Anti-Inflammatory Activity of Pyrazolo[1,5-a]quinazolines

Chronic inflammation contributes to a number of diseases. Therefore, control of the inflammatory response is an important therapeutic goal. To identify novel anti-inflammatory compounds, we synthesized and screened a library of 80 pyrazolo[1,5-a]quinazoline compounds and related derivatives. Screening of these compounds for their ability to inhibit lipopolysaccharide (LPS)-induced nuclear factor κB (NF-κB) transcriptional activity in human THP-1Blue monocytic cells identified 13 compounds with anti-inflammatory activity (IC50 < 50 µM) in a cell-based test system, with two of the most potent being compounds 13i (5-[(4-sulfamoylbenzyl)oxy]pyrazolo[1,5-a]quinazoline-3-carboxamide) and 16 (5-[(4-(methylsulfinyl)benzyloxy]pyrazolo[1,5-a]quinazoline-3-carboxamide). Pharmacophore mapping of potential targets predicted that 13i and 16 may be ligands for three mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 2 (ERK2), p38α, and c-Jun N-terminal kinase 3 (JNK3). Indeed, molecular modeling supported that these compounds could effectively bind to ERK2, p38α, and JNK3, with the highest complementarity to JNK3. The key residues of JNK3 important for this binding were identified. Moreover, compounds 13i and 16 exhibited micromolar binding affinities for JNK1, JNK2, and JNK3. Thus, our results demonstrate the potential for developing lead anti-inflammatory drugs based on the pyrazolo[1,5-a]quinazoline and related scaffolds that are targeted toward MAPKs.


Introduction
Inflammation is an essential process that protects the host from harmful pathogens or irritants and can be acute, lasting for a short period of time, or chronic and lasting much longer [1,2].Notably, chronic, low-grade inflammation has been shown to contribute to a variety of diseases, including cardiovascular disease [3], cancer [4], type 2 diabetes [5], Alzheimer's disease [6], arthritis, and many other chronic inflammatory conditions [5].Thus, it is essential that effective anti-inflammatory therapeutics are developed to help control chronic inflammation and the onset or progression of these diseases [7].The current therapeutics for treating inflammation generally focus on suppressing, blocking, or inhibiting proinflammatory mediators of inflammation, such as prostaglandins, leukotrienes, and cytokines [8].While many of these treatments are effective, it is evident that chronic inflammation continues to be a major component associated with the pathogenesis of chronic inflammatory diseases and that new therapeutic interventions with fewer adverse effects need to be developed.Indeed, the pipeline of new anti-inflammatory therapeutics targeting additional pathways other than those being currently targeted is quite limited.Nevertheless, recent work on the development of new resolving mediators has been a success [9,10].
Whether acute or chronic, inflammation involves the activation and/or recruitment of inflammatory leukocytes to sites of infection or injury.The acute inflammatory response is initiated by resident phagocytes, such as macrophages, dendritic cells, and mast cells, but soon results in the recruitment of large numbers of neutrophils, which are the primary leukocytes involved in acute inflammatory responses [11,12].If the acute response is not resolved, chronic inflammation can occur, which lasts much longer and primarily involves macrophages, as well as lymphocytes and plasma cells, which are able to produce a variety of bioactive inflammatory mediators that can cause cell and tissue damage [13].Thus, targeting inflammatory responses of leukocytes, such as neutrophils and macrophages, represents a reasonable approach to treating chronic inflammation.
Current anti-inflammatory therapeutics focus mainly on reducing the production or activity of inflammatory eicosanoids or certain cytokines or blocking their receptors, while others can block lymphocyte trafficking into tissues, prevent the binding of monocytelymphocyte costimulatory molecules, or reduce the number of circulating B lymphocytes [14][15][16].In addition, the potential of targeting several biochemical pathways and multiple enzymes involved in inflammation, including neuroinflammation, has been reported [17].For example, we have synthesized and characterized a number of compounds with anti-inflammatory activity that inhibit mitogen-activated protein kinase (MAPK) pathways, especially the c-Jun N-terminal kinase (JNK) pathway [18,19]; antagonize N-formyl peptide chemotactic receptors (FPRs) [20]; and inhibit human neutrophil elastase [21].
In addition, we have identified a number of pyridazinone-like compounds with antiinflammatory activity from a large compound library, suggesting that the pyridazinone scaffold could be useful for the development of novel anti-inflammatory therapeutics [22].
As indicated above, our research group has been investigating a number of biologically active polyheterocycles, and we have created a large library of compounds, including both final products and synthetic intermediates.Here, we selected 80 nitrogen (poly)heterocycles derivatives, which were mainly pyrazolo [1,5-a]quinazolines (Figure 1).These compounds were selected based on our previous work in this field, as well as recent publications reporting examples of anti-inflammatory agents with similar nitrogen polycyclic scaffolds [23][24][25][26].For example, the pyrazolo [5,1-b]quinazoline A (designated as 3j in the original publication [23], Figure 2) is a very potent cyclooxygenase 2 (COX-2) inhibitor (IC 50 = 47 nM), which had about 14-fold selectivity toward COX-2 versus COX-1 but also inhibited 5lipoxygenase (5-LOX), with an IC 50 of 2.3 µM.In an in vivo carrageenan-induced paw edema model, 10 mg/kg of compound A reduced edema by 39% and did not exhibit gastric ulcerogenic effects.Another recently reported polyheterocyclic anti-inflammatory compound is the purine derivative B (designated as 9j in the original publication [25], Figure 2), which was reported to be a potent dual inhibitor of Janus 2 tyrosine kinase and bromodomain-containing protein 4 (JAK2/BRD4) with IC 50 values of 22 and 13 nM, respectively, and also downregulated the NF-κB pathway.In vivo studies in an acute ulcerative model demonstrated that 60 mg/kg of compound B was able to relieve the symptom of ulcerative colitis with minimal adverse effects.
Molecules 2024, 29, x FOR PEER REVIEW 2 of 26 limited.Nevertheless, recent work on the development of new resolving mediators has been a success [9,10].Whether acute or chronic, inflammation involves the activation and/or recruitment of inflammatory leukocytes to sites of infection or injury.The acute inflammatory response is initiated by resident phagocytes, such as macrophages, dendritic cells, and mast cells, but soon results in the recruitment of large numbers of neutrophils, which are the primary leukocytes involved in acute inflammatory responses [11,12].If the acute response is not resolved, chronic inflammation can occur, which lasts much longer and primarily involves macrophages, as well as lymphocytes and plasma cells, which are able to produce a variety of bioactive inflammatory mediators that can cause cell and tissue damage [13].Thus, targeting inflammatory responses of leukocytes, such as neutrophils and macrophages, represents a reasonable approach to treating chronic inflammation.
Current anti-inflammatory therapeutics focus mainly on reducing the production or activity of inflammatory eicosanoids or certain cytokines or blocking their receptors, while others can block lymphocyte trafficking into tissues, prevent the binding of monocytelymphocyte costimulatory molecules, or reduce the number of circulating B lymphocytes [14][15][16].In addition, the potential of targeting several biochemical pathways and multiple enzymes involved in inflammation, including neuroinflammation, has been reported [17].For example, we have synthesized and characterized a number of compounds with antiinflammatory activity that inhibit mitogen-activated protein kinase (MAPK) pathways, especially the c-Jun N-terminal kinase (JNK) pathway [18,19]; antagonize N-formyl peptide chemotactic receptors (FPRs) [20]; and inhibit human neutrophil elastase [21].In addition, we have identified a number of pyridazinone-like compounds with anti-inflammatory activity from a large compound library, suggesting that the pyridazinone scaffold could be useful for the development of novel anti-inflammatory therapeutics [22].
As indicated above, our research group has been investigating a number of biologically active polyheterocycles, and we have created a large library of compounds, including both final products and synthetic intermediates.Here, we selected 80 nitrogen (poly)heterocycles derivatives, which were mainly pyrazolo[1,5-a]quinazolines (Figure 1).These compounds were selected based on our previous work in this field, as well as recent publications reporting examples of anti-inflammatory agents with similar nitrogen polycyclic scaffolds [23][24][25][26].For example, the pyrazolo[5,1-b]quinazoline A (designated as 3j in the original publication [23], Figure 2) is a very potent cyclooxygenase 2 (COX-2) inhibitor (IC50 = 47 nM), which had about 14-fold selectivity toward COX-2 versus COX-1 but also inhibited 5-lipoxygenase (5-LOX), with an IC50 of 2.3 M.In an in vivo carrageenan-induced paw edema model, 10 mg/kg of compound A reduced edema by 39% and did not exhibit gastric ulcerogenic effects.Another recently reported polyheterocyclic anti-inflammatory compound is the purine derivative B (designated as 9j in the original publication [25], Figure 2), which was reported to be a potent dual inhibitor of Janus 2 tyrosine kinase and bromodomain-containing protein 4 (JAK2/BRD4) with IC50 values of 22 and 13 nM, respectively, and also downregulated the NF-κB pathway.In vivo studies in an acute ulcerative model demonstrated that 60 mg/kg of compound B was able to relieve the symptom of ulcerative colitis with minimal adverse effects.For the biological screening of the selected library of compounds, we evaluated their effects on lipopolysaccharide (LPS)-induced NF-κB transcriptional activity in THP-1Blue monocyte/macrophages, since NF-κB activation is an important component of many inflammatory responses [27,28].The complete list and structures of all 80 selected and screened compounds can be found in the Supplementary Materials (Supplementary Tables S1-S3).Two of the most potent compounds identified by our biological screen (13i and 16) were then evaluated in silico to identify potential targets using PharmMapper, which suggested that ERK2, JNK3, and p38 MAPK could be likely biotargets.Furthermore, molecular docking of compounds 13i and 16 into the binding sites of these kinases using Rose a docking suggested high affinity binding interactions, confirming that these MAPKs are likely targets for these anti-inflammatory compounds.

Synthesis
We report here the procedures for the synthesis of 65 new compounds that were included in the screening library.For the 15 compounds in screening library that were already published, we provide the appropriate reference (chemical structures of all compounds and relevant references are presented in Supplementary Tables S1-S3) [29][30][31][32].As mentioned above, most of the compounds selected for this study are tricycles with a pyrazolo [1,5-a]quinazoline scaffold (abbreviated below as PQ), and their synthesis is outlined in Schemes 1-9.The synthesis of the other compounds with pyrazolo[1,5-a]pyrido [3,4e]pyrimidine and pyrazolo [1,5-a]pyrimido [5,4-e]pyrimidine nuclei, as well as the pyrazolo derivatives, is described in Scheme 10.

Synthesis
We report here the procedures for the synthesis of 65 new compounds that were included in the screening library.For the 15 compounds in screening library that were already published, we provide the appropriate reference (chemical structures of all compounds and relevant references are presented in Supplementary Tables S1-S3) [29][30][31][32].As mentioned above, most of the compounds selected for this study are tricycles with a pyrazolo [1,5-a]quinazoline scaffold (abbreviated below as PQ), and their synthesis is outlined in Schemes 1-9.The synthesis of the other compounds with pyrazolo[1,5a]pyrido [3,4e]pyrimidine and pyrazolo [1,5-a]pyrimido [5,4-e]pyrimidine nuclei, as well as the pyrazolo derivatives, is described in Scheme 10.
Further studies using NMR techniques such as HSQC and HMBC (see Supplementary Figures S60 and S61) were performed on compound 7b, which again allowed us to correctly assign a structure to the other N-alkylated compounds of type 7.The methylthio group of compounds 7b and 7f was oxidized to form the sulfoxide derivatives (-SOMe) with iodic acid/acetone (compounds 9, 10), but in the case of the 3-unsubstituted derivative 7f, even the iodination at position 3 occurred (9).Alternatively, the treatment of 7f with OXONE ® /water/methanol yielded the corresponding 4-sulfonylmethylbenzyl (-SO2Me) derivative 8.In Scheme 2, we show that when COOEt, CN, or H are present in position 3 of the PQ scaffold, the alkylation reactions only yielded the corresponding 4-N-alkylates.In contrast, when a CONH2 group was present in position 3 (5-oxo-4,5-dihydro-PQ-3-carboxamide), the same reaction (dry DMF/Cs2CO3/ArBr) was generally regiospecific toward the 5-O position (see Scheme 3).In fact, a mixture of the two regioisomers (5O-R, 13a, c and 4N-R, 14a, c) was achieved only when methyl iodide and 4-methylthiobenzyl bromide were used as alkylating reagents.Even in this case, an in-depth spectroscopic study was performed on compound 14c to assign the correct structures to the two isomers.The eval-   ).To confirm that alkylation always occurred on the nitrogen at position 4 under these conditions, we synthesized the isomer of 7a (i.e., the 5-O-methyl derivative 12 (ethyl 5-methoxy-PQ-3-carboxylate) [42]).Treatment of 4a with POCl 3 /PCl 5 resulted in the 5-chloro derivative 11, which, in turn, was transformed into the easily recovered 12 with dry DMF/t-BuOK/methanol.Through this reaction, we assigned the correct structure to the final compound 7a.Further studies using NMR techniques such as HSQC and HMBC (see Supplementary Figures S60 and S61) were performed on compound 7b, which again allowed us to correctly assign a structure to the other N-alkylated compounds of type 7.The methylthio group of compounds 7b and 7f was oxidized to form the sulfoxide derivatives (-SOMe) with iodic acid/acetone (compounds 9, 10), but in the case of the 3-unsubstituted derivative 7f, even the iodination at position 3 occurred (9).Alternatively, the treatment of 7f with OXONE ® /water/methanol yielded the corresponding 4-sulfonylmethylbenzyl (-SO 2 Me) derivative 8.

Biological Activity
All compounds (see complete list in Supplementary Tables S1-S3) were scree their ability to inhibit NF-κB/AP-1 reporter activity in THP-1Blue cells, which is a m of their anti-inflammatory activity since this pathway is essential to the inflamma sponse [45,46].Although we evaluated a considerable number of compounds, o In Scheme 2, we show that when COOEt, CN, or H are present in position 3 of the PQ scaffold, the alkylation reactions only yielded the corresponding 4-N-alkylates.In contrast, when a CONH 2 group was present in position 3 (5-oxo-4,5-dihydro-PQ-3carboxamide), the same reaction (dry DMF/Cs 2 CO 3 /ArBr) was generally regiospecific toward the 5-O position (see Scheme 3).In fact, a mixture of the two regioisomers (5O-R, 13a, c and 4N-R, 14a, c) was achieved only when methyl iodide and 4-methylthiobenzyl bromide were used as alkylating reagents.Even in this case, an in-depth spectroscopic study was performed on compound 14c to assign the correct structures to the two isomers.The evaluation of the monodimensional and bidimensional spectra (HSQC and HMBC) is included in Supplementary Figures S62 and S63.In all other cases, the 5-O-isomer was exclusively obtained (compounds 13b, d-i).Additionally, we obtained chemical confirmation of the 5-O-methylation in compound 13a by transforming 3a into the 5-[4-(methanesulphonyl)phenylmethoxy] derivative 15, which when treated with methanol/t-BuOK, resulting in 13a.The 5-(4-methylthiobenzyloxy) derivative, 13c, was oxidized in the presence of iodic acid/acetone or OXONE ® /methanol, and the two final products (sulfoxide) 16 and (sulfone) 17 were recovered in good yield.Likewise, the 5-benzyloxy derivative 13b was converted into the N-(dimethylamino)methylidene 3-carboxamide 18 (as a mixture of E and Z isomers, as indicated by TLC) by treating it with DMF-DMA, and the further cyclization to 1,2,4-triazole with hydrazine hydrate in acetic acid resulted in compound 19.

Biological Activity
All compounds (see complete list in Supplementary Tables S1-S3) were screened for their ability to inhibit NF-κB/AP-1 reporter activity in THP-1Blue cells, which is a measure of their anti-inflammatory activity since this pathway is essential to the inflammatory response [45,46].Although we evaluated a considerable number of compounds, only 13 were able to inhibit NF-κB/AP-1 activity with IC 50 values <50 µM, and they are shown in Table 1.As an example, the dose-dependent inhibition of LPS-induced NF-κB/AP-1 reporter activity by compounds 13i and 16 is shown in Figure 3.
Although no true structure-activity relationships could be identified, it was nevertheless possible to make general observations on some structural aspects of these compounds that seem important for activity.All compounds exhibiting some inhibitory activity in THP-1Blue cells were pyrazolo [1,5-a]quinazolines, while all the other tricyclics, bicyclics, and pyrazole derivatives tested were completely inactive, indicating that the pyrazolo[1,5a]quinazoline scaffold was the only appropriate structure among those that we tested.Moreover, it seems that among the three possible forms of the pyrazolo[1,5-a]quinazoline scaffold, the heteroaromatic form was most effective, as the 5-oxo-4,5-dihydropyrazolo[1,5a]quinazoline (26) and 4,5-dihydropyrazolo[1,5-a]quinazoline (58c) [47] nuclei were much less potent (IC 50 = 49.3 and 39.1 µM, respectively).Indeed, the heteroaromatic scaffold was present in eleven compounds exhibiting anti-inflammatory activity (IC 50 = 4.8-30.1 µM).For these products, the primary amide group at position 3 of the heteroaromatic scaffold appeared to be the best, although moderate activity was retained even when a CN group (compound 42a) or a 3-thienyl (compound 20) were present.On the other hand, ester or very bulky groups were not compatible with this biological activity.Regarding the substituent bonded to the oxygen at position 5, an increased size of the substituent was favorable for activity (e.g., 13a versus 13b with IC 50 = 24.4 and 4.81 µM, respectively), while the insertion of a chlorine atom or a nitro group at R7/R8 resulted in the maintenance of or a slight increase in activity.

Table 1. Summary of compound cytotoxic activity and inhibitory effects on LPS-induced NF-κB/AP-1 transcriptional activity in THP-1Blue cells.
their ability to inhibit NF-κB/AP-1 reporter activity in THP-1Blue cells, which is a measure of their anti-inflammatory activity since this pathway is essential to the inflammatory response [45,46].Although we evaluated a considerable number of compounds, only 13 were able to inhibit NF-κB/AP-1 activity with IC50 values <50 µM, and they are shown in Table 1.As an example, the dose-dependent inhibition of LPS-induced NF-κB/AP-1 reporter activity by compounds 13i and 16 is shown in Figure 3.Although no true structure-activity relationships could be identified, it was nevertheless possible to make general observations on some structural aspects of these compounds that seem important for activity.All compounds exhibiting some inhibitory activity in THP-1Blue cells were pyrazolo[1,5-a]quinazolines, while all the other tricyclics, bicyclics, and pyrazole derivatives tested were completely inactive, indicating that the pyrazolo[1,5-a]quinazoline scaffold was the only appropriate structure among those that we tested.Moreover, it seems that among the three possible forms of the pyrazolo[1,5a]quinazoline scaffold, the heteroaromatic form was most effective, as the 5-oxo-4,5-Figure 3. Effect of compounds 13i and 16 on NF-κB/AP-1 activity.THP-1Blue cells were pretreated with the indicated concentrations of compounds 13i (red symbols) and 16 (blue symbols) or DMSO control for 30 min, followed by the addition of 250 ng/mL of LPS or buffer for 24 h.NF-κB/AP-1 activity was monitored by measuring secreted alkaline phosphatase activity spectrophotometrically in the cell supernatants (absorbance at 655 nm).The data in each panel are presented as the mean ± S.D. of triplicate samples from one experiment that is representative of three independent experiments.

Identification of Potential Protein Targets for Compounds 13i and 16
We selected two of the most potent compounds for further characterization.To identify potential protein targets for 13i and 16, we performed reverse-pharmacophore mapping on the molecular structures of these compounds.PharmMapper compared a database of pharmacophore patterns with these compounds and generated target information, such as pharmacophoric characteristics and normalized fitness scores.The chemical structures of the compounds were submitted to the PharmMapper server, as mapping explicitly accounts for the three-dimensional structure of a molecule.The 30 top-ranked potential targets found by PharmMapper are shown in Supplementary Table S4 and only the kinase targets are shown in Table 2, as the PharmMapper analysis indicated that three MAPKs (ERK2, p38α MAPK, and JNK1/3) were among the potential targets for compounds 13i and 16.Indeed, MAPK signaling plays an important role in phagocyte/macrophage signal transduction cascades [48], and studies have shown that JNK and the p38 MAPK families of proteins are activated in response to phagocyte/macrophage priming/activation (reviewed in [49]).

Molecular Docking
According to the PharmMapper results, ERK2, JNK3, and p38α MAPK were among the potential biotargets for the investigated compounds.Thus, we performed a more sophisticated docking study of compounds 13i and 16 into the binding sites of these kinases using the ROSIE web server [50][51][52].In the PharmMapper database, the retrieved enzymes are represented by the PDB structures 1PME (ERK2), 1PMV (JNK3), and 1W7H (p38α).However, the 1PME structure corresponds to a mutant of ERK2 [53].Therefore, we used the non-mutated ERK2 (PDB code: 1TVO [54]) for the docking computations.Importantly, the Rosetta docking methodology implemented in ROSIE accounts for the flexibility of the side chains and backbone of the protein in the vicinity of the docked ligand.
The docking poses obtained with the lowest interface energies in the binding sites of the kinases for molecules 13i and 16 are shown in Figure 4. Notably, compounds 13i and 16 are anchored to the enzymes by a number of H bonds formed with the participation of different functional groups of the ligands (Table 3).
the non-mutated ERK2 (PDB code: 1TVO [54]) for the docking computations.Importantly, the Rose a docking methodology implemented in ROSIE accounts for the flexibility of the side chains and backbone of the protein in the vicinity of the docked ligand.
The docking poses obtained with the lowest interface energies in the binding sites of the kinases for molecules 13i and 16 are shown in Figure 4. Notably, compounds 13i and 16 are anchored to the enzymes by a number of H bonds formed with the participation of different functional groups of the ligands (Table 3).The H-bonding patterns of molecules 13i and 16 have similar structural features.Thus, protonated lysine residues contained in the binding sites of the investigated kinases (Lys114 in ERK2, Lys93 in JNK3, and Lys53 in p38α) form strong H bonds with the heteroatoms in both ligands.In the case of ERK2 and p38α, the deprotonated Glu71 and neutral Gln75, respectively, participate in H bonding with both 13i and 16.It should be noted that the sulfonamide moiety of molecule 13i and the sulfoxide group of 16 are H bonded to ERK2 and JNK3.The sulfonamide group of 13i also forms H bonds with p38α (Figure 3).These interactions promote the binding of the ligands to the kinases.The interface energy scores for the docking poses obtained for molecules 13i and 16 are presented in Table 3.The noticeably negative values of the interface energy scores indicate high affinities of these compounds to ERK2, JNK3, and p38α in accordance with the predictions of PharmMapper (see above).Nevertheless, the interaction of molecules 13i and 16 with JNK3, according to the ROSIE docking results, should be more prominent than with the other two kinases, in spite of the higher ranking of ERK2 and p38α in the more approximate PharmMapper data.
In addition to the 1PMV structure of JNK3 retrieved by PharmMapper that was used for the comparative docking with different kinases on the ROSIE server, there is another, more recent structure of JNK3 complexed with a pyrazole-containing ligand in the Protein Data Bank (PDB: 4WHZ [55]).Therefore, this structure was also used for docking molecules 13i and 16.For this purpose, the compounds were inserted into the active site with the AUTODOCK 4.1 program, and the conformation with the most favorable binding energy was selected for each complex.The JNK3-inhibitor complexes were further subject to full geometry optimization with the amber99sb force-field.The results were analyzed with a focus on H-bonding interactions (see Supplementary Table S5).It should be noted that Lys93 was found to be involved in H-bonding interactions with the investigated inhibitors using the 4WHZ protein structure, analogous to the results obtained with the 1PMV biotarget (Table 3).Additionally, compounds 13i and 16 have common H-bonding patterns with these two biotargets (Gln75, Lys93, and Asn152 for ligand 13i; Gln75, Lys93, and Asn194 for ligand 16).

Affinity of 13i and 16 for JNK1-3
To confirm the effectiveness of the predictions based on molecular modeling, compounds 13i and 16 were evaluated for their ability to bind to JNK1-3 using the KINOMEscan ATP site-dependent binding assay, which reflects the biologically relevant behavior of protein kinases [56].We found that both compounds bound to JNK1, JNK2, and JNK3, with K d values in the micromolar range (Table 4).Although compound 13b exhibited relatively high activity in THP-1Blue cells (IC 50 = 4.8 µM), this compound had a low solubility in DMSO and was not able to be tested in the binding assay.

Materials and Methods
All compound melting points were determined on a Büchi apparatus (New Castle, DE, USA) and are uncorrected.Extracts were dried over Na 2 SO 4 , and the solvents were removed under reduced pressure.Merck F-254 commercial plates (Merck, Durham, NC, USA) were used for analytical TLC to follow the course of the reactions.Silica gel 60 (Merck 70-230 mesh, Merck, Durham, NC, USA) was used for column chromatography. 1 H-NMR, 13 C-NMR, HSQC, and HMBC spectra were recorded on an Avance 400 instrument (Bruker Biospin Version 002 with SGU, Bruker Inc., Billerica, MA, USA).Chemical shifts (d) are given in parts per million (ppm), approximated to the nearest 0.01 ppm using the solvent as the internal standard.Coupling constants (J) are in Hz and were calculated by Top Spin 3.1 and approximated to 0.1 Hz.Data are reported as follows: chemical shift, multiplicity (exch, exchange; br, broad; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; or a combination of those, e.g., dd), integral, assignments, and coupling constant.Mass spectra (m/z) were recorded on a Varian 1200L ESI-MS triple quadrupole (Varian Inc., Walnut Creek, CA, USA) system in positive ion mode by injecting a 10 mg/L solution of each analyte dissolved in a mixture of mQ H 2 O/acetonitrile 1:1 v/v.All new compounds exhibited a purity >95%.Microanalyses indicated by the element symbols were performed with a Perkin-Elmer 260 elemental analyzer (Perkin-Elmer, Waltham, MA, USA) for C, H, and N, and they were within ±0.4% of the theoretical values.

General Procedure for Synthesizing Compounds 13b, i
A suspension of 4,5-dihydro-5-oxo-pyrazolo[1,5-a]quinazoline-3-carboxyamide 3a [57] (150 mg or 0.66 mmol in 3.0 mL of anhydrous DMF and 2.64 mmol anhydrous K 2 CO 3 ) was incubated at room temperature for 15 min.The appropriate substituted benzyl halide (0.99 mmol) was then added, and the reaction was heated at 50 • C for 2 h.After cooling, 20 mL of ice-cold water was added, and the precipitate formed was recovered by vacuum filtration and washed first with water, then ethanol, and finally with diethyl ether to obtain the desired compounds, which were purified by crystallization from the suitable solvent.To a solution of 176 mg (1 mmol) of HIO 3 in 11 mL of acetone/H 2 O (10:1), a small amount of tetra-n-butylammonium bromide (TBAB) was added while stirring for 5 min.Then, 0.25 mmol of compound 13c (the synthesis of 13c is reported in Supplementary Materials) was added, and the mixture was stirred at 80  was suspended in 10 mL of methylene chloride and 0.70 mmol of 4-toluenesulfonyl chloride, and 0.6 mL (in excess) of triethylamine was added.The reaction was maintained at reflux temperature for 2-3 h, and then the solvent was evaporated to dryness.The residue was dissolved in isopropyl alcohol and crystallized with the same solvent.Yield 97%, mp 203 • C; TLC eluent: toluene/ethyl acetate/acetic acid 8/2/1.5 v/v/v.A suspension of 8-chloro-5-oxo-4,5-dihydropyrazolo[1,5-a]quinazoline-3-carboxyamide 3b (the synthesis of 3b is reported in Supplementary Materials) (150 mg or 0.66 mmol in 3.0 mL of anhydrous DMF and 2.64 mmol of anhydrous K 2 CO 3 ) was incubated at room temperature for 15 min.The appropriate substituted halide (0.99 mmol) was then added, and the reaction was heated to 50 • C for 2 h.After cooling, 20 mL of ice-cold water was added, and the precipitate formed was recovered by vacuum filtration and purified by flash column chromatography using dichloromethane/methanol 10:0.5 (for 23a) or water/acetic acid 1:1 (for 23c) as the eluent.To a solution of 0.21 mmol of 3b in 3 mL of anhydrous toluene and 0.3 mL of dry DMF, 0.76 mmol (0.10 mL) of DMF-DMA was added.The reaction was refluxed for 2 h.After cooling, the precipitate formed was recovered by vacuum filtration to obtain compound 26.Yield 83%, mp 288-290 Compounds 3c or 3d (the synthesis of 3c and 3d is reported in Supplementary Materials) (0.35 mmol) were treated in 2.5 mL of anhydrous DMF and 0.35 mmol of K 2 CO 3 with stirring at room temperature for 15 min.Methyl iodide (0.70 mmol) was then added, and the reaction was heated at 80 • C for 1 h.After cooling, 20 mL of ice-cold water was added, and the precipitate formed was recovered by vacuum filtration.The crude compounds were purified by flash column chromatography using dichloromethane/methanol/acetic acid 97/3/03 v/v/v (for 33a) or toluene/ethyl acetate/acetic acid 8/2/1 v/v/v (for 33b) as the eluent.

Analysis of AP-1/NF-κB Activation
THP-1 cells are a human monocyte cell line that was developed from a monocyte isolated from the peripheral blood of an acute monocytic leukemia patient.This cell line is used as a monocyte/macrophage model in immunology research.The THP-1Blue cells obtained from InvivoGen (San Diego, CA, USA) are THP-1 cells that were stably transfected with a secreted embryonic alkaline phosphatase gene that is under control of a NF-κB/AP-1-inducible promoter.For this study, THP-1Blue cells were cultured at 37 • C in a humidified atmosphere containing 5% CO 2 in in RPMI 1640 medium (Mediatech Inc., Herndon, VA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 µg/mL streptomycin, 100 U/mL penicillin, 100 µg/mL phleomycin (Zeocin), and 10 µg/mL blasticidin S (all from Sigma-Aldrich, St. Louis, MO, USA).
To measure the activation of AP-1/NF-κB, the THP-1Blue cells (2 × 10 5 cells/well) were pretreated with the test compounds or dimethyl sulfoxide (DMSO; 1% final concentration) for 30 min, followed by the addition of 250 ng/mL of lipopolysaccharide (LPS; from Escherichia coli strain 0111:B4) for 24 h, and alkaline phosphatase activity was measured in the cell supernatants using the QUANTI-Blue mix (InvivoGen) as the absorbance at 655 nm and compared with positive control samples (LPS).The concentration of the compound that caused 50% inhibition of the NF-κB reporter activity (IC 50 ) was 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.THP-1Blue cells were treated with varying concentrations of the test compounds (up to 50 µM) and cultivated for 24 h.After treatment, the cells were allowed to equilibrate to room temperature for 30 min, the substrate was added, and the samples were analyzed with a Fluoroscan Ascent FL (Thermo Fisher Scientific, Waltham, MA, USA).

Kinase K d Determination
Compounds 13i and 16 were submitted for dissociation constant (K d ) determination toward JNK1-3 using KINOMEscan (Eurofins Pharma Discovery, San Diego, CA, USA), as described previously [56].In brief, JNK1-3 were produced and displayed on T7 phages or expressed in HEK-293 cells.Binding reactions were performed at room temperature for 1 h, and the fraction of kinase not bound to the test compound was determined by capture with an immobilized affinity ligand and quantified by quantitative polymerase chain reaction.The primary screening at fixed concentrations of the compound was performed in duplicate.For dissociation constant K d determination, a 12-point half-log dilution series (a maximum concentration of 33 µM) was used.Assays were performed in duplicate, and their average mean value is displayed.

PharmMapper Modeling
The PharmMapper Server [58] was used to identify potential protein targets for compounds 13i and 16.PharmMapper recognizes potential targets based on reverse pharmacophore mapping.The protein biotargets are represented by sets of pharmacophore points in reference databases incorporated in the software.The structures of 13i and 16 were uploaded in SDF format into PharmMapper.The system automatically generated up to 300 conformers of each compound based on the software option.We performed pharmacophore mapping using the "Human Protein Targets Only" database, which contained 2241 targets.We retrieved the top 250 potential targets for each compound evaluated.The potential targets were sorted by normalized fit score.

Molecular Docking
Docking of compounds 13i and 16 into the binding sites of kinases ERK2, JNK3, and p38α MAPK (structures 1TVO [54], 1PMV [59], and 1W7H [60], respectively, from Protein Data Bank) was performed with the use of the ROSIE server [52].The docking areas were chosen around the geometric centers of the co-crystallized ligands, each occupying the binding site of the corresponding enzyme in the 1TVO, 1PMV, or 1W7H structure.For each of the docked compounds, generation of up to 1000 ligand conformers with the BCL algorithm [61] was switched on.The number of intermediately generated docking poses was set to 2000.Other options were set to the default settings within the ROSIE ligand docking protocol, which accounts for the full flexibility of the main chain and side chains of residues in the vicinity of the docking area [50].Upon finishing the computation jobs, PDB files containing the best poses obtained for compounds 13i and 16 docked into ERK2, JNK3, and p38α were downloaded from the server, and imported into the Molegro Virtual Docker (MVD) program for visualization and analysis using the built-in "Pose Organizer" tool of MVD.
The docking of compounds 13i and 16 into the JNK3 binding site (PDB structure 4WHZ) was performed with AUTODOCK 4.1.For each investigated complex, the conformation with the most favorable binding energy was selected.The JNK3-ligand complex was further minimized in a vacuum using the amber99sb force-field implemented in GRO-MACS 5.1.No constraints were applied, and a conjugate gradient algorithm for energy minimization was used.The minimization was converged when the maximum force was smaller than 10.0 kJ•mol −1 •nm −1 .

Figure 1 .
Figure 1.Chemical structures of scaffolds of the screened compounds.

Figure 1 .
Figure 1.Chemical structures of scaffolds of the screened compounds.

Figure 2 .
Figure 2. New polyheterocycle anti-inflammatory compounds reported in the literature.

Figure 2 .
Figure 2. New polyheterocycle anti-inflammatory compounds reported in the literature.

Figure 3 .
Figure3.Effect of compounds 13i and 16 on NF-κB/AP-1 activity.THP-1Blue cells were pretreated with the indicated concentrations of compounds 13i (red symbols) and 16 (blue symbols) or DMSO control for 30 min, followed by the addition of 250 ng/mL of LPS or buffer for 24 h.NF-κB/AP-1 activity was monitored by measuring secreted alkaline phosphatase activity spectrophotometrically in the cell supernatants (absorbance at 655 nm).The data in each panel are presented as the mean ± S.D. of triplicate samples from one experiment that is representative of three independent experiments.

Table 2 .
Potential human protein kinase targets for compounds 13i and 16 identified by PharmMapper.

Table 3 .
Interface energy scores and H-bonding interactions obtained from the docking of ligands 13i and 16 into the binding sites of ERK2, JNK3, and p38α obtained using the ROSIE web server.

Table 4 .
JNK binding affinity of the compounds 13i and 16.