Design and Synthesis of Novel 5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidine Derivatives as VCP/p97 Inhibitors for the Treatment of Acute Myeloid Leukemia (AML)

Introduction

Acute myeloid leukemia (AML) is the most aggressive adult acute leukemia, characterised by clonal expansion of abnormally differentiated blasts of myeloid lineage. AML is one of the most common types of leukemia and has a poor prognosis. It causes more than 80,000 deaths worldwide each year, a number that is expected to double in the next 20 years.¹ With the continuous research, people have a better understanding of the molecular pathophysiology of AML. In recent years, multiple targeted therapeutic drugs were approved by FDA for the treatment of newly diagnosed or relapsed/refractory AML.^2–9 Contrary to traditional broad-spectrum cytotoxic chemotherapy drugs, the treatment of AML is becoming increasingly personalized.^10,11 However, despite significant progress in the treatment of AML over the past few decades and the approval of various new drugs, most patients still faced a dismal prognosis. In particular, for AML patients aged 60 years and older, current treatment strategies can only induce remission, and frequent recurrence results in a 2-year survival rate of only 10%–50%, which is still a huge unmet clinical need.¹²

The ubiquitin proteasome system (UPS) is the main protein degradation system that play an important role in regulating cell cycle progression, transcriptional regulation, apoptosis, genome integrity, immune response, tumorigenesis, angiogenesis, and chemotherapy resistance.^13–17 The uncontrolled rapid growth of tumor cells produces a large number of misfolded proteins, which are more dependent on UPS. Due to the poor prognosis of leukemia patients, targeting the components of the ubiquitin proteasome system for the treatment of leukemia has also received attention. For example, the MDM2 (E3 ligase) antagonist RG7112 exerts anti leukemia effects by stabilizing p53.¹⁸ Pevonedistat (TAK-924 or MLN4924) inhibits Cullin RING E3 ligase by inhibiting the stability of developmental downregulated protein 8 activating enzyme, leading to inhibition of ubiquitin activation and reduced AML growth.¹⁹

Valosin-containing protein (VCP/p97) is an ATPase that converts chemical energy into mechanical energy to perform a series of functions. The most extensively studied role of p97 is in endoplasmic reticulum associated degradation (ERAD).^20,21 In this process, misfolded polyubiquitinated proteins in the endoplasmic reticulum must first be transferred to the cytoplasm under the action of p97 before being delivered to proteasomes for degradation.^22,23 Since p97 also plays an important role in the UPS, researchers used the p97 inhibitor CB-5083 to screen a total of 131 human cancer cell lines from 16 cancer types. Compared with other cancer subtypes, AML cell lines were more sensitive to CB-5083, indicating that AML cell lines were highly dependent on p97.²⁴ In addition, p97 inhibition leads to the accumulation of ubiquitinated proteins in AML cells, activates the unfolded protein response, induces apoptosis, and inhibit the proliferation of various AML cells at nanomolar concentrations.²⁵

Although multiple p97 inhibitors have been developed (Figure 1), only CB-5083 and CB-5339 have conducted Phase I clinical trials. However, the clinical trial of CB-5083 has been terminated due to off-target effects causing vision problems.²⁶ CB-5339, as a second-generation p97 inhibitor, has higher selectivity and has been validated in multiple AML models, making it the only p97 inhibitor currently under clinical development. Herein, we report our design and synthesis of a series of novel 5,6,7, 8-tetrahydropyridine [2,3-d] pyrimidine derivatives as p97 inhibitors, and then biologically evaluated in vitro and in vivo. Among the inhibitors, compared to compound CB-5339, compounds V12 and V13 exhibited better p97 inhibitory activities. In the screening of 11 kinds of tumor cells, compound V12 and its metabolite V13 showed strong proliferative inhibitory activities against a variety of cell lines, especially leukemia cell lines. In vivo studies have shown that compound V12 showed good pharmacokinetic properties, good anti-tumor efficacy and lower toxicity. These data further indicated that compound V12 had a good therapeutic effect on leukemia.

Figure 1 Representative P97 small molecule inhibitor.

Results and Discussion

Design Strategy of Novel p97 Inhibitors

Since compound CB-5083 showed off-target toxicity in clinical trials, replacing the 7,8-dihydro-5H-pyran[4,3-d]pyrimidine core with 5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine to obtain the second-generation p97 inhibitor CB-5339 can avoid off-target toxicity.²⁴ Therefore, we have retained the 5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine core in this paper. The specific design strategy of novel p97 inhibitors were shown in Figure 2. Molecular docking studies showed that the end of the cavity where the benzylamine substituent bound to p97 was the residue Cys522. So, we introduced hydroxyl groups into the benzene ring or replace benzylamine N atoms with O and S atoms to investigate the effect on the activity. The amide substituent of indole forms a strong hydrogen bond with the hydroxyl group of threonine (T688). According to the principle of electron iso-platoon, we introduced different polar substituents to the indole ring in the hope of obtaining compounds with better activity. In addition, substituting the aromatic C atom with N atom could improve the water solubility of the compounds, and may also improve the biological activity of the compounds. Therefore, we introduced N atom at the A-F position of benzylamine benzene ring or indole ring. Herein, we present the discovery of compound V12, a potent and orally bioavailable p97 inhibitor.

Figure 2 Design of VCP/p97 inhibitors.

Chemistry

The syntheses of key intermediates 5, 6a-6c, 12 and 18a-18j have been summarized in Scheme 1. 1a-1e reacted with benzenesulfonyl chloride to give compounds 2a-2e (yield 85–93%). Intermediate 2 reacted with iodomethane to introduce a methyl at the 2-position of the indole, and then deprotected to obtain the intermediate 4a-4e (yield 47–66%). Compound 5 was prepared from 4b via palladium-catalyzed carbonyl intercalation reaction (yield 79%). 4c-4e reacted with zinc cyanide under the catalysis of Pd₂(dba)₃ to obtain 6a-6c (yield 53–78%). Intermediate 7 reacted with benzenesulfonyl chloride under the action of NaH to obtain 8 (yield 95%). Compound 8 was oxidized by m-CPBA to obtain 9, and then chlorinated with indole at position 4 under the action of phosphorus oxychloride to obtain intermediate 10 (with a total yield of 55% in two steps). A methyl group was introduced, and then deprotected to obtain the intermediate 11 (with a two-step total yield of 51%). 11 reacted with zinc cyanide under the catalysis of Pd₂(dba)₃ to obtain 12 (yield 53%). Commercially available 13a-13b reacted with urea and then produced 14a-14b in the presence of sodium hydroxide (yield 76–81%). Compound 14a-14b were reduced to obtain 15a-15b, followed by chlorination to obtain the intermediates 16a-16b. The introduction of Boc protection group on the amino group and replacement of 4-chloride with amines, alcohol or sulfhydryl group to give 18a-18j (yield 45–79%).

Scheme 1 Syntheses of intermediates 5, 6a-6c, 12 and 18a-18j.a aReagents and conditions: (a) PhSO2Cl, NaH, THF, rt; (b) Methyl iodide, LDA, Tetrahydrofuran, −50 °C; (c) Sodium hydroxide, Ethanol, H2O, 55 oC; (d) 4b, Carbon monoxide, Palladium acetate, 1,3-Bis(diphenylphosphino)propane, 80 °C; (e) Zinc cyanide, Pd2(dba)3, dppf, Zn, H2O, DMF, 120 °C; (f) m-CPBA, dioxane, rt; (g) POCl3, MeCN/dioxane, 90 °C; (h) Zinc cyanide, Pd2(dba)3, dppf, Zn, NMP, 120 °C; (i) urea, NaOH, 195 oC; (j) Pd(OH)2/C, AcOH, H2O, 70 °C; (k) PCl5, POCl3, 130 °C; (l) Boc2O, DMAP, DCM, rt; (m) ArNH2, TEA, CH3CN, 70 °C; (n) Benzyl mercaptan, K2CO3, DMF, rt; (o) BnOH, NaH, DMF, −20 °C.

The syntheses of target molecules V1-V13 have been summarized in Scheme 2. The intermediates 18a-18j were coupled with 4a under palladium catalysis to obtain compound 19a-19j (yield 62–0%). The nitrile group was then hydrolyzed to formamide, and then deprotected to obtain the target molecules V1-V8 and V10-V11 (yield 52–68%). Target molecule V1 reacted with Lawesson’s reagent to give molecule V9 (yield 80%). Intermediate 18a was coupled with compound 5 to give methyl ester 20, saponification of which afforded carboxylic acid 21 (yield 70%). Amide-coupling reactions were performed on acid 21, and then deprotected to give inhibitors V12 (yield 62%). In addition, the Boc protective group of carboxylic acid 21 was directly removed to give the target molecule V13 (yield 85%).

Scheme 2 Syntheses of target compounds V1-V13.a aReagents and conditions: (a) Tris(dibenzylideneacetone)dipalladium, Cesium carbonate, X-Phos, 1,4-Dioxane, 105 oC; (b) UHP, K2CO3, DMSO, H2O, 45 oC; (c) TFA, DCM, rt; (d) V1, Lawesson’s reagent, THF, 80 °C; (e) LiOH∙H2O, THF, MeOH, H2O, 60 oC; (f) Methoxyammonium chloride, EDCI, HOBt, DIPEA, DCM, rt.

The syntheses of target molecules V14-V20 have been summarized in Scheme 3. Similar to the synthesis method of molecules V1, an identical synthetic sequence was performed with 18a and indole 6a-c or 12 as starting materials, leading to compounds V14-V17. Finally, we chose three indoles with substituents at position 3 as raw materials. Similar to the synthesis route of compounds V1 or V12, intermediate 18a and indole 22a-22c were used as starting materials to obtain the target molecules V18-V20.

Scheme 3 Syntheses of target compounds V14-V20.a aReagents and conditions: (a) Tris(dibenzylideneacetone)dipalladium, Cesium carbonate, X-Phos, 1,4-Dioxane, 105 oC; (b) UHP, K2CO3, DMSO, H2O, 45 oC; (c) TFA, DCM, rt; (d) LiOH∙H2O, THF, MeOH, H2O, 60 oC; (e) Methoxyammonium chloride, EDCI, HOBt, DIPEA, DCM, rt.

Biological Evaluation

Enzymatic

We first tested the inhibitory activities of all the target compounds against human p97 enzyme. CB-5339 was used as the standard. IC₅₀ values of the target compounds V2-V20 and CB-5339 were shown in Table 1.

Table 1 Inhibitory Activities of Target Compounds on p97 Enzyme

When the H atom at the R¹ position (CB-5339, 44 nM) was replaced by a methyl group (2, 39 nM) showed potent p97 activity. Subsequently, a hydroxyl group was introduced into the benzene ring of the benzyl amino groups, and the activity of 2-hydroxybenzyl (3, 53 nM) was slightly reduced. When the hydroxyl group was located at position 4, the activity of the compound 5 was completely lost. Interestingly, 3-hydroxybenzyl (4, 14 nM) showed better inhibitory activity to CB-5339. Subsequently, substituting benzene ring with pyridine group (6–8, 115 nM-381 nM) did not improve the inhibitory activity of p97. In addition, we replace the N atom on benzylamine with an O atom (10, 185.2 nM) or S atom (11, 448.2 nM), the activity was reduced greatly. These results indicated that the benzylamine structure was necessary for maintaining activity. Attention was next focused on indole. When formamide (CB-5339) was replaced with thioformamide (V9, 80 nM), its activity decreased. Continuing to improve the activity, we introduce methoxy on formamide to obtain compound 12 (26 nM), which showed good inhibitory activity. In addition, replacing formamide with carboxylic acid (V13, 32 nM) also has good inhibitory activity. Subsequently, the effect of introducing N atoms into the indole benzene ring on the biological activity was investigated. The results show that the inhibitory activities of compounds 14 (121 nM) and 15 (82 nM) were decreased potencies in a certain extent, and the activity of compound 16 (1200 nM) was reduced by more than 25 times. Compared with compound CB-5339, the introduction of F atom at the 6-position of indole (V17, 35 nM) does not affect the inhibitory activity against p97. Finally, we transferred the indole 4 substituent of compound CB-5339 and V12 to the indole 3 position to obtain compounds V18 (393 nM) and V19 (1643 nM), and the activity decreased sharply. However, when we extended one carbon atom between the indole 3 substituent of compound V19 and the benzene ring to obtain compound V20 (177 nM), the inhibitory activity against p97 was enhanced, but still lower than V12. This may be due to the transfer of the 4-position substituent of indole to the 3-position, increasing the distance between the substituents and the amino acid residues at the p97 binding site, leading to a decrease in activity.

Cell

We selected compounds with IC₅₀ value less than 200 nM for p97 inhibition to further evaluate their anti-proliferation activity against multiple myeloma cell line RPMI-8226 and colorectal cancer cell line HCT-116 (Table 2). The results showed that multiple compounds exhibited strong inhibitory effects on HCT-116 cells and RPMI8226 cells. In the proliferation inhibition results of multiple myeloma cell line RPMI-8226, compounds V4 (IC₅₀, 0.3 μM), V12 (IC₅₀, 0.5 μM) and V13 (IC₅₀, 0.8 μM) showed better inhibitory activity, superior to the positive control CB-5339 (IC₅₀, 0.9 μM). And the proliferation inhibition experiment results of colorectal cancer cell line HCT-116, the inhibitory activities of compounds V4 (0.7 μM) and V12 (0.7 μM) were the same as CB-5339 (0.7 μM), while compound V13 exhibits better inhibitory activity, with an IC₅₀ value of only 0.4 μM.

Table 2 Representative Compounds in vitro Anti-Proliferative Activities (IC50, μM, Mean ± SD)

In subsequent PK studies, we found that compound V12 will rapidly metabolized to V13 in SD rats. Therefore, we further tested the proliferative inhibitory activity of compounds V12 and V13 on various tumor cell lines (Table 3). The results showed that compound V13 showed strong inhibitory activities against all tested hematoma cell lines and pancreatic cancer cell line BXPC-3, with IC₅₀ value less than 1 μM. Furthermore, it exhibits comparable or even better cell inhibitory activity than CB-5339. So, based on the above results, compound V12 and V13 were selected to be further evaluated the drug availability.

Table 3 Antiproliferative Activities of Compounds V12 and V13 on Various Cell Lines (IC50, μM, Mean ± SD)

Binding Mode of Compound V12 and V13

To provide a structural basis for the competitive inhibition mechanism of p97 inhibitors, we attached V12 and V13 pairs to the p97 protein (PDB 6mck). As shown in Figure 3. The docking results suggested that the carbonyl group on indole 4 formamide formed strong hydrogen bond with the Thr688. In addition, the N atom at the benzylamine position forms a strong hydrogen bond with the Asp478. In addition, the Ile656 forms hydrophobic interactions with the indole-phenyl ring. Amino acid residues of Leu526, Leu527, Leu482, and Ile479 form a hydrophobic pocket, which forms a hydrophobic interaction with the benzene ring of benzylamine.

Figure 3 Molecular modeling of compounds V12 (a) and V13 (b) in complex with VCP (PDB 6mck). Hydrogen bonds are highlighted as yellow dashed lines and Hydrophobic interactions are highlighted as blue dashed lines.

Microsome Stabilities

Based on the above results, we further tested the stability of the compounds V12 and V13 in five liver microsomes (Table 4). Compound V12 undergoes rapid metabolism in five species of liver microsomes, the T_1/2 was only 13.3 minutes in human liver microsomes and even less than 10 minutes in the mouse, rat, and monkey liver microsomes. The T_1/2 of compound V13 in the human, dog, and monkey liver microsomes were greater than 120 minutes, demonstrating good stability.

Table 4 The Microsomal Stability Profiles of Compound V12 and V13a

Pharmacokinetic

In order to investigate the oral absorption and metabolism of compound V12 in vivo, we further investigated the pharmacokinetics of V12 in SD rats, and detected both V12 and V13 in plasma (Table 5). The results showed that after gavage or tail vein injection of V12, the C_max and AUC_0-inf values in plasma were very low. We speculate that the indole 4 substituent of compound V12 may be metabolized into carboxylic acid (V13) in rats. Therefore, we then tested the plasma concentration of V13 in the same batch of plasma samples. The results indicate that after gavage or tail vein injection of V12, V12 rapidly metabolizes into V13. The oral T_1/2 of compound V12 reached 3.52 h, almost three times that of the CB-5339. The oral T_max, and C_max of V13 were 0.25 h and 1070 ng/mL, respectively, and the plasma AUC_0-inf value reached 1412 ng•h/mL, showed good PK properties. In addition, compound V12 had an oral absorption bioavailability of 29.7%, which has the potential to be developed as an oral drug.

Table 5 Single-Dose Pharmacokinetic Data of Compounds V12 and CB-5339 in SD Rats

Because of its potent activity and good pharmacokinetic properties, compound V12 was further evaluated for in vivo antitumor efficacy. We first examined the effects of compound V12 in nude mice weight (Figure 4a). In our previous study, the oral administration of CB-5339 at 100 mg/kg showed serious side effects in nude mice, and death occurred on day 7.²⁷ Therefore, we chose an oral dosage of 50 mg/kg for compound CB-5339. The oral doses of compound V12 were 100 mg/kg and 300 mg/kg. The body weight of the nude mice in the administration group was comparable to that of the control group, and the nude mice were in good condition, even at a dose of 300 mg/kg, indicating better safety. Subsequently, we evaluated the in vivo antitumor efficacy of compound V12 in the Molm-13 nude mouse xenograft model, and the results were shown in Figure 4b. The CB-5339 was used as the standard. The results showed that compound V12 could significantly inhibit tumor growth, and the efficacy in vivo was comparable to that of the CB-5339. These results indicate that compound V12 has lower toxicity and a wider treatment window.

Figure 4 In vivo efficacy of compounds V12 and CB-5339 in Molm-13 nude mouse xenograft model. (a) body weight change of Molm-13 nude mouse xenograft model after oral administration; (b) tumor volume change of Molm-13 nude mouse xenograft model after oral administration.

Conclusion

In summary, in order to develop novel AML therapeutics, we designed and synthesized a series of novel p97 inhibitors based on core 5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine. Then, all target compounds were subsequently biologically investigated in vitro and in vivo. Among them, compound V12 and its metabolite V13 showed excellent in vitro antitumor activities against a variety of solid tumor and hematoma cell lines. Subsequently, we further investigated the liver microsomal metabolism of compound V12 and its absorption and metabolism after oral administration in rats. The results showed that compound V12 was metabolized rapidly in liver microsomes while its metabolite V13 was almost not metabolized. The pharmacokinetic results of SD rats showed that compound V12 was rapidly metabolized into V13 in the body, and the oral bioavailability was 29.7%. In addition, in the Molm-13 nude mouse xenograft model, compound V12 showed good in vivo efficacy and safety. These results showed that compound V12 could be developed as an oral drug for the treatment of AML.

Experimental Section

General Methods

Purchased reagents were used directly without treatment. The reaction was monitored by silica gel chromatography plate or LCMS (Shimadzu, LCMS-2020). Intermediates and target compounds were purified by silica gel column chromatography (300–400 mesh). The purity of the target compounds was detected by HPLC (Agilent, 1100 Series). NMR spectra were acquired on a Bruker Avance 400 spectrometer (¹H NMR at 400 MHz, ¹³C NMR at 100 MHz). High-resolution mass spectra (HRMS) were recorded on a Bruker Solarix Fourier transform ion cyclotron resonance mass spectrometer.

Chemistry

2-Methyl-1H-Indole-4-Carbonitrile (4a)

Under ice bath conditions, intermediate 1a obtained by purchase (5 g, 35.17 mmol) was dissolved in THF (200 mL). The NaH (1.3 g, 52.75 mmol) was added slowly, and stirred for 30 min under the condition of ice bath. The 4-Benzenesulfonic acid chloride (6.5 g, 42.21 mmol) was added. The reaction was then transferred to room temperature and continued for 1 h. The reaction was quenched with 5% ammonium chloride solution and then extracted by EA. The organic phase was dried and concentrated, and the product 2a was purified by recrystallization (EtOAc/PE) (7.4 g, yield 75%). ¹H NMR (400 MHz, Chloroform-d) δ 8.26 (d, J = 8.4 hz, 1H), 7.94–7.88 (m, 2H), 7.78 (d, J = 3.7 hz, 1H), 7.64–7.57 (m, 2H), 7.51 (dd, J = 8.5, 7.2 hz, 2H), 7.41 (t, J = 8.0 hz, 1H), 6.92 (d, J = 3.7 hz, 1H). MS (ESI): m/z 283.1 (MH⁺).

In a three-mouth flask, DIPA (5.9 g, 58.3 mmol) was dissolved in anhydrous THF (50 mL), then cooled to −78°C and n-Butyllithium solution (2.5 M in THF, 23.3 mL, 58.3 mmol) was slowly added. The reaction continued at −78°C for 1 h. The intermediate 2a (9.8 g, 29.2 mmol) was dissolved in anhydrous THF (10 mL) and slowly added to the reaction solution. The reaction continued at −78°C for 1 h and iodomethane (8.3 g, 58.3 mmol) was added. The reaction was then transferred to ambient temperature and continued for 2 h. The reaction was quenched with 5% ammonium chloride solution and then extracted by EA. The organic phase was dried and concentrated, and the product 3a was purified by recrystallization (EtOAc/PE), (6.8 g, yield 66%), white solid. MS (ESI): m/z 297.4 (MH⁺).

The intermediate 3a (5.4 g, 18.19 mmol) was dissolved in ethanol (100 mL) and then 4 mol/L of sodium hydroxide aqueous solution (18.2 mL) was added. Concentration to remove ethanol after reaction at 40°C for 12 hours, then added water (100 mL) and extracted with ethyl acetate. The organic phase was dried and concentrated, and the product 4a was purified by column chromatography (2.6 g, yield 90%), white solid. ¹H NMR (400 MHz, Chloroform-d) δ 8.29 (s, 1H), 7.52 (dt, J = 8.1, 1.0 hz, 1H), 7.43 (dd, J = 7.5, 0.9 hz, 1H), 7.15 (t, J = 7.8 hz, 1H), 6.47 (dt, J = 2.1, 1.0 hz, 1H), 2.53 (s, 1H). MS (ESI): m/z 157.3 (MH⁺).

Methyl 2-Methyl-1H-Indole-4-Carboxylate (5)

In a high-pressure reactor, 4b (25.6 g, 121.8 mmol) was dissolved in methanol (200 mL), and palladium acetate (2.7 g, 12.2 mmol), 1,3-Bis (diphenylphosphino) propane (5.0 g, 12.2 mmol) and triethylamine (24.6 g, 243.6 mmol) was added. The reaction was degassed and reacted in carbon monoxide atmosphere at 80°C for 24 h. The reaction solution was concentrated and re-dissolved in EA. The organic phase was washed with water, dried and concentrated. And then, the intermediate 5 was purified by silica gel column (18.5 g, yield 80%), yellow solid. ¹H NMR (400 MHz, Chloroform-d) δ 8.17 (s, 1H), 7.88 (d, J = 7.6 hz, 1H), 7.49 (d, J = 7.9 hz, 1H), 7.17 (t, J = 7.8 hz, 1H), 6.92–6.86 (m, 1H), 4.00 (s, 3H), 2.51 (s, 3H). MS (ESI): m/z 190.3 (MH⁺).

2-Methyl-1H-Pyrrolo[2,3-C]pyridine-4-Carbonitrile (6b)

At ambient temperature, to a solution of intermediate (4d) (280 mg, 1.3 mmol) in DMF (10 mL) was added ultrapure water (0.3 mL), and then exhaust with ultrasound. Subsequently, dppf (72 mg, 0.13 mmol), Pd₂(dba)₃ (60 mg, 0.065 mmol), and zinc cyanide (160 mg, 1.36 mmol) were added, and then stirred at 120°C for 1.5 h. 50 mL of water was added to the reaction solution, and extracted with ethyl acetate. The organic phase was washed with brine, dried and concentrated. And then, the intermediate 6b was purified by silica gel column chromatography (160 mg, yield 78%). MS ESI: m/z 158.1 (MH⁺).

2-Methyl-1H-Pyrrolo[3,2-C]pyridine-4-Carbonitrile (12)

According to the synthesis method of intermediate 2a, intermediate 8 was synthesized from commercially available 1H-pyrrolo[3, 2-C]pyridine (yield 95%, white solid). ¹H NMR (400 MHz, CDCl₃) δ 8.90 (s, 1H), 8.51 (d, J = 5.8 hz, 1H), 7.92 (m, 3H), 7.65–7.58 (m, 2H), 7.51 (dd, J = 8.5, 7.0 hz, 2H), 6.77 (d, J = 3.7 hz, 1H). MS ESI: m/z 259.0 (MH⁺)。

Compound 8 (4.2 g, 16.1 mmol) was dissolved in dioxane (40 mL) and m-CPBA (3.1 g, 17.7 mmol) was slowly added, then stirred for 5 h at ambient temperature. The saturated solution of sodium thiosulfate was added to quench the reaction, followed by the addition of an appropriate amount of water, and then extracted with DCM. The organic phase were washed with brine, dried and evaporated in vacuo. The intermediate 9 was purified by silica gel column chromatography (3 g, yield 68%), yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.52 (dd, J = 1.7, 0.7 hz, 1H), 8.19 (dd, J = 7.2, 1.8 hz, 1H), 7.90 (m, 3H), 7.71–7.62 (m, 2H), 7.55 (m, 2H), 6.66 (dd, J = 3.7, 0.8 hz, 1H). MS ESI: m/z 275.0 (MH⁺).

At room temperature, compound 9 (3 g, 10.9 mmol) was dissolved in MeCN/dioxane (30/30, v/v) and POCl₃ (1.8 g, 12 mmol) was slowly added. The solution was stirred for 18 h at 90°C. The reaction mixture was concentrated to remove POCl₃, and extracted with ethyl acetate. The organic phase were washed with brine, dried and concentrated. Purification by column chromatography to provide intermediate 10 (2.6 g, yield 81%), yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.27 (d, J = 5.8 hz, 1H), 7.92 (d, J = 1.4 hz, 1H), 7.87 (dd, J = 5.8, 0.9 hz, 1H), 7.68–7.60 (m, 2H), 7.53 (m, 2H), 6.82 (dd, J = 3.7, 0.9 hz, 1H). MS ESI: m/z 292.0 (MH⁺)。

In accordance with the synthesis of 4a in Scheme 1., intermediate 10 reacted with iodomethane and was then deprotected to give target compound 11. Whereafter, compound 11 was dissolved in NMP (10 mL) and then dppf (134 mg, 0.24 mmol), Pd₂(dba)₃ (114 mg, 0.13 mmol), zinc cyanide (78 mg, 0.66 mmol) and zinc powder were added. After the reaction displacement of nitrogen for 3 times, the reaction was conducted at 120°C for 18 h. Water (10 mL) was then added to the reaction solution, which was then filtered, and the filtrate was extracted with EA. The organic phase were washed with brine, dried with Na₂SO₄, filtered and concentrated. Purification by silica gel column chromatography to provide compound 12 (50 mg, yield 53%). MS ESI: m/z 158.1 (MH⁺).

Tert-butyl 4-(Benzylamino)-2-Chloro-6,7-Dihydropyrido[2,3-D]pyrimidine-8(5H)- Carboxylate (18a)

The urea (150 g, 2497 mmol) was added to the eggplant shaped bottle, heated to 195°C and melted, and then 13a (69 g, 499 mmol) was slowly added. After the reaction continued for 1.5 hours, the reaction temperature was lowered to 100°C. The NaOH aqueous solution was added (600 mL, 499 mmol) and stirred at 100°C for 1 h. The reaction mixture was then cooled down to ambient temperature and the pH of the mixture was adjusted to 4 with the addition of hydrochloric acid aqueous solution (1 mol/L), then filtered. The filter cake was dried to obtain intermediate 14a (66 g, 81% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 11.68 (s, 1H), 11.47 (s, 1H), 8.60 (dd, J = 4.8, 1.9 hz, 1H), 8.26 (dd, J = 7.8, 1.9 hz, 1H), 7.25 (dd, J = 7.7, 4.8 hz, 1H). MS ESI: m/z 164.1 (MH⁺).

To a solution of intermediate 14a (66 g, 6 mmol) in acetic acid (900 mL) and water (600 mL), Pd(OH)₂/C (6.6 g, 10% wt) was added. After the reaction displacement of H₂ for 3 times, the reaction was refluxed at 70°C for 18 h under hydrogen atmosphere. Filtered and concentrated, and then dried under vacuum at 60°C to provide intermediate 15a (54 g, crude). 15a was used directly in the next step. ¹H NMR (400 MHz, DMSO-d₆) δ 10.10 (d, J = 6.1 hz, 2H), 5.97 (d, J = 2.7 hz, 1H), 3.17 (m, 2H), 2.17 (t, J = 6.2 hz, 2H), 1.74–1.56 (m, 2H). MS ESI: m/z 168.1 (MH⁺).

The intermediate 15a (30 g, 179.4 mmoL) and PCl₅ (18.7 g, 89.7 mmoL) were dissolved in POCl₃ (300 mL). The reaction mixture was then reacted at 130°C for 12 hours. Then, the reaction solution was concentrated to remove POCl₃ and quenched by adding ice water (300 mm), extracted with EA. The organic phase were washed with brine, dried with Na₂SO₄, filtered and concentrated. Purification by silica gel column chromatography to give intermediate 16a (13.5 g, 36.8% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.01 (s, 1H), 3.50 (m, 2H), 2.74 (t, J = 6.4 hz, 2H), 2.04–1.87 (m, 2H). MS ESI: m/z 204.1 (MH⁺).

At ambient temperature, intermediate 16a (13.5 g, 66.2 mmol), DMAP (1.6 g, 13.2 mmoL) and (Boc)₂O (15.9 g, 72.8 mmol) were dissolved in DCM (200 mL). Then the reaction was stirred for 3 h. The reaction solution was washed with water and brine, dried and concentrated. Purification by silica gel column chromatography to give intermediate 17a (18 g, 89.4% yield). ¹H NMR (400 MHz, CDCl₃) δ 3.83–3.76 (m, 2H), 2.79 (t, J = 6.7 hz, 2H), 2.08–1.96 (m, 2H), 1.58 (s, 9H). MS ESI: m/z 305.3 (MH⁺).

TEA (34.9 g, 345 mm) was added to a solution of intermediate 17a (35 g, 115 mmoL) and benzylamine (18.5 g, 173 mm) in isopropyl alcohol (500 mL) and the reaction solution was refluxed at 70°C for 12 h. Then concentrated, and the crude product was dissolved with ethyl acetate and the organic phase was washed with water and brine, dried and concentrated. The crude product was purified by silica gel column chromatography to obtain the key intermediate 18a (27 g, 62.8% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.43–7.31 (m, 5H), 4.81 (t, J = 5.4 hz, 1H), 4.71 (d, J = 5.3 hz, 2H), 3.78–3.69 (m, 2H), 2.33 (t, J = 6.8 hz, 2H), 2.05–1.94 (m, 2H), 1.57 (s, 9H). MS ESI: m/z 375.1 (MH⁺).

Tert-Butyl 4-(Benzyloxy)-2-Chloro-6,7-Dihydropyrido[2,3-D]pyrimidine-8(5H)- Carboxylate (18i)

At 0°C, NaH (40 mg, 0.99 mm) and benzyl alcohol (78 mg, 0.73 mm) were added to a solution of intermediate 17a (200 mg, 0.66 mmol) in DMF (5 mL). Then reacted at ambient temperature for 3 h. Water (50 mL) was added to quench the reaction, and extracted 3 times with EA. The organic phase were washed with brine, dried with Na₂SO₄. Concentrated and purification by column chromatography to provide intermediate 18i (112 mg, yield 45%), white solid. ¹H NMR (400 MHz, Chloroform-d) δ 7.55–7.33 (m, 5H), 5.44 (s, 2H), 3.79–3.70 (m, 2H), 2.62 (m, 2H), 1.98–1.89 (m, 2H), 1.57 (s, 9H). MS ESI: m/z 376.1 (MH⁺)。

Tert-Butyl 4-(Benzylthio)-2-Chloro-6,7-Dihydropyrido[2,3-D]pyrimidine-8(5H)- Carboxylate (18j)

At ambient temperature, to a solution of intermediate 17a (200 mg, 0.66 mmol) in DMF (5 mL) were added benzyl mercaptan (98 mg, 0.79 mm) and K₂CO₃ (182 mg, 1.32 mm). Then the reaction was stirred at ambient temperature for 3 h. Water (50 mL) was added to quench the reaction, and extracted 3 times with EA. The organic phase were washed with brine, dried with Na₂SO₄. Concentrated and purification by column chromatography to provide intermediate 18j (310 mg, yield 79%), yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 7.46–7.43 (m, 2H), 7.37–7.31 (m, 2H), 7.28 (s, 1H), 4.47 (s, 2H), 3.80–3.68 (m, 2H), 2.52 (t, J = 6.7 hz, 2H), 2.01–1.88 (m, 2H), 1.57 (s, 9H). MS ESI: m/z 392.1 (MH⁺)。

1-(4-(Benzylamino)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl)-2-Methyl-1H -Indole-4-Carboxamide (V1, CB-5339)

18a (200 mg, 0.53 mmol), 4a (83 mg, 1.8 mmol), Cs₂CO₃ (261 mg, 0.8 mmol), Pd₂(dba)₃ (73 mg, 0.08 mmol) and X-Phos (38 mg, 0.08 mmol) were dissolved in 1,4-dioxane (10 mL). Then the mixture was replaced with nitrogen for 3 times, and refluxed at 105°C for 4 h. The reaction solution was filtered, concentrated and then dissolved in EA, organic phase washed with water and brine, dried with Na₂SO₄. Concentrated and purification by column chromatography to provide intermediate 19a (235 mg, yield 89.7%). MS ESI: m/z 495.2 (MH⁺).

Urea hydrogen peroxide (219 mg, 2.38 mmol) and K₂CO₃ (66 mg, 0.48 mmol) were dissolved in water and then added to a DMSO solution of intermediate 19a (235 mg, 0.48 mmol), and reacted at 40°C for 4 h. Water (50 mL) was then added to the reaction solution, and extracted 3 times with EA. The organic phase were washed with brine, dried with Na₂SO₄. Concentrated and purification by column chromatography to provide intermediate (220 mg, 89.3% yield). MS ESI: m/z 513.2 (MH⁺).

The intermediate of the previous step (220 mg, 0.43 mmol) was dissolved in DCM (5 mL), TFA (1 mL) was added under ice bath conditions, and then the reaction continues at ambient temperature for 4 h. The reaction solution was concentrated to remove most of the TFA, then water was added, the pH was adjusted to 9 with 5% Na₂CO₃, and extracted with EA. The organic phase were washed with brine, dried with Na₂SO₄. Concentrated and purification by column chromatography to provide compound V1 (CB-5339) (120 mg, yield 67.6%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.83 (d, J = 8.2 hz, 1H), 7.73 (s, 1H), 7.42 (d, J = 7.4 hz, 1H), 7.31 (p, J = 7.4 hz, 4H), 7.23 (d, J = 6.3 hz, 2H), 7.09 (d, J = 6.1 hz, 1H), 6.93–6.82 (m, 2H), 6.79 (s, 1H), 4.58 (s, 2H), 3.39 (s, 2H), 3.25 (d, J = 6.7 hz, 2H), 2.46 (s, 3H), 1.96–1.81 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.02, 160.13, 159.83, 154.49, 141.53, 138.81, 137.38, 128.66, 127.14, 126.92, 126.80, 125.44, 120.57, 116.69, 104.76, 87.57, 44.06 (d, J = 10.6 hz), 21.14, 20.18, 16.10.HRMS calcd for C₂₄H₂₄N₆O [M + Na]⁺ 435.1904, found 435.1909。

1-(4-(Benzylamino)-7-Methyl-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl)-2- Methyl-1H-Indole-4-Carboxamide (V2)

Using intermediates 18b and 4a as raw materials, compound V2 was synthesized according to the preparation method of compound V1. ¹H NMR (400 MHz, DMSO-d₆) δ 7.88 (d, J = 8.2 hz, 1H), 7.71 (s, 1H), 7.42 (d, J = 7.4 hz, 1H), 7.36–7.26 (m, 5H), 7.23 (dd, J = 11.3, 4.7 hz, 2H), 7.06 (q, J = 4.7, 3.2 hz, 1H), 6.90 (t, J = 7.9 hz, 1H), 6.78 (s, 1H), 4.59 (d, J = 6.1 hz, 2H), 3.49–3.42 (m, 2H), 2.74–2.60 (m, 1H), 2.47 (s, 3H), 2.05–1.88 (m, 1H), 1.53 (m, 1H), 1.20 (d, J = 6.3 hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.05, 160.20, 159.85, 154.64, 141.51, 138.84, 137.40, 128.65, 127.16, 126.95, 126.80, 125.46, 120.57, 116.79, 104.79, 87.39, 55.39, 46.07, 44.14, 21.82, 19.35, 16.16. HRMS calcd for C₂₅H₂₆N₆O [M + Na]⁺ 449.2060, found 449.2076.

1-(4-((2-Hydroxybenzyl)amino)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl) −2-Methyl-1H-Indole-4-Carboxamide (V3)

Using intermediates 18c and 4a as raw materials, compound V3 was synthesized according to the preparation method of compound V1. ¹H NMR (400 MHz, DMSO-d₆) δ 7.89 (d, J = 8.2 hz, 1H), 7.70 (s, 1H), 7.41 (d, J = 7.4 hz, 1H), 7.19 (s, 1H), 7.09 (d, J = 7.6 hz,1H), 7.04 (t, J = 7.7 hz, 1H), 6.94–6.69 (m, 6H), 4.51 (d, J = 5.3 hz, 2H), 3.26 (s, 2H), 2.47 (s, 3H), 1.88 (d, J = 7.7 hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.01, 160.15, 159.82, 155.12, 154.42, 138.79, 137.43, 127.74, 127.12, 125.46, 120.59 (d, J = 6.5 hz), 119.14, 116.67, 115.25, 104.81, 87.52, 21.14, 20.13, 16.01. HRMS calcd for C₂₄H₂₄N₆O₂ [M + Na]⁺ 451.1853, found 451.1860.

1-(4-((3-Hydroxybenzyl)amino)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl) −2-Methyl-1H-Indole-4-Carboxamide (V4)

Using intermediates 18d and 4a as raw materials, compound V4 was synthesized according to the preparation method of compound V1. ¹H NMR (400 MHz, DMSO-d₆) δ 7.87 (d, J = 8.2 hz, 1H), 7.70 (s, 1H), 7.42 (d, J = 7.4 hz, 1H), 7.18 (s, 1H), 7.10 (t, J = 7.9 hz, 1H), 7.00 (t, J = 6.2 hz, 1H), 6.91 (t, J = 7.9 hz, 1H), 6.79 (d, J = 8.5 hz, 2H), 6.71 (d, J = 6.4 hz, 2H), 6.65–6.58 (m, 1H), 5.76 (s, 1H), 4.51 (d, J = 5.5 hz, 2H), 3.26 (t, J = 5.3 hz, 3H), 2.49 (s, 6H), 1.89 (p, J = 6.1 hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 160.19, 159.85, 157.78, 154.55, 143.09, 138.83, 137.41, 129.59, 127.14, 125.42, 120.58 (d, J = 5.4 hz), 117.50, 116.82, 113.65 (d, J = 3.9 hz), 104.77, 87.52, 55.38, 44.01, 21.18, 20.19, 16.14. HRMS calcd for C₂₄H₂₄N₆O₂ [M + Na]⁺ 451.1853, found 451.1861.

1-(4-((4-Hydroxybenzyl)amino)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl) −2-Methyl-1H-Indole-4-Carboxamide (V5)

Using intermediates 18e and 4a as raw materials, compound V5 was synthesized according to the preparation method of compound V1. ¹H NMR (400 MHz, DMSO-d₆) δ 8.18 (s, 1H), 7.95 (dd, J = 8.0, 1.3 hz, 1H), 7.70 (s, 1H), 7.26 (d, J = 2.3 hz, 1H), 7.12–6.99 (m, 2H), 6.97–6.92 (m, 2H), 6.88–6.71 (m, 2H), 6.59–6.52 (m, 2H), 6.22–6.18 (m, 1H), 5.76 (s, 1H), 4.09 (s, 2H), 3.23 (dt, J = 7.3, 3.6 hz, 2H), 2.47 (s, 3H), 2.38 (t, J = 6.3 hz, 2H), 1.83 (p, J = 6.3 hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 172.05, 161.55, 160.48, 155.27, 154.51, 136.74, 135.32, 132.93, 129.84, 129.63, 126.58, 124.50, 121.19, 120.12 (d, J = 10.6 hz), 115.14, 114.42, 113.94, 87.38 (d, J = 3.6 hz), 55.38, 29.33, 21.14, 20.33, 13.12. HRMS calcd for C₂₄H₂₄N₆O₂ [M + Na]⁺ 451.1853, found 451.1860.

2-Methyl-1-(4-((Pyridin-2-Ylmethyl)amino)-5,6,7,8-Tetrahydropyrido[2,3-D] Pyrimidin-2-Yl)-1H-Indole-4-Carboxamide (V6)

Using intermediates 18f and 4a as raw materials, compound V6 was synthesized according to the preparation method of compound V1. ¹H NMR (400 MHz, DMSO-d₆) δ 8.52 (dd, J = 5.3, 1.8 hz, 1H), 7.80 (d, J = 8.3 hz, 1H), 7.75 (td, J = 7.7, 1.8 hz, 1H), 7.70 (s, 1H), 7.41 (dd, J = 7.5, 0.9 hz, 1H), 7.30 (d, J = 7.9 hz, 1H), 7.26 (m, 1H), 7.19 (s, 1H), 7.10 (t, J = 6.1 hz, 1H), 6.87 (t, J = 7.9 hz, 2H), 6.76 (s, 1H), 4.65 (d, J = 5.3 hz, 2H), 3.36 (s, 2H), 3.27 (dd, J = 7.1, 3.8 hz, 2H), 2.40–2.36 (s, 3H), 1.90 (p, J = 6.0 hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.98, 160.78, 160.11, 159.89, 154.52, 149.27, 138.80, 137.36, 137.05, 127.14, 125.46, 122.20, 120.72, 120.56, 116.61, 104.77, 88.08–86.71, 46.29, 21.14, 20.15, 15.99. HRMS calcd for C₂₃H₂₃N₇O [M + Na]⁺ 436.1856, found 436.1862.

2-Methyl-1-(4-((Pyridin-3-Ylmethyl)amino)-5,6,7,8-Tetrahydropyrido[2,3-D] Pyrimidin-2-Yl)-1H-Indole-4-Carboxamide (V7)

Using intermediates 18g and 4a as raw materials, compound V7 was synthesized according to the preparation method of compound V1. ¹H NMR (400 MHz, DMSO-d₆) δ 8.51 (d, J = 2.2 hz, 1H), 8.45 (dd, J = 4.8, 1.6 hz, 1H), 7.81 (d, J = 8.3 hz, 1H), 7.74–7.72 (m, 1H), 7.68 (dd, J = 6.0, 4.0 hz, 1H), 7.43 (d, J = 7.4 hz, 1H), 7.35 (dd, J = 7.9, 4.8 hz, 1H), 7.21 (s, 1H), 7.11 (t, J = 6.1 hz, 1H), 6.91 (t, J = 7.9 hz, 1H), 6.88 (d, J = 2.8 hz, 1H), 6.79 (s, 1H), 4.60 (d, J = 5.5 hz, 2H), 3.26 (p, J = 3.1 hz, 2H), 2.47 (d, J = 6.6 hz, 2H), 2.45 (s, 3H), 1.88 (p, J = 6.0 hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.03, 159.95, 154.49, 148.70, 148.11, 138.75, 137.35, 136.89, 134.91, 127.14, 125.53, 123.90, 120.59, 116.53, 104.78, 87.80, 55.39, 42.02, 21.09, 20.14, 16.06. HRMS calcd for C₂₃H₂₃N₇O [M + Na]⁺ 436.1856, found 436.1864.

2-Methyl-1-(4-((Pyridin-4-Ylmethyl)amino)-5,6,7,8-Tetrahydropyrido[2,3-D] Pyrimidin-2-Yl)-1H-Indole-4-Carboxamide (V8)

Using intermediates 18h and 4a as raw materials, compound V8 was synthesized according to the preparation method of compound V1. ¹H NMR (400 MHz, DMSO-d₆) δ 8.54–8.48 (m, 2H), 7.74–7.68 (m, 2H), 7.44–7.38 (m, 1H), 7.32–7.27 (m, 2H), 7.20 (s, 1H), 7.14 (t, J = 6.1 hz, 1H), 6.90 (d, J = 2.5 hz, 1H), 6.84 (t, J = 7.9 hz, 1H), 6.77 (s, 1H), 4.58 (d, J = 5.5 hz, 2H), 3.27 (m, 2H), 2.52 (d, J = 6.5 hz, 2H), 2.39 (d, J = 1.0 hz, 3H), 1.90 (t, J = 5.9 hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.02, 159.98, 154.47, 151.02, 149.82, 138.73, 137.33, 127.13, 125.48, 122.15, 120.53, 116.42, 104.76, 87.85, 67.49, 55.38, 43.45, 25.59, 21.09, 20.15, 15.94. HRMS calcd for C₂₃H₂₃N₇O [M + Na]⁺ 436.1856, found 436.1863.

1-(4-(Benzylamino)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl)-2-Methyl- 1H-Indole-4-Carbothioamide (V9)

Dissolve the target product V1 (100 mg, 0.24 mmol) in anhydrous THF (20 mL) and then Lawesson’s reagent (146.7 mg, 0.36 mmol) was added. The mixture was refluxed for 12 h and concentrated. The crude product was extracted with DCM after adding water. The organic phase were washed with brine, dried and concentrated. The crude product was purified by silica gel column chromatography to provide compound V9 (83 mg, yield 80%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.81 (s, 1H,), 9.27 (s, 1H), 7.78 (d, J = 8.3 hz, 1H), 7.37–7.19 (m, 6H), 7.07 (t, J = 6.1 hz, 1H), 6.92–6.80 (m, 2H), 6.61 (s, 1H), 4.58 (d, J = 6.0 hz, 2H), 3.29–3.23 (m, 2H), 2.45 (s, 3H), 1.88 (p, J = 6.1, 5.7 hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 201.99, 160.16, 159.86, 154.48, 141.54, 138.47, 136.96, 133.07, 128.65, 126.93, 126.78, 125.16, 120.61, 120.18, 115.88, 104.02, 87.64, 44.13, 21.14, 20.19, 16.08. HRMS calcd for C₂₄H₂₄N₆S [M + H]⁺ 429.1856, found 429.1872.

1-(4-(Benzyloxy)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl)-2-Methyl-1H -Indole-4-Carboxamide (V10)

Using intermediates 18i and 4a as raw materials, compound V10 was synthesized according to the preparation method of compound V1. ¹H NMR (400 MHz, DMSO-d₆) δ 8.11 (d, J = 8.3 hz, 1H), 7.78 (s, 1H), 7.50 (d, J = 7.5 hz, 2H), 7.45–7.36 (m, 4H), 7.35–7.30 (m, 1H), 7.26 (s, 1H), 7.07 (t, J = 7.9 hz, 1H), 6.90 (s, 1H), 5.42 (s, 2H), 3.30 (q, J = 3.9, 3.4 hz, 2H), 2.61 (s, 3H), 2.58 (s, 2H), 1.83 (p, J = 5.7 hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.94, 165.40, 162.03, 154.12, 138.87, 137.85, 137.36, 128.92, 128.13, 127.54, 127.46, 125.74, 121.09 (d, J = 10.3 hz), 116.80, 105.71, 91.35, 67.51, 20.40, 19.41, 16.44. HRMS calcd for C₂₄H₂₃N₅O₂ [M + Na]⁺ 436.1744, found 436.1748.

1-(4-(Benzylthio)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl)-2-Methyl-1H-Indole-4-Carboxamide (V11)

Using intermediates 18j and 4a as raw materials, compound V11 was synthesized according to the preparation method of compound V1. ¹H NMR (400 MHz, DMSO-d₆) δ 8.10 (d, J = 8.2 hz, 1H), 7.78 (s, 1H), 7.73 (d, J = 2.6 hz, 1H), 7.50 (dd, J = 7.5, 0.9 hz, 1H), 7.41–7.36 (m, 2H), 7.35–7.28 (m, 2H), 7.27–7.22 (m, 2H), 7.07 (t, J = 7.9 hz, 1H), 6.91 (t, J = 0.9 hz, 1H), 4.48 (s, 2H), 3.30 (dd, J = 7.1, 4.0 hz, 2H), 2.62 (d, J = 1.0 hz, 3H), 1.86 (p, J = 5.7, 5.1 hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.92, 162.95, 159.71, 154.44, 138.74, 137.96, 137.41, 129.26, 128.97, 127.53 (d, J = 10.8 hz), 125.82 (d, J = 3.3 hz), 121.07 (d, J = 6.1 hz), 116.51, 105.61, 104.67, 33.09, 22.09, 20.38, 16.26. HRMS calcd for C₂₄H₂₃N₅OS [M + Na]⁺ 452.1515, found 452.1524.

1-(4-(Benzylamino)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl)-N-Methoxy-2-Methyl-1H-Indole-4-Carboxamide (V12)

Using intermediates 18a and 5 as raw materials, compound V12 was synthesized according to the preparation method of intermediate 19a. ¹H NMR (400 MHz, DMSO-d₆) δ 8.28 (d, J = 8.3 hz, 1H), 7.71 (ddd, J = 8.6, 5.9, 3.4 hz, 2H), 7.38–7.29 (m, 4H), 7.25 (td, J = 6.0, 2.6 hz, 1H), 6.99 (t, J = 7.9 hz, 1H), 6.89–6.86 (m, 1H), 4.67 (d, J = 5.9 hz, 2H), 3.88 (s, 3H), 3.77–3.69 (m, 2H), 2.56 (s, 2H), 2.54–2.54 (m, 3H), 2.04–1.91 (m, 2H), 1.43 (s, 9H). MS ESI: m/z 528.3 (MH⁺).

The LiOH⋅H₂O (2.38 g, 56.9 mmol) was dissolved in water (5 mL) and added to the THF/MeOH (15 mL/5 mL) solution of intermediate 20 (5 g, 9.48 mmol). The solution was refluxed at 60°C for 6 h, and then concentrated. The crude product was extracted with methyl tert-butyl ether after adding water. Then the pH of the water phase was adjusted to 4 and extracted with EA. The organic phase were washed with brine, dried with Na₂SO₄, and then concentrated. The crude product 21 was used directly for the next step without purification (4.4 g, yield 90%).

21 (200 mg, 0.39 mmol) was dissolved in THF (10 mL), and then methoxyammonium chloride (33 mg, 0.39 mmol), DIPEA (202 mg, 1.56 mmol), EDCI (112 mg, 0.59 mmol) and HOBt (80 mg, 0.59 mmol) were added, and then stirred at ambient temperature overnight. The reaction was quenched with water, extracted with ethyl acetate, dried with Na₂SO₄, and concentrated. The residue was purified by column chromatography (160 mg, yield 75%). The resulting product was deprotected with TFA and purified to give compound V12 (80 mg, yield 62%). ¹H NMR (400 MHz, DMSO-d₆) δ 11.45 (s, 1H), 7.84 (d, J = 8.3 hz, 1H), 7.30 (dq, J = 16.4, 8.2 hz, 5H), 7.10 (t, J = 6.3 hz, 1H), 6.89 (t, J = 8.0 hz, 1H), 6.86 (s, 1H), 6.65 (s, 1H), 5.77 (d, J = 1.5 hz, 1H), 4.59 (d, J = 5.6 hz, 2H), 3.72 (d, J = 1.6 hz, 3H), 3.37 (dd, J = 9.5, 1.6 hz, 2H), 3.26 (t, J = 5.3 hz, 2H), 2.47 (s, 3H), 1.88 (p, J = 5.8 hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.06, 160.15, 159.82, 154.42, 141.52, 139.17, 137.23, 128.66, 126.85, 123.22, 120.69, 120.00, 116.98, 104.09, 87.63, 63.63, 55.40, 44.12, 21.13, 20.18, 16.08. HRMS calcd for C₂₅H₂₆N₆O₂ [M + H]⁺ 443.2190, found 443.2202。

1-(4-(Benzylamino)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl)-2-Methyl- 1H-Indole-4-Carboxylic Acid (V13)

Intermediate 21 was deprotected under TFA to give the target product V13, brown powder. ¹H NMR (400 MHz, DMSO-d₆) δ 7.91 (d, J = 8.2 hz, 1H), 7.67 (d, J = 7.5 hz, 1H), 7.39–7.27 (m, 4H), 7.23 (m, 1H), 6.93 (t, J = 7.9 hz, 1H), 6.85 (s, 1H), 4.59 (d, J = 5.4 hz, 2H), 3.35 (s, 2H), 3.26 (t, J = 3.6 hz, 2H), 2.48 (s, 3H), 1.89 (t, J = 5.8 hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.80, 160.15 (d, J = 6.8 hz), 159.84 (d, J = 5.5 hz), 154.39, 141.50, 139.92, 137.51, 128.67, 128.35, 126.90, 126.81, 123.75, 120.68, 118.51, 104.89, 44.06 (d, J = 10.5 hz), 21.12, 20.17, 16.05. HRMS calcd for C₂₄H₂₃N₅O₂ [M + Na]⁺ 436.1744, found 436.1759.

1-(4-(Benzylamino)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl)-2-Methyl-1H-Pyrrolo[3,2-C]pyridine-4-Carboxamide (V14)

Using intermediates 18a and 12 as raw materials, compound V14 was synthesized according to the preparation method of compound V1. ¹H NMR (400 MHz, DMSO-d₆) δ 8.06 (d, J = 3.1 hz, 1H), 7.99 (d, J = 5.6 hz, 1H), 7.70 (d, J = 5.6 hz, 1H), 7.47 (d, J = 3.0 hz, 1H), 7.37–7.28 (m, 4H), 7.26–7.21 (m, 1H), 7.15 (t, J = 6.1 hz, 1H), 7.05 (s, 1H), 6.92 (d, J = 2.8 hz, 1H), 4.59 (d, J = 5.5 hz, 2H), 3.27 (p, J = 3.3 hz, 2H), 2.52–2.51 (m, 3H), 1.89 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.19, 160.13, 159.81, 154.01, 142.43, 141.43, 141.25, 141.15, 139.49, 128.73, 126.88, 123.82, 111.55, 104.52, 88.78–87.48 (m), 44.16, 21.05, 20.17, 16.02. HRMS calcd for C₂₃H₂₃N₇O [M + Na]⁺ 436.1856, found 436.1876.

1-(4-(Benzylamino)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl)-2-Methyl-1H-Pyrrolo[2,3-C]pyridine-4-Carboxamide (V15)

Using intermediates 18a and 6b as raw materials, compound V15 was synthesized according to the preparation method of compound V1. ¹H NMR (400 MHz, DMSO-d₆) δ 9.40 (s, 1H), 8.56 (s, 1H), 7.96 (s, 1H), 7.46 (s, 1H), 7.30 (d, J = 4.5 hz, 4H), 7.23–7.15 (m, 1H), 7.11 (t, J = 6.2 hz, 1H), 7.00 (d, J = 2.6 hz, 1H), 6.85 (s, 1H), 4.62 (d, J = 5.5 hz, 2H), 3.27 (q, J = 3.7 hz, 2H), 2.54 (s, 3H), 2.47 (d, J = 7.7 hz, 2H), 1.88 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.35, 160.12, 159.81, 154.13, 144.13, 141.28, 138.82, 137.87, 134.04, 132.42, 128.66, 126.93, 120.80, 104.77, 87.75, 44.22, 21.07, 20.17, 16.65. HRMS calcd for C₂₃H₂₃N₇O [M + Na]⁺ 436.1856, found 436.1848.

1-(4-(Benzylamino)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl)-2-Methyl- 1H-Pyrrolo[2,3-B]pyridine-4-Carboxamide (V16)

Using intermediates 18a and 6c as raw materials, compound V16 was synthesized according to the preparation method of compound V1. ¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (s, 1H), 8.15 (d, J = 4.9 hz, 1H), 7.99 (s, 1H), 7.57 (s, 1H), 7.39 (d, J = 5.0 hz, 1H), 7.33–7.26 (m, 4H), 7.19 (m, 1H), 7.03 (t, J = 6.1 hz, 1H), 6.84 (d, J = 2.6 hz, 1H), 6.65 (d, J = 1.2 hz, 1H), 4.51 (d, J = 5.3 hz, 2H), 3.25 (dt, J = 6.9, 3.5 hz, 2H), 2.46 (d, J = 6.3 hz, 2H), 2.18 (d, J = 1.0 hz, 3H), 1.88 (t, J = 5.7 hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.50, 160.78–159.34 (m), 153.16, 150.17, 141.58, 141.40, 139.64, 132.47, 128.52, 127.43, 126.81, 118.30, 114.72, 99.89, 88.94, 44.00, 20.97, 20.19, 13.78. HRMS calcd for C₂₃H₂₃N₇O [M + Na]⁺ 436.1856, found 436.1748.

1-(4-(Benzylamino)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl)-6-Fluoro- 2-Methyl-1H-Indole-4-Carboxamide (V17)

Using intermediates 18a and 6a as raw materials, compound V17 was synthesized according to the preparation method of compound V1. ¹H NMR (400 MHz, DMSO-d₆) δ 7.90 (dd, J = 10.7, 2.4 hz, 1H), 7.83 (d, J = 5.3 hz, 1H), 7.44–7.24 (m, 5H), 7.20 (dp, J = 6.4, 4.2 hz, 1H), 7.05 (dt, J = 13.2, 6.2 hz, 1H), 6.92 (d, J = 2.9 hz, 1H), 6.79 (s, 1H), 5.76 (s, 1H), 4.61 (d, J = 6.0 hz, 2H), 3.31–3.22 (m, 2H), 2.48 (s, 2H), 2.45 (s, 3H), 1.88 (t, J = 5.8 hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.76, 160.15, 159.81, 159.10, 156.78, 154.48, 141.36, 139.58, 137.27, 128.65, 126.88, 125.96, 124.04, 108.40, 104.78, 103.64, 87.55, 55.38, 44.12, 21.13, 20.18, 16.46. HRMS calcd for C₂₄H₂₃FN₆O [M + H]⁺ 431.1990, found 431.1924.

1-(4-(Benzylamino)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl)-2-Methyl-1H-Indole-3-Carboxamide (V18)

Using intermediates 18a and 22a as raw materials, compound V18 was synthesized according to the preparation method of compound V1. ¹H NMR (400 MHz, DMSO-d₆) δ 7.70 (d, J = 7.9 hz, 1H), 7.49 (d, J = 8.3 hz, 1H), 7.31 (m, 4H), 7.25–7.20 (m, 1H), 7.18 (s, 1H), 7.11 (t, J = 6.1 hz, 1H), 7.08–7.04 (m, 1H), 6.92 (m, 1H), 6.86 (d, J = 2.6 hz, 1H), 4.57 (d, J = 5.4 hz, 2H), 3.27 (p, J = 3.4 hz, 2H), 2.54 (s, 3H), 1.90 (q, J = 5.9 hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.47, 160.23, 159.98, 153.88, 141.43, 139.62, 135.67, 128.65, 126.99, 126.80, 126.22, 122.10, 121.30, 119.75, 113.41, 111.41, 88.21, 44.03, 21.03, 20.18, 14.11. HRMS calcd for C₂₄H₂₄N₆O [M + Na]⁺ 435.1904, found 435.1913.

1-(4-(Benzylamino)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl)-N-Methoxy-2-Methyl-1H-Indole-3-Carboxamide (V19)

Using intermediates 18a and 22b as raw materials, compound V19 was synthesized according to the preparation method of compound V12. ¹H NMR (400 MHz, DMSO-d₆) δ 11.01 (s, 1H), 7.64 (d, J = 7.9 hz, 1H), 7.51 (d, J = 8.3 hz, 1H), 7.36–7.26 (m, 4H), 7.25–7.20 (m, 1H), 7.14 (t, J = 6.1 hz, 1H), 7.08 (t, J = 7.5 hz, 1H), 6.94 (t, J = 7.7 hz, 1H), 6.88 (s, 1H), 4.57 (d, J = 5.3 hz, 2H), 3.72 (s, 3H), 3.27 (t, J = 5.6 hz, 2H), 2.51 (s, 3H), 2.51 (s, 2H), 1.89 (t, J = 5.8 hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 160.22, 159.90, 153.74, 141.39, 139.76, 135.71, 128.66, 126.98, 126.82, 125.90, 122.40, 121.49, 119.69, 113.56, 88.27, 63.67, 43.99 (d, J = 10.0 hz), 21.00, 20.17, 14.17. HRMS calcd for C₂₅H₂₆N₆O₂ [M + Na]⁺ 465.2009, found 465.2016。

2-(1-(4-(Benzylamino)-5,6,7,8-Tetrahydropyrido[2,3-D]pyrimidin-2-Yl)-2- Methyl-1H-Indol-3-Yl)-N-Methoxyacetamide (V20)

Using intermediates 18a and 22c as raw materials, compound V20 was synthesized according to the preparation method of compound V12. ¹H NMR (400 MHz, DMSO-d₆) δ 11.25 (s, 1H), 7.68 (d, J = 8.2 hz, 1H), 7.47 (d, J = 7.8 hz, 1H), 7.32 (h, J = 6.2 hz, 4H), 7.23 (td, J = 6.6, 2.3 hz, 1H), 7.05–6.95 (m, 2H), 6.91–6.84 (m, 1H), 6.77 (d, J = 2.4 hz, 1H), 4.60 (d, J = 6.0 hz, 2H), 3.56 (s, 3H), 3.35 (s, 2H), 3.31–3.20 (m, 2H), 2.47 (s, 2H), 2.40 (s, 3H), 1.89 (p, J = 6.0 hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.59, 160.19, 159.89, 154.67, 141.57, 135.82, 134.96, 128.63, 126.95, 126.77, 121.61, 120.31, 117.94, 113.84, 108.18, 87.42, 63.61, 44.06, 28.96, 21.21, 20.20, 13.39. HRMS calcd for C₂₆H₂₈N₆O₂ [M + Na]⁺ 479.2166, found 479.2168.

Biological Activity Assay

In vitro p97 Enzyme Inhibition Assay

The inhibitory activities of the compounds against p97 enzyme were tested as follows. The compounds to be tested were dissolved in DMSO and serially diluted 3-fold from 10 μM, each concentration point repeat 3 times. P97 hexamer enzyme (60 μg/mL, purchased from the Public Protein/Plasmid Library, China) and ATP (100 μM) were added to the 384-well plate with a total volume of 4 μL. After mixing, the mixture was incubated at 25°C for 60 min. According to the instructions, ADP Glo reagents 1 and 2 (Promega, Madison, WI) were added. CLARIO Star Plate Reader was used to detect luminescence values of each test hole and calculate the inhibition rate. The IC₅₀ values of each compound were calculated by fitting the nonlinear curves with GraphPad Prism 8.

In vitro Antitumor Activity Assay

The tumor cell lines used in the cell proliferation inhibition experiment were all from Jiangsu Chia Tai Fenghai Pharmaceutical Co., Ltd. In 384-well plate, 18 μL medium (1000 cells) was added to each well. After 24 h incubation at 37°C, 5% CO₂, the compounds to be tested were dissolved in DMSO, and then 2μL of the compounds were added to each well (the content of DMSO was less than 0.1%), the initial concentration of the compounds was 10 μM, and diluted twice, with a total of 10 concentration points. After 72 hours of continuous culture, 10 μL Cell-Titer glo reagent was added to each well, and the absorbance of each well at 450 nm was tested. The inhibition rates of each concentration point were calculated according to the absorbance, and the IC₅₀ values were calculated by fitting the sigmoid curve with GraphPad software.

Molecular Docking

The three-dimensional (3D) crystal structure of VCP protein (PDB ID: 6MCK) was retrieved from the RCSB PDB database (URL: http://www.rcsb.org/). The protein was then pretreated by the protein preparation workflow module in the Maestro software. The molecular docking procedure was conducted utilizing the Glide module within Maestro software under extra precision (XP) mode. The docking box was centered on the native ligand CB5083 extracted from the crystal structure. Optimal docking poses were selected based on the Glide scoring function (Gscore) and docking score parameters.

In vitro Liver Microsome Stabilities

The 100 μL total reaction system used to test the stability of the liver microsomes of the compounds contained PB buffer (100 mm, pH 7.4), MgCl₂-PB buffer (3 mm), compounds (1 μM), microsomal protein (0.5 mg/mL, Corning) and β-NADPH (1 mm). The control group without β-NADPH. The samples were incubated at 37°C and sampled at 0, 5, 15, 30, and 60 min. To each sample, 300 μL of ice-cold acetonitrile containing internal standards was added, mixed, and centrifuged at 5500 g for 10 minutes, and the supernatant was collected. 150 μL of supernatant was aspirated and 150 μL of water was added. After mixing, the content of the compounds to be tested was detected by LC-MS/MS and the curve was fitted.

Rat Pharmacokinetics

The male rats were obtained from Hunan SJA Laboratory Animal Co., Ltd. Twelve male sd rats (240–280 g) were randomly divided into 4 groups with 3 rats in each group. The compounds were dissolved in DMSO and castor oil, and then normal saline was added (DMSO/castor oil/NaCl=5%/5%/90%). The compounds were administered intravenously at a dose of 1mg/kg and orally at a dose of 10 mg/kg. Blood was collected from the fundus venous plexus at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h, and then added to a centrifuge tube containing heparin sodium. Whole blood was centrifuged at 6800 rpm for 6 min at 4°C to obtain plasma. The concentration of compounds in plasma was analyzed by LC-MS/MS, and non-ventricular pharmacokinetic parameters were analyzed by WinNonlin 6.3 program.

In vivo Antitumor Activity Study

BALB/c nude mice (female, 6–8 W, 18–22 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. All animal experiments were approved by the Laboratory Animal Ethics Committee of Nanjing Comer Pharma (protocol number: KMYY-AW-2021). Throughout the experiment, animal body weight and tumor volume were measured twice a week, and the clinical signs of the animals were observed and recorded daily. Molm-13 cells in logarithmic growth stage were collected and injected subcutaneously into Balb/c nude mice with 5 × 10⁶ cells per mice. When the average tumor volume reached 80 ~ 120 mm³, the animals were randomly grouped (n = 5), negative control group, 50 mg/kg CB-5339 (positive control) group and 100 mg/kg V12 group. The compounds was intragastric once a day at a volume of 0.1 mL/10 g animal body weight. The tumor volume was calculated using the formula: V = length (mm) × [width (mm)]²×0.5. The male Balb/c nude mice were housed in SPF (Specific Pathogen Free) laboratory animal centers.

Statistical Analysis

All quantitative data were expressed as the mean ± SD of three independent replicates. Data analysis was performed using GraphPad Prism 8.0.2, with analysis of variance (ANOVA) used for statistical analysis. Statistical significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.001.