Rational design and development of novel NAE inhibitors for the treatment of pancreatic cancer

Abstract

Pancreatic cancer remains clinically challenging because of the lack of efficient targeted therapies and high aggressiveness. NEDD8 activating enzyme (NAE) plays a critical role in various cellular functions in cancers. Herein, we report the synthesis, optimization, and evaluation of a new series of pyrido[2,3-d]pyrimidin-7(8H)-one derivative as highly selective and efficacious NAE inhibitors, enabling rapid degradation of related substrates and potent inhibition of BxPC-3 cell proliferation. Moreover, western blot assays demonstrated that compound 51 could inhibit NAE activity, resulting in apoptosis in BxPC-3 cells. Furthermore, compound 51 induced cell apoptosis in vitro and inhibited tumor growth in BxPC-3 xenograft models. Our work established that 51 was a highly potent and efficacious NAE inhibitor, representing an effective strategy and great potential as a new targeted therapy for pancreatic cancer.

Introduction

Pancreatic cancer is a highly malignant tumor of the digestive system, and its etiology and pathogenesis are still unclear. As reported, pancreatic cancer has the poorest prognosis of any common solid malignancy, with a 5-year overall survival rate of ~10%, lagging far behind the improved survival of other solid tumors [1]. Despite being relatively uncommon, pancreatic cancer is expected to become the second leading cause of cancer death by the end of the decade [2]. Disappointingly, pancreatic cancer exhibits resistance to many antineoplastic therapies due to rapid progression and low rates of pathologic complete response even with the most effective systemic agents and radiotherapy [3]. To significantly prolong the nonprogressive survival of patients with pancreatic cancer, developing new anti-pancreatic cancer drugs is extremely important.

The ubiquitin-proteasome system (UPS) is a multi-component system responsible for protein degradation, which participates in the degradation of more than 80% of intracellular proteins [4,5,6,7,8]. Dysregulation of UPS can lead to abnormal activation or inhibition of signaling pathways, which may lead to tumors or other disease progressions [9]. The ubiquitinylation pathway is carried out by three distinct enzymatic steps. Initially, ubiquitin is activated by the ubiquitin-activating enzyme (E1) in an ATP-dependent reaction. The ubiquitinylation pathway is executed. Ubiquitin-activating enzyme E1 delivers activated ubiquitin molecules to ubiquitin-conjugating enzyme E2, and then E2-bound ubiquitin is linked to target proteins by ubiquitin ligase E3 (as shown in Fig. 1A). The UPS pathway has been intensively studied as a therapeutic target, although medications targeting the three enzymes (E1, E2, and E3) are still in the early stages of development [10]. Bortezomib [11], is the first FDA-approved proteasome inhibitor drug targeting the ubiquitin-proteasome pathway, but Bortezomib is not very effective due to its poor selectivity in substrate degradation [12].

Fig. 1

A Summary of the UPS. B The NEDD8 conjugation pathway

In addition to the ubiquitin proteins, Ubiquitin-like proteins conjugation pathways, including NEDD8 and SUMO, have also been identified as critical players in the protein degradation process [13]. It covalently couples the ubiquitin-like small molecule NEDD8 to the substrate protein through a tertiary enzyme-linked reaction, thereby affecting the stability, conformation, and function of the substrate protein. (as shown in Fig. 1B). NEDD8 is initially activated by NAE in an ATP-dependent reaction and then transferred to the NEDD8 conjugating enzyme, Ubc12 (also known as UBE2M). Finally, with the coordinated action of Ubc12, NEDD8 is attached to cullin proteins, which are the central scaffold of cullin-RING ubiquitin E3 ligases (CRLs). Therefore, NAE mediates the degradation of proteins regulated by CRLs [10]. The substantial evidence indicated that the neddylation pathway is involved in a broad array of cellular functions and dysregulation of neddylation can lead to tumorigenesis [14,15,16,17,18,19,20,21]. Meanwhile, overactive neddylation has been reported in a variety of malignancies [21,22,23,24]. Hence, inhibiting NAE represents a potential approach to treating human cancers. MLN4924 [25] (shown in Fig. 2) is the first and only inhibitor of NAE that entered clinical trials to treat several types of cancer, especially hematologic malignancies [26,27,28,29,30,31,32]. However, clinical trials have shown that MLN4924 has a limited scope of application and general efficacy, which underlines the urgent need to develop novel NAE inhibitors [33, 34].

Fig. 2

Structure of MLN4924 and M22

In our previous work, M22 (as shown in Fig. 2) was identified as a novel reversible inhibitor of NAE through high-throughput virtual screening [35, 36]. As with A549 (lung cancer) cells, M22 induced apoptosis in AGS (gastric cancer) cells in nude mice, inhibited tumor growth in multiple cancer cell lines with IC₅₀ values in the nanomolar range, and showed low toxicity in a zebrafish model, which represents an ideal lead structure for the design of new antitumor agents. A schematic of the overall receptor-based virtual screening procedure according to our previous work is presented in Fig. 3 [35]. To screen and obtain the scaffold with drug-like properties, we downloaded 7941 molecules from the DrugBank Database and screened out 2259 marketed drug molecules through Prepare ligands in Discovery Studio 2016 (DS 2016). These small molecules were docked into the MLN4924 binding pocket (PDB ID: 3GZN) of NAE by the CDOCKER molecular docking module. The docking poses were scored by the scoring functions CDOCKER_INTERATION_ENERGY, Ligscore, PLP, and Jain. Finally, 124 compounds were divided into 10 categories by calculating the Tanimoto Coefficient (Tc) with ECFP_6. Finally, 22 compounds with diverse structures (as shown in Fig. S1) were obtained by manual selection. According to our experience in medicinal chemistry, the pyrido[2,3-d]pyrimidin-7(8H)-one structure was determined to replace the N-benzyl group in M22 to obtain the general structural formula of our target compounds. As shown in Fig. 4, the general formula could be accommodated in the ATP binding site of NAE. Predicted ligand-protein interaction showed that the pyrimidinone ring could form π-alkyl with Ile148, Ala171, Met101, and Ile170, and form a hydrogen bond with Asp102, Asp167, and Gln149. Compared with N-benzyl, the pyrimidinone ring can increase the interaction force, improving NAE inhibition. Then a series of structural optimizations were carried out based on the general structural formula. Almost 65 compounds exhibited suppression of BxPC-3 cell proliferation with IC₅₀ values in the low dose range. Among those compounds, compound 51 had excellent potency against BxPC-3 cells (IC₅₀ = 0.47 μM). In further investigation of the antitumor mechanism, compound 51 could directly inhibit NAE in the enzyme-based assay, induce apoptosis in BxPC-3 cells, and show antitumor efficacy in BxPC-3 xenograft mouse models. Moreover, compound 51 has low toxicity and a reasonable Log D value.

Fig. 3

The workflow of the virtual screening and the drug design of NAE inhibitors. 22 hits were filtered from DrugBank by docking scores and Cluster Ligands, then pyrido[2,3-d]pyrimidin-7(8H)-one structure was selceted to general structural formula and optimizated to our target compounds

Fig. 4

A Docking diagram of structural formula and NAE. B Interaction between structural formula and NAE

Results and discussion

Chemistry

The synthetic strategy used to prepare the target compounds 13-31, 36-77 is outlined in Scheme 1 and the typical intermediate 12 was prepared as described formerly [36]. Starting from compound 3, intermediates 4a−4i were obtained by a nucleophilic substitution reaction with amines. Intermediates 5a-5i were obtained by a simple hydrolysis reaction. Then 7a-7i were obtained by activating of carboxyl group by condensation with benzotriazole, the substitution of tert-butyl malonate, and removal of the Boc group. Under the alkaline condition, 7a-7i cyclized to form intermediates 8a-8i, which reacted with trifluoromethane sulfonic anhydride to form the critical intermediate trifluoromethanesulfonate 9a–9i. From 9a–9i, intermediates 10aa–10iz were prepared by the Suzuki coupling reaction or Sonogashira coupling reaction. After oxidation and coupling with the previously synthesized compound 12, target compounds 13–31, 36–77 were obtained by removing the Boc group.

Scheme 1

Synthetic Routes of Compounds 13-31, 36-77 ^a. ^aReagents and conditions: a Amine compounds, Et₃N, THF, rt, 1 h, 60–91% yield; b 1 M NaOH, reflux, EtOH, 1 h, 75%-80% yield; c EDCI, HOBt, benzotriazole, DCM, Rt, 8 h, 57–74% yield; d (I) NaH, ethyl tert-butyl malonate, THF, 0 °C, 2 h, (II) TFA, DCM, rt, 64–75% yield over two steps; e DBU, EtOH, reflux, 2 h, 74–79% yield; f (TfO)₂, Et₃N, DCM, 0 °C, 1 h, 72–74% yield; g Arylboronic acid, Pd(PPh₃)₄, Cs₂CO₃, THF, reflux, 73%-79% yield; h Terminal alkynes, Pd(PPh₃)₄, CuI, Et₃N, THF, reflux, 69.2–73.4% yield; i m-CPBA, DCM, rt, 84–89% yield; j Intermediate 12, Toluene, reflux, 79–86% yield; j TFA, DCM, rt, 89–92% yield

Chemical syntheses of compounds 32 and 35 are depicted in Scheme 2. The key intermediate 9a was coupled by Sonogashira reaction, and then intermediate 79 was oxidized and coupled with side chain 12 to obtain intermediate 80, which condensed with sodium azide to form intermediate 81. Finally, the trimethylsilyl and Boc were removed to obtain the target compound 32. Compound 35 was obtained by removing trimethylsilyl and the Boc group from intermediate 80.

Scheme 2

Synthetic Routes of Compounds 32, 35 ^a. ^aReagents and conditions: a Trimethylsilyl acetylene, Pd(PPh₃)₄, CuI, Et₃N, THF, reflux, 84% yield; b m-CPBA, DCM, rt, 85% yield; c Toluene, reflux, 87% yield; d NaN₃, DMSO, 100 °C, 3 h, 89% yield; e TBAF, THF, rt, 5 min; f TFA, DCM, rt, 89–92% yield over two steps

The synthetic route of compound 33 was described in Scheme 3. The key intermediate 9a was coupled with 3-(N-Boc-amino) phenylboronic acid by Suzuki reaction to obtain compound 84, followed by removal of the Boc protective group and condensed with acryloyl chloride to form intermediate 86. Then the target compound 33 was obtained as described previously.

Scheme 3

Synthetic Routes of Compounds 33 ^a. ^aReagents and conditions: a 3-(N-Boc-amino) phenylboronic acid, Pd(PPh₃)₄, Na₂CO₃, THF, reflux, 73% yield; b TFA, DCM, rt, and 89% yield; c Acryloyl chloride, NaHCO₃, CH₃CN, rt; d m-CPBA, DCM, rt, 84% yield; e Intermediate 12, toluene, reflux, 88% yield; f TFA, DCM, rt, 92% yield

The syntheses of compound 34 were outlined in Scheme 4. The key intermediate 8a was condensed with benzyl bromide and then the remaining synthesis steps were the same as Scheme 1.

Scheme 4

Synthetic Routes of Compounds 34 ^a. ^aReagents and conditions: a Benzyl bromide, NaHCO₃, CH₃CN, reflux, 83% yield; b m-CPBA, DCM, rt, 76% yield; c Intermediate 12, toluene, reflux, 89% yield; d TFA, DCM, rt, 90% yield

Structure-based molecular design strategy

As shown in Fig. 4A, a hydrophobic pocket was found above the 5-position of pyrido[2,3-d]pyrimidin-7(8H)-one. Therefore, hydrophobic substituents were introduced to pyrido[2,3-d]pyrimidin-7(8H)-one to occupy the hydrophobic region. Besides these, biaryl structures are privileged structures in antitumor drugs due to their rotatable bonds, suitable stiffness, bulk and shape, and suitable for binding [36, 37]. Therefore, hydrophilic and hydrophobic aryl rings were introduced at the 5-positions of pyrido[2,3-d]pyrimidin-7(8H)-one (3-35, shown in Fig. 5 and Table 1). Biological evaluation results are summarized in Table 1 and Table S1. It was discovered that modifying the 5-positions of pyrido[2,3-d]pyrimidin-7(8H)-one with phenyl group (13–29) increased anti-proliferation activities against BxPC-3 (pancreatic cancer), HCT-116 (colorectal carcinoma), and A549 (lung cancer), comparing with M22. Cancer cells used in this study were neddylation upregulated [38, 39]. Compounds 30-41 also showed more potent anti-proliferation activities than M22. It was disclosed that when introducing substituents such as fluorine to the meta- or para-position of the benzene ring, the resulting compound 15 or 16 had increased inhibition against BxPC-3 with IC₅₀ values of 1.45 μM and 0.96 μM, respectively. When an electron-withdrawing group such as −CF₃ or electron-donating group such as −OCH₃ was installed at the benzene ring, the cancer cells’ inhibitory activities of compounds 19–22 were dramatically increased compared to M22. However, these optimized compounds have similar activities, it seems that antiproliferative activity depends on general structural formula and the aromatic group modification could not improve the activity a lot.

Fig. 5

SAR of designed NAE Inhibitors

Table 1 Compounds 13−45 and their anti-tumor Activity against BxPC-3

Because of its rigidity and hydrophobicity structure, the ethynyl group was introduced to pyrido[2,3-d]pyrimidin-7(8H)-one with rings in this study. Therefore, compounds 35-45 (shown in Table 1) were designed, synthesized, and tested. As shown in Tables 1 and S1, those 5-ethynyl substituted derivatives had increased antiproliferation activities, indicating that introducing ethynyl groups on the 5-position of pyrido[2,3-d]pyrimidin-7(8H)-one would also contribute to increased antiproliferation activity. Compounds 45 and 39 had the lowest IC₅₀ value for inhibiting the proliferation of BxPC-3 and HCT-116 cells.

To obtain more effective antitumor drugs, the dominant group of the first round of structural modification was retained. The structural modification was conducted focusing on the 8-position of pyrido[2,3-d]pyrimidin-7(8H)-one. Through the strategy of group replacement, the 8-position alkane was changed. The length and volume of the base chain could change the position of the compound in the binding site, evaluate the binding effect of different groups, and design a series of new compounds (as shown in Fig. 5). We introduced linear and cyclic alkanes of different sizes to investigate the effect of volume on compound activity. As shown in Tables 2 and S2, compounds 51, 59, 67, and 75 with cyclopropyl structure generally showed comparable activity to the positive drugs in BxPC-3 cell assays, and the volume is just right. In addition, cyclopropane has directionality, and the H of cyclopropane acts as a nonclassical hydrogen bond donor to form hydrogen bonds.

Table 2 Compounds 46−77 and their anti-tumor activity against BxPC-3

In vitro cytotoxicity of selected compounds

NAE is widely expressed in non-cancerous cells, but it is expressed at a low level. NAE inhibitors may have some effect on non-cancer cells at high concentrations. Based on cell proliferative assay results, 51, 58, 59, 64, 67, and 72 were chosen to investigate their toxicity against normal cells L-02 (Human Liver Cells). On average, tested compounds exhibited a 3–10 times antiproliferative effect on BxPC-3 cells compared to normal cells, (shown in Fig. 6 and Table 3) suggesting that the proliferation inhibition on BxPC-3 cells was due to NAE inhibition rather than general toxicity.

Fig. 6

The IC₅₀ and LC₅₀ dose–response curves of tested compounds

Table 3 LC₅₀ values of tested compounds (μM, n = 3)^a

Half-life and intrinsic clearance of 51, 59, 64, 72

During the early discovery phase of compounds, the human-derived liver microsomes in vitro incubation method were used to assess the risk of compounds being metabolized in the body. As usual, half-life (T_1/2) and intrinsic clearance (CL_int) were used to evaluate the druggability of compounds 51, 59, 64, and 72. As shown in Table 4, 51 has a better half-life and inherent clearance rate than MLN4924.

Table 4 The metabolic stability of 51, 59, 64, 72

51 was chosen for further validation because it had the lowest IC₅₀ value against BxPC-3 cells and was moderately selective for normal cells and had a longer half-life. In addition, 51 is a relatively high binding force in molecular docking. Molecular docking (Fig. 3B) demonstrated that compound 51 had similar binding mode with the structural formula and formed three key hydrogen bonds with Asp100, Ile148 and Gln149 and a hydrogen bond with Asp167 which is similar to the co-crystal structure of MLN4924 (Fig. 7).

Fig. 7

A Merging between top-ranked 51 pose and MLN4924conformation (shown in yellow) from NAE crystal (PDB ID:3GZN). B Docking modes of compounds 51 with NAE

Compound 51 inhibited NAE activity in the enzyme-based assay

An enzymatic assay using an E2-mediated thioester product of Ubc12-NEDD8 was used to determine the level of NAE-mediated production in a cell-free system. NAE activity is reduced when the Ubc12-NEDD8 conjugation level is lower. Recombinant human NAE was incubated with NEDD8, Ubc12, and ATP in the presence of compound 51 for 1 h. Then levels of Ubc12-NEDD8 thioester product were detected by western blot. The intensity of Ubc12-NEDD8 bands was reduced in a concentration-dependent manner. The formation of Ubc12-NEDD8 was inhibited at 3.33 M (as shown in Fig. 8), indicating that compound 51 was an effective NAE inhibitor.

Fig. 8

Compound 51 inhibited Ubc12-NEDD8 formation in a dose-dependent manner in an enzyme assay

Effects of 51 on NAE-associated signaling pathway

Compound 51 was tested for its ability to inhibit NAE activity in BxPC-3 cells by western blot in the following study. As shown in Fig. 9A, NAE activated NEDD8 and then transferred it to Ubc12, resulting in an accumulation of the Ubc12-NEDD8 complex in the control group. After treatment with 51 for 24 h, the NEDD8 combined Ubc12 was noticeably decreased concentration-dependently, indicating that NAE activity was inhibited by 51. Inhibition of NAE can lead to the inactivation of CRL, causing the accumulation of substrates such as p27, CDT1, and NRF2, resulting in cell cycle arrest and induction of apoptosis. BxPC-3 cells were treated with 51 for 24 h, p27, CDT1, and NRF2 began to accumulate, suggesting that 51 could stabilize CRL substrates by suppressing neddylation.

Fig. 9

A Compound 51 could inhibit neddylation and CRL substrate degradation in BxPC-3 cells. B The relative protein expression level of P27, CDT1, NRF2, Ubc12-NEDD8. Total cell lysates were subjected to Western blot analysis. GAPDH was used as a loading control. **P < 0.01, *P < 0.05

Apoptosis induced by 51 in BxPC-3 Cells

Flow cytometry was used to assess the effect of compound 51 on inducing apoptosis in BxPC-3 cells. The results showed that in 51-treated BxPC-3 cells, apoptotic cells appeared in a dose-dependent pattern (Fig. 10). The apoptotic rates (Annexin V-EGFP) of BxPC-3 cells were increased to 82.87 percent of total cells (15 μM, 24 h) after treatment with 51, whereas only a few cells were observed as apoptotic in the control group. BxPC-3 cells went through the process of transitioning from early to late apoptosis as the concentration of 51 increased. These findings suggested that compound 51 could trigger apoptosis in a concentration-dependent manner.

Fig. 10

Flow cytometric analysis of the apoptotic effect of 51 in BxPC-3 cells through Annexin-V-EGFP/PI staining assay

The occurrence of most tumors is accompanied by the abnormal expression of the tumor suppressor p53. When cells are damaged, the level of p53 in the cells will increase rapidly and induce cell apoptosis. In cell apoptosis, characteristic apoptotic bodies are formed, which are regulated by Bcl-2. When the cell is apoptotic, the expression of Bax becomes more substantial, and the expression of Bcl-2 becomes weaker. The ratio of Bax/Bcl-2 determines the direction of cell apoptosis. The ratio of Bax/Bcl-2 is upregulated to promote cell apoptosis, and the ratio of Bax/Bcl-2 is down-regulated to inhibit cell apoptosis. This experiment investigated the effect of compound 51 on intracellular p53, Caspase 3, Bax, and Bcl-2. After BxPC-3 cells were treated with 51 for 24 h, the protein content was detected by Western blot. Results are shown in Fig. 11. As the dosage increased, p53 protein rose, the ratio of Bax/Bcl-2 increased, cell apoptosis increased, and apoptosis markers gradually accumulated.

Fig. 11

A Expression of apoptosis protein induced by Compound 51 B Relative protein expression level of Bax/Bcl-2. **P < 0.01, *P < 0.05

Those results further supported that compound 51 could induce apoptosis in a concentration-dependent manner.

In vivo study of compound 51

We investigated the antitumor activity of the new NAE inhibitor 51 in vivo based on its promising in vitro potency. Animals were given subcutaneous injections of vehicle or compound 51 for 21 days. In our preliminary study, the maximum tolerated single dose for compound 51 was determined as 160 mg/kg. Therefore, compound 51 was given at 90 mg/kg, 120 mg/kg, or 160 mg/kg once daily (QD) consecutively. As shown in Fig. 12, 51-treated mice showed significant suppression of tumor growth in a dose-dependent manner, compared to vehicle-treated mice. No significant change in body weight was observed at those doses, indicating that the treatments have no apparent toxicity in the xenograft models. Results show that compound 51 has a certain antitumor effect and has the potential to treat pancreatic cancer.

Fig. 12

In vivo activities of compound 51 in cell-derived xenografts. Nude mice bearing subcutaneous xenografts derived from BxPC-3 were injected subcutaneously daily with 90, 120, or 160 mg/kg compound 51, respectively for 21 consecutive days. A Anatomical nude mice’s tumor tissues untreated or treated with 51. B Relative rate of tumor proliferation. C The tumor weight values were measured daily. D The growing curves of mice’s body weight. Data are shown as mean ± SEM, n = 6; *P < 0.05

Conclusion

The NEDD8-activating enzyme (NAE), which regulates the degradation and turnover of cancer-related proteins by activating the cullin-RING E3 ubiquitin ligases, is an emerging target for cancer therapy. This research conducted a structural hopping strategy and synthesized a series of compounds by structural optimization. Candidates with potent anticancer activities were discovered during in vitro activity testing in three cancer cells, particularly in BxPC-3 cells. In a preliminary investigation, the best candidate compound 51 showed the least undesirable side effects and fulfilled lipophilicity after cytotoxicity testing. In the in vitro incubation experiment of liver microsomes, candidate compound 51 showed the best half-life. In addition, compound 51 induced a dose–response drop in the level of Ubc12-NEDD8, indicating that it was an NAE inhibitor. Further research revealed that compound 51 induced apoptosis in BxPC-3 cells and reduced tumor development. In general, compound 51 is a novel and highly effective NAE inhibitor for pancreatic cancer, worthy of further analysis.

Experimental

Chemistry general

All reactions were performed in glassware containing a Teflon-coated stir bar. Commercially available solvents and reagents were obtained from Adamas, Accela, Aikonchem, Sinopharm Chemical, Energy Chemical, Aladdin, Macklin, Meyer, Leyan, MCE, Bidepharm, Sigma-Aldrich, J&K, and TCI, and used without further purification. ¹H and ¹³C NMR spectra were recorded on a Bruker 300, 400, or 500 MHz NMR spectrometer. Chemical shifts (δ) were reported in ppm downfield from an internal tetramethylsilane standard. High-resolution mass spectrometry (HRMS) analysis was recorded on an HP1100 spectrometer. Flash column chromatography on silica gel (200−300 mesh) was used for the routine purification of reaction products. All reactions were monitored by thin-layer chromatography on silica gel plates (15 mm × 50 mm) and spots were visualized under UV light at 254 nm. HPLC analysis was conducted for all compounds on a Shimadzu LC system (Inertsil ODS-SP C18 column, 4.6 mm × 250 mm, 5 μM). Most compounds are >95% pure by HPLC analysis, compounds 28, 30, 36, 39, 44, 49, 53, and 62 are >93% pure by HPLC analysis.

Synthetic procedures

General method for the synthesis of 4a-4i

Raw material 3 (50 mmol), corresponding amine (75 mmol), and triethylamine (150 mmol) were added into a round bottom flask with 150 mL THF in turn, the mixture was stirred at 35 °C for 1 h. Excess solids were filtered out, and the filtrate was evaporated under reduced pressure to remove THF. Then the round bottom flask was reconstituted with 150 mL ethyl acetate, washed with saturated NH₄Cl solution (100 mL × 3), and then washed with saturated NaCl solution three times (100 mL × 3), anhydrous sulfuric acid. The organic phase was dried with sodium and filtered. The filtrate was rotated under reduced pressure to remove the solvent, 15 mL of toluene was added, and the solvent was removed under reduced pressure by rotary evaporation to obtain a yellow oil. The crude product was used for the next step without any purification.

General method for the synthesis of 5a–5i

The prepared product 4a–4i (50 mmol) and 30 mL 1 mol L⁻¹ sodium hydroxide solution were added into a 250 mL round bottom flask with 25 mL ethanol. The mixture was heated to reflux for 5 h. The ethanol was removed by rotary evaporation, the pH was adjusted to 3–4 with 1 mol L⁻¹ hydrochloric acid, a large amount of white solid was precipitated, filtered by suction, the filter cake was washed with a small amount of water, n-hexane, and dried to obtain white solid. The crude product was used for the next step without any purification.

General method for the Synthesis of 6a–6i

The prepared product 5a–5i (50 mmol), benzotriazole (100 mmol), EDCI (60 mmol), and HOBt (150 mmol) were added into a 250 mL round bottom flask with 150 mL dichloromethane. The mixture was stirred at 35 °C for 16 h, and the mixture gradually became clear. After the reaction was completed, silica gel was added to make sand and purified by column chromatography (PE:EA = 15:1) to obtain white crystals, the product was used for the next step.

General method for the synthesis of 7a-7i

NaH (75 mmol) was added into a 250 mL round-bottom flask with 100 mL anhydrous THF under an N₂ atmosphere. The mixture was stirred in an ice bath for 30 min. When the internal temperature dropped to 0–5 °C, propylene glycol Ethyl tert-butyl ester (75 mmol) was added and the mixture was stirred at 0 °C for 30 min. 70 mL of prepared product 6a–6i (50 mmol) in THF was added into a round-bottom flask under an N₂ atmosphere, the mixture was stirred in an ice bath for 3 h. After the reaction was completed, 200 mL of saturated aqueous ammonium chloride solution was added, most of the THF was distilled under reduced pressure, and the mixture was extracted three times with EA (100 mL × 3), and washed with saturated aqueous NaCl (100 mL × 3), dried over sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain a crude product. the crude product was reconstituted with 50 mL of dichloromethane, stirred magnetically, and 25 mL of trifluoroacetic acid was added. An irritating gas was generated. After the reaction was completed, the mixture was evaporated under reduced pressure with 25 mL toluene and purified and separated by column chromatography (PE:EA = 10:1) to obtain yellow oil, the product was used for the next step.

General method for the synthesis of 8a-8i

The prepared products 7a–7i (50 mmol) and DBU (100 mmol) were added into a 100 mL round bottom flask with 20 mL absolute ethanol. The mixture was heated to reflux for 2 h. The temperature was cooled down naturally, and most of the ethanol in the system was removed by rotary evaporation under reduced pressure. 1 mol L⁻¹ hydrochloric acid was added to adjust the pH of the reaction system to 2–3. A large amount of white solid precipitated, filtered, and filtered. The cake was washed with a small amount of water and dried by blowing to obtain a white solid, the product was used for the next step.

General method for the synthesis of 9a–9i

The prepared product 8a–8i (50 mmol) was added into a 250 mL round bottom flask with 70 mL anhydrous dichloromethane, and triethylamine (75 mmol) was added. The mixture was cooled to 0 °C, and then trifluoromethane sulfonic anhydride (75 mmol) was added dropwise with a constant pressure dropping funnel under the atmosphere of N₂. The reaction system first turned wine red and then yellow. After the reaction was completed, the mixture was purified by column chromatography (PE:EA = 24:1) to obtain a white solid, the product was used for the next step.

General method for the synthesis of 10aa-10iz

The prepared product 9a-9i (10 mmol), Cs₂CO₃ (20 mmol), Pd(PPh₃)₄ (0.5 mmol), and Phenylboronic acid compounds (15 mmol) were added into a 50 mL round bottom flask with 20 mL anhydrous THF. The mixture was heated to reflux for 1.5 h under an N₂ atmosphere. After the reaction was completed, the mixture was purified by column chromatography (PE:EA = 5:1) to obtain a solid product. Or the prepared products 9a-9i (10 mmol), triethylamine (30 mmol), CuI (1 mmol), Pd(PPh₃)₄ (1 mmol), and alkynes compounds (15 mmol) were added into a 50 mL round bottom flask with 20 mL anhydrous THF. The mixture was heated to reflux for 1.5 h under an N₂ atmosphere. After the reaction was completed, the mixture was purified by column chromatography (PE:EA = 50:1) to obtain the solid product, the product was used for the next step.

General method for the synthesis of 11aa-11iz

The prepared product 10aa-10iz (1 mmol) and m-CPBA (3 mmol) were added into a 50 mL round-bottom flask with 20 mL dichloromethane. The mixture was stirred at room temperature for 1 h. 5 mL saturated Na₂SO₃ aqueous solution was added to quench the reaction, the mixture was washed with saturated Na₂CO₃ aqueous solution (10 mL × 3) and saturated NaCl aqueous solution (10 mL × 3), and dried with anhydrous sodium sulfate. The mixture was filtered and evaporated under reduced pressure to obtain oil, the product was used for the next step.

8-Cyclopropyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(4-(trifluoromethyl)phenyl)pyrido[2,3-d]pyrimidin-7(8H)-one (51)

The prepared oil 11gi (5 mmol) and compound 12 were added into a 25 mL round bottom flask with 15 mL toluene. The mixture was heated to reflux for 2 h under N₂ atmosphere. After the reaction was completed, the mixture was cooled to room temperature, and the mixture was purified by column chromatography (PE:EA = 30:1) to obtain solid product. Then white solid (0.5 mmol) and 4 mL of trifluoroacetic acid were added into a 25 mL round bottom flask with 20 mL of dichloromethane, stirred magnetically. The mixture was stirred at room temperature for 1 h. After the reaction was completed, 10 mL of toluene was added, and the solvent was distilled off under reduced pressure. An oily substance was obtained, which was slurried by adding 20 mL of ether, filtered, the filter cake was washed with ether, and dried to obtain 173 mg of white solid powder, 98% yield, 98.8% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.73 (s, 2H), 8.25 (s, 1H), 7.55 (d, J = 4.9 Hz, 2H), 7.51 (d, J = 4.8 Hz, 2H), 7.29–7.16 (m, 4H), 6.28 (s, 1H), 5.02 (d, J = 8.1 Hz, 2H), 3.31 (s, 1H), 3.20 (s, 2H), 3.05-2.96 (m, 4H), 2.83–2.79 (m, 1H), 2.20 (d, J = 6.9 Hz, 2H), 1.84 (s, 1H), 1.80–1.73 (m, 2H), 1.19 (q, J = 12.7 Hz, 2H), 0.89 (d, J = 4.3 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 164.1, 159.9, 157.7, 157.2, 147.4, 138.9, 136.0, 131.3 (q, J_CF = 32.6 Hz), 129.0, 128.9, 128.5, 127.3, 125.8, 125.7 (q, J_CF = 10.8 Hz), 122.0, 118.5, 117.6, 114.6, 104.9, 55.8, 46.1, 42.3, 32.5, 28.2, 25.1, 9.4. HRMS (ESI) (m/z): calculated for C₃₀H₃₁F₃N₅O [M + H]⁺ 534.2404; found, 534.2481.

8-Cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)-5-phenylpyrido[2,3-d]pyrimidin-7(8H)-one (13)

The title compound was prepared from 3 according to the preparation of 51. 158 mg, 18% yield, 99.4% HPLC Purity. ¹H NMR (500 MHz, CDCl₃) δ 9.75 (s, 2H), 8.37 (s, 1H), 7.46 (t, J = 5.0 Hz, 3H), 7.39 (t, J = 7.5 Hz, 2H), 7.27 (q, J = 14.5 Hz, 2H), 7.22 (t, J = 14.0 Hz, 1H), 7.15 (d, J = 7.0 Hz, 2H), 6.30 (s, 1H), 5.84 (t, J = 18.0 Hz, 1H), 4.93 (d, J = 13.5 Hz, 2H), 3.31 (s, 1H), 3.20 (s, 2H), 3.04–2.96 (m, 4H), 2.40–2.35 (m, 2H), 2.18 (d, J = 11.5 Hz, 2H), 1.98 (s, 2H), 1.82 (t, J = 9.0 Hz, 2H), 1.80–1.64 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ 163.6, 159.7, 157.9, 156.5, 148.6, 136.1, 135.3, 129.1, 129.0, 128.7, 128.6, 128.5, 127.3, 117.6, 105.6, 55.9, 53.5, 46.1, 42.4, 32.5, 28.2, 27.9, 25.6. HRMS (ESI) (m/z): calculated for C₃₁H₃₅N₅O [M + H]⁺ 494.2842; found, 494.2923.

8-Cyclopentyl-5-(3,4-dimethylphenyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (14)

The title compound was prepared from 3 according to the preparation of 51. 56 mg, 22% yield, 97.8% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.75 (s, 1H), 8.44 (s, 2H), 7.31–7.23 (m, 4H), 7.22–7.11 (m, 4H), 6.29 (s, 1H), 5.96–5.80 (m, 1H), 4.95 (d, J = 13.5 Hz, 2H), 3.31 (s, 1H), 3.18 (s, 2H), 3.06–2.92 (m, 4H), 2.37 (d, J = 10.8 Hz, 1H), 2.31 (d, J = 5.7 Hz, 6H), 2.18 (d, J = 10.8 Hz, 2H), 1.96 (d, J = 4.8 Hz, 2H), 1.87 (m, 3H), 1.81–1.63 (m, 6H). ¹³C NMR (125 MHz, CDCl₃) δ 163.7, 159.6, 158.0, 156.5, 148.7, 137.8, 137.1, 136.1, 132.8, 129.9, 129.8, 129.0, 128.5, 127.3, 126.1, 117.4, 105.7, 55.9, 53.4, 46.1, 42.4, 32.5, 28.2, 27.9, 25.6, 19.8, 19.6. HRMS (ESI) (m/z): calculated for C₃₃H₃₉N₅O [M + H]⁺ 522.3155; found, 522.3229.

8-Cyclopentyl-5-(3-fluoro-4-methylphenyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (15)

The title compound was prepared from 3 according to the preparation of 51. 156 mg, 30% yield, 94.8% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.75 (s, 2H), 8.38 (s, 1H), 7.25 (t, J = 19.5 Hz, 3H), 7.21 (d, J = 6.5 Hz, 1H), 7.15 (d, J = 7.0 Hz, 2H), 7.06 (t, J = 17.0 Hz, 2H), 6.27 (s, 1H), 5.83 (t, J = 18.0 Hz, 1H), 4.94 (d, J = 12.0 Hz, 2H), 3.31 (s, 1H), 3.19 (s, 2H), 3.04-2.94 (m, 4H), 3.34 (s, 5H), 2.19 (d, J = 11.5 Hz, 2H), 1.98 (s, 2H), 1.81-1.75 (m, 4H), 1.64 (d, J = 3.5 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 163.4, 162.2 (d, J_CF = 245.0 Hz), 159.7, 157.7, 156.5, 147.3, 136.1, 134.6 (d, J_CF = 7.5 Hz), 131.9, 131.8, 128.9, 128.5, 127.3, 126.1 (d, J_CF = 15.0 Hz), 124.1, 117.6, 115.4, 115.3, 105.3, 55.8, 53.5, 46.1, 42.4, 32.5, 28.2, 27.8, 25.6, 14.4, 14.4. HRMS (ESI) (m/z): calculated for C₃₂H₃₆FN₅O [M + H]⁺ 526.2904; found, 526.2979.

8-Cyclopentyl-5-(4-fluoro-3-methylphenyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (16)

The title compound was prepared from 7 according to the preparation of 51. 230 mg, 44% yield, 99.8% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.73 (s, 2H), 8.36 (s, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.30–7.06 (m, 8H), 6.26 (s, 1H), 5.88–5.76 (m, 1H), 4.92 (d, J = 14.1 Hz, 2H), 3.30 (s, 1H), 3.20 (s, 2H), 3.05–2.92 (m, 4H), 2.37 (s, 2H), 2.32 (s, 3H), 2.17 (d, J = 11.7 Hz, 2H), 1.98 (s, 2H), 1.80–1.63 (m, 6H). ¹³C NMR (125 MHz, DMSO-d₆ + CDCl₃) δ 162.6, 161.0 (d, J_CF = 246.3 Hz), 159.1, 157.1, 155.8, 147.1, 136.2, 136.1, 131.2, 131.1, 130.4 (d, J_CF = 3.8 Hz), 128.2, 128.1, 127.2, 127.1 126.4, 124.8 (d, J_CF = 17.5 Hz), 116.6, 114.9, 114.7, 104.5, 54.7, 52.7, 51.8, 45.4, 45.1, 41.8, 41.3, 31.7, 31.7, 27.5, 27.2, 25.0, 24.6, 14.0, 13.9. HRMS (ESI) (m/z): calculated for C₃₂H₃₆FN₅O [M + H]⁺ 526.2904; found, 522. 526.2989.

5-(8-Cyclopentyl-7-oxo-2-(4-(phenethylamino)piperidin-1-yl)-7,8-dihydropyrido[2,3-d]pyrimidin-5-yl)-2-fluorobenzaldehyde (17)

The title compound was prepared from 3 according to the preparation of 51. 91 mg, 19% yield, 99.8% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 10.04 (s, 1H), 9.74 (s, 2H), 8.24 (s, 1H), 7.91 (q, J = 8.6 Hz, 1H), 7.65–7.62 (m, 1H), 7.34–7.15 (m, 6H), 6.27 (s, 1H), 5.86-5.79 (m, 1H), 4.93 (d, J = 13.2 Hz, 2H), 3.31 (s, 1H), 3.20 (s, 2H), 3.04–2.95 (m, 4H), 2.38–2.33 (m, 2H), 2.19 (d, J = 11.8 Hz, 2H), 1.98 (t, J = 13.8 Hz, 2H), 1.83–1.72 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 186.3 (q, J_CF = 5.9 Hz), 166.5, 163.1, 158.1 (d, J_CF = 240.9 Hz), 157.2, 146.1, 136.3, 136.2, 136.0, 132.2, 129.0, 128.8, 128.5, 127.3, 124.4 (d, J_CF = 8.9 Hz), 118.2, 117.6, 117.3, 105.1, 55.8, 53.6, 46.1, 42.4, 32.5, 28.2, 27.8, 25.6. HRMS (ESI) (m/z): calculated for C₃₂H₃₄FN₅O₂ [M + H]⁺ 540.2697; found, 540.2729.

3-(8-Cyclopentyl-7-oxo-2-(4-(phenethylamino)piperidin-1-yl)-7,8-dihydropyrido[2,3-d]pyrimidin-5-yl)benzaldehyde (18)

The title compound was prepared from 3 according to the preparation of 51. 105 mg, 20% yield, 97.6% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 10.07 (s, 1H), 9.74 (s, 2H), 8.28 (s, 1H), 8.00 (q, J = 7.5 Hz, 1H), 7.90 (s, 1H), 7.68 (t, J = 12.6 Hz, 2H), 7.30–7.14 (m, 5H), 6.31 (s, 1H), 5.84 (t, J = 17.7 Hz, 1H), 4.93 (d, J = 12.9 Hz, 2H), 3.31 (s, 1H), 3.19 (s, 2H), 3.05-2.93 (m, 4H), 2.35 (t, J = 15.0 Hz, 2H), 2.18 (d, J = 11.6 Hz, 2H), 1.98 (s, 2H), 1.85–1.63 (m, 6H). ¹³C NMR (125 MHz, CDCl₃) δ 191.4, 163.2, 162.1, 161.8, 159.8, 157.4, 156.6, 147.1, 136.8, 136.3, 136.0, 134.3, 130.2, 129.6, 128.9, 128.5, 127.3, 118.1, 105.2, 55.8, 53.6, 46.1, 42.4, 32.4, 28.1, 27.8, 25.6. HRMS (ESI) (m/z): calculated for C₃₂H₃₅N₅O₂ [M + H]⁺ 522.2791; found, 522.2867.

8-Cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(2-(trifluoromethyl)phenyl)pyrido[2,3-d]pyrimidin-7(8H)-one (19)

The title compound was prepared from 3 according to the preparation of 51. 184 mg, 33% yield, 99.4% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.74 (s, 2H), 8.26 (s, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.66–7.57 (m, 3H), 7.29–7.21 (m, 4H), 7.15 (d, J = 7.5 Hz, 2H), 6.30 (s, 1H), 5.84 (t, J = 18.0 Hz, 1H), 4.93 (d, J = 13.5 Hz, 2H), 3.30 (s, 1H), 3.19 (s, 2H), 3.04-2.95 (m, 4H), 2.39-2.34 (m, 2H), 2.19 (d, J = 12.0 Hz, 2H), 1.83-1.80 (m, 4H), 1.77–1.64 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ 163.0, 159.7, 157.7, 155.8, 145.7, 136.1, 133.5, 131.7, 130.8, 129.1, 129.0, 128.9, 128.8, 128.6, 128.5, 127.3, 126.5, 126.4, 124.7, 122.5, 118.9, 116.5 (q, J_CF = 290.0 Hz), 115.37, 113.04, 106.6, 55.8, 53.6, 46.1, 42.3, 32.4, 28.1, 27.9, 25.6. HRMS (ESI) (m/z): calculated for C₃₂H₃₄F₃N₅O [M + H]⁺ 562.2715; found, 562.2796.

8-Cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(3-(trifluoromethyl)phenyl)pyrido[2,3-d]pyrimidin-7(8H)-one (20)

The title compound was prepared from 3 according to the preparation of 51. 150 mg, 27% yield, 99.1% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.75 (s, 2H), 8.27 (s, 1H), 7.74 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.28–7.21 (m, 3H), 7.15 (d, J = 7.5 Hz, 2H), 6.30 (s, 1H), 5.87–5.80 (m, 1H), 4.94 (d, J = 13.0 Hz, 2H), 3.32 (s, 1H), 3.20 (s, 2H), 3.04–2.95 (m, 4H), 2.38 (t, J = 11.0 Hz, 2H), 2.19 (d, J = 11.0 Hz, 2H), 1.99 (s, 2H), 1.84–1.73 (m, 4H), 1.64 (d, J = 5.0 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 163.2, 159.8, 157.4, 156.5, 147.0, 138.9, 136.0, 131.7, 131.4 (q, J_CF = 32.5 Hz), 129.1, 128.9, 128.5, 127.3, 127.1, 125.8 (d, J_CF = 68.7 Hz), 124.9, 122.8, 118.0, 117.7, 115.4, 105.1, 55.8, 53.6, 46.3, 42.4, 32.4, 28.1, 27.8, 25.6. HRMS (ESI) (m/z): calculated for C₃₂H₃₄F₃N₅O [M + H]⁺ 562.2715; found, 562.2796.

8-Cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(4-(trifluoromethyl)phenyl)pyrido[2,3-d]pyrimidin-7(8H)-one (21)

The title compound was prepared from 3 according to the preparation of 51. 150 mg, 27% yield, 99.1% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.75 (s, 2H), 8.27 (s, 1H), 7.74 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.28–7.21 (m, 3H), 7.15 (d, J = 7.5 Hz, 2H), 6.30 (s, 1H), 5.87–5.80 (m, 1H), 4.94 (d, J = 13.0 Hz, 2H), 3.32 (s, 1H), 3.20 (s, 2H), 3.04–2.95 (m, 4H), 2.38 (t, J = 11.0 Hz, 2H), 2.19 (d, J = 11.0 Hz, 2H), 1.99 (s, 2H), 1.84–1.73 (m, 4H), 1.64 (d, J = 5.0 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 163.2, 159.8, 157.4, 156.5, 147.0, 138.9, 136.0, 131.3 (d, J_CF = 32.5 Hz), 129.1, 129.0, 128.5, 127.3, 127.1, 125.8, 125.8, 124.9, 122.8, 118.0, 117.7, 115.4, 105.1, 55.8, 53.6, 46.1, 42.4, 32.4, 28.1, 27.8, 25.6. HRMS (ESI) (m/z): calculated for C₃₂H₃₄F₃N₅O [M + H]⁺ 562.2715; found, 562.2796.

5-(3-Chloro-4-(trifluoromethyl)phenyl)-8-cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (22)

The title compound was prepared from 3 according to the preparation of 51. 158 mg, 27% yield, 99.7% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.72 (s, 2H), 8.24 (s, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.55 (s, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.29–7.21 (m, 2H), 7.15 (d, J = 7.0 Hz, 2H), 6.28 (s, 1H), 5.86–5.79 (m, 1H), 4.94 (d, J = 13.0 Hz, 2H), 3.32 (s, 1H), 3.19 (d, J = 8.5 Hz, 2H), 3.04–2.95 (m, 4H), 2.36 (s, 2H), 2.19 (d, J = 11.5 Hz, 2H), 1.99 (s, 2H), 1.84–1.72 (m, 6H), 1.65 (d, J = 4.5 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 163.0, 159.8, 157.1, 156.5, 145.5, 140.3, 136.0, 133.0, 131.4, 129.1, 128.9, 128.9, 128.5, 128.1, 128.0, 128.0, 127.3, 126.9, 125.8, 123.7, 121.5, 118.2, 104.7, 55.8, 53.7, 46.1, 42.4, 32.4, 28.1, 27.8, 25.6. HRMS (ESI) (m/z): calculated for C₃₂H₃₃ClF₃N₅O [M + H]⁺ 596.2326; found, 596.2404.

8-Cyclopentyl-5-(2-methoxyphenyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (23)

The title compound was prepared from 3 according to the preparation of 51. 135 mg, 11% yield, 98.9% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.74 (s, 2H), 8.08 (s, 1H), 7.42 (t, J = 14.5 Hz, 1H), 7.27 (t, J = 14.5 Hz, 2H), 7.21 (t, J = 15.5 Hz, 2H), 7.15 (d, J = 7.5 Hz, 2H), 7.05 (t, J = 14.5 Hz, 1H), 6.98 (d, J = 8.0 Hz, 2H), 6.27 (s, 1H), 5.87–5.80 (m, 1H), 4.93 (d, J = 13.5 Hz, 2H), 3.74 (s, 3H), 3.30 (s, 1H), 3.20 (s, 2H), 3.03 (t, J = 16.5 Hz, 2H), 2.95 (t, J = 21.0 Hz, 2H), 2.39 (d, J = 2.5 Hz, 2H), 2.18 (d, J = 11.0 Hz, 2H), 1.97 (s, 2H), 1.83–1.74 (m, 4H), 1.63 (d, J = 5.0 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 163.9, 159.6, 158.4, 156.4, 155.9, 146.3, 136.1, 130.6, 130.5, 128.9, 128.5, 127.3, 124.2, 121.0, 118.7, 111.0, 105.7, 55.9, 55.5, 53.4, 46.1, 42.4, 32.4, 28.2, 27.9, 25.6. HRMS (ESI) (m/z): calculated for C₃₂H₃₇N₅O₂ [M + H]⁺ 524.2947; found, 524.3030.

8-Cyclopentyl-5-(3-methoxyphenyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (24)

The title compound was prepared from 3 according to the preparation of 51. 187 mg, 36% yield, 99.8% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.75 (s, 2H), 8.41 (s, 1H), 7.37 (t, J = 16.0 Hz, 1H), 7.28–7.23 (m, 3H), 7.20 (d, J = 20.0 Hz, 2H), 7.15 (d, J = 7.0 Hz, 2H), 7.00–6.91 (m, 1H), 6.31 (s, 1H), 5.87–5.80 (m, 1H), 4.94 (d, J = 13.0 Hz, 2H), 3.83 (s, 3H), 3.31 (s, 1H), 3.20 (s, 2H), 3.04–2.94 (m, 4H), 2.38 (t, J = 26.5 Hz, 2H), 2.18 (d, J = 11.5 Hz, 2H), 1.98 (s, 2H), 1.83–1.72 (m, 4H), 1.64 (d, J = 5.0 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 163.6, 159.8, 159.7, 157.9, 156.5, 148.5, 136.6, 136.1, 129.8, 129.0, 128.5, 127.3, 121.0, 117.5, 114.8, 114.2, 105.5, 55.9, 55.3, 53.5, 46.1, 42.4, 32.4, 28.2, 27.8, 25.6. HRMS (ESI) (m/z): calculated for C₃₂H₃₇N₅O₂ [M + H]⁺ 524.2947; found, 524.3026.

8-Cyclopentyl-5-(4-methoxyphenyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (25)

The title compound was prepared from 3 according to the preparation of 51. 155 mg, 30% yield, 99.7% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.76 (s, 2H), 8.43 (s, 1H), 7.32 (t, J = 8.5 Hz, 2H), 7.27 (t, J = 14.0 Hz, 2H), 7.22 (t, J = 14.0 Hz, 1H), 7.15 (d, J = 7.0 Hz, 2H), 6.98 (d, J = 8.5 Hz, 2H), 6.27 (s, 1H), 5.85–5.81 (m, 1H), 4.94 (d, J = 13.0 Hz, 2H), 3.86 (s, 3H), 3.31 (s, 1H), 3.20 (s, 2H), 3.03–2.94 (m, 4H), 2.37 (s, 2H), 2.19 (d, J = 11.5 Hz, 2H), 2.19 (d, J = 11.5 Hz, 2H), 1.97 (s, 2H), 1.77 (t, J = 30.0 Hz, 4H), 1.63 (d, J = 4.0 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 163.7, 160.4, 159.6, 157.9, 156.5, 148.3, 136.1, 130.0, 128.9, 128.5, 127.5, 127.3, 117.2, 114.2, 105.7, 55.9, 55.4, 53.4, 46.1, 42.4, 32.4, 28.2, 27.8, 25.6. HRMS (ESI) (m/z): calculated for C₃₂H₃₇N₅O₂ [M + H]⁺ 524.2947; found, 524.3016.

5-([1,1’-Biphenyl]-4-yl)-8-cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (26)

The title compound was prepared from 3 according to the preparation of 51. 137 mg, 28% yield, 99.7% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.76 (s, 2H), 8.45 (s, 1H), 7.69–7.62 (m, 4H), 7.47 (t, J = 15.5 Hz, 4H), 7.39 (t, J = 17.5 Hz,1H), 7.27 (d, J = 7.5 Hz, 1H), 7.26–7.15 (m, 5H), 6.35 (s, 1H), 5.87–5.82 (m, 4H), 2.39 (d, J = 2.5 Hz, 2H), 1.99 (s, 2H), 1.84–1.65 (m, 4H), 1.20 (t, J = 14.0 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 163.6, 159.7, 157.9, 156.6, 148.2, 142.1, 140.2, 136.1, 134.1, 129.1, 129.0, 128.9, 128.5, 127.8, 127.4, 127.3, 127.1, 117.6, 105.2, 55.9, 53.5, 46.1, 42.4, 32.5, 28.2, 27.9, 25.6. HRMS (ESI) (m/z): calculated for C₃₇H₃₉N₅O [M + H]⁺ 570.3155; found, 570.3241.

8-Cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(4-phenoxyphenyl)pyrido[2,3-d]pyrimidin-7(8H)-one (27)

The title compound was prepared from 3 according to the preparation of 51. 127 mg, 23% yield, 99.7% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.76 (s, 2H), 8.42 (s, 1H), 7.39–7.34 (m, 4H), 7.27 (t, J = 15.0 Hz, 3H), 7.23–7.15 (m, 3H), 7.07 (d, J = 7.0 Hz, 4H), 6.29 (s, 1H), 5.83 (t, J = 18.0 Hz, 1H), 4.94 (d, J = 13.0 Hz, 2H), 3.31 (s, 1H), 3.19 (s, 2H), 3.04–2.94 (m, 4H), 2.37 (s, 2H), 2.19 (d, J = 11.0 Hz, 2H), 1.98 (s, 2H), 1.82–1.72 (m, 4H), 1.63 (d, J = 4.5 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 163.6, 159.7, 158.6, 157.8, 156.6, 148.0, 136.1, 130.2, 129.9, 129.8, 129.0, 127.3, 124.0, 119.6, 118.5, 117.5, 105.6, 55.9, 53.5, 46.1, 42.4, 32.5, 28.2, 27.9, 25.6. HRMS (ESI) (m/z): calculated for C₃₇H₃₉N₅O₂ [M + H]⁺ 586.3104; found, 586.3188.

5-(4-(Tert-butyl)phenyl)-8-cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (28)

The title compound was prepared from 3 according to the preparation of 51. 140 mg, 26% yield. ¹H NMR (300 MHz, CDCl₃) δ 9.75 (s, 2H), 8.43 (s, 1H), 7.47 (d, J = 8.1 Hz, 2H), 7.34–7.22 (m, 5H), 7.15 (d, J = 6.9 Hz, 2H), 6.31 (s, 1H), 5.84 (t, J = 17.7 Hz, 1H), 4.93 (d, J = 13.5 Hz, 2H), 3.31 (s, 1H), 3.19 (s, 2H), 3.05–2.92 (m, 4H), 2.37 (d, J = 3.0 Hz, 2H), 2.18 (d, J = 9.9 Hz, 2H), 1.97 (s, 2H), 1.77 (t, J = 8.4 Hz, 4H), 1.65 (t, J = 10.8 Hz, 2H), 1.37 (s, 9H). ¹³C NMR (125 MHz, CDCl₃) δ 163.7, 159.6, 158.0, 156.5, 152.4, 148.7, 136.1, 132.3, 128.9, 128.5, 128.4, 127.3, 125.7, 117.4, 105.7, 55.9, 53.5, 46.1, 42.4, 34.8, 32.5, 31.3, 28.2, 27.9, 25.6. HRMS (ESI) (m/z): calculated for C₃₅H₄₃N₅O [M + H]⁺ 550.3468; found, 550.3543.

8-Cyclopentyl-5-(4-(methylsulfonyl)phenyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (29)

The title compound was prepared from 3 according to the preparation of 51. 93 mg, 17% yield, 98.3% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.72 (s, 2H), 8.23 (s, 1H), 8.06 (d, J = 8.0 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H), 7.29–7.22 (m, 3H), 7.15 (d, J = 7.0 Hz, 2H), 6.30 (s, 1H), 5.83 (t, J = 18.0 Hz, 1H), 4.94 (d, J = 13.5 Hz, 2H), 3.31 (s, 1H), 3.20 (d, J = 24.5 Hz, 2H), 3.05 (s, 3H), 3.03–2.95 (m, 4H), 2.36 (t, J = 27.5 Hz, 2H), 2.19 (d, J = 11.0 Hz, 2H), 1.98 (s, 2H), 1.84–1.72 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 163.1, 159.8, 157.3, 156.5, 146.5, 141.3, 140.9, 136.0, 129.7, 129.0, 128.5, 127.9, 127.4, 118.1, 104.9, 55.7, 53.7, 46.1, 44.5, 42.4, 32.4, 28.1, 27.8, 25.6. HRMS (ESI) (m/z): calculated for C₃₂H₃₇N₅O₂ [M + H]⁺ 572.2617; found, 572.2692.

8-Cyclopentyl-5-(4-methoxyphenyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (30)

The title compound was prepared from 3 according to the preparation of 51. 165 mg, 40% yield. ¹H NMR (500 MHz, CDCl₃) δ 9.76 (s, 2H), 8.64 (s, 1H), 7.68 (s, 1H), 7.55 (t, J = 3.0 Hz, 1H), 7.29–7.21 (m, 3H), 7.16 (d, J = 7.0 Hz, 2H), 6.60 (d, J = 0.5 Hz, 1H), 6.35 (s, 1H), 5.80 (t, J = 18.0 Hz, 1H), 4.94 (d, J = 13.5 Hz, 2H), 3.32 (s, 1H), 3.20 (s, 2H), 3.05–2.95 (m, 4H), 2.37 (s, 3H), 2.35 (t, J = 11.5 Hz, 2H), 2.31 (d, J = 7.5 Hz, 2H), 2.20 (d, J = 11.5 Hz, 2H), 1.98–1.93 (m, 4H), 1.79 (q, J = 15.0 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 163.6, 159.7, 157.2, 156.5, 143.9, 141.4, 1391, 128.9, 128.5, 127.3, 120.4, 116.8, 110.7, 105.2, 55.8, 53.5, 46.1, 42.4, 32.5, 28.2, 27.8, 25.6. HRMS (ESI) (m/z): calculated for C₂₉H₃₃N₅O₂ [M + H]⁺ 484.2634; found, 484.2790.

8-Cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(thiophen-3-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (31)

The title compound was prepared from 3 according to the preparation of 51. 160 mg, 26% yield, 99.6% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.76 (s, 2H), 8.55 (s, 1H), 7.68 (s, 1H), 7.46–7.44 (m, 2H), 7.29–7.20 (m, 4H), 7.17 (t, J = 20.5 Hz, 2H), 6.36 (s, 1H), 5.82 (t, J = 17.0 Hz, 1H), 4.94 (d, J = 14.0 Hz, 2H), 3.31 (s, 1H), 3.20 (s, 2H), 3.04–2.94 (m, 4H), 2.38–2.33 (m, 2H), 2.19 (d, J = 11.5 Hz, 2H), 1.97 (d, J = 8.0 Hz, 2H), 1.81–1.74 (m, 6H). ¹³C NMR (125 MHz, CDCl₃) δ 163.6, 159.7, 157.6, 156.4, 143.2, 136.0, 135.8, 128.9, 128.5, 127.9, 127.3, 126.7, 125.2, 117.2, 105.5, 55.8, 53.5, 46.1, 42.4, 32.4, 28.1, 27.8, 25.6. HRMS (ESI) (m/z): calculated for C₂₉H₃₃N₅OS [M + H]⁺ 500.2406; found, 500.2476.

8-Cyclopentyl-2-(methylthio)-5-((trimethylsilyl)ethynyl)pyrido[2,3-d]pyrimidin-7(8H)-one (78)

Compound 9a (0.5 g, 1.22 mmol), Et₃N (0.37 g, 3.06 mmol), CuI (0.019 g, 0.122 mmol), Pd(PPh₃)₄ (0.071 g, 0.061 mmol), trimethylsilylacetylene (0.3 g, 3.05 mmol) were added to a 25 mL round-bottomed flask with 10 mL THF. The mixture was heated to reflux for 6 h under the atmosphere of N₂. After the reaction was completed, the resulting mixture was purified and separated by column chromatography (PE:EA = 30:1) to obtain 0.47 g of a yellow solid product.

8-Cyclopentyl-2-(methylsulfonyl)-5-((trimethylsilyl)ethynyl)pyrido[2,3-d]pyrimidin-7(8H)-one (79)

Compound 78 (0.47 g, 1.32 mmol) and m-CPBA (0.71 g, 3.95 mmol) was added into a 50 mL round bottom flask with 25 mL DCM. The reaction was carried out at room temperature for 2 h. After the reaction was completed, 10 mL of saturated sodium sulfite was added for quenching. The resulting mixture was washed with saturated sodium carbonate (15 mL × 3) and saturated brine (15 mL × 3). The separated organic layer was dried over anhydrous sodium sulfate, filtered, and distilled under a reduced pressure to obtain 0.61 g yellow solid.

Tert-butyl (1-(8-cyclopentyl-7-oxo-5-((trimethylsilyl)ethynyl)-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)piperidine-4-yl)(phenethyl)carbamate (80)

Compound 79 (0.61 g, 1.57 mmol), compound 12 (0.95 g, 3.13 mmol), and 10 mL toluene were added into a 25 mL round bottom flask. The reaction system was heated to reflux for 3 h under an atmosphere of N2. After the reaction was completed, the mixture was cooled to room temperature and subjected to column chromatography (PE:EA = 32:1) to obtain 0.45 g of white solid. 47% yield.

Tert-butyl (1-(8-cyclopentyl-7-oxo-5-(5-(trimethylsilyl)-1H-1,2,3-triazole-4-yl)-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)piperidine-4-yl)(phenethyl)carbamate (81)

Compound 80 (0.22 g, 0.36 mmol) and 10 mL toluene were added into a 25 mL round bottom flask. After dissolution, NaN₃ (0.05 g, 0.72 mmol) was added into bottom. The reaction system was heated to 100 °C for 3 h under an atmosphere of N₂. After the reaction was completed, the resulting mixture was added to 70 mL ethyl acetate and washed with water (30 mL × 3), and the separated organic layer was dried over anhydrous sodium sulfate, filtered, and distilled under a reduced pressure to provide 0.2 g product.

Tert-butyl (1-(8-cyclopentyl-7-oxo-5-(1H-1,2,3-triazol-4-yl)-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)piperidin-4-yl)(phenethyl)carbamate (82)

Compound 81 (0.22 g, 0.3 mmol) and 10 mL THF were added into a 25 mL round bottom flask. After dissolution, 0.49 mL 1 M TBAF tetrahydrofuran solution was added. The mixture was stirred under atmosphere of N₂ for 10 min, and distilled under a reduced pressure to obtain 0.2 g product.

8-Cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(1H-1,2,3-triazol-4-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (32)

Compound 82 (0.2 g, 0.34 mmol) and 20 mL of dichloromethane were added to a 25 mL round bottom flask. After dissolution, trifluoroacetic acid (4 mL, 54.4 mmol) was added, and the mixture was stirred at room temperature for 1 h. After the reaction was completed, 5 mL of toluene was added into and distilled under reduced pressure. Then 10 mL ether was added into stir for 16 h. The resulting residue was filtered, and the filter cake was wash with ether, dried to obtain 22 mg pale yellow solid product, 2% yield, 98.8% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.69 (s, 1H), 7.99 (s, 1H), 7.31 (t, J = 15.0 Hz, 2H), 7.22 (q, J = 16.5 Hz, 3H), 6.66 (s, 1H), 5.92–5.84 (m, 1H), 4.75 (d, J = 12.5 Hz, 2H), 3.18 (t, J = 23.5 Hz, 1H), 3.04 (t, J = 14.0 Hz, 2H), 2.93–2.87 (m, 3H), 2.41 (d, J = 8.5 Hz, 2H), 2.03 (s, 4H), 1.85 (d, J = 8.5 Hz, 2H), 1.69 (d, J = 5.0 Hz, 2H), 1.37 (q, J = 30.5 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 191.4, 163.3, 159.7, 157.4, 1566, 147.12, 136.8, 136.0, 134.3, 130.2, 129.7, 129.0, 128.5, 127.35, 118.0, 105.2, 55.8, 53.6, 46.1, 42.4, 32.5, 28.2, 27.9, 25.6. HRMS (ESI) (m/z): calculated for C₃₃H₄₁N₅O₂ [M + H]⁺ 485.2699; found, 485.2780.

5-(3-Aminophenyl)-8-cyclopentyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (85)

Compound 9a (0.45 g, 1.10 mmol), Na₂CO₃ (0.23 g, 2.12 mmol), Pd(PPh₃)₄ (0.102 g, 0.088 mmol) and 3-Boc-aminophenyl boronic acid (0.39 g, 1.65 mmol) were added into a 50 mL round-bottomed flask with 20 mL anhydrous THF. The reaction system was heated to reflux for 2 h under the atmosphere of N₂. After the reaction was completed, the resulting residue was subjected to column chromatography (PE:EA = 5:1) to obtain a 0.45 g oily substance. Then 5 mL of dichloromethane and trifluoroacetic acid (4 mL, 54.4 mmol) was added to the round bottom flask. The mixture was stirred at room temperature for 1 h. After the reaction was completed, 5 mL toluene was added and the mixture was distilled under a reduced pressure to obtain a 0.48 g product.

N-(3-(8-cyclopentyl-2-(methylthio)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-5-yl)phenyl)acrylamide (86)

Compound 85 (0.48 g, 1.03 mmol) and NaHCO₃ (0.26 g, 3.08 mmol) were added into a 50 mL round bottom flask with 20 mL MeCN, then acryloyl chloride (0.14 g, 1.54 mmol) was slowly added dropwise under ice bath. The mixture was stirred at room temperature for 30 min. After the completion of the reaction, the mixture was quenched by adding 50 mL of water, extracted with ethyl acetate (30 mL × 3), The separated organic layer was dried over anhydrous sodium sulfate, filtered, and distilled under a reduced pressure. The resulting residue was subjected to column chromatography (PE:EA = 6:1) to obtain 0.24 g oily substance. ¹H NMR (300 MHz, CDCl₃) δ 8.60 (s, 1H), 7.74 (d, J = 5.6 Hz, 2H), 7.69 (s, 1H), 7.47 (t, J = 15.8 Hz, 1H), 7.14 (d, J = 7.6 Hz, 1H), 6.48 (d, J = 12.9 Hz, 1H), 6.42 (s, 1H), 6.30–6.24 (m, 1H), 6.04–5.92 (m, 1H), 5.78 (d, J = 10.1 Hz, 1H), 2.62 (s, 3H), 2.40 (q, J = 19.2 Hz, 2H), 2.09 (t, J = 7.2 Hz, 2H), 1.93 (t, J = 9.2 Hz, 2H), 1.72 (t, J = 11.2 Hz, 2H). MS (ESI) (m/z): calculated for C₂₂H₂₂N₄O₂S [M + H]⁺ 407.15; found, 407.16.

N-(3-(8-cyclopentyl-7-oxo-2-(4-(phenethylamino)piperidin-1-yl)-7,8-dihydropyrido[2,3-d]pyrimidin-5-yl)phenyl)acrylamide (33)

The title compound was prepared according to the preparation of 51. 93 mg, 7% yield, 97.3% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.48 (s, 2H), 8.57 (s, 1H), 8.24 (s, 1H), 7.69 (s, 1H), 7.43 (t, J = 15.9 Hz, 1H), 7.26–7.16 (m, 5H), 7.07 (d, J = 7.5 Hz, 1H), 6.55–6.45 (m, 2H), 6.32 (s, 1H), 5.75 (d, J = 10.8 Hz, 1H),5.65 (t, J = 17.9 Hz, 1H), 4.93 (s, 2H), 3.56 (s, 1H), 3.26 (s, 2H), 3.12-3.09 (t, J = 15.0 Hz, 2H), 2.98 (t, J = 25.6 Hz, 2H), 2.25 (s, 2H), 1.97 (s, 2H), 1.81 (s, 1H), 1.73 (s, 2H), 1.54 (d, J = 10.4 Hz, 2H), 1.46 (s, 2H), 1.36 (s, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 191.4, 163.3, 159.7, 157.4, 156.6, 147.1, 136.8, 136.3, 136.0, 134.3, 130.2, 129.7, 129.0, 128.5, 127.3, 118.0, 105.2, 55.8, 53.6, 46.1, 42.4, 32.4, 28.2, 27.9, 25.6. HRMS (ESI) (m/z): calculated for C₃₄H₃₈N₆O₂ [M + H]⁺ 563.3056; found, 563.3146.

5-(Benzyloxy)-8-cyclopentyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (89)

Compound 8a (1 g, 3.6 mmol) K₂CO₃ (1 g, 7.2 mmol), and benzyl bromide (1.23 g, 7.2 mmol) were added into a 50 mL round-bottom flask with 10 mL acetonitrile. The mixture was heated to reflux. After the reaction was completed, the mixture was filtered, cooled, and crystallized, filtered to obtain a 0.3 g product, the filtrate was purified by column chromatography (PE:EA = 10: 1) to obtain 0.33 g of white solid, a total of 0.63 g of product was obtained. 47.6% yield.

5-(Benzyloxy)-8-cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (34)

The title compound was prepared according to the preparation of 51. 52 mg, 15% yield, 98.9% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.86 (s, 2H), 8.76 (s, 1H), 8.66 (d, J = 4.5 Hz, 1H), 7.83 (t, J = 14.0 Hz, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.42–7.35 (m, 5H), 7.28–7.16 (m, 5H), 5.80 (s, 1H), 5.70–5.63 (m, 1H), 5.14 (s, 2H), 4.92 (d, J = 12.0 Hz, 2H), 3.34 (s, 1H), 3.21 (s, 2H), 3.04 (t, J = 16.0 Hz, 2H), 2.95 (t, J = 25.0 Hz, 2H), 2.18 (d, J = 10.0 Hz, 4H), 1.87 (s, 2H), 1.71–1.65 (m, 4H), 1.57 (d, J = 4.5 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 165.3, 161.2, 160.3, 155.8, 154.9, 136.2, 135.2, 128.9, 128.7, 128.6, 128.5, 127.5, 127.3, 101.6, 95.0, 70.5, 55.8, 53.1, 46.1, 42.5, 32.5, 28.1, 27.9, 25.5. HRMS (ESI) (m/z): calculated for C₃₂H₃₇N₅O₂ [M + H]⁺ 524.2947; found, 524.3027.

8-Cyclopentyl-5-ethynyl-2-(4-(phenethylamine)piperidine-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (35)

Compound 78 (0.19 g, 0.31 mmol) and 0.50 mL 1 M TBAF were added to a 25 mL round bottom flask with 10 mL THF. The resulting mixture was stirred for 5 min under an atmosphere of N2. The resulting residue was subjected to column chromatography (PE:EA = 9:1) to obtain 0.17 g of oil. 20 mL dichloromethane and trifluoroacetic acid (4 mL, 54.4 mmol) was added to the round bottom flask and stirred at room temperature for 1 h. After the reaction was completed, 5 mL toluene was added and distilled under reduced pressure. Then 10 mL ether was added to stir for 16 h. The resulting residue was filtered, and the filter cake was washed with ether and dried to obtain 70 mg of white solid powder, 23.8% yield, 92.9% HPLC purity.

8-Cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(phenylethynyl)pyrido[2,3-d]pyrimidin-7(8H)-one (36)

The title compound was prepared according to the preparation of 51. 20 mg, 48% yield, 94.2% HPLC purity. ¹H NMR (300 MHz, DMSO-d₆) δ 9.93 (s, 1H), 8.70 (s, 2H), 7.75 (q, J = 9.3 Hz, 2H), 7.53–7.46 (m, 3H), 7.38–7.25 (m, 5H), 6.52 (s, 1H), 5.82–5.76 (m, 1H), 4.79 (d, J = 12.0 Hz, 2H), 3.49 (s, 1H), 3.42–3.34 (m, 3H), 3.24 (s, 2H), 3.10 (t, J = 24.6 Hz, 2H), 2.94 (t, J = 16.5 Hz, 2H), 2.28 (s, 2H), 2.19 (d, J = 10.8 Hz, 2H), 1.95 (s, 2H), 1.81 (t, J = 9.3 Hz, 2H), 1.67 (t, J = 10.2 Hz, 2H), 1.51 (d, J = 9.0 Hz, 2H), 1.10 (t, J = 14.1 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 163.3, 160.1, 157.7, 156.0, 136.0, 132.0, 129.9, 129.7, 129.0, 128.6, 128.5, 127.3, 121.6, 120.9, 105.4, 99.3, 82.4, 55.9, 53.5, 46.2, 42.4. HRMS (ESI) (m/z): calculated for C₃₃H₃₅N₅O [M + H]⁺ 517.2842; found, 518.2921.

8-Cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(m-tolylethynyl)pyrido[2,3-d]pyrimidin-7(8H)-one (37)

The title compound was prepared according to the preparation of 51. 108 mg, 21% yield, 99.7% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.76 (s, 2H), 8.85 (s, 1H), 7.39 (t, J = 16.0 Hz, 2H), 7.28 (t, J = 19.0 Hz, 5H), 7.23–7.16 (m, 2H), 6.51 (s, 1H), 5.81–5.74 (m, 1H), 4.96 (d, J = 12.5 Hz, 2H), 3.31 (s, 1H), 3.20 (s, 2H), 3.03–2.95 (m, 4H), 2.37 (s, 3H), 2.32 (d, J = 9.0 Hz, 2H), 2.20 (d, J = 11.5 Hz, 2H), 1.97 (s, 2H), 1.77 (d, J = 11.5 Hz, 4H), 1.63 (d, J = 4.5 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 163.3, 160.1, 157.8, 156.0, 138.4, 136.0, 132.6, 130.6, 130.0, 129.1, 129.0, 128.5, 128.5, 127.4, 121.4, 120.9, 105.4, 99.6, 82.0, 55.9, 53.4, 46.2, 42.4, 32.5, 28.2, 27.9, 25.6, 21.2. HRMS (ESI) (m/z): calculated for C₃₄H₃₇N₅O [M + H]⁺ 532.2998; found, 532.3082.

8-Cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(p-tolylethynyl)pyrido[2,3-d]pyrimidin-7(8H)-one (38)

The title compound was prepared according to the preparation of 51. 159 mg, 31% yield, 99.2% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.73 (s, 2H), 8.84 (s, 1H), 7.45 (d, J = 7.9 Hz, 2H), 7.31–7.15 (m, 7H), 6.51 (s, 1H), 5.80–5.74 (m, 1H), 4.94 (d, J = 14.1 Hz, 2H), 3.31 (s, 1H), 3.18 (d, J = 6.8 Hz, 2H), 3.06–2.94 (m, 4H), 2.38 (s, 3H), 2.30 (d, J = 7.5 Hz, 2H), 2.19 (d, J = 11.3 Hz, 2H), 1.97 (s, 2H), 1.82–1.62 (m, 6H). ¹³C NMR (125 MHz, DMSO-d₆ + CDCl₃) δ 162.4, 159.5, 157.1, 155.4, 139.6, 136.2, 136.1, 131.4, 129.5, 128.9, 128.2, 128.1, 126.5, 119.8, 119.6, 117.8, 117.5, 115.2, 104.4, 99.1, 81.3, 54.7, 52.7, 51.9, 45.4, 45.2, 41.9, 41.4, 40.0, 39.8, 39.7, 39.5, 39.3, 39.2, 39.0, 31.8, 31.7, 27.6, 27.3, 25.0, 24.6, 21.0. HRMS (ESI) (m/z): calculated for C₃₄H₃₇N₅O [M + H]⁺ 532.2998; found, 532.3097.

5-((3-Chlorophenyl)ethynyl)-8-cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (39)

The title compound was prepared according to the preparation of 51. 118 mg, 22% yield, 93.8% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.76 (s, 2H), 8.80 (s, 1H), 7.56 (s, 1H), 7.43 (t, J = 30.0 Hz 1H), 7.34 (d, J = 23.0 Hz, 1H), 7.31 (t, J = 8.5 Hz, 1H), 7.29 (t, J = 15.0 Hz, 1H), 7.25 (t, J = 15.0 Hz, 4H), 7.19 (t, J = 15.0 Hz, 2H), 6.52 (s, 1H), 5.77 (q, J = 25.0 Hz 1H), 4.95 (d, J = 12.5 Hz, 2H), 3.31 (s, 1H), 3.20 (s, 2H), 3.03–2.96 (m, 4H), 2.31 (t, J = 10.5 Hz, 2H), 2.20 (d, J = 11.0 Hz, 2H), 1.97 (s, 2H), 1.77 (q, J = 30.0 Hz, 4H), 1.63 (d, J = 4.5 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 163.1, 160.1, 157.6, 156.1, 136.0, 134.5, 131.8, 130.1, 129.9, 129.8, 129.3, 129.0, 128.5, 127.4, 123.3, 121.3, 105.2, 97.4, 83.6, 77.2, 55.8, 53.5, 46.2, 42.4, 32.5, 28.2, 27.9, 25.6. HRMS (ESI) (m/z): calculated for C₃₂H₃₇N₅O₂ [M + H]⁺ 552.2452; found, 552.2529.

5-((4-Chlorophenyl)ethynyl)-8-cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (40)

The title compound was prepared according to the preparation of 51. 100 mg, 15% yield, 99.4% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.76 (s, 2H), 8.80 (s, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 7.29 (t, J = 15.0 Hz, 3H), 7.23–7.15 (m, 2H), 6.52 (s, 1H), 5.83–5.74 (m, 1H), 4.94 (d, J = 12.9 Hz, 2H), 3.31 (s, 1H), 3.19 (s, 2H), 3.06–2.94 (m, 4H), 2.29 (t, J = 7.8 Hz, 2H), 2.20 (d, J = 11.4 Hz, 2H), 1.97 (s, 2H), 1.82–1.70 (m, 4H), 1.64 (t, J = 10.2 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 163.1, 160.1, 157.6, 156.0, 136.0, 135.9, 133.2, 129.5, 129.0, 129.0, 128.5, 127.3, 121.1, 120.0, 105.2, 97.9, 83.3, 55.8, 53.5, 46.2, 42.4, 32.5, 28.2, 27.9, 25.6. HRMS (ESI) (m/z): calculated for C₃₃H₃₄ClN₅O [M + H]⁺ 552.2452; found, 552.2529.

8-Cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(pyridin-2-ylethynyl)pyrido[2,3-d]pyrimidin-7(8H)-one (41)

The title compound was prepared according to the preparation of 51. 76 mg, 12% yield, 97.6% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.68 (s, 2H), 8.85 (s, 1H), 8.80 (s, 1H), 8.66 (d, J = 3.5 Hz, 2H), 7.97 (d, J = 8.0 Hz, 1H), 7.44 (q, J = 12.5 Hz, 1H), 7.31–7.22 (m, 3H), 7.16 (t, J = 15.5 Hz, 2H), 6.57 (s, 1H), 5.80–5.76 (t, J = 18.0 Hz, 1H), 4.96 (d, J = 12.0 Hz, 2H), 3.32 (s, 1H), 3.22–3.15 (m, 2H), 3.05–2.97 (m, 4H), 2.35–2.31 (m, 2H), 2.20 (d, J = 11.5 Hz, 2H), 1.98 (s, 2H), 1.82–1.73 (m, 6H). ¹³C NMR (125 MHz, CDCl₃) δ 163.0, 160.1, 157.5, 156.1, 151.1, 148.3, 140.3, 136.0, 129.0, 128.9, 128.5, 127.4, 123.9, 121.7, 105.0, 94.4, 86.2, 55.9, 54.6, 53.6, 46.3, 46.2, 43.8, 42.4, 36.0, 32.5, 28.2, 27.9, 25.7. HRMS (ESI) (m/z): calculated for C₃₂H₃₄N₆O [M + H]⁺ 519.2794; found, 519.2863.

8-Cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(pyridin-3-ylethynyl)pyrido[2,3-d]pyrimidin-7(8H)-one (42)

The title compound was prepared according to the preparation of 51. 56 mg, 8.8% yield, 99.1% HPLC purity. ¹H NMR (500 MHz, DMSO-d₆ + CDCl₃) δ 9.57 (s, 2H), 8.89 (s, 1H), 8.66 (d, J = 4.5 Hz, 1H), 7.83 (t, J = 14.0 Hz, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.41 (q, J = 12.0 Hz, 1H), 7.32 (t, J = 15.0 Hz, 2H), 7.25 (d, J = 7.5 Hz, 3H), 6.52 (s, 1H), 5.82 (t, J = 15.0 Hz, 1H), 4.94 (s, 2H), 3.41 (s, 1H), 3.20 (s, 2H), 3.04 (d, J = 9.0 Hz, 4H), 2.32 (d, J = 8.0 Hz, 2H), 2.22 (d, J = 11.5 Hz, 2H), 2.01 (s, 2H), 1.85–1.84 (t, J = 8.0 Hz, 2H), 1.69 (d, J = 10.5 Hz, 4H). ¹³C NMR (125 MHz, DMSO-d₆ + CDCl₃) δ 162.0, 159.6, 157.1, 155.4, 149.8, 141.1, 136.3, 136.1, 128.3, 128.2, 128.1, 127.5, 126.4, 123.7, 120.6, 104.1, 97.1, 80.7, 54.5, 52.7, 45.1, 41.8, 40.0, 39.8, 39.7, 39.5, 39.3, 39.2, 39.0, 31.7, 27.52, 27.2, 25.0. HRMS (ESI) (m/z): calculated for C₃₂H₃₄N₆O [M + H]⁺ 519.2794; found, 519.2864.

8-Cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(thiophen-2-ylethynyl)pyrido[2,3-d]pyrimidin-7(8H)-one (43)

The title compound was prepared according to the preparation of 51. 167 mg, 26% yield, 98.9% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.73 (s, 2H), 8.79 (s, 1H), 7.40 (d, J = 5.9 Hz, 2H) 7.30–7.21 (m, 3H), 7.16 (d, J = 7.2 Hz, 2H), 7.06 (d, J = 3.7 Hz, 1H), 6.50 (s, 1H), 5.79–5.75 (t, J = 20.0 Hz, 1H), 4.95 (d, J = 12.3 Hz, 2H), 3.31 (s, 1H), 3.20 (s, 2H), 3.05–2.95 (m, 4H), 2.31 (s, 2H), 2.20 (d, J = 11.3 Hz, 2H), 1.97 (s, 2H), 1.79 (s, 4H), 1.63 (d, J = 4.1 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 162.4, 159.4, 156.9, 155.3, 136.1, 133.4, 129.0, 128.9, 128.1, 128.1, 128.0, 127.0, 126.4, 120.5, 119.3, 104.1, 91.9, 85.6, 54.7, 52.7, 51.8, 45.4, 45.2, 41.8, 41.4, 39.7, 39.5, 39.4, 39.2, 39.0, 31.70, 31.6, 27.5, 27.2, 25.0, 24.5. HRMS (ESI) (m/z): calculated for C₃₁H₃₃N₅OS [M + H]⁺ 524.2406; found, 524.2505.

5-(Cyclohexylethynyl)-8-cyclopentyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (44)

The title compound was prepared according to the preparation of 51. 128 mg, 20% yield. ¹H NMR (300 MHz, CDCl₃) δ 9.75 (s, 2H), 8.73 (s, 1H), 7.31–7.15 (m, 5H), 6.39 (s, 1H), 5.75 (t, J = 17.7 Hz, 1H), 4.93 (d, J = 13.5 Hz, 2H), 3.30 (s, 1H), 3.19 (s, 2H), 3.05–2.92 (m, 4H), 2.72–2.66 (m, 1H), 2.26 (q, J = 25.2 Hz, 4H), 1.93 (t, J = 13.5 Hz, 4H), 1.76 (t, J = 8.7 Hz, 6H), 1.60 (t, J = 17.4 Hz, 5H), 1.39 (d, J = 6.9 Hz, 3H). ¹³C NMR (125 MHz, CDCl₃) δ 136.0, 130.7, 128.9, 128.5, 127.3, 120.7, 105.9, 105.6, 74.1, 55.9, 53.3, 46.1, 42.4, 32.5, 32.2, 29.9, 28.2, 27.9, 25.7, 25.6, 24.7. HRMS (ESI) (m/z): calculated for C₃₃H₄₁N₅O [M + H]⁺ 524.3311; found, 524.3388.

8-Cyclopentyl-5-((1-hydroxycyclohexyl)ethynyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (45)

The title compound was prepared according to the preparation of 51. 140 mg, 21% yield, 99.3% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.74 (s, 2H), 8.66 (s, 1H), 7.29 (q, J = 17.1 Hz, 3H), 7.20 (q, J = 21.9 Hz, 2H), 6.44 (s, 1H), 5.74 (t, J = 17.7 Hz, 1H), 4.86 (d, J = 12.6 Hz, 2H), 3.30 (s, 1H), 3.20 (s, 2H), 3.01 (q, J = 7.2 Hz, 4H), 2.62 (s, 1H), 3.34–2.19 (m, 4H), 2.06–1.97 (m, 4H), 1.79–1.54 (m, 14H), 1.32 (d, J = 10.5 Hz, 1H). ¹³C NMR (125 MHz, CDCl₃) δ 163.4, 156.0, 157.6, 155.8, 136.0, 129.8, 128.9, 128.5, 127.3, 121.3, 105.4, 104.1, 76.7, 76.6, 68.9, 56.0, 53.5, 46.4, 42.3, 39.6, 32.5, 28.2, 27.9, 25.6, 25.1, 23.3. HRMS (ESI) (m/z): calculated for C₃₃H₄₁N₅O₂ [M + H]⁺ 540.3260; found, 540.3338.

8-Methyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(4-(trifluoromethyl)phenyl)pyrido[2,3-d]pyrimidine-7(8H)-one (46)

The title compound was prepared with methylamine hydrochloride as the starting material. The synthesis method is similar to 51. White solid, 115 mg, 8% yield, 99.0% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.74 (s, 2H), 8.28 (s, 1H), 7.75 (d, J = 4.8 Hz, 2H), 7.53 (d, J = 4.8 Hz, 2H), 7.29–7.15 (m, 5H), 6.35 (s, 1H), 5.00 (d, J = 8.2 Hz, 2H), 3.61 (s, 1H), 3.31 (s, 1H), 3.20 (s, 2H), 3.04–2.95 (m, 5H), 2.20 (d, J = 6.9 Hz, 2H), 1.87 (s, 1H), 1.79–1.72 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 163.1, 160.1, 157.3, 156.2, 147.5, 138.9, 136.0, 131.4 (q, J_CF = 32.6 Hz), 129.1, 128.9, 128.5, 127.3, 125.8, 125.8, 125.6, 116.8, 104.6, 55.8, 46.2, 42.2, 32.5, 28.2, 27.6. HRMS (ESI) (m/z): calculated for C₂₈H₂₈F₃N₅O [M + H]⁺ 508.2246; found, 508.2339.

8-Ethyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(4-(trifluoromethyl)phenyl)pyrido[2,3-d]pyrimidine-7(8H)-one (47)

The title compound was prepared with ethylamine aqueous solution as the starting material. The synthesis method is similar to 51. White solid, 119 mg, 7.8% yield, 98.9% HPLC purity. ¹H NMR (500 MHz, CDCl₃) δ 9.72 (s, 1H), 8.41 (s, 1H), 7.46 (s, 1H), 7.38 (s, 1H), 7.27–7.34 (m, 1H), 7.25 (s, 2H), 7.23 (d, J = 7.5 Hz, 1H), 7.16 (t, J = 7.7 Hz, 3H), 5.84–5.92 (m, 1H), 4.97 (d, J = 14.0 Hz, 2H), 3.30 (s, 1H), 3.20 (s, 2H), 3.04 (t, J = 8.2 Hz, 2H), 2.97 (t, J = 12.2 Hz, 2H), 2.37 (q, J = 6.7 Hz, 2H), 2.29 (d, J = 10.0 Hz, 6H), 2.21 (d, J = 11.5 Hz, 2H), 1.99 (t, J = 6.5 Hz, 2H), 1.76–1.86 (m, 4H), 1.64 (d, J = 5.5 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 162.6, 160.2, 157.38, 155.7, 147.4, 139.1, 136.1, 131.5 (q, J_CF = 19.6 Hz), 129.1, 129.0, 128.5, 127.4, 125.8, 125.8, 122.8, 117.3, 104.8, 55.8, 46.2, 42.3, 36.0, 32.5, 28.2, 12.7. HRMS (ESI) (m/z): calculated for C₃₃H₃₉N₅O [M + H]⁺ 522.3163; found, 522.3236.

8-Butyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(4-(trifluoromethyl)phenyl)pyrido[2,3-d]pyrimidine-7(8H)-one (48)

The title compound was prepared with N-butylamine as the starting material. The synthesis method is similar to 51. White solid, 162 mg, 11% yield, 98.1% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.72 (s, 2H), 8.28 (s, 1H), 7.75 (d, J = 4.8 Hz, 2H), 7.74 (d, J = 4.8 Hz, 2H), 7.29–7.21 (m, 3H), 7.16 (d, J = 4.3 Hz, 2H), 6.34 (s, 1H), 4.97 (d, J = 8.1 Hz, 2H), 4.31 (t, J = 8.97 Hz, 3H), 3.31 (s, 1H), 1.79–1.66 (m, 4H), 1.39 (q, J = 13.4 Hz, 2H), 0.95 (t, J = 4.4 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 164.0, 159.5, 158.3, 156.4, 155.6, 146.4, 136.1, 130.6, 130.5, 129.0, 128.5, 127.3, 124.2, 121.0, 118.6, 111.0, 105.7, 55.9, 55.5, 46.1, 45.3, 42.4, 32.44, 28.2, 19.4. HRMS (ESI) (m/z): calculated for C₃₁H₃₄F₃N₅O [M + H]⁺ 550.2715; found, 550.2791.

8-Isopropyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(4-(trifluoromethyl)phenyl)pyrido[2,3-d]pyrimidine-7(8H)-ketone (49)

The title compound was prepared with isopropylamine as the starting material. The synthesis method is similar to 51. White solid, 138 mg, 9% yield, 94.6% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.72 (s, 2H), 8.27 (s,1H), 7.74 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.1 Hz, 2H), 7.30–7.15 (m, 6H), 6.30 (s, 1H), 5.77–5.68 (s, 1H), 4.96 (d, J = 13.8 Hz, 2H), 3.31–3.17 (m, 3H), 3.06–2.94 (m, 4H), 2.21 (d, J = 11.4 Hz, 2H), 1.82–1.73 (m, 5H), 1.58 (d, J = 6.9 Hz, 6H). ¹³C NMR (125 MHz, CDCl₃) δ 163.2, 159.7, 157.4, 156.2, 147.1, 138.9, 136.2, 129.1, 129.0, 128.5, 127.4, 125.8, 125.8, 118.0, 105.1, 55.8, 46.1, 45.6, 42.4, 32.5, 28.2, 19.4. HRMS (ESI) (m/z): calculated for C₃₀H₃₂F₃N₅O [M + H]⁺ 536.2559; found, 536.2645.

8-Isobutyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(4-(trifluoromethyl)phenyl)pyrido[2,3-d]pyrimidine-7(8H)-one (50)

The title compound was prepared with isobutylamine as the starting material. The synthesis method is similar to 51. White solid, 136 mg, 9% yield, 98.1% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.73 (s, 2H), 8.29 (s,1H), 7.75 (d, J = 8.1 Hz, 2H), 7.54 (d, J = 8.0 Hz, 2H), 7.3–7.23 (m,5H), 7.16 (d, J = 6.7 Hz, 2H), 6.35 (s, 1H), 4.97 (d, J = 13.5 Hz, 2H), 4.17 (d, J = 7.2 Hz, 2H), 3.49–2.93 (m, 7H), 1.76 (q, J = 33.5 Hz, 2H), 0.93 (t, J = 6.7 Hz, 6H). ¹³C NMR (125 MHz, CDCl₃) δ 163.1, 162.4, 162.2, 161.9, 160.0, 157.4, 156.3, 147.4, 139.0, 131.4 (q, J_CF = 32.5 Hz), 129.1, 129.0, 128.5, 127.4, 127.1, 125.8, 125.8, 125.8, 124.9, 122.8, 120.6, 117.7, 111.3, 105.4, 104.6, 55.8, 47.7, 46.2, 42.3, 32.5, 28.2, 27.3, 20.4. HRMS (ESI) (m/z): calculated for C₃₁H₃₄F₃N₅O [M + H]⁺ 550.2715; found, 550.2799.

8-Cyclohexyl-2-(4-(phenethylamino)piperidin-1-yl)-5-(4-(trifluoromethyl)phenyl)pyrido[2,3-d]pyrimidine-7(8H)-one (52)

The title compound was prepared with cyclohexylamine as the starting material. The synthesis method is similar to 51. White solid, 192 mg, 13% yield, 99.1% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.73 (s, 1H), 8.25 (s, 1H), 7.73 (d, J = 8.1 Hz, 2H), 7.51 (t, J = 8.0 Hz, 2H), 7.28 (t, J = 11.9 Hz, 4H), 7.18 (q, J = 22.5 Hz, 2H), 6.30 (s, 1H), 5.32 (t, J = 24.6 Hz, 1H), 4.95 (d, J = 13.4 Hz, 1H), 3.25 (d, J = 38.7 Hz, 3H), 3.00 (q, J = 31 Hz, 4H), 2.66 (q, J = 33.8 Hz, 2H), 2.21 (d, J = 11.6 Hz, 2H), 1.87–1.66 (m, 8H), 1.38 (q, J = 34.3 Hz, 2H), 1.22 (t, J = 23.2 Hz, 1H). ¹³C NMR (125 MHz, CDCl₃) δ 163.4, 159.6, 157.4, 156.4, 147.1, 138.9, 136.0, 131.2, 129.1, 129.0, 128.5, 127.4, 125.8, 125.8, 122.8, 105.1, 55.8, 46.1, 42.5, 32.4, 28.6, 28.1, 26.6, 25.8. HRMS (ESI) (m/z): calculated for C₃₃H₃₆F₃N₅O [M + H]⁺ 576.2872; found, 576.2937.

2-(4-(Phenethylamino)piperidin-1-yl)-8-phenyl-5-(4-(trifluoromethyl)phenyl)pyrido[2,3-d]pyrimidine-7(8H)-one (53)

The title compound was prepared with aniline as the starting material. The synthesis method is similar to 51. White solid, 138 mg, 9% yield, 94.1% HPLC purity. ¹ H NMR (300 MHz, CDCl₃) δ 9.56 (s, 2H), 8.31 (s, 1H), 7.78 (d, J = 8.0 Hz, 2H), 7.59 (d, J = 8.0 Hz, 2H), 7.46–7.20 (m, 9H), 7.12 (d, J = 6.9 Hz, 2H), 4.52 (s, 2H), 3.11 (s, 3H), 2.95 (d, J = 15.6 Hz, 2H), 2.71 (s, 1H), 1.99 (d, J = 9.5 Hz, 2H), 1.55 (d, J = 9.8 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃ + DMSO-d₆) δ 162.0, 159.1, 156.6, 156.3, 147.7, 138.3, 136.0, 135.4, 130.4 (t, J_CF = 32.5 Hz), 128.4, 128.3, 128.0, 127.9, 127.6, 126.3, 125.1, 125.1, 124.3, 122.1, 116.3, 103.5, 55.5, 45.0, 41.2, 39.9, 39.7, 39.5, 39.4, 39.2, 39.0, 38.9, 31.6, 27.3. HRMS (ESI) (m/z): calculated for C₃₃H₃₀F₃N₅O [M + H]⁺ 570.2402; found, 570.2487.

5-(2-Methoxyphenyl)-8-methyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (54)

The title compound was prepared with methylamine hydrochloride as the starting material. The synthesis method is similar to 51. White solid, 210 mg, 14% yield, 99.7% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.73 (s, 2H), 8.09 (s, 1H), 7.43 (t, J = 9.1 Hz, 1H), 7.28–7.15 (m, 4H), 7.05 (t, J = 8.8 Hz, 1H), 6.99 (d, J = 5.0 Hz, 1H), 6.33 (s, 1H), 4.99 (d, J = 8.1 Hz, 2H), 3.73 (s, 3H), 3.60 (s, 3H), 3.29 (s, 1H), 3.19 (s, 2H), 3.04 (t, J = 9.7 Hz, 2H), 2.95 (t, J = 15.0 Hz, 2H), 2.19 (d, J = 6.7 Hz, 2H), 1.76 (d, J = 6.0 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 163.8, 162.4, 161.9, 159.9, 158.2, 156.3, 155.5, 146.7, 136.1, 130.6, 130.4, 128.8, 128.4, 127.2, 124.2, 120.9, 118.5, 117.3, 114.6, 111.0, 105.2, 55.8, 55.4, 46.1, 42.2, 32.4, 28.2, 27.4. HRMS (ESI) (m/z): calculated for C₂₈H₃₁N₅O₂ [M + H]⁺ 470.2478; found, 470.2558.

8-Ethyl-5-(2-methoxyphenyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (55)

The title compound was prepared with ethylamine aqueous solution as the starting material. The synthesis method is similar to 51. White solid, 148 mg, 10% yield, 98.8% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.75 (s, 2H), 8.10 (s, 1H), 7.44 (t, J = 15.4 Hz, 1H), 7.30–7.15 (m, 4H), 7.07–6.98 (m, 3H), 6.32 (s, 1H), 4.98 (d, J = 13.6 Hz, 2H), 4.35 (q, J = 20.7 Hz, 2H), 3.74 (s, 3H), 3.29 (s, 1H), 3.19 (s, 1H), 3.05–2.91 (m, 4H), 2.19 (d, J = 11.9 Hz, 2H), 1.76 (q, J = 10.4 Hz, 2H), 1.27 (t, J = 13.9 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 163.3, 162.4, 162.1, 160.0, 158.3, 156.3, 155.0, 146.7, 136.1, 130.6, 130.5, 128.9, 128.5, 127.3, 124.3, 120.9, 117.8, 115.4, 111.0, 105.3, 55.9, 55.5, 46.1, 42.2, 35.8, 32.4, 28.2, 12.8. HRMS (ESI) (m/z): calculated for C₂₉H₃₃N₅O₂ [M + H]⁺ 484.2634; found, 484.2714.

8-Butyl-5-(2-methoxyphenyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (56)

The title compound was prepared with N-butylamine as the starting material. The synthesis method is similar to 51. White solid, 130 mg, 9% yield, 98.4% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.73 (s, 2H), 8.09 (s, 1H), 7.43 (t, J = 9.4 Hz, 1H), 7.28–7.15 (m, 4H), 7.05 (t, J = 8.9 Hz, 2H), 7.00 (s, 1H), 6.98 (s, 1H), 6.31 (s, 1H), 4.97 (d, J = 8.2 Hz, 2H), 4.30 (t, J = 9.0 Hz, 2H), 3.74 (s, 3H), 3.29 (s, 1H), 3.19 (s, 1H), 3.05-2.92 (m, 4H), 2.18 (d, J = 7.0 Hz, 2H), 2.06 (s, 1H), 1.76–1.67 (m, 4H), 1.39 (q, J = 13.9 Hz, 2H), 1.21 (t, J = 8.4 Hz, 3H).¹³C NMR (75 MHz, CDCl₃) δ 163.4, 162.5, 159.9, 158.2, 156.3, 155.1, 146.6, 136.1, 130.6, 130.5, 128.9, 128.4, 127.2, 124.2, 120.9, 117.7, 111.0, 105.2, 55.9, 55.4, 46.1, 42.2, 40.52, 32.41, 29.7, 28.1, 20.4, 13.8. HRMS (ESI) (m/z): calculated for C₃₁H₃₇N₅O₂ [M + H]⁺ 512.2947; found, 512.3026.

8-isopropyl-5-(2-methoxyphenyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (57)

The title compound was prepared with isopropylamine as the starting material. The synthesis method is similar to 51. White solid, 185 mg, 13% yield, 98.8% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.73 (s, 2H), 8.08 (s, 1H), 7.43 (t, J = 5.2 Hz, 1H), 7.41–6.98 (m, 8H), 6.27 (s, 1H), 5.73 (s, 1H), 4.95 (d, J = 8.2 Hz, 2H), 3.74 (s, 3H), 3.29 (s, H), 3.19 (s, 2H), 3.04–2.92 (m, 4H), 1.79-1.72 (m, 2H), 1.58 (d, J = 4.2 Hz, 6H). ¹³C NMR (125 MHz, DMSO-d₆) δ 162.6, 159.3, 158.0, 156.0, 155.1, 146.2, 137.0, 130.8, 130.1, 128.6, 128.5, 126.7, 123.3, 120.9, 117.3, 111.6, 104.3, 55.5, 54.2, 44.7, 44.4, 42.1, 40.3, 40.1, 39.8, 39.5, 39.2, 38.9, 38.7, 31.7, 27.6, 19.2. HRMS (ESI) (m/z): calculated for C₃₀H₃₅N₅O₂ [M + H]⁺ 498.2791; found, 498.2867.

8-Isobutyl-5-(2-methoxyphenyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (58)

The title compound was prepared with isobutylamine as the starting material. The synthesis method is similar to 51. White solid, 155 mg, 10% yield, 96.4% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.74 (s, 2H), 8.09 (s, 1H), 7.43 (t, J = 14.1 Hz, 1H), 7.30–6.98 (m, 11H), 6.32 (s, 1H), 4.96 (d, J = 13.7 Hz, 2H), 4.16 (d, J = 7.1 Hz, 2H), 3.74 (s, 3H), 3.29–2.90 (m, 7H), 2.32–2.07 (m, 3H), 1.80–1.73 (m, 2H), 0.93 (q, J = 6.7 Hz, 6H). ¹³C NMR (125 MHz, CDCl₃) δ 163.8, 159.8, 158.3, 156.4, 155.6, 146.6, 136.1, 130.6, 130.5, 128.9, 128.5, 127.3, 124.3, 121.0, 117.9, 111.0, 105.2, 55.9, 55.5, 47.6, 46.1, 42.2, 32.4, 28.2, 27.3, 20.5. HRMS (ESI) (m/z): calculated for 498.2867 [M + H]⁺ 512.2947; found, 512.3022.

8-Cyclopropyl-5-(2-methoxyphenyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (59)

The title compound was prepared with cyclopropyl as the starting material. The synthesis method is similar to 51. White solid, 182 mg, 12% yield, 98.6%. ¹H NMR (300 MHz, CDCl₃) δ 9.73 (s, 2H), 8.06 (s, 1H), 7.44 (t, J = 5.2 Hz, 1H), 7.29–7.15 (m, 6H), 7.05–6.98 (m, 2H), 6.26 (s, 1H), 5.02 (d, J = 8.0 Hz, 2H), 3.74 (s, 3H), 3.30 (s, 1H), 3.19 (3 s, 2H), 3.03 (t, J = 10.1 Hz, 2H), 2.96 (t, J = 14.8 Hz, 2H), 2.79 (s, 1H), 2.19 (d, J = 7.0 Hz, 2H), 1.96 (s, 1H), 1.76 (d, J = 6.3 Hz, 2H), 1.18 (d, J = 4.2 Hz, 2H), 0.88 (d, J = 2.1 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 164.7, 162.4, 162.0, 159.7, 158.1, 157.0, 156.3, 146.7, 136.1, 130.6, 130.4, 128.9, 128.5, 127.2, 124.2, 121.0, 118.1, 110.9, 105.4, 55.9, 55.4, 46.1, 42.2, 32.4, 28.2, 24.9, 9.3. HRMS (ESI) (m/z): calculated for C₃₀H₃₃N₅O₂ [M + H]⁺ 496.2634; found, 496.2724.

8-Cyclohexyl-5-(2-methoxyphenyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (60)

The title compound was prepared with cyclohexylamine as the starting material. The synthesis method is similar to 51. White solid, 165 mg, 11% yield, 99.4% HPLC purity. ¹ H NMR (300 MHz, CDCl₃) δ 9.73 (s, 1H), 8.07 (s, 1H), 7.44 (t, J = 9.0 Hz, 1H), 7.45 (t, J = 8.0 Hz, 2H), 7.3–7.06 (m, 7H), 7.04–6.97 (m, 2H), 6.28 (s, 1H), 5.32 (t, J = 24.6 Hz, 1H), 4.94 (d, J = 13.2 Hz, 2H), 3.74 (s, 1H), 3.30–3.18 (m, 3H), 3.06–2.92 (m, 4H), 2.69 (d, J = 12.0 Hz, 2H), 2.20 (d, J = 11.1 Hz 2H), 1.86–1.66 (m, 5H), 1.41–1.26 (m, 3H). ¹³C NMR (125 MHz, CDCl₃) δ 162.5, 159.2, 158.0, 155.9, 155.2, 146.2, 137.0, 130.8, 130.1, 128.6, 128.5, 126.7, 123.3, 120.8, 111. 6, 104.3, 55.4, 54.2, 44.7, 42.1, 40.0, 39.8, 39.7, 39.5, 39.3, 39.2, 39.0, 31.7, 28.2, 27.6, 26.1, 25.3. HRMS (ESI) (m/z): calculated for C₃₃H₃₉N₅O₂ [M + H]⁺ 538.3104; found, 538.3190.

5-(2-Methoxyphenyl)-8-phenyl-2-(4-(3-phenylpropyl)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (61)

The title compound was prepared with aniline as the starting material. The synthesis method is similar to 51. White solid, 85 mg, 6% yield, 96.4% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.57 (s, 2H), 8.14 (s, 1H), 7.49-7.01 (m, 17 H), 6.42 (s, 1H), 3.78 (s, 3H), 2.97 (d, J = 9.6 Hz, 2H), 2.68 (t, J = 18.9 Hz, 2H), 1.99 (d, J = 9.9 Hz, 2H), 1.75 (s, 4H), 1.54 (d, J = 8.4 Hz, 2H).¹³C NMR (75 MHz, DMSO-d₆) δ 162.2, 159.3, 157.8, 156.0, 156.0, 147.2, 137.0, 136.2, 130.9, 130.2, 128.8, 128.7, 128.6, 128.5, 127.9, 126.7, 123.4, 120.9, 117.0, 111.7, 103.9, 55.5, 54.0, 44.6, 41.5, 40.3, 40.1, 39.8, 39.5, 39.2, 38.9, 38.7, 31.6, 27.4. HRMS (ESI) (m/z): calculated for C₃₃H₃₃N₅O₂ [M + H]⁺ 532.2634; found, 532.2712.

5-((3-chlorophenyl)ethynyl)-8-methyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (62)

The title compound was prepared with methylamine hydrochloride as the starting material. The synthesis method is similar to 51. Yellow solid, 97 mg, 7% yield, 94.7% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.75 (s, 2H), 8.79 (s, 1H), 7.57 (s, 1H), 7.46 (d, J = 3.3 Hz, 1H), 7.43 (d, J = 14.8 Hz, 1H), 7.39–7.22 (m, 4H), 7.17 (d, J = 4.4 Hz, 2H), 6.56 (s, 1H), 5.01 (d, J = 7.8 Hz, 2H), 3.56 (s, 3H), 3.31 (s, 1H), 3.20 (s, 2H), 3.06–2.96 (m, 4H), 2.22 (d, J = 7.0 Hz, 2H), 1.95 (s, 1H), 1.81–1.75 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 162.9, 160.3, 157.4, 155.6, 136.1, 134.5, 131.8, 130.1, 123.0, 129.8, 129.6, 128.9, 128.5, 127.3, 123.2, 120.0, 104.8, 97.5, 83.4, 55.8, 46.2, 42.3, 32.5, 28.2, 27.5. HRMS (ESI) (m/z): calculated for C₂₉H₂₈ClN₅O [M + H]⁺ 498.1982; found, 498.2053.

5-((3-chlorophenyl)ethynyl)-8-ethyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (63)

The title compound was prepared with ethylamine aqueous solution as the starting material. The synthesis method is similar to 51. Yellow solid, 203 mg, 14% yield, 97.4% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 8.83 (s, 1H), 7.83 (s, 1H), 7.65 (d, J = 7.2 Hz, 1H), 7.56–7.18 (m, 8H), 6.44 (s, 1H), 4.55 (d, J = 9.0 Hz, 2H), 4.20 (d, J = 6.9 Hz, 2H), 3.22–3.15 (m, 8H), 2.65 (dd, J = 6.3 Hz, J = 6.6 Hz, 6H), 1.91 (d, J = 10.8 Hz, 2H), 1.27–1.16 (m, 6H). ¹³C NMR (75 MHz, DMSO-d₆) δ 161.2, 160.0, 157.3, 154.6, 140.2, 133.4, 131.5, 130.6, 130.5, 130.0, 128.9, 128.5, 128.1, 125.7, 122.7, 118.7, 103.2, 96.8, 83.5, 53.8, 47.7, 42.3, 40.3, 40.1, 39.8, 39.5, 39.2, 38.9, 38.7, 36.0, 35.0, 31.5, 12.5. HRMS (ESI) (m/z): calculated for C₃₀H₃₀ClN₅O [M + H]⁺ 512.2139; found, 512.2216.

8-Butyl-5-((3-chlorophenyl)ethynyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (64)

The title compound was prepared with N-butylamine as the starting material. The synthesis method is similar to 51. Yellow solid, 144 mg, 10% yield, 96.3% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.76 (s, 2H), 8.80 (s, 1H), 7.56 (s, 1H), 7.46 (d, J = 4.6 Hz, 1H), 7.40–7.12 (m, 8H), 6.56 (s, 1H), 4.99 (d, J = 7.4 Hz, 2H), 4.26 (t, J = 8.9 Hz, 2H), 3.31 (s, 1H), 3.20 (s, 2H), 3.14-2.96 (m, 4H), 2.21 (d, J = 6.8 Hz, 2H), 1.78 (q, J = 19.3 Hz, 2H), 1.68–1.62 (m, 2H), 1.38 (q, J = 13.3 Hz, 2H), 0.98 (t, J = 4.4 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 162.6, 162.5, 162.0, 160.3, 157.4, 155.3, 136.0, 134.5, 131.8, 130.8, 130.1, 129.9, 129.8, 130.7, 129.9, 129.8, 129.6, 129.3, 128.9, 128.5, 127.3, 123.2, 120.3, 118.5, 114.6, 104.8, 97.4, 83.4, 55.8, 46.1, 42.2, 40.5, 32.4, 29.6, 28.1, 20.3, 13.7. HRMS (ESI) (m/z): calculated for C₃₂H₃₄ClN₅O [M + H]⁺ 539.2452; found, 540.2525.

5-((3-chlorophenyl)ethynyl)-8-isopropyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (65)

The title compound was prepared with isopropylamine as the starting material. The synthesis method is similar to 51. Yellow solid, 88 mg, 6% yield, 96.7% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.76 (s, 2H), 8.79 (s, 1H), 7.56 (s, 1H), 7.45 (d, J = 4.5 Hz, 1H), 7.39 (d, J = 4.8 Hz, 1H), 7.33–7.16 (m, 8H), 6.51 (s, 1H), 5.65 (q, J = 12.3 Hz, 1H), 4.98 (d, J = 7.5 Hz, 2H), 3.47 (q, J = 12.6 Hz, 1H), 3.33 (s, 1H), 3.20 (d, J = 4.5 Hz, 2H), 3.05–2.97 (m, 4H), 2.22 (d, J = 7.2 Hz, 2H), 1.81–1.74 (m, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 163.1, 162.4, 162.1, 156.0, 157.6, 155.7, 136.0, 134.5, 131.8, 130.1, 129.9, 129.8, 129.4, 128.9, 128.5, 127.3, 123.2, 121.2, 105.1, 97.3, 83.3, 65.8, 55.8, 46.1, 45.5, 42.4, 32.4, 28.1, 19.3, 15.2. HRMS (ESI) (m/z): calculated for C₃₁H₃₂ClN₅O₂ [M + H]⁺ 526.2295; found, 526.2383.

5-((3-chlorophenyl)ethynyl)-8-isobutyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (66)

The title compound was prepared with isobutylamine as the starting material. The synthesis method is similar to 51. Yellow solid, 110 mg, 8% yield, 96.3% HPLC purity. ¹H NMR (300 MHz, DMSO-d₆) δ 8.89 (s, 2H), 7.88 (s, 1H), 7.68 (d, J = 6.9 Hz, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.51 (d, J = 8.4 Hz, 3H), 7.27–7.18 (m, 6H), 6.49 (s, 1H), 4.52 (s, 3H), 4.04 (d, J = 6.0 Hz, 3H), 3.25–3.18 (m, 7H), 2.79 (t, J = 30.3 Hz, 6H), 2.51 (s, 1H), 2.17 (t, J = 12.0 Hz, 2H), 1.89 (d, J = 10.5 Hz, 3H), 1.23 (d, J = 8.1 Hz, 3H), 0.87 (d, J = 5.7 Hz, 8H). ¹³C NMR (125 MHz, DMSO-d₆) δ 161.8, 159.9, 157.5, 155.3, 133.4, 131.6, 130.7, 130.6, 130.1, 129.0, 128.5, 128.1, 125.7, 122.7, 118.8, 103.2, 96.9, 83.5, 53.8, 47.9, 46.7, 42.3, 39.8, 39.5, 39.2, 38.9, 38.7, 36.1, 31.7, 26.7, 20.1. HRMS (ESI) (m/z): calculated for C₃₂H₃₄ClN₅O₂ [M + H]⁺ 540.2452; found, 540.2544.

5-((3-chlorophenyl)ethynyl)-8-cyclopropyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (67)

The title compound was prepared with cyclopropyl as the starting material. The synthesis method is similar to 51. Yellow solid, 77 mg, 5% yield, 98.3% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.74 (s, 2H), 8.77 (s, 1H), 7.56 (d, J = 1.0 Hz, 1H), 7.55–7.16 (m, 9H), 6.51 (s, 1H), 5.04 (d, J = 8.5 Hz, 2H), 3.32 (s, 1H), 3.20 (s, 2H), 3.06–2.97 (m, 4H), 2.79–2.76 (m, 1H), 2.21 (d, J = 6.5 Hz, 2H), 1.81–1.74 (m, 4H), 1.21–1.15 (m, 2H), 0.85 (s, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 164.0, 160.2, 157.3, 136.0, 134.5, 131.8, 130.1, 130.0, 129.8, 129.7, 129.0, 128.5, 127.3, 123.2, 120.8, 104.8, 97.6, 83.3, 55.9, 46.2, 42.3, 32.5, 28.2, 25.0, 9.3. HRMS (ESI) (m/z): calculated for C₃₁H₃₀ClN₅O [M + H]⁺ 524.2139; found, 524.2219.

5-((3-chlorophenyl)ethynyl)-8-cyclohexyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (68)

The title compound was prepared with cyclohexylamine as the starting material. The synthesis method is similar to 51. Yellow solid, 200 mg, 14% yield, 98.3% HPLC purity. ¹ H NMR (300 MHz, CDCl₃) δ 9.73 (s, 2H), 8.78 (s, 1H), 7.56 (d, J = 3.3 Hz, 2H), 7.47–7.16 (m, 7 H), 6.53 (s, 1H), 5.25 (t, J = 24.3 Hz, 2H), 4.97 (d, J = 12.9 Hz, 2H), 3.32(s, 1H), 3.21 (d, J = 8.4 Hz, 2H), 3.06–2.97 (m, 4H), 2.63 (q, J = 34.5 Hz, 2H), 2.23 (d, J = 10.2 Hz, 2H), 1.85–1.62 (m, 10H), 1.35 (t, J = 12.6 Hz, 2H), 1.21 (t, J = 14.1 Hz, 1H). ¹³C NMR (125 MHz, CDCl₃ + CD₃OD) δ 163.6, 157.6, 155.7, 136.0, 131.7, 130.0, 129.9, 129.8, 128.8, 128.4, 127.2, 105.2, 97.6, 83.0, 55.4, 52.0, 49.6, 49.3, 49.0, 48.7, 48.4, 45.9, 45.7, 42.4, 41.9, 32.2, 28.5, 28.0, 26.4, 25.6, 24.7. HRMS (ESI) (m/z): calculated for C₃₄H₃₆ClN₅O [M + H]⁺ 566.2608; found, 566.2886.

5-((3-chlorophenyl)ethynyl)-2-(4-(phenethylamino)piperidin-1-yl)-8-phenylpyrido[2,3-d]pyrimidine-7(8H)-one (69)

The title compound was prepared with aniline as the starting material. The synthesis method is similar to 51. Yellow solid, 200 mg, 14% yield, 98.3% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 8.89 (s, 1H), 7.79 (s, 1H), 7.65 (d, J = 7.2 Hz, 1H), 7.53–7.41 (m, 6H), 7.25–7.16 (m, 8H), 6.54 (s, 1H), 4.53 (s, 1H), 3.87 (s, 1H), 3.42–3.39 (m, 6H), 2.78 (d, J = 6.6 Hz, 4H), 2.71–2.52 (m, 4H), 1.76 (s, 3H), 1.12–1.10 (m, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 162.6, 161.6, 159.7, 157.1, 156.1, 140.0, 135.8, 133.5, 131.4, 130.4, 130.2, 129.8, 128.5, 128.4, 128.2, 127.9, 127.7, 125.5, 122.6, 119.1, 103.2, 97.0, 83.4, 53.6, 47.6, 41.9, 40.3, 40.1, 39.8, 39.5, 39.2, 39.0, 38.7, 36.0, 31.5. HRMS (ESI) (m/z): calculated for C₃₄H₃₀ClN₅O [M + H]⁺ 560.2139; found, 560.2222.

5-((1-hydroxycyclohexyl)ethynyl)-8-methyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (70)

The title compound was prepared with methylamine hydrochloride as the starting material. The synthesis method is similar to 51. White solid, 280 mg, 19% yield, 99.0% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.73 (s, 2H), 8.69 (s, 1H), 7.31–7.17 (m, 5H), 6.47 (s, 1H), 4.93 (d, J = 7.3 Hz, 2H), 3.57 (s, 3H), 3.30 (s, 1H), 3.22 (s, 2H), 3.06–2.97 (m, 6H), 2.22 (d, J = 6.8 Hz, 3H), 2.13–2.04 (m, 6H), 1.80–1.71 (m, 4H), 1.61–1.54 (m, 2H), 1.33 (d, J = 5.7 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃ + CD₃OD) δ 163.5, 156.0, 157.5, 155.1, 135.7, 130.6, 128.5, 128.1, 126.9, 118.6, 104.8, 104.3, 76.3, 68.9, 68.1, 55.2, 48.7, 48.5, 48.3, 48.2, 48.0, 47.8, 47.6, 45.5, 41.9, 39.0, 31.9, 27.8, 27.0, 24.6, 22.9. HRMS (ESI) (m/z): calculated for C₂₉H₃₅N₅O₂ [M + H]⁺ 486.2791; found, 486.2887.

8-Ethyl-5-((1-hydroxycyclohexyl)ethynyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (71)

The title compound was prepared with N-butylamine as the starting material. The synthesis method is similar to 51. White solid, 136 mg, 9% yield, 98.8% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.73 (s, 2H), 8.67 (s, 1H), 7.30–7.16 (m, 5H), 6.46 (s, 1H), 4.92 (d, J = 7.4 Hz, 2H), 4.28 (q, J = 12.3 Hz, 2H), 3.31 (s, 1H), 3.21 (s, 2H), 3.05–2.96 (m, 5H), 2.21 (d, J = 6.9 Hz, 2H), 2.05 (d, J = 7.4 Hz, 2H), 1.78–1.71 (m, 6H), 1.60–1.53 (m,3H), 1.33–1.30 (m, 1H), 1.24 (t, J = 4.2 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ 161.3, 160.1, 157.2, 154.7, 137.0, 129.6, 128.6, 128.5, 126.7, 118.6, 105.5, 104.1, 75.8, 67.2, 54.1, 44.7, 41.9, 38.9, 35.1, 31.7, 27.63, 24.7, 22.6, 12.5. HRMS (ESI) (m/z): calculated for C₃₀H₃₇N₅O₂ [M + H]⁺ 499.2947; found, 500.3027.

8-Butyl-5-((1-hydroxycyclohexyl)ethynyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7 (8H)-one (72)

The title compound was prepared with N-butylamine as the starting material. The synthesis method is similar to 51. Yellow solid, 75 mg, 5% yield, 96.1% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.74 (s, 2H), 8.69 (s, 1H), 7.30–7.16 (m, 5H), 6.47 (s, 1H), 4.92 (d, J = 8.3 Hz, 2H), 4.25 (t, J = 8.9 Hz, 2H), 3.30 (s, 1H), 3.21 (s, 2H), 3.05–2.98 (m, 4H), 2.54 (s, 1H), 2.20 (d, J = 7.3 Hz, 2H), 2.05 (d, J = 7.6 Hz, 2H), 1.78–1.56 (m, 12H), 1.38–1.34 (m, 3H), 0.95 (t, J = 4.4 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 161.5, 160.2, 157.2, 154.9, 137.1, 129.6, 128.6, 128.5, 126.7, 118.5, 105.5, 104.0, 75.9, 67.2, 54.1, 44.7, 42.0, 31.7, 29.1, 27.6, 24.7, 22.7, 19.7, 13.6. HRMS (ESI) (m/z): calculated for C₃₂H₄₁N₅O₂ [M + H]⁺ 528.3260; found, 528.3345.

5-((1-Hydroxycyclohexyl)ethynyl)-8-isopropyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (73)

The title compound was prepared with isopropylamine as the starting material. The synthesis method is similar to 51. White solid, 88 mg, 6% yield, 96.4% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.73 (s, 2H), 8.66 (s, 1H), 7.30–7.16 (m, 5H), 6.44 (s, 1H), 5.66–5.61 (m, 1H), 4.91 (t, J = 7.8 Hz, 2H), 3.31 (s, 1H), 3.21 (d, J = 4.7 Hz, 2H), 3.05–2.95 (m, 4H), 2.21 (d, J = 6.8 Hz, 2H), 2.04 (d, J = 8.2 Hz, 2H), 1.79–1.69 (m, 6H), 1.60–1.33 (m, 10H), 1.32 (d, J = 4.3 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 163.5, 162.4, 159.8, 157.6, 155.4, 136.0, 123.0, 128.9, 128.5, 127.3, 121.0, 105.4, 104.1, 68.8, 55.9, 46.3, 45.5, 42.3, 39.6, 32.4, 28.1, 25.0, 23.2, 19.3. HRMS (ESI) (m/z): calculated for C₃₁H₃₉N₅O₂ [M + H]⁺ 514.3104; found, 514.3195.

5-((1-Hydroxycyclohexyl)ethynyl)-8-isobutyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-ketone (74)

The title compound was prepared with isobutylamine as the starting material. The synthesis method is similar to 51. White solid, 93 mg, 6% yield, 97.1% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.73 (s, 2H), 8.71 (s, 1H), 7.30–7.16 (m, 5H), 6.48 (s, 1H), 4.93 (d, J = 7.6 Hz, 2H), 4.11 (d, J = 4.3 Hz, 2H), 3.30 (s, 1H), 3.20 (s, 2H), 3.04–2.96 (m, 4H), 2.69 (s, 2H), 2.19 (d, J = 12.4 Hz, 3H), 2.05 (d, J = 7.1 Hz, 2H), 1.79–1.70 (m, 6H), 1.61-1.54 (m, 3H), 1.33 (d, J = 12.8 Hz, 1H), 0.89 (s, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 163.3, 162.5, 162.0, 160.1, 157.6, 155.5, 136.0, 130.1, 128.9, 128.5, 127.3, 120.2, 105.0, 104.1, 76.6, 68.9, 55.9, 47.5, 46.3, 42.1, 39.6, 32.4, 28.2, 27.2, 25.1, 23.3, 20.3. HRMS (ESI) (m/z): calculated for C₃₂H₄₁N₅O₂ [M + H]⁺ 527.3260; found, 528.3345.

5-((1-Hydroxycyclohexyl)ethynyl)-8-cyclopropyl-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-ketone (75)

The title compound was prepared with cyclopropyl as the starting material. The synthesis method is similar to 51. White solid, 107 mg, 7% yield, 95.0% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.72 (s, 2H), 8.67 (s, 1H), 7.32–7.16 (m, 5H), 6.40 (s, 1H), 4.97 (d, J = 16.2 Hz, 2H), 3.32–3.22 (m, 3H), 3.01 (q, J = 33.6 Hz, 4H), 2.85–2.73 (m, 1H), 2.43 (s, 1H), 2.22 (d, J = 40.8 Hz, 2H), 2.04 (t, J = 15.9 Hz, 2H), 1.80–1.61 (m, 6H), 1.56 (q, J = 29.1 Hz, 3H), 1.35 (d, J = 10.8 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 162.8, 159.9, 156.9, 136.9, 129.4, 128.6, 128.6, 126.8, 119.3, 105.6, 104.3, 75.9, 67.2, 54.3, 44.7, 42.0, 31.8, 27.7, 24.7, 22.7, 9.0. HRMS (ESI) (m/z): calculated for C₃₁H₃₇N₅O₂ [M + H]⁺ 512.2947; found, 512.3033.

8-Cyclohexyl-5-((1-hydroxycyclohexyl)ethynyl)-2-(4-(phenethylamino)piperidin-1-yl)pyrido[2,3-d]pyrimidine-7(8H)-one (76)

The title compound was prepared with cyclohexylamine as the starting material. The synthesis method is similar to 51. White solid, 78 mg, 5% yield, 98.1% HPLC purity. ¹H NMR (300 MHz, DMSO-d₆) δ 8.74 (s,1H), 7.31–7.16 (m, 5H), 6.28 (s, 1H), 5.70 (s, 1H), 5.14 (s, 1H), 4.54 (d, J = 10.5 Hz, 2H), 3.32–3.19 (m, 3H), 2.86–2.60 (m, 6H), 2.50 (s, 2H), 1.91–1.82 (m, 7H), 1.67–1.46 (m, 12H), 1.42–1.19 (m, 8H). ¹³C NMR (125 MHz, CDCl₃ + CD₃OD) δ 158.7, 156.5, 136.9, 131.4, 129.7, 129.3, 128.1, 78.3, 69.3, 56.3, 50.0, 49.8, 49.7, 49.5, 49.3, 49.2, 49.0, 46.6, 43.3, 40.2, 33.1, 29.4, 28.9, 27.2, 25.8, 24.0. HRMS (ESI) (m/z): calculated for C₃₄H₄₃N₅O₂ [M + H]⁺ 554.3417; found, 554.3497.

5-((1-hydroxycyclohexyl)ethynyl)-2-(4-(phenethylamino)piperidin-1-yl)-8-phenylpyrido[2,3-d]pyrimidine-7(8H)-one (77)

The title compound was prepared with aniline as the starting material. The synthesis method is similar to 51. White solid, 77 mg, 5% yield, 97.1% HPLC purity. ¹H NMR (300 MHz, CDCl₃) δ 9.58 (s, 2H), 8.74 (s, 1H), 7.48–7.35 (m, 7H), 7.31–7.12 (m, 5H), 6.57 (s, 1H), 4.55 (s, 2H), 3.12 (s, 3H), 2.98–2.93 (m, 3H), 2.75 (s, 2H), 2.06–2.00 (m, 9H), 1.81–1.57 (m, 11H), 1.34–1.20 (m, 1H). ¹³C NMR (125 MHz, CDCl₃ + CD₃OD) δ 163.4, 156.0, 157.7, 156.3, 136.0, 135.5, 131.7, 128.9, 128.8, 128.4, 128.3, 127.2, 119.7, 105.0, 105.0, 68.5, 55.3, 49.6, 49.3, 49.0, 48.8, 48.5, 48.2, 45.7, 41.9, 39.3, 32.1, 27.8, 24.9, 23.2. HRMS (ESI) (m/z): calculated for C₃₄H₃₇N₅O₂, [M + H]⁺ 548.2947; found, 548.3033.

Tested compounds

All synthesized target compounds were characterized by ¹H NMR, ¹³C NMR, HRMS, IR, and HPLC. The purity of all tested compounds was all over 90%. (Supplementary Figs. S2–S326).

Cytotoxicity assay

The cytotoxicity assay was completed using the MTT (sharp) assay. Human cell line L-02 was seeded in 96-well plates at a density of 3000 cells/well in a 100 μL culture medium. The cells were allowed to settle and attach for 12 h. Then the cell line was exposed to different concentrations of tested compounds in serial dilutions in a 150 μL medium and incubated for 72 h. Then 15 μL sterile MTT (5 mg/mL, dissolved in PBS) was added to each well and incubated with cells for 4 h at 37 °C. Purple formazan product was resuspended in DMSO (150 μL/well) and measured in a microplate reader at 490 nm wavelength. The experiment was repeated three times with three wells for each concentration of the tested agents. LC₅₀ values were calculated from the inhibition curves by normalized nonlinear regression analysis using GraphPad Prism 8 software.

Enzyme-based NAE activity assay

The NEDD8 Conjugation Initiation Kit (K-800, R&D) was performed in the enzyme-based assay according to the instructions of the manufacturer. 2 μL NAE (2.5 μM), 2 μL NEDD8 (250 μM), 2 μL Ubc12 (50 μM), 2 μL reaction buffer and 10 μL water solution of 51 (0 μM, 0.12 μM, 0.37 μM, 1.11 μM, 3.33 μM, 10 μM) were added into 100 μL reaction tubes in order. Reaction solutions were incubated at 37 °C for 10 min. The reaction was initiated by the addition of 2 μL Mg²⁺-ATP solution (10 mM), and the mixture was incubated at 37 °C for 60 min. In the negative control group, 2 μL ddH₂O was added instead of ATP solution. The reaction was quenched by the addition of 2 μL EDTA (1 M), and protein samples were electrophoresed under non-reducing conditions on a 12 % SDS-PAGE gel. The Ubc12-NEDD8 levels were determined by western blot analysis.

Cell-based NAE activity assay

To validate whether 51 could inhibit NAE in BxPC-3 cells, BxPC-3 cells which were treated with 51 under 0, 4, 8, 12 μM for 24 h were lysed and protein extracts were analyzed by western blot. The NEDD8 combined Ubc12 was noticeably decreased in a concentration-dependent manner, indicating that NAE activity was inhibited and that neddylation was suppressed by 51.

Western blot analysis

Protein samples were transferred to a PVDF membrane. The membrane was blocked with 5% (w/v) milk for 1 h at room temperature and probed with primary antibody diluted (1:1000, v/v) in 5% (w/v) milk overnight at 4 °C. The membrane was washed with TBS/0.1% (v/v) Tween 20 (TBST) and incubated with horseradish peroxide-conjugated secondary antibody diluted (1:4000, v/v) in 5% (w/v) milk for 1.5 h at room temperature. Protein bands were detected using SignalFire™ ECL Reagent (6883, Cell Signaling Technology, CST). In this assay anti-Ubc12 antibody (Abcam) was used to measure Ubc12-NEDD8, Densitometry analysis of the Western blot was conducted by using Image J. All experiment was repeated two times.

Metabolic stability assay of liver microsomes

This assay was performed using the Human liver microsome mixed metabolic stability kit (0111013, PHASE) according to the instructions of the manufacturer. 10 μL NADPH regeneration system A solution (20×), 2 μL NADPH regeneration system B solution (100×), 1 μL tested compound (20 mM), 182 μL 0.1 M PBS buffer were added into 1 mL reaction tubes in order, reaction solutions were incubated at 37 °C for 5 min. The reaction was initiated by the addition of 5 μL liver particles (20 mg/mL). 35 μL reaction solution was taken out each time and 165 μL of pre-cooled acetonitrile was added into the incubation system to stop the reaction at the set incubation time point of 0, 15, 30, 60, 90 min. After the sample was vortexed for 5 minutes, centrifuged at 12,000 rpm at 4 °C for 10 min at 12,000 rpm, and the supernatant was taken and injected with HPLC to detect the concentration of the drug. The concentration of the compound to be tested at the 0 min incubation time point is regarded as 100%, and other incubation time points are converted into the percentage remaining amount, and the natural logarithm of the percentage remaining amount at each time point is linear regression to the incubation time, and the slope is calculated k.

$${{{\mathrm{T}}}}_{1/2} = \frac{{ - 0.693}}{{{{\mathrm{k}}}}},{{{\mathrm{CL}}}}_{{{{\mathrm{int}}}}} = \frac{{0.693}}{{{{{\mathrm{T}}}}1/2\left( {\min } \right)}} \ast \frac{{{{{\mathrm{Incubation}}}}\,{{{\mathrm{system}}}}\,\left( {{{{\mathrm{mL}}}}} \right)}}{{{{{\mathrm{Liver}}}}\,{{{\mathrm{microsomes}}}}\left( {{{{\mathrm{mg}}}}} \right)}}$$

Apoptosis assay

Apoptosis was evaluated using Annexin V-EGFP Apoptosis Detection Kit (KGA102, KeyGEN BioTECH) following the manufacturer’s instructions. BxPC-3 cells were seeded at 3 × 10⁵ cells per well in six-well plates and allowed to attach in 12 h. Cells were treated with different concentrations of 51 (0 μM, 4 μM, 10 μM and 15 μM) for 12 h. Afterward, the cells were collected and washed twice with ice-cold PBS, and then resuspended in 500 μL 1× binding buffer, followed by the addition of 5 μL Annexin V-EGFP staining solution and 5 μL PI staining solution. After incubation at room temperature in the dark for 15 min, stained cells were analyzed immediately by flow cytometry (ACEA NovoCyte). In each sample, 20,000 cells were examined, and fluorescence was measured at an excitation wavelength of 488 nm through FL-1 (530 nm) and FL-3 (630 nm) filters.

In vivo anticancer activity experiments

BxPC-3 cells were established by inoculating 5 × 10⁶ cells in nude mice, respectively. The efficacy study was initiated when tumors had reached ~90 mm³. Tumor-bearing mice were randomly assigned to control and treatment groups and received the corresponding treatments as indicated. Tumor measurements were performed twice a week. All procedures for animal studies were approved by the Institutional Animal Care and Use Committee of the Jiangsu Institute of Materia Medica.

Log D measurement

Log D values of compounds were measured using the shake-flask method. Each compound was dissolved in 1 mL PBS (PBS was saturated with 1-octanol), and then an equal volume of 1-octanol (1-octanol was saturated with PBS) was added. The tube was shaken for 24 h at 37 °C to allow the compound to partition into each phase. After being centrifugated at 5000 rpm, a compound in each phase was determined by HPLC (Inertsil ODS-SP C18 column, 4.6 × 250 mm, 5 μm, SHIMADZU) at 254 nm. All the experiments were carried out in triplicate. The partition coefficient was calculated using the equation below.

$${{{\mathrm{Log}}}}\,{{{\mathrm{D}}}} = {{{\mathrm{Log}}}}\frac{{{\it{AUC}}_{{\it{octanol}}}}}{{{\it{AUC}}_{{\it{PBS}}}}}$$

Supplemetary information

Supplemetary Information

FigureS2–326