Discovery of small-molecule inhibitors of RUVBL1/2 ATPase

Abstract

RUVBL1 and RUVBL2 are highly conserved AAA ATPases (ATPases Associated with various cellular Activities) and highly relevant to the progression of cancer, which makes them attractive targets for novel therapeutic anticancer drugs. In this work, docking-based virtual screening was performed to identify compounds with activity against the RUVBL1/2 complex. Seven compounds showed inhibitory activity against the complex in both enzymatic and cellular assays. A series of pyrazolo[1,5-a]pyrimidine-3-carboxamide analogs were synthesized based on the scaffold of compound 15 with inhibitory activity and good potential for structural manipulation. Analysis of the structure-activity relationship identified the benzyl group on R₂ and aromatic ring-substituted piperazinyl on R₄ as essential for inhibitory activity against the RUVBL1/2 complex. Of these, compound 18, which has IC₅₀ values of 6.0 ± 0.6 μM and 7.7 ± 0.9 μM against RUVBL1/2 complex and RUVBL1 respectively, showed the most potent inhibition in cell lines A549, H1795, HCT116, and MDA-MB-231 with IC₅₀ values of 15 ± 1.2 μM, 15 ± 1.8 μM, 11 ± 1.0 μM, and 8.9 ± 0.9 μM respectively. A docking study of the compound was performed to predict the binding mode of pyrazolo[1,5-a]pyrimidine-3-carboxamides. Furthermore, mass spectrometry-based proteomic analysis was employed to explore cellular proteins dysregulated by treatment with compounds 16, 18, and 19. Together, the data from these analyses suggest that that compound 18 could serve as a starting point for structural modifications in order to improve potency, selectivity, and pharmacokinetic parameters of potential therapeutic molecules.

Graphical Abstract

1. Introduction

RUVBL proteins RUVBL1 (TIP49, TIP49a, Rvb1, Pontin) and RUVBL2 (TIP48, TIP49b, Rvb2, Reptin) are present in the Archaea and Eukarya domains, including in humans and fungi. RUVBL proteins are highly conserved AAA proteins (ATPases Associated with various cellular Activities), known to be involved in transcription regulation, DNA damage repair, cell cycle control, and stress adaptation [1-3]. RUVBLs assemble as heteromeric complexes that form an integral component of chromatin remodeling complexes TIP60 [4], INO80 [5] and SWR1/SRCAP [6], as well as of the R2TP chaperone complex [7] and PAQosome [8]. The RuvBL1/2 complex functions as a chaperone itself during the biogenesis of telomerase, H/ACA RNPs and other supramolecular complexes [2, 4, 9, 10]. As well, RuvBL1 and RuvBL2 function independently and even antagonistically in many instances [11-13]. Numerous structural studies indicate that the assembled complex can occur in both hexameric and dodecameric (stacked double hexamer) arrangements [9, 14-17].

There is increasing evidence that the presence of RUVBL1 and RUVBL2 contributes to cancer progression. Overexpression of RUVBL1 and RUVBL2 is observed in many cancer types, including liver, colorectal, breast, gastric, and non-small cell lung cancer [3, 18-22]. Furthermore, multiple studies have suggested that depletion of RUVBLs can hinder cancer cell growth and progression in both in vitro and in vivo models [2, 23, 24]. Combined, this data indicates that RUVBL1 and RUVBL2 may be good targets for anticancer treatment.

Elkaim et al. used virtual screen and enzymatic assays to identify four small-molecule inhibitors of RUVBL1 (Akt1/2 inhibitor, PTP1B inhibitor, Rottlerin, and Pranlukast, see Figure 1) [25], which inhibit RUVBL1 with IC₅₀ values of 24, 15, 10 and 13 μM, respectively. The mechanism by which their action against RUVBL1 occurs needs to be determined in further study. Encouraged by previously published data from RUVBL1 inhibitors, Elkaim et al. focused on two scaffolds, 4-hydroxy-2-pyridone and 4-hydroxy-2-quinolone, and synthesized derivatives; compounds 5 and 6 (Figure 1) inhibited RUVBL1 activity in an ATP-dependent manner with IC₅₀ values of 9 and 18 μM, respectively. Compound 5 demonstrated antiproliferative activity against KB and HL60 cell lines with IC₅₀s of 9 and 15 μM, manifested as apoptotic cell death together with necrosis [26].

Figure 1.

Chemical Structures of known inhibitors against RUVBL proteins

Compound 7 (Figure 1) was initially identified as an RUVBL1/2 complex inhibitor with an IC₅₀ of 59 nM [27]. In cellular assays, compound 7 depleted PIKK-family members DNA-PKcs (PRKDC), ATM, and ATP, all of which require RUVBL1/2 ATPase activity for stability [28]. Compound 7 was shown to arrest NSCLC cells in S phase, which eventually underwent apoptosis, indicated by the sub-G1 population in DNA content analysis. Sorafenib (Figure 1, Compound 8) was identified as a RUVBL2 inhibitor with a K_d value of about 22 μM by high-throughput screening [29]. Sorafenib is shown to be an oral multi-kinase inhibitor used to treat advanced renal cell, liver and thyroid cancer [30]. Sorafenib inhibited RUVBL2 in a mixed non-competitive mode and inhibited activity of the RUVBL1/2 complex. Anderson and co-workers identified CB-6644 as a selective, noncompetitive inhibitor of the RUVBL1/2 complex with a IC₅₀ value of 15 nM [31]. However, CB-6644 showed a maximal inhibition of only ~50%. Herein, we report the discovery of five pyrazolo[1,5-a]pyrimidine-3-carboxamides with the inhibition of RUVBL1/2 ATPase activity and the IC₅₀ value of compound 18 is about 6 μM. Mass spectrometry-based proteomic analysis of cells treated with compounds 16, 18, or 19 were completed to compare the cellular pathways affected.

2. Results and Discussion

2.1. Docking-Based virtual Screening

In order to identify novel compounds with anti-RUVBL1/2 activity, docking-based virtual screening was performed using approximately 260,000 compounds from the NCI open compound library. The protein PDB file 5oaf was downloaded from Protein Data Bank (rcsb.org), followed by removal of water molecules and addition of hydrogens in AutoDock 4.2.6 [32]. The coordinates of the Grid center and box size were generated in AutoDock 4.2.6 and the docking results were ranked by binding affinity. Eighty compounds were selected from the top 3000 binding poses after visualization using the BIOVIA Discovery Studio Visualizer 4.5 (Biovia Inc.). In parallel, ligand-based similarity searches in the Swiss Similarity database [33] were performed to screen small molecules from the ChemDiv compound set (around 1,746,000) using CB-6644 as the template molecule. Seventeen compounds (including compounds 16-20) were selected from the top 500 compounds after evaluating their structures.

2.2. Biological evaluation of compounds from virtual screening

All 97 of the selected compounds were evaluated using an ATPase Assay. Seven of these compounds showed inhibition activity against the RUVBL1/2 complex at an enzymatic level in this work, including compounds 8 and 10-15 (Figure 2, Table 1). Interestingly, after we checked the structures of these compound, we found 8 is known as sorafenib, which was reported as an RUVBL2 inhibitor previously [29]. In addition to activity against the RUVBL1/2 complex, compounds 11, 13, 14, and 15 showed inhibitory activity against RUVBL1. Compound 15 was the only compound to show inhibition towards RUVBL1, RUVBL2, and the RUVBL1/2 complex. Next, we used four cancer cell lines to evaluate anti-proliferative effects of these compounds. However, no compound exhibited more potent inhibition than CB-6644 on any of these cell lines (Figure 3). Compounds 8, 12, 13, 14, and 15 showed moderate inhibitory activity against the RUVBL1/2 complex in enzymatic assays. CB-6644 showed the most potent inhibition in cell lines A549, HCT116, H1795 and MDA-MB-231 (Figure 3). Compared to 14, compound 15 showed comparable inhibition activity in A549, HCT116, and MDA-MB-231 and more potent inhibition in the H1795 cell line.

Figure 2.

Chemical Structure of compounds active against RUVBL1/2 complex

Table 1.

The inhibitory activity of active compounds from virtual screening

Compounds	IC50 (μM)
	RUVBL1/2	RUVBL1	RUVBL2
CB-6644	6.1 ± 0.5	> 66	> 66
8	55 ± 9.0	> 66	> 66
10	77 ± 24	> 66	> 66
11	46 ± 1.1	39 ± 7.7	> 66
12	68 ± 11	> 66	> 66
13	47± 2.0	26 ± 7.2	> 66
14	35 ± 6.4	31 ± 10	> 66
15	36 ± 2.1	65 ± 11	29 ± 7.4

Figure 3.

Anti-proliferative effects of active molecules from virtual screening. Compounds CB6644, 8 (Sorafenib), 10, 11, 12, 13, 14, and 15 were evaluated for their anti-proliferative effects on breast cancer cell line MDA-MB-231, non-small cell lung cancer cell lines A549 and H1795, as well as human colon cancer cell line HCT116

2.3. Synthesis of pyrazolo[1,5-a]pyrimidine analogs

Due to the in vitro activity and the scaffold that compounds 14 and 15 are sharing, we selected compound 15 as the target compound on which to perform further structural optimization. The pyrazolo[1,5-a]pyrimidine-3-carboxamides were synthesized in a three-step sequence of reactions that included cyclization of ethyl pyrazolo[1,5-a]pyrimidine-3-carboxylate core, hydrolysis of the ester moiety and amidation (Scheme 1). Specifically, ethyl pyrazolo[1,5-a]pyrimidine-3-carboxylate was synthesized by the reaction of ethyl 3-amino-1H-pyrazole-4-carboxylate with a series of diketone or dialdehyde compounds (1a-n) under acidic conditions. Hydrolysis of these intermediates with sodium hydroxide produced the corresponding carboxylic acids in good yields. For the synthesis of 3a, boration and benzylation were performed prior to saponification of the ethyl ester of 2a. Due to the difficulty of its isolation, (3-(ethoxycarbonyl)pyrazolo[1,5-a]pyrimidin-6-yl)boronic acid (2ab) was used directly for the next step without further purification. To deliver the final target compounds (21-48), the substituted pyrazolo[1,5-a]pyrimidine-3-carboxylic acids were coupled with substituted amines. Compound 47 was obtained by deprotection of Boc in the presence of hydrogen chloride; it was further derivatized into compound 48 through propargylation reactions with propargyl bromide.

Scheme 1.

Representative Synthetic Route of pyrazolo[1,5-a]pyrimidine-3-carboxamides. Reagents and conditions: (i) ethyl 3-amino-1H-pyrazole-4-carboxylate, EtOH, AcOH, 70 °C , 3h; (ii) Bis(pinacolato)diboron, KOAc, PdCl₂(dppf)(DCM), Dioxane, 80 °C, overnight; (iii) Benzyl bromide, K₂CO₃, PdCl₂(dppf)(DCM), Dioxane, H₂O, 50 °C, overnight; (iv) 2 M NaOH, MeOH, THF, 56 °C, 3 h; (v) substituted amines 6a-k, HATU, DIEA, DMF, rt, overnight; (vi) 4M HCl / EtOAc; (vii) propargyl bromide, K₂CO₃, DCM; (viii) N-Boc piperazine, Pd(OAc)₂, XPhos, KOtBu, Dioxane, 90°C, 10h.

2.4. Structure-Activity Relationship of pyrazolo[1,5-a]pyrimidine-3-carboxamide analogs

All of the pyrazolo[1,5-a]pyrimidine-3-carboxamides, including 16-20 from the ChemDiv compound library and the synthesized target compounds 21-48, were evaluated for the inhibitory activity against the RUVBL1/2 complex (Table S1). Among these compounds, Compound 18 showed the most potent inhibition against RUVBL1/2 with an IC₅₀ value of 6.0 ± 0.6 μM. Additionally, 18 could inhibit RUVBL1 with an IC₅₀ value of 7.7 ± 0.9 μM. Methyl groups on R₁ and R₃ were favorable for inhibitory activity; their replacement by H led to the loss of inhibition (Compound 23). As the volume of the hydrophobic group increases (CH₃ → CH₂CH₃), the inhibitory potency decreases (Compounds 18 → 22 → 21). The substituted benzyl on R₂ is essential to the inhibitory activity as the H atom and alkyl groups on R₂ resulted in the loss of the inhibitory activity (compounds 20, 31, and 32). The methyl group on the ortho-position of benzyl group led to the loss of the activity against RUVBL1/2 (compound 28). The aromatic-substituted piperazinyl on R₄ is essential to the inhibitory activity. However, the heteroaromatic ring-substituted on piperazine decrease the activity compared to the phenyl ring (17 vs 16). When R₄ is the amino group substituted by H (compound 42), alky (compounds 43 and 45) or aryl (compound 44) groups, the activity of these compounds decreases greatly. The CF₃-substituted on the ortho-position of phenyl ring (on piperazinyl) led to the loss of the activity (compound 34).

2.5. Proposed binding mode of compound 18

To investigate the binding mode of pyrazolo[1,5-a]pyrimidine-3-carboxamide analogs, we performed a docking study of compound 18 with the RUVBL1/2 complex. Residue Arg378 showed interactions with compound 18 by hydrogen bonds. His18 interacted with the pyrimidine ring of compound 18 by a cation-π interaction. Arg404 showed cation-π interactions with the pyrazolo and pyrimidine of compound 18. There was a potential halogen bond (with 2.9 Å) between the CF₃ group and Val40. Multiple residues, such as Leu39, Ala78, Leu79, and the carbon atoms on the side chains of Glu317 and Arg353, showed hydrophobic interactions with these two terminal phenyl rings of compound 18, which is consistent with the study of the structure-activity relationship described above.

2.6. Cytotoxicity of pyrazolo[1,5-a]pyrimidine-3-carboxamide analogs

To determine the cellular activity of pyrazolo[1,5-a]pyrimidine-3-carboxamides, we performed the CellTiter-Glo Luminescent Cell Viability Assay. CB-6644 showed more potent cytotoxicity in the nanomolar IC₅₀ range than did compounds 16-19. Among the pyrazolo[1,5-a]pyrimidine-3-carboxamides, compound 18 was identified as the compound with the most potent cytotoxicity against cell lines A549, HCT116, H1795, and MDA-MB-231 (Table 2 and Figure S1).

Table 2.

The anti-proliferative effects of pyrazolo[1,5-a]pyrimidine-3-carboxamides

Cmpds	IC50 (μM)
	A549	HCT116	H1795	MDA-MB-231
CB-6644	0.3 ± 0.07	0.08 ± 0.01	0.26 ± 0.07	0.70 ± 0.29
16	26 ± 5.5	21 ± 4.5	25 ± 12	22 ± 4.3
17	32 ± 1.6	32 ± 3.8	46 ± 5.5	37 ± 8.2
18	15 ± 1.2	11 ± 1.0	15 ± 1.8	8.9 ± 0.9
19	17 ± 1.7	17 ± 5.4	19 ± 4.1	18 ± 3.8

2.7. Quantitative Proteomics Analysis of compounds 16, 18, and 19 in treatment of HCT116 human colon cancer cells

To further study the cellular effects of compounds 16, 18, and 19 and determine which proteins are selectively altered upon treatment, we performed mass spectrometry-based proteomics analysis in HCT116 cells. We evaluated proteome changes using DMSO as negative control after 24 h treatment with either compound 16, 18 or 19. We identified a total of 7693 proteins, among which 268, 229 and 164 were dysregulated by compounds 16, 18 and 19, respectively (P < 0.05, ∣Log₂FC∣>0.5) (Figure 5A-C). Of these dysregulated proteins, 193, 112, and 117 were downregulated (Figure 5E). We performed heatmap analysis for differentially expressed proteins identified from two out of three compounds treated groups (p < 0.05, ∣Log₂FC∣>0.5) (Figure 5F). The dysregulated proteins from compounds 16, 18, and 19 showed the same trend in the heatmap analysis (Figure 5F). Comparative analysis showed 13 and 30 proteins respectively that were exclusively upregulated and downregulated by pyrazolo[1,5-a]pyrimidine-3-carboxamides 16, 18, and 19 (Figure 5D, Tables S2 and S3).

Figure 5.

Proteomic Effects of Compound Treatment. A-C) Volcano plots of the significantly differentially expressed proteins in HCT116 cells treated with 16, 18 and 19, respectively. D-E) Venn diagrams of upregulated (D) and down-regulated (E) proteins (p < 0.05, ∣Log₂FC∣>0.5) following treatment with 16, 18 and 19, respectively. F) Heatmap of differentially regulated proteins from at least two compound-treated groups (p < 0.05, ∣Log₂FC∣>0.5).

2.8. GO and KEGG pathway analysis of differentially expressed proteins

To better understand the biological functions of the differentially expressed proteins, we performed GO enrichment and KEGG pathway analyses (Figure 6). The four most significant processes identified by GO analysis annotations of biological process (BP) were regulation of mitotic cell cycle, cell division, cell proliferation, and response to cytokine. The two most significant processes identified among the annotations of cellular component (CC) was nucleus and extracellular space. Two most significant processes identified among annotations of molecular function (MF) were kinase activity and DNA binding. Signaling pathway enrichment analysis of the differentially expressed proteins identified two meaningful pathways with P-value<0.05, namely proteoglycans in cancer and the HIF-1 signaling pathway. Our pathway analysis results were consistent with the various roles of RUVBL1/2 ATPase in regulating cell proliferation, cell cycle [34], regulate chromatin complexes [35] and DNA replication [36]. In our recent review [37], several published studies are referenced to discuss that RUVBL1 and 2 are involved in many cellular activities that are also enriched in our inhibitors induced pathway analysis.

Figure 6.

GO and KEGG pathway enrichment analysis of the proteins dysregulated in at least two compound-treated samples. BP: biological progress; CC: cellular component; MF: molecular function.

3. Conclusion

RUVBL1 and RUVBL2 are well-known drug targets that assemble into heterohexameric rings. Beginning with docking-based virtual screening, 97 compounds were selected and their inhibition activity against RUVBL1/2 complex was evaluated. Seven compounds showed inhibitory activity against the RUVBL1/2 complex in both enzymatic and cellular assays. Due to the chemical space and observed activity, compound 15 was selected as the hit compound for further structural optimization due to its ability to inhibit RUVBL1 and RUVBL2 in addition to the RUVBL1/2 complex. In order to study the structure-activity relationship, a series of pyrazolo[1,5-a]pyrimidine-3-carboxamides (28 synthesized compounds and 10 selected compounds purchased from ChemDiv) were evaluated. Structure-activity relationship analysis suggested that the substituted benzyl on R₂ and the aromatic-substituted piperazinyl on R₄are essential to the RUVBL1/2 inhibition. Compounds 16, 18, and 19 showed potent inhibition of both enzymatic and cellular activity. The proposed binding mode of compound 18 by docking implied the involvement of hydrophobic interactions between the active residues and the substituents on R₂ andR₄. MS-based proteomics identified 13 and 30 proteins were up- and down-regulated respectively by pyrazolo[1,5-a]pyrimidine-3-carboxamides 16, 18, and 19. Compound 18 could be used as a lead compound for further therapeutic development.

4. Materials and Methods

4.1. Chemical Synthesis

4.1.1. Materials

All solvents and chemical reagents were purchased from commercial sources and used directly without further purification. The progress of all reactions was monitored by analytical thin-layer chromatography on silica gel 60 F₂₅₄ plates (Merck). The purification by column chromatography was performed using 200-300 mesh silica gel (Qingdao Haiyang Chemical Co. Ltd.). ¹H-NMR spectra were recorded on a Bruker AVANCE 300 or 500 MHz spectrometer in DMSO-d₆. ¹³C-NMR spectra were recorded on a Bruker AVANCE 75 or 125 MHz spectrometer in DMSO-d₆. Chemical shift values were reported in δ values parts per million (ppm), referenced to dimethyl sulfoxide (DMSO) using the residual solvent signal as an internal reference. High-resolution mass spectrometry data (HRMS) were recorded on the ICR (Fourier transform ion cyclotron resonance, FTICR) analyzer using an ESI source. Infrared spectra (IR) were recorded on a Thermo Scientific Nicolet iS5 FT-IR spectrophotometer and reported as wavelength numbers (cm⁻¹). Melting points were determined with an X-4A microscopic melting point apparatus, which was uncorrected. All of the test compounds exhibited >95% purity as determined by HPLC. HPLC analysis was performed using an Agilent HPLC 1200 system (column, Agilent C18, 4.6 mm × 150 mm, 5 μm).

4.1.2. General Procedure for the Synthesis of ethyl pyrazolo[1,5-a]pyrimidine-3-carboxylates (2a-n).

The mixture of ethyl 3-amino-1H-pyrazole-4-carboxylate (775 mg, 5.0 mmol, 1.0 eq.) and diketone or dialdehyde compounds (5.25 mmol, 1.05 eq.) in AcOH (4 mL) and EtOH (4 mL) was stirred at 70 °C for 3 h. The mixture was concentrated in vacuo and then partitioned between 50 mL EtOAc and 50 mL NaHCO₃ aqueous solution. The organic layer was collected, and the aqueous phase was extracted with 30 mL EtOAc. The combined organic phases were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residues were purified by column chromatography (silica gel, petroleum ether (b.p. 60-90 °C)/ethyl acetate, 20:1 to 3:1) to obtain the intermediates.

Ethyl 6-bromopyrazolo[1,5-a]pyrimidine-3-carboxylate (2aa).

Yield: 60%. Yellow solid. M.P. 140.7-142.8 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 9.75 (d, J = 2.1 Hz, 1 H), 8.89 (d, J = 2.1 Hz, 1 H), 8.59 (s, 1 H), 4.28 (q, J = 7.0 Hz, 2 H), 1.29 (t, J = 7.0 Hz, 3 H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 161.4, 153.7, 147.7, 145.2, 137.4, 105.7, 102.3, 59.8, 14.4. IR ν_max (neat): 3062, 2976, 1708, 1539, 1375, 1259, 1209, 1195, 1050, 902, 806, 782, 695. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₁₈H₁₆Br₂N₆O₄Na⁺, 560.9492; found, 560.9492 (error 0 ppm).

Ethyl 6-benzylpyrazolo[1,5-a]pyrimidine-3-carboxylate (2a).

A mixture of ethyl 6-bromopyrazolo[1,5-a]pyrimidine-3-carboxylate 2aa (2.0 g, 7.4 mmol, 1.0 eq.), Bis(pinacolato)diboron (2.26 g, 8.8 mmol, 1.2 eq.), KOAc (2.18 g, 22.2 mmol, 3.0 eq.) and 1,1'-bis(diphenylphosphino)ferrocene-palladium(II)dichloride complex with DCM (0.42 g, 0.52 mmol, 0.07 eq.) in dioxane (40 mL) was stirred at 80 °C overnight. The reaction mixture was allowed to cool to room temperature and partitioned between 50 mL EtOAc and 50 mL 2M HCl. The aqueous layer was extracted twice with 30 mL EtOAc and the combined organic layers were concentrated in vacuo. The residue was dissolved in 50 mL EtOAc and 50 mL 2M NaOH was added. After separation, the aqueous layer was acidified with hydrochloric acid and extracted with EtOAc (50 mL×3). The combined organic layer was dried over Na₂SO₄ and concentrated in vacuo to give the crude product 2ab (1.13 g, 4.8 mmol, 65%) as white solid, which was subjected to the next reaction without further purification.

A mixture of (3-(ethoxycarbonyl)pyrazolo[1,5-a]pyrimidin-6-yl)boronic acid 2ab (1.13 g, 4.8 mmol,1.0 eq.), benzyl bromide (0.84 g, 4.8 mmol, 1.0 eq.), and 1,1'-bis(diphenylphosphino)ferrocene-palladium(II)dichloride complex with DCM (0.39 g, 0.48 mmol, 0.1 eq.) in dioxane (30 mL) was treated with a solution of K₂CO₃ (1.32 g, 9.6 mmol, 2.0 eq.) in H₂O (6 mL). The reaction mixture was stirred at 50 °C overnight. The reaction mixture was allowed to cool to room temperature and partitioned between 50 mL EtOAc and 50 mL H₂O. The aqueous layer was extracted twice with 30 mL EtOAc. The combined organic layers were dried over anhydrous Na₂SO₄ followed by filtration and solvent removal in vacuo. The residue was purified by column chromatography (silica gel, petroleum ether (b.p. 60-90 °C)/ethyl acetate, 20:1 to 2:1) to give 2a. (643 mg, 2.28 mmol) Yield: 47%. White solid. M.P. 121.3-123.0 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 9.23 (s, 1H), 8.78 (m, 1H), 8.57 (s, 1H), 7.28-7.35 (m, 4H), 7.19-7.24 (m, 1H), 4.27 (q, J = 7.0 Hz, 2H), 4.08 (s, 2H), 1.28 (t, J = 7.0 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 161.6, 155.0, 147.1, 145.7, 139.4, 135.1, 128.7, 128.6, 126.5, 123.9, 101.6, 59.5, 34.5, 14.4. IR ν_max (neat): 3051, 2989, 2952, 2930, 1802, 1704, 1629, 1553, 1513, 1389, 1198, 1058, 786, 699. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₃₂H₃₀N₆O₄Na⁺, 585.2221; found, 585.2210 (error 1.84 ppm).

Ethyl 6-ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylate (2b).

Yield: 51%. White solid. M.P. 107.9-109.4 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.46 (s, 1H), 4.26 (q, J = 7.0 Hz, 2H), 2.67-2.73 (m, 5H), 2.60 (s, 3H), 1.30 (t, J = 7.0 Hz, 3H), 1.11 (t, J = 7.4 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 161.8, 161.7, 145.9, 145.2, 143.8, 122.5, 100.6, 59.3, 23.3, 20.7, 14.4, 13.5, 13.0. IR ν_max (neat): 2971, 2940, 2879, 1705, 1616, 1546, 1527, 1371, 1191, 1106, 1025, 786, 639. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₂₆H₃₄N₆O₄Na⁺, 517.2534; found, 517.2534 (error 0 ppm).

Ethyl 5,7-dimethyl-6-phenylpyrazolo[1,5-a]pyrimidine-3-carboxylate (2c).

Yield: 72%. White solid. M.P. 109.9-110.9 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.58 (s, 1H), 7.49-7.55 (m, 3H), 7.39-7.41 (m, 2H), 4.29 (q, J = 7.0 Hz, 2H), 2.47 (s, 3H), 2.31 (s, 3H), 1.32 (t, J = 7.0 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 161.8, 161.0, 146.6, 145.8, 144.3, 134.7, 129.9, 128.9, 128.4, 123.5, 101.0, 59.4, 24.7, 15.0, 14.4. IR ν_max (neat): 3112, 3051, 2977, 1693, 1612, 1386, 1373, 1289, 1207, 1108, 779, 705, 649. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₃₄H₃₄N₆O₄Na⁺, 613.2534; found, 613.2523 (error 1.75 ppm).

Ethyl 6-benzyl-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylate (2d).

Yield: 92%. White solid. M.P. 170.8-172.3 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.54 (s, 1H), 7.26-7.31 (m, 2H), 7.18-7.22 (m, 1H), 7.11-7.13 (m, 2H), 4.27 (q, J = 7.0 Hz, 2H), 4.19 (s, 2H), 2.78 (s, 3H), 2.48 (s, 3H), 1.30 (t, J = 7.0 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.4, 161.8, 146.1, 145.5, 145.2, 138.3, 128.7, 127.9, 126.4, 119.4, 100.9, 59.3, 32.6, 23.8, 14.5, 13.8. IR ν_max (neat): 3108, 3083, 3002, 2981, 1679, 1617, 1544, 1196, 1121, 781, 724. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₃₆H₃₈N₆O₄Na⁺, 641.2847; found, 641.2847 (error 0 ppm).

Ethyl 5,7-dimethyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (2e).

Yield: 66%. White solid. M.P. 103.7-105.9 °C. ¹H NMR (300 MHz, DMSO-d6) δ (ppm): 8.51 (s, 1H), 7.06 (d, J = 7.9 Hz, 2H), 6.97 (d, J = 7.9 Hz, 2H), 4.27 (q, J = 7.0 Hz, 2H), 4.10 (s, 2H), 2.75 (s, 3H), 2.46 (s, 3H), 2.22 (s, 3H), 1.30 (t, J = 7.0 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d6) δ (ppm): 162.3, 161.8, 146.0, 145.5, 145.0, 135.0, 129.2, 127.6, 119.5, 100.9, 59.2, 32.2, 23.7, 20.5, 14.4, 13.6. IR ν_max (neat): 2973, 1720, 1686, 1617, 1550, 1513, 1422, 1370, 1223, 1180, 1107, 974, 838, 780. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₃₈H₄₂N₆O₄Na⁺, 669.3160; found, 669.3160 (error 0 ppm).

Ethyl 5,7-dimethyl-6-(3-methylbenzyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (2f).

Yield: 82%. White solid. M.P. 115.3-116.7 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.51 (s, 1H), 7.12-7.17 (m, 1H), 6.98-7.01 (m, 1H), 6.87-6.92 (m, 2H), 4.27 (q, J = 7.0 Hz, 2H), 4.12 (s, 2H), 2.76 (s, 3H), 2.47 (s, 3H), 2.22 (s, 3H), 1.30 (t, J = 7.0 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.3, 161.8, 146.0, 145.5, 145.1, 138.1, 137.8, 128.5, 128.3, 127.0, 124.9, 119.4, 100.9, 59.3, 32.6, 23.8, 20.9, 14.4, 13.7. IR ν_max (neat): 2967, 1712, 1615, 1548, 1372, 1355, 1295, 1246, 1190, 1109, 1027, 975, 836, 792, 690. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₉H₂₂N₃O₂ ⁺, 324.1707; found, 324.1715 (error −2.61 ppm).

Ethyl 5,7-dimethyl-6-(2-methylbenzyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (2g).

Yield: 83%. White solid. M.P. 153.4-154.5 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.54 (s, 1H), 7.22-7.25 (m, 1H), 7.09-7.14 (m, 1H), 6.97-7.02 (m, 1H), 6.50-6.53 (m, 1H), 4.28 (q, J = 7.0 Hz, 2H), 4.05 (s, 2H), 2.68 (s, 3H), 2.43 (s, 3H), 2.411 (s, 3H), 1.31 (t, J = 7.0 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.4, 161.8, 146.0, 145.6, 145.4, 136.1, 130.0, 126.2, 126.1, 125.8, 118.8, 100.9, 59.3, 30.1, 23.4, 19.3, 14.4, 13.5. IR ν_max (neat): 2984, 1678, 1618, 1544, 1477, 1381, 1229, 1198, 1122, 1022, 785, 750. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₉H₂₂N₃O₂⁺, 324.1707; found, 324.1715 (error −2.61 ppm).

Ethyl 6-(4-isopropylbenzyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylate (2h).

Yield: 87%. White solid. M.P. 121.5-123.4 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.52 (s, 1H), 7.13 (d, J = 8.0 Hz, 2H), 7.01 (d, J = 8.0 Hz, 2H), 4.27 (q, J = 7.0 Hz, 2H), 4.12 (s, 2H), 2.78-2.87 (m, J = 6.8Hz, 1H), 2.76 (s, 3H), 2.48 (s, 3H), 1.30 (t, J = 7.0 Hz, 3H), 1.14 (d, J = 6.9 Hz, 6H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.3, 161.8, 146.4, 146.1, 145.5, 145.1, 135.5, 127.7, 126.5, 119.5, 100.9, 59.3, 32.9, 32.2, 23.8, 14.4, 13.7. IR ν_max (neat): 3107, 2959, 1678, 1614, 1541, 1515, 1379, 1224, 1198, 1124, 1026, 847, 782. HRMS (ESI+): m/z [M+H]⁺calcd. for C₂₁H₂₆N₃O₂⁺, 352.2020; found, 352.2028 (error −2.40 ppm).

Ethyl 6-(4-methoxybenzyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylate (2i).

Yield: 83%. White solid. M.P. 115.0-116.6 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.53 (s, 1H), 7.03 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 4.27 (q, J = 7.0 Hz, 2H), 4.10 (s, 2H), 3.70 (s, 3H), 2.77 (s, 3H), 2.48 (s, 3H), 1.30 (t, J = 7.0 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.4, 161.8, 157.8, 146.1, 145.5, 145.0, 129.9, 128.9, 119.8, 114.1, 100.8, 59.3, 55.0, 31.8, 23.8, 14.4, 13.7. IR ν_max (neat): 2977, 2936, 1720, 1615, 1549, 1514, 1383, 1370, 1116, 1027, 842, 785. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₃₈H₄₂N₆O₆Na⁺, 701.3058; found, 701.3058 (error 0 ppm).

Ethyl 6-(4-fluorobenzyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylate (2j).

Yield: 73%. White solid. M.P. 119.9-122.3 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.52 (s, 1H), 7.14-7.19 (m ,2H), 7.06-7.12 (m, 2H), 4.27 (q, J = 7.0 Hz, 2H), 4.17 (s, 2H), 2.77 (s, 3H), 2.47 (s, 3H), 1.30 (t, J = 7.0 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.2, 161.7, 160.7 (d, J = 240.7 Hz), 146.0, 145.4, 145.2, 134.2 (d, J = 2.8 Hz), 129.6 (d, J = 7.9 Hz), 119.2, 115.2 (d, J = 21.0 Hz), 59.2, 31.8, 23.7, 14.4, 13.7. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −116.7. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₈H₁₉FN₃O₂⁺, 328.1456; found, 328.1463 (error −2.19 ppm).

Ethyl 6-(4-chlorobenzyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylate (2k).

Yield: 83%. White solid. M.P. 137.8-140.0 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.52 (s, 1H), 7.32 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 8.3 Hz, 2H), 4.27 (q, J = 7.0 Hz, 2H), 4.17 (s, 2H), 2.76 (s, 3H), 2.46 (s, 3H), 1.30 (t, J = 7.0 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.2, 161.8, 146.1, 145.6, 145.4, 137.3, 131.0, 129.7, 128.5, 119.0, 100.9, 59.3, 32.0, 23.8, 14.4, 13.7. IR ν_max (neat): 2983, 2897, 1709, 1614, 1550, 1372, 1292, 1242, 1187, 1109, 984, 796, 783. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₃₆H₃₆Cl₂N₆O₄Na⁺, 709.2067; found, 709.2067 (error 0 ppm).

Ethyl 5,7-dimethyl-6-(4-(trifluoromethyl)benzyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (2l).

Yield: 84%. White solid. M.P. 143.8-144.4 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.54 (s, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 4.24-4.30 (m, 4H), 2.78 (s, 3H), 2.47 (s, 3H), 1.30 (t, J = 7.0 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.2, 161.7, 146.1, 145.6, 145.5, 143.3, 128.7, 127.1 (q, J = 31.5 Hz), 125.3 (q, J = 3.5 Hz), 124.2 (q, J = 270.2 Hz), 118.5, 100.9, 59.2, 32.5, 23.8, 14.4, 13.7. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −60.9. IR ν_max (neat): 2984, 1714, 1616, 1551, 1522, 1374, 1320, 1244, 1163, 1108, 813, 783. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₉H₁₉F₃N₃O₂⁺, 378.1424; found, 378.1433 (error −2.41 ppm).

Ethyl 5-ethyl-7-methyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (2m).

Yield: 58%. White solid. M.P. 121.3-124.1 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.54 (s, 1H), 7.09 (d, J = 7.8 Hz, 2H), 6.98 (d, J = 7.8 Hz, 2H), 4.27 (q, J = 7.0 Hz, 2H), 4.14 (s, 2H), 3.20 (q, J = 7.3 Hz, 2H), 2.46 (s, 3H), 2.24 (s, 3H), 1.30 (t, J = 7.0 Hz, 3H), 1.14 (t, J = 7.3 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm):162.9, 161.7, 149.3, 146.1, 145.6, 135.3, 129.2, 127.6, 118.6, 100.8, 59.2, 31.8, 23.8, 20.6, 20.4, 14.4, 10.9. IR ν_max (neat): 2969, 2936, 2873, 1716, 1609, 1548, 1514, 1370, 1347, 1291, 1190, 1121, 1090, 788. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₂₀H₂₄N₃O₂⁺, 338.1863; found, 338.1870 (error −2.06 ppm).

Ethyl 5,7-diethyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (2n).

Yield: 55%. White solid. M.P. 82.9-84.8 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.54 (s, 1H), 7.07 (d, J = 7.8 Hz, 2H), 6.96 (d, J = 7.8 Hz, 2H), 4.26 (q, J = 7.0 Hz, 2H), 4.15 (s, 2H), 3.19 (q, J = 7.2 Hz, 2H), 2.76 (q, J = 7.2 Hz, 2H), 2.23 (s, 3H), 1.30 (t, J = 7.0 Hz, 3H), 1.11-1.20 (m, 6H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 166.1, 161.8, 149.2, 146.1, 145.8, 135.6, 135.3, 129.1, 127.5, 118.1, 101.0, 59.2, 31.2, 28.3, 20.7, 20.5, 14.3, 11.3, 10.9. IR ν_max (neat): 2969, 1716, 1611, 1544, 1513, 1467, 1369, 1349, 1237, 1120, 1091, 928, 911, 795, 780. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₂₁H₂₆N₃O₂⁺, 352.2020; found, 352.2019 (error 0.15 ppm).

4.1.3. General Procedure for the Synthesis of pyrazolo[1,5-a]pyrimidine-3-carboxylic acids (3a-n)

To a solution of ethyl pyrazolo[1,5-a]pyrimidine-3-carboxylates (4.0 mmol, 1.0 eq.) in THF (3.0 mL) and MeOH (3.0 mL) was added 2 M NaOH aqueous solution (3.0 mL, 6.0 mmol, 1.5 eq.). The mixture was stirred at 56 °C for 3 h and then concentrated in vacuo. The residue was suspended in water, and 2 M HCl aqueous solution (3.0 mL, 6.0 mmol, 1.5 eq.) was added. The resulting solid was collected by filtration, rinsed with water, and dried to afford the intermediates as off-white to yellowish solids.

6-benzylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (3a).

Yield: 81%. Pale yellow solid. M.P. 237.3-240.1 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 12.39 (s, 1H), 9.20 (s, 1H), 8.74 (s, 1H), 8.54 (s, 1H), 7.28-7.33 (m, 4H), 7.21-7.23 (m, 1H), 4.08 (s, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 163.1, 154.7, 147.4, 145.8, 139.5, 135.0, 128.7, 128.6, 126.6, 123.7, 102.4, 34.5. IR ν_max (neat): 3079, 2921, 1698, 1626, 1558, 1521, 1413, 1386, 1269, 1196, 1049, 778, 721, 641. HRMS (ESI−): m/z [2M-H]⁻ calcd. for C₂₈H₂₁N₆O₄⁻, 505.1630; found, 505.1620 (error 1.94 ppm).

6-ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (3b).

Yield: 88%. Pale yellow solid. M.P. 179.5-180.9 °C. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 12.11 (s, 1H), 8.43 (s, 1H), 2.70-2.74 (m, 5H), 2.61 (s, 3H), 1.13 (t, J = 7.5 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm): 163.2, 161.2, 146.2, 145.1, 143.7, 122.2, 101.3, 23.1, 20.6, 13.4, 12.9. IR ν_max (neat): 2972, 2579, 1717, 1643, 1613, 1538, 1523, 1361, 1232, 1190, 784, 733, 657, 640. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₂₂H₂₆N₆O₄Na⁺, 461.1908; found, 461.1908 (error 0 ppm).

5,7-dimethyl-6-phenylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (3c).

Yield: 82%. White solid. M.P. 220.0-221.9 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 12.31 (brs, 1H), 8.54 (s, 1H), 7.45-7.56 (m, 3H), 7.38-7.41 (m, 2H), 2.47 (s, 3H), 2.30 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 163.4, 160.6, 147.0, 145.8, 144.3, 134.9, 129.9, 128.9, 128.4, 123.2, 101.9, 24.6, 15.0. IR ν_max (neat): 3061, 2697, 2631, 2577, 1714, 1658, 1610, 1513, 1386, 1233, 1198, 788, 702. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₃₀H₂₆N₆O₄Na⁺, 557.1908; found, 557.1908 (error 0 ppm).

6-benzyl-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (3d).

Yield: 85%. White solid. M.P. 180.5-182.9 °C. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 12.16 (s, 1H), 8.49 (s, 1H), 7.26-7.29 (m, 2H), 7.19 (t, J = 7.3 Hz, 1H), 7.11-7.12 (m, 2H), 4.18 (s, 2H), 2.77 (s, 3H), 2.47 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm): 163.3, 162.0, 146.4, 145.5, 145.1, 138.3, 128.6, 127.8, 126.3, 119.2, 101.6, 32.6, 23.7, 13.7. IR ν_max (neat): 2945, 1727, 1660, 1615, 1553, 1493, 1362, 1248, 1198, 784, 736, 701. HRMS (ESI−): m/z [M-H]⁻ calcd. for C₁₆H₁₄N₃O₂⁻, 280.1092; found, 280.1092 (error 0 ppm).

5,7-dimethyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (3e).

Yield: 90%. White solid. M.P. 192.0-193.8 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 12.58 (brs, 1H), 8.48 (s, 1H), 7.07 (d, J = 7.7 Hz, 2H), 6.98 (d, J = 7.7 Hz, 2H), 4.11 (s, 2H), 2.76 (s, 3H), 2.46 (s, 3H), 2.23 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 163.8, 161.8, 146.4, 145.3, 144.9, 135.3, 135.1, 129.2, 127.6, 119.1, 102.4, 32.2, 23.6, 20.5, 13.7. IR ν_max (neat): 2921, 1660, 1616, 1548, 1515, 1493, 1386, 1360, 1248, 1195, 944, 785. HRMS (ESI−): m/z [M-H]⁻ calcd. for C₁₇H₁₆N₃O₂ ⁻, 294.1248; found, 294.1253 (error −1.70 ppm).

5,7-dimethyl-6-(3-methylbenzyl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (3f).

Yield: 84%. White solid. M.P. 180.5-183.1 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 12.37 (brs, 1H), 8.49 (s, 1H), 7.16 (m, 1H), 6.99-7.01 (m, 1H), 6.88-6.93 (m, 2H), 4.13 (s, 2H), 2.77 (s, 3H), 2.47 (s, 3H), 2.23 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 163.5, 162.0, 146.4, 145.4, 145.0, 138.2, 137.8, 128.5, 128.3, 127.0, 124.9, 119.1, 101.9, 32.6, 23.7, 20.9, 13.7. IR ν_max (neat): 3391, 2941, 1672, 1615, 1548, 1521, 1486, 1374, 1231, 1193, 1143, 982, 785, 732. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₇H₁₈N₃O₂⁺, 296.1394; found, 296.1394 (error 0 ppm).

5,7-dimethyl-6-(2-methylbenzyl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (3g).

Yield: 96%. White solid. M.P. >300 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 12.60 (brs, 1H), 8.49 (s, 1H), 7.22-7.25 (m, 1H), 7.11 (m, 1H), 6.99 (m, 1H), 6.49-6.52 (m, 1H), 4.05 (s, 2H), 2.68 (s, 3H), 2.43 (s, 3H), 2.40 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 163.8, 161.9, 146.4, 145.5, 145.2, 136.2, 136.1, 130.0, 126.2.126.1, 125.8, 118.3, 102.6, 30.0, 23.2, 19.3, 13.5. IR ν_max (neat): 3479, 3020, 1654, 1618, 1551, 1532, 1373, 1196, 1137, 981, 845, 789, 741. HRMS (ESI−): m/z [M-H]⁻ calcd. for C₁₇H₁₆N₃O₂⁻, 294.1248; found, 294.1237 (error 3.74 ppm).

6-(4-isopropylbenzyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (3h).

Yield: 95%. White solid. M.P. 210.3-211.9 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 12.61 (brs, 1H), 8.48 (s, 1H), 7.13 (d, J = 7.9 Hz, 2H), 7.01 (d, J = 7.9 Hz, 2H), 4.12 (s, 2H), 2.76-2.85 (m, 4H), 2.47 (s, 3H), 1.13 (d, J = 6.8 Hz, 6H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 163.8, 161.8, 146.4, 146.3, 145.3, 144.9, 135.5, 127.7, 126.5, 119.1, 102.6, 32.9, 32.2, 23.8, 23.6, 13.7. IR ν_max (neat): 3464, 2957, 1683, 1619, 1548, 1523, 1377, 1364, 1299, 1192, 1144, 849, 787, 736. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₃₈H₄₂N₆O₄Na⁺, 669.3160; found, 669.3168 (error −1.23 ppm).

6-(4-methoxybenzyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (3i).

Yield: 91%. White solid. M.P. 213.2-214.8 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 12.25 (brs, 1H), 8.49 (s, 1H), 7.03 (d, J = 8.5 Hz, 2H), 6.83 (d, J = 8.5 Hz, 2H), 4.10 (s, 2H), 3.70 (s, 3H), 2.77 (s, 3H), 2.47 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 163.3, 162.0, 157.8, 146.4, 145.4, 144.9, 130.0, 128.8, 119.5, 114.0, 101.7, 55.0, 31.8, 23.6, 13.7. IR ν_max (neat): 2937, 1661, 1611, 1548, 1511, 1426, 1246, 1190, 1021, 844, 785, 744. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₇H₁₈N₃O₃⁺, 312.1343; found, 312.1348 (error −1.70 ppm).

6-(4-fluorobenzyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (3j).

Yield: 96%. White solid. M.P. 201.0-202.5 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.48 (s, 1H), 7.06-7.18 (m, 4H), 4.16 (s, 2H), 2.77 (s, 3H), 2.46 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 163.8, 161.6, 160.7 (d, J = 240.8 Hz), 146.4, 145.3, 145.0, 134.4 (d, J = 2.8 Hz), 129.6 (d, J = 7.8 Hz), 118.8, 115.2 (d, J = 21.0 Hz), 102.7, 31.8, 23.6, 13.7. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −116.7. IR ν_max (neat): 3063, 2585, 1659, 1615, 1550, 1509, 1385, 1362, 1249, 1196, 945, 852, 786, 770, 747. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₃₂H₂₈F₂N₆O₄Na⁺, 621.2032; found, 621.2032 (error 0 ppm).

6-(4-chlorobenzyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (3k).

Yield: 95%. White solid. M.P. >300 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 12.52 (brs, 1H), 8.49 (s, 1H), 7.33 (d, J = 8.3 Hz, 2H), 7.16 (d, J = 8.3 Hz, 2H), 4.18 (s, 2H), 2.77 (s, 3H), 2.46 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 163.7, 161.6, 146.5, 145.4, 145.2, 137.4, 130.9, 129.7, 128.5, 118.5, 102.7, 32.0, 23.6, 13.7. IR ν_max (neat): 3228, 1615, 1549, 1525, 1386, 1373, 1247, 1189, 1093, 1013, 908, 848, 797, 744. HRMS (ESI−): m/z [M-H]⁻ calcd. for C₁₆H₁₃ClN₃O₂ ⁻, 314.0702; found, 314.0691 (error 3.44 ppm).

5,7-dimethyl-6-(4-(trifluoromethyl)benzyl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (3l).

Yield: 90%. White solid. M.P. >300 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.46 (s, 1H), 7.63 (d, J = 7.8 Hz, 2H), 7.36 (d, J = 7.8 Hz, 2H), 4.30 (s, 2H), 2.77 (s, 3H), 2.47 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 161.2, 146.5, 145.2, 143.5, 128.6, 127.0 (q, J = 31.3 Hz), 125.4 (q, J = 3.6 Hz), 124.2 (q, J = 270.1 Hz), 117.8, 32.5, 23.6, 13.7. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −60.8. IR ν_max (neat): 3475, 1615, 1551, 1407, 1375, 1328, 1186, 1109, 1067, 902, 808. HRMS (ESI−): m/z [M-H]⁻ calcd. for C₁₇H₁₃F₃N₃O₂⁻, 348.0965; found, 348.0964 (error 0.39 ppm).

5-ethyl-7-methyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (3m).

Yield: 91%. White solid. M.P. >300 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.27 (s, 1H), 7.07 (d, J = 7.5 Hz, 2H), 6.95 (d, J = 7.5 Hz, 2H), 4.10 (s, 2H), 3.19 (q, J = 7.2 Hz, 2H), 2.43 (s, 3H), 2.24 (s, 3H), 1.14 (t, J = 7.2 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 148.4, 146.4, 144.5, 135.8, 135.2, 129.1, 127.5, 116.5, 31.8, 23.4, 20.5, 11.0. IR ν_max (neat): 3374, 2936, 1613, 1544, 1514, 1408, 1389, 1308, 1189, 808, 772. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₃₆H₃₈N₆O₄Na⁺, 641.2847; found, 641.2847 (error 0 ppm).

5,7-diethyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (3n).

Yield: 80%. Faint yellow solid. M.P. 158.1-160.5 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 12.13 (brs, 1H), 8.51 (s, 1H), 7.08 (d, J = 7.7 Hz, 2H), 6.97 (d, J = 7.7 Hz, 2H), 4.15 (s, 2H), 3.19 (q, J = 7.1 Hz, 2H), 2.75 (q, J = 7.3 Hz, 2H), 2.24 (s, 3H), 1.12-1.17 (m, 6H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 166.1, 163.2, 149.3, 146.6, 145.8, 135.7, 135.3, 129.2, 127.6, 117.8, 101.7, 31.2, 28.4, 20.7, 20.5, 11.8, 10.9. IR ν_max (neat): 3451, 2983, 2939, 1666, 1617, 1543, 1372, 1236, 1193, 1148, 787, 736. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₉H₂₂N₃O₂⁺, 324.1707; found, 324.1714 (error −2.30 ppm).

4.1.4. General Procedure for the Synthesis of tert-butyl 4-phenylpiperazine-1-carboxylates (5a-k).

A mixture of N-Boc piperazine (5.0 mmol, 1.0 eq.) and substituted bromo benzenes 4 (5.5 mmol, 1.1 eq.) in the presence of palladium acetate (0.5 mmol, 0.1 eq.), 2-Dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (1.0 mmol, 0.2 eq.) and potassium t-butoxide (6.0 mmol, 1.2 eq.) in dioxane (15 ml) was stirred in at 90 °C for 10 h. The reaction mixture was allowed to cool to room temperature, and 100 ml ethyl acetate and 100 ml water were added. The thus-obtained mixture was filtered through a celite pad. The organic layer was collected, and the aqueous phase was extracted with 50 mL EtOAc. The combined organic phases were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residues were purified by column chromatography (silica gel, petroleum ether (b.p. 60-90 °C)/ethyl acetate, 100:1 to 20:1) to afford the intermediates.

tert-Butyl 4-phenylpiperazine-1-carboxylate (5a).

Yield: 40%. Light brown solid. M.P. 68.0-69.1 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 7.19-7.24 (m, 2H), 6.92-6.95 (m, 2H), 6.80 (t, J = 7.2 Hz, 1H), 3.43-3.45 (m, 4H), 3.05-3.08 (m, 4H), 1.42 (s, 9H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 153.8, 150.9, 128.9, 119.3, 116.0, 78.9, 48.4, 42.8 (br), 28.0. IR ν_max (neat): 2979, 2929, 2859, 1684, 1597, 1427, 1389, 1364, 1235, 1156, 753, 689. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₅H₂₃N₂O₂⁺, 263.1754; found, 263.1754 (error 0 ppm).

tert-Butyl 4-(3-(trifluoromethyl)phenyl)piperazine-1-carboxylate (5b).

Yield: 37%. Light brown solid. M.P. 45.3-47.6 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 7.39-7.41 (m, J = 7.9 Hz, 1H), 7.17-7.22 (m, 2H), 7.06-7.09 (m, 1H), 3.44-3.46 (m, 4H), 3.18-3.20 (m, 4H), 1.42 (s, 9H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 153.8, 151.1, 129.9 (q, J = 30.8 Hz), 129.8, 124.3 (q, J = 270.7 Hz), 119.1, 114.9 (q, J = 3.7 Hz), 111.3 (q, J = 3.7 Hz), 78.9, 47.6, 43.0 (br), 28.0. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −61.1. IR ν_max (neat): 2979, 2933, 2828, 1684, 1607, 1420, 1389, 1362, 1234, 1153, 1109, 942, 857, 782, 691. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₆H₂₂F₃N₂O₂⁺, 331.1628; found, 331.1628 (error 0 ppm).

tert-Butyl 4-(2-(trifluoromethyl)phenyl)piperazine-1-carboxylate (5c).

Yield: 51%. White solid. M.P. 46.8-48.5 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 7.62-7.67 (m, 2H), 7.54-7.56 (m, 1H), 7.32-7.37 (m, 1H), 3.43 (br, 4H), 2.78-2.81 (m, 4H), 1.42 (s, 9H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm): 153.8, 152.0, 133.6, 126.8 (q, J = 5.3 Hz), 125.7 (q, J = 27.7 Hz), 125.5, 124.7, 124.0 (q, J = 271.5 Hz), 78.9, 52.9, 44.1 (br), 43.3 (br), 28.0. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −58.8. IR ν_max (neat): 2979, 2858, 1682, 1424, 1388, 1366, 1311, 1248, 1123, 780, 767. HRMS (ESI+): m/z [M+Na]⁺ calcd. for C₁₆H₂₁F₃N₂O₂Na⁺, 353.1447; found, 353.1442 (error 1.51 ppm).

tert-Butyl 4-(4-(trifluoromethyl)phenyl)piperazine-1-carboxylate (5d).

Yield: 63%. Light brown solid. M.P. 125.9-127.0 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 7.50 (d, J = 8.7 Hz, 2H), 7.05 (d, J = 8.7 Hz, 2H), 3.43-3.47 (m, 4H), 3.23-3.27 (m, 4H), 1.41 (s, 9H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 153.9, 153.1, 126.1 (q, J = 3.5 Hz), 124.9 (q, J = 268.6 Hz), 118.1 (q, J = 31.8 Hz), 114.5, 79.1, 46.9, 42.8 (br), 28.0. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −59.5. IR ν_max (neat): 3001, 2931, 2820, 1672, 1614, 1418, 1392, 1367, 1326, 1232, 1137, 1107, 831, 632. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₆H₂₂F₃N₂O₂⁺, 331.1628; found, 331.1623 (error 1.47 ppm).

tert-Butyl 4-(3-fluorophenyl)piperazine-1-carboxylate (5e).

Yield: 48%. White solid. M.P. 44.0-45.7 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 7.17-7.25 (m, 1H), 6.71-6.76 (m, 2H), 6.53-6.58 (m, 1H), 3.42-3.43 (m, 4H), 3.11-3.14 (m, 4H), 1.41 (s, 9H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 163.1 (d, J = 239.0 Hz), 153.8, 152.5 (d, J = 10.0 Hz), 130.3 (d, J = 10.0 Hz), 111.2, 104.9 (d, J = 21.2 Hz), 102.1 (d, J = 25.0 Hz), 78.9, 47.6, 42.9 (br), 28.0. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −112.3. IR ν_max (neat): 2981, 2859, 1682, 1609, 1581, 1497, 1424, 1390, 1364, 1243, 1157, 1124, 996, 967, 759, 682. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₅H₂₂FN₂O₂⁺, 281.1660; found, 281.1660 (error 0 ppm).

tert-Butyl 4-(3-chlorophenyl)piperazine-1-carboxylate (5f).

Yield: 51%. White solid. M.P. 64.6-65.8 °C. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 7.19-7.22 (m, 1H), 6.94-6.95 (m, 1H), 6.87-6.89 (m, 1H), 6.78-6.80 (m, 1H), 3.42-3.44 (m, 4H), 3.11-3.13 (m, 4H), 1.41 (s, 9H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm): 153.8, 152.1, 133.8, 130.4, 118.5, 115.0, 114.1, 79.0, 47.7, 43.2 (br), 28.0. IR ν_max (neat): 2976, 2861, 1679, 1592, 1424, 1391, 1364, 1235, 1158, 1124, 933, 760, 679. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₅H₂₂ClN₂O₂⁺, 297.1364; found, 297.1365 (error −0.23 ppm).

tert-Butyl 4-(3-(difluoromethyl)phenyl)piperazine-1-carboxylate (5g).

Yield: 51%. Light brown solid. M.P. 61.1-62.8 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 7.32-7.37 (m, 1H), 7.10 (m, 2H), 6.97-6.99 (m, 1H), 6.91 (t, J = 56.0 Hz, 1H), 3.44-3.46 (m, 4H), 3.13-3.16 (m, 4H), 1.42 (s, 9H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 153.8, 151.0, 134.9 (t, J = 21.6 Hz), 129.5, 117.9, 115.9 (t, J =5.8 Hz), 115.2 (t, J = 234.6 Hz), 112.6 (t, J = 6.3 Hz), 79.0, 47.9, 43.0 (br), 28.0. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −112.3. IR ν_max (neat): 2977, 2862, 1682, 1425, 1388, 1364, 1244, 1165, 1129, 1018, 950, 867, 801, 751. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₆H₂₃F₂N₂O₂⁺, 313.1722; found, 313.1720 (error 0.67 ppm).

tert-Butyl 4-(3-cyanophenyl)piperazine-1-carboxylate (5h).

Yield: 28%. White solid. M.P. 130.1-131.2 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 7.33-7.41 (m, 2H), 7.25-7.28 (m, 1H), 7.15-7.18 (m, 1H), 3.42-3.44 (m, 4H), 3.17-3.20 (m, 4H), 1.41 (s, 9H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 153.8, 150.9, 130.1, 121.9, 120.0, 119.2, 118.0, 112.0, 79.0, 47.3, 42.8 (br), 28.0. IR ν_max (neat): 2979, 2866, 2222, 1674, 1598, 1573, 1425, 1393, 1364, 1241, 1157, 1123, 992, 952, 783, 683. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₆H₂₂N₃O₂⁺, 288.1707; found, 288.1709 (error −0.86 ppm).

tert-Butyl 4-(m-tolyl)piperazine-1-carboxylate (5i).

Yield: 51%. Light brown solid. M.P. 66.3-67.1 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 7.07-7.12 (m, 1H), 6.71-6.75 (m, 2H), 6.61-6.63 (m, 1H), 3.42-3.45 (m, 4H), 3.03-3.06 (m, 4H), 2.25 (s, 3H), 1.42 (s, 9H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 153.8, 150.9, 137.9, 128.7, 120.1, 116.6, 113.1, 78.8, 48.4, 43.2 (br), 28.0, 21.3. IR ν_max (neat): 2973, 2917, 2820, 1694, 1599, 1423, 1385, 1363, 1421, 1157, 1118, 948, 767, 688. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₆H₂₅N₂O₂⁺, 277.1911; found, 277.1911 (error 0 ppm).

tert-Butyl 4-(3-isopropylphenyl)piperazine-1-carboxylate (5j).

Yield: 72%. White solid. M.P. 39.7-40.5 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 7.10-7.15 (m, 1H), 6.81 (s, 1H), 6.68-6.75 (m, 2H), 3.43-3.45 (m, 4H), 3.04-3.07 (m, 4H), 2.81 (hept, J = 6.8 Hz, 1H), 1.42 (s, 9H), 1.18 (d, J = 6.8 Hz, 6H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 153.8, 151.0, 149.1, 128.7, 117.4, 114.3, 113.5, 78.8, 48.6, 43.1 (br), 33.7, 28.0, 23.9. IR ν_max (neat): 2958, 2861, 2822, 1687, 1605, 1421, 1364, 1339, 1213, 1160, 1121, 953, 778, 703. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₈H₂₉N₂O₂⁺, 305.2224; found, 305.2224 (error 0 ppm).

tert-Butyl 4-(3-methoxyphenyl)piperazine-1-carboxylate (5k).

Yield: 36%. White solid. M.P. 59.8-61.8 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 7.09-7.14 (m, 1H), 6.38-6.53 (m, 3H), 3.71 (s, 3H), 3.42-3.45 (m,4H), 3.05-3.09 (m, 4H), 1.42 (s, 9H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 160.2, 153.8, 152.2, 129.6, 108.5, 104.6, 102.0, 78.9, 54.8, 48.3, 43.1 (br), 28.0. IR ν_max (neat): 2973, 2923, 2873, 1690, 1585, 1417, 1369, 1245, 1162, 995, 945, 819, 760, 684. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₁₆H₂₅N₂O₃⁺, 293.1860; found, 293.1858 (error 0.58 ppm).

4.1.5. General Procedure for the Synthesis of Pyrazolo[1,5-a]pyrimidine-3-carboxamides (21-48).

The Boc protected 4-phenylpiperazines (1.5 mmol) were dissolved in 8 ml of 4M HCl/EtOAc and the reaction mixture was stirred at room temperature for 2 h. Then the reaction mixture was evaporated in vacuo to give the crude piperazine hydrochlorides as near white solid, which was subjected to the next reaction without further purification.

To a mixture of pyrazolo[1,5-a]pyrimidine-3-carboxylic acids (1.0 mmol, 1.0 eq.) and amine (1.1 mmol, 1.1 eq.) in DMF (8 mL) was added HATU (1.5 mmol, 1.5 eq.), followed by DIEA (3.0 mmol, 3.0 eq.). The mixture was stirred at room temperature overnight and then diluted with 80 mL EtOAc and washed with 30 mL saturated aqueous NaCl. The organic layer was dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by column chromatography (silica gel, petroleum ether (b.p.60-90°C)/ethyl acetate, 20:1 to 1:3) to afford the desired compounds as solid.

(5,7-diethyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidin-3-yl)(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)methanone (21).

Yield: 87%. White solid. M.P. 64.4-66.0 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.40 (s, 1H), 7.40-7.45 (m, 1H), 7.21-7.25 (m, 2H), 7.08-7.10 (m, 3H), 6.98-7.01 (m, 2H), 4.15 (s, 2H), 3.78 (s, 4H), 3.34 (s, 4H), 3.21 (q, J = 7.4 Hz, 2H), 2.75 (q, J = 7.3 Hz, 2H), 2.24 (s, 3H), 1.13-1.20 (m, 6H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 164.7, 162.7, 151.1, 148.9, 145.8, 142.7, 135.7, 135.3, 130.0, 129.9 (q, J = 30.8 Hz), 129.2, 127.6, 124.3 (q, J = 270.8 Hz), 119.0, 117.0, 114.9 (q, J = 3.6 Hz), 111.2 (q, J = 3.7 Hz), 104.8, 48.2 (br), 31.2, 28.3, 20.7, 20.5, 11.5, 10.9. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −61.1. IR ν_max (neat): 2935, 1732, 1610, 1544, 1514, 1449, 1350, 1306, 1232, 1158, 1116, 994, 944, 844, 786, 695. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₃₀H₃₃F₃N₅O⁺, 536.2632; found, 536.2632 (error 0 ppm).

(5-ethyl-7-methyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidin-3-yl)(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)methanone (22).

Yield: 84%. White solid. M.P. 62.3-63.7 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.39 (s, 1H), 7.39-7.44 (m, 1H), 7.21-7.24 (m, 2H), 7.07-7.10 (m, 3H), 6.98-7.01 (m, 2H), 4.12 (s, 2H), 3.75 (s, 4H), 3.34 (m, 4H), 3.21 (q, J = 7.2 Hz, 2H), 2.43 (s, 3H), 2.23 (s, 3H), 1.18 (t, J = 7.2 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.6, 161.1, 151.0, 148.8, 145.4, 142.8, 135.5, 135.3, 130.1, 129.9 (q, J = 30.8 Hz), 129.2, 127.6, 124.3 (q, J = 270.7 Hz), 118.9, 117.4, 114.8 (q, J = 3.6 Hz), 111.2 (q, J = 3.7 Hz), 104.6, 48.0 (br), 31.8, 23.7, 20.6, 20.4, 10.9. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −61.1. IR ν_max (neat): 2938, 1613, 1547, 1514, 1449, 1391, 1318, 1306, 1232, 1159, 1116, 993, 944, 857, 782, 696. HRMS (ESI+): m/z [M+Na]⁺ calcd. for C₂₉H₃₀F₃N₅ONa⁺, 544.2295; found, 544.2292 (error 0.49 ppm).

(6-benzylpyrazolo[1,5-a]pyrimidin-3-yl)(4-phenylpiperazin-1-yl)methanone (23).

Yield: 85%. White solid. M.P. 120.7-122.8 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 9.18 (s, 1H), 8.66 (m, 1H), 8.41 (s, 1H), 7.29-7.38 (m, 4H), 7.18-7.23 (m, 3H), 6.92-6.95 (m, 2H), 6.77-6.81 (m, 1H), 4.06 (s, 2H), 3.70 (brs, 4H), 3.17 (brs, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.1, 153.5, 150.8, 146.0, 143.0, 139.6, 134.5, 128.9, 128.7, 126.5, 122.9, 119.3, 115.9, 105.4, 48.8, 34.5. IR ν_max (neat): 3059, 2919, 2853, 1669, 1627, 1494, 1384, 1231, 1205, 1007, 842, 761, 698. HRMS (ESI+): m/z [2M+H]⁺ calcd. for C₄₈H₄₇N₁₀O₂⁺, 795.3878; found, 795.3876 (error 0.25 ppm).

(6-(4-fluorobenzyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)methanone (24).

Yield: 69%. White solid. M.P. 60.6-62.2 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.39 (s, 1H), 7.40-7.45 (m, 1H), 7.17-7.25 (m, 4H), 7.07-7.13 (m, 3H), 4.17 (s, 2H), 3.75 (s, 4H), 3.34 (s, 4H), 2.79 (s, 3H), 2.45 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.6, 160.8 (d, J = 240.8 Hz), 160.6, 151.0, 145.4, 144.7, 142.7, 134.5 (d, J = 2.8 Hz), 130.0, 129.9 (q, J = 30.7 Hz), 129.7, 129.6, 124.3 (q, J = 270.8 Hz), 118.9, 118.1, 115.4, 115.1, 114.8 (q, J = 3.7 Hz), 111. 2 (q, J = 3.7 Hz), 104.6, 48.0 (br), 31.8, 23.7, 13.6. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −61.1, −116.8. IR ν_max (neat): 2916, 1731, 1614, 1548, 1507, 1429, 1352, 1318, 1306, 1220, 1156, 1115, 989, 943, 845, 784, 695. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₂₇H₂₆F₄N₅O⁺, 512.2068; found, 512.2062 (error 1.17 ppm).

(6-(4-chlorobenzyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)methanone (25).

Yield: 67%. White solid. M.P. 180.1-181.8 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.39 (s 1H), 7.40-7.45 (m, 1H), 7.32-7.35 (m, 2H), 7.17-7.26 (m, 4H), 7.07-7.09 (m, 1H), 4.18 (s, 2H), 3.74 (s, 4H), 3.35 (s, 4H), 2.78 (s, 3H), 2.44 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.6, 160.6, 151.1, 145.4, 144.9, 142.8, 137.6, 130.9, 130.0, 129.9 (q, J = 31.0 Hz), 129.7, 128.5, 124.4 (q, J = 270.7 Hz), 119.0, 117.9, 114.8 (q, J = 3.6 Hz), 111.3 (q, J = 3.6 Hz), 104.7, 48.1 (br), 32.0, 23.8, 13.7. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −61.1. IR ν_max (neat): 3103, 2816, 1608, 1543, 1447, 1431, 1382, 1352, 1319, 1157, 1100, 992, 942, 845, 784, 772. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₂₇H₂₆ClF₃N₅O⁺, 528.1773; found, 528.1762 (error 1.98 ppm).

(6-(4-methoxybenzyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)methanone (26).

Yield: 90%. White solid. M.P. 53.7-55.5 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.38 (s, 1H), 7.39-7.44 (m, 1H), 7.21-7.24 (m, 2H), 7.03-7.08 (m, 3H), 6.82-6.85 (m, 2H), 4.08 (s, 2H), 3.74 (s, 4H), 3.69 (s, 3H), 3.34 (s, 4H), 2.78 (s, 3H), 2.45 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.7, 160.7, 157.8, 151.1, 145.4, 144.5, 142.7, 130.1, 130.0, 129.9 (q, J = 30.8 Hz), 128.8, 124.4 (q, J = 270.8 Hz), 119.0, 118.7, 114.9 (q, J = 3.7 Hz), 114.1, 111.3 (q, J = 3.7 Hz), 104.6, 55.0, 48.1 (br), 31.8, 23.7, 13.7. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −61.1. IR ν_max (neat): 1613, 1583, 1510, 1445, 1383, 1312, 1245, 1113, 1025, 990, 839, 790, 699. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₂₈H₂₉F₃N₅O₂⁺, 524.2268; found, 524.2264 (error 0.73 ppm).

(5,7-dimethyl-6-(3-methylbenzyl)pyrazolo[1,5-a]pyrimidin-3-yl)(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)methanone (27).

Yield: 95%. White solid. M.P. 62.5-65.0 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.39 (s, 1H), 7.39-7.45 (m, 1H), 7.14-7.25 (m, 3H), 7.07-7.09 (m, 1H), 6.97-7.02 (m, 2H), 6.89-6.92 (m, 1H), 4.13 (s, 2H), 3.74 (s, 4H), 3.34 (s, 4H), 2.78 (s, 3H), 2.45 (s, 3H), 2.24 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.6, 160.7, 151.1, 145.3, 144.7, 142.7, 138.3, 137.8, 130.0, 129.9 (q, J = 30.8 Hz), 128.5, 128.4, 127.0, 124.8, 124.3 (q, J = 270.6 Hz), 119.0, 118.3, 114.8 (q, J = 3.4 Hz), 111.3 (q, J = 3.5 Hz), 104.6, 48.1 (br), 32.6, 23.8, 21.0, 13.7. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −61.1. IR ν_max (neat): 2925, 1613, 1550, 1446, 1383, 1315, 1160, 1114, 1025, 990, 838, 791, 771, 734, 702. HRMS (ESI+): m/z [M+Na]⁺ calcd. for C₂₈H₂₈F₃N₅ONa⁺, 530.2138; found, 530.2135 (error 0.59 ppm).

(5,7-dimethyl-6-(2-methylbenzyl)pyrazolo[1,5-a]pyrimidin-3-yl)(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)methanone (28).

Yield: 84%. White solid. M.P. 91.1- 93.5 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.40 (s, 1H), 7.40-7.45 (m, 1H), 7.23-7.26 (m, 3H), 7.07-7.14 (m, 2H), 6.98-7.03 (m, 1H), 6.55-6.57 (m, 1H), 4.06 (s, 2H), 3.77 (s, 4H), 3.38 (s ,4H), 2.69 (s, 3H), 2.44 (s, 3H), 2.39 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.7, 160.8, 151.1, 145.4, 144.9, 142.9, 136.3, 136.1, 130.0, 129.9 (q, J = 30.8 Hz), 126.2, 126.2, 125.8, 124.4 (q, J = 270.9 Hz), 119.0, 117.7, 114.8 (q, J = 3.6 Hz), 111.3 (q, J = 3.6 Hz), 104.7, 48.0 (br), 30.0, 23.4, 19.4, 13.5. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −61.1. IR ν_max (neat): 2914, 1615, 1548, 1432, 1352, 1318, 1307, 1159, 1117, 1027, 991, 944, 840, 765, 696. HRMS (ESI+): m/z [M+Na]⁺ calcd. for C₂₈H₂₈F₃N₅ONa⁺, 530.2138; found, 530.2130 (error 1.54 ppm).

(6-(4-isopropylbenzyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)methanone (29).

Yield: 65%. White solid. M.P. 176.4-177.9 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.38 (s, 1H), 7.40-7.45 (m, 1H), 7.21-7.25 (m, 2H), 7.14-7.17 (m, 2H), 7.04-7.09 (m, 3H), 4.14 (s, 2H), 3.74 (s, 4H), 3.34 (s, 4H), 2.79-2.88 (m, 4H), 2.46 (s, 3H), 1.15 (d, J = 6.8 Hz, 6H). 13C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.6, 160.7, 151.0, 146.3, 145.3, 144.5, 142.7, 135.6, 130.0, 129.8 (q, J = 31.1 Hz), 127.7, 126.5, 124.3 (q, J = 270.8 Hz), 118.9, 118.4, 114.8 (q, J = 3.7 Hz), 111.2 (q, J = 3.7 Hz), 104.6, 48.0 (br), 32.9, 32.2, 23.8, 13.7. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −61.1. IR νmax (neat): 2959, 1614, 1541, 1447, 1428, 1376, 1351, 1235, 1156, 1118, 1026, 992, 846, 774, 691. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₃₀H₃₃F₃N₅O⁺, 536.2632; found, 536.2631 (error 0.13 ppm).

(5,7-dimethyl-6-(4-(trifluoromethyl)benzyl)pyrazolo[1,5-a]pyrimidin-3-yl)(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)methanone (30).

Yield: 60%. White solid. M.P. 202.6- 203.6 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.40 (s, 1H), 7.63-7.66 (m, 2H), 7.38-7.45 (m, 3H), 7.21-7.26 (m, 2H), 7.07-7.10 (m, 1H), 4.31 (s, 2H), 3.75 (s, 4H), 3.35 (s, 4H), 2.80 (s, 3H), 2.45 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.5, 160.5, 151.0, 145.4, 145.0, 143.5, 142.8, 129.9, 129.8 (q, J = 30.8 Hz), 128.7, 127.0 (q, J = 31.5 Hz), 125.3 (q, J = 3.6 Hz), 124.3 (q, J = 270.7 Hz), 124.2 (q, J = 270.3 Hz), 118.9, 117.4, 114.8 (q, J = 3.7 Hz), 111.2 (q, J = 3.7 Hz), 104.7, 48.0 (br), 32.5, 23.7, 13.7. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −60.8, −61.1. IR ν_max (neat): 2816, 1609, 1447, 1432, 1383, 1319, 1159, 1111, 1066, 1018, 993, 943, 849, 788, 691. HRMS (ESI+): m/z [M+Na]⁺ calcd. for C₂₈H₂₅F₆N₅ONa⁺, 584.1856; found, 584.1851 (error 0.77 ppm).

(6-ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)(4-phenylpiperazin-1-yl)methanone (31).

Yield: 73%. White solid. M.P. 143.0-144.4 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.33 (s, 1H), 7.19-7.25 (m, 2H), 6.95-6.98 (m, 2H), 6.77-6.82 (m, 1H), 3.72 (brs, 4H), 3.22 (brs, 4H), 2.69-2.74 (m, 5H), 2.60 (s, 3H), 1.13 (t, J = 7.3 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.6, 160.0, 150.9, 145.1, 143.3, 142.4, 129.0, 121.4, 119.2, 115.8, 104.4, 48.8 (br), 23.3, 20.6, 13.6, 12.9. IR ν_max (neat): 2966, 2923, 2864, 1617, 1596, 1542, 1429, 1370, 1227, 1191, 837, 754, 689. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₄₂H₅₀N₁₀O₂Na⁺, 749.4010; found, 749.4010 (error 0 ppm).

(5,7-dimethyl-6-phenylpyrazolo[1,5-a]pyrimidin-3-yl)(4-phenylpiperazin-1-yl)methanone (32).

Yield: 84%. White solid. M.P. 97.0-98.5 °C. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 8.43 (s, 1H), 7.53-7.56 (m, 2H), 7.48-7.50 (m, 1H), 7.38-7.40 (m, 2H), 7.21-7.24 (m, 2H), 6.96-6.98 (m, 2H), 6.80 (t, J = 7.2 Hz, 1H), 3.77 (s, 4H), 3.24-3.26 (m, 4H), 2.48 (s, 3H), 2.29 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm): 162.4, 159.2, 150.9, 145.7, 143.8, 143.0, 134.9, 134.8, 129.9, 128.9, 128.8, 128.2, 122.4, 119.2, 115.8, 104.8, 48.8, 24.6, 14.8. IR ν_max (neat): 3056, 2920, 2850, 1722, 1614, 1597, 1494, 1385, 1228, 839, 757, 704, 692. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₅₀H₅₀N₁₀O₂Na⁺, 845.4010; found, 845.4010 (error 0 ppm).

(5,7-dimethyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidin-3-yl)(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)methanone (33).

Yield: 92%. White solid. M.P. 76.5-78.1 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.39 (s, 1H), 7.50 (d, J = 8.6 Hz, 2H), 7.00-7.10 (m, 6H), 4.11 (s, 2H), 3.74 (s, 4H), 3.40 (s, 4H), 2.78 (s, 3H), 2.45 (s, 3H), 2.24 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.6, 160.6, 153.0, 145.3, 144.5, 142.7, 135.3, 135.2, 129.2, 127.7, 126.1 (q, J = 3.5 Hz), 124.8 (q, J = 268.7 Hz), 118.4, 118.0 (q, J = 31.8 Hz), 114.3, 104.6, 47.3 (br), 32.2, 23.7, 20.5, 13.6. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −59.5. IR ν_max (neat): 2920, 2855, 1613, 1547, 1522, 1428, 1385, 1326, 1230, 1193, 1104, 1069, 988, 824. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₂₈H₂₉F₃N₅O⁺, 508.2319; found, 508.2302 (error 3.29 ppm).

(5,7-dimethyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidin-3-yl)(4-(2-(trifluoromethyl)phenyl)piperazin-1-yl)methanone (34).

Yield: 91%. White solid. M.P. 171.4-173.4 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.38 (s, 1H), 7.58-7.69 (m, 3H), 7.33-7.38 (m, 1H), 7.08 (d, J = 7.7 Hz, 2H), 7.01 (d, J = 7.7 Hz, 2H), 4.12 (s, 2H), 3.72 (s, 4H), 2.93 (s ,4H), 2.77 (s, 3H), 2.46 (s, 3H), 2.24 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.5, 160.6, 151.9, 145.2, 144.5, 142.7, 135.3, 135.2, 133.6, 129.2, 127.7, 126.8 (q, J = 5.2 Hz), 125.7 (q, J = 27.8 Hz), 125.4, 124.7, 124.0 (q, J = 271.6 Hz), 118.4, 104.7, 53.4 (br), 32.2, 23.7, 20.5, 13.6. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −58.7. IR ν_max (neat): 2916, 2810, 1620, 1548, 1514, 1481, 1430, 1377, 1312, 1120, 1107, 1036, 1025, 988, 771. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₂₈H₂₉F₃N₅O⁺, 508.2319; found, 508.2304 (error 2.89 ppm).

(4-(3-(difluoromethyl)phenyl)piperazin-1-yl)(5,7-dimethyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidin-3-yl)methanone (35).

Yield: 89%. White solid. M.P. 82.3-84.3 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.38 (s, 1H), 7.32-7.37 (m, 1H), 7.07-7.13 (m, 4H), 6.96-7.03 (m, 3H), 6.92 (t, J = 56.0 Hz, 1H), 4.12 (s, 2H), 3.74 (s, 4H), 3.30 (s, 4H), 2.78 (s, 3H), 2.45 (s, 3H), 2.24 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.6, 160.7, 150.9, 145.3, 144.5, 142.7, 135.3, 135.2, 134.8 (t, J = 21.7 Hz), 129.6, 129.2, 127.7, 118.4, 118.3, 117.8 (br), 115.7 (t, J = 5.7 Hz), 115.2 (t, J = 234.7 Hz), 112.5 (t, J = 6.3 Hz), 112.1, 104.6, 48.3 (br), 32.2, 23.7, 20.5, 13.6. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −109.0. IR ν_max (neat): 2921, 1614, 1548, 1480, 1432, 1377, 1236, 1192, 1026, 992, 840, 792, 774, 699. HRMS (ESI+): m/z [M+Na]⁺ calcd. for C₂₈H₂₉F₂N₅ONa⁺, 512.2232; found, 512.2232 (error 0 ppm).

3-(4-(5,7-dimethyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidine-3-carbonyl)piperazin-1-yl)benzo nitrile (36).

Yield: 94%. White solid. M.P. 83.8-85.8 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.38 (s, 1H), 7.37-7.42 (m, 2H), 7.29-7.31 (m, 1H), 7.16-7.19 (m, 1H), 7.09 (d, J = 7.8 Hz, 2H), 7.02 (d, J = 7.8 Hz, 2H), 4.13 (s, 2H), 3.72 (s, 4H), 3.34 (s, 4H), 2.78 (s, 3H), 2.45 (s, 3H), 2.25 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.6, 160.7, 150.8, 145.3, 144.6, 142.7, 135.3, 135.2, 130.2, 129.2, 127.7, 121.8, 119.9, 119.2, 118.4, 117.9, 112.0, 104.6, 47.7 (br), 32.2, 23.8, 20.5, 13.7. IR ν_max (neat): 2921, 2227, 1615, 1595, 1547, 1430, 1376, 1236, 1193, 1028, 991, 840, 772. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₂₈H₂₉N₆O⁺, 465.2397; found, 465.2401 (error −0.78 ppm).

(5,7-dimethyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidin-3-yl)(4-(3-fluorophenyl)piperazin-1-yl)methanone (37).

Yield: 96%. White solid. M.P. 62.0-63.4 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.38 (s, 1H), 7.17-7.25 (m, 1H), 7.09 (d, J = 7.8 Hz, 2H), 7.02 (d, J = 7.8 Hz, 2H), 6.74-6.79 (m, 2H), 6.53-6.58 (m, 1H), 4.12 (s, 2H), 3.72 (s, 4H), 3.28 (s, 4H), 2.78 (s, 3H), 2.45 (s, 3H), 2.25 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 163.2 (d, J = 238.9 Hz), 162.6, 160.7, 152.5 (d, J = 10.0 Hz), 145.3, 144.5, 142.7, 135.3, 135.2, 130.3 (d, J = 10.0 Hz), 129.2, 127.7, 118.4, 111.1, 104.9 (d, J = 21.2 Hz), 104.6, 102.0 (d, J = 25.0 Hz), 48.1 (br), 32.2, 23.7, 20.5, 13.6. ¹⁹F NMR (282 MHz, DMSO-d₆) δ (ppm): −112.3. IR ν_max (neat): 2919, 1668, 1611, 1582, 1547, 1494, 1434, 1385, 1243, 1160, 992, 968, 834, 772. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₅₄H₅₆F₂N₁₀O₂Na⁺, 937.4448; found, 937.4448 (error 0 ppm).

(4-(3-chlorophenyl)piperazin-1-yl)(5,7-dimethyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidin-3-yl)methanone (38).

Yield: 70%. White solid. M.P. 70.6-72.5 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.37 (s, 1H), 7.18-7.23 (m, 1H), 7.08 (d, J = 7.8 Hz, 2H), 7.01 (d, J = 7.8 Hz, 2H), 6.89-6.96 (m, 2H), 6.77-6.80 (m, 1H), 4.11 (s, 2H), 3.72 (s, 4H), 3.28 (s, 4H), 2.78 (s, 3H), 2.45 (s, 3H), 2.24 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.5, 160.6, 152.0, 145.3, 144.5, 142.6, 135.3, 135.2, 133.8, 130.4, 129.2, 127.7, 118.4, 118.3, 114.9, 113.9, 104.6, 48.0 (br), 32.2, 23.7, 20.5, 13.6. IR ν_max (neat): 2916, 1618, 1589, 1550, 1479, 1429, 1383, 1192, 1025, 989, 774. HRMS (ESI+): m/z [M+H]⁺calcd. for C₂₇H₂₉ClN₅O⁺, 474.2055; found, 474.2058 (error −0.60 ppm).

(5,7-dimethyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidin-3-yl)(4-(3-methoxyphenyl)piperazin-1-yl)methanone (39).

Yield: 85%. White solid. M.P. 60.1-61.8 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.38 (s, 1H), 7.07-7.13 (m, 3H), 7.01 (d, J = 7.8 Hz, 2H), 6.48-6.54 (m, 2H), 6.36-6.39 (m, 1H), 4.10 (s, 2H), 3.70 (m, 7H), 3.22 (s, 4H), 2.77 (s, 3H), 2.44 (s, 3H), 2.23 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.5, 160.6, 160.2, 152.2, 145.3, 144.5, 142.6, 135.3, 135.2, 129.6, 129.2, 127.7, 118.4, 108.3, 104.7, 104.5, 101.9, 54.8, 48.7 (br), 32.2, 23.7, 20.5, 13.6. IR ν_max (neat): 2920, 1615, 1594, 1549, 1481, 1445, 1382, 1256, 1191, 1163, 1024, 991, 840, 774. HRMS (ESI+): m/z [M+Na]⁺ calcd. for C₂₈H₃₁N₅O₂Na⁺, 492.2370; found, 492.2366 (error 0.80 ppm).

(5,7-dimethyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidin-3-yl)(4-(m-tolyl)piperazin-1-yl)methano ne (40).

Yield: 79%. White solid. M.P. 78.9-80.5 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.37 (s, 1H), 7.07-7.11 (m, 3H), 7.02 (d, J = 7.8 Hz, 2H), 6.74-6.78 (m, 2H), 6.60-6.63 (m, 1H), 4.12 (s, 2H), 3.71 (s, 4H), 3.20 (s ,4H), 2.78 (s, 3H), 2.45 (s, 3H), 2.24-2.25 (m, 6H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.5, 160.6, 150.9, 145.3, 144.5, 142.7, 138.0, 135.3, 135.2, 129.2, 128.8, 127.7, 120.0, 118.4, 116.5, 113.0, 104.7, 48.8 (br), 32.2, 23.8, 21.4, 20.5, 13.7. IR ν_max (neat): 2920, 1669, 1615, 1600, 1547, 1433, 1385, 1241, 1193, 991, 836, 772, 693. HRMS (ESI+): m/z [M+Na]⁺ calcd. for C₂₈H₃₁N₅ONa⁺, 476.2421; found, 476.2408 (error 2.69 ppm).

(5,7-dimethyl-6-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidin-3-yl)(4-(3-isopropylphenyl)piperazin-1-yl)methanone (41).

Yield: 75%. White solid. M.P. 78.9-80.5 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.38 (s, 1H), 7.08-7.15 (m, 3H), 7.02 (d, J = 7.7 Hz, 2H), 6.83 (s, 1H), 6.74-6.77 (m, 1H), 6.67-6.70 (m, 1H), 4.12 (s, 2H), 3.72 (s, 4H), 3.20 (s 4H), 2.78-2.84 (m, 4H), 2.45 (s, 3H), 2.24 (s, 3H), 1.16 (d, J = 6.8 Hz, 6H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.5, 160.6, 151.0, 149.2, 145.3, 144.5, 142.7, 135.3, 135.2, 129.2, 128.8, 127.7, 118.4, 117.3, 114.3, 113.4, 104.7, 49.0 (br), 33.7, 32.2, 23.9, 23.7, 20.5, 13.7. IR ν_max (neat): 2958, 1668, 1615, 1599, 1548, 1434, 1385, 1237, 1196, 993, 835, 776, 700. HRMS (ESI+): m/z [M+Na]⁺ calcd. for C₃₀H₃₅N₅ONa⁺, 504.2734; found, 504.2732 (error 0.36 ppm).

6-benzyl-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxamide (42).

Yield: 60%. White solid. M.P. 245.5-248.0 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.46 (s, 1H), 7.57 (s, 1H), 7.42 (s, 1H), 7.26-7.31 (m, 2H), 7.18-7.23 (m, 1H), 7.12-7.15 (m, 2H), 4.20 (s, 2H), 2.79 (s, 3H), 2.50 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 163.0, 161.5, 145.5, 144.8, 144.0, 138.3, 128.7, 127.9, 126.4, 118.7, 104.4, 32.6, 23.7, 13.7. IR ν_max (neat): 3396, 3314, 3272, 3155, 2940, 1651, 1614, 1541, 1524, 1485, 1267, 1196, 797, 704, 635. HRMS (ESI+): m/z [2M+H]⁺ calcd. for C₃₂H₃₃N₈O₂⁺, 561.2721; found, 561.2708 (error 2.31 ppm).

6-benzyl-N-cyclohexyl-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxamide (43).

Yield:57%. White solid. M.P. 180.1-181.8 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.46 (s, 1H), 8.07 (d, J = 7.7 Hz, 1H), 7.26-7.31 (m, 2H), 7.17-7.22 (m, 1H), 7.12-7.14 (m, 2H), 4.19 (s, 2H), 3.86 (m, 1H), 2.79 (s, 3H), 2.50 (s, 3H), 1.84-1.87 (m, 2H), 1.66 (m, 2H), 1.52 (m, 1H), 1.27-1.40 (m, 5H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 161.3, 160.4, 145.7, 144.4, 143.7, 138.2, 128.6, 127.8, 126.4, 118.7, 104.3, 46.4, 32.6, 32.4, 25.2, 24.0, 13.7. IR ν_max (neat): 3314, 2929, 2851, 1643, 1612, 1548, 1517, 1427, 1194, 778, 743, 703, 645. HRMS (ESI+): m/z [2M+H]⁺ calcd. for C₄₄H₅₃N₈O₂⁺, 725.4286; found, 725.4286 (error 0 ppm).

6-benzyl-5,7-dimethyl-N-phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (44).

Yield: 75%. White solid. M.P. 211.3-212.6 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 10.19 (s, 1H), 8.64 (s, 1H), 7.73-7.76 (m, 2H), 7.34-7.39 (m, 2H), 7.28-7.31 (m, 2H), 7.22-7.24 (m, 1H), 7.16-7.19 (m, 2H), 7.09 (m, 1H), 4.25 (s, 2H), 2.85 (s, 3H), 2.62 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.0, 159.8, 146.1, 144.9, 143.7, 138.8, 138.2, 129.0, 128.7, 127.9, 126.4, 123.3, 119.3, 119.1, 104.0, 32.6, 23.9, 13.8. IR ν_max (neat): 3297, 3256, 3197, 3146, 1779, 1662, 1600, 1543, 1499, 1481, 1381, 1312, 1257, 1196, 744, 706. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₂₂H₂₁N₄O⁺, 357.1710; found, 357.1713 (error −0.88 ppm).

(6-benzyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)(piperidin-1-yl)methanone (45).

Yield: 96%. White solid. M.P. 69.2-71.0 °C. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 8.30 (s, 1H), 7.27-7.30 (m, 2H), 7.18-7.21 (m, 1H), 7.12-7.14 (m, 2H), 4.17 (s, 2H), 3.53 (brs, 4H), 2.77 (s, 3H), 2.44 (s, 3H), 1.59-1.61 (m, 2H), 1.55-1.56 (m, 4H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm): 162.3, 160.3, 144.9, 144.4, 142.6, 138.5, 128.6, 127.8, 126.3, 118.0, 105.3, 48.2 (br), 42.6 (br), 32.6, 25.8 (br), 24.1, 23.7, 13.7. IR ν_max (neat): 2933, 2853, 1733, 1674, 1614, 1547, 1434, 1386, 1252, 982, 835, 770, 734, 703, 655. HRMS (ESI+): m/z [2M+Na]⁺ calcd. for C₄₂H₄₈N₈O₂Na⁺, 719.3792; found, 719.3792 (error 0 ppm).

tert-Butyl 4-(6-benzyl-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carbonyl)piperazine-1-carboxylate (46).

Yield: 86%. White solid. M.P. 186.3-188.8 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.35 (s, 1H); 7.27-7.31 (m, 2H), 7.21-7.23 (m, 1H), 7.13-7.15 (m, 2H), 4.18 (s, 2H), 3.55(s, 4H), 3.44(s, 4H), 2.78 (s, 3H), 2.46 (s, 3H), 1.41 (s, 9H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.7, 160.7, 153.8, 145.4, 144.7, 142.7, 138.4, 128.7, 127.9, 126.3, 118.3, 104.6, 79.1, 43.8, 32.6, 28.0, 23.8, 13.7. IR ν_max (neat): 3100, 2990, 2851, 1697, 1609, 1541, 1418, 1364, 1237, 1166, 837, 772, 742, 708, 657. HRMS (ESI): m/z [M+H]⁺ calcd. for C₂₅H₃₂N₅O₃⁺, 450.2500; found, 450.2508 (error −1.85 ppm).

(6-benzyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)(piperazin-1-yl)methanone (47).

tert-Butyl 4-(6-benzyl-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carbonyl)piperazine-1-carboxylate 46 (600 mg, 1.33 mmol, 1.0 eq.) was dissolved in 6 mL of 4 M HCl in EtOAc. The solution was stirred at room temperature for 4h, and then the solvent was removed in vacuo. The residue was partitioned between 30 mL DCM and 30 mL saturated sodium bicarbonate. The aqueous layer was extracted with DCM (20 mL×2). The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated in vacuo to give compound 47 (173 mg, 0.49 mmol). Yield: 37%. White solid. M.P. 53.7-55.4 °C. ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.32 (s, 1H), 7.26-7.30 (m, 2H), 7.17-7.22 (m, 1H), 7.12-7.14 (m, 2H), 4.16 (s, 2H), 3.49 (s, 4H), 2.88-2.98 (m, 1H), 2.77 (s, 3H), 2.74 (s, 4H), 2.44 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ (ppm): 162.4, 160.3, 145.0, 144.5, 142.6, 138.4, 128.6, 127.8, 126.3, 118.0, 105.0, 45.9, 32.6, 23.7, 13.6. IR ν_max (neat): 3491, 3311, 2918, 2852, 1732, 1614, 1547, 1427, 1386, 1230, 1192, 1027, 988, 769, 735, 705. HRMS (ESI+): m/z [M+H]⁺ calcd. for C₂₀H₂₄N₅O⁺, 350.1975; found, 350.1981 (error −1.61 ppm).

(6-benzyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)(4-(prop-2-yn-1-yl)piperazin-1-yl)methanone (48).

(6-benzyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)(piperazin-1-yl)methanone 47 (240 mg, 0.69 mmol, 1.0 eq.) was dissolved in anhydrous DCM (10 mL), and anhydrous K₂CO₃ (477 mg, 3.45 mmol, 5.0 eq.) was added. The mixture was vigorously stirred at room temperature, and neat propargyl bromide (204 mg, 1.72 mmol, 2.5 eq.) was added. The reaction was stirred at room temperature overnight, K₂CO₃ was filtered, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, Dichloromethane/Methanol, 200:1 to 30:1) to give compound 48. (36 mg, 0.09 mmol) Yield: 13%. White solid. M.P. 177.2-179.3 °C. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 8.34 (s, 1H), 7.29 (m, 2H), 7.20 (m, 1H), 7.13-7.14 (m, 2H), 4.18 (s, 2H), 3.59 (brs, 4H), 3.32 (d, J = 2.2 Hz, 2H), 3.18 (t, J = 2.2 Hz, 1H), 2.77 (s, 3H), 2.52-2.54 (m, 4H), 2.45 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm): 162.5, 160.6, 145.2, 144.6, 142.7, 138.5, 128.7, 127.9, 126.4, 118.2, 104.8, 79.1, 76.0, 51.6, 47.1, 46.0, 41.8, 32.6, 23.8, 13.7. IR ν_max (neat): 3290, 3023, 2993, 2922, 2856, 2828, 1614, 1552, 1451, 1430, 1376, 1234, 1193, 1138, 1000, 888, 709. HRMS (ESI+): m/z [2M+H]⁺ calcd. for C₄₆H₅₁N₁₀O₂⁺, 775.4191; found, 775.4191 (error 0 ppm).

4.2. Docking-Based Virtual Screening

The NCI compound library containing approximately 260,000 compounds was screened through the MTiOpen screen automated virtual screening platform using the integrated Vina AutoDock program against RUVBL1/2 complex (PDB code: 5oaf) [38, 39]. The duplicate compounds were removed using Mona [40]. The 3D structure of all the compounds were generated and the energy minimization was performed by Frog2 [41]. The settings for compound library filters were as follows: the number of hydrogen bond acceptors < 10, the number of hydrogen bonds donors < 5, LogP < 5, molecular weight < 500, the number of rotatable bonds < 10, the topological polar surface area <140 [42]. The Grid center coordinates were 14.5666, 189.849, and 166.659. The sizes of the search space [Å] was 50 × 50 × 50. The top 3,000 results were visualized, and 80 compounds were selected to evaluate the inhibition activity against RUVBL1/2 complex.

4.3. Molecular cloning and Sequencing

The cDNA sequences of RUVBL1 and RUVBL2 were amplified using Platinum SuperFi PCR Master Mix (Thermofisher, Cat. No. 12358-010). PCR product was purified after electrophoresis through 0.8% Agarose and extracted using QIAquick Gel Extraction kit (QIAGEN, Cat. No. 28115). Both RUVBL1 and RUVBL2 were inserted into pCOLA-duet vector by Gibson assembly. Molecular cloning was performed using a NEB PCR Cloning Kit (Biolabs, NEB #E1202) according to the manufacturer’s protocol. After overnight incubation, 5 clones were picked and plasmids were purified using a Plasmid miniprep kit (Bioland, Cat. No. PD01-01). Purified plasmids were sequenced by Sanger sequencing. PCR and sequencing primers are listed in Tables S4 and S5.

4.4. Expression and Purification of Recombinant Protein

Full-length recombinant His6x-tagged human RUVBL1 and RUVBL2 were expressed in E. coli Rosetta cells. The Rosetta cells were suspended in lysis buffer composed of 20 mM Tris, pH 8.0, 200 mM NaCl, 4 mM MgCl₂, 20 mM imidazole, β-mercaptoethanol, 50 mL/pill protease inhibitor, and 5% glycerol. The cell suspension was sonicated for three rounds at 65, 70 and 70 amp, respectively. The mixture was then centrifuged for 1.5 hours at 15000 x g. The resulting supernatant was loaded onto a Ni-NTA column and eluted with a linear gradient of imidazole (0 min, 20 mM Tris pH 8.0, 300 mM NaCl, 4 mM MgCl₂, 20 mM Imidazole, 1 mM DTT, and 5% glycerol; 50 min, 20 mM Tris pH 8.0, 300 mM NaCl, 4 mM MgCl₂, 400 mM Imidazole, 1 mM DTT, and 5% glycerol; 60 min, 20 mM Tris pH 8.0, 300 mM NaCl, 4 mM MgCl₂, 400 mM Imidazole, 1 mM DTT, and 5% glycerol). SDS-Page was employed to probe the fractions containing RUVBL1/2 complex. The RUVBL1/2 solution was condensed and further purified by size-exclusion chromatography in 20 mM Tris pH 7.5, 300 mM sodium chloride, 1 mM magnesium chloride, 1 mM DTT, and 10% glycerol using a Superose 6 Increase 10/300 GL column (GE Healthcare), and stored in aliquots at −80 °C with glycerol.

4.5. ATPase Assay

The purified protein was assayed in 50 μL ATPase assay buffer (50 mM Tris pH 7.4, 20 mM MgCl₂, 0.1% Glycerol, and 0.01% Triton X-100) containing 200 μM ATP. After 70 min incubation at 37 °C, 50 μL Biomol Green reagent (Enzo Life Sciences) was added to quench the reaction, and the absorbance at 635 nm was measured using a BioTek Synergy Neo 2 plate reader. The eight-dose titrations (0, 2.00, 6.58, 9.88, 14.81, 22.22, 33.33, 50.00 μM) were performed of the compound to the reaction to determine the IC₅₀ values of the compounds. In this assay, the concentration of RUVBL1/2, RUVBL, and RUVBL2 were 0.05 μM, 1.0 μM, and 3.0 μM, respectively. The results were calculated from six replicates using GraphPad Prism 7.0.

4.6. Antiproliferative assay

Cell viability was measured using CellTiter Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer’s procedure. RMPI1640 or DMEM containing 5% FBS and 1% Penicillin Streptomycin (Thermo Fisher) was used as cell viability assay medium. To find the linear relationship between the relative luminescence unit and the number of viable cells, a standard curve for each cell line was generated. Generally, 30 μL of cell suspension was plated in 384-well white plates (Greiner) with serial 2-fold dilution (from 30000 to 284 cells per well). Twenty-four hours after seeding, 8 μL of assay media containing 5% DMSO was added into each well and plates were incubated for an additional 48 h at 37 °C in a 5% CO₂ incubator. To test the anti-proliferative activity of RUVBL1/2 complex inhibitors, cells were seeded at 750 to 3000 cells per well according to the linear range determined from the standard curve of each cell line. Twenty-four hours after seeding, cells were treated with the compounds (three-fold dilution, eight concentrations). After 48 hours of treatment, cell viability was measured using CellTiter Glo and IC₅₀ values were calculated using the percentage of growth of treated cells versus the DMSO control.

4.7. Proteomics

HCT116 cells were treated with DMSO, 15 μM or 30 μM tested compound for 24 h, and pellets were harvested. The LC-MS samples were prepared by following the instructions of Thermo EasyPep Mini MS Sample Prep Kit (REF A4006) and peptide concentration was tested through Pierce Quantitative Fluorometric Peptide Assay (cat# 23290).

The LC-MS/MS experiments were performed using an EASY-nLC 1000 (ThermoFisher Scientific, San Jose, CA) connected to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA). The sample (1 μg) in 0.1% FA solution was loaded onto an Aurora UHPLC Column (25 cm x 75 μm, 1.6 μm C18, AUR2-25075C18A, Ion Opticks) and separated over 136 min at a flow rate of 0.35 μL/min with the following gradient: 2-6% Solvent B (7.5 min), 6-25% B (82.5 min), 25-40% B (30 min), 40-98% B (1 min), and 98% B (15 min). Solvent A consisted of 97.9% H₂O, 2% ACN, and 0.1% formic acid, and solvent B consisted of 19.9% H₂O, 80% ACN, and 0.1% formic acid. An MS1 scan was acquired in the Orbitrap at 120,000 resolution with a scan range of 350-1500 m/z. The AGC target was 4x10⁵, and the maximum injection time was 50 mins. Dynamic exclusion was set to exclude features after 1 time for 60 s with a 10-ppm mass tolerance. Higher-energy collisional dissociation (HCD) fragmentation was performed with 35% collision energy after quadruple isolation of features using a 1.6 m/z isolation window, 5×10⁴ AGC target, and 35 ms maximum injection time. MS2 scans were then also acquired by the Orbitrap with 50,000 resolution. Ion source settings were as follows: ion source type, NSI; spray voltage, 2400 V; ion transfer tube temperature, 275 °C. System control and data collection were performed using Xcalibur software.

The proteomic analysis was performed through Proteome Discoverer 2.4 (Thermo Scientific) using the Uniprot human database and the Byonic search algorithm (Protein Metrics). Percolator FDRs were set at 0.001 (strict) and 0.01 (relaxed). Peptide FDRs were set at 0.001 (strict) and 0.01 (relaxed), with medium confidence and a minimum peptide length of 6. Normalization was performed on the total peptide amount. The limma analysis was performed using R studio following the user guide [43]. The volcano and heatmap figures were plotted by GraphPad Prism 7.0. The Venn diagram was plotted using an online tool (http://bioinformatics.psb.ugent.be/webtools/Venn/).

4.8. GO and KEGG pathway analysis of the differentially expressed proteins

The DAVID 6.8 database (https://david.ncifcrf.gov/) was employed to perform GO functional and KEGG pathway analysis of the integrated differentially expressed proteins [44]. The GO functional analysis of the integrated differentially expressed proteins involves three parts: biological process, cellular component and molecular function. The bubble graph was plotted by http://www.bioinformatics.com.cn, an online platform for data analysis and visualization.

Figure 4.

Docking study of compound 18 with RUVBL1/2 (PDB code 5OAF). Compound 18 is presented as a cyan ball and stick model. Active residues are presented as gray sticks. Hydrogen bond, cation-π interaction, and halogen bond are presented as dashed yellow, magenta, and green lines, respectively.