°C. The reaction mixture was stirred at this
temperature during 1h, then quenched with cold water. The crude residue was extracted with
EtOAc (3 x 75 ml) and dried over anhydrous MgSO4. After removal of the solvent in vacuo, the
crude residue was dissolved in THF (50 ml), then CDI (0.78 g, 4.80 mmol, 1.2 equiv) and Et3N
(0.84 ml, 6.00 mmol, 1.5 equiv) were added and the reaction mixture was heated under reflux
during 12h, then cooled at rt and concentrated in vacuo. The reaction mixture was then diluted
with 1N HCl, extracted with EtOAc (3 x 75 ml), washed with brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was purified by flash chromatography on silica
gel (Cyclohexane:EtOAc, 70:30) to obtain compound 33 (0.67 g, 70% yield) as a solid.
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N-(Benzyl)-benzoxazol-2-thione (5). Yield: 90% (0.87 g). 1H NMR (300 MHz, CDCl3): δ 7.42ꢀ
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7ꢀ31 (m, 6H), 7.25ꢀ7.16 (m, 2H), 7.00ꢀ6.94 (m, 1H), 5.43 (s, 2H). C NMR (75 MHz, CDCl3): δ
181.0, 147.2, 133.9, 131.7, 129.0 (2C), 128.4, 127.7 (2C), 124.9, 124.3, 110.4, 109.9, 49.5.
HRMS (ESI) calcd m/z for [M + H]+ C14H12NOS 242.0640, found 242.0634.
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N-(Benzyl)-4-methylbenzoxazol-2-thione (6). Yield: 70% (0.71 g). H NMR (300 MHz,
CDCl3): δ 7.37ꢀ7.23 (m, 4H), 7.18ꢀ7.11 (m, 3H), 6.96 (d, J = 7.8 Hz, 1H), 5.69 (s, 2H), 2.34 (s,
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3H). C NMR (75 MHz, CDCl3): δ 181.7, 147.4, 135.1, 130.0, 129.0 (2C), 127.8, 125.9 (2C),
124.3, 121.2, 108.3 (2C), 50.3, 17.4. HRMS (ESI) calcd m/z for [M + H]+ C15H14NOS 256.0796,
found 256.0792.
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N-(Benzyl)-5-methylbenzoxazol-2-thione (7). Yield: 85% (0.87 g). H NMR (300 MHz,
CDCl3): δ 7.41ꢀ7.31 (m, 5H), 7.22 (d, J = 8.4 Hz, 1H), 7.01 (dd, J = 8.1, 0.9 Hz, 1H), 6.77 (s,
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1H), 5.41 (s, 2H), 2.35 (s, 3H). δ C NMR (75 MHz, CDCl3): δ 181.1, 145.4, 135.1, 134.0,
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131.7, 129.0 (2C), 128.3, 127.6 (2C), 125.0, 110.1, 109.9, 49.4, 21.5. HRMS (ESI) calcd m/z for
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[M + H]+ C15H14NOS 256.0796, found 256.0800.
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N-(Benzyl)-5-isopropylbenzoxazol-2-thione (8). Yield: 60% (0.45g). H NMR (300 MHz,
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CDCl3): δ 7.42 (m, 5H); 7.25 (d, J = 6.9 Hz, 1H); 7.08 (d, J = 8.4 Hz, 1H); 6.80 (s, 1H); 2.90
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(sept, J = 6.6 Hz, 1H); 1.20 (d, J = 1.5 Hz, 3H); 1.18 (d, J = 1.2 Hz, 3H). C NMR (75 MHz,
CDCl3): δ 181.1; 146.4; 145.5; 133.9; 131.6; 128.9 (2C); 128.3; 127.7 (2C); 122.5; 109.9; 107.7;
49.4; 34.1; 24.1 (2C). HRMS (ESI) calcd m/z for [M+H]+ C17H18NOS 284.1109, found
284.1113.
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N-(Benzyl)-5-tert-butylbenzoxazol-2-thione (9). Yield: 58% (0.52 g). H NMR (300 MHz,
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CDCl3): δ 7.44ꢀ7.32 (m, 5H); 7.26 (s, 2H); 6.95 (s, 1H); 5.44 (s, 2H); 1.27 (s, 9H). C NMR (75
MHz, CDCl3): δ 181.1; 148.7; 145.2; 134.0; 131.4; 128.9 (2C); 128.4; 127.8 (2C); 121.55; 109.6;
106.9; 49.4; 34.9; 31.5 (3C). HRMS (ESI) calcd m/z for [M+H]+ C18H20NOS 298.1266, found
298.1264
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N-(Benzyl)-5-trifluoromethylbenzoxazol-2-thione (10). Yield: 70% (0.86 g). H NMR (300
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MHz, CDCl3): δ 7.52 (d, J = 8.4 Hz, 1H), 7.44ꢀ7.32 (m, 6H), 7.18 (s, 1H), 5.44 (s, 2H). C
NMR (75 MHz, CDCl3): δ 181.4, 148.9, 133.1, 132.1, 129.1 (2C), 128.7, 127.7 (2C), 127.6 (q,
JCꢀF = 34.9 Hz), 127.1 (q, JCꢀF = 270.0 Hz), 121.7 (q, JCꢀF = 3.3 Hz), 110.5, 107.0 (q, JCꢀF = 3.2
Hz), 49.6. HRMS (ESI) calcd m/z for [M + H]+ C15H11F3NOS 310.0513, found 310.0514.
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N-(Benzyl)-5-fluorobenzoxazol-2-thione (11). Yield: 78% (0.83 g). H NMR (300 MHz,
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DMSOꢀd6): δ 7.65ꢀ7.58 (m, 1H), 7.48ꢀ7.29 (m, 6H), 7.22ꢀ7.13 (m, 1H), 5.44 (s, 2H). C NMR
(75 MHz, DMSOꢀd6): δ 180.8, 159.5 (d, JCꢀF = 238.9 Hz), 142.8, 134.3, 132.3 (d, JCꢀF = 14.2 Hz),
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128.7 (2C), 128.1, 127.8 (2C), 111.3 (d, JCꢀF= 9.8 Hz), 111.1 (d, JCꢀF = 26.2 Hz), 98.9 (d, JCꢀF
29.5 Hz), 48.5. HRMS (ESI) calcd m/z for [M + H]+ C14H11FNOS 260.0545, found 260.0545.
=
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N-(Benzyl)-5-chlorobenzoxazol-2-thione (12). Yield: 80% (0.88 g). Purity (HPLC/UV λ at 254
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nm): 91.8%. H NMR (300 MHz, CDCl3): δ 7.41ꢀ7.36 (m, 5H), 7.28 (d, J = 4.2 Hz, 1H), 7.20
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(dd, J = 8.4, 1.8 Hz, 1H), 6.95 (d, J = 1.5 Hz, 1H), 5.40 (s, 2H). C NMR (75 MHz, CDCl3): δ
181.4, 145.7, 133.4, 132.7, 130.7, 129.2 (2C), 128.7, 127.7 (2C), 124.4, 111.0, 110.1, 49.6.
HRMS (ESI) calcd m/z for [M + H]+ C14H11ClNOS 276.0250, found 276.0247.
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N-(Benzyl)-6-methylbenzoxazol-2-thione (13). Yield: 83% (0.84 g). H NMR (300 MHz,
CDCl3): δ 7.40ꢀ7.30 (m, 5H), 7.16 (s, 1H), 6.99 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 8.1 Hz, 1H),
5.41 (s, 2H), 2.40 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 180.7, 147.4, 134.9, 133.9, 129.3 (2C),
128.9, 128.4, 127.7 (2C), 125.6, 110.8, 109.5, 49.5, 21.4. HRMS (ESI) calcd m/z for [M + H]+
C15H14NOS 256.0796, found 256.0793.
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N-(2-Hydroxybenzyl)-5-methylbenzoxazol-2-thione (14). Yield: 80% (0.87 g). H NMR (300
MHz, DMSOꢀd6): δ 10.01 (s, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.14ꢀ7.11 (m, 3H), 6.99 (d, J = 6,6
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Hz, 1H), 6.88 (d, J = 8.1 Hz, 1H), 6.72 (app t, J = 6.9 Hz, 1H), 5.34 (s, 2H), 2.33 (s, 3H). C
NMR (75 MHz, DMSOꢀd6): δ 180.0, 155.0, 144.7, 134.8, 131.6, 129.0, 128.0, 125.0, 120.3,
119.1, 115.3, 110.9, 109.7, 43.8, 21.0. HRMS (ESI) calcd m/z for [M + H]+ C15H14NO2S
272.0745, found 272.0747.
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N-(2-Methoxybenzyl)-5-methylbenzoxazol-2-thione (15). Yield: 88% (1.00 g). H NMR (300
MHz, CDCl3): δ 7.31ꢀ7.24 (m, 2H), 7.21 (d, J = 8.4 Hz, 1H), 7.02 (dd, J = 8.1, 0.9 Hz, 1H), 6.91
(app t, J = 7.8 Hz, 2H), 6.86 (s, 1H), 5.42 (s, 2H), 3.91 (s, 3H), 2.36 (s, 3H). 13C NMR (75 MHz,
CDCl3): δ 181.1, 156.8, 145.3, 134.9, 131.9, 129.4, 128.9, 124.8, 121.9, 120.7, 110.6, 110.4,
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109.6, 55.3, 44.0, 21.5. HRMS (ESI) calcd m/z for [M + H]+ C16H16NO2S 286.0902, found
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N-(2-Fluorobenzyl)-5-methylbenzoxazol-2-thione (16). Yield: 84% (0.92 g). H NMR (300
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MHz, DMSOꢀd6): δ 7.48 (d, J = 7.8 Hz, 1H), 7.42ꢀ7.35 (m, 1H), 7.30ꢀ7.13 (m, 5H), 5.48 (s, 2H),
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131.5, 130.1 (d, JCꢀF = 7.6 Hz), 129.2 (d, JCꢀF = 3.2 Hz), 125.1, 124.7, 121.2 (d, JCꢀF = 14.2 Hz)
115.6 (d, JCꢀF = 20.7 Hz), 110.7, 109.9, 42.7 (d, JCꢀF = 3.3 Hz), 20.9. HRMS (ESI) calcd m/z for
[M + H]+ C15H13FNOS 274.0702, found 274.0703.
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N-(2-Chlorobenzyl)-5-methylbenzoxazol-2-thione (17). Yield: 82% (0.95 g). H NMR (300
MHz, CDCl3): δ 7.47 (d, J = 8.1 Hz, 1H), 7.31ꢀ7.18 (m, 3H), 7.12 (d, J = 7.2 Hz, 1H), 7.07 (d, J
= 8.1 Hz, 1H), 6.77 (s, 1H), 5.56 (s, 2H), 2.37 (s, 3H).13C NMR (75 MHz, CDCl3): δ 181.30,
145.3, 135.4, 132.8, 131.5, 131.2, 129.8, 129.4, 128.0, 127.4, 125.2, 110.1, 109.9, 46.4, 21.4.
HRMS (ESI) calcd m/z for [M + H]+ C15H13ClNOS 290.0406, found 290.0413.
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N-(2-Nitrobenzyl)-5-methylbenzoxazol-2-thione (18). Yield: 72% (0.86 g). H NMR (300
MHz, DMSOꢀd6): δ 8.23 (d, J = 7.8 Hz, 1H), 7.68ꢀ7.56 (m, 2H), 7.51 (d, J = 7.8 Hz, 1H), 7.31
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(brs, 1H), 7.15 (d, J = 8.7 Hz, 1H), 6.92 (d, J = 7.5 Hz, 1H), 5.80 (s, 2H), 2.3 (s, 3H). C NMR
(75 MHz, DMSOꢀd6): δ 180.4, 147.6, 144.8, 135.1, 134.5, 131.5, 129.7, 128.9, 126.9, 125.5,
125.2, 111.0, 109.9, 46.4, 20.8. HRMS (ESI) calcd m/z for [M + H]+ C15H13N2O3S 301.0647,
found 301.0648.
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N-(2-Methylbenzyl)-5-methylbenzoxazol-2-thione (19). Yield: 82% (0.88 g). H NMR (300
MHz, CDCl3): δ 7.24ꢀ7.22 (m, 1H), 7.24ꢀ7.20 (m, 2H), 7.17ꢀ7.10 (m, 1H), 7.02 (d, J = 8.4 Hz,
1H), 6.96 (d, J = 7.5 Hz, 1H), 6.58 (s, 1H), 5.40 (s, 2H), 2.41 (s, 3H), 2.31 (s, 3H). 13C NMR (75
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MHz, CDCl3): δ 181.1, 145.4, 135.8, 135.1, 131.8, 131.4, 130.8, 128.1, 126.5, 126.4, 125.0,
110.4, 109.9, 47.8, 21.4, 19.4. HRMS (ESI) calcd m/z for [M + H]+ C16H16NOS 270.0953, found
270.0963.
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N-(3-Hydroxybenzyl)-5-methylbenzoxazol-2-thione (20). Yield: 86% (0.93 g). H NMR (300
MHz, DMSOꢀd6): δ 9.47 (s, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.20ꢀ7.12 (m, 3H), 6.82 (d, J = 7.2
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Hz, 1H), 6.75 (brs, 1H), 6.69 (dd, J = 8.1, 2.1 Hz, 1H), 5.36 (s, 2H), 2.35 (s, 3H). C NMR (75
MHz, CDCl3): δ 181.1, 156.1, 145.3, 135.6, 135.3, 131.6, 130.2, 125.1, 119.9, 115.5, 114.4,
110.2, 109.9, 49.1, 21.4. HRMS (ESI) calcd m/z for [M + H]+ C15H14NO2S 272.0745, found
272.0741.
N-(3-Methoxybenzyl)-5-methylbenzoxazol-2-thione (21). Yield: 87% (0.99 g). Purity
(HPLC/UV λ at 254 nm): 82.6%. 1H NMR (300 MHz, CDCl3): δ 7.29ꢀ7.20 (m, 2H), 7.01 (d, J =
8.1 Hz, 1H), 6.96ꢀ6.94 (m, 2H), 6.84 (dd, J = 8.4, 2.1 Hz, 1H), 6.78 (brs, 1H), 5.37 (s, 2H), 3.77
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(s, 3H), 2.35 (s, 3H). C NMR (75 MHz, CDCl3): δ 181.1, 160.0, 145.3, 135.5, 135.2, 131.6,
130.0, 125.0, 119.8, 113.6, 113.3, 110.2, 109.9, 55.2, 49.3, 21.5. HRMS (ESI) calcd m/z for [M +
H]+ C16H16NO2S 286.0902, found 286.0902.
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N-(3-Fluorobenzyl)-5-methylbenzoxazol-2-thione (22). Yield: 72% (0.79 g). H NMR (300
MHz, CDCl3): δ 7.40ꢀ7.33 (m, 1H), 7.29 (d, J = 3.3 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 7.14ꢀ7.02
(m, 3H), 6.80 (s, 1H), 5.43 (s, 2H), 2.41 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 181.1, 163.0 (d,
JCꢀF = 245.4 Hz), 145.3, 136.3 (d, JCꢀF = 6.5 Hz), 135.3, 131.5, 130.6 (d, JCꢀF = 8.7 Hz), 125.2,
123.2, 115.4 (d, JCꢀF = 20.7 Hz), 114.6 (d, JCꢀF = 21.8 Hz), 110.0, 109.9, 48.7, 21.5. HRMS (ESI)
calcd m/z for [M + H]+ C15H13FNOS 274.0702, found 274.0707.
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N-(3-Nitrobenzyl)-5-methylbenzoxazol-2-thione (23). Yield: 85% (1.02 g). Purity (HPLC/UV
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λ at 254 nm): 84.9%. H NMR (300 MHz, DMSOꢀd6): δ 8.32 (s, 1H), 8.17 (d, J = 7.8 Hz, 1H),
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7.81 (d, J = 7.5 Hz, 1H), 7.66 (app t, J = 7.5 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.34 (s, 1H), 7.14
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(d, J = 8.1 Hz, 1H), 5.59 (s, 2H), 2.35 (s, 3H). C NMR (75 MHz, DMSOꢀd6): δ 180.1, 147.9,
144.8, 136.8, 135.2, 134.0, 131.2, 130.3, 125.3, 123.0, 122.4, 110.7, 110.0, 47.4, 20.9. HRMS
(ESI) calcd m/z for [M + H]+ C15H13N2O3S 301.0647, found 301.0652.
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N-(3-Methylbenzyl)-5-methylbenzoxazol-2-thione (24). Yield: 86% (0.92 g). H NMR (300
MHz, CDCl3): δ 7.29ꢀ7.13 (m, 5H), 7.04 (d, J = 8.4 Hz, 1H), 6.80 (s, 1H), 5.39 (s, 2H), 2.38 (s,
3H), 2.35 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 181.1, 145.4, 138.8, 135.1, 133.9, 131.7, 129.1,
128.8, 128.2, 125.0, 124.6, 110.2, 109.9, 49.4, 21.5, 21.4. HRMS (ESI) calcd m/z for [M + H]+
C16H16NOS 270.0953, found 270.0951.
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N-(4-Methoxybenzyl)-5-methylbenzoxazol-2-thione (25). Yield: 68% (0.77 g). H NMR (300
MHz, CDCl3): δ 7.35 (d, J = 8.7 Hz, 2H), 7.20 (d, J = 8.7 Hz, 1H), 7.00 (d, J = 8.1 Hz, 1H), 6.87
13
(d, J = 8.4 Hz, 2H), 6.80 (s, 1H), 5.34 (s, 2H), 3.78 (s, 3H), 2.36 (s, 3H). C NMR (75 MHz,
CDCl3): δ 180.9, 159.6, 145.3, 135.1, 131.6, 129.2 (2C), 126.0, 125.0, 114.3 (2C), 110.2, 109.8,
55.2, 48.9, 21.5. HRMS (ESI) calcd m/z for [M + H]+ C16H16NO2S 286.0902, found 286.0902.
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N-(4-Fluorobenzyl)-5-methylbenzoxazol-2-thione (26). Yield: 70% (0.76 g). H NMR (300
MHz, CDCl3): δ 7.45 (dd, J= 8.4, 5.4 Hz, 2H), 7.28 (app t, J = 8.4 Hz, 1H), 7.12ꢀ7.06 (m, 3H),
13
6.83 (s, 1H), 5.42 (s, 2H), 2.43 (s, 3H). C NMR (75 MHz, CDCl3): δ 181.0, 162.6 (d, JCꢀF
=
245.5 Hz), 145.3, 135.2, 131.4, 129.8 (d, JCꢀF = 2.2 Hz), 129.5 (d, JCꢀF = 8.8 Hz, 2C), 125.1,
115.9 (d, JCꢀF = 21.8 Hz, 2C), 110.0, 109.9, 48.6, 21.5. HRMS (ESI) calcd m/z for [M + H]+
C15H13FNOS 274.0702, found 274.0703.
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N-(4-Chlorobenzyl)-5-methylbenzoxazol-2-thione (27). Yield: 72% (0.83 g). H NMR (300
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MHz, CDCl3): δ 7.37 (brs, 4H), 7.30 (d, J = 5.1 Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 6.79 (s, 1H),
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5.41 (s, 2H), 2.42 (s, 3H). C NMR (75 MHz, CDCl3): δ 181.1, 145.4, 135.3, 134.3, 132.5,
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131.5, 129.2 (2C), 129.0 (2C), 125.2, 110.1, 109.9, 48.6, 21.5. HRMS (ESI) calcd m/z for [M +
H]+ C15H13ClNOS 290.0406, found 290.0408.
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N-(4-Nitrobenzyl)-5-methylbenzoxazol-2-thione (28). Yield: 75% (0.90 g). H NMR (300
MHz, CDCl3): δ 8.22 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.7 Hz, 2H), 7.28 (d, J = 3.9 Hz, 1H), 7.07
(d, J = 8.4 Hz, 1H), 6.75 (s, 1H), 5.51 (s, 2H), 2.39 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 181.1,
147.9, 145.3, 141.1, 135.6, 131.2, 128.3 (2C), 125.5, 124.3 (2C), 110.3, 109.6, 48.4, 21.5.
HRMS (ESI) calcd m/z for [M + H]+ C15H13N2O3S 341.0647, found 341.0650.
N-(4-Methylbenzyl)-5-methylbenzoxazol-2-thione (29). Yield: 74% (0.80 g). Purity
1
(HPLC/UV λ at 254 nm): 94.7%. H NMR (300 MHz, CDCl3): δ 7.34ꢀ7.30 (m, 2H), 7.23ꢀ7.17
(m, 3H), 7.04 (d, J = 8.1 Hz, 1H), 6.82 (s, 1H), 5.40 (s, 2H), 2.40 (s, 3H), 2.36 (s, 3H). 13C NMR
(75 MHz, CDCl3): δ 181.1, 145.4, 138.2, 135.1, 131.7, 130.9, 129.6 (2C), 127.6 (2C), 125.0,
110.2, 109.8, 49.1, 21.5, 21.1. HRMS (ESI) calcd m/z for [M + H]+ C16H16NOS 270.0953, found
270.0947.
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N-(3-Hydroxy-4-fluorobenzyl)-5-methylbenzoxazol-2-thione (30). Yield: 85% (0.98 g). H
NMR (300 MHz, DMSOꢀd6): δ 9.92 (s, 1H), 7.45 (d, J =8.4 Hz, 1H), 7.23 (s, 1H), 7.14ꢀ7.08 (m,
13
2H), 6.99 (dd, J = 8.4, 2.1 Hz, 1H), 6.89ꢀ6.83 (m, 1H), 5.34 (s, 2H), 2.35 (s, 3H). C NMR (75
MHz, CDCl3): δ 181.0, 150.8 (d, JCꢀF = 237.5 Hz), 154.3, 143.9 (d, JCꢀF = 14.7 Hz), 135.3, 131.5,
130.9 (d, JCꢀF = 3.8 Hz), 125.2, 120.1 (d, JCꢀF = 6.8 Hz), 116.6, 116.0 (d, JCꢀF = 18.6 Hz), 110.0
(2C), 48.6, 21.5. HRMS (ESI) calcd m/z for [M + H]+ C15H13FNO2S 290.0651, found 290.0654.
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N-(3-Hydroxy-4-fluorobenzyl)-5-trifluoromethylbenzoxazol-2-thione (31). Yield: 80% (1.10
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g). H NMR (300 MHz, (CH3)2COꢀd6): δ 8.81 (d, J = 1.2 Hz, 1H), 7.71ꢀ7.69 (m, 3H), 7.17ꢀ7.12
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(m, 1H), 7.10ꢀ7.06 (m, 1H), 7.03ꢀ6.98 (m, 1H), 5.51 (s, 2H). C NMR (75 MHz, (CH3)2COꢀd6):
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δ 182.3, 150.6 (d, JCꢀF = 238.9 Hz), 150.0, 146.0 (d, JCꢀF = 13.1 Hz), 133.3, 131.8 (d, JCꢀF = 3.2
Hz), 127.7 (q, JCꢀF = 32.7 Hz), 124.9 (q, JCꢀF = 269.4 Hz), 122.6 (q, JCꢀF = 3.3 Hz), 120.3 (d, JCꢀF
= 6.5 Hz), 118.1, 117.0 (d, JCꢀF = 18.5 Hz), 111.5, 108.5 (q, JCꢀF = 3.2 Hz), 49. HRMS (ESI)
calcd m/z for [M + H]+ C15H10F4NO2S 344.0368, found 344.0359.
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N-(3-Hydroxybenzyl)-5-methoxybenzoxazol-2-thione (32). Yield: 72% (0.83 g). H NMR
(300 MHz, CDCl3): δ 7.23ꢀ7.18 (m, 2H), 6.92 (d, J = 7.8 Hz, 1H), 6.86 (brs, 1H), 6.78 (dd, J =
8.4, 2.4 Hz, 1H), 6.73 (dd, J = 8.7, 2.4 Hz, 1H), 6.49 (d, J = 2.7 Hz, 1H), 5.33 (s, 2H), 3.75 (s,
3H). 13C NMR (75 MHz, CDCl3): δ 181.4, 157.5, 156.2, 141.4, 135.4, 132.3, 130.2, 119.9,
115.6, 114.4, 110.6, 109.8, 96.3, 56.0, 49.2. HRMS (ESI) calcd m/z for [M + H]+ C15H14NO3S
288.0694, found 288.0689.
N-(Benzyl)-5-methylbenzoxazol-2-one (33). Yield: 70% (0.67 g). 1H NMR (300 MHz, CDCl3):
δ 7.36ꢀ7.32 (m, 5H), 7.08 (d, J = 8.1 Hz, 1H), 6.88 (d, J = 7.8 Hz, 1H), 6.66 (s, 1H), 4.98 (s, 2H),
2.32 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 155.0, 140.7, 134.8, 133.8, 130.8, 128.9 (2C), 128.1,
127.5 (2C), 122.9, 109.5, 109.3, 45.9, 21.4. HRMS (ESI) calcd m/z for [M+H]+ C15H14NO2
240.1025, found 240.1023.
N-(3-Hydroxy-4-fluorobenzyl)-5-trifluoromethylbenzoxazol-2-one (34). Yield: 65% (0.85 g).
Purity (HPLC/UV λ at 300 nm): 92.6 %. 1H NMR (300 MHz, CD3OD): 7.52ꢀ7.40 (m, 3H), 7.10ꢀ
13
6.98 (m, 2H), 6.91ꢀ6.86 (m, 1H), 5.04 (s, 2H). C NMR (75 MHz, CD3OD): δ 156.2, 153.1 (d,
JCꢀF = 240.0 Hz), 147.0 (d, JCꢀF = 13.0 Hz), 146.6, 133.1, 132.9 (d, JCꢀF = 3.3 Hz), 127.9 (q, JCꢀF
=
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32.7 Hz), 125.7 (q, JCꢀF = 269.4 Hz), 121.6 (q, JCꢀF =3.3 Hz), 120.4 (d, JCꢀF = 6.6 Hz), 118.3 (d,
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JCꢀF = 2.2 Hz), 117.6 (d, JCꢀF =18.5 Hz), 111.5, 110.8 (q, JCꢀF = 3.2 Hz), 46.6. HRMS (ESI) calcd
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m/z for [M+Na]+ C15H9F4NO3Na 350.0416, found 350.0415.
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(S,R)-3-(4-Hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (35). Compound
35 has been synthesized according to the procedure described in the reference 34.34 Yield: 45%
(0.90 g). 1 H NMR (300 MHz, (CH3)2COꢀd6): δ 8.71 (s, 1H), 7.54 (d, J = 8.7 Hz, 2H), 6.88 (d, J
= 9.0 Hz, 2H), 5.06ꢀ4.95 (m, 1H), 3.65 (s, 3H), 3.52 (dd, J = 16.8, 10.2 Hz, 1H), 3.14 (dd, J =
16.8, 7.2 Hz, 1H), 2.74 (dd, J = 15.9, 6.9 Hz, 1H), 2.67 (dd, J = 14.7, 5.1 Hz, 1H). 13C NMR (75
MHz, (CH3)2COꢀd6): δ 171.2, 159.9, 156.8, 129.1 (2C), 123.3, 136.3 (2C), 77.6, 51.8, 40.7, 40.2.
HRMS (ESI) calcd m/z for [M+H]+ C12H14NO4 236.0923, found 236.0916.
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Methods
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Tested compounds
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All the tested compounds were synthesized by the biotechnology company MIFCARE.
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Molecular docking
Xꢀray structures of three different MIF coꢀcrystals were retrieved from the PDB33 (accession
codes 1CA7, 1GCZ and 1LJT)30ꢀ32 and prepared by missing atoms addition, and ligand
extraction from one of the binding sites. Coordinates for compounds 1 and 2 were generated
using MarvinSketch from JChem v15.12.7 suite. Ligands were then freely docked in the
prepared binding site with GOLD v5.2.2 software package35 using CHEMPLP with default
parameters as an objective function.36 Structures of complexes were exported and loaded in
Chimera v1.11 for examination (including hydrogen bond detection, close contact analysis, and
representation of solventꢀaccessible surface).
MIF tautomerase assay using 4-hydroxyphenylpyruvate (4-HPP)
Tautomerase activity was assessed using the substrate 4ꢀhydroxyphenylpyruvate (4ꢀHPP). 4ꢀHPP
was dissolved in 50 mM ammonium acetate and allowed to equilibrate at +4°C overnight before
use. MIF (100nM; rhMIF; RayBiotech, Le Perray en Yvelines, France) was preꢀincubated with 8
concentrations of hit compounds ranging from 0 to 10ꢁM for 15 min. Tautomerase activity was
performed at room temperature, by adding 4ꢀHHP to a 96ꢀwell plate containing 0.435 M boric
acid at pH 6.6 and the rhMIF with or without tested compound, and by measuring the increase in
absorbance at 305 nm over 0–360s using the FlexStation 3 MultiꢀMode Microplate Reader
(Molecular Devices). The assay was replicated at least 3 times independently.
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Cell culture
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DUꢀ145 cell line from ATCC was grown in RPMI medium (Fisher, Illkirch Cedex, France)
supplemented with 10% fetal calf serum (FCS), penicillinꢀstreptomycin solution, glutamine,
HEPES as recommended by the commercial company. Pulmonary ECs were isolated and
cultured from lung specimens of patients with idiopathic pulmonary arterial hypertension (iPAH
ECs) as previously described.5, 37ꢀ42 The isolated iPAHꢀECs were strongly positive for acetylated
lowꢀdensity lipoprotein coupled to Alexa 488 (Alexa488ꢀAcꢀLDL), von Willebrand factor
(vWF), CD31, and for Ulex europaeus agglutininꢀ1 (UEAꢀ1) and negative for alphaꢀsmooth
muscle actin (αꢀSMA). iPAHꢀECs were grown in MCDB131 medium (Fisher, Illkirch Cedex,
France) supplemented with 10% fetal calf serum (FCS), penicillinꢀstreptomycin solution,
glutamine, HEPES and amphotericine. This study was approved by the local ethics committee
(CPP IleꢀdeꢀFrance VII, Le KremlinꢀBicêtre, France). All patients gave informed consent before
the study. To study the ERK and AKT activation states, iPAHꢀECs were exposed to 50ng/mL of
recombinant MIF (Raybiotech, Tebu, France) that contains an extremely low endotoxin level
(less than 0.07 EU per µg protein as determined by LAL test).
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MTT colorimetric assay
To determine the effect of our compounds on cell survival, the methyl tetrazolium (MTT) assay
was performed of DUꢀ145 cell line and iPAHꢀECs. To this aim, cells were plated in a 96ꢀwell
plate and cultured in RPMI medium (Fisher, Illkirch Cedex, France) or MCDB131 medium
(Fisher, Illkirch Cedex, France), respectively, with 10% of FCS for 24h. Then, DUꢀ145 and
iPAHꢀECs monolayers were washed and 10, 50 or 100µM of tested compounds were added with
0% of FCS for 24h. Afterwards, cells were incubated with 5 mg/mL of MTT for 6h of
incubation. At the end of the incubation time, medium was removed, dimethylsulfoxide (DMSO)
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was added in each well and absorbance was recorded at 590nm using the microplate
spectrophotometer system EnVision (PerkinElmer, Waltam, USA).
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AnnexinV-FITC Assay
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Floating DUꢀ145 cells were collected and combined with adherent cells harvested by
trypsin/EDTA treatment and then stained with the Annexin VꢀFITC (BD Biosciences, Pontꢀdeꢀ
Claix, France). In each sample, at least 10,000 cells were counted by FACS analysis.
Cell proliferation Assays
DUꢀ145 cell line proliferation was measured by 5ꢀbromoꢀ2ꢀdeoxyuridine (BrdU) incorporation.
BrdU staining was measured by the DELFIA Cell proliferation kit (PerkinElmer, Courtaboeuf,
France) and a timeꢀresolved fluorometer EnVision TM Multilabel Reader (PerkinElmer).
Caspase 3/7 activity and TUNEL assay
Caspase 3/7 activity of DUꢀ145 cell line was measured by ApoꢀONE Kit (Promega,
Charbonnieres, France), after 24 hours of treatment with MIF/CD74 antagonist. The fluorescence
was measured by EnVision TM Multilabel Reader (PerkinElmer). In addition, we conducted
terminal deoxynucleotidyl transferase–mediated dUTP nick endꢀlabeling (TUNEL) using the
ApopTag red in situ apoptosis detection kit (Merk Millipore, Molsheim, France).
Western blot, ELISA, and Immunostaining
Cells were homogenized and sonicated in RIPA buffer containing protease and phosphatase
inhibitors and 30 µg of protein was used to detect pERK1/2, ERK, pAKT, and AKT as
previously described.39, 41ꢀ43
Rodent model of pulmonary hypertension (PH)
Animal studies were approved by the administrative panel on animal care at the University Parisꢀ
Sud, Le PlessisꢀRobinson, France (n°02229.01). Young male Wistar rats (100g, Janvier) were
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studied 3 weeks after a single subcutaneous injection of monocrotaline (60 mg/kg) (Sigmaꢀ
Aldrich,SaintꢀQuentin Fallavier, France), or vehicle. Two weeks after a single subcutaneous
injection of monocrotaline (MCT), rats were randomly divided into several groups and daily
treated for 2 weeks with MIF antagonists by gavage, or vehicle (33% of DMSO (Dimethyl
sulfoxide) in a solution of propylene glycol:saline (68:32) for per os administrations) at a dose of
30 mg.kg.dayꢀ1. Animals were anesthetized with isoflurane and a polyvinyl catheter was
introduced into the right jugular vein and pushed through the right ventricle into the pulmonary
artery. Cardiac output in rat was measured using the thermodilution method. After measurement
of hemodynamic parameters, the thorax was opened and the left lung immediately removed and
frozen. The right lung was fixed in the distended state with formalin buffer. The right ventricular
hypertrophy (RVH) (Fulton index) and the percentage of muscularized vessels were determined
as previously described.39, 41ꢀ43
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Cardiac fibrosis analyses
Heart from MCTꢀinjected rats or control rats were deparaffinized, and stained for collagen using
Picrosirius red staining. Three randomly selected fields from transversally sectioned tissue were
used. Fibrosis areas of both right and left ventricle myocardium were quantified using ImageJ
software and expressed as collagen area fraction.5
Statistical Analyses
Values of each variable are expressed as mean ± SEM. Statistical significance was tested using
the nonparametric MannꢀWhitney test or twoꢀway analysis of variance with Bonferroni post hoc
tests. Significant difference was assumed at a P value of less than 0.05.
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ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website at DOI:
Molecular formula strings and the associated biological data (CSV)
AUTHOR INFORMATION
Corresponding Authors
*Phone : +33 614 210 542. Email: gael.jalce@mifcare.com
*Phone : +33 140 948 833. Fax : +33 140 942 522. Email: christophe.guignabert@inserm.fr
Author Contributions
G.J. designed the chemical synthesis; GB performed the molecular docking studies; L.T. and
C.G. designed the biological experiments; B.A. and G.J. performed the chemical synthesis;
M.L.H., L.T., R.T., C.P. and C.G. performed the biological experiments; G.J. and C.G prepared
the manuscript. All authors have given approval to the final version of the manuscript.
Notes
M.L.H., L.T., C.G., B.A. and G.J. are the inventors on patent WO2015173433. G.B., R.T., A.H.,
C.P. have no conflicts of interest to disclose. G.S. reports grants, personal fees and nonꢀfinancial
support from Actelion, Bayer, GSK, Gilead, Pfizer. M.H. has served as a consultant for Actelion,
Bayer, GSK, Novartis, and Pfizer.
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ACKNOWLEDGMENTS
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The authors thank Jennifer Bordenave and EtienneꢀMarie Jutant for their help in the study. We
also thank Karine Leblanc (Univ. ParisꢀSud, BioCIS) for the HPLC and HRMS analyses. This
work was partly supported by the French National Research Agency (ANR) through the EPINE
grant no. ANRꢀ12ꢀJSV1ꢀ0004ꢀ01. M.L.H. was supported by the LabEx LERMIT (grant no ANRꢀ
10ꢀLABXꢀ0033ꢀLERMIT). C.P. was supported by the French Fonds de Dotation Recherche en
Santé Respiratoire–Fondation du Souffle.
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ABBREVIATIONS USED
CD, cluster of differentiation; CDI, 1,1′ꢀCarbonyldiimidazole; CO; cardiac output; IL,
interleukin; iPAH, idiopathic pulmonary hypertension; MAPK/ERK1/2, mitogenꢀactivated
protein kinase/extracellular signal regulated kinase 1/2; MCT, monocrotaline; MTT, methyl
thiazolyl tetrazolium; NFꢀκB; nuclear factor kappa B; PAP; pulmonary arterial pressure; PH,
pulmonary hypertension; PVR, pulmonary vascular resistance; RFU, relative fluorescence units;
rhMIF, recombinant MIF; RVH, right ventricular hypertrophy; SAR, structureꢀactivity
relationship; TNF, tumor necrosis factor; TPVR, total pulmonary vascular resistance.
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