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Abstract

In a five-step procedure, 18 new esters (20a–i and 21a–i) of ataluren and its analogs with dihydroartemisinin and zerumbone were synthesized from methyl 3-cyanobenzoate. The screening for their cytotoxic activity against two cancer cell lines, HepG2 and MCF-7, showed that most esters exhibit activity against the tested cell lines, with IC₅₀ values in the range of 1.61–36.52 µM. Among the tested compounds, ester 21f containing 4-methoxy in structure R did not show cytotoxic activity. The interactions of these new derivatives with the epidermal growth factor receptor protein were also investigated by molecular docking studies. The obtained binding conformation revealed several notable results, demonstrating similarities between molecular modeling theory and experiment. These results contributed to interpreting the in vitro cytotoxicity of esters and proposed the basis for predicting the mechanism of their cytotoxic action.

Keywords

ataluren cytotoxicity ester molecular docking oxadiazole

Introduction

Oxadiazole is a general name referring to a five-membered ring that contains an oxygen atom and two nitrogen atoms.¹ The position of heteroatoms on the ring results in the specific name of isomers starting from oxygen, including 1,3,4-, 1,2,6-, and 1,2,4-oxadiazole (1).² 1,2,4-oxadiazoles constitute a small group belonging to the oxadiazole family of heterocyclic chemistry.³ In addition, 1,3,4-oxadiazoles and 1,2,4-oxadiazoles also demonstrate a wide spectrum of biological activities, such as antimicrobial, anticancer, anti-inflammatory, and antiviral activities.^4–7 Furthermore, ataluren (2) (Figure 1), a member of the 1,2,4-oxadiazole family, holds a special pharmacological property applied to the treatment of Duchenne muscular dystrophy (DMD) disease.⁸ In pathology, DMD is a muscle-wasting condition that causes progressive muscle weakness, and ataluren is approved as a specific drug for this disease.⁹

Figure 1.

The structures of 1,2,4-oxadiazole 1, ataluren 2, aspirin 3, enalapril 4, nitazoxanide 5, artesunate 6, and zerumbone and DHA conjugates 7, 8, 9, and 10.

In pharmaceutical chemistry and drug development, esters are often used as prodrugs with the purpose of enhancing some properties of parent drugs, such as solubility, oral adsorption, membrane permeability, duration of action, and reducing side effects.^10–14 Some typical examples given approve the structural diversity of esters in commercially available medicines in the world, including aspirin (3), enalapril (4), nitazoxanide (5), and artesunate (6) (Figure 1). Structurally, esters are composed of two components, including carboxylic acids and alcohols/phenols, that are usually present in natural compounds or their derivatives; therefore, natural alcohols/phenols or carboxylic acids are always preferred compounds for ester formation. In such cases, natural compounds exhibit the role of leading agents to the known targets of the parent compounds.¹⁵ In this field, dihydroartemisinin (DHA) and zerumbone are suitable for incorporation with other substrates due to their impressive antimalarial and anticancer activities. Some motifs 7, 8, 9, and 10 (Figure 1) of zerumbone and DHA conjugates and their bioactivities have also been documented.^16–19 In our previous studies, the cytotoxic activity of DHA and zerumbone thioethers containing imidazole, benzimidazole, and 1,3,4-oxadiazole has been significantly improved compared to DHA and zerumbone alone. Conjugates 7 and 8 among the mentioned thioethers exhibit stronger cytotoxic activity than zerumbone, approximately 4- to 20-folds.^16,20 However, no studies on the synthesis and biological activity evaluation of DHA and zerumbone with 1,2,4-oxadiazole are available at the moment. Therefore, the ongoing study is aimed at further exploiting the cytotoxicity of novel esters obtained from DHA, zerumbone, and acids of 1,2,4-oxadiazoles.

A new era of selective chemotherapeutic hits has emerged with the advent of targeted therapies, specifically the signaling-pathways-targeting chemotherapeutic agents. These agents can target cancer cells or the tumor microenvironment that promotes tumor growth, leading to increased efficacy and minimal side effects on healthy cells. One of the most principal signaling pathways was the tyrosine kinase (TK) epidermal growth factor receptor (EGFR) which belongs to the TK receptor family and is present in the epithelial cell membrane.²¹ Its overexpression has been proven to play an important role in the proliferation of tumor cells, invasiveness, and metastasis in various cancer types, including breast, colon, and ovarian subtypes.²² Conformational alterations brought on by the endogenous EGFR ligand’s binding contacts with the EGFR extracellular binding domain activate the EGFR. Consequently, a number of tyrosine residues undergo autophosphorylation, which triggers the mitogen-activated protein kinase (MAPK) pathway by phosphorylating the subsequent proteins in the route. Profound mutations in several MAPK pathway proteins, including EGFR, have been connected to a number of cancer forms, including non–small cell lung cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, and breast cancer. This clarifies the reason why searching for new EGFR inhibitors is one of the most effective pathways for cancer therapy.²³

Recently, computer-aided drug design (CADD) has been widely used to decrease both time and cost for discovering potent compounds that are able to inhibit the biological function of a specific enzyme.²⁴ In the CADD, the binding-free energies (ΔG) can be computed over atomistic simulations. This metric discloses the binding affinity between ligand and protein through docking simulation, and their potential binding conformations were investigated. In this work, the physical insights into the EGFR protein target and synthesized ester binding were clarified using AutoDock4. The achieved results are significant contributions toward seeking new candidates for targeted cancer therapy and providing valuable guidance for further drug development studies.

Results and discussion

The esters 20a–i and 21a–i of 1,2,4-oxadiazoles with DHA and zerumbone were synthesized from two parts, including acid 15a–i moieties, bromides 17 and 19, which were prepared from methyl 3-cyanobenzoate 11, DHA 16, and zerumbone 18, respectively. In the first part, ataluren 15d and its derivatives 15a–i were synthesized from methyl 3-cyano benzoate 11 according to the previously reported methods.^25,26 First, ester 11 was converted into (Z)-3-(N′-hydroxycarbamimidoyl)benzoate 12 in 86% yield by the reaction with hydroxylamine hydrochloride in methanol in the presence of potassium tert-butoxide. Next, methyl esters 14a–i were obtained in 60%–70% yields from two successive reactions, including O-acylation with acyl chlorides 13a–i in acetone and the cyclization reaction in water, which were carried out in a one-pot reaction. Last but not least, the hydrolysis of esters 14a–i by potassium hydroxide (KOH) in DMF-H₂O overnight at room temperature gave ataluren and its analogs with 80%–87% yields (Scheme 1). In this step, the aim of the study was to introduce a simple, mild, and effective hydrolysis of the ester 14a–i to yield ataluren and its analogs 15a–i without the use of BBr₃²⁶ or THF-H₂O.²⁷ At first, a KOH/CH₃OH/H₂O system was selected for this reaction; however, the hydrolysis did not occur at room temperature, even in reflux conditions. The hydrolysis of esters 14a–i was completed using a KOH/DMF/H₂O system with 2 equiv. of KOH overnight at room temperature to obtain ataluren and its derivatives 15a–i in 80%–87% yields. The end of hydrolysis was recognized when the suspension of the reaction mixture became a clear solution. The potassium salts of acids 15a–i were acidified to pH = 3 by dilute HCl to give acids 15a–i as designed. Although acids 15a–i were known compounds, acid 15i was still chosen as a representative for checking its structure by ¹H NMR spectrum. The ¹H NMR of 15i gave rise to five signals of aromatic protons in the downfield region, including a triplet at 8.63 ppm (J = 1.5 Hz) of the H-2′, two double triplet signals at 8.33 and 8.17 ppm (J₁ = 1.5 Hz, J₂ = 7.8 Hz) attributed to H-4′ and H-6′, respectively, and another triplet at 7.74 ppm assigned to H-5′. Next, the resonance of 2 equiv. protons, H-2′′′ and H-6′′′, gave rise to a singlet observed at 7.47 ppm. In the upfield region, two singlets at 3.94 and 3.80 ppm belonged to (3′′′-OCH₃, 5′′′-OCH₃) and 4′′′-OCH₃, respectively. Therefore, the ¹H NMR data of 15i agreed well with its structure. In the second part, 2-(bromoethyl)ether of DHA 17 and bromide 19 of zerumbone was prepared from the corresponding DHA 16 and zerumbone 18 as described in the study by Liu et al.²⁸ and Kitayama et al.,²⁹ respectively, and in our previous study by Tran et al.¹⁶ Finally, the ethers 20a–i and 21a–i were obtained by the reaction of 15a–i with 17 and 19, respectively, in the presence of potassium tert-butoxide in DMF overnight at room temperature to yield two designed series 20a–i and 21a–i in moderate yields. Here, potassium tert-butoxide was employed and stirred with acids 15a–i first to form carboxylate ions as weak nucleophilic agents that reacted with 17 and 19 to yield the designed ethers 20a–i and 21a–i, and minimize byproducts formed by substitution reaction and Michael addition of tert-butoxide ion to the pentadienone group of zerumbone. Consequently, the esters 20a–i and 21a–i were obtained as designed (Scheme 1). Although the yields of esters prepared by this method could be lower than that of traditional method, this method had advantages, such as reaction at room temperature or no requirement for conversion of 17 and 19 into their corresponding alcohols.

Scheme 1.

The synthesis of esters 20a–i and 21a–i.

The structures of esters 20a–i and 21a–i were elucidated by analysis of their ¹H NMR, ¹³C NMR, and (for esters 20d and 21e) spectra. In each series 20a–i and 21a–i, the ¹H NMR and ¹³C NMR spectra in each of the series 20a–i and 21a–i were identical in terms of signal position, multiplicity, and integral intensity, with the exception of R moieties that have different substituents, which caused changes in the signal in their NMR spectra. An example of how these structures were interpreted is the structural elucidation of 20d. The ¹H NMR of 20d showed that eight aromatic proton signals appear in the range of 7.50–8.63 ppm. Among the mentioned protons, the signals of H-2′, H-4′, H-6′, and H-5′ were found at the position with δ_H values of 8.63 ppm (t, J = 1.5 Hz), 8.37 ppm (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz), 8.20 ppm (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz), and 7.78 ppm (t, J = 7.8 Hz), respectively. These δ_H values were completely similar to the corresponding protons in acid 16d. Subsequently, the signals of the remaining protons in the fluorinated aromatic ring (structure R) appeared at 8.25 ppm (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz), 7.56 ppm (ddd, J₁ = 1.5 Hz, J₂ = 2.4 Hz, J₃ = 7.8 Hz), and 7.50 ppm (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz) attributed to H-6′′′, H-4′′′, H-3′′′, and H-5′′′, respectively. Based on the interactions of protons and carbon in its HSQC spectrum, all carbons in this moiety were assigned as follows: C-2′ (127.5 ppm), C-4′ (131.6 ppm), C-6′ (132.1 ppm), C-5′ (130.0 ppm), C-6′′′ (130.9 ppm), C-4′′′ (135.9 ppm, d, J = 9.0 Hz), C-3′′′ (117.3 ppm, d, J = 21.0 Hz), and C-5′′′ (125.5 ppm, d, J = 3.0 Hz). Last but not least, other carbons in this moiety include C-1′, C-3′, C-5′′, C-3′′, C-1′′′, and C-2′′′, which were also determined by the peaks at 130.7, 126.6, 172.8, 167.3, 111.6 (d, J = 10.5 Hz), and 160.0 ppm (d, J = 255.0 Hz), respectively. In the upfield region, the signals of protons in the DHA structure were exhibited in the area of 0.57–5.25 ppm. In addition, the signals of all carbons in this moiety were also fully displayed, with δ_C values ranging from 12.6 to 103.2 ppm. The assignment of proton and carbon signals in the DHA moiety has been specifically completed in our previous study by Tran et al.¹⁶ Consequently, the structure of 20d has been interpreted accurately. The structure of other compounds in series 20a–i has been elucidated based on the structure of 20d. For series 21a–i, the structure in detail of the zerumbone moiety has been documented.¹⁶ Finally, the NMR spectral data of the two series 20a–i and 21a–i are in excellent agreement with their structures.

Biology

The synthesized esters 20a–i and 21a–i were screened for their cytotoxicity against two human cancer cell lines, HepG2 and MCF-7, with the highest tested concentration of 20 µg mL⁻¹. The tested compounds had no activity when the IC₅₀ > 20 µg mL⁻¹. The cytotoxic data for 20a–i and 21a–i are given in Table 1.

Table 1.

Cytotoxicity of the prepared esters 20a–i and 21a–i.

No	Compounds	IC₅₀ values^a (µM)
No	Compounds	Hep-G2	MCF-7
1	Ellipticine^b	3.29 ± 0.04	8.62 ± 0.24
2	20a	8.97 ± 0.17	36.52 ± 6.80
3	20b	2.29 ± 0.06	29.81 ± 4.00
4	20c	1.71 ± 0.02	29.89 ± 4.99
5	20d	1.61 ± 0.03	26.03 ± 3.26
6	20e	3.97 ± 0.08	–
7	20f	18.97 ± 1.65	–
8	20g	1.73 ± 0.01	29.30 ± 1.80
9	20h	1.74 ± 0.05	29.46 ± 3.73
10	20i	26.47 ± 1.74	–
11	DHA	35.88 ± 4.82	–
12	21a	5.21 ± 1.19	6.76 ± 0.05
13	21b	5.91 ± 0.22	6.85 ± 0.41
14	21c	4.73 ± 0.08	10.51 ± 0.85
15	21d	5.58 ± 0.10	5.36 ± 0.08
16	21e	9.32 ± 0.37	10.00 ± 1.78
17	21f	–	–
18	21g	6.29 ± 1.81	4.47 ± 1.62
19	21h	3.85 ± 0.06	12.62 ± 1.75
20	21i	3.79 ± 0.12	10.05 ± 1.45
21	Zerumbone	61.19 ± 9.08	44.35 ± 4.81

DHA: dihydroartemisinin.

Data represent the mean ± standard deviation of three independent wells.

Ellipticine was used as a positive control.

(–): No activity.

The IC₅₀ in Table 1 showed that most esters 20a–i and 21a–i displayed their cytotoxic activity against the tested cell lines. However, their cytotoxic effect had a significant difference between the two cell lines and depends on the structure of the R moiety. First, most of the esters 20a–i and 21a–i exhibited activity against HepG2 cell lines stronger than that of MCF-7 cell lines. Next, the presence of a methoxy group in the R moiety resulted in their reduced cytotoxicity, even loss of activity as in the case of 21f, which had a methoxy at the C-4′′′ position. The observation of cytotoxicity to the HepG2 cell line revealed that the esters 20a–i and 21a–i of DHA and zerumbone showed strong activity against this cell line with IC₅₀ values within the region of 1.61–9.32 µM, except for 20f and 20i, which exhibited weak cytotoxic activity with IC₅₀ values of 18.97 and 27.47 µM, respectively. Comparing the cytotoxicity of the series, 21a–i with two cancer cell lines HepG2 and MCF-7 through the IC₅₀ values showed that the difference between two these cell lines was insignificant.

Among the esters in the two series, esters 20d and 21i were found to have the strongest activity against HepG2 cells, with IC₅₀ values of 1.61 and 3.79 µM, respectively. For the MCF-7 cell line, four esters (20e, 20f, 20i, and 21f) showed no activity. The cytotoxic effect toward this cell line of the remaining esters in both series showed a significant difference. Specifically, while esters 21a–i showed impressive cytotoxic activities with IC₅₀ values ranging from 4.47 to 12.62 µM, esters 20a–i expressed weaker activities than those of 21a–i with IC₅₀ ranging from 26.03 to 36.52 µM, even inactive as the cases mentioned above. Therefore, the results in Table 1 led to the conclusion that the esters of the two series revealed more selective cytotoxic activity toward HepG2 than MCF-7. In addition, zerumbone esters exhibited significantly stronger activity against MCF-7 cells than that of DHA esters.

Docking studies

Re-docking validation

EGFR is critical for controlling the growth and survival of epithelial cells; thus, EGFR inhibitors have been used for the treatment of various cancer patients, for example, gefitinib for non–small lung cancer, erlotinib for pancreatic cancer, lapatinib and neratinib for breast cancer, and so on. However, it is reported that patients treated with these drugs might respond to treatment initially, but most of them will develop resistance within a year.³⁰ Due to this burden, there is still an urgent need for scientists to find novel compounds with significant inhibition activities. Recent studies have shown that tyrosine kinase inhibitors (TKIs) associated with mutations in EGFR are used to treat initial lung cancer. In this context, molecular docking simulation was conducted to clarify the synthesized esters against the inactive EGFR TK domain (PDB ID: 4HJO). It is reported by Gohlke et al.³¹ that ligand-calculated partial charge with the B3LYP method has been shown to greatly increase docking accuracy and cluster population of the most accurate docking.

The co-crystallized ligand, erlotinib, was re-docked to the binding site of the targeted enzyme to validate the accuracy of the docking procedure. Molecular docking is a useful tool to quickly find, for each protein interacting with the ligand, the optimal value of the score function. The objective of any docking calculation is to find the best pose, which corresponds to the lowest energy.³² The docking result is considered reliable when the root-mean-square deviation (RMSD) value does not exceed 2.0 Å. In this study, the docked pose of erlotinib with the lowest binding-free energy was compared with its native structure based on its RMSD value, which was 0.655 Å (Figure 2). This result suggests that the procedure and the setting parameters were reproducible and appropriate for further docking simulation.

Figure 2.

Dock pose of the studied erlotinib (green) and native erlotinib (magenta) in the active site of EGFR protein (PDB ID: 4HJO).

Docking results

We further investigated the EGFR inhibitory effect of the studied compounds by revealing how they interact with the active site of this protein through molecular docking studies. AutoDock4 is one of the most common docking tools that has been widely used with more than 6400 citations.³³ According to the ranking criteria of AutoDock, the more negative docking energy suggests a higher binding affinity of the compound toward the targeted receptor.³⁴ The binding-free energies, interaction type, and residues participating in interaction are tabulated in Table 2.

Table 2.

Binding-free energy and residue interactions of the studied compounds against EGFR protein.

Ligand	Binding-free energy (kcal mol⁻¹)	No. of hydrogen bonds	Interacting residues
20a	−13.24	3	Leu694, Lys721, Met742, Cys751, Leu764, Thr766, Thr830, Asp831, Phe832
20b	−12.44	4	Val702, Lys721, Met742, Cys751, Leu753, Asp813, Arg817, Asn818, Thr830, Leu834
20c	−13.06	1	Leu694, Val702, Lys721, Met742, Gly772, Thr830, Asp831, Leu834
20d	−13.33	2	Leu694, Gly695, Val702, Ala719, Lys721, Leu764, Thr766, Cys773, Arg817, Leu820, Thr830
20e	−12.91	2	Val702, Lys721, Met742, Leu753, Leu764, Leu820, Thr830, Asp831, Phe832, Leu834
20f	−10.69	1	Leu694, Val702, Lys721, Met742, Leu753, Leu764, Leu820, Phe832, Leu834
20g	−13.64	1	Val702, Ala719, Lys721, Met742, Val750, Cys751, Leu753, Leu764, Thr766, Gly772, Cys773, Thr830
20h	−12.85	3	Leu694, Val702, Lys721, Met742, Leu753, Leu764, Cys773, Thr830, Leu834
20i	−9.76	2	Leu694, Gly695, Val702, Ala719, Lys721, Leu764, Thr766, Gly772, Cys773, Arg817, Leu820, Asp831, Leu834
21a	−13.35	3	Val702, Lys721, Met742, Cys751, Leu764, Thr766, Leu820, Thr830, Asp831, Phe832
21b	−13.53	2	Leu694, Val702, Lys721, Met742, Leu753, Gly772, Leu820, Thr830, Leu834
21c	−14.38	2	Val702, Ala719, Lys721, Met742, Leu753, Cys773, Thr830, Leu834
21d	−13.75	1	Val702, Lys721, Met742, Leu753, Leu820, Thr830, Asp831, Leu834
21e	−14.37	2	Leu694, Gly695, Val702, Lys721, Met742, Leu764, Cys773, Arg817, Asn818, Thr830, Asp831, Phe832, Leu834
21f	−13.23	1	Leu694, Val702, Ala719, Leu768, Met769, Leu820, Asp831
21g	−14.70	2	Leu694, Val702, Ala719, Lys721, Leu768, Met769, Leu820, Thr830, Asp831
21h	−13.30	2	Val702, Lys721, Met742, Leu753, Leu820, Thr830, Asp831
21i	−13.84	3	Leu694, Ala719, Lys721, Leu768, Met769, Pro770, Cys773, Leu820, Thr830
Erlotinib	−11.84	1	Leu694, Val702, Ala719, Lys721, Leu764, Gln767, Met769, Leu820, Thr830

In this study, the obtained dock score of the reference compound erlotinib was −11.84 kcal mol⁻¹; thus, any ligands with docking energy close to this threshold would be assumed to exhibit high binding affinity toward the targeted protein. The docking results showed that most of the synthesized esters exhibit higher binding affinity toward EGFR than the reference compound, with binding-free energies varying within the range of −12.44 to −14.70 kcal mol⁻¹, except for compounds 20f (−10.69 kcal mol⁻¹) and 20i (−9.76 kcal mol⁻¹). Also, the absolute value dock scores of compounds 21a–i are slightly higher than those of compounds 20a–i. These results are in good agreement with the antiproliferative activities of cytotoxic assays.

In Figure 3 and Supplemental Material 2, the docking pose of the synthesized esters within the active site of the protein under study was shown. It has been reported in previous studies that the active site of the targeted receptor was mainly composed of residues Phe699, Gly700, Lys721, Met769, Arg817, Thr830, and Asp831.^35,36

Figure 3.

Docking conformation of studied compounds in the binding site of the inactive EGFR TK domain suggested by molecular docking studies.

The hydrophobic pockets formed with compound 21g are revealed in Figure 3, involving residues Leu694, Val702, Alaa719, Leu768, and Leu820. The interaction is further strengthened through two conventional hydrogen bonds with Lys721 and Met769. Binding orientation analysis of compound 21c indicated Val702, Ala719, Met742, Leu753, Cys773, and Leu834 were the main residues involved in weak interactions; meanwhile, Lys721 and Thr830 bonded with the compound for two hydrogen bonds. An array of hydrophobic interactions was observed, as contributed by Leu694, Gly695, Val702, Met742, Leu764, Cys773, Arg817, Asn818, Asp831, Phe832, and Leu834 to the binding with compound 21e. The interaction was further stabilized through two hydrogen bonds with Lys721 and Thr830 (Figure 3). It should be noted that the 1,2,4-oxadiazole ring usually forms hydrogen bonds with Thr830 and Lys721, which suggests that these are important residues that contribute to the inhibitory potential and significant cytotoxicity activity of these derivatives.

Compound 20f was observed to form an H-bond with Lys721; however, Leu694, Val702, Met742, Leu753, Leu764, Leu820, Phe832, and Leu834 were the key residues involved in hydrophobic interactions. In the dock pose of compound 20i, Leu694, Gly695, Val702, Ala719, Leu764, Thr766, Gly772, Arg817, Leu820, Asp831, and Leu834 were the key residues involved in hydrophobic interactions, and there were two H-bonds formed with Lys721 and Cys773 (Figure 3). Meanwhile, compound 21f also showed only an H-bonding interaction with Met769 (Figure 3). The analysis of docking simulation data further clarified the relationship between the structure of 20f, 20i, and 21f and their cytotoxicity. These esters containing the methoxy group at positions 4′′′ in the R structure had a binding affinity lower than that of the remaining esters in the same series (Table 2). In addition, the number of their hydrogen bonds with crucial amino acids Thr830 and Lys721 was less than that of other ligand-4HJO complexes, resulting in a reduction in their cytotoxic activity and even a loss of cytotoxicity as in the case of 21f which had no hydrogen bond with these amino acids.

Conclusion

A five-step procedure was applied for the synthesis of esters 20a–i and 21a–i of ataluren and its analogs with DHA and zerumbone. Among the mentioned steps, a significant improvement in the step of methyl ester hydrolysis was introduced using a KOH/DMF/H₂O system at room temperature to obtain ataluren and its analogs in good yields. Subsequently, the ester linkage was used to couple ataluren and its analogs with the bromide derivatives of DHA and zerumbone to yield two series, 20a–i and 21a–i, respectively. Screening for cytotoxic activity against two human cancer cell lines, HepG2 and MCF-7, showed that the cytotoxicity of esters 20a–i and 21a–i against the tested cell lines was improved compared to DHA and zerumbone. However, the cytotoxic activity of esters 21a–i was stronger than that of esters 20a–i and depended on the tested cell line and the structure of the R moiety. Specifically, the esters 20a–i exhibited better activity against the HepG2 cell line than that of MCF-7. Moreover, the presence of the methoxy –OCH₃ group at position 4′′′ in the R structure was not beneficial for cytotoxic activity. Finally, docking simulation for the studied esters highlights that compounds 21c, 21e, and 21g were the most potential inhibitors based on binding affinity and docking conformation analysis. These candidates could be considered for further studies in the drug development stage.

Experimental

Chemistry

DHA and zerumbone were prepared as described in our previous study by Tran et al.¹⁶ Other chemicals were purchased from Sigma Aldrich (Singapore) and used without further purification. ¹H NMR and ¹³C NMR spectra were recorded at ambient temperature on a Bruker Avance 600 MHz spectrometer (BioSpin, Germany) in DMSO-d₆. Chemical shifts δ are quoted in parts per million (ppm) referenced to the residual solvent peak (DMSO at 2.50, 3.32, and 39.5 ppm) relative to TMS. Mass spectra were recorded using an Agilent LC/MSD Trap SL. Thin-layer chromatography was performed on precoated Silica Gel 60 F254 aluminum sheets (Merck, Germany), and products were visualized under a UV lamp at 254 nm. Column chromatography was carried out on silica gel (40–230 mesh).

The esters 14a–i were prepared from methyl 3-cyano benzoate 11, hydroxylamine chloride, and corresponding various acyl chlorides: acetyl chloride 13a, isobutyryl chloride 13b, benzoyl chloride 13c, 2-fluorobenzoyl chloride 13d, 3-methoxybenzoyl chloride 13e, 4-methoxybenzoyl chloride 13f, 3-methylbenzoyl chloride 13g, 4-methylbenzoyl chloride 13h, and 3,4,5-trimethoxybenzoyl chloride 13i based on known procedures as reported in the study by Lentini et al.²⁵ and Zakeri et al.²⁶

The 2-(bromo)ethyl ether of DHA 17 was prepared from DHA according to a recipe as described in the study by Liu et al.²⁸ while the intermediate 19 was obtained from zerumbone by a method reported in the study by Kitayama et al.²⁹

General procedure for the hydrolysis of esters 14a–i

A suspension of each ester 14a–i (1 mmol) and KOH (118 mg, 2 mmol) in DMF (3 mL) was stirred at room temperature. Next, a few drops of water were added, and the resulting mixture was stirred at room temperature and was kept in the room overnight until the mixture becomes clear. The solution is diluted with water and filtered through a Buchner funnel. The filtrate was acidified to pH 3 with the dilute HCl, and the precipitate was filtered off and dried in a vacuum to give acids 15a–i that were pure enough for the next step without further purification.

3-(5-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazol-3-yl)benzoic acid 15i

Yield 84%. ¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.63 (t, J = 1.5 Hz, 1H, H-2′), 8.33 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-4′), 8.17 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-6′), 7.74 (t, J = 7.8 Hz, 1H, H-5′), and 7.47 (s, 2H, H-3′′′, H-5′′′).

General procedure for the synthesis of esters 20a–i and 21a–i

Compounds 17 or 19 (1.0 mmol) were added to a stirred solution of each acid 15a–i (1.0 mmol) and potassium tert-butoxide (152.0 mg, 1.1 mmol) in dry dimethylformamide (4 mL) at room temperature. The mixture was stirred at room temperature overnight until TLC (n-hexane: acetone) indicated the absence of acid. The mixture was poured into cool water and acidified by the dilute solution to a pH of 3. The precipitate was filtered on a Buchner funnel, washed thoroughly with water, and dried in a vacuum. Crude 20a–i and 21a–i were purified by column chromatography on silica gel eluted by n-hexane: acetone.

2-(((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)ethyl 3-(5-methyl-1,2,4-oxadiazol-3-yl)benzoate (20a)

Yield 52%, colorless oil; R_f = 0.41 (n-hexane/acetone 2.5:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.61 (t, J = 1.5 Hz, 1H, H-2′), 8.31 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-4′), 8.20 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-6′), 7.78 (t, J = 7.8 Hz, 1H, H-5′), 5.27 (s, 1H, H-12), 4.82 (d, J = 3.0 Hz, 1H, H-10), 4.69 (m, 1H, 10-O-CH_2a-), 4.48 (m, 1H, 10–O-CH_2b-), 4.11 (m, 1H, 10-O-CH₂-CH_2a-), 3.75 (m, 1H, 10-O-CH₂-CH_2b-), 2.74 (s, 3H, 5′′-CH₃), 2.43 (m, 1H, H-9), 2.14 (m, 1H, H-4₁), 1.95 (m, 1H, H-4₂), 1.67 (m, 2H, H-8₁, H-5₁), 1.58 (m, 1H, H-8₂), 1.33 (m, 1H, H-8a), 1.30 (s, 3H, H-15), 1.24 (m, 1H, H-7₁), 1.14 (m, 1H, H-5₂), 1.06 (m, 1H, H-5a), 0.87 (d, J = 7.2 Hz, 3H, H-13), 0.76 (m, 2H, H-6, H-7₂), 0.65 (d, J = 6.0 Hz, 3H, H-14). NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 177.8 (C-5′′), 166.9 (C-3′′), 164.8 (1′-COO-), 131.8 (C-6′), 131.4 (C-4′), 130.6 (C-1′), 129.9 (C-5′), 127.4 (C-2′), 126.9 (C-3′), 103.2 (C-3), 100.6 (C-10), 86.8 (C-12), 80.3 (C-12a), 65.0 (10-O-CH₂-), 64.0 (10-O-CH2-CH₂-), 51.8 (C-5a), 43.7 (C-8a), 36.5 (C-6), 35.9 (C-4), 33.9 (C-7), 30.3 (C-9), 25.5 (C-13), 24.0 (C-5), 23.8 (C-8), 19.9 (C-14), 12.6 (C-15), 11.9 (5′′-CH₃).

2-(((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)ethyl 3-(5-isopropyl-1,2,4-oxadiazol-3-yl)benzoate (20b)

Yield 53%, colorless oil; R_f = 0.41 (n-hexane/acetone 2.5:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.57 (t, J = 1.5 Hz, 1H, H-2′), 8.28 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-4′), 8.16 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-6′), 7.74 (t, J = 7.8 Hz, 1H, H-5′), 5.23 (s, 1H, H-12), 4.78 (d, J = 3.0 Hz, 1H, H-10), 4.66 (m, 1H, 10-O-CH_2a-), 4.44 (m, 1H, 10-O-CH_2b-), 4.06 (m, 1H, 10-O-CH₂-CH_2a-), 3.69 (m, 1H, 10-O-CH₂-CH_2b-), 3.39 (m, 1H, H-1′′′), 2.38 (m, 1H, H-9), 2.12 (m, 1H, H-4₁), 1.90 (m, 1H, H-4₂), 1.63 (m, 2H, H-8₁, H-5₁), 1.54 (m, 1H, H-8₂), 1.40 (d, J = 7.2 Hz, 6H, H-2′′′, H-3′′′), 1.29 (m, 1H, H-8a), 1.26 (s, 3H, H-15), 1.18 (m, 1H, H-7₁), 1.09 (m, 1H, H-5₂), 1.01 (m, 1H, H-5a), 0.83 (d, J = 7.2 Hz, 3H, H-13), 0.77 (m, 1H, H-6), 0.70 (m, 1H, H-7₂), 0.60 (d, J = 6.0 Hz, 3H, H-14). NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 184.5 (C-5′′), 166.7 (C-3′′), 164.8 (1′-COO-), 131.8 (C-6′), 131.4 (C-4′), 130.6 (C-1′), 129.9 (C-5′), 127.4 (C-2′), 127.0 (C-3′), 103.2 (C-3), 100.6 (C-10), 86.8 (C-12), 80.3 (C-12a), 64.9 (10-O-CH₂-), 64.0 (10-O-CH2-CH₂-), 51.7 (C-5a), 43.7 (C-8a), 36.5 (C-6), 35.9 (C-4), 33.8 (C-7), 30.3 (C-9), 25.8 (C-1′′′), 25.5 (C-13), 24.0 (C-5), 23.8 (C-8), 19.8 (C-14, C-2′′′, C-3′′′), 12.6 (C-15). ESI-HRMS calculated for C₂₉H₃₉N₂O₈: [M+H]⁺ (m/z): 543.2701, found: 543.2647.

2-(((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)ethyl 3-(5-phenyl-1,2,4-oxadiazol-3-yl)benzoate (20c)

Yield 63%, white powder, m.p. 75 °C–77 °C; R_f = 0.43 (n-hexane/acetone 2:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.66 (t, J = 1.5 Hz, 1H, H-2′), 8.37 (dt, J₁ = 1.5 Hz, J₂ = 7.5 Hz, 1H, H-4′), 8.21 (m, 3H, H-6′, H-2′′′, H-6′′′), 7.77 (m, 2H, H-5′, H-4′′′), 7.69 (m, 2H, H-3′′′, H-5′′′), 5.26 (s, 1H, H-12), 4.79 (d, J = 3.0 Hz, 1H, H-10), 4.68 (m, 1H, 10-O-CH_2a-), 4.44 (m, 1H, 10-O-CH_2b-), 4.09 (m, 1H, 10-O-CH₂-CH_2a-), 3.71 (m, 1H, 10-O-CH₂-CH_2b-), 2.39 (m, 1H, H-9), 2.08 (m, 1H, H-4₁), 1.80 (m, 1H, H-4₂), 1.57 (m, 3H, H-8₁, H-5₁, H-8₂), 1.28 (m, 1H, H-8a), 1.24 (s, 3H, H-15), 1.16 (m, 1H, H-7₁), 1.08 (m, 1H, H-5₂), 0.98 (m, 1H, H-5a), 0.83 (d, J = 7.2 Hz, 3H, H-13), 0.78 (m, 1H, H-6), 0.68 (m, 1H, H-7₂), 0.57 (d, J = 6.0 Hz, 3H, H-14). NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 175.7 (C-5′′), 167.6 (C-3′′), 164.8 (1′-COO-), 133.5 (C-4′′′), 132.0 (C-6′), 131.5 (C-4′), 130.6 (C-1′), 129.9 (C-5′), 129.6 (C-3′′′, C-5′′′), 127.9 (C-2′′′, C-6′′′), 127.5 (C-2′), 126.7 (C-3′), 123.1 (C-1′′′), 103.2 (C-3), 100.5 (C-10), 86.8 (C-12), 80.2 (C-12a), 64.9 (10-O-CH₂-), 64.0 (10-O-CH2-CH₂-), 51.7 (C-5a), 43.6 (C-8a), 36.5 (C-6), 35.9 (C-4), 33.8 (C-7), 30.3 (C-9), 25.5 (C-13), 23.9 (C-5), 23.8 (C-8), 19.7 (C-14), 12.6 (C-15). ESI-HRMS calculated for C₃₂H₃₇N₂O₈: [M+H]⁺ (m/z): 577.2544, found: 577.2529.

2-(((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)ethyl 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoate (20d)

Yield 45%, white powder, m.p. 75 °C–77 °C; R_f = 0.41 (n-hexane/acetone 2:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.65 (t, J = 1.5 Hz, 1H, H-2′), 8.37 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-4′), 8.25 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-6′′′), 8.20 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-6′), 7.82 (m, 1H, H-4′′′), 7.78 (t, J = 7.8 Hz, 1H, H-5′), 7.56 (ddd, J₁ = 1.5 Hz, J₂ = 2.4 Hz, J₃ = 7.8 Hz, 1H, H-3′′′), 7.50 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-5′′′), 5.25 (s, 1H, H-12), 4.78 (d, J = 3.0 Hz, 1H, H-10), 4.66 (m, 1H, 10-O-CH_2a-), 4.44 (10-O-CH_2b-), 4.07 (m, 1H, 10-O-CH₂-CH_2a-), 3.69 (m, 1H, 10-O-CH₂-CH_2b-), 2.38 (m, 1H, H-9), 2.08 (t, J = 3.9 Hz, 1H, H-4₁), 1.81 (m, 1H, H-4₂), 1.57 (m, 3H, H-8₁, H-5₁, H-8₂), 1.28 (m, H-8a), 1.23 (s, 3H, H-115), 1.17 (m, 1H, H-7₁), 1.07 (s, H-5₂), 0.98 (m, 1H, H-5a), 0.82 (d, J = 7.8 Hz, 3H, H-13), 0.77 (m, 1H, H-6), 0.68 (m, 1H, H-7₂), 0.57 (d, J = 6.0 Hz, 1H, H-14). NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 172.8 (C-5′′), 167.3 (C-3′′), 164.8 (1′-COO-), 160.0 (d, J = 255.0 Hz, C-2′′′), 135.9 (d, J = 9.0 Hz, C-4′′′), 132.1 (C-6′), 131.6 (C-4′), 130.9 (C-6′′′), 130.7 (C-1′), 130.0 (C-5′), 127.5 (C-2′), 126.6 (C-3′), 125.5 (d, J = 3.0 Hz, C-5′′′), 117.3 (d, J = 21.0 Hz, C-3′′′), 111.6 (d, J = 10.5 Hz, C-1′′′), 103.2 (C-3), 100.5 (C-10), 64.9 (10-O-CH2-CH₂-), 64.0 (10-O-CH₂-), 51.7 (C-5a), 43.6 (C-8a), 36.5 (C-6), 35.9 (C-4), 33.8 (C-7), 30.3 (C-9), 25.5 (C-15′), 23.9 (C-5), 23.8 (C-8), 19.7 (C-13), 12.6 (C-14). ESI-HRMS calculated for C₃₂H₃₆FN₂O₈: [M+H]⁺ (m/z): 595.2450, found: 595.2463.

2-(((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)ethyl 3-(5-(3-methoxyphenyl)-1,2,4-oxadiazol-3-yl)benzoate (20e)

Yield 67%, white powder, m.p. 72 °C–74 °C; R_f = 0.36 (n-hexane/acetone 2:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.66 (t, J = 1.5 Hz, 1H, H-2′), 8.37 (dt, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-4′), 8.20 (dt, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-6′), 7.79 (m, 2H, H-5′′′, H-6′′′), 7.68 (m, 1H, H-2′′′), 7.60 (t, J = 7.8 Hz, 1H, H-5′), 7.33 (dt, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-4′′′), 5.26 (s, 1H, H-12), 4.79 (d, J = 3.0 Hz, 1H, H-10), 4.68 (m, 1H, 10-O-CH_2a-), 4.44 (m, 1H, 10-O-CH_2b-), 4.09 (m, 1H, 10-O-CH₂-CH_2a-), 3.90 (s, 3H, 3′′′-OCH₃), 3.70 (m, 1H, 10-O-CH₂-CH_2b-), 2.38 (m, 1H, H-9), 2.08 (m, 1H, H-4₁), 1.80 (m, 1H, H-4₂), 1.58 (m, 3H, H-8₁, H-5₁, H-8₂), 1.28 (m, 1H, H-8a), 1.24 (s, 3H, H-15), 1.15 (m, 1H, H-7₁), 1.08 (m, 1H, H-5₂), 0.98 (m, 1H, H-5a), 0.83 (d, J = 7.2 Hz, 3H, H-13), 0.78 (m, 1H, H-6), 0.69 (m, 1H, H-7₂), 0.56 (d, J = 6.0 Hz, 3H, H-14). NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 175.6 (C-5′′), 167.6 (C-3′′), 164.8 (1′-COO-), 159.7 (C-3′′′), 132.1 (C-6′), 131.5 (C-4′), 130.9 (C-5′′′), 130.6 (C-1′), 130.0 (C-5′), 127.5 (C-2′), 126.7 (C-3′), 124.5 (C-1′′′), 120.2 (C-6′′′), 119.5 (C-4′′′), 112.6 (C-2′′′), 103.2 (C-3), 100.5 (C-10), 86.8 (C-12), 80.3 (C-12a), 64.9 (10-O-CH₂-), 64.0 (10-O-CH2-CH₂-), 55.5 (3′′′-OCH₃), 51.7 (C-5a), 43.6 (C-8a), 36.5 (C-6), 35.9 (C-4), 33.8 (C-7), 30.3 (C-9), 25.4 (C-13), 23.9 (C-5), 23.8 (C-8), 19.7 (C-14), 12.6 (C-15). ESI-HRMS calculated for C₃₃H₃₉N₂O₉: [M+H]⁺ (m/z): 607.2650, found: 607.2636.

2-(((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)ethyl 3-(5-(4-methoxyphenyl)-1,2,4-oxadiazol-3-yl)benzoate (20f)

Yield 64%, white powder, m.p. 121 °C–123 °C; R_f = 0.37 (n-hexane/acetone 2:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.64 (t, J = 1.5 Hz, 1H, H-2′), 8.34 (dd, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-4′), 8.18 (d, J = 7.8 Hz, 1H, H-6′), 8,15 (m, 2H, H-2′′′, H-6′′′), 7.77 (t, J = 7.8 Hz, 1H, H-5′), 7.20 (m, 2H, H-3′′′, H-5′′′), 5.26 (s, 1H, H-12), 4.79 (d, J = 3.0 Hz, 1H, H-10), 4.67 (m, 1H, 10-O-CH_2a-), 4.44 (m, 1H, 10-O-CH_2b-), 4.09 (m, 1H, 10-O-CH₂-CH_2a-), 3.89 (s, 3H, 4′′′-OCH₃), 3.70 (m, 1H, 10-O-CH₂-CH_2b-), 2.39 (m, 1H, H-9), 2.08 (m, 1H, H-4₁), 1.81 (m, 1H, H-4₂), 1.57 (m, 3H, H-8₁, H-5₁, H-8₂), 1.28 (m, 1H, H-8a), 1.24 (s, 3H, H-15), 1.17 (m, 1H, H-7₁), 1.08 (m, 1H, H-5₂), 0.98 (m, 1H, H-5a), 0.83 (d, J = 7.2 Hz, 3H, H-13), 0.78 (m, 1H, H-6), 0.68 (m, 1H, H-7₂), 0.57 (d, J = 6.0 Hz, 3H, H-14). NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 175.5 (C-5′′), 167.4 (C-3′′), 164.8 (1′-COO-), 163.2 (C-4′′′), 131.9 (C-6′), 131.5 (C-4′), 130.6 (C-1′), 130.0 (C-2′′′, C-6′′′), 129.9 (C-5′), 127.5 (C-2′), 126.9 (C-3′), 115.5 (C-1′′′), 115.0 (C-3′′′, C-5′′′), 103.2 (C-3), 100.5 (C-10), 86.8 (C-12), 80.2 (C-12a), 64.9 (10-O-CH₂-), 64.0 (10-O-CH2-CH₂-), 55.6 (4′′′-OCH₃), 51.7 (C-5a), 43.6 (C-8a), 36.5 (C-6), 35.9 (C-4), 33.8 (C-7), 30.3 (C-9), 25.5 (C-13), 23.9 (C-5), 23.8 (C-8), 19.7 (C-14), 12.6 (C-15). ESI-HRMS calculated for C₃₃H₃₉N₂O₉: [M+H]⁺ (m/z): 607.2650, found: 607.2636.

2-(((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)ethyl 3-(5-(m-tolyl)-1,2,4-oxadiazol-3-yl)benzoate (20g)

Yield 57%, white powder, m.p. 73 °C–75 °C; R_f=0.40 (n-hexane/acetone 2:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.65 (t, J = 1.5 Hz, 1H, H-2′), 8.36 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-4′), 8.19 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-6′), 8.00 (m, 2H, H-2′′′, H-5′′′), 7.77 (t, J = 7.8 Hz, 1H, H-5′), 7.56 (m, 2H, H-4′′′, H-6′′′), 5.26 (s, 1H, H-12), 4.79 (d, J = 3.0 Hz, 1H, H-10), 4.68 (m, 1H, 10-O-CH_2a-), 4.44 (m, 1H, 10-O-CH_2b-), 4.09 (m, 1H, 10-O-CH₂-CH_2a-), 3.71 (m, 1H, 10-O-CH₂-CH_2b-), 2.45 (s, 3H, 3′′′-CH₃), 2.39 (m, 1H, H-9), 2.08 (m, 1H, H-4₁), 1.82 (m, 1H, H-4₂), 1.58 (m, 3H, H-8₁, H-5₁, H-8₂), 1.28 (m, 1H, H-8a), 1.24 (s, 3H, H-15), 1.17 (m, 1H, H-7₁), 1.08 (m, 1H, H-5₂), 0.99 (m, 1H, H-5a), 0.84 (d, J = 7.2 Hz, 3H, H-13), 0.78 (m, 1H, H-6), 0.68 (m, 1H, H-7₂), 0.57 (d, J = 6.0 Hz, 3H, H-14). NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 175.8 (C-5′′), 167.5 (C-3′′), 164.8 (1′-COO-), 139.1 (C-3′′′), 134.1 (C-2′′′), 132.0 (C-6′), 131.5 (C-4′), 130.6 (C-1′), 129.9 (C-5′), 129.4 (C-5′′′), 128.1 (C-4′′′), 127.5 (C-2′), 126.8 (C-3′), 125.1 (C-1′′′), 123.1 (C-6′′′), 103.2 (C-3), 100.6 (C-10), 86.8 (C-12), 80.2 (C-12a), 64.9 (10-O-CH₂-), 64.0 (10-O-CH2-CH₂-), 51.7 (C-5a), 43.7 (C-8a), 36.5 (C-6), 35.9 (C-4), 33.8 (C-7), 30.3 (C-9), 25.5 (C-13), 24.0 (C-5), 23.8 (C-8), 20.7 (3′′′-CH₃), 19.7 (C-14), 12.6 (C-15). ESI-HRMS calculated for C₃₃H₃₉N₂O₈: [M+H]⁺ (m/z): 591.2701, found: 591.2694.

2-(((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)ethyl 3-(5-(p-tolyl)-1,2,4-oxadiazol-3-yl)benzoate (20h)

Yield 60%, white powder, m.p. 78 °C–80 °C; R_f = 0.40 (n-hexane/acetone 2:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.65 (t, J = 1.5 Hz, 1H, H-2′), 8.37 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-4′), 8.20 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-6′), 8.11 (d, J = 7.8 Hz, 2H, H-2′′′, H-6′′′), 7.78 (t, J = 7.8 Hz, 1H, H-5′), 7.49 (d, J = 7.8 Hz, 2H, H-3′′′, H-5′′′), 5.26 (s, 1H, H-12), 4.79 (d, J = 3.0 Hz, 1H, H-10), 4.68 (m, 1H, 10-O-CH_2a-), 4.44 (m, 1H, 10-O-CH_2b-), 4.09 (m, 1H, 10-O-CH₂-CH_2a-), 3.70 (m, 1H, 10-O-CH₂-CH_2b-), 2.45 (s, 3H, 4′′′-CH₃), 2.38 (m, 1H, H-9), 2.08 (m, 1H, H-4₁), 1.80 (m, 1H, H-4₂), 1.57 (m, 3H, H-8₁, H-5₁, H-8₂), 1.28 (m, 1H, H-8a), 1.24 (s, 3H, H-15), 1.16 (m, 1H, H-7₁), 1.07 (m, 1H, H-5₂), 0.99 (m, 1H, H-5a), 0.82 (d, J = 7.2 Hz, 3H, H-13), 0.76 (m, 1H, H-6), 0.68 (m, 1H, H-7₂), 0.56 (d, J = 6.0 Hz, 3H, H-14). NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 175.8 (C-5′′), 167.5 (C-3′′), 164.8 (1′-COO-), 144.0 (C-4′′′), 132.0 (C-6′), 131.5 (C-4′), 130.6 (C-1′), 130.1 (C-2′′′, C-6′′′), 130.0 (C-5′), 127.9 (C-3′′′, C-5′′′), 127.5 (C-2′), 126.8 (C-3′), 120.4 (C-1′′′), 103.2 (C-3), 100.5 (C-10), 86.8 (C-12), 80.3 (C-12a), 64.9 (10-O-CH₂-), 64.0 (10-O-CH2-CH₂-), 51.7 (C-5a), 43.6 (C-8a), 36.5 (C-6), 35.9 (C-4), 33.8 (C-7), 30.3 (C-9), 25.5 (C-13), 23.9 (C-5), 23.8 (C-8), 21.2 (4′′′-CH₃), 19.7 (C-14), 12.6 (C-15). ESI-HRMS calculated for C₃₃H₃₉N₂O₈: [M+H]⁺ (m/z): 591.2701, found: 591.2678.

2-(((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)oxy)ethyl 3-(5-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazol-3-yl)benzoate (20i)

Yield 58%, white powder, m.p. 186 °C–188 °C; R_f = 0.34 (n-hexane/acetone 2:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.65 (t, J = 1.5 Hz, 1H, H-2′), 8.38 (dt, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-4′), 8.20 (dt, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-6′), 7.78 (t, J = 7.8 Hz, 1H, H-5′), 7.47 (s, 2H, H-2′′′, H-6′′′), 5.25 (s, 1H, H-12), 4.79 (d, J = 3.6 Hz, 1H, H-10), 4.67 (m, 1H, 10-O-CH_2a-), 4.45 (m, 1H, 10-O-CH_2b-), 4.08 (m, 1H, 10-O-CH₂-CH_2a-), 3.94 (s, 6H, 3′′′-OCH₃, 5′′′-OCH₃), 3.80 (s, 3H, 4′′′-OCH₃), 3.70 (m, 1H, 10-O-CH₂-CH_2b-), 2.38 (m, 1H, H-9), 2.09 (m, 1H, H-4₁), 1.82 (m, 1H, H-4₂), 1.60 (m, 2H, H-8₁, H-5₁), 1.53 (m, 1H, H-8₂), 1.28 (m, 1H, H-8a), 1.24 (s, 3H, H-15), 1.16 (m, 1H, H-7₁), 1.07 (m, 1H, H-5₂), 0.99 (m, 1H, H-5a), 0.83 (d, J = 7.2 Hz, 3H, H-13), 0.79 (m, 1H, H-6), 0.68 (m, 1H, H-7₂), 0.58 (d, J = 6.0 Hz, 3H, H-14). NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 175.6 (C-5′′), 167.6 (C-3′′), 164.8 (1′-COO-), 153.5 (C-3′′′, C-5′′′), 141.9 (C-4′′′), 132.0 (C-6′), 131.6 (C-4′), 130.6 (C-1′), 130.0 (C-5′), 127.5 (C-2′), 126.7 (C-3′), 118.2 (C-1′′′), 105.4 (C-2′′′, C-6′′′), 103.2 (C-3), 100.6 (C-10), 86.8 (C-12), 80.3 (C-12a), 65.0 (10-O-CH₂-), 64.0 (10-O-CH2-CH₂-), 60.3 (4′′′-OCH₃), 56.3 (3′′′-OCH₃, 5′′′-OCH₃), 51.7 (C-5a), 43.7 (C-8a), 36.5 (C-6), 35.9 (C-4), 33.8 (C-7), 30.3 (C-9), 25.5 (C-13), 24.0 (C-5), 23.8 (C-8), 19.7 (C-14), 12.6 (C-15). ESI-HRMS calculated for C₃₅H₄₃N₂O₁₁: [M+H]⁺ (m/z): 667.2861, found: 667.2839.

((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methyl-3-(5-methyl-1,2,4-oxadiazol-3-yl)benzoate (21a)

Yield 57%, colorless oil; R_f = 0.42 (n-hexane/acetone 3:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.54 (t, J = 1.5 Hz, 1H, H-2′), 8.27 (m, 1H, H-4′), 8.16 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-6′), 7.74 (t, J = 7.8 Hz, 1H, H-5′), 6.05 (t, J = 5.7 Hz, 1H, H-3), 5.97 (d, J = 16.2 Hz, 1H, H-10), 5.91 (d, J = 16.2 Hz, 1H, H-11), 5.59 (t, J = 8.1 Hz, 1H, H-7), 4.97 (s, 1H, H-15a), 4.80 (s, 1H, H-15b), 2.67 (s, 3H, 5′′-CH₃), 2.59 (m, 2H, H5a, H-8a), 2.48 (m, 1H, H-4a), 2.28 (m, 2H, H-5b, H-4b), 2.03 (m, 1H, H-8b), 1.55 (s, 3H, H-12), 1.23 (s, 3H, H-13 or H-14), 1.09 (s, 3H, H-14 or H-13).¹³C NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 202.5 (C-1), 177.9 (C-5′′), 166.8 (C-3′′), 164.9 (1′-COO-), 159.3 (C-10), 148.9 (C-3), 137.6 (C-2), 134.0 (C-6), 131.9 (C-6′), 131.5 (C-7), 131.4 (C-4′), 130.6 (C-1′), 130.0 (C-5′), 127.4 (C-2′), 126.94 (C-11), 126.88 (C-3′), 61.8 (C-15), 41.6 (C-8), 37.0 (C-9), 34.7 (C-5), 28.9 (C-13 or C-14), 24.4 (C-4), 23.6 (C-14 or C-13), 20.7 (3′′′-CH₃), 11.9 (5′′-CH₃), 11.6 (C-12). ESI-HRMS calculated for C₂₅H₂₉N₂O₄: [M+H]⁺ (m/z): 421.2122, found: 421.2105.

((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methyl-3-(5-isopropyl-1,2,4-oxadiazol-3-yl)benzoate (21b)

Yield 54%, colorless oil; R_f = 0.42 (n-hexane/acetone 3:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.55 (t, J = 1.5 Hz, 1H, H-2′), 8.28 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-4′), 8.16 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-6′), 7.75 (t, J = 7.8 Hz, 1H, H-5′), 6.05 (t, J = 5.4 Hz, 1H, H-3), 5.97 (d, J = 16.2 Hz, 1H, H-10), 5.92 (d, J = 16.2 Hz, 1H, H-11), 5.60 (t, J = 8.1 Hz, 1H, H-7), 4.98 (s, 1H, H-15a), 4.81 (s, 1H, H-15b), 3.38 (m, 1H, H-1′′′), 2.58 (m, 2H, H5a, H-8a), 2.47 (m, 1H, H-4a), 2.28 (m, 2H, H-5b, H-4b), 2.03 (m, 1H, H-8b), 1.55 (s, 3H, H-12), 1.23 (s, 3H, H-13 or H-14), 1.10 (s, 3H, H-14 or H-13).¹³C NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 202.5 (C-1), 184.5 (C-5′′), 166.7 (C-3′′), 164.9 (1′-COO-), 159.3 (C-10), 148.9 (C-3), 137.6 (C-2), 134.0 (C-6), 131.9 (C-6′), 131.54 (C-7), 131.49 (C-4′), 130.6 (C-1′), 130.0 (C-5′), 127.4 (C-2′), 126.96 (C-11), 126.94 (C-3′), 61.8 (C-15), 41.6 (C-8), 37.0 (C-9), 34.7 (C-5), 28.9 (C-13 or C-14), 26.8 (C-1′′′), 24.4 (C-4), 23.6 (C-14 or C-13), 19.7 (C-2′′′, C-3′′′), 11.7 (C-12). ESI-HRMS calculated for C₂₇H₃₃N₂O₄: [M+H]⁺ (m/z): 449.2435, found: 449.2415.

((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methyl-3-(5-phenyl-1,2,4-oxadiazol-3-yl)benzoate (21c)

Yield 60%, white powder, m.p. 68 °C–70 °C; R_f = 0.39 (n-hexane/acetone 3:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.64 (t, J = 1.5 Hz, 1H, H-2′), 8.37 (dt, J₁ = 1.5 Hz, J₂ = 7.5 Hz, 1H, H-4′), 8.20 (m, 3H, H-6′, H-2′′′, H-6′′′), 7.77 (m, 2H, H-5′, H-4′′′), 7.68 (m, 2H, H-3′′′, H-5′′′), 6.06 (t, J = 5.7 Hz, 1H, H-3), 5.98 (d, J = 16.2 Hz, 1H, H-10), 5.93 (d, J = 16.2 Hz, 1H, H-11), 5.61 (t, J = 8.1 Hz, 1H, H-7), 5.00 (s, 1H, H-15a), 4.82 (s, 1H, H-15b), 2.61 (m, 2H, H5a, H-8a), 2.48 (m, 1H, H-4a), 2.29 (m, 2H, H-5b, H-4b), 2.05 (m, 1H, H-8b), 1.56 (s, 3H, H-12), 1.24 (s, 3H, H-13 or H-14), 1.11 (s, 3H, H-14 or H-13).¹³C NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 202.5 (C-1), 175.8 (C-5′′), 167.5 (C-3′′), 164.9 (1′-COO-), 159.3 (C-10), 148.9 (C-3), 137.6 (C-2), 134.0 (C-6), 133.5 (C-1′′′), 132.1 (C-6′), 131.6 (C-4′), 130.6 (C-1′), 130.1 (C-5′), 129.6 (C-3′′′, C-5′′′), 128.0 (C-2′′′, C-6′′′), 127.5 (C-2′), 126.9 (C-11), 126.8 (C-3′), 123.2 (C-1′′′), 61.8 (C-15), 41.7 (C-8), 37.0 (C-9), 34.7 (C-5), 28.9 (C-13 or C-14), 28.9 (C-13 or C-14), 24.4 (C-4), 23.6 (C-14 or C-13), 11.7 (C-12). ESI-HRMS calculated for C₃₀H₃₁N₂O₄: [M+H]⁺ (m/z): 483.2278, found: 483.2254.

((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methyl-3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoate (21d)

Yield 28%, colorless oil; R_f = 0.38 (n-hexane/acetone 3:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.64 (t, J = 1.5 Hz, 1H, H-2′), 8.38 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-4′), 8.26 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-6′′′), 8.21 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-6′), 7.82 (m, 2H, H-4′′′, H-5′), 7.57 (t, J = 7.8 Hz, 1H, H-3′′′), 7.52 (m, 1H, H-5′′′), 6.06 (t, J = 5.4 Hz, 1H, H-3), 5.98 (d, J = 16.5 Hz, 1H, H-10), 5.93 (d, J = 16.5 Hz, 1H, H-11), 5.61 (t, J = 8.1 Hz, 1H, H-7), 5.01 (s, 1H, H-15a), 4.82 (s, 1H, H-15b), 2.62 (m, 3H, H5a, H-8a, H-4a), 2.30 (m, 2H, H-5b, H-4b), 2.05 (m, 1H, H-8b), 1.56 (s, 3H, H-12), 1.24 (s, 3H, H-13 or H-14), 1.11 (s, 3H, H-14 or H-13). ¹³C NMR (125 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 212.0 (C-1), 176.8 (C-5′), 174.4 (C-3′), 169.5 (d, J = 257.5 Hz, C-2′′′), 168.9 (1′-COO-), 158.4 (C-10), 147.1 (C-3), 145.4 (C-4′′′), 143.6 (C-6), 141.7 (C-6′), 141.2 (C-4′), 141.0 (C-7), 140.4 (C-1′′′), 140.2 (C-1′), 139.7 (C-5′), 137.1 (C-2′), 136.5 (C-11), 136.1 (C-3′), 135.0 (C-5′′′), 126.8 (d, J = 21.15 Hz, C-3′′′), 121.1 (C-6′′′), 71.4 (C-15), 51.2 (C-8), 46.5 (C-9), 44.2 (C-6), 38.4 (C-13 or C-14), 33.9 (C-4), 33.3 (C-14 or C-13), 21.2 (C-12). ESI-HRMS calculated for C₃₀H₃₀FN₂O₄: [M+H]⁺ (m/z): 501.2198, found: 501.2176.

((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methyl-3-(5-(3-methoxyphenyl)-1,2,4-oxadiazol-3-yl)benzoate (21e)

Yield 55%, white powder, m.p. 82 °C–84 °C; R_f = 0.36 (n-hexane/acetone 3:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.61 (t, J = 1.8 Hz, 1H, H-2′), 8.34 (dt, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-4′), 8.18 (dt, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-6′), 7.77 (m, 2H, H-5′′′, H-6′′′), 7.65 (t, J = 1.8 Hz, 1H, H-2′′′), 7.58 (t, J = 7.8 Hz, 1H, H-5′), 7.31 (dt, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-4′′′), 6.05 (t, J = 5.4 Hz, 1H, H-3), 5.97 (d, J = 16.2 Hz, 1H, H-10), 5.92 (d, J = 16.2 Hz, 1H, H-11), 5.60 (t, J = 8.4 Hz, 1H, H-7), 4.99 (s, 1H, H-15a), 4.80 (s, 1H, H-15b), 3.89 (s, 3H, 3′′′-OCH₃), 2.60 (m, 2H, H5a, H-8a), 2.49 (m, 1H, H-4a), 2.29 (m, 2H, H-5b, H-4b), 2.03 (m, 1H, H-8b), 1.57 (s, 3H, H-12), 1.24 (s, 3H, H-13 or H-14), 1.11 (s, 3H, H-14 or H-13). NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 202.4 (C-1), 175.6 (C-5′′), 167.5 (C-3′′), 164.9 (1′-COO-), 159.7 (C-3′′′), 159.3 (C-10), 148.9 (C-3), 137.6 (C-2), 134.0 (C-6), 132.1 (C-6′), 131.6 (C-4′), 131.5 (C-7), 130.8 (C-1′), 130.6 (C-5′), 130.0 (C-5′′′), 127.5 (C-2′), 127.0 (C-11), 126.7 (C-3′), 124.3 (C-1′′′), 120.3 (C-6′′′), 119.5 (C-4′′′), 112.5 (C-2′′′), 61.8 (C-15), 55.5 (3′′′-OCH₃), 41.7 (C-8), 37.0 (C-9), 34.7 (C-5), 28.9 (C-13 or C-14), 24.4 (C-4), 23.6 (C-14 or C-13), 11.7 (C-12). ESI-HRMS calculated for C₃₁H₃₃N₂O₅: [M+H]⁺ (m/z): 513.2384, found: 513.2360.

((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methyl-3-(5-(4-methoxyphenyl)-1,2,4-oxadiazol-3-yl)benzoate (21f)

Yield 54%, white powder, m.p. 109 °C–111 °C; R_f=0.36 (n-hexane/acetone 2:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.60 (t, J = 1.5 Hz, 1H, H-2′), 8.33 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-4′), 8.17 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-6′), 8.13 (m, 2H, H-2′′′, H-6′′′), 7.76 (t, J = 7.8 Hz, 1H, H-5′), 7.19 (m, 2H, H-3′′′, H-5′′′), 6.04 (t, J = 5.7 Hz, 1H, H-3), 5.97 (d, J = 16.2 Hz, 1H, H-10), 5.91 (d, J = 16.2 Hz, 1H, H-11), 5.59 (t, J = 8.4 Hz, 1H, H-7), 4.98 (s, 1H, H-15a), 4.80 (s, 1H, H-15b), 3.88 (s, 3H, 4′′′-OCH₃), 2.60 (m, 2H, H5a, H-8a), 2.49 (m, 1H, H-4a), 2.29 (m, 2H, H-5b, H-4b), 2.04 (m, 1H, H-8b), 1.55 (s, 3H, H-12), 1.23 (s, 3H, H-13 or H-14), 1.10 (s, 3H, H-14 or H-13). NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 201.9 (C-1), 175.4 (C-5′′), 167.2 (C-3′′), 164.7 (1′-COO-), 163.1 (C-4′′′), 158.7 (C-10), 148.3 (C-3), 137.3 (C-2), 133.8 (C-6), 131.6 (C-6′), 131.3 (C-4′), 131.2 (C-7), 130.5 (C-1′), 129.7 (C-2′′′, C-6′′′), 129.6 (C-5′), 127.3 (C-2′), 126.8 (C-11), 126.7 (C-3′), 115.4 (C-1′′′), 114.8 (C-3′′′, C-5′′′), 61.6 (C-15), 55.4 (4′′′-OCH₃), 41.5 (C-8), 36.7 (C-9), 34.6 (C-5), 24.1 (C-4), 11.3 (C-12). ESI-HRMS calculated for C₃₁H₃₃N₂O₅: [M+H]⁺ (m/z): 513.2384, found: 513.2367.

((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methyl-3-(5-(m-tolyl)-1,2,4-oxadiazol-3-yl)benzoate (21g)

Yield 45%, white powder, m.p. 88 °C–90 °C; R_f = 0.38 (n-hexane/acetone 3:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.60 (s, 1H, H-2′), 8.33 (d, J = 7.8 Hz, 1H, H-4′), 8.17 (d, J = 7.8 Hz, 1H, H-6′), 7.97 (m, 2H, H-2′′′, H-5′′′), 7.76 (t, J = 7.8 Hz, 1H, H-5′), 7.54 (m, 2H, H-4′′′, H-6′′′), 6.05 (t, J = 5.4 Hz, 1H, H-3), 5.97 (d, J = 16.5 Hz, 1H, H-10), 5.93 (d, J = 16.5 Hz, 1H, H-11), 5.60 (t, J = 8.4 Hz, 1H, H-7), 4.99 (s, 1H, H-15a), 4.80 (s, 1H, H-15b), 2.60 (m, 2H, H5a, H-8a), 2.48 (m, 4H, H-4a, 3′′′-CH₃), 2.29 (m, 2H, H-5b, H-4b), 2.03 (m, 1H, H-8b), 1.57 (s, 3H, H-12), 1.24 (s, 3H, H-13 or H-14), 1.11 (s, 3H, H-14 or H-13). NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 202.4 (C-1), 175.8 (C-5′′), 167.4 (C-3′′), 164.8 (1′-COO-), 159.7 (C-3′′′), 159.3 (C-10), 148.9 (C-3), 137.6 (C-2), 134.1 (C-2′′′), 134.0 (C-6), 132.0 (C-6′), 131.54 (C-7), 131.51 (C-4′), 130.6 (C-1′), 130.0 (C-5′), 129.4 (C-5′′′), 128.2 (C-4′′′), 127.5 (C-2′), 127.0 (C-11), 126.8 (C-3′), 125.1 (C-1′′′), 123.1 (C-6′′′), 61.8 (C-15), 41.7 (C-8), 37.0 (C-9), 34.7 (C-5), 28.9 (C-13 or C-14), 24.4 (C-4), 23.6 (C-14 or C-13), 20.7 (3′′′-CH₃), 11.6 (C-12). ESI-HRMS calculated for C₃₁H₃₃N₂O₄: [M+H]⁺ (m/z): 497.2435, found: 497.2417.

((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methyl-3-(5-(p-tolyl)-1,2,4-oxadiazol-3-yl)benzoate (21h)

Yield 48%, white powder, m.p. 118 °C–120 °C; R_f = 0.38 (n-hexane/acetone 2:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.61 (t, J = 1.5 Hz, 1H, H-2′), 8.33 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-4′), 8.17 (dt, J₁ = 1.5 Hz, J₂ = 7.8 Hz, 1H, H-6′), 8.08 (d, J = 7.8 Hz, 2H, H-2′′′, H-6′′′), 7.77 (t, J = 7.8 Hz, 1H, H-5′), 7.47 (d, J = 7.8 Hz, 2H, H-3′′′, H-5′′′), 6.05 (t, J = 5.7 Hz, 1H, H-3), 5.97 (d, J = 16.2 Hz, 1H, H-10), 5.93 (d, J = 16.2 Hz, 1H, H-11), 5.60 (t, J = 8.4 Hz, 1H, H-7), 4.99 (s, 1H, H-15a), 4.81 (s, 1H, H-15b), 2.60 (m, 2H, H5a, H-8a), 2.43 (m, 4H, H-4a, 4′′′-CH₃), 2.29 (m, 2H, H-5b, H-4b), 2.04 (m, 1H, H-8b), 1.56 (s, 3H, H-12), 1.24 (s, 3H, H-13 or H-14), 1.11 (s, 3H, H-14 or H-13). NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 202.4 (C-1), 175.8 (C-5′′), 167.4 (C-3′′), 164.9 (1′-COO-), 159.3 (C-10), 148.9 (C-3), 137.6 (C-2), 134.0 (C-6), 132.0 (C-6′), 131.6 (C-4′), 131.5 (C-7, C-4′′′), 130.6 (C-1′), 130.1 (C-2′′′, C-6′′′), 130.0 (C-5′), 127.9 (C-3′′′, C-5′′′), 127.5 (C-2′), 127.0 (C-11), 126.8 (C-3′), 120.4 (C-1′′′), 61.8 (C-15), 41.7 (C-8), 37.0 (C-9), 34.7 (C-5), 28.9 (C-13 or C-14), 24.4 (C-4), 23.6 (C-14 or C-13), 11.7 (C-12). ESI-HRMS calculated for C31H33N2O4: [M+H]+ (m/z): 497.2435, found: 497.2414.

((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methyl-3-(5-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazol-3-yl)benzoate (21i)

Yield 50%, white powder, m.p. 154 °C–156 °C; R_f = 0.33 (n-hexane/acetone 3:1 v/v);¹H NMR (600 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 8.60 (t, J = 1.5 Hz, 1H, H-2′), 8.34 (dt, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-4′), 8.17 (dt, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-6′), 7.76 (t, J = 7.8 Hz, 1H, H-5′), 7.42 (s, 2H, H-2′′′, H-6′′′), 6.05 (t, J = 5.7 Hz, 1H, H-3), 5.97 (d, J = 16.8 Hz, 1H, H-10), 5.91 (d, J = 16.8 Hz, 1H, H-11), 5.59 (t, J = 8.1 Hz, 1H, H-7), 4.99 (s, 1H, H-15a), 4.80 (s, 1H, H-15b), 3.92 (s, 6H, 3′′′-CH₃, 5′′′-OCH₃), 3.80 (s, 3H, 4′′′-OCH₃), 2.59 (m, 2H, H5a, H-8a), 2.49 (m, 1H, H-4a), 2.29 (m, 2H, H-5b, H-4b), 2.04 (m, 1H, H-8b), 1.58 (s, 3H, H-12), 1.23 (s, 3H, H-13 or H-14), 1.10 (s, 3H, H-14 or H-13). NMR (150 MHz, DMSO-d₆, 25 °C, TMS, δ (ppm)): 202.4 (C-1), 175.5 (C-5′′), 167.5 (C-3′′), 164.9 (1′-COO-), 159.2 (C-10), 153.4 (C-3′′′, C-5′′′), 148.9 (C-3), 141.8 (C-4′′′), 137.6 (C-2), 134.0 (C-6), 132.0 (C-6′), 131.7 (C-4′), 131.4 (C-7), 130.6 (C-1′), 130.0 (C-5′), 127.5 (C-2′), 127.0 (C-11), 118.1 (C-1′′′), 105.3 (C-3′′′, C-5′′′), 61.8 (C-15), 60.2 (4′′′-OCH₃), 56.2 (3′′′-OCH₃, 5′′′-OCH₃), 41.6 (C-8), 37.0 (C-9), 34.7 (C-5), 28.9 (C-13 or C-14), 24.4 (C-4), 23.6 (C-14 or C-13), 11.7 (C-12). ESI-HRMS calculated for C₃₃H₃₇N₂O₇: [M+H]⁺ (m/z): 573.2595, found: 573.2571.

Biology

The in vitro cytotoxic evaluation of the synthesized compounds against Hep-G2 and MCF-7 cancer cell lines was carried out according to the described protocols.^37,38

Molecular docking

The three-dimensional structures of compounds were prepared using MarvinSketch version 19.27.0 and PyMOL version 1.3r1.³⁹ Energy minimization of the studied ligands was conducted using the MM2 force field, and quantum chemical calculations were performed at the B3LYP/6-31g(d, p) level implemented in Gaussian 09.⁴⁰ Erlotinib, a well-known inhibitor of the EGFR protein, was selected as a reference ligand.⁴¹

The X-ray crystal structure of the EGFR protein was obtained from the Protein Data Bank archive (PDB) with entry ID: 4HJO.⁴² It is assumed that the protein is rigid, and the conformational space of the ligands to analyze the inductive effect of the compounds is considered. The Graphical User Interface program named AutoDock Tools 1.5.6rc3 (ADT) was employed to set up input data.³³ Details of the molecular docking simulation are given in Supplementary Material 2.

Supplemental Material

sj-pdf-1-chl-10.1177_17475198231219451 – Supplemental material for Dihydroartemisinin and zerumbone esters of ataluren and its analogs as anticancer agents and EGFR inhibitors

Supplemental material, sj-pdf-1-chl-10.1177_17475198231219451 for Dihydroartemisinin and zerumbone esters of ataluren and its analogs as anticancer agents and EGFR inhibitors by Duc Quan Tran, Ngoc Hung Truong, Thi Hoang Anh Nguyen, Thi Thuy Trinh, Thi Cham Ba, Thi Thuy Linh Nguyen, Van Tu Ngo, Thi Inh Cam, Minh Quan Pham and Van Chinh Luu in Journal of Chemical Research

Supplemental Material

sj-pdf-2-chl-10.1177_17475198231219451 – Supplemental material for Dihydroartemisinin and zerumbone esters of ataluren and its analogs as anticancer agents and EGFR inhibitors

Supplemental material, sj-pdf-2-chl-10.1177_17475198231219451 for Dihydroartemisinin and zerumbone esters of ataluren and its analogs as anticancer agents and EGFR inhibitors by Duc Quan Tran, Ngoc Hung Truong, Thi Hoang Anh Nguyen, Thi Thuy Trinh, Thi Cham Ba, Thi Thuy Linh Nguyen, Van Tu Ngo, Thi Inh Cam, Minh Quan Pham and Van Chinh Luu in Journal of Chemical Research

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this paper.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this paper: This study was financially supported by the Vietnam Academy of Science and Technology under the project with the code: KHCBHH.02/21-23.

ORCID iDs

Ngoc Hung Truong

Thi Thuy Trinh

Van Chinh Luu

Supplemental material

Supplemental material for this paper is available online.

References

Boström

Hogner

Llinàs

, et al. J Med Chem 2012; 55: 1817–1830.

Biernacki

Daśko

Ciupak

, et al. Pharmaceuticals (Basel) 2020; 13: 111.

Atmaram

Roopan

. Appl Microbiol Biotechnol 2022; 106: 3489–3505.

Dhameliya

Chudasma

Patel

, et al. Mol Divers 2022; 26: 2967–2980.

Bommera

Kethireddy

Govindapur

, et al. BMC Chemistry 2021; 15: 30.

Singhai

Gupta

. Research J Pharm Tech 2020; 13(12): 5898–5902.

Simurova

Maiboroda

. Chem Heterocycl Comp 2021; 57: 420–422.

Michael

Sofou

Wahlgren

, et al. BMC Musculoskelet Disord 2021; 22: 837.

Berger

, et al. J Cell Mol Med 2020; 24(12): 6680–6689.

10.

Liu

Sun

Zhao

, et al. Exp Ther Med 2016; 12: 1611–1617.

11.

De Souza

HMR

Guedes

Freitas

RHCN

, et al. J Enzyme Inhib Med Chem 2022; 37: 718–727.

12.

Beaumont

Webster

Gardner

, et al. CDM 2003; 4: 461–485.

13.

Markovic

Ben-Shabat

Dahan

. Pharmaceutics 2020; 12: 1031.

14.

Abualhasan

Al-Masri

Manasara

, et al. Scientifica 2020; 2020: 1–6.

15.

Eichler

Klausner

, et al. Molecules 2022; 27: 8280.

16.

Tran

Truong

Nguyen

THA

, et al. J Chem Res 2023; 47: 17475198231199428.

17.

Hieu

TTH

Chi

, et al. LOC 2022; 19: 1062–1069.

18.

Zhang

, et al. Bioorganic Med Chem Lett 2019; 29: 1138–1142.

19.

Zhang

Fortin

Barnoin

, et al. Angew Chem Int Ed 2020; 59: 12062–12068.

20.

Hieu

TTH

Chi

, et al. LOC 2022; 19: 1062–1069.

21.

Santos

EDS

Nogueira

KAB

Fernandes

LCC

, et al. Int J Pharm 2021; 592: 120082.

22.

Sigismund

Avanzato

Lanzetti

. Mol Oncol 2018; 12: 3–20.

23.

Zubair

Bandyopadhyay

. Int J Mol Sci 2023; 24(3): 2651.

24.

Cao

Huong Doan

Pham

, et al. RSC Adv 2021; 11: 20173–20179.

25.

Lentini

Melfi

Di Leonardo

, et al. Mol Pharmaceutics 2014; 11: 653–664.

26.

Zakeri

Heravi

Abouzari-Lotf

. J Chem Sci 2013; 125: 731–735.

27.

Epple

. Compounds and methods for modulating g protein-coupled receptors. Patent WO2008103501A1, USA, 2008.

28.

Liu

Shi

, et al. Molecules 2013; 18: 2864–2877.

29.

Kitayama

Nakahira

Yamasaki

, et al. Tetrahedron 2013; 69: 10152–10160.

30.

Troiani

Martinelli

Capasso

, et al. CDT 2012; 13: 802–810.

31.

Gohlke

Hendlich

Klebe

. J Mol Biol 2000; 295: 337–356.

32.

Ben Issa

Sagaama

Issaoui

. Comput Biol Chem 2020; 86: 107268.

33.

Morris

Huey

Lindstrom

, et al. J Comput Chem 2009; 30: 2785–2791.

34.

Quan

Anh

HBQ

Hang

NTN

, et al. Reg Stud Mar Sci 2022; 56: 102619.

35.

Thanh

Hai

Thi Huyen

, et al. J Mol Struct 2023; 1271: 133932.

36.

Kumar

Ali

Abbas

, et al. J Biomol Struct Dyn 2023; 1–23.

37.

Likhitwitayawuid

Angerhofer

Cordell

, et al. J Nat Prod 1993; 56: 30–38.

38.

Skehan

Storeng

Scudiero

, et al. J Natl Cancer Inst 1990; 82: 1107–1112.

39.

DeLano

. The PyMOL molecular graphics system, http://wwwpymolorg/

40.

Frisch

Trucks

Schlegel

, et al. Gaussian 09, revision D. 01. Wallingford, CT: Gaussian, Inc, http://www.gaussian.com

41.

Buckley

Yoon

Yee

, et al. J Transl Med 2007; 5: 49.

42.

Park

Liu

Lemmon

, et al. Biochem J 2012; 448: 417–423.

Supplementary Material

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