Discovery of 1-Pyrimidinyl-2-Aryl-4,6-Dihydropyrrolo [3,4-d]Imidazole-5(1H)-Carboxamide as a Novel JNK Inhibitor

We designed and synthesized 1-pyrimidinyl-2-aryl-4, 6-dihydropyrrolo [3,4-d] imidazole-5(1H)-carboxamide derivatives as selective inhibitors of c-Jun-N-terminal Kinase 3 (JNK3), a target for the treatment of neurodegenerative diseases. Based on the compounds found in previous studies, a novel scaffold was designed to improve pharmacokinetic characters and activity, and compound 18a, (R)-1-(2-((1-(cyclopropanecarbonyl)pyrrolidin-3-yl)amino)pyrimidin-4-yl)-2-(3,4-dichlorophenyl)-4,6-dihydro pyrrolo [3,4-d]imidazole-5(1H)-carboxamide, showed the highest IC50 value of 2.69 nM. Kinase profiling results also showed high selectivity for JNK3 among 38 kinases, having mild activity against JNK2, RIPK3, and GSK3β, which also known to involve in neuronal apoptosis.


Introduction
Alzheimer's disease (AD) is one of the most common neurodegenerative diseases and features both amyloid β plaques and neurofibrillary tangles (NFTs) as pathological hallmarks [1]. Although the cause of AD, which affects many, is not clearly identified, many therapeutic agents have been studied to inhibit the formation of these two hallmarks, and we aim to develop a treatment for Alzheimer's disease that targets and inhibits JNK3, which is deeply involved in the formation of amyloid β protein and NFT [2].
Most importantly, JNK3 expressed in the brain phosphorylates Thr668 of the amyloid precursor protein (APP), so that APP is located on the cell membrane and cleaved by β-secretase and γ-secretase to induce the formation of amyloid β protein [12,13]. The produced amyloid β protein is known to form amyloid plaques, causing neuronal cell apoptosis, and the amyloid β protein also causes positive feedback to reactivate JNK3 [12]. In addition, JNK3 phosphates Ser422 of the tau protein to form NFTs. The formed NFTs disrupt the transport of neurotransmitters by breaking down the structure of microfabrication in neurons, leading to the apoptosis of nerve cells [14].
We studied protein kinase inhibitors targeting JNK3 to develop effective treatments for Alzheimer's disease by impeding these mechanisms.
In order to form the next imidazoline (7) ring, the corresponding aryl imidate was reacted with diamino-N-Boc pyrrolidine; then, the Swern oxidation was accomplished to synthesize the 2,5-dihydropyrrolo imidazole core (8). Next, 4-chloro-2-methylthio-pyrimidine was introduced to the core through S N Ar reaction under microwave irradiation (9). The methyl sulfide was oxidized to methyl sulfone (10) by potassium peroximonosulfate and substituted with the amide-coupled amine group through another S N Ar (11a-d, 12a-d, 13a-d, 14a, 14c and 15a). The final products (17a-d, 18a-d, 19a-d, 20a, 20c and 21a) were obtained after Boc deprotection by HCl and phenylcarbamate treatment. Another final product (22a) was obtained using 4-nitrophenyl chloroformate. After synthesis of all the compounds (17a-d, 18a-d, 19a-d, 20a, 20c and 21a), the JNK3 inhibitory activity of each compound was evaluated ( Table 1). Most of the synthesized compounds exhibited good activity against JNK3. In particular, 18a showed the most potent activity against JNK3, with an IC 50 value of 2.69 nM. Structure activity relationships (SARs) were inferred from potency data. First, when comparing the activity by the aryl group substitution, the compounds with the relatively large groups such as naphtyl and dichlorophenyl groups showed good inhibitory activity toward JNK3, rather than those with dioxolphenyl and dihydrobenzofuranphenyl groups (a and b vs. c and d). We think that the aryl group occupied a larger hydrophobic space under the roof and induced hydrophobic interaction. This was assumed from the docking studies of the previous inhibitor of JNK3. Moreover, the napthyl and dichlorophenyl rings have higher electron densities, so could form stronger interactions with the surrounded residues, supporting better activities. Secondly, when the piperidin-4-ol (17a) was substituted in the position of the carboxamide in 2, 5-dihydropyrrolo-1-carboxamide, the activity falls to half that of the corresponding carboxamide (17a vs. 22a). Next, when the cyclopropyl group in the solvent exposure part was replaced with a cyclobutyl or cyclopentyl group, the inhibitory activity decreased approximately two-to three-fold (17a vs. 20a, 20c, and 21a). In an effort to reduce the molecular weight, the piperidine ring was diversified into pyrrolidine with less carbon atoms (n = 2). Surprisingly, when (R)-aminopyrrolidine was coupled to the pyrimidyl group instead of the (S)-aminopiperidine, the activities were increased by approximately seven-to ten-fold (17 vs. 19). Interestingly, when (R)-aminopyrrolidine was introduced, the activity was significantly increased by approximately four-to five-fold (17 vs. 18). This also suggested that the size and configuration of the amino group in the ring should be considered important for binding, even in the solvent exposure part for optimal extra-hydrogen bonding. The extra hydrogen bonding seemed more plausible in (R)-pyrrolidine (18) than in the cases of (S)-piperidine (17) and (S)-pyrrolidine (19).
A docking study was conducted to understand the binding mode of the novel JNK3 inhibitor 18a ( Figure 2). When we performed the docking experiment of 18a with a known JNK3 structure (3OY1), it was shown that many of the interactions could contribute to complex stabilization. First, the amino pyrimidine is a hinge binder and forms two hydrogen interactions with the Met149 of JNK3. The oxygen of cyclopropyl carboxamide in compound 18a could form hydrogen bond interaction with Gln155 in the extended hinge region. The two hydrogen bonds were monitored between the oxygen in the carboxamide of 2,5-dihydropyrrole and the side chain of Asn152, and between the NH 2 in the carboxamide of 2,5-dihydropyrrole-1-carboxamide and the side chain of Ser72. Finally, the dichlorophenyl ring of compound 18a fits into the hydrophobic pocket and forms a halogen bond with Ala93.  Next, we performed kinases panel screening in duplicate for compound 18a on 38 different protein kinases at a single-dose concentration of 10µM ( Table 2). The compound 18a was indeed a selective JNK3 inhibitor with an excellent selectivity profile, only having slight activities on JNK2, GSK3β, and RIPK3 more than 50%. When we further determine the IC 50 of 18a on these three protein kinases and compared with it on JNK3, the selectivity was still maintained. And since the GSK3β and RIPK3 are said to be associated with neurodegenerative disease caused by all neuronal apoptosis [18][19][20][21][22][23], we could manipulate these characters of 18a for further developments. Table 2. Percentages of enzymatic inhibition exerted by 10 µM of 18a on 38 selected protein kinases [24] and enzymatic activities on selected protein kinases.

General Chemical Methods
All chemicals were of reagent grade and were purchased from Sigma-Aldrich, Inc. (Seoul, Korea). Separation of the compounds by column chromatography was carried out with silica gel 60 (200-300 mesh ASTM, E. Merck, Darmstadt, Germany). The quantity of silica gel used was 50-100 times the weight charged on the column. Thin layer chromatography (TLC) was run on silica gel-coated aluminum sheets (silica gel 60 GF254, E. Merck, Darmstadt, Germany) and visualized under ultraviolet (UV) light (254 nm). Both 1 H Nuclear Magnetic Resonance (NMR )and 13 C NMR spectra were recorded on a Bruker model digital AVANCE III 400 MHz spectrometer (Billerica, MA, USA) at 25 • C using tetramethylsilane (TMS) as an internal standard. High-resolution Mass Spectra (HR/MS) experiments were conducted with a Finnigan LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific Inc., New York, NY, USA) operated in positive-ion electrospray mode. (2) Compound 1 (4.65 mmol) was dissolved in dimethyl formamide (5 mL), and then sodium hydride (10.23 mmol) was added at 0 • C and stirred for 10 min. Compound 1 was added to the mixture and stirred at room temperature for 15 min, followed by stirring at 65 • C for 4 to 6 h. It was then cooled to ambient temperature, extracted with an organic layer (Ethylacetate:n-Hexane (EA:HEX) = 1:4), and washed with water. This was followed by drying with anhydrous magnesium sulfate and evaporation of the solvent to obtain compound 3 as a yellow oil. (45%); 1  Compound 2 (6.3 mmol) was dissolved in tetrahydrofuran (15.8 mL) and we then slowly dropped the mixture of osmium tetroxide (0.113 mmol) and N-methylmorpholine-N-oxide (8.29 mmol) in 15.8 mL of water. The mixture was stirred at room temperature for 3 to 5 h. It was then concentrated in vacuo, extracted with ethyl acetate, and washed with water. This was followed by drying with anhydrous magnesium sulfate, evaporation of the solvent, and then purification of the product by column chromatography on silica gel using a mobile phase of EA: HEX (3: 1) to obtain compound 3 as a yellow oil. (59%); 1 (4) Compound 3 (0.96 mmol) was dissolved in dichloromethane (4.8 mL), and then methanesulfonyl chloride (2.11 mmol) and triethylamine (2.11 mmol) were added and stirred at room temperature for 30 min to 1 h. The reaction mixture was washed with water and brine. This was followed by drying with anhydrous magnesium sulfate and evaporation of the solvent to obtain compound 4 as a white solid. Oxalyl chloride (0.59 mmol) and dimethyl sulfoxide (1.18 mmol) were dissolved in 7 mL of dichloromethane, stirred at −78 • C for 10 min, and then slowly added to compound 7a (0.59 mmol) dissolved in 5 mL of dichloromethane at −78 • C for 30 min. Then, triethylamine (5.9 mmol) was added slowly and stirred at room temperature for 1 h 30 min. The reaction mixture was washed with water and brine, followed by drying with anhydrous magnesium sulfate and concentration of the solvent in vacuo to obtain compound 8a as a white solid. (50%); 1
Methanol was concentrated and extracted with ethyl acetate and washed with water and brine, followed by drying with anhydrous magnesium sulfate and concentration of the solvent in vacuo to obtain compound 10a as a white solid. Next, (S)-(3-aminopiperidin-1-yl)(cyclopropyl)methanone (0.32 mmol) was dissolved in 1 mL of dimethylformamide, and 44.5 µL (0.32 mmol) of triethylamine were added. Then, compound 10a (0.14 mmol) dissolved in 3 mL of tetrahydrofuran was added and stirred at 80 • C for 24 h. Tetrahydrofuran was concentrated in vacuo, extracted with ethyl acetate, and washed with water and brine. After drying over anhydrous magnesium sulfate and concentration, the product was purified by column chromatography on silica gel using a mobile phase of EA:HEX (5:1) to obtain compound 11a as a yellow solid. Compound 9a (0.16 mmol) was dissolved in 2 mL of methanol, and then potassium peroxomonosulfate (0.8 mmol) dissolved in 2 mL of water was added, followed by stirring at ambient temperature for 2 h. Methanol was concentrated and extracted with ethyl acetate and washed with water and brine. This was followed by drying with anhydrous magnesium sulfate and concentration of the solvent in vacuo to obtain compound 10a as white solid. Next, (R)-(3-aminopirrolidine-1-yl)(cyclopropyl)methanone (0.32 mmol) was dissolved in 1 mL of dimethylformamide, and 44.5 µl (0.32 mmol) of triethylamine were added. Then, compound 10a (0.14 mmol) dissolved in 3 mL of tetrahydrofuran was added and stirred at 80 • C for 24 h. Tetrahydrofuran was concentrated in vacuo, extracted with ethyl acetate, and washed with water and brine. After drying over anhydrous magnesium sulfate and concentration, the product was purified by column chromatography on silica gel using a mobile phase of EA: HEX ( Compound 9a (0.16 mmol) was dissolved in 2 mL of methanol, and then potassium peroxomonosulfate (0.8 mmol) dissolved in 2 mL of water was added, followed by stirring at ambient temperature for 2 h. Methanol was concentrated and extracted with ethyl acetate and washed with water and brine, followed by drying with anhydrous magnesium sulfate and concentration of the solvent in vacuo to obtain compound 10a as white solid. (S)-(3-aminopirrolidine-1-yl)(cyclopropyl)methanone (0.32 mmol) was dissolved in 1 mL of dimethylformamide, and 44.5 µL (0.32 mmol) of triethylamine were added. Then, compound 10a (0.14 mmol) dissolved in 3 mL of tetrahydrofuran was added and stirred at 80 • C for 24 h. Tetrahydrofuran was concentrated in vacuo, extracted with ethyl acetate, and washed with water and brine. After drying over anhydrous magnesium sulfate and concentration, the product was purified by column chromatography on silica gel using a mobile phase of EA: HEX (5: 1) to obtain compound 13a as a yellow solid. (32%); 1  Compound 9a (0.16 mmol) was dissolved in 2 mL of methanol, and then potassium peroxomonosulfate (0.8 mmol) dissolved in 2 mL of water was added, followed by stirring at ambient temperature for 2 h. Methanol was concentrated and extracted with ethyl acetate and washed with water and brine, followed by drying with anhydrous magnesium sulfate and concentration of the solvent in vacuo to obtain compound 10a as white solid. (S)-(3-aminopiperidin-1-yl)(cyclobutyl)methanone (0.32 mmol) was dissolved in 1 mL of dimethylformamide, and 44.5 µl (0.32 mmol) of triethylamine were added. Then, compound 10a (0.14 mmol) dissolved in 3 mL of tetrahydrofuran was added and stirred at 80 • C for 24 h. Tetrahydrofuran was concentrated in vacuo, extracted with ethyl acetate, and washed with water and brine. After drying over anhydrous magnesium sulfate and concentration, the product was purified by column chromatography on silica gel using a mobile phase of EA: HEX (5: will be highly useful in the development of JNK3-selective inhibitors as therapeutic agents for neurodegenerative diseases.