Design, synthesis, and biological evaluation of novel pyrimidin-2-amine derivatives as potent PLK4 inhibitors

Abstract

Serine/threonine protein kinase PLK4 is a master regulator of centriole duplication, which is significant for maintaining genome integrity. Accordingly, due to the detection of PLK4 overexpression in a variety of cancers, PLK4 has been identified as a candidate anticancer target. Thus, it is a very meaningful to find effective and safe PLK4 inhibitors for the treatment of cancer. However, the reported PLK4 inhibitors are scarce and have potential safety issues. In this study, a series of novel and potent PLK4 inhibitors with an aminopyrimidine core was obtained utilizing the scaffold hopping strategy. The in vitro enzyme activity results showed that compound 8h (PLK4 IC₅₀ = 0.0067 μM) displayed high PLK4 inhibitory activity. In addition, compound 8h exhibited a good plasma stability (t_1/2 > 289.1 min), liver microsomal stability (t_1/2 > 145 min), and low risk of DDIs. At the cellular level, it presented excellent antiproliferative activity against breast cancer cells. Taken together, these results suggest that compound 8h has potential value in the further research of PLK4-targeted anticancer drugs.

A structurally novel and highly efficient PLK4 inhibitor was synthesized.

1. Introduction

Polo-like kinases (PLKs) are a conserved family of serine/threonine kinases that play a role in the process of mitosis, DNA damage pathways and maintaining gene stability.^1–3 Mammalian cells contain five PKL proteins, including PLK1, Snk/PLK2, Prk/Fnk/PLK3, Sak/PLK4, and PLK5.⁴ Among them, PLK4 has only one polo-box and an active site, which is highly homologous to Aurora kinases.⁵ PLK4 is located in the centriole and is a key regulatory factor to regulate centriole duplication. PLK4 dysregulation is the main cause of mitotic catastrophe, including chromosomal mis-segregation and cytokinesis failure, which is closely related to tumorigenesis and progression.⁶ Thus, the aberrant expression of PLK4 is closely associated with the development and progression of cancer.^7–9 It has been reported the that overexpression of PLK4 is detected in multiple cancers,¹⁰ such as colorectal,¹¹ breast,¹² melanoma,¹³ bone,^14,15 lymphoma,¹⁶ lung,¹⁷ osteosarcoma,¹⁸ pediatric embryonal brain tumors,¹⁹ and pancreatic cancer.²⁰ Surprisingly, PLK4 knockdown caused breast cancer cell death, while normal cells remained unaffected.^6,7,21,22 Therefore, the discovery of PLK4 inhibitors can provide a new avenue for targeting mitotic failure in the treatment of breast cancer.

The typical aminopyrazole and indazole PLK4 inhibitors are listed in Fig. 1. In 2004, VX680 was first as an pan-Aurora kinase inhibitor (K_i values for Aurora A/B/C were 0.65 nM, 3.36 nM, and 4.6 nM, respectively). It also inhibited PLK4 with a K_i value of 7.66 nM.^23,24 To develop a selective PLK4 inhibitor, Oegema et al. selected VX680 as a template to develop the PLK4 inhibitor centrinone (LCR-263, PLK4 IC₅₀ = 2.71 nM), which led to p53-dependent cell cycle arrest in the G1 phase. In addition, the X-ray diffraction analysis of the co-crystal structure of centrinone and PLK4 (PDB code 4YUR, 2.65 Å) provided a basis for subsequent research.^19,25 Pauls et al. designed a series of PLK4 inhibitors with a 1H indazole core, including CFI-400437 and CFI-400945, exhibiting IC₅₀ values of 0.6 nM and 0.26 nM, respectively. Subsequently, CFI-400945 entered Phase II clinical trials.^2,26,27 Yu et al. analysed the co-crystallization structure (PDB code 4JXF) and utilized a simplified structural strategy to obtain YLT-11, which exhibited moderate PLK4 inhibitory activity with an IC₅₀ value of 22 nM.²⁸

Fig. 1. Structures of potent PLK4 inhibitors.

To the best of our knowledge, the reported scaffolds of PLK4 inhibitors are scarce. Furthermore, although some molecules such as centrinone display good inhibitory activity against PLK4, they failed to enter clinical trials.²⁵ In our previous studies, centrinone showed poor liver microsomal stability (t_1/2 = 5.8 min) and strong cytochrome P450 (CYPs) inhibition (Tables S1 and S2†), which suggested that its drug-like properties need to be further optimized.²⁹ In addition, centrinone contains nitrobenzene, resulting in a potential safety issue, which may be one of the reasons why it has not entered clinical research. Therefore, we hope to obtain novel, safe and effective small molecule PLK4 inhibitors through the structural modification of centrinone (Fig. 2). According to the literature, aminopyrimidines are one of the most widely naturally occurring heterocyclic molecules and exhibit antitumor property. To date, several small molecules kinase inhibitors with an aminopyrimidine structure have entered clinical trials.^30–32 Therefore, we utilized scaffold hopping and computer aided drug design strategies to obtain compounds with an aminopyrimidine core. Finally, the potent PLK4 inhibitor 8h with good drug-like properties was discovered. The design, synthesis, biological evaluation, and docking study of the obtained inhibitors are described herein.

Fig. 2. Design strategies for novel PLK4 inhibitors.

2. Results and discussion

2.1. Chemistry

Compounds 3a–3m and 3q–3u were synthesized as described in Scheme 1. Intermediates 2a–2r were prepared via the nucleophilic substitution of commercially available 1 and various nucleophiles. In addition, compounds 3a–3m and 3q–3u were obtained using 4-morpholinoaniline, which nucleophilic attacked intermediates 2a–2r.

Scheme 1. Synthesis of target compounds 3a–3m and 3q–3u. Reagents and conditions: (a) Various nucleophiles, TEA, CHCl₃, r.t., 4 h. (b) 4-Morpholinoaniline, 12 M HCl, EtOH, 110 °C, 8 h.

The synthetic pathway used to achieve compounds 3n–3p and 3v–3x is outlined in Scheme 2. First, THP protection of 4 was performed with 3,4-dihydro-2H-pyran to support intermediate 5. Various iodinated aromatic rings and intermediate 5 underwent Ullmann coupling reaction with ethylene dithiol, and then affinity substitution with the commercially available 1 to obtain intermediates 6a–6f. Subsequently, intermediates 6a–6f were treated through substitution reaction and deprotection with hydrochloric acid to yield compounds 3n–3p and 3v–3x.

Scheme 2. Synthesis of target compounds 3n–3p and 3v–3x. Reagents and conditions: (a) DHP, p-toluenesulfonic acid, DCM/THF (1 : 1), r.t., 10 h. (b) 5 and various iodine-substituted aromatic rings, 1,2-ethanedithiol, KOH, CuSO₄·5H₂O, DMSO/H₂O (4 : 1), 90 °C – r.t., 8 h. (c) 4-Morpholinoaniline, 12 M HCl, EtOH, 110 °C, 8 h.

Scheme 3 shows the conditions for the synthesis of compounds 8a–8g and 8i. Compounds 8a–8e, 8i, 9a, and 9b were prepared using 2r and various amines via Buchwald–Hartwig coupling reaction. Compounds 8f and 8g were deprotected with hydrochloric acid to yield the corresponding compounds 9a and 9b, respectively.

Scheme 3. Synthesis of target compounds 8a–8g and 8i. Reagents and conditions: (a) various amines, K₂CO₃, X-Phos, Pd₂(dba)₃, n-BuOH, 85 °C, 8 h and (b) 4 M HCl in ethyl acetate, r.t., 2 h.

The preparation of compounds 8j and 8k is described in Scheme 4. Commercial 10 reacted with the corresponding amines to produce 11a and 11b, respectively. Then, intermediates 12a and 12b were obtained by catalytic hydrogenation. Subsequently, intermediates 13a and 13b were prepared using 2r and intermediates 12a and 12bvia Buchwald–Hartwig coupling reaction and deprotection to get compounds 8j and 8k, respectively.

Scheme 4. Synthesis of target compounds 8j and 8k. Reagents and conditions: (a) various amines, K₂CO₃, DMSO, 80 °C, 4 h; (b) NH₂–NH₂·H₂O, Pa/C, EtOH, 80 °C, 2 h; (c) 2r, K₂CO₃, X-Phos, Pd₂(dba)₃, n-BuOH, 85 °C, 8 h; and (d) 4 M HCl in ethyl acetate, r.t., 2 h.

Intermediates 15a and 15b were furnished using the commercially available 14a and 14b and tetrahydro-2H-pyran-4-amine by affinity substitution reaction, respectively. Then, intermediates 16a and 16b were obtained by catalytic hydrogenation. Furthermore, compounds 8h and 8l were acquired taking advantage of 2r, which nucleophilic attacked intermediates 16a and 16b, respectively (Scheme 5).

Scheme 5. Synthesis of target compounds 8h and 8l. Reagents and conditions: (a) tetrahydro-2H-pyran-4-amine, K₂CO₃, DMSO, 80 °C, 4 h; (b) NH₂–NH₂·H₂O, Pa/C, EtOH, 80 °C, 2 h; and (c) 2r, K₂CO₃, X-Phos, Pd₂(dba)₃, n-BuOH, 85 °C, 8 h.

2.2. Pharmacology/biology

All the designed compounds were evaluated for PLK4 enzyme inhibitory activity in vitro using the LanthaScreen Eu kinase binding assay (Thermo Fisher Scientific, Carlsbad, USA). Centrinone was employed as a positive control. Under our experimental conditions, centrinone strongly inhibited PLK4 with an IC₅₀ value of 0.003 μM, which is consistent with previous reports.³³

Based on the reasonable design of novel PLK4 inhibitors, we primarily analysed the binding mode of centrinone and PLK4 (PDB code 4YUR) (Fig. 3a and d). Specifically, the aminopyrazole moiety forms key hydrogen bonds with hinge residues Glu-90 and Cys-92, the morpholine ring extends to the solvent region, and the pyrimidine right substituent extends to the DFG motif and nitro groups, forming a hydrogen-bond interaction with Lys-41. Based on the above analysis, to keep the anchoring effect with the hinge region, the scaffold hopping strategy was adopted to transition part of the aminopyrazole moiety to the aminopyrimidine kinase inhibitor star framework. Also, the morpholine ring stretching to the solvent region was preserved. Subsequently, combined with previous work, we learned that the hydrophobic cavity next to the DFG motif has a significant impact on PLK4 inhibitor activity.^33,34 Given that the complex substituent on the centrinone right side cannot be well embedded in the hydrophobic cavity, we expected to replace this group with an appropriate aromatic ring to reach the hydrophobic cavity. Compounds 3a–3d were synthesized firstly. According to the results of the enzyme activity (Table 1), compound 3b (PLK4 IC₅₀ = 0.0312 μM) showed moderate inhibitory activity. Then, we verified the binding mode of compound 3b and PLK4 (PDB code 4YUR) by molecular docking. The docking results showed that the aminopyrimidine core formed key interactions with the hinge region, where the morpholine ring extends towards the solvent region and the right substituent group extends to the hydrophobic cavity next to the DFG motif (Fig. 3b and e). Thus, the enzyme inhibition and molecular docking results indicated that the design strategy was expected.

Fig. 3. (a) Binding mode of centrinone in the ATP-binding site of PLK4 (PDB code: 4YUR, colored grey). Interactions are illustrated with yellow dashed lines. Ligands are depicted by the element with carbons in green. (b) Binding mode of 3b in the ATP-binding site of PLK4 (PDB code: 4YUR, colored grey). Interactions are illustrated with yellow dashed lines. Ligands are depicted by the element with carbons in green. (c) Binding mode of 3r in the ATP-binding site of PLK4 (PDB code: 4YUR, colored grey). Interactions are illustrated with yellow dashed lines. Ligands are depicted by the element with carbons in green. (d) Binding mode of centrinone in the ATP-binding site of PLK4 (PDB code: 4YUR, surface mode). (e) Binding mode of 3b in the ATP-binding site of PLK4 (PDB code: 4YUR, surface mode). (f) Binding mode of 3r in the ATP-binding site of PLK4 (PDB code: 4YUR, surface mode).

The inhibitory activity (IC₅₀) of compounds 3a–3x against PLK4 in vitro.

Entry	R1	IC50/μM	Entry	R1	IC50/μM
3a		0.0807	3n		0.1255
3b		0.0312	3o		0.0463
3c		0.0654	3p		0.1771
3d		0.1775	3q		0.1021
3e		0.1983	3r		0.0174
3f		0.0788	3s		0.1525
3g		0.1360	3t		0.2974
3h		0.1499	3u		0.0714
3i		1.4260	3v		0.3130
3j		0.1422	3w		0.0565
3k		0.1894	3x		0.0352
3l		2.3760	Centrinone		0.0030
3m		0.2190

Under the guidance of the docking study (Fig. 3e), we found that the hydrophobic cavity adjacent to the DFG motif was not fully occupied by the naked benzene ring of compound 3b. To increase the hydrophobic cavity matching and enhance the inhibitory activity, we planned to modify the phenyl part of 3b through the following three strategies: (1) performing “halogen walk” to explore the hydrophobic cavity; (2) filling the hydrophobic cavity with bicyclic rings instead of monocyclic benzene to further occupy the hydrophobic cavity; and (3) introducing suitable groups to form interactions with the surrounding amino acid residues.

The in vitro PLK4 kinase activity of compounds 3e–3m is depicted in Table 1. Unfortunately, the activity of the halogen-substituted compounds did not improve. Subsequently, utilizing the second strategy, compounds 3n–3p were synthesized. However, the PLK4 inhibitory activity was not enhanced. Combined with the molecular docking study (Fig. S1†), we speculated that to improve the PLK4 inhibitory activity, it is necessary to extend the substituent to the hydrophobic cavity, forming a stronger interaction. Thus, a hydrogen bond donor was introduced in the benzene ring to increase the binding, and compounds 3q–3t were obtained. Considering the enzyme activity result (Table 1), it was gratifying that compound 3r (PLK4 IC₅₀ = 0.0174 μM) showed strong PLK4 inhibitory activity. Also, the molecular docking results (Fig. 3c and f) indicated that compared to compound 3b, the amino group of compound 3r formed a hydrogen bond with residues Glu-96 and Ser-140 in the hydrophobic cavity, which confirmed our hypothesis. Thus, to verify the contribution of the hydrogen bond interaction to the activity, compounds 3u and 3v were designed and synthesized. The enzyme activity (Table 1) of all three compounds decreased, indicating the importance of hydrogen bonding. According to the molecular docking results (Fig. S2†), compound 3u (PLK4 IC₅₀ = 0.0714 μM) retained a hydrogen bond interaction with Glu-96, while there was no hydrogen bond interaction in compound 3v (PLK4 IC₅₀ = 0.313 μM), which is consistent with the decrease in its enzyme activity. Based on compound 3r, a fluorine atom was introduced in the benzene ring (3w and 3x), but this did not improve its activity (Table 1). We speculated that the fluorine atom changed the molecular charge distribution, which resulted in the disappearance of the hydrogen bond interaction and a decrease in activity (Fig. S3†).

Besides the right hydrophobic group, which has an important influence on the activity, the hydrophilic morpholine moiety of 3r was observed to orient to the solvent area, and thus can be diversified to yield analogues possessing different profiles with regard to isoform stability, and cellular activity without sacrificing the potency of the compound. Therefore, we hoped to further enhance its activity and stability by optimizing its hydrophilic segments. The in vitro test results are described in Table 2. Firstly, compound 8a lacking a hydrophilic moiety in the solvent region was designed and synthesized. As predicted, the activity of compound 8a (PLK4 IC₅₀ = 0.5196 μM) decreased by nearly 30 times compared to compound 3r (PLK4 IC₅₀ = 0.0174 μM). Given that the hydrophilic group in this position influenced the activity, different types of hydrophilic heterocycles (8b–8g) were explored. According to the enzyme activity results, we concluded that para-substitution activity is slightly better than meta with the same substituent group, and the activity of the morpholine ring-substituted compounds in the same sites is better. Based on these observations, it was hypothesized that the introduction of different chain-linked hydrophilic fragments in opposite positions of the phenyl group may be a feasible strategy to improve the potency and stability of the inhibitor. Compounds 8h–8k were synthesized. According to the enzyme activity test information, it was found that compound 8h (PLK4 IC₅₀ = 0.0067 μM) exhibited nearly three-times stronger PLK4 inhibitory activity than compound 3r (PLK4 IC₅₀ = 0.0174 μM). It was found that compound 8h maintained the original hydrogen bond interaction with 3r (Fig. 4a). In addition, the 8h hydrophilic fragment can be better adapted to the solvent domain protein through the surface pattern of the eutectic structure (Fig. 4b), which may be the reason for the inhibited improvement in activity. In addition, alternatives to block the benzene ring were introduced and compound 8l was designed and synthesized. Unfortunately, its activity was not further improved. We preliminarily evaluated the liver microsomal stability of compounds 8h and 3r (Table 3). The half-life (t_1/2) of compound 8h was greater than 145 min, which was significantly improved compared to that of compound 3r (t_1/2 = 58.3 min).

The inhibitory activity (IC₅₀) of compounds 8a–8l against PLK4 in vitro.

Entry	R2	IC50/μM	Entry	R2	IC50/μM
8a		0.5196	8h		0.0067
8b		1.1020	8i		0.0389
8c		0.0639	8j		0.0569
8d		0.0454	8k		0.0803
8e		0.1369	8l		6.6200
8f		0.1392	Centrinone		0.0030
8g		0.3793

Fig. 4. (a) Binding mode of 8h in the ATP-binding site of PLK4 (PDB code: 4YUR, colored grey). (b) Binding mode of 3r (carbons in pick) and 8h (carbons in green) in the ATP-binding site of PLK4 (PDB code: 4YUR, surface mode). Interactions are illustrated with yellow dashed lines.

Liver microsomal stability of compounds 3r and 8h.

Compound	R 2	t 1/2	CLint(mic) (μL min−1 mg−1)	CLint(liver) (μL min−1 mg−1)	Remaining (%) (T = 60 min)	Remaining (%) (NCF,aT = 60 min)
3r	0.9604	58.3	23.8	21.4	48.7	94.4
8h	0.0291	>145	<9.6	<8.6	100.0	103.8

^aNCF: abbreviation of no co-factor. No NADPH was added to the NCF samples (replaced by buffer) during the 60 min incubation. If the NCF remaining is less than 60%, then there is the possibly of non-NADPH dependent metabolism.

2.3. Antiproliferative activity

The antiproliferation activities of seven compounds (3b, 3o, 3r, 3x, 8d, 8h and 8i) with IC₅₀ values of <50 nM on three breast cancer cell lines (MCF-7, MDA-MB-231, and BT474) were evaluated in this experiment.³³ Centrinone was used as a control. According to the test results (Table 4), compounds 3x, 8h, and 8i all exhibited comparable antiproliferative activity to the positive control centrinone against the three cell lines. Among them, the IC₅₀ value of compound 8h on the MCF-7 and MDA-MB-231 cell lines was 2-times and 4-times higher than that of centrinone, respectively. Therefore, compound 8h was selected for further evaluation.

Antiproliferative activity (IC₅₀,^a μM) of selected compounds against MCF-7, BT474, and MDA-MB-231 cells.

Compound	MCF-7	BT474	MDA-MB-231
3b	>20	>20	7.57 ± 0.55
3o	>20	>20	6.48 ± 0.58
3r	8.72 ± 0.69	>20	5.11 ± 0.42
3x	3.45 ± 0.22	9.32 ± 0.69	8.08 ± 0.72
8d	>20	12.9 ± 1.04	5.15 ± 0.39
8h	1.44 ± 0.11	7.81 ± 0.58	2.13 ± 0.19
8i	3.86 ± 0.29	16.2 ± 1.21	4.47 ± 0.42
Centrinone	2.27 ± 0.04	10.52 ± 0.73	8.69 ± 0.39

^aIC₅₀: concentration of the compound (μM) producing 50% cell growth inhibition. The mean values of three independent experiments ± SE are reported.

2.4. In vitro plasma stability and cytochrome P450 (CYP450) inhibition

Metabolic stability in vitro is an important factor when evaluating the drug-likeness of compounds. Therefore, the plasma stability of compound 8h was determined and presented in Table 5. The in vitro plasma stability studies showed that compound 8h had excellent plasma stability and a half-life (t_1/2) value of >289.1 min.

Plasma stability of compound 8h.

Time point (min)	0	10	30	60	120
Remaining (%)	100.0	105.1	113.9	107.4	103.5
t 1/2 (min)	>289.1

Cytochrome P450s (CYPs) enzymes are mainly involved in the synthesis or degradation of endogenous and exogenous compounds, such as steroids, cholesterol, and fatty acids. Drug–drug interactions (DDIs) are mainly related to the inhibition of CYPs.^35,36 Thus, compound 8h was evaluated using the main drug-metabolizing enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) (Table 6). The test results indicated that at the concentration of 10.0 μM, 8h had no significant inhibitory effect on CYPs, indicating the low risk of DDIs between compound 8h and other drugs.

In vitro CYP inhibition of compound 8h^a.

Isozyme	CYP1A2	CYP2C9	CYP2C19	CYP2D6	CYP3A4
Inhibition at 10 μM (%)	33.1	42.9	48.7	8.0	14.4

^aα-Naphthoflavone (CYP1A2, 83.5% inhibition), sulfaphenazole (CYP2C9, 85.1% inhibition), (+)-N-3-benzylnirvanol (CYP2C19, 82.6% inhibition), quinidine (CYP2D6, 94.8% inhibition), and ketoconazole (CYP3A4, 98.8% inhibition) were used as the positive controls.

2.5. Prediction of molecular properties and drug-likeness

As research on the physical and chemical properties of drugs progresses, evaluating the drug-like properties of target compounds has become crucial. This is achieved by detecting and optimizing ligand efficiency metrics instead of solely relying on potency.^37–39 The oil–water partition coefficient (c Log P), topological polar surface area (TPSA) is important criterion for evaluating the drug-likeness of target compounds. The TPSA, cLogP, and drug-likeness score were predicted making use of Home ADME Prediction (https://admet.scbdd.com/) and Molsoft (https://www.molsoft.com/). The ligand efficiency (LE), ligand lipophilicity efficiency (LLE), and ligand efficiency-dependent lipophilicity (LELP) were calculated based on several other physical and chemical properties according to the corresponding formula.⁴⁰ The evaluation results for drug-like properties are displayed in Table 7 and Fig. 5. With centrinone as the control, the results for compound 8h were all within the reasonable range, and TPSA and LE were optimized (TPSA values < 120, LE values > 0.3). According to the results, the drug-likeness score of 8h (score = 0.58) is greater than 0.5, which may show better absorption and bioavailability than centrinone (score = 0.33), suggesting that compound 8h has further research value.⁴¹

Molecular properties and drug-likeness score of compounds 8h and centrinone.

Compound	M.V.	pIC50	c Log P	Topological polar surface area (TPSA)	Ligand efficiency (LE)	Ligand lipophilicity efficiency (LLE)	Ligand efficiency-dependent lipophilicity (LELP)	Drug-likeness score
8h	393.16	8.17	3.13	85.09	0.40	5.04	7.83	0.58
Centrinone	633.65	8.52	3.40	165.47	0.27	5.12	12.59	0.33

Fig. 5. (a) Prediction of drug-like properties of compound 8h. (b) Prediction of drug-like properties of centrinone.

3. Conclusions

In summary, a series of novel aminopyrimidine derivatives was synthesized as potent PLK4 inhibitors using a structure-based design strategy starting from the lead compound centrinone. Starting with the hydrophobic pocket occupying the side of the DFG motif, the ensuing SAR led to the discovery of compound 8h, which exhibited a promising effect on PLK4 activity (IC₅₀ = 0.0067 μM) and potent antiproliferative activity against breast cancer cells (MCF-7, BT474, and MDA-MB-231 cells), with IC₅₀ values of 1.44, 7.81, and 2.13 μM, respectively. Remarkably, compound 8h exhibited good plasma stability, liver microsomal stability, and a lower risk of DDIs. In addition to good drug-like properties, compound 8h also has the advantage of simple and easy synthesis. These data indicate that this fragment of aminopyrimidine is a promising point for the development of PLK4 inhibitors and 8h is an attractive lead compound for further optimization and evaluation.

4. Experimental

For the experiment, all reagents and solvents were purchased from commercial suppliers and used without further purification. Anhydrous solvents were prepared and stored using standard procedures. All moisture- and air-sensitive reactions were carried out under an atmosphere of dry argon with heat-dried glassware and standard syringe techniques. Thin-layer chromatography (TLC) on HSGF-254 (20–25 μm) silica gel plates was used to monitor all reactions, which was observed under UV light. For conventional purification, preparative thin-layer chromatography (PTLC) was performed on HSGF-254 (40–50 μm). All target compounds were characterized through melting point determination, nuclear magnetic resonance (NMR), and high-resolution accurate mass spectrometry (HRMS). Melting points were determined using a BüCHI Melting Point B-540 apparatus. NMR data were obtained using a Bruker ARX-600 NMR spectrometer with TMS as the internal standard in DMSO-d₆. HRMS was performed on a Bruker micromass time of flight mass spectrometer with an electrospray ionization (ESI) detector for all target compounds.

4.1. Chemistry

4.1.1. General procedure for the synthesis of compounds 2a–2r

To a solution of 2,4-dichloropyrimidine (1, 1 equiv.) in chloroform (10 mL), various nucleophiles (1 equiv.) and TEA (1.3 equiv.) were added at room temperature and the mixture was stirred for 4 h. The reaction progress was monitored by TLC. Volatile components were removed in vacuum and the residue was purified by silica chromatography to give a white solid in 89–95% yield.

4.1.2. General procedure for the synthesis of compounds 3a–3m and 3q–3u

To a stirring solution of intermediates 2a–2r (1.0 equiv.) in dry EtOH (2 mL), 12 M HCl (16 μL) and 4-morpholinoaniline (1.2 equiv.) were successively added in a sealed tube at 110 °C for 8 h. The reaction was monitored by TLC. Upon cooling, the target compound gradually precipitated from solution, and then the precipitate was collected by filtration. The filter cake was washed with methanol (0.5 mL) to obtain compounds 3a–3m and 3q–3u in 83%–92% yield.

N-(4-Morpholinophenyl)-4-phenoxypyrimidin-2-amine (3a)

White solid, 89% yield, m.p. 154.5–155.0 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.34 (s, 1H), 8.30 (d, J = 5.5 Hz, 1H), 7.50 (t, J = 7.7 Hz, 2H), 7.36 (s, 2H), 7.33 (t, J = 12 Hz, 1H), 7.24 (d, J = 6 Hz, 2H), 6.71 (d, J = 7.1 Hz, 2H), 6.33 (d, J = 5.5 Hz, 1H), 3.73–3.71 (m, 4H), 2.99–2.97 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.8, 160.4, 160.3, 152.9, 146.5, 133.1, 130.2, 125.9, 122.4, 120.5, 115.8, 97.9, 66.6, 49.7. HRMS (ESI, m/z) calcd for C₂₀H₂₀N₄O₂, [M + H]⁺: 349.1659; found: 349.1696.

N-(4-Morpholinophenyl)-4-(phenylthio)pyrimidin-2-amine (3b)

White solid, 85% yield, m.p. 186.7–187.3 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.44 (s, 1H), 8.12 (d, J = 5.3 Hz, 1H), 7.66 (t, J = 7.0 Hz, 2H), 7.61 (t, J = 7.4 Hz, 1H), 7.57 (t, J = 7.6 Hz, 2H), 7.35 (d, J = 6.1 Hz, 2H), 6.78 (s, 2H), 6.30 (s, 1H), 3.76–3.74 (m, 4H), 3.03 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 171.6, 159.6, 157.6, 146.4, 136.3, 133.3, 130.6, 130.4, 128.1, 120.4, 116.2, 107.7, 66.6, 49.9. HRMS (ESI, m/z) calcd for C₂₀H₂₀N₄OS, [M + H]⁺: 365.1431; found: 365.1449.

N ²-(4-Morpholinophenyl)-N⁴-phenylpyrimidine-2,4-diamine (3c)

White solid, 88% yield, m.p. 289.1–190.3 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 11.18 (s, 1H), 10.57 (s, 1H), 7.95 (s, 1H), 7.69 (s, 2H), 7.34 (s, 4H), 7.17 (s, 1H), 7.00 (s, 2H), 6.55 (s, 1H), 3.76 (s, 4H), 3.13 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 161.3, 153.1, 149.2, 143.1, 138.1, 129.2, 128.4, 125.3, 124.7, 122.0, 115.9, 99.9, 66.5, 49.1. HRMS (ESI, m/z) calcd for C₂₀H₂₁N₅O, [M + H]⁺: 348.1819; found: 348.1851.

N ⁴-Benzyl-N²-(4-morpholinophenyl)pyrimidine-2,4-diamine (3d)

White solid, 86% yield, m.p. 279.5–280.1 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 10.59 (s, 1H), 9.72 (s, 1H), 7.82 (s, 1H), 7.36 (s, 2H), 7.32 (d, J = 5.4 Hz, 4H), 7.29 (t, J = 7.2 Hz, 1H), 6.94 (d, J = 8.3 Hz, 2H), 6.30 (d, J = 7.2 Hz, 1H), 4.57 (d, J = 5.6 Hz, 2H), 3.75–3.73 (m, 4H), 3.10–3.08 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 162.7, 152.5, 148.7, 141.8, 138.1, 129.1, 129.0, 128.1, 127.8, 123.2, 115.9, 98.6, 66.5, 49.1, 44.5. HRMS (ESI, m/z) calcd for C₂₁H₂₃N₅O, [M + H]⁺: 362.1975; found: 362.2000.

4-((4-Fluorophenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3e)

Yellow solid, 85% yield, m.p. 249.0–249.9 °C.¹H NMR (600 MHz, DMSO-d₆) δ 9.95 (s, 1H), 8.21 (d, J = 5.4 Hz, 1H), 7.72 (dd, J = 8.6, 5.4 Hz, 2H), 7.54 (s, 2H), 7.42 (t, J = 8.8 Hz, 4H), 6.49 (s, 1H), 4.00 (s, 4H), 3.40 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 172.4, 164.7, 163.0, 158.6, 156.7, 140.0, 138.9, 123.5, 120.2, 117.7, 117.6, 108.7, 64.8, 53.2. HRMS (ESI, m/z) calcd for C₂₀H₁₉FN₄OS, [M + H]⁺: 383.1336; found: 383.1362.

4-((3-Fluorophenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3f)

White solid, 85% yield, m.p. 241.8–242.5 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.74 (s, 1H), 8.20 (d, J = 5.3 Hz, 1H), 7.63–7.60 (m, 1H), 7.57–7.55 (m, 1H), 7.53–7.50 (m, 2H), 7.42 (s, 2H), 7.16 (s, 2H), 6.53 (s, 1H), 3.91 (s, 4H), 3.25 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 170.9, 163.5, 161.9, 159.2, 157.5, 132.5, 132.1 (d, J = 8.2 Hz), 130.0 (d, J = 8.2 Hz), 123.0 (d, J = 10.1 Hz), 120.1, 118.9, 118.4, 117.8 (d, J = 20.9 Hz), 108.7, 65.4, 52.0. HRMS (ESI, m/z) calcd for C₂₀H₁₉FN₄OS, [M + H]⁺: 383.1336; found: 383.1369.

4-((2-Fluorophenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3g)

White solid, 83% yield, m.p. 249.0–249.6 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.93 (s, 1H), 8.26 (d, J = 14.1 Hz, 1H), 7.80–7.74 (m 2H), 7.52–7.41 (m, 6H), 6.69 (s, 1H), 4.03 (s, 4H), 3.42 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.9, 163.7, 162.1, 159.0, 157.3, 138.5, 134.1, 126.3, 120.6, 119.8, 117.2(d, J = 22.6 Hz), 114.9 (d, J = 18.2 Hz), 108.9, 64.7, 53.4. HRMS (ESI, m/z) calcd for C₂₀H₁₉FN₄OS, [M + H]⁺: 383.1336; found: 383.1350.

4-((4-Chlorophenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3h)

Yellow solid, 87% yield, m.p. 266.8–267.4 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.95 (s, 1H), 8.22 (d, J = 5.5 Hz, 1H), 7.68–7.67 (m, 2H), 7.65–7.63 (m, 2H), 7.49 (s, 2H), 7.36 (s, 2H), 6.57 (s, 1H), 3.99 (s, 4H), 3.39 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 170.8, 157.4, 155.6, 155.4, 137.1, 134.8, 129.4, 125.7, 119.1, 107.9, 63.7, 52.1. HRMS (ESI, m/z) calcd for C₂₀H₁₉ClN₄OS, [M + H]⁺: 399.1041; found: 399.1078.

4-((3-Chlorophenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3i)

Purple solid, 89% yield, m.p. 261.1–261.7 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.93 (s, 1H), 8.24 (d, J = 5.4 Hz, 1H), 7.77–7.75 (m, 2H), 7.64 (dd, J = 6.4, 1.4 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.45 (s, 4H), 6.63 (s, 1H), 4.02 (s, 4H), 3.41 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 171.2, 158.9, 157.2, 157.1, 135.7, 135.1, 134.6, 134.5, 132.0, 130.9, 129.9, 120.5, 119.9, 109.1, 64.7, 53.4. HRMS (ESI, m/z) calcd for C₂₀H₁₉ClN₄OS, [M + H]⁺: 399.1041; found: 399.1057.

4-((2-Chlorophenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3j)

Purple solid, 89% yield, m.p. 254.9–255.7 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.90 (s, 1H), 8.24 (d, J = 5.4 Hz, 1H), 7.84–7.83 (m, 1H), 7.78–7.71 (m, 3H), 7.54–7.52 (m, 1H), 7.39 (s, 3H), 6.64 (s, 1H), 4.01 (s, 4H), 3.40 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 167.0, 159.0, 157.4, 157.3, 139.5, 139.1, 133.1131.1, 131.0, 129.0, 127.1, 120.6, 119.7, 109.1, 64.7, 53.4. HRMS (ESI, m/z) calcd for C₂₀H₁₉ClN₄OS, [M + H]⁺: 399.1041; found: 399.1058.

4-((4-Bromophenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3k)

Yellow solid, 88% yield, m.p. 260.3–261.2 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.92 (s, 1H), 8.22 (d, J = 5.4 Hz, 1H), 7.78–7.77 (m, 2H), 7.61–7.59 (m, 2H), 7.46 (s, 2H), 7.31 (s, 2H), 6.59 (s, 1H), 3.98 (s, 4H), 3.38 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 171.6, 158.7, 158.6, 156.7, 138.4, 133.4, 133.3, 132.3, 127.3, 124.6, 120.1, 108.9, 64.9, 52.9. HRMS (ESI, m/z) calcd for C₂₀H₁₉BrN₄OS, [M + H]⁺: 443.0536, 445.0517; found: 443.0554, 445.0536.

4-((3-Bromophenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3l)

Purple solid, 88% yield, m.p. 250.9–260.4 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.89 (s, 1H), 8.23 (d, J = 5.4 Hz, 1H), 7.88 (dd, J = 9.7, 4.9 Hz, 2H), 7.68 (d, J = 7.8 Hz, 1H), 7.54 (d, J = 7.9 Hz, 1H), 7.39 (d, J = 49.3 Hz, 4H), 6.62 (s, 1H), 3.99 (s, 4H), 3.38 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 171.2, 158.9, 157.1, 138.4, 135.5, 133.7, 132.3, 130.2, 122.9, 122.9, 120.0, 109.1, 64.9, 53.14. HRMS (ESI, m/z) calcd for C₂₀H₁₉BrN₄OS, [M + H]⁺: 445.0517; found: 445.0540.

4-((2-Bromophenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3m)

White solid, 86% yield, m.p. 192.2–192.7 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.43 (s, 1H), 8.15 (d, J = 5.3 Hz, 1H), 7.91 (dd, J = 5.8, 3.5 Hz, 1H), 7.83 (dd, J = 5.8, 3.5 Hz, 1H), 7.56–7.54 (m, 2H), 7.26 (s, 2H), 6.70 (d, J = 7.3 Hz, 2H), 6.38 (s, 1H), 3.74–3.72 (m, 4H), 3.01–2.99 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.5, 159.7, 157.8, 146.5, 138.9, 134.4, 133.0, 132.9, 132.8, 131.1, 129.5, 120.2, 115.9, 107.8, 66.6, 49.7. HRMS (ESI, m/z) calcd for C₂₀H₁₉BrN₄OS, [M + H]⁺: 445.0517; found: 445.0554.

N-Methyl-2-((2-((4-morpholinophenyl)amino)pyrimidin-4-yl)thio)benzamide (3q)

White solid, 91% yield, m.p. 167.3–168.0 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.36 (s, 1H), 8.23 (d, J = 4.4 Hz, 1H), 8.10 (d, J = 5.3 Hz, 1H), 7.68 (s, 1H), 7.58 (s, 1H), 7.55 (s, 2H), 7.39 (d, J = 7.4 Hz, 2H), 6.77 (s, 2H), 6.24 (s, 1H), 3.73 (s, 4H), 3.01 (s, 4H), 2.69 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 171.3, 168.3, 159.5, 157.5, 146.6, 143.3, 137.4, 133.0, 130.7, 130.3, 128.7, 126.1, 120.6, 116.0, 108.2, 66.6, 49.7, 26,5. HRMS (ESI, m/z) calcd for C₂₂H₂₃N₅O₂S, [M + H]⁺: 422.1645; found: 422.1674.

4-((4-Aminophenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3r)

Yellow solid, 88% yield, m.p. 269.8–270.5 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.83 (s, 1H), 8.16 (d, J = 5.4 Hz, 1H), 7.50 (d, J = 8.5 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.29 (s, 2H), 6.97 (d, J = 4.6 Hz, 2H), 6.45 (s, 1H), 3.95 (s, 6H), 3.35 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 173.9, 158.6, 156.3, 146.5, 140.7, 137.8, 137.5, 120.1, 119.4, 118.2, 115.5, 108.3, 65.2, 52.5. HRMS (ESI, m/z) calcd for C₂₀H₂₁N₅OS, [M + H]⁺: 380.1540; found: 380.1559.

4-((3-Aminophenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3s)

Grey solid, 90% yield, m.p. 204.9–206.0 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.40 (s, 1H), 8.11 (d, J = 5.3 Hz, 1H), 7.45 (d, J = 8.7 Hz, 2H), 7.18 (d, J = 7.8 Hz, 1H), 6.82 (d, J = 2.0 Hz, 2H), 6.81 (s, 1H), 6.74 (dd, J = 7.2, 0.9 Hz, 2H), 6.25 (d, J = 3.9 Hz, 1H), 5.42 (s, 2H), 3.74 (s, 4H), 3.01 (d, J = 4.8 Hz, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 172.6, 159.6, 157.6, 150.6, 146.6, 133.1, 130.8, 128.0, 122.8, 120.8, 120.5, 116.1, 115.9, 107.4, 66.6, 49.7. HRMS (ESI, m/z) calcd for C₂₀H₂₁N₅OS, [M + H]⁺: 380.1540; found: 380.1548.

4-((2-Aminophenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3t)

Yellow solid, 87% yield, m.p. 241.1–241.7 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 8.55 (s, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.30 (d, J = 9.2 Hz, 2H), 7.26–7.20 (m, 1H), 7.08 (s, 2H), 6.23 (d, J = 12.6 Hz, 1H), 3.82 (s, 2H), 3.78 (s, 4H), 3.15 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 174.8, 166.9, 159.4, 153.2, 147.9, 131.4, 130.1, 130.0, 125.9, 121.3, 121.2, 120.6, 118.2, 116.0, 66.6, 49.4. HRMS (ESI, m/z) calcd for C₂₀H₂₁N₅OS, [M + H]⁺: 380.1540; found: 380.1344.

4-((4-(Methylamino)phenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3u)

Yellow solid, 92% yield, m.p. 264.4–265.0 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.72 (s, 1H), 8.12 (d, J = 5.5 Hz, 1H), 7.49 (d, J = 7.0 Hz, 2H), 7.32 (s, 2H), 7.11 (s, 2H), 6.70 (s, 2H), 6.33 (s, 1H), 3.88 (s, 4H), 3.22 (s, 4H), 2.51–2.50 (m, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 173.8, 157.5, 155.1, 155.1, 150.6, 136.7, 119.2, 117.23, 117.0, 112.3, 110.0, 106.7, 64.6, 50.5, 28.9. HRMS (ESI, m/z) calcd for C₂₁H₂₃N₅OS, [M + H]⁺: 394.1696; found: 394.1720.

4.1.3. Synthesis of 5-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (5)

To a solution of 4 (300 mg, 1.23 mmol, 1 equiv.) and p-toluenesulfonic acid (105.6 mg, 0.61 mmol, 0.5 equiv.) in DCM/THF (1 : 1, v/v), 3,4-dihydro-2H-pyran was carefully added (242 μL, 3.69 mmol). Then the mixture was stirred at room temperature for 10 h. The solvent was removed under vacuum and the solid residue was purified by PLC to give a white solid in 91% yield. ¹H NMR (600 MHz, DMSO-d₆) δ 8.20 (dd, J = 1.5, 0.6 Hz, 1H), 8.07–8.06 (m, 1H), 7.66 (d, J = 7.2 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 5.84 (d, J = 7.1 Hz, 1H), 4.11 (m, 6H), 3.35 (s, 2H).

4.1.4. General procedure for the synthesis of compounds 6a–6f

To a stirring solution of various iodine-substituted aromatic rings (1 equiv.) in DMSO/H₂O (4 : 1, v/v), 1,2-ethanedithiol (160 μL, 2 mmol, 2 equiv.), KOH (265 mg, 4.7 mmol, 5 equiv.) and CuSO₄·5H₂O (2.4 mg, 0.009 mmol, 0.01 equiv.) were added in a flask. Then the mixture was degassed for 5 min. The resulting suspension was heated at 90 °C for 6 h in the flask under a nitrogen atmosphere. The reaction mixture was monitored by TLC. The reaction was cooled to room temperature, and then 2,4-dichloropyrimidine (1, 141 mg, 0.9 mmol, 1 equiv.) was added to the mixture at room temperature for 2 h. The reaction liquid was poured into 10-times volume of water, and the resulting mixture was extracted with DCM thrice. The combined organic layers were washed with brine and dried over Na₂SO₄, filtered and the solvent removed under reduced pressure. The resulting crude product was purified by PLC to obtain a colourless oil in 89% yield.

4.1.5. General procedure for the synthesis of compounds 3n–3p and 3v–3x

To a stirring solution of intermediates 6a–6f (1.0 equiv.) in dry EtOH (2 mL), 12 M HCl (16 μL) and 4-morpholinoaniline (1.2 equiv.) were successively added in a sealed tube at 110 °C for 8 h. The reaction mixture was monitored with TLC. The reaction was cooled to room temperature, and then filtered. The filter cake was washed with methanol (0.5 mL) to obtain compounds 3n–3q and 3v–3x in 85–89% yield.

4-((1H-Indazol-5-yl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3n)

White solid, 87% yield, m.p. 259.6–260.4 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 13.54 (s, 1H), 9.37 (s, 1H), 8.19 (s, 1H), 8.12 (s, 1H), 8.09 (d, J = 5.3 Hz, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.50 (d, J = 8.6 Hz, 1H), 7.15 (s, 2H), 6.38 (s, 3H), 3.73 (s, 4H), 2.92 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 172.2, 159.5, 157.3, 146.1, 140.7, 134.5, 133.7, 133.1, 130.2, 124.5, 120.0, 118.6, 115.6, 112.1, 107.7, 66.6, 49.7. HRMS (ESI, m/z) calcd for C₂₁H₂₀N₆OS, [M + H]⁺: 405.1492; found: 405.1504.

N-(4-Morpholinophenyl)-4-(quinolin-5-ylthio)pyrimidin-2-amine (3o)

Yellow solid, 89% yield, m.p. 257.9–261.7 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.69 (s, 1H), 9.10 (dd, J = 4.3, 1.2 Hz, 1H), 8.76 (d, J = 8.5 Hz, 1H), 8.50 (d, J = 8.5 Hz, 1H), 8.18 (d, J = 1.7 Hz, 1H), 8.17 (s, 1H), 8.05–8.02 (m, 1H), 7.76 (dd, J = 8.6, 4.4 Hz, 1H), 7.03 (s, 4H), 6.58 (s, 1H), 3.93 (s, 4H), 3.26 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 170.4, 159.0, 157.3, 150.1, 146.2, 137.8, 137.2, 131.6, 130.6, 130.5, 126.4, 123.6, 123.3, 119.8, 118.9, 116.7, 108.9, 65.3, 52.2. HRMS (ESI, m/z) calcd for C₂₃H₂₁N₅OS, [M + H]⁺: 416.1540; found: 416.1575.

N-(4-Morpholinophenyl)-4-(naphthalen-1-ylthio)pyrimidin-2-amine (3p)

White solid, 86% yield, m.p. 202.7–203.3 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.37 (s, 1H), 8.24 (s, 1H), 8.23 (s, 1H), 8.12–8.12 (m, 1H), 8.04 (d, J = 4.9 Hz, 1H), 7.98 (d, J = 6.9 Hz, 1H), 7.66 (t, J = 9 Hz, 1H), 7.63–7.61 (m, 2H), 7.10 (s, 2H), 6.60 (s, 2H), 6.19 (s, 1H), 3.73 (s, 4H), 2.97 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 171.0, 159.6, 157.6, 146.4, 136.8, 134.7, 136.6, 132.8, 132.0, 129.4, 128.3, 127.3, 126.8, 125.7, 125.4, 120.2, 115.8, 107.8, 66.6, 49.8. HRMS (ESI, m/z) calcd for C₂₄H₂₂N₄OS, [M + H]⁺: 415.1587; found: 415.1604.

4-((4-(Dimethylamino)phenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3v)

Yellow solid, 86% yield, m.p. 247.9–248.6 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.74 (s, 1H), 8.12 (d, J = 5.5 Hz, 1H), 7.51 (s, 2H), 7.39 (d, J = 8.7 Hz, 2H), 7.09 (s, 2H), 6.84 (d, J = 8.7 Hz, 2H), 6.29 (s, 1H), 3.88 (s, 4H), 3.21 (s, 4H), 3.01 (s, 6H). ¹³C NMR (151 MHz, DMSO-d₆) δ 173.8, 157.5, 155.2, 155.1, 150.6, 136.7, 119.2, 117.3, 117.0, 112.3, 111.2, 106.7, 64.6, 50.5, 28.9. HRMS (ESI, m/z) calcd for C₂₂H₂₅N₅OS, [M + H]⁺: 408.1853; found: 408.1859.

4-((4-Amino-3-fluorophenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3w)

Purple solid, 85% yield, m.p. 205.2–206.1 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.37 (s, 1H), 8.09 (d, J = 5.3 Hz, 1H), 7.35 (d, J = 7.4 Hz, 2H), 7.25 (dd, J = 11.2, 1.5 Hz, 1H), 7.12 (dd, J = 8.2, 1.6 Hz, 1H), 6.88 (t, J = 8.8 Hz, 1H), 6.77 (d, J = 8.7 Hz, 2H), 6.31 (s, 1H), 5.79 (s, 2H), 3.74–3.72 (m, 4H), 3.03–3.00 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 172.9, 159.6, 157.4, 150.6, 146.4, 139.4 (d, J = 12.7 Hz), 133.8, 133.1, 123.0 (d, J = 18.3 Hz), 120.2, 116.9, 115.9, 111.32 (d, J = 6.7 Hz), 107.3, 66.7, 49.7. HRMS (ESI, m/z) calcd for C₂₀H₂₀FN₅OS, [M + H]⁺: 398.1445; found: 398.1465.

4-((4-Amino-2-fluorophenyl)thio)-N-(4-morpholinophenyl)pyrimidin-2-amine (3x)

Yellow solid, 88% yield, m.p. 281.2–282.0 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.95 (s, 1H), 8.19 (d, J = 5.4 Hz, 1H), 7.52 (d, J = 8.0 Hz, 2H), 7.43 (s, 2H), 7.27 (s, 1H), 6.60 (s, 1H), 6.59 (s, 2H), 6.58 (s, 1H), 4.01 (s, 4H), 3.42 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 171.6, 163.9, 162.3, 156.4, 152.7, 138.0, 119.7, 119.0, 118.4, 110.8, 107.3, 100.4 (d, J = 25.3 Hz), 96.8, 63.7, 52.2. HRMS (ESI, m/z) calcd for C₂₀H₂₀FN₅OS, [M + H]⁺: 398.1445; found: 398.1470.

4.1.6. General procedure for the synthesis of compounds 8a–8g and 8i

K₂CO₃ (2.0 equiv.) was added to a solution of 2r (1.0 equiv.) and various amines (1.0 equiv.) in n-BuOH (5 mL). After degasification with nitrogen for 5 min, X-Phos (0.1 equiv.) and Pd₂(dba)₃ (0.05 equiv.) were added, and the mixture was degassed again for 5 min. The resulting suspension was heated at 85 °C for 8 h in a sealed flask under a nitrogen atmosphere. The precipitate was removed by filtration through a Celite pad, and then washed with dichloromethane/methanol (10 : 1, v/v). The crude product was purified by PLC to obtain 8a–8e, 8l, 9a, and 9b. Then 9a, 9b and 4 M HCl were mixed in ethyl acetate and stirred at room temperature for 2 h. The solvent was removed under reduced pressure to give 8f and 8j.

4-((4-Aminophenyl)thio)-N-phenylpyrimidin-2-amine (8a)

Grey solid, 92% yield, m.p. 111.0–111.9 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.59 (s, 1H), 8.13 (d, J = 5.2 Hz, 1H), 7.59 (d, J = 7.8 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 7.7 Hz, 2H), 6.90 (d, J = 7.1 Hz, 1H), 6.69 (d, J = 8.2 Hz, 2H), 6.27 (d, J = 4.5 Hz, 1H), 5.70 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 173.2, 158.4, 156.2, 150.3, 139.7, 136.7, 127.8, 120.6, 118.0, 114.2, 109.8, 106.8. HRMS (ESI, m/z) calcd for C₁₆H₁₄N₄S, [M + H]⁺: 295.1012; found: 295.1039.

4-((4-Aminophenyl)thio)-N-(4-thiomorpholinophenyl)pyrimidin-2-amine (8b)

Grey solid, 90% yield, m.p.205.9–206.8 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.34 (s, 1H), 8.06 (d, J = 5.1 Hz, 1H), 7.39 (d, J = 7.4 Hz, 2H), 7.22 (s, 2H), 6.79 (d, J = 8.4 Hz, 2H), 6.68 (d, J = 8.1 Hz, 2H), 6.21 (s, 1H), 5.70 (s, 2H), 3.39 (s, 4H), 2.68 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 174.0, 159.5, 157.2, 151.3, 146.3, 137.8, 133.2, 120.4, 117.6, 115.2, 111.0, 107.2, 52.4, 26.5. HRMS (ESI, m/z) calcd for C₂₀H₂₁N₅S₂, [M + H]⁺: 396.1335; found: 396.1335.

4-((4-Aminophenyl)thio)-N-(4-(morpholinomethyl)phenyl)pyrimidin-2-amine (8c)

White solid, 89% yield, m.p.179.6–180.2 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.57 (s, 1H), 8.11 (d, J = 5.2 Hz, 1H), 7.54 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 8.2 Hz, 2H), 7.12 (d, J = 8.0 Hz, 2H), 6.68 (d, J = 8.3 Hz, 2H), 6.21 (d, J = 4.7 Hz, 1H), 5.69 (s, 2H), 3.56 (s, 4H), 3.37 (s, 2H), 2.32 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 173.3, 158.4, 156.3, 150.3, 138.6, 136.6, 128.5, 117.9, 114.2, 113.0, 109.8, 106.6, 65.6, 61.5, 52.5. HRMS (ESI, m/z) calcd for C₂₁H₂₃N₅OS, [M + H]⁺: 394.1696; found: 394.1720.

4-((4-Aminophenyl)thio)-N-(3-morpholinophenyl)pyrimidin-2-amine (8d)

White solid, 89% yield, m.p. 216.0–216.8 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.44 (s, 1H), 8.11 (s, 1H), 7.30 (s, 1H), 7.23 (d, J = 7.7 Hz, 2H), 7.17 (d, J = 7.7 Hz, 1H), 7.06 (t, J = 7.8 Hz, 1H), 6.67 (d, J = 7.7 Hz, 2H), 6.53 (d, J = 7.6 Hz, 1H), 6.13 (d, J = 4.3 Hz, 1H), 5.69 (s, 2H), 3.74 (s, 4H), 3.05 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 174.6, 159.5, 157.4, 151.8, 151.4, 141.5, 137.6, 129.4, 115.3, 110.8, 110.7, 109.3, 107.5, 106.2, 66.6, 49.1. HRMS (ESI, m/z) calcd for C₂₀H₂₁N₅OS, [M + H]⁺: 380.1540; found: 380.1561.

4-((4-Aminophenyl)thio)-N-(3-(morpholinomethyl)phenyl)pyrimidin-2-amine (8e)

White solid, 86% yield, m.p. 129.3–130.0 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.57 (s, 1H), 8.12 (d, J = 5.3 Hz, 1H), 7.56 (s, 1H), 7.55 (s, 1H), 7.24 (d, J = 8.3 Hz, 2H), 7.15 (s, 1H), 6.86 (d, J = 7.3 Hz, 1H), 6.68 (d, J = 8.4 Hz, 2H), 6.18 (d, J = 5.2 Hz, 1H), 5.70 (s, 2H), 3.58 (s, 4H), 3.39 (s, 2H), 2.35 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 173.5, 158.4, 156.3, 150.3, 139.6, 137.3, 136.6, 127.6, 121.3, 118.6, 116.9, 114.2, 109.8, 106.6, 65.6, 62.0, 52.6. HRMS (ESI, m/z) calcd for C₂₁H₂₃N₅OS, [M + H]⁺: 394.1696; found: 394.1716.

4-((4-Aminophenyl)thio)-N-(4-(piperazin-1-yl)phenyl)pyrimidin-2-amine (8f)

White solid, 87% yield, m.p. 264.2–265.0 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 10.40 (s, 1H), 9.67 (s, 2H), 9.60 (s, 1H), 8.24 (d, J = 5.8 Hz, 1H), 7.67 (d, J = 8.3 Hz, 2H), 7.46 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 5.5 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H), 6.73 (s, 1H), 3.54 (s, 4H), 3.31 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 175.3, 172.5, 152.4, 143.8, 137.5, 133.6, 124.5, 123.4, 121.2, 118.1, 117.1, 108.4, 47.6, 42.5. HRMS (ESI, m/z) calcd for C₂₀H₂₂N₆S, [M + H]⁺: 379.1699; found: 379.1712.

4-((4-Aminophenyl)thio)-N-(4-(piperazin-1-ylmethyl)phenyl)pyrimidin-2-amine (8g)

White solid, 87% yield, m.p. 395.2–397.6 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 10.00 (s, 1H), 9.95 (1H), 8.25 (d, J = 5.4 Hz, 1H), 7.77 (d, J = 7.9 Hz, 1H), 7.68 (d, J = 7.1 Hz, 2H), 7.47 (s, 2H), 7.43 (s, 4H), 6.72 (s, 1H), 4.43 (s, 1H), 4.41 (s, 2H), 3.48 (s, 8H). ¹³C NMR (151 MHz, DMSO-d₆) δ 171.4, 169.7, 157.7, 155.6, 140.4, 136.8, 132.4, 131.4, 122.4, 120.7, 117.6, 108.2, 59.2, 57.2, 46.4. HRMS (ESI, m/z) calcd for C₂₁H₂₄N₆S, [M + H]⁺: 393.1856; found: 393.1873.

(4-((4-((4-Aminophenyl)thio)pyrimidin-2-yl)amino)phenyl)(morpholino)methanone (8i)

Yellow solid, 88% yield, m.p. 179.9–180.4 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.85 (s, 1H), 8.17 (d, J = 3.9 Hz, 1H), 7.64 (d, J = 6.7 Hz, 2H), 7.27 (d, J = 7.7 Hz, 2H), 7.24 (d, J = 7.4 Hz, 2H), 6.69 (d, J = 7.1 Hz, 2H), 6.36 (s, 1H), 5.74 (s, 2H), 3.60 (s, 4H), 3.50 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 170.9, 168.6, 158.2, 156.2, 150.3, 141.1, 136.7, 127.4, 126.9, 117.0, 114.2, 109.6, 107.4, 65.5. HRMS (ESI, m/z) calcd for C₂₁H₂₁N₅O₂S, [M + H]⁺: 408.1489; found: 408.1515.

4.1.7. General procedure for the synthesis of compounds 11a and 11b

Briefly, 10 (1 equiv.), various amines (1.2 equiv.) and K₂CO₃ (2 equiv.) were added to anhydrous DMSO sequentially. Then the mixture was heated to 80 °C for 4 h. The reaction mixture was monitored by TLC, and then cooled to room temperature. The reaction mixture was slowly poured into water and stirred to form a yellow solid, and then filtered. The filter cake was dried to give a yellow solid in 92–93% yield.

4.1.8. General procedure for the synthesis of compounds 12a and 12b

To a stirring solution of intermediates 11a and 11b (1.0 equiv.) in dry EtOH (2 mL), Pa/C was added (0.01 equiv.). After degasification with nitrogen for 10 min, the resulting suspension was heated at 80 °C. Then, NH₂–NH₂·H₂O was slowly added dropwise to the mixture at 80 °C for 2 h. The reaction mixture was monitored by TLC. The precipitate was removed by filtering through a pad of Celite, and then washed with dichloromethane/methanol (10 : 1, v/v) to give a colourless oil in 91–93% yield.

4.1.9. General procedure for the synthesis of compounds 8j and 8k

K₂CO₃ (2.0 equiv.) was added to a solution of 2r (1.0 equiv.) and the corresponding amines (1.0 equiv.) in n-BuOH (5 mL). After degasification with nitrogen for 5 min, X-Phos (0.1 equiv.) and Pd₂(dba)₃ (0.05 equiv.) were added, and the mixture was degassed again for 5 min. The resulting suspension was heated at 85 °C for 8 h in a sealed flask under a nitrogen atmosphere. The precipitate was removed by filtration through a Celite pad, and then washed with dichloromethane/methanol (10 : 1, v/v). The crude product was purified by PLC to obtain 13a and 13b. Then 13a and 13b and HCl were mixed in ethyl acetate and stirred at room temperature for 2 h. The solvent was removed under reduced pressure to give 8j and 8k in 88–92% yield.

N ¹-(4-((4-Aminophenyl)thio)pyrimidin-2-yl)-N⁴-(piperidin-4-yl)benzene-1,4-diamine (8j)

White solid, 90% yield, m.p. 251.5–252.1 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 10.09 (s, 1H), 9.41 (s, 1H), 8.93 (d, J = 9.8 Hz, 1H), 8.25 (d, J = 5.4 Hz, 1H), 7.69 (d, J = 7.4 Hz, 2H), 7.48 (d, J = 6.7 Hz, 4H), 7.37 (d, J = 8.8 Hz, 2H), 6.74 (s, 1H), 3.88 (s, 1H), 3.37 (d, J = 12.0 Hz, 2H), 2.95 (m, 2H), 2.11 (d, J = 11.7 Hz, 2H), 1.98 (d, J = 9.5 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 172.5, 172.5, 172.0, 158.5, 156.5, 140.5, 137.9, 127.9, 124.3, 123.6, 119.9, 109.4, 55.8, 41.9, 25.5. HRMS (ESI, m/z) calcd for C₂₁H₂₄N₆S, [M + H]⁺: 393.1856; found: 393.1882.

N ¹-(4-((4-Aminophenyl)thio)pyrimidin-2-yl)-N⁴-(pyrrolidin-3-yl)benzene-1,4-diamine (8k)

Yellow solid, 89% yield, m.p. 247.7–248.6 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 10.13 (s, 1H), 9.70 (s, 1H), 9.48 (s, 1H), 8.20 (s, 1H), 7.59 (s, 2H), 7.36–7.28 (m, 6H), 7.02 (s, 2H), 6.64 (s, 1H), 4.27 (s, 1H), 3.48 (s, 2H), 3.26 (s, 2H), 2.25–2.21 (m, 1H), 2.08 (s, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 174.9, 165.5, 156.7, 153.8, 137.7, 134.2, 130.1, 123.0, 122.8, 121.1, 119.7, 108.6, 67.6, 56.6, 47.9, 44.1. HRMS (ESI, m/z) calcd for C₂₀H₂₂N₆S, [M + H]⁺: 379.1699; found: 379.1716.

4.1.10. General procedure for the synthesis of compounds 15a and 15b

Briefly, 14a and 14b (1 equiv.), tetrahydro-2H-pyran-4-amine (1.2 equiv.) and K₂CO₃ (2 equiv.) were added to anhydrous DMSO sequentially. Then the mixture was heated to 80 °C for 4 h. The reaction mixture was monitored by TLC, and then cooled to room temperature. The reaction mixture was slowly poured into water and stirred, and then filtered. The filter cake was dried to obtain a yellow solid in 92–95% yield.

4.1.11. General procedure for the synthesis of compounds 16a and 16b

To a stirring solution of intermediates 15a and 15b (1.0 equiv.) in dry EtOH (2 mL), Pa/C was added (0.01 equiv.). After degasification with nitrogen for 10 min, the resulting suspension was heated at 80 °C. Then NH₂–NH₂·H₂O was slowly added dropwise to the mixture at 80 °C for 2 h. The reaction mixture was monitored by TLC. The precipitate was removed by filtering through a pad of Celite, and then washed with dichloromethane/methanol (10 : 1, v/v) to give a colourless oil in 91–93% yield.

4.1.12. General procedure for the synthesis of compounds 8h and 8l

K₂CO₃ (2.0 equiv.) was added to a solution of 2r (1.0 equiv.) and 16a and 16b (1.0 equiv.) in n-BuOH (5 mL). After degasification with nitrogen for 5 min, X-Phos (0.1 equiv.) and Pd₂(dba)₃ (0.05 equiv.) were added, and the mixture was degassed again for 5 min. The resulting suspension was heated at 85 °C for 8 h in a sealed flask under a nitrogen atmosphere. The precipitate was removed by filtering through a pad of Celite, and then washed with dichloromethane/methanol (10 : 1, v/v). The crude product was purified by PLC to obtain 8h and 8l in 91%–93% yield.

N ¹-(4-((4-Aminophenyl)thio)pyrimidin-2-yl)-N⁴-(tetrahydro-2H-pyran-4-yl)benzene-1,4-diamine (8h)

White solid, 90% yield, m.p. 192.9–193.3 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.12 (s, 1H), 8.01 (d, J = 5.3 Hz, 1H), 7.26 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 8.4 Hz, 2H), 6.51 (d, J = 8.4 Hz, 2H), 6.06 (s, 1H), 5.67 (s, 2H), 5.15 (s, 1H), 3.86 (d, J = 11.4 Hz, 2H), 3.40 (s, 2H), 1.87 (d, J = 12.0 Hz, 2H), 1.34 (d, J = 9.2 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 174.2, 159.7, 157.3, 151.3, 143.1, 137.7, 130.0, 121.4, 115.3, 113.2, 111.2, 106.5, 66.5, 48.9, 33.4. HRMS (ESI, m/z) calcd for C₂₁H₂₃N₅OS, [M + H]⁺: 394.1696; found: 394.1713.

Methyl 5-((4-((4-aminophenyl)thio)pyrimidin-2-yl)amino)-2-((tetrahydro-2H-pyran-4-yl)amino)benzoate (8l)

Grey solid, 88% yield, m.p. 197.5–198.5 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 9.30 (s, 1H), 8.04 (d, J = 5.3 Hz, 1H), 8.01 (s, 1H), 7.63 (d, J = 6.8 Hz, 1H), 7.42 (d, J = 7.5 Hz, 1H), 7.22 (d, J = 8.2 Hz, 2H), 6.76 (d, J = 9.1 Hz, 1H), 6.68 (d, J = 8.2 Hz, 2H), 6.11 (s, 1H), 5.69 (s, 2H), 3.85 (d, J = 11.5 Hz, 4H), 3.79 (s, 3H), 3.67 (s, 1H), 3.49 (t, J = 10.5 Hz, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 173.3, 167.6, 158.6, 156.3, 150.2, 144.6, 136.6, 127.6, 127.3, 121.2, 114.2, 111.6, 110.0, 108.0, 105.9, 65.1, 51.0, 46.6, 32.3. HRMS (ESI, m/z) calcd for C₂₃H₂₅N₅O₃S, [M/2 + H]⁺: 226.5839; found: 226.5920.

4.2. Pharmacological assays

4.2.1. PLK4 LanthaScreen Eu kinase binding assay

The reagents required for in vitro enzyme activity testing were purchased from Thermo Fisher Scientific (USA) including PLK4 recombinant human protein, kinase tracer 236 and LanthaScreen Eu-anti-GST antibody. The LanthaScreen Eu Kinase Binding assays were performed according to the manufacturer's instructions. Binding of the tracer to the kinase was detected using a europium-labeled anti-tag antibody, which binds to the kinase of interest. The high degree of FRET (fluorescence resonance energy transfer) from the europium (Eu) donor fluorophore to the Alexa Fluor® 647 receptor fluorophore on the kinase tracer is due to the simultaneous binding of the tracer and antibody with kinase. The binding of an inhibitor to kinase competes for binding with the tracer, resulting in the loss of FRET. In the first step, a stock solution of the compound to be tested with a concentration of 4 mM was diluted with 100% DMSO and 1 × kinase buffer A. Next, 1 nM kinase, 2 nM Eu-anti-GST antibody and 1 nM Kinase Tracer 236 were prepared by diluting with 1 × kinase buffer A. Then, the compound, kinase/antibody mixture and tracer were added into the analysis wells in turn according to the ratio of 4 μL, 8 μL and 4 μL, and incubated at 25 °C for 1 h. Then, the signal was obtained by reading the plate with an Infinite F500 microplate reader (Tecan, Switzerland). The “emission ratio” was calculated by dividing the receptor/tracer emission (665 nM) by the antibody/donor emission (615 nM). Finally, GraphPad Prism 8 was used to analyze the data. At least two parallel tests were performed.

4.2.2. Cell proliferation assay

The three cell lines (MCF-7, BT474, and MDA-MB-231) were cultured for 24 h in 96-well plates in 5% CO₂ incubators at 37 °C with a suitable density (2000–6000 cells/well). After incubation for 24 h, the cells were exposed to different concentrations of compounds or 0.1% DMSO for 5 days. The MTT was diluted to the final concentration of 5 mg mL⁻¹ with phosphate-buffered saline (PBS), and the freshly prepared MTT solution was added to each well, and the cells were cultured at 37 °C for 4 h. The culture solution was removing and the formazan crystals in each well were dissolved in 150 μL DMSO, and the absorbance of each test was measured at λ₅₇₀ nm and λ₆₃₀ nm using an Infinite® F500 microplate reader (Tecan, Switzerland). Finally, the IC₅₀ values were calculated by fitting the concentration-response curve using GraphPad prism 8.

4.2.3. Plasma stability assay

The pooled frozen plasma (BioreclamationIVT, batch No. BRH1569252) was thawed in a water bath at 37 °C prior to the experiment. Plasma was centrifuged at 4000 rpm for 5 min and the clots were removed, if any. Propantheline bromide solution (100 μM) solution was prepared by diluting 5 μL of 10 mM stock solution with 495 μL H₂O as a positive control. Use same method was used to prepare the other test compound solutions. To 98 μL of blank plasma, 2 μL of the dosing solution (100 μM) was added to bring the final concentration to 2 μM (double portion). All the reaction plates containing mixtures of compound and plasma were incubated at 37 °C in a water bath and the timer was started. At the end of incubation, 500 μL of stop solution (200 ng mL⁻¹ tolbutamide and 200 ng mL⁻¹ labetalol in ACN) was added to precipitate protein and mixed thoroughly. Each plate was sealed and shaken for 20 min. After shaking, each plate was centrifuged at 4000 rpm and 4 °C for 20 min. After centrifugation, the supernatant was sealed and shaken for 10 min. The final experimental data were obtained by LC–MS/MS analysis.

4.2.4. Liver microsomal stability assay

Empty T60 and NCF60 ‘Incubation’ plates were pre-warmed for 10 min. Liver microsomes were diluted to 0.56 mg mL⁻¹ in 100 mM phosphate buffer. Then, 445 μL microsome working solution (0.56 mg mL⁻¹) was transferred to the T60 and NCF60 pre-warmed ‘Incubation’ plates, and then they were pre-incubated for 10 min at 37 °C with constant shaking. Next, 54 μL liver microsomes was transferred to a blank plate, then 6 μL NAPDH cofactor was added, and then 180 μL quenching solution was added to the blank plate. Subsequently, 5 μL compound working solution (100 μM) was added to the ‘incubation’ plates (T60 and NCF60) containing microsomes and mixed 3 times thoroughly. For the NCF60 plate, 50 μL buffer was added and mixed 3 times thoroughly. Initially, the plate was incubated at 37 °C for 60 min while shaking. In the T0 ‘quenching’ plate, 180 μL quenching solution and 6 μL NAPDH cofactor were added. It is important to ensure that the plates were frozen to prevent evaporation. For the T60 plate, it was mixed 3 times thoroughly, and immediately 54 μL mixture was removed at the 0-min time point and added to the ‘quenching’ plate. Then 44 μL NAPDH cofactor was added to the incubation plate (T60). Initially, the plate was incubated at 37 °C for 60 min while shaking. At 5, 15, 30, 45, and 60 min, 180 μL quenching solution was added to the ‘quenching’ plates, mixed once, and 60 μL sample serially transferred from the T60 plate per time point to the ‘quenching’ plates. For NCF60, it was mixed once, and 60 μL sample transferred to the ‘quenching’ plate containing quenching solution at the 60 min time point. All sampling plates were shaken for 10 min, and then centrifuged at 4000 rpm for 20 min at 4 °C. Then 80 μL supernatant was transferred to 240 μL HPLC water and mixed on a plate shaker for 10 min. Each bioanalysis plate was sealed and shaken for 10 min prior to LC–MS/MS analysis.

4.2.5. Cytochrome P450 inhibition assay

Firstly, the test compounds and standard inhibitor working solutions were prepared (100×). Then, 20 μL of the substrate solutions was added to the corresponding wells. Also, 20 μL PB was added to the blank wells. 2 μL of the test compounds and positive control working solution were added to the corresponding wells. 2 μL solvent was added to the no-inhibitor wells, and 2 μL PB added to the blank wells. Then, the HLM working solution was prepared. 158 μL of the HLM working solution was added to all the wells in the incubation plate and pre-warmed for about 10 min at 37 °C in a water bath. Afterwards, the NADPH cofactor solution was prepared. 20 μL NADPH cofactor was added to all the incubation wells, mixed and incubated for 10 min at 37 °C in a water bath. At a certain time point, the reaction was terminated by adding 400 μL cold stop solution. The samples were centrifuged at 4000 rpm for 20 min to precipitate protein. Then 200 μL supernatant was diluted with 100 μL water and shaken for 10 min. Finally, the samples were ready for LC–MS/MS analysis.

4.3. Molecular docking

In this experiment, the molecular docking research was performed using the Schrödinger Maestro software. Firstly, the Protein Preparation Wizard function in the software was used to optimize the crystal structure of PLK4 (downloaded from the protein database: https://www.rcsb.org/), including adding hydrogen atoms and removing solvent water molecules, adjusting the bond order with Prime, and optimizing the hydrogen atoms in the crystal structure with the OPLS_2005 force field. Then the Receptor Grid Generation project in the Schrödinger suite was used to prepare the docking box. The ligand molecules in the eutectic structure were defined as binding sites, and the size of the docking grid box was 20 Å × 20 Å × 20 Å. Based on the OPLS_2005 force field, the lattice of the PLK4 crystal structure was generated. It should be noted that the Glu 90 and Cys92 amino acid residues were restricted to interact with the ligand molecules under the Constraints function. Then, the ligand molecules to be docked were optimized by the LigPrep and OPLS_2005 force fields. Finally, the Ligand Docking function was used to complete molecular docking.

Abbreviations

DCM

Dichloromethane

CHCl₃

Chloroform

DMSO

Dimethyl sulfoxide

EtOH

Ethanol

n-BuOH

1-Butanol

THP

Tetrahydropyran

DHP

3,4-Dihydro-2H-pyran

TEA

Triethylamine

Pd₂(dba)₃

Tris(dibenzylideneacetone)dipalladium

Conflicts of interest

The authors declare no conflicts of interest.