Scaffold Hopping-Guided Design of Novel PIM-1 Inhibitors with Anticancer Activities

TL;DR

Researchers designed new PIM-1 kinase inhibitors that show strong anticancer activity and drug-like properties.

Contribution

A scaffold-hopping strategy led to the development of a novel PIM-1 inhibitor with potent activity and favorable stability.

Findings

Compound C2 inhibits PIM-1 kinase with an IC50 of 33.02 ± 1.31 nM.

Compound C2 effectively inhibits MM.1S cell proliferation with an IC50 of 1.87 μM.

Compound C2 shows good stability in simulated gastrointestinal fluids and rat plasma.

Abstract

PIM kinases, as members of the serine/threonine kinase family, regulate key cellular processes such as proliferation, apoptosis, and metabolism by phosphorylating multiple substrates, making them important therapeutic targets for cancer treatment. In this study, we reported a series of structurally novel PIM-1 kinase inhibitors based on a scaffold-hopping strategy. After multiple rounds of structural optimization, the highly active compound C2 was obtained, exhibiting an IC50 of 33.02 ± 1.31 nM against PIM-1 kinase. Molecular docking results revealed that compound C2 stably bound to the hydrophobic cavity of the PIM-1 protein and formed hydrogen bond interactions with polar residues in the hinge region, thereby effectively inhibiting kinase activity. In vitro antitumor assessment demonstrated significant proliferation inhibition of the hematological tumor cell line MM.1S (IC50 = 1.87 μM), comparable to the positive control SGI-1776 (IC50 = 1.71 μM). In addition, compound C2 possessed favorable drug-like properties and excellent stability in simulated gastrointestinal fluids and rat plasma. This study provides promising lead compounds for the development of novel PIM-1-targeted anticancer drugs, which can be further optimized.

Figures

Full text

1. Introduction

The latest statistics from the International Agency for Research on Cancer (IARC) show that 20 million new cancer cases and 9.7 million cancer-related deaths were registered globally in 2022. Projections indicate that the global incidence of new cancer cases will surge to 35 million by 2050, representing a 77% increase relative to 2022 [1,2]. Thus, developing novel and efficient cancer therapeutic strategies has become a pressing priority in global biomedical research [3,4,5]. Driven by the rapid advancement of biomedical technologies, our understanding of the molecular mechanisms underlying tumorigenesis and progression has been greatly advanced in recent years [6,7,8,9]. Among such advances, precision-targeted therapeutic strategies against tumor-specific molecular targets have emerged as a mainstream approach in cancer therapy, owing to their high specificity and minimal off-target toxicity [10,11,12,13,14].

The Moloney murine leukemia virus proviral integration site kinase (PIM kinase) family constitutes a highly conserved group of serine/threonine kinases, encompassing three subtypes: PIM-1, PIM-2, and PIM-3 [15,16,17,18]. These kinases extensively regulate a spectrum of critical biological processes—including cellular transcription, translation, proliferation, apoptosis, and metabolism—through the phosphorylation of diverse substrate proteins [19,20]. Notably, PIM-1 and PIM-2, two key subtypes of this family, are frequently overexpressed in various hematologic malignancies (e.g., diffuse large B-cell lymphoma [DLBCL], multiple myeloma, and leukemia) [21] as well as solid tumors (e.g., prostate cancer, colorectal cancer, pancreatic cancer, hepatocellular carcinoma, and breast cancer) [22]. The dysregulated expression significantly amplifies oncogenic signaling cascades by phosphorylating downstream effector proteins of the PI3K/AKT/mTOR pathway, thereby driving the sustained proliferation of tumor cells, evasion of apoptosis, and invasive metastasis [23,24,25]. Given their pivotal regulatory role in tumorigenesis and progression, Pim kinases have emerged as highly promising targets for anticancer drug development [26,27].

PIM kinases play a critical role in various cancers, making the discovery and development of inhibitors for treating PIM kinase-dependent diseases of significant clinical importance. Unlike other protein kinases, the proline residue at position 123 in the hinge region of PIM kinases is a unique structural feature, providing a structural basis for developing highly selective pan-PIM kinase inhibitors [28,29]. Currently, multiple PIM inhibitors have advanced into clinical research phases. Among these, SGI-1776 was the first PIM inhibitor to enter clinical trials (NCT01239108), initially used for the treatment of relapsed/refractory leukemia before development was halted due to hERG toxicity concerns [30]. Additionally, TP-3654 is undergoing clinical evaluation in patients with advanced solid tumors (NCT03715504) [31,32], while PIM-447 (5-fluoropicolinamide) is currently in Phase I clinical trials for relapsed multiple myeloma (NCT01456689) [33]. Furthermore, compound ETH-155008 (structure undisclosed) has also entered clinical studies due to its demonstrated potent inhibitory activity in lymphoma (NCT05758610). SEL24 is a dual PIM/FLT3 kinase inhibitor currently being evaluated in Phase 2 clinical trials for the treatment of relapsed/refractory (R/R) acute myeloid leukemia (AML) [34]. The above developments provide important references and risk warnings for PIM inhibitor development, though most projects remain in Phase I or II clinical trials [35] (Figure 1). Therefore, further exploration of structurally diverse novel PIM inhibitors holds significant importance for advancing the research and development of highly selective PIM kinase inhibitors.

In summary, despite the recognized importance of Pim kinase as a therapeutic target, no drugs targeting this pathway have yet been approved for clinical use. This unmet clinical need drives ongoing exploration for novel, potent, and selective Pim kinase inhibitors [35,36]. Compound C28 (Figure 2A), reported by Amgen, is a potent pan-Pim kinase inhibitor that demonstrated tumor-suppressive activity in the KMS-12-BM mouse xenograft model. However, no further clinical development progress has been reported. Analysis of the PIM-1/C28 co-crystal structure reveals that the carbonyl oxygen of pyrrolidone in C28 forms a direct hydrogen bond with the conserved residue Lys67, while its pyrrolidone NH engages in water-mediated hydrogen-bonding interactions with Asn172 and Asp186. Additionally, the carbonyl oxygen of C28 quinazolinone moiety establishes a water-bridged hydrogen bond network with Pro123, whereas no additional hydrogen bonds are formed with the ATP-binding hinge region of Pim kinase (Figure 2B). In the C28 structure, the methylcyclopropyl group projects toward the solvent-exposed region. Though it does not engage in direct interactions with the active site pocket, its unique structural characteristics are critical for stabilizing the binding conformation [37].

Based on the analysis of the above binding mode, we employed a scaffold-hopping strategy to modify the quinazolinone ring of C28 by ring-opening. Subsequently, we restricted the molecular conformation through a ring-closing operation between the benzene ring and pyrrole ring, leading to the design and synthesis of a series of structurally novel PIM-1 inhibitors (Figure 2C). This study aims to obtain novel molecules with both good target selectivity and drug-like properties, providing new structural foundations and research clues for the development of PIM kinase inhibitors.

2. Results

2.1. Synthesis of the Designed Compounds

2.1.1. Synthesis of Compounds A1~A5

The synthesis of compounds A1~A5 is illustrated in Scheme 1. Intermediate 9, synthesized according to previously reported procedures [12,38], was coupled with commercially available intermediate 10 via a Suzuki coupling reaction to afford intermediate 11. Subsequent ring closure was mediated by bis(trichloromethyl) carbonate (triphosgene) to yield intermediate 12. The methyl ester moiety of intermediate 12 was hydrolyzed in a mixed methanol/water solution with lithium hydroxide to afford intermediate 13. The protecting group of intermediate 13 was then removed using trifluoroacetic acid (TFA) to yield intermediate 14. Finally, intermediate 14 underwent condensation with commercially available intermediates 15a–15e in the presence of the coupling reagent 2-(7-azabenzotriazol-1-yl)-N, N, N′, N′-tetramethyluronium hexafluorophosphate (HATU) to give the target compounds A1~A5.

2.1.2. Synthesis of Compounds B1~B8

The synthetic routes of compounds B1~B8 are illustrated in Scheme 2. With intermediate 11 (obtained from Scheme 1) as the starting material, it undergoes a ring-closing reaction with N, N-dimethylformamide dimethyl acetal (DMF-DMA) to afford intermediate 16. This intermediate is subjected to ester hydrolysis in an aqueous solution of methanol/lithium hydroxide to yield intermediate 17. Subsequently, intermediate 17 undergoes condensation with commercially available intermediates 15b–15c and 18a–18f in the presence of the coupling reagent HATU, giving rise to intermediates 19a–19h. Finally, the protecting groups of intermediates 19a–19h are removed by treatment with TFA, thereby affording the target compounds B1~B8.

2.1.3. Synthesis of Compounds C1~C8

The synthetic route for compound C1~C8 is illustrated in Scheme 3. Starting from commercially available compound 20, intermediate 21 was obtained via nitration with concentrated nitrosulfuric acid. Subsequently, intermediate 22 was reduced in an iron powder reduction system. Intermediate 22 underwent a Suzuki coupling reaction with intermediate 9 (prepared via Scheme 1) to construct the skeleton, yielding intermediate 23. This intermediate reacted with DMF-DMA to form intermediate 24 via a ring-closing reaction, which was then subjected to methyl ester hydrolysis in a lithium hydroxide solution in methanol/water mixture to afford intermediate 25. Intermediate 25 underwent condensation reactions with commercially available intermediates 15b–15c, 18d, and 26a–26e, mediated by the condensing agent HATU, yielding intermediates 27a–27h. Finally, intermediates 27a–27h underwent TFA deprotection to afford the target compounds C1~C8.

2.2. Target Activity Measurement

2.2.1. Target Inhibition Activity of Compounds A1~A5 on PIM-1

Based on the structural characteristics of the lead compound C28—whose methylcyclopropyl side chain forms hydrophobic interactions with the solvent-exposed region of the PIM-1 kinase active site and serves as a key structural moiety for sustaining its kinase inhibitory activity—we thus took the methylcyclopropyl group as the core reference, performed structural optimization focusing on the cycloalkyl hydrophobic skeleton, and first designed and synthesized the series A compounds (A1–A5). The inhibitory activity of these compounds against PIM-1 kinase was evaluated using homogeneous time-resolved fluorescence (HTRF) technology, with results summarized in Table 1. Activity data indicate that when the R group is methylcyclopropyl (A2), the compound exhibits preliminary inhibitory activity against PIM-1; however, when the R group is substituted with other hydrophobic groups, activity is significantly reduced or completely lost. This result suggests that although the introduction of the pyrimidinone ring is generally detrimental to activity, the limited activity exhibited by A2 indicated that the ring-closing strategy itself remains feasible.

2.2.2. Target Inhibition Activity of Compounds B1~B8 on PIM-1

Based on preliminary results, we further designed and synthesized the B series compounds (B1~B8) featuring a Pyrroloquinoline skeleton. Activity testing results are shown in Table 2. When the R group was methylcyclopropyl (B1), its inhibitory activity against PIM-1 kinase was significantly enhanced compared to the A series, with an IC_50_ value of 235.71 nM. Since the R group of the scaffold of our newly designed PIM-1 kinase inhibitors extends into the lower hinge region of the PIM protein (Figure 2C)—a region enriched in polar amino acid residues—replacing this group with saturated nitrogen-containing heterocycles (e.g., piperidine or piperazine derivatives) allowed the designed compounds to generally retain inhibitory activity against PIM-1, with IC_50_ values ranging from 170 nM to 1709 nM. Notably, compound B6, bearing a bipiperidinyl moiety, exhibited the most potent PIM-1 inhibition among the B-series compounds (IC_50_ = 172.28 nM).

Given the potent inhibitory activity of compound B6 against PIM-1 kinase, we employed molecular docking approaches to investigate its binding mode with PIM-1. As illustrated in Figure 3, B6 effectively occupies the active site cavity of PIM-1 and forms hydrogen bonding interactions with multiple key residues. Specifically, the pyrrolo[3,4-b]pyrrolidone scaffold establishes direct or water-mediated hydrogen bonds with Asp186, Lys67, and Asn172; the amide carbonyl group in the hinge region also forms direct or water-bridged hydrogen bonds with Arg122 and Glu124. In the lower hinge region, the nitrogen atoms on the bipiperidinyl ring further construct a hydrogen bond network with Asp182, Asp131, and Glu47. These coordinated interactions are likely to serve as the structural basis for the potent inhibitory activity of B6.

Based on the structural analysis, a discernible unoccupied space was observed between the benzene ring of compound B6 and the surface of the PIM-1 active site. This spatial feature suggested that introducing appropriate substituents on the benzene ring could optimize interactions within the binding pocket and potentially enhance inhibitory potency. Guided by this insight, we systematically introduced varied substituents at this position, aiming to strengthen complementary contacts with the target and generate new derivatives with improved activity and further optimization potential.

2.2.3. Target Inhibition Activity of Compounds C1~C8 on PIM-1

Based on raw material availability and synthetic feasibility, we introduced a chlorine atom at the para position of the benzene ring in compound B6 and systematically investigated the effect of different R substituents on activity, obtaining derivatives C1–C8. Activity testing revealed (Table 3) that, based on the chlorinated benzene ring, replacing the R group with a saturated nitrogen-containing heterocyclic ring and piperidine or piperazine derivatives (C1–C4) significantly enhanced PIM-1 inhibitory activity. Among these, C2 (R = bipiperidine group) exhibited the most prominent activity with an IC_50_ of 33.02 nM. Conversely, activity generally decreased when the R group was substituted with purely hydrophobic alkanes (C5–C8). This suggests that hydrophobic alkanes may fail to form critical hydrogen bonds with polar residues in the lower hinge region (e.g., Asp131, Glu47), thereby weakening binding affinity. To elucidate the structural basis underlying the enhanced activity of C2, we performed molecular docking analysis. The results revealed that the chlorine atom on its benzene ring penetrates deep into the upper hinge region of the ATP-binding pocket, forming favorable interactions with hydrophobic residues (Figure 4A), which likely underlies its superior inhibitory potency. Overlay docking indicated that C2 and B6 exhibit broadly consistent binding modes within the active site cavity (Figure 4B).

In summary, through multiple rounds of structure-based computer-aided design and optimization, we successfully obtained a series of novel PIM-1 inhibitors, including C2, exhibiting nanomolar-level inhibitory activity against PIM-1. We preliminarily elucidated the structural basis for their high activity, providing valuable lead compounds for subsequent development.

2.3. In Vitro Antitumor Activity Evaluation

To evaluate the in vitro antitumor activity of the compounds, we selected compounds with favorable target activity from Series B and Series C and tested their antiproliferative activity in two hematologic tumor cell lines (MV4-11, MOLM-13) and two myeloma cell lines (RPMI 8226, MM.1S). C28 and SGI-1776 served as positive controls. Activity results are shown in Table 4. Compounds from Series B exhibited weaker activity, with B2 showing no significant inhibition in any tested cell line (IC_50_ > 100 μM), while B6 exhibited only marginal activity against MV4-11 (IC_50_ = 98.37 ± 11.38 μM) and MM.1S (IC_50_ = 90.82 ± 17.51 μM). In contrast, the inhibitory activity of the C series compounds showed significant improvement. Compounds C1–C4 exhibited varying degrees of antiproliferative effects across multiple cell lines, with C2 demonstrating notable antiproliferative activity in all four cell types. Notably, C2 activity in MM.1S cells (IC_50_ = 1.87 ± 0.68 μM) was comparable to the positive control SGI-1776 (IC_50_ = 1.72 ± 0.97 μM), suggesting its anti-tumor potential in this model is comparable to that of the clinical candidate. The positive control C28 also exhibited potent inhibition across all cell lines (IC_50_ = 0.047–1.37 μM), while SGI-1776 demonstrated typical PIM inhibitory activity, collectively validating the reliability of the experimental system. Thus, this study identified C2, a novel PIM-1 kinase inhibitor with activity comparable to SGI-1776 in MM.1S cells. This compound serves as a lead compound with potential for further development, enabling subsequent structural optimization and research.

2.4. Prediction of Drug-like Properties for Compound C2

To evaluate the drug potential of lead compound C2, we conducted a systematic analysis of its pharmacokinetic properties using ADMET prediction software (Version 1.0), with results shown in Table 5. Prediction data indicate that C2 exhibits favorable water solubility, low hERG inhibition risk, and hepatocellular microsomal stability and absorption risk are within favorable ranges, indicating a solid pharmacokinetic foundation. Notably, C2 exhibits relatively low membrane permeability, potentially attributed to its multiple polar groups. Additionally, the CYP risk value indicates potential CYP inhibition, warranting attention during subsequent optimization. Overall, C2 demonstrates favorable performance across multiple key drugability parameters and holds potential for further development.

We determined the aqueous solubility of compound C2 and its stability in simulated gastrointestinal fluid, rat plasma, and rat liver microsomes, with the corresponding data summarized in Table 6. C2 displayed favorable aqueous solubility, with a solubility value of 317 mg/mL in water. Notably, after 12 h of incubation in simulated gastrointestinal fluid, the residual concentration of C2 exceeded 80%; following 6 h of incubation in rat plasma, its residual concentration remained above 60%, indicative of good stability in both solution and biological matrices. In contrast, C2 exhibited poor metabolic stability in rat liver microsomes, with an in vitro half-life (T_1/2_) of merely 18 min. This finding suggests that the compound may be susceptible to rapid first-pass metabolism in vivo, necessitating subsequent structural modification strategies to improve its metabolic stability.

3. Materials and Methods

3.1. Chemistry

All solvents and starting materials were commercially purchased from Bidepharm (Shanghai, China) and Aladdin (Shanghai, China) and used without further purification. Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mm silica gel GF-254 plates and visualized under UV light at 254 and 365 nm. ^1^H NMR and ^13^C NMR spectra were acquired on a Bruker AVANCE 300 spectrometer (Bruker Co., Leipzig, Germany) in deuterated solvents with tetramethylsilane (TMS) as the internal standard. ESI-MS and high-resolution mass spectrometry (HRMS) data were recorded on a Waters Q-Tof micro mass spectrometer (Waters Corporation, Milford, MA, USA). Compound purity was determined by high-performance liquid chromatography (HPLC; Shimadzu LC-20AT, Shimadzu Corporation, Kyoto, Japan) on an InertSustain C18 column (4.6 mm × 250 mm, 5 μm, GL Sciences Inc. Japan) using a methanol/water mobile phase at a flow rate of 1 mL/min, monitored at 254 nm. All target compounds exhibited a purity of >95% as confirmed by HPLC; the structural confirmation spectra of the target compounds are provided in the Supplementary Information.

3.1.1. General Procedure for Synthesis of Compounds A1–A5

The synthetic routes of the target compounds are shown in Scheme 1, Scheme 2 and Scheme 3. Intermediate 9 was synthesized according to the reported literature procedure [12,37].

tert-butyl(R)-2-(2-amino-3-(methoxycarbonyl)phenyl)-6-methyl-4-oxo-4,6-dihydr opyrrolo [3,4-b]pyrrole-5(1H)-carboxylate (11).

Intermediate 10 (2.00 g, 8.69 mmol) was dissolved in a mixture of dioxane (20 mL) and water (4 mL) in a sealed tube. Potassium phosphate (3.69 g, 17.38 mmol), [2-(2′-amino-1,1′-biphenyl)]palladium(II) (0.70 g, 0.42 mmol), and intermediate 9 (4.72 g, 13.04 mmol) were added sequentially. The tube was purged with argon for 5 min and then sealed. The reaction was stirred at 100 °C for 5 h and monitored by TLC for completion. After cooling, the mixture was diluted with water (30 mL) and extracted with ethyl acetate (3 × 60 mL). The combined organic layers were washed with saturated brine (60 mL), dried over anhydrous Na_2_SO_4_, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane/methanol, 50:1, v/v) to afford intermediate 11 (2.00 g, 5.19 mmol) in 59.7% yield. ^1^H NMR (300 MHz, DMSO-d6) δ (ppm) 11.89(s, 1H), 10.76 (s, 1H), 8.53 (s, 1H), 7.87–7.89 (m, 1H), 7.79 (s, 1H), 7.67 (s, 2H), 5.18 (s, 1H), 3.92 (s, 3H), 1.58 (s, 3H), 1.46 (s, 9H). ESI-MS: m/z 386.2 [M + H]^+^.

9-(tert-butyl)4-methyl(R)-8-methyl-6,10-dioxo-5,6,8,10-tetrahydro-9H-pyrrolo [3’,4’:4,5] pyrrolo[1,2-c] quinazoline-4,9-dicarboxylate (12).

Intermediate 11 (2.00 g, 5.19 mmol) was dissolved in dichloromethane (50 mL). Bis(trichloromethyl) carbonate (0.77 g, 2.60 mmol) was added portionwise under a nitrogen atmosphere, and the mixture was stirred at 40 °C for 4 h (reaction monitored by TLC). After cooling, the solution was concentrated under reduced pressure. Purification by silica gel column chromatography (petroleum ether/ethyl acetate = 10:1, v/v) afforded intermediate 12 as a pale yellow solid (2.00 g, 4.86 mmol, 93.7% yield). ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 10.66 (s, 1H), 8.03 (s, 1H), 7.88–7.82 (m, 1H), 7.76 (s, 1H), 7.57 (s, 1H), 5.13 (s, 1H), 3.87 (s, 3H), 1.64 (s, 3H), 1.42 (s, 9H). ESI-MS: m/z 412.4 [M + H]^+^.

(R)-9-(tert-butoxycarbonyl)-8-methyl-6,10-dioxo-5,8,9,10-tetrahydro-6H-pyrrolo [3′,4′:4,5] pyrrolo[1,2-c] quinazoline-4-carboxylic acid (13).

Intermediate 12 (2.00 g, 4.86 mmol) was dissolved in methanol (20 mL). To this solution, a solution of lithium hydroxide (0.70 g, 29.16 mmol) in water (5 mL) was added dropwise. The mixture was stirred at room temperature for 12 h and monitored by TLC. Upon completion, the pH was adjusted to weakly acidic with 2 M HCl, and the mixture was diluted with water (50 mL). The precipitated solid was collected by filtration, washed with water (5 × 10 mL), and dried in a vacuum desiccator overnight to afford intermediate 13 (1.50 g, 3.77 mmol, 77.7% yield). ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 11.05 (s, 1H), 10.66 (s, 1H), 7.95 (s, 1H), 7.85 (s, 1H), 7.76 (s, 1H), 7.48 (s, 1H), 5.13 (s, 1H), 1.64 (s, 3H), 1.42 (s, 9H). ESI-MS: m/z 398.1 [M + H]^+^.

(R)-8-methyl-6,10-dioxo-5,8,9,10-tetrahydro-6H-pyrrolo[3′,4′:4,5]pyrrolo [1,2-c] quinazoline -4-carboxylic acid (14).

Intermediate 13 (1.50 g, 3.77 mmol) was dissolved in dichloromethane (15 mL), and trifluoroacetic acid (5 mL) was added dropwise at room temperature. After stirring for 3 h (monitored by TLC), the mixture was concentrated under reduced pressure to afford crude intermediate 14 (1.01 g, 3.40 mmol, 90% yield), which was used directly in the next step without further purification. ESI-MS: m/z 298.1 [M + H]^+^.

Intermediate 14 (0.10 g, 0.34 mmol) was dissolved in anhydrous DMF (10 mL) and cooled in an ice bath. To this solution, N,N-diisopropylethylamine (0.18 mL, 1.02 mmol) and HATU (0.13 g, 0.41 mmol) were added sequentially. After stirring for 10 min, commercially available intermediates 15a–15e (each 0.68 mmol) were introduced. The ice bath was removed, and the reaction was allowed to proceed at room temperature under a nitrogen atmosphere for 4 h (monitored by TLC). Upon completion, the mixture was quenched with saturated aqueous NaHCO_3_, stirred for 5 min, and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with saturated aqueous NH_4_Cl (100 mL) and brine (100 mL), dried over anhydrous Na_2_SO_4_, filtered, and concentrated under reduced pressure. Purification by silica gel column chromatography (eluent: dichloromethane/methanol, 50:1, v/v) afforded the target compounds A1–A5.

(R)-N,8-dimethyl-6,10-dioxo-5,8,9,10-tetrahydro-6H-pyrrolo[3′,4′:4,5]pyrrolo [1,2-c]quin azoline-4-carboxamide (A1): Off-white powder, yield 60.4%, m.p. 166.2–168.4 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 11.85 (s, 1H), 7.89 (m, 2H), 7.42 (m, 2H), 7.01 (d, J = 3.0 Hz, 1H), 6.17 (d, J = 2.9 Hz, 1H), 5.78 (s, 1H), 5.27 (m, 1H), 2.39 (s, 3H), 1.66 (d, J = 6.4 Hz, 3H). HRMS(ESI): calcd. For C_16_H_14_N_4_O_3_ [M + H]^+^ 311.1066 found 311.1064.

(R)-8-methyl-N-(1-methylcyclopropyl)-6,10-dioxo-5,8,9,10-tetrahydro-6H-pyrrolo[3′,4′:4,5] pyrrolo[1,2-c]quinazoline-4-carboxamide (A2): Off-white powder, yield 52.6%, m.p. 168.3–171.8 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 12.40 (s, 1H), 9.21 (s, 1H), 8.30–8.18 (m, 2H), 7.95–7.87 (m, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.23 (s, 1H), 4.90 (q, J = 6.6 Hz, 1H), 1.58 (d, J = 6.6 Hz, 3H), 1.45 (s, 3H), 0.86–0.83 (m, 2H), 0.71 (t, J = 3.3 Hz, 2H).. HRMS(ESI): calcd. For C_19_H_18_N_4_O_3_ [M + H]^+^ 351.1379 found 351.1390.

(R)-N-(cyclopropylmethyl)-8-methyl-6,10-dioxo-5,8,9,10-tetrahydro-6H-pyrrolo [3′,4′:4,5] pyrrolo[1,2-c]quinazoline-4-carboxamide (A3): Off-white powder, yield 55.2%, m.p. 165.1–167.6 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 12.31 (s, 1H), 8.77 (d, J = 7.8 Hz, 1H), 8.24 (s, 2H), 7.97 (d, J = 7.2 Hz, 1H), 7.36 (t, J = 7.9 Hz, 1H), 7.24 (s, 1H), 4.89 (m, 1H), 2.93 (s, 2H), 1.88 (s, 1H), 1.57 (d, J = 6.6 Hz, 3H), 1.40–1.35 (m, 4H). HRMS(ESI): calcd. For C_19_H_18_N_4_O_3_ [M + H]^+^ 351.1379 found 351.1338.

(R)-N-(cyclobutylmethyl)-8-methyl-6,10-dioxo-5,8,9,10-tetrahydro-6H-pyrrolo [3′,4′:4,5] pyrrolo[1,2-c] quinazoline-4-carboxamide (A4): Off-white powder, yield 46.9%, m.p. 172.8–175.1 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 12.20 (s, 1H), 8.39 (s, 1H), 8.20 (d, J = 7.5 Hz, 1H), 8.04 (d, J = 7.7 Hz, 1H), 7.70 (s, 1H), 7.56 (t, J = 7.8 Hz, 1H), 6.97 (s, 1H), 4.57 (m, 1H), 3.31 (m, 2H), 1.43 (d, J = 6.6 Hz, 3H), 1.28 (m, 3H), 1.10 (m, 2H), 1.05–0.97 (m, 2H). HRMS(ESI): calcd. For C_20_H_20_N_4_O_3_ [M + H]^+^ 365.1535 found 365.1541.

(R)-N-cyclohexyl-8-methyl-6,10-dioxo-5,8,9,10-tetrahydro-6H-pyrrolo [3′,4′:4,5] pyrrolo [1,2-c]quinazoline-4-carboxamide (A5): Off-white powder, yield 51.1%, m.p. 171.1–173.6 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 13.03 (s, 1H), 8.60 (m, 1H), 8.18 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.43 (m, 1H), 7.25 (t, J = 7.7 Hz, 1H), 4.74 (m, 1H), 3.16 (m, 1H), 1.76 (m, 8H), 1.44 (d, J = 6.7 Hz, 3H), 1.23 (m, 2H). HRMS(ESI): calcd. For C_21_H_22_N_4_O_3_ [M + H]^+^ 378.1692 found 379.1701.

3.1.2. General Procedure for Synthesis of Compounds B1–B8

9-(tert-butyl)4-methyl(R)-8-methyl-10-oxo-8,10-dihydro-9H-pyrrolo[3′,4′:4,5] pyrrolo [1,2-c] quinazoline-4,9-dicarboxylate (16).

Intermediate 11 (2.00 g, 5.19 mmol) was dissolved in anhydrous DMF (5 mL). Under a nitrogen atmosphere, N,N-dimethylformamide dimethyl acetal (1.24 g, 10.38 mmol) was added portionwise. The mixture was stirred at 100 °C for 12 h (monitored by TLC). After cooling, the reaction was quenched with water, stirred for 5 min, and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with saturated aqueous NH_4_Cl (100 mL) and brine (100 mL), dried over anhydrous Na_2_SO_4_, filtered, and concentrated. Purification by silica-gel column chromatography (CH_2_Cl_2_/MeOH, 50:1, v/v) afforded intermediate 16 as a pale yellow solid (1.45 g, 3.66 mmol, 70.5% yield). ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 9.22 (s, 1H), 8.88 (m, 1H), 7.82 (d, J = 6.8 Hz, 1H), 7.78–7.70 (m, 1H), 5.76 (s, 1H), 5.32 (d, J = 6.5 Hz, 1H), 3.92 (s, 3H), 1.68 (d, J = 6.5 Hz, 3H), 1.55 (s, 9H). ESI-MS: m/z 396.1 [M + H]^+^.

(R)-9-(tert-butoxycarbonyl)-8-methyl-10-oxo-9,10-dihydro-8H-pyrrolo[3′,4′:4,5] pyrrolo [1,2-c] quinazoline-4-carboxylic acid (17).

Using intermediate 16 (1.00 g, 2.43 mmol) as the starting material, the reaction was conducted under conditions analogous to those for the synthesis of intermediate 13, yielding intermediate 17 (0.72 g, 1.89 mmol, 77.7% yield). ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 11.09 (s, 1H), 9.03 (s, 1H), 8.27–8.22 (m, 2H), 7.81 (s, 1H), 7.13 (s, 1H), 5.22 (s, 1H) 1.61 (s, 3H), 1.48 (s, 9H). ESI-MS: m/z 382.1 [M + H]^+^.

Synthesis of Intermediates 19a–19h: Under reaction conditions analogous to those employed for compound A1, intermediate 17 was employed as the starting material and subjected to condensation reactions with 15b–15c and 18a–18f, respectively, affording target intermediates 19a–19h.

Intermediates 19a–19h were reacted under conditions analogous to those employed for the synthesis of intermediate 14, affording target products B1–B8.

(R)-8-methyl-N-(1-methylcyclopropyl)-10-oxo-9,10-dihydro-8H-pyrrolo[3′,4′:4,5] pyrrolo[1,2-c]quinazoline-4-carboxamide (B1): Light white solid, yield 86.1%, m.p. 165.4–167.6 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 11.98 (s, 1H), 8.59 (s, 1H), 7.72 (m, 3H), 7.50 (d, J = 8.3 Hz, 1H), 6.69 (s, 1H), 4.54 (d, J = 6.9 Hz, 1H), 1.37 (d, J = 6.6 Hz, 3H), 1.25 (s, 3H), 0.50–0.44 (m, 2H), 0.28 (m, 2H). HRMS(ESI): calcd. For C_19_H_18_N_4_O_2_ [M + Na]^+^ 357.1430 found 357.1322.

(R)-N-(cyclopropylmethyl)-8-methyl-10-oxo-9,10-dihydro-8H-pyrrolo[3′,4′:4,5] pyrrolo[1,2-c]quinazoline-4-carboxamide (B2): Light white solid, yield 81.7%, m.p. 166.8–169.1 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 12.17 (s, 1H), 8.54 (s, 1H), 8.20 (m, 1H), 8.07 (m, 1H), 7.68 (s, 1H), 7.57 (t, J = 7.8 Hz, 1H), 6.95 (d, J = 1.5 Hz, 1H), 4.57 (m, 1H), 3.93 (m, 2H), 1.44 (d, J = 6.6 Hz, 3H), 1.27 (m 1H), 0.60–0.53 (m, 2H), 0.49 (t, J = 4.0 Hz, 2H). ^13^C NMR (101 MHz, DMSO-d6) δ 167.68, 152.88, 143.18, 143.08, 138.65, 131.56, 129.10, 125.66, 120.91, 119.96, 114.09, 113.85, 105.08, 104.95, 104.30, 48.94, 36.41, 20.19. HRMS(ESI): calcd. For C_19_H_18_N_4_O_2_ [M + Na]^+^ 357.1430 found 357.1317.

(R)-N-isopropyl-8-methyl-10-oxo-9,10-dihydro-8H-pyrrolo[3′,4′:4,5]pyrrolo[1,2-c]quinazo line-4-carboxamide (B3): Light white solid, yield 73.5%, m.p. 162.2–165.8 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 12.14 (s, 1H), 8.52 (s, 1H), 8.15 (d, J = 6.6 Hz, 1H), 8.01 (d, J = 7.5 Hz, 1H), 7.63 (s, 1H), 7.53 (d, J = 7.3 Hz, 1H), 6.91 (s, 1H), 5.00 (s, 1H), 4.61–4.39 (m, 1H), 1.45 (m, 6H), 1.38 (d, J = 5.8 Hz, 3H). HRMS(ESI): calcd. For C_18_H_18_N_4_O_2_ [M + Na]^+^ 345.1430 found 345.1318.

(R)-8-methyl-N-(2-(4-methylpiperazin-1-yl)ethyl)-10-oxo-9,10-dihydro-8H-pyrrolo[3′,4′:4,5] pyrrolo[1,2-c]quinazoline-4-carboxamide (B4): Light white solid, yield 78.3%, m.p. 179.6–182.5 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 12.15 (s, 1H), 8.39 (s, 1H), 8.18 (m, 1H), 8.03 (d, J = 7.6 Hz, 1H), 7.67 (s, 1H), 7.55 (m, 1H), 6.95 (s, 1H), 4.56 (m, 1H), 4.14 (s, 2H), 2.64 (d, J = 5.8 Hz, 2H), 2.50–2.38 (m, 4H), 2.28 (s, 4H), 2.13 (s, 3H), 1.42 (m, 3H). HRMS(ESI): calcd. For C_22_H_26_N_6_O_2_ [M + Na]^+^ 407.2117 found 407.2189.

(R)-8-methyl-N-(2-(1-methylpiperidin-4-yl)ethyl)-10-oxo-9,10-dihydro-8H-pyrrolo [3′,4′:4,5] pyrrolo[1,2-c]quinazoline-4-carboxamide (B5): Light white solid, yield 80.2%, m.p. 178.2–180.9 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 12.17 (s, 1H), 8.51 (s, 1H), 8.20 (d, J = 7.6 Hz, 1H), 8.05 (d, J = 7.2 Hz, 1H), 7.72 (s, 1H), 7.57 (s, 1H), 6.96 (s, 1H), 4.57 (d, J = 5.7 Hz, 1H), 4.08 (s, 2H), 2.75 (d, J = 9.5 Hz, 2H), 2.15 (s, 3H), 1.83 (s, 1H), 1.69 (s, 4H), 1.43 (d, J = 5.8 Hz, 3H), 1.26 (s, 4H). HRMS(ESI): calcd. For C_23_H_27_N_5_O_2_ [M + H]^+^ 406.2165 found 406.2232.

(R)-N-([1,4′-bipiperidin]-4-yl)-8-methyl-10-oxo-9,10-dihydro-8H-pyrrolo [3′,4′:4,5] pyrrolo [1,2-c]quinazoline-4-carboxamide (B6): Light white solid, yield 71.4%, m.p. 181.2–184.7 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 12.21 (s, 1H), 8.59 (s, 1H), 8.23 (d, J = 7.0 Hz, 1H), 8.11 (d, J = 7.7 Hz, 1H), 7.76 (s, 1H), 7.62 (m, 1H), 7.02 (s, 1H), 4.65 (m, 2H), 3.07 (m, 5H), 2.43 (m, 5H), 2.09 (d, J = 11.7 Hz, 2H), 1.95 (d, J = 9.2 Hz, 2H), 1.76 (d, J = 11.6 Hz, 2H), 1.47 (d, J = 6.6 Hz, 3H), 1.39 (m, 2H). ^13^C NMR (101 MHz, DMSO-d6) δ 178.83, 167.54, 159.81, 154.58, 152.89, 146.23, 144.26, 139.05, 136.61, 114.77, 103.27, 50.09, 48.96, 47.36, 46.81, 43.54, 33.16, 28.90, 21.12, 20.15. HRMS(ESI): calcd. For C_25_H_30_N_6_O_2_ [M + H]^+^ 447.2430 found 447.2501.

(R)-8-methyl-N-(1-((4-methylpiperazin-1-yl)methyl)cyclopropyl)-10-oxo-9,10-dihydro-8H-pyrrolo[3′,4′:4,5]pyrrolo[1,2-c]quinazoline-4-carboxamide (B7): Light white solid, yield 74.5%, m.p. 183.1–185.4 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ(ppm) 12.16 (s, 1H), 8.32 (s, 1H), 8.17 (d, J = 7.0 Hz, 1H), 8.00 (d, J = 7.3 Hz, 1H), 7.71 (s, 1H), 7.53 (m, 1H), 6.96 (s, 1H), 4.55 (m, 1H), 2.26 (s, 5H),2.24 (s, 4H), 2.10 (s, 4H), 1.41 (d, J = 6.5 Hz, 3H), 1.24–1.11 (m, 2H), 1.03 (s, 2H). HRMS(ESI): calcd. For C_24_H_28_N_6_O_2_ [M + H]^+^ 433.2274 found 433.2342.

(R)-8-methyl-N-(1-(((1-methylpiperidin-4-yl)amino)methyl)cyclopropyl)-10-oxo-9,10-dihy dro-8H-pyrrolo[3′,4′:4,5]pyrrolo[1,2-c]quinazoline-4-carboxamide (B8): Light white solid, yield 68.2%, m.p. 183.3–186.8 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ(ppm) 12.18 (s, 1H), 8.33 (s, 1H), 8.19 (d, J = 7.7 Hz, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.71 (s, 1H), 7.55 (m, 1H), 6.98 (s, 1H), 4.57 (q, J = 6.6 Hz, 1H), 2.26 (s, 5H), 2.24 (s, 4H), 2.12 (s, 4H), 1.42 (d, J = 6.6 Hz, 3H), 1.26–1.15 (m, 2H), 1.04 (s, 3H). HRMS(ESI): calcd. For C_25_H_30_N_6_O_2_ [M + H]^+^ 447.2430 found 447.2500.

3.1.3. General Procedure for Synthesis of Compounds C1–C8

Intermediate 20 (5.00 g, 20.2 mmol) was added to a 100 mL round-bottom flask. Concentrated sulfuric acid (10 mL) was added dropwise under stirring at room temperature. Subsequently, the mixture was cooled in an ice bath, and concentrated nitric acid (10 mL) was added dropwise thereto. After being removed from the ice bath, the mixture was further stirred at room temperature for 10 min. The reaction mixture was then heated at 60 °C under a nitrogen atmosphere for 4 h, with reaction progress monitored by thin-layer chromatography (TLC). After cooling to room temperature, the reaction mixture was slowly poured into a 10 M aqueous NaOH solution (100 mL), stirred for 5 min, and extracted with ethyl acetate (3 × 100 mL). The combined organic extracts were washed successively with saturated aqueous ammonium chloride (NH_4_Cl, 100 mL) and brine (100 mL), dried over anhydrous sodium sulfate (Na_2_SO_4_), filtered, and concentrated under reduced pressure. Purification of the residue by silica gel column chromatography (eluent: ethyl acetate/petroleum ether = 50:1, v/v) afforded intermediate 21 as a solid (1.52 g, 5.19 mmol, yield: 25.7%). ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 7.21 (d, J = 2.2 Hz, 1H), 6.96 (d, J = 2.2 Hz, 1H), 3.84 (s, 3H). ESI-MS: m/z 293.9 [M + H]^+^.

Intermediate 21 (1.50 g, 5.13 mmol) was added to a 100 mL eggplant-shaped flask and dissolved in 20 mL of anhydrous ethanol. Reduced iron powder (2.85 g, 51.3 mmol) was added portionwise under stirring, followed by the dropwise addition of acetic acid (3.08 g, 51.3 mmol). The reaction apparatus was then heated to 90 °C under a nitrogen atmosphere for 12 h. Reaction completion was confirmed by TLC. The reaction mixture was subjected to suction filtration, and the filter cake was washed repeatedly with anhydrous ethanol. The filtrate was collected and concentrated under reduced pressure. Purification of the residue by silica gel column chromatography (eluent: ethyl acetate/petroleum ether = 50:1, v/v) afforded the target intermediate 22 (1.30 g, 4.91 mmol, yield: 95.8%). ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 7.59 (d, J = 8.1 Hz, 1H), 6.90 (d, J = 8.1 Hz, 1H), 5.74 (s, 1H), 5.63 (s, 1H), 3.87 (s, 3H). ESI-MS: m/z 263.9 [M + H]^+^.

Intermediates 27a–27h were synthesized from starting materials 9 and 22 using the same sequence of Suzuki coupling, demethylation, and condensation as described for intermediates 19a–19h. Subsequent Boc deprotection with trifluoroacetic acid afforded the final target compounds C1–C8.

(R)-N-([1,4′-bipiperidin]-4-yl)-3-chloro-8-methyl-10-oxo-9,10-dihydro-8H-pyrrolo [3′,4′:4,5] pyrrolo[1,2-c]quinazoline-4-carboxamide (C1): Light yellow solid, yield 63.5%, m.p. 188.3–191.8 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 12.17 (s, 1H), 8.51 (s, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.69 (s, 1H), 7.54 (d, J = 8.5 Hz, 1H), 6.92 (s, 1H), 4.54 (d, J = 6.8 Hz, 2H), 3.23 (s, 2H), 3.00 (s, 4H), 2.85 (m, 2H), 2.42 (s, 1H), 2.34 (d, J = 11.9 Hz, 4H), 1.98 (s, 1H), 1.89 (s, 2H), 1.68 (m, 2H), 1.39 (m, 3H). ^13^C NMR (101 MHz, DMSO-d_6_) δ 168.00, 156.03, 152.98, 148.26, 139.11, 131.54, 131.47, 126.52, 124.91, 118.44, 118.11, 105.03, 104.89, 54.79, 49.06, 48.88, 46.17, 45.76, 19.94. HRMS(ESI): calcd. For C_25_H_29_ClN_6_O_2_ [M + H]^+^ 481.2041 found 481.2108.

(R)-3-chloro-8-methyl-N-(1′-methyl-[1,4′-bipiperidin]-4-yl)-10-oxo-9,10-dihydro-8H-pyrrol o[3′,4′:4,5]pyrrolo[1,2-c]quinazoline-4-carboxamide (C2): Light yellow solid, yield 77.3%, m.p. 186.7–190.1 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ (ppm) 12.19 (s, 1H), 8.56 (s, 1H), 8.11 (d, J = 8.5 Hz, 1H), 7.74 (s, 1H), 7.60 (d, J = 8.4 Hz, 1H), 6.97 (s, 1H), 4.59 (m, 2H), 3.09 (d, J = 11.0 Hz, 2H), 2.85 (d, J = 10.9 Hz, 2H), 2.34 (m, 3H), 2.19 (s, 3H), 2.02 (d, J = 11.4 Hz, 2H), 1.96–1.87 (m, 4H), 1.76 (m, 2H), 1.44 (d, J = 6.6 Hz, 3H), 1.29 (s, 2H). ^13^C NMR (101 MHz, DMSO-d6) δ 168.98, 163.18, 149.48, 145.08, 137.96, 135.30, 133.60, 127.92, 124.89, 122.08, 119.81, 118.61, 102.15, 49.62, 45.32, 48.63, 32.02, 31.50, 21.05, 20.57, 18.01, 16.22. HRMS(ESI): calcd. For C_26_H_31_ClN_6_O_2_ [M + H]^+^ 495.2197 found 495.2151.

(R)-3-chloro-N-(1′-ethyl-[1,4′-bipiperidin]-4-yl)-8-methyl-10-oxo-9,10-dihydro-8H-pyrrolo [3′,4′:4,5]pyrrolo[1,2-c]quinazoline-4-carboxamide (C3): Light yellow solid, yield 72.8%, m.p. 189.2–192.5 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ(ppm) 12.15 (s, 1H), 8.51 (s, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.69 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 6.92 (s, 1H), 4.55 (m, 2H), 3.04 (m, 2H), 2.91 (d, J = 11.2 Hz, 2H), 2.33 (d, J = 3.6 Hz, 3H), 2.27 (s, 2H), 1.99–1.95 (m, 2H), 1.90–1.82 (m, 4H), 1.77–1.71 (m, 2H), 1.39 (m, 3H), 1.23 (s, 2H), 0.99 (m, 3H). HRMS(ESI): calcd. For C_27_H_33_ClN_6_O_2_ [M + H]^+^ 509.2354 found 509.2327.

(8R)-3-chloro-8-methyl-N-(1-(1-methylpiperidin-4-yl)pyrrolidin-3-yl)-10-oxo-9,10-dihydro-8H-pyrrolo[3′,4′:4,5]pyrrolo[1,2-c]quinazoline-4-carboxamide (C4): Light yellow solid, yield 68.1%, m.p. 184.1–187.4 °C. ^1^H NMR (300 MHz, DMSO-d6) δ (ppm) 12.26 (s, 1H), 8.71 (m, 1H), 8.12 (m, 1H), 7.73 (s, 1H), 7.53 (d, J = 8.5 Hz, 1H), 6.93 (t, J = 2.1 Hz, 1H), 5.30 (s, 1H), 4.64–4.43 (m, 1H), 3.18 (m, 2H), 2.80 (s, 2H), 2.65–2.59 (m, 1H), 2.48–2.40 (m, 1H), 2.30 (s, 1H), 2.20 (m, 3H), 2.06–1.96 (m, 3H), 1.88 (m, 3H), 1.57–1.48 (m, 2H), 1.42 (d, J = 6.6 Hz, 3H). HRMS(ESI): calcd. For C_25_H_29_ClN_6_O_2_ [M + H]^+^ 481.2041 found 481.2044.

(R)-3-chloro-8-methyl-N-(1-methylcyclopropyl)-10-oxo-9,10-dihydro-8H-pyrrolo [3′,4′:4,5] pyrrolo[1,2-c]quinazoline-4-carboxamide (C5): Light yellow solid, yield 83.1%, m.p. 167.9–169.8 °C. ^1^H NMR (300 MHz, DMSO-d6) δ (ppm) 12.33 (s, 1H), 9.03 (s, 1H), 8.05 (d, J = 3.9 Hz, 1H), 7.99 (d, J = 2.3 Hz, 1H), 7.82 (m, 1H), 7.04 (m, 1H), 4.89 (d, J = 6.8 Hz, 1H), 1.77 (s, 3H), 1.72 (m, 3H), 1.11 (m, 2H), 0.98 (m, 2H). HRMS(ESI): calcd. For C_19_H_17_ClN_4_O_2_ [M + H]^+^ 369.1040 found 369.1104.

(R)-3-chloro-N-(cyclopropylmethyl)-8-methyl-10-oxo-9,10-dihydro-8H-pyrrolo [3′,4′:4,5] pyrrolo[1,2-c]quinazoline-4-carboxamide (C6): Light yellow solid, yield 78.6%, m.p. 165.1–168.7 °C. ^1^H NMR (300 MHz, DMSO-d6) δ(ppm) 12.14 (s, 1H), 8.52 (s, 1H), 8.09 (d, J = 8.5 Hz, 1H), 7.68 (s, 1H), 7.55 (d, J = 8.3 Hz, 1H), 6.91 (s, 1H), 4.53 (m, 1H), 3.86 (m, 2H), 1.40 (d, J = 6.5 Hz, 3H), 1.23 (s, 1H), 0.54 (m, 2H), 0.45 (m, 2H). HRMS(ESI): calcd. For C_19_H_17_ClN_4_O_2_ [M + Na]^+^ 391.1040 found 391.0932.

(R)-3-chloro-N-((S)-1-cyclopropylethyl)-8-methyl-10-oxo-9,10-dihydro-8H-pyrrolo [3′,4′:4,5] pyrrolo[1,2-c]quinazoline-4-carboxamide (C7): Light yellow solid, yield 78.6%, m.p. 169.2–173.6 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ(ppm) 12.16 (m, 1H), 8.68 (d, J = 4.0 Hz, 1H), 8.08 (m, 1H), 7.69 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 6.91 (s, 1H), 4.53 (m, 1H), 4.09 (m, 1H), 1.50 (d, J = 6.9 Hz, 3H), 1.40 (d, J = 6.5 Hz, 3H), 1.24 (s, 1H), 0.71 (s, 1H), 0.50 (s, 2H), 0.29 (s, 1H). HRMS(ESI): calcd. For C_20_H_19_ClN_4_O_2_ [M + Na]^+^ 405.1197 found 405.1087.

(R)-3-chloro-N-(cyclobutylmethyl)-8-methyl-10-oxo-9,10-dihydro-8H-pyrrolo [3′,4′:4,5] pyrrolo[1,2-c]quinazoline-4-carboxamide (C8): Light yellow solid, yield 83.1%, m.p. 168.1–170.8 °C. ^1^H NMR (300 MHz, DMSO-d_6_) δ(ppm) 12.13 (s, 1H), 8.48 (s, 1H), 8.08 (d, J = 8.5 Hz, 1H), 7.68 (s, 1H), 7.54 (d, J = 8.5 Hz, 1H), 6.90 (s, 1H), 4.53 (m, 1H), 4.03 (m, 2H), 2.76 (s, 1H), 1.99 (s, 2H), 1.84 (s, 4H), 1.40 (d, J = 6.5 Hz, 3H). HRMS(ESI): calcd. For C_20_H_19_ClN_4_O_2_ [M + H]^+^ 383.1197 found 383.1157.

3.2. Biological Studies

3.2.1. PIM-1 Inhibition Assay

The PIM-1 kinase inhibitory activity was assessed by Beijing Aisiyipu Bio-technology Co. Ltd. (China) using the HTRF^®^ KinEASE™-STK assay for PIM-1 [12]. Briefly, compounds dissolved in DMSO were serially diluted and transferred to a 384-well plate. Following the addition of enzyme buffer and PIM-1 kinase solution, the plate was centrifuged (1000 rpm, 1 min) and incubated at room temperature for 40 min. A mixture of STK3 substrate and ATP was then added, and the incubation continued for another 60 min. For detection, a buffer containing Streptavidin-XL665 (PerKinElmer, cat# 62ST1PEJ) and STK Antibody-Cryptate (PerKinElmer, cat# 61ST3BLC) was added. After centrifugation, fluorescence signals (emission at 665 nm and 615 nm) were measured on a Biotek instrument, and IC_50_ values were calculated using GraphPad Prism 8.0 software.

3.2.2. Cell Lines and Cell Culture

The MV4-11, MOLM-13, MM.1S, and RPMI 8226 cell lines were obtained from the Shanghai Cell Bank, Chinese Academy of Sciences. All cell lines were cultured in RPMI 1640 or IMDM medium supplemented with 10% fetal bovine serum (FBS) and maintained at 37 °C in a humidified 5% CO_2_ incubator.

3.2.3. CellTiter-Lumi™ Luminescent Cell Viability Assay

Cells were seeded into 96-well cell culture plates at a density of 7000 cells/well. After treatment with various concentrations of the target compound serially diluted in medium for 96 h, cell viability was detected using the CellTiter-Lumi™ Steady Luminescent Cell Viability Assay Kit (CTL steady). Briefly, 100 μL of CTL steady reagent was added to each well and incubated at room temperature for an additional 10 min. The optical density values were then measured using a Thermo Multiskan Spectrum. Cell viability was calculated using the following formula: Cell viability (%) = (reading of experimental well/reading of control well) × 100%. IC_50_ values of compounds were determined using GraphPad Prism 8.0 software.

3.2.4. Aqueous Solubility Study

The solubility of compound C2 was assessed using a reported HPLC-based method [39]. A standard curve was established by analyzing a series of known concentrations (0.23–500 µg/mL) and plotting peak area against concentration. For the sample, an excess of C2 was equilibrated in 1 mL of buffer at 37 °C to form a saturated solution. After centrifugation at 3000 rpm, the clear supernatant was analyzed under the same HPLC conditions (Shimadzu LC-20AT, Shimadzu Corporation, Kyoto, Japan). The solubility was calculated by fitting the sample peak area to the standard curve.

3.2.5. Prediction of Drug-Like Properties

Predicting the ADMET properties of a compound using ADMET 10.0 follows a four-step process. First, accurate compound structures are prepared and imported, with SMILES strings or mol/sdf files recommended. Next, in the ADMET Prediction module, key parameters relevant to kinase inhibitors—including absorption, metabolism, and toxicity—are selected, and the small molecule inhibitor-specific ADMET prediction model is adopted; other parameters are kept at their default settings. Subsequently, the calculation task is submitted. Finally, the predicted results are screened and analyzed in accordance with drugability criteria.

3.2.6. Simulated Gastric and Intestinal Fluid Stability

The SGF and SIF stabilities were measured according to the previously reported method [40,41]. A solution of C2 (10 μL, 10 mM in DMSO) was added to 990 μL of SGF or SIF. The mixture was incubated at 37 °C. Samples (90 μL) were collected at 0, 0.25, 0.5, 1, 1.5, 2, 4, 8 and 12 h and analyzed using HPLC (Shimadzu LC-20AT). This assay was performed in triplicate.

3.2.7. Rat Plasma Stability

The stability of C2 in rat and human plasma was tested using the previously reported method [42]. C2 was dissolved in DMSO at a concentration of 10 mM, and 4 μL of the compound solution was incubated with 996 μL of prewarmed rat or human plasma at 37 °C. A 5-fold volume of cold acetonitrile was added to terminate the reaction at 0, 0.25, 0.5, 1, 1.5, 2, 4 and 6h. The samples were centrifuged, and the supernatant was transferred to a new 96-well plate and mixed with purified water (v:v = 1:2). The compound concentration was quantified using LC-MS/MS (LCMS-8050).

3.2.8. RLM Stability

Procedures were performed according to the previously reported method [12]. C2 was preincubated with rat liver microsomes (RLMs, 0.5 mg/mL) for 5 min at 37 °C in phosphate buffer (100 mM, pH 7.4). The reaction was initiated by adding 1 mM NADPH. After incubation at 37 °C for different times (0, 0.25, 0.5, 1, 1.5 and 2 h), cold acetonitrile was added to precipitate the protein. The samples were centrifuged, and the supernatants were analyzed by LC-MS/MS (LCMS-8050).

3.2.9. Molecular Docking

The PIM X-ray structure (6MT0) was downloaded from the Protein Data Bank (http://www.rcsb.org, accessed on 11 March 2024). Molecular docking was performed using the XP mode of the Glide program in Schrödinger Suite. Protein structures were prepared using the Protein Preparation Wizard module (default settings), which included the addition of hydrogen atoms, deletion of water molecules, and generation of het states using Epik, as well as optimization and minimization using the Maestro/Protein Preparation Wizard. Binding pockets of proteins were generated using the lattice generation tool with default parameters. Molecular structures were generated using Maestro/LigPrep. Molecular docking between target proteins and prepared molecules was performed using the GLIDE docking wizard. Binding patterns were analyzed using Pymol.

4. Conclusions

In summary, a series of novel tetracyclic small-molecule compounds were designed and synthesized based on a scaffold-hopping strategy, which exhibited favorable inhibitory activity against PIM-1 kinase. Through iterative structural optimization, compound C2 was identified as a compound with further optimization potential, exhibiting significant inhibitory activity against PIM-1 kinase (IC_50_ = 33.02 ± 1.31 nM). Molecular docking studies revealed that C2 occupies the ATP-binding pocket of PIM-1 and forms an extensive hydrogen-bond network with key residues (Lys67, Asp186, Asn172, Arg122, Glu124), which likely underlies its favorable inhibitory efficacy. In vitro antitumor evaluation demonstrated that C2 possesses notable antiproliferative activity against hematological tumor cell lines, with potency comparable to the clinical candidate SGI-1776 in the MM.1S myeloma model (IC_50_ = 1.87 μM vs. 1.72 μM). Preliminary ADMET predictions and stability assays indicated that C2 exhibits favorable drug-like properties, including good aqueous solubility and stability in simulated gastrointestinal fluids and plasma, though its metabolic stability in liver microsomes requires further improvement. Collectively, this study provides a structurally novel and pharmacologically active PIM-1 inhibitor, C2, which serves as a valuable lead compound for subsequent optimization and development of targeted anticancer therapies. Future work will focus on enhancing metabolic stability and evaluating in vivo efficacy in relevant tumor models.