Rational Design and Synthesis of Matrine Containing Coumarin Derivatives as Hsp90 (NTD&amp;CTD) Isoform selective Inhibitors for the Treatment of Lung Carcinoma

doi:10.21203/rs.3.rs-4632508/v1

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Rational Design and Synthesis of Matrine Containing Coumarin Derivatives as Hsp90 (NTD&CTD) Isoform selective Inhibitors for the Treatment of Lung Carcinoma

https://doi.org/10.21203/rs.3.rs-4632508/v1

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Matrine serves as the molecular backbone, targeting the Hsp90 protein N-terminal domain (NTD) and C-terminal domain (CTD), both highly expressed in lung tumor cells. In this study, Matrine Contains Coumarins derivatives were designed and synthesized based on our previously reported compound C. Employing primary structure-activity relationships and docking analysis, a series of derivatives were biologically assessed for their antiproliferative effects against three cancer cell lines: A549, HepG-2, and HeLa cells. Based on the bioactivity results, derivative 5a emerged as the most potent, significantly enhancing antiproliferation against A549, HepG-2, and HeLa cells, with IC₅₀ values of 7.35 ± 0.097, 7.727 ± 0.10, and 8.02 ± 0.065 µM, respectively. Subsequent mechanistic investigations confirmed 5a's ability to inhibit A549 cell proliferation and suppress colony formation and migration. In in vivo studies utilizing a xenograft mouse model inoculated with A549 cells in female Balb/c nude mice, compound 5a displayed superior antitumor activity compared to reference compounds 5-Fluorouracil and Matrine. Notably, the tumor growth inhibition (TGI) values for 5a, 5-Fluorouracil, and Matrine were 72.4%, 64.3%, and 46.8%, respectively.

Physical sciences/Chemistry/Biochemistry

Physical sciences/Chemistry/Biosynthesis

Biological sciences/Biochemistry

Biological sciences/Cell biology

Biological sciences/Chemical biology

Biological sciences/Drug discovery

Biological sciences/Evolution

Biological sciences/Molecular biology

Physical sciences/Chemistry

Matrine-based Coumarin

Anticancer Agents

Hsp90 Inhibitors

Lung Carcinoma

Cancer represents a significant global health risk, despite the remarkable strides made in the field of medical science for cancer treatment. Consequently, substantial health concerns persist among individuals affected by this devastating disease[1]. The incidence and mortality of cancer have continued to rise over time [2]. Chemotherapy remains an essential component in the clinical treatment of solid tumors. However, various challenges, including drug resistance and unfavorable side effects, limit the widespread application of chemotherapy. It is vitally necessary to develop new anticancer medicines that are more effective and less toxic to healthy cells. Type II programmed cell death or autophagy is new and vital for preserving cellular homeostasis via lysosome-dependent degradation of cytoplasmic proteins and organelles. Recent research has demonstrated that activating autophagic cell death has a significant effect on cancer treatment [3] and autophagic cell death has become more and more widely used in the field of tumor treatment [4].

Long identified as an anticancer target, Hsp90 is an ATP-dependent chaperone necessary for various protein assembly and folding processes. Recently, scientists discovered that Hsp90 plays a significant role in inflammation, neurodegenerative disease and viral infection. Developing drugs that inhibit this chaperone might help treat these previously untreatable conditions. Numerous client proteins implicated in cancer pathogenesis have been identified several of which possess carcinogenic properties. Notable examples include the tumor protein 53 (p53), the human epidermal growth factor receptor 2 (HER2/neu), signal transduction proteins Braf and Akt, and cell cycle regulators CDK4/6. These proteins play pivotal roles in critical cellular processes associated with cancer development, progression, and therapeutic response. [5, 6]. A promising target for anticancer medicines has been identified as Hsp90 due to its involvement in crucial cancer activities [7]. Hsp90 is essential for the development of tumors and the expression of such an enzyme is significantly correlated with tumor malignancy and a poor prognosis. Hsp90 inhibitors affect more than 30 oncoproteins directly impacting several oncogenic processes [8]. As a result of Hsp90's prominence, it has become an attractive drug target for treating multiple diseases [9]. Several studies have discovered that it can be used as a potential strategy for cancer treatment [10]. According to various interaction sites, Hsp90 inhibitors are generally categorized as N-terminal, C-terminal and middle-domain inhibitors. The main clinical downside of Hsp90 N-terminal domain (NTD) inhibitors is the heat shock response (HSR). This pro-survival mechanism is activated by N-terminal Hsp90 inhibitors leading to increased Hsp70 transcription [11]. Due to NTD inhibitors' disadvantages, CTD and MD inhibitors were developed. The issues posed by NTD inhibitors motivated the development of C-terminal domain (CTD) and middle domain (MD) inhibitors. The discovery of the Hsp90 CTD ATP-binding site has led to the discovery of specific CTD inhibitors as potent anticancer therapeutic options including Novobiocin, a natural drug that contains coumarin [12, 13] and Gambogic acid was also a selective inhibitor that binds to Hsp90 middle domain Fig. 1. [14]. Twenty Hsp90 inhibitors have enrolled in clinical studies since this time [15]. Therefore, the imperative to create new Hsp90 inhibitors is still very important [16].

Traditional Chinese medicine is a rich natural product library containing several natural medicines with pharmacological effects. Matrine, a primary constituent found in leguminous plants like Sophora flavescens and Sophora alopecuroides, has demonstrated clinical potential. Studies have indicated that the combination of matrine with other anticancer drugs notably enhances patients' immune capacity and treatment efficacy [17]. Currently, the scientific literature on the synthesis of matrine derivatives is significant, indicating that matrine has various pharmacological properties and clinical potential. Moreover, matrine has several advantages over traditional Chinese medicine injections in terms of quality control as a one-component drug [18]. Synthesis of anticancer drugs such as matrine-containing coumarins derivatives is very attractive[19]. Previously, our group focused on matrine derivatives bearing benzo-α-pyrone structures scaffold, a privileged structure with a broad pharmacological activity that exerted potent anticancer activity by inhibiting lung cancer cell proliferation in vitro and in vivo with no apparent side effects [20]. This study builds upon the findings of our previous investigations, as we continue our research efforts in designing and synthesizing novel matrine-containing coumarin derivatives with a specific focus on targeting Hsp90 inhibitors for the treatment of Lung Carcinoma. The primary objective of our current work is to enhance the antiproliferative efficacy of these compounds against cancer cells. To achieve this, we have implemented strategic structural modifications to the matrine scaffold, aiming to optimize their pharmacological properties and bolster the potency of these Hsp90 inhibitors in combating cancer, as illustrated in Fig. 2.

The importance of coumarin has led to many efforts to develop new synthetic procedures. There is a wide range of applications of coumarins in biology, medicine, and polymer science [21]. Modern and recent medicine uses several coumarins as anticancer activities[22]. Organic and medicinal chemists have synthesized bioactive natural products containing coumarin heterocyclic nucleus. A variety of methods can be used to synthesize coumarins including the Perkin reaction [23], Knoevenagel condensation[24], Pechmann condensation [25], Wittig reaction, Baylis Hillman reaction, Claisen rearrangement, Vilsmeier-Haack and Suzuki cross‐coupling reaction[26]. The medicinal properties of coumarins have been studied by several authors[27].

In our quest to develop Hsp90 inhibitory autophagy activators with heightened anticancer potential, we undertook a series of modifications on matrine-based coumarin compounds. These alterations encompassed substituting the 12-N atom with various benzoyl halides, acyl halides, or sulfonyl halides, alongside exploring variations in the carboxyl group at the C-11 position. To evaluate the cytotoxicity of the synthesized compounds, we conducted the 3-(4,5-dimethylthiazol-2-diphenyltetrazolium) bromide (MTT) assay using three distinct cancer cell lines: human cervical carcinoma (HeLa), human hepatocellular carcinoma (HepG-2), and human lung adenocarcinoma (A549) cells. Additionally, we carried out structure-activity relationship (SAR) analysis, providing valuable insights into the correlation between compound structure and anticancer activity, as illustrated in Fig. 3[28]. To deepen our comprehension of the most effective compound's anticancer mechanism, we explored its potential as an autophagy-inducing agent. We further evaluated the compound's impact on colony formation, cellular migration, and apoptosis, as well as investigated in vivo antitumor efficacy and toxicity. Moreover, docking studies were conducted to assess the design of the target compounds (a, b, and c). Importantly, to the best of our knowledge [29].

Employing matrine as the foundational scaffold, our investigation was centered on targeting the N-terminal domain (NTD) and C-terminal domain (CTD) of the Hsp90 protein. Informed by insights derived from the two-dimensional crystal structures of the Hsp90 protein NTD (PDB code: 3T0Z) and CTD (PDB code: 2CG9), recognized for their upregulated expression in tumor cells, a specific array of matrine inhibitors was meticulously crafted to interact with both the NTD and CTD domains. This deliberate strategy resulted in the development of a group of matrine compounds intricately designed to engage with and modulate both the NTD and CTD regions of the Hsp90 protein.

3.1. Chemistry

As a part of this investigation, we synthesized different series of new compounds containing other various moieties in one structure. To obtain primary groups important for Hsp90 inhibitory activity target compounds, we first designed three matrine-based coumarin derivatives as shown in Fig. 2. The matrinic acid derivatives were efficiently synthesized according to the protocol outlined in Scheme 1. The detailed chemical structures of compounds are organized in Tables 1, 2, and 3. The experimental section describes a novel target compound synthesis using commercially available matrine as the initial material. The primary synthetic pathway involved the hydrolysis of matrine 1 with aqueous KOH to obtain intermediate 2, which was then subjected to esterification through the addition of SOCl₂ and an alcohol reaction, resulting in intermediate 3. Subsequent substitution reactions with acyl, sulfonyl, or benzoyl halides and anhydrous potassium carbonate yielded intermediate 4, further hydrolyzed to produce intermediate 5 with a 90% yield [30].

Reduction reactions were instrumental in transforming functional groups, with selective reduction of the carbonyl group yielding allyl alcohol compounds 6, 9, and 10 using reducing LiAlH₄ agent. The drop-wise addition of LiAlH₄ to the esters significantly enhanced product yield, as validated by literature reports [31]. Additionally, matrinic acid 2 was processed to yield protected 12 N-Fmoc Matrinic acid 7 and 12 N-Boc Matrinic acid 8 with a higher yield of 85% [32] as depicted in Scheme 1.

In another synthesis pathway, 3-aminocoumarins 14 were synthesized through Dakin's procedure from N-acetylglycine 11 and various salicylaldehyde derivatives 12. Hydrolysis of 3-acetamidocoumarins 13 was conducted using H₂SO₄, with 70% H₂SO₄ exclusively yielding 3-aminocoumarin within 15 minutes [33, 34]. Coumarin-3-carboxylic acids 16 were efficiently synthesized through Knoevenagel condensation between salicylaldehyde derivatives 12 and Meldrum's acid 15 utilizing sodium azide or potassium carbonate as catalysts [35, 36] as depicted in Scheme 2.

The synthesis of target compounds (a) involved the reaction of matrinic acid (5, 7, 8) with 3-aminocoumarins 14 in the presence of EDC, HOBt, and NMM, resulting in targets (1a-21a) [37, 38]. Furthermore, DCC esterification emerged as a pivotal methodology in the esterification reactions of Coumarin-3-carboxylic acids 16 and matrine alcohols (6, 9, 10). The addition of a small quantity of 4-methylaminopyridine (DMAP) as a catalyst in the reaction system accelerated the reaction, enhancing the conversion rate and yielding compound (1b-8b) effectively [30]. Additionally, the synthesis of compound (c) from matrinic acid (5, 7, 8) and 2-hydroxybenzaldehyde derivatives using anhydrous potassium acetate and acetic anhydride yielded target compounds (1c-16c) with high yields of 60–70% as depicted in Scheme 3[39].

3.2. Biological evaluation and SAR study

3.2.1. In vitro biological activity

In the context of in vitro cell growth activities, all synthesized matrine-based coumarin derivatives (a, b, and c) were evaluated against three human cancer cell lines, including HeLa (human cervical cancer cell line), A549 (Human lung cancer cells) and HepG-2 (Human Hepatoma Cells) serving as subjects for the MTT assay, with matrine employed as the positive control. The IC₅₀ values of target matrine derivatives (a, b and c) were illustrated in Tables 1, 2 and 3. According to Table 1, the parent matrine displayed unsatisfactory growth activities across these three cancer cell lines, all target compounds exhibited moderate to potent growth activities against the tested cell lines. In an effort to identify more potent inhibitors of Hsp90 a series of matrine-based coumarin derivatives were synthesized and initial structure-activity relationships (SARs) were established. The SARs observed between matrine-based coumarin compounds and their Hsp90 inhibitory activity is summarized in Fig. 3. The SAR investigation involved modifications inspired by previous studies [30, 40] particularly the opening of the D-ring from matrine. Chemical modifications of the nitrogen atoms at the 12-N position were substituted with benzoyl halides, acyl halides, or sulfonyl halides and the focus shifted to variations of the carboxyl group at the C-11 attachment aiming to enhance the activity of matrine [41, 42]. Significantly, among these derivatives, compounds 5a and 4c, modified by 4-tert-butylbenzenesulfonyl chloride at the 12-N atom and carboxyl group by 3-aminocoumarins for compound 5a, and 2-hydroxy-4-methylbenzaldehyde for compound 4c, exhibited the most potent cytotoxicity with IC₅₀ values of A549, HepG-2, and HeLa (7.35 ± 0.097, 7.727 ± 0.10, 8.02 ± 0.065 µM) and (10.75 ± 0.88, 12.66 ± 1.36, 14.62 ± 2.3), respectively. Additionally, compound 1b, modified by 4-bromobenzoyl chloride at the 12-N atom and carboxyl group by coumarin-3-carboxylic acids, also demonstrated significant cytotoxicity with IC₅₀ values of A549, HepG-2, and HeLa (8.94 ± 0.58, 10.65 ± 0.07, 11.22 ± 1.25) respectively. Among the compounds tested derivative 5a exhibited the highest potency. Considering these observations, the antiproliferative efficacy of the three newly designed matrine-based coumarin derivatives on the aforementioned cell lines prompted further investigation of their cytotoxicity profile against additional cancer types. Furthermore, the most effective compound, 5a was evaluated for its cytotoxic effect on normal lung epithelial cells (BEAS-2B). As presented in Table 5S, 5a exhibited a substantially higher IC₅₀ value against normal lung epithelial cells (BEAS-2B) indicating its exceptional selectivity between A549 (human lung cancer cells) and normal lung epithelial cells (BEAS-2B). Given its potent efficacy and increased selectivity for cancer cells compound 5a was selected for further investigation of its mechanism of growth inhibition in cancer cells.

3.2.1.1. Cell viability of compound 5a against A549 cell lines

In the MTT assay, the A549 cells were studied to determine the potential anticancer mechanism of compound 5a. To evaluate its effectiveness, different concentrations of drug 5a (0, 1, 2, 4, 8, 16, and 32 µM) were administered to the A549 cells for durations of 24, 48, and 72 h. The results are depicted in Fig. 4 and Table 4S. demonstrate that the proliferation of human lung cancer cells A549 was considerably diminished in a dose and time-dependent manner by 5a.

3.2.1.2. Antiproliferative activity of compound 5a on the normal cell line BEAS-2B and A549

The objective of using drugs to cure malignant tumors is to destroy the cancer cells without causing harm or toxicity to the healthy cells or overall health. Consequently, the effect of 5a on the survival of normal lung epithelial cells (BEAS-2B) and lung cancer cells (A549) was assessed by the MTT assay at various concentrations (0, 1, 2, 4, 8, 16 and 32 µM) after a 48 h treatment. As illustrated in Fig. 5 and Table 5S. the findings establish that 5a is comparatively less harmful to normal cells in contrast to lung cancer cells.

3.2.1.3. Effect of compound 5a on cancer cell colony formation of A549 cell lines

Cancer cell colony formation was widely used to estimate neoplastic transformation. After being exposed to 5a at various concentrations for 48 h human lung cancer cell A549 exhibited evidence of cell cloning. According to Fig. 6, the number of cell clones in A549 cells was significantly reduced after treatment with 5a at various concentrations (0, 2, 4, 8, 16, and 32 µM) compared to the negative control and the inhibitory effect grew more assertive with the increase in concentration.

3.2.1.4. Antimigration activity of compound 5a on A549 cell lines

Several matrine inhibitors demonstrated anti-angiogenesis activities against tumor endothelium and angiogenesis is crucial for the progression development and metastasis of human cancer. Therefore, utilizing human lung cancer A549 cells as a model, we evaluated 5a at various concentrations (0, 2, 4 and 8 µM) for 24 and 48 h to see how it affected endothelial cells. We also observed cell migration and calculated the scratch area which is a standard method to confirm anti-angiogenesis effects. We first evaluated 5a ability to inhibit A549 migration which is a crucial stage in the development of new blood vessels as shown in Fig. 7 and Table 6S. The study's findings illustrated that within 48 hours, untreated cells repopulated the initially scraped area. Conversely, compound 5a distinctly and dose-dependently inhibited the migration of A549 cells, as indicated by the results [3].

3.2.1.5. Cell apoptosis assay of 5a on A549.

We first evaluated its anticancer activities to determine whether derivative 5a inhibitory effects on the A549 cell lines resulted from apoptosis by DAPI staining and apoptosis morphological analysis. Derivative 5a caused apoptosis after 48 h of incubation with it at various concentrations (0, 1, 2, 4, 6 and 8 µM) in DAPI staining, as demonstrated by the micronuclei of proliferating A549 cells. 5a caused a significant amount of dense blue staining and the development of apoptotic bodies as the nuclear membrane was ruptured, indicating that 5a had an anticancer activity on A549 cells as illustrated in Fig. 8A.

The specific cytotoxic effects of A549 cells were also evaluated utilizing flow cytometry for the apoptotic analysis method and double stained with annexin V-FITC/PI. As illustrated in Fig. 8B and Table 7S. we evaluated the apoptosis induced by 5a in A549 and compared it with the standard. As a result, different concentrations of 5a (0, 2, 4 and 8 µM) were used to analyze human lung cancer A549 cells for apoptosis using Annexin V-FITC. The fluorescence of Annexin V labeled with FITC can be detected by flow cytometry when it binds to necrotic cells; PI (propidium iodide) is a nucleic acid dye and normal cells and early apoptosis can be detected. PI cannot penetrate the cell membrane in normal cells and early apoptosis. However, it can penetrate the membrane of necrotic and apoptotic cells to form a red color. Annexin V-FITC and PI can be used to detect early apoptosis, late apoptosis, and necrosis. As a result, flow cytometry can be used to quantitatively determine the apoptosis rate of A549 cells. Based on the flow cytometry results shown in Fig. 8B, 5a can induce early and late apoptosis in A549 cells. With the increase of dose, the proportion of A549 cell apoptosis induced by 5a low medium and high dose groups was 6.89% ±1.12%, 8.645 ± 1.12%, and 58.79 ± 7.35%, which were significantly different from the control group 4.244 ± 0.80% (P < 0.001) (* p < 0.05, ** p < 0.01, *** p < 0.001).

3.2.1.6. The effect of compound 5a on the apoptosis-related client proteins Hsp90, Bcl-2 and Bax.

Human lung cancer cells A549 were treated with 5a (0, 2, 4 and 8 µM) for 48 h and apoptosis-related proteins were determined to understand the effects of various concentrations of drugs on related proteins. The client protein Hsp90, Bcl-2 and Bax family members play a crucial role in the process of cell apoptosis, which can be divided into two main categories. The changes in the expression levels of Hsp90, Bcl-2 and Bax in the A549 cells were detected by Western blot. As shown in Fig. 9 and Table 8S. the experiment confirmed significant changes in the expression levels. The expression of Bcl-2 and Hsp90 proteins that inhibit apoptosis decreased and the presentation of Bax a protein that promotes apoptosis increased.

3.2.2. In Vivo antiproliferative activity

3.2.2.1. Acute Toxicity Study of Compound 5a

According to the Sub-Acute toxicity assay results, compound 5a did not cause death in mice and the body weight of the treatment group gradually increased while remaining identical to that of the vehicle, Matrine and 5-Fluorouracil groups as positive control Fig. 10A and Table 9S. In addition, HE staining revealed that mice receiving 40 mg/kg of the drug did not exhibit overt toxicity in their hearts, spleens, kidneys, lungs, livers and tumors Fig. 10B. Overall, molecule 5a had reasonable pharmacokinetic parameters and was safe for oral administration in vivo.

3.2.2.2. Compound 5a inhibited A549 tumor growth in vivo

To evaluate compound 5a antitumor activity in vivo we established the A549 cell inoculated xenograft mouse model in which female Balb/c nude mice were divided into four groups: vehicle, 5-Fluorouracil, Matrine and compound 5a (40 mg/kg intraperitoneal QD). In each group, mice were given an oral treatment after three days. Following 28 days of continuous treatment as shown in Fig. 11A, B compound 5a exhibited more potent antitumor activity than 5-Fluorouracil and Matrine as the dosage increased its antitumor activity improved. The tumor growth inhibition (TGI) value of 5a, Matrine and 5-Fluorouracil was 72.4%, 64.3% and 46.8% respectively Fig. 11C. In contrast the body weight of the 5a-treated group did not differ from that of the vehicle, 5-Fluorouracil and Matrine groups further demonstrating the good safety profile of compound 5a in vivo. The relationship curves of tumor volumes at various times of treatments over 28 days after administration as shown in Fig. 11D.

In order to comprehensively assess the potency of Matrine Contains Coumarins derivatives and to provide guidance for further Structure-Activity Relationship (SAR) studies, [29] conducted molecular docking of the target compounds, elucidating their action mechanisms on the N-terminal domain (NTD) and C-terminal domain (CTD) action targets of the Hsp90 protein.[29]. Molecular docking of various designed compounds with corresponding action targets is established to establish the basis of various inhibitor molecules [40, 41]. Demonstrates compound docking into the Hsp90 ATP-binding site of NTD (PDB ID: 3T0Z) the poses of compounds 5a into the active site of protein were generated based on the scores. As seen in Fig. 12A; B the resulting docking model with minimum relative binding energy is -10.5 kcal mol^− 1 indicates that compound 5a has interaction with NTD action targets of Hsp90. The interaction between compound 5a and protein was formed through hydrogen bonds from GLY137; GLY135; ASN51 and LYS112 in protein with the carbonyl group of the amide bond in compound 5a as well as CH-π interaction and also the interaction between compound 5a and protein was formed through N-H donor bond from GLY135. The binding model of compound 5a into the Hsp90 ATP-binding site of NTD revealed several molecular interactions thought to be responsible for the observed affinity: pi–anion interaction between the benzene ring of coumarin and ASP 54 and other interactions, including Alkyl MET 98; LEU 107; ALA 55 and Van der Waals. As seen in Fig. 12C; D compound 5a docking into the Hsp90 ATP-binding active sites of CTD (PDB ID: 2CG9) formed interactions with the key amino acid residues, the resulting docking model with minimum relative binding energy is -10.2 kcal mol^− 1 indicates that compound 5a has interaction with CTD action targets of Hsp90. The interaction between compound 5a and protein was formed through hydrogen bonds from LEU454; GLU453; ASN446 and SER419 in protein with carbonyl group of the amide bond in compound 5a as well as CH-π interaction, and interaction also was formed through N-H donor bond from GLU415. The binding model of compound 5a into Hsp90 ATP-binding active sites revealed several molecular interactions thought to be responsible for the observed affinity: including carbon-hydrogen bond -OCH₃ from ASP503 and Van der Waals. This result suggests that 4-tert-Butylbenzenesulfonyl introduced to the nitrogen atom at the 12-position and 3-amino-5-methoxy-coumarin through the carboxyl group might be beneficial for anticancer activity.

In summary, Matrine Contains Coumarins derivatives were designed, synthesized and evaluated for their cytotoxic activity against three cancer cell lines (A549, HepG-2 and HeLa cells) as Hsp90 inhibitors for the treatment of lung carcinoma. SAR studies indicated that compound 5a with 4-tert-butylbenzenesulfonyl chloride at the 12-N atom and carboxyl group by 3-amino-5-methyl-2H-chromen-2-one, had the best anti-cancer activity. Based on the bioactivity results derivative 5a was the most potent compound exhibiting excellent antiproliferative activity against A549, HepG-2 and Hela cells with IC₅₀ values of 7.35 ± 0.097, 7.727 ± 0.10 and 8.02 ± 0.065 µM respectively. Additionally, derivative 5a demonstrated the most potent activity against A549 cells and was non-toxic to normal lung epithelial cells BEAS-2B. Further mechanism investigations confirmed that 5a inhibited A549 cell proliferation, and suppressed colony formation and migration. In vivo, experiments showed that 5a possessed different mechanisms against A549 cancer cells and exhibited more potent antitumor activity than Matrine and 5-Fluorouracil with a tumor growth inhibition (TGI) value of 72.4% compared to 64.3% and 46.8% respectively. Based on the in vitro and in vivo experiments, compound 5a showed promise as an antitumor agent and further studies including pharmacokinetic profiles and prophase tests are encouraged. Molecular docking analysis revealed that the interactions between 5a functionalities and residues were crucial for binding compounds to proteins. Enhancing these interactions could positively impact the inhibitory activity of the Hsp90 ATP-binding at both CTD and NTD sites. This work provides useful information for further structural modifications of these compounds and the synthesis of new, potent antitumor agents.

6.1. General Chemistry

Reagents and solvents were purchased from commercial sources and used without further purification. Reactions were monitored by thin-layer chromatography (TLC) using precoated silica gel plates (silica gel GF/UV 254), and spots were visualized under UV light (254 nm). The products were purified by flash column chromatography equipped with commercial silica gel (300–400 mesh). All the compounds were characterized with ¹H-NMR, ¹³C-NMR and MS. ¹H and ¹³C nuclear magnetic resonance (NMR) data were recorded on Bruker Avance 500 spectrometers operating at ¹H and ¹³C frequencies of 500 and 126 MHz respectively, in the indicated solvents (DMSO-d6 or CDCl₃, TMS as internal standard). Chemical shifts (δ) are in ppm relative to the residual solvent signal (DMSO-d6 with 2.48 and 39.52 ppm and CDCl₃ with 7.26 and 77.16 ppm for ¹H and ¹³C, respectively). As an internal standard, chemical shifts were given in ppm (d) relative to SiMe4. Coupling constants (J) were in hertz (Hz) and signals were designated as follows: s, singlet; d, doublet; t, triplet; m, multiple; br, broad singlet, etc. Mass spectra were obtained from a ThermoFisher LCQ Fleet (ESI). Melting points were determined in open capillary tubes on the X-4 melting point apparatus without correction. Synthesis of intermediate 1–14 is shown in the supplementary information file.

6.1.1. Synthesis of compound (1a-20a)

Matrinic acid derivatives (5,7,8) (2.10 mmol), HOBt (2.10 mmol) and EDCI (2.10 mmol) were dissolved in DCM (10 mL). After 10 min of stirring NMM (3.15 mmol) and 3-aminocoumarins 12 (3.15 mmol) were added and stirred for 12 h. and monitored the reaction by TLC. Their action mixture was poured into water and extracted with EtOAc. The organic layer was washed with saturated bicarbonate solution followed by brine solution. It was dried under anhydrous Na₂SO₄ and the solvent was evaporated. The gained residue was purified by flash column chromatography on silica gel with CH₂Cl₂ /CH₃OH as the eluent to afford the title compounds 1a-21a[44].

tert-butyl1-(4-((6-chloro-2-oxo-2H-chromen-3-yl)amino)-4-oxobutyl)octahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridine-2(3H)-carboxylate (1a)Yield 64%; Mp 185–188°C. ¹H NMR (500 MHz, CDCl₃) δ 9.29 (s, 1H, NH), 8.02 (d, J = 1.4 Hz, 1H, CH), 7.99 (s, 1H, ArH), 7.39 (dd, J = 7.5, 1.4 Hz,1H, ArH), 7.30 (d, J = 7.5 Hz, 1H, ArH), 4.29 (dd, J = 12.4, 7.7 Hz, 1H, CH), 3.21 (dd, J = 12.4, 7.7 Hz, 1H), 2.80 (dt, J = 10.8, 4.0 Hz, 1H), 2.51 (dt, J = 12.3, 5.5 Hz, 2H), 2.41 (dt, J = 12.4, 5.4 Hz, 2H), 2.30–2.23 (m, 1H), 2.19–2.09 (m, 1H), 2.04 (dd, J = 5.0 Hz, 3H), 1.92–1.84 (m, 2H), 1.83–1.78 (m, 1H), 1.62 (td, J = 7.8, 4.1 Hz, 3H), 1.57–1.49 (m, 2H), 1.47–1.43 (m, 2H), 1.42 (s, 9H, C-(CH₃)₃), 1.40–1.28 (m, 2H).¹³C NMR (126 MHz, CDCl₃) δ 174.53, 157.65, 155.50, 149.07, 128.88, 125.69, 125.46, 124.19, 123.65, 117.56, 114.16, 81.20, 67.56, 54.76, 53.21, 46.17, 40.85, 37.70, 36.88, 30.89, 28.31, 26.28, 23.44, 23.25, 22.61. HRMS (ESI): Calcd. C₂₉H₃₈ClN₃O₅, [M + H]⁺m/z: 543.2500, found 544.2949. Anal: C, 64.02; H, 7.04; Cl, 6.52; N, 7.72; O, 14.70.

4-(2-((4-(tert-butyl)phenyl)sulfonyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)-N-(6-nitro-2-oxo-2H-chromen-3-yl)butanamide(2a)Yield 60%; Light yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 9.33 (s, 1H, NH), 8.58 (d, J = 2.9 Hz,1H, ArH), 8.21 (dd, J = 14.9, 3.0 Hz, 1H, ArH), 8.05 (s, 1H, ArH), 7.71–7.50 (m, 5H, Ar-5H), 3.47 (dd, J = 24.7, 13.7 Hz, 1H), 2.91–2.81 (m, 2H), 2.51 (dt, J = 24.8, 10.9 Hz, 2H), 2.41 (dt, J = 24.8, 10.8 Hz, 2H), 2.04 (td, J = 16.2, 1.8 Hz, 2H), 1.96–1.82 (m, 2H), 1.82–1.69 (m, 3H),1.63 (t, J = 21.8 Hz, 1H), 1.57–1.49 (m, 2H), 1.50–1.43 (m, 4H), 1.44–1.35 (m, 2H), 1.32 (s, 9H, C-(CH₃)₃), 1.32–1.26 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 174.53, 155.50, 154.56, 154.40, 144.74, 133.92, 128.79, 128.32, 126.05, 125.20, 124.51, 121.90, 117.69, 113.24, 67.56, 59.57, 53.21, 48.99, 41.06, 37.70, 36.19, 34.58, 31.36, 29.06, 28.28, 26.28, 23.44, 23.25, 22.61. HRMS (ESI): Calcd. C₃₄H₄₂N₄O₇S, [M + H]⁺m/z: 650.2774, found 650.2900. Anal: C, 62.75; H, 6.51; N, 8.61; O, 17.21; S, 4.93.

tert-butyl1-(4-((6-nitro-2-oxo-2H-chromen-3-yl)amino)-4-oxobutyl)octahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridine-2(3H)-carboxylate(3a)Yield 49%; brown oil. ¹H NMR (500 MHz, CDCl₃) δ 9.27 (s, 1H, NH), 8.59 (d, J = 2.9 Hz, 1H, ArH), 8.24 (dd, J = 15.0, 2.9 Hz, 1H, ArH), 8.04 (s, 1H, CH), 7.62 (d, J = 15.0 Hz, 1H, ArH), 4.37 (dd, J = 24.8, 14.7 Hz,1H, CH), 3.04 (dd, J = 24.8, 14.6 Hz, 1H, CH), 2.51 (dt, J = 24.8, 10.9 Hz, 3H), 2.41 (dt, J = 24.8, 10.8 Hz, 2H), 2.07 (dd, J = 15.6, 2.7 Hz, 3H), 1.95 (dt, J = 16.2, 5.8 Hz, 1H), 1.86–1.76 (m, 2H), 1.77–1.68 (m, 2H), 1.67–156 (m, 3H), 1.55–1.49 (m, 2H), 1.48–1.43 (m, 2H), 1.42 (s, 9H, C-(CH₃)₃), 1.39–1.27 (m, 2H).¹³C NMR (126 MHz, CDCl₃) δ 174.53, 157.65, 155.50, 154.56, 144.74, 126.05, 125.20, 124.51, 121.90, 117.69, 113.24, 81.20, 67.56, 54.76, 53.21, 46.17, 40.85, 37.70, 36.88, 30.89, 28.31, 26.28, 23.44, 23.25, 22.61 (s). HRMS (ESI): Calcd. C₂₉H₃₈N₄O₇, [M + H]⁺m/z: 554.2740, found 555.2471. Anal: C, 62.80; H, 6.91; N, 10.10; O, 20.19.

4-(2-cinnamoyldecahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)-N-(5-methoxy-2-oxo-2H-chromen-3-yl)butanamide(4a)Yield 57%; Mp 189–192°C. ¹H NMR (500 MHz, DMSO) δ 9.22 (s, 1H, NH), 7.97 (s, 1H, CH), 7.92 (dt, J = 4.7, 2.1 Hz, 1H, ArH), 7.90 (dd, J = 3.1, 1.5 Hz, 1H, ArH), 7.59 (t, J = 13.7 Hz, 2H), 7.50 (ddd, J = 17.6, 6.3, 3.2 Hz, 1H), 7.40 (s, 1H, ArH), 7.36 (dd, J = 20.0 Hz, 1H, ArH), 7.32 (s, 1H, CH), 7.23 (dd, J = 13.6, 4.4 Hz, 1H, ArH), 7.03 (d, J = 30.2 Hz, 1H, CH), 3.83 (s, 3H, Ar-OCH₃), 3.61 (dd, J = 24.8, 14.6 Hz, 1H), 3.05 (dd, J = 24.8, 14.5 Hz, 1H), 2.63 (dt, J = 22.2, 8.2 Hz, 1H), 2.57–2.48 (m, 2H), 2.47–2.37 (m, 2H), 2.34–2.22 (m, 1H), 2.21–2.11 (m, 1H), 2.10–2.06 (m, 1H), 2.04 (dd, J = 16.3, 12.4 Hz, 1H), 1.90–1.71 (m, 3H), 1.66–1.55 (m, 3H), 1.55–1.47 (m, 2H), 1.46–1.40 (m, 2H), 1.38–1.25 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 171.53, 163.97, 155.06, 145.89, 139.85, 138.48, 136.15, 128.70 (d, J = 16.4 Hz), 128.05, 124.68, 124.14, 123.52, 119.86, 119.20, 115.18, 114.05, 67.56, 58.77, 56.83, 53.21, 46.87, 40.85, 37.70, 36.88, 30.89, 28.28, 26.28, 23.44, 23.25, 22.61. HRMS (ESI): Calcd. C₃₄H₃₉N₃O₅, [M + H]⁺m/z: 569.2890, found 569.2653. Anal: C, 71.68; H, 6.90; N, 7.38; O, 14.04.

4-(2-((4-(tert-butyl)phenyl)sulfonyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)-N-(5-methoxy-2-oxo-2H-chromen-3-yl)butanamide(5a)Yield 59%; Mp 195–196°C. 1H NMR (500 MHz, CDCl₃) δ 9.21 (s, 1H, NH), 7.93 (s, 1H, CH), 7.65–7.58 (m, 4H, ArH), 7.42–7.31 (m, 2H, ArH), 7.04 (dd, J = 15.0 Hz, 1H, Ar-1H), 3.83 (s, 3H, Ar-OCH₃), 3.82 (dd, J = 24.9, 13.7 Hz, 1H, CH), 3.37 (dd, J = 24.8, 13.7 Hz, 1H), 3.17 (dt, J = 22.0, 8.0 Hz, 1H), 2.51 (dt, J = 23.2, 12.4 Hz, 2H), 2.41 (dt, J = 14.0, 10.8 Hz, 2H), 2.05 (dd, J = 15.7, 5.4 Hz, 2H),1.85–1.70 (m, 4H), 1.60 (d, J = 21.8 Hz, 1H), 1.54–1.48 (m, 3H), 1.47–1.42 (m, 3H), 1.41–1.35 (m, 2H), 1.33 (s, 9H), 1.28 (dd, J = 11.7, 4.1 Hz, 1H).¹³C NMR (126 MHz, CDCl₃) δ 174.53, 161.08, 155.50, 154.40, 150.91, 133.92, 128.79, 128.32, 127.48, 124.19, 116.58, 114.16, 111.15, 102.14, 67.56, 59.57, 56.08, 53.21, 48.99, 41.06, 37.70, 36.19, 34.58, 31.36, 29.06, 28.28, 26.28, 23.44, 23.25, 22.61. HRMS (ESI): Calcd. C₃₅H₄₅N₃O₆S, [M + H]⁺m/z: 635.3029, found 635.2773. Anal: C, 66.12; H, 7.13; N, 6.61; O, 15.10; S, 5.04.

4-(2-(4-fluorobenzoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)-N-(2-oxo-2H-chromen-3-yl)butanamide (6a) Yield 55%; Mp 175–178°C. ¹H NMR (500 MHz, CDCl₃) δ 9.25 (s, 1H, NH), 8.12 (dd, J = 7.5, 5.2 Hz, 2H, Ar-2H), 7.95 (s, 1H, CH), 7.68 (dd, J = 7.5, 1.4 Hz,1H, ArH), 7.55 (td, J = 7.5, 1.5 Hz, 1H, ArH), 7.44 (dd, J = 8.6, 6.9 Hz, 2H, Ar-2H), 7.29 (dd, J = 7.0, 5.6 Hz, 2H, Ar-2H), 3.36 (dd, J = 12.4, 7.3 Hz, 1H), 2.99 (dd, J = 12.4, 7.3 Hz, 1H), 2.68 (q, J = 11.0, 4.1 Hz, 1H), 2.51 (dt, J = 12.3, 5.4 Hz, 2H), 2.41 (dt, J = 12.4, 5.4 Hz, 2H), 2.34–2.23 (m, 1H), 2.21–2.10 (m, 1H), 2.04 (dd, J = 5.0 Hz, 3H), 1.87–1.80 (m, 2H), 1.80–1.72 (m, 1H), 1.72–1.60 (m, 3H), 1.57–1.49 (m, 2H), 1.48–1.38 (m, 2H), 1.38–1.27 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 174.53, 173.57, 153.33, 149.07, 133.61, 131.63, 128.88, 125.69, 125.46, 124.19, 123.65, 117.56, 115.98, 114.16, 67.56, 58.99, 53.21, 47.57, 40.85, 37.70, 36.88, 30.89, 28.28, 26.28, 23.44, 23.25, 22.61. HRMS (ESI): Calcd. C₃₁H₃₄FN₃O₄, [M + H]⁺m/z: 531.2533, found 532.2344. Analysis: C, 70.04; H, 6.45; F, 3.57; N, 7.90; O, 12.04.

4-(decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)-N-(2-oxo-2H-chromen-3-yl)butanamide (7a) Yield 50%; Mp 190–192°C. ¹H NMR (500 MHz, DMSO) δ 9.25 (s, 1H, NH), 7.96 (s, 1H, CH), 7.68 (dd, J = 14.3, 2.8 Hz, 1H, ArH), 7.61–7.50 (m, 1H),1H, ArH), 7.38–7.30 (m, 2H, Ar-2H), 3.01 (dd, J = 24.8, 14.9 Hz, 1H), 2.51 (dt, J = 12.3, 5.5 Hz, 2H), 2.46 (d, J = 9.3 Hz, 1H), 2.41 (dt, J = 12.4, 5.4 Hz, 2H), 2.17–2.07 (m, 2H), 2.04 (dd, J = 6.0, 4.6 Hz, 2H), 1.86–1.66 (m, 5H), 1.61–1.48 (m, 2H), 1.51–1.45 (m, 4H), 1.45–1.29 (m, 4H).¹³C NMR (126 MHz, DMSO) δ 171.53, 155.50, 149.07, 128.88, 125.69, 125.46, 124.19, 123.65, 117.56, 114.16, 68.36, 58.08, 53.21, 47.89, 41.90, 37.70, 36.11, 32.24, 28.36, 26.36, 23.44, 23.25, 21.56. HRMS (ESI): Calcd. C₂₄H₃₁N₃O₃, [M + H]⁺m/z: 409.2365, found 409.7183. Anal: C, 70.39; H, 7.63; N, 10.26; O, 11.72.

4-(2-(4-cyanobenzoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)-N-(2-oxo-2H-chromen-3-yl)butanamide (8a) Yield 49%; Light yellow oil. ¹H NMR (500 MHz, DMSO) δ 9.25 (s, 1H, NH), 8.19 (dd, J = 15.4, 2.9 Hz, 2H, Ar-2H), 8.09 (dd, J = 15.4, 2.9 Hz, 2H, Ar-2H), 7.95 (s, 1H, Ar-CH), 7.68 (dd, J = 14.5, 2.7 Hz, 1H, Ar-H), 7.55 (dd, J = 14.6, 12.2 Hz, 1H, Ar-H), 7.36 (dt, J = 14.0, 3.1 Hz, 2H, Ar-H), 3.34 (dd, J = 24.8, 14.4 Hz, 1H), 3.00 (dd, J = 24.7, 14.3 Hz, 1H), 2.69 (dt, J = 22.0, 8.1 Hz, 1H), 2.51 (dt, J = 24.8, 10.8 Hz, 2H), 2.41 (dt, J = 24.8, 10.8 Hz, 2H), 2.34–2.14 (m, 2H), 2.10–1.97, 1.88–1.72 (m, 3H), 1.72–1.57 (m, 3H), 1.59–1.44 (m, 3H), 1.44–1.22 (m, 3H). ¹³C NMR (126 MHz, DMSO) δ 171.53, 170.91, 155.50, 149.07, 139.16, 132.92, 129.44, 128.88, 125.69, 125.46, 124.19, 123.65, 118.94, 117.56, 114.76, 114.16, 67.56, 58.99, 53.21, 47.57, 40.85, 37.70, 36.88, 30.89, 28.28, 26.28, 23.44, 23.25, 22.61 HRMS (ESI): Calcd. C₃₂H₃₄N₄O₄, [M + H]⁺m/z: 538.2580, found 538.6747. Anal: C, 71.35; H, 6.36; N, 10.40; O, 11.88.

4-(2-((4-(tert-butyl)phenyl)sulfonyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)-N-(6-methyl-2-oxo-2H-chromen-3-yl)butanamide (9a) Yield 56%; Mp 190–193°C. ¹H NMR (500 MHz, CDCl₃) δ 9.28 (s, 1H, NH), 7.96 (s, 1H, CH), 7.66–7.54 (m, 4H, Ar-4H), 7.52 (d, J = 2.9 Hz, 1H, Ar-H), 7.28 (d, J = 14.9 Hz, 1H, Ar-H), 7.19 (dd, J = 14.9, 2.8 Hz, 1H, Ar-H), 3.31 (dd, J = 24.9, 13.7 Hz, 1H, CH), 2.91 (dd, J = 24.8, 13.7 Hz, 1H), 2.60–2.51 (m, 1H), 2.51–2.41 (m, 2H), 2.41 (s, 3H, Ar-CH₃), 2.38 (dd, J = 17.7, 7.0 Hz, 1H), 2.19–2.13 (m, 3H), 2.04 (t, J = 10.6 Hz, 3H), 1.87–1.78 (m, 1H), 1.75–1.66 (m, 3H), 1.63– 158 (m, 3H), 1.57–1.47 (m, 2H), 1.47–1.41 (m, 1H), 1.40–1.35 (m, 1H), 1.32 (s, 9H, C-(CH₃)₃), 1.29 (dd, J = 13.1, 8.9 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 174.53, 155.50, 154.40, 148.30, 134.77, 133.92, 128.79, 128.32, 127.63, 125.71, 124.51, 121.57, 117.43, 113.24, 67.56, 59.57, 53.21, 48.99, 41.06, 37.70, 36.19, 34.58, 31.36, 29.06, 28.28, 26.28, 23.44, 23.25, 22.61, 21.23. HRMS (ESI): Calcd. C₃₅H₄₅N₃O₅S, [M + H]⁺m/z: 619.3080, found 620.3302. Anal: C, 67.82; H, 7.32; N, 6.78; O, 12.91; S, 5.17.

N-(6-chloro-2-oxo-2H-chromen-3-yl)-4-(2-(3-chlorobenzoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)butanamide (10a) Yield 47%; Mp 188–190°C. ¹H NMR (500 MHz, CDCl₃) δ 9.25 (s, 1H, NH), 8.02 (d, J = 2.9 Hz, 1H, ArH), 8.01 (s, 1H, CH), 7.87 (t, J = 2.9 Hz,1H, ArH), 7.85 (dt, J = 14.7, 3.2 Hz, 1H, ArH), 7.69 (dt, J = 15.0, 3.1 Hz, 1H, ArH), 7.58 (t, J = 14.8 Hz, 1H, ArH), 7.37 (dd, J = 15.0, 2.9 Hz, 1H, ArH), 7.30 (d, J = 14.9 Hz, 1H, ArH), 3.42 (dd, J = 24.8, 14.4 Hz, 1H, CH), 3.10–2.86 (m, 2H), 2.51 (dt, J = 24.7, 10.8 Hz, 2H), 2.41 (dt, J = 24.8, 10.6 Hz, 2H), 2.24–2.10 (m, 1H), 2.04 (q, J = 12.9 Hz, 3H), 2.00–1.85 (m, 1H), 1.76 (t, J = 7.1 Hz, 3H), 1.65 (ddd, J = 8.3 Hz, 3H), 1.62–1.54 (m, 2H), 1.45–1.39 (m, 1H), 1.37–1.26 (m, 3H). ¹³C NMR (126 MHz, CDCl3) δ 174.53, 169.06, 155.50, 148.29, 137.27, 134.68, 132.01, 130.13, 129.96, 129.61, 126.77, 126.37, 124.51, 123.35, 122.38, 118.80, 113.24, 67.56, 58.99, 53.21, 47.57, 40.85, 37.70, 36.88, 30.89, 28.28, 26.28, 23.44, 23.25, 22.61. HRMS (ESI): Calcd. C₃₁H₃₃Cl₂N₃O₄, [M + H]⁺m/z: 581.1848, found 581.2474. Anal: C, 67.94; H, 6.25; Cl, 6.47; N, 7.67; O, 11.68.

4-(2-(3-chlorobenzoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)-N-(2-oxo-2H-chromen-3-yl)butanamide (11a) Yield 45%; Mp 178–180°C. ¹H NMR (500 MHz, CDCl₃) δ 9.25 (s, 1H, NH), 7.95 (s, 1H, CH), 7.86 (dt, J = 12.7, 3.0 Hz, 1H, ArH), 7.82 (t, J = 3.2 Hz, 1H, ArH), 7.70 (dd, J = 7.2, 3.1 Hz, 1H, ArH), 7.67 (dd, J = 6.6, 3.4 Hz, 1H, ArH), 7.59 (d, J = 14.8 Hz, 1H, ArH), 7.54 (dd, J = 14.3, 2.9 Hz, 1H, ArH), 7.34 (ddd, J = 18.0, 8.2, 2.0 Hz, 2H, Ar-2H), 3.38 (dd, J = 24.8, 14.4 Hz, 1H, CH), 2.98 (dd, J = 24.8, 14.3 Hz, 1H), 2.68 (dt, J = 22.0, 8.2 Hz, 1H), 2.51 (dt, J = 24.8, 10.9 Hz, 2H), 2.41 (dt, J = 24.8, 10.7 Hz, 2H), 2.35–2.12 (m, 2H), 2.11–1.98 (m, 3H), 1.88–1.73 (m, 3H), 1.71–1.61 (m, 3H), 1.60–1.53 (m, 2H), 1.52–1.41 (m, 2H), 1.42–1.27 (m, 2H).¹³C NMR (126 MHz, CDCl₃) δ 174.53, 169.06, 155.50, 149.07, 137.27, 134.68, 130.13, 129.96 (s), 129.61, 128.88, 126.37, 125.69, 125.46, 124.19, 123.65, 117.56, 114.16, 67.56, 58.99, 53.21, 47.57, 40.85, 37.70, 36.88, 30.89, 28.28, 26.28, 23.44, 23.25, 22.61. HRMS (ESI): Calcd. C₃₁H₃₄ClN₃O₄, [M + H]⁺m/z: 547.2238, found 548.2740. Anal: C, 67.94; H, 6.25; Cl, 6.47; N, 7.67; O, 11.68.

4-(2-(3-bromobenzoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)-N-(2-oxo-2H-chromen-3-yl)butanamide (12a) Yield 39%; Mp 187–188°C. ¹H NMR (500 MHz, DMSO) δ 9.25 (s, 1H, NH), 8.14 (t, J = 3.0 Hz, 1H, ArH), 7.95 (s, 1H, CH), 7.89 (dt, J = 14.8, 2.9 Hz, 1H, ArH), 7.79 (dt, J = 14.8, 3.0 Hz, 1H, ArH), 7.68 (dd, J = 14.3, 2.8 Hz, 1H, ArH), 7.57 (dd, J = 13.4, 2.4 Hz, 1H, ArH), 7.50 (t, J = 14.9 Hz, 1H, ArH) 7.37–7.30 (m, 2H, Ar-2H), 3.35 (dd, J = 24.7, 14.5 Hz, 1H, CH), 2.96 (dd, J = 24.8, 14.4 Hz, 1H), 2.66 (dt, J = 22.0, 8.1 Hz, 1H), 2.51 (dt, J = 24.8, 10.9 Hz, 2H), 2.41 (dt, J = 24.8, 10.7 Hz, 2H), 2.28–2.11 (m, 2H), 2.08 (dd, J = 7.1, 2.7 Hz, 1H), 2.03 (dd, J = 16.1, 12.2 Hz, 2H), 1.88–1.73 (m, 3H), 1.71–1.61 (m, 3H), 1.60–1.53 (m, 2H), 1.52–1.41 (m, 2H), 1.42–1.27 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 171.53, 169.06, 155.50, 149.07, 136.35, 133.80, 131.78, 130.77, 128.88, 126.18, 125.69, 125.46, 124.19, 123.65, 123.31, 117.56, 114.16, 67.56, 58.99, 53.21, 47.57, 40.85, 37.70, 36.88, 30.89, 28.28, 26.28, 23.44, 23.25, 22.61. HRMS (ESI): Calcd. C₃₁H₃₄BrN₃O₄, [M + H]⁺m/z: 591.1733, found 592.2801. Anal: C, 62.84; H, 5.78; Br, 13.49; N, 7.09; O, 10.80.

N-(2-oxo-2H-chromen-3-yl)-4-(2-(phenylsulfonyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)butanamide (13a) Yield 51%; Mp 199–201°C. ¹H NMR (500 MHz, DMSO) δ 9.33 (s, 1H, NH), 7.92 (s, 1H, CH), 7.89 (dd, J = 5.8, 2.5 Hz, 1H, ArH), 7.68 (dd, J = 14.3, 2.8 Hz, 1H, ArH), 7.64 (dd, J = 9.3, 3.2 Hz, 3H, Ar-3H), 7.59–7.52 (m, 1H, ArH), 7.38–7.30 (m, 2H, Ar-2H), 3.33 (dd, J = 24.9, 13.8 Hz, 1H, CH), 2.93 (dd, J = 24.9, 13.7 Hz, 1H), 2.51 (dt, J = 50.0 Hz, 2H), 2.49 (dt, J = 20.3, 6.2 Hz, 2H), 2.41 (dt, J = 24.8, 20.9, 10.8 Hz, 2H), 2.20–2.09 (m, 1H), 2.05 (t, J = 10.6 Hz, 2H), 1.87–1.77 (m, 4H), 1.65 (d, J = 21.6 Hz, 2H), 1.52–1.47 (m, 2H), 1.46–1.42 (m, 2H), 1.41–1.35 (m, 2H), 1.36–1.27 (m, 2H).¹³C NMR (126 MHz, DMSO) δ 171.53, 155.50, 149.07, 138.88, 134.48, 129.76, 129.05, 128.88, 125.69, 125.46, 124.19, 123.65, 117.56, 114.16, 67.56, 59.57, 53.21, 48.99, 41.06, 37.70, 36.19, 29.06, 28.28, 26.28, 23.44, 23.25, 22.61. HRMS (ESI): Calcd. C₃₀H₃₅N₃O₅S, [M + H] + m/z: 549.2297, found 549.2999. Anal: C, 65.55; H, 6.42; N, 7.64; O, 14.55; S, 5.83.

4-(2-benzoyldecahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)-N-(2-oxo-2H-chromen-3-yl)butanamide (14a) Yield 61%; Mp 196–199°C. ¹H NMR (500 MHz, CDCl₃) δ 9.25, (s, 1H, NH), 7.97 (d, J = 3.3 Hz, 1H, ArH), 7.96 (s, 1H, Ar-1H), 7.93(dd, J = 3.3, 1.7 Hz,1H, CH ), 7.68 (dd, J = 14.6, 3.1 Hz, 1H, ArH), 7.60 (ddd, J = 8.2, 7.3, 4.0 Hz, 1H, ArH), 7.57–7.55 (m, 1H, Ar-1H), 7.52 (dd, J = 6.9, 2.7 Hz, 1H, Ar-1H), 7.38–7.30 (m, 2H), 3.44 (dd, J = 24.8, 14.5 Hz, 1H, CH), 3.08–2.94 (m, 2H), 2.51 (dt, J = 24.7, 10.8 Hz, 2H), 2.41 (dt, J = 24.8, 10.6 Hz, 2H), 2.14 (ddd, J = 17.0, 11.7, 1.5 Hz, 1H), 2.05 (t, J = 12.2 Hz, 2H), 2.00–1.88 (m, 3H), 1.80–1.71 (m, 3H), 1.69–1.63 (m, 2H), 1.58 (dd, J = 11.9, 8.7 Hz, 2H), 1.50 (dd, J = 20.9, 10.2 Hz, 1H), 1.45–1.40 (m, 1H), 1.39–1.28 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 174.53, 170.91, 155.50, 149.07, 135.71, 130.18, 128.88, 128.26, 125.69, 125.46, 124.19, 123.65, 117.56, 114.16, 67.56, 58.99, 53.21, 47.57, 40.85, 37.70, 36.88, 30.89, 28.28, 26.28, 23.44, 23.25, 22.61. HRMS (ESI): Calcd. C₃₁H₃₅N₃O₄, [M + H]⁺m/z: 513.2628, found 513.1701. Anal: C, 72.49; H, 6.87; N, 8.18; O, 12.46.

4-(2-(4-cyanobenzoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)-N-(6-nitro-2-oxo-2H-chromen-3-yl)butanamide (15a) Yield 53%; Mp 198–200°C. ¹H NMR (500 MHz, CDCl₃) δ 9.14 (s, 1H, NH), 8.58 (d, J = 3.1 Hz, 1H, ArH), 8.25 (d, J = 2.9 Hz, 1H, ArH), 8.21 (dd, J = 6.1, 2.7 Hz, 1H, ArH), 8.18–8.17 (m, 1H), 8.09 (dd, J = 15.5, 2.8 Hz, 2H, ArH), 8.01 (s, 1H, CH), 7.62 (d, J = 15.0 Hz, 1H, ArH), 3.66 (dd, J = 24.9, 11.4 Hz, 1H), 3.50 (dt, J = 16.8, 13.6 Hz, 1H), 2.51 (dt, J = 2.0 Hz, 2H), 2.48 (d, J = 10.9 Hz, 1H), 2.41 (dt, J = 24.8, 20.9, 10.8 Hz, 2H), 2.17–1.96 (m, 4H), 1.86 (s, 1H), 1.78–1.67 (m, 1H), 1.68–1.59 (m, 4H), 1.61–1.51 (m, 2H), 1.50–1.42 (m, 2H), 1.41–1.33 (m, 2H).¹³C NMR (126 MHz, CDCl₃) δ 174.53, 170.91, 155.50, 154.56, 144.74, 139.16, 132.92, 129.44, 126.05, 125.20, 124.51, 121.90, 118.94, 117.69, 114.76, 113.24, 67.56, 58.99, 53.21, 47.57, 40.85, 37.70, 36.88, 30.89, 28.28, 26.28, 23.44, 23.25, 22.61. HRMS (ESI): Calcd. C₃₂H₃₃N₅O₆, [M + H]⁺m/z: 583.2431, found 584.2520. Anal: C, 65.85; H, 5.70; N, 12.00; O, 16.45.

4-(2-((4-(tert-butyl)phenyl)sulfonyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)-N-(6-chloro-2-oxo-2H-chromen-3-yl)butanamide (16a) Yield 49%; Mp 195–198°C. ¹H NMR (500 MHz, CDCl₃) δ 9.28 (s, 1H, NH), 8.00 (d, J = 2.9 Hz,, 1H, ArH), 7.98 (s, 1H, CH), 7.67–7.57 (m, 4H, Ar-4H), 7.38 (dd, J = 14.8, 2.9 Hz, 1H, ArH), 7.28 (d, J = 15.0 Hz, 1H, ArH), 3.32 (dd, J = 24.8, 13.8 Hz, 1H, CH), 2.91 (dd, J = 24.8, 13.7 Hz, 1H), 2.51 (dt, J = 24.8, 10.9 Hz, 2H), 2.49 (t, J = 10.8 Hz, 1H), 2.41 (dt, J = 24.8, 10.7 Hz, 2H), 2.18–2.09 (m, 1H), 2.05 (td, J = 10.7, 0.6 Hz, 2H), 1.87–1.76 (m, 2H), 1.76–1.66 (m, 2H), 1.60 (d, J = 2.1 Hz, 1H), 1.56–1.50 (m, 2H), 1.56–1.50 (m, 2H), 1.49–1.45 (m, 3H), 1.41–1.34 (m, 3H), 1.33 (s, 9H, C-(CH₃)₃), 1.31–1.23 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 174.53, 155.50, 154.40, 148.29, 133.92, 132.01, 128.79, 128.32, 126.77, 124.51, 123.35, 122.38, 118.80, 113.24, 67.56, 59.57, 53.21, 48.99, 41.06, 37.70, 36.19, 34.58, 31.36, 29.06, 28.28, 26.28, 23.44, 23.25, 22.61. HRMS (ESI): Calcd. C₃₄H₄₂ClN₃O₅S, [M + H]⁺m/z: 639.2534, found 640.3938. Anal: C, 63.78; H, 6.61; Cl, 5.54; N, 6.56; O, 12.49; S, 5.01.

N-(6-chloro-2-oxo-2H-chromen-3-yl)-4-(2-(4-fluorobenzoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)butanamide (17a) Yield 44%; Mp 187–189°C. ¹H NMR (500 MHz, CDCl₃) δ 9.28 (s, 1H, NH), 8.12 (dd, J = 15.0, 9.9 Hz, 2H, Ar-2H), 8.02 (d, J = 2.9 Hz, 1H, ArH), 8.00 (s, 1H, CH), 7.40 (q, J = 3.2 Hz, 1H, ArH), 7.36 (dd, J = 8.9, 6.0 Hz, 2H, Ar-2H), 7.29 (d, J = 14.9 Hz, 1H, ArH), 3.40 (dd, J = 24.7, 14.5 Hz, 1H, CH), 3.11–2.94 (m, 2H), 2.51 (dt, J = 24.7, 10.7 Hz, 2H), 2.41 (dt, J = 24.8, 10.6 Hz, 2H), 2.19–2.10 (m, 1H), 2.05 (dd, J = 15.8, 9.1 Hz, 3H), 2.00–1.86 (m, 1H), 1.81–1.71 (m, 3H), 1.72–1.62 (m, 3H), 1.62–1.46 (m, 3H), 1.46–1.39 (m, 2H), 1.39–1.29 (m, 2H).¹³C NMR (126 MHz, CDCl₃) δ 174.53, 173.30, 154.16, 148.29, 133.61, 132.01, 131.63, 126.77, 124.51, 123.35, 122.38, 118.80, 115.98, 113.24, 67.56, 58.99 (s), 53.21 (s), 47.57 (s), 40.85 (s), 37.70 (s), 36.88 (s), 30.89 (s), 28.28 (s), 26.28 (s), 23.44 (s), 23.25 (s), 22.61 (s). HRMS (ESI): Calcd. C₃₁H₃₃ClFN₃O₄, [M + H] + m/z: 565.2144, found 566.2206. Anal: C, 65.78; H, 5.88; Cl, 6.26; F, 3.36; N, 7.42; O, 11.31.

4-(decahydro-1H,4H-pyrido[3,2,1-ij][, ]naphthyridin-1-yl)-N-(6-methyl-2-oxo-2H-chromen-3-yl)butanamide

(18a) Yield 39%; Mp 200–202°C. ¹H NMR (500 MHz, CDCl₃) δ 9.24 (s, 1H, NH), 7.97 (s, 1H, CH), 7.52 (d, J = 2.9 Hz, 1H, ArH), 7.31 (d, J = 15.0 Hz, 1H, ArH), 7.22 (dd, J = 15.0, 2.9 Hz, 1H, ArH), 3.01 (dd, J = 24.8, 14.9 Hz, 1H, CH), 2.51 (dt, J = 24.7, 10.7 Hz, 2H), 2.42 (s, 3H, CH₃), 2.41 (dt, J = 24.8, 10.6 Hz, 2H), 2.11 (ddd, J = 15.9, 13.8, 8.0 Hz, 2H), 2.03 (dd, J = 16.4, 9.6 Hz, 2H), 1.82–1.68 (m, 5H), 1.56–1.49 (m, 2H), 1.46 (t, J = 5.5 Hz, 3H), 1.43–1.40 (m, 2H), 1.39–1.31 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 174.53, 155.50, 148.30, 134.77, 127.63, 125.71, 124.51, 121.57, 117.43, 113.24, 68.36, 58.08, 53.21, 47.89, 41.90, 37.70, 36.11, 32.24, 28.36, 26.36, 23.44, 23.25, 21.56, 21.23. HRMS (ESI): Calcd. C₂₅H₃₃N₃O₃, [M + H]⁺m/z: 423.2522, found 423.3215. Anal: C, 70.89; H, 7.85; N, 9.92; O, 11.33.

tert-butyl 1-(4-oxo-4-((2-oxo-2H-chromen-3-yl)amino)butyl)octahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridine-2(3H)-carboxylate (19a)Yield 47%; Light yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 9.30 (s, 1H, NH), 7.96 (s, 1H, CH), 7.68 (dd, J = 14.6, 3.1 Hz, 1H, ArH), 7.59–7.52 (m, 1H, ArH), 7.34 (td, J = 14.9, 7.6 Hz, J = 10.0 Hz, 2H, Ar-2H), 4.30 (dd, J = 24.8, 15.2 Hz, 1H), 3.21 (dd, J = 24.8, 15.2 Hz, 1H), 2.83 (dt, J = 21.6, 8.0 Hz, 1H), 2.57–2.46 (m, 2H), 2.46–2.35 (m, 2H), 2.24–2.10 (m, 2H), 2.10–1.98 (m, 3H), 1.93–1.78 (m, 3H), 1.68–1.59 (m, 3H), 1.60–1.50 (m, 2H), 1.51–1.43 (m, 2H), 1.42 (s, 9H), 1.40–1.25 (m, 2H).¹³C NMR (126 MHz, CDCl₃) δ 174.53, 157.65, 155.50, 149.07, 128.88, 125.69, 125.46, 124.19, 123.65, 117.56, 114.16, 81.20, 67.56, 54.76, 53.21, 46.17, 40.85, 37.70, 36.88, 30.89, 28.31, 26.28, 23.44, 23.25, 22.61. HRMS (ESI): Calcd. C₂₉H₃₉N₃O₅, [M + H]⁺m/z: 509.2890, found 509.8866. Anal: C, 68.35; H, 7.71; N, 8.25; O, 15.70.

(9H-fluoren-9-yl)methyl1-(4-((5-chloro-2-oxo-2H-chromen-3-yl)amino)-4-oxobutyl)octahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridine-2(3H)-carboxylate(20a)Yield 52%; Mp 196–199°C. ¹H NMR (500 MHz, CDCl₃) δ 9.29 (s, 1H, NH), 8.27 (s, 1H, CH), 7.90 (dd, J = 14.7, 3.2 Hz, 2H, Ar-2H), 7.57 (t, J = 15.0 Hz, 1H, ArH), 7.41 (dd, J = 14.6, 3.4 Hz, 2H, Ar-2H), 7.34 (td, J = 14.9, 3.4 Hz, 2H, Ar-2H),7.26 (dd, J = 7.1, 3.1 Hz, 1H, ArH), 7.24 (d, J = 3.1 Hz, 2H, Ar-2H), 7.17 (dd, J = 14.9, 3.0 Hz, 1H, ArH), 5.31 (d, J = 11.8 Hz, 2H, CH2), 4.46 (dd, J = 24.8, 15.3 Hz, 1H, CH), 3.99 (t, J = 11.8 Hz, 1H), 3.37 (dd, J = 24.8, 15.4 Hz, 1H), 2.82 (dt, J = 21.6, 8.1 Hz, 1H), 2.51 (dt, J = 50.0 Hz, 2H), 2.41 (dt, J = 50.0 Hz, 2H), 2.28–2.13 (m, 2H), 2.07–1.98 (m, 3H), 1.94–1.79 (m, 3H), 1.71–1.56 (m, 3H), 1.55–1.53 (m, 2H), 1.55–1.39 (m, 3H), 1.38–1.24 (m, 1H).¹³C NMR (126 MHz, CDCl₃) δ 174.53 (s), 157.88 (s), 155.50 (s), 149.92 (s), 143.41 (s), 139.81 (s), 128.10 (s), 127.80 (s), 127.52 (s), 126.50–126.07 (m), 125.07 (s), 120.89 (s), 117.47 (s), 116.92 (s), 116.65 (s), 67.56 (s), 67.18 (s), 54.76 (s), 53.21 (s), 48.95 (s), 46.17 (s), 40.85 (s), 37.70 (s), 36.88 (s), 30.89 (s), 28.28 (s), 26.28 (s), 23.44 (s), 23.25 (s), 22.61 (s). HRMS (ESI): Calcd. C₃₉H₄₀ClN₃O₅, [M + H]⁺m/z: 665.2656, found 666.4602. Anal: C, 70.31; H, 6.05; Cl, 5.32; N, 6.31; O, 12.01.

(9H-fluoren-9-yl)methyl1-(4-((5-fluoro-2-oxo-2H-chromen-3-yl)amino)-4-oxobutyl)octahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridine-2(3H)-carboxylate (21a)Yield 56%; Mp 195–198°C. ¹H NMR (500 MHz, CDCl₃) δ 9.25 (s, 1H, NH), 8.06 (s, 1H, CH), 7.90 (dd, J = 14.7, 3.2 Hz, 2H, ArH), 7.61 (td, J = 15.0, 10.0 Hz, 1H, ArH), 7.41 (dt, J = 12.1, 6.0 Hz, 2H, ArH), 7.33 (dd, J = 14.8, 3.4 Hz, 2H, ArH), 7.24 (td, J = 14.8, 3.2 Hz, 2H, ArH), 7.13 (dd, J = 15.0, 3.0 Hz, 1H, ArH), 6.91 (ddd, J = 15.9, 14.9, 2.9 Hz, 1H, ArH), 5.31 (d, J = 11.8 Hz, 2H, CH₂), 4.46 (dd, J = 24.8, 15.4 Hz, 1H, CH), 3.99 (t, J = 11.8 Hz, 1H, CH), 3.37 (dd, J = 24.8, 15.3 Hz, 1H), 2.82 (dt, J = 21.4, 8.0 Hz, 1H), 2.51 (dt, J = 50.0 Hz, 2H), 2.41 (dt, J = 50.0 Hz, 2H), 2.24–2.10 (m, 2H), 2.10–2.00 (m, 3H), 1.94–1.78 (m, 3H), 1.72–1.57 (m, 3H), 1.57–1.49 (m, 2H), 1.48–1.42 (m, 2H), 1.40–1.23 (m, 2H).¹³C NMR (126 MHz, CDCl₃) δ 174.53, 157.88, 155.50, 153.38, 149.70, 143.41, 139.81, 127.52, 126.18, 125.07, 120.89, 116.42, 115.03, 112.00, 110.97, 67.56, 67.18, 54.76, 53.21, 48.95, 46.17, 40.85, 37.70, 36.88, 30.89, 28.28, 26.28, 23.44, 23.25, 22.61. HRMS (ESI): Calcd. C₃₉H₄₀FN₃O₅, [M + H]⁺m/z: 649.2952, found 650.7940. Anal: C, 72.09; H, 6.21; F, 2.92; N, 6.47; O, 12.31.

6.1.2. Synthesis of compound (1b-7b)

The solution of Matrinic alcohol derivatives (6,9,10) (1.0 eq) and coumarin-3-carboxylic acids derivatives 14 (2.0 eq) in 10 mL of anhydrous dichloromethane (DCM) was cooled to 0°C. DCC (77 mg, 1.5 eq) and pyridine (0.168 mL 2.0 eq) were added to this, and a catalytic amount of DMAP (0.1 eq). The reaction mixture was stirred overnight at room temperature diluted with DCM and washed with water and brine solution. The dried organic layer on column chromatography yielded the desired product [40].

4-(2-(4-bromobenzoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)butyl2-oxo-2H-chromene-3-carboxylate (1b) Yield 58%; Light yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 8.55 (s, 1H, CH), 7.96 (d, J = 2.5 Hz, 2H, Ar-2H), 7.93 (d, J = 2.3 Hz, 2H), 7.69 (d, J = 2.5 Hz, 1H), 7.67 (dd, J = 4.2, 2.4 Hz, 1H, ArH), 7.65 (dd, J = 3.8, 3.2 Hz, ArH, 1H), 7.39–7.28 (m, 3H, Ar-3H), 3.97 (t, J = 9.9 Hz, 2H), 3.36 (dt, J = 21.6, 9.2 Hz, 1H), 3.18 (dd, J = 24.7, 14.7 Hz, 1H), 2.65 (dd, J = 24.7, 14.7 Hz, 1H), 2.51 (dt, J = 24.7, 10.8 Hz, 2H), 2.41 (dt, J = 24.7, 10.7 Hz, 2H), 2.16 (t, J = 58.8 Hz, 1H), 2.08–1.99 (m, 1H), 1.84–1.70 (m, 2H), 1.71–1.67 (m, 3H), 1.64 (d, J = 8.3 Hz, 2H), 1.62–1.54 (m, 2H), 1.43 (ddd, J = 19.2 Hz, 2H), 1.35 (dt, J = 23.5, 7.5 Hz, 2H), 1.30–1.19 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 170.91, 163.31, 160.42, 152.43, 136.97, 132.91, 131.51, 131.20, 130.81, 128.55, 125.32, 124.45, 122.09, 120.12, 117.43, 67.56, 66.85, 58.99, 53.21, 47.57, 40.85, 36.88, 32.94, 29.77, 28.28, 26.28, 24.17, 23.44, 23.25. HRMS (ESI): Calcd. C₃₂H₃₅BrN₂O₅, [M + H]⁺m/z: 606.1729, found 606.3915. Anal: C, 63.26; H, 5.81; Br, 13.15; N, 4.61; O, 13.17.

4-(2-(2-naphthoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)butyl2-oxo-2H-chromene-3-carboxylate(2b)Yield 57%; Light yellow oil ¹H NMR (500 MHz, CDCl₃) δ 8.55 (s, 1H, CH), 8.40 (t, J = 1.4 Hz, 1H, ArH), 8.15 (dd, J = 7.5, 1.4 Hz, 1H, ArH), 7.90 (ddt, J = 6.7, 4.9, 1.5 Hz, 2H, Ar-2H), 7.83 (dd, J = 7.5, 1.6 Hz, 1H, ArH), 7.68 (dd, J = 7.5, 1.4 Hz, 1H, ArH), 7.64 (dd, J = 7.4, 1.5 Hz, 1H, ArH), 7.60 (td, J = 7.4, 1.6 Hz, 1H, ArH), 7.56 (ddd, J = 9.0, 8.4, 1.6 Hz, 1H, ArH), 7.36 (dd, J = 6.4, 1.2 Hz, 1H, ArH), 7.34 (dd, J = 10.0 Hz, 1H, ArH), 3.97 (t, J = 7.5 Hz, 2H), 3.49 (dd, J = 12.3, 7.4 Hz, 1H), 3.02 (dd, J = 12.5, 7.3 Hz, 1H), 2.72 (dt, J = 11.0, 4.1 Hz, 1H), 2.51 (dt, J = 12.3, 5.4 Hz, 2H), 2.41 (dt, J = 12.4, 5.4 Hz, 2H), 2.27–2.16 (m, 1H), 2.04 (t, J = 11.2 Hz, 1H), 1.96 (ddt, J = 12.0, 8.2, 5.9 Hz, 1H), 1.80–1.71 (m, 2H), 1.66 (ddd, J = 11.8, 7.2, 3.5 Hz, 2H), 1.63–1.59 (m, 2H), 1.53 (dq, J = 11.1, 5.6 Hz, 2H), 1.43 (tt, J = 10.2, 5.2 Hz, 2H), 1.39–1.30 (m, 2H), 1.25 (p, J = 7.8 Hz, 2H).¹³C NMR (126 MHz, CDCl₃) δ 169.44, 163.31, 160.42, 152.43, 134.48, 134.11, 132.91, 131.20, 129.46, 128.60, 127.71, 127.31, 126.64, 125.32, 122.09, 120.12, 118.49, 117.43, 67.56, 66.85, 58.99, 53.21, 47.57, 40.85, 36.88, 32.94, 29.77, 28.28, 26.28, 24.17, 23.44, 23.25. HRMS (ESI): Calcd. C₃₆H₃₈N₂O₅, [M + H]⁺m/z: 578.2781, found 579.1513. Anal: C, 74.72; H, 6.62; N, 4.84; O, 13.82.

4-(2-(2-methylbenzoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)butyl-5-fluoro-2-oxo-2H-chromene-3-carboxylate(3b)Yield 49%; Mp 201–203°C. ¹H NMR (500 MHz, CDCl₃) δ 8.55 (s, 1H, CH), 7.76 (dd, J = 14.9, 2.9 Hz, 1H, ArH), 7.61 (td, J = 15.0, 10.0 Hz, 1H, ArH), 7.34 (td, J = 14.8, 2.9 Hz, 1H, ArH), 7.24 (dd, J = 15.0, 3.3 Hz, 1H, ArH), 7.11 (ddd, J = 18.0, 10.6, 3.1 Hz, Ar-2H), 6.94 (ddd, J = 15.8, 15.2, 3.0 Hz, 1H, ArH), 3.97 (t, J = 15.0 Hz, 2H), 3.33 (dd, J = 24.8, 14.9 Hz, 1H), 2.87 (dd, J = 24.8, 14.8 Hz, 1H), 2.69 (dt, J = 21.4, 8.2 Hz, 1H), 2.51 (dt, J = 50.0 Hz, 2H), 2.41 (dt, J = 50.0 Hz, 2H), 2.22 (s, 3H, Ar-CH₃), 2.16–2.02 (m, 2H), 2.00–1.84 (m, 2H), 1.81–1.71 (m, 1H), 1.73–1.66 (m, 4H), 1.66–1.60 (m, 2H), 1.48–1.35 (m, 2H), 1.33–1.27 (m, 2H), 1.26–1.18 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 171.46, 164.39, 160.30, 153.40, 138.49, 136.46, 130.96, 130.26, 130.06, 128.72, 125.57, 124.80, 115.39, 110.48, 67.56, 66.85, 58.99, 53.21, 47.57, 40.85, 36.88, 32.94, 29.77, 28.28, 26.28, 24.17, 23.44, 23.25, 20.26. HRMS (ESI): Calcd. C₃₃H₃₇FN₂O₅, [M + H]⁺m/z: 560.2687, found 560.2420. Anal: C, 70.69; H, 6.65; F, 3.39; N, 5.00; O, 14.27.

4-(2-(phenylsulfonyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)butyl-5-chloro-2-oxo-2H-chromene-3-carboxylate(4b)Yield 59%; Mp 199–201°C. ¹H NMR (500 MHz, CDCl₃) δ 8.52 (s, 1H, CH), 7.85 (dd, J = 6.6, 2.8 Hz, 2H, ArH), 7.63–7.59 (m, 3H, Ar-3H), 7.55 (dd, J = 15.5, 14.3 Hz, 1H, ArH), 7.21 (d, J = 1.7 Hz, 2H, Ar-2H), 3.96 (t, J = 14.8 Hz, 2H), 3.76 (dd, J = 24.8, 14.6 Hz, 1H), 3.33 (dd, J = 24.8, 14.6 Hz, 1H), 2.96 (dt, J = 21.8, 12.5 Hz, 1H), 2.51 (dt, J = 50.0 Hz, 3H), 2.41 (dt, J = 50.0 Hz, 3H), 1.85–1.72 (m, 2H), 1.65 (d, J = 22.3 Hz, 1H), 1.59–1.51 (m, 4H), 1.50–1.42 (m, 4H), 1.43–1.35 (m, 2H), 1.34–1.19 (m, 2H).¹³C NMR (126 MHz, CDCl₃) δ 163.31, 160.42, 152.74, 138.88, 138.69, 134.48, 131.17, 129.74, 129.05, 126.27, 125.07, 117.40, 116.61, 67.56, 66.85, 59.57, 53.21, 48.99, 41.06, 36.19, 31.85, 29.77, 28.28, 26.28, 24.17, 23.44, 23.25. HRMS (ESI): Calcd. C₃₁H₃₅ClN₂O₆S, [M + H]⁺m/z: 598.1904, found 599.1418. Anal: C, 62.15; H, 5.89; Cl, 5.92; N, 4.68; O, 16.02; S, 5.35.

4-(2-(2-methylbenzoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)butyl-2-oxo-2H-chromene-3-carboxylate(5b)Yield 67%; Mp 200–202°C. ¹H NMR (500 MHz, CDCl₃) δ 8.53 (s, 1H, CH), 7.74 (dd, J = 14.9, 3.0 Hz, 1H, ArH), 7.66 (dd, J = 14.3, 2.8 Hz, 1H, ArH), 7.57–7.50 (m, 1H, ArH), 7.38–7.31 (m, 2H, Ar-2H), 7.29 (t, J = 2.8 Hz, 1H), 7.22 (dd, J = 14.9, 3.4 Hz, 1H, ArH), 7.09 (td, J = 14.7, 3.3 Hz, 1H, ArH), 3.96 (t, J = 14.9 Hz, 2H), 3.35 (dd, J = 24.7, 14.7 Hz,1H), 2.83 (dd, J = 24.7, 14.7 Hz, 1H), 2.62 (dt, J = 21.8, 8.0 Hz, 1H), 2.51 (dt, J = 50.0 Hz, 2H), 2.41 (dt, J = 50.0 Hz, 2H), 2.21 (s, 3H, Ar-CH₃), 2.17–2.01 (m, 2H), 2.01–1.88 (m, 1H), 1.79–1.70 (m, 1H), 1.69–1.60 (m, 5H), 1.58–1.49 (m, 2H), 1.49–1.42 (m, 2H), 1.41–1.29 (m, 2H), 1.30–1.21 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 171.46, 163.31, 160.42, 152.43, 136.46, 132.91, 131.20, 130.96, 130.26, 128.72, 128.55, 125.57, 125.32, 122.09, 120.12, 117.43, 67.56, 66.85, 58.99, 53.21, 47.57, 40.85, 36.88, 32.94, 29.77, 28.28, 26.28, 24.17, 23.44, 23.25, 20.26. HRMS (ESI): Calcd. C₃₃H₃₈N₂O₅, [M + H]⁺m/z: 542.2781, found 542.2996. Anal: C, 73.04; H, 7.06; N, 5.16; O, 14.74.

4-(2-(2-methylbenzoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)butyl-6-methyl-2-oxo-2H-chromene-3-carboxylate(6b)Yield 48%; Mp 200–203°C. ¹H NMR (500 MHz, CDCl₃) δ 8.55 (s, 1H, CH), 7.76 (dd, J = 14.9, 2.9 Hz, 1H, ArH), 7.52 (d, J = 2.7 Hz, 1H, ArH), 7.36 (dd, J = 15.0, 2.5 Hz, 2H, ArH), 7.31 (dd, J = 9.0, 6.0 Hz, 1H, ArH),, 7.24 (dd, J = 15.0, 3.3 Hz, 1H, ArH), 7.11 (td, J = 14.7, 3.3 Hz, 1H, ArH), 3.97 (t, J = 14.9 Hz, 2H), 3.35 (dd, J = 24.8, 14.8 Hz, 1H), 2.84 (dd, J = 24.8, 14.8 Hz, 1H), 2.63 (dt, J = 21.8, 8.0 Hz, 1H), 2.52–2.44 (m, 3H), 2.42 (s, 3H, Ar-CH₃), 2.39 (dd, J = 17.8, 7.0 Hz, 1H), 2.22 (s, 3H, Ar-CH₃), 2.13–2.02 (m, 2H), 2.01–1.89 (m, 1H), 1.79–1.71 (m, 3H), 1.70–1.63 (m, 4H), 1.48–1.42 (m, 3H), 1.41–1.37 (m, 2H), 1.29–1.21 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 171.46, 163.31, 160.42, 151.23, 136.46, 135.61, 131.32, 130.89, 130.26, 128.72, 128.18, 125.57, 123.00, 120.49, 117.16, 67.56, 66.85, 58.99, 53.21, 47.57, 40.85, 36.88, 32.94, 29.77, 28.28, 26.28, 24.17, 23.44, 23.25, 21.23, 20.26. HRMS (ESI): Calcd. C₃₄H₄₀N₂O₅, [M + H]⁺m/z: 556.2937, found 557.4050. Anal: C, 73.36; H, 7.24; N, 5.03; O, 14.37.

4-(2-((4-(tert-butyl)phenyl)sulfonyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)butyl-5-bromo-2-oxo-2H-chromene-3-carboxylate(7b)Yield 51%; Mp 195–198°C. ¹H NMR (500 MHz, CDCl₃) δ 8.55 (s, 1H, CH), 7.67–7.60 (m, 4H, Ar-4H), 7.52 (t, J = 14.8 Hz, 1H, ArH), 7.37 (dd, J = 15.0, 3.3 Hz, 1H, ArH), 7.30 (dd, J = 14.7, 3.2 Hz, 1H, ArH), 3.97 (t, J = 14.9 Hz, 2H), 3.88 (dd, J = 24.1, 14.0 Hz, 1H), 3.33 (dd, J = 24.4, 14.3 Hz, 1H), 3.05 (dt, J = 21.4, 13.6 Hz, 1H), 2.51 (dt, J = 50.0 Hz, 2H), 2.41 (dt, J = 50.0 Hz, 2H), 1.87–1.79 (m, 2H), 1.69–1.60 (m, 4H), 1.54–1.49 (m, 3H), 1.47–1.41 (m, 4H), 1.41–1.34 (m, 2H), 1.33 (s, 9H, C-(CH₃)₃), 1.32–1.18 (m, 2H).¹³C NMR (126 MHz, CDCl₃) δ 163.31, 160.42, 154.40, 151.68, 133.92, 133.24, 131.14, 128.86, 128.32, 123.66, 118.81, 118.30, 117.05, 67.56, 66.85, 59.57, 53.21, 48.99, 41.06, 36.19, 34.58, 31.85, 31.36, 29.77, 28.28, 26.28, 24.17, 23.44, 23.25. HRMS (ESI): Calcd. C₃₅H₄₃BrN₂O₆S, [M + H]⁺m/z: 698.2025, found 699.4138. Anal: C, 60.08; H, 6.19; Br, 11.42; N, 4.00; O, 13.72; S, 4.58.

6.1.3. Synthesis of compound (1c-11c)

A solution containing anhydrous potassium acetate (CH₃CO₂K, 2.94 mmol) the conveniently substituted matrinic acid derivatives (5,7,8) (1.67 mmol) and the corresponding 2-hydroxybenzaldehyde (1.67 mmol) in acetic anhydride (Ac₂O, 10 mL) was refluxed (138°C) for 16 h. The reaction mixture was cooled, neutralized with 10% aqueous sodium bicarbonate (NaHCO₃), and extracted (3 × 30 mL) with ethyl acetate (EtOAc). The organic layers were combined and washed with distilled water dried with anhydrous sodium sulfate (Na₂SO₄) and evaporated under reduced pressure. The product was purified by recrystallization in ethanol (EtOH) and dried to afford the desired compound [39].

3-(2-(2-(4-fluorobenzoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)ethyl)-6-nitro-2H-chromen-2-one (1c)Yield 53%; Mp 173–174°C. ¹H NMR (500 MHz, CDCl₃) δ 8.76 (d, J = 2.9 Hz, 1H), 8.28 (dd, J = 15.0, 2.9 Hz, 1H, ArH), 8.22 (s, 1H, ArH), 8.12 (dd, J = 14.9, 10.0 Hz, 2H, Ar-2H), 7.62 (d, J = 15.0 Hz, 1H, ArH), 7.42–7.32 (m, 2H, Ar-2H), 3.72 (td, J = 13.4, 12.0 Hz, 1H), 3.49 (dd, J = 24.7, 14.3 Hz, 1H), 2.64 (td, J = 11.6, 2.0 Hz, 2H), 2.51 (dt, J = 24.8, 15.4, 12.6 Hz, 3H), 2.41 (dt, J = 24.8, 20.9, 10.0 Hz, 2H), 2.22–2.10 (m, 1H), 2.04 (t, J = 21.7 Hz, 1H), 1.91–1.75 (m, 4H), 1.72 (dt, J = 13.4, 7.2 Hz, 2H), 1.53 (dd, J = 12.8, 11.4 Hz, 2H), 1.50–1.38 (m, 2H), 1.39–1.20 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 167.32, 160.95, 156.78, 145.89, 144.19, 133.61, 131.63, 127.46, 127.16, 126.86, 122.40, 117.90, 115.98, 67.56, 58.76, 53.21, 47.57, 40.85, 36.88, 29.51, 28.28, 27.54, 26.28, 23.44, 23.25. HRMS (ESI): Calcd. C₂₉H₃₀FN₃O₅, [M + H]⁺m/z: 519.2169, found 519.3663. Anal: C, 67.04; H, 5.82; F, 3.66; N, 8.09; O, 15.40.

3-(2-(2-((4-(tert-butyl)phenyl)sulfonyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)ethyl)-6-nitro-2H-chromen-2-one (2c)Yield 59%; Mp 175–177°C. ¹H NMR (500 MHz, CDCl₃) δ 8.56 (s, 1H, CH), 8.28 (dd, J = 14.9, 3.0 Hz, 1H, ArH), 7.68–7.59 (m, 6H, Ar-6H), 3.62 (dd, J = 24.8, 14.7 Hz, 1H), 3.20 (dd, J = 24.8, 14.7 Hz, 1H), 3.09 (dt, J = 21.4, 12.4 Hz, 1H), 2.63–2.55 (m, 1H), 2.51 (dt, J = 24.8, 15.4, 12.6 Hz, 2H), 2.41 (dt, J = 24.8, 20.9, 10.0 Hz, 2H), 2.39–2.32 (m, 1H), 2.26 (ddd, J = 12.5, 1.9 Hz, 2H), 1.90–1.72 (m, 2H), 1.63 (d, J = 22.3 Hz, 1H), 1.59–1.52 (m, 4H), 1.46–1.41 (m, 1H), 1.39–1.34 (m, 2H), 1.33 (s, 9H, C-(CH₃)₃ ), 1.31–1.21 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 160.95, 156.78, 154.40, 145.89, 144.19, 133.92, 128.79, 128.32, 127.46, 127.16, 126.86, 122.40, 117.90, 67.56, 57.82, 53.21, 48.99, 41.06, 36.19, 34.58, 31.36, 29.51, 28.28, 26.67, 26.28, 23.44, 23.25. HRMS (ESI): Calcd. C₃₂H₃₉N₃O₆S, [M + H]⁺m/z: 593.2560, found 593.1752. Anal: C, 64.73; H, 6.62; N, 7.08; O, 16.17; S, 5.40.

3-(2-(2-((4-(tert-butyl)phenyl)sulfonyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)ethyl)-8-methoxy-2H-chromen-2-one(3c)Yield 45%; Mp 170–173°C. 1H NMR (500 MHz, CDCl3) δ 7.65 (t, J = 10.6 Hz, 4H), 7.42 (d, J = 15.0 Hz, 1H), 7.36 (dd, J = 15.9, 5.3 Hz, 1H), 7.25 (s, 1H), 7.23 (dd, J = 12.8, 3.6 Hz, 1H), 3.83 (s, 3H, Ar-OCH3), 3.79 (d, J = 14.5 Hz, 1H), 3.42 (dd, J = 24.8, 14.6 Hz, 1H), 3.15 (dt, J = 21.6, 12.3 Hz, 1H), 2.51 (dt, J = 24.8, 15.4, 12.6 Hz, 2H), 2.41 (dt, J = 24.8, 20.9, 10.0 Hz, 2H), 2.11 (td, J = 14.2, 0.6 Hz, 2H), 1.86–1.71 (m, 2H), 1.68–1.62 (m, 2H), 1.61 (t, J = 22.5 Hz, 1H), 1.48 (dd, J = 13.7, 8.7 Hz, 6H), 1.39–1.34 (m, 2H), 1.33 (s, 9H, C-(CH₃)₃).¹³C NMR (126 MHz, CDCl₃) δ 160.48, 154.40, 145.53, 144.99, 143.50, 133.92, 128.79, 128.32, 127.13, 125.21, 122.84, 121.33, 115.54, 67.56, 57.82, 56.83, 53.21, 48.99, 41.06, 36.19, 34.58, 31.36, 29.51, 28.28, 26.67, 26.28, 23.44, 23.25. HRMS (ESI): Calcd. C₃₃H₄₂N₂O₅S, [M + H]⁺m/z: 578.2814, found 578.3625. Anal: C, 68.48; H, 7.31; N, 4.84; O, 13.82; S, 5.54.

3-(2-(2-((4-(tert-butyl)phenyl)sulfonyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)ethyl)-6-methyl-2H-chromen-2-one(4c)Yield 59%; Mp 180–183°C. ¹H NMR (500 MHz, CDCl₃) δ 7.80 (s, 1H, Ar-H), 7.63 (t, J = 15.0 Hz, 4H, Ar-4H), 7.52 (d, J = 2.5 Hz, 1H, Ar-H), 7.29 (t, J = 16.6 Hz, 2H, Ar-2H), 3.36 (dd, J = 24.9, 13.9 Hz, 1H), 2.97 (dd, J = 24.8, 13.9 Hz, 1H), 2.69 (t, J = 8.0 Hz, 1H), 2.69–2.61 (m, 2H), 2.59–2.51 (m, 1H), 2.51 (dt, J = 24.8, 15.4, 12.6 Hz, 2H), 2.42 (s, 3H, Ar-CH₃), 2.41 (dt, J = 24.8, 20.9, 10.0 Hz, 2H), 2.14–2.02 (m, 1H), 1.92–1.74 (m, 2H), 1.70–1.62 (m, 2H), 1.74–1.63 (m, 4H), 1.48–1.42 (m, 2H), 1.41–1.34 (m, 3H), 1.33 (s, 9H, C-(CH₃)₃). ¹³C NMR (126 MHz, CDCl₃) δ 160.95, 154.40, 150.53, 145.89, 135.05, 133.92, 129.82, 128.79, 128.32, 127.46, 127.13, 122.25, 116.94, 67.56, 57.82, 53.21, 48.99, 41.06, 36.19, 34.58, 31.36, 29.51, 28.28, 26.67, 26.28, 23.44, 23.25, 21.23. HRMS (ESI): Calcd. C₃₃H₄₂N₂O₄S, [M + H]⁺m/z: 562.2865, found 563.5492. Anal: C, 70.43; H, 7.52; N, 4.98; O, 11.37; S, 5.70.

3-(2-(2-((4-(tert-butyl)phenyl)sulfonyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)ethyl)-2H-chromen-2-one(5c)Yield 57%; Mp 176–178°C. ¹H NMR (500 MHz, CDCl₃) δ 7.68 (s, 1H, ArH), 7.63 (t, J = 4.4 Hz, 3H, Ar-3H), 7.61–7.54 (m, 1H, ArH), 7.53 (tdt, J = 24.0 Hz, 2H, Ar-2H), 7.35 (ttd, J = 10.0 Hz, 2H, Ar-2H), 3.83 (dd, J = 24.9, 14.6 Hz, 1H), 3.69 (dd, J = 24.9, 14.5 Hz, 1H), 3.23 (dt, J = 21.6, 12.5 Hz, 1H), 2.51 (dt, J = 24.7, 10.8 Hz, 1H), 2.41 (dt, J = 10.0, 7.5 Hz, 1H), 2.39 (dt, J = 10.0, 7.5 Hz, 2H), 2.10 (td, J = 10.7, 1.8 Hz, 2H), 1.87–1.71 (m, 2H), 1.63 (t, J = 22.3 Hz, 1H), 1.54 (ddd, J = 10.8, 6.1, 3.1 Hz, 4H), 1.43 (ddd, J = 11.7, 7.1, 3.2 Hz, 2H), 1.43–1.36 (m, 2H), 1.33 (s, 9H, C-(CH₃)₃), 1.30–1.16 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 160.95, 154.40, 151.59, 146.28, 133.92, 131.84, 128.79, 128.32, 127.52, 126.97, 124.91, 120.68, 117.32, 67.56, 57.82, 53.21, 48.99, 41.06, 36.19, 34.58, 31.36, 29.51, 28.28, 26.67, 26.28, 23.44, 23.25. HRMS (ESI): Calcd. C₃₂H₄₀N₂O₄S, [M + H]⁺m/z: 548.2709, found 549.2999. Anal: C, 70.04; H, 7.35; N, 5.11; O, 11.66; S, 5.84.

tert-butyl-1-(2-(6-methyl-2-oxo-2H-chromen-3-yl)ethyl)octahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridine-2(3H)-carboxylate(6c)Yield 55%; Light brown viscous oil. ¹H NMR (500 MHz, CDCl₃) δ 7.91 (s, 1H, ArH), 7.52 (d, J = 2.5 Hz, 1H, ArH), 7.31 (d, J = 15.0 Hz, 1H, ArH), 7.27 (dd, J = 19.3 Hz, 1H, ArH), 4.32 (dd, J = 24.8, 15.1 Hz, 1H), 3.24 (dd, J = 24.7, 15.2 Hz, 1H), 2.87 (dt, J = 32.5 Hz, 3H), 2.80 (td, J = 15.6, 2.0 Hz, 2H), 2.51 (dt, J = 24.7, 10.8 Hz, 1H), 2.42 (s, 3H, Ar-CH₃), 2.41 (dt, J = 10.0, 7.5 Hz, 1H), 2.26–2.11 (m, 2H), 2.04 (t, J = 21.8 Hz,1H), 1.92–1.74 (m, 1H), 1.74–1.63 (m, 3H), 1.61–1.49 (m, 2H), 1.49–1.43 (m, 2H), 1.42 (s, 9H, C-(CH₃)₃), 1.39–1.28 (m, 2H).¹³C NMR (126 MHz, CDCl₃) δ 160.95, 157.65, 150.53, 145.89, 135.05, 129.82, 127.46, 127.13, 122.25, 116.94, 81.20, 67.56, 54.22, 53.21, 46.17, 40.85, 36.88, 29.51, 28.31, 27.54, 26.28, 23.44, 23.25, 21.23. HRMS (ESI): Calcd. C₂₈H₃₈N₂O₄, [M + H]⁺m/z: 466.2832, found 466.2039. Anal: C, 72.07; H, 8.21; N, 6.00; O, 13.71.

(9H-fluoren-9-yl)methyl 1-(2-(2-oxo-2H-chromen-3-yl)ethyl)octahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridine-2(3H)-carboxylate(7c)Yield 49%; Mp 166–169°C. ¹H NMR (500 MHz, CDCl₃) δ 7.90 (dd, J = 14.7, 3.2 Hz, 2H, Ar-2H), 7.77 (s, 1H, CH), 7.68 (dd, J = 14.6, 3.1 Hz, 1H, ArH), 7.56 (ddd, J = 18.5, 10.7, 3.2 Hz, 3H, Ar-3H), 7.37 (d, J = 3.2 Hz, 1H, ArH), 7.33 (dd, J = 14.8, 3.3 Hz, 1H, ArH), 7.24 (td, J = 14.9, 3.3 Hz, 2H, Ar-2H), 5.33 (d, J = 11.4 Hz, 2H, CH₂), 4.15 (dd, J = 24.8, 16.2 Hz, 1H), 4.05 (t, J = 11.4 Hz, 1H), 3.56 (q, J = 12.3 Hz, 1H), 3.44 (dd, J = 24.8, 16.1 Hz, 1H), 2.71 (td, J = 15.8, 1.9 Hz, 2H), 2.51 (dt, J = 24.7, 10.9 Hz, 2H), 2.41 (dt, J = 24.7, 10.9 Hz, 2H), 2.28–2.12 (m, 1H), 2.04 (t, J = 6.3 Hz, 2H), 2.13–1.79 (m, 2H), 1.68 (td, J = 15.7, 12.2 Hz, 2H), 1.58–1.47 (m, 4H), 1.46–1.33 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 160.95, 157.88, 151.59, 146.28, 143.41, 139.81, 131.84, 127.52, 126.97, 126.18, 125.07, 124.91, 120.89, 120.68, 117.32, 67.56, 67.18, 54.22, 53.21, 48.95, 46.17, 40.85, 36.88, 29.51, 28.28, 27.54, 26.28, 23.44, 23.25. HRMS (ESI): Calcd. C₃₇H₃₈N₂O₄ [M + H]⁺m/z: 574.2832, found 574.2695. Anal: C, 77.33; H, 6.66; N, 4.87; O, 11.14.

3-(2-(2-(4-fluorobenzoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)ethyl)-2H-chromen-2-one(8c)Yield 54%; Mp 181–183°C. ¹H NMR (500 MHz, CDCl₃) δ 8.17–8.08 (m, 2H, Ar-2H), 7.73 (s, 1H, CH), 7.68 (dd, J = 14.7, 3.0 Hz, 1H, ArH), 7.55 (dtd, J = 11.8 Hz, 1H, ArH), 7.42–7.28 (m, 2H, Ar-2H), 3.39 (dd, J = 24.8, 14.4 Hz, 1H), 3.02 (dd, J = 24.8, 14.4 Hz, 1H), 2.75 (dt, J = 22.0, 8.2 Hz, 1H), 2.68 (td, J = 15.6, 1.9 Hz, 1H), 2.51 (dt, J = 24.7, 10.8 Hz, 2H), 2.41 (dt, J = 24.7, 10.6 Hz, 2H), 2.25–2.09 (m, 2H), 2.04 (t, J = 22.0 Hz, 1H), 1.81–1.58 (m, 4H), 1.60–1.48 (m, 2H), 1.48–1.42 (m, 2H), 1.41– 1.31(m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 170.91, 162.34, 151.59, 146.28, 133.61, 131.84, 131.63, 127.52, 126.97, 124.91, 120.68, 117.32, 115.98, 67.56, 58.76, 53.21, 47.57, 40.85, 36.88, 29.51, 28.28, 27.54, 26.28, 23.44, 23.25. HRMS (ESI): Calcd. C₂₉H₃₁FN₂O₃ [M + H]⁺m/z: 474.2319, found 475.3319. Analysis: C, 73.40; H, 6.58; F, 4.00; N, 5.90; O, 10.11.

3-(2-(2-(4-fluorobenzoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)ethyl)-8-methoxy-2H-chromen-2-one(9c)Yield 49%; Mp 173–176°C. ¹H NMR (500 MHz, CDCl₃) δ 8.12 (dd, J = 14.9, 10.0 Hz, 2H, Ar-2H ), 7.74 (s, 1H, CH), 7.40 (dd, J = 4.0, 1.8 Hz, 1H, ArH), 7.37 (dd, J = 4.5, 1.7 Hz, 2H, Ar-2H), 7.34 (t, J = 3.1 Hz, 1H, ArH ), 7.23 (dd, J = 13.6, 4.4 Hz, 1H, ArH), 3.83 (s, 3H, Ar-OCH₃), 3.39 (dd, J = 24.8, 14.4 Hz, 1H), 3.01 (dd, J = 24.8, 14.4 Hz, 1H), 2.75 (dt, J = 22.0, 8.2 Hz, 1H), 2.67 (ddd, J = 15.6, 1.9 Hz, 2H), 2.51 (dt, J = 24.7, 10.8 Hz, 2H), 2.41 (dt, J = 24.7, 10.7 Hz, 2H), 2.25–2.09 (m, 2H), 2.04 (t, J = 22.0 Hz, 1H), 1.83–1.72 (m, 2H), 1.69 (dt, J = 7.7, 4.7 Hz, 2H), 1.64–1.51 (m, 2H), 1.48–1.38 (m, 2H), 1.37–1.29 (m, 2H).¹³C NMR (126 MHz, CDCl₃) δ 172.31, 164.23, 163.33, 160.25, 145.53, 144.99, 143.50, 133.61, 131.63, 127.13, 125.21, 122.84, 121.33, 115.98, 115.54, 67.56, 58.76, 56.83, 53.21, 47.57, 40.85, 36.88, 29.51, 28.28, 27.54, 26.28, 23.44, 23.25. HRMS (ESI): Calcd. C₃₀H₃₃FN₂O₄, [M + H]⁺m/z: 504.2424, found 504.2653. Anal: C, 71.41; H, 6.59; F, 3.77; N, 5.55; O, 12.68.

tert-butyl-1-(2-(6-methoxy-2-oxo-2H-chromen-3-yl)ethyl)decahydro-1H-benzo[de]isoquinoline-2(3H)-carboxylate( 10c )Yield 53%; Light yellow oil; ¹H NMR (500 MHz, CDCl₃) δ 7.92 (s, 1H, CH), 7.85 (d, J = 15.0 Hz, 1H, ArH), 7.23 (dd, J = 15.0, 2.9 Hz, 1H, ArH), 6.98 (d, J = 2.9 Hz, 1H, ArH), 4.32 (dd, J = 24.8, 15.2 Hz, 1H), 3.77 (s, 3H, Ar-OCH₃), 3.24 (dd, J = 24.8, 15.1 Hz, 1H), 2.90–2.83 (m, 1H), 2.80 (td, J = 15.4, 2.0 Hz, 2H), 2.51 (td, J = 23.2, 12.5 Hz, 2H), 2.41 (td, J = 23.2, 12.5 Hz, 2H), 2.22–2.09 (m, 2H), 2.04 (t, J = 21.8 Hz, 1H), 1.89–1.72 (m, 1H), 1.72–1.62 (m, 3H), 1.60–1.50 (m, 2H), 1.49–1.44 (m, 2H), 1.42 (s, 9H, C-(CH₃)₃), 1.39–1.27 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 160.95, 157.65, 156.09, 146.95, 145.89, 127.46, 122.22, 117.51, 116.26, 109.66, 81.20, 67.56, 56.08, 54.22, 53.21, 46.17, 40.85, 36.88, 29.51, 28.31, 27.54, 26.28, 23.44, 23.25. HRMS (ESI): Calcd. C₂₈H₃₈N₂O₅, [M + H]⁺m/z: 482.2781, found 483.2837. Anal: C, 69.68; H, 7.94; N, 5.80; O, 16.58.

3-(2-(2-(3-chlorobenzoyl)decahydro-1H,4H-pyrido[3,2,1-ij][1, 6]naphthyridin-1-yl)ethyl)-2H-chromen-2-on(11c)Yield 59%; Mp 189–192°C. ¹H NMR (500 MHz, CDCl₃) δ 7.82 (dt, J = 12.5, 3.0 Hz, 1H, ArH), 7.78 (t, J = 3.2 Hz, 1H, ArH), 7.70 (s, 1H, ArH), 7.67 (dd, J = 7.2, 3.2 Hz, 1H, ArH), 7.64 (dd, J = 7.2, 3.2 Hz, 1H, Ar-1H), 7.56 (dd, J = 6.6, 3.4 Hz, 1H, CH), 7.50 (dd, J = 10.0 Hz, 1H, Ar-1H), 7.34 (d, J = 14.8 Hz, 1H), 7.29 (td, J = 1.9 Hz, 1H), 3.40 (dd, J = 24.7, 14.4 Hz,, 1H), 2.99 (dd, J = 24.7, 14.5 Hz, 1H), 2.73 (td, J = 13.8, 8.2 Hz, 1H), 2.66 (ddd, J = 15.5, 1.8 Hz, 2H), 2.51 (td, J = 23.2, 12.5 Hz, 2H), 2.41 (td, J = 23.2, 12.5 Hz, 2H), 2.25–2.10 (m, 2H), 2.03 (t, J = 22.0 Hz, 1H), 1.84–1.67 (m, 4H), 1.53 (ddd, J = 20.0, 11.2, 6.8 Hz, 2H), 1.48–1.41 (m, 2H), 1.40–1.27 (m, 2H).

¹³C NMR (126 MHz, CDCl₃) δ 169.06, 160.95, 151.59, 146.28, 137.27, 134.68, 131.84, 130.13, 129.96, 129.61, 127.52, 126.97, 126.37, 124.91, 120.68, 117.32, 67.56, 58.76, 53.21, 47.57, 40.85, 36.88, 29.51, 28.28, 27.54, 26.28, 23.44, 23.25. HRMS (ESI): Calcd. C₂₉H₃₁ClN₂O₃ [M + H]⁺m/z: 490.2023, found 491.2933. Anal: C, 70.94; H, 6.36; Cl, 7.22; N, 5.71; O, 9.77.

6.2. Biological evaluation assay

6.2.1. Cell culture

A549 (Human lung cancer cells), HepG-2 (Human Hepatoma Cells), and HeLa (human cervical cancer cell line) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 (Solarbio) medium containing 10% Fetal Bovine Serum (FBS, GIBCO), 100U/ml Penicillin, and 100 mg/ml Streptomycin at 37°C under a 5% CO₂ humidified atmosphere.

6.2.2. Antiproliferative activity

A549, HeLa and HepG-2 cells were subjected to an MTT assay to evaluate the investigated compounds' antiproliferative activity. In a CO₂ incubator at 37°C cells were cultured in RPMI-1640 or DMEM complete medium with 10% fetal bovine serum. 96-well plates with a cell density of 2 * 10³ cells per well were employed to plate exponentially growing cells which were then incubated for 24 h at 37°C to evaluate for attachment. The test compound was dissolved in DMSO. Different concentrations of compounds (100, 50, 25, 12.5, and 6.25µM, respectively) were used to treat the cells for 48 h. Each group consisted of three replicate wells, and subsequent incubation of the plate was conducted at a temperature of 37 ◦C for a duration of 48 hours. MTT solution was added to each well and incubated at 37 ◦C for 4 h. Absorbance of each well was recorded at a wavelength of 570 nm with the multi-function microplate reader. Finally, the data are expressed as means of three independent experiments ± standard deviation (SD). the growth inhibition data represented as IC₅₀ values were calculated through the prism statistical package [45, 46].

6.2.3. Cell cycle assays

A549 and BEAS-2B cell lines were added to a 6-well plate at a final concentration of 2 * 10⁵ cells per well and various concentrations of 5a (0, 1, 2, 4, 8, 16, and 32 µM) were subsequently applied. After 48 h the wells were detached utilizing a trypsin/EDTA solution fixed using 70% ice-cold ethanol for 12 h and subsequently exposed to a 50 µL RNase (50 g/ml) treatment for an hour at 37°C. After that, 250 µl 50 g/ml PI was added and the cells were incubated at 4°C for 4 h. Cell Quest software (BD) and Flow Jo 4.8 were used to evaluate the effects on the cell cycle using a FACS Calibur (BD). In this manner, the cell cycle phase of 2 * 10⁴ cells was examined.

6.2.4. Colony formation assay

A549 cells were seeded in 6-well plates with a density of 1 * 10³ cells/well and incubated for 48 h before the medium was shifted to a fresh medium containing 5a at concentrations of (0, 2, 4, 8, 16 and 32 µM). The cells were treated for 7 days before being washed twice with PBS, fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and imaged under a microscope (Nikon). After being dissolved in alcohol the violet crystals were read by a microplate reader (BioTek, USA) at an absorbance of 595 nm. GraphPad Prism 7.0 was utilized to analyze the readers.

6.2.5. Cell migration assay

A549 cells were cultured on a six-well plate allowing space to adhere to other cells. With a 200 µL pipette tip, a wound was created by drawing a line through the middle of each well. Following three PBS washes to remove floating cells the wells were grown in DMEM with 2% FBS as an addition. After adding various doses of 5a (0, 2, 4 and 8 µM) to the wells the plate was incubated for 48 h. Following the removal of the medium three distinct areas were chosen randomly for imaging using a microscope (Nikon, Japan) (×100 magnification). The following formula was used to determine the scratch area healing rate: Scratch area healing rate is calculated as follows: [initial scratch area - scratch area at a specific time point initial scratch area 100%].

6.2.6. 4,6-Diamidino-2-phenylindole (DAPI) staining analysis.

Cells were seeded in 12-well plates, and exposed to 5a at a preset concentration (0, 1, 2, 4, 6 and 8) for 48 h and then the nutrient supernatant was discarded. The uncovered cells were fixed in 4% paraformaldehyde for 15 min and washed with PBS twice. Finally, the cells were stained in DAPI dye liquor (Southern Biotech Company, USA) at the final concentration of 1 mg/L for 15 min and washed in PBS once again. The samples were observed and photographed by fluorescence microscope (FM) (Olympus BX51, Olympus, Japan).

6.2.7. Annexin V-FITC/PI apoptosis assay

For apoptosis determination, 3 * 10⁵ cells/well were seeded in a six-well plate and treated with various concentrations of 5a (0, 2, 4 and 8 µM). After 24 h, the medium was removed and the cells were washed once with PBS, followed by detachment using trypsin/EDTA solution. The cells were stained with annexin V‐FITC/PI solution, and viability was measured on a FACS Calibur (BD) using Cell Quest software (BD) and analyzed using Flow Jo (Flow Jo LLC) as described before.

6.2.8. Western blot analysis

To examine the effect of 5a on the protein expression level in A549 cells the various concentrations (0, 2, 4 and 8 µM) of 5a-treated cells were harvested and lysed in sodium dodecyl sulfate (SDS) buffer for 30 min at 4°C. The supernatant obtained by centrifugation at 12000 g for 20 min was used to determine protein concentration by the bicinchoninic acid assay. Proteins (20–50 µg) were separated by SDS polyacrylamide gel electrophoresis in 8%-12% gels and transferred onto polyvinylidene difluoride membranes (Millipore) at a low temperature. After blocking the membrane for 1 h at room temperature in 5% skim milk in Tris‐buffered saline (TBS) containing 0.1% of Tween‐20 (TBST), they were incubated with an appropriate primary antibody (1:1000) at 4°C for 12 h. The membranes were then washed with TBST and probed with secondary antibodies (1:1000) for 1 h at room temperature. After washing protein expression was measured using an enhanced chemiluminescence kit.

6.3. Animal Experiments

6.3.1 Ethics Approval

This study was conducted by the guidelines of the Institutional Animal Care and Use Committee (IACUC). The protocol was approved by Animal Experimental Ethical Committee of Guangxi University under the No: Gxu-2024-258.

6.3.2. Acute-Toxicity Assessment

The Balb/c nude mice were divided randomly (six mice for each group) into one treatment group and the control group. Compound 5a (40 mg/kg, 10% DMSO/10% Tween-80/80% normal saline) and Vehicle (10% DMSO/10% Tween 80/80% normal saline) were administered one day. Subsequently, mice were observed for 28 days and the Mice were placed in a chamber gradually filled with CO₂ gas until unconsciousness and death occurred and the weight was recorded daily.

6.3.3. A549 Xenograft Tumor Model

Prior to implantation, A549 cell suspension was collected at a concentration of 1 * 10⁷ cells/ml, in PBS and 0.1 ml of each cell was inoculated subcutaneously in the right armpit of the Balb/c nude mice. The diameter of transplanted tumors in mice was measured with a vernier caliper, and when the tumors grew to 100 mm³ the animals were randomly divided into 6 groups. At the same time, the mice in each group started to be administered. The method of measuring the tumor diameter was used to observe the anti-tumor effect of the test samples dynamically. Immediately after the end of the experiment, the mice were sacrificed and the tumor mass was surgically removed and weighed. Body weight and tumor size were measured every 3 days for 28 days after treatment. The formula for calculating tumor volume (TV) is as follows: TV = 1/2×a×b² where a and b represent length and width, respectively [47].

6.3.4. Euthanasia study

The experimental mice were introduced into a controlled environment within a CO₂ anesthesia chamber. The flow of CO₂ agent was regulated to gradually induce unconsciousness in the mice. Upon achieving unconsciousness, the concentration of CO₂ was elevated to 100%. During this phase, observations were made to confirm the absence of finger pinching reflex and muscle tone, indicative of complete unconsciousness. Following verification, ventilation was sustained for an additional 2 minutes to ensure the cessation of vital signs, thereby confirming the animals' death.

6.3.5. Histological examination

The hearts, spleens, kidneys, lungs, livers, and tumor tissues were isolated to observe the integrity and injury in different groups by HE staining. Briefly, after ischemia and reperfusion, mice hearts, spleens, kidneys, lungs, livers, and tumors were isolated and the tissues were fixed in 4% paraformaldehyde overnight at room temperature, and then dehydrated by passing through gradient concentrations of ethanol (80% for 2 h, 90% for 2 h, 95% overnight, 100% for 0.5, 0.5 and 1 h) at room temperature, followed by embedding in paraffin wax. The paraffin‑embedded samples were then sectioned at 4 µm for staining with Mayer's Hematoxylin (H8070, Solarbio Life Sciences, Beijing, China) for 10 min and then by 0.5% aqueous eosin (DH0050, Leigene Biotech, Beijing, China) for 3 min at room temperature. With this method, the nucleus and other acidic structures are stained blue, while the cytoplasm is stained red. Images were acquired using a light microscope at x400 magnification [48].

6.4. Molecular docking studies

The CTD (PDB ID: 3T0Z) and NTD (PDB ID: 2CG9) atomic coordinates have been retrieved from the RCSB PDB (protein data bank) database [49] charge assignment, solution measurements, and fragment volumes to the protein have been performed using Autodock Vina PyRx and Discovery Studio 2019 Client for docking analysis. The ligand's 2D structure was drawn and analyzed using ChemDraw, standardized, and converted into PDBQT format using the PyRx Virtual Screening Tool. To show the binding modes 2D, 3D and ligand interaction we carried out using the Discovery Studio 2019 Client.

Hsp90 Heat Shock Protein 90

NTD N-terminal domain

MD Middle Domain

CTD C-Terminal Domain

DMAP Dimethylaminopyridine;

EDCI 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride;

DCC N, N-dicyclohexylcarbodiimide;

DCM Dichloromethane

STD Saturation Transfer Difference

HSR Heat Shock Response

NMR Nuclear Magnetic Resonance

DMSO Dimethyl Sulfoxide;

IC₅₀ Half Maximal Inhibitory Concentration

H&E Hematoxylin and Eosin

Author Contribution

W.L. Supervision, Funding acquisition, Writing - review & editing, Project administration.K.J. Conceptualization, Methodology, Formal analysis, Writing - original draft, Visualization. G.CH. Conceptualization, Methodology, Formal analysis, Visualization.J.M, G.R, L.Y, H.K, Q.G and Y.S Investigation, Data curation, review & editing.L.X. Software, Validation, Resources, Writing - review & editing.Each author contributed significantly to the study's conception, design, data analysis, and interpretation. All authors have read and approved the final manuscript.

Acknowledgement

This work was supported by the local funding project for scientific and technological development under the guidance of the central government (GuiKe ZY21195012) and Guangxi Innovation-Driven Development Project (GuiKe AA18242040).

Data Availability

The manuscript and supplementary information file contain the data. The original raw data can be obtained by contacting the Corresponding Author.

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Tables 1-3 are available in the Supplementary Files section.

Schemes1-3 are available in the Supplementary Files section.

No competing interests reported.