Design and synthesis of doublecortin-like kinase 1 inhibitors and their bioactivity evaluation

Abstract

Doublecortin-like kinase 1 (DCLK) is a microtubule-associated serine/threonine kinase that is upregulated in a wide range of cancers and is believed to be related to tumour growth and development. Upregulated DCLK1 has been used to identify patients at high risk of cancer progression and tumours with chemotherapy-resistance. Moreover, DCLK1 has been identified as a cancer stem cell (CSC) biomarker in various cancers, which has received considerable attention recently. Herein, a series of DCLK1 inhibitors were prepared based on the previously reported XMD8-92 structure. Among all the synthesised compounds, D1, D2, D6, D7, D8, D12, D14, and D15 showed higher DCLK1 inhibitory activities (IC₅₀ 40–74 nM) than XMD8-92 (IC₅₀ 161 nM). Compounds D1 and D2 were selective DCLK1 inhibitors as they showed a rather weak inhibitory effect on LRRK2. The antiproliferative activities of these compounds were also preliminarily evaluated. The structure-activity relationship revealed by our compounds provides useful guidance for the further development of DCLK1 inhibitors.

Keywords:

• Doublecortin-like kinase 1 (DCLK1)
• kinase inhibitor
• antitumor
• colorectal cancer (CRC)

Introduction

Doublecortin-like kinase 1 (DCLK1) was first identified in the brain as a microtubule-associated protein (MAP) and it was found to play a crucial role in early neurogenesis¹. Accumulating studies indicated that DCLK1 is related to tumour development. DCLK1 isoforms are overexpressed in many types of cancer cells, especially in colorectal cancer, which worsens the survival outcomes of colorectal cancer (CRC) patients^2,3. Epithelial-mesenchymal transition (EMT) plays a key role in cancer invasion and metastasis, especially in pancreatic cancer. It was shown that small-interfering RNA (siRNA)-mediated knockdown of DCLK-1 in human pancreatic cancer cells induced the downregulation of EMT-associated transcription factors⁴. Moreover, pharmacological inhibition of DCLK1 prevented the development of DCLK1⁺ pancreatic ductal adenocarcinoma (PDAC) in clinically relevant patient-derived PDAC organoid models⁵.

Additionally, DCLK1 is recognised as a regulator of type II immune response and linked with functional regulation of the tumour microenvironment. DCLK1 is strongly linked to the infiltration of multiple immune cell types, especially tumour-associated macrophages (TAMs) and regulatory T-cells (Treg). And DCLK1 might contribute to TAM-mediated inhibition of CD8⁺ T-cells to enhance tumour growth in the tumour microenvironment⁶. In addition, it was reported that the overexpression of miR-539 enhanced the sensitivity of cisplatin (DDP)-resistant NSCLC cells to DDP by directly targeting DCLK1⁷. Considering DCLK1 played key roles in tumour growth, EMT, tumour immunity, and drug resistance, targeting DCLK1 is a potential strategy for treating cancers.

Cancer stem cells (CSCs) exhibit unique self-renewal properties and are regarded as the key initiator in tumour formation, development, metastasis, and recurrence^8,9. Because CSCs have strong resistance to traditional drug treatment, new strategies targeting CSCs have been sought^10,11. DCLK1 is regarded as a marker of various cancer stem cells and it regulates pro-survival signalling and self-renewal of tumour cells^12–14 (Figure 1). In the adenomatous polyposis coli (APC) mouse model, the expression of DCLK1 is positively correlated with other stem cell markers, and the enteroids formed from the intestinal DCLK⁺ cells display higher pluripotency and pro-survival signalling. DCLK1 knockdown in APC^Min/+ mice reduced the ability of colon cancer cells to self-renew and survive¹⁴. Furthermore, DCLK1 marks a population of tumour stem-like cells in renal cell carcinoma (RCC). A novel monoclonal antibody targeting DCLK1 alternative splice variants-positive cells blocked RCC tumorigenesis in vivo and siRNA mediated knockdown of DCLK1 resulted in decreased expression of EMT and pluripotency factors in RCC^15,16. In addition, small extracellular vesicle (sEV/exosome) biogenesis and EMT are inhibited in gastric cancer (GC)¹⁷ by a specific small molecule inhibitor of DCLK1.

Figure 1. The role of DCLK1 in tumours.

The crystal structure of DCLK1 has been reported, which provides the necessary structure information for the design of DCLK1 inhibitors¹⁸. However, DCLK1 and the Leucine-rich repeat kinase 2 (LRRK2) share a similar inhibitor profile. And XMD8-92 and LRRK-IN-1, which are widely used DCLK1 inhibitors, do not have good selectivity^19,20. Therefore, we conducted chemical modifications of the structures of XMD8-92 and LRRK2-IN-1 (Figure 2), and various effective DCLK1 inhibitors with higher selectivity profiles were synthesised. Their structure–activity relationship was also investigated.

Figure 2. Crystal structure of DCLK1-KD in complex with DCLK1-IN-1 (PDB: 7kxw) and molecular design.

Results and discussion

The molecular design

DCLK1-IN-1 was an efficient DCLK1 selective inhibitor developed by Gray et al⁵. XMD8-92, LRRK-IN-1, and DCLK1-IN-1 share similar scaffold structures, namely the benzopyrimido diazepine moiety. As shown in Figure 2, we plan to introduce substitution groups on the A-ring, such as electron withdrawing or electron donating groups, to enhance the interactions with surrounding amino acid residues. On the B-ring, we plan to replace the R² and R³ with more bulky groups because there is still space in the kinase pocket adjacent to them. The C-ring has a strong interaction with VAL468, and we intend to keep it. G¹ and G² are located at the interface between protein and solvent, and we hope to introduce hydrophilic groups to improve the physicochemical properties of the resulting compounds and their selectivity to DCLK1.

Chemistry

The synthesis method for XMD8-92 analogues reported by Gray’s et al.^20,21 was adopted and modified in this study. As shown in Scheme 1, substituted anthranilates 1-1–1-10 were transformed into N-methylated products 2-1–2-10 in the presence of dimethyl carbonate and NaY molecular sieves²². Compounds 3-1–3-10 were synthesised through the nucleophilic substitution of 2,4-dichloro-5-nitropyrimidine by the secondary amine of compounds 2-1–2-10. The benzopyrimido diazepines 4-1–4-10 were achieved by reduction of the nitro group of compounds 3-1–3-10 followed by the spontaneous closure of the seven membered ring. Next, compounds 5-1–5-10 were obtained via methylation of compounds 4-1–4-10 by methyl iodide and NaH in anhydrous THF. Finally, A1–10 was obtained via Pd-catalysed Buchwald-Hartwig cross coupling reaction in low to moderate yields. As shown in Scheme 2, the synthesis of the B series is similar to that of the A series. The series C and D compounds were modified at the G¹ and G² positions (Figure 2).

Scheme 1. Synthesis of compounds A1–A10. Reagents and conditions: (a) NaY, 150 °C, dimethyl carbonate, 23–55%; (b) DIEA, 1,4-dioxane, 50 °C, 33–89%; (c) SnCl₂·2H₂O, EtOAc, 70 °C, 41–89%; (d) CH₃I, NaH, THF, 0 °C, 48–100%; (e) Pd(OAc)₂, XPhos, Cs₂CO₃, t-BuOH, 110 °C, 15–41%.

Scheme 2. Synthesis of compounds B1–B3. (a) DIEA, 1,4-dioxane, 50 °C, 70–85%; (c) SnCl₂·2H₂O, EtOAc, 70 °C, 43–95%; (d) R²I, NaH, THF, 0 °C, 57–100%; (e) Pd(OAc)₂, XPhos, Cs₂CO₃, t-BuOH, 110 °C, 15–41%.

The synthesis of series C compounds proceeded from the synthesis of G¹ and G² groups before carrying out coupling reaction with compound 13 (Scheme 3). Most of the compounds of series D were also prepared via similar procedure. Alternatively, compounds D1, D2, D5, D7, and D8 were obtained by coupling reaction of G¹/G² moiety, followed by further modification (Scheme 4). It is worth mentioning that compound D8 was synthesised with a longer linker, which could be used as a probe for DCLK1. Furthermore, some compounds were introduced with hydrophilic groups, such as compounds D5 and D6 with hydrophilic sulfoxide or sulphone groups, respectively, and compound D7 with a fructose group via the Maillard reaction²³. In addition, compounds with lipophilic G² groups were also synthesised, such as D10–D14. In total, more than 30 compounds were synthesised to evaluate their DCLK1 inhibitory activity.

Scheme 3. Synthesis of compounds C1, C2. (a) K₂CO₃, DMF, 50 °C, 80–96%; (b) SnCl₂·2H₂O, EtOAc, 70 °C, 52–78%; (c) Pd(OAc)₂, XPhos, Cs₂CO₃, t-BuOH, 110 °C, 35–46%.

Scheme 4. Synthesis of compounds D1–D16. (a) Pd(OAc)₂, XPhos, Cs₂CO₃, t-BuOH, 110 °C, 30–48%; (b)H₂, Pd/C, r.t., 100%; (c) Et₃N, CH₂Cl₂, r.t.; (d) LiOH (2 M), THF, 90% over two steps; (e) CH₃COSK, DMF, 100 °C, 38%; (f) K₂CO₃, DMF, 90 °C, 91%; (g) H₂O₂, AcOH, r.t., 95%; (h) TFA, CH₂Cl₂, r.t., 100%; (i) AcOH, EtOH, reflux, 82%.

In vitro evaluation of DCLK1 inhibitory activity

All compounds were tested for their kinase inhibitory activity at the concentration of 1 μM. As shown in Table 1, the kinase activity of DCLK1 and LRRK2 after drug treatment was presented as a percentage, with 100% indicating no inhibitory activity and 0% indicating complete inhibition. Compounds D1, D2, D5-D8, D11, D12, D14, and D15 exhibited higher DCLK1 kinase inhibitory activity than XMD8-92. In addition, at the same concentration, compounds A6, A9, B3, D1, D2, and D7 exhibited higher selectivity towards DCLK1 comparing with LRRK2. Compounds with high activity or good selectivity were selected for further IC₅₀ determination (Table 2). The IC₅₀ values of XMD8-92 for DCLK1 and LRRK2 kinases are similar, which indicates poor selectivity. Most compounds exhibited good DCLK1 inhibitory activity, and nine compounds exhibited higher DCLK1 inhibitory activity than XDM8-92. It is worth noting that compounds D1, D2, and D8 had significantly increased DCLK1 kinase selectivity with enhanced kinase inhibitory activity.

Table 1. DCLK1 and LRRK2 inhibitory activities of the synthesised compounds at the concentration of 1 uM.

Entry	DCLK1 activity (%)	LRRK2 activity (%)	Selectivity (DCLK1 activity/LRRK2 activity)
XMD8-92	14	18	0.78
A1	25	8	3.13
A2	103	100	1.03
A3	85	88	0.97
A4	89	83	1.07
A5	87	89	0.98
A6	19	51	0.37
A7	109	109	1.00
A8	102	93	1.10
A9	17	50	0.34
A10	96	98	0.98
B1	102	76	1.34
B2	59	72	0.82
B3	28	70	0.40
C1	56	99	0.57
C2	15	7	2.14
D1	7	56	0.13
D2	10	49	0.20
D3	20	34	0.59
D4	18	33	0.55
D5	12	21	0.57
D6	9	19	0.47
D7	6	21	0.29
D8	8	22	0.36
D9	26	51	0.51
D10	51	81	0.63
D11	11	18	0.61
D12	5	10	0.50
D13	59	72	0.82
D14	7	16	0.44
D15	4	9	0.44
D16	15	33	0.45

Table 2. DCLK1 and LRRK2 inhibitory IC₅₀ values of the synthesised compounds.

Entry	IC50 of DCLK1 (nM)	IC50 of LRRK2 (nM)	Selectivity (IC50 of DCLK1/IC50 of LRRK2)
XMD8-92	161	219	0.74
A6	365	>1000	<0.37
A9	338	>1000	<0.34
B3	324	>1000	<0.32
D1	47	>1000	<0.05
D2	70	>1000	<0.07
D3	340	>1000	<0.34
D4	271	425	0.64
D5	146	320	0.46
D6	74	195	0.38
D7	46	154	0.30
D8	40	228	0.18
D11	100	266	0.38
D12	47	99	0.47
D14	53	132	0.40
D15	52	89	0.58

Structure–activity relationships

We analysed the structure–activity relationship based on the kinase inhibitory data of the synthesised compounds (Figure 3). Firstly, any substituents on the A-ring led to decreased or lost activity, even the smallest F atom. Secondly, when the R² substituent was ethyl, the DCLK1 selectivity of compound B3 was improved, indicating that this position may accommodate larger groups, which was also mentioned in Gray’s reports^5,24. The DCLK1 inhibitory activity of compound B2 decreased, indicating that the R³ cannot be H atom. Compared with compound C2 with a pyridine ring structure, the DCLK1 inhibitory activity of compound C1 with an aliphatic ring was weakened, indicating the necessity of G¹ being an aromatic ring. The introduction of carboxyl groups at G2 increased the DCLK1 selectivity for compounds D1 and D2. The introduction of difluorinated compound D14 also has higher DCLK1 selectivity than monofluorinated compounds D10 and D13. Therefore, we believe that the G2 group is more important for the selectivity of DCLK1. In addition, compared with D10–D14 with hydrophobic groups, D5–D7 carrying hydrophilic G2 groups exhibited little effects on the activity and selectivity of DCLK1. Interestingly, when the G2 moiety was glucose (D7) or long-chain group (D8), enhanced DCLK1 inhibitory activity and selectivity were observed, indicating the potential in developing more potent and selective DCLK1 inhibitors with this class of compounds.

Figure 3. Structure–activity relationship analysis of DCLK1 inhibitors.

Anti-proliferation activity assay

To determine whether DCLK1 was required for tumour cell proliferation, the cytotoxicity of compounds was evaluated in colorectal cancer cell line HCT116. HCT116 was treated with compounds for 72 h, and the cell viability was determined using CCK8 assay (Figure 4). The antiproliferative effects of most compounds on HCT116 were correlated with their DCLK1 inhibitory activities, except for compounds D1 and D2. The poor cytotoxicity of compounds D1 and D2 may be attributed to their poor cell membrane permeability due to the negatively charged carboxylates. Compounds D5–8, D11–12, and D14–15 showed higher anti-proliferate activity than the positive control XMD8-92. Overall, targeting DCLK1 inhibited the proliferation of HCT116, indicating the importance of DCLK1 in tumour growth.

Figure 4. The antiproliferative effects of most compounds on HCT116.

Molecular docking study

Due to the excellent kinase inhibitory activity exhibited by compounds D1 and D8, the molecular docking study was performed to predict the possible binding mode with DCLK1 subunit¹⁸ (PDB ID: 5JZN). Through docking software, compounds D1 and D8 were successfully localised in the binding pocket of DCLK1 subunit. Similar to the crystal structures of DCLK1-KD in complex with reported inhibitors²⁵, the benzodiazepine scaffolds are located on the inner side of the hydrophobic pocket. As shown in Figure 5, compound D1 exhibited three hydrogen bonds. The carbonyl group on the diazepine ring formed a hydrogen bond with LYS419. Notably, two hydrogen bonds were formed by the carboxyl group with residues LYS469 and ARG394. In addition, the carboxyl group formed a salt bridge with LYS469 which enhanced the interaction between D1 and DCLK1. The docking results of compounds D8 and D1 were similar. Compound D8 formed two hydrogen bonds with residues LYS469 and ARG394. The hydrophobic interaction of benzodiazepine rings matched the surrounding hydrophobic amino acids. And the benzene ring had a p–π conjugation effect with LYS419. These interactions maintained the activity of compound D8 against DCLK1 even with a long “tail”. Compared to the crystal structure of DCLK1-IN-1 in Figure 2, compounds D1 and D8 gave up hydrogen bonding with residue VAL468 and chose to form more hydrogen bonds with residues LYS469 and ARG394, which may be more advantageous in enhancing the binding force and thus enhancing the inhibitory activity against DCLK1. Interestingly, compound D8 with a long-chain group replacing the carboxylic group did not undermine the binding to DCLK1, which also suggests that compound D8 may be suitable for developing a probe targeting DCLK1, such as proteolysis-targeting chimaeras (PROTACs)²⁶. The docking results somewhat explain the elevation of DCLK1 kinase activity by compounds D1 and D8 as well as validate the discussion in the structure–activity relationship analysis.

Figure 5. Docking of compounds D1 and D8 to the residues of DCLK1 subunit (PDB ID: 5JZN).

Conclusion

DCLK1 is an important marker of cancer stem cell and an important anti-tumour target. In this study, we synthesised a series of derivatives of XMD8-92 which is widely used as a non-selective DCLK1 inhibitor. We found that compounds D1, D2, D5, D6, D7, D8, D12, D14, and D15 exhibited higher activity than XMD8-92, among which compounds D1, D2, and D8 exhibited good DCLK1 selectivity. The cytotoxicity assay demonstrated that this series of compounds are suitable leads for developing new anti-tumour drugs.

Experimental section

Chemistry

All reagents and solvents were obtained from commercial suppliers (Bide Pharmatech, Energy Chemical, Alfa, etc.) without further purification unless noted. Reactions were monitored by TLC. Thin layer chromatography was carried out using TLC silica gel 60 F254 plates. Flash column chromatography was performed with 200–300 mesh silica gel. The NMR spectrum was recorded on a Bruker-400 NMR spectrometer, with TMS as an internal standard and chemical shifts reported in ppm (δ). Coupling constants (J) are reported in Hz. The melting point was measured by an X-5 micro melting point metre. High-resolution mass spectra (HRMS) were obtained on a Shimadzu LCMS-IT-TOF mass spectrometer.

Preparation of intermediate 2-1–2-10

A sealed tube (100 ml of internal volume) was charged with a solution of compound 1-1–1-10 (0.12 M) and NaY (1:1 w/w) in dimethyl carbonate (30 ml). Air in the reaction mixture was replaced by N₂ at room temperature. The sealed tube was then heated at 150 °C for 15 h. After the reaction was completed, the sealed tube was cooled to room temperature and opened slowly. The NaY was filtered out and the filtrate was concentrated in vacuo. Purification by silica gel column chromatography (petroleum ether/EtOAc, 20:1 v/v) gave intermediate 2-1–2-10 (23–55%).

Methyl 4-methyl-2-(methylamino)benzoate (2–4)

White solid; yield 51%; m.p. 49–50 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.80 (d, J = 8.1 Hz, 1H), 7.62 (s, 1H), 6.49 (s, 1H), 6.44 (d, J = 8.3 Hz, 1H), 3.86 (s, 3H), 2.93 (d, J = 5.1 Hz, 3H), 2.35 (s, 3H).¹³C NMR (101 MHz, CDCl₃) δ 169.06, 152.04, 145.41, 131.49, 115.76, 110.95, 107.45, 51.28, 29.54, 22.19.

Preparation of intermediate 3-1–3-10 or 7-1–7-3

2 or 6 (1 eq), 2,4-dichloro-5-nitropyrimidine (1.5 eq), and DIEA (1.5 eq) were dissolved in 1,4-dioxane and heated at 50 °C for 6–10 h. The solvent was concentrated, and the residue was diluted with water (50 ml) and extracted with DCM (50 ml) × 3. The organics were combined and washed with brine (20 ml), dried over Na₂SO₄, filtered, and concentrated in vacuo. Purification by silica gel column chromatography (eluant: petroleum ether/EtOAc, 10:1 v/v) gave intermediate 3-1–3-10 or 7-1–7-3 (33–89%).

Methyl 3-chloro-2-((2-chloro-5-nitropyrimidin-4-yl)amino)benzoate (3-1)

Yellow solid; yield 33%; m.p. 115–116 °C; ¹H NMR (400 MHz, DMSO-d6) δ 10.71 (s, 1H), 9.24 (s, 1H), 7.91 (t, J = 8.2 Hz, 2H), 7.56 (t, J = 8.0 Hz, 1H), 3.73 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 165.27, 162.65, 158.52, 154.90, 134.15, 134.04, 133.60, 130.23, 129.88, 129.58, 127.83, 53.00. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₂H₈Cl₂N₄O₄: 342.9995, found: 342.9993.

Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-3-methylbenzoate (3-2)

Yellow solid; yield 62%; m.p. 167–168 °C; ¹H NMR (400 MHz, DMSO-d6) δ 10.57 (s, 1H), 9.19 (s, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 3.70 (s, 3H), 2.24 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 166.32, 162.67, 158.19, 154.98, 137.50, 135.15, 135.07, 128.60, 128.02, 127.99, 52.63, 18.45. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₁ClN₄O₄: 323.0542, found: 323.0543.

Methyl 4-chloro-2-((2-chloro-5-nitropyrimidin-4-yl)(methyl)amino)benzoate (3-3)

White solid; yield 51%; m.p. 135–136 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.55 (s, 1H), 8.00 (d, J = 8.5 Hz, 1H), 7.43 (dd, J = 8.5, 2.1 Hz, 1H), 7.24 (d, J = 2.1 Hz, 1H), 3.81 (s, 3H), 3.57 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.99, 161.00, 155.55, 154.64, 144.11, 140.10, 133.67, 131.71, 129.11, 127.99, 52.68, 41.97. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₀Cl₂N₄O₄: 357.0152, found: 357.0151.

Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)(methyl)amino)-4-methylbenzoate (3-4)

Yellow solid; yield 85%; m.p. 143–144 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.45 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 8.1 Hz, 1H), 6.98 (s, 1H), 3.81 (s, 3H), 3.57 (s, 3H), 2.40 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 164.69, 160.80, 155.06, 154.54, 145.54, 142.95, 132.70, 131.94, 129.60, 127.78, 122.77, 52.35, 41.91, 21.39. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₄H₁₃ClN₄O₄: 337.0698, found: 337.0696.

Methyl 5-chloro-2-((2-chloro-5-nitropyrimidin-4-yl)(methyl)amino)benzoate (3-5)

Brown solid; yield 73%; m.p. 198–199 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.49 (s, 1H), 7.99 (d, J = 2.5 Hz, 1H), 7.54 (dd, J = 8.5, 2.5 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 3.80 (s, 3H), 3.52 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.60, 160.89, 155.49, 154.63, 141.47, 134.59, 134.23, 132.51, 131.69, 129.07, 127.36, 52.81, 42.02. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₀Cl₂N₄O₄: 357.0152, found: 357.0161.

Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)(methyl)amino)-5-fluorobenzoate (3-6)

White solid; yield 72%; m.p. 118–119 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.48 (s, 1H), 7.75 (dd, J = 8.7, 2.9 Hz, 1H), 7.36–7.29 (m, 1H), 7.23 (dd, J = 8.8, 4.9 Hz, 1H), 3.84 (d, J = 1.0 Hz, 3H), 3.56 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.60, 162.68, 160.86, 160.18, 155.28, 154.76, 138.96, 129.90, 129.82, 121.44, 119.61, 52.82, 42.10. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₀ClFN₄O₄: 341.0447, found: 341.0446.

Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-5-(trifluoromethyl)benzoate (3-7)

Yellow solid; yield 46%; m.p. = 149–150 °C; ¹H NMR (400 MHz, CDCl₃) δ 12.89 (s, 1H), 9.28 (s, 1H), 8.82 (d, J = 8.8 Hz, 1H), 8.41 (d, J = 2.3 Hz, 1H), 7.91 (dd, J = 8.9, 2.3 Hz, 1H), 4.07 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 166.49, 163.63, 157.80, 153.13, 141.08, 130.45, 128.64, 127.94, 126.87, 124.68, 123.51, 119.06, 53.21. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₈ClF₃N₄O₄: 377.0259, found: 377.0258.

Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)(methyl)amino)-5-methylbenzoate (3-8)

White solid; yield 89%; m.p. 96–97 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.46 (s, 1H), 7.86 (s, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.09 (d, J = 8.1 Hz, 1H), 3.82 (s, 3H), 3.57 (s, 3H), 2.42 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 164.90, 160.79, 155.11, 154.73, 140.45, 139.16, 134.83, 133.07, 131.90, 127.32, 125.55, 52.44, 42.03, 21.07. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₄H₁₃ClN₄O₄: 337.0698, found: 337.0695.

Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)(methyl)amino)-6-fluorobenzoate (3-9)

Yellow solid; yield 73%; m.p. 91–92 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.60 (s, 1H), 7.52 (q, J = 7.8 Hz, 1H), 7.17 (t, J = 8.9 Hz, 1H), 7.03 (d, J = 8.0 Hz, 1H), 3.81 (s, 3H), 3.56 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.21, 162.26, 161.07, 159.70, 155.82, 154.72, 143.11, 133.48, 131.71, 122.47, 116.95, 52.89, 42.31. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₀ClFN₄O₄: 341.0447, found: 341.0445.

Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)(methyl)amino)-6-methylbenzoate (3-10)

Yellow solid; yield 85%; m.p. 122–123 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.55 (s, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.24 (d, J = 7.7 Hz, 1H), 7.08 (d, J = 7.9 Hz, 1H), 3.74 (s, 3H), 3.55 (s, 3H), 2.37 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.13, 160.79, 155.50, 154.74, 141.01, 138.72, 131.97, 131.50, 130.87, 129.77, 124.26, 52.30, 42.25, 20.17. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₄H₁₃ClN₄O₄: 337.0698, found: 337.0695.

Preparation of intermediate 4-1–4-10 or 8-1–8-3

3-1–3-10 or 7-1–7-10 (1 eq) and SnCl₂·2H₂O (2 eq) in EtOAc (10 ml) was heated at 70 °C for 5–8 h. The solvent was concentrated, and the residue diluted with water (50 ml) and extracted with EtOAc (50 ml) × 3. The organics were combined and washed with brine (20 ml), dried over Na₂SO₄, filtered, and concentrated in vacuo to give the white or yellow powder, washed with MeOH and air dried to give intermediate 4-1–4-10 or 8-1–8-3 (43–95%).

2,10-Dichloro-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (4-1)

White solid; yield 52%; m.p. 246–247 °C; ¹H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.24 (s, 1H), 8.12 (s, 1H), 7.78 (dd, J = 8.0, 1.6 Hz, 1H), 7.68 (dd, J = 7.9, 1.6 Hz, 1H), 7.09 (t, J = 7.9 Hz, 1H). ¹³C NMR (101 MHz, DMSO) δ 165.54, 157.00, 153.20, 149.80, 139.13, 134.75, 132.36, 124.51, 124.12, 123.16, 122.61. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₁H₆Cl₂N₄O: 280.9991, found: 280.9992.

2-Chloro-10-methyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (4-2)

White solid; yield 58%; m.p. 210–211 °C; ¹H NMR (400 MHz, DMSO-d6) δ 10.23 (s, 1H), 8.30 (s, 1H), 8.06 (s, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 7.3 Hz, 1H), 6.98 (t, J = 7.6 Hz, 1H), 2.34 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 167.15, 158.41, 153.17, 149.23, 141.40, 136.08, 130.99, 128.54, 123.70, 123.29, 122.77, 18.45. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₂H₉ClN₄O: 261.0532, found: 261.0537.

2,9-Dichloro-11-methyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (4-3)

White solid; yield 71%; m.p. 241–242 °C; ¹H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.18 (s, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.34 (s, 1H), 7.28 (d, J = 8.4 Hz, 1H), 3.35 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 166.66, 160.88, 153.48, 150.13, 148.97, 138.89, 133.65, 124.62, 124.56, 124.31, 120.13, 37.35. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₂H₈Cl₂N₄O: 295.0148, found: 295.0150.

2-Chloro-9,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (4-4)

White solid; yield 82%; m.p. 238–239 °C; ¹H NMR (400 MHz, DMSO-d6) δ 10.36 (s, 1H), 8.13 (s, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.09 (s, 1H), 7.02 (d, J = 8.0 Hz, 1H), 3.33 (s, 3H), 2.34 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 167.52, 161.41, 153.35, 149.61, 147.69, 144.76, 131.96, 125.27, 124.42, 123.01, 120.46, 37.25, 21.51. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₁ClN₄O: 275.0694, found: 275.0692.

2,8-Dichloro-11-methyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (4-5)

White solid; yield 68%; m.p. 230–231 °C; ¹H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.16 (s, 1H), 7.66 (s, 1H), 7.63 (s, 1H), 7.30 (s, 1H), 3.32 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 166.27, 161.11, 153.57, 150.12, 146.76, 133.70, 131.10, 128.58, 127.49, 124.16, 122.28, 37.35. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₂H₈Cl₂N₄O: 295.0148, found: 295.0148.

2-Chloro-8-fluoro-11-methyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (4-6)

White solid; yield 70%; m.p. 216–217 °C; ¹H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.16 (s, 1H), 7.50–7.43 (m, 2H), 7.33 (dd, J = 9.9, 4.6 Hz, 1H), 2.09 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 166.32, 161.40, 159.74, 153.58, 149.93, 144.15, 127.71, 124.19, 122.41, 121.02, 117.88, 37.42. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₂H₈ClFN₄O: 279.0443, found: 279.0442.

2-Chloro-8-(trifluoromethyl)-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (4-7)

Yellow solid; yield 41%; m.p. = 241–242 °C; ¹H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 1H), 10.25 (s, 1H), 8.09 (d, J = 2.4 Hz, 1H), 7.98 (s, 1H), 7.73 (dd, J = 8.7, 2.4 Hz, 1H), 7.26 (d, J = 8.5 Hz, 1H). ¹³C NMR (101 MHz, DMSO) δ 164.36, 155.50, 153.01, 148.59, 146.06, 131.27, 130.53, 125.68, 122.64, 121.53, 121.06, 120.18. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₂H₆ClF₃N₄O: 315.0225, found: 315.0225.

2-Chloro-8,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (4-8)

White solid; yield 89%; m.p. 218–219 °C; ¹H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.12 (s, 1H), 7.53 (s, 1H), 7.39 (d, J = 8.3 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 3.33 (s, 3H), 2.28 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 167.64, 161.56, 153.39, 149.56, 145.42, 134.71, 133.87, 132.02, 125.61, 124.30, 120.03, 37.18, 20.33. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₁ClN₄O: 257.0694, found: 257.0693.

2-Chloro-7-fluoro-11-methyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (4-9)

White solid; yield 43%; m.p. 215–216 °C; ¹H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 8.20 (s, 1H), 7.55 (q, J = 7.8, 7.4 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 7.08 (t, J = 9.4 Hz, 1H), 3.33 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 163.77, 162.34, 153.59, 150.77, 150.24, 133.89, 133.78, 124.31, 115.99, 113.10, 112.88, 37.13. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₂H₈ClFN₄O: 279.0443, found: 279.0444.

2-Chloro-7,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (4-10)

Yellow solid; yield 49%; m.p. 145–146 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.63 (s, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.21 (d, J = 7.6 Hz, 1H), 6.93 (d, J = 7.8 Hz, 1H), 3.89 (s, 3H), 2.38 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.91, 165.01, 154.59, 151.69, 149.62, 140.86, 130.81, 128.96, 127.81, 126.28, 115.68, 36.00, 21.08, 0.01. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₁ClN₄O: 275.0694, found: 275.0699.

Preparation of intermediate 5-1–5-10 or 9-1–9-3

4-1–4-10 or 8-1–8-3 (1 eq), MeI (6 eq) and NaH (2 eq, 60% suspension in mineral oil) was added in THF (10 ml) at −5 °C. After the reaction was complete as monitored by TLC, the solvent was diluted with water (30 ml) and extracted with EtOAc (30 ml) × 3. The organics were combined, dried over Na₂SO₄, filtered, and concentrated in vacuo. Purification by silica gel column chromatography (eluant: petroleum ether/EtOAc, 4:1 v/v) gave intermediate 5-1–5-10 or 9-1–9-3 (48–100%).

2,10-Dichloro-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (5-1)

Yellow solid; yield 48%; m.p. 178–179 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.94 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.13 (s, 1H), 6.99 (t, J = 8.2 Hz, 1H), 3.86 (s, 3H), 3.38 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 166.93, 151.53, 145.55, 142.63, 136.51, 134.35, 132.95, 132.03, 128.90, 127.22, 124.22, 39.63, 36.79. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₀Cl₂N₄O: 309.0304, found: 309.0303.

2-Chloro-5,10,11-trimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (5-2)

Yellow solid; yield 68%; m.p. 188–189 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.88 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 7.4 Hz, 1H), 7.04 (s, 1H), 6.99 (t, J = 7.6 Hz, 1H), 3.74 (s, 3H), 3.38 (s, 3H), 2.34 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.07, 149.81, 145.19, 143.70, 136.49, 135.37, 134.93, 131.26, 128.80, 125.75, 124.16, 39.56, 36.36, 19.77, 0.03. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₄H₁₃ClN₄O: 289.0851, found: 289.0847.

2,9-Dichloro-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (5-3)

White solid; yield 100%; m.p. 165–166 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.28 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.17 (dd, J = 8.4, 1.9 Hz, 1H), 7.11 (d, J = 1.9 Hz, 1H), 3.52 (s, 3H), 3.43 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 166.74, 163.33, 154.90, 151.60, 149.27, 139.30, 133.89, 128.32, 124.62, 124.13, 118.35, 38.29, 36.35. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₀Cl₂N₄O: 309.0304, found: 309.0302.

2-Chloro-5,9,11-trimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (5-4)

White solid; yield 100%; m.p. 162–163 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.23 (s, 1H), 7.76 (d, J = 7.9 Hz, 1H), 7.00 (d, J = 8.0 Hz, 0H), 6.90 (s, 1H), 3.51 (s, 3H), 3.43 (s, 3H), 2.39 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.69, 163.94, 154.60, 151.17, 148.26, 143.94, 132.57, 128.55, 125.25, 123.00, 118.47, 38.27, 36.26, 21.67. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₄H₁₃ClN₄O: 289.0851, found: 289.0849.

2,8-Dichloro-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (5-5)

White solid; yield 96%; m.p. 179–180 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.27 (s, 1H), 7.82 (d, J = 2.6 Hz, 1H), 7.42 (dd, J = 8.9, 2.6 Hz, 1H), 7.05 (d, J = 8.8 Hz, 1H), 3.52 (s, 3H), 3.41 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 166.37, 163.54, 154.88, 151.62, 146.88, 132.81, 132.18, 129.96, 128.16, 127.17, 119.40, 38.47, 36.36. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₀Cl₂N₄O: 309.0304, found: 309.0305.

2-Chloro-8-fluoro-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (5-6)

White solid; yield 92%; m.p. 168–169 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.25 (s, 1H), 7.53 (dd, J = 8.7, 3.1 Hz, 1H), 7.17 (m, 1H), 7.07 (dd, J = 9.1, 4.4 Hz, 1H), 3.51 (s, 3H), 3.39 (s, 3H).¹³C NMR (101 MHz, CDCl₃) δ 166.41, 163.84, 160.19, 157.76, 154.84, 151.53, 144.44, 128.23, 127.48, 120.03, 118.86, 38.50, 36.44. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₀ClFN₄O: 293.0600, found: 293.0596.

2-Chloro-5,11-dimethyl-8-(trifluoromethyl)-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(5-7)

White solid; yield 90%; m.p. = 167–168 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.31 (s, 1H), 8.15 (d, J = 2.3 Hz, 1H), 7.72 (dd, J = 8.7, 2.3 Hz, 1H), 7.23 (d, J = 8.7 Hz, 1H), 3.55 (s, 3H), 3.48 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 166.39, 163.10, 155.01, 151.87, 151.14, 130.25, 129.71, 128.12, 126.80, 126.08, 124.78, 118.47, 38.36, 36.47. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₄H₁₀ClF₃N₄O: 343.0568, found: 343.0565.

2-Chloro-5,8,11-trimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (5-8)

White solid; yield 100%; m.p. 173–174 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.22 (s, 1H), 7.65 (d, J = 2.2 Hz, 1H), 7.27 (dd, J = 8.3, 2.8 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 3.51 (s, 3H), 3.40 (s, 3H), 2.33 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.85, 164.09, 154.62, 151.20, 145.98, 134.15, 133.72, 132.65, 128.42, 125.54, 117.87, 38.45, 36.17, 20.38. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₄H₁₃ClN₄O: 289.0851, found: 289.0848.

2-Chloro-7-fluoro-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (5-9)

White solid; yield 97%; m.p. 172–173 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.33 (s, 1H), 7.40 (m, 1H), 6.98–6.88 (m, 2H), 3.53 (s, 3H), 3.41 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 164.28, 163.50, 160.78, 154.81, 152.27, 150.56, 132.60, 128.49, 115.63, 113.67, 112.81, 37.96, 36.32. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₀ClFN₄O: 293.0600, found: 293.0597.

2-Chloro-5,7,11-trimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (5-10)

White solid; yield 81%; m.p. 132–133 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.29 (s, 1H), 7.29 (t, J = 8.1 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 3.53 (s, 3H), 3.39 (s, 3H), 2.45 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.91, 165.01, 154.59, 151.69, 149.62, 140.86, 130.81, 128.96, 127.81, 126.28, 115.68, 38.29, 36.00, 21.08. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₄H₁₃ClN₄O: 289.0851, found: 289.0848.

General procedure for synthesis of compound A1–A10 and B1–B3

A sealed tube with a mixture of 5-1–5-10 or 9-1–9-3 (1 eq), 1–(4-amino-3-ethoxyphenyl)piperidin-4-ol (1 eq), Pd(OAc)₂ (0.05 eq), XPhos (0.1 eq), and Cs₂CO₃ (2 eq) in t-BuOH (5.0 ml) was heated at 110 °C for 8–16 h. The reaction was then filtered through celite and eluted with dichloromethane. The dichloromethane was removed in vacuo and the resulting crude product was purified by silica gel column chromatography (eluant: DCM/MeOH, 40:1 v/v) to afford the target product (15–41%).

10-Chloro-2-((2-ethoxy-4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(A1)

Yellow solid; yield 21%; m.p. 130–131 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.32 (d, J = 8.6 Hz, 1H), 8.15 (s, 1H), 7.68 (dd, J = 7.7, 1.6 Hz, 1H), 7.52 (s, 1H), 7.45 (dd, J = 8.0, 1.7 Hz, 1H), 7.15 (t, J = 7.8 Hz, 1H), 6.65–6.56 (m, 2H), 4.13 (q, J = 6.9 Hz, 2H), 3.90–3.83 (m, 1H), 3.65 (s, 3H), 3.54–3.47 (m, 5H), 2.95–2.87 (m, 2H), 2.10–2.00 (m, 2H), 1.81–1.72 (m, 2H), 1.49 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.62, 163.36, 156.20, 151.67, 148.04, 147.11, 144.56, 134.46, 132.83, 129.92, 128.70, 126.04, 122.38, 121.63, 119.04, 108.72, 102.09, 67.85, 64.23, 48.46, 38.56, 38.45, 34.43, 14.99. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₆H₂₉ClN₆O₃: 509.2062, found: 509.2059.

2-((2-Ethoxy-4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5,10,11-trimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(A2)

Brown solid; yield 28%; m.p. 177–178 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.98 (d, J = 8.5 Hz, 1H), 7.86 (d, J = 7.9 Hz, 1H), 7.28 (s, 1H), 7.22 (s, 1H), 6.95–6.87 (m, 2H), 6.60–6.55 (m, 2H), 4.13 (q, J = 7.0 Hz, 2H), 3.89–3.82 (m, 1H), 3.73 (s, 3H), 3.53–3.47 (s, 2H), 3.37 (s, 3H), 2.96–2.85 (m, 2H), 2.36 (s, 3H), 2.08–1.99 (m, 2H), 1.77–1.67 (m, 2H), 1.49 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.97, 152.28, 149.03, 148.59, 148.50, 146.33, 139.66, 135.64, 134.21, 130.99, 126.07, 122.61, 121.94, 121.77, 120.19, 108.75, 101.62, 67.74, 64.22, 48.11, 39.93, 34.25, 29.70, 20.05, 15.00. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₇H₃₂N₆O₃: 489.2609, found: 489.2608.

9-Chloro-2-((2-ethoxy-4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(A3)

Brown solid; yield 31%; m.p. 162–163 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.22 (d, J = 8.5 Hz, 1H), 8.14 (s, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.50 (s, 1H), 7.14–7.07 (m, 2H), 6.63–6.58 (m, 2H), 4.12 (q, J = 6.9 Hz, 2H), 3.91–3.82 (m, 1H), 3.54–3.47 (m, 5H), 3.41 (s, 3H), 2.95–2.87 (m, 2H), 2.06–2.01 (m, 2H), 1.81–1.70 (m, 2H), 1.48 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.42, 163.13, 156.15, 151.75, 150.41, 148.18, 147.20, 138.41, 133.53, 124.95, 123.81, 122.19, 120.51, 119.20, 117.75, 108.59, 102.15, 67.75, 64.25, 48.43, 38.11, 35.97, 34.36, 14.97. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₆H₂₉ClN₆O₃: 509.2062, found: 509.2063.

2-((2-Ethoxy-4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5,9,11-trimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(A4)

White solid; yield 40%; m.p. 171–172 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.26 (d, J = 8.6 Hz, 1H), 8.11 (s, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.48 (s, 1H), 6.95 (d, J = 7.9 Hz, 1H), 6.89 (s, 1H), 6.64–6.55 (m, 2H), 4.12 (q, J = 7.0 Hz, 2H), 3.90–3.83 (m, 1H), 3.55–3.44 (m, 5H), 3.42 (s, 3H), 2.94–2.86 (m, 2H), 2.36 (s, 3H), 2.08–2.01 (m, 2H), 1.80–1.70 (m, 2H), 1.48 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.38, 163.75, 156.04, 151.33, 149.39, 148.07, 147.09, 142.98, 132.21, 124.50, 123.75, 122.44, 120.87, 119.05, 117.91, 108.61, 102.17, 67.78, 64.23, 48.48, 38.11, 35.91, 34.40, 21.64, 14.98. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₇H₃₂N₆O₃: 489.2609, found: 489.2602.

8-Chloro-2-((2-ethoxy-4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(A5)

Yellow solid; yield 28%; m.p. 173–174 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.21 (d, J = 8.6 Hz, 1H), 8.12 (s, 1H), 7.81 (s, 1H), 7.50 (s, 1H), 7.35 (d, J = 8.9 Hz, 1H), 7.00 (d, J = 9.1 Hz, 1H), 6.60 (s, 1H), 6.58 (s, 1H), 4.11 (q, J = 7.0 Hz, 2H), 3.89–3.80 (m, 1H), 3.52–3.44 (m, 5H), 3.38 (s, 3H), 2.93–2.83 (m, 2H), 2.07–2.00 (m, 2H), 1.79–1.68 (m, 2H), 1.47 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.05, 163.40, 156.12, 151.75, 148.17, 148.05, 147.28, 132.05, 131.82, 129.06, 127.92, 122.10, 120.31, 119.19, 118.82, 108.55, 102.09, 67.67, 64.24, 48.41, 38.31, 35.98, 34.35, 14.97. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₆H₂₉ClN₆O₃: 509.2062, found: 509.2061.

2-((2-Ethoxy-4-(4-hydroxypiperidin-1-yl)phenyl)amino)-8-fluoro-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(A6)

Yellow solid; yield 23%; m.p. 166–167 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.21 (d, J = 8.5 Hz, 1H), 8.11 (s, 1H), 7.57–7.49 (m, 2H), 7.14–7.06 (m, 1H), 7.02 (dd, J = 9.2, 4.4 Hz, 1H), 6.61–6.56 (m, 2H), 4.10 (q, J = 6.9 Hz, 2H), 3.87–3.78 (m, 1H), 3.53–3.42 (m, 5H), 3.37 (s, 3H), 2.92–2.83 (m, 2H), 2.06–1.98 (m, 2H), 1.78–1.68 (m, 2H), 1.46 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.13, 163.75, 159.87, 157.45, 156.13, 151.63, 148.16, 147.27, 145.62, 145.59, 128.06, 122.12, 120.39, 119.20, 118.21, 108.55, 102.09, 67.58, 64.24, 48.44, 38.35, 36.06, 34.34, 14.97. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₆H₂₉FN₆O₃: 493.2358, found: 493.2353.

2-((2-Ethoxy-4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5,11-dimethyl-8-(trifluoromethyl)-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(A7)

Yellow solid; yield 15%; m.p. 119–120 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.27–8.12 (m, 3H), 7.66 (d, J = 8.7 Hz, 1H), 7.52 (s, 1H), 7.19 (d, J = 8.7 Hz, 1H), 6.61 (s, 1H), 6.59 (s, 1H), 4.12 (q, J = 6.9 Hz, 2H), 3.90–3.83 (m, 1H), 3.60–3.44 (m, 8H), 2.96–2.87 (m, 2H), 2.10–1.99 (m, 2H), 1.79–1.74 (m, 2H), 1.48 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.08, 162.91, 156.18, 152.26, 152.00, 148.22, 147.34, 129.88, 129.56, 128.95, 126.79, 125.95, 122.02, 120.21, 119.22, 117.82, 108.53, 102.09, 67.76, 64.25, 48.37, 38.21, 36.09, 34.35, 14.96. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₇H₂₉F₃N₆O₃: 543.2326, found: 543.2322.

2-((2-Ethoxy-4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5,8,11-trimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(A8)

Yellow solid; yield 41%; m.p. 170–171 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.23 (d, J = 8.5 Hz, 1H), 8.08 (s, 1H), 7.62 (d, J = 2.2 Hz, 1H), 7.48 (s, 1H), 7.20 (dd, J = 8.5, 2.2 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.60–6.56 (m, 2H), 4.09 (q, J = 7.0 Hz, 2H), 3.86–3.77 (m, 1H), 3.49–3.44 (m, 5H), 3.37 (s, 3H), 2.90–2.82 (m, 2H), 2.30 (s, 3H), 2.04–1.97 (m, 2H), 1.78–1.67 (m, 2H), 1.45 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.52, 163.95, 156.01, 151.33, 148.08, 147.15, 147.13, 133.21, 133.01, 132.29, 126.22, 122.34, 120.68, 119.09, 117.28, 108.60, 102.14, 67.57, 64.23, 48.49, 38.29, 35.82, 34.36, 20.38, 14.97. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₇H₃₂N₆O₃: 489.2609, found: 489.2602.

2-((2-Ethoxy-4-(4-hydroxypiperidin-1-yl)phenyl)amino)-7-fluoro-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(A9)

Yellow solid; yield 23%; m.p. 174–175 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.21 (d, J = 8.9 Hz, 1H), 8.18 (s, 1H), 7.51 (s, 1H), 7.37–7.29 (m, 1H), 6.91–6.83 (m, 2H), 6.59 (s, 1H), 6.57 (s, 1H), 4.11 (q, J = 6.9 Hz, 2H), 3.89–3.80 (m, 1H), 3.54–3.44 (m, 5H), 3.38 (s, 3H), 2.93–2.85 (m, 2H), 2.10–2.00 (m, 2H), 1.79–1.68 (m, 2H), 1.47 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 164.20, 163.31, 160.77, 156.05, 152.42, 151.85, 148.17, 147.30, 131.93, 122.05, 120.52, 119.21, 116.01, 113.10, 111.99, 108.53, 102.05, 67.67, 64.23, 48.39, 37.80, 35.98, 34.34, 14.96. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₆H₂₉FN₆O₃: 493.2358, found: 493.2354.

2-((2-Ethoxy-4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5,7,11-trimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(A10)

Yellow solid; yield 25%; m.p. = 184–185 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.25 (d, J = 8.6 Hz, 1H), 8.16 (s, 1H), 7.48 (s, 1H), 7.22 (t, J = 7.9 Hz, 1H), 6.98 (dd, J = 12.0, 7.9 Hz, 2H), 6.64–6.55 (m, 2H), 4.12 (q, J = 7.0 Hz, 2H), 3.89–3.81 (m, 1H), 3.53–3.46 (m, 5H), 3.37 (s, 3H), 2.94–2.86 (m, 2H), 2.47 (s, 3H), 2.10–1.99 (m, 2H), 1.80–1.69 (m, 2H), 1.48 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.46, 164.94, 155.99, 151.90, 150.75, 148.09, 147.13, 140.40, 130.12, 127.03, 126.90, 122.38, 121.12, 119.11, 115.04, 108.63, 102.15, 67.77, 64.24, 48.45, 38.12, 35.67, 34.40, 21.06, 14.97. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₇H₃₂N₆O₃: 489.2609, found: 489.2606.

2-((2-Ethoxy-4-(4-hydroxypiperidin-1-yl)phenyl)amino)-11-methyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(B1)

Yellow solid; yield 29%; m.p. 165–166 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.83 (s, 1H), 8.26 (d, J = 8.6 Hz, 1H), 8.01 (s, 1H), 7.91 (dd, J = 7.8, 1.7 Hz, 1H), 7.54–7.48 (m, 1H), 7.47 (s, 1H), 7.20–7.09 (m, 2H), 6.63–6.57 (m, 2H), 4.12 (q, J = 7.0 Hz, 2H), 3.90–3.83 (m, 1H), 3.53–3.47 (m, 2H), 3.46 (s, 3H), 2.94–2.85 (m, 2H), 2.10–2.01 (m, 2H), 1.79–1.73 (m, 2H), 1.48 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 169.51, 161.75, 156.61, 149.25, 149.08, 148.02, 146.96, 133.33, 132.05, 125.53, 123.65, 122.60, 118.93, 118.71, 115.15, 108.64, 102.22, 67.84, 64.22, 48.56, 36.67, 34.41, 14.99. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₅H₂₈N₆O₃: 461.2296, found: 461.2293.

2-((2-Ethoxy-4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5-methyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(B2)

Yellow solid; yield 20%; m.p. 194–195 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.16–8.09 (m, 2H), 7.95 (dd, J = 7.9, 1.6 Hz, 1H), 7.39–7.30 (m, 2H), 7.07 (t, J = 7.6 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 6.57 (s, 1H), 6.55 (s, 1H), 6.39 (s, 1H), 4.08 (q, J = 7.0 Hz, 2H), 3.88–3.81 (m, 1H), 3.49–3.45 (m, 5H), 2.91–2.83 (m, 2H), 2.06–1.99 (m, 2H), 1.74–1.70 (m, 2H), 1.44 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.61, 160.63, 156.30, 151.81, 148.40, 147.45, 145.07, 133.27, 132.86, 123.46, 123.18, 121.96, 119.63, 119.10, 118.89, 108.57, 102.15, 67.79, 64.19, 48.41, 38.51, 34.37, 14.97. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₅H₂₈N₆O₃: 461.2296, found: 461.2292.

2-((2-Ethoxy-4-(4-hydroxypiperidin-1-yl)phenyl)amino)-5-ethyl-11-methyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(B3)

Brown solid; yield 25%; m.p. 162–163 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.25 (d, J = 8.7 Hz, 1H), 8.19 (s, 1H), 7.80 (dd, J = 7.8, 1.7 Hz, 1H), 7.50 (s, 1H), 7.40 (t, J = 7.9 Hz, 1H), 7.13 (t, J = 7.7 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 6.62–6.57 (m, 2H), 4.12 (q, J = 6.9 Hz, 2H), 3.99 (q, J = 6.9 Hz, 2H), 3.87–3.81 (m, 1H), 3.52–3.46 (m, 2H), 3.41 (s, 3H), 2.93–2.84 (m, 2H), 2.06–2.01 (m, 2H), 1.78–1.70 (m, 2H), 1.47 (t, J = 7.0 Hz, 3H), 1.30 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.26, 164.80, 156.05, 151.89, 149.62, 148.08, 147.15, 132.02, 131.89, 127.29, 123.72, 122.30, 119.27, 119.05, 117.27, 108.59, 102.12, 67.73, 64.20, 48.52, 46.19, 35.79, 34.37, 14.99, 13.87. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₇H₃₂N₆O₃: 489.2609, found: 489.2603.

General procedure for synthesis of compounds C1 and C2

The compounds C1 and C2 are synthesised by a similar method using the above-described procedure for the reaction of compound A1.

2-((4-(4-Hydroxypiperidin-1-yl)cyclohexyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(C1)

Yellow solid; yield 45%; m.p. 203–204 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.99 (s, 1H), 7.82 (dd, J = 7.8, 1.6 Hz, 1H), 7.40 (td, J = 7.9, 1.7 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 4.08–4.03 (m, 1H), 3.82–3.62 (m, 3H), 3.45 (s, 3H), 3.29 (s, 3H), 2.96–2.87 (m, 2H), 2.46–2.37 (m, 2H), 2.00–1.93 (m, 4H), 1.78–1.73 (m, 2H), 1.64–1.58 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 168.40, 164.19, 158.13, 151.46, 149.54, 132.20, 132.17, 126.59, 123.53, 117.20, 67.28, 62.30, 46.54, 46.22, 38.22, 35.42, 34.19, 29.18, 23.62. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₄H₃₂N₆O₂: 437.2660, found: 437.2657.

2-((6-(4-Hydroxypiperidin-1-yl)pyridin-3-yl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(C2)

Yellow solid; yield 62%; m.p. 189–190 °C; ¹H NMR (400 MHz, Methanol-d4) δ 8.55 (d, J = 2.3 Hz, 1H), 8.32 (s, 1H), 8.18 (dd, J = 9.9, 2.4 Hz, 1H), 7.83 (dd, J = 7.9, 1.5 Hz, 1H), 7.65–7.57 (m, 1H), 7.54 (d, J = 9.8 Hz, 1H), 7.34 (d, J = 8.3 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 4.09–4.01 (m, 3H), 3.68–3.60 (m, 2H), 3.56 (s, 3H), 3.52 (s, 3H), 2.12–2.04 (m, 2H), 1.79–1.68 (m, 2H). ¹³C NMR (101 MHz, CD₃OD) δ 167.73, 166.34, 150.90, 149.47, 146.33, 142.22, 139.36, 133.09, 131.78, 127.68, 125.59, 125.04, 124.80, 121.19, 118.90, 113.44, 64.77, 43.93, 38.04, 36.46, 32.72. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₃H₂₅N₇O₂: 432.2142, found: 432.2137.

Preparation of intermediate 14

A sealed tube with a mixture of 13 (1 eq), 2-Methoxy-4-nitroaniline (1 eq), Pd(OAc)₂ (0.05 eq), XPhos (0.1 eq),and Cs₂CO₃ (2 eq) in t-BuOH was heated at 110 °C for 10 h. The reaction was then filtered through celite and eluted with dichloromethane. The dichloromethane was removed in vacuo and the resulting crude product was purified by silica gel column chromatography (eluant: DCM/MeOH, 40:1 v/v) to afford the title intermediate 14 (36%).

2-((2-Methoxy-4-nitrophenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(14)

Yellow solid; yield 36%; m.p. 215–216 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.71 (d, J = 9.1 Hz, 1H), 8.23 (s, 1H), 8.01 (dd, J = 9.0, 2.3 Hz, 2H), 7.88 (dd, J = 7.8, 1.8 Hz, 1H), 7.78 (d, J = 2.5 Hz, 1H), 7.51–7.44 (m, 1H), 7.18 (td, J = 7.5, 1.0 Hz, 1H), 7.13 (dd, J = 8.4, 1.0 Hz, 1H), 4.05 (s, 3H), 3.54 (s, 3H), 3.48 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.07, 163.65, 154.71, 151.14, 149.03, 146.73, 141.17, 135.67, 132.60, 132.44, 126.37, 124.01, 123.04, 118.07, 117.50, 115.56, 105.09, 56.33, 38.30, 36.07. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₀H₁₈N₆O₄: 407.1462, found: 407.1461.

Preparation of intermediate 15

A mixture of 14 (1 eq), Pd/C (0.05 eq), and methanol was stirred at room temperature for 12 h under a hydrogen atmosphere. The reaction mixture was filtered and the residue obtained by evaporation of the solvent of the filtrate under reduced pressure. This residue is prepared as the hydrochloride salt to afford the intermediate 15 (100%).

2-((4-Amino-2-methoxyphenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(15)

Brown solid; yield 100%; m.p. 199–200 °C; ¹H NMR (400 MHz, Methanol-d4) δ 8.28 (s, 1H), 8.14–8.07 (m, 1H), 7.85 (dd, J = 7.8, 1.6 Hz, 1H), 7.66–7.60 (m, 1H), 7.37 (d, J = 8.3 Hz, 1H), 7.33 (t, J = 7.7 Hz, 1H), 7.21 (s, 1H), 7.16 (dd, J = 8.5, 2.3 Hz, 1H), 4.02 (s, 3H), 3.58 (s, 3H), 3.51 (s, 3H). ¹³C NMR (101 MHz, CD₃OD) δ 167.49, 167.19, 152.44, 149.74, 145.43, 138.96, 133.23, 131.85, 128.83, 125.51, 125.43, 125.08, 120.65, 119.19, 114.98, 106.56, 55.73, 38.19, 36.51. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₀H₂₀N₆O₂: 377.1721, found: 377.1715.

General procedure for synthesis of compounds D1 and D2

To a solution of 18 (1 eq) and triethylamine (2 eq) in DCM ethyl 3-chloro-3-oxopropanoate/ethyl 4-chloro-4-oxobutanoate (1.1 eq) in DCM was added dropwise. The reaction mixture was stirred at RT for 1.5 h. After the reaction was complete as monitored by TLC, water was added to the solution. The organic phase is washed with saturated NaHCO₃ and dried over anhydrous Na₂SO₄. After filtration, the combined organic solution is concentrated to dryness under reduced pressure. The crude product was dissolved in THF and treated with 2 N LiOH. The reaction was stirred for 1 h. The solution was added with 1 N HCl to make the pH = 6. This mixture was then washed with EtOAc (three times), and the combined extracts were washed with brine, dried with Na₂SO₄, and evaporated under reduced pressure to afford the crude product. The resulting crude product was purified by silica gel column chromatography (eluant: DCM/MeOH, 40:1 v/v) to afford the compound D1, D2 (70%).

3-((4-((5,11-Dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-2-yl)amino)-3-methoxyphenyl)amino)-3-oxopropanoic acid(D1)

White solid; yield 70%; m.p. 187–188 °C; ¹H NMR (400 MHz, DMSO-d6) δ 12.59 (br, 1H), 10.11 (s, 1H), 8.35 (s, 1H), 8.05 (s, 1H), 7.94 (d, J = 8.7 Hz, 1H), 7.68 (dd, J = 7.7, 1.7 Hz, 1H), 7.52–7.45 (m, 1H), 7.40 (d, J = 2.2 Hz, 1H), 7.23 (d, J = 8.4, 1.0 Hz, 1H), 7.19–7.12 (m, 2H), 3.81 (s, 3H), 3.39 (s, 3H), 3.35 (s, 2H), 3.29 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 169.75, 167.52, 164.73, 163.50, 152.76, 150.03, 149.70, 135.15, 132.90, 132.11, 126.65, 124.43, 123.89, 121.50, 120.89, 118.21, 111.17, 103.05, 56.05, 44.45, 37.90, 35.95. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₃H₂₂N₆O₅: 463.1724, found: 463.1726.

4-((4-((5,11-Dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-2-yl)amino)-3-methoxyphenyl)amino)-4-oxobutanoic acid(D2)

White solid; yield 70%; m.p. 237–238 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.12 (s, 1H), 9.94 (s, 1H), 8.34 (s, 1H), 8.03 (s, 1H), 7.90 (d, J = 8.6 Hz, 1H), 7.68 (dd, J = 7.8, 1.7 Hz, 1H), 7.53–7.45 (m, 1H), 7.45 (d, J = 2.2 Hz, 1H), 7.23 (dd, J = 8.4, 1.0 Hz, 1H), 7.19–7.14 (m, 1H), 7.12 (dd, J = 8.7, 2.2 Hz, 1H), 3.80 (s, 3H), 3.39 (s, 3H), 3.28 (s, 3H), 2.60–2.52 (m, 4H). ¹³C NMR (101 MHz, DMSO) δ 174.30, 170.25, 167.52, 163.50, 156.53, 152.76, 150.08, 149.71, 135.63, 132.90, 132.10, 126.66, 123.95, 123.89, 121.58, 120.82, 118.20, 110.96, 102.97, 56.01, 37.89, 35.93, 31.48, 29.28. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₄H₂₄N₆O₅: 477.1881, found: 477.1875.

Preparation of intermediate 16

The intermediate 16 are synthesised by a similar method using the above-described procedure for the reaction of compounds D1 and D2.

6-Bromo-N-(4-((5,11-dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-2-yl)amino)-3-methoxyphenyl)hexanamide(16)

White solid; yield 87%; m.p. 137–138 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.30 (d, J = 8.4 Hz, 2H), 8.11 (s, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.59 (s, 1H), 7.51 (s, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 8.3 Hz, 1H), 6.97 (d, J = 8.7 Hz, 1H), 3.84 (s, 3H), 3.47 (s, 3H), 3.40–3.34 (m, 5H), 2.35 (t, J = 7.5 Hz, 2H), 1.90–1.80 (m, 2H), 1.73 (m, 2H), 1.52–1.43 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 171.37, 168.45, 163.71, 155.85, 151.39, 149.43, 147.94, 132.95, 132.46, 132.17, 126.41, 125.29, 123.69, 121.11, 118.12, 117.44, 111.71, 103.32, 55.78, 38.28, 37.14, 35.87, 33.68, 32.46, 27.77, 24.71. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₆H₂₉BrN₆O₃: 553.1557, found: 553.1553.

General procedure for the synthesis of compound D8

A mixture of 16 (1 eq) and potassium ethanethioate (6 eq) in DMF was heated to 100 °C for 6 h. After the reaction was complete as monitored by TLC, the solvent was diluted with water (20 ml) and extracted with EtOAc (20 ml) × 3. The organics were combined, dried over Na₂SO₄, filtered, and concentrated in vacuo. Purification by silica gel column chromatography (eluant: DCM/MeOH, 50:1 v/v) gave compound D8 (72%).

S-(6-((4-((5,11-dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-2-yl)amino)-3-methoxyphenyl)amino)-6-oxohexyl) ethanethioate(D8)

Brown solid; yield 72%; m.p. 159–160 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.35 (d, J = 8.7 Hz, 1H), 8.14 (s, 1H), 7.86 (dd, J = 7.8, 1.7 Hz, 1H), 7.60 (s, 1H), 7.56 (d, J = 2.3 Hz, 1H), 7.46–7.40 (m, 1H), 7.38 (s, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.09 (d, J = 8.3 Hz, 1H), 6.87 (dd, J = 8.7, 2.3 Hz, 1H), 4.10 (t, J = 6.7 Hz, 2H), 3.92 (s, 3H), 3.51 (s, 3H), 3.42 (s, 3H), 2.39 (t, J = 7.4 Hz, 2H), 2.06 (s, 3H), 1.84–1.76 (m, 2H), 1.75–1.66 (m, 2H), 1.53–1.40 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 171.23, 170.91, 168.34, 163.75, 155.82, 151.35, 149.40, 148.01, 132.53, 132.37, 132.26, 126.53, 125.51, 123.71, 121.26, 118.08, 117.38, 111.36, 103.18, 64.26, 55.84, 38.25, 37.46, 35.88, 28.42, 25.61, 25.14, 21.02. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₈H₃₂N₆O₄S: 549.2279, found: 549.2286.

Preparation of intermediate 18

A mixture of 17 (1.5 eq), 5-fluoro-2-nitrophenyl ethyl ether (1 eq), and K₂CO₃ in DMF was heated to 90 °C for 8–10 h. After the reaction was complete as monitored by TLC, the solution was diluted with water (50 ml) and extracted with EtOAc (50 ml) × 3. The organics were combined, dried over Na₂SO₄, filtered, and concentrated in vacuo. Purification by silica gel column chromatography (eluant: petroleum ether/EtOAc, 5:1 v/v) gave intermediate 18 (62–91%).

4-(3-Ethoxy-4-nitrophenyl)thiomorpholine (18-4)

Yellow solid; yield 90%; m.p. 118–119 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.98 (d, J = 9.3 Hz, 1H), 6.38 (dd, J = 9.4, 2.5 Hz, 1H), 6.29 (d, J = 2.5 Hz, 1H), 4.15 (q, J = 6.9 Hz, 2H), 3.85–3.78 (m, 4H), 2.76–2.68 (m, 4H), 1.51 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 155.84, 154.33, 129.86, 128.89, 105.77, 98.80, 65.35, 50.44, 25.93, 14.65. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₂H₁₆N₂O₃S: 269.0954, found: 269.0953.

Preparation of intermediate 19

The intermediate 19 are synthesised by similar method using the above-described procedure for the reaction of intermediate 15.

1-(4-Amino-3-ethoxyphenyl)piperidine-4-carboxylic acid (19-3)

White solid; yield 100%; m.p. 146–147 °C; ¹H NMR (400 MHz, DMSO-d6) δ 7.64 (d, J = 14.6 Hz, 1H), 7.51–7.46 (m, 1H), 7.41–7.33 (m, 1H), 4.18 (q, J = 7.0 Hz, 2H), 3.66 (s, 2H), 3.56 (s, 4H), 2.24–2.08 (m, 4H), 1.38 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 175.25, 174.12, 151.96, 123.95, 113.07, 109.98, 106.59, 65.40, 52.27, 37.99, 26.13, 14.79. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₄H₂₀N₂O₃: 265.1547, found: 265.1544.

General procedure for synthesis of compounds D3, D4, D6, D9–D16

The compounds D3, D4, D6, D9–D16 are synthesised by a similar method using the above-described procedure for the reaction of compound A1.

1-(4-((5,11-Dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-2-yl)amino)-3-ethoxyphenyl)piperidine-4-carboxylic acid(D3)

White solid; yield 48%; m.p. 159–160 °C; ¹H NMR (400 MHz, Methanol-d4) δ 8.38 (d, J = 8.8 Hz, 1H), 8.32 (s, 1H), 7.80 (dd, J = 7.8, 1.7 Hz, 1H), 7.63–7.57 (m, 1H), 7.46 (s, 1H), 7.41 (dd, J = 8.8, 2.5 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.28 (t, J = 7.5 Hz, 1H), 4.30 (q, J = 6.9 Hz, 2H), 3.88–3.72 (m, 5H), 3.53 (s, 3H), 3.50 (s, 3H), 2.45–2.38 (m, 2H), 2.33–2.26 (m, 2H), 1.51 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CD₃OD) δ 175.52, 174.12, 168.40, 165.84, 152.07, 149.96, 147.16, 144.57, 137.80, 133.26, 131.70, 128.29, 125.47, 124.65, 121.05, 118.59, 112.76, 104.89, 65.34, 55.50, 55.38, 38.07, 37.14, 36.06, 25.84, 25.74, 13.61. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₇H₃₀N₆O₄: 503.2401, found: 503.2397.

2-((2-Ethoxy-4-thiomorpholinophenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(D4)

Yellow solid; yield 38%; m.p. 190–191 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.28 (d, J = 8.8 Hz, 1H), 8.13 (s, 1H), 7.86 (dd, J = 7.8, 1.7 Hz, 1H), 7.50 (s, 1H), 7.46–7.40 (m, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.58 (dd, J = 8.8, 2.6 Hz, 1H), 6.54 (d, J = 2.5 Hz, 1H), 4.12 (q, J = 7.0 Hz, 2H), 3.51 (s, 3H), 3.49–3.44 (m, 4H), 3.43 (s, 3H), 2.85–2.77 (m, 4H), 1.49 (t, J = 7.0 Hz, 3H). 13 C NMR (101 MHz, CDCl₃) δ 168.35, 163.77, 156.01, 151.47, 149.48, 148.15, 147.49, 132.29, 132.25, 126.60, 123.66, 122.97, 120.86, 119.06, 117.36, 109.55, 102.99, 64.29, 53.41, 38.23, 35.90, 27.45, 14.96. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₅H₂₈N₆O₂S: 477.2067, found: 477.2065.

2-((4-(1,1-Dioxidothiomorpholino)-2-ethoxyphenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(D6)

White solid; yield 32%; m.p. 157–158 °C; ¹H NMR (400 MHz, Methanol-d4) δ 8.12 (s, 1H), 7.83 (dd, J = 7.8, 1.7 Hz, 1H), 7.69–7.62 (m, 1H), 7.52 (s, 1H), 7.40–7.31 (m, 2H), 6.80–6.70 (m, 2H), 4.16 (q, J = 7.0 Hz, 2H), 3.99–3.89 (m, 4H), 3.55 (s, 3H), 3.49 (s, 3H), 3.25–3.20 (m, 4H), 1.35 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CD₃OD) δ 168.01, 167.57, 145.24, 133.56, 131.79, 125.25, 125.07, 119.95, 119.23, 107.95, 101.15, 64.33, 49.99, 47.17, 38.65, 36.48, 13.81. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₅H₂₈N₆O₄S: 509.1966, found: 509.1962.

2-((2-Ethoxy-4-(4-hydroxy-4-methylpiperidin-1-yl)phenyl)amino)-5-ethyl-11-methyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(D9)

White solid; yield 30%; m.p. 178–179 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.25 (d, J = 8.7 Hz, 1H), 8.18 (s, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.50 (s, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H), 6.66–6.56 (m, 2H), 4.26–3.71 (m, 4H), 3.40 (s, 3H), 3.32–3.22 (m, 2H), 3.21–3.09 (m, 2H), 1.85–1.70 (m, 4H), 1.46 (t, J = 7.0 Hz, 3H), 1.38–1.22 (m, 7H). ¹³C NMR (101 MHz, CDCl₃) δ 168.24, 164.80, 156.10, 151.89, 149.63, 148.14, 147.36, 132.00, 131.89, 127.30, 123.69, 122.17, 119.23, 119.17, 117.26, 108.66, 102.06, 77.45, 77.13, 76.81, 67.65, 64.23, 47.16, 46.16, 38.59, 35.76, 29.87, 14.98, 13.85. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₈H₃₄N₆O₃: 503.2771, found: 503.2767.

2-((2-Ethoxy-4-(4-fluoro-4-methylpiperidin-1-yl)phenyl)amino)-5-ethyl-11-methyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(D10)

White solid; yield 33%; m.p. 189–190 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.28 (d, J = 8.7 Hz, 1H), 8.20 (s, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.51 (s, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.07 (d, J = 8.3 Hz, 1H), 6.67–6.57 (m, 2H), 4.21–3.75 (m, 4H), 3.47–3.33 (m, 5H), 3.11 (t, J = 11.9 Hz, 2H), 2.01–1.77 (m, 4H), 1.53–1.37 (m, 6H), 1.31 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.20, 164.78, 156.09, 151.90, 149.63, 148.14, 147.10, 131.98, 131.90, 127.35, 123.69, 122.44, 119.34, 119.15, 117.25, 108.83, 102.18, 92.92, 91.25, 77.41, 77.09, 76.78, 64.24, 46.97, 46.94, 46.14, 36.45, 36.24, 35.76, 26.88, 14.97, 13.85. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₈H₃₃FN₆O₂: 505.2702, found: 505.2719.

2-((2-Methoxy-4-(piperidin-1-yl)phenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(D11)

White solid; yield 45%; m.p. 166–167 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.22 (d, J = 9.3 Hz, 1H), 8.13 (s, 1H), 7.86 (dd, J = 7.8, 1.7 Hz, 1H), 7.48–7.38 (m, 2H), 7.19–7.05 (m, 2H), 6.60 (d, J = 7.4 Hz, 2H), 3.90 (s, 3H), 3.51 (s, 3H), 3.42 (s, 3H), 3.17–3.08 (m, 4H), 1.81–1.72 (m, 4H), 1.65–1.55 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 168.36, 163.77, 156.19, 151.47, 149.52, 148.92, 148.34, 132.23, 126.64, 123.60, 121.95, 120.76, 119.32, 117.33, 108.56, 101.21, 77.33, 77.02, 76.70, 55.65, 51.83, 38.19, 35.84, 26.06, 24.28. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₅H₂₈N₆O₂: 445.2352, found: 445.2346.

2-((2-Methoxy-4-(4-methoxypiperidin-1-yl)phenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(D12)

White solid; yield 47%; m.p. 179–180 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.22 (d, J = 8.5 Hz, 1H), 8.12 (s, 1H), 7.86 (dd, J = 7.8, 1.8 Hz, 1H), 7.47–7.38 (m, 2H), 7.18–7.05 (m, 2H), 6.64–6.56 (m, 2H), 3.90 (s, 3H), 3.54–3.44 (m, 5H), 3.43–3.34 (m, 7H), 2.97–2.86 (m, 2H), 2.12–2.00 (m, 2H), 1.81–1.70 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 168.34, 163.76, 156.15, 151.46, 149.50, 148.91, 147.42, 132.25, 132.23, 126.62, 123.60, 122.17, 120.81, 119.32, 117.33, 108.64, 101.19, 77.36, 77.04, 76.73, 76.06, 55.67, 55.59, 48.40, 38.19, 35.84, 30.84. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₆H₃₀N₆O₃: 475.2458, found: 475.2450.

2-((4-(4-Fluoropiperidin-1-yl)-2-methoxyphenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(D13)

White solid; yield 33%; m.p. 160–161 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.17 (s, 1H), 8.15 (s, 1H), 7.87 (dd, J = 7.8, 1.7 Hz, 1H), 7.49–7.39 (m, 2H), 7.20–7.06 (m, 2H), 6.56 (s, 1H), 5.86–5.71 (m, 1H), 5.08–4.97 (m, 2H), 4.40 (s, 2H), 3.90 (s, 3H), 3.52 (s, 3H), 3.45 (s, 3H), 3.24 (t, J = 7.4 Hz, 2H), 2.29 (q, J = 7.2 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 168.34, 163.76, 156.20, 151.56, 149.47, 148.14, 136.49, 132.27, 132.26, 126.66, 123.67, 122.44, 120.71, 120.55, 117.27, 115.93, 101.15, 77.35, 77.03, 76.71, 55.76, 51.76, 38.22, 35.85, 32.64. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₅H₂₇FN₆O₂: 463.2252, found: 463.2261.

2-((4-(4,4-Difluoropiperidin-1-yl)-2-methoxyphenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(D14)

White solid; yield 32%; m.p. 171–172 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.27 (d, J = 8.5 Hz, 1H), 8.14 (s, 1H), 7.87 (d, J = 7.5 Hz, 1H), 7.51–7.39 (m, 2H), 7.20–7.06 (m, 2H), 6.67–6.56 (m, 2H), 3.91 (s, 3H), 3.55–3.36 (m, 6H), 3.35–3.23 (m, 4H), 2.23–2.05 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 168.30, 163.77, 156.07, 151.44, 149.46, 148.93, 146.07, 132.27, 126.62, 123.65, 122.96, 121.01, 119.26, 117.34, 109.13, 101.51, 77.37, 77.05, 76.74, 55.73, 47.99, 47.94, 47.89, 38.19, 35.85, 34.12, 33.89, 33.67. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₅H₂₆F₂N₆O₂: 481.2164, found: 481.2155.

2-((4-(4-Hydroxy-4-methylpiperidin-1-yl)-2-methoxyphenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(D15)

White solid; yield 37%; m.p. 162–163 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.22 (d, J = 8.6 Hz, 1H), 8.12 (s, 1H), 7.89–7.82 (m, 1H), 7.47–7.38 (m, 2H), 7.19–7.05 (m, 2H), 6.67–6.58 (m, 2H), 3.90 (s, 3H), 3.50 (s, 3H), 3.42 (s, 3H), 3.35–3.25 (m, 2H), 3.23–3.11 (m, 2H), 1.91–1.80 (m, 2H), 1.76–1.71 (m, 2H), 1.33 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.40, 163.80, 156.16, 151.46, 149.51, 148.95, 147.48, 132.28, 132.23, 126.59, 123.63, 122.12, 120.79, 119.38, 117.36, 108.72, 101.15, 77.35, 77.03, 76.72, 67.82, 55.69, 47.18, 38.66, 38.23, 35.85, 29.88. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₆H₃₀N₆O₃: 475.2458, found: 475.2451.

2-((4-(4,4-Dimethylpiperidin-1-yl)-2-methoxyphenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(D16)

White solid; yield 40%; m.p. 181–182 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.22 (d, J = 8.5 Hz, 1H), 8.12 (s, 1H), 7.90–7.82 (m, 1H), 7.47–7.39 (m, 2H), 7.18–7.04 (m, 2H), 6.66–6.57 (m, 2H), 3.90 (s, 3H), 3.50 (s, 3H), 3.41 (s, 3H), 3.19–3.11 (m, 4H), 1.61–1.53 (m, 4H), 1.02 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 168.35, 163.76, 156.20, 151.48, 149.52, 148.96, 147.94, 132.25, 132.22, 126.63, 123.59, 121.75, 120.73, 119.40, 117.33, 108.22, 100.77, 77.39, 77.07, 76.75, 55.65, 47.03, 38.67, 38.19, 35.84, 28.43, 27.94. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₇H₃₂N₆O₂: 473.2665, found: 473.2658.

General procedure for the synthesis of compound D5

D4 (1 eq) was dissolved in acetic acid, and hydrogen peroxide (1 eq) was added dropwise. After the reaction was complete as monitored by TLC, the residue was obtained by evaporation of solvent under reduced pressure. Purification by silica gel column chromatography (eluant: DCM/MeOH, 60:1 v/v) gave compound D5 (95%).

2-((2-ethoxy-4-(1-Oxidothiomorpholino)phenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(D5)

White solid; yield 95%; m.p. 163–164 °C; ¹H NMR (400 MHz, Methanol-d4) δ 8.19 (s, 1H), 7.83 (dd, J = 7.8, 1.7 Hz, 1H), 7.76 (s, 1H), 7.68–7.61 (m, 1H), 7.40–7.29 (m, 2H), 7.08–6.94 (m, 2H), 4.22 (q, J = 7.0 Hz, 2H), 4.16–4.05 (m, 2H), 3.83–3.76 (m, 2H), 3.56 (s, 3H), 3.50 (s, 3H), 3.31–3.24 (m, 2H), 3.14–3.04 (m, 2H), 1.40 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CD₃OD) δ 167.89, 167.31, 152.63, 150.25, 145.43, 138.51, 133.48, 131.80, 125.17, 120.20, 119.19, 109.55, 102.47, 64.61, 43.36, 42.38, 38.53, 36.51, 13.75. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₅H₂₈N₆O₃S: 493.2016, found: 493.2011.

Preparation of intermediate 20

The intermediate 20 are synthesised by a similar method using the above-described procedure for the reaction of compound A1.

Tert-butyl 4-(4-((5,11-dimethyl-6-oxo-6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4] diazepin-2-yl)amino)-3-ethoxyphenyl)piperazine-1-carboxylate(20)

White solid; yield 29%; m.p. 237–238 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.29 (d, J = 8.5 Hz, 1H), 8.13 (s, 1H), 7.86 (dd, J = 7.8, 1.7 Hz, 1H), 7.49 (s, 1H), 7.46–7.40 (m, 1H), 7.14 (t, J = 7.5 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 6.63–6.54 (m, 2H), 4.12 (q, J = 6.9 Hz, 2H), 3.61 (t, J = 5.1 Hz, 4H), 3.51 (s, 3H), 3.43 (s, 3H), 3.09 (t, J = 5.1 Hz, 4H), 1.52–1.46 (m, 12H). ¹³C NMR (101 MHz, CDCl₃) δ 168.32, 163.75, 156.01, 154.72, 151.46, 149.48, 148.10, 146.92, 132.27, 132.25, 126.61, 123.65, 123.06, 120.87, 119.02, 117.34, 108.68, 102.15, 79.89, 64.27, 50.59, 38.21, 35.89, 28.45, 14.95. HRMS-ESI (m/z): [M + H]⁺ calcd for C₃₀H₃₇N₇O₄: 560.2980, found: 560.2978.

Preparation of intermediate 21

A mixture of 21 (1 eq), trifluoroacetic acid (10 eq), and DCM was stirred at room temperature for 1.5 h. After the reaction was complete as monitored by TLC, the residue was obtained by evaporation of solvent under reduced pressure. This residue is prepared as the hydrochloride salt to afford the title intermediate 21 (100%).

2-((2-Ethoxy-4-(piperazin-1-yl)phenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(21)

Yellow solid; yield 100%; m.p. 185–186 °C; ¹H NMR (400 MHz, Methanol-d4) δ 8.15 (s, 1H), 7.85 (dd, J = 7.8, 1.7 Hz, 1H), 7.67–7.56 (m, 1.7 Hz, 2H), 7.38 (d, J = 8.0 Hz, 1H), 7.34 (t, J = 7.6 Hz, 1H), 6.92 (d, J = 2.5 Hz, 1H), 6.84 (dd, J = 8.7, 2.5 Hz, 1H), 4.20 (q, J = 7.0 Hz, 2H), 3.64–3.59 (m, 4H), 3.57 (s, 3H), 3.50 (s, 3H), 3.49–4.46 (m, 4H), 1.38 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CD₃OD) δ 167.59, 167.46, 145.13, 137.19, 133.26, 131.81, 125.34, 125.09, 120.16, 119.20, 108.85, 102.33, 64.31, 46.87, 43.07, 38.30, 36.44, 13.68. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₅H₂₉N₇O₂: 460.2455, found: 460.2449.

Preparation of compound D7

A mixture of 21 (1 eq), glucose (1.5 eq), and acetic acid (1 eq) in ethanol was heated to reflux for 8 h. After the reaction was complete as monitored by TLC, the residue was obtained by evaporation of solvent under reduced pressure. Purification by silica gel column chromatography (eluant: DCM/MeOH, 30:1 v/v) gave compound D7 (82%).

2-((2-Ethoxy-4-(4-(((2R,3S,4R,5R)-2,3,4,5-tetrahydroxytetrahydro-2H-pyran-2-yl)methyl)piperazin-1-yl)phenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one(D7)

Yellow solid; yield 82%; m.p. 126–127 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.25 (d, J = 8.5 Hz, 1H), 8.12 (s, 1H), 7.85 (dd, J = 7.8, 1.7 Hz, 1H), 7.54 (s, 1H), 7.46–7.40 (m, 1H), 7.14 (t, J = 7.3 Hz, 1H), 7.09 (d, J = 8.3 Hz, 1H), 6.59–5.64 (m, 2H), 4.11 (q, J = 7.0 Hz, 2H), 4.03 (d, J = 12.4 Hz, 2H), 3.92–3.69 (m, 7H), 3.57 (d, J = 9.4 Hz, 1H), 3.50 (s, 3H), 3.42 (s, 3H), 3.19–3.10 (m, 5H), 2.97 (d, J = 13.3 Hz, 1H), 2.76–2.66 (m, 2H), 2.08 (s, 1H), 1.47 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.42, 163.82, 155.99, 151.37, 149.45, 148.21, 146.76, 132.34, 132.24, 126.52, 123.69, 122.70, 120.76, 119.22, 117.38, 108.17, 101.68, 96.71, 71.56, 70.89, 69.27, 64.28, 62.79, 61.16, 54.32, 50.42, 38.29, 35.90, 14.95. HRMS-ESI (m/z): [M + H]⁺ calcd for C₃₁H₃₉N₇O₇: 622.2984, found: 622.2996.

Biological evaluation

In vitro kinase inhibition assay

Eurofins (Belgium) conducted in vitro DCLK1 and LRRK2 kinase activity assays. Compounds were sent as dry powders to Eurofins.

In vitro cytotoxicity assay

A 96-well plate (3599, Corning, USA) was inoculated with 100 μL of complete medium containing 10 000 cells, and the experimental, control, and blank groups were established. Three parallel wells were used per concentration. After 10 h, media from the experimental and control groups were replaced with a complete medium containing the indicated concentration of the compound. Cells were incubated for 72 h before 10 μL of CCK8 reagent (CA1210, Solarbio, China) was added to each well and incubated for 1 h. The absorbance at 450 nm was measured with a microplate reader (Tecan, Switzerland). The IC₅₀ was determined using GraphPad Prism 8 and nonlinear regression curve fitting.

Molecular modelling

Docking results were carried out by Schrödinger Maestro and Pymol. All parameters follow the default values. The DCLK1 crystal structure was acquired from RCSB Protein Data Bank (http://www.rcsb.org, PDB ID: 5JZN). The compound was prepared with ChemDraw and Schrödinger Maestro.

Consent form

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Disclosure statement

The authors report no conflicts of interest.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (Grant Nos. 81872738 and 81772565) and the National Key R&D Program of China (2022YFF1203005 and 2022YFC2303700).