Novel Tryptanthrin Derivatives with Selectivity as c–Jun N–Terminal Kinase (JNK) 3 Inhibitors

The c-Jun N-terminal kinase (JNK) family includes three proteins (JNK1-3) that regulate many physiological processes, including cell proliferation and differentiation, cell survival, and inflammation. Because of emerging data suggesting that JNK3 may play an important role in neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease, as well as cancer pathogenesis, we sought to identify JNK inhibitors with increased selectivity for JNK3. A panel of 26 novel tryptanthrin-6-oxime analogs was synthesized and evaluated for JNK1-3 binding (Kd) and inhibition of cellular inflammatory responses. Compounds 4d (8-methoxyindolo[2,1-b]quinazolin-6,12-dione oxime) and 4e (8-phenylindolo[2,1-b]quinazolin-6,12-dione oxime) had high selectivity for JNK3 versus JNK1 and JNK2 and inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 (NF-κB/AP-1) transcriptional activity in THP-1Blue cells and interleukin-6 (IL-6) production by MonoMac-6 monocytic cells in the low micromolar range. Likewise, compounds 4d, 4e, and pan-JNK inhibitor 4h (9-methylindolo[2,1-b]quinazolin-6,12-dione oxime) decreased LPS-induced c-Jun phosphorylation in MonoMac-6 cells, directly confirming JNK inhibition. Molecular modeling suggested modes of binding interaction of these compounds in the JNK3 catalytic site that were in agreement with the experimental data on JNK3 binding. Our results demonstrate the potential for developing anti-inflammatory drugs based on these nitrogen-containing heterocyclic systems with selectivity for JNK3.


Introduction
JNKs are members of the mitogen-activated protein kinase (MAPK) family that regulates many physiological processes [1,2]. The JNK pathway is a highly complex pathway within the MAPK signaling network [3]. Activation of JNKs can be induced by a number of proteins, such as other MAPKs and G protein-coupled receptors (GPCRs), which transmit information into the JNK signaling pathway [4]. Despite their name, activated JNKs do not only phosphorylate c-Jun, and close to 100 JNK substrates are known [5]. However, c-Jun is a major substrate for JNKs, and its phosphorylation is closely tied to activator protein 1 (AP-1) activation. Previous studies have shown that JNKs play an important role in the regulation of signaling pathways involved in inflammation, apoptosis, and necrosis [6][7][8][9]. Indeed, JNK signaling is involved in a wide range of diseases, including multiple sclerosis, rheumatoid arthritis, osteoarthritis, insulin resistance, inflammatory bowel diseases, cancer, stroke, renal ischemia, essential hypertension, Alzheimer's disease, and Parkinson's disease [10][11][12][13][14][15][16][17][18][19][20][21]. Therefore, JNKs have become important therapeutic targets and many groups have sought to develop inhibitors for these targets [22][23][24]. Nevertheless, further development of novel JNK inhibitors with clinical value is essential.
The human genome contains three closely related JNK genes (JNK1, JNK2, and JNK3), with each gene encoding multiple isoforms [25]. While JNK1 and JNK2 are expressed in a variety of tissues, JNK3 expression has been reported to be limited mainly to neuronal tissue and, to a lesser extent, heart and testes [26,27]. Thus, most early studies on JNK3 function have focused primarily on its role in brain and neural tissues. However, more recent studies suggest that JNK3 is involved in a wider range of tissues and pathologies. For example, JNK3 has been reported to be expressed in human and mouse pancreatic βcells [28], human CD14 + monocytes [29], normal human epithelial cells [30], normal human MRC-5 fibroblasts, and human thyroid cells and tissue [31]. While the function of JNK3 in these tissues is still being defined, several reports have suggested important regulatory functions. JNK3 has been reported to play an important role in the circulatory system, including in angiogenesis [32]. Likewise, it has been reported that β-arrestin 2 can regulate macrophage function via JNK3 [33]. Additional studies on the patterns of JNK3 expression in various tissues may find that JNK3 has both compensatory and opposing functions that affect the ability of JNK1 and JNK2 to carry out their characteristic activities [34].
Recent research has indicated that JNK3 may play an important role in cancer. For example, JNK3 was found to be expressed in human ovarian cancer SKOV3/DDP cells [35], human hepatocellular carcinoma cells [36], human small-cell lung cancer cells [37], human esophageal squamous cell carcinoma cells [38], and HeLa cells [39]. Notably, Gorogh et al. [40] reported that JNK3 was capable of conveying chemotherapy resistance and survival in HNSCC (head and neck squamous cell carcinoma) cells. Likewise, analysis of prostate cancer clinical samples indicated that high intratumor JNK3 levels correlated with a worse prognosis [41]. In human breast cancer samples and tumor cell lines, JNK3, but not the other JNK isoforms, responded to amyloid precursor protein (APP) signaling, and subsequent JNK3 phosphorylation facilitated epithelial-mesenchymal transition of breast cancer cells [42]. Finally, high basal levels of JNK3 expression were present in oral keratinocyte cell lines, and invasion by these cells was found to be primarily JNK3-dependent [43].

Synthesis of Tryptanthrin Derivatives
Our prior analysis of indenoquinoxaline and tryptanthrin derivatives demonstrated that an oxime group was essential for JNK inhibition, and compounds lacking the oxime had little or no effect on JNK activity [49,50,53,54]. Therefore, we examined the effect of

Synthesis of Tryptanthrin Derivatives
Our prior analysis of indenoquinoxaline and tryptanthrin derivatives demonstrated that an oxime group was essential for JNK inhibition, and compounds lacking the oxime had little or no effect on JNK activity [49,50,53,54]. Therefore, we examined the effect of adding different substituents to the tetracyclic scaffold of tryptanthrin while maintaining the oxime group. JNK binding and inhibitory activity of these TRYP-Ox derivatives were evaluated.
For compound synthesis, substituted isatins 1a-l and isatoic anhydrides 2a-g (see Table 1) were refluxed in boiling toluene (Scheme 1, path A) to obtain the corresponding ketones 3a-n. Poor yields were observed when electron-withdrawing substituents were present in position 8 (NO 2 , Br), and only traces of target compounds were obtained when starting with 7-substituted isatins.

Synthesis of Tryptanthrin Derivatives
Our prior analysis of indenoquinoxaline and tryptanthrin derivatives de that an oxime group was essential for JNK inhibition, and compounds lackin had little or no effect on JNK activity [49,50,53,54]. Therefore, we examined t adding different substituents to the tetracyclic scaffold of tryptanthrin while m the oxime group. JNK binding and inhibitory activity of these TRYP-Ox deriv evaluated.
For compound synthesis, substituted isatins 1a-l and isatoic anhydride Table 1) were refluxed in boiling toluene (Scheme 1, path A) to obtain the cor ketones 3a-n. Poor yields were observed when electron-withdrawing substi present in position 8 (NO2, Br), and only traces of target compounds we when starting with 7-substituted isatins.

Synthesis of Tryptanthrin Derivatives
Our prior analysis of indenoquinoxaline and tryptanthrin derivatives demonstrated that an oxime group was essential for JNK inhibition, and compounds lacking the oxime had little or no effect on JNK activity [49,50,53,54]. Therefore, we examined the effect of adding different substituents to the tetracyclic scaffold of tryptanthrin while maintaining the oxime group. JNK binding and inhibitory activity of these TRYP-Ox derivatives were evaluated.
For compound synthesis, substituted isatins 1a-l and isatoic anhydrides 2a-g (see Table 1) were refluxed in boiling toluene (Scheme 1, path A) to obtain the corresponding ketones 3a-n. Poor yields were observed when electron-withdrawing substituents were present in position 8 (NO2, Br), and only traces of target compounds were obtained when starting with 7-substituted isatins.
The procedure shown in path B, described previously in [55], was used for synthesis of pseudo-symmetric ketones 3o-u. Corresponding target compounds were obtained in low yields, although low yields were compensated for by simplicity of the procedure. Using 7-methyl, 7-methoxy, or 5-nitro substituted isatins for synthesis via path A gave just traces of the corresponding target products.
Oximes 4a-u were synthesized from ketones 3a-u by refluxing in pyridine and then treating with excess of hydroxylamine hydrochloride. The general yields of this procedure were high. Minor impurities and traces of starting compounds were removed by washing with water or boiling toluene, and with recrystallization from 1,4-dioxane.
We also considered the possibility of obtaining active tryptanthrin derivatives by modifying the oxime fragment using O-substitution [50]. One of the methods for obtaining aryl (Ar) derivatives with an isonitroso group (Ar=N-OR) is the reaction of tryptanthrin with O-substituted hydroxylamine hydrochlorides. However, this reaction is limited in the choice of reagents in so far as O-substituted hydroxylamines mainly have nonionic groups (e.g., alkyl and halogen derivatives) [56]. It is worth noting that the dependence of reaction mechanisms on the substrates used and reaction conditions has not been previously described in the literature. The procedure shown in path B, described previously in [55], was used for synthesis of pseudo-symmetric ketones 3o-u. Corresponding target compounds were obtained in low yields, although low yields were compensated for by simplicity of the procedure. Using 7-methyl, 7-methoxy, or 5-nitro substituted isatins for synthesis via path A gave just traces of the corresponding target products.
Oximes 4a-u were synthesized from ketones 3a-u by refluxing in pyridine and then treating with excess of hydroxylamine hydrochloride. The general yields of this procedure were high. Minor impurities and traces of starting compounds were removed by washing with water or boiling toluene, and with recrystallization from 1,4-dioxane.
We also considered the possibility of obtaining active tryptanthrin derivatives by modifying the oxime fragment using O-substitution [50]. One of the methods for obtaining aryl (Ar) derivatives with an isonitroso group (Ar=N-OR) is the reaction of tryptanthrin with O-substituted hydroxylamine hydrochlorides. However, this reaction is limited in the choice of reagents in so far as O-substituted hydroxylamines mainly have nonionic groups (e.g., alkyl and halogen derivatives) [56]. It is worth noting that the de- O-substitution reactions were performed to obtain compounds 5a-e in a superbasic medium (KOH/DMSO), which revealed dependence of the reactivity of oximes on these conditions. We considered a number of acylating and alkylating reagents, such as methyl chloroformate, propargyl chloroformate, piperonyl chloride, ethyl ester of monochloroacetic acid, and diethyl ester of bromomalonic acid. Synthesis of oxime analogues 5c-e by acyl substitution in the KOH/DMSO system (Scheme 2, path A) failed. Presumably, this was due to the fact that DMSO, being an aprotic solvent, easily solvates potassium cations, while anions are only slightly solvated, which in turn leads to superbasic properties of the medium [57] and a decrease in stability of the intermediate in the acyl substitution reaction. Another reason why it was not possible to carry out reactions with chloroformates in the KOH/DMSO system may be the basic hydrolysis of formates to salts of organic acids and reaction products to the starting oximes. However, the acyl substitution occurred under milder conditions, such as in pyridine, which simultaneously acts as a solvent and a base (Scheme 2, path B). properties of the medium [57] and a decrease in stability of the intermediate in the acyl substitution reaction. Another reason why it was not possible to carry out reactions with chloroformates in the KOH/DMSO system may be the basic hydrolysis of formates to salts of organic acids and reaction products to the starting oximes. However, the acyl substitution occurred under milder conditions, such as in pyridine, which simultaneously acts as a solvent and a base (Scheme 2, path B).

Compd. R 5a
-CH2COOEt 5b Nucleophilic substitution in the synthesis of compounds 5a-b was facilitated in the superbasic medium, probably due to an easier formation of the oximate nucleophile. Completion of the reactions shown in Scheme 2 was monitored with TLC (eluent: chloroform).

Binding Affinity of the Compounds for JNK1-3
All compounds were evaluated for their ability to bind to JNK1-3 using the KI-NOMEscan platform [58]. This method was validated for the structurally related 11Hindeno[1,2-b]quinoxalin-11-one JNK inhibitor IQ-1S by an independent fluorescence polarization-based competition binding assay [59].
One compound (4h) had high affinity for JNK, with Kd values in the submicromolar range for all three JNK isoforms, whereas seven compounds (4a, 4d, 4e, 4i, 4k, 4m, and 4n) were selective for JNK3 and did not bind to either JNK1 or JNK2 ( Table 2). Note that four compounds with relatively high selectivity (selectivity index > 7) had a single substituent at R2 (i.e., NO2, Br, OMe, and phenyl in compounds 4a, 4b, 4d, and 4e, respec- Nucleophilic substitution in the synthesis of compounds 5a-b was facilitated in the superbasic medium, probably due to an easier formation of the oximate nucleophile. Completion of the reactions shown in Scheme 2 was monitored with TLC (eluent: chloroform).

Binding Affinity of the Compounds for JNK1-3
All compounds were evaluated for their ability to bind to JNK1-3 using the KINOMEscan platform [58]. This method was validated for the structurally related 11H-indeno[1,2b]quinoxalin-11-one JNK inhibitor IQ-1S by an independent fluorescence polarization-based competition binding assay [59].
One compound (4h) had high affinity for JNK, with K d values in the submicromolar range for all three JNK isoforms, whereas seven compounds (4a, 4d, 4e, 4i, 4k, 4m, and 4n) were selective for JNK3 and did not bind to either JNK1 or JNK2 ( Table 2). Note that four compounds with relatively high selectivity (selectivity index > 7) had a single substituent at R 2 (i.e., NO 2 , Br, OMe, and phenyl in compounds 4a, 4b, 4d, and 4e, respectively). Moving the methyl group in compound 4g, which had relatively high selectivity for JNK3, from R 1 to R 3 led to compound 4h, which had low selectivity but high binding activity for all JNK isoforms. Moving NO 2 from R 2 (compound 4a) to R 5 (compound 4n) or R 6 (compound 4m) decreased binding affinity for JNK3 and reduced selectivity. Introduction of an additional OMe group in compound 4d or an additional Me in compound 4g led to inactive compounds 4q and 4p, respectively. Similarly, introduction of additional Br in compounds 4a, 4b, and 4k, led to inactive compounds 4f, 4t, and 4s, respectively, which is probably due to the bulkiness of the molecule and decreased complementarity to the binding site. tional Br in compounds 4a, 4b, and 4k, led to inactive compounds 4f, 4t, and 4s, respectively, which is probably due to the bulkiness of the molecule and decreased complementarity to the binding site.
Among the O-substituted TRYP-Ox derivatives, compounds 5a and 5b were inactive (5a) or had low binding activity (5b) for all JNK isoforms, whereas the other three compounds 5c-e had relatively high affinity, with Kd values in the submicromolar range for all three JNK isoforms, indicating low JNK isoform selectivity (Table 2).

Activity of Compounds in Monocytic Cells
Prior to evaluation of the compounds in cell-based assays, we measured their cytotoxicity in human monocytic THP-1Blue and MonoMac-6 cells during a 24 h incubation. Seven compounds (4a, 4f, 4i, 4j, and 4r-t) were cytotoxic in both cell lines, with IC50 values ranging from 0.17 to 49.1 μM (Table 3). Compounds 4k and 4n exhibited cytotoxicity in THP-1Blue cells, and compounds 4c and 5e were cytotoxic for MonoMac-6 cells. Thus, these compounds were all excluded from subsequent testing in these cell lines.
The JNK pathway can be activated through Toll-like receptor 4 (TLR4), leading to the activation of transcription factors NF-κB and AP-1 [60,61]. Thus, to assess the antiinflammatory activity of our derivatives, the remaining non-cytotoxic compounds were Molecules 2023, 28, x FOR PEER REVIEW 7 of 26

Activity of Compounds in Monocytic Cells
Prior to evaluation of the compounds in cell-based assays, we measured their cytotoxicity in human monocytic THP-1Blue and MonoMac-6 cells during a 24 h incubation. Seven compounds (4a, 4f, 4i, 4j, and 4r-t) were cytotoxic in both cell lines, with IC50 values ranging from 0.17 to 49.1 μM (Table 3). Compounds 4k and 4n exhibited cytotoxicity in THP-1Blue cells, and compounds 4c and 5e were cytotoxic for MonoMac-6 cells. Thus, these compounds were all excluded from subsequent testing in these cell lines.
The JNK pathway can be activated through Toll-like receptor 4 (TLR4), leading to the activation of transcription factors NF-κB and AP-1 [60,61]. Thus, to assess the antiinflammatory activity of our derivatives, the remaining non-cytotoxic compounds were evaluated for their ability to inhibit lipopolysaccharide (LPS)-induced NF-κB/AP-1 reporter activity and IL-6 production in THP-1Blue and MonoMac-6 cells, respectively. Compounds 4c-e, which contain ethyl, methoxy, and phenyl groups at R2, and com- Among the O-substituted TRYP-Ox derivatives, compounds 5a and 5b were inactive (5a) or had low binding activity (5b) for all JNK isoforms, whereas the other three compounds 5c-e had relatively high affinity, with K d values in the submicromolar range for all three JNK isoforms, indicating low JNK isoform selectivity (Table 2).

Activity of Compounds in Monocytic Cells
Prior to evaluation of the compounds in cell-based assays, we measured their cytotoxicity in human monocytic THP-1Blue and MonoMac-6 cells during a 24 h incubation. Seven compounds (4a, 4f, 4i, 4j, and 4r-t) were cytotoxic in both cell lines, with IC 50 values ranging from 0.17 to 49.1 µM (Table 3). Compounds 4k and 4n exhibited cytotoxicity in THP-1Blue cells, and compounds 4c and 5e were cytotoxic for MonoMac-6 cells. Thus, these compounds were all excluded from subsequent testing in these cell lines. Table 3. Summary of compound cytotoxic activity and their inhibitory effects on LPS-induced NF-κB/AP-1 transcriptional activity in THP-1Blue cells and IL-6 production in MonoMac-6 cells. Compd.

IL-6 Production in MonoMac-6
Cytotoxicity in MonoMac-6 The JNK pathway can be activated through Toll-like receptor 4 (TLR4), leading to the activation of transcription factors NF-κB and AP-1 [60,61]. Thus, to assess the antiinflammatory activity of our derivatives, the remaining non-cytotoxic compounds were evaluated for their ability to inhibit lipopolysaccharide (LPS)-induced NF-κB/AP-1 reporter activity and IL-6 production in THP-1Blue and MonoMac-6 cells, respectively. Compounds 4c-e, which contain ethyl, methoxy, and phenyl groups at R 2 , and compound 4h, which has a methyl group at R 3 , were the most potent inhibitors of NF-κB/AP-1 reporter activity in THP-1Blue cells, with IC 50 values < 2 µM (Table 3). As an example, the dose-dependent inhibition of LPS-induced NF-κB/AP-1 reporter activity by compounds 4d and 4e is shown in Figure 2A.
olecules 2023, 28, x FOR PEER REVIEW Alkaline Phosphatase (A655) IL-6 (pg/ml) Figure 2. Effect of compounds 4d and 4e on NF-κB/AP-1 activity and IL-6 production. Pan THP-1Blue cells were pretreated with the indicated compounds or DMSO for 30 min, follow addition of 250 ng/mL LPS or buffer for 24 h. NF-kB/AP-1 activity was monitored by meas secreted alkaline phosphatase activity spectrophotometrically in the cell supernatants (absor at 655 nm). Panel (B) MonoMac-6 cells were pretreated with the indicated compounds or D for 30 min, followed by addition of 250 ng/mL LPS or buffer for 24 h. Production of IL-6 in t pernatants was evaluated with ELISA. The data in each panel are presented as the mean ± S triplicate samples from one experiment that is representative of three independent experimen Six of the compounds (4d, 4e, 4h, 4k, 4l, and 4o) were also potent inhibitors o production in MonoMac-6 cells, with IC50 values < 1 μM. As an example, the dependent inhibition of LPS-induced IL-6 production by compounds 4d and 4e is sh in Figure 2B. Note that 4d and 4e were highly specific for JNK3, whereas compoun Six of the compounds (4d, 4e, 4h, 4k, 4l, and 4o) were also potent inhibitors of IL-6 production in MonoMac-6 cells, with IC 50 values < 1 µM. As an example, the dosedependent inhibition of LPS-induced IL-6 production by compounds 4d and 4e is shown in Figure 2B. Note that 4d and 4e were highly specific for JNK3, whereas compound 4h was a pan-JNK inhibitor. Thus, these three compounds were selected for subsequent analysis in MonoMac-6 cells for their ability to inhibit LPS-induced c-Jun phosphorylation.
MonoMac-6 cells were pretreated with compounds 4d, 4e, 4h, or a structural analog IQ-1S with demonstrated potent JNK inhibitory activity as a positive control [53]. After 30 min pretreatment with the compounds, the cells were stimulated with LPS (500 ng/mL), and the level of phospho-c-Jun (S63) was determined by a sandwich ELISA. All of these compounds dose-dependently inhibited c-Jun phosphorylation (Figure 3), directly confirming JNK inhibition.
ecules 2023, 28, x FOR PEER REVIEW Figure 3. Effect of the compounds 4d, 4e, 4h, and IQ-1S on LPS-induced c-Jun phospho Human MonoMac-6 monocytic cells were pretreated with the indicated concentrations pounds or 0.5% DMSO for 30 min, followed by treatment with LPS (500 ng/mL) or contro for another 30 min. The cells were lysed, and the lysates were analyzed with ELISA for pho Jun (Ser63) and total c-Jun. The data are represented as the ratio of phospho-c-Jun to tot The data are presented as the mean ± S.D. of triplicate samples from one experiment that sentative of three independent experiments. * Significant differences (p < 0.05) with LPS con

Molecular Modeling
To gain insight into interactions of the investigated compounds with JNK plain some of the observations made in our structure-activity relationship analy performed molecular docking of selected compounds and TRYP-Ox into the JNK ing site (PDB: 1PMV) using Molegro Virtual Docker 6.0 (MVD) software. Note th compounds exist as mixtures of Z and E isomers with respect to the exocyclic C=N and these geometric isomers are prone to interconversion [62,63]. MolDock d scores for the docking poses of both isomers of these compounds are shown in Ta   Table 4. MolDock docking scores (DS) for the best docking poses of compounds into the site of JNK3 (PDB: 1PMV).

Molecular Modeling
To gain insight into interactions of the investigated compounds with JNK and explain some of the observations made in our structure-activity relationship analysis, we performed molecular docking of selected compounds and TRYP-Ox into the JNK3 binding site (PDB: 1PMV) using Molegro Virtual Docker 6.0 (MVD) software. Note that these compounds exist as mixtures of Z and E isomers with respect to the exocyclic C=N bond, and these geometric isomers are prone to interconversion [62,63]. MolDock docking scores for the docking poses of both isomers of these compounds are shown in Table 4. Compounds 4a (Z), 4b (Z), and 4d (E) had the highest docking scores and were located within the JNK3 binding site, similar to the co-crystallized pan-JNK inhibitor SP600125. As an example, see the docking pose of the methoxy derivative 4d (E) in Figure 4A, where it forms a strong H-bond to Lys93 with its oxime oxygen atom. In this binding mode, the quinazolone moiety coincides with the experimentally determined position of SP600125 in PDB structure 1PMV [64]. In contrast, the inactive compound 4q (Z), which contains two methoxy groups and did not have affinity for any of the three JNK isoforms, had a different location in the JNK3 binding site. As shown in Figure 4B, molecular docking results suggested that compound 4q (Z) could be H-bonded to Ser193 and deviate significantly from the plane of SP600125. This difference is likely due to steric effects introduced by adding the second methoxy group in the tryptanthrin scaffold. In the docking pose of compound 4d (E), the benzene ring in the azaindene moiety is located near Met149, Asp150, and Ala151 of JNK ( Figure 4A), and the additional methoxy substituent in this ring would cause a steric clash with neighboring residues. Hence, compound 4q (Z) should be shifted and rotated within the binding site with respect to compound 4d (E), which leads to a lower DS value (Table 4). We also conducted molecular docking of the compounds found to be most po in the cell-based assays (4c-e,h; see Table 3) to evaluate their relative positions within JNK3 binding site. The superimposed final docking poses for these compounds shown in Figure 5 and show that these compounds occupy approximately the same of space in binding to the enzyme. Additionally, they overlap significantly with the p tion of co-crystallized SP600125 present in the PDB 1PMV structure. We also conducted molecular docking of the compounds found to be most potent in the cell-based assays (4c-e,h; see Table 3) to evaluate their relative positions within the JNK3 binding site. The superimposed final docking poses for these compounds are shown in Figure 5 and show that these compounds occupy approximately the same area of space in binding to the enzyme. Additionally, they overlap significantly with the position of co-crystallized SP600125 present in the PDB 1PMV structure. Molecules 2023, 28, x FOR PEER REVIEW 13 of 26 To evaluate the relative energies of geometric isomers and the barrier for Z,Eisomerization, we performed DFT calculations of relative Gibbs free energies for Z-and E-isomers of compound 4d in DMSO using the M06-2X functional, which is suitable for evaluation of thermochemical properties of organic compounds [65]. We found that the E-isomer had a slightly higher thermodynamic stability, with its Gibbs energy being 0.96 kcal/mol lower than that of the Z-isomer. Using the DFT method, we also found that E→Z isomerization occurs via in-plane inversion of the oxime nitrogen atom, similar to other molecules containing a C=N bond [66]. With the small difference in Gibbs energies indicated above, both isomers can be present in the solution [67]. However, the calculated isomerization barrier ΔG ≠ for oxime 4d was 49.5 kcal/mol in DMSO, indicating that isomerization in solution should be a slow process in accordance with one of the isomers predominating in the synthesized samples (see the NMR data in Section 3). However, isomerization may occur during interaction of the investigated tryptanthrin oximes with the JNK binding site. Z,E-isomerization of these oximes in solution and on binding to the enzyme is a subject of our future studies.

Chemistry
Starting materials for the synthesis of 4a-u were obtained from Enamine (Kiev, Ukraine), while starting materials for synthesis of 5a-e were obtained from Merck. Tryptanthrin (indolo[2,1-b]quinazolin-6,12-dione) was purchased from Combi-Blocks (San Diego, CA, USA). Reaction progress was monitored by thin-layer chromatography (TLC) with UV detection using pre-coated silica gel 60, F254 plates (Merck, Rahway, NJ, USA). Structures of the synthesized compounds were confirmed on the basis of analytical and spectral data. The melting points (mp) were determined using an electrothermal Mel-Temp capillary melting point apparatus and an SMP30 melting point apparatus, with a heating rate of 3.0 °C/min. Elemental analysis was performed with a Carlo Erba instrument. LC-HRMS analysis was performed on an Agilent chromatograph with a 6538 UHD Accurate-Mass Q-TOF detector (ESI in positive mode). EI-MS spectra were obtained from a MX-1321 instrument (Tsourse = 220 °C, Eionisation = 70 eV). NMR spectra were recorded on Bruker spectrometers (operating frequencies: 400 and 600 MHz for 1 H, 100 and 150 MHz for 13 C, and 280 MHz for 19 F). IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer with KBr pellets. To evaluate the relative energies of geometric isomers and the barrier for Z,E-isomerization, we performed DFT calculations of relative Gibbs free energies for Zand E-isomers of compound 4d in DMSO using the M06-2X functional, which is suitable for evaluation of thermochemical properties of organic compounds [65]. We found that the E-isomer had a slightly higher thermodynamic stability, with its Gibbs energy being 0.96 kcal/mol lower than that of the Z-isomer. Using the DFT method, we also found that E→Z isomerization occurs via in-plane inversion of the oxime nitrogen atom, similar to other molecules containing a C=N bond [66]. With the small difference in Gibbs energies indicated above, both isomers can be present in the solution [67]. However, the calculated isomerization barrier ∆G = for oxime 4d was 49.5 kcal/mol in DMSO, indicating that isomerization in solution should be a slow process in accordance with one of the isomers predominating in the synthesized samples (see the NMR data in Section 3). However, isomerization may occur during interaction of the investigated tryptanthrin oximes with the JNK binding site. Z,E-isomerization of these oximes in solution and on binding to the enzyme is a subject of our future studies.

Chemistry
Starting materials for the synthesis of 4a-u were obtained from Enamine (Kiev, Ukraine), while starting materials for synthesis of 5a-e were obtained from Merck. Tryptanthrin (indolo[2,1-b]quinazolin-6,12-dione) was purchased from Combi-Blocks (San Diego, CA, USA). Reaction progress was monitored by thin-layer chromatography (TLC) with UV detection using pre-coated silica gel 60, F254 plates (Merck, Rahway, NJ, USA). Structures of the synthesized compounds were confirmed on the basis of analytical and spectral data. The melting points (mp) were determined using an electrothermal Mel-Temp capillary melting point apparatus and an SMP30 melting point apparatus, with a heating rate of 3.0 • C/min. Elemental analysis was performed with a Carlo Erba instrument. LC-HRMS analysis was performed on an Agilent chromatograph with a 6538 UHD Accurate-Mass Q-TOF detector (ESI in positive mode). EI-MS spectra were obtained from a MX-1321 instrument (T sourse = 220 • C, E ionisation = 70 eV). NMR spectra were recorded on Bruker spectrometers (operating frequencies: 400 and 600 MHz for 1 H, 100 and 150 MHz for 13 C, and 280 MHz for 19 F). IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer with KBr pellets.

Analysis of AP-1/NF-κB Activation
Activation of AP-1/NF-κB was measured using an alkaline phosphatase reporter gene assay in THP1-Blue cells. Human monocytic THP-1Blue cells were stably transfected with a secreted embryonic alkaline phosphatase gene that was under the control of a promoter inducible by AP-1/NF-κB. THP-1Blue cells (2 × 10 5 cells/well) were pretreated with the test compound or DMSO (1% final concentration) for 30 min, followed by addition of 250 ng/mL LPS (from Escherichia coli K-235; Sigma Chemical Co., St. Louis, MO, USA) for 24 h, and alkaline phosphatase activity was measured in cell supernatants using QUANTI-Blue mix (InvivoGen) with absorbance at 655 nm and compared with positive control samples (LPS). The concentrations of compound that caused 50% inhibition of the AP-1/NF-κB reporter activity (IC50) were calculated.

Cytotoxicity Assay
Cytotoxicity was analyzed with a CellTiter-Glo Luminescent Cell Viability Assay Kit from Promega (Madison, WI, USA), according to the manufacturer's protocol. Cells were treated with the compound under investigation and cultivated for 24 h. After treatment, the cells were allowed to equilibrate to room temperature for 30 min, substrate was added, and the luminescence measured using a Fluoroscan Ascent FL (Thermo Fisher Scientific, Waltham, MA, USA). The cell IC 50 values were calculated by plotting the percentage inhibition against the logarithm of inhibitor concentration (at least five points).

Analysis of c-Jun Phosphorylation
MonoMac-6 cell lysates were prepared using Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA). The phospho-c-Jun content was measured with ELISA using the PathScan Phospho-c-Jun (Ser63) Sandwich ELISA Kit II (Cell Signaling Technology, USA), and the data are shown as the ratio of phospho-c-Jun vs. total c-Jun, which was measured using the FastScanTM Total c-Jun ELISA kit (Cell Signaling Technology).

Molecular Docking
The geometry of JNK3 was obtained by downloading its crystal structure from the Protein Data Bank (PDB entry code 1PMV) into Molegro 6.0 software (Molegro ApS, Aarhus, Denmark). All of the solvent molecules were removed, and the search space was chosen to be a sphere centered on the co-crystallized ligand present in the corresponding PDB structure. The radius of the sphere was 10 Å, which completely encompassed the cocrystallized ligand and the JNK3 binding site. Side chains of all JNK amino acid residues within the corresponding sphere were regarded as flexible during docking. The number of such residues was 39. The flexible residues were treated with the default settings of the "Setup Sidechain Flexibility" tool in Molegro, and a softening parameter of 0.7 was applied during flexible docking, according to the standard protocol using the Molegro Virtual Docker 6.0 (MVD). Before docking, structures of compounds were pre-optimized using HyperChem 7 software (HyperCube, Gainesville, FL, USA) with the MM+ force field, and saved in the Tripos MOL2 format (Tripos, St. Louis, MO, USA). The ligand structures were imported into MVD. The options "Create explicit hydrogens", "Assign charges (calculated by MVD)", and "Detect flexible torsions in ligands" were enabled during importing. Appropriate protonation states of the ligands were also automatically generated at this step. Each ligand was subjected to 30 docking runs with respect to the JNK3 structure using MVD software. The docking pose with the lowest MolDock docking score [69] was selected for each ligand and analyzed using the built-in tools of MVD.

DFT Calculations
ORCA 5.0 computational chemistry software [70] was used for density functional theory (DFT) calculations of compound 4d. Geometry optimizations were performed using the M06-2X functional [65] with the 6-311++G(2d,2p) basis set. Normal vibration analysis was performed for the optimized geometries to establish the nature of the stationary points (i.e., minimum or transition state). Transition state (TS) geometry was obtained using the climbing image nudged elastic band (CI-NEB) approach [71] with the PBEh-3c composite method [72] followed by TS optimization at the M06-2X/6-311++G(2d,2p) level of theory. During CI-NEB calculations, eight intermediate images on the isomerization path were used. All calculations were performed using the conductor-like polarizable continuum solvation model (CPCM) [73] with DMSO as the solvent.

Conclusions
Based on a tryptanthrin scaffold, we designed and synthesized a series of substituted TRYP-Ox derivatives in an effort to obtain novel JNK3-selective inhibitors. As determined in our JNK1-3 binding assays, we were successful in identifying at least two highly selective JNK3 inhibitors. These compounds inhibited LPS-induced NF-κB/AP-1 transcription activity in THP-1Blue cells and IL-6 production in MonoMac-6 monocytic cells in the low micromolar range. In addition, the JNK3-selective inhibitors 4d and 4e decreased LPSinduced c-Jun phosphorylation in MonoMac-6 cells, directly confirming JNK inhibition. Molecular docking confirmed the binding interaction of the active compounds in the JNK3 catalytic site. These compounds may be useful for investigating the role of JNK3 in various models of pathologies such as Alzheimer's disease, Parkinson's disease, and cancer.