Synthesis and Biological Evaluation of Chromenylurea and Chromanylurea Derivatives as Anti-TNF-α agents that Target the p38 MAPK Pathway

A series of 1-aryl-3-(2H-chromen-5-yl)urea and 1-aryl-3-(chroman-5-yl)urea derivatives were designed, synthesized and evaluated for their inhibitory activities towards TNF-α production in lipopolysaccharide-stimulated THP-1 cells. The most active compound, 40g, inhibited TNF-α release with an IC50 value of 0.033 μM, which is equipotent to that of BIRB796 (IC50 = 0.032 μM).


Introduction
The p38 mitogen-activated protein kinase (p38MAPK) plays a key role in inflammatory responses through the production of cytokines and inflammatory mediators such as TNF-α and IL-1β [1]. At least four distinct homologues, standardized in the nomenclature as p38α, β, γ and δ, have been identified. The inhibition of p38MAPKα is considered to be a promising therapeutic strategy for chronic inflammatory diseases such as rheumatoid arthritis [2], psoriasis, inflammatory bowel disease [2], and OPEN ACCESS chronic obstructive pulmonary disease [3]. Recent studies have also revealed that p38MAPKα inhibitors may have therapeutic potential in the treatment of cancer [4,5], neuropathic pain [6] and periodontal diseases [7]. Consequently, considerable effort has been directed toward the development of p38MAPKα inhibitors as potential anti-inflammatory and anticancer drug.
p38MAPKα inhibitors, like many other kinase inhibitors, can be classified into two types based on their mode of action: ATP-competitive inhibitors, which bind to an ATP-binding site, and non-ATP-competitive or allosteric inhibitors. Allosteric inhibitors utilize the ATP binding cleft and a hydrophobic allosteric pocket created when the activation loop adopts the inactive "Asp-Phe-Gly (DFG)-out" conformation. Because allosteric inhibitors do not compete directly with ATP or substrate, they can offer a significant kinetic advantage over ATP competitive inhibitors. In addition, because the allosteric pocket is less conserved than the ATP binding region, allosteric inhibitors usually have better kinase selectivity profiles than ATP competitive inhibitors [8]. BIRB796 is a typical allosteric p38MAPKα inhibitor with an N-pyrazole-N'-naphthyl urea scaffold. The crystal structure of the p38MAPKα/BIRB796 complex shows that BIRB796 fits well into the DFG-out conformation by forming several tight interactions. (Figure 1 graphically depicts a two-dimensional p38MAPKα/ BIRB796 interaction map.) By taking advantage of these interactions, BIRB796 achieves potent p38MAPKα inhibition with a Kd value of 0.1 nM [9]. The major drawback of BIRB796 is its hepatotoxicity [10], which may be caused by one of its metabolic intermediates, naphthalene epoxide [11]. We therefore performed scaffold modification and structure-activity relationship (SAR) investigations of BIRB796 and its analogues to discover novel p38MAPKα inhibitors with drug-like properties. Asp168, while the NH from the urea linkage forms hydrogen bonds to the Glu71 side chain. Such a hydrogen bonding network is essential to maintain the p38MAPKα inhibition activity, so we preserved this urea linkage in the compounds we designed. The naphthyl ring of BIRB796 pushes very deep into the hydrophobic gatekeeper pocket. It is known that such a hydrophobic interaction is essential to obtain high activity and selectivity for a p38MAPKα inhibitor [12]. A pi-cation interaction has also been observed between the naphthyl ring and the cationic amino groups in the side chains of Lys53. To preserve the activity and selectivity, and to eliminate the hepatotoxicity caused by the naphthyl ring, we attempted to replace the naphthalene ring with two other aromatic hydrophobic scaffolds: 2H-chromene and chromane. In BIRB796, the morpholinoethoxy group occupies the adenine binding site, and forms hydrogen bond interactions with the residue of Met109 and Gly110. According to SAR information of kinase inhibitors, a variety of functional groups could be well tolerated by this adenine binding site. We therefore attempted to either replace this morpholinoethoxy moiety with a 2-morpholino-2-oxoethoxy moiety, or to replace the morpholine ring with a larger aliphatic group (2,6-dimethylmorpholine) or aromatic hydrogen bond-accepting moieties (triazole, pyridine or imidazole) with the aim of increasing the binding affinity. BIRB796 also utilizes a unique allosteric pocket created when the activation loop adopts the "DFG-out" conformation. This allosteric pocket can be divided into two selectivity sites: the lower selectivity site, occupied by the t-butyl group, and the upper selectivity site, occupied by the p-tolyl group. It has been reported that the t-butyl group fits well with the highly conserved lower selectivity site, while the upper selectivity site is less conserved and offers a unique position to enable p38MAPKα inhibitory activity and kinase selectivity [13]. On the basis of this knowledge, the t-butyl group was preserved in our compounds, while we attempted to substitute the 4-tolyl group with another substituted phenyl to investigate the SAR around the phenyl ring unit. It was also of interest to us that whether substituting the pyrazole ring with its isosteres, such as oxazole and imidazole, would increase the inhibitory activity for p38MAPKα. On the basis of the above information, a series of N-aryl-N'-chromenyl urea and N-aryl-N'-chromanyl urea derivatives was designed, synthesized and evaluated for their inhibitory activity against TNF-α release.
Finally, the target N-aryl-N'-chromenylurea and N-aryl-N'-chromanylurea derivatives were prepared as shown in Scheme 5. The 5-alkoxy-2H-chromen-8-amines 10a-c or the 5-alkoxychroman-8-amines 17a-f reacted with triphosgene in dichloromethane at −15 °C to form the corresponding isocyanate compounds [22], which then reacted with aryl amines 25a-p in the presence of triethylamine at room temperature for 12-24 h to yield the target compounds 39a-j and 40a-i. The structures of 39a-j and 40a-i are listed in Table 1.
The potential inhibitors 39a-j and 40a-r were profiled for their ability to inhibit TNF-α release in lipopolysaccharide (LPS)-stimulated THP-1 cells [18], with BIRB796 as positive control. The results are shown in Table 2 and Table 3. Among our potential inhibitors, the 2H-chromen-5-ylurea compounds 40a, 40k, 40l and 40m exhibited approximately an order of magnitude more potent anti-TNF-α activity than their corresponding chroman-5-ylurea analogues 39a, 39c, 39d and 39e. Compounds 39a and 40a were identical to BIRB796, except the 2H-chromen-5-ylurea ring in BIRB796 was changed to a 2H-chromen and chroman moiety, respectively. The TNF-α inhibitory activities decreased in sequence of BIRB796 (IC 50 = 0.032 μM), 40a (IC 50 = 0.050 μM) and 39a (IC 50 = 0.31 μM), which is consistent with the downward tendency of the length of the conjugated system of the naphthyl, 2H-chromen and chroman rings. We presume that the conjugated system length of the naphthyl ring, 2H-chromen and chroman is related to the strength of the π-cation interaction between the conjugated system and the cationic amino groups in the side chains of Lys53, which in turn affects the anti-TNF-α activity of the corresponding derivatives. The activity of 2H-chromen-5-yl urea compounds was very close to that of the the corresponding naphthyl urea analogues, which indicates that 3-(2H-chromen-5-yl)ureas may serve as a novel chemotype for the development of p38MAPKα inhibitors.
When the left side of our molecules utilized the 1-aryl-3-tert-butyl-1H-pyrazol-5-amine group from BIRB796 (compounds 39f-j and compounds 40q-r), an obvious decrease in TNF-α inhibition activity was seen when the morpholine group was replaced with 1,2,4-triazole (39h, 40r) or pyridine (39j). In contrast, a relatively small decrease in activity was observed with the replacement of the morpholine group with 2,6-dimethylmorpholine (39g, 40q) and 1,3-imidazole (39i). These results, taken together, indicate that the TNF-α inhibitory activity of our compounds was very sensitive to substitutions in this region. Aromatic hydrogen bond accepting moieties such as 1,2,4-triazole, pyridine and 1,3-imidazole are inferior to the aliphatic morpholine. Introducing two methyl groups to the position ortho to the oxygen atom of morpholine is also not conducive to TNF-α inhibitory activity. In our molecules, we also attempted to modify the ethoxy linker of the morpholinoethoxy group with a 2-oxoethoxy. Unfortunately, the resultant compound 39f exhibited little activity against TNF-α release, which may indicate that a hydrophobic linkage is more suitable than a hydrophilic one for this region. Table 2. Structure and TNF-α inhibitory activity of 1-aryl-3-(chroman-5-yl)urea compounds 40a-i.

General Information
All the reagents were commercially available and used without further purification. 1 H-NMR spectra were measured using a Bruker-400 (Bruker Company, karlsruhe, Germany) or YS-300 instrument. Mass spectra were obtained from VG300, ZAD-2F or API3000 instruments. (2). To a stirred solution of 5-fluoro-2-nitrophenol (30 g, 191 mmol) in DMF (300 mL) anhydrous K 2 CO 3 (52.83 g, 382 mmol) and 3-bromopropyne (27.27 g, 229 mmol) were successively added The mixture was stirred at room temperature for 5 h and then poured into ice water (1,500 mL). A buff precipitate separated out upon standing overnight. The solid was collected by filtration and air-dried to give compound 2 as a buff solid (33.91 g, yield: 85%), mp: 50-52 °C, 1 (4). To a stirred solution of (4-methoxyphenyl)methanol (22.63 g, 163.8 mmol) in anhydrous DMF (200 mL), 60% sodium hydride (8.74 g, 218.4 mmol) was added in portions. The mixture was stirred at ambient temperature until no further gas was released, then cooled to −35 °C. To this solution, compound 2 (21.84 g, 109.2 mmol) was added in one batch. The reaction mixture was stirred for a further 6 h at −35 °C under nitrogen, and then was poured into ice water (1,000 mL). The precipitate was collected, washed with water and airdried to give compound 4 as a buff solid (27.82 g, yield: 93%), mp: 53-54 °C, 1 (5). Compound 4 (22 g, 71.8 mmol) was dissolved in N,N-diethylaniline (330 mL). The reaction mixture was heated to 195 °C and kept at this temperature for 1 h. After cooling to room temperature, the solvent was distilled off under reduced pressure. The residue was purified by column chromatography on silica gel with ethyl acetate/petroleum ether (3:2) as eluent to give compound 5 as a yellow solid (6.47 g, yield: 29%). 1  8-Nitro-2H-chromen-5-ol (6). To a solution of compound 5 (18.2 g, 58 mmol) in dichloromethane (200 mL), trifluoroethylacetic acid (10.0 mL) was added dropwise with stirring at −10 °C. The reaction mixture was stirred at this temperature for 8 h, then quenched by addition of ice water (5 mL). The aqueous solution was then adjusted to pH = 10 with 1 N sodium hydroxide and the two phases were separated. The water phase was extracted twice with dichloromethane. The organic phases were combined and washed with water and brine, dried with Na 2 SO 4 and the solvent removed under vacuum to yield the crude product, which was purified by column chromatography (dichloromethane/methanol 100:2) to give compound 6 as a yellow solid (7.5 g, yield: 67%). 1 (7). To a stirred solution of compound 6 (5.97 g, 31 mmol) in acetonitrile (150 mL) potassium carbonate (5.13 g, 37 mmol) and 1,2-dibromoethane (23.23 g, 124 mmol) were continuously added. The resulting mixture was heated to reflux for 2.5 h and then concentrated. The residue was partitioned between water (50 mL) and ethyl acetate (50 mL). The organic layer was separated, and the aqueous phase extracted with several additional portions of ethyl acetate. The combined organic phase was washed with brine, dried (MgSO 4 ) and concentrated to dryness. The residue was separated by column chromatography on silica gel with ethyl acetate/petroleum ether (1/1) as eluent to give compound 7 as a yellow solid (4.69 g, yield: 34%).
Step 2: preparation of N-boc-5-(2-morpholino-2-oxoethoxy)-8-aminochromane (16f). A solution of compound 12 (1.86 g, 5.0 mmol), anhydrous potassium carbonate (0.83 g, 6.0 mmol) and 4-(2chloroacetyl)-morpholine (20, 982 mg, 6.0 mmol) in DMF (20 mL) was heated to 80 °C for 2 h. The resulting mixture was cooled to room temperature, poured onto cold water and extracted three times with ethyl acetate. The combined ethyl acetate layers were washed with water, brine, and then dried over Na 2 SO 4 , filtrated and concentrated to dryness. The residue was separated by column chromatography on silica gel with ethyl acetate/petroleum ether (1/1) as eluent to give 1.35 g (69% yield) of compound 16f as a white solid. 1  Step 3: preparation of 5-(morpholinoethoxy)-8-aminochromane (17a). To a solution of compound 16a (1.48 g, 4.0 mmol) in CH 2 Cl 2 (40 mL), precooled trifluoroacetic acid (4.0 mL) was added at 0-4 °C, and the reaction was stirred at this temperature for 5 h. After evaporation, water was added to the residue, and the pH of the mixture was adjusted to 10 by addition of 1 M aqueous NaOH solution. The aqueous layer was extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated to give 930 mg (86% yield) of compound 17a as a grey solid. This product is unstable and was therefore used without delay for the next step.

5-(tert-Butyl)-3-aminoisoxazole (25s)
. To a solution of pivaloylacetonitrile (3 g, 23.97 mmol) in water (20 mL), NaOH (1.06 g, 26.4 mmol) and hydroxylamine hydrochloride (1.83 g, 26.4 mmol) were added continuously with stirring. The resulting solution was stirred for approximately 30 min at room temperature, and the pH adjusted to 10-11 with 1 M NaOH. After stirring for 10 h or more at 50 °C, the mixture was cooled and washed two to three times with carbon tetrachloride. The aqueous layer was acidified with concentrated HC1 until the pH = 4-5, and then further stirred for approximately 3 h at 50 °C. The reaction mixture was cooled to room temperature, and adjusted to pH 12 by adding an aqueous solution of 1 N NaOH. The resulting solid was filtered, washed with distilled water, and dried in air to obtain compound 25s as a white solid (2.0 g, yield: 70%). 1 H-NMR (DMSO-d 6 , 300 MHz), δ: 5.49 (s, 1H), 5.40 (s, 2H), 1.21 (s, 9H).

General Procedure for the Preparation of Chromanylureas (39a-j) and 2H-Chromenylureas (40a-r)
A solution of compounds 10a-d or compounds 17a-d (1.0 mmol) in dichloromethane (10 mL) was slowly added to a stirred solution of triphosgene (109 mg, 0.36 mmol) in dichloromethane (50 mL) over a period of 30 min using a syringe. After stirring for a further 30 min, a solution of compound 25a-r (0.6 mmol) and triethylamine (0.4 mL, 2.77 mmol) in dichloromethane (10 mL) was added in one portion. The reaction mixture was stirred for 2 h at room temperature. After completion of the reaction, the reaction was poured into water (50 mL) and extracted three times with dichloromethane. The organic layer was washed with water (5 mL), sat. NaCl solution (5 mL), and dried over Na 2 SO 4 . After evaporation of solvent under vacuum, the residue was purified by silica gel chromatography to give the desired chromanylurea or 2H-chromenylurea compounds.